WO2022093641A1 - Anti-nkg2a agents and compositions and methods for making and using the same - Google Patents

Anti-nkg2a agents and compositions and methods for making and using the same Download PDF

Info

Publication number
WO2022093641A1
WO2022093641A1 PCT/US2021/056218 US2021056218W WO2022093641A1 WO 2022093641 A1 WO2022093641 A1 WO 2022093641A1 US 2021056218 W US2021056218 W US 2021056218W WO 2022093641 A1 WO2022093641 A1 WO 2022093641A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
nkg2a
amino acid
sequence
agent
Prior art date
Application number
PCT/US2021/056218
Other languages
French (fr)
Inventor
Wei An
Takatoku Oida
David Soper
Xifeng Yang
Original Assignee
BioLegend, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by BioLegend, Inc. filed Critical BioLegend, Inc.
Publication of WO2022093641A1 publication Critical patent/WO2022093641A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • the technology relates in part to agents that bind the NK inhibitory receptor CD94/NK group 2 member A (NKG2A), particularly monoclonal antibodies, antibody fragments, and antibody derivatives specifically reactive to NKG2A under physiological and/or in vitro conditions.
  • Such agents can be useful for laboratory/research purposes (e.g., flow cytometry), and may be used in the treatment and/or prevention of various diseases or disorders through the delivery of pharmaceutical or other compositions that contain such agents.
  • Natural Killer (NK) cells play an important role in host immune regulation to a variety of pathological challenges, from viral infections to cancer. Natural Killer cells show a broad array of tissue distribution and phenotypic variability, and therefore express several activating and inhibitory receptors in order to control their function. Natural-killer group 2, member A (NKG2A) is a C-type lectin family inhibitory receptor that forms a heterodimer with CD94. NKG2A specifically recognizes the non-classical MHC molecule HLA-E, and acts as an inhibitory checkpoint receptor for HLA-E. Although HLA-E is ubiquitously expressed, it has also been associated with overexpression in several tumors. Blocking of NKG2A-HLA-E interactions could be of interest in the development of immunotherapies.
  • NKG2A a monoclonal antibody to NKG2A that provide superior target specificity, signal-to-noise ratios, and the like, as compared to other reported anti-NKG2A antibodies, as well as antigen-binding fragments thereof, that meet such needs.
  • anti-NKG2A agents that bind NKG2A, including anti-NKG2A antibodies, NKG2A-binding antibody fragments, derivatives, and variants of such antibodies and antibody fragments or antigen-binding fragments thereof (including immunoconjugates, labeled antibodies and antigen-binding antibody fragments, and the like), diagnostic reagents that comprise such agents, containers and kits that include any of the anti-NKG2A agents provided herein, and methods of making and using the same.
  • each immunoglobulin heavy chain variable domain of the anti-NKG2A agent comprises first, second, and third heavy chain complementarity determining regions (CDRs), where the first heavy chain CDR (CDRH1) comprises an amino acid sequence that is at least 80 percent identical to the amino acid sequence X 1 YX 3 MX 5 (SEQ ID NO: 22), where X 1 is S or D, X 3 is W or Y, and X 5 is N or Y; the second heavy chain CDR (CDRH2) comprises an amino acid sequence that is at least 80 percent identical to the amino acid sequence X 1 IX 3 X 4 X 5 X 6 X 7 X 8 TX 10 YX 12 X 13 X 14 X 15 KX 17 (SEQ ID NO: 23), where X
  • first anti-NKG2A agent that binds NKG2A under laboratory or physiological conditions, where the first agent competitively binds with a second anti-NKG2A agent, which the second agent comprises at least one immunoglobulin heavy chain variable domain and at least one immunoglobulin light chain variable domain, where a) each immunoglobulin heavy chain variable domain of the second agent comprises first, second, and third heavy chain complementarity determining regions (CDRs), where the first heavy chain CDR (CDRH1) comprises an amino acid sequence that is at least 80 percent identical to the amino acid sequence X 1 YX 3 MX 5 (SEQ ID NO: 22), where X 1 is S or D, X 3 is W or Y, and X 5 is N or Y; the second heavy chain CDR (CDRH2) comprises an amino acid sequence that is at least 80 percent identical to the amino acid sequence
  • X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 YX 11 X 12 X 13 X 14 X 15 (SEQ ID NO: 24), where X 1 is Q or D, X 2 is G or R, X 3 is D or no amino acid, X 4 is T or no amino acid, X 5 is S or no amino acid, X 6 is V or no amino acid, X 7 is I or Y, X 8 is W or V, X 9 is D or N, X 11 is D or Y, X 12 is G or T, X 13 is F or M, X 14 is A or D, and
  • each immunoglobulin light chain variable domain of the anti-NKG2A agent comprises first, second, and third light chain CDRs, where the first light chain CDR (CDRL1) comprises an amino acid sequence that is at least 80 percent identical to the amino acid sequence
  • X 1 ASX 4 SVX 7 X 8 X 9 X 10 X 11 X 12 YX 14 H (SEQ ID NO: 25), where X 1 is S or R, X 4 is S or K, X 7 is T or S, X 8 is T or no amino acid, X 9 is S or no amino acid, X 1 0 is G or no amino acid, X 11 is Y or no amino acid, X 12 is S or no amino acid, and X 14 is M or I;
  • the second light chain CDR (CDRL2) comprises an amino acid sequence that is at least 80 percent identical to the amino acid sequence X ⁇ SNLX 6 S (SEQ ID NO: 26), where X 1 is S or L, X 2 is T or A, and X 6 is A or E; and the third light chain CDR (CDRL3) comprises an amino acid sequence that is at least 80 percent identical to the amino acid sequence QX 2 X 3 X 4 XsX 6 PX 8 T (SEQ ID NO: 27), where X 2
  • first anti-NKG2A agent that binds NKG2A under laboratory or physiological conditions, where the first agent binds to the same epitope as a second anti-NKG2A agent, which the second agent comprises at least one immunoglobulin heavy chain variable domain and at least one immunoglobulin light chain variable domain, where a) each immunoglobulin heavy chain variable domain of the second agent comprises first, second, and third heavy chain complementarity determining regions (CDRs), where the first heavy chain CDR (CDRH1) comprises an amino acid sequence that is ⁇ t least 80 percent identical to the amino acid sequence X 1 YX 3 MX 5 (SEQ ID NO: 22), where X 1 is S or D, X 3 is W or Y, and X 5 is N or Y; the second heavy chain CDR (CDRH2) comprises an amino acid sequence that is at least 80 percent identical to the amino acid sequence X 1 IX 3 X 4 X 5 X 6 X 7 X 8 TX 10 YX 12
  • anti-NKG2A agents for detecting NKG2A in a heterogeneous population of immune cells, where NKG2A is detected at a significant level in NK cells and NK T- lymphocytes in the population, and NKG2A is not significantly detected in other immune cells in the population.
  • methods of detecting NKG2A in a heterogeneous population of immune cells comprising contacting the population with an anti-NKG2A agent provided herein, wherein NKG2A is not significantly detected in other immune cells in the population.
  • isolated, non-naturally occurring anti-NKG2A agents particularly antibodies, or antigen-binding fragments or derivatives thereof, that bind NKG2A under physiological conditions.
  • such molecules generally comprise two immunoglobulin heavy chain variable domains and two immunoglobulin light chain variable domains.
  • each of the immunoglobulin heavy and light chain variable domains comprise first, second, and third chain complementarity determining regions (CDRs) arrayed as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • the first heavy chain CDR (CDRH1) comprises an amino acid sequence that has a sequence identity of at least 65 percent, optionally a sequence identity of at least 80 percent, at least 90 percent, at least 95 percent, and 100 percent identity with the amino acid sequence SYWMN and DYYMY (SEQ ID NOS: 9 and 16, respectively)
  • the second heavy chain CDR (CDRH2) comprises an amino acid sequence that has a sequence identity of at least 65 percent, optionally a sequence identity of at least 80 percent, at least 90 percent, at least 95 percent, and 100 percent identity with the amino acid sequence RIYFEDGDTNYNGKFKG. RIYFEDGDTNYNGKFKD.
  • CDRH3 comprises an amino acid sequence that has a sequence identity of at least 65 percent, optionally a sequence identity of at least 80 percent, at least 90 percent, at least 95 percent, and 100 percent identity with the amino acid sequence QGIWDYDGFAD or DRDTSVYVNYYTMDY (SEQ ID NOS: 11 and 18, respectively).
  • the first light chain CDR (CDRL1) comprises an amino acid sequence that has a sequence identity of at least 65 percent, optionally a sequence identity of at least 80 percent, at least 90 percent, at least 95 percent, and 100 percent identity with the amino acid sequence SASSSVTYMH or RASKS VSTSGYSYIH (SEQ ID NOS: 12 and 19, respectively)
  • the second light chain CDR (CDRL2) comprises an amino acid sequence that has a sequence identity of at least 65 percent, optionally a sequence identity of at least 80 percent, at least 90 percent, at least 95 percent, and 100 percent identity with the amino acid sequence STSNLAS or LASNLES (SEQ ID NOS: 13 and 20, respectively)
  • the third light chain CDR (CDRL3) comprises an amino acid sequence that has a sequence identity of at least 65 percent, optionally a sequence identity of at least 80 percent, at least 90 percent, at least 95 percent, and 100 percent identity with the amino acid sequence QQRSSYPYT or QHSRELPLT (SEQ ID NOSEQ ID NOS
  • the isolated, non-naturally occurring anti-NKG2A antibodies, or NKG2A- binding fragments thereof including a first heavy chain CDR having the amino acid sequence SYWMN (SEQ ID NO: 9), the second heavy chain CDR has the amino acid sequence RIYFEDGDTNYNGKFKG (SEQ ID NO: 10), the third heavy chain CDR has the amino acid sequence QGIWDYDGFAD (SEQ ID NO: 11), the first light chain CDR has the amino acid sequence SASSSVTYMH (SEQ ID NO: 12), the second light chain CDR has the amino acid sequence STSNLAS (SEQ ID NO: 13), and the third light chain CDR has the amino acid sequence QQRSSYPYT (SEQ ID NO: 14).
  • the isolated anti-NKG2A agent comprises a non-naturally occurring anti- NKG2A antibody (mAb) comprising two immunoglobulin heavy chain variable domains comprising first, second, and third heavy chain complementarity determining regions (CDRH1-3, respectively) and two immunoglobulin light chain variable domains comprising first, second, and third light chain complementarity determining regions (CDRL1-3, respectively).
  • mAb non-naturally occurring anti- NKG2A antibody
  • CDRL1-3 two immunoglobulin heavy chain variable domains comprising first, second, and third heavy chain complementarity determining regions
  • CDRL1-3 light chain complementarity determining regions
  • the isolated anti-NKG2A agent comprises a non-naturally occurring anti- NKG2A antibody (mAb) comprising two immunoglobulin heavy chain variable domains and two immunoglobulin light chain variable domains, where the immunoglobulin heavy chain variable domains have an amino acid sequence selected from amongst SEQ ID NOS: 1, 3 and 4, or an amino acid sequence having at least 65%-95% or more sequence identity with any such heavy chain variable domain sequence and the immunoglobulin light chain variable domains are selected from amongst SEQ ID NOS: 5 or 8 or an amino acid sequence having at least 65%-95% or more sequence identity with any such light chain variable domain sequence.
  • mAb non-naturally occurring anti- NKG2A antibody
  • the antibodies (or fragments thereof) are monoclonal antibodies, and may be camel, human, humanized, mouse, rabbit, or other mammalian antibodies or antigen-binding antibody fragments.
  • the antibody or antigen-binding antibody fragment thereof is an IgG.
  • the IgG is an IgGl, IgG2a or IgG2b, or IgG3, or IgG4.
  • anti-NKG2A antibodies and antigen-binding antibody fragments thereof that are other than fully human antibodies (i.e., antibodies produced or derived from a mammal capable of producing all or a portion of the human antibody repertoire)
  • the molecules are chimeric or humanized anti-NKG2A antibodies and antigen-binding antibody fragments.
  • the anti-NKG2A antibody, antigen-binding antibody fragment, or derivative or variant thereof includes a detectable label.
  • the anti-NKG2A agent for example, an anti-NKG2A antibody, antigenbinding antibody fragment, or derivative or variant thereof, is part of an immunoconjugate that further includes a cytotoxic agent, for example, a nucleic acid, a peptide, a polypeptide, a small molecule, or an aptamer.
  • a cytotoxic agent for example, a nucleic acid, a peptide, a polypeptide, a small molecule, or an aptamer.
  • compositions that include an anti-NKG2A agent that is an isolated, non-naturally occurring anti-NKG2A antibody or antigen-binding antibody fragment according to the technology described herein.
  • compositions typically also include a carrier, for example, a pharmaceutically acceptable carrier.
  • a carrier for example, a pharmaceutically acceptable carrier.
  • Such compositions may be packaged in containers, which in some embodiments, are further packaged into kits that also include instructions for use.
  • kits instructions are a package insert containing not only instructions or use but also information about the pharmaceutically active ingredient (e.g., the anti-NKG2A antibody, antigen-binding antibody fragment, or derivative or variant thereof).
  • kits include a diagnostic reagent that includes an anti-NKG2A agent described herein, for example, an anti-NKG2A antibody, antigen-binding antibody fragment, or derivative or variant thereof conjugated with detectable reagents such as fluorophores or enzyme substrates and/or immobilized on a solid support.
  • detectable reagents such as fluorophores or enzyme substrates and/or immobilized on a solid support.
  • Still other aspects of the technology provided herein concern the manufacture of an anti-NKG2A agent described herein.
  • anti-NKG2A antibodies or antigen-binding antibody fragments or derivatives or variants thereof
  • one such aspect concerns isolated nucleic acid molecules that encode polypeptides provided herein.
  • such nucleic acids encode an immunoglobulin heavy chain variable domain having a first heavy chain CDR (CDRH1) that includes an amino acid sequence that has a sequence identity of at least 65%, optionally a sequence identity of at least 80%, at least 90%, at least 95%, and 100% identity with the amino acid sequence SYWMN and DYYMY (SEQ ID NOS: 9 and 16, respectively),
  • the second heavy chain CDR (CDRH2) comprises an amino acid sequence that has a sequence identity of at least 65 percent, optionally a sequence identity of at least 80 percent, at least 90 percent, at least 95 percent, and 100 percent identity with the amino acid sequence RIYFEDGDTNYNGKFKG.
  • CDRH3 comprises an amino acid sequence that has a sequence identity of at least 65 percent, optionally a sequence identity of at least 80 percent, at least 90 percent, at least 95 percent, and 100 percent identity with the amino acid sequence QGIWDYDGFAD or DRDTSVYVNYYTMDY (SEQ ID NOS: 11 and 18, respectively).
  • Such nucleic acids may also encode an immunoglobulin light chain variable domain where a first light chain CDR (CDRL1) that includes an amino acid sequence that has a sequence identity of at least 65%, optionally a sequence identity of at least 80%, at least 90%, at least 95%, and 100% identity with the amino acid sequence SASSSVTYMH or RASKSVSTSGYSYIH (SEQ ID NOS: 12 and 19, respectively),
  • the second light chain CDR (CDRL2) comprises an amino acid sequence that has a sequence identity of at least 65 percent, optionally a sequence identity of at least 80%, at least 90%, at least 95%, andl00% identity with the amino acid sequence STSNLAS or LASNLES (SEQ ID NOS: 13 and 20, respectively)
  • the third light chain CDR (CDRL3) comprises an amino acid sequence that has a sequence identity of at least 65%, optionally a sequence identity of at least 80%, at least 90%, at least 95%, and 100% identity with the amino acid sequence QQRSSYPYT or QHSRE
  • nucleic acid molecules provided herein encode an immunoglobulin heavy chain variable domain having an amino acid sequence selected from among SEQ ID NOS: 1, 3 and 4 or an amino acid sequence having at least 65%-95% or more sequence identity with any such immunoglobulin heavy chain variable domain sequence and an immunoglobulin light chain variable domain having an amino acid sequence selected from among SEQ ID NOS: 5 and 8 or an amino acid sequence having at least 65%-95% or more sequence identity with any such immunoglobulin light chain variable domain sequence.
  • aspects of the technology provided herein concern methods of treating or preventing a disease or disorder associated with aberrant levels of NKG2A. Such methods include administering to a subject in need of such treatment an anti-NKG2A agent provided herein (e.g., an anti-NKG2A antibody or antigen-binding fragment, derivative, or variant thereof) in an amount sufficient to effect treatment, thereby treating or preventing the disease or disorder.
  • an anti-NKG2A agent provided herein (e.g., an anti-NKG2A antibody or antigen-binding fragment, derivative, or variant thereof) in an amount sufficient to effect treatment, thereby treating or preventing the disease or disorder.
  • an anti-NKG2A agent provided herein e.g., an anti-NKG2A antibody or antigen-binding fragment, derivative, or variant thereof
  • an adjuvant e.g., for vaccines.
  • Fig. 1 shows the amino acid sequence of a representative full-length human NKG2A protein (SEQ ID NO: 28).
  • the amino acid residues 1-70 comprise the cytoplasmic domain
  • amino acid residues 71-93 comprise the transmembrane domain
  • amino acid residues 94-233 comprise the extracellular domain. See, e.g., NCBI Reference Sequence: NP_002250.2.
  • Fig. 2 shows the amino acid sequences of the variable domains of the immunoglobulin heavy (SEQ ID NOS: 1, 3 and 4) and light (SEQ ID NOS: 5 and 8) chains of the different anti-NKG2A antibodies (AB 1-4) provided herein.
  • the CDR regions of each of the heavy and light chains are shown in bold and underlined.
  • Fig. 3 lists the source, isotype and format for anti-NKG2A agents described herein.
  • Figs. 4A-4D depict the gating strategy used for the detection of cells expressing NKG2A, as assessed by flow cytometry.
  • Fig. 4A shows the identification of singlets based on side scatter.
  • Fig. 4B shows the forward and side scatter of singlets.
  • Fig. 4C shows lymphocytes co-stained with CD3 and CD19.
  • Fig. 4D shows CD3+CD19- lymphocytes co-stained with CD4 and CD8.
  • Figs. 5A and 5B depict results for surface expression of NKG2A on lymphocytes co-stained with CD56 and AB2.
  • Fig. 5A depicts a dot plot showing lymphocytes co-stained with AB2 and CD56.
  • Fig. 5B shows a histogram of lymphocytes stained with AB2.
  • Figs. 6A-6C depict dot plots of lymphocytes co-stained with CD56 and AB2 (Fig. 6A), Commercial ABI (Fig. 6B), and Commercial ABII (Fig. 6C).
  • Figs. 7A-7I depict the staining of subsets of CD3+CD19- lymphocytes including CD4+CD8-, CD4- CD8-, and CD4-CD8+ with AB2 (Figs.7A-7C), Commercial ABI (Figs.7D-7F), and Commercial ABII (Figs.7G-7I), respectively.
  • Figs. 8A-8H depict dot plots of lymphocytes co-stained with CD94 and ABI (Fig. 8A), CD94 and AB2 (Fig. 8B), CD94 and AB3 (Fig. 8C), and CD94 and AB4 (Fig. 8D), and CD3-CD19- lymphocytes co-stained with CD56 and ABI (Fig. 8E), CD56 and AB2 (Fig. 8F), CD56 and AB3 (Fig. 8G), and CD56 and AB4 (Fig. 8H).
  • Figs. 9A-9D depict histograms of mouse splenocytes stained with ABI (Fig. 9A), AB2 (Fig. 9B), AB3 (Fig. 9C), and AB4 (Fig. 9D).
  • Figs. 10A and 10B depict results of the anti-NKG2A antibodies described herein in a cell-mediated cytotoxicity assay.
  • Fig. 10A depicts the percent of DAPI positive cells in a population of purified primary NK and K562 cells at different effector to target ratios (E:T), with no effector used as control.
  • Fig . 1 OB depicts the percent of DAPI positive cells after treatment with AB 1 -AB4 antibodies or noantibody at lOpg/well at an E:T of 2: 1, or T only as control.
  • agents that bind NKG2A are antibodies, fragments thereof or antigen binding fragments thereof, that bind to NKG2A.
  • antibodies, fragments thereof or antigen binding fragments thereof that bind to NKG2A.
  • particular monoclonal antibodies to NKG2A that provide superior target specificity, signal-to-noise ratios, and the like, as compared to other reported anti-NKG2A antibodies, as well as antigen-binding fragments thereof that bind NKG2A, are provided herein.
  • NKG2 (Killer Cell Lectin-Like Receptor), also known as Cluster of Differentiation 159 (CD 159), is a family of type II transmembrane protein receptors.
  • the human NKG2 family comprises seven members referred to as NKG2A, NKG2B, NKG2C, NKG2D, NKG2E, NKG2F, and NKG2H. With the exception ofNKG2D, all other NKG2 members share substantial sequence homology.
  • NKG2A, NKG2B, NKG2C, NKG2E and NKG2H have been shown to form disulfide-linked heterodimers with the invariant Cluster of Differentiation 94 (CD94) protein, to form functionally distinct heterodimeric receptors.
  • CD94 Cluster of Differentiation 94
  • CD94 associated NKG2 family members are comprised of C-type lectin receptors that are predominantly expressed on the surface of NK cells, as well as a subset of CD8 + T-lymphocytes.
  • human CD94/NKG2 family has been found to recognize and interact with the same ligand, Human Leukocyte Antigen (HLA)-E, on target cells, different receptor family members can transmit opposing signals via their intracellular domains.
  • HLA Human Leukocyte Antigen
  • NKG2A and NKG2B receptors contain immunoreceptor tyrosine-based inhibition motifs (ITAM) and transmit inhibitory signals
  • the NKG2C, NKG2D, NKG2E, and NKG2H receptors are characterized by the presence of a charged amino acid residue in the transmembrane domain mediating interaction with DAP-12, an adapter molecule containing an immunoreceptor tyrosine-based activation motif (ITAM), that transmit activating signals.
  • ITAM immunoreceptor tyrosine-based activation motif
  • the murine CD94-NKG2 receptors recognize the class Ib molecule Qa-1b.
  • the CD94/NK group 2-member A (NKG2A) heterodimeric receptor is one of the most prominent NK inhibitory receptors (Lazetic et al J Immunol.1996;157(11):4741-4757), and is also expressed on a subset of T cells ( ⁇ / ⁇ and ⁇ / ⁇ ). It binds to the nonclassical minimally polymorphic HLA class I molecule (HLA-E), which presents peptides derived from leader peptide sequences of other HLA class I molecules, such as HLA-G (Braud et al. Nature 1998;391(6669):795-799; Lee et al. Proc Natl Acad SciUSA 1998;95(9):5199-5204; Miller et al.
  • HLA-E nonclassical minimally polymorphic HLA class I molecule
  • Anti-NKG2A agents e.g., anti-NKG2A antibodies
  • the anti-NKG2A agents provided herein bind all or a portion of the NKG2A receptor, or a fragment thereof. In some embodiments, the antibody or antigen-binding fragment thereof provided herein bind the NKG2A receptor or a fragment thereof. In some embodiments, the anti-NKG2A agents provided herein bind a portion of the NKG2A receptor that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:28.
  • the antibody or antigen-binding fragment thereof provided herein bind the NKG2A receptor or a fragment thereof that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:28.
  • the anti-NKG2A agents provided herein bind all or a portion of the NKG2A receptor described in SEQ ID NO:28.
  • the antibody or antigen-binding fragment thereof provided herein bind the NKG2A receptor or a fragment thereof comprising the sequence of amino acids described in SEQ ID NO:28.
  • Precursor human NKG2A protein sequence (UniProtKB - P26715 (NKG2A HUMAN)/ NCBI Reference Sequence: NP 002250,2) (SEQ ID NO: 28)
  • the human NKG2A protein sequence comprises a cytoplasmic domain, a transmembrane domain, and an extracellular domain.
  • the human NKG2A protein sequence comprises a cytoplasmic domain corresponding to amino acid residues 1-70 of SEQ ID NO:28. In some embodiments, the cytoplasmic domain corresponds to the sequence of amino acids of SEQ ID NO:2. In some embodiments, the human NKG2A protein sequence comprises a transmembrane domain corresponding to amino acid residues 71-93 of SEQ ID NO:28. In some embodiments, the transmembrane domain corresponds to the sequence of amino acids of SEQ ID NO:6. In some embodiments, the human NKG2A protein sequence comprises an extracellular domain corresponding to amino acid residues 94-233 of SEQ ID NO:28. In some embodiments, the extracellular domain corresponds to the sequence of amino acids of SEQ ID NO:7.
  • the anti-NKG2A agents provided herein bind all or a portion of the NKG2A receptor extracellular domain. In some embodiments, the anti-NKG2A agents provided herein bind all or a portion of the NKG2A receptor extracellular domain corresponding to amino acid residues 94- 233 of SEQ ID NO:28. In some embodiments, the anti-NKG2A agents provided herein bind all or a portion of the NKG2A receptor extracellular domain described in SEQ ID NO: 7.
  • the anti-NKG2A agents provided herein bind all or a portion of the NKG2A receptor extracellular domain that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid residues 94-233 of SEQ ID NO:28.
  • the anti- NKG2A agents provided herein bind all or a portion of the NKG2A receptor extracellular domain that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:7.
  • the antibody or antigen-binding fragment thereof provided herein bind the NKG2A receptor extracellular domain described in SEQ ID NO:7.
  • anti-NKG2A agents may be chimeric antibodies.
  • anti- NKG2A agents may be humanized antibodies.
  • anti-NKG2A agents may be variant antibodies.
  • Antibodies may have beneficial properties from a therapeutic perspective.
  • Assays for determining the activity of anti-NKG2A antibodies provided herein include, for example, cell-based ELISA (e.g., to measure relative avidity of the antibody for the target on cells), flow cytometry (e.g., to measure cell specificity of the antibody), and cytotoxicity (e.g., to measure potential to mediate direct or indirect killing of NKG2A-expressing target cells).
  • a humanized or variant antibody fails to elicit an immunogenic response upon administration of a therapeutically effective amount of the antibody to a human patient.
  • the response may be such that the antibody still provides a therapeutic benefit to the patient treated therewith.
  • the anti-NKG2A agents bind the same epitope.
  • a cross-blocking assay such as that described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988), can be performed.
  • epitope mapping e.g., as described in Champe et al., J. Biol. Chem.
  • the anti-NKG2A agents provided herein may include anti-NKG2A antibodies, anti-NKG2A antibody fragments (e.g., anti-NKG2A antigen binding fragments), and anti-NKG2A antibody derivatives.
  • the agent is isolated (e.g., separated from a component of its natural environment (e.g. an animal, a biological sample)).
  • the agent is non-naturally occurring (e.g., produced by human intervention).
  • the agent is a humanized antibody, or an antigen binding fragment thereof.
  • the agent is a derivative of a humanized antibody that binds NKG2A.
  • the agent binds NKG2A under laboratory conditions (e.g., binds NKG2A in vitro, binds NKG2A in a flow cytometry assay, binds NKG2A in an ELISA). In some embodiments, the agent binds NKG2A under physiological conditions (e.g., binds NKG2A in a cell in a subject).
  • Antibodies such as those described herein may have an immunoglobulin heavy chain variable domain comprising an amino acid sequence represented by the formula: ERH1-CDRH1-ERH2-CDRH2- ERH3-CDRH3-ERH4, where “ERH1-4” represent the four heavy chain framework regions and “CDRH1-3” represent the three hypervariable regions of an anti-NKG2A antibody variable heavy domain.
  • FRH1-4 may be derived from a consensus sequence (for example the most common amino acids of a class, subclass or subgroup of heavy or light chains of human immunoglobulins) or may be derived from an individual human antibody framework region or from a combination of different framework region sequences. Many human antibody framework region sequences are compiled in Kabat et al.
  • variable heavy FR is provided by a consensus sequence of a human immunoglobulin subgroup as compiled by Kabat et al., supra.
  • the human variable heavy FR sequence may have substitutions therein, e.g. where the human FR residue is replaced by a corresponding nonhuman residue (by “corresponding nonhuman residue” is meant the nonhuman residue with the same Kabat positional numbering as the human residue of interest when the human and nonhuman sequences are aligned), but replacement with the nonhuman residue is not necessary.
  • a replacement FR residue other than the corresponding nonhuman residue may be selected by phage display.
  • Antibodies such as those described herein may have an immunoglobulin light chain variable domain comprising an amino acid sequence represented by the formula: FRL1-CDRL1-FRL2-CDRL2-FRL3- CDRL3-FRL4, where “FRL1-4” represents the four framework regions and “CDRL1-3” represents the three hypervariable regions of an anti-NKG2A antibody variable light domain.
  • FRL1-4 may be derived from a consensus sequence (for example the most common amino acids of a class, subclass or subgroup of heavy or light chains of human immunoglobulins) or may be derived from an individual human antibody framework region or from a combination of different framework region sequences.
  • the immunoglobulin variable light FR is provided by a consensus sequence of a human immunoglobulin subgroup as compiled by Kabat et al., supra.
  • the human immunoglobulin variable light FR sequence may have substitutions therein, e.g. where the human FR residue is replaced by a corresponding mouse residue, but replacement with the nonhuman residue is not necessary. For example, a replacement residue other than the corresponding nonhuman residue may be selected by phage display.
  • the anti-NKG2A agent provided herein comprises at least one immunoglobulin heavy chain variable domain and at least one immunoglobulin light chain variable domain.
  • an anti-NKG2A agent herein comprises two immunoglobulin heavy chain variable domains and two immunoglobulin light chain variable domains.
  • each immunoglobulin heavy chain variable domain of the anti-NKG2A agent comprises first, second, and third heavy chain complementarity determining regions (CDRs; CDRH1, CDRH2, CDRH3)
  • each immunoglobulin light chain variable domain of the anti-NKG2A agent comprises first, second, and third light chain CDRs (CDRL1, CDRL2, CDRL3).
  • the first heavy chain CDR (CDRH1) of an anti-NKG2A agent provided herein comprises an amino acid sequence that is at least 80% identical to the amino acid sequence
  • the CDRH1 comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 22. In some embodiments, the CDRH1 comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 22. In some embodiments, the CDRH1 comprises an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO: 22. In some embodiments, the CDRH1 comprises the sequence of amino acids set forth in SEQ ID NO: 22.
  • the amino acid X 1 of SEQ ID NO: 22 may be substituted with any amino acid.
  • the amino acid X 1 of SEQ ID NO: 22 is substituted with a conservative amino acid (e.g., conservative to S and/or D).
  • the amino acid X 1 of SEQ ID NO: 22 is substituted with a hydrophilic amino acid.
  • the amino acid X 1 of SEQ ID NO: 22 is substituted with an acidic amino acid.
  • the amino acid X 3 of SEQ ID NO: 22 may be substituted with any amino acid.
  • the amino acid X 3 of SEQ ID NO: 22 is substituted with a conservative amino acid (e.g., conservative to W and/or Y).
  • the amino acid X 3 of SEQ ID NO: 22 is substituted with an aromatic amino acid.
  • the amino acid X 5 of SEQ ID NO: 22 may be substituted with any amino acid.
  • the amino acid X 5 of SEQ ID NO: 22 is substituted with a conservative amino acid (e.g., conservative to N and/or Y).
  • the amino acid X 5 of SEQ ID NO: 22 is substituted with a basic amino acid.
  • the amino acid X 5 of SEQ ID NO: 22 is substituted with an aromatic amino acid.
  • the CDRH1 of any of the anti-NKG2A agents provided herein comprises an amino acid sequence chosen from SYWMN (SEQ ID NO: 9) and DYYMY (SEQ ID NO: 16).
  • the CDRH1 of any of the anti-NKG2A agents provided herein comprises the sequence of amino acids SYWMN (SEQ ID NO: 9); or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:9.
  • the CDRH1 of any of the anti-NKG2A agents provided herein comprises the sequence of amino acids SYWMN (SEQ ID NO: 9).
  • the CDRH1 of any of the anti-NKG2A agents provided herein comprises the sequence of amino acids DYYMY (SEQ ID NO: 16); or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:9 or SEQ ID NO: 16.
  • the CDRH1 of any of the anti-NKG2A agents provided herein comprises the sequence of amino acids DYYMY (SEQ ID NO: 16).
  • the second heavy chain CDR (CDRH2) of an anti-NKG2A agent provided herein comprises an amino acid sequence that is at least 80 percent identical to the amino acid sequence X 1 D ⁇ XsX 6 XvX 6 TX 10 YX 11 XBX 11 X 1 sKX 11 (SEQ ID NO: 23), where X 1 is R or T; X 3 is Y or S; X 4 is F or Y; X 5 is E or G; X 6 is D or G; X 7 is G or I; X 6 is D or Y; X 1 0 is N or Y; X 12 is N or P;
  • the CDRH2 comprises an amino acid sequence that is at least 90 percent identical to the amino acid sequence of SEQ ID NO: 23. In some embodiments, the CDRH2 comprises an amino acid sequence that is at least 95 percent identical to the amino acid sequence of SEQ ID NO: 23. In some embodiments, the CDRH2 comprises an amino acid sequence that is 100 percent identical to the amino acid sequence of SEQ ID NO: 23. In some embodiments, the CDRH2 comprises the sequence of amino acids set forth in SEQ ID NO: 23.
  • the amino acid X 1 of SEQ ID NO: 23 may be substituted with any amino acid. In some embodiments, the amino acid X 1 of SEQ ID NO: 23 is substituted with a conservative amino acid (e.g., conservative to R and/or T). In some embodiments, the amino acid X 1 of SEQ ID NO: 23 is substituted with a basic amino acid. In some embodiments, the amino acid X 1 of SEQ ID NO: 23 is substituted with a neutral amino acid.
  • a conservative amino acid e.g., conservative to R and/or T
  • the amino acid X 1 of SEQ ID NO: 23 is substituted with a basic amino acid. In some embodiments, the amino acid X 1 of SEQ ID NO: 23 is substituted with a neutral amino acid.
  • the amino acid X 3 of SEQ ID NO: 23 may be substituted with any amino acid.
  • the amino acid X 3 of SEQ ID NO: 23 is substituted with a conservative amino acid (e.g., conservative to Y and/or S).
  • the amino acid X 3 of SEQ ID NO: 23 is substituted with an aromatic amino acid.
  • the amino acid X 3 of SEQ ID NO: 23 is substituted with a neutral amino acid.
  • the amino acid X 4 of SEQ ID NO: 23 may be substituted with any amino acid. In some embodiments, the amino acid X 4 of SEQ ID NO: 23 is substituted with a conservative amino acid (e.g., conservative to F and/or Y). In some embodiments, the amino acid X 4 of SEQ ID NO: 23 is substituted with an aromatic amino acid.
  • the amino acid X 5 of SEQ ID NO: 23 may be substituted with any amino acid. In some embodiments, the amino acid X 5 of SEQ ID NO: 23 is substituted with a conservative amino acid (e.g., conservative to E and/or G). In some embodiments, the amino acid X 5 of SEQ ID NO: 23 is substituted with an acidic amino acid.
  • the amino acid X 5 of SEQ ID NO: 23 is substituted with an amino acid residue that influences chain orientation.
  • the amino acid X 6 of SEQ ID NO: 23 may be substituted with any amino acid.
  • the amino acid X 6 of SEQ ID NO: 23 is substituted with a conservative amino acid (e.g., conservative to D and/or G).
  • the amino acid of SEQ ID NO: 23 is substituted with an acidic amino acid.
  • the amino acid X 6 of SEQ ID NO: 23 is substituted with an amino acid residue that influences chain orientation.
  • the amino acid X 7 of SEQ ID NO: 23 may be substituted with any amino acid.
  • the amino acid X 7 of SEQ ID NO: 23 is substituted with a conservative amino acid (e.g., conservative to G and/or I). In some embodiments, the amino acid X 7 of SEQ ID NO: 23 is substituted with an amino acid residue that influences chain orientation. In some embodiments, the amino acid X 7 of SEQ ID NO: 23 is substituted with a hydrophobic amino acid.
  • the amino acid X 8 of SEQ ID NO: 23 may be substituted with any amino acid. In some embodiments, the amino acid X 8 of SEQ ID NO: 23 is substituted with a conservative amino acid (e.g., conservative to D and/or Y).
  • the amino acid X 8 of SEQ ID NO: 23 is substituted with an acidic amino acid. In some embodiments, the amino acid X 8 of SEQ ID NO: 23 is substituted with an aromatic amino acid.
  • the amino acid X 10 of SEQ ID NO: 23 may be substituted with any amino acid. In some embodiments, the amino acid X 10 of SEQ ID NO: 23 is substituted with a conservative amino acid (e.g., conservative to N and/or Y). In some embodiments, the amino acid X 10 of SEQ ID NO: 23 is substituted with a basic amino acid. In some embodiments, the amino acid X 10 of SEQ ID NO: 23 is substituted with an aromatic amino acid.
  • the amino acid X 12 of SEQ ID NO: 23 may be substituted with any amino acid. In some embodiments, the amino acid X 12 of SEQ ID NO: 23 is substituted with a conservative amino acid (e.g., conservative to N and/or P). In some embodiments, the amino acid of SEQ ID NO: 23 is substituted with a basic amino acid. In some embodiments, the amino acid is substituted with an amino acid residue that influences chain orientation.
  • the amino acid X 13 of SEQ ID NO: 23 may be substituted with any amino acid. In some embodiments, the amino acid X 13 of SEQ ID NO: 23 is substituted with a conservative amino acid (e.g., conservative to G and/or D). In some embodiments, the amino acid X 13 of SEQ ID NO: 23 is substituted with an amino acid residue that influences chain orientation. In some embodiments, the amino acid X 13 of SEQ ID NO: 23 is substituted with an acidic amino acid.
  • the amino acid X 14 of SEQ ID NO: 23 may be substituted with any amino acid.
  • the amino acid X 14 of SEQ ID NO: 23 is substituted with a conservative amino acid (e.g., conservative to K and/or S).
  • the amino acid X 14 of SEQ ID NO: 23 is substituted with a basic amino acid.
  • the amino acid X 14 of SEQ ID NO: 23 is substituted with a neutral amino acid.
  • the amino acid X 15 of SEQ ID NO: 23 may be substituted with any amino acid.
  • the amino acid X 15 of SEQ ID NO: 23 is substituted with a conservative amino acid (e.g., conservative to F and/or V).
  • the amino acid X 15 of SEQ ID NO: 23 is substituted with an aromatic amino acid.
  • the amino acid X 15 of SEQ ID NO: 23 is substituted with a hydrophobic amino acid.
  • the amino acid X 17 of SEQ ID NO: 23 may be substituted with any amino acid.
  • the amino acid X 17 of SEQ ID NO: 23 is substituted with a conservative amino acid (e.g., conservative to G and/or D).
  • the amino acid X 17 of SEQ ID NO: 23 is substituted with an amino acid residue that influences chain orientation.
  • the amino acid X 17 of SEQ ID NO: 23 is substituted with an acidic amino acid.
  • the CDRH2 of any of the anti-NKG2A agents provided herein comprises an amino acid sequence chosen from IUYFEDGDTNYNGKFKG (SEQ ID NO: 10), RIYFEDGDTNYNGKFKD (SEQ ID NO: 15), and TISYGGIYTYYPDSVKG (SEQ ID NO: 17).
  • the CDRH2 of any of the anti-NKG2A agents provided herein comprises the sequence of amino acids IUYFEDGDTNYNGKFKG (SEQ ID NO: 10); or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
  • the CDRH2 of any of the anti-NKG2A agents provided herein comprises the sequence of amino acids IUYFEDGDTNYNGKFKG (SEQ ID NO: 10).
  • the CDRH2 of any of the anti-NKG2A agents provided herein comprises the sequence of amino acids IUYFEDGDTNYNGKFKD (SEQ ID NO: 15); or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
  • the CDRH2 of any of the anti-NKG2A agents provided herein comprises the sequence of amino acids RIYFEDGDTNYNGKFKD (SEQ ID NO: 15).
  • the CDRH2 of any of the anti-NKG2A agents provided herein comprises the sequence of amino acids TISYGGIYTYYPDSVKG (SEQ ID NO: 17); or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 17.
  • the CDRH2 of any of the anti-NKG2A agents provided herein comprises the sequence of amino acids TISYGGIYTYYPDSVKG (SEQ ID NO: 17).
  • the third heavy chain CDR (CDRH3) of an anti-NKG2A agent provided herein comprises an amino acid sequence that is at least 80 percent identical to the amino acid sequence X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 YX 11 X 12 X 13 X 14 X 15 (SEQ ID NO: 24), where X 1 is Q or D; X 2 is G or R; X 3 is D or no amino acid; X 4 is T or no amino acid; X 5 is S or no amino acid; X 6 is V or no amino acid; X 7 is I or Y; X 8 is W or V; X 9 is D or N; X 11 is D or Y; X 12 is G or T; X 13 is F or M; X 14 is A or D; and X 15 is D or Y.
  • the third heavy chain CDR (CDRH3) of an anti-NKG2A agent provided herein comprises an amino acid sequence that is at least 90 percent identical to the amino acid sequence of SEQ ID NO: 24. In some embodiments, the CDRH3 comprises an amino acid sequence that is at least 95 percent identical to the amino acid sequence of SEQ ID NO: 24. In some embodiments, the CDRH3 comprises an amino acid sequence of SEQ ID NO: 24. In some embodiments, the CDRH3 comprises an amino acid sequence that is 100 percent identical to the amino acid sequence of SEQ ID NO: 24. In some embodiments, the CDRH3 comprises the sequence of amino acids set forth in SEQ ID NO: 24. The amino acid X 1 of SEQ ID NO: 24 may be substituted with any amino acid.
  • the amino acid X 1 of SEQ ID NO: 24 is substituted with a conservative amino acid (e.g., conservative to Q and/or D). In some embodiments, the amino acid X 1 of SEQ ID NO: 24 is substituted with a basic amino acid. In some embodiments, the amino acid X 1 of SEQ ID NO: 24 is substituted with an acidic amino acid. The amino acid X 2 of SEQ ID NO: 24 may be substituted with any amino acid. In some embodiments, the amino acid X 2 of SEQ ID NO: 24 is substituted with a conservative amino acid (e.g., conservative to G and/or R). In some embodiments, the amino acid X 2 of SEQ ID NO: 24 is substituted with an amino acid residue that influences chain orientation.
  • a conservative amino acid e.g., conservative to Q and/or D
  • the amino acid X 1 of SEQ ID NO: 24 is substituted with a basic amino acid. In some embodiments, the amino acid X 1 of SEQ ID NO: 24 is substituted with an
  • the amino acid X 2 of SEQ ID NO: 24 is substituted with a basic amino acid.
  • the amino acid X 3 of SEQ ID NO: 24 may be substituted with any amino acid.
  • the amino acid X 3 of SEQ ID NO: 24 is substituted with a conservative amino acid (e.g., conservative to D and/or no amino acid).
  • the amino acid X 3 of SEQ ID NO: 24 is substituted with an acidic amino acid.
  • X 3 of SEQ ID NO: 24 is no amino acid.
  • the amino acid X 4 of SEQ ID NO: 24 may be substituted with any amino acid. In some embodiments, the amino acid X 4 of SEQ ID NO: 24 is substituted with a conservative amino acid (e.g., conservative to T and/or no amino acid). In some embodiments, the amino acid X 4 of SEQ ID NO: 24 is substituted with a neutral amino acid. In some embodiments, the amino acid X 4 of SEQ ID NO: 24 is no amino acid.
  • the amino acid X 5 of SEQ ID NO: 24 may be substituted with any amino acid. In some embodiments, the amino acid X 5 of SEQ ID NO: 24 is substituted with a conservative amino acid (e.g., conservative to S and/or no amino acid). In some embodiments, the amino acid X 5 of SEQ ID NO: 24 is substituted with a neutral amino acid. In some embodiments, the amino acid X 5 of SEQ ID NO: 24 is no amino acid.
  • the amino acid X 6 of SEQ ID NO: 24 may be substituted with any amino acid. In some embodiments, the amino acid X 6 of SEQ ID NO: 24 is substituted with a conservative amino acid (e.g., conservative to V and/or no amino acid). In some embodiments, the amino acid X 6 of SEQ ID NO: 24 is substituted with a hydrophobic amino acid. In some embodiments, the amino acid X 6 of SEQ ID NO: 24 is no amino acid.
  • the amino acid X 7 of SEQ ID NO: 24 may be substituted with any amino acid.
  • the amino acid X 7 of SEQ ID NO: 24 is substituted with a conservative amino acid (e.g., conservative to I and/or Y).
  • the amino acid X 7 of SEQ ID NO: 24 is substituted with a hydrophobic amino acid.
  • the amino acid X 7 of SEQ ID NO: 24 is substituted with an aromatic amino acid.
  • the amino acid X 6 of SEQ ID NO: 24 may be substituted with any amino acid.
  • the amino acid X 6 of SEQ ID NO: 24 is substituted with a conservative amino acid (e.g., conservative to W and/or V).
  • the amino acid X 6 of SEQ ID NO: 24 is substituted with an aromatic amino acid.
  • the amino acid X 6 of SEQ ID NO: 24 is substituted with a hydrophobic amino acid.
  • the amino acid X 9 of SEQ ID NO: 24 may be substituted with any amino acid. In some embodiments, the amino acid X 9 of SEQ ID NO: 24 is substituted with a conservative amino acid (e.g., conservative to D and/or N). In some embodiments, the amino acid X 9 of SEQ ID NO: 24 is substituted with an acidic amino acid. In some embodiments, the amino acid X 9 of SEQ ID NO: 24 is substituted with a basic amino acid.
  • the amino acid X 11 of SEQ ID NO: 24 may be substituted with any amino acid. In some embodiments, the amino acid X 11 of SEQ ID NO: 24 is substituted with a conservative amino acid (e.g., conservative to D and/or Y). In some embodiments, the amino acid X 11 of SEQ ID NO: 24 is substituted with an acidic amino acid. In some embodiments, the amino acid X 11 of SEQ ID NO: 24 is substituted with an aromatic amino acid.
  • the amino acid X 11 of SEQ ID NO: 24 may be substituted with any amino acid.
  • the amino acid XB of SEQ ID NO: 24 is substituted with a conservative amino acid (e.g., conservative to G and/or T).
  • the amino acid X of SEQ ID NO: 24 is substituted with an amino acid residue that influences chain orientation.
  • the amino acid XB of SEQ ID NO: 24 is substituted with a neutral amino acid.
  • the amino acid XB of SEQ ID NO: 24 may be substituted with any amino acid.
  • the amino acid XB of SEQ ID NO: 24 is substituted with a conservative amino acid (e.g., conservative to F and/or M).
  • the amino acid X of SEQ ID NO: 24 is substituted with an aromatic amino acid.
  • the amino acid XB of SEQ ID NO: 24 is substituted with a hydrophobic amino acid.
  • the amino acid X 14 of SEQ ID NO: 24 may be substituted with any amino acid.
  • the amino acid X 11 of SEQ ID NO: 24 is substituted with a conservative amino acid (e.g., conservative to A and/or D).
  • the amino acid X 11 of SEQ ID NO: 24 is substituted with a hydrophobic amino acid.
  • the amino acid X 11 of SEQ ID NO: 24 is substituted with an acidic amino acid.
  • the amino acid XB of SEQ ID NO: 24 may be substituted with any amino acid.
  • the amino acid X of SEQ ID NO: 24 is substituted with a conservative amino acid (e.g., conservative to D and/or Y).
  • the amino acid XB of SEQ ID NO: 24 is substituted with an acidic amino acid.
  • the amino acid XB of SEQ ID NO: 24 is substituted with an aromatic amino acid.
  • the CDRH3 of any of the anti-NKG2A agents provided herein comprises an amino acid sequence chosen from QGIWDYDGFAD (SEQ ID NO: 11) and DRDTSVYVNYYTMDY (SEQ ID NO: 18).
  • the CDRH3 of any of the anti-NKG2A agents provided herein comprises the sequence of amino acids QGIWDYDGFAD (SEQ ID NO: 11); or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 11.
  • the CDRH3 of any of the anti-NKG2A agents provided herein comprises the sequence of amino acids QGIWDYDGFAD (SEQ ID NO: 11).
  • the CDRH3 of any of the anti-NKG2A agents provided herein comprises the sequence of amino acids DRDTSVYVNYYTMDY (SEQ ID NO: 18); or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 18.
  • the CDRH3 of any of the anti-NKG2A agents provided herein comprises the sequence of amino acids DRDTSVYVNYYTMDY (SEQ ID NO: 18).
  • the first light chain CDR (CDRL1) of an anti-NKG2A agent provided herein comprises an amino acid sequence that is at least 80 percent identical to the amino acid sequence
  • X 1 ASX 4 SVX 7 X 8 X 9 X 1 0X 11 X 12 YX 14 H (SEQ ID NO: 25), where XI is S or R; X 4 is S or K; X 7 is T or S; X 8 is T or no amino acid; X 9 is S or no amino acid; X 1 0 is G or no amino acid; XI 1 is Y or no amino acid; X 1 2 is S or no amino acid; and X 1 4 is M or I.
  • the CDRL1 comprises an amino acid sequence that is at least 90 percent identical to the amino acid sequence of SEQ ID NO: 25.
  • the CDRL1 comprises an amino acid sequence that is at least 95 percent identical to the amino acid sequence of SEQ ID NO: 25. In some embodiments, the CDRL1 comprises an amino acid sequence that is 100 percent identical to the amino acid sequence of SEQ ID NO: 25. In some embodiments, the CDRL1 comprises the sequence of amino acids set forth in SEQ ID NO: 25.
  • the amino acid X 1 of SEQ ID NO: 25 may be substituted with any amino acid.
  • the amino acid X 1 of SEQ ID NO: 25 is substituted with a conservative amino acid (e.g., S and/or R).
  • the amino acid X 1 of SEQ ID NO: 25 is substituted with a neutral amino acid.
  • the amino acid X 1 of SEQ ID NO: 25 is substituted with a basic amino acid.
  • the amino acid X 4 of SEQ ID NO: 25 may be substituted with any amino acid.
  • the amino acid X 4 of SEQ ID NO: 25 is substituted with a conservative amino acid (e.g., S and/or K).
  • the amino acid X 4 of SEQ ID NO: 25 is substituted with a neutral amino acid.
  • the amino acid X 4 of SEQ ID NO: 25 is substituted with a basic amino acid.
  • the amino acid X 7 of SEQ ID NO: 25 may be substituted with any amino acid. In some embodiments, the amino acid X 7 of SEQ ID NO: 25 is substituted with a conservative amino acid (e.g., T and/or S). In some embodiments, the amino acid X 7 of SEQ ID NO: 25 is substituted with a neutral amino acid. The amino acid X 8 of SEQ ID NO: 25 may be substituted with any amino acid. In some embodiments, the amino acid X 8 of SEQ ID NO: 25 is substituted with a conservative amino acid (e.g., T and/or no amino acid). In some embodiments, the amino acid X 8 of SEQ ID NO: 25 is substituted with a neutral amino acid.
  • a conservative amino acid e.g., T and/or S
  • the amino acid X 8 of SEQ ID NO: 25 is substituted with a neutral amino acid.
  • the amino acid X 8 of SEQ ID NO: 25 is no amino acid.
  • the amino acid X 9 of SEQ ID NO: 25 may be substituted with any amino acid.
  • the amino acid X 9 of SEQ ID NO: 25 is substituted with a conservative amino acid (e.g., S and/or no amino acid).
  • the amino acid X 9 of SEQ ID NO: 25 is substituted with a neutral amino acid.
  • the amino acid X 9 of SEQ ID NO: 25 is no amino acid.
  • the amino acid X 10 of SEQ ID NO: 25 may be substituted with any amino acid.
  • the amino acid X 10 of SEQ ID NO: 25 is substituted with a conservative amino acid (e.g., G and/or no amino acid). In some embodiments, the amino acid X 10 of SEQ ID NO: 25 is substituted with an amino acid residue that influences chain orientation. In some embodiments, the amino acid X 10 of SEQ ID NO: 25 is no amino acid.
  • the amino acid X 11 of SEQ ID NO: 25 may be substituted with any amino acid. In some embodiments, the amino acid X 11 of SEQ ID NO: 25 is substituted with a conservative amino acid (e.g., Y and/or no amino acid). In some embodiments, the amino acid X 11 of SEQ ID NO: 25 is substituted with an aromatic amino acid.
  • the amino acid X 11 of SEQ ID NO:25 is no amino acid.
  • the amino acid X 12 of SEQ ID NO: 25 may be substituted with any amino acid.
  • the amino acid X 12 of SEQ ID NO: 25 is substituted with a conservative amino acid (e.g., S and/or no amino acid).
  • the amino acid X 12 of SEQ ID NO: 25 is substituted with a neutral amino acid.
  • the amino acid X 12 of SEQ ID NO: 25 is no amino acid.
  • the amino acid X14 of SEQ ID NO: 25 may be substituted with any amino acid.
  • the amino acid X14 of SEQ ID NO: 25 is substituted with a conservative amino acid (e.g., M and/or I). In some embodiments, the amino acid X14 of SEQ ID NO: 25 is substituted with a hydrophobic amino acid.
  • the CDRL1 of any of the anti-NKG2A agents provided herein comprises an amino acid sequence chosen from SASSSVTYMH (SEQ ID NO: 12) and RASKSVSTSGYSYIH (SEQ ID NO: 19).
  • the CDRL1 of any of the anti-NKG2A agents provided herein comprises the sequence of amino acids SASSSVTYMH (SEQ ID NO: 12); or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
  • the CDRL1 of any of the anti-NKG2A agents provided herein comprises the sequence of amino acids SASSSVTYMH (SEQ ID NO: 12).
  • the CDRL1 of any of the anti-NKG2A agents provided herein comprises the sequence of amino acids RASKSVSTSGYSYIH (SEQ ID NO: 19); or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 19 .
  • the CDRL1 of any of the anti-NKG2A agents provided herein comprises the sequence of amino acids RASKSVSTSGYSYIH (SEQ ID NO: 19).
  • the second light chain CDR (CDRL2) of an anti-NKG2A agent provided herein comprises an amino acid sequence that is at least 80 percent identical to the amino acid sequence X ⁇ SNLX 6 S (SEQ ID NO: 26), where XI is S or L, X 2 is T or A, and X 6 is A or E.
  • the CDRL2 comprises an amino acid sequence that is at least 90 percent identical to the amino acid sequence of SEQ ID NO: 26.
  • the CDRL2 comprises an amino acid sequence that is at least 95 percent identical to the amino acid sequence of SEQ ID NO: 26.
  • the CDRL2 comprises an amino acid sequence of SEQ ID NO: 26.
  • the CDRL2 comprises an amino acid sequence that is 100 percent identical to the amino acid sequence of SEQ ID NO: 26.
  • the CDRL2 comprises the sequence of amino acids set forth in SEQ ID NO: 26.
  • the amino acid X 1 of SEQ ID NO: 26 may be substituted with any amino acid. In some embodiments, the amino acid X 1 of SEQ ID NO: 26 is substituted with a conservative amino acid (e.g., S and/or L). In some embodiments, the amino acid X 1 of SEQ ID NO: 26 is substituted with a neutral amino acid. In some embodiments, the amino acid X 1 of SEQ ID NO: 26 is substituted with a hydrophobic amino acid.
  • a conservative amino acid e.g., S and/or L
  • the amino acid X 1 of SEQ ID NO: 26 is substituted with a neutral amino acid. In some embodiments, the amino acid X 1 of SEQ ID NO: 26 is substituted with a hydrophobic amino acid.
  • the amino acid X 2 of SEQ ID NO: 26 may be substituted with any amino acid. In some embodiments, the amino acid X 2 of SEQ ID NO: 26 is substituted with a conservative amino acid (e.g., T and/or A). In some embodiments, the amino acid X 2 of SEQ ID NO: 26 is substituted with a neutral amino acid. In some embodiments, the amino acid X 2 of SEQ ID NO: 26 is substituted with a hydrophobic amino acid. The amino acid X 6 of SEQ ID NO: 26 may be substituted with any amino acid. In some embodiments, the amino acid X 6 of SEQ ID NO: 26 is substituted with a conservative amino acid (e.g., A and/or E).
  • a conservative amino acid e.g., T and/or A
  • the amino acid X 6 of SEQ ID NO: 26 is substituted with a hydrophobic amino acid. In some embodiments, the amino acid X 6 of SEQ ID NO: 26 is substituted with an acidic amino acid.
  • the CDRL2 of any of the anti-NKG2A agents provided herein comprises an amino acid sequence chosen from STSNLAS (SEQ ID NO: 13) and LASNLES (SEQ ID NO: 20).
  • the CDRL2 of any of the anti-NKG2A agents provided herein comprises the sequence of amino acids STSNLAS (SEQ ID NO: 13); or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 13.
  • the CDRL2 of any of the anti-NKG2A agents provided herein comprises the sequence of amino acids STSNLAS (SEQ ID NO: 13).
  • the CDRL2 of any of the anti-NKG2A agents provided herein comprises the sequence of amino acids LASNLES (SEQ ID NO: 20); or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 20.
  • the CDRL2 of any of the anti-NKG2A agents provided herein comprises the sequence of amino acids LASNLES (SEQ ID NO: 20).
  • the third light chain CDR (CDRL3) of an anti-NKG2A agent provided herein comprises an amino acid sequence that is at least 80 percent identical to the amino acid sequence QX 2 X 3 X 4 X 5 X 6 PX 8 T (SEQ ID NO: 27), where X2 is Q or H, X3 is R or S, X4 is S or R, X5 is S or E, X6 is Y or L, and X8 is Y or L.
  • the CDRL3 comprises an amino acid sequence that is at least 90 percent identical to the amino acid sequence of SEQ ID NO: 27.
  • the CDRL3 comprises an amino acid sequence that is at least 95 percent identical to the amino acid sequence of SEQ ID NO: 27. In some embodiments, the CDRL3 comprises an amino acid sequence that is 100 percent identical to the amino acid sequence of SEQ ID NO: 27. In some embodiments, the CDRL3 comprises the sequence of amino acids set forth in SEQ ID NO: 27.
  • the amino acid X 2 of SEQ ID NO: 27 may be substituted with any amino acid. In some embodiments, the amino acid X 2 of SEQ ID NO: 27 is substituted with a conservative amino acid (e.g., Q and/or H). In some embodiments, the amino acid X 2 of SEQ ID NO: 27 is substituted with a basic amino acid.
  • the amino acid X 3 of SEQ ID NO: 27 may be substituted with any amino acid. In some embodiments, the amino acid X 3 of SEQ ID NO: 27 is substituted with a conservative amino acid (e.g., R and/or S). In some embodiments, the amino acid X 3 of SEQ ID NO: 27 is substituted with a basic amino acid. In some embodiments, the amino acid X 3 of SEQ ID NO: 27 is substituted with a neutral amino acid.
  • a conservative amino acid e.g., R and/or S
  • the amino acid X 3 of SEQ ID NO: 27 is substituted with a basic amino acid. In some embodiments, the amino acid X 3 of SEQ ID NO: 27 is substituted with a neutral amino acid.
  • the amino acid X 4 of SEQ ID NO: 27 may be substituted with any amino acid. In some embodiments, the amino acid X 4 of SEQ ID NO: 27 is substituted with a conservative amino acid (e.g., S and/or R). In some embodiments, the amino acid X 4 of SEQ ID NO: 27 is substituted with a neutral amino acid. In some embodiments, the amino acid X 4 of SEQ ID NO: 27 is substituted with a basic amino acid.
  • a conservative amino acid e.g., S and/or R
  • the amino acid X 4 of SEQ ID NO: 27 is substituted with a neutral amino acid. In some embodiments, the amino acid X 4 of SEQ ID NO: 27 is substituted with a basic amino acid.
  • the amino acid X 5 of SEQ ID NO: 27 may be substituted with any amino acid. In some embodiments, the amino acid X 5 of SEQ ID NO: 27 is substituted with a conservative amino acid (e.g., S and/or E). In some embodiments, the amino acid X 5 of SEQ ID NO: 27 is substituted with a neutral amino acid. In some embodiments, the amino acid X 5 of SEQ ID NO: 27 is substituted with an acidic amino acid.
  • a conservative amino acid e.g., S and/or E
  • the amino acid X 5 of SEQ ID NO: 27 is substituted with a neutral amino acid. In some embodiments, the amino acid X 5 of SEQ ID NO: 27 is substituted with an acidic amino acid.
  • the amino acid X 6 of SEQ ID NO: 27 may be substituted with any amino acid.
  • the amino acid X 6 of SEQ ID NO: 27 is substituted with a conservative amino acid (e.g., Y and/or L).
  • the amino acid X 6 of SEQ ID NO: 27 is substituted with an aromatic amino acid.
  • the amino acid X 6 of SEQ ID NO: 27 is substituted with a hydrophobic amino acid.
  • the amino acid X 6 of SEQ ID NO: 27 may be substituted with any amino acid.
  • the amino acid X 6 of SEQ ID NO: 27 is substituted with a conservative amino acid (e.g., Y and/or L).
  • the amino acid X 6 of SEQ ID NO: 27 is substituted with an aromatic amino acid.
  • the amino acid X 6 of SEQ ID NO: 27 is substituted with a hydrophobic amino acid.
  • the CDRL3 of any of the anti-NKG2A agents provided herein comprises an amino acid sequence chosen from QQRSSYPYT (SEQ ID NO: 14) and QHSRELPLT (SEQ ID NO: 21).
  • the CDRL3 of any of the anti-NKG2A agents provided herein comprises the sequence of amino acids QQRSSYPYT (SEQ ID NO: 14); or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 14.
  • the CDRL3 of any of the anti-NKG2A agents provided herein comprises the sequence of amino acids QQRSSYPYT (SEQ ID NO: 14).
  • the CDRL3 of any of the anti-NKG2A agents provided herein comprises the sequence of amino acids QHSRELPLT (SEQ ID NO: 21); or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 21.
  • the CDRL3 of any of the anti-NKG2A agents provided herein comprises the sequence of amino acids QHSRELPLT (SEQ ID NO: 21).
  • any of the anti-NKG2A agents described herein comprises an immunoglobulin heavy chain variable domain comprising a set of CDRs (i.e., CDRH1, CDRH2, CDRH3); and an immunoglobulin light chain variable domain comprising a set of CDRs (i.e., CDRL1, CDRL2, CDRL3).
  • any of the antibodies or antigen-binding fragments thereof that bind the NKG2A receptor or fragment thereof described herein comprises an immunoglobulin heavy chain variable domain comprising a set of CDRs (i.e., CDRH1, CDRH2, CDRH3); and an immunoglobulin light chain variable domain comprising a set of CDRs (i.e., CDRL1, CDRL2, CDRL3).
  • any of the anti-NKG2A agents provided herein comprises two immunoglobulin heavy chain variable domains each comprising a set of CDRs (i.e., CDRH1, CDRH2, CDRH3); and two immunoglobulin light chain variable domains each comprising a set of CDRs (i.e., CDRL1, CDRL2, CDRL3).
  • any of the antibodies or antigen-binding fragments thereof that bind the NKG2A receptor or fragment thereof described herein comprise one or more immunoglobulin heavy chain variable domains each comprising a set of CDRs (i.e., CDRH1, CDRH2, CDRH3); and one or more immunoglobulin light chain variable domains each comprising a set of CDRs (i.e., CDRL1, CDRL2, CDRL3).
  • any of the antibodies or antigen-binding fragments thereof that bind the NKG2A receptor or fragment thereof described herein comprise two immunoglobulin heavy chain variable domains each comprising a set of CDRs (i.e., CDRH1, CDRH2, CDRH3); and two immunoglobulin light chain variable domains each comprising a set of CDRs (i.e., CDRL1, CDRL2, CDRL3).
  • Sets of CDRs may comprise any combination of CDR amino acid sequences (i.e., CDRH1, CDRH2, CDRH3; and CDRL1, CDRL2, CDRL3) provided herein.
  • an immunoglobulin heavy chain variable domain comprises a set of CDRH1, CDRH2 and CDRH3 amino acid sequences
  • an immunoglobulin light chain variable domain comprises a set of CDRL1, CDRL2 and CDRL3 amino acid sequences chosen from sets 1-4 provided in the table below.
  • each immunoglobulin heavy chain variable domain may comprise a set of CDRH1, CDRH2 and CDRH3 amino acid sequences
  • each immunoglobulin light chain variable domain may comprise a set of CDRL1, CDRL2 and CDRL3 amino acid sequences chosen from sets 1-4 provided in Table 2.
  • the anti-NKG2A agent contains CDRs corresponding to the same set.
  • each immunoglobulin heavy chain variable domain may comprise a set of CDRH1, CDRH2 and CDRH3 amino acid sequences from set 1
  • each immunoglobulin light chain variable domain may comprise a set of CDRL1, CDRL2 and CDRL3 amino acid sequences from set 1.
  • the anti-NKG2A agent contains CDRs corresponding to different sets.
  • each immunoglobulin heavy chain variable domain may comprise a set of CDRH1, CDRH2 and CDRH3 amino acid sequences from set 1
  • each immunoglobulin light chain variable domain may comprise a set of CDRL1, CDRL2 and CDRL3 amino acid sequences from set 2.
  • one immunoglobulin heavy chain variable domain may comprise a set of CDRH1, CDRH2 and CDRH3 amino acid sequences from set 1 and the other immunoglobulin heavy chain variable domain may comprise a set of CDRH1, CDRH2 and CDRH3 amino acid sequences from set 2; and one immunoglobulin light chain variable domain may comprise a set of CDRL1, CDRL2 and CDRL3 amino acid sequences from set 1 and the other immunoglobulin light chain variable domain may comprise a set of CDRL1, CDRL2 and CDRL3 amino acid sequences from set 2.
  • the agent that binds the NKG2A receptor or a fragment thereof comprises one or more of: a) an immunoglobulin heavy chain variable domain comprising:
  • CDRH1 heavy chain complementary determining region 1
  • X 1 YX 3 MX 5 (SEQ ID NO: 22), wherein X 1 is S or D; X 3 is W or Y, and X 5 is N or Y;
  • CDRH2 heavy chain complementary determining region 2
  • a heavy chain complementary determining region 3 comprising the sequence XIX 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 YXIIXI2XI3XI4XI5 (SEQ ID NO: 24), wherein X 1 is Q or D; X 2 is G or R; X 3 is D or no amino acid; X 4 is T or no amino acid; X 5 is S or no amino acid; X 6 is V or no amino acid; X 7 is I or Y; X 8 is W or V; X 9 is D or N; X 11 is D or Y; X 12 is G or T; X 13 is F or M; X 14 is A or D; and
  • X 15 is D or Y; and b) an immunoglobulin light chain variable domain comprising:
  • CDRL1 a light chain complementary determining region 1
  • X 1 ASX 4 SVX 7 X 8 X 9 X 10 X 11 X 12 YX 14 H (SEQ ID NO: 25), wherein X 1 is S or R; X 4 is S or K; X 7 is T or S; X 8 is T or no amino acid; X 9 is S or no amino acid; X 1 0 is G or no amino acid; X 11 is Y or no amino acid; X 12 is S or no amino acid; and X 14 is M or I;
  • CDRL2 light chain complementary determining region 2
  • X 1 X 2 SNLX 6 S (SEQ ID NO: 26), wherein X 1 is S or L; X 2 is T or A; and X 6 is A or E; and
  • a light chain complementary determining region 3 comprising the sequence QX 2 X 3 X 4 X 5 X 6 PX 8 T (SEQ ID NO: 27), wherein X 2 is Q or H; X 3 is R or S; X 4 is S or R; X 5 is S or E; X 6 is Y or L; and X 8 is Y or L.
  • the agent that binds the NKG2A receptor or a fragment thereof comprises an immunoglobulin heavy chain variable domain comprising: a CDRH1 comprising the sequence of amino acids set forth in SEQ ID NO: 9 or SEQ ID NO: 16, or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
  • CDRH2 comprising the sequence of amino acids set forth in SEQ ID NO: 10, 15 or 17, or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 10, 15 or 17; and a CDRH3 comprising the sequence of amino acids set forth in SEQ ID NO: 11 or SEQ ID
  • the agent that binds the NKG2A receptor or a fragment thereof comprises an immunoglobulin heavy chain variable domain that comprises a CDRH1 comprising the sequence of amino acids set forth in SEQ ID NO: 9 or SEQ ID NO: 16; a CDRH2 comprising the sequence of amino acids set forth in SEQ ID NO: 10, 15 or 17; and a CDRH3 comprising the sequence of amino acids set forth in SEQ ID NO: 11 or SEQ ID NO: 18.
  • the agent that binds the NKG2A receptor or a fragment thereof comprises an immunoglobulin light chain variable domain comprising a CDRL1 comprising the sequence of amino acids set forth in SEQ ID NO: 12 or SEQ ID NO: 19, or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
  • CDRL2 comprising the sequence of amino acids set forth in SEQ ID NO: 13 or SEQ ID NO:20, or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
  • the agent that binds the NKG2A receptor or a fragment thereof comprises an immunoglobulin light chain variable domain comprising a CDRL1 comprising a sequence of amino acids set forth in SEQ ID NO: 12 or SEQ ID NO: 19; a CDRL2 comprising a sequence of amino acids set forth in SEQ ID NO: 13 or SEQ ID NO:20; and a CDRL3 comprising a sequence of amino acids set forth in SEQ ID NO: 14 or SEQ ID NO:21.
  • the agent that binds the NKG2A receptor or a fragment thereof comprises a CDRH1 comprising the sequence set forth in SEQ ID NO: 9, or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
  • a CDRH2 comprising the sequence set forth in SEQ ID NO: 10 ,or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 10; and a CDRH3 comprising the sequence set forth in SEQ ID NO: 11, or a sequence of amino acids that exhibits at least 80%, 81%,
  • the agent that binds the NKG2A receptor or a fragment thereof comprises a CDRH1 comprising the sequence set forth in SEQ ID NO: 9, the CDRH2 comprises the sequence set forth in SEQ ID NO: 10; and the CDRH3 comprises the sequence set forth in SEQ ID NO: 11.
  • the antibody or antigen-binding fragment thereof that binds the NKG2A receptor or a fragment thereof comprises a CDRH1 comprising the sequence set forth in SEQ ID NO: 9, or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:9; a CDRH2 comprising the sequence set forth in SEQ ID NO: 15 or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
  • CDRH3 comprising the sequence set forth in SEQ ID NO: 11 or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
  • the agent that binds the NKG2A receptor or a fragment thereof comprises a CDRH1 comprising the sequence set forth in SEQ ID NO: 9, a CDRH2 comprising the sequence set forth in SEQ ID NO: 15; and a CDRH3 comprising the sequence set forth in SEQ ID NO: 11.
  • the agent that binds the NKG2A receptor or a fragment thereof comprises a CDRH1 comprising the sequence set forth in SEQ ID NO: 16, or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
  • the CDRH3 comprises the sequence set forth in SEQ ID NO: 18 or a sequence of amino acids that exhibits at least 80%, 81%,
  • the agent that binds the NKG2A receptor or a fragment thereof comprises a CDRH1 comprising the sequence set forth in SEQ ID NO: 16, a CDRH2 comprising the sequence set forth in SEQ ID NO: 17; and a CDRH3 comprising the sequence set forth in SEQ ID NO: 18.
  • the agent that binds the NKG2A receptor or a fragment thereof comprises a CDRL1 comprising the sequence set forth in SEQ ID NO: 12, or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
  • the agent that binds the NKG2A receptor or a fragment thereof comprises a CDRL1 comprising the sequence set forth in SEQ ID NO: 12, a CDRL2 comprising the sequence set forth in SEQ ID NO: 13; and a CDRL3 comprising the sequence set forth in SEQ ID NO: 14.
  • the agent that binds the NKG2A receptor or a fragment thereof comprises a CDRL1 comprising the sequence set forth in SEQ ID NO: 19 or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
  • a CDRL2 comprising the sequence set forth in SEQ ID NO: 20 or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
  • the agent that binds the NKG2A receptor or a fragment thereof comprises a CDRL1 comprising the sequence set forth in SEQ ID NO: 19, a CDRL2 comprises the sequence set forth in SEQ ID NO: 20; and a CDRL3 comprises the sequence set forth in SEQ ID NO: 21.
  • the agent binds the NKG2A receptor or a fragment thereof comprises a CDRH1 comprising the sequence of amino acids set forth in SEQ ID NO: 9 or SEQ ID NO: 16, or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
  • CDRL2 comprising a sequence of amino acids set forth in SEQ ID NO: 13 or SEQ ID NO:20, or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
  • the agent that binds the NKG2A receptor or a fragment thereof comprises a CDRH1 comprising the sequence of amino acids set forth in SEQ ID NO: 9 or SEQ ID NO: 16; a CDRH2 comprising the sequence of amino acids set forth in SEQ ID NO: 10, 15 or 17; a CDRH3 comprising the sequence of amino acids set forth in SEQ ID NO: 11 or SEQ ID NO: 18; a CDRL1 comprising the sequence of amino acids set forth in SEQ ID NO: 12 or SEQ ID NO: 19; a CDRL2 comprising the sequence of amino acids set forth in SEQ ID NO: 13 or SEQ ID NO:20; and a CDRL3 comprising the sequence of amino acids set forth in SEQ ID NO: 14 or SEQ ID NO:21.
  • the agent that binds the NKG2A receptor or a fragment thereof comprises a CDRH1 comprising the sequence set forth in SEQ ID NO: 9 or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
  • a CDRL1 comprising the sequence set forth in SEQ ID NO: 12 or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
  • a CDRL2 comprising the sequence set forth in SEQ ID NO: 13 or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
  • the agent that binds the NKG2A receptor or a fragment thereof comprises a CDRH1 comprising the sequence set forth in SEQ ID NO: 9, a CDRH2 comprising the sequence set forth in SEQ ID NO: 10; a CDRH3 comprising the sequence set forth in SEQ ID NO: 11; a CDRL1 comprising the sequence set forth in SEQ ID NO: 12, a CDRL2 comprising the sequence set forth in SEQ ID NO: 13; and a CDRL3 comprising the sequence set forth in SEQ ID NO: 14.
  • the agent that binds the NKG2A receptor or a fragment thereof comprises a CDRH1 comprising the sequence set forth in SEQ ID NO: 9 or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
  • a CDRL1 comprising the sequence set forth in SEQ ID NO: 12 or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
  • a CDRL2 comprising the sequence set forth in SEQ ID NO: 13 or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
  • the agent that binds the NKG2A receptor or a fragment thereof comprises a CDRH1 comprising the sequence set forth in SEQ ID NO: 9, a CDRH2 comprising the sequence set forth in SEQ ID NO: 15; a CDRH3 comprising the sequence set forth in SEQ ID NO: 11; a CDRL1 comprising the sequence set forth in SEQ ID NO: 12, a CDRL2 comprising the sequence set forth in SEQ ID NO: 13; and a CDRL3 comprising the sequence set forth in SEQ ID NO: 14.
  • the agent that binds the NKG2A receptor or a fragment thereof comprises a CDRH1 comprising the sequence set forth in SEQ ID NO: 16 or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
  • SEQ ID NO: 19 or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%,
  • a CDRL2 comprising the sequence set forth in SEQ ID NO: 20 or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
  • CDRL3 comprising the sequence set forth in SEQ ID NO: 21 or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
  • the agent that binds the NKG2A receptor or a fragment thereof comprises a CDRH1 comprising the sequence set forth in SEQ ID NO: 16, a CDRH2 comprising the sequence set forth in SEQ ID NO: 17; a CDRH3 comprising the sequence set forth in SEQ ID NO: 18; a CDRL1 comprising the sequence set forth in SEQ ID NO: 19, a CDRL2 comprising the sequence set forth in SEQ ID NO: 20; and a CDRL3 comprising the sequence set forth in SEQ ID NO: 21.
  • the agents that binds the NKG2A receptor or fragment thereof is an antibody or antigen-binding fragment thereof.
  • the anti-NKG2A agent provided herein may comprise an immunoglobulin heavy chain variable domain (VH).
  • the immunoglobulin heavy chain variable domain (VH) of any of the anti-NKG2A agents provided herein comprises a polypeptide that is at least 80 percent identical to the amino acid sequence QVQLQQSGPELVKPGASVKISCKASGYVFSSYWMNWVKQRPGQGLEWIGRIYFEDGDTNY NGKFKGKATLTADKSSSTAYMQLSSLTSVDSAVYFCARQGIWDYDGFADWGQGTLVTVSA (SEQ ID NO: 1), e.g., 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more,
  • a VH comprises a polypeptide that is at least 90 percent identical to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the VH comprises a polypeptide that is at least 95 percent identical to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the VH comprises a polypeptide that is 100 percent identical to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the VH comprises the sequence of amino acids set forth in SEQ ID NO: 1.
  • the immunoglobulin heavy chain variable domain (VH) of an anti-NKG2A agent provided herein comprises a polypeptide that is at least 80 percent identical to the amino acid sequence QVQLQQSGPELVKPGASVKISCKASGYVFSSYWMNWVKQRPGQGLEWIGRIYFEDGDTNY NGKFKDKATLTADKSSSTAYMQLSSLTSVDSAVYFCARQGIWDYDGFADWGQGTLVTVSA (SEQ ID NO: 3), e.g., 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, identical to the amino acid sequence of SEQ ID NO: 3.
  • the VH comprises a polypeptide that is at least 90 percent identical to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the VH comprises a polypeptide that is at least 95 percent identical to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the VH comprises a polypeptide that is 100 percent identical to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the VH comprises the sequence of amino acids set forth in SEQ ID NO: 3.
  • the immunoglobulin heavy chain variable domain (VH) of an anti-NKG2A agent provided herein comprises a polypeptide that is at least 80 percent identical to the amino acid sequence
  • SEQ ID NO: 4 EVQLVESGGGLVKPGGSLKLSCAASGFTFSDYYMYWVRQTPEKRLEWVATISYGGIYTYY PDSVKGRFTISRDTAKNNLYLQMSSLKSEDTAVYYCARDRDTSVYVNYYTMDYWGQGTSV TVSS (SEQ ID NO: 4), e.g., 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, identical to the amino acid sequence of SEQ ID NO: 4.
  • the VH comprises a polypeptide that is at least 90 percent identical to the amino acid sequence of SEQ ID NO: 4. In some embodiments, the VH comprises a polypeptide that is at least 95 percent identical to the amino acid sequence of SEQ ID NO: 4. In some embodiments, the VH comprises a polypeptide that is 100 percent identical to the amino acid sequence of SEQ ID NO: 4. In some embodiments, the VH comprises the sequence of amino acids set forth in SEQ ID NO: 4.
  • the VH of an anti-NKG2A agent provided herein comprises a polypeptide chosen from SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 4.
  • VH of any of the anti-NKG2A agents provided herein further comprises a signal sequence.
  • VH signal sequence of any of the anti-NKG2A agents provided herein comprises the sequence of amino acids set forth in SEQ ID NO:49 or SEQ ID NO:53.
  • VH signal sequence of any of the anti-NKG2A agents provided herein comprises the sequence of amino acids set forth in SEQ ID NO:49.
  • VH signal sequence of any of the anti-NKG2A agents provided herein comprises the sequence of amino acids set forth in SEQ ID NO:53.
  • the anti-NKG2A agent provided herein may comprise an immunoglobulin light chain variable domain (VL).
  • the immunoglobulin light chain variable domain (VL) of an anti-NKG2A agent provided herein comprises a polypeptide that is at least 80 percent identical to the amino acid sequence QIVFTQSPAIMSASPGEKVTITCSASSSVTYMHWFQQKPGTSPKLWIYSTSNLAS GVPARFSGSGSGTSYSLTISRMEAEDAATYYCQQRSSYPYTFGGGTKLEIK (SEQ ID NO: 5), e.g., 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more
  • the VL comprises a polypeptide that is at least 90 percent identical to the amino acid sequence of SEQ ID NO: 5. In some embodiments, the VL comprises a polypeptide that is at least 95 percent identical to the amino acid sequence of SEQ ID NO: 5. In some embodiments, the VL comprises a polypeptide that is 100 percent identical to the amino acid sequence of SEQ ID NO: 5. In some embodiments, the VL comprises the sequence of amino acids set forth in SEQ ID NO: 5.
  • the immunoglobulin light chain variable domain (VL) of an anti-NKG2A agent provided herein comprises a polypeptide that is at least 80 percent identical to the amino acid sequence DIVLTQSPVSLTVSLGQRATISCRASKSVSTSGYSYIHWYQQRPGQPPKLLIYLASNLES GVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRELPLTFGAGTKLELK (SEQ ID NO: 8), e.g., 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, identical to the amino acid sequence of SEQ ID NO: 8.
  • the VL comprises a polypeptide that is at least 90 percent identical to the amino acid sequence of SEQ ID NO: 8. In some embodiments, the VL comprises a polypeptide that is at least 95 percent identical to the amino acid sequence of SEQ ID NO: 8. In some embodiments, the VL comprises a polypeptide that is 100 percent identical to the amino acid sequence of SEQ ID NO: 8. In some embodiments, the VL comprises the sequence of amino acids set forth in SEQ ID NO: 8.
  • the VL of an anti-NKG2A agent provided herein comprises a polypeptide chosen from SEQ ID NO: 5 and SEQ ID NO: 8.
  • VL of any of the anti-NKG2A agents provided herein further comprises a signal sequence.
  • VL signal sequence of any of the anti-NKG2A agents provided herein comprises the sequence of amino acids set forth in SEQ ID NO:47 or SEQ ID NO:51.
  • VL signal sequence of any of the anti-NKG2A agents provided herein comprises the sequence of amino acids set forth in SEQ ID NO:47.
  • VL signal sequence of any of the anti-NKG2A agents provided herein comprises the sequence of amino acids set forth in SEQ ID NO:51.
  • any of the anti-NKG2A agents provided herein may comprise a fragment crystallizable region (Fc region).
  • Fc region typically forms the tail of an antibody and can interact with certain cell surface receptors and certain components of the complement system.
  • An Fc region may include, for example, two polypeptides, each derived from the second (CH2) and third (CH3) constant domains of an antibody heavy chain.
  • amino acid sequence of a wild-type CH2-CH3 portion of an Fc region is provided below (positioning is as in the EU index as in Kabat et al. (1992) SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, National Institutes of Health Publication No. 91-3242) (SEQ ID NO:29).
  • an Fc region includes one or more modifications (e.g., one or more amino acid substitutions, insertions, or deletions relative to a comparable wild-type Fc region).
  • Agents comprising modified Fc regions typically have altered phenotypes relative to agents comprising wild-type Fc regions.
  • a variant agent phenotype may be expressed as altered serum halflife, altered stability, altered susceptibility to cellular enzymes, or altered effector function (e.g., as assayed in an NK dependent or macrophage dependent assay).
  • Fc region modifications that alter effector function may include modifications that increase binding to activating receptors (e.g., FcyRIIA (CD16A)) and reduce binding to inhibitory receptors (e.g., FcyRIIB (CD32B)) (see, e.g., Stavenhagen, J.B. et al. (2007) Cancer Res. 57(18):8882-8890).
  • FcyRIIA activating receptors
  • FcyRIIB CD32B
  • Examples of variants of human IgGl Fc regions with reduced binding to CD32B and/or increased binding to CD16A contain F243L, R292P, Y300L, V305I and/or P396L substitutions. Amino acid positions correspond to the amino acid numbering of the CH2-CH3 domain provided above.
  • an Fc region includes one or more modifications that reduce or abrogate binding of the Fc to Fc receptors.
  • modifications may include amino acid substitutions at positions 234, 235, 265 and 297 (see e.g., U.S. Patent No 5,624,821, which is incorporated by reference herein).
  • Example substitutions include one or more ofL234A, L235A, D265A and N297Q. Amino acid positions correspond to the amino acid numbering of the CH2-CH3 domain provided above.
  • an Fc region includes one or more modifications that alter (relative to a wildtype Fc region) the Ratio of Affinities of the modified Fc region to an activating FcyR (such as FcyRIIA or FcyRIIIA) relative to an inhibiting FcyR (such as FcyRIIB):
  • an anti-NKG2A agent herein may have particular use in providing a therapeutic or prophylactic treatment of a disease, disorder, or infection, or the amelioration of a symptom thereof, where an enhanced efficacy of effector cell function (e.g., ADCC) mediated by FcyR is desired, e.g., cancer or infectious disease.
  • ADCC effector cell function
  • an anti-NKG2A agent herein may have particular use in providing a therapeutic or prophylactic treatment of a disease or disorder, or the amelioration of a symptom thereof, where a decreased efficacy of effector cell function mediated by FcyR is desired, e.g., autoimmune or inflammatory disorders.
  • Table 3 lists example single, double, triple, quadruple and quintuple amino acid substitutions having a Ratio of Affinities greater than 1 or less than 1 (see e.g., PCT Publication Nos.
  • anti-NKG2A agents that competitively bind, or are capable of competitively binding, with one or more anti-NKG2A agents described herein.
  • anti-NKG2A agents that compete, or are capable of competing, with one or more anti-NKG2A agents described herein for binding to NKG2A.
  • Such agents that compete, or are capable of competing, with anti-NKG2A agents described herein may be referred to as competitor agents.
  • an agent i.e., competitor agent
  • an agent i.e., competitor agent
  • an agent may be considered to compete for binding to NKG2A when the competitor binds to the exact same region of NKG2A as an anti-NKG2A agent described herein (e.g., exact same peptide (linear epitope) or exact same surface amino acids (conformational epitope)).
  • an agent i.e., competitor agent
  • an agent may be considered capable of competing for binding to NKG2A when the competitor binds to the same general region of NKG2A as an anti-NKG2A agent described herein (i.e., extracellular region or leucine-rich binding domain) under suitable assay conditions.
  • an agent i.e., competitor agent
  • an agent may be considered capable of competing for binding to NKG2A when the competitor binds to the exact same region of NKG2A as an anti-NKG2A agent described herein (e.g., exact same peptide (linear epitope) or exact same surface amino acids (conformational epitope)) under suitable assay conditions.
  • an agent i.e., competitor agent
  • an agent may be considered to compete for binding to NKG2A when the competitor blocks the binding of one or more anti-NKG2A agents described herein to NKG2A.
  • an agent i.e., competitor agent
  • an agent may be considered capable of competing for binding to NKG2A when the competitor blocks the binding of one or more anti- NKG2A agents described herein to NKG2A under suitable assay conditions.
  • Whether a competitor blocks the binding of one or more anti-NKG2A agents described herein to NKG2A may be determined using a suitable competition assay or blocking assay, such as, for example, a blocking assay as described in Example 5 herein.
  • a competitor agent may block binding of one or more anti- NKG2A agents described herein to NKG2A in a competition or blocking assay by 50% or more, and conversely, one or more anti-NKG2A agents described herein may block binding of the competitor agent to NKG2A in a competition or blocking assay by about 50% or more.
  • an agent i.e., competitor agent
  • one or more anti-NKG2A agents described herein may block binding of the competitor agent to NKG2A in a competition or blocking assay by about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.
  • an agent i.e., competitor agent
  • an agent may be considered to compete for binding to NKG2A when the competitor binds to NKG2A with a similar affinity as one or more anti-NKG2A agents described herein.
  • an agent i.e., competitor agent
  • an agent may be considered capable of competing for binding to NKG2A when the competitor binds to NKG2A with a similar affinity as one or more anti-NKG2A agents described herein under suitable assay conditions.
  • an agent i.e., competitor agent
  • an agent i.e., competitor agent
  • a competitor agent may comprise any feature described herein for anti-NKG2A agents.
  • anti-NKG2A agents that bind to, or are capable of binding to, the same epitope as one or more anti-NKG2A agents described herein.
  • anti- NKG2A agents that compete with one or more anti-NKG2A agents described herein for binding to the same epitope on NKG2A.
  • Such agents that bind the same epitope may be referred to as epitope competitors.
  • an epitope competitor may bind to the exact same region of NKG2A as an anti-NKG2A agent described herein (e.g., exact same peptide (linear epitope) or exact same surface amino acids (conformational epitope)).
  • an epitope competitor blocks the binding of one or more anti-NKG2A agents described herein to NKG2A.
  • An epitope competitor may block binding of one or more anti-NKG2A agents described herein to NKG2A in a competition assay by about 50% or more, and conversely, one or more anti-NKG2A agents described herein may block binding of the epitope competitor to NKG2A in a competition assay by 50% or more.
  • an epitope competitor binds to NKG2A with a similar affinity as one or more anti-NKG2A agents described herein.
  • an epitope competitor binds to NKG2A with an affinity that is at least about 50% of the affinity of one or more anti-NKG2A agents described herein.
  • an epitope competitor may bind to NKG2A with an affinity that is at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the affinity of one or more anti-NKG2A agents described herein.
  • An epitope competitor may comprise any feature described herein for anti-NKG2A agents.
  • an anti-NKG2A agent is an antibody.
  • any of the anti- NKG2A agents provided herein is an antibody or antigen-binding fragment thereof. Methods for generating anti-NKG2A antibodies, antigen-binding fragments thereof and variants of anti-NKG2A antibodies are described in the Examples below.
  • an anti-NKG2A agent is a humanized antibody, or an antigen binding fragment thereof.
  • an anti-NKG2A agent is a humanized antibody, or a derivative thereof that binds NKG2A. Humanized anti-NKG2A antibodies may be prepared based on a nonhuman anti-NKG2A antibody.
  • Fully human antibodies may also be prepared, e.g., in a genetically engineered (i.e., transgenic) mouse (e.g., from Medarex) that, when presented with an immunogen, can produce a human antibody that does not necessarily require CDR grafting.
  • These antibodies are fully human (100% human protein sequences) from animals such as mice in which the non-human antibody genes are suppressed and replaced with human antibody gene expression.
  • Antibodies may be generated against NKG2A when presented to these genetically engineered mice or other animals that can to produce human frameworks for the relevant CDRs.
  • the parent antibody is prepared.
  • Example techniques for generating such nonhuman antibody and parent antibodies are described in the following sections.
  • the antigen for production of antibodies may be, e.g., intact NKG2A, particularly expressed in cells, or a portion of NKG2A (e.g. N-terminal domain, C-terminal domain, a cytoplasmic domain, an intra- organelle domain, a transmembrane domain, an extracellular domain, an ectodomain, a TIR domain, a leucine-rich domain, or a NKG2A fragment comprising a desired epitope).
  • N-terminal domain, C-terminal domain, a cytoplasmic domain, an intra- organelle domain, a transmembrane domain, an extracellular domain, an ectodomain, a TIR domain, a leucine-rich domain, or a NKG2A fragment comprising a desired epitope e.g., intact NKG2A, particularly expressed in cells, or a portion of NKG2A (e.g. N-terminal domain, C-terminal domain, a cytoplasmic domain, an
  • Polyclonal antibodies may be raised in animals (vertebrate or invertebrates, including mammals, birds and fish, including cartilaginous fish) by multiple subcutaneous (sc) or intraperitoneal (ip) injections of a relevant antigen and an adjuvant.
  • animals vertebrate or invertebrates, including mammals, birds and fish, including cartilaginous fish
  • sc subcutaneous
  • ip intraperitoneal
  • Non-protein carriers e.g., colloidal gold
  • a protein or other carrier that is immunogenic in the species to be immunized e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor
  • Non-protein carriers e.g., colloidal gold
  • animals are immunized against the antigen, immunogenic conjugates, or derivatives by combining, e.g., 100 pg or 5 pg of the protein or conjugate (for rabbits or mice, respectively) with three volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites.
  • the animals are boosted with one-fifth to one-tenth of the original amount of peptide or conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites.
  • Seven to 14 days later the animals are bled and the serum is assayed for antibody titer. Animals are boosted until the titer plateaus.
  • the animal is boosted with the conjugate of the same antigen, but conjugated to a different protein and/or through a different cross-linking reagent.
  • Conjugates also can be made in recombinant cell culture as protein fusions. Also, aggregating agents such as alum are suitably used to enhance the immune response.
  • Monoclonal antibodies may be made using the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or may be made by other methods such as recombinant DNA methods (U.S. Pat. No. 4,816,567).
  • a mouse or other appropriate host animal such as a hamster or macaque monkey, is immunized as hereinabove described to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization.
  • lymphocytes may be immunized in vitro.
  • Lymphocytes then are fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103 (Academic Press, 1986)).
  • a suitable fusing agent such as polyethylene glycol
  • the hybridoma cells are seeded and grown in a suitable culture medium that may contain one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells.
  • the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.
  • HGPRT hypoxanthine guanine phosphoribosyl transferase
  • Preferred myeloma cells are those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium.
  • preferred myeloma cell lines are murine myeloma lines, such as SP-2 or X 6 3- Ag8-653 cells available from the American Type Culture Collection, Rockville, Md. USA.
  • Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987).
  • Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen.
  • the binding specificity of monoclonal antibodies produced by hybridoma cells may be determined by immunoprecipitation, by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbant assay (ELISA), or by flow cytometric analysis of cells expressing the membrane antigen.
  • the binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson et al., Anal. Biochem., 107:220 (1980).
  • the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103 (Academic Press, 1986)). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium.
  • the hybridoma cells may be grown in vivo as ascites tumors in an animal.
  • the monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A- Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
  • DNA encoding the monoclonal antibodies is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the monoclonal antibodies).
  • cDNA may be prepared from mRNA and the cDNA then subjected to DNA sequencing.
  • the hybridoma cells serve as a preferred source of such genomic DNA or RNA for cDNA preparation.
  • the DNA may be placed into expression vectors, which are well known in the art, and which are then transfected into host cells such as E coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. Recombinant production of antibodies will be described in more detail below.
  • Amino acid sequence variants of the anti-NKG2A antibody are prepared by introducing appropriate nucleotide changes into the anti-NKG2A antibody DNA, or by peptide synthesis.
  • Such variants include, for example, deletions from, and/or insertions into and/or substitutions of, residues within the amino acid sequences of the anti-NKG2A antibodies of the examples herein. Any combination of deletion, insertion, and substitution is made to arrive at the final construct, provided that the final construct possesses the desired characteristics.
  • the amino acid changes also may alter post- translational processes of the humanized or variant anti-NKG2A antibody, such as changing the number or position of glycosylation sites.
  • alanine scanning mutagenesis One method for identification of certain residues or regions of the anti-NKG2A antibody that are preferred locations for mutagenesis is called “alanine scanning mutagenesis,” as described by Cunningham and Wells Science, 244: 1081-1085 (1989).
  • a residue or group of target residues are identified (e.g., charged residues such as arg, asp, his, lys, and glu) and replaced by a neutral or negatively charged amino acid (most preferably alanine or polyalanine) to affect the interaction of the amino acids with NKG2A antigen.
  • Those amino acid locations demonstrating functional sensitivity to the substitutions then are refined by introducing further or other variants at, or for, the sites of substitution.
  • Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intra-sequence insertions of single or multiple amino acid residues.
  • terminal insertions include an N- terminal methionyl residue or the antibody fused to an epitope tag.
  • Other insertional variants include the fusion of an enzyme or a polypeptide that increases the serum half-life of the antibody to the N- or C-terminus of the antibody.
  • variants Another type of variant is an amino acid substitution variant. These variants have at least one amino acid residue removed from the antibody molecule and a different residue inserted in its place.
  • the sites of greatest interest for substitutional mutagenesis include the hypervariable regions, but FR alterations are also contemplated. Conservative substitutions are preferred, but more substantial changes may be introduced and the products may be screened. Examples of substitutions are listed below:
  • Substantial modifications in the biological properties of an antibody are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.
  • Naturally occurring residues are divided into groups based on common side-chain properties:
  • hydrophobic norleucine, met, ala, val, leu, ile
  • Non-conservative substitutions will entail exchanging a member of one of these classes for another class.
  • cysteine residues not involved in maintaining the proper conformation of the antibody also may be substituted, to improve the oxidative stability of the molecule and prevent aberrant crosslinking.
  • cysteine bond(s) may be added to the antibody to improve its stability (particularly where the antibody is an antibody fragment such as an Fv fragment).
  • substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g. a humanized or human antibody).
  • a parent antibody e.g. a humanized or human antibody
  • the resulting variant(s) selected for further development will have improved biological properties relative to the parent antibody from which they are generated.
  • a convenient way for generating such substitutional variants is affinity maturation using phage display. Briefly, several hypervariable region sites (e.g. 6-7 sites) are mutated to generate all possible amino substitutions at each site.
  • the antibody variants thus generated are displayed in a monovalent fashion from filamentous phage particles as fusions to the gene III product of M13 packaged within each particle. The phage-displayed variants are then screened for their biological activity (e.g. binding affinity) as herein disclosed.
  • alanine-scanning mutagenesis can be performed to identify hypervariable region residues contributing significantly to antigen binding.
  • Another type of amino acid variant of the antibody alters the original glycosylation pattern of the antibody. By altering is meant deleting one or more carbohydrate moieties found in the antibody, and/or adding one or more glycosylation sites that are not present in the antibody.
  • Glycosylation of antibodies is typically either N-linked and/or or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue.
  • the tripeptide sequences asparagine- X-serine and asparagine-X-threonine, where X is any amino acid except proline, are the most common recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain.
  • O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5 -hydroxyproline or 5 -hydroxy lysine may also be used.
  • glycosylation sites to the antibody is conveniently accomplished by altering the amino acid sequence such that it contains one or more of the above-described tripeptide sequences (for N- linked glycosylation sites).
  • the alteration may also be made by the addition of, or substitution by, one or more serine or threonine residues to the sequence of the original antibody (for O-linked glycosylation sites).
  • Nucleic acid molecules encoding amino acid sequence variants of anti-NKG2A antibodies herein are prepared by a variety of methods known in the art. These methods include, but are not limited to, isolation from a natural source (in the case of naturally occurring amino acid sequence variants) or preparation by oligonucleotide-mediated (or site -directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared variant or a non-variant version of an anti-NKG2A antibody.
  • human antibodies can be generated.
  • transgenic animals e.g., mice
  • transgenic animals e.g., mice
  • a homozygous deletion of the antibody heavy-chain joining region (JH) gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production.
  • JH antibody heavy-chain joining region
  • Transfer of the human germ-line immunoglobulin gene array into such germ-line mutant mice can result in the production of human antibodies upon antigen challenge (see, e.g., Jakobovits et al., Proc. Natl. Acad. Sci.
  • Human antibodies also can be derived from phage-display libraries (Hoogenboom et al., J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581-597 (1991); and U.S. Pat. Nos. 5,565,332 and 5,573,905). Human antibodies also may be generated by in vitro activated B cells (see U.S. Pat. Nos. 5,567,610 and 5,229,275).
  • an anti-NKG2A agent is an antibody fragment or antigen-binding fragment that retains at least one desired activity, including antigen binding.
  • Various techniques have been developed for the production of antibody fragments or antigen-binding fragments. In some instances, these fragments are derived via proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24: 107-117(1992) and Brennan et al., Science 229:81 (1985)). In some instances, these fragments are produced directly by recombinant host cells. For example, Fab'-SH fragments can be directly recovered from E.
  • F(ab')2 is formed using the leucine zipper GCN4 to promote assembly of the F(ab')2 molecule.
  • Fv, Fab or F(ab')2 fragments can be isolated directly from recombinant host cell culture. Other techniques for the production of antibody fragments or antigenbinding fragments will be apparent to the skilled practitioner.
  • an anti-NKG2A agent comprises a first binding moiety and a second binding moiety, where the first binding moiety is specifically reactive with a first molecule that is NKG2A and the second binding moiety is specifically reactive with a second molecule that is a molecular species different from the first molecule.
  • Such agents may comprise a plurality of first binding moieties, a plurality of second binding moieties, or a plurality of first binding moieties and a plurality of second binding moieties.
  • the ratio of first binding moieties to second binding moieties is about 1: 1, although it may range from about 1000: 1 to about 1 : 1000, where the ratio may be measured in terms of valency.
  • the second binding moiety may also be an antibody.
  • the first and second moieties are linked via a linker moiety, which may have two to many 100’s or even thousands of valencies for attachment of first and second binding moieties by one or different chemistries.
  • linker moiety which may have two to many 100’s or even thousands of valencies for attachment of first and second binding moieties by one or different chemistries.
  • bispecific antibodies include those that are reactive against two different epitopes; in some instances, one epitope is a NKG2A epitope and the second epitope is on an unrelated soluble molecule.
  • the bispecific antibody is reactive against an epitope on NKG2A and against an epitope on a different molecule found on the surface of the same cell.
  • the bispecific antibody is reactive against an epitope on NKG2A and against an epitope on a different molecule found on the surface of a different cell.
  • Compositions herein may also comprise a first agent and a second agent, where the first agent comprises a first binding moiety specifically reactive with a first molecule (e.g., NKG2A) and the second agent comprises a second binding moiety specifically reactive with a second molecule that is a molecular species different than the first molecule.
  • the first and/or second agent may be an antibody.
  • the ratio of first agent to second agent may range from about 1,000: 1 to 1: 1,000, although the preferred ratio is about 1: 1.
  • Certain bispecific antibodies may bind to two different epitopes of NKG2A.
  • Bispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., F(ab')2 bispecific antibodies).
  • the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers that are recovered from recombinant cell culture.
  • the preferred interface comprises at least a part of the CH3 domain of an antibody constant domain.
  • one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan).
  • Compensatory “cavities” of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers (see e.g., WO96/27011 published Sep. 6, 1996).
  • Bispecific antibodies include cross-linked or “heteroconjugate” antibodies.
  • one of the antibodies in the heteroconjugate can be coupled to avidin, the other to biotin.
  • Heteroconjugate antibodies may be made using any convenient cross-linking methods. Suitable cross-linking agents are well known in the art, and are disclosed in U.S. Pat. No. 4,676,980, along with a number of crosslinking techniques.
  • bispecific antibodies can be prepared using chemical linkage.
  • intact antibodies are proteolytically cleaved to generate F(ab')2 fragments (see e.g., Brennan et al., Science 229:81 (1985), which is incorporated by reference herein). These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation.
  • the Fab' fragments generated are then converted to thionitrobenzoate (TNB) derivatives.
  • Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody.
  • Fab'-SH fragments directly recovered from E. coli can be chemically coupled in vitro to form bispecific antibodies (see e.g., Shalaby et al., J. Exp. Med. 175:217-225 (1992), which is incorporated by reference herein).
  • bispecific antibodies have been produced using leucine zippers (see e.g., Kostelny et al., J. Immunol. 148(5): 1547-1553 (1992), which is incorporated by reference herein).
  • the leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion.
  • the antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers.
  • a bispecific antibody may be a “linear antibody” produced as described in Zapata et al. Protein Eng. 8(10): 1057-1062 (1995), which is incorporated by reference herein.
  • Antibodies with two valencies or more are contemplated herein.
  • An antibody (or polymer or polypeptide) herein comprising one or more binding sites per arm or fragment thereof will be referred to herein as “multivalent” antibody.
  • a “bivalent” antibody herein comprises two binding sites per Fab or fragment thereof whereas a “trivalent” polypeptide herein comprises three binding sites per Fab or fragment thereof.
  • the two or more binding sites per Fab may be binding to the same or different antigens.
  • the two or more binding sites in a multivalent polypeptide herein may be directed against the same antigen, for example against the same parts or epitopes of said antigen or against two or more same or different parts or epitopes of said antigen; and/or may be directed against different antigens; or a combination thereof.
  • a bivalent polypeptide herein may comprise two identical binding sites, may comprise a first binding sites directed against a first part or epitope of an antigen and a second binding site directed against the same part or epitope of said antigen or against another part or epitope of said antigen; or may comprise a first binding sites directed against a first part or epitope of an antigen and a second binding site directed against a different antigen.
  • a multivalent polypeptide herein may comprise any number of binding sites directed against the same or different antigens.
  • the multivalent polypeptide comprises at least two ligand binding elements, one of which contains one or more CDR peptide sequences shown herein.
  • the multivalent polypeptide comprises three ligand binding sites, each independently selected from the CDR sequences disclosed herein.
  • At least one of the ligand binding elements binds NKG2A. In one embodiment, at least one of the ligand binding elements binds another target. In one embodiment, there are up to 10,000 binding elements in a multivalent binding molecule, and the ligand binding elements may be linked to a scaffold.
  • an antibody herein that contains at least two binding sites per Fab or fragment thereof, in which at least one binding site is directed against a first antigen and a second binding site directed against a second antigen different from the first antigen, may also be referred to as “multispecific.”
  • a “bispecific” polymer comprises at least one site directed against a first antigen and at least one second site directed against a second antigen
  • a “trispecific” is a polymer that comprises at least one binding site directed against a first antigen, at least one further binding site directed against a second antigen, and at least one further binding site directed against a third antigen; and the like.
  • a bispecific polypeptide herein is a bivalent polypeptide (per Fab) of the technology provided herein.
  • the technology herein is not limited thereto, in the sense that a multispecific polypeptide herein may comprise any number of binding sites directed against two or more different antigens.
  • an anti-NKG2A agent e.g., technology herein also pertains to immunoconjugates comprising an antibody described herein (e.g., an anti-NKG2A antibody) conjugated to a cytotoxic agent such as a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant or animal origin, or fragments thereof), or a radioactive isotope (for example, a radioconjugate), or a cytotoxic drug.
  • a cytotoxic agent such as a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant or animal origin, or fragments thereof), or a radioactive isotope (for example, a radioconjugate), or a cytotoxic drug.
  • ADC agent-drug conjugates
  • Conjugates are made using a variety of bifiinctional protein coupling agents such as N-succinimidyl-3-(2 -pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p- azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)- ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as l,5-difluoro-2,4-dinitrobenzene).
  • SPDP N-succinimidyl-3-(
  • any of the anti-NKG2A agents may be formulated as immunoliposomes.
  • Liposomes containing an antibody are prepared by methods known in the art, such as described in Epstein et al., Proc. Natl. Acad. Sci. USA 82:3688 (1985); Hwang et al., Proc. Natl Acad. Sci. USA 77:4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545.
  • Eiposomes with enhanced circulation time are disclosed in U.S. Pat. No. 5,013,556.
  • liposomes can be generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through fdters of defined pore size to yield liposomes with the desired diameter.
  • Fab' fragments of an antibody provided herein can be conjugated to the liposomes as described in Martin et al., J. Biol. Chem. 257:286-288 (1982) via a disulfide interchange reaction. Another active ingredient is optionally contained within the liposome.
  • Enzymes or other polypeptides can be covalently bound to an anti-NKG2A agent (e.g., anti-NKG2A antibody) by techniques well known in the art such as the use of the heterobifunctional cross-linking reagents discussed above.
  • fusion proteins comprising at least the antigen binding region of an antibody provided herein linked to at least a functionally active portion of an enzyme can be constructed using recombinant DNA techniques well known in the art (see, e.g., Neuberger et al., Nature 312:604-608 (1984)).
  • an antibody fragment rather than an intact antibody, to increase penetration of target tissues and cells, for example.
  • Covalent modifications of an anti-NKG2A agent are also included within the scope of this technology. For example, modifications may be made by chemical synthesis or by enzymatic or chemical cleavage of an anti-NKG2A antibody.
  • Other types of covalent modifications of an antibody are introduced into the molecule by reacting targeted amino acid residues of the antibody with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C-terminal residues.
  • Example covalent modifications of polypeptides are described in U.S. Pat. No. 5,534,615, specifically incorporated herein by reference.
  • a preferred type of covalent modification of the antibody comprises linking the antibody to one of a variety of non- proteinaceous polymers, e.g., polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, in the manner set forth in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.
  • non- proteinaceous polymers e.g., polyethylene glycol, polypropylene glycol, or polyoxyalkylenes
  • nucleic acids Nucleic acids, vectors, host cells, and recombinant methods
  • nucleic acids encodingany of the anti-NKG2A agents (e.g., anti-NKG2A antibody) described herein, and vectors and host cells comprising any of the nucleic acids provided herein, and recombinant techniques for the production of the agent or antibody.
  • a nucleic acid herein may include one or more subsequences, each referred to as a polynucleotide.
  • nucleic acids e.g., isolated nucleic acids
  • a nucleic acid encodes an immunoglobulin heavy chain variable domain of an anti-NKG2A agent provided herein.
  • the immunoglobulin heavy chain variable domain is encoded by the sequence of nucleotides set forth in any of SEQ ID NOs: 30-37, or sequence of nucleotides having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of SEQ ID NOs: 30-37.
  • the immunoglobulin heavy chain variable domain is encoded by the sequence of nucleotides set forth in any of SEQ ID NOs: 30-37.
  • the immunoglobulin heavy chain variable domain contains a signal sequence and is encoded by the sequence of nucleotides set forth in any of SEQ ID NOs:30-33. In some embodiments, the immunoglobulin heavy chain variable domain is encoded by the sequence of nucleotides set forth in SEQ ID NO: 30. In some embodiments, the immunoglobulin heavy chain variable domain is encoded by the sequence of nucleotides set forth in SEQ ID NO: 31. In some embodiments, the immunoglobulin heavy chain variable domain is encoded by the sequence of nucleotides set forth in SEQ ID NO: 32. In some embodiments, the immunoglobulin heavy chain variable domain is encoded by the sequence of nucleotides set forth in SEQ ID NO: 33. In some embodiments, the heavy chain signal sequence is encoded by the sequence of nucleotides set forth in SEQ ID NO: 48 or SEQ ID NO: 52.
  • the immunoglobulin heavy chain variable domain does not contain a signal sequence, and is encoded by the sequence of nucleotides set forth in any of SEQ ID NOs:34-37, or sequence of nucleotides having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of SEQ ID NOs: 34-37.
  • the immunoglobulin heavy chain variable domain does not contain a signal sequence, and is encoded by the sequence of nucleotides set forth in any of SEQ ID NOs:34-37.
  • the immunoglobulin heavy chain variable domain is encoded by the sequence of nucleotides set forth in SEQ ID NO: 34. In some embodiments, the immunoglobulin heavy chain variable domain is encoded by the sequence of nucleotides set forth in SEQ ID NO: 35. In some embodiments, the immunoglobulin heavy chain variable domain is encoded by the sequence of nucleotides set forth in SEQ ID NO: 36. In some embodiments, the immunoglobulin heavy chain variable domain is encoded by the sequence of nucleotides set forth in SEQ ID NO: 37.
  • a nucleic acid encodes an immunoglobulin light chain variable domain of an anti-NKG2A agent provided herein.
  • the immunoglobulin light chain variable domain is encoded by the sequence of nucleotides set forth in any of SEQ ID NOs:38-45, or sequence of nucleotides having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of SEQ ID NOs: 38- 45.
  • the immunoglobulin light chain variable domain is encoded by the sequence of nucleotides set forth in any of SEQ ID NOs:38-45. In some embodiments, the immunoglobulin light chain variable domain contains a signal sequence and is encoded by the sequence of nucleotides set forth in any of SEQ ID NOs:38-41. In some embodiments, the immunoglobulin light chain variable domain is encoded by the sequence of nucleotides set forth in SEQ ID NO: 38. In some embodiments, the immunoglobulin light chain variable domain is encoded by the sequence of nucleotides set forth in SEQ ID NO: 39.
  • the immunoglobulin light chain variable domain is encoded by the sequence of nucleotides set forth in SEQ ID NO: 40. In some embodiments, the immunoglobulin light chain variable domain is encoded by the sequence of nucleotides set forth in SEQ ID NO: 41. In some embodiments, the light chain signal sequence is encoded by the sequence of nucleotides set forth in SEQ ID NO: 46 or SEQ ID NO: 50.
  • the immunoglobulin light chain variable domain does not contain a signal sequence, and is encoded by the sequence of nucleotides set forth in any of SEQ ID NOs:42-45, or sequence of nucleotides having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of SEQ ID NOs:42-45.
  • the immunoglobulin light chain variable domain does not contain a signal sequence, and is encoded by the sequence of nucleotides set forth in any of SEQ ID NOs:42- 45.
  • the immunoglobulin light chain variable domain is encoded by the sequence of nucleotides set forth in SEQ ID NO: 42. In some embodiments, the immunoglobulin light chain variable domain is encoded by the sequence of nucleotides set forth in SEQ ID NO: 43. In some embodiments, the immunoglobulin light chain variable domain is encoded by the sequence of nucleotides set forth in SEQ ID NO: 44. In some embodiments, the immunoglobulin light chain variable domain is encoded by the sequence of nucleotides set forth in SEQ ID NO: 45.
  • a nucleic acid encodes an immunoglobulin heavy chain variable domain and an immunoglobulin light chain variable domain of an anti-NKG2A agent provided herein.
  • a nucleic acid comprises a nucleotide sequence that encodes an amino acid sequence of any one of SEQ ID NOS: 1, 3-5, or 8-21.
  • a nucleic acid may comprise a nucleotide sequence that encodes a CDR amino acid sequence of any one of SEQ ID NOS: 9-21.
  • a nucleic acid may comprise a nucleotide sequence that encodes an immunoglobulin heavy chain variable domain amino acid sequence of any one of SEQ ID NOS: 1, 3 or 4.
  • a nucleic acid may comprise a nucleotide sequence that encodes an immunoglobulin light chain variable domain amino acid sequence of any one of SEQ ID NOS: 5 or 8.
  • a nucleic acid encoding the anti- NKG2A agent or antibody may be isolated and inserted into a replicable vector for further cloning (amplification of the DNA) or for expression.
  • an anti-NKG2A agent or antibody may be produced by homologous recombination, e.g. as described in U.S. Pat. No. 5,204,244, specifically incorporated herein by reference.
  • DNA encoding an anti-NKG2A agent or antibody can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody). Many vectors are available.
  • the vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence, e.g., as described in U.S. Pat. No. 5,534,615 issued Jul. 9, 1996 and specifically incorporated herein by reference.
  • Suitable host cells for cloning or expressing DNA in vectors herein are prokaryote, yeast, or higher eukaryote cells.
  • Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli such as B. subtilis and B. licheniformis (e.g., B.
  • E. coli cloning host is E. coli 294 (ATCC 31,446), although other strains such as E. coli B, E. coli X 1 776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are suitable. These examples are illustrative rather than limiting.
  • eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for anti-NKG2A agent/antibody-encoding vectors.
  • Saccharomyces cerevisiae or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms.
  • a number of other genera, species, and strains are commonly available and useful herein, such as Schizosaccharomyces pombe; Kluyveromyces hosts such as, e.g., K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K.
  • waltii ATCC 56,500
  • K. drosophilarum ATCC 36,906
  • K. thermotolerans K. marxianus
  • yarrowia EP 402,226
  • Pichia pastoris EP 183,070
  • Candida Trichoderma reesia
  • Neurospora crassa Schwanniomyces such as Schwanniomyces occidentalis
  • filamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as A. nidulans and A. niger.
  • Suitable host cells for the expression of anti-NKG2A agents/antibodies are derived from multicellular organisms.
  • invertebrate cells include plant and insect cells.
  • Numerous baculoviral strains and variants and corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruitfly), and Bombyx mori (silk moth) have been identified.
  • a variety of viral strains for transfection are publicly available, e.g., the L-l variant of Autographa califomica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be used as the virus herein according to the present technology, particularly for transfection of Spodoptera frugiperda cells.
  • Plant cell cultures of cotton, com, potato, soybean, petunia, tomato, and tobacco can also be utilized as hosts.
  • Suitable host cells for the expression of anti-NKG2A agents/antibodies also may include vertebrate cells (e.g., mammalian cells). Vertebrate cells may be propagated in culture (tissue culture).
  • Examples of useful mammalian host cell lines include monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse Sertoli cells (TM4, Mather, Biol. Reprod.
  • COS-7 monkey kidney CV1 line transformed by SV40
  • human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)
  • baby hamster kidney cells BHK, ATCC CCL 10
  • Chinese hamster ovary cells/-DHFR CHO, Urlaub et al., Proc. Nat
  • monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL- 1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).
  • Host cells may be transformed with the above-described expression or cloning vectors for antibody production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.
  • Host cells used to produce an agent/antibody herein may be cultured in a variety of media.
  • Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing the host cells.
  • any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMY CINTM), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art.
  • the culture conditions such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.
  • an agent/antibody can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the antibody is produced intracellularly, as a first step, the particulate debris, either host cells or lysed fragments, is removed, for example, by centrifugation or ultrafiltration. Carter et al., Bio/Technology 10: 163-167 (1992) describe a procedure for isolating antibodies that are secreted to the periplasmic space of E. coli. Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min.
  • sodium acetate pH 3.5
  • EDTA EDTA
  • PMSF phenylmethylsulfonylfluoride
  • Cell debris can be removed by centrifugation.
  • supernatants from such expression systems are generally first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit.
  • a protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious contaminants.
  • the agent/antibody composition prepared from the cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being the preferred purification technique.
  • affinity chromatography is the preferred purification technique.
  • the suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain that is present in the antibody. Protein A can be used to purify antibodies that are based on human heavy chains (Lindmark et al., J. Immunol. Meth. 62: 1-13 (1983)). Protein G is recommended for all mouse isotypes and for human y3 (Guss et al., EMBO J. 5: 15671575 (1986)).
  • the matrix to which the affinity ligand is attached is most often agarose, but other matrices are available.
  • Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose.
  • Bakerbond ABX.TM. resin J. T. Baker, Phillipsburg, N.J. is useful for purification.
  • the mixture comprising the agent or antibody of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5-4.5, and may be performed at low salt concentrations (e.g., from about 0-0.25M salt).
  • the present technology provides anti-NKG2A agents and antibodies, fragments or antigen-binding fragments thereof and related compositions, which may be useful for elimination of NKG2A expressing cells from the body, for example, and for identification and quantification of the number of NKG2A expressing cells in tissue samples, for example.
  • NKG2A- based Therapeutic methods and compositions of the present technology may be referred to as “NKG2A- based” in order to indicate that these therapies can change the relative or absolute numbers of undesirable or toxic NKG2A expressing cells such as lymphomas or autoimmune B lymphocytes.
  • one way to control the amount of undesirable NKG2A expressing cells in a patient is by providing a composition that comprises one or more anti-NKG2A antibodies, fragments or antigen-binding fragments thereof to cause cytotoxic activity towards the NKG2A-expressing cells, for example.
  • any of the anti-NKG2A agents or anti-NKG2A antibodies described herein are capable of binding a cell.
  • the cell is a natural killer (NK) cell.
  • the cell is a lymphocyte.
  • the lymphocyte is a CD8+ lymphocyte.
  • the cell selected from B cells, plasmacytoid dendritic cells (pDCs), lymphocytes, leukocytes, T cells, monocytes, macrophages, neutrophils, myeloid dendritic cells (mDCs), innate lymphoid cells, mast cells, eosinophils, basophils, natural killer cells, and the like.
  • the cell is selected from a heterogeneous population of immune cells including peripheral blood mononuclear cells (PBMCs) which may include, for example, T cells, B cells, natural killer cells, and monocytes.
  • PBMCs peripheral blood mononuclear cells
  • the cell bound by any of the anti-NKG2A agents or anti-NKG2A antibodies interacts with a target cell that expresses HLA-E.
  • the target cell is a cancer cell.
  • the target cell is a cancer cell that expresses HLA-E.
  • the target cell is a cancer cell that over-expresses HLA-E.
  • the HLA-E is expressed on the surface of the target or cancer cell.
  • any of the anti-NKG2A agents or anti-NKG2A antibodies described herein specifically bind NKG2A on a cell. In some embodiments, any of the anti-NKG2A agents or anti- NKG2A antibodies described herein specifically bind NKG2A and do not specifically bind NKG2C or NKG2E. In some embodiments, any of the anti-NKG2A agents or anti-NKG2A antibodies described herein block the binding of HLA-E to NKG2A.
  • the cell is a cell with a disease or disorder. In some embodiments, the disease or disorder is a cancer, a viral infection, an autoimmune disease, an inflammatory disease or a bacterial infection.
  • the disease or disorder is a cancer including but not limited to acute myeloid leukemia, acute lymphoblastic leukemia, colorectal, ovarian, gynecologic, liver, glioblastoma, Hodgkin lymphoma, chronic lymphocytic leukemia, esophagus, gastric, pancreas, colon, kidney, head and neck, lung and melanoma.
  • any of the ani-NKG2A agents provided herein can be used to treat a disease or disorder by blocking the NKG2A receptor on NKG2A expressing cells in a subject.
  • any of the anti-NKG2A agents provided herein are provided as a composition.
  • the composition increases the cytotoxic activity of NKG2A-expressing cells.
  • the composition restores the cytotoxic activity of NKG2A-expressing cells.
  • the composition causes cytotoxic activity towards cells targeted by NKG2A-expressing cells.
  • one way to control the amount of undesirable NKG2A expressing cells in a patient is by providing a composition that comprises one or more anti-NKG2A agents, antibodies, fragments or antigen-binding fragments thereof to cause cytotoxic activity towards cells targeted by NKG2A-expressing cells.
  • the disease or disorder is associates with aberrant levels of HLA-E.
  • the disease or disorder is a cancer.
  • the disease or disorder is a cancer that expresses HLA-E.
  • the disease or disorder is a cancer, a viral infection, an autoimmune disease, an inflammatory disease or a bacterial infection.
  • the disease or disorder is a cancer including but not limited to acute myeloid leukemia, acute lymphoblastic leukemia, colorectal, ovarian, gynecologic, liver, glioblastoma, Hodgkin lymphoma, chronic lymphocytic leukemia, esophagus, gastric, pancreas, colon, kidney, head and neck, lung and melanoma.
  • the disease or disorder is a condition characterized by NK cell hyperactivity or NK cell hyperproliferation.
  • the binding of NKG2A to HLA-E to on a target cell is blocked. In some embodiments, the binding of any of the anti-NKG2A agents or anti-NKG2A antibodies described herein to NKG2A on a cell, blocks to ability of the NKG2A on the cell from interacting with HLA-E to on a target cell. In some embodiments, when any of the anti-NKG2A agents or anti- NKG2A antibodies described herein bind NKG2A on a cell, the cell causes the target cell to lyse.
  • Anti-NKG2A agents/antibodies, antibody fragments or antigen-binding fragments thereof may be formulated in a pharmaceutical composition that is useful for a variety of purposes, including the treatment of diseases, disorders or physical trauma.
  • Pharmaceutical compositions comprising one or more anti-NKG2A agents/antibodies, antibody fragments or antigen-binding fragments thereof herein may be incorporated into kits and medical devices for such treatment. Medical devices may be used to administer pharmaceutical compositions herein to a patient in need thereof, and according to one embodiment of the technology, kits are provided that include such devices. Such devices and kits may be designed for routine administration, including self-administration, of the pharmaceutical compositions herein.
  • Therapeutic formulations of an agent or antibody may be prepared for storage by mixing the agent/antibody having the desired degree of purity with optional physiologically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions.
  • Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m- cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine,
  • Active ingredients may also be entrapped in microcapsule prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatinmicrocapsule and poly-(methylmethacylate) microcapsule, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions.
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules
  • Formulations for in vivo administration generally are sterile. This may be accomplished for instance by filtration through sterile filtration membranes, for example.
  • Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the agent/antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsule. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinyl alcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and .gamma.
  • sustained-release preparations include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinyl alcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and .gamma.
  • ethyl-L-glutamate non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the Lupron Depot.TM. (injectable microspheres composed of lactic acid- glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3 -hydroxybutyric acid. While polymers such as ethylene -vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods.
  • encapsulated agents/antibodies When encapsulated agents/antibodies remain in the body for a long time, they may denature or aggregate as a result of exposure to moisture at 37° C, resulting in a loss of biological activity and possible changes in immunogenicity. Rational strategies can be devised for stabilization depending on the mechanism involved. For example, if the aggregation mechanism is discovered to be intermolecular S— S bond formation through thio-disulfide interchange, stabilization may be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions.
  • anti-NKG2A agents e.g., antibodies, provided herein are administered to a mammal, e.g., a human, in a pharmaceutically acceptable dosage form such as those discussed above, including those that may be administered to a human intravenously as a bolus or by continuous infusion over a period of time, or by intramuscular, intraperitoneal, intra-cerebrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes.
  • a mammal e.g., a human
  • a pharmaceutically acceptable dosage form such as those discussed above, including those that may be administered to a human intravenously as a bolus or by continuous infusion over a period of time, or by intramuscular, intraperitoneal, intra-cerebrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes.
  • the appropriate dosage of agent/antibody will depend on the type of disease to be treated, as defined above, the severity and course of the disease, whether the antibody is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the discretion of the attending physician.
  • the antibody is suitably administered to the patient at one time or over a series of treatments.
  • about 1 pg/kg to about 50 mg/kg (e.g., 0.1-20 mg/kg) of antibody may be an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion.
  • a typical daily or weekly dosage might range from about 1 pg/kg to about 20 mg/kg or more, depending on the factors mentioned above.
  • the treatment is repeated until a desired suppression of disease symptoms occurs.
  • other dosage regimens may be useful.
  • the progress of this therapy is easily monitored by conventional techniques and assays, including, for example, radiographic imaging. Detection methods using the antibody to determine NKG2A levels in bodily fluids or tissues may be used in order to optimize patient exposure to the therapeutic antibody.
  • a composition comprising an anti-NKG2A agent herein is administered as a monotherapy, and in some embodiments, the composition comprising the anti-NKG2A agent is administered as part of a combination therapy.
  • the effectiveness of the agent, agent/antibody, antibody or antigen-binding fragment thereof in preventing or treating disease may be improved by administering the agent/antibody serially or in combination with another agent that is effective for those purposes, such as a chemotherapeutic drug for treatment of cancer or a microbial infection.
  • the anti-NKG2A agent, antibody or antigen-binding fragment thereof may serve to enhance or sensitize cells to chemotherapeutic treatment, thus permitting efficacy at lower doses and with lower toxicity.
  • Certain combination therapies include, in addition to administration of the composition comprising an agent that reduces the number of NKG2A expressing cells, delivering a second therapeutic regimen selected from the group consisting of administration of a chemotherapeutic agent, radiation therapy, surgery, and a combination of any of the foregoing.
  • compositions comprising any of the anti-NKG2A agents described herein.
  • the composition comprising any of the anti-NKG2A agents described herein includes a pharmaceutically acceptable excipient.
  • diagnostic reagents comprising an anti-NKG2A agent, antibody or antigenbinding fragment thereof described herein.
  • any of the anti-NKG2A agents, e.g., antibodies or antigen-binding fragments thereof, provided herein may be used to detect and/or purify NKG2A, e.g., from bodily fluid(s) or expressed on cells in bodily fluids or tissues. Also provided herein are methods for detecting NKG2A.
  • a method may comprise contacting a sample (e.g., a biological sample known or suspected of to contain NKG2A) with an anti-NKG2A agent provided herein, and, if the sample contains NKG2A, detecting NKG2A: anti-NKG2A complexes.
  • a sample e.g., a biological sample known or suspected of to contain NKG2A
  • an anti-NKG2A agent provided herein
  • reagents comprising an anti-NKG2A agent described herein and methods for detecting NKG2A for research purposes.
  • Anti-NKG2A antibodies may be useful in diagnostic assays for NKG2A, e.g., detecting its presence in specific cells, tissues, or bodily fluids. Such diagnostic methods may be useful in diagnosis, e.g., of a hyperproliferative disease or disorder.
  • the disease or disorder is a cancer, a viral infection, an autoimmune disease, an inflammatory disease or a bacterial infection.
  • the disease or disorder is a cancer including but not limited to acute myeloid leukemia, acute lymphoblastic leukemia, colorectal, ovarian, gynecologic, liver, glioblastoma, Hodgkin lymphoma, chronic lymphocytic leukemia, esophagus, gastric, pancreas, colon, kidney, head and neck, lung and melanoma.
  • any of the anti-NKG2A agents or anti-NKG2A antibodies described herein are capable of binding a cell.
  • the cell is a natural killer (NK) cell.
  • the cell is a lymphocyte.
  • the lymphocyte is a CD8+ lymphocyte.
  • any of the anti-NKG2A agents or anti-NKG2A antibodies described herein specifically bind NKG2A on a cell.
  • any of the anti-NKG2A agents or anti- NKG2A antibodies described herein specifically bind NKG2A and do not specifically bind NKG2C or NKG2E.
  • an anti-NKG2A agent/antibody comprises a detectable marker or label.
  • an anti-NKG2A agent/antibody is conjugated to a detectable marker or label.
  • an anti-NKG2A agent/antibody may be labeled with a detectable moiety. Numerous labels are available which are generally grouped into the following categories:
  • Radioisotopes such as 35 S, 14 C, 125 1, 3 H, and 13 4.
  • the antibody can be labeled with the radioisotope using the techniques described in Current Protocols in Immunology, Volumes 1 and 2, Coligen et al., Ed. Wiley-Interscience, New York, N.Y., Pubs. (1991), for example, and radioactivity can be measured using scintillation counting.
  • Fluorescent labels such as rare earth chelates (europium chelates) or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, Lissamine, phycoerythrin, Texas Red and Brilliant VioletTM are available.
  • the fluorescent labels can be conjugated to the antibody using the techniques disclosed in Current Protocols in Immunology, supra, for example. Fluorescence can be quantified using a flow cytometer, imaging microscope or fluorimeter.
  • the enzyme generally catalyzes a chemical alteration of the chromogenic substrate that can be measured using various techniques. For example, the enzyme may catalyze a color change in a substrate, which can be measured spectrophotometrically. Alternatively, the enzyme may alter the fluorescence or chemiluminescence of the substrate. Techniques for quantifying a change in fluorescence are described above.
  • the chemiluminescent substrate becomes electronically excited by a chemical reaction and may then emit light that can be measured (using a chemilluminometer, for example) or donates energy to a fluorescent acceptor.
  • enzymatic labels include luciferases (e.g., firefly luciferase and bacterial luciferase; U.S. Pat. No. 4,737,456), luciferin, 2,3- dihydrophthalazinediones, malate dehydrogenase, urease, peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase, beta-galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclicoxidases (such as uricase and xanthine oxidase), lactoperoxidase, microperoxidase, and the like.
  • luciferases e.g., firefly luciferase and bacterial luciferase;
  • HRP Horseradish peroxidase
  • OPD orthophenylene diamine
  • TMB 3, 3', 5,5'- tetramethyl benzidine hydrochloride
  • alkaline phosphatase AP
  • para-Nitrophenyl phosphate as chromogenic substrate
  • iii alkaline phosphatase (AP) with para-Nitrophenyl phosphate as chromogenic substrate
  • iii).beta.-D-galactosidase (.beta.-D-Gal) with a chromogenic substrate (e.g., p-nitrophenyl-p-D- galactosidase) or Anorogenic substrate 4-methylumbelliferyl-.beta.-D-galactosidase.
  • a chromogenic substrate e.g., p-nitrophenyl-p-D- galactosidase
  • Anorogenic substrate 4-methylumbelliferyl-.beta.-D-galactosidase.
  • the label is indirectly conjugated with the agent, agent/antibody, antibody or antigen-binding fragment thereof.
  • an antibody can be conjugated with biotin and any of the three broad categories of labels mentioned above can be conjugated with avidin, or vice versa. Biotin binds selectively to avidin and thus, the label can be conjugated with the antibody in this indirect manner.
  • the antibody is conjugated with a small hapten (e.g., digoxin) and one of the different types of labels mentioned above is conjugated with an anti-hapten antibody (e.g., anti-digoxin antibody).
  • a small hapten e.g., digoxin
  • an anti-hapten antibody e.g., anti-digoxin antibody
  • an anti-NKG2A agent, agent/antibody, antibody or antigen-binding fragment thereof need not be labeled, and the presence thereof can be detected, e.g., using a labeled antibody which binds to an anti-NKG2A antibody.
  • an anti-NKG2A agent, agent/antibody, antibody or antigen-binding fragment thereof herein is immobilized on a solid support or substrate.
  • an anti-NKG2A agent/antibody herein is non-diffusively immobilized on a solid support (e.g., the anti-NKG2A agent/antibody does not detach from the solid support).
  • a solid support or substrate can be any physically separable solid to which an anti-NKG2A agent, agent/antibody, antibody or antigenbinding fragment thereof can be directly or indirectly attached including, but not limited to, surfaces provided by microarrays and wells, and particles such as beads (e.g., paramagnetic beads, magnetic beads, microbeads, nanobeads), microparticles, and nanoparticles.
  • Solid supports also can include, for example, chips, columns, optical fibers, wipes, filters (e.g., flat surface filters), one or more capillaries, glass and modified or functionalized glass (e.g., controlled-pore glass (CPG)), quartz, mica, diazotized membranes (paper or nylon), polyformaldehyde, cellulose, cellulose acetate, paper, ceramics, metals, metalloids, semiconductive materials, quantum dots, coated beads or particles, other chromatographic materials, magnetic particles; plastics (including acrylics, polystyrene, copolymers of styrene or other materials, polybutylene, polyurethanes, TEFLONTM, polyethylene, polypropylene, polyamide, polyester, polyvinylidenedifluoride (PVDF), and the like), polysaccharides, nylon or nitrocellulose, resins, silica or silica-based materials including silicon, silica gel, and modified silicon, Sephadex®, Sepharose®, carbon, metals (
  • the solid support or substrate may be coated using passive or chemically-derivatized coatings with any number of materials, including polymers, such as dextrans, acrylamides, gelatins or agarose. Beads and/or particles may be free or in connection with one another (e.g., sintered).
  • a solid support or substrate can be a collection of particles.
  • the particles can comprise silica, and the silica may comprise silica dioxide.
  • the silica can be porous, and in certain embodiments the silica can be non-porous.
  • the particles further comprise an agent that confers a paramagnetic property to the particles.
  • the agent comprises a metal
  • the agent is a metal oxide, (e.g., iron or iron oxides, where the iron oxide contains a mixture of Fe2+ and Fe3+).
  • An anti- NKG2A agent/antibody may be linked to a solid support by covalent bonds or by non-covalent interactions and may be linked to a solid support directly or indirectly (e.g., via an intermediary agent such as a spacer molecule or biotin).
  • Agents and antibodies provided herein may be employed in any known assay method, such as flow cytometry, immunohistochemistry, immunofluorescence, mass cytometry (e.g., Cytof instrument), competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays.
  • assay method such as flow cytometry, immunohistochemistry, immunofluorescence, mass cytometry (e.g., Cytof instrument), competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays.
  • the method comprises the use of a single primary antibody to specifically identify the presence of the target molecule expressed on the surface of a dispersed suspension of individual cells.
  • Flow cytometry and mass cytometry assays generally involve the use of a single primary antibody to specifically identify the presence of the target molecule expressed on the surface of a dispersed suspension of individual cells.
  • the dispersed cells are typically obtained from a biological fluid sample, e.g., blood, but may also be obtained from a dispersion of single cells prepared from a solid tissue sample such as spleen or tumor biopsy.
  • the primary antibody may be directly conjugated with a detectable moiety, e.g., a fluorophore such as phycoerythrin for flow cytometry or a heavy metal chelate for mass cytometry.
  • a detectable moiety e.g., a fluorophore such as phycoerythrin for flow cytometry or a heavy metal chelate for mass cytometry.
  • the primary antibody may be unlabeled or labeled with an undetectable tag such as biotin, and the primary antibody is then detected by a detectably labeled secondary antibody that specifically recognizes the primary antibody itself or the tag on the primary antibody.
  • the labeled cells are then analyzed in an instrument capable of single cell detection, e.g., flow cytometer, mass cytometer, fluorescence microscope or brightfield light microscope, to identify those individual cells in the dispersed population or tissue sample that express the target recognized by the primary antibody.
  • a detectable moiety e.g., a fluor
  • the method comprises the use of two antibodies, each capable of binding to a different immunogenic portion, or epitope.
  • Sandwich assays involve the use of two antibodies, each capable of binding to a different immunogenic portion, or epitope, of the protein that is detected.
  • the test sample analyte is bound by a first antibody that is immobilized on a solid support, and thereafter a second antibody binds to the analyte, thus forming an insoluble three-part complex. See, e.g., U.S. Pat. No.4,376,110.
  • the second antibody may itself be labeled with a detectable moiety (direct sandwich assays) or may be measured using an anti-immunoglobulin antibody that is labeled with a detectable moiety (indirect sandwich assay).
  • sandwich assay is an ELISA assay, in which case the detectable moiety is an enzyme.
  • the target cell population may be attached to the solid support using antibodies first attached to the support and that recognize different cell surface proteins. These first antibodies capture the cells to the support. NKG2A on the surface of the cells can then be detected by adding anti-NKG2A antibody to the captured cells and detecting the amount of NKG2A antibody attached to the cells.
  • the blood or tissue sample may be fresh or frozen or may be embedded in paraffin and fixed with a preservative such as formalin, for example.
  • the agents, agents/antibodies, antibodies or antigen-binding fragments thereof described herein also may be used for in vivo diagnostic assays.
  • the antibody is labeled with a radionuclide (such as 111 In, 99 Tc, 14 C, 131 I, 125 I, 3 H, 32 P, or 35 S) so that the bound target molecule can be localized using immunoscintillography.
  • a radionuclide such as 111 In, 99 Tc, 14 C, 131 I, 125 I, 3 H, 32 P, or 35 S
  • Detection of NKG2A in immune cells may refer to detection on the surface of immune cells (e.g., by surface staining) and/or inside immune cells (e.g., by intracellular staining). In some embodiments, any of the agents provided herein may be used for detection of NKG2A on the surface of immune cells. In some embodiments, any of the agents provided herein may be used for detection of intracellular NKG2A in immune cells.
  • the immune cell is selected from B cells, plasmacytoid dendritic cells (pDCs), lymphocytes, leukocytes, T cells, monocytes, macrophages, neutrophils, myeloid dendritic cells (mDCs), innate lymphoid cells, mast cells, eosinophils, basophils, natural killer cells, and the like.
  • a heterogeneous population of immune cells comprises peripheral blood mononuclear cells (PBMCs) which may include, for example, T cells, B cells, natural killer cells, and monocytes.
  • PBMCs peripheral blood mononuclear cells
  • agents and methods are provided for detecting NKG2A in a heterogeneous population of immune cells.
  • a heterogeneous population of immune cells may comprise two or more types of immune cells.
  • a heterogeneous population of immune cells may comprise two or more of B cells, plasmacytoid dendritic cells (pDCs), lymphocytes, leukocytes, T cells, monocytes, macrophages, neutrophils, myeloid dendritic cells (mDCs), innate lymphoid cells, mast cells, eosinophils, basophils, natural killer cells, and the like.
  • a heterogeneous population of immune cells comprises peripheral blood mononuclear cells (PBMCs) which may include, for example, T cells, B cells, natural killer cells, and monocytes.
  • PBMCs peripheral blood mononuclear cells
  • NKG2A is detected at a significant level in certain immune cells by an anti-NKG2A agent, antibody or antigen-binding fragment thereof described herein.
  • NKG2A may be detected at a significant level by an anti-NKG2A agent, antibody or antigen-binding fragment thereof described herein in certain immune cells and not significantly detected in other immune cells.
  • the level of NKG2A detection in certain immune cells may vary according to certain factors such as, for example, type of detection assay, type of detection reagent (e.g., type of dye), antibody concentration, donor cell variability, and the like.
  • Detection of NKG2A at a significant level may refer to a particular signal to noise (S:N) ratio (e.g., threshold or range) measured in a flow cytometry assay.
  • S:N signal to noise
  • NKG2A is detected at a significant level in NK cells, as well as a subset of CD8 + T-lymphocytes.
  • NK cells may be identified by the expression of CD56.
  • NKG2A is detected at a significant level in CD56+ NK cells.
  • CD56 also referred to as neural cell adhesion molecule (NCAM), NCAM1, MSK39, neural cell adhesion molecule 1, is a hemophilic binding glycoprotein expressed on the surface of neurons, glia, skeletal muscle, and also in the hematopoietic system. CD56 is most associated with natural killer cells, but is also detected on y5 T cells, activated CD8+ T cells, and dendritic cells.
  • NCAM is a glycoprotein of the Immunoglobulin (Ig) superfamily, with 27 isoforms produced by alternative mRNA splicing.
  • the three main isoforms of NCAM vary only in their cytoplasmic domain: NCAM-120 kDa (GPI anchored), NCAM-140 kDa (short cytoplasmic domain), and NCAM- 180 kDa (long cytoplasmic domain).
  • the extracellular domain of NCAM consists of five immunoglobulin-like (Ig) domains followed by two fibronectin type III (FNIII) domains.
  • the Ig domains of NCAM are involved in hemophilic binding to NCAM, while the FNIII domains are involved in signaling that leads to neurite outgrowth.
  • CD56 is detected on tumors such as myeloma, myeloid leukemia, neuroendocrine tumors, Wilm’s tumor, neuroblastoma, extranodal NK/T cell lymphoma, nasal type, pancreatic acinar cell carcinoma, pheochromocytoma, paraganglioma, small cell lung carcinoma, and Ewing’s sarcoma family of tumors.
  • tumors such as myeloma, myeloid leukemia, neuroendocrine tumors, Wilm’s tumor, neuroblastoma, extranodal NK/T cell lymphoma, nasal type, pancreatic acinar cell carcinoma, pheochromocytoma, paraganglioma, small cell lung carcinoma, and Ewing’s sarcoma family of tumors.
  • NKG2A is not significantly detected in certain lymphocytes by an anti- NKG2A agent described herein.
  • NKG2A may not be significantly detected in certain lymphocytes (e.g., plasmacytoid dendritic cells (pDCs) and/or B cells). No significant detection of NKG2A in certain lymphocytes (e.g., CD304 + plasmacytoid dendritic cells and/or CD19 + CD3- B cells).
  • No significant detection of NKG2A in certain lymphocytes may refer to a particular signal to noise (S:N) ratio (e.g., threshold or range) measured in a flow cytometry assay.
  • S:N signal to noise
  • no significant detection of noise (N) in certain lymphocytes refers to a signal to noise (S:N) ratio of about 1.5 or less.
  • kits incorporating anti-NKG2A agents herein An anti-NKG2A agent (e.g., an anti-NKG2A antibody) herein may be provided in a kit, for example, a packaged combination of reagents in predetermined amounts with instructions for use (e.g., instructions for performing a diagnostic assay; instructions for performing a laboratory assay).
  • the kit is a diagnostic kit configured to detect NKG2A in a sample (e.g., a biological sample).
  • the kit may include an identical isotype negative control irrelevant antibody to control for non-specific binding of the anti- NKG2A agent.
  • the kit may include substrates and cofactors required by the enzyme (e.g., a substrate precursor which provides the detectable chromophore or fluorophore). Additional additives may be included such as stabilizers, buffers (e.g., a block buffer or lysis buffer), and the like. The relative amounts of the various reagents may be varied widely to provide for concentrations in solution of the reagents that substantially optimize the sensitivity of the assay.
  • reagents may be provided as dry powders (e.g., lyophilized powder), including excipients that on dissolution with provide a reagent solution having the appropriate concentration.
  • An article of manufacture containing materials useful for the treatment, or diagnosis, of the disorders described above is provided.
  • An article of manufacture may comprise a container and a label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. Containers may be formed from a variety of materials such as glass or plastic.
  • a container may hold a composition that is effective for treating a condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
  • An active anti-NKG2A agent in the composition may be an anti-NKG2A antibody.
  • a label on, or associated with, the container indicates that the composition is used for treating, or diagnosing, a condition of choice.
  • the article of manufacture may further comprise a second container comprising a pharmaceutically acceptable buffer, such as phosphate-buffered saline, Ringer’s solution and dextrose solution; and may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, fdters, needles, syringes, and package inserts with instructions for use.
  • a pharmaceutically acceptable buffer such as phosphate-buffered saline, Ringer’s solution and dextrose solution
  • agent generally refers to an antibody, antibody fragment, antigen binding fragment thereof, derivative, variants of such antibodies and antibody fragments or antigen-binding fragments thereof (including immunoconjugates, labeled antibodies and antigen-binding antibody fragments, and the like).
  • acceptor human framework generally refers to a framework comprising the amino acid sequence of a immunoglobulin heavy chain variable domain (VH) framework or a immunoglobulin light chain variable domain (VL) framework derived from a human immunoglobulin framework or a human consensus framework, as defined herein.
  • An acceptor human framework “derived from” a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence thereof, or it may contain amino acid sequence changes.
  • the number of framework amino acid changes are 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less.
  • the VH and/or VL acceptor human framework(s) is(are) identical in sequence to the VH and/or VL human immunoglobulin framework amino acid sequence or human consensus framework amino acid sequence.
  • “Framework” or “FR” generally refers to variable domain residues other than hypervariable region (HVR) residues.
  • the FR of a variable domain generally consists of four FR domains: FR1; FR2; FR3; and FR4. Accordingly, the HVR and FR sequences generally appear in the following sequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.
  • a “human consensus framework” generally refers to a framework that represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences.
  • the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences.
  • the subgroup of sequences is a subgroup as in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91- 3242, Bethesda Md. (1991), vols. 1-3.
  • the subgroup is subgroup kappa I as in Kabat et al., supra.
  • the subgroup is subgroup III as in Kabat et al., supra.
  • hypervariable region generally refers to each of the regions of an antibody variable domain that are hypervariable in sequence and/or form structurally defined loops (“hypervariable loops”).
  • native four-chain antibodies comprise six HVRs; three in the VH (Hl, H2, H3), and three in the VL (LI, L2, L3).
  • HVRs generally comprise amino acid residues from the hypervariable loops and/or from the “complementarity determining regions” (CDRs), the latter being of highest sequence variability and/or involved in antigen recognition.
  • CDRs complementarity determining regions
  • the boundaries of a given CDR or FR may vary depending on the scheme used for identification.
  • the Kabat scheme is based on structural alignments
  • the Chothia scheme is based on structural information. Numbering for both the Kabat and Chothia schemes is based upon the most common antibody region sequence lengths, with insertions accommodated by insertion letters, for example, “30a” and deletions appearing in some antibodies. The two schemes place certain insertions and deletions (“indels”) at different positions, resulting in differential numbering.
  • the Contact scheme is based on analysis of complex crystal structures and is similar in many respects to the Chothia numbering scheme.
  • Table 4 lists exemplary position boundaries of CDRH1, CDRH2, CDRH3, and CDRL1, CDRL2, and CDRL3 as identified by Kabat, Chothia, and Contact schemes, respectively.
  • residue numbering is listed using both the Kabat and Chothia numbering schemes.
  • FRs are located between CDRs, for example, with FRH1 located between CDRH1 and CDRH2, and so forth. It is noted that because the shown Kabat numbering scheme places insertions at H35A and H35B, the end of the Chothia CDRH1 loop when numbered using the shown Kabat numbering convention varies between H32 and H34, depending on the length of the loop.
  • CDRs also comprise “specificity determining residues,” or “SDRs,” which are residues that contact a particular antigen. Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al., supra.
  • variable region or “variable domain” generally refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen.
  • the variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). See, e.g., Kindt et al. Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007).
  • a single VH or VL domain may be sufficient to confer antigen-binding specificity.
  • antibodies that bind a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).
  • Bind generally refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen).
  • binding affinity refers to intrinsic binding affinity, which reflects a 1 : 1 interaction between members of a binding pair (e.g., antibody and antigen).
  • the affinity of a molecule X for its partner Y can generally be represented by a dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Specific illustrative and example embodiments for measuring binding affinity are described elsewhere herein.
  • antibodies herein bind to a target (e.g., NKG2A) with a high affinity, e.g., a K d value of no more than about 1 x 10 -7 M; preferably no more than about 1 x 10 -8 M; and preferably no more than about 5 x 10 -9 M.
  • a target e.g., NKG2A
  • An “affinity matured” antibody generally refers to an antibody with one or more alterations in one or more hypervariable regions (HVRs), compared to a parent antibody that does not possess such alterations. Preferably, such alterations result in improved affinity of the antibody for its target antigen.
  • anti-NKG2A agent generally refers to a molecule that is, or comprises, one or more anti- NKG2A antibodies, NKG2A-binding antibody fragments, or NKG2A-binding antibody derivatives.
  • anti-NKG2A antibody and “an antibody that binds to NKG2A” generally refer to an antibody that is capable of binding NKG2A with sufficient affinity and/or specificity such that the antibody is useful as a research tool, diagnostic agent and/or therapeutic agent in targeting NKG2A.
  • the extent of binding of an anti-NKG2A antibody (or antigen-binding fragment thereof) to an unrelated, non-NKG2A protein is less than about 10% of the binding of the antibody to NKG2A as measured, e.g., by a radioimmunoassay (RIA) or by Scatchard analysis or by surface plasmon resonance, such as, for example, Biacore.
  • RIA radioimmunoassay
  • Scatchard analysis or by surface plasmon resonance, such as, for example, Biacore.
  • an antibody that binds to NKG2A has a dissociation constant (kD) of 0.1 ⁇ M, 100 nM, 10 nM, 1 nM, 0.1 nM, 0.01 nM, or 0.001 nM (e.g., 10 -7 M or less, e.g., from 10 -7 M to 10 -13 M).
  • an anti-NKG2A antibody binds to an epitope of NKG2A that is conserved among NKG2A from different species.
  • antibody herein is used in the broadest sense and includes polyclonal and monoclonal antibodies, including intact antibodies and functional (antigen-binding) antibody fragments, including fragment antigen binding (Fab) fragments, F(ab') 2 fragments, Fab’ fragments, Fv fragments, recombinant IgG (rIgG) fragments, variable heavy chain (V H ) regions capable of specifically binding the antigen, single chain antibody fragments, including single chain variable fragments (scFv), and single domain antibodies (e.g., sdAb, sdFv, nanobody) fragments.
  • Fab fragment antigen binding
  • rIgG recombinant IgG
  • V H variable heavy chain
  • the term encompasses genetically engineered and/or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific, e.g., bispecific, antibodies, diabodies, triabodies, and tetrabodies, tandem di-scFv, tandem tri-scFv.
  • antibody should be understood to encompass functional antibody fragments thereof.
  • the term also encompasses intact or full-length antibodies, including antibodies of any class or sub-class, including IgG and sub-classes thereof, IgM, IgE, IgA, and IgD.
  • antibody derivative generally refers to a molecule other than an intact antibody that comprises a portion derived from an intact antibody (or antigen-binding fragment thereof) and that binds the antigen to which the intact antibody (or antigen-binding fragment thereof) binds.
  • antibody derivatives include but are not limited to single chain variable fragments (scFv), diabodies, triabodies, and the like, aptamers comprising multiple antigen-binding antibody fragments, single chain variable fragments, diabodies, triabodies, and the like.
  • antibody fragment or “antigen-binding antibody fragment” generally refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds.
  • antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab')2 and multispecific antibodies formed from antibody fragments.
  • full length antibody “intact antibody,” and “whole antibody” are used herein interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure or having heavy chains that contain an Fc region as defined herein.
  • Fc region generally refers to a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region.
  • the term includes native sequence Fc regions and variant Fc regions.
  • a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxyl-terminus of the heavy chain.
  • the C-terminal lysine (Lys447) of the Fc region may or may not be present.
  • numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991.
  • an “antibody that binds to the same epitope” as a reference antibody generally refers to an antibody that blocks binding of the reference antibody to its antigen in a competition assay by 50% or more, and conversely, the reference antibody blocks binding of the antibody to its antigen in a competition assay by 50% or more.
  • chimeric antibody generally refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species.
  • a “human antibody” generally refers to an antibody that possesses an amino acid sequence corresponding to that of an antibody produced by a human or a human cell or derived from a nonhuman source that utilizes human antibody repertoires or other human antibody-encoding sequences. This definition of a human antibody specifically excludes a “humanized” antibody comprising nonhuman antigen-binding residues.
  • a “humanized” antibody generally refers to a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs.
  • a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody.
  • a humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody.
  • a humanized antibody when aligned with the antibody from which the acceptor framework regions were derived, includes one or more amino acid substitutions (or deletions or insertions) at desired locations.
  • the amino acid residue(s) substituted (or inserted or deleted) at a particular position in the human (or other) or other FR corresponds to the amino acid residue(s) at the corresponding location(s) in the parent antibody (i.e., the non-human antibody from which the CDRs or HVRs were derived).
  • a “humanized form” of an antibody e.g., a non-human antibody, refers to an antibody that has undergone humanization.
  • agent-drug conjugate is an anti-NKG2A agent (e.g., an anti- NKG2A antibody or NKG2A-binding fragment or derivative thereof) conjugated to one or more heterologous molecule(s), including, but not limited to, a cytotoxic agent.
  • cytotoxic agent generally refers to a substance that inhibits or prevents a cellular function and/or causes cell death or destruction.
  • Cytotoxic agents include, but are not limited to, radioactive isotopes (e.g., At 211 , 1 131 , 1 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 , and radioactive isotopes of Lu); chemotherapeutic agents or drugs (e.g., methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents); growth inhibitory agents; enzymes and fragments thereof such as nucleolytic enzymes; antibiotics; toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant
  • “Effector functions” generally refer to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: Clq binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibodydependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g. B cell receptor); and B cell activation.
  • an “effective amount” of an agent generally refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.
  • epitope generally refers to the particular site on an antigen molecule to which an antibody binds.
  • host cell refers to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells.
  • Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.
  • rabbit antibody generally refers to an antibody that possesses an amino acid sequence that corresponds to that of an antibody produced by a rabbit or a rabbit cell or derived from a non-rabbit source that utilizes rabbit antibody repertoires or other rabbit antibody-encoding sequences.
  • immunoconjugate generally refers to an antibody (or antigen-binding fragment or derivative thereof) conjugated to one or more heterologous molecule(s), including, but not limited to, a cytotoxic agent.
  • An immunoconjugate is equivalent to the term “antibody drug conjugate” (ADC).
  • mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats).
  • domesticated animals e.g., cows, sheep, cats, dogs, and horses
  • primates e.g., humans and non-human primates such as monkeys
  • rabbits e.g., mice and rats
  • rodents e.g., mice and rats
  • an “isolated” molecule generally refers to a molecule that has been separated from a component of its original environment (e.g., the natural environment if it is naturally occurring, or a host cell if expressed exogenously), and thus is altered by human intervention (e.g., "by the hand of man") from its original environment.
  • an antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC).
  • An isolated nucleic acid may refer to a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extra-chromosomally or at a chromosomal location that is different from its natural chromosomal location.
  • an isolated nucleic acid can be provided with fewer non-nucleic acid components (e.g., protein, lipid) than the amount of components present in a source sample.
  • a composition comprising isolated nucleic acid can be about 50% to greater than 99% free of non-nucleic acid components.
  • a composition comprising isolated nucleic acid can be about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater than 99% free of non-nucleic acid components.
  • isolated nucleic acid encoding an anti-NKG2A antibody or “isolated polynucleotide encoding an anti-NKG2A antibody” generally refers one or more nucleic acid molecules encoding antibody heavy and light chains (or fragments thereof), including such nucleic acid molecule(s) in a single vector or separate vectors, and such nucleic acid molecule(s) present at one or more locations in a recombinant host cell.
  • NKG2A generally refers to any native, mature NKG2A that results from processing of a NKG2A precursor protein in a cell.
  • the term includes NKG2A from any vertebrate source, including mammals such as primates (e.g. humans and cynomolgus or rhesus monkeys) and rodents (e.g., mice and rats), unless otherwise indicated.
  • the term also includes naturally occurring variants of NKG2A, e.g., splice variants or allelic variants.
  • the amino acid sequence of an exemplary human NKG2A protein is shown in Fig. 1 (SEQ ID NO: 28).
  • NKG2A-positive cell generally refers to any cell that expresses NKG2A on its surface or on an intracellular membrane or organelle (e.g., endosome, ER, Golgi apparatus, lysosome, and the like). Some cells, including those infected by a microbe or associated with some cancer types and tumors, exhibit up-regulation of NKG2A expression.
  • an intracellular membrane or organelle e.g., endosome, ER, Golgi apparatus, lysosome, and the like.
  • the term “monoclonal antibody” generally refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical (as assessed at the level of Ig heavy and/or light chain amino acid sequence) and/or bind the same epitope, except for possible variant antibodies, e.g., containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts.
  • polyclonal antibody preparations which typically include different antibodies directed against different determinants (epitopes)
  • each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen.
  • the modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibodies to be used in accordance with the present technology may be made by a variety of techniques, including, but not limited to, the hybridoma method, recombinant DNA methods, phage-display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other example methods for making monoclonal antibodies being described herein.
  • package insert generally refers to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and/or warnings concerning the use of such therapeutic products.
  • Percent (%) amino acid sequence identity with respect to a reference polypeptide sequence generally refers to the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • composition generally refers to a preparation that is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.
  • pharmaceutically acceptable carrier generally refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject.
  • a pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.
  • treatment generally refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology.
  • Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.
  • antibodies herein are used to delay development of a disease or to slow the progression of a disease.
  • vector generally refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked.
  • the term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced.
  • Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors.”
  • This Example describes the preparation of hybridomas that secrete monoclonal antibodies that react with NKG2A as expressed in vivo.
  • the resulting anti-NKG2A antibodies are suitable for a variety of purposes, including in diagnostic assays, examples of which are provided in the examples below.
  • mice e.g., BALB/c or C57/BL6, or rats, e.g., Sprague Dawley
  • hybridomas were formed using standard protocols to fuse myeloma cells with spleen, and for draining lymph node cells harvested from the animals.
  • fusions were cloned in HAT medium and plated to approximately one cell/well in microtiter plates. Culture supernatants were subsequently tested against NKG2A-expressing cell transfectants, e.g., HEK 293 or RBL, by flow cytometry.
  • hybridoma subclone candidates were then sub-cultured into larger vessels until sufficient cells were present to allow for subcloning. Further characterization of the hybridoma subclone candidates was performed by flow cytometry using NKG2A-transfected cells. Selected candidate clones were further screened, by flow cytometry against human blood cells divided into distinct subsets (e.g., lymphocytes) as well as against one or more cell lines generated from diseased and/or infected human cells. As compared to an isotype control, the percentage of positive cells in each blood cell subset was quantified.
  • distinct subsets e.g., lymphocytes
  • the percentage of positive cells in each blood cell subset was quantified.
  • Basis for clone selection included strong reactivity against immune cells that express NKG2A (e.g., natural killer cells, natural killer T cells, and activated CD8+ T cells) but no appreciable reactivity against other blood cell populations (e.g., certain lymphocytes, B cells); ability to detect NKG2A on the cell surface; ability to detect NKG2A on the cell surface of natural killer (NK) cells and natural killer T cells (NK T cells; ability to detect NKG2A on the cell surface of CD8+ T cells; ability to detect intracellular NKG2A in NK cells; ability to detect intracellular NKG2A in NK T cells; and/or ability to detect intracellular NKG2A in CD8+ T cells.
  • NKG2A e.g., natural killer cells, natural killer T cells, and activated CD8+ T cells
  • CDRs and Framework regions Amino acid sequences of the individual variable domains (CDRs and Framework regions), including the CDR1, CDR2, and CDR3 regions, for both the heavy and light chains for four different antibodies (clones), designated AB 1-4 (also referred to herein as antibodies 1-4, and clones 1-4), are shown in Fig. 2.
  • AB 1-4 also referred to herein as antibodies 1-4, and clones 1-4
  • Table 5 The various heavy and light chain CDR sequences are shown in Table 5, below.
  • This example describes the flow cytometry-based detection of human peripheral blood mononuclear cells (PBMCs) using exemplary anti-NKG2A monoclonal antibodies provided herein.
  • PBMCs peripheral blood mononuclear cells
  • PBMCs Human peripheral blood mononuclear cells
  • PBMCs were counted and adjusted in Cell Staining Buffer (BioLegend, cat. #420201) to a cell density of 0.7-lxl0 7 cells/mL.
  • a 100 pL cell suspension (0.7-lxl0 6 total cells) was stained with various cell surface markers (e.g., CD3, CD4, CD8, CD19, NCAM-1/CD56, CD94, and/or NKG2A/CD 159a) for 15 minutes in the dark. All reagents used are from Bio Legend unless otherwise indicated.
  • Single cells were identified through flow-cytometry by SSC-H and SSC-A (Fig. 4A), and lymphocytes, monocytes, and granulocytes were identified based on their forward (FSC-A) and side scatter (SSC-A) profiles (Fig. 4B).
  • Subsets of lymphocytes including CD3-CD19+ B lymphocytes, CD3+CD19- T lymphocytes, CD3-CD19- lymphocytes, were identified as shown in Fig. 4C. Of the CD3+CD19- lymphocytes, additional subsets of CD4-CD8+ lymphocytes, CD4-CD8- lymphocytes orCD4+CD8- lymphocytes were identified as represented in the dot plots of Fig. 4D.
  • lymphocytes were co-stained with CD56 and AB2, which were used to identify both NK cells and total lymphocytes positive for CD56 (Fig. 5A and Fig. 5B).
  • AB2 showed a similar staining pattern as commercially available anti-NKG2A antibodies, ABI and ABII in total lymphocytes (Fig. 6A-6C).
  • AB2 specifically stained CD3+CD19- lymphocyte subsets (CD4+CD8-, CD4-CD8-, and CD4-CD8+) at a similar level of that of the commercially available anti-NKG2A antibodies (ABI and ABII) (Figs. 7A-7I).
  • ABI, AB2, AB3, and AB4 for NKG2A was supported by co-staining total lymphocytes with CD94/KLRD1, which is reported to form a heterodimer with NKG2 (Figs. 8A-D).
  • NK cells were specifically identified within CD3-CD19- lymphocytes by co-staining with CD56 and each of ABI, AB2, AB3, and AB4 (Figs. 8E-8H).
  • the anti-NKG2A antibodies, ABI, AB2, AB3, and AB4 did not cross react with NKG2A expressed on mouse splenocytes (Figs. 9A-9D).
  • This example describes a functional assay based on NKG2A detection in cells that highly express the target.
  • K562 cells were labeled with CFSE (BioLegend Cat. #4423801). PBMCs were isolated and purified. Purified primary NK cells (effector) were purified by MojoSort Isolation Kit (BioLegend Cat. #480054). A standard was set up with the effector:target ratio of 4: 1, 2: 1, 1: 1, and 1:2 (Fig. 10A). To identify the function of anti-NKG2A antibody in NK cell-mediated cytotoxicity, an E:T ratio of 2: 1 was selected. Antibodies were mixed with target cells and effector cells at 440 nM (lOpg/well). Cells were then incubated at 37°C for 4 hours.
  • exemplary anti-NKG2A antibodies were capable of inhibiting the cytotoxicity of NK cells, as compared to no antibody control.
  • This example describes the ability of the exemplary anti-NKG2A antibodies described herein to block the binding of a ligand to the cell surface receptor.
  • PBMCs Human peripheral blood mononuclear cells
  • PBMCs were incubated with 5 pg purified AB1, AB2, AB3, AB4, or Commercial ABI antibodies, or 1.5 pg ofNKG2E recombinant protein for 15 minutes in the dark.
  • ABI, AB2, AB3, AB4, and Commercial ABI antibodies, and recombinant NKG2E protein were used as blocking agents. In some instances, no blocking agents were used.
  • cells were incubated with PE-conjugated antibody or isotype control for 15 minutes. Cells were washed, acquired on a BD Canto 11, and analyzed using FlowJo software.
  • Percentage original MFI was calculated by dividing the MFI of samples blocked with ABI, AB2, AB3, AB4, and Commercial ABI antibodies, and recombinant NKG2E protein by the MFI of samples blocked with isotype MOPC-21. This value was subtracted from 100 to get a blocking percentage. The formula is shown below:
  • Table 7 lists CDR sequences and SEQ ID NOs for exemplaryanti-NKG2A antibodies provided herein.
  • Precursor human NKG2A protein sequence (UniProtKB - P26715 (NKG2A HUMAN)/ NCBI Reference Sequence: NP 002250,2) (SEQ ID NO: 28)
  • each immunoglobulin heavy chain variable domain of the anti-NKG2A agent comprises first, second, and third heavy chain complementarity determining regions (CDRs), wherein the first heavy chain CDR (CDRH1) comprises an amino acid sequence that is at least 80 percent identical to the amino acid sequence X 1 YX 3 MX 5 (SEQ ID NO: 22), wherein
  • the second heavy chain CDR comprises an amino acid sequence that is at least 80 percent identical to the amino acid sequence X 1 IX 3 X 4 X 5 X 6 X 7 X 8 TX 1 0YX 12 X 13 X 14 X 15 KX 17 (SEQ ID NO: 23), wherein
  • X 17 G or D; and the third heavy chain CDR (CDRH3) comprises an amino acid sequence that is at least 80 percent identical to the amino acid sequence X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 Y X 11 X 12 X 13 X 14 X 15 (SEQ ID NO: 24), wherein
  • X 2 G or R
  • X 3 D or no amino acid
  • X 4 T or no amino acid
  • X 5 S or no amino acid
  • X 6 V or no amino acid
  • each immunoglobulin light chain variable domain of the anti-NKG2A agent comprises first, second, and third light chain CDRs, wherein the first light chain CDR (CDRL1) comprises an amino acid sequence that is at least 80 percent identical to the amino acid sequence X 1 ASX 4 SVX 7 X 8 X 9 X 1 0X 11 X 12 YX 14 H (SEQ ID NO: 25), wherein
  • X 1 S or R
  • X 4 S or K
  • X 7 T or S
  • Xs T or no amino acid
  • the second light chain (CDRL2) comprises an amino acid sequence that is at least 80 percent identical to the amino acid sequence X 1 X 2 SNLX 6 S (SEQ ID NO: 26), wherein
  • the anti-NKG2A agent of embodiment Al wherein the CDRH1 comprises and amino acid sequence that is at least 85 percent identical to the amino acid sequence of SEQ ID NO: 22.
  • the anti-NKG2A agent of embodiment Al wherein the CDRH1 comprises an amino acid sequence that is at least 90 percent identical to the amino acid sequence of SEQ ID NO: 22.
  • the anti-NKG2A agent of embodiment Al wherein the CDRH1 comprises an amino acid sequence that is at least 95 percent identical to the amino acid sequence of SEQ ID NO: 22.
  • A6 The anti-NKG2A agent of any one of embodiments Al to A5, wherein the CDRH2 comprises an amino acid sequence that is at least 85 percent identical to the amino acid sequence of SEQ ID NO: 23.
  • A7 The anti-NKG2A agent of any one of embodiments Al to A5, wherein the CDRH2 comprises an amino acid sequence that is at least 90 percent identical to the amino acid sequence of SEQ ID NO:
  • A8 The anti-NKG2A agent of any one of embodiments Al to A5, wherein the CDRH2 comprises an amino acid sequence that is at least 95 percent identical to the amino acid sequence of SEQ ID NO:
  • a 10 The anti-NKG2A agent of any one of embodiments Al to A9, wherein the CDRH3 comprises an amino acid sequence that is at least 85 percent identical to the amino acid sequence of SEQ ID NO:
  • a 12 The anti-NKG2A agent of any one of embodiments Al to A9, wherein the CDRH3 comprises an amino acid sequence that is at least 95 percent identical to the amino acid sequence of SEQ ID NO:
  • A13 The anti-NKG2A agent of any one of embodiments Al to A9, wherein the CDRH3 comprises an amino acid sequence that is 100 percent identical to the amino acid sequence of SEQ ID NO: 24.
  • a 14 The anti-NKG2A agent of any one of embodiments Al to A 13, wherein the CDRL1 comprises an amino acid sequence that is at least 85 percent identical to the amino acid sequence of SEQ ID NO:
  • a 16 The anti-NKG2A agent of any one of embodiments Al to A 13, wherein the CDRL1 comprises an amino acid sequence that is at least 95 percent identical to the amino acid sequence of SEQ ID NO:
  • a 17 The anti-NKG2A agent of any one of embodiments Al to A 13, wherein the CDRL1 comprises an amino acid sequence that is 100 percent identical to the amino acid sequence of SEQ ID NO: 25.
  • the CDRL2 comprises an amino acid sequence that is at least 85 percent identical to the amino acid sequence of SEQ ID NO:
  • a 19 The anti-NKG2A agent of any one of embodiments Al to A 17, wherein the CDRL2 comprises an amino acid sequence that is at least 90 percent identical to the amino acid sequence of SEQ ID NO:
  • A21 The anti-NKG2A agent of any one of embodiments Al to A 17, wherein the CDRL2 comprises an amino acid sequence that is 100 percent identical to the amino acid sequence of SEQ ID NO: 26.
  • A22 The anti-NKG2A agent of any one of embodiments Al to A21, wherein the CDRL3 comprises an amino acid sequence that is at least 85 percent identical to the amino acid sequence of SEQ ID NO:
  • A27 The anti-NKG2A agent of any one of embodiments Al to A26, wherein the CDRH2 comprises an amino acid sequence chosen from IUYFEDGDTNYNGKFKG (SEQ ID NO: 10), RIYFEDGDTNYNGKFKD (SEQ ID NO: 15), and TISYGGIYTYYPDSVKG (SEQ ID NO: 17).
  • A28 The anti-NKG2A agent of any one of embodiments Al to A27 , wherein the CDRH3 comprises an amino acid sequence chosen from QGIWDYD GF AD (SEQ ID NO: 11) and DRDTSVYVNYYTMDY (SEQ ID NO: 18).
  • CDRL1 comprises an amino acid sequence chosen from SASSSVTYMH (SEQ ID NO: 12) and RASKSVSTSGYSYIH (SEQ ID NO: 19).
  • A30 The anti-NKG2A agent of any one embodiments Al to A29, wherein CDRL2 comprises an amino acid sequence chosen from STSNLAS (SEQ ID NO: 13) and LASNLES (SEQ ID NO: 20).
  • CDRL3 comprises an amino acid sequence chosen from QQRSSYPYT (SEQ ID NO: 14) and QHSRELPLT (SEQ ID NO: 21).
  • anti-NKG2A agent of any one of embodiments Al to A31 which comprises two immunoglobulin heavy chain variable domains and two immunoglobulin light chain variable domains.
  • A33 The anti-NKG2A agent of embodiment A32, wherein the two immunoglobulin heavy chain variable domains each comprise a set of CDRH1, CDRH2, and CDRH3 amino acid sequences.
  • A34 The anti-NKG2A agent of embodiment A32 and A33, wherein the two immunoglobulin light chain variable domains each comprise a set of CDRL1, CDRL2, and CDRL3 amino acid sequences.
  • A35 The anti-NKG2A agent of embodiment A32, wherein the two immunoglobulin heavy chain variable domains each comprise a set of CDRL1, CDRL2, and CDRL3 amino acid sequences.
  • each immunoglobulin heavy chain variable domain comprises a set of CDRH1, CDRH2, CDRH3 amino acid sequences and each immunoglobulin light chain variable domain comprises a set of CDRL1, CDRL2, and CDRL3 amino acid sequences chosen from sets 1-4:
  • A36 The anti-NKG2A agent of embodiment A35, wherein all CDR sequences are from the same set.
  • A37. The anti-NKG2A agent of any one of embodiments Al to A36, wherein the agent is isolated.
  • A38. The anti-NKG2A agent of any one of embodiments Al to A37 wherein the agent is non- naturally occurring.
  • anti-NKG2A agent of any one of embodiments Al to A38, wherein the agent is an antibody, or antigen-binding fragment thereof.
  • anti-NKG2A agent of any one of embodiments Al to A38, wherein the agent is an antibody, or derivative thereof.
  • A41 The anti-NKG2A agent of any one of embodiments Al to A40, wherein the agent is a humanized antibody, or an antigen binding fragment thereof.
  • A42 The anti-NKG2A agent of any one of embodiments Al to A40, wherein the agent is a derivative of a humanized antibody that binds NKG2A.
  • anti-NKG2A agent of any one of embodiments Al to A42, wherein the agent comprises a detectable marker or label.
  • anti-NKG2A agent of any one of embodiments Al to A43, wherein the agent is conjugated to a detectable marker or label.
  • anti-NKG2A agent of any one of embodiments Al to A44, wherein the agent is non- diffusively immobilized on a solid support.
  • a diagnostic reagent comprising the anti-NKG2A agent of any one of embodiments Al to A45.
  • A47. A kit comprising the anti-NKG2A agent of any one of embodiments Al to A45 or the diagnostic reagent of embodiment A46.
  • a diagnostic kit configured to detect NK inhibitory receptor CD94/NK group 2 member A (NKG2A) in a biological sample, wherein the kit comprises the anti-NKG2A agent of any one of embodiments Al to A45 or the diagnostic reagent of embodiment A46.
  • A49 An isolated nucleic acid molecule comprising a nucleotide sequence that encodes an immunoglobulin heavy chain variable domain of the anti-NKG2A agent of any one of embodiments Al to A45.
  • A50 An isolated nucleic acid molecule comprising a nucleotide sequence that encodes an immunoglobulin light chain variable domain of the anti-NKG2A agent of any one of embodiments Al to A45.
  • a recombinant expression vector comprising a first expression cassette and a second expression cassette, wherein the first expression cassette comprises a promoter and a nucleic acid molecule comprising a nucleotide sequence that encodes an immunoglobulin heavy chain variable domain of the anti-NKG2A agent of any one of embodiments Al to A45, and the second expression cassette comprises a promoter and a nucleic acid molecule comprising a nucleotide sequence that encodes an immunoglobulin light chain variable domain of the anti-NKG2A agent of any one of embodiments Al to A45.
  • A52 A recombinant host cell transfected with the recombinant expression vector of embodiment A51.
  • A53. A method of detecting NKG2A, comprising contacting a sample known or suspected to contain NKG2A with the anti-NKG2A agent of any one of embodiments Al to A45, and, if the sample contains NKG2A, detecting NKG2A:anti-NKG2A complexes.
  • B 1 A first anti-NKG2A agent that binds NKG2A under laboratory or physiological conditions, wherein the first agent competitively binds, or is capable of competitively binding, with a second anti- NKG2A agent, which second agent is the anti-NKG2A agent of any one of embodiments Al to A36.
  • B2. A first anti-NKG2A agent that binds NKG2A under laboratory or physiological conditions, wherein the first agent binds to, or is capable of binding to, the same epitope as a second anti-NKG2A agent, which second agent is the anti-NKG2A agent of any one of embodiments Al to A36.
  • BIO The first anti-NKG2A agent of any one of embodiments Bl to B9, wherein the first agent is conjugated to a detectable marker or label.
  • Bl 1.
  • a diagnostic reagent comprising the first anti-NKG2A agent of any one of embodiments Bl to Bl l.
  • a kit comprising the first anti-NKG2A agent of any one of embodiments B 1 to B 11 or the diagnostic reagent of embodiment B 12.
  • a diagnostic kit configured to detect NK inhibitory receptor CD94/NK group 2 member A (NKG2A) in a biological sample, wherein the kit comprises the first anti-NKG2A agent of any one of embodiments B 1 to B 11 or the diagnostic reagent of embodiment B 12.
  • Bl An isolated nucleic acid molecule comprising a nucleotide sequence that encodes an immunoglobulin heavy chain variable domain of the first anti-NKG2A agent of any one of embodiments B 1 to B 11.
  • Bl An isolated nucleic acid molecule comprising a nucleotide sequence that encodes an immunoglobulin light chain variable domain of the first anti-NKG2A agent of any one of embodiments B 1 to B 11.
  • a recombinant expression vector comprising a first expression cassette and a second expression cassette, wherein the first expression cassette comprises a promoter and a nucleic acid molecule comprising a nucleotide sequence that encodes an immunoglobulin heavy chain variable domain of the first anti-NKG2A agent of any one of embodiments Bl to Bl 1, and the second expression cassette comprises a promoter and a nucleic acid molecule comprising a nucleotide sequence that encodes an immunoglobulin light chain variable domain of the first anti-NKG2A agent of any one of embodiments B 1 to B 11.
  • Bl 8 A recombinant host cell transfected with the recombinant expression vector of embodiment Bl 7. B19.
  • a method of detecting NKG2A comprising contacting a sample known or suspected to contain NKG2A with the first anti-NKG2A agent of any one of embodiments Bl to Bl 1, and, if the sample contains NKG2A, detecting NKG2A:anti-NKG2A complexes.
  • An anti-NKG2A agent for detecting NKG2A in a heterogeneous population of immune cells wherein NKG2A is detected at a significant level in natural killer (NK) cells and/or CD8 + T lymphocytes in the population, and NKG2A is not significantly detected in other immune cells in the population.
  • NK natural killer
  • each immunoglobulin heavy chain variable domain of the anti-NKG2A agent comprises first, second, and third heavy chain complementarity determining regions (CDRs); and ii) each immunoglobulin light chain variable domain of the anti-NKG2A agent comprises first, second, and third light chain CDRs.
  • X 1 is S or D
  • X 3 is W or Y
  • X 5 is N orY.
  • X 1 is R or T
  • X 3 is Y or S
  • X 4 is F or Y
  • X 5 is E or G
  • X 6 is D or G
  • X 7 is G or I
  • X 6 is D or Y
  • X 10 is N or Y
  • X 12 is N or P
  • X 13 is G or D
  • X 14 is K or S
  • X 15 is F or V
  • X 17 is G or D.
  • X 1 is Q or D
  • X 2 is G or R
  • X 3 is D or no amino acid
  • X 4 is T or no amino acid
  • X 5 is S or no amino acid
  • X 6 is V or no amino acid
  • X 7 is I or Y
  • X 8 is W or V
  • X 9 is D or N
  • X 11 is D or Y
  • X 12 is G or T
  • X 13 is F or M
  • X 14 is A or D
  • X 15 is D or Y.
  • X 1 is S or R
  • X 4 is S or K
  • X 7 is T or S
  • X 8 is T or no amino acid
  • X 9 is S or no amino acid
  • X 10 is G or no amino acid
  • X 11 is Y or no amino acid
  • X 12 is S or no amino acid
  • X 14 M or I.
  • C7 The anti-NKG2A agent of any one of embodiments C2 to C6, wherein the second light chain CDR (CDRL2) comprises an amino acid sequence that is at least 80 percent identical to the amino acid sequence X 1 X 2 SNLX 6 S (SEQ ID NO: 26), wherein
  • X 1 is S or L
  • X 2 is T or A, and X 6 is A or E.
  • X 2 is Q or H
  • X 3 is R or S
  • X 4 is S or R
  • X 5 is S or E
  • X 6 is Y or L
  • X 6 is Y or L.
  • C12 The anti-NKG2A agent of any one of embodiments C2 to C8, wherein the CDRH1 comprises an amino acid sequence that is 100 percent identical to the amino acid sequence of SEQ ID NO: 22.
  • C16 The anti-NKG2A agent of any one of embodiments C2 to C12, wherein the CDRH2 comprises an amino acid sequence that is 100 percent identical to the amino acid sequence of SEQ ID NO: 23.
  • C17 The anti-NKG2A agent of any one of embodiments C2 to C16, wherein the CDRH3 comprises an amino acid sequence that is at least 85 percent identical to the amino acid sequence SEQ ID NO:
  • CDRL2 comprises an amino acid sequence that is at least 90 percent identical to the amino acid sequence of SEQ ID NO:
  • C35 The anti-NKG2A agent of any one of embodiments C2 to C34, wherein the CDRH3 comprises an amino acid sequence chosen from QGIWDYD GF AD (SEQ ID NO: 11) and DRDTSVYVNYYTMDY (SEQ ID NO: 18).
  • CDRL1 comprises an amino acid sequence chosen from SASSSVTYMH (SEQ ID NO: 12) and RASKSVSTSGYSYIH (SEQ ID NO: 19).
  • CDRL2 comprises an amino acid sequence chosen from STSNLAS (SEQ ID NO: 13) and LASNLES (SEQ ID NO: 20).
  • CDRL3 comprises an amino acid sequence chosen from QQRSSYPYT (SEQ ID NO: 14) and QHSRELPLT (SEQ ID NO: 21).
  • the anti-NKG2A agent of any one of embodiments Cl to C38 which comprises two immunoglobulin heavy chain variable domains and two immunoglobulin light chain variable domains.
  • C40 The anti-NKG2A agent of embodiment C39, wherein the two immunoglobulin heavy chain variable domains each comprise a set of CDRH1, CDRH2, and CDRH3 amino acid sequences.
  • each immunoglobulin heavy chain variable domain comprises a set of CDRH1, CDRH2, and CDRH3 amino acid sequences and each immunoglobulin light chain variable domain comprises a set of CDRL1, CDRL2, and CDRL3 amino acid sequences chosen from sets 1-4:
  • the anti-NKG2A agent o any one of embodiments Cl to C47, wherein NKG2A is not significantly detected in other immune cells in the population, wherein the other immune cells comprise one or more of plasmacytoid dendritic cells, B cells, and monocytes.
  • C49 The anti-NKG2A agent of any one of embodiments Cl to C48, wherein the agent is isolated.
  • C50. The anti-NKG2A agent of any one of embodiments Cl to C49, wherein the agent is non- naturally occurring.
  • C51. The anti-NKG2A agent of any one of embodiments Cl to C50, wherein the agent is an antibody, or antigen-binding fragment thereof.
  • anti-NKG2A agent of any one of embodiments Cl to C50, wherein the agent is an antibody, or derivative thereof.
  • anti-NKG2A agent of any one of embodiments Cl to C52, wherein the agent is a humanized antibody, or an antigen binding fragment thereof.
  • anti-NKG2A agent of any one of embodiments Cl to C52, wherein the agent is a derivative of a humanized antibody that bind NKG2A.
  • anti-NKG2A agent of any one of embodiments Cl to C54, wherein the agent comprises a detectable marker or label.
  • anti-NKG2A agent of any one of embodiments Cl to C55, wherein the agent is conjugated to a detectable marker or label.
  • a diagnostic reagent comprising the anti-NKG2A agent of any one of embodiments Cl to C57.
  • C59 A kit comprising the anti-NKG2A agent of any one of embodiments Cl to C57 or the diagnostic reagent of embodiment C58.
  • a diagnostic kit configured to detect NKG2A in a biological sample, wherein the kit comprises the anti-NKG2A agent of any one of embodiments Cl to C57 or the diagnostic reagent of embodiment C58.
  • An isolated nucleic acid molecule comprising a nucleotide sequence that encodes an immunoglobulin heavy chain variable domain of the anti-NKG2A agent of any one of embodiments Cl to C57.
  • An isolated nucleic acid molecule comprising a nucleotide sequence that encodes an immunoglobulin light chain variable domain of the anti-NKG2A agent of any one of embodiments C 1 to C57.
  • a recombinant expression vector comprising a first expression cassette and a second expression cassette, wherein the first expression cassette comprises a promoter and a nucleic acid molecule comprising a nucleotide sequence that encodes an immunoglobulin heavy chain variable domain of the anti-NKG2A agent of any one of embodiments Cl to C57, and the second expression cassette comprises a promoter and a nucleic acid molecule comprising a nucleotide sequence that encodes an immunoglobulin light chain variable domain of the anti-NKG2A agent of any one of embodiments C 1 to C57.
  • a method of detecting NKG2A comprising contacting a sample known or suspected to contain NKG2A with the anti-NKG2A agent of any one of embodiments Cl to C57, and, if the sample contains NKG2A, detecting NKG2A:anti-NKG2A complexes.
  • a method of detecting NKG2A in a heterogeneous population of immune cells comprising contacting the population with an anti-NKG2A agent of any one of embodiments Cl to C57, and, if the population contains cells expressing NKG2A, detecting NKG2A:anti-NKG2A complexes.
  • An anti-NKG2A agent that binds the NKG2A receptor or a fragment thereof, comprising one or more of: a) an immunoglobulin heavy chain variable domain comprising:
  • CDRH1 heavy chain complementary determining region 1
  • X 1 YX 3 MX 5 (SEQ ID NO: 22), wherein X 1 is S or D; X 3 is W or Y, and X 5 is N or Y;
  • CDRH2 heavy chain complementary determining region 2
  • a heavy chain complementary determining region 3 comprising the sequence XIX 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 YXIIXI2XI3XI4XI5 (SEQ ID NO: 24), wherein X 1 is Q or D; X 2 is G or R; X 3 is D or no amino acid; X 4 is T or no amino acid; X 5 is S or no amino acid; X 6 is V or no amino acid; X 7 is I or Y; X 8 is W or V; X 9 is D or N; X 11 is D or Y; X 12 is G or T; X 13 is F or M; X 14 is A or D; and
  • X 15 is D or Y; and b) an immunoglobulin light chain variable domain comprising:
  • CDRL1 a light chain complementary determining region 1
  • X 1 ASX 4 SVX 7 X 8 X 9 X 10 X 11 X 12 YX 14 H (SEQ ID NO: 25), wherein X 1 is S or R; X 4 is S or K; X 7 is T or S; X 8 is T or no amino acid; X 9 is S or no amino acid; X 1 0 is G or no amino acid; X 11 is Y or no amino acid; X 12 is S or no amino acid; and X 14 is M or I;
  • CDRL2 light chain complementary determining region 2
  • X 1 X 2 SNLX 6 S (SEQ ID NO: 26), wherein X 1 is S or L; X 2 is T or A; and X 6 is A or E; and
  • a light chain complementary determining region 3 comprising the sequence QX 2 X 3 X 4 X 5 X 6 PX 8 T (SEQ ID NO: 27), wherein X 2 is Q or H; X 3 is R or S; X 4 is S or R; X 5 is S or E; X 6 is Y or L; and X 8 is Y or L.
  • the anti-NKG2A agent of embodiment DI wherein the immunoglobulin heavy chain variable domain comprises: a CDRH1 comprising the sequence of amino acids set forth in SEQ ID NO: 9 or SEQ ID NO: 16, or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:9 or SEQ ID NO: 16; a CDRH2 comprising the sequence of amino acids set forth in SEQ ID NO: 10, 15 or 17, or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 10, 15 or 17;
  • the anti-NKG2A agent of embodiment D 1 or embodiment D2, wherein the immunoglobulin heavy chain variable domain comprises: a CDRH1 comprising the sequence of amino acids set forth in SEQ ID NO: 9 or SEQ ID NO: 16; a CDRH2 comprising the sequence of amino acids set forth in SEQ ID NO: 10, 15 or 17; and a CDRH3 comprising the sequence of amino acids set forth in SEQ ID NO: 11 or SEQ ID NO: 18.
  • the anti-NKG2A agent of any of embodiments D1-D3, wherein the immunoglobulin light chain variable domain comprises: a CDRL1 comprising the sequence of amino acids set forth in SEQ ID NO: 12 or SEQ ID NO: 19, or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 12 or SEQ ID NO: 19; a CDRL2 comprising the sequence of amino acids set forth in SEQ ID NO: 13 or SEQ ID NO:20, or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ
  • the anti-NKG2A agent of any of embodiments D1-D4, wherein the immunoglobulin light chain variable domain comprises: a CDRL1 comprising a sequence of amino acids set forth in SEQ ID NO: 12 or SEQ ID NO: 19; a CDRL2 comprising a sequence of amino acids set forth in SEQ ID NO: 13 or SEQ ID NO:20; and a CDRL3 comprising a sequence of amino acids set forth in SEQ ID NO: 14 or SEQ ID NO:21.
  • the CDRH1 comprises the sequence set forth in SEQ ID NO: 9, or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 9,
  • the CDRH2 comprises the sequence set forth in SEQ ID NO: 10 ,or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 10; and the CDRH3 comprises the sequence set forth in SEQ ID NO: 11, or a sequence of amino acids that exhibits at least 80%,
  • D7 The anti-NKG2A agent of any of embodiments D1-D6, wherein the CDRH1 comprises the sequence set forth in SEQ ID NO: 9, the CDRH2 comprises the sequence set forth in SEQ ID NO: 10; and the CDRH3 comprises the sequence set forth in SEQ ID NO: 11. D8.
  • D11 The anti-NKG2A agent of any of embodiments D1-D5 and D10, wherein the CDRH1 comprises the sequence set forth in SEQ ID NO: 16, the CDRH2 comprises the sequence set forth in SEQ ID NO: 17; and the CDRH3 comprises the sequence set forth in SEQ ID NO: 18. D12.
  • the CDRL1 comprises the sequence set forth in SEQ ID NO: 12, or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:12;
  • the CDRL2 comprises the sequence set forth in SEQ ID NO: 13 or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 13;
  • the CDRL3 comprises the sequence set forth in SEQ ID NO: 14 or a sequence of amino acids that exhibits at least 80%, 81%, 82%,
  • the CDRL2 comprises the sequence set forth in
  • SEQ ID NO: 20 or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%,
  • the CDRL3 comprises the sequence set forth in SEQ ID NO: 21 or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
  • the anti-NKG2A agent of any of embodiments DI -DI 1 and D14 wherein the CDRL1 comprises the sequence set forth in SEQ ID NO: 19, the CDRL2 comprises the sequence set forth in SEQ ID NO: 20; and the CDRL3 comprises the sequence set forth in SEQ ID NO: 21.
  • DI 6 The anti-NKG2A agent of any of embodiments DI -DI 5, wherein the CDRH1 comprises the sequence of amino acids set forth in SEQ ID NO: 9 or SEQ ID NO: 16, or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
  • CDRH2 comprises a sequence of amino acids set forth in SEQ ID NO: 10, 15 or 17, or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,
  • the CDRH3 comprises a sequence of amino acids set forth in SEQ ID NO: 11 or SEQ ID NO: 18, or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
  • the CDRL1 comprises a sequence of amino acids set forth in SEQ ID NO: 11 or SEQ ID NO: 18; the CDRL1 comprises a sequence of amino acids set forth in SEQ ID NO:
  • the CDRL2 comprises a sequence of amino acids set forth in SEQ ID NO: 13 or SEQ ID NO:20, or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
  • the CDRL3 comprises a sequence of amino acids set forth in SEQ ID NO: 14 or SEQ ID NO:21, or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,
  • the CDRH1 comprises the sequence of amino acids set forth in SEQ ID NO: 9 or SEQ ID NO: 16;
  • the CDRH2 comprises the sequence of amino acids set forth in SEQ ID NO: 10, 15 or 17;
  • the CDRH3 comprises the sequence of amino acids set forth in SEQ ID NO: 11 or SEQ ID NO: 18;
  • the CDRL1 comprises the sequence of amino acids set forth in SEQ ID NO: 12 or SEQ ID NO: 19;
  • the CDRL2 comprises the sequence of amino acids set forth in SEQ ID NO: 13 or SEQ ID NO:20;
  • the CDRL3 comprises the sequence of amino acids set forth in SEQ ID NO:21.
  • the CDRH1 comprises the sequence set forth in SEQ ID NO: 9 or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:9
  • the CDRH2 comprises the sequence set forth in SEQ ID NO: 10 or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 10
  • the CDRH3 comprises the sequence set forth in SEQ ID NO: 11 or a sequence of amino acids that exhibits at least 80%, 81%
  • the CDRL2 comprises the sequence set forth in SEQ ID NO: 13 or a sequence of amino acids that exhibits at least 80%,
  • the CDRL3 comprises the sequence set forth in SEQ ID NO: 14 or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%,
  • the anti-NKG2A agent of any of embodiments D1-D18 wherein the CDRH1 comprises the sequence set forth in SEQ ID NO: 9, the CDRH2 comprises the sequence set forth in SEQ ID NO: 10; the CDRH3 comprises the sequence set forth in SEQ ID NO: 11; the CDRL1 comprises the sequence set forth in SEQ ID NO: 12, the CDRL2 comprises the sequence set forth in SEQ ID NO: 13; and the CDRL3 comprises the sequence set forth in SEQ ID NO: 14.
  • D20 The anti-NKG2A agent of any of embodiments DI -DI 7, wherein the CDRH1 comprises the sequence set forth in SEQ ID NO: 9 or a sequence of amino acids that exhibits at least 80%, 81%,
  • the CDRH2 comprises the sequence set forth in
  • SEQ ID NO: 15 or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%,
  • the CDRH3 comprises the sequence set forth in SEQ ID NO: 11 or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
  • the CDRL1 comprises the sequence set forth in SEQ ID NO: 12 or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
  • the CDRL2 comprises the sequence set forth in SEQ ID NO: 13 or a sequence of amino acids that exhibits at least 80%,
  • the CDRL3 comprises the sequence set forth in SEQ ID NO: 14 or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%,
  • the anti-NKG2A agent of any of embodiments DI -DI 7 and D20 wherein the CDRH1 comprises the sequence set forth in SEQ ID NO: 9, the CDRH2 comprises the sequence set forth in SEQ ID NO: 15; the CDRH3 comprises the sequence set forth in SEQ ID NO: 11; the CDRL1 comprises the sequence set forth in SEQ ID NO: 12, the CDRL2 comprises the sequence set forth in SEQ ID NO: 13; and the CDRL3 comprises the sequence set forth in SEQ ID NO: 14.
  • the CDRH2 comprises the sequence set forth in
  • SEQ ID NO: 17 or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%,
  • the CDRH3 comprises the sequence set forth in SEQ ID NO: 18 or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
  • the CDRL1 comprises the sequence set forth in SEQ ID NO: 19 or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
  • the CDRL2 comprises the sequence set forth in SEQ ID NO: 20 or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:20; and the CDRL3 comprises the sequence set forth in SEQ ID NO: 21 or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:21.
  • the anti-NKG2A agent of any of embodiments DI -DI 7 and D22 wherein the CDRH1 comprises the sequence set forth in SEQ ID NO: 16, the CDRH2 comprises the sequence set forth in SEQ ID NO: 17; the CDRH3 comprises the sequence set forth in SEQ ID NO: 18; the CDRL1 comprises the sequence set forth in SEQ ID NO: 19, the CDRL2 comprises the sequence set forth in SEQ ID NO: 20; and the CDRL3 comprises the sequence set forth in SEQ ID NO: 21.
  • the anti-NKG2A agent of any of embodiments D1-D23, wherein the immunoglobulin heavy chain variable domain comprises the amino acid sequence set forth in any of SEQ ID NOS: 1, 3 or 4, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to any of SEQ ID NOS: 1, 3 or 4.
  • the anti-NKG2A agent of any of embodiments D1-D24, wherein the immunoglobulin heavy chain variable domain comprises the amino acid sequence set forth in any of SEQ ID NOS: 1, 3 or 4.
  • D26 The anti-NKG2A agent of any of embodiments D1-D25, wherein the immunoglobulin heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 1 or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 1.
  • the anti-NKG2A agent of any of embodiments D1-D26, wherein the immunoglobulin heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 1.
  • the anti-NKG2A agent of any of embodiments D1-D25, wherein the immunoglobulin heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 3, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:3.
  • the anti-NKG2A agent of any of embodiments D1-D25 and D28, wherein the immunoglobulin heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 3.
  • the anti-NKG2A agent of any of embodiments D1-D25, wherein the immunoglobulin heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 4, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:4.
  • the anti-NKG2A agent of any of embodiments D1-D32, wherein the immunoglobulin light chain variable domain comprises the amino acid sequence set forth in any of SEQ ID NOS: 5 or 8.
  • D34 The anti-NKG2A agent of any of embodiments D1-D33, wherein the immunoglobulin light chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 5, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:5.
  • the anti-NKG2A agent of any of embodiments D1-D34, wherein the immunoglobulin light chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 5.
  • the anti-NKG2A agent of any of embodiments D1-D33, wherein the immunoglobulin light chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 8, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 8.
  • the anti-NKG2A agent of any of embodiments D1-D33 and D36, wherein the immunoglobulin light chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 8.
  • the immunoglobulin heavy chain variable domain comprises the amino acid sequence set forth in any of SEQ ID NOS: 1, 3 or 4, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to any of SEQ ID NOS: 1, 3 or 4, and the immunoglobulin light chain variable domain comprises the amino acid sequence set forth in any of SEQ ID NOS: 5 or 8, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to any of SEQ ID NOS: 5 or 8.
  • the immunoglobulin heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 1, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 1; and the immunoglobulin light chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 5, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:5.
  • the immunoglobulin heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 3, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 3; and the immunoglobulin light chain variable domain comprises the amino acid sequence set forth in SEQ ID NOS: 5, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:5.
  • the immunoglobulin heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 4, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 4; and the immunoglobulin light chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 8, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:8.
  • the anti-NKG2A agent of any of embodiments D1-D45 comprising one immunoglobulin heavy chain variable domain and one immunoglobulin light chain variable domain.
  • D47 The anti-NKG2A agent of any of embodiments D1-D45, comprising two immunoglobulin heavy chain variable domains and two immunoglobulin light chain variable domains.
  • D48 The anti-NKG2A agent of any of embodiments D1-D47, wherein the immunoglobulin heavy chain variable domain further comprises a signal sequence.
  • the anti-NKG2A agent of embodiment D50, wherein the signal sequence comprises the sequence set forth in SEQ ID NO: 46 or SEQ ID NO: 50.
  • D54 The anti-NKG2A agent of any of embodiments D1-D52, wherein the anti-NKG2A agent is humanized.
  • the anti-NKG2A agent of embodiment D57 wherein the detectable moiety is a radioisotope, fluorescent label or enzyme-substrate label.
  • D62 The anti-NKG2A agent of any of embodiments D1-D61, for use in the detection of NKG2A in a sample.
  • D63 The anti-NKG2A agent of any of embodiments D1-D62, wherein the anti-NKG2A agent binds to an NKG2A expressing cell in a sample.
  • D65 The anti-NKG2A of any of embodiments D62-D64, wherein the sample comprises a heterogenous population of immune cells.
  • pDCs plasmacytoid dendritic cells
  • mDCs myeloid dendritic cells
  • innate lymphoid cells mast cells
  • eosinophils basophils
  • natural killer cells and peripheral blood mononuclear cells (PBMCs).
  • PBMCs peripheral blood mononuclear cells
  • D68 The anti-NKG2A of any of embodiments D62-D66, wherein the detection comprises the use of two anti-NKG2A agents, each capable of binding to a different portion of NKG2A.
  • D70 The anti-NKG2A of any of embodiments D62-D69, wherein NKG2A is detected intracellularly.
  • D71 The anti-NKG2A of any of embodiments D62-D70, wherein the detection of NKG2A indicates the presence or absence of a disease or disorder.
  • D72 The anti-NKG2A of any of embodiments D62-D71, wherein the detection is performed in vitro.
  • D73 The anti-NKG2A of any of embodiments D62-D72, wherein the detection is performed in vivo.
  • D74 The anti-NKG2A agent of any of embodiments D1-D73, wherein the anti-NKG2A agent binds to an NKG2A expressing cell, and blocks the binding of NKG2A to HLA-E on a target cell.
  • the anti-NKG2A agent of any of embodiments D63-D75, wherein the anti-NKG2A agent bound to the NKG2A expressing cell increases the cytotoxic activity of the NKG2A expressing cell.
  • the anti-NKG2A agent of any of embodiments D63-D76, wherein the anti-NKG2A agent bound to the NKG2A expressing cell induces lysis of a target cell.
  • D78 The anti-NKG2A agent of any of embodiments D74-D77, wherein the target cell is a cell that expresses HLA-E.
  • D79 The anti-NKG2A agent of any of embodiments D74-D77, wherein the target cell is a cell associated with a disease or disorder.
  • the anti-NKG2A agent of embodiment D79 wherein the disease or disorder is a cancer, a viral infection, an autoimmune disease, an inflammatory disease or a bacterial infection.
  • D82 The anti-NKG2A agent of embodiment D87, wherein the cancer is acute myeloid leukemia, acute lymphoblastic leukemia, colorectal, ovarian, gynecologic, liver, glioblastoma, Hodgkin lymphoma, chronic lymphocytic leukemia, esophagus, gastric, pancreas, colon, kidney, head and neck, lung and melanoma.
  • D83 The anti-NKG2A agent of embodiment D79 or embodiment D80, wherein the disease or disorder is an autoimmune disease.
  • D84 The anti-NKG2A agent of any of embodiments D79-D83, wherein the disease or disorder is a condition characterized by NK cell hyperactivity or NK cell hyperproliferation.
  • a diagnostic reanti-NKG2A agent comprising the anti-NKG2A agent of any of embodiments D1-D84.
  • D86.A kit comprising the anti-NKG2A agent of any of embodiments D1-D84, or the diagnostic reagent of embodiment D85.
  • D87.A composition comprising the anti-NKG2A agent of any of embodiments D1-D84, and a pharmaceutically acceptable excipient.
  • the isolated nucleic acid of embodiment D89, wherein the immunoglobulin heavy chain variable domain comprises the sequence of nucleotides set forth in any of SEQ ID NOs: 30-37.
  • the isolated nucleic acid of embodiment D89 or embodiment D90, wherein the immunoglobulin heavy chain variable domain comprises the sequence of nucleotides set forth in SEQ ID NO: 30.
  • the isolated nucleic acid of embodiment D89 or embodiment D90, wherein the immunoglobulin heavy chain variable domain comprises the sequence of nucleotides set forth in SEQ ID NO: 34.
  • the isolated nucleic acid of embodiment D89 or embodiment D90, wherein the immunoglobulin heavy chain variable domain comprises the sequence of nucleotides set forth in SEQ ID NO: 36.
  • the isolated nucleic acid of embodiment D89 or embodiment D90, wherein the immunoglobulin heavy chain variable domain comprises the sequence of nucleotides set forth in SEQ ID NO: 37.
  • An isolated nucleic acid comprising a nucleotide sequence that encodes the immunoglobulin light chain variable domain of the agent of any of embodiments D1-D84.
  • the isolated nucleic acid of embodiment D99 or embodiment DI 00, wherein the immunoglobulin light chain variable domain comprises the sequence of nucleotides set forth in SEQ ID NO: 39.
  • the isolated nucleic acid of embodiment D99 or embodiment DI 00, wherein the immunoglobulin light chain variable domain comprises the sequence of nucleotides set forth in SEQ ID NO: 41.
  • the isolated nucleic acid of embodiment D99 or embodiment DI 00, wherein the immunoglobulin light chain variable domain comprises the sequence of nucleotides set forth in SEQ ID NO: 44.
  • the isolated nucleic acid of embodiment D99 or embodiment DI 00, wherein the immunoglobulin light chain variable domain comprises the sequence of nucleotides set forth in SEQ ID NO: 45.
  • D109 An isolated nucleic acid comprising a nucleotide sequence that encodes the immunoglobulin heavy chain variable domain and the immunoglobulin light chain variable domain of the anti-NKG2A agent of any of embodiments D1-D84.
  • DI 11 The isolated nucleic acid of embodiment DI 10, wherein the nucleotide sequence that encodes the immunoglobulin heavy chain variable domain comprises the sequence set forth in SEQ ID NO: 30; and the immunoglobulin light chain variable domain comprises the sequence of amino acids set forth in SEQ ID NO: 38.
  • DI 12 The isolated nucleic acid of embodiment DI 10, wherein the nucleotide sequence that encodes the immunoglobulin heavy chain variable domain comprises the sequence set forth in SEQ ID NO: 31 ; and the immunoglobulin light chain variable domain comprises the sequence of amino acids set forth in SEQ ID NO: 39.
  • a recombinant expression vector comprising a first expression cassette and a second expression cassette, wherein the first expression cassette comprises a nucleic acid molecule comprising a nucleotide sequence that encodes the immunoglobulin heavy chain variable domain of any one of embodiments D1-D84, and the second expression cassette comprises a nucleic acid molecule comprising a nucleotide sequence that encodes an immunoglobulin light chain variable domain of the anti-NKG2A agent of any one of embodiments D1-D84.
  • a recombinant expression vector comprising a first expression cassette and a second expression cassette, wherein the first expression cassette comprises a nucleic acid molecule comprising the nucleotide sequence of any of embodiments D89-D118, and the second expression cassette comprises a nucleic acid molecule comprising the nucleotide sequence of any of embodiments D89-D118.
  • DI 23 A method of detecting NKG2A, comprising a) contacting a sample with the anti-NKG2A agent of any of embodiments 1-33, under conditions to bind said anti-NKG2A to an NKG2A receptor on said sample, wherein the binding generates the production of a receptor/anti-NKG2A complex; b) detecting the presence of the receptor/anti-NKG2A complexes; c) wherein the detecting comprises the presence or absence of the NKG2A receptor on said sample.
  • a method of treating or preventing a disease or disorder associated with NKG2A in a subject comprising: a) contacting a sample known or suspected to contain NKG2A with the anti-NKG2A agent of any of embodiments D1-D84, b) detecting the presence of complexes comprising NKG2A and the anti-NKG2A agent; wherein the presence of the complexes indicates the presence of a disease or disorder; and c) administering to the subject the anti-NKG2A agent of any of embodiments D1-D84.
  • D126.A method of treating or preventing a disease or disorder in a subject comprising a) detecting the formation of complexes comprising NKG2A and the anti-NKG2A agent of any of embodiments D1-D84 in a sample from the subject; b) administering to a subject in need thereof the anti-NKG2A agent of any of embodiments D1-D84.
  • D127 The method of any of embodiments D123-D126, wherein the sample comprises immune cells.
  • D128. The method of embodiment DI 27, wherein the immune cell is selected from B cells, plasmacytoid dendritic cells (pDCs), lymphocytes, leukocytes, T cells, monocytes, macrophages, neutrophils, myeloid dendritic cells (mDCs), innate lymphoid cells, mast cells, eosinophils, basophils, natural killer cells, and peripheral blood mononuclear cells (PBMCs).
  • pDCs plasmacytoid dendritic cells
  • mDCs myeloid dendritic cells
  • innate lymphoid cells mast cells
  • eosinophils basophils
  • natural killer cells and peripheral blood mononuclear cells (PBMCs).
  • PBMCs peripheral blood mononuclear cells
  • a or “an” can refer to one of or a plurality of the elements it modifies (e.g., “a reagent” can mean one or more reagents) unless it is contextually clear either one of the elements or more than one of the elements is described.
  • the term “about” as used herein refers to a value within 10% of the underlying parameter (i.e., plus or minus 10%), and use of the term “about” at the beginning of a string of values modifies each of the values (i.e., “about 1, 2, and 3” refers to about 1, about 2, and about 3).
  • a weight of “about 100 grams” can include weights between 90 grams and 110 grams.

Landscapes

  • Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Peptides Or Proteins (AREA)

Abstract

Compositions and methods for making and using anti-NKG2A agents, for example, monoclonal antibodies, NKG2A-binding antibody fragments, and derivatives are described, as are kits, nucleic acids encoding such molecules, diagnostic reagents and kits that include anti-NKG2A agents, and methods of making and using the same.

Description

ANTI-NKG2A AGENTS AND COMPOSITIONS AND METHODS FOR MAKING AND USING
THE SAME
Cross Reference to Related Applications
This application claims priority from U.S. provisional application No. 63/107,812, filed October 30, 2020, entitled "ANTI-NKG2A AGENTS AND COMPOSITIONS AND METHODS FOR MAKING AND USING THE SAME" the contents of each which are incorporated herein by reference in their entirety.
Incorporation by Reference of Sequence Listing
The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled BLD013PCTlSeqList.TXT, created October 1, 2021, which is 31,900 bytes in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety.
Field
The technology relates in part to agents that bind the NK inhibitory receptor CD94/NK group 2 member A (NKG2A), particularly monoclonal antibodies, antibody fragments, and antibody derivatives specifically reactive to NKG2A under physiological and/or in vitro conditions. Such agents can be useful for laboratory/research purposes (e.g., flow cytometry), and may be used in the treatment and/or prevention of various diseases or disorders through the delivery of pharmaceutical or other compositions that contain such agents.
Background
The following description includes information that may be useful in understanding the present technology. It is not an admission that any of the information provided herein, or any publication specifically or implicitly referenced herein, is prior art, or even particularly relevant, to the presently claimed technology.
Natural Killer (NK) cells play an important role in host immune regulation to a variety of pathological challenges, from viral infections to cancer. Natural Killer cells show a broad array of tissue distribution and phenotypic variability, and therefore express several activating and inhibitory receptors in order to control their function. Natural-killer group 2, member A (NKG2A) is a C-type lectin family inhibitory receptor that forms a heterodimer with CD94. NKG2A specifically recognizes the non-classical MHC molecule HLA-E, and acts as an inhibitory checkpoint receptor for HLA-E. Although HLA-E is ubiquitously expressed, it has also been associated with overexpression in several tumors. Blocking of NKG2A-HLA-E interactions could be of interest in the development of immunotherapies. However, there remains a need for reagents, devices and methods of detecting NKG2A for both pre- clinical and clinical application settings. Described herein are particular monoclonal antibodies to NKG2A that provide superior target specificity, signal-to-noise ratios, and the like, as compared to other reported anti-NKG2A antibodies, as well as antigen-binding fragments thereof, that meet such needs. Summary Provided herein, in some aspects, are anti-NKG2A agents that bind NKG2A, including anti-NKG2A antibodies, NKG2A-binding antibody fragments, derivatives, and variants of such antibodies and antibody fragments or antigen-binding fragments thereof (including immunoconjugates, labeled antibodies and antigen-binding antibody fragments, and the like), diagnostic reagents that comprise such agents, containers and kits that include any of the anti-NKG2A agents provided herein, and methods of making and using the same. Provided herein, in certain aspects is an anti-NKG2A agent that binds NKG2A under laboratory or physiological conditions, where the agent comprises at least one immunoglobulin heavy chain variable domain and at least one immunoglobulin light chain variable domain, where a) each immunoglobulin heavy chain variable domain of the anti-NKG2A agent comprises first, second, and third heavy chain complementarity determining regions (CDRs), where the first heavy chain CDR (CDRH1) comprises an amino acid sequence that is at least 80 percent identical to the amino acid sequence X1YX3MX5 (SEQ ID NO: 22), where X1 is S or D, X3 is W or Y, and X5 is N or Y; the second heavy chain CDR (CDRH2) comprises an amino acid sequence that is at least 80 percent identical to the amino acid sequence X1IX3X4X5X6X7X8TX10YX12X13X14X15KX17 (SEQ ID NO: 23), where X1 is R or T, X3 is Y or S, X4 is F or Y, X5 is E or G, X6 is D or G, X7 is G or I, X8 is D or Y, X10 is N or Y, X12 is N or P, X13 is G or D, X14 is K or S, X15 is F or V, and X17 is G or D; and the third heavy chain CDR (CDRH3) comprises an amino acid sequence that is at least 80 percent identical to the amino acid sequence X1X2X3X4X5X6X7X8X9YX11X12X13X14X15 (SEQ ID NO: 24), where X1 is Q or D, X2 is G or R, X3 is D or no amino acid, X4 is T or no amino acid, X5 is S or no amino acid, X6 is V or no amino acid, X7 is I or Y, X8 is W or V, X9 is D or N, X11 is D or Y, X12 is G or T, X13 is F or M, X14 is A or D, and X15 is D or Y; and b) each immunoglobulin light chain variable domain of the anti-NKG2A agent comprises first, second, and third light chain CDRs, where the first light chain CDR (CDRL1) comprises an amino acid sequence that is at least 80 percent identical to the amino acid sequence X1ASX4SVX7X8X9X10X11X12YX14H (SEQ ID NO: 25), where X1 is S or R, X4 is S or K, X7 is T or S, X8 is T or no amino acid, X9 is S or no amino acid, X10 is G or no amino acid, X11 is Y or no amino acid, X12 is S or no amino acid, and X14 is M or I; the second light chain CDR (CDRL2) comprises an amino acid sequence that is at least 80 percent identical to the amino acid sequence X1X2SNLX6S (SEQ ID NO: 26), where X1 is S or L, X2 is T or A, and X6 is A or E; and the third light chain CDR (CDRL3) comprises an amino acid sequence that is at least 80 percent identical to the amino acid sequence QX2X3X4X5X6PX8T (SEQ ID NO: 27), where X2 is Q or H, X3 is R or S, X4 is S or R, X5 is S or E, X6 is Y or L, and X6 is Y or L.
Also provided in certain aspects is a first anti-NKG2A agent that binds NKG2A under laboratory or physiological conditions, where the first agent competitively binds with a second anti-NKG2A agent, which the second agent comprises at least one immunoglobulin heavy chain variable domain and at least one immunoglobulin light chain variable domain, where a) each immunoglobulin heavy chain variable domain of the second agent comprises first, second, and third heavy chain complementarity determining regions (CDRs), where the first heavy chain CDR (CDRH1) comprises an amino acid sequence that is at least 80 percent identical to the amino acid sequence X1YX3MX5 (SEQ ID NO: 22), where X1 is S or D, X3 is W or Y, and X5 is N or Y; the second heavy chain CDR (CDRH2) comprises an amino acid sequence that is at least 80 percent identical to the amino acid sequence
X1IX3X4X5X6X7X8TX10YX12X13X14X15KX17 (SEQ ID NO: 23), where X1 is R or T, X3 is Y or S, X4 is F or Y, X5 is E or G, X6 is D or G, X7 is G or I, X8 is D or Y, X10 is N or Y, X12 is N or P, X13 is G or D, X14 is K or S, X15 is F or V, and X17 is G or D; and the third heavy chain CDR (CDRH3) comprises an amino acid sequence that is at least 80 percent identical to the amino acid sequence
X1X2X3X4X5X6X7X8X9YX11X12X13X14X15 (SEQ ID NO: 24), where X1 is Q or D, X2 is G or R, X3 is D or no amino acid, X4 is T or no amino acid, X5 is S or no amino acid, X6 is V or no amino acid, X7 is I or Y, X8 is W or V, X9 is D or N, X11 is D or Y, X12 is G or T, X13 is F or M, X14 is A or D, and
X15 is D or Y; and b) each immunoglobulin light chain variable domain of the anti-NKG2A agent comprises first, second, and third light chain CDRs, where the first light chain CDR (CDRL1) comprises an amino acid sequence that is at least 80 percent identical to the amino acid sequence
X1ASX4SVX7X8X9X10X11X12YX14H (SEQ ID NO: 25), where X1 is S or R, X4 is S or K, X7 is T or S, X8 is T or no amino acid, X9 is S or no amino acid, X10 is G or no amino acid, X11 is Y or no amino acid, X12 is S or no amino acid, and X14 is M or I; the second light chain CDR (CDRL2) comprises an amino acid sequence that is at least 80 percent identical to the amino acid sequence X^SNLX6S (SEQ ID NO: 26), where X1 is S or L, X2 is T or A, and X6 is A or E; and the third light chain CDR (CDRL3) comprises an amino acid sequence that is at least 80 percent identical to the amino acid sequence QX2X3X4XsX6PX8T (SEQ ID NO: 27), where X2 is Q or H, X3 is R or S, X4 is S or R, X5 is S or E, X6 is Y or L, and X8 is Y or L.
Also provided in certain aspects is a first anti-NKG2A agent that binds NKG2A under laboratory or physiological conditions, where the first agent binds to the same epitope as a second anti-NKG2A agent, which the second agent comprises at least one immunoglobulin heavy chain variable domain and at least one immunoglobulin light chain variable domain, where a) each immunoglobulin heavy chain variable domain of the second agent comprises first, second, and third heavy chain complementarity determining regions (CDRs), where the first heavy chain CDR (CDRH1) comprises an amino acid sequence that is ^t least 80 percent identical to the amino acid sequence X1YX3MX5 (SEQ ID NO: 22), where X1 is S or D, X3 is W or Y, and X5 is N or Y; the second heavy chain CDR (CDRH2) comprises an amino acid sequence that is at least 80 percent identical to the amino acid sequence X1IX3X4X5X6X7X8TX10YX12X13X14X15KX17 (SEQ ID NO: 23), where X1 is R or T, X3 is Y or S, X4 is F or Y, X5 is E or G, X6 is D or G, X7 is G or I, X8 is D or Y, X10 is N or Y, X12 is N or P, X13 is G or D, X14 is K or S, X15 is F or V, and X17 is G or D; and the third heavy chain CDR (CDRH3) comprises an amino acid sequence that is at least 80 percent identical to the amino acid sequence X1X2X3X4X5X6X7X8X9YX11X12X13X14X15 (SEQ ID NO: 24), where X1 is Q or D, X2 is G or R, X3 is D or no amino acid, X4 is T or no amino acid, X5 is S or no amino acid, X6 is V or no amino acid, X7 is I or Y, X8 is W or V, X9 is D or N, X11 is D or Y, X12 is G or T, X13 is F or M, X14 is A or D, and X15 is D or Y; and b) each immunoglobulin light chain variable domain of the anti-NKG2A agent comprises first, second, and third light chain CDRs, where the first light chain CDR (CDRL1) comprises an amino acid sequence that is at least 80 percent identical to the amino acid sequence X1ASX4SVX7X8X9X10X11X12YX14H (SEQ ID NO: 25), where X1 is S or R, X4 is S or K, X7 is T or S, X8 is T or no amino acid, X9 is S or no amino acid, X10 is G or no amino acid, X11 is Y or no amino acid, X12 is S or no amino acid, and X14 is M or I; the second light chain CDR (CDRL2) comprises an amino acid sequence that is at least 80 percent identical to the amino acid sequence X1X2SNLX6S (SEQ ID NO: 26), where X1 is S or L, X2 is T or A, and X6 is A or E; and the third light chain CDR (CDRL3) comprises an amino acid sequence that is at least 80 percent identical to the amino acid sequence QX2X3X4X5X6PX8T (SEQ ID NO: 27), where X2 is Q or H, X3 is R or S, X4 is S or R, X5 is S or E, X6 is Y or L, and X8 is Y or L. Also provided in certain aspects are anti-NKG2A agents for detecting NKG2A in a heterogeneous population of immune cells, where NKG2A is detected at a significant level in NK cells and NK T- lymphocytes in the population, and NKG2A is not significantly detected in other immune cells in the population. Also provided in certain aspects are methods of detecting NKG2A in a heterogeneous population of immune cells, comprising contacting the population with an anti-NKG2A agent provided herein, wherein NKG2A is not significantly detected in other immune cells in the population. In one aspect, provided herein are isolated, non-naturally occurring anti-NKG2A agents, particularly antibodies, or antigen-binding fragments or derivatives thereof, that bind NKG2A under physiological conditions. In the context of anti-NKG2A antibodies or antigen-binding fragments, such molecules generally comprise two immunoglobulin heavy chain variable domains and two immunoglobulin light chain variable domains. In such molecules, each of the immunoglobulin heavy and light chain variable domains comprise first, second, and third chain complementarity determining regions (CDRs) arrayed as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
In the heavy chain variable domain portions, the first heavy chain CDR (CDRH1) comprises an amino acid sequence that has a sequence identity of at least 65 percent, optionally a sequence identity of at least 80 percent, at least 90 percent, at least 95 percent, and 100 percent identity with the amino acid sequence SYWMN and DYYMY (SEQ ID NOS: 9 and 16, respectively), the second heavy chain CDR (CDRH2) comprises an amino acid sequence that has a sequence identity of at least 65 percent, optionally a sequence identity of at least 80 percent, at least 90 percent, at least 95 percent, and 100 percent identity with the amino acid sequence RIYFEDGDTNYNGKFKG. RIYFEDGDTNYNGKFKD. or TISYGGIYTYYPDSVKG (SEQ ID NOS: 10, 15, 17, respectively), and the third heavy chain CDR (CDRH3) comprises an amino acid sequence that has a sequence identity of at least 65 percent, optionally a sequence identity of at least 80 percent, at least 90 percent, at least 95 percent, and 100 percent identity with the amino acid sequence QGIWDYDGFAD or DRDTSVYVNYYTMDY (SEQ ID NOS: 11 and 18, respectively).
In the immunoglobulin light chain variable domain portions, the first light chain CDR (CDRL1) comprises an amino acid sequence that has a sequence identity of at least 65 percent, optionally a sequence identity of at least 80 percent, at least 90 percent, at least 95 percent, and 100 percent identity with the amino acid sequence SASSSVTYMH or RASKS VSTSGYSYIH (SEQ ID NOS: 12 and 19, respectively), the second light chain CDR (CDRL2) comprises an amino acid sequence that has a sequence identity of at least 65 percent, optionally a sequence identity of at least 80 percent, at least 90 percent, at least 95 percent, and 100 percent identity with the amino acid sequence STSNLAS or LASNLES (SEQ ID NOS: 13 and 20, respectively), and the third light chain CDR (CDRL3) comprises an amino acid sequence that has a sequence identity of at least 65 percent, optionally a sequence identity of at least 80 percent, at least 90 percent, at least 95 percent, and 100 percent identity with the amino acid sequence QQRSSYPYT or QHSRELPLT (SEQ ID NOS: 14 and 21, respectively).
In some embodiments, the isolated, non-naturally occurring anti-NKG2A antibodies, or NKG2A- binding fragments thereof, including a first heavy chain CDR having the amino acid sequence SYWMN (SEQ ID NO: 9), the second heavy chain CDR has the amino acid sequence RIYFEDGDTNYNGKFKG (SEQ ID NO: 10), the third heavy chain CDR has the amino acid sequence QGIWDYDGFAD (SEQ ID NO: 11), the first light chain CDR has the amino acid sequence SASSSVTYMH (SEQ ID NO: 12), the second light chain CDR has the amino acid sequence STSNLAS (SEQ ID NO: 13), and the third light chain CDR has the amino acid sequence QQRSSYPYT (SEQ ID NO: 14). In some embodiments, the isolated anti-NKG2A agent comprises a non-naturally occurring anti- NKG2A antibody (mAb) comprising two immunoglobulin heavy chain variable domains comprising first, second, and third heavy chain complementarity determining regions (CDRH1-3, respectively) and two immunoglobulin light chain variable domains comprising first, second, and third light chain complementarity determining regions (CDRL1-3, respectively). In some embodiments, the antibody comprises immunoglobulin heavy chain variable domains and immunoglobulin light chain variable domains having sets of CDRH1-3 and CDRL1-3 selected from the sequences set forth in Table 1.
Figure imgf000008_0001
In some embodiments, the isolated anti-NKG2A agent comprises a non-naturally occurring anti- NKG2A antibody (mAb) comprising two immunoglobulin heavy chain variable domains and two immunoglobulin light chain variable domains, where the immunoglobulin heavy chain variable domains have an amino acid sequence selected from amongst SEQ ID NOS: 1, 3 and 4, or an amino acid sequence having at least 65%-95% or more sequence identity with any such heavy chain variable domain sequence and the immunoglobulin light chain variable domains are selected from amongst SEQ ID NOS: 5 or 8 or an amino acid sequence having at least 65%-95% or more sequence identity with any such light chain variable domain sequence.
In some embodiments, where the anti-NKG2A agents are antibodies, or antigen-binding fragments thereof, the antibodies (or fragments thereof) are monoclonal antibodies, and may be camel, human, humanized, mouse, rabbit, or other mammalian antibodies or antigen-binding antibody fragments. In some embodiments, the antibody or antigen-binding antibody fragment thereof is an IgG. In some embodiments, the IgG is an IgGl, IgG2a or IgG2b, or IgG3, or IgG4.
In certain embodiments of anti-NKG2A antibodies and antigen-binding antibody fragments thereof that are other than fully human antibodies (i.e., antibodies produced or derived from a mammal capable of producing all or a portion of the human antibody repertoire), the molecules are chimeric or humanized anti-NKG2A antibodies and antigen-binding antibody fragments.
In some embodiments, the anti-NKG2A antibody, antigen-binding antibody fragment, or derivative or variant thereof includes a detectable label.
In some embodiments, the anti-NKG2A agent, for example, an anti-NKG2A antibody, antigenbinding antibody fragment, or derivative or variant thereof, is part of an immunoconjugate that further includes a cytotoxic agent, for example, a nucleic acid, a peptide, a polypeptide, a small molecule, or an aptamer.
A related aspect of technology described herein concerns compositions that include an anti-NKG2A agent that is an isolated, non-naturally occurring anti-NKG2A antibody or antigen-binding antibody fragment according to the technology described herein. In addition to containing an anti-NKG2A antibody or an antigen-binding antibody fragment thereof described herein, such compositions typically also include a carrier, for example, a pharmaceutically acceptable carrier. Such compositions may be packaged in containers, which in some embodiments, are further packaged into kits that also include instructions for use. In the context of pharmaceutical compositions, such kits instructions are a package insert containing not only instructions or use but also information about the pharmaceutically active ingredient (e.g., the anti-NKG2A antibody, antigen-binding antibody fragment, or derivative or variant thereof).
Another related aspect concerns diagnostics configured to detect NKG2A in a biological sample, often a biological sample taken from a subject. Such kits include a diagnostic reagent that includes an anti-NKG2A agent described herein, for example, an anti-NKG2A antibody, antigen-binding antibody fragment, or derivative or variant thereof conjugated with detectable reagents such as fluorophores or enzyme substrates and/or immobilized on a solid support. Still other aspects of the technology provided herein concern the manufacture of an anti-NKG2A agent described herein. In the context of anti-NKG2A antibodies (or antigen-binding antibody fragments or derivatives or variants thereof), one such aspect concerns isolated nucleic acid molecules that encode polypeptides provided herein. In some embodiments, such nucleic acids encode an immunoglobulin heavy chain variable domain having a first heavy chain CDR (CDRH1) that includes an amino acid sequence that has a sequence identity of at least 65%, optionally a sequence identity of at least 80%, at least 90%, at least 95%, and 100% identity with the amino acid sequence SYWMN and DYYMY (SEQ ID NOS: 9 and 16, respectively), the second heavy chain CDR (CDRH2) comprises an amino acid sequence that has a sequence identity of at least 65 percent, optionally a sequence identity of at least 80 percent, at least 90 percent, at least 95 percent, and 100 percent identity with the amino acid sequence RIYFEDGDTNYNGKFKG. RIYFEDGDTNYNGKFKD, or TISYGGIYTYYPDSVKG (SEQ ID NOS: 10, 15, 17, respectively), and the third heavy chain CDR (CDRH3) comprises an amino acid sequence that has a sequence identity of at least 65 percent, optionally a sequence identity of at least 80 percent, at least 90 percent, at least 95 percent, and 100 percent identity with the amino acid sequence QGIWDYDGFAD or DRDTSVYVNYYTMDY (SEQ ID NOS: 11 and 18, respectively). Such nucleic acids may also encode an immunoglobulin light chain variable domain where a first light chain CDR (CDRL1) that includes an amino acid sequence that has a sequence identity of at least 65%, optionally a sequence identity of at least 80%, at least 90%, at least 95%, and 100% identity with the amino acid sequence SASSSVTYMH or RASKSVSTSGYSYIH (SEQ ID NOS: 12 and 19, respectively), the second light chain CDR (CDRL2) comprises an amino acid sequence that has a sequence identity of at least 65 percent, optionally a sequence identity of at least 80%, at least 90%, at least 95%, andl00% identity with the amino acid sequence STSNLAS or LASNLES (SEQ ID NOS: 13 and 20, respectively), and the third light chain CDR (CDRL3) comprises an amino acid sequence that has a sequence identity of at least 65%, optionally a sequence identity of at least 80%, at least 90%, at least 95%, and 100% identity with the amino acid sequence QQRSSYPYT or QHSRELPLT (SEQ ID NOS: 14 and 21, respectively).
In certain embodiments, nucleic acid molecules provided herein encode an immunoglobulin heavy chain variable domain having an amino acid sequence selected from among SEQ ID NOS: 1, 3 and 4 or an amino acid sequence having at least 65%-95% or more sequence identity with any such immunoglobulin heavy chain variable domain sequence and an immunoglobulin light chain variable domain having an amino acid sequence selected from among SEQ ID NOS: 5 and 8 or an amino acid sequence having at least 65%-95% or more sequence identity with any such immunoglobulin light chain variable domain sequence.
Related aspects concern plasmids, and expression cassettes and vectors, that carry nucleic acids provided herein, as well as recombinant host cells transfected with such nucleic acid molecules. Still other aspects of the technology provided herein concern methods of treating or preventing a disease or disorder associated with aberrant levels of NKG2A. Such methods include administering to a subject in need of such treatment an anti-NKG2A agent provided herein (e.g., an anti-NKG2A antibody or antigen-binding fragment, derivative, or variant thereof) in an amount sufficient to effect treatment, thereby treating or preventing the disease or disorder. In certain instances, an anti-NKG2A agent provided herein (e.g., an anti-NKG2A antibody or antigen-binding fragment, derivative, or variant thereof) may be used as an adjuvant or in conjunction with an adjuvant (e.g., for vaccines).
Further aspects of the technology provided herein concern diagnostic methods of using an anti- NKG2A agent provided herein, for example, in vitro or in vivo diagnostic assays to detect the presence ofNKG2A.
Certain embodiments are described further in the following description, examples, claims and drawings.
Brief Description of the Drawings
The drawings illustrate certain embodiments of the technology and are not limiting. For clarity and ease of illustration, the drawings are not made to scale and, in some instances, various aspects may be shown exaggerated or enlarged to facilitate an understanding of particular embodiments.
Fig. 1 shows the amino acid sequence of a representative full-length human NKG2A protein (SEQ ID NO: 28). The amino acid residues 1-70 comprise the cytoplasmic domain, amino acid residues 71-93 comprise the transmembrane domain, and amino acid residues 94-233 comprise the extracellular domain. See, e.g., NCBI Reference Sequence: NP_002250.2.
Fig. 2 shows the amino acid sequences of the variable domains of the immunoglobulin heavy (SEQ ID NOS: 1, 3 and 4) and light (SEQ ID NOS: 5 and 8) chains of the different anti-NKG2A antibodies (AB 1-4) provided herein. The CDR regions of each of the heavy and light chains are shown in bold and underlined. In each of the alignments, three characters (“*”, and “.”) are used: “*” indicates positions that have a single, fully conserved residue; indicates that one of the following “strong” residue groups is fully conserved: STA; NEQK; NHQK; NDEQ; QHRK; MILV; MILF; HY; and FYW; and indicates that one of the following “weaker” residue groups is fully conserved: CSA; ATV; SAG; STNK; STPA; SGND; SNDEQK; NDEQHK; NEQHRK; FVLIM; and HFY. These are all the positively scoring residue groups that occur in the Gonnet Pam250 matrix. The “strong” and “weak” residue groups are defined as “strong” score > 0.5 and “weak” score < 0.5, respectively.
Fig. 3 lists the source, isotype and format for anti-NKG2A agents described herein.
Figs. 4A-4D, depict the gating strategy used for the detection of cells expressing NKG2A, as assessed by flow cytometry. Fig. 4A shows the identification of singlets based on side scatter. Fig. 4B shows the forward and side scatter of singlets. Fig. 4C shows lymphocytes co-stained with CD3 and CD19.
Fig. 4D shows CD3+CD19- lymphocytes co-stained with CD4 and CD8.
Figs. 5A and 5B depict results for surface expression of NKG2A on lymphocytes co-stained with CD56 and AB2. Fig. 5A depicts a dot plot showing lymphocytes co-stained with AB2 and CD56. Fig. 5B shows a histogram of lymphocytes stained with AB2.
Figs. 6A-6C, depict dot plots of lymphocytes co-stained with CD56 and AB2 (Fig. 6A), Commercial ABI (Fig. 6B), and Commercial ABII (Fig. 6C).
Figs. 7A-7I, depict the staining of subsets of CD3+CD19- lymphocytes including CD4+CD8-, CD4- CD8-, and CD4-CD8+ with AB2 (Figs.7A-7C), Commercial ABI (Figs.7D-7F), and Commercial ABII (Figs.7G-7I), respectively.
Figs. 8A-8H, depict dot plots of lymphocytes co-stained with CD94 and ABI (Fig. 8A), CD94 and AB2 (Fig. 8B), CD94 and AB3 (Fig. 8C), and CD94 and AB4 (Fig. 8D), and CD3-CD19- lymphocytes co-stained with CD56 and ABI (Fig. 8E), CD56 and AB2 (Fig. 8F), CD56 and AB3 (Fig. 8G), and CD56 and AB4 (Fig. 8H).
Figs. 9A-9D, depict histograms of mouse splenocytes stained with ABI (Fig. 9A), AB2 (Fig. 9B), AB3 (Fig. 9C), and AB4 (Fig. 9D).
Figs. 10A and 10B depict results of the anti-NKG2A antibodies described herein in a cell-mediated cytotoxicity assay. Fig. 10A depicts the percent of DAPI positive cells in a population of purified primary NK and K562 cells at different effector to target ratios (E:T), with no effector used as control. Fig . 1 OB depicts the percent of DAPI positive cells after treatment with AB 1 -AB4 antibodies or noantibody at lOpg/well at an E:T of 2: 1, or T only as control.
Detailed Description
Provided herein are agents that bind NKG2A. In some aspects, the agents that bind NKG2A are antibodies, fragments thereof or antigen binding fragments thereof, that bind to NKG2A. In some embodiments, particular monoclonal antibodies to NKG2A that provide superior target specificity, signal-to-noise ratios, and the like, as compared to other reported anti-NKG2A antibodies, as well as antigen-binding fragments thereof that bind NKG2A, are provided herein. Also provided herein are methods for producing anti-NKG2A agents, particularly anti-NKG2A antibodies, with desirable properties including affinity and/or specificity for NKG2A and/or its variants.
NKG2 (Killer Cell Lectin-Like Receptor), also known as Cluster of Differentiation 159 (CD 159), is a family of type II transmembrane protein receptors. The human NKG2 family comprises seven members referred to as NKG2A, NKG2B, NKG2C, NKG2D, NKG2E, NKG2F, and NKG2H. With the exception ofNKG2D, all other NKG2 members share substantial sequence homology. NKG2A, NKG2B, NKG2C, NKG2E and NKG2H have been shown to form disulfide-linked heterodimers with the invariant Cluster of Differentiation 94 (CD94) protein, to form functionally distinct heterodimeric receptors. The CD94 associated NKG2 family members (CD94/NKG2) are comprised of C-type lectin receptors that are predominantly expressed on the surface of NK cells, as well as a subset of CD8+ T-lymphocytes. Although the human CD94/NKG2 family has been found to recognize and interact with the same ligand, Human Leukocyte Antigen (HLA)-E, on target cells, different receptor family members can transmit opposing signals via their intracellular domains. While the NKG2A and NKG2B receptors contain immunoreceptor tyrosine-based inhibition motifs (ITAM) and transmit inhibitory signals, in contrast, the NKG2C, NKG2D, NKG2E, and NKG2H receptors are characterized by the presence of a charged amino acid residue in the transmembrane domain mediating interaction with DAP-12, an adapter molecule containing an immunoreceptor tyrosine-based activation motif (ITAM), that transmit activating signals. The murine CD94-NKG2 receptors recognize the class Ib molecule Qa-1b. The CD94/NK group 2-member A (NKG2A) heterodimeric receptor is one of the most prominent NK inhibitory receptors (Lazetic et al J Immunol.1996;157(11):4741-4757), and is also expressed on a subset of T cells (α/β and γ/δ). It binds to the nonclassical minimally polymorphic HLA class I molecule (HLA-E), which presents peptides derived from leader peptide sequences of other HLA class I molecules, such as HLA-G (Braud et al. Nature 1998;391(6669):795-799; Lee et al. Proc Natl Acad SciUSA 1998;95(9):5199-5204; Miller et al. J Immunol.2003;171(3):1369-1375). Upon ligation by peptide-loaded HLA-E, NKG2A transduces inhibitory signaling through 2 inhibitory immune-receptor tyrosine-based inhibition motifs, thus suppressing NK cytokine secretion and cytotoxicity. Although HLA-E transcripts are ubiquitously expressed, they have also been associated with overexpression in several tumors (Kayima et al. The Journal of Clinical Investigation.2019 10.1172/JCI123955). Anti-NKG2A Agents Anti-NKG2A agents (e.g., anti-NKG2A antibodies) provided herein may have a strong binding affinity and/or specificity for NKG2A. In some embodiments, the anti-NKG2A agents provided herein bind all or a portion of the NKG2A receptor, or a fragment thereof. In some embodiments, the antibody or antigen-binding fragment thereof provided herein bind the NKG2A receptor or a fragment thereof. In some embodiments, the anti-NKG2A agents provided herein bind a portion of the NKG2A receptor that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:28. In some embodiments, the antibody or antigen-binding fragment thereof provided herein bind the NKG2A receptor or a fragment thereof that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:28. In some embodiments, the anti-NKG2A agents provided herein bind all or a portion of the NKG2A receptor described in SEQ ID NO:28. In some embodiments, the antibody or antigen-binding fragment thereof provided herein bind the NKG2A receptor or a fragment thereof comprising the sequence of amino acids described in SEQ ID NO:28.
Precursor human NKG2A protein sequence (UniProtKB - P26715 (NKG2A HUMAN)/ NCBI Reference Sequence: NP 002250,2) (SEQ ID NO: 28)
MDNQGVIYSD LNLPPNPKRQ QRKPKGNKNS ILATEQEITYAELNLQKASQDFQG NDKTYHCKDLPSAPEK LIVGILGIIC LILMASVVTI VVIPSTLIQR HNNSSLNTRT QKARHCGHCPEEWITYSNSC YYIGKERRTW EESLLACTSK NSSLLSIDNE EEMKFLSIIS PSSWIGVFRNSSHHPWVTMN GLAFKHEIKD SDNAELNCAV LQVNRLKSAQ CGSSIIYHCK HKL
In some embodiments, the human NKG2A protein sequence comprises a cytoplasmic domain, a transmembrane domain, and an extracellular domain.
In some embodiments, the human NKG2A protein sequence comprises a cytoplasmic domain corresponding to amino acid residues 1-70 of SEQ ID NO:28. In some embodiments, the cytoplasmic domain corresponds to the sequence of amino acids of SEQ ID NO:2. In some embodiments, the human NKG2A protein sequence comprises a transmembrane domain corresponding to amino acid residues 71-93 of SEQ ID NO:28. In some embodiments, the transmembrane domain corresponds to the sequence of amino acids of SEQ ID NO:6. In some embodiments, the human NKG2A protein sequence comprises an extracellular domain corresponding to amino acid residues 94-233 of SEQ ID NO:28. In some embodiments, the extracellular domain corresponds to the sequence of amino acids of SEQ ID NO:7.
In some embodiments, the anti-NKG2A agents provided herein bind all or a portion of the NKG2A receptor extracellular domain. In some embodiments, the anti-NKG2A agents provided herein bind all or a portion of the NKG2A receptor extracellular domain corresponding to amino acid residues 94- 233 of SEQ ID NO:28. In some embodiments, the anti-NKG2A agents provided herein bind all or a portion of the NKG2A receptor extracellular domain described in SEQ ID NO: 7.
In some embodiments, the anti-NKG2A agents provided herein bind all or a portion of the NKG2A receptor extracellular domain that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid residues 94-233 of SEQ ID NO:28. In some embodiments, the anti- NKG2A agents provided herein bind all or a portion of the NKG2A receptor extracellular domain that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:7. In some embodiments, the antibody or antigen-binding fragment thereof provided herein bind the NKG2A receptor extracellular domain described in SEQ ID NO:7.
In some embodiments, anti-NKG2A agents may be chimeric antibodies. In some embodiments, anti- NKG2A agents may be humanized antibodies. In some embodiments, anti-NKG2A agents may be variant antibodies. Antibodies, for example, may have beneficial properties from a therapeutic perspective. Assays for determining the activity of anti-NKG2A antibodies provided herein include, for example, cell-based ELISA (e.g., to measure relative avidity of the antibody for the target on cells), flow cytometry (e.g., to measure cell specificity of the antibody), and cytotoxicity (e.g., to measure potential to mediate direct or indirect killing of NKG2A-expressing target cells). In certain instances, a humanized or variant antibody fails to elicit an immunogenic response upon administration of a therapeutically effective amount of the antibody to a human patient. In certain instances, if an immunogenic response is elicited, the response may be such that the antibody still provides a therapeutic benefit to the patient treated therewith.
In some embodiments, the anti-NKG2A agents (e.g., anti-NKG2A antibodies, humanized anti- NKG2A antibodies) provided herein bind the same epitope. To screen for antibodies that bind to an epitope on NKG2A bound by an antibody of interest (e.g., those that block binding of the antibody to NKG2A), a cross-blocking assay such as that described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988), can be performed. In certain instances, epitope mapping, e.g., as described in Champe et al., J. Biol. Chem. 270: 1388-1394 (1995), in Cunningham and Wells, Science 244: 1081 - 1085 (1989), or in Davidson and Doranz, Immunology 143: 13 - 20 (2014), can be performed to determine whether the antibody binds an epitope of interest.
In some embodiments, the anti-NKG2A agents provided herein may include anti-NKG2A antibodies, anti-NKG2A antibody fragments (e.g., anti-NKG2A antigen binding fragments), and anti-NKG2A antibody derivatives. In some embodiments, the agent is isolated (e.g., separated from a component of its natural environment (e.g. an animal, a biological sample)). In some embodiments, the agent is non-naturally occurring (e.g., produced by human intervention). In some embodiments, the agent is a humanized antibody, or an antigen binding fragment thereof. In some embodiments, the agent is a derivative of a humanized antibody that binds NKG2A.
In some embodiments, the agent binds NKG2A under laboratory conditions (e.g., binds NKG2A in vitro, binds NKG2A in a flow cytometry assay, binds NKG2A in an ELISA). In some embodiments, the agent binds NKG2A under physiological conditions (e.g., binds NKG2A in a cell in a subject).
Antibodies such as those described herein, may have an immunoglobulin heavy chain variable domain comprising an amino acid sequence represented by the formula: ERH1-CDRH1-ERH2-CDRH2- ERH3-CDRH3-ERH4, where “ERH1-4” represent the four heavy chain framework regions and “CDRH1-3” represent the three hypervariable regions of an anti-NKG2A antibody variable heavy domain. FRH1-4 may be derived from a consensus sequence (for example the most common amino acids of a class, subclass or subgroup of heavy or light chains of human immunoglobulins) or may be derived from an individual human antibody framework region or from a combination of different framework region sequences. Many human antibody framework region sequences are compiled in Kabat et al. (1992) SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, National Institutes of Health Publication No. 91-3242, for example. In one embodiment, the variable heavy FR is provided by a consensus sequence of a human immunoglobulin subgroup as compiled by Kabat et al., supra.
The human variable heavy FR sequence may have substitutions therein, e.g. where the human FR residue is replaced by a corresponding nonhuman residue (by “corresponding nonhuman residue” is meant the nonhuman residue with the same Kabat positional numbering as the human residue of interest when the human and nonhuman sequences are aligned), but replacement with the nonhuman residue is not necessary. For example, a replacement FR residue other than the corresponding nonhuman residue may be selected by phage display.
Antibodies such as those described herein, may have an immunoglobulin light chain variable domain comprising an amino acid sequence represented by the formula: FRL1-CDRL1-FRL2-CDRL2-FRL3- CDRL3-FRL4, where “FRL1-4” represents the four framework regions and “CDRL1-3” represents the three hypervariable regions of an anti-NKG2A antibody variable light domain. FRL1-4 may be derived from a consensus sequence (for example the most common amino acids of a class, subclass or subgroup of heavy or light chains of human immunoglobulins) or may be derived from an individual human antibody framework region or from a combination of different framework region sequences. In some embodiments, the immunoglobulin variable light FR is provided by a consensus sequence of a human immunoglobulin subgroup as compiled by Kabat et al., supra.
The human immunoglobulin variable light FR sequence may have substitutions therein, e.g. where the human FR residue is replaced by a corresponding mouse residue, but replacement with the nonhuman residue is not necessary. For example, a replacement residue other than the corresponding nonhuman residue may be selected by phage display. Methods for generating humanized anti-NKG2A antibodies of interest herein are elaborated in more detail below.
Generally, the anti-NKG2A agent provided herein comprises at least one immunoglobulin heavy chain variable domain and at least one immunoglobulin light chain variable domain. In some embodiments, an anti-NKG2A agent herein comprises two immunoglobulin heavy chain variable domains and two immunoglobulin light chain variable domains. Typically, each immunoglobulin heavy chain variable domain of the anti-NKG2A agent comprises first, second, and third heavy chain complementarity determining regions (CDRs; CDRH1, CDRH2, CDRH3), and each immunoglobulin light chain variable domain of the anti-NKG2A agent comprises first, second, and third light chain CDRs (CDRL1, CDRL2, CDRL3).
CDRH1
In some embodiments, the first heavy chain CDR (CDRH1) of an anti-NKG2A agent provided herein comprises an amino acid sequence that is at least 80% identical to the amino acid sequence
X1YX3MX5 (SEQ ID NO: 22), where X1 is S or D; X3 is W or Y; and X5 is N or Y. In some embodiments, the CDRH1 comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 22. In some embodiments, the CDRH1 comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 22. In some embodiments, the CDRH1 comprises an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO: 22. In some embodiments, the CDRH1 comprises the sequence of amino acids set forth in SEQ ID NO: 22.
The amino acid X1 of SEQ ID NO: 22 may be substituted with any amino acid. In some embodiments, the amino acid X1 of SEQ ID NO: 22 is substituted with a conservative amino acid (e.g., conservative to S and/or D). In some embodiments, the amino acid X1 of SEQ ID NO: 22 is substituted with a hydrophilic amino acid. In some embodiments, the amino acid X1 of SEQ ID NO: 22 is substituted with an acidic amino acid.
The amino acid X3 of SEQ ID NO: 22 may be substituted with any amino acid. In some embodiments, the amino acid X3 of SEQ ID NO: 22 is substituted with a conservative amino acid (e.g., conservative to W and/or Y). In some embodiments, the amino acid X3 of SEQ ID NO: 22 is substituted with an aromatic amino acid.
The amino acid X5 of SEQ ID NO: 22 may be substituted with any amino acid. In some embodiments, the amino acid X5 of SEQ ID NO: 22 is substituted with a conservative amino acid (e.g., conservative to N and/or Y). In some embodiments, the amino acid X5 of SEQ ID NO: 22 is substituted with a basic amino acid. In some embodiments, the amino acid X5 of SEQ ID NO: 22 is substituted with an aromatic amino acid.
In some embodiments, the CDRH1 of any of the anti-NKG2A agents provided herein comprises an amino acid sequence chosen from SYWMN (SEQ ID NO: 9) and DYYMY (SEQ ID NO: 16). In some embodiments, the CDRH1 of any of the anti-NKG2A agents provided herein comprises the sequence of amino acids SYWMN (SEQ ID NO: 9); or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:9. In some embodiments, the CDRH1 of any of the anti-NKG2A agents provided herein comprises the sequence of amino acids SYWMN (SEQ ID NO: 9).
In some embodiments, the CDRH1 of any of the anti-NKG2A agents provided herein comprises the sequence of amino acids DYYMY (SEQ ID NO: 16); or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:9 or SEQ ID NO: 16. In some embodiments, the CDRH1 of any of the anti-NKG2A agents provided herein comprises the sequence of amino acids DYYMY (SEQ ID NO: 16).
CDRH2
In some embodiments, the second heavy chain CDR (CDRH2) of an anti-NKG2A agent provided herein comprises an amino acid sequence that is at least 80 percent identical to the amino acid sequence X1D^XsX6XvX6TX10YX11XBX11X1sKX11 (SEQ ID NO: 23), where X1 is R or T; X3 is Y or S; X4 is F or Y; X5 is E or G; X6 is D or G; X7 is G or I; X6 is D or Y; X10 is N or Y; X12 is N or P;
X13 is G or D; X14 is K or S; X15 is F or V; and X17 is G or D. In some embodiments, the CDRH2 comprises an amino acid sequence that is at least 90 percent identical to the amino acid sequence of SEQ ID NO: 23. In some embodiments, the CDRH2 comprises an amino acid sequence that is at least 95 percent identical to the amino acid sequence of SEQ ID NO: 23. In some embodiments, the CDRH2 comprises an amino acid sequence that is 100 percent identical to the amino acid sequence of SEQ ID NO: 23. In some embodiments, the CDRH2 comprises the sequence of amino acids set forth in SEQ ID NO: 23.
The amino acid X1 of SEQ ID NO: 23 may be substituted with any amino acid. In some embodiments, the amino acid X1 of SEQ ID NO: 23 is substituted with a conservative amino acid (e.g., conservative to R and/or T). In some embodiments, the amino acid X1 of SEQ ID NO: 23 is substituted with a basic amino acid. In some embodiments, the amino acid X1 of SEQ ID NO: 23 is substituted with a neutral amino acid.
The amino acid X3 of SEQ ID NO: 23 may be substituted with any amino acid. In some embodiments, the amino acid X3 of SEQ ID NO: 23 is substituted with a conservative amino acid (e.g., conservative to Y and/or S). In some embodiments, the amino acid X3 of SEQ ID NO: 23 is substituted with an aromatic amino acid. In some embodiments, the amino acid X3 of SEQ ID NO: 23 is substituted with a neutral amino acid.
The amino acid X4 of SEQ ID NO: 23 may be substituted with any amino acid. In some embodiments, the amino acid X4 of SEQ ID NO: 23 is substituted with a conservative amino acid (e.g., conservative to F and/or Y). In some embodiments, the amino acid X4 of SEQ ID NO: 23 is substituted with an aromatic amino acid. The amino acid X5 of SEQ ID NO: 23 may be substituted with any amino acid. In some embodiments, the amino acid X5 of SEQ ID NO: 23 is substituted with a conservative amino acid (e.g., conservative to E and/or G). In some embodiments, the amino acid X5 of SEQ ID NO: 23 is substituted with an acidic amino acid. In some embodiments, the amino acid X5 of SEQ ID NO: 23 is substituted with an amino acid residue that influences chain orientation. The amino acid X6 of SEQ ID NO: 23 may be substituted with any amino acid. In some embodiments, the amino acid X6 of SEQ ID NO: 23 is substituted with a conservative amino acid (e.g., conservative to D and/or G). In some embodiments, the amino acid of SEQ ID NO: 23 is substituted with an acidic amino acid. In some embodiments, the amino acid X6 of SEQ ID NO: 23 is substituted with an amino acid residue that influences chain orientation. The amino acid X7 of SEQ ID NO: 23 may be substituted with any amino acid. In some embodiments, the amino acid X7 of SEQ ID NO: 23 is substituted with a conservative amino acid (e.g., conservative to G and/or I). In some embodiments, the amino acid X7 of SEQ ID NO: 23 is substituted with an amino acid residue that influences chain orientation. In some embodiments, the amino acid X7 of SEQ ID NO: 23 is substituted with a hydrophobic amino acid. The amino acid X8 of SEQ ID NO: 23 may be substituted with any amino acid. In some embodiments, the amino acid X8 of SEQ ID NO: 23 is substituted with a conservative amino acid (e.g., conservative to D and/or Y). In some embodiments, the amino acid X8 of SEQ ID NO: 23 is substituted with an acidic amino acid. In some embodiments, the amino acid X8 of SEQ ID NO: 23 is substituted with an aromatic amino acid. The amino acid X10 of SEQ ID NO: 23 may be substituted with any amino acid. In some embodiments, the amino acid X10 of SEQ ID NO: 23 is substituted with a conservative amino acid (e.g., conservative to N and/or Y). In some embodiments, the amino acid X10 of SEQ ID NO: 23 is substituted with a basic amino acid. In some embodiments, the amino acid X10 of SEQ ID NO: 23 is substituted with an aromatic amino acid. The amino acid X12 of SEQ ID NO: 23 may be substituted with any amino acid. In some embodiments, the amino acid X12 of SEQ ID NO: 23 is substituted with a conservative amino acid (e.g., conservative to N and/or P). In some embodiments, the amino acid of SEQ ID NO: 23 is substituted with a basic amino acid. In some embodiments, the amino acid is substituted with an amino acid residue that influences chain orientation. The amino acid X13 of SEQ ID NO: 23 may be substituted with any amino acid. In some embodiments, the amino acid X13 of SEQ ID NO: 23 is substituted with a conservative amino acid (e.g., conservative to G and/or D). In some embodiments, the amino acid X13 of SEQ ID NO: 23 is substituted with an amino acid residue that influences chain orientation. In some embodiments, the amino acid X13 of SEQ ID NO: 23 is substituted with an acidic amino acid.
The amino acid X14 of SEQ ID NO: 23 may be substituted with any amino acid. In some embodiments, the amino acid X14 of SEQ ID NO: 23 is substituted with a conservative amino acid (e.g., conservative to K and/or S). In some embodiments, the amino acid X14 of SEQ ID NO: 23 is substituted with a basic amino acid. In some embodiments, the amino acid X14 of SEQ ID NO: 23 is substituted with a neutral amino acid.
The amino acid X15 of SEQ ID NO: 23 may be substituted with any amino acid. In some embodiments, the amino acid X15 of SEQ ID NO: 23 is substituted with a conservative amino acid (e.g., conservative to F and/or V). In some embodiments, the amino acid X15 of SEQ ID NO: 23 is substituted with an aromatic amino acid. In some embodiments, the amino acid X15 of SEQ ID NO: 23 is substituted with a hydrophobic amino acid.
The amino acid X17 of SEQ ID NO: 23 may be substituted with any amino acid. In some embodiments, the amino acid X17 of SEQ ID NO: 23 is substituted with a conservative amino acid (e.g., conservative to G and/or D). In some embodiments, the amino acid X17 of SEQ ID NO: 23 is substituted with an amino acid residue that influences chain orientation. In some embodiments, the amino acid X17 of SEQ ID NO: 23 is substituted with an acidic amino acid.
In some embodiments, the CDRH2 of any of the anti-NKG2A agents provided herein comprises an amino acid sequence chosen from IUYFEDGDTNYNGKFKG (SEQ ID NO: 10), RIYFEDGDTNYNGKFKD (SEQ ID NO: 15), and TISYGGIYTYYPDSVKG (SEQ ID NO: 17).
In some embodiments, the CDRH2 of any of the anti-NKG2A agents provided herein comprises the sequence of amino acids IUYFEDGDTNYNGKFKG (SEQ ID NO: 10); or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 10. In some embodiments, the CDRH2 of any of the anti-NKG2A agents provided herein comprises the sequence of amino acids IUYFEDGDTNYNGKFKG (SEQ ID NO: 10).
In some embodiments, the CDRH2 of any of the anti-NKG2A agents provided herein comprises the sequence of amino acids IUYFEDGDTNYNGKFKD (SEQ ID NO: 15); or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 15. In some embodiments, the CDRH2 of any of the anti-NKG2A agents provided herein comprises the sequence of amino acids RIYFEDGDTNYNGKFKD (SEQ ID NO: 15). In some embodiments, the CDRH2 of any of the anti-NKG2A agents provided herein comprises the sequence of amino acids TISYGGIYTYYPDSVKG (SEQ ID NO: 17); or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 17. In some embodiments, the CDRH2 of any of the anti-NKG2A agents provided herein comprises the sequence of amino acids TISYGGIYTYYPDSVKG (SEQ ID NO: 17). CDRH3 In some embodiments, the third heavy chain CDR (CDRH3) of an anti-NKG2A agent provided herein comprises an amino acid sequence that is at least 80 percent identical to the amino acid sequence X1X2X3X4X5X6X7X8X9YX11X12X13X14X15 (SEQ ID NO: 24), where X1 is Q or D; X2 is G or R; X3 is D or no amino acid; X4 is T or no amino acid; X5 is S or no amino acid; X6 is V or no amino acid; X7 is I or Y; X8 is W or V; X9 is D or N; X11 is D or Y; X12 is G or T; X13 is F or M; X14 is A or D; and X15 is D or Y. In some embodiments, the third heavy chain CDR (CDRH3) of an anti-NKG2A agent provided herein comprises an amino acid sequence that is at least 90 percent identical to the amino acid sequence of SEQ ID NO: 24. In some embodiments, the CDRH3 comprises an amino acid sequence that is at least 95 percent identical to the amino acid sequence of SEQ ID NO: 24. In some embodiments, the CDRH3 comprises an amino acid sequence of SEQ ID NO: 24. In some embodiments, the CDRH3 comprises an amino acid sequence that is 100 percent identical to the amino acid sequence of SEQ ID NO: 24. In some embodiments, the CDRH3 comprises the sequence of amino acids set forth in SEQ ID NO: 24. The amino acid X1 of SEQ ID NO: 24 may be substituted with any amino acid. In some embodiments, the amino acid X1 of SEQ ID NO: 24 is substituted with a conservative amino acid (e.g., conservative to Q and/or D). In some embodiments, the amino acid X1 of SEQ ID NO: 24 is substituted with a basic amino acid. In some embodiments, the amino acid X1 of SEQ ID NO: 24 is substituted with an acidic amino acid. The amino acid X2 of SEQ ID NO: 24 may be substituted with any amino acid. In some embodiments, the amino acid X2 of SEQ ID NO: 24 is substituted with a conservative amino acid (e.g., conservative to G and/or R). In some embodiments, the amino acid X2 of SEQ ID NO: 24 is substituted with an amino acid residue that influences chain orientation. In some embodiments, the amino acid X2 of SEQ ID NO: 24 is substituted with a basic amino acid. The amino acid X3 of SEQ ID NO: 24 may be substituted with any amino acid. In some embodiments, the amino acid X3 of SEQ ID NO: 24 is substituted with a conservative amino acid (e.g., conservative to D and/or no amino acid). In some embodiments, the amino acid X3 of SEQ ID NO: 24 is substituted with an acidic amino acid. In some embodiments, X3 of SEQ ID NO: 24 is no amino acid.
The amino acid X4 of SEQ ID NO: 24 may be substituted with any amino acid. In some embodiments, the amino acid X4 of SEQ ID NO: 24 is substituted with a conservative amino acid (e.g., conservative to T and/or no amino acid). In some embodiments, the amino acid X4 of SEQ ID NO: 24 is substituted with a neutral amino acid. In some embodiments, the amino acid X4 of SEQ ID NO: 24 is no amino acid.
The amino acid X5 of SEQ ID NO: 24 may be substituted with any amino acid. In some embodiments, the amino acid X5 of SEQ ID NO: 24 is substituted with a conservative amino acid (e.g., conservative to S and/or no amino acid). In some embodiments, the amino acid X5 of SEQ ID NO: 24 is substituted with a neutral amino acid. In some embodiments, the amino acid X5 of SEQ ID NO: 24 is no amino acid.
The amino acid X6 of SEQ ID NO: 24 may be substituted with any amino acid. In some embodiments, the amino acid X6 of SEQ ID NO: 24 is substituted with a conservative amino acid (e.g., conservative to V and/or no amino acid). In some embodiments, the amino acid X6 of SEQ ID NO: 24 is substituted with a hydrophobic amino acid. In some embodiments, the amino acid X6 of SEQ ID NO: 24 is no amino acid.
The amino acid X7 of SEQ ID NO: 24 may be substituted with any amino acid. In some embodiments, the amino acid X7 of SEQ ID NO: 24 is substituted with a conservative amino acid (e.g., conservative to I and/or Y). In some embodiments, the amino acid X7 of SEQ ID NO: 24 is substituted with a hydrophobic amino acid. In some embodiments, the amino acid X7 of SEQ ID NO: 24 is substituted with an aromatic amino acid.
The amino acid X6 of SEQ ID NO: 24 may be substituted with any amino acid. In some embodiments, the amino acid X6 of SEQ ID NO: 24 is substituted with a conservative amino acid (e.g., conservative to W and/or V). In some embodiments, the amino acid X6 of SEQ ID NO: 24 is substituted with an aromatic amino acid. In some embodiments, the amino acid X6 of SEQ ID NO: 24 is substituted with a hydrophobic amino acid.
The amino acid X9 of SEQ ID NO: 24 may be substituted with any amino acid. In some embodiments, the amino acid X9 of SEQ ID NO: 24 is substituted with a conservative amino acid (e.g., conservative to D and/or N). In some embodiments, the amino acid X9 of SEQ ID NO: 24 is substituted with an acidic amino acid. In some embodiments, the amino acid X9 of SEQ ID NO: 24 is substituted with a basic amino acid. The amino acid X11 of SEQ ID NO: 24 may be substituted with any amino acid. In some embodiments, the amino acid X11 of SEQ ID NO: 24 is substituted with a conservative amino acid (e.g., conservative to D and/or Y). In some embodiments, the amino acid X11 of SEQ ID NO: 24 is substituted with an acidic amino acid. In some embodiments, the amino acid X11 of SEQ ID NO: 24 is substituted with an aromatic amino acid.
The amino acid X11 of SEQ ID NO: 24 may be substituted with any amino acid. In some embodiments, the amino acid XB of SEQ ID NO: 24 is substituted with a conservative amino acid (e.g., conservative to G and/or T). In some embodiments, the amino acid X of SEQ ID NO: 24 is substituted with an amino acid residue that influences chain orientation. In some embodiments, the amino acid XB of SEQ ID NO: 24 is substituted with a neutral amino acid.
The amino acid XB of SEQ ID NO: 24 may be substituted with any amino acid. In some embodiments, the amino acid XB of SEQ ID NO: 24 is substituted with a conservative amino acid (e.g., conservative to F and/or M). In some embodiments, the amino acid X of SEQ ID NO: 24 is substituted with an aromatic amino acid. In some embodiments, the amino acid XB of SEQ ID NO: 24 is substituted with a hydrophobic amino acid.
The amino acid X14 of SEQ ID NO: 24 may be substituted with any amino acid. In some embodiments, the amino acid X11 of SEQ ID NO: 24 is substituted with a conservative amino acid (e.g., conservative to A and/or D). In some embodiments, the amino acid X11 of SEQ ID NO: 24 is substituted with a hydrophobic amino acid. In some embodiments, the amino acid X11 of SEQ ID NO: 24 is substituted with an acidic amino acid.
The amino acid XB of SEQ ID NO: 24 may be substituted with any amino acid. In some embodiments, the amino acid X of SEQ ID NO: 24 is substituted with a conservative amino acid (e.g., conservative to D and/or Y). In some embodiments, the amino acid XB of SEQ ID NO: 24 is substituted with an acidic amino acid. In some embodiments, the amino acid XB of SEQ ID NO: 24 is substituted with an aromatic amino acid.
In some embodiments, the CDRH3 of any of the anti-NKG2A agents provided herein comprises an amino acid sequence chosen from QGIWDYDGFAD (SEQ ID NO: 11) and DRDTSVYVNYYTMDY (SEQ ID NO: 18).
In some embodiments, the CDRH3 of any of the anti-NKG2A agents provided herein comprises the sequence of amino acids QGIWDYDGFAD (SEQ ID NO: 11); or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 11. In some embodiments, the CDRH3 of any of the anti-NKG2A agents provided herein comprises the sequence of amino acids QGIWDYDGFAD (SEQ ID NO: 11). In some embodiments, the CDRH3 of any of the anti-NKG2A agents provided herein comprises the sequence of amino acids DRDTSVYVNYYTMDY (SEQ ID NO: 18); or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 18. In some embodiments, the CDRH3 of any of the anti-NKG2A agents provided herein comprises the sequence of amino acids DRDTSVYVNYYTMDY (SEQ ID NO: 18).
CDRL1
In some embodiments, the first light chain CDR (CDRL1) of an anti-NKG2A agent provided herein comprises an amino acid sequence that is at least 80 percent identical to the amino acid sequence
X1ASX4SVX7X8X9X10X11X12YX14H (SEQ ID NO: 25), where XI is S or R; X4 is S or K; X7 is T or S; X8 is T or no amino acid; X9 is S or no amino acid; X1 0 is G or no amino acid; XI 1 is Y or no amino acid; X12 is S or no amino acid; and X14 is M or I. In some embodiments, the CDRL1 comprises an amino acid sequence that is at least 90 percent identical to the amino acid sequence of SEQ ID NO: 25. In some embodiments, the CDRL1 comprises an amino acid sequence that is at least 95 percent identical to the amino acid sequence of SEQ ID NO: 25. In some embodiments, the CDRL1 comprises an amino acid sequence that is 100 percent identical to the amino acid sequence of SEQ ID NO: 25. In some embodiments, the CDRL1 comprises the sequence of amino acids set forth in SEQ ID NO: 25.
The amino acid X1 of SEQ ID NO: 25 may be substituted with any amino acid. In some embodiments, the amino acid X1 of SEQ ID NO: 25 is substituted with a conservative amino acid (e.g., S and/or R). In some embodiments, the amino acid X1 of SEQ ID NO: 25 is substituted with a neutral amino acid. In some embodiments, the amino acid X1 of SEQ ID NO: 25 is substituted with a basic amino acid.
The amino acid X4 of SEQ ID NO: 25 may be substituted with any amino acid. In some embodiments, the amino acid X4 of SEQ ID NO: 25 is substituted with a conservative amino acid (e.g., S and/or K). In some embodiments, the amino acid X4 of SEQ ID NO: 25 is substituted with a neutral amino acid. In some embodiments, the amino acid X4 of SEQ ID NO: 25 is substituted with a basic amino acid.
The amino acid X7 of SEQ ID NO: 25 may be substituted with any amino acid. In some embodiments, the amino acid X7 of SEQ ID NO: 25 is substituted with a conservative amino acid (e.g., T and/or S). In some embodiments, the amino acid X7 of SEQ ID NO: 25 is substituted with a neutral amino acid. The amino acid X8 of SEQ ID NO: 25 may be substituted with any amino acid. In some embodiments, the amino acid X8 of SEQ ID NO: 25 is substituted with a conservative amino acid (e.g., T and/or no amino acid). In some embodiments, the amino acid X8 of SEQ ID NO: 25 is substituted with a neutral amino acid. In some embodiments, the amino acid X8 of SEQ ID NO: 25 is no amino acid. The amino acid X9 of SEQ ID NO: 25 may be substituted with any amino acid. In some embodiments, the amino acid X9 of SEQ ID NO: 25 is substituted with a conservative amino acid (e.g., S and/or no amino acid). In some embodiments, the amino acid X9 of SEQ ID NO: 25 is substituted with a neutral amino acid. In some embodiments, the amino acid X9 of SEQ ID NO: 25 is no amino acid. The amino acid X10 of SEQ ID NO: 25 may be substituted with any amino acid. In some embodiments, the amino acid X10 of SEQ ID NO: 25 is substituted with a conservative amino acid (e.g., G and/or no amino acid). In some embodiments, the amino acid X10 of SEQ ID NO: 25 is substituted with an amino acid residue that influences chain orientation. In some embodiments, the amino acid X10 of SEQ ID NO: 25 is no amino acid. The amino acid X11 of SEQ ID NO: 25 may be substituted with any amino acid. In some embodiments, the amino acid X11 of SEQ ID NO: 25 is substituted with a conservative amino acid (e.g., Y and/or no amino acid). In some embodiments, the amino acid X11 of SEQ ID NO: 25 is substituted with an aromatic amino acid. In some embodiments, the amino acid X11 of SEQ ID NO:25 is no amino acid. The amino acid X12 of SEQ ID NO: 25 may be substituted with any amino acid. In some embodiments, the amino acid X12 of SEQ ID NO: 25 is substituted with a conservative amino acid (e.g., S and/or no amino acid). In some embodiments, the amino acid X12 of SEQ ID NO: 25 is substituted with a neutral amino acid. In some embodiments, the amino acid X12 of SEQ ID NO: 25 is no amino acid. The amino acid X14 of SEQ ID NO: 25 may be substituted with any amino acid. In some embodiments, the amino acid X14 of SEQ ID NO: 25 is substituted with a conservative amino acid (e.g., M and/or I). In some embodiments, the amino acid X14 of SEQ ID NO: 25 is substituted with a hydrophobic amino acid. In some embodiments, the CDRL1 of any of the anti-NKG2A agents provided herein comprises an amino acid sequence chosen from SASSSVTYMH (SEQ ID NO: 12) and RASKSVSTSGYSYIH (SEQ ID NO: 19). In some embodiments, the CDRL1 of any of the anti-NKG2A agents provided herein comprises the sequence of amino acids SASSSVTYMH (SEQ ID NO: 12); or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 12. In some embodiments, the CDRL1 of any of the anti-NKG2A agents provided herein comprises the sequence of amino acids SASSSVTYMH (SEQ ID NO: 12).
In some embodiments, the CDRL1 of any of the anti-NKG2A agents provided herein comprises the sequence of amino acids RASKSVSTSGYSYIH (SEQ ID NO: 19); or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 19 .In some embodiments, the CDRL1 of any of the anti-NKG2A agents provided herein comprises the sequence of amino acids RASKSVSTSGYSYIH (SEQ ID NO: 19).
CDRL2
In some embodiments, the second light chain CDR (CDRL2) of an anti-NKG2A agent provided herein comprises an amino acid sequence that is at least 80 percent identical to the amino acid sequence X^SNLX6S (SEQ ID NO: 26), where XI is S or L, X2 is T or A, and X6 is A or E. In some embodiments, the CDRL2 comprises an amino acid sequence that is at least 90 percent identical to the amino acid sequence of SEQ ID NO: 26. In some embodiments, the CDRL2 comprises an amino acid sequence that is at least 95 percent identical to the amino acid sequence of SEQ ID NO: 26. In some embodiments, the CDRL2 comprises an amino acid sequence of SEQ ID NO: 26. In some embodiments, the CDRL2 comprises an amino acid sequence that is 100 percent identical to the amino acid sequence of SEQ ID NO: 26. In some embodiments, the CDRL2 comprises the sequence of amino acids set forth in SEQ ID NO: 26.
The amino acid X1 of SEQ ID NO: 26 may be substituted with any amino acid. In some embodiments, the amino acid X1 of SEQ ID NO: 26 is substituted with a conservative amino acid (e.g., S and/or L). In some embodiments, the amino acid X1 of SEQ ID NO: 26 is substituted with a neutral amino acid. In some embodiments, the amino acid X1 of SEQ ID NO: 26 is substituted with a hydrophobic amino acid.
The amino acid X2 of SEQ ID NO: 26 may be substituted with any amino acid. In some embodiments, the amino acid X2 of SEQ ID NO: 26 is substituted with a conservative amino acid (e.g., T and/or A). In some embodiments, the amino acid X2 of SEQ ID NO: 26 is substituted with a neutral amino acid. In some embodiments, the amino acid X2 of SEQ ID NO: 26 is substituted with a hydrophobic amino acid. The amino acid X6 of SEQ ID NO: 26 may be substituted with any amino acid. In some embodiments, the amino acid X6 of SEQ ID NO: 26 is substituted with a conservative amino acid (e.g., A and/or E). In some embodiments, the amino acid X6 of SEQ ID NO: 26 is substituted with a hydrophobic amino acid. In some embodiments, the amino acid X6 of SEQ ID NO: 26 is substituted with an acidic amino acid. In some embodiments, the CDRL2 of any of the anti-NKG2A agents provided herein comprises an amino acid sequence chosen from STSNLAS (SEQ ID NO: 13) and LASNLES (SEQ ID NO: 20). In some embodiments, the CDRL2 of any of the anti-NKG2A agents provided herein comprises the sequence of amino acids STSNLAS (SEQ ID NO: 13); or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 13. In some embodiments, the CDRL2 of any of the anti-NKG2A agents provided herein comprises the sequence of amino acids STSNLAS (SEQ ID NO: 13). In some embodiments, the CDRL2 of any of the anti-NKG2A agents provided herein comprises the sequence of amino acids LASNLES (SEQ ID NO: 20); or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 20. In some embodiments, the CDRL2 of any of the anti-NKG2A agents provided herein comprises the sequence of amino acids LASNLES (SEQ ID NO: 20). CDRL3 In some embodiments, the third light chain CDR (CDRL3) of an anti-NKG2A agent provided herein comprises an amino acid sequence that is at least 80 percent identical to the amino acid sequence QX2X3X4X5X6PX8T (SEQ ID NO: 27), where X2 is Q or H, X3 is R or S, X4 is S or R, X5 is S or E, X6 is Y or L, and X8 is Y or L. In some embodiments, the CDRL3 comprises an amino acid sequence that is at least 90 percent identical to the amino acid sequence of SEQ ID NO: 27. In some embodiments, the CDRL3 comprises an amino acid sequence that is at least 95 percent identical to the amino acid sequence of SEQ ID NO: 27. In some embodiments, the CDRL3 comprises an amino acid sequence that is 100 percent identical to the amino acid sequence of SEQ ID NO: 27. In some embodiments, the CDRL3 comprises the sequence of amino acids set forth in SEQ ID NO: 27. The amino acid X2 of SEQ ID NO: 27 may be substituted with any amino acid. In some embodiments, the amino acid X2 of SEQ ID NO: 27 is substituted with a conservative amino acid (e.g., Q and/or H). In some embodiments, the amino acid X2 of SEQ ID NO: 27 is substituted with a basic amino acid. The amino acid X3 of SEQ ID NO: 27 may be substituted with any amino acid. In some embodiments, the amino acid X3 of SEQ ID NO: 27 is substituted with a conservative amino acid (e.g., R and/or S). In some embodiments, the amino acid X3 of SEQ ID NO: 27 is substituted with a basic amino acid. In some embodiments, the amino acid X3 of SEQ ID NO: 27 is substituted with a neutral amino acid.
The amino acid X4 of SEQ ID NO: 27 may be substituted with any amino acid. In some embodiments, the amino acid X4 of SEQ ID NO: 27 is substituted with a conservative amino acid (e.g., S and/or R). In some embodiments, the amino acid X4 of SEQ ID NO: 27 is substituted with a neutral amino acid. In some embodiments, the amino acid X4 of SEQ ID NO: 27 is substituted with a basic amino acid.
The amino acid X5 of SEQ ID NO: 27 may be substituted with any amino acid. In some embodiments, the amino acid X5 of SEQ ID NO: 27 is substituted with a conservative amino acid (e.g., S and/or E). In some embodiments, the amino acid X5 of SEQ ID NO: 27 is substituted with a neutral amino acid. In some embodiments, the amino acid X5 of SEQ ID NO: 27 is substituted with an acidic amino acid.
The amino acid X6 of SEQ ID NO: 27 may be substituted with any amino acid. In some embodiments, the amino acid X6 of SEQ ID NO: 27 is substituted with a conservative amino acid (e.g., Y and/or L). In some embodiments, the amino acid X6 of SEQ ID NO: 27 is substituted with an aromatic amino acid. In some embodiments, the amino acid X6 of SEQ ID NO: 27 is substituted with a hydrophobic amino acid.
The amino acid X6 of SEQ ID NO: 27 may be substituted with any amino acid. In some embodiments, the amino acid X6 of SEQ ID NO: 27 is substituted with a conservative amino acid (e.g., Y and/or L). In some embodiments, the amino acid X6 of SEQ ID NO: 27 is substituted with an aromatic amino acid. In some embodiments, the amino acid X6 of SEQ ID NO: 27 is substituted with a hydrophobic amino acid.
In some embodiments, the CDRL3 of any of the anti-NKG2A agents provided herein comprises an amino acid sequence chosen from QQRSSYPYT (SEQ ID NO: 14) and QHSRELPLT (SEQ ID NO: 21).
In some embodiments, the CDRL3 of any of the anti-NKG2A agents provided herein comprises the sequence of amino acids QQRSSYPYT (SEQ ID NO: 14); or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 14. In some embodiments, the CDRL3 of any of the anti-NKG2A agents provided herein comprises the sequence of amino acids QQRSSYPYT (SEQ ID NO: 14). In some embodiments, the CDRL3 of any of the anti-NKG2A agents provided herein comprises the sequence of amino acids QHSRELPLT (SEQ ID NO: 21); or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 21. In some embodiments, the CDRL3 of any of the anti-NKG2A agents provided herein comprises the sequence of amino acids QHSRELPLT (SEQ ID NO: 21).
CDR sets
In some embodiments, any of the anti-NKG2A agents described herein comprises an immunoglobulin heavy chain variable domain comprising a set of CDRs (i.e., CDRH1, CDRH2, CDRH3); and an immunoglobulin light chain variable domain comprising a set of CDRs (i.e., CDRL1, CDRL2, CDRL3). In some embodiments, any of the antibodies or antigen-binding fragments thereof that bind the NKG2A receptor or fragment thereof described herein comprises an immunoglobulin heavy chain variable domain comprising a set of CDRs (i.e., CDRH1, CDRH2, CDRH3); and an immunoglobulin light chain variable domain comprising a set of CDRs (i.e., CDRL1, CDRL2, CDRL3). In some embodiments, any of the anti-NKG2A agents provided herein comprises two immunoglobulin heavy chain variable domains each comprising a set of CDRs (i.e., CDRH1, CDRH2, CDRH3); and two immunoglobulin light chain variable domains each comprising a set of CDRs (i.e., CDRL1, CDRL2, CDRL3). In some embodiments, any of the antibodies or antigen-binding fragments thereof that bind the NKG2A receptor or fragment thereof described herein comprise one or more immunoglobulin heavy chain variable domains each comprising a set of CDRs (i.e., CDRH1, CDRH2, CDRH3); and one or more immunoglobulin light chain variable domains each comprising a set of CDRs (i.e., CDRL1, CDRL2, CDRL3). In some embodiments, any of the antibodies or antigen-binding fragments thereof that bind the NKG2A receptor or fragment thereof described herein comprise two immunoglobulin heavy chain variable domains each comprising a set of CDRs (i.e., CDRH1, CDRH2, CDRH3); and two immunoglobulin light chain variable domains each comprising a set of CDRs (i.e., CDRL1, CDRL2, CDRL3). Sets of CDRs may comprise any combination of CDR amino acid sequences (i.e., CDRH1, CDRH2, CDRH3; and CDRL1, CDRL2, CDRL3) provided herein. In some embodiments, an immunoglobulin heavy chain variable domain comprises a set of CDRH1, CDRH2 and CDRH3 amino acid sequences, and an immunoglobulin light chain variable domain comprises a set of CDRL1, CDRL2 and CDRL3 amino acid sequences chosen from sets 1-4 provided in the table below. For an anti-NKG2A agent comprising two immunoglobulin heavy chain variable domains and two immunoglobulin light chain variable domains, each immunoglobulin heavy chain variable domain may comprise a set of CDRH1, CDRH2 and CDRH3 amino acid sequences, and each immunoglobulin light chain variable domain may comprise a set of CDRL1, CDRL2 and CDRL3 amino acid sequences chosen from sets 1-4 provided in Table 2.
Figure imgf000030_0001
In some embodiments, the anti-NKG2A agent contains CDRs corresponding to the same set. For example, for an anti-NKG2A agent comprising two immunoglobulin heavy chain variable domains and two immunoglobulin light chain variable domains, each immunoglobulin heavy chain variable domain may comprise a set of CDRH1, CDRH2 and CDRH3 amino acid sequences from set 1, and each immunoglobulin light chain variable domain may comprise a set of CDRL1, CDRL2 and CDRL3 amino acid sequences from set 1.
In some embodiments, the anti-NKG2A agent contains CDRs corresponding to different sets. For example, for an anti-NKG2A agent comprising two immunoglobulin heavy chain variable domains and two immunoglobulin light chain variable domains, each immunoglobulin heavy chain variable domain may comprise a set of CDRH1, CDRH2 and CDRH3 amino acid sequences from set 1, and each immunoglobulin light chain variable domain may comprise a set of CDRL1, CDRL2 and CDRL3 amino acid sequences from set 2. In another example, for an anti-NKG2A agent comprising two immunoglobulin heavy chain variable domains and two immunoglobulin light chain variable domains, one immunoglobulin heavy chain variable domain may comprise a set of CDRH1, CDRH2 and CDRH3 amino acid sequences from set 1 and the other immunoglobulin heavy chain variable domain may comprise a set of CDRH1, CDRH2 and CDRH3 amino acid sequences from set 2; and one immunoglobulin light chain variable domain may comprise a set of CDRL1, CDRL2 and CDRL3 amino acid sequences from set 1 and the other immunoglobulin light chain variable domain may comprise a set of CDRL1, CDRL2 and CDRL3 amino acid sequences from set 2.
In some embodiments, the agent that binds the NKG2A receptor or a fragment thereof, comprises one or more of: a) an immunoglobulin heavy chain variable domain comprising:
(i) a heavy chain complementary determining region 1 (CDRH1) comprising the sequence
X1YX3MX5 (SEQ ID NO: 22), wherein X1 is S or D; X3 is W or Y, and X5 is N or Y;
(ii) a heavy chain complementary determining region 2 (CDRH2) comprising the sequence
X1IX3X4X5X6X7X8TX10YX12X13X14X15KX17 (SEQ ID NO: 23), wherein X1 is R or T; X3 is Y or S; X4 is F or Y; X5 is E or G; X6 is D or G; X7 is G or I; X8 is D or Y; X10 is N or Y; X12 is N or P; X13 is G or D; X14 is K or S; X15 is F or V, and X17 is G or D; and
(iii) a heavy chain complementary determining region 3 (CDRH3) comprising the sequence XIX2X3X4X5X6X7X8X9YXIIXI2XI3XI4XI5 (SEQ ID NO: 24), wherein X1 is Q or D; X2 is G or R; X3 is D or no amino acid; X4 is T or no amino acid; X5 is S or no amino acid; X6 is V or no amino acid; X7 is I or Y; X8 is W or V; X9 is D or N; X11 is D or Y; X12 is G or T; X13 is F or M; X14 is A or D; and
X15 is D or Y; and b) an immunoglobulin light chain variable domain comprising:
(i) a light chain complementary determining region 1 (CDRL1) comprising the sequence
X1ASX4SVX7X8X9X10X11X12YX14H (SEQ ID NO: 25), wherein X1 is S or R; X4 is S or K; X7 is T or S; X8 is T or no amino acid; X9 is S or no amino acid; X10 is G or no amino acid; X11 is Y or no amino acid; X12 is S or no amino acid; and X14 is M or I;
(ii) a light chain complementary determining region 2 (CDRL2) comprising the sequence
X1X2SNLX6S (SEQ ID NO: 26), wherein X1 is S or L; X2 is T or A; and X6 is A or E; and
(iii) a light chain complementary determining region 3 (CDRL3) comprising the sequence QX2X3X4X5X6PX8T (SEQ ID NO: 27), wherein X2 is Q or H; X3 is R or S; X4 is S or R; X5 is S or E; X6 is Y or L; and X8 is Y or L.
In some embodiments, the agent that binds the NKG2A receptor or a fragment thereof, comprises an immunoglobulin heavy chain variable domain comprising: a CDRH1 comprising the sequence of amino acids set forth in SEQ ID NO: 9 or SEQ ID NO: 16, or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:9 or SEQ ID NO: 16; a
CDRH2 comprising the sequence of amino acids set forth in SEQ ID NO: 10, 15 or 17, or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 10, 15 or 17; and a CDRH3 comprising the sequence of amino acids set forth in SEQ ID NO: 11 or SEQ ID
NO: 18, or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%,
87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 11 or SEQ ID NO: 18.
In some embodiments, the agent that binds the NKG2A receptor or a fragment thereof, comprises an immunoglobulin heavy chain variable domain that comprises a CDRH1 comprising the sequence of amino acids set forth in SEQ ID NO: 9 or SEQ ID NO: 16; a CDRH2 comprising the sequence of amino acids set forth in SEQ ID NO: 10, 15 or 17; and a CDRH3 comprising the sequence of amino acids set forth in SEQ ID NO: 11 or SEQ ID NO: 18.
In some embodiments, the agent that binds the NKG2A receptor or a fragment thereof, comprises an immunoglobulin light chain variable domain comprising a CDRL1 comprising the sequence of amino acids set forth in SEQ ID NO: 12 or SEQ ID NO: 19, or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 12 or SEQ ID NO: 19; a
CDRL2 comprising the sequence of amino acids set forth in SEQ ID NO: 13 or SEQ ID NO:20, or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID
NO: 13 or SEQ ID NO:20; and a CDRL3 comprising the sequence of amino acids set forth in SEQ ID
NO: 14 or SEQ ID NO:21, or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%,
84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 14 or SEQ ID NO:21.
In some embodiments, the agent that binds the NKG2A receptor or a fragment thereof, comprises an immunoglobulin light chain variable domain comprising a CDRL1 comprising a sequence of amino acids set forth in SEQ ID NO: 12 or SEQ ID NO: 19; a CDRL2 comprising a sequence of amino acids set forth in SEQ ID NO: 13 or SEQ ID NO:20; and a CDRL3 comprising a sequence of amino acids set forth in SEQ ID NO: 14 or SEQ ID NO:21.
In some embodiments, the agent that binds the NKG2A receptor or a fragment thereof, comprises a CDRH1 comprising the sequence set forth in SEQ ID NO: 9, or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:9, a CDRH2 comprising the sequence set forth in SEQ ID NO: 10 ,or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 10; and a CDRH3 comprising the sequence set forth in SEQ ID NO: 11, or a sequence of amino acids that exhibits at least 80%, 81%,
82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
99% or more sequence identity to SEQ ID NO: 11.
In some embodiments, the agent that binds the NKG2A receptor or a fragment thereof, comprises a CDRH1 comprising the sequence set forth in SEQ ID NO: 9, the CDRH2 comprises the sequence set forth in SEQ ID NO: 10; and the CDRH3 comprises the sequence set forth in SEQ ID NO: 11.
In some embodiments, the antibody or antigen-binding fragment thereof that binds the NKG2A receptor or a fragment thereof, comprises a CDRH1 comprising the sequence set forth in SEQ ID NO: 9, or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:9; a CDRH2 comprising the sequence set forth in SEQ ID NO: 15 or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID
NO: 15; and a CDRH3 comprising the sequence set forth in SEQ ID NO: 11 or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 11.
In some embodiments, the agent that binds the NKG2A receptor or a fragment thereof, comprises a CDRH1 comprising the sequence set forth in SEQ ID NO: 9, a CDRH2 comprising the sequence set forth in SEQ ID NO: 15; and a CDRH3 comprising the sequence set forth in SEQ ID NO: 11.
In some embodiments, the agent that binds the NKG2A receptor or a fragment thereof, comprises a CDRH1 comprising the sequence set forth in SEQ ID NO: 16, or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 16; a CDRH2 comprising the sequence set forth in SEQ ID NO: 17 or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO; 17; and the CDRH3 comprises the sequence set forth in SEQ ID NO: 18 or a sequence of amino acids that exhibits at least 80%, 81%,
82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
99% or more sequence identity to SEQ ID NO: 18. In some embodiments, the agent that binds the NKG2A receptor or a fragment thereof, comprises a CDRH1 comprising the sequence set forth in SEQ ID NO: 16, a CDRH2 comprising the sequence set forth in SEQ ID NO: 17; and a CDRH3 comprising the sequence set forth in SEQ ID NO: 18.
In some embodiments, the agent that binds the NKG2A receptor or a fragment thereof, comprises a CDRL1 comprising the sequence set forth in SEQ ID NO: 12, or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 12; a CDRL2 comprising the sequence set forth in SEQ ID NO: 13 or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 13; and a CDRL3 comprising the sequence set forth in SEQ ID NO: 14 or a sequence of amino acids that exhibits at least 80%, 81%,
82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
99% or more sequence identity to SEQ ID NO: 14.
In some embodiments, the agent that binds the NKG2A receptor or a fragment thereof, comprises a CDRL1 comprising the sequence set forth in SEQ ID NO: 12, a CDRL2 comprising the sequence set forth in SEQ ID NO: 13; and a CDRL3 comprising the sequence set forth in SEQ ID NO: 14.
In some embodiments, the agent that binds the NKG2A receptor or a fragment thereof, comprises a CDRL1 comprising the sequence set forth in SEQ ID NO: 19 or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 19, a CDRL2 comprising the sequence set forth in SEQ ID NO: 20 or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:20; and a CDRL3 comprising the sequence set forth in SEQ ID NO: 21 or a sequence of amino acids that exhibits at least 80%, 81%,
82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
99% or more sequence identity to SEQ ID NO:21.
In some embodiments, the agent that binds the NKG2A receptor or a fragment thereof, comprises a CDRL1 comprising the sequence set forth in SEQ ID NO: 19, a CDRL2 comprises the sequence set forth in SEQ ID NO: 20; and a CDRL3 comprises the sequence set forth in SEQ ID NO: 21. In some embodiments, the agent binds the NKG2A receptor or a fragment thereof, comprises a CDRH1 comprising the sequence of amino acids set forth in SEQ ID NO: 9 or SEQ ID NO: 16, or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID
NO:9 or SEQ ID NO: 16; a CDRH2 comprising a sequence of amino acids set forth in SEQ ID NO:
10, 15 or 17, or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 10, 15 or 17; a CDRH3 comprising a sequence of amino acids set forth in
SEQ ID NO: 11 or SEQ ID NO: 18, or a sequence of amino acids that exhibits at least 80%, 81%,
82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
99% or more sequence identity to SEQ ID NO: 11 or SEQ ID NO: 18; a CDRL1 comprising a sequence of amino acids set forth in SEQ ID NO: 12 or SEQ ID NO: 19, or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 12 or SEQ ID NO: 19; a
CDRL2 comprising a sequence of amino acids set forth in SEQ ID NO: 13 or SEQ ID NO:20, or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID
NO: 13 or SEQ ID NO:20; and a CDRL3 comprising a sequence of amino acids set forth in SEQ ID
NO: 14 or SEQ ID NO:21, or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%,
84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 14 or SEQ ID NO:21.
In some embodiments, the agent that binds the NKG2A receptor or a fragment thereof, comprises a CDRH1 comprising the sequence of amino acids set forth in SEQ ID NO: 9 or SEQ ID NO: 16; a CDRH2 comprising the sequence of amino acids set forth in SEQ ID NO: 10, 15 or 17; a CDRH3 comprising the sequence of amino acids set forth in SEQ ID NO: 11 or SEQ ID NO: 18; a CDRL1 comprising the sequence of amino acids set forth in SEQ ID NO: 12 or SEQ ID NO: 19; a CDRL2 comprising the sequence of amino acids set forth in SEQ ID NO: 13 or SEQ ID NO:20; and a CDRL3 comprising the sequence of amino acids set forth in SEQ ID NO: 14 or SEQ ID NO:21.
In some embodiments, the agent that binds the NKG2A receptor or a fragment thereof, comprises a CDRH1 comprising the sequence set forth in SEQ ID NO: 9 or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:9, a CDRH2 comprising the sequence set forth in SEQ ID NO: 10 or a sequence of amino acids that exhibits at least 80%,
81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99% or more sequence identity to SEQ ID NO: 10; a CDRH3 comprising the sequence set forth in SEQ ID NO: 11 or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%,
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 11; a CDRL1 comprising the sequence set forth in SEQ ID NO: 12 or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID
NO: 12, a CDRL2 comprising the sequence set forth in SEQ ID NO: 13 or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 13; and a CDRL3 comprising the sequence set forth in SEQ ID NO: 14 or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 14.
In some embodiments, the agent that binds the NKG2A receptor or a fragment thereof, comprises a CDRH1 comprising the sequence set forth in SEQ ID NO: 9, a CDRH2 comprising the sequence set forth in SEQ ID NO: 10; a CDRH3 comprising the sequence set forth in SEQ ID NO: 11; a CDRL1 comprising the sequence set forth in SEQ ID NO: 12, a CDRL2 comprising the sequence set forth in SEQ ID NO: 13; and a CDRL3 comprising the sequence set forth in SEQ ID NO: 14.
In some embodiments, the agent that binds the NKG2A receptor or a fragment thereof, comprises a CDRH1 comprising the sequence set forth in SEQ ID NO: 9 or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:9; a CDRH2 comprising the sequence set forth in SEQ ID NO: 15 or a sequence of amino acids that exhibits at least 80%,
81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99% or more sequence identity to SEQ ID NO: 15; a CDRH3 comprising the sequence set forth in SEQ ID NO: 11 or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%,
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 11; a CDRL1 comprising the sequence set forth in SEQ ID NO: 12 or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID
NO: 12, a CDRL2 comprising the sequence set forth in SEQ ID NO: 13 or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 13; and a CDRL3 comprising the sequence set forth in SEQ ID NO: 14 or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 14.
In some embodiments, the agent that binds the NKG2A receptor or a fragment thereof, comprises a CDRH1 comprising the sequence set forth in SEQ ID NO: 9, a CDRH2 comprising the sequence set forth in SEQ ID NO: 15; a CDRH3 comprising the sequence set forth in SEQ ID NO: 11; a CDRL1 comprising the sequence set forth in SEQ ID NO: 12, a CDRL2 comprising the sequence set forth in SEQ ID NO: 13; and a CDRL3 comprising the sequence set forth in SEQ ID NO: 14.
In some embodiments, the agent that binds the NKG2A receptor or a fragment thereof, comprises a CDRH1 comprising the sequence set forth in SEQ ID NO: 16 or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 16, a CDRH2 comprising the sequence set forth in SEQ ID NO: 17 or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 17; a CDRH3 comprising the sequence set forth in SEQ ID NO: 18 or a sequence of amino acids that exhibits at least 80%, 81%,
82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
99% or more sequence identity to SEQ ID NO: 18; a CDRL1 comprising the sequence set forth in
SEQ ID NO: 19 or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 19, a CDRL2 comprising the sequence set forth in SEQ ID NO: 20 or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID
NO:20; and a CDRL3 comprising the sequence set forth in SEQ ID NO: 21 or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:21.
In some embodiments, the agent that binds the NKG2A receptor or a fragment thereof, comprises a CDRH1 comprising the sequence set forth in SEQ ID NO: 16, a CDRH2 comprising the sequence set forth in SEQ ID NO: 17; a CDRH3 comprising the sequence set forth in SEQ ID NO: 18; a CDRL1 comprising the sequence set forth in SEQ ID NO: 19, a CDRL2 comprising the sequence set forth in SEQ ID NO: 20; and a CDRL3 comprising the sequence set forth in SEQ ID NO: 21. In some of any embodiments, the agents that binds the NKG2A receptor or fragment thereof is an antibody or antigen-binding fragment thereof.
VH
In some embodiments, the anti-NKG2A agent provided herein may comprise an immunoglobulin heavy chain variable domain (VH). In some embodiments, the immunoglobulin heavy chain variable domain (VH) of any of the anti-NKG2A agents provided herein comprises a polypeptide that is at least 80 percent identical to the amino acid sequence QVQLQQSGPELVKPGASVKISCKASGYVFSSYWMNWVKQRPGQGLEWIGRIYFEDGDTNY NGKFKGKATLTADKSSSTAYMQLSSLTSVDSAVYFCARQGIWDYDGFADWGQGTLVTVSA (SEQ ID NO: 1), e.g., 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, identical to the amino acid sequence of SEQ ID NO: 1. In some embodiments, a VH comprises a polypeptide that is at least 90 percent identical to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the VH comprises a polypeptide that is at least 95 percent identical to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the VH comprises a polypeptide that is 100 percent identical to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the VH comprises the sequence of amino acids set forth in SEQ ID NO: 1.
In some embodiments, the immunoglobulin heavy chain variable domain (VH) of an anti-NKG2A agent provided herein comprises a polypeptide that is at least 80 percent identical to the amino acid sequence QVQLQQSGPELVKPGASVKISCKASGYVFSSYWMNWVKQRPGQGLEWIGRIYFEDGDTNY NGKFKDKATLTADKSSSTAYMQLSSLTSVDSAVYFCARQGIWDYDGFADWGQGTLVTVSA (SEQ ID NO: 3), e.g., 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, identical to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the VH comprises a polypeptide that is at least 90 percent identical to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the VH comprises a polypeptide that is at least 95 percent identical to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the VH comprises a polypeptide that is 100 percent identical to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the VH comprises the sequence of amino acids set forth in SEQ ID NO: 3.
In some embodiments, the immunoglobulin heavy chain variable domain (VH) of an anti-NKG2A agent provided herein comprises a polypeptide that is at least 80 percent identical to the amino acid sequence
EVQLVESGGGLVKPGGSLKLSCAASGFTFSDYYMYWVRQTPEKRLEWVATISYGGIYTYY PDSVKGRFTISRDTAKNNLYLQMSSLKSEDTAVYYCARDRDTSVYVNYYTMDYWGQGTSV TVSS (SEQ ID NO: 4), e.g., 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, identical to the amino acid sequence of SEQ ID NO: 4. In some embodiments, the VH comprises a polypeptide that is at least 90 percent identical to the amino acid sequence of SEQ ID NO: 4. In some embodiments, the VH comprises a polypeptide that is at least 95 percent identical to the amino acid sequence of SEQ ID NO: 4. In some embodiments, the VH comprises a polypeptide that is 100 percent identical to the amino acid sequence of SEQ ID NO: 4. In some embodiments, the VH comprises the sequence of amino acids set forth in SEQ ID NO: 4.
In some embodiments, the VH of an anti-NKG2A agent provided herein comprises a polypeptide chosen from SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 4.
In some embodiments, the VH of any of the anti-NKG2A agents provided herein further comprises a signal sequence. In some embodiments, VH signal sequence of any of the anti-NKG2A agents provided herein comprises the sequence of amino acids set forth in SEQ ID NO:49 or SEQ ID NO:53. In some embodiments, VH signal sequence of any of the anti-NKG2A agents provided herein comprises the sequence of amino acids set forth in SEQ ID NO:49. In some embodiments, VH signal sequence of any of the anti-NKG2A agents provided herein comprises the sequence of amino acids set forth in SEQ ID NO:53.
VL
In some embodiments, the anti-NKG2A agent provided herein may comprise an immunoglobulin light chain variable domain (VL). In some embodiments, the immunoglobulin light chain variable domain (VL) of an anti-NKG2A agent provided herein comprises a polypeptide that is at least 80 percent identical to the amino acid sequence QIVFTQSPAIMSASPGEKVTITCSASSSVTYMHWFQQKPGTSPKLWIYSTSNLAS GVPARFSGSGSGTSYSLTISRMEAEDAATYYCQQRSSYPYTFGGGTKLEIK (SEQ ID NO: 5), e.g., 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, identical to the amino acid sequence of SEQ ID NO: 5. In some embodiments, the VL comprises a polypeptide that is at least 90 percent identical to the amino acid sequence of SEQ ID NO: 5. In some embodiments, the VL comprises a polypeptide that is at least 95 percent identical to the amino acid sequence of SEQ ID NO: 5. In some embodiments, the VL comprises a polypeptide that is 100 percent identical to the amino acid sequence of SEQ ID NO: 5. In some embodiments, the VL comprises the sequence of amino acids set forth in SEQ ID NO: 5.
In some embodiments, the immunoglobulin light chain variable domain (VL) of an anti-NKG2A agent provided herein comprises a polypeptide that is at least 80 percent identical to the amino acid sequence DIVLTQSPVSLTVSLGQRATISCRASKSVSTSGYSYIHWYQQRPGQPPKLLIYLASNLES GVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRELPLTFGAGTKLELK (SEQ ID NO: 8), e.g., 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, identical to the amino acid sequence of SEQ ID NO: 8. In some embodiments, the VL comprises a polypeptide that is at least 90 percent identical to the amino acid sequence of SEQ ID NO: 8. In some embodiments, the VL comprises a polypeptide that is at least 95 percent identical to the amino acid sequence of SEQ ID NO: 8. In some embodiments, the VL comprises a polypeptide that is 100 percent identical to the amino acid sequence of SEQ ID NO: 8. In some embodiments, the VL comprises the sequence of amino acids set forth in SEQ ID NO: 8.
In some embodiments, the VL of an anti-NKG2A agent provided herein comprises a polypeptide chosen from SEQ ID NO: 5 and SEQ ID NO: 8.
In some embodiments, the VL of any of the anti-NKG2A agents provided herein further comprises a signal sequence. In some embodiments, VL signal sequence of any of the anti-NKG2A agents provided herein comprises the sequence of amino acids set forth in SEQ ID NO:47 or SEQ ID NO:51. In some embodiments, VL signal sequence of any of the anti-NKG2A agents provided herein comprises the sequence of amino acids set forth in SEQ ID NO:47. In some embodiments, VL signal sequence of any of the anti-NKG2A agents provided herein comprises the sequence of amino acids set forth in SEQ ID NO:51.
Fc
In some embodiments, any of the anti-NKG2A agents provided herein may comprise a fragment crystallizable region (Fc region). An Fc region typically forms the tail of an antibody and can interact with certain cell surface receptors and certain components of the complement system. An Fc region may include, for example, two polypeptides, each derived from the second (CH2) and third (CH3) constant domains of an antibody heavy chain.
The amino acid sequence of a wild-type CH2-CH3 portion of an Fc region is provided below (positioning is as in the EU index as in Kabat et al. (1992) SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, National Institutes of Health Publication No. 91-3242) (SEQ ID NO:29).
APELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT
231 240 250 260 270 280
<-( H2 | CH3->
KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA K GQPREPQVY
290 300 310 320 330 340
TLPPSREEMT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK
350 360 370 380 390 400
<-( H3 |
LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGK
410 420 430 440
In some embodiments, an Fc region includes one or more modifications (e.g., one or more amino acid substitutions, insertions, or deletions relative to a comparable wild-type Fc region). Agents comprising modified Fc regions (variant agents) typically have altered phenotypes relative to agents comprising wild-type Fc regions. A variant agent phenotype may be expressed as altered serum halflife, altered stability, altered susceptibility to cellular enzymes, or altered effector function (e.g., as assayed in an NK dependent or macrophage dependent assay). Fc region modifications that alter effector function may include modifications that increase binding to activating receptors (e.g., FcyRIIA (CD16A)) and reduce binding to inhibitory receptors (e.g., FcyRIIB (CD32B)) (see, e.g., Stavenhagen, J.B. et al. (2007) Cancer Res. 57(18):8882-8890). Examples of variants of human IgGl Fc regions with reduced binding to CD32B and/or increased binding to CD16A contain F243L, R292P, Y300L, V305I and/or P396L substitutions. Amino acid positions correspond to the amino acid numbering of the CH2-CH3 domain provided above.
In some embodiments, an Fc region includes one or more modifications that reduce or abrogate binding of the Fc to Fc receptors. Such modifications may include amino acid substitutions at positions 234, 235, 265 and 297 (see e.g., U.S. Patent No 5,624,821, which is incorporated by reference herein). Example substitutions include one or more ofL234A, L235A, D265A and N297Q. Amino acid positions correspond to the amino acid numbering of the CH2-CH3 domain provided above.
In some embodiments, an Fc region includes one or more modifications that alter (relative to a wildtype Fc region) the Ratio of Affinities of the modified Fc region to an activating FcyR (such as FcyRIIA or FcyRIIIA) relative to an inhibiting FcyR (such as FcyRIIB):
Figure imgf000042_0001
Where a modified Fc region has a Ratio of Affinities greater than 1, an anti-NKG2A agent herein may have particular use in providing a therapeutic or prophylactic treatment of a disease, disorder, or infection, or the amelioration of a symptom thereof, where an enhanced efficacy of effector cell function (e.g., ADCC) mediated by FcyR is desired, e.g., cancer or infectious disease. Where a modified Fc region has a Ratio of Affinities less than 1, an anti-NKG2A agent herein may have particular use in providing a therapeutic or prophylactic treatment of a disease or disorder, or the amelioration of a symptom thereof, where a decreased efficacy of effector cell function mediated by FcyR is desired, e.g., autoimmune or inflammatory disorders. Table 3 lists example single, double, triple, quadruple and quintuple amino acid substitutions having a Ratio of Affinities greater than 1 or less than 1 (see e.g., PCT Publication Nos. WO 04/063351; WO 06/088494; WO 07/024249; WO 06/113665; WO 07/021841; WO 07/106707; WO 2008/140603, each of which is incorporated by reference herein). Amino acid positions correspond to the amino acid numbering of the CH2-CH3 domain provided above.
Figure imgf000042_0002
Figure imgf000043_0001
Figure imgf000044_0001
Agents that competitively bind with an anti-NKG2A agent
Provided herein are anti-NKG2A agents that competitively bind, or are capable of competitively binding, with one or more anti-NKG2A agents described herein. In particular, provided herein are anti-NKG2A agents that compete, or are capable of competing, with one or more anti-NKG2A agents described herein for binding to NKG2A. Such agents that compete, or are capable of competing, with anti-NKG2A agents described herein may be referred to as competitor agents. In certain instances, an agent (i.e., competitor agent) may be considered to compete for binding to NKG2A when the competitor binds to the same general region of NKG2A as an anti-NKG2A agent described herein (i.e., extracellular region or leucine-rich binding domain). In certain instances, an agent (i.e., competitor agent) may be considered to compete for binding to NKG2A when the competitor binds to the exact same region of NKG2A as an anti-NKG2A agent described herein (e.g., exact same peptide (linear epitope) or exact same surface amino acids (conformational epitope)). In certain instances, an agent (i.e., competitor agent) may be considered capable of competing for binding to NKG2A when the competitor binds to the same general region of NKG2A as an anti-NKG2A agent described herein (i.e., extracellular region or leucine-rich binding domain) under suitable assay conditions. In certain instances, an agent (i.e., competitor agent) may be considered capable of competing for binding to NKG2A when the competitor binds to the exact same region of NKG2A as an anti-NKG2A agent described herein (e.g., exact same peptide (linear epitope) or exact same surface amino acids (conformational epitope)) under suitable assay conditions.
In certain instances, an agent (i.e., competitor agent) may be considered to compete for binding to NKG2A when the competitor blocks the binding of one or more anti-NKG2A agents described herein to NKG2A. In certain instances, an agent (i.e., competitor agent) may be considered capable of competing for binding to NKG2A when the competitor blocks the binding of one or more anti- NKG2A agents described herein to NKG2A under suitable assay conditions. Whether a competitor blocks the binding of one or more anti-NKG2A agents described herein to NKG2A may be determined using a suitable competition assay or blocking assay, such as, for example, a blocking assay as described in Example 5 herein. A competitor agent may block binding of one or more anti- NKG2A agents described herein to NKG2A in a competition or blocking assay by 50% or more, and conversely, one or more anti-NKG2A agents described herein may block binding of the competitor agent to NKG2A in a competition or blocking assay by about 50% or more. For example, an agent (i.e., competitor agent) may block binding of one or more anti-NKG2A agents described herein to NKG2A in a competition or blocking assay by about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, and conversely, one or more anti-NKG2A agents described herein may block binding of the competitor agent to NKG2A in a competition or blocking assay by about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.
In certain instances, an agent (i.e., competitor agent) may be considered to compete for binding to NKG2A when the competitor binds to NKG2A with a similar affinity as one or more anti-NKG2A agents described herein. In certain instances, an agent (i.e., competitor agent) may be considered capable of competing for binding to NKG2A when the competitor binds to NKG2A with a similar affinity as one or more anti-NKG2A agents described herein under suitable assay conditions. In some embodiments, an agent (i.e., competitor agent) is considered to compete for binding to NKG2A when the competitor binds to NKG2A with an affinity that is at least about 50% of the affinity of one or more anti-NKG2A agents described herein. For example, an agent (i.e., competitor agent) may be considered to compete for binding to NKG2A when the competitor binds to NKG2A with an affinity that is at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the affinity of one or more anti-NKG2A agents described herein. A competitor agent may comprise any feature described herein for anti-NKG2A agents.
Also provided herein are anti-NKG2A agents that bind to, or are capable of binding to, the same epitope as one or more anti-NKG2A agents described herein. In particular, provided herein are anti- NKG2A agents that compete with one or more anti-NKG2A agents described herein for binding to the same epitope on NKG2A. Such agents that bind the same epitope may be referred to as epitope competitors. In certain instances, an epitope competitor may bind to the exact same region of NKG2A as an anti-NKG2A agent described herein (e.g., exact same peptide (linear epitope) or exact same surface amino acids (conformational epitope)). In certain instances, an epitope competitor blocks the binding of one or more anti-NKG2A agents described herein to NKG2A. An epitope competitor may block binding of one or more anti-NKG2A agents described herein to NKG2A in a competition assay by about 50% or more, and conversely, one or more anti-NKG2A agents described herein may block binding of the epitope competitor to NKG2A in a competition assay by 50% or more. In certain instances, an epitope competitor binds to NKG2A with a similar affinity as one or more anti-NKG2A agents described herein. In some embodiments, an epitope competitor binds to NKG2A with an affinity that is at least about 50% of the affinity of one or more anti-NKG2A agents described herein. For example, an epitope competitor may bind to NKG2A with an affinity that is at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the affinity of one or more anti-NKG2A agents described herein. An epitope competitor may comprise any feature described herein for anti-NKG2A agents.
Antibody Preparation
In some embodiments, an anti-NKG2A agent is an antibody. In some embodiments, any of the anti- NKG2A agents provided herein is an antibody or antigen-binding fragment thereof. Methods for generating anti-NKG2A antibodies, antigen-binding fragments thereof and variants of anti-NKG2A antibodies are described in the Examples below. In some embodiments, an anti-NKG2A agent is a humanized antibody, or an antigen binding fragment thereof. In some embodiments, an anti-NKG2A agent is a humanized antibody, or a derivative thereof that binds NKG2A. Humanized anti-NKG2A antibodies may be prepared based on a nonhuman anti-NKG2A antibody. Fully human antibodies may also be prepared, e.g., in a genetically engineered (i.e., transgenic) mouse (e.g., from Medarex) that, when presented with an immunogen, can produce a human antibody that does not necessarily require CDR grafting. These antibodies are fully human (100% human protein sequences) from animals such as mice in which the non-human antibody genes are suppressed and replaced with human antibody gene expression. Antibodies may be generated against NKG2A when presented to these genetically engineered mice or other animals that can to produce human frameworks for the relevant CDRs.
Where a variant is generated, the parent antibody is prepared. Example techniques for generating such nonhuman antibody and parent antibodies are described in the following sections.
Antigen Preparation
The antigen for production of antibodies may be, e.g., intact NKG2A, particularly expressed in cells, or a portion of NKG2A (e.g. N-terminal domain, C-terminal domain, a cytoplasmic domain, an intra- organelle domain, a transmembrane domain, an extracellular domain, an ectodomain, a TIR domain, a leucine-rich domain, or a NKG2A fragment comprising a desired epitope). Other forms of antigens useful for generating antibodies will be apparent to those skilled in the art.
Polyclonal Antibodies
Polyclonal antibodies may be raised in animals (vertebrate or invertebrates, including mammals, birds and fish, including cartilaginous fish) by multiple subcutaneous (sc) or intraperitoneal (ip) injections of a relevant antigen and an adjuvant. It may be useful to conjugate the relevant antigen to a protein or other carrier that is immunogenic in the species to be immunized, e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor using a bifimctional or derivatizing agent, for example, maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine residues), N-hydroxy succinimide (through lysine residues), glutaraldehyde, succinic anhydride, SOCI2, or R1N=C=NR, where R and R1 are different alkyl groups. Non-protein carriers (e.g., colloidal gold) also may be used for antibody production.
In some embodiments, animals are immunized against the antigen, immunogenic conjugates, or derivatives by combining, e.g., 100 pg or 5 pg of the protein or conjugate (for rabbits or mice, respectively) with three volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites. One month later the animals are boosted with one-fifth to one-tenth of the original amount of peptide or conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites. Seven to 14 days later the animals are bled and the serum is assayed for antibody titer. Animals are boosted until the titer plateaus. Often, the animal is boosted with the conjugate of the same antigen, but conjugated to a different protein and/or through a different cross-linking reagent. Conjugates also can be made in recombinant cell culture as protein fusions. Also, aggregating agents such as alum are suitably used to enhance the immune response.
Monoclonal Antibodies
Monoclonal antibodies may be made using the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or may be made by other methods such as recombinant DNA methods (U.S. Pat. No. 4,816,567). In the hybridoma method, a mouse or other appropriate host animal, such as a hamster or macaque monkey, is immunized as hereinabove described to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization. Alternatively, lymphocytes may be immunized in vitro. Lymphocytes then are fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103 (Academic Press, 1986)). In some embodiments, the hybridoma cells are seeded and grown in a suitable culture medium that may contain one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.
Preferred myeloma cells are those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. Among these, preferred myeloma cell lines are murine myeloma lines, such as SP-2 or X63- Ag8-653 cells available from the American Type Culture Collection, Rockville, Md. USA. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987).
Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen. The binding specificity of monoclonal antibodies produced by hybridoma cells may be determined by immunoprecipitation, by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbant assay (ELISA), or by flow cytometric analysis of cells expressing the membrane antigen. The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson et al., Anal. Biochem., 107:220 (1980).
After hybridoma cells are identified that produce antibodies of the desired specificity, affinity, and/or activity, the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103 (Academic Press, 1986)). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, the hybridoma cells may be grown in vivo as ascites tumors in an animal. The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A- Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
DNA encoding the monoclonal antibodies is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the monoclonal antibodies). Alternatively, cDNA may be prepared from mRNA and the cDNA then subjected to DNA sequencing. The hybridoma cells serve as a preferred source of such genomic DNA or RNA for cDNA preparation. Once isolated, the DNA may be placed into expression vectors, which are well known in the art, and which are then transfected into host cells such as E coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. Recombinant production of antibodies will be described in more detail below.
Humanization and Amino Acid Sequence Variants
General methods for humanization of antibodies are described, for example, in U.S. Patent Nos. 5861155, 6479284, 6407213, 6639055, 6500931, 5530101, 5585089, 5693761, 5693762, 6180370, 5714350, 6350861, 5777085, 5834597, 5882644, 5932448, 6013256, 6129914, 6210671, 6329511, 5225539, 6548640, and 5624821, each of which is incorporated by reference herein. In certain embodiments, it may be desirable to generate amino acid sequence variants of these humanized antibodies, particularly where these improve the binding affinity or other biological properties of the antibody.
Amino acid sequence variants of the anti-NKG2A antibody are prepared by introducing appropriate nucleotide changes into the anti-NKG2A antibody DNA, or by peptide synthesis. Such variants include, for example, deletions from, and/or insertions into and/or substitutions of, residues within the amino acid sequences of the anti-NKG2A antibodies of the examples herein. Any combination of deletion, insertion, and substitution is made to arrive at the final construct, provided that the final construct possesses the desired characteristics. The amino acid changes also may alter post- translational processes of the humanized or variant anti-NKG2A antibody, such as changing the number or position of glycosylation sites.
One method for identification of certain residues or regions of the anti-NKG2A antibody that are preferred locations for mutagenesis is called “alanine scanning mutagenesis,” as described by Cunningham and Wells Science, 244: 1081-1085 (1989). Here, a residue or group of target residues are identified (e.g., charged residues such as arg, asp, his, lys, and glu) and replaced by a neutral or negatively charged amino acid (most preferably alanine or polyalanine) to affect the interaction of the amino acids with NKG2A antigen. Those amino acid locations demonstrating functional sensitivity to the substitutions then are refined by introducing further or other variants at, or for, the sites of substitution. Thus, while the site for introducing an amino acid sequence variation is predetermined, the nature of the mutation per se need not be predetermined. For example, to analyze the performance of a mutation at a given site, alanine scanning or random mutagenesis is conducted at the target codon or region and the expressed anti-NKG2A antibody variants are screened for the desired activity. Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intra-sequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an N- terminal methionyl residue or the antibody fused to an epitope tag. Other insertional variants include the fusion of an enzyme or a polypeptide that increases the serum half-life of the antibody to the N- or C-terminus of the antibody.
Another type of variant is an amino acid substitution variant. These variants have at least one amino acid residue removed from the antibody molecule and a different residue inserted in its place. The sites of greatest interest for substitutional mutagenesis include the hypervariable regions, but FR alterations are also contemplated. Conservative substitutions are preferred, but more substantial changes may be introduced and the products may be screened. Examples of substitutions are listed below:
Example Amino Acid Residue Substitutions
Ala (A) val; leu; ile; val
Arg (R) lys; gin; asn; lys
Asn (N) gin; his; asp, lys; gin; arg
Asp (D) glu; asn
Cys (C) ser; ala Gin (Q) asn; glu Glu (E) asp; gin Gly (G) ala His (H) asn; gin; lys; arg lie (I) leu; val; met; ala; leu; phe; norleucine Leu (L) norleucine; ile; val; ile; met; ala; phe Lys (K) arg; gin; asn Met (M) leu; phe; ile
Phe (F) leu; val; ile; ala; tyr
Pro (P) ala
Ser (S) thr
Thr (T) ser
Trp (W) tyr; phe
Tyr (Y) trp; phe; thr; ser Vai (V) ile; leu; met; phe; ala; norleucine
Substantial modifications in the biological properties of an antibody are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Naturally occurring residues are divided into groups based on common side-chain properties:
(1) hydrophobic: norleucine, met, ala, val, leu, ile;
(2) neutral hydrophilic: cys, ser, thr;
(3) acidic: asp, glu;
(4) basic: asn, gin, his, lys, arg;
(5) residues that influence chain orientation: gly, pro; and
(6) aromatic: trp, tyr, phe.
Non-conservative substitutions will entail exchanging a member of one of these classes for another class.
Any cysteine residue not involved in maintaining the proper conformation of the antibody also may be substituted, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine bond(s) may be added to the antibody to improve its stability (particularly where the antibody is an antibody fragment such as an Fv fragment).
One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g. a humanized or human antibody). Generally, the resulting variant(s) selected for further development will have improved biological properties relative to the parent antibody from which they are generated. A convenient way for generating such substitutional variants is affinity maturation using phage display. Briefly, several hypervariable region sites (e.g. 6-7 sites) are mutated to generate all possible amino substitutions at each site. The antibody variants thus generated are displayed in a monovalent fashion from filamentous phage particles as fusions to the gene III product of M13 packaged within each particle. The phage-displayed variants are then screened for their biological activity (e.g. binding affinity) as herein disclosed. In order to identify candidate hypervariable region sites for modification, alanine-scanning mutagenesis can be performed to identify hypervariable region residues contributing significantly to antigen binding. Alternatively, or in addition, it may be beneficial to analyze a crystal structure of the antigen-antibody complex to identify contact points between the antibody and antigen. Such contact residues and neighboring residues are candidates for substitution according to the techniques elaborated herein. Once such variants are generated, the panel of variants is subjected to screening as described herein and antibodies with superior properties in one or more relevant assays may be selected for further development.
Another type of amino acid variant of the antibody alters the original glycosylation pattern of the antibody. By altering is meant deleting one or more carbohydrate moieties found in the antibody, and/or adding one or more glycosylation sites that are not present in the antibody. Glycosylation of antibodies is typically either N-linked and/or or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine- X-serine and asparagine-X-threonine, where X is any amino acid except proline, are the most common recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5 -hydroxyproline or 5 -hydroxy lysine may also be used.
Addition of glycosylation sites to the antibody is conveniently accomplished by altering the amino acid sequence such that it contains one or more of the above-described tripeptide sequences (for N- linked glycosylation sites). The alteration may also be made by the addition of, or substitution by, one or more serine or threonine residues to the sequence of the original antibody (for O-linked glycosylation sites).
Nucleic acid molecules encoding amino acid sequence variants of anti-NKG2A antibodies herein are prepared by a variety of methods known in the art. These methods include, but are not limited to, isolation from a natural source (in the case of naturally occurring amino acid sequence variants) or preparation by oligonucleotide-mediated (or site -directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared variant or a non-variant version of an anti-NKG2A antibody.
Human Antibodies
As an alternative to humanization, human antibodies can be generated. For example, transgenic animals (e.g., mice) may be generated that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, a homozygous deletion of the antibody heavy-chain joining region (JH) gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human germ-line immunoglobulin gene array into such germ-line mutant mice can result in the production of human antibodies upon antigen challenge (see, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258(1993); Bruggermann et al., Year in Immuno., 7:33 (1993); and U.S. Pat. Nos. 5,591,669, 5,589,369 and 5,545,807). Human antibodies also can be derived from phage-display libraries (Hoogenboom et al., J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581-597 (1991); and U.S. Pat. Nos. 5,565,332 and 5,573,905). Human antibodies also may be generated by in vitro activated B cells (see U.S. Pat. Nos. 5,567,610 and 5,229,275).
Antigen-Binding Antibody Fragments
In certain embodiments, an anti-NKG2A agent is an antibody fragment or antigen-binding fragment that retains at least one desired activity, including antigen binding. Various techniques have been developed for the production of antibody fragments or antigen-binding fragments. In some instances, these fragments are derived via proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24: 107-117(1992) and Brennan et al., Science 229:81 (1985)). In some instances, these fragments are produced directly by recombinant host cells. For example, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab')2 fragments (Carter et al., Bio/Technology 10: 163-167 (1992)). In some instances, the F(ab')2 is formed using the leucine zipper GCN4 to promote assembly of the F(ab')2 molecule. According to another approach, Fv, Fab or F(ab')2 fragments can be isolated directly from recombinant host cell culture. Other techniques for the production of antibody fragments or antigenbinding fragments will be apparent to the skilled practitioner.
Multispecific Antibodies and other Agents
In some embodiments, an anti-NKG2A agent comprises a first binding moiety and a second binding moiety, where the first binding moiety is specifically reactive with a first molecule that is NKG2A and the second binding moiety is specifically reactive with a second molecule that is a molecular species different from the first molecule. Such agents may comprise a plurality of first binding moieties, a plurality of second binding moieties, or a plurality of first binding moieties and a plurality of second binding moieties. Preferably, the ratio of first binding moieties to second binding moieties is about 1: 1, although it may range from about 1000: 1 to about 1 : 1000, where the ratio may be measured in terms of valency.
In those embodiments where the first moiety is an antibody, the second binding moiety may also be an antibody. In some embodiments, the first and second moieties are linked via a linker moiety, which may have two to many 100’s or even thousands of valencies for attachment of first and second binding moieties by one or different chemistries. Examples of bispecific antibodies include those that are reactive against two different epitopes; in some instances, one epitope is a NKG2A epitope and the second epitope is on an unrelated soluble molecule. In some embodiments, the bispecific antibody is reactive against an epitope on NKG2A and against an epitope on a different molecule found on the surface of the same cell. In some embodiments, the bispecific antibody is reactive against an epitope on NKG2A and against an epitope on a different molecule found on the surface of a different cell.
Compositions herein may also comprise a first agent and a second agent, where the first agent comprises a first binding moiety specifically reactive with a first molecule (e.g., NKG2A) and the second agent comprises a second binding moiety specifically reactive with a second molecule that is a molecular species different than the first molecule. The first and/or second agent may be an antibody. The ratio of first agent to second agent may range from about 1,000: 1 to 1: 1,000, although the preferred ratio is about 1: 1. In some embodiments, it may be desirable to generate multispecific (e.g. bispecific) anti-NKG2A antibodies having binding specificities for at least two different epitopes. Certain bispecific antibodies may bind to two different epitopes of NKG2A. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., F(ab')2 bispecific antibodies).
According to one for making bispecific antibodies, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers that are recovered from recombinant cell culture. The preferred interface comprises at least a part of the CH3 domain of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). Compensatory “cavities” of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers (see e.g., WO96/27011 published Sep. 6, 1996).
Bispecific antibodies include cross-linked or “heteroconjugate” antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin, the other to biotin. Heteroconjugate antibodies may be made using any convenient cross-linking methods. Suitable cross-linking agents are well known in the art, and are disclosed in U.S. Pat. No. 4,676,980, along with a number of crosslinking techniques.
Any suitable technique may be used for generating bispecific antibodies from antibody fragments. For example, bispecific antibodies can be prepared using chemical linkage. In certain methods, intact antibodies are proteolytically cleaved to generate F(ab')2 fragments (see e.g., Brennan et al., Science 229:81 (1985), which is incorporated by reference herein). These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab' fragments generated are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody. In yet a further embodiment, Fab'-SH fragments directly recovered from E. coli can be chemically coupled in vitro to form bispecific antibodies (see e.g., Shalaby et al., J. Exp. Med. 175:217-225 (1992), which is incorporated by reference herein).
Any suitable technique for making and isolating bispecific antibody fragments directly from recombinant cell culture may be used. For example, bispecific antibodies have been produced using leucine zippers (see e.g., Kostelny et al., J. Immunol. 148(5): 1547-1553 (1992), which is incorporated by reference herein). The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers. The “diabody” technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a heavychain variable domain (VH) connected to a light-chain variable domain (VL) by a linker that is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites. Another strategy for making bispecific antibody fragments by the use of single-chain Fv (scFv) dimers (see e.g., Gruber et al., J. Immunol. 152:5368 (1994), which is incorporated by reference herein). In certain instances, a bispecific antibody may be a “linear antibody” produced as described in Zapata et al. Protein Eng. 8(10): 1057-1062 (1995), which is incorporated by reference herein.
Antibodies with two valencies or more are contemplated herein. An antibody (or polymer or polypeptide) herein comprising one or more binding sites per arm or fragment thereof will be referred to herein as “multivalent” antibody. For example, a “bivalent” antibody herein comprises two binding sites per Fab or fragment thereof whereas a “trivalent” polypeptide herein comprises three binding sites per Fab or fragment thereof. In a multivalent polymer herein, the two or more binding sites per Fab may be binding to the same or different antigens. For example, the two or more binding sites in a multivalent polypeptide herein may be directed against the same antigen, for example against the same parts or epitopes of said antigen or against two or more same or different parts or epitopes of said antigen; and/or may be directed against different antigens; or a combination thereof. Thus, a bivalent polypeptide herein, for example, may comprise two identical binding sites, may comprise a first binding sites directed against a first part or epitope of an antigen and a second binding site directed against the same part or epitope of said antigen or against another part or epitope of said antigen; or may comprise a first binding sites directed against a first part or epitope of an antigen and a second binding site directed against a different antigen. However, as will be clear from the description hereinabove, the technology herein is not limited thereto, in the sense that a multivalent polypeptide herein may comprise any number of binding sites directed against the same or different antigens. In one embodiment the multivalent polypeptide comprises at least two ligand binding elements, one of which contains one or more CDR peptide sequences shown herein. In another embodiment the multivalent polypeptide comprises three ligand binding sites, each independently selected from the CDR sequences disclosed herein.
In certain embodiments, at least one of the ligand binding elements binds NKG2A. In one embodiment, at least one of the ligand binding elements binds another target. In one embodiment, there are up to 10,000 binding elements in a multivalent binding molecule, and the ligand binding elements may be linked to a scaffold.
An antibody (or polymer or polypeptide) herein that contains at least two binding sites per Fab or fragment thereof, in which at least one binding site is directed against a first antigen and a second binding site directed against a second antigen different from the first antigen, may also be referred to as “multispecific.” Thus, a “bispecific” polymer comprises at least one site directed against a first antigen and at least one second site directed against a second antigen, whereas a “trispecific” is a polymer that comprises at least one binding site directed against a first antigen, at least one further binding site directed against a second antigen, and at least one further binding site directed against a third antigen; and the like. Accordingly, in their simplest form, a bispecific polypeptide herein is a bivalent polypeptide (per Fab) of the technology provided herein. However, as will be clear from the description hereinabove, the technology herein is not limited thereto, in the sense that a multispecific polypeptide herein may comprise any number of binding sites directed against two or more different antigens.
Other Modifications
Other modifications of an anti-NKG2A agent are contemplated. For example, technology herein also pertains to immunoconjugates comprising an antibody described herein (e.g., an anti-NKG2A antibody) conjugated to a cytotoxic agent such as a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant or animal origin, or fragments thereof), or a radioactive isotope (for example, a radioconjugate), or a cytotoxic drug. Such conjugates are sometimes referred to as “agent-drug conjugates” or “ADC”. Conjugates are made using a variety of bifiinctional protein coupling agents such as N-succinimidyl-3-(2 -pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p- azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)- ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as l,5-difluoro-2,4-dinitrobenzene).
In some embodiments, any of the anti-NKG2A agents (e.g., anti-NKG2A antibodies) disclosed herein may be formulated as immunoliposomes. Liposomes containing an antibody are prepared by methods known in the art, such as described in Epstein et al., Proc. Natl. Acad. Sci. USA 82:3688 (1985); Hwang et al., Proc. Natl Acad. Sci. USA 77:4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545. Eiposomes with enhanced circulation time are disclosed in U.S. Pat. No. 5,013,556. For example, liposomes can be generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through fdters of defined pore size to yield liposomes with the desired diameter. Fab' fragments of an antibody provided herein can be conjugated to the liposomes as described in Martin et al., J. Biol. Chem. 257:286-288 (1982) via a disulfide interchange reaction. Another active ingredient is optionally contained within the liposome.
Enzymes or other polypeptides can be covalently bound to an anti-NKG2A agent (e.g., anti-NKG2A antibody) by techniques well known in the art such as the use of the heterobifunctional cross-linking reagents discussed above. In some embodiments, fusion proteins comprising at least the antigen binding region of an antibody provided herein linked to at least a functionally active portion of an enzyme can be constructed using recombinant DNA techniques well known in the art (see, e.g., Neuberger et al., Nature 312:604-608 (1984)).
In certain embodiments, it may be desirable to use an antibody fragment, rather than an intact antibody, to increase penetration of target tissues and cells, for example. In such instances, it may be desirable to modify the antibody fragment in order to increase its serum half-life. This may be achieved, for example, by incorporation of a salvage receptor binding epitope into the antibody fragment (e.g., by mutation of the appropriate region in the antibody fragment or by incorporating the epitope into a peptide tag that is then fused to the antibody fragment at either end or in the middle, e.g., by DNA or peptide synthesis; see, e.g., WO96/32478 published Oct. 17, 1996).
Covalent modifications of an anti-NKG2A agent (e.g., anti-NKG2A antibody) are also included within the scope of this technology. For example, modifications may be made by chemical synthesis or by enzymatic or chemical cleavage of an anti-NKG2A antibody. Other types of covalent modifications of an antibody are introduced into the molecule by reacting targeted amino acid residues of the antibody with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C-terminal residues. Example covalent modifications of polypeptides are described in U.S. Pat. No. 5,534,615, specifically incorporated herein by reference. A preferred type of covalent modification of the antibody comprises linking the antibody to one of a variety of non- proteinaceous polymers, e.g., polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, in the manner set forth in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.
Nucleic acids, vectors, host cells, and recombinant methods
Provided herein are isolated nucleic acids encodingany of the anti-NKG2A agents (e.g., anti-NKG2A antibody) described herein, and vectors and host cells comprising any of the nucleic acids provided herein, and recombinant techniques for the production of the agent or antibody. A nucleic acid herein may include one or more subsequences, each referred to as a polynucleotide.
Provided herein are nucleic acids (e.g., isolated nucleic acids) comprising a nucleotide sequence that encodes an anti-NKG2A agent or antibody, or fragment thereof. In some embodiments, a nucleic acid encodes an immunoglobulin heavy chain variable domain of an anti-NKG2A agent provided herein. In some embodiments, the immunoglobulin heavy chain variable domain is encoded by the sequence of nucleotides set forth in any of SEQ ID NOs: 30-37, or sequence of nucleotides having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of SEQ ID NOs: 30-37. In some embodiments, the immunoglobulin heavy chain variable domain is encoded by the sequence of nucleotides set forth in any of SEQ ID NOs: 30-37. In some embodiments, the immunoglobulin heavy chain variable domain contains a signal sequence and is encoded by the sequence of nucleotides set forth in any of SEQ ID NOs:30-33. In some embodiments, the immunoglobulin heavy chain variable domain is encoded by the sequence of nucleotides set forth in SEQ ID NO: 30. In some embodiments, the immunoglobulin heavy chain variable domain is encoded by the sequence of nucleotides set forth in SEQ ID NO: 31. In some embodiments, the immunoglobulin heavy chain variable domain is encoded by the sequence of nucleotides set forth in SEQ ID NO: 32. In some embodiments, the immunoglobulin heavy chain variable domain is encoded by the sequence of nucleotides set forth in SEQ ID NO: 33. In some embodiments, the heavy chain signal sequence is encoded by the sequence of nucleotides set forth in SEQ ID NO: 48 or SEQ ID NO: 52.
In some embodiments, the immunoglobulin heavy chain variable domain does not contain a signal sequence, and is encoded by the sequence of nucleotides set forth in any of SEQ ID NOs:34-37, or sequence of nucleotides having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of SEQ ID NOs: 34-37. In some embodiments, the immunoglobulin heavy chain variable domain does not contain a signal sequence, and is encoded by the sequence of nucleotides set forth in any of SEQ ID NOs:34-37. In some embodiments, the immunoglobulin heavy chain variable domain is encoded by the sequence of nucleotides set forth in SEQ ID NO: 34. In some embodiments, the immunoglobulin heavy chain variable domain is encoded by the sequence of nucleotides set forth in SEQ ID NO: 35. In some embodiments, the immunoglobulin heavy chain variable domain is encoded by the sequence of nucleotides set forth in SEQ ID NO: 36. In some embodiments, the immunoglobulin heavy chain variable domain is encoded by the sequence of nucleotides set forth in SEQ ID NO: 37.
In some embodiments, a nucleic acid encodes an immunoglobulin light chain variable domain of an anti-NKG2A agent provided herein. In some embodiments, the immunoglobulin light chain variable domain is encoded by the sequence of nucleotides set forth in any of SEQ ID NOs:38-45, or sequence of nucleotides having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of SEQ ID NOs: 38- 45. In some embodiments, the immunoglobulin light chain variable domain is encoded by the sequence of nucleotides set forth in any of SEQ ID NOs:38-45. In some embodiments, the immunoglobulin light chain variable domain contains a signal sequence and is encoded by the sequence of nucleotides set forth in any of SEQ ID NOs:38-41. In some embodiments, the immunoglobulin light chain variable domain is encoded by the sequence of nucleotides set forth in SEQ ID NO: 38. In some embodiments, the immunoglobulin light chain variable domain is encoded by the sequence of nucleotides set forth in SEQ ID NO: 39. In some embodiments, the immunoglobulin light chain variable domain is encoded by the sequence of nucleotides set forth in SEQ ID NO: 40. In some embodiments, the immunoglobulin light chain variable domain is encoded by the sequence of nucleotides set forth in SEQ ID NO: 41. In some embodiments, the light chain signal sequence is encoded by the sequence of nucleotides set forth in SEQ ID NO: 46 or SEQ ID NO: 50.
In some embodiments, the immunoglobulin light chain variable domain does not contain a signal sequence, and is encoded by the sequence of nucleotides set forth in any of SEQ ID NOs:42-45, or sequence of nucleotides having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of SEQ ID NOs:42-45. In some embodiments, the immunoglobulin light chain variable domain does not contain a signal sequence, and is encoded by the sequence of nucleotides set forth in any of SEQ ID NOs:42- 45. In some embodiments, the immunoglobulin light chain variable domain is encoded by the sequence of nucleotides set forth in SEQ ID NO: 42. In some embodiments, the immunoglobulin light chain variable domain is encoded by the sequence of nucleotides set forth in SEQ ID NO: 43. In some embodiments, the immunoglobulin light chain variable domain is encoded by the sequence of nucleotides set forth in SEQ ID NO: 44. In some embodiments, the immunoglobulin light chain variable domain is encoded by the sequence of nucleotides set forth in SEQ ID NO: 45.
In some embodiments, a nucleic acid encodes an immunoglobulin heavy chain variable domain and an immunoglobulin light chain variable domain of an anti-NKG2A agent provided herein.
In some embodiments, a nucleic acid comprises a nucleotide sequence that encodes an amino acid sequence of any one of SEQ ID NOS: 1, 3-5, or 8-21. For example, a nucleic acid may comprise a nucleotide sequence that encodes a CDR amino acid sequence of any one of SEQ ID NOS: 9-21. A nucleic acid may comprise a nucleotide sequence that encodes an immunoglobulin heavy chain variable domain amino acid sequence of any one of SEQ ID NOS: 1, 3 or 4. A nucleic acid may comprise a nucleotide sequence that encodes an immunoglobulin light chain variable domain amino acid sequence of any one of SEQ ID NOS: 5 or 8.
For recombinant production of an anti-NKG2A agent or antibody, a nucleic acid encoding the anti- NKG2A agent or antibody may be isolated and inserted into a replicable vector for further cloning (amplification of the DNA) or for expression. In certain instances, an anti-NKG2A agent or antibody may be produced by homologous recombination, e.g. as described in U.S. Pat. No. 5,204,244, specifically incorporated herein by reference. DNA encoding an anti-NKG2A agent or antibody can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody). Many vectors are available. The vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence, e.g., as described in U.S. Pat. No. 5,534,615 issued Jul. 9, 1996 and specifically incorporated herein by reference.
Suitable host cells for cloning or expressing DNA in vectors herein are prokaryote, yeast, or higher eukaryote cells. Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli such as B. subtilis and B. licheniformis (e.g., B. licheniformis 41P disclosed in DD 266,710 published 12 Apr. 1989), Pseudomonas such as P. aeruginosa, and Streptomyces. One preferred E. coli cloning host is E. coli 294 (ATCC 31,446), although other strains such as E. coli B, E. coli X1 776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are suitable. These examples are illustrative rather than limiting.
In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for anti-NKG2A agent/antibody-encoding vectors. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms. A number of other genera, species, and strains are commonly available and useful herein, such as Schizosaccharomyces pombe; Kluyveromyces hosts such as, e.g., K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans, and K. marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces such as Schwanniomyces occidentalis; and filamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as A. nidulans and A. niger.
Suitable host cells for the expression of anti-NKG2A agents/antibodies (e.g., glycosylated anti- NKG2A agents/antibodies) are derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains and variants and corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruitfly), and Bombyx mori (silk moth) have been identified. A variety of viral strains for transfection are publicly available, e.g., the L-l variant of Autographa califomica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be used as the virus herein according to the present technology, particularly for transfection of Spodoptera frugiperda cells. Plant cell cultures of cotton, com, potato, soybean, petunia, tomato, and tobacco can also be utilized as hosts.
Suitable host cells for the expression of anti-NKG2A agents/antibodies also may include vertebrate cells (e.g., mammalian cells). Vertebrate cells may be propagated in culture (tissue culture).
Examples of useful mammalian host cell lines include monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL- 1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).
Host cells may be transformed with the above-described expression or cloning vectors for antibody production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. Host cells used to produce an agent/antibody herein may be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing the host cells. In addition, any of the media described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem.102:255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or U.S. Pat. Re. 30,985 may be used as culture media for the host cells. Any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMY CIN™), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art. The culture conditions, such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.
When using recombinant techniques, an agent/antibody can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the antibody is produced intracellularly, as a first step, the particulate debris, either host cells or lysed fragments, is removed, for example, by centrifugation or ultrafiltration. Carter et al., Bio/Technology 10: 163-167 (1992) describe a procedure for isolating antibodies that are secreted to the periplasmic space of E. coli. Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min. Cell debris can be removed by centrifugation. Where the antibody is secreted into the medium, supernatants from such expression systems are generally first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. A protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious contaminants.
The agent/antibody composition prepared from the cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being the preferred purification technique. The suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain that is present in the antibody. Protein A can be used to purify antibodies that are based on human heavy chains (Lindmark et al., J. Immunol. Meth. 62: 1-13 (1983)). Protein G is recommended for all mouse isotypes and for human y3 (Guss et al., EMBO J. 5: 15671575 (1986)). The matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. Where the antibody comprises a CJB domain, Bakerbond ABX.TM. resin (J. T. Baker, Phillipsburg, N.J.) is useful for purification. Other techniques for protein purification, such as fractionation on an ion-exchange column, ethanol precipitation, Reverse Phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE™, chromatography on an anion or cation exchange resin (such as a polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are also available depending on the antibody to be recovered.
Following any preliminary purification step(s), the mixture comprising the agent or antibody of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5-4.5, and may be performed at low salt concentrations (e.g., from about 0-0.25M salt).
Pharmaceutical formulations, dosing, and routes of administration
The present technology provides anti-NKG2A agents and antibodies, fragments or antigen-binding fragments thereof and related compositions, which may be useful for elimination of NKG2A expressing cells from the body, for example, and for identification and quantification of the number of NKG2A expressing cells in tissue samples, for example.
Therapeutic methods and compositions of the present technology may be referred to as “NKG2A- based” in order to indicate that these therapies can change the relative or absolute numbers of undesirable or toxic NKG2A expressing cells such as lymphomas or autoimmune B lymphocytes.
In some embodiments, one way to control the amount of undesirable NKG2A expressing cells in a patient is by providing a composition that comprises one or more anti-NKG2A antibodies, fragments or antigen-binding fragments thereof to cause cytotoxic activity towards the NKG2A-expressing cells, for example.
In some embodiments, any of the anti-NKG2A agents or anti-NKG2A antibodies described herein are capable of binding a cell. In some embodiments, the cell is a natural killer (NK) cell. In some embodiments, the cell is a lymphocyte. In some embodiments, the lymphocyte is a CD8+ lymphocyte. In some embodiments, the cell selected from B cells, plasmacytoid dendritic cells (pDCs), lymphocytes, leukocytes, T cells, monocytes, macrophages, neutrophils, myeloid dendritic cells (mDCs), innate lymphoid cells, mast cells, eosinophils, basophils, natural killer cells, and the like. In some embodiments, the cell is selected from a heterogeneous population of immune cells including peripheral blood mononuclear cells (PBMCs) which may include, for example, T cells, B cells, natural killer cells, and monocytes. In some embodiments, the cell bound by any of the anti-NKG2A agents or anti-NKG2A antibodies interacts with a target cell that expresses HLA-E. In some embodiments, the target cell is a cancer cell. In some embodiments, the target cell is a cancer cell that expresses HLA-E. In some embodiments, the target cell is a cancer cell that over-expresses HLA-E. In some of any embodiments, the HLA-E is expressed on the surface of the target or cancer cell.
In some embodiments, any of the anti-NKG2A agents or anti-NKG2A antibodies described herein specifically bind NKG2A on a cell. In some embodiments, any of the anti-NKG2A agents or anti- NKG2A antibodies described herein specifically bind NKG2A and do not specifically bind NKG2C or NKG2E. In some embodiments, any of the anti-NKG2A agents or anti-NKG2A antibodies described herein block the binding of HLA-E to NKG2A. In some embodiments, the cell is a cell with a disease or disorder. In some embodiments, the disease or disorder is a cancer, a viral infection, an autoimmune disease, an inflammatory disease or a bacterial infection. In some embodiments, the disease or disorder is a cancer including but not limited to acute myeloid leukemia, acute lymphoblastic leukemia, colorectal, ovarian, gynecologic, liver, glioblastoma, Hodgkin lymphoma, chronic lymphocytic leukemia, esophagus, gastric, pancreas, colon, kidney, head and neck, lung and melanoma.
In some embodiments, any of the ani-NKG2A agents provided herein can be used to treat a disease or disorder by blocking the NKG2A receptor on NKG2A expressing cells in a subject. In some embodiments, any of the anti-NKG2A agents provided herein are provided as a composition. In some embodiments, the composition increases the cytotoxic activity of NKG2A-expressing cells. In some embodiments, the composition restores the cytotoxic activity of NKG2A-expressing cells. In some embodiments, the composition causes cytotoxic activity towards cells targeted by NKG2A-expressing cells. In some embodiments, one way to control the amount of undesirable NKG2A expressing cells in a patient is by providing a composition that comprises one or more anti-NKG2A agents, antibodies, fragments or antigen-binding fragments thereof to cause cytotoxic activity towards cells targeted by NKG2A-expressing cells.
In some embodiments, the disease or disorder is associates with aberrant levels of HLA-E. In some embodiments, the disease or disorder is a cancer. In some embodiments, the disease or disorder is a cancer that expresses HLA-E. In some embodiments, the disease or disorder is a cancer, a viral infection, an autoimmune disease, an inflammatory disease or a bacterial infection. In some embodiments, the disease or disorder is a cancer including but not limited to acute myeloid leukemia, acute lymphoblastic leukemia, colorectal, ovarian, gynecologic, liver, glioblastoma, Hodgkin lymphoma, chronic lymphocytic leukemia, esophagus, gastric, pancreas, colon, kidney, head and neck, lung and melanoma. In some embodiments, the disease or disorder is a condition characterized by NK cell hyperactivity or NK cell hyperproliferation.
In some embodiments, when any of the anti-NKG2A agents or anti-NKG2A antibodies described herein bind NKG2A on a cell, the binding of NKG2A to HLA-E to on a target cell is blocked. In some embodiments, the binding of any of the anti-NKG2A agents or anti-NKG2A antibodies described herein to NKG2A on a cell, blocks to ability of the NKG2A on the cell from interacting with HLA-E to on a target cell. In some embodiments, when any of the anti-NKG2A agents or anti- NKG2A antibodies described herein bind NKG2A on a cell, the cell causes the target cell to lyse.
Anti-NKG2A agents/antibodies, antibody fragments or antigen-binding fragments thereof may be formulated in a pharmaceutical composition that is useful for a variety of purposes, including the treatment of diseases, disorders or physical trauma. Pharmaceutical compositions comprising one or more anti-NKG2A agents/antibodies, antibody fragments or antigen-binding fragments thereof herein may be incorporated into kits and medical devices for such treatment. Medical devices may be used to administer pharmaceutical compositions herein to a patient in need thereof, and according to one embodiment of the technology, kits are provided that include such devices. Such devices and kits may be designed for routine administration, including self-administration, of the pharmaceutical compositions herein.
Therapeutic formulations of an agent or antibody may be prepared for storage by mixing the agent/antibody having the desired degree of purity with optional physiologically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m- cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG). Formulations herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Such molecules are suitably present in combination in amounts that are effective for the purpose intended.
Active ingredients may also be entrapped in microcapsule prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatinmicrocapsule and poly-(methylmethacylate) microcapsule, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).
Formulations for in vivo administration generally are sterile. This may be accomplished for instance by filtration through sterile filtration membranes, for example.
Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the agent/antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsule. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinyl alcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and .gamma. ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the Lupron Depot.TM. (injectable microspheres composed of lactic acid- glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3 -hydroxybutyric acid. While polymers such as ethylene -vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods. When encapsulated agents/antibodies remain in the body for a long time, they may denature or aggregate as a result of exposure to moisture at 37° C, resulting in a loss of biological activity and possible changes in immunogenicity. Rational strategies can be devised for stabilization depending on the mechanism involved. For example, if the aggregation mechanism is discovered to be intermolecular S— S bond formation through thio-disulfide interchange, stabilization may be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions.
For therapeutic applications, anti-NKG2A agents, e.g., antibodies, provided herein are administered to a mammal, e.g., a human, in a pharmaceutically acceptable dosage form such as those discussed above, including those that may be administered to a human intravenously as a bolus or by continuous infusion over a period of time, or by intramuscular, intraperitoneal, intra-cerebrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes. For the prevention or treatment of disease, the appropriate dosage of agent/antibody will depend on the type of disease to be treated, as defined above, the severity and course of the disease, whether the antibody is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the discretion of the attending physician. The antibody is suitably administered to the patient at one time or over a series of treatments.
Depending on the type and severity of the disease, about 1 pg/kg to about 50 mg/kg (e.g., 0.1-20 mg/kg) of antibody may be an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. A typical daily or weekly dosage might range from about 1 pg/kg to about 20 mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment is repeated until a desired suppression of disease symptoms occurs. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays, including, for example, radiographic imaging. Detection methods using the antibody to determine NKG2A levels in bodily fluids or tissues may be used in order to optimize patient exposure to the therapeutic antibody.
In some embodiments, a composition comprising an anti-NKG2A agent herein (e.g., an mAb or antigen-binding fragments thereof that interferes with NKG2A activity) is administered as a monotherapy, and in some embodiments, the composition comprising the anti-NKG2A agent is administered as part of a combination therapy. In some cases, the effectiveness of the agent, agent/antibody, antibody or antigen-binding fragment thereof in preventing or treating disease may be improved by administering the agent/antibody serially or in combination with another agent that is effective for those purposes, such as a chemotherapeutic drug for treatment of cancer or a microbial infection. In other cases, the anti-NKG2A agent, antibody or antigen-binding fragment thereof may serve to enhance or sensitize cells to chemotherapeutic treatment, thus permitting efficacy at lower doses and with lower toxicity. Certain combination therapies include, in addition to administration of the composition comprising an agent that reduces the number of NKG2A expressing cells, delivering a second therapeutic regimen selected from the group consisting of administration of a chemotherapeutic agent, radiation therapy, surgery, and a combination of any of the foregoing.
Such other agents may be present in the composition being administered or may be administered separately. Also, the anti-NKG2A agent, antibody or antigen-binding fragment thereof may be suitably administered serially or in combination with the other agent or modality, e.g., chemotherapeutic drug or radiation for treatment of cancer, infection, and the like, or an immunosuppressive drug. Provided herein are compositions comprising any of the anti-NKG2A agents described herein. In some embodiments, the composition comprising any of the anti-NKG2A agents described herein includes a pharmaceutically acceptable excipient.
Research and diagnostic, including clinical diagnostic, uses for anti-NKG2A agents herein
Provided herein are diagnostic reagents comprising an anti-NKG2A agent, antibody or antigenbinding fragment thereof described herein. For example, any of the anti-NKG2A agents, e.g., antibodies or antigen-binding fragments thereof, provided herein may be used to detect and/or purify NKG2A, e.g., from bodily fluid(s) or expressed on cells in bodily fluids or tissues. Also provided herein are methods for detecting NKG2A. For example, a method may comprise contacting a sample (e.g., a biological sample known or suspected of to contain NKG2A) with an anti-NKG2A agent provided herein, and, if the sample contains NKG2A, detecting NKG2A: anti-NKG2A complexes. Also provided herein are reagents comprising an anti-NKG2A agent described herein and methods for detecting NKG2A for research purposes.
Anti-NKG2A antibodies, for example, may be useful in diagnostic assays for NKG2A, e.g., detecting its presence in specific cells, tissues, or bodily fluids. Such diagnostic methods may be useful in diagnosis, e.g., of a hyperproliferative disease or disorder. In some embodiments, the disease or disorder is a cancer, a viral infection, an autoimmune disease, an inflammatory disease or a bacterial infection. In some embodiments, the disease or disorder is a cancer including but not limited to acute myeloid leukemia, acute lymphoblastic leukemia, colorectal, ovarian, gynecologic, liver, glioblastoma, Hodgkin lymphoma, chronic lymphocytic leukemia, esophagus, gastric, pancreas, colon, kidney, head and neck, lung and melanoma.
In some embodiments, any of the anti-NKG2A agents or anti-NKG2A antibodies described herein are capable of binding a cell. In some embodiments, the cell is a natural killer (NK) cell. In some embodiments, the cell is a lymphocyte. In some embodiments, the lymphocyte is a CD8+ lymphocyte. In some embodiments, any of the anti-NKG2A agents or anti-NKG2A antibodies described herein specifically bind NKG2A on a cell. In some embodiments, any of the anti-NKG2A agents or anti- NKG2A antibodies described herein specifically bind NKG2A and do not specifically bind NKG2C or NKG2E.
Thus, clinical diagnostic uses as well as research uses are comprehended herein.
In some embodiments, an anti-NKG2A agent/antibody comprises a detectable marker or label. In some embodiments, an anti-NKG2A agent/antibody is conjugated to a detectable marker or label. For example, for research and diagnostic applications, an anti-NKG2A agent/antibody may be labeled with a detectable moiety. Numerous labels are available which are generally grouped into the following categories:
(a) Radioisotopes, such as 35S, 14C, 1251, 3H, and 134. The antibody can be labeled with the radioisotope using the techniques described in Current Protocols in Immunology, Volumes 1 and 2, Coligen et al., Ed. Wiley-Interscience, New York, N.Y., Pubs. (1991), for example, and radioactivity can be measured using scintillation counting.
(b) Fluorescent labels such as rare earth chelates (europium chelates) or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, Lissamine, phycoerythrin, Texas Red and Brilliant Violet™ are available. The fluorescent labels can be conjugated to the antibody using the techniques disclosed in Current Protocols in Immunology, supra, for example. Fluorescence can be quantified using a flow cytometer, imaging microscope or fluorimeter.
(c) Various enzyme-substrate labels are available and U.S. Pat. No. 4,275,149 provides a review of some of these. The enzyme generally catalyzes a chemical alteration of the chromogenic substrate that can be measured using various techniques. For example, the enzyme may catalyze a color change in a substrate, which can be measured spectrophotometrically. Alternatively, the enzyme may alter the fluorescence or chemiluminescence of the substrate. Techniques for quantifying a change in fluorescence are described above. The chemiluminescent substrate becomes electronically excited by a chemical reaction and may then emit light that can be measured (using a chemilluminometer, for example) or donates energy to a fluorescent acceptor. Examples of enzymatic labels include luciferases (e.g., firefly luciferase and bacterial luciferase; U.S. Pat. No. 4,737,456), luciferin, 2,3- dihydrophthalazinediones, malate dehydrogenase, urease, peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase, beta-galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclicoxidases (such as uricase and xanthine oxidase), lactoperoxidase, microperoxidase, and the like. Techniques for conjugating enzymes to antibodies are described in O'Sullivan et al., Methods for the Preparation of Enzyme-Antibody Conjugates for use in Enzyme Immunoassay, in Methods in Enzym. (ed J. Langone & H. Van Vunakis), Academic press, New York, 73: 147-166 (1981). Examples of enzymesubstrate combinations include, for example:
(i) Horseradish peroxidase (HRP) with hydrogen peroxidase as a substrate, where the hydrogen peroxidase oxidizes a dye precursor (e.g., orthophenylene diamine (OPD) or 3, 3', 5,5'- tetramethyl benzidine hydrochloride (TMB));
(ii) alkaline phosphatase (AP) with para-Nitrophenyl phosphate as chromogenic substrate; and (iii).beta.-D-galactosidase (.beta.-D-Gal) with a chromogenic substrate (e.g., p-nitrophenyl-p-D- galactosidase) or Anorogenic substrate 4-methylumbelliferyl-.beta.-D-galactosidase.
Numerous other enzyme-substrate combinations may be used (e.g., U.S. Pat. Nos. 4,275,149 and 4,318,980, each of which is incorporated by reference herein). In certain instances, the label is indirectly conjugated with the agent, agent/antibody, antibody or antigen-binding fragment thereof. The skilled artisan will be aware of various techniques for achieving this. For example, an antibody can be conjugated with biotin and any of the three broad categories of labels mentioned above can be conjugated with avidin, or vice versa. Biotin binds selectively to avidin and thus, the label can be conjugated with the antibody in this indirect manner. Alternatively, to achieve indirect conjugation of the label with the antibody, the antibody is conjugated with a small hapten (e.g., digoxin) and one of the different types of labels mentioned above is conjugated with an anti-hapten antibody (e.g., anti-digoxin antibody). Thus, indirect conjugation of the label with the antibody can be achieved.
In some embodiments, an anti-NKG2A agent, agent/antibody, antibody or antigen-binding fragment thereof need not be labeled, and the presence thereof can be detected, e.g., using a labeled antibody which binds to an anti-NKG2A antibody.
In some embodiments, an anti-NKG2A agent, agent/antibody, antibody or antigen-binding fragment thereof herein is immobilized on a solid support or substrate. In some embodiments, an anti-NKG2A agent/antibody herein is non-diffusively immobilized on a solid support (e.g., the anti-NKG2A agent/antibody does not detach from the solid support). A solid support or substrate can be any physically separable solid to which an anti-NKG2A agent, agent/antibody, antibody or antigenbinding fragment thereof can be directly or indirectly attached including, but not limited to, surfaces provided by microarrays and wells, and particles such as beads (e.g., paramagnetic beads, magnetic beads, microbeads, nanobeads), microparticles, and nanoparticles. Solid supports also can include, for example, chips, columns, optical fibers, wipes, filters (e.g., flat surface filters), one or more capillaries, glass and modified or functionalized glass (e.g., controlled-pore glass (CPG)), quartz, mica, diazotized membranes (paper or nylon), polyformaldehyde, cellulose, cellulose acetate, paper, ceramics, metals, metalloids, semiconductive materials, quantum dots, coated beads or particles, other chromatographic materials, magnetic particles; plastics (including acrylics, polystyrene, copolymers of styrene or other materials, polybutylene, polyurethanes, TEFLON™, polyethylene, polypropylene, polyamide, polyester, polyvinylidenedifluoride (PVDF), and the like), polysaccharides, nylon or nitrocellulose, resins, silica or silica-based materials including silicon, silica gel, and modified silicon, Sephadex®, Sepharose®, carbon, metals (e.g., steel, gold, silver, aluminum, silicon and copper), inorganic glasses, conducting polymers (including polymers such as polypyrole and polyindole); micro or nanostructured surfaces such as nucleic acid tiling arrays, nanotube, nanowire, or nanoparticulate decorated surfaces; or porous surfaces or gels such as methacrylates, acrylamides, sugar polymers, cellulose, silicates, or other fibrous or stranded polymers. In some embodiments, the solid support or substrate may be coated using passive or chemically-derivatized coatings with any number of materials, including polymers, such as dextrans, acrylamides, gelatins or agarose. Beads and/or particles may be free or in connection with one another (e.g., sintered). In some embodiments, a solid support or substrate can be a collection of particles. In some embodiments, the particles can comprise silica, and the silica may comprise silica dioxide. In some embodiments the silica can be porous, and in certain embodiments the silica can be non-porous. In some embodiments, the particles further comprise an agent that confers a paramagnetic property to the particles. In certain embodiments, the agent comprises a metal, and in certain embodiments the agent is a metal oxide, (e.g., iron or iron oxides, where the iron oxide contains a mixture of Fe2+ and Fe3+). An anti- NKG2A agent/antibody may be linked to a solid support by covalent bonds or by non-covalent interactions and may be linked to a solid support directly or indirectly (e.g., via an intermediary agent such as a spacer molecule or biotin).
Agents and antibodies provided herein may be employed in any known assay method, such as flow cytometry, immunohistochemistry, immunofluorescence, mass cytometry (e.g., Cytof instrument), competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays. Zola, Monoclonal Antibodies: A Manual of Techniques, pp.147-158 (CRC Press, Inc. 1987).
In some embodiments, the method comprises the use of a single primary antibody to specifically identify the presence of the target molecule expressed on the surface of a dispersed suspension of individual cells. Flow cytometry and mass cytometry assays generally involve the use of a single primary antibody to specifically identify the presence of the target molecule expressed on the surface of a dispersed suspension of individual cells. The dispersed cells are typically obtained from a biological fluid sample, e.g., blood, but may also be obtained from a dispersion of single cells prepared from a solid tissue sample such as spleen or tumor biopsy. The primary antibody may be directly conjugated with a detectable moiety, e.g., a fluorophore such as phycoerythrin for flow cytometry or a heavy metal chelate for mass cytometry. Alternatively, the primary antibody may be unlabeled or labeled with an undetectable tag such as biotin, and the primary antibody is then detected by a detectably labeled secondary antibody that specifically recognizes the primary antibody itself or the tag on the primary antibody. The labeled cells are then analyzed in an instrument capable of single cell detection, e.g., flow cytometer, mass cytometer, fluorescence microscope or brightfield light microscope, to identify those individual cells in the dispersed population or tissue sample that express the target recognized by the primary antibody. Detailed description of the technological basis and practical application of flow cytometry principles may be found in, e.g., Shapiro, Practical Flow Cytometry, 4th Edition, Wiley, 2003.
In some embodiments, the method comprises the use of two antibodies, each capable of binding to a different immunogenic portion, or epitope. Sandwich assays involve the use of two antibodies, each capable of binding to a different immunogenic portion, or epitope, of the protein that is detected. In a sandwich assay, the test sample analyte is bound by a first antibody that is immobilized on a solid support, and thereafter a second antibody binds to the analyte, thus forming an insoluble three-part complex. See, e.g., U.S. Pat. No.4,376,110. The second antibody may itself be labeled with a detectable moiety (direct sandwich assays) or may be measured using an anti-immunoglobulin antibody that is labeled with a detectable moiety (indirect sandwich assay). For example, one type of sandwich assay is an ELISA assay, in which case the detectable moiety is an enzyme. In a cell ELISA, the target cell population may be attached to the solid support using antibodies first attached to the support and that recognize different cell surface proteins. These first antibodies capture the cells to the support. NKG2A on the surface of the cells can then be detected by adding anti-NKG2A antibody to the captured cells and detecting the amount of NKG2A antibody attached to the cells. In certain instances, fixed and permeabilized cells may be used, and in such instances, surface NKG2A and intracellular NKG2A may be detected. For immunohistochemistry, the blood or tissue sample may be fresh or frozen or may be embedded in paraffin and fixed with a preservative such as formalin, for example. The agents, agents/antibodies, antibodies or antigen-binding fragments thereof described herein also may be used for in vivo diagnostic assays. Generally, the antibody is labeled with a radionuclide (such as 111In, 99Tc, 14C, 131I, 125I, 3H, 32P, or 35S) so that the bound target molecule can be localized using immunoscintillography. Detection of NKG2A in immune cells Provided herein are agents and methods for detecting NKG2A in immune cells. Detection of NKG2A in immune cells may refer to detection on the surface of immune cells (e.g., by surface staining) and/or inside immune cells (e.g., by intracellular staining). In some embodiments, any of the agents provided herein may be used for detection of NKG2A on the surface of immune cells. In some embodiments, any of the agents provided herein may be used for detection of intracellular NKG2A in immune cells. In some embodiments, the immune cell is selected from B cells, plasmacytoid dendritic cells (pDCs), lymphocytes, leukocytes, T cells, monocytes, macrophages, neutrophils, myeloid dendritic cells (mDCs), innate lymphoid cells, mast cells, eosinophils, basophils, natural killer cells, and the like. In some embodiments, a heterogeneous population of immune cells comprises peripheral blood mononuclear cells (PBMCs) which may include, for example, T cells, B cells, natural killer cells, and monocytes. In some embodiments, agents and methods are provided for detecting NKG2A in a heterogeneous population of immune cells. A heterogeneous population of immune cells may comprise two or more types of immune cells. For example, a heterogeneous population of immune cells may comprise two or more of B cells, plasmacytoid dendritic cells (pDCs), lymphocytes, leukocytes, T cells, monocytes, macrophages, neutrophils, myeloid dendritic cells (mDCs), innate lymphoid cells, mast cells, eosinophils, basophils, natural killer cells, and the like. In some embodiments, a heterogeneous population of immune cells comprises peripheral blood mononuclear cells (PBMCs) which may include, for example, T cells, B cells, natural killer cells, and monocytes.
Generally, cells are contacted with an anti-NKG2A agent described herein (e.g., in a flow cytometry assay as described in Example 3; or any suitable protein or cell detection assay). In some embodiments, NKG2A is detected at a significant level in certain immune cells by an anti-NKG2A agent, antibody or antigen-binding fragment thereof described herein. NKG2A may be detected at a significant level by an anti-NKG2A agent, antibody or antigen-binding fragment thereof described herein in certain immune cells and not significantly detected in other immune cells. The level of NKG2A detection in certain immune cells may vary according to certain factors such as, for example, type of detection assay, type of detection reagent (e.g., type of dye), antibody concentration, donor cell variability, and the like.
Detection of NKG2A at a significant level may refer to a particular signal to noise (S:N) ratio (e.g., threshold or range) measured in a flow cytometry assay.
In some embodiments, NKG2A is detected at a significant level in NK cells, as well as a subset of CD8+ T-lymphocytes. In certain instances, NK cells may be identified by the expression of CD56. Accordingly, in some embodiments, NKG2A is detected at a significant level in CD56+ NK cells. CD56, also referred to as neural cell adhesion molecule (NCAM), NCAM1, MSK39, neural cell adhesion molecule 1, is a hemophilic binding glycoprotein expressed on the surface of neurons, glia, skeletal muscle, and also in the hematopoietic system. CD56 is most associated with natural killer cells, but is also detected on y5 T cells, activated CD8+ T cells, and dendritic cells.
NCAM is a glycoprotein of the Immunoglobulin (Ig) superfamily, with 27 isoforms produced by alternative mRNA splicing. The three main isoforms of NCAM vary only in their cytoplasmic domain: NCAM-120 kDa (GPI anchored), NCAM-140 kDa (short cytoplasmic domain), and NCAM- 180 kDa (long cytoplasmic domain). The extracellular domain of NCAM consists of five immunoglobulin-like (Ig) domains followed by two fibronectin type III (FNIII) domains. The Ig domains of NCAM are involved in hemophilic binding to NCAM, while the FNIII domains are involved in signaling that leads to neurite outgrowth.
In addition to NK cells and activated T cells, CD56 is detected on tumors such as myeloma, myeloid leukemia, neuroendocrine tumors, Wilm’s tumor, neuroblastoma, extranodal NK/T cell lymphoma, nasal type, pancreatic acinar cell carcinoma, pheochromocytoma, paraganglioma, small cell lung carcinoma, and Ewing’s sarcoma family of tumors.
In some embodiments, NKG2A is not significantly detected in certain lymphocytes by an anti- NKG2A agent described herein. For example, NKG2A may not be significantly detected in certain lymphocytes (e.g., plasmacytoid dendritic cells (pDCs) and/or B cells). No significant detection of NKG2A in certain lymphocytes (e.g., CD304+ plasmacytoid dendritic cells and/or CD19+CD3- B cells). No significant detection of NKG2A in certain lymphocytes (e.g., CD304+ plasmacytoid dendritic cells and/or CD19+CD3- B cells) may refer to a particular signal to noise (S:N) ratio (e.g., threshold or range) measured in a flow cytometry assay. In some embodiments, no significant detection of noise (N) in certain lymphocytes (e.g., CD304+ plasmacytoid dendritic cells and/or CD19+CD3- B cells) refers to a signal to noise (S:N) ratio of about 1.5 or less. In some embodiments, no significant detection of NKG2A in certain lymphocytes (e.g., CD304+ plasmacytoid dendritic cells and/or CD19+CD3- B cells) refers to a signal to noise (S:N) ratio of about 1 or less. Kits incorporating anti-NKG2A agents herein An anti-NKG2A agent (e.g., an anti-NKG2A antibody) herein may be provided in a kit, for example, a packaged combination of reagents in predetermined amounts with instructions for use (e.g., instructions for performing a diagnostic assay; instructions for performing a laboratory assay). In some embodiments, the kit is a diagnostic kit configured to detect NKG2A in a sample (e.g., a biological sample). Where the anti-NKG2A agent is labeled with a fluorophore, the kit may include an identical isotype negative control irrelevant antibody to control for non-specific binding of the anti- NKG2A agent. Where the anti-NKG2A agent is labeled with an enzyme, the kit may include substrates and cofactors required by the enzyme (e.g., a substrate precursor which provides the detectable chromophore or fluorophore). Additional additives may be included such as stabilizers, buffers (e.g., a block buffer or lysis buffer), and the like. The relative amounts of the various reagents may be varied widely to provide for concentrations in solution of the reagents that substantially optimize the sensitivity of the assay. In certain instances, reagents may be provided as dry powders (e.g., lyophilized powder), including excipients that on dissolution with provide a reagent solution having the appropriate concentration. Articles of Manufacture In another aspect of the present technology, an article of manufacture containing materials useful for the treatment, or diagnosis, of the disorders described above is provided. An article of manufacture may comprise a container and a label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. Containers may be formed from a variety of materials such as glass or plastic. A container may hold a composition that is effective for treating a condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). An active anti-NKG2A agent in the composition may be an anti-NKG2A antibody. A label on, or associated with, the container indicates that the composition is used for treating, or diagnosing, a condition of choice. The article of manufacture may further comprise a second container comprising a pharmaceutically acceptable buffer, such as phosphate-buffered saline, Ringer’s solution and dextrose solution; and may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, fdters, needles, syringes, and package inserts with instructions for use.
Certain terms used herein
An “agent” generally refers to an antibody, antibody fragment, antigen binding fragment thereof, derivative, variants of such antibodies and antibody fragments or antigen-binding fragments thereof (including immunoconjugates, labeled antibodies and antigen-binding antibody fragments, and the like).
An “acceptor human framework” generally refers to a framework comprising the amino acid sequence of a immunoglobulin heavy chain variable domain (VH) framework or a immunoglobulin light chain variable domain (VL) framework derived from a human immunoglobulin framework or a human consensus framework, as defined herein. An acceptor human framework “derived from” a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence thereof, or it may contain amino acid sequence changes. In some embodiments, the number of framework amino acid changes are 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VH and/or VL acceptor human framework(s) is(are) identical in sequence to the VH and/or VL human immunoglobulin framework amino acid sequence or human consensus framework amino acid sequence.
“Framework” or “FR” generally refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain generally consists of four FR domains: FR1; FR2; FR3; and FR4. Accordingly, the HVR and FR sequences generally appear in the following sequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.
A “human consensus framework” generally refers to a framework that represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Generally, the subgroup of sequences is a subgroup as in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91- 3242, Bethesda Md. (1991), vols. 1-3. In some embodiments, for the VL, the subgroup is subgroup kappa I as in Kabat et al., supra. In some embodiments, for the VH, the subgroup is subgroup III as in Kabat et al., supra.
The term “hypervariable region” or “HVR” generally refers to each of the regions of an antibody variable domain that are hypervariable in sequence and/or form structurally defined loops (“hypervariable loops”). Generally, native four-chain antibodies comprise six HVRs; three in the VH (Hl, H2, H3), and three in the VL (LI, L2, L3). HVRs generally comprise amino acid residues from the hypervariable loops and/or from the “complementarity determining regions” (CDRs), the latter being of highest sequence variability and/or involved in antigen recognition.
The precise amino acid sequence boundaries of a given CDR or FR can be readily determined using any number of well-known schemes, including those described in Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (“Kabat” numbering scheme), Al-Lazikani et al., (1997) JMB 273,927-948 (“Chothia” numbering scheme), MacCallum et al., J. Mol. Biol. 262:732-745 (1996), “Antibody-antigen interactions: Contact analysis and binding site topography,” J. Mol. Biol. 262, 732-745,” (“Contact” numbering scheme), Martin et al., Proc. Natl. Acad. Sci., 86:9268-9272 (1989) (“AbM” numbering scheme), Lefranc MP et al., “IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Dev Comp Immunol, 2003 Jan;27(l):55-77 (“IMGT” numbering scheme), and Honegger A and Pltickthun A, “Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool,” J Mol Biol, 2001 Jun 8;309(3):657-70, (“Aho” numbering scheme).
The boundaries of a given CDR or FR may vary depending on the scheme used for identification. For example, the Kabat scheme is based on structural alignments, while the Chothia scheme is based on structural information. Numbering for both the Kabat and Chothia schemes is based upon the most common antibody region sequence lengths, with insertions accommodated by insertion letters, for example, “30a” and deletions appearing in some antibodies. The two schemes place certain insertions and deletions (“indels”) at different positions, resulting in differential numbering. The Contact scheme is based on analysis of complex crystal structures and is similar in many respects to the Chothia numbering scheme.
Table 4, below, lists exemplary position boundaries of CDRH1, CDRH2, CDRH3, and CDRL1, CDRL2, and CDRL3 as identified by Kabat, Chothia, and Contact schemes, respectively. For CDRH1, residue numbering is listed using both the Kabat and Chothia numbering schemes. FRs are located between CDRs, for example, with FRH1 located between CDRH1 and CDRH2, and so forth. It is noted that because the shown Kabat numbering scheme places insertions at H35A and H35B, the end of the Chothia CDRH1 loop when numbered using the shown Kabat numbering convention varies between H32 and H34, depending on the length of the loop.
Figure imgf000077_0001
1 - Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD
2 - Al-Lazikani et al., (1997) JMB 273,927-948
CDRs also comprise “specificity determining residues,” or “SDRs,” which are residues that contact a particular antigen. Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al., supra.
The term “variable region” or “variable domain” generally refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). See, e.g., Kindt et al. Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).
“Affinity” generally refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, “binding affinity” refers to intrinsic binding affinity, which reflects a 1 : 1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by a dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Specific illustrative and example embodiments for measuring binding affinity are described elsewhere herein. In some instances, antibodies herein bind to a target (e.g., NKG2A) with a high affinity, e.g., a Kd value of no more than about 1 x 10-7 M; preferably no more than about 1 x 10-8 M; and preferably no more than about 5 x 10-9 M. An “affinity matured” antibody generally refers to an antibody with one or more alterations in one or more hypervariable regions (HVRs), compared to a parent antibody that does not possess such alterations. Preferably, such alterations result in improved affinity of the antibody for its target antigen. The term “anti-NKG2A agent” generally refers to a molecule that is, or comprises, one or more anti- NKG2A antibodies, NKG2A-binding antibody fragments, or NKG2A-binding antibody derivatives. The terms “anti-NKG2A antibody” and “an antibody that binds to NKG2A” generally refer to an antibody that is capable of binding NKG2A with sufficient affinity and/or specificity such that the antibody is useful as a research tool, diagnostic agent and/or therapeutic agent in targeting NKG2A. In some embodiments, the extent of binding of an anti-NKG2A antibody (or antigen-binding fragment thereof) to an unrelated, non-NKG2A protein is less than about 10% of the binding of the antibody to NKG2A as measured, e.g., by a radioimmunoassay (RIA) or by Scatchard analysis or by surface plasmon resonance, such as, for example, Biacore. In certain embodiments, an antibody that binds to NKG2A has a dissociation constant (kD) of 0.1 µM, 100 nM, 10 nM, 1 nM, 0.1 nM, 0.01 nM, or 0.001 nM (e.g., 10-7 M or less, e.g., from 10-7 M to 10-13 M). In certain embodiments, an anti-NKG2A antibody binds to an epitope of NKG2A that is conserved among NKG2A from different species. The term “antibody” herein is used in the broadest sense and includes polyclonal and monoclonal antibodies, including intact antibodies and functional (antigen-binding) antibody fragments, including fragment antigen binding (Fab) fragments, F(ab')2 fragments, Fab’ fragments, Fv fragments, recombinant IgG (rIgG) fragments, variable heavy chain (VH) regions capable of specifically binding the antigen, single chain antibody fragments, including single chain variable fragments (scFv), and single domain antibodies (e.g., sdAb, sdFv, nanobody) fragments. The term encompasses genetically engineered and/or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific, e.g., bispecific, antibodies, diabodies, triabodies, and tetrabodies, tandem di-scFv, tandem tri-scFv. Unless otherwise stated, the term “antibody” should be understood to encompass functional antibody fragments thereof. The term also encompasses intact or full-length antibodies, including antibodies of any class or sub-class, including IgG and sub-classes thereof, IgM, IgE, IgA, and IgD. An “antibody derivative” generally refers to a molecule other than an intact antibody that comprises a portion derived from an intact antibody (or antigen-binding fragment thereof) and that binds the antigen to which the intact antibody (or antigen-binding fragment thereof) binds. Examples of antibody derivatives include but are not limited to single chain variable fragments (scFv), diabodies, triabodies, and the like, aptamers comprising multiple antigen-binding antibody fragments, single chain variable fragments, diabodies, triabodies, and the like.
An “antibody fragment” or “antigen-binding antibody fragment” generally refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab')2 and multispecific antibodies formed from antibody fragments.
The terms “full length antibody,” “intact antibody,” and “whole antibody” are used herein interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure or having heavy chains that contain an Fc region as defined herein.
The term “Fc region” generally refers to a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxyl-terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991.
An “antibody that binds to the same epitope” as a reference antibody (e.g., an antibody that binds NKG2A) generally refers to an antibody that blocks binding of the reference antibody to its antigen in a competition assay by 50% or more, and conversely, the reference antibody blocks binding of the antibody to its antigen in a competition assay by 50% or more.
The term “chimeric” antibody generally refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species. A “human antibody” generally refers to an antibody that possesses an amino acid sequence corresponding to that of an antibody produced by a human or a human cell or derived from a nonhuman source that utilizes human antibody repertoires or other human antibody-encoding sequences. This definition of a human antibody specifically excludes a “humanized” antibody comprising nonhuman antigen-binding residues.
A “humanized” antibody generally refers to a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. In some embodiments, a humanized antibody (or antigen-binding fragment or derivative thereof), when aligned with the antibody from which the acceptor framework regions were derived, includes one or more amino acid substitutions (or deletions or insertions) at desired locations. In some such embodiments, the amino acid residue(s) substituted (or inserted or deleted) at a particular position in the human (or other) or other FR corresponds to the amino acid residue(s) at the corresponding location(s) in the parent antibody (i.e., the non-human antibody from which the CDRs or HVRs were derived). A “humanized form” of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.
The term “antibody drug conjugate” (ADC) or “immunoconjugate” generally refers to a particular class of agent-drug conjugates. Here, “agent-drug conjugate” is an anti-NKG2A agent (e.g., an anti- NKG2A antibody or NKG2A-binding fragment or derivative thereof) conjugated to one or more heterologous molecule(s), including, but not limited to, a cytotoxic agent.
The term “cytotoxic agent” generally refers to a substance that inhibits or prevents a cellular function and/or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioactive isotopes (e.g., At211, 1131, 1125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212, and radioactive isotopes of Lu); chemotherapeutic agents or drugs (e.g., methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents); growth inhibitory agents; enzymes and fragments thereof such as nucleolytic enzymes; antibiotics; toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof; and the various antitumor or anticancer agents disclosed below. A “diagnostic reagent” generally refers to a compound, e.g., a target-specific antibody (or antigenbinding thereof) used to perform a diagnostic assay.
“Effector functions” generally refer to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: Clq binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibodydependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g. B cell receptor); and B cell activation.
An “effective amount” of an agent, e.g., a pharmaceutical formulation, generally refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.
The term “epitope” generally refers to the particular site on an antigen molecule to which an antibody binds.
The terms “host cell”, “host cell line”, and “host cell culture” are used interchangeably and generally refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.
A “rabbit antibody” generally refers to an antibody that possesses an amino acid sequence that corresponds to that of an antibody produced by a rabbit or a rabbit cell or derived from a non-rabbit source that utilizes rabbit antibody repertoires or other rabbit antibody-encoding sequences.
An “immunoconjugate” generally refers to an antibody (or antigen-binding fragment or derivative thereof) conjugated to one or more heterologous molecule(s), including, but not limited to, a cytotoxic agent. An immunoconjugate is equivalent to the term “antibody drug conjugate” (ADC).
An “individual” or “patient” or “subject” generally is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human. An “isolated” molecule (e.g., nucleic acid, antibody) generally refers to a molecule that has been separated from a component of its original environment (e.g., the natural environment if it is naturally occurring, or a host cell if expressed exogenously), and thus is altered by human intervention (e.g., "by the hand of man") from its original environment. In some embodiments, for example, an antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC). An isolated nucleic acid may refer to a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extra-chromosomally or at a chromosomal location that is different from its natural chromosomal location. In some embodiments, an isolated nucleic acid can be provided with fewer non-nucleic acid components (e.g., protein, lipid) than the amount of components present in a source sample. A composition comprising isolated nucleic acid can be about 50% to greater than 99% free of non-nucleic acid components. A composition comprising isolated nucleic acid can be about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater than 99% free of non-nucleic acid components.
“Isolated nucleic acid encoding an anti-NKG2A antibody” or “isolated polynucleotide encoding an anti-NKG2A antibody” generally refers one or more nucleic acid molecules encoding antibody heavy and light chains (or fragments thereof), including such nucleic acid molecule(s) in a single vector or separate vectors, and such nucleic acid molecule(s) present at one or more locations in a recombinant host cell.
The term “NKG2A” generally refers to any native, mature NKG2A that results from processing of a NKG2A precursor protein in a cell. The term includes NKG2A from any vertebrate source, including mammals such as primates (e.g. humans and cynomolgus or rhesus monkeys) and rodents (e.g., mice and rats), unless otherwise indicated. The term also includes naturally occurring variants of NKG2A, e.g., splice variants or allelic variants. The amino acid sequence of an exemplary human NKG2A protein is shown in Fig. 1 (SEQ ID NO: 28).
The term “NKG2A-positive cell” generally refers to any cell that expresses NKG2A on its surface or on an intracellular membrane or organelle (e.g., endosome, ER, Golgi apparatus, lysosome, and the like). Some cells, including those infected by a microbe or associated with some cancer types and tumors, exhibit up-regulation of NKG2A expression. The term “monoclonal antibody” generally refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical (as assessed at the level of Ig heavy and/or light chain amino acid sequence) and/or bind the same epitope, except for possible variant antibodies, e.g., containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present technology may be made by a variety of techniques, including, but not limited to, the hybridoma method, recombinant DNA methods, phage-display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other example methods for making monoclonal antibodies being described herein.
The term “package insert” generally refers to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and/or warnings concerning the use of such therapeutic products.
“Percent (%) amino acid sequence identity” with respect to a reference polypeptide sequence generally refers to the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
The term “pharmaceutical composition” generally refers to a preparation that is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered. A “pharmaceutically acceptable carrier” generally refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.
As used herein, “treatment” (and grammatical variations thereof such as “treat” or “treating”) generally refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology.
Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, antibodies herein are used to delay development of a disease or to slow the progression of a disease.
The term “vector” generally refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors.”
Examples
The examples set forth below illustrate certain embodiments and do not limit the technology.
Example 1 : Creation and Characterization of anti-NKG2A Hyhridomas
This Example describes the preparation of hybridomas that secrete monoclonal antibodies that react with NKG2A as expressed in vivo. The resulting anti-NKG2A antibodies are suitable for a variety of purposes, including in diagnostic assays, examples of which are provided in the examples below.
Common strains of laboratory mice, e.g., BALB/c or C57/BL6, or rats, e.g., Sprague Dawley, were immunized with an NKG2A immunogen. Following immunization of mice, hybridomas were formed using standard protocols to fuse myeloma cells with spleen, and for draining lymph node cells harvested from the animals. Following selection, fusions were cloned in HAT medium and plated to approximately one cell/well in microtiter plates. Culture supernatants were subsequently tested against NKG2A-expressing cell transfectants, e.g., HEK 293 or RBL, by flow cytometry. Wells with successful staining profdes were then sub-cultured into larger vessels until sufficient cells were present to allow for subcloning. Further characterization of the hybridoma subclone candidates was performed by flow cytometry using NKG2A-transfected cells. Selected candidate clones were further screened, by flow cytometry against human blood cells divided into distinct subsets (e.g., lymphocytes) as well as against one or more cell lines generated from diseased and/or infected human cells. As compared to an isotype control, the percentage of positive cells in each blood cell subset was quantified.
Basis for clone selection included strong reactivity against immune cells that express NKG2A (e.g., natural killer cells, natural killer T cells, and activated CD8+ T cells) but no appreciable reactivity against other blood cell populations (e.g., certain lymphocytes, B cells); ability to detect NKG2A on the cell surface; ability to detect NKG2A on the cell surface of natural killer (NK) cells and natural killer T cells (NK T cells; ability to detect NKG2A on the cell surface of CD8+ T cells; ability to detect intracellular NKG2A in NK cells; ability to detect intracellular NKG2A in NK T cells; and/or ability to detect intracellular NKG2A in CD8+ T cells.
Example 2: Sequencing of the Anti- NKG2A Antibody Variable Regions
Cells from the anti-NKG2A hybridoma cell lines described in Example 1 above were grown in standard mammalian tissue culture media. Total RNA was isolated from hybridoma cells from various clones expressing anti-NKG2A monoclonal antibodies, using a procedure based on the RNeasy Mini kit (Qiagen). Briefly, the RNA was used to generate first strand cDNA, and both light chain and heavy chain variable domain cDNAs were amplified by a 5 ’-RACE technique. Positive clones were prepared by PCR and then subjected to DNA sequencing of multiple clones.
Amino acid sequences of the individual variable domains (CDRs and Framework regions), including the CDR1, CDR2, and CDR3 regions, for both the heavy and light chains for four different antibodies (clones), designated AB 1-4 (also referred to herein as antibodies 1-4, and clones 1-4), are shown in Fig. 2. The various heavy and light chain CDR sequences are shown in Table 5, below.
Figure imgf000085_0001
Figure imgf000086_0001
Example 3: Detection of Cells Expressing NKG2A
This example describes the flow cytometry-based detection of human peripheral blood mononuclear cells (PBMCs) using exemplary anti-NKG2A monoclonal antibodies provided herein.
PBMC Isolation
Human peripheral blood mononuclear cells (PBMCs) were isolated under aseptic conditions through a gradient of Ficoll-Paque PLUS (GE Healthcare) according manufacturer instructions.
Surface Staining
PBMCs were counted and adjusted in Cell Staining Buffer (BioLegend, cat. #420201) to a cell density of 0.7-lxl07 cells/mL. A 100 pL cell suspension (0.7-lxl06 total cells) was stained with various cell surface markers (e.g., CD3, CD4, CD8, CD19, NCAM-1/CD56, CD94, and/or NKG2A/CD 159a) for 15 minutes in the dark. All reagents used are from Bio Legend unless otherwise indicated.
Flow Cytometry/Gating Hierarchy
Single cells (singlets) were identified through flow-cytometry by SSC-H and SSC-A (Fig. 4A), and lymphocytes, monocytes, and granulocytes were identified based on their forward (FSC-A) and side scatter (SSC-A) profiles (Fig. 4B). Subsets of lymphocytes including CD3-CD19+ B lymphocytes, CD3+CD19- T lymphocytes, CD3-CD19- lymphocytes, were identified as shown in Fig. 4C. Of the CD3+CD19- lymphocytes, additional subsets of CD4-CD8+ lymphocytes, CD4-CD8- lymphocytes orCD4+CD8- lymphocytes were identified as represented in the dot plots of Fig. 4D.
Results
For surface expression of NKG2A, lymphocytes were co-stained with CD56 and AB2, which were used to identify both NK cells and total lymphocytes positive for CD56 (Fig. 5A and Fig. 5B). AB2 showed a similar staining pattern as commercially available anti-NKG2A antibodies, ABI and ABII in total lymphocytes (Fig. 6A-6C). Additionally, AB2 specifically stained CD3+CD19- lymphocyte subsets (CD4+CD8-, CD4-CD8-, and CD4-CD8+) at a similar level of that of the commercially available anti-NKG2A antibodies (ABI and ABII) (Figs. 7A-7I). The specificity of ABI, AB2, AB3, and AB4 for NKG2A was supported by co-staining total lymphocytes with CD94/KLRD1, which is reported to form a heterodimer with NKG2 (Figs. 8A-D). Similarly, NK cells were specifically identified within CD3-CD19- lymphocytes by co-staining with CD56 and each of ABI, AB2, AB3, and AB4 (Figs. 8E-8H). Finally, the anti-NKG2A antibodies, ABI, AB2, AB3, and AB4, did not cross react with NKG2A expressed on mouse splenocytes (Figs. 9A-9D).
Example 4: Functional Assay
This example describes a functional assay based on NKG2A detection in cells that highly express the target.
K562 cells (target) were labeled with CFSE (BioLegend Cat. #4423801). PBMCs were isolated and purified. Purified primary NK cells (effector) were purified by MojoSort Isolation Kit (BioLegend Cat. #480054). A standard was set up with the effector:target ratio of 4: 1, 2: 1, 1: 1, and 1:2 (Fig. 10A). To identify the function of anti-NKG2A antibody in NK cell-mediated cytotoxicity, an E:T ratio of 2: 1 was selected. Antibodies were mixed with target cells and effector cells at 440 nM (lOpg/well). Cells were then incubated at 37°C for 4 hours. After 4-hour incubation, cells were washed once and DAPI (BioLegend Cat. #422801) was added to the cells to label dead cells, and samples were collected for flow cytometry analysis. As shown in Fig. 10B, all exemplary anti-NKG2A antibodies were capable of inhibiting the cytotoxicity of NK cells, as compared to no antibody control.
Example 5: Blocking assay
This example describes the ability of the exemplary anti-NKG2A antibodies described herein to block the binding of a ligand to the cell surface receptor.
PBMC isolation
Human peripheral blood mononuclear cells (PBMCs) were isolated through a gradient of Ficoll-Paque PLUS (GE Healthcare) according to manufacturer instructions and as described above.
Blocking Assay
PBMCs were incubated with 5 pg purified AB1, AB2, AB3, AB4, or Commercial ABI antibodies, or 1.5 pg ofNKG2E recombinant protein for 15 minutes in the dark. ABI, AB2, AB3, AB4, and Commercial ABI antibodies, and recombinant NKG2E protein were used as blocking agents. In some instances, no blocking agents were used. After incubation with or without blocking agent, cells were incubated with PE-conjugated antibody or isotype control for 15 minutes. Cells were washed, acquired on a BD Canto 11, and analyzed using FlowJo software.
Calculations for % Blocking
Percentage original MFI was calculated by dividing the MFI of samples blocked with ABI, AB2, AB3, AB4, and Commercial ABI antibodies, and recombinant NKG2E protein by the MFI of samples blocked with isotype MOPC-21. This value was subtracted from 100 to get a blocking percentage. The formula is shown below:
Figure imgf000088_0001
The results for calculated MFI and % blocking are described in Table 6.
Figure imgf000089_0001
Figure imgf000090_0001
As shown in Table 6, all exemplary anti-NKG2A antibodies were capable of blocking AB1, AB2, AB3, AB4 and commercial antibody ABI, with minimal to no blocking of MOPC-21 and NKG2E.
Example 7: Examples of sequences
Provided below are examples of amino acid sequences related to the technology described herein.
Table 7 lists CDR sequences and SEQ ID NOs for exemplaryanti-NKG2A antibodies provided herein.
Figure imgf000090_0002
Figure imgf000091_0001
CDRH1
X1YX3MX5 (SEQ ID NO: 22)
X1 = S or D
X3=WorY
X5 = NorY
CDRH2
X1IX3X4X5X6X7X8T X10YX12X13X14X15KX17 (SEQ ID NO: 23)
X1 = RorT
X3=YorS
X4 = ForY
X5 = E or G
X6 = D or G
X7 = G or I
X8 = DorY
X10 = NorY
X12 = NorP
X13 = GorD
X14 = K or S
X15 = ForV X17 = GorD CDRH3
X1X2X3X4X5X6X7X8X9YX11X12X13X14X15 (SEQ ID NO: 24)
X1 = Q or D
X2 = G or R
X3 = D or no amino acid
X4 = T or no amino acid
X5 = S or no amino acid
X6 = V or no amino acid
X7 = I orY
X8 = W or V
X9 = D orN
X11 = D orY
X12 = G or T
X13 = F or M
X14 = A or D
X15 = D orY
CDRL1
X1ASX4SVX7X8X9X10X11X12YX14H (SEQ ID NO: 25)
X1 = S or R
X4 = S or K
X7 = T or S
X8 = T or no amino acid
X9 = S or no amino acid
X10 = G or no amino acid
X11 = Y or no amino acid
X12 = S or no amino acid
X14 = M or I
CDRL2
X1X2SNLX6S (SEQ ID NO: 26)
X1 = S or L X2 = T or A
X6 = A or E
CDRL3
QX2X3X4X5X6PX8T (SEQ ID NO: 27)
X2 = Q or H
X3 = R or S X4 = S or R
X5 = S or E
X6 = Y or L
X8 = Y or L
Figure imgf000093_0001
Figure imgf000094_0001
Precursor human NKG2A protein sequence (UniProtKB - P26715 (NKG2A HUMAN)/ NCBI Reference Sequence: NP 002250,2) (SEQ ID NO: 28)
1 mdnqgviysd Inlppnpkrq qrkpkgnkns ilateqeity aelnlqkasq dfqgndktyh 61 ckdlpsapek livgilgiic lilmasvvti vvipstliqr hnnsslntrt qkarhcghcp 121 eewitysnsc yyigkerrtw eesllactsk nssllsidne eemkflsiis psswigvfm 181 sshhpwvtmn glafkheikd sdnaelncav Iqvnrlksaq cgssiiyhck hkl Example 8: Examples of embodiments
The examples set forth below illustrate certain embodiments and do not limit the technology.
Al . An anti-NKG2A agent that binds NK inhibitory receptor CD94/NK group 2 member A (NKG2A) under laboratory or physiological conditions, wherein the agent comprises at least one immunoglobulin heavy chain variable domain and/or at least one immunoglobulin light chain variable domain, wherein: a) each immunoglobulin heavy chain variable domain of the anti-NKG2A agent comprises first, second, and third heavy chain complementarity determining regions (CDRs), wherein the first heavy chain CDR (CDRH1) comprises an amino acid sequence that is at least 80 percent identical to the amino acid sequence X1YX3MX5 (SEQ ID NO: 22), wherein
X1 = S or D, X3 = W orY, and X5 = N orY; the second heavy chain CDR (CDRH2) comprises an amino acid sequence that is at least 80 percent identical to the amino acid sequence X1IX3X4X5X6X7X8TX10YX12X13X14X15KX17 (SEQ ID NO: 23), wherein
X1 = R or T,
X3=Yor S,
X4 = ForY,
X5 = E or G,
X6 = D or G,
X7 = G or I,
X8 = DorY,
X10 = NorY,
X12 = NorP,
X13 = GorD,
X14 = K or S,
X15 = F or V, and
X17 = G or D; and the third heavy chain CDR (CDRH3) comprises an amino acid sequence that is at least 80 percent identical to the amino acid sequence X1 X2X3X4X5X6 X7 X8 X9Y X11X12X13X14X15 (SEQ ID NO: 24), wherein
X1 = Q or D,
X2 = G or R, X3 = D or no amino acid, X4 = T or no amino acid, X5 = S or no amino acid, X6 = V or no amino acid,
X7 = IorY,
X8 = WorV,
X9 = DorN,
X11 = DorY,
X12 = GorT,
X13 = F or M,
X14 = A or D, and
X15 = D or Y; and/or b) each immunoglobulin light chain variable domain of the anti-NKG2A agent comprises first, second, and third light chain CDRs, wherein the first light chain CDR (CDRL1) comprises an amino acid sequence that is at least 80 percent identical to the amino acid sequence X1ASX4SVX7X8X9X10X11X12YX14H (SEQ ID NO: 25), wherein
X1 = S or R, X4 = S or K, X7 = T or S, Xs = T or no amino acid,
X9 = S or no amino acid,
X10 = G or no amino acid,
X11 = Y or no amino acid,
X12 = S or no amino acid,
X14 = M or I; the second light chain (CDRL2) comprises an amino acid sequence that is at least 80 percent identical to the amino acid sequence X1X2SNLX6S (SEQ ID NO: 26), wherein
X1 = S or L,
X2 = T or A, and X6 = A or E; and the third light chain CDR (CDRL3) comprises an amino acid sequence that is at least 80 percent identical to the amino acid sequence QX2X3X4X5X6PX8T (SEQ ID NO: 27), wherein X2 = Q or H, X3 = R or S, X4 = S or R, X5 = S or E, X6 = Y or L, and Xs = Y or L.
A2. The anti-NKG2A agent of embodiment Al, wherein the CDRH1 comprises and amino acid sequence that is at least 85 percent identical to the amino acid sequence of SEQ ID NO: 22. A3. The anti-NKG2A agent of embodiment Al, wherein the CDRH1 comprises an amino acid sequence that is at least 90 percent identical to the amino acid sequence of SEQ ID NO: 22. A4. The anti-NKG2A agent of embodiment Al, wherein the CDRH1 comprises an amino acid sequence that is at least 95 percent identical to the amino acid sequence of SEQ ID NO: 22. A5. The anti-NKG2A agent of embodiment Al, wherein the CDRH1 comprises an amino acid sequence that is 100 percent identical to the amino acid sequence of SEQ ID NO: 22.
A6. The anti-NKG2A agent of any one of embodiments Al to A5, wherein the CDRH2 comprises an amino acid sequence that is at least 85 percent identical to the amino acid sequence of SEQ ID NO: 23. A7. The anti-NKG2A agent of any one of embodiments Al to A5, wherein the CDRH2 comprises an amino acid sequence that is at least 90 percent identical to the amino acid sequence of SEQ ID NO:
23.
A8. The anti-NKG2A agent of any one of embodiments Al to A5, wherein the CDRH2 comprises an amino acid sequence that is at least 95 percent identical to the amino acid sequence of SEQ ID NO:
23.
A9. The anti-NKG2A agent of any one of embodiments Al to A5, wherein the CDRH2 comprises an amino acid sequence that is 100 percent identical to the amino acid sequence of SEQ ID NO: 23.
A 10. The anti-NKG2A agent of any one of embodiments Al to A9, wherein the CDRH3 comprises an amino acid sequence that is at least 85 percent identical to the amino acid sequence of SEQ ID NO:
24.
Al l. The anti-NKG2A agent of any one of embodiments Al to A9, wherein the CDRH3 comprises an amino acid sequence that is at least 90 percent identical to the amino acid sequence of SEQ ID NO:
24.
A 12. The anti-NKG2A agent of any one of embodiments Al to A9, wherein the CDRH3 comprises an amino acid sequence that is at least 95 percent identical to the amino acid sequence of SEQ ID NO:
24.
A13. The anti-NKG2A agent of any one of embodiments Al to A9, wherein the CDRH3 comprises an amino acid sequence that is 100 percent identical to the amino acid sequence of SEQ ID NO: 24. A 14. The anti-NKG2A agent of any one of embodiments Al to A 13, wherein the CDRL1 comprises an amino acid sequence that is at least 85 percent identical to the amino acid sequence of SEQ ID NO:
25.
A15. The anti-NKG2A agent of any one of embodiments Al to A 13, wherein the CDRL1 comprises an amino acid sequence that is at least 90 percent identical to the amino acid sequence of SEQ ID NO:
25.
A 16. The anti-NKG2A agent of any one of embodiments Al to A 13, wherein the CDRL1 comprises an amino acid sequence that is at least 95 percent identical to the amino acid sequence of SEQ ID NO:
25.
A 17. The anti-NKG2A agent of any one of embodiments Al to A 13, wherein the CDRL1 comprises an amino acid sequence that is 100 percent identical to the amino acid sequence of SEQ ID NO: 25. Al 8. The anti-NKG2A agent of any one of embodiments Al to A 17, wherein the CDRL2 comprises an amino acid sequence that is at least 85 percent identical to the amino acid sequence of SEQ ID NO:
26.
A 19. The anti-NKG2A agent of any one of embodiments Al to A 17, wherein the CDRL2 comprises an amino acid sequence that is at least 90 percent identical to the amino acid sequence of SEQ ID NO:
26. A20. The anti-NKG2A agent of any one of embodiments Al to A 17, wherein the CDRL2 comprises an amino acid sequence that is at least 95 percent identical to the amino acid sequence of SEQ ID NO:
26.
A21. The anti-NKG2A agent of any one of embodiments Al to A 17, wherein the CDRL2 comprises an amino acid sequence that is 100 percent identical to the amino acid sequence of SEQ ID NO: 26. A22. The anti-NKG2A agent of any one of embodiments Al to A21, wherein the CDRL3 comprises an amino acid sequence that is at least 85 percent identical to the amino acid sequence of SEQ ID NO:
27.
A23. The anti-NKG2A agent of any one of embodiments Al to A21, wherein the CDRL3 comprises an amino acid sequence that is at least 90 percent identical to the amino acid sequence of SEQ ID NO: 27.
A24. The anti-NKG2A agent of any one of embodiments Al to A21, wherein the CDRL3 comprises an amino acid sequence that is at least 95 percent identical to the amino acid sequence of SEQ ID NO: 27.
A25. The anti-NKG2A agent of any one of embodiments Al to A21, wherein the CDRL3 comprises an amino acid sequence that is 100 percent identical to the amino acid sequence of SEQ ID NO: 27. A26. The anti-NKG2A agent of any one embodiments Al to A25, wherein the CDRH1 comprises an amino acid sequence chosen from SYWMN (SEQ ID NO: 9) and DYYMY (SEQ ID NO: 16).
A27. The anti-NKG2A agent of any one of embodiments Al to A26, wherein the CDRH2 comprises an amino acid sequence chosen from IUYFEDGDTNYNGKFKG (SEQ ID NO: 10), RIYFEDGDTNYNGKFKD (SEQ ID NO: 15), and TISYGGIYTYYPDSVKG (SEQ ID NO: 17). A28. The anti-NKG2A agent of any one of embodiments Al to A27 , wherein the CDRH3 comprises an amino acid sequence chosen from QGIWDYD GF AD (SEQ ID NO: 11) and DRDTSVYVNYYTMDY (SEQ ID NO: 18).
A29. The anti-NKG2A agent of any one of embodiments Al to A28, wherein CDRL1 comprises an amino acid sequence chosen from SASSSVTYMH (SEQ ID NO: 12) and RASKSVSTSGYSYIH (SEQ ID NO: 19).
A30. The anti-NKG2A agent of any one embodiments Al to A29, wherein CDRL2 comprises an amino acid sequence chosen from STSNLAS (SEQ ID NO: 13) and LASNLES (SEQ ID NO: 20). A31. The anti-NKG2A agent of any one of embodiments Al to A30, wherein CDRL3 comprises an amino acid sequence chosen from QQRSSYPYT (SEQ ID NO: 14) and QHSRELPLT (SEQ ID NO: 21).
A32. The anti-NKG2A agent of any one of embodiments Al to A31, which comprises two immunoglobulin heavy chain variable domains and two immunoglobulin light chain variable domains.
A33. The anti-NKG2A agent of embodiment A32, wherein the two immunoglobulin heavy chain variable domains each comprise a set of CDRH1, CDRH2, and CDRH3 amino acid sequences. A34. The anti-NKG2A agent of embodiment A32 and A33, wherein the two immunoglobulin light chain variable domains each comprise a set of CDRL1, CDRL2, and CDRL3 amino acid sequences. A35. The anti-NKG2A agent of any one of embodiments Al to A34, wherein each immunoglobulin heavy chain variable domain comprises a set of CDRH1, CDRH2, CDRH3 amino acid sequences and each immunoglobulin light chain variable domain comprises a set of CDRL1, CDRL2, and CDRL3 amino acid sequences chosen from sets 1-4:
Figure imgf000099_0001
A36. The anti-NKG2A agent of embodiment A35, wherein all CDR sequences are from the same set. A37. The anti-NKG2A agent of any one of embodiments Al to A36, wherein the agent is isolated. A38. The anti-NKG2A agent of any one of embodiments Al to A37 wherein the agent is non- naturally occurring.
A39. The anti-NKG2A agent of any one of embodiments Al to A38, wherein the agent is an antibody, or antigen-binding fragment thereof.
A40. The anti-NKG2A agent of any one of embodiments Al to A38, wherein the agent is an antibody, or derivative thereof.
A41. The anti-NKG2A agent of any one of embodiments Al to A40, wherein the agent is a humanized antibody, or an antigen binding fragment thereof. A42. The anti-NKG2A agent of any one of embodiments Al to A40, wherein the agent is a derivative of a humanized antibody that binds NKG2A.
A43. The anti-NKG2A agent of any one of embodiments Al to A42, wherein the agent comprises a detectable marker or label.
A44. The anti-NKG2A agent of any one of embodiments Al to A43, wherein the agent is conjugated to a detectable marker or label.
A45. The anti-NKG2A agent of any one of embodiments Al to A44, wherein the agent is non- diffusively immobilized on a solid support.
A46. A diagnostic reagent comprising the anti-NKG2A agent of any one of embodiments Al to A45. A47. A kit comprising the anti-NKG2A agent of any one of embodiments Al to A45 or the diagnostic reagent of embodiment A46.
A48. A diagnostic kit configured to detect NK inhibitory receptor CD94/NK group 2 member A (NKG2A) in a biological sample, wherein the kit comprises the anti-NKG2A agent of any one of embodiments Al to A45 or the diagnostic reagent of embodiment A46.
A49. An isolated nucleic acid molecule comprising a nucleotide sequence that encodes an immunoglobulin heavy chain variable domain of the anti-NKG2A agent of any one of embodiments Al to A45.
A50. An isolated nucleic acid molecule comprising a nucleotide sequence that encodes an immunoglobulin light chain variable domain of the anti-NKG2A agent of any one of embodiments Al to A45.
A51. A recombinant expression vector comprising a first expression cassette and a second expression cassette, wherein the first expression cassette comprises a promoter and a nucleic acid molecule comprising a nucleotide sequence that encodes an immunoglobulin heavy chain variable domain of the anti-NKG2A agent of any one of embodiments Al to A45, and the second expression cassette comprises a promoter and a nucleic acid molecule comprising a nucleotide sequence that encodes an immunoglobulin light chain variable domain of the anti-NKG2A agent of any one of embodiments Al to A45.
A52. A recombinant host cell transfected with the recombinant expression vector of embodiment A51. A53. A method of detecting NKG2A, comprising contacting a sample known or suspected to contain NKG2A with the anti-NKG2A agent of any one of embodiments Al to A45, and, if the sample contains NKG2A, detecting NKG2A:anti-NKG2A complexes.
B 1. A first anti-NKG2A agent that binds NKG2A under laboratory or physiological conditions, wherein the first agent competitively binds, or is capable of competitively binding, with a second anti- NKG2A agent, which second agent is the anti-NKG2A agent of any one of embodiments Al to A36. B2. A first anti-NKG2A agent that binds NKG2A under laboratory or physiological conditions, wherein the first agent binds to, or is capable of binding to, the same epitope as a second anti-NKG2A agent, which second agent is the anti-NKG2A agent of any one of embodiments Al to A36.
B3. The first anti-NKG2A agent of embodiment B 1 or B2, wherein the first agent and/or second agent is isolated.
B4. The first anti-NKG2A agent of any one of embodiments Bl to B3, wherein the first agent and/or second agent is non-naturally occurring.
B5. The first anti-NKG2A agent of any one of embodiments Bl to B4, wherein the first agent and/or second agent is an antibody, or derivative.
B6. The first anti-NKG2A agent of any one of embodiments Bl to B4, wherein the first agent and/or second agent is an antibody, or derivative thereof.
B7. The first anti-NKG2A agent of any one of embodiments Bl to B6, wherein the first agent and/or second agent is a humanized antibody, or an antigen binding fragment thereof.
B8. The first anti-NKG2A agent of any one of embodiments Bl to B6, wherein the first agent and/or second agent is a derivative of a humanized antibody that binds NKG2A.
B9. The first anti-NKG2A agent of any one of embodiments Bl to B8, comprising a detectable marker or label.
BIO. The first anti-NKG2A agent of any one of embodiments Bl to B9, wherein the first agent is conjugated to a detectable marker or label.
Bl 1. The first anti-NKG2A agent of any one of embodiments Bl to BIO, wherein the first agent is non-diffusively immobilized on a solid support.
B12. A diagnostic reagent comprising the first anti-NKG2A agent of any one of embodiments Bl to Bl l.
B 13. A kit comprising the first anti-NKG2A agent of any one of embodiments B 1 to B 11 or the diagnostic reagent of embodiment B 12.
B14. A diagnostic kit configured to detect NK inhibitory receptor CD94/NK group 2 member A (NKG2A) in a biological sample, wherein the kit comprises the first anti-NKG2A agent of any one of embodiments B 1 to B 11 or the diagnostic reagent of embodiment B 12.
Bl 5. An isolated nucleic acid molecule comprising a nucleotide sequence that encodes an immunoglobulin heavy chain variable domain of the first anti-NKG2A agent of any one of embodiments B 1 to B 11.
Bl 6. An isolated nucleic acid molecule comprising a nucleotide sequence that encodes an immunoglobulin light chain variable domain of the first anti-NKG2A agent of any one of embodiments B 1 to B 11.
Bl 7. A recombinant expression vector comprising a first expression cassette and a second expression cassette, wherein the first expression cassette comprises a promoter and a nucleic acid molecule comprising a nucleotide sequence that encodes an immunoglobulin heavy chain variable domain of the first anti-NKG2A agent of any one of embodiments Bl to Bl 1, and the second expression cassette comprises a promoter and a nucleic acid molecule comprising a nucleotide sequence that encodes an immunoglobulin light chain variable domain of the first anti-NKG2A agent of any one of embodiments B 1 to B 11.
Bl 8. A recombinant host cell transfected with the recombinant expression vector of embodiment Bl 7. B19. A method of detecting NKG2A, comprising contacting a sample known or suspected to contain NKG2A with the first anti-NKG2A agent of any one of embodiments Bl to Bl 1, and, if the sample contains NKG2A, detecting NKG2A:anti-NKG2A complexes.
Cl . An anti-NKG2A agent for detecting NKG2A in a heterogeneous population of immune cells, wherein NKG2A is detected at a significant level in natural killer (NK) cells and/or CD8+ T lymphocytes in the population, and NKG2A is not significantly detected in other immune cells in the population.
C2. The anti-NKG2A agent of embodiment Cl, wherein the anti-NKG2A agent comprises at least one immunoglobulin heavy chain variable domain and at least one immunoglobulin light chain variable domain, wherein: i) each immunoglobulin heavy chain variable domain of the anti-NKG2A agent comprises first, second, and third heavy chain complementarity determining regions (CDRs); and ii) each immunoglobulin light chain variable domain of the anti-NKG2A agent comprises first, second, and third light chain CDRs.
C3. The anti-NKG2A agent of embodiment C2, wherein the first heavy chain CDR (CDRH1) comprises an amino acid sequence that is at least 80 percent identical to the amino acid sequence
X1YX3MX5 (SEQ ID NO: 22), wherein
X1 is S or D, X3 is W or Y, and
X5 is N orY.
C4. The anti-NKG2A agent of embodiment C2 or C3, wherein the second heavy chain CDR (CDRH2) comprises an amino acid sequence that is at least 80 percent identical to the amino acid sequence X1IX3X4X5X6X7X8TX10YX12X13X14X15KX17 (SEQ ID NO: 23), wherein
X1 is R or T, X3 is Y or S, X4 is F or Y, X5 is E or G, X6 is D or G,
X7 is G or I, X6 is D or Y, X10 is N or Y,
X12 is N or P,
X13 is G or D,
X14 is K or S,
X15 is F or V, and
X17 is G or D.
C5. The anti-NKG2A agent of embodiment C2, C3, or C4, wherein the third heavy chain CDR (CDRH3) comprises an amino acid sequence that is at least 80 percent identical to the amino acid sequence X1X2X3X4X5X6X7X8X9YX11X12X13X14X15 (SEQ ID NO: 24), wherein
X1 is Q or D,
X2 is G or R, X3 is D or no amino acid,
X4 is T or no amino acid, X5 is S or no amino acid, X6 is V or no amino acid,
X7 is I or Y,
X8 is W or V,
X9 is D or N,
X11 is D or Y,
X12 is G or T,
X13 is F or M,
X14 is A or D, and
X15 is D or Y.
C6. The anti-NKG2A agent of any one of embodiments C2 to C5, wherein the first light chain CDR (CDRL1) comprises an amino acid sequence that is at least 80 percent identical to the amino acid sequence X1ASX4SVX7X8X9X10 X11X12YX14H (SEQ ID NO: 25), wherein
X1 is S or R,
X4 is S or K,
X7 is T or S,
X8 is T or no amino acid,
X9 is S or no amino acid,
X10 is G or no amino acid,
X11 is Y or no amino acid,
X12 is S or no amino acid, and
X14 = M or I. C7. The anti-NKG2A agent of any one of embodiments C2 to C6, wherein the second light chain CDR (CDRL2) comprises an amino acid sequence that is at least 80 percent identical to the amino acid sequence X1X2SNLX6S (SEQ ID NO: 26), wherein
X1 is S or L,
X2 is T or A, and X6 is A or E.
C8. The anti-NKG2A agent of any one of embodiments C2 to C7, wherein the third light chain CDR (CDRL3) comprises an amino acid sequence that is at least 80 percent identical to the amino acid sequence QX2X3X4X5X6PX8T (SEQ ID NO: 27), wherein
X2 is Q or H, X3 is R or S, X4 is S or R, X5 is S or E, X6 is Y or L, and X6 is Y or L.
C9. The anti-NKG2A agent of any one of embodiments C2 to C8, wherein the CDRH1 comprises an amino acid sequence that is at least 85 percent identical to the amino acid sequence of SEQ ID NO:
22.
CIO. The anti-NKG2A agent of any one of embodiments C2 to C8, wherein the CDRH1 comprises an amino acid sequence that is at least 90 percent identical to the amino acid sequence of SEQ ID NO:
22.
Cl 1. The anti-NKG2A agent of any one of embodiments C2 to C8, wherein the CDRH1 comprises an amino acid sequence that is at least 95 percent identical to the amino acid sequence of SEQ ID NO:
22.
C12. The anti-NKG2A agent of any one of embodiments C2 to C8, wherein the CDRH1 comprises an amino acid sequence that is 100 percent identical to the amino acid sequence of SEQ ID NO: 22.
C13. The anti-NKG2A agent of any one of embodiments C2 to C12, wherein the CDRH2 comprises an amino acid sequence that is at least 85 percent identical to the amino acid sequence of SEQ ID NO:
23.
C14. The anti-NKG2A agent of any one of embodiments C2 to C12, wherein the CDRH2 comprises an amino acid sequence that is at least 90 percent identical to the amino acid sequence of SEQ ID NO:
23.
C15. The anti-NKG2A agent of any one of embodiments C2 to C12, wherein the CDRH2 comprises an amino acid sequence that is at least 95 percent identical to the amino acid sequence of SEQ ID NO:
23. C16. The anti-NKG2A agent of any one of embodiments C2 to C12, wherein the CDRH2 comprises an amino acid sequence that is 100 percent identical to the amino acid sequence of SEQ ID NO: 23. C17. The anti-NKG2A agent of any one of embodiments C2 to C16, wherein the CDRH3 comprises an amino acid sequence that is at least 85 percent identical to the amino acid sequence SEQ ID NO:
24.
Cl 8. The anti-NKG2A agent of any one of embodiments C2 to Cl 6, wherein the CDRH3 comprises an amino acid sequence that is at least 90 percent identical to the amino acid sequence of SEQ ID NO: 24.
Cl 9. The anti-NKG2A agent of any one of embodiments C2 to Cl 6, wherein the CDRH3 comprises an amino acid sequence that is at least 95 percent identical to the amino acid sequence of SEQ ID NO:
24.
C20. The anti-NKG2A agent of any one of embodiments C2 to C16, wherein the CDRH3 comprises an amino acid sequence that is 100 percent identical to the amino acid sequence of SEQ ID NO: 24. C21. The anti-NKG2A agent of any one of embodiments C2 to C20, wherein the CDRL1 comprises an amino acid sequence that is at least 85 percent identical to the amino acid sequence of SEQ ID NO:
25.
C22. The anti-NKG2A agent of any one of embodiments C2 to C20, wherein the CDRL1 comprises an amino acid sequence that is at least 90 percent identical to the amino acid sequence of SEQ ID NO:
25.
C23. The anti-NKG2A agent of any one of embodiments C2 to C20, wherein the CDRL1 comprises an amino acid sequence that is at least 95 percent identical to the amino acid sequence of SEQ ID NO:
25.
C24. The anti-NKG2A agent of any one of embodiments C2 to C20, wherein the CDRL1 comprises an amino acid sequence that is 100 percent identical to the amino acid sequence of SEQ ID NO: 25. C25. The anti-NKG2A agent of any one of embodiments C2 to C24, wherein the CDRL2 comprises an amino acid sequence that is at least 85 percent identical to the amino acid sequence of SEQ ID NO:
26.
C26. The anti-NKG2A agent of any one of embodiments C2 to C24, wherein the CDRL2 comprises an amino acid sequence that is at least 90 percent identical to the amino acid sequence of SEQ ID NO:
26.
C27. The anti-NKG2A agent of any one of embodiments C2 to C24, wherein the CDRL2 comprises an amino acid sequence that is at least 95 percent identical to the amino acid sequence of SEQ ID NO: 26.
C28. The anti-NKG2A agent of any one of embodiments C2 to C24, wherein the CDRL2 comprises an amino acid sequence that is 100 percent identical to the amino acid sequence of SEQ ID NO: 26. C29. The anti-NKG2A agent of any one of embodiments C2 to C28, wherein the CDRL3 comprises an amino acid sequence that is at least 85 percent identical to the amino acid sequence of SEQ ID NO: 27.
C30. The anti-NKG2A agent of any one of embodiments C2 to C28, wherein the CDRL3 comprises an amino acid sequence that is at least 90 percent identical to the amino acid sequence of SEQ ID NO: 27.
C31. The anti-NKG2A agent of any one of embodiments C2 to C28, wherein the CDRL3 comprises an amino acid sequence that is at least 95 percent identical to the amino acid sequence of SEQ ID NO: 27.
C32. The anti-NKG2A agent of any one of embodiments C2 to C28, wherein the CDRL3 comprises an amino acid sequence that is 100 percent identical to the amino acid sequence of SEQ ID NO: 27. C33. The anti-NKG2A agent of any one of embodiments C2 to C32, wherein the CDRH1 comprises an amino acid sequence chosen from SYWMN (SEQ ID NO: 9) and DYYMY (SEQ ID NO: 16). C34. The anti-NKG2A agent of any one of embodiments C2 to C33, wherein the CDRH2 comprises an amino acid sequence chosen from IUYFEDGDTNYNGKFKG (SEQ ID NO: 10), RIYFEDGDTNYNGKFKD (SEQ ID NO: 15), and TISYGGIYTYYPDSVKG (SEQ ID NO: 17). C35. The anti-NKG2A agent of any one of embodiments C2 to C34, wherein the CDRH3 comprises an amino acid sequence chosen from QGIWDYD GF AD (SEQ ID NO: 11) and DRDTSVYVNYYTMDY (SEQ ID NO: 18).
C36. The anti-NKG2A agent of any one of embodiments C2 to C35, wherein the CDRL1 comprises an amino acid sequence chosen from SASSSVTYMH (SEQ ID NO: 12) and RASKSVSTSGYSYIH (SEQ ID NO: 19).
C37. The anti-NKG2A agent of any one of embodiments C2 to C36, wherein the CDRL2 comprises an amino acid sequence chosen from STSNLAS (SEQ ID NO: 13) and LASNLES (SEQ ID NO: 20). C38. The anti-NKG2A agent of any one of embodiments C2 to C37, wherein the CDRL3 comprises an amino acid sequence chosen from QQRSSYPYT (SEQ ID NO: 14) and QHSRELPLT (SEQ ID NO: 21).
C39. The anti-NKG2A agent of any one of embodiments Cl to C38, which comprises two immunoglobulin heavy chain variable domains and two immunoglobulin light chain variable domains.
C40. The anti-NKG2A agent of embodiment C39, wherein the two immunoglobulin heavy chain variable domains each comprise a set of CDRH1, CDRH2, and CDRH3 amino acid sequences. C41. The anti-NKG2A agent of embodiment C39 or C40, wherein the two immunoglobulin light chain variable domains each comprise a set of CDRL1, CDRL2, and CDRL3 amino acid sequences. C42. The anti-NKG2A agent of any one of embodiments C2 to C41, wherein each immunoglobulin heavy chain variable domain comprises a set of CDRH1, CDRH2, and CDRH3 amino acid sequences and each immunoglobulin light chain variable domain comprises a set of CDRL1, CDRL2, and CDRL3 amino acid sequences chosen from sets 1-4:
Figure imgf000107_0001
C43. The anti-NKG2A agent of embodiment C42, wherein all CDR sequences are from the same set. C44. The anti-NKG2A agent of any one of embodiments Cl to C43, wherein NKG2A is detected at a significant level in NK cells.
C45. The anti-NKG2A agent of embodiment C44, wherein NKG2A is detected in NK cells using a flow cytometry assay.
C46. The anti-NKG2A agent of embodiment Cl to C43, wherein NKG2A is detected at a significant level in CD8+ T cells.
C47. The anti-NKG2A agent of embodiment C46, wherein NKG2A is detected in CD8+ T cells using a flow cytometry assay.
C48. The anti-NKG2A agent o any one of embodiments Cl to C47, wherein NKG2A is not significantly detected in other immune cells in the population, wherein the other immune cells comprise one or more of plasmacytoid dendritic cells, B cells, and monocytes.
C49. The anti-NKG2A agent of any one of embodiments Cl to C48, wherein the agent is isolated. C50. The anti-NKG2A agent of any one of embodiments Cl to C49, wherein the agent is non- naturally occurring. C51. The anti-NKG2A agent of any one of embodiments Cl to C50, wherein the agent is an antibody, or antigen-binding fragment thereof.
C52. The anti-NKG2A agent of any one of embodiments Cl to C50, wherein the agent is an antibody, or derivative thereof.
C53. The anti-NKG2A agent of any one of embodiments Cl to C52, wherein the agent is a humanized antibody, or an antigen binding fragment thereof.
C54. The anti-NKG2A agent of any one of embodiments Cl to C52, wherein the agent is a derivative of a humanized antibody that bind NKG2A.
C55. The anti-NKG2A agent of any one of embodiments Cl to C54, wherein the agent comprises a detectable marker or label.
C56. The anti-NKG2A agent of any one of embodiments Cl to C55, wherein the agent is conjugated to a detectable marker or label.
C57. The anti-NKG2A agent of any one of embodiments Cl to C56, wherein the agent is non- diffiisively immobilized on a solid support.
C58. A diagnostic reagent comprising the anti-NKG2A agent of any one of embodiments Cl to C57. C59. A kit comprising the anti-NKG2A agent of any one of embodiments Cl to C57 or the diagnostic reagent of embodiment C58.
C60. A diagnostic kit configured to detect NKG2A in a biological sample, wherein the kit comprises the anti-NKG2A agent of any one of embodiments Cl to C57 or the diagnostic reagent of embodiment C58.
C61. An isolated nucleic acid molecule comprising a nucleotide sequence that encodes an immunoglobulin heavy chain variable domain of the anti-NKG2A agent of any one of embodiments Cl to C57.
C62. An isolated nucleic acid molecule comprising a nucleotide sequence that encodes an immunoglobulin light chain variable domain of the anti-NKG2A agent of any one of embodiments C 1 to C57.
C63. A recombinant expression vector comprising a first expression cassette and a second expression cassette, wherein the first expression cassette comprises a promoter and a nucleic acid molecule comprising a nucleotide sequence that encodes an immunoglobulin heavy chain variable domain of the anti-NKG2A agent of any one of embodiments Cl to C57, and the second expression cassette comprises a promoter and a nucleic acid molecule comprising a nucleotide sequence that encodes an immunoglobulin light chain variable domain of the anti-NKG2A agent of any one of embodiments C 1 to C57.
C64. A recombinant host cell transfected with the recombinant expression vector of embodiment C63. C65. A method of detecting NKG2A, comprising contacting a sample known or suspected to contain NKG2A with the anti-NKG2A agent of any one of embodiments Cl to C57, and, if the sample contains NKG2A, detecting NKG2A:anti-NKG2A complexes.
C66. A method of detecting NKG2A in a heterogeneous population of immune cells, comprising contacting the population with an anti-NKG2A agent of any one of embodiments Cl to C57, and, if the population contains cells expressing NKG2A, detecting NKG2A:anti-NKG2A complexes.
D 1. An anti-NKG2A agent that binds the NKG2A receptor or a fragment thereof, comprising one or more of: a) an immunoglobulin heavy chain variable domain comprising:
(i) a heavy chain complementary determining region 1 (CDRH1) comprising the sequence
X1YX3MX5 (SEQ ID NO: 22), wherein X1 is S or D; X3 is W or Y, and X5 is N or Y;
(ii) a heavy chain complementary determining region 2 (CDRH2) comprising the sequence
X1IX3X4X5X6X7X8TX10YX12X13X14X15KX17 (SEQ ID NO: 23), wherein X1 is R or T; X3 is Y or S; X4 is F or Y; X5 is E or G; X6 is D or G; X7 is G or I; X8 is D or Y; X10 is N or Y; X12 is N or P; X13 is G or D; X14 is K or S; X15 is F or V, and X17 is G or D; and
(iii) a heavy chain complementary determining region 3 (CDRH3) comprising the sequence XIX2X3X4X5X6X7X8X9YXIIXI2XI3XI4XI5 (SEQ ID NO: 24), wherein X1 is Q or D; X2 is G or R; X3 is D or no amino acid; X4 is T or no amino acid; X5 is S or no amino acid; X6 is V or no amino acid; X7 is I or Y; X8 is W or V; X9 is D or N; X11 is D or Y; X12 is G or T; X13 is F or M; X14 is A or D; and
X15 is D or Y; and b) an immunoglobulin light chain variable domain comprising:
(i) a light chain complementary determining region 1 (CDRL1) comprising the sequence
X1ASX4SVX7X8X9X10X11X12YX14H (SEQ ID NO: 25), wherein X1 is S or R; X4 is S or K; X7 is T or S; X8 is T or no amino acid; X9 is S or no amino acid; X10 is G or no amino acid; X11 is Y or no amino acid; X12 is S or no amino acid; and X14 is M or I;
(ii) a light chain complementary determining region 2 (CDRL2) comprising the sequence
X1X2SNLX6S (SEQ ID NO: 26), wherein X1 is S or L; X2 is T or A; and X6 is A or E; and
(iii) a light chain complementary determining region 3 (CDRL3) comprising the sequence QX2X3X4X5X6PX8T (SEQ ID NO: 27), wherein X2 is Q or H; X3 is R or S; X4 is S or R; X5 is S or E; X6 is Y or L; and X8 is Y or L.
D2. The anti-NKG2A agent of embodiment DI, wherein the immunoglobulin heavy chain variable domain comprises: a CDRH1 comprising the sequence of amino acids set forth in SEQ ID NO: 9 or SEQ ID NO: 16, or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:9 or SEQ ID NO: 16; a CDRH2 comprising the sequence of amino acids set forth in SEQ ID NO: 10, 15 or 17, or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 10, 15 or 17; and a CDRH3 comprising the sequence of amino acids set forth in SEQ ID NO: 11 or SEQ ID NO: 18, or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 11 or SEQ ID NO: 18.
D3. The anti-NKG2A agent of embodiment D 1 or embodiment D2, wherein the immunoglobulin heavy chain variable domain comprises: a CDRH1 comprising the sequence of amino acids set forth in SEQ ID NO: 9 or SEQ ID NO: 16; a CDRH2 comprising the sequence of amino acids set forth in SEQ ID NO: 10, 15 or 17; and a CDRH3 comprising the sequence of amino acids set forth in SEQ ID NO: 11 or SEQ ID NO: 18.
D4.The anti-NKG2A agent of any of embodiments D1-D3, wherein the immunoglobulin light chain variable domain comprises: a CDRL1 comprising the sequence of amino acids set forth in SEQ ID NO: 12 or SEQ ID NO: 19, or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 12 or SEQ ID NO: 19; a CDRL2 comprising the sequence of amino acids set forth in SEQ ID NO: 13 or SEQ ID NO:20, or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 13 or SEQ ID NO:20; and a CDRL3 comprising the sequence of amino acids set forth in SEQ ID NO: 14 or SEQ ID NO:21, or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 14 or SEQ ID NO:21.
D5. The anti-NKG2A agent of any of embodiments D1-D4, wherein the immunoglobulin light chain variable domain comprises: a CDRL1 comprising a sequence of amino acids set forth in SEQ ID NO: 12 or SEQ ID NO: 19; a CDRL2 comprising a sequence of amino acids set forth in SEQ ID NO: 13 or SEQ ID NO:20; and a CDRL3 comprising a sequence of amino acids set forth in SEQ ID NO: 14 or SEQ ID NO:21.
D6. The anti-NKG2A agent of any of embodiments D1-D5, wherein the CDRH1 comprises the sequence set forth in SEQ ID NO: 9, or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 9, the CDRH2 comprises the sequence set forth in SEQ ID NO: 10 ,or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 10; and the CDRH3 comprises the sequence set forth in SEQ ID NO: 11, or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:11. D7. The anti-NKG2A agent of any of embodiments D1-D6, wherein the CDRH1 comprises the sequence set forth in SEQ ID NO: 9, the CDRH2 comprises the sequence set forth in SEQ ID NO: 10; and the CDRH3 comprises the sequence set forth in SEQ ID NO: 11. D8. The anti-NKG2A agent of any of embodiments D1-D5, wherein the CDRH1 comprises the sequence set forth in SEQ ID NO: 9, or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:9; the CDRH2 comprises the sequence set forth in SEQ ID NO: 15 or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:15; and the CDRH3 comprises the sequence set forth in SEQ ID NO: 11 or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:11. D9. The anti-NKG2A agent of any of embodiments D1-D5 and D8, wherein the CDRH1 comprises the sequence set forth in SEQ ID NO: 9, the CDRH2 comprises the sequence set forth in SEQ ID NO: 15; and the CDRH3 comprises the sequence set forth in SEQ ID NO: 11. D10. The anti-NKG2A agent of any of embodiments D1-D5, wherein the CDRH1 comprises the sequence set forth in SEQ ID NO: 16, or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:16; the CDRH2 comprises the sequence set forth in SEQ ID NO: 17 or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:17; and the CDRH3 comprises the sequence set forth in SEQ ID NO: 18 or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:18. D11. The anti-NKG2A agent of any of embodiments D1-D5 and D10, wherein the CDRH1 comprises the sequence set forth in SEQ ID NO: 16, the CDRH2 comprises the sequence set forth in SEQ ID NO: 17; and the CDRH3 comprises the sequence set forth in SEQ ID NO: 18. D12. The anti-NKG2A agent of any of embodiments D1-D11, wherein the CDRL1 comprises the sequence set forth in SEQ ID NO: 12, or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:12; the CDRL2 comprises the sequence set forth in SEQ ID NO: 13 or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 13; and the CDRL3 comprises the sequence set forth in SEQ ID NO: 14 or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID
NO: 14.
D13. The anti-NKG2A agent of any of embodiments DI -DI 2, wherein the CDRL1 comprises the sequence set forth in SEQ ID NO: 12, the CDRL2 comprises the sequence set forth in SEQ ID NO: 13; and the CDRL3 comprises the sequence set forth in SEQ ID NO: 14.
D14. The anti-NKG2A agent of any of embodiments Dl-Dl 1, wherein the CDRL1 comprises the sequence set forth in SEQ ID NO: 19 or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
99% or more sequence identity to SEQ ID NO: 19, the CDRL2 comprises the sequence set forth in
SEQ ID NO: 20 or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:20; and the CDRL3 comprises the sequence set forth in SEQ ID NO: 21 or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID
NO:21.
D15. The anti-NKG2A agent of any of embodiments DI -DI 1 and D14, wherein the CDRL1 comprises the sequence set forth in SEQ ID NO: 19, the CDRL2 comprises the sequence set forth in SEQ ID NO: 20; and the CDRL3 comprises the sequence set forth in SEQ ID NO: 21.
DI 6. The anti-NKG2A agent of any of embodiments DI -DI 5, wherein the CDRH1 comprises the sequence of amino acids set forth in SEQ ID NO: 9 or SEQ ID NO: 16, or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:9 or SEQ ID NO: 16; the
CDRH2 comprises a sequence of amino acids set forth in SEQ ID NO: 10, 15 or 17, or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 10, 15 or 17; the CDRH3 comprises a sequence of amino acids set forth in SEQ ID NO: 11 or SEQ ID NO: 18, or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID
NO: 11 or SEQ ID NO: 18; the CDRL1 comprises a sequence of amino acids set forth in SEQ ID NO:
12 or SEQ ID NO: 19, or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%,
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 12 or SEQ ID NO: 19; the CDRL2 comprises a sequence of amino acids set forth in SEQ ID NO: 13 or SEQ ID NO:20, or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 13 or SEQ ID NO:20; the CDRL3 comprises a sequence of amino acids set forth in SEQ ID NO: 14 or SEQ ID NO:21, or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 14 or SEQ ID NO:21.
DI 7. The anti-NKG2A agent of any of embodiments DI -DI 6, wherein the CDRH1 comprises the sequence of amino acids set forth in SEQ ID NO: 9 or SEQ ID NO: 16; the CDRH2 comprises the sequence of amino acids set forth in SEQ ID NO: 10, 15 or 17; the CDRH3 comprises the sequence of amino acids set forth in SEQ ID NO: 11 or SEQ ID NO: 18; the CDRL1 comprises the sequence of amino acids set forth in SEQ ID NO: 12 or SEQ ID NO: 19; the CDRL2 comprises the sequence of amino acids set forth in SEQ ID NO: 13 or SEQ ID NO:20; and the CDRL3 comprises the sequence of amino acids set forth in SEQ ID NO: 14 or SEQ ID NO:21.
DI 8. The anti-NKG2A agent of any of embodiments DI -DI 7, wherein the CDRH1 comprises the sequence set forth in SEQ ID NO: 9 or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:9, the CDRH2 comprises the sequence set forth in SEQ ID NO: 10 or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 10; the CDRH3 comprises the sequence set forth in SEQ ID NO: 11 or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 11; the CDRL1 comprises the sequence set forth in SEQ ID NO: 12 or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 12, the CDRL2 comprises the sequence set forth in SEQ ID NO: 13 or a sequence of amino acids that exhibits at least 80%,
81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99% or more sequence identity to SEQ ID NO: 13; and the CDRL3 comprises the sequence set forth in SEQ ID NO: 14 or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%,
84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 14.
D19. The anti-NKG2A agent of any of embodiments D1-D18, wherein the CDRH1 comprises the sequence set forth in SEQ ID NO: 9, the CDRH2 comprises the sequence set forth in SEQ ID NO: 10; the CDRH3 comprises the sequence set forth in SEQ ID NO: 11; the CDRL1 comprises the sequence set forth in SEQ ID NO: 12, the CDRL2 comprises the sequence set forth in SEQ ID NO: 13; and the CDRL3 comprises the sequence set forth in SEQ ID NO: 14. D20. The anti-NKG2A agent of any of embodiments DI -DI 7, wherein the CDRH1 comprises the sequence set forth in SEQ ID NO: 9 or a sequence of amino acids that exhibits at least 80%, 81%,
82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
99% or more sequence identity to SEQ ID NO:9; the CDRH2 comprises the sequence set forth in
SEQ ID NO: 15 or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 15; the CDRH3 comprises the sequence set forth in SEQ ID NO: 11 or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID
NO: 11; the CDRL1 comprises the sequence set forth in SEQ ID NO: 12 or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 12, the CDRL2 comprises the sequence set forth in SEQ ID NO: 13 or a sequence of amino acids that exhibits at least 80%,
81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99% or more sequence identity to SEQ ID NO: 13; and the CDRL3 comprises the sequence set forth in SEQ ID NO: 14 or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%,
84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 14.
D21. The anti-NKG2A agent of any of embodiments DI -DI 7 and D20, wherein the CDRH1 comprises the sequence set forth in SEQ ID NO: 9, the CDRH2 comprises the sequence set forth in SEQ ID NO: 15; the CDRH3 comprises the sequence set forth in SEQ ID NO: 11; the CDRL1 comprises the sequence set forth in SEQ ID NO: 12, the CDRL2 comprises the sequence set forth in SEQ ID NO: 13; and the CDRL3 comprises the sequence set forth in SEQ ID NO: 14.
D22. The anti-NKG2A agent of any of embodiments DI -DI 7, wherein the CDRH1 comprises the sequence set forth in SEQ ID NO: 16 or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
99% or more sequence identity to SEQ ID NO: 16, the CDRH2 comprises the sequence set forth in
SEQ ID NO: 17 or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 17; the CDRH3 comprises the sequence set forth in SEQ ID NO: 18 or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID
NO: 18; the CDRL1 comprises the sequence set forth in SEQ ID NO: 19 or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 19, the CDRL2 comprises the sequence set forth in SEQ ID NO: 20 or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:20; and the CDRL3 comprises the sequence set forth in SEQ ID NO: 21 or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:21.
D23. The anti-NKG2A agent of any of embodiments DI -DI 7 and D22, wherein the CDRH1 comprises the sequence set forth in SEQ ID NO: 16, the CDRH2 comprises the sequence set forth in SEQ ID NO: 17; the CDRH3 comprises the sequence set forth in SEQ ID NO: 18; the CDRL1 comprises the sequence set forth in SEQ ID NO: 19, the CDRL2 comprises the sequence set forth in SEQ ID NO: 20; and the CDRL3 comprises the sequence set forth in SEQ ID NO: 21.
D24. The anti-NKG2A agent of any of embodiments D1-D23, wherein the immunoglobulin heavy chain variable domain comprises the amino acid sequence set forth in any of SEQ ID NOS: 1, 3 or 4, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to any of SEQ ID NOS: 1, 3 or 4.
D25. The anti-NKG2A agent of any of embodiments D1-D24, wherein the immunoglobulin heavy chain variable domain comprises the amino acid sequence set forth in any of SEQ ID NOS: 1, 3 or 4. D26. The anti-NKG2A agent of any of embodiments D1-D25, wherein the immunoglobulin heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 1 or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 1.
D27. The anti-NKG2A agent of any of embodiments D1-D26, wherein the immunoglobulin heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 1.
D28. The anti-NKG2A agent of any of embodiments D1-D25, wherein the immunoglobulin heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 3, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:3.
D29. The anti-NKG2A agent of any of embodiments D1-D25 and D28, wherein the immunoglobulin heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 3.
D30. The anti-NKG2A agent of any of embodiments D1-D25, wherein the immunoglobulin heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 4, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:4.
D31. The anti-NKG2A agent of any of embodiments D 1 -D25 and D30, wherein the immunoglobulin heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 4. D32. The anti-NKG2A agent of any of embodiments D1-D31, wherein the immunoglobulin light chain variable domain comprises the amino acid sequence set forth in any of SEQ ID NOS: 5 or 8, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to any of SEQ ID NOS:5 or 8.
D33. The anti-NKG2A agent of any of embodiments D1-D32, wherein the immunoglobulin light chain variable domain comprises the amino acid sequence set forth in any of SEQ ID NOS: 5 or 8. D34. The anti-NKG2A agent of any of embodiments D1-D33, wherein the immunoglobulin light chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 5, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:5.
D35. The anti-NKG2A agent of any of embodiments D1-D34, wherein the immunoglobulin light chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 5.
D36. The anti-NKG2A agent of any of embodiments D1-D33, wherein the immunoglobulin light chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 8, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 8.
D37. The anti-NKG2A agent of any of embodiments D1-D33 and D36, wherein the immunoglobulin light chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 8.
D38. The anti-NKG2A agent of any of embodiments D1-D37, wherein the immunoglobulin heavy chain variable domain comprises the amino acid sequence set forth in any of SEQ ID NOS: 1, 3 or 4, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to any of SEQ ID NOS: 1, 3 or 4, and the immunoglobulin light chain variable domain comprises the amino acid sequence set forth in any of SEQ ID NOS: 5 or 8, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to any of SEQ ID NOS: 5 or 8.
D39. The anti-NKG2A agent of any of embodiments D1-D38, wherein the immunoglobulin heavy chain variable domain comprises the amino acid sequence set forth in any of SEQ ID NOS: 1, 3 or 4, and the immunoglobulin light chain variable domain comprises the amino acid sequence set forth in any of SEQ ID NOS: 5 or 8.
D40. The anti-NKG2A agent of any of embodiments D1-D39, wherein the immunoglobulin heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 1, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 1; and the immunoglobulin light chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 5, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:5.
D41. The anti-NKG2A agent of any of embodiments D1-D40, wherein the immunoglobulin heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 1, and the immunoglobulin light chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 5.
D42. The anti-NKG2A agent of any of embodiments D1-D39, wherein the immunoglobulin heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 3, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 3; and the immunoglobulin light chain variable domain comprises the amino acid sequence set forth in SEQ ID NOS: 5, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:5.
D43. The anti-NKG2A agent of any of embodiments D1-D39 and D42, wherein the immunoglobulin heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 3, and the immunoglobulin light chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 5.
D44. The anti-NKG2A agent of any of embodiments D1-D39, wherein the immunoglobulin heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 4, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 4; and the immunoglobulin light chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 8, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:8.
D45. The anti-NKG2A agent of any of embodiments D1-D39 and D44, wherein the immunoglobulin heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 4, and the immunoglobulin light chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 8.
D46. The anti-NKG2A agent of any of embodiments D1-D45, comprising one immunoglobulin heavy chain variable domain and one immunoglobulin light chain variable domain.
D47. The anti-NKG2A agent of any of embodiments D1-D45, comprising two immunoglobulin heavy chain variable domains and two immunoglobulin light chain variable domains. D48. The anti-NKG2A agent of any of embodiments D1-D47, wherein the immunoglobulin heavy chain variable domain further comprises a signal sequence.
D49. The anti-NKG2A agent of embodiment D48, wherein the signal sequence comprises the sequence set forth in SEQ ID NO: 48 or SEQ ID NO: 52.
D50. The anti-NKG2A agent of any of embodiments D1-D49, wherein the immunoglobulin light chain variable domain further comprises a signal sequence.
D51. The anti-NKG2A agent of embodiment D50, wherein the signal sequence comprises the sequence set forth in SEQ ID NO: 46 or SEQ ID NO: 50.
D52. The anti-NKG2A agent of any of any of embodiments D1-D51, wherein the anti-NKG2A agent is an antibody or antigen-binding fragment thereof.
D53. The anti-NKG2A agent of any of embodiments D1-D51, wherein the anti-NKG2A agent is isolated.
D54. The anti-NKG2A agent of any of embodiments D1-D52, wherein the anti-NKG2A agent is humanized.
D55. The anti-NKG2A agent of any of embodiments D1-D54, wherein the anti-NKG2A agent is conjugated.
D56.The anti-NKG2A agent of embodiment D55, wherein the anti-NKG2A agent is conjugated to a detectable marker or label.
D57. The anti-NKG2A agent of embodiment D55 or embodiment D56, wherein the detectable marker or label comprises a detectable moiety.
D58.The anti-NKG2A agent of embodiment D57, wherein the detectable moiety is a radioisotope, fluorescent label or enzyme-substrate label.
D59. The anti-NKG2A agent of any of embodiments D1-D58, wherein the anti-NKG2A agent is non- diffusively immobilized on a solid support.
D60. The anti-NKG2A agent of any of embodiments D1-D59, that is a single chain fragment.
D61.The anti-NKG2A agent of embodiment D60, wherein the single chain fragment is a single chain variable fragment (scFv).
D62.The anti-NKG2A agent of any of embodiments D1-D61, for use in the detection of NKG2A in a sample.
D63.The anti-NKG2A agent of any of embodiments D1-D62, wherein the anti-NKG2A agent binds to an NKG2A expressing cell in a sample.
D64.The anti-NKG2A agent of embodiment D62 or embodiment D63, wherein the sample comprises immune cells.
D65.The anti-NKG2A of any of embodiments D62-D64, wherein the sample comprises a heterogenous population of immune cells. D66.The anti-NKG2A of embodiment D64 or embodiment D65, wherein the immune cell is selected from B cells, plasmacytoid dendritic cells (pDCs), lymphocytes, leukocytes, T cells, monocytes, macrophages, neutrophils, myeloid dendritic cells (mDCs), innate lymphoid cells, mast cells, eosinophils, basophils, natural killer cells, and peripheral blood mononuclear cells (PBMCs).
D67.The anti-NKG2A of any of embodiments D62-D66, wherein the detection comprises the use of a single anti-NKG2A agent to identify the presence of a complex comprising NKG2A and the anti- NKG2A agent.
D68.The anti-NKG2A of any of embodiments D62-D66, wherein the detection comprises the use of two anti-NKG2A agents, each capable of binding to a different portion of NKG2A.
D69.The anti-NKG2A of any of embodiments D62-D68, wherein the detection of NKG2A is on the surface of immune cells.
D70. The anti-NKG2A of any of embodiments D62-D69, wherein NKG2A is detected intracellularly. D71.The anti-NKG2A of any of embodiments D62-D70, wherein the detection of NKG2A indicates the presence or absence of a disease or disorder.
D72. The anti-NKG2A of any of embodiments D62-D71, wherein the detection is performed in vitro. D73. The anti-NKG2A of any of embodiments D62-D72, wherein the detection is performed in vivo. D74.The anti-NKG2A agent of any of embodiments D1-D73, wherein the anti-NKG2A agent binds to an NKG2A expressing cell, and blocks the binding of NKG2A to HLA-E on a target cell.
D75.The anti-NKG2A agent of any of embodiments D1-D74, wherein the anti-NKG2A agent blocks the binding of NKG2A on an NKG2A expressing cell to HLA-E on a target cell.
D76.The anti-NKG2A agent of any of embodiments D63-D75, wherein the anti-NKG2A agent bound to the NKG2A expressing cell increases the cytotoxic activity of the NKG2A expressing cell.
D77.The anti-NKG2A agent of any of embodiments D63-D76, wherein the anti-NKG2A agent bound to the NKG2A expressing cell induces lysis of a target cell.
D78.The anti-NKG2A agent of any of embodiments D74-D77, wherein the target cell is a cell that expresses HLA-E.
D79.The anti-NKG2A agent of any of embodiments D74-D77, wherein the target cell is a cell associated with a disease or disorder.
D80.The anti-NKG2A agent of embodiment D79, wherein the disease or disorder is a cancer, a viral infection, an autoimmune disease, an inflammatory disease or a bacterial infection.
D81. The anti-NKG2A agent of embodiment D79 or embodiment D80, wherein the disease or disorder is a cancer.
D82.The anti-NKG2A agent of embodiment D87, wherein the cancer is acute myeloid leukemia, acute lymphoblastic leukemia, colorectal, ovarian, gynecologic, liver, glioblastoma, Hodgkin lymphoma, chronic lymphocytic leukemia, esophagus, gastric, pancreas, colon, kidney, head and neck, lung and melanoma. D83. The anti-NKG2A agent of embodiment D79 or embodiment D80, wherein the disease or disorder is an autoimmune disease.
D84.The anti-NKG2A agent of any of embodiments D79-D83, wherein the disease or disorder is a condition characterized by NK cell hyperactivity or NK cell hyperproliferation.
D85.A diagnostic reanti-NKG2A agent comprising the anti-NKG2A agent of any of embodiments D1-D84.
D86.A kit comprising the anti-NKG2A agent of any of embodiments D1-D84, or the diagnostic reagent of embodiment D85.
D87.A composition comprising the anti-NKG2A agent of any of embodiments D1-D84, and a pharmaceutically acceptable excipient.
D88.The composition of embodiment D87, wherein the anti-NKG2A is used as an adjuvant or in conjunction with an adjuvant.
D89.An isolated nucleic acid comprising a nucleotide sequence that encodes the immunoglobulin heavy chain variable domain of the agent of any of embodiments D 1-D84.
D90.The isolated nucleic acid of embodiment D89, wherein the immunoglobulin heavy chain variable domain comprises the sequence of nucleotides set forth in any of SEQ ID NOs: 30-37.
D91.The isolated nucleic acid of embodiment D89 or embodiment D90, wherein the immunoglobulin heavy chain variable domain comprises the sequence of nucleotides set forth in SEQ ID NO: 30.
D92. The isolated nucleic acid of embodiment D89 or embodiment D90, wherein the immunoglobulin heavy chain variable domain comprises the sequence of nucleotides set forth in SEQ ID NO: 31.
D93. The isolated nucleic acid of embodiment D89 or embodiment D90, wherein the immunoglobulin heavy chain variable domain comprises the sequence of nucleotides set forth in SEQ ID NO: 32.
D94. The isolated nucleic acid of embodiment D89 or embodiment D90, wherein the immunoglobulin heavy chain variable domain comprises the sequence of nucleotides set forth in SEQ ID NO: 33.
D95. The isolated nucleic acid of embodiment D89 or embodiment D90, wherein the immunoglobulin heavy chain variable domain comprises the sequence of nucleotides set forth in SEQ ID NO: 34.
D96. The isolated nucleic acid of embodiment D89 or embodiment D90, wherein the immunoglobulin heavy chain variable domain comprises the sequence of nucleotides set forth in SEQ ID NO: 35.
D97. The isolated nucleic acid of embodiment D89 or embodiment D90, wherein the immunoglobulin heavy chain variable domain comprises the sequence of nucleotides set forth in SEQ ID NO: 36.
D98. The isolated nucleic acid of embodiment D89 or embodiment D90, wherein the immunoglobulin heavy chain variable domain comprises the sequence of nucleotides set forth in SEQ ID NO: 37.
D99. An isolated nucleic acid comprising a nucleotide sequence that encodes the immunoglobulin light chain variable domain of the agent of any of embodiments D1-D84.
DI 00. The isolated nucleic acid molecule of embodiment D99, wherein the immunoglobulin light chain variable domain comprises the sequence of nucleotides set forth in any of SEQ ID NOs: 38-45. D101. The isolated nucleic acid of embodiment D99 or embodiment DI 00, wherein the immunoglobulin light chain variable domain comprises the sequence of nucleotides set forth in SEQ ID NO: 38.
DI 02. The isolated nucleic acid of embodiment D99 or embodiment DI 00, wherein the immunoglobulin light chain variable domain comprises the sequence of nucleotides set forth in SEQ ID NO: 39.
DI 03. The isolated nucleic acid of embodiment D99 or embodiment DI 00, wherein the immunoglobulin light chain variable domain comprises the sequence of nucleotides set forth in SEQ ID NO: 40.
DI 04. The isolated nucleic acid of embodiment D99 or embodiment DI 00, wherein the immunoglobulin light chain variable domain comprises the sequence of nucleotides set forth in SEQ ID NO: 41.
DI 05. The isolated nucleic acid of embodiment D99 or embodiment DI 00, wherein the immunoglobulin light chain variable domain comprises the sequence of nucleotides set forth in SEQ ID NO: 42.
DI 06. The isolated nucleic acid of embodiment D99 or embodiment DI 00, wherein the immunoglobulin light chain variable domain comprises the sequence of nucleotides set forth in SEQ ID NO: 43.
DI 07. The isolated nucleic acid of embodiment D99 or embodiment DI 00, wherein the immunoglobulin light chain variable domain comprises the sequence of nucleotides set forth in SEQ ID NO: 44.
DI 08. The isolated nucleic acid of embodiment D99 or embodiment DI 00, wherein the immunoglobulin light chain variable domain comprises the sequence of nucleotides set forth in SEQ ID NO: 45.
D109. An isolated nucleic acid comprising a nucleotide sequence that encodes the immunoglobulin heavy chain variable domain and the immunoglobulin light chain variable domain of the anti-NKG2A agent of any of embodiments D1-D84.
DI 10. The isolated nucleic acid of any of embodiments D89-D109, wherein the nucleotide sequence that encodes the immunoglobulin heavy chain variable domain comprises the sequence set forth in any of SEQ ID NOs: 30-37; and the immunoglobulin light chain variable domain comprises the sequence of amino acids set forth in any of SEQ ID NOs: 38-45.
DI 11. The isolated nucleic acid of embodiment DI 10, wherein the nucleotide sequence that encodes the immunoglobulin heavy chain variable domain comprises the sequence set forth in SEQ ID NO: 30; and the immunoglobulin light chain variable domain comprises the sequence of amino acids set forth in SEQ ID NO: 38. DI 12. The isolated nucleic acid of embodiment DI 10, wherein the nucleotide sequence that encodes the immunoglobulin heavy chain variable domain comprises the sequence set forth in SEQ ID NO: 31 ; and the immunoglobulin light chain variable domain comprises the sequence of amino acids set forth in SEQ ID NO: 39.
DI 13. The isolated nucleic acid of embodiment DI 10, wherein the nucleotide sequence that encodes the immunoglobulin heavy chain variable domain comprises the sequence set forth in SEQ ID NO: 32; and the immunoglobulin light chain variable domain comprises the sequence of amino acids set forth in SEQ ID NO: 40.
DI 14. The isolated nucleic acid of embodiment DI 10, wherein the nucleotide sequence that encodes the immunoglobulin heavy chain variable domain comprises the sequence set forth in SEQ ID NO: 33; and the immunoglobulin light chain variable domain comprises the sequence of amino acids set forth in SEQ ID NO: 41.
DI 15. The isolated nucleic acid of embodiment DI 10, wherein the nucleotide sequence that encodes the immunoglobulin heavy chain variable domain comprises the sequence set forth in SEQ ID NO: 34; and the immunoglobulin light chain variable domain comprises the sequence of amino acids set forth in SEQ ID NO: 42.
DI 16. The isolated nucleic acid of embodiment DI 10, wherein the nucleotide sequence that encodes the immunoglobulin heavy chain variable domain comprises the sequence set forth in SEQ ID NO: 35; and the immunoglobulin light chain variable domain comprises the sequence of amino acids set forth in SEQ ID NO: 43.
DI 17. The isolated nucleic acid of embodiment DI 10, wherein the nucleotide sequence that encodes the immunoglobulin heavy chain variable domain comprises the sequence set forth in SEQ ID NO: 36; and the immunoglobulin light chain variable domain comprises the sequence of amino acids set forth in SEQ ID NO: 44.
DI 18. The isolated nucleic acid of embodiment DI 10, wherein the nucleotide sequence that encodes the immunoglobulin heavy chain variable domain comprises the sequence set forth in SEQ ID NO: 37; and the immunoglobulin light chain variable domain comprises the sequence of amino acids set forth in SEQ ID NO: 45.
DI 19. A recombinant expression vector comprising a first expression cassette and a second expression cassette, wherein the first expression cassette comprises a nucleic acid molecule comprising a nucleotide sequence that encodes the immunoglobulin heavy chain variable domain of any one of embodiments D1-D84, and the second expression cassette comprises a nucleic acid molecule comprising a nucleotide sequence that encodes an immunoglobulin light chain variable domain of the anti-NKG2A agent of any one of embodiments D1-D84.
D120. A recombinant expression vector comprising a first expression cassette and a second expression cassette, wherein the first expression cassette comprises a nucleic acid molecule comprising the nucleotide sequence of any of embodiments D89-D118, and the second expression cassette comprises a nucleic acid molecule comprising the nucleotide sequence of any of embodiments D89-D118.
D121. The recombinant expression vector of embodiment DI 19 or embodiment DI 20, wherein the first and second expression cassettes comprise a promoter.
D122. A host cell transfected with the recombinant expression vector of any of embodiments DI 19- D121.
DI 23. A method of detecting NKG2A, comprising a) contacting a sample with the anti-NKG2A agent of any of embodiments 1-33, under conditions to bind said anti-NKG2A to an NKG2A receptor on said sample, wherein the binding generates the production of a receptor/anti-NKG2A complex; b) detecting the presence of the receptor/anti-NKG2A complexes; c) wherein the detecting comprises the presence or absence of the NKG2A receptor on said sample. D124.The method of embodiment DI 23, wherein the method is performed in vitro.
DI 25. A method of treating or preventing a disease or disorder associated with NKG2A in a subject, comprising: a) contacting a sample known or suspected to contain NKG2A with the anti-NKG2A agent of any of embodiments D1-D84, b) detecting the presence of complexes comprising NKG2A and the anti-NKG2A agent; wherein the presence of the complexes indicates the presence of a disease or disorder; and c) administering to the subject the anti-NKG2A agent of any of embodiments D1-D84.
D126.A method of treating or preventing a disease or disorder in a subject, comprising a) detecting the formation of complexes comprising NKG2A and the anti-NKG2A agent of any of embodiments D1-D84 in a sample from the subject; b) administering to a subject in need thereof the anti-NKG2A agent of any of embodiments D1-D84.
D127. The method of any of embodiments D123-D126, wherein the sample comprises immune cells. D128. The method of embodiment DI 27, wherein the immune cell is selected from B cells, plasmacytoid dendritic cells (pDCs), lymphocytes, leukocytes, T cells, monocytes, macrophages, neutrophils, myeloid dendritic cells (mDCs), innate lymphoid cells, mast cells, eosinophils, basophils, natural killer cells, and peripheral blood mononuclear cells (PBMCs).
Figure imgf000123_0001
The entirety of each patent, patent application, publication and document referenced herein hereby is incorporated by reference. Citation of the above patents, patent applications, publications and documents is not an admission that any of the foregoing is pertinent prior art, nor does it constitute any admission as to the contents or date of these publications or documents. Their citation is not an indication of a search for relevant disclosures. All statements regarding the date(s) or contents of the documents is based on available information and is not an admission as to their accuracy or correctness.
Modifications may be made to the foregoing without departing from the basic aspects of the technology. Although the technology has been described in substantial detail with reference to one or more specific embodiments, those of ordinary skill in the art will recognize that changes may be made to embodiments specifically disclosed in this application, yet these modifications and improvements are within the scope and spirit of the technology.
The technology illustratively described herein suitably may be practiced in the absence of any element(s) not specifically disclosed herein. Thus, for example, in each instance herein any of the terms “comprising,” “consisting essentially of,” and “consisting of’ may be replaced with either of the other two terms. The terms and expressions which have been employed are used as terms of description and not of limitation, and use of such terms and expressions do not exclude nay equivalents of the features shown and described or portions thereof, and various modifications are possible within the scope of the technology claimed. The term “a” or “an” can refer to one of or a plurality of the elements it modifies (e.g., “a reagent” can mean one or more reagents) unless it is contextually clear either one of the elements or more than one of the elements is described. The term “about” as used herein refers to a value within 10% of the underlying parameter (i.e., plus or minus 10%), and use of the term “about” at the beginning of a string of values modifies each of the values (i.e., “about 1, 2, and 3” refers to about 1, about 2, and about 3). For example, a weight of “about 100 grams” can include weights between 90 grams and 110 grams. Further, when a listing of values is described herein (e.g., about 50%, 60%, 70%, 80%, 85%, or 86%) the listing included all intermediate and fractional values thereof (e.g., 54%, 85.4%). Thus, it should be understood that although the present technology has been specifically disclosed by representative embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and such modifications and variations are considered within the scope of this technology.
Certain embodiments of the technology are set forth in the claim(s) that follow(s).

Claims

What is claimed is: 1. An anti-NKG2A agent that binds the NKG2A receptor or a fragment thereof, comprising one or more of: a) an immunoglobulin heavy chain variable domain comprising: (i) a heavy chain complementary determining region 1 (CDRH1) comprising the sequence X1YX3MX5 (SEQ ID NO: 22), wherein X1 is S or D; X3 is W or Y, and X5 is N or Y; (ii) a heavy chain complementary determining region 2 (CDRH2) comprising the sequence X1IX3X4X5X6X7X8TX10YX12X13X14X15KX17 (SEQ ID NO: 23), wherein X1 is R or T; X3 is Y or S; X4 is F or Y; X5 is E or G; X6 is D or G; X7 is G or I; X8 is D or Y; X10 is N or Y; X12 is N or P; X13 is G or D; X14 is K or S; X15 is F or V, and X17 is G or D; and (iii) a heavy chain complementary determining region 3 (CDRH3) comprising the sequence X1X2X3X4X5X6X7X8X9YX11X12X13X14X15 (SEQ ID NO: 24), wherein X1 is Q or D; X2 is G or R; X3 is D or no amino acid; X4 is T or no amino acid; X5 is S or no amino acid; X6 is V or no amino acid; X7 is I or Y; X8 is W or V; X9 is D or N; X11 is D or Y; X12 is G or T; X13 is F or M; X14 is A or D; and X15 is D or Y; and b) an immunoglobulin light chain variable domain comprising: (i) a light chain complementary determining region 1 (CDRL1) comprising the sequence X1ASX4SVX7X8X9X10X11X12YX14H (SEQ ID NO: 25), wherein X1 is S or R; X4 is S or K; X7 is T or S; X8 is T or no amino acid; X9 is S or no amino acid; X10 is G or no amino acid; X11 is Y or no amino acid; X12 is S or no amino acid; and X14 is M or I; (ii) a light chain complementary determining region 2 (CDRL2) comprising the sequence X1X2SNLX6S (SEQ ID NO: 26), wherein X1 is S or L; X2 is T or A; and X6 is A or E; and (iii) a light chain complementary determining region 3 (CDRL3) comprising the sequence QX2X3X4X5X6PX8T (SEQ ID NO: 27), wherein X2 is Q or H; X3 is R or S; X4 is S or R; X5 is S or E; X6 is Y or L; and X8 is Y or L.
2. The anti-NKG2A agent of claim 1, wherein the immunoglobulin heavy chain variable domain comprises: a CDRH1 comprising the sequence of amino acids set forth in SEQ ID NO: 9 or SEQ ID NO:16, or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:9 or SEQ ID NO:16; a CDRH2 comprising the sequence of amino acids set forth in SEQ ID NO: 10, 15 or 17, or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 10, 15 or 17; and a CDRH3 comprising the sequence of amino acids set forth in SEQ ID NO: 11 or SEQ ID NO:18, or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 11 or SEQ ID NO: 18.
3. The anti-NKG2A agent of claim 1 or claim 2, wherein the immunoglobulin light chain variable domain comprises: a CDRL1 comprising the sequence of amino acids set forth in SEQ
ID NO: 12 or SEQ ID NO: 19, or a sequence of amino acids that exhibits at least 80%, 81%, 82%,
83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 12 or SEQ ID NO: 19; a CDRL2 comprising the sequence of amino acids set forth in SEQ ID NO: 13 or SEQ ID NO:20, or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 13 or SEQ ID NO:20; and a CDRL3 comprising the sequence of amino acids set forth in SEQ ID NO: 14 or SEQ ID NO:21, or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 14 or
SEQ ID NO:21.
4. The anti-NKG2A agent of any one of claims 1-3, wherein the CDRH1 comprises the sequence of amino acids set forth in SEQ ID NO: 9 or SEQ ID NO: 16, or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:9 or SEQ ID NO: 16; the
CDRH2 comprises a sequence of amino acids set forth in SEQ ID NO: 10, 15 or 17, or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 10, 15 or 17; the CDRH3 comprises a sequence of amino acids set forth in SEQ ID NO: 11 or SEQ ID NO: 18, or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID
NO: 11 or SEQ ID NO: 18; the CDRL1 comprises a sequence of amino acids set forth in SEQ ID NO:
12 or SEQ ID NO: 19, or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%,
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 12 or SEQ ID NO: 19; the CDRL2 comprises a sequence of amino acids set forth in SEQ ID NO: 13 or SEQ ID NO:20, or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 13 or SEQ ID NO:20; the CDRL3 comprises a sequence of amino acids set forth in SEQ ID NO: 14 or SEQ ID NO:21, or a sequence of amino acids that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 14 or SEQ ID NO:21.
5. The anti-NKG2A agent of any one of claims 1 -4, wherein the immunoglobulin heavy chain variable domain comprises the amino acid sequence set forth in any of SEQ ID NOS: 1, 3 or 4, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to any of SEQ ID NOS: 1, 3 or 4.
6. The anti-NKG2A agent of any one of claims 1-5, wherein the immunoglobulin light chain variable domain comprises the amino acid sequence set forth in any of SEQ ID NOS: 5 or 8, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to any of SEQ ID
NOS:5 or 8.
7. The anti-NKG2A agent of any one of claims 1-6, wherein the immunoglobulin heavy chain variable domain comprises the amino acid sequence set forth in any of SEQ ID NOS: 1, 3 or 4, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to any of SEQ ID
NOS: 1, 3 or 4, and the immunoglobulin light chain variable domain comprises the amino acid sequence set forth in any of SEQ ID NOS: 5 or 8, or a sequence having at least 80%, 81%, 82%, 83%,
84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to any of SEQ ID NOS:5 or 8.
8. The anti-NKG2A agent of any one of claims 1-7, comprising one immunoglobulin heavy chain variable domain and one immunoglobulin light chain variable domain.
9. The anti-NKG2A agent of any one of claims 1-8, comprising two immunoglobulin heavy chain variable domains and two immunoglobulin light chain variable domains.
10. The anti-NKG2A agent of any of any one of claims 1-9, wherein the anti-NKG2A agent is an antibody or antigen-binding fragment thereof.
11. The anti-NKG2A agent of any one of claims 1-10, wherein the anti-NKG2A agent is isolated.
12. The anti-NKG2A agent of any one of claims 1-11, wherein the anti-NKG2A agent is humanized.
13. The anti-NKG2A agent of any one of claims 1-12, wherein the anti-NKG2A agent is conjugated to a detectable marker or label.
14. The anti-NKG2A agent of claim 13, wherein the detectable marker or label comprises a detectable moiety.
15. The anti-NKG2A agent of any one of claims 1-14, wherein the anti-NKG2A agent is non-diffusively immobilized on a solid support.
16. The anti-NKG2A agent of any one of claims 1-15, that is a single chain fragment.
17. The anti-NKG2A agent of claim 16, wherein the single chain fragment is a single chain variable fragment (scFv).
18. The anti-NKG2A agent of any one of claims 1-17, for use in the detection of NKG2A in a sample.
19. The anti-NKG2A agent of any one of claims 1-18, wherein the anti-NKG2A agent binds to an NKG2A expressing cell in a sample.
20. The anti-NKG2A agent of claim 18 or claim 19, wherein the sample comprises immune cells.
21. The anti-NKG2A of claim 20, wherein the immune cell is selected from B cells, plasmacytoid dendritic cells (pDCs), lymphocytes, leukocytes, T cells, monocytes, macrophages, neutrophils, myeloid dendritic cells (mDCs), innate lymphoid cells, mast cells, eosinophils, basophils, natural killer cells, and peripheral blood mononuclear cells (PBMCs).
22. The anti-NKG2A of any one of claims 18-21, wherein the detection of NKG2A is on the surface of immune cells.
23. The anti-NKG2A of any one of claims 18-22, wherein NKG2A is detected intracellularly.
24. The anti-NKG2A of any one of claims 18-23, wherein the detection is performed in vitro.
25. The anti-NKG2A of any one of claims 18-23, wherein the detection is performed in vivo.
26. The anti-NKG2A agent of any one of claims 1-25, wherein the anti-NKG2A agent binds to an NKG2A expressing cell, and blocks the binding of NKG2A to HLA-E on a target cell.
27. The anti-NKG2A agent of any one of claims 1-26, wherein the anti-NKG2A agent blocks the binding of NKG2A on an NKG2A expressing cell to HLA-E on a target cell.
28. The anti-NKG2A agent of any one of claims 19-27, wherein the anti-NKG2A agent bound to the NKG2A expressing cell increases the cytotoxic activity of the NKG2A expressing cell.
29. The anti-NKG2A agent of any one of claims 19-28, wherein the anti-NKG2A agent bound to the NKG2A expressing cell induces lysis of a target cell.
30. The anti-NKG2A agent of any one of claims 26-29, wherein the target cell is a cell that expresses HLA-E.
31. The anti-NKG2A agent of any one of claims 26-30, wherein the target cell is a cell associated with a disease or disorder.
32. The anti-NKG2A agent of claim 31, wherein the disease or disorder is a cancer, a viral infection, an autoimmune disease, an inflammatory disease or a bacterial infection.
33. The anti-NKG2A agent of claim 32, wherein the cancer is acute myeloid leukemia, acute lymphoblastic leukemia, colorectal, ovarian, gynecologic, liver, glioblastoma, Hodgkin lymphoma, chronic lymphocytic leukemia, esophagus, gastric, pancreas, colon, kidney, head and neck, lung and melanoma.
34. A diagnostic reagent comprising the anti-NKG2A agent of any one of claims 1-33.
35. A kit comprising the anti-NKG2A agent of any one of claims 1-33, or the diagnostic reagent of claim 34.
36. A composition comprising the anti-NKG2A agent of any one of claims 1-33, and a pharmaceutically acceptable excipient.
37. An isolated nucleic acid comprising a nucleotide sequence that encodes the immunoglobulin heavy chain variable domain of the antibody or antigen-binding fragment of any one of claims 1-33.
38. The isolated nucleic acid of claim 37, wherein the immunoglobulin heavy chain variable domain comprises the sequence of nucleotides set forth in any of SEQ ID NOs: 30-37.
39. An isolated nucleic acid comprising a nucleotide sequence that encodes the immunoglobulin light chain variable domain of the anti-NKG2A agent of any one of claims 1-33.
40. The isolated nucleic acid molecule of claim 39, wherein the immunoglobulin light chain variable domain comprises the sequence of nucleotides set forth in any of SEQ ID NOs: 38-45.
41. An isolated nucleic acid comprising a nucleotide sequence that encodes the immunoglobulin heavy chain variable domain and the immunoglobulin light chain variable domain of the anti-NKG2A agent of any one of claims 1-33.
42. The isolated nucleic acid of claim 41, wherein the nucleotide sequence that encodes the immunoglobulin heavy chain variable domain comprises the sequence set forth in any of SEQ ID NOs: 30-37; and the immunoglobulin light chain variable domain comprises the sequence of amino acids set forth in any of SEQ ID NOs: 38-45.
43. A recombinant expression vector comprising a first expression cassette and a second expression cassette, wherein the first expression cassette comprises a nucleic acid molecule comprising a nucleotide sequence that encodes the immunoglobulin heavy chain variable domain of any one of claims 1-33, and the second expression cassette comprises a nucleic acid molecule comprising a nucleotide sequence that encodes an immunoglobulin light chain variable domain of the anti-NKG2A agent of any one of claims 1-33.
44. The recombinant expression vector of claim 43, wherein the first and second expression cassettes comprise a promoter.
45. A host cell transfected with the recombinant expression vector of claim 43 or claim
44.
46. A method of detecting NKG2A in a sample, comprising, a) contacting a sample with the anti-NKG2A agent of any one of claims 1-33, under conditions to bind said anti-NKG2A to an NKG2A receptor on said sample, wherein the binding generates the production of a receptor/anti-NKG2A complex; b) detecting the presence of the receptor/anti-NKG2A complexes; c) wherein the detecting comprises the presence or absence of the NKG2A receptor on said sample.
47. A method of treating or preventing a disease or disorder associated with NKG2A in a subject, comprising: a) contacting a sample with the anti-NKG2A agent of any one of claims 1-33, b) detecting the presence of complexes comprising NKG2A and the anti-NKG2A agent; wherein the presence of the complexes indicates the presence of a disease or disorder; and c) administering to the subject the anti-NKG2A agent of any one of claims 1-33.
PCT/US2021/056218 2020-10-30 2021-10-22 Anti-nkg2a agents and compositions and methods for making and using the same WO2022093641A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202063107812P 2020-10-30 2020-10-30
US63/107,812 2020-10-30

Publications (1)

Publication Number Publication Date
WO2022093641A1 true WO2022093641A1 (en) 2022-05-05

Family

ID=78650085

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2021/056218 WO2022093641A1 (en) 2020-10-30 2021-10-22 Anti-nkg2a agents and compositions and methods for making and using the same

Country Status (1)

Country Link
WO (1) WO2022093641A1 (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024056010A1 (en) * 2022-09-16 2024-03-21 北京三诺佳邑生物技术有限责任公司 Antibody specifically recognizing nkg2a and use thereof
WO2024140820A1 (en) * 2022-12-28 2024-07-04 浙江博锐生物制药有限公司 Anti-nkg2a antibody and use thereof

Citations (64)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3773919A (en) 1969-10-23 1973-11-20 Du Pont Polylactide-drug mixtures
US4179337A (en) 1973-07-20 1979-12-18 Davis Frank F Non-immunogenic polypeptides
US4275149A (en) 1978-11-24 1981-06-23 Syva Company Macromolecular environment control in specific receptor assays
US4301144A (en) 1979-07-11 1981-11-17 Ajinomoto Company, Incorporated Blood substitute containing modified hemoglobin
US4318980A (en) 1978-04-10 1982-03-09 Miles Laboratories, Inc. Heterogenous specific binding assay employing a cycling reactant as label
USRE30985E (en) 1978-01-01 1982-06-29 Serum-free cell culture media
US4376110A (en) 1980-08-04 1983-03-08 Hybritech, Incorporated Immunometric assays using monoclonal antibodies
US4485045A (en) 1981-07-06 1984-11-27 Research Corporation Synthetic phosphatidyl cholines useful in forming liposomes
US4496689A (en) 1983-12-27 1985-01-29 Miles Laboratories, Inc. Covalently attached complex of alpha-1-proteinase inhibitor with a water soluble polymer
US4544545A (en) 1983-06-20 1985-10-01 Trustees University Of Massachusetts Liposomes containing modified cholesterol for organ targeting
US4560655A (en) 1982-12-16 1985-12-24 Immunex Corporation Serum-free cell culture medium and process for making same
EP0183070A2 (en) 1984-10-30 1986-06-04 Phillips Petroleum Company Transformation of yeasts of the genus pichia
WO1987000195A1 (en) 1985-06-28 1987-01-15 Celltech Limited Animal cell culture
US4640835A (en) 1981-10-30 1987-02-03 Nippon Chemiphar Company, Ltd. Plasminogen activator derivatives
US4657866A (en) 1982-12-21 1987-04-14 Sudhir Kumar Serum-free, synthetic, completely chemically defined tissue culture media
US4670417A (en) 1985-06-19 1987-06-02 Ajinomoto Co., Inc. Hemoglobin combined with a poly(alkylene oxide)
US4676980A (en) 1985-09-23 1987-06-30 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Target specific cross-linked heteroantibodies
EP0244234A2 (en) 1986-04-30 1987-11-04 Alko Group Ltd. Transformation of trichoderma
US4737456A (en) 1985-05-09 1988-04-12 Syntex (U.S.A.) Inc. Reducing interference in ligand-receptor binding assays
US4767704A (en) 1983-10-07 1988-08-30 Columbia University In The City Of New York Protein-free culture medium
US4791192A (en) 1986-06-26 1988-12-13 Takeda Chemical Industries, Ltd. Chemically modified protein with polyethyleneglycol
US4816567A (en) 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
DD266710A3 (en) 1983-06-06 1989-04-12 Ve Forschungszentrum Biotechnologie Process for the biotechnical production of alkaline phosphatase
WO1990003430A1 (en) 1988-09-23 1990-04-05 Cetus Corporation Cell culture medium for enhanced cell growth, culture longevity and product expression
US4927762A (en) 1986-04-01 1990-05-22 Cell Enterprises, Inc. Cell culture medium with antioxidant
EP0402226A1 (en) 1989-06-06 1990-12-12 Institut National De La Recherche Agronomique Transformation vectors for yeast yarrowia
US5013556A (en) 1989-10-20 1991-05-07 Liposome Technology, Inc. Liposomes with enhanced circulation time
US5122469A (en) 1990-10-03 1992-06-16 Genentech, Inc. Method for culturing Chinese hamster ovary cells to improve production of recombinant proteins
US5204244A (en) 1987-10-27 1993-04-20 Oncogen Production of chimeric antibodies by homologous recombination
US5225539A (en) 1986-03-27 1993-07-06 Medical Research Council Recombinant altered antibodies and methods of making altered antibodies
US5229275A (en) 1990-04-26 1993-07-20 Akzo N.V. In-vitro method for producing antigen-specific human monoclonal antibodies
US5530101A (en) 1988-12-28 1996-06-25 Protein Design Labs, Inc. Humanized immunoglobulins
US5534615A (en) 1994-04-25 1996-07-09 Genentech, Inc. Cardiac hypertrophy factor and uses therefor
US5545807A (en) 1988-10-12 1996-08-13 The Babraham Institute Production of antibodies from transgenic animals
WO1996027011A1 (en) 1995-03-01 1996-09-06 Genentech, Inc. A method for making heteromultimeric polypeptides
US5565332A (en) 1991-09-23 1996-10-15 Medical Research Council Production of chimeric antibodies - a combinatorial approach
WO1996032478A1 (en) 1995-04-14 1996-10-17 Genentech, Inc. Altered polypeptides with increased half-life
US5567610A (en) 1986-09-04 1996-10-22 Bioinvent International Ab Method of producing human monoclonal antibodies and kit therefor
US5573905A (en) 1992-03-30 1996-11-12 The Scripps Research Institute Encoded combinatorial chemical libraries
US5589369A (en) 1992-02-11 1996-12-31 Cell Genesys Inc. Cells homozygous for disrupted target loci
US5591669A (en) 1988-12-05 1997-01-07 Genpharm International, Inc. Transgenic mice depleted in a mature lymphocytic cell-type
US5624821A (en) 1987-03-18 1997-04-29 Scotgen Biopharmaceuticals Incorporated Antibodies with altered effector functions
US5714350A (en) 1992-03-09 1998-02-03 Protein Design Labs, Inc. Increasing antibody affinity by altering glycosylation in the immunoglobulin variable region
US5777085A (en) 1991-12-20 1998-07-07 Protein Design Labs, Inc. Humanized antibodies reactive with GPIIB/IIIA
US5834597A (en) 1996-05-20 1998-11-10 Protein Design Labs, Inc. Mutated nonactivating IgG2 domains and anti CD3 antibodies incorporating the same
US5861155A (en) 1993-12-08 1999-01-19 Astra Ab Humanized antibodies and uses thereof
US5882644A (en) 1996-03-22 1999-03-16 Protein Design Labs, Inc. Monoclonal antibodies specific for the platelet derived growth factor β receptor and methods of use thereof
US5932448A (en) 1991-11-29 1999-08-03 Protein Design Labs., Inc. Bispecific antibody heterodimers
US6013256A (en) 1996-09-24 2000-01-11 Protein Design Labs, Inc. Method of preventing acute rejection following solid organ transplantation
US6129914A (en) 1992-03-27 2000-10-10 Protein Design Labs, Inc. Bispecific antibody effective to treat B-cell lymphoma and cell line
US6210671B1 (en) 1992-12-01 2001-04-03 Protein Design Labs, Inc. Humanized antibodies reactive with L-selectin
US6329511B1 (en) 1998-12-01 2001-12-11 Protein Design Labs, Inc. Humanized antibodies to γ-interferon
US6407213B1 (en) 1991-06-14 2002-06-18 Genentech, Inc. Method for making humanized antibodies
US6479284B1 (en) 1998-03-13 2002-11-12 Dana-Farber Cancer Institute, Inc. Humanized antibody and uses thereof
US6500931B1 (en) 1992-11-04 2002-12-31 Medarex, Inc. Humanized antibodies to Fc receptors for immunoglobulin G on human mononuclear phagocytes
US6548640B1 (en) 1986-03-27 2003-04-15 Btg International Limited Altered antibodies
WO2004063351A2 (en) 2003-01-09 2004-07-29 Macrogenics, Inc. IDENTIFICATION AND ENGINEERING OF ANTIBODIES WITH VARIANT Fc REGIONS AND METHODS OF USING SAME
WO2006088494A2 (en) 2004-07-12 2006-08-24 Macrogenics, Inc. Identification and engineering of antibodies with variant fc regions and methods of using the same
WO2006113665A2 (en) 2005-04-15 2006-10-26 Macrogenics, Inc. Covalent diabodies and uses thereof
WO2007021841A2 (en) 2005-08-10 2007-02-22 Macrogenics, Inc. Identification and engineering of antibodies with variant fc regions and methods of using same
WO2007024249A2 (en) 2004-11-10 2007-03-01 Macrogenics, Inc. Engineering fc antibody regions to confer effector function
WO2007106707A2 (en) 2006-03-10 2007-09-20 Macrogenics, Inc. Identification and engineering of antibodies with variant heavy chains and methods of using same
WO2008140603A2 (en) 2006-12-08 2008-11-20 Macrogenics, Inc. METHODS FOR THE TREATMENT OF DISEASE USING IMMUNOGLOBULINS HAVING FC REGIONS WITH ALTERED AFFINITIES FOR FCγR ACTIVATING AND FCγR INHIBITING
WO2020094071A1 (en) * 2018-11-07 2020-05-14 上海怀越生物科技有限公司 Nkg2a antibody, preparation method therefor and application thereof

Patent Citations (70)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3773919A (en) 1969-10-23 1973-11-20 Du Pont Polylactide-drug mixtures
US4179337A (en) 1973-07-20 1979-12-18 Davis Frank F Non-immunogenic polypeptides
USRE30985E (en) 1978-01-01 1982-06-29 Serum-free cell culture media
US4318980A (en) 1978-04-10 1982-03-09 Miles Laboratories, Inc. Heterogenous specific binding assay employing a cycling reactant as label
US4275149A (en) 1978-11-24 1981-06-23 Syva Company Macromolecular environment control in specific receptor assays
US4301144A (en) 1979-07-11 1981-11-17 Ajinomoto Company, Incorporated Blood substitute containing modified hemoglobin
US4376110A (en) 1980-08-04 1983-03-08 Hybritech, Incorporated Immunometric assays using monoclonal antibodies
US4485045A (en) 1981-07-06 1984-11-27 Research Corporation Synthetic phosphatidyl cholines useful in forming liposomes
US4640835A (en) 1981-10-30 1987-02-03 Nippon Chemiphar Company, Ltd. Plasminogen activator derivatives
US4560655A (en) 1982-12-16 1985-12-24 Immunex Corporation Serum-free cell culture medium and process for making same
US4657866A (en) 1982-12-21 1987-04-14 Sudhir Kumar Serum-free, synthetic, completely chemically defined tissue culture media
US4816567A (en) 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
DD266710A3 (en) 1983-06-06 1989-04-12 Ve Forschungszentrum Biotechnologie Process for the biotechnical production of alkaline phosphatase
US4544545A (en) 1983-06-20 1985-10-01 Trustees University Of Massachusetts Liposomes containing modified cholesterol for organ targeting
US4767704A (en) 1983-10-07 1988-08-30 Columbia University In The City Of New York Protein-free culture medium
US4496689A (en) 1983-12-27 1985-01-29 Miles Laboratories, Inc. Covalently attached complex of alpha-1-proteinase inhibitor with a water soluble polymer
EP0183070A2 (en) 1984-10-30 1986-06-04 Phillips Petroleum Company Transformation of yeasts of the genus pichia
US4737456A (en) 1985-05-09 1988-04-12 Syntex (U.S.A.) Inc. Reducing interference in ligand-receptor binding assays
US4670417A (en) 1985-06-19 1987-06-02 Ajinomoto Co., Inc. Hemoglobin combined with a poly(alkylene oxide)
WO1987000195A1 (en) 1985-06-28 1987-01-15 Celltech Limited Animal cell culture
US4676980A (en) 1985-09-23 1987-06-30 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Target specific cross-linked heteroantibodies
US5225539A (en) 1986-03-27 1993-07-06 Medical Research Council Recombinant altered antibodies and methods of making altered antibodies
US6548640B1 (en) 1986-03-27 2003-04-15 Btg International Limited Altered antibodies
US4927762A (en) 1986-04-01 1990-05-22 Cell Enterprises, Inc. Cell culture medium with antioxidant
EP0244234A2 (en) 1986-04-30 1987-11-04 Alko Group Ltd. Transformation of trichoderma
US4791192A (en) 1986-06-26 1988-12-13 Takeda Chemical Industries, Ltd. Chemically modified protein with polyethyleneglycol
US5567610A (en) 1986-09-04 1996-10-22 Bioinvent International Ab Method of producing human monoclonal antibodies and kit therefor
US5624821A (en) 1987-03-18 1997-04-29 Scotgen Biopharmaceuticals Incorporated Antibodies with altered effector functions
US5204244A (en) 1987-10-27 1993-04-20 Oncogen Production of chimeric antibodies by homologous recombination
WO1990003430A1 (en) 1988-09-23 1990-04-05 Cetus Corporation Cell culture medium for enhanced cell growth, culture longevity and product expression
US5545807A (en) 1988-10-12 1996-08-13 The Babraham Institute Production of antibodies from transgenic animals
US5591669A (en) 1988-12-05 1997-01-07 Genpharm International, Inc. Transgenic mice depleted in a mature lymphocytic cell-type
US6180370B1 (en) 1988-12-28 2001-01-30 Protein Design Labs, Inc. Humanized immunoglobulins and methods of making the same
US5530101A (en) 1988-12-28 1996-06-25 Protein Design Labs, Inc. Humanized immunoglobulins
US5693761A (en) 1988-12-28 1997-12-02 Protein Design Labs, Inc. Polynucleotides encoding improved humanized immunoglobulins
US5585089A (en) 1988-12-28 1996-12-17 Protein Design Labs, Inc. Humanized immunoglobulins
US5693762A (en) 1988-12-28 1997-12-02 Protein Design Labs, Inc. Humanized immunoglobulins
EP0402226A1 (en) 1989-06-06 1990-12-12 Institut National De La Recherche Agronomique Transformation vectors for yeast yarrowia
US5013556A (en) 1989-10-20 1991-05-07 Liposome Technology, Inc. Liposomes with enhanced circulation time
US5229275A (en) 1990-04-26 1993-07-20 Akzo N.V. In-vitro method for producing antigen-specific human monoclonal antibodies
US5122469A (en) 1990-10-03 1992-06-16 Genentech, Inc. Method for culturing Chinese hamster ovary cells to improve production of recombinant proteins
US6639055B1 (en) 1991-06-14 2003-10-28 Genentech, Inc. Method for making humanized antibodies
US6407213B1 (en) 1991-06-14 2002-06-18 Genentech, Inc. Method for making humanized antibodies
US5565332A (en) 1991-09-23 1996-10-15 Medical Research Council Production of chimeric antibodies - a combinatorial approach
US5932448A (en) 1991-11-29 1999-08-03 Protein Design Labs., Inc. Bispecific antibody heterodimers
US5777085A (en) 1991-12-20 1998-07-07 Protein Design Labs, Inc. Humanized antibodies reactive with GPIIB/IIIA
US5589369A (en) 1992-02-11 1996-12-31 Cell Genesys Inc. Cells homozygous for disrupted target loci
US6350861B1 (en) 1992-03-09 2002-02-26 Protein Design Labs, Inc. Antibodies with increased binding affinity
US5714350A (en) 1992-03-09 1998-02-03 Protein Design Labs, Inc. Increasing antibody affinity by altering glycosylation in the immunoglobulin variable region
US6129914A (en) 1992-03-27 2000-10-10 Protein Design Labs, Inc. Bispecific antibody effective to treat B-cell lymphoma and cell line
US5573905A (en) 1992-03-30 1996-11-12 The Scripps Research Institute Encoded combinatorial chemical libraries
US6500931B1 (en) 1992-11-04 2002-12-31 Medarex, Inc. Humanized antibodies to Fc receptors for immunoglobulin G on human mononuclear phagocytes
US6210671B1 (en) 1992-12-01 2001-04-03 Protein Design Labs, Inc. Humanized antibodies reactive with L-selectin
US5861155A (en) 1993-12-08 1999-01-19 Astra Ab Humanized antibodies and uses thereof
US5534615A (en) 1994-04-25 1996-07-09 Genentech, Inc. Cardiac hypertrophy factor and uses therefor
WO1996027011A1 (en) 1995-03-01 1996-09-06 Genentech, Inc. A method for making heteromultimeric polypeptides
WO1996032478A1 (en) 1995-04-14 1996-10-17 Genentech, Inc. Altered polypeptides with increased half-life
US5882644A (en) 1996-03-22 1999-03-16 Protein Design Labs, Inc. Monoclonal antibodies specific for the platelet derived growth factor β receptor and methods of use thereof
US5834597A (en) 1996-05-20 1998-11-10 Protein Design Labs, Inc. Mutated nonactivating IgG2 domains and anti CD3 antibodies incorporating the same
US6013256A (en) 1996-09-24 2000-01-11 Protein Design Labs, Inc. Method of preventing acute rejection following solid organ transplantation
US6479284B1 (en) 1998-03-13 2002-11-12 Dana-Farber Cancer Institute, Inc. Humanized antibody and uses thereof
US6329511B1 (en) 1998-12-01 2001-12-11 Protein Design Labs, Inc. Humanized antibodies to γ-interferon
WO2004063351A2 (en) 2003-01-09 2004-07-29 Macrogenics, Inc. IDENTIFICATION AND ENGINEERING OF ANTIBODIES WITH VARIANT Fc REGIONS AND METHODS OF USING SAME
WO2006088494A2 (en) 2004-07-12 2006-08-24 Macrogenics, Inc. Identification and engineering of antibodies with variant fc regions and methods of using the same
WO2007024249A2 (en) 2004-11-10 2007-03-01 Macrogenics, Inc. Engineering fc antibody regions to confer effector function
WO2006113665A2 (en) 2005-04-15 2006-10-26 Macrogenics, Inc. Covalent diabodies and uses thereof
WO2007021841A2 (en) 2005-08-10 2007-02-22 Macrogenics, Inc. Identification and engineering of antibodies with variant fc regions and methods of using same
WO2007106707A2 (en) 2006-03-10 2007-09-20 Macrogenics, Inc. Identification and engineering of antibodies with variant heavy chains and methods of using same
WO2008140603A2 (en) 2006-12-08 2008-11-20 Macrogenics, Inc. METHODS FOR THE TREATMENT OF DISEASE USING IMMUNOGLOBULINS HAVING FC REGIONS WITH ALTERED AFFINITIES FOR FCγR ACTIVATING AND FCγR INHIBITING
WO2020094071A1 (en) * 2018-11-07 2020-05-14 上海怀越生物科技有限公司 Nkg2a antibody, preparation method therefor and application thereof

Non-Patent Citations (49)

* Cited by examiner, † Cited by third party
Title
"Antibodies, A Laboratory Manual", 1988, COLD SPRING HARBOR LABORATORY
"Antibody-antigen interactions: Contact analysis and binding site topography", J. MOL. BIOL., vol. 262, pages 732 - 745
"NCBI", Database accession no. NP 002250.2
"Remington's Pharmaceutical Sciences", 1980
AL-LAZIKANI ET AL., JMB, vol. 273, 1997, pages 927 - 948
BARNES ET AL., ANAL. BIOCHEM., vol. l02, 1980, pages 255
BRAUD ET AL., NATURE, vol. 391, no. 6669, 1998, pages 795 - 799
BRENNAN ET AL., SCIENCE, vol. 229, 1985, pages 81
BRODEUR ET AL.: "Monoclonal Antibody Production Techniques and Applications", 1987, MARCEL DEKKER, INC., pages: 147 - 158
BRUGGERMANN ET AL., YEAR IN IMMUNO., vol. 7, 1993, pages 33
CARTER ET AL., BIO/TECHNOLOGY, vol. 10, 1992, pages 163 - 167
CHAMPE ET AL., J. BIOL. CHEM., vol. 270, 1995, pages 1388 - 1394
CLARKSON ET AL., NATURE, vol. 1, 2, 1991, pages 624 - 628
CUNNINGHAMWELLS, SCIENCE, vol. 244, 1989, pages 1081 - 1085
DAVIDSONDORANZ, IMMUNOLOGY, vol. 143, 2014, pages 13 - 20
EPSTEIN ET AL., PROC. NATL. ACAD. SCI. USA, vol. 82, 1985, pages 3688
GRAHAM ET AL., J. GEN VIROL., vol. 36, 1977, pages 59
GRUBER ET AL., J. IMMUNOL., vol. 152, 1994, pages 5368
GUSS ET AL., EMBO J., vol. 5, 1986, pages 15671575 - 103
HAM ET AL., METH. ENZ., vol. 58, 1979, pages 44
HONEGGER APLIICKTHUN A: "Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool", J MOL BIOL, vol. 309, no. 3, 8 June 2001 (2001-06-08), pages 657 - 70, XP004626893, DOI: 10.1006/jmbi.2001.4662
HOOGENBOOM ET AL., J. MOL. BIOL., vol. 222, 1991, pages 581 - 597
HWANG ET AL., PROC. NATL ACAD. SCI. USA, vol. 77, 1980, pages 4030
JAKOBOVITS ET AL., NATURE, vol. 362, 1993, pages 255 - 258
JAKOBOVITS ET AL., PROC. NATL. ACAD. SCI. USA, vol. 90, 1993, pages 6444 - 6448
KAYIMA ET AL., THE JOURNAL OF CLINICAL INVESTIGATION, 2019
KINDT ET AL.: "Kuby Immunology", 2007, W.H. FREEMAN AND CO., pages: 91
KOHLER ET AL., NATURE, vol. 256, 1975, pages 495
KOSTELNY ET AL., J. IMMUNOL., vol. 148, no. 5, 1992, pages 1547 - 1553
KOZBOR, J. IMMUNOL., vol. 133, 1984, pages 3001
LAZETIC ET AL., J IMMUNOL., vol. 157, no. 11, 1996, pages 4741 - 4757
LEE ET AL., PROC NATL ACAD SCIUSA, vol. 95, no. 9, 1998, pages 5199 - 5204
LEFRANC MP ET AL.: "IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains", DEV COMP IMMUNOL, vol. 27, no. l, January 2003 (2003-01-01), pages 55 - 77, XP055585227, DOI: 10.1016/S0145-305X(02)00039-3
LINDMARK ET AL., J. IMMUNOL. METH., vol. 62, 1983, pages 1 - 13
MACCALLUM ET AL., J. MOL. BIOL., vol. 262, 1996, pages 732 - 745
MARTIN ET AL., J. BIOL. CHEM., vol. 257, 1982, pages 286 - 288
MARTIN ET AL., PROC. NATL. ACAD. SCI., vol. 86, 1989, pages 9268 - 9272
MATHER ET AL., ANNALS N.Y. ACAD. SCI., vol. 383, 1982, pages 44 - 68
MATHER, BIOL. REPROD., vol. 23, 1980, pages 243 - 251
MILLER ET AL., J IMMUNOL., vol. 171, no. 3, 2003, pages 1369 - 1375
MORIMOTO ET AL., JOURNAL OF BIOCHEMICAL AND BIOPHYSICAL METHODS, vol. 24, 1992, pages 107 - 117
NEUBERGER ET AL., NATURE, vol. 312, 1984, pages 604 - 608
O'SULLIVAN ET AL.: "Methods for the Preparation of Enzyme-Antibody Conjugates for use in Enzyme Immunoassay, in Methods in Enzym", vol. 73, 1981, ACADEMIC PRESS, pages: 147 - 166
PORTOLANO ET AL., J. IMMUNOL., vol. 150, 1993, pages 880 - 887
SHALABY ET AL., J. EXP. MED., vol. 175, 1992, pages 217 - 225
SHAPIRO: "Practical Flow Cytometry", 2003, WILEY
STAVENHAGEN, J.B. ET AL., CANCER RES., vol. 57, no. 18, 2007, pages 8882 - 8890
URLAUB ET AL., PROC. NATL. ACAD. SCI. USA, vol. 77, 1980, pages 4216
ZAPATA ET AL., PROTEIN ENG, vol. 8, no. 10, 1995, pages 1057 - 1062

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024056010A1 (en) * 2022-09-16 2024-03-21 北京三诺佳邑生物技术有限责任公司 Antibody specifically recognizing nkg2a and use thereof
WO2024140820A1 (en) * 2022-12-28 2024-07-04 浙江博锐生物制药有限公司 Anti-nkg2a antibody and use thereof

Similar Documents

Publication Publication Date Title
JP7155126B2 (en) Antibody that specifically binds to human IL-15 and use thereof
AU2017232160B2 (en) Fc Receptor Binding Proteins
JP6416630B2 (en) Compositions and methods for using CSF1R inhibitors
JP6938383B2 (en) Antibodies to canine interleukin-4 receptor alpha
KR102038310B1 (en) Anti il-36r antibodies
JP2023538782A (en) CCR8 antibody and its uses
JP2022023058A (en) HUMANIZED ANTI-COMPLEMENT FACTOR C1q ANTIBODIES AND USES THEREOF
TW200918558A (en) Humanized anti-CXCR5 antibodies, derivatives thereof and their use
WO2022093640A1 (en) Anti-nkg2c agents and compositions and methods for making and using the same
WO2022093641A1 (en) Anti-nkg2a agents and compositions and methods for making and using the same
US20240360228A1 (en) Anti-ccr8 antibodies, antigen-binding fragments thereof, and agents and compositions and methods for making and using the same
US20210206873A1 (en) Anti-tlr9 agents and compositions and methods for making and using the same
JP2024505674A (en) Anti-IL1RAP antibody
US12006363B2 (en) Anti-tetraspanin 33 agents and compositions and methods for making and using the same
WO2022187050A1 (en) Anti-cd117 agents and compositions and methods for making and using the same
EP4448581A2 (en) Cd28 binding antibodies and antigen binding fragments thereof
US20230054169A1 (en) Anti-tlr7 agents and compositions and methods for making and using the same
WO2022182562A1 (en) ANTI-SARS-CoV-2 SPIKE GLYCOPROTEIN S1 AGENTS AND COMPOSITIONS
WO2024020051A1 (en) Anti-cd157 antibodies, antigen-binding fragments thereof and compositions and methods for making and using the same
WO2024040114A2 (en) Anti-axl antibodies, antigen-binding fragments thereof and methods for making and using the same
WO2023133470A2 (en) Tmprss2 binding antibodies and antigen binding fragments thereof
CN118613505A (en) CD28 binding antibodies and antigen binding fragments thereof
NZ618046B2 (en) Fc RECEPTOR BINDING PROTEINS
NZ715057B2 (en) Fc Receptor Binding Proteins

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21810207

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21810207

Country of ref document: EP

Kind code of ref document: A1