WO2017193104A1 - Immunothérapie de lymphocytes t spécifique de mart-1 - Google Patents

Immunothérapie de lymphocytes t spécifique de mart-1 Download PDF

Info

Publication number
WO2017193104A1
WO2017193104A1 PCT/US2017/031453 US2017031453W WO2017193104A1 WO 2017193104 A1 WO2017193104 A1 WO 2017193104A1 US 2017031453 W US2017031453 W US 2017031453W WO 2017193104 A1 WO2017193104 A1 WO 2017193104A1
Authority
WO
WIPO (PCT)
Prior art keywords
amino acid
acid sequence
cell
seq
tcr
Prior art date
Application number
PCT/US2017/031453
Other languages
English (en)
Inventor
Aude CHAPUIS
Philip Greenberg
Thomas Schmitt
Cassian Yee
Original Assignee
Fred Hutchinson Cancer Research Center
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 Fred Hutchinson Cancer Research Center filed Critical Fred Hutchinson Cancer Research Center
Priority to EP17724686.5A priority Critical patent/EP3452514A1/fr
Publication of WO2017193104A1 publication Critical patent/WO2017193104A1/fr

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
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3053—Skin, nerves, brain
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39541—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against normal tissues, cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4632—T-cell receptors [TCR]; antibody T-cell receptor constructs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464436—Cytokines
    • A61K39/46444—Interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464454—Enzymes
    • A61K39/464456—Tyrosinase or tyrosinase related proteinases [TRP-1 or TRP-2]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/46449—Melanoma antigens
    • A61K39/464491—Melan-A/MART
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • 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
    • C07K16/2818—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 against CD28 or CD152
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/57—Skin; melanoma
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • 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
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • 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

  • Agents for treating stage IV melanoma include BRAF inhibitors, such as vemurafenib (Zelboraf), dabrafenib (Tafmlar), trametinib (Mekinist), and cobimetimb (CoteUic), for those cancers expressing a BRAF mutation; c-KIT inhibitors, such as imatinib (Gleevec) and nilotinib (Tasigna)); alkylating agents such as DTIC (or dacarbazine); interleukin-2 (IL-2) administered in high doses; and more recently, immunotherapies, such as ipilimumab (Yervoy).
  • BRAF inhibitors such as vemurafenib (Zelboraf), dabrafenib (Tafmlar), trametinib (Mekinist), and cobimetimb (CoteUic
  • c-KIT inhibitors such as imat
  • TIL tumor-infiltrating lymphocyte
  • Endogenous antigen-specific CTL can also be obtained and expanded from peripheral blood (PB) and infused with lower-dose conditioning and a very tolerable safety profile, but have effectively reduced tumor burdens in only a limited number of patients, due in part to the short persistence of the transferred cells (Chapuis et al., 2012, Proc. Natl. Acad. Sci. USA 109:4592-7; Hunder et al., 2008, N. Engl. J. Med. 358:2698-703; Wallen et al., 2009, PLoS One 4:e4749; and Yee et al., 2002, Proc. Natl. Acad. Sci. USA 99: 16168- 73).
  • PB peripheral blood
  • Figures 1A-1D Tumor regressions following melanoma-reactive polyclonal CTL combined with anti-CTLA4.
  • A Timeline of successive therapies in "Patient Z.”
  • B Kinetics of response for 3 target lesions (y-axis) spanning 5 years (x-axis).
  • C Serial PET (leftmost image) and CT images at indicated time-points. Arrows indicate the location of the right hilar (upper row) and subcarinal (lower row) masses.
  • D Tumor regressions following melanoma-reactive polyclonal CTL combined with anti-CTLA4.
  • Figures 2A-2F Kinetics, clonality, phenotype and function of monoclonal and polyclonal CTL in vivo.
  • A Percent multimer + CD8 + T cells (left y-axis) in peripheral blood mononuclear cells (PBMCs) (solid circles) collected before and at defined timepoints after monoclonal (dashed line) and polyclonal (solid line) CTL infusions (indicated). Grey shaded areas indicate anti-CTLA4 treatment.
  • B, C Inset pie plots represent individual clonotypes composing the monoclonal (B) and polyclonal (C) infused CTL.
  • Graphs track the corresponding unique (B) and sum of clonotypes (C) as a percent of total CD8 + T cells (y-axis). Time-points in which the corresponding clones were assessed but not detected (nd) are indicated. *Only clone TCR-13 was detected immediately prior to the polyclonal infusion with a frequency of 0.054%.
  • D Percent expression of CD28, CD62L, CCR7 (long-lived memory markers - left), PD1 (activation/exhaustion marker - middle), IFNy, T Fa, and IL2 (functional markers - right) on polyclonal (upper graph) and monoclonal (lower graph) infused CTL.
  • E, F The same analysis performed on multimer + cells 1 day (E) and 86 days (F) in vivo after infusion.
  • Figure 3 Reactivity to non-targeted epitopes. Heat-map summarizing responses of CD8 + and CD4 + T cells independent of HLA restriction to pools of 20-30 peptides spanning MARTI, NY-ESOl, gplOO, tyrosinase, and MAGE- A3.
  • the shading scale (light to dark) reflects the response magnitude at indicated timepoints before and after administration of monoclonal and polyclonal CTL during the patients' treatment course (upper schema). Inset numbers indicate IFNy spots per 10 5 PBMC for each peptide pool.
  • Figures 4A and 4B Purity and phenotype of infused monoclonal and polyclonal products.
  • A Plots to the left: percent expression of CD8 + cells (x-axis) binding the specific HLA A*0201 -restricted MART- I 26-35 multimer (y-axis) within infused lymphocytes.
  • Figure 5 Patient Characteristics. Tumor size by mWHO in response to ipilimumab alone (Stromnes et al., 2014, Immunol. Rev. 257: 145-64): +8% at 12 weeks and +63% at 28 weeks (Rosenberg et al.,2011, Clin. Cancer Res. 17:4550-7). +43% at 12 weeks (Yang, 2013, Clin. Dermatol. 31 :209-19). Received a 1 st course of ipilimumab with no evaluable disease in the neo-adjuvant setting immediately after 1 st surgery, second course: +56% at 12 weeks. (Chapuis et al., 2012, Proc. Natl.
  • Leukapheresis performed 3 or 4 weeks after last surgery and CTL product generated and frozen in anticipation of progressive disease (Wallen et al., 2009, PLoS One 4:e4749). No treatment for 6 weeks between Leukapheresis and the start of
  • LN lymph node
  • s.c subcutaneous
  • HD High-dose
  • Figures 6A and 6B Tumor regressions following melanoma-reactive polyclonal CTL combined with anti-CTLA-4.
  • A Spider plot of all treated patients showing changes from baseline in the tumor burden (y-axis), measured as the products of the perpendicular diameters of all target lesions, assessed weeks after the CTL infusion (x-axis). The dashed lines above the solid baseline indicate 25% progression (mWHO PD) and the dashed line below the solid baseline indicate 50% reduction
  • Figures 7A and 7B Kinetics of in vivo persistence of melanoma-reactive polyclonal CTL.
  • A Percent multimer + CD8 + T-cells (left y-axis) in PBMCs (solid circles) collected 7 days ⁇ +1-2 days) before and at defined time-points after infusions are shown for patients who achieved CR, PR or SD after treatment.
  • Vertical downward arrows indicate CTL infusions
  • vertical arrows pointing upwards indicate anti-CTLA-4 infusions
  • asterisks indicate the start of an alternate treatment
  • pound signs indicate comfort care
  • horizontal arrows indicate concurrent corticosteroid therapy
  • horizontal arrows indicate ongoing monitoring.
  • B The same analysis performed for patients who progressed after treatment.
  • Figures 8A-8D Phenotypic and functional characteristics of transferred melanoma-reactive CTL.
  • A Expression of CD27, CD28, CD127, CD62L, CCR7, CD57 and PD1 (y-axis) on gated multimer + cells for CD8 + CTL products immediately before infusion and at after 3, 6, 9 and 23 weeks in vivo for patients who achieved CR, PR or SD.
  • B The same analysis performed for patients who progressed.
  • C Open symbols indicate patients who achieved CR, OR or SD (left column), solid symbols indicate patients who had PD (right column).
  • Figures 9A and 9B Reactivity to non-targeted epitopes.
  • B who progressed after receiving the treatment.
  • Pound (#) signs indicate patients who had received prior ipilimumab
  • vertical downward arrows indicate CTL infusions
  • horizontal lines indicate ipilimumab administration with the total number of doses received indicated immediately above.
  • Figure 10 Treatment plan. All patients received polyclonal MARTI -specific CTL lines primed with IL-21 on day 0 (10 10 cells/m 2 ) preceded by 300mg/m 2 of Cyclophosphamide on day -2. Infusions were followed by low-dose subcutaneous IL-2 for 14 days (2.5 x 10 5 R7 twice daily) starting within 6 hours of the CTL infusion.
  • Ipilimumab 3mg/m 2 intavenous was administered once every 3 weeks for a total of 4 doses starting on day 1 after the CTL infusion. Patients were evaluated for responses 6 and 12 weeks after infusion (on study) and then as clinically indicated.
  • Figures 11A and B Phenotypic and functional characteristics of infused MARTl-specific CTL products.
  • A From the left: Production of IFNy in response to T2 B-LCL targets pulsed with the HLA-A*0201 -restricted MARTl 26 -35 peptide, and lysis of HLA A*0201 + MARTI -expressing MEL-526 cell line (solid circles) and the HLA A*0201 + MARTI -negative MEL-375 cell line (open circles) at decreasing effector to target ratios in 51 Cr-release assays.
  • B Expression of CD45RO, CD27, CD28 and CD127 (upper row), and CD62L, CCR7, CD57 and PD1 (lower row) by MARTl-specific infused CTL for all patients.
  • Figures 12A and 12B Cutaneous regression of metastatic melanoma.
  • A The right inguinal region of Patient 9 before (left), 24 (middle) and 31 (right) weeks after the CTL infusion.
  • B Close-up of Patient 9's lesions before (left) and 31 weeks (right) after the CTL infusion.
  • Figure 13 Kaplan-Meier curve for progression-free survival and overall survival. As of November 1, 2015, the median follow-up for overall survival was 187 weeks (range 220-141 weeks); 5 patients are alive, 2 of whom continue in CR (*) and did not receive additional anti-tumor treatment.
  • Figure 14 Functional characteristics of transferred melanoma-reactive CTL.
  • Left column percent CD8 + T cells (x axis) producing IFNy (y axis) before infusions, by the CTL product, and at indicated time points in PBMC after the CTL infusion for Pt 10 (representative) in response to MARTI peptide.
  • Middle and right columns respective percentages of TNFa and IL2 (x-axis) producing cells amongst IFNy + cells (y axis).
  • Figure 15 Phenotype and function of products infused in patients who presented with disease control versus progression. Comparative surface expression of CD27, CD28, CD127, CD62L, CCR7, CD57 and PD1; intracellular production of IFNy, TNFa and IL-2 in response to cognate antigen; and intranuclear expression of Ki- 67 for products infused in patients who presented with a CR, PR or SD to the combined treatment or with PD.
  • Figures 16A and 16B Reactivity to melanoma-associated antigens in patients with melanoma and patients who received monoclonal CTL products.
  • Figures 17A-17C depict characteristics of monoclonal CTL products and concurrence of clonotype frequencies determined by TCR ⁇ PCR, multimer stains and HTTCS in vivo.
  • A Scatter plot showing binding of the monoclonal cells to CD8 (x- axis) and HLA A*0201 -restricted MARTI27-35 (y-axis).
  • B Pie graphs showing the percent of individual clonotypes composing the monoclonal CTL products. The frequency of the specific clone is overlayed on the plots. The total number of sequences detected in the products is stated above each plot.
  • Figures 18A and 18B depict characteristics of polyclonal products and concurrence of clonotype frequencies determined by HTTCS and multimer staining.
  • Figure 19 depicts a bar graph showing naive vs antigen-experienced phenotype based on clonotype frequency in PBMC.
  • the propensity of clonotypes present in 3 normal donor PBMC above or below the threshold of 0.001% with a CD3 + CD45RO + (antigen experienced) versus a CD3 + CD45RA + (naive) phenotype was determined by tracking clonotypes across whole PBMCs, sorted CD3 + CD45RA + and sorted
  • CD3 + CD45RO + populations using HTTCS The proportion of clonotypes with PBMC frequencies ⁇ 0.001% (bar to the left) and >0.001% (bar to the right) that were also in the CD3 + CD45RA + sorted population are shown.
  • Figures 20A-20C depict the composition of infused products
  • A Percent clonotypes detected at any time-point after infusions (open circles). The individual percent contribution of the most prevalent 25 antigen-specific clonotypes in the infused products (left y axis) are shown (colored lines) as well as the sum of the remaining sequences in the products (grey lines), before (left-most timepoint) and at selected time-points after infusions (day 0). Arrows indicate the immunodominant clonotypes for Pts P2225-1 and 7 who achieved a CR (indicated) as best response.
  • B Half-lives of clonotypes for each Pt grouped according to their best response.
  • Figures 21A and 21B depict persistence/frequency of different clonotype subsets in vivo.
  • A Pie graphs showing the number (top row) and percentage (bottom row) of clonotypes composing the infused polyclonal products. Clonotypes in the infusion products which were below the limit of detection either before or at any time- point after infusions (grey area). Clonotypes detected at any time-point after infusions which were not detected in PBMC before infusions (white area). The number of clonotypes composing the blue area for each patient are shown on the top row and their respective percentage are shown on the bottom row. Sum of clonotypes detected in PBMC before infusions expanded in the infusion product (orange area).
  • TCRs T cell receptors
  • MHC major histocompatibility complex
  • HLA human leukocyte antigen
  • PB-derived antigen-specific T cells can increase the frequency of melanoma-specific T cells, with a very tolerable safety profile (Chapuis et al., 2012, Proc. Natl. Acad. Sci. USA 109:4592-7; Wallen et al., 2009, PLoS One 4:e4749; Yee et al., 2002, Proc Natl Acad Sci USA 99: 16168-16173).
  • compositions and methods described herein will in certain embodiments have therapeutic utility for the treatment of diseases and conditions associated with MART-1 overexpression (e.g., detectable MART-1 expression at a level that is greater in magnitude, in a statistically significant manner, than the level of MART-1 expression that is detectable in a normal or disease-free cell).
  • diseases include various forms of hyperproliferative disorders, such as melanoma.
  • Non-limiting examples of these and related uses are described herein and include in vitro, ex vivo and in vivo stimulation of MART-1 antigen-specific T cell responses, such as by the use of recombinant T cells expressing a TCR specific for a MART-1 peptide (e.g., EAAGIGILTV (SEQ ID NO: 1)) ⁇
  • any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated.
  • any number range recited herein relating to any physical feature, such as polymer subunits, size or thickness are to be understood to include any integer within the recited range, unless otherwise indicated.
  • the term “about” means ⁇ 20% of the indicated range, value, or structure, unless otherwise indicated. It should be understood that the terms “a” and “an” as used herein refer to “one or more" of the enumerated components.
  • a protein domain, region, or module e.g., a binding domain, hinge region, linker module
  • a protein which may have one or more domains, regions, or modules
  • an "immune system cell” means any cell of the immune system that originates from a hematopoietic stem cell in the bone marrow, which gives rise to two major lineages, a myeloid progenitor cell (which give rise to myeloid cells such as monocytes, macrophages, dendritic cells, meagakaryocytes and granulocytes) and a lymphoid progenitor cell (which give rise to lymphoid cells such as T cells, B cells and natural killer (NK) cells).
  • myeloid progenitor cell which give rise to myeloid cells such as monocytes, macrophages, dendritic cells, meagakaryocytes and granulocytes
  • lymphoid progenitor cell which give rise to lymphoid cells such as T cells, B cells and natural killer (NK) cells.
  • Exemplary immune system cells include a CD4+ T cell, a CD8+ T cell, a CD4- CD8- double negative T cell, a ⁇ T cell, a regulatory T cell, a natural killer cell, and a dendritic cell.
  • Macrophages and dendritic cells may be referred to as "antigen presenting cells” or "APCs,” which are specialized cells that can activate T cells when a major histocompatibility complex (MHC) receptor on the surface of the APC complexed with a peptide interacts with a TCR on the surface of a T cell.
  • MHC major histocompatibility complex
  • MHC Major histocompatibility complex
  • MHC class I molecules are heterodimers having a membrane spanning a chain (with three a domains) and a non-covalently associated ⁇ 2 microglobulin.
  • MHC class II molecules are composed of two transmembrane glycoproteins, a and ⁇ , both of which span the membrane. Each chain has two domains.
  • MHC class I molecules deliver peptides originating in the cytosol to the cell surface, where a peptide:MHC complex is recognized by CD8 + T cells.
  • MHC class II molecules deliver peptides originating in the vesicular system to the cell surface, where they are recognized by CD4 + T cells.
  • Human MHC is referred to as human leukocyte antigen (HLA).
  • T cell is an immune system cell that matures in the thymus and produces T cell receptors (TCRs), which can be obtained (enriched or isolated) from, for example, peripheral blood mononuclear cells (PBMCs) and are referred to herein as "bulk” T cells.
  • TCRs T cell receptors
  • PBMCs peripheral blood mononuclear cells
  • CD4+ helper T cells
  • T cells can be naive (not exposed to antigen; increased expression of CD62L, CCR7, CD28, CD3, CD 127, and CD45RA, and decreased expression of CD45RO as compared to TC M ), memory T cells (T M ) (antigen-experienced and long-lived), and effector cells (antigen-experienced, cytotoxic).
  • T M can be further divided into subsets of central memory T cells (TC M , increased expression of CD62L, CCR7, CD28, CD127, CD45RO, and CD95, and decreased expression of CD54RA as compared to naive T cells) and effector memory T cells (T EM , decreased expression of CD62L, CCR7, CD28, CD45RA, and increased expression of CD127 as compared to naive T cells or T CM )- Effector T cells (T E ) refers to antigen-experienced CD8+ cytotoxic T lymphocytes that has decreased expression of CD62L ,CCR7, CD28, and are positive for granzyme and perforin as compared to T CM - Helper T cells (T H ) are CD4+ cells that influence the activity of other immune cells by releasing cytokines.
  • T M central memory T cells
  • T EM effector memory T cells
  • T E refers to antigen-experienced CD8+ cytotoxic T lymphocytes that has decreased
  • CD4+ T cells can activate and suppress an adaptive immune response, and which action is induced will depend on presence of other cells and signals.
  • T cells can be collected in accordance with known techniques, and the various subpopulations or combinations thereof can be enriched or depleted by known techniques, such as by affinity binding to antibodies, flow cytometry, or
  • T cell receptor refers to an immunoglobulin superfamily member (having a variable binding domain, a constant domain, a transmembrane region, and a short cytoplasmic tail; see, e.g., Janeway et al., Immunobiology: The Immune System in Health and Disease, 3 rd Ed., Current Biology Publications, p. 4:33, 1997) capable of specifically binding to an antigen peptide bound to a MHC receptor.
  • a TCR can be found on the surface of a cell or in soluble form and generally is comprised of a heterodimer having a and ⁇ chains (also known as TCRa and TCR , respectively), or ⁇ and ⁇ chains (also known as TCRy and TCR6, respectively).
  • TCRa and TCR also known as TCRa and TCR , respectively
  • ⁇ and ⁇ chains also known as TCRy and TCR6, respectively.
  • the extracellular portion of TCR chains ⁇ e.g., a-chain, ⁇ -chain
  • variable domains a variable domain ⁇ e.g., a-chain variable domain or V a , ⁇ - chain variable domain or Vp; typically amino acids 1 to 116 based on Kabat numbering Kabat et al., "Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services, Public Health Service National Institutes of Health, 1991, 5 th ed.) at the N-terminus, and one constant domain ⁇ e.g., a-chain constant domain or C a , typically amino acids 117 to 259 based on Kabat, ⁇ -chain constant domain or C , typically amino acids 117 to 295 based on Kabat) adjacent to the cell membrane. Also like
  • variable domains contain complementary determining regions (CDRs) separated by framework regions (FRs) ⁇ see, e.g., Jores et al., Proc. Nat'lAcad. Sci. U.S.A. 57:9138, 1990; Chothia et al, EMBO J. 7:3745, 1988; see also Lefranc et al., Dev. Comp. Immunol. 27:55, 2003).
  • a TCR is found on the surface of T cells (or T lymphocytes) and associates with the CD3 complex.
  • the source of a TCR as used in the present disclosure may be from various animal species, such as a human, mouse, rat, rabbit or other mammal.
  • variable region refers to the domain of a TCR a-chain or ⁇ -chain (or ⁇ chain and ⁇ chain for ⁇ TCRs) that is involved in binding of the TCR to antigen.
  • the variable domains of the a-chain and ⁇ -chain (V a and V a , respectively) of a native TCR generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three CDRs.
  • the V a domain is encoded by two separate DNA segments, the variable gene segment and the joining gene segment (V-J);
  • the ⁇ ⁇ domain is encoded by three separate DNA segments, the variable gene segment, the diversity gene segment, and the joining gene segment (V-D- J).
  • V a or V domain may be sufficient to confer antigen-binding specificity.
  • TCRs that bind a particular antigen may be isolated using a V a or V domain from a TCR that binds the antigen to screen a library of complementary V a or V domains, respectively.
  • CDR complementarity determining region
  • HVR hypervariable region
  • CD3 is known in the art as a multi-protein complex of six chains (see, Abbas and Lichtman, 2003; Janeway et al., pl72 and 178, 1999). In mammals, the complex comprises a CD3y chain, a CD35 chain, two CD3s chains, and a homodimer of CD3 ⁇ chains.
  • the CD3y, CD35, and CD3s chains are highly related cell surface proteins of the immunoglobulin superfamily containing a single immunoglobulin domain.
  • the transmembrane regions of the CD3y, CD35, and CD3s chains are negatively charged, which is a characteristic that allows these chains to associate with the positively charged T cell receptor chains.
  • the intracellular tails of the CD3y, CD35, and CD3s chains each contain a single conserved motif known as an immunoreceptor tyrosine- based activation motif or ITAM, whereas each 0)3 ⁇ chain has three.
  • ITAMs are important for the signaling capacity of a TCR complex.
  • CD3 as used in the present disclosure may be from various animal species, including human, mouse, rat, or other mammals.
  • TCR complex refers to a complex formed by the association of CD3 with TCR.
  • a TCR complex can be composed of a CD3y chain, a CD35 chain, two CD3s chains, a homodimer of CD3 ⁇ chains, a TCRa chain, and a
  • a TCR complex can be composed of a CD3y chain, a CD35 chain, two CD3s chains, a homodimer of 0)3 ⁇ chains, a TCRy chain, and a TCR5 chain.
  • a “component of a TCR complex,” as used herein, refers to a TCR chain (i.e., TCRa, TCRp, TCRy or TCR5), a CD3 chain (i.e., CO3j, CD35, CD3s or CO3Q, or a complex formed by two or more TCR chains or CD3 chains (e.g., a complex of TCRa and TCRP, a complex of TCRy and TCR5, a complex of CD3s and CD35, a complex of CD3y and CD3s, or a sub-TCR complex of TCRa, TCRp, CD3y, CD35, and two CD3s chains).
  • TCR chain i.e., TCRa, TCRp, TCRy or TCR5
  • a CD3 chain i.e., CO3j, CD35, CD3s or CO3Q
  • a complex formed by two or more TCR chains or CD3 chains e.g., a complex of TCRa
  • Antigen refers to an immunogenic molecule that provokes an immune response. This immune response may involve antibody production, activation of specific immunologically-competent cells (e.g., T cells), or both.
  • An antigen immunologically-competent cell
  • An antigen immunologically-competent molecule
  • An antigen immunologically-competent molecule
  • An antigen immunologically-competent molecule
  • An antigen immunologically-competent cells
  • glycopeptide polypeptide, glycopolypeptide, polynucleotide, polysaccharide, lipid or the like. It is readily apparent that an antigen can be synthesized, produced
  • Exemplary biological samples that can contain one or more antigens include tissue samples, tumor samples, cells, biological fluids, or combinations thereof.
  • Antigens can be produced by cells that have been modified or genetically engineered to express an antigen.
  • Exemplary antigens include MART- 1.
  • epitope includes any molecule, structure, amino acid sequence or protein determinant that is recognized and specifically bound by a cognate binding molecule, such as an immunoglobulin, T cell receptor (TCR), chimeric antigen receptor, or other binding molecule, domain or protein.
  • Epitopic determinants generally contain chemically active surface groupings of molecules, such as amino acids or sugar side chains, and can have specific three dimensional structural characteristics, as well as specific charge characteristics.
  • a MART-1 protein or fragment thereof may be an antigen that contains one or more antigenic epitopes.
  • binding domain refers to a molecule or portion thereof (e.g., peptide, oligopeptide, polypeptide, protein) that possesses the ability to specifically and non-covalently associate, unite, or combine with a target (e.g., MART-1, MART-1 peptide:MHC complex).
  • a binding domain includes any naturally occurring, synthetic, semisynthetic, or recombinantly produced binding partner for a biological molecule, a molecular complex (i.e., complex comprising two or more biological molecules), or other target of interest.
  • Exemplary binding domains include single chain
  • immunoglobulin variable regions e.g., scTCR, scFv
  • receptor ectodomains e.g., ligands (e.g., cytokines, chemokines), or synthetic polypeptides selected for their specific ability to bind to a biological molecule, a molecular complex or other target of interest.
  • ligands e.g., cytokines, chemokines
  • synthetic polypeptides selected for their specific ability to bind to a biological molecule, a molecular complex or other target of interest.
  • binding protein e.g., TCR receptor
  • binding domain or fusion protein thereof
  • target molecule e.g., MART-1 peptide: :HLA or a tetramer such an HLA complex
  • K a i.e., an equilibrium association constant of a particular binding interaction with units of 1/M
  • 10 5 M "1 which equals the ratio of the on-rate [k on ] to the off-rate [k 0 ff] for this association reaction
  • Binding proteins or binding domains may be classified as “high affinity” binding proteins or binding domains (or fusion proteins thereof) or as “low affinity” binding proteins or binding domains (or fusion proteins thereof).
  • “High affinity” binding proteins or binding domains refer to those binding proteins or binding domains having a K a of at least 10 7 M “1 , at least 10 8 M “1 , at least 10 9 M “1 , at least 10 10 M “1 , at least 10 11 M “1 , at least 10 12 M “1 , or at least 10 13 M “1 .
  • Bind affinity binding proteins or binding domains refer to those binding proteins or binding domains having a K a of up to 10 7 M “1 , up to 10 6 M “1 , up to 10 5 M “1 .
  • affinity may be defined as an equilibrium dissociation constant (K d ) of a particular binding interaction with units of M (e.g., 10 "5 M to 10 "13 M).
  • a receptor or binding domain may have "enhanced affinity," which refers to selected or engineered receptors or binding domains with stronger binding to a target antigen than a wild type (or parent) binding domain.
  • enhanced affinity may be due to a K a (equilibrium association constant) for the target antigen that is higher than the wild type binding domain, due to a K d
  • enhanced affinity TCRs may be codon optimized to enhance expression in a particular host cell, such as T cells (Scholten et al., Clin. Immunol. 779: 135, 2006).
  • binding domains of the present disclosure that specifically bind a particular target, as well as determining binding domain or fusion protein affinities, such as Western blot, ELISA, analytical
  • MART-1 -specific binding protein refers to a protein or polypeptide that specifically binds to MART-1 or a peptide or fragment thereof.
  • a protein or polypeptide binds to MART-1 or a peptide thereof, such as a MART-1 peptide in complexed with an MHC or HLA molecule, e.g., on a cell surface, with at or at least about a particular affinity.
  • a MART-1- specific binding protein binds a MART-1 -derived peptide:HLA complex (or MART-1- derived peptide:MHC complex) with a 3 ⁇ 4 of less than about 10 "8 M, less than about 10 "9 M, less than about 10 "10 M, less than about 10 "11 M, less than about 10 "12 M, or less than about 10 "13 M, or with an affinity that is about the same as, at least about the same as, or is greater than at or about the affinity exhibited by an exemplary MART-1 specific binding protein provided herein, such as any of the MART-1 -specific TCRs provided herein, for example, as measured by the same assay.
  • a MART-1 -specific binding protein comprises a MART-1 -specific immunoglobulin superfamily binding protein or binding portion thereof.
  • apparent affinity for a TCR is measured by assessing binding to various concentrations of tetramers, for example, by flow cytometry using labeled tetramers.
  • apparent K D of a TCR is measured using 2-fold dilutions of labeled tetramers at a range of concentrations, followed by determination of binding curves by non-linear regression, apparent K D being determined as the concentration of ligand that yielded half-maximal binding.
  • MART-1 binding domain or "MART-1 binding fragment” refer to a domain or portion of a MART-1 -specific binding protein responsible for the specific MART-1 binding.
  • a MART-1 -specific binding domain alone i.e., without any other portion of a MART-1 -specific binding protein
  • Exemplary MART-1 -specific binding domains include MART-1 -specific scTCR (e.g., single chain o ⁇ TCR proteins such as Va-L- ⁇ , ⁇ -L-Va, Va-Ca-L-Va, or Va-L-V -C , wherein Va and ⁇ are TCRa and ⁇ variable domains respectively, Ca and C are TCRa and ⁇ constant domains, respectively, and L is a linker) and scFv fragments as described herein, which can be derived from an anti-MART-1 TCR or antibody.
  • MART-1 -specific scTCR e.g., single chain o ⁇ TCR proteins such as Va-L- ⁇ , ⁇ -L-Va, Va-Ca-L-Va, or Va-L-V -C , wherein Va and ⁇ are TCRa and ⁇ variable domains respectively, Ca and C are TCRa and ⁇ constant domains, respectively, and L is a linker
  • APC antigen presenting cells
  • MHC major histocompatibility complex
  • processed antigen peptides originating in the cytosol are generally from about 7 amino acids to about 1 1 amino acids in length and will associate with class I MHC molecules
  • peptides processed in the vesicular system e.g., bacterial, viral
  • peptides processed in the vesicular system will vary in length from about 10 amino acids to about 25 amino acids and associate with class II MHC molecules.
  • MART-1 antigen or " MART-1 peptide antigen” refer to a naturally or synthetically produced portion of a MART-1 protein ranging in length from about 7 amino acids to about 15 amino acids, which can form a complex with a MHC (e.g., HLA) molecule and such a complex can bind with a TCR specific for a MART-1 peptide:MHC (e.g., HLA) complex.
  • MART-1 antigen peptides are presented in the context of class I MHC. In particular embodiments, a MART-1 peptide is
  • EAAGIGILTV (SEQ ID NO: 1), which is known to associate with human class I HLA (and, more specifically, associates with allele HLA-A*0201).
  • a “linker” refers to an amino acid sequence that connects two proteins, polypeptides, peptides, domains, regions, or motifs and may provide a spacer function compatible with interaction of the two sub-binding domains so that the resulting polypeptide retains a specific binding affinity (e.g., scTCR) to a target molecule or retains signaling activity (e.g., TCR complex).
  • a linker is comprised of about two to about 35 amino acids, for instance, or about four to about 20 amino acids or about eight to about 15 amino acids or about 15 to about 25 amino acids.
  • “Junction amino acids” or “junction amino acid residues” refer to one or more (e.g., about 2-10) amino acid residues between two adjacent motifs, regions or domains of a polypeptide, such as between a binding domain and an adjacent constant domain or between a TCR chain and an adjacent self-cleaving peptide. Junction amino acids may result from the construct design of a fusion protein (e.g., amino acid residues resulting from the use of a restriction enzyme site during the construction of a nucleic acid molecule encoding a fusion protein).
  • altered domain refers to a motif, region, domain, peptide, polypeptide, or protein with a non-identical sequence identity to a wild type motif, region, domain, peptide, polypeptide, or protein (e.g., a wild type TCRa chain, TCR chain, TCRa constant domain, TCR constant domain) of at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%).
  • nucleic acid or “nucleic acid molecule” refers to any of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), oligonucleotides, fragments generated, for example, by the polymerase chain reaction (PCR) or by in vitro translation, and fragments generated by any of ligation, scission, endonuclease action, or exonuclease action.
  • the nucleic acids of the present disclosure are produced by PCR.
  • Nucleic acids may be composed of monomers that are naturally occurring nucleotides (such as deoxyribonucleotides and ribonucleotides), analogs of naturally occurring nucleotides (e.g., a-enantiomeric forms of naturally- occurring nucleotides), or a combination of both. Modified nucleotides can have modifications in or replacement of sugar moieties, or pyrimidine or purine base moieties. Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such linkages.
  • Analogs of phosphodiester linkages include phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate, phosphoramidate, and the like. Nucleic acid molecules can be either single stranded or double stranded.
  • isolated means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring).
  • a naturally occurring nucleic acid or polypeptide present in a living animal is not isolated, but the same nucleic acid or polypeptide, separated from some or all of the co-existing materials in the natural system, is isolated.
  • Such nucleic acid could be part of a vector and/or such nucleic acid or polypeptide could be part of a composition (e.g., a cell lysate), and still be isolated in that such vector or composition is not part of the natural environment for the nucleic acid or polypeptide.
  • gene means the segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region "leader and trailer” as well as intervening sequences (introns) between individual coding segments (exons).
  • the term “recombinant” or “genetically engineered” refers to a cell, microorganism, nucleic acid molecule, or vector that has been genetically modified by human intervention - that is, modified by introduction of an exogenous or heterologous nucleic acid molecule, or refers to a cell or microorganism that has been altered such that expression of an endogenous nucleic acid molecule or gene is controlled, deregulated or constitutive.
  • Human generated genetic alterations may include, for example, modifications that introduce nucleic acid molecules (which may include an expression control element, such as a promoter) that encode one or more proteins or enzymes, or other nucleic acid molecule additions, deletions, substitutions, or other functional disruption of or addition to a cell's genetic material.
  • modifications include those in coding regions or functional fragments thereof of heterologous or homologous polypeptides from a reference or parent molecule.
  • mutation refers to a change in the sequence of a nucleic acid molecule or polypeptide molecule as compared to a reference or wild-type nucleic acid molecule or polypeptide molecule, respectively.
  • a mutation can result in several different types of change in sequence, including substitution, insertion or deletion of nucleotide(s) or amino acid(s).
  • a mutation is a substitution of one or three codons or amino acids, a deletion of one to about 5 codons or amino acids, or a combination thereof.
  • a "conservative substitution” is recognized in the art as a substitution of one amino acid for another amino acid that has similar properties.
  • Exemplary conservative substitutions are well known in the art (see, e.g., WO 97/09433 at page 10; Lehninger, Biochemistry, 2 m Edition; Worth Publishers, Inc. NY, NY, pp.71-77, 1975; Lewin, Genes IV, Oxford University Press, NY and Cell Press, Cambridge, MA, p. 8, 1990).
  • construct refers to any polynucleotide that contains a recombinant nucleic acid molecule.
  • a construct may be present in a vector (e.g., a bacterial vector, a viral vector) or may be integrated into a genome.
  • a "vector” is a nucleic acid molecule that is capable of transporting another nucleic acid molecule.
  • Vectors may be, for example, plasmids, cosmids, viruses, a RNA vector or a linear or circular DNA or RNA molecule that may include chromosomal, non-chromosomal, semi-synthetic or synthetic nucleic acid molecules.
  • Exemplary vectors are those capable of autonomous replication (episomal vector) or expression of nucleic acid molecules to which they are linked (expression vectors).
  • Viral vectors include retrovirus, adenovirus, parvovirus (e.g., adeno-associated viruses), coronavirus, negative strand RNA viruses such as ortho-myxovirus (e.g., influenza virus), rhabdovirus (e.g., rabies and vesicular stomatitis virus), paramyxovirus (e.g., measles and Sendai), positive strand RNA viruses such as picornavirus and alphavirus, and double-stranded DNA viruses including adenovirus, herpesvirus (e.g., Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxvirus (e.g., vaccinia, fowlpox and canarypox).
  • ortho-myxovirus e.g., influenza virus
  • rhabdovirus e.g., rabies and vesicular stomatitis virus
  • viruses include Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, and hepatitis virus, for example.
  • retroviruses include avian leukosis-sarcoma, mammalian C- type, B-type viruses, D type viruses, HTLV-BLV group, lentivirus, spumavirus (Coffin, J. M., Retroviridae: The viruses and their replication, In Fundamental Virology, Third Edition, B. N. Fields et al., Eds., Lippincott-Raven Publishers, Philadelphia, 1996).
  • Lentiviral vector means HIV-based lentiviral vectors for gene delivery, which can be integrative or non-integrative, have relatively large packaging capacity, and can transduce a range of different cell types. Lentiviral vectors are usually generated following transient transfection of three (packaging, envelope and transfer) or more plasmids into producer cells. Like HIV, lentiviral vectors enter the target cell through the interaction of viral surface glycoproteins with receptors on the cell surface. On entry, the viral RNA undergoes reverse transcription, which is mediated by the viral reverse transcriptase complex. The product of reverse transcription is a double-stranded linear viral DNA, which is the substrate for viral integration into the DNA of infected cells.
  • operably-linked refers to the association of two or more nucleic acid molecules on a single nucleic acid fragment so that the function of one is affected by the other.
  • a promoter is operably-linked with a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter).
  • Unlinked means that the associated genetic elements are not closely associated with one another and the function of one does not affect the other.
  • expression vector refers to a DNA construct containing a nucleic acid molecule that is operably-linked to a suitable control sequence capable of effecting the expression of the nucleic acid molecule in a suitable host.
  • control sequences include a promoter to effect transcription, an optional operator sequence to control such transcription, a sequence encoding suitable mRNA ribosome binding sites, and sequences which control termination of transcription and translation.
  • the vector may be a plasmid, a phage particle, a virus, or simply a potential genomic insert. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or may, in some instances, integrate into the genome itself.
  • "plasmid,” “expression plasmid,” “virus” and “vector” are often used interchangeably.
  • expression refers to the process by which a polypeptide is produced based on the encoding sequence of a nucleic acid molecule, such as a gene.
  • the process may include transcription, post-transcriptional control, post-transcriptional modification, translation, post-translational control, post- translational modification, or any combination thereof.
  • the term "introduced” in the context of inserting a nucleic acid molecule into a cell means “transfection", or 'transformation” or “transduction” and includes reference to the incorporation of a nucleic acid molecule into a eukaryotic or prokaryotic cell wherein the nucleic acid molecule may be incorporated into the genome of a cell (e.g., chromosome, plasmid, plastid, or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).
  • a cell e.g., chromosome, plasmid, plastid, or mitochondrial DNA
  • transiently expressed e.g., transfected mRNA
  • heterologous or exogenous nucleic acid molecule, construct or sequence refers to a nucleic acid molecule or portion of a nucleic acid molecule that is not native to a host cell, but may be homologous to a nucleic acid molecule or portion of a nucleic acid molecule from the host cell.
  • the source of the heterologous or exogenous nucleic acid molecule, construct or sequence may be from a different genus or species.
  • a heterologous or exogenous nucleic acid molecule is added (i.e., not endogenous or native) to a host cell or host genome by, for example, conjugation, transformation, transfection, electroporation, or the like, wherein the added molecule may integrate into the host genome or exist as extra-chromosomal genetic material (e.g., as a plasmid or other form of self-replicating vector), and may be present in multiple copies.
  • heterologous refers to a non-native enzyme, protein or other activity encoded by an exogenous nucleic acid molecule introduced into the host cell, even if the host cell encodes a homologous protein or activity.
  • heterologous or exogenous nucleic acid molecule can be introduced into a host cell as separate nucleic acid molecules, as a plurality of individually controlled genes, as a polycistronic nucleic acid molecule, as a single nucleic acid molecule encoding a fusion protein, or any combination thereof.
  • a host cell can be modified to express two or more heterologous or exogenous nucleic acid molecules encoding desired TCR specific for a MART-1 antigen peptide (e.g., TCRa and TCR ).
  • the two or more exogenous nucleic acid molecules can be introduced as a single nucleic acid molecule (e.g., on a single vector), on separate vectors, integrated into the host chromosome at a single site or multiple sites, or any combination thereof.
  • the number of referenced heterologous nucleic acid molecules or protein activities refers to the number of encoding nucleic acid molecules or the number of protein activities, not the number of separate nucleic acid molecules introduced into a host cell.
  • endogenous refers to a gene, protein, or activity that is normally present in a host cell. Moreover, a gene, protein or activity that is mutated, overexpressed, shuffled, duplicated or otherwise altered as compared to a parent gene, protein or activity is still considered to be endogenous or native to that particular host cell.
  • an endogenous control sequence from a first gene e.g., promoter, translational attenuation sequences
  • a second native gene or nucleic acid molecule may be used to alter or regulate expression of a second native gene or nucleic acid molecule, wherein the expression or regulation of the second native gene or nucleic acid molecule differs from normal expression or regulation in a parent cell.
  • homologous refers to a molecule or activity found in or derived from a host cell, species or strain.
  • a heterologous or exogenous nucleic acid molecule may be homologous to a native host cell gene, and may optionally have an altered expression level, a different sequence, an altered activity, or any combination thereof.
  • Sequence identity refers to the percentage of amino acid residues in one sequence that are identical with the amino acid residues in another 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.
  • the percentage sequence identity values can be generated using the NCBI BLAST2.0 software as defined by Altschul et al. (1997) "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25:3389-3402, with the parameters set to default values.
  • hematopoietic progenitor stem cell refers to undifferentiated hematopoietic cells that are capable of self-renewal either in vivo, essentially unlimited propagation in vitro, and capable of differentiation to other cell types including cells of the T cell lineage.
  • Hematopoietic stem cells may be isolated, for example, but not limited to, from fetal liver, bone marrow, cord blood.
  • hematopoietic progenitor cell is a cell that can be derived from hematopoietic stem cells or fetal tissue and is capable of further differentiation into mature cells types ⁇ e.g., immune system cells).
  • exemplary hematopoietic progenitor cells include those with a CD24 Lo Lin " CD117 + phenotype or those found in the thymus (referred to as progenitor thymocytes).
  • a host refers to a cell ⁇ e.g., T cell) or microorganism targeted for genetic modification with a heterologous or exogenous nucleic acid molecule to produce a polypeptide of interest (e.g., high or enhanced affinity anti- MART-1 TCR).
  • a host cell may optionally already possess or be modified to include other genetic modifications that confer desired properties related or unrelated to biosynthesis of the heterologous or exogenous protein (e.g., inclusion of a detectable marker; deleted, altered or truncated endogenous TCR; increased co- stimulatory factor expression).
  • a host cell is a human hematopoietic progenitor cell transduced with a heterologous or exogenous nucleic acid molecule encoding a TCRP, TCRa chain or both, specific for a MART-1 antigen peptide.
  • a host cells are autologous, allogeneic or syngeneic to subject to receive the host cells containing a polynucleotide encoding a binding protein of this disclosure.
  • hyperproliferative disorder refers to excessive growth or proliferation as compared to a normal or undiseased cell.
  • exemplary hyperproliferative disorders include tumors, cancers, neoplastic tissue, carcinoma, sarcoma, malignant cells, pre-malignant cells, as well as non-neoplastic or non-malignant hyperproliferative disorders (e.g., adenoma, fibroma, lipoma, leiomyoma, hemangioma, fibrosis, restenosis, as well as autoimmune diseases such as rheumatoid arthritis, osteoarthritis, psoriasis, inflammatory bowel disease, or the like).
  • a hyperproliferative disorder is a melanoma. Binding Proteins Specific for MART-1 Antigen
  • MART-1 or "Protein melan-A” or “melanoma antigen recognized by T cells 1” refers to a 118 amino acid transmembrane protein encoded by the MLANA gene.
  • the transcript sequence for human MLANA is set forth in NCBI Reference identifier
  • MART-1 is an antigen specific for cells of the melanocyte lineage, found in skin, the retina, and melanocytes, but not in other normal tissues. MART-1 is an attractive target for tumor therapy because it is the antigen most commonly expressed by melanoma tumors, and MART-1 epitopes presented in the HLA-A*0201 context are most frequently recognized by tumor infiltrating lymphocytes (TILs) from melanoma patients (Labarriere et al., 1998, Int. J. Cancer 78:209-215; Benlalam et al. 2001, Eur.
  • TILs tumor infiltrating lymphocytes
  • the present disclosure provides a binding protein (e.g., an immunoglobulin superfamily binding protein or portion thereof), comprising (a) a T cell receptor (TCR) a-chain variable (V a ) domain, and a TCR ⁇ -chain variable domain comprising a CDR3 amino acid sequence set forth in any one of SEQ ID NOS:4-16 and 24-151, wherein the binding protein is capable of specifically binding to a MART-1 peptide (SEQ ID NO: l):HLA complex.
  • the ⁇ CDR3 amino acid sequence consists of the amino acid sequence set forth in any one of SEQ ID NOS:4-16 and 24-151.
  • a ⁇ CDR3 amino acid sequence comprises or consists of the amino acid sequence set forth in any one of SEQ ID
  • the ⁇ ⁇ CDR3 amino acid sequence comprises or consists of the amino acid sequence of SEQ ID NO:4.
  • An exemplary ⁇ amino acid sequence that comprises the CDR3 amino acid sequence of SEQ ID NO:4 is set forth in SEQ ID NO: 173.
  • An exemplary TCR ⁇ -chain amino acid sequence that comprises the ⁇ ⁇ amino acid sequence of SEQ ID NO: 173 is set forth in SEQ ID NO: 172.
  • a binding protein e.g., an immunoglobulin
  • superfamily binding protein or portion thereof as described herein includes variant polypeptide species that have up to five amino acid substitutions, insertions, or deletions in the amino acid sequence of any one of SEQ ID NOS:4-16 and 24-151 as presented herein, provided that the binding protein retains or substantially retains its specific binding function to MART-1 peptide EAAGIGILTV (SEQ ID NO: 1).
  • a variant binding protein has one, two, three, four, or five amino acids substitutions, insertions, or deletions in the amino acid sequence of any one of SEQ ID NOS:4-16 and 24-151, provided that the binding protein retains or substantially retains its ability to specificaly bind to MART-1 peptide EAAGIGILTV (SEQ ID NO: 1):HLA complex.
  • a binding protein provided herein may comprise a variant TCR ⁇ domain having an amino acid sequence with at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 1
  • binding protein retains or substantially retains its binding function to MART-1 peptide (SEQ ID NO: 1):HLA complex.
  • a binding protein provided herein may comprise a variant TCR ⁇ -chain having an amino acid sequence with at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 172, provided that the binding protein retains or substantially retains its ability to specifically bind to MART-1 peptide (SEQ ID NO: l):HLA complex.
  • Binding proteins provided herein that comprise a ⁇ CDR3 amino acid sequence comprising of the amino acid sequence of SEQ ID NO:4, or a ⁇ ⁇ domain comprising an amino acid sequence set forth in SEQ ID NO: 173, may be paired with a V a domain comprising a CDR3 amino acid sequence as set forth in Table 1 A (e.g., any one of SEQ ID NOS: 155, 157, 159, 161, 163, 165, 167, and 169).
  • an exemplary binding protein comprises: (a) a T cell receptor (TCR) a-chain variable (V a ) domain comprising a CDR3 amino acid sequence set forth in any one of SEQ ID NOS: 155, 157, 159, 161, 163, 165, 167, and 169; and a TCR ⁇ -chain variable ( ⁇ ⁇ ) domain comprising a CDR3 amino acid sequence set forth in SEQ ID NO:4, or a ⁇ ⁇ domain comprising an amino acid sequence set forth in SEQ ID NO: 173, wherein the binding protein is capable of specifically binding to a MART-1 peptide (SEQ ID NO: 1):HLA complex.
  • TCR T cell receptor
  • V a a-chain variable domain comprising a CDR3 amino acid sequence set forth in any one of SEQ ID NOS: 155, 157, 159, 161, 163, 165, 167, and 169
  • TCR ⁇ -chain variable ( ⁇ ⁇ ) domain comprising a CDR3 amino acid
  • a binding protein as described herein comprises a ⁇ CDR3 amino acid sequence comprising SEQ ID NO:4 or a ⁇ ⁇ domain comprising an amino acid sequence set forth in SEQ ID NO: 173, and a V a CDR3 amino acid sequence of SEQ ID NO: 155; a ⁇ ⁇ CDR3 amino acid sequence comprising SEQ ID NO:4 or a ⁇ ⁇ domain comprising an amino acid sequence set forth in SEQ ID NO: 173, and a V a CDR3 amino acid sequence of SEQ ID NO: 157; a ⁇ CDR3 amino acid sequence comprising SEQ ID NO:4 or a ⁇ ⁇ domain comprising an amino acid sequence set forth in SEQ ID NO: 173, and a V a CDR3 amino acid sequence of SEQ ID NO: 159; a ⁇ ⁇ CDR3 amino acid sequence comprising SEQ ID NO:4 or a ⁇ ⁇ domain comprising an amino acid sequence set forth in SEQ ID NO: 173, and a V a CDR3 amino
  • binding proteins provided herein that may comprise a V a domain comprising a variant of the CDR3 amino acid sequence set forth in any one of SEQ ID NOS: 155, 157, 159, 161, 163, 165, 167, and 169 that has up to five (one, two, three, four, or five) amino acid substitutions, insertions, or deletions in the amino acid sequence of any one of SEQ ID NO S: 155, 157, 159, 161, 163, 165, 167, and 169, respectively, provided that the binding protein retains or substantially retains its ability to specifically bind to MART-1 peptide (SEQ ID NO: 1):HLA complex.
  • SEQ ID NO: 1 MART-1 peptide
  • the present disclosure further provides a binding protein (e.g., an immunoglobulin superfamily binding protein or portion thereof), comprising (a) a T cell receptor (TCR) a-chain variable (V a ) domain, and a TCR ⁇ -chain variable ( ⁇ 3 ⁇ 4) domain comprising a CDR3 amino acid sequence encoded by a nucleotide sequence contained in SEQ ID NO: 153, wherein the binding protein is capable of specifically binding to a Tyrosinase peptide (SEQ ID NO:23):HLA complex.
  • a binding protein e.g., an immunoglobulin superfamily binding protein or portion thereof
  • TCR T cell receptor
  • V a a-chain variable
  • ⁇ 3 ⁇ 4 domain comprising a CDR3 amino acid sequence encoded by a nucleotide sequence contained in SEQ ID NO: 153, wherein the binding protein is capable of specifically binding to a Tyrosinase peptide (SEQ ID NO:23):HLA complex.
  • Tyrosinase refers to refers to a 529 amino acid protein encoded by the TYR gene.
  • the transcript sequence for human TYR is set forth in NCBI Reference identifier NM_000372.4, and protein sequence is set forth in NCBI Reference identifier
  • Tyrosinase is an oxidase that is involved in melanin synthesis.
  • Tyrosinase is an attractive target for melanoma therapy because it an antigen commonly expressed by melanoma tumors.
  • a binding protein ⁇ e.g., an immunoglobulin superfamily binding protein or portion thereof) as described herein includes variant polypeptide species that have up to five (one, two, three, four, or five) amino acid substitutions, insertions, or deletions in the amino acid sequence of ⁇ CDR3 encoded by the nucleotide acid sequence contained in SEQ ID NO: 153 as presented herein, provided that the binding protein retains or substantially retains its ability to specifically bind to Tyrosinase peptide SEIWRDIDF (SEQ ID NO:23).
  • Peptide-MHC complexes such as MART-1 peptide (SEQ ID NO: 1):MHC complexes are recognized by and bound through the TCR Va and TCR ⁇ domains.
  • Va exons are assembled from different variable and joining gene segments (V-J)
  • ⁇ exons are assembled from different variable, diversity, and joining gene segments (V-D-J).
  • the TCRa chromosomal locus has 70-80 variable gene segments and 61 joining gene segments.
  • the TCRP chromosomal locus has 52 variable gene segments, and two separate clusters of each containing a single diversity gene segment, together with six or seven joining gene segments.
  • Functional Va and ⁇ gene exons are generated by the recombination of a variable gene segment with a joining gene segment for Va, and a variable gene segment with a diversity gene segment and a joining gene segment for ⁇ .
  • the Va and ⁇ domains each comprise three hypervariable loops, also referred to as complementary determining regions (CDRs) that contact the peptide-MHC complex.
  • CDR1 and CDR2 are encoded within the variable gene segment
  • CDR3 is encoded by the region spanning the variable and joining segments for Va, or the region spanning variable, diversity, and joining segments for ⁇ .
  • the identity of the variable gene segment of a Va or ⁇ is known, the sequences of their corresponding CDR1 and CDR2 can be deduced.
  • CDR3 is significantly more diverse because of the addition and loss of nucleotides during the recombination process.
  • TCR variable domain sequences can be aligned to a numbering scheme (Kabat, Chothia, Enhanced Chothia, and Aho), allowing equivalent residue positions to be annotated and for different molecules to be compared using ANARCI software tool (2016, Bioinformatics 15:298-300).
  • a numbering scheme provides a standardized delineation of framework regions and CDRs in the TCR variable domains.
  • Table 1A provides the identities of the variable gene segment and joining gene segment for a TCR Va comprising a CDR3 comprising an amino acid sequence of any one of SEQ ID NOS: 155, 157, 159, 161, 163, 165, 167, and 169 and the identities of the variable gene segment and joining gene segment for a TCR ⁇ comprising a CDR3 comprising an amino acid sequence of any one of SEQ ID NOS:4-16. Accordingly, the CDR1 and CDR2 sequences may be deduced from the corresponding variable gene segments.
  • a TCR Va comprising a CDR3 comprising an amino acid sequence of SEQ ID NO: 155 is contained in an amino acid sequence encoded by a variable gene segment of TCRAV12-02 and a joining gene segment of TCRAJ34- 01 *01. Accordingly, the TCR Va comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 155 and a CDR1 and CDR2 sequence encoded by a
  • the TCR Va comprising a CDR3 comprising an amino acid sequence of SEQ ID NO: 157 is contained in an amino acid sequence encoded by a variable gene segment of TCRAV29-01 and a joining gene segment of TCRAJ26-01 *01. Accordingly, the TCR Va comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 157 and a CDRl and CDR2 sequence encoded by a TCRAV29-01 gene.
  • the TCR Va comprising a CDR3 comprising an amino acid sequence of SEQ ID NO: 159 is contained in an amino acid sequence encoded by a variable gene segment of TCRAV12-02 and a joining gene segment of TCRAJ30-01 *01.
  • the TCR Va comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 159 and a CDRl and CDR2 sequence encoded by a TCRAV12-02 gene.
  • the TCR Va comprising a CDR3 comprising an amino acid sequence of SEQ ID NO: 161 is contained in an amino acid sequence encoded by a variable gene segment of TCRAV12-01 and a joining gene segment of TCRAJ24-01.
  • the TCR Va comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 161 and a CDRl and CDR2 sequence encoded by a TCRAV12-01 gene.
  • the TCR Va comprising a CDR3 comprising an amino acid sequence of SEQ ID NO: 163 is contained in an amino acid sequence encoded by a variable gene segment of TCRAV13-02 and a joining gene segment of TCRAJ38-01 *01. Accordingly, the TCR Va comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 163 and a CDRl and CDR2 sequence encoded by a TCRAV13-02 gene. In another embodiment, the TCR Va comprising a CDR3 comprising an amino acid sequence of SEQ ID NO: 165 is contained in an amino acid sequence encoded by a variable gene segment of TCRAV12-02 and a joining gene segment of TCRAJ17-01 *01.
  • the TCR Va comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 165 and a CDRl and CDR2 sequence encoded by TCRAV 12-02.
  • the TCR Va comprising a CDR3 comprising an amino acid sequence of SEQ ID NO: 167 is contained in an amino acid sequence encoded by a variable gene segment of TCRAV25-01 *01 and a joining gene segment of a TCRAJ28-01 *01 gene.
  • the TCR Va comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 167 and a CDRl and CDR2 sequence encoded by a TCRAV25-01 *01 gene.
  • the TCR Va comprising a CDR3 comprising an amino acid sequence of SEQ ID NO: 169 is contained in an amino acid sequence encoded by a a variable gene segment of
  • the TCR Va comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 169 and a CDRl and CDR2 sequence encoded by a TCRAV29-01 gene.
  • the TCR ⁇ comprising a CDR3 comprising an amino acid sequence of SEQ ID NO:4 is contained in an amino acid sequence encoded by a variable gene segment of TCRBV28-01 and a joining gene segment of TCRBJ02- 07. Accordingly, the TCR ⁇ comprises a CDR3 comprising an amino acid sequence of SEQ ID NO:4 and a CDRl and CDR2 sequence encoded by a TCRBV28-01 gene. In some embodiments, the TCR ⁇ comprising a CDR3 comprising an amino acid sequence of SEQ ID NO:5 is contained in an amino acid sequence encoded by a variable gene segment of TCRBV06-05 and a joining gene segment of TCRBJOl-05.
  • the TCR ⁇ comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 5 and a CDRl and CDR2 sequence encoded by TCRBV06-05.
  • the TCR ⁇ comprising a CDR3 comprising an amino acid sequence of SEQ ID NO:6 is contained in an amino acid sequence encoded by a variable gene segment of TCRBV28-01 and a joining gene segment of TCRBJ02-01.
  • the TCR ⁇ comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 6 and a CDRl and CDR2 sequence encoded by TCRBV28-01.
  • the TCR ⁇ comprising a CDR3 comprising an amino acid sequence of SEQ ID NO:7 is contained in an amino acid sequence encoded by a variable gene segment of TCRBV04-01 and a joining gene segment of TCRBJOl-06. Accordingly, the TCR ⁇ comprises a CDR3 comprising an amino acid sequence of SEQ ID NO:7 and a CDRl and CDR2 sequence encoded by TCRBV04-01. In some embodiments, the TCR ⁇ comprising a CDR3 comprising an amino acid sequence of SEQ ID NO:8 is contained in an amino acid sequence encoded by a variable gene segment of TCRBV05-05 and a joining gene segment of TCRBJ02-04.
  • the TCR ⁇ comprises a CDR3 comprising an amino acid sequence of SEQ ID NO:8 and a CDRl and CDR2 sequence encoded by TCRBV05-05.
  • the TCR ⁇ comprising a CDR3 comprising an amino acid sequence of SEQ ID NO:9 is contained in an amino acid sequence encoded by a variable gene segment of TCRBV28-01 and a joining gene segment of TCRBJ02-03.
  • the TCR ⁇ comprises a CDR3 comprising an amino acid sequence of SEQ ID NO:9 and a CDRl and CDR2 sequence encoded by TCRBV28-01.
  • the TCR ⁇ comprising a CDR3 comprising an amino acid sequence of SEQ ID NO: 10 is contained in an amino acid sequence encoded by a variable gene segment of TCRB V28-01 and a j oining gene segment of TCRB JO 1 - 05. Accordingly, the TCR ⁇ comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10 and a CDRl and CDR2 sequence encoded by a TCRBV28-01 gene.
  • the TCR ⁇ comprising a CDR3 comprising an amino acid sequence of SEQ ID NO: 11 is contained in an amino acid sequence encoded by a variable gene segment of TCRB V06-05 and a j oining gene segment of TCRB JO 1 -05. Accordingly, the TCR ⁇ comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 11 and a CDRl and CDR2 sequence encoded by a TCRBV06-05 gene. In some embodiments, the TCR ⁇ comprising a CDR3 comprising an amino acid sequence of SEQ ID NO: 12 is contained in an amino acid sequence encoded by a variable gene segment of TCRBV05-04 and a joining gene segment of TCRBJ02-01.
  • the TCR ⁇ comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 12 and a CDRl and CDR2 sequence encoded by a TCRBV05-04 gene.
  • the TCR ⁇ comprising a CDR3 comprising an amino acid sequence of SEQ ID NO: 14 is contained in an amino acid sequence encoded by a variable gene segment of TCRB V27-01 and a j oining gene segment of TCRB J02-03.
  • the TCR ⁇ comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 14 and a CDRl and CDR2 sequence encoded by a TCRBV27-01 gene.
  • the TCR ⁇ comprising a CDR3 comprising an amino acid sequence of SEQ ID NO: 15 is contained in an amino acid sequence encoded by a a variable gene segment of TCRBV06-05 and a joining gene segment of TCRBJ02-02. Accordingly, the TCR ⁇ comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 15 and a CDRl and CDR2 sequence encoded by a TCRBV06-05 gene.
  • the TCR ⁇ comprising a CDR3 comprising an amino acid sequence of SEQ ID NO: 16 is contained in an amino acid sequence encoded by a variable gene segment of TCRBV06-06 and a joining gene segment of TCRB JO 1-05. Accordingly, the TCR ⁇ comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 16 and a CDR1 and CDR2 sequence encoded by a TCRBV06-06 gene.
  • V a domain for the MART- 1 specific ⁇ domains described herein may be identified using methods known in the art.
  • amino acids are well known and may occur naturally or may be introduced when the binding protein or TCR is recombinantly produced.
  • Amino acid substitutions, deletions, and additions may be introduced into a protein using mutagenesis methods known in the art (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, NY, 2001).
  • Oligonucleotide-directed site-specific (or segment specific) mutagenesis procedures may be employed to provide an altered polynucleotide that has particular codons altered according to the substitution, deletion, or insertion desired.
  • random or saturation mutagenesis techniques such as alanine scanning mutagenesis, error prone polymerase chain reaction mutagenesis, and oligonucleotide- directed mutagenesis may be used to prepare immunogen polypeptide variants (see, e.g., Sambrook et al., supra).
  • amino acid that is substituted at a particular position in a peptide or polypeptide is conservative (or similar).
  • a similar amino acid or a conservative amino acid substitution is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain.
  • amino acids with basic side chains e.g., lysine, arginine, histidine
  • amino acids with acidic side chains e.g., aspartic acid, glutamic acid
  • amino acids with uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, histidine
  • amino acids with nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
  • amino acids with beta-branched side chains e.g., threonine, valine, isoleucine
  • amino acids with aromatic side chains e.g., tyrosine, phenylalanine, tryptophan
  • Proline which is considered more difficult to classify, shares properties with amino acids that have aliphatic side chains (e.g., leucine, valine, isoleucine, and alanine).
  • substitution of glutamine for glutamic acid or asparagine for aspartic acid may be considered a similar substitution in that glutamine and asparagine are amide derivatives of glutamic acid and aspartic acid, respectively.
  • "similarity" between two polypeptides is determined by comparing the amino acid sequence and conserved amino acid substitutes thereto of the polypeptide to the sequence of a second polypeptide (e.g., using GE EWORKS, Align, the BLAST algorithm, or other algorithms described herein and practiced in the art).
  • any of the MART-1 specific binding proteins disclosed herein is capable of specifically binding to MART-1 "EAAGIGILTV” peptide (SEQ ID NO: l): HLA-A*0201 complex.
  • any of the Tyrosinase specific binding proteins disclosed herein is capable of specifically binding to Tyrosinase "SEIWRDIDF” peptide (SEQ ID NO:23): HLA-B*4402 complex.
  • any of the MART-1 or Tyrosinase specific binding proteins disclosed herein are each a T cell receptor (TCR), a chimeric antigen receptor or an antigen-binding fragment of a TCR, any of which can be chimeric, humanized or human.
  • an antigen-binding fragment of the TCR comprises a single chain TCR (scTCR) or a chimeric antigen receptor (CAR).
  • a MART-1 or Tyrosinase specific binding protein is a TCR.
  • a MART-1 or Tyrosinase specific binding protein is an enhanced affinity or high affinity TCR.
  • Binding proteins according to the present disclosure may further comprise a TCR constant domain, e.g., joined to the C-terminus of a V a domain, a ⁇ domain, or both.
  • a TCR ⁇ -chain constant domain may be encoded by a TRBCl gene or TRBC2 gene.
  • the TCR ⁇ -chain constant domain is a TRBC2 constant domain having an amino acid sequence as set forth in SEQ ID NO: 1
  • a TCR a-chain constant domain may be encoded by a TRAC gene.
  • a cell expressing a high affinity or enhanced affinity TCR specific for a MART- 1 peptide is capable of binding to a MART-1 :HL
  • a complex or Tyrosinase:HLA complex
  • Methods of generating enhanced affinity TCRs for use in gene therapy are known in the art, and include techniques involving generation of libraries of TCR mutants that have undergone rounds of mutagenesis and subsequent screening for mutations that confer higher affinity for the target peptide/MHC ligand (Richman & Kranz, 2007, Biomol. Eng.
  • any of the MART-1 binding proteins disclosed herein is capable of specifically binding to MART-1 "EAAGIGILTV” peptide (SEQ ID NO: 1): HLA-A complex with a Kd of less than or equal to 10 "8 M.
  • composition comprising a MART-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N
  • compositions disclosed herein can further comprise at least one additional binding protein comprising a V a domain and a ⁇ ⁇ domain, wherein the additional binding protein is capable of specifically binding to a MART-1 peptide EAAGIGILTV (SEQ ID NO: l):HLA complex.
  • the composition can comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 20, 25, 30, 35, 40, 45, 50 or more additional binding proteins comprising a V a domain and a ⁇ ⁇ domain, wherein the additional binding protein is capable of specifically binding to a MART-1 peptide EAAGIGILTV (SEQ ID NO: 1):HLA complex.
  • the additional binding proteins within the composition are also binding proteins provided herein, e.g., comprising a V a domain, and a ⁇ ⁇ domain comprising CDR3 of any one of the sequences set forth in SEQ ID NOS:4-16 and 24-151.
  • Each of the binding domains present in the composition can be at a frequency of at least 0.1% in the composition. Moreover, the frequency of each of the binding domains may be unevenly distributed in the composition.
  • compositions comprising more than one MART-1 specific binding protein represent polyclonal compositions.
  • a polyclonal composition is composed of at least two or more binding domains that are produced by different host cell (e.g., T cell) lineages.
  • a polyclonal composition is composed of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 75,
  • a monoclonal composition is composed of a binding domain that is produced from a single host cell lineage.
  • TCR may include obtaining supernatants from suitable host cell/vector systems that secrete the recombinant soluble TCR into culture media and then concentrating the media using a commercially available filter. Following concentration, the concentrate may be applied to a single suitable purification matrix or to a series of suitable matrices, such as an affinity matrix or an ion exchange resin. One or more reverse phase HPLC steps may be employed to further purify a recombinant polypeptide. These purification methods may also be employed when isolating an immunogen from its natural environment. Methods for large scale production of one or more of the isolated/recombinant soluble TCR described herein include batch cell culture, which is monitored and controlled to maintain appropriate culture conditions. Purification of the soluble TCR may be performed according to methods described herein and known in the art and that comport with laws and guidelines of domestic and foreign regulatory agencies.
  • nucleic acid molecules encoding an immunoglobulin superfamily binding protein or high affinity TCR specific for MART-1 are used to transfect/transduce a host cell (e.g., T cells) for use in adoptive transfer therapy, wherein the host cells are autologous, allogeneic or syngeneic to the subject to receive the adoptive transfer therapy.
  • a host cell e.g., T cells
  • Advances in TCR sequencing have been described (e.g., Robins et al, Blood 114:4099, 2009; Robins et al, Sci. Translat. Med. 2:47ra64, 2010; Robins et al, J Immunol. Methods 375: 14-9, 2012; Warren et al, Genome Res.
  • the MART-1 -specific binding proteins or domains as described herein may be functionally characterized according to any of a large number of art accepted methodologies for assaying T cell activity, including determination of T cell binding, activation or induction and also including determination of T cell responses that are antigen-specific. Examples include determination of T cell proliferation, T cell cytokine release, antigen-specific T cell stimulation, MHC restricted T cell stimulation, CTL activity ⁇ e.g., by detecting 51 Cr release from pre-loaded target cells), changes in T cell phenotypic marker expression, and other measures of T-cell functions.
  • MHC-peptide tetramer staining refers to an assay used to detect antigen- specific T cells, which features a tetramer of MHC molecules, each comprising an identical peptide having an amino acid sequence that is cognate (e.g., identical or related to) at least one antigen (e.g., MART-1), wherein the complex is capable of binding T cell receptors specific for the cognate antigen.
  • MHC molecules may be tagged with a biotin molecule.
  • Biotinylated MHC/peptides are tetramerized by the addition of streptavidin, which can be fluorescently labeled. The tetramer may be detected by flow cytometry via the fluorescent label.
  • an MHC- peptide tetramer assay is used to detect or select enhanced affinity TCRs of the instant disclosure.
  • cytokines may be determined according to methods described herein and practiced in the art, including for example, ELISA, ELISPOT, intracellular cytokine staining, and flow cytometry and combinations thereof (e.g., intracellular cytokine staining and flow cytometry).
  • Immune cell proliferation and clonal expansion resulting from an antigen-specific elicitation or stimulation of an immune response may be determined by isolating lymphocytes, such as circulating lymphocytes in samples of peripheral blood cells or cells from lymph nodes, stimulating the cells with antigen, and measuring cytokine production, cell proliferation and/or cell viability, such as by incorporation of tritiated thymidine or non-radioactive assays, such as MTT assays and the like.
  • lymphocytes such as circulating lymphocytes in samples of peripheral blood cells or cells from lymph nodes
  • stimulating the cells with antigen and measuring cytokine production, cell proliferation and/or cell viability, such as by incorporation of tritiated thymidine or non-radioactive assays, such as MTT assays and the like.
  • Thl cytokines such as IFN- ⁇ , IL-12, IL-2, and TNF- ⁇
  • Type 2 cytokines such as IL-4, IL-5, IL-9, IL-10, and IL-13.
  • binding protein e.g., immunoglobulin superfamily binding protein
  • TCR T cell receptor
  • Construction of an expression vector that is used for recombinantly producing a binding protein or high affinity engineered TCR specific for a MART-1 or Tyrosinase peptide of interest can be accomplished by using any suitable molecular biology engineering techniques known in the art, including the use of restriction endonuclease digestion, ligation, transformation, plasmid purification, and DNA sequencing as described in, for example, Sambrook et al. (1989 and 2001 editions; Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY) and Ausubel et al. (Current Protocols in Molecular Biology, 2003).
  • a polynucleotide in each recombinant expression construct includes at least one appropriate expression control sequence (also called a regulatory sequence), such as a leader sequence and particularly a promoter operably ⁇ i.e., operatively) linked to the nucleotide sequence encoding the immunogen.
  • a regulatory sequence also called a regulatory sequence
  • nucleic acids that encode the polypeptides contemplated herein, for instance, binding proteins or high affinity engineered TCRs specific for MART-1 or Tyrosinase.
  • a nucleic acid may refer to a single- or a double-stranded DNA, cDNA or RNA in any form, and may include a positive and a negative strand of the nucleic acid which complement each other, including anti-sense DNA, cDNA and RNA.
  • isolated polynucleotides that encode a binding protein or high affinity engineered TCR of this disclosure specific for a MART-1 peptide or a MART-1 peptide :HLA complex.
  • a polynucleotide encodes a V a domain
  • a polynucleotide encodes a ⁇ domain that comprises a nucleotide sequence that encodes a CDR3 as set forth in any one of SEQ ID NOS:4-16 and 24-151.
  • a polynucleotide encodes two or more binding proteins according to any of the embodiments disclosed herein.
  • a polynucleotide that encodes a binding protein or high affinity engineered TCR of this disclosure specific for a Tyrosinase peptide.
  • a polynucleotide encodes a V a domain
  • a polynucleotide encodes a ⁇ domain and comprises a ⁇ CDR3 nucleotide sequence contained or set forth in SEQ ID NO: 153.
  • a polynucleotide encoding a binding protein of the instant disclosure is codon optimized for efficient expression in a target host cell.
  • An exemplary codon optimized polynucleotide sequence of a TCR ⁇ domain is set forth in SEQ ID NO: 174.
  • An exemplary codon optimized polynucleotide sequence of a TCR ⁇ -2 chain constant domain is set forth in SEQ ID NO: 176.
  • An exemplary codon optimized polynucleotide sequence of a TCR ⁇ -chain is set forth in SEQ ID NO: 171.
  • a polynucleotide encoding a binding protein of the instant disclosure encodes an additional sequence disposed between the TCR a-chain encoding polynucleotide and a TCR ⁇ -chain encoding polynucleotide, or TCR ⁇ - chain polynucleotide and TCR ⁇ -chain encoding polynucleotide, allowing multicistronic expression.
  • Sequences that may be used for multicistronic expression include protease sites, viral self-cleaving 2A peptides, furin cleavage sites, and internal ribosome entry sites (IRES). Examples of self-cleaving peptides include those encoded by any one of SEQ ID NOS: 17-21.
  • Standard techniques may be used for recombinant (i.e., engineered) DNA, peptide and oligonucleotide synthesis, immunoassays and tissue culture and
  • Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. These and related techniques and procedures may be generally performed according to conventional methods well- known in the art and as described in various general and more specific references in microbiology, molecular biology, biochemistry, molecular genetics, cell biology, virology and immunology techniques that are cited and discussed throughout the present specification.
  • Certain embodiments include nucleic acid molecules contained in a vector.
  • An exemplary vector may comprise a nucleic acid molecule capable of transporting another nucleic acid molecule to which it has been linked, or which is capable of replication in a host organism.
  • Some examples of vectors include plasmids, viral vectors, cosmids, and others.
  • Some vectors may be capable of autonomous replication in a host cell into which they are introduced (e.g. bacterial vectors having a bacterial origin of replication and episomal mammalian vectors), whereas other vectors may be integrated into the genome of a host cell or promote integration of the polynucleotide insert upon introduction into the host cell and thereby replicate along with the host genome (e.g., lentiviral vector)). Additionally, some vectors are capable of directing the expression of genes to which they are operatively linked (these vectors may be referred to as "expression vectors").
  • agents e.g., polynucleotides encoding binding proteins or high affinity recombinant TCRs specific for MART-1, or variants thereof, as described herein
  • each agent may reside in separate or the same vectors, and multiple vectors (each containing a different agent or the same agent) may be introduced to a cell or cell population or administered to a subject.
  • the nucleic acid encoding binding proteins or high affinity recombinant binding proteins (e.g., TCRs) specific for MART-1 or Tyrosinase may be operatively linked to certain elements of a vector.
  • polynucleotide sequences that are needed to effect the expression and processing of coding sequences to which they are ligated may be operatively linked.
  • Expression control sequences may include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and possibly sequences that enhance protein secretion.
  • Expression control sequences may be operatively linked if they are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest.
  • polynucleotides encoding binding proteins of the instant disclosure are contained in an expression vector that is a viral vector, such as a lentiviral vector or a ⁇ -retroviral vector.
  • a polynucleotide encoding a binding protein or recombinant expression vector comprising a polynucleotide encoding a binding protein is delivered to an appropriate cell, for example, a T cell or an antigen-presenting cell, i.e., a cell that displays a peptide/MHC complex on its cell surface ⁇ e.g., a dendritic cell) and lacks CD8.
  • the host cell is a hematopoietic progenitor cell, hematopoietic stem cell, or a human immune system cell.
  • the immune system cell can be a CD4+ T cell, a CD8+ T cell, a CD4- CD8- double negative T cell, a ⁇ T cell, a natural killer cell, a dendritic cell, or any combination thereof .
  • a T cell is the host
  • the T cell can be naive, a central memory T cell, an effector memory T cell, or any combination thereof.
  • the recombinant expression vectors may therefore also include, for example, lymphoid tissue-specific transcriptional regulatory elements (TREs), such as a B lymphocyte, T lymphocyte, or dendritic cell specific TREs.
  • TREs lymphoid tissue-specific transcriptional regulatory elements
  • Lymphoid tissue specific TREs are known in the art ⁇ see, e.g., Thompson et al., Mol. Cell. Biol. 72: 1043, 1992); Todd et al., J. Exp. Med. 777: 1663, 1993); Penix et al, J. Exp. Med. 775: 1483, 1993).
  • certain embodiments relate to host cells modified ⁇ i.e., genetically engineered) to contain a heterologous polynucleotide encoding a binding protein ⁇ e.g., TCR) or a vector comprising a heterologous polynucleotide encoding a binding protein ⁇ e.g., TCR) that are presently disclosed.
  • a modified or genetically engineered host cell comprising a heterologous polynucleotide encoding at least one binding protein expresses on its cell surface at least one binding protein of the instant disclosure.
  • Host cells can be modified ex vivo or in vivo.
  • a host cell may include any individual cell or cell culture that may receive a vector or the incorporation of a nucleic acid or protein, as well as any progeny cells. The term also encompasses progeny of the host cell, whether genetically or phenotypically the same or different. Suitable host cells may depend on the vector and may include mammalian cells, animal cells, human cells, simian cells, insect cells, yeast cells, and bacterial cells. These cells may be induced to incorporate the vector or other material by use of a viral vector,
  • a host cell containing a polynucleotide encoding a binding protein of this disclosure are comprised of cells that are autologous, allogeneic or syngeneic to the subject receiving the modified host cells, such as in an adoptive immunotherapy procedure.
  • a binding protein of the instant disclosure is expressed at a higher level on the surface of a host T cell containing a polynucleotide encoding the binding protein ⁇ e.g., TCR) as compared to an endogenous TCR.
  • a host cell comprising one or more polynucleotides encoding one or more binding proteins of this disclosure may express a single binding protein or two or more different binding proteins of the instant disclosure.
  • the instant disclosure is directed to methods for treating a hyperproliferative disorder or a condition characterized by MART-1 overexpression by administering to human subject in need thereof an effective amount of a composition comprising a binding protein or high affinity recombinant TCR specific for human MART-1 according to any of the binding proteins or TCRs of this disclosure.
  • the instant disclosure is directed to methods for treating a hyperproliferative disorder or a condition characterized by Tyrosinase overexpression by administering to human subject in need thereof an effective amount of a composition comprising a binding protein or high affinity recombinant TCR specific for human Tyrosinase according to any of the binding proteins or TCRs of this disclosure.
  • the presence of a hyperproliferative disorder or malignant condition in a subject refers to the presence of dysplastic, cancerous and/or transformed cells in the subject, including, for example neoplastic, tumor, non-contact inhibited or oncogenically transformed cells, or the like ⁇ e.g., solid cancers; hematologic cancers including lymphomas and leukemias, such as acute myeloid leukemia, chronic myeloid leukemia, etc.), which are known in the art and for which criteria for diagnosis and classification are established ⁇ e.g., Hanahan and Weinberg, Cell 144:646, 2011; Hanahan and Weinberg, Cell 100:57, 2000; Cavallo et al., Cane. Immunol. Immunother. 60:319, 2011; Kyrigideis et al., J. Carcinog. 9:3, 2010).
  • such cancer cells may be melanoma cells or metastatic melanoma.
  • treat and “treatment,” refer to medical management of a disease, disorder, or condition of a subject ⁇ i.e., patient, host, who may be a human or non-human animal) ⁇ see, e.g.,
  • an appropriate dose and treatment regimen provide one or more of a binding protein or high affinity recombinant TCR specific for human MART-1 ⁇ e.g., comprising ⁇ CDR3 of any one of the sequences in Table IB, such as SEQ ID NO S: 4- 16, and variants thereof) or a host cell expressing the same, and optionally an adjunctive therapy ⁇ e.g., a cytokine such as IL-2, IL-15, IL-21 or any combination thereof), in an amount sufficient to provide therapeutic or prophylactic benefit.
  • a binding protein or high affinity recombinant TCR specific for human MART-1 ⁇ e.g., comprising ⁇ CDR3 of any one of the sequences in Table IB, such as SEQ ID NO S: 4- 16, and variants thereof
  • an adjunctive therapy e.g., a cytokine such as IL-2, IL-15, IL-21 or any combination thereof
  • Therapeutic or prophylactic benefit resulting from therapeutic treatment or prophylactic or preventative methods include, for example an improved clinical outcome, wherein the object is to prevent or retard or otherwise reduce ⁇ e.g., decrease in a statistically significant manner relative to an untreated control) an undesired physiological change or disorder, or to prevent, retard or otherwise reduce the expansion or severity of such a disease or disorder.
  • Beneficial or desired clinical results from treating a subject include abatement, lessening, or alleviation of symptoms that result from or are associated the disease or disorder to be treated; decreased occurrence of symptoms; improved quality of life; longer disease-free status ⁇ i.e., decreasing the likelihood or the propensity that a subject will present symptoms on the basis of which a diagnosis of a disease is made); diminishment of extent of disease; stabilized ⁇ i.e., not worsening) state of disease; delay or slowing of disease progression; amelioration or palliation of the disease state; and remission (whether partial or total), whether detectable or undetectable; or overall survival.
  • Treatment can also mean prolonging survival when compared to expected survival if a subject were not receiving treatment.
  • Subjects in need of the methods and compositions described herein include those who already have the disease or disorder, as well as subjects prone to have or at risk of developing the disease or disorder.
  • Subjects in need of prophylactic treatment include subjects in whom the disease, condition, or disorder is to be prevented ⁇ i.e., decreasing the likelihood of occurrence or recurrence of the disease or disorder).
  • compositions and preparations comprising the compositions and methods described herein can be evaluated by design and execution of in vitro assays, preclinical studies, and clinical studies in subjects to whom administration of the compositions is intended to benefit, as described in the examples.
  • Genetically engineered or modified host cells comprising a heterologous polynucleotide and expressing the binding protein or high affinity recombinant TCR specific for human MART-1 as described herein may be administered to a subject in a pharmaceutically or physiologically acceptable or suitable excipient or carrier.
  • compositions are biologically compatible vehicles, e.g., physiological saline, which are described in greater detail herein, that are suitable for administration to a human or other non-human mammalian subject.
  • genetically modified host cells expressing the binding proteins disclosed herein promote an antigen-specific T cell response against human MART-1 in a class I HLA-restricted manner.
  • the HLA- restricted response is transporter-associated with antigen processing (TAP)
  • TAP independent pathways for MHCI class presentation of endogenous peptides can be advantageous where tumors inhibit TAP function to avoid immune detection.
  • an antigen-specific T cell response induced by the methods disclosed herein comprises at least one of a CD4+ helper lymphocyte (Th) response and a CD8+ cytotoxic T lymphocyte (CTL) response.
  • the CTL response is directed against a MART-1 overexpressing cell.
  • T cells Genetically engineered host cells ⁇ e.g., T cells) expressing the binding proteins described herein can be exposed ex vivo to IL-21. Priming the MART-1 specific T cells with IL-21 (Hinrichs et al., 2008, Blood 111 :5326-33) can promote expansion of CTLs that phenotypically exhibit a less terminally differentiated phenotype, with a majority of cells expressing CD28 after ex vivo culture (Li et al., 2005, J. Immunol. 175:2261-9; Li et al., 2008, Blood 111 :229-35) and exhibiting enhanced persistence after adoptive transfer.
  • IL-21 Priming the MART-1 specific T cells with IL-21 (Hinrichs et al., 2008, Blood 111 :5326-33) can promote expansion of CTLs that phenotypically exhibit a less terminally differentiated phenotype, with a majority of cells expressing CD28 after ex vivo culture (Li
  • the genetically engineered host cells may be cultured for a shorter duration of time, e.g., no more than 6 weeks, which is significantly less than the standard >12 week production time.
  • a clinical grade sorter (Pollack et al., 2014, Journal for Immunotherapy of Cancer 2:36, incorporated by reference in its entirety) can be used to select CTLs for a polyclonal composition, thereby limiting the ex vivo expansion steps usually needed to achieve target CTL numbers for adoptive therapy.
  • Genetically engineered host cells used in adoptive transfer may represent a monoclonal composition or a polyclonal composition.
  • genetically engineered host cells may be further distinguished by their (a) expression of CD28 prior to infusion; (b) production of IFNy, TNFa, IL-2 or any combination thereof prior to infusion; (c) low expression of PD-1 or CD57, or both prior to infusion; or any combination of (a), (b), and (c).
  • a therapeutically effective dose is an amount of host cells (expressing a binding protein or high affinity recombinant TCR specific for human MART-1) used in adoptive transfer that is capable of producing a clinically desirable result (i.e., a sufficient amount to induce or enhance a specific T cell immune response against cells overexpressing MART-1 (e.g., a cytotoxic T cell response) in a statistically significant manner) in a treated human or non-human mammal.
  • a clinically desirable result i.e., a sufficient amount to induce or enhance a specific T cell immune response against cells overexpressing MART-1 (e.g., a cytotoxic T cell response) in a statistically significant manner
  • the dosage for any one patient depends upon many factors, including the patient's size, weight, body surface area, age, the particular therapy to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently.
  • a preferred dose for administration of a host cell comprising a recombinant expression vector as described herein is about 10 7 cells/m 2 , about 5 x 10 7 cells/m 2 , about 108 cells/m2 , about 5 x 108 cells/m2 , about 109 cells/m2 , about 5 x 10 9 cells/m 2 , about 10 10 cells/m 2 , about 5 x 10 10 cells/m 2 , or about 10 11 cells/m 2 .
  • compositions may be administered in a manner appropriate to the disease or condition to be treated (or prevented) as determined by persons skilled in the medical art.
  • An appropriate dose and a suitable duration and frequency of administration of the compositions will be determined by such factors as the health condition of the patient, size of the patient (i.e., weight, mass, or body area), the type and severity of the patient's disease, the particular form of the active ingredient, and the method of administration.
  • an appropriate dose and treatment regimen provide the composition(s) in an amount sufficient to provide therapeutic and/or prophylactic benefit (such as described herein, including an improved clinical outcome, such as more frequent complete or partial remissions, or longer disease-free and/or overall survival, or a lessening of symptom severity).
  • a dose should be sufficient to prevent, delay the onset of, or diminish the severity of a disease associated with disease or disorder.
  • Prophylactic benefit of the immunogenic compositions administered according to the methods described herein can be determined by performing pre-clinical (including in vitro and in vivo animal studies) and clinical studies and analyzing data obtained therefrom by appropriate statistical, biological, and clinical methods and techniques, all of which can readily be practiced by a person skilled in the art.
  • a condition associated with MART-1 overexpression includes any disorder or condition in which underactivity, over-activity or improper activity of a MART-1 cellular or molecular event is present, and typically results from unusually high (with statistical significance) levels of MART-1 expression in afflicted cells ⁇ e.g., melanoma cells), relative to normal cells.
  • a subject having such a disorder or condition would benefit from treatment with a composition or method of the presently described embodiments.
  • Some conditions associated with MART -1 overexpression thus may include acute as well as chronic disorders and diseases, such as those pathological conditions that predispose the subject to a particular disorder.
  • hyperproliferative disorders refer to states of activated and/or proliferating cells (which may also be transcriptionally overactive) in a subject including tumors, neoplasms, cancer, malignancy, etc.
  • the hyperproliferative disorder may also include an aberration or dysregulation of cell death processes, whether by necrosis or apoptosis.
  • aberration of cell death processes may be associated with a variety of conditions, including cancer (including primary, secondary malignancies as well as metastasis), or other conditions.
  • cancer may refer to any accelerated proliferation of cells, including solid tumors, ascites tumors, blood or lymph or other malignancies; connective tissue malignancies; metastatic disease; minimal residual disease following transplantation of organs or stem cells; multi-drug resistant cancers, primary or secondary malignancies, angiogenesis related to malignancy, or other forms of cancer. Also contemplated within the presently disclosed embodiments are specific embodiments wherein only one of the above types of disease is included, or where specific conditions may be excluded regardless of whether or not they are characterized by MART-1 overexpression.
  • Certain methods of treatment or prevention contemplated herein include administering a host cell (which may be autologous, allogeneic or syngeneic to the subject being treated) comprising a desired heterologous nucleic acid molecule encoding a bidning protein as described herein that is stably integrated into the chromosome of the cell.
  • a host cell which may be autologous, allogeneic or syngeneic to the subject being treated
  • a desired heterologous nucleic acid molecule encoding a bidning protein as described herein that is stably integrated into the chromosome of the cell.
  • a cellular composition may be generated ex vivo using autologous, allogeneic or syngeneic immune system cells (e.g., T cells, antigen-presenting cells, natural killer cells) in order to administer a desired,
  • MART-1 -targeted T-cell composition to a subject as an adoptive immunotherapy.
  • administration of a composition or therapy refers to delivering the same to a subject, regardless of the route or mode of delivery. Administration may be effected continuously or intermittently, and parenterally. Administration may be for treating a subject already confirmed as having a recognized condition, disease or disease state, or for treating a subject susceptible to or at risk of developing such a condition, disease or disease state.
  • Co-administration with an adjunctive therapy may include simultaneous and/or sequential delivery of multiple agents in any order and on any dosing schedule (e.g., MART-1 specific recombinant (i.e., engineered) host cells with one or more cytokines (e.g., JL-2, IL-15, IL-21); alkylating agents, such as cyclophosphamide, an inhibitor of an immune checkpoint molecule, or any combination thereof).
  • MART-1 specific recombinant i.e., engineered
  • cytokines e.g., JL-2, IL-15, IL-21
  • alkylating agents such as cyclophosphamide, an inhibitor of an immune checkpoint molecule, or any combination thereof.
  • a plurality of doses of a genetically engineered host cell containing a heterologous polynucleotide encoding a binding protein as described herein is administered to the subject, which may be administered at intervals between administrations of about two to about four weeks.
  • a cytokine e.g., IL-2, IL-15, IL-21
  • IL-2, IL-15, IL-21 is administered sequentially, provided that the subject was administered the genetically engineered host cell containing a heterologous
  • the cytokine is administered subcutaneously (e.g., IL-2, IL-15, IL-21).
  • the subject being treated is further receiving a chemotherapy (e.g., dacarbazine or temozolomide), a BRAF inhibitor, a c-KIT inhibitor, an inhibitor of an immune checkpoint molecule or any combination thereof.
  • a chemotherapy e.g., dacarbazine or temozolomide
  • a BRAF inhibitor e.g., a BRAF inhibitor
  • a c-KIT inhibitor e.g., an inhibitor of an immune checkpoint molecule blocks the activity or expression of the immune checkpoint molecule.
  • An immune checkpoint molecule can be CTLA-4, A2AR, B7- H3, B7-H4, BTLA, HVEM, GAL9, IDO, KIR, LAG-3, PD-1, PD-L1, Tim-3, VISTA, TIGIT, LAIR1, CD 160, 2B4, TGFR beta, CEACAM-1, CEACAM-3, CEACAM-5, CD244, or any combination thereof.
  • an immune checkpoint inhibitor molecule is CTLA-4, PD-1, PD-L1, or a combinatin thereof.
  • an inhibitor of an immune checkpoint molecule is an antibody or antigen binding fragment thereof, a fusion protein, a small molecule, an RNAi molecule, (e.g., siRNA, shRNA, miRNA) a ribozyme, an aptamer, or an antisense oligonucleotide.
  • RNAi molecule e.g., siRNA, shRNA, miRNA
  • the genetically modified host cells are further modified to reduce or inhibit expression of the gene encoding the immune checkpoint inhibitor molecule, for example, by using an RNA-guided endonuclease (e.g., CRISPR/Cas system), a zinc finger nuclease, a Transcription activator-like effector nuclease (TALEN), an RNAi molecule, or an antisense oligonucleotide.
  • the inhibitor of the immune checkpoint molecule is ipilimumab or tremelimumab.
  • the inhibitor of the immune checkpoint molecule is nivolumab or pembrolizumab.
  • the inhibitor of the immune checkpoint molecule is durvalumab or atezolizumab. In still further embodiments, the inhibitor of the immune checkpoint inhibitor molecule is administered concurrently or subsequent to administration of the genetically engineered host cell (e.g., within a week) and may comprise multiple doses administered at intervals of about two to four weeks.
  • methods of treatment involving a polyclonal T cell composition specific for MART-1 may be capable of inducing a de novo antigen-specific T cell response to a non-targeted antigen (e.g., NY-ESOl, gplOO, MAGE A3, tyrosinase, or any combination thereof).
  • a non-targeted antigen e.g., NY-ESOl, gplOO, MAGE A3, tyrosinase, or any combination thereof.
  • This phenomenon of "epitope spreading” may be advantageous for treating melanoma, which is a highly mutated tumor. Multivalent responses induced by epitope spreading may block outgrowth of antigen-loss tumor variants and improve tumor eradication.
  • Polyclonal T cell compositions specific for MART-1 comprising two or more different binding proteins according to the embodiments disclosed herein may be particularly useful for treating subjects afflicted with a proliferative disorder, e.g., melanoma, more particularly metastatic melanoma, that is refractory to monotherapy with MART-1 specific monoclonal adoptive immunotherapy, monotherapy with an inhibitor of an immune checkpoint inhibitor molecule, or a combination thereof.
  • a proliferative disorder e.g., melanoma, more particularly metastatic melanoma
  • MART-1 specific monoclonal adoptive immunotherapy monotherapy with an inhibitor of an immune checkpoint inhibitor molecule, or a combination thereof.
  • An effective amount of a therapeutic or pharmaceutical composition refers to an amount sufficient, at dosages and for periods of time needed, to achieve the desired clinical results or beneficial treatment, as described herein.
  • An effective amount may be delivered in one or more administrations. If the administration is to a subject already known or confirmed to have a disease or disease-state, the term "therapeutic amount” may be used in reference to treatment, whereas “prophylactically effective amount” may be used to describe administrating an effective amount to a subject that is susceptible or at risk of developing a disease or disease-state (e.g., recurrence) as a preventative course.
  • a disease or disease-state e.g., recurrence
  • the level of a CTL immune response may be determined by any one of numerous immunological methods described herein and routinely practiced in the art.
  • the level of a CTL immune response may be determined prior to and following administration of any one of the herein described MART-1 -specific binding proteins expressed by, for example, a T cell.
  • Cytotoxicity assays for determining CTL activity may be performed using any one of several techniques and methods routinely practiced in the art (see, e.g., Henkart et al., "Cytotoxic T-Lymphocytes" in Fundamental Immunology, Paul (ed.) (2003 Lippincott Williams & Wilkins, Philadelphia, PA), pages 1127-50, and references cited therein).
  • Antigen-specific T cell responses are generally determined by comparisons of observed T cell responses according to any of the herein described T cell functional parameters (e.g., proliferation, cytokine release, CTL activity, altered cell surface marker phenotype, etc.) that may be made between T cells that are exposed to a cognate antigen in an appropriate context (e.g., the antigen used to prime or activate the T cells, when presented by immunocompatible antigen-presenting cells) and T cells from the same source population that are exposed instead to a structurally distinct or irrelevant control antigen.
  • a cognate antigen e.g., the antigen used to prime or activate the T cells, when presented by immunocompatible antigen-presenting cells
  • a response to the cognate antigen that is greater, with statistical significance, than the response to the control antigen signifies antigen-specificity.
  • a biological sample may be obtained from a subject for determining the presence and level of an immune response to a MART- 1 -derived antigen peptide as described herein.
  • a "biological sample” as used herein may be a blood sample (from which serum or plasma may be prepared), biopsy specimen, body fluids (e.g., lung lavage, ascites, mucosal washings, synovial fluid), bone marrow, lymph nodes, tissue explant, organ culture, or any other tissue or cell preparation from the subject or a biological source.
  • Biological samples may also be obtained from the subject prior to receiving any immunogenic composition, which biological sample is useful as a control for establishing baseline (i.e., pre-immunization) data.
  • compositions described herein may be presented in unit- dose or multi-dose containers, such as sealed ampoules or vials. Such containers may be frozen to preserve the stability of the formulation until.
  • a unit dose comprises a genetically engineered host cell as described herein at a dose of about 10 7 cells/m 2 to about 10 11 cells/m 2 .
  • the composition may also include sterile aqueous or oleaginous solution or suspension.
  • suitable non-toxic parenterally acceptable diluents or solvents include water, Ringer's solution, isotonic salt solution, 1,3-butanediol, ethanol, propylene glycol or polythethylene glycols in mixtures with water.
  • Aqueous solutions or suspensions may further comprise one or more buffering agents, such as sodium acetate, sodium citrate, sodium borate or sodium tartrate.
  • any material used in preparing any dosage unit formulation should be pharmaceutically pure and substantially non-toxic in the amounts employed.
  • the active compounds may be incorporated into sustained-release preparation and formulations.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit may contain a predetermined quantity of recombinant cells or active compound calculated to produce the desired therapeutic effect in association with an appropriate pharmaceutical carrier.
  • an appropriate dosage and treatment regimen provides the active molecules or cells in an amount sufficient to provide therapeutic or prophylactic benefit.
  • Such a response can be monitored by establishing an improved clinical outcome (e.g., more frequent remissions, complete or partial, or longer disease-free survival) in treated subjects as compared to non-treated subjects.
  • Increases in preexisting immune responses to a tumor protein generally correlate with an improved clinical outcome.
  • Such immune responses may generally be evaluated using standard proliferation, cytotoxicity or cytokine assays, which are routine in the art and may be performed using samples obtained from a subject before and after treatment.
  • Treatment Plans Enrolled in protocol #2271, Patient Z received 10 10 /m 2 monoclonal (Wallen et al., 2009, PLoS One 4:e4749; Yee et al., 2002, Proc Natl Acad Sci USA 99: 16168-16173) A*0201 -restricted MARTI -specific T cells 28 days apart. The second infusion was preceded by Ontak (18mcg/kg i.v. 6, 4, and 2 days prior to infusion), and both infusions were followed by low-dose IL-2 (250,000U/m 2 s.c. twice daily x 14 days).
  • Protocol #2225 Patient Z received CY (300mg/m 2 iv) before the infusion of 10 10 polyclonal, IL-21 primed (Pollack et al., 2014, J. Immunother, Cancer 2:36) MARTI 26 -35 CTL/m 2 , immediately followed by low-dose subcutaneous IL-2 and ipilimumab (3mg/kg every 3 weeks x 4 doses) (Hodi et al., 2010, N. Engl. J. Med. 363 :711-723). Radiologic responses were evaluated according to the mWHO-based irRC Criteria (Wolchok et al., 2009, Cancer Res. 15:7412-7420).
  • PBMCs were collected by leukapheresis and all ensuing ex vivo manipulations were performed in the clinical Good Manufacturing Practices (cGMP) Cell Processing Facility of the FHCRC.
  • Donor PBMC were stimulated three times for 7-10 day cycles with autologous dendritic cells (DC) pulsed with the HLA*0201 -restricted MART-1 26 - 35 (EAAGIGILTV - SEQ ID NO: 1) peptide (Anaspec) at a DC to effector ratio of 1 :2- 10 to obtain sufficient frequencies (>5%) of MARTI -reactive CD8 + T-cells.
  • DC autologous dendritic cells
  • PBMCs were depleted of CD25 + T cells (Miltenyi Biotec Inc.) to eliminate regulatory T cells, and stimulated twice for seven days with autologous DC pulsed with MARTI 26 -35 (EAAGIGILTV - SEQ ID NO: l).
  • DC Dendritic Cell
  • Cultures that contained > 5% specific CD8 + T cells were clinical-grade sorted (BD Influx cell sorter, BD Biosciences) and stimulated twice using the Rapid Expansion Protocol. The total production time was 6 weeks.
  • the purity and phenotype as well as the ⁇ repertoire of each CTL product immediately before infusion are shown in Figure 4 and Table IB, respectively. There was no overlap in the ⁇ repertoire of both products.
  • MHC peptide-Major Histocompatibility Complex
  • HTTCS T-cell tracking by HTTCS.
  • the HTTCS limit of detection was set at 0.001% of all TCR reads, below which frequencies could not be reliably determined (Robins et al., 2012, J Immunol Methods, 375:14-9).
  • the HTTCS frequency of each clonotype is based on all TCR ⁇ chain reads, from both CD4 + and CD8 + T cells.
  • HTTCS results are reported as a percent of CD8 + cells using the formula: (HTTCS frequencies) x ([%total CD8 + T cells]+[%total CD4 + T cells] / [%total CD8 + T cells in each sample]).
  • PBMCs post-transfer were identified by binding to a specific pMHC multimer, and analyzed by flow cytometry after staining with fluorochrome-conjugated mAbs to CD4, CD 16, CD 19 (exclusion channel), CD8, CD28, CD62L, CCR7, and PDl (BD- Pharmingen).
  • Assessments of the intracellular cytokine expression of IFNy, TNFa and IL-2 in response to cognate antigen were performed, as previously described (Papagno et al., 2007).
  • Cells were analyzed on an LSRII instrument (Becton Dickinson) using fluorescence-activated cell sorting (FACS)-Diva software.
  • Enzyme -Linked Immunospot (ELISpots) Assays Amino acid (aa) peptides, 15 aa in length offset by 5 aas, were grouped into pools of 20-30 peptides spanning
  • MARTI NY-ESO-1, gplOO, tyrosinase, and MAGE A3 (2, 2, 5, 4 and 3 pools, respectively)
  • Peptide pools were used to stimulate PBMC at indicated time-points and T cell reactivity was quantified using a human IFNy ELISpot assay, as previously described (Scheibenbogen et al., 2000, J Immunol
  • IL-2 low-dose subcutaneous (s.c.) IL-2 (250,000 U/m 2 every 12h).
  • the second infusion was preceded by selective CD25 T-regulatory cell depletion (denileukin diftitox); the melanoma progressed.
  • the patient received anti- CTLA4 monoclonal antibody (ipilimumab 3 mg/kg x 4 doses) that partially slowed tumor growth initially, but four months later he developed new metastases.
  • CY cyclophosphamide
  • the patient had bulky disease in the paratracheal, supraclavicular and subcarinal regions at the time the combined therapy with IL-21 primed, polyclonal MART-1 -specific T cells plus ipilimumab was initiated. Twelve weeks (day 384) after the start of the combined treatment, progressive tumor reduction was associated with the development of vitiligo ( Figure ID), manifest as depigmentation of the eyebrows and eyelashes.
  • the patient achieved a CR by Response Evaluation Criteria In Solid Tumors (RECIST) and immune-related response criteria (irRC) (Wolchok et al., 2009, Cancer Res. 15:7412-7420) at year 3, and remains disease-free five years later, with no additional therapy or long-term immune side effects other than persistent vitiligo.
  • RECIST Solid Tumors
  • irRC immune-related response criteria
  • the polyclonal CTL product expressed CD28 (55.1%), low PDl (12%) and produced IFNy, TNFa and IL-2 ( Figure 2D, upper graph), whereas monoclonal CTL expressed none of the memory markers, a higher fraction of PD1 (44%), and produced only IFNy in response to cognate antigen ( Figure 2D, lower graph).
  • the polyclonal CTL additionally expressed CD62L in vivo ( Figure 2E).
  • polyclonal IL-21 -primed CTL achieved higher peak frequencies and longer persistence in vivo, compared to identical doses of monoclonal CTL.
  • Reduced ex vivo manipulation ⁇ 6 weeks vs >12 weeks
  • IL-21 addition during priming (Pollack et al., 2014, J. Immunother. Cancer 2:36) generated CTL that had undergone fewer divisions and had characteristics associated with enhanced survival.
  • expression of CD28 and a retained capacity to secrete IL-2 after exposure to cognate antigen likely facilitated the robust persistence of transferred tumor-specific cells.
  • ipilimumab exposure likely enhanced the antitumor activity of the transferred cells.
  • CD28 + CTL subset may have preferentially survived/expanded through continued production/secretion of autocrine IL-2.
  • CTL examined in vivo months after transfer nearly all expressed CD28 + , retained the capacity to secrete IL-2 in response to cognate antigen and had low PD1 expression (Freeman et al., 2000, J. Exp. Med. 192: 1027-1034).
  • CD28 " , IL-2 " and PD1 M monoclonal cells did not survive beyond one day post-transfer without ipilimumab exposure.
  • the transferred cells With ipilimumab, the transferred cells further acquired the canonical markers of long-lived memory cells CD62L and CCR7, suggesting that the remaining cells were now programmed to persist (Unsoeld et al., 2002, J. Immunol. 169:638-641; Wolfl et al., 2010, Cancer Immunol. Immunother. 60: 173-186).
  • epitope spreading was observed when both the targeted immune response provided by tumor-specific CTL, plus the pro-inflammatory context fostered by anti-CTLA4 blockade, were present (Ribas et al., 2003, Trends Immunol. 24:58-61). While epitope spreading has been demonstrated in some patients receiving anti-CTLA4 antibody monotherapy (Kvistborg et al., 2014, Sci. Transl. Med. 6:254ral28), no evidence of antigen spreading or a clinical response was evident in this patient prior to receiving the combination therapy. Although delayed responses can occur after ipilimumab alone, this usually occurs by 3 months (Wolchok et al., 2009, Cancer Res. 15:7412-7420).
  • cyclophosphamide 300mg/m 2 48 hours before the infusion of 10 10 melanoma- specific CTL/m 2 (determined safe from previous studies) (Wallen et al., 2009, PLoS One 4:e4749), followed by low-dose s.c. IL-2 (250,000 IU/m 2 twice daily for 14 days) to enhance the survival of transferred T-cells. (Yee et al., 2002, Proc. Natl. Acad. Sci.
  • irRC irRC ; RECIST ; Received 2 or 3 of 4 planned ipilimumab doses due to progressive disease 4Patient initiated alternate treatment modality; ND: not done; 5 Received 3/4 planned ipilimumab doses due to ipilimumab-induced hypophysitis 6
  • X patient died due to progressive disease; (mets): development of brain metastasis. Isolation and expansion of polyclonal MARTI -specific CTL.
  • PBMCs were collected by leukapheresis and all ensuing ex vivo manipulations involving processing of products destined for infusion were performed in the cGMP Cell Processing Facility of the FHCRC based on a protocol established for in vitro enrichment of low frequency antigen-specific CTL.
  • PBMCs were depleted of CD25 + T cells (Miltenyi Biotec Inc.) to eliminate regulatory T cells, and stimulated twice for 7 days with autologous dendritic cells pulsed with the HLA*0201 -restricted MART-l 26- 35 (EAAGIGILTV) peptide (SEQ ID NO: l) (Anaspec, San Jose, CA).
  • DC stimulations were supplemented with the y c -chain cytokines IL-2 (10 IU/mL), IL-7 (5 ng/mL), and IL-21 (30 ng/mL).
  • Cytotoxicity assays Cytotoxic activity of infusion products was examined by assessing the capacity of the CTL to lyse HLA A*0201 + MART1 + MEL-526 compared to MARTl ⁇ MEL-375 melanoma cells at an E:T ratio ranging from 100: 1 to 1 : 1 (Suppl. Figure 11). (Wallen et al., 2009, PLoS One 4:e4749)
  • MARTI -specific pMHC multimers produced by the FHCRC immune monitoring core facility were used to detect transferred CTL in PBMCs collected after infusions, with a staining sensitivity of 0.05% of total CD8 + T-cells, as previously described. (Yee et al., 2002, Proc. Natl. Acad. Sci. USA 99: 16168-73)
  • ELISpots For each patient, reactivity of whole PBMC to peptides 15 amino- acids (aa) in length offset by 5 aa and spanning MARTI, NY-ESO-1, Tyrosinase, gplOO and MAGE A3 (Sigma- Aldrich, St. Louis, Mo) was quantified using a human IFNy ELISpot assay as described. (Klebanoff et al., 2012, J. Immunother. 35:651-60) Results are presented as the number of spot forming cells/10 5 PBMCs.
  • Polyclonal IL-21 -primed MARTI -specific CTL demonstrate ex vivo anti-tumor activity and express phenotypic characteristics associated with in vivo survival.
  • All polyclonal CTL products demonstrated intracellular production of IFNy in response to antigen-presenting targets (HLA-A*0201 + , transporter for antigen presentation (TAP)- deficient, B-lymphoblastoid cells, or T2 B-LCL), pulsed with the HLA-A*0201- restricted MARTl 26 -35 peptide (SEQ ID NO: 1) (Figure 11 A, left columns), as well as specific lysis (range 27-66%, median 54%) of the HLA A*0201 + MART1 + MEL-526 cell line 31 ( Figure.
  • Adoptive transfer of MARTI -specific CTL with concurrent anti-CTLA-4 can produce sustained clinical responses.
  • Two of 10 patients reached a sustained CR as defined by irRC and RECIST criteria,(Kawakami et al., 1994, J. Exp. Med. 180:347-52; Ho et al., 2006, J. Immunol. Methods 310:40-52) at 12 and 104 weeks following the CTL infusion respectively (Table 2, Fig. 6A green lines, Fig. 6B).
  • Patient 1 had progressed after salvage ipilimumab monotherapy initiated 7 months before (Example 1).
  • Polyclonal MARTI -specific IL-21-primed CTL persist in vivo when transferred with concurrent anti-CTLA-4.
  • Persistence of the infused CTL was documented for 10/10 patients at 6 weeks and for 7/7 evaluable patients at 12 weeks, with median frequencies of 1.6% (range 0.3-2.9%) and 1.1% (range 0.3-2.2%), respectively (Fig. 7).
  • the transferred CTL could be detected for as long as the patients donated PBMC for analysis, regardless of their tumor response at 12 weeks, with the exceptions of patients 9 and 5 who each received the equivalent of lmg/kg prednisone. Both experienced a gradual decline in the frequency of transferred cells to undetectable levels at 6 and 17 weeks, respectively.
  • Transferred CTL detected in PB exhibit/acquire phenotypic and functional characteristics of long-lived memory T-cells in patients who achieved CR PR or SD.
  • CTL tracking in patients who achieved CR, PR or SD documented a significant increase in the frequency of multimer + cells expressing CD28 (p ⁇ 0.05), CD27 (p ⁇ 0.05), CD127 (p ⁇ 0.005), CD62L (p ⁇ 0.05) and CCR7 (p ⁇ 0.005) at 12 weeks (Fig. 8A, left column).
  • the functional profile of transferred CTL was determined by gating for ⁇ FNy + cells (Fig. 8C). Before CY infusions, ⁇ 0.2% of IFNy-producing cells could be detected in PBMC, including in patients with low but detectable levels of MARTI -specific cells, attesting to the lack of pre-existing, functional PB CTL. Consistent with the
  • infused CTLs in this study consisted of polyclonal, pMHC multimer-sorted T-cell lines that had undergone shorter ex vivo manipulation ( ⁇ 6 weeks) and fewer cell divisions.
  • CD28 + cells may have gained proliferative/survival advantages related to Bcl-X L expression and autocrine production of IL-2. (McAdam et al., 1998, Immunol. Rev. 165:231-47) In contrast, no IFNy-secreting cells could be detected post-transfer in patients with PD.
  • T-cell responses towards wild-type- or non-evaluated tumor-specific mutations, (Schreiber et al., 2011, Science 331 : 1565-70) induced the decrease in tumor size observed in some patients cannot be ascertained. Yet the combination may represent a strategy to specifically increase the number and strength of T-cells targeting multiple antigens of the patient's own tumor, which may be particularly relevant when targeting non-oncogenic antigens such as MART-1. (Chandran et al., 2015, Clin. Cancer Res. 21 :534-43) EXAMPLE 3
  • Protocol #2140 Patients M-2140-1 and M-2140-2 with metastatic melanoma received cyclophosphamide (CY) 4000mg/m 2 administered over 2 days before the infusion of 10 10 monoclonal melanoma-specific CTL/m 2 followed by low-dose subcutaneous (s.c.) IL-2 (250,000 U/m 2 ) twice daily for 14 days (NCT 00438984) (Chapuis et al. 2012, Proc. Natl. Acad. Sci. USA 109:4592-7). Pts Ml and M2 received CTL specific for HLA A*0201 -restricted MARTl 26-3 5
  • IL-21 For protocol #2140, which involved monoclonal tumor-specific cells, cell processing was as previously described (Chapuis et al. 2012, Proc. Natl. Acad. Sci. USA 109:4592-7). Briefly, PBMCs were collected by leukapheresis and all ensuing ex vivo manipulations were performed in the clinical Good Manufacturing Practices (cGMP) Cell Processing Facility of the FHCRC. (Pollack et al., J. Immunother. Cancer 2:36) Donor PBMC were stimulated three times for 7-10 day cycles with autologous dendritic cells (DC) pulsed with the HLA*0201 -restricted MART-l 2 6- 35
  • DC autologous dendritic cells
  • EAAGIGILTV EAAGIGILTV peptide (SEQ ID NO: 1) (Anaspec) at a DC to effector ratio of 1 :2-10 to obtain sufficient frequencies (>5%) of MARTI -reactive CD8 + T cells.
  • the y c -chain cytokines, IL-2 (12.5IU/ml), IL-7 (5ng/ml) and IL-15 (lng/ml) were added. Cultures that contained > 5% specific CD8 + T cells, assessed by multimer analysis, were cloned by limiting dilution and then stimulated twice using the Rapid Expansion Protocol. (Ho et al. 2006, J. Immunol. Methods 310:40-52) CTL products were frozen, thawed and washed before infusion, for a total production time of 12-13 weeks.
  • CTL tracking by peptide-MHC multimer s The sensitivity of multimer staining was fixed at 0.1% of total CD8 + T cells for monoclonal products (Chapuis et al. 2012, Proc. Natl. Acad. Sci. USA 109:4592-7), and at 0.05% for polyclonal products (Chapuis et al., 2016, J. Clin. Oncol. 34:3787-3795), as previously described.
  • DNA extraction and immunosequencing DNA was extracted from CTL products and whole PBMC using Qiagen Maxi DNA isolation kits (QIAGEN Inc.). TCRP CDR3 regions were amplified and 750 ng of extracted DNA sequenced by Adaptive Biotechnologies Corp (Seattle, WA) using the "deep" resolution ImmunoSEQ assay, as previously described (Sherwood et al., 2011, Sci Transl. Med. 3 :90ra61). Raw sequence data was filtered using the Adaptive bioinformatic website based on the TCRP V, D and J gene definitions provided by the International ImMunoGeneTics
  • HTTCS CTL tracking by HTTCS. Only cells that bound pMHC multimers were selected by flow cytometry before DNA isolation for HTTCS. The limit of detection of HTTCS was set at 0.001% of all TCR reads below which frequency could not be reliably determined (Robins et al., 2012, J Immunol Methods, 375: 14-9). Only clonotypes present in the CTL products were tracked in PBMC obtained after infusions. The frequency of each clonotype detected by HTTCS was based on all TCR ⁇ reads from CD4 + and CD8 + T cells.
  • PBMC Normal Donor Sorts: PBMC were collected from three healthy adult donors, and processed into three populations for TCR sequencing: i.e., whole PBMC, flow- sorted CD3 + CD45RA + and CD3 D45RO , representing the naive and antigen- experienced T cell populations, respectively. TCRs from ⁇ 1 million cells from each population were sequenced by HTTCS. Clonotypes were tracked in the original PBMC samples and considered part of a CD3 CD45RA versus an CD3 + CD45RO + phenotype if it was observed in one population but not the other, or if its abundance was ten-fold greater in one population compared to the other.
  • the cell product included a total of 868 clonotypes of which the most prevalent single clonotype represented 94% of all TCR reads ( Figure 17B, lower pie plot).
  • the most prevalent HTTCS-detected clonotype was confirmed as the infused CTL clone by TCR ⁇ quantitative PCR (Table 7).
  • the post-transfer in vivo frequencies of the most prevalent clonotype in each infusion product were compared, as assessed by HTTCS, p- HLA multimer binding and/or TCR ⁇ quantitative PCR.
  • HTTCS frequencies yielded results concordant to TCR ⁇ -specific PCR and pMHC multimer binding, suggesting HTTCS can be used to quantitatively track the frequency of infused monoclonal CD8 + T cells in vivo.
  • Table 6 Total, roductive and uni ue se uences in each sam le.
  • Pt P2225-1 Pre-infusion: Day -6 3710481 64057 3164580 54593
  • Pt P2225-4 Pre-infusion: Day -1 1 13665366 130151 12038950 1 13781
  • Protocol #2225 Patients P2225-1-P10 with metastatic melanoma received CY 300mg/m 2 before the infusion of 10 10 polyclonal A*0201- restricted MART127-35 CTL/m 2 , immediately followed by low-dose s.c. IL-2 and ipilimumab (anti- Cytotoxic T-Lymphocyte Associated Protein 4, Yervoy ® , Bristol
  • CTL tracking by peptide-MHC multimers The sensitivity of multimer staining was fixed at 0.1% of total CD8 + T cells for monoclonal products (Chapuis et al. 2012, Proc. Natl. Acad. Sci. USA 109:4592-7), and at 0.05% for polyclonal products (Chapuis et al., 2016, J. Clin. Oncol. 34:3787-3795), as previously described.
  • DNA extraction and immunosequencing DNA was extracted from CTL products and whole PBMC using Qiagen Maxi DNA isolation kits (QIAGEN Inc.). TCRP CDR3 regions were amplified and 750 ng of extracted DNA sequenced by Adaptive Biotechnologies Corp (Seattle, WA) using the "deep" resolution ImmunoSEQ assay, as previously described (Sherwood et al., 2011, Sci Transl. Med. 3 :90ra61). Raw sequence data was filtered using the Adaptive bioinformatic website based on the TCRP V, D and J gene definitions provided by the International ImMunoGeneTics
  • IMGT IMGT-based Bioinformatics 20 Suppl l :i379-85
  • IMGT database www.imgt.org
  • Productive nucleotide sequences were used for all tracking experiments. The data was further filtered to exclude sequences with no identifiable V and J removing PCR errors such as primer dimer and mispriming, as well as sequences with a raw read count ⁇ 2, removing nucleotide sequencing errors (Robins et al., 2009, Blood 114:4099-4107).
  • HTTCS CTL tracking by HTTCS. Only cells that bound pMHC multimers were selected by flow cytometry before DNA isolation for HTTCS. The limit of detection of HTTCS was set at 0.001% of all TCR reads below which frequency could not be reliably determined (Robins et al., 2012, J Immunol Methods, 375: 14-9). Only clonotypes present in the CTL products were tracked in PBMC obtained after infusions. The frequency of each clonotype detected by HTTCS is based on all TCR ⁇ reads from CD4 + and CD8 + T cells.
  • PBMC Normal Donor Sorts: PBMC were collected from three healthy adult donors, and processed into three populations for TCR sequencing: i.e. whole PBMC, flow- sorted CD3 + CD45RA + and CD3 D45RO , representing the naive and antigen- experienced T cell populations, respectively. TCRs from ⁇ 1 million cells from each population were sequenced by HTTCS. Clonotypes were tracked in the original PBMC samples and considered part of a CD3 CD45RA versus an CD3 D45RCT phenotype if it was observed in one population but not the other, or if its abundance was ten-fold greater in one population compared to the other.
  • the most prevalent clonotype comprised between 4% and 77% of the total cells in each infusion (mean 33.3%), and the 25 most prevalent clonotypes together comprised between 35.0% and 99.9% (mean 78.4%) of the total cell products.
  • This HTTCS analysis was performed on infusion products selected for binding pMHC multimers with >99% purity.
  • the limit of pMHC detection is typically >0.01% of CD8 + T cells, which is at least 10-fold less sensitive than HTTCS.
  • HTTCS of infusion products that have been selected by pMHC multimer-binding is likely to also include
  • HTTCS appears to accurately track the in vivo frequencies of infused polyclonal CTL.
  • Clonotype half-lives (t1 ⁇ 2, see Statistical Analysis) were determined for each patient and grouped according to best clinical response ( Figure 20B). For patients who achieved a CR (Pts P2225-1 and 7), the immunodominant clonotypes
  • Table 9 Clinical responses of patients with metastatic melanoma who received
  • Patient initiated alternate treatment modality 2 Patient had peripheral disease regression but new sub- centimeter brain metastasis. Overall, at 16 weeks, the volume of disease was lower than at baseline, but the location of the brain lesions eventually caused death. 3 Patient developed brain metastasis at 46 weeks. X: Patient died due to progressive disease. Table 10: Ki67 expression of transferred multimer+ CTL in patients who achieved a complete remission.
  • Pre-existing clonotype frequencies provide insights into the nature of persisting CTL.
  • the number of clonotypes comprising the polyclonal cell products for Pts. P2225-1 to 10 was between 56 and 2036, with a median of 262.5 clonal sequences (Table 7).
  • most clonotypes present in cell products range 43 to 1275, median 160
  • a clonotype with a frequency in the CTL product that was less than its corresponding frequency in the pre-infusion PBMC was defined as having not expanded during the culture process (Table 7, bottom row). These clones comprised a minor fraction of the final infusion product, with a median of 0.02% (range 0%-0.61%) ( Figure 21 A, purple areas), and were not further monitored.
  • Clonotypes detected in any post-infusion PBMC sample, but not detected in pre-infusion samples were the dominant components of the final cell product (range 74.68%-98.97%, median 97.67%) (Figure 21A, blue areas), implying preferential expansion from individual parental clonotypes with frequencies ⁇ 0.001%; i.e., a very low frequency (VLF) population.
  • VLF very low frequency
  • Clonotypes expanded in the cell infusion product that were detectable in PBMC before infusion were less prevalent (range 0-6, median 2 sequences), comprising ⁇ 10% (range 0-9.22%, median 0.03%) of the final cell products that were infused ( Figure 21 A, orange areas).
  • the frequencies of all expanded clonotypes were summed at multiple timepoints after infusion (Figure 2 IB, solid red circles) and further subdivided into previously undetected (Figure 2 IB, open blue circles) versus previously detected clonotypes (Figure 2 IB orange open circles).
  • Previously undetected clonotypes represented the majority of cells in both infusion products and the detected clonotypes in post-transfer PBMC, whereas previously detected clonotypes comprised a small fraction of infused products and a minority of post-transfer clonotypes.
  • CD3 + CD45RA + (primarily naive T cells, T N ) VLF population than from a
  • CD3 + CD45RO + (mostly antigen-experienced cells) population ( Figure 19).

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Cell Biology (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Organic Chemistry (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Epidemiology (AREA)
  • Molecular Biology (AREA)
  • Oncology (AREA)
  • Biophysics (AREA)
  • Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biomedical Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Neurology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Zoology (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne des récepteurs de lymphocytes T spécifiques d'un antigène de mélanome humain reconnu par des épitopes de lymphocytes T 1 (MART-1), destinés à être utilisés dans le traitement de maladies ou de troubles, tels que des cellules cancéreuses qui surexpriment MART-1.
PCT/US2017/031453 2016-05-06 2017-05-05 Immunothérapie de lymphocytes t spécifique de mart-1 WO2017193104A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP17724686.5A EP3452514A1 (fr) 2016-05-06 2017-05-05 Immunothérapie de lymphocytes t spécifique de mart-1

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201662333117P 2016-05-06 2016-05-06
US62/333,117 2016-05-06

Publications (1)

Publication Number Publication Date
WO2017193104A1 true WO2017193104A1 (fr) 2017-11-09

Family

ID=58739367

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2017/031453 WO2017193104A1 (fr) 2016-05-06 2017-05-05 Immunothérapie de lymphocytes t spécifique de mart-1

Country Status (2)

Country Link
EP (1) EP3452514A1 (fr)
WO (1) WO2017193104A1 (fr)

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019222427A1 (fr) * 2018-05-16 2019-11-21 Fred Hutchinson Cancer Research Center Tcr spécifiques d'un antigène t de polyomavirus à cellules de merkel et leurs utilisations
WO2020010565A1 (fr) * 2018-07-12 2020-01-16 深圳华大生命科学研究院 Récepteur de lymphocytes t spécifique d'un épitope mart-1(27-35)
CN110894241A (zh) * 2018-09-13 2020-03-20 中国科学院生物物理研究所 一种靶向抗原特异性t细胞诱导其向记忆干性细胞分化的融合蛋白
CN111961648A (zh) * 2019-05-20 2020-11-20 河南省肿瘤医院 一种肿瘤特异性t细胞的分离培养方法及由其获得的产品
WO2021108500A1 (fr) * 2019-11-26 2021-06-03 University Of Pittsburgh - Of The Commonwealth System Of Higher Education Récepteurs de lymphocytes t ciblant des mutations dans des facteurs d'épissage d'arn
CN112912387A (zh) * 2018-08-22 2021-06-04 弗雷德哈钦森癌症研究中心 靶向kras或her2抗原的免疫疗法
WO2022132836A2 (fr) 2020-12-14 2022-06-23 Fred Hutchinson Cancer Research Center Compositions et méthodes pour l'immunothérapie cellulaire
EP4161555A4 (fr) * 2020-06-09 2024-06-19 Board of Regents, The University of Texas System Récepteurs de lymphocytes t modifiés et leurs procédés d'utilisation

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5283173A (en) 1990-01-24 1994-02-01 The Research Foundation Of State University Of New York System to detect protein-protein interactions
WO1997009433A1 (fr) 1995-09-06 1997-03-13 Icos Corporation Genes de point de controle a cycle cellulaire
US20040087025A1 (en) 1995-06-07 2004-05-06 The United States Of America As Represented By The Secretary Of The Navy Methods for transfecting T cells
US20110189141A1 (en) 2009-05-19 2011-08-04 Max-Delbrück-Centrum für Molekulare Medizin Multiple target t cell receptor
US20110243972A1 (en) 2005-01-13 2011-10-06 Johns Hopkins University Prostate stem cell antigen vaccines and uses thereof
WO2013166321A1 (fr) 2012-05-03 2013-11-07 Fred Hutchinson Cancer Research Center Récepteurs de lymphocyte t à affinité augmentée et procédés pour fabriquer ceux-ci
WO2016161273A1 (fr) 2015-04-01 2016-10-06 Adaptive Biotechnologies Corp. Procédé d'identification des récepteurs de lymphocytes t spécifiques à compatibilité humaine pour une cible antigénique

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5283173A (en) 1990-01-24 1994-02-01 The Research Foundation Of State University Of New York System to detect protein-protein interactions
US5468614A (en) 1990-01-24 1995-11-21 The Research Foundation Of State University Of New York System to detect protein-protein interactions
US20040087025A1 (en) 1995-06-07 2004-05-06 The United States Of America As Represented By The Secretary Of The Navy Methods for transfecting T cells
WO1997009433A1 (fr) 1995-09-06 1997-03-13 Icos Corporation Genes de point de controle a cycle cellulaire
US20110243972A1 (en) 2005-01-13 2011-10-06 Johns Hopkins University Prostate stem cell antigen vaccines and uses thereof
US20110189141A1 (en) 2009-05-19 2011-08-04 Max-Delbrück-Centrum für Molekulare Medizin Multiple target t cell receptor
WO2013166321A1 (fr) 2012-05-03 2013-11-07 Fred Hutchinson Cancer Research Center Récepteurs de lymphocyte t à affinité augmentée et procédés pour fabriquer ceux-ci
WO2016161273A1 (fr) 2015-04-01 2016-10-06 Adaptive Biotechnologies Corp. Procédé d'identification des récepteurs de lymphocytes t spécifiques à compatibilité humaine pour une cible antigénique

Non-Patent Citations (145)

* Cited by examiner, † Cited by third party
Title
"Current Protocols in Molecular Biology", July 2008, JOHN WILEY AND SONS
"Immobilized Cells And Enzymes", 1986, IRL PRESS
"Methods In Enzymology", ACADEMIC PRESS, INC.
"PCR Protocols, 3rd Edition,", 2010, HUMANA PRESS
"Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology", GREENE PUB. ASSOCIATES AND WILEY-INTERSCIENCE
ALTMAN ET AL., SCIENCE, vol. 274, 1996, pages 94 - 96
ALTSCHUL ET AL.: "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", NUCLEIC ACIDS RES., vol. 25, 1997, pages 3389 - 3402, XP002905950, DOI: doi:10.1093/nar/25.17.3389
ANAND: "Techniques for the Analysis of Complex Genomes", 1992, ACADEMIC PRESS
AUDE G. CHAPUIS ET AL: "Combined IL-21?primed polyclonal CTL plus CTLA4 blockade controls refractory metastatic melanoma in a patient", THE JOURNAL OF EXPERIMENTAL MEDICINE, vol. 213, no. 7, 30 May 2016 (2016-05-30), US, pages 1133 - 1139, XP055384321, ISSN: 0022-1007, DOI: 10.1084/jem.20152021 *
AUSUBEL ET AL., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, 2003
B. HAMES & S. HIGGINS,: "Nucleic Acid Hybridization", 1985
B. HAMES & S. HIGGINS,: "Transcription and Translation", 1984
B. N. FIELDS ET AL.,: "Fundamental Virology, Third Edition,", 1996, IPPINCOTT-RAVEN PUBLISHERS, article COFFIN, J. M. ET AL.: "Retroviridae: The viruses and their replication"
BENLALAM ET AL., EUR. J. IMMUNOL., vol. 31, 2001, pages 2007 - 2015
BERNSEN ET AL., BR. J. CANCER, vol. 88, 2003, pages 424 - 31
BIOINFORMATICS, vol. 15, 2016, pages 298 - 300
BUTLER ET AL., SCI. TRANSL. MED., vol. 3, 2011, pages 80RA34
CAVALLO ET AL., CANC. IMMUNOL. IMMUNOTHER., vol. 60, 2011, pages 319
CHANDRAN ET AL., CLIN. CANCER RES., vol. 21, 2015, pages 534 - 43
CHAPUIS AUDE G ET AL: "Abstract LB-136: IL-21-derived melanoma-reactive CTL combined with anti-CTLA4 persist, acquire central memory characteristics, and mediate tumor regression in patients with metastatic melanoma", CANCER RESEARCH, vol. 72, no. Suppl. 8, April 2012 (2012-04-01), & 103RD ANNUAL MEETING OF THE AMERICAN-ASSOCIATION-FOR-CANCER-RESEARCH; CHICAGO, IL, USA; MARCH 31 -APRIL 04, 2012, XP002771463, DOI: 10.1158/1538-7445.AM2012-LB-136 *
CHAPUIS ET AL., J. CLIN. ONCOL., vol. 34, 2016, pages 3787 - 3795
CHAPUIS ET AL., J. EXP. MED., vol. 213, pages 1133 - 9
CHAPUIS ET AL., PROC. NATL. ACAD. SCI. USA, vol. 109, 2012, pages 4592 - 7
CHAPUIS ET AL., SCI. TRANSL. MED., vol. 5, 2013, pages 174RA27
CHAPUIS ET AL., SCIENCE TRANSLATIONAL MEDICINE, vol. 5, 2013, pages 174RAL27
CHOTHIA ET AL., EMBO J., vol. 7, 1988, pages 3745
CHRISTOPHER A. KLUG, AND CRAIG T. JORDAN: "Hematopoietic Stem Cell Protocols", 2001
CUI ET AL., IMMUNITY, vol. 35, 2011, pages 792 - 805
D. K. COLE ET AL: "Modification of MHC Anchor Residues Generates Heteroclitic Peptides That Alter TCR Binding and T Cell Recognition", THE JOURNAL OF IMMUNOLOGY, vol. 185, no. 4, 16 July 2010 (2010-07-16), US, pages 2600 - 2610, XP055385851, ISSN: 0022-1767, DOI: 10.4049/jimmunol.1000629 *
D. M. WEIR ANDCC BLACKWELL,: "Handbook Of Experimental Immunology", vol. I-IV, 1986
DARWIN J. PROCKOP, DONALD G. PHINNEY, AND BRUCE A. BUNNELL: "Mesenchymal Stem Cells: Methods and Protocols", 2008
DOSSETT ET AL., MOL. THER., vol. 17, 2009, pages 742
EISENHAUER ET AL., EUR. J. CANCER, vol. 45, 2009, pages 228 - 47
FREEMAN ET AL., J. EXP. MED., vol. 192, 2000, pages 1027 - 1034
GLOVER: "DNA Cloning: A Practical Approach", vol. I, II, 1985, IRL PRESS, OXFORD UNIV. PRESS
GREEN; REED, SCIENCE, vol. 281, 1998, pages 1309
GUTHRIE; FINK: "Guide to Yeast Genetics and Molecular Biology", 1991, ACADEMIC PRESS
HANAHAN; WEINBERG, CELL, vol. 100, 2000, pages 57
HANAHAN; WEINBERG, CELL, vol. 144, 2011, pages 646
HARDING ET AL., N. ENGL. J. MED., vol. 366, 2012, pages 866 - 8
HARLOW; LANE: "Antibodies", 1998, COLD SPRING HARBOR LABORATORY PRESS
HENKART ET AL.: "Fundamental Immunology", 2003, LIPPINCOTT WILLIAMS & WILKINS, article "Cytotoxic T-Lymphocytes", pages: 1127 - 50
HERSEY ET AL., INT. J. CANCER, vol. 48, 1994, pages 527 - 32
HINRICHS ET AL., BLOOD, vol. 111, 2008, pages 5326 - 33
HO ET AL., J. IMMUNOL. METHODS, vol. 310, 2006, pages 40 - 52
HODI ET AL., N. ENGL. J. MED., vol. 363, 2010, pages 711 - 723
HUNDER ET AL., N. ENGL. J. MED., vol. 358, 2008, pages 2698 - 703
HUNDER ET AL., NEW ENGL. M. MED., vol. 358, 2008, pages 2698 - 2703
J. H. MILLER AND M. P. CALOS: "Gene Transfer Vectors For Mammalian Cells", 1987, COLD SPRING HARBOR LABORATORY
JANEWAY ET AL.: "Immunobiology: The Immune System in Health and Disease, 3rd Ed.,", vol. 33, 1997, CURRENT BIOLOGY PUBLICATIONS, pages: 4
JANITZ: "Next-Generation Genome Sequencing", 2008, WILEY-VCH
JOHN E. COLIGAN, ADA M. KRUISBEEK, DAVID H. MARGULIES, ETHAN M. SHEVACH, WARREN STROBER: "Current Protocols in Immunology", 2001, JOHN WILEY & SONS
JORES ET AL., PROC. NAT'L ACAD. SCI. U.S.A., vol. 87, 1990, pages 9138
JULIE LOGAN, KIRSTIN EDWARDS AND NICK SAUNDERS,: "Real-Time PCR: Current Technology and Applications", 2009, ACADEMIC PRESS
KABAT ET AL.: "Sequences of Proteins of Immunological Interest", 1991, PUBLIC HEALTH SERVICE NATIONAL INSTITUTES OF HEALTH
KAECH ET AL., NAT. REV. IMMUNOL., vol. 2, 2002, pages 251 - 62
KATHERINE JANE ADAMS: "REDIRECTED T CELL ACTIVITY BY HIGH AFFINITY TCR-ANTI-CD3 BISPECIFIC CANDIDATE THERAPEUTICS", 3 April 2013 (2013-04-03), pages 1 - 270, XP055136123, Retrieved from the Internet <URL:https://orca.cf.ac.uk/47578/1/2013ADAMSKIPHD.pdf> [retrieved on 20140822] *
KAWAKAMI ET AL., J. EXP. MED., vol. 180, 1994, pages 347 - 52
KEVIN D. BUNTING: "Hematopoietic Stem Cell Protocols", 2008
KIM ET AL., PLOS ONE, vol. 7, 2012, pages E37338
KIMURA ET AL., NAT. IMMUNOL., vol. 14, 2013, pages 143 - 51
KLEBANOFF ET AL., J. IMMUNOTHER., vol. 35, 2012, pages 651 - 60
KUBALL ET AL., BLOOD, vol. 109, 2007, pages 2331
KURSTAD TURKSEN,: "Embryonic Stem Cell Protocols: Volume I: Isolation and Characterization", vol. I, 2006
KURSTAD TURKSEN,: "Embryonic Stem Cell Protocols: Volume II: Differentiation Models", vol. II, 2006
KURSTAD TURKSEN,: "Embryonic Stem Cells: Methods and Protocols", 2002
KURSTAD TURKSEN,: "Human Embryonic Stem Cell Protocols", 2006
KVISTBORG ET AL., SCI. TRANSL. MED., vol. 6, 2014, pages 254RA128
KYRIGIDEIS ET AL., J. CARCINOG., vol. 9, 2010, pages 3
LABARRIERE ET AL., INT. J. CANCER, vol. 78, 1998, pages 209 - 215
LEEN ET AL., ANN. REV. IMMUNOL., vol. 25, 2007, pages 243
LEFKOVITS: "Immunology Methods Manual: The Comprehensive Sourcebook of Techniques", 1998
LEFRANC ET AL., DEV. COMP. IMMUNOL., vol. 27, 2003, pages 55
LEHNINGER: "Biochemistry, 2nd Edition;", 1975, WORTH PUBLISHERS, INC., pages: 71 - 77
LESLIE P. WEINER: "Neural Stem Cells: Methods and Protocols", 2008
LEWIN: "Genes IV", 1990, OXFORD UNIVERSITY PRESS, NY AND CELL PRESS, pages: 8
LI ET AL., BLOOD, vol. 111, 2008, pages 229 - 35
LI ET AL., J. IMMUNOL., vol. 175, 2005, pages 2261 - 9
MAYER AND WALKER,: "Immunochemical Methods In Cell And Molecular Biology", 1987, ACADEMIC PRESS
MCADAM ET AL., IMMUNOL. REV., vol. 165, 1998, pages 231 - 47
MISHELL AND SHIGII: "Selected Methods in Cellular Immunology", 1979, FREEMAN PUBLISHING
MONOD ET AL., BIOINFORMATICS, vol. 20, no. 1, 2004, pages I379 - 85
MUNSON ET AL., PROC. NATL. ACAD. SCI., vol. 113, 2016, pages 8272 - 7
MURPHY: "Janeway's Immunobiology (8th Ed.)", 2011, GARLAND SCIENCE
N. GAIT: "Oligonucleotide Synthesis", 1984
NATHANIEL LIDDY ET AL: "Monoclonal TCR-redirected tumor cell killing", NATURE MEDICINE, vol. 18, no. 6, 6 May 2012 (2012-05-06), pages 980 - 987, XP055241791, ISSN: 1078-8956, DOI: 10.1038/nm.2764 *
PENIX ET AL., J. EXP. MED., vol. 178, 1993, pages 1483
PERBAL: "A Practical Guide to Molecular Cloning", 1984
POLLACK ET AL., J. IMMUNOTHER, CANCER, vol. 2, 2014, pages 36
POLLACK ET AL., J. IMMUNOTHER. CANCER, vol. 2, 2014, pages 36
POLLACK ET AL., J. IMMUNOTHER. CANCER, vol. 2, pages 36
POLLACK ET AL., JOURNAL FOR IMMUNOTHERAPY OF CANCER, vol. 2, 2014, pages 36
R. FRESHNEY,: "Animal Cell Culture", 1986
RAGHEB ET AL., J. IMMUNOL., vol. 163, 1999, pages 120 - 9
RANSOHOFF ET AL., NAT. REV. IMMUNOL., vol. 12, 2012, pages 623 - 35
REDMOND ET AL., GENOME MED., vol. 8, 2016, pages 80
RESTIFO ET AL., NAT REV IMMUNOL, vol. 12, 2012, pages 269 - 281
RIBAS ET AL., N. ENGL. J. MED., vol. 368, 2013, pages 1365 - 6
RIBAS ET AL., TRENDS IMMUNOL., vol. 24, 2003, pages 58 - 61
RICHMAN; KRANZ, BIOMOL. ENG., vol. 24, 2007, pages 361 - 373
RIDDELL ET AL., SCIENCE, vol. 257, 1992, pages 238 - 241
ROBERT ET AL., CLIN. CANCER RES., vol. 20, 2014, pages 2424 - 32
ROBINS ET AL., BLOOD, vol. 114, 2009, pages 4099
ROBINS ET AL., BLOOD, vol. 114, 2009, pages 4099 - 4107
ROBINS ET AL., J IMMUNOL METHODS, vol. 375, 2012, pages 14 - 9
ROBINS ET AL., J IMMUNOL. METHODS, vol. 375, 2012, pages 14 - 9
ROBINS ET AL., SCI. TRANSLAT. MED., vol. 2, 2010, pages 47RA64
ROITT: "Essential Immunology, 6th Edition,", 1988, BLACKWELL SCIENTIFIC PUBLICATIONS
ROSENBERG ET AL., CLIN. CANCER RES., vol. 17, 2011, pages 4550 - 7
SAMBROOK ET AL.: "Molecular Cloning: A Laboratory Manual 2d ed.", 1989, COLD SPRING HARBOR LABORATORY
SAMBROOK ET AL.: "Molecular Cloning: A Laboratory Manual", 1989, COLD SPRING HARBOR LABORATORY PRESS
SAMBROOK ET AL.: "Molecular Cloning: A Laboratory Manual, 3rd ed.,", 2001, COLD SPRING HARBOR LABORATORY PRESS
SAMBROOK ET AL.: "Molecular Cloning: A Laboratory Manual, 3rd ed.,", COLD SPRING HARBOR LABORATORY PRESS
SCATCHARD ET AL., ANN. N.Y. ACAD. SCI., vol. 51, 1949, pages 660
SCHEIBENBOGEN ET AL., J IMMUNOL METHODS, vol. 244, 2000, pages 81 - 89
SCHMITT ET AL., HUM. GEN., vol. 20, 2009, pages 1240
SCHOLTEN ET AL., CLIN. IMMUNOL., vol. 119, 2006, pages 135
SCHOLZEN ET AL., J. CELL PHYSIOL., vol. 182, 2000, pages 311 - 22
SCHREIBER ET AL., SCIENCE, vol. 331, 2011, pages 1565 - 1570
SCHREIBER ET AL., SCIENCE, vol. 331, 2011, pages 1565 - 70
SHERWOOD ET AL., SCI TRANSL. MED., vol. 3, 2011, pages 90RA61
STROMNES ET AL., IMMUNOL. REV., vol. 257, 2014, pages 145 - 64
T. CHODON ET AL: "Adoptive Transfer of MART-1 T-Cell Receptor Transgenic Lymphocytes and Dendritic Cell Vaccination in Patients with Metastatic Melanoma", CLINICAL CANCER RESEARCH, vol. 20, no. 9, 1 May 2014 (2014-05-01), US, pages 2457 - 2465, XP055384670, ISSN: 1078-0432, DOI: 10.1158/1078-0432.CCR-13-3017 *
THOMPSON ET AL., MOL. CELL. BIOL., vol. 12, 1992, pages 1043
TILL ET AL., BLOOD, vol. 112, 2008, pages 2261
TODD ET AL., J. EXP. MED., vol. 177, 1993, pages 1663
TOPP ET AL., J. EXP. MED., vol. 198, 2003, pages 947 - 955
TSAO ET AL., N. ENGL. J. MED., vol. 351, 2004, pages 998 - 1012
UDYAVAR ET AL., J. IMMUNOL., vol. 182, 2009, pages 4439 - 4447
UNSOELD ET AL., J. IMMUNOL., vol. 169, 2002, pages 638 - 641
WALLEN ET AL., PLOS ONE, vol. 4, 2009, pages E4749
WANG ET AL., HUM. GENE THER., vol. 18, 2007, pages 712
WARREN ET AL., GENOME RES., vol. 21, 2011, pages 790
WEBER ET AL., J. CLIN. ONCOL., vol. 30, 2012, pages 2691 - 7
WEIR: "Handbook of Experimental Immunology", 1986, BLACKWELL SCIENTIFIC
WILSON, SCIENCE, vol. 295, 2002, pages 2103
WOLCHOK ET AL., CANCER RES., vol. 15, 2009, pages 7412 - 7420
WOLCHOK ET AL., CLIN. CANCER RES., vol. 15, 2009, pages 7412 - 20
WOLCHOK ET AL., CLIN. CANCER RES., vol. 15, 2009, pages 7412 - 7420
WOLFF ET AL., CANCER RES., vol. 53, 1993, pages 2560
WOLFL ET AL., CANCER IMMUNOL. IMMUNOTHER., vol. 60, 2010, pages 173 - 186
YANG, CLIN. DERMATOL., vol. 31, 2013, pages 209 - 19
YEE ET AL., PROC NATL ACAD SCI USA, vol. 99, 2002, pages 16168 - 16173
YEE ET AL., PROC. NATL. ACAD. SCI. USA, vol. 99, 2002, pages 16168 - 73
ZHAO ET AL., J. IMMUNOL., vol. 179, 2007, pages 5845 - 5854

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019222427A1 (fr) * 2018-05-16 2019-11-21 Fred Hutchinson Cancer Research Center Tcr spécifiques d'un antigène t de polyomavirus à cellules de merkel et leurs utilisations
WO2020010565A1 (fr) * 2018-07-12 2020-01-16 深圳华大生命科学研究院 Récepteur de lymphocytes t spécifique d'un épitope mart-1(27-35)
US12134637B2 (en) 2018-07-12 2024-11-05 Bgi Shenzhen MART-1(27-35) epitope-specific T cell receptor
CN112469733B (zh) * 2018-07-12 2022-04-19 深圳华大生命科学研究院 Mart-1(27-35)表位特异性t细胞受体
CN112469733A (zh) * 2018-07-12 2021-03-09 深圳华大生命科学研究院 Mart-1(27-35)表位特异性t细胞受体
CN112912387A (zh) * 2018-08-22 2021-06-04 弗雷德哈钦森癌症研究中心 靶向kras或her2抗原的免疫疗法
CN110894241B (zh) * 2018-09-13 2022-01-18 中国科学院生物物理研究所 一种靶向抗原特异性t细胞诱导其向记忆干性细胞分化的融合蛋白
CN110894241A (zh) * 2018-09-13 2020-03-20 中国科学院生物物理研究所 一种靶向抗原特异性t细胞诱导其向记忆干性细胞分化的融合蛋白
CN111961648B (zh) * 2019-05-20 2022-03-29 河南省肿瘤医院 一种肿瘤特异性t细胞的分离培养方法及由其获得的产品
CN111961648A (zh) * 2019-05-20 2020-11-20 河南省肿瘤医院 一种肿瘤特异性t细胞的分离培养方法及由其获得的产品
WO2021108500A1 (fr) * 2019-11-26 2021-06-03 University Of Pittsburgh - Of The Commonwealth System Of Higher Education Récepteurs de lymphocytes t ciblant des mutations dans des facteurs d'épissage d'arn
EP4161555A4 (fr) * 2020-06-09 2024-06-19 Board of Regents, The University of Texas System Récepteurs de lymphocytes t modifiés et leurs procédés d'utilisation
WO2022132836A2 (fr) 2020-12-14 2022-06-23 Fred Hutchinson Cancer Research Center Compositions et méthodes pour l'immunothérapie cellulaire

Also Published As

Publication number Publication date
EP3452514A1 (fr) 2019-03-13

Similar Documents

Publication Publication Date Title
EP3177314B1 (fr) Immunothérapie par cellules t spécifiques de la wt-1
EP3212223B1 (fr) Compositions et procédés pour intensifier l&#39;efficacité de l&#39;immunothérapie cellulaire adoptive
US11026969B2 (en) High affinity T cell receptors and uses thereof
WO2017193104A1 (fr) Immunothérapie de lymphocytes t spécifique de mart-1
CN113286811A (zh) 改善过继性细胞疗法的效力和安全性
CA3117539A1 (fr) Recepteurs de lymphocytes t de ny-eso-1 et leurs procedes d&#39;utilisation
JP2017500869A (ja) ヒトメソテリンキメラ抗原受容体およびその使用
CN114585646B (zh) Mage-a4 t细胞受体及其使用方法
US11534461B2 (en) High affinity merkel cell polyomavirus T antigen-specific TCRS and uses thereof
CN115135674A (zh) 树突细胞激活性嵌合抗原受体和其用途
EP3841113A1 (fr) Immunothérapie ciblant les antigènes kras ou her2
JP2021512637A (ja) サイクリンａ１特異的ｔ細胞受容体およびその使用
EP4253410A1 (fr) Peptide épitope mutant ras et récepteur de lymphocyte t reconnaissant un mutant ras
Kerr Modulation of T Cells to Promote an Anti-Tumor Response: Activation And Inhibition Of T Cells For Efficacy In T-Cell Lymphomas And Melanoma
TW202246511A (zh) 靶向ny-eso-1之增強免疫細胞療法
WO2018148180A2 (fr) Matériaux et méthodes pour l&#39;identification et le traitement du cancer

Legal Events

Date Code Title Description
NENP Non-entry into the national phase

Ref country code: DE

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

Ref document number: 17724686

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2017724686

Country of ref document: EP

Effective date: 20181206