WO2011161266A1 - Domaines variables simples améliorés de l'immunoglobuline et leurs produits de recombinaison dirigés contre cxcr4 - Google Patents

Domaines variables simples améliorés de l'immunoglobuline et leurs produits de recombinaison dirigés contre cxcr4 Download PDF

Info

Publication number
WO2011161266A1
WO2011161266A1 PCT/EP2011/060738 EP2011060738W WO2011161266A1 WO 2011161266 A1 WO2011161266 A1 WO 2011161266A1 EP 2011060738 W EP2011060738 W EP 2011060738W WO 2011161266 A1 WO2011161266 A1 WO 2011161266A1
Authority
WO
WIPO (PCT)
Prior art keywords
amino acid
acid sequence
protein
polypeptide
seq
Prior art date
Application number
PCT/EP2011/060738
Other languages
English (en)
Inventor
Peter Schotte
Ann Brige
Maarten Dewilde
Hilde Stals
Beatrijs Strubbe
Original Assignee
Ablynx Nv
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 Ablynx Nv filed Critical Ablynx Nv
Publication of WO2011161266A1 publication Critical patent/WO2011161266A1/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
    • C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/35—Valency
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to amino acid sequences that are directed against CXCR4; as well as protein, constructs and compounds comprising the same; and also nucleic acids encoding the same.
  • WO 09/138519 or in the prior art cited in WO 09/138519.
  • a method or technique it can be performed as described in WO 09/138519 (or in the prior art cited in WO 09/138519).
  • the term "Nanobody” is as defined in WO 09/138519, and thus in a specific aspect generally denotes a VHH, a humanized VHH or a cameiized VH (such as a camelized human VH) or generally a sequence optimized VHH (such as e.g. optimized for chemical stability and/or solubility, maximum overlap with known human framework regions and maximum expression).
  • WO 09/138519 describe a number of amino acid sequences and in particular VHHs and constructs thereof that are directed against human CXCR4 (see for example the amino acid sequences mentioned such as SEQ ID NO: 238 and SEQ ID NO: 239 in Table B-l.l of WO 09/138519).
  • WO 09/138519 also describes multivalent, muitispecific and/or biparatopic constructs (as defined in WO 09/138519) that are directed against human CXCR4. Reference is for example made to the constructs referred to in Example 4 of WO 09/138519 such as SEQ ID NO: 264 in Table B-5 of WO 09/138519).
  • amino acid sequence against human CXCR4 from WO 09/138519 is the sequence called 238D2 (see SEQ ID NO: 238 in WO 09/138519):
  • amino acid sequence against the human CXCR4 from WO 09/138519 is the sequence called 238D2-20GS-238D4 (see SEQ ID NO: 264 in WO 09/138519):
  • the anti-human CXCR4 amino acid sequences and constructs from WO 09/138519 show excellent biological activity and other desired properties. However, this does not mean that an anti- human CXCR4 amino acid sequence that would have (even further) improved properties would not be a valuable addition to the art.
  • the invention provides such improved anti-human CXCR4 amino acid sequences, and in particular (even further) improved variants of the sequences 238D2 ⁇ SEQ ID NO:l), 238D4 (SEQ ID NO:2) and 238D2-20GS-238D4 (SEQ ID O:3).
  • the amino acid sequence provided by the invention is a variant of 238D2-20GS-238D4 that comprises, at position 5 of the 238D4 building block (numbering according to Figure 5 of this application), a valine instead of the original methionine residue.
  • an amino acid sequence of the invention may be a variant of 238D2-20GS-238D4, also referred to herein as 4CXCR100 (SEQ ID NO: 4) that comprises, at position 5 of the 238D4 building block (numbering according to Figure 5 of this application), a valine residue:
  • the amino acid sequence provided by the invention is a variant of 238D2-20GS- 238D4 that comprises i) at position 5 of the 238D4 building block (numbering according to Figure 1 of this application), a va!ine instead of the original methionine residue; and ii) at position 77 of the 238D2 building block (numbering according to Figure 1 of this application), a threonine instead of the original methionine residue.
  • an amino acid sequence of the invention may be a variant of 238D2-20GS-238D4, also referred to herein as 4CXCR101 (SEQ ID NO: 5 ⁇ that comprises i) at position 5 of the 238D4 building block (numbering according to Figure 5 of this application), a valine residue; and ii) at position 77 of the 238D2 building block ⁇ numbering according to Figure 5 of this application), a threonine:
  • an "optimized variant" of an amino acid sequence according to the invention is a variant that comprises one or more beneficial substitutions such as a substitutions increasing i) the degree of "humanization", ii) the chemical stability, and/or til) the levei of expression; while the potency (measured e.g. by the potency assay as described in the experimental part of WO 09/138519 or in this application ⁇ remains comparable (i.e.
  • an amino acid sequence of the invention contains at least one such substitution, and preferably at ieast two such substitutions, and preferably at least three humanizing substitutions and preferably at least 10 such humanizing substitutions.
  • the amino acid sequences of the invention preferably comprise a maximum of 20 substitutions, and preferably a total of 15, 13, 11 or 10 substitutions (although the maximum number may in some cases not be critical, depending on the substitutions chosen).
  • substitutions include, without limitation, for the 238D2 building block: T14P, M77T, Y82aN, K83R, and/or Q108L; and for the 238D4 building block: M5V, A14P, R39Q, K83R, T91Y, and/or Q108L (numbering according to Figure 5 of this application ).
  • amino acid sequences of the invention may contain one or more other/further substitutions.
  • other/further substitutions wiil become clear from the further description herein, and for example may include (and preferably essentially consist of) one or more of the following substitutions (also referred to herein as "substitutions (a) to
  • substitutions in which a "camelid" amino acid residue at a certain position is replaced by a different “camelid” amino acid residue that occurs at said position (for which reference is for example made to Figures 1-4).
  • Some non-!imiting examples of such substitutions are V5L, IVI43K (substitution to the residue that is most prevalent in this position in both human VH's as well as VHH's), S49A and/or A74S; and/or
  • substitutions that improve the (other) properties of the protein such as substitutions that improve the long-term stability and/or properties under storage of the protein.
  • substitutions that prevent or reduce oxidation events for example, of methionine residues
  • substitutions that prevent or reduce pyroglutamate formation for example, of DG, DS, NG or NS motifs.
  • substitutions For such substitutions, reference is for example made to the International application WO 09/095235, which is generaliy directed to methods for stabilizing single immunoglobulin variable domains by means of such substitutions, and also gives some specific example of suitable substitutions (see for example pages 4 and 5 and pages 10 to 15).
  • One example of such substitution may be to replace an NS motif at positions 82a and 82b with an NN motif;
  • amino acid sequences of the invention contain at least one "amino acid difference” compared to each of the sequences of 238D2-20GS-238D4, respectively (in which the term "amino acid difference” is used herein in the same meaning as defined in WO 09/138519, namely as an insertion, deletion or substitution of a single amino acid residue on a position of the first sequence, compared to the second sequence; it being understood that two amino a id sequences can contain one, two, 5, 10, 11, 12 or more such amino acid differences. In the context of the present invention, any amino acid difference is preferably a substitution).
  • amino acid sequences of the invention contain at least one substitution (as defined herein), and may optionally contain one or more further substitutions (such as any one of, or any suitable combination of any two or more of, the further substitutions (a) to (c) as mentioned herein).
  • the amino acid sequences of the invention contain compared to the sequence 238D2-20GS-238D4 at ieast the substitutions: for the 238D2 building biock: T14P, IV177T, Y82aN, K83R, and Q108L and for the 238D4 building block: M5V (numbering according to Figure 5 of this application); also referred to herein as 4CXCR103 (SEQ ID NO: 6):
  • the amino acid sequences of the invention contain a total of between 6 and 15, preferably between 9 and 13, such as 10, 11 or 12 amino acid substitutions compared to the wild-type sequence 238D2-20GS-238D4.
  • these differences preferably at Ieast comprise one and preferably both of the substitutions M5V in the 238D4 building block and/or M77T in the 238D2 building block, and at Ieast one, preferably at Ieast two, such as three, four or five or ten humanizing substitutions, and may optionally comprise one or more further substitutions (such as any one of, or any suitable combination of any two or more of, the further substitutions (a) to (c) as mentioned herein).
  • the skilled person will be able to select (a suitable combination of) one or more such suitable humanizing and/or further substitutions.
  • the amino acid sequences of the invention contain a total of between 1 and 15, such as one, 5 or 10 amino acid differences compared to the sequence of 4CXCR100, in which at least one of these amino acid differences is the substitution M77T in the 238D2 building block and the other substitutions may for example be, and preferably are, either one or more further beneficial substitutions (better expression and better chemical stability) and/or one or more further substitutions (such as any one of, or any suitable combination of any two or more of, the further substitutions (a) to (c) as mentioned herein).
  • the skilled person will be able to select (a suitable combination of) one or more such suitable humanizing and/or further substitutions.
  • these amino acid differences compared to 238D2-20GS-238D4 and/or 4CXCR100 are most preferably located in the framework regions (defined according to Kabat, reference is again made to WO 09/138519), although it is not fully excluded that a very limited number of these amino acid differences (such as for example only one or two) may be present in the CDR's (as long as these do not detract (too much) from the desired affinity, on-rate or off-rate (for example, such amino acid differences in the CDR's may be introduced as a result of affinity maturation).
  • a preferred, but non-limiting aspect of an amino acid sequence of the invention is the amino acid sequences of the invention that contain compared to the sequence 238D2-20GS-238D4 at least the substitutions: for the 238D2 building block: T14P, M77T, Y82aN, K83R, and Q108L and for the 238D4 building biock: M5V, A14P, R39Q, K83R, T91Y, and Q108L (numbering according to Figure 5 of this application); also referred to herein as 4CXCR104 (SEQ ID NO: 7):
  • amino acid sequences of the invention may for example contain a total of between one and five, such as one, two or three amino acid differences compared to the sequence of 4CXCR104 (while retaining all substitutions as defined above for 4CXCR104), in which such amino acid differences may for example be, and most preferably are, either one or more further humanizing substitutions and/or any one of, or any suitable combination of any two or more of, the further substitutions (a) to (c) as mentioned herein.
  • the invention relates to an amino acid sequence (i.e. an amino acid sequence of the invention) that is a variant of 238D2-20GS-238D4 (SEQ. ID NO: 3) that comprises, compared to the amino acid sequence of 238D2-20GS-238D4, (i) the M5V mutation in the 238D4 building block; and (ii) the M77T mutation in the 238D2 building block; and optionally (iii) at least one to twenty, preferably at least one to eleven, and more preferably four or eleven substitutions (as defined herein i.e. one or more humanizing substitutions as well as one or more further suitable amino acid substitutions (preferably, any one of, or any suitable combination of any two or more of, the further substitutions (a) to (c) as mentioned herein).
  • amino acid sequence i.e. an amino acid sequence of the invention
  • SEQ. ID NO: 3 that comprises, compared to the amino acid sequence of 238D2-20GS-238D4, (i) the M5V mutation
  • such a variant of 238D2-20GS-238D4 preferably contains (i) a total of between 7 and 15, preferably between 9 and 13, such as 10, 11 or 12 amino acid differences compared to the wild- type sequence 238D2-20GS-238D4.
  • the invention relates to an amino acid sequence (i.e. an amino acid sequence of the invention) that is a variant of 4CXCR101 (SEQ ID NO: 5) that comprises, compared to the amino acid sequence of 4CXCR101, at least the substitutions: for the 238D2 building block: T14P, M77T, Y82aN, K83R, and Q108L and for the 238D4 building block: M5V (also referred herein as 4CXCR103 or SEQ. ID NO: 6); and optionally (iii) as we!l one or more further suitable amino acid substitutions (preferably, any one of, or any suitable combination of any two or more of, the further substitutions (a) to (c) as mentioned herein ⁇ .
  • Such a variant of 4CXCR103 preferably contains (i) a total of between 1 and 10, preferably 5 amino acid substitutions compared to the sequence of 4CXCR103 (although the maximum number may in some cases not be critical, depending e.g. on the humanizing substitutions chosen).
  • the invention relates to an amino acid sequence (i.e. an amino acid sequence of the invention) that is a variant of 4CXCR103 that comprises, compared to the amino acid sequence of 4CXCR103, (i) for the 238D4 building block: A14P, R39Q, K83R, T91Y, and Q108L (also referred to herein as 4CXCR104 or SEQ ID NO: 7); and optionally (ii) as well one or more further suitable amino acid substitutions, in which said amino acid differences are preferably substitutions and more preferably substitutions that are chosen from one or more further humanizing substitutions (compared to the humanizing substitutions already present in 4CXCR104) and/or from any one of, or any suitable combination of any two or more of, the further substitutions (a ⁇ to (c) as mentioned herein.
  • amino acid sequence i.e. an amino acid sequence of the invention
  • the invention aiso relates to proteins and polypeptides that comprise or essentially consist of an amino acid sequence of the invention.
  • a humanizing substitution can generally be defined as a substitution whereby an amino acid residue that occurs in a framework regions of a cameiid V HH domain is replaced by a different amino acid that occurs at the same position in the framework region of a human V H domain (and preferably, a human V H 3 domain).
  • particularly suited and/or optimal humanizing substitutions may generally be determined by limited trial and error, i.e. by introducing one or more envisaged humanizing substitutions and testing the humanized variants thus obtained for one or more desired properties, such as melting temperature, affinity, potency, properties upon formatting, expression levels in a desired host organism, and/or other desired properties for VHH domains or Nanobodies or proteins/polypepttdes comprising the same, for which again reference is made to WO 09/138519 and the further patent applications by Ablynx N.V. mentioned therein).
  • desired properties such as melting temperature, affinity, potency, properties upon formatting, expression levels in a desired host organism, and/or other desired properties for VHH domains or Nanobodies or proteins/polypepttdes comprising the same, for which again reference is made to WO 09/138519 and the further patent applications by Ablynx N.V. mentioned therein).
  • any substitutions at any of the camelid "Hallmark residues” should not be counted as a "humanizing substitution”.
  • Such substitutions at any of the Hallmark residues may or may not be present, and which when present may or may not be a substitution in which an amino acid residue in a VHH is replaced by an amino acid residue that occurs at the same position of a human VH sequence.
  • such a substitution at a Hallmark residue may for example also be a substitution in which an amino acid residue that occurs at the Hallmark position is replaced by another amino acid residue that occurs at said position in camelid V HH sequences (reference is again made to Figures 1-4).
  • amino acid sequences of the invention are directed against human CXCR4 and are improved variants for 238D2-20GS-238D4 as described in WO 09/138519.
  • the amino acid sequences of the invention can be used for the same purposes, uses and applications as described in WO 09/138519, for example to inhibit signaling that is mediated by human CXCR4 and/or its iigand(s); and/or in the prevention or treatment of diseases associated with an increased signalling of CXCR4, such as the various diseases in the group of cancer such as hematopoietic cancers like CLL, AML, ALL, MM, Non-Hodgkin lymphoma, solid tumors such as breast cancer, lung cancer, brain tumors, ovarian cancer, stromal chemoreststance of tumors, leukemia and other cancers, disrupting adhesive stromal interactions that confer tumor cell survival and drug resistance, mobilizing tumour cells from tissue sites and making them better accessible to conventional therapy, inhibiting
  • diseases associated with an increased signalling of CXCR4
  • the amino acid sequences of the invention can be used for stem cell mobilization in various patients in need of stem cells after X-ray radiation such as e.g. cancer patients after radiation treatment to replenish the stem ceil pooi after radiation in cancer patients, or in patients in need of more stem cells, e.g. in patients with ischemic diseases such as myocardial infarction (Ml), stroke and/or diabetes (i.e. patients in need of tissue repair) wherein more stem eel! would be re-transfused (after mobilization, screening, selection for lineage in need (e.g. cardiac, vascular lineages) and ex-vivo expansion of patient's own stem cells).
  • X-ray radiation such as e.g. cancer patients after radiation treatment to replenish the stem ceil pooi after radiation in cancer patients, or in patients in need of more stem cells, e.g. in patients with ischemic diseases such as myocardial infarction (Ml), stroke and/or diabetes (i.e. patients in need of tissue repair) wherein more
  • the amino acid sequences of the invention are very potent (i.e. EC50 vaiues as measured e.g. in the experimental part in the pM range) antagonists of human CXCR4 and/or are inverse agonists in certain continuously active human CXCR4 mutants (see e.g. Example 5 of WO 09/138519).
  • Example 5 and 6 Reference is for example made to Example 5 and 6 on pages 222ff of WO 09/138519, as well as the further general disclosure of WO 09/138519.
  • the amino acid sequences of the invention may be used as an improved alternative to 238D2-20GS-238D4, and thus may in particular be used for the same purposes as described in WO 09/138519 for 238D2-20GS- 238D4.
  • one of these applications of 238D2-20GS-238D4 (and thus for the amino acid sequences of the invention) is as a building block in compounds or constructs thai comprise, next to one or more amino acid sequences of the invention, and one or more other groups, residues, moieties, binding domains or binding units (as described in WO 09/138519).
  • such one or more further binding domains or binding units may be other immunoglobulin single variable domains, VHH's, (single) domain antibodies, Nanobodies or dAb's, and these may for example be directed against a protein or binding unit that provides for increased half -life (for example, albumin or a binding unit or binding peptide that can bind to a serum protein such as albumin).
  • a protein or binding unit that provides for increased half -life for example, albumin or a binding unit or binding peptide that can bind to a serum protein such as albumin.
  • Such multispecific constructs are preferably proteins and polypeptides (i.e. encoded by a nucleotide sequence and/or capable of being expressed by a host or host cell), as also generally described in WO 09/138519.
  • polypeptides of the invention may be essentially as described for the “polypeptides of the invention” described in WO 09/138519 that comprise 238D2-20GS-238D4 and one or more substitutions (e.g. as disclosed herein) and are in particular and preferred 4CXCR103 and 4CXCR104, and most preferred are 4CXCR104.
  • amino acid sequences and Nanobodies of the invention and polypeptides of the invention are capable of undergoing essentially the same binding interactions as described in WO 09/138519 for 238D2-20GS-238D4, and polypeptides comprising 238D2-20GS-238D4.
  • amino acid sequences of the invention preferably have a functional activity (as measured using the cAMP Hunter express CXCR4 assay described in Example 2 in the experimental part) that is essentially the same than the functional activity of 238D2-20GS-238D4.
  • amino acid sequences of the invention preferably have a inhibiting activity in the chemotaxis assay (see Example 4 in the experimental part ⁇ that is essentially the same than the activity of 238D2-20GS-238D4.
  • Polypeptides of the invention preferably have a functional activity as measured using the cAMP Hunter express CXCR4 assay described in Example 3 in the experimental part that has an EC50 value that is lower than about ⁇ such as between ⁇ and 50nM or lower, more preferably that is 50nM or lower (see Example 4).
  • Poiypeptides of the invention preferably have an inhibiting activity in the chemotaxis assay (see Example 4 in the experimental part ⁇ that has an IC50 value that is lower than 0.8 n such as between 0.6 nM and 0.8 nM (see Example 4 ⁇ .
  • the invention in another aspect, relates to a nucleic acid that encodes an amino acid sequence of the invention, a Nanobody of the invention or a polypeptide of the invention (or a suitable fragment thereof).
  • a nucleic acid will also be referred to herein as a "nucleic acid of the invention” and may for example be as essentially further described in WO 09/138519; and may in particular be in the form of a genetic construct, again as essentially further described in WO 09/138519 and may be the nucleic acid sequences as disclosed herein (SEQ, ID NO: 11 to 14).
  • the invention relates to a host or host cell that expresses (or that under suitable circumstances is capable of expressing ⁇ an amino acid sequence of the invention, a Nanobody of the invention and/or a polypeptide of the invention; and/or that contains a nucleic acid of the invention.
  • a host or host cell may again generally be as described in WO 09/138519 and may be Pichia Pastoris as disclosed in the experimental part of this application.
  • the invention also relates to methods for the production/expression of the amino acid sequences, Nanobodies and poiypeptides of the invention.
  • Such methods may generally comprise the steps of (i) the expression, in a suitable host cell or host organism or in another suitable expression system of a nucleic acid that encodes an amino acid sequence, a Nanobody or polypeptide of the invention, optionally followed by: (ii) isolating and/or purifying the amino acid sequence, Nanobody or polypeptide of the invention thus obtained, in particular, such a method may comprise the steps of 0) cultivating and/or maintaining a host of the invention under conditions that are such that said host of the invention expresses and/or produces at least one amino acid sequence, Nanobody and/or polypeptide of the invention; optionally followed by (ii) isolating and/or purifying the amino acid sequence, Nanobody or polypeptide of the invention thus obtained.
  • These methods again may essentially be performed as described in WO 09/138519.
  • the invention in another aspect relates to a method for producing an amino acid sequence, a Nanobody or protein or polypeptide according to the invention by the methods as described in WO201Q/125187, which is incorporated herein by its entirety.
  • the invention relates to a method for producing an amino acid sequence, a Nanobody or protein or polypeptide according to the invention, said method at least comprising the steps of culturing a host cell to produce said amino acid sequence, a Nanobody or protein or polypeptide according to the invention comprising: a ⁇ cultivating said host cell in a culture medium under conditions that are such that said host cell will multiply;
  • the invention further relates to a method as described herein, further applying conditions that promote the formation of disulfide bridges in and/or after step a), step b), step c), and/or step d),
  • the conditions that promote the formation of disulfide brides are selected from one or more of the following:
  • oxidizing agents preferably oxidizing metal ions, preferably one or more selected from Cu2+, Fe2+, Fe3+ and Zn2+;
  • the oxidizing agent preferably oxidizing metal ions, more preferably one or more selected from Cu2+, Fe2+, Fe3+ and Zn2+, are added to at least one production step of the an amino acid sequence, a Nanobody or protein or polypeptide according to the invention, preferably seiected from: cuituring the host to produce the amino acid sequence, Nanobody or protein or polypeptide according to the invention, the culture supernatant comprising the amino acid sequence, Nanobody or protein or polypeptide according to the invention after removal of the host, any step of purifying the amino acid sequence, Nanobody or protein or polypeptide according to the invention, or the purified amino acid sequence, Nanobody or protein or polypeptide according to the invention .
  • the present invention relates to a method as described herein, wherein said addition of oxidizing agents, preferably oxidizing metal ions, preferably Cu2+, Fe2+, Fe3+ and Zn2+, more preferably 1-10 mM Cu2+ is performed alone, or in combination with one or more of the conditions according to claim 2 b ⁇ to e) and/or in combination with one or more of the conditions as described herein.
  • oxidizing agents preferably oxidizing metal ions, preferably Cu2+, Fe2+, Fe3+ and Zn2+, more preferably 1-10 mM Cu2+
  • the present invention relates to a method a s described herein, wherein said thiol isomerase is selected from PDI, calsequestrin and other PDI-related proteins comprising, but not limited to E p72, ERp57, ERp60, ERp44, ERp5, ERp27 and PDI , preferably PDI, or coexpressing genes (such as HAC1P, Bip/Kar2p) that increase the basal expression level of foldases and chaperones.
  • PDI a s described herein, wherein said thiol isomerase is selected from PDI, calsequestrin and other PDI-related proteins comprising, but not limited to E p72, ERp57, ERp60, ERp44, ERp5, ERp27 and PDI , preferably PDI, or coexpressing genes (such as HAC1P, Bip/Kar2p) that increase the basal expression level of fold
  • the present invention relates to a method as described herein, wherein said cuituring temperature is lowered by 5°C as compared to the standard cuituring temperature for the host organism.
  • the present invention relates to a method as described herein, wherein said methanol feed is lowered by 30-80% as compared to the standard methanol feed for the respective host.
  • the present invention relates to a method as described herein, wherein said conductivity of the culture medium is lowered by 30% to 80% as compared to the standard medium for the respective host.
  • the present invention relates to a method as described herein, wherein yeast extract and/or peptone are added to the culture medium at a concentration in the feed of 0 to 20%.
  • the present invention relates to a method as described herein, wherein refolding the amino acid sequence, Nanobody or protein or polypeptide according to the invention in the presence of denaturant and redox-buffer is performed using 2M guanidinium hydrochloride and 1:5 mM/mM cystamine/cysteamine.
  • the present invention relates to a method as described herein, wherein the amino acid sequence, IManobody or protein or polypeptide according to the invention is treated by increasing the temperature to 40-60°C, preferably 55°C, increasing pH to pH 8-9, and/or high pressure to 250- 5000 bar, preferably about 1000-2000 bar, optionaily combined with oxygenation by purging with oxygen.
  • the present invention relates to a method as described herein, wherein the amino acid sequence, Nanobody or protein or polypeptide according to the invention is attached to a stationary phase of a chromatographic column.
  • the present invention relates to a method as described herein, wherein said eukaryotic host is selected from insect ceils, mammalian cells, and lower eukaryotic hosts comprising yeasts such as Pichia, Hansenula, Saccharomyces, Kluyveromyces, Candida, Tomlopsis, Toruiaspora, Schizosaccharomyces, Citeromyces, Pachysolen, Debaromyces, ivletschunikowia, Rhodosporidium, Leucosporidium, Botryoascus, Sporidiobo!us, Endomycopsis, preferably Pichia pastoris.
  • yeasts such as Pichia, Hansenula, Saccharomyces, Kluyveromyces, Candida, Tomlopsis, Toruiaspora, Schizosaccharomyces, Citeromyces, Pachysolen, Debaromyces, ivletschunikowia, Rhodosporidium,
  • the invention relates to methods for the production/expression of the amino acid sequences, Nanobodies and polypeptides of the invention wherein the production/expression is improved (i.e. 2 to 3.5 higher expression of the selected optimized amino acid sequences, Nanobodies and polypeptides of the invention) compared to the reference compound 4CXCR100 while having at the same time essentially the same activity than 4CXCR100, i.e. has an iC50 value in the chemotaxis assay of about 0.8 ntvl or lower (see Example 4) and/or an EC50 value in the cAMP assay of about 55pM or lower (see Example 3).
  • Such methods may generally comprise the steps of (i) the expression, in a suitable host ceil or host organism or in another suitable expression system of a nucleic acid that encodes an amino acid sequence, Nanobody or polypeptide of the invention (and in particular4CXCR104 or 4CXC 103, or most preferred 4CXCR104), optionally followed by: (ii) isolating and/or purifying the amino acid sequence, Nanobody or polypeptide of the invention thus obtained.
  • such a method may comprise the steps of ⁇ i ⁇ cultivating and/or maintaining a host of the invention under conditions that are such that said host of the invention expresses and/or produces at least one amino acid sequence and/or polypeptide of the invention; optionally followed by (ii) isolating and/or purifying the amino acid sequence, Nanobody or polypeptide of the invention thus obtained.
  • the invention further relates to a product or composition containing or comprising at least one amino acid sequence of the invention, at least one polypeptide of the invention (or a suitable fragment thereof ⁇ and/or at least one nucleic acid of the invention, and optionally one or more further components of such compositions known per se, i.e. depending on the intended- use of the composition.
  • a product or composition may for example be a pharmaceutical composition (as described herein), a veterinary composition or a product or composition for diagnostic use (as also described herein).
  • the invention also relates to the use of an amino acid sequence, Nanobody or polypeptide of the invention, or of a composition comprising the same, in ⁇ methods or compositions for) modulating and in particular inhibiting (as defined herein and/or in WO 09/138519) CXCR4 and/or CXCR4 - mediated signalling, either in vitro (e.g. in an in vitro or cellular assay) or in vivo (e.g. in an a single cell or in a multicellular organism, and in particular in a mammal, and more in particular in a human being, such as in a human being that is at risk of or suffers from a disease or disorder associated with CXCR4 and/or its ligands).
  • in vitro e.g. in an in vitro or cellular assay
  • in vivo e.g. in an a single cell or in a multicellular organism, and in particular in a mammal, and more in particular in a human being, such as in
  • the invention also relates to methods for modulating and in particular inhibiting (as defined herein and/or in WO 09/138519) CXCR4 and/or CXCR4 -mediated signalling, either in vitro (e.g. in an in vitro or cellular assay) or in vivo (e.g.
  • CXCR4 in an a single cell or multicellular organism, and in particular in a mammal, and more in particular in a human being, such as in a human being that is at risk of or suffers from a disease or disorder associated with CXCR4 and/or its ligands
  • method comprises at least the step of contacting CXCR4 with at least one amino acid sequence, Nanobody or polypeptide of the invention, or with a com position comprising the same, in a manner and in an amount suitable to moduiate and in particular to block CXCR4 and/or CXCR4 -mediated signalling.
  • the invention also relates to the use of an one amino acid sequence, Nanobody or polypeptide of the invention in the preparation of a composition (such as, without lim itation, a pha rmaceutical composition or preparation as further described herein) for modulating and in particular inhibiting (as defined herein and/or in WO 09/138519) CXCR4 and/or CXCR4 -mediated signa lling, either in vitro (e.g. in an in vitro or celiular assay) or in vivo (e.g.
  • Figures 1-4 Ta bles giving sequence comparisons of the framework regions of hu man V H 3 domains and V HH sequences (data taken from WO 09/138519) .
  • Figure 5 Alignment of clones of interest with their hu ma nized counterparts vs. VH3-23/JH5 with germline counterparts and residues substituted during optimization underlined
  • Figure 6 Expression level analysis of 3 clones of respectively 4CXCR101, 4CXCR103 and 4CXCR104 was compared to one clone of the wild type construct 4CXC 100.
  • Figure 7 The functional activity of the Nanobody construct variants 4CXCR101, 4CXCR103 and 4CXCR104 was compared to the parental Nanobody construct 4CXCR100 in a cAMP assay
  • Figure 8 The functiona l activity of the Nanobody construct variants 4CXCR101, 4CXCR103 and 4CXCR104 was compared to the parental Nanobody construct 4CXCR100 in a chemotaxis assay
  • Figure 9 RP- HPLC chromatograms of 4CXCR100 (panel A), 4CXCR103 (panel B), 4CXCR104 (panel C), and 4CXCR101 (panel D) before a nd after treatment with 20 m H z Oz for 3 hou rs at room temperature.
  • Example 1 Sequence optimization of the two building blocks of 238D2-20GS-238D4
  • Nanobodies Purified, monovalent material was produced from nucleic acid encoding for 4CXCR238D2, 4CXCR0003, 4CXCR238D4 and 4CXCR0024. These materials (also referred to here as Nanobodies) were then characterized in a number of assays, like binding ELISA and ligand displacement and others (see Table B-2). in the binding ELISA assay, CXCR4 lipoparticles were immobilized per well on 96-well Maxisorp plates by overnight coating at 4°C. Following inhibiting with 4% Marvel in PBS, the Nanobodies were added and bound Nanobody (harbouring a c-Myc tag) were detected via sequential mouse anti-Myc and rabbit anti-mouse-HRP detection.
  • Aspecific binding of the radio-labeled ligand to the membrane extracts was determined by addition of excess unlabelled SDF- 1 (100 nM) to compete all radio-ligand from the CXCR4 receptor, The aspecific binding value for each plate was subtracted from the total binding (cpm in absence of Nanobody) and the cpm values obtained for each Nanobody, and % residual [ 125 l] SDF-1 binding in presence of Nanobody was calculated.
  • the variants were analyzed in the thermal shift assay (TSA) and by differential scanning calorimetry (DSC) as well.
  • Nanobodies (0.2 mg/ml) at different pH (range 3.5-9.0) were subjected to a heating cycle (37 -> 90 * C, 0.05 °C/min ⁇ in presence of Sypro Orange.
  • a heating cycle 37 -> 90 * C, 0.05 °C/min ⁇ in presence of Sypro Orange.
  • samples started to unfold and Sypro Orange got un- quenched by binding to the hydrophobic patches of the Nanobody which became surface exposed.
  • the fluorescence signal of Sypro Orange was monitored during the heating cycle.
  • the first derivative of the signal was calculated. The maximum peak corresponded to the melting temperature of the Nanobody in a particular buffer.
  • samples 0.5 mg/ml for Tm determination, 0.3 mg/ml for reversibility studies
  • samples were analyzed with the IVltcroCa! Automated VP-capillary Differential Scanning Calorimeter.
  • Samples were heated (1 °C/min) tili 95°C for Tm determination.
  • samples were heated (1 °C/min) till 5°C above Tm and cooled down (1 "C/min) again.
  • the percentage reversibility was calculated by looking at the ratio of Cp at the melting temperature and a baseiine point.
  • all variants were tested by analytical size exclusion chromatography (SEC) to check on unwanted multimerization (Phenomenex column (BioSep-Sec2000, flow 200 ⁇ /min in PBS) ⁇ .
  • SEC analytical size exclusion chromatography
  • Table B-2 Overview characterization data parenteral compound and sequence optimized variants.
  • the variants showed comparable binding and ligand inhibiting characteristics compared to their parental counterparts.
  • the Tm of 4CXCR0024 increased by around 10% upon sequence optimization.
  • These sequence optimized building biocks were used then to make the biparatopic constructs 4CXCR101 (SEQ ID NO: 5), 4CXCR103 (SEQ ID NO: 6) and 4CXCR104 (SEQ ID NO: 7), the only difference being position five of each building block which was valine in the final construct for which there is no expectation that this mutation (L5V) has an influence on the behavior of the final construct.
  • Example 2 Production at smai! scale and medium scale of 4CXC 0101, 4CXCR103 and 4CXCR104 a) Cloning, copy number determination a nd expression analysis.
  • the 4 different Nanobody constructs were produced after recombinant expression in Pichia pastoris expression system, based on Pichia pastoris optim ized nucleic acid sequences and the commercia lly available system from I nvitrogen/RTC using X-33 as a host strain.
  • the genes coding for the different variants 4CXCR101, 4CXCR103 and 4CXCR104 were designed in correspondence to their amino acid sequence.
  • the genes were synthesized at Geneart and codon use favorable for Pichia expression was introduced ⁇ see Table B-3).
  • Table B-3 Nucleic acid sequences encoding protein products 4CXCR100, 4CXCR101, 4CXCR103 and 4CXCR104
  • Transformation of the X-33 strain was done with the obtained expression vectors, and clones were selected on zeocin containing plates. Clones were picked ad random and were streaked on a new zeocin plate. A qPCR was performed to rank the clones according to their copy numbers. For each Nanobody construct 4 different clones with a high copy number (>3) were selected based on a qPCR copy number screening assay. Next the respective clones of each construct were tested for their expression level in shake flask.
  • Figure 6 shows the relative expression levels versus the parental construct after SDS-Page analysis of a medium sample. All clones from one construct showed equal expression levels. The expression level of the different constructs could be ranked according to the level of sequence optimization, which was the highest for 4CXCR104, followed by 4CXCR103, and the lowest expression level for 4CXCR101 and the parental Nanobody 4CXCR100. Hence, it appears that the M77T mutation in the 4CXCR238D2 (SEQ. ID NO: 1) building block, introduced for sequence optimization, resulted in a reduced expression level.
  • the different constructs 4CXCR100, 4CXCR101, 4CXCR103 and 4CXCR104 were further evaluated for their expression level at 2L fermentor scale.
  • Baseline high cell density Pichia pastoris (X33) fermentation conditions were used with the following pa rameter settings: temperatu re set constant to 30°C, pH 5 du ring the biomass production and the following induction phase, dissolved oxygen set constant to 30%, and antifoam A204 (Sigma) for foam control.
  • Cell biomass was accumulated during the first batch and glycerol fed batch phase, followed by the MeOH induction phase, sta rting at Sow MeOH feed rate (4 mL/L.h with adaptation phase), during which the Nanobody was secreted into the fermentation maxim m.
  • the estimated expression titres of the 3 different constructs in the Ably/Hyp- A medium are shown in B-4, confirm that also at fermentation scale production, 4CXCR104 had the highest expression levels, followed by 4CXCR103 and the parental 4CXCR100, The lowest expression was again observed for 4CXCR101, which was 35% lower compared to the parental construct 4CXCR100.
  • the production yields of the sequence optimized constructs 4CXCR103 and 4CXCR104 were 2 to 3.5 times higher.
  • Table S-4 Overview of the estimated expression yields for the different anti-CXCR4 Nanobody constructs after SDS-Page analysis
  • the parental 4CXCR100 Nanobody, expressed in Pichia pastoris was purified using a downstream process consisting of five DSP steps; centrifuged cu lture broth was cla rified by a microfiitration-TFF step (Hydrosart, 0.2 ⁇ " ⁇ cassette ⁇ , followed by an u!trafi!tratlon-TFF step (Hydrosart !OkDa, Sartocon, Sartorius).
  • the product was captured on an SP Sepharose FF equilibrated in 1/10 PBS, pH 6.5 and eluted with PBS pH7.3, 1M NaCI, followed by a polish step using Poros 50HS, equilibrated in 25m!Vt citric acid pH4.0, and eluted using PBS, 1M NaCI pH 7.3 to remove product related impurities such as degradation products.
  • SEC size exclusion chromatogra phy
  • the Nanobody* was concentrated via NFF using Vivaspin (5kDa MWCO, 20m L Sartorius). DNA and endotoxins were removed via an anion exchange chromatography (AEX) step in flow-through mode, using Source 30G. in PBS.
  • AEX anion exchange chromatography
  • 4CXCR101, 4CXCR103 and 4CXCR104 were pu rified via a 2-step purification proceedu re to provide rapidly a limited amount of product, with low HCP and endotoxin content.
  • part of the spent medium was clarified via a mtcrofiitration-TFF step ⁇ Hydrosart, 0.2 ⁇ cassette) followed by a capture step using Toyopearl GigaCapS-650M equilibrated in 25m M NaPi, pH6.8 and eluted with 250m NaCI.
  • LPS-removal was done by OGP (N-octyl--D ⁇ glucopyranoside) treatment followed by SEC in PBS.
  • Nanobody construct variants 4CXCR101, 4CXCR103 and 4CXCR104 were com pared to the parental Nanobody construct 4CXCR100 (238D2-20GS-23SD4 with 5V, SEQ ID NO: 4) in a cAMP assay (cAMP HunterTM express CXCR4 Assay, DiscoveRx).
  • cAMP HunterTM express CXCR4 Assay, DiscoveRx cAMP HunterTM express CXCR4 Assay, DiscoveRx.
  • cells were seeded in a 96-well plate at a density of 30,000 cells/well in OCC2 medium and incubated overnight in a 37°C, 5% C ⁇ 3 ⁇ 4, humidified incubator. The medium was aspirated the next day and 45 HBSS/10 m Hepes/Antibody Reagent mix was added.
  • This mix was composed of 1/3 Antibody Reagent and 2/3 HBSS/10 mM Hepes.
  • a 1 ⁇ 2 serial dilution of Nanobody, together with 25 nM SDF-1 (R&D Systems) and 20 ⁇ forskoSin (provided with kit) was added to the plate and incubated for 30 minutes at 37°C.
  • 60 ⁇ xl cAMP Detection Reagent/ cAMP Solution D mixture composed of 1 part Substrate Reagent 2, 5 parts Substrate Reagent 1 and 19 parts cAMP lysis buffer, was transferred to the wells and incubated for 1 hour at room temperature protected from light.
  • 60 ⁇ cAMP solution A was added to each well and incubated for 3 hours at room temperature protected from light.
  • the functional activity of the three Nanobody variants 4CXCR101, 4CXCR103 and 4CXCR104 was compared to the parental Nanobody 4CXCR100 in a chemotaxis assay using the Jurkat cell line (Jurkat E6- 1; ATCC). For this, cells were seeded 1 day before the experiment at a cell concentration of 0.5xlQ 6 cell/ml in complete medium (RPMI1640 + 10% FBS). The following day, SDF-1 (200pM final concentration, R&D Systems) and serially diluted Nanobodies were added to the bottom of a small chemotaxis plate (Neuprobe 106-5) in a total volume of 29 ⁇ .
  • a chemotaxis filter membrane (ChemoTx ® Disposibla, pore size 5 ⁇ ) was placed on top of the wells, ensuring that the membrane was in contact with the solution in the wells beiow.
  • Nanobody dilution (10 ⁇ at 5X the serially diluted final concentration as below the membrane in each well) was added on top of the membrane, followed by 40 ⁇ of Jurkat cell suspension (6.25xl0 6 cell/ml). The plates were incubated for 3 hours at 37°C in a humidified incubator (5% C0 2 ). After incubation, the filters were carefully removed and the cells in the well below were resuspended in the existing solution. The complete cell suspension was transferred to the corresponding wells of white polystyrene Costar plates.
  • Table B-6 The functional activity of the three Nanobody variants 4CXCR101, 4CXCR103 and 4CXCR104 was compared to the parental Nanobody 4CXCR100 in a chemotaxis assay using the Jurkat cell line
  • Example 5 Chemical stability of 4CXCR0101, 4CXCR103 and 4CXCR104 in oxidation assay
  • the molecules were subjected to a forced oxidation reaction. Briefly, the four Nanobodies were diluted to 1 mg/mL in D-PBS and incubated for 3 hours at room temperature in the presence of 20 mM H2O2. After incubation, the samples were desalted and analyzed on RP-HPLC. The resulting chromatograms are shown in Figure 9 and the integration data are summarized in B-7.
  • RP-HPLC experiments were carried out on an Agilent 1200 series instrument from Agilent Technologies ⁇ Palo Alto, USA ⁇ .
  • the RP- HPLC conditions were as follows:
  • CXCR4 Upon ligand binding, CXCR4 is stabilized in a conformation that activates heterotrimeric G -protein, of which Gi is a major component.
  • Gi is a major component.
  • other G-proteins and non-G-proteins mediated pathways are also used, A wide variety of downstream effector pathways are activated such as adenylate cyclase and phospholipase C that influence intracellular concentrations of second messengers (cyclic AM P, diacylglycerol, inositol 1,4,5 triphosphate and Ca2+) but also mediate Extracellular Signa l Regu lated kinase (ERKl/2) phosphorylation.
  • cyclic AM P diacylglycerol, inositol 1,4,5 triphosphate and Ca2+
  • ERKl/2 Signa l Regu lated kinase
  • Activation of Ga i/o, Gas, G q/11 or Get 12/13 modulates ERKl/2 a ctivation via nu merous mechan isms.
  • both a and ⁇ -subunits of G proteins can stimulate ERKl/2 phosphorylation through transactivation of receptor tyrosine kinases (RT s).
  • RT s receptor tyrosine kinases
  • GPCRs have a lso been shown to mediate ERKl/2 activaiion in a G protein-independent but ⁇ -arrestin dependent manner.
  • Cisbio catalog nr 64ER PEI
  • 10 4 stable CXCR4 transfected CHO-K1 cells were plated in 96 well Tissue Culture Treated Plates (white bottom ⁇ (Corning // Cat#: 3917 // 18210022) according to plate layout, and incu bated for 24h in a humid chamber (wet tissues) at 37°C in C02 incubator. 24h after plating of the ceils, the medium was replaced by assay medium, comprising SDF- ⁇ or SDF-l and the test compounds.
  • 4CXCR104 was compared to AMD3100 (plerixafor, mozobil) in an ERK phosphorylation assay, a cAMP assay and in a migration assay.
  • AMD3100 is a bicyciam com pou nd that binds CXCR4 (see e.g., Antimicrob Agents Chemother 2000, 44: 1667-11673 ; AMD3100 is also known as plerixafor or mozobit).
  • the cAM P was performed essentially as described in Example 3, but with 111.5 nM SDFla .
  • the chemotaxis ⁇ or migration ⁇ assay was performed essentially as described in Example 4, but with 1 nM SDFla.
  • the Erk assay was performed essentially as described in Example 6, but with 10 nM SDFla.
  • Nanobodies ® have significantly better potency in the cAMP assay, the migration assay and the ERK phosphorylation assay at physiologically relevant !igand concentrations, as compared with the SME benchmark molecule.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Animal Behavior & Ethology (AREA)
  • Engineering & Computer Science (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Veterinary Medicine (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne des séquences d'acides aminés qui sont dirigées contre CXCR4 ; ainsi que des protéines, des produits de recombinaison et des composés les contenant ; et également des acides nucléiques codant pour ceux-ci.
PCT/EP2011/060738 2010-06-25 2011-06-27 Domaines variables simples améliorés de l'immunoglobuline et leurs produits de recombinaison dirigés contre cxcr4 WO2011161266A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US35849510P 2010-06-25 2010-06-25
US61/358,495 2010-06-25

Publications (1)

Publication Number Publication Date
WO2011161266A1 true WO2011161266A1 (fr) 2011-12-29

Family

ID=44627662

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2011/060738 WO2011161266A1 (fr) 2010-06-25 2011-06-27 Domaines variables simples améliorés de l'immunoglobuline et leurs produits de recombinaison dirigés contre cxcr4

Country Status (1)

Country Link
WO (1) WO2011161266A1 (fr)

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017137579A1 (fr) 2016-02-12 2017-08-17 Ablynx Nv Procédé de production de domaines variables uniques d'immunoglobuline
EP3248986A2 (fr) 2014-05-16 2017-11-29 Ablynx NV Domaines variables d'immunoglobuline
WO2017220988A1 (fr) 2016-06-20 2017-12-28 Kymab Limited Anticorps multispécifiques pour l'immuno-oncologie
CN113018244A (zh) * 2021-03-11 2021-06-25 苏州大学 生物活性组合物及其制备方法和应用
US11492383B2 (en) 2011-06-24 2022-11-08 Stephen D. Gillies Light chain immunoglobulin fusion proteins and methods of use thereof
WO2024133935A1 (fr) 2022-12-23 2024-06-27 Ablynx Nv Excipients de conjugaison protéiques
WO2024170756A1 (fr) 2023-02-17 2024-08-22 Ablynx N.V. Polypeptides se liant au récepteur fc néonatal

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008060367A2 (fr) * 2006-10-02 2008-05-22 Medarex, Inc. Anticorps humains qui se lient au cxcr4, et leurs utilisations
WO2009095235A1 (fr) 2008-01-29 2009-08-06 Ablynx N.V. Procédés de stabilisation de protéines et de polypeptides
WO2009138519A1 (fr) 2008-05-16 2009-11-19 Ablynx Nv Séquences d'acides aminés dirigées contre cxcr4 et autres gpcr et composés renfermant ces dernières
WO2010043650A2 (fr) * 2008-10-14 2010-04-22 Ablynx Nv Séquences d'acides aminés dirigées contre des récepteurs cellulaires pour des virus et des bactéries
WO2010125187A2 (fr) 2009-04-30 2010-11-04 Ablynx Nv Procédé de production d'anticorps à domaines

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008060367A2 (fr) * 2006-10-02 2008-05-22 Medarex, Inc. Anticorps humains qui se lient au cxcr4, et leurs utilisations
WO2009095235A1 (fr) 2008-01-29 2009-08-06 Ablynx N.V. Procédés de stabilisation de protéines et de polypeptides
WO2009138519A1 (fr) 2008-05-16 2009-11-19 Ablynx Nv Séquences d'acides aminés dirigées contre cxcr4 et autres gpcr et composés renfermant ces dernières
WO2010043650A2 (fr) * 2008-10-14 2010-04-22 Ablynx Nv Séquences d'acides aminés dirigées contre des récepteurs cellulaires pour des virus et des bactéries
WO2010125187A2 (fr) 2009-04-30 2010-11-04 Ablynx Nv Procédé de production d'anticorps à domaines

Non-Patent Citations (13)

* Cited by examiner, † Cited by third party
Title
"Data sheet of monoclonal antibody MAB173- anti CXCR4", 13 November 2007 (2007-11-13), XP002458711, Retrieved from the Internet <URL:https://www.rndsystems.com/pdf/mab173.pdf> [retrieved on 20071113] *
ANTIMICROB AGENTS CHEMOTHER, vol. 44, 2000, pages 1667 - 11673
BARIBAUD F ET AL: "Antigenically distinct conformations of CXCR4.", JOURNAL OF VIROLOGY OCT 2001 LNKD- PUBMED:11533159, vol. 75, no. 19, October 2001 (2001-10-01), pages 8957 - 8967, XP002518542, ISSN: 0022-538X *
BRELOT A ET AL: "Identification of residues of CXCR4 critical for human immunodeficiency virus coreceptor and chemokine receptor activities.", THE JOURNAL OF BIOLOGICAL CHEMISTRY 4 AUG 2000 LNKD- PUBMED:10825158, vol. 275, no. 31, 4 August 2000 (2000-08-04), pages 23736 - 23744, XP002611000, ISSN: 0021-9258 *
BURGER JAN A ET AL: "Potential of CXCR4 antagonists for the treatment of metastatic lung cancer", EXPERT REVIEW OF ANTICANCER THERAPY, FUTURE DRUGS LTD, UK, vol. 11, no. 4, 1 April 2011 (2011-04-01), pages 621 - 630, XP009152669, ISSN: 1744-8328 *
CARNEC XAVIER ET AL: "Anti-CXCR4 monoclonal antibodies recognizing overlapping epitopes differ significantly in their ability to inhibit entry of human immunodeficiency virus type 1.", JOURNAL OF VIROLOGY FEB 2005 LNKD- PUBMED:15650218, vol. 79, no. 3, February 2005 (2005-02-01), pages 1930 - 1933, XP002234668, ISSN: 0022-538X *
HARMSEN M M ET AL: "Properties, production, and applications of camelid single-domain antibody fragments.", APPLIED MICROBIOLOGY AND BIOTECHNOLOGY NOV 2007 LNKD- PUBMED:17704915, vol. 77, no. 1, November 2007 (2007-11-01), pages 13 - 22, XP002611003, ISSN: 0175-7598 *
JÄHNICHEN S: "CXCR4 nanobodies (VHH-based single variable domains) potently inhibit chemotaxis and HIV-1 replication and mobilize stem cells.", PROC NATL ACAD SCI U S A, vol. 107, no. 47, November 2010 (2010-11-01), pages 20565 - 20570, XP002611004 *
KHAN ABID ET AL: "Small molecule CXCR4 chemokine receptor antagonists: developing drug candidates.", CURRENT MEDICINAL CHEMISTRY 2007 LNKD- PUBMED:17896975, vol. 14, no. 21, 2007, pages 2257 - 2277, XP002611002, ISSN: 0929-8673 *
ROOVERS R C ET AL: "Nanobodies in therapeutic applications", CURRENT OPINION IN MOLECULAR THERAPEUTICS, CURRENT DRUGS, LONDON, GB, vol. 9, no. 4, 1 January 2007 (2007-01-01), pages 327 - 335, XP009093747, ISSN: 1464-8431 *
VADAY GAYLE G ET AL: "CXCR4 and CXCL12 (SDF-1) in prostate cancer: inhibitory effects of human single chain Fv antibodies", CLINICAL CANCER RESEARCH, THE AMERICAN ASSOCIATION FOR CANCER RESEARCH, US, vol. 10, no. 16, 15 August 2004 (2004-08-15), pages 5630 - 5639, XP002397863, ISSN: 1078-0432, DOI: DOI:10.1158/1078-0432.CCR-03-0633 *
WONG REBECCA S Y ET AL: "Comparison of the potential multiple binding modes of bicyclam, monocylam, and noncyclam small-molecule CXC chemokine receptor 4 inhibitors.", MOLECULAR PHARMACOLOGY DEC 2008 LNKD- PUBMED:18768385, vol. 74, no. 6, December 2008 (2008-12-01), pages 1485 - 1495, XP002611001, ISSN: 1521-0111 *
XU CHEN ET AL: "Human anti-CXCR4 antibodies undergo VH replacement, exhibit functional V-region sulfation, and define CXCR4 antigenic heterogeneity.", JOURNAL OF IMMUNOLOGY (BALTIMORE, MD. : 1950) 15 AUG 2007 LNKD- PUBMED:17675502, vol. 179, no. 4, 15 August 2007 (2007-08-15), pages 2408 - 2418, XP002610999, ISSN: 0022-1767 *

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11492383B2 (en) 2011-06-24 2022-11-08 Stephen D. Gillies Light chain immunoglobulin fusion proteins and methods of use thereof
EP3693386A1 (fr) 2014-05-16 2020-08-12 Ablynx NV Domaines variables d'immunoglobuline
EP3248986A2 (fr) 2014-05-16 2017-11-29 Ablynx NV Domaines variables d'immunoglobuline
EP3702369A1 (fr) 2014-05-16 2020-09-02 Ablynx NV Domaines variables d'immunoglobuline
US11851480B2 (en) 2016-02-12 2023-12-26 Ablynx N.V. Method for the production of immunoglobulin single variable domains
WO2017137579A1 (fr) 2016-02-12 2017-08-17 Ablynx Nv Procédé de production de domaines variables uniques d'immunoglobuline
JP7540877B2 (ja) 2016-02-12 2024-08-27 アブリンクス エン．ヴェー． 免疫グロブリン単一可変ドメインの生成方法
WO2017220990A1 (fr) 2016-06-20 2017-12-28 Kymab Limited Anticorps anti-pd-l1
WO2017220989A1 (fr) 2016-06-20 2017-12-28 Kymab Limited Anti-pd-l1 et cytokines il-2
WO2017220988A1 (fr) 2016-06-20 2017-12-28 Kymab Limited Anticorps multispécifiques pour l'immuno-oncologie
CN113018244A (zh) * 2021-03-11 2021-06-25 苏州大学 生物活性组合物及其制备方法和应用
WO2024133935A1 (fr) 2022-12-23 2024-06-27 Ablynx Nv Excipients de conjugaison protéiques
WO2024170756A1 (fr) 2023-02-17 2024-08-22 Ablynx N.V. Polypeptides se liant au récepteur fc néonatal

Similar Documents

Publication Publication Date Title
WO2011161266A1 (fr) Domaines variables simples améliorés de l&#39;immunoglobuline et leurs produits de recombinaison dirigés contre cxcr4
JP6034023B2 (ja) Ｃｘｃｒ４及び他のｇｐｃｒに指向性を有するアミノ酸配列及びそれを含む化合物
AU2020217351B2 (en) CXCR2 binding polypeptides
CN102906118B (zh) 与her3相关的生物材料
JP5647222B2 (ja) Ｉｌ−６ｒ関連疾患及び障害の治療のためのｉｌ−６ｒに指向性を有する改善されたアミノ酸配列及びこれを含むポリペプチド
AU2013257642B2 (en) Biparatopic binding polypeptides for CXCR2 and uses thereof
ES2774192T3 (es) Variantes mejoradas de unión de anti-albúmina sérica
CN110214147A (zh) IL15/IL15Rα异源二聚体FC-融合蛋白
CN101128487A (zh) 靶向血清白蛋白和glp-1或pyy的双特异性结构域抗体
KR20120127729A (ko) 엑센딘-4 및 이의 유사체의 융합 단백질, 제조방법 및 이의 용도
JP2014503209A5 (fr)
JP2013529080A (ja) 改良された抗血清アルブミン結合変異体
AU2016208161B2 (en) IL-17A-binding polypeptides
EP2691418A1 (fr) Domaines variables uniques d&#39;immunoglobulines anti-cxcr7 bispécifiques
CN114835810B (zh) 一种抗pd-1纳米抗体及其应用
CN116143926A (zh) 一种抗IL1RAcP的单域抗体及其用途
KR20230137342A (ko) 반감기 연장 모이어티 및 그의 사용 방법
KR20240019034A (ko) 변형된 융합 단백질 및 이의 용도
CN117843782A (zh) 靶向人lilrb4的纳米抗体及其应用
CN118702820A (zh) 一种靶向Frizzled-7及PD-L1的双特异性抗体及其制备方法与应用
TWI314558B (en) Antibodies to human il-1beta

Legal Events

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

Ref document number: 11728263

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 11728263

Country of ref document: EP

Kind code of ref document: A1