WO2023143537A1 - Gprc5d抗体及其应用 - Google Patents

Gprc5d抗体及其应用 Download PDF

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Publication number
WO2023143537A1
WO2023143537A1 PCT/CN2023/073614 CN2023073614W WO2023143537A1 WO 2023143537 A1 WO2023143537 A1 WO 2023143537A1 CN 2023073614 W CN2023073614 W CN 2023073614W WO 2023143537 A1 WO2023143537 A1 WO 2023143537A1
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seq
amino acid
antibody
acid sequence
variable region
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PCT/CN2023/073614
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French (fr)
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WO2023143537A9 (zh
Inventor
周亮
王鹏
任鹏举
蒋华
李宗海
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恺兴生命科技(上海)有限公司
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Publication of WO2023143537A1 publication Critical patent/WO2023143537A1/zh
Publication of WO2023143537A9 publication Critical patent/WO2023143537A9/zh

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    • 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
    • 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
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/64—General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • This application relates to the field of tumor immunotherapy or diagnosis, and more specifically, relates to antibodies specifically binding to GPRC5D and applications thereof.
  • the clinical targeted therapy for multiple myeloma mainly includes immunomodulatory drugs (thalidomide, lenalidomide, pomalidomide), proteasome inhibitors (bortezomib, carfilzomib, escarcinamide, etc.) Zomi) and anti-CD38 monoclonal antibody, etc.
  • immunomodulatory drugs thalidomide, lenalidomide, pomalidomide
  • proteasome inhibitors bortezomib, carfilzomib, escarcinamide, etc.
  • Zomi anti-CD38 monoclonal antibody
  • GPRC5D is a relatively new target, and as a multi-transmembrane receptor, the development of its antibody has a certain technical threshold. Therefore, the GPRC5D antibody developed in this application, especially the fully human GPRC5D antibody, has high application value.
  • the purpose of this application is to provide an antibody that specifically recognizes GPRC5D.
  • the present application also relates to a method for preparing an anti-GPRC5D specific antibody, including phage display technology and hybridoma technology.
  • This application also relates to the research on the characteristics and specificity of anti-GPRC5D antibodies (including but not limited to Fab, scFv, scFv-Fc forms).
  • the present application provides a chimeric antigen receptor (CAR) targeting GPRC5D and a preparation method thereof.
  • the application also provides isolated nucleic acids encoding the anti-GPRC5D antibodies and chimeric antigen receptors targeting GPRC5D of the application.
  • the present application also provides a host cell comprising the nucleic acid of the present application.
  • the method also includes culturing the host cell of the present application to produce the antibody or the CAR.
  • the antibody and/or CAR of the present application are used to treat tumors or diagnose tumors.
  • the application provides an antibody recognizing GPRC5D, wherein the antibody is selected from any of the following groups:
  • an antibody comprising a heavy chain variable region comprising HCDR1 shown in any one of SEQ ID NO: 1, 11, 17, 70 or 71, and/or comprising SEQ ID NO: 2 , 12, 18, 72 or 73 any one of the HCDR2 shown, and/or comprise SEQ ID NO: 3, 7, 9, 13 or 19 any one of the HCDR3 shown in;
  • An antibody comprising a light chain variable region comprising any one of SEQ ID NO: 4, 14, 20 or 74 LCDR1 shown, and/or LCDR2 shown in any one of SEQ ID NO: 5, 15, 21 or 75, and/or LCDR3 shown in any one of SEQ ID NO: 6, 8, 10, 16 or 22;
  • an antibody comprising (1) the heavy chain variable region of the antibody and (2) the light chain variable region of the antibody;
  • it comprises at least one CDR in a heavy chain variable region comprising any one of SEQ ID NO: 23, 27, 31, 35, 39, 76 or 80
  • the antibody comprises HCDR1, HCDR2, HCDR3 in a heavy chain variable region comprising SEQ ID NO: 23, 27, 31, 35, 39, 76 or 80
  • the light chain variable region comprises an amino acid sequence or a variant thereof as shown in any one of SEQ ID NO: 25, 29, 33, 37, 41 or 78, or comprises at least 80% similarity with any of the above sequences amino acid sequence.
  • the CDR region of the antibody heavy chain variable region and/or the CDR region of the light chain variable region is selected from any of the following sequences or variants thereof:
  • an antibody comprising HCDR1 shown in SEQ ID NO:1, HCDR2 shown in SEQ ID NO:2 and HCDR3 shown in SEQ ID NO:3; LCDR1 shown in SEQ ID NO:4, SEQ ID NO LCDR2 shown in :5 and LCDR3 shown in SEQ ID NO:6; or
  • Antibody comprising HCDR1 shown in SEQ ID NO:1, HCDR2 shown in SEQ ID NO:2 and HCDR3 shown in SEQ ID NO:7; LCDR1 shown in SEQ ID NO:4, SEQ ID NO LCDR2 shown in :5 and LCDR3 shown in SEQ ID NO:8; or
  • Antibody comprising HCDR1 shown in SEQ ID NO:1, HCDR2 shown in SEQ ID NO:2 and HCDR3 shown in SEQ ID NO:9; LCDR1 shown in SEQ ID NO:4, SEQ ID NO LCDR2 shown in :5 and LCDR3 shown in SEQ ID NO:10; or
  • Antibody comprising HCDR1 shown in SEQ ID NO:11, HCDR2 shown in SEQ ID NO:12 and HCDR3 shown in SEQ ID NO:13; LCDR1 shown in SEQ ID NO:14, SEQ ID NO LCDR2 shown in :15 and LCDR3 shown in SEQ ID NO:16; or
  • Antibody comprising HCDR1 shown in SEQ ID NO:17, HCDR2 shown in SEQ ID NO:18 and HCDR3 shown in SEQ ID NO:19; LCDR1 shown in SEQ ID NO:20, SEQ ID NO LCDR2 shown in :21 and LCDR3 shown in SEQ ID NO:22;
  • Antibody comprising HCDR1 shown in SEQ ID NO:71, HCDR2 shown in SEQ ID NO:73 and HCDR3 shown in SEQ ID NO:3; LCDR1 shown in SEQ ID NO:4, SEQ ID NO LCDR2 shown in :5 and LCDR3 shown in SEQ ID NO:6; or
  • Antibody comprising HCDR1 shown in SEQ ID NO:1, HCDR2 shown in SEQ ID NO:2 and HCDR3 shown in SEQ ID NO:3; LCDR1 shown in SEQ ID NO:74, SEQ ID NO LCDR2 shown in :75 and LCDR3 shown in SEQ ID NO:6;
  • the antibody is selected from any of the following sequences or variants thereof:
  • An antibody whose heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 23 or at least 80%, 90%, 91%, 92%, 93%, 94% with SEQ ID NO: 23 %, 95%, 96%, 97%, 98%, 99% similarity amino acid sequence
  • the light chain variable region has the amino acid sequence shown in SEQ ID NO: 25 or has at least 80% with SEQ ID NO: 25
  • An antibody whose heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 39 or at least 80%, 90%, 91%, 92%, 93%, 94% with SEQ ID NO: 39 %, 95%, 96%, 97%, 98%, 99% similarity amino acid sequence
  • the light chain variable region has the amino acid sequence shown in SEQ ID NO: 41 or has at least 80% with SEQ ID NO: 41
  • An antibody whose heavy chain variable region has the amino acid sequence shown in SEQ ID NO:76 or at least 80%, 90%, 91%, 92%, 93%, 94% of SEQ ID NO:76 %, 95%, 96%, 97%, 98%, 99% similarity amino acid sequence
  • the light chain variable region has the amino acid sequence shown in SEQ ID NO: 25 or has at least 80% with SEQ ID NO: 25 Amino acid sequences of %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% similarity;
  • An antibody whose heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 80 or has at least 80%, 90%, 91%, 92%, 93%, 94% of SEQ ID NO: 80 %, 95%, 96%, 97%, 98%, 99% similarity amino acid sequence
  • the light chain variable region has the amino acid sequence shown in SEQ ID NO: 25 or has at least 80% with SEQ ID NO: 25 Amino acid sequences of %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% similarity;
  • An antibody whose heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 23 or at least 80%, 90%, 91%, 92%, 93%, 94% with SEQ ID NO: 23 %, 95%, 96%, 97%, 98%, 99% similarity amino acid sequence
  • the light chain variable region has the amino acid sequence shown in SEQ ID NO:78 or has at least 80% with SEQ ID NO:78 Amino acid sequences of %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% similarity.
  • the antibody is selected from a whole antibody, scFv, single domain antibody, Fab fragment, Fab' fragment, Fv fragment, F(ab')2 fragment, Fd fragment, dAb fragment, multifunctional antibody or scFv - Fc antibody, hybridoma antibody, chimeric antibody, humanized antibody, fully human antibody or monoclonal antibody.
  • the antibody is a hybridoma antibody selected from:
  • An antibody whose heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 35 or at least 80%, 90%, 91%, 92%, 93%, 94% with SEQ ID NO: 35 %, 95%, 96%, 97%, 98%, 99% similarity amino acid sequence
  • the light chain variable region has the amino acid sequence shown in SEQ ID NO: 37 or has at least 80% with SEQ ID NO: 37 Amino acid sequences of %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% similarity;
  • the antibody is a fully human antibody selected from:
  • An antibody whose heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 23 or at least 80%, 90%, 91%, 92%, 93%, 94% with SEQ ID NO: 23 %, 95%, 96%, 97%, 98%, 99% similarity amino acid sequence
  • the light chain variable region has the amino acid sequence shown in SEQ ID NO: 25 or has at least 80% with SEQ ID NO: 25
  • an antibody whose heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 31 or with SEQ ID NO: 31
  • Amino acid sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% similarity, the light chain variable region having SEQ
  • An antibody whose heavy chain variable region has the amino acid sequence shown in SEQ ID NO:76 or has at least 80%, 90%, 91%, 92%, 93%, 94% with SEQ ID NO:76 %, 95%, 96%, 97%, 98%, 99% similarity amino acid sequence
  • the light chain variable region has the amino acid sequence shown in SEQ ID NO: 25 or has at least 80% with SEQ ID NO: 25
  • An antibody whose heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 80 or at least 80%, 90%, 91%, 92%, 93%, 94% with SEQ ID NO: 23 %, 95%, 96%, 97%, 98%, 99% similarity amino acid sequence
  • the light chain variable region has the amino acid sequence shown in SEQ ID NO: 25 or has at least 80% with SEQ ID NO: 25
  • An antibody whose heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 23 or at least 80%, 90%, 91%, 92%, 93%, 94% with SEQ ID NO: 23 %, 95%, 96%, 97%, 98%, 99% similarity amino acid sequence
  • the light chain variable region has the amino acid sequence shown in SEQ ID NO:78 or has at least 80% with SEQ ID NO:78 Amino acid sequences of %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% similarity;
  • the antibody is selected from:
  • Antibodies the heavy chain of which has the amino acid sequence shown in SEQ ID NO:45 or at least 80%, 90%, 91%, 92%, 93%, 94%, 95% with SEQ ID NO:45 %, 96%, 97%, 98%, 99% similarity amino acid sequence, said light chain has the amino acid sequence shown in SEQ ID NO:47 or has at least 80%, 90%, 91% with SEQ ID NO:47 Amino acid sequences of %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% similarity;
  • Antibodies the heavy chain of which has the amino acid sequence shown in SEQ ID NO: 49 or at least 80%, 90%, 91%, 92%, 93%, 94%, 95% with SEQ ID NO: 49 %, 96%, 97%, 98%, 99% similarity amino acid sequence, said light chain has the amino acid sequence shown in SEQ ID NO:51 or has at least 80%, 90%, 91% with SEQ ID NO:51 Amino acid sequences of %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% similarity;
  • Antibodies the heavy chain of which has the amino acid sequence shown in SEQ ID NO:53 or at least 80%, 90%, 91%, 92%, 93%, 94%, 95% with SEQ ID NO:53 %, 96%, 97%, 98%, 99% similarity amino acid sequence, said light chain has the amino acid sequence shown in SEQ ID NO:55 or has at least 80%, 90%, 91% with SEQ ID NO:55 Amino acid sequences of %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% similarity;
  • any of the antibodies in the first aspect is a hybridoma antibody, a chimeric antibody, a humanized antibody, or a fully human antibody; or the antibody is a monoclonal antibody; or the antibody is a fully antibody , scFv, single domain antibody, Fab fragment, Fab' fragment, Fv fragment, F(ab') 2 fragment, Fd fragment, dAb fragment, multifunctional antibody or scFv-Fc antibody.
  • any of the antibodies in the first aspect is a hybridoma antibody selected from:
  • the heavy chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO:35, and the light chain variable region has the amino acid sequence shown in SEQ ID NO:37;
  • the heavy chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO:39, and the light chain variable region has the amino acid sequence shown in SEQ ID NO:41.
  • any one of the antibodies in the first aspect is a fully human antibody selected from:
  • the heavy chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO:23, and the light chain variable region has the amino acid sequence shown in SEQ ID NO:25;
  • the heavy chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO:27, and the light chain variable region has the amino acid sequence shown in SEQ ID NO:29;
  • the heavy chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO: 31, and the light chain variable region has the amino acid sequence shown in SEQ ID NO: 33;
  • any one of the antibodies in the first aspect is a fully human antibody selected from:
  • the heavy chain of the antibody has the amino acid sequence shown in SEQ ID NO:45, and the light chain has the amino acid sequence shown in SEQ ID NO:47;
  • the heavy chain of the antibody has the amino acid sequence shown in SEQ ID NO:49, and the light chain has the amino acid sequence shown in SEQ ID NO:51;
  • the heavy chain of the antibody has the amino acid sequence shown in SEQ ID NO:53, and the light chain has the amino acid sequence shown in SEQ ID NO:55;
  • the present application provides an immunoconjugate, which includes the antibody described in any one of the first aspect, and a functional molecule linked thereto.
  • the present application provides a chimeric receptor, the extracellular domain of the chimeric receptor comprises the antibody described in any one of the first aspects, and the chimeric receptor includes: chimeric antigen receptor (CAR ), a chimeric T cell receptor, a T cell antigen coupler (TAC), or a combination thereof.
  • CAR chimeric antigen receptor
  • TAC T cell antigen coupler
  • the chimeric receptor is a chimeric antigen receptor (CAR).
  • CAR chimeric antigen receptor
  • the CAR comprises the antibody of any one of the first aspect, a transmembrane region and an intracellular signaling region.
  • the chimeric receptor comprises sequentially connected: the antibody of any one of the first aspect, the transmembrane region and intracellular signaling region.
  • the intracellular signal region is selected from: CD3 ⁇ , Fc ⁇ RI ⁇ , CD27, CD28, CD137, CD134, MyD88, CD40 intracellular signal region sequences or combinations thereof; and/or the transmembrane The region comprises the transmembrane region of CD8 or CD28.
  • the CD8 transmembrane region comprises the amino acid sequence shown in SEQ ID NO:60.
  • the CD137 intracellular signaling region comprises the amino acid sequence shown in SEQ ID NO: 61.
  • the CD3 ⁇ intracellular signaling region comprises the amino acid sequence shown in SEQ ID NO: 62.
  • the chimeric receptors include: any one of the antibodies described in the first aspect, the transmembrane region of CD8/CD28, and CD3 ⁇ ; or any of the antibodies described in the first aspect, CD8/CD28 The transmembrane region of CD137, the intracellular signal region of CD137 and CD3 ⁇ ; or the antibody described in any one of the first aspect, the transmembrane region of CD8/CD28, the intracellular signal region of CD28 and CD3 ⁇ ; or any one of the first aspect The antibody of CD8/CD28, the transmembrane region of CD28, the intracellular signal region of CD28, CD137 and CD3 ⁇ .
  • the chimeric receptor further includes a hinge region.
  • the hinge region comprises a CD8 hinge region.
  • the CD8 hinge region comprises the amino acid sequence shown in SEQ ID NO: 59.
  • the chimeric receptor further includes a signal peptide.
  • the signal peptide comprises a CD8 signal peptide.
  • the CD8 signal peptide comprises the amino acid sequence shown in SEQ ID NO: 58.
  • the chimeric receptor comprises an amino acid sequence as shown in any one of SEQ ID NO: 43, 82, 84 or 86.
  • amino acid sequence of the chimeric receptor is shown in any one of SEQ ID NO: 63, 88, 89, or 90.
  • the present application provides a nucleic acid encoding the antibody of any one of the first aspect, the immunoconjugate of the second aspect, or the chimeric receptor of the third aspect.
  • the nucleic acid comprises a sequence as shown in any of SEQ ID NO: 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 77, 79 or 81 or its combination.
  • the nucleic acid comprises a sequence as shown in any one of SEQ ID NO: 46, 48, 50, 52, 54 or 56 or a combination thereof.
  • the nucleic acid comprises a sequence as shown in any of SEQ ID NO: 44, 83, 85 or 87.
  • the present application provides a vector comprising the nucleic acid described in the fourth aspect.
  • the present application provides a virus comprising the expression vector described in the fifth aspect.
  • the present application provides a composition, comprising the immunoconjugate described in the second aspect, and/or the chimeric receptor described in the third aspect, the composition has a cellular effect on cells expressing GPRC5D toxicity.
  • the cells expressing GPRC5D are tumor cells and/or pathogen cells.
  • the present application provides a cell comprising the antibody described in the first aspect, the immunoconjugated antibody described in the second aspect compound, the chimeric receptor of the third aspect, the nucleic acid of the fourth aspect, and/or the vector of the fifth aspect.
  • the cell is a host cell, which comprises the vector of the fifth aspect or has the nucleic acid of the fourth aspect integrated in its genome.
  • the cell/host cell expresses the chimeric receptor of the third aspect.
  • the cell/host cell is a T cell, natural killer cell, natural killer T cell, NK92 cell, stem cell-derived immune effector cell, or a combination thereof.
  • the T cells include cytotoxic T lymphocytes, DNT cells and/or regulatory T cells.
  • the T cells are derived from natural T cells and/or T cells induced by pluripotent stem cells.
  • the T cells are autologous/allogeneic T cells.
  • the T cells are primary T cells.
  • the T cells are derived from the human's own T cells.
  • the T cells comprise memory stem cell-like T cells (Tscm cells), central memory T cells (Tcm), effector T cells (Tef), regulatory T cells (Tregs), effector memory T cells cells (Tem), ⁇ T cells, or a combination thereof.
  • the host cell binds cells that express GPRC5D and does not significantly bind cells that do not express GPRC5D.
  • the host cell also carries the coding sequence of an exogenous cytokine; and/or also expresses a non-targeting GPRC5D chimeric receptor; and/or also expresses a chemokine; and/or also expresses expresses a chemokine receptor; and/or also expresses a safety switch.
  • the host cell further carries coding sequences for exogenous cytokines including IL-7, IL-12, IL-15, IL-18, IL-21, or type I interferon.
  • the host cell also expresses chemokines including CCL19 or CCL21.
  • the host cell also expresses chemokine receptors including CCR2, CCR4, CCR5, CXCR2, CXCR4, or CXCR5.
  • the host cell also expresses a safety switch comprising iCaspase-9, Truancated EGFR, or RQR8.
  • the present application provides a pharmaceutical composition, which comprises the antibody of any one of the first aspect, the immunoconjugate of the second aspect, the chimeric receptor of the third aspect, the fourth aspect
  • the present application provides a drug combination, the antibody described in any one of the first aspect, the immunoconjugate described in the second aspect, the chimeric receptor described in the third aspect, the immunoconjugate described in the seventh aspect,
  • the composition, the cell according to the eighth aspect, and the pharmaceutical composition according to the ninth aspect are administered in combination with agents that enhance their functions, preferably, in combination with chemotherapeutic drugs; Combined administration of agents with side effects; and/or with chimeric antigens expressing targets other than GPRC5D
  • the host cells of the recipient are co-administered.
  • the present application provides a method for preparing the antibody described in any one of the first aspect, the immunoconjugate described in the second aspect, the chimeric receptor described in the third aspect, and the immunoconjugate described in the seventh aspect.
  • the present application provides a kit, which includes the antibody described in any one of the first aspect, the immunoconjugate described in the second aspect, the chimeric receptor described in the third aspect, the fourth aspect.
  • the kit includes:
  • the container and the antibody or the nucleic acid encoding the antibody according to any one of the first aspect located in the container; or the immunoconjugate described in the second aspect or the nucleic acid encoding the immunoconjugate; or the nucleic acid encoding the immunoconjugate described in the third aspect or the nucleic acid encoding the chimeric receptor; or the host cell of the eighth aspect.
  • the present application provides a method for treating/diagnosing a disease, which includes administering an effective amount of the antibody as described in any one of the first aspect or the immunoconjugate as described in the fourth aspect to a subject in need.
  • said disease is selected from inflammatory disorders, infections, autoimmune diseases and tumors;
  • said subject is a human
  • said host cell is an autologous or allogeneic T cell to said subject.
  • the present application provides the antibody described in any one of the first aspect or the immunoconjugate as described in the second aspect or the host cell as described in the eighth aspect or the drug combination as described in the ninth aspect
  • Figure 1 shows the EC50 of hybridoma antibodies AB1, AB2 combined with CHOK1-huGPRC5D cells
  • Figure 2 shows that the hybridoma antibodies AB1 and AB2 are significantly combined with human GPRC5D and mouse GPRC5D;
  • Figure 3 shows the specific binding of recombinant hybridoma antibodies AB1 (scFv-mFc) and AB2 (scFv-mFc) to cell lines expressing human GPRC5D;
  • Figure 4 shows that antibodies AB3, AB4, and AB5 all specifically bind to human GPRC5D;
  • Figure 5 shows that antibodies AB3, AB4, and AB5 all specifically bind to 293T cells overexpressing human GPRC5D;
  • Figure 6 shows the EC50 of antibodies AB3, AB4, AB5 binding to human GPRC5D
  • Figure 7 shows that antibodies AB3, AB4, and AB5 all bind significantly to cells expressing human GPRC5D (CHOK1-huGPRC5D, 293T-huGPRC5D, MM.1S and NCI-H929), but do not bind to cells that do not express GPRC5D (CHOK1, 293T and Daudi );
  • Figure 8 shows the EC50 of antibodies AB3, AB4, AB5 combined with CHOK1-huGPRC5D cells
  • Figure 9 shows the EC50 of antibodies AB3, AB4, AB5 binding to MM.1S cells
  • Figure 10 shows that antibody AB3 binds to CHOK1-muGPRC5D cells
  • Figure 11 shows the in vitro killing effect of AB3-BBZ CAR T cells on target cells
  • Figure 12 shows the effect of AB3-BBZ CAR T cells and 18-BBZ CAR T cells in inhibiting tumor growth in tumor-bearing mice;
  • Figure 13 shows the body weight changes of tumor-bearing mice treated with AB3-BBZ CAR T cells and 18-BBZ CAR T cells;
  • Figure 14 shows the binding results of antibodies AB6, AB7 and AB8 to human GPRC5D
  • Figure 15 shows the detection results of the binding of antibodies AB3, AB6, AB7 and AB8 to different species of GPRC5D cell lines;
  • Figure 16 shows the EC50 detection results of antibodies AB3, AB6, AB7 and AB8 binding to 293T-huGPRC5D and MM.1S cells;
  • Figure 17 shows the affinity test results of antibodies AB3, AB7 and Bio-GPRC5D
  • Figure 18 shows the in vitro killing effect of AB3-BBZ, AB6-BBZ, AB7-BBZ, AB8-BBZ CAR T cells on target cells.
  • the applicant obtained hybridoma antibodies and fully human antibodies that specifically recognize GPRC5D.
  • the antibody of the present application can be applied to the preparation of targeted anti-tumor drugs and drugs for diagnosing tumors.
  • any concentration range, percentage range, ratio range or integer range recited herein should be understood to include any integer within the stated range, and, where appropriate, fractions thereof (e.g., one-tenth of an integer and percent).
  • GPRC5D refers to group 5 classified into G protein-coupled receptor family C, and is one of human GPCR proteins newly discovered through homology search of EST database using amino acid sequences of a series of human GPCRs. This protein has been registered under Genbank accession numbers: AF209923, NM_018654 and NP_0611124. GPRC5D is an orphan receptor, There is no known ligand or function in humans and human cancers. Exemplarily, the full-length amino acid sequence of human GPRC5D is shown in SEQ ID NO:65, and the full-length amino acid sequence of mouse GPRC5D is shown in SEQ ID NO:66.
  • polypeptide peptide
  • protein protein
  • protein protein
  • polymer may be linear, cyclic or branched, it may comprise modified amino acids, especially conservatively modified amino acids, and it may be interrupted by non-amino acids.
  • amino acid polymers that have been modified, for example, by sulfation, glycosylation, lipidation, acetylation, phosphorylation, iodination, methylation, oxidation, proteolytic processing, prenylation, elimination Amino acid polymers modified by spinylation, selenoylation, transfer-RNA-mediated amino addition such as arginylation, ubiquitination, or any other manipulation such as conjugation with labeling components.
  • amino acid refers to natural and/or unnatural or synthetic amino acids, including glycine and D or L optical isomers, as well as amino acid analogs and peptidomimetics.
  • a polypeptide or amino acid sequence "derived from" a specified protein refers to the source of the polypeptide.
  • the term also includes polypeptides expressed from a specified nucleic acid sequence.
  • antibody is used herein in the broadest sense and includes various antibody structures including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired The desired antigen-binding activity is sufficient.
  • Antibody fragment refers to a molecule other than an intact antibody that comprises the portion of the intact antibody that binds the antigen to which the intact antibody binds.
  • antibody fragments include, but are not limited to (i) Fab fragments consisting of VL, VH, CL and CH1 domains, including Fab' and Fab'-SH, (ii) Fd fragments consisting of VH and CH1 domains, (iii) ) Fv fragments consisting of the VL and VH domains of a single antibody; (iv) dAb fragments consisting of a single variable domain (Ward et al., 1989, Nature 341:544-546); (v) F(ab')2 Fragment, bivalent fragment comprising 2 linked Fab fragments; (vi) antigen binding site of single chain Fv molecule; (vii) bispecific single chain Fv dimer (PCT/US92/09965); (viii)" Dimeric” or "trimeric", multivalent or multispecific fragments constructed by
  • the "class" of an antibody refers to the type of constant domain or region that its heavy chain possesses.
  • the heavy-chain constant domains that correspond to the different classes of immunoglobulins are called ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
  • variable region or variable domain refers to the domains of an antibody heavy or light chain that participate in antibody antigen binding.
  • the heavy and light chain variable domains (VH and VL, respectively) of native antibodies generally have similar structures, with each domain comprising four conserved FRs and three CDRs. (See, eg, Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman & Co., p. 91 (2007)).
  • a single VH or VL domain may be sufficient to confer antigen binding specificity.
  • antibodies that bind a particular antigen can be isolated using the VH or VL domains from antibodies that bind the antigen to screen libraries of complementary VL or VH domains, respectively. See, eg, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).
  • hypervariable region or “complementarity determining region” or “CDR” refers to an antibody variable domain that is hypervariable in sequence and/or forms structurally defined loops ("hypervariable loops") and/or contains antigen-contacting residues ("antigenic contacts").
  • the CDR Can be identified by a numbering system selected from: Kabat, Chothia, IMGT, Gelfand, Aho and Martin. For example, it can be identified by the Kabat numbering system.
  • an antibody may contain six CDRs: three in the VH (HCDR1, HCDR2, HCDR3) and three in the VL (LCDR1, LCDR2, LCDR3).
  • Fc region or “Fc” is used to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region.
  • the term includes native sequence Fc regions and variant Fc regions.
  • FR Framework (FR) refers to variable domain residues that are distinct from hypervariable region (CDR) residues.
  • the FR of a variable domain usually consists of four FR domains: FR1, FR2, FR3 and FR4.
  • CDR residues and other residues in the variable domain are numbered according to Kabat et al. above.
  • natural antibody refers to naturally occurring immunoglobulin molecules of various structures.
  • native IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 Daltons consisting of two identical light chains and two identical heavy chains bonded by disulfide bonds.
  • each heavy chain has a variable region (VH), also known as a variable heavy domain or a heavy chain variable domain, followed by three constant domains (CH1, CH2 and CH3 ).
  • VH variable region
  • VL variable region
  • CL light chain constant domain
  • the light chains of antibodies can be assigned to one of two types, called kappa ( ⁇ ) and lambda ( ⁇ ), based on the amino acid sequence of their constant domains.
  • whole antibody “full-length antibody”, “intact antibody” are used interchangeably and refer to having a structure substantially similar to that of a native antibody or having a heavy chain comprising an Fc region as defined herein or comprising having an antigen binding region of the intact full-length antibody.
  • single domain antibody refers to a type of antibody that lacks the light chain of the antibody and only has the variable region of the heavy chain. Because of its small molecular weight, it is also called a nanobody (Nanobody).
  • single domain antibody refers to an antibody comprising all or part of the heavy chain variable domain or all or part of the light chain variable domain.
  • the single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, eg, US Patent No. 6248516).
  • mAb refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising said population are identical and/or bind the same epitope, except where possible
  • variant antibodies for example, containing naturally occurring mutations or arising during the preparation of monoclonal antibody preparations, such variants usually exist in minor amounts.
  • polyclonal antibody preparations which typically include different antibodies directed against different determinants (epitopes)
  • each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant on the antigen.
  • the designation "monoclonal” indicates that the nature of the antibody is that it was obtained from a substantially homogeneous population of antibodies, and is not considered to require that the antibody be prepared by any particular method.
  • they can be produced by a variety of techniques including, but not limited to, the hybridoma method, recombinant DNA methods, phage display methods, and methods using transgenic animals containing all or part of the human immunoglobulin loci.
  • the monoclonal antibodies of the present application can be produced by the hybridoma method, which can be formed by isolating stimulated immune cells, such as those from the spleen of an inoculated animal. These cells, such as myeloma cells or transformed cells, can then be fused with immortalized cells, which are capable of replicating indefinitely in cell culture, thereby producing an immortal, secretory immune globulin cell line.
  • the immortalized cell lines utilized are selected (whether they lack enzymes necessary for the utilization of certain nutrients).
  • TK thymidine kinase
  • HGPRT hypoxanthine-guanine phosphoribosyltransferase
  • chimeric antibody refers to an antibody in which a portion of the heavy and/or light chain of an antibody is derived from a particular source or species, and the remaining portion of the heavy and/or light chain is derived from a different source or species.
  • chimeric antibodies comprise non-human variable regions (eg, variable regions derived from a mouse, rat, hamster, rabbit, or non-human primate such as a monkey) and human constant regions.
  • chimeric antibodies are "class-switched" antibodies, in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.
  • chimeric antibodies are "humanized antibodies.”
  • humanized is used for non-human antibodies, such as rodents or primates, etc., that are hybrid immunoglobulins, immunoglobulin chains or fragments thereof that contain minimal sequence derived from non-human immunoglobulins.
  • a “humanized antibody” refers to a chimeric antibody comprising amino acid residues from non-human CDRs and amino acid residues from human FRs.
  • a humanized antibody will comprise substantially all of at least one (typically two) variable domains, wherein all or substantially all of the CDRs correspond to those of a non-human antibody, and all or substantially all of the variable domains All FRs above correspond to FRs of human antibodies.
  • a humanized antibody optionally can comprise at least a portion of an antibody constant region derived from a human antibody.
  • a "humanized antibody” may include mutations, for example, introduced by random or site-directed mutagenesis in vitro or by somatic mutation in vivo.
  • fully human antibody is an antibody having an amino acid sequence corresponding to that of an antibody produced by a human or human cell or derived from a non-human source using human antibody repertoires or other human antibody coding sequences.
  • the definition of fully human specifically excludes humanized antibodies that contain non-human antigen-binding residues.
  • the antibodies provided herein are "fully human antibodies” generated by phage display technology.
  • Antibodies of the present application can be isolated by screening combinatorial libraries of antibodies possessing the desired activity or activities. For example, various methods are known in the art for generating phage display libraries and screening the libraries for antibodies with desired binding properties. Such methods are reviewed, e.g., in Hoogenboom et al., Methods in Molecular Biology 178:1-37 (O'Brien et al., Human Press, Totowa, NJ, 2001) and further described, e.g., in McCafferty et al., Nature 348:552-554 ; Clackson et al., Nature 352:624-628 (1991); Marks et al., J.Mol.Biol.222:581-597 (1992); Marks, Meth.Mol.Biol., 248:161-175 (Lo, ed ., Human Press, Totowa, NJ, 2003); Sidhu et al., J.Mol.Biol.338(2):299
  • VH and VL gene repertoires are cloned separately by polymerase chain reaction (PCR) and randomly recombined in a phage library, which can then be screened for antigen-binding phage, as in Winter et al., Ann. Rev. Immunol. 12:433-455 (1994).
  • Phage typically display antibody fragments as single-chain Fv (scFv) fragments or Fab fragments. Libraries from immunized sources provide high affinity antibodies to the immunogen without the need to construct hybridomas.
  • antibodies or antibody fragments isolated from fully human antibody libraries are considered fully human antibodies or fully human antibody fragments.
  • parent antibody refers to the antibody provided in this application or the antibody obtained after undergoing mutation or affinity maturation according to the antibody provided in this application.
  • the parent antibody may be a naturally occurring antibody, or a variant or engineered version of a naturally occurring antibody.
  • a parent antibody may refer to the antibody itself, a composition comprising said parent antibody, or an amino acid sequence encoding it.
  • affinity matured antibody refers to an antibody that has one or more alterations in one or more hypervariable regions (CDRs) compared to the parent antibody, such alterations resulting in an increase in the affinity of the antibody for the antigen.
  • variant refers to a polypeptide having substantially the same amino acid sequence or one or more activities encoded by the substantially same nucleotide sequence as the sequence of the antibody provided herein.
  • the variant has the same or similar activity as the antibody provided in the examples of the present application.
  • a variant can be a variant antibody or antibody variant based on the amino acid sequence of an antibody provided herein.
  • variant antibody or “antibody variant” includes antibody sequences that differ from a parent antibody sequence by at least one amino acid modification compared to the parent.
  • the variant antibody sequences herein preferably have at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% amino acid sequence identity.
  • Antibody variants may refer to the antibody itself or to a composition comprising the antibody variant.
  • Amino acid sequence variants of antibodies can be prepared by introducing appropriate modifications to the nucleotide sequence encoding the antibody or by peptide synthesis.
  • amino acid modification includes amino acid substitution, addition and/or deletion
  • amino acid substitution means to replace the amino acid at a specific position in the parental polypeptide sequence with another amino acid
  • amino acid insertion means to replace an amino acid at a specific position in the parental polypeptide sequence
  • amino acid insertion means An amino acid is added at a specific position in a polypeptide sequence
  • amino acid deletion means the removal of an amino acid at a specific position in a parental polypeptide sequence. Any combination of deletions, insertions and substitutions can be made to arrive at the final construct provided that the final construct possesses the desired characteristics, eg binding of antigen.
  • Modification refers to a change in the state or structure of the protein or polypeptide of the present application. Modifications can be chemical, structural and functional.
  • conservative modification or “conservative sequence modification” means an amino acid modification that does not significantly affect or alter the binding characteristics of an antibody comprising the amino acid sequence. Such conservative modifications include amino acid substitutions, insertions and deletions. Modifications can be introduced into the antibodies of the present application by standard techniques known in the art, such as site-directed mutagenesis, PCR-mediated mutagenesis. Families of amino acid residues with similar side chains have been defined in the art, as shown in Table 1.
  • one or more amino acid residues in the CDR region or in the framework region of the antibody of the present application can be replaced with other amino acid residues of the same side chain family, and the function retained by the changed antibody (variant antibody) can be tested.
  • Non-conservative substitutions entail exchanging a member of one of these groups for a member of another group.
  • a substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (eg, a humanized or human antibody).
  • a parent antibody eg., a humanized or human antibody
  • the resulting variant selected for further study will have an altered (e.g., improved) certain biological property (e.g., increased affinity, reduced immunogenicity) relative to the parent antibody and/or will be substantially Some biological properties of the parent antibody are maintained.
  • Exemplary substitutional variants are affinity matured antibodies, which can be routinely prepared, eg, using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more CDR residues are mutated and the variant antibodies displayed on phage and screened for specific biological activity (eg, binding affinity).
  • Alterations can be made in the CDR regions, eg, to increase antibody affinity. Such changes can be made in CDR "hot spots", i.e. residues encoded by codons that are mutated with high frequency during the process of somatic cell maturation (see, e.g., Chowdhury, Methods Mol. Biol.
  • affinity maturation by any of a variety of methods (e.g. , error-prone PCR, strand shuffling, or oligonucleotide-directed mutagenesis) introduce diversity into the variable gene species selected for maturation.
  • a secondary library is then generated.
  • the library is then screened to identify any antibodies with the desired affinity Variants.
  • Another method of introducing diversity includes the CDR-directed approach, in which several CDR residues (eg, 4-6 residues at a time) are randomized.
  • substitutions, insertions, or deletions may occur within one or more CDRs, so long as such alterations do not significantly reduce the ability of the antibody to bind antigen.
  • conservative changes that do not significantly reduce binding affinity eg, the conservative modifications described herein
  • Such alterations may be, for example, outside of the residues in the CDRs that contact the antigen.
  • each CDR is unchanged, or contains no more than one, two or three amino acid substitutions.
  • anti-GPRC5D antibody refers to an antibody capable of binding to GPRC5D with sufficient affinity, which is useful as a diagnostic agent for targeting GPRC5D and / or therapeutic agent.
  • the anti-GPRC5D antibody binds to an irrelevant, non-GPRC5D protein to an extent that is less than about 10% of the antibody's binding to GPRC5D, as determined by an enzyme-linked immunosorbent assay (ELISA).
  • ELISA enzyme-linked immunosorbent assay
  • an anti-GPRC5D antibody binds an epitope of GPRC5D that is conserved among GPRC5D derived from different species.
  • chimeric T cell receptor includes recombinant polypeptides derived from various polypeptides that make up the TCR, which are capable of binding to surface antigens on target cells, and interact with other polypeptides of the complete TCR complex, usually co-localized at T cell surface.
  • a chimeric T cell receptor consists of a TCR subunit and an antigen-binding domain composed of a human or humanized antibody domain, wherein the TCR subunit includes at least part of the TCR extracellular domain, transmembrane domain, TCR cellular The stimulation domain of the intracellular signaling domain of the internal domain; the TCR subunit is operably linked to the antibody domain, wherein the extracellular, transmembrane, and intracellular signaling domain of the TCR subunit is derived from CD3 ⁇ or CD3 ⁇ , and , the chimeric T cell receptor is integrated into the TCR expressed on T cells.
  • T cell antigen coupler includes three functional domains: 1. Antigen binding domain, including single-chain antibody, designed ankyrin repeat protein (designed ankyrin repeat protein, DARPin) or other targeting groups; 2, the extracellular region domain, the single-chain antibody that binds to CD3, so that the TAC receptor and the TCR receptor are close; 3, the transmembrane region and the intracellular region of the CD4 co-receptor, wherein , the intracellular domain-linked protein kinase LCK, catalyzes the phosphorylation of immunoreceptor tyrosine-activating motifs (ITAMs) of the TCR complex as an initial step in T cell activation.
  • ITAMs immunoreceptor tyrosine-activating motifs
  • chimeric antigen receptor includes an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain.
  • Intracellular signaling domains may include functional signaling domains of stimulatory molecules (also referred to as stimulatory molecules, primary signaling molecules) and/or co-stimulatory molecules.
  • the stimulatory molecule can be a delta chain that binds to a T cell receptor complex; for example, the cytoplasmic signaling domain further includes a functional signaling domain of one or more co-stimulatory molecules, such as 4-1BB( ie CD137), CD27 and/or CD28.
  • CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain, which contains a domain derived from Stimulatory molecule's functional signaling domain.
  • the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain containing functions derived from co-stimulatory molecules Sexual signaling domains and functional signaling domains derived from stimulatory molecules.
  • the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain comprising At least two functional signaling domains of and a functional signaling domain derived from a stimulatory molecule.
  • the CAR comprises an optional leader sequence at the amino acid (ND terminus) of the CAR fusion protein.
  • the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen recognition domain, wherein the leader sequence is optionally cleaved from the antigen recognition domain (eg, scFv) during the cellular processing and localization of the CAR to the cell membrane.
  • primary signaling molecule or "stimulatory molecule” regulates the initial activation of the TCR complex in a stimulatory manner.
  • primary signals are elicited by, for example, the binding of the TCR/CD3 complex to peptide-loaded MHC molecules, thereby mediating T cell responses (including, but not limited to, proliferation, activation, differentiation, etc.).
  • Primary signaling molecules that act in a stimulatory manner can To contain the signaling motif of the immunoreceptor tyrosine activation motif or ITAM.
  • Examples of functional signaling domains (primary signaling domains) of ITAM-containing primary signaling molecules that are particularly useful in the present application include, but are not limited to, those derived from CD3 ⁇ , FcR ⁇ , FcR ⁇ , CD3 ⁇ , CD3 ⁇ , CD5, CD22, CD79a , the sequences of CD79b, CD278 (also referred to as "ICOS") and CD66d, in a specific CAR of the present application, in any one or more of the CARs of the present application, the intracellular signaling domain comprises an intracellular signaling sequence, such as CD3 ⁇ primary signal domain.
  • co-stimulatory signaling domain generally refers to the intracellular domain of co-stimulatory molecules that can combine with cell-stimulating signaling molecules, such as TCR/CD3, to combine signals leading to T cell proliferation and/or up-regulation or down-regulation of key molecules.
  • a costimulatory molecule is typically a cognate binding partner on a T cell that specifically binds a costimulatory ligand, thereby mediating a costimulatory response of the T cell, eg, but not limited to proliferation.
  • Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are required for an effective immune response.
  • Costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA and Toll ligand receptors, and OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18) and 4-1BB (CD137 ).
  • CD3 delta also known as CD3 Zeta
  • CD3 Zeta is defined as the protein provided by GenBan Accession No. BAG36664.1, or the equivalent residues from non-human species such as mouse, rodent, monkey, ape, and the like.
  • CD3 delta domain is defined as amino acid residues from the cytoplasmic domain of the zeta chain sufficient to functionally transmit the initial signal required for T cell activation.
  • the cytoplasmic domain of ⁇ comprises residues 52 to 164 of GenBan accession number BAG36664.1, a functional ortholog thereof—from a non-human species such as mouse, rodent, monkey, ape, etc. Valence residues.
  • hybridoma antibodies are obtained by immunizing mice using conventional hybridoma antibody preparation techniques in the art, and the construction of recombinant hybridoma antibodies (scFv-Fc) is also described.
  • Fab antigen-binding fragment
  • Fabs antigen binding proteins
  • the human GPRC5D antigen Kaijia Biology
  • the antibody only recognizes GPRC5D, and 293T cells, CHO-K1 cells, MM.1S cells and NCI-H929 cells that overexpress GPRC5D, but does not recognize cells that do not express GPRC5D.
  • GPRC5D herein refers to human GPRC5D or mouse GPRC5D.
  • the present application includes antibodies having a Fab sequence fused to one or more heavy chain constant regions to form an antibody having the Fc region of a human immunoglobulin to generate a bivalent protein, thereby increasing the antibody's Overall affinity and stability.
  • the Fc portion allows direct conjugation of other molecules (including but not limited to fluorescent dyes, cytotoxins, radioisotopes, etc.) to antibodies for example in antigen quantification studies, for immobilization of antibodies for affinity measurements, for targeted delivery of therapeutics Drugs, testing Fc-mediated cytotoxicity using immune effector cells and many other applications.
  • the antibodies or antibody fragments of the present application are based on the use of phage display to identify and select antigen-binding fragments (Fabs) whose amino acid sequence confers specificity to the antibody or antibody fragment against GPRC5D and forms the basis of all antigen-binding proteins of the disclosure .
  • the Fab can be used to design a range of different "antibodies or antibody fragments" including, for example, full-length antibodies, fragments thereof such as F(ab')2, fusion proteins, multivalent antibodies, scFv-Fc antibodies, i.e., antibodies with against the same antigen or not Antibodies with more than one specificity to the antigen, e.g., bispecific T cell-binding antibody (BiTE), triabody, etc. (see Cuesta et al., Multivalent antibodies: when design surprises evolution, Trends in Biotechnology 28:355-362 ,2010).
  • the application provides full-length antibodies, the heavy and light chains of which can be full-length (for example, the antibody can include at least one, preferably two, complete heavy chains, and at least one, preferably two, complete light chain) or may include an antigen binding portion (Fab, F(ab')2, Fv or scFv).
  • the antibody heavy chain constant region is selected from, for example, IgGl, IgG2, IgG3, IgG4, IgM, IgAl, IgA2, IgD, and IgE.
  • the choice of antibody type will depend on the immune effector function the antibody is designed to elicit.
  • the appropriate amino acid sequences for the constant regions of the various immunoglobulin isotypes and methods for producing the broad class of antibodies are known to those skilled in the art.
  • the application provides an antibody recognizing GPRC5D, which comprises at least one CDR in the heavy chain variable region, and the heavy chain variable region comprises any of SEQ ID NO:23, 27, 31, 35, 39, 76 or 80 An amino acid sequence or a variant thereof, or an amino acid sequence comprising at least 80% similarity to any of the above sequences; and/or comprising at least one CDR in the variable region of a light chain that can
  • the variable region comprises an amino acid sequence as shown in any one of SEQ ID NO: 25, 29, 33, 37, 41 or 78 or a variant thereof, or comprises an amino acid sequence having at least 80% similarity to any of the above sequences.
  • the heavy chain variable region comprises an amino acid sequence or a variant thereof as shown in any one of SEQ ID NO: 23, 27, 31, 35 or 39, or comprises an amino acid sequence at least 80% similar to any of the above sequences.
  • the light chain variable region comprises an amino acid sequence or a variant thereof as shown in any one of SEQ ID NO: 25, 29, 33, 37 or 41, or comprises any of the above Amino acid sequences having at least 80% similarity in sequence.
  • the heavy chain variable region comprises an amino acid sequence as shown in any one of SEQ ID NO: 23, 76 or 80 or a variant thereof, or an amino acid sequence having at least 80% similarity to any of the above sequences and/or the light chain variable region comprises an amino acid sequence or a variant thereof as shown in any one of SEQ ID NO: 25 or 78, or comprises an amino acid sequence having at least 80% similarity to any of the above sequences .
  • the CDRs can be identified by a numbering system selected from: Kabat, Chothia, IMGT, Gelfand, Aho and Martin.
  • the antibody that recognizes GPRC5D provided in this application may contain a certain CDR sequence or a combination thereof determined by any of the above-mentioned numbering systems, and the CDRs in the heavy chain and/or light chain that make up the antibody do not have to be determined by the same numbering, for example,
  • One or some CDRs in an antibody can be identified by the Kabat system, other CDRs can be identified by any one or combination of the above numbering systems.
  • the CDRs of the antibody can be identified by a numbering system, e.g., by the Kabat numbering system; e.g., by the Chothia numbering system; e.g., by the IMGT numbering system; e.g. , can be identified by the Gelfand numbering system; for example, can be identified by the Aho numbering system; for example, can be identified by the Martin numbering system.
  • the antibody may comprise one CDR, two CDRs, or three CDRs in the heavy chain variable region.
  • the antibody may comprise one CDR, two CDRs, or three CDRs in the light chain variable region.
  • the antibody may comprise one CDR, two CDRs or three CDRs in the variable region of the heavy chain and one CDR, two CDRs or three CDRs in the variable region of the light chain.
  • one CDR in the variable region of the heavy chain may be included, and one CDR, two CDRs, or three CDRs in the variable region of the light chain may be included;
  • two CDRs in the variable region of the heavy chain may be included, and comprising one CDR, two CDRs or three CDRs in the light chain variable region; for example, may comprise Three CDRs in the variable region of the heavy chain, also comprising one CDR, two CDRs, or three CDRs in the variable region of the light chain; for example, can comprise three CDRs in the variable region of the heavy chain, also comprising Three CDRs in the variable region.
  • the heavy chain variable region comprises the amino acid sequence shown in any one of SEQ ID NO: 23, 27, 31, 35, 39, 76 or 80, or a variant thereof.
  • the light chain variable region comprises the amino acid sequence shown in any one of SEQ ID NO: 25, 29, 33, 37, 41 or 78, or a variant thereof.
  • the heavy chain variable region comprises an amino acid sequence as shown in any one of SEQ ID NO: 23, 27, 31, 35, 39, 76 or 80 or a variant thereof, and the The light chain variable region comprises the amino acid sequence shown in any one of SEQ ID NO: 25, 29, 33, 37, 41 or 78, or a variant thereof.
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 23 or a variant thereof, and the light chain variable region comprises SEQ ID NO: 25, 29, 33, 37, 41 or The amino acid sequence shown in any one of 78 or its variant;
  • described heavy chain variable region comprises the aminoacid sequence shown in SEQ ID NO:27 or its variant, and described light chain variable region comprises An amino acid sequence as shown in any one of SEQ ID NO:25, 29, 33, 37, 41 or 78 or a variant thereof;
  • the heavy chain variable region comprises an amino acid as shown in SEQ ID NO:31 sequence or a variant thereof, and the light chain variable region comprises an amino acid sequence or a variant thereof as shown in any one of SEQ ID NO: 25, 29, 33, 37, 41 or 78;
  • the heavy chain The chain variable region comprises an amino acid sequence as shown in SEQ ID NO:35 or a variant thereof, and the light chain variable region comprises any one of SEQ ID NO:25, 29, 33, 37, 41 or 78
  • the heavy chain variable region comprises the amino acid sequence shown in any one of SEQ ID NO: 23, 27, 31, 35 or 39, or a variant thereof.
  • the light chain variable region comprises the amino acid sequence shown in any one of SEQ ID NO: 25, 29, 33, 37 or 41 or a variant thereof.
  • the heavy chain variable region comprises an amino acid sequence as shown in any one of SEQ ID NO: 23, 27, 31, 35 or 39 or a variant thereof, and the light chain variable The region comprises an amino acid sequence as set forth in any one of SEQ ID NO: 25, 29, 33, 37 or 41 or a variant thereof.
  • the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 23 or a variant thereof, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 25, 29, 33, 37 or 41
  • the amino acid sequence shown in any one or its variant For example, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 27 or its variant, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:27
  • described heavy chain variable region comprises the aminoacid sequence shown in SEQ ID NO:31 or its variant and said light chain variable region comprises an amino acid sequence or a variant thereof as shown in any one of SEQ ID NO: 25, 29, 33, 37 or 41
  • the The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 35 or a variant thereof, and the light chain variable region comprises any one of SEQ ID NO: 25, 29, 33, 37 or 41
  • the amino acid sequence shown or its variant
  • the heavy chain variable region comprises the amino acid sequence shown in any one of SEQ ID NO: 23, 76 or 80, or a variant thereof.
  • the light chain variable region comprises the amino acid sequence shown in any one of SEQ ID NO: 25 or 78, or a variant thereof.
  • the heavy chain variable region comprises an amino acid sequence as shown in any one of SEQ ID NO: 23, 76 or 80 or a variant thereof
  • the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: The amino acid sequence shown in any one of ID NO:25 or 78 or its variant.
  • the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 23 or a variant thereof, and the light chain variable region comprises an amino acid sequence as shown in any one of SEQ ID NO: 25 or 78
  • An amino acid sequence or a variant thereof for example, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 76 or a variant thereof, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 25 or 78
  • described heavy chain variable region comprises the aminoacid sequence shown in SEQ ID NO:80 or its variant, and described light chain variable region comprises as shown in The amino acid sequence shown in any one of SEQ ID NO: 25 or 78, or a variant thereof.
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:23, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:25.
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:27, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:29.
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:31, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:33.
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:35, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:37.
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:39, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:41.
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:76, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:25.
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:80, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:25.
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:23, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:78.
  • said antibody may comprise said heavy chain variable region and/or said light chain variable region.
  • the heavy chain variable region may comprise a heavy chain CDR1 (HCDR1), which may comprise an amino acid sequence as shown in any one of SEQ ID NO: 1, 11, 17, 70 or 71.
  • the heavy chain variable region may comprise a heavy chain CDR2 (HCDR2), which may comprise an amino acid sequence as shown in any one of SEQ ID NO:2, 12, 18, 72 or 73.
  • the heavy chain variable region may comprise a heavy chain CDR3 (HCDR3), which may comprise an amino acid sequence as shown in any one of SEQ ID NO: 3, 7, 9, 13 or 19.
  • the heavy chain variable region may comprise HCDR1 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in any of SEQ ID NO: 1, 11, 17, 70 or 71, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: NO: The amino acid sequence shown in any one of 3, 7, 9, 13 or 19.
  • the heavy chain variable region may comprise HCDR1 and HCDR2, the HCDR1 may comprise the amino acid sequence shown in any one of SEQ ID NO: 1, 11, 17, 70 or 71, and the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: The amino acid sequence shown in any one of NO: 2, 12, 18, 72 or 73.
  • the heavy chain variable region may comprise HCDR2 and HCDR3, the HCDR2 may comprise an amino acid sequence as shown in any of SEQ ID NO: 2, 12, 18, 72 or 73, and the HCDR3 may comprise an amino acid sequence such as SEQ ID NO: 2, 12, 18, 72 or 73 NO: the amino acid sequence shown in any one of 3, 7, 9, 13 or 19.
  • the heavy chain variable region may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in any of SEQ ID NO: 1, 11, 17, 70 or 71, and the HCDR2 may comprise the amino acid sequence shown in The amino acid sequence shown in any one of SEQ ID NO: 2, 12, 18, 72 or 73, the HCDR3 may comprise the amino acid sequence shown in any one of SEQ ID NO: 3, 7, 9, 13 or 19.
  • the light chain variable region may comprise a light chain CDR1 (LCDR1), and the LCDR1 may comprise an amino acid sequence as shown in any one of SEQ ID NO: 4, 14, 20 or 74.
  • the light chain variable region may comprise a light chain CDR2 (LCDR2), and the LCDR2 may comprise an amino acid sequence as shown in any one of SEQ ID NO:5, 15, 21 or 75.
  • the light chain variable region may comprise a light chain CDR3 (LCDR3), and the LCDR3 may comprise an amino acid sequence as shown in any one of SEQ ID NO: 6, 8, 10, 16 or 22.
  • the light chain variable region may comprise LCDR1 and LCDR3, the LCDR1 may comprise an amino acid sequence as shown in any of SEQ ID NO: 4, 14, 20 or 74, and the LCDR3 may comprise an amino acid sequence such as SEQ ID NO: The amino acid sequence shown in any one of 6, 8, 10, 16 or 22.
  • the light chain variable region may comprise LCDR1 and LCDR2, the LCDR1 may comprise an amino acid sequence as shown in any of SEQ ID NO: 4, 14, 20 or 74, and the LCDR2 may comprise an amino acid sequence such as SEQ ID NO: 5, 15, 21 or 75 any amino acid sequence shown.
  • the light chain variable region may comprise LCDR2 and LCDR3, the LCDR2 may comprise an amino acid sequence as shown in any of SEQ ID NO: 5, 15, 21 or 75, and the LCDR3 may comprise an amino acid sequence such as SEQ ID NO: The amino acid sequence shown in any one of 6, 8, 10, 16 or 22.
  • the light chain variable region may comprise LCDR1, LCDR2 and LCDR3, the LCDR1 may comprise the amino acid sequence shown in any of SEQ ID NO: 4, 14, 20 or 74, and the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: The amino acid sequence shown in any one of SEQ ID NO: 5, 15, 21 or 75, and the LCDR3 may include the amino acid sequence shown in any one of SEQ ID NO: 6, 8, 10, 16 or 22.
  • said antibody may comprise said heavy chain variable region and/or said light chain variable region.
  • the heavy chain variable region may comprise heavy chain CDR1 (HCDR1), which may comprise an amino acid sequence as shown in any one of SEQ ID NO: 1, 11 or 17.
  • the heavy chain variable region may comprise a heavy chain CDR2 (HCDR2), which may comprise an amino acid sequence as shown in any one of SEQ ID NO: 2, 12 or 18.
  • the heavy chain variable region may comprise a heavy chain CDR3 (HCDR3), which may comprise an amino acid sequence as shown in any one of SEQ ID NO: 3, 7, 9, 13 or 19.
  • the heavy chain variable region may comprise HCDR1 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in any one of SEQ ID NO: 1, 11 or 17, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 3, The amino acid sequence shown in any one of 7, 9, 13 or 19.
  • the heavy chain variable region may comprise HCDR1 and HCDR2, the HCDR1 may comprise an amino acid sequence as shown in any of SEQ ID NO: 1, 11 or 17, and the HCDR2 may comprise an amino acid sequence such as SEQ ID NO: 2, 12 or 18 any one of the amino acid sequences shown.
  • the heavy chain variable region may comprise HCDR2 and HCDR3, the HCDR2 may comprise the amino acid sequence shown in any of SEQ ID NO: 2, 12 or 18, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 3, 7.
  • the heavy chain variable region may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise an amino acid sequence as shown in any of SEQ ID NO: 1, 11 or 17, and the HCDR2 may comprise an amino acid sequence such as SEQ ID NO: 2, 12 or 18, the HCDR3 may comprise any amino acid sequence as shown in SEQ ID NO: 3, 7, 9, 13 or 19.
  • the light chain variable region may comprise a light chain CDR1 (LCDR1), and the LCDR1 may comprise an amino acid sequence as shown in any one of SEQ ID NO:4, 14 or 20.
  • the light chain variable region may comprise a light chain CDR2 (LCDR2), and the LCDR2 may comprise an amino acid sequence as shown in any one of SEQ ID NO:5, 15 or 21.
  • the light chain variable region may comprise a light chain CDR3 (LCDR3), and the LCDR3 may comprise an amino acid sequence as shown in any one of SEQ ID NO: 6, 8, 10, 16 or 22.
  • the light chain variable region may comprise LCDR1 and LCDR3, the LCDR1 may comprise an amino acid sequence as shown in any of SEQ ID NO: 4, 14 or 20, and the LCDR3 may comprise an amino acid sequence such as SEQ ID NO: 6, The amino acid sequence shown in any one of 8, 10, 16 or 22.
  • the light chain variable region may comprise LCDR1 and LCDR2, the LCDR1 may comprise an amino acid sequence as shown in any of SEQ ID NO: 4, 14 or 20, and the LCDR2 may comprise an amino acid sequence such as SEQ ID NO: 5, 15 or 21 any amino acid sequence shown.
  • the light chain variable region may comprise LCDR2 and LCDR3, the LCDR2 may comprise the amino acid sequence shown in any of SEQ ID NO: 5, 15 or 21, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 6, The amino acid sequence shown in any one of 8, 10, 16 or 22.
  • the light chain variable region may comprise LCDR1, LCDR2 and LCDR3, the LCDR1 may comprise an amino acid sequence as shown in any of SEQ ID NO: 4, 14 or 20, and the LCDR2 may comprise an amino acid sequence such as SEQ ID NO: 5, 15 or 21, the LCDR3 may comprise the amino acid sequence shown in any of SEQ ID NO: 6, 8, 10, 16 or 22.
  • said antibody may comprise said heavy chain variable region and/or said light chain variable region.
  • the heavy chain variable region may comprise a heavy chain CDR1 (HCDR1), which may comprise an amino acid sequence as shown in any one of SEQ ID NO: 1, 70 or 71.
  • the heavy chain variable region may comprise a heavy chain CDR2 (HCDR2), which may comprise an amino acid sequence as shown in any one of SEQ ID NO:2, 72 or 73.
  • the heavy chain variable region may comprise heavy chain CDR3 (HCDR3), and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:3.
  • the heavy chain variable region may comprise HCDR1 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in any of SEQ ID NO: 1, 70 or 71, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 3
  • the amino acid sequence shown may comprise HCDR1 and HCDR2, the HCDR1 may comprise an amino acid sequence as shown in any of SEQ ID NO: 1, 70 or 71, and the HCDR2 may comprise an amino acid sequence such as SEQ ID NO: 2, 72 or the amino acid sequence shown in either of 73.
  • the heavy chain variable region may comprise HCDR2 and HCDR3, the HCDR2 may comprise the amino acid sequence shown in any of SEQ ID NO: 2, 72 or 73, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 3
  • the heavy chain variable region may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise an amino acid sequence as shown in any of SEQ ID NO: 1, 70 or 71, and the HCDR2 may comprise an amino acid sequence such as SEQ ID NO: 2, the amino acid sequence shown in any one of 72 or 73, the HCDR3 may include the amino acid sequence shown in SEQ ID NO:3.
  • the light chain variable region may comprise a light chain CDR1 (LCDR1), and the LCDR1 may comprise an amino acid sequence as shown in any one of SEQ ID NO:4 or 74.
  • the light chain variable region may comprise a light chain CDR2 (LCDR2), and the LCDR2 may comprise an amino acid sequence as shown in any one of SEQ ID NO:5 or 75.
  • the light chain variable region can comprise a light chain CDR3 (LCDR3), which can comprise Contains the amino acid sequence shown in SEQ ID NO:6.
  • the light chain variable region may comprise LCDR1 and LCDR3, the LCDR1 may comprise the amino acid sequence shown in any of SEQ ID NO: 4 or 74, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 6. amino acid sequence.
  • the light chain variable region may comprise LCDR1 and LCDR2, the LCDR1 may comprise the amino acid sequence shown in any of SEQ ID NO: 4 or 74, and the LCDR2 may comprise the amino acid sequence shown in any of SEQ ID NO: 5 or 75.
  • the light chain variable region may comprise LCDR2 and LCDR3, the LCDR2 may comprise the amino acid sequence shown in any of SEQ ID NO: 5 or 75, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 6. amino acid sequence.
  • the light chain variable region may comprise LCDR1, LCDR2 and LCDR3, the LCDR1 may comprise an amino acid sequence as shown in any of SEQ ID NO: 4 or 74, and the LCDR2 may comprise an amino acid sequence such as SEQ ID NO: 5 or 75, the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:6.
  • the antibody can comprise the heavy chain variable region and the light chain variable region
  • the heavy chain variable region can comprise the HCDR1, the HCDR2, the HCDR3, and the light chain can
  • a variable region may comprise the LCDR1, the LCDR2, the LCDR3.
  • the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:1
  • the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:2
  • the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:3
  • the LCDR1 can include the amino acid sequence shown in SEQ ID NO:4
  • the LCDR2 can include the amino acid sequence shown in SEQ ID NO:5
  • the LCDR3 can include the amino acid sequence shown in SEQ ID NO:6 The amino acid sequence shown.
  • the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:1, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:2, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:7 Amino acid sequence, and the LCDR1 can include the amino acid sequence shown in SEQ ID NO:4, the LCDR2 can include the amino acid sequence shown in SEQ ID NO:5, and the LCDR3 can include the amino acid sequence shown in SEQ ID NO:8 The amino acid sequence shown.
  • the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:1, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:2, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:9.
  • the LCDR1 can include the amino acid sequence shown in SEQ ID NO:4, the LCDR2 can include the amino acid sequence shown in SEQ ID NO:5, and the LCDR3 can include the amino acid sequence shown in SEQ ID NO:10
  • the amino acid sequence shown may comprise the amino acid sequence shown in SEQ ID NO: 11, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 12, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 13.
  • the LCDR1 can include the amino acid sequence shown in SEQ ID NO: 14
  • the LCDR2 can include the amino acid sequence shown in SEQ ID NO: 15
  • the LCDR3 can include the amino acid sequence shown in SEQ ID NO: 16 The amino acid sequence shown.
  • the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 17, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 18, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 19 Amino acid sequence, and the LCDR1 can include the amino acid sequence shown in SEQ ID NO:20, the LCDR2 can include the amino acid sequence shown in SEQ ID NO:21, and the LCDR3 can include the amino acid sequence shown in SEQ ID NO:22 The amino acid sequence shown.
  • the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:70
  • the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:72
  • the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:3 Amino acid sequence
  • the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO:4
  • the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:5
  • the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: The amino acid sequence shown in 6.
  • the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:71
  • the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:73
  • the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:3 Amino acid sequence
  • the LCDR1 can include the amino acid sequence shown in SEQ ID NO:4
  • the LCDR2 can include the amino acid sequence shown in SEQ ID NO:5
  • the LCDR3 can include the amino acid sequence shown in SEQ ID NO:6 The amino acid sequence shown.
  • the antibody comprises a heavy chain variable region comprising the heavy chain CDR1 of any one of the amino acid sequences shown in SEQ ID NO: 1, 11 or 17, and/or comprising SEQ ID NO: 1, 11 or 17.
  • the application provides an antibody that recognizes GPRC5D comprising: light chain CDR1 comprising any amino acid sequence shown in SEQ ID NO: 4, 14 or 20, and/or comprising SEQ ID NO: 5, 15 or 21, the light chain CDR2 of any one of the amino acid sequences shown, and/or the light chain CDR3 comprising any of the amino acid sequences shown in SEQ ID NO: 6, 8, 10, 16 or 22.
  • the application provides an antibody that recognizes GPRC5D comprising: heavy chain CDR1 comprising any amino acid sequence shown in SEQ ID NO: 1, 11 or 17, and/or comprising SEQ ID NO: 2, 12 or 18 heavy chain CDR2 of any amino acid sequence shown, and/or heavy chain CDR3 comprising any amino acid sequence shown in SEQ ID NO: 3, 7, 9, 13 or 19, and/or comprising SEQ ID NO: 4,
  • the light chain CDR1 of any amino acid sequence shown in 14 or 20 and/or the light chain CDR2 comprising any amino acid sequence shown in SEQ ID NO:5, 15 or 21, and/or comprising SEQ ID NO:6,8,
  • the antibody that recognizes GPRC5D includes: heavy chain CDR1 comprising any of the amino acid sequences shown in SEQ ID NO: 1, 11 or 17, and heavy chain CDR1 comprising any of the amino acid sequences shown in SEQ ID NO: 2, 12 or 18 chain CDR2, and the heavy chain CDR3 comprising any amino acid sequence shown in SEQ ID NO:3, 7, 9, 13 or 19, and/or the light chain CDR3 comprising any amino acid sequence shown in SEQ ID NO:4, 14 or 20 Chain CDR1, and light chain CDR2 comprising any amino acid sequence shown in SEQ ID NO:5, 15 or 21, and light chain CDR3 comprising any amino acid sequence shown in SEQ ID NO:6, 8, 10, 16 or 22 .
  • the antibody that recognizes GPRC5D includes: heavy chain CDR1 comprising any of the amino acid sequences shown in SEQ ID NO: 1, 11 or 17, and heavy chain CDR1 comprising any of the amino acid sequences shown in SEQ ID NO: 2, 12 or 18 Chain CDR2, and heavy chain CDR3 comprising any amino acid sequence shown in SEQ ID NO:3, 7, 9, 13 or 19, and light chain CDR1 comprising any amino acid sequence shown in SEQ ID NO:4, 14 or 20 , and light chain CDR2 comprising any one of the amino acid sequences shown in SEQ ID NO:5, 15 or 21, and light chain CDR3 comprising any one of the amino acid sequences shown in SEQ ID NO:6, 8, 10, 16 or 22.
  • the antibody that recognizes GPRC5D includes: HCDR1 shown in SEQ ID NO:1, HCDR2 shown in SEQ ID NO:2 and HCDR3 shown in SEQ ID NO:3; LCDR1 shown in SEQ ID NO:4 , LCDR2 shown in SEQ ID NO:5 and LCDR3 shown in SEQ ID NO:6; or HCDR1 shown in SEQ ID NO:1, HCDR2 shown in SEQ ID NO:2 and shown in SEQ ID NO:7 HCDR3 shown in SEQ ID NO:4; LCDR1 shown in SEQ ID NO:4, LCDR2 shown in SEQ ID NO:5 and LCDR3 shown in SEQ ID NO:8; or HCDR1 shown in SEQ ID NO:1, SEQ ID NO:2 HCDR2 shown and HCDR3 shown in SEQ ID NO:9; LCDR1 shown in SEQ ID NO:4, LCDR2 shown in SEQ ID NO:5, and LCDR3 shown in SEQ ID NO:10.
  • said antibody may comprise said heavy chain variable region and/or said light chain variable region.
  • the heavy chain variable region may comprise an amino acid sequence as shown in any one of SEQ ID NO: 23, 27, 31, 35, 39, 76 or 80.
  • the light chain variable region may comprise an amino acid sequence as shown in any one of SEQ ID NO: 25, 29, 33, 37, 41 or 78.
  • said antibody may comprise said heavy chain variable region and/or said light chain variable region.
  • the heavy chain variable region may comprise the amino acid sequence shown in any one of SEQ ID NO: 23, 27, 31, 35 or 39.
  • the light chain variable region may comprise the amino acid sequence shown in any one of SEQ ID NO: 25, 29, 33, 37 or 41.
  • said antibody may comprise said heavy chain variable region and/or said light chain variable region.
  • the heavy chain variable region may comprise the amino acid sequence shown in any one of SEQ ID NO: 23, 76 or 80.
  • the light chain variable region may comprise the amino acid sequence shown in either of SEQ ID NO: 25 or 78.
  • the antibody can comprise the heavy chain variable region and the light chain variable region.
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:23, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:25.
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:27, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:29.
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:31, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:33.
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:35, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:37.
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:39, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:41.
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:76, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:25.
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:80, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:25.
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:23, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:78.
  • the application provides an antibody recognizing GPRC5D, comprising a heavy chain variable region, the heavy chain variable region comprising any amino acid sequence shown in SEQ ID NO: 23, 27, 31, 35 or 39, or variants of the above sequences.
  • the application provides an antibody recognizing GPRC5D, comprising a light chain variable region, the light chain variable region comprising any amino acid sequence shown in SEQ ID NO: 25, 29, 33, 37 or 41, or Variants of the above sequences.
  • the present application provides an antibody recognizing GPRC5D, comprising the above-mentioned heavy chain variable region and light chain variable region.
  • the heavy chain and light chain variable region sequences can bind GPRC5D
  • the heavy chain and light chain variable region sequences can be "mixed and matched" to generate anti-GPRC5D binding molecules of the present application.
  • the application provides variants of antibodies or fragments thereof that bind GPRC5D.
  • the present application thus provides antibodies or fragments thereof having heavy and/or light chain variable regions that are at least 80% identical to the variable region sequences of the heavy or light chains.
  • the amino acid sequence identity of the heavy and/or light chain variable regions is at least 85%, more preferably at least 90%, most preferably at least 95%, especially 96%, more particularly 97%, even more particularly 98% , most particularly 99%, including for example 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%.
  • Variants can use the antibody described in this application as the parent antibody, and pass yeast library screening, phage library screening obtained by selection and point mutation.
  • the present application provides an antibody that recognizes the same epitope as the aforementioned anti-GPRC5D antibody.
  • the present application provides an antibody that competitively binds to GPRC5D with the aforementioned anti-GPRC5D antibody.
  • the application provides an antibody specifically binding to GPRC5D, said antibody being a whole antibody, scFv, single domain antibody, Fab fragment, Fab' fragment, Fv fragment, F(ab') 2 fragment, Fd fragment, dAb fragments, multifunctional antibodies or scFv-Fc antibodies.
  • Anti-GPRC5D antibodies provided herein can be identified, screened for or characterized for their physical/chemical properties and/or biological activity by a variety of assays known in the art. Includes, for example, ELISA, biacore, Western blot and flow cytometry analysis. Suitable assays are described in detail in the Examples.
  • affinity refers to the sum of the forces of non-covalent interactions between a single binding site of a molecule (eg, an antibody) and its binding partner (eg, an antigen).
  • binding affinity refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (eg, antibody and antigen).
  • the affinity of a molecule X for its ligand Y can generally be represented by a dissociation constant (Kd). Affinity can be measured by conventional methods known in the art, including the determination of antibody affinity using Biacore as described herein.
  • the "affinity" of an antibody for GPRC5D is expressed herein as the KD of the antibody.
  • the KD of an antibody refers to the equilibrium dissociation constant of the antibody-antigen interaction. The greater the KD value of an antibody for binding its antigen, the weaker its binding affinity for that particular antigen.
  • EC50 half maximum effect concentration (concentration for 50% of maximal effect, EC50) refers to the concentration that can cause 50% of the maximum effect.
  • antigen refers to a substance recognized and specifically bound by an antigen-binding unit.
  • Antigens may include peptides, proteins, glycoproteins, polysaccharides and lipids, portions thereof and combinations thereof.
  • Non-limiting exemplary antigens include tumor antigens or pathogen antigens.
  • Antigen can also refer to a molecule that elicits an immune response. This immune response may involve antibody production or the activation of specific immunologically-competent cells, or both. Those skilled in the art will appreciate that any macromolecule, including virtually all proteins or peptides, can serve as an antigen.
  • epitope refers to an antigen or part of an antigen that can be recognized by an antibody, B cell, T cell or engineered cell.
  • an epitope can be a tumor epitope or a pathogen epitope recognized by an antibody; an antibody recognizes multiple epitopes within an antigen. Epitopes can also be mutated.
  • antigenic epitope also known as “antigenic epitope” or “epitope” or “antigenic determinant” includes any determinant or region capable of being bound by an antibody.
  • An epitope is a region of an antigen that is bound by an antibody targeting the antigen, comprising specific amino acids that make direct contact with the antibody.
  • the antigenic epitope may consist of a continuous sequence of the GPRC5D protein sequence, or may consist of a discontinuous three-dimensional structure of the GPRC5D protein sequence.
  • the antigen used herein is human or murine GPRC5D.
  • the application also provides immunoconjugates, said immune conjugates comprising the antibodies described herein, and linked thereto Linked functional molecules.
  • the antibody provided in this application has been described above, and the immunoconjugate provided in this application includes all technical solutions thereof.
  • the antibody and the functional molecule can form a conjugate through covalent connection, coupling, attachment, cross-linking and the like.
  • Link or “fusion” are used interchangeably herein. It generally refers to the joining together of two or more chemical elements or modules by any means including chemical conjugation or recombinant methods.
  • In-frame fusion refers to the joining of two or more ORFs to form a contiguous longer ORF in a manner that maintains the correct reading frame of the original open reading frame (ORF).
  • ORF open reading frame
  • the resulting recombinant fusion protein is a single protein containing two or more segments corresponding to the polypeptide encoded by the original ORF (the segments are not normally so linked in nature).
  • the reading frames are thus contiguous throughout the fusion fragments, the fragments may be separated physically or spatially by, for example, in-frame linking sequences (eg, "flexons").
  • Another aspect of the application provides a nucleic acid molecule encoding at least one antibody, functional variant or immunoconjugate thereof of the application.
  • the present application also relates to vectors comprising the above-mentioned appropriate DNA sequences and appropriate promoter or control sequences. These vectors can be used to transform appropriate host cells so that they express the protein.
  • the host cell may be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
  • the present application also provides a chimeric receptor, which generally refers to the expression product of a fusion molecule formed by linking DNA fragments from different sources or corresponding cDNAs of proteins using gene recombination technology, and may include extracellular domains, transmembrane domains, and intracellular domains. area. Wherein the extracellular domain comprises the antibody provided in this application, which has been described in detail above, and the chimeric receptor provided in this application includes all technical solutions thereof.
  • the chimeric receptors include, but are not limited to: chimeric antigen receptor (CAR), chimeric T cell receptor, T cell antigen coupler (TAC).
  • the chimeric receptor of the present application is a chimeric antigen receptor (CAR).
  • CAR chimeric antigen receptor
  • the extracellular antigen-binding region (or extracellular domain) of the CAR is derived from a mouse or humanized or human monoclonal antibody.
  • the chimeric antigen receptor typically comprises an extracellular antigen binding domain or an antibody.
  • the extracellular antigen binding region can be fully human. In other cases, the extracellular antigen binding region can be humanized. In other cases, the extracellular antigen binding region may be of murine origin, or a chimera in said extracellular antigen binding region may consist of amino acid sequences from at least two different animals. In some embodiments, the extracellular antigen binding region may be non-human.
  • the chimeric antigen receptor can also be designed to comprise a variety of antigen-binding regions, including single-chain variable fragments (scFv) derived from antibodies, fragment antigen-binding regions (Fab) selected from libraries , single domain fragments, or natural ligands that engage their cognate receptors.
  • the extracellular antigen binding region may comprise scFv, Fab or natural ligand, and any derivatives thereof.
  • An extracellular antigen binding region can refer to a molecule other than an intact antibody, which can comprise a portion of an intact antibody and can bind to the antigen to which the intact antibody binds.
  • antibody fragments may include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') 2 ; diabodies, linear antibodies; single chain antibody molecules (e.g. scFv); Multispecific Antibodies.
  • Extracellular antigen binding domains such as scFv, Fab or natural ligands, can Is part of the CAR that determines antigen specificity.
  • the extracellular antigen binding domain can bind any complementary target.
  • Extracellular antigen binding regions can be derived from antibodies of known variable region sequences.
  • Extracellular antigen binding regions can be derived from antibody sequences obtained from available mouse hybridomas. Alternatively, extracellular antigen binding regions can be obtained from total excision sequencing of tumor cells or primary cells such as tumor infiltrating lymphocytes (TILs).
  • TILs tumor infiltrating lymphocytes
  • the binding specificity of the extracellular antigen-binding region of the CAR can be determined by complementarity determining regions or CDRs, such as light chain CDRs or heavy chain CDRs. In many cases, binding specificity can be determined by the light and heavy chain CDRs.
  • the extracellular antigen binding region of the CAR includes a hinge or a spacer, and the hinge and spacer can be used interchangeably.
  • the hinge can be considered as part of the CAR used to provide flexibility to the extracellular antigen-binding region.
  • the hinge can be used to detect a CAR on the cell surface of a cell, particularly when antibodies to detect the extracellular antigen binding region are ineffective or unavailable.
  • the hinge may not belong to an immunoglobulin, but to another molecule, such as the native hinge of the CD8 ⁇ molecule.
  • the CD8 ⁇ hinge may contain cysteine and proline residues known to play a role in the interaction of CD8 coreceptors and MHC molecules.
  • the hinge can be adjusted depending on the extracellular antigen binding region used.
  • Hinges can be of any length.
  • the hinge can comprise the amino acid sequence shown in SEQ ID NO:59.
  • the transmembrane domain (or structural region) of CAR can anchor the CAR on the plasma membrane of the cell.
  • the native transmembrane portion of CD28 can be used in CAR. In other cases, it is also possible to use the native transmembrane portion of CD8 ⁇ in the CAR.
  • CD8 may be a protein having at least 85, 90, 95, 96, 97, 98, 99 or 100% identity to NCBI reference number: NP_001759 or a fragment thereof having stimulatory activity.
  • CD8 nucleic acid molecule may be a polynucleotide encoding a CD8 polypeptide. In some cases, the transmembrane region may be the natural transmembrane part of CD28.
  • a fragment is at least 85, 90, 95, 96, 97, 98, 99 or 100% identical to a protein.
  • a "CD28 nucleic acid molecule” may be a polynucleotide encoding a CD28 polypeptide.
  • the transmembrane portion may comprise a CD8 ⁇ region.
  • the transmembrane domain can comprise the amino acid sequence shown in SEQ ID NO:60.
  • the (cellular) signaling region of the CAR may be responsible for activating at least one of the effector functions of an immune response cell comprising said CAR.
  • CAR can induce the effector function of T cells, for example, the effector function is cytolytic activity or auxiliary activity, including the secretion of cytokines, such as IL-2, TNF- ⁇ , ⁇ -IFN, etc.
  • the term intracellular signaling region refers to the portion of a protein that transduces the signal for effector functions and directs the cell to specific functions. While usually the entire intracellular signaling region can be used, in many cases it is not necessary to use the entire chain of signaling domains. In some embodiments, truncated portions of intracellular signaling regions are used. In some embodiments, the term intracellular signaling region is thus intended to include any truncated portion of an intracellular signaling region sufficient to transduce an effector function signal.
  • a preferred example of a signaling domain (or called a structural region) used in a CAR may include the cytoplasmic sequence of the T cell receptor (TCR) and a co-receptor that cooperates to initiate signal transduction following target-receptor binding, As well as any derivative or variant sequences thereof and any synthetic sequences of these sequences having the same functionality.
  • TCR T cell receptor
  • the intracellular signaling region of the CAR may contain a known signaling motif of an immunoreceptor tyrosine activation motif (ITAM).
  • ITAMs containing cytoplasmic signaling sequences include those derived from CD3 ⁇ , FcR ⁇ , Those of FcR ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD5, CD22, CD79a, CD79b and CD66d.
  • the intracellular signaling domain is derived from the CD3 delta chain.
  • CD3 delta domain also known as the T cell receptor CD3 delta chain or CD247.
  • This domain is part of the T cell receptor-CD3 complex and plays an important role in coupling antigen recognition of several intracellular signal transduction pathways with activation of main effectors of T cells.
  • CD3delta primarily refers to human CD3delta and its isoforms, as known from Swissprot entry P20963, including proteins with substantially identical sequences.
  • the full T cell receptor CD3 delta chain is not required and any derivative thereof comprising the signaling domain of the T cell receptor CD3 delta chain is suitable, including any functional equivalents thereof.
  • the signal domain of the CD3 ⁇ chain may comprise the amino acid sequence shown in SEQ ID NO:62.
  • the intracellular signaling domain (or called structural region) of CAR can be selected from any co-stimulatory domain in Table 2.
  • a domain can be modified such that the identity to a reference domain can be from about 50% to about 100%. Any one of the domains of Table 1 may be modified such that the modified form may comprise about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or up to about 100% identity.
  • the intracellular signaling region of the CAR may further comprise one or more co-stimulatory domains.
  • the intracellular signaling domain may comprise a single co-stimulatory domain, such as the delta chain (first generation CAR) or its combination with CD28 or 4-1BB (second generation CAR).
  • the intracellular signaling domain may comprise two co-stimulatory domains, such as CD28/OX40 or CD28/4-1BB (third generation).
  • the co-stimulatory domain of the 4-1BB may comprise the amino acid sequence shown in SEQ ID NO:61.
  • these co-stimulatory domains can generate downstream activation of kinase pathways to support gene transcription and functional cellular responses.
  • the co-stimulatory domain of CAR can activate the CD28 (phosphatidylinositol-4,5-bisphosphate 3-kinase) or 4-1BB/OX40 (TNF-receptor-associated factor adapter protein) pathways and activation of MAPK and Akt Proximal signaling protein.
  • signals generated by CARs may be combined with auxiliary or co-stimulatory signals.
  • chimeric antigen receptor-like complexes can be designed to contain several possible co-stimulatory signaling domains. It is well known in the art that in naive T cells, binding of the T cell receptor alone is not sufficient to induce full activation of the T cell into a cytotoxic T cell. A second co-stimulatory signal is required for full productive T cell activation.
  • T cell activation Several receptors have been reported to provide co-stimulation to T cell activation, including but not limited to CD28, OX40, CD27, CD2, CD5, ICAM-1, LFA-1 (CD11a/CD18), 4-1BBL, MyD88, and 4- 1BB.
  • the signaling pathways used by these co-stimulatory molecules all work synergistically with primary T cell receptor activation signals.
  • the signals provided by these co-stimulatory signaling domains can cooperate with main effector activation signals derived from one or more ITAM motifs (such as CD3zeta signaling domains) and can fulfill the requirements of T cell activation.
  • adding costimulatory domains to chimeric antigen receptor-like complexes can enhance the efficacy and durability of engineered cells.
  • the T cell signaling domain and the co-stimulatory domain are fused to each other to constitute the signaling domain.
  • cells are transduced with a CAR-encoding viral vector.
  • the viral vector is a lentiviral vector.
  • the cells can stably express the CAR.
  • the GPRC5D-binding portion of the CAR is a scFv that retains equivalent affinity binding compared to the Fab antibody from which it is derived, e.g., it binds the same antigen with comparable efficacy.
  • the antibody fragment is functional in that it provides a biochemical response, such as activation of an immune response, inhibition of signaling initiation from its target antigen, inhibition of kinase activity, and the like.
  • the scFv may comprise a sequence as shown in any of SEQ ID NO: 43, 82, 84 or 86.
  • the anti-GPRC5D antigen binding domain of the CAR is a fully human antibody fragment.
  • the CAR of the present application combines the antigen-binding domain of a specific antibody with an intracellular signaling molecule.
  • intracellular signaling molecules include, but are not limited to, CD3 delta chain, 4-1BB and CD28 signaling modules and combinations thereof.
  • the GPRC5D-CAR comprises at least one intracellular signaling domain selected from a CD137(4-1BB) signaling domain, a CD28 signaling domain, a CD3delta signaling domain, and any combination thereof. In one aspect, the GPRC5D-CAR comprises at least one intracellular signaling domain derived from one or more co-stimulatory molecules other than CD137(4-1BB) or CD28.
  • sequence of GPRC5D-CAR can be:
  • SEQ ID NO:43 It has an extracellular domain shown in SEQ ID NO:43, a hinge domain shown in SEQ ID NO:59, a transmembrane domain shown in SEQ ID NO:60, a co-stimulatory signal domain shown in SEQ ID NO:61, and SEQ ID NO: NO: primary signal domain shown in 62 (AB3-BBZ).
  • SEQ ID NO:82 It has an extracellular domain shown in SEQ ID NO:82, a hinge domain shown in SEQ ID NO:59, a transmembrane domain shown in SEQ ID NO:60, a co-stimulatory signal domain shown in SEQ ID NO:61, and SEQ ID NO: Primary signaling domain (AB6-BBZ) shown in 62.
  • SEQ ID NO:84 It has an extracellular domain shown in SEQ ID NO:84, a hinge domain shown in SEQ ID NO:59, a transmembrane domain shown in SEQ ID NO:60, a co-stimulatory signal domain shown in SEQ ID NO:61, and SEQ ID NO: Primary signaling domain (AB7-BBZ) shown in 62.
  • SEQ ID NO: 86 It has an extracellular domain shown in SEQ ID NO: 86, a hinge domain shown in SEQ ID NO: 59, a transmembrane domain shown in SEQ ID NO: 60, a co-stimulatory signal domain shown in SEQ ID NO: 61, and SEQ ID NO: Primary signaling domain (AB8-BBZ) shown in 62.
  • amino acid sequence of the chimeric antigen receptor is shown in any one of SEQ ID NO: 63, 88, 89, or 90.
  • transmembrane domain and intracellular domain of the chimeric antigen receptor above can be replaced by conventional transmembrane domains and intracellular domains by those skilled in the art, and all of them fall within the protection scope of the present application.
  • nucleic acid molecule encoding refers to the sequence or sequence of deoxyribonucleotides along a deoxyribose nucleic acid chain. The order of these deoxyribonucleotides determines the order of amino acids along the polypeptide (protein) chain. Thus, a nucleic acid sequence encodes an amino acid sequence.
  • sequence when used to refer to a nucleotide sequence includes DNA or RNA, and may be single- or double-stranded.
  • target sequence refers to a sequence that is complementary to a guide sequence, and the complementary pairing between the target sequence and the guide sequence promotes the formation of a CRISPR complex.
  • a target sequence can comprise any polynucleotide, such as DNA or RNA polynucleotides.
  • the target sequence is located in the nucleus or cytoplasm of the cell.
  • sequence identity determines the percent identity by comparing two best-matched sequences over a comparison window (e.g., at least 20 positions), wherein portions of the polynucleotide or polypeptide sequence within the comparison window may contain additions or deletions (ie gaps), eg, 20% or less (eg, 5 to 15%, or 10 to 12%) gaps for the two sequences that best match compared to the reference sequence (which contains no additions or deletions).
  • a comparison window e.g., at least 20 positions
  • portions of the polynucleotide or polypeptide sequence within the comparison window may contain additions or deletions (ie gaps), eg, 20% or less (eg, 5 to 15%, or 10 to 12%) gaps for the two sequences that best match compared to the reference sequence (which contains no additions or deletions).
  • Percentages are generally calculated by determining the number of positions where the same nucleic acid base or amino acid residue occurs in the two sequences to yield the number of correctly matched positions, dividing the number of correctly matched positions by the total number of positions in the reference sequence ( ie window size), and multiply the result by 100 to yield the percent sequence identity.
  • transfection refers to the introduction of exogenous nucleic acid into a eukaryotic cell. Transfection can be achieved by various means known in the art, including calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, Liposome fusion, lipofection, protoplast fusion, retroviral infection and biolistics.
  • expression vector refers to a vector comprising a recombinant polynucleotide comprising a An expression control sequence operably linked to the sequence.
  • Expression vectors contain sufficient cis-acting elements for expression; other elements for expression may be provided by the host cell or by an in vitro expression system.
  • Expression vectors include all those known in the art, such as plasmids, viruses (eg, lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses).
  • vector is a composition comprising an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell.
  • vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids and viruses.
  • vector includes autonomously replicating plasmids or viruses.
  • Non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells may also be included, such as polylysine compounds, liposomes, and the like.
  • lentivirus refers to a genus of the Retroviridae family. Retroviruses are unique among retroviruses in their ability to infect non-dividing cells; they can deliver large amounts of genetic information into the host cell's DNA, so they are one of the most efficient methods of gene delivery vectors. HIV, SIV and FIV are all examples of lentiviruses. Vectors derived from lentiviruses provide the means to achieve significant levels of gene transfer in vivo.
  • endogenous means that a nucleic acid molecule or polypeptide etc. is derived from the organism itself.
  • exogenous refers to a function of a nucleic acid molecule or polypeptide, cell, tissue, etc. that is not expressed endogenously in the organism itself, or the expression level is insufficient to achieve overexpression.
  • exogenous protein used herein may be a protein introduced into cells exogenously that recognizes a target antigen, such as an exogenous receptor (ie, the aforementioned “chimeric receptor” herein).
  • host refers to a recipient receiving a transplant, and in some embodiments, may be an individual, such as a human, receiving exogenous cells implanted.
  • isolated means separated from cellular components or other components in which polynucleotides, peptides, polypeptides, proteins, antibodies or fragments thereof are normally associated in their natural state.
  • a non-naturally occurring polynucleotide, peptide, polypeptide, protein, antibody or fragment thereof does not need to be “isolated” to distinguish it from its naturally occurring counterpart.
  • a “concentrated”, “isolated” or “diluted” polynucleotide, peptide, polypeptide, protein, antibody or fragment thereof can be distinguished from its naturally occurring counterpart because the concentration or number of molecules per volume is greater than that of ("concentrated ") or less than ("dilute") the concentration of its naturally occurring counterpart.
  • the degree of enrichment can be measured on an absolute basis, such as weight per solution volume, or can be measured relative to another potential interferent present in the source mixture.
  • the preferred enrichment degree of the technical solution of the present application is higher.
  • 2-fold enrichment is preferred
  • 10-fold enrichment is more preferred
  • 100-fold enrichment is more preferred
  • 1000-fold enrichment is more preferred.
  • An "isolated" substance can also be provided by artificial means of assembly, for example, by chemical synthesis or recombinant expression.
  • the present application provides an isolated nucleic acid encoding an antibody or a fragment thereof that recognizes GPRC5D, a vector, and a host cell comprising the nucleic acid or vector.
  • Nucleic acids can be located in intact cells, in cell lysates, or in partially or substantially purified form.
  • Nucleic acids of the present application can be obtained using standard molecular biology techniques, for example, cDNAs encoding antibody light and heavy chains or encoding VH and VL segments can be obtained by standard PCR amplification or cDNA cloning techniques. For antibodies obtained from immunoglobulin gene libraries (e.g., using phage display technology), one or more genes encoding the antibodies can be recovered from the library. nucleic acid. Methods for introducing exogenous nucleic acid into host cells are generally known in the art and may vary with the host cell used.
  • the nucleic acid molecule of the present application is selected from SEQ ID NO: 24, 28, 32, 36, 40, 77 or 81 encoding the variable region of the heavy chain, and/or selected from the SEQ ID NO encoding the variable region of the light chain :26, 30, 34, 38, 42 or 79.
  • it is a nucleic acid molecule comprising the heavy chain variable region sequence of SEQ ID NO:24, and the light chain variable region sequence comprising SEQ ID NO:26; or comprising SEQ ID NO:28 A heavy chain variable region sequence comprising SEQ ID NO:30, and a light chain variable region sequence comprising SEQ ID NO:30; or a heavy chain variable region sequence comprising SEQ ID NO:32, and a light chain variable region comprising SEQ ID NO:34 or a heavy chain variable region sequence comprising SEQ ID NO:36, and a light chain variable region sequence comprising SEQ ID NO:38; or a heavy chain variable region sequence comprising SEQ ID NO:40, and comprising The light chain variable region sequence of SEQ ID NO:42; or the heavy chain variable region sequence comprising SEQ ID NO:77, and the light chain variable region sequence comprising SEQ ID NO:26; or comprising SEQ ID NO:81 A heavy chain variable region sequence comprising SEQ ID NO:26, and a light chain variable region sequence comprising SEQ
  • one or more vectors comprising the nucleic acids described above are provided.
  • cell refers to a cell of human or non-human animal origin.
  • host cell refers to a cell into which exogenous nucleic acid has been introduced, including the progeny of such cells.
  • Host cells include “transformants” and “transformed cells,” which include transformed primary cells and progeny derived therefrom (regardless of the number of passages).
  • the nucleic acid content of the progeny may not be identical to that of the parental cells and may contain mutations. Mutant progeny having the same function or biological activity as screened or selected for in the originally transformed cell are included herein.
  • GPRC5D-positive host cell refers to a host cell expressing GPRC5D on the cell surface, which can be detected, for example, by flow cytometry using antibodies that specifically recognize epitopes on GPRC5D.
  • the host cells are immune effector cells.
  • immune effector cells refers to cells that participate in the immune response and produce immune effects, such as T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, dendritic cells, CIK cells, macrophages , mast cells, etc.
  • the immune effector cells are T cells, NK cells, NKT cells.
  • the T cells may be autologous T cells, allogeneic T cells, allogeneic T cells.
  • the NK cells may be allogeneic NK cells.
  • immune effector function or immune effector response refers to immune effector cells, eg, functions or responses that enhance or facilitate immune attack of target cells.
  • immune effector function or response refers to the properties of T cells or NK cells that promote killing of target cells or inhibit growth or proliferation.
  • artificially engineered cell with immune effector cell function refers to a cell or cell line that does not have immune effector after being artificially modified or stimulated by a stimulant, the cell acquires immune effector cell function.
  • 293T cells are artificially modified to have the function of immune effector cells; for example, stem cells are induced in vitro to differentiate into immune effector cells.
  • T cells may be pluripotent stem cells derived from bone marrow that differentiate and mature into immunocompetent mature T cells within the thymus.
  • a "T cell” may be a cell population with specific phenotypic characteristics, or a mixed cell population with different phenotypic characteristics, such as a “T cell” may be a cell comprising at least one T cell subset: Memory stem cell-like T cells (stem cell-like memory T cells, Tscm cells), central memory T cells (Tcm), effector T cells (Tef, Teff), regulatory T cells (tregs) and/or effector memory T cells Cell (Tem).
  • T cells may be a specific subtype of T cells, such as ⁇ T cells.
  • T cells can be obtained from many sources, including PBMC, bone marrow, lymph node tissue, cord blood, thymus tissue, and tissue from sites of infection, ascites, pleural effusion, spleen tissue, and tumors.
  • T cells can be obtained from blood collected from an individual using any number of techniques known to those of skill in the art, such as Ficoll(TM) isolation.
  • the cells from the circulating blood of the individual are obtained by apheresis.
  • Apheresis products usually contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets.
  • cells collected by apheresis can be washed to remove plasma molecules and placed in a suitable buffer or culture medium for subsequent processing steps.
  • T cells can be obtained from a healthy donor, or from cells derived from a patient diagnosed with a tumor.
  • peripheral blood mononuclear cell refers to cells with a single nucleus in peripheral blood, including lymphocytes, monocytes and the like.
  • activation and “activation” are used interchangeably and can refer to the process by which a cell transitions from a quiescent state to an active state.
  • the process can include responses to antigenic, phenotypic or genetic changes in migration and/or functional activity status.
  • activation may refer to the process of gradual activation of NK cells and T cells.
  • T cell activation or “T cell activation” refers to the state of a T cell that is sufficiently stimulated to induce detectable cell proliferation, cytokine production, and/or detectable effector function.
  • chemokine is a polypeptide with a molecular weight of 8-10kDa, which is the largest family of cytokines, and its main function is to recruit monocytes, neutrophils, lymphocytes, etc. in the blood into specific lymphoid organs and tissues and the site of infection.
  • Chemokine receptors are seven transmembrane G protein-coupled receptors (GPCRs) that mediate the function of chemokines, and are usually expressed on the cell membranes of immune cells, neutrophils, and endothelial cells. Chemokines and chemokine receptors play an important role in mediating cell migration, proliferation and resisting pathogen invasion, and are closely related to the occurrence and development of inflammation and cancer in the immune environment.
  • safety switch is intended to improve the safety of CAR-T therapy by designing a rapid and reversible "off” or “on” safety switch for CAR-T cells to minimize treatment-related toxicity.
  • CAR-T cell therapy has excellent clinical characteristics, when the tumor load is unpredictable and the activity of T cells is not controlled, it will cause potentially fatal side effects such as severe CRS.
  • severe CRS requires appropriate monitoring using the CRS grading system as a guide and precise regulation using small molecule-based safety switches.
  • a host cell comprising the nucleic acid described above.
  • the host cell comprises (e.g., transduced with): (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) comprising an amino acid sequence encoding an amino acid comprising the VL of the antibody A first vector comprising a nucleic acid of an amino acid sequence, and a second vector comprising a nucleic acid encoding an amino acid sequence comprising an antibody VH.
  • the host cell is eukaryotic, eg, Chinese Hamster Ovary (CHO) cells, 293T cells, NIH3T3 cells, or lymphocytes (eg, YO, NSO, Sp20 cells).
  • the host cell expresses a chimeric receptor described herein.
  • the host cells include T cells, natural killer cells, cytotoxic T lymphocytes, natural killer T cells, DNT cells, regulatory T cells, NK92 cells, and/or stem cell-derived immune effector cells .
  • the T cells are derived from natural T cells and/or T cells induced by pluripotent stem cells; preferably, the T cells are autologous/allogeneic T cells; preferably, The T cells are primary T cells; preferably, the T cells are derived from human autologous T cells.
  • the T cells comprise memory stem cell-like T cells (Tscm cells), central memory T cells (Tcm), effector T cells (Tef), regulatory T cells (Tregs), effector memory T cells cells (Tem), ⁇ T cells, or a combination thereof.
  • the host cell binds cells expressing GPRC5D and does not significantly bind cells that do not express GPRC5D.
  • the host cell also carries coding sequences of exogenous cytokines; and/or also expresses non-targeting GPRC5D chimeric receptors; and/or also expresses chemokines; and/or also expresses expresses a chemokine receptor; and/or also expresses a safety switch.
  • the host cell further carries coding sequences of exogenous cytokines including IL-7, IL-12, IL-15, IL-18, IL-21, or type I interferon.
  • the host cell may also express a chimeric receptor that binds another antigen in addition to the above-mentioned receptor that binds GPRC5D.
  • the host cell also expresses chemokines including CCL19 or CCL21. In another embodiment, the host cell also expresses chemokine receptors including CCR2, CCR4, CCR5, CXCR2, CXCR4, or CXCR5. In another embodiment, the host cell also expresses a safety switch comprising iCaspase-9, Truancated EGFR or RQR8.
  • a method for preparing an anti-GPRC5D antibody comprises culturing a host cell comprising a nucleic acid encoding the antibody under conditions suitable for expressing the antibody as described above, and optionally extracting from the host cell (or host cell culture medium) to recover the antibody.
  • the nucleic acid encoding the antibody of the present application can be integrated into an expression vector.
  • expression vectors are available for protein expression.
  • Expression vectors may include self-replicating extrachromosomal vectors, or vectors that integrate into the host genome.
  • Expression vectors for use in this application include, but are not limited to, those that enable protein expression in mammalian cells, bacteria, insect cells, yeast, and in vitro systems.
  • a variety of expression vectors are available commercially or otherwise, as known in the art. can be used in this application to express antibodies.
  • the host cells are administered in combination with agents that enhance their functions, preferably, chemotherapy drugs; and/or the host cells are administered in combination with agents that improve one or more side effects associated therewith and/or the host cell is administered in combination with a host cell expressing a chimeric antigen receptor targeting other than GPRC5D.
  • Antibodies of the present application immune conjugates containing the antibodies, chimeric receptors, and host cells can be applied to the preparation of pharmaceutical compositions compounds or diagnostic reagents.
  • the composition may also contain a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable means that the molecular entities and compositions do not produce adverse, allergic or other adverse reactions when properly administered to animals or humans.
  • the composition includes another therapeutic agent.
  • the other therapeutic agent is a chemotherapeutic agent, such as those described in US20140271820 and/or a pharmaceutically acceptable salt or analog thereof.
  • the therapeutic agents include, but are not limited to, mitotic inhibitors (vinca alkaloids), including vincristine, vinblastine, vindesine, and novibine(TM) (vinorelbine, 5' - dehydrohydrogen sulfide); topoisomerase I inhibitors, such as camptothecin compounds, including CamptosarTM (irinotecan HCL), HycamtinTM (topotecan HCL) and others derived from camptothecin and its analogs Compounds; podophyllotoxin derivatives such as etoposide, teniposide, and midoxezoz; alkylating agents cisplatin, cyclophosphamide, nitrogen mustard, trimethylenethiophosphoramide, carmustine ,
  • the additional therapeutic agent is selected from one or more of epirubicin, oxaliplatin, and 5-fluorouracil.
  • additional therapeutic agents include, but are not limited to, anti-angiogenic agents, including anti-VEGF antibodies (including humanized and chimeric antibodies, anti-VEGF aptamers and antisense oligonucleotides), and other vascular Occurrence inhibitors, such as angiostatin, endostatin, interferon, interleukin 1 (including alpha and beta) interleukin 12, retinoic acid and tissue inhibitors of metalloproteinase-1 and -2, etc.
  • anti-angiogenic agents including anti-VEGF antibodies (including humanized and chimeric antibodies, anti-VEGF aptamers and antisense oligonucleotides), and other vascular Occurrence inhibitors, such as angiostatin, endostatin, interferon, interleukin 1 (including alpha and beta) interleukin 12, retinoic acid and tissue
  • sugars such as lactose, glucose, and sucrose
  • starches such as corn starch and potato starch
  • cellulose and its derivatives such as carboxymethylcellulose sodium, ethylcellulose, and methylcellulose
  • tragacanth powder malt
  • gelatin talc
  • solid lubricants such as stearic acid and magnesium stearate
  • calcium sulfate vegetable oils such as peanut oil, cottonseed oil, Sesame oil, olive oil, corn oil, and cocoa butter
  • polyols such as propylene glycol, glycerin, sorbitol, mannitol, and polyethylene glycol
  • alginic acid emulsifiers, such as Tween
  • wetting agents such as sodium lauryl sulfate
  • Coloring agents flavoring agents; tableting agents, stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic sa
  • compositions described herein may comprise one or more pharmaceutically acceptable salts.
  • “Pharmaceutically acceptable salt” refers to a salt that retains the desired biological activity of the parent compound and does not produce any adverse toxicological effects (see, e.g., Berge, S.M et al., 1977, J.Pharm.Sci. 66:1-19). Examples of such salts include acid addition salts and base addition salts.
  • Acid addition salts include those derived from nontoxic inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphorous acid, and the like, as well as those derived from nontoxic organic acids such as aliphatic monocarboxylic acids and Salts of dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids, etc.
  • nontoxic inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphorous acid, and the like
  • nontoxic organic acids such as aliphatic monocarboxylic acids and Salts of dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids, etc
  • Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium, and the like, as well as those derived from nontoxic organic amines, such as N,N'-dibenzylethylenediamine, N-methylglucose, Sugar amine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine, etc.
  • antioxidants include, but are not limited to: water-soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, ⁇ -tocopherol, etc.; and metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid etc.
  • water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.
  • oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin,
  • composition of the present application can be made into various dosage forms according to needs, and can be administered by the doctor according to the patient's type, age, body weight and general disease condition, administration method and other factors to determine the dosage beneficial to the patient.
  • the mode of administration can be, for example, parenteral administration (such as injection) or other therapeutic modes.
  • parenteral administration of immunogenic compositions includes, for example, subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.) or intrasternal injection or infusion techniques.
  • compositions may be isotonic, ie, they may have the same osmotic pressure as blood and tear fluid.
  • the desired isotonicity of the compositions of the present application can be achieved using sodium chloride or other pharmaceutically acceptable agents such as dextrose, boric acid, sodium tartrate, propylene glycol or other inorganic or organic solutes.
  • the viscosity of the composition can be maintained at a selected level using a pharmaceutically acceptable thickener, if desired.
  • Suitable thickeners include, for example, methylcellulose, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, carbomer, and the like.
  • the preferred concentration of thickener will depend on the agent chosen. Obviously, the choice of suitable carriers and other additives will depend on the exact route of administration and the nature of the particular dosage form, eg a liquid dosage form.
  • kits comprising the antibodies, immunoconjugates, chimeric receptors, nucleic acids or host cells described herein.
  • a kit can include a therapeutic or prophylactic composition comprising an effective amount of an antibody, chimeric receptor, nucleic acid, or host cell described herein comprising one or more unit dosage forms.
  • the kit comprises a sterile container that may contain a therapeutic or prophylactic composition; such container may be a box, ampoule, bottle, vial, tube, bag, blister pack, or other suitable container known in the art. container form.
  • Such containers may be made of plastic, glass, laminated paper, foil, or other materials suitable for holding medications.
  • the kit comprises an antibody, immune conjugate, chimeric receptor, nucleic acid or host cell described herein, and an antibody, immune conjugate, chimeric receptor, Instructions for administering the nucleic acid or host cell to an individual.
  • the instructions generally include methods of treating or preventing cancer or tumors using the antibodies, immunoconjugates, chimeric receptors, nucleic acids or host cells described herein.
  • the kit comprises a host cell described herein, and can include from about 1 x 104 cells to about 1 x 106 cells.
  • the kit can include at least about 1 x 10 5 cells, at least about 1 x 10 6 cells, at least about 1 x 10 7 cells, at least about 4 x 10 7 cells, at least about 5 x 107 cells, at least about 6 x 107 cells, at least about 6 x 107 cells, 8 x 107 cells, at least about 9 x 107 cells, at least about 1 x 108 cells, at least about 2 x 108 cells, at least about 3 x 108 cells, at least about 4 x 108 cells, at least about 5 x 108 cells, at least about 6 x 108 cells, at least about 6 x 108 cells , at least about 8 x 10 8 cells, at least about 9 x 10 8 cells, at least about 1 x 10 9 cells, at least about 2 x 10 9 cells, at least about 3 x 10 9 cells, at least about 4 x 10 9 cells, at least about 5 x 109 cells, at least about 6 x 109 cells, at least about 8 x 109
  • the kit can include allogeneic cells.
  • a kit can include a cell that can contain a genomic modification.
  • a kit may comprise "ready-to-use" cells.
  • a kit can include cells that can be expanded for clinical use. In some cases, kits may contain contents intended for research purposes.
  • the instructions include at least one of: a description of the therapeutic agent; dosage regimen and administration for the treatment or prevention of tumors or symptoms thereof; precautions, warnings, contraindications, overdose information, adverse reactions, animal Pharmacology, clinical research, and/or literature citations. Instructions may be printed directly on the container (if present), or as a label on the container, or as a separate sheet, booklet, card or folder provided within or in the container. In some embodiments, the instructions provide methods of administering the antibodies described herein for treating or preventing tumors. In certain instances, the instructions provide methods of administering an antibody of the present application before, after, or concurrently with administration of a chemotherapeutic agent.
  • module refers to positive or negative changes. Examples of adjustments include 1%, 2%, 10%, 25%, 50%, 75%, or 100% changes. In a specific embodiment, refers to a negative change.
  • treatment refers to interventions in an attempt to modify the disease process, either prophylaxis or intervention in the clinicopathological process.
  • Therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of the disease, relieving symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing down the progression of the disease, improving or relieving the disease, remission or improving the prognosis, etc.
  • prevention refers to interventions that are attempted in advance of a disease such as rejection of a cell transplant.
  • tumor antigen refers to an antigen emerging or overexpressed during the onset, progression of a hyperproliferative disease.
  • the hyperproliferative disorder of the present application refers to a tumor.
  • the tumor antigens described in this application may be solid tumor antigens or blood tumor antigens.
  • the tumor antigens of the present application include, but are not limited to: thyroid-stimulating hormone receptor (TSHR); CD171; CS-1; C-type lectin-like molecule-1; ganglioside GD3; Tn antigen; CD19; CD20; CD 22; CD 30; CD 70; CD 123; CD 138; CD33; CD44; CD44v7/8; CD38; CD44v6; B7H3(CD276), B7H6; KIT(CD117); 11 receptor alpha (IL-11R ⁇ ); prostate stem cell antigen (PSCA); prostate-specific membrane antigen (PSMA); carcinoembryonic antigen (CEA); NY-ESO-1; HIV-1 Gag; MART-1; gp100; Tyrosinase; Mesothelin; EpCAM; Protease Serine 21 (PRSS21); Vascular Endothelial Growth Factor Receptor, Vascular Endothelial growth factor receptor 2 (VEGFR2); Lewis (Y) antigen; CD24;
  • Pathogen antigens are selected from: antigens of viruses, bacteria, fungi, protozoa, or parasites; virus antigens are selected from: cytomegalovirus antigens, Epstein-Barr virus antigens, human immunodeficiency virus antigens, or influenza virus antigens.
  • subject refers to any animal, such as a mammal or a marsupial.
  • Subjects of the present application include, but are not limited to, humans, non-human primates (such as rhesus monkeys or other types of macaques), mice, pigs, horses, donkeys, cows, sheep, rats, and poultry of any kind.
  • the term "effective amount” refers to an amount that provides a therapeutic or prophylactic benefit.
  • anti-GPRC5D antibodies any of the anti-GPRC5D antibodies, immunoconjugates, chimeric receptor-modified host cells, pharmaceutical compositions or kits provided herein can be used in methods of treatment.
  • any anti-GPRC5D antibody, immunoconjugate, chimeric receptor modified host cell, pharmaceutical composition or kit for use as a medicament is provided.
  • any anti-GPRC5D antibody, immunoconjugate, chimeric receptor modified host cell, pharmaceutical composition or kit for use in treating a disease is provided.
  • any anti-GPRC5D antibody, immunoconjugate, chimeric receptor-modified host cell, pharmaceutical composition, or kit for use in a method of treatment is provided.
  • the application provides any anti-GPRC5D antibody, immunoconjugate, chimeric receptor-modified host cell, pharmaceutical composition, or kit for use in a method of treating an individual suffering from a disease, so
  • the methods include administering to the individual an effective amount of any anti-GPRC5D antibody, immunoconjugate, chimeric receptor-modified host cell, pharmaceutical composition, or kit.
  • the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent.
  • the "individual" is preferably a human.
  • the present application provides the use of any anti-GPRC5D antibody, immunoconjugate, chimeric receptor-modified host cell, pharmaceutical composition or kit in the preparation or preparation of medicine.
  • the medicament is used to treat a disease.
  • the medicament is for use in a method of treating a disease comprising administering an effective amount of the medicament to a diseased individual.
  • the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent.
  • the "individual" is preferably a human.
  • the present application provides methods for treating a disease.
  • the method comprises administering to an individual having a disease expressing GPRC5D an effective amount of any anti-GPRC5D antibody, immunoconjugate, chimeric receptor modified host cell, pharmaceutical composition or kit.
  • the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent.
  • the "individual" is preferably a human.
  • the present application provides, for example, a medicament comprising any anti-GPRC5D antibody, immunoconjugate, chimeric receptor modified host cell, pharmaceutical composition or kit provided herein for use in any of the above-mentioned treatment methods preparation.
  • the pharmaceutical preparation comprises any anti-GPRC5D antibody, immunoconjugate, chimeric receptor-modified host cell, pharmaceutical composition or kit and pharmaceutical carrier provided herein.
  • the pharmaceutical formulation comprises any of the anti-GPRC5D antibodies, immunoconjugates, chimeric receptor modified host cells, pharmaceutical compositions or kits provided herein and at least one additional therapeutic agent .
  • the pharmaceutical formulation is used to treat a disease.
  • the pharmaceutical formulation is administered to a diseased individual.
  • An "individual" according to any of the above embodiments is preferably a human.
  • the present application provides a method for preparing a drug or a pharmaceutical preparation, the method comprising using any of the anti-GPRC5D antibodies, immunoconjugates, chimeric receptor modified host cells, and pharmaceutical compositions provided herein or reagent
  • the cartridge is in admixture with a pharmaceutically acceptable carrier, eg, for use in any of the methods of treatment described above.
  • the method for preparing a medicament or pharmaceutical formulation further comprises adding at least one additional therapeutic agent to the medicament or pharmaceutical formulation.
  • any of the anti-GPRC5D antibodies, immunoconjugates, chimeric receptor-modified host cells, pharmaceutical compositions or kits of the present application can be used alone or in combination with other agents for therapy.
  • any of the anti-GPRC5D antibodies, immunoconjugates, chimeric receptor-modified host cells, pharmaceutical compositions or kits of the application can be co-administered with at least one additional therapeutic agent.
  • the above-mentioned combination therapy includes combined administration (wherein two or more therapeutic agents are contained in the same or separate preparations) and separate administration, in this case, any anti-GPRC5D antibody, immunoconjugate, Administration of the chimeric receptor-modified host cell, pharmaceutical composition or kit can occur before, concurrently with, and/or after administration of the additional therapeutic agent or agents.
  • administration of any anti-GPRC5D antibody, immunoconjugate, chimeric receptor-modified host cell, pharmaceutical composition or kit of the application and administration of the additional therapeutic agent occur within about one month of each other within, or within about one, two, or three weeks, or within about one, two, three, four, five, or six days.
  • Any anti-GPRC5D antibody, immunoconjugate, chimeric receptor-modified host cell, pharmaceutical composition or kit (and any additional therapeutic agent) of the application may be administered by any suitable means, including parenteral administration, Intrapulmonary or intranasal administration, and, if indicated for treatment, intralesional administration.
  • Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Administration may be by any suitable route, for example, by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is transient or chronic.
  • a variety of dosing regimens are contemplated herein, including, but not limited to, a single administration or multiple administrations at multiple time points, bolus administration, and pulse infusion.
  • a formulation comprising a population of immunoreactive cells administered to an individual comprises a plurality of immunoreactive cells effective to treat and/or prevent a particular indication or disease.
  • a therapeutically effective population of immunoreactive cells can be administered to an individual.
  • a formulation comprising about 1 x 104 to about 1 x 1010 immunoreactive cells is administered.
  • the formulation will contain from about 1 x 105 to about 1 x 109 immunoreactive cells, from about 5 x 105 to about 5 x 108 immunoreactive cells, or from about 1 x 106 to Approximately 1 x 10 7 immunoreactive cells.
  • the number of CAR immunoreactive cells administered to an individual will vary between wide ranges. Your doctor will ultimately determine the proper dosage to use.
  • chimeric receptors are used to stimulate host cell-mediated immune responses.
  • a T cell-mediated immune response is an immune response that involves the activation of T cells.
  • Activated antigen-specific cytotoxic T cells are capable of inducing apoptosis in target cells displaying foreign antigen epitopes on their surface, such as cancer cells displaying tumor antigens.
  • chimeric antigen receptors are used to provide anti-tumor immunity in mammals. Due to the T cell-mediated immune response, the subject will develop anti-tumor immunity.
  • methods of treating a subject with a tumor may involve administering to a subject in need of treatment one or more host cells described herein.
  • the host cells can bind tumor target molecules and induce cancer cell death.
  • the present application also provides a method of treating a pathogenic infection in an individual, comprising administering to the individual a therapeutically effective amount of the subject of the present application.
  • the frequency of administration of the immunoreactive cells of the present application will depend on factors including the disease being treated, the elements of the particular immunoreactive cells and the mode of administration. For example, it can be administered 4 times, 3 times, 2 times a day or once a day, every other day, every three days, every four days, every five days, once every six days, once a week, once every eight days, once every Doses are given once every nine days, every ten days, once a week, or twice a month.
  • the immune response cells of the present application not only can they be administered in lower therapeutically effective amounts than similar immune response cells that do not express exogenous type I interferon, but they can also Dosing is less frequent to obtain at least similar, and preferably more pronounced, therapeutic effects.
  • This application provides a fully human antibody that specifically recognizes GPRC5D, and the CAR T cells prepared from this antibody show a good killing effect on target cells both in vivo and in vitro.
  • Human GPRC5D (the amino acid sequence is shown in SEQ ID NO: 65) was transfected into CHOK1 (ATCC), 293T (ATCC), and NIH3T3 cells (ATCC) that did not express endogenous GPRC5D through conventional molecular biology techniques, respectively. Positive single clones were selected by limiting dilution method, and CHOK1-huGPRC5D, 293T-huGPRC5D, NIH3T3-huGPRC5D stably transfected cell lines were constructed.
  • Mouse GPRC5D (amino acid sequence shown in SEQ ID NO: 66) was transfected into CHOK1 cells by lentivirus using conventional molecular biology techniques, and positive clones were selected by limiting dilution to construct a CHOK1-muGPRC5D stably transfected cell line.
  • the anti-GPRC5D hybridoma antibody was prepared by conventional hybridoma antibody preparation techniques in the art.
  • the full-length gene encoding human GPRC5D (SEQ ID No: 65) was constructed in the eukaryotic expression pCAGGS plasmid to form the eukaryotic expression plasmid pCAGGS-GPRC5D. Then, Balb/c mice (Victoria, female, 6-8 weeks old) were immunized four times with plasmid pCAGGS-GPRC5D and NIH3T3-huGPRC5D cells.
  • plasmid pCAGGS-GPRC5D diluted in normal saline, total volume 2 mL
  • 2 ⁇ 10 7 NIH3T3-huGPRC5D cells were used for intraperitoneal booster immunization
  • the mouse spleen was taken and fused according to the published standard protocol (Kohler, Milstein, 1975). After specific screening by HAT and flow cytometry, two hybridoma antibodies mIgG:AB1 (IgG2b-k) binding to human GPRC5D were obtained. , AB2 (IgG3-k).
  • Example 3 Determination of EC50 binding of anti-GPRC5D hybridoma antibody to CHOK1-huGPRC5D cells
  • CHOK1 cells (as negative control cells), CHOK1-huGPRC5D cells, and CHOK1-muGPRC5D cells were respectively taken, and the cross-reactivity of AB1 and AB2 with human GPRC5D and mouse GPRC5D was determined according to the method in Example 3.
  • RNA was extracted from hybridoma cells AB1 and AB2 using conventional molecular biology techniques, and cDNA was synthesized by reverse transcription.
  • UPM mIgG2b-outer (SEQ ID NO: 67), mIgG3-outer (SEQ ID NO: 68), mK-outer (SEQ ID NO: 69) primers were used for PCR, cloned and sequenced to obtain the AB1-VH gene sequence ( SEQ ID NO: 36), AB1-VL gene sequence (SEQ ID NO: 38), AB2-VH gene sequence (SEQ ID NO: 40), AB2-VL gene sequence (SEQ ID NO: 42).
  • the amino acid sequence of HCDR1 of AB1 is shown in SEQ ID NO:11
  • the amino acid sequence of HCDR2 is shown in SEQ ID NO:12
  • the amino acid sequence of HCDR3 is shown in SEQ ID NO:13
  • the amino acid sequence of LCDR1 is shown in SEQ ID NO: 14
  • the amino acid sequence of LCDR2 is shown in SEQ ID NO:15
  • the amino acid sequence of LCDR3 is shown in SEQ ID NO:16.
  • the amino acid sequence of the heavy chain variable region of AB1 is shown in SEQ ID NO:35
  • the amino acid sequence of the light chain variable region is shown in SEQ ID NO:37.
  • the amino acid sequence of HCDR1 of AB2 is shown in SEQ ID NO:17
  • the amino acid sequence of HCDR2 is shown in SEQ ID NO:18
  • the amino acid sequence of HCDR3 is shown in SEQ ID NO:19
  • the amino acid sequence of LCDR1 is shown in SEQ ID NO:
  • the amino acid sequence of LCDR2 is shown in SEQ ID NO:21
  • the amino acid sequence of LCDR3 is shown in SEQ ID NO:22.
  • the amino acid sequence of the heavy chain variable region of AB2 is shown in SEQ ID NO:39
  • the amino acid sequence of the light chain variable region is shown in SEQ ID NO:41.
  • Example 6 Determination of the binding activity of recombinant hybridoma scFv-mFc antibody to cell lines expressing GPRC5D
  • the heavy chain and light chain gene sequences of AB1 and AB2 antibodies were respectively cloned into eukaryotic expression vectors containing mFc (SEQ ID NO: 64), and V152S-AB1-mFc and V152S-AB2-mFc plasmids were constructed. Then, 293F cells (Thermo Company) were transfected and cultured for 7 days, and purified by affinity chromatography to obtain anti-GPRC5D recombinant antibodies AB1 (scFv-mFc) and AB2 (scFv-mFc).
  • CHOK1-huGPRC5D, 293T-huGPRC5D, NIH3T3-huGPRC5D, and CHOK1 cells were taken respectively, and the recombinant antibodies AB1 (scFv-mFc) and AB2 (scFv-mFc) and AB2 (scFv-mFc) expressed human GPRC5D were determined according to the method in Example 3. Binding activity of cell lines.
  • the phage display library used in this application is a phage library constructed by our company with a capacity of 1E+11.
  • human GPRC5D antigen Kaijia Biology
  • the Fab fragments that specifically bind to human GPRC5D were obtained through screening methods known to those skilled in the art, and finally three antibodies specifically binding to human GPRC5D were obtained, named AB3, AB4, and AB5.
  • the amino acid sequence of HCDR1 of AB3 is shown in SEQ ID NO:1, the amino acid sequence of HCDR2 is shown in SEQ ID NO:2, the amino acid sequence of HCDR3 is shown in SEQ ID NO:3, and the amino acid sequence of LCDR1 is shown in SEQ ID NO: 4, the amino acid sequence of LCDR2 is shown in SEQ ID NO:5, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:6.
  • the amino acid sequence of the heavy chain variable region of AB3 is shown in SEQ ID NO:23, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:25.
  • the amino acid sequence of the heavy chain of AB3 is shown in SEQ ID NO: 45, and the amino acid sequence of the light chain is shown in SEQ ID NO: 47.
  • the amino acid sequence of HCDR1 of AB4 is shown in SEQ ID NO:1, the amino acid sequence of HCDR2 is shown in SEQ ID NO:2, the amino acid sequence of HCDR3 is shown in SEQ ID NO:7, and the amino acid sequence of LCDR1 is shown in SEQ ID NO: 4, the amino acid sequence of LCDR2 is shown in SEQ ID NO:5, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:8.
  • the amino acid sequence of the heavy chain variable region of AB4 is shown in SEQ ID NO:27, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:29.
  • the amino acid sequence of the AB4 heavy chain is shown in SEQ ID NO: 49, and the amino acid sequence of the light chain is shown in SEQ ID NO: 51.
  • the amino acid sequence of HCDR1 of AB5 is shown in SEQ ID NO:1, the amino acid sequence of HCDR2 is shown in SEQ ID NO:2, the amino acid sequence of HCDR3 is shown in SEQ ID NO:9, and the amino acid sequence of LCDR1 is shown in SEQ ID NO: 4, the amino acid sequence of LCDR2 is shown in SEQ ID NO:5, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:10.
  • the amino acid sequence of the heavy chain variable region of AB5 is shown in SEQ ID NO: 31, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 33.
  • the amino acid sequence of the AB5 heavy chain is shown in SEQ ID NO: 53, and the amino acid sequence of the light chain is shown in SEQ ID NO: 55.
  • Example 8 Using ELISA to check the EC50 of anti-GPRC5D-antibody binding to antigen
  • the clones producing antibodies AB3, AB4, and AB5 in Example 7 were respectively subjected to prokaryotic expression and purification through nickel columns to obtain antibodies AB3, AB4, and AB5. Then the antibody was diluted 3-fold (8 gradients) from a concentration of 10 ⁇ g/ml, incubated with human GPRC5D antigen (coating concentration: 2 ⁇ g/ml) for 1 h at room temperature, and then passed through the secondary antibody anti-Flag-HRP (Sigma, 1: 4000 dilutions) were incubated at room temperature for 1 h to detect the bound antibody; then TMB was added for color development and the OD450 value was read with a microplate reader. Using the GraphPad Prism5 software, the primary antibody concentration was used as the abscissa, and the OD450 value was used as the ordinate to perform four-parameter fitting to calculate the EC50 value.
  • Antibodies AB3, AB4, and AB5 all significantly combined with human GPRC5D antigen, with EC50 values of 0.07382 ⁇ g/ml, 0.1424 ⁇ g/ml and 0.1396 ⁇ g/ml, respectively.
  • Human lymphoma cells Daudi (Cell Bank, Chinese Academy of Sciences) are cells that do not express endogenous GPRC5D; multiple myeloma cells MM.1S (Cell Bank, Chinese Academy of Sciences), NCI-H929 (ATCC) are cells that endogenously express GPRC5D .
  • the AB3, AB4 and AB5 antibodies (10 ⁇ g/ml) described in Example 7 were co-incubated with CHOK1, CHOK1-huGPRC5D, 293T, 293T-huGPRC5D, MM.1S, NCI-H929, and Daudi cells (no antibody was added to the blank group) , ie NA), the antibody bound to the cells was fluorescently labeled with the secondary antibody anti-F(ab') 2 -488 (Jackson ImmunoResearch, used at 1:200 dilution), and then the fluorescence intensity was detected by flow cytometry, the experimental data The mean fluorescence intensity (MFI) was calculated with FlowJo analysis software.
  • MFI mean fluorescence intensity
  • AB3, AB4 and AB5 antibodies all significantly bind to cells expressing human GPRC5D (CHOK1-huGPRC5D, 293T-huGPRC5D, MM.1S and NCI-H929), but do not bind to cells that do not express GPRC5D (CHOK1 , 293T and Daudi).
  • CHOK1-huGPRC5D cells and MM.1S cells were counted and laid on U-shaped bottom plates respectively, with about 2 ⁇ 105 cells per well, and then the primary antibody (AB3, AB4 or AB5 antibody described in Example 7: starting from 30 ⁇ g/mL Initially, 3-fold serial dilution of 8 gradients) and secondary antibody (Anti-Fab-FITC: 1:200, Jackson ImmunoResearch) were incubated, followed by fluorescence intensity detection with a flow cytometer.
  • the experimental data was calculated with the FlowJo analysis software and the mean fluorescence intensity (MFI), and then the four-parameter fitting was performed using the GraphPad Prism5 software with the primary antibody concentration as the abscissa and the calibrated mean fluorescence intensity (MFI) as the ordinate to calculate the EC50 value.
  • the AB3, AB4 and AB5 antibodies (5 ⁇ g/ml) described in Example 7 were co-incubated with CHOK1-muGPRC5D cells (the blank group did not add antibodies, ie NA), and the secondary antibody anti-F (ab') 2-488 (Jackson ImmunoResearch (1:200 dilution) was used for fluorescent labeling, and then the fluorescence intensity was detected by a flow cytometer, and the experimental data was calculated with the FlowJo analysis software for mean fluorescence intensity (MFI).
  • MFI mean fluorescence intensity
  • test results are shown in Figure 10, the AB3 antibody can bind to mouse GPRC5D.
  • PRRLSIN-cPPT.EF-1 ⁇ as a carrier, construct a lentiviral plasmid expressing the second generation chimeric antigen receptor of AB3 scFv (SEQ ID NO: 43) and control antibody 18 scFv (SEQ ID NO: 57, sequence from patent CN107428829A) . It contains CD8 ⁇ signal peptide (SEQ ID NO: 58), CD8 hinge region (SEQ ID NO: 59) and transmembrane region (SEQ ID NO: 60), CD137 intracellular signaling domain (SEQ ID NO: 61) and CD3 ⁇ (SEQ ID NO: 62); the amino acid sequence of AB3-CAR is shown in SEQ ID NO: 63.
  • the calcium phosphate method was used to package the lentivirus, and the viral supernatant was purified with PEG8000/NaCl, and then infected with T cells activated by CD3/CD28 magnetic beads for 48 hours to obtain AB3-BBZ CAR-T cells expressing AB3-BBZ , 18-BBZ CAR-T cells expressing 18-BBZ, T cells not transfected with virus were regarded as UTD.
  • the CAR positive rate was detected by FACS method.
  • the primary antibody was Biotin-anti-F(ab') 2 -488 (Jackson ImmunoResearch, used at 1:100), and the secondary antibody was SA-PE (eBioscience, 1:100). 200 used).
  • Example 13 In vitro killing experiment of GPRC5D CAR-T cells on target cells
  • AB3-BBZ CAR-T has no killing effect on 293T cells; however, it has obvious killing effects on 293T-huGPRC5D and MM.1S cells expressing GPRC5D (killing rate at 3:1 up to about 60%), and the killing effect is positively correlated with the effect-to-target ratio.
  • the results showed that AB3-BBZ CAR-T had a specific in vitro killing effect on cells expressing GPRC5D.
  • Example 14 In vivo anti-tumor ability of GPRC5D CAR-T cells
  • 3 ⁇ 10 6 human multiple myeloma cells MM.1S were inoculated subcutaneously in the right axillary of NPG female mice, and the mice were divided into 3 groups (AB3-BBZ CAR T group, 18-BBZ CAR T group, UTD group). Group of 8. The day of tumor cell inoculation was recorded as D0. On the 16th day after inoculation, the average tumor volume was about 200 mm 3 , and AB3-BBZ CAR T, 18-BBZ CAR T, and UTD cells were injected into the tail vein respectively, and the injection dose was 3 ⁇ 10 6 cells/ mice. On day 20 after CAR T cell injection (the day after tumor inoculation 36 days), the tumor volume of mice in the UTD group exceeded 2000 mm 3 , and the experiment was terminated.
  • the light chain or heavy chain sequence of AB3 was randomly mutated, a phage library was constructed, and AB6, AB7, and AB8 antibodies were obtained by screening with human GPRC5D antigen (Kaijia Biology). ELISA detection showed that AB6, AB7, and AB8 had significant binding to human GPRC5D ( Figure 14).
  • the 3 clones selected in Example 15 were prokaryotically expressed and affinity-purified using a nickel column to obtain purified single-chain antibodies (scFv) (such as SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO: 86), the C-terminus of the single-chain antibody contains a his tag and a flag tag.
  • scFv purified single-chain antibodies
  • CHO-K1-huGPRC5D and CHO-K1-muGPRC5D stably transfected cell lines were constructed according to the method of Example 1.
  • the above purified scFv (10ug/ml) was co-incubated with CHO-K1-huGPRC5D and CHO-K1-muGPRC5D cells and untransfected CHO-K1 cells, using the secondary antibody anti-Flag-488 (R&D, diluted 1:200) ) to fluorescently label the scFv bound to cells, and then detect the fluorescence intensity by a flow cytometer, and calculate the mean fluorescence intensity (MFI) of the experimental data with FlowJo analysis software. The detection results are shown in Figure 15.
  • the binding ability of AB6, AB7 and AB8 to cell lines expressing human GPRC5D and mouse GPRC5D was significantly higher than that of AB3, and they did not bind to CHO-K1 cells not expressing GPRC5D. It shows that antibodies AB6, AB7 and AB8 have good species binding properties and cell binding specificity.
  • the EC50 values of the antibodies described in Example 15 were determined according to the method of Example 10, the primary antibodies AB3 (Fab), AB6 (scFv ), AB7 (scFv) and AB8 (scFv): 30 ⁇ g/mL initial 3-fold serial dilution 12 gradients and secondary antibody (Anti-Flag-488:1:200, R&D) incubation, the results are shown in Figure 16, based on The modified antibodies of AB3 all have good cell binding activity, among which AB6 and AB7 had lower EC50s than AB3 on both cells.
  • lentiviral plasmids expressing second-generation chimeric antigen receptors of AB6, AB7, and AB8 were constructed.
  • the amino acid sequence of AB6-BBZ is shown in SEQ ID NO: 88
  • the amino acid sequence of AB7-BBZ is shown in SEQ ID NO: 89
  • the amino acid sequence of AB8-BBZ is shown in SEQ ID NO: 90.
  • AB3-BBZ CAR-T cells AB6-BBZ CAR-T cells, AB7-BBZ CAR-T cells, AB8-BBZ CAR-T cells, and AB8-BBZ CAR-T cells were prepared according to the method in Example 12.
  • T cells, T cells not transfected with virus are UTD.
  • the cell killing experiment was carried out according to the method of Example 13.
  • AB3 CAR-T, AB6 CAR-T, AB7 CAR-T, and AB8 CAR-T had no killing effect on 293T cells that did not express GPRC5D; and For MM.1S expressing GPRC5D, 293T-huGPRC5D cells showed obvious killing effect, and the killing effect was positively correlated with the effect-to-target ratio. It shows that AB3 CAR-T, AB6 CAR-T, AB7 CAR-T and AB8 CAR-T have specific killing effect on cells expressing human GPRC5D.

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Abstract

本申请涉及结合GPRC5D的抗体及其应用。还涉及包含本申请涉及的结合GPRC5D的抗体的细胞、药物组合物、联合用药，以及制备抗体的方法。

Description

GPRC5D抗体及其应用
相关申请
本专利申请要求于2022年1月29日递交的申请号为202210111504.5的中国专利申请的优先权，要求2022年8月17日递交的申请号为202210985594.0的中国专利申请的优先权。
同时提交的序列表文件
下列XML文件的全部内容通过整体引用并入本文：计算机可读格式(CRF)的序列表(名称：FF00719PCT-sequence listing.xml，日期：20230122，大小：95.6KB)。
技术领域
本申请涉及肿瘤免疫治疗或诊断领域，更具体地，涉及特异性结合GPRC5D抗体及其应用。
背景技术
目前临床上多发性骨髓瘤的靶向治疗方法主要包括免疫调节药物(沙利度胺、来那度胺、泊马度胺)、蛋白酶体抑制剂(硼替佐米、卡非佐米、伊沙佐米)及抗CD38单抗等。尽管前线疗法已取得了良好的疗效，但末线疗效十分有限。靶向BCMA的CAR-T细胞疗法在临床试验中展示了很好的疗效，但对于BCMA低表达或经BCMA CAR-T治疗后复发的病例，亟需开发新的治疗手段。有研究表明65％的多发性骨髓瘤患者表达GPRC5D，因此开发靶向GPRC5D的治疗路径，临床价值凸显。由于GPRC5D是一个较新的靶点，而且作为多次跨膜受体，其抗体的开发具有一定技术门槛。因此本申请开发的GPRC5D抗体，尤其是全人源GPRC5D抗体具有较高应用价值。
发明内容
本申请目的在于提供特异性识别GPRC5D的抗体。本申请还涉及制备抗GPRC5D特异性抗体的方法，包括噬菌体展示技术和杂交瘤技术。本申请还涉及抗GPRC5D抗体(包括但不限于Fab，scFv,scFv-Fc形式)的特性及特异性的研究。本申请提供了靶向GPRC5D的嵌合抗原受体(CAR)及其制备方法。本申请还提供分离的编码本申请的抗GPRC5D抗体和靶向GPRC5D的嵌合抗原受体的核酸。本申请还提供宿主细胞，所述宿主细胞包含本申请的核酸。所述方法还包括培养本申请的宿主细胞以制备所述抗体或所述CAR。本申请的抗体和/或CAR用于治疗肿瘤或肿瘤诊断。
在第一方面，本申请提供了识别GPRC5D的抗体，所述抗体选自如下任意组：
(1)抗体，其包含重链可变区，所述重链可变区包含SEQ ID NO：1、11、17、70或71任一所示的HCDR1，和/或包含SEQ ID NO：2、12、18、72或73任一所示的HCDR2，和/或包含SEQ ID NO：3、7、9、13或19任一所示的HCDR3；
(2)抗体，其包含轻链可变区，所述轻链可变区包含SEQ ID NO：4、14、20或74任一所 示的LCDR1，和/或包含SEQ ID NO：5、15、21或75任一所示的LCDR2，和/或包含SEQ ID NO：6、8、10、16或22任一所示的LCDR3；
(3)抗体，包含(1)所述抗体的重链可变区及(2)所述抗体的轻链可变区；
(4)抗体，(1)～(3)中任一项所述的抗体的变体，且具备与(1)～(3)中任一项所述的抗体相同或相似的活性。
在某些实施方式中，其包含重链可变区中的至少一个CDR，所述重链可变区包含如SEQ ID NO:23、27、31、35、39、76或80中任一项所示的氨基酸序列或其变体，或包含与上述任一序列具有至少80％相似性的氨基酸序列；和/或其包含轻链可变区中的至少一个CDR，所述轻链可变区包含如SEQ ID NO:25、29、33、37、41或78中任一项所示的氨基酸序列或其变体，或包含与上述任一序列具有至少80％相似性的氨基酸序列。
在某些实施方式中，所述抗体包含重链可变区中的HCDR1、HCDR2、HCDR3，所述重链可变区包含如SEQ ID NO:23、27、31、35、39、76或80中任一项所示的氨基酸序列或其变体，或包含与上述任一序列具有至少80％相似性的氨基酸序列；和/或其包含轻链可变区中的LCDR1、LCDR2、LCDR3，所述轻链可变区包含如SEQ ID NO:25、29、33、37、41或78中任一项所示的氨基酸序列或其变体，或包含与上述任一序列具有至少80％相似性的氨基酸序列。
在某些实施方式中，所述抗体重链可变区的CDR区和/或轻链可变区的CDR区选自如下任意组的序列或其变体：
(1)抗体，其包含SEQ ID NO:1所示的HCDR1，SEQ ID NO:2所示的HCDR2和SEQ ID NO:3所示的HCDR3；SEQ ID NO:4所示的LCDR1，SEQ ID NO:5所示的LCDR2和SEQ ID NO:6所示的LCDR3；或
(2)抗体，其包含SEQ ID NO:1所示的HCDR1，SEQ ID NO:2所示的HCDR2和SEQ ID NO:7所示的HCDR3；SEQ ID NO:4所示的LCDR1，SEQ ID NO:5所示的LCDR2和SEQ ID NO:8所示的LCDR3；或
(3)抗体，其包含SEQ ID NO:1所示的HCDR1，SEQ ID NO:2所示的HCDR2和SEQ ID NO:9所示的HCDR3；SEQ ID NO:4所示的LCDR1，SEQ ID NO:5所示的LCDR2和SEQ ID NO:10所示的LCDR3；或
(4)抗体，其包含SEQ ID NO:11所示的HCDR1，SEQ ID NO:12所示的HCDR2和SEQ ID NO:13所示的HCDR3；SEQ ID NO:14所示的LCDR1，SEQ ID NO:15所示的LCDR2和SEQ ID NO:16所示的LCDR3；或
(5)抗体，其包含SEQ ID NO:17所示的HCDR1，SEQ ID NO:18所示的HCDR2和SEQ ID NO:19所示的HCDR3；SEQ ID NO:20所示的LCDR1，SEQ ID NO:21所示的LCDR2和SEQ ID NO:22所示的LCDR3；
(6)抗体，其包含SEQ ID NO:70所示的HCDR1，SEQ ID NO:72所示的HCDR2和SEQ ID NO:3所示的HCDR3；SEQ ID NO:4所示的LCDR1，SEQ ID NO:5所示的LCDR2和SEQ ID NO: 6所示的LCDR3；或
(7)抗体，其包含SEQ ID NO:71所示的HCDR1，SEQ ID NO:73所示的HCDR2和SEQ ID NO:3所示的HCDR3；SEQ ID NO:4所示的LCDR1，SEQ ID NO:5所示的LCDR2和SEQ ID NO:6所示的LCDR3；或
(8)抗体，其包含SEQ ID NO:1所示的HCDR1，SEQ ID NO:2所示的HCDR2和SEQ ID NO:3所示的HCDR3；SEQ ID NO:74所示的LCDR1，SEQ ID NO:75所示的LCDR2和SEQ ID NO:6所示的LCDR3；
(9)抗体，(1)～(8)中任一项所述的抗体的变体，且具备与(1)～(8)中任一项所述的抗体相同或相似的活性。
在某些实施方式中，所述抗体选自如下任意组的序列或其变体：
(1)抗体，所述抗体的重链可变区具有SEQ ID NO:23所示的氨基酸序列或与SEQ ID NO:23具有至少80％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％相似性的氨基酸序列，所述轻链可变区具有SEQ ID NO:25所示的氨基酸序列或与SEQ ID NO:25具有至少80％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％相似性的氨基酸序列；
(2)抗体，所述抗体的重链可变区具有SEQ ID NO:27所示的氨基酸序列或与SEQ ID NO:27具有至少80％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％相似性的氨基酸序列，所述轻链可变区具有SEQ ID NO:29所示的氨基酸序列或与SEQ ID NO:29具有至少80％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％相似性的氨基酸序列；
(3)抗体，所述抗体的重链可变区具有SEQ ID NO:31所示的氨基酸序列或与SEQ ID NO:31具有至少80％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％相似性的氨基酸序列，所述轻链可变区具有SEQ ID NO:33所示的氨基酸序列或与SEQ ID NO:33具有至少80％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％相似性的氨基酸序列；
(4)抗体，所述抗体的重链可变区具有SEQ ID NO:35所示的氨基酸序列或与SEQ ID NO:35具有至少80％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％相似性的氨基酸序列，所述轻链可变区具有SEQ ID NO:37所示的氨基酸序列或与SEQ ID NO:37具有至少80％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％相似性的氨基酸序列；
(5)抗体，所述抗体的重链可变区具有SEQ ID NO:39所示的氨基酸序列或与SEQ ID NO:39具有至少80％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％相似性的氨基酸序列，所述轻链可变区具有SEQ ID NO:41所示的氨基酸序列或与SEQ ID NO:41具有至少80％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％相似性的氨基酸序列；
(6)抗体，所述抗体的重链可变区具有SEQ ID NO:76所示的氨基酸序列或与SEQ ID NO:76具有至少80％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％相似性的氨基酸序列，所述轻链可变区具有SEQ ID NO:25所示的氨基酸序列或与SEQ ID NO:25具有至少80％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％相似性的氨基酸序列；
(7)抗体，所述抗体的重链可变区具有SEQ ID NO:80所示的氨基酸序列或与SEQ ID NO:80具有至少80％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％相似性的氨基酸序列，所述轻链可变区具有SEQ ID NO:25所示的氨基酸序列或与SEQ ID NO:25具有至少80％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％相似性的氨基酸序列；
(8)抗体，所述抗体的重链可变区具有SEQ ID NO:23所示的氨基酸序列或与SEQ ID NO:23具有至少80％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％相似性的氨基酸序列，所述轻链可变区具有SEQ ID NO:78所示的氨基酸序列或与SEQ ID NO:78具有至少80％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％相似性的氨基酸序列。
(9)抗体，(1)～(8)中任一项所述的抗体的变体，且具备与(1)～(8)中任一项所述的抗体相同或相似的活性。
在某些实施方式中，所述抗体选自全抗、scFv、单域抗体、Fab片段、Fab’片段、Fv片段、F(ab’)2片段、Fd片段、dAb片段、多功能抗体或scFv-Fc抗体、杂交瘤抗体、嵌合抗体、人源化抗体、全人源抗体或单克隆抗体。
在某些实施方式中，所述抗体是杂交瘤抗体，选自：
(1)抗体，所述抗体的重链可变区具有SEQ ID NO:35所示的氨基酸序列或与SEQ ID NO:35具有至少80％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％相似性的氨基酸序列，所述轻链可变区具有SEQ ID NO:37所示的氨基酸序列或与SEQ ID NO:37具有至少80％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％相似性的氨基酸序列；
(2)抗体，所述抗体的重链可变区具有SEQ ID NO:39所示的氨基酸序列或与SEQ ID NO:39具有至少80％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％相似性的氨基酸序列，所述轻链可变区具有SEQ ID NO:41所示的氨基酸序列或与SEQ ID NO:41具有至少80％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％相似性的氨基酸序列；
(3)抗体，(1)～(2)中任一项所述的抗体的变体，且具备与(1)～(2)中任一项所述的抗体相同或相似的活性。
在某些实施方式中，所述抗体是全人源抗体，选自：
(1)抗体，所述抗体的重链可变区具有SEQ ID NO:23所示的氨基酸序列或与SEQ ID NO:23具有至少80％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％相似性的氨基酸序列，所述轻链可变区具有SEQ ID NO:25所示的氨基酸序列或与SEQ ID NO:25具有至少80％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％相似性的氨基酸序列；
(2)抗体，所述抗体的重链可变区具有SEQ ID NO:27所示的氨基酸序列或与SEQ ID NO:27具有至少80％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％相似性的氨基酸序列，所述轻链可变区具有SEQ ID NO:29所示的氨基酸序列或与SEQ ID NO:29具有至少80％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％相似性的氨基酸序列；
(3)抗体，所述抗体的重链可变区具有SEQ ID NO:31所示的氨基酸序列或与SEQ ID NO: 31具有至少80％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％相似性的氨基酸序列，所述轻链可变区具有SEQ ID NO:33所示的氨基酸序列或与SEQ ID NO:33具有至少80％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％相似性的氨基酸序列；
(4)抗体，所述抗体的重链可变区具有SEQ ID NO:76所示的氨基酸序列或与SEQ ID NO:76具有至少80％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％相似性的氨基酸序列，所述轻链可变区具有SEQ ID NO:25所示的氨基酸序列或与SEQ ID NO:25具有至少80％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％相似性的氨基酸序列；
(5)抗体，所述抗体的重链可变区具有SEQ ID NO:80所示的氨基酸序列或与SEQ ID NO:23具有至少80％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％相似性的氨基酸序列，所述轻链可变区具有SEQ ID NO:25所示的氨基酸序列或与SEQ ID NO:25具有至少80％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％相似性的氨基酸序列；
(6)抗体，所述抗体的重链可变区具有SEQ ID NO:23所示的氨基酸序列或与SEQ ID NO:23具有至少80％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％相似性的氨基酸序列，所述轻链可变区具有SEQ ID NO:78所示的氨基酸序列或与SEQ ID NO:78具有至少80％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％相似性的氨基酸序列；
(7)抗体，(1)～(6)中任一项所述的抗体的变体，且具备与(1)～(6)中任一项所述的抗体相同或相似的活性。
在某些实施方式中，所述抗体选自：
(1)抗体，所述抗体的重链具有SEQ ID NO:45所示的氨基酸序列或与SEQ ID NO:45具有至少80％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％相似性的氨基酸序列，所述轻链具有SEQ ID NO:47所示的氨基酸序列或与SEQ ID NO:47具有至少80％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％相似性的氨基酸序列；
(2)抗体，所述抗体的重链具有SEQ ID NO:49所示的氨基酸序列或与SEQ ID NO:49具有至少80％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％相似性的氨基酸序列，所述轻链具有SEQ ID NO:51所示的氨基酸序列或与SEQ ID NO:51具有至少80％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％相似性的氨基酸序列；
(3)抗体，所述抗体的重链具有SEQ ID NO:53所示的氨基酸序列或与SEQ ID NO:53具有至少80％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％相似性的氨基酸序列，所述轻链具有SEQ ID NO:55所示的氨基酸序列或与SEQ ID NO:55具有至少80％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％相似性的氨基酸序列；
(4)抗体，(1)～(3)中任一项所述的抗体的变体，且具备与(1)～(3)中任一项所述的抗体相同或相似的活性。
在某些实施方式中，第一方面任一所述抗体是杂交瘤抗体、嵌合抗体、人源化抗体或全人源抗体；或者所述抗体是单克隆抗体；或者所述抗体是全抗、scFv、单域抗体、Fab片段、Fab’ 片段、Fv片段、F(ab’)2片段、Fd片段、dAb片段、多功能抗体或scFv-Fc抗体。
在某些实施方式中，第一方面任一所述抗体是杂交瘤抗体，选自：
(1)抗体，所述抗体的重链可变区具有SEQ ID NO:35所示的氨基酸序列，所述轻链可变区具有SEQ ID NO:37所示的氨基酸序列；
(2)抗体，所述抗体的重链可变区具有SEQ ID NO:39所示的氨基酸序列，所述轻链可变区具有SEQ ID NO:41所示的氨基酸序列。
(3)抗体，(1)～(2)中任一项所述的抗体的变体，且具备与(1)～(2)中任一项所述的抗体相同或相似的活性。
在某些实施方式中，第一方面任一所述抗体是全人源抗体，选自：
(1)抗体，所述抗体的重链可变区具有SEQ ID NO:23所示的氨基酸序列，所述轻链可变区具有SEQ ID NO:25所示的氨基酸序列；
(2)抗体，所述抗体的重链可变区具有SEQ ID NO:27所示的氨基酸序列，所述轻链可变区具有SEQ ID NO:29所示的氨基酸序列；
(3)抗体，所述抗体的重链可变区具有SEQ ID NO:31所示的氨基酸序列，所述轻链可变区具有SEQ ID NO:33所示的氨基酸序列；
(4)抗体，(1)～(3)中任一项所述的抗体的变体，且具备与(1)～(3)中任一项所述的抗体相同或相似的活性。
在某些实施方式中，第一方面任一所述抗体是全人源抗体，选自：
(1)抗体，所述抗体的重链具有SEQ ID NO:45所示的氨基酸序列，所述轻链具有SEQ ID NO:47所示的氨基酸序列；
(2)抗体，所述抗体的重链具有SEQ ID NO:49所示的氨基酸序列，所述轻链具有SEQ ID NO:51所示的氨基酸序列；
(3)抗体，所述抗体的重链具有SEQ ID NO:53所示的氨基酸序列，所述轻链具有SEQ ID NO:55所示的氨基酸序列；
(4)抗体，(1)～(3)中任一项所述的抗体的变体，且具备与(1)～(3)中任一项所述的抗体相同或相似的活性。
在第二方面，本申请提供一种免疫缀合物，所述的免疫辍合物包括第一方面任一所述的抗体，以及与之连接的功能性分子。
在第三方面，本申请提供一种嵌合受体，所述嵌合受体的胞外域包含第一方面任一所述的抗体，所述嵌合受体包括：嵌合抗原受体(CAR)、嵌合T细胞受体、T细胞抗原耦合器(TAC)或其组合。
在某些实施方式中，所述嵌合受体是嵌合抗原受体(CAR)。
在某些实施方式中，所述CAR包含第一方面任一所述的抗体、跨膜区和胞内信号区。
在某些实施方式中，所述嵌合受体包含顺序连接的：第一方面任一所述的抗体、跨膜 区和胞内信号区。
在某些实施方式中，所述的胞内信号区选自：CD3δ、FcεRIγ、CD27、CD28、CD137、CD134、MyD88、CD40的胞内信号区序列或其组合；和/或所述的跨膜区包含CD8或CD28的跨膜区。
在某些实施方式中，所述CD8跨膜区包含如SEQ ID NO：60所示的氨基酸序列。
在某些实施方式中，所述CD137胞内信号传区包含如SEQ ID NO：61所示的氨基酸序列。
在某些实施方式中，所述CD3δ胞内信号传区包含如SEQ ID NO：62所示的氨基酸序列。
在某些实施方式中，所述的嵌合受体包括：第一方面任一所述的抗体、CD8/CD28的跨膜区和CD3δ；或第一方面任一所述的抗体、CD8/CD28的跨膜区、CD137的胞内信号区和CD3δ；或第一方面任一所述的抗体、CD8/CD28的跨膜区、CD28的胞内信号区和CD3δ；或第一方面任一所述的抗体、CD8/CD28的跨膜区、CD28的胞内信号区、CD137和CD3δ。
在某些实施方式中，所述的嵌合受体还包括铰链区。
在某些实施方式中，所述铰链区包括CD8铰链区。
在某些实施方式中，所述CD8铰链区包含如SEQ ID NO：59所示的氨基酸序列。
在某些实施方式中，所述的嵌合受体还包括信号肽。
在某些实施方式中，所述信号肽包括CD8信号肽。
在某些实施方式中，所述CD8信号肽包含如SEQ ID NO：58所示的氨基酸序列。
在某些实施方式中，所述的嵌合受体包含如SEQ ID NO：43、82、84或86任一所示的氨基酸序列。
在某些实施方式中，所述的嵌合受体的氨基酸序列如SEQ ID NO：63、88、89、90任一所示。
在第四方面，本申请提供一种编码第一方面任一所述的抗体、第二方面所述的免疫缀合物、第三方面所述的嵌合受体的核酸。
在某些实施方式中，所述核酸包含如SEQ ID NO：24、26、28、30、32、34、36、38、40、42、77、79或81中任一所示的序列或其组合。
在某些实施方式中，所述核酸包含如SEQ ID NO：46、48、50、52、54或56中任一所示的序列或其组合。
在某些实施方式中，所述核酸包含如SEQ ID NO：44、83、85或87中任一所示的序列。
在第五方面，本申请提供一种载体，其包含第四方面所述的核酸。
在第六方面，本申请提供一种病毒，其包含第五方面所述的表达载体。
在第七方面，本申请提供一种组合物，包括第二方面所述的免疫缀合物、和/或第三方面所述的嵌合受体，所述组合物对表达GPRC5D的细胞具有细胞毒性。
在某些实施方式中，所述表达GPRC5D的细胞是肿瘤细胞和/或病原体细胞。
在第八方面，本申请提供了细胞，其包含第一方面所述的抗体、第二方面所述的免疫缀 合物、第三方面所述的嵌合受体、第四方面所述的核酸、和/或第五方面所述的载体。
在某些实施方式中，所述细胞为宿主细胞，其包含第五方面所述的载体或基因组中整合有第四方面所述的核酸。
在某些实施方式中，所述细胞/宿主细胞表达第三方面所述的嵌合受体。
在某些实施方式中，所述细胞/宿主细胞是T细胞、自然杀伤细胞、自然杀伤T细胞、NK92细胞、干细胞衍生的免疫效应细胞或其组合。
在某些实施方式中，所述T细胞包括细胞毒性T淋巴细胞、DNT细胞和/或调节性T细胞。
在某些实施方式中，所述T细胞为来源于天然的T细胞和/或经多能干细胞诱导产生的T细胞。
在某些实施方式中，所述T细胞为自体/同种异体T细胞。
在某些实施方式中，所述T细胞为原代T细胞。
在某些实施方式中，所述T细胞来源于人的自体T细胞。
在某些实施方式中，所述T细胞包含记忆性干细胞样T细胞(Tscm细胞)、中心记忆T细胞(Tcm)、效应性T细胞(Tef)、调节性T细胞(Tregs)，效应记忆T细胞(Tem)、γδT细胞或其组合。
在某些实施方式中，所述宿主细胞结合表达GPRC5D的细胞，不显著结合不表达GPRC5D的细胞。
在某些实施方式中，所述宿主细胞还携带外源的细胞因子的编码序列；和/或还表达非靶向GPRC5D的嵌合受体；和/或还表达趋化因子；和/或还表达趋化因子受体；和/或还表达安全开关。
在某些实施方式中，所述宿主细胞还携带外源的细胞因子包括IL-7、IL-12、IL-15、IL-18、IL-21、或I型干扰素的编码序列。
在某些实施方式中，所述宿主细胞还表达趋化因子包括CCL19或CCL21。
在某些实施方式中，所述宿主细胞还表达趋化因子受体包括CCR2、CCR4、CCR5、CXCR2、CXCR4或CXCR5。
在某些实施方式中，所述宿主细胞还表达安全开关包括iCaspase-9、Truancated EGFR或RQR8。
在第九方面，本申请提供了药物组合物，其包含第一方面任一所述的抗体、第二方面所述的免疫缀合物、第三方面所述的嵌合受体、第四方面所述核酸、第五方面所述的载体、第六方面所述的病毒、第七方面所述的组合物和/或第八方面所述的细胞，以及药学上可接受的佐剂。
在第十方面，本申请提供了联合用药，第一方面任一所述的抗体、第二方面所述的免疫缀合物、第三方面所述的嵌合受体、第七方面所述的组合物、第八方面所述的细胞、第九方面所述的药物组合物与增强其功能的药剂组合施用，优选地，与化疗药物联用；和/或与改善其相关的一种或多种副作用的药剂联合施用；和/或与表达靶向GPRC5D之外的嵌合抗原 受体的宿主细胞联合施用。
在第十一方面，本申请提供一种制备第一方面任一所述的抗体、第二方面所述的免疫缀合物、第三方面所述的嵌合受体、第七方面所述的组合物、第九方面所述的药物组合物的方法，所述方法包含在适于表达所述抗体、免疫缀合物、嵌合受体的条件下培养第八方面所述的宿主细胞，以及分离出由所述宿主细胞表达的所述抗体、免疫缀合物、嵌合受体、组合物和/或药物组合物。
在第十二方面，本申请提供一种试剂盒，其包括第一方面任一所述的抗体、第二方面所述的免疫缀合物、第三方面所述的嵌合受体、第四方面所述核酸、第五方面所述的载体、第六方面所述的病毒、第七方面所述的组合物、第八方面所述的细胞和/或第九方面所述的药物组合物。
在某些实施方式中，所述试剂盒包括：
容器，以及位于容器中的第九方面所述的药物组合物；或
容器，以及位于容器中的第一方面任一所述的抗体或编码该抗体的核酸；或第二方面所述的免疫缀合物或编码该免疫缀合物的核酸；或第三方面所述的嵌合受体或编码该嵌合受体的核酸；或第八方面所述的宿主细胞。
第十三方面，本申请提供一种治疗/诊断疾病的方法，其包括向有需要的受试者给予有效量的如第一方面任一所述的抗体或如第四方面所述的免疫缀合物或如第八方面所述的细胞或如第九方面所述的药物组合物或如第十二方面所述的试剂盒。
在具体的实施方式中，所述疾病选自炎性病症、感染、自身免疫性疾病和肿瘤；
在具体的实施方式中，所述受试者是人；
在具体的实施方式中，其中所述宿主细胞对于所述受试者是自体的或同种异体的T细胞。
在第十四方面，本申请提供第一方面任一所述的抗体或如第二方面所述的免疫缀合物或如第八方面所述的宿主细胞或如第九方面所述的药物组合物或如第十二方面所述的试剂盒在制备用于治疗/诊断疾病的药物中的用途，其特征在于，所述疾病包括表达GPRC5D；优选地，所述疾病选自炎性病症、感染、自身免疫性疾病和肿瘤。
应理解，在本申请范围内中，本申请的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了杂交瘤抗体AB1、AB2与CHOK1-huGPRC5D细胞结合的EC50；
图2显示了杂交瘤抗体AB1、AB2均与人GPRC5D和鼠GPRC5D显著结合；
图3显示了重组杂交瘤抗体AB1(scFv-mFc)和AB2(scFv-mFc)与表达人GPRC5D的细胞系特异性结合；
图4显示了抗体AB3、AB4、AB5均特异性结合人GPRC5D；
图5显示了抗体AB3、AB4、AB5均特异性结合过表达人GPRC5D的293T细胞；
图6显示了抗体AB3、AB4、AB5与人GPRC5D结合的EC50；
图7显示了抗体AB3、AB4、AB5均与表达人GPRC5D的细胞(CHOK1-huGPRC5D，293T-huGPRC5D，MM.1S和NCI-H929)显著结合，不结合不表达GPRC5D的细胞(CHOK1，293T和Daudi)；
图8显示了抗体AB3、AB4、AB5与CHOK1-huGPRC5D细胞结合的EC50；
图9显示了抗体AB3、AB4、AB5与MM.1S细胞结合的EC50；
图10显示了抗体AB3与CHOK1-muGPRC5D细胞结合；
图11显示了AB3-BBZ CAR T细胞对靶细胞的体外杀伤效果；
图12显示了AB3-BBZ CAR T细胞和18-BBZ CAR T细胞在荷瘤小鼠体内抑制肿瘤生长的效果；
图13显示了AB3-BBZ CAR T细胞和18-BBZ CAR T细胞治疗荷瘤小鼠的体重变化情况；
图14显示了抗体AB6、AB7和AB8与人GPRC5D的结合结果；
图15显示了抗体AB3、AB6、AB7和AB8与不同种属GPRC5D细胞系结合的检测结果；
图16显示了抗体AB3、AB6、AB7和AB8与293T-huGPRC5D、MM.1S细胞结合的EC50检测结果；
图17显示了抗体AB3、AB7与Bio-GPRC5D亲和力检测结果；
图18显示了AB3-BBZ、AB6-BBZ、AB7-BBZ、AB8-BBZ CAR T细胞对靶细胞的体外杀伤效果。
具体实施方式
本申请人经过深入的研究筛选，获得了特异性识别GPRC5D的杂交瘤抗体和全人源抗体。本申请的抗体可以被应用于制备靶向抗肿瘤药物以及诊断肿瘤的药物。
术语
除非专门定义，本文所用的所有技术和科学术语具有在基因治疗，生物化学、遗传学和分子生物学领域内的技术人员通常理解的相同含义。类似或等效于本文中描述的所有方法和材料都可以在本申请的实践或测试中使用，其中，本文描述的是合适的方法和材料。本文提及的所有出版物、专利申请、专利和其他参考文献都以其全部内容结合于本文中作为参考。在冲突的情况下，以本说明书，包括定义为准。此外，除非另有规定，材料、方法和实施例仅是说明性的，而并非旨在进行限制。
除非另有说明，本申请的实践将采用细胞生物学、细胞培养、分子生物学、转基因生物学、微生物学、重组DNA和免疫学的传统技术，这都属于本领域的技术范围。这些技术充分解释于文献中。参见，例如，Current Protocols in Molecular Biology(FrederickM.AUSUBEL，2000，Wileyand sonInc，Library of Congress，USA)；Molecular Cloning:A Laboratory Manual，Third  Edition，(Sambrooketal，2001，Cold Spring Harbor，NewYork:Cold Spring Harbor Laboratory Press)；Oligonucleotide Synthesis(M.J.Gaited.，1984)；Mullis et al.U.S.Pat.No.4,683,195；Nucleic Acid Hybridization(B.D.Harries&S.J.Higginseds.1984)；Transcription And Translation(B.D.Hames&S.J.Higginseds.1984)；Culture Of Animal Cells(R.I.Freshney，Alan R.Liss，Inc.，1987)；Immobilized Cells And Enzymes(IRL Press，1986)；B.Perbal，A Practical Guide To Molecular Cloning(1984)；the series，Methods In ENZYMOLOGY(J.Abelson和M.Simon，eds.-in-chief，Academic Press，Inc.，New York)，尤其是Vols.154和155(Wuetal.eds.)和Vol.185，“Gene Expression Technology”(D.Goeddel，ed.)；Gene Transfer Vectors For Mammalian Cells(J.H.Miller和M.P.Caloseds.，1987，Cold Spring Harbor Laboratory)；Immunochemical Methods In Cell And Molecular Biology(Mayer和Walker，eds.，Academic Press，London，1987)；Hand book Of Experimental Immunology，卷I-IV(D.M.Weir和C.C.Blackwell，eds.，1986)；和Manipulating the Mouse Embryo(Cold Spring Harbor Laboratory Press，Cold Spring Harbor，N.Y.，1986)。
公开内容中，请求保护的主题的各个方面均以范围形式呈现。应当理解，范围形式的描述仅仅是为了方便和简洁，并且不应被解释为对所要求保护的主题的范围的硬性限制。因此，范围的描述应当被认为已经具体公开了所有可能的子范围以及该范围内的单个数值。例如，在提供值的范围的情况下，应当理解，在该范围的上限和下限之间的每个中间值以及在所述范围内的任何其他所述的或中间的值均被包括在要求保护的主题内，所述范围的上下限也属于请求保护的主题的范围。所述较小范围内可独立地包含这些较小范围的上下限，它们也属于请求保护的主题的范围，除非明确地排除所述范围的上下限。设定范围包含一个或两个限值时，请求保护的主题也包括排除所述限值之一个或两个的范围。这适用而无关范围的宽度。
术语“约”是指本技术领域技术人员容易知晓的各值的通常误差范围。本文中述及“约”值或参数，包括(并描述)指向该值或参数本身的实施方式。例如，关于“约X”的描述包括“X”的描述。例如，“约”或“包含”可意指按照在该领域中的实际的标准偏差在1以内或多于1。或者“约”或“包含”可意指至多10％(即±10％)的范围。例如，约5uM可包括在4.5uM与5.5uM之间的任何数目。当在申请案与申请专利范围中提供特定值或组成时，除非另外指出，否则“约”或“包含”应假定为在该特定值或组成的可接受误差范围内。
除非另外指出，本文中所述任何浓度范围、百分比范围、比例范围或整数范围应理解为包括在所述范围内的任何整数，以及在合适情况下，其分数(例如整数的十分之一与百分之一)的数值。
为了更易于理解本申请，首先定义一些术语。
术语“GPRC5D”是指被分类至G蛋白偶联受体家族C的组5，并且是使用一系列人GPCR的氨基酸序列通过EST数据库的同源性搜索新发现的人GPCR蛋白之一。该蛋白已在Genbank登录号：AF209923、NM_018654和NP_0611124下登记。GPRC5D是一种孤儿受体， 在人和人癌症中没有已知的配体或功能。示例性，人GPRC5D全长的氨基酸序列如SEQ ID NO:65所示，鼠GPRC5D全长的氨基酸序列如SEQ ID NO:66所示。
术语“多肽”、“肽”、“蛋白”和“蛋白质”可互换使用，指任何长度的氨基酸的聚合物。聚合物可以是直链、环状或支链的，它可以包含修饰的氨基酸，特别是保守修饰的氨基酸，并且它可以被非氨基酸中断。该术语还包括改性的氨基酸聚合物例如已经通过硫酸化、糖基化、脂化、乙酰化、磷酸化、碘化、甲基化、氧化、蛋白水解加工、异戊二烯化、外消旋化、硒酰化、转移-RNA介导的氨基加成如精氨酸化、泛在化、或任何其他操作如与标记组分缀合等改性的氨基酸聚合物。如本文所用，术语“氨基酸”是指天然和/或非天然或合成氨基酸，包括甘氨酸以及D或L光学异构体，以及氨基酸类似物和肽模拟物。“衍生自”指定的蛋白质的多肽或氨基酸序列是指多肽的来源。该术语还包括由指定的核酸序列表达的多肽。
术语“抗体”在本文中以最广义使用并且包括各种抗体结构，包括但不限于单克隆抗体、多克隆抗体、多特异性抗体(例如，双特异性抗体)和抗体片段，只要其显示所需的抗原结合活性即可。
“抗体片段”是指不同于完整抗体的分子，其包含完整抗体结合完整抗体所结合的抗原的部分。抗体片段的实例包括但不限于(i)由VL、VH、CL和CH1结构域组成的Fab片段，包括Fab’和Fab’-SH，(ii)VH和CH1结构域组成的Fd片段，(iii)由单个抗体的VL和VH结构域组成的Fv片段；(iv)由单个可变区组成的dAb片段(Ward等，1989，Nature 341:544-546)；(v)F(ab’)2片段，包含2个连接的Fab片段的二价片段；(vi)单链Fv分子抗原结合位点；(vii)双特异性单链Fv二聚体(PCT/US92/09965)；(viii)“二体”或“三体”，通过基因融合构建的多价或多特异性片段；和(ix)与相同或不同抗体遗传融合的scFv。
抗体的“分类”是指其重链所具有的恒定结构域或恒定区的类型。主要有五类抗体：IgA、IgD、IgE、IgG和IgM，并且这些中的一些可以被进一步划分成亚类(同种异型)，例如，IgG1、IgG2、IgG3、IgG4、IgA1和IgA2。对应于不同的类型的免疫球蛋白的重链恒定结构域被分别称为α，δ，ε，γ，和μ。
术语“可变区或可变结构域”是指参与抗体抗原结合的抗体重链或轻链的结构域。天然抗体的重链和轻链可变结构域(分别为VH和VL)通常具有相似的结构，其中各结构域包含四个保守的FR和三个CDR。(参见，例如，Kindt等，KubyImmunology,6th ed.,W.H.Freeman&Co.,第91页(2007))。单个VH或VL结构域可足以给予抗原结合特异性。此外，结合特定抗原的抗体可以分别使用来自与所述抗原结合的抗体的VH或V L结构域筛选互补VL或VH结构域的文库来分离。参见，例如，Portolano等,J.Immunol.150:880-887(1993)；Clarkson等,Nature352:624-628(1991)。
术语“高变区”或“互补决定区”或“CDR”是指抗体可变结构域中序列高变和/或形成结构确定的环(“高变环”)和/或含有与抗原接触的残基(“抗原触点”)的各区域。在某些实施方式中，CDR 可通过选自如下的编号系统确定：Kabat、Chothia、IMGT、Gelfand、Aho和Martin。例如，可通过Kabat编号系统确定。例如，抗体可以包含六个CDR：VH中的三个(HCDR1，HCDR2，HCDR3)和VL中的三个(LCDR1，LCDR2，LCDR3)。
术语“Fc区”或“Fc”被用于限定含有恒定区的至少一部分的免疫球蛋白重链的C-端区域。该术语包括天然序列Fc区和变体Fc区。
“框架(FR)”是指不同于高变区(CDR)残基的可变结构域残基。可变结构域的FR通常由四个FR结构域组成：FR1，FR2，FR3和FR4。因此，在VH(或VL)中CDR和FR序列通常按以下顺序出现：FR1-HCDR1(LCDR1)-FR2-HCDR2(LCDR2)-FR3-HCDR3(LCDR3)-FR4。
除非另外指出，在本文中，CDR残基和可变结构域中的其他残基(例如，FR残基)根据以上Kabat等编号。
术语“天然抗体”是指天然存在的具有多种结构的免疫球蛋白分子。例如，天然IgG抗体是约150,000道尔顿的异源四聚糖蛋白，由通过二硫键键合的两个相同的轻链和两个相同的重链组成。从N端到C端，各重链具有可变区(VH)，其也被称为可变重链结构域或重链可变结构域，之后是三个恒定结构域(CH1，CH2和CH3)。类似地，从N端到C端，各轻链具有可变区(VL)，其也被称为可变轻链结构域或轻链可变结构域，之后是轻链恒定(CL)结构域。抗体的轻链基于其恒定结构域的氨基酸序列可以被分配至两种类型之一，被称为κ(κ)和λ(λ)。
术语“全抗”、“全长抗体”、“完整抗体”可交换使用，是指具有与天然抗体结构基本类似的结构或具有含有如本文中所限定的Fc区的重链或包括具有抗原结合区域的完整的全长抗体。
术语“单域抗体(Single domain antibody，sdAb)”是指缺失抗体轻链而只有重链可变区的一类抗体，因其分子量小，也被称为纳米抗体(Nanobody)。
术语“单结构域抗体”是指包含抗体的全部或部分的重链可变结构域或全部或部分的轻链可变结构域。在某些实施方案中，单结构域抗体是人单结构域抗体(Domantis,Inc.,Waltham,MA；参加，例如，美国专利号6248516)。
术语“单克隆抗体”、“单抗”是指获自基本上同源的抗体的群体的抗体，即，包括所述群体的个体抗体是相同的和/或结合相同的表位，除可能的变体抗体以外，例如，含天然存在的突变或在单克隆抗体制剂的制备过程中产生，此种变体通常少量存在。对比于多克隆抗体制剂(通常包括针对不同的决定簇(表位)的不同抗体)，单克隆抗体制剂中的各单克隆抗体是针对抗原上的单个决定簇。因此，定于“单克隆”指示抗体的性质为获得自基本上同源的抗体群体，并且不视为要求通过任何特定的方法制备所述抗体。例如，可以通过多种技术制备，包括但不限于杂交瘤方法、重组DNA法、噬菌体展示法，以及利用含有所有或部分的人免疫球蛋白基因座的转基因动物的方法。
示例性，本申请的单克隆抗体可以由杂交瘤方法生产，杂交瘤可通过分离出受刺激的免疫细胞(诸如来自被接种动物的脾脏的那些)形成。然后可以将这些细胞(诸如骨髓瘤细胞或经转化细胞)与永生化细胞融合，它们能够在细胞培养物中无限复制，由此生产出永生的、分泌免疫 球蛋白的细胞系。所利用的永生细胞系被选出(是否缺乏对利用某些营养物所必需的酶)。许多这样的细胞系(诸如骨髓瘤)是本领域技术人员已知的，并且包括例如：胸苷激酶(TK)或次黄嘌呤-鸟嘌呤磷酸核糖转移酶(HGPRT)。这些缺陷允许了：根据融合细胞在例如次黄嘌呤氨基喋呤胸腺嘧啶核苷培养基(HAT)上生长的能力选择出融合细胞。
术语“嵌合抗体”是指抗体重链和/或轻链的一部分来源于特定来源或物种，而重链和/或轻链的剩余部分来源于不同的来源或物种的抗体。在某些实施方案中，嵌合抗体包含非人可变区(例如，来源于小鼠、大鼠、仓鼠、兔或非人灵长类动物如猴的可变区)和人恒定区。在另外的实施方案中，嵌合抗体是“类型转换”抗体，其中类型或亚类已经由亲本抗体的类型或亚类改变。嵌合抗体包括其抗原结合片段。在某些实施方案中，嵌合抗体是“人源化抗体”。
术语“人源化”用于非人抗体，例如啮齿动物或灵长类动物等，是含有来源于非人免疫球蛋白的最小序列的杂合免疫球蛋白，免疫球蛋白链或其片段。“人源化抗体”是指一种嵌合抗体，其包含来自非人CDR的氨基酸残基和来自人FR的氨基酸残基。在某些实施例中，人源化抗体将包含基本上全部的至少一个(一般为两个)可变结构域，其中所有或基本上所有的CDR对应于非人抗体的CDR，并且所有或基本上所有的FR对应于人抗体的FR。人源化抗体任选地可以包含来源于人抗体的抗体恒定区的至少一部分。
在一些实施方案中，“人源化抗体”可包括突变，例如通过体外随机或定点诱变或通过体内体细胞突变引入的突变。
术语“全人源抗体”是一种抗体，其具有的氨基酸序列对应于由人或人细胞产生的抗体或来源于利用人抗体库或其他人抗体编码序列的非人来源的抗体的氨基酸序列。全人源体的定义明确排除了包含非人抗原结合残基的人源化抗体。在某些实施方案中，本文中提供的抗体是“全人源抗体”，是由噬菌体展示技术生成。
本申请的抗体可以通过筛选具有所需一种或多种活性的抗体的组合文库来分离。例如，本领域中已知多种方法用于生成噬菌体展示文库并且针对具有所需结合性质的抗体筛选所述文库。此种方法被综述于例如Hoogenboom等，Methods in Molecular Biology 178:1-37(0’Brien等，Human Press,Totowa,NJ,2001)中并被进一步描述于例如McCafferty等，Nature 348：552-554；Clackson等，Nature 352：624-628(1991)；Marks等，J.Mol.Biol.222:581-597(1992)；Marks,Meth.Mol.Biol.,248:161-175(Lo,ed.,Human Press,Totowa,NJ,2003)；Sidhu等,J.Mol.Biol.338(2):299-310(2004)；Lee等，J.Mol.Biol.340(5):1073-1093(2004)；Fellouse,Proc.Natl.Acad.Sci.USA101(34):12467-12472(2004)；Lee等，J.Immunol.Methods 284(1-2):119-132(2004)中。
在某些噬菌体展示方法中，VH和VL基因库通过聚合酶链式反应(PCR)分别克隆并且在噬菌体文库中随机重组，然后可以针对结合抗原的噬菌体筛选所述文库，如Winter等，Ann.Rev.Immunol.12:433-455(1994)中所述。噬菌体典型地将抗体片段展示位单链Fv(scFv)片段或Fab片段。来自被免疫的来源的文库向免疫原提供高亲和力抗体而不需要构建杂交瘤。备选地，可 以克隆天然库(例如，由人)从而向多种非自身抗原以及自身抗原提供单一来源的抗体而不需要进行任何免疫，如Griffiths等，EMBO J,12:725-734(1993)所述。最后，也可以通过以下方式合成制备天然文库：自干细胞克隆未重排的V-基因区段，并且使用含随机序列的PCR引物以编码高变CDR3区并且在体外实现重排，如Hoogenboom，J.Mol.Biol.227:381-388(1992)所述。
在本文中，分离自全人源抗体文库的抗体或抗体片段被认为是全人源抗体或全人源抗体片段。
在某些实施方案中，本文中提供抗体的氨基酸序列变体。术语“亲本抗体”指本申请所提供的抗体或依据本申请所提供的抗体进行突变、或亲和力成熟等处理后所得的抗体。所述亲本抗体可以是天然存在的抗体，或者天然存在的抗体的变体或改造版本。亲本抗体可以指抗体本身，包含所述亲本抗体的组合物，或其编码氨基酸序列。
术语“亲和力成熟”抗体是指相比于亲本抗体在一个或多个高变区(CDR)中具有一个或多个改变的抗体，此种改变导致抗体对抗原的亲和力提高。
术语“变体”指与本申请所提供的抗体的序列具有基本上相同氨基酸序列或由基本上相同的核苷酸序列编码的一种或多种活性的多肽。所述变体与本申请实施例中所提供的抗体具有相同或相似的活性。例如，变体可以是基于本申请提供的抗体的氨基酸序列的变体抗体或抗体变体。
术语“变体抗体”或“抗体变体”包括由于相比亲本的至少一个氨基酸修饰，而不同于亲本抗体序列的抗体序列。本文中的变体抗体序列优选的具有与亲本抗体序列至少约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％％、91％、92％、93％、94％、95％％、96％、97％、98％、99％的氨基酸序列同一性。抗体变体可以指抗体本身，也可以指包含所述抗体变体的组合物。抗体的氨基酸序列变体可以通过向编码所述抗体的核苷酸序列引入合适的修饰或通过肽合成来制备。术语“氨基酸修饰”包括氨基酸取代、添加和/或缺失，“氨基酸取代”、“氨基酸置换”意指用另一种氨基酸替换亲本多肽序列中特定位置上的氨基酸，“氨基酸插入”意指在亲本多肽序列中的特定位置添加氨基酸，“氨基酸缺失”意指去除亲本多肽序列中特定位置上的氨基酸。可以进行缺失、插入和取代的任意组合以获得最终的构建体，前提是最终的构建体具有所需的特征，例如：结合抗原。
术语“修饰”一词是指本申请的蛋白或多肽的状态或结构的改变。修饰的方式可以是化学的、结构的和功能上的。
术语“保守修饰”或“保守序列修饰”意指不显著影响或改变含有所述氨基酸序列的抗体的结合特征的氨基酸修饰。此类保守修饰包括氨基酸取代、插入和缺失。可通过本领域已知的标准技术将修饰导入本申请的抗体中，例如定点诱变、PCR介导的诱变。本领域已经定义了具有相似侧链的氨基酸残基家族，如表1所示。
表1：具有相似侧链的氨基酸残基家族
因而，可以用其他相同侧链家族的氨基酸残基替换本申请抗体的CDR区中或框架区中的一个或多个氨基酸残基，并可以测试所改变的抗体(变体抗体)保留的功能。
非保守置换需要将这些组之一的成员换成另一组的成员。
一种置换变体包括置换亲本抗体(例如，人源化或人抗体)的一个或多个高变区残基。通常，被选择用于进一步研究的所得的变体相对于亲本抗体将具有某些生物学性质(例如，增加的亲和力、减小的免疫原性)的改变(例如，提高)和/或将基本上保持亲本抗体的某些生物学性质。示例性替换变体是亲和力成熟抗体，其可以例如，使用基于噬菌体展示的亲和力成熟技术(如本文中描述的那些)常规制备。简言之，将一个或多个CDR残基突变并将变体抗体展示在噬菌体上并且筛选特定的生物学活性(例如，结合亲和力)。
改变(例如，置换)可以在CDR区进行，例如，以提高抗体亲和力。此种改变可以在CDR“热点”中进行，所述“热点”即在体细胞成熟过程期间以高频率进行突变的密码子编码的残基(参见，例如，Chowdhury,Methods Mol.Biol.207:179-196(2008)，和/或解除抗原的残基，测试所得的变体VH或VL的结合亲和力。通过构建二级文库并自其进行再选择的亲和力成熟已被描述于例如Hoogenboom等，Methods in Molecular Biology 178:1-37(0’Brien等，ed.,Human press,Totowa,NJ,(2001))中。在亲和力成熟的一些实施方案中，通过多种方法中任一种(例如，易错PCR、链改组或寡核苷酸定向诱变)将多样性引入选择用于成熟的可变基因种。然后产生二级文库。然后筛选所述文库以鉴定任何具有所需亲和力的抗体变体。引入多样性的另一种方法包括CDR定向方法，其中若干CDR残基(例如，同时4-6个残基)被随机化。
在某些实施方案种，置换、插入或缺失可以发生在一个或多个CDR内，只要这样的改变不显著减小抗体结合抗原的能力。例如，不显著减小结合亲和力的保守改变(例如，本文中所述的保守修饰)可以在CDR中进行。此种改变可以例如在CDR中接触抗原的残基的外部。在上述提供的变体VH和VL序列的某些实施方案中，各CDR是未改变的，或含不超过一个、两个或三个氨基酸置换。
术语“抗GPRC5D抗体”、“结合GPRC5D的抗体”、“GPRC5D抗体”、“识别GPRC5D的抗体”是指能够以足够的亲和力结合GPRC5D的抗体，所述抗体可用作用于靶向GPRC5D的诊断剂和 /或治疗剂。在一个实施方案中，抗GPRC5D抗体与不相关的、非GPRC5D蛋白的结合程度小于所述抗体与GPRC5D的约10％，如通过酶联免疫吸附实验(ELISA)测定。在某些实施方案中，抗GPRC5D抗体结合GPRC5D的表位，所述表位在来源于不同物种的GPRC5D之间是保守的。
术语“嵌合T细胞受体”，包括构成TCR的各种多肽衍生的重组多肽，其能够结合到靶细胞上的表面抗原，和与完整的TCR复合物的其他多肽相互作用，通常同定位在T细胞表面。嵌合T细胞受体由一个TCR亚基与人或人源化抗体结构域组成的一个抗原结合结构域组成，其中，TCR亚基包括至少部分TCR胞外结构域、跨膜结构域、TCR胞内结构域的胞内信号结构域的刺激结构域；该TCR亚基和该抗体结构域有效连接，其中，TCR亚基的胞外、跨膜、胞内信号结构域来源于CD3ε或CD3γ，并且，该嵌合T细胞受体整合进T细胞上表达的TCR。
术语“T细胞抗原耦合器(T cell antigen coupler，TAC)”，包括三个功能结构域：1、抗原结合结构域，包括单链抗体、设计的锚蛋白重复蛋白(designed ankyrin repeat protein，DARPin)或其他靶向基团；2、胞外区结构域，与CD3结合的单链抗体，从而使得TAC受体与TCR受体靠近；3、跨膜区和CD4共受体的胞内区，其中，胞内区连接蛋白激酶LCK，催化TCR复合物的免疫受体酪氨酸活化基序(ITAMs)磷酸化作为T细胞活化的初始步骤。
术语“嵌合抗原受体”(CAR)包括胞外抗原结合结构域、跨膜结构域和胞内信号传导结构域。胞内信号传导结构域可以包括刺激分子(也可称作刺激性分子、初级信号分子)和/或共刺激性分子的功能信号传导结构域。例如，刺激性分子可以是与T细胞受体复合体结合的δ链；例如，细胞质信号传导结构域进一步包括一种或多种共刺激性分子的功能性信号传导结构域，例如4-1BB(即CD137)、CD27和/或CD28。
在本申请中，一方面，CAR包含嵌合融合蛋白，所述蛋白包含胞外抗原识别结构域、跨膜结构域、和胞内信号传导结构域，所述胞内信号传导结构域含有源自刺激分子的功能信号传导结构域。一方面，CAR包含嵌合融合蛋白，所述蛋白包含胞外抗原识别结构域、跨膜结构域、和胞内信号传导结构域，所述胞内信号传导结构域含有源自共刺激分子的功能性信号传导结构域和源自刺激分子的功能性信号传导结构域。一方面，CAR包含嵌合融合蛋白，所述蛋白包含胞外抗原识别结构域、跨膜结构域和胞内传导结构域，所述胞内信号传导结构域包含源自一个或多个共刺激分子的至少两个功能性信号传导结构域和源自刺激分子的功能性信号传导结构域。一方面，CAR在CAR融合蛋白的氨基酸(ND端)包含可选的前导序列。一方面，CAR在胞外抗原识别结构域的N端还包含前导序列，其中前导序列任选地在CAR的细胞加工和定位至细胞膜的过程中从抗原识别结构域(例如scFv)切下。
术语“初级信号分子”或“刺激分子”以刺激性方式调节TCR复合物的初始活化。通常，初级信号由例如TCR/CD3复合物与加载了肽的MHC分子的结合而引发，由此介导T细胞反应(包括但不限于，增殖、活化、分化等)。以刺激性方式起作用的初级信号分子可 以包含免疫受体酪氨酸激活基序或ITAM的信号传导基序。在本申请中尤其有用的包含ITAM的初级信号分子的功能信号传导结构域(初级信号域)的例子包括但不限于，源自CD3δ、FcRγ、FcRβ、CD3γ、CD3δ、CD3ε，CD5，CD22，CD79a，CD79b，CD278(也称作“ICOS”)和CD66d的序列，在特例的本申请CAR中，在任何一个或多个本申请CAR中胞内信号传导结构域包含胞内信号传导序列，例如CD3δ的初级信号域。
术语“共刺激信号域”通常是指能够与细胞刺激信号分子，例如TCR/CD3结合，组合导致T细胞增殖和/或关键分子的上调或下调的信号的共刺激分子的胞内结构域。共刺激分子通常为T细胞上的关连结合性配偶体，其特异性结合共刺激配体，由此介导T细胞的共刺激反应，例如，但不限于增殖。共刺激分子是有效免疫反应所需的、非抗原受体的细胞表面分子或其配体。共刺激分子包括但不限于，MHC I类分子、BTLA和Toll配体受体、以及OX40、CD2、CD27、CD28、CDS、ICAM-1、LFA-1(CD11a/CD18)和4-1BB(CD137)。
术语“CD3δ(也称为CD3 Zeta)”定义为GenBan登录号BAG36664.1提供的蛋白质、或来自非人类物种例如小鼠、啮齿类动物、猴、猿等的等价残基。“CD3δ结构域”定义为来自ξ链的胞质结构域的氨基酸残基，其足以功能性地传递T细胞活化所需的初始信号。一方面，ξ的胞质结构域包含GenBan登录号BAG36664.1的残基52至164、其功能性直向同源物—来自非人物种例如小鼠、啮齿类动物、猴、猿等的等价残基。
发明详述
抗体
在本文中，描述了利用本领域常规杂交瘤抗体制备技术，通过免疫小鼠获得杂交瘤抗体，以及构建重组杂交瘤抗体(scFv-Fc)，还描述了具有基于Fab(抗原结合片段)的抗原结合区域的抗原结合蛋白，包括抗体Fab。其中使用人GPRC5D抗原(恺佧生物)，从全人的天然的Fab噬菌体文库选择Fab。这些分子展示了精细的特异性。例如，该抗体仅识别GPRC5D，及过表达GPRC5D的293T细胞、CHO-K1细胞、MM.1S细胞和NCI-H929细胞，不识别不表达GPRC5D的细胞。本申请中如果没有特别说明，本文GPRC5D指人的GPRC5D或鼠的GPRC5D。
在一些实施方案中，本申请包括具有Fab序列的抗体，所述Fab序列与一个或多个重链恒定区域融合以形成具有人免疫球蛋白Fc区的抗体以产生双价蛋白，从而增加抗体的总体亲和力和稳定性。此外，Fc部分允许将其他分子(包括但不限于荧光染料、细胞毒素、放射性同位素等)与例如用于抗原定量研究中的抗体直接缀合，以便固定抗体用于亲和力测量、用于定向递送治疗药、使用免疫效应细胞测试Fc介导的细胞毒性和许多其它应用。
本文提供的结果突出本申请抗体在靶向GPRC5D时的特异性、灵敏性和效用。
本申请的抗体或抗体片段基于使用噬菌体展示鉴定和选择抗原结合片段(Fab)，所述抗原结合片段的氨基酸序列赋予抗体或抗体片段针对GPRC5D的特异性并且形成本公开的全部抗原结合蛋白的基础。因此，所述Fab可以用来设计一系列不同“抗体或抗体片段”，包括例如全长抗体、其片段如F(ab’)2、融合蛋白、多价抗体、scFv-Fc抗体，即，具有针对相同抗原或不 同抗原的多于一种特异性的抗体，例如，双特异性T细胞结合抗体(BiTE)、三抗体等(见Cuesta等人，Multivalent antibodies:when design surpasses evolution,Trends in Biotechnology 28:355-362,2010)。
在具体实施例中，本申请提供全长抗体，其重链和轻链可以是全长(例如，抗体可以包括至少一条，优选地两条，完整重链，和至少一条，优选地两条，完整轻链)或可以包括抗原结合部分(Fab、F(ab’)2、Fv或scFv)。在其他实施方案中，抗体重链恒定区选自例如IgG1、IgG2、IgG3、IgG4、IgM、IgA1、IgA2、IgD和IgE。抗体类型的选择将取决于所设计的抗体欲引发的免疫效应子功能。在构建重组免疫球蛋白时，各种免疫球蛋白同种型的恒定区的适宜氨基酸序列和用于产生广泛种类抗体的方法是本领域技术人员已知的。
本申请提供了识别GPRC5D的抗体，其包含重链可变区中的至少一个CDR，所述重链可变区包含如SEQ ID NO:23、27、31、35、39、76或80中任一项所示的氨基酸序列或其变体，或包含与上述任一序列具有至少80％相似性的氨基酸序列；和/或其包含轻链可变区中的至少一个CDR，所述轻链可变区包含如SEQ ID NO:25、29、33、37、41或78中任一项所示的氨基酸序列或其变体，或包含与上述任一序列具有至少80％相似性的氨基酸序列。例如，所述重链可变区包含如SEQ ID NO:23、27、31、35或39中任一项所示的氨基酸序列或其变体，或包含与上述任一序列具有至少80％相似性的氨基酸序列；和/或所述轻链可变区包含如SEQ ID NO:25、29、33、37或41中任一项所示的氨基酸序列或其变体，或包含与上述任一序列具有至少80％相似性的氨基酸序列。例如，所述重链可变区包含如SEQ ID NO:23、76或80中任一项所示的氨基酸序列或其变体，或包含与上述任一序列具有至少80％相似性的氨基酸序列；和/或所述轻链可变区包含如SEQ ID NO:25或78中任一项所示的氨基酸序列或其变体，或包含与上述任一序列具有至少80％相似性的氨基酸序列。其中，所述CDR可通过选自如下的编号系统确定：Kabat、Chothia、IMGT、Gelfand、Aho和Martin。本申请所提供的识别GPRC5D的抗体可以包含通过上述任意编号系统确定的某个CDR序列或其组合，组成抗体的重链和/或轻链中的CDR不必是通过相同的编号确定的，例如，抗体中的一个或一些CDR可通过Kabat系统确定，其他CDR可通过上述编号系统的任意一种或其组合确定。在某些实施方式中，所述抗体的CDR可以是通过一种编号系统确定的，例如，可通过Kabat编号系统确定；例如，可通过Chothia编号系统确定；例如，可通过IMGT编号系统确定；例如，可通过Gelfand编号系统确定；例如，可通过Aho编号系统确定；例如，可通过Martin编号系统确定。
在某些实施方式中，所述抗体可以包含重链可变区中的一个CDR、两个CDR或三个CDR。
在某些实施方式中，所述抗体可以包含轻链可变区中的一个CDR、两个CDR或三个CDR。
在某些实施方式中，所述抗体可以包含重链可变区中的一个CDR、两个CDR或三个CDR，还包含轻链可变区中的一个CDR、两个CDR或三个CDR。例如，可以包含重链可变区中的一个CDR，还包含轻链可变区中的一个CDR、两个CDR或三个CDR；例如，可以包含重链可变区中的两个CDR，还包含轻链可变区中的一个CDR、两个CDR或三个CDR；例如，可以包含 重链可变区中的三个CDR，还包含轻链可变区中的一个CDR、两个CDR或三个CDR；例如，可以包含重链可变区中的三个CDR，还包含轻链可变区中的三个CDR。
在某些实施方式中，所述重链可变区包含如SEQ ID NO:23、27、31、35、39、76或80中任一项所示的氨基酸序列或其变体。
在某些实施方式中，所述轻链可变区包含如SEQ ID NO:25、29、33、37、41或78中任一项所示的氨基酸序列或其变体。
在某些实施方式中，所述重链可变区包含如SEQ ID NO:23、27、31、35、39、76或80中任一项所示的氨基酸序列或其变体，且所述轻链可变区包含如SEQ ID NO:25、29、33、37、41或78中任一项所示的氨基酸序列或其变体。例如，所述重链可变区包含如SEQ ID NO:23所示的氨基酸序列或其变体，且所述轻链可变区包含如SEQ ID NO:25、29、33、37、41或78中任一项所示的氨基酸序列或其变体；例如，所述重链可变区包含如SEQ ID NO:27所示的氨基酸序列或其变体，且所述轻链可变区包含如SEQ ID NO:25、29、33、37、41或78中任一项所示的氨基酸序列或其变体；例如，所述重链可变区包含如SEQ ID NO:31所示的氨基酸序列或其变体，且所述轻链可变区包含如SEQ ID NO:25、29、33、37、41或78中任一项所示的氨基酸序列或其变体；例如，所述重链可变区包含如SEQ ID NO:35所示的氨基酸序列或其变体，且所述轻链可变区包含如SEQ ID NO:25、29、33、37、41或78中任一项所示的氨基酸序列或其变体；例如，所述重链可变区包含如SEQ ID NO:39所示的氨基酸序列或其变体，且所述轻链可变区包含如SEQ ID NO:25、29、33、37、41或78中任一项所示的氨基酸序列或其变体；例如，所述重链可变区包含如SEQ ID NO:76所示的氨基酸序列或其变体，且所述轻链可变区包含如SEQ ID NO:25、29、33、37、41或78中任一项所示的氨基酸序列或其变体；例如，所述重链可变区包含如SEQ ID NO:80所示的氨基酸序列或其变体，且所述轻链可变区包含如SEQ ID NO:25、29、33、37、41或78中任一项所示的氨基酸序列或其变体。
在某些实施方式中，所述重链可变区包含如SEQ ID NO:23、27、31、35或39中任一项所示的氨基酸序列或其变体。
在某些实施方式中，所述轻链可变区包含如SEQ ID NO:25、29、33、37或41中任一项所示的氨基酸序列或其变体。
在某些实施方式中，所述重链可变区包含如SEQ ID NO:23、27、31、35或39中任一项所示的氨基酸序列或其变体，且所述轻链可变区包含如SEQ ID NO:25、29、33、37或41中任一项所示的氨基酸序列或其变体。例如，所述重链可变区包含如SEQ ID NO:23所示的氨基酸序列或其变体，且所述轻链可变区包含如SEQ ID NO:25、29、33、37或41中任一项所示的氨基酸序列或其变体；例如，所述重链可变区包含如SEQ ID NO:27所示的氨基酸序列或其变体，且所述轻链可变区包含如SEQ ID NO:25、29、33、37或41中任一项所示的氨基酸序列或其变体；例如，所述重链可变区包含如SEQ ID NO:31所示的氨基酸序列或其变体，且所述轻链可变区包含如SEQ ID NO:25、29、33、37或41中任一项所示的氨基酸序列或其变体；例如，所 述重链可变区包含如SEQ ID NO:35所示的氨基酸序列或其变体，且所述轻链可变区包含如SEQ ID NO:25、29、33、37或41中任一项所示的氨基酸序列或其变体；例如，所述重链可变区包含如SEQ ID NO:39所示的氨基酸序列或其变体，且所述轻链可变区包含如SEQ ID NO:25、29、33、37或41中任一项所示的氨基酸序列或其变体。
在某些实施方式中，所述重链可变区包含如SEQ ID NO:23、76或80中任一项所示的氨基酸序列或其变体。
在某些实施方式中，所述轻链可变区包含如SEQ ID NO:25或78中任一项所示的氨基酸序列或其变体。
在某些实施方式中，所述重链可变区包含如SEQ ID NO:23、76或80中任一项所示的氨基酸序列或其变体，且所述轻链可变区包含如SEQ ID NO:25或78中任一项所示的氨基酸序列或其变体。例如，所述重链可变区包含如SEQ ID NO:23所示的氨基酸序列或其变体，且所述轻链可变区包含如SEQ ID NO:25或78中任一项所示的氨基酸序列或其变体；例如，所述重链可变区包含如SEQ ID NO:76所示的氨基酸序列或其变体，且所述轻链可变区包含如SEQ ID NO:25或78中任一项所示的氨基酸序列或其变体；例如，所述重链可变区包含如SEQ ID NO:80所示的氨基酸序列或其变体，且所述轻链可变区包含如SEQ ID NO:25或78中任一项所示的氨基酸序列或其变体。
例如，所述重链可变区包含如SEQ ID NO:23所示的氨基酸序列，且所述轻链可变区包含如SEQ ID NO:25所示的氨基酸序列。例如，所述重链可变区包含如SEQ ID NO:27所示的氨基酸序列，且所述轻链可变区包含如SEQ ID NO:29所示的氨基酸序列。例如，所述重链可变区包含如SEQ ID NO:31所示的氨基酸序列，且所述轻链可变区包含如SEQ ID NO:33所示的氨基酸序列。例如，所述重链可变区包含如SEQ ID NO:35所示的氨基酸序列，且所述轻链可变区包含如SEQ ID NO:37所示的氨基酸序列。例如，所述重链可变区包含如SEQ ID NO:39所示的氨基酸序列，且所述轻链可变区包含如SEQ ID NO:41所示的氨基酸序列。例如，所述重链可变区包含如SEQ ID NO:76所示的氨基酸序列，且所述轻链可变区包含如SEQ ID NO:25所示的氨基酸序列。例如，所述重链可变区包含如SEQ ID NO:80所示的氨基酸序列，且所述轻链可变区包含如SEQ ID NO:25所示的氨基酸序列。例如，所述重链可变区包含如SEQ ID NO:23所示的氨基酸序列，且所述轻链可变区包含如SEQ ID NO:78所示的氨基酸序列。
在某些实施方式中，所述抗体可以包含所述重链可变区和/或所述轻链可变区。例如，所述重链可变区可以包含重链CDR1(HCDR1)，所述HCDR1可以包含如SEQ ID NO:1、11、17、70或71任一所示的氨基酸序列。例如，所述重链可变区可以包含重链CDR2(HCDR2)，所述HCDR2可以包含如SEQ ID NO:2、12、18、72或73任一所示的氨基酸序列。例如，所述重链可变区可以包含重链CDR3(HCDR3)，所述HCDR3可以包含如SEQ ID NO:3、7、9、13或19任一所示的氨基酸序列。例如，所述重链可变区可以包含HCDR1和HCDR3，所述HCDR1可以包含如SEQ ID NO:1、11、17、70或71任一所示的氨基酸序列，所述HCDR3可以包含如SEQ ID NO: 3、7、9、13或19任一所示的氨基酸序列。例如，所述重链可变区可以包含HCDR1和HCDR2，所述HCDR1可以包含如SEQ ID NO:1、11、17、70或71任一所示的氨基酸序列，所述HCDR2可以包含如SEQ ID NO:2、12、18、72或73任一所示的氨基酸序列。例如，所述重链可变区可以包含HCDR2和HCDR3，所述HCDR2可以包含如SEQ ID NO:2、12、18、72或73任一所示的氨基酸序列，所述HCDR3可以包含如SEQ ID NO:3、7、9、13或19任一所示的氨基酸序列。例如，所述重链可变区可以包含HCDR1，HCDR2和HCDR3，所述HCDR1可以包含如SEQ ID NO:1、11、17、70或71任一所示的氨基酸序列，所述HCDR2可以包含如SEQ ID NO:2、12、18、72或73任一所示的氨基酸序列，所述HCDR3可以包含如SEQ ID NO:3、7、9、13或19任一所示的氨基酸序列。例如，所述轻链可变区可以包含轻链CDR1(LCDR1)，所述LCDR1可以包含如SEQ ID NO:4、14、20或74任一所示的氨基酸序列。例如，所述轻链可变区可以包含轻链CDR2(LCDR2)，所述LCDR2可以包含如SEQ ID NO:5、15、21或75任一所示的氨基酸序列。例如，所述轻链可变区可以包含轻链CDR3(LCDR3)，所述LCDR3可以包含如SEQ ID NO:6、8、10、16或22任一所示的氨基酸序列。例如，所述轻链可变区可以包含LCDR1和LCDR3，所述LCDR1可以包含如SEQ ID NO:4、14、20或74任一所示的氨基酸序列，所述LCDR3可以包含如SEQ ID NO:6、8、10、16或22任一所示的氨基酸序列。例如，所述轻链可变区可以包含LCDR1和LCDR2，所述LCDR1可以包含如SEQ ID NO:4、14、20或74任一所示的氨基酸序列，所述LCDR2可以包含如SEQ ID NO:5、15、21或75任一所示的氨基酸序列。例如，所述轻链可变区可以包含LCDR2和LCDR3，所述LCDR2可以包含如SEQ ID NO:5、15、21或75任一所示的氨基酸序列，所述LCDR3可以包含如SEQ ID NO:6、8、10、16或22任一所示的氨基酸序列。例如，所述轻链可变区可以包含LCDR1、LCDR2和LCDR3，所述LCDR1可以包含如SEQ ID NO:4、14、20或74任一所示的氨基酸序列，所述LCDR2可以包含如SEQ ID NO:5、15、21或75任一所示的氨基酸序列，所述LCDR3可以包含如SEQ ID NO:6、8、10、16或22任一所示的氨基酸序列。
在某些实施方式中，所述抗体可以包含所述重链可变区和/或所述轻链可变区。例如，所述重链可变区可以包含重链CDR1(HCDR1)，所述HCDR1可以包含如SEQ ID NO:1、11或17任一所示的氨基酸序列。例如，所述重链可变区可以包含重链CDR2(HCDR2)，所述HCDR2可以包含如SEQ ID NO:2、12或18任一所示的氨基酸序列。例如，所述重链可变区可以包含重链CDR3(HCDR3)，所述HCDR3可以包含如SEQ ID NO:3、7、9、13或19任一所示的氨基酸序列。例如，所述重链可变区可以包含HCDR1和HCDR3，所述HCDR1可以包含如SEQ ID NO:1、11或17任一所示的氨基酸序列，所述HCDR3可以包含如SEQ ID NO:3、7、9、13或19任一所示的氨基酸序列。例如，所述重链可变区可以包含HCDR1和HCDR2，所述HCDR1可以包含如SEQ ID NO:1、11或17任一所示的氨基酸序列，所述HCDR2可以包含如SEQ ID NO:2、12或18任一所示的氨基酸序列。例如，所述重链可变区可以包含HCDR2和HCDR3，所述HCDR2可以包含如SEQ ID NO:2、12或18任一所示的氨基酸序列，所述HCDR3可以包含如SEQ ID NO:3、7、 9、13或19任一所示的氨基酸序列。例如，所述重链可变区可以包含HCDR1，HCDR2和HCDR3，所述HCDR1可以包含如SEQ ID NO:1、11或17任一所示的氨基酸序列，所述HCDR2可以包含如SEQ ID NO:2、12或18任一所示的氨基酸序列，所述HCDR3可以包含如SEQ ID NO:3、7、9、13或19任一所示的氨基酸序列。例如，所述轻链可变区可以包含轻链CDR1(LCDR1)，所述LCDR1可以包含如SEQ ID NO:4、14或20任一所示的氨基酸序列。例如，所述轻链可变区可以包含轻链CDR2(LCDR2)，所述LCDR2可以包含如SEQ ID NO:5、15或21任一所示的氨基酸序列。例如，所述轻链可变区可以包含轻链CDR3(LCDR3)，所述LCDR3可以包含如SEQ ID NO:6、8、10、16或22任一所示的氨基酸序列。例如，所述轻链可变区可以包含LCDR1和LCDR3，所述LCDR1可以包含如SEQ ID NO:4、14或20任一所示的氨基酸序列，所述LCDR3可以包含如SEQ ID NO:6、8、10、16或22任一所示的氨基酸序列。例如，所述轻链可变区可以包含LCDR1和LCDR2，所述LCDR1可以包含如SEQ ID NO:4、14或20任一所示的氨基酸序列，所述LCDR2可以包含如SEQ ID NO:5、15或21任一所示的氨基酸序列。例如，所述轻链可变区可以包含LCDR2和LCDR3，所述LCDR2可以包含如SEQ ID NO:5、15或21任一所示的氨基酸序列，所述LCDR3可以包含如SEQ ID NO:6、8、10、16或22任一所示的氨基酸序列。例如，所述轻链可变区可以包含LCDR1、LCDR2和LCDR3，所述LCDR1可以包含如SEQ ID NO:4、14或20任一所示的氨基酸序列，所述LCDR2可以包含如SEQ ID NO:5、15或21任一所示的氨基酸序列，所述LCDR3可以包含如SEQ ID NO:6、8、10、16或22任一所示的氨基酸序列。
在某些实施方式中，所述抗体可以包含所述重链可变区和/或所述轻链可变区。例如，所述重链可变区可以包含重链CDR1(HCDR1)，所述HCDR1可以包含如SEQ ID NO:1、70或71任一所示的氨基酸序列。例如，所述重链可变区可以包含重链CDR2(HCDR2)，所述HCDR2可以包含如SEQ ID NO:2、72或73任一所示的氨基酸序列。例如，所述重链可变区可以包含重链CDR3(HCDR3)，所述HCDR3可以包含如SEQ ID NO:3所示的氨基酸序列。例如，所述重链可变区可以包含HCDR1和HCDR3，所述HCDR1可以包含如SEQ ID NO:1、70或71任一所示的氨基酸序列，所述HCDR3可以包含如SEQ ID NO:3所示的氨基酸序列。例如，所述重链可变区可以包含HCDR1和HCDR2，所述HCDR1可以包含如SEQ ID NO:1、70或71任一所示的氨基酸序列，所述HCDR2可以包含如SEQ ID NO:2、72或73任一所示的氨基酸序列。例如，所述重链可变区可以包含HCDR2和HCDR3，所述HCDR2可以包含如SEQ ID NO:2、72或73任一所示的氨基酸序列，所述HCDR3可以包含如SEQ ID NO:3所示的氨基酸序列。例如，所述重链可变区可以包含HCDR1，HCDR2和HCDR3，所述HCDR1可以包含如SEQ ID NO:1、70或71任一所示的氨基酸序列，所述HCDR2可以包含如SEQ ID NO:2、72或73任一所示的氨基酸序列，所述HCDR3可以包含如SEQ ID NO:3所示的氨基酸序列。例如，所述轻链可变区可以包含轻链CDR1(LCDR1)，所述LCDR1可以包含如SEQ ID NO:4或74任一所示的氨基酸序列。例如，所述轻链可变区可以包含轻链CDR2(LCDR2)，所述LCDR2可以包含如SEQ ID NO:5或75任一所示的氨基酸序列。例如，所述轻链可变区可以包含轻链CDR3(LCDR3)，所述LCDR3可以包 含如SEQ ID NO:6所示的氨基酸序列。例如，所述轻链可变区可以包含LCDR1和LCDR3，所述LCDR1可以包含如SEQ ID NO:4或74任一所示的氨基酸序列，所述LCDR3可以包含如SEQ ID NO:6所示的氨基酸序列。例如，所述轻链可变区可以包含LCDR1和LCDR2，所述LCDR1可以包含如SEQ ID NO:4或74任一所示的氨基酸序列，所述LCDR2可以包含如SEQ ID NO:5或75任一所示的氨基酸序列。例如，所述轻链可变区可以包含LCDR2和LCDR3，所述LCDR2可以包含如SEQ ID NO:5或75任一所示的氨基酸序列，所述LCDR3可以包含如SEQ ID NO:6所示的氨基酸序列。例如，所述轻链可变区可以包含LCDR1、LCDR2和LCDR3，所述LCDR1可以包含如SEQ ID NO:4或74任一所示的氨基酸序列，所述LCDR2可以包含如SEQ ID NO:5或75任一所示的氨基酸序列，所述LCDR3可以包含如SEQ ID NO:6所示的氨基酸序列。
例如，所述抗体可以包含所述重链可变区和所述轻链可变区，所述重链可变区可以包含所述HCDR1、所述HCDR2、所述HCDR3，且所述轻链可变区可以包含所述LCDR1、所述LCDR2、所述LCDR3。例如，所述HCDR1可以包含如SEQ ID NO:1所示的氨基酸序列、所述HCDR2可以包含如SEQ ID NO:2所示的氨基酸序列、所述HCDR3可以包含如SEQ ID NO:3所示的氨基酸序列，且所述LCDR1可以包含如SEQ ID NO:4所示的氨基酸序列、所述LCDR2可以包含如SEQ ID NO:5所示的氨基酸序列、所述LCDR3可以包含如SEQ ID NO:6所示的氨基酸序列。例如，所述HCDR1可以包含如SEQ ID NO:1所示的氨基酸序列、所述HCDR2可以包含如SEQ ID NO:2所示的氨基酸序列、所述HCDR3可以包含如SEQ ID NO:7所示的氨基酸序列，且所述LCDR1可以包含如SEQ ID NO:4所示的氨基酸序列、所述LCDR2可以包含如SEQ ID NO:5所示的氨基酸序列、所述LCDR3可以包含如SEQ ID NO:8所示的氨基酸序列。例如，所述HCDR1可以包含如SEQ ID NO:1所示的氨基酸序列、所述HCDR2可以包含如SEQ ID NO:2所示的氨基酸序列、所述HCDR3可以包含如SEQ ID NO:9所示的氨基酸序列，且所述LCDR1可以包含如SEQ ID NO:4所示的氨基酸序列、所述LCDR2可以包含如SEQ ID NO:5所示的氨基酸序列、所述LCDR3可以包含如SEQ ID NO:10所示的氨基酸序列。例如，所述HCDR1可以包含如SEQ ID NO:11所示的氨基酸序列、所述HCDR2可以包含如SEQ ID NO:12所示的氨基酸序列、所述HCDR3可以包含如SEQ ID NO:13所示的氨基酸序列，且所述LCDR1可以包含如SEQ ID NO:14所示的氨基酸序列、所述LCDR2可以包含如SEQ ID NO:15所示的氨基酸序列、所述LCDR3可以包含如SEQ ID NO:16所示的氨基酸序列。例如，所述HCDR1可以包含如SEQ ID NO:17所示的氨基酸序列、所述HCDR2可以包含如SEQ ID NO:18所示的氨基酸序列、所述HCDR3可以包含如SEQ ID NO:19所示的氨基酸序列，且所述LCDR1可以包含如SEQ ID NO:20所示的氨基酸序列、所述LCDR2可以包含如SEQ ID NO:21所示的氨基酸序列、所述LCDR3可以包含如SEQ ID NO:22所示的氨基酸序列。例如，所述HCDR1可以包含如SEQ ID NO:70所示的氨基酸序列、所述HCDR2可以包含如SEQ ID NO:72所示的氨基酸序列、所述HCDR3可以包含如SEQ ID NO:3所示的氨基酸序列，且所述LCDR1可以包含如SEQ ID NO:4所示的氨基酸序列、所述LCDR2可以包含如SEQ ID NO:5所示的氨基酸序列、所述LCDR3可以包含如SEQ ID NO: 6所示的氨基酸序列。例如，所述HCDR1可以包含如SEQ ID NO:71所示的氨基酸序列、所述HCDR2可以包含如SEQ ID NO:73所示的氨基酸序列、所述HCDR3可以包含如SEQ ID NO:3所示的氨基酸序列，且所述LCDR1可以包含如SEQ ID NO:4所示的氨基酸序列、所述LCDR2可以包含如SEQ ID NO:5所示的氨基酸序列、所述LCDR3可以包含如SEQ ID NO:6所示的氨基酸序列。
在某些实施方式中，所述抗体包含重链可变区，所述重链可变区包含SEQ ID NO:1、11或17任一所示氨基酸序列的重链CDR1，和/或包含SEQ ID NO:2、12或18任一所示氨基酸序列的重链CDR2，和/或包含SEQ ID NO:3、7、9、13或19任一所示氨基酸序列的重链CDR3。在某些实施方式中，本申请提供了识别GPRC5D的抗体包括：包含SEQ ID NO:4、14或20任一所示氨基酸序列的轻链CDR1，和/或包含SEQ ID NO:5、15或21任一所示氨基酸序列的轻链CDR2，和/或包含SEQ ID NO:6、8、10、16或22任一所示氨基酸序列的轻链CDR3。在某些实施方式中，本申请提供了识别GPRC5D的抗体包括：包含SEQ ID NO:1、11或17任一所示氨基酸序列的重链CDR1，和/或包含SEQ ID NO:2、12或18任一所示氨基酸序列的重链CDR2，和/或包含SEQ ID NO:3、7、9、13或19任一所示氨基酸序列的重链CDR3，和/或包含SEQ ID NO:4、14或20任一所示氨基酸序列的轻链CDR1，和/或包含SEQ ID NO:5、15或21任一所示氨基酸序列的轻链CDR2，和/或包含SEQ ID NO:6、8、10、16或22任一所示氨基酸序列的轻链CDR3。例如，所述识别GPRC5D的抗体包括：包含SEQ ID NO:1、11或17任一所示氨基酸序列的重链CDR1，和包含SEQ ID NO:2、12或18任一所示氨基酸序列的重链CDR2，和包含SEQ ID NO:3、7、9、13或19任一所示氨基酸序列的重链CDR3，和/或包含SEQ ID NO:4、14或20任一所示氨基酸序列的轻链CDR1，和包含SEQ ID NO:5、15或21任一所示氨基酸序列的轻链CDR2，和包含SEQ ID NO:6、8、10、16或22任一所示氨基酸序列的轻链CDR3。例如，所述识别GPRC5D的抗体包括：包含SEQ ID NO:1、11或17任一所示氨基酸序列的重链CDR1，和包含SEQ ID NO:2、12或18任一所示氨基酸序列的重链CDR2，和包含SEQ ID NO:3、7、9、13或19任一所示氨基酸序列的重链CDR3，和包含SEQ ID NO:4、14或20任一所示氨基酸序列的轻链CDR1，和包含SEQ ID NO:5、15或21任一所示氨基酸序列的轻链CDR2，和包含SEQ ID NO:6、8、10、16或22任一所示氨基酸序列的轻链CDR3。例如，所述识别GPRC5D的抗体包括：包含SEQ ID NO:1所示的HCDR1，SEQ ID NO:2所示的HCDR2和SEQ ID NO:3所示的HCDR3；SEQ ID NO:4所示的LCDR1，SEQ ID NO:5所示的LCDR2和SEQ ID NO:6所示的LCDR3；或包含SEQ ID NO:1所示的HCDR1，SEQ ID NO:2所示的HCDR2和SEQ ID NO:7所示的HCDR3；SEQ ID NO:4所示的LCDR1，SEQ ID NO:5所示的LCDR2和SEQ ID NO:8所示的LCDR3；或包含SEQ ID NO:1所示的HCDR1，SEQ ID NO:2所示的HCDR2和SEQ ID NO:9所示的HCDR3；SEQ ID NO:4所示的LCDR1，SEQ ID NO:5所示的LCDR2和SEQ ID NO:10所示的LCDR3。
在某些实施方式中，所述抗体可以包含所述重链可变区和/或所述轻链可变区。例如，所述重链可变区可以包含如SEQ ID NO:23、27、31、35、39、76或80任一所示的氨基酸序列。 例如，所述轻链可变区可以包含如SEQ ID NO:25、29、33、37、41或78任一所示的氨基酸序列。
在某些实施方式中，所述抗体可以包含所述重链可变区和/或所述轻链可变区。例如，所述重链可变区可以包含如SEQ ID NO:23、27、31、35或39任一所示的氨基酸序列。例如，所述轻链可变区可以包含如SEQ ID NO:25、29、33、37或41任一所示的氨基酸序列。
在某些实施方式中，所述抗体可以包含所述重链可变区和/或所述轻链可变区。例如，所述重链可变区可以包含如SEQ ID NO:23、76或80任一所示的氨基酸序列。例如，所述轻链可变区可以包含如SEQ ID NO:25或78任一所示的氨基酸序列。
例如，所述抗体可以包含所述重链可变区和所述轻链可变区。例如，所述重链可变区包含如SEQ ID NO:23所示的氨基酸序列，且所述轻链可变区包含如SEQ ID NO:25所示的氨基酸序列。例如，所述重链可变区包含如SEQ ID NO:27所示的氨基酸序列，且所述轻链可变区包含如SEQ ID NO:29所示的氨基酸序列。例如，所述重链可变区包含如SEQ ID NO:31所示的氨基酸序列，且所述轻链可变区包含如SEQ ID NO:33所示的氨基酸序列。例如，所述重链可变区包含如SEQ ID NO:35所示的氨基酸序列，且所述轻链可变区包含如SEQ ID NO:37所示的氨基酸序列。例如，所述重链可变区包含如SEQ ID NO:39所示的氨基酸序列，且所述轻链可变区包含如SEQ ID NO:41所示的氨基酸序列。例如，所述重链可变区包含如SEQ ID NO:76所示的氨基酸序列，且所述轻链可变区包含如SEQ ID NO:25所示的氨基酸序列。例如，所述重链可变区包含如SEQ ID NO:80所示的氨基酸序列，且所述轻链可变区包含如SEQ ID NO:25所示的氨基酸序列。例如，所述重链可变区包含如SEQ ID NO:23所示的氨基酸序列，且所述轻链可变区包含如SEQ ID NO:78所示的氨基酸序列。
在另一方面，本申请提供了识别GPRC5D的抗体，包含重链可变区，所述重链可变区包含SEQ ID NO:23、27、31、35或39任一所示的氨基酸序列、或上述序列的变体。
在另一方面，本申请提供了识别GPRC5D的抗体，包含轻链可变区，该轻链可变区包含SEQ ID NO:25、29、33、37或41任一所示的氨基酸序列、或上述序列的变体。
在另一方面，本申请提供了识别GPRC5D的抗体，包含上述重链可变区及轻链可变区的抗体。
考虑到这些重链可变区和轻链可变区序列各自可以结合GPRC5D，可以“混合和匹配”重链和轻链可变区序列来产生本申请的抗GPRC5D的结合分子。
在另一个方面，本申请提供了结合GPRC5D的抗体的变体或其片段的变体。因而本申请提供了抗体或其片段，具有与重链或轻链的可变区序列至少80％相同的重链和/或轻链可变区。优选的，重链和/或轻链可变区的氨基酸序列同一性是至少85％，更优选至少90％，最优选至少95％，特别是96％，更特别97％，甚至更特别98％，最特别99％，包括例如80％，81％，82％，83％，84％，85％，86％，87％，88％，89％，90％，91％，92％，93％，94％，95％，96％，97％，98％，99％和100％。变体可以以本申请所述的抗体为母本抗体，通过酵母库筛选、噬菌体库筛 选、点突变等方法得到。
在另一个方面，本申请提供了与前述的抗GPRC5D的抗体识别相同的抗原决定部位的抗体。
在另一个方面，本申请提供了与前述的抗GPRC5D的抗体竞争性结合GPRC5D的抗体。
在另一个方面，本申请提供了特异性结合GPRC5D的抗体，所述抗体是全抗、scFv、单域抗体、Fab片段、Fab’片段、Fv片段、F(ab’)2片段、Fd片段、dAb片段、多功能抗体或scFv-Fc抗体。
抗体测定
通过本领域已知的多种测定可以鉴定、筛选本文中提供的抗GPRC5D抗体或表征其物理/化学性质和/或生物学活性。包括例如ELISA、biacore、Western印迹和流式细胞仪分析。合适的测定详细描述在实施例中。
术语“亲和力”是指分子(例如：抗体)的单个结合位点及其结合配体(例如：抗原)之间非共价相互作用的力量总和。除非另外指出，如本文中使用的“结合亲和力”是指固有结合亲和力，其反应结合对的成员(例如：抗体和抗原)之间1：1相互作用。分子X对其配体Y的亲和力通常可以由解离常数(Kd)代表。亲和力可以通过本领域中已知的常规方法测量，所述方法包括本文中所述的利用Biacore测定抗体的亲和力。本文中的抗体对GPRC5D的“亲和力”以抗体的KD表示。抗体的KD是指抗体——抗原相互作用的平衡解离常数。抗体结合其抗原的KD值越大，其对所述具体抗原的结合亲和力越弱。
术语“EC50”，半最大效应浓度(concentration for 50％of maximal effect,EC50)是指能引起50％最大效应的浓度。
抗原
术语“抗原”是指被抗原结合单元识别并特异性结合的物质。抗原可以包括肽、蛋白质、糖蛋白、多糖和脂质，其部分及其组合。非限制性示例性抗原包括肿瘤抗原或病原体抗原。“抗原”也可以指引发免疫反应的分子。这种免疫反应可能涉及抗体产生或特定免疫活性细胞(immunologically-competent cells)的活化，或两者兼有。本领域技术人员将理解，任何大分子，包括实际上所有的蛋白质或肽，都可以作为抗原。
术语“表位”指可被抗体、B细胞、T细胞或工程化细胞识别的抗原或部分抗原。例如，表位可以是被抗体识别的肿瘤表位或病原体表位；抗体识别抗原内的多个表位。表位也可以突变。
术语“抗原决定部位”又称“抗原表位”或“表位”或“抗原决定簇”，包括任何能够被抗体结合的决定簇或区域。抗原表位是抗原中被靶向所述抗原的抗体结合的区域，包括与抗体直接接触的特定氨基酸。示例性，抗原表位可以由GPRC5D蛋白序列的连续序列组成，也可以由GPRC5D蛋白序列不连续的三维结构组成。示例性，本文中使用的抗原是人或鼠的GPRC5D。
免疫缀合物
本申请还提供了免疫缀合物，所述的免疫辍合物包括本文所述的抗体，以及与之连 接的功能性分子。本申请提供的所述抗体已在前文描述，本申请提供的免疫缀合物包含其全部技术方案。所述抗体与所述功能性分子可以通过共价连接、偶联、附着、交联等方式构成缀合物。
“连接”或“融合”在本文中可互换使用。其通常是指通过包括化学缀合或重组方法的任何手段将两个以上化学元件或组件连接在一起。“框内融合”是指以维持原始开放阅读框(ORF)的正确阅读框的方式连接两个或更多个ORF以形式连续的较长的ORF。因此，所得到的重组融合蛋白是含有两个或更多个片段的单一蛋白质，这些片段对应于由原始ORF编码的多肽(这些片段在自然状态通常不是如此连接)。尽管阅读框因此在整个融合片段中是连续的，但这些片段可以在物理上或空间上通过例如框内连接序列(例如“flexon”)分开。
本申请另一方面提供了编码本申请的至少一种抗体、其功能变体或免疫缀合物的核酸分子。一旦获得了有关的序列，就可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。
本申请还涉及包含上述的适当DNA序列以及适当启动子或者控制序列的载体。这些载体可以用于转化适当的宿主细胞，以使其能够表达蛋白质。宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如哺乳动物细胞。
嵌合受体
本申请还提供了嵌合受体，其通常是指用基因重组技术将不同来源的DNA片段或蛋白质相应的cDNA连接而成的融合分子的表达产物，可以包括胞外域、跨膜域和胞内域。其中所述胞外域包含本申请提供的抗体，所述抗体已在前文详细描述，本申请提供的嵌合受体包含其全部技术方案。所述嵌合受体包括但不限于：嵌合抗原受体(CAR)、嵌合T细胞受体、T细胞抗原耦合器(TAC)。
在一些实施方案中，本申请的嵌合受体是嵌合抗原受体(CAR)。在某些实施方式中，CAR的胞外抗原结合区(或称为胞外域)来源于小鼠或人源化或人的单克隆抗体。
所述嵌合抗原受体通常包含胞外抗原结合区或者抗体。在一些实施例中，胞外抗原结合区可以是完全人的。在其他情况下，胞外抗原结合区可以是人源化的。在其他情况下，胞外抗原结合区可以是鼠源的，或者所述胞外抗原结合区中的嵌合体由来自至少两种不同动物的氨基酸序列组成。在一些实施例中，所述胞外抗原结合区可以是非人的。
在某些实施方式中，所述嵌合抗原受体还可以设计为包含多种抗原结合区，包括衍生自抗体的单链可变片段(scFv)、选自文库的片段抗原结合区(Fab)、单结构域片段或与接合其同源受体的自然配体。在一些实施例中，胞外抗原结合区可以包含scFv、Fab或天然配体，以及它们的任何衍生物。胞外抗原结合区可以指除完整抗体之外的分子，其可以包含完整抗体的一部分并且可以与完整抗体所结合的抗原结合。抗体片段的实例可以包括但不限于Fv、Fab、Fab'、Fab'-SH、F(ab')2；双功能抗体、线性抗体；单链抗体分子(例如scFv)；和由抗体片段形成的多特异性抗体。胞外抗原结合区，例如scFv、Fab或天然配体，可以 是确定抗原特异性的CAR的一部分。胞外抗原结合区可以结合任何互补靶。胞外抗原结合区可以衍生自已知可变区序列的抗体。胞外抗原结合区可以从获自可获得的小鼠杂交瘤的抗体序列中得到。或者，可以从肿瘤细胞或原代细胞例如肿瘤浸润淋巴细胞(TIL)的全外切割测序获得胞外抗原结合区。
在一些实施例中，CAR的胞外抗原结合区的结合特异性可以通过互补决定区或CDR，如轻链CDR或重链CDR来确定。在许多情况下，结合特异性可以通过轻链CDR和重链CDR来确定。
在一些实施例中，CAR的胞外抗原结合区包括铰链或间隔区，铰链和间隔区可以互换使用。铰链可以被认为是用于向胞外抗原结合区提供柔性的CAR的一部分。在一些实施例中，铰链可用于检测细胞的细胞表面上的CAR，特别是当检测胞外抗原结合区的抗体不起作用或不可用时。在一些实施例中，铰链可能不属于免疫球蛋白，而是属于另一种分子，如CD8α分子的天然铰链。CD8α铰链可以含有已知在CD8辅助受体和MHC分子的相互作用中起作用的半胱氨酸和脯氨酸残基。可以根据所使用的胞外抗原结合区来调节铰链。铰链可以是任何长度的。例如，所述铰链可以包含如SEQ ID NO:59所示的氨基酸序列。
CAR的跨膜结构域(或称为结构区)可以将CAR锚定在细胞的质膜上。CD28的天然跨膜部分可用于CAR。在其他情况下，也可以在CAR中使用CD8α的天然跨膜部分。“CD8”可以是与NCBI参考号：NP_001759或其具有刺激活性的片段具有至少85、90、95、96、97、98、99或100％同一性的蛋白质。“CD8核酸分子”可以是编码CD8多肽的多核苷酸，在某些情况下，跨膜区可以是CD28的天然跨膜部分，“CD28”可以指与NCBI参考号：NP_006130或其具有刺激活性的片段具有至少85、90、95、96、97、98、99或100％同一性的蛋白质。“CD28核酸分子”可以是编码CD28多肽的多核苷酸。在一些实施例中，跨膜部分可以包含CD8α区。例如，所述跨膜结构域可以包含如SEQ ID NO:60所示的氨基酸序列。
CAR的(细)胞内信号传导区可以负责活化包含所述CAR的免疫应答细胞的效应子功能中的至少一种。CAR可以诱导T细胞的效应子功能，例如，所述效应子功能是细胞溶解活性或辅助活性，包括细胞因子的分泌，如IL-2，TNF-α,γ-IFN等。因此，术语细胞内信号传导区是指转导效应子功能信号并引导细胞进行特异功能的蛋白质部分。虽然通常可以使用整个细胞内信号传导区，但是在许多情况下，不必使用信号结构域的整个链。在一些实施例中，使用细胞内信号传导区的截短部分。在一些实施例中，术语细胞内信号传导区因此意在包括足以转导效应子功能信号的细胞内信号传导区的任何截短部分。
在CAR中使用的信号结构域(或称为结构区)的优选实例可以包括T细胞受体(TCR)的细胞质序列和协同作用以在靶-受体结合之后启动信号转导的共同受体，以及它们的任何衍生物或变体序列和这些序列的具有相同功能性的任何合成序列。
在一些实施例中，所述CAR的细胞内信号传导区可以含有已知的免疫受体酪氨酸激活基序(ITAM)的信号基序。含有细胞质信号传导序列的ITAM的实例包括衍生自CD3δ、FcRγ、 FcRβ、CD3γ、CD3δ、CD3ε、CD5、CD22、CD79a、CD79b和CD66d的那些。然而，在优选的实施例中，细胞内信号结构域衍生自CD3δ链。
含有一个或多个ITAM基序的T细胞信号结构域的实例是CD3δ结构域，也称为T细胞受体CD3δ链或CD247。该结构域是T细胞受体-CD3复合物的一部分，并且在将几种细胞内信号转导途径的抗原识别与T细胞的主效应激活相结合方面起重要作用。如本文所用，CD3δ主要是指人类CD3δ及其同种型，如从Swissprot条目P20963所知的，包括具有基本相同序列的蛋白质。作为嵌合抗原受体的一部分，不需要全T细胞受体CD3δ链，并且其包含T细胞受体CD3δ链的信号结构域的任何衍生物都是合适的，包括其任何功能等同物。例如，所述CD3δ链的信号结构域可以包含如SEQ ID NO:62所示的氨基酸序列。
在某些实施方式中，CAR的细胞内信号传导结构域(或称为结构区)可以选自表2的任何一个共刺激结构域。在一些实施例中，可以修饰结构域，使得与参考结构域的同一性可以为约50％至约100％。可以修饰表1的任何一个结构域，使得修饰形式可以包含约50％、60％、70％、80％、90％、95％、96％、97％、98％、99％或至多约100％的同一性。
在某些实施方式中，CAR的细胞内信号传导区可以进一步包含一个或多个共刺激结构域。细胞内信号传导区可以包含单个共刺激结构域，例如δ链(第一代CAR)或其与CD28或4-1BB(第二代CAR)。在其他实例中，细胞内信号传导区可以包含两个共刺激结构域，例如CD28/OX40或CD28/4-1BB(第三代)。例如，所述4-1BB的共刺激结构域可以包含如SEQ ID NO:61所示的氨基酸序列。
在某些实施方式中，与细胞内信号结构域如CD8一起，这些共刺激结构域可以产生激酶途径的下游激活，从而支持基因转录和功能性细胞反应。CAR的共刺激结构域可以激活与CD28(磷脂酰肌醇-4,5-二磷酸3-激酶)或4-1BB/OX40(TNF-受体相关因子衔接蛋白)途径以及MAPK和Akt激活相关的近端信号蛋白。
在某些情况下，通过CAR产生的信号可能与辅助或共刺激信号相结合。对于共刺激信号结构域，嵌合抗原受体样复合物可被设计成包含若干可能的共刺激信号结构域。在本领域众所周知的，在幼稚T细胞中，T细胞受体的单独结合不足以诱导T细胞的完全活化为细胞毒性T细胞。完整的生产性T细胞激活需要第二共刺激信号。已经报道对T细胞活化提供共刺激的几种受体，包括但不限于CD28、OX40、CD27、CD2、CD5、ICAM-1、LFA-1(CD11a/CD18)、4-1BBL、MyD88和4-1BB。这些共刺激分子使用的信号传导途径均能与主T细胞受体激活信号协同作用。这些共刺激信号传导区提供的信号可以与源自一个或多个ITAM基序(例如CD3zeta信号转导域)的主效应激活信号协同作用，并且可以完成T细胞激活的要求。
在一些实施方案中，向嵌合抗原受体样复合物添加共刺激结构域可以增强工程细胞的功效和耐久性。在另一些实施方案中，T细胞信号结构域和共刺激结构域彼此融合从而构成信号传导区。
表2.共刺激结构域
在一些实施案例中，用编码CAR的病毒载体转导细胞(例如T细胞)。在一些实施案例中，病毒载体是慢病毒载体。一些实施方案中，细胞可以稳定地表达CAR。
在某些实施方式中，CAR的GPRC5D结合部分是scFv，与其所来自的Fab抗体相比，保持等价的亲和结合力，例如其以相当的功效结合相同抗原。该抗体片段是功能性的，其由此提供生物化学反应，例如激活免疫反应、抑制从其靶抗原的信号传导起始、抑制激酶活性等。例如，所述scFv可以包含如SEQ ID NO：43、82、84或86任一所示的序列。
在某些实施方式中，CAR的抗GPRC5D抗原结合结构域是全人源抗体片段。
在某些实施方式中，本申请CAR将特定抗体的抗原结合结构域和胞内信号传导分子组合在一起。例如，胞内信号传导分子包括但不限于，CD3δ链、4-1BB和CD28信号传导模块及其组合。
在某些实施方式中，GPRC5D-CAR包含至少一个胞内信号传导结构域，其选择CD137(4-1BB)信号传导结构域、CD28信号传导结构域、CD3δ信号结构域，及其任何组合。一方面，GPRC5D-CAR包含至少一个胞内信号传导结构域，其来自一个或多个非CD137(4-1BB)或CD28的共刺激分子。
作为示例性的，GPRC5D-CAR的序列可以是：
具有SEQ ID NO:43所示的胞外域、SEQ ID NO：59所示的铰链域、SEQID NO：60所示的跨膜域、SEQ ID NO：61所示的共刺激信号域、以及SEQ ID NO：62所示的初级信号域 (AB3-BBZ)。
具有SEQ ID NO:82所示的胞外域、SEQ ID NO：59所示的铰链域、SEQID NO：60所示的跨膜域、SEQ ID NO：61所示的共刺激信号域、以及SEQ ID NO：62所示的初级信号域(AB6-BBZ)。
具有SEQ ID NO:84所示的胞外域、SEQ ID NO：59所示的铰链域、SEQID NO：60所示的跨膜域、SEQ ID NO：61所示的共刺激信号域、以及SEQ ID NO：62所示的初级信号域(AB7-BBZ)。
具有SEQ ID NO:86所示的胞外域、SEQ ID NO：59所示的铰链域、SEQID NO：60所示的跨膜域、SEQ ID NO：61所示的共刺激信号域、以及SEQ ID NO：62所示的初级信号域(AB8-BBZ)。
示例性的，嵌合抗原受体的氨基酸序列如SEQ ID NO：63、88、89、90任一所示。
上述嵌合抗原受体的跨膜域和胞内域，本领域技术人员可以选择常规的跨膜域和胞内域进行替换，且均落入本申请的保护范围。
核酸、载体、病毒、宿主细胞
术语“核酸分子编码”、“编码DNA序列”和“编码DNA”是指沿着脱氧核糖核酸链的脱氧核糖核苷酸的顺序或顺序。这些脱氧核糖核苷酸的顺序决定了沿着多肽(蛋白质)链的氨基酸的顺序。因此，核酸序列编码氨基酸序列。
当用于指核苷酸序列时，本文所用的术语“序列”包括DNA或RNA，并且可以是单链或双链。
术语“靶序列”是指与指导序列具有互补性的序列，靶序列与指导序列之间互补配对促进CRISPR复合物的形成。一个靶序列可以包含任何多核苷酸，如DNA或RNA多核苷酸。在一些实施例中，靶序列位于细胞的细胞核或细胞质中。
术语序列“同一性”通过在比较窗口(例如至少20个位置)上比较两个经最佳匹配的序列来确定同一性百分比，其中比较窗口中多核苷酸或多肽序列的部分可以包含添加或缺失(即间隙)，例如对于最佳匹配的两个序列而言与参考序列(其不包含添加或缺失)相比20％或更少的间隙(例如5至15％、或10至12％)。通常通过确定在两个序列中发生相同的核酸碱基或氨基酸残基的位置的数目来计算百分比，以产生正确匹配的位置的数目，将正确匹配位置的数目除以参考序列中的位置总数(即窗口大小)，并将结果乘以100，以产生序列同一性的百分比。
术语“转染”是指将外源核酸引入真核细胞。转染可以通过本领域已知的各种手段来实现，包括磷酸钙-DNA共沉淀、DEAE-葡聚糖介导的转染、聚凝胺介导的转染、电穿孔、显微注射、脂质体融合、脂质转染、原生质体融合、逆转录病毒感染和生物弹道技术(biolistics)。
本文所用的术语“表达载体”是指包含重组多核苷酸的载体，其包含与待表达的核苷酸 序列有效连接的表达调控序列。表达载体包含用于表达的足够的顺式作用元件(cis-acting elements)；用于表达的其它元件可以由宿主细胞或体外表达系统提供。表达载体包括本领域所有已知的那些，如质粒、病毒(例如，慢病毒、逆转录病毒、腺病毒和腺相关病毒)。
本文使用的术语“载体”是包含分离的核酸并可用于将分离的核酸递送至细胞内部的组合物。在本领域中已知许多载体，包括但不限于线性多核苷酸、与离子或两亲化合物相关的多核苷酸、质粒和病毒。因此，术语“载体”包括自主复制的质粒或病毒。还可以包括促进核酸转移到细胞中的非质粒和非病毒化合物，例如聚赖氨酸化合物、脂质体等。
本文使用的术语“慢病毒”是指逆转录病毒科的属。逆转录病毒在能够感染非分裂细胞方面是逆转录病毒中独特的；它们可以将大量的遗传信息递送到宿主细胞的DNA中，因此它们是基因递送载体最有效的方法之一。HIV、SIV和FIV都是慢病毒的实例。源自慢病毒的载体提供了在体内实现显著水平的基因转移的手段。
术语“内源”是指一个核酸分子或多肽等来自生物体自身。
本文所用的术语“外源”指的是一个核酸分子或多肽、细胞、组织等没有在生物体自身内源性表达，或表达水平不足以实现过表达时具有的功能。
本文所用的术语“外源蛋白”可以是识别靶抗原的外源转入细胞的蛋白，如外源受体(即本文中前述“嵌合受体”)。
本文中使用的术语“宿主”是指接受移植物移植的受体，在一些实施方式中，可以是接受外源细胞植入的个体，如人。
术语“分离的”是指与细胞成分或其他成分相分离，在这些成分中，多核苷酸、肽、多肽、蛋白质、抗体或其片段在自然状态通常是相关联的。如本领域技术人员将理解，非天然存在的多核苷酸、肽、多肽、蛋白质、抗体或其片段不需要“分离”以将其与天然存在的对应物区分开。此外，“浓缩”、“分离”或“稀释”的多核苷酸、肽、多肽、蛋白质、抗体或其片段可与其天然存在的对应物区分开，因为每体积分子的浓度或数量大于(“浓缩”)或小于(“稀释”)其天然存在的配对物的浓度。富集程度可以以绝对基础测量，例如每溶液体积的重量，或者可以相对于存在于源混合物中的另一潜在的干扰物测量。在一些实施方案中，本申请的技术方案优选的富集程度更高。因此，例如，优选2倍富集、更优选10倍富集、更优选100倍富集、更优选1000倍富集。也可以通过人工组装的方法，例如通过化学合成或重组表达，从而提供“分离的”物质。
本申请提供了编码识别GPRC5D的抗体或其片段的分离的核酸、载体以及包含所述核酸或载体的宿主细胞。核酸可位于完整细胞中、细胞裂解液中或者以部分纯化的或基本纯化的形式。
可以使用标准的分子生物学技术获得本申请的核酸，例如可以通过标准的PCR扩增或cDNA克隆技术，获得编码抗体的轻链和重链或者编码VH和VL区段的cDNA。对于从免疫球蛋白基因文库获得的抗体(例如，使用噬菌体展示技术)，可以从文库回收编码抗体的一种或多种 核酸。向宿主细胞中导入外源核酸的方法是本领域普遍已知的，并可随所使用的宿主细胞而变化。
优选的，本申请核酸分子是选自编码重链可变区的SEQ ID NO:24、28、32、36、40、77或81，和/或选自编码轻链可变区的SEQ ID NO:26、30、34、38、42或79。更优选的，是这样的核酸分子，所述核酸分子包含SEQ ID NO:24的重链可变区序列，和包含SEQ ID NO:26的轻链可变区序列；或者包含SEQ ID NO:28的重链可变区序列，和包含SEQ ID NO:30的轻链可变区序列；或者包含SEQ ID NO:32的重链可变区序列，和包含SEQ ID NO:34的轻链可变区序列；或者包含SEQ ID NO:36的重链可变区序列，和包含SEQ ID NO:38的轻链可变区序列；或者包含SEQ ID NO:40的重链可变区序列，和包含SEQ ID NO:42的轻链可变区序列；或者包含SEQ ID NO:77的重链可变区序列，和包含SEQ ID NO:26的轻链可变区序列；或者包含SEQ ID NO:81的重链可变区序列，和包含SEQ ID NO:26的轻链可变区序列；或者包含SEQ ID NO:24的重链可变区序列，和包含SEQ ID NO:79的轻链可变区序列。
在一个实施方案中，提供一种或多种包含上述核酸的载体(例如，表达载体)。
术语“细胞”指人或非人动物来源的细胞。
术语“宿主细胞”指被引入外源核酸的细胞，包括此种细胞的后代。宿主细胞包括“转化体”和“转化的细胞”，其包括转化的原代细胞及来源于其的后代(不考虑传代次数)。后代的核酸内容可以与亲本细胞不完全相同，并且可以含有突变。本文中包括具有与对于原始转化的细胞中筛选或选择的相同的功能或生物学活性的突变体后代。
术语“GPRC5D阳性宿主细胞”是指在细胞表面上表达GPRC5D的宿主细胞，这些细胞可以通过例如使用抗体的流式细胞术来检测，这些抗体特异性识别GPRC5D上的表位。
在一些实施方案中，所述宿主细胞是免疫效应细胞。
术语“免疫效应细胞”是指参与免疫应答，产生免疫效应的细胞，如T细胞、B细胞、自然杀伤(NK)细胞、自然杀伤T(NKT)细胞、树突细胞、CIK细胞、巨噬细胞、肥大细胞等。在一些实施方案中，所述的免疫效应细胞为T细胞、NK细胞、NKT细胞。在一些实施方案中，所述T细胞可以是自体T细胞、异种T细胞、同种异体T细胞。在一些实施方案中，所述的NK细胞可以是同种异体NK细胞。“免疫效应功能或免疫效应应答”是指免疫效应细胞，例如增强或促进靶细胞的免疫攻击的功能或反应。例如，免疫效应功能或应答是指促进靶细胞的杀伤或者抑制生长或增殖的T细胞或NK细胞的属性。
术语“经人工改造的具有免疫效应细胞功能的细胞”是指不具有免疫效应的细胞或细胞系经人工改造或接受刺激物刺激后，该细胞获得了免疫效应细胞功能。如293T细胞，经人工改造，使其具有免疫效应细胞的功能；如干细胞，经体外诱导，使其分化成免疫效应细胞。
在一些情况下，“T细胞”可以是来自骨髓的多能干细胞，在胸腺内分化成熟成为具有免疫活性的成熟的T细胞。在一些情况下，“T细胞”可以是具有特定表型特征的细胞群，或不同表型特征的混合细胞群体，如“T细胞”可以是包含至少一种T细胞亚群的细胞：记 忆性干细胞样T细胞(stem cell-like memory T cells，Tscm细胞)、中心记忆T细胞(Tcm)、效应性T细胞(Tef、Teff)、调节性T细胞(tregs)和/或效应记忆T细胞(Tem)。在一些情况下，“T细胞”可以是某种特定亚型的T细胞，如γδT细胞。
T细胞可以从许多来源获得，包括PBMC、骨髓、淋巴结组织、脐带血、胸腺组织和来自感染部位、腹水、胸腔积液、脾组织和肿瘤的组织。在某些情况下，可以使用任何数量的本领域技术人员已知的技术，例如FicollTM分离，从个体收集的血液获得T细胞。在一个实施方案中，通过单采血获得来自个体的循环血液的细胞。单采制品通常含有淋巴细胞，包括T细胞、单核细胞、粒细胞、B细胞、其他有核白细胞、红细胞和血小板。在一个实施方案中，可以洗涤通过单采采集收集的细胞以除去血浆分子并将细胞置于合适的缓冲液或培养基中用于随后的加工步骤。在一个实施方案中，可以从健康供体，或来自诊断患有肿瘤的患者衍生细胞获得T细胞。
术语“外周血单个核细胞”(peripheral blood mononuclear cell,PBMC)是指外周血中具有单个核的细胞，包含淋巴细胞、单核细胞等。
术语“激活”和“活化”可互换使用，可以指细胞从静止状态转变为活性状态的过程。该过程可以包括对抗原、迁移和/或功能活性状态的表型或遗传变化的响应。例如，术语“激活”可以指NK细胞、T细胞逐步活化的过程。
术语“T细胞活化”或“T细胞激活”指被充分刺激以诱导可检测的细胞增殖、细胞因子产生和/或可检测的效应物功能的T细胞的状态。
术语“趋化因子”是一种分子量为8～10kDa的多肽，是最大的细胞因子家族，主要功能是招募血液中的单核细胞、中性粒细胞、淋巴细胞等进入特定的淋巴器官和组织以及感染发生的部位。“趋化因子受体”是一类介导趋化因子行使功能的七次跨膜G蛋白偶联受体(GPCR)，通常表达于免疫细胞、中性粒细胞、内皮细胞等细胞膜上。趋化因子及趋化因子受体在介导细胞迁移、增殖和抵御病原体入侵过程中发挥重要作用，并且与免疫环境中炎症和癌症的发生发展密切相关。
术语“安全开关”是为了提高CAR-T疗法的安全性，为CAR-T细胞设计一个快速且可逆的“关闭”或“开启”安全开关，最大限度地减少与治疗相关的毒性。尽管CAR-T细胞疗法具有出色的临床特征，但当肿瘤负荷不可预测且T细胞的活性不受控制时，引发严重的CRS等潜在致命副作用。为了控制毒性，严重的CRS需要使用CRS分级系统作为指导进行适当监测，并使用基于小分子的安全开关进行精确调节。
在另一实施方案中，提供包含上述核酸的宿主细胞。宿主细胞包含(例如，转导有)：(1)载体，所述载体包含核酸，所述核酸编码包含抗体VL的氨基酸序列和包含抗体VH的氨基酸序列，或(2)包含编码包含抗体VL的氨基酸序列的核酸的第一载体，和包含编码包含抗体VH的氨基酸序列的核酸的第二载体。在一个实施方案中，宿主细胞是真核的，例如，中国仓鼠卵巢(CHO)细胞、293T细胞、NIH3T3细胞或淋巴细胞(例如，YO、NSO、Sp20细胞)。
在另一实施方案中，所述宿主细胞表达本申请所述的嵌合受体。
在另一实施方案中，所述宿主细胞包括T细胞、自然杀伤细胞、细胞毒性T淋巴细胞、自然杀伤T细胞、DNT细胞、调节性T细胞、NK92细胞、和/或干细胞衍生的免疫效应细胞。
在另一实施方案中，所述T细胞为来源于天然的T细胞和/或经多能干细胞诱导产生的T细胞；优选地，所述T细胞为自体/同种异体T细胞；优选地，所述T细胞为原代T细胞；优选地，所述T细胞来源于人的自体T细胞。
在另一实施方案中，所述T细胞包含记忆性干细胞样T细胞(Tscm细胞)、中心记忆T细胞(Tcm)、效应性T细胞(Tef)、调节性T细胞(Tregs)，效应记忆T细胞(Tem)、γδT细胞或其组合。
在另一实施方案中，所述宿主细胞结合表达GPRC5D的细胞，不显著结合不表达GPRC5D的细胞。
在另一实施方案中，所述宿主细胞还携带外源的细胞因子的编码序列；和/或还表达非靶向GPRC5D的嵌合受体；和/或还表达趋化因子；和/或还表达趋化因子受体；和/或还表达安全开关。
在另一实施方案中，所述宿主细胞还携带外源的细胞因子包括IL-7、IL-12、IL-15、IL-18、IL-21、或I型干扰素的编码序列。
在另一实施方案中，所述宿主细胞还可以表达除了上述结合GPRC5D的受体以外的结合另一种抗原的嵌合受体。
在另一实施方案中，所述宿主细胞还表达趋化因子包括CCL19或CCL21。在另一实施方案中，所述宿主细胞还表达趋化因子受体包括CCR2、CCR4、CCR5、CXCR2、CXCR4或CXCR5。在另一实施方案中，所述宿主细胞还表达安全开关包括iCaspase-9、Truancated EGFR或RQR8。
在一个实施例中，提供制备抗GPRC5D抗体的方法，其中所述方法包括在适合于表达如上所述的抗体的条件下培养包含编码所述抗体的核酸的宿主细胞，和任选地从宿主细胞(或宿主细胞培养基)回收抗体。
为了表达蛋白质，可以将编码本申请抗体的核酸整合到表达载体中。多种表达载体可用于蛋白质表达。表达载体可包括自我复制的染色体外载体，或整合到宿主基因组中的载体。用于本申请的表达载体包括但不限于使蛋白质能够在哺乳动物细胞、细菌、昆虫细胞、酵母和体外系统中表达的那些。如本领域已知的，多种表达载体是可商业或其他方式获得的。可用于本申请中来表达抗体。
在一优选例中，所述宿主细胞与增强其功能的药剂组合施用，优选地，与化疗药物联用；和/或所述宿主细胞与改善其相关的一种或多种副作用的药剂联合施用；和/或所述宿主细胞与表达靶向GPRC5D之外的嵌合抗原受体的宿主细胞联合施用。
药物组合物
本申请的抗体、包含该抗体的免疫辍合物、嵌合受体、宿主细胞可以应用于制备药物组 合物或诊断试剂。所述的组合物除了包括有效量的所述抗体、免疫辍合物、嵌合受体、核酸或宿主细胞，还可包含药学上可接受的载体。
术语“药学上可接受的”是指当分子本体和组合物适当地给予动物或人时，它们不会产生不利的、过敏的或其它不良反应。
在一些实施方案中，所述组合物包含另一治疗剂。在一些实施方案中，所述另一治疗剂是化疗剂，如US20140271820中记载的那些和/或其药学上可接受的盐或类似物。在一些实施方案中，所述治疗剂包括但不限于有丝分裂抑制剂(长春花生物碱)，包括长春新碱、长春花碱、长春地辛和诺维宾(TM)(长春瑞滨，5'-去氢硫化氢)；拓扑异构酶I抑制剂，例如喜树碱化合物，包括CamptosarTM(伊立替康HCL)、HycamtinTM(托泊替康HCL)和衍生自喜树碱及其类似物的其它化合物；鬼臼毒素衍生物，例如依托泊苷、替尼泊苷和米多昔佐兹；烷基化剂顺铂、环磷酰胺、氮芥、三亚甲基硫代磷酰胺、卡莫司汀、白消安、苯丁酸氮芥、布列喹嗪、尿嘧啶芥末、氯洛芬和达卡巴嗪；抗代谢物，包括阿糖胞苷、5-氟尿嘧啶、甲氨蝶呤、巯嘌呤、硫唑嘌呤和丙卡巴肼；抗生素，包括但不限于多柔比星、博来霉素、更生霉素、柔红霉素、霉素霉素、丝裂霉素、肉瘤霉素C和道诺霉素；以及其它化疗药物，包括但不限于抗肿瘤抗体、达卡巴嗪、氮胞苷、阿姆沙康、美法仑、异环磷酰胺和米托蒽醌。在一些实施方案中，所述另外的治疗剂选自表柔比星、奥沙利铂和5-氟尿嘧啶中的一种或多种。在一些实施方案中，所述另外的治疗剂包括但不限于抗血管生成剂，包括抗VEGF抗体(包括人源化和嵌合抗体、抗VEGF适体和反义寡核苷酸)以及其他血管发生抑制剂，例如血管抑素、内皮抑制素、干扰素、白细胞介素1(包括α和β)白介素12、视黄酸和金属蛋白酶-1和-2的组织抑制剂等。
可作为药学上可接受的载体或其组分的一些物质的具体例子是糖类，如乳糖、葡萄糖和蔗糖；淀粉，如玉米淀粉和土豆淀粉；纤维素及其衍生物，如羧甲基纤维素钠、乙基纤维素和甲基纤维素；西黄蓍胶粉末；麦芽；明胶；滑石；固体润滑剂，如硬脂酸和硬脂酸镁；硫酸钙；植物油，如花生油、棉籽油、芝麻油、橄榄油、玉米油和可可油；多元醇，如丙二醇、甘油、山梨糖醇、甘露糖醇和聚乙二醇；海藻酸；乳化剂，如Tween；润湿剂，如月桂基硫酸钠；着色剂；调味剂；压片剂、稳定剂；抗氧化剂；防腐剂；无热原水；等渗盐溶液；和磷酸盐缓冲液等。
本文所述的药物组合物可包含一种或多种药学可接受的盐。“药学可接受的盐”指这样一种盐，其保留亲本化合物的期望生物学活性且不产生任何不利的毒物学效果(参见例如，Berge,S.M等人.,1977,J.Pharm.Sci.66:1-19)。此类盐的例子包括酸加成盐和碱加成盐。
酸加成盐包括衍生自无毒无机酸，诸如盐酸、硝酸、磷酸、硫酸、氢溴酸、氢碘酸、亚磷酸等的盐，以及衍生自无毒有机酸，诸如脂肪族单羧酸和二羧酸、苯基取代的链烷酸、羟基链烷酸、芳香族酸、脂肪族和芳香族磺酸等的盐。碱加成盐包括衍生自碱土金属(诸如钠、钾、镁、钙等)的盐，以及衍生自无毒有机胺的盐，诸如N,N'-二苄乙二胺、N-甲基葡糖胺、氯普鲁卡因、胆碱、二乙醇胺、乙二胺、普鲁卡因等。
本文所述的药物组合物还可包含抗氧化剂。抗氧化剂的实例包括但不限于:水溶性抗氧化剂，诸如抗坏血酸、盐酸半胱氨酸、硫酸氢钠、焦亚硫酸钠、亚硫酸钠等；油溶性抗氧化剂，诸如抗坏血酸棕榈酸酯、丁基化羟基茴香醚(BHA)、丁基化羟基甲苯(BHT)，卵磷脂、没食子酸丙酯、α-生育酚等；和金属螯合剂，诸如柠檬酸、乙二胺四乙酸(EDTA)、山梨醇、酒石酸、磷酸等。
本申请的组合物可根据需要制成各种剂型，并可由医师根据患者种类、年龄、体重和大致疾病状况、给药方式等因素确定对病人有益的剂量进行施用。给药方式例如可以采用肠胃外给药(如注射)或其它治疗方式。
免疫原性组合物的“肠胃外”施用包括例如皮下(s.c.)、静脉内(i.v.)、肌内(i.m.)或胸骨内注射或输注技术。
在一些实施方案中，组合物可以是等渗的，即它们可以具有与血液和泪液相同的渗透压。本申请组合物的期望等渗性可以使用氯化钠或其它药学上可接受的试剂如葡萄糖、硼酸、酒石酸钠、丙二醇或其它无机或有机溶质来实现。如果需要，组合物的粘度可以使用药学上可接受的增稠剂维持在选定的水平。合适的增稠剂包括，例如，甲基纤维素、黄原胶、羧甲基纤维素、羟丙基纤维素、卡波姆等。增稠剂的优选浓度将取决于所选择的试剂。显然，合适的载体和其它添加剂的选择将取决于确切的给药途径和特定剂型的性质，例如液体剂型。
试剂盒
本申请还提供了包含本文所述的抗体、免疫缀合物、嵌合受体、核酸或宿主细胞的试剂盒。在一些实施方案中，试剂盒可以包括含有有效量的包含一种或多种单位剂型的本文所述的抗体、嵌合受体、核酸或宿主细胞的治疗或预防组合物。在一些实施方案中，试剂盒包含可含有治疗或预防性组合物的无菌容器；这样的容器可以是盒、安瓿、瓶、小瓶、管、袋、泡罩包装或本领域已知的其它合适的容器形式。这种容器可以由塑料、玻璃、层压纸、金属箔或其他适合于保持药物的材料制成。在一些实施方案中，所述试剂盒包含本文所述的抗体、免疫辍合物、嵌合受体、核酸或宿主细胞，以及将本文所述的抗体、免疫辍合物、嵌合受体、核酸或宿主细胞给予个体的说明书。说明书中通常包含使用本文所述的抗体、免疫辍合物、嵌合受体、核酸或宿主细胞来治疗或预防癌症或肿瘤的方法。在一些实施方案中，试剂盒包含本文所述的宿主细胞，并且可以包括约1×104个细胞至约1×106个细胞。在一些实施方案中，试剂盒可以包括至少约1×105个细胞，至少约1×106个细胞，至少约1×107个细胞，至少约4×107个细胞，至少约5×107个细胞，至少约6×107个细胞，至少约6×107个细胞，8×107个细胞，至少约9×107个细胞，至少约1×108个细胞，至少约2×108个细胞，至少约3×108个细胞，至少约4×108个细胞，至少约5×108个细胞，至少约6×108个细胞，至少约6×108细胞，至少约8×108个细胞，至少约9×108细胞，至少约1×109个细胞，至少约2×109个细胞，至少约3×109个细胞，至少约4×109个细胞，至少约5×109个细胞，至少约6×109个细胞，至少约8×109个细胞， 至少约9×109个细胞，至少约1×1010个细胞，至少约2×1010个细胞，至少约3×1010个细胞，至少约4×1010个细胞，至少约5×1010个细胞，至少约6×1010个细胞，至少约7×1010个细胞、至少约8×1010个细胞、至少约9×1010个细胞，至少约1×1011个细胞，至少约2×1011个细胞，至少约3×1011个细胞，至少约4×1011个细胞，至少约5×1011个细胞，至少约8×1011个细胞，至少约9×1011个细胞，或至少约1×1012个细胞。例如，可以在试剂盒中包括大约5×1010个细胞。在另一个实例中，试剂盒可以包括3×106个细胞；细胞可以扩增至约5×1010个细胞并施用于受试者。
在一些实施方案中，试剂盒可以包括同种异体细胞。在一些实施方案中，试剂盒可以包括可以包含基因组修饰的细胞。在一些实施方案中，试剂盒可以包含“现成的”细胞。在一些实施方案中，试剂盒可以包括可以扩展用于临床使用的细胞。在某些情况下，试剂盒可能包含用于研究目的的内容物。
在一些实施方案中，说明书包括以下中的至少一个：治疗剂的描述；用于治疗或预防肿瘤或其症状的剂量方案和给药；预防措施、警示、禁忌症、过量信息、不良反应、动物药理学、临床研究、和/或引用文献。说明书可以直接打印在容器上(如果有的话)，或作为容器上的标签，或作为容器内或容器中提供的单独的纸张、小册子、卡片或文件夹打印。在一些实施方案中，说明书提供施用本申请所述的抗体用于治疗或预防肿瘤的方法。在某些情况下，说明书提供了施用化学治疗剂之前、之后或同时给与本申请的抗体的方法。
用于诊断/检测/治疗的方法
术语“调控”是指正向或负向改变。调节范例包括1％、2％、10％、25％、50％、75％、或100％变化。在一具体实施方式中，是指负向改变。
术语“治疗”是指在试图改变疾病过程的干预措施，既可以进行预防也可以在临床病理过程干预。治疗效果包括但不限于，防止疾病的发生或复发、减轻症状、减少任何疾病直接或间接的病理后果、防止转移、减慢疾病的进展速度、改善或缓解病情、缓解或改善预后等。
术语“预防”是指在试图在疾病(如细胞移植产生的排斥反应)产生前进行的干预措施。
术语“肿瘤抗原”指的是过度增生性疾病发生、发展过程中新出现的或过度表达的抗原。在某些方面，本申请的过度增生性病症是指肿瘤。
本申请所述的肿瘤抗原可以是实体瘤抗原，也可以是血液瘤抗原。
本申请的肿瘤抗原包括但不限于：促甲状腺激素受体(TSHR)；CD171；CS-1；C型凝集素样分子-1；神经节苷脂GD3；Tn抗原；CD19；CD20；CD 22；CD 30；CD 70；CD 123；CD 138；CD33；CD44；CD44v7/8；CD38；CD44v6；B7H3(CD276)，B7H6；KIT(CD117)；白介素13受体亚单位α(IL-13Rα)；白介素11受体α(IL-11Rα)；前列腺干细胞抗原(PSCA)；前列腺特异性膜抗原(PSMA)；癌胚抗原(CEA)；NY-ESO-1；HIV-1 Gag；MART-1；gp100；酪氨酸酶；间皮素；EpCAM；蛋白酶丝氨酸21(PRSS21)；血管内皮生长因子受体，血管 内皮生长因子受体2(VEGFR2)；路易斯(Y)抗原；CD24；血小板衍生生长因子受体β(PDGFR-β)；阶段特异性胚胎抗原-4(SSEA-4)；细胞表面相关的粘蛋白1(MUC1)，MUC6；表皮生长因子受体家族及其突变体(EGFR，EGFR2，ERBB3，ERBB4，EGFRvIII)；神经细胞粘附分子(NCAM)；碳酸酐酶IX(CAIX)；LMP2；肝配蛋白A型受体2(EphA2)；岩藻糖基GM1；唾液酸基路易斯粘附分子(sLe)；神经节苷脂GM3；TGS5；高分子量黑素瘤相关抗原(HMWMAA)；邻乙酰基GD2神经节苷脂(OAcGD2)；叶酸受体；肿瘤血管内皮标记1(TEM1/CD248)；肿瘤血管内皮标记7相关的(TEM7R)；Claudin 6，Claudin18.2、Claudin18.1；ASGPR1；CDH16；5T4；8H9；αvβ6整合素；B细胞成熟抗原(BCMA)；CA9；κ轻链(kappa light chain)；CSPG4；EGP2，EGP40；FAP；FAR；FBP；胚胎型AchR；HLA-A1，HLA-A2；MAGEA1，MAGE3；KDR；MCSP；NKG2D配体；PSC1；ROR1；Sp17；SURVIVIN；TAG72；TEM1；纤连蛋白；腱生蛋白；肿瘤坏死区的癌胚变体；G蛋白偶联受体C类5组-成员D(GPRC5D)；X染色体开放阅读框61(CXORF61)；CD97；CD179a；间变性淋巴瘤激酶(ALK)；聚唾液酸；胎盘特异性1(PLAC1)；globoH glycoceramide的己糖部分(GloboH)；乳腺分化抗原(NY-BR-1)；uroplakin 2(UPK2)；甲型肝炎病毒细胞受体1(HAVCR1)；肾上腺素受体β3(ADRB3)；pannexin 3(PANX3)；G蛋白偶联受体20(GPR20)；淋巴细胞抗原6复合物基因座K9(LY6K)；嗅觉受体51E2(OR51E2)；TCRγ交替阅读框蛋白(TARP)；肾母细胞瘤蛋白(WT1)；ETS易位变异基因6(ETV6-AML)；精子蛋白17(SPA17)；X抗原家族成员1A(XAGE1)；血管生成素结合细胞表面受体2(Tie2)；黑素瘤癌睾丸抗原-1(MAD-CT-1)；黑素瘤癌睾丸抗原-2(MAD-CT-2)；Fos相关抗原1；p53突变体；人端粒酶逆转录酶(hTERT)；肉瘤易位断点；细胞凋亡的黑素瘤抑制剂(ML-IAP)；ERG(跨膜蛋白酶丝氨酸2(TMPRSS2)ETS融合基因)；N-乙酰葡糖胺基转移酶V(NA17)；配对盒蛋白Pax-3(PAX3)；雄激素受体；细胞周期蛋白B1；V-myc鸟髓细胞瘤病病毒癌基因神经母细胞瘤衍生的同源物(MYCN)；Ras同源物家族成员C(RhoC)；细胞色素P450 1B1(CYP1B1)；CCCTC结合因子(锌指蛋白)样(BORIS)；由T细胞识别的鳞状细胞癌抗原3(SART3)；配对盒蛋白Pax-5(PAX5)；proacrosin结合蛋白sp32(OYTES1)；淋巴细胞特异性蛋白酪氨酸激酶(LCK)；A激酶锚定蛋白4(AKAP-4)；滑膜肉瘤X断点2(SSX2)；CD79a；CD79b；CD72；白细胞相关免疫球蛋白样受体1(LAIR1)；IgA受体的Fc片段(FCAR)；白细胞免疫球蛋白样受体亚家族成员2(LILRA2)；CD300分子样家族成员f(CD300LF)；C型凝集素结构域家族12成员A(CLEC12A)；骨髓基质细胞抗原2(BST2)；含有EGF样模块粘蛋白样激素受体样2(EMR2)；淋巴细胞抗原75(LY75)；磷脂酰肌醇蛋白聚糖-3(GPC3)；Fc受体样5(FCRL5)；免疫球蛋白λ样多肽1(IGLL1)。优选的，所述肿瘤抗原为CS1、Claudin18.2、GPC3、BCMA或者CD19。
病原体抗原选自：病毒、细菌、真菌、原生动物，或寄生虫的抗原；病毒抗原选自：巨细胞病毒抗原、爱泼斯坦-巴尔病毒抗原、人类免疫缺陷病毒抗原,或流感病毒抗原。
本文中使用的术语“个体”是指任何动物，例如哺乳动物或有袋动物。本申请的个体包括但不限于人类、非人类灵长类动物(例如恒河猴或其他类型的猕猴)、小鼠、猪、马、驴、牛、绵羊、大鼠和任何种类的家禽。
本文所用的术语“有效量”是指提供治疗或预防益处的量。
本文中提供的任一抗GPRC5D抗体、免疫缀合物、嵌合受体修饰的宿主细胞、药物组合物或试剂盒都可以用于治疗方法中。
在一个方面，提供用作药物的任一抗GPRC5D抗体、免疫缀合物、嵌合受体修饰的宿主细胞、药物组合物或试剂盒。在另一方面，提供用于治疗疾病的任一抗GPRC5D抗体、免疫缀合物、嵌合受体修饰的宿主细胞、药物组合物或试剂盒。在某些实施方案中，提供用于治疗方法的任一抗GPRC5D抗体、免疫缀合物、嵌合受体修饰的宿主细胞、药物组合物或试剂盒。在某些实施方案中，本申请提供任一抗GPRC5D抗体、免疫缀合物、嵌合受体修饰的宿主细胞、药物组合物或试剂盒，其用于治疗患有疾病的个体的方法，所述方法包括向个体施用有效量的任一抗GPRC5D抗体、免疫缀合物、嵌合受体修饰的宿主细胞、药物组合物或试剂盒。在一个实施方案中，所述方法还包括向个体施用有效量的至少一种另外的治疗剂。所述“个体”优选是人。
在另一个方面，本申请提供任一抗GPRC5D抗体、免疫缀合物、嵌合受体修饰的宿主细胞、药物组合物或试剂盒在制备或配制药物中的用途。在一个实施方案中，所述药物用于治疗疾病。在另一个实施方案中，所述药物用于治疗疾病的方法，所述方法包括向患病个体施用有效量的药物。在一个实施方案中，所述方法还包括向个体施用有效量的至少一种另外的治疗剂。所述“个体”优选是人。
在另一个方面，本申请提供用于治疗疾病的方法。在一个实施方案中，所述方法包括向患有表达GPRC5D的疾病的个体施用有效量的任一抗GPRC5D抗体、免疫缀合物、嵌合受体修饰的宿主细胞、药物组合物或试剂盒。在一个实施方案中，所述方法还包括向个体施用有效量的至少一种另外的治疗剂。所述“个体”优选是人。
在另一个方面，本申请提供例如用于任一上述治疗方法的包含本文中提供的任一抗GPRC5D抗体、免疫缀合物、嵌合受体修饰的宿主细胞、药物组合物或试剂盒的药物制剂。在一个实施方案中，所述药物制剂包含本文中提供的任一抗GPRC5D抗体、免疫缀合物、嵌合受体修饰的宿主细胞、药物组合物或试剂盒和药用载体。在另一个实施方案中，所述药物制剂包含本文中提供的任一抗GPRC5D抗体、免疫缀合物、嵌合受体修饰的宿主细胞、药物组合物或试剂盒和至少一种另外的治疗剂。
在另一个方面，所述药物制剂用于治疗疾病。在一个实施方案中，向患病个体施用所述药物制剂。根据任一以上实施方案的“个体”优选是人。
在另一个方面，本申请提供用于制备药物或药物制剂的方法，所述方法包括将本文中提供的任一抗GPRC5D抗体、免疫缀合物、嵌合受体修饰的宿主细胞、药物组合物或试剂 盒与药用载体混合，例如，以用于任一上述治疗方法。在一个实施方案中，用于制备药物或药物制剂的方法还包括添加至少一种另外的治疗剂至药物或药物制剂。
本申请的任一抗GPRC5D抗体、免疫缀合物、嵌合受体修饰的宿主细胞、药物组合物或试剂盒可以单独地用于治疗或与其他试剂组合地用于治疗。或者，本申请的任一抗GPRC5D抗体、免疫缀合物、嵌合受体修饰的宿主细胞、药物组合物或试剂盒可以与至少一种另外的治疗剂共同施用。
上述的此种组合治疗包括组合施用(其中两种以上治疗剂被包含在同一或分开的制剂中)和分开施用，在此种情况中，本申请的任一抗GPRC5D抗体、免疫缀合物、嵌合受体修饰的宿主细胞、药物组合物或试剂盒的施用可以发生在另外的治疗剂或试剂的施用之前、同时、和/或之后。在一个实施方案中，本申请的任一抗GPRC5D抗体、免疫缀合物、嵌合受体修饰的宿主细胞、药物组合物或试剂盒的施用和另外的治疗剂的施用彼此发生在约一个月以内、或在约一周、两周或三周以内，或在约一天、两天、三天、四天、五天或六天以内。
本申请的任一抗GPRC5D抗体、免疫缀合物、嵌合受体修饰的宿主细胞、药物组合物或试剂盒(以及任意另外的治疗剂)可以通过任何合适的手段施用，包括肠胃外施用、肺内施用或鼻内施用，以及，如果具备治疗需要，病变内施用。肠胃外输注包括肌肉内施用、静脉内施用、动脉内施用、腹膜内施用或皮下施用。用药可以是通过任何合适的途径，例如，通过注射，如静脉内或皮下注射，这部分取决于施用是短暂的还是长期的。本文中考虑多种用药方案，包括但不限于单次施用或在多个时间点的多次施用、推注施用，和脉冲注入。
给予个体的包含免疫反应性细胞群体的制剂包含有效治疗和/或预防特定适应症或疾病的多个免疫反应性细胞。因此，可以向个体施用免疫反应性细胞的治疗有效群体。通常，施用包含约1×104至约1×1010个免疫反应性细胞的制剂。在大多数情况下，制剂将包含约1×105至约1×109个免疫反应性细胞、约5×105至约5×108个免疫反应性细胞、或约1×106至约1×107个免疫反应性细胞。然而，根据肿瘤的位置、来源、身份、程度和严重程度、待治疗的个体的年龄和身体状况等，对个体施用的CAR免疫反应性细胞的数量将在宽的范围之间变化。医生将最终确定要使用的适当剂量。
在一些实施方案中，使用嵌合受体来刺激宿主细胞介导的免疫应答。例如，T细胞介导的免疫应答是涉及T细胞活化的免疫应答。活化的抗原特异性细胞毒性T细胞能够在表面上显示外源抗原表位的靶细胞中诱导细胞凋亡，例如显示肿瘤抗原的癌细胞。在另一些实施方案中，使用嵌合抗原受体在哺乳动物中提供抗肿瘤免疫。由于T细胞介导的免疫应答，受试者将产生抗肿瘤免疫。
在某些情况下，治疗患有肿瘤的受试者的方法可以涉及向需要治疗的受试者施用一种或多种本申请所述的宿主细胞。所述宿主细胞可结合肿瘤靶分子并诱导癌细胞死亡。如 前文所述，本申请还提供治疗个体中的病原体感染的方法，包括向所述个体施用治疗有效量的本申请的宿主。
本申请的免疫反应性细胞的给药频率将根据包括所治疗疾病的因素、特定免疫反应性细胞的元件和给药方式。例如可以每日给药4次、3次、2次或每日一次、每隔一天、每三天、每四天、每五天、每六天一次、每周一次、每八天一次、每九天一次、每十天、每周一次、或者每月两次给药。如本文所述，由于本申请的免疫应答细胞具有改善的活力，从而可以不仅以与类似的但不表达外源性I型干扰素的免疫应答细胞更低的治疗有效的量给药，并且可以以更低的频率给药，以获得至少类似、并且优选更加显著的疗效。
本申请的优点：
本申请提供了特异性识别GPRC5D的全人源抗体，由该抗体制备的CAR T细胞在体内外均表现出对靶细胞较好的杀伤效果。
下面结合具体实施例，进一步阐述本申请。应理解，这些实施例仅用于说明本申请而不用于限制本申请的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如J.萨姆布鲁克等编著，分子克隆实验指南，第三版，科学出版社，2002中所述的条件，或按照制造厂商所建议的条件。
实施例1.构建表达GPRC5D的细胞系
利用常规分子生物学技术通过慢病毒将人GPRC5D(氨基酸序列如SEQ ID NO:65所示)分别转染内源性GPRC5D不表达的CHOK1(ATCC)、293T(ATCC)、NIH3T3细胞(ATCC)，通过有限稀释法挑选阳性单克隆，构建CHOK1-huGPRC5D、293T-huGPRC5D、NIH3T3-huGPRC5D稳转细胞系。
利用常规分子生物学技术通过慢病毒将鼠GPRC5D(氨基酸序列如SEQ ID NO:66所示)转染CHOK1细胞，通过有限稀释法挑选阳性克隆，构建CHOK1-muGPRC5D稳转细胞系。
实施例2.抗GPRC5D杂交瘤细胞株产生
利用本领域常规杂交瘤抗体制备技术制备抗GPRC5D杂交瘤抗体。
将编码人GPRC5D(SEQ ID No：65)全长基因构建于真核表达pCAGGS质粒中，形成真核表达质粒pCAGGS-GPRC5D。然后利用质粒pCAGGS-GPRC5D和NIH3T3-huGPRC5D细胞对Balb/c小鼠(维通利华，雌性，6-8周龄)进行4次免疫。前三次免疫使用100μg质粒pCAGGS-GPRC5D(生理盐水稀释，总体积2mL)按尾静脉大剂量注射法在5-8s内完成免疫；第四次使用2×107个NIH3T3-huGPRC5D细胞腹腔加强免疫，3天后取小鼠脾脏按公开的标准方案(Kohler、Milstein，1975)进行融合，经HAT及流式细胞术特异性筛选后得到2个结合人GPRC5D的杂交瘤抗体mIgG：AB1(IgG2b-k)、AB2(IgG3-k)。
实施例3.抗GPRC5D杂交瘤抗体与CHOK1-huGPRC5D细胞结合EC50测定
取CHOK1-huGPRC5D细胞2×105 cells/孔于96孔圆底培养板中，用含1％FBS的PBS清洗2次；之后加入杂交瘤抗体，起始浓度为100μg/mL，然后3倍梯度稀释11次，4℃孵育45min； 500g离心5min，弃上清后用含1％FBS的PBS清洗2次；之后加入Goat-anti-Mouse FITC(KANFCHEN，1:200使用)100μL/孔，4℃孵育45min；500g离心5min，弃上清后用含1％FBS的PBS清洗2次，之后加入200μL含1％FBS的PBS重悬细胞，用流式细胞仪进行检测，结果用FlowJo v.X.0.7统计及GraphPad Prism8.0作图。
结果如图1所示，两个杂交瘤抗体AB1和AB2分别与CHOK1-huGPRC5D细胞有较强的结合活性，EC50分别为1.692nM和1.930nM。说明AB1和AB2均能显著结合人GPRC5D。
实施例4.抗GPRC5D杂交瘤抗体与人GPRC5D和鼠GPRC5D交叉反应测定
分别取CHOK1细胞(作为阴性对照细胞)、CHOK1-huGPRC5D细胞、CHOK1-muGPRC5D细胞，按照实施例3的方法测定AB1和AB2与人GPRC5D和鼠GPRC5D的交叉反应。
结果如图2所示，两个杂交瘤抗体AB1和AB2均与人GPRC5D和鼠GPRC5D显著结合。
实施例5.抗GPRC5D杂交瘤抗体基因序列获得
利用常规分子生物学技术从杂交瘤细胞AB1和AB2中提取RNA并反转录合成cDNA。使用UPM、mIgG2b-outer(SEQ ID NO：67)、mIgG3-outer(SEQ ID NO：68)、mK-outer(SEQ ID NO：69)引物进行PCR，克隆并测序，得到AB1-VH基因序列(SEQ ID NO：36)、AB1-VL基因序列(SEQ ID NO：38)、AB2-VH基因序列(SEQ ID NO：40)、AB2-VL基因序列(SEQ ID NO：42)。
AB1的HCDR1的氨基酸序列如SEQ ID NO:11所示、HCDR2的氨基酸序列如SEQ ID NO:12所示、HCDR3的氨基酸序列如SEQ ID NO:13所示、LCDR1的氨基酸序列如SEQ ID NO:14所示、LCDR2的氨基酸序列如SEQ ID NO:15所示、LCDR3的氨基酸序列如SEQ ID NO:16所示。AB1的重链可变区的氨基酸序列如SEQ ID NO:35所示、轻链可变区的氨基酸序列如SEQ ID NO:37所示。
AB2的HCDR1的氨基酸序列如SEQ ID NO:17所示、HCDR2的氨基酸序列如SEQ ID NO:18所示、HCDR3的氨基酸序列如SEQ ID NO:19所示、LCDR1的氨基酸序列如SEQ ID NO:20所示、LCDR2的氨基酸序列如SEQ ID NO:21所示、LCDR3的氨基酸序列如SEQ ID NO:22所示。AB2的重链可变区的氨基酸序列如SEQ ID NO:39所示、轻链可变区的氨基酸序列如SEQ ID NO:41所示。
实施例6.重组杂交瘤scFv-mFc抗体与表达GPRC5D细胞系结合活性测定
使用分子克隆技术将AB1、AB2抗体重链及轻链基因序列分别克隆至含有mFc(SEQ ID NO：64)的真核表达载体中，构建成V152S-AB1-mFc和V152S-AB2-mFc质粒，然后转染293F细胞(Thermo公司)培养7天后经亲和层析纯化得到抗GPRC5D重组抗体AB1(scFv-mFc)和AB2(scFv-mFc)。
分别取CHOK1-huGPRC5D、293T-huGPRC5D、NIH3T3-huGPRC5D、CHOK1细胞，按照实施例3的方法测定重组抗体AB1(scFv-mFc)和AB2(scFv-mFc)与表达人GPRC5D的 细胞系的结合活性。
结果如图3所示，两个重组抗体AB1(scFv-mFc)和AB2(scFv-mFc)与表达人GPRC5D的细胞系特异性结合。
实施例7.GPRC5D抗体的筛选和鉴定
1、使用全人噬菌体展示文库筛选结合人GPRC5D的抗体
本申请使用的噬菌体展示文库为本公司构建的噬菌体文库，库容为1E+11。利用人GPRC5D抗原(恺佧生物)，通过本领域技术人员已知的筛选方法得到特异结合人GPRC5D的Fab片段，最终得到三个特异性结合人GPRC5D的抗体，命名为AB3、AB4、AB5。
AB3的HCDR1的氨基酸序列如SEQ ID NO:1所示、HCDR2的氨基酸序列如SEQ ID NO:2所示、HCDR3的氨基酸序列如SEQ ID NO:3所示、LCDR1的氨基酸序列如SEQ ID NO:4所示、LCDR2的氨基酸序列如SEQ ID NO:5所示、LCDR3的氨基酸序列如SEQ ID NO:6所示。AB3的重链可变区的氨基酸序列如SEQ ID NO:23所示、轻链可变区的氨基酸序列如SEQ ID NO:25所示。AB3的重链的氨基酸序列如SEQ ID NO：45所示，轻链的氨基酸序列如SEQ ID NO：47所示。
AB4的HCDR1的氨基酸序列如SEQ ID NO:1所示、HCDR2的氨基酸序列如SEQ ID NO:2所示、HCDR3的氨基酸序列如SEQ ID NO:7所示、LCDR1的氨基酸序列如SEQ ID NO:4所示、LCDR2的氨基酸序列如SEQ ID NO:5所示、LCDR3的氨基酸序列如SEQ ID NO:8所示。AB4的重链可变区的氨基酸序列如SEQ ID NO:27所示、轻链可变区的氨基酸序列如SEQ ID NO:29所示。AB4重链的氨基酸序列如SEQ ID NO：49所示，轻链的氨基酸序列如SEQ ID NO：51所示。
AB5的HCDR1的氨基酸序列如SEQ ID NO:1所示、HCDR2的氨基酸序列如SEQ ID NO:2所示、HCDR3的氨基酸序列如SEQ ID NO:9所示、LCDR1的氨基酸序列如SEQ ID NO:4所示、LCDR2的氨基酸序列如SEQ ID NO:5所示、LCDR3的氨基酸序列如SEQ ID NO:10所示。AB5的重链可变区的氨基酸序列如SEQ ID NO:31所示、轻链可变区的氨基酸序列如SEQ ID NO:33所示。AB5重链的氨基酸序列如SEQ ID NO：53所示，轻链的氨基酸序列如SEQ ID NO：55所示。
2.通过标准ELISA检测抗体的特异性
各取产生抗体AB3、AB4、AB5的克隆诱导表达上清50μl与2μg/ml人GPRC5D抗原室温共孵育1h，然后通过二抗anti-Flag-HRP(Sigma，1:4000稀释使用)室温孵育1h检测结合的抗体；随后加入TMB显色并用酶标仪读数OD450值。
结果如图4所示(空白组不添加Fab抗体，即NA)，显示抗体AB3、AB4、AB5均特异性结合人GPRC5D抗原。
3.利用FACs测定抗体与靶细胞结合的特异性
各取产生抗体AB3、AB4、AB5的克隆诱导表达上清50μl与2×105个293T细胞和 293T-huGPRC5D细胞共孵育1h，然后通过二抗anti-F(ab')2-488(Jackson ImmunoResearch，1:200稀释使用)对结合细胞的抗体进行荧光标记和检测，实验数据用FlowJo分析软件计算平均荧光强度(MFI)。
结果如图5所示(空白组不添加Fab抗体，即NA)，显示抗体AB3、AB4、AB5均特异性结合过表达人GPRC5D的293T细胞，不结合293T细胞。
实施例8.利用ELISA检查抗GPRC5D-抗体与抗原结合EC50
将实施例7中产生抗体AB3、AB4、AB5的克隆分别通过镍柱进行原核表达纯化，得到抗体AB3、AB4、AB5。然后将抗体从10μg/ml浓度3倍梯度稀释(8个梯度)，与人GPRC5D抗原(包被浓度:2μg/ml)室温共孵育1h，然后通过二抗anti-Flag-HRP(Sigma，1:4000稀释使用)室温孵育1h检测结合的抗体；随后加入TMB显色并用酶标仪读数OD450值。通过GraphPad Prism5软件以一抗浓度为横坐标，OD450值为纵坐标进行四参数拟合，计算EC50值。
结果如图6显示，抗体AB3、AB4、AB5与人GPRC5D抗原均有显著结合，EC50分别为0.07382μg/ml，0.1424μg/ml和0.1396μg/ml。
实施例9.抗GPRC5D抗体与靶细胞特异性结合活性检测
人淋巴瘤细胞Daudi(中国科学院细胞库)是内源性GPRC5D不表达的细胞；多发性骨髓瘤细胞MM.1S(中国科学院细胞库)、NCI-H929(ATCC)是内源性表达GPRC5D的细胞。
将实施例7中描述的AB3、AB4和AB5抗体(10μg/ml)分别与CHOK1、CHOK1-huGPRC5D、293T、293T-huGPRC5D、MM.1S、NCI-H929、Daudi细胞共孵育(空白组不添加抗体，即NA)，利用二抗anti-F(ab')2-488(Jackson ImmunoResearch，1:200稀释使用)对结合细胞的抗体进行荧光标记，然后通过流式分析仪进行荧光强度检测，实验数据用FlowJo分析软件计算平均荧光强度(MFI)。
检测结果如图7所示，AB3、AB4和AB5抗体均与表达人GPRC5D的细胞(CHOK1-huGPRC5D，293T-huGPRC5D，MM.1S和NCI-H929)显著结合，不结合不表达GPRC5D的细胞(CHOK1，293T和Daudi)。
实施例10.抗GPRC5D抗体与表达人GPRC5D细胞的EC50结合活性检测
取CHOK1-huGPRC5D细胞和MM.1S细胞计数并分别铺U型底板，每孔约2×105个细胞，然后依次进行一抗(实施例7描述的AB3、AB4或AB5抗体:30μg/mL起始，3倍梯度稀释8个梯度)和二抗(Anti-Fab-FITC:1:200,Jackson ImmunoResearch)孵育，随后用流式分析仪进行荧光强度检测。实验数据用FlowJo分析软件计算平均荧光强度(MFI)，随后通过GraphPad Prism5软件以一抗浓度为横坐标，校准平均荧光强度(MFI)为纵坐标进行四参数拟合，计算EC50值。
结果如图8和图9示，AB3、AB4、AB5抗体与CHOK1-huGPRC5D细胞结合的EC50分别为3.869μg/ml，5.064μg/ml和4.196μg/ml；AB3、AB4和AB5与MM.1S细胞结合的EC50分 别为0.8512μg/ml，0.6578μg/ml和0.8047μg/ml。
实施例11.抗GPRC5D抗体与CHOK1-muGPRC5D细胞结合活性检测
将实施例7描述AB3、AB4和AB5抗体(5μg/ml)分别与CHOK1-muGPRC5D细胞共孵育(空白组不添加抗体，即NA)，利用二抗anti-F(ab')2-488(Jackson ImmunoResearch，1:200稀释使用)进行荧光标记，然后通过流式分析仪进行荧光强度检测，实验数据用FlowJo分析软件计算平均荧光强度(MFI)。
检测结果如图10所示，AB3抗体可以结合鼠GPRC5D。
实施例12.GPRC5D CAR-T细胞的制备
1.CAR载体构建
以PRRLSIN-cPPT.EF-1α为载体，构建表达AB3 scFv(SEQ ID NO：43)和对照抗体18scFv(SEQ ID NO：57，序列来自专利CN107428829A)的二代嵌合抗原受体的慢病毒质粒。其中包含CD8α信号肽(SEQ ID NO：58)，CD8铰链区(SEQ ID NO：59)和跨膜区(SEQ ID NO：60)，CD137胞内信号传导结构域(SEQ ID NO：61)以及CD3δ(SEQ ID NO：62)；AB3-CAR的氨基酸序列如SEQ ID NO：63所示。
2.GPRC5D CAR-T细胞的制备
用磷酸钙法进行慢病毒的包装，病毒上清用PEG8000/NaCl进行纯化，纯化后感染CD3/CD28磁珠活化48小时后的T细胞，分别得到表达AB3-BBZ的AB3-BBZ CAR-T细胞，表达18-BBZ的18-BBZ CAR-T细胞，未转染病毒的T细胞视为UTD。感染后第5天用FACS法检测CAR阳性率，检测一抗为Biotin-anti-F(ab')2-488(Jackson ImmunoResearch，1:100使用)，二抗为SA-PE(eBioscience，1:200使用)。
实施例13.GPRC5D CAR-T细胞对靶细胞的体外杀伤实验
首先将靶细胞MM.1S，293T-huGPRC5D和293T细胞分别用AIM-V培养基(AIM-V+2％ABS)调整其密度至0.2x106/mL，96孔细胞培养板每孔加入50μl，根据效靶比3：1、1：1、1：3分别加入50μl效应细胞(CAR-T细胞，UTD做对照)，37度共孵育16小时后取上清采用LDH试剂盒进行检测，最后用酶标仪进行OD490读数。
结果如图11所示，AB3-BBZ CAR-T对293T细胞没有杀伤作用；而对于表达GPRC5D的293T-huGPRC5D及MM.1S细胞皆表现出明显的杀伤作用(效靶比3:1时杀伤率高达60％左右)，且杀伤效果与效靶比呈正相关。结果表明AB3-BBZ CAR-T对表达GPRC5D的细胞具有特异性的体外杀伤作用。
实施例14.GPRC5D CAR-T细胞的体内抗肿瘤能力
接种3×106个人多发性骨髓瘤细胞MM.1S于NPG雌性小鼠右侧腋部皮下，小鼠分3组(AB3-BBZ CAR T组、18-BBZ CAR T组、UTD组)，每组8只。肿瘤细胞接种当天记为D0，接种后第16天，肿瘤平均体积约200mm3，尾静脉分别注射AB3-BBZ CAR T、18-BBZ CAR T、UTD细胞，注射剂量为3×106个细胞/只小鼠。CAR T细胞注射后第20天(肿瘤接种后第 36天)，UTD组小鼠肿瘤体积超过2000mm3，实验终止。
结果如图12所示，AB3-BBZ CAR-T组在CAR-T注射后第9天(肿瘤接种后第25天)小鼠肿瘤出现消退，到CAR-T注射后第13天(肿瘤接种后第29天)组内8只小鼠肿瘤全部消退。作为对照的18-BBZ CAR-T组在CAR-T注射后第20天(肿瘤接种第36天)时，没有小鼠出现肿瘤消退，其相对UTD组抑瘤率仅为46.02％。该结果表明，AB3-BBZ CAR-T比18-BBZ CAR-T具有更优异的体内抗肿瘤效果(P<0.0001)。同时检测各治疗组小鼠体重变化，结果如图13所示。
实施例15.基于AB3的修饰抗体
AB3的轻链或重链序列进行随机突变，构建噬菌体文库，利用人GPRC5D抗原(恺佧生物)筛选获得AB6、AB7和AB8抗体，ELISA检测显示AB6、AB7和AB8与人GPRC5D有显著的结合(图14)。测序结果显示，AB6的重链可变区如SEQ ID NO:76所示，AB7的重链可变区如SEQ ID NO:23所示，AB8的重链可变区如SEQ ID NO:80所示，AB6和AB8的轻链可变区如SEQ ID NO:25所示，AB7的轻链可变区如SEQ ID NO:78所示；AB6的HCDR1如SEQ ID NO:70所示，HCDR2如SEQ ID NO:72所示，AB7的HCDR1如SEQ ID NO:1所示，HCDR2如SEQ ID NO:2所示，AB8的HCDR1如SEQ ID NO:71所示，HCDR2如SEQ ID NO:73所示，AB6、AB7和AB8的HCDR3如SEQ ID NO:3所示；AB6和AB8的LCDR1如SEQ ID NO:4所示，LCDR2如SEQ ID NO:5所示，AB7的LCDR1如SEQ ID NO:74所示，LCDR2如SEQ ID NO:75所示，AB6、AB7和AB8的LCDR3如SEQ ID NO:6所示。
实施例16.修饰抗体与不同种属GPRC5D细胞系的结合
将实施例15中选取的3个克隆经原核表达并利用镍柱进行亲和纯化，得到纯化的单链抗体(scFv)(分别如SEQ ID NO:82、SEQ ID NO:84、SEQ ID NO:86所示)，该单链抗体的C端含有his标签及flag标签。按照实施例1的方式构建CHO-K1-huGPRC5D和CHO-K1-muGPRC5D稳转细胞系。将上述纯化scFv(10ug/ml)与CHO-K1-huGPRC5D和CHO-K1-muGPRC5D细胞以及未转染的CHO-K1细胞共孵育，利用二抗anti-Flag-488(R&D，1:200稀释使用)对结合细胞的scFv进行荧光标记，然后通过流式分析仪进行荧光强度检测，实验数据用FlowJo分析软件计算平均荧光强度(MFI)。检测结果如图15所示，AB6，AB7和AB8与表达人GPRC5D以及鼠GPRC5D细胞系的结合能力较AB3显著提升，与不表达GPRC5D的CHO-K1细胞不结合。表明抗体AB6，AB7和AB8具有良好的种属结合特性和细胞结合特异性。
实施例17.抗GPRC5D抗体与表达GPRC5D细胞系结合的EC50检测
使用实施例1记载的293T-huGPRC5D细胞以及内源性表达GPRC5D的细胞系MM.1S，按照实施例10的方法测定实施例15记载的抗体的EC50值，一抗AB3(Fab)，AB6(scFv)，AB7(scFv)和AB8(scFv):30μg/mL起始3倍梯度稀释12个梯度和二抗(Anti-Flag-488:1:200,R&D)孵育，结果如图16所示，基于AB3的修饰抗体均具有较好的细胞结合活性，其中AB6 和AB7在两个细胞上的EC50均低于AB3。
实施例18抗GPRC5D抗体scFv-Fc形式构建及其单体率检测
将抗GPRC5D抗体(scFv)AB3，AB6，AB7和AB8与人IgG的Fc(huFc，如SEQ ID NO：91所示)段进行融合表达，通过293Fectin转染试剂转染293F细胞，第7天收集上清，采用Protein A填料进行亲和纯化，纯化后蛋白通过GE XK16/40空层析柱，收集单体峰，并进行单体率检测，结果显示抗体单体率都很高，没有明显差异。抗体蛋白用截流量为10KD的millipore超滤管进行浓缩，通过OD280/消光系数测浓度，具体结果见表3。
表3抗GPRC5D抗体scFv-Fc形式纯化结果
实施例19抗GPRC5D抗体亲和力测定
使用CYTIVA提供的SA chip(Cat:BR100531)，捕获Bio-GPRC5D(Acro,Cat.No.GPD-H82D6)741.4Ru。目标抗体(AB3-huFc、AB7-huFc)150nM起3倍梯度稀释做为流动相，Gly.Hcl PH1.7再生180s。检测完成后使用Evaluation Software拟合得到亲和力结果，AB3-huFc与Bio-GPRC5D亲和力KD为4.68nM；AB7-huFc与Bio-GPRC5D亲和力KD为2.14nM(图17)。
实施例20嵌合抗原受体(CAR)质粒的构建
按照实施例12的方法构建表达AB6、AB7、AB8的二代嵌合抗原受体的慢病毒质粒。AB6-BBZ的氨基酸序列如SEQ ID NO：88所示，AB7-BBZ的氨基酸序列如SEQ ID NO：89所示，AB8-BBZ的氨基酸序列如SEQ ID NO：90所示。
实施例21制备CAR-T细胞并检测其体外杀伤效果
利用实施例12和20制备的表达CAR的质粒，按照实施例12的方法制备获得AB3-BBZ CAR-T细胞、AB6-BBZ CAR-T细胞、AB7-BBZ CAR-T细胞、AB8-BBZ CAR-T细胞,未转染病毒的T细胞为UTD。按照实施例13的方法进行细胞杀伤实验，结果如图18所示，AB3 CAR-T、AB6 CAR-T、AB7 CAR-T、AB8 CAR-T对不表达GPRC5D的293T细胞都没有杀伤作用；而对于表达GPRC5D的MM.1S，293T-huGPRC5D细胞皆表现出明显的杀伤作用，且杀伤效果与效靶比呈正相关。表明AB3 CAR-T、AB6 CAR-T、AB7 CAR-T和AB8 CAR-T对表达人GPRC5D的细胞具有特异性的杀伤作用。
在本申请提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本申请的上述讲授内容之后，本领域技术人员可以对本申请作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。
本申请涉及的序列如下表所示：
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Claims (26)

  1. 识别GPRC5D的抗体，其特征在于，所述抗体选自如下任意组：
    (1)抗体，其包含重链可变区，所述重链可变区包含SEQ ID NO：1、11、17、70或71任一所示的HCDR1，和/或包含SEQ ID NO：2、12、18、72或73任一所示的HCDR2，和/或包含SEQ ID NO：3、7、9、13或19任一所示的HCDR3；
    (2)抗体，其包含轻链可变区，所述轻链可变区包含SEQ ID NO：4、14、20、或74任一所示的LCDR1，和/或包含SEQ ID NO：5、15、21或75任一所示的LCDR2，和/或包含SEQ ID NO：6、8、10、16或22任一所示的LCDR3；
    (3)抗体，包含(1)所述抗体的重链可变区及(2)所述抗体的轻链可变区；
    (4)抗体，(1)～(3)中任一项所述的抗体的变体，且具备与(1)～(3)中任一项所述的抗体相同或相似的活性。
  2. 如权利要求1所述的抗体，其包含重链可变区中的至少一个CDR，所述重链可变区包含如SEQ ID NO:23、27、31、35、39、76或80中任一项所示的氨基酸序列或其变体，或包含与上述任一序列具有至少80％相似性的氨基酸序列；和/或其包含轻链可变区中的至少一个CDR，所述轻链可变区包含如SEQ ID NO:25、29、33、37、41或78中任一项所示的氨基酸序列或其变体，或包含与上述任一序列具有至少80％相似性的氨基酸序列。
  3. 如权利要求1所述的抗体，其包含重链可变区中的HCDR1、HCDR2、HCDR3，所述重链可变区包含如SEQ ID NO:23、27、31、35、39、76或80中任一项所示的氨基酸序列或其变体，或包含与上述任一序列具有至少80％相似性的氨基酸序列；和/或其包含轻链可变区中的LCDR1、LCDR2、LCDR3，所述轻链可变区包含如SEQ ID NO:25、29、33、37、41或78中任一项所示的氨基酸序列或其变体，或包含与上述任一序列具有至少80％相似性的氨基酸序列。
  4. 如权利要求1所述的抗体，其特征在于，所述抗体重链可变区的CDR区和/或轻链可变区的CDR区选自如下任意组的序列：
    (1)抗体，其包含SEQ ID NO:1所示的HCDR1，SEQ ID NO:2所示的HCDR2和SEQ ID NO:3所示的HCDR3；SEQ ID NO:4所示的LCDR1，SEQ ID NO:5所示的LCDR2和SEQ ID NO:6所示的LCDR3；或
    (2)抗体，其包含SEQ ID NO:1所示的HCDR1，SEQ ID NO:2所示的HCDR2和SEQ ID NO:7所示的HCDR3；SEQ ID NO:4所示的LCDR1，SEQ ID NO:5所示的LCDR2和SEQ ID NO:8所示的LCDR3；或
    (3)抗体，其包含SEQ ID NO:1所示的HCDR1，SEQ ID NO:2所示的HCDR2和SEQ ID NO:9所示的HCDR3；SEQ ID NO:4所示的LCDR1，SEQ ID NO:5所示的LCDR2和SEQ ID NO:10所示的LCDR3；或
    (4)抗体，其包含SEQ ID NO:11所示的HCDR1，SEQ ID NO:12所示的HCDR2和SEQ ID NO: 13所示的HCDR3；SEQ ID NO:14所示的LCDR1，SEQ ID NO:15所示的LCDR2和SEQ ID NO:16所示的LCDR3；或
    (5)抗体，其包含SEQ ID NO:17所示的HCDR1，SEQ ID NO:18所示的HCDR2和SEQ ID NO:19所示的HCDR3；SEQ ID NO:20所示的LCDR1，SEQ ID NO:21所示的LCDR2和SEQ ID NO:22所示的LCDR3；或
    (6)抗体，其包含SEQ ID NO:70所示的HCDR1，SEQ ID NO:72所示的HCDR2和SEQ ID NO:3所示的HCDR3；SEQ ID NO:4所示的LCDR1，SEQ ID NO:5所示的LCDR2和SEQ ID NO:6所示的LCDR3；或
    (7)抗体，其包含SEQ ID NO:71所示的HCDR1，SEQ ID NO:73所示的HCDR2和SEQ ID NO:3所示的HCDR3；SEQ ID NO:4所示的LCDR1，SEQ ID NO:5所示的LCDR2和SEQ ID NO:6所示的LCDR3；或
    (8)抗体，其包含SEQ ID NO:1所示的HCDR1，SEQ ID NO:2所示的HCDR2和SEQ ID NO:3所示的HCDR3；SEQ ID NO:74所示的LCDR1，SEQ ID NO:75所示的LCDR2和SEQ ID NO:6所示的LCDR3；
    (9)抗体，(1)～(8)中任一项所述的抗体的变体，且具备与(1)～(8)中任一项所述的抗体相同或相似的活性。
  5. 如权利要求2或3所述的抗体，其特征在于，所述抗体选自如下任意组的序列：
    (1)抗体，所述抗体的重链可变区具有SEQ ID NO:23所示的氨基酸序列或与SEQ ID NO:23具有至少80％相似性的氨基酸序列，所述轻链可变区具有SEQ ID NO:25所示的氨基酸序列或与SEQ ID NO:25具有至少80％相似性的氨基酸序列；
    (2)抗体，所述抗体的重链可变区具有SEQ ID NO:27所示的氨基酸序列或与SEQ ID NO:27具有至少80％相似性的氨基酸序列，所述轻链可变区具有SEQ ID NO:29所示的氨基酸序列或与SEQ ID NO:29具有至少80％相似性的氨基酸序列；
    (3)抗体，所述抗体的重链可变区具有SEQ ID NO:31所示的氨基酸序列或与SEQ ID NO:31具有至少80％相似性的氨基酸序列，所述轻链可变区具有SEQ ID NO:33所示的氨基酸序列或与SEQ ID NO:33具有至少80％相似性的氨基酸序列；
    (4)抗体，所述抗体的重链可变区具有SEQ ID NO:35所示的氨基酸序列或与SEQ ID NO:35具有至少80％相似性的氨基酸序列，所述轻链可变区具有SEQ ID NO:37所示的氨基酸序列或与SEQ ID NO:37具有至少80％相似性的氨基酸序列；
    (5)抗体，所述抗体的重链可变区具有SEQ ID NO:39所示的氨基酸序列或与SEQ ID NO:39具有至少80％相似性的氨基酸序列，所述轻链可变区具有SEQ ID NO:41所示的氨基酸序列或与SEQ ID NO:40具有至少80％相似性的氨基酸序列；
    (6)抗体，所述抗体的重链可变区具有SEQ ID NO:76所示的氨基酸序列或与SEQ ID NO:76具有至少80％相似性的氨基酸序列，所述轻链可变区具有SEQ ID NO:25所示的氨基酸序列 或与SEQ ID NO:25具有至少80％相似性的氨基酸序列；
    (7)抗体，所述抗体的重链可变区具有SEQ ID NO:80所示的氨基酸序列或与SEQ ID NO:80具有至少80％相似性的氨基酸序列，所述轻链可变区具有SEQ ID NO:25所示的氨基酸序列或与SEQ ID NO:25具有至少80％相似性的氨基酸序列；
    (8)抗体，所述抗体的重链可变区具有SEQ ID NO:23所示的氨基酸序列或与SEQ ID NO:23具有至少80％相似性的氨基酸序列，所述轻链可变区具有SEQ ID NO:78所示的氨基酸序列或与SEQ ID NO:78具有至少80％相似性的氨基酸序列；
    (9)抗体，(1)～(8)中任一项所述的抗体的变体，且具备与(1)～(8)中任一项所述的抗体相同或相似的活性。
  6. 如权利要求1-5任一项所述的抗体，其特征在于，所述抗体选自全抗、scFv、单域抗体、Fab片段、Fab’片段、Fv片段、F(ab’)2片段、Fd片段、dAb片段、多功能抗体或scFv-Fc抗体、杂交瘤抗体、嵌合抗体、人源化抗体、全人源抗体、单克隆抗体。
  7. 如权利要求6所述的抗体，其特征在于，所述抗体是杂交瘤抗体，选自：
    (1)抗体，所述抗体的重链可变区具有SEQ ID NO:35所示的氨基酸序列或与SEQ ID NO:35具有至少80％相似性的氨基酸序列，所述轻链可变区具有SEQ ID NO:37所示的氨基酸序列或与SEQ ID NO:37具有至少80％相似性的氨基酸序列；
    (2)抗体，所述抗体的重链可变区具有SEQ ID NO:39所示的氨基酸序列或与SEQ ID NO:39具有至少80％相似性的氨基酸序列，所述轻链可变区具有SEQ ID NO:41所示的氨基酸序列或与SEQ ID NO:41具有至少80％相似性的氨基酸序列；
    (3)抗体，(1)～(2)中任一项所述的抗体的变体，且具备与(1)～(2)中任一项所述的抗体相同或相似的活性。
  8. 如权利要求6所述的抗体，其特征在于，所述抗体是全人源抗体，选自：
    (1)抗体，所述抗体的重链可变区具有SEQ ID NO:23所示的氨基酸序列或与SEQ ID NO:23具有至少80％相似性的氨基酸序列，所述轻链可变区具有SEQ ID NO:25所示的氨基酸序列或与SEQ ID NO:25具有至少80％相似性的氨基酸序列；
    (2)抗体，所述抗体的重链可变区具有SEQ ID NO:27所示的氨基酸序列或与SEQ ID NO:27具有至少80％相似性的氨基酸序列，所述轻链可变区具有SEQ ID NO:29所示的氨基酸序列或与SEQ ID NO:29具有至少80％相似性的氨基酸序列；
    (3)抗体，所述抗体的重链可变区具有SEQ ID NO:31所示的氨基酸序列或与SEQ ID NO:31具有至少80％相似性的氨基酸序列，所述轻链可变区具有SEQ ID NO:33所示的氨基酸序列或与SEQ ID NO:33具有至少80％相似性的氨基酸序列；
    (4)抗体，所述抗体的重链可变区具有SEQ ID NO:76所示的氨基酸序列或与SEQ ID NO:76具有至少80％相似性的氨基酸序列，所述轻链可变区具有SEQ ID NO:25所示的氨基酸序列或与SEQ ID NO:25具有至少80％相似性的氨基酸序列；
    (5)抗体，所述抗体的重链可变区具有SEQ ID NO:80所示的氨基酸序列或与SEQ ID NO:80具有至少80％相似性的氨基酸序列，所述轻链可变区具有SEQ ID NO:25所示的氨基酸序列或与SEQ ID NO:25具有至少80％相似性的氨基酸序列；
    (6)抗体，所述抗体的重链可变区具有SEQ ID NO:23所示的氨基酸序列或与SEQ ID NO:23具有至少80％相似性的氨基酸序列，所述轻链可变区具有SEQ ID NO:78所示的氨基酸序列或与SEQ ID NO:78具有至少80％相似性的氨基酸序列；
    (7)抗体，(1)～(6)中任一项所述的抗体的变体，且具备与(1)～(6)中任一项所述的抗体相同或相似的活性。
  9. 如权利要求8所述的抗体，其特征在于，所述抗体选自：
    (1)抗体，所述抗体的重链具有SEQ ID NO:45所示的氨基酸序列或与SEQ ID NO:45具有至少80％相似性的氨基酸序列，所述轻链具有SEQ ID NO:47所示的氨基酸序列或与SEQ ID NO:47具有至少80％相似性的氨基酸序列；
    (2)抗体，所述抗体的重链具有SEQ ID NO:49所示的氨基酸序列或与SEQ ID NO:49具有至少80％相似性的氨基酸序列，所述轻链具有SEQ ID NO:51所示的氨基酸序列或与SEQ ID NO:51具有至少80％相似性的氨基酸序列；
    (3)抗体，所述抗体的重链具有SEQ ID NO:53所示的氨基酸序列或与SEQ ID NO:53具有至少80％相似性的氨基酸序列，所述轻链具有SEQ ID NO:55所示的氨基酸序列或与SEQ ID NO:55具有至少80％相似性的氨基酸序列；
    (4)抗体，(1)～(3)中任一项所述的抗体的变体，且具备与(1)～(3)中任一项所述的抗体相同或相似的活性。
  10. 免疫缀合物，其特征在于，所述免疫缀合物包括：权利要求1-9任一所述的抗体，以及与之连接的功能性分子。
  11. 嵌合受体，其特征在于，所述嵌合受体的胞外域包含权利要求1-9任一所述的抗体，所述嵌合受体包括：嵌合抗原受体(CAR)、嵌合T细胞受体、T细胞抗原耦合器(TAC)或其组合。
  12. 如权利要求11所述嵌合受体，其特征在于，所述嵌合受体为嵌合抗原受体(CAR)，其包含权利要求1-9任一所述的抗体、跨膜区和胞内信号区。
  13. 如权利要求12所述的嵌合受体，所述的胞内信号区选自：CD3δ、FcεRIγ、CD27、CD28、CD137、CD134、MyD88、CD40的胞内信号区序列或其组合；和/或所述的跨膜区包含CD8或CD28的跨膜区。
  14. 如权利要求13所述的嵌合受体，其特征在于，所述的嵌合受体选自如下中的任意：
    权利要求1-9任一所述的抗体、CD8/CD28的跨膜区和CD3δ；或
    权利要求1-9任一所述的抗体、CD8/CD28的跨膜区、CD137的胞内信号区和CD3δ；或
    权利要求1-9任一所述的抗体、CD8/CD28的跨膜区、CD28的胞内信号区和CD3δ；或
    权利要求1-9任一所述的抗体、CD8/CD28的跨膜区、CD28的胞内信号区、CD137和CD3δ。
  15. 如权利要求11所述的嵌合受体，其特征在于，所述的嵌合受体的氨基酸序列如SEQ ID NO：63、88、89、90任一所示。
  16. 一种生物材料，其为如下中的任意一种：
    1)编码权利要求1-9任一所述的抗体、权利要求10所述的免疫缀合物、权利要求11-15任一所述的嵌合受体的核酸；
    2)载体，其包含1)所述的核酸；
    3)病毒，其包含1)或2)所述核酸或载体。
  17. 细胞，其包含权利要求1-9任一所述的抗体、权利要求10所述的免疫缀合物、权利要求11-15任一所述的嵌合受体、权利要求16中1)所述核酸、和/或权利要求16中2)所述的载体。
  18. 如权利要求17所述的细胞，其特征在于，所述细胞包括T细胞、自然杀伤细胞、自然杀伤T细胞、NK92细胞、干细胞衍生的免疫效应细胞或其组合。
  19. 如权利要求18所述的细胞，其特征在于，所述T细胞包括天然的T细胞和/或经多能干细胞诱导产生的T细胞；
    优选地，所述T细胞包括自体T细胞和/或同种异体T细胞；
    优选地，所述T细胞为原代T细胞；
    优选地，所述T细胞来源于人的自体T细胞。
  20. 如权利要求17-19任一所述的细胞，其特征在于，其还携带外源的细胞因子的编码序列；和/或
    其还表达非靶向GPRC5D的嵌合受体；和/或
    其还表达趋化因子；和/或
    其还表达趋化因子受体；和/或
    其还表达安全开关。
  21. 药物组合物，其包含权利要求1-9任一所述的抗体、权利要求10所述的免疫缀合物、权利要求11-15任一所述的嵌合受体、权利要求16中1)所述核酸、权利要求16中2)所述的载体和/或权利要求17-20任一所述的细胞，以及药学上可接受的佐剂。
  22. 联合用药，其特征在于，权利要求1-9任一所述的抗体、权利要求10所述的免疫缀合物、权利要求11-15任一所述的嵌合受体、权利要求17-20任一所述的细胞、权利要求21所述的药物组合物与增强其功能的药剂组合施用，优选地，与化疗药物联用；
    和/或与改善其相关的一种或多种副作用的药剂联合施用；
    和/或与表达靶向GPRC5D之外的嵌合抗原受体的细胞联合施用。
  23. 一种制备权利要求1-9任一所述的抗体、权利要求10所述的免疫缀合物、权利要求11-15任一所述的嵌合受体的方法，所述方法包含在适于表达所述抗体、免疫缀合物、嵌合受体的条件下培养权利要求17-20任一所述的细胞，以及分离出由所述细胞表达的所述抗体、免疫缀合物、嵌合受体。
  24. 一种试剂盒，其特征在于，其包括权利要求1-9任一所述的抗体、权利要求10所述的免疫缀合物、权利要求11-15任一所述的嵌合受体、权利要求16中1)所述核酸、权利要求16中2)所述的载体、权利要求17-20任一所述的细胞和/或权利要求21所述药物组合物。
  25. 一种治疗/诊断疾病的方法，其包括向有需要的受试者给予有效量的如权利要求1-9任一所述的抗体或如权利要求10所述的免疫缀合物或如权利要求17-2\0任一所述的细胞或如权利要求21所述的药物组合物或如权利要求24所述的试剂盒；
    优选地，所述疾病选自炎性病症、感染、自身免疫性疾病和肿瘤；
    优选地，所述受试者是人；
    优选地，其中所述细胞对于所述受试者是自体的或同种异体的T细胞。
  26. 如权利要求1-9任一所述的抗体、权利要求10所述的免疫缀合物、权利要求17-20任一所述的细胞、权利要求21所述的药物组合物和/或权利要求24所述的试剂盒在制备用于治疗/诊断疾病的药物中的用途，其特征在于，所述疾病包含表达GPRC5D；优选地，所述疾病选自炎性病症、感染、自身免疫性疾病和肿瘤。
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