WO2024046239A1 - 靶向人gprc5d的重组人源化单克隆抗体及其应用 - Google Patents

靶向人gprc5d的重组人源化单克隆抗体及其应用 Download PDF

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WO2024046239A1
WO2024046239A1 PCT/CN2023/115089 CN2023115089W WO2024046239A1 WO 2024046239 A1 WO2024046239 A1 WO 2024046239A1 CN 2023115089 W CN2023115089 W CN 2023115089W WO 2024046239 A1 WO2024046239 A1 WO 2024046239A1
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antibody
antigen
seq
cells
binding fragment
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PCT/CN2023/115089
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English (en)
French (fr)
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马东晖
曾楷楷
何安涛
洪淑娟
梅芬
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苏州缔码生物科技有限公司
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Publication of WO2024046239A1 publication Critical patent/WO2024046239A1/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
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • 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

  • the invention belongs to the field of biotechnology, and specifically relates to recombinant humanized monoclonal antibodies targeting human GPRC5D and their applications.
  • GPRC5D is an orphan receptor of G protein-coupled receptors (GPCRs). It is subtype D of the C5 family of G protein-coupled receptors and is a 7-transmembrane protein. Orphan receptors refer to receptors that are structurally similar to other confirmed receptors, but whose endogenous ligands have not yet been discovered. GPRC5D is highly expressed on the surface of primary multiple myeloma cells, while the expression in normal tissues is limited to the hair follicle area. Studies have shown that 65% of multiple myeloma patients have GPRC5D exceeding the 50% expression threshold. With this characteristic, GPRC5D has become potential targets for the treatment of MM.
  • GPCRs G protein-coupled receptors
  • GPRC5D is highly expressed on the surface of plasma cells and multiple myeloma cells and is not expressed on other normal tissue cells and hematopoietic stem cells, it can be used as an ideal target for the diagnosis and treatment of multiple myeloma (MM).
  • the purpose of the present invention is to provide a specific recombinant humanized monoclonal antibody targeting human GPRC5D.
  • the specific recombinant humanized monoclonal antibody targeting human GPRC5D provided by the invention has high affinity, good specificity, and can compensate for Diagnostic and therapeutic applications of recombinant humanized monoclonal antibodies targeting human GPRC5D protein on the market.
  • the GPRC5D antibody sequence can be constructed into chimeric antigen receptor T cell immunotherapy (CART) or bispecific antibodies or antibody conjugates, which can be used for subsequent treatment of tumor diseases.
  • a first aspect of the present invention provides an antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region, the heavy chain variable region comprising the following CDRs:
  • VH-CDR2 shown in SEQ ID NO:45,59,79,12,34,68,2,23,54 or 88, and
  • VH-CDR3 shown in SEQ ID NO: 46, 60, 13, 35, 69, 3, 24, 55 or 89;
  • said antibody or antigen-binding fragment thereof specifically binds GPRC5D, preferably human GPRC5D.
  • the heavy chain variable region of the antibody or antigen-binding fragment thereof includes the following 3 CDR:
  • VH-CDR2 shown in SEQ ID NO:45, and
  • VH-CDR3 shown in SEQ ID NO:46.
  • the heavy chain variable region of the antibody or antigen-binding fragment thereof includes the following 3 CDRs:
  • VH-CDR2 shown in SEQ ID NO:59, and
  • VH-CDR3 shown in SEQ ID NO:60.
  • the heavy chain variable region of the antibody or antigen-binding fragment thereof includes the following 3 CDRs:
  • VH-CDR2 shown in SEQ ID NO:79, and
  • VH-CDR3 shown in SEQ ID NO:60.
  • the heavy chain variable region of the antibody or antigen-binding fragment thereof includes the following 3 CDRs:
  • VH-CDR1 shown in SEQ ID NO:1,
  • VH-CDR2 shown in SEQ ID NO:12, and
  • VH-CDR3 shown in SEQ ID NO:13.
  • the antibody or antigen-binding fragment thereof further includes a light chain variable region, and the light chain variable region includes the following CDRs:
  • VL-CDR1 shown in SEQ ID NO:47,61,80,14,36,70,4,25,47 or 90,
  • VL-CDR2 shown in SEQ ID NO:37,15,71,5 or 26, and
  • VL-CDR3 shown in SEQ ID NO:48,81,16,38,72,6,27,48 or 91.
  • the light chain variable region of the antibody or antigen-binding fragment thereof includes the following 3 CDRs:
  • VL-CDR1 shown in SEQ ID NO:47,
  • VL-CDR2 shown in SEQ ID NO:37, and
  • VL-CDR3 shown in SEQ ID NO:48.
  • the light chain variable region of the antibody or antigen-binding fragment thereof includes the following 3 CDRs:
  • VL-CDR1 shown in SEQ ID NO:61
  • VL-CDR2 shown in SEQ ID NO:37, and
  • VL-CDR3 shown in SEQ ID NO:48.
  • the light chain variable region of the antibody or antigen-binding fragment thereof includes the following 3 CDRs:
  • VL-CDR1 shown in SEQ ID NO:80
  • VL-CDR2 shown in SEQ ID NO:37, and
  • VL-CDR3 shown in SEQ ID NO:81.
  • the light chain variable region of the antibody or antigen-binding fragment thereof includes the following 3 CDRs:
  • VL-CDR1 shown in SEQ ID NO:14,
  • VL-CDR2 shown in SEQ ID NO:15, and
  • VL-CDR3 shown in SEQ ID NO:16.
  • the antibody or antigen-binding fragment thereof comprises a heavy chain variable region CDR and a light chain variable region CDR selected from the following group:
  • the antibody or antigen-binding fragment thereof comprises at least 80%, 85%, 90% of SEQ ID NO: 49, 62, 82, 17, 39, 73, 7, 28, 56 or 92 , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity of the heavy chain variable region of the amino acid sequence.
  • the antibody or antigen-binding fragment thereof comprises at least 80%, 85%, 90%, 91 A light chain variable region with an amino acid sequence that is %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical.
  • the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region selected from the following group:
  • the antibody or antigen-binding fragment thereof comprises a heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 49, and a light chain variable region of the amino acid sequence shown in SEQ ID NO: 51. Change area.
  • the antibody or antigen-binding fragment thereof comprises a heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 62, and a light chain variable region of the amino acid sequence shown in SEQ ID NO: 64. Change area.
  • the antibody or antigen-binding fragment thereof comprises a heavy chain variable region of the amino acid sequence shown in SEQ ID NO:82, and a light chain variable region of the amino acid sequence shown in SEQ ID NO:84. Change area.
  • the antibody or antigen-binding fragment thereof comprises a heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 17, and a light chain variable region of the amino acid sequence shown in SEQ ID NO: 19. Change area.
  • the antibody includes a single-chain antibody (scFv), a diabody, a monoclonal antibody, a chimeric antibody (such as a human-mouse chimeric antibody), an animal-derived antibody, a fully human antibody, or a humanized antibody.
  • scFv single-chain antibody
  • diabody a diabody
  • monoclonal antibody a monoclonal antibody
  • a chimeric antibody such as a human-mouse chimeric antibody
  • an animal-derived antibody such as a human-mouse chimeric antibody
  • a fully human antibody such as a humanized antibody.
  • the antibody is a humanized antibody.
  • the antibody is a monoclonal antibody.
  • the light chain constant region of the antibody is a kappa chain
  • the heavy chain constant region is of IgG type.
  • the antibody is a bispecific antibody or a multispecific antibody.
  • a second aspect of the present invention provides a recombinant protein, said recombinant protein comprising:
  • the tag sequence is selected from the following group: 6 ⁇ His tag, GGGS sequence, and FLAG tag.
  • a third aspect of the invention provides a polynucleotide encoding a polypeptide selected from the group consisting of:
  • a fourth aspect of the present invention provides a vector containing the polynucleotide described in the third aspect of the present invention.
  • the vector includes: bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, adeno-associated virus, retrovirus, lentiviral vector or other vectors.
  • the fifth aspect of the present invention provides a genetically engineered host cell, the host cell contains the vector as described in the fourth aspect of the present invention, or the polynucleotide as described in the third aspect of the present invention is integrated into the genome. .
  • a sixth aspect of the present invention provides an immunoconjugate, the immunoconjugate comprising:
  • a conjugation moiety selected from the group consisting of a detectable marker, a drug, a toxin, a cytokine, a radionuclide, or an enzyme.
  • the conjugate is selected from: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computerized X-ray tomography) contrast agents, or can produce detectable Product enzymes, radionuclides, biotoxins, cytokines (such as IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles/nanorods, virus particles, liposomes, nanomagnetic particles, pro- Drug-activated enzymes (eg, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), chemotherapeutic agents (eg, cisplatin), or any form of nanoparticles, etc.
  • DTD DT-diaphorase
  • BPHL biphenyl hydrolase-like protein
  • a seventh aspect of the present invention provides a chimeric antigen receptor, the antigen-binding domain of the chimeric antigen receptor comprising the antibody or antigen-binding fragment thereof as described in the first aspect of the present invention.
  • An eighth aspect of the present invention provides an immune cell that expresses or has the antibody or antigen-binding fragment thereof as described in the first aspect of the present invention expressed or exposed outside the cell membrane.
  • the immune cells include T cells, NK cells or macrophages.
  • the immune cells are CAR-T cells.
  • a ninth aspect of the present invention provides a pharmaceutical composition containing:
  • Active ingredient is selected from the group consisting of: an antibody or an antigen-binding fragment thereof as described in the first aspect of the present invention, a recombinant protein as described in the second aspect of the present invention, a recombinant protein as described in the sixth aspect of the present invention
  • the pharmaceutical composition is a liquid preparation.
  • the pharmaceutical composition is an injection.
  • the pharmaceutical composition is used to prepare drugs for treating diseases related to high expression of GPRC5D.
  • the disease associated with high expression of GPRC5D is a tumor, such as multiple myeloma.
  • a tenth aspect of the present invention provides the use of an active ingredient selected from the following group: the antibody or antigen-binding fragment thereof as described in the first aspect of the present invention, the antibody as described in the second aspect of the present invention
  • Recombinant proteins, immunoconjugates as described in the sixth aspect of the present invention or immune cells as described in the eighth aspect of the present invention, or combinations thereof, are used for preparing diagnostic reagents or kits, or for preparing treatments for GPRC5D hypertensive disorders.
  • Drugs for expression-related diseases are used for preparing diagnostic reagents or kits, or for preparing treatments for GPRC5D hypertensive disorders.
  • the disease associated with high expression of GPRC5D is a tumor, such as multiple myeloma.
  • An eleventh aspect of the present invention provides a method for in vitro non-diagnostic detection of GPRC5D protein in a sample, including the steps:
  • a twelfth aspect of the present invention provides a method for preparing the antibody or antigen-binding fragment thereof as described in the first aspect of the present invention, or the recombinant protein as described in the second aspect of the present invention, comprising the steps: (si) Cultivate the host cell as described in the fifth aspect of the present invention under conditions suitable for protein expression; (sii) isolate the antibody or antigen-binding fragment thereof as described in the first aspect of the present invention from the cell culture, or as described herein The recombinant protein according to the second aspect of the invention.
  • a thirteenth aspect of the present invention provides a method for treating diseases associated with high GPRC5D expression, the method comprising administering an antibody or an antigen-binding fragment thereof as described in the first aspect of the present invention to a subject in need , the recombinant protein as described in the second aspect of the present invention, the immunoconjugate as described in the sixth aspect of the present invention, the chimeric antigen receptor as described in the seventh aspect of the present invention, as described in the eighth aspect of the present invention immune cells or the pharmaceutical composition according to the ninth aspect of the present invention.
  • the disease associated with high expression of GPRC5D is a tumor, such as multiple myeloma.
  • the subject in need is a human or non-human mammal.
  • a fourteenth aspect of the present invention provides a bispecific antibody comprising the antibody or antigen-binding fragment thereof as described in the first aspect of the present invention.
  • the bispecific antibody further includes an antibody targeting a second antigen or an antigen-binding fragment thereof, and the second antigen is selected from the group consisting of: CD3, CD16, CD32, and 41BB.
  • the bispecific antibody comprises an antibody targeting CD3 or an antigen-binding fragment thereof.
  • the CD3-targeting antibody or antigen-binding fragment thereof comprises a heavy chain variable region as shown in SEQ ID NO: 97, and a light chain variable region as shown in SEQ ID NO: 98 .
  • the bispecific antibody includes the following three peptide chains:
  • Chain 3 CD3 VL-CL; or
  • Chain 2 CD3 VH-CH1-Fc Hole
  • Chain 3 CD3 VL-CL;
  • the GPRC5D scFv is a scFv targeting GPRC5D
  • CD3 VH is the heavy chain variable region of an antibody targeting CD3
  • CD3 VL is the light chain variable region of an antibody targeting CD3
  • CH1 is the heavy chain of an IgG antibody.
  • Constant region 1 CL is the light chain constant region of the antibody.
  • amino acid sequence of the Fc Knob is shown in SEQ ID NO: 106
  • amino acid sequence of the Fc Knob is shown in SEQ ID NO: 107.
  • the CL is the constant region of human Kappa light chain.
  • the GPRC5D scFv is selected from the group consisting of: SEQ ID NO: 53, 66 or 86.
  • the bispecific antibody includes the following three peptide chains: chain 1 as shown in any one of SEQ ID NO: 102-104, chain 2 as shown in SEQ ID NO: 101, including Chain 3 of the amino acid sequence shown in SEQ ID NO:98.
  • the present invention also provides polynucleotides encoding the bispecific antibodies according to the fourteenth aspect of the present invention.
  • Vectors comprising the polynucleotides, and host cells containing the vectors.
  • the present invention also provides immunoconjugates and pharmaceutical compositions comprising the bispecific antibody according to the fourteenth aspect of the present invention.
  • the present invention also provides the use of the bispecific antibody described in the fourteenth aspect of the present invention in preparing drugs for treating diseases related to high expression of GPRC5D, and the use of the bispecific antibody to treat diseases related to high expression of GPRC5D. method of disease.
  • Figure 1 shows two structures of humanized antibodies: native structure and scFv-hFc structure.
  • Figure 2 shows the affinity of native structure humanized antibodies to K562-GPRC5D cells.
  • Figure 3 shows the affinity of scFv-hFc structured humanized antibodies to K562-GPRC5D ( Figure 3a) and RPMI 8226 cells ( Figure 3b).
  • Figure 4 shows the binding activity of the scFv-hFc structure humanized antibody to MM.1S ( Figure 4a) and NCI-H929 ( Figure 4b).
  • Figure 5 shows the vector insert sequence structure of GPRC5D CAR.
  • Figure 6 shows the GPRC5D CAR jurkat nfat luc cells with K562-GPRC5D ( Figure 6a) or Self-activation and antigen-dependent activation after co-culture of RPMI 8226 cells (Fig. 6b).
  • Figure 7 shows the flow cytometry results of transfection efficiency of GPRC5D CAR-T cells.
  • Figure 8 shows the detection results of the concentration of inflammatory factors produced by GPRC5D CAR-T cells killing RPMI 8226 cells;
  • Figure 8a is the detection result of IL-2 concentration,
  • Figure 8b is the detection result of TNF- ⁇ concentration, and
  • Figure 8c is the detection result of IFN- ⁇ concentration. result.
  • Figure 9 shows the comparison results of inflammatory factor concentration detection produced by GPRC5D CAR-T cells co-cultured with MM.1S or K562 cells, and CAR-T cells cultured alone;
  • Figure 9a shows the IL-2 concentration detection results, and
  • Figure 9b shows TNF - ⁇ concentration detection results,
  • Figure 9c shows the IFN- ⁇ concentration detection results.
  • Figure 10 shows the cell proliferation flow cytometry results of GPRC5D CAR-T cells co-cultured with NCI-H929 or K562 cells, and CAR-T cells cultured alone.
  • Figure 11 shows that GPRC5D CAR-T cells with different efficacy-target ratios were compared with K562-luc (Figure 11a), K562-GPRC5D-luc ( Figure 11b), NCI-H929-luc ( Figure 11c), and MM.1S-luc ( Figure 11c).
  • Figure 12 shows the chemiluminescence detection results of B-NDG tumor-bearing mice treated with different administration groups.
  • Figure 13 shows the K562-GPRC5D endocytosis effect mediated by G05, G07, and G09 antibodies.
  • Figure 14 shows the in vitro binding activity of the GPRC5D&CD3 bispecific antibody
  • Figure 14a shows the binding activity of the GPRC5D&CD3 bispecific antibody to H929 cells
  • Figure 14b shows the binding activity of the GPRC5D&CD3 bispecific antibody to Jurkat cells.
  • Figure 15 shows the in vitro killing efficiency of the GPRC5D&CD3 bispecific antibody
  • Figure 15a shows the 48h killing efficiency of the GPRC5D&CD3 bispecific antibody on the 8226 cell line
  • Figure 15b shows the 48h killing efficiency of the GPRC5D&CD3 bispecific antibody on the H929 cell line
  • Figure 15 15c shows the 48h killing efficiency of the GPRC5D&CD3 bispecific antibody on the MM.1S cell line
  • Figure 15d shows the 48h killing efficiency of the GPRC5D&CD3 bispecific antibody on the K562 cell line.
  • antibody or "immunoglobulin” is a heterotetrameric protein of approximately 150,000 daltons with the same structural characteristics, consisting of two identical light chains (L) and two identical heavy chains (H) Composition. Each light chain is connected to the heavy chain by a covalent disulfide bond, and the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable domain (VH) at one end, followed by multiple constant domains.
  • VH variable domain
  • Each light chain has a variable region (VL) at one end and a constant region at the other end; the constant region of the light chain is opposite to the first constant region of the heavy chain, and the variable region of the light chain is opposite to the variable region of the heavy chain. .
  • Special amino acid residues form the interface between the variable regions of the light and heavy chains.
  • variable means that certain portions of the variable regions of an antibody differ in sequence and contribute to the binding and specificity of each particular antibody to its particular antigen. However, variability is not evenly distributed throughout the antibody variable region. It is concentrated in three segments in the variable regions of the light and heavy chains called complementarity determining regions (CDRs) or hypervariable regions. The more conserved part of the variable region is called the framework region (FR).
  • CDRs complementarity determining regions
  • FR framework region
  • the variable regions of natural heavy and light chains each contain four FR regions, which are generally in a ⁇ -sheet configuration and are connected by three CDRs forming a connecting loop. In some cases, a partial ⁇ -sheet structure can be formed.
  • the CDRs in each chain are held closely together by the FR region and together with the CDRs of the other chain form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Volume I, pp. 647-669 (1991)). Constant regions are not directly involved in the binding of the antibody to the antigen, but they exhibit different effector functions, such as involvement in antibody-dependent cytotoxicity of the antibody.
  • the "light chains" of vertebrate antibodies can be assigned to one of two distinct classes (termed kappa and lambda) based on the amino acid sequence of their constant regions.
  • Immunoglobulins can be divided into different classes based on the amino acid sequence of their heavy chain constant region.
  • the heavy chain constant regions corresponding to different classes of immunoglobulins are called ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
  • the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known to those in the art.
  • the term "monoclonal antibody (mAb)” refers to an antibody obtained from a population that is substantially homogeneous, that is, the individual antibodies contained in the population are identical except for a few naturally occurring mutations that may be present. Monoclonal antibodies target a single antigenic site with high specificity. Furthermore, unlike conventional polyclonal antibody preparations, which typically have different antibodies directed against different determinants, each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies have the benefit of not being contaminated by other immunoglobulins. The modifier "monoclonal" indicates the nature of the antibody as having been obtained from a homogeneous population of antibodies and should not be construed as requiring any special method to produce the antibody.
  • the recombinant humanized monoclonal antibodies targeting human GPRC5D provided by the invention are selected from one or more of the following recombinant humanized monoclonal antibodies:
  • Monoclonal antibody named G01 the amino acid sequences of its heavy chain complementarity determining regions CDR1, CDR2, and CDR3 are the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 respectively; and the light chain
  • the amino acid sequences of the complementarity determining regions CDR1, CDR2 and CDR3 are the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6 respectively;
  • the amino acid sequence of the humanized heavy chain variable region is SEQ ID NO:7; Its humanized heavy chain variable region nucleic acid sequence is SEQ ID NO:8; Its humanized light chain variable region amino acid sequence is SEQ ID NO:9; Its humanized light chain variable region nucleic acid
  • the sequence is SEQ ID NO:10; its humanized scFv amino acid sequence is SEQ ID NO:11;
  • Monoclonal antibody named G02 the amino acid sequences of its heavy chain complementarity determining regions CDR1, CDR2 and CDR3 are the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:12 and SEQ ID NO:13 respectively; and the light chain
  • the amino acid sequences of the complementarity determining regions CDR1, CDR2, and CDR3 are the amino acid sequences shown in SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16 respectively;
  • the amino acid sequence of the humanized heavy chain variable region is SEQ ID NO: 17; its humanized heavy chain variable region nucleic acid sequence is SEQ ID NO: 18; its humanized light chain variable region amino acid sequence is SEQ ID NO: 19; its humanized light chain variable region nucleic acid
  • the sequence is SEQ ID NO:20; its humanized scFv amino acid sequence is SEQ ID NO:21;
  • Monoclonal antibody named G03 the amino acid sequences of its heavy chain complementarity determining regions CDR1, CDR2, and CDR3 are the amino acid sequences shown in SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24 respectively; and the light chain The amino acid sequences of the complementarity determining regions CDR1, CDR2, and CDR3 are the amino acid sequences shown in SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27 respectively; the amino acid sequence of the humanized heavy chain variable region is SEQ ID NO: 28; its humanized heavy chain variable region nucleic acid sequence is SEQ ID NO: 29; its humanized light chain variable region amino acid sequence is SEQ ID NO: 30; its humanized light chain variable region nucleic acid The sequence is SEQ ID NO:31; its humanized scFv amino acid sequence is SEQ ID NO:32;
  • Monoclonal antibody named G04 the amino acid sequences of its heavy chain complementarity determining regions CDR1, CDR2, and CDR3 are the amino acid sequences shown in SEQ ID NO:33, SEQ ID NO:34, and SEQ ID NO:35 respectively; and the light chain
  • the amino acid sequences of the complementarity determining regions CDR1, CDR2, and CDR3 are the amino acid sequences shown in SEQ ID NO:36, SEQ ID NO:37, and SEQ ID NO:38 respectively;
  • the amino acid sequence of the humanized heavy chain variable region is SEQ ID NO: 39; its humanized heavy chain variable region nucleic acid sequence is SEQ ID NO: 40; its humanized light chain variable region amino acid sequence is SEQ ID NO: 41; its humanized light chain variable region nucleic acid
  • the sequence is SEQ ID NO:42; its humanized scFv amino acid sequence is SEQ ID NO:43;
  • the monoclonal antibody named G05 the amino acid sequences of its heavy chain complementarity determining regions CDR1, CDR2, and CDR3 are the amino acid sequences shown in SEQ ID NO: 44, SEQ ID NO: 45, and SEQ ID NO: 46 respectively; and the light chain
  • the amino acid sequences of the complementarity determining regions CDR1, CDR2 and CDR3 are the amino acid sequences shown in SEQ ID NO:47, SEQ ID NO:37 and SEQ ID NO:48 respectively; the amino acids of the humanized heavy chain variable region
  • the sequence is SEQ ID NO: 49; the nucleic acid sequence of its humanized heavy chain variable region is SEQ ID NO: 50; the amino acid sequence of its humanized light chain variable region is SEQ ID NO: 51; its humanized light chain
  • the nucleic acid sequence of the variable region is SEQ ID NO: 52; the amino acid sequence of its humanized scFv is SEQ ID NO: 53;
  • Monoclonal antibody named G06 the amino acid sequences of its heavy chain complementarity determining regions CDR1, CDR2, and CDR3 are the amino acid sequences shown in SEQ ID NO:44, SEQ ID NO:54, and SEQ ID NO:55 respectively; and the light chain The amino acid sequences of the complementarity determining regions CDR1, CDR2, and CDR3 are the amino acid sequences shown in SEQ ID NO:47, SEQ ID NO:37, and SEQ ID NO:48 respectively; the amino acid sequence of the humanized heavy chain variable region is SEQ ID NO: 56; its humanized heavy chain variable region nucleic acid sequence is SEQ ID NO: 57; its humanized light chain variable region amino acid sequence is SEQ ID NO: 51; its humanized light chain variable region nucleic acid The sequence is SEQ ID NO:52; its humanized scFv amino acid sequence is SEQ ID NO:58;
  • Monoclonal antibody named G07 the amino acid sequences of its heavy chain complementarity determining regions CDR1, CDR2, and CDR3 are the amino acid sequences shown in SEQ ID NO:44, SEQ ID NO:59, and SEQ ID NO:60 respectively; and the light chain The amino acid sequences of the complementarity determining regions CDR1, CDR2, and CDR3 are the amino acid sequences shown in SEQ ID NO:61, SEQ ID NO:37, and SEQ ID NO:48 respectively; the amino acid sequence of the humanized heavy chain variable region is SEQ ID NO: 62; its humanized heavy chain variable region nucleic acid sequence is SEQ ID NO: 63; its humanized light chain variable region amino acid sequence is SEQ ID NO: 64; its humanized light chain variable region nucleic acid The sequence is SEQ ID NO:65; its humanized scFv amino acid sequence is SEQ ID NO:66;
  • Monoclonal antibody named G08 the amino acid sequences of its heavy chain complementarity determining regions CDR1, CDR2 and CDR3 are the amino acid sequences shown in SEQ ID NO:67, SEQ ID NO:68 and SEQ ID NO:69 respectively; and the light chain
  • the amino acid sequences of the complementarity determining regions CDR1, CDR2, and CDR3 are the amino acid sequences shown in SEQ ID NO:70, SEQ ID NO:71, and SEQ ID NO:72 respectively;
  • the amino acid sequence of the humanized heavy chain variable region is SEQ ID NO:73; its humanized heavy chain variable region nucleic acid sequence is SEQ ID NO:74; its humanized light chain variable region amino acid sequence is SEQ ID NO:75; its humanized light chain variable region nucleic acid
  • the sequence is SEQ ID NO:76; its humanized scFv amino acid sequence is SEQ ID NO:77;
  • Monoclonal antibody named G09 the amino acid sequences of its heavy chain complementarity determining regions CDR1, CDR2, and CDR3 are the amino acid sequences shown in SEQ ID NO:78, SEQ ID NO:79, and SEQ ID NO:60 respectively; and the light chain
  • the amino acid sequences of the complementarity determining regions CDR1, CDR2, and CDR3 are the amino acid sequences shown in SEQ ID NO:80, SEQ ID NO:37, and SEQ ID NO:81 respectively;
  • the amino acid sequence of the humanized heavy chain variable region is SEQ ID NO: 82; its humanized heavy chain variable region nucleic acid sequence is SEQ ID NO: 83; its humanized light chain variable region amino acid sequence is SEQ ID NO: 84; its humanized light chain variable region nucleic acid sequence
  • the sequence is SEQ ID NO:85; its humanized scFv amino acid sequence is SEQ ID NO:86;
  • Monoclonal antibody named G10 amino acids of its heavy chain complementarity determining regions CDR1, CDR2, and CDR3
  • the acid sequences are the amino acid sequences shown in SEQ ID NO:87, SEQ ID NO:88 and SEQ ID NO:89 respectively; and the amino acid sequences of the light chain complementarity determining regions CDR1, CDR2 and CDR3 are respectively SEQ ID NO:90 and SEQ
  • the light chain constant region of the recombinant humanized monoclonal antibody is a kappa chain
  • the heavy chain constant region is of the IgG type
  • the present invention includes not only complete monoclonal antibodies, but also immunologically active antibody fragments, such as Fab or (Fab”) 2 fragments; scFv; antibody heavy chain; antibody light chain.
  • immunologically active antibody fragments such as Fab or (Fab”) 2 fragments; scFv; antibody heavy chain; antibody light chain.
  • the invention also provides other proteins or fusion expression products with the antibodies of the invention.
  • the invention includes any protein or protein conjugates and fusion expression products (i.e., immunoconjugates and fusion expression products) having heavy and light chains containing variable regions, as long as the variable regions are identical to the antibodies of the invention
  • the variable regions of the heavy and light chains are identical or at least about 90% homologous, preferably at least about 95% homologous.
  • variable regions of the heavy and/or light chains of the antibodies of the invention are of particular interest since at least part of them are involved in binding the antigen. Therefore, the present invention includes those molecules having monoclonal antibody light and heavy chain variable regions with CDRs as long as their CDRs are more than 90% (preferably more than 95%, optimally 98%) identical to the CDRs identified herein above) homology.
  • fragment refers to polypeptides that retain substantially the same biological function or activity of the antibodies of the invention.
  • the polypeptide fragment, derivative or analog of the present invention may be (i) a polypeptide in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) a polypeptide having substituent groups in one or more amino acid residues, or (iii) a mature polypeptide combined with another compound (such as a compound that extends the half-life of the polypeptide, e.g.
  • polyethylene glycol or (iv) a polypeptide formed by fusion of an additional amino acid sequence to this polypeptide sequence (such as a leader sequence or secretion sequence or a sequence used to purify this polypeptide or a protein sequence, or with Fusion protein formed by 6His tag).
  • additional amino acid sequence such as a leader sequence or secretion sequence or a sequence used to purify this polypeptide or a protein sequence, or with Fusion protein formed by 6His tag.
  • the antibody of the present invention refers to a polypeptide that has human GPRC5D protein-binding activity and includes the above-mentioned CDR region.
  • the term also includes variant forms of polypeptides containing the above-described CDR regions that have the same function as the antibodies of the invention. These variant forms include (but are not limited to): deletion of one or more (usually 1-50, preferably 1-30, more preferably 1-20, optimally 1-10) amino acids , insertion and/or substitution, and at the end of C One or several (usually within 20, preferably within 10, more preferably within 5) amino acids are added to the terminal and/or N-terminus. For example, in the art, substitutions with amino acids with similar or similar properties generally do not alter the function of the protein. As another example, adding one or more amino acids to the C-terminus and/or N-terminus usually does not change the function of the protein.
  • the term also includes active fragments and active derivatives of the antibodies of the invention.
  • Variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, and DNA that can hybridize with the DNA encoding the antibody of the invention under high or low stringency conditions.
  • the invention also provides other polypeptides, such as fusion proteins comprising human antibodies or fragments thereof.
  • the invention also encompasses fragments of the antibodies of the invention.
  • the fragment has at least about 50 contiguous amino acids of an antibody of the invention, preferably at least about 60 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100 contiguous amino acids.
  • “conservative variants of the antibody of the present invention” means that compared with the amino acid sequence of the antibody of the present invention, there are at most 10, preferably at most 8, more preferably at most 5, optimally at most 3 Amino acids are replaced by amino acids with similar or similar properties to form a polypeptide. These conservative variant polypeptides are preferably produced by amino acid substitutions according to Table 1.
  • the present invention also provides polynucleotide molecules encoding the above-mentioned antibodies or fragments or fusion proteins thereof.
  • the polynucleotides of the invention may be in DNA form or RNA form. Forms of DNA include cDNA, genomic DNA, or synthetic DNA. DNA can be single-stranded or double-stranded. DNA can be a coding strand or a non-coding strand.
  • the coding region sequence encoding the mature polypeptide can be the same as, for example, the coding region sequence shown in SEQ ID NO: 50, 52, or a degenerate variant.
  • degenerate variant in the present invention refers to a nucleic acid sequence encoding an amino acid sequence identical to the polypeptide of the present invention, but different from the coding region sequence shown in, for example, SEQ ID NO: 50 and 52. .
  • Polynucleotides encoding mature polypeptides of the present invention include: coding sequences encoding only mature polypeptides; coding sequences for mature polypeptides and various additional coding sequences; coding sequences for mature polypeptides (and optional additional coding sequences) and non-coding sequences .
  • polynucleotide encoding a polypeptide may include polynucleotides encoding such polypeptides, or may also include polynucleotides that also include additional coding and/or non-coding sequences.
  • the invention also relates to polynucleotides that hybridize to the sequences described above and have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences.
  • the invention particularly relates to polynucleotides that hybridize under stringent conditions to the polynucleotides of the invention.
  • stringent conditions refer to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 ⁇ SSC, 0.1% SDS, 60°C; or (2) adding There are denaturants, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42°C, etc.; or (3) only the identity between the two sequences is at least 90%, more It is best when hybridization occurs only when the ratio is above 95%.
  • the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO: 49 and SEQ ID NO: 51, for example.
  • the full-length nucleotide sequence of the antibody of the present invention or its fragment can usually be obtained by PCR amplification, recombinant or artificial synthesis.
  • a feasible method is to use artificial synthesis to synthesize the relevant sequences, especially when the fragment length is short. Often, fragments with long sequences are obtained by first synthesizing multiple small fragments and then ligating them.
  • the coding sequence of the heavy chain and the expression tag (such as 6His) can also be fused together to form a fusion protein.
  • Biomolecules (nucleic acids, proteins, etc.) involved in the present invention include biomolecules in isolated form.
  • the DNA sequence encoding the protein of the present invention (or its fragments, or its derivatives) can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells. In addition, mutations can also be introduced into the protein sequence of the invention through chemical synthesis.
  • the invention also relates to vectors comprising the appropriate DNA sequences as described above and appropriate promoter or control sequences. These vectors can be used to transform appropriate host cells to enable expression of the protein.
  • the host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
  • a prokaryotic cell such as a bacterial cell
  • a lower eukaryotic cell such as a yeast cell
  • a higher eukaryotic cell such as a mammalian cell.
  • Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; animal cells of CHO, COS7, 293 cells, etc.
  • Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art.
  • competent cells capable of taking up DNA can be harvested after the exponential growth phase and treated with the CaCl2 method, using steps well known in the art. Another method is to use MgCl 2 .
  • transformation can also be performed by electroporation.
  • DNA transfection methods can be used: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.
  • the obtained transformants can be cultured using conventional methods to express the polypeptide encoded by the gene of the present invention.
  • the medium used in culture can be selected from various conventional media. Cultivate under conditions suitable for host cell growth. After the host cells have grown to an appropriate cell density, the selected promoter is induced using an appropriate method (such as temperature shift or chemical induction), and the cells are cultured for a further period of time.
  • the recombinant polypeptide in the above method can be expressed within the cell, or on the cell membrane, or secreted outside the cell.
  • the recombinant protein can be isolated and purified by various separation methods utilizing its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitating agents (salting out method), centrifugation, osmotic sterilization, ultratreatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • the antibodies of the invention may be used alone, or may be combined or coupled to a detectable label (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modifying moiety, or a combination of any of these.
  • Detectable markers for diagnostic purposes include, but are not limited to: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or capable of producing a detectable product of enzymes.
  • Therapeutic agents that can be coupled include, but are not limited to: insulin, IL-2, interferons, calcitonin, GHRH peptides, intestinal peptide analogs, albumin, antibody fragments, cytokines, and hormones.
  • DTD DT-diaphorase
  • BPHL biphenyl hydrolase-like protein
  • the invention also provides a chimeric antigen receptor (CAR), which includes an extracellular domain, a transmembrane domain, and an intracellular domain.
  • CAR chimeric antigen receptor
  • the extracellular domain includes target-specific binding elements (also called antigen-binding domains).
  • the intracellular domain includes costimulatory signaling regions and zeta chain portions.
  • a costimulatory signaling domain refers to the portion of the intracellular domain that includes costimulatory molecules. Costimulatory molecules are cell surface molecules that are required for effective lymphocyte response to antigen, rather than antigen receptors or their ligands.
  • Linkers can be incorporated between the extracellular and transmembrane domains of the CAR, or between the cytoplasmic and transmembrane domains of the CAR.
  • the term "linker” generally refers to any oligopeptide or polypeptide that serves to connect a transmembrane domain to the extracellular or cytoplasmic domain of a polypeptide chain.
  • the linker may comprise 0-300 amino acids, preferably 2 to 100 amino acids and most preferably 3 to 50 amino acids.
  • the extracellular domain of the CAR provided by the present invention includes an antigen-binding domain targeting human GPRC5D.
  • the CAR of the present invention When expressed in T cells, the CAR of the present invention is capable of antigen recognition based on antigen-binding specificity. When it binds to its cognate antigen, it affects tumor cells, causing the tumor cells to fail to grow, be driven to death, or otherwise affected, and cause the patient's tumor burden to shrink or be eliminated.
  • the antigen binding domain is preferably fused to an intracellular domain from one or more of a costimulatory molecule and a zeta chain.
  • the antigen binding domain is fused to the intracellular domain in combination with the 4-1BB signaling domain and the CD3 ⁇ signaling domain.
  • antigen-binding domain and “single-chain antibody fragment” both refer to a Fab fragment, a Fab' fragment, an F(ab')2 fragment, or a single Fv fragment with antigen-binding activity.
  • Fv antibodies are the smallest antibody fragments that contain an antibody heavy chain variable region, a light chain variable region, but no constant region, and have all antigen-binding sites. Typically, Fv antibodies also contain a polypeptide linker between the VH and VL domains and are capable of forming the structure required for antigen binding.
  • the antigen-binding domain is usually scFv (single-chain variable fragment). The size of scFv is generally 1/6 of a complete antibody.
  • Single chain antibodies are preferably one amino acid chain sequence encoded by one nucleotide chain.
  • the antigen-binding domain includes an antibody that specifically recognizes human GPRC5D, preferably a single-chain antibody.
  • the CAR can be designed to include the transmembrane domain fused to the extracellular domain of the CAR.
  • a transmembrane domain naturally associated with one of the domains in the CAR is used.
  • transmembrane domains may be selected or modified by amino acid substitutions to avoid binding such domains to the transmembrane domains of the same or different surface membrane proteins, thus minimizing interaction with the receptor complex. Interactions with other members.
  • the CAR-T cells constructed using humanized anti-GPRC5D scFv can further improve their killing effect and tumor clearance ability.
  • the present invention also provides a pharmaceutical composition, which contains the above-mentioned antibody or active fragment thereof or fusion protein thereof or immune cells expressing the antibody, and a pharmaceutically acceptable carrier.
  • these materials may be formulated in a nontoxic, inert, and pharmaceutically acceptable aqueous carrier medium, usually at a pH of about 5-8, preferably at a pH of about 6-8, although the pH may vary. It will vary depending on the nature of the substance formulated and the condition to be treated.
  • the formulated pharmaceutical composition can be administered via conventional routes, including but not limited to: oral, respiratory, intratumoral, intraperitoneal, intravenous, or topical administration.
  • the pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80wt%) of the above-mentioned monoclonal antibody of the present invention (or its conjugate) and pharmaceutical acceptable carrier or excipient.
  • Such carriers include, but are not limited to: saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof.
  • the drug formulation should match the mode of administration.
  • the pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions should be manufactured under sterile conditions.
  • the active ingredient is administered in a therapeutically effective amount, for example, about 1 microgram/kg body weight to about 10 mg/kg body weight per day. Additionally, the antibodies of the invention may be used with other therapeutic agents.
  • a safe and effective amount of the immunoconjugate is administered to the mammal, wherein the safe and effective amount is generally at least about 10 micrograms per kilogram of body weight, and in most cases does not exceed about 8 mg per kilogram of body weight, Preferably the dosage is about 10 micrograms/kg body weight to about 1 mg/kg body weight.
  • the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the skill of a skilled physician.
  • the invention also provides the antibody or antigen-binding fragment thereof of the invention, the chimeric antigen receptor of the invention and the engineered immune cells expressing the chimeric antigen receptor, or the drug conjugate or drug combination of the invention.
  • an agent in the prevention and/or treatment of cancer eg, multiple myeloma
  • tumor cells expressing GPRC5D e.g, multiple myeloma
  • the present invention has the following advantages and beneficial effects:
  • the present invention uses B cell cloning technology to provide recombinant humanized monoclonal antibodies targeting human GPRC5D. What is important is that the specific recombinant humanized monoclonal antibodies targeting human GPRC5D have high affinity and good specificity, which can make up for the market. Diagnosis of recombinant humanized monoclonal antibodies targeting human GPRC5D protein Applications related to diagnosis and treatment.
  • the GPRC5D antibody sequence can be constructed into CAR-T or bispecific antibodies or antibody conjugates, which can be used for subsequent treatment of tumor diseases.
  • the present invention constructed a recombinant protein expressed in mammalian cells expressing the N-terminal extracellular region of human GPRC5D as an immunogen, and used New Zealand white rabbits for immunization. After the immunization was completed, Antigen-coated magnetic beads are used to screen B lymphocytes. After expanding and culturing single B lymphocytes, the expression of GPRC5D antibody is detected by flow cytometry. The positive clones are genetically sequenced to obtain the gene sequence of the rabbit monoclonal antibody. Then, the rabbit monoclonal antibody gene sequence is obtained.
  • the humanization transformation project resulted in 10 humanized GPRC5D monoclonal antibodies, named G01, G02, G03, G04, G05, G06, G07, G08, G09 and G10 respectively.
  • G00 ⁇ G09 were constructed into humanized IgG1 antibody vector plasmids, and the recombinant vector plasmids were transfected into 293 cells. The supernatant was harvested to purify and concentrate the antibodies (the structure is shown in the left picture of Figure 1), and was used for flow cytometric detection experiments after quantification.
  • G02, G05, G07, and G09 have good binding activity to K562-GPRC5D, which is better than the control antibody G00.
  • Example 2 Affinity flow cytometric detection of humanized antibodies with scFv-hFc structure
  • G00 ⁇ G09 were constructed into scFv-hFc humanized IgG1 vector plasmids.
  • the recombinant vector plasmids were transfected into 293 cells, and the supernatant was harvested to purify and concentrate the antibodies (the structure is shown in the right picture of Figure 1). After quantification, it was used Flow cytometry experiments.
  • G02, G05, G07, and G09 have good affinity to K562-GPRC5D (Figure 3a) and 8226 cells (Figure 3b), which is better than the control antibody G00.
  • Example 3 Flow cytometric detection of binding activity of humanized antibodies with scFv-hFc structure to MM.1S and H929 cells
  • MM.1S and NCI-H929 are both natural multiple myeloma cells and express GPRC5D.
  • G02, G05, G07, and G09 have good binding activities to MM.1S cells (Figure 4a) and NCI-H929 cells (Figure 4b), which are significantly better than the control antibody G00.
  • the scFvs of G00 to G09 were constructed into CAR lentiviral vectors, and the vector was used for lentiviral packaging.
  • the lentiviral supernatant was obtained for concentration and purification, and then infected into jurkat-nfat-luc cells to obtain Jurkat-nfat-luc cells after infection, namely CAR-jurkat-nfat-luc cells.
  • Each CAR-jurkat-nfat-luc cell was co-cultured with K562, K562-GPRC5D, and RPMI 8226 cells for 48 hours and then the bioluminescence value was detected.
  • Figure 6a shows the results of co-culture of CAR-jurkat-nfat-luc cells with K562-GPRC5D or K562 cells
  • Figure 6b shows the results of co-culture of CAR-jurkat-nfat-luc cells with RPMI 8226 or K562. Results; It can be seen that CAR-jurkat-nfat-luc cells have a lower self-activation background and a higher antigen-dependent activation signal, especially CAR-jurkat-nfat-luc cells expressing G02, G05, G07 and G09 scFv.
  • the CD3-positive cell population was obtained by sorting the CD3 nanomagnetic beads and Miltenyi magnetic bead sorting system.
  • the obtained CD3-positive cells were counted and activated using CD3/CD28 magnetic beads. They were cultured for 24 hours and then infected with G00 ⁇ G09 CAR lentivirus respectively. The cells were washed overnight, and the expression efficiency of CAR was detected by flow cytometry 48 hours later.
  • G00 ⁇ G09 CAR-T cells all have high transfection efficiency.
  • the cultured G00 ⁇ G09 CAR-T cells and T cells prepared in Example 5 were washed with PBS respectively and then resuspended in culture medium for sampling and counting.
  • the CAR-T cell concentration and T cell concentration were adjusted to 1*10E5 according to the cell concentration. cells/ml, add CAR-T cells and T cells to the labeled 96-well plate respectively, with 100ul of cell suspension in each well.
  • the cultured G00 ⁇ G09 CAR-T cells and T cells prepared in Example 5 were washed with PBS respectively and then resuspended in culture medium for sampling and counting. According to the cell concentration, the CAR-T concentration and T cell concentration were adjusted to 1*10E5 cells. /ml, add CAR-T cells and T cells to the labeled 96-well plate respectively, with 100ul of cell suspension in each well.
  • the 24-well plate label sets 4 reaction condition groups:
  • A CAR-T/T+NCI-H929, CAR-T cells or T cells co-cultured with multiple myeloma cells NCI-H929
  • C CAR-T/T only, CAR-T cells or T cells are cultured alone
  • CAR-T/T+IL-2 CAR-T cells or T cells are cultured with 300U/ml IL-2 added.
  • the cultured G00 ⁇ G09 CAR-T cells and T cells prepared in Example 5 were washed with PBS respectively and then resuspended in the culture medium for sampling and counting.
  • the CAR-T cell concentration and the T cell concentration were adjusted to 5 to 10 according to the cell concentration.
  • CAR-T cells select CD3-positive cells, select CAR-positive cells, and superimpose the FITC peaks; T cells select CD3-positive cells and superimpose the FITC peaks.
  • test product GPRC5D-CAR-T was intravenously administered to NOD-Prkdcscid Il2rgtm1/Bcgen mice (B-NDG mice) transplanted with human myeloma cells MM1.S to evaluate its inhibitory effect on tumor cell proliferation in vivo. .
  • mice were intravenously inoculated with 1E6/mouse MM1.S-Luc (luciferase-labeled human MM1.S cells) and administered on the fifth day after inoculation.
  • 1E6/mouse MM1.S-Luc luciferase-labeled human MM1.S cells
  • mice in the blank control group were given vehicle solution; mice in the MOCK T group were given negative control MOCK-T cells, and mice in the G05 1E6 group were given 1E6/mouse of the test product G05 CAR-T cells. ; The mice in the G05 5E6 group were given 5E6/mouse of the test product G05 CAR-T cells; the mice in the G07 1E6 group were given 1E6/mouse of the test product G07 CAR-T cells; the mice in the G07 5E6 group were given 5E6/mouse of the test product G07 CAR-T cells.
  • Test sample G07 CAR-T cells mice in the G09 1E6 group were given 1E6/mouse of the test product G09 CAR-T cells; mice in the G09 5E6 group were given 5E6/mouse of the test product G09 CAR-T cells; take the first dose The day was D0, and all animals were given a single injection into the tail vein.
  • Detection indicators Chemiluminescence was captured on all animals on D-5 (5 days before administration), D-1 (1 day before administration), D3, D6, D10, D13, D18 and D24 using a Bruker small animal imager. Signal.
  • the antibody sequence comes from the patent of Shanghai Lixin Pharmaceutical R&D Co., Ltd. (CN 115038720 A); the irrelevant control antibody BM138 is the antibody zolbetuximab targeting CLDN18.2.
  • BCA After purifying the produced antibody, use BCA to detect the concentration and then dilute the antibody: use culture medium to prepare antibody dilutions (40ug/ml, 4ug/ml, 0.4ug/ml) 25ul per well, and set 3 duplicate holes for each dilution concentration. Use culture medium to set wells without antibody as control wells.
  • Labeling reagent Zenon pHrodo iFL green IgG dilution Use culture medium to prepare labeling reagent dilutions (40ug/ml, 4ug/ml, 0.4ug/ml), and add each concentration of dilution to the antibody dilution wells of equal concentration.
  • the CD3 antibody used in this example comes from the humanized antibody of mouse hybridoma SP34.
  • the sequence of the antibody can be found in CD3B219 VH and CD3B219 VL in patent CN109715667A.
  • the sequence information was codon optimized and gene synthesized into CD3 VH and CD3VL.
  • CD3VH was cloned into the expression vector pTT5-hlgG1FC-Knob
  • CD3VL was cloned into the expression vector pTT5-hKappa.
  • the ScFv of GPRC5D antibody G05, G07, G09, and G00 were cloned into the expression vector pTT5-hlgG1FC-Hole.
  • pTT5-CD3VH-Knob and pTT5-CD3VL were co-transfected with four plasmids: pTT5-G05-Hole, pTT5-G07-Hole, pTT5-G09-Hole and pTT5-G00-Hole respectively into 293F cells for antibody expression and collected separately.
  • the cell supernatants expressing bispecific antibodies of G05 BsAb, G07 BsAb, G09 BsAb and G00 BsAb were subjected to ProteinA affinity chromatography, and the protein concentration was determined after PBS dialysis to remove impurities.
  • NCI-H929 and Jurkat cells were recovered and cultured for 48 hours, cells were counted and cells were plated at 2 ⁇ 10 ⁇ 5 in a 96-well V-shaped plate.
  • the bispecific antibody was serially diluted in PBS, and Talquetamab was used as a positive control.
  • the starting concentration is 100 ⁇ g/ml, and then diluted 1:5, 1:2, 1:2, 1:2, 1:2, 1:2, and 1:2, with a total of 8 tests. concentration.
  • BsAb and cells were incubated for 1 hour at 4°C. Cells were then washed three times with PBS and 0.2% FBS.
  • APC-conjugated anti-human IgGmAb was added and the cells were incubated with the secondary antibody for 1 hour at 4°C. Cells were then washed three times with PBS and 0.2% FBS. Cells were washed again with PBS and 0.2% FBS and subsequently analyzed on a flow cytometer.
  • NCI-H929-GFP-LUC, MM.1S-GFP-LUC, RPMI-8826-GFP-LUC were used to analyze the T-cell-mediated cytotoxicity of bispecific antibodies.
  • Target cells NCI-H929-GFP-LUC, MM.1S-GFP-LUC, RPMI826-GFP-LUC
  • Cells were washed twice in X-Vivo killing medium containing 5% FBS. Cells were diluted to 2 ⁇ 10 ⁇ 5/mL with X-Vivo killing culture and incubated at 37°C until use.
  • T cell complete culture medium (X-Vivo+5% FBS+200IU/ml IL- 2+1% PS) to resuspend the cells so that the T cell concentration is between 1.5 and 2.5 ⁇ 10 ⁇ 6/ml.
  • T cell complete culture medium X-Vivo+5% FBS+200IU/ml IL- 2+1% PS
  • 2 ⁇ 10 ⁇ 4 target cells were added to a 96-well cell culture and 6 ⁇ 10 ⁇ 4 T cells were added to the wells (3:1 effector:target ratio).
  • After mixing target cells and T cells add 20 ⁇ l of GPRC5D ⁇ CD3 bispecific antibody dilution to each well.
  • the GPRC5D&CD3 bispecific antibody was diluted with PBS to a total concentration of 20 ⁇ g/m in the target cell and T cell mixing wells.
  • Titers were prepared in 96-well plates with 5-fold serial dilutions in PBS. The last column is reserved for PBS alone (vehicle control). Add to the target cells and T cell mixing wells in sequence. Plates were incubated at 37°C and 5% CO2 for 48 hours.

Abstract

本发明涉及靶向人GPRC5D的特异性重组人源化单克隆抗体及其应用，可用于ELISA和流式检测方法中，可用于靶向GPRC5D蛋白的诊断与治疗。该单克隆抗体包括重链可变区和轻链可变区，且该单克隆抗体的氨基酸序列和核苷酸序列可以单独使用或双链组合使用。此外，靶向人GPRC5D的特异性重组人源化单克隆抗体的序列可以构建成单抗，抗体偶联药，双特异性抗体，CAR-免疫细胞等生物材料或制剂，可用于GPRC5D相关疾病的诊断和治疗。

Description

靶向人GPRC5D的重组人源化单克隆抗体及其应用 技术领域
本发明属于生物技术领域，具体地涉及靶向人GPRC5D的重组人源化单克隆抗体及其应用。
背景技术
GPRC5D属于G蛋白偶联受体(GPCRs)的一种孤儿受体，是G蛋白偶联受体C5家族亚型D，为7次跨膜蛋白。孤儿受体(Orphan Receptor)是指与其它已确认的受体结构明显相似，但其内源配体还未发现的受体。GPRC5D在原代多发性骨髓瘤细胞表面高表达，而在正常组织的表达仅限于毛囊区域，有研究表明65％的多发性骨髓瘤患者GPRC5D有超过50％的表达阈值，凭借这一特点，GPRC5D成为了治疗MM的潜在靶标。
由于GPRC5D高表达于浆细胞和多发性骨髓瘤细胞表面，其他正常组织细胞和造血干细胞中均不表达，因此可以作为多发性骨髓瘤(MM)的诊断和治疗用的理想靶点。
发明内容
本发明目的在于提供靶向人GPRC5D的特异性重组人源化单克隆抗体，本发明所提供的靶向人GPRC5D的特异性重组人源化单克隆抗体亲和力高，具有良好的特异性，可以弥补市场上靶向人GPRC5D蛋白的重组人源化单克隆抗体的诊断与治疗相关的应用。另外，该GPRC5D抗体序列可以构建成嵌合抗原受体T细胞免疫疗法(CART)或双特异性抗体或抗体偶联药，可用于后续的肿瘤疾病的治疗。
本发明的第一方面，提供了一种抗体或其抗原结合片段，其包含重链可变区，所述重链可变区包含以下CDR：
SEQ ID NO:44,78,1,33,67,22,或87所示的VH-CDR1，
SEQ ID NO:45,59,79,12,34,68,2,23,54或88所示的VH-CDR2，和
SEQ ID NO:46,60,13,35,69,3,24,55或89所示的VH-CDR3；
其中所述抗体或其抗原结合片段特异性地结合GPRC5D，优选人GPRC5D。
在另一优选例中，所述抗体或其抗原结合片段的重链可变区包含以下3个 CDR：
SEQ ID NO:44所示的VH-CDR1，
SEQ ID NO:45所示的VH-CDR2，和
SEQ ID NO:46所示的VH-CDR3。
在另一优选例中，所述抗体或其抗原结合片段的重链可变区包含以下3个CDR：
SEQ ID NO:44所示的VH-CDR1，
SEQ ID NO:59所示的VH-CDR2，和
SEQ ID NO:60所示的VH-CDR3。
在另一优选例中，所述抗体或其抗原结合片段的重链可变区包含以下3个CDR：
SEQ ID NO:78所示的VH-CDR1，
SEQ ID NO:79所示的VH-CDR2，和
SEQ ID NO:60所示的VH-CDR3。
在另一优选例中，所述抗体或其抗原结合片段的重链可变区包含以下3个CDR：
SEQ ID NO:1所示的VH-CDR1，
SEQ ID NO:12所示的VH-CDR2，和
SEQ ID NO:13所示的VH-CDR3。
在另一优选例中，所述抗体或其抗原结合片段还包含轻链可变区，所述轻链可变区包含以下CDR：
SEQ ID NO:47,61,80,14,36,70,4,25,47或90所示的VL-CDR1，
SEQ ID NO:37,15,71,5或26所示的VL-CDR2，和
SEQ ID NO:48,81,16,38,72,6,27,48或91所示的VL-CDR3。
在另一优选例中，所述抗体或其抗原结合片段的轻链可变区包含以下3个CDR：
SEQ ID NO:47所示的VL-CDR1，
SEQ ID NO:37所示的VL-CDR2，和
SEQ ID NO:48所示的VL-CDR3。
在另一优选例中，所述抗体或其抗原结合片段的轻链可变区包含以下3个CDR：
SEQ ID NO:61所示的VL-CDR1，
SEQ ID NO:37所示的VL-CDR2，和
SEQ ID NO:48所示的VL-CDR3。
在另一优选例中，所述抗体或其抗原结合片段的轻链可变区包含以下3个CDR：
SEQ ID NO:80所示的VL-CDR1，
SEQ ID NO:37所示的VL-CDR2，和
SEQ ID NO:81所示的VL-CDR3。
在另一优选例中，所述抗体或其抗原结合片段的轻链可变区包含以下3个CDR：
SEQ ID NO:14所示的VL-CDR1，
SEQ ID NO:15所示的VL-CDR2，和
SEQ ID NO:16所示的VL-CDR3。
在另一优选例中，所述抗体或其抗原结合片段包含选自下组的重链可变区CDR和轻链可变区CDR：
在另一优选例中，所述抗体或其抗原结合片段包含与SEQ ID NO:49、62、82、17、39、73、7、28、56或92具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列的重链可变区。
在另一优选例中，所述抗体或其抗原结合片段包含与SEQ ID NO:51、64、84、19、41、75、9、30或94具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列的轻链可变区。
在另一优选例中，所述抗体或其抗原结合片段包含选自下组的重链可变区和轻链可变区：
在另一优选例中，所述抗体或其抗原结合片段包含如SEQ ID NO:49所示的氨基酸序列的重链可变区，和如SEQ ID NO:51所示的氨基酸序列的轻链可变区。
在另一优选例中，所述抗体或其抗原结合片段包含如SEQ ID NO:62所示的氨基酸序列的重链可变区，和如SEQ ID NO:64所示的氨基酸序列的轻链可变区。
在另一优选例中，所述抗体或其抗原结合片段包含如SEQ ID NO:82所示的氨基酸序列的重链可变区，和如SEQ ID NO:84所示的氨基酸序列的轻链可变区。
在另一优选例中，所述抗体或其抗原结合片段包含如SEQ ID NO:17所示的氨基酸序列的重链可变区，和如SEQ ID NO:19所示的氨基酸序列的轻链可变区。
在另一优选例中，所述抗体包括单链抗体(scFv)、双链抗体、单克隆抗体、嵌合抗体(如人鼠嵌合抗体)、动物源抗体、全人抗体或人源化抗体。
在另一优选例中，所述抗体为人源化抗体。
在另一优选例中，所述抗体为单克隆抗体。
在另一优选例中，所述抗体的轻链恒定区为κ链，重链恒定区为IgG型。
在另一优选例中，所述抗体为双特异性抗体或多特异性抗体。
本发明的第二方面，提供了一种重组蛋白，所述重组蛋白包含：
(i)如本发明第一方面所述的抗体或其抗原结合片段；和
(ii)任选的协助表达和/或纯化的标签序列。
在另一优选例中，所述的标签序列选自下组：6×His标签、GGGS序列、FLAG标签。
本发明的第三方面，提供了一种多核苷酸，所述多核苷酸编码选自下组的多肽：
(1)如本发明第一方面所述的抗体或其抗原结合片段；或
(2)如本发明第二方面所述的重组蛋白。
本发明的第四方面，提供了一种载体，所述载体含有本发明第三方面所述的多核苷酸。
在另一优选例中，所述的载体包括：细菌质粒、噬菌体、酵母质粒、植物细胞病毒、哺乳动物细胞病毒如腺病毒、腺相关病毒、逆转录病毒、慢病毒载体或其他载体。
本发明的第五方面，提供了一种遗传工程化的宿主细胞，所述宿主细胞含有如本发明第四方面所述的载体，或基因组中整合有本发明第三方面所述的多核苷酸。
本发明的第六方面，提供了一种免疫偶联物，所述免疫偶联物包含：
(a)如本发明第一方面所述的抗体或其抗原结合片段、或如本发明第二方面所述的重组蛋白；和
(b)选自下组的偶联部分：可检测标记物、药物、毒素、细胞因子、放射性核素、或酶。
在另一优选例中，所述偶联物选自：荧光或发光标记物、放射性标记物、MRI(磁共振成像)或CT(电子计算机X射线断层扫描技术)造影剂、或能够产生可检测产物的酶、放射性核素、生物毒素、细胞因子(如IL-2等)、抗体、抗体Fc片段、抗体scFv片段、金纳米颗粒/纳米棒、病毒颗粒、脂质体、纳米磁粒、前药激活酶(例如，DT-心肌黄酶(DTD)或联苯基水解酶-样蛋白质(BPHL))、化疗剂(例如，顺铂)或任何形式的纳米颗粒等。
本发明的第七方面，提供了一种嵌合抗原受体，所述嵌合抗原受体的抗原结合结构域包含如本发明第一方面所述的抗体或其抗原结合片段。
本发明的第八方面，提供了一种免疫细胞，所述免疫细胞表达或在细胞膜外暴露有如本发明第一方面所述的抗体或其抗原结合片段。
在另一优选例中，所述免疫细胞包括T细胞，NK细胞或巨噬细胞。
在另一优选例中，所述免疫细胞为CAR-T细胞。
本发明的第九方面，提供了一种药物组合物，所述药物组合物含有：
(z1)活性成分，所述活性成分选自下组：如本发明第一方面所述的抗体或其抗原结合片段、如本发明第二方面所述的重组蛋白、如本发明第六方面所述的免疫偶联物或如本发明第八方面所述的免疫细胞、或其组合；以及
(z2)药学上可接受的载体。
在另一优选例中，所述的药物组合物为液态制剂。
在另一优选例中，所述的药物组合物为注射剂。
在另一优选例中，所述药物组合物用于制备治疗与GPRC5D高表达相关的疾病的药物。
在另一优选例中，所述与GPRC5D高表达相关的疾病为肿瘤，例如多发性骨髓瘤。
本发明的第十方面，提供了一种活性成分的用途，所述活性成分选自下组：如本发明第一方面所述的抗体或其抗原结合片段、如本发明第二方面所述的重组蛋白、如本发明第六方面所述的免疫偶联物或如本发明第八方面所述的免疫细胞、或其组合，用于制备诊断试剂或试剂盒，或用于制备治疗与GPRC5D高表达相关的疾病的药物。
在另一优选例中，所述与GPRC5D高表达相关的疾病为肿瘤，例如多发性骨髓瘤。
本发明的第十一方面，提供了一种体外非诊断性检测样品中GPRC5D蛋白的方法，包括步骤：
(s1)在体外，将所述样品与如本发明的第一方面所述的抗体接触；
(s2)检测是否形成抗原-抗体复合物，其中形成复合物就表示样品中存在 GPRC5D蛋白。
本发明的第十二方面，提供了一种制备如本发明第一方面所述的抗体或其抗原结合片段，或如本发明第二方面所述的重组蛋白的方法，包括步骤：(si)在适合表达蛋白的条件下，培养如本发明第五方面所述的宿主细胞；(sii)从细胞培养物中分离出如本发明第一方面所述的抗体或其抗原结合片段，或如本发明第二方面所述的重组蛋白。
本发明的第十三方面，提供了一种治疗与GPRC5D高表达相关的疾病的方法，所述方法包括向有需要的受试者施用如本发明第一方面所述的抗体或其抗原结合片段、如本发明第二方面所述的重组蛋白、如本发明第六方面所述的免疫偶联物、如本发明第七方面所述的嵌合抗原受体、如本发明第八方面所述的免疫细胞或如本发明第九方面所述的药物组合物。
在另一优选例中，所述与GPRC5D高表达相关的疾病为肿瘤，例如多发性骨髓瘤。
在另一优选例中，所述有需要的受试者为人类或非人类哺乳动物。
本发明的第十四方面，提供了一种双特异性抗体，其包含如本发明第一方面所述的抗体或其抗原结合片段。
在另一优选例中，所述双特异性抗体还包含靶向第二抗原的抗体或其抗原结合片段，所述第二抗原选自：CD3、CD16、CD32、41BB。
在另一优选例中，所述双特异性抗体包含靶向CD3的抗体或其抗原结合片段。
在另一优选例中，所述靶向CD3的抗体或其抗原结合片段包含如SEQ ID NO:97所示的重链可变区，和如SEQ ID NO:98所示的轻链可变区。
在另一优选例中，所述双特异性抗体包含以下三条肽链：
链1：GPRC5D scFv-Fc Hole
链2：CD3 VH-CH1-Fc Knob
链3：CD3 VL-CL；或
链1：GPRC5D scFv-Fc Knob
链2：CD3 VH-CH1-Fc Hole
链3：CD3 VL-CL；
其中，所述GPRC5D scFv为靶向GPRC5D的scFv，CD3 VH为靶向CD3的抗体的重链可变区，CD3 VL为靶向CD3的抗体的轻链可变区，CH1为IgG抗体的重链恒定区1，CL为抗体的轻链恒定区。
在另一优选例中，所述Fc Knob的氨基酸序列如SEQ ID NO:106所示，所述Fc Knob的氨基酸序列如SEQ ID NO:107所示。
在另一优选例中，所述CL为人Kappa轻链的恒定区。
在另一优选例中，所述GPRC5D scFv选自下组：SEQ ID NO:53、66或86。
在另一优选例中，所述双特异性抗体包含以下三条肽链：如SEQ ID NO:102-104中任一项所示的链1，如SEQ ID NO:101所示的链2，包含如SEQ ID NO:98所示的氨基酸序列的链3。
本发明还提供了编码如本发明第十四方面所述的双特异性抗体的多核苷酸。包含所述多核苷酸的载体，以及含有所述载体的宿主细胞。
本发明还提供了包含如本发明第十四方面所述的双特异性抗体的免疫偶联物和药物组合物。
本发明还提供了本发明第十四方面所述的双特异性抗体的在制备治疗与GPRC5D高表达相关的疾病的药物中的用途，以及使用所述双特异性抗体治疗与GPRC5D高表达相关的疾病的方法。
应理解，在本发明范围中，本发明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显示了人源化抗体的两种结构：天然结构和scFv-hFc结构。
图2显示了天然结构人源化抗体与K562-GPRC5D细胞的亲和性。
图3显示了scFv-hFc结构人源化抗体与K562-GPRC5D(图3a)和RPMI 8226细胞(图3b)的亲和性。
图4显示了scFv-hFc结构人源化抗体与MM.1S(图4a)和NCI-H929(图4b)的结合活性。
图5显示了GPRC5D CAR的载体插入序列结构。
图6显示了GPRC5D CAR jurkat nfat luc细胞与K562-GPRC5D(图6a)或 RPMI 8226细胞(图6b)共培养后的自激活和抗原依赖激活。
图7显示了GPRC5D CAR-T细胞的转染效率流式检测结果。
图8显示了GPRC5D CAR-T细胞杀伤RPMI 8226细胞产生的炎症因子浓度检测结果；其中图8a为IL-2浓度检测结果，图8b为TNF-α浓度检测结果，图8c为IFN-γ浓度检测结果。
图9显示了GPRC5D CAR-T细胞与MM.1S或K562细胞共培养，以及CAR-T细胞单独培养产生的炎症因子浓度检测比较结果；其中图9a为IL-2浓度检测结果，图9b为TNF-α浓度检测结果，图9c为IFN-γ浓度检测结果。
图10显示了GPRC5D CAR-T细胞与NCI-H929或K562细胞共培养，以及CAR-T细胞单独培养产生细胞增殖流式检测结果。
图11显示了不同效靶比的GPRC5D CAR-T细胞分别与K562-luc(图11a)、K562-GPRC5D-luc(图11b)、NCI-H929-luc(图11c)、MM.1S-luc(图11d)、RPMI 8226-luc(图11e)靶细胞共培养24h后的杀伤效果。
图12显示了B-NDG荷瘤小鼠经不同给药组处理后的化学发光检测结果。
图13显示了G05、G07、G09抗体介导的K562-GPRC5D内吞效应。
图14显示了GPRC5D&CD3双特异性抗体的体外结合活性；其中图14a显示了GPRC5D&CD3双特异性抗体与H929细胞的结合活性；图14b显示了GPRC5D&CD3双特异性抗体与Jurkat细胞的结合活性。
图15显示了GPRC5D&CD3双特异性抗体的体外杀伤效率；其中图15a显示了GPRC5D&CD3双特异性抗体对8226细胞株48h杀伤效率；图15b显示了GPRC5D&CD3双特异性抗体对H929细胞株48h杀伤效率；图15c显示了GPRC5D&CD3双特异性抗体对MM.1S细胞株48h杀伤效率；图15d显示了GPRC5D&CD3双特异性抗体对K562细胞株48h杀伤效率。
具体实施方式
本发明人经过广泛而深入的研究，经过大量的实验筛选和人源化改造，首次意外地获得了一组具有全新氨基酸序列的特异性结合人GPRC5D的人源化单克隆抗体。实验结果表明，本发明的人源化抗体与细胞表面的GPRC5D蛋白具有较强的亲和力，且具有很好的刺激T细胞激活的活性；基于本发明的人源化抗体序列制备的CAR-T细胞表现出优异的靶细胞特异性杀伤活性；各方面性能均显著优于现有的抗GPRC5D抗体。在此基础上完成了本发明。
如本文所用，术语“抗体”或“免疫球蛋白”是有相同结构特征的约150000道尔顿的异四聚糖蛋白，其由两个相同的轻链(L)和两个相同的重链(H)组成。每条轻链通过一个共价二硫键与重链相连，而不同免疫球蛋白同种型的重链间的二硫键数目不同。每条重链和轻链也有规则间隔的链内二硫键。每条重链的一端有可变区(VH)，其后是多个恒定区。每条轻链的一端有可变区(VL)，另一端有恒定区；轻链的恒定区与重链的第一个恒定区相对，轻链的可变区与重链的可变区相对。特殊的氨基酸残基在轻链和重链的可变区之间形成界面。
如本文所用，术语“可变”表示抗体中可变区的某些部分在序列上有所不同，它形成了各种特定抗体对其特定抗原的结合和特异性。然而，可变性并不均匀地分布在整个抗体可变区中。它集中于轻链和重链可变区中称为互补决定区(CDR)或超变区中的三个片段中。可变区中较保守的部分称为构架区(FR)。天然重链和轻链的可变区中各自包含四个FR区，它们大致上呈β-折叠构型，由形成连接环的三个CDR相连，在某些情况下可形成部分β折叠结构。每条链中的CDR通过FR区紧密地靠在一起并与另一链的CDR一起形成了抗体的抗原结合部位(参见Kabat等，NIH Publ.No.91-3242，卷I，647-669页(1991))。恒定区不直接参与抗体与抗原的结合，但是它们表现出不同的效应功能，例如参与抗体的依赖于抗体的细胞毒性。
脊椎动物抗体(免疫球蛋白)的“轻链”可根据其恒定区的氨基酸序列归为明显不同的两类(称为κ和λ)中的一类。根据其重链恒定区的氨基酸序列，免疫球蛋白可以分为不同的种类。主要有5类免疫球蛋白：IgA,IgD,IgE,IgG和IgM，其中一些还可进一步分成亚类(同种型)，如IgG1,IgG2,IgG3,IgG4,IgA和IgA2。对应于不同类免疫球蛋白的重链恒定区分别称为α、δ、ε、γ、和μ。不同类免疫球蛋白的亚单位结构和三维构型是本领域人员所熟知的。
如本文所用，术语“单克隆抗体(单抗)”指从一类基本均一的群体获得的抗体，即该群体中包含的单个抗体是相同的，除少数可能存在的天然发生的突变外。单克隆抗体高特异性地针对单个抗原位点。而且，与常规多克隆抗体制剂(通常是具有针对不同决定簇的不同抗体)不同，各单克隆抗体是针对抗原上的单个决定簇。除了它们的特异性外，单克隆抗体的好处还在于不会被其它免疫球蛋白污染。修饰语“单克隆”表示了抗体的特性，是从均一的抗体群中获得的，这不应被解释成需要用任何特殊方法来生产抗体。
本发明提供的靶向人GPRC5D的重组人源化单克隆抗体，选自以下重组人源化单克隆抗体中的一种或多种：
命名为G01的单克隆抗体：其重链互补决定区CDR1、CDR2、CDR3的氨基酸序列分别为SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3所示的氨基酸序列；且轻链互补决定区CDR1、CDR2、CDR3的氨基酸序列分别为SEQ ID NO:4，SEQ ID NO:5、SEQ ID NO:6所示的氨基酸序列；其人源化重链可变区氨基酸序列为SEQ ID NO:7；其人源化重链可变区核酸序列为SEQ ID NO:8；其人源化轻链可变区氨基酸序列为SEQ ID NO:9；其人源化轻链可变区核酸序列为SEQ ID NO:10；其人源化scFv氨基酸序列为SEQ ID NO:11；
命名为G02的单克隆抗体：其重链互补决定区CDR1、CDR2、CDR3的氨基酸序列分别为SEQ ID NO:1、SEQ ID NO:12、SEQ ID NO:13所示的氨基酸序列；且轻链互补决定区CDR1、CDR2、CDR3的氨基酸序列分别为SEQ ID NO:14，SEQ ID NO:15、SEQ ID NO:16所示的氨基酸序列；其人源化重链可变区氨基酸序列为SEQ ID NO:17；其人源化重链可变区核酸序列为SEQ ID NO:18；其人源化轻链可变区氨基酸序列为SEQ ID NO:19；其人源化轻链可变区核酸序列为SEQ ID NO:20；其人源化scFv氨基酸序列为SEQ ID NO:21；
命名为G03的单克隆抗体：其重链互补决定区CDR1、CDR2、CDR3的氨基酸序列分别为SEQ ID NO:22、SEQ ID NO:23、SEQ ID NO:24所示的氨基酸序列；且轻链互补决定区CDR1、CDR2、CDR3的氨基酸序列分别为SEQ ID NO:25，SEQ ID NO:26、SEQ ID NO:27所示的氨基酸序列；其人源化重链可变区氨基酸序列为SEQ ID NO:28；其人源化重链可变区核酸序列为SEQ ID NO:29；其人源化轻链可变区氨基酸序列为SEQ ID NO:30；其人源化轻链可变区核酸序列为SEQ ID NO:31；其人源化scFv氨基酸序列为SEQ ID NO:32；
命名为G04的单克隆抗体：其重链互补决定区CDR1、CDR2、CDR3的氨基酸序列分别为SEQ ID NO:33、SEQ ID NO:34、SEQ ID NO:35所示的氨基酸序列；且轻链互补决定区CDR1、CDR2、CDR3的氨基酸序列分别为SEQ ID NO:36，SEQ ID NO:37、SEQ ID NO:38所示的氨基酸序列；其人源化重链可变区氨基酸序列为SEQ ID NO:39；其人源化重链可变区核酸序列为SEQ ID NO:40；其人源化轻链可变区氨基酸序列为SEQ ID NO:41；其人源化轻链可变区核酸序列为SEQ ID NO:42；其人源化scFv氨基酸序列为SEQ ID NO:43；
命名为G05的单克隆抗体：其重链互补决定区CDR1、CDR2、CDR3的氨基酸序列分别为SEQ ID NO:44、SEQ ID NO:45、SEQ ID NO:46所示的氨基酸序列；且轻链互补决定区CDR1、CDR2、CDR3的氨基酸序列分别为SEQ ID NO:47，SEQ ID NO:37、SEQ ID NO:48所示的氨基酸序列；其人源化重链可变区氨基酸 序列为SEQ ID NO:49；其人源化重链可变区核酸序列为SEQ ID NO:50；其人源化轻链可变区氨基酸序列为SEQ ID NO:51；其人源化轻链可变区核酸序列为SEQ ID NO:52；其人源化scFv氨基酸序列为SEQ ID NO:53；
命名为G06的单克隆抗体：其重链互补决定区CDR1、CDR2、CDR3的氨基酸序列分别为SEQ ID NO:44、SEQ ID NO:54、SEQ ID NO:55所示的氨基酸序列；且轻链互补决定区CDR1、CDR2、CDR3的氨基酸序列分别为SEQ ID NO:47，SEQ ID NO:37、SEQ ID NO:48所示的氨基酸序列；其人源化重链可变区氨基酸序列为SEQ ID NO:56；其人源化重链可变区核酸序列为SEQ ID NO:57；其人源化轻链可变区氨基酸序列为SEQ ID NO:51；其人源化轻链可变区核酸序列为SEQ ID NO:52；其人源化scFv氨基酸序列为SEQ ID NO:58；
命名为G07的单克隆抗体：其重链互补决定区CDR1、CDR2、CDR3的氨基酸序列分别为SEQ ID NO:44、SEQ ID NO:59、SEQ ID NO:60所示的氨基酸序列；且轻链互补决定区CDR1、CDR2、CDR3的氨基酸序列分别为SEQ ID NO:61，SEQ ID NO:37、SEQ ID NO:48所示的氨基酸序列；其人源化重链可变区氨基酸序列为SEQ ID NO:62；其人源化重链可变区核酸序列为SEQ ID NO:63；其人源化轻链可变区氨基酸序列为SEQ ID NO:64；其人源化轻链可变区核酸序列为SEQ ID NO:65；其人源化scFv氨基酸序列为SEQ ID NO:66；
命名为G08的单克隆抗体：其重链互补决定区CDR1、CDR2、CDR3的氨基酸序列分别为SEQ ID NO:67、SEQ ID NO:68、SEQ ID NO:69所示的氨基酸序列；且轻链互补决定区CDR1、CDR2、CDR3的氨基酸序列分别为SEQ ID NO:70，SEQ ID NO:71、SEQ ID NO:72所示的氨基酸序列；其人源化重链可变区氨基酸序列为SEQ ID NO:73；其人源化重链可变区核酸序列为SEQ ID NO:74；其人源化轻链可变区氨基酸序列为SEQ ID NO:75；其人源化轻链可变区核酸序列为SEQ ID NO:76；其人源化scFv氨基酸序列为SEQ ID NO:77；
命名为G09的单克隆抗体：其重链互补决定区CDR1、CDR2、CDR3的氨基酸序列分别为SEQ ID NO:78、SEQ ID NO:79、SEQ ID NO:60所示的氨基酸序列；且轻链互补决定区CDR1、CDR2、CDR3的氨基酸序列分别为SEQ ID NO:80，SEQ ID NO:37、SEQ ID NO:81所示的氨基酸序列；其人源化重链可变区氨基酸序列为SEQ ID NO:82；其人源化重链可变区核酸序列为SEQ ID NO:83；其人源化轻链可变区氨基酸序列为SEQ ID NO:84；其人源化轻链可变区核酸序列为SEQ ID NO:85；其人源化scFv氨基酸序列为SEQ ID NO:86；
命名为G10的单克隆抗体：其重链互补决定区CDR1、CDR2、CDR3的氨基 酸序列分别为SEQ ID NO:87、SEQ ID NO:88、SEQ ID NO:89所示的氨基酸序列；且轻链互补决定区CDR1、CDR2、CDR3的氨基酸序列分别为SEQ ID NO:90，SEQ ID NO:26、SEQ ID NO:91所示的氨基酸序列；其人源化重链可变区氨基酸序列为SEQ ID NO:92；其人源化重链可变区核酸序列为SEQ ID NO:93；其人源化轻链可变区氨基酸序列为SEQ ID NO:94；其人源化轻链可变区核酸序列为SEQ ID NO:95；其人源化scFv氨基酸序列为SEQ ID NO:96；
在上述技术方案的基础上，其重组人源化单克隆抗体的轻链恒定区为κ链，重链恒定区为IgG型。
本发明不仅包括完整的单克隆抗体，还包括具有免疫活性的抗体片段，如Fab或(Fab”)2片段；scFv；抗体重链；抗体轻链。
本发明还提供了具有本发明抗体的其他蛋白质或融合表达产物。具体地，本发明包括具有含可变区的重链和轻链的任何蛋白质或蛋白质偶联物及融合表达产物(即免疫偶联物及融合表达产物)，只要该可变区与本发明抗体的重链和轻链的可变区相同或至少约90％同源性，较佳地至少约95％同源性。
本发明抗体的重链和/或轻链的可变区特别令人感兴趣，因为它们中至少部分涉及结合抗原。因此，本发明包括那些具有带CDR的单克隆抗体轻链和重链可变区的分子，只要其CDR与此处鉴定的CDR具有90％以上(较佳地95％以上，最佳地98％以上)的同源性。
如本文所用，术语“片段”、“衍生物”和“类似物”是指基本上保持本发明抗体相同的生物学功能或活性的多肽。本发明的多肽片段、衍生物或类似物可以是(i)有一个或多个保守或非保守性氨基酸残基(优选保守性氨基酸残基)被取代的多肽，而这样的取代的氨基酸残基可以是也可以不是由遗传密码编码的，或(ii)在一个或多个氨基酸残基中具有取代基团的多肽，或(iii)成熟多肽与另一个化合物(比如延长多肽半衰期的化合物，例如聚乙二醇)融合所形成的多肽，或(iv)附加的氨基酸序列融合到此多肽序列而形成的多肽(如前导序列或分泌序列或用来纯化此多肽的序列或蛋白原序列，或与6His标签形成的融合蛋白)。根据本文的教导，这些片段、衍生物和类似物属于本领域熟练技术人员公知的范围。
本发明抗体指具有人GPRC5D蛋白结合活性的、包括上述CDR区的多肽。该术语还包括具有与本发明抗体相同功能的、包含上述CDR区的多肽的变异形式。这些变异形式包括(但并不限于)：一个或多个(通常为1-50个，较佳地1-30个，更佳地1-20个，最佳地1-10个)氨基酸的缺失、插入和/或取代，以及在C末 端和/或N末端添加一个或数个(通常为20个以内，较佳地为10个以内，更佳地为5个以内)氨基酸。例如，在本领域中，用性能相近或相似的氨基酸进行取代时，通常不会改变蛋白质的功能。又比如，在C末端和/或N末端添加一个或数个氨基酸通常也不会改变蛋白质的功能。该术语还包括本发明抗体的活性片段和活性衍生物。
该多肽的变异形式包括：同源序列、保守性变异体、等位变异体、天然突变体、诱导突变体、在高或低的严紧度条件下能与本发明抗体的编码DNA杂交的DNA所编码的蛋白、以及利用抗本发明抗体的抗血清获得的多肽或蛋白。
本发明还提供了其他多肽，如包含人抗体或其片段的融合蛋白。除了几乎全长的多肽外，本发明还包括了本发明抗体的片段。通常，该片段具有本发明抗体的至少约50个连续氨基酸，较佳地至少约60个连续氨基酸，更佳地至少约80个连续氨基酸，最佳地至少约100个连续氨基酸。
在本发明中，“本发明抗体的保守性变异体”指与本发明抗体的氨基酸序列相比，有至多10个，较佳地至多8个，更佳地至多5个，最佳地至多3个氨基酸被性质相似或相近的氨基酸所替换而形成多肽。这些保守性变异多肽最好根据表1进行氨基酸替换而产生。
表1
本发明还提供了编码上述抗体或其片段或其融合蛋白的多核苷酸分子。本发明的多核苷酸可以是DNA形式或RNA形式。DNA形式包括cDNA、基因组DNA或人工合成的DNA。DNA可以是单链的或是双链的。DNA可以是编码链或非编码链。编码成熟多肽的编码区序列可以与例如SEQ ID NO:50、52,所示的编码区序列相同或者是简并的变异体。如本文所用，“简并的变异体”在本发明中是指编码具有与本发明的多肽相同的氨基酸序列，但与例如SEQ ID NO:50、52所示的编码区序列有差别的核酸序列。
编码本发明的成熟多肽的多核苷酸包括：只编码成熟多肽的编码序列；成熟多肽的编码序列和各种附加编码序列；成熟多肽的编码序列(和任选的附加编码序列)以及非编码序列。
术语“编码多肽的多核苷酸”可以是包括编码此多肽的多核苷酸，也可以是还包括附加编码和/或非编码序列的多核苷酸。
本发明还涉及与上述的序列杂交且两个序列之间具有至少50％，较佳地至少70％，更佳地至少80％相同性的多核苷酸。本发明特别涉及在严格条件下与本发明所述多核苷酸可杂交的多核苷酸。在本发明中，“严格条件”是指：(1)在较低离子强度和较高温度下的杂交和洗脱，如0.2×SSC,0.1％SDS,60℃；或(2)杂交时加有变性剂,如50％(v/v)甲酰胺，0.1％小牛血清/0.1％Ficoll，42℃等；或(3)仅在两条序列之间的相同性至少在90％以上,更好是95％以上时才发生杂交。并且,可杂交的多核苷酸编码的多肽与例如SEQ ID NO:49、SEQ ID NO:51所示的成熟多肽有相同的生物学功能和活性。
本发明的抗体的核苷酸全长序列或其片段通常可以用PCR扩增法、重组法或人工合成的方法获得。一种可行的方法是用人工合成的方法来合成有关序列，尤其是片段长度较短时。通常，通过先合成多个小片段，然后再进行连接可获得序列很长的片段。此外，还可将重链的编码序列和表达标签(如6His)融合在一起，形成融合蛋白。
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。本发明所涉及的生物分子(核酸、蛋白等)包括以分离的形式存在的生物分子。
目前，已经可以完全通过化学合成来得到编码本发明蛋白(或其片段，或其衍生物)的DNA序列。然后可将该DNA序列引入本领域中已知的各种现有的 DNA分子(或如载体)和细胞中。此外，还可通过化学合成将突变引入本发明蛋白序列中。
本发明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。代表性例子有：大肠杆菌，链霉菌属；鼠伤寒沙门氏菌的细菌细胞；真菌细胞如酵母；果蝇S2或Sf9的昆虫细胞；CHO、COS7、293细胞的动物细胞等。
用重组DNA转化宿主细胞可用本领域技术人员熟知的常规技术进行。当宿主为原核生物如大肠杆菌时，能吸收DNA的感受态细胞可在指数生长期后收获，用CaCl2法处理，所用的步骤在本领域众所周知。另一种方法是使用MgCl2。如果需要，转化也可用电穿孔的方法进行。当宿主是真核生物，可选用如下的DNA转染方法：磷酸钙共沉淀法，常规机械方法如显微注射、电穿孔,脂质体包装等。
获得的转化子可以用常规方法培养，表达本发明的基因所编码的多肽。根据所用的宿主细胞，培养中所用的培养基可选自各种常规培养基。在适于宿主细胞生长的条件下进行培养。当宿主细胞生长到适当的细胞密度后，用合适的方法(如温度转换或化学诱导)诱导选择的启动子，将细胞再培养一段时间。
在上面的方法中的重组多肽可在细胞内、或在细胞膜上表达、或分泌到细胞外。如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并不限于：常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超处理、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。
本发明的抗体可以单独使用，也可与可检测标记物(为诊断目的)、治疗剂、PK(蛋白激酶)修饰部分或任何以上这些物质的组合结合或偶联。
用于诊断目的的可检测标记物包括但不限于：荧光或发光标记物、放射性标记物、MRI(磁共振成像)或CT(电子计算机X射线断层扫描技术)造影剂、或能够产生可检测产物的酶。
可偶联的治疗剂包括但不限于：胰岛素、IL-2、干扰素、降钙素、GHRH肽、肠肽类似物、白蛋白、抗体片段、细胞因子、和激素。
此外还可与本发明抗体结合或偶联的治疗剂包括但不限于：1.放射性核素；2.生物毒；3.细胞因子如IL-2等；4.金纳米颗粒/纳米棒；5.病毒颗粒； 6.脂质体；7.纳米磁粒；8.前药激活酶(例如，DT-心肌黄酶(DTD)或联苯基水解酶-样蛋白质(BPHL))；10.化疗剂(例如，顺铂)或任何形式的纳米颗粒等。
本发明还提供了一种嵌合抗原受体(CAR)，包括细胞外结构域、跨膜结构域、和细胞内结构域。胞外结构域包括靶-特异性结合元件(也称为抗原结合结构域)。细胞内结构域包括共刺激信号传导区和ζ链部分。共刺激信号传导区指包括共刺激分子的细胞内结构域的一部分。共刺激分子为淋巴细胞对抗原的有效应答所需要的细胞表面分子，而不是抗原受体或它们的配体。
在CAR的胞外结构域和跨膜结构域之间，或在CAR的胞浆结构域和跨膜结构域之间，可并入接头。如本文所用的，术语“接头”通常指起到将跨膜结构域连接至多肽链的胞外结构域或胞浆结构域作用的任何寡肽或多肽。接头可包括0-300个氨基酸，优选地2至100个氨基酸和最优选地3至50个氨基酸。
在本发明的一个较佳的实施方式中，本发明提供的CAR的胞外结构域包括靶向人GPRC5D的抗原结合结构域。本发明的CAR当在T细胞中表达时，能够基于抗原结合特异性进行抗原识别。当其结合其关联抗原时，影响肿瘤细胞，导致肿瘤细胞不生长、被促使死亡或以其他方式被影响，并导致患者的肿瘤负荷缩小或消除。抗原结合结构域优选与来自共刺激分子和ζ链中的一个或多个的细胞内结构域融合。优选地，抗原结合结构域与4-1BB信号传导结构域、和CD3ζ信号结构域组合的细胞内结构域融合。
如本文所用，“抗原结合结构域”、“单链抗体片段”均指具有抗原结合活性的Fab片段，Fab’片段，F(ab’)2片段，或单一Fv片段。Fv抗体含有抗体重链可变区、轻链可变区，但没有恒定区，并具有全部抗原结合位点的最小抗体片段。一般的，Fv抗体还包含VH和VL结构域之间的多肽接头，且能够形成抗原结合所需的结构。抗原结合结构域通常是scFv(single-chain variable fragment)。scFv的大小一般是一个完整抗体的1/6。单链抗体优选是由一条核苷酸链编码的一条氨基酸链序列。作为本发明的优选方式，所述抗原结合结构域包含特异性识别人GPRC5D的抗体，较佳地为单链抗体。
对于绞链区和跨膜区(跨膜结构域)，CAR可被设计以包括融合至CAR的胞外结构域的跨膜结构域。在一个实施方式中，使用天然与CAR中的结构域之一相关联的跨膜结构域。在一些例子中，可选择跨膜结构域，或通过氨基酸置换进行修饰，以避免将这样的结构域结合至相同或不同的表面膜蛋白的跨膜结构域，从而最小化与受体复合物的其他成员的相互作用。
在本发明的一个优选实施方式中，本发明利用人源化抗GPRC5D的scFv构建的CAR-T细胞，可以进一步提高其杀伤效果和肿瘤清除能力。
本发明还提供了一种药物组合物，它含有上述的抗体或其活性片段或其融合蛋白或表达所述抗体的免疫细胞，以及药学上可接受的载体。通常，可将这些物质配制于无毒的、惰性的和药学上可接受的水性载体介质中，其中pH通常约为5-8，较佳地pH约为6-8，尽管pH值可随被配制物质的性质以及待治疗的病症而有所变化。配制好的药物组合物可以通过常规途径进行给药，其中包括(但并不限于)：口服、呼吸道、瘤内、腹膜内、静脉内、或局部给药。
本发明的药物组合物含有安全有效量(如0.001-99wt％,较佳地0.01-90wt％，更佳地0.1-80wt％)的本发明上述的单克隆抗体(或其偶联物)以及药学上可接受的载体或赋形剂。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、及其组合。药物制剂应与给药方式相匹配。本发明的药物组合物可以被制成针剂形式，例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。药物组合物如针剂、溶液宜在无菌条件下制造。活性成分的给药量是治疗有效量，例如每天约1微克/千克体重-约10毫克/千克体重。此外，本发明的抗体还可与其他治疗剂一起使用。
使用药物组合物时，是将安全有效量的免疫偶联物施用于哺乳动物，其中该安全有效量通常至少约10微克/千克体重，而且在大多数情况下不超过约8毫克/千克体重，较佳地该剂量是约10微克/千克体重-约1毫克/千克体重。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是熟练医师技能范围之内的。
本发明还提供了如本发明的抗体或其抗原结合片段，本发明的嵌合抗原受体以及表达所述嵌合抗原受体的工程化免疫细胞，或本发明的药物偶联物或药物组合物在预防和/或治疗含有表达GPRC5D的肿瘤细胞的癌症(例如多发性骨髓瘤)中的用途。
与现有技术相比，本发明具有以下优点和有益效果：
本发明利用B细胞克隆技术提供靶向人GPRC5D的重组人源化单克隆抗体，重要的是靶向人GPRC5D的特异性重组人源化单克隆抗体亲和力高，具有良好的特异性，可以弥补市场上靶向人GPRC5D蛋白的重组人源化单克隆抗体的诊 断与治疗相关的应用。另外，该GPRC5D抗体序列可以构建成CAR-T或双特异性抗体或抗体偶联药，可用于后续的肿瘤疾病的治疗。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。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，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。以下实施例中所用的实验材料和试剂如无特别说明均可从市售渠道获得。
实施例1天然结构人源化抗体的亲和性流式检测
设置Eureka公司开发的抗GPRC5D抗体(MCARH109)为对照抗体G00，其scFv序列来自Eureka公司的专利(CN107428829B)，并根据实验需要将该对照抗体构建成人源化抗体，scFv-hFc抗体，CAR-T，CAR-jurkat-Nfat-Luc细胞等形式。
为了获得靶向人GPRC5D蛋白的人重组单克隆抗体，本发明构建表达了人GPRC5D N端胞外区的哺乳动物细胞表达的重组蛋白作为免疫原，利用新西兰大白兔进行免疫，免疫完成后，用抗原包被的磁珠进行筛选B淋巴细胞，将单B淋巴细胞扩大培养后，流式检测GPRC5D抗体的表达，阳性克隆进行基因测序，即可获得兔单克隆抗体的基因序列，之后通过兔抗人源化改造工程得到10株人源化GPRC5D单克隆抗体，分别命名为G01、G02、G03、G04、G05、G06、G07、G08、G09和G10。
将G00～G09构建成人源化IgG1型抗体载体质粒，将重组的载体质粒转染293细胞，收获上清纯化浓缩抗体(结构如图1左图所示)，定量后用于流式检测实验。
流式检测按照以下步骤进行：
(1)将G00～G09人源化抗体进行等浓度稀释：40ug/ml，20ug/ml，10ug/ml，5ug/ml,2.5ug/ml,1.25ug/ml,0.625ug/ml,0.375ug/ml,0ug/ml,
(2)将培养着的K562-GPRC5D稳转细胞系(稳定表达GPRC5D的K562细胞)取出使用PBS洗涤一次后，去除上清，加入PBS重悬细胞取样计数，将细胞密度调整为3*10^6个/ml,将细胞悬液以每孔50ul加入到标记好的96孔板中；
(3)向96孔板中加入稀释的各浓度人源化抗体稀释液孵育30min,
(4)向孔板中加入PBS洗涤2次，离心去除上清，
(5)向孔板中加入50ul二抗进行孵育30min,孔板细胞再次洗涤后加入PBS重悬，将孔板放流式仪上进行检测分析。
如图2所示，G02,G05,G07,G09对K562-GPRC5D有良好的结合活性，优于对照抗体G00。
实施例2scFv-hFc结构人源化抗体的亲和性流式检测
将G00～G09构建成scFv-hFc人源化IgG1构型的载体质粒，将重组的载体质粒转染293细胞，收获上清纯化浓缩抗体(结构如图1右图所示)，定量后用于流式检测实验。
流式检测按照以下步骤进行：
(1)将G00～G09scFv-hFc IgG1构型人源化抗体进行等浓度稀释：40ug/ml，20ug/ml，10ug/ml，5ug/ml,2.5ug/ml,1.25ug/ml,0.625ug/ml,0.375ug/ml,0ug/ml,
(2)将培养着的K562-GPRC5D稳转细胞和RPMI 8226细胞(天然的多发性骨髓瘤细胞，表达GPRC5D)取出使用PBS洗涤一次后，去除上清，加入PBS重悬细胞取样计数，将细胞密度调整为3*10^6个/ml,将细胞悬液以每孔50ul加入到标记好的96孔板中；
(3)向96孔板中加入稀释的各浓度抗体稀释液孵育30min,
(4)向孔板中加入PBS洗涤2次，离心去除上清，
(5)向孔板中加入50ul二抗进行孵育30min,孔板细胞再次洗涤后加入PBS重悬，将孔板放流式仪上进行检测分析。
如图3所示，G02,G05,G07,G09对K562-GPRC5D(图3a)和8226细胞(图3b)有良好的亲和性，优于对照抗体G00。
实施例3 scFv-hFc结构人源化抗体对MM.1S和H929细胞结合活性流式检测
检测实施例2中制备的scFv-hFc结构人源化抗体对MM.1S和NCI-H929细胞(MM.1S与NCI-H929均是天然的多发性骨髓瘤细胞，表达GPRC5D)的结合活性。按照以下步骤进行：
(1)将G00～G09scFv-hFc IgG1构型人源化抗体进行浓度稀释至20ug/ml；
(2)将培养着的MM.1S细胞和NCI-H929细胞取出使用PBS洗涤一次后，去除上清，加入PBS重悬细胞取样计数，将细胞密度调整为3*10^6个/ml,将细胞悬 液以每孔50ul加入到标记好的96孔板中；
(3)向96孔板中加入稀释的抗体稀释液孵育30min,
(4)向孔板中加入PBS洗涤2次，离心去除上清，
(5)向孔板中加入50ul二抗进行孵育30min,孔板细胞再次洗涤后加入PBS重悬，将孔板放流式仪上进行检测分析；
如图4所示，G02,G05,G07,G09对MM.1S细胞(图4a)和NCI-H929细胞(图4b)有良好的结合活性，显著优于对照抗体G00。
实施例4 CAR-jurkat-nfat-luc细胞自激活和抗原依赖的激活检测
如图5所示，将G00～G09的scFv分别构建到CAR的慢病毒载体中，将载体用于慢病毒包装，得到慢病毒上清进行浓缩纯化，然后感染jurkat-nfat-luc细胞，从而获得感染后的jurkat-nfat-luc细胞，即CAR-jurkat-nfat-luc细胞。将各CAR-jurkat-nfat-luc细胞与分别与K562、K562-GPRC5D、RPMI 8226细胞进行共培养48h后检测生物发光值。
如图6所示，图6a显示了CAR-jurkat-nfat-luc细胞与K562-GPRC5D或K562细胞共培养的结果，图6b显示了CAR-jurkat-nfat-luc细胞与RPMI 8226或K562共培养的结果；可见，CAR-jurkat-nfat-luc细胞有较低的自激活背景和较高的抗原依赖激活信号，尤其是表达G02、G05、G07和G09的scFv的CAR-jurkat-nfat-luc细胞。
实施例5 CAR-T细胞制备
用肝素钠抗凝采血管抽取人外周血30ml，离心600g*10min，吸取上层黄色血浆灭活备用，将剩余血液成分加PBS混匀后铺到含淋巴细胞分离液的离心管中，离心1000g*10min，小心吸取白膜层细胞，白膜层细胞悬液用PBS洗涤后离心去除上清，加PBS重悬计数；离心去除上清，加入分选buffer重悬细胞，根据细胞计数结果使用抗人CD3纳米磁珠和美天旎磁珠分选系统分选得到CD3阳性细胞群，将得到的CD3阳性细胞计数后使用CD3/CD28磁珠进行激活培养24h后分别感染G00～G09的CAR慢病毒，感染过夜后洗涤细胞，48h后流式检测CAR的表达效率。
如图7所示，G00～G09 CAR-T细胞均有高的转染效率。
实施例6 CAR-T细胞杀伤RPMI-8226细胞因子检测
将培养的实施例5中制备的G00～G09 CAR-T细胞和T细胞分别用PBS洗涤后使用培养基重悬取样计数，根据细胞浓度将CAR-T细胞浓度和T细胞浓度调整为1*10E5个/ml，将CAR-T细胞和T细胞分别加入到标记好的96孔板中，每孔100ul细胞悬液。
将培养中的RPMI-8226细胞用PBS洗涤后使用培养基重悬，取样计数，将细胞浓度调整为1*10E5个/ml，将RPMI-8226细胞悬液加入到96孔板对应孔中，每孔100ul细胞悬液。
将CAR-T细胞与RPMI-8226细胞混合悬液混匀后放二氧化碳培养箱过夜培养24h；将共培养的孔板取出，离心300g*10min，取出150ul上清转到新的96孔板中，使用ELISA试剂盒检测上清中的IL-2、TNF-α、IFN-γ浓度。
如图8所示，分别显示了上清中IL-2(图8a)，TNF-α(图8b)，IFN-γ(图8c)的浓度；可见G02、G05、G07、G09 CAR-T细胞能够特异杀伤RPMI-8226细胞并产生较高水平的炎症因子。
实施例7 CAR-T细胞杀伤MM.1S和K562细胞因子检测
将培养的实施例5中制备的G00～G09 CAR-T细胞和T细胞分别用PBS洗涤后使用培养基重悬取样计数，根据细胞浓度将CAR-T浓度和T细胞浓度调整为1*10E5个/ml,将CAR-T细胞和T细胞分别加入到标记好的96孔板中，每孔100ul细胞悬液。
将培养中的MM.1S和K562细胞分别用PBS洗涤后使用培养基重悬，取样计数，将细胞浓度调整为1*10E5个/ml，将MM.1S和K562细胞分别加入到96孔板对应孔中，每孔100ul细胞悬液。
将CAR-T细胞或T细胞与MM.1S或K562细胞混合液混匀后放二氧化碳培养箱过夜培养24h；将共培养的孔板取出，离心300g*10min，取出150ul培养上清转到新的96孔板中，使用ELISA试剂盒检测上清中的IL-2,TNF-α,IFN-γ浓度。
如图9所示，分别显示了上清中IL-2(图9a),TNF-α(图9b),IFN-γ(图9c)的浓度；可见G02，G04，G05，G07，G08，G09 CAR-T细胞能够特异杀伤MM.1S细胞并产生较高水平的炎症因子,所有CAR-T或T细胞都不能杀伤K562细胞，说明CAR-T细胞杀伤的特异性。
实施例8 CAR-T细胞特异增殖实验
24孔板标记设置4个反应条件组：
A：CAR-T/T+NCI-H929，CAR-T细胞或T细胞与多发性骨髓瘤细胞NCI-H929共培养
B：CAR-T/T+K562，CAR-T细胞或T细胞与K562细胞共培养
C：CAR-T/T only，CAR-T细胞或者T细胞单独培养
D：CAR-T/T+IL-2，CAR-T细胞或T细胞添加300U/ml IL-2培养。
将培养的实施例5中制备的G00～G09 CAR-T细胞和T细胞分别用PBS洗涤后使用培养基重悬取样计数，根据细胞浓度将CAR-T细胞浓度和T细胞浓度调整为5～10*10E6个/ml,向细胞悬液中加入CFSE染色10min后加入FBS终止染色，离心去除上清后加入完全培养基多次洗涤细胞，加入培养基重悬细胞，取样计数，将细胞悬液CAR阳性细胞或T细胞浓度调整为1*10E6个/ml；将CAR-T细胞和T细胞分别加入到标记好的24孔板中，每孔100ul细胞悬液。
将培养中的NCI-H929细胞和K562细胞用PBS洗涤后使用培养基重悬，取样计数，将细胞浓度调整为1*10E6个/ml，将NCI-H929细胞悬液加入到24孔板A组CAR-T/T细胞对应孔中，每孔200ul细胞悬液，将K562细胞悬液加入到24孔板B组CAR-T/T细胞对应孔中，每孔200ul细胞悬液，将CAR-T/T细胞C组每孔补加不含IL-2的培养基200ul，CAR-T/T细胞D组每孔补加含IL-2的培养基200ul，将孔板中各细胞孔悬液混匀后放二氧化碳培养箱过夜培养120h。
将培养的孔板中细胞悬液取出，用PBS洗涤细胞后分别进行流式染色，流式染色CD3和CAR，CAR-T细胞选中CD3阳性细胞，选中其中CAR阳性细胞，对FITC峰进行叠加；T细胞选中CD3阳性细胞，对FITC峰进行叠加。
如图10所示，分别显示了各CAR-T细胞与靶细胞共培养后的CFSE荧光减弱情况，说明G02、G04、G05、G07、G09 CAR-T细胞能够特异识别NCI-H929细胞并发生特异性增殖；G01 CAR-T和T细胞不能特异识别NCI-H929细胞，也就不能产生特异增殖，G08 CAR-T细胞在培养过程中，无论培养基中有无IL-2，无论与K562细胞还是与NCI-H929细胞共培养后，均发生增殖，G08 CAR-T细胞中scFv导致的细胞自激活程度很高。
实施例9 CAR-T细胞特异杀伤实验
将培养的G00～G09的CAR-T细胞和T细胞分别用PBS洗涤后使用培养基重悬取样计数，根据细胞浓度将CAR阳性细胞浓度和T细胞浓度调整为3*10E5个/ml、1*10E5个/ml、0.3*10E5个/ml、0.1*10E5个/ml，将稀释后的CAR-T细胞或T细胞加入到96孔板对应孔中，每孔100ul。
将培养中的靶细胞：K562-luc、K562-GPRC5D-luc、NCI-H929-luc、RPMI8226-luc、MM.1S-luc用PBS洗涤后使用培养基重悬，取样计数，将细胞浓度调整为1*10E5个/ml,将靶细胞悬液加入到96孔板应孔中，每孔100ul细胞悬液，将孔板放二氧化碳培养箱过夜培养24h。
取出各孔板加入荧光素钾盐溶液，将孔板放多功能酶标仪进行生物发光值检测。
结果如图11所示，G00～G09 CAR-T中的G00、G02、G05、G07、G09 CAR-T细胞可以很好地对GPRC5D阳性靶细胞进行杀伤。
实施例10 GPRC5D-CAR-T的体内药效试验
本实验以供试品GPRC5D-CAR-T静脉注射给予移植人骨髓瘤细胞MM1.S的NOD-Prkdcscid Il2rgtm1/Bcgen小鼠(B-NDG小鼠)，以评价其体内对肿瘤细胞增殖的抑制效果。
方法：
1)筛选和分组：40只雌性B-NDG小鼠静脉接种MM1.S-Luc(荧光素酶标记的人MM1.S细胞)后5天，随机分为8组，空白对照组(Vehicle组)、阴性对照MOCK T组(T mock组),供试品G05 1E6治疗组，供试品G05 5E6治疗组，供试品G07 1E6治疗组，供试品G07 5E6治疗组，供试品G09 1E6治疗组，供试品G095E6治疗组，共八组，每组五只小鼠。
2)接种：每只小鼠静脉注射接种1E6/只MM1.S-Luc(荧光素酶标记的人MM1.S细胞)，接种后第五天给药。
3)给药方式：空白对照组(Vehicle组)小鼠给予溶媒溶液；MOCK T组小鼠给予阴性对照MOCK-T细胞，G05 1E6组小鼠给予1E6/只的供试品G05 CAR-T细胞；G05 5E6组小鼠给予5E6/只的供试品G05 CAR-T细胞；G07 1E6组小鼠给予1E6/只的供试品G07 CAR-T细胞；G07 5E6组小鼠给予5E6/只的供试品G07 CAR-T细胞；G09 1E6组小鼠给予1E6/只的供试品G09 CAR-T细胞；G09 5E6组小鼠给予5E6/只的供试品G09 CAR-T细胞；以首次给药当日为D0，所有动物均为尾静脉单次注射给药。
4)检测指标：在D-5(给药前5天)、D-1(给药前1天)、D3、D6、D10、D13、D18和D24所有动物用Bruker小动物成像仪拍摄化学发光信号。
结论：
结果如图12所示，相比低剂量组(G05 1E6、G07 1E6、G09 1E6)，高剂 量组(G05 5E6、G07 5E6、G09 5E6组)显示出更优的治疗效果。
实施例11 GPRC5D抗体内吞效应实验
设置参照克隆LX，抗体序列来自上海礼新医药研发有限公司的专利(CN 115038720 A)；无关对照抗体BM138，即靶向CLDN18.2的抗体zolbetuximab。将生产的抗体纯化后使用BCA检测浓度后进行抗体稀释：使用培养基配制抗体稀释液(40ug/ml，4ug/ml，0.4ug/ml)每孔25ul，每个稀释浓度设置3个复孔，用培养基设置不加抗体的孔为对照孔。标记试剂Zenon pHrodo iFL green IgG稀释：使用培养基配制标记试剂稀释液(40ug/ml，4ug/ml，0.4ug/ml)，将每个浓度的稀释液分别加入到等浓度的抗体稀释液孔中，每孔25ul，混匀，设置加入标记试剂不同浓度稀释液但无抗体的对照孔，室温孵育10min，每孔加入50ul K562-GPRC5D细胞悬液，每孔细胞数设置为50000个，将孔板放培养箱24小时后使用流式仪检测各孔细胞MFI值，作图统计不同抗体不同浓度孔的MFI值。
如图13所示，G05-mAb，G07-mAb，G09-mAb的MFI值曲线图显示出这3个克隆有在不同的抗体浓度下均有较好的内吞效果。
实施例12 GPRC5D&CD3双特异性抗体的体外功能鉴定
12.1 GPRC5D&CD3双特异性抗体载体的生产与检测
本实施例中使用的CD3的抗体来自小鼠杂交瘤SP34的人源化抗体，抗体的序列参见专利CN109715667A中CD3B219 VH和CD3B219 VL，序列信息进行密码子优化和基因合成为CD3 VH和CD3VL。CD3VH克隆至表达载体pTT5-hIgG1FC-Knob上，CD3VL克隆至表达载体pTT5-hKappa上。将GPRC5D抗体的ScFv：G05、G07、G09、G00克隆至表达载体pTT5-hIgG1FC-Hole上。进行PCR扩增、酶切连接转化、克隆鉴定以及质粒制备，共得到6个表达质粒分别为：pTT5-CD3VH-Knob、pTT5-CD3VL、pTT5-G05-Hole、pTT5-G07-Hole、pTT5-G09-Hole以及pTT5-G00-Hole。
将pTT5-CD3VH-Knob、pTT5-CD3VL分别与pTT5-G05-Hole、pTT5-G07-Hole、pTT5-G09-Hole以及pTT5-G00-Hole四个质粒共转染至293F细胞进行抗体表达，分别收集G05 BsAb、G07 BsAb、G09 BsAb和G00 BsAb的双特异性抗体表达的细胞上清，进行ProteinA亲和层析，PBS透析去除杂质后测定蛋白浓度。
12.2双特异性抗体的体外功能鉴定
12.2.1流式细胞术测定双特异性抗体的结合活性
NCI-H929及Jurkat细胞复苏培养48h后，细胞计数与细胞铺板2×10^5于96孔V型板中。用PBS对双特性抗体进行梯度稀释，Talquetamab作为阳性对照。起始浓度为100μg/ml，之后1:5、1:2、1:2、1:2、1:2、1:2、1:2、1:2进行倍比稀释，共设8个检测浓度。将100ul的蛋白质与每孔200,000个细胞混合。使BsAb与细胞在4℃温育下1小时。然后用PBS和0.2％FBS将细胞洗涤三次。随后，加入缀合有APC的抗人IgGmAb使细胞与第二抗体在4℃温育下1小时。然后用PBS和0.2％FBS将细胞洗涤三次。将细胞用PBS和0.2％FBS再次洗涤并随后在流式细胞仪上进行分析。
结果如图14a和图14b所示。观察到所有双特异性抗体均以剂量依赖性方式于内源性表达GPRC5D蛋白的NCI-H929细胞结合。其中G05、G07的抗体亲和力显著高于G09、G00以及Talquetamab。
12.2.2双特异性抗体介导T细胞杀伤肿瘤细胞
使用NCI-H929-GFP-LUC，MM.1S-GFP-LUC，RPMI-8826-GFP-LUC，分析双特异性抗体在T-细胞介导的细胞毒性。靶细胞(NCI-H929-GFP-LUC，MM.1S-GFP-LUC，RPMI826-GFP-LUC)进行计数将细胞在含有5％FBS的X-Vivo杀伤培养基中洗涤两次。用X-Vivo杀伤培养将细胞稀释至2×10^5/mL并在37℃下温育直至使用。
正常供体的PBMC细胞在37℃水浴中解冻复苏后，使用CD3磁珠分选出CD3阳性T细，根据计数结果加入T细胞完全培养基(X-Vivo+5％FBS+200IU/ml IL-2+1％PS)重悬细胞，使得T细胞浓度在1.5～2.5×10^6/ml。扩增培养1周后，弃去上清液并以1.0×10^6/mL浓度重悬于X-Vivo杀伤培养基中。
将2×10^4个靶细胞加入96孔细胞培养中，向孔中加入6×10^4个T细胞(3:1的效应子：靶比率)。混合靶细胞和T细胞后，将20μl的GPRC5D×CD3双特异性抗体稀释液加入每个孔中。将GPRC5D&CD3双特异性抗体用PBS稀释至起始终浓度为20μg/m于靶细胞和T细胞混合孔中。在96孔板中用PBS中5倍系列稀释度制备滴定液。最后一列留为单独的PBS(载体对照)。依次加入到靶细胞和T细胞混合孔中。将平板在37℃和5％CO2下温育48小时。
两天之后(48小时)，平板离心后并将100μL的上清液于-80℃储存以供细胞因子释放测定。加入100ul浓度为30mg/ml荧光素钾盐，37℃孵育5min后，酶标仪检测荧光素酶值。通过荧光素酶数值分析细胞的双特异抗体对靶细胞的杀伤效果。使用具有可变斜率(四个参数)函数的非线性回归并使用最小二乘法在 GraphPad Prism 8中完成数据的作图和拟合。
结果如图15a-d所示。观察到所有的GPRC5D&CD3双特异性抗体均以剂量依赖性方式杀伤内源性表达GPRC5D蛋白的多发性骨髓瘤细胞株，其中G05、G07杀伤效率明显优于G00、G09以及Talquetamab。所有GPRC5D&CD3双特异性抗体对阴性对照K562细胞株无明显杀伤。
本发明的序列







在本发明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，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，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。

Claims (15)

  1. 一种抗体或其抗原结合片段，其包含重链可变区，其特征在于，所述重链可变区包含以下CDR：
    SEQ ID NO:44,78,1,33,67,22,或87所示的VH-CDR1，
    SEQ ID NO:45,59,79,12,34,68,2,23,54或88所示的VH-CDR2，和
    SEQ ID NO:46,60,13,35,69,3,24,55或89所示的VH-CDR3；
    其中所述抗体或其抗原结合片段特异性地结合GPRC5D，优选人GPRC5D。
  2. 如权利要求1所述的抗体或其抗原结合片段，其特征在于，所述抗体或其抗原结合片段还包含轻链可变区，所述轻链可变区包含以下CDR：
    SEQ ID NO:47,61,80,14,36,70,4,25,47或90所示的VL-CDR1，
    SEQ ID NO:37,15,71,5或26所示的VL-CDR2，和
    SEQ ID NO:48,81,16,38,72,6,27,48或91所示的VL-CDR3。
  3. 如权利要求1所述的抗体或其抗原结合片段，其特征在于，所述抗体或其抗原结合片段包含与SEQ ID NO:49、62、82、17、39、73、7、28、56或92具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列的重链可变区。
  4. 如权利要求2所述的抗体或其抗原结合片段，其特征在于，所述抗体或其抗原结合片段包含与SEQ ID NO:51、64、84、19、41、75、9、30或94具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列的轻链可变区。
  5. 一种重组蛋白，所述重组蛋白包含：
    (i)如权利要求1-4任一项所述的抗体或其抗原结合片段；和
    (ii)任选的协助表达和/或纯化的标签序列。
  6. 一种多核苷酸，其特征在于，所述多核苷酸编码选自下组的多肽：
    (1)如权利要求1-4任一项所述的抗体或其抗原结合片段；或
    (2)如权利要求5所述的重组蛋白。
  7. 一种载体，其特征在于，所述载体含有权利要求6所述的多核苷酸。
  8. 一种免疫偶联物，其特征在于，所述免疫偶联物包含：
    (a)如权利要求1-4任一项所述的抗体或其抗原结合片段、或如权利要求5所述的重组蛋白；和
    (b)选自下组的偶联部分：可检测标记物、药物、毒素、细胞因子、放射 性核素、或酶。
  9. 一种嵌合抗原受体，其特征在于，所述嵌合抗原受体的抗原结合结构域包含如权利要求1-4任一项所述的抗体或其抗原结合片段。
  10. 一种免疫细胞，所述免疫细胞表达或在细胞膜外暴露有如权利要求1-4任一项所述的抗体或其抗原结合片段。
  11. 一种双特异性抗体，其包含如权利要求1-4任一项所述的抗体或其抗原结合片段。
  12. 如权利要求11所述的双特异性抗体，其特征在于，所述双特异性抗体包含靶向CD3的抗体或其抗原结合片段。
  13. 一种活性成分的用途，所述活性成分选自下组：如权利要求1-4任一项所述的抗体或其抗原结合片段、如权利要求5所述的重组蛋白、如权利要求8所述的免疫偶联物、如权利要求10所述的免疫细胞、如权利要求11所述的双特异性抗体、或其组合，用于制备诊断试剂或试剂盒，或用于制备治疗与GPRC5D高表达相关的疾病的药物。
  14. 一种药物组合物，其含有：
    (z1)活性成分，所述活性成分选自下组：如权利要求1-4任一项所述的抗体或其抗原结合片段、如权利要求5所述的重组蛋白、如权利要求8所述的免疫偶联物、如权利要求10所述的免疫细胞、如权利要求11所述的双特异性抗体、或其组合；以及
    (z2)药学上可接受的载体。
  15. 一种治疗与GPRC5D高表达相关的疾病的方法，所述方法包括向有需要的受试者施用如权利要求1-4任一项所述的抗体或其抗原结合片段、如权利要求5所述的重组蛋白、如权利要求8所述的免疫偶联物、如权利要求10所述的免疫细胞、如权利要求11所述的双特异性抗体、或如权利要求14所述的药物组合物。
PCT/CN2023/115089 2022-08-30 2023-08-25 靶向人gprc5d的重组人源化单克隆抗体及其应用 WO2024046239A1 (zh)

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