WO2024087731A1 - 一种抗gprc5d的多特异性抗体 - Google Patents

一种抗gprc5d的多特异性抗体 Download PDF

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WO2024087731A1
WO2024087731A1 PCT/CN2023/105979 CN2023105979W WO2024087731A1 WO 2024087731 A1 WO2024087731 A1 WO 2024087731A1 CN 2023105979 W CN2023105979 W CN 2023105979W WO 2024087731 A1 WO2024087731 A1 WO 2024087731A1
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antigen
seq
antibody
variable region
chain variable
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French (fr)
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赵文祥
魏海涛
宁婷婷
李亚男
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上海祥耀生物科技有限责任公司
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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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • 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
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • 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
    • C12N5/12—Fused cells, e.g. hybridomas
    • C12N5/16—Animal cells
    • C12N5/163—Animal cells one of the fusion partners being a B or a T lymphocyte
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/91—Cell lines ; Processes using cell lines

Definitions

  • the invention relates to a multispecific antibody against GPRC5D, belonging to the field of biomedicine.
  • MM Multiple Myeloma
  • WHO global cancer statistics report released by WHO, there were 20,066 new cases of multiple myeloma in China in 2018, 14,665 deaths, and a total of 44,643 cases in 5 years.
  • the incidence and mortality of patients gradually increase with age.
  • the total number of patients will gradually increase, and the clinical demand is huge.
  • immunomodulators the treatment options for multiple myeloma in my country can be divided into three categories: immunomodulators, proteasome inhibitors and biological molecular targeted therapies.
  • immunomodulators and proteasome inhibitors are mainly used for first-line combination therapy before stem cell transplantation and maintenance therapy after transplantation for patients who meet the conditions for stem cell transplantation; for example, in the past decade, the application of immunomodulators represented by thalidomide and its derivative lenalidomide and small molecule proteasome inhibitors represented by bortezomib has greatly improved the remission rate and survival of MM patients.
  • the biological molecular targeted drugs for MM mainly involve three major targets: plasma cell surface protein CD38, signaling lymphocytes activating molecule family member 7 (Signaling Lymphocytes Activating Molecule Factor 7, SLAMF7) and B cell maturation antigen (B Cell Maturation Antigen, BCMA).
  • plasma cell surface protein CD38 signaling lymphocytes activating molecule family member 7 (Signaling Lymphocytes Activating Molecule Factor 7, SLAMF7)
  • B cell maturation antigen B Cell Maturation Antigen
  • anti-CD38 monoclonal antibodies for example, Daratumumab, which was conditionally approved and imported for registration and marketing by the National Medical Products Administration (NMPA) in 2019
  • NMPA National Medical Products Administration
  • BCMA protein has also become a research hotspot in this field due to its high expression specificity on MM cells.
  • the US FDA accelerated the approval of the BMCA-targeted antibody-drug conjugate (ADC) Blenrep for the treatment of patients with a median of 7 lines of relapse; The overall response rate of the drug can still reach 31%, and the median duration of response (DoR) is greater than 6 months.
  • the anti-tumor efficacy of CAR-T or CD3 bispecific antibodies targeting BCMA by mediating T cells is also significant.
  • GPRC5D belongs to the G protein-coupled receptor (GPCR) family; specifically, it is the G protein-coupled receptor C5 family subtype D, which is the orphan SARS C class GPCR first identified in 2001 (Brauner-Osborne, H., et al. Cloning and characterization of a human orphan family C G-protein coupled receptor GPRC5D. Biochim Biophys Acta, 2001. 1518(3): p.237-48).
  • GPCR G protein-coupled receptor
  • GPRC5D has been previously identified in cells from multiple myeloma patients, it has not been used in clinical development due to the lack of protein expression profile studies. Until 2019, research reports showed that GPRC5D is highly expressed in plasma cells of multiple myeloma, mostly not expressed in normal tissues, and only expressed in hair follicles with immune privilege (Smith, E.L., et al., GPRC5D is a target for the immunotherapy of multiple myeloma with rationally designed CAR T cells. Science Translational Medicine, 2019.11(485)).
  • the inventors of the present invention have developed a novel antibody and antigen-binding molecule thereof that specifically binds to GPRC5D based on the GPRC5D antigen, and have further developed bi/multi-specific antibodies that bind to the GPRC5D antigen on target cells and activated T cell antigens (such as CD3) on T cells.
  • Antibodies and antigen-binding molecules thereof; the bi-/multi-specific antibodies and antigen-binding molecules thereof of the present invention simultaneously bind to target cells and T cells and allow them to interact with each other, thereby causing activation of cytotoxic T cells and lysis of target cells.
  • the first aspect of the present invention provides a multispecific antibody or antigen-binding molecule thereof, wherein:
  • the multispecific antibody or antigen-binding molecule thereof comprises a first antigen-binding moiety and a second antigen-binding moiety;
  • first antigen binding moiety binds to GPRC5D, and the second antigen binding moiety binds to CD3 or CD3 ⁇ ;
  • the first antigen binding module comprises a heavy chain variable region i-VH and a light chain variable region i-VL, wherein the heavy chain variable region i-VH comprises i-HCDR1 as shown in SEQ ID NO.1, i-HCDR2 as shown in SEQ ID NO.2, and i-HCDR3 as shown in SEQ ID NO.3; the light chain variable region i-VL comprises i-LCDR1 as shown in SEQ ID NO.4, i-LCDR2 having an amino acid sequence of SAS, and i-LCDR3 as shown in SEQ ID NO.5;
  • the second antigen binding module comprises a heavy chain variable region ii-VH and a light chain variable region ii-VL, wherein the heavy chain variable region ii-VH comprises ii-HCDR1 as shown in SEQ ID NO.6, ii-HCDR2 as shown in SEQ ID NO.7 and ii-HCDR3 as shown in SEQ ID NO.8; the light chain variable region ii-VL comprises ii-LCDR1 as shown in SEQ ID NO.9, ii-LCDR2 having an amino acid sequence of GTN and ii-LCDR3 as shown in SEQ ID NO.10.
  • the sequence of the heavy chain variable region i-VH of the first antigen binding moiety is as shown in SEQ ID NO.11, or it has more than 80% sequence homology with the sequence shown in SEQ ID NO.11.
  • the sequence of the heavy chain variable region i-VH of the first antigen binding moiety has 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence homology with the sequence shown in SEQ ID NO.11.
  • the sequence of the light chain variable region i-VL of the first antigen binding moiety is as shown in SEQ ID NO.12, or it has more than 80% sequence homology with the sequence shown in SEQ ID NO.12.
  • the sequence of the light chain variable region i-VL of the first antigen binding moiety has 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence homology with the sequence shown in SEQ ID NO.12.
  • the sequence of the heavy chain variable region ii-VH of the second antigen binding moiety is as shown in SEQ ID NO.13, or it has more than 80% sequence homology with the sequence shown in SEQ ID NO.13.
  • the sequence of the heavy chain variable region ii-VH of the second antigen binding moiety has 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence homology with the sequence shown in SEQ ID NO.13.
  • the sequence of the light chain variable region ii-VL of the second antigen binding moiety is as shown in SEQ ID NO.14, or it has more than 80% sequence homology with the sequence shown in SEQ ID NO.14.
  • the sequence of the light chain variable region ii-VL of the second antigen binding moiety has 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence homology with the sequence shown in SEQ ID NO.14.
  • sequence homology with respect to amino acid sequences is determined by determining the number of amino acid residues present in the two sequences to produce the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to produce the percentage of sequence homology. Sequence homology is also known as sequence identity.
  • the heavy chain variable region i-VH and the light chain variable region i-VL of the above-mentioned first antigen binding module can be subjected to a small amount of amino acid deletion, insertion or amino acid mutation based on the sequences shown in SEQ ID NO.11 and SEQ ID NO.12, respectively, to obtain an amino acid sequence with a homology of more than 80%.
  • variants obtained by substitution of a small amount of amino acids (deletion or insertion, or amino acid mutation, or substitution of similar amino acids), especially conservative amino acid substitution in the framework region, retain the original properties and functions of the heavy chain variable region and the light chain variable region, that is, the antibody properties and functions of specific binding to GPRC5D, then these variants also fall within the scope of protection of the present invention.
  • the heavy chain variable region ii-VH and light chain variable region ii-VL of the above-mentioned second antigen binding module can be subjected to a small amount of amino acid deletion, insertion or amino acid mutation based on the sequences shown in SEQ ID NO.13 and SEQ ID NO.14, respectively, to obtain an amino acid sequence with a homology of more than 80%.
  • amino acid deletion, insertion or amino acid mutation based on the sequences shown in SEQ ID NO.13 and SEQ ID NO.14, respectively.
  • framework region refers to the amino acid sequence located between CDRs, including the framework region of the heavy chain variable region and the framework region of the light chain variable region.
  • the first antigen binding moiety is selected from any one of Fv, Fab, F(ab')2, Fab', dsFv, scFv, sc(Fv)2 or a single-chain antibody;
  • the second antigen binding moiety is selected from any one of Fv, Fab, F(ab')2, Fab', dsFv, scFv, sc(Fv)2 or a single-chain antibody.
  • the first antigen binding moiety and/or the second antigen binding moiety is a Fab molecule.
  • the first antigen binding moiety and the second antigen binding moiety are Fab molecules.
  • Fab molecules refer to protein molecules composed of the VH and CH1 domains of the Fab heavy chain and the VL and CL domains of the Fab light chain.
  • Conventional Fab molecules refer to natural Fab molecules, in which the Fab heavy chain is VH-CH1 from N to C-terminus, and the Fab light chain is VL-CL from N to C-terminus.
  • the Fab heavy chain variable region and the Fab light chain variable region can be exchanged/replaced with each other; or, in the same Fab molecule, the Fab heavy chain constant region and the Fab light chain constant region can be exchanged/replaced with each other.
  • the Fab molecule contains a peptide chain consisting of VL-CH1 (N to C-terminus) and a peptide chain consisting of VH-CL (N to C-terminus).
  • the peptide chain containing the heavy chain constant domain CH1 is called the Fab heavy chain.
  • the first antigen binding module is a Fab molecule, wherein the amino acid at position 124 of the heavy chain constant region can be independently substituted with lysine (K), arginine (R) or histidine (H) (numbering according to Kabat) and the amino acid at position 123 can be independently substituted with lysine (K), arginine (R) or histidine (H) (numbering according to Kabat), and the amino acid at position 147 in the heavy chain constant region CH1 can be independently substituted with glutamic acid (E) or aspartic acid (D) (numbering according to Kabat EU index) and the amino acid at position 213 can be independently substituted with glutamic acid (E) or aspartic acid (D) (numbering according to Kabat EU index).
  • the first antigen binding moiety and the second antigen binding moiety are fused via a linker peptide.
  • the first antigen binding moiety and the second antigen binding moiety are Fab molecules, and the C-terminus of the Fab heavy chain of the first antigen binding moiety is fused to the N-terminus of the Fab heavy chain of the second antigen binding moiety, or the C-terminus of the Fab heavy chain of the second antigen binding moiety is fused to the N-terminus of the Fab heavy chain of the first antigen binding moiety.
  • the linker peptide can adopt a conventional linker peptide sequence, such as a (Gly 4 Ser) 3 sequence.
  • the multispecific antibody or antigen binding molecule thereof of the present invention may further comprise more antigen binding moieties, which may be coupled to the first/second antigen binding moiety.
  • the antigen-binding moiety may be the same as or different from the antigen-binding moiety, for example, it may be an antigen-binding moiety that binds to another antigen.
  • the first/second antigen binding moiety is selected from a murine antibody, a human antibody or a chimeric antibody.
  • the multispecific antibody or antigen-binding molecule thereof comprises a light chain constant region; the light chain constant region is preferably a human light chain constant region; preferably, the light chain constant region is a human ⁇ or ⁇ light chain constant region.
  • the multispecific antibody or antigen-binding molecule thereof comprises a heavy chain constant region; the heavy chain constant region is preferably a heavy chain constant region of human IgG1, 2, 3, or 4.
  • the Fc domain of the heavy chain constant region is a mutant Fc domain.
  • the amino acid residues in the CH3 region of the first subunit of the mutant Fc domain are replaced with amino acid residues having a larger side chain volume, thereby forming a protruding structure;
  • the amino acid residues in the CH3 region of the second subunit of the mutant Fc domain are replaced with amino acid residues having a smaller side chain volume, thereby forming a cavity structure;
  • the cavity structure accommodates the protruding structure so that the first subunit and the second subunit combine to form a heterodimer.
  • the above-mentioned mutant Fc domain may have reduced binding to Fc receptors and/or effector function.
  • the mutation scheme of the mutated Fc domain is selected from: (a) knob mutation T366W, and hole mutation T366S, L368A or Y407V; (b) knob mutation S354C, T366W, and hole mutation Y349C T366S, L368A or Y407V.
  • the addition or deletion of glycosylation sites of an antibody can be facilitated by altering the amino acid sequence, for example, altering the amino acid sequence of the Fc domain, such that one or more glycosylation sites are created or eliminated.
  • the multispecific antibody or antigen-binding molecule thereof of the present invention can simultaneously bind to the GPRC5D protein of the target cell and the antigen CD3 of the activated T cell, so that the target cell and the T cell interact with each other, thereby causing the activation of the cytotoxic T cell and the lysis of the target cell.
  • the multispecific antibody or antigen-binding molecule thereof of the present invention can have cross-reactivity with monkey GPRC5D in terms of species cross-reactivity, which is conducive to subsequent preclinical experiments and facilitates the subsequent development of products with therapeutic uses; in addition, the multispecific antibody or antigen-binding molecule thereof of the present invention has antibody endocytosis activity in cells and is suitable for the development of ADC drugs.
  • the second aspect of the invention provides a nucleic acid molecule encoding a multispecific antibody or antigen-binding molecule thereof as described above.
  • the nucleic acid molecule may be an isolated nucleic acid molecule.
  • the third aspect of the present invention provides a vector comprising the above-mentioned nucleic acid molecule, that is, a vector comprising a nucleic acid molecule encoding the above-mentioned multispecific antibody or its antigen-binding molecule, in particular an expression vector expressing the above-mentioned multispecific antibody or its antigen-binding molecule.
  • vector refers to a nucleic acid delivery vehicle into which a polynucleotide encoding a protein can be inserted and the protein can be expressed.
  • the vector can be transformed, transduced or transfected into a host cell so that the genetic material elements it carries can be expressed in the host cell.
  • the vector can contain a variety of elements that control expression, such as a promoter sequence, a transcription start sequence, an enhancer sequence, a selection element, and a reporter gene.
  • the vector may also contain a replication initiation site.
  • the vector may also include components that assist it in entering the cell, such as viral particles, liposomes, or protein shells, but not only these substances.
  • the vector can be selected from, but not limited to: plasmids, phagemids, cosmids, artificial chromosomes (such as yeast artificial chromosomes YAC, bacterial artificial chromosomes BAC, or P1-derived artificial chromosomes PAC), phages (such as lambda phages or M13 phages), and animal viruses used as vectors, for example, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, papillomaviruses (such as SV40).
  • retroviruses including lentiviruses
  • adenoviruses such as yeast artificial chromosomes YAC, bacterial artificial chromosomes BAC, or P1-derived artificial chromosomes PAC
  • the fourth aspect of the present invention provides a host cell comprising the above nucleic acid molecule or the above vector.
  • the host cell is a eukaryotic cell, preferably a mammalian cell.
  • prokaryotic cells such as Escherichia coli or Bacillus subtilis
  • fungal cells such as yeast cells or Aspergillus
  • insect cells such as S2 Drosophila cells or Sf9
  • animal cell models such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells, etc.
  • the host cell is a HEK293 cell.
  • a method for producing an antibody binding to GPRC5D comprising the following steps: first, culturing the above-mentioned host cell under conditions suitable for the expression of the above-mentioned multispecific antibody or its antigen-binding molecule, and then recovering the multispecific antibody or its antigen-binding molecule.
  • the multispecific antibody or antigen-binding molecule of the present invention can be prepared by using the above-mentioned recombinant It can be produced by the method of hybridoma.
  • aspects of the present invention also provide glycosylation variants of the above-mentioned multispecific antibodies or antigen-binding molecules thereof, antibody variants engineered with cysteine, antibody derivatives, and immunoconjugates.
  • the sixth aspect of the present invention provides a recombinant protein, which includes the above-mentioned multispecific antibody or antigen-binding molecule thereof.
  • the seventh aspect of the present invention provides an immunoconjugate, which comprises the above-mentioned multispecific antibody or antigen-binding molecule thereof.
  • the conjugated part of the immunoconjugate is one or more heterologous molecules, for example, a heterologous molecule with cytotoxicity that can be applied to an immunoconjugate.
  • the eighth aspect of the present invention provides a pharmaceutical composition, wherein the pharmaceutical composition comprises the above-mentioned multispecific antibody or its antigen-binding molecule, or comprises the above-mentioned nucleic acid molecule, or comprises the above-mentioned vector, or comprises the above-mentioned host cell, or comprises the above-mentioned recombinant protein.
  • the ninth aspect of the present invention provides a detection product, wherein the detection product comprises the above-mentioned multispecific antibody or its antigen-binding molecule, or comprises the above-mentioned nucleic acid molecule, or comprises the above-mentioned vector, or comprises the above-mentioned host cell, or comprises the above-mentioned recombinant protein, or comprises the above-mentioned immunoconjugate.
  • the detection product is used to detect the presence or level of GPRC5D in a sample.
  • the detection product includes, but is not limited to, a detection reagent, a detection kit, a detection chip or a test paper, etc.
  • the tenth aspect of the present invention provides the use of the above-mentioned multispecific antibody or antigen-binding molecule, or the above-mentioned nucleic acid molecule, or the above-mentioned vector, or the above-mentioned host cell, or the above-mentioned recombinant protein, or the above-mentioned immunoconjugate, or the above-mentioned pharmaceutical composition in the preparation of a drug for treating or preventing a disease; preferably, the disease is cancer or an autoimmune disease; preferably, the disease is multiple myeloma.
  • the disease is systemic lupus erythematosus and/or rheumatoid arthritis.
  • the present invention relates to a multispecific antibody and an antigen-binding molecule thereof that binds to GPRC5D and CD3 antigens, a nucleic acid molecule encoding the multispecific antibody and the antigen-binding molecule thereof, a vector comprising the nucleic acid molecule, a host cell comprising the vector, a recombinant protein comprising the multispecific antibody and the antigen-binding molecule thereof, and the use of the same in the preparation of a drug for treating or preventing a disease, in particular treating multiple myeloma. Its pharmaceutical uses and its application in testing products.
  • Figure 1 shows the effect of dual antibody GPRC5D ⁇ CD3 on the tumor volume of NCI-H929 xenograft tumors in Example 1;
  • FIG. 2 shows the effect of the dual antibody GPRC5D ⁇ CD3 of Example 1 on the tumor weight of NCI-H929 xenograft tumors.
  • Example embodiments will now be described more fully. However, example embodiments can be implemented in many forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that the present invention will be comprehensive and complete and fully convey the concept of the example embodiments to those skilled in the art.
  • the materials and reagents used in the following examples can be obtained from commercial sources. If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field (for example, refer to "Molecular Cloning Experiment Guide” 3rd edition, written by J. Sambrook et al., translated by Huang Peitang et al., Science Press) or the product instructions are used.
  • the bispecific antibody of Example 1 comprises a first antigen binding moiety and a second antigen binding moiety, wherein the first antigen binding moiety binds to GPRC5D and the second antigen binding moiety binds to CD3.
  • the first antigen binding moiety comprises a heavy chain variable region i-VH and a light chain variable region i-VL, the heavy chain
  • the variable region i-VH comprises i-HCDR1 as shown in SEQ ID NO.1, i-HCDR2 as shown in SEQ ID NO.2 and i-HCDR3 as shown in SEQ ID NO.3;
  • the light chain variable region i-VL comprises i-LCDR1 as shown in SEQ ID NO.4, i-LCDR2 with an amino acid sequence of SAS and i-LCDR3 as shown in SEQ ID NO.5.
  • the second antigen binding module comprises a heavy chain variable region ii-VH and a light chain variable region ii-VL, wherein the heavy chain variable region ii-VH comprises ii-HCDR1 as shown in SEQ ID NO.6, ii-HCDR2 as shown in SEQ ID NO.7 and ii-HCDR3 as shown in SEQ ID NO.8; the light chain variable region ii-VL comprises ii-LCDR1 as shown in SEQ ID NO.9, ii-LCDR2 having an amino acid sequence of GTN and ii-LCDR3 as shown in SEQ ID NO.10.
  • the sequence of the heavy chain variable region i-VH of the first antigen binding module is shown as SEQ ID NO.11; the sequence of the light chain variable region i-VL of the first antigen binding module is shown as SEQ ID NO.12; the sequence of the heavy chain variable region ii-VH of the second antigen binding module is shown as SEQ ID NO.13; the sequence of the light chain variable region ii-VL of the second antigen binding module is shown as SEQ ID NO.14.
  • Example 1 The preparation process of the bispecific antibody GPRC5D ⁇ CD3 is as follows:
  • the sequences of the heavy chain variable region i-VH, light chain variable region i-VL, heavy chain variable region ii-VH and light chain variable region ii-VL were synthesized separately (conventionally synthesized by Sangon Biotech (Shanghai) Co., Ltd.).
  • Expression vector of the first heavy chain clone the heavy chain variable region i-VH by homologous recombination into the pTT5-hIgG1.CH vector containing the hIgG1 heavy chain constant region (which includes the Knob mutation of the Fc domain) to obtain the chimeric antibody expression vector pTT5-iVH-hIgG1.CH (Knob) of the first heavy chain;
  • Expression vector of the second heavy chain The heavy chain variable region ii-VH was cloned into the pTT5-hIgG1.CH vector containing the hIgG1 heavy chain constant region (including the Hole mutation of the Fc domain) by homologous recombination to obtain the chimeric antibody expression vector pTT5-iiVH-hIgG1.CH (Hole) of the second heavy chain;
  • the mutation schemes can be selected from: (a) knob mutation T366W, and hole mutations T366S, L368A or Y407V; (b) knob mutations S354C, T366W, and hole mutations Y349C T366S, L368A or Y407V.
  • the mutation scheme adopted for the mutant Fc domain is the above-mentioned scheme (b).
  • S354C and T366W are used for Knob mutation
  • Y349C, T366S, L368A, and Y407V are used for Hole mutation
  • H435R is used for Hole mutation to reduce the binding of the Hole chain Fc of hIgG1 with ProteinA (ProteinA purification medium used for expression and purification of chimeric antibodies)
  • L234A, L235A, P329G, D356E, and L358M mutations are present in both the Knob mutant Fc and the Hole mutant Fc.
  • Expression vector of the first light chain The light chain variable region i-VL was cloned into the vector pTT5-hKappa.CL containing the human ⁇ light chain constant region by homologous recombination to obtain the chimeric antibody expression vector pTT5-iVL-hKappa.CL of the first light chain;
  • Expression vector of the second light chain The light chain variable region ii-VL was cloned into the vector pTT5-hKappa-CL containing the human ⁇ light chain constant region by homologous recombination to obtain the chimeric antibody expression vector pTT5-iiVL-hKappa.CL of the second light chain;
  • the above-mentioned CrossMab technology was used, that is, the CH1 in the hIgG1 heavy chain constant region in the expression vector of the second heavy chain was replaced with the CL of the human ⁇ light chain constant region, and the CL of the human ⁇ light chain constant region in the expression vector of the second light chain was replaced with the CH1 in the hIgG1 heavy chain constant region.
  • Expression vector of the first heavy chain pTT5-iVH-CH1-hinge-CH2-CH3 (Knob);
  • Expression vector of the second heavy chain pTT5-iiVH-CL-hinge-CH2-CH3 (Hole);
  • Expression vector of the first light chain pTT5-iVL-CL
  • Expression vector of the second light chain pTT5-iiVL-CH1.
  • CH1 and CL in the first heavy chain and the first light chain expression vectors may also be interchanged; generally speaking, if CH1/CL in the first heavy chain/light chain expression vector are interchanged, then CL/CH1 in the second heavy chain/light chain expression vector are also interchanged.
  • 293F cells purchased from ThermoFisher in the logarithmic growth phase with good growth status were collected and inoculated into 1L cell culture flasks and cultured in 300mL culture medium.
  • the four expression vectors (expression vectors of the first heavy chain, the second heavy chain, the first light chain, and the second light chain) obtained above were transfected into 293F cells using the PEI co-transfection method.
  • the cell supernatant on the 7th day after transfection was collected, centrifuged and filtered using a 0.45 ⁇ M filter.
  • the filtered cell supernatant was initially purified using Protein A purification medium to obtain the purified antibody, and then Fractogel EMD COOM cationic medium was used for fine purification; wherein, the equilibrium solution A of the cationic chromatography was 50mM Ac-NaAc buffer, pH 5.3, Cond 3.2 mS/cm, and the eluent B was 50mM Ac-NaAc buffer + 0.5M sodium chloride, pH 5.3, Cond 50 mS/cm.
  • the antibody concentration and purity of the four elution peaks were determined by measuring absorbance using Nanodrop, and the purity was checked by sodium dodecyl sulfate gel electrophoresis and Coomassie staining.
  • the antibody concentration of the first elution peak was determined to be 0.37 mg/mL. From the non-reducing electrophoresis pattern, there were obvious bands at 130 kDa, 95 kDa and 50 kDa, and the purity was about 80%. Preliminary analysis showed that the protein at 130 kDa lacked a light chain; the protein at 95 kDa lacked two light chains and only contained two heavy chains of the antibody fragment; the protein at 50 kDa was a protein fragment of an antibody heavy chain.
  • the antibody concentration of the second elution peak is 0.98 mg/mL. From the non-reducing electrophoresis pattern, there is an obvious band at 130 kDa, and there are bands at 95 kDa and 50 kDa (the bands are not clear), and the purity is about 85%.
  • the antibody concentration of the third elution peak is 0.93 mg/mL. From the non-reducing electrophoresis spectrum, there is an obvious band at 130 kDa and a band at 50 kDa (the band is not obvious), and the purity is about 90%.
  • the antibody concentration of the fourth elution peak was 0.12 mg/mL, and the peak was 130 kDa from the non-reducing electrophoresis spectrum. There are obvious bands at 50kDa and 50kDa, and the contents are higher than those of the first three elution peak proteins, so the purity is about 70%.
  • the binding activity of the four elution peak products (the bispecific antibody GPRC5D ⁇ CD3 of Example 1 of the present invention) collected after the above purification to HEK293-GPRC5D-ZSGreen1 stably transfected cells overexpressing the human GPRC5D antigen was detected by FACS.
  • Positive control bispecific antibody bispecific antibody GC5B596D in patent document US10562968B2; specifically, the inventors prepared the positive control bispecific antibody GC5B596D according to the light/heavy chain variable region sequences recorded in the document and the preparation method of Example 7 in patent US10562968B2.
  • Negative control bispecific antibody The bispecific antibody targeting SARS-CoV-2 ⁇ CD3 was prepared by the preparation method of Example 7 in patent US10562968B2.
  • the antibody sequence targeting SARS-CoV-2 was derived from the neutralizing antibody HTS0483 in patent CN113402602A, and the bispecific antibody HTS0483 ⁇ CD3 was prepared according to the preparation method of Example 7 in patent US10562968B2.
  • the detection method is briefly described as follows: 0.2 ml of HEK293-GPRC5D-ZSGreen1 cell culture medium with a concentration of 2.5 ⁇ 10 6 cells/ml was added to each well of a 96-well V-shaped microplate, centrifuged at 1500r/min for 1 min, and the supernatant was discarded; gradient dilution of antibodies (the above 4 elution peak products, positive control antibody), 50 ⁇ L per well, incubated on ice for 30 min; then 150 ⁇ L PBS was added to each well, centrifuged at 1500r/min for 1 min, the supernatant was discarded, and the plate was washed 4 times; 50uL 200nM CD3E protein (6 ⁇ His tag) was added to each well to resuspend the cells and incubated on ice for 30 min.
  • the EC50 value of elution peak 3 is closest to that of the positive control bispecific antibody, proving that the binding activity of elution peak 3 to stably transfected cells overexpressing human GPRC5D antigen is optimal.
  • the antibody in elution peak 3 is the target product (Example 1 bispecific antibody GPRC5D ⁇ CD3).
  • H929, MM1.S and the above-mentioned stably transfected cells overexpressing human GPRC5D antigen were used as target cells, and human PBMCs were used as effector cells; the efficacy of the bispecific antibody GPRC5D ⁇ CD3 of Example 1 (i.e., elution peak 3 prepared in Example 1) on T-cell-mediated cytotoxicity was determined and analyzed.
  • the specific operation was as follows:
  • RPMI-1640 medium containing 1% FBS as killing test medium. Resuscitate the frozen PBMC effector cells, transfer the PBMC cells to a 15mL centrifuge tube, centrifuge at 1500rpm for 5min, and discard the supernatant. Wash the cells twice with the culture medium, resuspend the cells, count the cells using a cell counter, adjust the effector cell density to 1.25 ⁇ 10 6 /mL, and place in a 37°C, 5% CO 2 incubator for use. Add 5mL of culture medium to a 15mL centrifuge tube, place the frozen target cells in a 37°C water bath for resuscitation, and then transfer to 5mL of culture medium. Centrifuge at 1500rpm for 5min.
  • Discard the supernatant wash the cells twice with the culture medium, resuspend the cells, count them using a cell counter, adjust the target cell density to 1.88 ⁇ 10 5 /mL, and place in a 37°C, 5% CO 2 incubator for use.
  • Antibody dilution The highest final concentration of the bispecific antibody GPRC5D ⁇ CD3 (elution peak 3) and the positive control bispecific antibody (GC5B596D bispecific antibody) in Example 1 was 0.23uM, and they were diluted 4-fold in sequence to obtain 12 gradient concentrations (since 60uL of the antibody was added to 60uL of effector-target mixed cells, when diluting the antibody, the dilution concentration of the antibody needed to be 3 times the final concentration, i.e. 0.7uM).
  • the cytotoxicity results against the target cell MM.1S are as follows:
  • the IC50 value of the dual antibody GPRC5D ⁇ CD3 in Example 1 is 0.9916, and the IC50 value of the positive control dual antibody (GC5B596D dual antibody) is 0.5064.
  • the cytotoxicity results against target cells H929 are as follows:
  • the IC50 value of the dual antibody GPRC5D ⁇ CD3 in Example 1 is 0.002706, and the IC50 value of the positive control dual antibody (GC5B596D dual antibody) is 0.002086.
  • the dual antibody GPRC5D ⁇ CD3 in Example 1 and the positive control dual antibody are active in T cell-mediated killing of MM.1S and H929 target cells, but the killing activity in different cells has certain differences.
  • the killing activity of the dual antibody in Example 1 is better than that of the dual antibody GC5B596D; in T cell-mediated killing of H929 target cells, the killing activity of the dual antibody in Example 1 is slightly worse than that of the dual antibody GC5B596D.
  • Effector cells redirect target cells under the mediation of bispecific antibodies (bispecific antibody GPRC5D ⁇ CD3 in Example 1 and positive control GC5B596D bispecific antibody), while killing target cells, releasing cells H929 cells were used as target cells and human PBMCs were used as effector cells.
  • bispecific antibodies bispecific antibody GPRC5D ⁇ CD3 in Example 1 and positive control GC5B596D bispecific antibody
  • the ELISA method was used to quantitatively detect the secreted cytokine content in the cell culture supernatant, including IL-6, IL2, IFN- ⁇ , and TNF- ⁇ .
  • the cell culture supernatant was collected and placed in a 96-well plate at -20°C for future use.
  • the frozen culture supernatant was taken out, thawed at room temperature, and centrifuged at 3500rpm for 10 minutes to collect the supernatant for ELISA experiment.
  • the kit Human IL-6 ELISA kit, Human IL-2 ELISA kit, Human IFN- ⁇ ELISA Kit, Human TNF- ⁇ ELISA kit.
  • the ELISA detection method for cytokine IL-6 is to add 25uL 2ug/mL of coated antibody to a 384-well ELISA plate and coat overnight at 4°C. Discard the coating solution and add 80uL 2% BSA in PBS to each well for blocking at room temperature for 1.5 hours. Discard the blocking solution and wash the plate 3 times using an automatic plate washer. Dilute the supernatant in the 96-well plate 4 times with 1% FBS 1640 culture medium, transfer 25ul/well to a 384-well plate, duplicate wells, and incubate at room temperature for 2 hours.
  • the ELISA detection method for cytokine IL-2 is to add 25uL 2ug/mL coatingantibody to a 384-well ELISA plate and coat overnight at 4°C. Discard the coating solution and add 80uL 2% BSA in PBS to each well for blocking at room temperature for 1.5 hours. Discard the blocking solution and wash the plate 3 times using an automatic plate washer. Dilute the supernatant in the 96-well plate 4 times with 1% FBS 1640 medium, transfer 25ul/well to a 384-well plate, duplicate wells, and incubate at room temperature for 2 hours.
  • the ELISA detection method for cytokine IFN- ⁇ is to add 25uL 2ug/mL coating antibody to a 384-well ELISA plate and coat it overnight at 4°C. Discard the coating solution and add 80uL 2% BSA in PBS to each well for blocking at room temperature for 1.5 hours. Discard the blocking solution and wash the plate 3 times using an automatic plate washer. Dilute the supernatant in the 96-well plate 4 times with 1% FBS 1640 culture medium, transfer 25ul/well to a 384-well plate, duplicate wells, and incubate at room temperature for 2 hours.
  • the ELISA detection method for cytokine TNF- ⁇ is to add 25uL 2ug/mL coatingantibody to a 384-well ELISA plate and coat overnight at 4°C. Discard the coating solution and add 80uL 2% BSA in PBS to each well for blocking at room temperature for 1.5 hours. Discard the blocking solution and wash the plate 3 times using an automatic plate washer. Dilute the supernatant in the 96-well plate 4 times with 1% FBS 1640 medium, transfer 25ul/well to a 384-well plate, duplicate wells, and incubate at room temperature for 2 hours.
  • the release results of IL6 were as follows: the EC50 value of the dual antibody GPRC5D ⁇ CD3 in Example 1 was 0.01054, and the EC50 value of the positive control dual antibody (GC5B596D dual antibody) was 0.07981.
  • the release results of IL2 were as follows: the EC50 value of the dual antibody GPRC5D ⁇ CD3 in Example 1 was 0.04965, and the EC50 value of the positive control dual antibody (GC5B596D dual antibody) was 0.7283 (approximate value).
  • the release results of IFN- ⁇ were as follows: the EC50 value of the dual antibody GPRC5D ⁇ CD3 in Example 1 was 0.02979, and the EC50 value of the positive control dual antibody (GC5B596D dual antibody) was 0.1291.
  • Example 1 Dual Antibody GPRC5D ⁇ CD3
  • the EC50 value was 0.1741
  • the EC50 value of the positive control dual antibody was 0.8140 (approximate value).
  • the anti-tumor effect of the dual antibody GPRC5D ⁇ CD3 of Test Example 1 was evaluated using the NCI-H929 xenograft tumor model of NOG mice (Shanghai Medicilon Biopharmaceuticals Co., Ltd.) reconstructed with human PBMC.
  • PBS containing 2 ⁇ 10 6 NCI-H929 cells was subcutaneously injected into the right back of 6-8 week old healthy NOG female mice to prepare for tumor bearing, and the size of tumor growth was monitored every three days.
  • human PBMCs frozen in liquid nitrogen were resuscitated and cultured in PRMI-1640 medium containing 10% HIFBS (FBS, 56°C ⁇ 30min), and incubated in a 37°C incubator containing 5% CO2 for 6h. After incubation, hPBMCs were collected and resuspended in PBS buffer to adjust the cell concentration to 2.5 ⁇ 10 7 /mL. Under sterile conditions, 200uL of cell suspension was intraperitoneally injected into the mouse, and the injection concentration was 5 ⁇ 10 6 PBMC cells.
  • mice When the tumor volume of the tumor-bearing mice reached about 100 mm3 , the mice were randomly divided into groups so that the difference in tumor volume between groups was less than 10% of the mean, and the drugs were administered according to the weight of the animals, and the drugs were administered through the tail vein twice a week for a total of 5 times.
  • Positive control group administration of positive control dual antibody GC5B596D, dosage: 6ug per mouse (marked as GC5B596D, 6ug/mouse in Figures 1 and 2);
  • Vehicle control group PBS was administered, and the dosage was 6ug per mouse (marked as PBS vehicle control in Figures 1 and 2);
  • FIG. 1 shows the effect of the dual antibody GPRC5D ⁇ CD3 in Example 1 on the tumor volume of NCI-H929 xenograft tumors
  • FIG. 2 shows the effect of the dual antibody GPRC5D ⁇ CD3 in Example 1 on the tumor weight of NCI-H929 xenograft tumors.
  • both the bispecific antibody GPRC5D ⁇ CD3 of Example 1 and the positive control bispecific antibody GC5B596D have significant anti-tumor effects on NCI-H929 xenograft tumors in PBMC humanized mice.
  • the average tumor volume of the vehicle control group was 1585.65 ⁇ 144.58 mm 3 .
  • the bispecific antibody GPRC5D ⁇ CD3 of Example 1 had a very significant tumor inhibition effect at both the low dose of 1.5ug/mouse and the high dose of 6ug/mouse, with average tumor volumes of 64.46 ⁇ 12.46mm 3 and 59.67 ⁇ 6.02mm 3 , respectively, and tumor inhibition rates of 95.93% (P ⁇ 0.001) and 96.24% (P ⁇ 0.001), respectively.
  • the positive control dual antibody GC5B596D showed significant anti-tumor effects, with an average tumor volume of 396.10 ⁇ 287.62 mm 3 and a tumor inhibition rate of 75.02% (P ⁇ 0.01).
  • the in vitro tumor weight data were well consistent with the tumor volume data.

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Abstract

提供一种结合GPRC5D和CD3抗原的多特异性抗体及其抗原结合分子，编码该多特异性抗体及其抗原结合分子的核酸分子，包含该核酸分子的载体，包含该载体的宿主细胞，包含该多特异性抗体及其抗原结合分子的重组蛋白或免疫缀合物，以及它们在在制备治疗或预防疾病，特别是治疗多发性骨髓瘤的药物方面的用途，以及在检测产品方面的应用。

Description

一种抗GPRC5D的多特异性抗体 技术领域
本发明涉及一种抗GPRC5D的多特异性抗体，属于生物医药领域。
背景技术
多发性骨髓瘤(Multiple Myeloma，MM)是全球仅次于非霍奇金氏淋巴瘤的第二常见血液肿瘤，约占血液系统恶性肿瘤的10％。根据WHO发布的全球癌症统计报告，2018年中国新发多发性骨髓瘤患者20,066例，死亡患者14,665例；5年累计发病人数44,643例。并且，患者的发病率和死亡率随着年龄增长而逐步上升，随着我国人口老龄化程度不断加剧，患病总人数也将逐渐增多，临床需求巨大。
目前我国针对多发性骨髓瘤的治疗方案可分为三类：免疫调节剂、蛋白酶体抑制剂和生物分子靶向性疗法。其中，免疫调节剂和蛋白酶体抑制剂主要用于符合干细胞移植条件的患者在移植前接受的一线联合治疗及移植后的维持治疗；例如，近十年来，以沙利度胺及其衍生物来那度胺为代表的免疫调节剂和以硼替佐米为代表的小分子蛋白酶体抑制剂的应用，极大地提高了MM患者的缓解率和生存期。
针对MM的生物分子靶向药物，主要涉及三大靶点：浆细胞表面蛋白CD38、信号淋巴细胞激活分子家族成员7(Signaling Lymphocytes Activating Molecule Factor 7，SLAMF7)和B细胞成熟抗原(B Cell Maturation Antigen，BCMA)，
其中，抗CD38单抗(例如，2019年国家药监局(NMPA)有条件批准并进口注册上市的Daratumumab)主要用于一线治疗复发或二线治疗难治性多发性骨髓瘤。
此外，BCMA蛋白因其在MM细胞上的高表达特异性也成为本领域的研究热点。2020年8月美国FDA加速批准了靶向BMCA的抗体偶联药物(Antibody-Drug Conjugate，ADC)Blenrep用于治疗中位7线复发的患者； 该药总体缓解率仍能达到31％，中位缓解持续时间(DoR)大于6个月。此外，靶向BCMA的CAR-T或CD3双特异性抗体通过介导T细胞起到的抗肿瘤疗效亦然显著。
然而，虽然靶向BCMA的ADC、双抗及CAR-T疗法已显示出了积极的临床效果，但有关BCMA阴性(或低表达)及相关的治疗后复发病例已见报道，MM的复发性和难治性仍然是MM临床治疗的难点，寻找新的有效靶点仍然是本领域亟待解决的问题。
已有研究报道，GPRC5D的高表达与多发性骨髓瘤的不良预后相关(Atamaniuk,J.,et al.,Overexpression of G protein-coupled receptor 5D in the bone marrow is associated with poor prognosis in patients with multiple myeloma.European Journal of Clinical Investigation,2012.42(9):p.953-960)。
GPRC5D属于G蛋白偶联受体(G protein-coupled receptor，GPCR)家族；具体的，其为G蛋白偶联受体C5家族亚型D，是最早于2001年鉴定出的孤儿非典C类GPCR(Brauner-Osborne,H.,et al.Cloning and characterization of a human orphan family C G-protein coupled receptor GPRC5D.Biochim Biophys Acta,2001.1518(3):p.237-48)。
虽然，GPRC5D先前已在多发性骨髓瘤患者的细胞中被鉴定发现，但因为缺少蛋白表达谱研究，一直未被应用于临床开发。直至2019年的研究报道显示，GPRC5D在多发性骨髓瘤的浆细胞中高表达，在正常组织中多不表达，仅在具有免疫赦免性的毛囊区域有表达(Smith,E.L.,et al.,GPRC5D is a target for the immunotherapy of multiple myeloma with rationally designed CAR T cells.Science Translational Medicine,2019.11(485))。更为让人惊讶的发现是，GPRC5D的表达谱与BCMA并不重叠——该发现让本领域研究人员意识到GPRC5D有望成为替代BCMA靶向治疗、或者治疗BCMA低/不表达的患者或者，承继BCMA治疗复发后的全新治疗靶点。
发明内容
本发明的发明人基于GPRC5D抗原开发了一种新的特异性结合GPRC5D的抗体及其抗原结合分子，并进一步开发了结合靶细胞上的GPRC5D抗原和T细胞上的活化性T细胞抗原(诸如CD3)的双/多特异性 抗体及其抗原结合分子；本发明的双/多特异性抗体及其抗原结合分子同时结合靶细胞和T细胞并使得它们相互作用，从而引起细胞毒性T细胞活化，靶细胞被裂解。
本发明的第一个方面提供了一种多特异性抗体或其抗原结合分子，其中，
所述多特异性抗体或其抗原结合分子包含第一抗原结合模块和第二抗原结合模块；
其中，所述第一抗原结合模块结合GPRC5D，所述第二抗原结合模块结合CD3或CD3ε；
所述第一抗原结合模块包含重链可变区i-VH和轻链可变区i-VL，所述重链可变区i-VH包含如SEQ ID NO.1所示的i-HCDR1、如SEQ ID NO.2所示的i-HCDR2和如SEQ ID NO.3所示的i-HCDR3；所述轻链可变区i-VL包含如SEQ ID NO.4所示的i-LCDR1、氨基酸序列为SAS的i-LCDR2和如SEQ ID NO.5所示的i-LCDR3；
所述第二抗原结合模块包含重链可变区ii-VH和轻链可变区ii-VL，所述重链可变区ii-VH包含如SEQ ID NO.6所示的ii-HCDR1、如SEQ ID NO.7所示的ii-HCDR2和如SEQ ID NO.8所示的ii-HCDR3；所述轻链可变区ii-VL包含如SEQ ID NO.9所示的ii-LCDR1、氨基酸序列为GTN的ii-LCDR2和如SEQ ID NO.10所示的ii-LCDR3。
在本发明的一个更优选实施方案中，所述第一抗原结合模块的重链可变区i-VH的序列如SEQ ID NO.11所示，或者其与SEQ ID NO.11所示序列有80％以上的序列同源性。例如，所述第一抗原结合模块的重链可变区i-VH的序列与SEQ ID NO.11所示序列具有80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的序列同源性。
在本发明的另一个更优选实施方案中，所述第一抗原结合模块的轻链可变区i-VL的序列如SEQ ID NO.12所示，或者其与SEQ ID NO.12所示序列有80％以上的序列同源性。例如，所述第一抗原结合模块的轻链可变区i-VL的序列与SEQ ID NO.12所示序列具有80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的序列同源性。
在本发明的一个更优选实施方案中，所述第二抗原结合模块的重链可变区ii-VH的序列如SEQ ID NO.13所示，或者其与SEQ ID NO.13所示序列有80％以上的序列同源性。例如，所述第二抗原结合模块的重链可变区ii-VH的序列与SEQ ID NO.13所示序列具有80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的序列同源性。
在本发明的另一个更优选实施方案中，所述第二抗原结合模块的轻链可变区ii-VL的序列如SEQ ID NO.14所示，或者其与SEQ ID NO.14所示序列有80％以上的序列同源性。例如，所述第二抗原结合模块的轻链可变区ii-VL的序列与SEQ ID NO.14所示序列具有80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的序列同源性。
关于氨基酸序列的“序列同源性”的百分比，是通过确定两个序列中存在的氨基酸残基的数目来产生匹配位置的数目，将匹配位置的数目除以比较窗口中的位置总数，将结果乘以100从而产生序列的同源性百分比。序列同源性，也被称为序列同一性。
在本发明的一个具体实施方案中，上述第一抗原结合模块的重链可变区i-VH和轻链可变区i-VL可以分别在SEQ ID NO.11和SEQ ID NO.12所示序列的基础之上进行少量氨基酸的缺失、插入或者氨基酸突变，获得同源性在80％以上的氨基酸序列。少量氨基酸的置换(缺失或插入，或者氨基酸突变，或者相似氨基酸的替代)，特别是在构架区部分的保守氨基酸置换所获得的变体，保留了重链可变区和轻链可变区原有的性质和功能，即特异性结合GPRC5D的抗体性质和功能，那么，这些变体也落入本发明的保护范围之内。同理，上述第二抗原结合模块的重链可变区ii-VH和轻链可变区ii-VL可以分别在SEQ ID NO.13和SEQ ID NO.14所示序列的基础之上进行少量氨基酸的缺失、插入或者氨基酸突变，获得同源性在80％以上的氨基酸序列，只要保留了重链可变区和轻链可变区原有的性质和功能，即特异性结合CD3或CD3ε的抗体性质和功能，那么，这些变体也落入本发明的保护范围之内。
其中，上述的“构架区”，是指位于CDR之间的氨基酸序列，包括重链可变区的构架区和轻链可变区的构架区。
在本发明的一个具体实施方案中，所述第一抗原结合模块选自Fv、Fab、F(ab’)2、Fab’、dsFv、scFv、sc(Fv)2或单链抗体中的任意一种；所述第二抗原结合模块选自Fv、Fab、F(ab’)2、Fab’、dsFv、scFv、sc(Fv)2或单链抗体中的任意一种。
在本发明的一个具体实施方案中，所述第一抗原结合模块和/或第二抗原结合模块是Fab分子。在本发明的一个优选实施方案中，所述第一抗原结合模块和所述第二抗原结合模块是Fab分子。
Fab分子是指有Fab重链的VH和CH1结构域以及Fab轻链的VL和CL结构域组成的蛋白质分子。常规的Fab分子是指天然形式的Fab分子，其中Fab重链从N到C端为VH-CH1，而Fab轻链从N到C端为VL-CL。
可选的，在同一个Fab分子中，Fab重链可变区与Fab轻链可变区可以相互交换/替换；或者说，在同一个Fab分子中，Fab重链恒定区与Fab轻链恒定区可以相互交换/替换。例如Fab分子包含由VL-CH1(N到C端)构成的肽链，以及由VH-CL(N到C端)构成的肽链。通常为了便于表述，包含重链恒定域CH1的肽链被称为Fab重链。
在另一个具体实施方案中，该第一抗原结合模块是Fab分子，其中在重链恒定区位置124处的氨基酸可以用赖氨酸(K)、精氨酸(R)或组氨酸(H)独立替代的(编号方式依照Kabat)且位置123处的氨基酸可以用赖氨酸(K)、精氨酸(R)或组氨酸(H)独立替代的(编号方式依照Kabat)，且在重链恒定区CH1中位置147处的氨基酸可以用谷氨酸(E)或天冬氨酸(D)独立替代的(编号方式依照Kabat EU索引)且位置213处的氨基酸是用谷氨酸(E)或天冬氨酸(D)独立替代的(编号方式依照Kabat EU索引)。
在本发明的一个替代实施方案中，所述第一抗原结合模块与所述第二抗原结合模块之间通过接头肽融合。优选的，所述第一抗原结合模块与所述第二抗原结合模块是Fab分子，并且第一抗原结合模块的Fab重链的C端融合至第二抗原结合模块Fab重链的N端，或者，第二抗原结合模块的Fab重链的C端融合至第一抗原结合模块Fab重链的N端。其中，所述接头肽可以采用常规的接头肽序列，例如(Gly4Ser)3序列。
在本发明的一个替代实施方案中，本发明的多特异性抗体或其抗原结合分子还可以包含更多的抗原结合模块，它们可以与第一/第二抗原结合模块相 同，也可以不同，例如可以是结合其他抗原的抗原结合模块。
在本发明的一个替代实施方案中，第一/第二抗原结合模块选自鼠源抗体、人源抗体或嵌合抗体。
在本发明的一个优选实施方案中，所述多特异性抗体或其抗原结合分子包括轻链恒定区；所述轻链恒定区优选人轻链恒定区；较佳的，所述轻链恒定区为人λ或κ轻链恒定区。
在本发明的一个优选实施方案中，所述多特异性抗体或其抗原结合分子包括重链恒定区；所述重链恒定区优选人IgG1、2、3、4的重链恒定区。
较佳的，所述重链恒定区的Fc结构域为突变Fc结构域。
较佳的，所述突变Fc结构域的第一亚基的CH3区中的氨基酸残基采用具有较大侧链体积的氨基酸残基替换，从而形成隆起结构；所述突变Fc结构域的第二亚基的CH3区中的氨基酸残基采用具有较小侧链体积的氨基酸残基替换，从而形成空腔结构；所述空腔结构容纳所述隆起结构从而使得所述所述第一亚基和第二亚基结合形成异二聚体。
在本发明的另一个实施方案中，上述的突变Fc结构域，其对于Fc受体的结合和/或效应器功能可以是降低的。
较佳的，所述突变Fc结构域的突变方案选自：(a)knob突变T366W，以及hole突变T366S、L368A或Y407V；(b)Knob突变S354C、T366W，以及hole突变Y349C T366S、L368A或Y407V。
在本发明的某些实施方案中，可以通过改变氨基酸序列，例如改变Fc结构域的氨基酸序列，使得创建或消除一个或多个糖基化位点来方便实现抗体的糖基化位点的添加或删除。
本发明的多特异性抗体或其抗原结合分子，能够同时结合靶细胞的GPRC5D蛋白和活化性T细胞的抗原CD3，使得靶细胞和T细胞相互作用，从而引起细胞毒性T细胞活化，靶细胞被裂解。
在本发明的一个具体实施方案中，本发明的多特异性抗体或其抗原结合分子，在物种交叉反应方面，能够与猴GPRC5D具有交叉反应性，利于开展后续的临床前实验，便于后续有治疗用途的产品开发；此外，本发明的多特异性抗体或其抗原结合分子在细胞中具有抗体内吞活性，适合ADC药物的开发。
发明的第二个方面提供了一种核酸分子，所述核酸分子编码如上述的多特异性抗体或其抗原结合分子的核酸分子。
在本发明的一个具体实施方案中，所述核酸分子可以为分离的核酸分子。
本发明的第三个方面提供了包含上述核酸分子的载体，即包含编码上述多特异性抗体或其抗原结合分子的核酸分子的载体，特别是表达上述的多特异性抗体或其抗原结合分子的表达载体。
术语“载体”一词指的是，可将编码某蛋白的多聚核苷酸插入其中并使该蛋白获得表达的一种核酸运载工具。载体可通过转化、转导或转染宿主细胞，使其携带的遗传物质元件在宿主细胞内得以表达。载体可以包含多种控制表达的元件，例如启动子序列、转录起始序列、增强子序列、选择元件及报告基因等。另外，载体还可含有复制起始位点。载体还有可能包括协助其进入细胞的成分，如病毒颗粒、脂质体或蛋白外壳，但不仅仅只有这些物质。在本发明的实施方案中，载体可以选自，但不限于：质粒、噬菌粒、柯斯质粒、人工染色体(如酵母人工染色体YAC、细菌人工染色体BAC或P1来源的人工染色体PAC)、噬菌体(如λ噬菌体或M13噬菌体)以及用作载体的动物病毒，例如，逆转录病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。
本发明的第四个方面提供了包含上述核酸分子或上述载体的宿主细胞。
在本发明的一个具体实施方案中，所述宿主细胞为真核细胞，优选的哺乳动物细胞。
关于“宿主细胞”，可以选择，但不限于：大肠杆菌或枯草菌等原核细胞，酵母细胞或曲霉菌等真菌细胞，S2果蝇细胞或Sf9等昆虫细胞，或者纤维原细胞、CHO细胞、COS细胞、NSO细胞、HeLa细胞、BHK细胞、HEK293细胞等动物细胞模型。
在本发明的一个优选实施例中，所述宿主细胞为HEK293细胞。
在本发明第五方面还提供了一种生产结合GPRC5D抗体的方法，其包括以下步骤：首先在适合于上述的多特异性抗体或其抗原结合分子表达的条件下培养上述的宿主细胞，然后回收该多特异性抗体或其抗原结合分子。
在本发明的上述的多特异性抗体或其抗原结合分子可以采用上述的重组 的方式进行生产，也可以采用杂交瘤的方式进行生产。
本发明的其他方面还提供了上述的多特异性抗体或其抗原结合分子的糖基化变体、经半胱氨酸工程化改造的抗体变体、抗体衍生物以及免疫缀合物等。
本发明的第六方面提供了一种重组蛋白，该重组蛋白包括上述的多特异性抗体或其抗原结合分子。
本发明的第七方面提供了一种免疫缀合物，该免疫缀合物包括上述的多特异性抗体或其抗原结合分子。
优选的，所述免疫缀合物的缀合部分采用1个或多个异源分子，例如采用可以应用于免疫缀合物的、具有细胞毒性的异源分子。
本发明的第八个方面提供了一种药物组合物，其中，所述药物组合物包含上述的多特异性抗体或其抗原结合分子，或者包含上述的核酸分子，或者包含上述载体，或者包含上述宿主细胞，或者包含上述的重组蛋白。
本发明的第九个方面提供了一种检测产品，其中，所述检测产品包含上述的多特异性抗体或其抗原结合分子，或者包含上述的核酸分子，或者包含上述载体，或者包含上述宿主细胞，或者包含上述的重组蛋白，或者包含上述免疫缀合物。
所述检测产品用于检测GPRC5D在样品中的存在或水平。
在本发明的一个具体实施方案中，所述检测产品包括，但不限于，检测试剂、检测试剂盒、检测芯片或试纸等。
本发明的第十个方面提供了上述的多特异性抗体或其抗原结合分子，或者上述的核酸分子，或者上述载体，或者上述宿主细胞，或者上述的重组蛋白，或者上述的免疫缀合物，或者上述药物组合物，在制备治疗或预防疾病的药物方面的用途；较佳的，所述疾病为癌症或自身免疫疾病；较佳的，所述疾病为多发性骨髓瘤。较佳的，所述疾病为系统性红斑狼疮和/或类风湿性关节炎。
本发明涉及一种结合GPRC5D和CD3抗原的多特异性抗体及其抗原结合分子，编码该多特异性抗体及其抗原结合分子的核酸分子，包含该核酸分子的载体，包含该载体的宿主细胞，包含该多特异性抗体及其抗原结合分子的重组蛋白，以及它们在在制备治疗或预防疾病，特别是治疗多发性骨髓瘤 的药物方面的用途，以及在检测产品方面的应用。
附图说明
图1为实施例1双抗GPRC5D×CD3对NCI-H929异种移植瘤肿瘤体积的影响；
图2为实施例1双抗GPRC5D×CD3对NCI-H929异种移植瘤肿瘤重量的影响。
具体实施方式
以下将结合具体实施方式对本发明进行详细描述。但这些实施方式并不限制本发明，本领域的普通技术人员根据这些实施方式所做出的结构、方法、或功能上的变换均包含在本发明的保护范围内。
现在将更全面地描述示例实施方式。然而，示例实施方式能够以多种形式实施，且不应被理解为限于在此阐述的实施方式；相反，提供这些实施方式使得本发明将全面和完整，并将示例实施方式的构思全面地传达给本领域的技术人员。
下面实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件(例如参考J.萨姆布鲁克等著，黄培堂等译的《分子克隆实验指南》第三版，科学出版社)或按照产品说明书进行。
关于人GPRC5D抗原表达载体pLVX-huGPRC5D-IRES-ZSGreen1和pTT5-huGPRC5D的制备，以及过表达人GPRC5D抗原的HEK293稳转细胞株的制备，具体参见本申请发明人于2021年5月23日向国家知识产权局递交的申请号为202110561819.5的专利申请书实施例1部分的内容，具体不再赘述。
实施例1
实施例1的双特异性抗体包括第一抗原结合模块和第二抗原结合模块，第一抗原结合模块结合GPRC5D，所述第二抗原结合模块结合CD3。
第一抗原结合模块包含重链可变区i-VH和轻链可变区i-VL，所述重链 可变区i-VH包含如SEQ ID NO.1所示的i-HCDR1、如SEQ ID NO.2所示的i-HCDR2和如SEQ ID NO.3所示的i-HCDR3；所述轻链可变区i-VL包含如SEQ ID NO.4所示的i-LCDR1、氨基酸序列为SAS的i-LCDR2和如SEQ ID NO.5所示的i-LCDR3。
第二抗原结合模块包含重链可变区ii-VH和轻链可变区ii-VL，所述重链可变区ii-VH包含如SEQ ID NO.6所示的ii-HCDR1、如SEQ ID NO.7所示的ii-HCDR2和如SEQ ID NO.8所示的ii-HCDR3；所述轻链可变区ii-VL包含如SEQ ID NO.9所示的ii-LCDR1、氨基酸序列为GTN的ii-LCDR2和如SEQ ID NO.10所示的ii-LCDR3。
具体在本实施例中，第一抗原结合模块的重链可变区i-VH的序列如SEQ ID NO.11所示；所述第一抗原结合模块的轻链可变区i-VL的序列如SEQ ID NO.12所示；所述第二抗原结合模块的重链可变区ii-VH的序列如SEQ ID NO.13所示；所述第二抗原结合模块的轻链可变区ii-VL的序列如SEQ ID NO.14所示。
具体序列如下表1和表2所示。
表1
表2
关于实施例1双特异性抗体的构建和制备，可以参考CrossMab技术(例如文献CN101896504B所采用的)和Knob-in-Hole技术(例如文献CN1176659A所采用的)。文献CN101896504B和CN1176659A在此并入本文。
实施例1双特异性抗体GPRC5D×CD3的制备过程如下：
1、双特异性抗体表达载体的构建
分别合成上述重链可变区i-VH、轻链可变区i-VL、重链可变区ii-VH和轻链可变区ii-VL的序列(由生工生物工程(上海)股份有限公司常规合成)。
附注：重链可变区i-VH和轻链可变区i-VL的序列与发明人开发的抗体“zw.HTS0375Z56”的对应序列一致(参见本申请发明人于2021年5月23日向国家知识产权局递交的申请号为202110561819.5的专利申请书)。
1)第一重链的表达载体：通过同源重组的方式将重链可变区i-VH克隆 到含有hIgG1重链恒定区(其中包含Fc结构域的Knob突变)的pTT5-hIgG1.CH的载体中获得第一重链的嵌合抗体表达载体pTT5-iVH-hIgG1.CH(Knob)；
2)第二重链的表达载体：通过同源重组的方式将重链可变区ii-VH克隆到含有hIgG1重链恒定区(其中包含Fc结构域的Hole突变)的pTT5-hIgG1.CH的载体中获得第二重链的嵌合抗体表达载体pTT5-iiVH-hIgG1.CH(Hole)；
关于上述第1)和2)的突变Fc结构域，其突变方案可以选自：(a)knob突变T366W，以及hole突变T366S、L368A或Y407V；(b)Knob突变S354C、T366W，以及hole突变Y349C T366S、L368A或Y407V。
在本实施例中，采用的突变Fc结构域采用的突变方案是上述的(b)方案。具体的，在本实施例中，Knob突变采用S354C和T366W，Hole突变采用Y349C、T366S、L368A、Y407V，为降低hIgG1的Hole链Fc与ProteinA(嵌合抗体的表达纯化所采用的ProteinA纯化介质)的结合，Hole突变采用H435R；此外，具体在本实施例中，为降低Fc结构域的效应，Knob突变的Fc和Hole突变的Fc中均存在L234A、L235A、P329G、D356E和L358M突变。
3)第一轻链的表达载体：通过同源重组的方式将轻链可变区i-VL克隆到含有人κ轻链恒定区的pTT5-hKappa.CL的载体中获得第一轻链的嵌合抗体表达载体pTT5-iVL-hKappa.CL；
4)第二轻链的表达载体：通过同源重组的方式将轻链可变区ii-VL克隆到含有人κ轻链恒定区的pTT5-hKappa-CL的载体中获得第二轻链的嵌合抗体表达载体pTT5-iiVL-hKappa.CL；
具体在本实施例中，为防止轻链错配，采用了上述的CrossMab技术，即将上述第二重链的表达载体中的hIgG1重链恒定区中的CH1替换成人κ轻链恒定区的CL，将上述第二轻链的表达载体中的人κ轻链恒定区的CL替换成hIgG1重链恒定区中的CH1。
为清楚展示，上述第1)-4)获得的表达载体如下所示：
第一重链的表达载体：pTT5-iVH-CH1-hinge-CH2-CH3(Knob)；
第二重链的表达载体：pTT5-iiVH-CL-hinge-CH2-CH3(Hole)；
第一轻链的表达载体：pTT5-iVL-CL
第二轻链的表达载体：pTT5-iiVL-CH1。
在实施例1的替代实施例中，第一重链、第一轻链表达载体中的CH1和CL也可以互换位置；一般来说，若第一重链/轻链表达载体中的CH1/CL互换，则若第二重链/轻链表达载体中的CL/CH1也互换位置。
2、双特异性抗体表达载体的表达和纯化
收集生长状态良好的对数增长期的293F细胞(购自ThermoFisher)接种至1L细胞培养瓶中并在300mL培养基中培养，采用PEI共转染法，将上述获得的四个表达载体(第一重链、第二重链、第一轻链、第二轻链的表达载体)，质粒各75μg，转染293F细胞。收集转染后培养第7天的细胞上清，离心并使用0.45μM滤器过滤。
对过滤后的细胞上清使用Protein A纯化介质初步纯化，获得纯化后的抗体，再使用Fractogel EMD COOM阳离子介质进行精纯操作；其中，阳离子层析的平衡液A为50mMHAc-NaAc缓冲液，pH5.3，Cond 3.2 mS/cm，洗脱液B为50mMHAc-NaAc缓冲液+0.5M氯化钠，pH5.3，Cond 50 mS/cm。上样完毕后至少冲洗5个柱体积平衡液A，后以100％洗脱液B线性洗脱20个柱体积，按照洗脱峰峰型切峰收集，收集到了4个洗脱峰产物。
分别通过Nanodrop测定吸光度来确定4个洗脱峰产物的抗体浓度和纯度，并通过十二烷基硫酸钠凝胶电泳和考马斯染色来检查纯度。
经测定，第一个洗脱峰的抗体浓度是0.37mg/mL，从非还原电泳图谱中在130kDa、95kDa和50kDa处都有明显的条带，纯度在80％左右。初步分析130kDa处的蛋白是缺少一个轻链；95kDa处的蛋白是缺少两个轻链，只含有两个重链的抗体片段；50kDa处的蛋白是一个抗体重链的蛋白片段。
第二个洗脱峰的抗体浓度是0.98mg/mL，从非还原电泳图谱中在130kDa处有明显的条带，在95kDa和50kDa处均有条带(条带不清晰)，纯度在85％左右。
第三个洗脱峰的抗体浓度是0.93mg/mL，从非还原电泳图谱中在130kDa有明显条带，在50kDa处有条带(条带不明显)，纯度在90％左右。
第四个洗脱峰的抗体浓度是0.12mg/mL，从非还原电泳图谱中在130kDa 和50kDa处都有明显的条带，且含量均比前三个洗脱峰蛋白的含量高，因此纯度在70％左右。
效果数据
一、实施例1的双特异性抗体与过表达人GPRC5D抗原稳转细胞的结合活性检测
通过FACS的方法，检测上述纯化后收集到的4个洗脱峰产物(本发明实施例1的双特异性抗体GPRC5D×CD3)与过表达人GPRC5D抗原的HEK293-GPRC5D-ZSGreen1稳转细胞的结合活性。
阳性对照双抗：专利文献US10562968B2中的双特异性抗体GC5B596D；具体的，发明人根据该文献中记载的轻/重链可变区序列，并按照专利US10562968B2中实施例7的制备方法制备获得了阳性对照双特异性抗体GC5B596D。
阴性对照双抗：采用专利US10562968B2中实施例7的制备方法制备靶向SARS-CoV-2×CD3的双特异性抗体，靶向SARS-CoV-2的抗体序列来源于专利CN113402602A中的中和抗体HTS0483，并按照专利US10562968B2中实施例7的制备方法制备获得双特异性抗体HTS0483×CD3。检测方法简述如下：每孔加入0.2ml，浓度为2.5×106个/ml的HEK293-GPRC5D-ZSGreen1细胞培养液于96孔V型微孔板中，1500r/min离心1min，弃上清；梯度稀释抗体(上述的4个洗脱峰产物、阳性对照抗体)，每孔50μL，冰上孵育30min；然后每孔加入150μL PBS，1500r/min离心1min，弃上清，重复洗板4次；每孔加入50uL 200nM CD3E蛋白(6×His标签)重悬细胞，冰上孵育30min。然后每孔加入150μL PBS，1500r/min离心1min，弃上清，重复洗板4次。加入APC标记的Anti-His二抗(Jackson，货号109-605-098，PBS 1:1000稀释)，每孔50μL，冰上孵育30min。用PBS洗板4次后每孔加入100μL PBS重悬细胞，用CytoFLEX进行检测(Beckman)，并对检测结果进行GraphPad8.0.2分析，结果参见下表1。
表1

从表1中可以看出，洗脱峰3的EC50值与阳性对照双抗的最为接近，证明了洗脱峰3与过表达人GPRC5D抗原稳转细胞的结合活性最优。综合上述的洗脱峰浓度、SDS-PAGE胶图结果，以及与GPRC5D抗原的结合活性结果可以确定：洗脱峰3的抗体是目标产物(实施例1双特异性抗体GPRC5D×CD3)。
二、实施例1的双特异性抗体的体外T细胞依赖性细胞毒性
使用H929，MM1.S和上述的过表达人GPRC5D抗原稳转细胞作为靶细胞，人PBMC作为效应细胞；测定、分析实施例1的双特异性抗体GPRC5D×CD3(即实施例1制备获得的洗脱峰3)对T-细胞介导的细胞毒性的效力。具体操作如下：
配制含有1％FBS的RPMI-1640培养基作为杀伤检测培养基。将冻存的PBMC效应细胞复苏，将PBMC细胞转移至15mL离心管中，1500rpm离心5min，弃上清。使用培养基洗细胞两遍，将细胞重悬，使用细胞计数仪对细胞计数，调整效应细胞密度为1.25×106/mL，放入37℃，5％CO2培养箱备用。在15mL离心管中加入5mL培养基，将冻存靶细胞置于37℃水浴锅进行复苏后转移至5mL培养基中。1500rpm离心5min。弃上清，使用培养基洗细胞两遍，将细胞重悬，使用细胞计数仪计数，调整靶细胞密度为1.88×105/mL，放入37℃，5％CO2培养箱备用。
抗体稀释：实施例1双抗GPRC5D×CD3(洗脱峰3)和阳性对照双抗(GC5B596D双抗)最高终浓度为0.23uM，依次4倍梯度稀释，获得12个梯度浓度(由于抗体是60uL加入到60uL效靶混合细胞中，所以稀释抗体时，抗体的稀释浓度需要是终浓度的3倍，即0.7uM)。
将备用的PBMC效应细胞与靶细胞取出，各取60uL/well加入96孔板中进行培养；使用排枪，将稀释后的抗体取60uL加入96孔板中，进行孵育。显微镜下观察细胞铺匀状态，对照孔中补加60uL培养基。将其放入37℃，5％CO2培养箱培养24h。在共培养结束前45分钟，向靶细胞最大LDH释放孔加入20uL 10×裂解液，混匀，37℃、5％CO2条件下培养细胞45分钟。将共培养后的细胞，3000rpm离心2分钟。转移10uL上清液至新的384孔酶标板中，每孔加入10uLCytoTox96试剂，1000rpm离心1分钟，shake1分钟。室温避光孵育30分钟，最大读数控制在1-2之间。每孔加入10uL终止液，shake 1分钟。记录发光值，保存原始读数并导出Excel读值整理实验结果。
针对靶细胞MM.1S的细胞毒性结果如下：
实施例1双抗GPRC5D×CD3的IC50值为0.9916，阳性对照双抗(GC5B596D双抗)的IC50值为0.5064。
针对靶细胞H929的细胞毒性结果如下：
实施例1双抗GPRC5D×CD3的IC50值为0.002706，阳性对照双抗(GC5B596D双抗)的IC50值为0.002086。
对比上述结果可以看出，实施例1双抗GPRC5D×CD3和阳性对照双抗(GC5B596D双抗)在T细胞介导的MM.1S和H929靶细胞杀伤中都有活性，但是在不同的细胞中杀伤活性具有一定的差异。在T细胞介导的MM.1S靶细胞杀伤中，实施例1双抗的杀伤活性优于GC5B596D双抗的；在T细胞介导的H929靶细胞杀伤中，实施例1双抗的杀伤活性略差于GC5B596D双抗的。
三、实施例1的双特异性抗体的体外效应细胞细胞因子释放评估
效应细胞在双特异抗体(实施例1双抗GPRC5D×CD3和阳性对照GC5B596D双抗)的介导下重定向靶细胞，在杀伤靶细胞的同时，释放细胞 因子。使用H929作为靶细胞，人PBMC作为效应细胞。
通过ELISA的方法定量检测细胞培养上清中的所分泌的细胞因子含量，包括IL-6、IL2、IFN-γ、TNF-α。
在体外杀伤实验的终点收集细胞培养上清，至96孔板中放置-20℃保存备用。ELISA实验时，取出冻存的培养上清，室温融化，3500rpm离心l0mins收集上清用于ELISA实验。ELISA步骤详见试剂盒中的说明书(Human IL-6ELISA kit、Human IL-2 ELISA kit、Human IFN-γELISA Kit、Human TNF-αELISA kit)。
细胞因子IL-6的ELISA检测方法是在384孔酶标板上加入25uL 2ug/mL的经包被处理的抗体，4℃过夜包被。弃去包被液，每孔加入80uL 2％BSA的PBS室温封闭1.5小时。弃去封闭液，使用自动洗板机洗版3次。将96孔板中上清用1％FBS的1640培养基4倍稀释，转移25ul/孔至384孔板，2个复孔，室温孵育2小时。将试剂盒中的Standard首孔350pg/mL，用1％FBS的1640培养基进行2倍梯度稀释8个点后，384孔板加入25ul/孔作为标准曲线，2个复孔，室温孵育2小时。弃去上清，使用PBST洗板3次。准备detection antibody(1:800 in 0.1％BSA&0.05％Tween20-PBS)，每孔板加入25ul/孔，室温孵育1小时。弃去二抗，使用PBST洗板3次。加入HRP底物TMB，25ul/孔，显色20分钟。加入终止液2M HCl，25ul/孔来终止显色。使用酶标仪读取450nm的吸光度值。保存原始文件和Excel文件。
细胞因子IL-2的ELISA检测方法是在384孔酶标板上加入25uL 2ug/mL的coatingantibody，4℃过夜包被。弃去包被液，每孔加入80uL 2％BSA的PBS室温封闭1.5小时。弃去封闭液，使用自动洗板机洗版3次。将96孔板中上清用1％FBS的1640培养基4倍稀释，转移25ul/孔至384孔板，2个复孔，室温孵育2小时。将试剂盒中的Standard首孔1000pg/mL，用1％FBS的1640培养基进行2倍梯度稀释8个点后，384孔板加入25ul/孔作为标准曲线，2个复孔，室温孵育2小时。弃去上清，使用PBST洗板3次。准备detection antibody(1:2500 in 0.1％BSA&0.05％Tween20-PBS)，每孔板加入25ul/孔，室温孵育1小时。弃去二抗，使用PBST洗板3次。加入HRP底物TMB，25ul/孔，显色20分钟。加入终止液2M HCl，25ul/孔来终止显色。使用酶标仪读取450nm的吸光度值。保存原始文件和Excel文件。
细胞因子IFN-γ的ELISA检测方法是在384孔酶标板上加入25uL2ug/mL的coatingantibody，4℃过夜包被。弃去包被液，每孔加入80uL 2％BSA的PBS室温封闭1.5小时。弃去封闭液，使用自动洗板机洗版3次。将96孔板中上清用1％FBS的1640培养基4倍稀释，转移25ul/孔至384孔板，2个复孔，室温孵育2小时。将试剂盒中的Standard首孔1400pg/mL，用1％FBS的1640培养基进行2倍梯度稀释8个点后，384孔板加入25ul/孔作为标准曲线，2个复孔，室温孵育2小时。弃去上清，使用PBST洗板3次。准备detection antibody(1:800 in 0.1％BSA&0.05％Tween20-PBS)，每孔板加入25ul/孔，室温孵育1小时。弃去二抗，使用PBST洗板3次。加入HRP底物TMB，25ul/孔，显色20分钟。加入终止液2M HCl，25ul/孔来终止显色。使用酶标仪读取450nm的吸光度值。保存原始文件和Excel文件。
细胞因子TNF-α的ELISA检测方法是在384孔酶标板上加入25uL2ug/mL的coatingantibody，4℃过夜包被。弃去包被液，每孔加入80uL2％BSA的PBS室温封闭1.5小时。弃去封闭液，使用自动洗板机洗版3次。将96孔板中上清用1％FBS的1640培养基4倍稀释，转移25ul/孔至384孔板，2个复孔，室温孵育2小时。将试剂盒中的Standard首孔2500pg/mL，用1％FBS的1640培养基进行2倍梯度稀释8个点后，384孔板加入25ul/孔作为标准曲线，2个复孔，室温孵育2小时。弃去上清，使用PBST洗板3次。准备detection antibody(1:667 in 0.1％BSA&0.05％Tween20-PBS)，每孔板加入25ul/孔，室温孵育1小时。弃去二抗，使用PBST洗板3次。加入HRP底物TMB，25ul/孔，显色20分钟。加入终止液2M HCl，25ul/孔来终止显色。使用酶标仪读取450nm的吸光度值。保存原始文件和Excel文件。
体外杀伤实验后，IL6的释放结果：实施例1双抗GPRC5D×CD3的EC50值为0.01054，阳性对照双抗(GC5B596D双抗)的EC50值为0.07981。
体外杀伤实验后，IL2的释放结果：实施例1双抗GPRC5D×CD3的EC50值为0.04965，阳性对照双抗(GC5B596D双抗)的EC50值为0.7283(近似值)。
体外杀伤实验后，IFN-γ的释放结果：实施例1双抗GPRC5D×CD3的EC50值为0.02979，阳性对照双抗(GC5B596D双抗)的EC50值为0.1291。
体外杀伤实验后，TNF-α的释放结果：实施例1双抗GPRC5D×CD3的 EC50值为0.1741，阳性对照双抗(GC5B596D双抗)的EC50值为0.8140(近似值)。
对比上述结果可以看出，实施例1双抗GPRC5D×CD3在PBMC和H929共存在时，在细胞水平上均能有效诱导PBMC分泌IL-6、IL-2、IFN-γ和TNF-α。
四、实施例1的双特异性抗体在人骨髓瘤NCI-H929细胞人源化小鼠异种移植肿瘤的抗肿瘤效应评估
利用人PBMC重建的NOG小鼠(上海美迪西生物医药股份有限公司)NCI-H929异种移植瘤模型来评估测试实施例1双抗GPRC5D×CD3的抗肿瘤效应。
将含有2×106个NCI-H929细胞的100uLPBS皮下注射接种到6-8周龄健康的NOG雌鼠的右侧背部皮下，准备荷瘤，每隔三天监控肿瘤生长的大小情况。肿瘤细胞接种后1天，复苏液氮冻存的人PBMC，培养于含10％HIFBS(FBS，56℃×30min)的PRMI-1640培养基中，在含5％CO2的37℃培养箱中孵育6h。收集孵育后hPBMC，重悬于PBS缓冲液中，调整细胞浓度至2.5×107/mL。在无菌条件下，腹腔注射200uL细胞悬液至小鼠体内，注射浓度为5×106个PBMC细胞。
当荷瘤小鼠肿瘤体积达到100mm3左右时将小鼠随机分组，使各组肿瘤体积差异小于均值的10％，并按照动物体重开始给药，均为每周2次尾静脉给药，共进行5次给药。实施例1的双特异性抗体组，GC5B596D组。
实验组(低剂量和高剂量)：给药实施例1双抗GPRC5D×CD3，给药剂量：每只老鼠1.5ug、6ug(图1和2中，低剂量和高剂量分别标注为“375Z56KC，1.5ug/mouse；375Z56KC，6ug/mouse”；
阳性对照组：给药阳性对照双抗GC5B596D，给药剂量：每只老鼠6ug(图1和图2中标注为GC5B596D，6ug/mouse)；
溶媒对照组：给药PBS，给药剂量：每只老鼠6ug(图1和图2中标注为PBS溶媒对照)；
图1为实施例1双抗GPRC5D×CD3对NCI-H929异种移植瘤肿瘤体积的影响；图2为实施例1双抗GPRC5D×CD3对NCI-H929异种移植瘤肿瘤重量的影响。
从图1和图2的结果可以看出，实施例1双抗GPRC5D×CD3和阳性对照双抗GC5B596D均对PBMC人源化小鼠中NCI-H929异种移植瘤肿瘤具有明显的抗肿瘤效应。实验结束时溶媒对照组平均肿瘤体积为1585.65±144.58mm3，与溶媒对照组相比，实施例1双抗GPRC5D×CD3在低剂量1.5ug/mouse和高剂量6ug/mouse中均具有极显著的抑瘤效应，平均肿瘤体积分别为64.46±12.46mm3和59.67±6.02mm3，抑瘤率分别为95.93％(P<0.001)和96.24％(P<0.001)。阳性对照双抗GC5B596D展现出明显的抗肿瘤效应，平均肿瘤体积为396.10±287.62mm3，抑瘤率为75.02％(P<0.01)。离体肿瘤重量数据与肿瘤体积数据有良好的一致性。
应当理解，虽然本说明书按照实施方式加以描述，但并非每个实施方式仅包含一个独立的技术方案，说明书的这种叙述方式仅仅是为清楚起见，本领域技术人员应当将说明书作为一个整体，各实施方式中的技术方案也可以经适当组合，形成本领域技术人员可以理解的其他实施方式。
上文所列出的一系列的详细说明仅仅是针对本发明的可行性实施方式的具体说明，它们并非用以限制本发明的保护范围，凡未脱离本发明技艺精神所作的等效实施方式或变更均应包含在本发明的保护范围之内。

Claims (15)

  1. 一种多特异性抗体或其抗原结合分子，其特征在于：
    所述多特异性抗体或其抗原结合分子包含第一抗原结合模块和第二抗原结合模块；
    其中，所述第一抗原结合模块结合GPRC5D，所述第二抗原结合模块结合CD3或CD3ε；
    所述第一抗原结合模块包含重链可变区i-VH和轻链可变区i-VL，所述重链可变区i-VH包含如SEQ ID NO.1所示的i-HCDR1、如SEQ ID NO.2所示的i-HCDR2和如SEQ ID NO.3所示的i-HCDR3；所述轻链可变区i-VL包含如SEQ ID NO.4所示的i-LCDR1、氨基酸序列为SAS的i-LCDR2和如SEQ ID NO.5所示的i-LCDR3；
    所述第二抗原结合模块包含重链可变区ii-VH和轻链可变区ii-VL，所述重链可变区ii-VH包含如SEQ ID NO.6所示的ii-HCDR1、如SEQ ID NO.7所示的ii-HCDR2和如SEQ ID NO.8所示的ii-HCDR3；所述轻链可变区ii-VL包含如SEQ ID NO.9所示的ii-LCDR1、氨基酸序列为GTN的ii-LCDR2和如SEQ ID NO.10所示的ii-LCDR3。
  2. 如权利要求1所述的多特异性抗体或其抗原结合分子，其特征在于：
    所述第一抗原结合模块的重链可变区i-VH的序列如SEQ ID NO.11所示，或者其与SEQ ID NO.11所示序列有80％以上的序列同源性；所述第一抗原结合模块的轻链可变区i-VL的序列如SEQ ID NO.12所示，或者其与SEQ ID NO.12所示序列有80％以上的序列同源性；
    所述第二抗原结合模块的重链可变区ii-VH的序列如SEQ ID NO.13所示，或者其与SEQ ID NO.13所示序列有80％以上的序列同源性；所述第二抗原结合模块的轻链可变区ii-VL的序列如SEQ ID NO.14所示，或者其与SEQ ID NO.14所示序列有80％以上的序列同源性。
  3. 如权利要求1或2所述的多特异性抗体或其抗原结合分子，其特征在于：所述第一抗原结合模块选自Fv、Fab、F(ab’)2、Fab’、dsFv、scFv、 sc(Fv)2或单链抗体中的任意一种；
    所述第二抗原结合模块选自Fv、Fab、F(ab’)2、Fab’、dsFv、scFv、sc(Fv)2或单链抗体中的任意一种。
  4. 如权利要求3所述的多特异性抗体或其抗原结合分子，其特征在于：所述第一抗原结合模块和所述第二抗原结合模块是Fab分子。
  5. 如权利要求3所述的多特异性抗体或其抗原结合分子，其特征在于：所述第一抗原结合模块与所述第二抗原结合模块之间通过接头肽融合。
  6. 如权利要求3所述的多特异性抗体或其抗原结合分子，其特征在于：
    所述多特异性抗体或其抗原结合分子包括轻链恒定区；所述轻链恒定区优选人轻链恒定区；较佳的，所述轻链恒定区为人λ或κ轻链恒定区。
  7. 如权利要求3所述的多特异性抗体或其抗原结合分子，其特征在于：
    所述多特异性抗体或其抗原结合分子包括重链恒定区；所述重链恒定区优选人IgG1、2、3、4的重链恒定区；
    较佳的，所述重链恒定区的Fc结构域为突变Fc结构域；
    较佳的，所述突变Fc结构域的第一亚基的CH3区中的氨基酸残基采用具有较大侧链体积的氨基酸残基替换，从而形成隆起结构；所述突变Fc结构域的第二亚基的CH3区中的氨基酸残基采用具有较小侧链体积的氨基酸残基替换，从而形成空腔结构；所述空腔结构容纳所述隆起结构从而使得所述所述第一亚基和第二亚基结合形成异二聚体；
    较佳的，所述突变Fc结构域的突变方案选自：(a)knob突变T366W，以及hole突变T366S、L368A或Y407V；(b)Knob突变S354C、T366W，以及hole突变Y349C T366S、L368A或Y407V。
  8. 一种核酸分子，所述核酸分子编码如权利要求1至7中任意一项所述的多特异性抗体或其抗原结合分子的核酸分子。
  9. 包含如权利要求8中所述核酸分子的载体。
  10. 包含如权利要求8所述核酸分子或权利要求9所述载体的宿主细胞。
  11. 一种重组蛋白，其特征在于：所述重组蛋白包含如权利要求1至7中任意一项所述的多特异性抗体或其抗原结合分子。
  12. 一种免疫缀合物，其包含如权利要求1至7中任意一项所述的多特异性抗体或其抗原结合分子。
  13. 一种药物组合物，其特征在于：所述药物组合物包含如权利要求1至7中任意一项所述的多特异性抗体或其抗原结合分子，或者包含如权利要求8所述的核酸分子，或者包含如权利要求10所述载体，或者包含如权利要求10所述宿主细胞，或者包含如权利要求12所述的重组蛋白，或者包含如权利要求12所述的免疫缀合物，以及药学上可接受的载剂。
  14. 一种检测产品，其特征在于：所述检测产品包含如权利要求1至7中任意一项所述的多特异性抗体或其抗原结合分子，或者包含如权利要求8所述的核酸分子，或者包含如权利要求9所述载体，或者包含如权利要求10所述宿主细胞，或者包含如权利要求11所述的重组蛋白，或者权利要求12所述的免疫缀合物。
  15. 如权利要求1至7中任意一项所述的多特异性抗体或其抗原结合分子，或者如权利要求8所述的核酸分子，或者如权利要求9所述载体，或者如权利要求10所述宿主细胞，或者如权利要求11所述的重组蛋白，或者权利要求12所述的免疫缀合物，或者如权利要求13所述药物组合物，在制备治疗或预防疾病的药物方面的用途；较佳的，所述疾病为癌症或自身免疫疾病；较佳的，所述疾病为多发性骨髓瘤。
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