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WO2023125888A1 - 一种gprc5d抗体及其应用 - Google Patents

一种gprc5d抗体及其应用 Download PDF

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Publication number
WO2023125888A1
WO2023125888A1 PCT/CN2022/143719 CN2022143719W WO2023125888A1 WO 2023125888 A1 WO2023125888 A1 WO 2023125888A1 CN 2022143719 W CN2022143719 W CN 2022143719W WO 2023125888 A1 WO2023125888 A1 WO 2023125888A1
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seq
antibody
variable region
chain variable
antigen
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PCT/CN2022/143719
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English (en)
French (fr)
Inventor
付雅媛
廖敬礼
殷博薇
曹卓晓
唐任宏
任晋生
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山东先声生物制药有限公司
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Priority to EP22915137.8A priority Critical patent/EP4458854A1/en
Priority to CN202280085894.1A priority patent/CN118510805A/zh
Publication of WO2023125888A1 publication Critical patent/WO2023125888A1/zh

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    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • 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
    • 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

Definitions

  • This application relates to the field of biomedicine, in particular, to a GPRC5D antibody and its application.
  • Multiple myeloma is a malignant tumor of plasma cells, which is characterized by the unlimited proliferation of plasma cells in the bone marrow like tumor cells, accompanied by the secretion of monoclonal immunoglobulin, which causes multiple lytic
  • a series of clinical manifestations such as bone damage, hypercalcemia, anemia, kidney damage, and repeated infection.
  • Combined treatment with hematopoietic stem cell transplantation and immunomodulators and protease inhibitors can significantly improve the prognosis.
  • CAR T therapy has made breakthroughs in the treatment of multiple myeloma, especially CAR T targeting B cell maturation antigen (BCMA) has shown positive clinical results in the treatment of multiple myeloma.
  • BCMA B cell maturation antigen
  • GPRC5D is a G protein-coupled receptor C5 family subtype D, which belongs to an orphan receptor and is a 7-transmembrane protein.
  • GPRC5D is a new multiple myeloma target after BCMA.
  • Tissue expression profiling studies have found that GPRC5D is specifically highly expressed in plasma cells of multiple myeloma, while low in normal tissues, limited to immune-privileged hair follicles area. And the expression of GPRC5D and BCMA did not overlap.
  • GPRC5D CAR-T still has therapeutic effect and overcomes tumor escape.
  • the dual targeting of GPRC5D and BCMA can also cover more people and lead to better clinical response. Therefore, there is a huge potential clinical demand for the development of GPRC5D-specific antibodies and related biological products.
  • the present application discloses an antibody specifically binding to G protein-coupled receptor C5 family subtype D (GPRC5D) or its antigen-binding fragment, multispecific antigen-binding molecule, nucleic acid fragment, vector, host cell, immune Effector cells, methods of preparation, pharmaceutical compositions, pharmaceutical uses and methods of treatment of tumors or cancers such as B-cell lymphoma or multiple myeloma.
  • GPRC5D G protein-coupled receptor C5 family subtype D
  • the application provides an antibody or an antigen-binding fragment thereof that specifically binds to G protein-coupled receptor C5 family subtype D (GPRC5D), wherein the antibody or an antigen-binding fragment thereof comprises a heavy chain variable region and light chain variable region, and where
  • the light chain variable region comprises LCDR1, LCDR2, and LCDR3, and the LCDR1 has any sequence of LCDR1 shown below or has 1, 2, 3 or more amino acid insertions compared to the sequence, A deleted and/or substituted sequence, the LCDR2 having any of the LCDR2 sequences shown below or a sequence having 1, 2, 3 or more amino acid insertions, deletions and/or substitutions compared to said sequence, and
  • the LCDR3 has any sequence of LCDR3 shown below or a sequence with 1, 2, 3 or more amino acid insertions, deletions and/or substitutions compared to the sequence:
  • the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, and the HCDR1 has any sequence of HCDR1 shown below or has 1, 2, 3 or more amino acid insertions compared to the sequence, A deleted and/or substituted sequence, the HCDR2 having any of the HCDR2 sequences shown below or a sequence having 1, 2, 3 or more amino acid insertions, deletions and/or substitutions compared to said sequence, and The HCDR3 has any sequence of HCDR3 shown below or a sequence with 1, 2, 3 or more amino acid insertions, deletions and/or substitutions compared to the sequence:
  • the present application provides a multispecific antigen-binding molecule, wherein the multispecific antigen-binding molecule comprises the aforementioned antibody or an antigen-binding fragment thereof, and an antigen-binding molecule that binds to an antigen other than GPRC5D, or Binds to a different epitope of GPRC5D than the aforementioned antibodies or antigen-binding fragments thereof.
  • the present application provides a chimeric antigen receptor (CAR), wherein the chimeric antigen receptor at least comprises a signal peptide, an extracellular antigen binding domain, a hinge region, a transmembrane domain and The intracellular signaling domain, the extracellular antigen-binding domain comprises the aforementioned GPRC5D antibody or an antigen-binding fragment thereof, or the aforementioned multispecific antigen-binding molecule.
  • CAR chimeric antigen receptor
  • the present application provides an immune effector cell, wherein the immune effector cell expresses the aforementioned chimeric antigen receptor, or comprises a nucleic acid fragment encoding the aforementioned chimeric antigen receptor.
  • the present application provides an isolated nucleic acid fragment encoding the aforementioned antibody or antigen-binding fragment thereof, the aforementioned multispecific antigen-binding molecule, or the aforementioned chimeric antigen receptor.
  • the present application provides a vector, wherein the vector comprises the aforementioned nucleic acid fragment.
  • the present application provides a host cell, wherein the host cell comprises the aforementioned vector.
  • the present application provides a method for preparing the aforementioned antibody or antigen-binding fragment thereof or the aforementioned multispecific antigen-binding molecule, wherein the method includes culturing the aforementioned cells, and isolating the antibodies expressed by the cells, Antigen-binding fragments or multispecific antigen-binding molecules.
  • the present application provides a method for preparing the aforementioned immune effector cells, wherein the method includes introducing the nucleic acid fragment encoding the aforementioned CAR into the immune effector cells.
  • the present application provides a pharmaceutical composition, wherein the pharmaceutical composition comprises the aforementioned antibody or antigen-binding fragment thereof, the aforementioned multispecific antigen-binding molecule, the aforementioned immune effector cell, the aforementioned nucleic acid fragment, the aforementioned The carrier or the product prepared according to the aforementioned method.
  • the present application provides a method for treating a tumor or cancer, wherein the method comprises administering to a subject an effective amount of the aforementioned antibody or antigen-binding fragment thereof, the aforementioned multispecific antigen-binding molecule, the aforementioned Immune effector cells, the aforementioned nucleic acid fragments, the aforementioned vectors, or products prepared according to the aforementioned methods, or the aforementioned pharmaceutical compositions; the tumor or cancer is a tumor or cancer expressing GPRC5D.
  • the present application provides the aforementioned antibody or antigen-binding fragment thereof, the aforementioned multispecific antigen-binding molecule, the aforementioned immune effector cell, the aforementioned nucleic acid fragment, the aforementioned carrier, or the product prepared according to the aforementioned method, or the aforementioned pharmaceutical composition Use in preparing a medicine for treating tumor or cancer; said tumor or cancer is a tumor or cancer expressing GPRC5D.
  • the present application provides the aforementioned antibody or antigen-binding fragment thereof, the aforementioned multispecific antigen-binding molecule, the aforementioned immune effector cell, the aforementioned nucleic acid fragment, the aforementioned carrier, or the product prepared according to the aforementioned method, or the aforementioned pharmaceutical composition , for treating a tumor or cancer;
  • the tumor or cancer is a tumor or cancer expressing GPRC5D.
  • the present application provides a kit, wherein the kit comprises the aforementioned antibody or antigen-binding fragment thereof, the aforementioned multispecific antigen-binding molecule, the aforementioned immune effector cell, the aforementioned nucleic acid fragment, the aforementioned vector or Prepare the obtained product or the aforementioned pharmaceutical composition according to the aforementioned method.
  • the present application provides a method for detecting the expression of GPRC5D in a biological sample, the method comprising allowing the biological A chemical sample is contacted with the antibody or antigen-binding fragment thereof.
  • the present application provides the use of the aforementioned antibodies or antigen-binding fragments thereof in the preparation of GPRC5D detection reagents.
  • Negative or low expression of BCMA is a common cause of relapse or refractory multiple myeloma.
  • This application focuses on another target of relapsed/refractory multiple myeloma, GPRC5D, and provides antibodies targeting human GPRC5D or its antigen-binding fragments , can bind human or monkey GPRC5D with higher affinity, but not bind or bind GPRC5A, GPRC5B or GPRC5C protein with low affinity, which is useful for the further development of antibodies (monoclonal antibodies or multispecific antibodies) and cell therapy products targeting GPRC5D is of great significance.
  • Figure 1 FACS detection of GPRC5D expression in endogenous cells.
  • FIG. 2A FACS detection results of the CHOK1 stable cell line expressing human GPRC5A protein
  • Figure 2B FACS detection results of the CHOK1 stable cell line expressing human GPRC5B protein
  • Figure 2C FACS detection results of the CHOK1 stable cell line expressing human GPRC5C protein
  • FIG. 2D FACS detection results of CHOK1 stably transfected cell line expressing human GPRC5D protein.
  • FIG. 1 FACS detection results of HEK293T stably transfected cell line expressing human GPRC5D protein.
  • FIG. 4A FACS detection results of CHOK1 stably transfected cell line expressing monkey GPRC5D protein
  • Figure 4B FACS detection results of HEK293T stably transfected cell line expressing monkey GPRC5D protein.
  • FIG. 5A FACS detection of the binding reaction of the control antibody to NCI-H929 tumor cells
  • Figure 5B FACS detection of the binding reaction of the control antibody to 293T-human-GPRC5D recombinant cells
  • Figure 5C FACS detection of the binding reaction of the control antibody to 293T-monkey-GPRC5D Cell binding response.
  • Figure 6A-6C Cell based ELISA detection of the binding reaction between chimeric antibody and CHOK1-hGPRC5D.
  • Figure 7A-7C Cell based ELISA detection of the binding reaction of chimeric antibody and CHOK1-cynoGPRC5D.
  • Figure 8A-8C Cell based ELISA detection of the binding reaction between chimeric antibody and CHOK1.
  • Figure 13A-13J Cell based ELISA detection of binding reaction of humanized antibody to CHOK1-hGPRC5D.
  • Fig. 14A-14J Cell based ELISA detection of binding reaction of humanized antibody to CHOK1-cynoGPRC5D.
  • GPRC5D refers to G protein-coupled receptor C5 family subtype D, which belongs to an orphan receptor and is a 7-transmembrane protein. GPRC5D is highly expressed on the surface of primary multiple myeloma cells, while its expression in normal tissues is limited to the hair follicle region. Studies have shown that 65% of multiple myeloma patients have an expression threshold of GPRC5D exceeding 50%. With this feature, GPRC5D has become a potential target for the treatment of MM.
  • an antigen-binding molecule eg, an antibody
  • an antigen-binding molecule specifically binds an antigen and substantially the same antigen with high affinity, typically, but does not bind an unrelated antigen with high affinity.
  • the equilibrium dissociation constant KD can be measured by methods known in the art, such as surface plasmon resonance (eg Biacore) or equilibrium dialysis.
  • antigen binding molecule is used herein in the broadest sense to refer to a molecule that specifically binds an antigen.
  • antigen binding molecules include, but are not limited to, antibodies or antibody mimetics.
  • Antibody mimic refers to an organic compound or binding domain that can specifically bind to an antigen, but has nothing to do with the structure of an antibody.
  • antibody mimics include but are not limited to affibody, affitin, affilin, designed ankyrin repeat proteins (DARPins), aptamers or Kunitz-type domain peptides.
  • antibody is used herein in the broadest sense to refer to a polypeptide comprising sufficient sequence from the variable region of an immunoglobulin heavy chain and/or sufficient sequence from the variable region of an immunoglobulin light chain to be capable of specifically binding to an antigen or peptide combinations.
  • Antibody herein encompasses various forms and various structures as long as they exhibit the desired antigen-binding activity.
  • Antibody herein includes alternative protein scaffolds or artificial scaffolds with grafted complementarity determining regions (CDRs) or CDR derivatives. Such scaffolds include antibody-derived scaffolds comprising mutations introduced, eg, to stabilize the three-dimensional structure of the antibody, as well as fully synthetic scaffolds comprising, eg, biocompatible polymers.
  • Such scaffolds may also include non-antibody-derived scaffolds, such as scaffold proteins known in the art to be useful for grafting CDRs, including but not limited to tenascin, fibronectin, peptide aptamers, and the like.
  • antibody includes whole antibodies and any antigen-binding fragment (ie, "antigen-binding portion") or single chains thereof.
  • Antibody refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding portion thereof.
  • Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region.
  • the heavy chain constant region consists of three domains, CH1, CH2 and CH3.
  • Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region.
  • the light chain constant region consists of one domain, CL.
  • the VH and VL regions can be further subdivided into hypervariable regions, called complementarity determining regions (CDRs), interspersed in more conserved regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • Each VH and VL consists of three CDRs and four FRs, which are arranged in the following order from the amino terminal to the carboxyl terminal: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions of the heavy and light chains contain binding domains that can interact with antigen.
  • the constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.
  • immunoglobulins can be divided into five classes, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE, and their corresponding heavy chains are the ⁇ chain and the delta chain, respectively. , ⁇ chain, ⁇ chain and ⁇ chain.
  • IgM, IgD, IgG, IgA, and IgE immunoglobulins
  • their corresponding heavy chains are the ⁇ chain and the delta chain, respectively.
  • ⁇ chain, ⁇ chain and ⁇ chain The same class of Ig can be divided into different subclasses according to the amino acid composition of its hinge region and the number and position of heavy chain disulfide bonds.
  • IgG can be divided into IgG1, IgG2, IgG3, IgG4, and IgA can be divided into IgA1 and IgA.
  • Light chains are classified as either kappa chains or lambda chains by difference in the constant region.
  • Each of the five Ig classes can have either a kappa chain or a lambda chain.
  • Antibody herein also includes antibodies that do not comprise light chains, for example, antibodies produced from Camelus dromedarius, Camelus bactrianus, Lama glama, Lama guanicoe and alpaca ( Heavy-chain antibodies (HCAbs) produced by camelids such as Vicugna pacos) and immunoglobulin new antigen receptors (Ig new antigen receptors, IgNAR) found in cartilaginous fishes such as sharks.
  • HCAbs Heavy-chain antibodies
  • Ig new antigen receptors Ig new antigen receptors, IgNAR
  • antibody herein may be derived from any animal, including but not limited to humans and non-human animals selected from primates, mammals, rodents and vertebrates, such as camelids, large sheep Camels, protozoans, alpacas, sheep, rabbits, mice, rats or cartilaginous fishes (eg sharks).
  • heavy chain antibody herein refers to an antibody that lacks the light chains of conventional antibodies.
  • the term specifically includes, but is not limited to, homodimeric antibodies comprising a VH antigen binding domain and CH2 and CH3 constant domains in the absence of a CH1 domain.
  • nanobody in this paper refers to the natural heavy chain antibody lacking the light chain in camels and other bodies, and its variable region can be cloned to obtain a single domain antibody consisting of only the variable region of the heavy chain, also known as VHH (Variable domain of heavy chain of heavy chain antibody), which is the smallest functional antigen-binding fragment.
  • VHH Very domain of heavy chain of heavy chain antibody
  • VHH domain and “nanobody” and “single domain antibody” (single domain antibody, sdAb) herein have the same meaning and are used interchangeably, referring to the variable region of a cloned heavy chain antibody, constructed A single domain antibody consisting of only one heavy chain variable region, which is the smallest fully functional antigen-binding fragment.
  • the variable region of the heavy chain of the antibody is cloned to construct a single domain antibody consisting of only one heavy chain variable region.
  • multispecific herein refers to the ability of an antibody or antigen-binding fragment thereof to bind, for example, different antigens or at least two different epitopes on the same antigen.
  • terms such as “bispecific”, “trispecific”, “tetraspecific” and the like refer to the number of different epitopes to which an antibody can bind.
  • conventional monospecific IgG-type antibodies have two identical antigen-binding sites (paratopes) and thus can only bind the same epitope (rather than bind different epitopes).
  • multispecific antibodies have at least two different types of paratopes/binding sites and thus can bind at least two different epitopes.
  • complementarity determining region refers to the antigen binding site of an antibody.
  • a single “specificity” may refer to one, two, three or more than three identical CDRs in a single antibody (the actual number of CDRs/binding sites in a single antibody molecule is referred to as " price").
  • a single native IgG antibody is monospecific and bivalent because it has two identical paratopes.
  • a multispecific antibody comprises at least two (different) complementarity determining regions/binding sites.
  • the term “multispecific antibody” refers to an antibody that has more than one paratope and has the ability to bind two or more different epitopes.
  • multispecific antibody includes in particular bispecific antibodies as defined above, but generally also proteins, e.g. antibodies, scaffolds which specifically bind three or more than three different epitopes, i.e. having three or more Antibodies with more than three paratopes/binding sites.
  • valence herein refers to the presence of a defined number of binding sites in an antibody/antigen binding molecule. Accordingly, the terms “monovalent”, “bivalent”, “tetravalent” and “hexavalent” denote one binding site, two binding sites, four binding sites and six binding sites in an antibody/antigen binding molecule, respectively. point of existence.
  • full-length antibody intact antibody
  • intact antibody intact antibody
  • Antigen-binding fragment and “antibody fragment” are used interchangeably herein, and do not possess the full structure of an intact antibody, but only include partial or partial variants of an intact antibody that possess the ability to bind Antigen capacity.
  • exemplary, "antigen-binding fragment” or “antibody fragment” herein includes, but is not limited to, Fab, F(ab')2, Fab', Fab'-SH, Fd, Fv, scFv, diabody and single domain Antibody.
  • chimeric antibody refers to an antibody that has variable sequences derived from immunoglobulins of one source organism (such as rat, mouse, rabbit or alpaca) and derived from a different organism (such as human ) of the immunoglobulin constant region.
  • Methods for producing chimeric antibodies are known in the art. See, e.g., Morrison, 1985, Science 229(4719):1202-7; Oi et al., 1986, Bio Techniques 4:214-221; Gillies et al., 1985 J Immunol Methods 125:191-202; incorporated by reference above and into this article.
  • humanized antibody herein refers to a genetically engineered non-human antibody whose amino acid sequence has been modified to increase sequence homology with a human antibody.
  • all or part of the CDR region of a humanized antibody is derived from a non-human antibody (donor antibody), and all or part of the non-CDR region (for example, variable region FR and/or constant region) is derived from a human Immunoglobulin (receptor antibody).
  • Humanized antibodies usually retain or partially retain the expected properties of the donor antibody, including but not limited to, antigen specificity, affinity, reactivity, ability to enhance immune cell activity or enhance immune response, etc.
  • Fully human antibody refers to antibodies having variable regions in which both the FRs and CDRs are derived from human germline immunoglobulin sequences. Furthermore, if the antibody comprises a constant region, the constant region also is derived from human germline immunoglobulin sequences. Fully human antibodies herein may include amino acid residues not encoded by human germline immunoglobulin sequences (eg, mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, "fully human antibodies” herein do not include antibodies in which CDR sequences derived from the germline of another mammalian species (eg, mouse) have been grafted onto human framework sequences.
  • another mammalian species eg, mouse
  • variable region herein refers to the region in the heavy or light chain of an antibody that is involved in making the antibody bind to an antigen
  • “heavy chain variable region” is used interchangeably with “VH” and “HCVR”
  • “light chain variable region”” can be used interchangeably with “VL” and “LCVR”.
  • the variable domains of the heavy and light chains of native antibodies generally have similar structures, with each domain comprising four conserved framework regions (FR) and three hypervariable regions (HVR). See, eg, Kindt et al., Kuby Immunology, 6 th ed., WH Freeman and Co., p. 91 (2007).
  • a single VH or VL domain may be sufficient to confer antigen binding specificity.
  • variable domains hypervariable regions
  • FR framework regions
  • amino acid positions representing the hypervariable regions of an antibody may vary according to the context and various definitions known in the art. Some positions within variable domains may be considered heterozygous hypervariable positions, as these positions may be considered within hypervariable regions under one set of criteria (such as IMGT or KABAT) but under a different set of criteria (such as KABAT or IMGT) outside the hypervariable region. One or more of these positions may also be found in extended hypervariable regions.
  • the present application includes antibodies comprising modifications in these hybrid hypervariable positions.
  • the heavy chain variable region CDR may be abbreviated as HCDR and the light chain variable region may be abbreviated as LCDR.
  • the variable domains of the native heavy and light chains each comprise four framework regions predominantly in a sheet configuration, connected by three CDRs (CDR1, CDR2, and CDR3) that form loops connecting the sheets , and in some cases form part of the lamellar structure.
  • the CDRs in each chain are held tightly together by the FR regions in the sequence FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and together with CDRs from other antibody chains contribute to the formation of the antigen-binding site of the antibody (see Kabat et al., Sequences of Protein of Immunological Interest, National Institute of Health, Bethesda, Md. 1987; which is incorporated herein by reference).
  • CDR CDR
  • Kabat et al. J.Biol.Chem., 252:6609-6616 (1977); Kabat et al., U.S. Department of Health and Human Services, "Sequences of proteins of immunological interest” (1991); Chothia et al., J.Mol.Biol.196:901-917 (1987); Al-Lazikani B. et al., J.Mol.Biol., 273:927-948 (1997); MacCallum et al., J.Mol. .Biol.262:732-745 (1996); Abhinandan and Martin, Mol. Immunol., 45:3832-3839 (2008); Lefranc M.P.
  • CDR herein can be marked and defined by methods known in the art, including but not limited to Kabat numbering system, Chothia numbering system or IMGT numbering system, and the tool websites used include but not limited to AbRSA website (http://cao.labshare.
  • CDRs herein include overlaps and subsets of amino acid residues defined in different ways.
  • Kabat numbering system herein generally refers to the immunoglobulin alignment and numbering system proposed by Elvin A. Kabat (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991).
  • Chothia numbering system generally refers to the immunoglobulin numbering system proposed by Chothia et al., which is a classical rule for identifying the boundaries of CDR regions based on the position of structural loop regions (see, for example, Chothia & Lesk (1987) J. Mol. Biol 196:901-917; Chothia et al. (1989) Nature 342:878-883).
  • IMGT numbering system herein generally refers to the numbering system based on the international ImMunoGeneTics information system (IMGT) initiated by Lefranc et al., see Lefranc et al., Dev.Comparat.Immunol.27 :55-77, 2003.
  • IMGT ImMunoGeneTics information system
  • heavy chain constant region herein refers to the carboxy-terminal portion of the heavy chain of an antibody that is not directly involved in binding the antibody to an antigen, but exhibits effector functions, such as interaction with Fc receptors, which are relative to the antibody's available Variable domains have more conserved amino acid sequences.
  • the “heavy chain constant region” may be selected from a CH1 domain, a hinge region, a CH2 domain, a CH3 domain, or variants or fragments thereof.
  • “Heavy chain constant region” includes "full-length heavy chain constant region” and “heavy chain constant region fragment", the former has a structure substantially similar to that of a natural antibody constant region, while the latter only includes “full-length heavy chain constant region” part".
  • a typical "full-length antibody heavy chain constant region” consists of a CH1 domain-hinge region-CH2 domain-CH3 domain; when the antibody is IgE, it also includes a CH4 domain; when the antibody is a heavy chain In the case of an antibody, it does not include a CH1 domain.
  • typical "heavy chain constant region fragments" can be selected from Fc or CH3 domains.
  • light chain constant region refers to the carboxy-terminal part of the antibody light chain, which is not directly involved in the binding of the antibody to the antigen, and the light chain constant region can be selected from a constant kappa domain or a constant lambda domain.
  • Fc region is used herein to define the C-terminal region of an antibody heavy chain that contains at least a portion of the constant region.
  • the term includes native sequence Fc regions and variant Fc regions.
  • a human IgG heavy chain Fc region can extend from Cys226 or Pro230 to the carboxyl terminus of the heavy chain.
  • antibodies produced by host cells may undergo post-translational cleavage whereby one or more, especially one or two amino acids are excised from the C-terminus of the heavy chain.
  • an antibody produced by a host cell by expression of a specific nucleic acid molecule encoding a full-length heavy chain may include the full-length heavy chain, or it may include cleavage variants of the full-length heavy chain.
  • the last two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, numbering according to the Kabat EU index).
  • the C-terminal lysine (Lys447), or the C-terminal glycine (Gly446) and lysine (Lys447) of the Fc region may or may not be present.
  • the IgG Fc region includes IgG CH2 and IgG CH3 domains, optionally, on this basis, it may also include a complete or partial hinge region, but does not include a CH1 domain.
  • the "CH2 domain" of a human IgG Fc region generally extends from an amino acid residue at about position 231 to an amino acid residue at about position 340.
  • the carbohydrate chain is attached to the CH2 domain.
  • the CH2 domain herein may be a native sequence CH2 domain or a variant CH2 domain.
  • a "CH3 domain” comprises the stretch of residues in the Fc region that is C-terminal to the CH2 domain (ie, from the amino acid residue at about position 341 to the amino acid residue at about position 447 of IgG).
  • the CH3 region herein may be a native sequence CH3 domain or a variant CH3 domain (e.g.
  • the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index, as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Described in Institutes of Health, Bethesda, MD, 1991.
  • Fc variant herein refers to changes in the structure or function of Fc caused by one or more amino acid substitutions, insertions or deletion mutations at appropriate positions on the Fc.
  • Interaction between Fc variants refers to the space-filling effect, electrostatic guidance, hydrogen bond interaction, hydrophobic interaction, etc. between Fc variants designed by mutation. Interactions between Fc variants contribute to the formation of stable heterodimeric proteins.
  • a preferred mutation design is a "Knob-into-Hole” style mutation design.
  • Fc variants have been widely used in the field to prepare bispecific antibodies or heterodimeric Fc fusion proteins.
  • the representative one is the "Knob-into-Hole" form proposed by Cater et al. (Protein Engineering vol.9 no.7 pp.617-621, 1996); Heterodimer form (US 20100286374 A1); Jonathan H.Davis et al.
  • the Knob/Hole structure on the Fc variant fragments described in the present application means that the two Fc fragments are mutated respectively, and after the mutations, they can be combined in the form of "Knob-into-Hole". It is preferable to use the "knob-into-hole" model of Cater et al. to carry out site mutation transformation on the Fc region, so that the obtained first Fc variant and the second Fc variant can be in the form of "knob-into-hole" Combine together to form heterodimers.
  • the selection of particular immunoglobulin Fc regions from particular immunoglobulin classes and subclasses is within the purview of those skilled in the art.
  • the Fc region of human antibody IgG1, IgG2, IgG3, IgG4 is preferred, and the Fc region of human antibody IgG1 is more preferred.
  • One of the first Fc variant or the second Fc variant is randomly selected for knob mutation and the other for hole mutation.
  • amino acid herein generally refers to amino acids that belong to the same class or have similar characteristics (eg, charge, side chain size, hydrophobicity, hydrophilicity, backbone conformation, and rigidity).
  • amino acids in each of the following groups belong to each other's conservative amino acid residues, and the substitution of amino acid residues in the group belongs to the conservative amino acid substitution:
  • identity may be calculated by aligning said sequences for optimal comparison purposes in order to determine the percent "identity" of two amino acid sequences or two nucleic acid sequences (for example, may be optimal alignment to introduce gaps in one or both of the first and second amino acid sequences or nucleic acid sequences or non-homologous sequences may be discarded for comparison purposes).
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
  • the percent identity between two sequences will vary with the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that need to be introduced to optimally align the two sequences.
  • the comparison of sequences and the calculation of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, using the Needlema and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm in the GAP program that has been integrated into the GCG software package (available at www.gcg.com), using the Blossum 62 matrix or The PAM250 matrix and gap weights of 16, 14, 12, 10, 8, 6 or 4 and length weights of 1, 2, 3, 4, 5 or 6 determine the percent identity between two amino acid sequences.
  • the GAP program in the GCG software package (available at www.gcg.com), using the NWSgapdna.CMP matrix with gap weights of 40, 50, 60, 70, or 80 and length weights of 1, 2, 3, 4, 5 or 6, determining the percent identity between two nucleotide sequences.
  • a particularly preferred parameter set (and one that should be used unless otherwise stated) is the Blossum62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5. It is also possible to use a PAM120 weighted remainder table, a gap length penalty of 12, and a gap penalty of 4, using the E. Meyers and W. Miller algorithm that has been incorporated into the ALIGN program (version 2.0), ((1989) CABIOS, 4:11-17 ) to determine the percent identity between two amino acid sequences or nucleotide sequences.
  • nucleic acid sequences and protein sequences described herein may further be used as "query sequences" to perform searches against public databases, eg, to identify other family member sequences or related sequences.
  • searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul et al., (1990) J. Mol. Biol. 215:403-10.
  • Gapped BLAST can be used as described in Altschul et al., (1997) Nucleic Acids Res. 25:3389-3402.
  • the default parameters of the respective programs eg, XBLAST and NBLAST
  • XBLAST and NBLAST can be used. See www.ncbi.nlm.nih.gov.
  • chimeric antigen receptor herein refers to an artificial cell surface receptor engineered to be expressed on immune effector cells and to specifically bind an antigen, comprising at least (1) an extracellular antigen-binding domain, such as an antibody The heavy chain variable region and/or the light chain variable region, (2) the transmembrane domain that anchors the CAR into immune effector cells, and (3) the intracellular signaling domain.
  • CARs are able to redirect T cells and other immune effector cells to a target of choice, such as cancer cells, in a non-MHC-restricted manner using an extracellular antigen-binding domain.
  • nucleic acid includes any compound and/or substance comprising a polymer of nucleotides.
  • Each nucleotide consists of a base, especially a purine or pyrimidine base (i.e. cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), a sugar (i.e. deoxyribose or ribose) and phosphate groups.
  • cytosine C
  • G guanine
  • A adenine
  • T thymine
  • U uracil
  • nucleic acid molecules are described by a sequence of bases, whereby the bases represent the primary structure (linear structure) of the nucleic acid molecule.
  • the sequence of bases is usually expressed 5' to 3'.
  • nucleic acid molecule encompasses deoxyribonucleic acid (DNA), including for example complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), especially messenger RNA (mRNA), synthetic forms of DNA or RNA, and synthetic forms of DNA or RNA comprising both Mixed polymers of one or more of these molecules.
  • Nucleic acid molecules can be linear or circular.
  • nucleic acid molecule includes both sense and antisense strands, as well as single- and double-stranded forms.
  • nucleic acid molecules described herein may contain naturally occurring or non-naturally occurring nucleotides.
  • Nucleic acid molecules also encompass DNA and RNA molecules suitable as vectors for direct expression of the antibodies of the present application in vitro and/or in vivo, for example in a host or patient.
  • DNA eg cDNA
  • RNA eg mRNA
  • Such DNA (eg cDNA) or RNA (eg mRNA) vectors may be unmodified or modified.
  • mRNA can be chemically modified to enhance the stability of the RNA vector and/or the expression of the encoded molecule, so that the mRNA can be injected into a subject to generate antibodies in vivo (see e.g. Stadler et al., Nature Medicine 2017, published online June 12, 2017, doi: 10.1038/nm.4356 or EP2101823B1).
  • isolated nucleic acid refers to a nucleic acid molecule that has been separated from components of its natural environment.
  • An isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but which is present extrachromosomally or at a chromosomal location other than its natural chromosomal location.
  • vector refers to a nucleic acid molecule capable of amplifying another nucleic acid to which it has been linked.
  • the term includes vectors that are self-replicating nucleic acid structures as well as vectors that integrate into the genome of a host cell into which the vector has been introduced.
  • Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors”.
  • host cell herein refers to a cell into which exogenous nucleic acid has been introduced, including the progeny of such a cell.
  • Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages. Progeny may not be identical to the parental cell in nucleic acid content, but may contain mutations. Mutant progeny having the same function or biological activity as screened or selected for in the originally transformed cell are included herein.
  • the term "pharmaceutical composition” refers to a preparation that is present in a form that permits the biological activity of the active ingredients contained therein to be effective and that does not contain substances that are unacceptably toxic to the subject to which the pharmaceutical composition is administered. additional ingredients.
  • the term "pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial, antifungal), isotonic agents, absorption delaying agents, Agents, salts, preservatives, drug stabilizers, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, etc., and combinations thereof, which are known to those skilled in the art (see For example, Remington's Pharmaceutical Sciences, 18th ed. Mack Printing Company, 1990, pp. 1289-1329). Except in cases of incompatibility with the active ingredient, any conventional carrier is contemplated for use in therapeutic or pharmaceutical compositions.
  • treatment refers to surgical or therapeutic treatment, the purpose of which is to prevent, slow down (reduce) undesired physiological changes or lesions, such as cancers and tumors, in the subject being treated.
  • beneficial or desired clinical outcomes include, but are not limited to, alleviation of symptoms, diminished extent of disease, stable disease state (i.e., not worsening), delay or slowing of disease progression, amelioration or palliation of disease state, and remission (whether partial response or complete response), whether detectable or undetectable.
  • Those in need of treatment include those already with the condition or disease as well as those prone to have the condition or disease or those in which the condition or disease is to be prevented.
  • slow down lessen, weaken, moderate, alleviate, etc., the meaning of eliminate, disappear, not occur, etc. is also included.
  • subject herein refers to an organism receiving treatment for a particular disease or condition as described herein.
  • a “subject” includes a mammal, such as a human, a primate (eg, monkey) or a non-primate mammal, receiving treatment for a disease or disorder.
  • an effective amount herein refers to an amount of a therapeutic agent effective to prevent or alleviate a disease condition or the progression of the disease when administered alone or in combination with another therapeutic agent to a cell, tissue or subject.
  • Effective amount also refers to an amount of a compound sufficient to alleviate symptoms, eg, treat, cure, prevent or alleviate the associated medical condition, or to increase the rate of treatment, cure, prevent or alleviate such condition.
  • a therapeutically effective dose refers to that ingredient alone.
  • a therapeutically effective dose refers to the combined amounts of the active ingredients that produce a therapeutic effect, whether administered in combination, sequentially or simultaneously.
  • cancer refers to or describes the physiological condition in mammals typically characterized by unregulated cell growth. Both benign and malignant cancers are included in this definition.
  • tumor or “neoplastic” herein refers to all neoplastic cell growth and proliferation, whether malignant or benign, and to all pre-cancerous and cancerous cells and tissues.
  • cancer and “tumor” are not mutually exclusive when referred to herein.
  • EC50 refers to the half-maximal effective concentration, which includes the antibody concentration that induces a response halfway between baseline and maximum after a specified exposure time. EC50 essentially represents the concentration of antibody at which 50% of its maximal effect is observed and can be measured by methods known in the art.
  • the present application provides an antibody or antigen-binding fragment thereof that specifically binds G protein-coupled receptor C5 family subtype D (GPRC5D), wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and light chain variable region, and where
  • the light chain variable region comprises LCDR1, LCDR2, and LCDR3, and the LCDR1 has any sequence of LCDR1 shown below or has 1, 2, 3 or more amino acid insertions compared to the sequence, A deleted and/or substituted sequence, the LCDR2 having any of the LCDR2 sequences shown below or a sequence having 1, 2, 3 or more amino acid insertions, deletions and/or substitutions compared to said sequence, and
  • the LCDR3 has any sequence of LCDR3 shown below or a sequence with 1, 2, 3 or more amino acid insertions, deletions and/or substitutions compared to the sequence:
  • the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, and the HCDR1 has any sequence of HCDR1 shown below or has 1, 2, 3 or more amino acid insertions compared to the sequence, A deleted and/or substituted sequence, the HCDR2 having any of the HCDR2 sequences shown below or a sequence having 1, 2, 3 or more amino acid insertions, deletions and/or substitutions compared to said sequence, and The HCDR3 has any sequence of HCDR3 shown below or a sequence with 1, 2, 3 or more amino acid insertions, deletions and/or substitutions compared to the sequence:
  • the antibody or antigen-binding fragment thereof comprises the sequence of six CDRs in the combination of the following heavy chain variable region and light chain variable region: L1+H1, L2+H2, L3+H3, L4+H4, L5+H5, L6+H6, L7+H7, L8+H8, L9+H9, L10+H10, L11+H11, L12+H12, L13+H13, L14+H14, L15+H15, L16+ H16, L3+H17, L3+H18, L5+H19, L5+H20, L17+H13, L18+H13, L19+H13, L20+H13 or L21+H13, or have a sequence compared with the six CDRs Sequences of the six CDRs with 1, 2, 3 or more amino acid insertions, deletions and/or substitutions.
  • the application provides such an antibody or antigen-binding fragment thereof, wherein:
  • the light chain variable region sequence comprises SEQ ID NO: 14, 16, 18, 20, 22, 24, 26, 28, 118 ⁇ 120, 130 ⁇ 131, 144 ⁇ 145, 160 ⁇ 163, 172 ⁇ 175, 184-187, 196-197, 206-208, 218-221 and 231-233, or have 80%, 85%, 90%, 95%, 96%, Sequences with 97%, 98%, 99% or higher identity;
  • the heavy chain variable region sequence comprises SEQ ID NO: 13, 15, 17, 19, 21, 23, 25, 27, 121-125, 132-137, 146-152, 164-166, 176- 178, 188-190, 198-201, 209-212, 222 and 234-238, or have 80%, 85%, 90%, 95%, 96%, 97% of the sequence %, 98%, 99% or higher identity sequences.
  • the antibody or antigen-binding fragment thereof has a light chain variable region and a heavy chain variable region as follows:
  • the light chain variable region and the heavy chain variable region respectively comprise the sequences shown in SEQ ID NO: 14 and SEQ ID NO: 13;
  • the light chain variable region and the heavy chain variable region respectively comprise the sequences shown in SEQ ID NO: 16 and SEQ ID NO: 15;
  • the light chain variable region and the heavy chain variable region respectively comprise the sequences shown in SEQ ID NO: 18 and SEQ ID NO: 17;
  • the light chain variable region and the heavy chain variable region respectively comprise the sequences shown in SEQ ID NO: 20 and SEQ ID NO: 19;
  • the light chain variable region and the heavy chain variable region respectively comprise the sequences shown in SEQ ID NO: 22 and SEQ ID NO: 21;
  • the light chain variable region and the heavy chain variable region respectively comprise the sequences shown in SEQ ID NO: 24 and SEQ ID NO: 23;
  • the light chain variable region and the heavy chain variable region respectively comprise the sequences shown in SEQ ID NO: 26 and SEQ ID NO: 25;
  • the light chain variable region and the heavy chain variable region respectively comprise the sequences shown in SEQ ID NO: 28 and SEQ ID NO: 27;
  • the light chain variable region comprises the sequence shown in any one of SEQ ID NO: 118-120, and the heavy chain variable region comprises the sequence shown in any one of SEQ ID NO: 121-125;
  • the light chain variable region comprises the sequence shown in any one of SEQ ID NO: 130-131, and the heavy chain variable region comprises the sequence shown in any one of SEQ ID NO: 132-137;
  • the light chain variable region comprises the sequence shown in any one of SEQ ID NO: 144-145, and the heavy chain variable region comprises the sequence shown in any one of SEQ ID NO: 146-152;
  • the light chain variable region comprises the sequence shown in any one of SEQ ID NO: 160-163, and the heavy chain variable region comprises the sequence shown in any one of SEQ ID NO: 164-166;
  • the light chain variable region comprises the sequence shown in any one of SEQ ID NO: 172-175, and the heavy chain variable region comprises the sequence shown in any one of SEQ ID NO: 176-178;
  • the light chain variable region comprises the sequence shown in any one of SEQ ID NO: 184-187, and the heavy chain variable region comprises the sequence shown in any one of SEQ ID NO: 188-190;
  • the light chain variable region comprises the sequence shown in any one of SEQ ID NO: 196-197, and the heavy chain variable region comprises the sequence shown in any one of SEQ ID NO: 198-201;
  • the light chain variable region comprises the sequence shown in any one of SEQ ID NO: 206-208, and the heavy chain variable region comprises the sequence shown in any one of SEQ ID NO: 209-212;
  • the light chain variable region comprises the sequence shown in any one of SEQ ID NO: 218-221, and the heavy chain variable region comprises the sequence shown in any one of SEQ ID NO: 222;
  • the light chain variable region comprises the sequence shown in any one of SEQ ID NO: 231-233, and the heavy chain variable region comprises the sequence shown in any one of SEQ ID NO: 234-238;
  • the light chain variable region comprises 80%, 85%, 90%, 95%, 96%, 97% of the light chain variable region shown in any one of (1) to (18) above %, 98%, 99% or higher sequence identity
  • the heavy chain variable region comprises 80%, 85% %, 90%, 95%, 96%, 97%, 98%, 99% or more identical sequences.
  • said antibody or antigen-binding fragment thereof is chimeric, humanized or fully human.
  • said antibody or antigen-binding fragment thereof is capable of binding to human or monkey GPRC5D.
  • the antibody or antigen-binding fragment thereof comprises any constant region sequence of a human or murine antibody IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; preferably a human or murine antibody IgG1 , IgG2, IgG3 or IgG4 constant region sequence, or comprising 80%, 85%, 90%, 95%, 96%, 97%, 98% of the constant region sequence of human or murine antibody IgG1, IgG2, IgG3 or IgG4 , 99% or higher identity sequence; further, the antibody or its antigen-binding fragment is also coupled with a therapeutic agent or tracer; preferably, the therapeutic agent is selected from radioisotopes, chemotherapeutics or immunomodulatory The tracer is selected from radiological contrast agents, paramagnetic ions, metals, fluorescent labels, chemiluminescence labels, ultrasound contrast agents and photosensitizers: more preferably, the cytotoxic agent is
  • the antigen-binding fragment is selected from one or more of F(ab') 2 , Fab', Fab, Fv, scFv, Nanobody or affibody.
  • the present application also provides a multispecific antigen-binding molecule, which comprises the aforementioned antibody or antigen-binding fragment thereof, and an antigen-binding molecule that binds to an antigen other than GPRC5D, or binds to GPRC5D epitopes distinct from the aforementioned antibodies or antigen-binding fragments thereof.
  • said other antigen-binding molecule is an antibody or an antigen-binding fragment thereof.
  • said multispecific antigen binding molecule may be bispecific, trispecific or tetraspecific.
  • the multispecific antigen binding molecule may be bivalent, trivalent, tetravalent, pentavalent or hexavalent.
  • the present application also provides a chimeric antigen receptor (CAR), which at least comprises a signal peptide, an extracellular antigen-binding domain, a hinge region, a transmembrane domain, and an intracellular
  • CAR chimeric antigen receptor
  • the signal transduction domain, the extracellular antigen-binding domain comprises any one of the aforementioned GPRC5D antibodies or antigen-binding fragments thereof, or the aforementioned multispecific antigen-binding molecules.
  • the present application also provides an immune effector cell expressing the aforementioned chimeric antigen receptor, or comprising a nucleic acid fragment encoding the aforementioned chimeric antigen receptor; preferably, the immune effector cell Cells are selected from T cells, NK cells (natural killer cell), NKT cells (natural killer T cell), DNT cells (double negative T cell), monocytes, macrophages, dendritic cells or mast cells, the T cells are preferably selected from cytotoxic T cells (CTL), regulatory T cells or helper T cells; preferably, the immune effector cells are autologous immune effector cells or allogeneic immune effector cells
  • the present application also provides an isolated nucleic acid fragment encoding the aforementioned antibody or an antigen-binding fragment thereof, a multispecific antigen-binding molecule or a chimeric antigen receptor.
  • the present application also provides a vector, the vector comprising the aforementioned nucleic acid fragment.
  • the present application also provides a host cell comprising the aforementioned vector; preferably, the cell is a prokaryotic cell or a eukaryotic cell, such as bacteria (Escherichia coli), fungi (yeast), Insect cells or mammalian cells (CHO cell line or 293T cell line).
  • the cell is a prokaryotic cell or a eukaryotic cell, such as bacteria (Escherichia coli), fungi (yeast), Insect cells or mammalian cells (CHO cell line or 293T cell line).
  • the present application also provides a method for preparing the aforementioned antibody or its antigen-binding fragment or multispecific antigen-binding molecule, the method comprising culturing the aforementioned cell, and isolating the antibody or antigen-binding fragment expressed by the cell or multispecific antigen-binding molecules.
  • the present application also provides a method for preparing the aforementioned immune effector cells, the method includes introducing the nucleic acid fragment encoding the aforementioned CAR into the immune effector cells, optionally, the method further includes activating the Immune effector cells express the aforementioned CAR.
  • the present application also provides a pharmaceutical composition, which comprises the aforementioned antibody or its antigen-binding fragment, multispecific antigen-binding molecule, immune effector cell, nucleic acid fragment, carrier or according to the aforementioned method Prepare the obtained product; optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, diluent or adjuvant; optionally, the pharmaceutical composition further comprises an additional antineoplastic agent.
  • the pharmaceutically acceptable carrier is a carrier that does not weaken the viability and function of immune cells, and does not affect the specific binding of the antibody or its antigen-binding fragment to the antigen, including but not limited to cell culture medium, buffer , normal saline and balanced salt solution.
  • buffers include isotonic phosphates, acetates, citrates, borates, carbonates, and the like.
  • the pharmaceutically acceptable carrier is phosphate buffered saline containing 1% serum.
  • the present application also provides a method for treating tumor or cancer, the method comprising administering to a subject an effective amount of the aforementioned antibody or antigen-binding fragment thereof, multispecific antigen-binding molecule, immune effector Cells, nucleic acid fragments, vectors, products or pharmaceutical compositions prepared according to the aforementioned methods.
  • the tumor or cancer is a tumor or cancer expressing GPRC5D, preferably B-cell lymphoma, more preferably multiple myeloma (MM).
  • GPRC5D preferably B-cell lymphoma, more preferably multiple myeloma (MM).
  • the present application also provides the aforementioned antibodies or antigen-binding fragments thereof, multispecific antigen-binding molecules, immune effector cells, nucleic acid fragments, vectors, products or pharmaceutical compositions prepared according to the aforementioned methods for the treatment of tumors or use in cancer drugs;
  • the tumor or cancer is a tumor or cancer expressing GPRC5D, preferably B-cell lymphoma, more preferably multiple myeloma (MM).
  • GPRC5D preferably B-cell lymphoma, more preferably multiple myeloma (MM).
  • the present application also provides a kit, the kit comprising the aforementioned antibody or its antigen-binding fragment, multispecific antigen-binding molecule, immune effector cells, nucleic acid fragment, carrier, prepared according to the aforementioned method The obtained product or pharmaceutical composition.
  • the present application also provides a method for detecting the expression of GPRC5D in a biological sample, the method comprising, under the condition that a complex can be formed between the aforementioned antibody or its antigen-binding fragment and GPRC5D, allowing the The biological sample is contacted with the antibody or antigen-binding fragment thereof; preferably, the method further comprises detecting the formation of the complex, indicating the presence or expression level of GPRC5D in the sample.
  • the present application also provides the use of the aforementioned antibody or antigen-binding fragment thereof in the preparation of a GPRC5D detection reagent.
  • JNJ7564 comes from International Patent Application No. WO2018017786A2
  • sequence of 5F11 comes from International Patent Application No. WO2019154890A1.
  • the cloned VH and VL sequences recognizing the human GPRC5D epitope were recombined into human IgG1 CH and CL expression vectors to obtain recombinant plasmids. See Table 1 for the control antibody, CH, CL and Fc sequences.
  • B1 is full of Mab affinity eluent (citric acid monohydrate 3.92 g/L (No. 10007118, Sinopharm Chemical Reagent Co., Ltd.), Trisodium citrate dihydrate 1.88g/L (No. 10019418, Sinopharm Chemical Reagent Co., Ltd.)),
  • A1 was filled with Mab affinity balance solution (20 ⁇ PBS Buffer added ddH 2 O diluted to 1 ⁇ PBS (No. B548117-050, Sangon Bioengineering Shanghai Co., Ltd.)), the Buffer pump was filled with Mab affinity balance solution (20 ⁇ PBS Buffer was diluted to 1 ⁇ PBS by adding ddH 2 O (No.
  • hinge region sequence in bold in the human IgG1 Fc sequence contains the C220S mutation and is underlined.
  • humanGPRC5A (Uniprot: Q8NFJ5-1, SEQ ID NO: 8), humanGPRC5B (Uniprot: Q9NZH0-1, SEQ ID NO: 9), humanGPRC5C (Uniprot: Q9NQ84, SEQ ID NO: 10) and humanGPRC5D (Uniprot: Q9NZD1,
  • the nucleotide sequence of the amino acid sequence of SEQ ID NO: 11) was respectively cloned into the pLVX lentiviral vector, and virus particles were prepared in HEK293T cells.
  • FAB6594G-100UG staining, CHOK1 transfected with humanGPRC5D using human anti-human GPRC5D antibody (JNJ7564, self-produced) and goat anti-human IgG (H+L) antibody (Jackson, catalog number: 109605088) for staining, and then sorted the positive cell population with high expression level on the flow cytometer FACS Aria III (purchased from BD Biosciences) to a 96-well plate, and placed it at 37 ° C, 5% (v/ v) CO2 culture, after about 2 weeks, select some cells for expansion and select positive cell populations with better growth, higher fluorescence intensity, and better uniformity to continue to expand and culture and freeze in liquid nitrogen.
  • human anti-human GPRC5D antibody JNJ7564, self-produced
  • goat anti-human IgG (H+L) antibody Jackson, catalog number: 109605088
  • results of expression levels are identified in the table 3 and Figures 2A-2D, the results show that the stable CHOK1 cell lines expressing human GPRC5A, GPRC5B, GPRC5C and GPRC5D after puromycin pressurized selection have a relatively single positive peak, which can be used for FACS detection of antibodies and human GPRC5A, GPRC5B , GPRC5C and GPRC5D protein binding activity.
  • Example 1.3 Infect HEK293T cell line with lentivirus packaging human GPRC5D plasmid vector, in DMEM Medium containing 2 ⁇ g/ml puromycin (Gibco, Cat. No. A1113803) containing 10% (v/v) fetal bovine serum (ExCell Bio, Cat. (Gibco, Cat. No. 10569044) culture medium for 2 weeks, using human anti-human GPRC5D antibody (JNJ7564, self-produced) and goat anti-human IgG (H+L) antibody (Jackson, Cat.
  • cynoGPRC5D For the specific method of preparing CHO-K1 recombinant cell line stably expressing monkey GPRC5D (cynoGPRC5D), refer to Example 1.3. cynoGPRC5D amino acid sequence, NCBI: XP_005570249.1 (SEQ ID NO: 12). Use human anti-cynoGPRC5D antibody (JNJ7564, self-produced) and goat anti-human IgG (H+L) antibody (Jackson, catalog number: 109605088) to detect in flow cytometer FACS CantoII (purchased from BD Biosciences), and the cell lines with better expression The expression identification results are shown in Table 5 and Figure 4A. The results show that CHOK1-cynoGPRC5D after puromycin pressure selection has a relatively single positive peak, which can be used for FACS detection of cross-activity between antibodies and monkey GPRC5D protein.
  • Example 1.4 For the preparation method of HEK293T recombinant cell line stably expressing monkey GPRC5D (cynoGPRC5D), see Example 1.4. See Table 6 and Figure 4B for the expression identification results of cell lines with better expression. The results show that HEK293T-cynoGPRC5D after puromycin pressurized selection has a relatively single positive peak, which can be used for FACS detection of crossover between antibodies and monkey GPRC5D protein active.
  • FACS detection of the binding activity of the control antibody to cells expressing human GPRC5D and monkey GPRC5D is shown in Figures 5A-5C, and the IgG subtype control is human IgG1.
  • JNJ7564-hIgG1 and 5F11-hIgG1 have good binding activity to NCI-H929 tumor cells expressing human GPRC5D protein, HEK293T-hGPRC5D and HEK293T-cynoGPRC5D recombinant cells
  • 5F11-hIgG1 has good binding activity to NCI-H929 cells and HEK293T-hGPRC5D recombinant cells
  • the binding activity of JNJ7564-hIgG1 and HEK293T-cynoGPRC5D recombinant cells is relatively stronger.
  • Embodiment 2 Preparation of anti-human GPRC5D hybridoma monoclonal antibody
  • Anti-human GPRC5D monoclonal antibody was produced by immunizing mice. 6-8 week-old female SJL mice (purchased from Shanghai Slack Experimental Animal Co., Ltd.) were used in the experiment, and the mice were fed under SPF conditions. After the mice were purchased, they were raised in a laboratory environment for 1 week, with a 12/12 hour light/dark cycle adjustment, a temperature of 20-25° C., and a humidity of 40-60%. The acclimatized mice were immunized according to the following scheme.
  • the human GPRC5D overexpression cell line (HEK293T-hGPRC5D, in-house) was washed twice with PBS, then mixed with oligonucleotide CpG (ODN 1826, synthesized from Shanghai Sangon Biotech), and each mouse First, 50 ⁇ l of emulsified Titer max (purchased from sigma, Cat. T2684) was injected intraperitoneally, and after waiting for 15 minutes, each mouse was injected intraperitoneally with 5 ⁇ 10 6 cells/100 ⁇ l of cells.
  • emulsified Titer max purchased from sigma, Cat. T2684
  • the human GPRC5D overexpression cell line was washed twice with PBS, mixed with CpG, and 5 ⁇ 10 6 cells/100 ⁇ l cells were injected intraperitoneally into each mouse. Subsequent booster immunizations were carried out alternately according to the method of initial immunization and first booster immunization, and the immunogens were alternately used monkey GPRC5D overexpression cell line (HEK293T-cynoGPRC5D, in-house) and human GPRC5D overexpression cell line (HEK293T-hGPRC5D).
  • the interval between each immunization was 7 days, and the blood collection operation of the mice was carried out on the 5th day after the second and fourth booster immunization respectively, and the serum was separated, and the cell based ELISA method was used The titer of specific antibody in serum was determined.
  • HEK293T-hGPRC5D, HEK293T-cynoGPRC5D, CHOK1-hGPRC5D, CHOK1-cynoGPRC5D recombinant cells and blank control cells obtained in Example 1 were inoculated into 96 Well cell plate (manufacturer: corning, product number: 3599), incubate overnight in a cell culture incubator, remove the supernatant medium, add 50 ⁇ l of fixative solution (manufacturer: Beyotime, product number: P0098-500ML) to each well, and store in a fume hood at room temperature Stand for 30 minutes, remove the supernatant fixative, wash twice with PBST, then add 0.5% skimmed milk (manufacturer: Sangon, article number: A600669-0250) to block at room temperature for 2 hours, pour off the blocking solution, wash the plate twice with PBST, and 50 ⁇ l/well
  • mice were injected intraperitoneally with 5 ⁇ 10 6 HEK293T-hGPRC5D cells, and the mice were sacrificed 3 days later to collect splenocytes and lymphocytes. After centrifugation at 1500 rpm, the supernatant was discarded, and ACK lysate (Gibco, Cat.A1049201) was added to the cells to lyse the erythrocytes doped in the cells to obtain a cell suspension, and the DMEM base medium (purchased from Gibco, Cat. No. 10569044) 1500 rpm, wash the cells 3 times, count the cells, then mix with mouse myeloma cells SP2/0 (purchased from ATCC, Cat. Cell fusion.
  • ACK lysate Gibco, Cat. No. 10569044
  • the fused cells were diluted to contain 20% (v/v) fetal bovine serum (purchased from ExCell Bio, Cat.FND500), 1 ⁇ HAT (purchased from sigma, Cat.H0262-10VL), bovine insulin (purchased from Yeason, Cat.40107ES25), NEAA (purchased from Gibco, Cat.11140050) in DMEM medium, then add 5 ⁇ 10 4 cells/200 ⁇ L per well into a 96-well cell culture plate, put in 5% (v/v) CO 2. Cultivate in a 37°C incubator.
  • the supernatant of the fusion plate was screened by cell based ELISA to confirm the binding activity to human GPRC5D overexpression cells; for the supernatant of positive clones, it was confirmed by cell based ELISA and monkey GPRC5D overexpression cells and FACS confirmed that it was compatible with endogenous cells NCI- Binding activity of H929.
  • the qualified positive clones were selected and subcloned with semi-solid medium (purchased from stemcell, Cat.03810).
  • semi-solid medium purchased from stemcell, Cat.03810
  • w/w Fetal bovine serum, 1 ⁇ HT (purchased from sigma, Cat.H0137-10VL) in DMEM medium for expansion culture, and after 1 day, use cell based ELISA for preliminary screening, select positive single clones and expand to 24-well plates Continue to cultivate. After 3 days, the culture supernatant was further detected to evaluate its binding activity to monkey GPRC5D overexpression cells and endogenous cells NCI-H929.
  • the optimal clone was selected and contained in 10% (v/v) FBS DMEM medium at 37° C., 5% (v/v) CO 2
  • the optimal clone was expanded and cultured, and frozen in liquid nitrogen to obtain the hybridoma cells of the present application.
  • the hybridoma cells in the logarithmic growth phase were collected, and the cells were fully lysed with Trizol (Invitrogen, Cat No. 15596-018) and stored at -80°C for testing.
  • the samples were entrusted to Suzhou Jinweizhi Biotechnology Co., Ltd. to complete the amino acid sequence determination of the light chain and heavy chain variable regions of hybridoma positive clones.
  • the sequencing results were analyzed using MOE software, and the evolutionary tree was constructed according to the amino acid sequence of the protein encoded by the variable region. After eliminating the sequences that were close to the evolutionary tree according to the sequence similarity, 8 clones were screened: GPRC5D-mab01 ⁇ 08( Sequences are shown in Table 7, SEQ ID NO: 13-28).
  • Example 2.1 For the specific detection method, refer to Example 2.1.
  • the CHOK1-hGPRC5D, CHOK1-cynoGPRC5D recombinant cells and CHOK1 cells obtained in Example 1 were fixed, and 50 ⁇ l/well was added with 100 nM as the initial concentration, and the chimeric antibody and control antibody diluted 3 times were detected.
  • GPRC5D protein binding activity cell based ELISA detection results are shown in Figures 6A-6C, 7A-7C and 8A-8C. The results showed that the purified chimeric antibody combined with human GPRC5D protein in different degrees at ELISA level.
  • the negative control antibody hIgG1 is the antibody anti-hel-hIgG1 against chicken egg lysozyme (purchased from Baiying, article number: B117901). The data in the figure are OD450nm values.
  • the chimeric antibody can be well bound to NCI-H929 (high expression), MolP-8 (medium expression), RPMI-8226 (low expression) cells.
  • Example 4.2 For the specific method.
  • the CHOK1-hGPRC5A, CHOK1-hGPRC5B, and CHOK1-hGPRC5C cells were taken, and the results are shown in Table 10.
  • the results showed that none of the chimeric antibodies bound to CHOK1-hGPRC5A, CHOK1-hGPRC5B, and CHOK1-hGPRC5C cells, and had no cross-binding with these subtypes.
  • competition ELISA was used to analyze the competition between the GPRC5D chimeric antibody and the positive control molecule 5F11 for binding to the antigen.
  • the positive molecule 5F11 was biotin-labeled, hGPRC5D-LVP protein (manufacturer: Kaikai, product number: GPR-HM05P) 2 ⁇ g/mL was coated on an ELISA plate, and the Biotin-5F11 antibody was serially diluted three times starting from 100nM, and the Biotin- The OD 450 value of the 5F11 antibody was used to fit the curve using GraphPad Prism software, and the EC80 concentration was calculated as the reference concentration in the competitive ELISA.
  • the hGPRC5D-LVP protein was diluted to 2 ⁇ g/mL, coated with 50 ⁇ L/well on a 96-well high-adsorption microtiter plate, and coated overnight at 4 °C with 250 ⁇ L of blocking solution (PBS containing 2% (w/v) BSA) for two times at room temperature.
  • the CDRs of the murine antibody were grafted into corresponding human templates to form a variable region sequence in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • FR region sequence backbone sequence
  • the CDR amino acid residues of the antibody are determined and annotated by the Kabat numbering system.
  • the humanized light chain templates of the murine antibody GPRC5D-mab01 are IGKV1-9*01 and IGKJ4*01, and the humanized heavy chain templates are IGHV2-26*01 and IGHJ6*01.
  • the specific mutation design is shown in Table 12.
  • Graft means that the mouse antibody CDR is implanted into the FR region sequence of the human germline template; L45P means that the 45th position of Graft is mutated into P, and so on. Mutant amino acids are numbered in natural order.
  • variable region of the GPRC5D-mab01 humanized antibody is as follows:
  • amino acid sequence of GPRC5D-mab01.VL1 is shown in SEQ ID NO: 118:
  • amino acid sequence of GPRC5D-mab01.VL2 is shown in SEQ ID NO: 119:
  • amino acid sequence of GPRC5D-mab01.VL3 is shown in SEQ ID NO: 120:
  • amino acid sequence of GPRC5D-mab01.VH1 is shown in SEQ ID NO: 121:
  • amino acid sequence of GPRC5D-mab01.VH2 is shown in SEQ ID NO: 122:
  • amino acid sequence of GPRC5D-mab01.VH3 is shown in SEQ ID NO: 123:
  • amino acid sequence of GPRC5D-mab01.VH4 is shown in SEQ ID NO: 124:
  • amino acid sequence of GPRC5D-mab01.VH5 is shown in SEQ ID NO: 125:
  • amino acid sequence of the humanized light chain template IGKV1-9*01 is shown in SEQ ID NO: 126:
  • amino acid sequence of the humanized light chain template IGKJ4*01 is shown in SEQ ID NO: 127:
  • amino acid sequence of the humanized heavy chain template IGHV2-26*01 is shown in SEQ ID NO: 128:
  • amino acid sequence of the humanized heavy chain template IGHJ6*01 is shown in SEQ ID NO: 129:
  • the method is the same as 5.1.
  • the humanized light chain templates of the murine antibody GPRC5D-mab02 are IGKV3-20*02 and IGKJ2*01, and the humanized heavy chain templates are IGHV1-46*01 and IGHJ6*01.
  • the specific mutation design is shown in Table 15.
  • Graft means that the murine antibody CDR is implanted into the FR region sequence of the human germline template; L45V means that the 45th position of Graft is mutated from L to V, and so on. Mutant amino acids are numbered in natural order.
  • variable region of the GPRC5D-mab02 humanized antibody is as follows:
  • amino acid sequence of GPRC5D-mab02.VL1 is shown in SEQ ID NO: 130:
  • amino acid sequence of GPRC5D-mab02.VL2 is shown in SEQ ID NO: 131:
  • amino acid sequence of GPRC5D-mab02.VH1 is shown in SEQ ID NO: 132:
  • amino acid sequence of GPRC5D-mab02.VH2 is shown in SEQ ID NO: 133:
  • amino acid sequence of GPRC5D-mab02.VH2a is shown in SEQ ID NO: 134:
  • amino acid sequence of GPRC5D-mab02.VH3 is shown in SEQ ID NO: 135:
  • amino acid sequence of GPRC5D-mab02.VH3a is shown in SEQ ID NO: 136:
  • amino acid sequence of GPRC5D-mab02.VH4 is shown in SEQ ID NO: 137:
  • amino acid sequence of the humanized light chain template IGKV3-20*02 is shown in SEQ ID NO: 138:
  • amino acid sequence of the humanized light chain template IGKJ2*01 is shown in SEQ ID NO: 139:
  • amino acid sequence of the humanized heavy chain template IGHV1-46*01 is shown in SEQ ID NO: 140:
  • amino acid sequence of the humanized heavy chain template IGHJ6*01 is shown in SEQ ID NO: 141:
  • This application selects different light chain and heavy chain sequences from the mutation design of the light chain and heavy chain variable regions of the humanized antibody of GPRC5D-mab02 mentioned above for cross-combination, and finally obtains a variety of GPRC5D-mab02 humanized antibodies , the amino acid sequences of the variable regions of each antibody are shown in Table 16 as follows:
  • the method is the same as 5.1.
  • the humanized light chain templates of the mouse antibody GPRC5D-mab03 are IGKV4-1*01/IGKV1-39*01 and IGKJ4*01, and the humanized heavy chain templates are IGHV1-3*01 and IGHJ6*01 .
  • the specific mutation design is shown in Table 18.
  • Graft means that the murine antibody CDR is implanted into the FR region sequence of the human germline template; A43S means that the 43rd A of Graft is mutated into S, and so on. Mutant amino acids are numbered in natural order.
  • variable region of the GPRC5D-mab03 humanized antibody is as follows:
  • amino acid sequence of GPRC5D-mab03.VL1 is shown in SEQ ID NO: 144:
  • amino acid sequence of GPRC5D-mab03.VL2 is shown in SEQ ID NO: 145:
  • amino acid sequence of GPRC5D-mab03.VH1 is shown in SEQ ID NO: 146:
  • amino acid sequence of GPRC5D-mab03.VH2 is shown in SEQ ID NO: 147:
  • amino acid sequence of GPRC5D-mab03.VH2a is shown in SEQ ID NO: 148:
  • amino acid sequence of GPRC5D-mab03.VH3 is shown in SEQ ID NO: 149:
  • amino acid sequence of GPRC5D-mab03.VH4 is shown in SEQ ID NO: 150:
  • amino acid sequence of GPRC5D-mab03.VH4a is shown in SEQ ID NO: 151:
  • amino acid sequence of GPRC5D-mab03.VH5 is shown in SEQ ID NO: 152:
  • amino acid sequence of the humanized light chain template IGKV4-1*01 is shown in SEQ ID NO: 153:
  • amino acid sequence of the humanized light chain template IGKV1-39*01 is shown in SEQ ID NO: 154:
  • amino acid sequence of the humanized light chain template IGKJ4*01 is shown in SEQ ID NO: 155:
  • amino acid sequence of the humanized heavy chain template IGHV1-3*01 is shown in SEQ ID NO: 156:
  • amino acid sequence of the humanized heavy chain template IGHJ6*01 is shown in SEQ ID NO: 157:
  • This application selects different light chain and heavy chain sequences from the mutation design of the light chain and heavy chain variable regions of the humanized antibody of GPRC5D-mab03 mentioned above for cross-combination, and finally obtains a variety of GPRC5D-mab03 humanized antibodies , the amino acid sequences of the variable regions of each antibody are shown in Table 19 as follows:
  • the method is the same as 5.1, the humanized light chain templates of mouse antibody GPRC5D-mab04 are IGKV1-39*01/IGKV2-28*01 and IGKJ4*01, and the humanized heavy chain templates are IGHV3-7*01 and IGHJ6*01 .
  • the specific mutation design is shown in Table 21.
  • Graft means that the murine antibody CDR is implanted into the FR region sequence of the human germline template; A43S means that the 43rd A of Graft is mutated into an S, and so on. Mutant amino acids are numbered in natural order.
  • variable region of the GPRC5D-mab04 humanized antibody is as follows:
  • amino acid sequence of GPRC5D-mab04.VL1 is shown in SEQ ID NO: 160:
  • amino acid sequence of GPRC5D-mab04.VL2 is shown in SEQ ID NO: 161:
  • amino acid sequence of GPRC5D-mab04.VL3 is shown in SEQ ID NO: 162:
  • amino acid sequence of GPRC5D-mab04.VH1 is shown in SEQ ID NO: 164:
  • amino acid sequence of GPRC5D-mab04.VH2 is shown in SEQ ID NO: 165:
  • amino acid sequence of GPRC5D-mab04.VH3 is shown in SEQ ID NO: 166:
  • amino acid sequence of the humanized light chain template IGKV1-39*01 is shown in SEQ ID NO: 167:
  • amino acid sequence of the humanized light chain template IGKV2-28*01 is shown in SEQ ID NO: 168:
  • amino acid sequence of the humanized light chain template IGKJ4*01 is shown in SEQ ID NO: 169:
  • amino acid sequence of the humanized heavy chain template IGHV3-7*01 is shown in SEQ ID NO: 170:
  • amino acid sequence of the humanized heavy chain template IGHJ6*01 is shown in SEQ ID NO: 171:
  • This application selects different light chain and heavy chain sequences from the mutation design of the light chain and heavy chain variable regions of the humanized antibody of GPRC5D-mab04 mentioned above for cross-combination, and finally obtains a variety of GPRC5D-mab04 humanized antibodies , the amino acid sequences of the variable regions of each antibody are shown in Table 22 as follows:
  • the method is the same as 5.1, the humanized light chain templates of mouse antibody GPRC5D-mab05 are IGKV6-21*01/IGKV3-11*01 and IGKJ4*01, and the humanized heavy chain templates are IGHV1-69*02 and IGHJ6*01 .
  • the specific mutation design is shown in Table 24.
  • Graft means that the murine antibody CDR is implanted into the FR region sequence of the human germline template; V58I means that the 58th V of Graft is mutated into I, and so on. Mutant amino acids are numbered in natural order.
  • variable region of the GPRC5D-mab05 humanized antibody is as follows:
  • amino acid sequence of GPRC5D-mab05.VL1 is shown in SEQ ID NO: 172:
  • amino acid sequence of GPRC5D-mab05.VL2 is shown in SEQ ID NO: 173:
  • amino acid sequence of GPRC5D-mab05.VL3 is shown in SEQ ID NO: 174:
  • amino acid sequence of GPRC5D-mab05.VL4 is shown in SEQ ID NO: 175:
  • amino acid sequence of GPRC5D-mab05.VH1 is shown in SEQ ID NO: 176:
  • amino acid sequence of GPRC5D-mab05.VH2 is shown in SEQ ID NO: 177:
  • amino acid sequence of GPRC5D-mab05.VH3 is shown in SEQ ID NO: 178:
  • amino acid sequence of the humanized light chain template IGKV6-21*01 is shown in SEQ ID NO: 179:
  • amino acid sequence of the humanized light chain template IGKV3-11*01 is shown in SEQ ID NO: 180:
  • amino acid sequence of the humanized light chain template IGKJ4*01 is shown in SEQ ID NO: 181:
  • amino acid sequence of the humanized heavy chain template IGHV1-69*02 is shown in SEQ ID NO: 182:
  • amino acid sequence of the humanized heavy chain template IGHJ6*01 is shown in SEQ ID NO: 183:
  • This application selects different light chain and heavy chain sequences for cross-combination from the mutation design of the light chain and heavy chain variable regions of the humanized antibody of GPRC5D-mab05 mentioned above, and finally obtains a variety of GPRC5D-mab05 humanized antibodies , the amino acid sequences of the variable regions of each antibody are shown in Table 25 as follows:
  • the method is the same as 5.1, the humanized light chain templates of mouse antibody GPRC5D-mab06 are IGKV4-1*01/IGKV1-27*01 and IGKJ2*01, and the humanized heavy chain templates are IGHV2-26*01 and IGHJ6*01 .
  • the specific mutation design is shown in Table 27.
  • Graft means that the murine antibody CDR is implanted into the FR region sequence of the human germline template; P43S means that the 43rd P of Graft is mutated into an S, and so on. Mutant amino acids are numbered in natural order.
  • variable region of the GPRC5D-mab06 humanized antibody is as follows:
  • amino acid sequence of GPRC5D-mab06.VL1 is shown in SEQ ID NO: 184:
  • amino acid sequence of GPRC5D-mab06.VL2 is shown in SEQ ID NO: 185:
  • amino acid sequence of GPRC5D-mab06.VL3 is shown in SEQ ID NO: 186:
  • amino acid sequence of GPRC5D-mab06.VL4 is shown in SEQ ID NO: 187:
  • amino acid sequence of GPRC5D-mab06.VH1 is shown in SEQ ID NO: 188:
  • amino acid sequence of GPRC5D-mab06.VH2 is shown in SEQ ID NO: 189:
  • amino acid sequence of GPRC5D-mab06.VH3 is shown in SEQ ID NO: 190:
  • amino acid sequence of the humanized light chain template IGKV4-1*01 is shown in SEQ ID NO: 191:
  • amino acid sequence of the humanized light chain template IGKV1-27*01 is shown in SEQ ID NO: 192:
  • amino acid sequence of the humanized light chain template IGKJ2*01 is shown in SEQ ID NO: 193:
  • amino acid sequence of the humanized heavy chain template IGHV2-26*01 is shown in SEQ ID NO: 194:
  • amino acid sequence of the humanized heavy chain template IGHJ6*01 is shown in SEQ ID NO: 195:
  • This application selects different light chain and heavy chain sequences from the mutation design of the first round of humanized antibody light chain and heavy chain variable regions of GPRC5D-mab06 for cross-combination, and finally obtains a variety of GPRC5D-mab06 human
  • the amino acid sequences of the variable regions of each antibody are shown in Table 28:
  • the method is the same as 5.1, the templates for the second round of humanized light chain of mouse antibody GPRC5D-mab06 are IGKV1-27*01 and IGKJ2*01, and the templates for the second round of humanized heavy chain are IGHV2-26*01 and IGHJ6*01 .
  • the specific mutation design is shown in Table 30.
  • Graft means that the murine antibody CDR is implanted into the FR region sequence of the human germline template; V43S means that the 43rd V of Graft is mutated into an S, and so on. Mutant amino acids are numbered in natural order.
  • variable region of the GPRC5D-mab06 humanized antibody is as follows:
  • amino acid sequence of GPRC5D-mab06.VL4 is shown in SEQ ID NO: 196:
  • amino acid sequence of GPRC5D-mab06.VL5 is shown in SEQ ID NO: 197:
  • amino acid sequence of GPRC5D-mab06.VH4 is shown in SEQ ID NO: 198:
  • amino acid sequence of GPRC5D-mab06.VH5 is shown in SEQ ID NO: 199:
  • amino acid sequence of GPRC5D-mab06.VH6 is shown in SEQ ID NO: 200:
  • amino acid sequence of GPRC5D-mab06.VH7 is shown in SEQ ID NO: 201:
  • amino acid sequence of the humanized light chain template IGKV1-27*01 is shown in SEQ ID NO: 202:
  • amino acid sequence of the humanized light chain template IGKJ2*01 is shown in SEQ ID NO: 203:
  • amino acid sequence of the humanized heavy chain template IGHV2-26*01 is shown in SEQ ID NO: 204:
  • the amino acid sequence of the humanized heavy chain template IGHJ6*01 is shown in SEQ ID NO: 205:
  • This application selects different light chain and heavy chain sequences from the second round of mutation design of the light chain and heavy chain variable regions of the humanized antibody of GPRC5D-mab06, and finally obtains a variety of GPRC5D-mab06 human
  • the amino acid sequences of the variable regions of each antibody are shown in Table 31:
  • the method is the same as 5.1, the humanized light chain templates of the murine antibody GPRC5D-mab07 are IGKV2-40*01 and IGKJ4*01, and the humanized heavy chain templates are IGHV1-69*02 and IGHJ6*01.
  • the specific mutation design is shown in Table 33.
  • Graft means that the mouse antibody CDR is implanted into the FR region sequence of the human germline template; Q50K means that the 50th position of Graft is mutated into K, and so on. Mutant amino acids are numbered in natural order.
  • variable region of the GPRC5D-mab07 humanized antibody is as follows:
  • amino acid sequence of GPRC5D-mab07.VL1 is shown in SEQ ID NO: 206:
  • amino acid sequence of GPRC5D-mab07.VL2 is shown in SEQ ID NO: 207:
  • amino acid sequence of GPRC5D-mab07.VL2a is shown in SEQ ID NO: 208:
  • amino acid sequence of GPRC5D-mab07.VH1 is shown in SEQ ID NO: 209:
  • amino acid sequence of GPRC5D-mab07.VH2 is shown in SEQ ID NO: 210:
  • amino acid sequence of GPRC5D-mab07.VH3 is shown in SEQ ID NO: 211:
  • amino acid sequence of GPRC5D-mab07.VH4 is shown in SEQ ID NO: 212:
  • amino acid sequence of the humanized light chain template IGKV2-40*01 is shown in SEQ ID NO: 213:
  • amino acid sequence of the humanized light chain template IGKJ4*01 is shown in SEQ ID NO: 214:
  • amino acid sequence of the humanized heavy chain template IGHV1-69*02 is shown in SEQ ID NO: 215:
  • amino acid sequence of the humanized heavy chain template IGHJ6*01 is shown in SEQ ID NO: 216:
  • the method is the same as 5.1, the templates for the second round of humanized light chain of mouse antibody GPRC5D-mab07 are IGKV2-40*01 and IGKJ4*01, and the templates for the second round of humanized heavy chain are IGHV1-69*02 and IGHJ6*01 .
  • the specific mutation design is shown in Table 36.
  • Graft means that the mouse antibody CDR is implanted into the FR region sequence of the human germline template; G43A means that the 43rd position of Graft is mutated into A, and so on. Mutant amino acids are numbered in natural order.
  • variable region of the GPRC5D-mab07 humanized antibody is as follows:
  • amino acid sequence of GPRC5D-mab07.VL1a is shown in SEQ ID NO: 218:
  • amino acid sequence of GPRC5D-mab07.VL1b is shown in SEQ ID NO: 219:
  • amino acid sequence of GPRC5D-mab07.VL1c is shown in SEQ ID NO: 220:
  • amino acid sequence of GPRC5D-mab07.VL1d is shown in SEQ ID NO: 221:
  • amino acid sequence of GPRC5D-mab07.VH4 is shown in SEQ ID NO: 222:
  • amino acid sequence of the humanized light chain template IGKV2-40*01 is shown in SEQ ID NO: 223:
  • amino acid sequence of the humanized light chain template IGKJ4*01 is shown in SEQ ID NO: 224:
  • amino acid sequence of the humanized heavy chain template IGHV1-69*02 is shown in SEQ ID NO: 225:
  • amino acid sequence of the humanized heavy chain template IGHJ6*01 is shown in SEQ ID NO: 226:
  • This application selects different light chain and heavy chain sequences from the second round of mutation design of the light chain and heavy chain variable regions of the humanized antibody of GPRC5D-mab07, and finally obtains a variety of GPRC5D-mab07 human
  • the amino acid sequences of the variable regions of each antibody are shown in Table 37:
  • the method is the same as 5.1.
  • the humanized light chain templates of the mouse antibody GPRC5D-mab08 are IGKV1-27*01/IGKV2-29*02 and IGKJ4*01, and the humanized heavy chain templates are IGHV1-3*01 and IGHJ6*01 .
  • the specific mutation design is shown in Table 39.
  • Graft means that the murine antibody CDR is implanted into the FR region sequence of the human germline template; L46A means that the 46th position of Graft is mutated into A, and so on. Mutant amino acids are numbered in natural order.
  • variable region of the GPRC5D-mab08 humanized antibody is as follows:
  • amino acid sequence of the humanized light chain template IGKV1-27*01 is shown in SEQ ID NO: 239:
  • amino acid sequence of the humanized light chain template IGKV2-29*02 is shown in SEQ ID NO: 240:
  • amino acid sequence of the humanized light chain template IGKJ4*01 is shown in SEQ ID NO: 241:
  • amino acid sequence of the humanized heavy chain template IGHV1-3*01 is shown in SEQ ID NO: 242:
  • the amino acid sequence of the humanized heavy chain template IGHJ6*01 is shown in SEQ ID NO: 243:
  • This application selects different light chain and heavy chain sequences from the mutation design of the light chain and heavy chain variable regions of the above-mentioned humanized antibody of GPRC5D-mab08 for cross-combination, and finally obtains a variety of GPRC5D-mab08 humanized antibodies , the amino acid sequences of the variable regions of each antibody are shown in Table 40:
  • Example 4 cell-based ELISA In order to detect the binding activity of the GPRC5D humanized antibody to the full-length human GPRC5D protein, the same detection method as in Example 4 cell-based ELISA was used to detect the binding of the humanized antibody to the human GPRC5D protein , the test results are shown in Figures 13A-13J, 14A-14J, and the results show that the purified humanized antibody binds to the full-length human GPRC5D protein at the ELISA level to varying degrees.

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Abstract

涉及针对G蛋白偶联受体C5家族亚型D(GPRC5D)抗体及其应用。具体而言，公开了一种特异性结合GPRC5D的抗体或其抗原结合片段，其编码核酸、表达载体和表达细胞、制备方法、药物组合物、以及它们用于制备治疗疾病的药物组合物中的用途，例如治疗肿瘤中的用途。对于GPRC5D抗体治疗药物和检测试剂的开发具有重要意义。

Description

一种GPRC5D抗体及其应用
相关专利申请的交叉引用
本申请要求2021年12月31日向中国国家知识产权局提交的，专利申请号为202111668463.1，发明名称为《一种GPRC5D抗体及其应用》的中国专利申请的优先权。上述在先申请的全文通过引用的方式并入本申请中。
技术领域
本申请涉及生物医药领域，具体而言，涉及一种GPRC5D抗体及其应用。
背景技术
多发性骨髓瘤(Multiple myeloma，MM)是一种浆细胞恶性肿瘤，其特征是骨髓中的浆细胞像肿瘤细胞一样无限制地增生，并伴有单克隆免疫球蛋白分泌，从而引起多发性溶骨性损害、高钙血症、贫血、肾脏损害、反复感染等一系列临床表现。造血干细胞移植和以免疫调节剂及蛋白酶抑制剂等联合治疗，显著改善预后。近年来CAR T疗法在多发性骨髓瘤治疗中取得突破性进展，尤其以B细胞成熟抗原(BCMA)为靶点的CAR T在治疗多发性骨髓瘤上显示出了积极的临床结果。但对于BCMA阴性或BCMA低表达的多发性骨髓瘤患者来说，在接受靶向BCMA CAR-T细胞治疗后仍会复发，因此，多发性骨髓瘤治疗还需寻找其他更具潜力的新靶点。
GPRC5D是G蛋白偶联受体C5家族亚型D，属于一种孤儿受体，为7次跨膜蛋白。GPRC5D是BCMA之后一个新的多发性骨髓瘤靶点，组织表达谱研究发现GPRC5D特异性地在多发性骨髓瘤的浆细胞高表达，而在正常组织中低表达，仅限于具免疫赦免性的毛囊区域。并且GPRC5D与BCMA的表达并不重合。对于BCMA丢失的肿瘤复发模型中，GPRC5D CAR-T仍有治疗效果，克服肿瘤逃逸。理论上针对GPRC5D和BCMA的双靶向也可以覆盖更多人群，并带来更好的临床应答。因此，开发GPRC5D特异性抗体及相关的生物制品具有巨大潜在的临床需求。
发明内容
本申请公开了一种特异性结合G蛋白偶联受体C5家族亚型D(GPRC5D)的抗体或其抗原结合片段、多特异性抗原结合分子、核酸片段、载体(vector)、宿主细胞、免疫效应细胞、制备方法、药物组合物、制药用途以及肿瘤或癌症(例如B细胞淋巴瘤或多发性骨髓瘤)的治疗方法。
在一方面，本申请提供了一种特异性结合G蛋白偶联受体C5家族亚型D(GPRC5D)的抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段包含重链可变区和轻链可变区，并且其中
(1)所述轻链可变区包含LCDR1、LCDR2和LCDR3，所述LCDR1具有以下所示的LCDR1的任一序列或者与所述序列相比具有1、2、3或更多个氨基酸插入、缺失和/或替换的序列，所述LCDR2具有以下所示的LCDR2的任一序列或者与所述序列相比具有1、2、3或更多个氨基酸插入、缺失和/或替换的序列，并且所述LCDR3具有以下所示的LCDR3的任一序列或者与所述序列相比具有1、2、3或更多个氨基酸插入、缺失和/或替换的序列：
编号 LCDR1 LCDR2 LCDR3
L1 SEQ ID NO：35 SEQ ID NO：36 SEQ ID NO：37
L2 SEQ ID NO：38 SEQ ID NO：39 SEQ ID NO：37
L3 SEQ ID NO：46 SEQ ID NO：47 SEQ ID NO：48
L4 SEQ ID NO：49 SEQ ID NO：50 SEQ ID NO：48
L5 SEQ ID NO：57 SEQ ID NO：58 SEQ ID NO：59
L6 SEQ ID NO：60 SEQ ID NO：61 SEQ ID NO：59
L7 SEQ ID NO：68 SEQ ID NO：69 SEQ ID NO：70
L8 SEQ ID NO：71 SEQ ID NO：72 SEQ ID NO：70
L9 SEQ ID NO：79 SEQ ID NO：80 SEQ ID NO：81
L10 SEQ ID NO：82 SEQ ID NO：83 SEQ ID NO：81
L11 SEQ ID NO：90 SEQ ID NO：91 SEQ ID NO：92
L12 SEQ ID NO：93 SEQ ID NO：94 SEQ ID NO：92
L13 SEQ ID NO：101 SEQ ID NO：102 SEQ ID NO：103
L14 SEQ ID NO：104 SEQ ID NO：105 SEQ ID NO：103
L15 SEQ ID NO：112 SEQ ID NO：113 SEQ ID NO：114
L16 SEQ ID NO：115 SEQ ID NO：116 SEQ ID NO：114
L17 SEQ ID NO：217 SEQ ID NO：102 SEQ ID NO：103
L18 SEQ ID NO：227 SEQ ID NO：102 SEQ ID NO：103
L19 SEQ ID NO：228 SEQ ID NO：102 SEQ ID NO：103
L20 SEQ ID NO：229 SEQ ID NO：102 SEQ ID NO：103
L21 SEQ ID NO：230 SEQ ID NO：102 SEQ ID NO：103
和，
(2)所述重链可变区包含HCDR1、HCDR2和HCDR3，所述HCDR1具有以下所示的HCDR1的任一序列或者与所述序列相比具有1、2、3或更多个氨基酸插入、缺失和/或替换的序列，所述HCDR2具有以下所示的HCDR2的任一序列或者与所述序列相比具有1、2、3或更多个氨基酸插入、缺失和/或替换的序列，并且所述HCDR3具有以下所示的HCDR3的任一序列或者与所述序列相比具有1、2、3或更多个氨基酸插入、缺失和/或替换的序列：
编号 HCDR1 HCDR2 HCDR3
H1 SEQ ID NO：29 SEQ ID NO：30 SEQ ID NO：31
H2 SEQ ID NO：32 SEQ ID NO：33 SEQ ID NO：34
H3 SEQ ID NO：40 SEQ ID NO：41 SEQ ID NO：42
H4 SEQ ID NO：43 SEQ ID NO：44 SEQ ID NO：45
H5 SEQ ID NO：51 SEQ ID NO：52 SEQ ID NO：53
H6 SEQ ID NO：54 SEQ ID NO：55 SEQ ID NO：56
H7 SEQ ID NO：62 SEQ ID NO：63 SEQ ID NO：64
H8 SEQ ID NO：65 SEQ ID NO：66 SEQ ID NO：67
H9 SEQ ID NO：73 SEQ ID NO：74 SEQ ID NO：75
H10 SEQ ID NO：76 SEQ ID NO：77 SEQ ID NO：78
H11 SEQ ID NO：84 SEQ ID NO：85 SEQ ID NO：86
H12 SEQ ID NO：87 SEQ ID NO：88 SEQ ID NO：89
H13 SEQ ID NO：95 SEQ ID NO：96 SEQ ID NO：97
H14 SEQ ID NO：98 SEQ ID NO：99 SEQ ID NO：100
H15 SEQ ID NO：106 SEQ ID NO：107 SEQ ID NO：108
H16 SEQ ID NO：109 SEQ ID NO：110 SEQ ID NO：111
H17 SEQ ID NO：40 SEQ ID NO：142 SEQ ID NO：42
H18 SEQ ID NO：40 SEQ ID NO：143 SEQ ID NO：42
H19 SEQ ID NO：51 SEQ ID NO：158 SEQ ID NO：53
H20 SEQ ID NO：51 SEQ ID NO：159 SEQ ID NO：53
。
在另一方面中，本申请提供了一种多特异性抗原结合分子，其中，所述多特异性抗原结合分子包含前述抗体或其抗原结合片段，以及结合GPRC5D以外其他抗原的抗原结合分子，或结合与前述抗体或其抗原结合片段不同的GPRC5D表位。
在另一方面中，本申请提供了一种嵌合抗原受体(CAR)，其中，所述嵌合抗原受体至少包含信号肽、胞外抗原结合结构域、铰链区、跨膜结构域和胞内信号传导结构域，所述胞外抗原结合结构域包含前述GPRC5D抗体或其抗原结合片段，或前述多特异性抗原结合分子。
在另一方面中，本申请提供了一种免疫效应细胞，其中，所述免疫效应细胞表达前述嵌合抗原受体，或包含编码前述嵌合抗原受体的核酸片段。
在另一方面中，本申请提供了一种分离的核酸片段，所述核酸片段编码前述抗体或其抗原结合片段、前述多特异性抗原结合分子或前述嵌合抗原受体。
在另一方面中，本申请提供了一种载体，其中，所述载体包含前述核酸片段。
在另一方面中，本申请提供了一种宿主细胞，其中，所述宿主细胞包含前述载体。
在另一方面中，本申请提供了一种制备前述抗体或其抗原结合片段或前述多特异性抗原结合分子的方法，其中，所述方法包括培养前述细胞，以及分离所述细胞表达的抗体、抗原结合片段或多特异性抗原结合分子。
在另一方面中，本申请提供了一种制备前述免疫效应细胞的方法，其中，所述方法包括将编码前述CAR的核酸片段导入免疫效应细胞。
在另一方面中，本申请提供了一种药物组合物，其中，所述药物组合物包含前述抗体或其抗原结合片段、前述多特异性抗原结合分子、前述免疫效应细胞、前述核酸片段、前述载体或根据前述方法制备获得的产品。
在另一方面中，本申请提供了一种治疗肿瘤或癌症的方法，其中，所述方法包括向受试者施用有效量的前述抗体或其抗原结合片段、前述多特异性抗原结合分子、前述免疫效应细胞、前述核酸片段、前述载体或根据前述方法制备获得的产品或前述药物组合物；所述肿瘤或癌症为表达GPRC5D的肿瘤或癌症。
在另一方面中，本申请提供了前述抗体或其抗原结合片段、前述多特异性抗原结合分子、前述免疫效应细胞、前述核酸片段、前述载体或根据前述方法制备获得的产品或前述药物组合物在制备治疗肿瘤或癌症药物中的用途；所述肿瘤或癌症为表达GPRC5D的肿瘤或癌症。
在另一方面中，本申请提供了前述抗体或其抗原结合片段、前述多特异性抗原结合分子、前述免疫效应细胞、前述核酸片段、前述载体或根据前述方法制备获得的产品或前述药物组 合物，用于治疗肿瘤或癌症；所述肿瘤或癌症为表达GPRC5D的肿瘤或癌症。
在另一方面中，本申请提供了一种试剂盒，其中，所述试剂盒包含前述抗体或其抗原结合片段、前述多特异性抗原结合分子、前述免疫效应细胞、前述核酸片段、前述载体或根据前述方法制备获得的产品或前述药物组合物。
在另一方面中，本申请提供了一种检测生物学样品中GPRC5D表达的方法，所述方法包括在前述抗体或其抗原结合片段与GPRC5D之间能够形成复合物的条件下，使所述生物学样品与所述的抗体或其抗原结合片段接触。
在另一方面中，本申请提供了前述抗体或其抗原结合片段在制备GPRC5D检测试剂中的用途。
BCMA阴性或低表达是多发性骨髓瘤复发或难治的常见原因，本申请聚焦于复发/难治性多发性骨髓瘤的另一靶点GPRC5D，提供了靶向人GPRC5D抗体或其抗原结合片段，能以较高亲和力结合人或猴GPRC5D，而不结合或以低亲和力结合GPRC5A、GPRC5B或GPRC5C蛋白，这对于进一步开发针对GPRC5D靶点的抗体(单抗或多特异性抗体)、细胞治疗产品具有重要意义。
附图说明
图1.FACS检测内源细胞GPRC5D表达量。
图2A.表达人GPRC5A蛋白的CHOK1稳转细胞系FACS检测结果；图2B.表达人GPRC5B蛋白的CHOK1稳转细胞系FACS检测结果；图2C.表达人GPRC5C蛋白的CHOK1稳转细胞系FACS检测结果；图2D.表达人GPRC5D蛋白的CHOK1稳转细胞系FACS检测结果。
图3.表达人GPRC5D蛋白的HEK293T稳转细胞系FACS检测结果。
图4A.表达猴GPRC5D蛋白的CHOK1稳转细胞系FACS检测结果；图4B.表达猴GPRC5D蛋白的HEK293T稳转细胞系FACS检测结果。
图5A.FACS检测对照抗体与NCI-H929肿瘤细胞的结合反应；图5B.FACS检测对照抗体与293T-人-GPRC5D重组细胞的结合反应；图5C.FACS检测对照抗体与293T-猴-GPRC5D重组细胞的结合反应。
图6A～6C.Cell based ELISA检测嵌合抗体与CHOK1-hGPRC5D的结合反应。
图7A～7C.Cell based ELISA检测嵌合抗体与CHOK1-cynoGPRC5D的结合反应。
图8A～8C.Cell based ELISA检测嵌合抗体与CHOK1的结合反应。
图9A～9C.FACS检测嵌合抗体与NCI-H929(高表达)细胞的结合反应。
图10A～10C.FACS检测嵌合抗体与MolP-8(中表达)细胞的结合反应。
图11A～11C.FACS检测嵌合抗体与RPMI-8226(低表达)细胞的结合反应。
图12A～12C.ELISA检测嵌合抗体与阳性对照抗体5F11竞争性结合反应。
图13A～13J.Cell based ELISA检测人源化抗体与CHOK1-hGPRC5D的结合反应。
图14A～14J.Cell based ELISA检测人源化抗体与CHOK1-cynoGPRC5D的结合反应。
图15A～15J.FACS检测GPRC5D人源化抗体与NCI-H929的结合反应。
发明的详细描述
术语定义和说明
除非本申请另外定义，与本申请相关的科学和技术术语应具有本领域普通技术人员所理解的含义。
此外，除非本文另有说明，本文单数形式的术语应包括复数形式，复数形式的术语应包括单数形式。更具体地，如在本说明书和所附权利要求中所使用的，除非另外明确指出，否则单数形式“一种”和“这种”包括复数指示物。
本文术语“包括”、“包含”和“具有”之间可互换使用，旨在表示方案的包含性，意味着所述方案可存在除所列出的元素之外的其他元素。同时应当理解，在本文中使用“包括”、“包含”和“具有”描述，也提供“由……组成”方案。
本文术语“和/或”在本文使用时，包括“和”、“或”和“由所属术语链接的要素的全部或任何其他组合”的含义。
本文术语GPRC5D是指G蛋白偶联受体C5家族亚型D，属于一种孤儿受体，为7次跨膜蛋白。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。
本文术语“特异性结合”是指抗原结合分子(例如抗体)通常以高亲和力特异性结合抗原和实质上相同的抗原，但不以高亲和力结合不相关抗原。可采用本领域周知的方法测量平衡解离常数KD，如表面等离子共振(例如Biacore)或平衡透析法测定。
本文术语“抗原结合分子”按最广义使用，是指特异性结合抗原的分子。示例性地，抗原结合分子包括但不限于抗体或抗体模拟物。“抗体模拟物”是指能够与抗原特异性结合，但与抗体结构无关的有机化合物或结合域，示例性地，抗体模拟物包括但不限于affibody、affitin、affilin、经设计的锚蛋白重复蛋白(DARPin)、核酸适体或Kunitz型结构域肽。
本文术语“抗体”按最广义使用，是指包含来自免疫球蛋白重链可变区的足够序列和/或来自免疫球蛋白轻链可变区的足够序列，从而能够特异性结合至抗原的多肽或多肽组合。本文“抗体”涵盖各种形式和各种结构，只要它们展现出期望的抗原结合活性。本文“抗体”包括具有移植的互补决定区(CDR)或CDR衍生物的替代蛋白质支架或人工支架。此类支架包括抗体衍生的支架(其包含引入以例如稳定化抗体三维结构的突变)以及包含例如生物相容性聚合物的全合成支架。参见，例如Korndorfer等人，2003,Proteins:Structure,Function,and Bioinformatics,53(1):121-129(2003)；Roque等人，Biotechnol.Prog.20:639-654(2004)。此类支架还可以包括非抗体衍生的支架，例如本领域已知可用于移植CDR的支架蛋白，包括但不限于肌腱蛋白、纤连蛋白、肽适体等。
术语“抗体”包括完整抗体及其任何抗原结合片段(即“抗原结合部分”)或单链。“抗体”是指包含通过二硫键互相连接在一起的至少两条重(H)链和两条轻(L)链的糖蛋白，或其抗原结合部分。每条重链由重链可变区(在此缩写为VH)和重链恒定区组成。重链恒定区由三个结构域CH1、CH2和CH3组成。每条轻链由轻链可变区(在此缩写为VL)和轻 链恒定区组成。轻链恒定区由一个结构域CL组成。VH和VL区可进一步再分为高变区，称为互补决定区(CDR)，CDR散布在被称为构架区(FR)的更加保守的区域中。每个VH和VL，均由三个CDR和四个FR组成，它们从氨基端向羧基端以如下顺序排列：FR1，CDR1，FR2，CDR2，FR3，CDR3，FR4。重链和轻链的可变区含有可与抗原相互作用的结合结构域。抗体的恒定区可以介导免疫球蛋白与宿主组织或因子的结合，该宿主组织或因子包括免疫系统的各种细胞(例如效应细胞)和经典补体系统的第一成分(C1q)。由于免疫球蛋白重链恒定区的氨基酸组成和排列顺序不同，故其抗原性也不同。据此，可将本文“免疫球蛋白”分为五类，或称为免疫球蛋白的同种型，即IgM、IgD、IgG、IgA和IgE，其相应的重链分别为μ链、δ链、γ链、α链和ε链。同一类Ig根据其铰链区氨基酸组成和重链二硫键的数目和位置的差别，又可分为不同的亚类，如IgG可分为IgG1、IgG2、IgG3、IgG4，IgA可分为IgA1和IgA2。轻链通过恒定区的不同分为κ链或λ链。五类Ig中每类Ig都可以有κ链或λ链。
本文“抗体”还包括不包含轻链的抗体，例如，由单峰驼(Camelus dromedarius)、双峰驼(Camelus bactrianus)、大羊驼(Lama glama)、原驼(Lama guanicoe)和羊驼(Vicugna pacos)等骆驼科动物产生的重链抗体(heavy-chain antibodies,HCAbs)以及在鲨等软骨鱼纲中发现的免疫球蛋白新抗原受体(Ig new antigen receptor,IgNAR)。
本文术语“抗体”可以来源于任何动物，包括但不限于人和非人动物，所述非人动物可选自灵长类动物、哺乳动物、啮齿动物和脊椎动物，例如骆驼科动物、大羊驼、原鸵、羊驼、羊、兔、小鼠、大鼠或软骨鱼纲(例如鲨)。
本文术语“重链抗体”是指缺乏常规抗体的轻链的抗体。该术语具体包括但不限于在不存在CH1结构域的情况下包含VH抗原结合结构域以及CH2和CH3恒定结构域的同型二聚体抗体。
本文术语“纳米抗体”是指骆驼等体内存在天然的缺失轻链的重链抗体，克隆其可变区可以得到只有重链可变区组成的单域抗体，也称为VHH(Variable domain of heavy chain of heavy chain antibody)，它是最小的功能性抗原结合片段。
本文术语“VHH结构域”和“纳米抗体(nanobody)”、“单域抗体”(single domain antibody,sdAb)具有相同的含义并可互换使用，是指克隆重链抗体的可变区，构建仅由一个重链可变区组成的单域抗体，它是具有完整功能的最小的抗原结合片段。通常先获得天然缺失轻链和重链恒定区1(CH1)的重链抗体后，再克隆抗体重链的可变区，构建仅由一个重链可变区组成的单域抗体。
关于“重链抗体”和“单域抗体”、“VHH结构域”和“纳米抗体”的进一步描述可参见：Hamers-Casterman等，Nature.1993；363；446-8；Muyldermans的综述文章(Reviews in Molecular Biotechnology 74：277-302，2001)；以及以下专利申请，其被作为一般背景技术提及：WO 94/04678，WO 95/04079和WO 96/34103；WO 94/25591，WO 99/37681，WO 00/40968，WO 00/43507，WO 00/65057，WO 01/40310，WO 01/44301，EP 1134231和WO 02/48193；WO97/49805，WO 01/21817，WO 03/035694，WO 03/054016和WO 03/055527；WO 03/050531；WO 01/90190；WO03/025020；以及WO 04/041867，WO 04/041862，WO 04/041865，WO 04/041863，WO 04/062551，WO 05/044858，WO 06/40153，WO 06/079372，WO 06/122786，WO 06/122787和WO 06/122825以及这些申请中提到的其他现有技术。
本文术语“多特异性”是指抗体或其抗原结合片段结合例如不同抗原或同一抗原上的至少两种不同表位的能力。因此，诸如“双特异性”、“三特异性”、“四特异性”等术语是指抗体可以结合的不同表位的数目。例如，常规的单特异性IgG型抗体具有两个相同的抗原结合位点(互补位)，因此仅可以结合相同的表位(而不是结合不同的表位)。相比之下，多特异性抗体具有至少两种不同类型的互补位/结合位点，因此可以结合至少两种不同的表位。如本文所述，“互补决定区”是指抗体的抗原结合位点。此外，单个“特异性”可以指单个抗体中的一个、两个、三个或多于三个相同的互补决定区(一个单个抗体分子中的互补决定区/结合位点的实际数量称为“价”)。例如，单个天然IgG抗体是单特异性和二价的，因为它具有两个相同的互补位。相应地，多特异性抗体包含至少两种(不同的)互补决定区/结合位点。因此，术语“多特异性抗体”是指具有多于一个互补位并具有结合两种或多于两种不同表位的能力的抗体。术语“多特异性抗体”特别地包括如上文所定义的双特异性抗体，但是通常还包括蛋白质，例如特别结合三种或多于三种不同的表位的抗体、支架，即具有三种或多于三种互补位/结合位点的抗体。
本文术语“价”表示抗体/抗原结合分子中规定数目的结合位点的存在。因此,术语“单价”、“二价”、“四价”和“六价”分别表示抗体/抗原结合分子中一个结合位点、两个结合位点、四个结合位点和六个结合位点的存在。
本文“全长抗体”、“完好抗体”和“完整抗体”在本文中可互换使用，是指具有基本上与天然抗体结构相似的结构。
本文“抗原结合片段”和“抗体片段”在本文中可互换使用，其不具备完整抗体的全部结构，仅包含完整抗体的局部或局部的变体，所述局部或局部的变体具备结合抗原的能力。示例性地，本文“抗原结合片段”或“抗体片段”包括但不限于Fab、F(ab’)2、Fab’、Fab’-SH、Fd、Fv、scFv、双抗体(diabody)和单域抗体。
本文术语“嵌合抗体”是指以下抗体，其具有源自一种来源生物(如大鼠、小鼠、兔或羊驼)的免疫球蛋白的可变序列以及源自不同生物体(例如人)的免疫球蛋白的恒定区。用于生产嵌合抗体的方法是本领域已知的。参见例如，Morrison,1985,Science 229(4719):1202-7；Oi等人，1986,Bio Techniques 4:214-221；Gillies等人，1985 J Immunol Methods 125:191-202；以上通过援引加入并入本文。
本文术语“人源化抗体”是指经基因工程改造的非人源抗体，其氨基酸序列经修饰以提高与人源抗体的序列的同源性。通常而言，人源化抗体的全部或部分CDR区来自于非人源抗体(供体抗体)，全部或部分的非CDR区(例如，可变区FR和/或恒定区)来自于人源免疫球蛋白(受体抗体)。人源化抗体通常保留或部分保留了供体抗体的预期性质，包括但不限于，抗原特异性、亲和性、反应性、提高免疫细胞活性的能力或增强免疫应答的能力等。
本文术语“全人抗体”是指具有其中FR和CDR二者都源自人种系免疫球蛋白序列的可变区的抗体。此外，如果抗体包含恒定区，则恒定区也源自人种系免疫球蛋白序列。本文全人抗体可以包括不由人种系免疫球蛋白序列编码的氨基酸残基(例如，通过体外随机或位点特异性诱变或通过体内体细胞突变引入的突变)。然而，本文“全人抗体”不包括其中来源于另一个哺乳动物物种(例如小鼠)的种系的CDR序列已被移植到人框架序列上的抗体。
本文术语“可变区”是指抗体重链或轻链中牵涉使抗体结合抗原的区域，“重链可变区”与“VH”、“HCVR”可互换使用，“轻链可变区”与“VL”、“LCVR”可互换使用。天然抗体的重链和轻链的可变域一般具有相似的结构，每个域包含四个保守的框架区(FR)和三个高变区(HVR)。参见例如Kindt等人，Kuby Immunology,6 th ed.,W.H.Freeman and Co.,p.91(2007)。单个VH或VL域可足以赋予抗原结合特异性。
本文术语“互补决定区”与“CDR”可互换使用，通常指在轻链和重链可变结构域中均发现的高变区(HVR)。可变结构域中更高保守性的部分称为框架区(FR)。如本领域所理解的，表示抗体的高变区的氨基酸位置可以根据上下文和本领域已知的各种定义而变化。可变结构域内的一些位置可以被视为杂合高变位置，因为这些位置可以被认为是在一组标准(如IMGT或KABAT)下的高变区之内，而被认为在不同组的标准(如KABAT或IMGT)下的高变区之外。这些位置中的一个或更多个也可以在延伸的高变区中找到。本申请包括在这些杂合高变的位置中包含修饰的抗体。重链可变区CDR可缩写为HCDR，轻链可变区可缩写为LCDR。天然重链和轻链的可变结构域各自包含主要采用片层构型的四个框架区，其通过三个CDR(CDR1、CDR2和CDR3)连接，这三个CDR形成连接片层结构的环，并且在一些情况下形成片层结构的一部分。每条链中的CDR通过FR区按顺序FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4紧密保持在一起，并且与来自其他抗体链的CDR促成了抗体的抗原结合位点的形成(参见Kabat等人，Sequences of Protein sofImmunological Interest,National Institute of Health,Bethesda,Md.1987；其通过援引加入并入本文)。
对于CDR的进一步描述，参考Kabat等人,J.Biol.Chem.,252:6609-6616(1977)；Kabat等人，美国卫生与公共服务部，“Sequences of proteins of immunological interest”(1991)；Chothia等人，J.Mol.Biol.196:901-917(1987)；Al-Lazikani B.等人，J.Mol.Biol.,273:927-948(1997)；MacCallum等人,J.Mol.Biol.262:732-745(1996)；Abhinandan和Martin,Mol.Immunol.,45:3832-3839(2008)；Lefranc M.P.等人，Dev.Comp.Immunol.,27:55-77(2003)；以及Honegger和Plückthun,J.Mol.Biol.,309:657-670(2001)。本文“CDR”可由本领域公知的方式加以标注和定义，包括但不限于Kabat编号系统、Chothia编号系统或IMGT编号系统，使用的工具网站包括但不限于AbRSA网站(http://cao.labshare.cn/AbRSA/cdrs.php)、abYsis网站(www.abysis.org/abysis/sequence_input/key_annotation/key_annotation.cgi)和IMGT网站(http://www.imgt.org/3Dstructure-DB/cgi/DomainGapAlign.cgi#results)。本文CDR包括不同定义方式的氨基酸残基的重叠(overlap)和子集。
本文术语“Kabat编号系统”通常是指由Elvin A.Kabat提出的免疫球蛋白比对及编号系统(参见，例如Kabat等人，Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md.,1991)。
本文术语“Chothia编号系统”通常是指由Chothia等人提出的免疫球蛋白编号系统，其是基于结构环区的位置鉴定CDR区边界的经典规则(参见，例如Chothia&Lesk(1987)J.Mol.Biol.196:901-917；Chothia等人(1989)Nature 342:878-883)。
本文术语“IMGT编号系统”通常是指基于由Lefranc等人发起的国际免疫遗传学信息系统(The international ImMunoGeneTics information system(IMGT))的编号系统，可参阅Lefranc等人，Dev.Comparat.Immunol.27:55-77,2003。
本文术语“重链恒定区”是指抗体重链的羧基端部分，其不直接参与抗体与抗原的结合，但是表现出效应子功能，诸如与Fc受体的相互作用，其相对于抗体的可变结构域具有更保守的氨基酸序列。“重链恒定区”可选自CH1结构域，铰链区，CH2结构域，CH3结构域，或其变体或片段。“重链恒定区”包括“全长重链恒定区”和“重链恒定区片段”，前者具有基本上与天然抗体恒定区基本相似的结构，而后者仅包括“全长重链恒定区的一部分”。示例性地，典型的“全长抗体重链恒定区”由CH1结构域-铰链区-CH2结构域-CH3结构域组成；当抗体为IgE时，其还包括CH4结构域；当抗体为重链抗体时，则其不包括CH1结构域。示例性地，典型的“重链恒定区片段”可选自Fc或CH3结构域。
本文术语“轻链恒定区”是指抗体轻链的羧基端部分，其不直接参与抗体与抗原的结合，所述轻链恒定区可选自恒定κ结构域或恒定λ结构域。
本文中的术语“Fc区”用于定义抗体重链中含有恒定区的至少一部分的C端区域。该术语包括天然序列Fc区和变体Fc区。示例性地，人IgG重链Fc区可自Cys226或Pro230延伸至重链的羧基末端。然而，由宿主细胞生成的抗体可经历翻译后切割，自重链的C端切除一个或多个，特别是一个或两个氨基酸。因此，通过编码全长重链的特定核酸分子的表达由宿主细胞生成的抗体可包括全长重链，或者它可包括全长重链的切割变体。当重链的最终两个C端氨基酸是甘氨酸(G446)和赖氨酸(K447，编号方式依照Kabat EU索引)时可能就是这种情况。因此，Fc区的C端赖氨酸(Lys447)，或C端甘氨酸(Gly446)和赖氨酸(Lys447)可以存在或不存在。典型地，IgG Fc区包含IgG CH2和IgG CH3域，可选地，在此基础上还可包含完整或部分铰链区，但不包含CH1域。人IgG Fc区的“CH2域”通常自约位置231处的氨基酸残基延伸至约位置340处的氨基酸残基。在一个实施方案中，碳水化合物链附着于CH2域。本文中的CH2域可以是天然序列CH2域或变体CH2域。“CH3域”包含Fc区中在CH2域C端的那段残基(即自IgG的约位置341处的氨基酸残基至约位置447处的氨基酸残基)。本文中的CH3区可以是天然序列CH3域或变体CH3域(例如具有在其一条链中引入的“隆起”“节”，(knob)和在其另一条链中相应引入的“空腔”“穴”，(hole)的CH3域；参见美国专利No.5,821,333，通过援引明确收入本文)。如本文中描述的，此类变体CH3域可用于促进两条不相同抗体重链的异二聚化。
除非本文中另有规定，Fc区或恒定区中的氨基酸残基的编号依照EU编号系统，也称作EU索引，如Kabat等人，Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,MD,1991中描述的。
本文术语“Fc变体”是指，通过在Fc上合适的位点处存在一个或者多个氨基酸替换、插入或缺失突变引起Fc结构或功能的变化。“Fc变体间作用”指的是经突变设计的Fc变体之间，可以形成空间填充效应、静电导引、氢键作用、疏水作用等。Fc变体间相互作用有助于形成稳定的异源二聚体蛋白。优选的突变设计为“Knob-into-Hole”形式的突变设计。
Fc变体的突变设计技术在本领域内已经较为广泛的应用于制备双特异性抗体或者异源二聚的Fc融合蛋白形式。代表性的有Cater等人(Protein Engineering vol.9 no.7 pp.617-621，1996)提出的“Knob-into-Hole”形式；Amgen公司技术人员利用静电导引(Electrostatic Steering)形成含Fc的异源二聚体形式(US 20100286374 A1)；Jonathan H.Davis等人(Protein Engineering，Design&Selection pp.1-8，2010)提出的通过IgG/Ig链交换形成的异源二聚体形式 (SEEDbodies)；Genmab公司DuoBody(Science，2007.317(5844))平台技术形成的双特异性分子；Xencor公司的技术人员综合结构计算及Fc氨基酸突变，综合不同作用方式形成异源二聚体蛋白形式(mAbs 3：6，546-557；November/December 2011)；苏州康宁杰瑞公司的基于电荷网络的Fc改造方法(CN201110459100.7)得到异源二聚体蛋白形式；以及其它基于Fc氨基酸变化或者功能改造手段，达到形成异源二聚体功能蛋白的基因工程方法。本申请所述的Fc变体片段上的Knob/Hole结构指两条Fc片段各自突变，突变后可以通过“Knob-into-Hole”形式进行结合。优选用Cater等人的“knob-into-hole”模型在Fc区上进行位点突变的改造，以使得到的第一Fc变体和第二Fc变体能以“knob-into-hole”的形式结合在一起形成异源二聚体。从特定的免疫球蛋白类别和亚类中选择特定的免疫球蛋白Fc区在本领域技术人员所掌握的范围之内。优选人类抗体IgG1、IgG2、IgG3、IgG4的Fc区，更优选人抗体IgG1的Fc区。随机任选第一Fc变体或第二Fc变体中一个做knob的突变，另一个做hole的突变。
本文术语“保守氨基酸”通常是指属于同一类或具有类似特征(例如电荷、侧链大小、疏水性、亲水性、主链构象和刚性)的氨基酸。示例性地，下述每组内的氨基酸属于彼此的保守氨基酸残基，组内氨基酸残基的替换属于保守氨基酸的替换：
1)丙氨酸(A)、丝氨酸(S)、苏氨酸(T)；
2)天冬氨酸(D)、谷氨酸(E)；
3)天冬酰胺(N)、谷氨酰胺(Q)；
4)精氨酸(R)、赖氨酸(K)、组氨酸(H)；
5)异亮氨酸(I)、亮氨酸(L)、甲硫氨酸(M)、缬氨酸(V)；和
6)苯丙氨酸(F)、酪氨酸(Y)、色氨酸(W)。
本文术语“同一性”可通过以下方式计算获得：为确定两个氨基酸序列或两个核酸序列的“同一性”百分数，将所述序列出于最佳比较目的比对(例如，可以为最佳比对而在第一和第二氨基酸序列或核酸序列之一或二者中引入空位或可以为比较目的而抛弃非同源序列)。随后比较在对应氨基酸位置或核苷酸位置处的氨基酸残基或核苷酸。当第一序列中的位置由第二序列中对应位置处的相同氨基酸残基或核苷酸占据时，则所述分子在这个位置处是相同的。考虑到为最佳比对这两个序列而需要引入的空位的数目和每个空位的长度，两个序列之间的同一性百分数随所述序列共有的相同位置变化而变化。
可以利用数学算法实现两个序列间的序列比较和同一性百分数的计算。例如，使用已经集成至GCG软件包的GAP程序中的Needlema和Wunsch((1970)J.Mol.Biol.48:444-453)算法(在www.gcg.com可获得)，使用Blossum 62矩阵或PAM250矩阵和空位权重16、14、12、10、8、6或4和长度权重1、2、3、4、5或6，确定两个氨基酸序列之间的同一性百分数。又例如，使用GCG软件包中的GAP程序(在www.gcg.com可获得)，使用NWSgapdna.CMP矩阵和空位权重40、50、60、70或80和长度权重1、2、3、4、5或6，确定两个核苷酸序列之间的同一性百分数。特别优选的参数集合(和除非另外说明否则应当使用的一个参数集合)是采用空位罚分12、空位延伸罚分4和移码空位罚分5的Blossum62评分矩阵。还可以使用PAM120加权余数表、空位长度罚分12，空位罚分4，利用已经并入ALIGN程序(2.0版)的E.Meyers和W.Miller算法,((1989)CABIOS,4:11-17)确定两个氨基酸序列或核苷酸序列之间的同一性百 分数。
额外地或备选地，可以进一步使用本申请所述的核酸序列和蛋白质序列作为“查询序列”以针对公共数据库执行检索，以例如鉴定其他家族成员序列或相关序列。例如，可以使用Altschul等人,(1990)J.Mol.Biol.215:403-10的NBLAST及XBLAST程序(版本2.0)执行此类检索。BLAST核苷酸检索可以用NBLAST程序，评分＝100、字长度＝12执行，以获得与本申请核酸分子同源的核苷酸序列。BLAST蛋白质检索可以用XBLAST程序、评分＝50、字长度＝3执行，以获得与本申请蛋白质分子同源的氨基酸序列。为了出于比较目的获得带空位的比对结果，可以如Altschul等人,(1997)Nucleic Acids Res.25:3389-3402中所述那样使用空位BLAST。当使用BLAST和空位BLAST程序时，可以使用相应程序(例如，XBLAST和NBLAST)的默认参数。参见www.ncbi.nlm.nih.gov。
本文术语“嵌合抗原受体(CAR)”是指经改造以在免疫效应细胞上表达并且特异性结合抗原的人工细胞表面受体，其至少包含(1)细胞外抗原结合结构域，例如抗体的重链可变区和/或轻链可变区，(2)锚定CAR进入免疫效应细胞的跨膜结构域，和(3)胞内信号传导结构域。CAR能够利用细胞外抗原结合结构域以非MHC限制性的方式将T细胞和其它免疫效应细胞重定向至所选择的靶标，例如癌细胞。
本文术语“核酸”包括包含核苷酸的聚合物的任何化合物和/或物质。每个核苷酸由碱基，特别是嘌呤或嘧啶碱基(即胞嘧啶(C)、鸟嘌呤(G)、腺嘌呤(A)、胸腺嘧啶(T)或尿嘧啶(U))、糖(即脱氧核糖或核糖)和磷酸基团组成。通常，核酸分子由碱基的序列描述，由此所述碱基代表核酸分子的一级结构(线性结构)。碱基的序列通常表示为5’至3’。在本文中，术语核酸分子涵盖脱氧核糖核酸(DNA)，包括例如互补DNA(cDNA)和基因组DNA、核糖核酸(RNA)，特别是信使RNA(mRNA)、DNA或RNA的合成形式，以及包含两种或更多种这些分子的混合的聚合物。核酸分子可以是线性的或环状的。此外，术语核酸分子包括有义链和反义链二者，以及单链和双链形式。而且，本文所述的核酸分子可含有天然存在的或非天然存在的核苷酸。非天然存在的核苷酸的例子包括具有衍生的糖或磷酸骨架键合或化学修饰的残基的修饰的核苷酸碱基。核酸分子还涵盖DNA和RNA分子，其适合作为载体用于在体外和/或体内，例如在宿主或患者中，直接表达本申请的抗体。此类DNA(例如cDNA)或RNA(例如mRNA)载体可以是未修饰的或修饰的。例如，可以对mRNA进行化学修饰以增强RNA载体的稳定性和/或被编码分子的表达，从而可以将mRNA注入到受试者内以在体内产生抗体(参见例如Stadler等人，Nature Medicine 2017，published online 2017年6月12日，doi：10.1038/nm.4356或EP2101823B1)。
本文“分离的”核酸指已经与其天然环境的组分分开的核酸分子。分离的核酸包括在下述细胞中含有的核酸分子，所述细胞通常含有该核酸分子，但该核酸分子存在于染色体外或存在于不同于其天然染色体位置的染色体位置处。
本文术语“载体”是指能够扩增与其连接的另一个核酸的核酸分子。该术语包括作为自我复制型核酸结构的载体以及整合入已引入该载体的宿主细胞的基因组中的载体。某些载体能够指导与它们可操作连接的核酸的表达。这样的载体在本文中称为“表达载体”。
本文术语“宿主细胞”是指细胞中引入外源核酸的细胞，包括这种细胞的后代。宿主细胞包括“转化体”和“经转化的细胞”，其包括原代的经转化的细胞和来源于其的后代，而 不考虑传代的次数。后代在核酸内容物上可能与亲本细胞不完全相同，而是可以包含突变。本文中包括具有与在初始转化的细胞中筛选或选择的相同功能或生物学活性的突变体后代。
本文术语“药物组合物”是指这样的制剂，其以允许包含在其中的活性成分的生物学活性有效的形式存在，并且不含有对施用所述药物组合物的受试者具有不可接受的毒性的另外的成分。
本文术语“药学上可接受的载体”包括任何和所有溶剂、分散介质、包衣材料、表面活性剂、抗氧化剂、防腐剂(例如，抗细菌剂、抗真菌剂)、等渗剂、吸收延迟剂、盐、防腐剂、药物稳定剂、粘合剂、赋形剂、崩解剂、润滑剂、甜味剂、矫味剂、染料等及其组合，其为本领域技术人员所知(参见例如，Remington's Pharmaceutical Sciences,第18版.MackPrinting Company,1990,第1289-1329页)。除了与活性成分不相容的情况之外，考虑任一常规载体在治疗或药物组合物中的应用。
本文术语“治疗”是指外科手术或药物处理(surgical or therapeutic treatment)，其目的是预防、减缓(减少)治疗对象中不希望的生理变化或病变，如癌症和肿瘤。有益的或所希望的临床结果包括但不限于症状的减轻、疾病程度减弱、疾病状态稳定(即，未恶化)、疾病进展的延迟或减慢、疾病状态的改善或缓和、以及缓解(无论是部分缓解或完全缓解)，无论是可检测的或不可检测的。需要治疗的对象包括已患有病症或疾病的对象以及易于患上病症或疾病的对象或打算预防病症或疾病的对象。当提到减缓、减轻、减弱、缓和、缓解等术语时，其含义也包括消除、消失、不发生等情况。
本文术语“受试者”是指接受对如本文所述的特定疾病或病症的治疗的生物体。示例性地，“受试者”包括接受疾病或病症治疗的哺乳动物，如人、灵长类动物(例如，猴)或非灵长类哺乳动物。
本文术语“有效量”指单独给予或与另一治疗剂组合给予细胞、组织或对象时能有效防止或缓解疾病病症或该疾病进展的治疗剂用量。“有效量”还指足以缓解症状，例如治疗、治愈、防止或缓解相关医学病症，或治疗、治愈、防止或缓解这些病症的速度增加的化合物用量。当将活性成分单独给予个体时，治疗有效剂量单指该成分。当应用某一组合时，治疗有效剂量指产生治疗作用的活性成分的组合用量，而无论是组合、连续或同时给予。
本文术语“癌症”指向或描述哺乳动物中典型地以不受调节的细胞生长为特征的生理状况。此定义中包括良性和恶性癌症。本文术语“肿瘤”或“瘤”是指所有赘生性(neoplastic)细胞生长和增殖,无论是恶性的还是良性的，及所有癌前(pre-cancerous)和癌性细胞和组织。术语“癌症”和“肿瘤”在本文中提到时并不互相排斥。
本文术语“EC50”是指半最大有效浓度，其包括在指定暴露时间之后诱导基线与最大值之间的半途响应的抗体浓度。EC50本质上代表其中观察到其最大作用的50％的抗体浓度，可通过本领域已知方法测量。
具体实施方式
第一方面，本申请提供了一种特异性结合G蛋白偶联受体C5家族亚型D(GPRC5D)的抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段包含重链可变区和轻链可变区，并且其中
(1)所述轻链可变区包含LCDR1、LCDR2和LCDR3，所述LCDR1具有以下所示的LCDR1的任一序列或者与所述序列相比具有1、2、3或更多个氨基酸插入、缺失和/或替换的序列，所述LCDR2具有以下所示的LCDR2的任一序列或者与所述序列相比具有1、2、3或更多个氨基酸插入、缺失和/或替换的序列，并且所述LCDR3具有以下所示的LCDR3的任一序列或者与所述序列相比具有1、2、3或更多个氨基酸插入、缺失和/或替换的序列：
编号 LCDR1 LCDR2 LCDR3
L1 SEQ ID NO：35 SEQ ID NO：36 SEQ ID NO：37
L2 SEQ ID NO：38 SEQ ID NO：39 SEQ ID NO：37
L3 SEQ ID NO：46 SEQ ID NO：47 SEQ ID NO：48
L4 SEQ ID NO：49 SEQ ID NO：50 SEQ ID NO：48
L5 SEQ ID NO：57 SEQ ID NO：58 SEQ ID NO：59
L6 SEQ ID NO：60 SEQ ID NO：61 SEQ ID NO：59
L7 SEQ ID NO：68 SEQ ID NO：69 SEQ ID NO：70
L8 SEQ ID NO：71 SEQ ID NO：72 SEQ ID NO：70
L9 SEQ ID NO：79 SEQ ID NO：80 SEQ ID NO：81
L10 SEQ ID NO：82 SEQ ID NO：83 SEQ ID NO：81
L11 SEQ ID NO：90 SEQ ID NO：91 SEQ ID NO：92
L12 SEQ ID NO：93 SEQ ID NO：94 SEQ ID NO：92
L13 SEQ ID NO：101 SEQ ID NO：102 SEQ ID NO：103
L14 SEQ ID NO：104 SEQ ID NO：105 SEQ ID NO：103
L15 SEQ ID NO：112 SEQ ID NO：113 SEQ ID NO：114
L16 SEQ ID NO：115 SEQ ID NO：116 SEQ ID NO：114
L17 SEQ ID NO：217 SEQ ID NO：102 SEQ ID NO：103
L18 SEQ ID NO：227 SEQ ID NO：102 SEQ ID NO：103
L19 SEQ ID NO：228 SEQ ID NO：102 SEQ ID NO：103
L20 SEQ ID NO：229 SEQ ID NO：102 SEQ ID NO：103
L21 SEQ ID NO：230 SEQ ID NO：102 SEQ ID NO：103
和，
(2)所述重链可变区包含HCDR1、HCDR2和HCDR3，所述HCDR1具有以下所示的HCDR1的任一序列或者与所述序列相比具有1、2、3或更多个氨基酸插入、缺失和/或替换的序列，所述HCDR2具有以下所示的HCDR2的任一序列或者与所述序列相比具有1、2、3或更多个氨基酸插入、缺失和/或替换的序列，并且所述HCDR3具有以下所示的HCDR3的任一序列或者与所述序列相比具有1、2、3或更多个氨基酸插入、缺失和/或替换的序列：
编号 HCDR1 HCDR2 HCDR3
H1 SEQ ID NO：29 SEQ ID NO：30 SEQ ID NO：31
H2 SEQ ID NO：32 SEQ ID NO：33 SEQ ID NO：34
H3 SEQ ID NO：40 SEQ ID NO：41 SEQ ID NO：42
H4 SEQ ID NO：43 SEQ ID NO：44 SEQ ID NO：45
H5 SEQ ID NO：51 SEQ ID NO：52 SEQ ID NO：53
H6 SEQ ID NO：54 SEQ ID NO：55 SEQ ID NO：56
H7 SEQ ID NO：62 SEQ ID NO：63 SEQ ID NO：64
H8 SEQ ID NO：65 SEQ ID NO：66 SEQ ID NO：67
H9 SEQ ID NO：73 SEQ ID NO：74 SEQ ID NO：75
H10 SEQ ID NO：76 SEQ ID NO：77 SEQ ID NO：78
H11 SEQ ID NO：84 SEQ ID NO：85 SEQ ID NO：86
H12 SEQ ID NO：87 SEQ ID NO：88 SEQ ID NO：89
H13 SEQ ID NO：95 SEQ ID NO：96 SEQ ID NO：97
H14 SEQ ID NO：98 SEQ ID NO：99 SEQ ID NO：100
H15 SEQ ID NO：106 SEQ ID NO：107 SEQ ID NO：108
H16 SEQ ID NO：109 SEQ ID NO：110 SEQ ID NO：111
H17 SEQ ID NO：40 SEQ ID NO：142 SEQ ID NO：42
H18 SEQ ID NO：40 SEQ ID NO：143 SEQ ID NO：42
H19 SEQ ID NO：51 SEQ ID NO：158 SEQ ID NO：53
H20 SEQ ID NO：51 SEQ ID NO：159 SEQ ID NO：53
。
在优选的实施方案中，所述抗体或其抗原结合片段包含以下重链可变区和轻链可变区的组合中的六个CDR的序列：L1+H1、L2+H2、L3+H3、L4+H4、L5+H5、L6+H6、L7+H7、L8+H8、L9+H9、L10+H10、L11+H11、L12+H12、L13+H13、L14+H14、L15+H15、L16+H16、L3+H17、L3+H18、L5+H19、L5+H20、L17+H13、L18+H13、L19+H13、L20+H13或L21+H13，或者与所述六个CDR的序列相比具有1、2、3或更多个氨基酸插入、缺失和/或替换的六个CDR的序列。
在一个具体的实施方案中，本申请提供这样一种抗体或其抗原结合片段，其中：
(1)所述轻链可变区序列包含SEQ ID NO：14、16、18、20、22、24、26、28、118～120、130～131、144～145、160～163、172～175、184～187、196～197、206～208、218～221和231～233中任一条所示的序列，或与所述序列具有80％、85％、90％、95％、96％、97％、98％、99％或更高一致性的序列；
和，
(2)所述重链可变区序列包含SEQ ID NO：13、15、17、19、21、23、25、27、121～125、132～137、146～152、164～166、176～178、188～190、198～201、209～212、222和234～238中任一项所示的序列，或与所述序列具有80％、85％、90％、95％、96％、97％、98％、99％或更高一致性的序列。
在优选的实施方案中，所述抗体或其抗原结合片段具有如下所示的轻链可变区和重链可变区：
(1)所述轻链可变区和所述重链可变区分别包含SEQ ID NO：14和SEQ ID NO：13所示的序列；
(2)所述轻链可变区和所述重链可变区分别包含SEQ ID NO：16和SEQ ID NO：15所示的序列；
(3)所述轻链可变区和所述重链可变区分别包含SEQ ID NO：18和SEQ ID NO：17所示的序列；
(4)所述轻链可变区和所述重链可变区分别包含SEQ ID NO：20和SEQ ID NO：19所 示的序列；
(5)所述轻链可变区和所述重链可变区分别包含SEQ ID NO：22和SEQ ID NO：21所示的序列；
(6)所述轻链可变区和所述重链可变区分别包含SEQ ID NO：24和SEQ ID NO：23所示的序列；
(7)所述轻链可变区和所述重链可变区分别包含SEQ ID NO：26和SEQ ID NO：25所示的序列；
(8)所述轻链可变区和所述重链可变区分别包含SEQ ID NO：28和SEQ ID NO：27所示的序列；
(9)所述轻链可变区包含SEQ ID NO：118～120中任一条所示的序列，并且所述重链可变区包含SEQ ID NO：121～125中任一条所示的序列；
(10)所述轻链可变区包含SEQ ID NO：130～131中任一条所示的序列，并且所述重链可变区包含SEQ ID NO：132～137中任一条所示的序列；
(11)所述轻链可变区包含SEQ ID NO：144～145中任一条所示的序列，并且所述重链可变区包含SEQ ID NO：146～152中任一条所示的序列；
(12)所述轻链可变区包含SEQ ID NO：160～163中任一条所示的序列，并且所述重链可变区包含SEQ ID NO：164～166中任一条所示的序列；
(13)所述轻链可变区包含SEQ ID NO：172～175中任一条所示的序列，并且所述重链可变区包含SEQ ID NO：176～178中任一条所示的序列；
(14)所述轻链可变区包含SEQ ID NO：184～187中任一条所示的序列，并且所述重链可变区包含SEQ ID NO：188～190中任一条所示的序列；
(15)所述轻链可变区包含SEQ ID NO：196～197中任一条所示的序列，并且所述重链可变区包含SEQ ID NO：198～201中任一条所示的序列；
(16)所述轻链可变区包含SEQ ID NO：206～208中任一条所示的序列，并且所述重链可变区包含SEQ ID NO：209～212中任一条所示的序列；
(17)所述轻链可变区包含SEQ ID NO：218～221中任一条所示的序列，并且所述重链可变区包含SEQ ID NO：222中任一条所示的序列；
(18)所述轻链可变区包含SEQ ID NO：231～233中任一条所示的序列，并且所述重链可变区包含SEQ ID NO：234～238中任一条所示的序列；
或(19)所述轻链可变区包含与上述(1)～(18)中任一项所示的轻链可变区具有80％、85％、90％、95％、96％、97％、98％、99％或更高一致性的序列，并且所述重链可变区包含与上述(1)～(18)中任一项所示的重链可变区具有80％、85％、90％、95％、96％、97％、98％、99％或更高一致性的序列。
在一个具体的实施方案中，所述抗体或其抗原结合片段是嵌合的、人源化的或全人源的。
在一个具体的实施方案中，所述抗体或其抗原结合片段能与人或猴GPRC5D结合。
在一个具体的实施方案中，所述抗体或其抗原结合片段，包含人或鼠抗体IgG1、IgG2、 IgG3、IgG4、IgA、IgM、IgE或IgD任一恒定区序列；优选包含人或鼠抗体IgG1、IgG2、IgG3或IgG4的恒定区序列，或包含与人或鼠抗体IgG1、IgG2、IgG3或IgG4的恒定区序列具有80％、85％、90％、95％、96％、97％、98％、99％或更高一致性的序列；进一步地，所述抗体或其抗原结合片段还偶联有治疗剂或示踪剂；优选地，所述治疗剂选自放射性同位素、化疗药或免疫调节剂，所述示踪剂选自放射学造影剂、顺磁离子、金属、荧光标记、化学发光标记、超声造影剂和光敏剂：更优选地，所述细胞毒性剂选自生物碱类(alkaloids)、甲氨蝶呤(methotrexate)、蒽环类抗生素(doxorubicin)、紫杉烷类(taxanes)、吡咯并苯并二氮杂
Figure PCTCN2022143719-appb-000001
(pyrrolobenzodiazepine，PBD)或毒素化合物。
在一个具体的实施方案中，所述抗原结合片段选自F(ab’) 2、Fab’、Fab、Fv、scFv、纳米抗体或affibody中的一种或多种。
在第二方面，本申请还提供了一种多特异性抗原结合分子，所述多特异性抗原结合分子包含前述的抗体或其抗原结合片段，以及结合GPRC5D以外其他抗原的抗原结合分子，或结合与前述抗体或其抗原结合片段不同的GPRC5D表位。
优选地，所述其他抗原结合分子为抗体或其抗原结合片段。
优选地，所述多特异性抗原结合分子可为双特异性、三特异性或四特异性。
优选地，所述多特异性抗原结合分子可为二价、三价、四价、五价或六价。
在第三方面，本申请还提供了一种嵌合抗原受体(CAR)，所述嵌合抗原受体至少包含信号肽、胞外抗原结合结构域、铰链区、跨膜结构域和胞内信号传导结构域，所述胞外抗原结合结构域包含前述任一项所述GPRC5D抗体或其抗原结合片段，或前述多特异性抗原结合分子。
在第四方面，本申请还提供了一种免疫效应细胞，所述免疫效应细胞表达前述的嵌合抗原受体，或包含编码前述嵌合抗原受体的核酸片段；优选地，所述免疫效应细胞选自T细胞、NK细胞(natural killer cell)、NKT细胞(natural killer T cell)、DNT细胞(double negative T cell)、单核细胞、巨噬细胞、树突状细胞或肥大细胞，所述T细胞优选自细胞毒性T细胞(CTL)、调节性T细胞或辅助性T细胞；优选地，所述免疫效应细胞为自体免疫效应细胞或同种异体免疫效应细胞
在第五方面，本申请还提供了一种分离的核酸片段，所述核酸片段编码前述抗体或其抗原结合片段、多特异性抗原结合分子或嵌合抗原受体。
在第六方面，本申请还提供了一种载体(vector)，所述载体包含前述的核酸片段。
在第七方面，本申请还提供了一种宿主细胞，所述宿主细胞包含前述的载体；优选地，所述细胞为原核细胞或真核细胞，例如细菌(大肠杆菌)、真菌(酵母)、昆虫细胞或哺乳动物细胞(CHO细胞系或293T细胞系)。
在第八方面，本申请还提供了一种制备前述抗体或其抗原结合片段或多特异性抗原结合分子的方法，所述方法包括培养前述细胞，以及分离所述细胞表达的抗体、抗原结合片段或多特异性抗原结合分子。
在第九方面，本申请还提供了一种制备前述免疫效应细胞的方法，所述方法包括将编码前述CAR的核酸片段导入所述免疫效应细胞，可选地，所述方法还包括启动所述免疫效应细 胞表达前述CAR。
在第十方面，本申请还提供了一种药物组合物，所述药物组合物包含前述的抗体或其抗原结合片段、多特异性抗原结合分子、免疫效应细胞、核酸片段、载体或根据前述方法制备获得的产品；可选地，所述药物组合物还包含药学上可接受的运载体(carrier)、稀释剂或助剂；可选地，所述药物组合物还包含额外的抗肿瘤剂。
在一些实施方式中，所述药学上可接受的运载体为不减弱免疫细胞活力以及功能、不影响抗体或其抗原结合片段与抗原特异性结合的载体，包括但不限于细胞培养基、缓冲液、生理盐水和平衡盐溶液等。缓冲液的实例包括等渗磷酸盐、醋酸盐、柠檬酸盐、硼酸盐以及碳酸盐等。在具体的实施方式中，所述药学上可接受的运载体为含1％血清的磷酸盐缓冲液。
在第十一方面，本申请还提供了一种治疗肿瘤或癌症的方法，所述方法包括向受试者施用有效量的前述的抗体或其抗原结合片段、多特异性抗原结合分子、免疫效应细胞、核酸片段、载体、根据前述方法制备获得的产品或药物组合物。
在优选的实施方案中，所述肿瘤或癌症为表达GPRC5D的肿瘤或癌症，优选B细胞淋巴瘤，更优选多发性骨髓瘤(MM)。
在第十二方面，本申请还提供了前述抗体或其抗原结合片段、多特异性抗原结合分子、免疫效应细胞、核酸片段、载体、根据前述方法制备获得的产品或药物组合物在制备治疗肿瘤或癌症药物中的用途；
在优选的实施方案中，所述肿瘤或癌症为表达GPRC5D的肿瘤或癌症，优选B细胞淋巴瘤，更优选多发性骨髓瘤(MM)。
在第十三方面，本申请还提供了一种试剂盒，所述试剂盒包含前述的抗体或其抗原结合片段、多特异性抗原结合分子、免疫效应细胞、核酸片段、载体、根据前述方法制备获得的产品或药物组合物。
在第十四方面，本申请还提供了一种检测生物学样品中GPRC5D表达的方法，所述方法包括在前述的抗体或其抗原结合片段与GPRC5D之间能够形成复合物的条件下，使所述生物学样品与所述的抗体或其抗原结合片段接触；优选地，所述方法还包括检测所述复合物的形成，指示样品中GPRC5D的存在或表达水平。
在第十五方面，本申请还提供了前述抗体或其抗原结合片段在制备GPRC5D检测试剂中的用途。
实施例
下面结合具体实施例来进一步描述本申请，本申请的优点和特点将会随着描述而更为清楚。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
本申请实施例仅是范例性的，并不对本申请的范围构成任何限制。本领域技术人员应该理解的是，在不偏离本申请的精神和范围下可以对本申请技术方案的细节和形式进行修改或替换，但这些修改和替换均落入本申请的保护范围内。
实施例1对照抗体制备、内源细胞鉴定和过表达细胞株的制备
1.1对照抗体的制备
JNJ7564的序列来自国际专利申请第WO2018017786A2号，5F11的序列来自国际专利申请第WO2019154890A1号。将识别人GPRC5D表位的克隆的VH和VL序列重组到人IgG1 CH和CL表达载体，获得重组质粒。对照抗体、CH、CL和Fc序列见表1。
将质粒和转染试剂PEI(Polysciences，货号：24765-1)加入OPTI-MEM(Gibco，货号：11058021)中混匀后静置15min，加入Expi293细胞(厂家：Thermofisher，货号：A14527)中，放入5％CO 2，120 rpm，37℃摇床培养。转染第二天，加入OPM-293 ProFeed(上海奥浦迈，货号：F081918-001)和6g/L葡萄糖(厂家：Sigma，货号:G7528)。转染第六天，收集细胞上清。
用0.22μm一次性过滤器对样品过滤，再用0.5M NaOH对系统进行消毒30min，0.1M NaOH对层析柱消毒5min，将AKTA pure150每个流路充满相应的缓冲液，顺序依次为A2充满Mab亲和淋洗液(无水磷酸氢二钠2.69g/L(编号20040618，国药集团化学试剂有限公司)，二水合磷酸二氢钠0.17g/L(编号20040718，国药集团化学试剂有限公司)，氯化钠58.44g/L(编号10019318，国药集团化学试剂有限公司))，B1充满Mab亲和洗脱液(一水合柠檬酸3.92 g/L(编号10007118，国药集团化学试剂有限公司)，二水合柠檬酸三钠1.88g/L(编号10019418，国药集团化学试剂有限公司))，A1充满Mab亲和平衡液(20×PBS Buffer加入ddH 2O稀释至1×PBS(编号B548117-050，生工生物工程上海股份有限公司))，Buffer泵充满Mab亲和平衡液(20×PBS Buffer加入ddH 2O稀释至1×PBS(编号B548117-050，生工生物工程上海股份有限公司))，S1进注样品，使用5mL/min的流速进行上样，上样结束后使用buffer泵进行wash1(Mab亲和平衡液)至UV降到10mAU以下，进口切至A2，高盐溶液进行wash2(Mab亲和淋洗液)除杂，进口切至A1，将流路中的高盐溶液置换掉，至电导降至20ms/cm左右，进口切至B1，进行目的蛋白洗脱，收集目标峰，添加1M Tris(121.2g/L，pH8.0)调节pH至中性，使用Nano drop 8000进行浓度测定待用。
表1对照抗体序列信息
Figure PCTCN2022143719-appb-000002
Figure PCTCN2022143719-appb-000003
注：人IgG1 Fc序列中加粗为hinge区序列，其包含C220S突变，用下划线表示。
1.2内源性表达人GPRC5D蛋白的细胞株鉴定
将内源性表达人GPRC5D蛋白的细胞在T-75细胞培养瓶中扩大培养至对数生长期，离心弃去培养基上清，细胞沉淀用PBS洗涤2次。用20nM JNJ7564-hIgG1和5F11-hIgG1抗体作为一抗，Alexa
Figure PCTCN2022143719-appb-000004
647 AffiniPure Goat Anti-Human IgG(H+L)作为二抗(购自Jackson Immuno，货号：109-605-088)经FACS(FACS CantoTM，购自BD公司)检测和分析。结果如表2和图1所示，说明NCI-H929、MolP-8和RPMI-8226细胞均内源表达人GPRC5D蛋白，且JNJ7564-hIgG1、5F11-hIgG1对照抗体与所有细胞均有结合活性。
表2 FACS检测肿瘤细胞内源GPRC5D表达量
Figure PCTCN2022143719-appb-000005
1.3表达人GPRC5A、GPRC5B、GPRC5C、GPRC5D蛋白的CHO-K1重组细胞株的制备
编码humanGPRC5A(Uniprot：Q8NFJ5-1，SEQ ID NO：8)、humanGPRC5B(Uniprot：Q9NZH0-1，SEQ ID NO：9)、humanGPRC5C(Uniprot：Q9NQ84，SEQ ID NO：10)和humanGPRC5D(Uniprot：Q9NZD1，SEQ ID NO：11)氨基酸序列的核苷酸序列，被分别克隆到pLVX慢病毒载体，并在HEK293T细胞中制备病毒颗粒。对CHOK1细胞系(购自中科院)进行慢病毒感染后，在含10μg/ml puromycin(Gibco,货号A1113803)的含10％(v/v)胎牛血清(ExCell Bio，货号FND500)的Advanced DMEM/F12 Medium(Gibco,货号12634028)培养基中选择性培养1周，分别用humanGPRC5A Alexa Fluor 488-conjugated antibody(R&D systems，货号IC5239G-100UG)、humanGPRC5B Alexa Fluor 488-conjugated antibody(R&D systems，货号FAB10253G-100UG)、humanGPRC5C Alexa Fluor 488-conjugated antibody(R&D systems，货号FAB6594G-100UG)染色，转染humanGPRC5D的CHOK1使用人抗人源GPRC5D抗体(JNJ7564，自产)和山羊抗人IgG(H+L)抗体(Jackson，货号：109605088)进行染色，随后在流式细胞仪FACS Aria III(购自BD Biosciences)上分选高表达水平阳性细胞群到96孔板，并置于37℃，5％(v/v)CO 2培养，大约2周后选择部分细胞进行扩增选择长势较好、荧光强度较高、均一性较好的阳性细胞群继续扩大培养并液氮冻存，表达量的结果鉴定见表3和图2A～2D，结果显示经过嘌呤霉素加压筛选后的表达人GPRC5A、GPRC5B、GPRC5C和GPRC5D的CHOK1稳转细胞系具有相对单一的阳性峰，可用于FACS检测抗体与人GPRC5A、GPRC5B、GPRC5C和GPRC5D蛋白的结合活性。
表3人GPRC5蛋白的CHOK1稳转细胞系FACS检测结果
Figure PCTCN2022143719-appb-000006
人GPRC5A全长氨基酸序列(Uniprot：Q8NFJ5-1，SEQ ID NO：8)：
Figure PCTCN2022143719-appb-000007
人GPRC5B全长氨基酸序列(Uniprot：Q9NZH0-1，SEQ ID NO：9)：
Figure PCTCN2022143719-appb-000008
人GPRC5C全长氨基酸序列(Uniprot：Q9NQ84，SEQ ID NO：10)：
Figure PCTCN2022143719-appb-000009
人GPRC5D全长氨基酸序列(Uniprot：Q9NZD1，SEQ ID NO：11)：
Figure PCTCN2022143719-appb-000010
1.4表达人GPRC5D蛋白的HEK293T重组细胞株的制备
具体方法参见实施例1.3。使用包装人GPRC5D质粒载体的慢病毒对HEK293T细胞系进行感染，在含2μg/ml puromycin(Gibco,货号A1113803)的含10％(v/v)胎牛血清(ExCell Bio，货号FND500)的DMEM Medium(Gibco,货号10569044)培养基中选择性培养2周，用人 抗人源GPRC5D抗体(JNJ7564，自产)和山羊抗人IgG(H+L)抗体(Jackson，货号：109605088)在流式细胞仪FACS CantoII(购自BD Biosciences)进行检测，表达较好的细胞株表达量的鉴定结果见表4和图3，显示经过嘌呤霉素加压筛选后的HEK293T-hGPRC5D具有相对单一的阳性峰，可用于FACS检测抗体与人GPRC5D蛋白的结合活性。
表4人GPRC5D蛋白的293T稳转细胞系FACS检测结果
Figure PCTCN2022143719-appb-000011
1.5表达猴GPRC5D蛋白的CHO-K1和HEK293T重组细胞株的制备
稳定表达猴GPRC5D(cynoGPRC5D)的CHO-K1重组细胞株制备具体方法参见实施例1.3。cynoGPRC5D氨基酸序列，NCBI：XP_005570249.1(SEQ ID NO：12)。用人抗cynoGPRC5D抗体(JNJ7564，自产)和山羊抗人IgG(H+L)抗体(Jackson，货号：109605088)在流式细胞仪FACS CantoII(购自BD Biosciences)进行检测，表达较好的细胞株表达量鉴定结果见表5和图4A，结果显示经过嘌呤霉素加压筛选后的CHOK1-cynoGPRC5D具有相对单一的阳性峰，可用于FACS检测抗体与猴GPRC5D蛋白的交叉活性。
表5 cynoGPRC5D蛋白的CHOK1稳转细胞系FACS检测结果
Figure PCTCN2022143719-appb-000012
稳定表达猴GPRC5D(cynoGPRC5D)的HEK293T重组细胞株制备具体方法参见实施例1.4。表达较好的细胞株的表达量鉴定结果见表6和图4B，结果显示经过嘌呤霉素加压筛选后的HEK293T-cynoGPRC5D具有相对单一的阳性峰，可用于FACS检测抗体与猴GPRC5D蛋白的交叉活性。
表6 cynoGPRC5D蛋白的HEK293T稳转细胞系FACS检测结果
Figure PCTCN2022143719-appb-000013
猴GPRC5D全长氨基酸序列(NCBI：XP_005570249.1，SEQ ID NO：12)：
Figure PCTCN2022143719-appb-000014
1.6重组细胞系与对照抗体的结合实验
FACS检测对照抗体与表达人GPRC5D和猴GPRC5D的细胞结合活性如图5A～5C所示，IgG亚型对照为人IgG1。JNJ7564-hIgG1和5F11-hIgG1与表达人GPRC5D蛋白的NCI-H929肿瘤细胞、HEK293T-hGPRC5D和HEK293T-cynoGPRC5D重组细胞有较好的结合活性，5F11-hIgG1与NCI-H929细胞和HEK293T-hGPRC5D的重组细胞的结合活性相对更强， JNJ7564-hIgG1与HEK293T-cynoGPRC5D重组细胞的结合活性相对更强。
实施例2抗人GPRC5D杂交瘤单克隆抗体的制备
2.1动物免疫
抗人GPRC5D单克隆抗体通过免疫小鼠产生。实验用6～8周龄的雌性SJL小鼠(购自上海斯莱克实验动物有限公司)，小鼠在SPF条件下饲养。小鼠购进后，实验室环境饲养1周，12/12小时光/暗周期调节，温度20-25℃；湿度40-60％。将已适应环境的小鼠按以下方案免疫。初次免疫时，将人GPRC5D过表达细胞系(HEK293T-hGPRC5D，in-house)用PBS清洗2遍后，与寡核苷酸CpG(ODN 1826，合成自上海生工生物)混合，每只小鼠先腹腔注射50μl乳化好的Titer max(购自sigma,Cat.T2684)，等待15分钟后每只小鼠腹腔注射5×10 6个/100μl细胞。第一次加强免疫时，将人GPRC5D过表达细胞系用PBS清洗2遍后，与CpG混合，每只小鼠腹腔注射5×10 6个/100μl细胞。后续加强免疫按照初次免疫和第一次加强免疫的方式交替进行，免疫原交替使用猴GPRC5D过表达细胞系(HEK293T-cynoGPRC5D，in-house)和人GPRC5D过表达细胞系(HEK293T-hGPRC5D)。每次免疫之间间隔7天，分别在第二次和第四次加强免疫后的第5天进行小鼠的采血操作，分离血清，使用基于细胞的酶联免疫吸附实验(cell based ELISA)方法测定血清中特异性抗体的滴度。
为了检测血清中特异性抗体的滴度，将实施例1获得的HEK293T-hGPRC5D、HEK293T-cynoGPRC5D、CHOK1-hGPRC5D、CHOK1-cynoGPRC5D重组细胞和空白对照细胞，每孔4×10 4个细胞接种到96孔细胞板(厂家：corning，货号：3599)中，细胞培养箱中孵育过夜，去掉上清培养基，每孔加入50μl固定液(厂家：Beyotime，货号：P0098-500ML)，在通风橱中室温放置30min，去掉上清固定液，PBST洗涤2次，再加入0.5％脱脂牛奶(厂家：生工，货号：A600669-0250)室温封闭2小时，倒掉封闭液，用PBST洗板2次，以50μl/孔加入1:100为起始稀释度，3倍梯度稀释的小鼠血清。室温孵育1小时后，用PBST洗板3次。加入HRP(辣根过氧化物酶)标记的二抗(购自Jackson，货号：109-035-088)，室温孵育1小时后，用PBST洗板5次。加入TMB底物50μl/孔，室温孵育5-10分钟后，加入终止液(1.0N HCl)50μl/孔。用ELISA读板机(Multimode Plate Reader，EnSight，购自Perkin Elmer)读取OD450nm数值，cell-based ELISA检测结果显示，经以上细胞免疫的小鼠的免疫后血清对免疫原均有不同程度的结合，呈现抗原抗体反应。
2.2脾细胞融合和杂交瘤筛选
向每只挑选出的小鼠腹腔注射5×10 6数量的HEK293T-hGPRC5D细胞，3天后处死小鼠，收集脾细胞和淋巴细胞。1500转每分离心后，弃掉上清，在细胞中加入ACK裂解液(Gibco,Cat.A1049201)，裂解细胞中掺杂的红细胞，获得细胞悬液，用DMEM基础培养基(购自Gibco，货号10569044)1500转每分清洗细胞3次，细胞计数，然后按活细胞数目2:1与小鼠骨髓瘤细胞SP2/0(购自ATCC，Cat.CRL-1581)混合，采用电融合方法进行细胞融合。融合后的细胞稀释到含20％(v/v)胎牛血清(购自ExCell Bio，Cat.FND500)、1×HAT(购自sigma，Cat.H0262-10VL)、牛胰岛素(购自Yeason，Cat.40107ES25)、NEAA(购自Gibco，Cat.11140050)的DMEM培养基中，然后按5×10 4个/200μL每孔加入96孔细胞培养板中，放入5％(v/v)CO 2、37℃培养箱中培养。7天后用cell based ELISA筛选融合板上清以确认对人GPRC5D过表达细胞的结合活性；对阳性克隆上清，通过cell based ELISA确认与猴GPRC5D过表达细胞和 FACS确认与内源性细胞NCI-H929的结合活性。
根据筛选结果，挑选符合条件的阳性克隆，用半固体培养基(购自stemcell，Cat.03810)进行亚克隆，7天后将长出的克隆逐个挑入96孔培养板中，在含10％(w/w)胎牛血清、1×HT(购自sigma，Cat.H0137-10VL)的DMEM培养基中扩大培养，1天后用cell based ELISA进行初步筛选，挑选阳性单克隆扩增到24孔板继续培养。3天后对培养上清进行进一步检测，评估其与猴GPRC5D过表达细胞和内源性细胞NCI-H929的结合活性，根据24孔板样品检测结果，挑选出最优的克隆，并于含10％(v/v)FBS的DMEM培养基中在37℃、5％(v/v)CO 2条件下将该最优的克隆进行扩大培养，液氮冻存即得本申请杂交瘤细胞。
实施例3杂交瘤阳性克隆轻重链可变区氨基酸序列测定
收集对数生长期杂交瘤细胞，用Trizol(Invitrogen，Cat No.15596-018)充分裂解细胞后于-80℃保存待测。样品委托苏州金唯智生物科技有限公司完成杂交瘤阳性克隆轻链和重链可变区氨基酸序列测定。对测序结果使用MOE软件进行分析，根据可变区编码蛋白氨基酸序列构建进化树，根据序列相似性剔除在进化树上距离较近的序列后，从中筛选获得8个克隆：GPRC5D-mab01～08(序列见表7，SEQ ID NO：13-28)。
表7 GPRC5D阳性克隆抗体轻重链可变区氨基酸序列信息
Figure PCTCN2022143719-appb-000015
Figure PCTCN2022143719-appb-000016
分别使用Kabat和IMGT编号系统对表7中序列进行CDRs划分，CDRs的序列见表8：
表8 GPRC5D阳性克隆CDR序列表
Figure PCTCN2022143719-appb-000017
Figure PCTCN2022143719-appb-000018
委托通用生物系统(安徽)有限公司合成编码上述8个克隆的重链及轻链可变区的核苷酸序列，并通过序列为GGGGSGGGGSGGGGS(SEQ ID NO：117)的linker连接，然后克隆到包含信号肽和人源抗体Fc的表达载体pTT5-huFc(C220S)上(重链恒定区序列SEQ ID NO：5)， 获得人鼠嵌合抗体的表达载体，表达纯化步骤参照实施例1.1。
实施例4 GPRC5D人鼠嵌合抗体的鉴定
4.1基于细胞的酶联免疫吸附实验(cell-based ELISA)检测嵌合抗体与人GPRC5D蛋白的结合
具体检测方法参照实施例2.1。将实施例1获得的CHOK1-hGPRC5D、CHOK1-cynoGPRC5D重组细胞和CHOK1细胞固定，以50μl/孔加入100nM为起始浓度，3倍梯度稀释的嵌合抗体和对照抗体进行检测，嵌合抗体与人GPRC5D蛋白的结合活性cell based ELISA检测结果如图6A～6C、7A～7C和8A～8C。结果说明，纯化生产后的嵌合抗体与人GPRC5D蛋白在ELISA水平上有不同程度的结合。其中阴性对照抗体hIgG1为针对鸡卵溶菌酶的抗体anti-hel-hIgG1(购自百英，货号：B117901)图中的数据为OD450nm值。
4.2流式细胞实验(FACS)检测嵌合抗体与内源性细胞的结合
将所需细胞在T-75细胞培养瓶中扩大培养至对数生长期，收取NCI-H929，MolP-8，RPMI-8226细胞，用PBS缓冲液洗涤2次，对细胞进行细胞计数后将细胞沉淀用[PBS+2％(w/v)BSA]封闭液重悬至2×10 6个细胞/毫升，按50μl/孔加入到96孔FACS反应板中，加入嵌合抗体待测样品50μl/孔，冰上孵育1小时。用PBS缓冲液离心洗涤3次，加入50μl/孔Alexa
Figure PCTCN2022143719-appb-000019
647 AffiniPure Goat Anti-Human IgG,Fcγfragment specific二抗(购自Jackson，货号：109-605-098)，冰上孵育1小时。用PBS缓冲液离心洗涤3次，用FACS(FACS CantoII，购自BD公司)检测和分析结果。通过软件(Flowjo)进行数据分析，得到细胞的平均荧光密度(MFI)。再通过软件(GraphPad Prism8)分析，分析结果如表9以及图9A～9C、10A～10C和11A～11C所示，嵌合抗体均可较好地结合NCI-H929(高表达)，MolP-8(中表达)，RPMI-8226(低表达)细胞。
表9 FACS检测嵌合抗体与细胞的结合反应
Figure PCTCN2022143719-appb-000020
Figure PCTCN2022143719-appb-000021
注：NB表示无结合。
4.3流式细胞实验(FACS)检测嵌合抗体与CHOK1-hGPRC5A、CHOK1-hGPRC5B和CHOK1-hGPRC5C结合活性
具体方法参照实施例4.2。取CHOK1-hGPRC5A、CHOK1-hGPRC5B和CHOK1-hGPRC5C细胞，结果如表10所示，结果说明嵌合抗体均不结合CHOK1-hGPRC5A、CHOK1-hGPRC5B、CHOK1-hGPRC5C细胞，与这些亚型无交叉结合。
表10 FACS检测嵌合抗体与CHOK1-hGPRC5A,CHOK1-hGPRC5B,CHOK1-hGPRC5C细胞的结合反应
Figure PCTCN2022143719-appb-000022
4.4嵌合抗体与阳性对照抗体5F11竞争力鉴定
为了鉴定嵌合抗体与抗原的结合能力，采用竞争ELISA的方法对GPRC5D嵌合抗体与阳性对照分子5F11竞争结合抗原进行分析。对阳性分子5F11进行生物素标记，hGPRC5D-LVP蛋白(厂家：恺佧生物，货号：GPR-HM05P)2μg/mL包被ELISA板，Biotin-5F11抗体从100nM开始三倍进行梯度稀释，测定Biotin-5F11抗体OD 450值，使用GraphPad Prism软件拟合曲线，计算出EC80浓度作为竞争性ELISA中的参考浓度。
hGPRC5D-LVP蛋白稀释到2μg/mL，以50μL/孔包被96孔高吸附酶标板，4℃过夜包被后用250μL封闭液(含有2％(w/v)BSA的PBS)进行室温两小时封闭，加入100nM开始、三倍梯度稀释的嵌合抗体后，再加入Biotin-5F11 EC80浓度的抗体(约为1nM)，孵育1.5小时，用PBS洗5次后加入HRP标记的Streptavidin Peroxidase Conjugate二抗(购自sigma-millipore，货号：189733)，孵育1小时，洗板5次。加入TMB底物50μL每孔，室温孵育10分钟后，加入终止液(1.0M HCl)50μL每孔。用ELISA读板机(Insight，购自PerkinElmer)读取OD450nm数值，根据OD450nm数值，结果如表11和图12A～12C所示，值越低，表示嵌合抗体的竞争力越大。结果显示8个嵌合抗体均与阳性分子5F11-hIgG1有表位竞争关系。
表11 ELISA检测嵌合抗体与阳性对照抗体5F11竞争力反应
Figure PCTCN2022143719-appb-000023
实施例5 GPRC5D鼠源抗体的人源化
5.1 GPRC5D-mab01的人源化
通过比对IMGT(http://imgt.cines.fr)人类抗体重轻链可变区种系基因数据库，分别挑选与鼠源抗体同源性高的重链和轻链可变区种系基因作为模板，将鼠源抗体的CDR分别移植到相应的人源模板中，形成次序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4的可变区序列。根据需要，将骨架序列(FR区序列)中关键氨基酸回复突变为鼠源抗体对应的氨基酸，以保证原有的亲和力，同时抗体存在的易发生化学修饰的位点，已对这些位点进行点突变以消除修饰风险，得到人源化单克隆抗体。其中抗体的CDR氨基酸残基由Kabat编号系统确定并注释。
鼠源抗体GPRC5D-mab01的人源化轻链模板为IGKV1-9*01和IGKJ4*01，人源化重链模板为IGHV2-26*01和IGHJ6*01。具体突变设计见表12。
表12 GPRC5D-mab01的人源化抗体突变设计
Figure PCTCN2022143719-appb-000024
Figure PCTCN2022143719-appb-000025
注：Graft代表将鼠源抗体CDR植入人种系模板FR区序列；L45P表示将Graft第45位L突变成P，其它依此类推。突变氨基酸的编号为自然顺序编号。
GPRC5D-mab01人源化抗体可变区具体序列如下：
GPRC5D-mab01.VL1氨基酸序列如SEQ ID NO：118所示：
Figure PCTCN2022143719-appb-000026
GPRC5D-mab01.VL2氨基酸序列如SEQ ID NO：119所示：
Figure PCTCN2022143719-appb-000027
GPRC5D-mab01.VL3氨基酸序列如SEQ ID NO：120所示：
Figure PCTCN2022143719-appb-000028
GPRC5D-mab01.VH1氨基酸序列如SEQ ID NO：121所示：
Figure PCTCN2022143719-appb-000029
GPRC5D-mab01.VH2氨基酸序列如SEQ ID NO：122所示：
Figure PCTCN2022143719-appb-000030
GPRC5D-mab01.VH3氨基酸序列如SEQ ID NO：123所示：
Figure PCTCN2022143719-appb-000031
GPRC5D-mab01.VH4氨基酸序列如SEQ ID NO：124所示：
Figure PCTCN2022143719-appb-000032
GPRC5D-mab01.VH5氨基酸序列如SEQ ID NO：125所示：
Figure PCTCN2022143719-appb-000033
人源化轻链模板IGKV1-9*01氨基酸序列如SEQ ID NO：126所示：
Figure PCTCN2022143719-appb-000034
人源化轻链模板IGKJ4*01氨基酸序列如SEQ ID NO：127所示：
Figure PCTCN2022143719-appb-000035
人源化重链模板IGHV2-26*01氨基酸序列如SEQ ID NO：128所示：
Figure PCTCN2022143719-appb-000036
人源化重链模板IGHJ6*01氨基酸序列如SEQ ID NO：129所示：
Figure PCTCN2022143719-appb-000037
本申请分别从上述GPRC5D-mab01的人源化抗体轻链和重链可变区的突变设计中，选择不同的轻链和重链序列进行交叉组合，最终获得多种GPRC5D-mab01人源化抗体，各抗体的可变区氨基酸序列如下见表13：
表13 GPRC5D-mab01抗体可变区对应氨基酸序列
Figure PCTCN2022143719-appb-000038
根据Kabat编号系统，上述人源化抗体VH和VL序列分析结果如表14所示。
表14 GPRC5D-mab01人源化抗体VH和VL序列的Kabat分析结果
Figure PCTCN2022143719-appb-000039
5.2 GPRC5D-mab02的人源化
方法同5.1，鼠源抗体GPRC5D-mab02的人源化轻链模板为IGKV3-20*02和IGKJ2*01，人源化重链模板为IGHV1-46*01和IGHJ6*01。具体突变设计见表15。
表15 GPRC5D-mab02的人源化抗体突变设计
Figure PCTCN2022143719-appb-000040
注：Graft代表将鼠源抗体CDR植入人种系模板FR区序列；L45V表示将Graft第45位L突变成V， 其它依此类推。突变氨基酸的编号为自然顺序编号。
GPRC5D-mab02人源化抗体可变区具体序列如下：
GPRC5D-mab02.VL1氨基酸序列如SEQ ID NO：130所示：
Figure PCTCN2022143719-appb-000041
GPRC5D-mab02.VL2氨基酸序列如SEQ ID NO：131所示：
Figure PCTCN2022143719-appb-000042
GPRC5D-mab02.VH1氨基酸序列如SEQ ID NO：132所示：
Figure PCTCN2022143719-appb-000043
GPRC5D-mab02.VH2氨基酸序列如SEQ ID NO：133所示：
Figure PCTCN2022143719-appb-000044
GPRC5D-mab02.VH2a氨基酸序列如SEQ ID NO：134所示：
Figure PCTCN2022143719-appb-000045
GPRC5D-mab02.VH3氨基酸序列如SEQ ID NO：135所示：
Figure PCTCN2022143719-appb-000046
GPRC5D-mab02.VH3a氨基酸序列如SEQ ID NO：136所示：
Figure PCTCN2022143719-appb-000047
GPRC5D-mab02.VH4氨基酸序列如SEQ ID NO：137所示：
Figure PCTCN2022143719-appb-000048
人源化轻链模板IGKV3-20*02氨基酸序列如SEQ ID NO：138所示：
Figure PCTCN2022143719-appb-000049
人源化轻链模板IGKJ2*01氨基酸序列如SEQ ID NO：139所示：
Figure PCTCN2022143719-appb-000050
人源化重链模板IGHV1-46*01氨基酸序列如SEQ ID NO：140所示：
Figure PCTCN2022143719-appb-000051
人源化重链模板IGHJ6*01氨基酸序列如SEQ ID NO：141所示：
Figure PCTCN2022143719-appb-000052
本申请分别从上述GPRC5D-mab02的人源化抗体轻链和重链可变区的突变设计中，选择不同的轻链和重链序列进行交叉组合，最终获得多种GPRC5D-mab02人源化抗体，各抗体的可变区氨基酸序列如下见表16：
表16 GPRC5D-mab02抗体可变区对应氨基酸序列
Figure PCTCN2022143719-appb-000053
根据Kabat编号系统，上述人源化抗体VH和VL序列分析结果如表17所示。
表17 GPRC5D-mab02人源化抗体VH和VL序列的Kabat分析结果
Figure PCTCN2022143719-appb-000054
5.3 GPRC5D-mab03的人源化
方法同5.1，鼠源抗体GPRC5D-mab03的人源化轻链模板为IGKV4-1*01/IGKV1-39*01和IGKJ4*01，人源化重链模板为IGHV1-3*01和IGHJ6*01。具体突变设计见表18。
表18 GPRC5D-mab03的人源化抗体突变设计
Figure PCTCN2022143719-appb-000055
Figure PCTCN2022143719-appb-000056
注：Graft代表将鼠源抗体CDR植入人种系模板FR区序列；A43S表示将Graft第43位A突变成S，其它依此类推。突变氨基酸的编号为自然顺序编号。
GPRC5D-mab03人源化抗体可变区具体序列如下：
GPRC5D-mab03.VL1氨基酸序列如SEQ ID NO：144所示：
Figure PCTCN2022143719-appb-000057
GPRC5D-mab03.VL2氨基酸序列如SEQ ID NO：145所示：
Figure PCTCN2022143719-appb-000058
GPRC5D-mab03.VH1氨基酸序列如SEQ ID NO：146所示：
Figure PCTCN2022143719-appb-000059
GPRC5D-mab03.VH2氨基酸序列如SEQ ID NO：147所示：
Figure PCTCN2022143719-appb-000060
GPRC5D-mab03.VH2a氨基酸序列如SEQ ID NO：148所示：
Figure PCTCN2022143719-appb-000061
GPRC5D-mab03.VH3氨基酸序列如SEQ ID NO：149所示：
Figure PCTCN2022143719-appb-000062
GPRC5D-mab03.VH4氨基酸序列如SEQ ID NO：150所示：
Figure PCTCN2022143719-appb-000063
GPRC5D-mab03.VH4a氨基酸序列如SEQ ID NO：151所示：
Figure PCTCN2022143719-appb-000064
GPRC5D-mab03.VH5氨基酸序列如SEQ ID NO：152所示：
Figure PCTCN2022143719-appb-000065
人源化轻链模板IGKV4-1*01氨基酸序列如SEQ ID NO：153所示：
Figure PCTCN2022143719-appb-000066
人源化轻链模板IGKV1-39*01氨基酸序列如SEQ ID NO：154所示：
Figure PCTCN2022143719-appb-000067
人源化轻链模板IGKJ4*01氨基酸序列如SEQ ID NO：155所示：
Figure PCTCN2022143719-appb-000068
人源化重链模板IGHV1-3*01氨基酸序列如SEQ ID NO：156所示：
Figure PCTCN2022143719-appb-000069
人源化重链模板IGHJ6*01氨基酸序列如SEQ ID NO：157所示：
Figure PCTCN2022143719-appb-000070
本申请分别从上述GPRC5D-mab03的人源化抗体轻链和重链可变区的突变设计中，选择不同的轻链和重链序列进行交叉组合，最终获得多种GPRC5D-mab03人源化抗体，各抗体的可变区氨基酸序列如下见表19：
表19 GPRC5D-mab03抗体可变区对应氨基酸序列
Figure PCTCN2022143719-appb-000071
根据Kabat编号系统，上述人源化抗体VH和VL序列分析结果如表20所示。
表20 GPRC5D-mab03人源化抗体VH和VL序列的Kabat分析结果
Figure PCTCN2022143719-appb-000072
5.4 GPRC5D-mab04的人源化
方法同5.1，鼠源抗体GPRC5D-mab04的人源化轻链模板为IGKV1-39*01/IGKV2-28*01和IGKJ4*01，人源化重链模板为IGHV3-7*01和IGHJ6*01。具体突变设计见表21。
表21 GPRC5D-mab04的人源化抗体突变设计
Figure PCTCN2022143719-appb-000073
Figure PCTCN2022143719-appb-000074
注：Graft代表将鼠源抗体CDR植入人种系模板FR区序列；A43S表示将Graft第43位A突变成S，其它依此类推。突变氨基酸的编号为自然顺序编号。
GPRC5D-mab04人源化抗体可变区具体序列如下：
GPRC5D-mab04.VL1氨基酸序列如SEQ ID NO：160所示：
Figure PCTCN2022143719-appb-000075
GPRC5D-mab04.VL2氨基酸序列如SEQ ID NO：161所示：
Figure PCTCN2022143719-appb-000076
GPRC5D-mab04.VL3氨基酸序列如SEQ ID NO：162所示：
Figure PCTCN2022143719-appb-000077
GPRC5D-mab04.VL4氨基酸序列如SEQ ID NO：163所示：
Figure PCTCN2022143719-appb-000078
GPRC5D-mab04.VH1氨基酸序列如SEQ ID NO：164所示：
Figure PCTCN2022143719-appb-000079
GPRC5D-mab04.VH2氨基酸序列如SEQ ID NO：165所示：
Figure PCTCN2022143719-appb-000080
GPRC5D-mab04.VH3氨基酸序列如SEQ ID NO：166所示：
Figure PCTCN2022143719-appb-000081
人源化轻链模板IGKV1-39*01氨基酸序列如SEQ ID NO：167所示：
Figure PCTCN2022143719-appb-000082
人源化轻链模板IGKV2-28*01氨基酸序列如SEQ ID NO：168所示：
Figure PCTCN2022143719-appb-000083
人源化轻链模板IGKJ4*01氨基酸序列如SEQ ID NO：169所示：
Figure PCTCN2022143719-appb-000084
人源化重链模板IGHV3-7*01氨基酸序列如SEQ ID NO：170所示：
Figure PCTCN2022143719-appb-000085
人源化重链模板IGHJ6*01氨基酸序列如SEQ ID NO：171所示：
Figure PCTCN2022143719-appb-000086
本申请分别从上述GPRC5D-mab04的人源化抗体轻链和重链可变区的突变设计中，选择不同的轻链和重链序列进行交叉组合，最终获得多种GPRC5D-mab04人源化抗体，各抗体的可变区氨基酸序列如下见表22：
表22 GPRC5D-mab04抗体可变区对应氨基酸序列
  GPRC5D-mab04.VH1 GPRC5D-mab04.VH2 GPRC5D-mab04.VH3
GPRC5D-mab04.VL1 GPRC5D-mab04VL1VH1 GPRC5D-mab04VL1VH2 GPRC5D-mab04VL1VH3
GPRC5D-mab04.VL2 GPRC5D-mab04VL2VH1 GPRC5D-mab04VL2VH2 GPRC5D-mab04VL2VH3
GPRC5D-mab04.VL3 GPRC5D-mab04VL3VH1 GPRC5D-mab04VL3VH2 GPRC5D-mab04VL3VH3
GPRC5D-mab04.VL4 GPRC5D-mab04VL4VH1 GPRC5D-mab04VL4VH2 GPRC5D-mab04VL4VH3
根据Kabat编号系统，上述人源化抗体VH和VL序列分析结果如表23所示。
表23 GPRC5D-mab04人源化抗体VH和VL序列的Kabat分析结果
Figure PCTCN2022143719-appb-000087
5.5 GPRC5D-mab05的人源化
方法同5.1，鼠源抗体GPRC5D-mab05的人源化轻链模板为IGKV6-21*01/IGKV3-11*01和IGKJ4*01，人源化重链模板为IGHV1-69*02和IGHJ6*01。具体突变设计见表24。
表24 GPRC5D-mab05的人源化抗体突变设计
Figure PCTCN2022143719-appb-000088
注：Graft代表将鼠源抗体CDR植入人种系模板FR区序列；V58I表示将Graft第58位V突变成I，其它依此类推。突变氨基酸的编号为自然顺序编号。
GPRC5D-mab05人源化抗体可变区具体序列如下：
GPRC5D-mab05.VL1氨基酸序列如SEQ ID NO：172所示：
Figure PCTCN2022143719-appb-000089
GPRC5D-mab05.VL2氨基酸序列如SEQ ID NO：173所示：
Figure PCTCN2022143719-appb-000090
GPRC5D-mab05.VL3氨基酸序列如SEQ ID NO：174所示：
Figure PCTCN2022143719-appb-000091
GPRC5D-mab05.VL4氨基酸序列如SEQ ID NO：175所示：
Figure PCTCN2022143719-appb-000092
GPRC5D-mab05.VH1氨基酸序列如SEQ ID NO：176所示：
Figure PCTCN2022143719-appb-000093
GPRC5D-mab05.VH2氨基酸序列如SEQ ID NO：177所示：
Figure PCTCN2022143719-appb-000094
GPRC5D-mab05.VH3氨基酸序列如SEQ ID NO：178所示：
Figure PCTCN2022143719-appb-000095
人源化轻链模板IGKV6-21*01氨基酸序列如SEQ ID NO：179所示：
Figure PCTCN2022143719-appb-000096
人源化轻链模板IGKV3-11*01氨基酸序列如SEQ ID NO：180所示：
Figure PCTCN2022143719-appb-000097
人源化轻链模板IGKJ4*01氨基酸序列如SEQ ID NO：181所示：
Figure PCTCN2022143719-appb-000098
人源化重链模板IGHV1-69*02氨基酸序列如SEQ ID NO：182所示：
Figure PCTCN2022143719-appb-000099
人源化重链模板IGHJ6*01氨基酸序列如SEQ ID NO：183所示：
Figure PCTCN2022143719-appb-000100
本申请分别从上述GPRC5D-mab05的人源化抗体轻链和重链可变区的突变设计中，选择不同的轻链和重链序列进行交叉组合，最终获得多种GPRC5D-mab05人源化抗体，各抗体的可变区氨基酸序列如下见表25：
表25 GPRC5D-mab05抗体可变区对应氨基酸序列
  GPRC5D-mab05.VH1 GPRC5D-mab05.VH2 GPRC5D-mab05.VH3
GPRC5D-mab05.VL1 GPRC5D-mab05VL1VH1 GPRC5D-mab05VL1VH2 GPRC5D-mab05VL1VH3
GPRC5D-mab05.VL2 GPRC5D-mab05VL2VH1 GPRC5D-mab05VL2VH2 GPRC5D-mab05VL2VH3
GPRC5D-mab05.VL3 GPRC5D-mab05VL3VH1 GPRC5D-mab05VL3VH2 GPRC5D-mab05VL3VH3
GPRC5D-mab05.VL4 GPRC5D-mab05VL4VH1 GPRC5D-mab05VL4VH2 GPRC5D-mab05VL4VH3
根据Kabat编号系统，上述人源化抗体VH和VL序列分析结果如表26所示。
表26 GPRC5D-mab05人源化抗体VH和VL序列的Kabat分析结果
Figure PCTCN2022143719-appb-000101
5.6 GPRC5D-mab06人源化
5.6.1 GPRC5D-mab06第一轮人源化设计
方法同5.1，鼠源抗体GPRC5D-mab06的人源化轻链模板为IGKV4-1*01/IGKV1-27*01和IGKJ2*01，人源化重链模板为IGHV2-26*01和IGHJ6*01。具体突变设计见表27。
表27 GPRC5D-mab06的人源化抗体突变设计
Figure PCTCN2022143719-appb-000102
注：Graft代表将鼠源抗体CDR植入人种系模板FR区序列；P43S表示将Graft第43位P突变成S，其它依此类推。突变氨基酸的编号为自然顺序编号。
GPRC5D-mab06人源化抗体可变区具体序列如下：
GPRC5D-mab06.VL1氨基酸序列如SEQ ID NO：184所示：
Figure PCTCN2022143719-appb-000103
GPRC5D-mab06.VL2氨基酸序列如SEQ ID NO：185所示：
Figure PCTCN2022143719-appb-000104
GPRC5D-mab06.VL3氨基酸序列如SEQ ID NO：186所示：
Figure PCTCN2022143719-appb-000105
GPRC5D-mab06.VL4氨基酸序列如SEQ ID NO：187所示：
Figure PCTCN2022143719-appb-000106
GPRC5D-mab06.VH1氨基酸序列如SEQ ID NO：188所示：
Figure PCTCN2022143719-appb-000107
GPRC5D-mab06.VH2氨基酸序列如SEQ ID NO：189所示：
Figure PCTCN2022143719-appb-000108
GPRC5D-mab06.VH3氨基酸序列如SEQ ID NO：190所示：
Figure PCTCN2022143719-appb-000109
人源化轻链模板IGKV4-1*01氨基酸序列如SEQ ID NO：191所示：
Figure PCTCN2022143719-appb-000110
人源化轻链模板IGKV1-27*01氨基酸序列如SEQ ID NO：192所示：
Figure PCTCN2022143719-appb-000111
人源化轻链模板IGKJ2*01氨基酸序列如SEQ ID NO：193所示：
Figure PCTCN2022143719-appb-000112
人源化重链模板IGHV2-26*01氨基酸序列如SEQ ID NO：194所示：
Figure PCTCN2022143719-appb-000113
人源化重链模板IGHJ6*01氨基酸序列如SEQ ID NO：195所示：
Figure PCTCN2022143719-appb-000114
本申请分别从上述GPRC5D-mab06的第一轮人源化抗体轻链和重链可变区的突变设计中，选择不同的轻链和重链序列进行交叉组合，最终获得多种GPRC5D-mab06人源化抗体，各抗体的可变区氨基酸序列如表28：
表28 GPRC5D-mab06抗体可变区对应氨基酸序列
  GPRC5D-mab06.VH1 GPRC5D-mab06.VH2 GPRC5D-mab06.VH3
GPRC5D-mab06.VL1 GPRC5D-mab06.VL1VH1 GPRC5D-mab06.VL1VH2 GPRC5D-mab06.VL1VH3
GPRC5D-mab06.VL2 GPRC5D-mab06.VL2VH1 GPRC5D-mab06.VL2VH2 GPRC5D-mab06.VL2VH3
GPRC5D-mab06.VL3 GPRC5D-mab06.VL3VH1 GPRC5D-mab06.VL3VH2 GPRC5D-mab06.VL3VH3
GPRC5D-mab06.VL4 GPRC5D-mab06.VL4VH1 GPRC5D-mab06.VL4VH2 GPRC5D-mab06.VL4VH3
根据Kabat编号系统，上述人源化抗体VH和VL序列分析结果如表29所示。
表29 GPRC5D-mab06人源化抗体VH和VL序列的Kabat分析结果
Figure PCTCN2022143719-appb-000115
Figure PCTCN2022143719-appb-000116
5.6.2 GPRC5D-mab06第二轮人源化设计
方法同5.1，鼠源抗体GPRC5D-mab06的第二轮人源化轻链模板为IGKV1-27*01和IGKJ2*01，第二轮人源化重链模板为IGHV2-26*01和IGHJ6*01。具体突变设计见表30。
表30 GPRC5D-mab06的人源化抗体突变设计
Figure PCTCN2022143719-appb-000117
注：Graft代表将鼠源抗体CDR植入人种系模板FR区序列；V43S表示将Graft第43位V突变成S，其它依此类推。突变氨基酸的编号为自然顺序编号。
GPRC5D-mab06人源化抗体可变区具体序列如下：
GPRC5D-mab06.VL4氨基酸序列如SEQ ID NO：196所示：
Figure PCTCN2022143719-appb-000118
GPRC5D-mab06.VL5氨基酸序列如SEQ ID NO：197所示：
Figure PCTCN2022143719-appb-000119
GPRC5D-mab06.VH4氨基酸序列如SEQ ID NO：198所示：
Figure PCTCN2022143719-appb-000120
GPRC5D-mab06.VH5氨基酸序列如SEQ ID NO：199所示：
Figure PCTCN2022143719-appb-000121
GPRC5D-mab06.VH6氨基酸序列如SEQ ID NO：200所示：
Figure PCTCN2022143719-appb-000122
GPRC5D-mab06.VH7氨基酸序列如SEQ ID NO：201所示：
Figure PCTCN2022143719-appb-000123
人源化轻链模板IGKV1-27*01氨基酸序列如SEQ ID NO：202所示：
Figure PCTCN2022143719-appb-000124
人源化轻链模板IGKJ2*01氨基酸序列如SEQ ID NO：203所示：
Figure PCTCN2022143719-appb-000125
人源化重链模板IGHV2-26*01氨基酸序列如SEQ ID NO：204所示：
Figure PCTCN2022143719-appb-000126
人源化重链模板IGHJ6*01氨基酸序列如SEQ ID NO：205所示：
Figure PCTCN2022143719-appb-000127
本申请分别从上述GPRC5D-mab06的第二轮人源化抗体轻链和重链可变区的突变设计中，选择不同的轻链和重链序列进行交叉组合，最终获得多种GPRC5D-mab06人源化抗体，各抗体的可变区氨基酸序列如表31所示：
表31 GPRC5D-mab06抗体可变区对应氨基酸序列
Figure PCTCN2022143719-appb-000128
根据Kabat编号系统，上述人源化抗体VH和VL序列分析结果如表32所示。
表32 GPRC5D-mab06人源化抗体VH和VL序列的Kabat分析结果
Figure PCTCN2022143719-appb-000129
5.7 GPRC5D-mab07人源化
5.7.1 GPRC5D-mab07第一轮人源化设计
方法同5.1，鼠源抗体GPRC5D-mab07的人源化轻链模板为IGKV2-40*01和IGKJ4*01，人源化重链模板为IGHV1-69*02和IGHJ6*01。具体突变设计见表33。
表33 GPRC5D-mab07的人源化抗体突变设计
Figure PCTCN2022143719-appb-000130
注：Graft代表将鼠源抗体CDR植入人种系模板FR区序列；Q50K表示将Graft第50位Q突变成K， 其它依此类推。突变氨基酸的编号为自然顺序编号。
GPRC5D-mab07人源化抗体可变区具体序列如下：
GPRC5D-mab07.VL1氨基酸序列如SEQ ID NO：206所示：
Figure PCTCN2022143719-appb-000131
GPRC5D-mab07.VL2氨基酸序列如SEQ ID NO：207所示：
Figure PCTCN2022143719-appb-000132
GPRC5D-mab07.VL2a氨基酸序列如SEQ ID NO：208所示：
Figure PCTCN2022143719-appb-000133
GPRC5D-mab07.VH1氨基酸序列如SEQ ID NO：209所示：
Figure PCTCN2022143719-appb-000134
GPRC5D-mab07.VH2氨基酸序列如SEQ ID NO：210所示：
Figure PCTCN2022143719-appb-000135
GPRC5D-mab07.VH3氨基酸序列如SEQ ID NO：211所示：
Figure PCTCN2022143719-appb-000136
GPRC5D-mab07.VH4氨基酸序列如SEQ ID NO：212所示：
Figure PCTCN2022143719-appb-000137
人源化轻链模板IGKV2-40*01氨基酸序列如SEQ ID NO：213所示：
Figure PCTCN2022143719-appb-000138
人源化轻链模板IGKJ4*01氨基酸序列如SEQ ID NO：214所示：
Figure PCTCN2022143719-appb-000139
人源化重链模板IGHV1-69*02氨基酸序列如SEQ ID NO：215所示：
Figure PCTCN2022143719-appb-000140
人源化重链模板IGHJ6*01氨基酸序列如SEQ ID NO：216所示：
Figure PCTCN2022143719-appb-000141
本申请分别从上述GPRC5D-mab07的第一轮人源化抗体轻链和重链可变区的回复突变设计中，选择不同的轻链和重链序列进行交叉组合，最终获得多种GPRC5D-mab07人源化抗体， 各抗体的可变区氨基酸序列如表34所示：
表34 GPRC5D-mab07抗体可变区对应氨基酸序列
Figure PCTCN2022143719-appb-000142
根据Kabat编号系统，上述人源化抗体VH和VL序列分析结果如表35所示。
表35 GPRC5D-mab07人源化抗体VH和VL序列的Kabat分析结果
Figure PCTCN2022143719-appb-000143
5.7.2 GPRC5D-mab07第二轮人源化设计
方法同5.1，鼠源抗体GPRC5D-mab07的第二轮人源化轻链模板为IGKV2-40*01和IGKJ4*01，第二轮人源化重链模板为IGHV1-69*02和IGHJ6*01。具体突变设计见表36。
表36 GPRC5D-mab07的人源化抗体突变设计
Figure PCTCN2022143719-appb-000144
注：Graft代表将鼠源抗体CDR植入人种系模板FR区序列；G43A表示将Graft第43位G突变成A，其它依此类推。突变氨基酸的编号为自然顺序编号。
GPRC5D-mab07人源化抗体可变区具体序列如下：
GPRC5D-mab07.VL1a氨基酸序列如SEQ ID NO：218所示：
Figure PCTCN2022143719-appb-000145
GPRC5D-mab07.VL1b氨基酸序列如SEQ ID NO：219所示：
Figure PCTCN2022143719-appb-000146
GPRC5D-mab07.VL1c氨基酸序列如SEQ ID NO：220所示：
Figure PCTCN2022143719-appb-000147
GPRC5D-mab07.VL1d氨基酸序列如SEQ ID NO：221所示：
Figure PCTCN2022143719-appb-000148
GPRC5D-mab07.VH4氨基酸序列如SEQ ID NO：222所示：
Figure PCTCN2022143719-appb-000149
人源化轻链模板IGKV2-40*01氨基酸序列如SEQ ID NO：223所示：
Figure PCTCN2022143719-appb-000150
人源化轻链模板IGKJ4*01氨基酸序列如SEQ ID NO：224所示：
Figure PCTCN2022143719-appb-000151
人源化重链模板IGHV1-69*02氨基酸序列如SEQ ID NO：225所示：
Figure PCTCN2022143719-appb-000152
人源化重链模板IGHJ6*01氨基酸序列如SEQ ID NO：226所示：
Figure PCTCN2022143719-appb-000153
本申请分别从上述GPRC5D-mab07的第二轮人源化抗体轻链和重链可变区的突变设计中，选择不同的轻链和重链序列进行交叉组合，最终获得多种GPRC5D-mab07人源化抗体，各抗体的可变区氨基酸序列如表37所示：
表37 GPRC5D-mab07抗体可变区对应氨基酸序列
  GPRC5D-mab07.VH4
GPRC5D-mab07.VL1a GPRC5D-mab07.VL1aVH4
GPRC5D-mab07.VL1b GPRC5D-mab07.VL1bVH4
GPRC5D-mab07.VL1c GPRC5D-mab07.VL1cVH4
GPRC5D-mab07.VL1d GPRC5D-mab07.VL1dVH4
根据Kabat编号系统，上述人源化抗体VH和VL序列分析结果如表38所示。
表38 GPRC5D-mab07人源化抗体VH和VL序列的Kabat分析结果
Figure PCTCN2022143719-appb-000154
Figure PCTCN2022143719-appb-000155
5.8 GPRC5D-mab08人源化
方法同5.1，鼠源抗体GPRC5D-mab08的人源化轻链模板为IGKV1-27*01/IGKV2-29*02和IGKJ4*01，人源化重链模板为IGHV1-3*01和IGHJ6*01。具体突变设计见表39。
表39 GPRC5D-mab08的人源化抗体突变设计
Figure PCTCN2022143719-appb-000156
注：Graft代表将鼠源抗体CDR植入人种系模板FR区序列；L46A表示将Graft第46位L突变成A，其它依此类推。突变氨基酸的编号为自然顺序编号。
GPRC5D-mab08人源化抗体可变区具体序列如下：
GPRC5D-mab08.VL4氨基酸序列如SEQ ID NO：231所示：
Figure PCTCN2022143719-appb-000157
GPRC5D-mab08.VL5氨基酸序列如SEQ ID NO：232所示：
Figure PCTCN2022143719-appb-000158
GPRC5D-mab08.VL6氨基酸序列如SEQ ID NO：233所示：
Figure PCTCN2022143719-appb-000159
GPRC5D-mab08.VH6氨基酸序列如SEQ ID NO：234所示：
Figure PCTCN2022143719-appb-000160
GPRC5D-mab08.VH7氨基酸序列如SEQ ID NO：235所示：
Figure PCTCN2022143719-appb-000161
GPRC5D-mab08.VH8氨基酸序列如SEQ ID NO：236所示：
Figure PCTCN2022143719-appb-000162
GPRC5D-mab08.VH9氨基酸序列如SEQ ID NO：237所示：
Figure PCTCN2022143719-appb-000163
Figure PCTCN2022143719-appb-000164
GPRC5D-mab08.VH10氨基酸序列如SEQ ID NO：238所示：
Figure PCTCN2022143719-appb-000165
人源化轻链模板IGKV1-27*01氨基酸序列如SEQ ID NO：239所示：
Figure PCTCN2022143719-appb-000166
人源化轻链模板IGKV2-29*02氨基酸序列如SEQ ID NO：240所示：
Figure PCTCN2022143719-appb-000167
人源化轻链模板IGKJ4*01氨基酸序列如SEQ ID NO：241所示：
Figure PCTCN2022143719-appb-000168
人源化重链模板IGHV1-3*01氨基酸序列如SEQ ID NO：242所示：
Figure PCTCN2022143719-appb-000169
人源化重链模板IGHJ6*01氨基酸序列如SEQ ID NO：243所示：
Figure PCTCN2022143719-appb-000170
本申请分别从上述GPRC5D-mab08的人源化抗体轻链和重链可变区的突变设计中，选择不同的轻链和重链序列进行交叉组合，最终获得多种GPRC5D-mab08人源化抗体，各抗体的可变区氨基酸序列如表40所示：
表40 GPRC5D-mab08抗体可变区对应氨基酸序列
Figure PCTCN2022143719-appb-000171
根据Kabat编号系统，上述人源化抗体VH和VL序列分析结果如表41所示。
表41 GPRC5D-mab08人源化抗体VH和VL序列的Kabat分析结果
Figure PCTCN2022143719-appb-000172
Figure PCTCN2022143719-appb-000173
实施例6 GPRC5D人源化抗体的鉴定
6.1基于细胞的酶联免疫吸附实验(cell-based ELISA)检测人源化抗体与人GPRC5D蛋白的结合
为了检测GPRC5D人源化抗体与人GPRC5D全长蛋白的结合活性，采用实施例4基于细胞的酶联免疫吸附实验(cell-based ELISA)同样的检测方法检测人源化抗体与人GPRC5D蛋白的结合，检测结果如图13A～13J、14A～14J，结果显示，纯化生产后的人源化抗体与人GPRC5D全长蛋白在ELISA水平上有不同程度的结合。
6.2流式细胞实验(FACS)检测人源化抗体与内源表达人GPRC5D细胞NCI-H929结合活性
为了检测GPRC5D人源化抗体与人GPRC5D全长蛋白的结合活性，采用实施例4中FACS同样的检测方法检测人源化抗体与人GPRC5D蛋白的结合，检测结果如图15A～15J，结果说明，人源化抗体均可不同程度的结合NCI-H929细胞，具有很好的特异性。

Claims (20)

  1. 一种特异性结合G蛋白偶联受体C5家族亚型D(GPRC5D)的抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段包含重链可变区和轻链可变区，并且其中
    (1)所述轻链可变区包含LCDR1、LCDR2和LCDR3，所述LCDR1具有以下所示的LCDR1的任一序列或者与所述序列相比具有1、2、3或更多个氨基酸插入、缺失和/或替换的序列，所述LCDR2具有以下所示的LCDR2的任一序列或者与所述序列相比具有1、2、3或更多个氨基酸插入、缺失和/或替换的序列，并且所述LCDR3具有以下所示的LCDR3的任一序列或者与所述序列相比具有1、2、3或更多个氨基酸插入、缺失和/或替换的序列：
    编号 LCDR1 LCDR2 LCDR3 L1 SEQ ID NO：35 SEQ ID NO：36 SEQ ID NO：37 L2 SEQ ID NO：38 SEQ ID NO：39 SEQ ID NO：37 L3 SEQ ID NO：46 SEQ ID NO：47 SEQ ID NO：48 L4 SEQ ID NO：49 SEQ ID NO：50 SEQ ID NO：48 L5 SEQ ID NO：57 SEQ ID NO：58 SEQ ID NO：59 L6 SEQ ID NO：60 SEQ ID NO：61 SEQ ID NO：59 L7 SEQ ID NO：68 SEQ ID NO：69 SEQ ID NO：70 L8 SEQ ID NO：71 SEQ ID NO：72 SEQ ID NO：70 L9 SEQ ID NO：79 SEQ ID NO：80 SEQ ID NO：81 L10 SEQ ID NO：82 SEQ ID NO：83 SEQ ID NO：81 L11 SEQ ID NO：90 SEQ ID NO：91 SEQ ID NO：92 L12 SEQ ID NO：93 SEQ ID NO：94 SEQ ID NO：92 L13 SEQ ID NO：101 SEQ ID NO：102 SEQ ID NO：103 L14 SEQ ID NO：104 SEQ ID NO：105 SEQ ID NO：103 L15 SEQ ID NO：112 SEQ ID NO：113 SEQ ID NO：114 L16 SEQ ID NO：115 SEQ ID NO：116 SEQ ID NO：114 L17 SEQ ID NO：217 SEQ ID NO：102 SEQ ID NO：103 L18 SEQ ID NO：227 SEQ ID NO：102 SEQ ID NO：103 L19 SEQ ID NO：228 SEQ ID NO：102 SEQ ID NO：103 L20 SEQ ID NO：229 SEQ ID NO：102 SEQ ID NO：103 L21 SEQ ID NO：230 SEQ ID NO：102 SEQ ID NO：103
    和，
    (2)所述重链可变区包含HCDR1、HCDR2和HCDR3，所述HCDR1具有以下所示的HCDR1的任一序列或者与所述序列相比具有1、2、3或更多个氨基酸插入、缺失和/或替换的序列，所述HCDR2具有以下所示的HCDR2的任一序列或者与所述序列相比具有1、2、3或更多个氨基酸插入、缺失和/或替换的序列，并且所述HCDR3具有以下所示的HCDR3的任一序列或者与所述序列相比具有1、2、3或更多个氨基酸插入、缺失和/或替换的序列：
    编号 HCDR1 HCDR2 HCDR3 H1 SEQ ID NO：29 SEQ ID NO：30 SEQ ID NO：31 H2 SEQ ID NO：32 SEQ ID NO：33 SEQ ID NO：34 H3 SEQ ID NO：40 SEQ ID NO：41 SEQ ID NO：42
    H4 SEQ ID NO：43 SEQ ID NO：44 SEQ ID NO：45 H5 SEQ ID NO：51 SEQ ID NO：52 SEQ ID NO：53 H6 SEQ ID NO：54 SEQ ID NO：55 SEQ ID NO：56 H7 SEQ ID NO：62 SEQ ID NO：63 SEQ ID NO：64 H8 SEQ ID NO：65 SEQ ID NO：66 SEQ ID NO：67 H9 SEQ ID NO：73 SEQ ID NO：74 SEQ ID NO：75 H10 SEQ ID NO：76 SEQ ID NO：77 SEQ ID NO：78 H11 SEQ ID NO：84 SEQ ID NO：85 SEQ ID NO：86 H12 SEQ ID NO：87 SEQ ID NO：88 SEQ ID NO：89 H13 SEQ ID NO：95 SEQ ID NO：96 SEQ ID NO：97 H14 SEQ ID NO：98 SEQ ID NO：99 SEQ ID NO：100 H15 SEQ ID NO：106 SEQ ID NO：107 SEQ ID NO：108 H16 SEQ ID NO：109 SEQ ID NO：110 SEQ ID NO：111 H17 SEQ ID NO：40 SEQ ID NO：142 SEQ ID NO：42 H18 SEQ ID NO：40 SEQ ID NO：143 SEQ ID NO：42 H19 SEQ ID NO：51 SEQ ID NO：158 SEQ ID NO：53 H20 SEQ ID NO：51 SEQ ID NO：159 SEQ ID NO：53
    优选地，所述抗体或其抗原结合片段包含以下轻链可变区和重链可变区的组合中的六个CDR的序列：L1+H1、L2+H2、L3+H3、L4+H4、L5+H5、L6+H6、L7+H7、L8+H8、L9+H9、L10+H10、L11+H11、L12+H12、L13+H13、L14+H14、L15+H15、L16+H16、L3+H17、L3+H18、L5+H19、L5+H20、L17+H13、L18+H13、L19+H13、L20+H13或L21+H13，或者与所述六个CDR的序列相比具有1、2、3或更多个氨基酸插入、缺失和/或替换的六个CDR的序列。
  2. 根据权利要求1所述的抗体或其抗原结合片段，其中：
    (1)所述轻链可变区序列包含SEQ ID NO：14、16、18、20、22、24、26、28、118～120、130～131、144～145、160～163、172～175、184～187、196～197、206～208、218～221和231～233中任一条所示的序列，或与所述序列具有80％、85％、90％、95％、96％、97％、98％、99％或更高一致性的序列；
    和，
    (2)所述重链可变区序列包含SEQ ID NO：13、15、17、19、21、23、25、27、121～125、132～137、146～152、164～166、176～178、188～190、198～201、209～212、222和234～238中任一条所示的序列，或与所述序列具有80％、85％、90％、95％、96％、97％、98％、99％或更高一致性的序列；
    优选地，所述抗体或其抗原结合片段具有如下所示的轻链可变区和重链可变区：
    (1)所述轻链可变区和所述重链可变区分别包含SEQ ID NO：14和SEQ ID NO：13所示的序列；
    (2)所述轻链可变区和所述重链可变区分别包含SEQ ID NO：16和SEQ ID NO：15所示的序列；
    (3)所述轻链可变区和所述重链可变区分别包含SEQ ID NO：18和SEQ ID NO：17所示的序列；
    (4)所述轻链可变区和所述重链可变区分别包含SEQ ID NO：20和SEQ ID NO：19所示的序列；
    (5)所述轻链可变区和所述重链可变区分别包含SEQ ID NO：22和SEQ ID NO：21所示的序列；
    (6)所述轻链可变区和所述重链可变区分别包含SEQ ID NO：24和SEQ ID NO：23所示的序列；
    (7)所述轻链可变区和所述重链可变区分别包含SEQ ID NO：26和SEQ ID NO：25所示的序列；
    (8)所述轻链可变区和所述重链可变区分别包含SEQ ID NO：28和SEQ ID NO：27所示的序列；
    (9)所述轻链可变区包含SEQ ID NO：118～120中任一条所示的序列，并且所述重链可变区包含SEQ ID NO：121～125中任一条所示的序列；
    (10)所述轻链可变区包含SEQ ID NO：130～131中任一条所示的序列，并且所述重链可变区包含SEQ ID NO：132～137中任一条所示的序列；
    (11)所述轻链可变区包含SEQ ID NO：144～145中任一条所示的序列，并且所述重链可变区包含SEQ ID NO：146～152中任一条所示的序列；
    (12)所述轻链可变区包含SEQ ID NO：160～163中任一条所示的序列，并且所述重链可变区包含SEQ ID NO：164～166中任一条所示的序列；
    (13)所述轻链可变区包含SEQ ID NO：172～175中任一条所示的序列，并且所述重链可变区包含SEQ ID NO：176～178中任一条所示的序列；
    (14)所述轻链可变区包含SEQ ID NO：184～187中任一条所示的序列，并且所述重链可变区包含SEQ ID NO：188～190中任一条所示的序列；
    (15)所述轻链可变区包含SEQ ID NO：196～197中任一条所示的序列，并且所述重链可变区包含SEQ ID NO：198～201中任一条所示的序列；
    (16)所述轻链可变区包含SEQ ID NO：206～208中任一条所示的序列，并且所述重链可变区包含SEQ ID NO：209～212中任一条所示的序列；
    (17)所述轻链可变区包含SEQ ID NO：218～221中任一条所示的序列，并且所述重链可变区包含SEQ ID NO：222中任一条所示的序列；
    (18)所述轻链可变区包含SEQ ID NO：231～233中任一条所示的序列，并且所述重链可变区包含SEQ ID NO：234～238中任一条所示的序列；
    或(19)所述轻链可变区包含与上述(1)～(18)中任一项所示的轻链可变区具有80％、85％、90％、95％、96％、97％、98％、99％或更高一致性的序列，并且所述重链可变区包含与上述(1)～(18)中任一项所示的重链可变区具有80％、85％、90％、95％、96％、97％、98％、99％或更高一致性的序列。
  3. 根据权利要求1或2所述的抗体或其抗原结合片段，其是嵌合的、人源化的或全人源的。
  4. 根据权利要求1～3任一项所述的抗体或其抗原结合片段，其能与人或猴GPRC5D结合。
  5. 根据权利要求1～4任一项所述抗原结合片段，其中，所述抗原结合片段选自F(ab) 2、Fab’、Fab、Fv、scFv、纳米抗体或affibody中的一种或多种。
  6. 根据权利要求1～5任一项所述抗体或其抗原结合片段，其还包含人或鼠抗体IgG1、IgG2、IgG3、IgG4、IgA、IgM、IgE和IgD中的任一恒定区序列；优选地，包含人或鼠抗体IgG1、IgG2、IgG3或IgG4的恒定区序列，或包含与人或鼠抗体IgG1、IgG2、IgG3或IgG4的恒定区序列具有80％、85％、90％、95％、96％、97％、98％、99％或更高一致性的序列；进一步地，所述抗体或其抗原结合片段还偶联有治疗剂或示踪剂；优选地，所述治疗剂选自放射性同位素、化疗药或免疫调节剂，所述示踪剂选自放射学造影剂、顺磁离子、金属、荧光标记、化学发光标记、超声造影剂和光敏剂：更优选地，所述细胞毒性剂选自生物碱类、甲氨蝶呤、蒽环类抗生素、紫杉烷类、吡咯并苯并二氮杂
    Figure PCTCN2022143719-appb-100001
    或毒素化合物。
  7. 一种多特异性抗原结合分子，其中，所述多特异性抗原结合分子包含权利要求1～6任一项所述的GPRC5D抗体或其抗原结合片段，以及结合GPRC5D以外其他抗原的抗原结合分子，或结合与权利要求1～6任一项所述的抗体或其抗原结合片段不同的GPRC5D表位；
    优选地，所述其他抗原结合分子为抗体或其抗原结合片段；
    优选地，所述多特异性抗原结合分子可为双特异性、三特异性或四特异性；
    优选地，所述多特异性抗原结合分子可为二价、三价、四价、五价或六价。
  8. 一种嵌合抗原受体(CAR)，其中，所述嵌合抗原受体至少包含信号肽、胞外抗原结合结构域、铰链区、跨膜结构域和胞内信号传导结构域，所述胞外抗原结合结构域包含权利要求1～6任一项所述的GPRC5D抗体或其抗原结合片段，或权利要求7所述的多特异性抗原结合分子。
  9. 一种免疫效应细胞，其中，所述免疫效应细胞表达权利要求8所述的嵌合抗原受体，或包含编码权利要求8所述嵌合抗原受体的核酸片段；优选地，所述免疫效应细胞选自T细胞、NK细胞、NKT细胞、DNT细胞、单核细胞、巨噬细胞、树突状细胞或肥大细胞，所述T细胞优选自细胞毒性T细胞(CTL)、调节性T细胞或辅助性T细胞；优选地，所述免疫效应细胞为自体免疫效应细胞或同种异体免疫效应细胞。
  10. 一种分离的核酸片段，其中，所述核酸片段编码权利要求1～6任一项所述的抗体或其抗原结合片段、权利要求7所述的多特异性抗原结合分子或权利要求8所述的嵌合抗原受体。
  11. 一种载体，其中，所述载体包含权利要求10所述的核酸片段。
  12. 一种宿主细胞，所述宿主细胞包含权利要求11所述的载体；优选地，所述细胞为原核细胞或真核细胞，例如细菌(例如大肠杆菌)、真菌(例如酵母)、昆虫细胞或哺乳动物细胞(例如CHO细胞系或293T细胞系)。
  13. 一种制备权利要求1～6任一项所述的抗体或其抗原结合片段或权利要求7所述的多特异性抗原结合分子的方法，其中，所述方法包括培养权利要求12所述的细胞，以及分离所述细胞表达的抗体、抗原结合片段或多特异性抗原结合分子。
  14. 一种制备前述免疫效应细胞的方法，其中，所述方法包括将编码权利要求8所述的CAR的核酸片段导入所述免疫效应细胞，可选地，所述方法还包括启动所述免疫效应细胞表达权利要求8所述的CAR。
  15. 一种药物组合物，其中，所述药物组合物包含权利要求1～6任一项所述的抗体或其抗原结合片段、权利要求7所述的多特异性抗原结合分子、权利要求9所述的免疫效应细胞、权利要求10所述的核酸片段、权利要求11所述的载体或根据权利要求13或14所述的方法制备获得的产品；可选地，所述药物组合物还包含药学上可接受的运载体、稀释剂或助剂；可选地，所述药物组合物还包含额外的抗肿瘤剂。
  16. 一种治疗肿瘤或癌症的方法，所述方法包括向受试者施用有效量的权利要求1～6任一项所述的抗体或其抗原结合片段、权利要求7所述的多特异性抗原结合分子、权利要求9所述的免疫效应细胞、权利要求10所述的核酸片段、权利要求11所述的载体、根据权利要求13或14所述的方法制备获得的产品或权利要求15所述的药物组合物；
    优选地，所述肿瘤或癌症为表达GPRC5D的肿瘤或癌症，优选B细胞淋巴瘤，更优选多发性骨髓瘤(MM)。
  17. 权利要求1～6任一项所述的抗体或其抗原结合片段、权利要求7所述的多特异性抗原结合分子、权利要求9所述的免疫效应细胞、权利要求10所述的核酸片段、权利要求11所述的载体、根据权利要求13或14所述的方法制备获得的产品或权利要求15所述的药物组合物在制备治疗肿瘤或癌症药物中的用途；
    优选地，所述肿瘤或癌症为表达GPRC5D的肿瘤或癌症，优选B细胞淋巴瘤，更优选多发性骨髓瘤(MM)。
  18. 一种试剂盒，所述试剂盒包含权利要求1～6任一项所述的抗体或其抗原结合片段、权利要求7所述的多特异性抗原结合分子、权利要求9所述的免疫效应细胞、权利要求10所述的核酸片段、权利要求11所述的载体、根据权利要求13或14所述的方法制备获得的产品或权利要求15所述的药物组合物。
  19. 一种检测生物学样品中GPRC5D表达的方法，所述方法包括在权利要求1～6任一项所述的抗体或其抗原结合片段与GPRC5D之间能够形成复合物的条件下，使所述生物学样品与所述的抗体或其抗原结合片段接触；优选地，所述方法还包括检测所述复合物的形成，指示样品中GPRC5D的存在或表达水平。
  20. 权利要求1～6任一项所述的抗体或其抗原结合片段在制备GPRC5D检测试剂中的用途。
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CN117510636A (zh) * 2023-11-17 2024-02-06 上海吉凯生物技术有限公司 Gprc5d抗体及其应用
CN117510636B (zh) * 2023-11-17 2024-05-28 上海吉凯生物技术有限公司 Gprc5d抗体及其应用

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