Anti-GPRC5D multispecific antibody

Abstract

The invention provides a multispecific antibody capable of binding GPRC5D and CD3 antigens and an antigen binding molecule thereof, a nucleic acid molecule for coding the multispecific antibody and the antigen binding molecule thereof, a carrier containing the nucleic acid molecule, a host cell containing the carrier, a recombinant protein or an immunoconjugate containing the multispecific antibody and the antigen binding molecule thereof, and a preparation method of the multispecific antibody and the antigen binding molecule thereof. The invention also relates to application of the compounds in preparation of drugs for treating or preventing diseases, especially for treating multiple myeloma, and application in detection of products.

Description

A multispecific antibody against GPRC5D Technical Field

[0001] The present invention relates to a multispecific antibody against GPRC5D, belonging to the field of biomedicine. Background Art

[0002] Multiple myeloma (MM) is the second most common hematologic malignancy worldwide, after non-Hodgkin's lymphoma, accounting for approximately 10% of all hematologic malignancies. According to the World Health Organization's Global Cancer Statistics Report, in 2018, there were 20,066 new cases of MM and 14,665 deaths in China, with a cumulative total of 44,643 cases over the past five years. Furthermore, the incidence and mortality rates of MM patients increase with age. As my country's population continues to age, the total number of patients with MM is expected to increase, creating a significant clinical need.

[0003] Currently, treatment options for multiple myeloma in my country can be divided into three categories: immunomodulators, proteasome inhibitors, and biomolecular targeted therapies. Immunomodulators and proteasome inhibitors are primarily used as first-line combination therapy before stem cell transplantation and as maintenance therapy after transplantation for patients eligible for stem cell transplantation. For example, over the past decade, the use of immunomodulators such as thalidomide and its derivative lenalidomide, and small molecule proteasome inhibitors such as bortezomib, has significantly improved the remission rate and survival of MM patients.

[0004] The biomolecular targeted drugs for MM mainly involve three major targets: plasma cell surface protein CD38, signaling lymphocytes activating molecule family member 7 (Signaling Lymphocytes Activating Molecule Factor 7, SLAMF7) and B cell maturation antigen (B Cell Maturation Antigen, BCMA).

[0005] Among them, anti-CD38 monoclonal antibodies (for example, Daratumumab, which was conditionally approved and imported for registration and marketing by the National Medical Products Administration (NMPA) in 2019) are mainly used for first-line treatment of relapsed or second-line treatment of refractory multiple myeloma.

[0006] In addition, BCMA protein has also become a research hotspot in this field due to its high expression specificity on MM cells. In August 2020, the U.S. FDA accelerated the approval of the BMCA-targeting antibody-drug conjugate (ADC) Blenrep for the treatment of adult patients with relapsed or refractory multiple myeloma (R / R MM) who have previously received at least 4 therapies and whose disease is refractory to at least one proteasome inhibitor / immunomodulator / CD38 monoclonal antibody and has been proven to have disease progression in the last treatment. The overall response rate of this drug can still reach 31%, and the median duration of response (DoR) is greater than 6 months. In addition, the anti-tumor effect of CAR-T or CD3 bispecific antibodies targeting BCMA by mediating T cells is also significant.

[0007] However, although ADC, bispecific antibodies and CAR-T therapies targeting BCMA have shown positive clinical effects, cases of BCMA negativity (or low expression) and related relapses after treatment have been reported. The recurrence and refractory nature of MM remain difficulties in the clinical treatment of MM, and finding new effective targets remains an urgent problem to be solved in this field.

[0008] Studies have reported that high expression of GPRC5D is associated with poor prognosis in multiple myeloma (Atamaniuk, J., et al., Overexpression of G protein-coupled receptor 5D in the bone marrow is associated with poor prognosis in patients with multiple myeloma. European Journal of Clinical Investigation, 2012. 42(9): p. 953-960).

[0009] GPRC5D belongs to the G protein-coupled receptor (GPCR) family; specifically, it is a G protein-coupled receptor C5 family subtype D, which was first identified as an orphan SARS-like C class GPCR in 2001 (Brauner-Osborne, H., et al. Cloning and characterization of a human orphan family C G-protein coupled receptor GPRC5D. Biochim Biophys Acta, 2001. 1518(3): p. 237-48).

[0010] Although GPRC5D has been previously identified in cells from multiple myeloma patients, it has not been used in clinical development due to the lack of protein expression profile studies. Until 2019, research reports showed that GPRC5D is highly expressed in plasma cells of multiple myeloma, mostly not expressed in normal tissues, and only expressed in hair follicles with immune privilege (Smith, EL, et al., GPRC5D is a target for the immunotherapy of multiple myeloma with rationally designed CAR T cells. Science Translational Medicine, 2019.11(485)). An even more surprising discovery is that the expression profile of GPRC5D does not overlap with BCMA - this discovery made researchers in this field realize that GPRC5D is expected to become a new therapeutic target to replace BCMA targeted therapy, or to treat patients with low / no BCMA expression, or to succeed BCMA treatment after relapse.

[0011] Summary of the Invention

[0012] The inventors of the present invention have developed a novel antibody and antigen-binding molecule thereof that specifically binds to GPRC5D based on the GPRC5D antigen, and further developed bi- / multi-specific antibodies and antigen-binding molecules thereof that bind to the GPRC5D antigen on target cells and activating T cell antigens (such as CD3) on T cells; the bi- / multi-specific antibodies and antigen-binding molecules thereof of the present invention simultaneously bind to target cells and T cells and enable them to interact with each other, thereby activating cytotoxic T cells and lysing the target cells.

[0013] The first aspect of the present invention provides a multispecific antibody or antigen-binding molecule thereof, wherein:

[0014] The multispecific antibody or antigen-binding molecule thereof comprises a first antigen-binding moiety and a second antigen-binding moiety;

[0015] wherein the first antigen binding moiety binds to GPRC5D and the second antigen binding moiety binds to CD3;

[0016] The first antigen binding moiety comprises a heavy chain variable region i-VH and a light chain variable region i-VL, wherein the heavy chain variable region i-VH comprises i-HCDR1 as shown in SEQ ID NO.1, i-HCDR2 as shown in SEQ ID NO.2, and i-HCDR3 as shown in SEQ ID NO.3; the light chain variable region i-VL comprises i-LCDR1 as shown in SEQ ID NO.4, i-LCDR2 with an amino acid sequence of SAS, and i-LCDR3 as shown in SEQ ID NO.5;

[0017] The second antigen binding moiety comprises a heavy chain variable region ii-VH and a light chain variable region ii-VL, wherein the heavy chain variable region ii-VH comprises ii-HCDR1 as shown in SEQ ID NO.6, ii-HCDR2 as shown in SEQ ID NO.7, and ii-HCDR3 as shown in SEQ ID NO.8; the light chain variable region ii-VL comprises ii-LCDR1 as shown in SEQ ID NO.9, ii-LCDR2 with an amino acid sequence of GTN, and ii-LCDR3 as shown in SEQ ID NO.10.

[0018] In a more preferred embodiment of the invention, the second antigen binding moiety binds CD3ε.

[0019] In a more preferred embodiment of the present invention, the heavy chain variable region i-VH of the first antigen binding moiety comprises the sequence shown in SEQ ID NO. 11, or comprises a sequence having more than 80% homology to the sequence shown in SEQ ID NO. 11. For example, the sequence of the heavy chain variable region i-VH of the first antigen binding moiety comprises a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology to the sequence shown in SEQ ID NO. 11.

[0020] In a more preferred embodiment of the present invention, the light chain variable region i-VL of the first antigen binding moiety comprises the sequence shown in SEQ ID NO. 12, or comprises a sequence having more than 80% homology to the sequence shown in SEQ ID NO. 12. For example, the sequence of the light chain variable region i-VL of the first antigen binding moiety comprises a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology to the sequence shown in SEQ ID NO. 12.

[0021] In a more preferred embodiment of the present invention, the heavy chain variable region ii-VH of the second antigen binding moiety comprises the sequence shown in SEQ ID NO. 13, or comprises a sequence having more than 80% homology to the sequence shown in SEQ ID NO. 13. For example, the sequence of the heavy chain variable region ii-VH of the second antigen binding moiety comprises a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology to the sequence shown in SEQ ID NO. 13.

[0022] In a more preferred embodiment of the present invention, the light chain variable region ii-VL of the second antigen binding moiety comprises the sequence shown in SEQ ID NO. 14, or comprises a sequence having more than 80% homology to the sequence shown in SEQ ID NO. 14. For example, the sequence of the light chain variable region ii-VL of the second antigen binding moiety comprises a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology to the sequence shown in SEQ ID NO. 14.

[0023] The percentage of "sequence homology" with respect to amino acid sequences is determined by determining the number of amino acid residues present in the two sequences to produce the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to produce the percentage of sequence homology. Sequence homology is also known as sequence identity.

[0024] In a specific embodiment of the present invention, the heavy chain variable region i-VH and light chain variable region i-VL of the above-mentioned first antigen binding moiety can be subjected to a small number of amino acid deletions, insertions or amino acid mutations based on the sequences shown in SEQ ID NO. 11 and SEQ ID NO. 12, respectively, to obtain an amino acid sequence with a homology of more than 80%. Variants obtained by a small number of amino acid substitutions (deletions or insertions, or amino acid mutations, or replacements of similar amino acids), especially conservative amino acid substitutions in the framework regions, retain the original properties and functions of the heavy chain variable region and light chain variable region, i.e., the properties and functions of antibodies that specifically bind to GPRC5D, then these variants also fall within the scope of protection of the present invention. Similarly, the heavy chain variable region ii-VH and light chain variable region ii-VL of the above-mentioned second antigen-binding moiety can be subjected to a small number of amino acid deletions, insertions or amino acid mutations based on the sequences shown in SEQ ID NO. 13 and SEQ ID NO. 14, respectively, to obtain amino acid sequences with a homology of more than 80%. As long as the original properties and functions of the heavy chain variable region and the light chain variable region are retained, that is, the antibody properties and functions of specific binding to CD3 or CD3ε, then these variants also fall within the scope of protection of the present invention.

[0025] The above-mentioned "framework region" refers to the amino acid sequence located between CDRs, including the framework region of the heavy chain variable region and the framework region of the light chain variable region.

[0026] In a specific embodiment of the present invention, the first antigen binding moiety is selected from any one of Fv, Fab, F(ab')2, Fab', dsFv, scFv, sc(Fv)2 or a single domain antibody; the second antigen binding moiety is selected from any one of Fv, Fab, F(ab')2, Fab', dsFv, scFv, sc(Fv)2 or a single domain antibody.

[0027] In a specific embodiment of the present invention, the first antigen binding moiety and / or the second antigen binding moiety is a Fab molecule. In a preferred embodiment of the present invention, the first antigen binding moiety and the second antigen binding moiety are Fab molecules.

[0028] A Fab molecule is a protein molecule composed of the VH and CH1 domains of the Fab heavy chain and the VL and CL domains of the Fab light chain. Conventional Fab molecules refer to naturally occurring Fab molecules, in which the Fab heavy chain is VH-CH1 from N-terminus to C-terminus, and the Fab light chain is VL-CL from N-terminus to C-terminus.

[0029] Alternatively, within the same Fab molecule, the Fab heavy chain variable region and the Fab light chain variable region can be interchanged / replaced; alternatively, within the same Fab molecule, the Fab heavy chain constant region and the Fab light chain constant region can be interchanged / replaced. For example, a Fab molecule comprises a peptide chain consisting of VL-CH1 (N to C-terminus) and a peptide chain consisting of VH-CL (N to C-terminus). For ease of description, the peptide chain comprising the heavy chain constant domain CH1 is generally referred to as the Fab heavy chain.

[0030] In another specific embodiment, the second antigen binding moiety is a Fab molecule in which the amino acid at position 124 of the light chain constant region can be independently substituted with lysine (K), arginine (R) or histidine (H) (numbering according to Kabat) and the amino acid at position 123 can be independently substituted with lysine (K), arginine (R) or histidine (H) (numbering according to Kabat), and in the heavy chain constant region CH1 the amino acid at position 147 can be independently substituted with glutamic acid (E) or aspartic acid (D) (numbering according to the EU index) and the amino acid at position 213 can be independently substituted with glutamic acid (E) or aspartic acid (D) (numbering according to the EU index).

[0031] In an alternative embodiment of the present invention, the multispecific antibody or antigen-binding molecule thereof may further comprise more antigen-binding moieties, which may be the same as or different from the first / second antigen-binding moieties, for example, antigen-binding moieties that bind to other antigens.

[0032] In an alternative embodiment of the present invention, the first / second antigen binding moiety is selected from a murine antibody, a humanized antibody or a chimeric antibody.

[0033] In a preferred embodiment of the present invention, the multispecific antibody or antigen-binding molecule thereof comprises a light chain constant region; the light chain constant region is preferably a human light chain constant region; preferably, the light chain constant region is a human λ or κ light chain constant region; preferably, the light chain constant region is a human κ light chain constant region.

[0034] In a preferred embodiment of the present invention, the multispecific antibody or antigen-binding molecule thereof comprises a heavy chain constant region; the heavy chain constant region is preferably a heavy chain constant region of human IgG1, 2, 3, or 4.

[0035] Preferably, the heavy chain constant region comprises a first subunit containing a CH3 region and a second subunit containing a CH3 region, which together constitute an Fc domain; the heavy chain constant region is human IgG1;

[0036] Preferably, the Fc domain of the heavy chain constant region is a mutant Fc domain;

[0037] Preferably, the amino acid residues in the CH3 region of the first subunit of the mutant Fc domain are replaced with amino acid residues having a larger side chain volume, i.e., knob mutation, thereby forming a protrusion structure; the amino acid residues in the CH3 region of the second subunit of the mutant Fc domain are replaced with amino acid residues having a smaller side chain volume, i.e., hole mutation, thereby forming a cavity structure; the cavity structure accommodates the protrusion structure so that the first subunit and the second subunit combine to form a heterodimer.

[0038] In another embodiment of the present invention, the aforementioned mutant Fc domain may have reduced Fc receptor binding and / or effector function.

[0039] More preferably, the mutation scheme of the Fc domain is selected from: (a) the knob mutation is T366W, and the hole mutation is T366S, L368A and Y407V; (b) the knob mutation is S354C and T366W, and the hole mutation is Y349C, T366S, L368A and Y407V; the above numbering is according to the EU index.

[0040] The Fc domain further comprises an H435R mutation in the CH3 region of the second subunit; and / or, L234A, L235A, P329G, D356E and L358M mutations in the second subunit; and / or, L234A, L235A, P329G, D356E and L358M mutations in the first subunit, the above numbering being according to the EU index.

[0041] In certain embodiments of the present invention, the addition or deletion of glycosylation sites of an antibody can be facilitated by altering the amino acid sequence, for example, altering the amino acid sequence of the Fc domain, such that one or more glycosylation sites are created or eliminated.

[0042] In a specific embodiment of the present invention, the heavy chain variable region ii-VH and the light chain variable region ii-VL in the second antigen binding module Fab molecule are exchanged by crossmab light and heavy chains, that is, ii-VH and ii-VL are replaced with each other, or the constant regions ii-CL and ii-CH1 are replaced with each other.

[0043] Preferably, the constant regions ii-CL and ii-CH1 in the second antigen-binding moiety Fab molecule are replaced with each other, and the heavy chain amino acid sequence containing the constant region ii-CL after replacement is shown in SEQ ID NO.36, and the light chain amino acid sequence containing ii-CH1 after replacement is shown in SEQ ID NO.37;

[0044] And / or, the heavy chain amino acid sequence of the first antigen binding moiety is shown as SEQ ID NO.34, and the light chain amino acid sequence is shown as SEQ ID NO.35.

[0045] In a specific embodiment of the invention, the Fab molecule of the first antigen binding moiety has the amino acid at position 123 of the constant region i-CL replaced by arginine (R) (numbering according to Kabat), the amino acid at position 124 is replaced by lysine (K) (numbering according to Kabat), and the amino acids at positions 147 and 213 of the constant region i-CH1 are replaced by glutamic acid (E), respectively (numbering according to the EU index); and the Fab molecule of the second antigen binding moiety has the amino acid at position 170 of the constant region ii-CL replaced by arginine (R) (numbering according to Kabat), and the amino acid at position 133 of the constant region ii-CH1 is replaced by glutamic acid (E) (numbering according to the EU index).

[0046] In a specific embodiment of the invention, the Fab molecule of the first antigen binding moiety has the amino acid at position 123 of the constant region i-CL replaced by arginine (R) (numbering according to Kabat), the amino acid at position 124 is replaced by lysine (K) (numbering according to Kabat), and the amino acids at positions 147 and 213 of the constant region i-CH1 are replaced by glutamic acid (E), respectively (numbering according to the EU index); and the Fab molecule of the second antigen binding moiety has the amino acid at position 170 of the constant region ii-CL replaced by lysine (K) (numbering according to Kabat), and the amino acid at position 133 of the constant region ii-CH1 is replaced by glutamic acid (E) (numbering according to the EU index).

[0047] In a specific embodiment of the invention, the Fab molecule of the first antigen binding moiety has the amino acid at position 176 of the constant region i-CL replaced by C (numbering according to Kabat), and the amino acid at position 183 of the constant region i-CH1 replaced by C (numbering according to the EU index).

[0048] In a specific embodiment of the invention, the Fab molecule of the first antigen binding moiety has the amino acid at position 162 of the constant region i-CL replaced by cysteine ​​(C) (numbering according to Kabat), and the amino acid at position 173 of the constant region i-CH1 replaced by cysteine ​​(C) (numbering according to the EU index).

[0049] In a specific embodiment of the invention, the Fab molecule of the first antigen binding moiety has the amino acid at position 121 of the constant region i-CL replaced by cysteine ​​(C) (numbering according to Kabat), and the amino acid at position 126 of the constant region i-CH1 replaced by cysteine ​​(C) (numbering according to the EU index).

[0050] In a specific embodiment of the invention, the Fab molecule of the first antigen binding moiety has the amino acid at position 121 of the constant region i-VL replaced by cysteine ​​(C) (numbering according to IGMT), and the amino acid at position 49 of the constant region i-VH1 replaced by cysteine ​​(C) (numbering according to IGMT).

[0051] In a specific embodiment of the present invention, disulfide bonds are formed between the amino acid sites substituted by cysteine ​​(C).

[0052] The multispecific antibody or antigen-binding molecule thereof of the present invention can simultaneously bind to the GPRC5D protein of target cells and the antigen CD3 of activated T cells, thereby enabling interaction between target cells and T cells, thereby activating cytotoxic T cells and lysing target cells.

[0053] In a specific embodiment of the present invention, the multispecific antibodies or antigen-binding molecules thereof of the present invention can cross-react with monkey GPRC5D in terms of species cross-reactivity, which is beneficial for conducting subsequent preclinical experiments and facilitating the subsequent development of products with therapeutic uses. In addition, the multispecific antibodies or antigen-binding molecules thereof of the present invention have antibody endocytosis activity in cells and are suitable for the development of ADC drugs.

[0054] The second aspect of the invention provides a nucleic acid molecule encoding the multispecific antibody or antigen-binding molecule thereof as described above.

[0055] In a specific embodiment of the present invention, the nucleic acid molecule may be an isolated nucleic acid molecule.

[0056] The third aspect of the present invention provides a vector comprising the above-mentioned nucleic acid molecule, that is, a vector comprising a nucleic acid molecule encoding the above-mentioned multispecific antibody or its antigen-binding molecule, in particular an expression vector expressing the above-mentioned multispecific antibody or its antigen-binding molecule.

[0057] The fourth aspect of the present invention provides a host cell comprising the above nucleic acid molecule or the above vector.

[0058] In a specific embodiment of the present invention, the host cell is a eukaryotic cell, preferably a mammalian cell.

[0059] Regarding "host cells", one can choose, but is not limited to: prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cell models such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells, etc.

[0060] In a preferred embodiment of the present invention, the host cell is a HEK293 cell.

[0061] In a fifth aspect, the present invention also provides a method for producing an antibody that binds to GPRC5D, comprising the following steps: first, culturing the above-mentioned host cell under conditions suitable for the expression of the above-mentioned multispecific antibody or its antigen-binding molecule, and then recovering the multispecific antibody or its antigen-binding molecule.

[0062] The multispecific antibodies or antigen-binding molecules thereof of the present invention can be produced by the aforementioned recombinant method or by a hybridoma method.

[0063] Other aspects of the present invention also provide glycosylation variants of the above-mentioned multispecific antibodies or antigen-binding molecules thereof, antibody variants engineered with cysteine ​​residues, antibody derivatives, and immunoconjugates.

[0064] The sixth aspect of the present invention provides a recombinant protein comprising the above-mentioned multispecific antibody or antigen-binding molecule thereof.

[0065] The seventh aspect of the present invention provides an immunoconjugate, which comprises the above-mentioned multispecific antibody or antigen-binding molecule.

[0066] Preferably, the conjugated portion of the immunoconjugate is one or more heterologous molecules, for example, a heterologous molecule with cytotoxicity that can be used in an immunoconjugate.

[0067] The eighth aspect of the present invention provides a pharmaceutical composition, wherein the pharmaceutical composition comprises the above-mentioned multispecific antibody or its antigen-binding molecule, or comprises the above-mentioned nucleic acid molecule, or comprises the above-mentioned vector, or comprises the above-mentioned host cell, or comprises the above-mentioned recombinant protein.

[0068] Preferably, the pharmaceutical composition of the present invention further contains other agents. In some embodiments, the other agents are selected from one or more of the group consisting of hormone preparations, targeted small molecule preparations, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, oncolytic drugs, cytotoxic agents, cytokines, activators of co-stimulatory molecules, inhibitors of inhibitory molecules, and vaccines.

[0069] The ninth aspect of the present invention provides a detection product, wherein the detection product comprises the above-mentioned multispecific antibody or its antigen-binding molecule, or comprises the above-mentioned nucleic acid molecule, or comprises the above-mentioned vector, or comprises the above-mentioned host cell, or comprises the above-mentioned recombinant protein, or comprises the above-mentioned immunoconjugate.

[0070] The detection product is used to detect the presence or level of GPRC5D in a sample.

[0071] In a specific embodiment of the present invention, the detection product includes, but is not limited to, a detection reagent, a detection kit, a detection chip or a test paper, etc.

[0072] The tenth aspect of the present invention provides the use of the above-mentioned multispecific antibody or its antigen-binding molecule, or the above-mentioned nucleic acid molecule, or the above-mentioned vector, or the above-mentioned host cell, or the above-mentioned recombinant protein, or the above-mentioned immunoconjugate, or the above-mentioned pharmaceutical composition in the preparation of a medicament for treating or preventing a disease; preferably, the disease is cancer or an autoimmune disease; preferably, the disease is multiple myeloma; preferably, the disease is systemic lupus erythematosus and / or rheumatoid arthritis.

[0073] The present invention relates to a multispecific antibody that binds to GPRC5D and CD3 antigens and an antigen-binding molecule thereof, a nucleic acid molecule encoding the multispecific antibody and the antigen-binding molecule thereof, a vector comprising the nucleic acid molecule, a host cell comprising the vector, a recombinant protein comprising the multispecific antibody and the antigen-binding molecule thereof, and their use in preparing drugs for treating or preventing diseases, in particular for treating multiple myeloma, and their use in detection products.

[0074] The bispecific antibodies of the present invention have the following advantages:

[0075] 1. It has high binding activity with human GPRC5D antigen, cross-reactivity with monkeys and mice, and high killing activity against tumor cells;

[0076] 2. It does not cause obvious cytokine storm and is highly safe;

[0077] 3. In vivo experimental results show that the bispecific antibody of the present invention has significant anti-tumor effects;

[0078] 4. The Fab structure of the bispecific antibody of the present invention was further optimized. The results showed that the optimized bispecific antibody had a significantly improved yield and resulted in a higher mortality rate of tumor cells. 5. The bispecific antibody of the present invention significantly increased T cell infiltration in tumor tissues after treatment compared to GC5B596D, had good drugability, and the bispecific antibody molecule had good thermal stability, pH, oxidation, and freeze-thaw stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0079] Figure 1 shows the effect of the dual antibody GPRC5D×CD3 (375Z56KC) in Example 1 on the tumor volume of NCI-H929 xenograft tumors;

[0080] FIG2 shows the effect of the dual antibody GPRC5D×CD3 (375Z56KC) in Example 1 on the tumor weight of NCI-H929 xenograft tumors.

[0081] FIG3 shows the results of the killing effect of PBMC mediated by the bispecific antibody molecule on H929 cells (A) and MM1.S cells (B). DETAILED DESCRIPTION

[0082] the term

[0083] The terms used herein are for the purpose of describing the embodiments only and are not intended to be limiting. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0084] The term "comprising" or "including" means including the stated elements, integers, or steps, but does not exclude any other elements, integers, or steps. In this document, when the term "comprising" or "including" is used, unless otherwise indicated, combinations of the stated elements, integers, or steps are also encompassed. For example, when referring to an antibody variable region "comprising" a specific sequence, it is intended to encompass an antibody variable region consisting of the specific sequence.

[0085] The term "and / or," such as "X and / or Y," should be understood to mean "X and Y" or "X or Y" and should be used to provide clear support for both meanings or either meaning.

[0086] The term "antibody" is used herein in the broadest sense to refer to a protein that contains an antigen binding site and encompasses natural and artificial antibodies of various structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), single-chain antibodies, whole antibodies, and antibody fragments.

[0087] The term "bispecific" refers to an antigen-binding molecule that is capable of specifically binding to at least two different antigenic determinants. Typically, a bispecific antigen-binding molecule comprises two antigen-binding sites, each specific for a different antigenic determinant. In certain embodiments, the bispecific antigen-binding molecule is capable of simultaneously binding to two antigenic determinants, particularly two antigenic determinants expressed on two different cells.

[0088] The term "CD3" refers to the human CD3 protein multi-subunit complex. The CD3 protein multi-subunit complex is composed of six different polypeptide chains. Unless otherwise indicated, the term "CD3" includes any CD3 variant, isoform, and species homolog that is naturally expressed by cells (including T cells) or is capable of being expressed on cells transfected with genes or cDNAs encoding those polypeptides.

[0089] The term "GPRC5D" refers to the tumor-associated antigen G protein-coupled receptor family C group 5 member D (e.g., human GPRC5D protein under accession number UniProt Q9NZD1). As used herein, "antigen binding specificity for GPRC5D" refers to an antibody or antibody fragment, such as an scFv or Fab, that specifically binds to GPRC5D. In one embodiment, the antigen binding site of an antibody molecule of the invention that binds to GPRC5D can have high affinity binding activity for cells expressing GPRC5D. In one embodiment, the antigen binding specificity has cross-reactivity to human and monkey GPRC5D.

[0090] As used herein, the term "antigen binding moiety" refers to a polypeptide molecule that specifically binds to an antigenic determinant. In one embodiment, the antigen binding moiety is capable of directing an entity to which it is attached (e.g., a second antigen binding moiety) to a target site, such as a specific type of tumor cell bearing the antigenic determinant. In another embodiment, the antigen binding moiety is capable of activating signaling via its target antigen, such as a T-cell receptor complex antigen. Antigen binding moieties include antibodies and fragments thereof as further defined herein. Specific antigen binding moieties include the antigen binding domain of an antibody, which comprises an antibody heavy chain variable region and an antibody light chain variable region. In certain embodiments, the antigen binding moiety may comprise an antibody constant region, as further defined herein and known in the art. Available heavy chain constant regions include any of the following five isotypes: α, δ, ε, γ, or μ. Available light chain constant regions include any of the following two isotypes: κ and λ.

[0091] The term "variable region" or "variable domain" refers to the domain of an antibody's heavy or light chain involved in antigen binding. VHH, VH, and VL each contain four conserved framework regions (FRs) and three complementarity-determining regions (CDRs). The term "complementarity-determining region" or "CDR" refers to the region of the variable domain that primarily contributes to antigen binding; "framework" or "FR" refers to the variable domain residues excluding the CDR residues. A VH or VHH contains three CDR regions: HCDR1, HCDR2, and HCDR3 for VHH, and VHH-CDR1, VHH-CDR2, and VHH-CDR3 for VHH. The VL contains three CDR regions: LCDR1, LCDR2, and LCDR3. Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. A single VH or VL may be sufficient to confer antigen-binding specificity.

[0092] In the present invention, "Fab" is composed of a light chain and the CH1 and variable regions of a heavy chain. "Fab'" contains a light chain and a portion encompassing the VH domain, CH1 domain, and the region between the CH1 and CH2 domains. The two heavy chains of two Fab' fragments can form an interchain disulfide bond to form an F(ab')2 molecule. The term "Fv" refers to an antibody fragment consisting of the VL and VH domains of a single arm of an antibody.

[0093] In the present invention, the scFv (single chain antibody fragment) refers to a polypeptide chain formed by connecting a VH domain and a VL domain via a linker (also known as a linker). The VL and VH domains are paired to form a monovalent molecule via a linker that enables them to be produced as a single polypeptide chain [see, for example, Bird et al., Science 242:423-426 (1988) and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988)]. Such scFv molecules may have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers are composed of repeated G4S amino acid sequences or variants thereof. For example, linkers having the amino acid sequence (G4S)4 or (G4S)3 may be used, but variants thereof may also be used.

[0094] The term "single-domain antibody (VHH)" generally refers to antibodies composed of only a single heavy chain variable region with antigen-binding activity. This refers to an antibody consisting of a single chain from C-terminus to N-terminus: FR4-VCDR3-FR3-VCDR2-FR2-VCDR1-FR1. These antibodies can be produced naturally in camels or through genetic engineering. Single-domain antibodies are the smallest known unit capable of binding to a target antigen.

[0095] The term "multispecific antibody" is used in its broadest sense to encompass antibodies with two or more epitope specificities, such as bispecific antibodies. These multispecific antibodies include, but are not limited to: antibodies comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH-VL unit has two or more epitope specificities; antibodies having two or more VL and VH regions, each VH-VL unit binding to a different target or a different epitope of the same target; antibodies having two or more single variable domains (e.g., VHH), each single variable domain binding to a different target or a different epitope of the same target.

[0096] As is well known to those skilled in the art, the CDRs of an antibody can be defined in the art by a variety of methods, such as Chothia (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), based on the three-dimensional structure of the antibody and the topology of the CDR loops; Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Edition, US Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), the international ImMunoGeneTics database (IMGT) (world wide web imgt.cines.fr / ), and the North CDR definition based on affinity propagation clustering using a large number of crystal structures. It will be understood by those skilled in the art that, unless otherwise specified, the terms "CDR" and "complementarity determining region" of a given antibody or region thereof (e.g., variable region) should be understood to encompass complementarity determining regions as defined by any of the above-mentioned known schemes described herein. Various numbering systems corresponding to CDRs are well known to those skilled in the art:

[0097] Note: In the table above, Laa-Lbb refers to the amino acid sequence starting from the N-terminus of the antibody light chain and following the corresponding numbering rules, from positions aa to bb; Haa-Hbb refers to the amino acid sequence starting from the N-terminus of the antibody heavy chain and following the corresponding numbering rules, from positions aa to bb. For example, L24-L34 in the second row, second column of the table above may refer to the amino acid sequence starting from the N-terminus of the antibody light chain variable region and following the Kabat numbering rules, from residues 24 to 34; and so on.

[0098] Unless otherwise specified, the variable region and CDR sequences in the disclosed examples are all numbered using the "IMGT" convention. The term "specific binding" refers to an antibody binding to an antigen or an epitope within the antigen with a higher affinity than to other antigens or epitopes. Typically, antibodies bind with an affinity of approximately 1×10 -7 M or less (e.g., about 1×10 -8 M or smaller, approximately 1×10 -9 M or smaller, approximately 1×10 -10 M or smaller, approximately 1×10 -11 M or smaller, or about 1×10 -12 The antibody binds to the antigen or an epitope within the antigen with an equilibrium dissociation constant (KD) of 1 M or less. In some embodiments, the KD of the antibody binding to the antigen is 10% or 1% of the KD of the antibody binding to a nonspecific antigen (e.g., BSA, casein). KD can be measured using standard procedures, for example, by However, an antibody that specifically binds to an antigen or an epitope within an antigen may have cross-reactivity to other related antigens, for example, to the same antigen from other species (homologous), such as humans or monkeys, e.g., Macaca fascicularis (cynomolgus, cyno), Pan troglodytes (chimpanzee, chimp), or Callithrix jacchus (common marmoset, marmoset).

[0099] A "humanized" antibody refers to a chimeric antibody that comprises amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise at least one, and typically two, substantially entire variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. Such variable domains are referred to herein as "humanized variable regions." Optionally, a humanized antibody may comprise at least a portion of an antibody constant region derived from a human antibody. In some embodiments, some FR residues in a humanized antibody are replaced with corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived), for example, to restore or improve antibody specificity or affinity. A "humanized form" of an antibody (e.g., a non-human antibody) refers to an antibody that has undergone humanization. Other forms of "humanized antibodies" encompassed by the present invention are those in which the constant region has been modified or altered from that of the original antibody to generate the properties according to the present invention (particularly with respect to C1q binding and / or Fc receptor (FcR) binding).

[0100] The Fc domain refers to a crystallizable segment (fragment crystallizable, Fc), which is equivalent to the CH2 and CH3 domains of Ig, and is the site where Ig interacts with effector molecules or cells. As used herein, the heavy chain constant region of the present invention includes a first subunit containing a CH3 region and a second subunit containing a CH3 region, both of which constitute the Fc domain. Unless otherwise specified herein, the amino acid residues in the Fc region or heavy chain constant region are numbered according to the EU numbering system (also known as the EU index) as described in Edelman, GM et al., Proc. Natl. Acad. USA, 63, 78-85 (1969) (https: / / pubmed.ncbi.nlm.nih.gov / 5257969 / ), see also http: / / www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html.

[0101] The term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide encoding a protein can be inserted and the protein expressed. A vector can be used to transform, transduce, or transfect host cells, allowing the genetic material it carries to be expressed in host cells. A vector may contain a variety of elements that control expression, such as promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, a vector may also contain a replication origin site. A vector may also include components that facilitate its entry into cells, such as viral particles, liposomes, or protein coats, but is not limited to these. In embodiments of the present invention, a vector may be selected from, but is not limited to, plasmids, phagemids, cosmids, artificial chromosomes (such as yeast artificial chromosomes YACs, bacterial artificial chromosomes BACs, or P1-derived artificial chromosomes PACs), bacteriophages (such as lambda phage or M13 phage), and animal viruses used as vectors, such as retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (such as SV40).

[0102] The vectors of the present invention can be any suitable recombinant expression vector that can be used to transform or transfect one or more genes or sequences of interest into any suitable host cell and preferably express the genes or sequences in the host cell. Suitable vectors include those designed for expansion and amplification, for expression, or both. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids, or phage vectors, DNA or RNA expression vectors associated with cationic coagulants, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as producer cells.

[0103] As used herein, the term "host cell" refers to any type of cell that can contain a nucleic acid or vector described herein. The host cell can be a eukaryotic cell, such as a plant, animal, fungus, or algae; or the host cell can be a prokaryotic cell, such as a bacterium or protozoa. As described herein, a host cell can be a cell that originates from or is obtained from an individual. The host cell can be derived from or is obtained from a mammal.

[0104] The term "pharmaceutical composition" refers to a composition that is in form permitting the biological activity of the active ingredient contained therein to be effective, and that contains no additional ingredients that are unacceptably toxic to a subject to which the composition would be administered.

[0105] The terms "treat," ...

[0106] The term "prevent" refers to the administration of a drug before signs or symptoms of cancer develop, particularly in a subject at risk for cancer.

[0107] The term "immunoconjugate" is an antibody conjugated to one or more other substances, including but not limited to a cytotoxic agent or a label.

[0108] In some embodiments, the pharmaceutical compositions comprising the bispecific antibodies described herein are formulated for parenteral, subcutaneous, intravenous, intramuscular, intraarterial, intrathecal, or intraperitoneal administration. In other embodiments, the pharmaceutical compositions are administered nasally, by spray, orally, by aerosol, rectally, or vaginally. The compositions can be administered by infusion, bolus injection, or by implantable device.

[0109] The present invention will be described in detail below with reference to specific embodiments. However, these embodiments do not limit the present invention, and any structural, methodological, or functional changes made by those skilled in the art based on these embodiments are all within the scope of protection of the present invention.

[0110] Example embodiments will now be described more fully. However, example embodiments can be implemented in many forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will be thorough and complete and will fully convey the concept of the example embodiments to those skilled in the art.

[0111] Unless otherwise specified, the materials and reagents used in the following examples were commercially available. Where specific techniques or conditions are not specified in the examples, the experiments were performed according to those described in the literature in the field (e.g., Molecular Cloning: A Laboratory Manual, 3rd edition, by J. Sambrook et al., translated by Huang Peitang et al., Science Press) or according to the product instructions.

[0112] Regarding the preparation of human GPRC5D antigen expression vectors pLVX-huGPRC5D-IRES-ZSGreen1 and pTT5-huGPRC5D, and the preparation of HEK293 stable cell lines overexpressing human GPRC5D antigen, please refer to the contents of Example 1 of the patent application with application number 202110561819.5 submitted by the inventor of this application to the State Intellectual Property Office on May 23, 2021. The details will not be repeated here.

[0113] Example 1 Preparation of bispecific antibody GPRC5D×CD3

[0114] Regarding the construction and preparation of the bispecific antibody in Example 1, reference may be made to CrossMab technology (e.g., that used in document CN101896504B) and Knob-in-Hole technology (e.g., that used in document CN1176659A). Documents CN101896504B and CN1176659A are incorporated herein.

[0115] 1. Construction of bispecific antibody expression vector

[0116] The sequences of the heavy chain variable region i-VH, light chain variable region i-VL, heavy chain variable region ii-VH, and light chain variable region ii-VL of the GPRC5D antigen binding moiety (i.e., the first antigen binding moiety) and the CD3 antigen binding moiety (i.e., the second antigen binding moiety) were synthesized respectively (conventionally synthesized by Sangon Biotech (Shanghai) Co., Ltd.).

[0117] Note: The sequences of the heavy chain variable region i-VH and light chain variable region i-VL of the GPRC5D antigen binding module are consistent with the corresponding sequences of the antibody "zw.HTS0375Z56" developed by the inventor (see the patent application number 202110561819.5 submitted by the inventor of this application to the State Intellectual Property Office on May 23, 2021).

[0118] 1) First heavy chain expression vector: The heavy chain variable region i-VH was cloned by homologous recombination into the pTT5-hIgG1.CH vector containing the hIgG1 heavy chain constant region (including the Knob mutation in the Fc domain) to obtain the first heavy chain chimeric antibody expression vector pTT5-iVH-hIgG1.CH (Knob);

[0119] 2) Second heavy chain expression vector: The heavy chain variable region ii-VH was cloned by homologous recombination into the pTT5-hIgG1.CH vector containing the hIgG1 heavy chain constant region (including the Hole mutation in the Fc domain) to obtain the second heavy chain chimeric antibody expression vector pTT5-iiVH-hIgG1.CH(Hole);

[0120] Regarding the mutant Fc domains of 1) and 2) above, the mutation schemes can be selected from: (a) knob mutation T366W, and hole mutations T366S, L368A and Y407V; (b) knob mutations S354C and T366W, and hole mutations Y349C, T366S, L368A and Y407V.

[0121] In this example, the mutation scheme used for the mutated Fc domain is the above-mentioned scheme (b).

[0122] In addition, specifically in this embodiment, in order to reduce the effect of the Fc domain, the Fc domain further includes the presence of an H435R mutation in the second subunit; and / or, the presence of L234A, L235A, P329G, D356E and L358M mutations in the second subunit; and / or, the presence of L234A, L235A, P329G, D356E and L358M mutations in the first subunit.

[0123] 3) First light chain expression vector: The light chain variable region i-VL was cloned into the pTT5-hKappa.CL vector containing the human kappa light chain constant region by homologous recombination to obtain the first light chain chimeric antibody expression vector pTT5-iVL-hKappa.CL;

[0124] 4) Second light chain expression vector: The light chain variable region ii-VL was cloned into the pTT5-hKappa-CL vector containing the human kappa light chain constant region by homologous recombination to obtain the second light chain chimeric antibody expression vector pTT5-iiVL-hKappa.CL;

[0125] Specifically in this embodiment, in order to prevent light chain mispairing, the above-mentioned CrossMab technology was used, that is, the CH1 in the hIgG1 heavy chain constant region in the expression vector of the above-mentioned second heavy chain was replaced with the CL in the human κ light chain constant region, and the CL in the human κ light chain constant region in the expression vector of the above-mentioned second light chain was replaced with the CH1 in the hIgG1 heavy chain constant region.

[0126] For clear demonstration, the expression vector obtained in steps 1)-4) above is shown below:

[0127] Expression vector for the first heavy chain: pTT5-iVH-CH1-hinge-CH2-CH3 (Knob);

[0128] Expression vector for the second heavy chain: pTT5-iiVH-CL-hinge-CH2-CH3 (Hole);

[0129] Expression vector of the first light chain: pTT5-iVL-CL

[0130] Expression vector of the second light chain: pTT5-iiVL-CH1.

[0131] The specific sequence information is shown in Tables 1 and 2:

[0132] Table 1

[0133] First antigen binding module (GPRC5D)

[0134] Table 2

[0135] Second antigen binding moiety (CD3)

[0136] In an alternative embodiment of Example 1, the CH1 and CL in the first heavy chain and the first light chain expression vectors can also be interchanged; generally speaking, if the CH1 / CL in the first heavy chain / light chain expression vector are interchanged, then the CL / CH1 in the second heavy chain / light chain expression vector should also be interchanged.

[0137] 2. Expression and purification of bispecific antibody expression vector

[0138] Well-growing 293F cells (purchased from ThermoFisher) in the logarithmic growth phase were harvested and inoculated into 1L cell culture flasks and cultured in 300mL of culture medium. Polyethylene glycol (PEI) co-transfection was used to transfect the 293F cells with 75μg of each of the four expression vectors obtained above (expression vectors for the first heavy chain, second heavy chain, first light chain, and second light chain). Cell supernatants were collected on day 7 of culture after transfection, centrifuged, and filtered through a 0.45μM filter.

[0139] The filtered cell supernatant was initially purified using Protein A purification medium to obtain the purified antibody, which was then polished using Fractogel EMD COOM cationic medium. For cationic chromatography, equilibration buffer A consisted of 50 mM HAc-NaAc buffer, pH 5.3, Cond 3.2 mS / cm, and eluent B consisted of 50 mM HAc-NaAc buffer + 0.5 M sodium chloride, pH 5.3, Cond 50 mS / cm. After sample loading, the antibody was rinsed with at least five column volumes of equilibration buffer A, followed by a linear elution with 100% eluent B for 20 column volumes. Four peaks were collected, based on the peak profile.

[0140] The antibody concentration and purity of the four elution peaks were determined by measuring absorbance using Nanodrop, and the purity was checked by sodium dodecyl sulfate gel electrophoresis and Coomassie staining.

[0141] The antibody concentration of the first elution peak was determined to be 0.37 mg / mL. The non-reducing electrophoresis pattern revealed distinct bands at 130 kDa, 95 kDa, and 50 kDa, with a purity of approximately 80%. Preliminary analysis indicated that the 130 kDa protein lacked one light chain; the 95 kDa protein was an antibody fragment lacking both light chains and containing only two heavy chains; and the 50 kDa protein was a fragment of an antibody heavy chain.

[0142] The antibody concentration of the second elution peak is 0.98 mg / mL. From the non-reducing electrophoresis pattern, there is a clear band at 130 kDa, and bands at 95 kDa and 50 kDa (the bands are not clear). The purity is about 85%.

[0143] The antibody concentration of the third elution peak is 0.93 mg / mL. From the non-reducing electrophoresis pattern, there is a clear band at 130 kDa and a band at 50 kDa (the band is not obvious), and the purity is about 90%.

[0144] The antibody concentration of the fourth elution peak is 0.12 mg / mL. From the non-reducing electrophoresis pattern, there are obvious bands at 130 kDa and 50 kDa. The content is higher than that of the first three elution peak proteins, and the purity is about 70%.

[0145] 3. Binding activity assay of bispecific antibodies to cells stably overexpressing human GPRC5D antigen

[0146] The binding activity of the four elution peak products collected after the above purification (the bispecific antibody GPRC5D×CD3 of Example 1 of the present invention) to HEK293-GPRC5D-ZSGreen1 stably transfected cells overexpressing the human GPRC5D antigen was detected by FACS.

[0147] Positive control bispecific antibody: bispecific antibody GC5B596D in patent document US10562968B2; specifically, the inventors prepared the positive control bispecific antibody GC5B596D based on the light / heavy chain variable region sequences recorded in the document and according to the preparation method of Example 7 in patent US10562968B2.

[0148] Negative control bispecific antibody: A bispecific antibody targeting SARS-CoV-2×CD3 was prepared using the preparation method of Example 7 in patent US10562968B2. The antibody sequence targeting SARS-CoV-2 was derived from the neutralizing antibody HTS0483 in patent CN113402602A. The bispecific antibody HTS0483×CD3 was prepared according to the preparation method of Example 7 in patent US10562968B2. The detection method is briefly described as follows: 0.2 ml was added to each well at a concentration of 2.5×10 6 HEK293-GPRC5D-ZSGreen1 cell culture medium (100 μg / ml) was placed in a 96-well V-shaped microplate and centrifuged at 1500 rpm for 1 minute, with the supernatant discarded. Serially diluted antibodies (the four elution peaks described above and the positive control antibody) were added at 50 μL per well and incubated on ice for 30 minutes. Then, 150 μL of PBS was added to each well and the cells were centrifuged at 1500 rpm for 1 minute, with the supernatant discarded. The plate was washed four times. The cells were resuspended in 50 μL of 200 nM CD3E protein (6×His tag) per well and incubated on ice for 30 minutes. Then, 150 μL of PBS was added to each well and centrifuged at 1500 rpm for 1 minute, with the supernatant discarded. The plate was washed four times. APC-conjugated Anti-His secondary antibody (Jackson, Cat. No. 109-605-098, diluted 1:1000 in PBS) was added at 50 μL per well and incubated on ice for 30 minutes. After washing the plate four times with PBS, 100 μL of PBS was added to each well to resuspend the cells. The cells were detected using CytoFLEX (Beckman) and the detection results were analyzed using GraphPad 8.0.2. The results are shown in Table 3 below.

[0149] Table 3

[0150] As shown in Table 3, the EC50 value of elution peak 3 is closest to that of the positive control bispecific antibody, demonstrating that elution peak 3 has the best binding activity to cells stably overexpressing the human GPRC5D antigen. Based on the above elution peak concentration, SDS-PAGE gel image results, and binding activity results to the GPRC5D antigen, it can be confirmed that the antibody in elution peak 3 is the target product (the bispecific antibody GPRC5D×CD3 in Example 1).

[0151] 4. In vitro T cell-dependent cytotoxicity of bispecific antibodies

[0152] H929, MM1.S, and the aforementioned cells stably transfected with overexpressing human GPRC5D antigen were used as target cells, and human PBMCs were used as effector cells. The efficacy of the bispecific antibody GPRC5D×CD3 (i.e., peak 3, prepared in Example 1) in T-cell-mediated cytotoxicity was determined and analyzed. The specific procedures were as follows:

[0153] Prepare RPMI-1640 medium containing 1% FBS as the killing assay medium. Resuscitate frozen PBMC effector cells and transfer them to a 15 mL centrifuge tube. Centrifuge at 1500 rpm for 5 minutes and discard the supernatant. Wash the cells twice with culture medium, resuspend them, and count them using a cell counter. Adjust the effector cell density to 1.25 × 10 6 / mL and place in a 37°C, 5% CO2 incubator until ready to use. Add 5mL of culture medium to a 15mL centrifuge tube, thaw the frozen target cells in a 37°C water bath, and transfer them to 5mL of culture medium. Centrifuge at 1500rpm for 5min. Discard the supernatant, wash the cells twice with culture medium, resuspend the cells, and count them using a cell counter. Adjust the target cell density to 1.88×10 5 / mL, and place in a 37°C, 5% CO2 incubator for later use.

[0154] Antibody dilution: The highest final concentration of the bispecific antibody GPRC5D×CD3 (elution peak 3) and the positive control bispecific antibody (GC5B596D bispecific antibody) in Example 1 was 0.23 uM, and they were diluted 4-fold in sequence to obtain 12 gradient concentrations (since 60 uL of the antibody was added to 60 uL of the effector-target mixed cells, when diluting the antibody, the dilution concentration of the antibody needed to be 3 times the final concentration, i.e. 0.7 uM).

[0155] Remove the reserved PBMC effector and target cells and add 60 μL / well of each to a 96-well plate for culture. Using a dispenser, add 60 μL of the diluted antibody to the 96-well plate and incubate. Observe the cells for uniformity under a microscope. Add 60 μL of culture medium to the control wells. Incubate the cells at 37°C, 5% CO₂ for 24 hours. 45 minutes before the end of the co-culture, add 20 μL of 10× lysis buffer to the wells showing maximum LDH release from the target cells. Mix thoroughly and incubate the cells at 37°C, 5% CO₂ for 45 minutes. Centrifuge the co-cultured cells at 3000 rpm for 2 minutes. Transfer 10 μL of the supernatant to a new 384-well microtiter plate. Add 10 μL of CytoTox96 reagent to each well, centrifuge at 1000 rpm for 1 minute, and shake for 1 minute. Incubate at room temperature, protected from light, for 30 minutes, maintaining a maximum reading between 1 and 2. Add 10 μL of stop solution to each well and shake for 1 minute. Record the luminescence value, save the original reading and export the reading to Excel to organize the experimental results.

[0156] The cytotoxicity results against target cells MM.1S are as follows:

[0157] The EC50 value of the dual antibody GPRC5D×CD3 in Example 1 was 0.02761, and the EC50 value of the positive control dual antibody (GC5B596D dual antibody) was 0.07887.

[0158] The cytotoxicity results against target cells H929 are as follows:

[0159] The EC50 value of the dual antibody GPRC5D×CD3 in Example 1 was 0.03121, and the EC50 value of the positive control dual antibody (GC5B596D dual antibody) was 0.1044.

[0160] Comparison of the above results shows that both the GPRC5D×CD3 bispecific antibody of Example 1 and the positive control bispecific antibody (GC5B596D bispecific antibody) are active in T cell-mediated killing of MM.1S and H929 target cells, but the killing activity varies among different cell types. In T cell-mediated killing of MM.1S and H929 target cells, the bispecific antibody of Example 1 exhibits superior killing activity to that of the GC5B596D bispecific antibody.

[0161] 5. In vitro evaluation of cytokine release from effector cells of bispecific antibodies

[0162] Effector cells redirect target cells under the mediation of bispecific antibodies (GPRC5D×CD3 bispecific antibody of Example 1 and positive control GC5B596D bispecific antibody), killing target cells and releasing cytokines. H929 cells were used as target cells and human PBMCs were used as effector cells.

[0163] The ELISA method was used to quantitatively detect the secreted cytokine levels in the cell culture supernatant, including IL-6, IL-2, IFN-γ, and TNF-α.

[0164] At the end point of the in vitro killing assay, cell culture supernatant was collected, transferred to a 96-well plate, and stored at -20°C until use. For ELISA assays, the frozen culture supernatant was removed, thawed at room temperature, and centrifuged at 3500 rpm for 10 minutes to collect the supernatant for ELISA. For detailed ELISA procedures, refer to the kit instructions (Human IL-6 ELISA kit, Human IL-2 ELISA kit, Human IFN-γ ELISA kit, Human TNF-α ELISA kit).

[0165] The IL-6 cytokine ELISA assay involves adding 25 μL of 2 μg / mL coated antibody to a 384-well microtiter plate and coating overnight at 4°C. Discard the coating solution, then add 80 μL of 2% BSA in PBS to each well for blocking at room temperature for 1.5 hours. Discard the blocking solution and wash the plate three times using an automated plate washer. Dilute the supernatant from the 96-well plate four-fold with 1640 medium containing 1% FBS, transfer 25 μL / well to a 384-well plate, in duplicate, and incubate at room temperature for 2 hours. Dilute the standard from the kit (350 pg / mL) in the first well two-fold using 1640 medium containing 1% FBS to eight points. Add 25 μL / well to a 384-well plate as a standard curve, in duplicate, and incubate at room temperature for 2 hours. Discard the supernatant and wash the plate three times with PBST. Prepare the detection antibody (1:800 in 0.1% BSA & 0.05% Tween 20-PBS) and add 25 μl / well to each plate. Incubate at room temperature for 1 hour. Discard the secondary antibody and wash the plate three times with PBST. Add 25 μl / well of the HRP substrate TMB and develop the color for 20 minutes. Add 25 μl / well of the stop solution 2M HCl to stop the color development. Read the absorbance at 450 nm using a microplate reader. Save the original file and the Excel file.

[0166] The ELISA assay for the cytokine IL-2 involves adding 25 μL of 2 μg / mL coating antibody to a 384-well microtiter plate and coating overnight at 4°C. Discard the coating solution and block the plate with 80 μL of 2% BSA in PBS per well at room temperature for 1.5 hours. Discard the blocking solution and wash the plate three times using an automated plate washer. Dilute the supernatant from the 96-well plate fourfold with 1% FBS-containing 1640 medium, transfer 25 μL / well to a 384-well plate, in duplicate, and incubate at room temperature for 2 hours. Dilute the standard from the kit (1000 pg / mL) in the first well two-fold to eight points with 1% FBS-containing 1640 medium. Add 25 μL / well to a 384-well plate as a standard curve, in duplicate, and incubate at room temperature for 2 hours. Discard the supernatant and wash the plate three times with PBST. Prepare the detection antibody (1:2500 in 0.1% BSA & 0.05% Tween 20-PBS) and add 25 μl / well to each plate. Incubate at room temperature for 1 hour. Discard the secondary antibody and wash the plate three times with PBST. Add 25 μl / well of the HRP substrate TMB and develop the color for 20 minutes. Add 25 μl / well of the stop solution 2M HCl to stop the color development. Read the absorbance at 450 nm using a microplate reader. Save the original file and the Excel file.

[0167] The ELISA assay for the cytokine IFN-γ involves adding 25 μL of 2 μg / mL coating antibody to a 384-well microtiter plate and coating overnight at 4°C. Discard the coating solution and block the plate with 80 μL of 2% BSA in PBS per well at room temperature for 1.5 hours. Discard the blocking solution and wash the plate three times using an automated plate washer. Dilute the supernatant from the 96-well plate fourfold with 1% FBS-containing 1640 medium, transfer 25 μL / well to a 384-well plate, in duplicate, and incubate at room temperature for 2 hours. Dilute the standard from the kit (1400 pg / mL) in the first well two-fold to eight points with 1% FBS-containing 1640 medium. Add 25 μL / well to a 384-well plate as a standard curve, in duplicate, and incubate at room temperature for 2 hours. Discard the supernatant and wash the plate three times with PBST. Prepare the detection antibody (1:800 in 0.1% BSA & 0.05% Tween 20-PBS) and add 25 μl / well to each plate. Incubate at room temperature for 1 hour. Discard the secondary antibody and wash the plate three times with PBST. Add 25 μl / well of the HRP substrate TMB and develop the color for 20 minutes. Add 25 μl / well of the stop solution 2M HCl to stop the color development. Read the absorbance at 450 nm using a microplate reader. Save the original file and the Excel file.

[0168] The ELISA assay for the cytokine TNF-α involves adding 25 μL of 2 μg / mL coating antibody to a 384-well microtiter plate and coating overnight at 4°C. Discard the coating solution and block the plate with 80 μL of 2% BSA in PBS per well at room temperature for 1.5 hours. Discard the blocking solution and wash the plate three times using an automated plate washer. Dilute the supernatant from the 96-well plate fourfold with 1% FBS-containing 1640 medium, transfer 25 μL / well to a 384-well plate, in duplicate, and incubate at room temperature for 2 hours. Dilute the standard in the kit (2500 pg / mL) in the first well two-fold to eight points with 1% FBS-containing 1640 medium. Add 25 μL / well to a 384-well plate as a standard curve, in duplicate, and incubate at room temperature for 2 hours. Discard the supernatant and wash the plate three times with PBST. Prepare the detection antibody (1:667 in 0.1% BSA & 0.05% Tween 20-PBS) and add 25 μl / well to each plate. Incubate at room temperature for 1 hour. Discard the secondary antibody and wash the plate three times with PBST. Add 25 μl / well of the HRP substrate TMB and develop the color for 20 minutes. Add 25 μl / well of the stop solution 2M HCl to stop the color development. Read the absorbance at 450 nm using a microplate reader. Save the original file and the Excel file.

[0169] After the in vitro killing experiment, the release results of IL-6 were as follows: the EC50 value of the dual antibody GPRC5D×CD3 in Example 1 was 0.01054, and the EC50 value of the positive control dual antibody (GC5B596D dual antibody) was 0.07981.

[0170] After the in vitro killing experiment, the release results of IL-2 were as follows: the EC50 value of the dual antibody GPRC5D×CD3 in Example 1 was 0.04965, and the EC50 value of the positive control dual antibody (GC5B596D dual antibody) was 0.7283 (approximate value).

[0171] After the in vitro killing experiment, the results of IFN-γ release were as follows: the EC50 value of the dual antibody GPRC5D×CD3 in Example 1 was 0.02979, and the EC50 value of the positive control dual antibody (GC5B596D dual antibody) was 0.1291.

[0172] After the in vitro killing experiment, the release results of TNF-α were as follows: the EC50 value of the dual antibody GPRC5D×CD3 in Example 1 was 0.1741, and the EC50 value of the positive control dual antibody (GC5B596D dual antibody) was 0.8140 (approximate value).

[0173] Comparison of the above results shows that the dual antibody GPRC5D×CD3 of Example 1 can effectively induce PBMC to secrete IL-6, IL-2, IFN-γ and TNF-α at the cellular level when PBMC and H929 co-exist.

[0174] 6. Evaluation of the anti-tumor effect of bispecific antibodies in humanized myeloma NCI-H929 cell xenografts in mice

[0175] The anti-tumor effect of the dual antibody GPRC5D×CD3 (hereinafter referred to as 375Z56KC) of Test Example 1 was evaluated using the NCI-H929 xenograft tumor model of NOG mice (Shanghai Medicilon Biopharmaceutical Co., Ltd.) reconstituted with human PBMC.

[0176] will contain 2×10 6 100uL of PBS containing NCI-H929 cells was subcutaneously injected into the right back of healthy NOG female mice aged 6-8 weeks to prepare for tumor bearing. The size of the tumor growth was monitored every three days. One day after tumor cell inoculation, human PBMCs frozen in liquid nitrogen were revived and cultured in PRMI-1640 medium containing 10% HIFBS (FBS, 56°C × 30min). They were incubated in a 37°C incubator with 5% CO2 for 6h. After incubation, hPBMCs were collected and resuspended in PBS buffer to adjust the cell concentration to 2.5×10 7 Under sterile conditions, 200 μL of cell suspension was intraperitoneally injected into mice at a concentration of 5×10 6 PBMC cells.

[0177] When the tumor volume of the tumor-bearing mice reached 100 mm 3 Around 4:00 p.m., mice were randomly divided into groups so that the difference in tumor volume between groups was less than 10% of the mean, and drug administration was started according to animal body weight, with tail vein administration twice a week for a total of 5 doses. The bispecific antibody group of Example 1, GC5B596D group.

[0178] Experimental groups (low dose and high dose): The dual antibody GPRC5D×CD3 of Example 1 was administered at a dose of 1.5 μg and 6 μg per mouse (in Figures 1 and 2 , the low dose and high dose are marked as “375Z56KC, 1.5 μg / mouse; 375Z56KC, 6 μg / mouse”, respectively);

[0179] Positive control group: administration of positive control dual antibody GC5B596D, dosage: 6 μg per mouse (marked as GC5B596D, 6 μg / mouse in Figures 1 and 2);

[0180] Vehicle control group: PBS was administered, and the dosage was 6 μg per mouse (marked as PBS vehicle control in Figures 1 and 2 );

[0181] Figure 1 shows the effect of the dual antibody GPRC5D×CD3 (375Z56KC) in Example 1 on the tumor volume of NCI-H929 xenograft tumors; Figure 2 shows the effect of the dual antibody GPRC5D×CD3 (375Z56KC) in Example 1 on the tumor weight of NCI-H929 xenograft tumors.

[0182] As can be seen from the results in Figures 1 and 2, both the dual antibody GPRC5D×CD3 in Example 1 and the positive control dual antibody GC5B596D had significant anti-tumor effects on NCI-H929 xenograft tumors in PBMC humanized mice. At the end of the experiment, the average tumor volume of the vehicle control group was 1585.65±144.58mm 3 Compared with the vehicle control group, the dual antibody GPRC5D×CD3 in Example 1 had a very significant tumor inhibitory effect at both the low dose of 1.5 μg / mouse and the high dose of 6 μg / mouse, with the average tumor volume being 64.46±12.46 mm 3 and 59.67±6.02mm 3 The tumor inhibition rates were 95.93% (P<0.001) and 96.24% (P<0.001), respectively. The positive control dual antibody GC5B596D showed a significant anti-tumor effect, with an average tumor volume of 396.10±287.62mm 3 The tumor inhibition rate was 75.02% (P<0.01). The in vitro tumor weight data were consistent with the tumor volume data.

[0183] Example 2 Optimization of bispecific antibody molecular structure and verification of its effect

[0184] The Fab structure of the dual-antibody GPRC5D×CD3 molecule (375Z56KC) in Example 1 was optimized, and the mutation sites are shown in Table 4:

[0185] Table 4 Structural optimization scheme

[0186] The mutated sequences are shown in Table 5 below, where the underlined sites are the mutated residues:

[0187] Table 5 Sequences after Fab mutation

[0188] The constant region sequence of the Fab molecule of 375Z56KC is shown in the following table:

[0189] Table 6 Constant region sequences in Fab molecules

[0190] 375Z56KC is shown in Table 7 below:

[0191] Table 7 Heavy and light chain sequences of 375Z56KC

[0192] 1. Then determine the peak of the optimized antibody molecule on the cation exchange column. The experimental steps are as follows:

[0193] Well-growing 293F cells (purchased from ThermoFisher) in the logarithmic growth phase were harvested and inoculated into 1L cell culture flasks and cultured in 300mL of culture medium. Polyethylene glycol (PEI) co-transfection was used to transfect the 293F cells with 75μg of each of the four expression vectors obtained above (expression vectors for the first heavy chain, second heavy chain, first light chain, and second light chain). Cell supernatants were collected on day 7 of culture after transfection, centrifuged, and filtered through a 0.45μM filter.

[0194] The filtered cell supernatant was initially purified using Protein A purification medium to obtain the purified antibody, which was then polished using Fractogel EMD COOM cationic medium. For cationic chromatography, equilibration buffer A consisted of 50 mM HAc-NaAc buffer, pH 5.3, Cond 3.2 mS / cm, and eluent B consisted of 50 mM HAc-NaAc buffer + 0.5 M sodium chloride, pH 5.3, Cond 50 mS / cm. After sample loading, the antibody was washed with equilibration buffer A for at least five column volumes, followed by a linear elution with 100% eluent B for 20 column volumes. The peak was cut and collected according to the elution peak profile.

[0195] The results are shown in Table 8:

[0196] Table 8 Cation exchange column peak results

[0197] As shown in Table 8, comparing the antibodies before and after optimization, the purity of the main products of the optimized antibody molecules 375Z56KC-2E and 375Z56KC-4E were significantly improved compared to the molecule 37Z56KC before optimization, among which 375Z56KC-2E increased from 27.09% before optimization to 90.13%, and the antibody molecule 375Z56KC-4E increased to 95.97%.

[0198] 2. The in vitro functional assay of the optimized 375Z56KC molecule was performed as follows:

[0199] Human myeloma cell lines H929 (ATCC, Catalog No. CRL-3580) and MM1.S (ATCC, Catalog No. CRL-3580) were resuspended in assay buffer (RMPI1640 + 10% FBS + 1% P / S) and seeded into 96-well plates at a density of 2E4 cells / well, with a volume of 100 μl per well. Six hours later, 2E5 cells / well of PBMCs resuspended in 50 μl of assay buffer were added, with a ratio of effector cells to target cells of 10:1. Subsequently, 50 μl of the antibody drug was added at the pre-determined concentration. After incubation for 24 hours, absorbance was measured using LDH reagent, and cell mortality was calculated.

[0200] The results are shown in Table 9 and Figure 3. The optimized antibody molecules 375Z56KC-2E and 375Z56KC-4E mediated PBMC killing of H929 and MM1.S cells and the activity was basically the same as that of 375Z56KC, and were significantly better than GC5B596D.

[0201] Table 9 Cell death rate determination

[0202] It should be understood that although this specification is described in terms of implementation methods, not every implementation method contains only one independent technical solution. This narrative method of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each implementation method can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

[0203] The series of detailed descriptions listed above are only specific descriptions of feasible implementation methods of the present invention. They are not intended to limit the scope of protection of the present invention. Any equivalent implementation methods or changes that do not deviate from the technical spirit of the present invention should be included in the scope of protection of the present invention.

Claims

1. A multispecific antibody or antigen-binding molecule thereof, characterized in that: The multispecific antibody or antigen-binding molecule thereof comprises a first antigen-binding moiety and a second antigen-binding moiety; wherein the first antigen binding moiety binds to GPRC5D and the second antigen binding moiety binds to CD3; The first antigen binding moiety comprises a heavy chain variable region i-VH and a light chain variable region i-VL, wherein the heavy chain variable region i-VH comprises i-HCDR1 as shown in SEQ ID NO.1, i-HCDR2 as shown in SEQ ID NO.2, and i-HCDR3 as shown in SEQ ID NO.3; the light chain variable region i-VL comprises i-LCDR1 as shown in SEQ ID NO.4, i-LCDR2 with an amino acid sequence of SAS, and i-LCDR3 as shown in SEQ ID NO.5; The second antigen binding moiety comprises a heavy chain variable region ii-VH and a light chain variable region ii-VL, wherein the heavy chain variable region ii-VH comprises ii-HCDR1 as shown in SEQ ID NO.6, ii-HCDR2 as shown in SEQ ID NO.7, and ii-HCDR3 as shown in SEQ ID NO.8; the light chain variable region ii-VL comprises ii-LCDR1 as shown in SEQ ID NO.9, ii-LCDR2 with an amino acid sequence of GTN, and ii-LCDR3 as shown in SEQ ID NO.

10.

2. The multispecific antibody or antigen-binding molecule thereof according to claim 1, wherein: The heavy chain variable region i-VH of the first antigen binding moiety comprises the sequence shown in SEQ ID NO.11, or comprises a sequence having more than 80% homology to the sequence shown in SEQ ID NO.11; the light chain variable region i-VL of the first antigen binding moiety comprises the sequence shown in SEQ ID NO.12, or comprises a sequence having more than 80% homology to the sequence shown in SEQ ID NO.12; The heavy chain variable region ii-VH of the second antigen binding moiety comprises the sequence shown in SEQ ID NO.13, or comprises a sequence having more than 80% homology to the sequence shown in SEQ ID NO.13; the light chain variable region ii-VL of the second antigen binding moiety comprises the sequence shown in SEQ ID NO.14, or comprises a sequence having more than 80% homology to the sequence shown in SEQ ID NO.

14.

3. The multispecific antibody or antigen-binding molecule thereof according to claim 1 or 2, wherein: The second antigen binding moiety is CD3ε.

4. The multispecific antibody or antigen-binding molecule according to any one of claims 1 to 3, wherein: The first antigen binding moiety is selected from any one of Fv, Fab, F(ab')2, Fab', dsFv, scFv, sc(Fv)2 or single domain antibody; The second antigen binding moiety is selected from any one of Fv, Fab, F(ab')2, Fab', dsFv, scFv, sc(Fv)2 or single domain antibody.

5. The multispecific antibody or antigen-binding molecule thereof according to claim 4, wherein: The first antigen binding moiety and the second antigen binding moiety are Fab molecules.

6. The multispecific antibody or antigen-binding molecule thereof according to claim 4, wherein: The multispecific antibody or antigen-binding molecule thereof comprises a light chain constant region; the light chain constant region is preferably a human light chain constant region; preferably, the light chain constant region is a human λ or κ light chain constant region; more preferably, the light chain constant region is a human κ light chain constant region.

7. The multispecific antibody or antigen-binding molecule thereof according to claim 4, wherein: The multispecific antibody or antigen-binding molecule thereof comprises a heavy chain constant region; the heavy chain constant region is preferably a heavy chain constant region of human IgG1, 2, 3, or 4.

8. The multispecific antibody or antigen-binding molecule thereof according to claim 7, wherein: The heavy chain constant region includes a first subunit containing a CH3 region and a second subunit containing a CH3 region, which together constitute an Fc domain; the heavy chain constant region is human IgG1; Preferably, amino acid residues in the CH3 region of the first subunit of the Fc domain are replaced with amino acid residues having a larger side chain volume, i.e., knob mutation, thereby forming a protrusion structure; amino acid residues in the CH3 region of the second subunit of the Fc domain are replaced with amino acid residues having a smaller side chain volume, i.e., hole mutation, thereby forming a cavity structure; the cavity structure accommodates the protrusion structure, thereby allowing the first subunit and the second subunit to combine to form a heterodimer; More preferably, the mutation scheme of the Fc domain is selected from: (a) the knob mutation is T366W, and the hole mutation is T366S, L368A and Y407V; (b) the knob mutation is S354C and T366W, and the hole mutation is Y349C, T366S, L368A and Y407V; the above numbering is according to the EU index.

9. The multispecific antibody or antigen-binding molecule thereof according to claim 8, wherein: The Fc domain further comprises an H435R mutation in the CH3 region of the second subunit; and / or, L234A, L235A, P329G, D356E and L358M mutations in the second subunit; and / or, L234A, L235A, P329G, D356E and L358M mutations in the first subunit, the above numbering being according to the EU index.

10. The multispecific antibody or antigen-binding molecule according to any one of claims 5 to 9, wherein: The heavy chain variable region ii-VH and the light chain variable region ii-VL in the second antigen binding moiety Fab molecule are replaced with each other, or the constant regions ii-CL and ii-CH1 are replaced with each other.

11. The multispecific antibody or antigen-binding molecule thereof according to claim 10, wherein: The constant regions ii-CL and ii-CH1 in the second antigen-binding moiety Fab molecule are replaced with each other, and the heavy chain amino acid sequence containing the constant region ii-CL after replacement is shown in SEQ ID NO.36, and the light chain amino acid sequence containing ii-CH1 after replacement is shown in SEQ ID NO.37; And / or, the heavy chain amino acid sequence of the first antigen binding moiety is shown as SEQ ID NO.34, and the light chain amino acid sequence is shown as SEQ ID NO.

35.

12. The multispecific antibody or antigen-binding molecule according to any one of claims 5 to 11, wherein: The Fab molecules of the first antigen binding moiety and the second antigen binding moiety are any of the following: (1) the Fab molecule of the first antigen binding moiety has the amino acid at position 123 of the constant region i-CL replaced by arginine (R) (numbering according to Kabat), the amino acid at position 124 replaced by lysine (K) (numbering according to Kabat), and the amino acids at positions 147 and 213 of the constant region i-CH1 replaced by glutamic acid (E), respectively (numbering according to the EU index); and the Fab molecule of the second antigen binding moiety has the amino acid at position 170 of the constant region ii-CL replaced by arginine (R) (numbering according to Kabat), and the amino acid at position 133 of the constant region ii-CH1 replaced by glutamic acid (E) (numbering according to the EU index); (2) The amino acid at position 123 of the constant region i-CL of the Fab molecule of the first antigen binding moiety is replaced by arginine (R) (numbering is according to Kabat), and the amino acid at position 124 is replaced by lysine (R). the amino acid at position 170 of the constant region ii-CL of the Fab molecule of the second antigen binding moiety is replaced by lysine (K) (numbering according to Kabat), and the amino acid at position 133 of the constant region ii-CH1 is replaced by glutamic acid (E) (numbering according to EU index); (3) the amino acid at position 176 of the constant region i-CL of the Fab molecule of the first antigen binding moiety is replaced by C (numbering according to Kabat), and the amino acid at position 183 of the constant region i-CH1 is replaced by C (numbering according to the EU index); (4) the amino acid at position 162 of the constant region i-CL of the Fab molecule of the first antigen binding moiety is replaced by cysteine ​​(C) (numbering according to Kabat), and the amino acid at position 173 of the constant region i-CH1 is replaced by cysteine ​​(C) (numbering according to the EU index); (5) the amino acid at position 121 of the constant region i-CL of the Fab molecule of the first antigen binding moiety is replaced by cysteine ​​(C) (numbering according to Kabat), and the amino acid at position 126 of the constant region i-CH1 is replaced by cysteine ​​(C) (numbering according to EU index); (6) The amino acid at position 121 of the constant region i-VL of the Fab molecule of the first antigen binding moiety is replaced by cysteine ​​(C) (numbering according to IGMT), and the amino acid at position 49 of the constant region i-VH1 is replaced by cysteine ​​(C) (numbering according to IGMT).

13. The multispecific antibody or antigen-binding molecule thereof according to claim 12, wherein: In any one of the above (3) to (7), a disulfide bond is formed between the amino acid sites substituted with cysteine ​​(C). 14 . A nucleic acid molecule encoding the multispecific antibody or antigen-binding molecule thereof according to claim 1 .

15. A vector comprising the nucleic acid molecule according to claim 14.

16. A host cell comprising the nucleic acid molecule of claim 14 or the vector of claim 15.

17. A recombinant protein, characterized in that: The recombinant protein comprises the multispecific antibody or antigen-binding molecule thereof according to any one of claims 1 to 113.

18. An immunoconjugate comprising the multispecific antibody or antigen-binding molecule thereof according to any one of claims 1 to 13.

19. A pharmaceutical composition, characterized in that: The pharmaceutical composition comprises the multispecific antibody or antigen-binding molecule thereof according to any one of claims 1 to 13, or the nucleic acid molecule according to claim 14, or the vector according to claim 15, or the host cell according to claim 16, or the recombinant protein according to claim 17, or the immunoconjugate according to claim 18, and a pharmaceutically acceptable carrier.

20. A detection product, characterized in that: The detection product comprises the multispecific antibody or antigen-binding molecule thereof according to any one of claims 1 to 13, or the nucleic acid molecule according to claim 14, or the vector according to claim 15, or the host cell according to claim 16, or the recombinant protein according to claim 17, or the immunoconjugate according to claim 18.

21. Use of the multispecific antibody or antigen-binding molecule thereof according to any one of claims 1 to 13, or the nucleic acid molecule according to claim 14, or the vector according to claim 15, or the host cell according to claim 16, or the recombinant protein according to claim 17, or the immunoconjugate according to claim 18, or the pharmaceutical composition according to claim 19, for preparing a medicament for treating or preventing a disease; preferably, the disease is cancer or an autoimmune disease; preferably, the disease is multiple myeloma.

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