CN117924485A

CN117924485A - Multi-specific antibody for resisting GPRC5D

Info

Publication number: CN117924485A
Application number: CN202211310077.XA
Authority: CN
Inventors: 赵文祥; 魏海涛; 宁婷婷; 李亚男; 董国良; 李秀兰
Original assignee: Shanghai Xiangyao Biotechnology Co ltd
Current assignee: Shanghai Xiangyao Biotechnology Co ltd
Priority date: 2022-10-25
Filing date: 2022-10-25
Publication date: 2024-04-26
Also published as: WO2024087731A1; WO2024088309A1

Abstract

The present invention relates to a multispecific antibody that binds to GPRC5D and CD3 antigens and antigen-binding molecules thereof, nucleic acid molecules encoding the multispecific antibody and antigen-binding molecules thereof, vectors comprising the nucleic acid molecules, host cells comprising the vectors, recombinant proteins or immunoconjugates comprising the multispecific antibody and antigen-binding molecules thereof, and their use in the preparation of a medicament for the treatment or prophylaxis of a disease, in particular for the treatment of multiple myeloma, and in the detection of a product.

Description

Multi-specific antibody for resisting GPRC5D

Technical Field

The invention relates to a multi-specific antibody for resisting GPRC5D, belonging to the field of biological medicine.

Background

Multiple myeloma (Multiple Myeloma, MM) is the second most common hematological tumor worldwide, next to non-hodgkin's lymphoma, accounting for about 10% of hematological malignancies. According to global cancer statistics report issued by WHO, 20,066 patients with new-onset multiple myeloma in China in 2018 and 14,665 patients with death; 44,643 cases of morbidity are accumulated in 5 years. In addition, the morbidity and mortality of patients gradually rise along with the increase of the age, and the total number of patients gradually increases along with the continuous aggravation of the aging degree of the population in China, so that the clinical demands are huge.

Current treatment regimens for multiple myeloma in our country can be divided into three categories: immunomodulators, proteasome inhibitors, and biomolecular targeted therapies. Wherein, the immunomodulator and the proteasome inhibitor are mainly used for the first-line combination treatment accepted by the patient meeting the stem cell transplantation condition before transplantation and the maintenance treatment after transplantation; for example, the use of immunomodulators, represented by thalidomide and its derivatives lenalidomide, and small molecule proteasome inhibitors, represented by bortezomib, has greatly increased remission rates and survival in MM patients over the last decade.

The biomolecular targeting drug for MM mainly relates to three major targets: plasma cell surface protein CD38, signaling lymphocyte activating molecule family member 7 (SIGNALING LYMPHOCYTES ACTIVATING MOLECULE FACTOR, SLAMF 7) and B cell maturation antigen (B Cell Maturation Antigen, BCMA),

Among them, anti-CD 38 mab (e.g., daratumumab conditionally approved by the national drug administration (NMPA) and registered for marketing in 2019) is mainly used for first-line treatment of relapsed or second-line treatment-refractory multiple myeloma.

In addition, BCMA protein is also a research hotspot in the art due to its high expression specificity on MM cells. U.S. FDA accelerated approval of BMCA-targeted Antibody-conjugated drugs (ADCs) Blenrep in 2020 for treatment of patients with median 7-line recurrence; the total remission rate of the medicine can still reach 31%, and the median remission duration (DoR) is more than 6 months. In addition, CAR-T or CD3 bispecific antibodies targeting BCMA play a significant role in anti-tumor efficacy by mediating T cells.

However, while BCMA-targeted ADC, diabodies and CAR-T therapies have shown positive clinical effects, it has been reported that BCMA-negative (or low-expression) and related post-treatment recurrent cases have been reported, that the recurrent and refractory nature of MM remains a challenge for MM clinical treatment, and that finding new effective targets remains a problem to be solved in the art.

Studies have reported that high expression of GPRC5D correlates with poor prognosis for 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).

GPRC5D belongs to the family of G-protein coupled receptors (GPCRs); specifically, it is subtype D of the G protein-coupled receptor C5 family, which was identified earlier in 2001 as the orphan class C GPCR(Brauner-Osborne,H.,et al.Cloning and characterization of a human orphan family C G-protein coupled receptor GPRC5D.Biochim Biophys Acta,2001.1518(3):p.237-48).

Although GPRC5D has been previously identified in cells of patients with multiple myeloma, it has not been applied to clinical development because of lack of protein expression profiling studies. Studies up to 2019 have reported that GPRC5D is highly expressed in plasma cells of multiple myeloma, is mostly not expressed in normal tissues, and that it is surprisingly found that expression (Smith,E.L.,et al.,GPRC5D is a target for the immunotherapy of multiple myeloma with rationally designed CAR T cells.Science Translational Medicine,2019.11(485)). is not overlapping with BCMA only in the area of hair follicles with immune privilege—this finding makes it possible for researchers in the field to realize that GPRC5D is expected to be a completely new therapeutic target for BCMA-targeted therapy, or for patients with low/non-expression of BCMA, or for patients with recurrence of BCMA therapy.

Disclosure of Invention

The present inventors developed a new antibody and antigen binding molecule thereof that specifically bind to GPRC5D based on GPRC5D antigen, and further developed a bispecific antibody and antigen binding molecule thereof that bind to GPRC5D antigen on target cells and an activating T cell antigen (such as CD 3) on T cells; the bispecific antibodies and antigen binding molecules thereof of the invention bind to both target cells and T cells and allow them to interact, thereby causing cytotoxic T cell activation and target cells to be lysed.

In a first aspect the invention provides a multispecific antibody or antigen-binding molecule thereof, wherein,

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

wherein the first antigen binding moiety binds GPRC5D and the second antigen binding moiety binds CD3 or CD3 epsilon;

The first antigen binding module comprises a heavy chain variable region i-VH comprising 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, and a light chain variable region i-VL; the light chain variable region i-VL comprises i-LCDR1 shown in SEQ ID NO.4, i-LCDR2 with an amino acid sequence of SAS and i-LCDR3 shown in SEQ ID NO. 5;

The second antigen binding module comprises a heavy chain variable region ii-VH comprising 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, and a light chain variable region ii-VL; the light chain variable region ii-VL comprises ii-LCDR1 shown in SEQ ID NO.9, ii-LCDR2 having an amino acid sequence of GTN, and ii-LCDR3 shown in SEQ ID NO. 10.

In a more preferred embodiment of the invention, the heavy chain variable region i-VH of the first antigen binding moiety has a sequence as shown in SEQ ID No.11, or has more than 80% sequence homology with 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 module has 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence homology to the sequence set forth in SEQ ID No. 11.

In another more preferred embodiment of the invention, the sequence of the light chain variable region i-VL of the first antigen binding moiety is as shown in SEQ ID NO.12 or has more than 80% sequence homology with 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 module has 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence homology to the sequence set forth in SEQ ID No. 12.

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

In another more preferred embodiment of the invention, the sequence of the light chain variable region ii-VL of the second antigen binding moiety is as shown in SEQ ID NO.14 or has more than 80% sequence homology with 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 has 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence homology to the sequence set forth in SEQ ID No. 14.

With respect to the percentage of "sequence homology" of an amino acid sequence, the percentage of homology of the sequence is generated by determining the number of amino acid residues present in both sequences to produce the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100. Sequence homology, also known as sequence identity.

In a specific embodiment of the present invention, the heavy chain variable region i-VH and the light chain variable region i-VL of the above-described first antigen binding module may be subjected to deletion, insertion or amino acid mutation of a small amount of amino acids based on the sequences shown in SEQ ID No.11 and SEQ ID No.12, respectively, to obtain an amino acid sequence having homology of 80% or more. Substitution of a small number of amino acids (deletions or insertions, or amino acid mutations, or substitutions of similar amino acids), particularly those resulting from conservative amino acid substitutions in portions of the framework regions, retains the original properties and functions of the heavy and light chain variable regions, i.e., those of antibodies that specifically bind GPRC5D, and thus, such variants are within the scope of the present invention. Similarly, the heavy chain variable region ii-VH and the light chain variable region ii-VL of the above-described second antigen binding moiety may be deleted, inserted or mutated in a small amount of amino acids based on the sequences shown in SEQ ID No.13 and SEQ ID No.14, respectively, to obtain an amino acid sequence having homology of 80% or more, so long as the original properties and functions of the heavy chain variable region and the light chain variable region, i.e., the properties and functions of an antibody that specifically binds CD3 or CD3 epsilon, are retained, and such variants are within the scope of the present invention.

Wherein, the above-mentioned "framework region" refers to an amino acid sequence located between CDRs, including a framework region of a heavy chain variable region and a framework region of a light chain variable region.

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

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

Fab molecules refer to protein molecules consisting of VH and CH1 domains of Fab heavy chains and VL and CL domains of Fab light chains. Conventional Fab molecules refer to the native form of the Fab molecule, wherein the Fab heavy chain is VH-CH1 from N to C-terminus and the Fab light chain is VL-CL from N to C-terminus.

Alternatively, the Fab heavy chain variable region and the Fab light chain variable region may be interchanged/replaced in the same Fab molecule; alternatively, the Fab heavy chain constant region and the Fab light chain constant region may be interchanged/replaced in the same Fab molecule. For example, fab molecules contain peptide chains consisting of VL-CH1 (N-to-C terminal) and peptide chains consisting of VH-CL (N-to-C terminal). Generally, for ease of presentation, a peptide chain comprising a heavy chain constant domain CH1 is referred to as a Fab heavy chain.

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

In an alternative embodiment of the invention, the first antigen binding moiety and the second antigen binding moiety are fused by a linker peptide. Preferably, the first antigen binding moiety and the second antigen binding moiety are Fab molecules, and the C-terminus of the Fab heavy chain of the first antigen binding moiety is fused to the N-terminus of the Fab heavy chain of the second antigen binding moiety, or the C-terminus of the Fab heavy chain of the second antigen binding moiety is fused to the N-terminus of the Fab heavy chain of the first antigen binding moiety. The linker peptide may be a conventional linker peptide sequence, for example (Gly ₄Ser)₃ sequence).

In an alternative embodiment of the invention, the multispecific antibodies or antigen-binding molecules thereof of the invention may further comprise additional antigen-binding moieties, which may be the same as the first/second antigen-binding moiety or may be different, e.g., may be antigen-binding moieties that bind to other antigens.

In an alternative embodiment of the invention, the first/second antigen binding moiety is selected from a murine, a human or a chimeric antibody.

In a preferred embodiment of the 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 lambda or kappa light chain constant region.

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

Preferably, the Fc domain of the heavy chain constant region is a mutant Fc domain.

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, thereby forming a raised structure; amino acid residues in the CH3 region of the second subunit of the mutant Fc domain are replaced with amino acid residues having a smaller side chain volume, thereby forming a cavity structure; the cavity structure accommodates the raised structure such that the first and second subunits combine to form a heterodimer.

In another embodiment of the invention, the mutant Fc domains described above may be reduced in binding to Fc receptors and/or effector function.

Preferably, the mutation scheme of the mutant Fc domain is selected from the group consisting of: (a) The knob mutation T366W, the hole mutation T366S, L368A or Y407V; (b) Knob mutation S354C, T366W, hole mutation Y349C T366S, L368A or Y407V.

In certain embodiments of the invention, the addition or deletion of glycosylation sites of an antibody can be conveniently accomplished by altering the amino acid sequence, e.g., altering the amino acid sequence of an Fc domain, such that one or more glycosylation sites are created or eliminated.

The multispecific antibody or antigen binding molecule thereof of the present invention is capable of simultaneously binding the GPRC5D protein of a target cell and the antigen CD3 of an activating T cell, such that the target cell and T cell interact, thereby causing cytotoxic T cell activation and target cell lysis.

In a specific embodiment of the invention, the multispecific antibody or antigen binding molecule thereof of the invention is capable of cross-reacting with monkey GPRC5D in terms of species cross-reactivity, facilitating subsequent preclinical experiments and facilitating subsequent product development for therapeutic use; in addition, the multispecific antibody or antigen binding molecule thereof of the present invention has antibody endocytosis activity in cells, and is suitable for development of ADC drugs.

In a second aspect of the invention there is provided a nucleic acid molecule encoding a multispecific antibody or antigen-binding molecule thereof as described above.

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

In a third aspect the invention provides a vector comprising a nucleic acid molecule as described above, i.e. a vector comprising a nucleic acid molecule encoding a multispecific antibody or antigen-binding molecule thereof as described above, in particular an expression vector expressing a multispecific antibody or antigen-binding molecule thereof as described above.

The term "vector" refers to a nucleic acid vector into which a polynucleotide encoding a protein can be inserted and the protein expressed. The vector may be transformed, transduced or transfected into a host cell to allow expression of the genetic material elements carried thereby within the host cell. The vector may contain various elements for controlling expression, such as promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes, etc. In addition, the vector may also contain a replication origin. It is also possible for the vector to include components that assist it in entering the cell, such as viral particles, liposomes or protein shells, but not just these. In embodiments of the present invention, the carrier may be selected from, but is not limited to: plasmids, phagemids, cosmids, artificial chromosomes (e.g., yeast artificial chromosome YAC, bacterial artificial chromosome BAC, or P1-derived artificial chromosome PAC), phages (e.g., lambda phage or M13 phage), and animal viruses used as vectors, for example, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, papilloma viruses (e.g., SV 40).

In a fourth aspect the invention provides a host cell comprising a nucleic acid molecule as described above or a vector as described above.

In a specific embodiment of the invention, the host cell is a eukaryotic cell, preferably a mammalian cell.

With respect to "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.

In a preferred embodiment of the invention, the host cell is a HEK293 cell.

In a fifth aspect of the invention there is also provided a method of producing an antibody that binds GPRC5D comprising the steps of: the host cells described above are first cultured under conditions suitable for expression of the multispecific antibodies or antigen-binding molecules thereof described above, and then the multispecific antibodies or antigen-binding molecules thereof are recovered.

The multispecific antibody or antigen-binding molecule thereof of the present invention may be produced by the recombinant method described above, or may be produced by hybridoma method.

Other aspects of the invention also provide glycosylated variants of the multispecific antibodies or antigen-binding molecules thereof described above, cysteine engineered antibody variants, antibody derivatives, immunoconjugates, and the like.

In a sixth aspect, the invention provides a recombinant protein comprising a multispecific antibody or antigen-binding molecule thereof as described above.

In a seventh aspect the invention provides an immunoconjugate comprising the multispecific antibody or antigen-binding molecule thereof described above.

Preferably, the conjugate moiety of the immunoconjugate employs 1 or more heterologous molecules, for example, a cytotoxic heterologous molecule that can be used in the immunoconjugate.

In an eighth aspect, the invention provides a pharmaceutical composition comprising a multispecific antibody or antigen-binding molecule thereof described above, or comprising a nucleic acid molecule described above, or comprising a vector described above, or comprising a host cell described above, or comprising a recombinant protein described above.

In a ninth aspect, the invention provides an assay product, wherein the assay product comprises a multispecific antibody or antigen-binding molecule thereof described above, or comprises a nucleic acid molecule described above, or comprises a vector described above, or comprises a host cell described above, or comprises a recombinant protein described above, or comprises an immunoconjugate described above.

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

In one embodiment of the invention, the detection product includes, but is not limited to, a detection reagent, a detection kit, a detection chip or test paper, and the like.

In a tenth aspect, the invention provides the use of a multispecific antibody or antigen-binding molecule thereof, or a nucleic acid molecule thereof, or a vector thereof, or a host cell thereof, or a recombinant protein thereof, or an immunoconjugate thereof, or a pharmaceutical composition thereof, as described above, for the manufacture of a medicament for the treatment or prophylaxis of 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.

The present invention relates to a multispecific antibody that binds to GPRC5D and CD3 antigens and antigen binding molecules thereof, nucleic acid molecules encoding the multispecific antibody and antigen binding molecules thereof, vectors comprising the nucleic acid molecules, host cells comprising the vectors, recombinant proteins comprising the multispecific antibody and antigen binding molecules thereof, and their use in the manufacture of a medicament for the treatment or prophylaxis of a disease, in particular for the treatment of multiple myeloma, and in the detection of a product.

Drawings

FIG. 1 is a graph showing the effect of dual anti-GPRC 5D×CD3 of example 1 on tumor volume of NCI-H929 xenograft tumors;

FIG. 2 is the effect of dual anti-GPRC 5D×CD3 of example 1 on tumor weight of NCI-H929 xenograft tumors.

Detailed Description

The present invention will be described in detail with reference to specific embodiments. These embodiments are not intended to limit the invention and structural, methodological, or functional modifications of these embodiments that may be made by one of ordinary skill in the art are included within the scope of the invention.

Example embodiments will now be described more fully. However, the exemplary embodiments can be embodied 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.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified. The specific techniques or conditions are not noted in the examples, and are carried out according to the techniques or conditions described in the literature in the art (for example, refer to J. Sam Brookfield et al, third edition, scientific Press, et al, compiled "molecular cloning Experimental guidelines" Huang Peitang et al) or according to the product specifications.

For the preparation of the human GPRC5D antigen expression vector pLVX-huGPRC5D-IRES-ZSGreen1 and pTT5-huGPRC D, and the preparation of HEK293 stably transformed cell lines overexpressing the human GPRC5D antigen, see in particular the contents of example 1 of application No. 202110561819.5, filed by the inventors of the present application to the national intellectual property office on day 5 and 23 of 2021, details are not repeated.

Example 1

The bispecific antibody of example 1 comprises a first antigen binding moiety that binds GPRC5D and a second antigen binding moiety that binds CD3.

The first antigen binding module comprises a heavy chain variable region i-VH comprising 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, and a light chain variable region i-VL; the light chain variable region i-VL comprises i-LCDR1 shown in SEQ ID NO.4, i-LCDR2 with an amino acid sequence of SAS and i-LCDR3 shown in SEQ ID NO. 5.

In particular, in this example, the sequence of the heavy chain variable region i-VH of the first antigen binding moiety is shown in SEQ ID No. 11; the sequence of the light chain variable region i-VL of the first antigen binding module is shown in SEQ ID NO. 12; the sequence of the heavy chain variable region ii-VH of the second antigen binding module is shown in SEQ ID NO. 13; the sequence of the light chain variable region ii-VL of the second antigen binding moiety is shown in SEQ ID NO. 14.

Specific sequences are shown in tables 1 and 2 below.

TABLE 1

TABLE 2

For the construction and preparation of bispecific antibodies of example 1, reference may be made to the cross mab technique (as used, for example, in document CN 101896504B) and the Knob-in-Hole technique (as used, for example, in document CN1176659 a). The documents CN101896504B and CN1176659a are incorporated herein.

Example 1 bispecific antibody GPRC5d×cd3 was prepared as follows:

1. Construction of bispecific antibody expression vectors

The sequences of the above 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 (conventionally synthesized by the division of Biotechnology (Shanghai)) were synthesized, respectively.

And (3) notes: the sequences of the heavy chain variable region i-VH and the light chain variable region i-VL are identical to the corresponding sequences of the antibody "zw.hts0375z56" developed by the inventors (see patent application No. 202110561819.5 filed by the inventors of the present application to the national intellectual property office at 5 month 23 of 2021).

1) Expression vector for the first heavy chain: cloning the heavy chain variable region i-VH into a pTT5-hIgG1.Ch vector containing a hIgG1 heavy chain constant region (wherein Knob mutations of the Fc domain are included) by homologous recombination to obtain a chimeric antibody expression vector of first heavy chain pTT 5-ihh-hIgG 1.Ch (Knob);

2) Expression vector for the second heavy chain: cloning the heavy chain variable region ii-VH into a pTT5-hIgG1.Ch vector containing a hIgG1 heavy chain constant region (containing therein a Hole mutation of the Fc domain) by homologous recombination to obtain a chimeric antibody expression vector of a second heavy chain, pTT5-iiVH-hIgG1.Ch (Hole);

Regarding the mutant Fc domains of 1) and 2) above, the mutation scheme may be selected from: (a) The knob mutation T366W, the hole mutation T366S, L368A or Y407V; (b) Knob the mutation S354C, T366W and the hole mutation Y349CT366S, L368A or Y407V.

In this example, the mutation scheme employed for the mutant Fc domain employed is the scheme (b) described above. Specifically, in this example, the Knob mutation was S354C and T366W, the Hole mutation was Y349C, T366S, L368A, Y V, and in order to reduce the binding of the Hole chain Fc of hig 1 to protein a (protein a purification medium used for expression purification of chimeric antibodies), the Hole mutation was H435R; furthermore, in particular in this example, to reduce the effects of the Fc domain, both the L234A, L235A, P329G, D E and L358M mutations were present in both the Knob mutated Fc and the Hole mutated Fc.

3) Expression vector for the first light chain: cloning the light chain variable region i-VL into a pTT5-hKappa.CL containing a human kappa light chain constant region by homologous recombination to obtain a chimeric antibody expression vector pTT5-iVL-hKappa.CL of the first light chain;

4) Expression vector for the second light chain: cloning the light chain variable region ii-VL into a vector pTT5-hKappa-CL containing a human kappa light chain constant region by homologous recombination to obtain a chimeric antibody expression vector pTT5-iiVL-hKappa.CL of a second light chain;

Specifically, in this example, to prevent light chain mismatches, the CrossMab technique described above was used, namely, replacing CH1 in the hIgG1 heavy chain constant region in the expression vector for the second heavy chain with CL in the adult kappa light chain constant region, and replacing CL in the human kappa light chain constant region in the expression vector for the second light chain with CH1 in the hIgG1 heavy chain constant region.

For clarity of presentation, the expression vectors obtained in 1) to 4) above are as follows:

Expression vector for the first heavy chain: pTT5-iVH-CH 1-range-CH 2-CH3 (Knob);

expression vector for the second heavy chain: pTT 5-iiVH-CL-change-CH 2-CH3 (Hole);

expression vector for the first light chain: pTT5-iVL-CL

Expression vector for the second light chain: pTT5-iiVL-CH1.

In an alternative embodiment of example 1, CH1 and CL in the first heavy chain, first light chain expression vector may also be interchanged; generally, if CH1/CL in the first heavy/light chain expression vector is exchanged, then if CL/CH1 in the second heavy/light chain expression vector is also exchanged.

2. Expression and purification of bispecific antibody expression vectors

293F cells (available from ThermoFisher) in a logarithmic growth phase, which had a good growth state, were collected, inoculated into 1L cell culture flasks and cultured in 300mL of medium, and the four expression vectors (first heavy chain, second heavy chain, first light chain, second light chain expression vectors) obtained above were each 75. Mu.g of plasmid was transfected into 293F cells by PEI cotransfection. Cell supernatants from day 7 post-transfection cultures were collected, centrifuged and filtered using a 0.45 μm filter.

The filtered cell supernatant is primarily purified by using a ProteinA purification medium to obtain a purified antibody, and then refined purification operation is carried out by using FractogelEMDCOOM cation medium; wherein, the balance A of the cationic chromatography is 50mMHAc-NaAc buffer, pH5.3, cond3.2mS/cm, and the eluent B is 50mMHAc-NaAc buffer+0.5M sodium chloride, pH5.3, cond50mS/cm. After the sample is completely loaded, at least 5 column volumes of balanced solution A are washed, then 20 column volumes are linearly eluted by 100% of eluent B, and 4 elution peak products are collected according to elution peak-to-peak cutting.

The antibody concentration and purity of the 4 eluted peak products were determined by Nanodrop measurement absorbance, respectively, and the purity was checked by sodium dodecyl sulfate gel electrophoresis and coomassie staining.

The concentration of the antibody in the first elution peak was determined to be 0.37mg/mL, and the antibody showed a distinct band at 130kDa, 95kDa and 50kDa from the non-reducing electropherogram, with a purity of about 80%. Preliminary analysis of the protein at 130kDa was the lack of a light chain; the 95kDa protein is an antibody fragment lacking two light chains and containing only two heavy chains; the protein at 50kDa is a protein fragment of the heavy chain of an antibody.

The antibody concentration of the second elution peak was 0.98mg/mL, and a distinct band was found at 130kDa from the non-reducing electropherogram, and bands (band unclear) were found at both 95kDa and 50kDa, with a purity of about 85%.

The antibody concentration of the third elution peak was 0.93mg/mL, and from the non-reducing electropherogram, there was a distinct band at 130kDa and a band at 50kDa (band was not distinct), with a purity of about 90%.

The antibody concentration of the fourth elution peak was 0.12mg/mL, and the bands were evident at both 130kDa and 50kDa from the non-reducing electropherograms, and the content was higher than that of the first three elution peak proteins, so the purity was about 70%.

Effect data

1. Binding Activity assay of bispecific antibody of example 1 and human GPRC5D antigen-overexpressing stably transduced cells

The binding activity of the 4 eluted peak products (bispecific antibody GPRC5D XCD 3 of example 1 of the present invention) collected after the above purification to HEK293-GPRC5D-ZSGreen1 stably transfected cells overexpressing human GPRC5D antigen was examined by FACS method.

Positive control diabody: bispecific antibody GC5B596D in patent document US10562968B 2; specifically, the inventors prepared a positive control bispecific antibody GC5B596D according to the light/heavy chain variable region sequence described in this document and following the preparation method of example 7 in patent US10562968B 2.

Negative control diabody: bispecific antibody targeting SARS-CoV-2×CD3 was prepared using the preparation method of example 7 of patent US10562968B2, the antibody sequence targeting SARS-CoV-2 was derived from neutralizing antibody HTS0483 of patent CN113402602A, and bispecific antibody HTS0483×CD3 was prepared according to the preparation method of example 7 of patent US10562968B 2. The detection method is briefly described as follows: adding 0.2ml HEK293-GPRC5D-ZSGreen cell culture solution with concentration of 2.5X10. 10 ⁶ cells/ml into 96-well V-shaped micro-pore plate, centrifuging at 1500r/min for 1min, and discarding supernatant; gradient diluted antibodies (4 elution peak products, positive control antibody above), 50 μl per well, incubated on ice for 30min; then 150 mu L of PBS is added into each hole, the centrifugation is carried out for 1min at 1500r/min, the supernatant is discarded, and the plate is repeatedly washed for 4 times; 50uL200nM CD3E protein (6 XHis tag) was added to each well to resuspend cells and incubated on ice for 30min. Then 150. Mu.L PBS was added to each well, centrifuged at 1500r/min for 1min, the supernatant was discarded, and the plate was washed 4 times. APC-labeled Anti-His secondary antibody (Jackson, cat. No. 109-605-098, PBS1:1000 dilution) was added and incubated for 30min on ice at 50. Mu.L per well. After washing the plates 4 times with PBS, 100. Mu.L of PBS was added to each well to resuspend the cells, detection was performed with CytoFLEX (Beckman), and the detection results were analyzed by GraphPad8.0.2, see Table 1 below.

TABLE 1

As can be seen from table 1, the EC50 value of elution peak 3 is closest to that of the positive control diabody, demonstrating that the binding activity of elution peak 3 to the human GPRC5D antigen overexpressing stably transformed cells is optimal. From a combination of the above elution peak concentrations, SDS-PAGE gel results, and binding activity to GPRC5D antigen, it was confirmed that: the antibody eluting peak 3 was the target product (bispecific antibody GPRC5d×cd3 of example 1).

2. In vitro T cell dependent cytotoxicity of bispecific antibodies of example 1

Human PBMCs were used as effector cells using H929, mm1.S and the above-described human GPRC5D antigen-overexpressing stably transformed cells as target cells; the efficacy of the bispecific antibody GPRC5d×cd3 of example 1 (i.e. elution peak 3 obtained as prepared in example 1) on T-cell mediated cytotoxicity was determined and analyzed. The specific operation is as follows:

RPMI-1640 medium containing 1% FBS was prepared as a killing test medium. Cryopreserved PBMC effector cells were resuscitated, PBMC cells were transferred to a 15mL centrifuge tube, centrifuged at 1500rpm for 5min, and the supernatant discarded. The cells were washed twice with medium, resuspended, counted with a cytometer, and effector cell density was adjusted to 1.25X10 ⁶/mL and placed in a 37℃5% CO ₂ incubator for use. 5mL of culture medium is added into a 15mL centrifuge tube, and frozen target cells are placed into a 37 ℃ water bath kettle for resuscitation and then transferred into 5mL of culture medium. Centrifuge at 1500rpm for 5min. The supernatant was discarded, the cells were washed twice with medium, resuspended, counted with a cytometer, the target cell density was adjusted to 1.88×10 ⁵/mL, and placed in a 37 ℃ 5% co ₂ incubator for use.

Antibody dilution: example 1 the highest final concentration of the diabody GPRC5d×cd3 (elution peak 3) and the positive control diabody (GC 5B596D diabody) was 0.23uM, followed by 4-fold gradient dilution to obtain 12 gradient concentrations (the diluted concentration of the antibody needs to be 3-fold of the final concentration, i.e. 0.7uM, since the antibody is 60uL added to 60uL of the target mix cells).

Taking out spare PBMC effector cells and target cells, and adding 60uL/well of each cell into a 96-well plate for culture; the diluted antibody was added to a 96-well plate at 60uL using a row gun, and incubated. The cell plating was observed under a microscope, and 60uL of medium was added to the control well. It was placed in a 37℃5% CO ₂ incubator for 24h. Before 45 minutes before the end of co-culture, 20uL of 10 Xlysate was added to the target cell maximum LDH release well, and the cells were incubated at 37℃for 45 minutes under 5% CO ₂. The co-cultured cells were centrifuged at 3000rpm for 2 minutes. Transfer 10uL of supernatant to a new 384 well elisa plate, add 10uLCytoTox reagents per well, centrifuge at 1000rpm for 1 min, shake for 1 min. Incubate for 30 min at room temperature in the dark, with maximum reading controlled between 1-2. 10uL of stop solution was added to each well and shake was performed for 1 minute. And recording a luminescence value, saving an original reading and deriving an Excel reading finishing experiment result.

Cytotoxicity results against target cells mm.1s were as follows:

Example 1 the IC50 value of the diabody GPRC5d×cd3 was 0.9916 and the IC50 value of the positive control diabody (GC 5B596D diabody) was 0.5064.

Cytotoxicity results against target cell H929 were as follows:

example 1 the IC50 value of the diabody GPRC5d×cd3 was 0.002706 and the IC50 value of the positive control diabody (GC 5B596D diabody) was 0.002086.

Comparing the above results, it can be seen that example 1 diabody GPRC5d×cd3 and the positive control diabody (GC 5B596D diabody) are active in T cell-mediated killing of both mm.1s and H929 target cells, but have a certain difference in killing activity among different cells. In T cell mediated killing of mm.1s target cells, the killing activity of example 1 diabody was superior to GC5B596D diabody; in T cell mediated killing of H929 target cells, the killing activity of example 1 diabody was slightly worse than GC5B596D diabody.

3. In vitro effector cytokine release assessment of bispecific antibodies of example 1

Effector cells redirect target cells under the mediation of bispecific antibodies (example 1 dual anti-GPRC 5d×cd3 and positive control GC5B596D dual antibody), releasing cytokines while killing target cells. H929 was used as target cells and human PBMC as effector cells.

The secreted cytokine content in the cell culture supernatant, including IL-6, IL2, IFN-gamma, TNF-alpha, was quantitatively detected by ELISA.

Cell culture supernatants were collected at the end of the in vitro killing experiments and placed in 96 well plates at-20℃for storage. In ELISA experiments, frozen culture supernatants were removed, thawed at room temperature, centrifuged at 3500rpm for l0mins and the supernatants were collected for ELISA experiments. The ELISA procedure is described in the Kit (Human IL-6 ELISA Kit, human IL-2 ELISA Kit, human IFN-. Gamma.ELISA Kit, human TNF-. Alpha.ELISA Kit).

ELISA detection of cytokine IL-6 was performed by adding 25uL 2ug/mL of the coated antibody to 384-well ELISA plates and coating overnight at 4 ℃. The coating was discarded and each well was blocked with 80uL of 2% BSA in PBS for 1.5 hours at room temperature. The blocking solution was discarded and the plate was washed 3 times using an automatic plate washer. Supernatants from 96-well plates were 4-fold diluted with 1640 medium of 1% fbs, transferred to 25 ul/well to 384-well plates, 2 replicate wells, and incubated for 2 hours at room temperature. Standard first well 350pg/mL in the kit was diluted 2-fold with 1640 medium of 1% FBS at 8 spots, and then 25 ul/well was added to 384-well plate as Standard curve, 2 duplicate wells were incubated at room temperature for 2 hours. The supernatant was discarded and the plates were washed 3 times with PBST. Detection antibody (1:800 in 0.1%BSA&0.05%Tween20-PBS) was prepared, 25 ul/well was added to each well plate and incubated for 1 hour at room temperature. The secondary antibodies were discarded and the plates were washed 3 times with PBST. HRP substrate TMB,25 ul/well, was added and developed for 20 min. Stop solution 2M HCl,25 ul/well was added to stop the color development. Absorbance values at 450nm were read using a microplate reader. The original file and Excel file are saved.

ELISA detection of cytokine IL-2 was performed by adding coatingantibody of 25uL 2ug/mL to 384-well ELISA plates and coating overnight at 4 ℃. The coating was discarded and each well was blocked with 80uL of 2% BSA in PBS for 1.5 hours at room temperature. The blocking solution was discarded and the plate was washed 3 times using an automatic plate washer. Supernatants from 96-well plates were 4-fold diluted with 1640 medium of 1% fbs, transferred to 25 ul/well to 384-well plates, 2 replicate wells, and incubated for 2 hours at room temperature. Standard first well 1000pg/mL in the kit was diluted 2-fold with 1640 medium of 1% FBS for 8 spots, and then 25 ul/well was added to 384-well plate as Standard curve, 2 duplicate wells were incubated at room temperature for 2 hours. The supernatant was discarded and the plates were washed 3 times with PBST. Detection antibody (1:2500 in 0.1%BSA&0.05%Tween20-PBS) was prepared, 25 ul/well was added to each well plate and incubated for 1 hour at room temperature. The secondary antibodies were discarded and the plates were washed 3 times with PBST. HRP substrate TMB,25 ul/well, was added and developed for 20 min. Stop solution 2M HCl,25 ul/well was added to stop the color development. Absorbance values at 450nm were read using a microplate reader. The original file and Excel file are saved.

ELISA detection method of cytokine IFN-gamma is carried out by adding coatingantibody of 25uL 2ug/mL on 384-well ELISA plate and coating at 4deg.C overnight. The coating was discarded and each well was blocked with 80uL of 2% BSA in PBS for 1.5 hours at room temperature. The blocking solution was discarded and the plate was washed 3 times using an automatic plate washer. Supernatants from 96-well plates were 4-fold diluted with 1640 medium of 1% fbs, transferred to 25 ul/well to 384-well plates, 2 replicate wells, and incubated for 2 hours at room temperature. Standard first well 1400pg/mL in the kit was diluted 2-fold with 1640 medium of 1% FBS for 8 spots, and then 25 ul/well was added to 384-well plate as Standard curve, 2 duplicate wells were incubated at room temperature for 2 hours. The supernatant was discarded and the plates were washed 3 times with PBST. Detection antibody (1:800 in 0.1%BSA&0.05%Tween20-PBS) was prepared, 25 ul/well was added to each well plate and incubated for 1 hour at room temperature. The secondary antibodies were discarded and the plates were washed 3 times with PBST. HRP substrate TMB,25 ul/well, was added and developed for 20 min. Stop solution 2M HCl,25 ul/well was added to stop the color development. Absorbance values at 450nm were read using a microplate reader. The original file and Excel file are saved.

ELISA detection of cytokine TNF-alpha was performed by adding 25uL 2ug/mL coatingantibody to 384-well ELISA plates and coating overnight at 4 ℃. The coating was discarded and each well was blocked with 80ul2% bsa in PBS for 1.5 hours at room temperature. The blocking solution was discarded and the plate was washed 3 times using an automatic plate washer. Supernatants from 96-well plates were 4-fold diluted with 1640 medium of 1% fbs, transferred to 25 ul/well to 384-well plates, 2 replicate wells, and incubated for 2 hours at room temperature. Standard head wells in the kit were 2500pg/mL, diluted 2-fold with 1640 medium of 1% FBS for 8 spots, and 25 ul/well was added to 384-well plates as Standard curve, 2 duplicate wells were incubated at room temperature for 2 hours. The supernatant was discarded and the plates were washed 3 times with PBST. Detection antibody (1:667 in 0.1%BSA&0.05%Tween20-PBS) was prepared, 25 ul/well was added to each well plate and incubated for 1 hour at room temperature. The secondary antibodies were discarded and the plates were washed 3 times with PBST. HRP substrate TMB,25 ul/well, was added and developed for 20 min. Stop solution 2M HCl,25 ul/well was added to stop the color development. Absorbance values at 450nm were read using a microplate reader. The original file and Excel file are saved.

In vitro killing experiments, release results of IL 6: example 1 the EC50 value of the diabody GPRC5d×cd3 was 0.01054 and the EC50 value of the positive control diabody (GC 5B596D diabody) was 0.07981.

In vitro killing experiments, release results of IL 2: example 1 the EC50 value of the diabody GPRC5d×cd3 was 0.04965 and the EC50 value of the positive control diabody (GC 5B596D diabody) was 0.7283 (approximation).

Release results of IFN- γ after in vitro killing experiments: example 1 the EC50 value of the diabody GPRC5d×cd3 was 0.02979 and the EC50 value of the positive control diabody (GC 5B596D diabody) was 0.1291.

In vitro killing experiments, TNF- α release results: example 1 the EC50 value of the diabody GPRC5d×cd3 was 0.1741 and the EC50 value of the positive control diabody (GC 5B596D diabody) was 0.8140 (approximation).

As can be seen from a comparison of the above results, the dual anti-GPRC 5D×CD3 of example 1 is effective in inducing secretion of IL-6, IL-2, IFN- γ and TNF- α by PBMC at the cellular level in the presence of both PBMC and H929.

4. Evaluation of anti-tumor Effect of bispecific antibody of example 1 on human myeloma NCI-H929 cell humanized mouse xenograft tumors

The anti-tumor effect of the dual anti-GPRC 5d×cd3 of test example 1 was evaluated using the NOG mice (division of biomedical sciences, middi, shanghai) NCI-H929 xenograft tumor model reconstituted with human PBMC.

100ULPBS containing 2X 10 ⁶ NCI-H929 cells was subcutaneously inoculated into the right back of 6-8 week old healthy NOG females, prepared to support tumors, and the size of tumor growth was monitored every three days. 1 day after tumor cell inoculation, liquid nitrogen frozen human PBMC were resuscitated, cultured in PRMI-1640 medium containing 10% HIFBS (FBS, 56 ℃ C. Times.30 min) and incubated for 6h in a 37 ℃ incubator containing 5% CO 2. After incubation hBMC was collected, resuspended in PBS buffer and the cell concentration was adjusted to 2.5X10 ⁷/mL. 200uL of cell suspension was injected intraperitoneally into mice under sterile conditions at a concentration of 5X 10 ⁶ PBMC cells.

When the tumor volume of the tumor-bearing mice reaches about 100mm ³, the mice are randomly grouped, so that the difference of the tumor volumes of the groups is less than 10% of the average value, and the animals begin to be dosed according to the body weight of the animals, namely, the animals are dosed by tail vein 2 times per week for 5 times. Bispecific antibody panel, GC5B596D panel of example 1.

Experimental group (low and high dose): example 1 dual anti-GPRC 5d×cd3 dosing: each mouse was 1.5ug, 6ug (low and high doses labeled "375z56kc,1.5ug/mouse;375z56kc,6ug/mouse", respectively, in fig. 1 and 2;

Positive control group: positive control dual anti GC5B596D was administered at doses: each mouse 6ug (labeled GC5B596D,6ug/mouse in fig. 1 and 2);

vehicle control group: PBS was administered, dose: 6ug per mouse (labeled PBS vehicle control in figures 1 and 2);

FIG. 1 is a graph showing the effect of dual anti-GPRC 5D×CD3 of example 1 on tumor volume of NCI-H929 xenograft tumors; FIG. 2 is the effect of dual anti-GPRC 5D×CD3 of example 1 on tumor weight of NCI-H929 xenograft tumors.

From the results of fig. 1 and 2, it can be seen that example 1 dual anti-GPRC 5d×cd3 and the positive control dual anti-GC 5B596D both have significant anti-tumor effects on NCI-H929 xenograft tumors in PBMC humanized mice. The average tumor volume of the vehicle control group at the end of the experiment was 1585.65 ± 144.58mm ³, and the dual anti-GPRC 5d×cd3 of example 1 had extremely remarkable tumor inhibition effects in both low dose 1.5ug/mouse and high dose 6ug/mouse, the average tumor volumes were 64.46±12.46mm ³ and 59.67±6.02mm ³, respectively, and the tumor inhibition rates were 95.93% (P < 0.001) and 96.24% (P < 0.001), respectively, compared to the vehicle control group. The positive control double anti-GC 5B596D shows obvious anti-tumor effect, the average tumor volume is 396.10 +/-287.62 mm ³, and the tumor inhibition rate is 75.02% (P < 0.01). The in vitro tumor weight data were well consistent with tumor volume data.

It should be understood that although the present disclosure describes embodiments, not every embodiment is provided with a separate embodiment, and that this description is for clarity only, and that the skilled artisan should recognize that the embodiments may be combined as appropriate to form other embodiments that will be understood by those skilled in the art.

The above list of detailed descriptions is only specific to practical embodiments of the present invention, and they are not intended to limit the scope of the present invention, and all equivalent embodiments or modifications that do not depart from the spirit of the present invention should be included in the scope of the present invention.

Claims

1. A multispecific antibody or antigen-binding molecule thereof, characterized in that:

2. The multispecific antibody, or antigen-binding molecule thereof, of claim 1, wherein:

the sequence of the heavy chain variable region i-VH of the first antigen binding module is shown as SEQ ID NO.11, or the sequence homology of the heavy chain variable region i-VH with the sequence shown as SEQ ID NO.11 is more than 80%; the sequence of the light chain variable region i-VL of the first antigen binding module is shown as SEQ ID NO.12, or the sequence homology of the light chain variable region i-VL and the sequence shown as SEQ ID NO.12 is more than 80%;

The sequence of the heavy chain variable region ii-VH of the second antigen binding module is shown as SEQ ID NO.13, or the sequence homology of the heavy chain variable region ii-VH with the sequence shown as SEQ ID NO.13 is more than 80%; the sequence of the light chain variable region ii-VL of the second antigen binding module is shown as SEQ ID NO.14, or the sequence homology of the second antigen binding module and the sequence shown as SEQ ID NO.14 is more than 80 percent.

3. The multispecific antibody or antigen-binding molecule thereof of claim 1 or 2, 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 chain antibody;

the second antigen binding moiety is selected from any one of Fv, fab, F (ab ') 2, fab', dsFv, scFv, sc (Fv) 2, or single chain antibody.

4. The multispecific antibody, or antigen-binding molecule thereof, of claim 3, wherein: the first antigen binding moiety and the second antigen binding moiety are Fab molecules.

5. The multispecific antibody, or antigen-binding molecule thereof, of claim 3, wherein: the first antigen binding moiety and the second antigen binding moiety are fused by a linker peptide.

6. The multispecific antibody, or antigen-binding molecule thereof, of claim 3, 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 lambda or kappa light chain constant region.

7. The multispecific antibody, or antigen-binding molecule thereof, of claim 3, 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;

preferably, the Fc domain of the heavy chain constant region is a mutant Fc domain;

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, thereby forming a raised structure; amino acid residues in the CH3 region of the second subunit of the mutant Fc domain are replaced with amino acid residues having a smaller side chain volume, thereby forming a cavity structure; the cavity structure accommodates the raised structure such that the first and second subunits combine to form a heterodimer;

8. A nucleic acid molecule encoding the multispecific antibody or antigen-binding molecule thereof of any one of claims 1 to 7.

9. A vector comprising the nucleic acid molecule of claim 8.

10. A host cell comprising the nucleic acid molecule of claim 8 or the vector of claim 9.

11. A recombinant protein, characterized in that: the recombinant protein comprising the multispecific antibody or antigen-binding molecule thereof of any one of claims 1 to 7.

12. An immunoconjugate comprising the multispecific antibody or antigen-binding molecule thereof of any one of claims 1 to 7.

13. A pharmaceutical composition characterized by: the pharmaceutical composition comprises the multispecific antibody or antigen-binding molecule thereof of any one of claims 1 to 7, or comprises the nucleic acid molecule of claim 8, or comprises the vector of claim 10, or comprises the host cell of claim 10, or comprises the recombinant protein of claim 12, or comprises the immunoconjugate of claim 12, and a pharmaceutically acceptable carrier.

14. A test product, characterized by: the detection product comprises the multispecific antibody or antigen-binding molecule thereof of any one of claims 1 to 7, or comprises the nucleic acid molecule of claim 8, or comprises the vector of claim 9, or comprises the host cell of claim 10, or comprises the recombinant protein of claim 11, or comprises the immunoconjugate of claim 12.

15. Use of a multispecific antibody or antigen-binding molecule thereof according to any one of claims 1 to 7, or a nucleic acid molecule according to claim 8, or a vector according to claim 9, or a host cell according to claim 10, or a recombinant protein according to claim 11, or an immunoconjugate according to claim 12, or a pharmaceutical composition according to claim 13, for the preparation of a medicament for the treatment or prophylaxis of a disease; preferably, the disease is cancer or an autoimmune disease; preferably, the disease is multiple myeloma.