Multispecific antibodies targeting B cell antigens and T cell antigens and their applications

Abstract

This invention provides a multispecific antibody targeting B-cell antigens and T-cell antigens, and its applications. Specifically, this invention provides a single-domain antibody targeting BCMA, and a multispecific antibody targeting BCMA, CD3, and CD20 constructed based on this single-domain antibody. The multispecific antibody of this invention can be used as a T-cell adaptor, connecting target cells (such as B cells and plasma cells) and T cells, promoting the killing effect of T cells on target cells. The multispecific antibody of this invention shows promise for applications in autoimmune diseases and anti-tumor fields.

Description

Technical Field

[0001] This invention relates to the field of biomedicine. Specifically, it relates to multispecific antibodies targeting B-cell antigens and T-cell antigens and their applications. Background Technology

[0002] Bispecific antibodies are genetically engineered antibodies created through artificial genetic engineering. They can simultaneously target two different antibody-binding epitopes, which can originate from different antigens or from the same antigen. Numerous studies have demonstrated the significant therapeutic potential of bispecific antibodies in treating diseases such as cancer, autoimmune diseases, and viral infections. Compared to monoclonal antibodies, the main advantage of bispecific antibodies lies in their ability to mediate the spatial effects of two recognizing epitopes and the synergistic effect of dual targeting, producing biological effects that cannot be achieved by combining two antibodies.

[0003] Bispecific antibodies, due to their complexity compared to monoclonal antibodies, have higher technical barriers and development costs. The development of bispecific antibodies requires comprehensive consideration of target selection, antibody engineering design, and subsequent production processes. Common challenges encountered during bispecific antibody construction include low expression levels, poor stability, and difficulties in establishing pharmacodynamic models. Traditional antibodies typically consist of four chains: two heavy chains and two light chains. Using traditional antibodies to construct bispecific antibodies can lead to light and heavy chain mismatches. Furthermore, whether the constructed bispecific antibody will possess the affinity of the parent antibody and produce the desired biological effects remains unknown.

[0004] T cell engagers are specialized bispecific antibodies that can activate endogenous T cells by bispecifically binding to target sites on the surface of tumor cells and T cells, leading to tumor cell lysis and thus achieving the goal of treating tumors. Micromet, a German company, was one of the first to develop the BiTE (Bispecific T cell engager) platform. BiTEs are formed by linking two scFv (single-chain variable fragments) with a linker peptide. Therefore, these molecules are bivalent, monovalent for each antigen, and typically have a molecular weight of 50-60 kDa. In 1998, Micromet developed Blinatumomab based on the BiTE platform. Subsequently, Blinatumomab successfully completed Phase I, II, and III clinical trials, becoming the first BiTE drug to receive FDA approval. Blinatumomab can simultaneously target both CD19 and CD3 proteins. CD19 is expressed on the surface of acute lymphoblastic leukemia and lymphoma tumor cells, serving as a target for tumor cells. CD3 includes multiple subunits (d, g, and e) and is an important surface marker of T cells. The CD3 subunit can non-covalently bind to the TCR to form the TCR-CD3 complex. CD3 antibodies generally recognize the ε chain of the CD3 complex, transmitting the activation signal generated after the TCR binds to the antigen into the cell, inducing T cell activation, and subsequently killing tumor cells. T cell connectors have been proven to be useful in cancer treatment, and subsequent T cell connectors targeting other targets such as CD20, BCMA, and CD19 are in clinical trials. Besides their significant applications in cancer treatment, these targets also have broad potential applications in autoimmune diseases (such as systemic lupus erythematosus, rheumatoid arthritis, and multiple sclerosis), especially in autoimmune diseases where B cells play a major role.

[0005] However, current bispecific or multispecific antibodies are structurally complex, difficult to prepare, and costly to produce. Furthermore, targeting only two targets is insufficient for addressing complex diseases such as autoimmune diseases and tumors. Therefore, there is a need in this field to develop novel structural antibodies that target multiple antigens and multispecific T-cell adaptors. Summary of the Invention

[0006] The purpose of this invention is to provide multispecific antibodies targeting B cell antigens and T cell antigens and their applications.

[0007] In a first aspect of the invention, a single-domain antibody against BCMA is provided, wherein the VHH chain of the single-domain antibody has a complementarity-determining region (CDR) selected from the group consisting of:

[0008] 1) CDR1 shown in SEQ ID NO:7, CDR2 shown in SEQ ID NO:8, and CDR3 shown in SEQ ID NO:9;

[0009] 2) CDR1 shown in SEQ ID NO:10, CDR2 shown in SEQ ID NO:11, and CDR3 shown in SEQ ID NO:12;

[0010] 3) CDR1 shown in SEQ ID NO:7, CDR2 shown in SEQ ID NO:13, and CDR3 shown in SEQ ID NO:14;

[0011] 4) CDR1 shown in SEQ ID NO:10, CDR2 shown in SEQ ID NO:15, and CDR3 shown in SEQ ID NO:16;

[0012] 5) CDR1 shown in SEQ ID NO:17, CDR2 shown in SEQ ID NO:18, and CDR3 shown in SEQ ID NO:19;

[0013] 6) CDR1 shown in SEQ ID NO:20, CDR2 shown in SEQ ID NO:21, and CDR3 shown in SEQ ID NO:22;

[0014] 7) CDR1 shown in SEQ ID NO:1, CDR2 shown in SEQ ID NO:2, and CDR3 shown in SEQ ID NO:3; and

[0015] 8) CDR1 shown in SEQ ID NO:4, CDR2 shown in SEQ ID NO:5, and CDR3 shown in SEQ ID NO:6.

[0016] In another preferred embodiment, the CDR region of the single-domain antibody VHH chain contains an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95% sequence similarity to any of the above sequences.

[0017] In another preferred embodiment, any of the above-mentioned amino acid sequences further includes a derivative sequence that has optionally been added, deleted, modified, and / or substituted at least one amino acid and is capable of retaining BCMA binding affinity.

[0018] In another preferred embodiment, the number of added, deleted, modified and / or substituted amino acids is 1-3, more preferably 1-2, and even more preferably 1.

[0019] In another preferred embodiment, the VHH chain of the single-domain antibody further includes a framework region (FR).

[0020] In another preferred embodiment, CDR1, CDR2 and CDR3 are separated by the frame regions FR1, FR2, FR3 and FR4 of the VHH chain.

[0021] In another preferred embodiment, the frame region FR is of human, mouse, rabbit, or camel origin.

[0022] In another preferred embodiment, the VHH chain of the single-domain antibody has an amino acid sequence that is ≥85%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% homology with the amino acid sequence shown in any one of SEQ ID NO:23-30.

[0023] In another preferred embodiment, the VHH chain of the single-domain antibody has one or more amino acid sequences as shown in any one of SEQ ID NO:23-30 (preferably, as shown in any one of SEQ ID NO:25-30).

[0024] In another preferred embodiment, the single-domain antibody is a monomer, a bivalent (bivalent antibody), or a multivalent (multivalent antibody).

[0025] In another preferred embodiment, the amino acid sequence of the VHH chain of the single-domain antibody is shown in any one of SEQ ID NO:23-30; more preferably, it is shown in any one of SEQ ID NO:25-30.

[0026] In a second aspect of the invention, a multispecific antibody is provided, the multispecific antibody comprising the anti-BCMA single-domain antibody as described in the first aspect of the invention.

[0027] In another preferred embodiment, the multispecific antibody is a T-cell adaptor that further comprises an antigen-binding fragment that specifically binds to T cells.

[0028] In another preferred embodiment, the multispecific antibody further comprises an antigen-binding fragment that specifically binds to other tumor antigens.

[0029] In another preferred embodiment, the multispecific antibody is a trispecific antibody, the trispecific antibody comprising:

[0030] 1) CD20 targeting binding domain, which contains one or more anti-CD20 antibody or antigen-binding fragments;

[0031] 2) A BCMA-targeting binding domain comprising one or more of the aforementioned anti-BCMA single-domain antibodies; and

[0032] 3) CD3 targeting binding domain, which contains one or more anti-CD3 antibody or antigen binding fragments.

[0033] In another preferred embodiment, the trispecific antibody comprises heavy chain 1, heavy chain 2, and light chain.

[0034] In another preferred embodiment, the trispecific antibody comprises two identical light chains.

[0035] In another preferred embodiment, heavy chain 1 comprises a heavy chain of anti-CD20 antibody; heavy chain 2 comprises a heavy chain of anti-CD3 antibody and the anti-BCMA single-domain antibody; and the light chain comprises a light chain of anti-CD20 antibody.

[0036] Preferably, in heavy chain 2, the anti-BCMA single-domain antibody is attached to the N-terminus of the anti-CD3 antibody heavy chain via a linker.

[0037] In another preferred embodiment, heavy chain 1 comprises the heavy chain of the anti-CD20 antibody and the anti-BCMA single-domain antibody; heavy chain 2 comprises the heavy chain of the anti-CD3 antibody and the anti-BCMA single-domain antibody; and the light chain comprises the light chain of the anti-CD20 antibody.

[0038] Preferably, in heavy chain 1, the anti-BCMA single-domain antibody is attached to the N-terminus of the anti-CD20 antibody heavy chain via a adapter; in heavy chain 2, the anti-BCMA single-domain antibody is attached to the N-terminus of the anti-CD3 antibody heavy chain via a adapter.

[0039] In another preferred embodiment, heavy chain 1 comprises a heavy chain of anti-CD20 antibody; heavy chain 2 comprises a heavy chain of anti-CD3 antibody; and the light chain comprises a light chain of anti-CD20 antibody and the anti-BCMA single-domain antibody.

[0040] Preferably, in the light chain, the anti-BCMA single-domain antibody is attached to the N-terminus of the anti-CD20 antibody light chain via a linker.

[0041] In another preferred embodiment, the heavy chain amino acid sequence of the anti-CD20 antibody is shown in SEQ ID NO:31.

[0042] In another preferred embodiment, the heavy chain amino acid sequence of the anti-CD3 antibody is shown in SEQ ID NO:32.

[0043] In another preferred embodiment, the light chain amino acid sequence of the anti-CD20 antibody is as shown in SEQ ID NO:33 or 34.

[0044] In another preferred embodiment, the amino acid sequence of the anti-BCMA single-domain antibody is as shown in any one of SEQ ID NO:25-30.

[0045] In another preferred embodiment, the linker amino acid sequence is as shown in GGGGS (SEQ ID NO:35).

[0046] In another preferred embodiment, the multispecific antibody is composed of heavy chain 1, heavy chain 2 and light chain as shown in any one of (1)-(18):

[0047] (1) Heavy chain 1 shown in SEQ ID NO:36, heavy chain 2 shown in SEQ ID NO:42, and light chain shown in SEQ ID NO:33;

[0048] (2) Heavy chain 1 shown in SEQ ID NO:37, heavy chain 2 shown in SEQ ID NO:43, and light chain shown in SEQ ID NO:33;

[0049] (3) Heavy chain 1 shown in SEQ ID NO:38, heavy chain 2 shown in SEQ ID NO:44, and light chain shown in SEQ ID NO:33;

[0050] (4) Heavy chain 1 shown in SEQ ID NO:39, heavy chain 2 shown in SEQ ID NO:45, and light chain shown in SEQ ID NO:33;

[0051] (5) Heavy chain 1 shown in SEQ ID NO:40, heavy chain 2 shown in SEQ ID NO:46, and light chain shown in SEQ ID NO:33;

[0052] (6) Heavy chain 1 shown in SEQ ID NO:41, heavy chain 2 shown in SEQ ID NO:47, and light chain shown in SEQ ID NO:33;

[0053] (7) Heavy chain 1 shown in SEQ ID NO:31, heavy chain 2 shown in SEQ ID NO:32, and light chain shown in SEQ ID NO:48;

[0054] (8) Heavy chain 1 shown in SEQ ID NO:31, heavy chain 2 shown in SEQ ID NO:32, and light chain shown in SEQ ID NO:49;

[0055] (9) Heavy chain 1 shown in SEQ ID NO:31, heavy chain 2 shown in SEQ ID NO:32, and light chain shown in SEQ ID NO:50;

[0056] (10) Heavy chain 1 shown in SEQ ID NO:31, heavy chain 2 shown in SEQ ID NO:32, and light chain shown in SEQ ID NO:51;

[0057] (11) Heavy chain 1 shown in SEQ ID NO:31, heavy chain 2 shown in SEQ ID NO:32, and light chain shown in SEQ ID NO:52;

[0058] (12) Heavy chain 1 shown in SEQ ID NO:31, heavy chain 2 shown in SEQ ID NO:32, and light chain shown in SEQ ID NO:53;

[0059] (13) Heavy chain 1 shown in SEQ ID NO:31, heavy chain 2 shown in SEQ ID NO:42, and light chain shown in SEQ ID NO:33;

[0060] (14) Heavy chain 1 shown in SEQ ID NO:31, heavy chain 2 shown in SEQ ID NO:43, and light chain shown in SEQ ID NO:33;

[0061] (15) Heavy chain 1 shown in SEQ ID NO:31, heavy chain 2 shown in SEQ ID NO:44, and light chain shown in SEQ ID NO:33;

[0062] (16) Heavy chain 1 shown in SEQ ID NO:31, heavy chain 2 shown in SEQ ID NO:45, and light chain shown in SEQ ID NO:33;

[0063] (17) Heavy chain 1 shown in SEQ ID NO:31, heavy chain 2 shown in SEQ ID NO:46, and light chain shown in SEQ ID NO:33;

[0064] (18) Heavy chain 1 shown in SEQ ID NO:31, heavy chain 2 shown in SEQ ID NO:47, and light chain shown in SEQ ID NO:33.

[0065] In a third aspect of the invention, a polynucleotide is provided that encodes a protein selected from the group consisting of: a single-domain antibody against BCMA as described in the first aspect of the invention, or a multispecific antibody as described in the second aspect of the invention, or a combination thereof.

[0066] In another preferred embodiment, the polynucleotide includes DNA, RNA, or cDNA.

[0067] In a fourth aspect of the invention, an expression vector is provided, the expression vector containing the polynucleotide as described in the third aspect of the invention.

[0068] In another preferred embodiment, the expression vector is selected from the group consisting of DNA, RNA, viral vectors, plasmids, transposons, other gene transfer systems, or combinations thereof.

[0069] In another preferred embodiment, the expression vector includes a viral vector, such as a lentivirus, adenovirus, AAV virus, or retrovirus.

[0070] In a fifth aspect of the invention, a host cell is provided, the host cell containing an expression vector as described in the fourth aspect of the invention, or having a genome containing polynucleotides as described in the third aspect of the invention.

[0071] In another preferred embodiment, the host cell includes a prokaryotic cell or a eukaryotic cell.

[0072] In another preferred embodiment, the host cell is selected from the group consisting of Escherichia coli, yeast cells, mammalian cells, bacteriophages, or combinations thereof.

[0073] In a sixth aspect of the invention, a method for generating a single-domain antibody against BCMA as described in the first aspect of the invention or a multi-specific antibody as described in the second aspect of the invention is provided, comprising the steps of:

[0074] (a) Under conditions suitable for antibody production, host cells as described in the fifth aspect of the invention are cultured to obtain a culture containing the said anti-BCMA single-domain antibody or multispecific antibody; and

[0075] (b) Isolating or recovering the anti-BCMA single-domain antibody or multispecific antibody from the culture; and

[0076] (c) Optionally, purify and / or modify the anti-BCMA single-domain antibody or multispecific antibody obtained in step (b).

[0077] In a seventh aspect of the invention, an immunoconjugate is provided, the immunoconjugate comprising:

[0078] (a) an anti-BCMA single-domain antibody as described in the first aspect of the invention, or a multispecific antibody as described in the second aspect of the invention; and

[0079] (b) The conjugation portion of the single-domain antibody, wherein the conjugation portion is selected from the group consisting of detectable markers, drugs, or combinations thereof.

[0080] In an eighth aspect of the invention, a pharmaceutical composition is provided comprising:

[0081] (i) an anti-BCMA single-domain antibody as described in the first aspect of the invention, or a multispecific antibody as described in the second aspect of the invention, or an immunoconjugate as described in the seventh aspect of the invention, or a combination thereof; and

[0082] (ii) Pharmaceutically acceptable carriers.

[0083] In another preferred embodiment, the pharmaceutical composition further contains other drugs for treating tumors and / or autoimmune diseases, such as cytotoxic drugs.

[0084] In another preferred embodiment, the pharmaceutical composition is an injectable dosage form.

[0085] In another preferred embodiment, the pharmaceutical composition is used to prepare a medicament for treating tumors and / or autoimmune diseases.

[0086] In another preferred embodiment, the tumor is a tumor that highly expresses BCMA and / or CD20.

[0087] In another preferred embodiment, the tumor is selected from the group consisting of: multiple myeloma, lymphocytic leukemia, lymphoma, acute myeloid leukemia, chronic myeloid leukemia, colorectal cancer, breast cancer, gallbladder cancer, gastric cancer, liver cancer, kidney cancer, head and neck tumors, brain tumors, glioblastoma, lung cancer, small bowel cancer, bone cancer, prostate cancer, ovarian cancer, cervical cancer, adrenal tumors, bladder tumors, sarcomas, or combinations thereof.

[0088] In another preferred embodiment, the autoimmune disease is selected from the group consisting of: systemic lupus erythematosus, rheumatoid arthritis, Sjögren's syndrome, ankylosing spondylitis, immune thrombocytopenic purpura, idiopathic inflammatory myopathy, systemic sclerosis, neuromyelitis optica spectrum disorder, myasthenia gravis, multiple sclerosis, celiac disease, type 1 diabetes, diffuse toxic goiter (also known as Graves' disease), inflammatory bowel disease, autoimmune hepatitis, or combinations thereof.

[0089] In a ninth aspect of the invention, there is provided a use of an active ingredient selected from the group consisting of: anti-BCMA single-domain antibodies as described in the first aspect of the invention, or multispecific antibodies as described in the second aspect of the invention, or immunoconjugates as described in the seventh aspect of the invention, or combinations thereof, wherein the active ingredient is used for (a) preparing a detection reagent, detection plate or kit; and / or (b) preparing a medicament for the prevention and / or treatment of a disease.

[0090] In another preferred embodiment, the detection reagent, detection plate, or kit is used for:

[0091] (1) Detect BCMA protein in the sample; and / or

[0092] (2) Detect tumor cells expressing BCMA protein.

[0093] In another preferred embodiment, the test reagent, test plate, or kit is used to diagnose BCMA-related diseases.

[0094] In another preferred embodiment, the disease is a tumor or autoimmune disease with high expression of BCMA and / or CD20.

[0095] In another preferred embodiment, the tumor is selected from the group consisting of: multiple myeloma, lymphocytic leukemia, lymphoma, acute myeloid leukemia, chronic myeloid leukemia, colorectal cancer, breast cancer, gallbladder cancer, gastric cancer, liver cancer, kidney cancer, head and neck tumors, brain tumors, glioblastoma, lung cancer, small bowel cancer, bone cancer, prostate cancer, ovarian cancer, cervical cancer, adrenal tumors, bladder tumors, sarcomas, or combinations thereof.

[0096] In another preferred embodiment, the autoimmune disease is selected from the group consisting of: systemic lupus erythematosus, rheumatoid arthritis, Sjögren's syndrome, ankylosing spondylitis, immune thrombocytopenic purpura, idiopathic inflammatory myopathy, systemic sclerosis, neuromyelitis optica spectrum disorder, myasthenia gravis, multiple sclerosis, celiac disease, type 1 diabetes, diffuse toxic goiter (also known as Graves' disease), inflammatory bowel disease, autoimmune hepatitis, or combinations thereof.

[0097] In a tenth aspect of the present invention, a method for detecting BCMA protein in a sample is provided, the method comprising the steps of:

[0098] (1) Contact the sample with the anti-BCMA single-domain antibody as described in the first aspect of the present invention;

[0099] (2) Detect whether an antigen-antibody complex is formed, where the formation of a complex indicates the presence of BCMA protein in the sample.

[0100] In an eleventh aspect of the present invention, a method for treating BCMA-related diseases is provided, the method comprising administering to a desired subject an anti-BCMA single-domain antibody as described in the first aspect of the present invention, a multispecific antibody as described in the second aspect of the present invention, an immunoconjugate as described in the seventh aspect of the present invention, or a pharmaceutical composition as described in the eighth aspect of the present invention.

[0101] In another preferred embodiment, the object includes a human or a non-human mammal.

[0102] In another preferred embodiment, the non-human mammals include rodents (such as mice and rabbits) and non-human primates (such as monkeys).

[0103] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0104] The following figures are used to illustrate specific embodiments of the present invention and are not intended to limit the scope of the invention as defined by the claims.

[0105] Figure 1 The study evaluated the cytotoxic effect of bispecific antibodies on Raji cells. In Figure A, representative flow cytometry data (5 nM concentration) show T cell killing of Raji cells mediated by bispecific antibodies; Figure B shows T cell killing of Raji cells mediated by bispecific antibodies.

[0106] Figure 2 The binding effect of the anti-BCMA monoclonal antibody on the human BCMA antigen was evaluated.

[0107] Figure 3 A schematic diagram of the structure of the trispecific antibody of the present invention is shown.

[0108] Figure 4 The diagrams show representative SDS-PAGE and SEC-HPLC purity assays for trispecific antibodies. A shows a representative SDS-PAGE result illustrating the purity of the trispecific antibody; B shows a representative SEC-HPLC result illustrating the purity of the trispecific antibody.

[0109] Figure 5 The images show the binding ability of trispecific antibodies to target cells. A shows the binding ability of trispecific antibodies to Raji cells. B shows the binding ability of trispecific antibodies to U266 cells.

[0110] Figure 6 The binding ability of the trispecific antibody to T cells was demonstrated.

[0111] Figure 7 This invention demonstrates how the trispecific antibody mediates T cell killing of target cells Raji and B cells. A shows T cell killing of Raji cells mediated by the trispecific antibody. B shows T cell killing of human primary B cells mediated by the trispecific antibody.

[0112] Figure 8 This diagram illustrates the activation of T cells by Raji cells and B cells mediated by the trispecific antibody of this invention. A is a schematic diagram of the activation of T cells by Raji cells mediated by the trispecific antibody. B shows the expression of CD137 (5 nM concentration) in T cells activated by Raji cells mediated by the trispecific antibody.

[0113] Figure 9 This diagram illustrates the T-cell killing of U266 cells mediated by the trispecific antibody of this invention. A is a flow cytometry schematic diagram of T-cell killing of U266 cells. B shows the efficient T-cell killing of U266 cells mediated by the trispecific antibody.

[0114] Figure 10 The assay showed the ability of the trispecific antibody of the present invention to directly activate human primary T cells (12ug / ml). Detailed Implementation

[0115] Through extensive and in-depth research, the inventors have developed, for the first time, a multispecific antibody targeting B-cell antigens and T-cell antigens, and its applications. This invention screened single-domain antibodies targeting BCMA and fused these single-domain antibodies with existing CD3 / CD20 bispecific antibodies to construct a trispecific antibody specifically targeting BCMA, CD3, and CD20. This trispecific antibody can be used as a T-cell adaptor, connecting target cells (such as B cells) and T cells to promote the killing effect of T cells on target cells. Based on this, the invention was completed.

[0116] This invention's trispecific antibody simultaneously binds to BCMA, CD20, and CD3 molecules, possessing advantages not found in bispecific antibodies targeting BCMA and CD3 or bispecific antibodies targeting CD20 and CD3. This is primarily reflected in the fact that the trispecific antibody can simultaneously mediate T cell killing of cells expressing BCMA (such as various types of plasma cells) and cells expressing CD20 (such as mature B cells and memory B cells). Since B cells and plasma cells play crucial roles in many autoimmune diseases (such as systemic lupus erythematosus, rheumatoid arthritis, Sjögren's syndrome, ankylosing spondylitis, immune thrombocytopenic purpura, idiopathic inflammatory myopathy, systemic sclerosis, neuromyelitis optica spectrum disorder, myasthenia gravis, multiple sclerosis, celiac disease, type 1 diabetes, diffuse toxic goiter (also known as Graves' disease), inflammatory bowel disease, and autoimmune hepatitis), simultaneously eliminating both B cells and plasma cells may achieve better therapeutic effects for autoimmune diseases. The trispecific antibody of this invention offers advantages for the treatment of autoimmune diseases that other bispecific antibody molecules lack, and has the potential to lead to better clinical treatment outcomes. Furthermore, the trispecific antibody of this invention has a simple structure, is molecularly stable, can be expressed in large quantities, and has high purity. Compared to existing bispecific and trispecific antibodies with complex structures, the trispecific antibody of this invention will have a significantly lower production cost in the future, offering a substantial production cost advantage.

[0117] The single-domain antibodies and multispecific antibodies of the present invention

[0118] As used herein, the terms "antibody" or "immunoglobulin" refer to heterotetraglycoproteins of approximately 150,000 Daltons with the same structural characteristics, consisting of two light chains (L) and two heavy chains (H). Each light chain is linked to the heavy chain by a covalent disulfide bond, and the number of disulfide bonds between heavy chains varies among different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by multiple constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other end; the constant regions of the light chains are opposite the first constant regions of the heavy chains, and the variable regions of the light chains are opposite the variable regions of the heavy chains. Specific amino acid residues form interfaces between the variable regions of the light and heavy chains.

[0119] As used herein, the terms "single-domain antibody," "nanobody," and "VHH" have the same meaning, referring to the variable region of the heavy chain of a monoclonal antibody. A nanobody (VHH) is the smallest antigen-binding fragment with complete function. Typically, an antibody lacking both the light chain and the constant region 1 (CH1) of the heavy chain is first obtained, and then the variable region of the antibody heavy chain is cloned to construct a nanobody (VHH) consisting of only one heavy chain variable region.

[0120] As used in this article, the term "multispecific antibody" refers to a molecule that can bind to multiple different types of epitopes simultaneously, which may be located on the same target cell or different target cells.

[0121] Based on the teachings of this invention, those skilled in the art will understand that the present invention is a multispecific antibody that is a T-cell adaptor type multispecific antibody; that is, a multispecific antibody (e.g., a trispecific antibody) that simultaneously targets tumor antigens and T-cell antigens.

[0122] In a specific embodiment, the multispecific antibody of the present invention is composed of an antibody fragment that specifically binds to tumor antigens CD20 and BCMA and an antibody fragment that specifically binds to T-cell antigen CD3.

[0123] The single-domain antibodies and multispecific antibodies of the present invention include not only complete antibodies, but also fragments, derivatives, and analogs of said antibodies. As used herein, the terms “fragment,” “derivative,” and “analyte” refer to polypeptides that substantially retain the same biological function or activity as the antibodies of the present invention. The polypeptide fragments, derivatives, or analogs of the present invention may be (i) polypeptides in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) polypeptides having substituent groups in one or more amino acid residues; or (iii) polypeptides formed by fusing a mature polypeptide with another compound (e.g., a compound that extends the half-life of the polypeptide, such as polyethylene glycol); or (iv) polypeptides formed by fusing an additional amino acid sequence to this polypeptide sequence (e.g., a leader sequence or secretion sequence, or a sequence used to purify this polypeptide, or a proteogenic sequence, or a fusion protein formed with a 6His tag). Based on the teachings herein, these fragments, derivatives, and analogs are within the scope of knowledge of those skilled in the art.

[0124] The single-domain antibodies and multispecific antibodies of the present invention also include variants of polypeptides containing the aforementioned CDR region having the same function as the antibodies described above. These variants include (but are not limited to): deletions, insertions, and / or substitutions of one or more amino acids (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10), and the addition of one or more amino acids (typically less than 20, preferably less than 10, more preferably less than 5) to the C-terminus and / or N-terminus. For example, in the art, substitution with amino acids of similar or comparable properties generally does not alter the function of the protein. Similarly, the addition of one or more amino acids to the C-terminus and / or N-terminus generally does not alter the function of the protein. This term also includes active fragments and active derivatives of the single-domain antibodies and multispecific antibodies of the present invention. The variant forms of the polypeptide include: homologous sequences, conserved variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize with the encoding DNA of the antibody of the present invention under high or low severity conditions, and polypeptides or proteins obtained using antiserum against the antibody of the present invention.

[0125] In addition to the nearly full-length polypeptide, the present invention also includes fragments of the single-domain antibody and multi-specific antibody of the present invention. Typically, the fragment has at least about 50 consecutive amino acids of the antibody of the present invention, preferably at least about 50 consecutive amino acids, more preferably at least about 80 consecutive amino acids, and most preferably at least about 100 consecutive amino acids.

[0126] In this invention, "a conserved variant of the antibody of the present invention" refers to a polypeptide formed by replacing up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids with amino acids of similar or analogous properties, compared to the amino acid sequence of the single-domain antibody or multispecific antibody of the present invention. These conserved variant polypeptides are preferably generated by amino acid substitutions according to Table 1.

[0127] Table 1

[0128] The initial residues Representative substitution Preferred replacement Ala(A) Val; Leu; Ile Val Arg(R) Lys;Gln;Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe Leu Leu(L) Ile; Val; Met; Ala; Phe Ile Lys(K) Arg;Gln;Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; Ala; Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Ala Leu

[0129] The present invention also provides polynucleotide molecules encoding the aforementioned antibodies or fragments thereof or fusion proteins thereof. The polynucleotides of the present invention may be in DNA or RNA form. DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. DNA may be single-stranded or double-stranded. DNA may be a coding strand or a non-coding strand. Polynucleotides encoding the mature polypeptides of the present invention include: a coding sequence encoding only the mature polypeptide; a coding sequence of the mature polypeptide and various additional coding sequences; a coding sequence of the mature polypeptide (and optional additional coding sequences) and a non-coding sequence.

[0130] The term "polynucleotide encoding a polypeptide" can refer to a polynucleotide that includes the polypeptide itself, or it can also include additional coding and / or non-coding sequences. This invention also relates to polynucleotides that hybridize with the aforementioned sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. This invention particularly relates to polynucleotides that hybridize with the polynucleotides described herein under stringent conditions. In this invention, "stringent conditions" means: (1) hybridization and elution at lower ionic strength and higher temperatures, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) hybridization with a denaturing agent, such as 50% (v / v) formamide, 0.1% fetal bovine serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization only occurs when the identity between the two sequences is at least 90%, more preferably at least 95%. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.

[0131] The full-length nucleotide sequence or fragments of the antibodies of this invention can generally be obtained by PCR amplification, recombinant methods, or artificial synthesis. One feasible method is to synthesize the relevant sequence artificially, especially when the fragment length is short. Typically, long fragments can be obtained by first synthesizing multiple small fragments and then ligating them. Alternatively, the coding sequence of the heavy chain and an expression tag (such as 6His) can be fused together to form a fusion protein. Once the relevant sequence is obtained, it can be obtained in large quantities using recombinant methods. This typically involves cloning it into a vector, transforming it into cells, and then isolating the relevant sequence from the proliferated host cells using conventional methods. The biomolecules (nucleic acids, proteins, etc.) involved in this invention include biomolecules existing in isolated forms.

[0132] Currently, the DNA sequence encoding the protein of this invention (or a fragment thereof, or a derivative thereof) can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. Furthermore, mutations can be introduced into the protein sequence of this invention through chemical synthesis.

[0133] This invention also relates to vectors comprising the aforementioned suitable DNA sequences and suitable promoters or control sequences. These vectors can be used to transform suitable host cells to enable them to express proteins. Host cells can be prokaryotic cells, such as bacterial cells; lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples include: *Escherichia coli*, *Streptomyces*; bacterial cells of *Salmonella typhimurium*; fungal cells such as yeast; insect cells of *Drosophila* S2 or Sf9; animal cells of CHO, COS7, 293, etc.

[0134] Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as *E. coli*, competent cells capable of uptake DNA can be harvested after the exponential growth phase and treated with CaCl2, the steps of which are well known in the art. Another method is to use MgCl2. If desired, transformation can also be performed using electroporation. When the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0135] The obtained transformants can be cultured using conventional methods to express the polypeptide encoded by the gene of this invention. Depending on the host cells used, the culture medium can be selected from various conventional media. Culture is carried out under conditions suitable for host cell growth. Once the host cells have grown to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature adjustment or chemical induction), and the cells are cultured for a further period.

[0136] The recombinant peptides used in the methods described above can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, the recombinant proteins can be separated and purified using various separation methods based on their physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitants (salting out), centrifugation, permeation, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods.

[0137] The single-domain antibodies and multispecific antibodies of the present invention can be used alone or in combination or conjugated with detectable markers (for diagnostic purposes), therapeutic agents, PK (protein kinase) modified moieties, or any combination of the above substances. Detectable markers for diagnostic purposes include, but are not limited to: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes capable of producing detectable products.

[0138] Therefore, the present invention also provides an immunoconjugate containing the single-domain antibody or multispecific antibody of the present invention, and a conjugation portion. Therapeutic agents that can bind to or conjugate with the single-domain antibody and multispecific antibody of the present invention include, but are not limited to: 1. radionuclides; 2. biotoxins; 3. cytokines, such as IL-2; 4. gold nanoparticles / nanorings; 5. viral particles; 6. liposomes; 7. magnetic nanoparticles; 8. drug-activated enzymes (e.g., DT-cardiac flavin enzyme (DTD) or biphenyl hydrolase-like protein (BPHL)); 9. therapeutic agents (e.g., cisplatin) or any form of nanoparticle, etc. In a preferred embodiment, the immunoconjugate is an antibody-drug conjugate (ADC).

[0139] Pharmaceutical Composition

[0140] The present invention also provides a pharmaceutical composition comprising the aforementioned single-domain antibody or multispecific antibody or its active fragment or fusion protein, and a pharmaceutically acceptable carrier. Typically, these substances are formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 5-8, preferably about 6-8, although the pH may vary depending on the nature of the formulated substance and the condition to be treated. The formulated pharmaceutical composition can be administered via conventional routes, including (but not limited to): intratumoral, intraperitoneal, intravenous, or local administration.

[0141] The pharmaceutical compositions of the present invention can be directly used to bind to the PD-1 protein, thereby blocking the interaction between PD-1 and PD-L1. The pharmaceutical compositions of the present invention can also be directly used to bind to other targets, thereby blocking the binding of said other targets to their receptors or ligands. Therefore, the pharmaceutical compositions of the present invention can be used to treat tumors. In a preferred embodiment, the tumors are selected from the group consisting of: multiple myeloma, lymphocytic leukemia, lymphoma, acute myeloid leukemia, chronic myeloid leukemia, colorectal cancer, breast cancer, gallbladder cancer, gastric cancer, liver cancer, kidney cancer, head and neck tumors, brain tumors, glioblastoma, lung cancer, small intestine cancer, bone cancer, prostate cancer, ovarian cancer, cervical cancer, adrenal tumors, bladder tumors, sarcomas, etc. Furthermore, the pharmaceutical compositions of the present invention can also be used in combination with other therapeutic agents.

[0142] The pharmaceutical compositions of the present invention contain a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the above-described single-domain antibody (or conjugate thereof) of the present invention, and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer solutions, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be matched to the route of administration. The pharmaceutical compositions of the present invention can be formulated into injectable forms, for example, prepared using conventional methods with physiological saline or an aqueous solution containing glucose and other excipients. Pharmaceutical compositions such as injections and solutions are preferably manufactured under aseptic conditions.

[0143] The dosage of the active ingredient is a therapeutically effective amount, such as approximately 10 micrograms / kg body weight to approximately 50 milligrams / kg body weight daily. When using a pharmaceutical composition, a safe and effective amount of the immunoconjugate is administered to mammals, where this safe and effective amount is generally at least approximately 10 micrograms / kg body weight and, in most cases, does not exceed approximately 50 milligrams / kg body weight; preferably, this dose is approximately 10 micrograms / kg body weight to approximately 10 milligrams / kg body weight. Of course, the specific dosage should also consider factors such as the route of administration and the patient's health condition, which are all within the scope of a skilled physician's expertise.

[0144] The main advantages of this invention include:

[0145] 1) The multispecific antibody of the present invention is simple to construct, has a large expression level and high purity, and is easy to prepare;

[0146] 2) The multispecific antibodies of the present invention possess excellent affinity for target cells and T cells, and can produce ideal biological effects;

[0147] 3) The multispecific antibody of the present invention targets B cells at different differentiation stages and can deeply remove B cells in vivo;

[0148] 4) The multispecific antibody of this invention has a high degree of safety;

[0149] 5) The multispecific antibody of the present invention lays a new material basis for the development of anti-tumor and autoimmune disease drugs.

[0150] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0151] Example 1. Maternal bispecific antibody-mediated T cell killing of B-cell tumor cells

[0152] This invention first evaluates the bispecific antibodies (hereinafter referred to as bispecific antibodies) targeting CD3 and CD20, as described in the authorized patent CN202210113943: LL206-1 and LL206-2, which mediate the killing activity of T cells against B-cell tumor cells. Both LL206-1 and LL206-2 molecules consist of three chains.

[0153] The amino acid sequence of LL206-1 is as follows:

[0154] Heavy chain 1: SEQ ID NO:31 (SEQ ID NO:11 in CN202210113943)

[0155] EVQLVESGGGLVQPGRSLRLSCAASGFTFNDYAMHWVRQAPGKGLEWVSTISWNSGSIGYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTALYYCAKDIQYGNYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0156] Heavy chain 2: SEQ ID NO:32 (SEQ ID NO:12 in CN202210113943)

[0157] QVQLVESGGGVVQPGRSLRLSCAASGFKFSGYGMHWVRQAPGKGLEWVAVIWYDGSKKYYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQMGYWHFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK

[0158] Light chain: SEQ ID NO:33 (SEQ ID NO:2 in CN202210113943)

[0159] EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPITFGQGTRLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0160] The amino acid sequence of LL206-2 is as follows:

[0161] Heavy chain 1: SEQ ID NO:31 (SEQ ID NO:11 in CN202210113943)

[0162] Heavy chain 2: SEQ ID NO:32 (SEQ ID NO:12 in CN202210113943)

[0163] Light chain: SEQ ID NO:34 (SEQ ID NO:4 in CN202210113943)

[0164] EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPLTFGGGTKVEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0165] First, two bispecific antibody molecules, 206-1 and 206-2, were prepared using conventional protein expression and purification methods. Flow cytometry was then used to detect the cytotoxic effects of these two antibodies on Raji cells (Chinese Academy of Sciences Cell Bank), a B-cell tumor cell line. The basic steps are as follows:

[0166] Cell co-culture. Adjust Raji cell concentration to 5 × 10⁶. 5 / ml, then seed Raji cells at 50ul per well into 96-well plates and temporarily incubate at 37°C. Adjust the concentration of human primary PBMCs (Peripheral blood mononucleus cells) to 25×10⁶ cells / ml. 5 / ml, then seed PBMCs cells at 50µl per well into 96-well plates and temporarily incubate at 37°C. The final PBMCs cell to tumor cell ratio is 5:1. Using 1640 complete medium, antibodies LL206-1 and LL206-2 were diluted 4-fold to nine concentration gradients: 20nM, 5nM, 1.25nM, 312.5pM, 78.1pM, 19.5pM, 4.9pM, 1.2pM, and 0.3pM. Then, 100µl of each of the nine diluted antibody gradients was added to 96-well plates containing Raji cells and T cells. The dilution ratio was 1:2. After thorough mixing, centrifuge at 500 rpm for 3 minutes. Incubate the cells at 37°C for 24 hours.

[0167] Antibody staining: Anti-CD19 antibody (Biolegend, catalog number: 302207) was added to the wells of the 96-well plate above at the concentration recommended by the supplier to label Raji cells. After mixing by pipetting, the plate was incubated at 4°C in the dark for 30 min.

[0168] Antibody washing: Add 1 ml wash buffer (1xPBS + 0.5% FBS) to the incubated antibody-cell mixture, centrifuge at 2000 rpm for 5 min and discard the supernatant;

[0169] Flow cytometry: Add 200 μL of wash buffer to each tube for resuspending, transfer to flow cytometry tubes, and perform analysis using a Beckman CytoFLEX flow cytometer.

[0170] from Figure 1 As shown in Figure A, the addition of the bispecific antibodies LL206-1 and LL206-1 resulted in the elimination of both the Raji cell population (labeled as Raji) and the B cell population (labeled as Primary B). Therefore, both LL206-1 and LL206-1 effectively mediated the killing of Raji cells and B cells by T cells. Statistical analysis revealed that the EC50 values ​​for Raji cell killing by LL206-1 and LL206-1 were 40 pM and 200 pM, respectively. Figure 1 Therefore, LL206-1 exhibits better Raji cell killing activity than LL206-2.

[0171] Example 2. Screening of anti-BCMA monoclonal antibodies

[0172] This invention, referencing the standard procedure for single-domain antibody screening, identified 30 unique anti-BCMA monoclonal antibodies through animal immunization and sequence optimization. Further screening and sequence optimization yielded 8 unique BCMA monoclonal antibodies: LLB-01, LLB-02, LLB-03, LLB-04, LLB-05, LLB-06, LLB-07, and LLB-08. The basic steps are as follows:

[0173] 1) Select healthy adult alpacas as experimental animals for immunization. Use recombinant human BCMA protein as the antigen to immunize the alpacas. Perform 4-5 immunizations in total.

[0174] 2) Peripheral blood samples were collected from alpacas at different time points and the immunogenicity was detected using the ELISA method.

[0175] 3) After immunization, peripheral blood was collected from alpacas, and mRNA from peripheral blood mononuclear cells was extracted and reverse transcribed to obtain cDNA.

[0176] 4) Use specific primers to amplify cDNA to obtain PCR products containing single-domain antibody gene fragments.

[0177] 5) Use electroporation to introduce PCR products and yeast library vectors into competent yeast cells to prepare yeast libraries.

[0178] 6) The yeast library was screened using recombinant protein antigens or tumor cells expressing BCMA. Monoclonal antibodies that specifically bind to positive antigens but not negative antigens were obtained through three rounds of positive screening and two rounds of negative screening.

[0179] 7) Obtain the full-length sequence of a single-domain antibody that specifically binds to the target protein by performing first-generation sequencing on the monoclonal antibody.

[0180] 8) Use an ELISA kit to assess the affinity of the screened monoclonal antibodies and identify candidate antibodies.

[0181] 9) Humanize the selected monoclonal antibodies. Basic steps: Input the monoclonal antibody sequence into the IMGT database for human antibody heavy chain sequence alignment. Based on the database sequence alignment results, select the human antibody heavy chain sequence with the most similar sequence as the humanization parent vector. Further, transplant the CDR region of the monoclonal antibody onto the human antibody heavy chain. Referring to published patents and experience, perform reversion mutations on the transplanted humanized antibody to ensure its affinity. Eight candidate humanized monoclonal antibodies were obtained through the above method. The CDR regions of the anti-BCMA monoclonal antibodies LLB-01 to LLB-8 of this invention are shown in Table 2.

[0182] Table 2. CDR sequences of anti-BCMA monoclonal antibodies

[0183] Antibody name CDR1 (SEQ ID NO:) CDR2(SEQ ID NO:) CDR3 (SEQ ID NO:) LLB-01 GGDGSSYLMA(1) NIRYERGTYYANGDKG(2) GSPAGGSVGSYKY(3) LLB-02 GRQLERVYAMG(4) AIGRISDQMLADSVKG(5) EGSRGDSGAYGMDY(6) LLB-03 GGTFSSYLMA(7) NYADGGGSYANSVKG(8) QTEGQDEGAGGGSY(9) LLB-04 GRTFSTYAMG(10) AIGWIGAYTNLADSVKG(11) SGGSGAGAYGMGDV(12) LLB-05 GGTFSSYLMA(7) NIRWSGDITYYANSVKG(13) ADPKTDYHAGYKY(14) LLB-06 GRTFSTYAMG(10) AIGWIGGYTNLADSVKG(15) SETGRESGAYGMDY(16) LLB-07 GRLFNTFSGA(17) VGTWSGAPTDYADSVKG(18) AGSWEDGRPYDY(19) LLB-08 GRLFNTFRVA(20) HITWSGGSTDYADSVKG(21) AYRSGSGYPRPYEY(22)

[0184] The full-length sequences of LLB-01 to LLB-8 are shown below:

[0185] LLB-01:

[0186] EVQLVESGGGLVQPGGSLRLSCAASGGDGSSYLMAWFRQAPGKEREIVANIRYERGTYYANGDKGRFTISRDNAKNTVYLQMNSLRAEDTAVYYCAAGSPAGGSVGSYKYWGQGTQVTVSS (SEQ ID NO:23).

[0187] LLB-02:

[0188] EVQLVESGGGLVQPGGSLRLSCAASGRQLERVYAMGWFRQAPGKERE FVGAIGRISDQMLADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCAA EGSRGDSGAYGMDYWGKGTLVTVSS (SEQ ID NO:24).

[0189] LLB-03:

[0190] EVQLVESGGGLVQPGGSLRLSCAASGGTFSSYLMAWFRQAPGKEREIV ANYADGGGSYANSVKGRFTISRDNAKNTVYLQMNSLRAEDTAVYYCAAQT EGQDEGAGGGSYWGQGTQVTVSS (SEQ ID NO:25). <00005于2>LLB-04:

[0192] EVQLVESGGGLVQPGGSLRLSCAASGRTFSTYAMGWFRQAPGKEREFV GAIGWIGAYTNLADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCAAS GGSGAGAYGMGDVWGKGTLVTVSS (SEQ ID NO:26).

[0193] LLB-05:

[0194] It seems there is a typo in the original text where "

[0191] " is followed by "<00005于2> ". I've translated it as best as possible with the given context. If this is incorrect, please provide the correct text.EVQLVESGGGLVQPGGSLRLSCAASGGTFSSYLMAWFRQAPGKEREIV ANIRWSGDITYYANSVKGRFTISRDNAKNTVYLQMNSLRAEDTAVYYCAAA DPKTDYHAGYKYWGQGTQVTVSS(SEQ ID NO:27)。

[0195] LLB-06:

[0196] EVQLVESGGGLVQPGGSLRLSCAASGRTFSTYAMGWFRQAPGKEREFV GAIGWIGGYTNLADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCAAS ETGRESGAYGMDYWGKGTLVTVSS(SEQ ID NO:28)。

[0197] LLB-07:

[0198] QVQLVESGGGLVQPGGSLRLSCAASGRLFNTFSGAWFRQAPGKEREFV AVGTWSGAPTDYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAS AGSWEDGRPYDYWGQGTQVTVSS(SEQ ID NO:29)。

[0199] LLB-08:

[0200] EVQLVESGGGLVQPGGSLRLSCAASGRLFNTFRVAWFRQAPGKEREFV AHITWSGGSTDYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCASA YRSGSGYPRPYEYWGQGTQVTVSS(SEQ ID NO:30)。

[0201] First, the above-mentioned anti-BCMA monoclonal antibody was prepared using conventional protein expression and purification methods. The antigen-binding activity of different antibodies against human BCMA protein was evaluated using ELISA. The basic steps are as follows: First, recombinant human BCMA protein (purchased from Beijing Baipusaisi, catalog number: BCA-H522y) was diluted to 1 μg / mL with 1x PBS (purchased from Hyclone). 100 μl / well was added to a 96-well ELISA plate for antigen coating and incubated at 37°C for 60 minutes. After incubation, the plate was washed three times with PBS solution, and 200 μl / well of PBS containing 2% BSA was added for blocking. The plate was then incubated at 37°C for 60 minutes. Simultaneously, the anti-BCMA monoclonal antibody of this invention was diluted to 100 ng / mL with dilution buffer (PBS containing 2% BSA), and then serially diluted three-fold to 0.05 ng / mL on the sample dilution plate. Then, 100 μl of sample was added to each well of a 96-well microplate and incubated at 37°C for 60 min. After incubation, the plate was washed, and the secondary antibody (goat anti-human IgG (Fc specific)-HRP antibody, purchased from Sigma, catalog number A-0170) was diluted 6000-fold with 2% BSA. 100 μl of this solution was added to each well and incubated at 37°C for 30 min. After incubation, the plate was washed three times, and 100 μl of the chromogenic reagent (100 μg / ml TMB, 3,3',5,5'-tetramethylbenzidine) was added to each well. The plate was then incubated at 37°C in the dark for 15 min. Finally, stop solution (2M / L hydrochloric acid solution) was added at 100μl / well and the absorbance at 450nm / 620nm was detected using a microplate reader (Thermo Fisher Scientific, Varioskan LUX). The results were then analyzed using software.

[0202] The results are as follows Figure 2 As shown in the figure. The results show that among the eight single-domain antibodies screened in this invention, six single-domain antibodies can bind well to the human BCMA protein antigen and have high affinity, with EC50 values ​​all less than 1 nM. Based on the evaluation of protein antigen affinity, these six BCMA monoclonal antibody molecules with good affinity—LLB-03, LLB-04, LLB-05, LLB-06, LLB-07, and LLB-08—were selected for further design and functional evaluation of trispecific antibodies.

[0203] Example 3. Design of Trispecific Antibody Sequences

[0204] This invention uses LL206-1 as the base antibody structure. Then, the six selected anti-BCMA monoclonal antibodies (LLB-03 to LLB-08) are sorted numerically, renumbered, and linked to different heavy and light chains of LL206-1. This invention designs a total of 18 trispecific antibodies.

[0205] After the selected anti-BCMA monoclonal antibodies were linked to the N-terminus of the LL206-1 molecular heavy chain 2 (SEQ ID NO:32), the trispecific antibodies designed were: LL206-TA101, LL206-TA102, LL206-TA103, LL206-TA104, LL206-TA105, and LL206-TA106.

[0206] By simultaneously ligating selected anti-BCMA monoclonal antibodies to the N-terminus of both heavy chain 1 (SEQ ID NO:31) and heavy chain 2 (SEQ ID NO:32) of the LL206-1 molecule, the designed trispecific antibodies are: LL206-TA201, LL206-TA202, LL206-TA203, LL206-TA204, LL206-TA205, and LL206-TA206. This series of trispecific antibody molecules contains two BCMA single-domain antibodies, therefore they are bivalent molecules.

[0207] After linking selected anti-BCMA monoclonal antibodies to the N-terminus of the common light chain (SEQ ID NO:33) of the LL206-1 molecule, the designed trispecific antibodies are: LL206-TA301, LL206-TA302, LL206-TA303, LL206-TA304, LL206-TA305, and LL206-TA306. This series of trispecific antibody molecules contains two BCMA single-domain antibodies, therefore they are bivalent molecules.

[0208] A simplified structural diagram of the trispecific antibody designed in this invention is shown below. Figure 3 The structures of the three specific antibodies LL206-TA101, LL206-TA102, LL206-TA103, LL206-TA104, LL206-TA105, and LL206-TA106 are shown below. Figure 3 The structures of the three specific antibodies LL206-TA201, LL206-TA202, LL206-TA203, LL206-TA204, LL206-TA205, and LL206-TA206 are shown in Figure A. Figure 3In B; trispecific antibodies: The structures of LL206-TA301, LL206-TA302, LL206-TA303, LL206-TA304, LL206-TA305, LL206-TA306 are shown in Figure 3 In C.

[0209] Table 3 Trispecific Antibody Sequences

[0210] name Heavy chain 1 (SEQ ID NO:) Heavy chain 2 (SEQ ID NO:) Light chain (SEQ ID NO:) LL206-TA101 31 42 33 LL206-TA102 31 43 33 LL206-TA103 31 44 33 LL206-TA104 31 45 33 LL206-TA105 31 46 33 LL206-TA106 31 47 33 LL206-TA201 36 42 33 LL206-TA202 37 43 33 LL206-TA203 38 44 33 LL206-TA204 39 45 33 LL206-TA205 40 46 33 LL206-TA206 41 47 33 LL206-TA301 31 32 48 LL206-TA302 31 32 49 LL206-TA303 31 32 50 LL206-TA304 31 32 51 LL206-TA305 31 32 52 LL206-TA306 31 32 53

[0211] SEQ ID NO:36

[0212] EVQLVESGGGLVQPGGSLRLSCAASGGTFSSYLMAWFRQAPGKEREIVANYADGGGSYANSVKGRFTISRDNAKNTVYLQMNSLRAEDTAVYYCAAQTEGQDEGAGGGSYWGQGTQVTVSSGGGGSEVQLVESGGGLVQPGRSLRLSCAASGFTFNDYAMHWVRQAPGKGLEWVSTISWNSGSIGYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTALYYCAKDIQYGNYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0213] SEQ ID NO:37

[0214] 。

[0215] SEQ ID NO:38

[0216] 。

[0217] SEQ ID NO:39

[0218] EVQLVESGGGLVQPGGSLRLSCAASGRTFSTYAMGWFRQAPGKEREFVGAIGWIGGYTNLADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCAASETGRESGAYGMDYWGKGTLVTVSSGGGGSEVQLVESGGGLVQPGRSLRLSCAASGFTFNDYAMHWVRQAPGKGLEWVSTISWNSGSIGYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTALYYCAKDIQYGNYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK。

[0219] SEQ ID NO:40

[0220] QVQLVESGGGLVQPGGSLRLSCAASGRLFNTFSGAWFRQAPGKEREFVAVGTWSGAPTDYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCASAGSWEDGRPYDYWGQGTQVTVSSGGGGSEVQLVESGGGLVQPGRSLRLSCAASGFTFNDYAMHWVRQAPGKGLEWVSTISWNSGSIGYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTALYYCAKDIQYGNYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK。

[0221] SEQ ID NO:41

[0222] 。

[0223] SEQ ID NO:42

[0224] EVQLVESGGGLVQPGGSLRLSCAASGGTFSSYLMAWFRQAPGKEREIVANYADGGGSYANSVKGRFTISRDNAKNTVYLQMNSLRAEDTAVYYCAAQTEGQDEGAGGGSYWGQGTQVTVSSGGGGSQVQLVESGGGVVQPGRSLRLSCAASGFKFSGYGMHWVRQAPGKGLEWVAVIWYDGSKKYYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQMGYWHFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK。

[0225] SEQ ID NO:43

[0226] EVQLVESGGGLVQPGGSLRLSCAASGRTFSTYAMGWFRQAPGKEREFVGAIGWIGAYTNLADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCAASGGSGAGAYGMGDVWGKGTLVTVSSGGGGSQVQLVESGGGVVQPGRSLRLSCAASGFKFSGYGMHWVRQAPGKGLEWVAVIWYDGSKKYYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQMGYWHFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK。

[0227] SEQ ID NO:44

[0228] EVQLVESGGGLVQPGGSLRLSCAASGGTFSSYLMAWFRQAPGKEREIVANIRWSGDITYYANSVKGRFTISRDNAKNTVYLQMNSLRAEDTAVYYCAAADPKTDYHAGYKYWGQGTQVTVSSGGGGSQVQLVESGGGVVQPGRSLRLSCAASGFKFSGYGMHWVRQAPGKGLEWVAVIWYDGSKKYYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQMGYWHFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK。

[0229] SEQ ID NO:45

[0230] EVQLVESGGGLVQPGGSLRLSCAASGRTFSTYAMGWFRQAPGKEREFVGAIGWIGGYTNLADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCAASETGRESGAYGMDYWGKGTLVTVSSGGGGSQVQLVESGGGVVQPGRSLRLSCAASGFKFSGYGMHWVRQAPGKGLEWVAVIWYDGSKKYYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQMGYWHFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK。

[0231] SEQ ID NO:46

[0232] QVQLVESGGGLVQPGGSLRLSCAASGRLFNTFSGAWFRQAPGKEREFVAVGTWSGAPTDYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCASAGSWEDGRPYDYWGQGTQVTVSSGGGGSQVQLVESGGGVVQPGRSLRLSCAASGFKFSGYGMHWVRQAPGKGLEWVAVIWYDGSKKYYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQMGYWHFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK。

[0233] SEQ ID NO:47

[0234] EVQLVESGGGLVQPGGSLRLSCAASGRLFNTFRVAWFRQAPGKEREFVAHITWSGGSTDYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCASAYRSGSGYPRPYEYWGQGTQVTVSSGGGGSQVQLVESGGGVVQPGRSLRLSCAASGFKFSGYGMHWVRQAPGKGLEWVAVIWYDGSKKYYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQMGYWHFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK。

[0235] SEQ ID NO:48

[0236] EVQLVESGGGLVQPGGSLRLSCAASGGTFSSYLMAWFRQAPGKEREIVANYADGGGSYANSVKGRFTISRDNAKNTVYLQMNSLRAEDTAVYYCAAQTEGQDEGAGGGSYWGQGTQVTVSSGGGGSEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。

[0237] SEQ ID NO:49

[0238] EVQLVESGGGLVQPGGSLRLSCAASGRTFSTYAMGWFRQAPGKEREFVGAIGWIGAYTNLADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCAASGGSGAGAYGMGDVWGKGTLVTVSSGGGGSEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。

[0239] SEQ ID NO:50

[0240] EVQLVESGGGLVQPGGSLRLSCAASGGTFSSYLMAWFRQAPGKEREIVANIRWSGDITYYANSVKGRFTISRDNAKNTVYLQMNSLRAEDTAVYYCAAADPKTDYHAGYKYWGQGTQVTVSSGGGGSEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。

[0241] SEQ ID NO:51

[0242] EVQLVESGGGLVQPGGSLRLSCAASGRTFSTYAMGWFRQAPGKEREFVGAIGWIGGYTNLADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCAASETGRESGAYGMDYWGKGTLVTVSSGGGGSEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。

[0243] SEQ ID NO:52

[0244] QVQLVESGGGLVQPGGSLRLSCAASGRLFNTFSGAWFRQAPGKEREFVAVGTWSGAPTDYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCASAGSWEDGRPYDYWGQGTQVTVSSGGGGSEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。

[0245] SEQ ID NO:53

[0246] EVQLVESGGGLVQPGGSLRLSCAASGRLFNTFRVAWFRQAPGKEREFVAHIITWSGGSTDYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCASAYRSGSGYPRPYEYWGQGTQVTVSSGGGGSEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQA PRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPITFGQGTRLEIKRTVAAPSVFIFPPSDEQSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0247] Example 4. Expression and purification of trispecific antibodies

[0248] The gene sequences of the trispecific antibodies were constructed into antibody expression vectors using molecular cloning technology. After successful vector construction, the 18 trispecific antibodies were expressed using the CHO cell protein expression system. The basic steps are as follows: The trispecific antibody genes were cloned into protein expression vectors (Shanghai Baiying Biotechnology Co., Ltd.). The vectors carrying the trispecific antibody gene sequences were transfected into CHO suspension cells using transient transfection. The cells were placed in a cell culture incubator for suspension culture. After expression, the cell culture supernatant was collected, and the antibodies were analyzed using a Protein A affinity chromatography column. After the trispecific antibodies were purified, the protein concentration and total protein amount of different antibodies were detected using ultraviolet spectrophotometry, and the expression level of each trispecific antibody was calculated based on the expression volume. Specifically, the absorbance value A280 of the sample solution was read at a wavelength of 280 nm using a NanoDrop 1000, and the protein concentration of different samples was calculated using the formula C (mg / mL) = A280 / ε (ε is 1.482 mL / mg·cm⁻¹).

[0249] This invention first uses SDS-PAGE gel electrophoresis to evaluate the purity of different antibodies. The basic steps are as follows: the sample is diluted to approximately 1 mg / mL, an appropriate amount of loading buffer is added, and after mixing, the mixture is heated at 90°C for approximately 3 minutes. Then, the samples are separated by electrophoresis using the standard SDS-PAGE gel electrophoresis method. The loading amount for each antibody is 2-10 μg. After electrophoresis, staining and destaining are performed according to the standard SDS-PAGE procedure. After destaining, images are taken using a gel imaging system, and the purity of the main band is calculated.

[0250] In addition, the purity of different antibodies was evaluated using size exclusion high-performance liquid chromatography (SEC-HPLC). The basic steps were as follows: the sample was diluted to approximately 1.0 mg / mL, a TSKgel G3000SWXL column was used, the column temperature was set to 25℃, the injection volume was 30–50 μL, isocratic elution was performed at a flow rate of 1.0 mL / min for 20 min, and detection was performed at a wavelength of 280 nm. The monomer content was obtained using the peak area normalization method.

[0251] The expression levels and purity of the trispecific antibodies designed in this study are shown in Table 4. The results of SEC-HPLC and SDS-PAGE confirm that the designed trispecific antibodies have intact structures and high purity and expression levels. Representative SDS-PAGE and SEC-HPLC results are shown in [Table 4]. Figure 4 Analysis of the results revealed that the expression levels and purity of LL206-TA101, LL206-TA102, LL206-TA201, LL206-TA202, LL206-TA301, and LL206-TA302 among the trispecific antibodies of this invention were low, with expression levels of approximately 10-30 mg / L. In contrast, LL206-TA103, LL206-TA104, LL206-TA105, LL206-TA106, LL206-TA203, LL206-TA204, LL206-TA205, LL206-TA206, LL206-TA303, LL206-TA304, LL206-TA305, and LL206-TA306 exhibited high expression levels and high purity, making them suitable for further biological function screening and evaluation.

[0252] Table 4. Expression levels and purity of trispecific antibodies

[0253]

[0254] Example 5. Detection of target binding activity of trispecific antibodies

[0255] This invention uses flow cytometry to detect the activity of different trispecific antibodies at the cellular level in binding to target sites. The basic steps are as follows:

[0256] 1. Cell preparation: Prepare Raji lymphoma cell line (highly expressing CD20), U266 multiple myeloma cell line (highly expressing BCMA), and human primary T cells (highly expressing CD3). Adjust the cell density to 2E6 / ml and aliquot them into 1.5ml centrifuge tubes, 50ul per tube (approximately 1E5 cells).

[0257] 2. Antibody dilution: The initial antibody concentration was determined to be 19.2 ug / ml according to experimental requirements. After a 1:1 dilution, the concentration was 9.6 ug / ml. The original antibody was adjusted to the initial concentration with PBS and then diluted 2-fold. A total of 8 concentration gradients were set: 19.2 ug / ml, 9.6 ug / ml, 4.8 ug / ml, 2.4 ug / ml, 1.2 ug / ml, 0.6 ug / ml, 0.3 ug / ml, and 0.15 ug / ml.

[0258] 3. Antibody incubation: Add 50 μL of the diluted antibody to the previously aliquoted cells at a dilution ratio of 1:1. Mix well by pipetting and incubate at 4°C in the dark for 30 min.

[0259] 4. Antibody washing: Add 1 ml of wash buffer (1x PBS + 0.5% FBS) to the incubated antibody-cell mixture, centrifuge at 2000 rpm for 5 min, and discard the supernatant.

[0260] 5. Secondary antibody incubation: Dilute the secondary antibody, anti-human IgG Fc-APC (BD Biosciences; catalog number: 550931), with PBS at a ratio of 1:200. Take 50 μL and add it to the cell pellet after discarding the supernatant. Mix well by pipetting and incubate at 4°C in the dark for 30 min.

[0261] 6. Secondary antibody washing: Add 1 ml wash buffer (1x PBS + 0.5% FBS) to the incubated antibody-cell mixture, centrifuge at 2000 rpm for 5 min and discard the supernatant.

[0262] 7. Analytical Testing: Resuspend each tube in 200 μL wash buffer, transfer to a flow cytometry tube, and analyze using a Beckman CytoFLEX analyzer. Calculate the mean fluorescence intensity (MFI) for each antibody staining, plot antibody binding curves based on the MFI values, and calculate the EC50 value of antibody binding. Figures 5-6 ).

[0263] The results are as follows Figure 5 As shown in A, the three specific antibodies of this invention: LL206-TA103, LL206-TA104, LL206-TA105, LL206-TA106, LL206-TA203, LL206-TA204, LL206-TA205, LL206-TA206, LL206-TA303, LL206-TA304, LL206-TA305, and LL206-TA306 can all bind well to Raji cells, with EC50 values ​​similar to the parent analysis, approximately 1.1 ug / ml.

[0264] The results are as follows Figure 5 As shown in B, the trispecific antibodies LL206-TA103, LL206-TA104, LL206-TA105, LL206-TA106, LL206-TA203, LL206-TA204, LL206-TA205, LL206-TA206, LL206-TA303, LL206-TA304, LL206-TA305, and LL206-TA306 of this invention exhibit significant differences in affinity for U266 cells. LL206-TA103, LL206-TA104, LL206-TA105, and LL206-TA106 are monovalent molecules with an EC50 of approximately 1.8 μg / ml. LL206-TA203, LL206-TA204, LL206-TA205, LL206-TA206, LL206-TA303, LL206-TA304, LL206-TA305, and LL206-TA306 are bivalent molecules with an EC50 of approximately 0.65 ug / ml.

[0265] Analysis revealed that ( Figure 6 The trispecific antibodies LL206-TA103, LL206-TA104, LL206-TA105, LL206-TA106, LL206-TA203, LL206-TA204, LL206-TA205, LL206-TA206, LL206-TA303, LL206-TA304, LL206-TA305, and LL206-TA306 of this invention exhibit similar affinity for T cells to the parent assay. The EC50 value is approximately 14 μg / ml. The relatively low affinity of these trispecific antibodies for T cells reduces the probability of non-specific T cell activation to some extent, suggesting that these trispecific antibodies have better safety.

[0266] In summary, the trispecific antibody of this invention exhibits excellent affinity for both target cells and T cells, and has the potential for further development.

[0267] Example 6. Trispecific antibodies mediate efficient killing of tumor cells and human primary B cells by T cells.

[0268] Referring to the method in Example 1, the killing effect of the trispecific antibody of the present invention on tumor cell lines: Raji cells and U266 cells (Chinese Academy of Sciences Cell Bank) and human primary B cells was evaluated. The basic steps are as follows: Cell co-culture. The concentration of Raji cells or U266 cells was adjusted to 5 × 10⁻⁶ cells / year. 5 / ml, then seed Raji cells or U266 cells at 50µl per well into 96-well plates and incubate temporarily at 37°C. Adjust the human PBMCs cell concentration to 25×10⁶. 5 / ml, then seed human PBMCs cells at 50µl per well into 96-well plates and temporarily incubate at 37°C. The final ratio of human PBMCs cells to tumor cells is 5:1. The trispecific antibody was diluted five-fold to eight concentration gradients using 1640 complete medium: 20nM, 4nM, 800pM, 160pM, 32pM, 6.4pM, 1.28pM, and 0.26pM. Then, 100µl of each of the eight diluted antibody gradients was added to each well of a 96-well plate containing tumor cells and PBMCs cells. The dilution ratio was 1:1. After thorough mixing, the plates were centrifuged at 500rpm for 3 minutes. The cells were then incubated at 37°C for 48 hours.

[0269] Antibody staining: Anti-CD19 antibody (Biolegend, catalog number: 302207) and anti-CD137 antibody (Biolegend, catalog number: 309809) were added to culture plates containing Raji cells at the concentrations recommended by the supplier to label Raji cells and detect T cell activation. Anti-CD138 antibody (Biolegend, catalog number: 987002) and anti-CD137 antibody (Biolegend, catalog number: 309809) were added to culture plates containing U266 cells at the concentrations recommended by the supplier to label U266 cells and detect T cell activation. After mixing by pipetting, the plates were incubated at 4°C in the dark for 30 min.

[0270] Antibody washing: Add 1 ml wash buffer (1xPBS + 0.5% FBS) to the incubated antibody-cell mixture, centrifuge at 2000 rpm for 5 min and discard the supernatant;

[0271] Flow cytometry: Add 200 μL of wash buffer to each tube for resuspending, transfer to flow cytometry tubes, and perform analysis using a Beckman CytoFLEX flow cytometer.

[0272] Statistical analysis showed that the trispecific antibody of this invention exhibited similar Raji cell killing activity to the parent antibody in Example 1, with an EC50 value of approximately 45 pM. (See [link]). Figure 7 In section A, the trispecific antibody of this invention exhibits similar killing activity against human primary B cells to the maternal antibody in Example 1, with an EC50 value of approximately 55 pM. Figure 7In the study, the activation of T cells by trispecific antibodies mediated by target cells was analyzed using the T cell activation marker CD137. The results showed that after the addition of the trispecific antibody, T cells highly expressed CD137, and the expression abundance was positively correlated with target cell killing. (See [reference needed]). Figure 8 Therefore, all three specific antibodies of this invention can mediate the activation of T cells by target cells.

[0273] The three specific antibodies of this invention showed significant differences in their killing effect on U266 cells. The results are as follows: Figure 9 As shown, after the addition of the trispecific antibody, the U266 cell population ( Figure 9 Since the A (CD138+ labeled) components were cleared, all three specific antibodies of this invention were able to mediate effective T cell killing of U266 cells. Statistical analysis showed that the EC50 of monovalent BCMA-bound trispecific antibody molecules (LL206-TA103, LL206-TA104, LL206-TA105, and LL206-TA106) against U266 cells was approximately 350 pM. The EC50 of bivalent BCMA-bound trispecific antibody molecules (LL206-TA203, LL206-TA204, LL206-TA205, and LL206-TA206) ​​linked to the heavy chain against U266 cells was approximately 69 pM. The bivalent BCMA linked to the light chain binds to trispecific antibody molecules: LL206-TA303, LL206-TA304, LL206-TA305, and LL206-TA306, with an EC50 of approximately 212 pM against U266 cells. (See...) Figure 9 Therefore, all three specific antibodies of this invention can mediate efficient killing of U266 target cells by T cells. Among them, the bivalent molecules linking the heavy chain: LL206-TA203, LL206-TA204, LL206-TA205, and LL206-TA206 have the most significant killing effect on U266 cells, and therefore these three specific antibody molecules have the potential to have better clinical activity.

[0274] Example 7. Safety evaluation of trispecific antibodies

[0275] Referring to the method in Example 1, the direct activation effect of the trispecific antibody of the present invention on human primary T cells in the absence of target cells was evaluated. The basic steps are as follows:

[0276] 1. Adjust the concentration of primary human T cells to 25 × 10⁻⁶. 5 / ml, and then seed human T cells into 96-well plates at 100ul per well, and temporarily incubate at 37°C.

[0277] 2. Using 1640 complete medium, dilute the trispecific antibody to six concentration gradients at a 5-fold dilution ratio: 12ug / ml, 2.4ug / ml, 0.48ug / ml, 0.0816ug / ml, 0.01632ug / ml, and 0.00232ug / ml. Then, add 100µl of each of the six diluted antibody concentrations to a 96-well plate containing T cells. The dilution ratio is 1:1. After thorough mixing, centrifuge at 500 rpm for 3 minutes. Incubate the cells at 37°C for 24 hours.

[0278] 3. Antibody staining: Anti-human CD137 antibody (Biolegend, catalog number: 309809) was added to the culture plate containing T cells at the concentration recommended by the supplier to detect T cell activation. After mixing by pipetting, the plate was incubated at 4°C in the dark for 30 min.

[0279] 4. Antibody washing: After centrifuging the incubated antibody-cell mixture (1600 rpm, 5 min) to remove the supernatant, add 1 ml wash buffer (1xPBS + 0.5% FBS), centrifuge again (1600 rpm, 5 min), and discard the supernatant.

[0280] 5. Detection on flow cytometer: Add 200ul of wash buffer to each tube for resuspending, transfer to flow cytometer tubes, and perform detection using a Beckman CytoFLEX flow cytometer.

[0281] After the addition of the three specific antibodies of this invention, CD137 was not expressed, indicating that T cells could not be nonspecifically activated. The results are as follows: Figure 10 As shown, even at the highest concentration (12 ug / ml), T cells were not activated. Therefore, the trispecific antibody of this invention cannot activate human primary T cells in the absence of target cells, indicating that the trispecific antibody of this invention has good safety.

[0282] Example 8: In vivo efficacy evaluation of the trispecific antibody

[0283] This invention uses transgenic humanized mice (Biocytogen, B-hCD3EDG / hCD20 / hBCMA mice) to evaluate the in vivo activity of a trispecific antibody. First, mice were divided into groups of three: a solvent control group and four drug test groups: LL206-TA203, LL206-TA204, LL206-TA205, and LL206-TA206. On day 1, the trispecific antibody of this invention was administered via tail vein at a dose of 10 μg / mouse. On days -2, 4, and 7, 50 μL of peripheral blood was collected, and the proportion and number of peripheral blood B cells (CD19-positive cells) were evaluated by flow cytometry. The study found that the trispecific antibody of this invention could efficiently remove peripheral blood B cells in mice on days 4 and 7. These results suggest that the trispecific antibody of this invention has significant clinical value in the treatment of autoimmune diseases and tumors.

[0284] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. An anti-BCMA single domain antibody, the VHH chain of the single domain antibody has the following CDRs: CDR1 as shown in SEQ ID NO: 20, CDR2 as shown in SEQ ID NO: 21, and CDR3 as shown in SEQ ID NO:

22.

2. The single domain antibody of claim 1, wherein, The amino acid sequence of the VHH chain of the single domain antibody is as shown in SEQ ID NO:

30.

3. A multispecific antibody comprising the anti-BCMA single domain antibody of claim 1.

4. The multispecific antibody of claim 3, wherein The multispecific antibody is a trispecific antibody, which comprises: 1) a CD20-targeting binding domain comprising one or more anti-CD20 antibodies or antigen binding fragments; 2) a BCMA-targeting binding domain comprising one or more of the anti-BCMA single domain antibodies; and 3) a CD3-targeting binding domain comprising one or more anti-CD3 antibodies or antigen binding fragments.

5. The multispecific antibody of claim 4, wherein The trispecific antibody comprises a heavy chain 1, a heavy chain 2, and two identical light chains, wherein: The heavy chain 1 comprises a heavy chain of an anti-CD20 antibody and the anti-BCMA single domain antibody; the heavy chain 2 comprises a heavy chain of an anti-CD3 antibody and the anti-BCMA single domain antibody; and the light chain comprises a light chain of an anti-CD20 antibody; or The heavy chain 1 comprises a heavy chain of an anti-CD20 antibody; the heavy chain 2 comprises a heavy chain of an anti-CD3 antibody; and the light chain comprises a light chain of an anti-CD20 antibody and the anti-BCMA single domain antibody; or The heavy chain 1 comprises a heavy chain of an anti-CD20 antibody; the heavy chain 2 comprises a heavy chain of an anti-CD3 antibody and the anti-BCMA single domain antibody; and the light chain comprises a light chain of an anti-CD20 antibody.

6. The multispecific antibody of claim 5, wherein The multispecific antibody consists of the heavy chain 1, the heavy chain 2, and the light chain as shown in any one of the following (1)-(3): (1) the heavy chain 1 as shown in SEQ ID NO: 41, the heavy chain 2 as shown in SEQ ID NO: 47, and the light chain as shown in SEQ ID NO: 33; (2) the heavy chain 1 as shown in SEQ ID NO: 31, the heavy chain 2 as shown in SEQ ID NO: 32, and the light chain as shown in SEQ ID NO: 53; (3) the heavy chain 1 as shown in SEQ ID NO: 31, the heavy chain 2 as shown in SEQ ID NO: 47, and the light chain as shown in SEQ ID NO:

33.

7. A polynucleotide encoding a protein selected from the group consisting of the anti-BCMA single domain antibody of claim 1, or the multispecific antibody of claim 3, or a combination thereof.

8. An expression vector containing the polynucleotide of claim 7.

9. A host cell containing the expression vector of claim 8, or having integrated into its genome the polynucleotide of claim 7.

10. A method of producing the anti-BCMA single domain antibody of claim 1 or the multispecific antibody of claim 3, comprising the steps of: ​ (a) culturing the host cell of claim 9 under conditions suitable for production of an antibody, thereby obtaining a culture comprising the anti-BCMA single domain antibody or multispecific antibody; and (b) isolating or recovering the anti-BCMA single domain antibody or multispecific antibody from the culture; and (c) optionally, purifying and / or modifying the anti-BCMA single domain antibody or multispecific antibody obtained in step (b).

11. An immunoconjugate comprising: (a) the anti-BCMA single domain antibody of claim 1, or the multispecific antibody of claim 3; and (b) a conjugate moiety detectable label conjugated to the single domain antibody moiety.

12. A pharmaceutical composition comprising: (i) the anti-BCMA single domain antibody of claim 1, or the multispecific antibody of claim 3, or the immunoconjugate of claim 11, or a combination thereof; and (ii) a pharmaceutically acceptable carrier.

13. Use of an active ingredient selected from the group consisting of the anti-BCMA single domain antibody of claim 1, or the multispecific antibody of claim 3, or the immunoconjugate of claim 11, or a combination thereof, for (a) the manufacture of a detection reagent, a detection plate or a kit; and / or (b) the manufacture of a medicament for the prevention and / or treatment of a disease; wherein, the disease being multiple myeloma.

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