Trispecific antibodies binding cd3, cd19 and bcma and uses thereof

Abstract

The application discloses a trispecific antibody combining CD3, CD19 and BCMA and application thereof. The trispecific antibody comprises a first antigen binding domain combining CD3, a second antigen binding domain combining CD19, a third antigen binding domain combining BCMA and a heterodimeric Fc region. The application recruits and activates T cells through the CD3 binding domain, and realizes broad-spectrum recognition and combination of the whole abnormal B cell lineage from early B cells to terminally differentiated plasma cells through the CD19 and BCMA double binding domains, can more thoroughly eliminate pathological B cells, overcomes the antigen escape or drug resistance problems possibly existing in single target treatment by using multiple synergies, and provides a novel antibody drug for diseases such as multiple myeloma, B cell lymphoma and B cell related autoimmune diseases.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology and antibody engineering, and relates to a trispecific antibody that binds CD3, CD19 and BCMA and its application. More specifically, it relates to an antibody against B cell maturation antigen (BCMA) and a trispecific antibody that simultaneously targets CD3, CD19 and BCMA and its application. Background Technology

[0002] B cells play a crucial role in the immune response. Their development begins with pre-B cells in the bone marrow, progressing through different stages before finally differentiating into plasma cells. When B cells undergo malignant mutations at a certain developmental stage, they can lead to various tumors, such as multiple myeloma (MM), B-cell lymphoma, and leukemia. Furthermore, in autoimmune diseases such as systemic lupus erythematosus (SLE), autoreactive B cells survive and differentiate, producing pathogenic autoantibodies that cause tissue damage. Currently, B-cell targeted therapy mainly focuses on eliminating abnormal B cells by blocking the BAFF / APRIL signaling pathway or using monoclonal antibodies against B cell surface antigens (such as CD20, CD19, and BCMA).

[0003] CD19 is widely expressed throughout the entire developmental process of B cells (except for the terminal plasma cell stage) and is an important target for B-cell malignancies and autoimmune diseases. However, tumor cells can escape CD19-targeted therapy by downregulating or deleting antigens, and this strategy has limited effectiveness against malignant plasma cells with low or no CD19 expression (such as multiple myeloma cells).

[0004] BCMA is specifically expressed on the surface of both normal and malignant plasma cells and plays a crucial role in diseases such as multiple myeloma, making it an important therapeutic target. However, BCMA is not expressed on early-stage B cells and carries the risk of drug resistance due to soluble shedding and antigen downregulation. Therefore, targeting BCMA alone is insufficient to eliminate early-stage abnormal B cells and minimal residual disease.

[0005] Given the high heterogeneity of abnormal cells in B-cell lineage tumors and autoimmune diseases, single-target strategies are insufficient for complete eradication. Therefore, there is an urgent need to develop novel therapeutic strategies that can broadly cover the entire spectrum of abnormal B cells, achieve deep eradication, and have synergistic effects, in order to improve efficacy and reduce recurrence. Summary of the Invention

[0006] To address the shortcomings of existing technologies and practical needs, this invention provides a trispecific antibody combining CD3, CD19, and BCMA, and its applications. The design of this trispecific antibody is based on the widespread expression of CD19 during B cell development and the specific high expression of BCMA on terminally differentiated plasma cells. By simultaneously targeting these two antigens, it can achieve comprehensive coverage of abnormal cells at all stages from early B cells to mature plasma cells, thereby more thoroughly eliminating pathological B cell lineages. Therefore, this trispecific antibody holds promise for treating multiple myeloma, B-cell lymphoma, and B-cell-related autoimmune diseases. Furthermore, this antibody recruits T cells through the CD3 arm to exert its immune-killing effect, and the dual targeting of CD19 and BCMA enhances the specificity and breadth of its binding. This multi-faceted synergistic effect is expected to overcome single-target resistance and antigen escape, exhibiting superior B-cell clearance efficacy and therapeutic potential compared to traditional bispecific antibodies.

[0007] To achieve this objective, the present invention adopts the following technical solution: In a first aspect, the present invention provides a trispecific antibody that binds CD3, CD19 and BCMA, the trispecific antibody comprising a first antigen-binding domain capable of binding CD3, a second antigen-binding domain capable of binding CD19, a third antigen-binding domain capable of binding BCMA, and a heterodimer Fc region; the first antigen-binding domain and the second antigen-binding domain are paired to form a diabody structure, and the third antigen-binding domain is selected from any of the following structures: Fab structure, single-domain antibody VHH structure, and bivalent structure formed by Fab and VHH in tandem.

[0008] This invention addresses the limitations of existing single-target therapies and the urgent need for more effective treatment strategies by developing a trispecific antibody that simultaneously targets CD3 (recruits and activates T cells), CD19 (covers early to mature B cells), and BCMA (targets terminal plasma cells), possessing significant scientific and clinical value. This innovative design promises to achieve comprehensive recognition and efficient elimination of abnormal B cell lineages through multi-target synergy and T cell-mediated cytotoxicity. On one hand, the CD3 binding domain can recruit the patient's own T cells, directing and activating them to attack target cells; on the other hand, the dual binding domains of CD19 and BCMA ensure broad recognition of the entire abnormal B cell lineage, from early B cells to terminal plasma cells. This multi-targeting strategy not only improves the breadth and depth of treatment but also effectively reduces the risk of treatment failure due to the loss of a single antigen. Therefore, this trispecific antibody holds promise as a novel antibody drug with superior therapeutic potential for various diseases such as multiple myeloma, B-cell lymphoma, and B-cell-related autoimmune diseases, driving significant progress in the field of immunotherapy.

[0009] Preferably, the trispecific antibody consists of three chains having the following structure: 1) The first heavy chain, from the N-terminus to the C-terminus, is VL2-VH1-Hinge-CH2-CH3. 2) The first light chain, from the N-terminus to the C-terminus, is VL1-VH2, and... 3) The second chain, from the N end to the C end, is VHH-Hinge-CH2-CH3.

[0010] In this structure, VL2 is the light chain variable region of the second antigen-binding domain (CD19 antibody), VH1 is the heavy chain variable region of the first antigen-binding domain (CD3 antibody), Hinge is the hinge region, CH2 and CH3 are the heavy chain constant regions, VL1 is the light chain variable region of the first antigen-binding domain (CD3 antibody), VH2 is the heavy chain variable region of the second antigen-binding domain (CD19 antibody), and VHH is the heavy chain variable region of the third antigen-binding domain (BCMA antibody). VL2-VH1 and VL1-VH2 pair up to form a Diabody structure.

[0011] Preferably, the trispecific antibody consists of four chains having the following structure: 1) The first heavy chain, from the N-terminus to the C-terminus, is VL2-VH1-Hinge-CH2-CH3. 2) The first light chain, from the N-terminus to the C-terminus, is VL1-VH2. 3) The second chain, from the N-terminus to the C-terminus, is VH3-CH1-Hinge-CH2-CH3, and... 4) The second light chain, from the N end to the C end, is VL3-CL.

[0012] In this structure, VL2 is the light chain variable region of the second antigen-binding domain (CD19 antibody), VH1 is the heavy chain variable region of the first antigen-binding domain (CD3 antibody), Hinge is the hinge region, CH1, CH2 and CH3 are the heavy chain constant regions, CL is the light chain constant region, VL1 is the light chain variable region of the first antigen-binding domain (CD3 antibody), VH2 is the heavy chain variable region of the second antigen-binding domain (CD19 antibody), VH3 is the heavy chain variable region of the third antigen-binding domain (BCMA antibody), and VL3 is the light chain variable region of the third antigen-binding domain (BCMA antibody). VL2-VH1 and VL1-VH2 pair with each other to form a Diabody structure, and VH3-CH1 and VL3-CL pair with each other to form a Fab structure.

[0013] Preferably, the trispecific antibody consists of four chains having the following structure: 1) The first heavy chain, from the N-terminus to the C-terminus, is VL2-VH1-Hinge-CH2-CH3. 2) The first light chain, from the N-terminus to the C-terminus, is VL1-VH2. 3) The second chain, from the N-terminus to the C-terminus, is VHH-VH3-CH1-Hinge-CH2-CH3, and... 4) The second light chain, from the N end to the C end, is VL3-CL.

[0014] In this structure, VL2 is the light chain variable region of the second antigen-binding domain (CD19 antibody), VH1 is the heavy chain variable region of the first antigen-binding domain (CD3 antibody), Hinge is the hinge region, CH1, CH2, and CH3 are the heavy chain constant regions, CL is the light chain constant region, VL1 is the light chain variable region of the first antigen-binding domain (CD3 antibody), and VH2 is the heavy chain variable region of the second antigen-binding domain (CD19 antibody). VL2-VH1 and VL1-VH2 pair up to form a diabody structure. VHH and VH3 are the heavy chain variable regions of the third antigen-binding domain (BCMA antibody), and VL3 is the light chain variable region of the third antigen-binding domain (BCMA antibody). The single-domain antibody VHH is connected via (G4S). n (n=2, 3, 4) The linker is tandemly linked with Fab formed by VH3-CH1 and VL3-CL to form a BCMA bivalent binding domain of single-domain antibody plus Fab.

[0015] Preferably, the first antigen-binding domain capable of binding CD3 includes a CD3 antibody; the second antigen-binding domain capable of binding CD19 includes a CD19 antibody; and the third antigen-binding domain capable of binding BCMA includes a BCMA antibody.

[0016] Preferably, the BCMA antibody includes human BCMA antibody and camel-derived BCMA nanobody.

[0017] Preferably, the human BCMA antibody comprises a human BCMA antibody heavy chain and a human BCMA antibody light chain.

[0018] Preferably, the amino acid sequences of CDR1, CDR2, and CDR3 of the human BCMA antibody heavy chain are shown in SEQ ID No. 2, SEQ ID No. 3, and SEQ ID No. 4, respectively, and the amino acid sequences of CDR1, CDR2, and CDR3 of the light chain are shown in SEQ ID No. 6, SEQ ID No. 7, and SEQ ID No. 8, respectively; or, the amino acid sequences of CDR1, CDR2, and CDR3 of the human BCMA antibody heavy chain are shown in SEQ ID No. 10, SEQ ID No. 11, and SEQ ID No. 12, respectively, and the amino acid sequences of CDR1, CDR2, and CDR3 of the light chain are shown in SEQ ID No. 14, SEQ ID No. 15, and SEQ ID No. 16, respectively; or, the amino acid sequences of CDR1, CDR2, and CDR3 of the human BCMA antibody heavy chain are shown in SEQ ID No. 18, SEQ ID No. 19, and SEQ ID No. 20, respectively, and the amino acid sequences of CDR1, CDR2, and CDR3 of the light chain are shown in SEQ ID No. 6, SEQ ID No. 7, and SEQ ID No. 8, respectively. The amino acid sequences of CDR1, CDR2, and CDR3 of the human BCMA antibody heavy chain are shown in SEQ ID No. 22, SEQ ID No. 23, and SEQ ID No. 24, respectively; or, the amino acid sequences of CDR1, CDR2, and CDR3 of the light chain are shown in SEQ ID No. 26, SEQ ID No. 27, and SEQ ID No. 28, respectively, and the amino acid sequences of CDR1, CDR2, and CDR3 of the light chain are shown in SEQ ID No. 30, SEQ ID No. 31, and SEQ ID No. 32, respectively; or, the amino acid sequences of CDR1, CDR2, and CDR3 of the human BCMA antibody heavy chain are shown in SEQ ID No. 34, SEQ ID No. 35, and SEQ ID No. 36, respectively, and the amino acid sequences of CDR1, CDR2, and CDR3 of the light chain are shown in SEQ ID No. 38, SEQ ID No. 39, and SEQ ID No. 40, respectively; or, the amino acid sequences of CDR1, CDR2, and CDR3 of the human BCMA antibody heavy chain are shown in SEQ ID No. 42, SEQ ID No. 43, and SEQ ID No. 24, respectively. As shown in No. 44, the amino acid sequences of CDR1, CDR2, and CDR3 of the light chain are shown in SEQ ID No. 46, SEQ ID No. 47, and SEQ ID No. 48, respectively; or, the amino acid sequences of CDR1, CDR2, and CDR3 of the human BCMA antibody heavy chain are shown in SEQ ID No. 50, SEQ ID No. 51, and SEQ ID No. 52, respectively, and the amino acid sequences of CDR1, CDR2, and CDR3 of the light chain are shown in SEQ ID No. 54, SEQ ID No. 48, and SEQ ID No. 49, respectively.The amino acid sequences of CDR1, CDR2, and CDR3 of the human BCMA antibody heavy chain are shown in SEQ ID No. 55 and SEQ ID No. 56; or, the amino acid sequences of CDR1, CDR2, and CDR3 of the light chain are shown in SEQ ID No. 60, SEQ ID No. 58, SEQ ID No. 59, and SEQ ID No. 60, respectively, and the amino acid sequences of CDR1, CDR2, and CDR3 of the light chain are shown in SEQ ID No. 62, SEQ ID No. 63, and SEQ ID No. 64, respectively; or, the amino acid sequences of CDR1, CDR2, and CDR3 of the human BCMA antibody heavy chain are shown in SEQ ID No. 66, SEQ ID No. 67, and SEQ ID No. 68, respectively, and the amino acid sequences of CDR1, CDR2, and CDR3 of the light chain are shown in SEQ ID No. 70, SEQ ID No. 71, and SEQ ID No. 72, respectively; or, the amino acid sequences of CDR1, CDR2, and CDR3 of the human BCMA antibody heavy chain are shown in SEQ ID No. 74, SEQ ID No. 75, and SEQ ID No. 76, respectively. As shown in No. 76, the amino acid sequences of CDR1, CDR2, and CDR3 of the light chain are shown in SEQ ID No. 78, SEQ ID No. 79, and SEQ ID No. 80, respectively; or, the amino acid sequences of CDR1, CDR2, and CDR3 of the human BCMA antibody heavy chain are shown in SEQ ID No. 82, SEQ ID No. 83, and SEQ ID No. 84, respectively, and the amino acid sequences of CDR1, CDR2, and CDR3 of the light chain are shown in SEQ ID No. 86, SEQ ID No. 87, and SEQ ID No. 88, respectively.

[0019] Preferably, the camel-derived BCMA nanobody comprises a camel-derived BCMA nanobody heavy chain.

[0020] Preferably, the amino acid sequences of CDR1, CDR2, and CDR3 of the camel-derived BCMA nanobody heavy chain are as shown in SEQ ID No. 90, SEQ ID No. 91, and SEQ ID No. 92, respectively; or, the amino acid sequences of CDR1, CDR2, and CDR3 of the camel-derived BCMA nanobody heavy chain are as shown in SEQ ID No. 94, SEQ ID No. 95, and SEQ ID No. 96, respectively; or, the amino acid sequences of CDR1, CDR2, and CDR3 of the camel-derived BCMA nanobody heavy chain are as shown in SEQ ID No. 98, SEQ ID No. 99, and SEQ ID No. 100, respectively; or, the amino acid sequences of CDR1, CDR2, and CDR3 of the camel-derived BCMA nanobody heavy chain are as shown in SEQ ID No. 102, SEQ ID No. 103, and SEQ ID No. 104, respectively; or, the amino acid sequences of CDR1, CDR2, and CDR3 of the camel-derived BCMA nanobody heavy chain are as shown in SEQ ID No. 106, SEQ ID No. 98, SEQ ID No. 99, and SEQ ID No. 100, respectively. As shown in ID No. 107 and SEQ ID No. 108.

[0021] In this invention, BCMA antibodies are screened from human and camel-derived BCMA antibody libraries. The heavy chain variable region CDR1-3 and the light chain variable region CDR1-3 of the obtained antibody jointly determine the antibody's specific recognition and binding ability to the antigen. The BCMA antibody of this invention can specifically bind to the BCMA antigen with high affinity and has strong killing activity against BCMA-positive target cells, which has important application value in the fields of immunotherapy and cancer treatment.

[0022] Preferably, the heavy chain variable region of the human BCMA antibody has the amino acid sequence shown in SEQ ID No. 1, and the light chain variable region has the amino acid sequence shown in SEQ ID No. 5; or, the heavy chain variable region of the human BCMA antibody has the amino acid sequence shown in SEQ ID No. 9, and the light chain variable region has the amino acid sequence shown in SEQ ID No. 13; or, the heavy chain variable region of the human BCMA antibody has the amino acid sequence shown in SEQ ID No. 17, and the light chain variable region has the amino acid sequence shown in SEQ ID No. 21; or, the heavy chain variable region of the human BCMA antibody has the amino acid sequence shown in SEQ ID No. 25, and the light chain variable region has the amino acid sequence shown in SEQ ID No. 29; or, the heavy chain variable region of the human BCMA antibody has the amino acid sequence shown in SEQ ID No. 33, and the light chain variable region has the amino acid sequence shown in SEQ ID No. 37; or, the heavy chain variable region of the human BCMA antibody has the amino acid sequence shown in SEQ ID No. 41, and the light chain variable region has the amino acid sequence shown in SEQ ID No. 13. The amino acid sequence shown in SEQ ID No. 45; or, the heavy chain variable region of the human BCMA antibody has the amino acid sequence shown in SEQ ID No. 49, and the light chain variable region has the amino acid sequence shown in SEQ ID No. 53; or, the heavy chain variable region of the human BCMA antibody has the amino acid sequence shown in SEQ ID No. 57, and the light chain variable region has the amino acid sequence shown in SEQ ID No. 61; or, the heavy chain variable region of the human BCMA antibody has the amino acid sequence shown in SEQ ID No. 65, and the light chain variable region has the amino acid sequence shown in SEQ ID No. 69; or, the heavy chain variable region of the human BCMA antibody has the amino acid sequence shown in SEQ ID No. 73, and the light chain variable region has the amino acid sequence shown in SEQ ID No. 77; or, the heavy chain variable region of the human BCMA antibody has the amino acid sequence shown in SEQ ID No. 81, and the light chain variable region has the amino acid sequence shown in SEQ ID No. 85.

[0023] Preferably, the heavy chain variable region of the camel-derived BCMA nanobody has an amino acid sequence as shown in SEQ ID No. 89, SEQ ID No. 93, SEQ ID No. 97, SEQ ID No. 101 or SEQ ID No. 105; Preferably, the camel-derived BCMA nanobody comprises a humanized camel-derived BCMA nanobody; the humanized camel-derived BCMA nanobody comprises heavy chain VHH.

[0024] Preferably, the heavy chain variable region of the humanized camel-derived BCMA nanobody has an amino acid sequence as shown in SEQ ID No. 113 or SEQ ID No. 125.

[0025] Preferably, the first antigen-binding domain, the second antigen-binding domain, and the third antigen-binding domain are each composed of the following sequences: 1) A first antigen-binding domain, wherein the heavy chain variable region of the first antigen-binding domain has an amino acid sequence as shown in SEQ ID No. 109, and the light chain variable region has an amino acid sequence as shown in SEQ ID No. 110; 2) A second antigen-binding domain, wherein the heavy chain variable region of the second antigen-binding domain has the amino acid sequence shown in SEQ ID No. 111, and the light chain variable region has the amino acid sequence shown in SEQ ID No. 112; or, the heavy chain variable region of the second antigen-binding domain has the amino acid sequence shown in SEQ ID No. 121, and the light chain variable region has the amino acid sequence shown in SEQ ID No. 122; and 3) A third antigen-binding domain, wherein the heavy chain variable region of the third antigen-binding domain has an amino acid sequence as shown in SEQ ID No. 49 and the light chain variable region has an amino acid sequence as shown in SEQ ID No. 53; or, the heavy chain variable region of the third antigen-binding domain has an amino acid sequence as shown in SEQ ID No. 93, SEQ ID No. 113 or SEQ ID No. 125.

[0026] Preferably, the trispecific antibody comprises: a first heavy chain of the amino acid sequence shown in SEQ ID No. 114, a first light chain of the amino acid sequence shown in SEQ ID No. 115, and a second heavy chain of the amino acid sequence shown in SEQ ID No. 116; or, a first heavy chain of the amino acid sequence shown in SEQ ID No. 123, a first light chain of the amino acid sequence shown in SEQ ID No. 124, and a second heavy chain of the amino acid sequence shown in SEQ ID No. 116.

[0027] Preferably, the trispecific antibody comprises: a first heavy chain of the amino acid sequence shown in SEQ ID No. 114, a first light chain of the amino acid sequence shown in SEQ ID No. 115, a second heavy chain of the amino acid sequence shown in SEQ ID No. 120, and a second light chain of the amino acid sequence shown in SEQ ID No. 118; Alternatively, the first heavy chain of the amino acid sequence shown in SEQ ID No. 123, the first light chain of the amino acid sequence shown in SEQ ID No. 124, the second heavy chain of the amino acid sequence shown in SEQ ID No. 120, and the second light chain of the amino acid sequence shown in SEQ ID No. 118.

[0028] Preferably, the trispecific antibody comprises: a first heavy chain of the amino acid sequence shown in SEQ ID No. 114, a first light chain of the amino acid sequence shown in SEQ ID No. 115, a second heavy chain of the amino acid sequence shown in SEQ ID No. 117, SEQ ID No. 126 or SEQ ID No. 127, and a second light chain of the amino acid sequence shown in SEQ ID No. 118; or, The first heavy chain of the amino acid sequence shown in SEQ ID No. 114, the first light chain of the amino acid sequence shown in SEQ ID No. 115, the second heavy chain of the amino acid sequence shown in SEQ ID No. 119, SEQ ID No. 128 or SEQ ID No. 129, and the second light chain of the amino acid sequence shown in SEQ ID No. 118; or, The first heavy chain of the amino acid sequence shown in SEQ ID No. 123, the first light chain of the amino acid sequence shown in SEQ ID No. 124, the second heavy chain of the amino acid sequence shown in SEQ ID No. 117, and the second light chain of the amino acid sequence shown in SEQ ID No. 118.

[0029] Preferably, the heavy chain FR2 of the second antigen-binding domain contains a G44C mutation, and the light chain FR4 contains a Q100C or G100C mutation (according to Kabat numbering).

[0030] Preferably, the Fc is derived from immunoglobulin IgG4 or IgG1.

[0031] Preferably, the CH3 of the first heavy chain contains P395D and P396D (according to EU numbering) mutations, and the CH3 of the second heavy chain contains P395K, P396K, and V397K (according to EU numbering) mutations.

[0032] Preferably, the CH2 of both the first and second heavy chains contains L234A and L235A mutations.

[0033] In a second aspect, the present invention provides a nucleic acid molecule that encodes the trispecific antibody that binds CD3, CD19 and BCMA as described in the first aspect.

[0034] Thirdly, the present invention provides an expression vector containing the nucleic acid molecule described in the second aspect.

[0035] Fourthly, the present invention provides an antibody conjugate comprising the trispecific antibody described in the first aspect, and a conjugated label.

[0036] Preferably, the marker includes any one or a combination of at least two of the following: cytotoxins, radioactive isotopes, fluorescent markers, luminescent substances, chromogenic substances, or enzymes.

[0037] Fifthly, the present invention provides a pharmaceutical composition comprising the trispecific antibody described in the first aspect or the antibody-drug conjugate described in the fourth aspect.

[0038] In a sixth aspect, the present invention provides the use of any one or a combination of at least two of the trispecific antibody combining CD3, CD19 and BCMA described in the first aspect, the antibody conjugate described in the fourth aspect, or the pharmaceutical composition described in the fifth aspect in the preparation of a medicament for the treatment of a disease.

[0039] Preferably, the disease is caused by B-cell lineage abnormalities, including any one or a combination of at least two of the following: multiple myeloma, Waldenström macroglobulinemia, light chain amyloidosis, B-cell lymphoma, leukemia, or autoimmune diseases.

[0040] Preferably, the B-cell lymphoma includes any one or a combination of at least two of the following: diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, or Burkitt lymphoma.

[0041] Preferably, the leukemia includes any one or a combination of at least two of the following: acute B-lymphoblastic leukemia, chronic lymphocytic leukemia, or B-prolymphoblastic leukemia.

[0042] Preferably, the autoimmune disease includes any one or a combination of at least two of the following: systemic lupus erythematosus, antineutrophil cytoplasmic antibody-associated vasculitis, pemphigus, rheumatoid arthritis, systemic sclerosis, myasthenia gravis, or Sjögren's syndrome.

[0043] Compared with the prior art, the present invention has the following beneficial effects: (1) The trispecific antibody that combines CD3, CD19 and BCMA described in this invention maintains the structure of natural IgG antibody or a similar structure, and therefore has stable physicochemical properties and a long antibody half-life.

[0044] (2) The trispecific antibody combining CD3, CD19 and BCMA described in this invention adopts a Diabody structure and introduces an additional pair of disulfide bonds between the heavy chain and the light chain of the antibody, which significantly enhances the stability of the antibody structure, solves the common mismatch problem between heavy chains and between heavy and light chains in the production process of multispecific antibodies, and achieves a high proportion of correct pairing, thereby improving the uniformity and stability of the product.

[0045] (3) The trispecific antibody that binds CD3, CD19 and BCMA described in this invention has a high binding affinity for CD19 positive tumor cells (e.g., Raji cells) and BCMA positive tumor cells (e.g., NCI-H929 cells), and can more effectively recognize and bind to CD19 or BCMA antigens on the surface of tumor cells.

[0046] (4) When the trispecific antibody combining CD3, CD19 and BCMA described in this invention is co-incubated with peripheral blood mononuclear cells (PBMCs) in vitro, it has a weak activating effect on T cells, a low level of cytokine release, and fewer toxic side effects, which is beneficial to improving clinical safety.

[0047] (5) The trispecific antibody combining CD3, CD19 and BCMA described in this invention has a stronger killing effect on CD19-positive Raji cells than the control antibody Blincyto; and the trispecific antibody has a stronger killing effect on BCMA-positive NCI-H929 cells than the control antibody Elranatamab.

[0048] (6) The trispecific antibody combining CD3, CD19 and BCMA described in this invention can promote the proliferation of T cells in humanized mice, effectively clear B cells in mice, and significantly reduce antibody production, suggesting that it has good therapeutic effects and application prospects in the treatment of B cell-related autoimmune diseases (such as systemic lupus erythematosus, antineutrophil cytoplasmic antibody-related vasculitis, pemphigus, rheumatoid arthritis, systemic sclerosis, myasthenia gravis, Sjögren's syndrome, etc.).

[0049] (7) The trispecific antibody combining CD3, CD19 and BCMA described in this invention showed significant tumor suppression effects in mouse tumor models, indicating that it has good therapeutic potential in treating B-cell-related tumors (such as diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, Burkitt lymphoma and leukemia with high CD19 expression, and multiple myeloma with high BCMA expression). Attached Figure Description

[0050] Figure 1AThe diagram shows the binding activity of human anti-BCMA antibodies 16F10, 14E5, 6G6, and 6G8 to BCMA antigen as detected by ELISA.

[0051] Figure 1B The diagram shows the binding activity of human anti-BCMA antibodies 13A8, 12A1, and 12C2 to BCMA antigen as detected by ELISA.

[0052] Figure 1C The graph shows the binding activity of human anti-BCMA antibodies 5A3 and 7E2 to BCMA antigen as detected by ELISA.

[0053] Figure 1D This is a graph showing the binding activity of human anti-BCMA antibodies 12H3 and 17B9 to BCMA antigen as detected by ELISA.

[0054] Figure 2A The diagram shows the binding activity of camel-derived anti-BCMA antibodies 5A5, 6B2, 5C2, and 6A3 to BCMA antigen as detected by ELISA.

[0055] Figure 2B This is a graph showing the binding activity of camel-derived anti-BCMA antibody 3E11 to BCMA antigen as detected by ELISA.

[0056] Figure 3 The graph shows the binding of human anti-BCMA antibodies 14E5, 12C2, 5A3, and 7E2 to 293T / BCMA cells by flow cytometry.

[0057] Figure 4 The graph shows the binding of camel-derived anti-BCMA antibodies 5A5, 5C2, 6A3, and 3E11 to 293T / BCMA cells by flow cytometry.

[0058] Figure 5A The results of PBMC killing of BCMA-positive 293T / BCMA cells mediated by human anti-BCMA antibodies 12A1, 7E2, 12C2, 5A3, and 14E5 are shown.

[0059] Figure 5B The graph shows the killing effect of human anti-BCMA antibodies 17B9 and 16E11 on PBMCs against BCMA-positive 293T / BCMA cells.

[0060] Figure 6 The results of the killing activity of PBMCs mediated by camel-derived BCMA nanobody against BCMA-positive 293T / BCMA cells are shown in the figure.

[0061] Figure 7A This is a schematic diagram of the CD3×CD19×BCMA(5A5) structure of the CD3×CD19×BCMA trispecific antibody.

[0062] Figure 7B This is a schematic diagram of the CD3×CD19×BCMA(14E5) structure of the CD3×CD19×BCMA trispecific antibody.

[0063] Figure 7C This is a schematic diagram of the CD3×CD19×BCMA(14E5+5A5) structure of the CD3×CD19×BCMA trispecific antibody.

[0064] Figure 8A The images show non-reduced and reduced SDS-PAGE electrophoresis images of CD3×CD19×BCMA trispecific antibodies. The samples in lanes 1 to 3 are CD3×CD19-1×BCMA(14E5+5A5), CD3×CD19-1×BCMA(5A5), and CD3×CD19-1×BCMA(14E5), respectively.

[0065] Figure 8B The images show the non-reduced and reduced SDS-PAGE electrophoresis images of the CD3×CD19×BCMA trispecific antibody. The samples in lanes 1 and 2 are CD3×CD19-1×BCMA(14E5+5A5) and CD3×CD19-1×BCMA(14E5+h5A5), respectively.

[0066] Figure 8C The images show non-reduced and reduced SDS-PAGE electrophoresis images of the CD3×CD19×BCMA trispecific antibody, where the sample in lane 1 is CD3×CD19-2×BCMA(14E5).

[0067] Figure 8D The images show non-reduced and reduced SDS-PAGE electrophoresis images of the CD3×CD19×BCMA trispecific antibody. The samples in lanes 1 and 2 are CD3×CD19-2×BCMA(5A5) and CD3×CD19-2×BCMA(14E5+5A5), respectively.

[0068] Figure 9A This is a diagram showing the binding activity of the CD3×CD19-1×BCMA trispecific antibody to BCMA.

[0069] Figure 9B This is a diagram showing the binding activity of the CD3×CD19-1×BCMA trispecific antibody against CD3.

[0070] Figure 9C This is a diagram showing the binding activity of camel-derived and humanized CD3×CD19-1×BCMA trispecific antibodies against BCMA.

[0071] Figure 9DThis is a diagram showing the binding activity of camel-derived and humanized CD3×CD19-1×BCMA to CD3.

[0072] Figure 9E This is a diagram showing the binding activity of camel-derived and humanized CD3×CD19-1×BCMA to cynomolgus monkey BCMA.

[0073] Figure 9F This is a diagram showing the binding activity of camel-derived and humanized CD3×CD19-1×BCMA to CD3 in cynomolgus monkeys.

[0074] Figure 9G This is a diagram showing the binding activity of CD3×CD19-2×BCMA to BCMA.

[0075] Figure 9H This is a diagram showing the binding activity of CD3×CD19-2×BCMA to CD3.

[0076] Figure 9I This is a diagram showing the binding activity of CD3×CD19-1×BCMA with different G4S linkers to BCMA.

[0077] Figure 9J This is a diagram showing the binding activity of CD3×CD19-1×BCMA with different G4S linkers to CD3.

[0078] Figure 10A This figure shows the binding of the CD3×CD19×BCMA trispecific antibody to CD19-positive Raji cells as detected by flow cytometry.

[0079] Figure 10B This is a flow cytometry diagram showing the binding of CD3×CD19-1×BCMA trispecific antibody to BCMA-positive NCI-H929 cells.

[0080] Figure 10C This is a flow cytometry diagram showing the binding of the CD3×CD19-1×BCMA trispecific antibody to CD3-positive Jurkat cells.

[0081] Figure 11A The diagram shows the activation effect of CD3×CD19-1×BCMA trispecific antibody on T cells under co-culture conditions with CD19-positive Raji cells.

[0082] Figure 11B The diagram shows the activation effect of CD3×CD19-1×BCMA trispecific antibody on T cells under co-culture conditions with CD19-positive Daudi cells.

[0083] Figure 11CThe diagram shows the activation effect of CD3×CD19-1×BCMA trispecific antibody on T cells under co-culture conditions with BCMA-positive NCI-H929 cells.

[0084] Figure 11D The diagram shows the activation effect of camel-derived and humanized CD3×CD19-1×BCMA trispecific antibodies on T cells under co-culture conditions with CD19-positive Raji cells.

[0085] Figure 11E The diagram shows the activation effect of camel-derived and humanized CD3×CD19-1×BCMA trispecific antibodies on T cells under co-culture conditions with BCMA-positive NCI-H929 cells.

[0086] Figure 11F The diagram shows the activation effect of the CD3×CD19-2×BCMA trispecific antibody on T cells under co-culture conditions with CD19-positive Raji cells.

[0087] Figure 11G The diagram shows the activation effect of the CD3×CD19-2×BCMA trispecific antibody on T cells under co-culture conditions with CD19-positive Daudi cells.

[0088] Figure 11H The diagram shows the activation effect of CD3×CD19-2×BCMA trispecific antibody on T cells under co-culture conditions with BCMA-positive NCI-H929 cells.

[0089] Figure 11I This diagram illustrates the activation effect of CD3×CD19-1×BCMA trispecific antibodies with different G4S linkers on T cells under co-culture conditions with BCMA-positive NCI-H929 cells.

[0090] Figure 12A For CD3×CD19-1×BCMA to Raji (CD19) + The kill activity map of ).

[0091] Figure 12B CD3×CD19-1×BCMA for NCI-H929 (BCMA) + The kill activity map of ).

[0092] Figure 12C For camel-derived and humanized CD3×CD19-1×BCMA, the pair of Raji (CD19) + The kill activity map of ).

[0093] Figure 12D For camel-derived and humanized CD3×CD19-1×BCMA to NCI-H929 (BCMA)+ The kill activity map of ).

[0094] Figure 12E For CD3×CD19-2×BCMA to Raji (CD19) + (Image of cell cytotoxic activity)

[0095] Figure 12F CD3×CD19-2×BCMA for NCI-H929 (BCMA) + The kill activity map of ).

[0096] Figure 12G For CD3×CD19-1×BCMA with different G4S linkers to NCI-H929 (BCMA) + The kill activity map of ).

[0097] Figure 13A The figure shows the IL-2 results of camel-derived and humanized CD3×CD19-1×BCMA trispecific antibodies.

[0098] Figure 13B The figure shows the IL-6 results of camel-derived and humanized CD3×CD19-1×BCMA trispecific antibodies.

[0099] Figure 13C The results of TNF-α assays using camel-derived and humanized CD3×CD19-1×BCMA trispecific antibodies are shown in the figure.

[0100] Figure 14 This diagram illustrates the in vitro clearance effect of CD3×CD19×BCMA trispecific antibodies on B cells in human PBMCs.

[0101] Figure 15A For trispecific antibodies against NCI-H929 (BCMA) + The results of the xenotransplantation model.

[0102] Figure 15B For trispecific antibodies against Raji (CD19) + The results of the xenotransplantation model.

[0103] Figure 16A The image shows the results of detecting trispecific antibodies in peripheral blood T cells of humanized mice.

[0104] Figure 16B The image shows the results of detecting trispecific antibodies in humanized mouse spleen T cells.

[0105] Figure 16C The figure shows the detection results of trispecific antibodies in the clearance of peripheral blood B cells in humanized mice.

[0106] Figure 16D The figure shows the detection results of trispecific antibodies in the clearance of B cells in the spleen of humanized mice.

[0107] Figure 16E The image shows the results of total IgG detection in humanized mouse plasma. Detailed Implementation

[0108] To further illustrate the technical means and effects of this invention, the following description, in conjunction with embodiments and accompanying drawings, provides a further explanation of the invention. It is understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it.

[0109] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0110] Unless otherwise defined, scientific and technical terms and their abbreviations used in conjunction with this invention shall have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. Some of the terms and abbreviations used in this invention are listed below.

[0111] Antibody: Ab; Immunoglobulin: Ig; Heavy chain: HC; Light chain: LC; Heavy chain variable domain: VH or VHH; Heavy chain constant domain (CH); Light chain variable domain (VL); Light chain constant domain (CL); Antigen-binding fragment (Fab); Hinge region; Framework region (FR); Fc region: fragment crystallizable region, Fc region; Monoclonal antibodies (mAbs) Antibody-dependent cell-mediated cytotoxicity (ADCC); Complement-dependent cytotoxicity (CDC); Natural killing cells (NK cells); Bispecific antibody: BsAb; Tri-specific antibodies (TsAb) T cell receptor (TCR); Complementarity determining region (CDR) refers to the antigen-complementary binding region of an antibody. Single-chain variable region antibody fragment (also known as single-chain antibody): scFv; Tumor inhibition rate: tumor growth inhibition (TGI); Burkitt lymphoma (BL) Systemic lupus erythematosus (SLE) Rheumatoid arthritis (RA) Myasthenia gravis, MG; Sjögren's syndrome (SS); Autoimmune diseases (ADs); B-cell activating factor (BAFF); A proliferation-inducing ligand (APRIL); B-cell maturation antigen (BCMA); Germinal center B cells (GC B cells); Soluble BCMA: soluble BCMA, sBCMA; Multiple myeloma (MM).

[0112] The operational procedures for molecular cloning, cell culture, protein purification, and animal model experiments described in this invention are standard procedures widely used in this field. Unless otherwise defined, the relevant terms used in this invention have the same meaning as commonly understood in this technical field.

[0113] The term "amino acid" refers to one of the 20 naturally occurring amino acids or any non-natural analogue that may be present at a specific defined position. The three-letter abbreviations for amino acids and the single-letter abbreviations for nucleotides used in this invention are the forms generally accepted in this technical field, and the single-letter abbreviations for amino acids are the forms recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Unless otherwise stated, the amino acid numbering of the antibody variable region described in this invention uses the coding scheme described by Kabat et al. in 1991, namely the "Kabat index" or "Kabat number" (Kabat, EA et al. Sequences of Proteins of Immunological Interest, 5th ed., NIH Publication No. 91-3242, Bethesda, MD.:1991). Unless otherwise stated, the amino acid numbering of the constant region of the antibody described in this invention uses the EU index (Edelman GM, et al. The Covalent Structure of an Entire γ G Immunoglobulin Molecule. Proc Natl Acad Sci USA 1969, 63:78-85.).

[0114] The term "amino acid mutation" as used in this invention refers to amino acid substitution, insertion, deletion, and modification in a polypeptide sequence, as well as any combination of such substitution, insertion, deletion, and modification. Substitution is the preferred amino acid modification described herein. In this invention, "amino acid substitution" or "replacement" refers to replacing an amino acid at a specific position in the parental polypeptide sequence with another amino acid. For example, C220S substitution refers to a mutant polypeptide with an amino acid substitution, wherein the aminocysteine ​​C at position 220 of the polypeptide has been replaced by the amino acid serine S. Amino acid mutations can be achieved through molecular cloning or chemical methods. Molecular cloning methods include PCR, site-directed mutagenesis, and whole-genome synthesis.

[0115] In this invention, singular terms include plurals and plural meanings include singular meanings. Unless otherwise specified, the nucleotide sequences described in this invention are arranged and written from left to right in the direction from the 5' end to the 3' end. Unless otherwise specified, the amino acid sequences described in this invention are arranged and written from left to right in the direction from the amino terminus (N-terminus) to the carboxyl terminus (C-terminus).

[0116] The terms "peptide chain," "polypeptide chain," and "protein" refer to molecules in which two or more amino acids are linked by peptide bonds, including natural proteins, artificial proteins, protein fragments, mutant proteins, fusion proteins, etc.

[0117] The term "domain" refers to a specific structural region in a biological macromolecule that has an independent function. A domain has an independent tertiary structure, and its function does not depend on the rest of the biological macromolecule. In this invention, the domain specifically refers to such regions in proteins, such as the VH domain of the heavy chain variable region and the VL domain of the light chain variable region. Domains can combine with each other to form a larger domain.

[0118] The term "antibody" refers to an immunoglobulin molecule that contains at least one antigen recognition site and can specifically bind to an antigen. Here, the term "antigen" refers to a substance in the body that can induce an immune response and specifically bind to an antibody, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, hapten, or a combination of the above. The binding of an antibody to an antigen is mediated by interactions formed between them, including hydrogen bonds, van der Waals forces, ionic bonds, and hydrophobic bonds. The term "antibody" as used in this invention includes monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, antibody fragments, multispecific antibodies (e.g., bispecific antibodies) containing at least two different epitope binding domains, human antibodies, humanized antibodies, post-translational modified antibodies, camel antibodies, chimeric antibodies, fusion proteins containing antibody antigenic determinants, and any other modified immunoglobulin molecules containing an antigen recognition site, provided that these antibodies exhibit the desired biological activity. Specifically, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules containing at least one antigen-binding site.

[0119] The terms “Fab,” “Fab region,” “Fab fragment,” or “Fab molecule” refer to antigen-binding fragments that contain the VH and CH1 domains of the immunoglobulin heavy chain and the VL and CL domains of the light chain. The first constant region domain CH1 of the heavy chain binds to the constant region domain CL of the light chain, and the variable region domain VH of the heavy chain binds to the variable region domain VL of the light chain.

[0120] The term "single-chain variable region antibody fragment" or "scFv" refers to a fusion protein of the variable region VH of the immunoglobulin heavy chain and the variable region VL of the light chain, including different combinations of N-terminal VH and N-terminal VL, which can be prepared using conventional molecular cloning methods for constructing recombinant proteins (Sambrook JF, EF et al. Molecular cloning: a laboratory manual. 4th ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York: 2012).

[0121] The terms "Fc," "Fc region," or "Fc fragment" refer to the effector region of an antibody, capable of inducing responses such as CDC, ADCC, ADCP, and cytokine release. Natural antibody Fcs are typically composed of two identical protein fragments, each containing two or three immunoglobulin constant domains. The Fc described in this invention includes both natural Fc and mutant Fc. Under experimental conditions, fragments generated from immunoglobulin monomers by papain cleavage are designated Fab and Fc, respectively.

[0122] The term "hinge" or "hinge region" refers to a flexible polypeptide containing amino acids between the first and second constant structural domains (CH1 and CH2) of an antibody.

[0123] The term "(G4S)" n "" refers to n sequences consisting of 4 glycine residues and 1 serine residue, where n is any integer from 1 to 10.

[0124] This invention further comprises nucleic acid sequences encoding these polypeptide chains. During antibody expression, the nucleic acid sequence is inserted into a suitable vector, including but not limited to: plasmids, phage expression vectors, Coase plasmids, artificial chromosomes, bacteriophages, and animal viruses. The expression vector contains elements for regulating expression, including but not limited to promoters, transcription initiation sequences, enhancers, and signal peptide sequences. Promoters include, but are not limited to, T7 promoters, T3 promoters, SP6 promoters, β-actin promoters, EF-1α promoters, CMV promoters, and SV40 promoters. The expression vector can be transferred into host cells using suitable methods known in the art, including but not limited to: calcium phosphate precipitation, liposome transfection, electroporation, and PEI (polyethyleneimine) transfection.

[0125] The term "monoclonal antibody" or "monoclonal antibody" refers to an antibody molecule that consists of a single molecule. Monoclonal antibodies have monovalent affinity and bind to the same epitope (the site at which the antibody recognizes the antigen).

[0126] The term "antigen binding site" refers to one or more amino acid residues that directly interact with an antigen in an antigen-binding molecule. The antigen binding site of an antibody is composed of the antigen complementarity-determining region (CDR). Natural immunoglobulin molecules typically contain two antigen binding sites, while Fab molecules typically contain one antigen binding site.

[0127] The terms “antigen complementarity-determining region (CDR)” or “hypervariant region (HVR)” refer to regions where the composition and sequence of amino acid residues at certain specific positions are highly variable.

[0128] The term "framework region (FR)" refers to the region in the V region other than the CDR. This part has a low amino acid substitution frequency, which helps stabilize the structure of the CDR. The CDR and FR in the variable region are arranged in the pattern "FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4".

[0129] The term "light chain" or "L-chain" refers to a polypeptide chain consisting of approximately 214 amino acid residues, with a molecular weight of about 24 kDa. Each light chain contains two cyclic peptides formed by intrachain disulfide bonds. L-chains can be classified into κ and λ types, and an antibody molecule can only contain one of these two types of light chains.

[0130] The term "heavy chain" or "H chain" refers to a polypeptide chain composed of approximately 450-550 amino acid residues, with a molecular weight of about 55 or 75 kDa. Based on differences in H chain antigenicity, they can be classified into five types: μ chain, γ chain, α chain, δ chain, and ε chain. Different H chains combined with L chains (κ or λ chains) to form complete Ig molecules are respectively called IgM, IgG, IgA, IgD, and IgE.

[0131] The term "CD19" refers to a transmembrane glycoprotein encoded by the CD19 gene located on chromosome 16p11.2, belonging to the immunoglobulin superfamily. CD19 is distributed on all B cells, malignant B cells such as hairy cell leukemia cells, and follicular dendritic cells. It is expressed at all stages of B cell maturation, but its expression is reduced or absent at the plasma cell stage.

[0132] The term "BCMA" refers to the B cell maturation antigen, also known as CD269 or tumor necrosis factor receptor superfamily member 17 (TNFRSF17), encoded by the TNFRSF17 gene located on chromosome 16p13.13, containing three exons. BCMA is highly expressed in mature B cells, plasma cells, and malignant plasma cells (such as multiple myeloma cells), but is almost not expressed in normal tissues. Unless explicitly specified, such as "cynomolgus monkey BCMA" or "mouse BCMA," "BCMA" as used in this invention refers to human BCMA.

[0133] The term "CD3" refers to differentiation antigen cluster-3 protein, a component of the T cell receptor complex (TCR) expressed on the surface of T cells. It is a homodimer composed of four polypeptide chains: CD3δ (human CD3δ UniProt Entry No. P0423), CD3ε (human CD3ε UniProt Entry No. P07766), CD3γ (human CD3γ UniProt Entry No. P09693), and CD3ζ (human CD3ζ UniProt Entry No. P20963). Unless explicitly specified, such as "cynomolgus monkey CD3" or "mouse CD3," "CD3" as used in this invention refers to human CD3.

[0134] The term "BAFF" refers to B cell activating factor, also known as B-lymphocyte stimulator (BLyS), a TNF superfamily cytokine encoded by the TNFSF13B gene. BAFF is mainly secreted by monocytes, macrophages, and dendritic cells, and is an essential signaling molecule for B cell survival and the transformation from transitioning B cells to mature B cells.

[0135] The term "APRIL" refers to proliferation-inducing ligand, a member of the tumor necrosis factor superfamily, also known as TNFSF13 or CD256. Encoded by the TNFSF13 gene, it regulates immune cell function by binding to specific receptors. This protein exists in a type II transmembrane form and, after enzymatic cleavage, forms a soluble active molecule, primarily involved in B lymphocyte proliferation and activation, antibody class switching, and plasma cell survival.

[0136] The term "sBCMA" refers to soluble B-cell maturation antigen, a soluble form formed by the shedding of the extracellular domain of BCMA. It is primarily released from the surface of plasma cells or multiple myeloma cells via γ-secretase-mediated proteolytic cleavage and enters the bloodstream. In diseases such as multiple myeloma, the high proliferative activity of tumor cells leads to a significant increase in BCMA expression and shedding, resulting in the accumulation of sBCMA levels in the blood. sBCMA consists of an extracellular domain and a portion of the transmembrane domain of BCMA. It not only reduces the density of the target antigen but also provides a soluble decoy, limiting the efficacy of currently developed anti-BCMA drugs.

[0137] The term "EC" 50 "The half-maximal effect concentration (50% of maximal effect) refers to the antibody concentration that produces a 50% maximum effect."

[0138] The term "TGI" stands for tumor growth inhibition rate, which is used to evaluate the inhibitory effect of a test drug on tumor growth in vivo (i.e., in animal experiments). TGI = (1 - tumor volume in the treatment group / tumor volume in the control group) × 100%.

[0139] This invention relates to pharmaceutical compositions comprising the antibodies or antibody fragments, bispecific antibodies or antibody-drug conjugates of the present invention, and optionally pharmaceutically acceptable carriers, surfactants, and / or diluents. In some embodiments, in addition to the antibodies, bispecific antibodies, or antibody-drug conjugates of the present invention, the pharmaceutical compositions further comprise one or more additional therapeutic agents. In some embodiments, the additional therapeutic agents include, but are not limited to, chemotherapeutic agents, growth inhibitors, cytotoxic agents, agents for radiotherapy, anti-angiogenic agents, apoptotic agents, anti-microtubule agents, and other agents for treating cancer.

[0140] The term "host cell" refers to the cell into which exogenous nucleic acid has been introduced and its progeny, which can express the exogenous polypeptide through nucleotide conversion or transfection encoding the polypeptide. The host cells described in this invention include, but are not limited to, 293F cells (human embryonic kidney cells), CHO cells (Chinese hamster ovary cells), BHK cells (Baby Hamster Kidney cells), myeloma cells, yeast, insect cells, or prokaryotic cells such as *Escherichia coli*. It should be noted that the term "host cell" in this invention refers not only to the cell into which exogenous nucleic acid has been introduced, but also to the progeny of that cell. While progeny cells may mutate during cell division, they still fall within the scope of the terminology used in this invention.

[0141] The term "ADs" stands for autoimmune diseases, which are disease states caused by the body's immune system's immune response against its own components. While an immune response to foreign antigens usually results in the clearance of those antigens, an immune response against antigens affecting one's own cells or tissues is less likely to be completely cleared by the immune system's effector cells and instead leads to continuous attack, resulting in a disease state. Based on the extent of involvement, ADs can be divided into organ-specific autoimmune diseases and systemic autoimmune diseases. Organ-specific autoimmune diseases mainly include myasthenia gravis, rheumatoid arthritis, and hyperthyroidism; systemic autoimmune diseases mainly include SLE, SS, systemic sclerosis, and polymyositis / dermatomyositis. Common treatment strategies include traditional immunosuppressants, targeted biologics, and symptomatic and supportive therapy.

[0142] The term "SLE" stands for systemic lupus erythematosus, an autoimmune disease characterized by the formation of pathogenic autoantibodies and immune complexes that mediate organ and tissue damage, primarily affecting women of reproductive age. The pathogenesis of SLE is complex, characterized by the involvement of multiple systems and immune abnormalities. A prominent feature of SLE is the production of autoantibodies, which form immune complexes that deposit at the vascular level, leading to multi-organ damage. The underlying mechanisms of autoantibody-induced tissue damage and systemic inflammation remain not fully understood. The disease can range from mild to severe, potentially life-threatening, and exhibits acute, subacute, and chronic progression patterns.

[0143] The term "RA" stands for rheumatoid arthritis, a systemic autoimmune disease characterized primarily by erosive arthritis. Clinically, it presents as symmetrical, persistent polyarthritis, mainly affecting small joints such as the hands and wrists. Pathologically, it involves chronic inflammation of the synovial membrane, pannus formation, and can lead to cartilage and bone destruction, ultimately resulting in joint deformities and loss of function. Its etiology is still under investigation, but research suggests it may be closely related to factors such as autoimmunity, genetics, and microbial infections.

[0144] The term "MG" stands for myasthenia gravis, an acquired autoimmune disease mediated by autoantibodies, dependent on cell-mediated immunity, and involving complement. Its core pathological change is impaired neuromuscular junction (NMJ) transmission. The core symptom is fluctuating skeletal muscle weakness, characterized by worsening with activity and relief with rest, and being more severe in the evening than in the morning. Commonly affected muscle groups include ocular muscles (ptosis, diplopia), facial muscles (masked face, weakness in chewing), pharyngeal muscles (dysphagia, hoarseness), and limb muscles (weakness in raising arms and legs). In severe cases, respiratory muscles may be affected, leading to myasthenic crisis and endangering life.

[0145] The term "SS" stands for Sjögren's syndrome, a chronic systemic autoimmune disease that primarily affects exocrine glands throughout the body. Its core symptoms are dry mouth and dry eyes due to impaired salivary and lacrimal gland function, and it can also affect internal organs and is accompanied by various positive autoantibodies. The disease is related to multiple factors, including infection, endocrine disorders, and genetics, but its exact cause remains unclear. Sjögren's syndrome is divided into primary and secondary types. Primary Sjögren's syndrome occurs when the patient does not have other connective tissue diseases that could trigger SS, such as rheumatoid arthritis or systemic lupus erythematosus. Secondary Sjögren's syndrome develops as a result of these underlying diseases.

[0146] Example 1: Preparation of BCMA antigen Based on the sequences of BCMA (Q02223) and cynomolgus monkey BCMA (A0A2K5UD97) from the UniProtKB database, the antigen sequences were cloned into the eukaryotic expression vector pcDNA3.1-TEV-cFc-His to construct recombinant eukaryotic expression plasmids hBCMA-TEV-cFc-His and cBCMA-TEV-cFc-His. The plasmids were transfected into 293F host cells using the PEI transfection method, and the cell supernatant was collected after 5 days of culture. The supernatant was purified by protein A affinity to obtain the BCMA-TEV-cFc-His fusion protein, which was then digested with TEV protease and purified by nickel column chromatography to obtain the BCMA and cynomolgus monkey BCMA antigens. The antigen sequences are as follows: BCMA antigen (SEQ ID No. 130): MLQMAGQCSQNEYFDSLLHACIPCQLRCSSNTPPLTCQRYCNASVTNSVKGTNAILWTCLGLSLIISLAVFVLMFLLRKINSEPLKDEFKNTGSGLLGMANIDLEKSRTGDEIILPRGLEYTVEECTCEDCIKSKPKVDSDHCFPLPAMEEGATILVTTKTNDYCKSLPAALSATEIEKSISAR.

[0147] BCMA antigen from cynomolgus monkeys (SEQ ID No. 131): MLQMARQCSQNEYFDSLLHDCKPCQLRCSSTPPLTCQRYCNASMTNSVKGMNAILWTCLGLSLIISLAVFVLTFLLRKMSSEPLKDEFKNTGSGLLGMANIDLEKGRTGDEIVLPRGLEYTVEECTCEDCIKNKPKVDSDHCFPLPAMEEGATILVTTKTNDYCNSLSAALSVTEIEKSISAR.

[0148] Example 2: Screening and preparation of human BCMA antibodies The human antibody library was released, packaged, and precipitated with phages. Screening was performed using BCMA antigen, and finally, TG1 strain was infected to obtain single clones with different sequences. Single clones were selected for phage-ELISA to obtain candidate clones. The candidate clone sequences are shown in Table 1 below. Table 1 Example 3: Generation and preparation of BCMA camel-derived antibodies 1. Immunization of camels with BCMA antigen The purified BCMA antigen was inoculated into camels in multiple immunizations, with each immunization spaced one week apart. The first immunization used Freund's complete adjuvant to emulsify the antigen, while subsequent immunizations used Freund's incomplete adjuvant. Serum titers were verified after immunization.

[0149] 2. Obtaining camel-derived anti-BCMA antibodies Peripheral blood was collected from camels, mononuclear cells were isolated, total RNA was extracted, and cDNA was obtained using a reverse transcription kit. Using this cDNA as a template, specific primers were designed for PCR to amplify the VHH fragment, obtaining the camel-derived VHH gene. The scFv gene was cloned into the pITa phage vector via restriction enzyme digestion and ligation, and transformed into host cells (TG1 Electrocompetent Cells: Lucigen.) to obtain a camel-derived VHH phage library.

[0150] After culturing, packaging, releasing, and purifying the camel-derived VHH phage library, positive clones were enriched through multiple rounds of screening using the BCMA extracellular region antigen. Following sequencing and functional verification, five camel-derived BCMA nanobodies were finally obtained. Their heavy chain variable region sequences are shown in Table 2 below.

[0151] Table 2 Example 4: Expression and purification of anti-BCMA antibody All anti-BCMA antibodies involved in this embodiment were expressed using transient transfection. First, the sequences of candidate antibodies were cloned into a pcDNA vector to prepare expression plasmids. The plasmids were transfected into 293F cells using PEI transfection, and the cell supernatant was collected after 6 days of culture. The supernatant was purified by protein A affinity to obtain anti-BCMA antibodies. An appropriate amount of the purified product was analyzed and identified by SDS-PAGE, while the remaining samples were stored at -80 °C.

[0152] Example 5: ELISA method for detecting the binding activity of candidate anti-BCMA antibodies to BCMA antigen BCMA antigen was diluted with NaHCO3 buffer and then coated and blocked. The sample was serially diluted with PBS buffer, a total of 8 dilutions. The diluted samples were added to the blocked ELISA plate. After washing, HRP-labeled secondary antibody was added. After TMB color development, the reaction was terminated with stop solution, and the absorbance (OD) at 450 nm was measured using an ELISA reader. 450 value).

[0153] Plot a dose-response curve with the logarithm of sample concentration on the X-axis and the absorbance OD value on the Y-axis, and calculate EC. 50 EC 50 This represents the activity of the sample in binding to the BCMA antigen. The experiment included a positive control anti-BCMA monoclonal antibody (sequence derived from the BCMA-binding arm of Elranatamab, denoted as BCMA PC Ab (positive control antibody)) and a negative control hIgG.

[0154] The results are as follows Figures 1A-1D and Figures 2A-2B As shown, all anti-BCMA antibodies can bind to BCMA. Among them, the human anti-BCMA antibody 14E5 and the camel-derived anti-BCMA nanobody 5A5 exhibit the strongest binding activity to BCMA.

[0155] Example 6: Flow cytometry detection of the binding activity of candidate anti-BCMA antibodies to BCMA-positive 293T / BCMA cells To further verify the binding activity of the candidate anti-BCMA antibody to the BCMA functional region, this invention constructed 293T / BCMA cells that stably express BCMA, and used flow cytometry to detect the binding activity of the candidate antibody to the 293T / BCMA cells.

[0156] The BCMA gene was cloned into a lentiviral vector and transfected into 293T cells. Stable 293T / BCMA cell lines were obtained through pressure selection. 293T and 293T / BCMA cells were added to 96-well culture plates and incubated with candidate antibodies, positive control samples, and negative control samples, respectively. After washing, secondary antibodies were added and incubated. After washing again, the cells were resuspended and analyzed by flow cytometry.

[0157] The results are as follows Figure 3 and Figure 4 As shown, anti-BCMA antibodies can bind to 293T / BCMA cells but not to 293T cells. This indicates that anti-BCMA antibodies have high binding activity and specific binding to 293T / BCMA. Among them, human anti-BCMA antibody 14E5 and camel-derived anti-BCMA nanobody 5A5 exhibit the strongest binding activity to BCMA.

[0158] Example 7: In vitro cytotoxicity assay mediated by candidate anti-BCMA antibody In this embodiment, 293T / BCMA cells were used as target cells and human PBMCs were used as effector cells.

[0159] The anti-BCMA antibody was serially diluted with PBS buffer. 293T / BCMA cells, PBMCs, and the serially diluted antibody samples were added sequentially to cell culture plates. After mixing, the 96-well plates were incubated at 37°C in a 5% CO2 incubator. The following day, the absorbance (OD) was measured using a lactate dehydrogenase cytotoxicity assay kit. 490 The logarithm of the antibody working concentration was used to calculate the relative kill value. A dose-response curve was plotted with the logarithm of the antibody working concentration on the X-axis and the relative kill value on the Y-axis, and the half-maximal effective concentration (EC50) was calculated. 50 ), with EC 50 The value represents the cytotoxic activity of the BCMA monoclonal antibody against 293T / BCMA cells.

[0160] The results are as follows Figures 5A-5B and Figure 6 As shown, the candidate anti-BCMA antibodies of the present invention all exhibit good killing effects on 293T / BCMA cells. Among them, the human BCMA antibody 14E5 and the camel-derived BCMA nanobody 5A5 both showed stronger killing activity than the positive control (BCMA PC Ab).

[0161] Example 8: Preparation of CD3×CD19×BCMA trispecific antibody (1) Sequence of CD3×CD19×BCMA trispecific antibody The Fc region of the antibody was modified according to the method disclosed in the applicant's PCT patent (WO2017034770A1). Specifically, the following mutations were introduced into the CH3 domain of the Fc region of the anti-CD19 and CD3 heavy chains: P395D and P396D (according to EU numbers), with the mutation marker OB and a negative charge; the following mutations were introduced into the CH3 domain of the Fc region of the anti-BCMA heavy chain: P395K, P396K, and V397K (according to EU numbers), with the mutation marker OA and a positive charge; to reduce the binding of Fc to its receptor FcγR, the L234A and L235A mutations were introduced into the CH2 domain of the Fc region of both heavy chains.

[0162] CD3 antibody: The heavy chain variable region sequence and the optimized light chain variable region sequence of the anti-CD3 antibody were selected from the applicant's authorized patent (CN110551221A). The amino acid sequence of the anti-CD3 heavy chain variable region is shown in SEQ ID No. 109, and the amino acid sequence of the anti-CD3 light chain variable region is shown in SEQ ID No. 110.

[0163] CD19 Antibodies: Two different CD19 antibodies were selected. The first was derived from the classic mouse anti-human CD19 monoclonal antibody BU12, which was humanized to become a humanized CD19 antibody, named CD19-1. Its antibody heavy chain variable region has the amino acid sequence shown in SEQ ID No. 111, and its light chain variable region has the amino acid sequence shown in SEQ ID No. 112. The second was Blincyto... ® The CD19 antibody used in this study was humanized to become a humanized CD19 antibody, named CD19-2. Its antibody heavy chain variable region has the amino acid sequence shown in SEQ ID No. 121, and the CD19-2 antibody light chain variable region has the amino acid sequence shown in SEQ ID No. 122.

[0164] BCMA antibodies: Two BCMA antibodies from different sources prepared above were selected. The first is the human anti-BCMA antibody 14E5; the second is the camel-derived anti-BCMA nanobody 5A5 and its humanized modified antibodies h5A5-1 or h5A5-2. BCMA-14E5 and BCMA-5A5 / h5A5 can be used to enhance binding or design bivalent binding structures. Specifically, the heavy chain variable region of BCMA-14E5 has the amino acid sequence shown in SEQ ID No. 49, and the light chain variable region of BCMA-14E5 has the amino acid sequence shown in SEQ ID No. 53; the heavy chain variable region of BCMA-5A5 has the amino acid sequence shown in SEQ ID No. 93; the heavy chain variable region of BCMA-h5A5-1 has the amino acid sequence shown in SEQ ID No. 113; and the heavy chain variable region of BCMA-h5A5-2 has the amino acid sequence shown in SEQ ID No. 125.

[0165] The sequences of the variable regions of the antibodies are shown in Table 3. In the two CD19 antibody heavy chain variable region sequences, the underlined regions are all FR2 regions, and the underlined regions are all FR4 regions.

[0166] Table 3 Based on the selected antibody sequences, three different CD3×CD19×BCMA trispecific antibody molecules were constructed. Each trispecific antibody includes a first antigen-binding domain that binds to CD3, a second antigen-binding domain that binds to CD19, a third antigen-binding domain that binds to BCMA, and a heterodimeric Fc region. The first and second antigen-binding domains pair to form a diabody structure, and the third antigen-binding domain has a Fab structure, a single-domain antibody VHH structure, and a bivalent structure formed by the tandem formation of Fab and VHH. A schematic diagram of the specific structures is shown below. Figures 7A-7C As shown.

[0167] The specific designs of the three structures are as follows: The first type is a three-chain structure ( Figure 7A It consists of the following three chains: 1) The first heavy chain, from the N-terminus to the C-terminus, is VL2-VH1-Hinge-CH2-CH3. 2) The first light chain, from the N-terminus to the C-terminus, is VL1-VH2, and... 3) The second chain, from the N end to the C end, is VHH-Hinge-CH2-CH3.

[0168] The above-mentioned VL2 is the light chain variable region of the second antigen-binding domain (CD19 antibody), and VH1 is the heavy chain variable region of the first antigen-binding domain (CD3 antibody). Hinge is the hinge region, CH2 and CH3 are the heavy chain constant regions. VL1 is the light chain variable region of the first antigen-binding domain (CD3 antibody), VH2 is the heavy chain variable region of the second antigen-binding domain (CD19 antibody), and VHH is the heavy chain variable region of the third antigen-binding domain (BCMA antibody). VL2-VH1 and VL1-VH2 pair up to form a Diabody structure.

[0169] Based on the above structure, the trispecific antibody is named CD3×CD19-1×BCMA(5A5), with its first heavy chain having the amino acid sequence shown in SEQ ID No. 114, its first light chain having the amino acid sequence shown in SEQ ID No. 115, and its second heavy chain having the amino acid sequence shown in SEQ ID No. 116. Another trispecific antibody is named CD3×CD19-2×BCMA(5A5), whose first heavy chain has the amino acid sequence shown in SEQ ID No. 123, the first light chain has the amino acid sequence shown in SEQ ID No. 124, and the second heavy chain has the amino acid sequence shown in SEQ ID No. 116.

[0170] The second type is a four-chain structure ( Figure 7B It consists of the following four chains: 1) the first heavy chain, from N to C end: VL2-VH1-Hinge-CH2-CH3; 2) the first light chain, from N to C end: VL1-VH2; 3) the second heavy chain, from N to C end: VH3-CH1-Hinge-CH2-CH3; and... 4) The second light chain, from the N end to the C end, is VL3-CL.

[0171] The above-mentioned VL2 is the light chain variable region of the second antigen-binding domain (CD19 antibody), VH1 is the heavy chain variable region of the first antigen-binding domain (CD3 antibody), Hinge is the hinge region, CH1, CH2 and CH3 are the heavy chain constant regions, CL is the light chain constant region, VL1 is the light chain variable region of the first antigen-binding domain (CD3 antibody), VH2 is the heavy chain variable region of the second antigen-binding domain (CD19 antibody), VH3 is the heavy chain variable region of the third antigen-binding domain (BCMA antibody), and VL3 is the light chain variable region of the third antigen-binding domain (BCMA antibody). VL2-VH1 and VL1-VH2 pair with each other to form a Diabody structure, and VH3-CH1 and VL3-CL pair with each other to form a Fab structure.

[0172] Based on the above structure, the trispecific antibody is named CD3×CD19-1×BCMA(14E5), whose first heavy chain has the amino acid sequence shown in SEQ ID No. 114, the first light chain has the amino acid sequence shown in SEQ ID No. 115, the second heavy chain has the amino acid sequence shown in SEQ ID No. 120, and the second light chain has the amino acid sequence shown in SEQ ID No. 118. Another trispecific antibody is named CD3×CD19-2×BCMA(14E5), whose first heavy chain has the amino acid sequence shown in SEQ ID No. 123, the first light chain has the amino acid sequence shown in SEQ ID No. 124, the second heavy chain has the amino acid sequence shown in SEQ ID No. 120, and the second light chain has the amino acid sequence shown in SEQ ID No. 118.

[0173] The third type is a four-chain cascade structure ( Figure 7C It consists of the following four chains: 1) The first heavy chain, from the N-terminus to the C-terminus, is VL2-VH1-Hinge-CH2-CH3. 2) The first light chain, from the N-terminus to the C-terminus, is VL1-VH2. 3) The second chain, from the N-terminus to the C-terminus, is VHH-VH3-CH1-Hinge-CH2-CH3, and... 4) The second light chain, from the N end to the C end, is VL3-CL.

[0174] The aforementioned VL2 is the light chain variable region of the second antigen-binding domain (CD19 antibody), VH1 is the heavy chain variable region of the first antigen-binding domain (CD3 antibody), Hinge is the hinge region, CH1, CH2, and CH3 are the heavy chain constant regions, CL is the light chain constant region, VL1 is the light chain variable region of the first antigen-binding domain (CD3 antibody), and VH2 is the heavy chain variable region of the second antigen-binding domain (CD19 antibody). VL2-VH1 and VL1-VH2 pair up to form a Diabody structure; VHH and VH3 are the heavy chain variable regions of the third antigen-binding domain (BCMA antibody), and VL3 is the light chain variable region of the third antigen-binding domain (BCMA antibody). The single-domain antibody VHH passes through (G4S). n (n=2, 3, 4) The linker is tandemly linked with Fab formed by VH3-CH1 and VL3-CL to form a BCMA bivalent binding domain of single-domain antibody plus Fab.

[0175] Based on the above structure, the following three-specific antibodies were constructed: The first trispecific antibody was named CD3×CD19-1×BCMA(5A5-(G4S)2-14E5), whose first heavy chain has the amino acid sequence shown in SEQ ID No. 114, the first light chain has the amino acid sequence shown in SEQ ID No. 115, the second heavy chain has the amino acid sequence shown in SEQ ID No. 126, and the second light chain has the amino acid sequence shown in SEQ ID No. 118. The second trispecific antibody is named CD3×CD19-1×BCMA(5A5-(G4S)3-14E5). Unless otherwise specified, CD3×CD19-1×BCMA(14E5+5A5) in this invention are all abbreviations of CD3×CD19-1×BCMA(5A5-(G4S)3-14E5). Its first heavy chain has the amino acid sequence shown in SEQ ID No. 114, its first light chain has the amino acid sequence shown in SEQ ID No. 115, its second heavy chain has the amino acid sequence shown in SEQ ID No. 117, and its second light chain has the amino acid sequence shown in SEQ ID No. 118. The third trispecific antibody is named CD3×CD19-1×BCMA(5A5-(G4S)4-14E5), whose first heavy chain has the amino acid sequence shown in SEQ ID No. 114, the first light chain has the amino acid sequence shown in SEQ ID No. 115, the second heavy chain has the amino acid sequence shown in SEQ ID No. 127, and the second light chain has the amino acid sequence shown in SEQ ID No. 118. The fourth trispecific antibody is named CD3×CD19-1×BCMA(h5A5-(G4S)2-14E5), whose first heavy chain has the amino acid sequence shown in SEQ ID No. 114, the first light chain has the amino acid sequence shown in SEQ ID No. 115, the second heavy chain has the amino acid sequence shown in SEQ ID No. 128, and the second light chain has the amino acid sequence shown in SEQ ID No. 118. The fifth trispecific antibody is named CD3×CD19-1×BCMA(h5A5-(G4S)3-14E5). Unless otherwise specified, CD3×CD19-1×BCMA(14E5+h5A5) in this invention are all abbreviations of CD3×CD19-1×BCMA(h5A5-(G4S)3-14E5). Its first heavy chain has the amino acid sequence shown in SEQ ID No. 114, its first light chain has the amino acid sequence shown in SEQ ID No. 115, its second heavy chain has the amino acid sequence shown in SEQ ID No. 119, and its second light chain has the amino acid sequence shown in SEQ ID No. 118. The sixth trispecific antibody is named CD3×CD19-1×BCMA(h5A5-(G4S)4-14E5), whose first heavy chain has the amino acid sequence shown in SEQ ID No. 114, the first light chain has the amino acid sequence shown in SEQ ID No. 115, the second heavy chain has the amino acid sequence shown in SEQ ID No. 129, and the second light chain has the amino acid sequence shown in SEQ ID No. 118. The seventh trispecific antibody is named CD3×CD19-2×BCMA(14E5+5A5). Its first heavy chain has the amino acid sequence shown in SEQ ID No. 123, its first light chain has the amino acid sequence shown in SEQ ID No. 124, its second heavy chain has the amino acid sequence shown in SEQ ID No. 117, and its second light chain has the amino acid sequence shown in SEQ ID No. 118.

[0176] (2) Molecular cloning of expression plasmids Using conventional molecular cloning methods, the BCMA antibody heavy chain gene was cloned into the vector pFUSE-hIgG4-Fc2 (InvivoGene) to obtain the expression plasmid, labeled pFUSE-BCMA-HC-OA; the BCMA antibody light chain gene was cloned into the vector pCDNA3.1(+) (Invitrogen) to obtain the expression plasmid, labeled pCDNA3.1-BCMA-LC; the CD19 antibody light chain and CD3 antibody heavy chain genes were cloned into the vector pFUSE-hIgG4-Fc2 to obtain the expression plasmid, labeled pFUSE-CD19 VL-CD3 VH-OB; and the CD3 antibody light chain and CD19 antibody heavy chain genes were cloned into the vector pCDNA3.1(+) to obtain the expression plasmid, labeled pCDNA3.1-CD3 VL-CD19 VH. Table 4 shows the plasmid combinations for transient transfection with trispecific antibodies, where the underlined sequence represents the CH2 region and the wavy line represents the CH3 region.

[0177] Table 4 SEQ ID No. 114: ENVLTQSPDFQSVTPKEKVTITCSASSSVSYMHWYQQKPDQSPKLWIYDTSKLASGVPSRFSGSGSGNSHTLTINSLEAEDAATYYCFQGSVYPFTFGCGTKLEIKGGGSGGGEVQLVESGGGLV QPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAMYYCVRHGNFGTSYVSWFAYWGQGTLVTVSSESKYGPPCPPCP APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFN WYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAK GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTDDVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

[0178] SEQ ID No. 115: QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTTKRAPGTPARFSGSLIGGKAALTLSGVQPEDEAEYYCALWYGNLWVFGGGTKLTVLGGGSGGGEVTLKESGPALVKPTQTLTLTCTFSGFSLSTSGMGVGWIRQPPGKCLEWLAHIWWDDDKRYNPALKSRLTISKDTSKNQVVLTMTNMDPVDTATYYCARMELWSYYFDYWGQGTTVTVSS。

[0179] SEQ ID No.116: QVQLQESGGNSVQAGGSLRLSCAASAYIDNTYCMGWFRQAPGKEREEVASINTDGITTYADSVKGRFTASRDNAKNTLYLQMNSLKPEDTAMYYCWVTAGSRGYCYTPARRWGQGTQVTVSSESKYGPPCPPCP APEAAGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKGLPSSIEKTISKAK GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTKKKLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK。

[0180] SEQ ID No.117: QVQLQESGGNSVQAGGSLRLSCAASAYIDNTYCMGWFRQAPGKEREEVASINTDGITTYADSVKGRFTASRDNAKNTLYLQMNSLKPEDTAMYYCWVTAGSRGYCYTPARRWGQGTQVTVSSGGGGSGGGGSGGGGSEVQLLETGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKVGYSSGWYQGFDYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCP APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAK GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTKKKLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK。

[0181] SEQ ID No.118: EIVLTQSPDSLSLSPGERATLSCRASQSVSSTFLAWYQQKPGQAPRLLIYGASSRATGIPARFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSLWTFGQGTKVEIKTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。

[0182] SEQ ID No.119: QVQLVESGGGVVQPGGSLRLSCAASAYIDNTYCMGWFRQAPGKEREEVASINTDGITYYADSVKGRFTASRDNAKNTLYLQMNSLRAEDTAVYYCWVTAGSRGYCYTPARRWGQGTMVTVSSGGGGSGGGGSGGGGSEVQLLETGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKVGYSSGWYQGFDYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCP APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAK GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTKKKLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK。

[0183] SEQ ID No.120: EVQLLETGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKVGYSSGWYQGFDYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCP APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKT KPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAK GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTKKKLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK。

[0184] SEQ ID No.123: DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGCGTKLEIKGGGSGGGEVQLVESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAMYYCVRHGNFGTSYVSWFAYWGQGTLVTVSSESKYGPPCPPCP APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDP EVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAK GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTDDVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK。

[0185] SEQ ID No.124: QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTTKRAPGTPARFSGSLIGGKAALTLSGVQPEDEAEYYCALWYGNLWVFGGGTKLTVLGGGSGGGQVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQCLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTTVTVSS。

[0186] SEQ ID No.126: QVQLQESGGNSVQAGGSLRLSCAASAYIDNTYCMGWFRQAPGKEREEVASINTDGITTYADSVKGRFTASRDNAKNTLYLQMNSLKPEDTAMYYCWVTAGSRGYCYTPARRWGQGTQVTVSSGGGGSGGGGSEVQLLETGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKVGYSSGWYQGFDYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCP APE AAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKGLPSSIEKTISKAK GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTKKKLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK。

[0187] SEQ ID No.127: QVQLQESGGNSVQAGGSLRLSCAASAYIDNTYCMGWFRQAPGKEREEVASINTDGITTYADSVKGRFTASRDNAKNTLYLQMNSLKPEDTAMYYCWVTAGSRGYCYTPARRWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSEVQLLETGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKVGYSSGWYQGFDYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCP APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNST YRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAK GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTKKKLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK。

[0188] SEQ ID No.128: QVQLVESGGGVVQPGGSLRLSCAASAYIDNTYCMGWFRQAPGKEREEVASINTDGITYYADSVKGRFTASRDNAKNTLYLQMNSLRAEDTAVYYCWVTAGSRGYCYTPARRWGQGTMVTVSSGGGGSGGGGSEVQLLETGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKVGYSSGWYQGFDYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCP APE AAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTKKKLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

[0189] SEQ ID No. 129: QVQLVESGGGVVQPGGSLRLSCAASAYIDNTYCMGWFRQAPGKEREEVASINTDGITYYADSVKGRFTASRDNAKNTLYLQMNSLRAEDTAVYYCWVTAGSRGYCYTPARRWGQGTMVTVSSGGGGSGGGGSGGGGSGGGGSEVQLLETGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEW VSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKVGYSSGWYQGFDYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCP APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNST YRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAK GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTKKKLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

[0190] (3) Transient transfection and expression purification Use the kit (EZNA) ® Plasmids were extracted using a Plasmid Midi Kit (purchased from Omega Bio-Tek). 293F cells were cultured to an appropriate density, the cell suspension was centrifuged, and the cells were resuspended in fresh culture medium. Co-transfection was performed using the plasmid combinations provided in Table 4. The transfected cell suspension was incubated at 37°C in the dark on a shaker. The supernatant was collected by centrifugation, and the antibody in the supernatant was captured using a pre-equilibrated protein A affinity chromatography column (MabSelect SuRe, purchased from GE Healthcare). After washing away non-specifically bound proteins with equilibration buffer, elution buffer was used, and the eluent was collected. The samples were then analyzed by SDS-PAGE.

[0191] The results are as follows Figures 8A-8D As shown. Among them, Figure 8A The samples in lanes 1 to 3 were CD3×CD19-1×BCMA(14E5+5A5), CD3×CD19-1×BCMA(5A5), and CD3×CD19-1×BCMA(14E5), respectively. Figure 8B The samples in lanes 1 and 2 were CD3×CD19-1×BCMA(14E5+5A5) and CD3×CD19-1×BCMA(14E5+h5A5), respectively. Figure 8C The sample in lane 1 was CD3×CD19-2×BCMA(14E5); Figure 8D The samples in lanes 1 and 2 were CD3×CD19-2×BCMA(5A5) and CD3×CD19-2×BCMA(14E5+5A5), respectively.

[0192] Example 9: ELISA detection of the binding activity of trispecific antibodies to BCMA, cynomolgus monkey BCMA, CD3, and cynomolgus monkey CD3. Coat CD3 antigen (Acro Biosystems) or BCMA antigen onto the ELISA plate and block. Dilute the trispecific antibody, positive control, and negative control hIgG with 1×PBS, creating eight concentration gradients. Add the diluted samples to the ELISA plate and incubate. After washing, add HRP-labeled secondary antibody. After TMB color development, terminate the reaction with stop solution. Measure the absorbance (OD) using an ELISA reader. 450 (Value). The absorbance OD is plotted with the logarithm of the sample concentration on the X-axis. 450 The value is on the Y-axis; plot the dose-response curve and calculate EC. 50 EC 50 This represents the ability of a trispecific antibody to bind to a target antigen.

[0193] The results are as follows Figures 9A-9J As shown, the CD3×CD19×BCMA trispecific antibody can bind to both BCMA antigen and cynomolgus monkey BCMA antigen, and its binding activity is similar to that of the positive control antibody Elranatamab; however, the binding activity of the CD3×CD19×BCMA trispecific antibody to CD3 antigen and cynomolgus monkey CD3 antigen is significantly weaker than that of Elranatamab, which is beneficial in avoiding excessive activation of T cells.

[0194] Example 10: Flow cytometry (FACS) detection of the binding of trispecific antibodies to CD3-positive, CD19-positive, and BCMA-positive cells, respectively. In this embodiment, Jurkat T lymphocytes, Raji Burkitt's lymphoma cell line, and NCI-H929 human myeloma cell line were selected as target cells to evaluate the binding activity of trispecific antibodies with CD3-positive Jurkat cells, CD19-positive Raji cells, and BCMA-positive NCI-H929 cells, respectively. The specific steps are as follows (taking Raji cells as an example).

[0195] Raji cells were collected into centrifuge tubes, centrifuged, washed, and then diluted with pre-chilled wash buffer to obtain CD3×CD19×BCMA samples, positive control antibody (Blincyto or Elranatamab), and negative control hIgG. Equal volumes of cells and diluted samples were added to 96-well plates, mixed, and incubated on ice. After washing, secondary antibody was added and incubated. After a second wash, the binding activity of the sample to Raji cells was detected by flow cytometry.

[0196] The results are as follows Figures 10A-10C As shown, both CD3×CD19×BCMA(14E5+5A5) and CD3×CD19×BCMA(14E5+h5A5) trispecific antibodies can bind to CD19-positive Raji cells, BCMA-positive NCI-H929 cells, and CD3-positive Jurkat cells.

[0197] Example 11: Activation effect of trispecific antibodies on T cells A detection system consisting of the Jurkat / NFAT-Luc reporter gene cell line and CD19-positive Raji and Daudi cells or BCMA-positive NCI-H929 cells was used to evaluate the activation effect of trispecific antibodies on T cells. In this system, the Jurkat / NFAT-Luc cell line served as effector cells, with its luciferase gene regulated by the NFAT (Nuclear factor of activated T-cells) transcription factor. Raji, Daudi, or NCI-H929 cells served as target cells. When Jurkat / NFAT-Luc cells were co-cultured with target cells, luciferase was not expressed, and no obvious fluorescent signal was produced. However, when the trispecific antibody was added, CD19 on Raji and Daudi cells or BCMA on NCI-H929 cells could activate the CD3 signaling pathway in Jurkat / NFAT-Luc cells, promoting luciferase expression. Therefore, the degree of Jurkat cell activation could be determined by detecting the level of luciferase expression in the cells.

[0198] In the experiment, target cells, Jurkat / NFAT-Luc cells, and diluted trispecific antibodies were added sequentially to 96-well cell culture plates. The plates were incubated at 37°C for 5 h, and then detection reagents were added. Chemiluminescence values ​​were measured using a multi-mode microplate reader. A four-parameter fitting was performed, with the logarithm of antibody concentration on the x-axis and the average chemiluminescence value on the y-axis, to plot dose-response curves and obtain the EC50 values ​​for each curve. 50 Value, in EC 50 To represent the activity of the three specific antibodies in activating T cells, the experiment included positive control antibodies Elranatamab and / or Blincyto.

[0199] Results: When Jurkat / NFAT-Luc cells were co-cultured with CD19-positive Raji and Daudi cells, the fluorescence signal value increased with increasing doses of the trispecific antibody and Blincyto, while the negative control antibody hIgG showed no obvious fluorescence signal. Figure 11A , Figure 11B , Figure 11D , Figure 11F and Figure 11G When Jurkat / NFAT-Luc cells were co-cultured with BCMA-positive NCI-H929 cells, the fluorescence signal increased with increasing doses of the trispecific antibody and Elranatamab, while the control antibodies hIgG and Blincyto showed no significant fluorescence signal. Figure 11C , Figure 11E , Figure 11H and Figure 11I Furthermore, the activation effect of trispecific antibodies on T cells was weaker than that of Blincyto and Elranatamab, suggesting that trispecific antibodies may have lower toxicity and higher safety in vivo.

[0200] Example 12: In vitro cytotoxicity assay mediated by trispecific antibodies In this embodiment, CD19-positive Raji cells and BCMA-positive NCI-H929 cells were used as target cells, and human PBMCs were used as effector cells. The trispecific antibody, positive control antibodies Elranatamab and Blincyto, and negative control hIgG were serially diluted with PBS buffer. Target cells, PBMCs, and the serially diluted samples were added sequentially to cell culture plates. After mixing, the 96-well plates were incubated at 37 °C in a 5% CO2 incubator, and the results were analyzed using a lactate dehydrogenase cytotoxicity assay kit. The absorbance (OD) was measured using a microplate reader. 490 The logarithm of the antibody working concentration was used to calculate the relative kill value. A dose-response curve was plotted with the logarithm of the antibody working concentration on the X-axis and the relative kill value on the Y-axis, and the half-maximal effective concentration (EC50) was calculated. 50 ), with EC50 The value represents the cytotoxic activity of the trispecific antibody against target cells.

[0201] The results are shown in Figure 12. The trispecific antibody had a stronger killing effect on CD19-positive Raji cells than or comparable to that of the positive antibody Blincyto. Figure 12A , Figure 12C and Figure 12E Furthermore, the trispecific antibody exhibits comparable cytotoxic effects on BCMA-positive NCI-H929 cells to the positive antibody Elranatamab. Figure 12B , Figure 12D , Figure 12F and Figure 12G ).

[0202] Example 13: In vitro cytokine release assay of trispecific antibodies In this embodiment, a trispecific antibody was co-incubated with human PBMC cells to evaluate its ability to induce cytokine release. The trispecific antibody, positive control Blincyto, Elranatamab, and negative control (hIgG) were serially diluted in RPMI 1640 medium to a total of eight concentration gradients. PBMCs and diluted samples were added to 96-well cell culture plates, mixed thoroughly, and incubated overnight at 37°C. The supernatant was used to detect the concentrations of cytokines such as IL-2, TNF-α, and IL-6.

[0203] (1) IL-2: Detected according to the instructions provided with the Human IL-2 Detection Kit (R&D Systems, Cat. DY202); (2) TNF-α: The test was performed according to the instructions provided with the Human TNF-α Detection Kit (R&D Systems, Cat. DY210); (3) IL-6: Detect according to the instructions provided with the Human IL-6 Detection Kit (R&D Systems, Cat. DY206).

[0204] The results are as follows Figures 13A-13C As shown, Blincyto can stimulate PBMCs to release high levels of various cytokines, including IL-2, TNF-α, and IL-6. In comparison, the ability of trispecific antibodies to stimulate T cells to produce cytokines is significantly weaker than that of Blincytoto, and it is expected to reduce the risk of cytokine storm and has lower toxicity. Due to the relatively small number of plasma cells in PBMCs, Elranatamab stimulates T cells to produce fewer cytokines or below the detection range.

[0205] Example 14: In vitro clearance experiment of B cells by trispecific antibodies In this embodiment, trispecific antibodies were co-incubated with human PBMC cells, and the ability of the trispecific antibodies to clear B cells was detected by flow cytometry. The trispecific antibodies, positive control Blincyto, Elranatamab, and negative control (hIgG) were serially diluted in RPMI 1640 medium to a total of 10 concentration gradients. PBMCs and diluted samples were added to 96-well cell culture plates, mixed, and incubated overnight at 37°C. The next day, cells were collected by centrifugation, incubated with Ofatumumab antibody (Anti-CD20 antibody), washed, and then incubated with secondary antibody. After washing again, the ability of the trispecific antibodies to clear B cells was detected by flow cytometry via the PE channel.

[0206] The results are as follows Figure 14 As shown, the trispecific antibody has a stronger ability to clear B cells in human PBMCs than the positive antibodies Blincyto and Elranatamab.

[0207] Example 15: Detection of antitumor activity of trispecific antibodies in mice (1) Pharmacodynamic study of trispecific antibodies in NCI-H929 human myeloma cell line xenograft model of NOD / SCID mice with peripheral blood mononuclear cells reconstructing human immune system Female NOD-SCID mice (purchased from Beijing Vital River Laboratory Animal Co., Ltd.) were selected. An appropriate amount of NCI-H929 cells mixed with PBMCs was subcutaneously inoculated into the right side of the mice. The day of inoculation was designated D-1. The day after cell inoculation, the mice were divided into 5 groups of 5 mice each according to their body weight: saline group (G1), CD3×CD19-1×BCMA(14E5+5A5) group (G2), CD3×CD19-1×BCMA(5A5) group (G3), CD3×CD19-1×BCMA(14E5) group (G4), and Elranatamab group (G5). The day of grouping was designated D0. Drugs were administered via tail vein injection on D0, D3, D7, and D10. Tumor volume was measured twice weekly, and body weight was also measured. The formula for calculating tumor volume (mm²) is: Tumor volume (mm²) = ... 3 = 0.5 × (tumor long diameter × tumor short diameter) 2 The tumor inhibition rate (TGI) was calculated using tumor volume. Treatment efficacy was evaluated based on the TGI.

[0208] This experiment ended on day 17 after group administration. Tumor volume changes in mice of different groups are shown below. Figures 15A-15B As shown. At the experimental endpoint, the mean tumor volume in the saline control group (G1) was 717.23 ± 46.64 mm. 3 (G1, Figure 15AThe mean tumor volume in the positive control Elranatamab group (G5) was 19.13 ± 1.78 mm. 3 (G5), the tumor inhibition rate (TGI) was 97.3% (G5, P <0.0001). The mean tumor volume of the test drug groups CD3×CD19-1×BCMA(14E5+5A5) group (G2), CD3×CD19-1×BCMA(5A5) group (G3), and CD3×CD19-1×BCMA(14E5) group (G4) was 17.75±4.03 mm. 3 (G2), 142.07±50.82 mm 3 (G3) and 418.00±132.29 mm 3 (G4), with corresponding TGIs of 97.5% (G2), 80.2% (G3), and 41.7% (G4). Both CD3×CD19-1×BCMA(14E5+5A5) and CD3×CD19-1×BCMA(5A5) exhibited strong tumor-suppressing effects, showing statistically significant differences compared to the saline control group. P <0.0001), and the former has an effect comparable to Elranatamab.

[0209] (2) Pharmacodynamic study of trispecific antibodies in a human Burkitt's lymphoma Raji xenograft model established subcutaneously in NOD / SCID mice with reconstructed human immune system by peripheral blood mononuclear cells. Female NOD-SCID mice (purchased from Beijing Vital River Laboratory Animal Co., Ltd.) were used. An appropriate amount of Raji cells mixed with PBMCs was subcutaneously injected into the right side of the mice. One day after injection, mice were randomly divided into 6 groups according to their body weight: saline group (G1), CD3×CD19-2×BCMA(14E5+5A5) group (G2), CD3×CD19-1×BCMA(14E5+5A5) group (G3), CD3×CD19-1×BCMA(14E5+h5A5) group (G4), Blincyto group (G5), and CD3×CD19-1×BCMA(5A5) group (G6). The day of grouping was designated as D0. The drugs were administered via tail vein injection on D0, D3, D7, D10, and D14. Tumor volume was measured twice weekly, and body weight was also measured. The formula for calculating tumor volume (mm²) is: Tumor volume (mm²) = ... 3 = 0.5 × (Tumor long diameter × Tumor short diameter) 2 The tumor inhibition rate (TGI) is calculated using tumor volume, and the treatment efficacy is evaluated based on the TGI.

[0210] The results are as follows Figure 15BAs shown, compared with the control group, each experimental group had a significant antitumor effect in mice. Compared with the saline group, the tumor inhibition rates of the drug-treated groups at the experimental endpoint were 95.3% (G2), 86.9% (G3), 92.0% (G4), 91.2% (G5), and 90.8% (G6), respectively.

[0211] Example 16: Evaluation of the efficacy of trispecific antibodies in humanized mice In this embodiment, humanized C57BL / 6 mice (hBCMA / hCD3EDG / hCD19) with CD19, BCMA, and CD3 (purchased from Shanghai Southern Model Biotechnology Co., Ltd.) were selected to evaluate the in vivo efficacy of the CD3×CD19×BCMA trispecific antibody.

[0212] hBCMA / hCD3EDG / hCD19 mice were randomly divided into three groups: a saline group (G1), a CD3×CD19-1×BCMA(14E5+5A5) group (G2), and a CD3×CD19-1×BCMA(14E5+h5A5) group (G3). The mice were administered the drug via tail vein on days 0, 3, 7, 10, and 14 at a dose of 2 mg / kg. The experiment was terminated one week after drug withdrawal. Peripheral blood was collected to detect changes in the proportion of T cells (hCD3). + ), B cell clearance status (mCD20) + Changes in total IgG levels in plasma. Spleen was collected, spleen cells were isolated, and changes in the proportion of T cells (hCD3) were detected. + ), B cell clearance status (mCD20) + ).

[0213] The results are as follows Figures 16A-16E As shown in Tables 5-7.

[0214] (1) T cell proliferation: Compared with the saline group, after administration of the trispecific antibody, the proportion of T cells in lymphocytes in the peripheral blood of mice changed from 46.89% to 70.53% (G2) and 55.29% (G3) (see Figure 16A The proportion of [specific component] in spleen tissue changed from 53.32% to 75.84% (G2) and 61.88% (G3) (see [reference]). Figure 16B Both increased significantly.

[0215] (2) B cell clearance: The proportion of B cells in the lymphocytes of peripheral blood of mice was reduced to almost 0 (the proportion in the saline group was about 18%) (see Figure 16C The clearance rate was close to 100% (see Table 5); the proportion of B cells in the spleen tissue of mice decreased to almost 0 (the proportion in the saline group was about 20%) (see Table 5). Figure 16D The clearance rate was 100% (see Table 6).

[0216] (3) Decreased antibody levels: The IgG content in mouse plasma changed from 1595.35 μg / mL (G1) to 548.87 μg / mL (G2) and 264.25 μg / mL (G3) (see Figure 16E The figures decreased by 65.6% and 83.4% respectively (see Table 7).

[0217] The above results indicate that the trispecific antibodies can promote the proliferation of T cells in humanized mice, effectively eliminate B cells (peripheral blood and tissues) in mice, and significantly reduce antibody production.

[0218] Table 5 Table 6 Table 7 In summary, this invention recruits and activates T cells through its CD3 binding domain, and simultaneously achieves broad-spectrum recognition and binding to the entire abnormal B cell lineage, from early B cells to terminally differentiated plasma cells, through its CD19 and BCMA dual binding domains. This design aims to more thoroughly eliminate pathological B cells, overcoming the antigen escape or drug resistance problems that may exist in single-target therapy through multiple synergistic effects. Therefore, it provides a novel antibody drug with superior B cell clearance efficacy and therapeutic potential for diseases such as multiple myeloma, B-cell lymphoma, and B-cell-related autoimmune diseases.

[0219] The applicant declares that the detailed method of the present invention is illustrated by the above embodiments, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A trispecific antibody binding CD3, CD19, and BCMA, characterized in that, The trispecific antibody includes a first antigen-binding domain that can bind CD3, a second antigen-binding domain that can bind CD19, a third antigen-binding domain that can bind BCMA, and a heterodimer Fc region; the first antigen-binding domain and the second antigen-binding domain pair up to form a diabody structure, and the third antigen-binding domain is selected from any of the following structures: Fab structure, single-domain antibody VHH structure, and bivalent structure formed by Fab and VHH in tandem.

2. The trispecific antibody according to claim 1, characterized in that, The trispecific antibody consists of three chains having the following structure: 1) The first heavy chain, from the N-terminus to the C-terminus, is VL2-VH1-Hinge-CH2-CH3. 2) The first light chain, from the N-terminus to the C-terminus, is VL1-VH2, and... 3) The second chain, from the N-terminus to the C-terminus, is VHH-Hinge-CH2-CH3; VL2 is the light chain variable region of the second antigen-binding domain (CD19 antibody), VH1 is the heavy chain variable region of the first antigen-binding domain (CD3 antibody), Hinge is the hinge region, CH2 and CH3 are the heavy chain constant regions, VL1 is the light chain variable region of the first antigen-binding domain (CD3 antibody), VH2 is the heavy chain variable region of the second antigen-binding domain (CD19 antibody), and VHH is the heavy chain variable region of the third antigen-binding domain (BCMA antibody). VL2-VH1 and VL1-VH2 pair up to form a Diabody structure. Preferably, the trispecific antibody consists of four chains having the following structure: 1) The first heavy chain, from the N-terminus to the C-terminus, is VL2-VH1-Hinge-CH2-CH3. 2) The first light chain, from the N-terminus to the C-terminus, is VL1-VH2. 3) The second chain, from the N-terminus to the C-terminus, is VH3-CH1-Hinge-CH2-CH3, and... 4) The second light chain, from the N-terminus to the C-terminus, is VL3-CL; In this structure, VL2 is the light chain variable region of the second antigen-binding domain (CD19 antibody), VH1 is the heavy chain variable region of the first antigen-binding domain (CD3 antibody), Hinge is the hinge region, CH1, CH2 and CH3 are the heavy chain constant regions, CL is the light chain constant region, VL1 is the light chain variable region of the first antigen-binding domain (CD3 antibody), VH2 is the heavy chain variable region of the second antigen-binding domain (CD19 antibody), VH3 is the heavy chain variable region of the third antigen-binding domain (BCMA antibody), and VL3 is the light chain variable region of the third antigen-binding domain (BCMA antibody). VL2-VH1 and VL1-VH2 pair with each other to form a Diabody structure, and VH3-CH1 and VL3-CL pair with each other to form a Fab structure. Preferably, the trispecific antibody consists of four chains having the following structure: 1) The first heavy chain, from the N-terminus to the C-terminus, is VL2-VH1-Hinge-CH2-CH3. 2) The first light chain, from the N-terminus to the C-terminus, is VL1-VH2. 3) The second chain, from the N-terminus to the C-terminus, is VHH-VH3-CH1-Hinge-CH2-CH3, and... 4) The second light chain, from the N-terminus to the C-terminus, is VL3-CL; In this structure, VL2 is the light chain variable region of the second antigen-binding domain (CD19 antibody), VH1 is the heavy chain variable region of the first antigen-binding domain (CD3 antibody), Hinge is the hinge region, CH1, CH2, and CH3 are the heavy chain constant regions, CL is the light chain constant region, VL1 is the light chain variable region of the first antigen-binding domain (CD3 antibody), and VH2 is the heavy chain variable region of the second antigen-binding domain (CD19 antibody). VL2-VH1 and VL1-VH2 pair up to form a diabody structure. VHH and VH3 are the heavy chain variable regions of the third antigen-binding domain (BCMA antibody), and VL3 is the light chain variable region of the third antigen-binding domain (BCMA antibody). The single-domain antibody VHH is connected via (G4S). n (n=2, 3, 4) The linker is tandemly linked with Fab formed by VH3-CH1 and VL3-CL to form a BCMA bivalent binding domain of single-domain antibody plus Fab.

3. The trispecific antibody according to claim 1 or 2, characterized in that, The first antigen-binding domain capable of binding CD3 includes a CD3 antibody; the second antigen-binding domain capable of binding CD19 includes a CD19 antibody; and the third antigen-binding domain capable of binding BCMA includes a BCMA antibody. Preferably, the BCMA antibody includes human BCMA antibody and camel-derived BCMA nanobody; Preferably, the human BCMA antibody comprises a human BCMA antibody heavy chain and a human BCMA antibody light chain; Preferably, the amino acid sequences of CDR1, CDR2, and CDR3 of the human BCMA antibody heavy chain are shown in SEQ ID No. 2, SEQ ID No. 3, and SEQ ID No. 4, respectively, and the amino acid sequences of CDR1, CDR2, and CDR3 of the light chain are shown in SEQ ID No. 6, SEQ ID No. 7, and SEQ ID No. 8, respectively. or, The amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain of the human BCMA antibody are shown in SEQ ID No. 10, SEQ ID No. 11 and SEQ ID No. 12, respectively, and the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain are shown in SEQ ID No. 14, SEQ ID No. 15 and SEQ ID No. 16, respectively. or, The amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain of the human BCMA antibody are shown in SEQ ID No. 18, SEQ ID No. 19 and SEQ ID No. 20, respectively, and the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain are shown in SEQ ID No. 22, SEQ ID No. 23 and SEQ ID No. 24, respectively. or, The amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain of the human BCMA antibody are shown in SEQ ID No. 26, SEQ ID No. 27 and SEQ ID No. 28, respectively, and the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain are shown in SEQ ID No. 30, SEQ ID No. 31 and SEQ ID No. 32, respectively. or, The amino acid sequences of CDR1, CDR2 and CDR3 of the human BCMA antibody heavy chain are shown in SEQ ID No. 34, SEQ ID No. 35 and SEQ ID No. 36, respectively, and the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain are shown in SEQ ID No. 38, SEQ ID No. 39 and SEQ ID No. 40, respectively. or, The amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain of the human BCMA antibody are shown in SEQ ID No. 42, SEQ ID No. 43 and SEQ ID No. 44, respectively, and the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain are shown in SEQ ID No. 46, SEQ ID No. 47 and SEQ ID No. 48, respectively. or, The amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain of the human BCMA antibody are shown in SEQ ID No. 50, SEQ ID No. 51 and SEQ ID No. 52, respectively, and the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain are shown in SEQ ID No. 54, SEQ ID No. 55 and SEQ ID No. 56, respectively. or, The amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain of the human BCMA antibody are shown in SEQ ID No. 58, SEQ ID No. 59 and SEQ ID No. 60, respectively, and the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain are shown in SEQ ID No. 62, SEQ ID No. 63 and SEQ ID No. 64, respectively. or, The amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain of the human BCMA antibody are shown in SEQ ID No. 66, SEQ ID No. 67 and SEQ ID No. 68, respectively, and the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain are shown in SEQ ID No. 70, SEQ ID No. 71 and SEQ ID No. 72, respectively. or, The amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain of the human BCMA antibody are shown in SEQ ID No. 74, SEQ ID No. 75 and SEQ ID No. 76, respectively, and the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain are shown in SEQ ID No. 78, SEQ ID No. 79 and SEQ ID No. 80, respectively. or, The amino acid sequences of CDR1, CDR2 and CDR3 of the human BCMA antibody heavy chain are shown in SEQ ID No. 82, SEQ ID No. 83 and SEQ ID No. 84, respectively, and the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain are shown in SEQ ID No. 86, SEQ ID No. 87 and SEQ ID No. 88, respectively. Preferably, the camel-derived BCMA nanobody comprises a camel-derived BCMA nanobody heavy chain; Preferably, the amino acid sequences of CDR1, CDR2 and CDR3 of the camel-derived BCMA nanobody heavy chain are shown in SEQ ID No. 90, SEQ ID No. 91 and SEQ ID No. 92, respectively; or, The amino acid sequences of CDR1, CDR2 and CDR3 of the camel-derived BCMA nanobody heavy chain are shown in SEQ ID No. 94, SEQ ID No. 95 and SEQ ID No. 96, respectively; or, The amino acid sequences of CDR1, CDR2 and CDR3 of the camel-derived BCMA nanobody heavy chain are shown in SEQ ID No. 98, SEQ ID No. 99 and SEQ ID No. 100, respectively; or, The amino acid sequences of CDR1, CDR2 and CDR3 of the camel-derived BCMA nanobody heavy chain are shown in SEQ ID No. 102, SEQ ID No. 103 and SEQ ID No. 104, respectively; or, The amino acid sequences of CDR1, CDR2 and CDR3 of the camel-derived BCMA nanobody heavy chain are shown in SEQ ID No. 106, SEQ ID No. 107 and SEQ ID No. 108, respectively.

4. The trispecific antibody according to claim 3, characterized in that, The heavy chain variable region of the human BCMA antibody has the amino acid sequence shown in SEQ ID No. 1, and the light chain variable region has the amino acid sequence shown in SEQ ID No. 5; or, The heavy chain variable region of the human BCMA antibody has an amino acid sequence as shown in SEQ ID No. 9, and the light chain variable region has an amino acid sequence as shown in SEQ ID No. 13; or, The heavy chain variable region of the human BCMA antibody has an amino acid sequence as shown in SEQ ID No. 17, and the light chain variable region has an amino acid sequence as shown in SEQ ID No. 21; or, The heavy chain variable region of the human BCMA antibody has an amino acid sequence as shown in SEQ ID No. 25, and the light chain variable region has an amino acid sequence as shown in SEQ ID No. 29; or, The heavy chain variable region of the human BCMA antibody has an amino acid sequence as shown in SEQ ID No. 33, and the light chain variable region has an amino acid sequence as shown in SEQ ID No. 37; or, The heavy chain variable region of the human BCMA antibody has an amino acid sequence as shown in SEQ ID No. 41, and the light chain variable region has an amino acid sequence as shown in SEQ ID No. 45; or, The heavy chain variable region of the human BCMA antibody has an amino acid sequence as shown in SEQ ID No. 49, and the light chain variable region has an amino acid sequence as shown in SEQ ID No. 53; or, The heavy chain variable region of the human BCMA antibody has an amino acid sequence as shown in SEQ ID No. 57, and the light chain variable region has an amino acid sequence as shown in SEQ ID No. 61; or, The heavy chain variable region of the human BCMA antibody has an amino acid sequence as shown in SEQ ID No. 65, and the light chain variable region has an amino acid sequence as shown in SEQ ID No. 69; or, The heavy chain variable region of the human BCMA antibody has an amino acid sequence as shown in SEQ ID No. 73, and the light chain variable region has an amino acid sequence as shown in SEQ ID No. 77; or, The heavy chain variable region of the human BCMA antibody has an amino acid sequence as shown in SEQ ID No. 81, and the light chain variable region has an amino acid sequence as shown in SEQ ID No. 85; Preferably, the heavy chain variable region of the camel-derived BCMA nanobody has an amino acid sequence as shown in SEQ ID No. 89, SEQ ID No. 93, SEQ ID No. 97, SEQ ID No. 101 or SEQ ID No. 105; Preferably, the camel-derived BCMA nanobody comprises a humanized camel-derived BCMA nanobody; the humanized camel-derived BCMA nanobody comprises heavy chain VHH; Preferably, the heavy chain variable region of the humanized camel-derived BCMA nanobody has an amino acid sequence as shown in SEQ ID No. 113 or SEQ ID No.

125.

5. The trispecific antibody according to any one of claims 1-4, characterized in that, The first antigen-binding domain, the second antigen-binding domain, and the third antigen-binding domain are each composed of the following sequences: 1) A first antigen-binding domain, wherein the heavy chain variable region of the first antigen-binding domain has an amino acid sequence as shown in SEQ ID No. 109, and the light chain variable region has an amino acid sequence as shown in SEQ ID No. 110; 2) A second antigen-binding domain, wherein the heavy chain variable region of the second antigen-binding domain has the amino acid sequence shown in SEQ ID No. 111, and the light chain variable region has the amino acid sequence shown in SEQ ID No. 112; or, the heavy chain variable region of the second antigen-binding domain has the amino acid sequence shown in SEQ ID No. 121, and the light chain variable region has the amino acid sequence shown in SEQ ID No. 122; and 3) A third antigen-binding domain, wherein the heavy chain variable region of the third antigen-binding domain has an amino acid sequence as shown in SEQ ID No. 49, and the light chain variable region has an amino acid sequence as shown in SEQ ID No. 53; or, the heavy chain variable region of the third antigen-binding domain has an amino acid sequence as shown in SEQ ID No. 93, SEQ ID No. 113, or SEQ ID No. 125; Preferably, the trispecific antibody comprises: The first heavy chain of the amino acid sequence shown in SEQ ID No.

114. The first light chain of the amino acid sequence shown in SEQ ID No. 115, and The second heavy chain of the amino acid sequence shown in SEQ ID No. 116; or, The first heavy chain of the amino acid sequence shown in SEQ ID No.

123. The first light chain of the amino acid sequence shown in SEQ ID No. 124, and The second heavy chain of the amino acid sequence shown in SEQ ID No. 116; Preferably, the trispecific antibody comprises: The first heavy chain of the amino acid sequence shown in SEQ ID No.

114. The first light chain of the amino acid sequence shown in SEQ ID No. 115, The second heavy chain of the amino acid sequence shown in SEQ ID No. 120, and The second light chain of the amino acid sequence shown in SEQ ID No. 118; or, The first heavy chain of the amino acid sequence shown in SEQ ID No.

123. The first light chain of the amino acid sequence shown in SEQ ID No. 124, The second heavy chain of the amino acid sequence shown in SEQ ID No. 120, and The second light chain of the amino acid sequence shown in SEQ ID No. 118; Preferably, the trispecific antibody comprises: The first heavy chain of the amino acid sequence shown in SEQ ID No.

114. The first light chain of the amino acid sequence shown in SEQ ID No. 115, The second heavy chain of the amino acid sequence shown in SEQ ID No. 117, SEQ ID No. 126, or SEQ ID No. 127, and The second light chain of the amino acid sequence shown in SEQ ID No. 118; or, The first heavy chain of the amino acid sequence shown in SEQ ID No.

114. The first light chain of the amino acid sequence shown in SEQ ID No. 115, The second chain of the amino acid sequence shown in SEQ ID No. 119, SEQ ID No. 128, or SEQ ID No. 129, and The second light chain of the amino acid sequence shown in SEQ ID No. 118; or, The first heavy chain of the amino acid sequence shown in SEQ ID No.

123. The first light chain of the amino acid sequence shown in SEQ ID No. 124, The second heavy chain of the amino acid sequence shown in SEQ ID No. 117, and The second light chain of the amino acid sequence shown in SEQ ID No. 118; Preferably, the heavy chain FR2 of the second antigen-binding domain contains a G44C mutation, and the light chain FR4 contains a Q100C or G100C mutation. Preferably, the Fc is derived from immunoglobulin IgG4 or IgG1; Preferably, the CH3 of the first heavy chain contains P395D and P396D mutations, and the CH3 of the second heavy chain contains P395K, P396K, and V397K mutations. Preferably, the CH2 of both the first and second heavy chains contains L234A and L235A mutations.

6. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the trispecific antibody that binds CD3, CD19 and BCMA as described in any one of claims 1-5.

7. An expression carrier, characterized in that, The expression vector contains the nucleic acid molecule as described in claim 6.

8. An antibody conjugate, characterized in that, The antibody conjugate includes the trispecific antibody as described in any one of claims 1-5, and the conjugated marker; The markers include any one or a combination of at least two of the following: cytotoxins, radioactive isotopes, fluorescent markers, luminescent substances, chromogenic substances, or enzymes.

9. A pharmaceutical composition, characterized in that, The pharmaceutical composition contains the trispecific antibody as described in any one of claims 1-5 or the antibody conjugate as described in claim 8.

10. The use of any one or a combination of at least two of the trispecific antibody combining CD3, CD19 and BCMA as described in any one of claims 1-5, the antibody conjugate as described in claim 8, or the pharmaceutical composition as described in claim 9 in the preparation of a medicament for the treatment of a disease; The disease is caused by B-cell lineage abnormalities, including: Multiple myeloma, Waldenström macroglobulinemia, light chain amyloidosis, B-cell lymphoma, leukemia, or autoimmune disease, or any combination of at least two of these conditions; The B-cell lymphoma includes any one or a combination of at least two of the following: diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, or Burkitt lymphoma; The leukemia includes any one or a combination of at least two of the following: acute B-lymphoblastic leukemia, chronic lymphocytic leukemia, or B-prolymphoblastic leukemia. The autoimmune diseases include any one or a combination of at least two of the following: systemic lupus erythematosus, antineutrophil cytoplasmic antibody-associated vasculitis, pemphigus, rheumatoid arthritis, systemic sclerosis, myasthenia gravis, or Sjögren's syndrome.

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