Trispecific antigen-binding molecule and use thereof

Abstract

The present disclosure provides a trispecific antigen-binding molecule targeting CD79b, CD20, and CD3, an antibody or an antigen-binding fragment thereof targeting CD79b or CD20, a pharmaceutical composition comprising the trispecific antigen-binding molecule and the antibody or the antigen-binding fragment thereof, and related use thereof in the treatment of tumors and autoimmune diseases.

Description

Trispecific antigen-binding molecules and their applications

[0001] This application claims priority to Chinese patent application 2025101118969, filed on January 22, 2025. The entire contents of the aforementioned Chinese patent application are incorporated herein by reference. Technical Field

[0002] This disclosure pertains to the field of immunology and relates to a trispecific antigen-binding molecule. It also relates to related encoding nucleic acids, vectors, host cells, drugs, and their applications in the treatment of cancer and autoimmune diseases. Background Technology

[0003] CD79b (also known as immunoglobulin β chain, Igβ) is a transmembrane protein and a key component of the B cell receptor (BCR) complex, participating in BCR signaling along with CD79a (Igα). CD79b is primarily expressed in B cells, forming the BCR complex by binding to membrane-bound immunoglobulins (mIg), mediating antigen recognition and downstream signal transduction. This signaling pathway plays a crucial role in B cell development, differentiation, and functional maintenance, and is also closely related to the development of various B cell-related diseases, such as B-cell lymphoma and leukemia. Due to its specific B cell expression characteristics, CD79b has become an important target for B cell-targeted therapy, showing broad application prospects, particularly in antibody-drug conjugates (ADCs) and monoclonal antibody therapy. Targeted drugs based on CD79b can effectively deliver cytotoxic drugs to malignant B cells while avoiding non-specific damage to other cells, thereby improving the precision and safety of treatment. Therefore, research on the structure, function, and application of CD79b in disease treatment has significant scientific and clinical value.

[0004] CD20 is a non-glycosylated transmembrane protein belonging to the four-transmembrane domain family. Encoded by the MS4A1 gene, it is primarily expressed during the late developmental stages of B cells (from pre-B cells to memory B cells), but not in naïve B cells and plasma cells. The biological functions of CD20 are not fully elucidated, but studies have shown that it plays a crucial role in B cell activation, proliferation, differentiation, and the regulation of calcium ion channels. Due to its highly specific expression on B cells and its high expression in hematologic malignancies, CD20 has become an important therapeutic target for B-cell-related diseases. Monoclonal antibodies targeting CD20 (such as rituximab) have been widely used in the treatment of B-cell non-Hodgkin's lymphoma (NHL), chronic lymphocytic leukemia (CLL), and other autoimmune diseases. These drugs kill diseased B cells through mechanisms such as antibody-dependent cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and direct apoptosis induction. The clinical success of targeting CD20 has spurred the development of more drug formulations targeting this molecule, including bispecific antibodies, antibody-drug conjugates (ADCs), and chimeric antigen receptor T-cell (CAR-T) therapy. The properties and broad clinical application potential of CD20 make it an important research direction for B-cell targeted therapy.

[0005] B-cell lymphomas are a group of malignant hematologic malignancies originating from B lymphocytes, broadly encompassing subtypes such as diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), and mantle cell lymphoma (MCL). According to global cancer statistics, non-Hodgkin lymphoma (NHL) is one of the most common malignant tumors, accounting for approximately 85%-90% of all cases. In the United States, there are approximately 80,000 new cases of non-Hodgkin lymphoma each year, while globally this number approaches 500,000. Although advances in targeted therapies and immunotherapy in recent years have significantly improved patient survival, overall response rates (ORR) and long-term progression-free survival (PFS) remain limited for some subtypes, especially for relapsed or refractory (R / R) patients. Current clinical treatments primarily include chemotherapy (such as the R-CHOP regimen), targeted monoclonal antibodies (such as rituximab), and emerging CAR-T cell therapies and antibody-drug conjugates (ADCs). However, some patients fail to benefit from existing treatments due to issues such as drug resistance, short-lived efficacy, or toxic side effects. Furthermore, treatment options remain limited for certain aggressive or rare subtypes (such as primary central nervous system lymphoma and double-hit lymphoma). Therefore, developing more efficient, precise, and less toxic treatments to meet unmet clinical needs is an important direction for B-cell lymphoma research.

[0006] B cells play a crucial role in the pathogenesis of autoimmune diseases, and their abnormal activation and dysfunction are key pathological features of many autoimmune disorders. B cells participate in disease development through multiple mechanisms, including the abnormal production of autoantibodies (such as rheumatoid factor in rheumatoid arthritis and antinuclear antibodies in systemic lupus erythematosus), antigen presentation, and the secretion of pro-inflammatory cytokines (such as TNF-α and IL-6), thereby inducing or exacerbating abnormal immune system responses. Currently, various B-cell-targeting therapeutic strategies are used clinically to treat autoimmune diseases. For example, CD20 monoclonal antibodies (such as rituximab) can alleviate symptoms by depleting B cells, showing efficacy in diseases such as rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), and multiple sclerosis (MS). However, existing treatments still have limitations, including significant fluctuations in efficacy due to individual patient differences, short duration of efficacy, and increased risk of treatment-related infections. Furthermore, for some refractory or rare autoimmune diseases, the selection of current therapies remains limited. Therefore, further development of novel treatments targeting B cells, especially innovative drugs that can precisely regulate B cell function and reduce non-specific immunosuppression, has become an important research direction for improving the treatment of autoimmune diseases and addressing unmet clinical needs.

[0007] T-cell-based therapies have demonstrated significant anti-tumor effects in numerous animal models, and many T-cell therapies have recently made significant progress in treating cancer and autoimmune diseases. Therefore, developing novel, highly effective, and low-toxicity T-cell multispecific antibodies is crucial to unlocking the potential of T cells. Multispecific antibodies targeting the CD3 antigen can spatially bring T cells closer to target cells, enabling specific killing of target cells by T cells; these antibodies are called T-cell-engagers (TCEs). Targeting dual-target TCEs can enhance the killing efficacy against cells expressing both antigens and can kill target cells expressing only one antigen. This allows for maximal elimination of target cells in the presence of heterogeneous cell populations, preventing drug resistance through tumor antigen escape and prolonging the time to clinical benefit. Nearly half of the candidates for bispecific antibodies in clinical trials target the CD3 antigen, and the safety and efficacy of TCE bispecific antibodies are being studied. Although some progress has been made in related research, many challenges remain, such as how to balance anti-tumor activity and safety. Therefore, developing novel, highly effective, and low-toxicity T-cell multispecific antibodies remains crucial.

[0008] Therefore, there is a need to develop new forms of trispecific antibodies and to utilize them to further develop highly effective and low-toxicity trispecific antibody drugs.

[0009] Invention Overview

[0010] To achieve the above objectives, this disclosure provides a trispecific antibody against CD79b, CD20 and CD3. This molecule can recruit T cells to the tumor site through the CD3 target and specifically kill tumor cells that highly express CD79b and / or CD20, making it possible for the cytotoxic effect of T cells to target cancer cells.

[0011] The first aspect of this disclosure is to provide a trispecific antigen-binding molecule comprising: a first binding site for a first antigen, a second binding site for a second antigen, and a third binding site for a third antigen, wherein the first antigen is CD79b, the second antigen is CD3, and the third antigen is CD20, preferably wherein the second antigen is CD3ε.

[0012] In one embodiment, the first binding site against the first antigen comprises an antigen-binding fragment Fab capable of specifically binding to the first antigen; and / or, the second binding site against the second antigen comprises a single-chain antibody (scFv) domain capable of specifically binding to the second antigen; and / or, the third binding site against the third antigen comprises a heavy chain single-domain antibody (VHH) domain capable of specifically binding to the third antigen or an antigen-binding fragment Fab capable of specifically binding to the third antigen.

[0013] In one embodiment, the first binding site against the first antigen includes an antigen-binding fragment Fab capable of specifically binding to the first antigen, and the second binding site against the second antigen includes a single-chain antibody (scFv) domain capable of specifically binding to the second antigen.

[0014] In one embodiment, the trispecific antigen-binding molecule of this disclosure comprises the following polypeptide:

[0015] The first polypeptide comprises: (i) a heavy chain domain of an antigen-binding fragment Fab capable of specifically binding to a first antigen, (ii) a single-chain antibody (scFv) domain capable of specifically binding to a second antigen, and (iii) a first Fc domain.

[0016] The second polypeptide comprises a light chain domain of the antigen-binding fragment Fab, which is capable of specifically binding to the first antigen.

[0017] The third polypeptide comprises: (i) a heavy chain single-domain antibody (VHH) domain capable of specifically binding to the third antigen or a heavy chain domain of an antigen-binding fragment Fab capable of specifically binding to the third antigen, and (ii) a second Fc domain.

[0018] When the third polypeptide contains a heavy chain domain of the Fab antigen-binding fragment capable of specifically binding to the third antigen, the trispecific antigen-binding molecule further contains a fourth polypeptide containing a Fab light chain domain capable of specifically binding to the third antigen.

[0019] The heavy chain domain of the antigen-binding fragment Fab of the first polypeptide, which specifically binds to the first antigen, and the light chain domain of the antigen-binding fragment Fab of the second polypeptide, which specifically binds to the first antigen, form a first binding site against the first antigen; the single-chain antibody (scFv) domain forms a second binding site against the second antigen; the first Fc domain and the second Fc domain associate with each other; the heavy chain single-domain antibody (VHH) domain forms a third binding site against the third antigen, or, the heavy chain domain of the antigen-binding fragment Fab, which specifically binds to the third antigen, and the light chain domain of the antigen-binding fragment Fab, which specifically binds to the third antigen, form a third binding site against the third antigen.

[0020] In one embodiment, the scFv structural domain includes a heavy chain variable region and a light chain variable region; preferably, the heavy chain variable region and the light chain variable region of the scFv structural domain are connected by a first connector.

[0021] In a preferred embodiment, the C-end of the heavy chain variable region is fused with the N-end of the first connector, and the C-end of the first connector is fused with the N-end of the light chain variable region.

[0022] More preferably, the first linker comprises an amino acid sequence (G4S)n, where n is any integer from 1 to 10. The sequence of G4S is SEQ ID NO:68.

[0023] In one embodiment, the first Fc domain comprises a first CH2 domain and a first CH3 domain of an immunoglobulin, wherein the C-terminus of the first CH2 domain is fused to the N-terminus of the first CH3 domain; the second Fc domain comprises a second CH2 domain and a second CH3 domain of an immunoglobulin, wherein the C-terminus of the second CH2 domain is fused to the N-terminus of the second CH3 domain.

[0024] Preferably, the first CH3 domain contains a "knob" structure, and the second CH3 domain contains a "hole" structure; more preferably, the "knob" structure contains amino acid substitutions of S354C and T366W, and the "hole" structure contains amino acid substitutions of Y349C, T366S, L368A, and Y407V.

[0025] Preferably, to reduce the ADCC activity of the antibody, the first and / or second Fc domains contain amino acid substitutions of L234A, L235A and / or G237A.

[0026] Preferably, the second Fc domain of the third polypeptide contains an amino acid substitution of H435R.

[0027] Preferably, the Fc domain is derived from IgG1.

[0028] In one embodiment, the heavy chain of the first polypeptide is designed as a "knob" structure, including amino acid substitutions at four sites: Q39E, T139K, K147R, and V185S; the second polypeptide includes amino acid substitutions at four sites: Q38K, S114D, S131D, and L135H; and the heavy chain of the third polypeptide is designed as a "hole" structure, with amino acid substitutions at three sites: Q39K, T139D, and K147D; and the fourth polypeptide includes amino acid substitutions at four sites: Q38E, S114K, S131R, and L135T (amino acid positions are numbered according to Kabat).

[0029] In one embodiment, the N-terminus of the first Fc domain is fused with the C-terminus of the scFv domain. Preferably, the N-terminus of the first Fc domain is fused with the C-terminus of the scFv domain via a second connector. Preferably, the C-terminus of the single-chain antibody (scFv) domain is fused with the N-terminus of the second connector, and the C-terminus of the second connector is fused with the N-terminus of the first Fc domain.

[0030] In one embodiment, the antigen-binding fragment Fab comprises a heavy chain domain and a light chain domain. The heavy chain domain of the antigen-binding fragment Fab comprises a heavy chain variable region and a CH1 domain of an immunoglobulin, with the C-terminus of the heavy chain variable region fused to the N-terminus of the CH1 domain. The light chain domain of the antigen-binding fragment Fab comprises a light chain variable region and a light chain constant region of an immunoglobulin, with the C-terminus of the light chain variable region fused to the N-terminus of the light chain constant region.

[0031] In one embodiment, the heavy chain domain of the antigen-binding fragment Fab of the first polypeptide is connected to the scFv domain via a third connector, wherein the C-terminus of the heavy chain domain of the antigen-binding fragment Fab is fused to the N-terminus of the third connector, and the C-terminus of the third connector is fused to the N-terminus of the scFv domain. Preferably, the third connector comprises the amino acid sequence (G4S)n, where n is any integer from 1 to 10. More preferably, the third connector comprises the amino acid sequence EPKSC(G4S)n, where n is any integer from 1 to 10. The sequence of EPKSC(G4S)n is SEQ ID NO:69.

[0032] In one embodiment, the C-terminus of the heavy chain single-domain antibody (VHH) domain of the third polypeptide, which is capable of specifically binding to the third antigen, is fused to the N-terminus of the second Fc domain. Preferably, the C-terminus of the VHH domain is fused to the N-terminus of the second Fc domain via a fourth linker. Preferably, the fourth linker comprises the amino acid sequence EPKSS (SEQ ID NO:70).

[0033] In one embodiment, the first polypeptide comprises the following structure: Fab heavy chain domain - third linker - scFv domain - second linker - first Fc domain.

[0034] Preferably, the first polypeptide comprises the following structure: Fab heavy chain variable region - Fab CH1 - third linker - scFv heavy chain variable region - first linker - scFv light chain variable region - second linker - first CH2 - first CH3.

[0035] In one embodiment, the second polypeptide comprises the following structure: Fab light chain variable region - light chain constant region.

[0036] In one embodiment, the third polypeptide comprises a VHH domain followed by a second Fc domain. Preferably, the third polypeptide comprises a VHH-fourth linker-second CH2-second CH3. In another embodiment, the third polypeptide comprises a Fab heavy chain domain followed by a second Fc domain, preferably comprising a Fab heavy chain variable region-Fab CH1-second CH2-second CH3, and the trispecific antigen-binding molecule comprises a fourth polypeptide comprising a Fab light chain variable region-light chain constant region.

[0037] In one embodiment, the antigen-binding fragment Fab capable of specifically binding to the first antigen includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes HCDR1, HCDR2, and HCDR3, and the light chain variable region includes LCDR1, LCDR2, and LCDR3. The antigen-binding fragment Fab capable of specifically binding to the first antigen comprises HCDR1 as shown in SEQ ID NO:20, HCDR2 as shown in SEQ ID NO:21, and HCDR3 as shown in SEQ ID NO:22, and / or LCDR1 as shown in SEQ ID NO:23, LCDR2 as shown in SEQ ID NO:24, and LCDR3 as shown in SEQ ID NO:25; or, the antigen-binding fragment Fab capable of specifically binding to the first antigen comprises HCDR1 as shown in SEQ ID NO:26, HCDR2 as shown in SEQ ID NO:27, and HCDR3 as shown in SEQ ID NO:28, and / or LCDR1 as shown in SEQ ID NO:29, LCDR2 as shown in SEQ ID NO:30, and LCDR3 as shown in SEQ ID NO:31. Alternatively, the heavy chain variable region and light chain variable region of the antigen-binding fragment Fab capable of specifically binding to the first antigen contain the same HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences as the heavy chain variable regions and light chain variable regions of any of the following groups: (1) SEQ ID NO:9 and SEQ ID NO:10; (2) SEQ ID NO:11 and SEQ ID NO:12.

[0038] Preferably, the antigen-binding fragment Fab capable of specifically binding to the first antigen includes a heavy chain variable region as shown in SEQ ID NO:9, and / or a light chain variable region as shown in SEQ ID NO:10; or, the antigen-binding fragment Fab capable of specifically binding to the first antigen includes a heavy chain variable region as shown in SEQ ID NO:11, and / or a light chain variable region as shown in SEQ ID NO:12.

[0039] More preferably, the antigen-binding fragment Fab capable of specifically binding to the first antigen comprises a heavy chain domain as shown in SEQ ID NO:53 and / or a light chain domain as shown in SEQ ID NO:3; or, the antigen-binding fragment Fab capable of specifically binding to the first antigen comprises a heavy chain domain sequence as shown in SEQ ID NO:68 and / or a light chain domain sequence as shown in SEQ ID NO:7.

[0040] In one embodiment, the single-chain antibody (scFv) domain includes HCDR1 as shown in SEQ ID NO:44, HCDR2 as shown in SEQ ID NO:45, HCDR3 as shown in SEQ ID NO:46, LCDR1 as shown in SEQ ID NO:47, LCDR2 as shown in SEQ ID NO:48, and LCDR3 as shown in SEQ ID NO:49.

[0041] Preferably, the single-chain antibody (scFv) domain comprises a heavy chain variable region as shown in SEQ ID NO:17 and a light chain variable region as shown in SEQ ID NO:18; more preferably, the single-chain antibody (scFv) domain comprises an amino acid sequence as shown in SEQ ID NO:19.

[0042] In one embodiment, the VHH domain comprises HCDR1 as shown in SEQ ID NO:32, HCDR2 as shown in SEQ ID NO:33, and HCDR3 as shown in SEQ ID NO:34; or, the VHH domain comprises HCDR1 as shown in SEQ ID NO:35, HCDR2 as shown in SEQ ID NO:36, and HCDR3 as shown in SEQ ID NO:37; or, the VHH domain comprises the same HCDR1, HCDR2, and HCDR3 sequences as those in the heavy chain variable region shown in SEQ ID NO:13 or SEQ ID NO:14; preferably, the VHH domain comprises the sequences shown in SEQ ID NO:13 or SEQ ID NO:14.

[0043] In another embodiment, the antigen-binding fragment Fab capable of specifically binding to the third antigen comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3. The antigen-binding fragment Fab capable of specifically binding to the third antigen comprises HCDR1 as shown in SEQ ID NO:38, HCDR2 as shown in SEQ ID NO:39, and HCDR3 as shown in SEQ ID NO:40, and / or LCDR1 as shown in SEQ ID NO:41, LCDR2 as shown in SEQ ID NO:42, and LCDR3 as shown in SEQ ID NO:43. Alternatively, the antigen-binding fragment Fab capable of specifically binding to the third antigen comprises HCDR1, HCDR2, and HCDR3 identical to those in the heavy chain variable region shown in SEQ ID NO:15, and LCDR1, LCDR2, and LCDR3 identical to those in the light chain variable region shown in SEQ ID NO:16.

[0044] Preferably, the antigen-binding fragment Fab capable of specifically binding to the third antigen comprises a heavy chain variable region as shown in SEQ ID NO:15, and / or a light chain variable region as shown in SEQ ID NO:16.

[0045] More preferably, the antigen-binding fragment Fab capable of specifically binding to the third antigen comprises a heavy chain domain as shown in SEQ ID NO:69, and / or a light chain domain as shown in SEQ ID NO:8.

[0046] In a preferred embodiment, the antigen-binding fragment Fab capable of specifically binding to the first antigen comprises HCDR1 as shown in SEQ ID NO:20, HCDR2 as shown in SEQ ID NO:21, and HCDR3 as shown in SEQ ID NO:22, and LCDR1 as shown in SEQ ID NO:23, LCDR2 as shown in SEQ ID NO:24, and LCDR3 as shown in SEQ ID NO:25; the single-chain antibody (scFv) domain comprises HCDR1 as shown in SEQ ID NO:44, HCDR2 as shown in SEQ ID NO:45, HCDR3 as shown in SEQ ID NO:46, and LCDR1 as shown in SEQ ID NO:47, LCDR2 as shown in SEQ ID NO:48, and LCDR3 as shown in SEQ ID NO:49; and the VHH domain comprises any one of the following sets of HCDR1, HCDR2, and HCDR3 sequences:

[0047] (1) SEQ ID NO:32, SEQ ID NO:33 and SEQ ID NO:34, or

[0048] (2) SEQ ID NO:35, SEQ ID NO:36 and SEQ ID NO:37.

[0049] Preferably, the antigen-binding fragment Fab capable of specifically binding to the first antigen comprises a heavy chain variable region as shown in SEQ ID NO:9 and a light chain variable region as shown in SEQ ID NO:10, the single-chain antibody (scFv) domain comprises a heavy chain variable region sequence as shown in SEQ ID NO:17 and a light chain variable region sequence as shown in SEQ ID NO:18, and the VHH domain comprises a sequence as shown in SEQ ID NO:13 or SEQ ID NO:14.

[0050] More preferably, the antigen-binding fragment Fab capable of specifically binding to the first antigen comprises a heavy chain domain as shown in SEQ ID NO:53 and a light chain domain as shown in SEQ ID NO:3, the single-chain antibody (scFv) domain comprises the amino acid sequence shown in SEQ ID NO:19, and the VHH domain comprises the sequence shown in SEQ ID NO:13 or SEQ ID NO:14.

[0051] In another preferred embodiment, the antigen-binding fragment Fab capable of specifically binding to the first antigen comprises HCDR1 as shown in SEQ ID NO:26, HCDR2 as shown in SEQ ID NO:27, and HCDR3 as shown in SEQ ID NO:28, and LCDR1 as shown in SEQ ID NO:29, LCDR2 as shown in SEQ ID NO:30, and LCDR3 as shown in SEQ ID NO:31; the single-chain antibody (scFv) domain comprises HCDR1 as shown in SEQ ID NO:44, HCDR2 as shown in SEQ ID NO:45, HCDR3 as shown in SEQ ID NO:46, LCDR1 as shown in SEQ ID NO:47, LCDR2 as shown in SEQ ID NO:48, and LCDR3 as shown in SEQ ID NO:49; and the antigen-binding fragment Fab capable of specifically binding to the third antigen comprises HCDR1 as shown in SEQ ID NO:38, HCDR2 as shown in SEQ ID NO:39, and HCDR3 as shown in SEQ ID NO:40, and... LCDR1 as shown in NO:41, LCDR2 as shown in SEQ ID NO:42, and LCDR3 as shown in SEQ ID NO:43.

[0052] Preferably, the antigen-binding fragment Fab capable of specifically binding to the first antigen comprises a heavy chain variable region as shown in SEQ ID NO:11 and a light chain variable region as shown in SEQ ID NO:12, the single-chain antibody (scFv) domain comprises a heavy chain variable region as shown in SEQ ID NO:17 and a light chain variable region as shown in SEQ ID NO:18, and the antigen-binding fragment Fab capable of specifically binding to the third antigen comprises a heavy chain variable region as shown in SEQ ID NO:15 and a light chain variable region as shown in SEQ ID NO:16.

[0053] More preferably, the antigen-binding fragment Fab capable of specifically binding to the first antigen comprises a heavy chain domain sequence as shown in SEQ ID NO:68 and a light chain domain sequence as shown in SEQ ID NO:7, the single-chain antibody (scFv) domain comprises an amino acid sequence as shown in SEQ ID NO:19, and the antigen-binding fragment Fab capable of specifically binding to the third antigen comprises a heavy chain domain sequence as shown in SEQ ID NO:69 and a light chain domain sequence as shown in SEQ ID NO:8.

[0054] In one embodiment, the first Fc domain comprises the amino acid sequence shown in SEQ ID NO:50, and the second Fc domain comprises the amino acid sequence shown in SEQ ID NO:51 or 55; preferably, when the trispecific antigen-binding molecule comprises a VHH domain capable of specifically binding to a third antigen, the second Fc domain comprises the amino acid sequence shown in SEQ ID NO:51, or when the trispecific antigen-binding molecule comprises a Fab domain capable of specifically binding to a third antigen, the second Fc domain comprises the amino acid sequence shown in SEQ ID NO:55.

[0055] In one embodiment, the trispecific antigen-binding molecule comprises: a first polypeptide comprising the sequence shown in SEQ ID NO:1, a second polypeptide comprising the sequence shown in SEQ ID NO:3, and a third polypeptide comprising the sequence shown in SEQ ID NO:2; or, a first polypeptide comprising the sequence shown in SEQ ID NO:1, a second polypeptide comprising the sequence shown in SEQ ID NO:3, and a third polypeptide comprising the sequence shown in SEQ ID NO:4; or, a first polypeptide comprising the sequence shown in SEQ ID NO:5, a second polypeptide comprising the sequence shown in SEQ ID NO:7, a third polypeptide comprising the sequence shown in SEQ ID NO:6, and a fourth polypeptide comprising the sequence shown in SEQ ID NO:8.

[0056] This disclosure also provides a nucleic acid molecule that encodes the three specific antigen-binding molecules described herein.

[0057] This disclosure also provides a carrier containing the aforementioned nucleic acid molecule.

[0058] In one embodiment, the carrier is an expression carrier.

[0059] This disclosure also provides a host cell containing the aforementioned nucleic acid molecule or vector; preferably, the host cell is a prokaryotic cell or a eukaryotic cell; the prokaryotic cell is preferably Escherichia coli; the eukaryotic cell is preferably a mammalian cell or yeast; more preferably, the mammalian cell is a CHO cell, an Expi293 cell, or a HEK293 cell.

[0060] This disclosure also provides a method for preparing a trispecific antigen-binding molecule, the method comprising: culturing the host cell under suitable conditions, preferably further comprising purifying the expression product from the cell.

[0061] This disclosure also provides antibody-drug conjugates, which are formed by conjugating the aforementioned trispecific antigen-binding molecule with other bioactive molecules; preferably, the other bioactive molecules are small molecule drugs; preferably, the trispecific antigen-binding molecule and the other bioactive molecules are connected by a linker.

[0062] This disclosure also provides pharmaceutical compositions comprising the aforementioned trispecific antigen-binding molecules, nucleic acid molecules, expression vectors, host cells, and / or antibody-drug conjugates. Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. Preferably, the pharmaceutical composition further comprises one or more additional therapeutic agents. Preferably, the pharmaceutical composition is intended for the treatment, relief, and / or prevention of autoimmune diseases or tumors. Preferably, the tumor is selected from: lymphomas such as B-cell lymphomas, including subtypes such as diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), and mantle cell lymphoma (MCL), and metastatic carcinomas of the above tumors.

[0063] This disclosure also provides the use of the aforementioned trispecific antigen-binding molecules, nucleic acid molecules, carriers, host cells, pharmaceutical compositions, and / or antibody-drug conjugates in the preparation of medicaments for treating, alleviating, and / or preventing autoimmune diseases or tumors. Preferably, the tumor is a CD79b-positive and / or CD20-positive tumor. Preferably, the tumor is selected from: lymphomas such as B-cell lymphomas, including subtypes such as diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), and mantle cell lymphoma (MCL), as well as metastatic tumors of the above-mentioned tumors.

[0064] This disclosure also provides a method for inducing cell death in cells expressing CD79b and / or CD20, the method comprising contacting the cells with the trispecific antigen-binding molecule, nucleic acid molecule, carrier, host cell, antibody-drug conjugate, and / or pharmaceutical composition, wherein the cells expressing CD79b and / or CD20 are immune cells or tumor cells. Preferably, the tumor is selected from: lymphomas such as B-cell lymphoma, including subtypes such as diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), and mantle cell lymphoma (MCL), and metastatic carcinomas of the above tumors.

[0065] In one embodiment, the method is in vivo or in vitro.

[0066] This disclosure also provides a method for treating a disease in a subject, the method comprising administering to a subject in need an effective amount of the trispecific antigen-binding molecule, nucleic acid molecule, carrier, host cell, antibody-drug conjugate, and / or pharmaceutical composition. Preferably, the disease is associated with expression of CD79b and / or CD20. Preferably, the disease is an autoimmune disease or a tumor. Preferably, the subject has relapsed or refractory history of treatment with a prior anticancer agent. In one embodiment, the method further comprises administering an additional therapeutic agent to the subject. Preferably, the tumor or tumor cells are selected from: lymphomas such as B-cell lymphoma, including subtypes such as diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), and mantle cell lymphoma (MCL), and metastatic carcinomas of the above tumors.

[0067] This disclosure also provides a method for treating an autoimmune disease in a subject, the method comprising administering to a subject in need an effective amount of the trispecific antigen-binding molecule, nucleic acid molecule, carrier, host cell, antibody-drug conjugate, and / or pharmaceutical composition.

[0068] A second aspect of this disclosure is to provide an anti-CD79b antibody or an antigen-binding fragment thereof. In one embodiment, the anti-CD79b antibody or the antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, and the light chain variable region comprising LCDR1, LCDR2, and LCDR3. The heavy chain variable region and the light chain variable region comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences identical to those of any of the following groups of heavy chain variable regions and light chain variable regions:

[0069] (1) SEQ ID NO: 9 and 10; or

[0070] (2) SEQ ID NO: 11 and 12;

[0071] Alternatively, the heavy chain variable region and the light chain variable region comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences selected from any one of the following:

[0072] (1) SEQ ID NO: 20, 21, 22, 23, 24 and 25;

[0073] (2) SEQ ID NO: 26, 27, 28, 29, 30 and 31; or

[0074] (3)SEQ ID NO:26, 27, 62, 29, 63 and 31.

[0075] In one embodiment, the anti-CD79b antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region each comprise an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of the following groups:

[0076] (1) SEQ ID NO: 9 and 10;

[0077] (2) SEQ ID NO: 58 and 59;

[0078] (3) SEQ ID NO: 11 and 12; or

[0079] (4) SEQ ID NO: 60 and 61.

[0080] In a preferred embodiment, the anti-CD79b antibody or its antigen-binding fragment further includes a heavy chain constant region and / or a light chain constant region. Preferably, the heavy chain constant region includes a natural Fc or a modified variant Fc. More preferably, the Fc is derived from a mouse or a human.

[0081] In a preferred embodiment, the anti-CD79b antibody or its antigen-binding fragment described in this disclosure is a murine antibody, a chimeric antibody, a fully human antibody, or a humanized antibody.

[0082] In a preferred embodiment, the anti-CD79b antibody or its antigen-binding fragment is a monoclonal antibody or its antigen-binding fragment, a bispecific antigen-binding molecule or its antigen-binding fragment, or a multispecific antigen-binding molecule or its antigen-binding fragment.

[0083] In a preferred embodiment, the anti-CD79b antibody is a full-length antibody.

[0084] In a preferred embodiment, the anti-CD79b antibody or its antigen-binding fragment described in this disclosure is in the form of IgG1, IgG2, IgG3 or IgG4.

[0085] In a preferred embodiment, the antigen-binding fragment for anti-CD79b described in this disclosure is Fab, Fv, scFv, F(ab')2, a linear antibody, or a heavy chain single-domain antibody.

[0086] In one embodiment, this disclosure provides a conjugate comprising the aforementioned anti-CD79b antibody or its antigen-binding fragment, and a capture or detection marker conjugated thereto. The detection marker includes, but is not limited to, radionuclides, luminescent substances (e.g., fluorescein), colored substances, or enzymes.

[0087] In one embodiment, this disclosure provides a fusion protein, wherein one fused portion comprises an antibody against CD79b of this disclosure or an antigen-binding fragment thereof.

[0088] In one embodiment, this disclosure provides a fusion protein comprising an antibody against CD79b of this disclosure or an antigen-binding fragment thereof.

[0089] In one embodiment, this disclosure provides a bispecific antibody or an antigen-binding fragment thereof, or a multispecific antibody or an antigen-binding fragment thereof, wherein one antigen-binding domain of the bispecific antibody or its antigen-binding fragment, or the multispecific antibody or its antigen-binding fragment thereof, comprises the anti-CD79b antibody or its antigen-binding fragment of this disclosure.

[0090] In one embodiment, this disclosure provides a bispecific antibody or an antigen-binding fragment thereof, or a multispecific antibody or an antigen-binding fragment thereof, wherein the bispecific antibody or antigen-binding fragment thereof or the multispecific antibody or antigen-binding fragment thereof comprises the anti-CD79b antibody or antigen-binding fragment thereof disclosed herein.

[0091] This disclosure also provides an antibody-drug conjugate (ADC) comprising the anti-CD79b antibody or its antigen-binding fragment of this disclosure, or the fusion protein of this disclosure, or the specific antibody or its antigen-binding fragment or multispecific antibody or its antigen-binding fragment of this disclosure, and other bioactive molecules conjugated thereto. Preferably, the other bioactive molecules are small molecule drugs, such as antitumor drugs, and more preferably, the antitumor drugs are antitumor compounds. Preferably, the anti-CD79b antibody or its antigen-binding fragment, the fusion protein of this disclosure, or the specific antibody or its antigen-binding fragment or multispecific antibody or its antigen-binding fragment of this disclosure are connected to the other bioactive molecules via a linker.

[0092] This disclosure also provides a chimeric antigen receptor (CAR) or a cell containing said chimeric antigen receptor (e.g., CAR-T cells) comprising the anti-CD79b antibody of this disclosure or its antigen-binding fragment, the fusion protein of this disclosure, or the bispecific antibody of this disclosure or its antigen-binding fragment, or the multispecific antibody or its antigen-binding fragment.

[0093] This disclosure also provides nucleic acids encoding the anti-CD79b antibody or its antigen-binding fragment, the fusion protein, or the bispecific antibody or its antigen-binding fragment, or the multispecific antibody or its antigen-binding fragment, as well as recombinant vectors containing the nucleic acids, and host cells containing the aforementioned nucleic acids or vectors. Preferably, the host cell is a prokaryotic cell (preferably Escherichia coli) or a eukaryotic cell (preferably mammalian cells or yeast; more preferably, the mammalian cell is a CHO cell or a HEK293 cell).

[0094] This disclosure also provides a method for preparing an anti-CD79b antibody or its antigen-binding fragment, the fusion protein, or the bispecific antibody or its antigen-binding fragment, or a multispecific antibody or its antigen-binding fragment, the method comprising: culturing the host cells under suitable conditions, and purifying the expression product from the cells.

[0095] This disclosure also provides the use of the described anti-CD79b antibody or its antigen-binding fragment in the preparation of detection or diagnostic reagents.

[0096] In one embodiment, the detection reagent is used to detect CD79b expression; preferably, the diagnostic reagent is used to diagnose tumors; preferably, the tumor is selected from: lymphomas such as B-cell lymphomas, including subtypes such as diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), and mantle cell lymphoma (MCL), as well as metastatic tumors of the above-mentioned tumors.

[0097] This disclosure also provides a method for detecting CD79b expression in a sample, the method comprising:

[0098] (1) Contact the sample with the CD79b antigen-binding molecule or its antigen-binding fragment disclosed herein;

[0099] (2) Detect the formation of a complex of the anti-CD79b antibody or its antigen-binding fragment with CD79b; optionally, the anti-CD79b antibody or its antigen-binding fragment is detectably labeled.

[0100] This disclosure also provides a pharmaceutical composition comprising the anti-CD79b antibody of this disclosure or its antigen-binding fragment, or comprising the fusion protein of this disclosure, or comprising a bispecific antibody or its antigen-binding fragment, or a multispecific antibody or its antigen-binding fragment, or comprising an antibody-drug conjugate of this disclosure, or comprising the fusion protein of this disclosure, or comprising CAR-T cells of this disclosure, or comprising the nucleic acid of this disclosure, or comprising the recombinant vector of this disclosure, or comprising the host cell of this disclosure. In one embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. Preferably, the pharmaceutical composition further comprises one or more additional therapeutic agents. Preferably, the pharmaceutical composition is intended for the treatment, relief, and / or prevention of autoimmune diseases or tumors. Preferably, the tumor is selected from: lymphomas such as B-cell lymphomas, including subtypes such as diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), and mantle cell lymphoma (MCL), and metastatic tumors of the above tumors.

[0101] This disclosure also provides the use of anti-CD79b antibodies or antigen-binding fragments thereof, or the pharmaceutical compositions described above, in the preparation of medicaments for treating or alleviating tumors or autoimmune diseases. Preferably, the disease is associated with CD79b expression. Preferably, the tumor is selected from: lymphomas such as B-cell lymphomas, including subtypes such as diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), and mantle cell lymphoma (MCL), and metastatic carcinomas of the above tumors.

[0102] This disclosure also provides a method of treating a disease in a subject, comprising administering to the subject in need an effective amount of the anti-CD79b antibody or its antigen-binding fragment, or the pharmaceutical composition described above. Preferably, the disease is a disease associated with CD79b expression. Preferably, the disease is a tumor or an autoimmune disease; preferably, the tumor is selected from lymphomas such as B-cell lymphomas, including subtypes such as diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), and mantle cell lymphoma (MCL), as well as metastatic tumors of the above-mentioned tumors. More preferably, it further comprises administering an additional therapeutic agent to the subject.

[0103] A third aspect of this disclosure is to provide an anti-CD20 antibody or an antigen-binding fragment thereof. In one embodiment, the anti-CD20 antibody or the antigen-binding fragment thereof comprises a heavy chain variable region, the heavy chain variable region comprising HCDR1, HCDR2, and HCDR3. The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 sequences identical to those of any of the following groups of heavy chain variable regions:

[0104] (1)SEQ ID NO:64;

[0105] (2)SEQ ID NO:13;

[0106] (3) SEQ ID NO:65; or

[0107] (4)SEQ ID NO:14.

[0108] Alternatively, the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 sequences selected from any of the following:

[0109] (1) SEQ ID NO: 66, 67 and 34;

[0110] (2) SEQ ID NO: 32, 33 and 34; or

[0111] (3)SEQ ID NO:35, 36 and 37.

[0112] In one embodiment, the anti-CD20 antibody or its antigen-binding fragment comprises a heavy chain variable region, the heavy chain variable region comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with a heavy chain variable region of any of the following groups:

[0113] (1)SEQ ID NO:64;

[0114] (2)SEQ ID NO:13;

[0115] (3) SEQ ID NO:14; or

[0116] (4)SEQ ID NO:65.

[0117] In a preferred embodiment, the anti-CD20 antibody or its antigen-binding fragment further includes a heavy chain constant region and / or a light chain constant region. Preferably, the heavy chain constant region includes a natural Fc or a modified variant Fc. More preferably, the Fc is derived from a mouse or a human.

[0118] In a preferred embodiment, the anti-CD20 antibody or its antigen-binding fragment described in this disclosure is a mouse antibody, an alpaca antibody, a chimeric antibody, a fully human antibody, or a humanized antibody.

[0119] In a preferred embodiment, the anti-CD20 antibody or its antigen-binding fragment is a monoclonal antibody or its antigen-binding fragment, a bispecific antigen-binding molecule or its antigen-binding fragment, or a multispecific antigen-binding molecule or its antigen-binding fragment.

[0120] In a preferred embodiment, the anti-CD20 antibody is a full-length antibody.

[0121] In a preferred embodiment, the anti-CD20 antibody or its antigen-binding fragment described in this disclosure is in the form of IgG1, IgG2, IgG3 or IgG4.

[0122] In a preferred embodiment, the antigen-binding fragment for anti-CD20 described in this disclosure is Fab, Fv, scFv, F(ab')2, a linear antibody, or a heavy chain single-domain antibody.

[0123] In one embodiment, this disclosure provides a conjugate formed by conjugating an anti-CD20 antibody or its antigen-binding fragment to a capture marker or detection marker. The detection marker includes, but is not limited to, radionuclides, luminescent substances (e.g., fluorescein), colored substances, or enzymes.

[0124] In one embodiment, this disclosure provides a fusion protein, wherein one fused portion comprises an antibody against CD20 of this disclosure or an antigen-binding fragment thereof.

[0125] In one embodiment, this disclosure provides a bispecific antibody or antigen-binding fragment thereof, or a multispecific antibody or antigen-binding fragment thereof, wherein an antigen-binding domain of the bispecific antibody or antigen-binding fragment thereof or the multispecific antibody or antigen-binding fragment thereof comprises an antibody or antigen-binding fragment thereof containing CD20 of this disclosure.

[0126] This disclosure also provides an antibody-drug conjugate (ADC), which is formed by conjugating the anti-CD20 antibody or its antigen-binding fragment, or the fusion protein of this disclosure, or the specific antibody or its antigen-binding fragment, or the multispecific antibody or its antigen-binding fragment, of this disclosure with other bioactive molecules; preferably, the other bioactive molecules are small molecule drugs, such as antitumor drugs, more preferably, the antitumor drugs are antitumor compounds; preferably, the anti-CD20 antibody or its antigen-binding fragment, the fusion protein of this disclosure, or the specific antibody or its antigen-binding fragment, or the multispecific antibody or its antigen-binding fragment, of this disclosure with the other bioactive molecules are connected by a linker.

[0127] This disclosure also provides a chimeric antigen receptor (CAR) or a cell containing said chimeric antigen receptor (e.g., CAR-T cells) comprising the anti-CD20 antibody of this disclosure or its antigen-binding fragment, the fusion protein of this disclosure, or the bispecific antibody of this disclosure or its antigen-binding fragment, or the multispecific antibody or its antigen-binding fragment.

[0128] This disclosure also provides nucleic acids encoding the anti-CD20 antibody or its antigen-binding fragment, the fusion protein, or the bispecific antibody or its antigen-binding fragment, or the multispecific antibody or its antigen-binding fragment, as well as recombinant vectors containing the nucleic acids, and host cells containing the aforementioned nucleic acids or vectors. Preferably, the host cell is a prokaryotic cell (preferably Escherichia coli) or a eukaryotic cell (preferably mammalian cells or yeast; more preferably, the mammalian cell is a CHO cell or a HEK293 cell).

[0129] This disclosure also provides a method for preparing an anti-CD20 antibody or an antigen-binding fragment thereof, the fusion protein, or the bispecific antibody or an antigen-binding fragment thereof, or a multispecific antibody or an antigen-binding fragment thereof, the method comprising: culturing the host cells under suitable conditions, and purifying the expression product from the cells.

[0130] This disclosure also provides the use of the described anti-CD20 antibody or its antigen-binding fragment in the preparation of detection or diagnostic reagents.

[0131] In one embodiment, the detection reagent is used to detect CD20 expression; the diagnostic reagent is used to diagnose a tumor; preferably, the tumor is selected from: lymphomas such as B-cell lymphoma, including subtypes such as diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), and mantle cell lymphoma (MCL), as well as metastatic tumors of the above-mentioned tumors.

[0132] This disclosure also provides a method for detecting CD20 expression in a sample, the method comprising:

[0133] (1) Contact the sample with the CD20 antigen-binding molecule or its antigen-binding fragment disclosed herein;

[0134] (2) Detect the formation of a complex of the anti-CD20 antibody or its antigen-binding fragment with CD20; optionally, the anti-CD20 antibody or its antigen-binding fragment is detectably labeled.

[0135] This disclosure also provides a pharmaceutical composition comprising the anti-CD20 antibody of this disclosure or its antigen-binding fragment, or comprising an antibody-drug conjugate of this disclosure, or comprising a fusion protein of this disclosure, or comprising a specific antibody or its antigen-binding fragment or a multispecific antibody or its antigen-binding fragment of this disclosure, or comprising CAR-T cells of this disclosure, or comprising nucleic acids of this disclosure, or comprising a recombinant vector of this disclosure, or comprising host cells of this disclosure. In one embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. Preferably, the pharmaceutical composition further comprises one or more additional therapeutic agents. Preferably, the pharmaceutical composition is intended for the treatment, relief, and / or prevention of autoimmune diseases or tumors. Preferably, the tumor is selected from: lymphomas such as B-cell lymphomas, including subtypes such as diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), and mantle cell lymphoma (MCL), and metastatic tumors of the above tumors.

[0136] This disclosure also provides the use of anti-CD20 antibodies or antigen-binding fragments thereof, or the above-described pharmaceutical compositions or antibody-drug conjugates, in the preparation of medicaments for treating or alleviating tumors or autoimmune diseases. Preferably, the disease is a disease associated with CD20 expression. Preferably, the tumor is selected from: lymphomas such as B-cell lymphomas, including subtypes such as diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), and mantle cell lymphoma (MCL), and metastatic carcinomas of the above tumors.

[0137] This disclosure also provides a method of treating a disease in a subject, comprising administering to the subject in need an effective amount of the anti-CD20 antibody or its antigen-binding fragment, or the pharmaceutical composition described above. Preferably, the disease is a disease associated with CD20 expression. Preferably, the disease is a tumor or an autoimmune disease; preferably, the tumor is selected from lymphomas such as B-cell lymphomas, including subtypes such as diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), and mantle cell lymphoma (MCL), as well as metastatic tumors of the above-mentioned tumors. More preferably, it further comprises administering an additional therapeutic agent to the subject. Attached Figure Description

[0138] The accompanying drawings further illustrate the novel features disclosed herein. A better understanding of the features and advantages disclosed herein will be achieved by referring to these drawings; however, it should be understood that these drawings are for illustrating specific embodiments of the principles disclosed herein and are not intended to limit the scope of the appended claims.

[0139] Figure 1 shows the structures of the three trispecific antibodies constructed in the example, namely triantibody 1, triantibody 2 and triantibody 3.

[0140] Figure 2A shows the binding of triple antibody 1, triple antibody 2 and triple antibody 2 to hCD20-expressing cells (hCD20-CHOK1 cells).

[0141] Figure 2B shows the binding of triple antibody 1, triple antibody 2, and triple antibody 2 to hCD79b-expressing cells (hCD79b-CHOK1 cells).

[0142] Figure 2C shows the binding of triple antibody 1, triple antibody 2, and triple antibody 2 to hCD3-expressing cells (Jurkat cells).

[0143] Figure 2D shows the binding of triple antibody 1, triple antibody 2 and triple antibody 2 to cells co-expressing CD20 and CD79b (SU-DHL-4 cells).

[0144] Figure 2E shows the binding of triple antibody 1, triple antibody 2, and triple antibody 2 to cells co-expressing CD20 and CD79b (OCI-LY19 cells).

[0145] Figure 3A shows the results of a PBMC-mediated cytotoxicity assay, with SU-DHL-4 cells as the target cells.

[0146] Figure 3B shows the results of the PBMC cell-mediated cytotoxicity assay, with OCI-LY19 cells as the target cells.

[0147] Figure 3C shows the release of cytokines under the condition of co-incubation of PBMC and SU-DHL-4 tumor cells with triple antibody molecules.

[0148] Figure 3D shows the release of cytokines under the condition of co-incubation of PBMC and OCI-LY19 tumor cells with triple antibody molecules.

[0149] Figure 3E shows the cell survival of the three anti-antibody molecules on Ramos tumor cells.

[0150] Figure 4 shows the preliminary pharmacokinetic blood concentration curves of the three-antibody molecule in cynomolgus monkeys.

[0151] Figure 5 shows the efficacy of the three-antibody molecules in a mouse model reconstructed with human immune cells (tumor cells were OCI-LY19).

[0152] Figure 6 shows the efficacy of the three-antibody molecules in a mouse model reconstructed with human immune cells (tumor cells were WSU-DLCL2).

[0153] Figure 7 shows the binding of the mouse-derived anti-CD79b chimeric antibody of this disclosure to CD79b-expressing cells (hCD79b-CHOK1 cells).

[0154] Figure 8 shows the binding of the alpaca-derived anti-CD20 chimeric antibody of this disclosure to CD20-expressing cells (hCD20-CHOK1 cells).

[0155] Figures 9A and 9B show the binding of the humanized anti-CD79b monoclonal antibody of this disclosure to CD79b-expressing cells (hCD79b-CHOK1 cells).

[0156] Figures 9C and 9D show the binding of the humanized anti-CD20 monoclonal antibody of this disclosure to CD20-expressing cells (hCD20-CHOK1 cells).

[0157] Invention Details

[0158] the term

[0159] Unless otherwise stated, the terms used herein have their general meanings within the technical field. A particular term or phrase should not be considered uncertain or unclear unless specifically defined, but should be understood in its ordinary sense. When trade names appear herein, they are intended to refer to the corresponding product or its active ingredient.

[0160] All publications, patents and patent applications mentioned in this specification are incorporated herein by reference as if specifically and individually indicated that each individual publication, patent or patent application is incorporated by reference.

[0161] Before this disclosure is described in detail below, it should be understood that this disclosure is not limited to the specific methodologies, procedures, and reagents described herein, as these can vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of this disclosure.

[0162] Some embodiments disclosed herein include numerical ranges, and certain aspects of this disclosure may be described using ranges. Unless otherwise stated, it should be understood that numerical ranges or descriptions using ranges are for purposes of brevity and convenience only and should not be considered as a strict limitation of the scope of this disclosure. Therefore, descriptions using ranges should be considered as specifically disclosing all possible subranges and all possible specific numerical points within those ranges, as these subranges and numerical points have been explicitly stated herein. The above principles apply equally regardless of the breadth of the numerical values. When a range description is used, the range includes the endpoints of the range. For example, a description of a range from 1 to 6 should be considered as specifically disclosing subranges from 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and specific numerical points within those ranges, such as 1, 2, 3, 4, 5, 6. The above principles apply equally regardless of the breadth of the numerical values. When a range description is used, the range includes the endpoints of the range.

[0163] When referring to measurable values ​​such as quantities, temporary durations, etc., the term “about” means a variation of ±20%, or in some cases ±10%, or in some cases ±5%, or in some cases ±1%, or in some cases ±0.1% of the specified value.

[0164] The terms “immunoglobulin β chain” and “CD79b” specifically include human CD79b protein, and include variants, subtypes, homologs and analogs of human CD79b that share at least one common epitope with CD79b (e.g., human CD79b). Exemplary human CD79b sequences are available in NCBI reference sequences: NM_000626.3 and NP_000617.1, UniProtKB / Swiss-Prot accession number P40259.

[0165] The term "CD20" specifically includes the human CD20 protein and includes variants, subtypes, homologs, and analogs of human CD20 that share at least one common epitope with CD20 (e.g., human CD20), encoded by the MS4A1 gene. Exemplary human CD20 sequences are available in NCBI reference sequences: NM_021950.3 and NP_068769.2, UniProtKB / Swiss-Prot accession number P11836.

[0166] The three-letter and single-letter codes for amino acids used in this article are as described in J. BIOL. CHEM, 243, P3558 (1968).

[0167] As used in this article, the term "antibody" typically refers to a Y-type tetrameric protein comprising two heavy (H) polypeptide chains and two light (L) polypeptide chains held together by covalent disulfide bonds and non-covalent interactions. Natural IgG antibodies possess this structure. Each light chain contains a light chain variable domain (VL) and a light chain constant domain (CL). Each heavy chain contains a heavy chain variable domain (VH) and a heavy chain constant domain (CH), or heavy chain constant region (CH).

[0168] The term "antibody" in this article may include complete antibodies (e.g., full-length monoclonal antibodies) and any antigen-binding fragment (i.e., antigen-binding part) or its single chain, and may also include products with antigen-specific binding ability formed by modifying complete antibodies or their antigen-binding fragments or their single chains (e.g., linking other peptides, rearranging functional units, etc.).

[0169] Five main classes of antibodies are known in this art: IgA, IgD, IgE, IgG, and IgM, with their corresponding heavy chain constant domains designated α, δ, ε, γ, and μ, respectively. IgG and IgA can be further subdivided into different subclasses; for example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4, and IgA into IgA1 and IgA2. The light chain of antibodies from any vertebrate species can be identified as one of two distinctly different types based on the amino acid sequence of their constant domains, termed κ and λ.

[0170] In the case of IgG, IgA, and IgD antibodies, this heavy chain constant region contains three domains called CH1, CH2, and CH3 (IgM and IgE have a fourth domain, CH4). In the IgG, IgA, and IgD classes, the CH1 and CH2 domains are separated by a flexible hinge region, which is a variable-length segment rich in proline and cysteine. Each class of antibody further contains interchain and intrachain disulfide bonds formed by paired cysteine ​​residues.

[0171] The term "variable region" or "variable domain" indicates a significant change in the amino acid composition from one antibody to another and is primarily responsible for antigen recognition and binding. The variable region of each light / heavy chain pair forms the antigen-binding site, giving the complete IgG antibody two binding sites (i.e., it is bivalent). The variable region (VH) of the heavy chain and the variable region (VL) of the light chain each contain three regions with extreme variability, referred to as hypervariable regions (HVR), or more commonly, complementarity-determining regions (CDR). Each VH and VL has four backbone regions (FR), denoted as FR1, FR2, FR3, and FR4, respectively. Therefore, the CDR and FR sequences typically appear in the following sequence of the heavy chain variable domain (VH) (or light chain variable domain (VL)): FR1-HCDR1(LCDR1)-FR2-HCDR2(LCDR2)-FR3-HCDR3(LCDR3)-FR4.

[0172] The term "Fc" is used herein to define the C-terminal region of the immunoglobulin heavy chain, which comprises at least a portion of the constant region. This term includes both native sequence Fc regions and variant Fc regions. Unless otherwise stated, the amino acid residues in the Fc region or constant region are numbered according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0173] In this article, "antibody" can be used in the broadest sense, including polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies and primate-derived antibodies, CDR-grafted antibodies, human antibodies (including recombinant human antibodies), recombinant antibodies, intracellular antibodies, multispecific antibodies, bispecific antibodies, monovalent antibodies, multivalent antibodies, anti-individual genotype antibodies, synthetic antibodies (including mutant proteins and their variants), etc.

[0174] The terms “full-length antibody,” “complete antibody,” and “intact antibody” may be used interchangeably in this document to refer to antibodies whose structure is substantially similar to that of natural antibodies or that contain the FC region.

[0175] The term "monoclonal antibody" (or "mAb") refers to a substantially homogeneous antibody produced from a single cell clone that targets only a specific antigenic epitope. Monoclonal antibodies can be prepared using a variety of techniques known in the art, including hybridoma technology, recombinant technology, phage display technology, transgenic animals, synthetic technology, or combinations of the above.

[0176] The term "chimeric antibody" refers to an antibody in which the variable region is derived from one species and the constant region is derived from another species, such as an antibody in which the variable region is derived from a mouse antibody and the constant region is derived from a human antibody.

[0177] The term "humanized antibody" refers to a hybrid immunoglobulin, immunoglobulin chain, or fragment thereof containing a minimal sequence derived from a non-human immunoglobulin. In most cases, humanized antibodies are human immunoglobulins (receptor antibodies) where residues of the receptor's core sequence (CDR) are replaced by residues of a CDR from a non-human species (donor antibody) possessing the desired specificity, affinity, and performance, such as mice, rats, rabbits, or primates. In some cases, framework region residues of the human immunoglobulin are replaced by corresponding non-human residues. In certain circumstances, "reversion mutations" can be introduced into humanized antibodies where residues in one or more frame regions (FRs) of the variable region of the recipient human antibody are replaced by corresponding residues from a non-human species donor antibody. Such reversion mutations can help maintain the appropriate three-dimensional conformation of one or more grafted CDRs and thus improve affinity and antibody stability. Antibodies from a variety of donor species can be used, including but not limited to mice, rats, rabbits, or non-human primates. Additionally, humanized antibodies may contain novel residues not found in the recipient antibody or the donor antibody to further improve antibody performance.

[0178] It should be noted that the division of the CDR and FR in the variable region of the monoclonal antibody disclosed herein is determined according to the Kabat definition. Other nomenclature and numbering systems, such as Chothia, IMGT, or AHo, are also known to those skilled in the art. Therefore, humanized antibodies containing one or more CDRs derived from any nomenclature system based on the monoclonal antibody sequence of this disclosure are explicitly kept within the scope of this disclosure.

[0179] The terms "sequence identity," "sequence similarity," or "sequence homology" refer to the percentage of amino acid residues in a candidate sequence that are identical to those in a reference polypeptide sequence after aligning the sequences (and, where necessary, introducing gaps) to obtain the maximum percentage sequence identity, without considering any conserved substitutions as part of the sequence identity. Sequence alignment can be performed using various methods in the art to determine the percentage amino acid sequence identity, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN (DNASTAR) software. Those skilled in the art can determine suitable parameters for measuring the alignment, including any algorithm required to obtain the maximum alignment of the full length of the sequences being compared.

[0180] The term "antibody fragment" includes at least a portion of a complete antibody. As used herein, a "fraction" of an antibody molecule includes an "antigen-binding fragment" of the antibody, and the term "antigen-binding fragment" refers to a polypeptide fragment of an immunoglobulin or antibody that specifically binds to or reacts with a selected antigen or its epitope, or a fusion protein product further derived from such fragment, or a product conjugated with other compounds, such as a single-chain antibody, an extracellular binding region in a chimeric antigen receptor, etc. Exemplary antibody fragments or their antigen-binding fragments include, but are not limited to: variable light chain fragments (VL), variable heavy chain fragments (VH), Fab fragments, F(ab')2 fragments, Fd fragments, Fv fragments, heavy chain single-domain antibodies, linear antibodies, single-chain antibodies (scFv), and bispecific or multispecific antibodies formed from antibody fragments, etc.

[0181] The term "Fab" or "Fab fragment" includes a heavy chain variable region and a light chain variable region, and further includes a first constant region CH1 of the heavy chain and a constant region CL of the light chain, which is a monovalent antibody fragment. The heavy chain domain of "Fab" or "Fab fragment" as used herein includes the heavy chain variable region and CH1, and the light chain domain of "Fab" or "Fab fragment" as used herein includes the light chain variable region and CL. The term "F(ab')2" or "F(ab')2 fragment" includes two Fab fragments and a hinge region, which is a bivalent antibody fragment.

[0182] The term "Fd fragment" generally includes the heavy chain variable region and the constant region CH1; the term "Fv fragment" contains the antibody heavy chain variable region and the light chain variable region, but no constant region, and is the smallest antibody fragment with all antigen binding sites.

[0183] The term "scFv" refers to a fusion protein comprising at least one antibody fragment including a variable region of a light chain and at least one antibody fragment including a variable region of a heavy chain, wherein the light and heavy chain variable regions are adjacent (e.g., via a synthetic linker, such as a short, flexible peptide linker) and are capable of being expressed as a single-chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it originates. Unless otherwise specified, the scFv may have the VL and VH variable regions in any order (e.g., relative to the N-terminus and C-terminus of the polypeptide), and the scFv may comprise VL-linker-VH or may comprise VH-linker-VL.

[0184] The “VHH domain,” also known as a heavy chain single-domain antibody, VHH, VHH antibody fragment, VHH antibody, or nanobody, is a variable domain of an antigen-binding immunoglobulin called a “heavy chain antibody” (i.e., an antibody lacking a light chain). The term “VHH domain” is used to distinguish this variable domain from the heavy chain variable domain (referred to herein as the “VH domain”) and light chain variable domain (referred to herein as the “VL domain”) present in conventional tetrapeptide chain antibody structures. The VHH domain specifically binds to an epitope without the need for other antigen-binding domains (unlike the VH or VL domains in conventional tetrapeptide chain antibody structures, where the epitope is recognized by both the VL and VH domains). The VHH domain is a small, stable, and highly efficient antigen-recognition unit formed by a single immunoglobulin domain. The terms “heavy chain single-domain antibody,” “VHH domain,” “VHH,” “VHH domain,” “VHH antibody fragment,” “VHH antibody,” and “heavy chain antibody variable region” are used interchangeably. The “VHH domain” includes, but is not limited to, natural antibodies produced by camelids, antibodies produced by camelids that have been humanized, or antibodies obtained through phage display technology.

[0185] The term "fusion protein" refers to a larger molecule formed by linking different polypeptides / proteins together through genetic recombination or chemical methods. Linkers can be used for this linking, or not.

[0186] The term "antigen-binding molecule," in its broadest sense, refers to a molecule that specifically binds to an antigen or antigenic determinant. Examples of antigen-binding molecules are immunoglobulins and their derivatives, such as antibodies or their antigen-binding fragments. The term "multispecific" refers to an antigen-binding molecule capable of specifically binding to multiple different antigens or antigenic determinants. An example of the term "multispecific antigen-binding molecule" or "multispecific binding molecule" is "trispecific antigen-binding molecule" or "trispecific binding molecule," which refers to a binding molecule (e.g., a trispecific antibody or its antigen-binding fragment, or a molecule containing a trispecific antibody or its antigen-binding fragment), particularly a trispecific antibody, that specifically binds to three different antigens (or antigenic determinants).

[0187] When preparing antibodies, binding molecules, bispecific antigen-binding molecules or multispecific antigen-binding molecules using the variable regions of this disclosure, the constant regions are not particularly limited. Constant regions known to those skilled in the art or obtained independently can be used. Amino acid mutations (e.g., mutations that increase or decrease the binding of Fc to receptors or FcRn) can also be introduced into the constant region.

[0188] The term "antigen" refers to a substance that is recognized and specifically bound by an antibody or its antigen-binding fragment. In a broad sense, an antigen can include any immunogenic fragment or determinant of a selected target, including single epitopes, multiple epitopes, single domains, multiple domains, or intact extracellular domains (ECDs) or proteins. Peptides, proteins, glycoproteins, polysaccharides, and lipids, as well as portions thereof, can constitute antigens. Non-limiting exemplary antigens include tumor antigens or pathogen antigens, etc. "Antigen" can also refer to a molecule that elicits an immune response. Any form of antigen, or cells or preparations containing that antigen, can be used to generate antibodies specific to the antigenic determinant. An antigen can be an isolated full-length protein, a cell surface protein (e.g., used for immunization with cells expressing at least a portion of the antigen on their surface), or a soluble protein (e.g., used for immunization with only the ECD portion of the protein), or a protein construct (e.g., an Fc antigen). The antigen can be produced in genetically modified cells. Any of the foregoing antigens can be used alone or in combination with one or more immunogenic adjuvants known in the art. The DNA encoding the antigen can be genomic or non-genomic (e.g., cDNA) and can encode at least a portion of the ECD sufficient to elicit an immunogenic response. Any vector can be used to transform cells expressing the antigen, including but not limited to adenoviral vectors, lentiviral vectors, plasmids, and non-viral vectors such as cationic lipids.

[0189] The term "epitope," also known as an "antigenic determinant," refers to a site on an antigen that specifically binds to an immunoglobulin or antibody. Epitopes can be formed from adjacent amino acids or from non-adjacent amino acids arranged side-by-side through the ternary folding of a protein. Epitopes formed from adjacent amino acids are typically retained after exposure to denaturing solvents, while epitopes formed through ternary folding are typically lost after treatment with denaturing solvents. Epitopes typically consist of 3–15 amino acid residues. Methods for determining the epitope bound to a given antibody are well known in the art, including immunoblotting and immunoprecipitation assays. Methods for determining the spatial conformation of an epitope include techniques in the art and those described herein, such as X-ray crystallography and two-dimensional nuclear magnetic resonance.

[0190] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. Polymers may be linear, cyclic, or branched, may contain modified amino acids, particularly conserved modified amino acids, and may be interrupted by non-amino acid components. The term also includes amino acid polymers that have been modified, for example, by glycosylation, esterification, acetylation, phosphorylation, methylation, or any other manipulation such as conjugation with a labeled component. As used herein, the term “amino acid” refers to natural and / or non-natural or synthetic amino acids, including glycine and its D or L optical isomers, as well as amino acid analogs and peptide mimics. “Derived from” a specified protein refers to the source of the polypeptide. The term also includes polypeptides expressed by a specified nucleic acid sequence.

[0191] The term "amino acid modification" (or "modified amino acid") includes amino acid substitutions, insertions, and / or deletions in a polypeptide sequence. "Amino acid substitution" or "replacement" means replacing an amino acid at a specific position in the parent polypeptide sequence with another amino acid. For example, substitution of S32A means that the serine at position 32 is replaced by alanine.

[0192] The sequence identity or homology between the variable region of the humanized antibody and the variable region of the human receptor can be determined as discussed herein, and when such a determination is made, preferably at least 60% or 65% sequence identity will be shared, more preferably at least 70%, 75%, 80%, 85%, or 90% sequence identity, and even more preferably at least 93%, 95%, 98%, or 99% sequence identity. Preferably, the different residue positions are due to conserved amino acid substitutions. A “conserved substitution” is an amino acid substitution in which one amino acid residue is replaced by another amino acid residue with a side chain (R group) having similar chemical properties (e.g., charge or hydrophobicity). Generally, conserved amino acid substitutions do not substantially alter the functional properties of the protein. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, one or more amino acid residues in the CDR region or frame region of the disclosed antibody can be replaced with amino acid residues of other similar side chains. In cases where two or more amino acid sequences differ from each other due to conserved substitutions, the sequence identity percentage or degree of similarity can be adjusted upwards to correct for the conservatism of the substitution.

[0193] During monoclonal antibody production, various physicochemical factors can easily generate post-translational modification (PTM) variants, such as glycosylation, oxidation, glycation, deamidation, isomerization, and terminal cyclization. These PTMs can cause changes in the physicochemical properties of antibodies, alter their interaction with the antibody Fc receptor, and affect their binding activity to the target antigen. Some PTMs can even reduce antibody stability and induce immunogenicity (JARASCH et al., JOURNAL OF PHARMACEUTICAL SCIENCES, 2015). The negative effects of PTMs can be eliminated by modifying the amino acid sites, such as through conserved substitutions. Amino acid substitutions of antibody CDRs for the purpose of modifying PTMs are also explicitly kept within the scope of this disclosure.

[0194] The antibodies disclosed herein may also include substitutions or modifications to constant regions (e.g., Fc), including but not limited to amino acid residue substitutions, mutations, and / or modifications, which produce compounds having preferred characteristics, including but not limited to: altered pharmacokinetics, increased serum half-life, increased binding affinity, decreased immunogenicity, increased yield, altered binding to Fc receptors (FcRs), enhanced or weakened ADCC or CDC, altered glycosylation and / or disulfide bonds, and modified binding specificity.

[0195] The multispecific antigen-binding molecules disclosed herein may also include substitutions or modifications to a constant region (e.g., Fc), including but not limited to amino acid residue substitutions, mutations, and / or modifications, which produce compounds having preferred characteristics, including but not limited to: altered pharmacokinetics, increased serum half-life, increased binding affinity, decreased immunogenicity, increased yield, altered Fc ligand binding to an Fc receptor (FcR), enhanced or weakened ADCC or CDC, altered glycosylation and / or disulfide bonds, and modified binding specificity. In some aspects, the antibody variants comprise an Fc region having one or more amino acid substitutions that weaken FcγR binding (e.g., substitutions at positions 234 and 235 of the Fc region). In one aspect, the substitutions are L234A and L235A.

[0196] The term "knob-into-Hole" refers to a modification used to promote the association of two polypeptide chains in a fungible cell (Fc), comprising a "knob" modification in one of the two polypeptide chains of Fc and a "hole" modification in the other. Generally, this method involves introducing a bulge ("knob") at the interface of the first polypeptide chain and a corresponding cavity ("hole") at the interface of the second polypeptide chain, such that the bulge can be placed within the cavity to promote heterodimer formation and inhibit homodimer formation. The bulge is constructed by replacing a small amino acid side chain from the interface of the first polypeptide chain with a larger side chain (e.g., tyrosine or tryptophan). A complementary cavity with the same or similar size as the bulge is created at the interface of the second polypeptide chain by replacing the large amino acid side chain with a smaller amino acid side chain (e.g., alanine or threonine).

[0197] Therefore, in one specific embodiment, in the CH3 domain of the first polypeptide chain of the Fc domain of the multispecific antigen-binding molecule of this disclosure, an amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby creating a bulge within the CH3 domain of the first polypeptide chain, which can be placed in a cavity within the CH3 domain of the second polypeptide chain. Furthermore, in the CH3 domain of the second polypeptide chain of the Fc domain, an amino acid residue is replaced with an amino acid residue having a smaller side chain volume, thereby creating a cavity within the CH3 domain of the second polypeptide chain, in which the bulge within the CH3 domain of the first polypeptide chain can be placed. Preferably, the amino acid residue having a larger side chain volume is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W). Preferably, the amino acid residue having a smaller side chain volume is selected from the group consisting of alanine (A), serine (S), threonine (T), and valine (V).

[0198] The term "connector" refers to any tool used to connect two different functional units (e.g., antigen-binding fragments). Types of connectors include, but are not limited to, chemical connectors and peptide connectors. The sequence of peptide connectors is not limited. Peptide connectors are preferably non-immunogenic and flexible, such as those containing serine and glycine sequences. Depending on the specific construct, connectors can be long or short.

[0199] According to this disclosure, the linker connecting different functional units preferably comprises a flexible peptide linker, such as a glycine-serine peptide linker. The linker connecting the VH and VL domains to form a VH-VL or VL-VH scFv domain preferably comprises a flexible peptide linker, such as a glycine-serine peptide linker. In one embodiment, the linker comprises an amino acid sequence (G4S). n Or (G4S) n A (SEQ ID NO:71), where n is any integer chosen from 1 to 10, preferably comprising the amino acid sequence (G4S)3 (SEQ ID NO:72) or (G4S)3A (SEQ ID NO:73). In one embodiment, the linker comprises the amino acid sequence EPKSC (G4S). n Or EPKSC (G4S) n A (SEQ ID NO:74), where n is any integer chosen from 1 to 10, preferably containing the amino acid sequence EPKSC(G4S)3 (SEQ ID NO:75) or EPKSC(G4S) (SEQ ID NO:76).

[0200] The term "affinity" or "binding affinity" refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). The term "KD" refers to the dissociation constant of a specific antibody-antigen interaction. Binding affinity can be determined using a variety of techniques known in the art, such as surface plasmon resonance, biolayer interferometry, bipolar interferometry, static light scattering, dynamic light scattering, isothermal titration calorimetry, ELISA, analytical ultracentrifugation, and flow cytometry.

[0201] The term "pharmaceutical composition" refers to a formulation or combination of formulations containing one, two, or more active ingredients, wherein the active ingredients contained herein are present in a biologically effective form and do not contain any additional ingredients that would have unacceptable toxicity to a subject administering the formulation. When a "pharmaceutical composition" exists as a combination of individual formulations containing two or more different active ingredients, it can be administered simultaneously, sequentially, separately, or at intervals, with the aim of exerting the biological activity of multiple active ingredients together for the treatment of a disease.

[0202] The term "antibody-drug conjugate" (ADC) refers to an antibody covalently conjugated to a bioactive molecule, such as a therapeutic active substance or active pharmaceutical ingredient (API), so that the therapeutic active substance or active pharmaceutical ingredient (API) can target the antibody's binding target to exhibit its pharmacological function. The therapeutic active substance or active pharmaceutical ingredient can be a cytotoxic agent capable of killing cells targeted by the ADC, preferably malignant or cancerous cells. The covalent linking of the therapeutic active substance, active pharmaceutical ingredient, or cytotoxic agent can be performed in a non-site-specific manner using standard chemical linkers that conjugate the payload to lysine or cysteine ​​residues, or preferably, the conjugation is performed in a site-specific manner, which allows complete control over the conjugation site and the drug-to-antibody ratio of the resulting ADC. The ADCs described herein can be used to deliver cytotoxic agents or other payloads to target sites (e.g., tumorigenic cells and / or cells expressing CDH6). As used herein, the terms "drug" and "warhead" are used interchangeably and will refer to a bioactive or detectable molecule or compound, including anticancer agents. A "payload" may comprise a drug or warhead in combination with an optional linker compound. The warhead can contain peptides, polypeptides, proteins, precursor drugs that are metabolized into active agents in the body, polymers, nucleic acid molecules, small molecules, binders, mimics, synthetic drugs, inorganic molecules, organic molecules, and radioactive isotopes.

[0203] The term "pharmaceutical acceptable" refers to compounds, materials, compositions, and / or dosage forms that, within the bounds of reliable medical judgment, are suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, in proportion to a reasonable benefit / risk ratio.

[0204] The term “pharmaceutical carrier” or “pharmaceuticalally acceptable carrier” refers to a diluent, adjuvant (e.g., Freund’s adjuvant (complete and incomplete)), excipient, or medium that is administered with a therapeutic agent.

[0205] The term "effective dose" refers to a dosage of a pharmaceutical formulation of the antibody or its antigen-binding fragment disclosed herein, which, when administered to a patient in a single or multiple doses, produces the intended effect in the treated patient. The effective dose can be readily determined by an attending physician skilled in the art by considering a variety of factors, such as: racial differences; weight, age, and health status; the specific disease involved; the severity of the disease; the individual patient's response; the specific antibody administered; the administration modality; the bioavailability characteristics of the administered formulation; the chosen dosing regimen; and the use of any concomitant therapies.

[0206] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells in which exogenous nucleic acids have been introduced, including the progeny of such cells. Host cells include “transformers” and “transformed cells,” which include the primary transformed cells and their progeny, regardless of the number of passages. Progeny may not be identical to the parent cells in terms of nucleic acid content and may contain mutations. This document includes mutant progeny that have the same function or biological activity as those screened or selected in the initially transformed cells.

[0207] As used in this article, the term "transfection" refers to the introduction of exogenous nucleic acids into eukaryotic cells. Transfection can be achieved through a variety of techniques known in the art, including calcium phosphate-DNA coprecipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipid transfection, protoplast fusion, retroviral infection, and biolistics.

[0208] The term "stable transfection" or "stable transformation" refers to the introduction and integration of exogenous nucleic acids, DNA, or RNA into the genome of transfected cells. The term "stable transfectant" refers to a cell in which foreign DNA is stably integrated into the genomic DNA.

[0209] The terms "isolated polynucleotide" or "isolated nucleic acid" refer to nucleic acid molecules, DNA, or RNA that have been removed from their natural environment. For example, for the purposes of this disclosure, a recombinant polynucleotide encoding a polypeptide contained in a vector is considered isolated. Other examples of isolated polynucleotides include recombinant polynucleotides maintained in a heterologous host cell or (partially or substantially) purified polynucleotides in solution. Isolated polynucleotides include polynucleotide molecules contained in cells that normally contain such polynucleotide molecules, but which are present outside the chromosome or at a chromosomal location other than their natural chromosomal location. Isolated RNA molecules include in vivo or in vitro RNA transcripts of this disclosure, as well as positive-stranded, negative-stranded, and double-stranded forms.

[0210] The terms "nucleic acid molecule encoding," "encoding DNA sequence," and "encoding DNA" refer to the sequence of deoxyribonucleotides along a deoxyribonucleic acid (DNA) chain. This sequence of deoxyribonucleotides determines the sequence of amino acids along a polypeptide (protein) chain. Therefore, a nucleic acid sequence encodes an amino acid sequence.

[0211] There are no particular limitations on the methods for obtaining the binding molecules, antigen-binding fragments, antibodies, bispecific antigen-binding molecules, or multispecific antigen-binding molecules of this disclosure, which are well known and available in the prior art and can be obtained by any method. The binding molecules, antigen-binding fragments, antibodies, bispecific antigen-binding molecules, or multispecific antigen-binding molecules of this disclosure can be prepared and purified using conventional methods. For example, cDNA sequences encoding heavy and light chains can be cloned and recombined into expression vectors. Recombinant immunoglobulin expression vectors can stably transfect CHO cells. As a more preferred prior art, mammalian expression systems lead to glycosylation of antibodies, particularly at the highly conserved N-terminus of the Fc region. Stable clones are obtained by expressing antibodies that specifically bind to human antigens. Positive clones are scaled up in serum-free medium in a bioreactor to produce antibodies. Culture media secreting antibodies can be purified and collected using conventional techniques. Antibodies can be concentrated by filtration using conventional methods. Soluble mixtures and polymers can also be removed using conventional methods, such as molecular sieving or ion exchange.

[0212] As used herein, the terms “individual” or “subject” refer to any animal, such as a mammal or marsupial. Individuals disclosed herein include, but are not limited to, humans, non-human primates (such as cynomolgus monkeys or rhesus monkeys or other types of macaques), mice, pigs, horses, donkeys, cattle, sheep, rats, and any kind of poultry.

[0213] As used herein, the terms “disease,” “symptom,” or “disorder,” etc., refer to any alteration or dysregulation that impairs or interferes with the normal function of cells, tissues, or organs. For example, “disease” includes, but is not limited to: tumors, pathogen infections, autoimmune diseases, T-cell dysfunction disorders, or deficiencies in immune tolerance (such as transplant rejection).

[0214] As used in this article, the term "tumor" refers to a disease characterized by the pathological proliferation of cells or tissues, and their subsequent migration or invasion into other tissues or organs. Tumor growth is typically uncontrolled and progressive, neither inducing nor inhibiting the proliferation of normal cells.

[0215] The term "autoimmune disease" refers to a disorder resulting from an autoimmune response. Autoimmune diseases are the result of an inappropriate or excessive response to self-antigens.

[0216] As used in this article, the term "treatment" refers to a clinical intervention in an attempt to alter an individual's or treat a disease caused by cells, which can be preventative or intervention in a clinicopathological process. Treatment effects include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or alleviating the condition, and alleviating or improving prognosis.

[0217] As used herein, the term "combination" refers to a treatment regimen that provides at least two or more different therapies to achieve a specified therapeutic effect. These therapies can be physical, such as radiation therapy, or chemical, such as administering a drug to the subject, including combination drugs. "Combination drugs" refers to a combination of two or more pharmaceutical preparations, each containing an active ingredient, that are administered to a subject in combination. The active ingredients may be mixed together to form a single dosing unit or may be administered separately as independent dosing units; during administration, the different pharmaceutical preparations may be administered substantially synchronously, simultaneously, or sequentially. Detailed Implementation

[0218] The present disclosure is further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the disclosure. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer.

[0219] Example 1: Design and Sequence of Anti-CD79b-CD20-CD3 Trispecific Antibody

[0220] The trispecific antigen-binding molecule constructed in this embodiment is specifically an anti-CD79b-CD20-CD3 trispecific antibody. One end consists of a heavy chain domain of the Fab fragment specifically binding to CD79b connected to the scFv domain of the human T-cell receptor subunit CD3ε via a flexible linker, and then connected to the Fc domain. The other end consists of a heavy chain domain of the Fab fragment specifically binding to CD20 or a VHH domain specifically binding to CD20 connected to the Fc domain. The heavy chain variable region sequence of the Fab fragment specifically binding to CD79b is shown in SEQ ID NO:9 or SEQ ID NO:11, and the light chain variable region sequence is shown in SEQ ID NO:10 or SEQ ID NO:12. The VHH domain specifically binding to CD20 includes the sequence shown in SEQ ID NO:13 or SEQ ID NO:14; or the reconnected variable region sequence of the Fab fragment specifically binding to CD20 is shown in SEQ ID NO:15, and the light chain variable region sequence is shown in SEQ ID NO:16. The heavy chain variable region sequence that specifically binds to the scFv domain of human CD3ε is shown in SEQ ID NO:17, and the light chain variable region sequence is shown in SEQ ID NO:18. To reduce the ADCC activity of the antibody, the Fc fragments of the finally constructed trispecific antibody were all substituted with amino acids L234A, L235A, and G237A.

[0221] The scFv that specifically binds to the human CD3ε structural domain is formed by linking the heavy chain variable region and the light chain variable region of the full-length anti-CD3 antibody via a flexible linker. Its structure is VH-(G4S)3-VL, as shown in SEQ ID NO:19. The scFv is then fused to the C-terminus of the heavy chain structural domain of the Fab fragment that specifically binds to CD79b via a flexible linker.

[0222] The N-terminus of the VH fragment of scFv is fused to the C-terminus of the heavy chain domain of the Fab fragment that specifically binds to CD79b, the C-terminus of the VL fragment of scFv is fused to the N-terminus of the Fc domain with a "node" structure, and the C-terminus of the VHH domain that specifically binds to CD20 is fused to the N-terminus of the Fc domain with a "hole" structure. The resulting trispecific antibody containing two heterologous heavy chains and one light chain is named triantibody 1 or triantibody 2, which respectively contain the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 or SEQ ID NO:1, SEQ ID NO:4 and SEQ ID NO:3. A schematic diagram is shown in Figure 1.

[0223] The N-terminus of the VH fragment of scFv was fused to the C-terminus of the heavy chain domain of the Fab fragment that specifically binds to CD79b, the C-terminus of the VL fragment of scFv was fused to the N-terminus of the Fc domain containing a "node" structure, and the C-terminus of the heavy chain domain of the Fab fragment that specifically binds to CD20 was fused to the N-terminus of the Fc domain containing a "hole" structure. The resulting trispecific antibody containing two heterologous heavy chains and two heterologous light chains was named Triantibody 3. It contains the amino acid sequences shown in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8. A schematic diagram is shown in Figure 1.

[0224] In either the trispecific antibody 1 or trispecific antibody 2, the heavy chain containing scFv is designed as a "knob" structure, with amino acid substitutions at the S354C and T366W sites. The heavy chain without scFv is designed as a "hole" structure, with amino acid substitutions at the Y349C, T366S, L368A, and Y407V sites. Furthermore, to facilitate the purification of the trispecific antibody, the "hole" structure heavy chain undergoes H435R substitution.

[0225] Of the two heterologous heavy chains in the trispecific antibody 3, the heavy chain containing scFv was designed as a "knob" structure, including amino acid substitutions at the S354C and T366W sites; the heavy chain without scFv was designed as a "hole" structure, including amino acid substitutions at the Y349C, T366S, L368A, and Y407V sites. Furthermore, to facilitate the purification of the trispecific antibody, the "hole" structure heavy chain also underwent H435R substitution.

[0226] To prevent mismatches, the heavy chain containing scFv in the triple antibody 3 has amino acid substitutions at four sites: Q39E, T139K, K147R, and V185S. ​​The light chain of the Fab fragment that specifically binds to CD79b has amino acid substitutions at four sites: Q38K, S114D, S131D, and L135H. The heavy chain without scFv is substituted with amino acids at three sites: Q39K, T139D, and K147D. The light chain of the Fab fragment that specifically binds to CD20 has amino acid substitutions at four sites: Q38E, S114K, S131R, and L135T (amino acid positions are numbered according to Kabat rules).

[0227] The structures and related amino acid sequences of the three antibodies 1, 2 and 3 are summarized in Tables 1 and 2, respectively.

[0228] Table 1. Structure of trispecific antibodies

[0229] Table 2. Amino acid sequences of trispecific antibodies

[0230] Example 2: Construction of anti-CD79b-CD20-CD3 trispecific antibody and its transient transfection expression in eukaryotic cells

[0231] The gene fragments encoding the aforementioned three specific antibody molecules were cloned into the pTT5 expression vector to prepare transfection-grade recombinant expression plasmids.

[0232] Expi293F cultured in serum-free medium TM Cells (Thermo Fisher Scientific) were seeded in shake flasks (Corning Inc.) and cultured on a shaker at 37°C with 8% CO2. Cell density was adjusted, and the recombinant expression plasmid containing the target gene fragment and PEI transfection reagent were mixed in an appropriate ratio and added to the cell culture shake flasks. After 6 days of cell culture, the expression supernatant was collected, centrifuged at high speed to remove cell debris, and then purified using a Protein A column for affinity purification. The column was washed with PBS until the A280 reading returned to baseline. The target protein was eluted with acidic elution buffer (pH 3.0-3.5) and neutralized with 1M Tris-HCl (pH 8.0-9.0). After appropriate concentration, the eluted sample was further purified using PBS-equilibrated Superdex 200 (GE) gel chromatography to remove aggregates. The monomer peak was collected, and the solution was transferred to PBS aliquots for further processing. The final purified antibody was analyzed by SDS-PAGE and HPLC for purity and A280 concentration determination.

[0233] Example 3: Affinity Detection Experiment of Anti-CD79b-CD20-CD3 Trispecific Antibody

[0234] A. Detection of the affinity of anti-CD79b-CD20-CD3 trispecific antibody for cells expressing hCD79b, CD20, and hCD3.

[0235] The binding of the anti-CD79b-CD20-CD3 trispecific antibody to hCD79b-CHOK1 cells expressing hCD79b (human CD79b), hCD20-CHOK1 cells expressing hCD20, natively hCD3-expressing T lymphocytes (Jurkat), and natively co-expressing hCD20 and hCD79b SU-DHL-4 cells (high expression) and OCI-LY19 cells (low expression) were detected using FACS.

[0236] hCD79b-CHOK1 cells (Shanghai Jiman Biotechnology), hCD20-CHOK1 cells (Shanghai Jiman Biotechnology), Jurkat cells (ATCC, TIB-152), SU-DHL-4 cells (Chinese Academy of Sciences Cell Bank, SCSP-5048), and OCI-LY19 cells (Nanjing Kebai, CBP60621) were cultured. The culture medium for hCD79b-CHOK1 and hCD20-CHOK1 cells was F12K + 10% FBS + 4ug / mL puromycin; the culture medium for Jurkat and SU-DHL-4 cells was RPMI 1640 + 10% FBS; and the culture medium for OCI-LY19 cells was MEMα + 20% FBS. All cells were cultured in T75 cell culture flasks at 37℃ in a 5% CO2 incubator. When the cells were ready for use, hCD79b-CHOK1 cells and hCD20-CHOK1 cells were digested with 0.25% trypsin and then placed into centrifuge tubes; Jurkat cells, SU-DHL-4 cells and OCI-LY19 cells were placed directly into 50mL centrifuge tubes without digestion.

[0237] Centrifuge the obtained cells at 1000 rpm at room temperature for 5 minutes, discard the supernatant, and resuspend the cells in 100 μL of 1% BSA (in PBS). Count the cells and adjust the cell density to 1E6 / mL. Plate the cells into 96-well round-bottom culture plates, centrifuge at 1500 rpm at 4°C for 5 minutes, discard the supernatant, resuspend the cells in 200 μL of 1% BSA (in PBS), centrifuge again at 1500 rpm at 4°C for 5 minutes, discard the supernatant, and store at 4°C for later use. Dilute the test antibody and negative control IgG1 AA (purchased from Baiying Biotechnology, catalog number B109802) with 1% BSA (in PBS), starting at 100 nM, and then dilute 5-fold down to 8 concentrations. Resuspend the cells with the diluted antibody, 100 μL / well, and incubate at 4°C for 1 hour. Centrifuge at 1500 rpm at 4°C for 5 minutes, and discard the supernatant. Resuspend and wash cells in 160 μL of 1% BSA (in PBS), centrifuge at 1500 rpm for 5 minutes at 4°C, and discard the supernatant. Dilute the secondary antibody (goat anti-human IgG Fc PE) 1:400 with 1% BSA (in PBS) according to the manufacturer's instructions, resuspend the cells in the diluted secondary antibody at 100 μL / well, and incubate at 4°C for 0.5 hours. Centrifuge at 1500 rpm for 5 minutes at 4°C, and discard the supernatant. Resuspend and wash cells in 200 μL of 1% BSA (in PBS), centrifuge at 1500 rpm for 5 minutes at 4°C, and discard the supernatant. Resuspend the cells in 100 μL of 1% BSA (in PBS), filter the cells through 300-mesh gauze, and detect the mean fluorescence intensity of the PE channel using an iQue3 flow cytometer.

[0238] The average fluorescence intensity (MFI) of the PE channel for each sample was analyzed using the software included with iQue3. The obtained average fluorescence intensity was then imported into Graphpad to analyze the half-maximal binding concentration (EC) of the antibody to cells. 50 The results of the highest mean fluorescence intensity (Top MFI) are shown in Table 3, Figure 2A (hCD20-CHOK1 cells), Figure 2B (hCD79b-CHOK1 cells), Figure 2C (Jurkat cells), Figure 2D (SU-DHL-4), and Figure 2E (OCI-LY19). The affinity ranking with hCD20 monoexpressing cell lines was: triple antibody 3 > triple antibody 1 > triple antibody 2; the affinity ranking with hCD79b monoexpressing cell lines was: triple antibody 1 ≈ triple antibody 2 > triple antibody 3; the affinity ranking with hCD3 monoexpressing cell line Jurkat was: triple antibody 1 > triple antibody 2 > triple antibody 3. The binding ability of triple antibodies 1 and 2 to Jurkat cell lines was slightly weaker than their binding ability to hCD20 monoexpressing cell lines; the binding ability of triple antibody 3 to Jurkat cell lines was weaker than its affinity for hCD20 and hCD79b. Literature reports that the expression levels of CD20 and CD79b in OCI-LY19 cells are lower than those in SU-DHL-4 cells. Therefore, the binding ability of the three triple antibody molecules to the OCI-LY19 cell line is weaker than that to the SU-DHL-4 cell line.

[0239] Table 3. Affinity of the trispecific antibodies to hCD20-CHOK1, hCD79b-CHOK1, Jurkat, SU-DHL-4, and OCI-LY19 cell lines.

[0240] B. In vitro protein binding affinity and kinetics of the anti-CD79b-CD20-CD3 trispecific antibody for recombinant proteins.

[0241] The affinity and kinetic properties of the anti-CD79b-CD20-CD3 trispecific antibody molecule with human CD79b and human / cynomolgus monkey CD3 were analyzed using a Biacore 8K instrument.

[0242] The affinity and kinetic properties between trispecific antibodies and human CD79b (ACRO, CDB-H52H3), human CD3 (ACRO, CDD-H52W1), and cynomolgus monkey CD3 (ACRO, CDD-C52W4) were detected using a Protein A chip and a capture assay. The trispecific antibodies were diluted to 1 μg / mL with HBS-EP+ (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% P20) buffer. Proteins were serially diluted twofold with HBS-EP+ (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% P20) buffer to serial concentrations: human CD79b protein (100 nM–0.39 nM), and human / cynomolgus monkey CD3 protein (800 nM–3.125 nM). The trispecific antibody was used to capture the protein at a flow rate of 10 μL / min for 60 s, and the protein was bound at a flow rate of 30 μL / min for 60 s and then dissociated for 60 s. After each round of experiments, the chip was washed with pH 1.5 Glycine solution at a flow rate of 30 μL / min for 60 s to remove the antigen protein molecules, thus completing the chip regeneration.

[0243] The obtained trispecific antibody affinity and kinetic experimental data are shown in Table 4.

[0244] Table 4. Binding affinity and kinetics of anti-CD79b-CD20-CD3 trispecific antibodies to human CD79b and human / cynomolgus monkey CD3 proteins.

[0245] The experimental results showed that the two triple antibody molecules, triple antibody 1 and triple antibody 2, both bound to human CD79b and human / cynomolgus monkey CD3, and their affinity was comparable; triple antibody 3 bound to human CD79b, but its affinity was weaker than that of triple antibody 1 and triple antibody 2; triple antibody 3 bound to both human and cynomolgus monkey CD3, and its affinity was similar to that of triple antibody 1 and triple antibody 2.

[0246] Example 4: In vitro functional experiment of anti-CD79b-CD20-CD3 trispecific antibody

[0247] A. PBMC cell-mediated cytotoxicity assay, target cells were SU-DHL-4 cells.

[0248] SU-DHL-4 cells were cultured in RPMI 1640 + 10% FBS medium. They were cultured in T75 cell culture flasks at 37°C in a 5% CO2 incubator. When ready for use, SU-DHL-4 cells were directly transferred to 50mL centrifuge tubes without digestion, centrifuged at 1000rpm for 5 minutes, and the supernatant was discarded. The cells were resuspended in complete medium and counted, adjusting the cell density to 4E5 / mL and seeded into 96-well plates at 100μL / well. PBMCs were placed in 50mL centrifuge tubes, centrifuged at 400g for 5 minutes, the supernatant was discarded, and the cells were resuspended in fresh RPMI 1640 + 10% FBS medium and counted, adjusting the cell density to 4E6 / mL. The cells were then seeded into 96-well cell culture plates (pre-seeded with target cells) at 50μL / well.

[0249] The antibody was diluted with RPMI 1640 medium in 10% FBS to a final concentration of 100 nM in the first well. A 5-fold serial dilution was then performed, and the diluted antibody was added to cell culture plates at 50 μL / well. The plates were incubated at 37°C with 5% CO2 for 48 hours. The cells were then centrifuged at 1000 rpm for 5 minutes, and 50 μL of the supernatant was transferred to a 96-well plate. 50 μL of LDH solution was added, and the plates were incubated for 10 minutes. The OD492 nM wavelength was then measured using EnVision.

[0250] The percentage of cell killing caused by PBMCs is calculated using the following formula:

[0251] Cell killing percentage = [(sample well - culture medium background) - (spontaneous release of target cells - culture medium background) - (spontaneous release of PBMC cells - culture medium background)] / [(maximum lysis of target cells - culture medium lysis correction) - (spontaneous release of target cells - culture medium background))] × 100% ([(Sample - Medium) - (Target only - Medium) - (PBMC only - Medium)] / [(Target lysis - Medium lysis) - (Target only - Medium)] x 100%, where all data are OD492nm wavelength data for the corresponding sample wells).

[0252] Import cell killing data into GraphPad Prism, plot cell killing / concentration curves, and calculate EC50. 50 The results are shown in Table 5 and Figure 3A. The killing activities of triple antibody 1 and triple antibody 3 against the CD79b and CD20 dual-high expression cell line SU-DHL-4 were similar, and the maximum killing value and half-maximal effective concentration were similar; the half-maximal effective concentration of triple antibody 2 was higher than that of triple antibody 1 and triple antibody 3, and its killing activity was weaker.

[0253] B. PBMC cell-mediated cytotoxicity assay, target cells were OCI-LY19 cells.

[0254] OCI-LY19 cells were cultured in MEMα + 20% FBS medium. They were cultured in T75 cell culture flasks at 37°C in a 5% CO2 incubator. When ready for use, OCI-LY19 cells were directly transferred to 50mL centrifuge tubes without digestion, centrifuged at 1000rpm for 5 minutes, and the supernatant was discarded. The cells were resuspended in complete medium and counted, adjusting the cell density to 4E5 / mL and seeded into 96-well plates at 100μL / well. PBMCs were placed in 50mL centrifuge tubes, centrifuged at 400g for 5 minutes, the supernatant was discarded, and the cells were resuspended in fresh MEMα + 20% FBS medium and counted, adjusting the cell density to 4E6 / mL. The cells were then seeded into 96-well cell culture plates (pre-seeded with target cells) at 50μL / well.

[0255] Antibodies were prepared using MEMα medium with 20% FBS, with an initial concentration of 100 nM (final concentration). A 5-fold serial dilution was performed, and the solutions were added to cell culture plates at 50 μL / well. The plates were incubated at 37°C with 5% CO2 for 48 hours. The cells were centrifuged at 1000 rpm for 5 minutes, and 50 μL of the supernatant was transferred to a 96-well plate. 50 μL of LDH solution was added, and the plates were incubated for 10 minutes. The OD492 nM wavelength was then measured using EnVision.

[0256] The percentage of cell killing induced by PBMCs is calculated using the following formula:

[0257] Cell killing % = [(sample well - culture medium background) - (spontaneous release of target cells - culture medium background) - (spontaneous release of PBMC cells - culture medium background)] / [(maximum lysis of target cells - culture medium lysis correction) - (spontaneous release of target cells - culture medium background))] × 100%.

[0258] Import cell killing data into GraphPad Prism, plot cell killing / concentration curves, and calculate EC50. 50 The results are shown in Table 5 and Figure 3B. Regarding the killing activity against the CD79b and CD20 dual-low expression cell line OCI-LY19 (CD20 is lower than CD79b), the maximum killing values ​​of the three triple antibody molecules were similar, with the half-maximal effective concentration (MCC) being: triple antibody 3 < triple antibody 1 < triple antibody 2; the killing activity was: triple antibody 3 was stronger than triple antibody 1, and stronger than triple antibody 2.

[0259] Table 5 Results of PBMC cell-mediated cytotoxicity assays using anti-CD79b-CD20-CD3 trispecific antibodies.

[0260] C. Cytokine release assay

[0261] SU-DHL-4 cells were cultured in RPMI 1640 medium with 10% FBS. OCI-LY19 cells were cultured in MEMα medium with 20% FBS. Both were cultured in T75 cell culture flasks at 37°C in a 5% CO2 incubator. When ready for use, SU-DHL-4 and OCI-LY19 cells were directly transferred to 50mL centrifuge tubes without digestion, centrifuged at 1000rpm for 5 minutes, the supernatant was discarded, and the cells were resuspended in their respective culture media. Cell counts were performed, and the cell density was adjusted to 4E5 / mL. Target cells were then seeded into 96-well plates at 100μL / well.

[0262] Purchase fresh PBMCs, count the cells, and adjust the cell density to 4E6 / mL using their respective culture media. Then, seed the cells into 96-well cell culture plates (with target cells already seeded), 50 μL / well.

[0263] The initial concentration was 100 nM (final concentration), and the antibody was serially diluted 5-fold. 50 μL of the diluted antibody was added to each well of a cell culture plate and incubated at 37°C with 5% CO2 for 48 hours.

[0264] Cells were centrifuged at 1000 rpm for 5 minutes, and the supernatant was transferred to new wells. The secretion levels of IL-2, IL-6, TNF-α, and IFN-γ in the supernatant were detected using the Biolegend ELISA kit (Cat.: 431804, 430516, 430904, 430104). Finally, the release levels of each cytokine in the corresponding samples were calculated and exported using a four-parameter method on a microplate reader. The results are shown in Figures 3C-3D.

[0265] Experimental results showed that when PBMCs were co-incubated with tumor cells, the cytokine release activity of the three anti-tumor molecules was positively correlated with their killing activity.

[0266] E. Antibody cell surface retention assay

[0267] Ramos cell lines were cultured in RPMI 1640 medium with 10% FBS using T75 cell culture flasks at 37°C in a 5% CO2 incubator. Cells were collected at passage 2-3, centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, and the cells were resuspended in complete culture medium. Cell counts were performed, and the cell density was adjusted to 1E6 / mL. Cells were then seeded into 96-well plates (100 μL / well) and pre-chilled on ice.

[0268] Pre-chill PBS containing 1% BSA and use this solution to prepare the antibody to be tested, with a final concentration of 100 nM. Centrifuge the cells at 1000 rpm for 5 minutes at 4°C, discard the supernatant, add 100 μL of antibody to each well and resuspend, and incubate at 4°C for 0.5 hours. Centrifuge the cells at 1000 rpm for 5 minutes at 4°C, discard the supernatant, add 180 μL of pre-chilled PBS containing 1% BSA, centrifuge again at 1000 rpm for 5 minutes at 4°C, add 100 μL of pre-chilled PBS containing 1% BSA, and incubate at 37°C in a 5% CO2 incubator. At 0h, 1h, 2h, 4h, and 24h, samples were transferred from the above-mentioned plates to new round-bottom plates. The plates were centrifuged at 1000 rpm for 5 minutes at 4°C, the supernatant was discarded, and 100 μL of fluorescent secondary antibody (goat anti-human IgG Fc PE, 109-15-098, Jackson) diluted in pre-chilled PBS containing 1% BSA was added. The plates were incubated at 4°C in the dark for 0.5 hours. The plates were then centrifuged at 1000 rpm for 5 minutes at 4°C, the supernatant was discarded, and the plates were resuspended in 200 μL of pre-chilled PBS containing 1% BSA. The plates were centrifuged at 1000 rpm for 5 minutes at 4°C, the supernatant was discarded, and this process was repeated once. Finally, 100 μL of PBS containing 1% BSA was added for resuspending, and the fluorescence intensity of the PE channel was detected by flow cytometry.

[0269] As shown in Figure 3E, the antibody surface retention rates of the three triple antibody molecules after 24 hours were: triple antibody 3 was better than triple antibody 1, which was better than triple antibody 2, and none of them showed significant internalization activity.

[0270] Example 5: Physical stability test of CD79b-CD20-CD3 trispecific antibody

[0271] The thermostability of different antibodies in pH 7.4 PBS buffer was detected using NanoDSF (differential fluorescence scanning technology). Sample concentrations were approximately 1 mg / mL, and detection was performed using a Prometheus NT.Plex (nano DSF). Before detection, each sample was centrifuged at 10000g for 10 minutes. 40 μL of sample was added to each well of the sample plate (instrument loading volume is 10 μL, with one replicate per sample). The scanning temperature ranged from 30℃ to 95℃, with a scan rate of 0.5℃ / min. The experimental results are shown in Table 6. All three triple antibody molecules exhibited good thermostability.

[0272] Table 6. NanoDSF detection results of anti-CD79b-CD20-CD3 trispecific antibody

[0273] Example 6: Pharmacokinetics of CD79b-CD20-CD3 Trispecific Antibody

[0274] Two cynomolgus monkeys were allocated to each group for the experiment, with free access to water. The trispecific antibody was administered at a dose of 1 mg / kg via intravenous infusion over 30 minutes. Blood samples were collected at the following time points: before administration (0 h), 5 min (±1 min), 4 h (±5 min), 8 h (±10 min), 24 h (±30 min), 36 h (±30 min), 48 h (±30 min), 72 h (±30 min), 96 h (±30 min), 168 h (±1 h), 240 h (±1 h), 336 h (±1 h), 504 h (±1 h), and 672 h (±1 h). 1–2 ml of whole blood was collected from suitable sites such as the cephalic vein or saphenous vein of the cynomolgus monkeys. The blood was incubated at 4°C for 30 min, centrifuged at 1000 rpm for 15 min at 4°C to collect serum, and immediately stored at -80°C for subsequent LBA (DELFIA) analysis to detect antibody concentration in the serum.

[0275] Total human IgG concentration detection method: Coat the test plate with 100 μL / well of 1 μg / mL Goat Anti-Human IgG, Monkey ads-UNLB (Southern Biotech-2049-1), refrigerate overnight, wash the test plate 3 times with PBST (0.05% Tween 20), 300 μL / well, add 200 μL / well of Casein (Pierce-37528) blocking buffer, incubate at 37℃ for 1 h, and wash the test plate 3 times; prepare sample dilution buffer - dilute Casein reagent 10 times with 1x PBS, add 100 μL / well of diluted standard curve and serum samples at different time points (MRD=50), incubate at 37℃ for 1 h, wash the test plate 6 times, add 100 μL / well of Goat Anti-Human IgG, Monkey ads-BIOT (1:5000, Southern Biotech-2049-1), After incubating at 37°C for 1 hour (Biotech-2049-08), the test plate was washed 6 times. Then, 100 μL / well EU-STREPTAVIDIN (1:1000, PerkinElmer-1244-360) was added and incubated at 37°C for 1 hour. After washing the test plate 6 times, 100 μL / well DELFIA Enhancement Solution (PerkinElmer-4001-0010) was added and incubated at room temperature for 30 minutes. The DELFIA module was then used to read the plate (laser excitation) on an EnVision microplate reader to read the fluorescence signal at 620 nm.

[0276] Pharmacokinetic parameters were calculated using a non-compartmental model. The results are shown in Figure 4, indicating that the three triple antibody molecules exhibited good pharmacokinetic properties.

[0277] Example 7: Pharmacokinetics of anti-CD79b-CD20-CD3 trispecific antibody with accompanying cytokine detection

[0278] The test kit is LEGENDplex TM For the NHP Th1 / Th2 Panel (8-plex) (Biolegend, catalog number 740393), before use, allow 20×Wash Buffer to dissolve at room temperature to fully dissolve the salts, then prepare 1×Wash Buffer using ddH2O. Dissolve the Standard in 250 μL of Lyphilized Standard Reconstitution Buffer, inverting several times to mix thoroughly, and let stand for 10 minutes. Dissolve Martix B in 5 mL of Assay Buffer, inverting several times to mix thoroughly, and let stand for 15 minutes to fully dissolve the powder. Vortex mix the detection beads, mix all detection beads in a 1:1 ratio, and dilute to the working concentration with Assay Buffer. Dilute the sample in a 1:1 ratio with Assay Buffer.

[0279] Add 25 μL Matrix B to each standard well, 25 μL Assay Buffer to each sample well, and 25 μL of each standard to its corresponding well. Add 25 μL of cynomolgus monkey plasma sample from each time point to each sample well (samples need to be diluted 40-fold when detecting IL-6). Add 25 μL of mixed beads to each well. Seal the plate with sealing film and incubate at 800 rpm at room temperature in the dark for 2 hours. Centrifuge at 500 g for 5 minutes, discard the supernatant, add 200 μL Wash Buffer to each well, centrifuge at 500 g for 5 minutes, discard the supernatant, and wash the plate twice. Add 25 μL of detection antibody to each well, seal the plate with sealing film, and incubate at 800 rpm at room temperature in the dark for 1 hour. Add 25 μL SA-PE to each well and incubate at 800 rpm at room temperature in the dark for 30 minutes. Centrifuge at 500g for 5 minutes, discard the supernatant, wash the plate twice, resuspend the beads in 200μL / well Wash Buffer, and analyze using a flow cytometer.

[0280] Export the FCS file and analyze the cytokine release levels of the samples. The results are shown in Table 7.

[0281] Table 7. Pharmacokinetic assay results of anti-CD79b-CD20-CD3 trispecific antibody, accompanied by cytokine detection.

[0282] Experimental results showed that the peak release of cytokines from the triple antibody occurred 4-8 hours after administration, primarily due to the release of IL-6, followed by a decline in release levels. The increased cytokine release was likely mainly due to the expression of CD79b and CD20 on peripheral blood B cells. Compared to the CD20 / CD3 bispecific antibody Mosunetuzumab, the triple antibody did not show an increase in cytokine release (data not shown), suggesting that the CRS risk of the triple antibody is not higher than that of Mosunetuzumab, indicating a better safety profile.

[0283] Example 8: In vivo efficacy experiment of CD79b-CD20-CD3 trispecific antibody

[0284] A. A mouse model reconstructed from human immune cells, with OCI-LY19 tumor cells.

[0285] All experimental animals were housed in individually ventilated, temperature- and humidity-controlled enclosures. The temperature ranged from 20.0 to 26.0°C, and the humidity from 40% to 70%. The day / night cycle was 12 hours. OCI-LY19 cells were cultured in MEMα medium containing 20% ​​fetal bovine serum. OCI-LY19 cells in the logarithmic growth phase were collected and resuspended in PBS to a suitable concentration for subcutaneous tumor inoculation in mice.

[0286] Recovery PBMC, 5×10 6 / Only intraperitoneal injection was performed on mice to establish a mouse model reconstructed with human immune cells; on the same day, 1×10⁻⁶ OCI-LY19 cells in logarithmic growth phase were injected into the peritoneal cavity of mice. 7 The mixture of one mouse and matrix gel (volume 1:1) was injected subcutaneously into the right forelimb of NOG mice (inoculation volume 200uL).

[0287] Before administration, all animals were weighed and tumor volume was measured using calipers. Given that tumor volume affects treatment efficacy, a randomized controlled trial was conducted, grouping mice according to their tumor volume to ensure similarity in tumor volume between groups. Grouping was performed using StudyDirector™ (version 3.1.399.19, vendor Studylog System, Inc., S. San Francisco, CA, USA). The day of mouse grouping was defined as day 0, and intraperitoneal administration began on day D0, administered every three days for a total of six doses. The hIgG1AA negative control group (purchased from BioNTech, catalog number B109802) and the grouping and administration regimens for the triple antibody molecular assay are shown in Table 8.

[0288] Table 8 Experimental Groups and Dosing Regimens

[0289] Animals were monitored daily for 22 days after drug administration. Throughout the experiment, the long and wide diameters of the tumor were measured every 3 days using calipers, and the tumor volume (mm²) was calculated according to the formula. 3 = 0.5 × (tumor long diameter × tumor short diameter) 2 Tumor volume was calculated. The relative tumor inhibition rate (TGI%) was calculated as follows: TGI% = (1 - T / C) × 100%. T / C% represents the relative tumor growth rate, which is the percentage of tumor volume or weight in the treatment group and the hIgG1 AA control group at a specific time point. T and C represent the tumor volume (TV) or tumor weight (TW) in the treatment group and the hIgG1 AA control group at a specific time point, respectively. The experimental results, including tumor volume, mouse body weight, and tumor weight in each group, are expressed as mean ± standard error (Mean ± SEM). An independent samples t-test was used to compare whether there were significant differences between the different treatment groups and the control group. Data were analyzed using statistical software. P < 0.05 was considered statistically significant. The experimental results are shown in Table 9 and Figure 5.

[0290] Both the 5mpk dose groups of Tri-antibody 1 and Tri-antibody 3 showed very significant tumor-inhibiting effects in the human diffuse large B-cell lymphoma OCI-LY19 subcutaneous transplantation NOG female mouse MiXeno model, and the mice tolerated the test drugs well, with no significant weight loss or toxicity observed.

[0291] Table 9. Tumor size and tumor inhibition rate in each treatment group

[0292] B. Mouse model reconstructed with human immune cells, tumor cells were WSU-DLCL2.

[0293] All experimental animals were housed in individually ventilated, temperature- and humidity-controlled enclosures. The temperature ranged from 20.0 to 26.0°C, and the humidity from 40% to 70%. The day / night cycle was 12 hours. WSU-DLCL2 cells (Nanjing Kebai, CBP60273) were cultured in RPMI 1640 medium containing 10% fetal bovine serum. WSU-DLCL2 cells in the logarithmic growth phase were collected and resuspended in PBS to a suitable concentration for subcutaneous tumor inoculation in mice.

[0294] Recovery PBMC, 5×10 6 / Human-derived immune cell reconstructed mouse model was established by intraperitoneal injection only into mice; on the same day, WSU-DLCL2 cells in logarithmic growth phase (1×10⁻⁶) were injected into the peritoneal cavity of mice; 7 The mixture of one mouse and matrix gel (volume 1:1) was injected subcutaneously into the right forelimb of NOG mice (inoculation volume 200uL).

[0295] Before administration, all animals were weighed and tumor volume was measured using calipers. Given that tumor volume affects treatment efficacy, a randomized controlled trial was conducted, grouping mice according to their tumor volume to ensure similarity in tumor volume between groups. Grouping was performed using StudyDirector™ (version 3.1.399.19, vendor Studylog System, Inc., S. San Francisco, CA, USA). The day of mouse grouping was defined as day 0, and intraperitoneal administration began on day D0, administered every three days for a total of six doses. The grouping and administration regimens for the hIgG1AA negative control group and the triple antibody molecular assay are shown in Table 10.

[0296] Table 10 Experimental Groups and Dosing Regimens

[0297] Animals were monitored daily for 22 days after drug administration. Throughout the experiment, the long and wide diameters of the tumor were measured twice a week using calipers, and the tumor volume (mm²) was calculated according to the formula. 3 = 0.5 × (tumor long diameter × tumor short diameter) 2 Tumor volume was calculated. The relative tumor inhibition rate (TGI%) was calculated as follows: TGI% = (1 - T / C) × 100%. T / C% represents the relative tumor growth rate, which is the percentage of tumor volume or weight in the treatment group and the hIgG1AA control group at a specific time point. T and C represent the tumor volume (TV) or tumor weight (TW) in the treatment group and the hIgG1AA control group at a specific time point, respectively. The experimental results, including tumor volume, mouse body weight, and tumor weight in each group, are expressed as mean ± standard error (Mean ± SEM). An independent samples t-test was used to compare whether there were significant differences between the different treatment groups and the control group. Data were analyzed using statistical software. P < 0.05 was considered statistically significant. The experimental results are shown in Table 11 and Figure 6.

[0298] The 0.5mpk and 5mpk dose treatment groups of triple antibody 1, and the 0.1mpk, 0.5mpk, and 5mpk dose treatment groups of triple antibody 3 all showed very significant tumor-inhibiting effects in the human diffuse large B-cell lymphoma WSU-DLCL2 subcutaneous transplantation NOG female mouse MiXeno model. The mice tolerated the test drugs well and did not show significant weight loss or toxicity.

[0299] Table 11 Tumor size and tumor inhibition rate in each treatment group

[0300] Example 9: Obtaining mouse-derived anti-CD79b antibody

[0301] Anti-CD79b monoclonal antibodies were generated by immunizing mice.

[0302] Female, 6-week-old Swiss Webster mice (Charles River Company) were used in the experiment. The mice were housed in an SPF-grade environment. After purchase, the mice were housed in a laboratory environment for one week with a 12 / 12-hour light / dark cycle, temperature 20-25°C, and humidity 40-60%. The immunogen was full-length human CD79b protein with a human Fc tag (ACRO, CDB-H5259, Uniprot: P40259-1). The initial immunization consisted of 50 μg of protein, followed by 25 μg immunizations on days 0, 14, 28, and 42. Three days before spleen cell fusion, a booster immunization was performed using CHOK1 cells (hCD79b-CHOK1) that highly expressed human CD79b. During this period, the antibody titer in mouse serum was determined using the FACS method. After the booster immunization, spleen cells from mice with high and plateauing antibody titers were harvested for SingleB selection.

[0303] Chok1 cells expressing high levels of human CD79b were used to screen for SingleB antibodies. The positive cells were then sequenced individually. After excluding duplicate sequences and sequences containing high-risk sites through computer sequence analysis, 10 monoclonal antibodies were obtained: S7, S20, S27, S36, S37, S39, S44, S45, S48, and S49. The amino acid sequences of the variable regions of S27 and S48 are shown in Table 12.

[0304] Table 12. Amino acid sequences of the variable region of mouse-derived anti-CD79b monoclonal antibodies.

[0305] Based on the above amino acid sequence, the CDR and FR of the antibody variable region were divided using the Kabat numbering rule. The CDR sequence composition of the CD79b monoclonal antibody is shown in Table 13 below.

[0306] Table 13 CDR sequences of mouse-derived anti-CD79b monoclonal antibodies

[0307] Example 10: Obtaining anti-CD20 antibodies derived from alpacas

[0308] Anti-CD20 monoclonal antibodies were generated by immunizing alpacas.

[0309] Healthy alpacas (Chengdu Apak) were used in the experiment. The immunogen was CHOK1 cells (hCD20-CHOK1) that highly expressed full-length human CD20. Immunization was performed every three weeks for a total of four immunizations. Alpaca serum was analyzed using FACS to determine antibody titers. PBMCs were collected after the third and fourth immunizations to construct an alpaca antibody VHH phage library with a library size of 1.04 × 10⁻⁶. 9 .

[0310] Cell-based ELISA was performed using CHOK1 cells (hCD79b-CHOK1) that highly express human CD20. Strains with positive antibodies were sequenced, and two monoclonal antibodies, B8-1 and 1B3, were obtained. The amino acid sequences of the variable regions are shown in Table 14.

[0311] Table 14. Amino acid sequences of the variable region of anti-CD20 monoclonal antibodies derived from alpacas.

[0312] Based on the above amino acid sequence, the CDR and FR of the antibody variable region were divided using the Kabat numbering rule. The CDR sequence composition of the CD20 monoclonal antibody is shown in Table 15 below.

[0313] Table 15 CDR sequences of anti-CD20 monoclonal antibodies derived from alpacas.

[0314] Example 11: Construction of anti-CD79b chimeric monoclonal antibody and CD20 chimeric monoclonal antibody and their transient transfection expression in eukaryotic cells

[0315] The target gene fragments generated by splicing the heavy chain variable region and light chain variable region coding nucleic acid sequences of the disclosed anti-CD79b monoclonal antibody (after sequencing) with the coding nucleic acid sequences of the IgG1 heavy chain constant region and κ light chain constant region, respectively, were cloned into the pTT5 expression vector; the target gene fragments generated by splicing the heavy chain variable region coding nucleic acid sequence of the disclosed anti-CD20 monoclonal antibody (after sequencing) with the coding nucleic acid sequence of the IgG1 Fc constant region were cloned into the pTT5 expression vector. Transfection-grade expression plasmids were prepared for both.

[0316] Expi293F cultured in serum-free medium TMCells (Thermo Fisher Scientific) were seeded in shake flasks (Corning Inc.) and cultured on a shaker at 37°C with 8% CO2. Cell density was adjusted, and the recombinant expression vector containing the target gene fragment and PEI transfection reagent were mixed in an appropriate ratio and added to the cell culture shake flasks. After 6 days of cell culture, the expression supernatant was collected, centrifuged at high speed to remove cell debris, and then purified using a Protein A column for affinity purification. The column was washed with PBS until the A280 reading returned to baseline. The target protein was eluted with acidic elution buffer (pH 3.0-3.5) and neutralized with 1M Tris-HCl (pH 8.0-9.0). The eluted sample was appropriately concentrated and transferred to PBS for aliquoting. The final purified chimeric antibody was analyzed for purity and A280 concentration by SDS-PAGE and HPLC.

[0317] Example 12: Binding of mouse-derived anti-CD79b chimeric antibody to CD79b-expressing cells

[0318] hCD79b-CHOK1 cells were cultured in a medium of F12K + 10% FBS + 4 μg / mL puromycin using T75 cell culture flasks in a 37°C, 5% CO2 incubator. Before use, the cells were washed twice with sterile DPBS, digested with 0.25% trypsin and EDTA for approximately 5 minutes, and then the culture was stopped with complete culture medium.

[0319] Centrifuge the obtained cells at 1000 rpm at room temperature for 5 minutes, discard the supernatant, and resuspend the cells in 100 μL of 1% BSA (in PBS). Count the cells and adjust the cell density to 1E6 / mL. Plate the cells into 96-well round-bottom culture plates, centrifuge at 1500 rpm at 4°C for 5 minutes, discard the supernatant, resuspend the cells in 200 μL of 1% BSA (in PBS), centrifuge again at 1500 rpm at 4°C for 5 minutes, discard the supernatant, and store at 4°C for later use. Dilute the antibody sample to be tested with 1% BSA (in PBS), starting at 100 nM, and then dilute 5-fold down to 8 concentrations. Resuspend the cells in the diluted antibody at 100 μL / well and incubate at 4°C for 1 hour. Centrifuge at 1500 rpm at 4°C for 5 minutes, discard the supernatant. Resuspend and wash the cells in 160 μL of 1% BSA (in PBS), centrifuge at 1500 rpm at 4°C for 5 minutes, and discard the supernatant. Dilute the secondary antibody (goat anti-human IgG Fc PE) 1:400 with 1% BSA (in PBS) according to the manufacturer's instructions. Resuspend the cells in the diluted secondary antibody at a rate of 100 μL / well and incubate at 4°C for 0.5 hours. Centrifuge at 1500 rpm for 5 minutes at 4°C and discard the supernatant. Resuspend and wash the cells in 200 μL of 1% BSA (in PBS), centrifuge at 1500 rpm for 5 minutes at 4°C and discard the supernatant. Resuspend the cells in 100 μL of 1% BSA (in PBS), filter the cells through 300-mesh gauze, and analyze the mean fluorescence intensity of the PE channel using an iQue3 flow cytometer.

[0320] The average fluorescence intensity of the PE channel for each sample was analyzed using the software included with iQue3. The obtained average fluorescence intensity was then imported into Graphpad to analyze the half-maximal binding concentration (EC50) of the antibody to cells. 50 The results of the screening of anti-CD79b chimeric antibodies and the highest mean fluorescence intensity (Top MFI) are shown in Table 16 and Figure 7. All the screened anti-CD79b chimeric antibodies showed good binding to human CD79b.

[0321] Table 16. Binding of mouse-derived anti-CD79b chimeric antibodies to human CD79b-expressing cells.

[0322] Example 13: Binding of alpaca-derived anti-CD20 chimeric antibody to CD20-expressing cells

[0323] hCD20-CHOK1 cells were cultured in a medium of F12K + 10% FBS + 4 μg / mL puromycin using T75 cell culture flasks in a 37°C, 5% CO2 incubator. Before use, the cells were washed twice with sterile DPBS, digested with 0.25% trypsin and EDTA for approximately 5 minutes, and then the culture was stopped with complete culture medium.

[0324] Centrifuge the obtained cells at 1000 rpm at room temperature for 5 minutes, discard the supernatant, and resuspend the cells in 100 μL of 1% BSA (in PBS). Count the cells and adjust the cell density to 1E6 / mL. Plate the cells into 96-well round-bottom culture plates, centrifuge at 1500 rpm at 4°C for 5 minutes, discard the supernatant, resuspend the cells in 200 μL of 1% BSA (in PBS), centrifuge again at 1500 rpm at 4°C for 5 minutes, discard the supernatant, and store at 4°C for later use. Dilute the antibody sample to be tested with 1% BSA (in PBS), starting at 100 nM, and then dilute 5-fold down to 8 concentrations. Resuspend the cells in the diluted antibody at 100 μL / well and incubate at 4°C for 1 hour. Centrifuge at 1500 rpm at 4°C for 5 minutes, discard the supernatant. Resuspend and wash the cells in 160 μL of 1% BSA (in PBS), centrifuge at 1500 rpm at 4°C for 5 minutes, and discard the supernatant. Dilute the secondary antibody (goat anti-human IgG Fc PE) 1:400 with 1% BSA (in PBS) according to the manufacturer's instructions. Resuspend the cells in the diluted secondary antibody at a rate of 100 μL / well and incubate at 4°C for 0.5 hours. Centrifuge at 1500 rpm for 5 minutes at 4°C and discard the supernatant. Resuspend and wash the cells in 200 μL of 1% BSA (in PBS), centrifuge at 1500 rpm for 5 minutes at 4°C and discard the supernatant. Resuspend the cells in 100 μL of 1% BSA (in PBS), filter the cells through 300-mesh gauze, and analyze the mean fluorescence intensity of the PE channel using an iQue3 flow cytometer.

[0325] The average fluorescence intensity of the PE channel for each sample was analyzed using the software included with iQue3. The obtained average fluorescence intensity was then imported into Graphpad to analyze the half-maximal binding concentration (MCC) of the antibody and the top average fluorescence intensity (Top MFI). The results are shown in Table 17 and Figure 8. Among the clones that bound to human CD20-expressing cells, clone B8-1 showed a stronger affinity for human CD20 than clone 1B3.

[0326] Table 17 Binding of alpaca-derived anti-CD20 chimeric antibodies to human CD20-expressing cells.

[0327] Example 14: Humanization design of mouse-derived anti-CD79b antibody and alpaca-derived anti-CD20 antibody

[0328] A. Humanization of mouse-derived anti-CD79b antibody

[0329] After expression, purification, and cell-level binding tests, two clones of the 10 chimeric antibodies against CD79b were further selected for humanization design.

[0330] Humanization of murine anti-human CD79b monoclonal antibodies was performed using methods published in numerous publications in the field. In short, a human constant domain was used to replace the parental (murine antibody) constant domain, and the human antibody sequence was selected based on the homology between the murine and human antibodies. Based on the obtained typical VH / VL CDR structure of the murine antibody, the heavy and light chain variable region sequences were compared with a human antibody germline database to obtain a highly homologous human germline template.

[0331] The CDR region of the murine antibody was transplanted onto a selected humanized template, replacing the humanized variable region, and then recombined with the IgG constant region (preferably IgG1 for the heavy chain and κ for the light chain). Then, based on the three-dimensional structure of the murine antibody, reverse mutations were performed on embedded residues, residues that directly interact with the CDR region, and residues that significantly affect the conformation of VL and VH, resulting in the design of antibodies composed of multiple humanized light and heavy chain variable region sequences. Humanized antibodies for S27 include 27H2L2, 27H2L3, 27H2L4, 27H2L5, 27H2L6, 27H2L7, 27H3L2, 27H3L3, 27H3L4, 27H3L5, 27H3L6, 27H3L7, 27H4L2, 27H4L3, 27H4L4, 27H4L5, 27H4L6, and 27H4L7. Humanized antibodies for S48 include 49H2L2, 48H2L3, 48H2L4, 48H2L5, 48H4L2, 48H4L3, 48H4L4, and 48H4L5. The sequences of 27H4L6 and 48H4L5 are shown in Table 18.

[0332] Table 18. Amino acid sequences of the variable regions of mouse-derived humanized antibodies 27H4L6 and 48H4L5

[0333] B. Humanization of alpaca-derived anti-CD20 antibodies

[0334] Humanization was carried out on two CD20-resistant alpaca VHH clones.

[0335] Humanization of alpaca-derived anti-human CD20 monoclonal antibodies was performed using methods published in numerous publications in the field. In short, human antibody sequences were selected based on the homology between alpaca-derived antibodies and human antibodies. Based on the obtained typical VHH structure of the alpaca antibody, the variable region sequence was compared with a human heavy chain antibody phylogenetic database to obtain a highly homologous human phylogenetic template.

[0336] The CDR region of the alpaca antibody was transplanted onto the selected corresponding humanized template, replacing the humanized variable region, and then recombined with the IgG1 constant region. Then, reverse mutations were performed on the embedded residues, residues that directly interact with the CDR region, and residues that have an important influence on the conformation of VHH. Antibodies composed of multiple humanized heavy chain variable region sequences were designed. The humanized antibodies of B8-1 include B8-H1, B8H2, B8H8, B8H9, B8H10, B8H11, B8H12, B8H13, B8H14, B8H15, and B8H16. The humanized antibodies of 1B3-1 include 1B3H1, 1B3H2, 1B3H3, 1B3H4, 1B3H5, ​​1B3H6, 1B3H3a, 1B3H4a, 1B3H4b, 1B3H3c, and 1B3H4c. The sequences of B8-H9 and 1B3-H4 are shown in Table 19.

[0337] Table 19. Amino acid sequences of the variable regions of humanized antibodies B8-H9 and 1B3-H4 derived from alpacas.

[0338] Example 15: Preparation of humanized antibodies against CD79b and CD20 monoclonal antibodies

[0339] Referring to the method described in Example 11, the target gene fragment generated by splicing the nucleic acid sequences encoding the heavy chain variable region and light chain variable region of the anti-CD79b humanized monoclonal antibody with the nucleic acid sequences encoding the IgG1 heavy chain constant region and κ light chain constant region, respectively, was cloned into the pTT5 expression vector; the target gene fragment generated by splicing the nucleic acid sequence encoding the VHH region of the heavy chain variable region of the anti-CD20 humanized antibody with the nucleic acid sequence encoding the IgG1 Fc constant region was cloned into the pTT5 expression vector. Transfection-grade expression plasmids were prepared for both methods.

[0340] Expi293F cultured in serum-free medium TM Cells (Thermo Fisher Scientific) were seeded in shake flasks (Corning Inc.) and cultured on a shaker at 37°C with 8% CO2. Cell density was adjusted, and the recombinant expression vector containing the target gene fragment and PEI transfection reagent were mixed in an appropriate ratio and added to the cell culture shake flasks. After 6 days of cell culture, the expression supernatant was collected, centrifuged at high speed to remove cell debris, and then purified using a Protein A column for affinity purification. The column was washed with PBS until the A280 reading returned to baseline. The target protein was eluted with acidic elution buffer (pH 3.0-3.5) and neutralized with 1M Tris-HCl (pH 8.0-9.0). The eluted sample was appropriately concentrated and transferred to PBS for aliquoting. The final purified antibody was analyzed for purity and A280 concentration by SDS-PAGE and HPLC.

[0341] Example 16: Binding of humanized antibodies to cells expressing human CD79b or human CD20

[0342] Both hCD79b-CHOK1 and hCD20-CHOK1 cells were cultured in F12K + 10% FBS + 4 μg / mL puromycin using T75 cell culture flasks in a 37°C, 5% CO2 incubator. Before use, the cells were washed twice with sterile DPBS, digested with 0.25% trypsin and EDTA for approximately 5 minutes, and then the culture was stopped with complete culture medium.

[0343] Centrifuge the obtained cells at 1000 rpm at room temperature for 5 minutes, discard the supernatant, and resuspend the cells in 100 μL of 1% BSA (in PBS). Count the cells and adjust the cell density to 1E6 / mL. Plate the cells into 96-well round-bottom culture plates, centrifuge at 1500 rpm at 4°C for 5 minutes, discard the supernatant, resuspend the cells in 200 μL of 1% BSA (in PBS), centrifuge again at 1500 rpm at 4°C for 5 minutes, discard the supernatant, and store at 4°C for later use. Dilute the antibody sample to be tested with 1% BSA (in PBS), starting at 100 nM, and then dilute 5-fold down to 8 concentrations. Resuspend the cells in the diluted antibody at 100 μL / well and incubate at 4°C for 1 hour. Centrifuge at 1500 rpm at 4°C for 5 minutes, discard the supernatant. Resuspend and wash the cells in 160 μL of 1% BSA (in PBS), centrifuge at 1500 rpm at 4°C for 5 minutes, and discard the supernatant. Dilute the secondary antibody (goat anti-human IgG Fc PE) 1:400 with 1% BSA (in PBS) according to the manufacturer's instructions. Resuspend the cells in the diluted secondary antibody at a rate of 100 μL / well and incubate at 4°C for 0.5 hours. Centrifuge at 1500 rpm for 5 minutes at 4°C and discard the supernatant. Resuspend and wash the cells in 200 μL of 1% BSA (in PBS), centrifuge at 1500 rpm for 5 minutes at 4°C and discard the supernatant. Resuspend the cells in 100 μL of 1% BSA (in PBS), filter the cells through 300-mesh gauze, and analyze the mean fluorescence intensity of the PE channel using an iQue3 flow cytometer.

[0344] The average fluorescence intensity of the PE channel for each sample was analyzed using the software included with iQue3. The obtained average fluorescence intensity was then imported into Graphpad to analyze the half-maximal binding concentration (MCC) and top average fluorescence intensity (Top MFI) of the antibody and cells. The results are shown in Table 20 and Figures 9A-9D. The binding ability of the humanized antibody clone 27H4L6 to hCD79b-CHOK1 cells was basically equivalent to that of the parent antibody S27; the binding ability of the humanized antibody clone 48H4L5 to hCD79b-CHOK1 cells was basically equivalent to that of the parent antibody S48; the binding ability of the humanized antibody clone B8-H9 to hCD20-CHOK1 cells was basically equivalent to that of the parent antibody B8-1; and the binding ability of the humanized antibody clone 1B3-H4 to hCD20-CHOK1 cells was basically equivalent to that of the parent antibody 1B3.

[0345] Table 20. Binding of humanized monoclonal antibodies with cells expressing CD79b or CD20

[0346] The embodiments described above are merely exemplary, and any person skilled in the art will recognize or be able to identify numerous equivalents of specific compounds, materials, and operations without the need for extraordinary experimentation. All such equivalents are within the scope of this disclosure and are encompassed by the claims.

Claims

1. A trispecific antigen-binding molecule comprising: a first binding site for a first antigen, a second binding site for a second antigen, and a third binding site for a third antigen, wherein the first antigen is CD79b, the second antigen is CD3, and the third antigen is CD20, preferably wherein the second antigen is CD3ε.

2. The trispecific antigen-binding molecule of claim 1, wherein the first binding site against the first antigen comprises an antigen-binding fragment Fab capable of specifically binding to the first antigen, and / or, the second binding site against the second antigen comprises a single-chain antibody (scFv) domain capable of specifically binding to the second antigen, and / or, the third binding site against the third antigen comprises a heavy chain single-domain antibody (VHH) domain capable of specifically binding to the third antigen or an antigen-binding fragment Fab capable of specifically binding to the third antigen.

3. The trispecific antigen-binding molecule as described in claim 2, wherein, The antigen-binding fragment Fab capable of specifically binding to the first antigen comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises HCDR1 as shown in SEQ ID NO:20, HCDR2 as shown in SEQ ID NO:21, and HCDR3 as shown in SEQ ID NO:22, and / or, the light chain variable region comprises LCDR1 as shown in SEQ ID NO:23, LCDR2 as shown in SEQ ID NO:24, and LCDR3 as shown in SEQ ID NO:25; or, the heavy chain variable region comprises HCDR1 as shown in SEQ ID NO:26, HCDR2 as shown in SEQ ID NO:27, and HCDR3 as shown in SEQ ID NO:28, and / or, the light chain variable region comprises LCDR1 as shown in SEQ ID NO:29, LCDR2 as shown in SEQ ID NO:30, and LCDR3 as shown in SEQ ID NO:

31. Alternatively, the heavy chain variable region and light chain variable region of the antigen-binding fragment Fab capable of specifically binding to the first antigen contain the same HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences as the heavy chain variable regions and light chain variable regions of any of the following groups: (1) SEQ ID NO:9 and SEQ ID NO:10; (2) SEQ ID NO:11 and SEQ ID NO:12; Preferably, the antigen-binding fragment Fab capable of specifically binding to the first antigen comprises a heavy chain variable region as shown in SEQ ID NO:9 and / or a light chain variable region as shown in SEQ ID NO:10; or, the antigen-binding fragment Fab capable of specifically binding to the first antigen comprises a heavy chain variable region as shown in SEQ ID NO:11 and / or a light chain variable region as shown in SEQ ID NO:

12. More preferably, the antigen-binding fragment Fab capable of specifically binding to the first antigen comprises a heavy chain domain as shown in SEQ ID NO:53 and / or a light chain domain as shown in SEQ ID NO:3; or, the antigen-binding fragment Fab capable of specifically binding to the first antigen comprises a heavy chain domain sequence as shown in SEQ ID NO:68 and / or a light chain domain sequence as shown in SEQ ID NO:

7.

4. The trispecific antigen-binding molecule of claim 2 or 3, wherein the single-chain antibody (scFv) domain comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising HCDR1 as shown in SEQ ID NO:44, HCDR2 as shown in SEQ ID NO:45, and HCDR3 as shown in SEQ ID NO:46, and the light chain variable region comprising LCDR1 as shown in SEQ ID NO:47, LCDR2 as shown in SEQ ID NO:48, and LCDR3 as shown in SEQ ID NO:

49. Preferably, the single-chain antibody (scFv) domain includes a heavy chain variable region as shown in SEQ ID NO:17 and a light chain variable region as shown in SEQ ID NO:18; More preferably, the single-chain antibody (scFv) domain comprises an amino acid sequence as shown in SEQ ID NO:

19.

5. The trispecific antigen-binding molecule according to any one of claims 2-4, wherein, The VHH domain includes HCDR1 as shown in SEQ ID NO:32, HCDR2 as shown in SEQ ID NO:33, and HCDR3 as shown in SEQ ID NO:34; or, the VHH domain includes HCDR1 as shown in SEQ ID NO:35, HCDR2 as shown in SEQ ID NO:36, and HCDR3 as shown in SEQ ID NO:

37. Alternatively, the VHH domain contains the same HCDR1, HCDR2, HCDR3 sequences as the heavy chain variable regions shown in SEQ ID NO:13 or SEQ ID NO:14; Preferably, the VHH domain comprises a sequence as shown in SEQ ID NO:13 or SEQ ID NO:

14.

6. The trispecific antigen-binding molecule according to any one of claims 2-4, wherein, The antigen-binding fragment Fab capable of specifically binding to the third antigen includes a heavy chain variable region and a light chain variable region. The heavy chain variable region includes HCDR1 as shown in SEQ ID NO:38, HCDR2 as shown in SEQ ID NO:39, and HCDR3 as shown in SEQ ID NO:40, and / or the light chain variable region includes LCDR1 as shown in SEQ ID NO:41, LCDR2 as shown in SEQ ID NO:42, and LCDR3 as shown in SEQ ID NO:

43. Alternatively, the antigen-binding fragment Fab capable of specifically binding to the third antigen comprises HCDR1, HCDR2, and HCDR3 identical to HCDR1, HCDR2, and HCDR3 of the heavy chain variable region as shown in SEQ ID NO:15, and LCDR1, LCDR2, and LCDR3 identical to LCDR1, LCDR2, and LCDR3 of the light chain variable region as shown in SEQ ID NO:

16. Preferably, the antigen-binding fragment Fab capable of specifically binding to the third antigen comprises a heavy chain variable region as shown in SEQ ID NO:15, and / or a light chain variable region as shown in SEQ ID NO:16; More preferably, the antigen-binding fragment Fab capable of specifically binding to the third antigen comprises a heavy chain domain as shown in SEQ ID NO:69, and / or a light chain domain as shown in SEQ ID NO:

8.

7. The trispecific antigen-binding molecule according to any one of claims 2-5, wherein the antigen-binding fragment Fab capable of specifically binding to the first antigen comprises HCDR1 as shown in SEQ ID NO:20, HCDR2 as shown in SEQ ID NO:21, and HCDR3 as shown in SEQ ID NO:22, and LCDR1 as shown in SEQ ID NO:23, LCDR2 as shown in SEQ ID NO:24, and LCDR3 as shown in SEQ ID NO:25, wherein the single-chain antibody (scFv) domain comprises HCDR1 as shown in SEQ ID NO:44, HCDR2 as shown in SEQ ID NO:45, HCDR3 as shown in SEQ ID NO:46, and LCDR1 as shown in SEQ ID NO:47, LCDR2 as shown in SEQ ID NO:48, and LCDR3 as shown in SEQ ID NO:49, and the VHH domain comprises any one of the following sets of HCDR1, HCDR2, and HCDR3 sequences: (1) SEQ ID NO:32, SEQ ID NO:33 and SEQ ID NO:34, or (2) SEQ ID NO:35, SEQ ID NO:36 and SEQ ID NO:37; Preferably, the antigen-binding fragment Fab capable of specifically binding to the first antigen comprises a heavy chain variable region as shown in SEQ ID NO:9 and a light chain variable region as shown in SEQ ID NO:10, the single-chain antibody (scFv) domain comprises a heavy chain variable region sequence as shown in SEQ ID NO:17 and a light chain variable region sequence as shown in SEQ ID NO:18, and the VHH domain comprises a sequence as shown in SEQ ID NO:13 or SEQ ID NO:14; More preferably, the antigen-binding fragment Fab capable of specifically binding to the first antigen comprises a heavy chain domain as shown in SEQ ID NO:53 and a light chain domain as shown in SEQ ID NO:3, the single-chain antibody (scFv) domain comprises the amino acid sequence shown in SEQ ID NO:19, and the VHH domain comprises the sequence shown in SEQ ID NO:13 or SEQ ID NO:

14.

8. The trispecific antigen-binding molecule according to any one of claims 2-4 and 6, wherein the antigen-binding fragment Fab capable of specifically binding to the first antigen comprises HCDR1 as shown in SEQ ID NO:26, HCDR2 as shown in SEQ ID NO:27, and HCDR3 as shown in SEQ ID NO:28, and LCDR1 as shown in SEQ ID NO:29, LCDR2 as shown in SEQ ID NO:30, and LCDR3 as shown in SEQ ID NO:31; the single-chain antibody (scFv) domain comprises HCDR1 as shown in SEQ ID NO:44, HCDR2 as shown in SEQ ID NO:45, HCDR3 as shown in SEQ ID NO:46, LCDR1 as shown in SEQ ID NO:47, LCDR2 as shown in SEQ ID NO:48, and LCDR3 as shown in SEQ ID NO:49; and the antigen-binding fragment Fab capable of specifically binding to the third antigen comprises HCDR1 as shown in SEQ ID NO:38, HCDR2 as shown in SEQ ID NO:39, and LCDR3 as shown in SEQ ID NO:

49. HCDR3 shown in NO:40, and LCDR1 with sequence as shown in SEQ ID NO:41, LCDR2 with sequence as shown in SEQ ID NO:42, and LCDR3 with sequence as shown in SEQ ID NO:43; Preferably, the antigen-binding fragment Fab capable of specifically binding to the first antigen comprises a heavy chain variable region as shown in SEQ ID NO:11 and a light chain variable region as shown in SEQ ID NO:12, the single-chain antibody (scFv) domain comprises a heavy chain variable region as shown in SEQ ID NO:17 and a light chain variable region as shown in SEQ ID NO:18, and the antigen-binding fragment Fab capable of specifically binding to the third antigen comprises a heavy chain variable region as shown in SEQ ID NO:15 and a light chain variable region as shown in SEQ ID NO:16; More preferably, the antigen-binding fragment Fab capable of specifically binding to the first antigen comprises a heavy chain domain sequence as shown in SEQ ID NO:68 and a light chain domain sequence as shown in SEQ ID NO:7, the single-chain antibody (scFv) domain comprises an amino acid sequence as shown in SEQ ID NO:19, and the antigen-binding fragment Fab capable of specifically binding to the third antigen comprises a heavy chain domain sequence as shown in SEQ ID NO:69 and a light chain domain sequence as shown in SEQ ID NO:

8.

9. The trispecific antigen-binding molecule according to any one of claims 1-8, comprising the following polypeptide: The first polypeptide comprises: (i) a heavy chain domain of an antigen-binding fragment Fab capable of specifically binding to a first antigen, (ii) a single-chain antibody (scFv) domain capable of specifically binding to a second antigen, and (iii) a first Fc domain. The second polypeptide comprises: a light chain domain of the antigen-binding fragment Fab capable of specifically binding to the first antigen, and, The third polypeptide comprises: (a) a heavy chain single-domain antibody (VHH) domain capable of specifically binding to the third antigen or a heavy chain domain of an antigen-binding fragment Fab capable of specifically binding to the third antigen, and (b) a second Fc domain. When the third polypeptide contains a heavy chain domain of an antigen-binding fragment Fab capable of specifically binding to the third antigen, the trispecific antigen-binding molecule further contains a fourth polypeptide containing a light chain domain of an antigen-binding fragment Fab capable of specifically binding to the third antigen. The heavy chain domain of the antigen-binding fragment Fab capable of specifically binding to the first antigen and the light chain domain of the antigen-binding fragment Fab capable of specifically binding to the first antigen form a first binding site against the first antigen; the single-chain antibody (scFv) domain capable of specifically binding to the second antigen forms a second binding site against the second antigen; the first Fc domain and the second Fc domain associate with each other; the heavy chain single-domain antibody (VHH) domain of the third polypeptide forms a third binding site against the third antigen, or the heavy chain domain of the antigen-binding fragment Fab capable of specifically binding to the third antigen and the light chain domain of the antigen-binding fragment Fab capable of specifically binding to the third antigen form a third binding site against the third antigen.

10. The trispecific antigen-binding molecule according to any one of claims 2-9, wherein the heavy chain variable region of the single-chain antibody (scFv) domain and the light chain variable region of the single-chain antibody (scFv) domain are connected by a first linker; preferably, the C-terminus of the heavy chain variable region of the single-chain antibody (scFv) domain is fused to the N-terminus of the first linker, and the C-terminus of the first linker is fused to the N-terminus of the light chain variable region of the single-chain antibody (scFv) domain; more preferably, the first linker comprises an amino acid sequence (G4S). n n is any integer from 1 to 10.

11. The trispecific antigen-binding molecule of claim 9 or 10, wherein the first Fc domain comprises a first CH2 domain and a first CH3 domain of an immunoglobulin, and the C-terminus of the first CH2 domain is fused to the N-terminus of the first CH3 domain; the second Fc domain comprises a second CH2 domain and a second CH3 domain of an immunoglobulin, and the C-terminus of the second CH2 domain is fused to the N-terminus of the second CH3 domain; Preferably, the N-terminus of the first Fc domain is fused with the C-terminus of the scFv domain; more preferably, the N-terminus of the first Fc domain is fused with the C-terminus of the scFv domain via a second connector; even more preferably, the C-terminus of the single-chain antibody (scFv) domain is fused with the N-terminus of the second connector, and the C-terminus of the second connector is fused with the N-terminus of the first Fc domain. More preferably, the Fc domain is derived from IgG1.

12. The trispecific antigen-binding molecule according to any one of claims 2-11, wherein the heavy chain domain of the antigen-binding fragment Fab capable of specifically binding the first antigen is connected to the scFv domain via a third connector, wherein the C-terminus of the heavy chain domain of the antigen-binding fragment Fab capable of specifically binding the first antigen is fused to the N-terminus of the third connector, and the C-terminus of the third connector is fused to the N-terminus of the scFv domain.

13. The trispecific antigen-binding molecule according to any one of claims 9-12, wherein the C-terminus of the heavy chain single-domain antibody (VHH) domain capable of specifically binding to the third antigen is fused to the N-terminus of the second Fc domain, preferably, the C-terminus of the VHH domain is fused to the N-terminus of the second Fc domain via a fourth linker; Alternatively, the heavy chain domain of the Fab structure capable of specifically binding to the third antigen may be fused with the N-terminus of the second Fc domain.

14. The trispecific antigen-binding molecule according to any one of claims 9-13, wherein the first polypeptide comprises the following structure: Fab heavy chain domain - scFv domain - first Fc domain, preferably the first polypeptide comprises the following structure: Fab heavy chain variable region - Fab CH1 - third linker - scFv heavy chain variable region - first linker - scFv light chain variable region - second linker - first CH2 - first CH3; The second polypeptide comprises the following structure: Fab light chain variable region - light chain constant region; And / or, wherein the third polypeptide comprises the following structure: VHH domain-second Fc domain, preferably, the third polypeptide comprises the following structure: VHH-fourth linker-second CH2-second CH3; or, the third polypeptide comprises the following structure: Fab heavy chain domain-second Fc domain, preferably comprising the following structure: Fab heavy chain variable region-Fab CH1-second CH2-second CH3, and the fourth polypeptide comprises the following structure: Fab light chain variable region-light chain constant region.

15. The trispecific antigen-binding molecule according to any one of claims 9-14, wherein the first Fc domain comprises the amino acid sequence shown in SEQ ID NO:50, and the second Fc domain comprises the amino acid sequence shown in SEQ ID NO:51 or 55; preferably, when the trispecific antigen-binding molecule comprises a VHH domain capable of specifically binding a third antigen, the second Fc domain comprises the amino acid sequence shown in SEQ ID NO:51, or when the trispecific antigen-binding molecule comprises a Fab domain capable of specifically binding a third antigen, the second Fc domain comprises the amino acid sequence shown in SEQ ID NO:

55.

16. The trispecific antigen-binding molecule according to any one of claims 1-15, wherein the trispecific antigen-binding molecule comprises: a first polypeptide with the sequence shown in SEQ ID NO:1, a second polypeptide with the sequence shown in SEQ ID NO:3, and a third polypeptide with the sequence shown in SEQ ID NO:2; or a first polypeptide with the sequence shown in SEQ ID NO:1, a second polypeptide with the sequence shown in SEQ ID NO:3, and a third polypeptide with the sequence shown in SEQ ID NO:4; or a first polypeptide with the sequence shown in SEQ ID NO:5, a second polypeptide with the sequence shown in SEQ ID NO:7, a third polypeptide with the sequence shown in SEQ ID NO:6, and a fourth polypeptide with the sequence shown in SEQ ID NO:

8.

17. A nucleic acid molecule encoding the trispecific antigen-binding molecule as described in any of the preceding claims.

18. An expression vector comprising the nucleic acid molecule as described in claim 17.

19. A host cell comprising the nucleic acid molecule of claim 17 or the expression vector of claim 18; preferably, the host cell is a prokaryotic cell or a eukaryotic cell; the prokaryotic cell is preferably Escherichia coli; the eukaryotic cell is preferably a mammalian cell or yeast; more preferably, the mammalian cell is a CHO cell, an Expi293 cell, or a HEK293 cell.

20. A method for preparing a trispecific antigen-binding molecule, wherein, The method includes culturing the host cell as described in claim 19 under suitable conditions.

21. A pharmaceutical composition comprising a trispecific antigen-binding molecule as described in any one of claims 1-16, a nucleic acid molecule as described in claim 17, an expression vector as described in claim 18, and / or a host cell as described in claim 19; preferably, the pharmaceutical composition is used to treat, alleviate, and / or prevent autoimmune diseases or tumors; preferably, it further comprises a pharmaceutically acceptable carrier.

22. The pharmaceutical composition of claim 21, further comprising one or more additional therapeutic agents.

23. An antibody-drug conjugate comprising the trispecific antigen-binding molecule as described in any one of claims 1-16, and other bioactive molecules; Preferably, the other bioactive molecules are small molecule drugs; preferably, the trispecific antigen-binding molecule is connected to the other bioactive molecules through a connector.

24. Use of a trispecific antigen-binding molecule as described in any one of claims 1-16, a nucleic acid molecule as described in claim 17, an expression vector as described in claim 18, a host cell as described in claim 19, a pharmaceutical composition as described in claim 21 or 22, or an antibody-drug conjugate as described in claim 23 in the preparation of a medicament for treating, alleviating, and / or preventing tumors or autoimmune diseases.

25. The use as described in claim 24, wherein the tumor is a CD20 and / or CD79b positive tumor.

26. The pharmaceutical composition of claim 21 or 22, or the antibody-drug conjugate of claim 23, or the use of claim 24 or 25, wherein the tumor is selected from: lymphomas such as B-cell lymphoma, including subtypes such as diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), and mantle cell lymphoma (MCL), and metastatic carcinomas of the above tumors.

27. A method for inducing cell death expressing CD20 and / or CD79b, wherein, The method includes contacting the cells with a trispecific antigen-binding molecule as described in any one of claims 1-16, a nucleic acid molecule as described in claim 17, an expression vector as described in claim 18, a host cell as described in claim 19, a pharmaceutical composition as described in claim 21 or 22, and / or an antibody-drug conjugate as described in claim 23. Preferably, the cells expressing CD20 and / or CD79b are immune cells or tumor cells.

28. The method of claim 27, wherein the tumor cells are selected from cells of tumors such as lymphomas such as B-cell lymphoma, including subtypes such as diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL) and mantle cell lymphoma (MCL), and metastatic carcinomas of the above tumors.

29. A method for treating a disease in a subject, wherein, The method includes administering to a subject in need an effective amount of the trispecific antigen-binding molecule as described in any one of claims 1-16, the nucleic acid molecule as described in claim 17, the expression vector as described in claim 18, the host cell as described in claim 19, the pharmaceutical composition as described in claim 21 or 22, and / or the antibody-drug conjugate as described in claim 23.

30. The method of claim 29, wherein the disease is a tumor or an autoimmune disease.

31. The method of claim 30, wherein the tumor is selected from: lymphomas such as B-cell lymphoma, including subtypes such as diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), and mantle cell lymphoma (MCL), and metastatic carcinomas of the above tumors.

32. The method of any one of claims 29-31, further comprising administering an additional therapeutic agent to the subject.

33. An anti-CD79b antibody or its antigen-binding fragment, wherein, The anti-CD79b antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein the heavy chain variable region and the light chain variable region comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences identical to those of any of the following groups of heavy chain variable regions and light chain variable regions: (1) SEQ ID NO: 9 and 10; (2) SEQ ID NO: 58 and 59; (3) SEQ ID NO: 11 and 12; or (4) SEQ ID NO: 60 and 61; Preferably, the heavy chain variable region and the light chain variable region each comprise an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of the following groups of heavy chain variable regions and light chain variable regions: (1) SEQ ID NO: 9 and 10; (2) SEQ ID NO: 58 and 59; (3) SEQ ID NO: 11 and 12; or (4) SEQ ID NO: 60 and 61; Alternatively, the heavy chain variable region and the light chain variable region comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences selected from any one of the following: (1) SEQ ID NO: 20, 21, 22, 23, 24 and 25; (2) SEQ ID NO: 26, 27, 28, 29, 30 and 31; or (3) SEQ ID NO: 26, 27, 62, 29, 63 and 31; Preferably, the heavy chain variable region and the light chain variable region each comprise an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of the following groups of heavy chain variable regions and light chain variable regions: (1) SEQ ID NO: 9 and 10; (2) SEQ ID NO: 58 and 59; (3) SEQ ID NO: 11 and 12; or (4) SEQ ID NO: 60 and 61.

34. An anti-CD79b antibody or its antigen-binding fragment, wherein, The anti-CD79b antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region each comprise an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of the following groups: (1) SEQ ID NO: 9 and 10; (2) SEQ ID NO: 58 and 59; (3) SEQ ID NO: 11 and 12; or (4) SEQ ID NO: 60 and 61.

35. The anti-CD79b antibody or its antigen-binding fragment as described in claim 33 or 34, further comprising one or more of the following characteristics: (1) It also includes a heavy chain constant region and / or a light chain constant region; preferably, the heavy chain constant region contains Fc; more preferably, Fc is derived from mice or humans; more preferably, the sequence of Fc is a natural or modified variant; (2) It is a mouse-derived antibody, a chimeric antibody, a humanized antibody, or a fully human antibody; (3) It is a monoclonal antibody, or a full-length antibody, or its antigen-binding fragment is Fab, Fv, scFv, F(ab')2, a linear antibody or a heavy chain single-domain antibody; (4) It is in the form of IgG1, IgG2, IgG3 or IgG4.

36. A conjugate comprising an anti-CD79b antibody or an antigen-binding fragment thereof as described in any one of claims 33-35 and a capture marker or detection marker conjugated thereto, wherein the detection marker includes, but is not limited to, a radionuclide, a luminescent substance (e.g., fluorescein), a colored substance, or an enzyme.

37. A fusion protein, wherein the fusion protein comprises an anti-CD79b antibody or an antigen-binding fragment thereof as described in any one of claims 33-35.

38. A bispecific antibody or its antigen-binding fragment thereof, or a multispecific antibody or its antigen-binding fragment thereof, wherein, The bispecific antibody or its antigen-binding fragment or the multispecific antibody or its antigen-binding fragment comprises the anti-CD79b antibody or its antigen-binding fragment as described in any one of claims 33-35.

39. An antibody-drug conjugate (ADC), wherein, The antibody-drug conjugate comprises an anti-CD79b antibody or its antigen-binding fragment as described in any one of claims 33-35, a fusion protein as described in claim 37, or a bispecific antibody or its antigen-binding fragment or a multispecific antibody or its antigen-binding fragment as described in claim 38, and other bioactive molecules conjugated thereto; preferably, the other bioactive molecules are small molecule drugs; more preferably, the anti-CD79b antibody or its antigen-binding fragment, the fusion protein or the bispecific antibody or its antigen-binding fragment or the multispecific antibody or its antigen-binding fragment are connected to the other bioactive molecules via a linker.

40. A chimeric antigen receptor (CAR) or a cell containing said chimeric antigen receptor (e.g., CAR-T cells) comprising an anti-CD79b antibody as described in any one of claims 33-35 or an antigen-binding fragment thereof, a fusion protein as described in claim 37, or a bispecific antibody as described in claim 38 or an antigen-binding fragment thereof, or a multispecific antibody or an antigen-binding fragment thereof.

41. A nucleic acid encoding an anti-CD79b antibody or its antigen-binding fragment as described in any one of claims 33-35, a fusion protein as described in claim 37, or a bispecific antibody or its antigen-binding fragment as described in claim 38, or a multispecific antibody or its antigen-binding fragment.

42. A recombinant vector comprising the nucleic acid as described in claim 41.

43. A host cell comprising the nucleic acid as described in claim 41 or the recombinant vector as described in claim 42.

44. The host cell as claimed in claim 43, wherein it is a prokaryotic cell, such as Escherichia coli; or a eukaryotic cell, such as yeast or a mammalian cell, wherein the mammalian cell is preferably a CHO cell or a HEK293 cell.

45. A method for preparing an anti-CD79b antibody or its antigen-binding fragment, a fusion protein, a bispecific antibody or its antigen-binding fragment, or a multispecific antibody or its antigen-binding fragment, wherein, The method includes: culturing the host cell as described in claim 43 or 44 under suitable conditions, and purifying the expression product from the cell.

46. ​​Use of the anti-CD79b antibody or its antigen-binding fragment as described in any one of claims 33-35, the fusion protein as described in claim 37, or the bispecific antibody or its antigen-binding fragment as described in claim 38, or the multispecific antibody or its antigen-binding fragment, in the preparation of a detection reagent or diagnostic reagent.

47. The use as described in claim 46, wherein, The detection reagent is used to detect the expression of CD79b; the diagnostic reagent is used to diagnose tumors; preferably, the tumor is selected from: lymphomas such as B-cell lymphoma, including diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL) and mantle cell lymphoma (MCL) subtypes, as well as metastatic cancers of the above tumors.

48. A pharmaceutical composition comprising an anti-CD79b antibody or an antigen-binding fragment thereof as described in any one of claims 33-35, an antibody-drug conjugate as described in claim 39, a fusion protein as described in claim 37, a bispecific antibody or an antigen-binding fragment thereof or a multispecific antibody or an antigen-binding fragment thereof as described in claim 38, a chimeric antigen receptor (CAR) or a cell containing said chimeric antigen receptor as described in claim 40, a nucleic acid as described in claim 41, a recombinant vector as described in claim 42, or a host cell as described in claim 43 or 44; preferably, the pharmaceutical composition is used for the treatment, relief, and / or prevention of autoimmune diseases or tumors; more preferably, the tumor is selected from: lymphomas such as B-cell lymphoma, including subtypes such as diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), and mantle cell lymphoma (MCL), and metastatic tumors of the above tumors.

49. The pharmaceutical composition of claim 48, further comprising a pharmaceutically acceptable carrier.

50. The pharmaceutical composition of claim 48 or 49, further comprising one or more additional therapeutic agents.

51. A method for treating a disease in a subject, wherein, The method includes administering an effective amount of the pharmaceutical composition as described in any one of claims 48-50 to a subject in need, preferably, the disease being a disease associated with CD79b expression.

52. The method of claim 51, wherein the disease is an autoimmune disease or a tumor.

53. The method of claim 52, wherein the tumor is selected from: lymphomas such as B-cell lymphoma, including subtypes such as diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), and mantle cell lymphoma (MCL), and metastatic carcinomas of the above tumors.

54. The method of any one of claims 51-53, further comprising administering additional therapeutic agents to the subject.

55. An anti-CD20 antibody or an antigen-binding fragment thereof, wherein, The anti-CD20 antibody or its antigen-binding fragment includes a heavy chain variable region, which includes HCDR1, HCDR2, and HCDR3. The heavy chain variable region contains HCDR1, HCDR2, and HCDR3 sequences identical to those of any of the following groups of heavy chain variable regions: (1)SEQ ID NO:64; (2)SEQ ID NO:13; (3) SEQ ID NO:65; or (4)SEQ ID NO:14; Preferably, the heavy chain variable regions each comprise an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the heavy chain variable regions of any of the following groups: (1)SEQ ID NO:64; (2)SEQ ID NO:13; (3) SEQ ID NO:65; or (4)SEQ ID NO:14; Alternatively, the heavy chain variable region may comprise HCDR1, HCDR2, and HCDR3 sequences selected from any of the following: (1) SEQ ID NO: 66, 67 and 34; (2) SEQ ID NO: 32, 33 and 34; or (3) SEQ ID NO: 35, 36 and 37; Preferably, the heavy chain variable regions each comprise an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the heavy chain variable regions of any of the following groups: (1)SEQ ID NO:64; (2)SEQ ID NO:13; (3) SEQ ID NO:65; or (4)SEQ ID NO:

14.

56. An anti-CD20 antibody or an antigen-binding fragment thereof, wherein, The anti-CD20 antibody or its antigen-binding fragment comprises a heavy chain variable region, wherein the heavy chain variable region comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of the following groups of heavy chain variable regions: (1)SEQ ID NO:64; (2)SEQ ID NO:13; (3) SEQ ID NO:65; or (4)SEQ ID NO:

14.

57. The anti-CD20 antibody or its antigen-binding fragment as described in claim 55 or 56, further comprising one or more of the following characteristics: (1) It also includes a heavy chain constant region and / or a light chain constant region; preferably, the heavy chain constant region contains Fc; more preferably, Fc is derived from mice or humans; more preferably, the sequence of Fc is a natural or modified variant; (2) It is a mouse-derived antibody, a chimeric antibody, a humanized antibody, or a fully human antibody; (3) It is a monoclonal antibody, or a full-length antibody, or its antigen-binding fragment is Fab, Fv, scFv, F(ab')2, linear antibody, or heavy chain single-domain antibody; (4) It is in the form of IgG1, IgG2, IgG3 or IgG4.

58. A conjugate comprising an anti-CD20 antibody or an antigen-binding fragment thereof as described in any one of claims 55-57, and a capture marker or detection marker conjugated thereto, wherein the detection marker includes, but is not limited to, a radionuclide, a luminescent substance (e.g., fluorescein), a colored substance, or an enzyme.

59. A fusion protein, wherein, The fusion protein comprises an anti-CD20 antibody or an antigen-binding fragment thereof as described in any one of claims 55-57.

60. A bispecific antibody or its antigen-binding fragment thereof, or a multispecific antibody or its antigen-binding fragment thereof, wherein, The bispecific antibody or its antigen-binding fragment or the multispecific antibody or its antigen-binding fragment comprises the anti-CD20 antibody or its antigen-binding fragment as described in any one of claims 55-57.

61. An antibody-drug conjugate (ADC), wherein, The antibody-drug conjugate comprises an anti-CD20 antibody or its antigen-binding fragment as described in any one of claims 55-57, a fusion protein as described in claim 59, or a bispecific antibody or its antigen-binding fragment or a multispecific antibody or its antigen-binding fragment as described in claim 60, and other bioactive molecules conjugated thereto; preferably, the other bioactive molecules are small molecule drugs; preferably, the anti-CD20 antibody or its antigen-binding fragment, the fusion protein or the bispecific antibody or its antigen-binding fragment or the multispecific antibody or its antigen-binding fragment are connected to the other bioactive molecules via a linker.

62. A chimeric antigen receptor (CAR) or a cell containing said chimeric antigen receptor (e.g., CAR-T cells) comprising an anti-CD20 antibody or an antigen-binding fragment thereof as described in any one of claims 55-57, a fusion protein as described in claim 59, or a bispecific antibody or an antigen-binding fragment thereof as described in claim 60, or a multispecific antibody or an antigen-binding fragment thereof.

63. A nucleic acid encoding an anti-CD20 antibody or an antigen-binding fragment thereof as described in any one of claims 55-57, a fusion protein as described in claim 59, or a bispecific antibody or an antigen-binding fragment thereof as described in claim 60, or a multispecific antibody or an antigen-binding fragment thereof.

64. A recombinant vector comprising the nucleic acid as described in claim 63.

65. A host cell comprising the nucleic acid as described in claim 63 or the recombinant vector as described in claim 64.

66. The host cell of claim 65, wherein it is a prokaryotic cell, such as Escherichia coli; or a eukaryotic cell, such as yeast or a mammalian cell, wherein the mammalian cell is preferably a CHO cell or a HEK293 cell.

67. A method for preparing an anti-CD20 antibody or its antigen-binding fragment, a fusion protein, a bispecific antibody or its antigen-binding fragment, or a multispecific antibody or its antigen-binding fragment, wherein, The method includes: culturing the host cell as described in claim 65 or 66 under suitable conditions, and purifying the expression product from the cell.

68. Use of an anti-CD20 antibody or antigen-binding fragment thereof as described in any one of claims 55-57, a fusion protein as described in claim 59, or a bispecific antibody or antigen-binding fragment thereof as described in claim 60, or a multispecific antibody or antigen-binding fragment thereof, in the preparation of a detection reagent or diagnostic reagent.

69. The use as described in claim 68, wherein the detection reagent is used to detect CD20 expression; the diagnostic reagent is used to diagnose tumors; preferably, the tumor is selected from: lymphomas such as B-cell lymphoma, including subtypes such as diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), and mantle cell lymphoma (MCL), and metastatic carcinomas of the above tumors.

70. A pharmaceutical composition comprising an anti-CD20 antibody or an antigen-binding fragment thereof as described in any one of claims 55-57, an antibody-drug conjugate as described in claim 61, a fusion protein as described in claim 59, or a bispecific antibody or an antigen-binding fragment thereof as described in claim 60, or a multispecific antibody or an antigen-binding fragment thereof, a chimeric antigen receptor (CAR) as described in claim 62, or a cell containing said chimeric antigen receptor, a nucleic acid as described in claim 63, a recombinant vector as described in claim 64, or a host cell as described in claim 65 or 66; preferably, the pharmaceutical composition is used for the treatment, relief, and / or prevention of autoimmune diseases or tumors; preferably, the tumor is selected from: lymphomas such as B-cell lymphoma, including subtypes such as diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), and mantle cell lymphoma (MCL), and metastatic tumors of the above tumors.

71. The pharmaceutical composition of claim 70, further comprising a pharmaceutically acceptable carrier.

72. The pharmaceutical composition of claim 70 or 71, further comprising one or more additional therapeutic agents.

73. A method for treating a disease in a subject, wherein, The method includes administering an effective amount of the pharmaceutical composition as described in any one of claims 70-72 to a subject in need, preferably, the disease being a disease associated with CD20 expression.

74. The method of claim 73, wherein, The disease in question is either an autoimmune disease or a tumor.

75. The method of claim 74, wherein, The tumors are selected from: lymphomas such as B-cell lymphomas, including diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), and mantle cell lymphoma (MCL), as well as metastatic cancers of the above tumors.

76. The method according to any one of claims 73-75, wherein, It also includes administering additional therapeutic agents to the subject.

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