Anti-CD20 / CD3 bispecific antibody and use thereof

Abstract

Provided are a CD20 / CD3 bispecific antibody and a use thereof. Said antibody has reduced binding activity to CD3, enhanced tumor killing ability, and lower cytokine release level.

Description

Anti-CD20 / CD3 bispecific antibodies and their applications Technical Field

[0001] This application belongs to the field of biomedicine, specifically relating to a bispecific antibody targeting CD20 / CD3 and its uses. Background Technology

[0002] Lymphoma is a malignant tumor originating from the lymphohematopoietic system. Based on the tumor cells, it is divided into non-Hodgkin lymphoma and Hodgkin lymphoma. Non-Hodgkin lymphoma accounts for approximately 90% of all lymphomas, and about 85%-90% of these are B-cell lymphomas. Diffuse large B-cell lymphoma (DLBCL) accounts for 30%-40% of all non-Hodgkin lymphomas, while follicular lymphoma accounts for about 20%. CD20 is expressed on the surface of normal and approximately 95% of malignant B lymphocytes, but not in hematopoietic stem cells, plasma cells, and other normal tissues. Therefore, CD20 is considered an ideal target for the treatment of B-cell lymphoma and autoimmune diseases.

[0003] CD20, as a surface antigen of B cells, is only found in the transition from pre-B cells to mature B cells, and is not expressed on hematopoietic stem cells, progenitor B cells, or mature plasma cells. Although attacking malignant B lymphocytes carrying CD20 protein may also mistakenly kill normal mature B cells, progenitor B cells can subsequently replenish mature B cells, and existing plasma cells can support the normal functioning of the immune system, making the side effects manageable. Rituximab and its-based regimens have been the first-line treatment for patients with B-cell lymphoma, bringing significant survival benefits. Rituximab has expanded its indications from primarily treating lymphoma to treating a variety of diseases, including rheumatoid arthritis, systemic lupus erythematosus, vasculitis, and autoimmune thrombocytopenic purpura. While most patients achieve complete remission with treatment, nearly 40% relapse or become refractory after initial treatment. For patients with multiple-line relapsed diffuse large B-cell lymphoma (DLBCL), treatment options remain limited, and the prognosis is relatively poor. There is an urgent need to develop new B-cell targeted therapeutic strategies with different mechanisms of action. Attached Figure Description

[0004] The novel features of this application are specifically set forth in the appended claims. To better understand the features and advantages of this application, detailed descriptions will be provided below through embodiments.

[0005] Figure 1 shows the results of the CD3 monoclonal antibody binding assay to Jurkat cells;

[0006] Figure 2 shows a schematic diagram of the structure of the CD20 / CD3 bispecific antibody;

[0007] Figure 3A shows the detection results of the binding of CD20 / CD3 bispecific antibody to human CD3;

[0008] Figure 3B shows the results of the detection of the binding of the CD20 / CD3 bispecific antibody to CD3 in cynomolgus monkeys;

[0009] Figure 4A shows the detection results of the binding of CD20 / CD3 bispecific antibody to human CD20;

[0010] Figure 4B shows the results of the detection of the binding of the CD20 / CD3 bispecific antibody to CD20 in cynomolgus monkeys;

[0011] Figure 5 shows the binding results of the CD20 / CD3 bispecific antibody to Raji cells;

[0012] Figure 6 shows the binding results of the CD20 / CD3 bispecific antibody to Jurkat cells;

[0013] Figure 7A shows the results of CD20 / CD3 bispecific antibody activation of the T cell reporter gene pathway;

[0014] Figure 7B shows the results of CD20 / CD3 bispecific antibody activation of the T cell reporter gene pathway;

[0015] Figure 7C shows the results of CD20 / CD3 bispecific antibody activation of the T cell reporter gene pathway;

[0016] Figure 7D shows the results of CD20 / CD3 bispecific antibody activation of the T cell reporter gene pathway;

[0017] Figure 8A shows the results of CD20 / CD3 bispecific antibody activating CD25 expression, a marker of CD4+ T cell activation, in the presence of Raji cells;

[0018] Figure 8B shows the results of CD20 / CD3 bispecific antibody activating CD69, a marker of CD4+ T cell activation, in the presence of Raji cells;

[0019] Figure 8C shows the results of CD20 / CD3 bispecific antibody activating CD25 expression, a marker of CD8+ T cell activation, in the presence of Raji cells;

[0020] Figure 8D shows the results of CD20 / CD3 bispecific antibody activating CD69, a marker of CD8+ T cell activation, in the presence of Raji cells;

[0021] Figure 9A shows the results of CD20 / CD3 bispecific antibody activating CD25 expression, a marker of CD4+ T cell activation, in the presence of Ramos cells;

[0022] Figure 9B shows the results of CD20 / CD3 bispecific antibody activating CD69, a marker of CD4+ T cell activation, in the presence of Ramos cells;

[0023] Figure 9C shows the results of CD20 / CD3 bispecific antibody activating CD25 expression, a marker of CD8+ T cell activation, in the presence of Ramos cells;

[0024] Figure 9D shows the results of CD20 / CD3 bispecific antibody activating CD69, a marker of CD8+ T cell activation, in the presence of Ramos cells;

[0025] Figure 10A shows the results of the experiment on the release of cytokine IL2 targeted by the CD20 / CD3 bispecific antibody;

[0026] Figure 10B shows the results of the experiment on the release of cytokine IFNγ targeted by the CD20 / CD3 bispecific antibody;

[0027] Figure 10C shows the results of the experiment on the release of TNFα cytokine targeted by the CD20 / CD3 bispecific antibody;

[0028] Figure 10D shows the results of the experiment on the release of cytokine IL6 targeted by the CD20 / CD3 bispecific antibody;

[0029] Figure 11A shows the effect of CD20 / CD3 bispecific antibody on IL2 secretion in PBMC cells;

[0030] Figure 11B shows the effect of CD20 / CD3 bispecific antibody on IFNγ secretion in PBMC cells;

[0031] Figure 11C shows the effect of CD20 / CD3 bispecific antibody on TNFα secretion in PBMC cells;

[0032] Figure 11D shows the effect of CD20 / CD3 bispecific antibody on IL6 secretion in PBMC cells;

[0033] Figure 12 shows the killing effect of CD20-positive tumor cells Raji cells mediated by CD20 / CD3 bispecific antibody by PBMCs.

[0034] Figure 13 shows the efficacy results of administration of the CD20 / CD3 bispecific antibody in the Raji local PBMC model. In the figure, B2 vs Vehicle: **P < 0.01, ***P < 0.001.

[0035] Figure 14 shows the body weight change curves after administration of the CD20 / CD3 bispecific antibody in the Raji local PBMC model.

[0036] Invention Details

[0037] definition

[0038] In this application, “AMG757-CD3” refers to the scFv form formed by the CD3 light and heavy chain variable region sequence in the AMG757 (see US20170037130A1, SEQ ID NO:520) bispecific antibody, the sequence of which can be found in Tables 3 and 4 of this application.

[0039] In this application, “AMG757” refers to the AMG757 bispecific antibody in US20170037130A1, the sequence of which can be found in SEQ ID NO:520 in US20170037130A1.

[0040] Glofitamab (trade name Columvi) is a bispecific T-cell engaging antibody (TCE) co-developed by Roche and Genentech, specifically designed to treat relapsed or refractory large B-cell lymphoma (LBCL). It binds to both cancer cells (CD20) and T cells (CD3), forcing them to approach each other, thereby activating T cells and inducing them to release cytotoxic substances to kill cancer cells. Compared to traditional monoclonal antibodies, it can more effectively recruit and activate T cells in the body.

[0041] Odronextamab, marketed as Baiying in Chinese, is an innovative bispecific antibody developed jointly by Regeneron Pharmaceuticals and BGI Genomics, targeting CD20 (B cell surface antigen) and CD3 (T cell surface antigen). It is an "off-the-shelf" immunotherapy designed to recruit the patient's own T cells directly to the vicinity of tumor cells, inducing rapid lysis and apoptosis of these cells. Detailed Implementation

[0042] The novel CD3 antibody or its antigen-binding fragment provided in this application has particularly advantageous properties, such as reduced binding activity to CD3. This application also provides a CD20 / CD3 bispecific antibody with particularly advantageous properties, including enhanced tumor-killing ability and lower cytokine release levels.

[0043] In a first aspect, this application provides a CD3 antibody or an antigen-binding fragment thereof, comprising a light chain variable region (VL) and a heavy chain variable region (VH), wherein the light chain variable region and the heavy chain variable region comprise:

[0044] HCDR1 shown in SEQ ID NO:7, HCDR2 shown in SEQ ID NO:8, HCDR3 shown in SEQ ID NO:9, LCDR1 shown in SEQ ID NO:10, LCDR2 shown in SEQ ID NO:11, and LCDR3 shown in SEQ ID NO:12; or

[0045] HCDR1 shown in SEQ ID NO:13, HCDR2 shown in SEQ ID NO:14, HCDR3 shown in SEQ ID NO:15, LCDR1 shown in SEQ ID NO:16, LCDR2 shown in SEQ ID NO:11 and LCDR3 shown in SEQ ID NO:17.

[0046] In one embodiment, the antigen-binding fragment is selected from Fab fragments, Fab′ fragments, Fab′-SH fragments, F(ab′)2 fragments, Fv fragments, and scFv fragments; optionally, the antigen-binding fragment is an antigen-binding fragment derived from a monoclonal antibody and / or a humanized antibody.

[0047] In one embodiment, the light chain variable region (VL) and heavy chain variable region (VH) of the CD3 antibody or its antigen-binding fragment include:

[0048] The VH contains or has an amino acid sequence that is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO:1, and the VL contains or has an amino acid sequence that is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO:2; or

[0049] The VH contains or has an amino acid sequence that is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO:3, and the VL contains or has an amino acid sequence that is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO:4.

[0050] Secondly, this application provides a bispecific antigen-binding molecule, comprising an immunoglobulin domain targeting CD3 and an immunoglobulin domain targeting an antigen expressed on tumor cells; optionally, the immunoglobulin domain targeting the antigen expressed on tumor cells is an antigen-binding fragment targeting CD20.

[0051] In one embodiment, the bispecific antigen-binding molecule includes (a) a first immunoglobulin domain targeting CD3, (b) a second immunoglobulin domain targeting CD20, and (c) a third immunoglobulin domain targeting CD20; optionally, the first immunoglobulin domain is an scFv structure, and the second and / or third immunoglobulin domain is a Fab structure.

[0052] In one embodiment, in the bispecific antigen-binding molecule, the CD20-targeting antigen-binding fragment includes a light chain variable region and a heavy chain variable region, wherein the light chain variable region (VL) and the heavy chain variable region (VH) include:

[0053] HCDR1 shown in SEQ ID NO:28, HCDR2 shown in SEQ ID NO:29, HCDR3 shown in SEQ ID NO:30, LCDR1 shown in SEQ ID NO:31, LCDR2 shown in SEQ ID NO:32 and LCDR3 shown in SEQ ID NO:33;

[0054] Optionally, the VH of the CD20-targeting antigen-binding fragment contains an amino acid sequence having at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:26, and the VL contains an amino acid sequence having at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:27.

[0055] In one embodiment, in the bispecific antigen-binding molecule, the first immunoglobulin domain targeting CD3 includes a light chain variable region and a heavy chain variable region, wherein the light chain variable region and the heavy chain variable region include: HCDR1 shown in SEQ ID NO:7, HCDR2 shown in SEQ ID NO:8, HCDR3 shown in SEQ ID NO:9, LCDR1 shown in SEQ ID NO:10, LCDR2 shown in SEQ ID NO:11, and LCDR3 shown in SEQ ID NO:12; or HCDR1 shown in SEQ ID NO:13, HCDR2 shown in SEQ ID NO:14, HCDR3 shown in SEQ ID NO:15, LCDR1 shown in SEQ ID NO:16, LCDR2 shown in SEQ ID NO:11, and LCDR3 shown in SEQ ID NO:17;

[0056] Optionally, the first immunoglobulin domain targeting CD3 includes a light chain variable region (VL) and a heavy chain variable region (VH), wherein the VH contains or has an amino acid sequence that is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO:1, and the VL contains or has an amino acid sequence that is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO:2; or the VH contains or has an amino acid sequence that is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO:3, and the VL contains or has an amino acid sequence that is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO:3, and the VH contains or has an amino acid sequence that is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO:1 ... The amino acid sequence shown in NO:4 has an amino acid sequence that is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical.

[0057] In one embodiment, in a bispecific antigen-binding molecule, the CD20-targeting antigen-binding fragment includes a light chain variable region and a heavy chain variable region, wherein the light chain variable region and the heavy chain variable region include:

[0058] HCDR1 shown in SEQ ID NO:28, HCDR2 shown in SEQ ID NO:29, HCDR3 shown in SEQ ID NO:30, LCDR1 shown in SEQ ID NO:31, LCDR2 shown in SEQ ID NO:32, and LCDR3 shown in SEQ ID NO:33; and

[0059] The CD3 immunoglobulin domain includes a light chain variable region and a heavy chain variable region, wherein the light chain variable region and the heavy chain variable region include: HCDR1 shown in SEQ ID NO:7, HCDR2 shown in SEQ ID NO:8, HCDR3 shown in SEQ ID NO:9, LCDR1 shown in SEQ ID NO:10, LCDR2 shown in SEQ ID NO:11, and LCDR3 shown in SEQ ID NO:12; or HCDR1 shown in SEQ ID NO:13, HCDR2 shown in SEQ ID NO:14, HCDR3 shown in SEQ ID NO:15, LCDR1 shown in SEQ ID NO:16, LCDR2 shown in SEQ ID NO:11, and LCDR3 shown in SEQ ID NO:17;

[0060] In one embodiment, in the bispecific antigen-binding molecule, the second immunoglobulin domain targeting CD20 and the third immunoglobulin domain targeting CD20 contain the same light chain variable region and heavy chain variable region, wherein the light chain variable region and heavy chain variable region include: HCDR1 shown in SEQ ID NO:28, HCDR2 shown in SEQ ID NO:29, HCDR3 shown in SEQ ID NO:30, LCDR1 shown in SEQ ID NO:31, LCDR2 shown in SEQ ID NO:32, and LCDR3 shown in SEQ ID NO:33; and

[0061] The first immunoglobulin domain targeting CD3 includes a light chain variable region and a heavy chain variable region, wherein the light chain variable region and the heavy chain variable region include: HCDR1 shown in SEQ ID NO:7, HCDR2 shown in SEQ ID NO:8, HCDR3 shown in SEQ ID NO:9, LCDR1 shown in SEQ ID NO:10, LCDR2 shown in SEQ ID NO:11, and LCDR3 shown in SEQ ID NO:12; or HCDR1 shown in SEQ ID NO:13, HCDR2 shown in SEQ ID NO:14, HCDR3 shown in SEQ ID NO:15, LCDR1 shown in SEQ ID NO:16, LCDR2 shown in SEQ ID NO:11, and LCDR3 shown in SEQ ID NO:17;

[0062] Optionally, both the second immunoglobulin domain targeting CD20 and the third immunoglobulin domain targeting CD20 are Fab domains and contain the same light chain variable region and heavy chain variable region, wherein the light chain variable region and heavy chain variable region include: HCDR1 shown in SEQ ID NO:28, HCDR2 shown in SEQ ID NO:29, HCDR3 shown in SEQ ID NO:30, LCDR1 shown in SEQ ID NO:31, LCDR2 shown in SEQ ID NO:32, and LCDR3 shown in SEQ ID NO:33; and

[0063] The first immunoglobulin domain targeting CD3 is an scFv structure, including a light chain variable region and a heavy chain variable region, wherein the light chain variable region and the heavy chain variable region include: HCDR1 shown in SEQ ID NO:7, HCDR2 shown in SEQ ID NO:8, HCDR3 shown in SEQ ID NO:9, LCDR1 shown in SEQ ID NO:10, LCDR2 shown in SEQ ID NO:11, and LCDR3 shown in SEQ ID NO:12; or HCDR1 shown in SEQ ID NO:13, HCDR2 shown in SEQ ID NO:14, HCDR3 shown in SEQ ID NO:15, LCDR1 shown in SEQ ID NO:16, LCDR2 shown in SEQ ID NO:11, and LCDR3 shown in SEQ ID NO:17.

[0064] In one embodiment, in the bispecific antigen-binding molecule, the second immunoglobulin domain targeting CD20 and the third immunoglobulin domain targeting CD20 contain the same light chain variable region (VL) and heavy chain variable region (VH), wherein the VH comprises, or has an amino acid sequence having at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:26, and the VL comprises, or has an amino acid sequence having at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:27; and

[0065] The first immunoglobulin domain targeting CD3 includes a light chain variable region (VL) and a heavy chain variable region (VH), wherein the VH contains or has an amino acid sequence that is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO:1, and the VL contains or has an amino acid sequence that is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO:2; or the VH contains or has an amino acid sequence that is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO:3, and the VL contains or has an amino acid sequence that is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO:3, and the VL contains or has an amino acid sequence that is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO:1. The amino acid sequence shown in NO:4 has an amino acid sequence with at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity;

[0066] Optionally, both the second immunoglobulin domain targeting CD20 and the third immunoglobulin domain targeting CD20 are Fab domains, containing the same light chain variable region and heavy chain variable region, wherein the VH contains or has an amino acid sequence that has at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:26, and the VL contains or has an amino acid sequence that has at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:27; and

[0067] The first immunoglobulin domain targeting CD3 is an scFv structure, wherein the VH contains or has an amino acid sequence that is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO:1, and the VL contains or has an amino acid sequence that is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO:2; or the VH contains or has an amino acid sequence that is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO:3, and the VL contains or has an amino acid sequence that is identical to the amino acid sequence shown in SEQ ID NO:1. The amino acid sequence shown in NO:4 has an amino acid sequence that is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical.

[0068] In one embodiment, the bispecific antigen-binding molecule comprises the following peptide chains (1)-(4):

[0069] Peptide chain (1)N'-CD20VL-CL-C';

[0070] peptide chain (2) N'-CD20VH-CH1-CD3VL-CD3VH-Fc-C';

[0071] peptide chain (3)N'-CD20VH-CH1-Fc-C'; and

[0072] peptide chain (4)N'-CD20VL-CL-C'

[0073] Wherein, VH represents the heavy chain variable region, CD20VH represents the heavy chain variable region of the antigen-binding fragment targeting CD20, and CD3VH represents the heavy chain variable region of the antigen-binding fragment targeting CD3; VL represents the light chain variable region, CD20VL represents the light chain variable region of the antigen-binding fragment targeting CD20, and CD3VL represents the light chain variable region of the antigen-binding fragment targeting CD3; Fc contains CH2 and CH3; CH1, CH2, and CH3 represent domains 1, 2, and 3 of the heavy chain constant region, respectively; CL represents the light chain constant region;

[0074] Optionally, the Fc region comprises a natural Fc sequence or a non-natural Fc sequence; optionally, the Fc region is a human Fc region; further optionally, the Fc region is the Fc region of an IgG1, IgG2, IgG3, or IgG4 antibody; even further optionally, the Fc region is an IgG1 Fc region; even further optionally, the Fc region comprises one or more mutations selected from those weakening the antibody ADCC effect, those weakening the antibody CDC effect, knocks-in-holes mutations, and disulfide bond mutations. Further, the Fc region comprises one or more of L234A, L235A, P329A, P331S, S354C, T366W, Y349C, T366S, L368A, and Y407V.

[0075] In one embodiment, in the bispecific antigen-binding molecule, CH1 comprises or has an amino acid sequence having at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:34.

[0076] The CL comprises, or has an amino acid sequence having at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:35; and / or

[0077] The Fc contains or has an amino acid sequence that is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in any of SEQ ID NO:36-37.

[0078] In one embodiment, the bispecific antigen-binding molecule comprises peptide chain (1)-peptide chain (4);

[0079] The peptide chain (1) comprises or has an amino acid sequence that is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:38;

[0080] The peptide chain (2) comprises or has an amino acid sequence that is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:39 or SEQ ID NO:41;

[0081] The peptide chain (3) comprises or has an amino acid sequence that is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:40;

[0082] The peptide chain (4) comprises or has an amino acid sequence that is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:38;

[0083] Optionally, the peptide chains (1)-(4) comprise or have an amino acid sequence that is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequences shown in SEQ ID NO:38, 39, 40, and 38, or the peptide chains (1)-(4) comprise or have an amino acid sequence that is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequences shown in SEQ ID NO:38, 41, 40, and 38.

[0084] Thirdly, this application provides an isolated nucleic acid comprising a nucleic acid sequence encoding the CD3 antibody or its antigen-binding fragment, or a bispecific antigen-binding molecule.

[0085] Fourthly, this application provides a vector containing the nucleic acid.

[0086] Fifthly, this application provides an isolated host cell containing the nucleic acid or vector.

[0087] In a sixth aspect, this application provides a composition comprising the CD3 antigen-binding fragment, the bispecific antigen-binding molecule, the nucleic acid, a vector, or a host cell; optionally, the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable vector.

[0088] In a seventh aspect, this application provides the use of the CD3 antibody or its antigen-binding fragment, the bispecific antigen-binding molecule, the nucleic acid, the vector, or the host cell in the manufacture of a medicament for treating or delaying the progression of an individual's cancer; optionally, the cancer is a CD20-positive cancer, such as lymphoma, including: Hodgkin lymphoma and non-Hodgkin lymphoma; further optionally, the cancer is a non-Hodgkin lymphoma, including: diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, and mucosa-associated lymphoid tissue lymphoma; even more optionally, the cancer is diffuse large B-cell lymphoma.

[0089] Eighthly, this application provides a method for treating or delaying cancer progression in a subject in need, wherein the method includes administering the bispecific antigen-binding molecule to the subject; optionally, wherein the cancer is a CD20-positive cancer, such as lymphoma, including: Hodgkin lymphoma and non-Hodgkin lymphoma; further optionally, the cancer is non-Hodgkin lymphoma, including: diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, mucosa-associated lymphoid tissue lymphoma; even further optionally, the cancer is diffuse large B-cell lymphoma.

[0090] The technical solution of this application has the following advantages:

[0091] 1. The CD3 antibody or its antigen-binding fragment has particularly advantageous properties, such as reduced binding activity to CD3 and cross-reactivity with cynomolgus monkey CD3 protein.

[0092] 2. The CD3 / CD20 bispecific antibody of this application has been shown in preclinical studies to have stronger killing activity and reduced or similar cytokine release levels compared to the marketed Odronextama.

[0093] 3. The CD20-CD3 bispecific antibody developed in this application, according to preclinical studies, has reduced affinity for CD3 antibodies compared to Glofitamab, resulting in a milder effect, target-dependent T-cell activation, and reduced toxicity while exhibiting durable anticancer activity. It is expected to reduce side effects and broaden the dosing window.

[0094] Example

[0095] Example 1: Design, production, and identification of anti-CD3 antibodies or their antigen-binding fragment sequences

[0096] 1.1: Design and production of anti-CD3 antibodies or their antigen-binding fragment sequences

[0097] The CDR region of a CD3 antibody capable of recognizing the CD3 receptor in humans and cynomolgus monkeys was mutated to obtain a CD3 monoclonal antibody variable region in scFv form, which was then constructed into a eukaryotic expression vector containing hIgG1. The Fc sequence of this vector incorporated amino acid mutations at L234A / L235A / P329A / P331S (according to Kabat's "EU" number). The CD3 light and heavy chain variable region sequences of the AMG757 (SEQ ID NO: 520 in patent number US20170037130A1) bispecific antibody were also constructed in scFv form and used as a positive control. The obtained eukaryotic expression vector was transiently transfected into CHO cells and cultured for 6 days. The cell supernatant was collected by centrifugation and purified using a Protein A column to obtain the target antibodies, named #6, #7, and AMG757-CD3, respectively. Their specific sequences are shown in Tables 1 and 2.

[0098] Table 1: Variable region sequences of CD3 antibody heavy and light chains (underlined regions are CDR regions, encoded using kabat encoding)

[0099] Table 2: Complete scFv sequences and Fc region sequences of #6, #7, and AMG757-CD3

[0100] 1.2: BLI assay to determine the affinity of CD3 monoclonal antibody for human and cynomolgus monkey CD3 protein

[0101] Using ProA biosensor ( ProABiosensors (Sartorius, catalog number 18-5012) immobilized CD3 monoclonal antibodies on biosensors to a binding response threshold of 0.3 nm. After a 120-s baseline step, the sensors were immersed in recombinant human CD3 protein (ACRO Biosystem, CDE-H5223) or cynomolgus monkey CD3 protein (ACRO Biosystem, CDE-C5226) diluted with 0.02% PBST buffer. The initial protein concentration was 100 nM, with 3-fold serial dilutions and 7 gradients. Binding and dissociation times were 120 s and 300 s, respectively. Affinity data for the antigen and antibody were calculated using regression analysis software based on the binding curves. AMG757-CD3 showed the best binding affinity to recombinant human or cynomolgus monkey CD3 protein, #7 showed a weaker binding affinity to recombinant human or cynomolgus monkey CD3 protein than AMG757-CD3 (CD3 in the AMG757 molecule), and #6 did not bind to recombinant human or cynomolgus monkey CD3 protein. The results are shown in Tables 3 and 4.

[0102] Table 3: Affinity of CD3 monoclonal antibody to recombinant human CD3 protein as determined by BLI

[0103] Table 4: Affinity of CD3 monoclonal antibody to recombinant cynomolgus CD3 protein as determined by BLI

[0104] 1.3: Binding activity of CD3 monoclonal antibody to CD3 protein on the surface of Jurkat cells

[0105] The binding affinity of CD3 monoclonal antibodies to CD3 on the cell surface was assessed using Jurkat cells. Jurkat cells were seeded at a density of 2E6 / mL, 100 μL / well in 96-well plates. CD3 antibody (0-250 nM) was added in 4-fold serial dilutions and incubated at 4°C for 1 h. Goat anti-human secondary antibody (Invitrogen, A11013) was added, and the cells were labeled at 4°C in the dark for 45 min. After cell collection and washing, the fluorescence intensity was read by flow cytometry. A stronger fluorescence signal indicated higher antibody affinity to the target. The experimental results are shown in Figure 1 and Table 5. The results show that the binding affinity of molecules #6 and #7 was lower than that of AMG757-CD3, while the binding affinity of #7 was higher than that of #6.

[0106] Table 5: Binding ability of CD3 antibody to CD3 on the surface of Jurkat cells

[0107] Example 2: Production and identification of anti-CD20 / CD3 bispecific antibodies

[0108] 2.1 Production of anti-CD20 / CD3 bispecific antibodies

[0109] Bispecific antibodies were constructed using the CD20 antibody sequence and the #6 and #7 CD30 monoclonal antibody molecules from Example 1. The structures of the bispecific antibodies are shown in Figure 2 and Table 6. The nucleic acid encoding the bispecific antibody molecules was constructed into the eukaryotic expression vector pCDNA3.4, transiently transfected into CHO cells, and cultured for 6 days. The cell supernatant was collected by centrifugation, and the harvested culture medium supernatant was purified by Protein A column to obtain the target antibodies, named B2 and B7, respectively. Their sequences are shown in Tables 7-9.

[0110] Table 6: Chain structure of CD20 / CD3 bispecific antibodies

[0111] Table 7: Antibody variable region sequences used in anti-CD20 / CD3 bispecific antibodies (underlined regions are CDR regions, encoded using kabat encoding).

[0112] Table 8: Constant region sequences used in anti-CD20 / CD3 bispecific antibodies

[0113] Table 9: Complete sequences of anti-CD20 / CD3 bispecific antibodies

[0114] 2.2: Identification of anti-CD20 / CD3 bispecific antibodies

[0115] 2.2.1: ELISA detection of the affinity of the penicillin antibody for human or cynomolgus monkey CD3 protein

[0116] The affinity of CD20 / CD3 bispecific antibodies for human CD3 protein (ACRO Biosystem, CDE-H5223) or cynomolgus monkey CD3 protein (ACRO Biosystem, CDE-C5226) was determined using ELISA. Human or cynomolgus monkey CD3 protein was diluted to 0.5 μg / mL and coated onto the ELISA plate. After blocking, a 5-fold serial dilution of CD20 / CD3 bispecific antibody (0-1000 pM) was added, and the plate was incubated at 37°C for 1 h. After incubation with secondary antibody (Jackson ImmunoResearch, 109-036-098), the results were analyzed and read. The affinity was fitted using GraphPad, and the results are shown in Figures 3A-3B and Table 10. Glofitamab (B21997601) was used as a positive control antibody. The results showed that the binding activity of bispecific antibodies B7 and B2 to CD3 protein in both humans and cynomolgus monkeys was weaker than that of Glofitamab, with the binding activity of bispecific antibody B2 being weaker than that of B7.

[0117] Table 10: ELISA detection of the affinity of CD20 / CD3 bispecific antibodies for human or cynomolgus monkey CD3 in ECMO cells 50 value

[0118] 2.2.2: ELISA detection of the affinity of CD20 / CD3 double antibody for human or cynomolgus monkey CD20 protein

[0119] The affinity of CD20 / CD3 bispecific antibodies for human CD20 protein (ACRO Biosystem, CD0-H52H3) or cynomolgus monkey CD20 protein (ACRO Biosystem, CD0-C52H8) was determined using ELISA. Human or cynomolgus monkey CD20 protein was diluted to 0.5 μg / mL and coated onto ELISA plates. After blocking, CD20 / CD3 bispecific antibodies (0-5000 pM) were serially diluted 5-fold and incubated at 37°C for 1 h. After incubation with secondary antibody (Jackson ImmunoResearch, 109-036-098), the affinities were measured. The results were fitted using GraphPad, and the affinities are shown in Figures 4A-4B and Table 11. For both human and cynomolgus monkey CD20 proteins, the affinity of bispecific antibodies B2 and B7 for CD20 protein was comparable, both weaker than that of Glofitamab.

[0120] Table 11: ELISA assay results for the affinity of CD20 / CD3 double antibody to human or cynomolgus monkey CD20 protein. 50 value

[0121] 2.2.3: Binding of CD20 / CD3 bispecific antibody to human CD20-expressing cells

[0122] To detect the binding ability of bispecific antibodies to CD20-expressing cells, Raji cells (Nanjing Kebai, CBP60272), a CD20-high expression cell line, were selected. Cells in logarithmic growth phase were adjusted to a density of 2E6 / mL, and 100 μL / well was added to each well of a 96-well plate. After centrifugation at 2000 rpm for 3 min, the supernatant was discarded, and serially diluted antibody (0-500 nM) was added three times. The plates were incubated at 4°C for 1 h. Then, 488 goat anti-human secondary antibody (Invitrogen, A11013) was added, and the plates were labeled at 4°C in the dark for 45 min. Cells were collected, washed, and their fluorescence intensity was read by flow cytometry. Glofitamab (Baiying, B21997601) and odronextamab (Baiying, B623801) were used as positive control antibodies. The experimental results are shown in Figure 5 and Table 12. The results show that both antibiotics B2 and B7 have a high binding capacity to CD20 expression cell line Raji. The binding capacity of B2 and B7 to the control Glofitamab is similar and higher than that of odronextamab.

[0123] Table 12: Binding assay of bispecific antibody to CD20 on the surface of human B lymphoma cells

[0124] 2.2.4: Binding of CD20 / CD3 bispecific antibody to CD3 on the surface of Jurkat cells

[0125] The binding affinity of CD20 / CD3 bispecific antibodies to CD3 on the cell surface was assessed using Jurkat cells. Cells were added to 96-well plates at a density of 2E6 / mL, 100 μL / well. After centrifugation at 2000 rpm for 3 min, the supernatant was discarded, and serially diluted CD20 / CD3 bispecific antibodies (0-1800 nM) were added 3-fold, and the cells were incubated at 4°C for 1 h. Goat anti-human secondary antibody (Invitrogen, A11013) was added, and the cells were labeled at 4°C in the dark for 45 min. Cells were collected, washed, and their fluorescence intensity was read by flow cytometry. The experimental results are shown in Figure 6 and Table 13. The results indicate that the affinity of B2 and B7 for CD3 was lower than that of the controls Glofitamab and odronextamab.

[0126] Table 13: Assay of bispecific antibody binding to CD3 on the surface of Jurkat cells

[0127] 2.2.5: Activation activity of CD20 / CD3 bispecific antibody against Jurkat reporter gene

[0128] Using reporter gene-expressing cell lines Jurkat-NFAT-Luc, CD20-expressing Raji cells (Nanjing Kebai, CBP60272), Ramos cells (Nanjing Kebai, CBP60263), and non-CD20-expressing A375 cells as target cells, the activity of CD20 / CD3 bispecific antibodies was detected using a reporter gene system. Jurkat-NFAT-Luc cells in logarithmic growth phase were centrifuged, and the cell density was adjusted to 4E6 / mL. 25 μL of each cell was added to each well of a 96-well plate with a white-walled, clear bottom. Target cells were added at a 2:1 effector cell to target cell ratio, adjusting the target cell density to 2E6 / mL, with 25 μL added to each well. 50 μL (0-40000 pM) of serially diluted CD20 / CD3 bispecific antibody sample was added, and the plates were incubated at 37°C in a 5% CO2 incubator for 6 hours. Add 50 μL of right-Glo Luciferase (Promega, E2620) to each well and incubate at room temperature for 3-5 min before reading the values ​​using a microplate reader. The results are shown in Figures 7A-7B and Table 14. The CD20 / CD3 bispecific antibody induced Jurkat reporter gene activation only in the presence of target Raji cells (CD20-high expressing cell lines) or Ramos cells (CD20-medium expressing cell lines). The B2 / B7 molecules showed lower activity than Glofitamab but higher activity than Odronextamab, with B7 exhibiting superior activity compared to B2. In the absence of positive CD20 cells (i.e., A375 cells + Jurkat cells, Figure 7C) or in the presence of only Jurkat cells (i.e., Jurkat cells present, Figure 7D), Jurkat reporter gene activation was not induced.

[0129] Table 14: Reporter Gene Activation Experiment with Bispecific Antibody Jurkat

[0130] Note: In Table 14, “ND” indicates no binding and no response value.

[0131] 2.2.6: Detection of T cell activation by CD20 / CD3 bispecific antibody

[0132] To measure T cell activation, PBMCs were used as effector cells and CD20-positive Raji and Ramos cells were used as target cells. Cells were incubated for 24 hours and then collected by centrifugation. The T cell activation detection antibody CD3-eFluor was added. TM 450 (Invitrogen, 48-0037-42), CD4-PE (Invitrogen, 12-0049-42), CD8-APC-eFluor TM780 (Invitrogen, 47-0088-42), CD25-APC (Invitrogen, 17-0257-42), and CD69-FITC (Invitrogen, 11-0699-42) were incubated at 4°C for 30 minutes, the supernatant was washed and discarded, and the results were analyzed by flow cytometry. The results are shown in Figures 8A-9D and Tables 15-16. In the presence of target cells, the CD20 / CD3 bispecific antibody could induce the activation of CD4+ and CD8+ T cells. The activity of B2 / B7 molecules was lower than that of Glofitamab but higher than that of Odronextamab, with B7 molecules showing better activity than B2.

[0133] Table 15: T cell activation assay with Raji bispecific antibody as the target cell

[0134] Table 16: T cell activation assay with Ramos bispecific antibody as target cells

[0135] 2.2.7: Cytokine release accompanying CD20 / CD3 bispecific antibody TDCC (T cell-mediated tumor cell killing)

[0136] Cytokines secreted by activated T cells influence immune responses both in vitro and in vivo. Some cytokines limit tumor cell growth through direct anti-proliferative or pro-apoptotic activities, or indirectly by stimulating the cytotoxic activity of immune cells against tumor cells. To determine the effect of a CD20 bispecific antibody on the production of effector cytokines in vitro, PBMCs were co-cultured with target Raji cells (E:T = 5:1) for 24 hours in the presence of serially diluted CD20 / CD3 bispecific antibody (starting at 1000 pM, 4-fold dilution). The culture plates were centrifuged at 300 g for 10 min, and the cell culture supernatant was collected and stored at -80°C before cytokine assays. The levels of four cytokines, including IL2 (biolegend, 431815), IL6 (biolegend, 430515), TNFα (biolegend, 430215), and IFNγ (biolegend, 430116), were measured using the Biolegend Cytokine Assay Kit. The results, shown in Figures 10A-10D and Table 17, indicate that the bispecific antibody-induced T cell cytokine secretion was mediated by CD20-positive cells. The B2 molecule concentration was lower than Glofitamab but higher than Odronextamab; the B7 molecule concentration was roughly equivalent to Glofitamab. It can be seen that, based on the EC50 concentrations of B2 and Odronextamab molecules during TDCC (T cell-mediated tumor killing) in Table 18 (i.e., 0.03686 nM of B2 antibody and 0.8497 nM of Odronextamab antibody), in the presence of Raji target cells, the 0.03686 nM B2 antibody induced lower IFNγ release from T cells than the 0.8497 nM Odronextamab antibody (Figure 10B). The release of IL2, TNFα, and IL6 was not significantly different, indicating that the B2 antibody in this application has stronger cell lysis activity (lower EC50), but its relative cytokine release level is lower or similar to that of Odronextamab.

[0137] Table 17: Bispecific antibody targeting cytokine release assay

[0138] 2.2.8: Non-specific activation of PBMCs by CD20 / CD3 bispecific antibodies

[0139] Antibody-induced cytokine release could be assessed by measuring cytokine release using fresh peripheral blood mononuclear cells (PBMCs) in a plate binding assay. 96-well cell culture plates were coated with 100 μL of serially diluted test antibody (100 nM starting concentration, 5-fold dilution), incubated overnight at 4°C, washed twice with PBS, and then 250 μL / well (2E5 PBMCs) was added. The plates were incubated at 37°C and 5% CO2 for 24 hours. After incubation, the plates were centrifuged at 300g for 10 min, and the cell culture supernatant was collected. Four cytokines, including IL2, IL6, TNFα, and IFNγ, were detected using the Biolegend Cytokine Detection Kit (same as 2.2.7). The results are shown in Figures 11A-11D. In the absence of target cells, B2 induced T cell cytokine release more mildly than Glofitamab and Odronextamab.

[0140] 2.2.9: CD20 / CD3 bispecific antibody mediates PBMC killing of CD20-positive tumor cells

[0141] The bispecific antibody was further analyzed to assess its ability to induce the killing of CD20-positive Raji cells (target cells) by human PBMCs (effect cells in this experiment). Raji cells were labeled with carboxyfluorescein succinimide (CFSE, Invitrogen, 65-0850-84), which exhibits green fluorescence. Specifically, Raji cells were resuspended in PBS, adjusted to a cell density of 5E6 / mL, labeled with 1 μM CFSE, and incubated at room temperature in the dark for 10 minutes. After labeling, the cells were resuspended in RPMI complete medium (RPMI + 10% FBS), and the cell density was adjusted to 8E5 / mL. Human PBMC cells were used, and the density was adjusted to 4E6 / mL. 50 μL of each cell type was added to 96-well plates (effect cell to target cell ratio 5:1), along with 100 μL of different concentrations of bispecific antibody. The cell and antibody mixture was incubated at 37°C and 5% CO2 for 72 hours.

[0142] The cytotoxic effect of Raji tumor cells was detected using the LIVE / DEAD fixed dead cell staining kit (Invitrogen, L34964). The kit stains dead cells with purple, and cytotoxicity is detected by calculating the dead target cells exhibiting both green and purple fluorescence. The cultured cell mixture was washed three times with PBS and then incubated with the LIVE / DEAD fixed dead cell stain at 4°C for 30 minutes. The cells were washed three times with PBS and analyzed by flow cytometry. The results are shown in Figure 12 and Table 18. The results indicate that B2 molecules mediate effective killing of T cells, with cytotoxic activity superior to Odronextamab.

[0143] Table 18: Bispecific antibody TDCC killing assay

[0144] 2.2.10 Efficacy of CD20 / CD3 bispecific antibody in Raji local PBMC model

[0145] To detect the antitumor efficacy of bispecific antibodies in vivo, human lymphoma Raji and human PBMC cells were subcutaneously inoculated into immunodeficient NCG mice (Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd., 6-week-old female NCG mice). The in vivo inoculation continued until the average tumor volume reached approximately 150 mm². 3 Mice were randomly assigned to groups and administered CD20 / CD3 bispecific antibody molecule 0.3 mg / kg via tail vein injection. Seven days later, mice were administered CD20 / CD3 bispecific antibody molecule 0.45 mg / kg via tail vein injection. The efficacy of the CD20 / CD3 bispecific antibody against Raji xenografts was evaluated by monitoring tumor growth in mice. The results, as shown in Figure 13, indicate that the B2 molecule significantly inhibited the growth of Raji tumors. Furthermore, as shown in Figure 14, the tumor-bearing mice tolerated the above doses well, with no adverse reactions such as weight loss.

Claims

1. A CD3 antibody or an antigen-binding fragment thereof, comprising a light chain variable region (VL) and a heavy chain variable region (VH), wherein the light chain variable region and the heavy chain variable region comprise: HCDR1 shown in SEQ ID NO:7, HCDR2 shown in SEQ ID NO:8, HCDR3 shown in SEQ ID NO:9, LCDR1 shown in SEQ ID NO:10, LCDR2 shown in SEQ ID NO:11, and LCDR3 shown in SEQ ID NO:12; or HCDR1 shown in SEQ ID NO:13, HCDR2 shown in SEQ ID NO:14, HCDR3 shown in SEQ ID NO:15, LCDR1 shown in SEQ ID NO:16, LCDR2 shown in SEQ ID NO:11 and LCDR3 shown in SEQ ID NO:17; Optionally, the antigen-binding fragment is selected from Fab fragment, Fab′ fragment, Fab′-SH fragment, F(ab′)2 fragment, Fv fragment and scFv fragment; optionally, the antigen-binding fragment is an antigen-binding fragment derived from monoclonal antibody and / or humanized antibody.

2. The CD3 antibody or its antigen-binding fragment according to claim 1, wherein... VH contains or has an amino acid sequence that is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO:1, and VL contains or has an amino acid sequence that is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO:2; or The VH contains or has an amino acid sequence that is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO:3, and the VL contains or has an amino acid sequence that is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO:

4.

3. A bispecific antigen-binding molecule comprising an immunoglobulin domain targeting CD3 and an immunoglobulin domain targeting an antigen expressed on tumor cells; wherein the immunoglobulin domain targeting CD3 is selected from the antigen-binding fragment of the CD3 antibody according to claim 1 or 2; Optionally, the immunoglobulin domain of the antigen expressed on the tumor cells is an antigen-binding fragment targeting CD20.

4. The bispecific antigen-binding molecule according to claim 3, wherein the bispecific antigen-binding molecule comprises (a) a first immunoglobulin domain targeting CD3, (b) a second immunoglobulin domain targeting CD20, and (c) a third immunoglobulin domain targeting CD20; optionally, the first immunoglobulin domain is an scFv structure, and the second and / or the third immunoglobulin domain is a Fab structure.

5. The bispecific antigen-binding molecule according to any one of claims 3-4, wherein the CD20-targeting antigen-binding fragment comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region and the heavy chain variable region comprise: HCDR1 shown in SEQ ID NO:28, HCDR2 shown in SEQ ID NO:29, HCDR3 shown in SEQ ID NO:30, LCDR1 shown in SEQ ID NO:31, LCDR2 shown in SEQ ID NO:32 and LCDR3 shown in SEQ ID NO:33; Optionally, the VH of the CD20-targeting antigen-binding fragment contains an amino acid sequence having at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:26, and the VL contains an amino acid sequence having at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:

27.

6. The bispecific antigen-binding molecule according to any one of claims 3-4, wherein the first immunoglobulin domain targeting CD3 comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region and the heavy chain variable region comprise: HCDR1 shown in SEQ ID NO:7, HCDR2 shown in SEQ ID NO:8, HCDR3 shown in SEQ ID NO:9, LCDR1 shown in SEQ ID NO:10, LCDR2 shown in SEQ ID NO:11, and LCDR3 shown in SEQ ID NO:12; or HCDR1 shown in SEQ ID NO:13, HCDR2 shown in SEQ ID NO:14, HCDR3 shown in SEQ ID NO:15, LCDR1 shown in SEQ ID NO:16, LCDR2 shown in SEQ ID NO:11, and LCDR3 shown in SEQ ID NO:17; Optionally, the first immunoglobulin domain targeting CD3 includes a light chain variable region (VL) and a heavy chain variable region (VH), wherein the VH contains or has an amino acid sequence that is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO:1, and the VL contains or has an amino acid sequence that is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO:2; or the VH contains or has an amino acid sequence that is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO:3, and the VL contains or has an amino acid sequence that is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO:3, and the VH contains or has an amino acid sequence that is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO:

1. The amino acid sequence shown in NO:4 has an amino acid sequence that is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical.

7. The bispecific antigen-binding molecule according to any one of claims 3-6, wherein the CD20-targeting antigen-binding fragment comprises a light chain variable region (VL) and a heavy chain variable region (VH), wherein the light chain variable region and the heavy chain variable region comprise: HCDR1 shown in SEQ ID NO:28, HCDR2 shown in SEQ ID NO:29, HCDR3 shown in SEQ ID NO:30, LCDR1 shown in SEQ ID NO:31, LCDR2 shown in SEQ ID NO:32, and LCDR3 shown in SEQ ID NO:33; and The CD3 immunoglobulin domain includes a light chain variable region and a heavy chain variable region, wherein the light chain variable region and the heavy chain variable region include: HCDR1 shown in SEQ ID NO:7, HCDR2 shown in SEQ ID NO:8, HCDR3 shown in SEQ ID NO:9, LCDR1 shown in SEQ ID NO:10, LCDR2 shown in SEQ ID NO:11, and LCDR3 shown in SEQ ID NO:12; or HCDR1 shown in SEQ ID NO:13, HCDR2 shown in SEQ ID NO:14, HCDR3 shown in SEQ ID NO:15, LCDR1 shown in SEQ ID NO:16, LCDR2 shown in SEQ ID NO:11, and LCDR3 shown in SEQ ID NO:

17.

8. The bispecific antigen-binding molecule according to any one of claims 4-6, wherein the second immunoglobulin domain targeting CD20 and the third immunoglobulin domain targeting CD20 contain the same light chain variable region and heavy chain variable region, wherein the light chain variable region and heavy chain variable region comprise: HCDR1 shown in SEQ ID NO:28, HCDR2 shown in SEQ ID NO:29, HCDR3 shown in SEQ ID NO:30, LCDR1 shown in SEQ ID NO:31, LCDR2 shown in SEQ ID NO:32, and LCDR3 shown in SEQ ID NO:33; and The first immunoglobulin domain targeting CD3 includes a light chain variable region and a heavy chain variable region, wherein the light chain variable region and the heavy chain variable region include: HCDR1 shown in SEQ ID NO:7, HCDR2 shown in SEQ ID NO:8, HCDR3 shown in SEQ ID NO:9, LCDR1 shown in SEQ ID NO:10, LCDR2 shown in SEQ ID NO:11, and LCDR3 shown in SEQ ID NO:12; or HCDR1 shown in SEQ ID NO:13, HCDR2 shown in SEQ ID NO:14, HCDR3 shown in SEQ ID NO:15, LCDR1 shown in SEQ ID NO:16, LCDR2 shown in SEQ ID NO:11, and LCDR3 shown in SEQ ID NO:17; Optionally, both the second immunoglobulin domain targeting CD20 and the third immunoglobulin domain targeting CD20 are Fab domains and contain the same light chain variable region and heavy chain variable region, wherein the light chain variable region and heavy chain variable region include: HCDR1 shown in SEQ ID NO:28, HCDR2 shown in SEQ ID NO:29, HCDR3 shown in SEQ ID NO:30, LCDR1 shown in SEQ ID NO:31, LCDR2 shown in SEQ ID NO:32, and LCDR3 shown in SEQ ID NO:33; and The first immunoglobulin domain targeting CD3 is an scFv structure, including a light chain variable region and a heavy chain variable region, wherein the light chain variable region and the heavy chain variable region include: HCDR1 shown in SEQ ID NO:7, HCDR2 shown in SEQ ID NO:8, HCDR3 shown in SEQ ID NO:9, LCDR1 shown in SEQ ID NO:10, LCDR2 shown in SEQ ID NO:11, and LCDR3 shown in SEQ ID NO:12; or HCDR1 shown in SEQ ID NO:13, HCDR2 shown in SEQ ID NO:14, HCDR3 shown in SEQ ID NO:15, LCDR1 shown in SEQ ID NO:16, LCDR2 shown in SEQ ID NO:11, and LCDR3 shown in SEQ ID NO:

17.

9. The bispecific antigen-binding molecule according to any one of claims 4-8, wherein the second immunoglobulin domain targeting CD20 and the third immunoglobulin domain targeting CD20 contain the same light chain variable region (VL) and heavy chain variable region (VH), wherein the VH comprises, or has an amino acid sequence having at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:26, and the VL comprises, or has an amino acid sequence having at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:27; and The first immunoglobulin domain targeting CD3 includes a light chain variable region (VL) and a heavy chain variable region (VH), wherein the VH contains or has an amino acid sequence that is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO:1, and the VL contains or has an amino acid sequence that is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO:2; or the VH contains or has an amino acid sequence that is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO:3, and the VL contains or has an amino acid sequence that is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO:3, and the VL contains or has an amino acid sequence that is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO:

1. The amino acid sequence shown in NO:4 has an amino acid sequence with at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity; Optionally, both the second immunoglobulin domain targeting CD20 and the third immunoglobulin domain targeting CD20 are Fab domains, containing the same light chain variable region (VL) and heavy chain variable region (VH), wherein the VH contains or has an amino acid sequence that is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO:26, and the VL contains or has an amino acid sequence that is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO:27; and The first immunoglobulin domain targeting CD3 is an scFv structure, wherein the VH contains or has an amino acid sequence that is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO:1, and the VL contains or has an amino acid sequence that is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO:2; or the VH contains or has an amino acid sequence that is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO:3, and the VL contains or has an amino acid sequence that is identical to the amino acid sequence shown in SEQ ID NO:

1. The amino acid sequence shown in NO:4 has an amino acid sequence that is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical.

10. The bispecific antigen-binding molecule according to any one of claims 3-9, comprising the following peptide chains (1)-(4): Peptide chain (1)N'-CD20VL-CL-C'; peptide chain (2) N'-CD20VH-CH1-CD3VL-CD3VH-Fc-C'; peptide chain (3)N'-CD20VH-CH1-Fc-C'; and peptide chain (4)N'-CD20VL-CL-C' Where VH represents the heavy chain variable region, CD20VH represents the heavy chain variable region of the antigen-binding fragment targeting CD20, and CD3VH represents the heavy chain variable region of the antigen-binding fragment targeting CD3; VL represents the light chain variable region, CD20VL represents the light chain variable region of the antigen-binding fragment targeting CD20, and CD3VL represents the light chain variable region of the antigen-binding fragment targeting CD3; Fc contains CH2 and CH3; CH1, CH2, and CH3 represent domains 1, 2, and 3 of the heavy chain constant region, respectively; CL represents the light chain constant region; Optionally, the Fc region comprises a natural Fc sequence or a non-natural Fc sequence; optionally, the Fc region is a human Fc region; further optionally, the Fc region is the Fc region of an IgG1, IgG2, IgG3, or IgG4 antibody; even further optionally, the Fc region is an IgG1 Fc region; even further optionally, the Fc region comprises one or more mutations selected from those that weaken the antibody ADCC effect, those that weaken the antibody CDC effect, knocks-into-holes mutations, and disulfide bond mutations.

11. The bispecific antigen-binding molecule according to claim 10, wherein the CH1 comprises or has an amino acid sequence having at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:34; The CL comprises, or has an amino acid sequence having at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:35; and / or The Fc contains or has an amino acid sequence that is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in any of SEQ ID NO:36-37.

12. The bispecific antigen-binding molecule according to any one of claims 10-11, wherein the bispecific antigen-binding molecule comprises peptide chain (1)-peptide chain (4); The peptide chain (1) comprises or has an amino acid sequence that is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:38; The peptide chain (2) comprises or has an amino acid sequence that is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:39 or SEQ ID NO:41; The peptide chain (3) comprises or has an amino acid sequence that is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:40; The peptide chain (4) comprises or has an amino acid sequence that is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:38; Optionally, the peptide chains (1)-(4) comprise or have an amino acid sequence that is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequences shown in SEQ ID NO:38, 39, 40, and 38, or the peptide chains (1)-(4) comprise or have an amino acid sequence that is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequences shown in SEQ ID NO:38, 41, 40, and 38.

13. An isolated nucleic acid comprising a nucleic acid sequence encoding a CD3 antibody or an antigen-binding fragment thereof of any one of claims 1-2 or a bispecific antigen-binding molecule of any one of claims 3-12.

14. A vector comprising the nucleic acid according to claim 13.

15. An isolated host cell comprising the nucleic acid of claim 13 or the vector of claim 14.

16. A composition comprising a CD3 antibody or an antigen-binding fragment thereof according to any one of claims 1-2, or a bispecific antigen-binding molecule according to any one of claims 3-12, or a nucleic acid according to claim 13, or a vector according to claim 14, or a host cell according to claim 15; optionally, the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable vector.

17. Use of the CD3 antibody or antigen-binding fragment thereof according to any one of claims 1-2, the bispecific antigen-binding molecule according to any one of claims 3-12, the nucleic acid according to claim 13, the vector according to claim 14, or the host cell according to claim 15 in the manufacture of a medicament for treating or delaying the progression of individual cancer; optionally, wherein said cancer is a CD20-positive cancer, such as lymphoma, comprising: Hodgkin's lymphoma and non-Hodgkin's lymphoma; Further optionally, the cancer is non-Hodgkin lymphoma, which includes: diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, and mucosa-associated lymphoid tissue lymphoma. Alternatively, the cancer may be diffuse large B-cell lymphoma.

18. A method of treating or delaying cancer progression in a subject in need, comprising administering to the subject the bispecific antigen-binding molecule of any one of claims 3-12; optionally, wherein said cancer is a CD20-positive cancer, such as lymphoma, comprising: Hodgkin's lymphoma and non-Hodgkin's lymphoma; Further optionally, the cancer is non-Hodgkin lymphoma, which includes: diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, and mucosa-associated lymphoid tissue lymphoma. Alternatively, the cancer may be diffuse large B-cell lymphoma.

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