Antibody having t-cell activating function

Abstract

Provided are a T-cell activating anti-CD3 antibody and a multispecific antibody comprising same, in particular a T-cell engager. Also provided are a composition comprising the antibody or multispecific antibody, and a therapeutic application thereof.

Description

Antibodies with T cell activation function

[0001] Cross-reference to related applications

[0002] This application was filed on December 18, 2025 as a PCT international patent application, claiming priority to PCT international patent application No. PCT / CN2024 / 140604, filed on December 19, 2024, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention relates to the field of antibody technology, and more specifically to T-cell activating anti-CD3 antibodies and multispecific antibodies comprising them, particularly T-cell connectors. This invention also relates to compositions containing said antibodies or multispecific antibodies and their therapeutic applications. Background Technology

[0004] T-cell engagers (TCEs) are an important molecular form in antibody therapy. Several TCE drugs have already been approved for marketing. For example, the 2:1 anti-CD3xCD20 monoclonal antibody Glofitamab has received accelerated approval as an effective treatment for adult patients with relapsed or refractory diffuse large B-cell lymphoma (DLBCL) and large B-cell lymphoma caused by follicular lymphoma (LBCL). The core mechanism of TCEs is to utilize T-cell-activating anti-CD3 antibodies to recruit the body's own T cells to target cells (e.g., tumor cells) that express target antigens (e.g., tumor-associated antigens), thereby selectively killing the target cells. Therefore, providing high-performance CD3 antibodies is a crucial step in TCE development.

[0005] Currently, the number of commercially available anti-CD3 antibodies is limited. Most generic anti-CD3 antibodies freely available to the public each have their drawbacks. The most common, OKT3, lacks immune cross-reactivity with monkey CD3, increasing the difficulty of toxicological studies for drugs based on this molecule. SP34, another commonly chosen anti-CD3 molecule, requires humanization for human drug development and has been found to have physicochemical defects, exhibiting weak non-specific binding and poor molecular stability, leading to CMC (Chemical Manufacturing and Control) issues in polyclonal antibody construction. Furthermore, existing CD3-targeted drug development has revealed that most traditional anti-CD3 antibodies, while recruiting T cells to kill tumor cells, can induce potentially serious toxicities related to T cell activation, especially cytokine release syndrome (CRS) and neurotoxicity, thus limiting clinical use. In addition, the success of anti-CD3 drugs in solid tumors is very limited, facing multiple challenges. See, Overcoming Challenges for CD3-Bispecific Antibody Therapy in Solid Tumors, Cancers (Basel). 2021Jan; 13(2):287, doi:10.3390 / cancers13020287.

[0006] Therefore, there remains a need in this field to develop novel anti-CD3 antibodies, as well as T-cell activating antibodies and TCE molecules based on them. In the development of CD3-based multi-(bi)specific antibody drugs, optimized solutions are achieved through appropriate target selection and the design and matching of binding sites in terms of epitope location, affinity range, molecular conformation, and druggability.

[0007] Invention Overview

[0008] The inventors have obtained a novel functional sequence for an anti-CD3 antibody de novo using hybridoma technology. This anti-CD3 sequence exhibits unique behavior. When applied alone, especially in monovalent form, its binding to CD3 on the cell surface is very weak. However, when assembled into a TCE (transmitted cytokine exchange), it demonstrates very strong T cell activation and killing capabilities, comparable to leading commercially available competitors. This characteristic of the anti-CD3 sequence suggests a potentially better efficacy window; the weak binding to CD3 prevents T cell exhaustion, while the strong killing effect on target cells ensures good antitumor efficacy.

[0009] Furthermore, the inventors have also derived a novel CD20 single-domain antibody from scratch. The anti-CD20 single-domain antibody of this invention possesses excellent CD20 binding properties and is suitable for tumor treatment applications.

[0010] Therefore, in a first aspect, this disclosure provides an anti-CD3 antibody. In some embodiments, the anti-CD3 antibody of the present invention comprises three CDRs (HCDR1, HCDR2, and HCDR3) or variants thereof contained in the heavy chain variable region selected from SEQ ID NOs:7, 15, 23, 31, 39, 47, and 67-69; and three CDRs (LCDR1, LCDR2, and LCDR3) or variants thereof contained in the light chain variable region selected from SEQ ID NOs:8, 16, 24, 32, 40, 48, 70-74, 78-82, and 87.

[0011] In a second aspect, this disclosure provides an anti-CD20 antibody. In some embodiments, the anti-CD20 antibody of the present invention comprises three CDRs (CDR1, CDR2, and CDR3) or variants thereof contained in the heavy chain variable region (VHH) of one of SEQ ID NO: 102 or 115-124.

[0012] In a third aspect, this disclosure provides a multispecific antibody comprising a CD3 antigen-binding domain, particularly a T-cell connector comprising a CD3 antigen-binding domain and a tumor-associated antigen (TAA)-binding domain. In some embodiments, the multispecific antibody according to the invention is a multispecific antibody comprising CD3 and CD20 binding specificity, particularly a bispecific antibody. In other embodiments, the multispecific antibody according to the invention is a multispecific antibody comprising CD3 and MSLN binding specificity, particularly a bispecific antibody.

[0013] In a fourth aspect, this disclosure provides a nucleic acid encoding the anti-CD3 antibody, anti-CD20 antibody, or multispecific antibody of the present invention, a host cell comprising the present invention, and a method for generating the anti-CD3 antibody, anti-CD20 antibody, or multispecific antibody of the present invention.

[0014] In a fifth aspect, this disclosure provides antigen-binding molecules comprising the anti-CD3 antibody, anti-CD20 antibody, or multispecific antibody of the present invention, such as immunoconjugates and immunofusions.

[0015] In a sixth aspect, this disclosure provides pharmaceutical compositions comprising the anti-CD3 antibody, anti-CD20 antibody, or multispecific antibody of the present invention.

[0016] In a seventh aspect, this disclosure provides the use of the anti-CD3 antibody, anti-CD20 antibody, or multispecific antibody of the present invention as a medicine or in the preparation of a medicine, and methods of treating or preventing cancer (including solid tumors and hematologic malignancies) using the anti-CD3 antibody, anti-CD20 antibody, or multispecific antibody of the present invention.

[0017] The invention is further illustrated in the following figures and specific embodiments. However, these figures and specific embodiments should not be considered as limiting the scope of the invention, and modifications readily apparent to those skilled in the art will be included within the spirit of the invention and the scope of protection of the appended claims.

[0018] Brief description of the attached diagram

[0019] Figure 1 shows the binding of hybridoma chimeric antibodies to Jurkat cells.

[0020] Figure 2 shows the binding of hybridoma chimeric antibody to Jurkat-CD3KO cells.

[0021] Figure 3 shows the binding of hybridoma chimeric antibodies to cynomolgus CD4+ T cells (cyno CD4+ T cells).

[0022] Figure 4 shows the binding of hybridoma chimeric antibodies to cynomolgus CD8+ T cells.

[0023] Figure 5 shows the activation of CD4+ T cells from the donor Donor1 by the hybridoma chimeric antibody.

[0024] Figure 6 shows the activation of CD8+ T cells from the donor Donor1 by the hybridoma chimeric antibody.

[0025] Figure 7 shows the activation of CD4+ T cells from the donor Donor2 by the hybridoma chimeric antibody.

[0026] Figure 8 shows the activation of CD8+ T cells from the donor Donor2 by the hybridoma chimeric antibody.

[0027] Figure 9 shows the binding of the humanized variant of 136G6.3 to Jurkat cells.

[0028] Figure 10 shows the binding of the humanized variant of 136G6.3 to Jurkat-CD3KO cells.

[0029] Figure 11 shows the binding of the PTM-removed variant of 136G6.3 to Jurkat cells.

[0030] Figure 12 shows the binding of the PTM-removed variant of 136G6.3 to Jurkat-CD3KO cells.

[0031] Figure 13 shows the binding of the deimmunogenic variant of 136G6.3-zom2 to Jurkat cells.

[0032] Figure 14 shows the binding of the deimmunogenic variant of 136G6.3-zom2 to Jurkat-CD3KO cells.

[0033] Figure 15 shows the binding of the humanized deimmunogenic PTM-removed variant 136G6.3M to Jurkat cells.

[0034] Figure 16 shows the binding of 136G6.3M to Jurkat-CD3KO cells.

[0035] Figure 17 shows the affinity of 136G6.3M for human CD3 (hCD3) and cynomolgus monkey CD3 (cynoCD3) antigens. 136G6.3M is represented in the figure as "136G6.3-pdzom2m21m11".

[0036] Figure 18A shows the binding activity of the anti-CD20-VHH antibody to human CD20-positive tumor cells Raji.

[0037] Figure 18B shows the binding activity of the anti-CD20-VHH antibody to human CD20-positive tumor cells Daudi.

[0038] Figure 19A shows the binding activity of anti-CD20-VHH antibody to HEK293-cynoCD20 cells expressing monkey CD20.

[0039] Figure 19B shows the binding activity of anti-CD20-VHH antibody to HEK293 cells that do not express CD20.

[0040] Figure 20 shows the binding activity of the humanized anti-CD20-VHH antibody to human CD20-positive tumor cells Daudi.

[0041] Figure 21A shows the binding activity of humanized anti-CD20-VHH antibody to HEK293-cynoCD20 cells expressing monkey CD20.

[0042] Figure 21B shows the binding activity of the humanized anti-CD20-VHH antibody to HEK293 cells that do not express CD20.

[0043] Figure 22A shows the endocytic activity of humanized anti-CD20-VHH antibody V-zn6D11.m2 on Ramos cells.

[0044] Figure 22B shows the endocytic activity of humanized anti-CD20-VHH antibody V-zn6D11.m2 on Daudi cells.

[0045] Figures 23A and 23B show schematic diagrams of the structure of CD3xTAA (CD20 or MSLN) bispecific antibodies (bsAb). In the figures, "LALA" represents the L234AL235A mutation located in the CH2 domain; k represents the Knob mutation located in the CH3 domain; and h represents the Hole mutation located in the CH3 domain corresponding to the knob mutation.

[0046] Figure 24 shows the binding of the CD3xCD20 bispecific antibody to Ramos cells.

[0047] Figure 25 shows the binding of the CD3xCD20 bispecific antibody to Jurkat cells.

[0048] Figure 26 shows the killing effect of Donor3 PBMCs on Ramos cells mediated by CD3xCD20 bispecific antibody.

[0049] Figure 27 shows the killing effect of Donor4 PBMCs on Ramos cells mediated by CD3xCD20 bispecific antibody.

[0050] Figure 28 shows CD3xCD20 dual antibody-mediated IL6 release accompanying Donor3 PBMC killing of Ramos.

[0051] Figure 29 shows the release of IFN-γ mediated by CD3xCD20 dual antibody, which accompanies Donor3 PBMC killing of Ramos.

[0052] Figure 30 shows the release of IL-6 mediated by CD3xCD20 dual antibody, accompanied by Donor4 PBMC killing of Ramos.

[0053] Figure 31 shows the release of IFN-γ mediated by CD3xCD20 dual antibody, which accompanies the killing of Ramos by Donor4 PBMC.

[0054] Figure 32 shows the binding of CD3xMSLN bispecific antibody to AsPC1 cells.

[0055] Figure 33 shows the binding of CD3xMSLN bispecific antibody to Jurkat cells.

[0056] Figure 34 shows the killing effect of Donor3 PBMCs on AsPC1 cells mediated by CD3xMSLN dual antibody.

[0057] Figure 35 shows the killing effect of Donor4 PBMCs on AsPC1 cells mediated by CD3xMSLN dual antibody.

[0058] Figure 36 shows the release of IFN-γ mediated by CD3xMSLN dual antibody, which accompanies the killing of target cells by Donor3 PBMCs.

[0059] Figure 37 shows the release of IL-6 mediated by CD3xMSLN dual antibody, which accompanies the killing of target cells by Donor3 PBMCs.

[0060] Figure 38 shows the release of TNF-α mediated by CD3xMSLN bispecific antibody, which accompanies the killing of target cells by Donor3 PBMCs.

[0061] Figure 39 shows the release of IFN-γ mediated by CD3xMSLN dual antibody, which accompanies the killing of target cells by Donor4 PBMCs.

[0062] Figure 40 shows the release of IL-6 mediated by CD3xMSLN dual antibody, which accompanies the killing of target cells by Donor4 PBMCs.

[0063] Figure 41 shows the release of TNF-α mediated by CD3xMSLN bispecific antibody, which accompanies the killing of target cells by Donor4 PBMCs.

[0064] Figure 42 shows the killing effect of Donor5 PBMCs on Ramos cells mediated by CD3xCD20 bispecific antibody.

[0065] Figure 43 shows the killing effect of Donor5 PBMCs on Raji cells mediated by CD3xCD20 bispecific antibody.

[0066] Figure 44 shows the release of IFN-γ mediated by CD3xCD20 dual antibody, which accompanies Donor5 PBMC killing of Ramos.

[0067] Figure 45 shows CD3xCD20 dual antibody-mediated IL6 release accompanying Donor5 PBMC killing of Ramos.

[0068] Figure 46 shows the release of IFN-γ mediated by CD3xCD20 dual antibody, which accompanies Donor5 PBMC killing of Raji.

[0069] Figure 47 shows CD3xCD20 dual antibody-mediated IL-6 release accompanying Donor5 PBMC killing of Raji.

[0070] Figure 48 shows CD4+ T cell activation mediated by CD3xCD20 dual antibody, accompanied by Donor5 PBMC killing of Ramos.

[0071] Figure 49 shows CD8+ T cell activation mediated by CD3xCD20 dual antibody, accompanied by Donor5 PBMC killing of Ramos.

[0072] Figure 50 shows the in vivo antitumor efficacy of the CD3xCD20 bispecific antibody in mice.

[0073] Invention Details

[0074] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. Furthermore, the materials, methods, and examples described herein are illustrative only and are not intended to be limiting. Other features, objects, and advantages of the invention will become apparent from this specification and the accompanying drawings, and from the appended claims.

[0075] definition

[0076] The term “about” when used in conjunction with a numeric value means to cover a range of numeric values ​​that have a lower limit of 5% less than the specified numeric value and an upper limit of 5% greater than the specified numeric value.

[0077] As used herein, the term "and / or", when used in conjunction with two or more options, means any one of the options or any two or more of the options.

[0078] In this document, when the terms “comprising” or “including” are used, unless otherwise specified, they also cover situations where the variable region consists of the mentioned elements, integers, or steps. For example, when referring to an antibody variable region that “comprising” a specific sequence, it is also intended to cover the antibody variable region consisting of that specific sequence.

[0079] In this document, the term "antigen-binding molecule" refers to a molecule, such as a protein or polypeptide, or a molecule derived therefrom, that contains an antigen-binding domain or antigen-binding site capable of binding to a target antigen. When the target antigen is CD3 and / or a tumor-associated antigen (TAA), an antigen-binding molecule that binds to CD3 and / or TAA is also referred to as a CD3-binding molecule, a TAA-binding molecule, or a CD3 / TAA-binding molecule. Antigen-binding molecules include, for example, antibodies and their antigen-binding fragments, as well as various fusions and conjugates constructed based on antibodies or antigen-binding fragments, such as immunoconjugates, antibody-drug conjugates (ADCs), multi- / bispecific antibodies, and chimeric antigen receptors (CARs). As will be apparent to those skilled in the art, the antigen-binding site of an antibody typically contains an amino acid residue from a "complementarity-determining region" or "CDR". In some aspects of the invention, antigen-binding molecules based on the anti-CD3 antibody or antigen-binding fragment of the invention, or based on the multi-specific antibody of the invention, are within the scope of consideration of the invention.

[0080] In this document, the term "antibody" refers to a polypeptide containing at least a light or heavy chain immunoglobulin variable region that specifically recognizes and binds to an antigen. This term encompasses a wide range of antibody structures, including, but not limited to, monoclonal antibodies, single-chain or multi-chain antibodies, monospecific or multispecific antibodies (e.g., bispecific antibodies), single-domain antibodies, heavy chain antibodies, murine antibodies, chimeric or humanized antibodies, intact antibodies, and antibody fragments, provided they exhibit the desired antigen-binding activity.

[0081] In this article, "intact antibody" and "full-length antibody" are used interchangeably, referring to an immunoglobulin molecule containing at least two heavy chains (H) and two light chains (L). Each heavy chain consists of a heavy chain variable region (abbreviated as VH) and a heavy chain constant region. The heavy chain constant region consists of 3 to 4 immunoglobulin domains, CH1, CH2, and CH3, and optionally CH4. Each light chain consists of a light chain variable region (abbreviated as VL) and a light chain constant region. The light chain constant region consists of one domain, CL.

[0082] In this document, the terms “antibody fragment” and “antigen-binding fragment” are used interchangeably to refer to a molecule distinct from the intact antibody that contains a portion of the intact antibody and is capable of binding the antigen bound by the intact antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, crossFab, Fab', Fab'-SH, F(ab')2; bibody; linear antibody; single-chain antibody (e.g., scFv, scFab); single-domain antibody; camelid antibody (heavy chain antibody) or fragments thereof (e.g., VHH); and monospecific, bispecific, or multispecific antibodies formed from antibody fragments. Unless otherwise stated herein or explicitly contradicted by the context, the term “antibody” is used in this document in the same way as “antibody or antibody fragment thereof.”

[0083] In this document, the terms "antigen binding site" and "antigen binding domain" are used interchangeably to refer to the region in an antibody molecule that actually binds to an antigen. In the multispecific antibodies of the present invention, antigen binding specificity is preferably provided by the antigen binding domain. In some embodiments, the CD3 antigen binding domain for the multispecific antibodies of the present invention is preferably provided by a heavy chain variable domain (VH) and a light chain variable domain (VL) from the anti-CD3 antibody according to the present invention. In some embodiments, the TAA antigen binding domain for the multispecific antibodies of the present invention is preferably provided by a variable domain (i.e., "VHH") from the heavy chain antibody.

[0084] In this document, the term "multispecific" refers to an antigen-binding molecule (e.g., an antibody) comprising two or more antigen-binding domains, wherein at least two of said antigen-binding domains bind to different antigenic epitopes, for example, to different epitopes on different antigens or different epitopes on the same antigen. Correspondingly, "single-specific" refers to the ability to bind to only one epitope. "Dual-specific" refers to the ability to bind to two different epitopes.

[0085] In this article, the antibody-related terms "valence" or "valence number" refer to the total number of antigen-binding sites in an antibody molecule, or the number of antigen-binding sites with the same antigen-binding specificity. For example, a trivalent antibody means that the antibody molecule contains a total of 3 antigen-binding sites; the antibody molecule can also be a "2+1" type bispecific antibody, that is, the antibody has two different antigen-binding specificities, with 2 antigen-binding sites for one antigen-binding specificity and only 1 antigen-binding site for the other antigen-binding specificity. Such bispecific antibodies can also be called 2:1 type bispecific antibodies based on the ratio of the number of antigen-binding domains of the two different specificities.

[0086] In this document, the terms “first,” “second,” “third,” and “fourth,” etc., when used in conjunction with elements such as Fc regions or antigen-binding sites, are intended to conveniently distinguish two elements belonging to the same category. However, it should be noted that, unless explicitly stated otherwise, the use of these terms is not intended to assign a specific order, orientation, or position to the elements.

[0087] In this paper, the term "CD3" refers to the cluster of differentiation 3 antigen expressed on the surface of T cells and interacting with the T cell receptor (TCR). CD3 has four major subunits: γ, δ, ε, and ζ, which respectively include the N-terminal extracellular domain, the transmembrane domain, and the cytoplasmic tail region containing the immunoreceptor tyrosine activation motif (ITAM). These subunits interact to form three different polypeptide dimers: γε, δε, and ζζ. The CD3 complex composed of these dimers binds to the α and β chains of the T cell receptor (TCR) to form the complete T cell receptor complex, which participates in the regulation of T cell antigen recognition, signal transduction, and T cell development. CD3 on the cell membrane surface, as a pan-T cell marker, is expressed on the surface of all mature T cells in peripheral blood and lymphoid tissues. An example of CD3-ε is the human CD3-ε protein containing the amino acid sequence UniProtKB-P07766; an example of CD3-δ is the human CD3-δ protein containing the amino acid sequence UniProtKB-P04234; an example of CD3-ζ is the human CD3-ζ protein containing the amino acid sequence UniProtKB-P20963; and an example of CD3-γ is the human CD3-γ protein containing the amino acid sequence UniProtKB-P09693. Unless explicitly specified as originating from a non-human species, all references to CD3 proteins, peptides, and protein fragments herein are intended to refer to the human version of the corresponding protein, peptide, or protein fragment. Therefore, unless specified as originating from a non-human species, such as “mouse CD3,” “monkey CD3,” etc., the expression “CD3” refers to human CD3. Accordingly, in this document, unless otherwise specified, the term “antigen-binding specificity against CD3,” that is, “antigen-binding domain that specifically binds to CD3,” refers to binding specificity against human CD3.

[0088] In this document, the terms "CD3-binding antibody" or "anti-CD3 antibody" encompass antibodies and their antigen-binding fragments that specifically recognize a single CD3 subunit (e.g., ε, δ, γ, or ζ), as well as antibodies and their antigen-binding fragments that specifically recognize dimeric complexes of two CD3 subunits (e.g., γ / ε, δ / ε, and ζ / ζ CD3 dimers). The antibodies and antigen-binding fragments of this invention can bind to soluble CD3 and / or CD3 expressed on the cell surface. Examples of soluble CD3 include recombinant CD3 protein variants, such as monomeric and dimeric CD3 constructs.

[0089] In this document, the term "cell surface expressed CD3" means a CD3 protein expressed on the surface of cells in vitro or in vivo, wherein at least a portion of the CD3 protein is exposed on the extracellular side of the cell membrane and is accessible to the antigen-binding portion of an antibody. "Cell surface expressed CD3" includes CD3 proteins that interact with functional T cell receptors within the cell membrane.

[0090] In this paper, the term "CD3 antigen-positive cells" refers to cells that express CD3 on their cell surface, either in vitro or in vivo, such as CD4+ and CD8+ T cells. The term "CD3 antigen-negative cells" refers to cells that do not express CD3 on their cell surface, either in vitro or in vivo, such as native B cells and recombinant T cells with CD3 knockout.

[0091] In this article, the term "tumor-associated antigen" (TAA), also known as TAA, refers to any antigen that is highly expressed in tumor cells or the tumor stroma. Typically, TAAs are expressed or overexpressed in the tumor cells to be treated, but expressed at low levels or not at all in normal cells. Examples of TAAs include, but are not limited to, CD20, MSLN, etc.

[0092] In this paper, the term "MSLN" refers to mesothelin. Mesothelin is a cell surface protein expressed by the mesothelin gene (MSLN). This protein is produced from the precursor protein encoded by the MSLN gene via furin protease hydrolysis and is anchored to the cell membrane surface by glycosylphosphatidylinositol. MSLN is highly expressed in malignant tumors but has limited expression in normal tissues, and has therefore been proposed as a candidate for tumor-targeted therapy. In this paper, unless otherwise stated, the term MSLN includes any variant of human MSLN, including sequence variants, especially naturally occurring variants, allelic variants, as well as post-translational modification variants and conformational variants, and encompasses its species homologs. In some cases, the term specifically refers to the MSLN antigen expressed on the surface of tumor cells. An example of MSLN is the human MSLN protein containing the amino acid sequence UniProtKB-Q13421. Accordingly, in this document, unless otherwise specified, the term "antigen binding specificity against MSLN", that is, "antigen binding domain that specifically binds to MSLN", refers to the binding specificity against human MSLN.

[0093] In this document, the term "MSLN-positive" cell refers to a cell that is positive for MSLN expression on its cell surface. The MSLN expression level on the cell surface can be determined by any conventional method known in the art for determining cell surface antigen expression levels, such as FACS detection or immunofluorescence staining. MSLN has significantly higher expression levels on a variety of tumor cells than on normal tissues / cells; for example, MSLN has been found to be overexpressed in 90% of mesotheliomas, 80%–85% of pancreatic cancers, 60%–65% of ovarian cancers, and 60%–65% of lung cancers. Preferably, in some embodiments, MSLN-positive cells are MSLN-positive tumor cells.

[0094] In this document, the term "CD20" refers to the B lymphocyte surface antigen CD20. This antigen has been proposed as a candidate for targeted therapy of B lymphocyte tumors. In this document, unless otherwise stated, the term CD20 includes any variant of human CD20, including sequence variants, especially naturally occurring variants, allelic variants, as well as post-translational modification variants and conformational variants, and encompasses its species homologs. In some cases, the term specifically refers to the CD20 antigen expressed on the surface of tumor cells. An example of CD20 is the human CD20 protein containing the amino acid sequence UniProtKB-P11836. Accordingly, in this document, unless specifically specified, the term "antigen-binding specificity against CD20," i.e., "antigen-binding domain specifically binding to CD20," refers to binding specificity against human CD20. In this document, the term "CD20-positive" cell refers to cells that are positive for CD20 expression on their cell surface, especially CD20-positive tumor cells.

[0095] In this document, the term "T-cell connector" (TCE) refers to a multi- (bi)specific antibody containing an antigen-binding domain that specifically targets T-cell surface antigens (such as CD3 or CD3 subunit complexes) and an antigen-binding domain that specifically targets target antigens (such as tumor-associated antigens). Through these antigen-binding domains with different specificities, the TCE can connect T cells and target cells (such as target tumor cells), forming an immune synapse between them, thereby inducing the killing effect of activated T cells on the target cells. It should be understood that in some cases, the TCE may contain other antigen-binding domains besides the CD3 and TAA binding domains to, for example, further enhance or assist the tumor-suppressive efficacy of the TCE. In some aspects, examples of other antigen-binding domains that may be mentioned are binding domains targeting co-stimulatory molecules.

[0096] In this document, the term "T cell activation" or "T cell activation" refers to one or more cellular responses of T lymphocytes, particularly cytotoxic T lymphocytes, selected from the following: proliferation, differentiation, cytokine secretion, release of cytotoxic effector molecules, cytotoxic activity, and expression of activation markers. In some embodiments, the anti-CD3 antibody and multispecific antibody according to the invention are T cell activating antibodies capable of inducing T cell activation. Suitable assays for measuring T cell activation include those described herein and those known in the art.

[0097] In this document, the terms "binding" or "specific binding" refer to the selective binding of an antigen-binding site to an antigenic epitope, which can be distinguished from unwanted or nonspecific interactions. The binding ability or binding specificity of an antigen-binding site to a specific antigenic epitope can be determined by conventional binding assays known in the art, including but not limited to, detecting antibody-antigen binding by ELISA, detecting antibody binding to cells expressing antigens by FACS, or characterizing the binding affinity constant KD by surface plasmon resonance (SPR) or thin-layer interferometry (BLI) techniques.

[0098] In this paper, 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 antigenic epitope). Binding affinity reflects the intrinsic binding affinity of a 1:1 interaction between members of a binding pair. Binding affinity is typically expressed as the binding dissociation equilibrium constant (KD) and can be measured using methods commonly known in the art, such as surface plasmon resonance (SPR) techniques.

[0099] In this paper, the term "affinity" or "binding affinity" refers to the combined strength of interactions between multiple binding sites of a molecule (e.g., an antibody) and the same target. Therefore, a necessary condition for affinity is the multivalent nature of the molecule (e.g., an antibody) to a target.

[0100] In this paper, the term "immunoglobulin" refers to a protein with a structure that contains naturally occurring antibodies. For example, IgG immunoglobulins are heterotetrameric glycoproteins of approximately 150,000 Daltons, composed of two light chains and two heavy chains linked by disulfide bonds. Each immunoglobulin heavy chain has a heavy chain variable region (VH), also called a heavy chain variable domain, from the N-terminus to the C-terminus, followed by a heavy chain constant region consisting of three heavy chain constant domains (CH1, CH2, and CH3). Similarly, each immunoglobulin light chain has a light chain variable region (VL), also called a light chain variable domain, from the N-terminus to the C-terminus, followed by a light chain constant region consisting of a light chain constant domain (CL). The heavy chains of immunoglobulins can be classified into one of five categories, referred to as α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), based on the type of their constant regions. Some of these categories can be further subdivided into subclasses, such as γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1), and α2 (IgA2). The light chains of immunoglobulins can also be classified into one of two types, referred to as κ and λ, based on the amino acid sequence of their constant domains. In some embodiments of the antibodies according to the invention having constant regions, the constant regions refer to the constant regions of immunoglobulins or sequence variants thereof.

[0101] In this document, the term "isotype" in relation to antibody type refers to the antibody type determined by the antibody heavy chain constant region. Antibodies according to the invention can be antibodies of the IgA (e.g., IgA1 or IgA2), IgG (e.g., IgG1, IgG2 (e.g., IgG2a or IgG2b), IgG3 or IgG4), IgE, IgM, and IgD isotypes, and have a heavy chain constant region of said immunoglobulin type. It should be understood herein that when referring to an antibody having a certain isotype, this includes not only antibodies having the native sequence constant region of that isotype, but also antibodies with a few mutations introduced into the native constant region sequence of that isotype.

[0102] In this document, the term "variable region" or "variable domain" refers to a domain of the heavy or light chain of an antibody involved in antibody-antigen binding. Typically, the pairing of a heavy chain variable domain (VH) with a light chain variable domain (VL) confers antigen-binding specificity; however, in some cases, a single VH domain (e.g., a single VHH domain from a heavy chain antibody) is sufficient to confer antigen-binding specificity. The heavy chain and light chain variable regions are identical, each containing four conserved framework regions (FRs) and three complementarity-determining regions (CDRs), arranged in the sequence FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. According to some aspects of the invention, one or more residues in the variable region of the antibody can be modified, for example, by modifying one or more CDR regions and / or one or more framework regions, particularly by substituting conserved residues, to obtain antibody variants that still substantially retain at least one biological property (e.g., antigen-binding ability) of the parent antibody. In other aspects, the antibody variable region can be modified by CDR transplantation. Since CDR sequences are responsible for most antibody-antigen interactions, antibody variants that mimic the properties of known antibodies can be constructed. In such antibody variants, CDR sequences from known antibodies are grafted onto the framework regions of different antibodies with different properties, and one to several residues can be mutated as needed, such as reverting mutations, to refine the desired properties of the antibody. In some cases, for the therapeutic application of antibodies or their derivatives, it is desirable to reduce their immunogenicity and improve their drugability. For this purpose, variable domains of antibodies can be engineered to construct humanized, deimmunogenic, and / or PTM (post-translational modification) de-modified variants. The properties of the mutated and / or modified antibodies, such as target antigen binding properties or other desired functional properties, such as T cell activation activity and / or tumor cell killing activity, can be determined and screened in vitro or in vivo using methods known in the art and described herein. It should be understood that such functional variants of any variable regions (e.g., VH and / or VL regions, VHH regions) given herein are within the scope of this invention.

[0103] In this paper, the terms "complementarity-determining region" and "CDR region," "CDR," and "hypervariant region" are used interchangeably. They refer to regions within the variable domain of an antibody that are highly variable in sequence and form structurally defined loops ("hypervariant loops") and / or contain antigen contact residues ("antigen contact sites"). CDRs are primarily responsible for binding to antigen epitopes. In the VH and VL domains, CDRs are sequentially numbered starting from the N-terminus and are typically referred to as HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3, respectively. In the VHH domain, CDRs are sequentially numbered starting from the N-terminus and are typically referred to as CDR1, CDR2, and CDR3, respectively. The CDR sequence within a specific variable region can be determined using schemes known in the art, such as the Kabat, AbM, Chothia, Contact, and IMGT schemes, or any combination thereof, to define the extent of the CDR. Kabat complementarity-determining regions (CDRs) are determined based on sequence variability and are the most commonly used scheme (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The Chothia scheme refers to the location of the structural loop (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). AbM HVR is a compromise between Kabat HVR and Chothia structural loops, used by the AbM antibody modeling software from Oxford Molecular. “Contact” HVR is based on the analysis of the available crystal structure of the complex.

[0104] The following are examples of CDR region ranges defined using the Kabat, AbM, IMGT, and Contact schemes.

[0105] Unless otherwise stated, in this disclosure, the term "CDR" or "CDR sequence" encompasses a CDR sequence determined in any of the foregoing methods and combinations thereof. Furthermore, a CDR may also be determined based on having the same Kabat number position as a reference CDR sequence (e.g., any of the exemplary CDRs of this invention). Moreover, it is understood in the art that although CDRs differ between antibodies, only a limited number of amino acid positions within a CDR are directly involved in antigen binding. Minimal overlapping regions can be determined using at least two of the Kabat, Chothia, AbM, and Contact methods, thereby providing a "minimum binding unit" for antigen binding. Such a minimum binding unit may be a sub-part of a CDR. The remaining residues of the CDR sequence, as will be apparent to those skilled in the art, can be determined by the antibody's structure and protein folding. Therefore, the invention also contemplates any variants of the CDRs given herein. For example, in a variant of a CDR, the amino acid residues of the minimum binding unit may remain unchanged, while the remaining CDR residues may be substituted.

[0106] In this document, unless otherwise stated, references to residue positions in the antibody variable region and CDR refer to their positions according to the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991)). Therefore, in this document, when referring to an amino acid substitution at a specific position in the antibody variable region sequence, the substitution is described as follows: [original amino acid residue / position / substituted amino acid residue]. For example, according to the Kabat numbering system, a serine residue at position 27E in the light chain variable region, if substituted with threonine, can be represented as S27eT; if substituted with valine, it can be represented as S27eV; and if substituted with isoleucine, it can be represented as S27eI. Similarly, according to the Kabat numbering system, amino acid substitution V51I on the variable region of the light chain means that valine at position 51 is replaced with isoleucine, amino acid substitution S52Q means that serine at position 52 is replaced with glutamine, amino acid substitution V55R means that valine at position 55 is replaced with arginine, and amino acid substitution S56I means that serine at position 56 is replaced with isoleucine.

[0107] In this document, the terms "Fab" and "Fab domain" are used interchangeably to refer to a structure similar to that in a conventional four-chain IgG antibody, formed by the pairing of the heavy chain variable region VH and the heavy chain constant region CH1 (VH-CH1) with the complementary light chain variable region VL and the light chain constant region CL (VL-CL). The term also encompasses structures in which CH1 and CL are exchanged, i.e., structures formed by the pairing of VH-CL and VL-CH1. In some embodiments, the Fab domain may be fused to the Fc region of an immunoglobulin, including but not limited to, by fusing a fragment containing VH to the N-terminus of the Fc region of an immunoglobulin; or by fusing a fragment containing VL to the N-terminus of the Fc region of an immunoglobulin.

[0108] In this document, the terms "scFv" and "scFv domain" are used interchangeably to refer to a single-chain polypeptide comprising a VH domain and a VL domain linked together by a flexible linker, wherein the VH domain and the VL domain located on the polypeptide chain pair to form an antigen-binding domain responsible for antigen binding. In some embodiments, the scFv domain may be fused to the Fc region of an immunoglobulin, for example, at the N-terminus or C-terminus of the Fc region.

[0109] In this document, the terms "VHH" and "VHH domain" are used interchangeably to refer to a heavy chain variable domain derived from a heavy chain antibody lacking a light chain, sometimes also called a single variable domain fragment (sVD). Therefore, a VHH differs from the conventional VH of a four-chain immunoglobulin in that it does not require pairing with a light chain variable domain to form an antigen-binding site. Such VHH molecules can be derived from antibodies produced in camelid species (e.g., camels, alpacas, dromedaries, llamas, and guanacos). Other species besides camelids may also produce naturally occurring heavy chain antibodies lacking a light chain, and such VHHs are also within the scope of this invention.

[0110] In this document, the term "half-life extension domain" refers to a chemical structure capable of conferring an increased circulating half-life to a molecule (e.g., an antibody) after administration to an animal. Such chemical structures include, for example, flexible hydrophilic molecules (e.g., carbohydrates or PEG (polyethylene glycol)), immunoglobulin Fc regions, serum albumin, serum albumin-binding domains (e.g., small organic molecules, fatty acids, peptides, and proteins capable of binding to serum albumin), or serum albumin-binding peptides. The half-life extension domain can be linked to the antibodies of the present invention via chemical conjugation or fusion, depending on its specific properties. In some embodiments, preferably, the half-life extension domain used in the anti-CD3 antibody or multispecific antibody such as TCE of the present invention is an immunoglobulin Fc region, but the present invention also contemplates the use of other half-life extension domains such as serum albumin-binding domains.

[0111] In this document, the term "immunoglobulin Fc region" is used interchangeably with "Fc region" and "Fc domain" to define the C-terminal region of the immunoglobulin heavy chain, which comprises at least a portion of the heavy chain constant region. According to this disclosure, "Fc region" or "Fc domain" does not include the heavy chain variable region VH and light chain variable region VL, or the heavy chain constant region CH1 and light chain constant region CL of immunoglobulin; but may include all or part of the immunoglobulin hinge region. The sequence constituting the Fc region can be a native sequence or a variant sequence. Therefore, the term "Fc region" encompasses both native sequence Fc regions and variant Fc regions. In this document, unless otherwise stated, the amino acid residues in the Fc region and the heavy chain 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 Interes, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0112] In this document, the term "natural sequence Fc region" encompasses the Fc region sequences of various naturally occurring immunoglobulins, such as the Fc region sequences of various Ig subtypes and their allotypes (Gestur Vidarsson et al., IgG subclasses and allotypes: from structure to effector functions, 20 October 2014, doi:10.3389 / fimmu.2014.00520.). In some embodiments, the human IgG heavy chain Fc region has an amino acid sequence extending from Cys226 or from Pro230 to the C-terminus of the heavy chain. In other embodiments, the human IgG heavy chain Fc region has an amino acid sequence extending from E216 to the C-terminus of the heavy chain. However, the C-terminal lysine (Lys447) or glycine-lysine (Gly446Lys447) of the Fc region may or may not be present.

[0113] In this document, the term "variant sequence Fc region" refers to a polypeptide containing a modified Fc region relative to the native Fc region sequence. The modification can be the addition, deletion, and / or substitution of amino acid residues. Substitution can include naturally occurring amino acid substitutions and non-naturally occurring amino acid substitutions. The purpose of the modification includes, but is not limited to, altering the binding of the Fc region to its receptor and the resulting effector function, preventing undesirable heavy chain mismatches, or site-directed introduction of amino acid mutations that can be used to alter interchain disulfide bond formation.

[0114] In this document, the term "effective function" refers to those biological activities attributable to the Fc region of immunoglobulins that vary with immunoglobulin isotype. Examples of immunoglobulin effector functions include Fc receptor binding, C1q binding and complement-dependent cytotoxicity (CDC), and antibody-dependent cell-mediated cytotoxicity (ADCC). Depending on the intended use of the antibody molecule, the Fc region of the antibody can be selected and / or modified to give it effector functions appropriate to said use, such as attenuated, reduced, or eliminated Fcγ receptor binding, ADCC activity, and / or CDC activity compared to the wild-type IgG1 Fc region.

[0115] In this paper, the term "co-stimulatory molecule" refers to the related partner of a co-stimulatory ligand, which specifically binds to the co-stimulatory ligand on T cells and thereby mediates a co-stimulatory response of T cells (e.g., but not limited to, T cell proliferation). Co-stimulatory molecules are cell surface molecules other than antigen receptors or their ligands required for an effective immune response. Co-stimulatory molecules include, but are not limited to, MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signal transduction lymphocyte activation molecules (SLAM proteins), NK cell activation receptors, CD8, OX40, CD40, GITR, 4-1BB (i.e., CD137), CD27, and CD28.

[0116] In this document, the terms “flexible linker peptide” or “linker peptide” or “linker peptide” are used interchangeably to refer to a short amino acid sequence consisting of amino acids, such as glycine (G) and / or serine (S) and / or threonine residues (T) used alone or in combination, or from the hinge region of an immunoglobulin.

[0117] In this document, the “percentage of identity (%)” for an amino acid sequence refers to the percentage of positions in the candidate sequence that share the same amino acid residues at the corresponding positions in the aligned specific amino acid sequence shown in this disclosure, after aligning the candidate sequence with the specific amino acid sequence shown in this disclosure and, if necessary, introducing vacancies to achieve the maximum percentage of sequence identity, without considering any conserved substitutions as part of sequence identity. In some embodiments, the invention contemplates variants of the antibody sequences of the invention that have a considerable degree of identity with respect to the antibody sequences specifically disclosed herein within a comparison window, for example, an identity of at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% or higher. In this document, if no comparison window (i.e., the antibody region of interest to be compared) is specified, the alignment is performed over the full length of the reference antibody sequence. In some embodiments, the variants may contain conserved modifications.

[0118] In this document, for a polypeptide sequence, “conserved modification” includes substitutions, deletions, or additions to the polypeptide sequence that do not significantly affect or alter the desired properties of the polypeptide containing the modification (e.g., binding characteristics and / or T cell activation characteristics). Tables of conserved substitutions for functionally similar amino acids are well known in the art. The following eight groups contain amino acids that are conservedly substituted for each other: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine ​​(C), methionine (M) (see, for example, Creighton, Proteins (1984)).

[0119] In this paper, the term "chimeric antibody" refers to an antibody in which one part (e.g., the variable region sequence) comes from one species and another part (e.g., the constant region sequence) comes from another species, such as an antibody in which the variable region sequence comes from a mouse antibody and the constant region sequence comes from a human antibody.

[0120] In this document, a “humanized” antibody refers to a chimeric antibody comprising amino acid residues from nonhuman CDRs and amino acid residues from human FRs. In some embodiments, all or substantially all of the CDRs (e.g., CDRs) in a humanized antibody correspond to those in nonhuman antibodies, and all or substantially all of the FRs correspond to those in human antibodies. A humanized antibody may optionally contain at least a portion of an antibody constant region derived from a human antibody. The “humanized form” of an antibody (e.g., a nonhuman antibody) refers to an antibody that has been humanized. In some embodiments, the humanized antibody of the present invention has a framework region sequence “derived” from a specific human lineage sequence. Here, “derived” means that the amino acid sequence of the antibody framework region has at least 85% or 90% identity with the corresponding framework region amino acid sequence encoded by the human lineage immunoglobulin gene, and that the antibody retains antigen-binding activity.

[0121] In this document, an antibody is described as "cross-reactive" to two different antigens or antigenic determinants if its amino acid sequence is specific to two different antigens or antigenic determinants (e.g., CD3 from different mammalian species, such as human CD3 and cynomolgus monkey CD3). Antibodies exhibiting human-monkey species cross-reactivity, particularly having similar human-monkey antigen-binding affinities, is advantageous, as this property can facilitate preclinical drug development of the antibody, such as toxicological assays of antigen-binding molecules composed of antibodies. In some embodiments, the antibodies of the present invention preferably exhibit human-monkey species cross-reactivity.

[0122] In this document, "isolated" antibody refers to artificial antibodies, recombinant antibodies, and antibodies that have been at least partially separated from components in the natural environment in which they originated. In some embodiments, the antibodies of the present invention are "isolated" antibodies. In some embodiments, the isolated antibodies are purified to a purity of more than 90%, 95%, or 99%, as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC).

[0123] In this document, the term "endocytosis" refers to the process by which a ligand / receptor complex is internalized and delivered into the cytosol or translocated to a suitable intracellular compartment, triggered by the binding of a ligand to a corresponding receptor on the cell surface. In some embodiments, the anti-CD20 antibody of the present invention initiates only weak endocytosis mediated by the CD20 receptor upon binding to CD20 expressed on the cell surface. In this document, endocytosis and endocytosis rate can be determined, for example, by the methods described in the examples, to characterize the endocytic activity of the antibody.

[0124] In this paper, the term "heavy-chain antibody (hcAb)" refers to an antibody that does not have a light chain and may contain VHH-CH2-CH3 or VHH-CH1-CH2-CH3 from the N-terminus to the C-terminus; it can form a homodimer, such as a heavy-chain dimer antibody that does not have a light chain.

[0125] In this document, the term "host cell" refers to a cell into which exogenous polynucleotides have been introduced, including progeny cells of this type. Host cells include "transformers" and "transformed cells," which include primary transformed cells and their derived progeny. Host cells can be any type of cell system that can be used to produce the antibody molecules of this invention, including eukaryotic cells, such as mammalian cells, insect cells, and yeast cells; and prokaryotic cells, such as *E. coli* cells. Host cells include cultured cells, as well as cells within transgenic animals, transgenic plants, or cultured plant or animal tissues.

[0126] In this document, the term "expression vector" refers to a vector capable of directing the expression of a nucleotide sequence operatively linked thereto. Expression vectors typically contain a cis-acting element for the expression of said nucleotide sequence; other elements for expression may be provided by a host cell or by an in vitro expression system. Expression vectors include, for example, but not limited to, entrapments, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses).

[0127] In this document, the terms "immunoconjugate" or "immunofusion" generally refer to molecules formed by conjugating or fusing one or more immunoglobulin-related molecules or fragments thereof (e.g., antibodies or fragments thereof) with one or more other molecules. These other molecules may be protein-like molecules, such as peptides, polypeptides, or proteins; or non-protein-like molecules, such as chemical toxins. In cases involving multiple other molecules, these molecules may be the same or different from each other.

[0128] In this document, the terms “individual” or “subject” are used interchangeably and refer to mammals. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., human and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, an individual specifically refers to a human individual.

[0129] In this article, the term "treatment" refers to a clinical intervention intended to alter the natural course of a disease in an individual receiving treatment. Desired therapeutic effects include, but are not limited to, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or mitigating the disease state, and alleviating or improving prognosis. In cases involving tumor or cancer treatment, "treatment" encompasses antitumor biological effects that can be induced by artificial intervention (e.g., through the administration of drugs), including but not limited to, reductions in tumor volume, number of tumor cells, proliferation, or survival.

[0130] In this document, the term "prevention" refers to a medical intervention implemented before the onset of at least one symptom of a disease to suppress, delay, or prevent the occurrence or development of the disease or specific disease symptoms. Therefore, in some implementations, prevention includes administering a drug to a subject before the onset of the disease or symptoms.

[0131] In this document, the terms “cancer” and “tumor” are used interchangeably to refer to or describe a physiological disorder in mammals characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinomas, solid tumors, and liquid tumors. In some embodiments, cancers suitable for treatment by the antibodies or immune conjugates or immune fusions of the present invention include CD20-positive and / or MSLN-positive tumors / cancers, including their metastatic forms.

[0132] The present invention will now be described in detail. Those skilled in the art will understand that, unless the context clearly indicates otherwise, any technical feature described in any of the following sections, subsections, or embodiments may be combined with any technical feature described in any other section, subsection, or embodiment, and such combinations are all within the scope of this invention.

[0133] I. Anti-CD3 antibody of the present invention

[0134] A first aspect of the present invention provides an antibody that specifically binds to CD3, preferably human CD3 protein, or an antigen-binding fragment thereof. The anti-CD3 antibody of the present invention is a T-cell activating antibody. In some embodiments, the antigen-binding fragment of the antibody of the present invention is selected from antibody fragments including: Fab, Fab', Fab'-SH, Fv, single-chain antibodies such as scFv and scFab, (Fab')2 fragments, and linear antibodies.

[0135] Antibody CDR region

[0136] The CDR region is the amino acid region in the antibody variable region that is primarily responsible for binding to the antigen epitope. Some exemplary VH and VL sequence combinations of the anti-CD3 antibodies of this invention are given in Table A below:

[0137] Table A

[0138] In some embodiments, the antibodies of the present invention comprise the HCDR1, HCDR2, and HCDR3 sequences contained in the VH of any antibody shown in Table A and the LCDR1, LCDR2, and LCDR3 sequences contained in the VL. In some embodiments, the present invention also contemplates including humanized, deimmunized, and / or PTM-removed antibodies in the said CDR sequences.

[0139] In some embodiments, the present invention provides an anti-CD3 antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment comprises: three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) contained in a heavy chain variable region (VH) sequence selected from SEQ ID NO:31 and 67-69, and three light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) contained in a light chain variable region (VL) sequence selected from SEQ ID NO:32 and 70-71. In some embodiments, the antibody or the antigen-binding fragment thereof optionally comprises the following amino acid substitutions: (a) amino acid substitutions selected from S27eT, S27eV, and S27eI in LCDR1, (b) amino acid substitutions selected from V51I, S52Q, V55R, and S56I in LCDR2, or a combination of (a) and (b), wherein the variable region amino acid residues are numbered according to the Kabat numbering system.

[0140] In some embodiments, the present invention provides an anti-CD3 antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment comprises: three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) contained in the heavy chain variable region (VH) sequence of SEQ ID NO:31, and three light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) contained in the light chain variable region (VL) sequences selected from SEQ ID NO:32 and 72-74. In some embodiments, the antibody or antigen-binding fragment thereof optionally comprises the following amino acid substitutions: (a) amino acid substitutions selected from S27eT, S27eV, and S27eI in LCDR1, (b) amino acid substitutions selected from V51I, S52Q, V55R, and S56I in LCDR2, or a combination of (a) and (b), wherein the variable region amino acid residues are numbered according to the Kabat numbering system.

[0141] In some embodiments, the present invention provides an anti-CD3 antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment comprises: three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) contained in a heavy chain variable region (VH) sequence selected from SEQ ID NO: 68, and three light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) contained in a light chain variable region (VL) sequence selected from SEQ ID NO: 70-74, 78-82, and 87. In some embodiments, the antibody or the antigen-binding fragment thereof optionally comprises the following amino acid substitutions: (a) amino acid substitutions selected from S27eT, S27eV, and S27eI in LCDR1, (b) amino acid substitutions selected from V51I, S52Q, V55R, and S56I in LCDR2, or a combination of (a) and (b), wherein the variable region amino acid residues are numbered according to the Kabat numbering system.

[0142] In some preferred embodiments, the present invention provides an anti-CD3 antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment comprises HCDR1-3 and LCDR1-3 contained in the following VH and VL sequence pairs:

[0143] (a) The VH sequence of SEQ ID NO:31 and the VL sequence of one of SEQ ID NOs:32 and 72-74;

[0144] (b) The VH sequence of one of SEQ ID NO: 67-69 and the VL sequence of one of SEQ ID NOs: 70-71;

[0145] (c) The VH sequence of SEQ ID NO:68 and the VL sequence of one of SEQ ID NOs:78-82; or

[0146] (d) The VH sequence of SEQ ID NO:68 and the VL sequence of SEQ ID NO:87.

[0147] In other embodiments, the present invention provides an anti-CD3 antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment comprises HCDR1-3 contained in the VH sequence of SEQ ID NO:47 and LCDR1-3 contained in the VL sequence of SEQ ID NO:48.

[0148] In other embodiments, the present invention provides an anti-CD3 antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment comprises HCDR1-3 contained in the VH sequence of SEQ ID NO:7 and LCDR1-3 contained in the VL sequence of SEQ ID NO:8. In other embodiments, the present invention provides an anti-CD3 antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment comprises HCDR1-3 contained in the VH sequence of SEQ ID NO:15 and LCDR1-3 contained in the VL sequence of SEQ ID NO:16. In other embodiments, the present invention provides an anti-CD3 antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment comprises HCDR1-3 contained in the VH sequence of SEQ ID NO:23 and LCDR1-3 contained in the VL sequence of SEQ ID NO:24. In other embodiments, the present invention provides an anti-CD3 antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment comprises HCDR1-3 contained in the VH sequence of SEQ ID NO:39 and LCDR1-3 contained in the VL sequence of SEQ ID NO:40.

[0149] The CDR according to the present invention can be determined using any CDR definition scheme known in the art. In some embodiments, it is defined according to AbM, Chothia, Kabat, IMGT, or any combination thereof. In other embodiments, preferably, the CDR according to the present invention is defined according to Kabat or AbM, or a combination thereof, and more preferably, according to AbM.

[0150] Table B below provides some exemplary CDR sequence combinations of the present invention:

[0151] Table C below provides some other exemplary CDR sequence combinations of the present invention:

[0152] In some embodiments, the anti-CD3 antibody of the present invention or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the antibody comprises:

[0153] (i) The six CDR sequences contained in the VH and VL sequences of any antibody listed in Table A; or

[0154] (ii) Any combination listed in Table B containing the six CDR sequences; or

[0155] (iii) The six CDR sequences contained in any combination listed in Table C.

[0156] In some preferred embodiments, the antibody or antigen-binding fragment of the present invention comprises:

[0157] (i) HCDR1-3 and LCDR1-3, respectively, comprising the amino acid sequences of SEQ ID NOs:25-27 and SEQ ID NOs:28-30;

[0158] (ii) HCDR1-3, which respectively contain the amino acid sequences of SEQ ID NOs:25-27 or composed thereof, LCDR1, which contains the amino acid sequences of SEQ ID NOs:75, 76 or 77 or composed thereof, and LCDR2-3, which respectively contain the amino acid sequences of SEQ ID NOs:29-30 or composed thereof.

[0159] (iii) HCDR1-3 comprising or consisting of the amino acid sequences of SEQ ID NOs:25-27, respectively; LCDR1 and LCDR3 comprising or consisting of the amino acid sequences of SEQ ID NOs:28 and 30, respectively; and LCDR2 comprising or consisting of the amino acid sequences of SEQ ID NOs:83, 84, 85, or 86, respectively; or

[0160] (iv) HCDR1-3 and LCDR1-3, respectively, comprising the amino acid sequences of SEQ ID NOs:25-27 and SEQ ID NOs:75, 86 and 30.

[0161] In a preferred embodiment, the antibody or antigen-binding fragment of the present invention comprises three complementarity-determining regions (HCDRs) of the heavy chain variable region and three complementarity-determining regions (LCDRs) of the light chain variable region, wherein,

[0162] -HCDR1 contains or consists of the amino acid sequence of SEQ ID NO:25.

[0163] -HCDR2 contains or consists of the amino acid sequence of SEQ ID NO:26.

[0164] -HCDR3 contains or consists of the amino acid sequence of SEQ ID NO:27.

[0165] -LCDR1 contains or consists of the amino acid sequence of SEQ ID NO:28.

[0166] -LCDR2 contains or consists of the amino acid sequence of SEQ ID NO:29, and

[0167] -LCDR3 contains or consists of the amino acid sequence of SEQ ID NO:30.

[0168] In a preferred embodiment, the antibody or antigen-binding fragment of the present invention comprises three complementarity-determining regions (HCDRs) of the heavy chain variable region and three complementarity-determining regions (LCDRs) of the light chain variable region, wherein,

[0169] -HCDR1 contains or consists of the amino acid sequence of SEQ ID NO:25.

[0170] -HCDR2 contains or consists of the amino acid sequence of SEQ ID NO:26.

[0171] -HCDR3 contains or consists of the amino acid sequence of SEQ ID NO:27.

[0172] -LCDR1 contains or consists of the amino acid sequence of SEQ ID NO:75.

[0173] -LCDR2 contains or consists of the amino acid sequence of SEQ ID NO:29, and

[0174] -LCDR3 contains or consists of the amino acid sequence of SEQ ID NO:30.

[0175] In a preferred embodiment, the antibody or antigen-binding fragment of the present invention comprises three complementarity-determining regions (HCDRs) of the heavy chain variable region and three complementarity-determining regions (LCDRs) of the light chain variable region, wherein,

[0176] -HCDR1 contains or consists of the amino acid sequence of SEQ ID NO:25.

[0177] -HCDR2 contains or consists of the amino acid sequence of SEQ ID NO:26.

[0178] -HCDR3 contains or consists of the amino acid sequence of SEQ ID NO:27.

[0179] -LCDR1 contains or consists of the amino acid sequence of SEQ ID NO:28.

[0180] -LCDR2 contains or consists of the amino acid sequence of SEQ ID NO:86, and

[0181] -LCDR3 contains or consists of the amino acid sequence of SEQ ID NO:30.

[0182] In a preferred embodiment, the antibody or antigen-binding fragment of the present invention comprises three complementarity-determining regions (HCDRs) of the heavy chain variable region and three complementarity-determining regions (LCDRs) of the light chain variable region, wherein,

[0183] -HCDR1 contains or consists of the amino acid sequence of SEQ ID NO:25.

[0184] -HCDR2 contains or consists of the amino acid sequence of SEQ ID NO:26.

[0185] -HCDR3 contains or consists of the amino acid sequence of SEQ ID NO:27.

[0186] -LCDR1 contains or consists of the amino acid sequence of SEQ ID NO:75.

[0187] -LCDR2 contains or consists of the amino acid sequence of SEQ ID NO:86, and

[0188] -LCDR3 contains or consists of the amino acid sequence of SEQ ID NO:30.

[0189] Antibody variable region

[0190] The variable region of an antibody consists of a CDR and a frame region. Since the antigen-binding properties of an antibody are primarily governed by the CDR sequence, different frame regions can be selected based on the CDR region according to the invention to construct various anti-CD3 antibodies of the present invention that bind CD3 with equivalent effectiveness. Therefore, in one embodiment, the present invention relates to an anti-CD3 antibody or its antigen-binding fragment comprising CDR sequences from the heavy and light chain variable regions of one of the antibodies shown in Table A, but with different frame region sequences. The frame region sequence used for this replacement can be obtained from a public DNA database, such as a germline DNA database of human heavy and light chain variable region genes. Sequence similarity search tools (e.g., Gapped BLAST) can be used to compare the antibody protein sequence with protein sequences in the database to find suitable frame candidates. Preferably, the frame sequence used for replacement has sequence identity with the original antibody's frame sequence, for example, at least 80%, 85%, or 90% sequence identity, or more preferably more than 95%, 96%, 97%, 98%, or 99% sequence identity.

[0191] In some embodiments, the present invention therefore provides an anti-CD3 antibody or an antigen-binding fragment thereof, wherein the antibody comprises a heavy chain variable region comprising or consisting of the VH sequence of any antibody listed in Table A or a variant thereof. In some embodiments, the present invention provides an anti-CD3 antibody or an antigen-binding fragment thereof, wherein the antibody comprises a light chain variable region comprising or consisting of the VL sequence of any antibody listed in Table A or a variant thereof. In one embodiment, the variant VH / VL sequence has at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher of amino acid sequence identity compared to a reference VH / VL sequence. In one embodiment, the variant VH / VL sequence comprises at least 30, 10 or 5, 4, 3, 2 amino acid modifications (preferably amino acid substitutions, preferably conserved substitutions) in the amino acid sequence compared to a reference VH / VL sequence. Preferably, the sequence modification does not occur in the CDR region.

[0192] In some embodiments, the anti-CD3 antibody of the present invention comprises a heavy chain variable region, wherein the heavy chain variable region (VH) comprises or is composed of a sequence selected from the amino acid sequence shown in SEQ ID NO:7, or a sequence having at least 80%, 85%, or 90%, or preferably at least 95%, 96%, 97%, 98%, or 99% sequence identity therewith. In some embodiments, the anti-CD3 antibody of the present invention comprises a light chain variable region, wherein the light chain variable region (VL) comprises or is composed of a sequence selected from the amino acid sequence shown in SEQ ID NO:8, or a sequence having at least 80%, 85%, or 90%, or preferably at least 95%, 96%, 97%, 98%, or 99% sequence identity therewith. In some embodiments, the anti-CD3 antibody of the present invention comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region (VH) comprises or is composed of the amino acid sequence shown in SEQ ID NO:7; and the light chain variable region (VL) comprises or is composed of the amino acid sequence shown in SEQ ID NO:8.

[0193] In some embodiments, the anti-CD3 antibody of the present invention comprises a heavy chain variable region, wherein the heavy chain variable region (VH) comprises or is composed of a sequence selected from the amino acid sequence shown in SEQ ID NO:15, or a sequence having at least 80%, 85%, or 90%, or preferably at least 95%, 96%, 97%, 98%, or 99% sequence identity therewith. In some embodiments, the anti-CD3 antibody of the present invention comprises a light chain variable region, wherein the light chain variable region (VL) comprises or is composed of a sequence selected from the amino acid sequence shown in SEQ ID NO:16, or a sequence having at least 80%, 85%, or 90%, or preferably at least 95%, 96%, 97%, 98%, or 99% sequence identity therewith. In some embodiments, the anti-CD3 antibody of the present invention comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region (VH) comprises or is composed of the amino acid sequence shown in SEQ ID NO:15; and the light chain variable region (VL) comprises or is composed of the amino acid sequence shown in SEQ ID NO:16.

[0194] In some embodiments, the anti-CD3 antibody of the present invention comprises a heavy chain variable region, wherein the heavy chain variable region (VH) comprises or is composed of the amino acid sequence shown in SEQ ID NO:23, or a sequence having at least 80%, 85%, or 90%, or preferably at least 95%, 96%, 97%, 98%, or 99% sequence identity therewith. In some embodiments, the anti-CD3 antibody of the present invention comprises a light chain variable region, wherein the light chain variable region (VL) comprises or is composed of the amino acid sequence shown in SEQ ID NO:24, or a sequence having at least 80%, 85%, or 90%, or preferably at least 95%, 96%, 97%, 98%, or 99% sequence identity therewith. In some embodiments, the anti-CD3 antibody of the present invention comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region (VH) comprises or is composed of the amino acid sequence shown in SEQ ID NO:23; and the light chain variable region (VL) comprises or is composed of the amino acid sequence shown in SEQ ID NO:24.

[0195] In some embodiments, the anti-CD3 antibody of the present invention comprises a heavy chain variable region, wherein the heavy chain variable region (VH) comprises or is composed of the amino acid sequence shown in SEQ ID NO:39, or a sequence having at least 80%, 85%, or 90%, or preferably at least 95%, 96%, 97%, 98%, or 99% sequence identity therewith. In some embodiments, the anti-CD3 antibody of the present invention comprises a light chain variable region, wherein the light chain variable region (VL) comprises or is composed of the amino acid sequence shown in SEQ ID NO:40, or a sequence having at least 80%, 85%, or 90%, or preferably at least 95%, 96%, 97%, 98%, or 99% sequence identity therewith. In some embodiments, the anti-CD3 antibody of the present invention comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region (VH) comprises or is composed of the amino acid sequence shown in SEQ ID NO:39; and the light chain variable region (VL) comprises or is composed of the amino acid sequence shown in SEQ ID NO:40.

[0196] In some embodiments, the anti-CD3 antibody of the present invention comprises a heavy chain variable region, wherein the heavy chain variable region (VH) comprises or is composed of the amino acid sequence shown in SEQ ID NO:47, or a sequence having at least 80%, 85%, or 90%, or preferably at least 95%, 96%, 97%, 98%, or 99% sequence identity therewith. In some embodiments, the anti-CD3 antibody of the present invention comprises a light chain variable region, wherein the light chain variable region (VL) comprises or is composed of the amino acid sequence shown in SEQ ID NO:48, or a sequence having at least 80%, 85%, or 90%, or preferably at least 95%, 96%, 97%, 98%, or 99% sequence identity therewith. In some embodiments, the anti-CD3 antibody of the present invention comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region (VH) comprises or is composed of the amino acid sequence shown in SEQ ID NO:47; and the light chain variable region (VL) comprises or is composed of the amino acid sequence shown in SEQ ID NO:48.

[0197] In some embodiments, the anti-CD3 antibody of the present invention comprises a heavy chain variable region, wherein the heavy chain variable region (VH) comprises or consists of the following sequence: an amino acid sequence shown in one of SEQ ID NO: 31 and 67-69, or a sequence having at least 80%, 85%, or 90%, or preferably at least 95%, 96%, 97%, 98%, or 99% sequence identity therewith. In some embodiments, the anti-CD3 antibody of the present invention comprises a light chain variable region, wherein the light chain variable region (VL) comprises or consists of the following sequence: an amino acid sequence shown in one of SEQ ID NO: 32, 70-74, 78-82, and 87, or a sequence having at least 80%, 85%, or 90%, or preferably at least 95%, 96%, 97%, 98%, or 99% sequence identity therewith. In some preferred embodiments, the anti-CD3 antibody of the present invention comprises a heavy chain variable region (VH), wherein the heavy chain variable region (VH) comprises or consists of the following sequence: the amino acid sequence shown in SEQ ID NO:31, or a sequence having at least 80%, 85%, or 90%, or preferably at least 95%, 96%, 97%, 98%, or 99% sequence identity therewith; and a light chain variable region (VL), wherein the light chain variable region (VL) comprises or consists of the following sequence: the amino acid sequence shown in one of SEQ ID NO:32 and 72-74, or a sequence having at least 80%, 85%, or 90%, or preferably at least 95%, 96%, 97%, 98%, or 99% sequence identity therewith. In some other preferred embodiments, the anti-CD3 antibody of the present invention comprises a heavy chain variable region (VH), wherein the heavy chain variable region (VH) comprises or consists of the following sequence: the amino acid sequence shown in SEQ ID NO: 67 or 69, or a sequence having at least 80%, 85%, or 90%, or preferably at least 95%, 96%, 97%, 98%, or 99% sequence identity therewith; and a light chain variable region (VL), wherein the light chain variable region (VL) comprises or consists of the following sequence: the amino acid sequence shown in SEQ ID NO: 70 or 71, or a sequence having at least 80%, 85%, or 90%, or preferably at least 95%, 96%, 97%, 98%, or 99% sequence identity therewith. In some other preferred embodiments, the anti-CD3 antibody of the present invention comprises a heavy chain variable region (VH), wherein the heavy chain variable region (VH) comprises or consists of the following sequence: the amino acid sequence shown in SEQ ID NO:68, or a sequence having at least 80%, 85%, or 90%, or preferably at least 95%, 96%, 97%, 98%, or 99% sequence identity therewith; and a light chain variable region (VL), wherein the light chain variable region (VL) comprises or consists of the following sequence: the amino acid sequence shown in one of SEQ ID NO:70-71, 78-82, and 87, or a sequence having at least 80%, 85%, or 90%, or preferably at least 95%, 96%, 97%, 98%, or 99% sequence identity therewith.

[0198] In some preferred embodiments, the present invention provides an anti-CD3 antibody or an antigen-binding fragment thereof comprising: a VH containing or consisting of the amino acid sequence shown in SEQ ID NO:31, and a VL containing or consisting of the amino acid sequence shown in SEQ ID NO:32.

[0199] In some preferred embodiments, the present invention provides an anti-CD3 antibody or an antigen-binding fragment thereof comprising: a VH containing or consisting of the amino acid sequence shown in SEQ ID NO:31, and a VL containing or consisting of the amino acid sequence shown in SEQ ID NO:72, 73 or 74.

[0200] In some preferred embodiments, the present invention provides an anti-CD3 antibody or an antigen-binding fragment thereof comprising: a VH containing or consisting of the amino acid sequence shown in SEQ ID NO: 67 or 69, and a VL containing or consisting of the amino acid sequence shown in SEQ ID NO: 70 or 71.

[0201] In some preferred embodiments, the present invention provides an anti-CD3 antibody or an antigen-binding fragment thereof comprising: a VH containing or consisting of the amino acid sequence shown in SEQ ID NO:68, and a VL containing or consisting of the amino acid sequence shown in SEQ ID NO:70 or 71.

[0202] In some preferred embodiments, the present invention provides an anti-CD3 antibody or an antigen-binding fragment thereof comprising: a VH containing or consisting of the amino acid sequence shown in SEQ ID NO:68, and a VL containing or consisting of the amino acid sequence shown in one of SEQ ID NO:78-82.

[0203] In some preferred embodiments, the present invention provides an anti-CD3 antibody or an antigen-binding fragment thereof comprising: a VH containing or consisting of the amino acid sequence shown in SEQ ID NO:68, and a VL containing or consisting of the amino acid sequence shown in SEQ ID NO:82.

[0204] In some preferred embodiments, the present invention provides an anti-CD3 antibody or an antigen-binding fragment thereof comprising: a VH containing or consisting of the amino acid sequence shown in SEQ ID NO:68, and a VL containing or consisting of the amino acid sequence shown in SEQ ID NO:87.

[0205] Antibody heavy chain and light chain

[0206] In some embodiments, the anti-CD3 antibody of the present invention comprises a heavy chain constant region, such as the Fc region of an IgG1, IgG2, IgG3, or IgG4 isotype. In some embodiments, the antibody of the present invention comprises a κ or λ light chain constant region, such as the human κ light chain constant region.

[0207] In some preferred embodiments, the anti-CD3 antibody of the present invention comprises an Fc region containing the amino acid sequence of SEQ ID NO:111, or an amino acid sequence containing at least 1, 2, or 3, but no more than 20, 10, or 5 amino acid modifications relative to the amino acid sequence of SEQ ID NO:111, or a sequence having at least 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:111. In some embodiments, the Fc region contains a mutation that reduces or eliminates the binding interaction between the Fc region and FcγR, for example, the L234AL235A mutation.

[0208] In some preferred embodiments, the anti-CD3 antibody of the present invention comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO:61, or an amino acid sequence comprising at least 1, 2, or 3, but no more than 20, 10, or 5 amino acid modifications relative to the amino acid sequence of SEQ ID NO:61, or a sequence having at least 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:61.

[0209] In some preferred embodiments, the anti-CD3 antibody of the present invention comprises a light chain constant region. In one preferred embodiment, the light chain constant region is a human κ light chain constant region. In a further preferred embodiment, the light chain constant region comprises the amino acid sequence of SEQ ID NO:62, or an amino acid sequence comprising at least 1, 2, or 3, but no more than 20, 10, or 5 amino acid modifications relative to the amino acid sequence of SEQ ID NO:62, or a sequence having at least 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:62.

[0210] In some embodiments, the anti-CD3 antibody of the present invention comprises a heavy chain, wherein the heavy chain comprises an amino acid sequence selected from SEQ ID NOs:49, 51, 53, 55, 57, and 59. In other embodiments, the anti-CD3 antibody of the present invention comprises a light chain, wherein the light chain comprises an amino acid sequence selected from SEQ ID NOs:50, 52, 54, 56, 58, and 60. In some embodiments, the anti-CD3 antibody of the present invention comprises a heavy chain / light chain pair selected from the following: heavy chain / light chain pairs of the amino acid sequences shown in SEQ ID NOs:49 / 50, 51 / 52, 53 / 54, 55 / 56, 57 / 58, and 59 / 60. In some embodiments, the anti-CD3 antibody according to the present invention is a full-length IgG1 antibody.

[0211] In some embodiments, the anti-CD3 antibody of the present invention comprises a heavy chain, wherein the heavy chain comprises a heavy chain variable region selected from SEQ ID NOs:31 and 67-69 and an immunoglobulin IgG heavy chain constant region. In some embodiments, the anti-CD3 antibody of the present invention comprises a light chain, wherein the light chain comprises a light chain variable region selected from SEQ ID NOs:32, 70-74, 78-82 and 87 and an immunoglobulin light chain constant region. In some embodiments, the anti-CD3 antibody of the present invention is a full-length antibody comprising the heavy chain and the light chain. In some embodiments, the anti-CD3 antibody of the present invention is a full-length IgG1 antibody. In some embodiments, the full-length anti-CD3 antibody of the present invention comprises the heavy chain constant region of SEQ ID NO:61 and the light chain constant region of SEQ ID NO:62.

[0212] Properties of the anti-CD3 antibody of this invention

[0213] In some embodiments, the anti-CD3 antibody or antigen-binding fragment of the present invention has one or more or all of the following features:

[0214] (a) It specifically binds to human CD3 antigen;

[0215] (b) It exhibits cross-reactivity with human and monkey CD3 antigens;

[0216] (c) It exhibits essentially no nonspecific binding to CD3 antigen-negative cells; and

[0217] (d) Activation of CD4+ and CD8+ T cells.

[0218] In some embodiments, the anti-CD3 antibody or antigen-binding fragment of the present invention specifically binds to the human CD3 antigen. In some embodiments, the binding affinity K of the anti-CD3 antibody of the present invention is measured, as by in vitro surface plasmon resonance (SPR) binding assay. DThe value is approximately 10x10 -7 M to approximately 1x10 -8 M, preferably approximately 5x10 -7 M to approximately 5x10 -8 M. Preferably, the binding affinity K D The values ​​were measured as described in Example 3.7.

[0219] In some embodiments, the anti-CD3 antibody or antigen-binding fragment of the present invention exhibits immunoreactivity with human and monkey CD3 antigens. In some embodiments, the binding affinity K to human and monkey CD3 antigens is measured, as indicated by in vitro surface plasmon resonance (SPR) binding assay. D The values ​​differ by no more than approximately 5 times, preferably no more than approximately 3 times. Preferably, the binding affinity K... D The values ​​were measured as described in Example 3.7.

[0220] In some embodiments, the anti-CD3 antibody of the present invention specifically binds to surface human CD3 antigen-positive T cells. In some embodiments, the EC50 of the positive T cells when the anti-CD3 antibody of the present invention binds in a bivalent form is determined by flow cytometry on Jurkat cells. 50 The value is approximately 0.01-10 nM, preferably 0.1-5 nM. In some embodiments, the anti-CD3 antibody of the present invention has lower cell-binding activity compared to the BMK8 reference positive antibody, preferably with a bivalent cell-binding EC50 value approximately 5-50 times or 10-30 times that of the reference antibody. Preferably, the assay is performed according to Example 2.2. In this document, "BMK8 reference positive antibody" refers to a reference antibody having the same antibody structure as the compared anti-CD3 antibody of the present invention, except for differences in VH and VL, wherein the VH and VL of the reference antibody comprise the amino acid sequences of SEQ ID NO: 63 and 64, respectively.

[0221] In some embodiments, the anti-CD3 antibody of the present invention exhibits substantially no nonspecific binding to CD3 antigen-negative cells. Preferably, the binding on CD3 knockout Jurkat cells is measured by flow cytometry, for example, as described in Example 2.2.

[0222] In some embodiments, the anti-CD3 antibody of the present invention specifically binds to surface cynomolgus monkey CD3 antigen-positive T cells. In some embodiments, flow cytometry analysis on cynomolgus monkey CD4+ or CD8+ T cells shows that when the anti-CD3 antibody of the present invention binds to said T cells in a bivalent form, its EC50 is significantly increased. 50The value is approximately 0.1-20 nM, preferably 1-10 nM. In some embodiments, in the assay, the antibody according to the invention exhibits lower cynomolgus monkey T-cell binding activity compared to the BMK8 reference positive antibody, preferably with an EC50 value approximately 5-50 times or 10-30 times that of the reference antibody. Preferably, the assay is performed according to Example 2.3.

[0223] In some embodiments, the anti-CD3 antibody of the present invention activates CD4+ and CD8+ T cells. In some embodiments, by a T cell activation assay, the bivalent form of the anti-CD3 antibody of the present invention has an intermediate T cell activation function compared to BMK8 and BMK9 reference positive antibodies. In some embodiments, the T cell activation assay is performed by detecting an increase in the percentage of antibody-induced CD25-positive CD4+ and CD8+ T cells. Preferably, the assay is performed according to Example 2.4.

[0224] II. Anti-CD20 antibody of the present invention

[0225] In a second aspect, the present invention provides an anti-CD20 antibody that binds to CD20. In some embodiments, the anti-CD20 antibody according to the present invention comprises a VHH domain. In some embodiments, the anti-CD20 VHH domain according to the present invention comprises CDR1, CDR2, and CDR3 sequences in a variable region having amino acid sequences selected from those shown in SEQ ID NOs:102 and 115-124. Preferably, the CDRs are defined according to AbM, Chothia, Kabat, IMGT, or any combination thereof. More preferably, the CDRs are defined according to Kabat or AbM, or combinations thereof, and more preferably, the CDRs are defined according to AbM. However, it should be understood that the CDRs may also be defined in any other manner known in the art. In some embodiments, the CD20 VHH domain according to the present invention comprises complementarity-determining regions CDR1, CDR2, and CDR3, wherein:

[0226] (i) The CDR1 contains or is composed of the amino acid sequence of SEQ ID NO: 103 or 137;

[0227] (ii) The CDR2 comprises or is composed of the amino acid sequence of SEQ ID NO:104;

[0228] (iii) The CDR3 contains or is composed of the amino acid sequence of SEQ ID NO:105.

[0229] Since the antigen-binding properties of antibodies are mainly responsible for the CDR sequence, different framework regions can be selected based on the CDR region of the present invention to construct a variety of anti-CD20 VHH domains of the present invention and anti-CD20 antibodies containing them that are equally effective at binding CD20. These anti-CD20 VHH domains and anti-CD20 antibodies are all within the scope of the present invention.

[0230] Accordingly, in one embodiment, the present invention provides an anti-CD20 VHH domain and an anti-CD20 antibody or antigen-binding fragment comprising therein, wherein the VHH domain comprises three CDR sequences from one of SEQ ID NOs:102 and 115-124, but with a different frame region sequence. The replacement frame sequence preferably has a certain sequence identity in the frame region with one of SEQ ID NOs:102 and 115-124, for example, at least 80%, 85%, or 90% sequence identity, or more preferably at least 95%, 96%, 97%, 98%, or 99% sequence identity. In some embodiments, the VHH domain is humanized.

[0231] In some specific embodiments, the present invention provides an anti-CD20 VHH domain and an anti-CD20 antibody or antigen-binding fragment comprising thereof, wherein the VHH domain comprises a variable region sequence having an amino acid sequence selected from SEQ ID Nos: 102 and 115-124. In still other embodiments, the anti-CD20 VHH domain according to the present invention comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% identity with an amino acid sequence selected from SEQ ID Nos: 102 and 115-124 and retaining the ability to specifically bind CD20. In still other embodiments, the anti-CD20 VHH domain according to the present invention comprises an amino acid sequence having one or more (preferably 1-10, more preferably 1-5) amino acid additions, deletions, and / or substitutions (e.g., conserved substitutions) compared to an amino acid sequence selected from SEQ ID Nos: 102 and 115-124 and retaining the ability to specifically bind CD20. Preferably, the addition, deletion, and / or substitution of the amino acids does not occur in the CDR region.

[0232] In some preferred embodiments, the present invention provides an anti-CD20 VHH domain and an anti-CD20 antibody or antigen-binding fragment comprising thereof, wherein the VHH domain comprises the CDR1, CDR2, and CDR3 sequences of the amino acid sequence of SEQ ID NO:102. In some embodiments, the CDRs are defined according to AbM, Chothia, Kabat, IMGT, or any combination thereof. In other embodiments, preferably, the CDRs are defined according to AbM. In some embodiments, CDR1 comprises or is composed of the amino acid sequence of SEQ ID NO:103; CDR2 comprises or is composed of the amino acid sequence of SEQ ID NO:104; and CDR3 comprises or is composed of the amino acid sequence of SEQ ID NO:105. In some embodiments, the VHH domain comprises the amino acid sequence of SEQ ID NO:102. In some embodiments, the VHH domain is composed of the amino acid sequence of SEQ ID NO:102.

[0233] In some embodiments, the anti-CD20 VHH domain according to the present invention has one or more of the following properties:

[0234] (i) It has strong binding specificity and affinity to human CD20;

[0235] (ii) Compared with traditional four-chain full-length antibodies, it has a smaller volume, higher stability and deeper tissue penetration;

[0236] (iii) It can recognize and precisely target CD20 antigens on the surface of tumor cells;

[0237] (iv) Weak endocytic activity.

[0238] In some aspects, the strong antigen-binding and weak endocytic properties of the anti-CD20 VHH domain according to the present invention make it suitable as a component for constructing full-length antibodies, bispecific antibodies, or multispecific antibodies, especially TCEs. In other aspects, the good tumor targeting properties of the anti-CD20 antibody according to the present invention make it suitable for use in tumor therapy in the form of nanobodies (VHHs), for example, conjugates designed to deliver payloads (e.g., drugs or radioisotopes), thereby facilitating the delivery of the carried payload to tumor tissue to provide better applications such as tumor killing, immunomodulation, or disease detection. Therefore, in some embodiments, the present invention provides an anti-CD20 antibody comprising the anti-CD20 VHH domain of the present invention, which includes an immunoglobulin Fc region linked to said VHH domain (i.e., having a VHH-Fc form). In other embodiments, the present invention provides an anti-CD20 antibody comprising the anti-CD20 VHH domain of the present invention, wherein said anti-CD20 antibody is a heavy chain antibody. In other embodiments, the present invention provides an anti-CD20 antibody comprising the anti-CD20 VHH domain of the present invention, wherein the anti-CD20 antibody is a nanobody. In other embodiments, the present invention provides an anti-CD20 antibody comprising the anti-CD20 VHH domain of the present invention, wherein the anti-CD20 antibody is a bispecific antibody or a multispecific antibody. In other embodiments, the present invention provides an anti-CD20 antibody comprising the anti-CD20 VHH domain of the present invention, wherein the anti-CD20 antibody is a TCE.

[0239] In addition to the antibody forms described above, it should be understood that the anti-CD20 antibody of the present invention, which contains the anti-CD20 VHH domain according to the present invention, may also have any other suitable antibody structure, such as, but not limited to, single-chain or multi-chain antibodies, monovalent or multivalent antibodies, linear antibodies; single-domain antibodies or multi-domain antibodies.

[0240] III. The multispecific antibody of the present invention

[0241] In a third aspect, the present invention provides a multispecific antibody comprising a CD3 antigen-binding domain. The multispecific antibody of the present invention comprises at least two different antigen-binding specificities. In some embodiments, in addition to CD3 binding specificity, the multispecific antibody of the present invention further comprises at least one different antigen-binding specificity. In some embodiments, in addition to CD3 binding specificity, the multispecific antibody of the present invention further comprises at least two, three, or four different antigen-binding specificities. The binding specificities that can be incorporated into the anti-CD3 multispecific antibody of the present invention can be selected from, for example, but not limited to, tumor-associated antigens (TAAs), other immune-associated molecules, and co-stimulatory molecules. Depending on the included binding specificities, the anti-CD3 multispecific antibody of the present invention can be a bispecific antibody, a trispecific antibody, a tetraspecific antibody, or an antibody with more specificities. In some embodiments, the present invention provides a multispecific antibody having CD3 and at least one (e.g., 1-3 different) TAA binding specificities. In other embodiments, the present invention provides a multispecific antibody having CD3 and at least one (e.g., 1-3 different) TAA binding specificities and having binding specificity against at least one (e.g., 1) co-stimulatory molecule. In some cases, the TAA can be selected from solid tumor surface antigens or hematologic tumor surface antibodies. In other cases, the co-stimulatory molecules may be selected from CD28, OX40, CD137, CD8, ICOS, CD27, GITR, CD2, IL-2RP, CD58, CD80, CD7, and MyD88 / CD40. In some particular aspects, the present invention provides T-cell connectives comprising a CD3 antigen-binding domain and a tumor-associated antigen (TAA)-binding domain. Preferably, in some embodiments, at least one CD3 antigen-binding domain of the multispecific antibody according to the present invention comprises or is derived from, or is composed of, an anti-CD3 antibody or its antigen-binding fragment according to the first aspect of the present invention.

[0242] The structure of the multispecific antibody of this invention

[0243] Therefore, in some embodiments, the present invention provides a multispecific antibody comprising one or more first antigen-binding domains that specifically bind to CD3 and second antigen-binding domains having different binding specificities (and optionally, third, fourth and / or fifth antigen-binding domains having different binding specificities), wherein the one or more CD3 antigen-binding domains comprise, or are composed of, an anti-CD3 antibody or an antigen-binding fragment thereof according to the first aspect of the present invention.

[0244] The second antigen-binding domain (and optionally, the third, fourth, and / or fifth antigen-binding domains) included in the multispecific antibody of the present invention is not particularly limited. In some embodiments, the second antigen-binding domain (and optionally, the third, fourth, and / or fifth antigen-binding domains) specifically binds to target cell antigens. In some embodiments, the target cell antigen is an antigenic determinant presented on the surface of target cells (e.g., tumor cells or cells in the tumor stroma, or T cells).

[0245] In some preferred aspects, the multispecific antibody according to the invention comprises first and second antigen-binding domains, wherein the first antigen-binding domain is a CD3 antigen-binding domain according to the invention, and the second antigen-binding domain is an antigen-binding domain that specifically binds to a tumor-associated antigen (TAA). The multispecific antibody of the invention, comprising a combination of a TAA antigen-binding domain and a CD3 binding domain, facilitates the formation of an immune synapse between target tumor cells expressing the TAA on their surface and T cells, thereby achieving the killing of the target tumor. The TAA that can be used in the invention can be a solid tumor cell surface antigen or a hematologic tumor cell surface antigen.

[0246] In some embodiments, the multispecific antibody of the present invention specifically binds to one or more TAAs, wherein each of the one or more TAAs is independently selected from CD19, BCMA, TSHR, CD171, CS-1, CLL-1, GD3, and Tn. Ag,FLT3,CD38,CD123,CD44v6,B7H3,B7H4,KIT,IL-13Ra2,IL-11Ra,PSCA,PSMA,PRSS21,VEGFR2,L ewisY,CD24,PDGFR-beta,SSEA-4,MUC1,EGFR,NCAM,CAIX,LMP2,EphA2,sLe,GM3,TGS5,HMWMAA,GD 2,FOLR1,FOLR2,TEM1 / CD248,TEM7R,CLDN6,GPRC5D,CXORF61,CD97,CD179a,ALK,PLAC1,GloboH,N Y-BR-1,UPK2,HAVCR1,ADRB3,PANX3,GPR20,LY6K,OR51E2,TAARP,WT1,ETV6-AML,SPA17,XAGE1,Tie 2, MAD-CT-1, MAD-CT-2, FOSL1, hTERT, ML-IAP, ERG, NA17, PAX3, AR, Cyclin B1,MYCN,RhoC,CYP1B1,BORIS,SART3,PAX5,OY-TES1,LCK,AKAP-4,SSX2,CD79a,CD79b,CD72,LAIR1,F CAR,LILRA2,CD300LF,CLEC12A,BST2,EMR2,LY75,GPC3,FCRL5,IGLL1,CD20,CD30,HER2,ROR1,FLT3,T AAG72,CD22,CD33,GD2,gp100Tn,FAP,TYR,EPCAM,CEA,IGF-1R,EphB2,MSLN,Claudin18.2,CDH17,CD3 2b, EGFRvIII, GPNMB, GPR64, HER3, LRP6, LYPD8, NKG2D, SLC34A2, SLC39A6, SLITRK6, GUCY2C and TACSTD2.

[0247] In some embodiments, the multispecific antibody of the present invention specifically binds to surface antigens of solid tumor cells, selected from, for example, mesothelin (MSLN), carcinoembryonic antigen (CEA), epithelial cell adhesion factor (EpCAM), human epidermal growth factor receptor-2 (HER2), and prostate-specific membrane antigen (PSMA), epidermal growth factor receptor (EGFR), Claudin 18.2, and CDH17. In other embodiments, the multispecific antibody of the present invention specifically binds to surface antigens of hematologic tumor cells, selected from, for example, CD19, CD20, CD79b, CD33, BCMA, and GPRC5D.

[0248] Therefore, in some embodiments, the present invention provides a multispecific antibody comprising a CD3 antigen-binding domain and a TAA antigen-binding domain. In some embodiments, the CD3 antigen-binding domain of the antibody according to the present invention has a weak monovalent binding affinity for CD3 (preferably human CD3), for example, a binding affinity K0. D The value is 10x10 -7 M to 1x10 -8 M, or 5x10 -7 M to 5x10 -8 M.

[0249] In some cases, to balance the effects of the affinity of the CD3 antigen-binding domain and the TAA antigen-binding domain, the ratio of the CD3 and TAA antigen-binding domains in the multispecific antibody of the present invention can advantageously be adjusted. In some embodiments, the present invention provides a multispecific antibody wherein the ratio of the number or valence of the CD3 antigen-binding domain to the TAA antigen-binding domain is 1:1 or 1:2. In some embodiments, the valence (i.e., the total number of antigen-binding domains) of the multispecific antibody according to the present invention is 2-4 valences, preferably 3 valences. In some embodiments, the multispecific antibody according to the present invention is a trivalent antibody comprising one CD3 binding domain with weak binding affinity and two TAA binding domains with medium to high affinity.

[0250] In some embodiments, the CD3 antigen-binding domain contained in the multispecific antibody of the present invention comprises or is composed of Fab, scFab, or scFv domains. In other embodiments, the TAA antigen-binding domain contained in the multispecific antibody of the present invention comprises or is composed of Fab, scFab, scFv, or VHH domains.

[0251] In some embodiments, the multispecific antibody according to the present invention may further comprise an immunoglobulin Fc region in addition to the aforementioned antigen-binding domain. In other embodiments, alternatively, it may comprise a half-life extension domain, such as serum albumin or a serum albumin-binding peptide, to adjust the circulating half-life of the antibody after administration to animals. In some embodiments, preferably, the antibody of the present invention comprises an immunoglobulin Fc region.

[0252] The immunoglobulin Fc region used in the multispecific antibody of the present invention can be an Fc region derived from any immunoglobulin. In some embodiments, the immunoglobulin Fc region comprises at least an immunoglobulin CH2 domain and a CH3 domain. In some embodiments, the immunoglobulin Fc region further comprises a hinge region or a partial hinge region. In some embodiments, the immunoglobulin Fc region comprises, or consists of, an immunoglobulin hinge region or a partial hinge region, a CH2 domain, and a CH3 domain from the N-terminus to the C-terminus. In some embodiments, the immunoglobulin Fc region comprises, or consists of, a CH2 domain and a CH3 domain from the N-terminus to the C-terminus. In some embodiments, the immunoglobulin Fc region is preferably derived from IgG1, IgG2, or IgG4, or a subtype thereof. Preferably, the immunoglobulin Fc region comprises an Fc region sequence derived from humans.

[0253] The immunoglobulin Fc region can be fused to the antigen-binding domain and / or other domains according to the invention via its N- or C-terminus. C-terminal fusion of the immunoglobulin Fc region can be direct fusion, but in some cases is preferred via linker fusion. N-terminal fusion of the immunoglobulin Fc region can be direct fusion, but in some cases is preferably performed via the immunoglobulin hinge region sequence.

[0254] The immunoglobulin Fc region of the multispecific antibody used in this invention can be the native Fc region sequence. Alternatively, the Fc region can contain mutations relative to the native Fc sequence. Mutations include substitutions, insertions, and / or deletions. Such mutations can be made for the purpose of introducing desired therapeutic properties. A Knob-into-Hole (KiH) mutation can be introduced into the CH3 domain to promote heterodimerization in order to facilitate proper antibody assembly. In the case of introducing a KiH mutation, one Fc chain will be designed to contain a large protruding residue (i.e., Knob), while the other Fc chain is designed to contain a complementary pocket (i.e., Hole). Suitable locations for KiH mutations are known in the art. Exemplary KiH mutations include, but are not limited to, combinations of Knob mutation T366W and Hole mutations T366S, L368A, Y407V; and combinations of Knob mutation T366Y and Hole mutation Y407T. When the multispecific antibody of the present invention comprises an asymmetric double-stranded structure, preferably, the Fc region contains a KiH mutation that promotes proper heterodimerization of the antibody polypeptide chain. Alternatively, a cysteine ​​mutation may be introduced into the Fc region to increase disulfide bond linkage in the dimerized Fc region; for example, the mutation S354C may be introduced into one Fc chain and the mutation Y349C may be introduced into the other Fc chain.

[0255] Furthermore, depending on the specific application of the antibody or antibody-based molecule according to the present invention, the Fc region may also contain mutations that alter effector function. For example, in the case of the multispecific antibody of the present invention being TCE, the Fc region preferably contains mutations that reduce or eliminate effector function, such as the LALA mutation where leucine (L) at positions 234 and 235 of the Fc region is replaced with alanine (A).

[0256] According to the specific application of the antibody or antibody-based molecule of the present invention, the Fc region may also contain other mutations, such as mutations to increase binding to FcRn and / or remove protease sites, and / or introduce amino acid modifications that can be used to couple active molecules. Additionally or alternatively, the Fc region may be mutated to remove or replace amino acids that may undergo post-translational modifications (e.g., glycosylation) to provide improved druggability and developability of the therapeutic antibody.

[0257] In some embodiments, the multispecific antibody according to the present invention comprises a first Fc and a second Fc that form a dimer. In some embodiments, the first and second Fc regions are Fc regions of IgG isotypes, for example, Fc regions of IgG1, IgG2, or IgG4 isotypes, preferably derived from the Fc regions of human IgG1 or human IgG4. In some embodiments, the first and second Fc regions contain amino acid mutations that promote the formation of the Fc dimer. In some embodiments, the first Fc region contains T336W and S354C, and the second Fc region contains T366S, L368A, Y407V, and Y349C, or vice versa. In some embodiments, the first and second Fc regions further contain mutations that reduce or eliminate the binding of the Fc region to FcγR, for example, the L234AL235A mutation. In some embodiments, the first and second Fc regions respectively contain the amino acid sequences of SEQ ID NO:108 and SEQ ID NO:107, or amino acid sequences that are at least 95%, 96%, 98%, or 99% identical to them.

[0258] In the multispecific antibody according to the invention, antibody components (i.e., the antigen-binding domain and optionally the immunoglobulin Fc or half-life-binding domain) can be linked using linkers. There are no specific limitations on the linkers that can be used in the antibodies of the invention. Linker sequences are generally flexible. They can consist primarily of amino acids with large side chains that do not limit flexibility, such as glycine, alanine, and serine. Alternatively, they can consist of sequences from the hinge region of immunoglobulins. Depending on the linking location and the components to be linked, those skilled in the art can readily determine the available linker sequences or optimal lengths. Suitable linker lengths can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids long, or longer. In some cases, the length of the linker sequence can be relatively short, for example less than about 20 or 15 amino acids, such as 2-15 amino acids or 5-10 amino acids. Suitable connective sequences include, but are not limited to, G4S (SEQ ID NO: 138); (G4S)2 (SEQ ID NO: 106); (G4S)3 (SEQ ID NO: 136); GGGSG (SEQ ID NO: 139); GGSGG (SEQ ID NO: 140); GSGGG (SEQ ID NO: 141); GSGGGP (SEQ ID NO: 142); GGEPS (SEQ ID NO: 143); GGEGGGP (SEQ ID NO: 144) and GGEGGGSEGGGS (SEQ ID NO: 145); and (G4S)n (SEQ ID NO: 112), where n is an integer equal to or greater than 1; TS(G4S)n (SEQ ID NO: 146), where n is an integer equal to or greater than 1; G(G4S)n (SEQ ID NO: 147), where n is an integer equal to or greater than 1; (G4)n (SEQ ID NO: 112); and (G4)n (SEQ ID NO: 112). NO:148), where n is an integer equal to or greater than 1; (GRPGS)n (SEQ ID NO:149), where n is an integer equal to or greater than 1. Suitable flexible linker peptides can be rationally designed using computer programs to simulate the three-dimensional structures of proteins and peptides, or through phage display methods. In some embodiments, the linker used in the antibody of the present invention is a flexible linker peptide of 5-50 amino acids, preferably comprising a linker peptide containing glycine (G) and / or serine (S) and / or threonine residues (T). In one embodiment, the linker has a length of 5-50 amino acids, for example, 5, 10, 15, 20, 25, or 30 amino acids, or an amino acid length falling between any two integers.In some embodiments, the linker contains an amino acid sequence (G4S)n (SEQ ID NO:112), where n is an integer equal to or greater than 1, for example, n is an integer from 1 to 7, such as n = 1, 2, 3, 4, 5, 6 or 7.

[0259] The multispecific antibody according to the invention can take any suitable form, such as single-chain or multi-chain. In some embodiments, the multispecific antibody according to the invention comprises a CD3 arm providing at least one CD3 antigen-binding domain and a TAA arm providing at least one TAA antigen-binding domain; and optionally at least one additional antigen-binding domain (e.g., at least one additional TAA antigen-binding domain) attached to said CD3 arm, TAA arm, or both.

[0260] In some embodiments, the multispecific antibody according to the present invention comprises:

[0261] -A first structural portion comprising, from the N-terminus to the C-terminus, the following components: a first antigen-binding domain, optionally a linker, and a first immunoglobulin Fc region; and

[0262] -A second structural portion from the N-terminus to the C-terminus comprising the following components: a second antigen-binding domain, optionally a linker, and a second immunoglobulin Fc region;

[0263] -Optionally, a third antigen-binding domain is connected to the N-terminus or C-terminus of the first or second structural portion via a linker;

[0264] The first and second immunoglobulin Fc regions dimerize to form an Fc dimer. In some embodiments, the first antigen-binding domain binds CD3 and comprises or is composed of Fab, scFab, or scFv domains. In some embodiments, the second and third antigen-binding domains bind TAA and comprise or are composed of Fab, scFab, scFv, or VHH domains. Preferably, the first antigen-binding domain is a Fab domain that binds CD3, and the second and third antigen-binding domains are VHH domains that bind TAA. In some embodiments, preferably, the third antigen-binding domain binds to the N-terminus of the first structural portion or to the N-terminus of the second structural portion. In some embodiments, the second and third antigen-binding domains bind the same TAA antigen. In some embodiments, the linker is 5-25 amino acids long or comprises the amino acid sequence of SEQ ID NO: 106, 136, or 112.

[0265] In some embodiments, the multispecific antibody according to the present invention is an anti-CD3xTAA multispecific antibody comprising the following structural moiety:

[0266] -A first structural portion comprising, from the N-terminus to the C-terminus, the following components: a CD3 antigen-binding domain, an optional linker, and a first immunoglobulin Fc region; and

[0267] -A second structural portion comprising the following components from the N-terminus to the C-terminus: a first TAA antigen-binding domain, optionally a linker, and a second immunoglobulin Fc region;

[0268] -Optionally, a second TAA antigen-binding domain connected to the first or second portion via a linker;

[0269] The first and second immunoglobulin Fc regions dimerize to form an Fc dimer. Preferably, the second TAA antigen-binding domain is attached to the N-terminus of the first or second structural portion; however, C-terminal attachment is also considered in this disclosure.

[0270] In some embodiments, the anti-CD3xTAA multispecific antibody comprises:

[0271] - A first structural portion comprising, from the N-terminus to the C-terminus, the following components: a second TAA antigen-binding domain, optionally a linker, a CD3 antigen-binding domain, optionally a linker, and a first immunoglobulin Fc region; and

[0272] -A second structural portion comprising the following components from the N-terminus to the C-terminus: a first TAA antigen-binding domain, optionally a linker, and a second immunoglobulin Fc region;

[0273] The first and second immunoglobulin Fc regions dimerize to form Fc dimers.

[0274] In some embodiments, the anti-CD3xTAA multispecific antibody comprises:

[0275] -A first structural portion comprising, from the N-terminus to the C-terminus, the following components: a CD3 antigen-binding domain, an optional linker, and a first immunoglobulin Fc region; and

[0276] - A second structural portion comprising the following components from the N-terminus to the C-terminus: a second TAA antigen-binding domain, optionally a linker, a first TAA antigen-binding domain, optionally a linker, and a second immunoglobulin Fc region;

[0277] The first and second immunoglobulin Fc regions dimerize to form Fc dimers.

[0278] In some embodiments of the anti-CD3xTAA multispecific antibody according to the present invention, preferably, the CD3 antigen-binding domain is a Fab domain that binds CD3, and the first and second TAA antigen-binding domains are VHH domains that bind TAA. In some embodiments, preferably, the linker is 5-25 amino acids in length, or contains the amino acid sequence SEQ ID NO: 106, 136, or 112. In some embodiments, the Fab domain is fused to the N-terminus of the Fc region via a fragment containing VH-CH1 or VL-CL. In other embodiments, the Fab domain is fused to the N-terminus of the Fc region via a fragment containing VH-CL or VL-CH1.

[0279] In some embodiments, the multispecific antibody according to the present invention comprises a first, second, and third polypeptide chain, wherein: the first polypeptide chain comprises, from the N-terminus to the C-terminus, a VHH domain, a linker, a VH domain, a CH1 domain, and a first Fc region; the second polypeptide chain comprises, from the N-terminus to the C-terminus, a VL domain and a CL domain; and the third polypeptide chain comprises, from the N-terminus to the C-terminus, a VHH domain and a second Fc region. In some embodiments, the VH domain and the VL domain pair to form a CD3 binding domain according to the present invention; and the VHH domain comprises or is composed of a TAA binding domain according to the present invention. Preferably, the linker is 10-25 amino acids in length, for example, about 15 amino acids in length, or comprises an amino acid sequence of SEQ ID NO: 136 or 112.

[0280] In some embodiments, the multispecific antibody according to the present invention comprises a first, second, and third polypeptide chain, wherein: the first polypeptide chain comprises a VH domain, a CH1 domain, and a first Fc region from the N-terminus to the C-terminus; the second polypeptide chain comprises a VL domain and a CL domain from the N-terminus to the C-terminus; and the third polypeptide chain comprises a VHH domain, a linker, a VHH domain, and a second Fc region from the N-terminus to the C-terminus. In some embodiments, the VH domain and the VL domain pair to form a CD3 binding domain according to the present invention; and the VHH domain comprises or is composed of a TAA binding domain according to the present invention. Preferably, the linker is 5-15 amino acids long, for example, about 10 amino acids long, or comprises the amino acid sequence of SEQ ID NO: 106 or 112.

[0281] Exemplary CD3xCD20 multispecific antibody

[0282] In some embodiments of the multispecific antibody according to the present invention, preferably, the multispecific antibody according to the present invention comprises an antigen-binding domain of a TAA on the surface of hematologic malignancies cells. In some embodiments, the TAA is CD20. In some embodiments, the TAA antigen-binding domain comprises or is composed of a VHH domain that specifically binds to CD20. In some embodiments, the anti-CD20 VHH domain comprises the CDR1, CDR2, and CDR3 sequences of one of the amino acid sequences of SEQ ID NO: 102 or 115-124; preferably, the CDR1, CDR2, and CDR3 sequences comprise or are composed of the amino acid sequences of SEQ ID NOs: 103, 104, and 105, respectively, or comprise or are composed of the amino acid sequences of SEQ ID NOs: 137, 104, and 105, respectively. In some embodiments, the anti-CD20 VHH domain comprises an amino acid sequence of one of SEQ ID NO: 102 or 115-124, or has at least 85%, 90%, 95% or 99% identity with respect to the amino acid sequence, or has an amino acid sequence having one or more (preferably 1-10, more preferably 1-5) added, deleted and / or substituted amino acids, or is composed of such amino acids.

[0283] In some embodiments, the multispecific antibody according to the invention is an anti-CD3 multispecific antibody comprising a CD3 antigen-binding domain combined with the anti-CD20 antigen-binding domain of any of the embodiments described above. In some embodiments, the CD3 antigen-binding domain may comprise any anti-CD3 antibody according to the invention or its antigen-binding fragment, or a combination comprising its six CDRs or its VH / VL combinations. In some embodiments, the CD3 antigen-binding domain comprises HCDR1-3 and LCDR1-3 contained in the VH and VL sequence pairs selected from:

[0284] (a) The VH sequence of SEQ ID NO:31 and the VL sequence of one of SEQ ID NOs:32 and 72-74;

[0285] (b) The VH sequence of one of SEQ ID NO: 67-69 and the VL sequence of one of SEQ ID NOs: 70-71;

[0286] (c) The VH sequence of SEQ ID NO:68 and the VL sequence of one of SEQ ID NOs:78-82; or

[0287] (d) The VH sequence of SEQ ID NO:68 and the VL sequence of SEQ ID NO:87.

[0288] In some embodiments, the anti-CD3 antigen-binding domain comprises HCDR1-3 contained in the VH sequence of SEQ ID NO:47 and LCDR1-3 contained in the VL sequence of SEQ ID NO:48. In some preferred embodiments, the CD3 antigen-binding domain comprises a VH / VL amino acid sequence pair selected from: SEQ ID NOs:31 and 32; one of SEQ ID NOs:67 and SEQ ID NOs:70-71; one of SEQ ID NOs:69 and SEQ ID NOs:70-71; one of SEQ ID NO:68 and SEQ ID NOs:70-71 and 78-82; SEQ ID NO:68 and SEQ ID NO:87. In some preferred embodiments, the CD3 antigen-binding domain comprises the VH sequence of SEQ ID NO:31 and the VL sequence of SEQ ID NO:32. In some preferred embodiments, the CD3 antigen-binding domain comprises the VH sequence of SEQ ID NO:68 and the VL sequence of SEQ ID NO:87. In some other preferred embodiments, the CD3 antigen-binding domain comprises the VH sequence of SEQ ID NO:47 and the VL sequence of SEQ ID NO:48.

[0289] In some embodiments, the multispecific antibody according to the present invention is a bispecific antibody against CD3 and CD20, preferably a CD3xCD20 bispecific T-cell connector. In some embodiments, the multispecific antibody according to the present invention comprises:

[0290] (i) The first, second, and third polypeptide chains respectively containing SEQ ID NOs: 88, 89, and 101, or having at least 95%, 96%, 97%, 98%, or 99% identity with them, or

[0291] (ii) First, second, and third polypeptide chains comprising, respectively, amino acid sequences of SEQ ID NOs: 90, 91, and 101, or having at least 95%, 96%, 97%, 98%, or 99% identity with them. In some preferred embodiments, the first, second, and third polypeptide chains comprise amino acid sequences of SEQ ID NOs: 88, 89, and 101, respectively.

[0292] In some embodiments, the multispecific antibody according to the present invention comprises:

[0293] (i) The first, second, and third polypeptide chains respectively comprising SEQ ID NOs: 130, 131, and 132, or having at least 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequences therewith, or

[0294] (ii) First, second, and third polypeptide chains comprising, respectively, amino acid sequences of SEQ ID NOs: 133, 134, and 135, or having at least 95%, 96%, 97%, 98%, or 99% identity with them. In some preferred embodiments, the first, second, and third polypeptide chains comprise, respectively, amino acid sequences of SEQ ID NOs: 130, 131, and 132; or amino acid sequences of SEQ ID NOs: 133, 134, and 135.

[0295] Example CD3xMSLN multispecific antibody

[0296] In some embodiments of the multispecific antibody according to the present invention, preferably, the multispecific antibody according to the present invention comprises an antigen-binding domain of a TAA on the surface of solid tumor cells. In some embodiments, the TAA is an MSLN. In some embodiments, the TAA antigen-binding domain comprises or is composed of a VHH domain that specifically binds to MSLN. In some embodiments, the anti-MSLN VHH domain comprises the CDR1, CDR2, and CDR3 sequences of the amino acid sequence of SEQ ID NO:97; preferably, the CDR1, CDR2, and CDR3 sequences comprise or are composed of the amino acid sequences of SEQ ID NOs:98, 99, and 100, respectively. In some embodiments, the anti-MSLN VHH domain comprises the amino acid sequence of SEQ ID NO:97, or has at least 85%, 90%, 95%, or 99% identity with the amino acid sequence, or has one or more (preferably 1-10, more preferably 1-5) amino acid sequences with additions, deletions, and / or substitutions, or is composed of the amino acid sequence.

[0297] In some embodiments, the multispecific antibody according to the invention is an anti-CD3 multispecific antibody comprising a CD3 antigen-binding domain combined with the anti-MSLN antigen-binding domain of any of the above embodiments. In some embodiments, the CD3 antigen-binding domain may comprise any anti-CD3 antibody according to the invention or its antigen-binding fragment, or a combination comprising its six CDRs, or its VH / VL combinations. In some embodiments, the CD3 antigen-binding domain comprises HCDR1-3 and LCDR1-3 contained in the following VH and VL sequence pairs:

[0298] (a) The VH sequence of SEQ ID NO:31 and the VL sequence of one of SEQ ID NOs:32 and 72-74;

[0299] (b) The VH sequence of one of SEQ ID NO: 67-69 and the VL sequence of one of SEQ ID NOs: 70-71;

[0300] (c) The VH sequence of SEQ ID NO:68 and the VL sequence of one of SEQ ID NOs:78-82; or

[0301] (d) The VH sequence of SEQ ID NO:68 and the VL sequence of SEQ ID NO:87.

[0302] In some embodiments, the anti-CD3 antigen-binding domain comprises HCDR1-3 contained in the VH sequence of SEQ ID NO:47 and LCDR1-3 contained in the VL sequence of SEQ ID NO:48. In some preferred embodiments, the CD3 antigen-binding domain comprises a VH / VL amino acid sequence pair selected from: SEQ ID NOs:31 and 32; one of SEQ ID NOs:67 and SEQ ID NOs:70-71; one of SEQ ID NOs:69 and SEQ ID NOs:70-71; one of SEQ ID NO:68 and SEQ ID NOs:70-71 and 78-82; SEQ ID NO:68 and SEQ ID NO:87. In some preferred embodiments, the CD3 antigen-binding domain comprises the VH sequence of SEQ ID NO:31 and the VL sequence of SEQ ID NO:32. In some preferred embodiments, the CD3 antigen-binding domain comprises the VH sequence of SEQ ID NO:47 and the VL sequence of SEQ ID NO:48.

[0303] In some embodiments, the multispecific antibody according to the present invention is a bispecific antibody against CD3 and MSLN, preferably a CD3xMSLN bispecific T-cell connector. In some embodiments, the multispecific antibody according to the present invention comprises:

[0304] (i) The first, second, and third polypeptide chains respectively containing SEQ ID NOs: 88, 89, and 96, or having at least 95%, 96%, 97%, 98%, or 99% identity with them, or

[0305] (ii) First, second, and third polypeptide chains comprising, respectively, amino acid sequences of SEQ ID NOs: 90, 91, and 96, or having at least 95%, 96%, 97%, 98%, or 99% identity with them. In some preferred embodiments, the first, second, and third polypeptide chains comprise amino acid sequences of SEQ ID NOs: 88, 89, and 96, respectively. In other embodiments, the first, second, and third polypeptide chains comprise amino acid sequences of SEQ ID NOs: 90, 91, and 96, respectively.

[0306] Properties of the multispecific antibodies of this invention

[0307] As illustrated in the embodiments, the anti-CD3 sequence of the present invention exhibits unique behavior. When applied alone, especially in a monovalent manner, its binding to CD3 on the cell surface is very weak. However, when spliced ​​into a TCE, it demonstrates very strong T cell activation and killing capabilities by means of the TAA arm binding to tumor cells. Therefore, in some embodiments, this disclosure provides a multispecific antibody according to the present invention, said multispecific antibody being a TCE. In some embodiments, the TCE according to the present invention has one or more of the following properties:

[0308] (i) Binding to TAA-positive tumor cells on the surface. In some embodiments, the binding activity of the TCE according to the invention is comparable to that of a corresponding TCE composed of a BMK8 reference antibody.

[0309] (ii) Exhibiting T-cell activation activity dependent on TAA-positive tumor cells. Through T-cell activation assays, in the absence of relevant tumor cells, the TCE molecule containing the CD3 domain of this invention exhibits only weak activation activity against T cells; however, in the presence of relevant tumor cells, the CD3 domain of this invention exhibits significant T-cell activation by binding to TAA-positive tumor cells via the anti-TAA arm. In some embodiments, the T-cell activation assay is performed by detecting T-cell-induced cytokine release or the tumor-killing activity of T cells.

[0310] In some further embodiments, the present invention TCE also exhibits one or more of the following properties:

[0311] (i) In the presence of target tumor cells without TAA expression, it weakly binds to surface CD3-positive T cells, preferably exhibiting extremely weak T cell binding as determined by flow cytometry on Jurkat cells. In some embodiments, this binding activity of the TCE according to the invention is comparable to that of a corresponding TCE composed of a BMK9 reference antibody. Preferably, the assay is performed according to Examples 5.2 and 6.2.

[0312] (ii) In the presence of surface TAA-positive tumor cells and T cells, induction of T cell killing activity against the tumor cells. In some embodiments, the tumor cells are hematologic malignancies. In other embodiments, the tumor cells are solid tumor cells. In some embodiments, the killing activity of the TCE according to the invention is comparable to or even stronger than that of a corresponding TCE composed of a BMK8 reference antibody. Preferably, the assay is performed according to the method described in Example 5.3 or 6.3.

[0313] (iii) At an antibody dose that induces half-kill of target tumor cells, the release of cytokines from T cells is not substantially induced. In some embodiments, the release of cytokines IL-6, IFNγ, TNF-α, or any combination thereof can be measured. In some embodiments, in the same assay, for the TCE according to the invention, the ratio of the cytokine release EC50 value to the tumor-killing EC50 value is greater than 1.2, 1.3, or 1.5, or greater than 3 in some embodiments, or 4 to 10, for example 5 to 7, in other embodiments. In some embodiments, the TCE drug according to the invention has a lower EC50 ratio and thus better safety compared to the corresponding TCE composed of BMK8, by separating tumor cell killing activity from cytokine release activity. In some embodiments, the tumor cells are hematologic malignancies. In other embodiments, the tumor cells are solid tumor cells. In some embodiments, the assay is performed according to Examples 5.3 or 6.3.

[0314] In some embodiments, the multispecific antibody according to the invention is a 2:1 asymmetric TCE bispecific antibody structure, such as the TCE molecule with the configuration shown in Figure 23. While not bound by any theory, it is believed that in the 2:1 form, since the CD3 antigen-binding domain provided by the anti-CD3 antibody of the present invention has only a weak monovalent binding affinity to T cells, in some cases, the bivalent affinity of the antibody for the TAA can balance antibody efficacy and specificity. On the other hand, while not bound by any theory, it is believed that the weak T cell binding activity of the monovalent CD3 antigen-binding domain of the present invention will help avoid excessive activation and attenuation of T cells, reducing the risk of adverse reactions such as cytokine release syndrome (CRS) and neurotoxicity.

[0315] IV. Production and purification of the antibodies of this invention

[0316] In a fourth aspect, the present invention provides a nucleic acid encoding an anti-CD3 antibody of the first aspect of the present invention, an anti-CD20 antibody of the second aspect of the present invention, or a multispecific antibody of the third aspect of the present invention, a host cell comprising the thereof, and a method for producing said anti-CD3 antibody, said anti-CD20 antibody, or multispecific antibody.

[0317] To generate the antibodies of the present invention, polypeptide chains of the antibodies of the present invention can be obtained, for example, through solid-state peptide synthesis (e.g., Merrifield solid-phase synthesis) or recombinant production, and assembled under suitable conditions. For recombinant production, polynucleotides encoding any one and / or multiple polypeptide chains of the antibody can be isolated and inserted into one or more vectors for further cloning and / or expression in host cells. The polynucleotides can be easily isolated and sequenced using conventional methods. In one embodiment, polynucleotides encoding one or more polypeptide chains of the antibodies of the present invention are provided. In yet another embodiment, the present invention provides vectors comprising one or more polynucleotides of the present invention, preferably expression vectors. Thus, in one embodiment, the present invention provides a method for producing the antibodies of the present invention, the method comprising: culturing host cells containing a polypeptide chain encoding the polypeptide chain under conditions suitable for expression of the polypeptide chain of the antibody; and assembling the polypeptide chain to produce the antibody under conditions suitable for assembly of the polypeptide chain into the antibody.

[0318] Expression vectors can be constructed using methods well known to those skilled in the art. Expression vectors include, but are not limited to, viruses, plasmids, visceral phages, or yeast artificial chromosomes (YACs).

[0319] In one embodiment, the present invention also provides a host cell comprising one or more of the polynucleotides of the present invention. In some embodiments, a host cell comprising the expression vector of the present invention is provided. Suitable host cells include prokaryotic microorganisms such as *Escherichia coli*, eukaryotic microorganisms such as filamentous fungi or yeast, or various eukaryotic cells such as Chinese hamster ovary cells (CHO), insect cells, etc. Mammalian cell lines suitable for suspension culture can be used. Examples of useful mammalian host cell lines include SV40-transformed monkey kidney CV1 line (COS-7), human embryonic kidney line (HEK293 or 293F cells), young hamster kidney cells (BHK), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical cancer cells (HELA), canine kidney cells (MDCK), Buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (HepG2), CHO cells, NSO cells, myeloma cell lines such as YO, NSO, P3X63, and Sp2 / O, etc. In a preferred embodiment, the host cell is a CHO or HEK293 cell.

[0320] The antibodies prepared by the methods described herein can be purified using known existing techniques such as high-performance liquid chromatography (HPLC), ion-exchange chromatography, gel electrophoresis, affinity chromatography, and size exclusion chromatography. After purification, the purity of the antibodies of this invention can be determined by any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, and HPLC. The physical / chemical properties and / or biological activity of the antibodies provided herein can be identified, screened, or characterized using various assays known in the art.

[0321] In a preferred embodiment, the antibody of the present invention exhibits good production properties when recombinantly produced in mammalian host cells such as CHO cells, particularly good expression yield and a good byproduct profile.

[0322] V. Immunofusions and Immunoconjugates

[0323] In a fifth aspect, the present invention provides antigen-binding molecules, such as immunofusions or immunoconjugates, generated by fusing or conjugating the antibodies of the present invention to a heterologous molecule.

[0324] In one embodiment, in the immunofusion, the antibody (or its antigen-binding fragment) of the present invention is linked directly or via an amino acid linker to a heterologous peptide or polypeptide molecule. Heterologous peptides or polypeptides that may be mentioned include, but are not limited to, proteins or polypeptides that confer another functional activity to the fusion, or tag peptides that facilitate the purification or detection of the immunofusion. For example, a chimeric antigen receptor (CAR) comprising the antibody of the present invention or its antigen-binding fragment is an example of an immunofusion according to the present invention.

[0325] In one embodiment, in the immunoconjugate, the antibody (or its antigen-binding fragment) of the present invention is conjugated to a conjugated portion (e.g., a therapeutic agent, diagnostic agent, or detectable agent). In the conjugate, linkers can be used to covalently link different entities of the conjugate. Suitable linkers include chemical linkers or peptide linkers. Advantageously, the linker is a "cleavable linker" that facilitates the release of the conjugated portion upon delivery of the conjugate to the target site. For example, acid-instable linkers, peptidase-sensitive linkers, photostable linkers, dimethyl linkers, or disulfide-containing linkers can be used.

[0326] In embodiments where a therapeutic agent is conjugated, the therapeutic agent suitable for the conjugation includes, but is not limited to, cytotoxins (e.g., cell growth inhibitors or cell killers), drugs, or radioisotopes.

[0327] In embodiments conjugated with diagnostic or detectable agents, such conjugates can be used as part of clinical testing methods (e.g., to determine the efficacy of a particular therapy) to monitor or predict the onset, development, progression, and / or severity of a disease or condition. Such diagnostics and detections can be achieved by conjugating antibodies to detectable agents, including but not limited to a variety of enzymes such as horseradish peroxidase; prosthetic groups such as streptavidin / biotin and avidin / biotin; fluorescent substances; luminescent substances; radioactive substances; and positron-emitting metal and non-radioactive paramagnetic metal ions used in various positron emission tomography (PET) imaging techniques.

[0328] VI. Pharmaceutical compositions, drug combinations, and reagent kits

[0329] In a sixth aspect, the present invention provides compositions, such as pharmaceutical compositions, comprising an antibody of the first, second, or third aspect of the present invention, or an antigen-binding molecule of the fifth aspect of the present invention (e.g., an immunoconjugate or immunofusion), formulated with a pharmaceutically acceptable carrier. As used herein, a "pharmaceutically acceptable carrier" includes any and all physiologically compatible solvents, dispersion media, isotonic agents, and absorption delay agents, etc. The pharmaceutical compositions of the present invention are suitable for intravenous, intramuscular, subcutaneous, parenteral, rectal, spinal, or epidermal administration (e.g., by injection or infusion). In some embodiments, the antibody or immunoconjugate or immunofusion of the present invention is the sole active ingredient in the pharmaceutical composition. In other embodiments, the pharmaceutical composition may comprise the antibody or immunoconjugate or immunofusion of the present invention described herein, along with other therapeutic agents.

[0330] In another aspect, the present invention also provides a pharmaceutical combination comprising the antibodies described herein or the immunoconjugates or immunofusions of the present invention with other therapeutic agents.

[0331] The therapeutic agents applicable to the pharmaceutical compositions and combinations thereof of the present invention may be therapeutic agents selected from any of the following categories (i)-(iv): (i) drugs that enhance antigen presentation (e.g., tumor antigen presentation); (ii) drugs that enhance effector cell responses (e.g., B cell and / or T cell activation and / or mobilization); (iii) drugs that reduce immunosuppression (e.g., anti-PD-L1 antibodies); and (iv) drugs that have tumor-suppressing effects.

[0332] The pharmaceutical compositions of the present invention may comprise the antibody of the present invention at a “therapeutic effective amount” or a “preventive effective amount.” A “therapeutic effective amount” refers to the amount that effectively achieves the desired therapeutic outcome at the required dose and for the required duration. The therapeutic effective amount can vary depending on various factors such as disease state, individual age, sex, and weight. A therapeutic effective amount is any amount in which any toxic or harmful effects are less than the beneficial therapeutic effect. Relative to untreated subjects, the “therapeutic effective amount” preferably inhibits a measurable parameter (e.g., tumor growth rate) by at least about 20%, more preferably at least about 40%, even more preferably at least about 60%, and still more preferably at least about 80%. The ability of the antibody of the present invention to inhibit a measurable parameter (e.g., tumor volume) can be evaluated in animal model systems that predict efficacy in human tumors. A “preventive effective amount” refers to the amount that effectively achieves the desired preventive outcome at the required dose and for the required duration. Typically, because the preventive dose is used in subjects before or at an earlier stage of the disease, the preventive effective amount is less than the therapeutic effective amount.

[0333] Kits containing the antibodies described herein are also within the scope of this invention. Kits may include one or more other elements, such as: instructions for use; other reagents, such as markers or conjugation agents; a pharmaceutically acceptable carrier; and a device or other material for administration to a subject.

[0334] VII. Uses and Methods

[0335] In a seventh aspect, the present invention provides the use and methods of antibodies according to the first, second and third aspects of the present invention or antigen-binding molecules (e.g., immune conjugates or fusions) according to the fifth aspect of the present invention in the treatment and prevention of CD3-related diseases and / or cancer, or CD20-related diseases and / or cancer.

[0336] Overexpression of tumor-associated antigens such as CD20 and MSLN on the surface of cancerous tissue cells makes them suitable targets for cancer immunotherapy. In one aspect, the present invention provides the use of a multispecific antibody or TCE of the present invention comprising a CD3 and TAA antigen-binding domain for the prevention and / or treatment of TAA-associated tumors (i.e., TAA-positive tumors) in a subject. In another aspect, the present invention provides the use of an anti-CD20 antibody or multispecific antibody of the present invention comprising a CD20 binding domain for the prevention and / or treatment of CD20-associated tumors (i.e., CD20-positive tumors) in a subject. In said applications, the antibody of the present invention may be administered to the subject as the sole active agent or may be administered to the subject in combination with other therapies or therapeutic agents. These other therapies and therapeutic agents include, for example, drugs that target antigens on the surface of tumor cells to eliminate tumors by binding to and / or blocking these molecules; and drugs that activate the subject's immune system to induce spontaneous elimination of tumors. In yet another aspect, the present invention also provides a method for the prevention or treatment of cancer in a subject, comprising administering the antibody of the present invention or its antigen-binding fragment to a subject in need.

[0337] The TAA-positive or CD20-positive tumors suitable for the methods and applications of this invention can be selected from various solid tumors or hematologic malignancies. In some embodiments, the TAA is an MSLN, and the tumor is selected from, for example, mesothelioma (such as malignant mesothelioma), pancreatic cancer, ovarian cancer, lung cancer (such as non-small cell lung cancer), and colorectal cancer. In some embodiments, the TAA is a CD20, and the tumor is selected from B-cell lymphomas (such as DLBCL and LBCL). The tumors suitable for the methods and applications of this invention can be early, intermediate, or late-stage or metastatic cancers. Furthermore, the tumors suitable for the methods and applications of this invention can be tumors that have previously received treatment and have experienced immune escape.

[0338] In any of the above embodiments of the method of the present invention, the administration of the antibody or fragment thereof according to the present invention may include 1) a therapeutic measure that cures, slows, alleviates, reduces or stops the progression of a diagnosed pathological condition or disease; or 2) a preventive or preventative measure that prevents and / or slows the development of a pathological condition or disease. Therefore, in the method of the present invention, the subject may be an individual who already has a disease, an individual susceptible to a disease, or an individual who wishes to prevent a disease. The individual will benefit from the therapeutic or preventative measures and, compared to an individual who has not received the treatment, exhibit a reduction or improvement in the occurrence, recurrence, or development of the disease, condition, symptom, and / or symptoms. In some embodiments, the present invention relates to the treatment of a disease or condition; in other embodiments, the present invention relates to the prevention of a disease or condition.

[0339] The antibodies or fragments thereof according to the invention, and optionally other therapeutic agents used in combination therewith, can be administered by any suitable method, including parenteral administration, intratumoral administration, and intranasal administration. Parenteral infusion includes intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. Various dosing schedules are covered herein, including, but not limited to, single-dose or multiple-dose administration at multiple time points, bolus administration, and pulsatile infusion.

[0340] For the prevention or treatment of disease, the appropriate dose of the antibody or fragment thereof according to the invention (when used alone or in combination with one or more other therapeutic agents) will depend on the type of disease to be treated, the specific type of drug used, the severity and course of the disease, whether the drug is administered for preventive or therapeutic purposes, previous treatments, the patient's clinical history and response to the antibody, and the judgment of the attending physician.

[0341] In some embodiments, the present invention also provides for the use of the antibodies or antibody fragments of the present invention as pharmaceuticals or for the preparation of pharmaceuticals. In some embodiments, the pharmaceutical is a medicament for use in the aforementioned treatment and prevention methods.

[0342] Any or all of the features described above and throughout this application may be combined in various embodiments of the invention. The following examples further illustrate the invention; however, it should be understood that the examples are for illustrative purposes only and should not be construed as constituting any limitation. Example

[0343] Example 1: Obtaining Immunoma and Hybridoma Clones

[0344] 1.1 Animal Immunization

[0345] Different strains of mice (Balb / c, SJL) and different forms of immunogens (huCD3ED dimer (ACRO,CAT#CDD-H52W1), huCD3EG dimer (ACRO,CAT#CDG-H52W5), huCD3E (ACRO,CAT#CDE-H5223), etc.) were used to implement various combinations of animal immunizations. Mice with antigen- or cell-specific serum antibody titers were selected for hybridoma fusion experiments by monitoring antigen or cell-specific antibody titers. Positive hybridoma clones were selected by ELISA and FACS screening; after subcloning, ELISA and FACS rescreening were performed to confirm the specific binding of the molecules to human and cynomolgus monkey CD3.

[0346] 1.2 Screening for hybridomas

[0347] RNA was extracted from hybridoma cells and reverse transcribed into cDNA using a reverse transcription kit. The cDNA was then amplified by PCR using degenerate primers (synthesized using Azenta primers). The PCR product was cloned into the pMD18-T vector (TaKaRa, Cat#6013), transformed, amplified, and sequenced. Six clones were selected based on the sequencing results: H4 (27E2.1, also known as 27E2-1D10), H45 (119D3.4), H10 (112F4.1), H36 (136G6.3), H54 (217B11.1), and H61 (250E4.1) to construct plasmids for expressing chimeric antibodies and identifying their functional activity. The CDR sequences and heavy and light chain variable region sequences of each antibody are shown in SEQ ID NOs:1-48 of the sequence listing.

[0348] Example 2: Expression and functional determination of hybridoma chimeric antibodies

[0349] 2.1 Construction and expression of chimeric antibodies

[0350] The hybridoma candidate clones obtained in Example 1 were used to retrieve the antibody light and heavy chain gene sequences and construct human-mouse chimeric antibodies. In short, the VH and VL genes of the murine monoclonal antibody were re-amplified using cloning primers containing appropriate restriction sites and cloned into the expression vector pcDNA3.4 to generate corresponding clones of chimeric antibodies with the constant region of human IgG1. The clones were transfected into Expi-CHOS cells for transient transfection expression, purified with Protein A, and the obtained proteins were identified. Chimeric antibodies V-HD-27E2.1, V-HD-119D3.4, V-HD-112F4.1, V-HD-136G6.3, V-HD-217B11.1, and V-HD-250E4.1 were obtained, and their full-length heavy and light chain sequences are shown in SEQ ID NOs:49-60 of the sequence listing.

[0351] 2.2 Flow cytometry binding assay

[0352] Freshly cultured Jurkat cells and CD3 knockout Jurkat-CD3KO cells (eBioscience™) culture suspensions were collected into 50 mL centrifuge tubes, centrifuged at 300 g for 5 minutes, washed once with 20 mL of pre-chilled 4°C FACS buffer (PBS + 2% FBS), and resuspended in 10 mL of FACS buffer. 1 mL of cell suspension was used for cell counting, and the cell suspension was diluted to 1E6 cells / mL. 100 μL of cell suspension was added to a 96-well U-plate, centrifuged at 300 g for 5 minutes, and the supernatant was discarded. 100 μL of serially diluted chimeric antibody was added to each well, and the plate was incubated at 4°C for 1 hour. The plate was washed three times with pre-chilled 4°C FACS buffer. Goat anti-Human IgG Fc,PE (eBioscience™, CAT#12-4998-82, 1:200 dilution) diluted in FACS buffer was added, and the plate was incubated at 4°C for 0.5 hours. Wash once with pre-cooled FACS buffer at 4°C, resuspend the cells in 100 μL of FACS buffer, and detect the cell surface fluorescence intensity using FACS.

[0353] The assay included the anti-CD3 antibody BMK8 as a positive control. The BMK8 sequence (SEQ ID NOs: 63 and 64, derived from patent US10781264B2) is the sequence of the anti-CD3 antibody used in Amgen's second-generation, long-half-life BITE, a drug with representative drugs such as IMDELLTRA. TM Features include strong CD3 binding activity and T cell activating ability. It has been reported that BMK8 exhibits good monkey CD3 cross-activity, unlike the CD3 sequence OKT3 used in Amgen's first-generation BITE. (See: A Bispecific DLL3 / CD3 IgG-Like T-Cell Engaging Antibody Induces Antitumor Responses in Small Cell Lung Cancer. Clin Cancer Res. 2020 Oct 1; 26(19):5258-5268. doi:10.1158 / 1078-0432.CCR-20-0926.) As a control antibody for testing, BMK8 contains the human IgG1 constant region of SEQ ID NO:61 and the human lambda light chain constant region of SEQ ID NO:114, and is expressed according to the procedure described in Example 2.1.

[0354] As shown in Figure 1, all chimeric antibodies can bind to Jurkat cells. As shown in Figure 2, among the antibodies tested, BMK8 and the chimeric antibody 250E4.1 showed some non-specific binding to CD3 knockout Jurkat cells.

[0355] 2.3 Flow cytometry determination of CynoCD3 cross-binding

[0356] Resuscitate frozen cynomolgus monkey PBMC (cynoPBMC) cells (from eBioscience), wash once with 20 mL of pre-chilled 4°C FACS buffer (PBS + 2% FBS), and resuspend in 10 mL of FACS buffer. Count cells using 1 mL of the cell suspension and dilute to 1E6 cells / mL. Add 100 μL of the cell suspension to a 96-well U-plate, centrifuge at 300g for 5 minutes, discard the supernatant, add 100 μL of serially diluted chimeric antibody to each well, and incubate at 4°C for 1 hour. Wash three times with pre-chilled 4°C FACS buffer. Add the fluorescent secondary antibody Goat anti-Human IgG Fc,PE diluted in FACS buffer (eBioscience). TM CAT#12-4998-82, 1:200 dilution); Directly labeled flow cytometry antibody anti-CD4, APC (BD Pharmaceuticals). TM Cells were incubated with anti-CD8 BV421 (CAT#551980, 1:200 dilution) and anti-CD8 BV421 (BioLegend, CAT#301036, 1:200). The cells were incubated at 4°C for 0.5 hours. After washing once with pre-chilled (4°C) FACS buffer, the cells were resuspended in 100 μL of FACS buffer, and the cell surface fluorescence intensity was detected using FACS. Anti-CD3 antibody BMK8 was included as a positive control in the assay.

[0357] As shown in Figures 3 and 4, all chimeric antibodies can specifically bind to cyno CD4. + T and CD8 + T cells.

[0358] 2.4 T cell activation assay

[0359] Further PBMC functional activation assays were performed on the selected chimeric antibodies. Specifically, frozen PBMCs from two healthy volunteers (Donor1 and Donor2, from Ausen Biotech) were revived and diluted to 1E6 cells / mL with RPMI 1640 complete medium. 100 μL of cell suspension was added to a 96-well U-plate, followed by 100 μL of serially diluted chimeric antibody per well. The 96-well plates were then incubated at 37°C in a 5% CO2 incubator for 40 hours. Staining solution was prepared using Human TruStain FCX.TM (BioLegend, CAT#422302,1:100); anti-human CD4, FITC (Biolegend, CAT#317408,1:200); anti-human CD8, APC (eBioscience TM Cells were analyzed using the following assays: anti-CD3 antibodies (CAT#17-0087-42, 1:200) and anti-human CD25, BV421 (Biolegend, CAT#302630, 1:200). Cells in 96-well plates were washed once with FACS buffer, and 100 μL of cell staining solution was added to each well. Cells were incubated at 4°C for 0.5 hours. After washing once with pre-chilled (4°C) FACS buffer, cells were resuspended in 100 μL of FACS buffer, and cell surface fluorescence intensity was detected using FACS. Anti-CD3 antibodies BMK8 and BMK9 (Teneobio-F2B) were included as positive controls in the assay. Teneobio-F2B is a CD3 antibody with weak T-cell binding and activation activity. When incorporated monovalently into the TCE molecule, it exhibits cytotoxic activity against hematologic malignancies, but it was ineffective against solid tumors in a recent clinical trial (NCT04740034) (J Clin Oncol 42,e14587(2024).DOI:10.1200 / JCO.2024.42.16_suppl.e14587). As a control antibody for testing, BMK9 contains the variable region sequence (SEQ ID NOs:65 and 66) of Teneobio-F2B, as well as the constant region of the human IgG1 heavy chain (SEQ ID NO:61) and the constant region of the human kappa light chain (SEQ ID NO:62), and is expressed according to the procedure described in Example 2.1.

[0360] As shown in Figures 5-8, 136G6.3 and 250E4.1 exhibited weaker activation functions compared to the control antibody BMK8. However, previous reports have shown that for CD3 antibodies, excessive T-cell activation similar to BMK8 may predict potential toxicity and the risk of T-cell exhaustion and activation-induced T-cell death (AICD) (Efficient tumor killing and minimal cytokine release with novel T-cell agonist bispecific antibodies. MAbs. 2019 May / Jun; 11(4):639-652.). Therefore, 136G6.3 and 250E4.1 are expected to have a better therapeutic window.

[0361] Example 3: Antibody engineering of 136G6.3

[0362] 3.1 Sequence Modification for 136G6.3 Humanization and PTM Removal

[0363] By comparing the human antibody variable region germline gene database in the Biophi (Humanize Antibody-BioPhi Antibody design platform (dichlab.org)) database, and using MOE (Molecular Operating Environment) software, germline genes of the heavy and light chain variable regions with high homology to V-HD-136G6.3 were selected as templates. The CDR sequences of the murine antibody, determined based on the AbM protocol, were then transplanted into the corresponding human templates, forming variable region sequences in the order "FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4". The humanization templates for the V-HD-136G6.3 murine antibody were IGHV1-18*01, IGHJ1*01 and IGKV2-30*01, IGKJ4*01. Some amino acids in the FR region sequence of the V-HD-136G6.3 humanized antibody were reverted to the amino acids corresponding to those in the murine antibody. Table 1 below shows the constructed humanized antibodies and their variable region versions and sequences.

[0364] Table 1. 136G6.3 Humanized Antibody

[0365] Based on the PTM risk assessment, the V-HD-136G6.3 antibody sequence was modified by post-translational modification (PTM) to create a PTM-removed variant. Table 2 below shows the constructed PTM-removed antibodies and their variable region versions and sequences.

[0366] Table 2. PTM removal antibody of 136G6.3

[0367] Humanized antibodies and their encoding genes were synthesized using the Azenta gene and PTM was used to remove the antibodies. The synthesized genes were cloned into an expression vector, expressed to produce humanized antibodies with the human IgG1 constant region, and purified using protein A.

[0368] 3.2 Cell binding assay of 136G6.3 humanized and PTM-degraded variants

[0369] Following the flow cytometry binding assay procedure of Example 2.2, the binding of each variant to the parent cell on Jurkat and Jurkat-CD3KO cells was compared. See Figures 9-12 for specific data.

[0370] 3.3 Immunogenic modification of 136G6.3-zom2

[0371] The immunogenicity of 136G6.3-zom2 was predicted using Wemol 3.0, and MOE software was used to design primers to ensure the original affinity and reduce the risk of immunogenicity. Primers with corresponding amino acid site mutations were designed based on the nucleotide sequence of 136G6.3-zom2, and PCR was performed using a vector containing the 136G6.3-zom2 fragment as a template. After PCR, Dpn I enzyme was added for digestion at 37°C for 1 hour, followed by 1% agarose gel electrophoresis to recover the target fragment, which was then transformed into DH5α competent cells. Colonies grew the next day, and single clones were selected for sequencing. Sequencing confirmed that the nucleotide sequence completely matched the target sequence, and plasmids were extracted, expressed, and purified. The resulting deimmunogenic antibodies and their variable region sequences are shown in Table 3 below, with the mutation introduced in the VL sequence (m1L, SEQ ID NO:70) of 136G6.3-zom2 shown in parentheses after m1L.

[0372] Table 3. Deimmunogenic variants of the 136G6.3 humanized antibody

[0373] 3.4 Cell binding assay of the immunogenic variant of 136G6.3-zom2

[0374] Following the flow cytometry binding assay procedure in Example 2.2, the binding of each variant to the parent cell on Jurkat and Jurkat-CD3KO cells was compared. See Figures 13-14 for specific data.

[0375] 3.5 Combination of 136G6.3-dzom2.m21 and 136G6.3-pom11 sequences

[0376] Primers for corresponding amino acid site mutations were designed based on the nucleotide sequence of 136G6.3-dzom2.m21. PCR was performed using a vector containing the 136G6.3-dzom2.m21 fragment as a template. After PCR, Dpn I enzyme was added for digestion at 37°C for 1 hour. The target fragment was recovered by 1% agarose gel electrophoresis and then transformed into DH5α competent cells. Colonies grew the next day, and single clones were selected for sequencing. Sequencing identification revealed that nucleotide sequences completely identical to the target sequence were selected for plasmid extraction, expression, and purification, yielding 136G6.3M (VH sequence: SEQ ID NO: 68; VL sequence: SEQ ID NO: 87; HCDR1-3: SEQ ID NOs: 25-27, LCDR1-3: SEQ ID NOs: 75, 86, and 30).

[0377] 3.6 Cell binding assay of 136G 6.3M

[0378] The binding of 136G6.3M to Jurkat and Jurkat-CD3KO cells was detected using the flow cytometry binding assay procedure described in Example 2.2. See Figures 15-16 for specific data.

[0379] 3.7 Antigen affinity detection of 136G6.3M

[0380] The affinity of 136G 6.3M antibody for human CD3 and cynoCD3 antigens was determined using the SPR method on a Biocore 8K instrument. Specifically, a fixed concentration of antibody (10 μg / mL) was captured using a protein A chip (Cytiva, CAT#10323089). Serially diluted antigens were used as the mobile phase. The binding time was set to 120 seconds, and the dissociation time to 200 seconds. After each dissociation, regeneration was performed with 20 mM glycine (pH 2.0). Data were analyzed using a 1:1 binding model.

[0381] As shown in Figure 17 and Table 4, 136G6.3M has comparable affinity for human CD3 and cyno CD3 antigens, with a difference of less than 3 times.

[0382] Table 4. Affinity of 136G6.3M to human CD3 and cyno CD3 antigens

[0383] Example 4: Screening and characterization of anti-CD20-VHH antibodies

[0384] 4.1 Screening, recombinant expression and identification of anti-CD20-VHH

[0385] Alpaca immunization and magnetic sorting techniques were used to screen for candidate VHH sequences binding to CD20 through preliminary characterization. In short, alpaca were alternately immunized using Raji (from ICON) and Daudi (from ICON) cells expressing human CD20 membrane protein, with an interval of 14 days. Peripheral blood was collected seven days after each immunization, starting from the second immunization, and serum titers were monitored using FACS experiments. Once the serum titer reached the standard for blood bank construction, peripheral blood was collected from immunized alpaca, and peripheral blood mononuclear cells (PBMCs) were isolated. Total RNA was extracted from PBMCs, and PrimeScript was used as a template for RNA extraction. TM II. Reverse transcription was performed using the 1st Strand cDNA Synthesis Kit (Takara) to prepare cDNA. Using the cDNA as a template, a first-round PCR amplification produced nucleic acid fragments of conventional IgG (containing VH) and pure heavy chain IgG lacking the CH1 domain (containing VHH). These two types of nucleic acids were separated on an agarose gel. The VHH-encoding nucleic acid was extracted and purified, followed by a second-round PCR amplification. The VHH fragment was then isolated and purified by gel electrophoresis. The recovered VHH gene fragment was mixed with the linearized yeast display vector pDisaplay and co-transformed into competent yeast cells by electroporation to generate a yeast display library displaying VHH antibodies on the yeast cell surface. Yeast cells bound to the target antigen were enriched from the constructed library by magnetic sorting using streptavidin beads that had been pre-incubated with and thus bound to the target antigen.

[0386] Yeast culture obtained after magnetic bead sorting was plated on SDCAA plates, and single-clonal cells were picked and cultured. After 48 hours of induction, the single-clonal cell cultures were incubated sequentially with biotin-antigen and PE-Streptavidin. After incubation, flow cytometry (FACS) was performed to identify positive single-clonal yeast cells that bound the target antigen. Genomic DNA was extracted from the cultures of the obtained positive yeast cell clones for PCR amplification of antibody sequences and sequencing.

[0387] Based on the sequencing results, candidate VHH sequences were selected and ligated into the expression vector pcDNA3.4 in the form of a C-terminal fusion with a human IgG1Fc sequence. After the vector was verified by sequencing, it was transiently transfected into HEK-293F cells (hereinafter referred to as "293F cells"). The culture supernatant was used to characterize the binding and endocytic properties of the expressed antibody, and finally, the anti-CD20 antibody V-n6D11 with the VHH sequence of SEQ ID NO:115 was obtained. The CDR1-3 sequence of this antibody, as defined by the AbM protocol, is shown in SEQ ID NOs:103-105.

[0388] 4.2 Expression and purification of candidate VHH-Fc antibodies

[0389] The encoding gene for the VHH antibody sequence described above was synthesized and inserted into the expression vector pcDNA3.4, fusing the hIgG1 Fc sequence (SEQ ID NO: 125) at its C-terminus. The constructed expression vector was transiently transfected into 293F cells. After continuous culturing of transfected cells for 7 days, the culture supernatant was collected and filtered through a 0.45 μm filter membrane. The filtrate was transferred to sterile centrifuge tubes, and the antibody was purified using a Protein A column. The purity of the antibody product was determined by SEC-HPLC.

[0390] 4.3 Flow cytometry analysis of human CD20-positive tumor cells

[0391] The binding of the anti-CD20 VHH-Fc candidate antibody molecule V-n6D11 to CD20-positive target cells was detected using a FACS binding assay. Specific experimental conditions were as follows: Raji / Daudi target cells (2 × 10⁶ cells / year). 5 ( / well) + VHH-Fc or reference antibody (375nM, 5× dilution, 4℃ 1h) + anti-hIgG Fc-PE secondary antibody (eBioscience / 12-4998-82, 1:500, 4℃ 0.5h). This experiment included reference antibodies Ofatumumab and Obinutuzumab as controls. Ofatumumab sequences (SEQ ID NOs: 126 and 127) were obtained from the IMGT database, and their sequences were retrieved as: IMGT / 3Dstructure-DB card, IMGT / 2Dstructure-DB card for INN: 8606. Obinutuzumab sequences (SEQ ID NOs: 128 and 129) were obtained from the IMGT database, and their sequences were retrieved as: IMGT / 3Dstructure-DB card, IMGT / 2Dstructure-DB card for INN: 9043.

[0392] In summary, following the experimental conditions described above, target cells were seeded into 96-well plates at the stated cell density and centrifuged at 300g for 5 minutes at 4°C. The test sample was added, and the cells were incubated at 4°C for 1 hour. After centrifugation at 4°C, the supernatant was removed, and the cells were washed twice with FACS buffer (1% BSA or 2% FBS in PBS), followed by centrifugation at 4°C. The aforementioned flow cytometry secondary antibody was added, and the cells were resuspended and incubated at 4°C in the dark for 0.5 hours. After washing twice with FACS buffer, the cells were resuspended in 100 μl / well of FACS buffer and analyzed using flow cytometry. The MFI of the cells was measured using a Beckman Coulter flow cytometer.

[0393] The FACS binding results are shown in Figure 18. The candidate antibodies exhibited good target cell binding properties on both cell lines tested. In the FACS binding assay of Raji cells (Figure 18A), V-n6D11 showed better maximum binding (Bmax) than Ofatumumab and Obinutuzumab; in the FACS binding assay of Daudi cells (Figure 18B), V-n6D11 had a similar EC50 value and maximum binding (Bmax) to the reference antibody Ofatumumab, and was better than Obinutuzumab in terms of maximum binding (Bmax).

[0394] 4.4 Flow cytometry determination of cross-binding of monkey CD20

[0395] The binding of the anti-CD20 VHH-Fc candidate antibody molecule V-n6D11 to cynoCD20 overexpressing cells was detected using a FACS binding assay. The FACS experimental procedure was basically as described in section 4.3. The experiment was conducted under the following conditions: HEK293 empty cells or HEK293-cynoCD20 target cells (2 × 10⁻⁶ cells). 5 / well) + VHH-Fc or reference antibody (375nM, 5× dilution, 4℃ 1h) + anti-hIgG Fc-PE secondary antibody (eBioscience / 12-4998-82, 1:500, 4℃ 0.5h).

[0396] The FACS binding results are shown in Figure 19. The candidate antibody showed good target cell binding properties on HEK293-cynoCD20 cells, with a better maximum binding (Bmax) than the control antibodies Ofatumumab and Obinutuzumab (Figure 19A); and there was no obvious binding signal with blank negative cells HEK293 that do not express CD20 (Figure 19B).

[0397] 4.5 Humanization modification to resist CD20-VHH

[0398] The original V-n6D11 VHH sequence was humanized using the "best-matching method." Amino acid sequences of the VHH framework region were aligned using a human germline V gene database to select the optimal germline sequence. The best-matching human CDR sequence was replaced with the VHH CDR sequence to generate the humanized VHH sequence. Analysis of the V-n6D11 sequence revealed no post-translational modifications (PTMs) requiring removal. The humanized sequence was reverse-translated and synthesized by Genewiz (Shanghai, China). It was then constructed into the pcDNA 3.4 expression vector, with the C-terminus fused with the hIgG1 Fc sequence (SEQ ID NO: 125) to express humanized VHHs in the form of human IgG1, thereby obtaining the VHH antibody protein. Table 5 below shows the VHH sequences of V-n6D11 and its humanized antibody.

[0399] Table 5. Anti-CD20 VHH sequences

[0400] 4.6 Cell binding assay of V-n6D11 humanized variant

[0401] As described in section 4.3, the obtained antibodies V-zn6D11.m1-m9 were subjected to FACS detection. The results, as shown in Figure 20, indicate that the binding of all humanized antibody variants to the target cells Daudi was similar to that of the parent antibody.

[0402] The obtained antibodies V-zn6D11.m1 to m9 were subjected to FACS assay to verify their human-monkey cross-reactivity. As shown in Figures 21A and 21B, the binding of the humanized antibodies to target cells HEK293-cynoCD20 (Figure 21A) was similar to that of the maternal antibodies, and neither bound to HEK293 negative cells (Figure 21B).

[0403] 4.7 Internalization determination of humanized variants of V-n6D11

[0404] The internalization capacity of the CD20 antibody molecule V-zn6D11.m2 and the control antibody Rituximab (derived from Bio-Engineering) on ​​different target cells, Ramos and Daudi cells, was tested using an endocytosis assay.

[0405] Specifically: target cells were spaced at 2 × 10⁶ cells per well. 5Seed cells onto plates, add the test sample diluted to the appropriate concentration, and incubate at 2-8°C for approximately 0.5 hours to allow the test sample to bind to the cells. Centrifuge at 400x g for 4 minutes at 4°C, remove the supernatant, and wash the cells 2-3 times with pre-chilled 200 μl / well FACS buffer to remove excess unbound test sample. Resuspend the cells in pre-chilled 100 μl / well FACS buffer. Divide the cells into two groups and incubate at 4°C and 37°C for 4 hours, respectively. After incubation, immediately add ice-cold FACS buffer to terminate the endocytosis experiment. Centrifuge at 400x g for 4 minutes at 4°C, and wash the cells 2-3 times with pre-chilled 200 μL / well FACS buffer. Immediately add 100 μl / well of flow cytometry secondary antibody diluted to the target dilution, resuspend the cells, and incubate at 2-8°C in the dark for approximately 30 minutes to 1 hour. Wash cells 2-3 times with 200 μL / well FACS buffer, then resuspend cells in 100 μL / well FACS buffer for flow cytometry analysis. Measure the MFI of cells using a Beckman Coulter flow cytometer. Calculate the internalization level of antibodies bound to the cell surface using the following formula:

[0406] Internalization rate (%) = 100% - (MFI of samples incubated at 37℃ / MFI of samples incubated at 4℃) × 100%

[0407] As shown in Figure 22, the endocytosis of V-zn6D11.m2 in both Ramos (Figure 22A) and Daudi (Figure 22B) cell lines was weaker than that of the control antibody Rituximab.

[0408] Based on the data from Example 4, V-n6D11 is a novel anti-CD20 nanobody (VHH). Nanobodies are small antibody molecules composed of a single heavy chain, exhibiting high specificity and affinity. Compared to conventional antibodies, they possess smaller size, higher stability, and deeper tissue penetration. This makes them highly promising for cancer therapy, enabling precise treatment by recognizing and targeting specific antigens on the surface of tumor cells and penetrating deep tumor tissues inaccessible to conventional antibodies. Nanobodies can be designed to deliver drugs or radioisotopes to tumor cells to kill them. Furthermore, nanobodies have a natural advantage in the construction of bispecific and multispecific antibodies, avoiding the problem of light and heavy chain mismatch. V-n6D11 exhibits superior binding affinity to human and monkey CD20 compared to the control antibodies Ofatumumab and Obinutuzumab; simultaneously, in tumor cell internalization experiments, it demonstrates significantly weaker endocytic activity than the control antibody Retuximab. The above-mentioned strong binding and weak endocytosis properties suggest that V-n6D11 is suitable for splicing bispecific and multispecific antibodies for TCE.

[0409] Example 5: CD3xCD20 dual antibody splicing and activity verification

[0410] 5.1 CD3xCD20 double anti-interference splicing

[0411] Using the CD20 VHH clone selected above, four CD3xCD20 bispecific antibodies were constructed as CD20 arms. These bispecific antibodies are bispecific antibodies against human IgG1 subtypes constructed using a knot-in-hole approach. The "CD3 arm" and "CD20 arm" are each linked to a human IgG1 region sequence via a hinge region, where human IgG1 exhibits L234A and L235A mutations, i.e., IgG1 LALA. The CD3xCD20 bispecific antibodies composed of 136G6.3, 250E4.1, BMK8, and BMK9 (Teneobio-F2B) as CD3 arms were named V-F1F1S1.4, V-F1F1S2.5, V-F1F1S3.6, and V-F1F1S4.7, respectively (see Table 6 below). A schematic diagram of the bispecific antibody structure is shown in Figure 23A.

[0412] Table 6. Composition of CD3xCD20 dual antibody

[0413] 5.2 Cell binding assays of CD3xCD20 bispecific antibody CD3 and CD20 arms

[0414] Following the flow cytometry binding assay procedure in Example 2.2, the cell binding activities of the CD3 and CD20 arms of the CD3xCD20 bispecific antibody were measured and compared. Jurkat cells were used as target cells in the CD3 arm binding assay. Ramos cells (derived from ICON Biotech) were used as target cells in the CD20 arm binding assay. A commercially available CD3xCD20 bispecific antibody analogue, glofitamab (derived from BioNTech), was selected as a positive control.

[0415] As shown in Figure 24, the binding of the four bispecific antibody molecules to the CD20 arm is comparable, and CD20 maintains good activity when paired with different CD3 molecules. However, as shown in Figure 25, the binding of the monovalent CD3 arms exhibits significant differences. The CD3 arm composed of the BMK8 antibody sequence shows the strongest CD3 binding, followed by the CD3 arm of the glofitamab analog. The monovalent Jurkat binding of the CD3 arm composed of the 136G6.3 sequence is extremely weak, as is the monovalent Jurkat binding of the CD3 arm composed of the control antibody BMK9 sequence.

[0416] 5.3 PBMC killing and cytokine release by CD3xCD20 bispecific antibodies

[0417] The commercially available CD3xCD20 bispecific antibody glofitamab analog (from SAILYBIO) was selected as a positive control. The target cell killing activity of four CD3xCD20 bispecific antibodies was compared, with PBMCs from two healthy human donors selected as effector cell sources (Donor3, a strong immune-responsive donor, and Donor4, a weak immune-responsive donor, from SAILYBIO). Specifically, Donor3 and Donor4 PBMCs were thawed and diluted to 4E6 cells / mL using RPMI 1640 complete medium. CD20-positive Ramos cells were selected as target cells, and the cell density was adjusted to 2E5 cells / mL. Serial dilutions of the CD3xCD20 bispecific antibody (4-fold) were prepared. 50 μL of PBMC suspension, 100 μL of target cell suspension, and 50 μL of bispecific antibody dilution were added to 96-well U-type plates, with an effector-to-target ratio of 10:1. The 96-well plates were incubated at 37°C in a 5% CO2 incubator for 48 hours. Aspirate 50 μL of culture supernatant and calculate target cell killing by detecting LDH release. Refer to the instruction manual for LDH usage (kit from Dojindo, CAT#CK12). Detect the release of cytokines such as IFN-γ (kit from Thermo, CAT#88-7316-88) and IL-6 (kit from Thermo, CAT#88-7066-86) in the culture supernatant using an ELISA kit.

[0418] As shown in Figures 26 and 27, regardless of whether Donor3 or Donor4 was used as the strong donor, the bispecific antibody V-F1F1S1.4 composed of 136G6.3 exhibited strong cytotoxic activity comparable to the glofitamab analogue, and superior to the bispecific antibodies V-F1F1S3.6 with the BMK8 sequence and V-F1F1S4.7 with the BMK9 sequence. Figures 28 to 31 show that the bispecific antibody composed of 136G6.3 was comparable to the glofitamab analogue in IL-6 secretion, but showed a lower level in IFNγ secretion, suggesting that the bispecific antibody composed of 136G6.3 has better safety.

[0419] Example 6: CD3xMSLN dual antibody splicing and activity verification

[0420] 6.1 CD3xMSLN double-antibody splicing

[0421] Three CD3xMSLN bispecific antibodies were constructed using the publicly disclosed mesothelin-binding (MSLN) antibody sequence 2A2 (from patent applications: WO2018209298(A1), WO2018209304). These bispecific antibodies are bispecific antibodies against human IgG1 subtypes constructed using a knock-in-hole approach. The "CD3 arm" and "MSLN arm" are each linked to a human IgG1 region sequence via a hinge region, where human IgG1 exhibits the L234A / L235A mutation, i.e., IgG1 LALA. The CD3xMSLN bispecific antibodies, with 136G6.3, BMK8, and BMK9 as the CD3 arms, were named V-T1T1S1.8, V-T1T1S3.9, and V-T1T1S4.10, respectively (see Table 7 below). A schematic diagram of the bispecific antibody structure is shown in Figure 23A.

[0422] Table 7. Composition of CD3x MSLN dual antibody

[0423] 6.2 Cell binding assays of CD3xMSLN bispecific antibody CD3 arm and MSLN arm

[0424] Following the flow cytometry binding assay procedure in Example 2.2, the cell binding activities of the CD3 and MSLN arms of the CD3xMSLN bispecific antibody were measured and compared. In the CD3 arm binding assay, Jurkat cells were used as target cells. In the MSLN arm binding assay, AsPC1 cells (derived from Nanjing Kebai) were used as target cells.

[0425] As shown in Figure 32, the three bispecific antibody molecules exhibited comparable binding to the MSLN arm, and the MSLN arm maintained good activity regardless of the CD3 molecule it paired with. However, as shown in Figure 33, the binding of the monovalent CD3 arm showed significant differences, with the BMK8 antibody sequence exhibiting the strongest CD3 binding. The 136G6.3 Jurkat monovalent binding was extremely weak.

[0426] 6.3 PBMC killing and cytokine release by CD3xMSLN bispecific antibody

[0427] Two healthy human PBMCs (Donor3, a strong immunoreactive donor, and Donor4, a weak immunoreactive donor, both derived from SAILYBIO) were selected as effector cell sources. The target cell killing activity of three CD3xMSLN bispecific antibodies was compared. Specifically, MLSN-positive AsPC-1 cells were selected as target cells, and the cell density was adjusted to 1E5 cells / mL. 100 μL of target cell suspension was added to a 96-well V plate and incubated at 37°C, 5% CO2 to allow adhesion. Donor3 and Donor4 PBMCs were thawed and cryopreserved, and diluted to 2E6 cells / mL with RPMI 1640 complete medium. Serial 4-fold dilutions of the CD3xMSLN bispecific antibodies were prepared. 50 μL of PBMC suspension and 50 μL of bispecific antibody dilution were added to a 96-well U plate, with an effector-to-target ratio of 10:1. The 96-well plate was incubated at 37°C, 5% CO2 for 48 hours. 50 μL of culture supernatant was aspirated, and the killing effect on target cells was calculated by detecting LDH release. The release of cytokines such as IFN-γ (Thermo, CAT#88-7316-88), TNF-α (Thermo, CAT#88-7346-86), and IL-6 (Thermo, CAT#88-7066-86) was detected in the culture supernatant using an ELISA kit.

[0428] As shown in Figures 34 and 35, regardless of whether it was the strong donor (Donor3) or the weak donor (Donor4), the cytotoxic effect of the bispecific antibody V-T1T1S1.8 composed of 136G6.3 was only slightly weaker than that of the bispecific antibody V-T1T1S3.9 composed of the BMK8 sequence; it was far superior to the bispecific antibody V-T1T1S4.10 composed of BMK9 (Teneobio-F2B). Figures 36-41 show that the bispecific antibody composed of 136G6.3 induced a lower level of cytokine secretion compared to the bispecific antibody composed of the BMK8 sequence, suggesting that 136G6.3 has better safety.

[0429] Based on the data from Examples 5 and 6, 136G6.3 is a moderately potent CD3 agonist suitable for both hematologic malignancy (TCE) and solid tumor (TCE) assembly. In vitro evaluations showed that 136G6.3 has the best potential among similar antibodies. For the hematologic malignancy target CD20, the bispecific antibody composed of 136G6.3 mediated superior killing compared to the bispecific antibody composed of BMK8, while exhibiting relatively lower cytokine release compared to the approved 2+1 bispecific antibody Golifitamab, under similar killing conditions. For the solid tumor target MSLN, the bispecific antibody composed of 136G6.3 effectively killed solid tumor cells as effectively as the bispecific antibody composed of BMK8, while the bispecific antibody composed of Teneobio-F2B failed to mediate effective killing of solid tumor cells due to its low affinity for CD3.

[0430] Example 7: Verification of the Intra- and Extracellular Activity of CD3xCD20 Bispecific Antibody

[0431] Based on the previous description, the CD3 arm in V-F1F1S1.4 was replaced with the 136G6.3M sequence to obtain V-F1S5.14, according to the structure shown in Figure 23A; at the same time, a new dual-antibody structure V-F1S5.15 was constructed. A schematic diagram of V-F1S5.15 is shown in Figure 23B.

[0432] Table 8. Composition of CD3xCD20 dual antibody

[0433] 7.1 Assay of PBMC killing, cytokine release and T cell activation by CD3xCD20 bispecific antibody

[0434] The commercially available CD3xCD20 bispecific antibody glofitamab analog (from SAILYBIO) was selected as a positive control. The target cell killing activity of the two CD3xCD20 bispecific antibodies was compared, with PBMCs from healthy human donors (Donor5, from SAILYBIO) selected as the effector cell source. Specifically, Donor5 PBMCs were thawed and diluted to 4E6 cells / mL with RPMI 1640 complete medium. CD20-positive Ramos and Raji cells were selected as target cells, and the cell density was adjusted to 2E5 cells / mL. A 6-fold serial dilution of the CD3xCD20 bispecific antibody was prepared. 50 μL of PBMC suspension, 100 μL of target cell suspension, and 50 μL of bispecific antibody dilution were added to 96-well U-type plates, with an effector-to-target ratio of 10:1. The 96-well plates were incubated at 37°C in a 5% CO2 incubator for 48 hours. Aspirate 50 μL of culture supernatant and calculate target cell killing by detecting LDH release. Refer to the instruction manual for LDH (kit from Dojindo, CAT#CK12) usage. Detect the release of cytokines such as IFN-γ (kit from Thermo, CAT#88-7316-88) and IL-6 (kit from Thermo, CAT#88-7066-86) in the culture supernatant using an ELISA kit. Co-cultured cells are then used to detect CD25 expression on CD4 and CD8 T cells. Specifically: Prepare staining solution: Human TruStain FcX. TM (BioLegend, CAT#422302,1:100); anti-human CD4, FITC (Biolegend, CAT#317408,1:200); anti-human CD8, APC (eBioscience TM (CAT#17-0087-42, 1:200); anti-human CD25, BV421 (Biolegend, CAT#302630, 1:200). Cells in 96-well plates were washed once with FACS buffer, and 100 μL of cell staining solution was added to each well. The plates were incubated at 4°C for 0.5 hours. Cells were then washed once with pre-chilled (4°C) FACS buffer, resuspended in 100 μL of FACS buffer, and the cell surface fluorescence intensity was detected using FACS.

[0435] As shown in Figures 42-49, the candidate CD3xCD20 bispecific antibody V-F1S5.14 exhibits stronger in vitro killing, cytokine release, and T cell activation than the glofitamab analogue. Although the candidate molecule V-F1S5.15 shows weaker in vitro killing than the glofitamab analogue due to the occlusion of the CD3 arm, its weaker cytokine release and T cell activation suggest better safety.

[0436] 7.2 Antitumor effect of CD3xCD20 bispecific antibody in subcutaneous transplantation of hPBMC-reconstructed mouse WSU-DLCL2 model

[0437] This experiment was used to evaluate the efficacy of the test products (including the CD3xCD20 bispecific antibody of the present invention) in the subcutaneous transplantation model of hPBMC-reconstructed NSG mice with WSU-DLCL2. The control antibodies were the bispecific antibodies V-F1F1S3.6 and V-F1F1S4.7 constructed in Example 5.

[0438] Model building and grouping

[0439] Model construction: 7-8 week old female NSG mice (NSG, from Shanghai Southern Model Biotechnology Co., Ltd.) were used. After acclimatization for 1 week, WSU-DLCL2 (CBP60273, Nanjing Kebai Biotechnology Co., Ltd.) was injected into the right scapula with PBS mixed with 50% matrix gel to resuspend the cells (1×10⁻⁶). 7 (cells / animals). When the tumor volume grows to 30-60mm... 3 Based on the average tumor volume, hPBMCs (8×10⁻⁶) were injected via the tail vein. 6 Immune reconstitution is performed using cells / animals. The tumor is allowed to grow to 100-200 mm in size. 3 The subjects were randomly assigned to groups (n = 6-8). The day of group assignment was defined as D0. The test drug was administered intravenously once a week for a total of 3 weeks.

[0440] Dosage volume: Adjusted according to mouse body weight (dosage volume for mice = 10 μL / g × mouse body weight (g))

[0441] Data collection: After the start of drug administration, the mice were weighed twice a week, the tumor volume was measured twice a week, and the animals were observed twice a day.

[0442] Trial endpoint: based on tumor volume (1500-2000 cm³) 3 The endpoint is determined by the animal's condition. At the endpoint, all surviving animals are euthanized and tumors are collected. The tumors are photographed, weighed, and then processed for further treatment.

[0443] Endpoint Analysis

[0444] At the end of the trial, the following indicators were analyzed: tumor volume change (TGI) TV (and weight changes).

[0445] TGI TV Calculation formula:

[0446] TGI TV ={1-[(Vt-V0) / (Ct-C0)]}

[0447] Vt: The average tumor volume of mice in the test drug administration group on day t;

[0448] V0: The average tumor volume of mice in the test drug administration group on day 0;

[0449] Ct: Average tumor volume of mice in the solvent group on day t;

[0450] C0: Average tumor volume of the solvent group mice on day 0.

[0451] Statistical analysis

[0452] Analysis, processing, and reporting: Quantitative indicators are described using mean ± standard error (Mean ± SEM / SD). One-way ANOVA or two-way ANOVA was used for analysis of quantitative indicators. For inter-group comparisons, the t-test was used, and p < 0.05 was considered statistically significant. Both statistical and biological significance were considered in the analysis of results.

[0453] As shown in Figure 50, the antitumor efficacy of the bispecific antibodies V-F1S5.14 and V-F1S5.15, composed of CD3 antibodies from this invention, is significantly superior to that of the control bispecific antibody composed of CD3 molecules. (TGI of each group) TV See Table 9.

[0454] Table 9. Antitumor effects of CD3xCD20 bispecific antibody in a mouse model of subcutaneous transplantation of hPBMC-reconstructed WSU-DLCL2.

[0455] Sequence List Overview

Claims

1. An anti-CD3 antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment comprises: - The three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) contained in the heavy chain variable region (VH) sequences selected from SEQ ID NOs:31 and 67-69, and the three light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) contained in the light chain variable region (VL) sequences selected from SEQ ID NOs:32 and 70-71, and optionally containing the following amino acid substitutions: amino acid substitutions selected from S27eT, S27eV, and S27eI in LCDR1, and / or amino acid substitutions selected from V51I, S52Q, V55R, and S56I in LCDR2, wherein the variable region amino acid residues are numbered according to the Kabat numbering system; -HCDR1-3 contained in the VH sequence of SEQ ID NO:7 and LCDR1-3 contained in the VL sequence of SEQ ID NO:8; -HCDR1-3 contained in the VH sequence of SEQ ID NO:15 and LCDR1-3 contained in the VL sequence of SEQ ID NO:16; -HCDR1-3 contained in the VH sequence of SEQ ID NO:23 and LCDR1-3 contained in the VL sequence of SEQ ID NO:24; -HCDR1-3 contained in the VH sequence of SEQ ID NO:39, and LCDR1-3 contained in the VL sequence of SEQ ID NO:40; or -HCDR1-3 contained in the VH sequence of SEQ ID NO:47 and LCDR1-3 contained in the VL sequence of SEQ ID NO:48; Preferably, the antibody or antigen-binding fragment comprises HCDR1-3 and LCDR1-3 contained in the following VH and VL sequence pairs: (a) The VH sequence of SEQ ID NO:31 and the VL sequence of one of SEQ ID NOs:32 and 72-74; (b) The VH sequence of one of SEQ ID NOs: 67-69 and the VL sequence of one of SEQ ID NOs: 70-71; (c) The VH sequence of SEQ ID NO:68 and the VL sequence of one of SEQ ID NOs:78-82; or (d) The VH sequence of SEQ ID NO:68 and the VL sequence of SEQ ID NO:

87.

2. The anti-CD3 antibody or its antigen-binding fragment according to claim 1, wherein the antibody or antigen-binding fragment comprises HCDR1-3 and LCDR1-3, wherein: -HCDR1-3 respectively contain or consist of the amino acid sequences of SEQ ID NOs:25, 26 and 27, and LCDR1 contains or consists of the amino acid sequences selected from SEQ ID NOs:28 and 75-77, LCDR2 contains or consists of the amino acid sequences selected from SEQ ID NOs:29 and 83-86, and LCDR3 contains or consists of the amino acid sequence of SEQ ID NO:30; -HCDR1-3 respectively contain or consist of the amino acid sequences of SEQ ID NOs:1, 2 and 3, and LCDR1-3 respectively contain or consist of the amino acid sequences of SEQ ID NOs:4, 5 and 6; -HCDR1-3 comprises or is composed of the amino acid sequences of SEQ ID NOs:9, 10 and 11, respectively, and LCDR1-3 comprises or is composed of the amino acid sequences of SEQ ID NOs:12, 13 and 14, respectively; -HCDR1-3 comprises or is composed of the amino acid sequences of SEQ ID NOs:17, 18 and 19, respectively, and LCDR1-3 comprises or is composed of the amino acid sequences of SEQ ID NOs:20, 21 and 22, respectively; -HCDR1-3 respectively comprises or is composed of the amino acid sequences of SEQ ID NOs:33, 34 and 35, and LCDR1-3 respectively comprises or is composed of the amino acid sequences of SEQ ID NOs:36, 37 and 38; or -HCDR1-3 comprises or is composed of the amino acid sequences of SEQ ID NOs:41, 42, and 43, respectively, and LCDR1-3 comprises or is composed of the amino acid sequences of SEQ ID NOs:44, 45, and 46, respectively. Preferably, the antibody or antigen-binding fragment comprises HCDR1-3 and LCDR1-3, wherein: (i)HCDR1-3 respectively contain or consist of the amino acid sequences of SEQ ID NOs:25, 26 and 27, and LCDR1-3 respectively contain or consist of the amino acid sequences of SEQ ID NOs:28, 29 and 30; (ii) HCDR1-3 respectively contain or consist of the amino acid sequences of SEQ ID NOs:25, 26 and 27, and LCDR1 contains or consists of the amino acid sequences of SEQ ID NOs:75, 76 or 77, and LCDR2-3 respectively contain or consist of the amino acid sequences of SEQ ID NOs:29 and 30. (iii) HCDR1-3 respectively contain or consist of the amino acid sequences of SEQ ID NOs:25, 26 and 27, and LCDR1 and LCDR3 respectively contain or consist of the amino acid sequences of SEQ ID NOs:28 and 30, and LCDR2 contains or consists of the amino acid sequences of SEQ ID NOs:83, 84, 85 or 86; or (iv)HCDR1-3 contain or consist of the amino acid sequences of SEQ ID NOs:25, 26 and 27 respectively, and LCDR1-3 contain or consist of the amino acid sequences of SEQ ID NOs:75, 86 and 30 respectively.

3. The anti-CD3 antibody or its antigen-binding fragment according to claim 1 or 2, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein: (a) The heavy chain variable region comprises an amino acid sequence selected from SEQ ID NOs:31, or has at least 85%, 90%, 95% or 99% identity with respect to the amino acid sequence, or has an amino acid sequence having one or more (preferably 1-10, more preferably 1-5) amino acid additions, deletions and / or substitutions, or is composed thereof; and / or the light chain variable region comprises an amino acid sequence selected from SEQ ID NOs:32 and 72-74, or has at least 85%, 90%, 95% or 99% identity with respect to the amino acid sequence, or has an amino acid sequence having one or more (preferably 1-10, more preferably 1-5) amino acid additions, deletions and / or substitutions, or is composed thereof; (b) The heavy chain variable region comprises an amino acid sequence selected from SEQ ID NOs:67-69, or has at least 85%, 90%, 95% or 99% identity with respect to the amino acid sequence, or has an amino acid sequence having one or more (preferably 1-10, more preferably 1-5) amino acid additions, deletions and / or substitutions, or is composed thereof; and / or the light chain variable region comprises an amino acid sequence selected from SEQ ID NOs:70-71 and SEQ ID NOs:78-82 and 87, or has at least 85%, 90%, 95% or 99% identity with respect to the amino acid sequence, or has an amino acid sequence having one or more (preferably 1-10, more preferably 1-5) amino acid additions, deletions and / or substitutions, or is composed thereof; (c) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO:7, or has at least 85%, 90%, 95% or 99% identity with respect to the amino acid sequence, or has an amino acid sequence having one or more (preferably 1-10, more preferably 1-5) added, deleted and / or substituted amino acids, or is composed of the thereof; and / or the light chain variable region comprises the amino acid sequence of SEQ ID NO:8, or has at least 85%, 90%, 95% or 99% identity with respect to the amino acid sequence, or has an amino acid sequence having one or more (preferably 1-10, more preferably 1-5) added, deleted and / or substituted amino acids, or is composed of the thereof; (d) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO:15, or has at least 85%, 90%, 95% or 99% identity with respect to the amino acid sequence, or has an amino acid sequence having one or more (preferably 1-10, more preferably 1-5) added, deleted and / or substituted amino acids, or is composed of the thereof; and / or the light chain variable region comprises the amino acid sequence of SEQ ID NO:16, or has at least 85%, 90%, 95% or 99% identity with respect to the amino acid sequence, or has an amino acid sequence having one or more (preferably 1-10, more preferably 1-5) added, deleted and / or substituted amino acids, or is composed of the thereof; (e) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO:23, or has at least 85%, 90%, 95% or 99% identity with respect to the amino acid sequence, or has an amino acid sequence having one or more (preferably 1-10, more preferably 1-5) added, deleted and / or substituted amino acids, or is composed thereof; and / or the light chain variable region comprises the amino acid sequence of SEQ ID NO:24, or has at least 85%, 90%, 95% or 99% identity with respect to the amino acid sequence, or has an amino acid sequence having one or more (preferably 1-10, more preferably 1-5) added, deleted and / or substituted amino acids, or is composed thereof; (f) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO:39, or has at least 85%, 90%, 95%, or 99% identity with respect to the amino acid sequence, or has an amino acid sequence having one or more (preferably 1-10, more preferably 1-5) added, deleted, and / or substituted amino acids, or is composed thereof; and / or the light chain variable region comprises the amino acid sequence of SEQ ID NO:40, or has at least 85%, 90%, 95%, or 99% identity with respect to the amino acid sequence, or has an amino acid sequence having one or more (preferably 1-10, more preferably 1-5) added, deleted, and / or substituted amino acids, or is composed thereof; or (g) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO:47, or has at least 85%, 90%, 95% or 99% identity with respect to the amino acid sequence, or has an amino acid sequence having one or more (preferably 1-10, more preferably 1-5) added, deleted and / or substituted amino acids, or is composed of the thereof; and / or the light chain variable region comprises the amino acid sequence of SEQ ID NO:48, or has at least 85%, 90%, 95% or 99% identity with respect to the amino acid sequence, or has an amino acid sequence having one or more (preferably 1-10, more preferably 1-5) added, deleted and / or substituted amino acids, or is composed of the thereof.

4. The anti-CD3 antibody or its antigen-binding fragment according to any one of claims 1-3, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein: (a) The heavy chain variable region comprises or is composed of the amino acid sequence of SEQ ID NO:31; and the light chain variable region comprises or is composed of the amino acid sequence selected from SEQ ID NOs:32 and 72-74. (b) The heavy chain variable region comprises, or is composed of, an amino acid sequence selected from SEQ ID NOs:67-69; and the light chain variable region comprises, or is composed of, an amino acid sequence selected from SEQ ID NOs:70-71, 78-82 and 87; (c) The heavy chain variable region contains or is composed of the amino acid sequence of SEQ ID NO:7; and the light chain variable region contains or is composed of the amino acid sequence of SEQ ID NO:8; (d) The heavy chain variable region contains or is composed of the amino acid sequence of SEQ ID NO:15; and the light chain variable region contains or is composed of the amino acid sequence of SEQ ID NO:

16. (e) The heavy chain variable region comprises or is composed of the amino acid sequence of SEQ ID NO:23; and the light chain variable region comprises or is composed of the amino acid sequence of SEQ ID NO:24; (f) The heavy chain variable region comprises, or is composed of, the amino acid sequence of SEQ ID NO:39; and the light chain variable region comprises, or is composed of, the amino acid sequence of SEQ ID NO:40; or (g) The heavy chain variable region comprises, or is composed of, the amino acid sequence of SEQ ID NO:47; and the light chain variable region comprises, or is composed of, the amino acid sequence of SEQ ID NO:

48. Preferably, the antibody or antigen-binding fragment comprises: (i) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:31 or thereof, and a light chain variable region comprising one of the amino acid sequences of SEQ ID NO:32 and 72-74; or (ii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:68 or thereof, and a light chain variable region comprising the amino acid sequence selected from SEQ ID NOs:70-71, 78-82 and 87 or thereof; or (iii) A heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 67 or 69 or thereof, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 70 or 71 or thereof.

5. The anti-CD3 antibody or its antigen-binding fragment according to any one of claims 1-4, wherein said antibody or antigen-binding fragment has one or more of the following characteristics: (a) Specific binding to human CD3 antigen, preferably, wherein the binding affinity K is measured by in vitro surface plasmon resonance (SPR) binding assay. D The value is approximately 10x10 -7 M to approximately 1x10 -8 M, preferably approximately 5x10 -7 M to approximately 5x10 -8 M; (b) exhibits immune cross-reactivity with human and monkey CD3 antigens, preferably wherein the binding affinity K for human and monkey CD3 antigens, as measured by in vitro surface plasmon resonance (SPR) binding assay, is [value missing]. D The values ​​should not differ by more than approximately 5 times, preferably not by more than approximately 3 times; (c) It exhibits essentially no nonspecific binding to CD3 antigen-negative cells; and (d) Activation of CD4+ and CD8+ T cells.

6. The anti-CD3 antibody or its antigen-binding fragment according to any one of claims 1-5, wherein: - The antibody or its antigen-binding fragment is murine, chimeric, or humanized; and / or - The antibody or its antigen-binding fragment is selected from full-length antibodies, Fab, Fab', Fab'-SH, Fv, single-chain antibodies (e.g., scFv and scFab), crossFab, F(ab')2 or linear antibodies.

7. The anti-CD3 antibody or its antigen-binding fragment according to any one of claims 1-6, wherein the antibody comprises an immunoglobulin Fc region, and preferably the Fc region is an IgG isotype, such as the human IgG1 or IgG4 isotype Fc region.

8. A multispecific antibody comprising at least two different antigen-binding specificities, wherein the first binding specificity is provided by a first antigen-binding domain that specifically binds to CD3, and wherein the CD3 antigen-binding domain comprises or is composed of an anti-CD3 antibody according to any one of claims 1-7 or an antigen-binding fragment thereof.

9. The multispecific antibody of claim 8, wherein, in addition to CD3 binding specificity, the multispecific antibody further comprises at least one, two, three, four or five different antigen binding specificities, optionally the antigens being independently selected from tumor-associated antigens (TAAs), other immune-associated molecules and co-stimulatory molecules.

10. The multispecific antibody of claim 9, wherein the multispecific antibody has binding specificity to CD3 and at least one (e.g., 1-3 different) TAAs, and optionally also has binding specificity to at least one (e.g., 1) co-stimulatory molecule.

11. The multispecific antibody according to any one of claims 9-10, wherein the TAA is a solid tumor cell surface antigen or a hematologic tumor cell surface antigen, and optionally selected from CD19, BCMA, TSHR, CD171, CS-1, CLL-1, GD3, Tn Ag,FLT3,CD38,CD123,CD44v6,B7H3,B7H4,KIT,IL-13Ra2,IL-11Ra,PSCA,PSMA,PRSS21,VEGFR2,L ewisY,CD24,PDGFR-beta,SSEA-4,MUC1,EGFR,NCAM,CAIX,LMP2,EphA2,sLe,GM3,TGS5,HMWMAA,GD 2,FOLR1,FOLR2,TEM1 / CD248,TEM7R,CLDN6,GPRC5D,CXORF61,CD97,CD179a,ALK,PLAC1,GloboH,N Y-BR-1,UPK2,HAVCR1,ADRB3,PANX3,GPR20,LY6K,OR51E2,TAARP,WT1,ETV6-AML,SPA17,XAGE1,Tie 2, MAD-CT-1, MAD-CT-2, FOSL1, hTERT, ML-IAP, ERG, NA17, PAX3, AR, Cyclin B1,MYCN,RhoC,CYP1B1,BORIS,SART3,PAX5,OY-TES1,LCK,AKAP-4,SSX2,CD79a,CD79b,CD72,LAIR1,F CAR,LILRA2,CD300LF,CLEC12A,BST2,EMR2,LY75,GPC3,FCRL5,IGLL1,CD20,CD30,HER2,ROR1,FLT3,T AAG72,CD22,CD33,GD2,gp100Tn,FAP,TYR,EPCAM,CEA,IGF-1R,EphB2,MSLN,Claudin18.2,CDH17,CD3 2b, EGFRvIII, GPNMB, GPR64, HER3, LRP6, LYPD8, NKG2D, SLC34A2, SLC39A6, SLITRK6, GUCY2C and TACSTD2.

12. The multispecific antibody according to any one of claims 9-11, wherein the co-stimulatory molecule is selected from CD28, OX40, CD137, CD8, ICOS, CD27, GITR, CD2, IL-2RP and MyD88 / CD40.

13. The multispecific antibody according to any one of claims 8-12, wherein the multispecific antibody comprises a second antigen-binding domain that specifically binds to a tumor-associated antigen (TAA), preferably, the TAA is a solid tumor cell surface antigen selected from, for example, MSLN, CEA, EpCAM, HER2, PSMA, EGFR, Claudin18.2 and CDH17, or the TAA is a hematologic tumor cell surface antigen selected from, for example, CD19, CD20, CD79b, CD33, BCMA and GPRC5D.

14. The multispecific antibody according to any one of claims 8-13, wherein the antibody comprises a CD3 antigen-binding domain and a TAA antigen-binding domain, and has one or more features selected from: (a) The valence ratio of the TAA antigen-binding domain to the CD3 antigen-binding domain is 1:1 or 2:1, preferably 2:1; (b) The antibody is a bivalent, trivalent, or quadrivalent bispecific antibody, preferably a trivalent bispecific antibody; and (c) The antibody is a T-cell adaptor (TCE).

15. The multispecific antibody according to any one of claims 8-14, wherein the antibody further comprises an Fc dimer having first and second immunoglobulin Fc regions, and optionally wherein: (i) The first Fc region and the second Fc region contain amino acid mutations that promote the formation of the Fc dimer; (ii) The first Fc region is a human IgG Fc region containing T336W and S354C, and the second Fc region is a human IgG Fc region containing T366S, L368A, Y407V, and Y349C. (iii) The first Fc region and the second Fc region respectively contain mutations that reduce or eliminate the binding interaction between the Fc region and FcγR, for example, L234A and L235A mutations. (iv) The first Fc region and the second Fc region are IgG1 or IgG4 isotypes; and / or (v) The first and second Fc regions respectively contain the amino acid sequences of SEQ ID NO:108 and SEQ ID NO:107, or amino acid sequences that are at least 95%, 96%, 98% or 99% identical to them.

16. The multispecific antibody according to any one of claims 8-15, wherein the antibody comprises -A first structural portion comprising the following components from the N-terminus to the C-terminus: a first antigen-binding domain, optionally a linker, and a first immunoglobulin Fc region; -A second structural portion from the N-terminus to the C-terminus comprising the following components: a second antigen-binding domain, an optional linker, and a second immunoglobulin Fc region; and -Optionally, a third antigen-binding domain is connected to the N-terminus or C-terminus of the first or second structural portion via a linker; The Fc regions of the first and second immunoglobulins dimerize to form Fc dimers. Preferably, the linker is 5-25 amino acids in length, or contains an amino acid sequence of SEQ ID NO: 106, 136 or 112.

17. The multispecific antibody according to any one of claims 8-16, wherein: - The first antigen-binding domain binds to CD3 and includes or is composed of Fab, scFab, or scFv domains; and / or - The second and third antigen-binding domains bind to TAAs and contain or consist of Fab, scFab, scFv, or VHH domains. Preferably, the first antigen-binding domain is a Fab domain that binds CD3, and the second and third antigen-binding domains are VHH domains that bind TAA.

18. The multispecific antibody according to any one of claims 8-17, wherein the antibody comprises a TAA antigen-binding domain, and The TAA antigen-binding domain specifically binds to CD20. Preferably, the CD20 antigen-binding domain comprises or is composed of a VHH domain. More preferably, the VHH domain comprises the CDR1, CDR2, and CDR3 sequences of one of the amino acid sequences in SEQ ID NO: 102 and 115-124; More preferably, the CDR1, CDR2 and CDR3 sequences respectively contain or are composed of the amino acid sequences of SEQ ID NOs:103,104 and 105, or respectively contain or are composed of the amino acid sequences of SEQ ID NOs:137,104 and 105; More preferably, the VHH domain comprises an amino acid sequence selected from SEQ ID NO: 102 and 115-124, or has at least 85%, 90%, 95%, or 99% identity with respect to the amino acid sequence, or has an amino acid sequence having one or more (preferably 1-10, more preferably 1-5) added, deleted, and / or substituted amino acids, or is composed of such an amino acid sequence. Alternatively, the TAA antigen-binding domain may specifically bind to MSLN; preferably, the MSLN antigen-binding domain comprises or is composed of a VHH domain. More preferably, the VHH domain comprises the CDR1, CDR2, and CDR3 sequences in the amino acid sequence of SEQ ID NO:97; More preferably, the CDR1, CDR2 and CDR3 sequences respectively contain or consist of the amino acid sequences of SEQ ID NOs:98,99 and 100; More preferably, the VHH domain comprises the amino acid sequence of SEQ ID NO:97, or has at least 85%, 90%, 95% or 99% identity with respect to the amino acid sequence, or has an amino acid sequence having one or more (preferably 1-10, more preferably 1-5) added, deleted and / or substituted amino acids, or is composed of the above.

19. The multispecific antibody according to any one of claims 8-18, wherein the antibody is a bispecific antibody against CD3 and CD20, and comprises: (i) First, second, and third polypeptide chains comprising SEQ ID NOs: 88, 89, and 101, respectively, or having an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity with them. (ii) First, second, and third polypeptide chains comprising SEQ ID NOs: 90, 91, and 101, respectively, or having at least 95%, 96%, 97%, 98%, or 99% identity with them. (iii) The first, second, and third polypeptide chains respectively containing SEQ ID NOs: 130, 131, and 132, or having at least 95%, 96%, 97%, 98%, or 99% identity with them, or (iv) First, second, and third polypeptide chains comprising SEQ ID NOs: 133, 134, and 135, respectively, or having at least 95%, 96%, 97%, 98%, or 99% identity with them. Or the antibody described therein is a bispecific antibody against CD3 and MSLN, and contains: (v) The first, second, and third polypeptide chains respectively containing SEQ ID NOs: 88, 89, and 96, or having an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity with them, or (vi) The first, second, and third polypeptide chains respectively contain SEQ ID NOs: 90, 91, and 96, or have an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity with them. Preferably, the first, second, and third polypeptide chains contain amino acid sequences of SEQ ID NOs:88, 89, and 96, respectively, or contain amino acid sequences of SEQ ID NOs:88, 89, and 101, respectively.

20. An anti-CD20 antibody or an antigen-binding fragment thereof, comprising a VHH domain that specifically binds to CD20, and The VHH domain contains the CDR1, CDR2, and CDR3 sequences of one of the amino acid sequences in SEQ ID NO: 102, 115-124; Preferably, the CDR1, CDR2 and CDR3 sequences respectively contain or are composed of the amino acid sequences of SEQ ID NOs:103,104 and 105, or respectively contain or are composed of the amino acid sequences of SEQ ID NOs:137,104 and 105; More preferably, the VHH domain comprises an amino acid sequence of one of SEQ ID NO: 102, 115-124, or has at least 85%, 90%, 95% or 99% identity with respect to the amino acid sequence, or has an amino acid sequence having one or more (preferably 1-10, more preferably 1-5) added, deleted and / or substituted amino acids. Most preferably, the VHH domain comprises, or is composed of, the amino acid sequence of SEQ ID NO:

102.

21. A polynucleotide encoding the anti-CD3 antibody or its antigen-binding fragment as claimed in any one of claims 1-7, the multispecific antibody as claimed in any one of claims 8-19, or the anti-CD20 antibody or its antigen-binding fragment as claimed in claim 20.

22. A vector, preferably an expression vector, comprising the polynucleotide of claim 21.

23. A host cell comprising the polynucleotide of claim 21 or the vector of claim 22, wherein the host cell is optionally a mammalian cell.

24. A method for producing the anti-CD3 antibody or its antigen-binding fragment according to any one of claims 1-7, the multispecific antibody according to any one of claims 8-19, or the anti-CD20 antibody or its antigen-binding fragment according to claim 20, the method comprising: Host cells containing polynucleotides encoding the polypeptide chains are cultured under conditions suitable for producing the antibody or its antigen-binding fragments or polypeptide chains of the multispecific antibody.

25. An antigen-binding molecule comprising, for example, an immunoconjugate or an immunofusion compound, the anti-CD3 antibody of any one of claims 1-7 or its antigen-binding fragment, the multispecific antibody of any one of claims 8-19 or the anti-CD20 antibody of claim 20 or its antigen-binding fragment.

26. A pharmaceutical composition comprising the anti-CD3 antibody of any one of claims 1-7 or an antigen-binding fragment thereof, the multispecific antibody of any one of claims 8-19, the anti-CD20 antibody of claim 20 or an antigen-binding fragment thereof, or the antigen-binding molecule of claim 25, and a pharmaceutically acceptable carrier.

27. The use of the anti-CD3 antibody or its antigen-binding fragment according to any one of claims 1-7, the multispecific antibody according to any one of claims 8-19, the anti-CD20 antibody according to claim 20 or its antigen-binding fragment, or the antigen-binding molecule according to claim 25 as a drug or for the preparation of a drug.

28. The use of claim 27, wherein the drug is used to treat and / or prevent cancer in an individual.

29. A method of treating or preventing cancer, comprising administering to an individual in need an effective amount of the anti-CD3 antibody of any one of claims 1-7 or an antigen-binding fragment thereof, the multispecific antibody of any one of claims 8-19, the anti-CD20 antibody of claim 20 or an antigen-binding fragment thereof, the antigen-binding molecule of claim 25, or the pharmaceutical composition of claim 26.

30. The use or method of claim 28 or 29, wherein the cancer is a solid tumor or hematologic malignancy, preferably wherein: The solid tumor is an MSLN-positive solid tumor, selected from, for example, mesothelioma (such as malignant mesothelioma), pancreatic cancer, ovarian cancer, lung cancer (such as non-small cell lung cancer), and colorectal cancer; The hematologic malignancy is a CD20-positive hematologic malignancy, selected from, for example, B-cell lymphomas (such as DLBCL and LBCL).

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