Anti-CD3 and Anti-CD20 bispecific antibody and use thereof

Abstract

The present invention provides a bispecific antibody targeting CD3 and CD20 and a composition comprising same. Further provided are a nucleic acid molecule encoding the bispecific antibody of the present invention, a vector and a host cell for expressing the bispecific antibody of the present invention, and a therapeutic and diagnostic method and a use of the antibody of the present invention or an antigen-binding fragment thereof.

Description

Anti-CD3 and anti-CD20 bispecific antibodies and uses thereof Technical Field

[0001] The present invention relates to the field of antibodies, and more specifically, to bispecific antibodies targeting CD3 and CD20 and compositions comprising the same. It also relates to nucleic acid molecules encoding the antibodies or antigen-binding fragments thereof of the present invention, vectors and host cells for expressing the antibodies or antigen-binding fragments thereof of the present invention, and therapeutic and diagnostic methods and uses of the antibodies or antigen-binding fragments thereof of the present invention. Background Art

[0002] CD3 (T cell surface glycoprotein CD3, signal transduction co-receptor of T cell receptor, which contains subunits γ, δ, ε and ζ) is a differentiation antigen expressed on the surface of all T lymphocytes, which mainly mediates the transduction of T cell activation signals. It plays an important role in the body's immune system's anti-infection immunity. The CD3 molecule forms a stable TCR-CD3 complex with the T cell antigen receptor (T cell receptor). Its extracellular region recognizes and binds to major histocompatibility complex class II molecules, enhancing the stability of the binding of the T cell antigen receptor (TCR) to the MHC molecule; the intracellular region enhances the activation signal transduced by leukocyte CD3, thereby participating in and regulating the activation of the immune system. The number of CD3-positive lymphocytes is an important indicator for measuring the body's cellular immunity.

[0003] CD20 is a marker molecule specific to the surface of B lymphocytes. It is expressed on mature B cells and most malignant B lymphocytes, but not on early B cell progenitors or late-mature plasma cells. This molecule consists of 297 amino acid residues and penetrates the cell membrane four times. Its antigenic epitope is a single, extracellularly exposed loop consisting of 43 amino acid residues without carbohydrate chains between the third and fourth transmembrane regions. The precise function of CD20 is still unclear, but CD20 may participate in B cell activation and differentiation and function as a calcium channel. Because CD20 is not internalized into cells after binding to antibodies and does not undergo significant cell surface shedding, it is an ideal antigen for treating B lymphocyte-related diseases. For example, rituximab and obinutuzumab are both anti-CD20 monoclonal antibodies with good efficacy. Given the potential for improvement in the immune cell-mediated killing of tumor cells by existing antibodies targeting CD20 and the advantages of bispecific antibodies, well-characterized anti-CD20 / CD3 bispecific antibodies can effectively mediate immune cell killing of tumor cells and hold great clinical promise.

[0004] Bispecific antibodies (BsAbs), also known as bifunctional antibodies, can simultaneously and specifically bind to two different antigens or two different antigenic epitopes. Due to their specificity and bifunctionality, they have promising applications and prospects in fields such as tumor immunotherapy and autoimmune diseases. Bispecific antibodies come in a variety of formats, and different formats have different effects on different tumor-associated antigens. Due to their unique functionality, they have broad application prospects in tumor immunotherapy.

[0005] There is currently no drug available on the market for anti-CD3×CD20 bispecific antibodies. The present invention aims to develop a novel bispecific antibody with better affinity, safety, and stability.

[0006] Summary of the Invention

[0007] This article discloses a novel bispecific antibody molecule constructed by antibody engineering methods.

[0008] The present invention provides a bispecific antibody that specifically binds to CD20 and CD3, comprising:

[0009] (1) an antigen-binding fragment having a CD20 binding domain, comprising a first heavy chain variable region (VH1) and a first light chain variable region (VL1); and

[0010] (2) An antigen-binding fragment having an anti-CD3 binding domain, which comprises a second heavy chain variable region (VH2) and a second light chain variable region (VL2).

[0011] In some embodiments, the bispecific antibody of the present invention comprises the following polypeptide chains:

[0012] (1) A polypeptide chain represented by formula (I) and formula (II) having a CD20 binding domain: VH1-CH1-Fc1 formula (I), VL1-CL formula (II), and

[0013] (2) A polypeptide chain represented by formula (III) and formula (II-2) having CD20 and CD3 binding domains: VH1-CH1-L-VL2-L-VH2-L-Fc2 formula (III); VL1-CL formula (II-2); or

[0014] A polypeptide chain represented by formula (IV) having a CD3 binding domain: VL2-L-VH2-L-Fc2 Formula (IV);

[0015] wherein VH represents the heavy chain variable region; VL represents the light chain variable region; Fc comprises CH2 and CH3; CH1, CH2, and CH3 represent domains 1, 2, and 3 of the heavy chain constant region, respectively; VL represents the light chain variable region; CL represents the light chain constant region; L represents a connecting peptide, each connecting peptide being the same or different; optionally, a hinge region is present between CH1 and Fc1;

[0016] Formulas (I), (II), (II-2), (III) and (IV) are connected sequentially from N-terminus to C-terminus;

[0017] The antigen binding site formed by VH1 and VL1 binds CD20, and the antigen binding site formed by VL2 and VH2 binds CD3.

[0018] In some embodiments, the connecting peptide of the present invention comprises an amino acid sequence (GGGGS)n, wherein n is independently selected from 1, 2, 3, 4, 5 or 6.

[0019] In some embodiments, the VH1 of the present invention comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively, and the VL1 comprises LCDR1, LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively.

[0020] In some embodiments, the bispecific antibody of the present invention, wherein:

[0021] The VH1 comprises the amino acid sequence of SEQ ID NO:7, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:7; and

[0022] The VL1 comprises the amino acid sequence shown in SEQ ID NO: 8; or comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 8.

[0023] In some embodiments, the bispecific antibody of the present invention, wherein the VH2 comprises HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11, respectively, and the VL2 comprises LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14, respectively.

[0024] In some embodiments, the bispecific antibody of the present invention, wherein:

[0025] The VH2 comprises the amino acid sequence of SEQ ID NO: 15, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 15; and

[0026] The VL2 comprises the amino acid sequence shown in SEQ ID NO: 16; or comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 16.

[0027] In some embodiments, the bispecific antibody of the present invention, wherein:

[0028] The polypeptide chain of formula (I) comprises the amino acid sequence as shown in SEQ ID NO: 17 or 23, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence as shown in SEQ ID NO: 17 or 23; and

[0029] The polypeptide chains of formula (II) and (II-2) comprise the amino acid sequence shown in SEQ ID NO:18, or comprise an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:18.

[0030] In some embodiments, the bispecific antibody of the present invention, wherein the polypeptide chain of formula (III) comprises the amino acid sequence as shown in SEQ ID NO: 19, or comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence as shown in SEQ ID NO: 19.

[0031] In some embodiments, the bispecific antibody of the present invention, wherein the polypeptide chain of formula (IV) comprises the amino acid sequence as shown in SEQ ID NO:20, or comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence as shown in SEQ ID NO:20.

[0032] In some embodiments, the bispecific antibody of the present invention comprises polypeptide chains represented by formula (I) and formula (II), and polypeptide chains represented by formula (III) and formula (II-2), wherein the amino acid sequence of the polypeptide chain of formula (I) is shown in SEQ ID NO: 23, the amino acid sequences of the polypeptide chains of formula (II) and formula (II-2) are shown in SEQ ID NO: 18, and the amino acid sequence of the polypeptide chain of formula (III) is shown in SEQ ID NO: 19.

[0033] In some embodiments, the bispecific antibody of the present invention comprises polypeptide chains represented by formula (I), formula (II), and a polypeptide chain represented by formula (IV), wherein the amino acid sequence of the polypeptide chain of formula (I) is shown in SEQ ID NO: 17, the amino acid sequence of the polypeptide chain of formula (II) is shown in SEQ ID NO: 18, and the amino acid sequence of the polypeptide chain of formula (IV) is shown in SEQ ID NO: 20.

[0034] In some embodiments, the bispecific antibody of the present invention, wherein the formula (I) and the formula (II), and the formula (III) and the formula (II-2) are connected by a disulfide bond; and the formula (I) and the formula (III) or the formula (IV) are connected by a disulfide bond and the Knob into Hole structure of the CH3 domain;

[0035] Preferably, Fc1 in formula (I) is knob-Fc, and Fc2 in formula (III) or (IV) is hole-Fc; or Fc1 in formula (I) is hole-Fc, and Fc2 in formula (III) or (IV) is knob-Fc.

[0036] In some embodiments, the bispecific antibody of the present invention, wherein the CH1-Fc1 in formula (I) and the Fc2 in formula (III) are in the form of IgG, such as IgG1, IgG2, IgG3 or IgG4, and / or the CL in formula (II) and formula (II-2) is from λ or κ chain.

[0037] In yet another aspect, the present invention provides isolated nucleic acids encoding any one or more polypeptide chains of the bispecific antibodies described herein.

[0038] In another aspect, the present invention provides an expression vector comprising the nucleic acid as described herein, preferably, the expression vector is a eukaryotic expression vector.

[0039] In another aspect, the present invention provides a host cell comprising the nucleic acid as described herein or the expression vector as claimed in claim 12, preferably, the host cell is a eukaryotic cell, more preferably a mammalian cell.

[0040] In yet another aspect, the present invention provides a method for preparing a bispecific antibody as described herein, the method comprising culturing a host cell as described herein under conditions suitable for expression of a nucleic acid as described herein, and recovering the bispecific antibody from the host cell.

[0041] In yet another aspect, the present invention provides a pharmaceutical composition comprising the bispecific antibody as described herein, the polynucleotide as described herein, the expression vector as described herein, and / or the host cell as described herein, and a pharmaceutically acceptable carrier or excipient.

[0042] In another aspect, the present invention provides use of the bispecific antibody described herein, the polynucleotide described herein, the expression vector described herein, the host cell described herein and / or the pharmaceutical composition described herein in the preparation of a medicament for preventing or treating cancer, preferably, the cancer is selected from acute B lymphocytic leukemia, diffuse large B-cell lymphoma, chronic lymphocytic leukemia, follicular lymphoma, non-Hodgkin's lymphoma, chronic myeloid leukemia or Burkitt lymphoma.

[0043] In yet another aspect, the present invention provides a method for preventing or treating cancer in a subject, comprising administering to a subject in need thereof a bispecific antibody as described herein, a polynucleotide as described herein, an expression vector as described herein, a host cell as described herein, and / or a pharmaceutical composition as described herein.

[0044] In yet another aspect, the present invention provides a bispecific antibody as described herein, a polynucleotide as described herein, an expression vector as described herein, a host cell as described herein and / or a pharmaceutical composition as described herein for use in treating cancer.

[0045] In some embodiments, the cancer of the present invention is selected from acute B-cell lymphocytic leukemia, diffuse large B-cell lymphoma, chronic lymphocytic leukemia, follicular lymphoma, non-Hodgkin's lymphoma, chronic myeloid leukemia or Burkitt lymphoma.

[0046] In yet another aspect, the present invention provides a pharmaceutical combination comprising an antibody or antigen-binding fragment thereof as described herein, a polynucleotide as described herein, an expression vector as described herein, a host cell as described herein and / or a pharmaceutical composition as described herein, and one or more additional therapeutic agents.

[0047] In yet another aspect, the present invention provides methods for detecting the presence of CD3 and / or CD20 in a sample using the bispecific antibodies described herein.

[0048] In yet another aspect, the present invention provides a kit comprising the antibody as described herein or the pharmaceutical composition as described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1: Schematic diagram of the molecular structure of anti-CD3×CD20 bispecific antibodies. 1a: Schematic diagram of the structure of TZT6; 1b: Schematic diagram of the structure of TZT7.

[0050] Figure 2: ELISA detection of the binding of bispecific antibodies to human CD3ε.

[0051] Figure 3: Binding of bispecific antibodies to Raji cells.

[0052] Figure 4: Binding of bispecific antibodies to Jurkat cells.

[0053] Figure 5: Binding of bispecific antibodies to cells overexpressing cynomolgus macaque CD3e.

[0054] Figure 6: Activity of bispecific antibodies in a luciferase reporter gene system.

[0055] Figure 7: T lymphocyte activation activity of bispecific antibodies.

[0056] Figure 8: Bispecific antibodies promote the activity of T cells in killing B lymphoma cells.

[0057] Figure 9: Inhibition of B16 OVA huCD20 tumor growth by bispecific antibodies TZT6 and TZT7.

[0058] FIG10 : Inhibition of B16 OVA huCD20 tumor growth by bispecific antibody TZT7.

[0059] Figure 11: Inhibitory effect of bispecific antibodies on the Raji Mixeno model of human lymphoma. DETAILED DESCRIPTION

[0060] definition

[0061] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art.

[0062] In order to make it easier to understand the present invention, certain scientific and technological terms are specifically defined as follows. Unless otherwise clearly defined in other parts of this article, the scientific and technological terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention belongs. Regarding the definitions and terms in this area, professionals can specifically refer to Current Protocols in Molecular Biology (Ausubel). The abbreviations of amino acid residues are standard 3-letter and / or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids. The singular form used herein (including the claims) includes its corresponding plural form, unless otherwise clearly provided in the text.

[0063] The term "about" when used in conjunction with a numerical value is meant to encompass the numerical value within a range having a lower limit that is 5% less than the specified numerical value and an upper limit that is 5% greater than the specified numerical value.

[0064] The term "and / or" should be understood to mean any one of the alternatives or a combination of any two or more of the alternatives.

[0065] The term "CD3" refers to a part of the T cell receptor complex, which consists of three different chains, CD3ε, CD3δ and CD3γ. The concentration of CD3 on T cells, for example, by the fixation of anti-CD3 antibodies, leads to the activation of T cells, similar to T cell receptor-mediated activation, but independent of the specificity of the TCR clone. The vast majority of anti-CD3 antibodies recognize the CD3ε chain. The term refers to any natural CD3 from any vertebrate (including mammals such as primates (e.g., humans)) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses "full-length" unprocessed CD3 and any form of CD3 or any fragment thereof produced by intracellular processing. The term also includes naturally occurring variants of CD3, for example, splice variants or allelic variants. In a preferred embodiment, CD3 refers to the full length or fragments thereof from humans and cynomolgus monkeys (such as mature fragments thereof lacking a signal peptide). In a preferred embodiment, CD3 refers to the full length or fragments thereof from mice / rat (such as mature fragments thereof lacking a signal peptide).

[0066] The term "human CD20" or "CD20" refers to human CD20 (UniProtKB / Swiss-Prot No. P11836) and includes any variants, isoforms, and species homologs of CD20 that are naturally expressed by cells (including tumor cells) or expressed on cells transfected with a CD20 gene or cDNA. Species homologs include rhesus macaque CD20 (macaca mulatta; UniProtKB / Swiss-Prot No H9YXP1) and cynomolgus macaque.

[0067] The term "percent (%) amino acid sequence identity," or simply "identity," is defined as the percentage of amino acid residues in a candidate amino acid sequence that are identical to the amino acid residues in a reference amino acid sequence, after aligning the amino acid sequences (and introducing gaps, if necessary) to achieve maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Sequence alignment to determine percent amino acid sequence identity can be performed using various methods in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN (DNASTAR) software. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm needed to achieve maximum alignment over the full length of the compared sequences.

[0068] The term "immune response" refers to the actions of, for example, lymphocytes, antigen-presenting cells, phagocytes, granulocytes, and soluble macromolecules (including antibodies, cytokines, and complement) produced by the above cells or the liver, which result in the selective damage, destruction, or elimination from the body of invading pathogens, cells or tissues infected with pathogens, cancer cells, or, in the case of autoimmunity or pathological inflammation, normal human cells or tissues.

[0069] The term "signal transduction pathway" or "signal transduction activity" refers to a biochemical cause-and-effect relationship, typically initiated by protein-protein interactions such as the binding of a growth factor to a receptor, that results in the transmission of a signal from one part of a cell to another. Typically, the transmission involves specific phosphorylation of one or more tyrosine, serine, or threonine residues on one or more proteins in a series of reactions that lead to signal transduction. The penultimate process typically involves nuclear events that result in changes in gene expression.

[0070] The terms "activity" or "biological activity," or the terms "biological property" or "biological characteristic" are used interchangeably herein and include, but are not limited to, epitope / antigen affinity and specificity, the ability to neutralize or antagonize CD20 activity in vivo or in vitro, IC50, in vivo stability of the antibody, and the immunogenic properties of the antibody. Other identifiable biological properties or characteristics of antibodies known in the art include, for example, cross-reactivity (i.e., cross-reactivity with non-human homologs of the target peptide, or with other proteins or tissues), and the ability to maintain high protein expression levels in mammalian cells. The aforementioned properties or characteristics can be observed, measured, or assessed using techniques known in the art, including, but not limited to, ELISA, FACS, or BIACORE plasmon resonance analysis, in vitro or in vivo neutralization assays, receptor binding, cytokine or growth factor production and / or secretion, signal transduction, and immunohistochemistry of tissue sections from various sources (including humans, primates, or any other source).

[0071] The term "antibody" refers to any form of antibody having the desired biological activity. Therefore, it is used in the broadest sense and specifically includes, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), humanized antibodies, fully human antibodies, chimeric antibodies, and camelized single domain antibodies.

[0072] The term "isolated antibody" refers to the purified state of the binding compound, and in this case means that the molecule is substantially free of other biomolecules, such as nucleic acids, proteins, lipids, sugars, or other substances such as cell debris and growth medium. The term "isolated" does not imply the complete absence of such substances or the absence of water, buffers, or salts unless they are present in amounts that significantly interfere with experimental or therapeutic applications of the binding compounds described herein.

[0073] The term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic epitope. In contrast, conventional (polyclonal) antibody preparations typically include a large number of antibodies directed against (or specific for) different epitopes. The modifier "monoclonal" indicates the character of the antibody as being obtained from a population of substantially homogeneous antibodies and is not to be construed as requiring production of the antibody by any particular method.

[0074] The term "bispecific antibody" refers to an antibody molecule that can bind to two independent antigens or has binding specificity for different epitopes within the same antigen. For example, in some embodiments, one arm of the bispecific antibody molecule binds to a tumor-associated antigen and the other arm binds to an immune cell-associated antigen (e.g., CD3 molecule), which can activate and initiate cellular immune mechanisms in tumor cells.

[0075] The term "full-length antibody" refers to an immunoglobulin molecule that, when naturally occurring, contains four peptide chains: two heavy (H) chains (approximately 50-70 kDa in full length) and two light (L) chains (approximately 25 kDa in full length) linked to each other by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH). The heavy chain constant region consists of three domains, CH1, CH2 and CH3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain, CL. The VH and VL regions can be further subdivided into highly variable complementarity determining regions (CDRs) separated by more conservative regions called framework regions (FRs). Each VH or VL region consists of 3 CDRs and 4 FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of antibodies mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0076] The term "antigen-binding fragment" of an antibody ("parent antibody") includes fragments or derivatives of the antibody, typically including at least one fragment of the antigen-binding region or variable region (e.g., one or more CDRs) of the parent antibody that retains at least some of the binding specificity of the parent antibody. Examples of antibody-binding fragments include, but are not limited to, Fab, Fab', F(ab')2 and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules, such as sc-Fv; nanobodies and multispecific antibodies formed from antibody fragments. When the binding activity of an antigen is expressed on a molar concentration basis, the binding fragment or derivative generally retains at least 10% of its antigen-binding activity. Preferably the binding fragment or derivative retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or greater of the antigen binding affinity of the parent antibody. It is also contemplated that antigen-binding fragments of an antibody may include conservative or non-conservative amino acid substitutions that do not significantly alter its biological activity (referred to as "conservative variants" or "functionally conserved variants" of the antibody). The term "binding compound" refers to both an antibody and its binding fragment.

[0077] The term "single-chain Fv" or "scFv" antibody refers to an antibody fragment comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. The Fv polypeptide generally further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding.

[0078] The term "Fc" or "Fc region" or "Fc fragment" refers to a polypeptide consisting of the CH2 and CH3 domains of IgA, IgD, and IgG, or the CH2, CH3, and CH4 domains of IgE and IgM, connected by a hinge region. Although the breakdown of the Fc fragment varies, the heavy chain Fc fragment of human IgG typically refers to the polypeptide from A231 to its carboxyl terminus.

[0079] The term "hinge region" refers to the proline-rich, easily stretchable and bendable portion of the polypeptide chain located between CH1 and CH2 in an antibody. The generally accepted IgG hinge region is comprised of amino acid residues 216 to 230.

[0080] The term "domain antibody" refers to an immunologically functional immunoglobulin fragment containing only the variable region of a heavy chain or a light chain. In some cases, two or more VH regions are covalently linked with a peptide linker to form a bivalent domain antibody. The two VH regions of a bivalent domain antibody can target the same or different antigens.

[0081] The term "epitope" refers to a protein determinant that is capable of specific binding to an antibody. An epitope is typically composed of surface clustered molecules, such as amino acids or sugar side chains, and typically has specific three-dimensional structural characteristics, as well as specific charge characteristics. The difference between conformational and non-conformational epitopes is that the binding of the former, but not the latter, is lost in the presence of a denaturing solvent. An epitope may include amino acid residues that are directly involved in binding and other amino acid residues that are not directly involved in binding, such as amino acid residues that are effectively blocked or covered by the specific antigen-binding peptide (in other words, the amino acid residue is within the footprint of the specific antigen-binding peptide).

[0082] The term "bivalent antibody" contains two antigen-binding sites. In some cases, the two binding sites have the same antigen-specificity. However, a bivalent antibody can be bispecific.

[0083] The term "diabody" refers to small antibody fragments with two antigen-binding sites, which contain a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH-VL or VL-VH). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and generate two antigen-binding sites.

[0084] The term "chimeric antibody" refers to an antibody having the variable domains of a first antibody and the constant domains of a second antibody, wherein the first antibody and the second antibody are from different species. Typically, the variable domains are obtained from an antibody such as a rodent ("parent antibody"), while the constant domain sequences are obtained from a human antibody, such that the resulting chimeric antibody is less likely to induce an adverse immune response in a human subject than the parent rodent antibody.

[0085] The term "humanized antibody" refers to an antibody form containing sequences from both human and non-human (e.g., mouse, rat) antibodies. In general, a humanized antibody comprises substantially all of at least one, usually two, variable domains, wherein all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, and all or substantially all of the framework (FR) regions are framework regions of human immunoglobulin sequences. Optionally, the humanized antibody may comprise at least a portion of a human immunoglobulin constant region (Fc).

[0086] The term "fully human antibody" refers to an antibody that contains only human immunoglobulin protein sequences. If produced in a mouse, in a mouse cell, or in a hybridoma derived from a mouse cell, a fully human antibody may contain rat carbohydrate chains. Similarly, a "mouse antibody" refers to an antibody that contains only mouse immunoglobulin sequences. Alternatively, if produced in a rat, in a rat cell, or in a hybridoma derived from a rat cell, a fully human antibody may contain rat carbohydrate chains. Similarly, a "rat antibody" refers to an antibody that contains only rat immunoglobulin sequences.

[0087] An "isotype" antibody refers to the class of antibody provided by the heavy chain constant region genes (e.g., IgM, IgE, IgG such as IgG1, IgG2, or IgG4). Isotypes also include modified forms of one of these classes, where the modifications have been made to alter Fc function, for example to enhance or diminish effector function or binding to an Fc receptor.

[0088] The term "epitope" refers to the region of an antigen to which an antibody binds. An epitope can be formed by contiguous amino acids or non-contiguous amino acids juxtaposed by tertiary folding of a protein.

[0089] "Affinity" or "binding affinity" refers to the inherent binding affinity that reflects the interaction between members of a binding pair. The affinity of a molecule X for its partner Y can generally be represented by the equilibrium dissociation constant (KD), which is the ratio of the dissociation rate constant and the association rate constant (kdis and kon respectively). Affinity can be measured by common methods known in the art. One specific method used to measure affinity is the ForteBio kinetic binding assay herein.

[0090] The term "not binding" to a protein or cell means that the protein or cell does not bind to the protein or cell, or does not bind to the protein or cell with high affinity, i.e., the KD of the binding protein or cell is 1.0×10 -6 M or higher, more preferably 1.0×10 -5 M or higher, more preferably 1.0×10 -4 M or higher, 1.0×10 -3 M or higher, more preferably 1.0×10 -2 M or higher.

[0091] The term "high affinity" for IgG antibodies refers to a KD of 1.0 × 10 -6 M or less, preferably 5.0×10 -8 M or less, more preferably 1.0×10 -8 M or lower, 5.0×10 -9 M or less, more preferably 1.0×10 -9 M or lower. For other antibody subtypes, “high affinity” binding may vary. For example, “high affinity” binding for the IgM subtype is defined as a KD of 10 -6 M or less, preferably 10 -7 M or less, more preferably 10 -8 M or lower.

[0092] The terms "antibody-dependent cellular cytotoxicity," "antibody-dependent cell-mediated cytotoxicity," or "ADCC" refer to a cell-mediated immune defense in which immune system effector cells actively lyse target cells, such as cancer cells, that are bound to cell membrane surface antigens and antibodies, such as Claudin18.2 antibodies.

[0093] The term "complement-dependent cytotoxicity" or "CDC" refers to the effector function of IgG and IgM antibodies, which, when bound to surface antigens, trigger the classic complement pathway, including formation of the membrane attack complex and target cell lysis. The antibodies of the present invention, when bound to CD20, trigger CDC against cancer cells.

[0094] The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single-stranded or double-stranded form. Unless explicitly limited, the term includes nucleic acids having similar binding properties to reference nucleic acids and analogs containing known natural nucleotides that are metabolized in a manner similar to naturally occurring nucleotides (see, U.S. Patent No. 8,278,036 to Kariko et al., which discloses mRNA molecules in which uridine is replaced by pseudouridine, methods for synthesizing the mRNA molecules, and methods for delivering therapeutic proteins in vivo). Unless otherwise indicated, a specific nucleic acid sequence also implicitly includes conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as sequences explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0095] "Construct" refers to any recombinant polynucleotide molecule (such as a plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, bacteriophage, or linear or circular single-stranded or double-stranded DNA or RNA polynucleotide molecule), derived from any source, capable of integrating with a genome or autonomously replicating, constituting a polynucleotide molecule in which one or more polynucleotide molecules have been linked (i.e., operably linked) in a functionally operable manner. Recombinant constructs will typically comprise a polynucleotide of the present invention operably linked to transcriptional initiation regulatory sequences that direct transcription of the polynucleotide in a host cell. Both heterologous and non-heterologous (i.e., endogenous) promoters can be used to direct expression of the nucleic acids of the present invention.

[0096] "Vector" refers to any recombinant polynucleotide construct that can be used for the purpose of transformation (i.e., introducing heterologous DNA into a host cell). One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be connected. Another type of vector is a viral vector, in which additional DNA segments can be connected to the viral genome. Certain vectors are capable of autonomous replication in the host cell into which they are introduced (e.g., bacterial vectors and episomal mammalian vectors with bacterial origins of replication). After being introduced into the host cell, other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of the host cell and are therefore replicated together with the host genome. In addition, certain vectors are capable of directing the expression of operatively connected genes. Such vectors are referred to herein as "expression vectors."

[0097] As used herein, the term "expression vector" refers to a nucleic acid molecule capable of replicating and expressing a gene of interest when transformed, transfected, or transduced into a host cell. Expression vectors contain one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and, if desired, to provide for amplification within the host.

[0098] "Activation," "stimulation," and "treatment" as applied to a cell or receptor may have the same meaning, e.g., a cell or receptor is activated, stimulated, or treated with a ligand, unless the context dictates otherwise or explicitly. "Ligand" includes natural and synthetic ligands, e.g., cytokines, cytokine variants, analogs, muteins, and binding compounds derived from antibodies. "Ligand" also includes small molecules, e.g., peptide mimetics of cytokines and peptide mimetics of antibodies. "Activation" may refer to cell activation regulated by internal mechanisms as well as external or environmental factors. "Response," e.g., a response of a cell, tissue, organ, or organism, includes a change in biochemical or physiological behavior (e.g., concentration, density, adhesion or migration within a biological compartment, rate of gene expression, or differentiation state), where the change is related to activation, stimulation, or treatment, or to internal mechanisms such as genetic programming.

[0099] As used herein, the terms "treatment" or "treating" of any disease or condition refer, in one embodiment, to ameliorating the disease or condition (i.e., slowing or preventing or reducing the progression of the disease or at least one of its clinical symptoms). In another embodiment, "treatment" or "treating" refers to alleviating or improving at least one physical parameter, including those physical parameters that may not be discerned by the patient. In another embodiment, "treatment" or "treating" refers to regulating the disease or condition physically (e.g., stabilization of discernible symptoms), physiologically (e.g., stabilization of physical parameters), or both. Unless expressly described herein, methods for assessing the treatment and / or prevention of a disease are generally known in the art.

[0100] "Subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. As used herein, the term "cyno" or "cynomolgus monkey" refers to a cynomolgus monkey.

[0101] Administration "in combination with" one or more additional therapeutic agents includes simultaneous (concurrent) and consecutive administration in either order.

[0102] "Therapeutically effective amount," "therapeutically effective dose," and "effective amount" refer to an amount of a CD20 antibody or antigen-binding fragment thereof of the present invention that, when administered alone or in combination with other therapeutic agents to a cell, tissue, or subject, is effective to prevent or ameliorate the symptoms of one or more diseases or conditions, or the progression of such diseases or conditions. A therapeutically effective dose also refers to an amount of an antibody or antigen-binding fragment thereof sufficient to result in amelioration of symptoms, e.g., to treat, cure, prevent, or ameliorate the relevant medical condition, or to increase the rate of treatment, cure, prevention, or amelioration of such a condition. When a single active ingredient is administered to an individual, a therapeutically effective dose refers only to that ingredient. When administered in combination, a therapeutically effective dose refers to the combined amounts of the active ingredients that result in a therapeutic effect, whether administered in combination, sequentially, or simultaneously. An effective amount of a therapeutic agent will result in an improvement in a diagnostic criterion or parameter by at least 10%; typically by at least 20%; preferably by at least about 30%; more preferably by at least 40%, and most preferably by at least 50%.

[0103] "Cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Included within this definition are both benign and malignant cancers, as well as dormant tumors or micrometastases. Examples of cancer include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, and leukemias. More specific examples of such cancers include squamous cell carcinoma, lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung), peritoneal cancer, hepatocellular carcinoma, gastric cancer or stomach cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer or renal cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, and various types of head and neck cancer, as well as B-cell lymphomas (including low-grade / follicular non-Hodgkin's lymphoma (NHL), small lymphocytic (SL) NHL, intermediate-grade / follicular NHL, intermediate-grade diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small non-cleaved cell NHL, storage diseases (bulky disease) NHL, mantle cell lymphoma, AIDS-related lymphoma, and Waldenstrom's macroglobulinemia), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia, and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses, edema (such as that associated with brain tumors), and Meigs syndrome.

[0104] Antibody

[0105] The present invention provides a novel antibody molecule, which can be used for the treatment, prevention and / or diagnosis of various diseases.

[0106] The bispecific antibodies of the present invention comprise a first antigen-binding site that specifically binds to CD3 and a second antigen-binding site that specifically binds to CD20. Such antibodies may be referred to herein as, for example, "anti-CD3 / anti-CD20" or "anti-CD3×CD20" or "CD3×CD20" bispecific molecules, or other similar terms.

[0107] The terms "anti-CD3 antibody," "anti-CD3," "CD3 antibody," or "binding to CD3" refer to antibodies that are capable of binding to CD3 protein or a fragment thereof with sufficient affinity such that the antibody can be used as a diagnostic and / or therapeutic agent targeting CD3.

[0108] The terms "anti-CD20 antibody," "anti-CD20," "CD20 antibody," or "binding to CD20" refer to antibodies that bind to CD20 protein or a fragment thereof with sufficient affinity such that the antibody can be used as a diagnostic and / or therapeutic agent targeting CD20.

[0109] In some embodiments, the bispecific antibody of the present invention comprises the following polypeptide chains:

[0110] (1) The polypeptide chains represented by formula (I) and formula (II) that bind to the CD20 domain: VH1-CH1-Fc1 formula (I), VL1-CL formula (II),

[0111] (2) A polypeptide chain represented by formula (III) and formula (II-2) that binds to the CD20 and CD3 domains: VH1-CH1-L-VL2-L-VH2-L-Fc2 Formula (III); VL1-CL Formula (II-2);

[0112] wherein VH represents the heavy chain variable region; VL represents the light chain variable region; Fc comprises CH2 and CH3; CH1, CH2, and CH3 represent domains 1, 2, and 3 of the heavy chain constant region, respectively; VL represents the light chain variable region; CL represents the light chain constant region; L represents a connecting peptide, each connecting peptide being the same or different; optionally, a hinge region is present between CH1 and Fc1;

[0113] Formulas (I), (II), (II-2) and (III) are connected sequentially from the N-terminus to the C-terminus;

[0114] The antigen binding site formed by VH1 and VL1 binds to CD20, and the antigen binding site formed by VL2 and VH2 binds to CD3. In some embodiments, the bispecific antibody TZT6 of the present invention has the above structure.

[0115] In some embodiments, the bispecific antibody of the present invention comprises the following polypeptide chains:

[0116] (1) The polypeptide chains represented by formula (I) and formula (II) that bind to the CD20 domain: VH1-CH1-Fc1 formula (I), VL1-CL formula (II),

[0117] (2) A polypeptide chain represented by formula (IV) that binds to the CD3 domain: VL2-L-VH2-L-Fc2 Formula (IV).

[0118] wherein VH represents the heavy chain variable region; VL represents the light chain variable region; Fc comprises CH2 and CH3; CH1, CH2, and CH3 represent domains 1, 2, and 3 of the heavy chain constant region, respectively; VL represents the light chain variable region; CL represents the light chain constant region; L represents a connecting peptide, each connecting peptide being the same or different; optionally, a hinge region is present between CH1 and Fc1;

[0119] Formulas (I), (II) and (IV) are connected sequentially from the N-terminus to the C-terminus;

[0120] The antigen binding site formed by VH1 and VL1 binds to CD20, and the antigen binding site formed by VL2 and VH2 binds to CD3. In some embodiments, the bispecific antibody TZT7 of the present invention has the above structure.

[0121] In some embodiments, the connecting peptide of the present invention comprises the amino acid sequence (GGGGS)n, wherein n is independently selected from 1, 2, 3, 4, 5 or 6; preferably, n is independently selected from 2, 3 or 4.

[0122] In some embodiments, the VH1 of the present invention comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively, and the VL1 comprises LCDR1, LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively.

[0123] In some embodiments, the bispecific antibody of the present invention, wherein:

[0124] The VH1 comprises the amino acid sequence of SEQ ID NO:7, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:7; and

[0125] The VL1 comprises the amino acid sequence shown in SEQ ID NO: 8; or comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 8.

[0126] In some embodiments, the bispecific antibody of the present invention, wherein the VH2 comprises HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11, respectively, and the VL2 comprises LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14, respectively.

[0127] In some embodiments, the bispecific antibody of the present invention, wherein:

[0128] The VH2 comprises the amino acid sequence of SEQ ID NO: 15, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 15; and

[0129] The VL2 comprises the amino acid sequence shown in SEQ ID NO: 16; or comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 16.

[0130] In some embodiments, the bispecific antibody of the present invention, wherein:

[0131] The polypeptide chain of formula (I) comprises the amino acid sequence as shown in SEQ ID NO: 17 or 23, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence as shown in SEQ ID NO: 17 or 23; and

[0132] The polypeptide chains of formula (II) and formula (II-2) comprise the amino acid sequence shown in SEQ ID NO:18, or comprise an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:18.

[0133] In some embodiments, the bispecific antibody of the present invention, wherein the polypeptide chain of formula (III) comprises the amino acid sequence as shown in SEQ ID NO: 19, or comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence as shown in SEQ ID NO: 19.

[0134] In some embodiments, the bispecific antibody of the present invention, wherein the polypeptide chain of formula (IV) comprises the amino acid sequence as shown in SEQ ID NO:20, or comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence as shown in SEQ ID NO:20.

[0135] In some embodiments, the bispecific antibody of the present invention, wherein the amino acid sequence of the polypeptide chain of formula (I) is shown as SEQ ID NO: 23, the amino acid sequences of the polypeptide chains of formula (II) and formula (II-2) are shown as SEQ ID NO: 18, and the amino acid sequence of the polypeptide chain of formula (III) is shown as SEQ ID NO: 19.

[0136] In some embodiments, the bispecific antibody of the present invention, wherein the amino acid sequence of the polypeptide chain of formula (I) is shown as SEQ ID NO: 17, the amino acid sequence of the polypeptide chain of formula (II) is shown as SEQ ID NO: 18, and the amino acid sequence of the polypeptide chain of formula (IV) is shown as SEQ ID NO: 20.

[0137] In some embodiments, the antibody molecules of the invention are humanized.Different methods for humanizing antibodies are known to the skilled person, as reviewed by Almagro & Fransson, the contents of which are incorporated herein by reference in their entirety (Almagro JC and Fransson J (2008) Frontiers in Bioscience 13: 1619-1633).

[0138] In some embodiments, the antibody molecules of the present invention are human antibodies or humanized antibodies. Human antibodies or humanized antibodies can be prepared using various techniques known in the art.

[0139] In some embodiments, the antibody molecules of the invention are chimeric antibodies.

[0140] In some embodiments, at least a portion of the framework sequence of an antibody molecule of the invention is a human consensus framework sequence.

[0141] In one embodiment, the antibody molecules of the present invention also encompass antigen-binding fragments thereof, such as the following antibody fragments: Fab, Fab', F(ab')2, Fv, scFv or sdAb.

[0142] The present invention conducts in-depth analysis and comparison of multiple anti-human CD3 antibodies, optimizes the sequences and performs humanization transformation to obtain the humanized antibody JSCD3.

[0143] The humanized anti-CD3 antibody described in this invention exhibits enhanced tumor-killing activity when used in the preparation of bispecific antibodies. This humanized sequence disclosed by the present invention offers advantages and features over humanized sequences from other companies. It exhibits enhanced tumor-killing activity when used in the preparation of new anti-tumor drugs, and its physicochemical stability is significantly improved, making it more suitable for the screening and development of anti-tumor drugs. This innovative achievement contributes to the beneficial effects and extremely high medical application value of this invention.

[0144] The precise amino acid sequence boundaries of the variable region CDRs of the antibodies of the invention can be determined using any of a number of well-known schemes, including Chothia (Chothia et al. (1989) Nature 342:877-883; Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), based on the three-dimensional structure of the antibody and the topology of the CDR loops; Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 4th ed., US Department of Health and Human Services, National Institutes of Health (1987)), based on the variability of antibody sequences; AbM (University of Bath); Contact (University College London); the international ImMunoGeneTics database (IMGT) (1999 Nucleic Acids Research, 27, 209-212), and the North CDR definition based on affinity propagation clustering using a large number of crystal structures.

[0145] Unless otherwise indicated, the boundaries of the CDRs of the antibodies of the invention can be determined by one skilled in the art according to any scheme in the art (eg, different assignment systems or combinations).

[0146] In some embodiments, the amino acid changes described herein include amino acid deletions, insertions, or substitutions. Preferably, the amino acid changes described herein are amino acid substitutions, preferably conservative substitutions.

[0147] In a preferred embodiment, the amino acid changes described herein occur in regions outside of the CDRs (e.g., in the FRs). More preferably, the amino acid changes described herein occur in regions outside of the heavy chain variable region and / or outside of the light chain variable region.

[0148] In some embodiments, the substitution is a conservative substitution. A conservative substitution refers to the substitution of one amino acid by another amino acid within the same class, such as an acidic amino acid by another acidic amino acid, a basic amino acid by another basic amino acid, or a neutral amino acid by another neutral amino acid.

[0149] In some embodiments, the bispecific antibodies or antigen-binding fragments thereof of the invention include those having amino acid sequences that have been mutated by amino acid deletions, insertions, or substitutions, but still have at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the above-described antibodies (particularly in the CDR regions depicted in the above sequences). In some embodiments, the antibodies of the invention have no more than 1, 2, 3, 4, or 5 amino acid mutations in the CDR regions by amino acid deletions, insertions, or substitutions when compared to the CDR regions depicted in the specific sequences.

[0150] In some embodiments, one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein to generate an Fc region variant. The Fc region variant can comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) comprising an amino acid modification (e.g., substitution, deletion, and insertion) at one or more amino acid positions.

[0151] In some embodiments, the number of cysteine ​​residues in an antibody can be altered to modify antibody properties. For example, the hinge region of CH1 can be modified to alter (eg, increase or decrease) the number of cysteine ​​residues in the hinge region.

[0152] The term "Knob into Hole structure" refers to mutating the hydrophobic amino acid of CH3 of the antibody Fc. The side chain amino acid of one CH3 chain is mutated to form a relatively large hydrophobic amino acid (knob) to strengthen the hydrophobic force; the other CH3 side chain amino acid is mutated to form a small amino acid (hole) to reduce steric hindrance; after mutation, the CH3 with Knob and the CH3 with Hole form Knob into in the form of hydrophobic interaction. Hole structure (KiH) is conducive to the formation of heavy chain heterodimers; KiH mutations mainly occur in the internal hydrophobic amino acids of the spatial structure of the CH3 domain. After the mutation, the exposed amino acids are almost unchanged, so it does not affect the effector function of Fc and the immunogenicity caused. The term "knob-Fc" refers to a point mutation containing T366W in the Fc region of an antibody to form a knob-like spatial structure. Correspondingly, "hole-Fc" refers to an antibody that contains point mutations of T366S, L368A, and Y407V in the Fc region to form a hole-like spatial structure. To further promote heterodimer formation, point mutations S354C and Y349C can be introduced into the knob-Fc and hole-Fc, respectively, to further promote heterodimer formation through disulfide bonds. Furthermore, to weaken binding to Protein A, point mutations H435R and Y436F can be introduced into the hole-Fc, respectively.

[0153] The bispecific antibodies of the present invention may comprise two Fc regions, each of which is part of a separate antibody heavy chain. Fc1 in Formula (I) and Fc2 in Formula (III) or Formula (IV) may have identical sequences, except for mutations in the CH3 domain intended to promote or facilitate purification of the heterodimeric (i.e., bispecific) molecule.

[0154] In some embodiments, in the bispecific antibody of the present invention, the compounds of formula (I) and (II), and the compounds of formula (III) and (II-2) are linked by disulfide bonds.

[0155] In some embodiments, the bispecific antibodies of formula (I) and formula (III) can be directly or indirectly linked to each other. In some embodiments, the bispecific antibodies of formula (I) and formula (III) can be linked to each other via a linker. In some embodiments, the linker is a peptide linker.

[0156] In some embodiments, the bispecific antibodies of the present invention are linked by a disulfide bond in the Fc region and a knob-in-hole structure in the CH3 domain. Preferably, Fc1 in Formula (I) is a knob-Fc, and Fc1 in Formula (III) or (IV) is a hole-Fc; or Fc1 in Formula (I) is a hole-Fc, and Fc2 in Formula (III) or (IV) is a knob-Fc.

[0157] In some embodiments, the bispecific antibody of the present invention, wherein the CH1-Fc1 in formula (I) and the Fc2 in formula (III) are in the form of IgG, such as IgG1, IgG2, IgG3 or IgG4, and / or the CL in formula (II) and formula (II-2) is from λ or κ chain.

[0158] The Fc region of the bispecific antibody of the invention may be a human Fc region. The Fc region of the bispecific antibodies of the invention can be of any isotype, including but not limited to IgGl, IgG2, IgG3 or IgG4. In some embodiments, the Fc regions of the first and second antibodies are both of the IgG1 isotype. In some embodiments, the Fc regions of the first and second antibodies are both of the IgG4 isotype. In some embodiments, one of the Fc regions of the antibody is of the IgG1 isotype and the other is of the IgG4 isotype. In the latter embodiment, the resulting bispecific antibody contains the Fc region of IgG1 and the Fc region of IgG4 and may therefore have interesting intermediate properties with respect to activation of effector functions.

[0159] In some embodiments, the antibodies provided herein can be further modified to contain other non-proteinaceous moieties known in the art and readily available. Suitable moieties for antibody derivatization include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), and dextran or poly (n-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.

[0160] Antibody expression

[0161] In yet another aspect, the present invention provides nucleic acids encoding any of the above antibodies or fragments thereof or any of their chains. In one embodiment, the polynucleotides may comprise polynucleotides encoding the amino acid sequences of the light chain variable region and / or heavy chain variable region of an antibody, or polynucleotides encoding the amino acid sequences of the light chain and / or heavy chain of an antibody.

[0162] In some embodiments, the nucleic acid of the present invention comprises a nucleic acid encoding an amino acid sequence selected from any one of SEQ ID NOs: 1-20, or a nucleic acid encoding an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from any one of SEQ ID NOs: 1-20.

[0163] In yet another aspect, the present invention provides an expression vector comprising a nucleic acid as described herein, preferably a eukaryotic expression vector. In some embodiments, the nucleic acid as described herein is contained in one or more expression vectors. Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phages, or yeast artificial chromosomes (YACs).

[0164] In yet another aspect, the present invention provides a host cell comprising a nucleic acid as described herein or an expression vector as described herein, preferably, the host cell is a eukaryotic cell, more preferably a mammalian cell (e.g., a CHO cell or a 293 cell). In another embodiment, the host cell is prokaryotic.

[0165] In one embodiment, the present invention provides a method for preparing an anti-CD3×CD20 bispecific antibody of the present invention, wherein the method comprises introducing an expression vector into a mammalian host cell and then culturing the host cell for a period of time sufficient to allow expression of the antibody in the host cell, or more preferably, secretion of the antibody into the culture medium in which the host cell is grown, to produce the antibody. The antibody can be recovered from the culture medium using standard protein purification methods.

[0166] The present invention provides mammalian host cells for expressing the recombinant antibodies of the present invention, including many immortalized cell lines available from the American Type Culture Collection (ATCC). These especially include Chinese hamster ovary (CHO) cells, NSO, SP2 / 0 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells, A549 cells, 293T cells and many other cell lines. Mammalian host cells include humans, mice, rats, dogs, monkeys, pigs, goats, cattle, horses and hamster cells. Particularly preferred cell lines are selected by determining which cell line has high expression levels.

[0167] Antibodies likely to be expressed by different cell lines or in transgenic animals may have different glycosylations from each other. However, all antibodies encoded by the nucleic acid molecules provided herein or comprising the amino acid sequences provided herein are part of the present invention, regardless of the glycosylation of the antibody. Similarly, in certain embodiments, afucosylated antibodies are advantageous because they generally have more potent efficacy in vitro and in vivo than their fucosylated counterparts and are unlikely to be immunogenic because their sugar structures are normal components of natural human serum IgG.

[0168] Pharmaceutical Compositions and Preparations

[0169] In yet another aspect, the present invention provides a pharmaceutical composition comprising an anti-CD3×CD20 bispecific antibody or an antigen-binding fragment thereof as described herein, and a pharmaceutically acceptable carrier or excipient.

[0170] It should be understood that the anti-CD3×CD20 antibody or its pharmaceutical composition provided by the present invention can incorporate suitable carriers, excipients, and other reagents in the formulation for co-administration, thereby providing improved metastasis, delivery, tolerance, etc.

[0171] The term "pharmaceutical composition" refers to a formulation that allows the biologically active form of the active ingredient contained therein to exist and does not contain additional ingredients that are unacceptably toxic to the subject to which the formulation is administered.

[0172] A pharmaceutical preparation comprising the anti-CD3×CD20 bispecific antibody described herein can be prepared by mixing the anti-CD3×CD20 bispecific antibody of the present invention having the desired purity with one or more optional pharmaceutical excipients (Remington's Pharmaceutical Sciences, 16th Edition, edited by Osol, A. (1980)), preferably in the form of an aqueous solution or a lyophilized preparation.

[0173] The pharmaceutical composition or preparation of the present invention may also comprise one or more other active ingredients that are required for the particular indication being treated, preferably those active ingredients having complementary activities that do not adversely affect each other. In some embodiments, the other active ingredients are chemotherapeutic agents, immune checkpoint inhibitors, growth inhibitors, antibiotics, or various known anti-tumor or anti-cancer agents, and the active ingredients are present in a suitable combination in an amount effective for the intended use.

[0174] In some embodiments, the pharmaceutical composition of the present invention further comprises a composition encoding the nucleic acid of the anti-CD3×CD20 bispecific antibody.

[0175] In yet another aspect, the present invention provides a pharmaceutical combination comprising the anti-CD3×CD20 bispecific antibody or antigen-binding fragment thereof, or the pharmaceutical composition described herein, and one or more additional therapeutic agents.

[0176] In yet another aspect, the present invention provides a kit comprising the antibody or antigen-binding fragment thereof as described herein, the polynucleotide as described herein, the expression vector as described herein, the host cell as described herein, or the pharmaceutical composition as described herein.

[0177] Medical uses

[0178] In yet another aspect, the present invention provides use of the anti-CD3×CD20 bispecific antibody or antigen-binding fragment thereof, or the pharmaceutical composition described herein, in the preparation of a medicament for preventing and / or treating cancer.

[0179] In yet another aspect, the present invention provides an anti-CD3×CD20 bispecific antibody or antigen-binding fragment thereof as described herein, or a pharmaceutical composition as described herein, for use in preventing and / or treating cancer.

[0180] In yet another aspect, the present invention provides a method for preventing and / or treating cancer, comprising administering the anti-CD3×CD20 bispecific antibody or the pharmaceutical composition described herein to a subject in need thereof.

[0181] In some embodiments, the cancer of the present invention is selected from acute B-cell lymphocytic leukemia, diffuse large B-cell lymphoma, chronic lymphocytic leukemia, follicular lymphoma, non-Hodgkin's lymphoma, chronic myeloid leukemia or Burkitt lymphoma.

[0182] The subject can be a mammal, e.g., a primate, preferably a higher primate, e.g., a human (e.g., a patient suffering from a disease described herein or at risk of suffering from a disease described herein). In one embodiment, the subject suffers from a disease described herein (e.g., a tumor described herein) or is at risk of suffering from a disease described herein. In certain embodiments, the subject receives or has received other treatments, e.g., chemotherapy and / or radiation therapy.

[0183] In some embodiments, the administration of the present invention includes, but is not limited to, oral, intravenous, subcutaneous, intramuscular, intraarterial, intraarticular (e.g., in arthritic joints), by inhalation, aerosol delivery, or intratumoral administration.

[0184] In some embodiments, the present invention provides for co-administering to a subject therapeutically effective amounts of one or more therapies (eg, treatment modalities and / or other therapeutic agents).

[0185] In some embodiments, the methods or uses provided herein further comprise administering one or more therapies (e.g., treatment modalities and / or other therapeutic agents) to an individual. The antibodies of the present invention may be used alone or in combination with other therapeutic agents in a therapy. For example, they may be co-administered with at least one additional therapeutic agent.

[0186] In some embodiments, the above treatment modalities include surgery, radiotherapy, local irradiation or focused irradiation, etc. In some embodiments, the above therapeutic agent is selected from chemotherapeutic agents, cytotoxic agents, vaccines, other antibodies, anti-infective agents or immunomodulators.

[0187] Methods for diagnosis and detection

[0188] In yet another aspect, the present invention provides methods for detecting the presence of CD3 or CD20 in a sample using any antibody or antigen-binding fragment thereof as described herein. The term "detection" as used herein includes quantitative or qualitative detection. Exemplary detection methods can involve immunohistochemistry, immunocytochemistry, flow cytometry (e.g., FACS), magnetic beads complexed with antibody molecules, ELISA assays, PCR-technology (e.g., RT-PCR). In some embodiments, the sample is a biological sample. In some embodiments, the biological sample is blood, serum, or other liquid samples of biological origin. In certain embodiments, the biological sample comprises cells or tissues. In some embodiments, the biological sample is from a hyperproliferative or cancerous lesion.

[0189] In one aspect, the present invention provides a diagnostic method for detecting the presence of CD3 or CD20 antigens in a biological sample, such as serum, semen, or urine, or a tissue biopsy sample (e.g., from a hyperproliferative or cancerous lesion), in vitro or in vivo. The diagnostic method comprises: ① contacting the sample (and optionally a control sample) with an antibody molecule as described herein or administering the antibody molecule to a subject under conditions that allow the interaction to occur and ② detecting the formation of a complex between the antibody molecule and the sample (and optionally a control sample). The formation of a complex indicates the presence of the relevant antigen and can indicate the suitability or need for treatment and / or prevention as described herein.

[0190] In some embodiments, the present invention provides a diagnostic kit comprising an antibody molecule or antigen-binding fragment thereof described herein or a pharmaceutical composition described herein and instructions for use.

[0191] The present invention includes all combinations of the specific embodiments described. Further embodiments of the present invention and the full scope of its applicability will become apparent from the detailed description provided below. However, it should be understood that although the detailed description and specific examples indicate preferred embodiments of the present invention, these descriptions and examples are provided by way of illustration only, as various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art from this detailed description. All publications, patents, and patent applications cited herein, including citations, are incorporated herein by reference in their entirety for all purposes.

[0192] The present invention uses the following abbreviations:

[0193] his-tag stands for histidine tag;

[0194] Fc tag stands for fragment crystallizable tag;

[0195] ECD stands for extracellular domain;

[0196] PEI stands for polyethyleneimine;

[0197] BSA stands for bovine serum albumin;

[0198] PBS stands for phosphate-buffered saline;

[0199] FBS stands for fetal bovine serum;

[0200] CFSE stands for carboxyfluorescein diacetate succinimidyl ester;

[0201] APC stands for ectopic phycocyanin;

[0202] NA-PE represents phycoerythrin-labeled neutravidin;

[0203] PE stands for phycoerythrin;

[0204] TMB stands for 3,3',5,5'-tetramethylbenzidine;

[0205] PBST stands for Phosphate Buffered Saline Tween.

[0206] Example

[0207] The present invention is illustrated by the following examples, but is not intended to be limiting thereof. The present invention has been described in detail herein, and specific embodiments thereof are disclosed. It will be apparent to those skilled in the art that various changes and modifications can be made to the specific embodiments of the present invention without departing from the spirit and scope of the invention.

[0208] Example 1. Construction of bispecific antibody expression vector

[0209] 1.1 Anti-CD3 Antibodies

[0210] The present invention analyzes multiple anti-human CD3 antibodies, optimizes the sequence of one of the mouse-derived antibodies, SP34, and performs humanization transformation to obtain a humanized anti-CD3 antibody, JSCD3.

[0211] SP34 is a murine monoclonal antibody against human CD3, the heavy chain amino acid sequence of which is SEQ ID NO: 24, and the light chain amino acid sequence of which is SEQ ID NO: 25.

[0212] The amino acid sequence of the humanized anti-CD3 antibody JSCD3 is as follows:

[0213] JSCD3VH: SEQ ID NO: 15; the amino acid sequences of its heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs: 9, 10 and 11, respectively.

[0214] JSCD3VL: SEQ ID NO: 16; the amino acid sequences of its light chain complementary determining regions LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs: 12, 13 and 14, respectively.

[0215] The full-length amino acid sequence of JSCD3 is as follows:

[0216] JSCD3 HC: SEQ ID NO: 21

[0217] JSCD3 LC: SEQ ID NO: 22

[0218] 1.2 Construction of bispecific antibody expression vector

[0219] Bispecific antibody molecule TZT6:

[0220] The expression vectors were constructed by transiently expressing HXT2-JS CD20 LC-2, HXT4s-JS CD20 HC-2b, and HX4-JSCD3ScFv NVL-Mut. The first expression vector, HXT2-JS CD20 LC-2, was synthesized by GenScript Biotech, which was digested with BSPQI and ligated into the HXT2 vector (a Junshi Biosciences proprietary vector derived from pCDNA3.1). The second expression vector, HXT4s-JS CD20 HC-2b, was synthesized by GenScript Biotech, which was digested with HindIII and NheI and ligated into HXT4s-Mut-b (a Junshi Biosciences proprietary vector derived from pCDNA3.1). GenScript Biotech was commissioned to synthesize the gene encoding JSCD3ScFv, which was digested with HindIII and NheI and ligated into the HX4-FC-mut-h vector (Junshi Biosciences' independently modified vector, derived from pCDNA3.1) to obtain the third expression vector HX4-JSCD3ScFv NVL-Mut h.

[0221] Bispecific antibody molecule TZT7:

[0222] The expression vectors were constructed by transiently expressing HXT2-JS CD20 LC-2, HXT4s-JS CD20HC-2 Mut h, and HX4-JS CD20 HC-2 JS CD3ScFv NVL-G4 FC b V2. The first expression vector, HXT2-JS CD20 LC-2, was synthesized by GenScript Biotech, which digested the gene encoding JSCD20 LC-2 with BSPQI and ligated into the HXT2 vector. The second expression vector, HXT4s-JS CD20 HC-2h, was synthesized by GenScript Biotech, which digested the gene encoding JSCD20 HC-2 with HindIII and NheI and ligated into HXT4s-Mut-h. GenScript Biotech was commissioned to synthesize the gene encoding JSCD3ScFv, which was digested with HindIII and NheI and ligated into the HX4-JSCD20HC-2-G4 FC mut b vector to obtain the third expression vector HX4-JS CD20 HC-2 JS CD3ScFv NVL-G4 FC b V2.

[0223] Example 2. Expression and purification of bispecific antibody molecules

[0224] 2.1 Expression of bispecific antibody molecules

[0225] Transient expression and purification of bispecific antibody molecules TZT6 and TZT7: The three plasmids constructed above were extracted using an endotoxin-controlled kit for subsequent mammalian cell expression. Count the CHO-K1 cells in culture (which have been modified at the genome level to make them suitable for transient expression). When the cell density is between 2 and 6*10 6 / mL, and then subculture and expand using CD CHO medium. The day before transfection, dilute the cell density to 1.5-2.0*10 6 / mL, the next day when the cell density reached about 3.5*10 6 Perform transfection at 1 / 10 the transfection volume of culture medium, then add 1-2 μg / mL of plasmid, and finally 3-14 μg / mL of PEI. Mix thoroughly and incubate at room temperature for no more than 30 minutes. Slowly add the transfection mixture to the pre-treated cells, mixing thoroughly. Incubate the transfection mixture in a shaker at 36.5°C, 120 rpm, and 7% CO2. The culture period is 6-10 days after transfection, with feeding every two days.

[0226] 2.2 Purification of bispecific antibody molecules

[0227] After the transfection mixture is incubated, the precipitate is discarded by centrifugation at 1000 g for 10 minutes, followed by centrifugation at 12000 g for 30 minutes to collect the cell supernatant and sterile filtration. Purification is performed using an AKTA Avant purifier. A column packed with Praesto Jetted A50 affinity medium is first subjected to CIP with 0.1 M NaOH for 15-20 minutes. The column is then equilibrated with PBS buffer for 3-5 CV before loading the sample. Following loading, the column is washed with affinity chromatography wash buffer and the target protein is eluted with acetic acid-sodium acetate buffer (pH 3.8). The sample is neutralized with 1 M Tris buffer, adjusted to pH 5.5, and a sample is retained for SEC-HPLC analysis to test the sample purity. The remaining samples were purified using the weakly cationic filler EMD COOM. The equilibrium solution was a pH 5.5 50mM acetic acid-sodium acetate system, and the eluent was a pH 5.5 50mM acetic acid-sodium acetate + 1M NaCl buffer system. A linear elution method was used to collect the target protein. The final SEC-HPLC monomer purity reached over 95%, yielding the bispecific antibody molecules TZT6 and TZT7, respectively. The schematic structure of the bispecific antibody molecule TZT6 is shown in Figure 1a, and the schematic structure of the bispecific antibody molecule TZT7 is shown in Figure 1b.

[0228] The amino acid sequence of the bispecific antibody molecule TZT6 is shown below:

[0229] HC1(HXT4s-JS CD20 HC-2b):SEQ ID NO:17

[0230] LC(HXT2-JS CD20 LC-2):SEQ ID NO:18

[0231] HC2(HX4-JSCD3ScFv NVL-Mut h):SEQ ID NO:20

[0232] The amino acid sequence of the bispecific antibody molecule TZT7 is shown below:

[0233] HC1(HXT4s-JS CD20HC-2 Mut h): SEQ ID NO:23

[0234] LC(HXT2-JS CD20 LC-2): SEQ ID NO:18

[0235] HC2 (HX4-JS CD20 HC-2 JS CD3ScFv NVL-G4 FC b V2): SEQ ID NO: 19.

[0236] The CDR and variable region amino acid sequences of the bispecific antibody molecules TZT6 and TZT7 are shown in Table 1 .

[0237] Table 1: CDR and variable region amino acid sequences of bispecific antibody molecules (KABAT scheme)

[0238] Example 3. Stability of bispecific antibodies

[0239] 3.1 Thermal stability of bispecific antibodies

[0240] 1. Test Purpose

[0241] The thermal stability of the bispecific antibody of the present invention was tested by using DSF (differential scanning fluorescence) to investigate the stability of the bispecific antibody in a conventional formulation (20 mM citric acid-sodium citrate buffer, 50 mM sodium chloride, 140 mM mannitol, pH 6.0).

[0242] 2. Test process and results

[0243] The antibody samples were added to the aforementioned buffer at a concentration of approximately 4 mg / ml and assayed using DSF. The results are shown in Table 2. In the aforementioned buffer, the thermal transition temperatures (Tm) of the bispecific antibodies TZT6 and TZT7 of the present invention were both above 60°C, demonstrating good thermal stability.

[0244] Table 2 Thermal stability determination of bispecific antibodies

[0245] 3.2 High-temperature stability of bispecific antibodies

[0246] 1. Test Purpose

[0247] The stability of bispecific antibodies was investigated under high temperature conditions.

[0248] 2. Test process and results

[0249] Add the antibody sample to a pH 6.0 buffer system (20mM citric acid-sodium citrate / 50mM sodium chloride / 140mM mannitol), control the sample concentration to approximately 4mg / ml, and dispense 500μl / tube into vials. Place in a 40°C thermostat to examine the stability of 0W, 2W and 4W, and send samples for testing according to Table 3. Stability was evaluated through the following parameters: (a) SEC-HPLC (size exclusion chromatography) to measure the content of antibody monomers, aggregates or fragments; (b) two CE-SDS (sodium dodecyl sulfate capillary electrophoresis) methods to detect the molecular weight of the antibody; (c) ELISA and reporter gene methods to detect the biological activity of the antibody (see Example 4 and Example 8 for experimental methods);

[0250] The test results showed that after being placed at 40°C for 4 weeks, the purity (SEC-HPLC method, R-CE-SDS (reducing electrophoresis method), NR-CE-SDS (non-reducing electrophoresis method)) and biological activity of the bispecific antibodies TZT6 and TZT7 of the present invention did not change significantly, and they had good thermal stability. The specific results are shown in Table 3.

[0251] Table 3 Stability of bispecific antibodies during 4 weeks at 40°C

[0252] Example 4: ELISA detection of binding of bispecific antibodies to human CD3ε

[0253] 1. Test Purpose

[0254] The binding of the bispecific antibody of the present invention to human CD3ε was detected by ELISA.

[0255] 2. Testing process

[0256] a. Coat a 96-well plate with 100 μl / well of recombinant human CD3ε (Novoprotein, C578) at a concentration of 1.0 μg / ml and incubate at 37°C for 1.5 hours.

[0257] b. Wash four times with 300 μl / well 1× PBST, add 200 μl / well 2% BSA, and block at 37°C for 1.5 hours.

[0258] c. Wash four times with 300 μl / well 1× PBST. Add serially diluted bispecific antibodies TZT6 and TZT7 and a negative control antibody (anti-KLH hIgG4) (starting concentration 10 μg / ml, 2.5-fold serial dilutions with 12 concentration points, 100 μl / well), and incubate at 37°C for 1 hour.

[0259] d. Wash four times with 300 μl / well 1× PBST.

[0260] e. Add 100 μl / well of 5000-fold diluted horseradish peroxidase (HRP)-conjugated mouse anti-human IgG4 Fc antibody (Southern Biotech, 9200-05) and incubate at 37°C for 1 hour.

[0261] f. Wash four times with 300 μl / well 1× PBST.

[0262] g. Add 0.1 mg / ml TMB to 100 μl / well, incubate at 37°C for 15 minutes, and then add 2 M hydrochloric acid solution to 100 μl / well to terminate the reaction.

[0263] h. The absorbance was measured at 450 nm and 620 nm on a microplate reader, and the data were analyzed using Graphpad Prism7.

[0264] 3. Test results

[0265] As shown in FIG2 , the bispecific antibodies TZT6 and TZT7 of the present invention have strong binding to human CD3ε, EC 50 21.4ng / mL and 85.63ng / mL respectively.

[0266] Example 5: Binding of bispecific antibodies to Raji cells

[0267] Raji cells (a human B lymphoma cell, CD20 positive) were incubated with different concentrations of bispecific antibodies TZT7 and TZT6 (starting at 100 μg / ml, 3-fold dilution, a total of 12 concentration gradients) at 4°C for 30 minutes, then washed and incubated with fluorescently labeled secondary antibodies at 4°C in the dark for 30 minutes. Finally, cells were collected using a flow cytometer (BD CantoⅡ) to detect fluorescent antibodies bound to the cell surface. The raw data were analyzed using FlowJo to obtain the MFI value, and the antibody dose-dependent binding curve was fitted using GraphPad (Figure 3) and the EC was calculated. 50 , the positive and negative controls were REGN1979 (Regeneron Pharmaceuticals) and anti-KLH hu-IgG4 antibody, respectively.

[0268] As shown in Figure 3, TZT7, TZT6, and REGN1979 can bind to CD20 on the surface of Raji cells with high affinity. 50 The concentrations of TZT7 and TZT6 were 0.5152 μg / mL, 3.673 μg / mL and 30.11 μg / mL respectively. The binding ability of TZT7 and TZT6 to Raji cells was significantly better than that of the positive control REGN1979.

[0269] Example 6: Binding of bispecific antibodies to Jurkat cells

[0270] Jurkat cells (a type of human T lymphoma cell, CD3 positive) were incubated with different concentrations of bispecific antibodies TZT6 and TZT67 (starting at 100 μg / ml, 3-fold dilution, a total of 12 concentration gradients) at 4°C for 30 minutes, then washed and incubated with fluorescently labeled secondary antibodies at 4°C in the dark for 30 minutes. Finally, cells were collected using a flow cytometer (BD CantoⅡ) to detect fluorescent antibodies bound to the cell surface. The raw data were analyzed using FlowJo to obtain the MFI value, and the antibody dose-dependent binding curve was fitted using GraphPad (Figure 4) and the EC was calculated. 50 , the positive and negative controls were REGN1979 (Regeneron Pharmaceuticals) and anti-KLH hu-IgG4 antibody, respectively.

[0271] As shown in Figure 4, REGN1979, TZT6, and TZT7 can all bind to human CD3 on the surface of Jurkat cells. 50 They were 4.698μg / mL, 4.442μg / mL and 26.59μg / mL respectively.

[0272] Example 7: Binding of bispecific antibodies to cells overexpressing cynomolgus monkey CD3e

[0273] CHO Cyno CD3e cells (overexpressing cynomolgus monkey CD3e on the surface of CHO cells) were incubated with different concentrations of bispecific antibodies TZT7 and TZT6 (starting concentration of 100 μg / ml, 3-fold dilution, a total of 12 concentration gradients) at 4°C for 30 minutes, then washed and incubated with fluorescently labeled secondary antibodies at 4°C in the dark for 30 minutes. Finally, the cells were collected by flow cytometry (BD CantoⅡ) and the fluorescent antibodies bound to the cell surface were detected. The raw data were analyzed by FlowJo to obtain the MFI value, and the antibody dose-dependent binding curve was fitted by GraphPad (Figure 5) and the EC was calculated. 50 , the positive and negative controls were JSCD3 (anti-CD3 antibody) and anti-KLH hu-IgG4 antibody, respectively.

[0274] As shown in FIG5 , both bispecific antibodies TZT7 and TZT6 were able to bind to cynomolgus monkey CD3e.

[0275] Example 8: Activity detection of bispecific antibodies in luciferase reporter gene system

[0276] Target cells Raji (a human B lymphoma cell line, CD20 positive) and effector cells Jurkat NFAT (stable expression of luc2P / NFAT-RE) were cultured at 5×10 4 cells and 1×10 5 The number of cells was added to a 96-well flat-bottom white plate (Corning, Cat#3917). Then, the bispecific antibodies TZT7, TZT6 and REGN1979 (starting concentration of 1 μg / ml, 3-fold dilution, a total of 12 concentration gradients) were added to the cell plate using experimental buffer (RPMI 1640 (1×) + 2% FBS) and incubated in a 37°C incubator for 4-6 hours. Finally, ONE-Glo luciferase detection reagent (Promega) was added to the cell-antibody mixture system and the chemiluminescent signal was detected using a multifunctional microplate reader (TECAN M1000pro). The four-parameter regression curve was fitted by GraphPad prism software to calculate the EC 50 value.

[0277] As shown in Figure 6, TZT7, TZT6, and REGN1979 (Regeneron Pharmaceuticals) have strong T cell activation activity in the luciferase reporter gene system composed of effector cells Jurkat NFAT and target cells Raji cells. 50 The T cell activation activity of TZT7 and TZT6 were significantly better than that of REGN1979, which were 0.2087 ng / mL, 0.6057 ng / mL and 3.688 ng / mL respectively.

[0278] Example 9: Study on the activation activity of bispecific antibodies on T lymphocytes

[0279] Anti-CD3 / CD20 bispecific antibodies can effectively promote the activation of T lymphocytes in peripheral blood mononuclear cells. Generally, upregulated expression of the cell surface marker CD69 is a sign of early T cell activation, while upregulated expression of the cell surface marker CD25 is a sign of late T cell activation. This experiment used the increased proportion of CD25 and CD69 double-positive populations to evaluate the activation effect of anti-CD3 / CD20 bispecific antibodies on T cells in this study.

[0280] Human total T cells were isolated and purified from commercial PBMC (Allcells, Cat#PB004F-C) using a human total T cell purification kit (Miltenyi Biotec, Cat#130-096-535); the purified human total T cells (1×10 per well) were then 5 cells) and Raji cells (2×10 4 Cells) and gradient dilutions of TZT7 and TZT6 or control samples (starting concentration of 10 μg / ml, 5-fold dilution, a total of 12 concentration gradients) were incubated in 96-well plates at 37°C for 24 hours. Finally, the cells were collected and stained with APC anti-human CD8a (Biolegend, Cat#301049), PE anti-human CD25 (Biolegend, Cat#302606), and BV421 anti-human CD69 (BD, Cat#562884), and then detected by flow cytometry (BD, CantoⅡ). CD8 was exported using FlowJo software. + CD25 on T cells + CD69 + The proportion of double-positive cell populations was calculated by fitting a four-parameter regression curve using GraphPad Prism software to calculate EC 50 value.

[0281] As shown in FIG7 , the anti-CD3 / CD20 bispecific antibodies TZT7 and TZT6 of the present invention and the positive control REGN1979 (Regeneron Pharmaceuticals) can effectively promote the expression of CD25 in CD8 T lymphocytes. + and CD69 + The increase in the proportion of double-positive cell populations indicates the promotion of T cell activation, EC 50 The T cell activation activities of TZT7 and TZT6 were significantly better than those of REGN1979.

[0282] Example 10: Study on the activity of bispecific antibodies in promoting T cells to kill B lymphoma cells

[0283] Flow cytometry was used to detect the ability of the anti-CD3 / CD20 bispecific antibody of the present invention to promote T cells to kill B lymphoma cells (Raji cells).

[0284] First, human total T cells were isolated and purified from commercial PBMC (Allcells, Cat#PB004F-C) using Human pan T cell isolation kit (Miltenyi Biotec, Cat#130-096-535); then Raji cells were labeled with CFSE cell proliferation kit (Invitrogen, Cat#C34554), and purified human total T cells (1×10 per well) were added to the culture medium. 5 cells) and CFSE-labeled Raji cells (2×10 4 Cells) and gradient dilutions of TZT7, TZT6, or control samples (starting at 10 μg / ml, 6-fold dilution, a total of 10 concentration gradients) were incubated in a 96-well plate at 37°C for 72 hours. Finally, the cells were collected and stained with 7-AAD (BD, Cat#559925) and collected by flow cytometry (BD, CantoⅡ). The proportion of the killed Raji cell population, i.e., CFSE expression, was analyzed using FlowJo software. + 7-AAD + The ratio of double-positive population (Percp-cy5.5 channel) was calculated, and the EC was calculated by fitting a four-parameter regression curve using GraphPad Prism software. 50 value.

[0285] As shown in FIG8 , the anti-CD3 / CD20 bispecific antibodies TZT7, TZT6 and REGN1979 (Regeneron Pharmaceuticals) of the present invention can effectively promote T cells to kill B lymphoma cells (ie, Raji cells), EC 50 The killing activity of TZT7 was significantly better than that of REGN1979, which was 0.9869 ng / mL, 41.40 ng / mL and 62.95 ng / mL respectively.

[0286] Example 11: Single-dose PK study in cynomolgus monkeys

[0287] 1. Test Purpose

[0288] PK study of a single dose of the bispecific antibody of the present invention in cynomolgus monkeys.

[0289] 2. Testing process

[0290] a. Coat a 96-well plate with 100 μl / well of recombinant human CD3ε (Novoprotein, C578) at a concentration of 1.0 μg / ml and incubate at 37°C for 1.5 hours.

[0291] b. Wash four times with 300 μl / well 1× PBST, add 200 μl / well 2% BSA, and block at 37°C for 1.5 hours.

[0292] c. Prepare a TZT7 standard curve using blank cynomolgus monkey serum, with a range of 8 μg / ml to 125 ng / ml. Prepare a TZT6 standard curve using blank cynomolgus monkey serum, with a range of 4 μg / ml to 62.5 ng / ml. Dilute the test sample with blank cynomolgus monkey serum to the standard curve range. Then, dilute the standard curve samples and test sample 10-fold with 2% BSA. Add 100 μl of the prepared test sample per well, incubate at 37°C for 1 hour, and wash the plate.

[0293] d. Wash four times with 300 μl / well 1× PBST.

[0294] e. Add 100 μl / well of 5000-fold diluted horseradish peroxidase (HRP)-conjugated mouse anti-human IgG4 Fc antibody (Southern Biotech, 9200-05) and incubate at 37°C for 1 hour.

[0295] f. Wash four times with 300 μl / well 1× PBST.

[0296] g. Add 0.1 mg / ml TMB to 100 μl / well, incubate at 37°C for 5-7 minutes, and then add 2 M hydrochloric acid solution to 100 μl / well to terminate the reaction.

[0297] h. The absorbance was measured at 450 nm and 620 nm on a microplate reader, and the data were analyzed using a four-parameter model in SoftMax Pro 5.4.1.

[0298] 3. Test results

[0299] As shown in Table 4, when the bispecific antibody TZT7 of the present invention was administered to cynomolgus monkeys at a dose of 1 mg / kg, the half-life was 19.1 h, C max When the dosage is 10 mg / kg, the half-life is 138.3 h, C max When the bispecific antibody TZT6 of the present invention is administered to cynomolgus monkeys at a dose of 1 mg / kg, the half-life is 164.2 h, C max When the dosage is 10 mg / kg, the half-life is 168.8 h, C max It is 244.9 mg / L.

[0300] Table 4 PK results of single administration in cynomolgus monkeys

[0301] Note: AUC (0-t) : area under the drug concentration-time curve; t1 / 2z : terminal elimination half-life; T max : peak time; C max : Peak drug concentration.

[0302] Example 12: Inhibitory Effect of Bispecific Antibodies on B16 OVA huCD20 Tumor Growth

[0303] 1. Test Purpose

[0304] The anti-tumor effects of TZT6 and TZT7 of the present invention were evaluated in a murine melanoma B16 OVA huCD20 subcutaneous transplant tumor model.

[0305] 2. Testing process

[0306] Female hCD3e humanized mice aged 6-8 weeks (purchased from Biocytogen Jiangsu Gene Biotechnology Co., Ltd.) were subcutaneously inoculated on the right side of the back with 2.5×10 5 B16 OVA huCD20 1F11 cells (transfected with human CD20 gene). The average tumor volume is about 52mm 3 When the tumor volume was quantified, appropriate animals were selected and randomly divided into three groups, with 6 animals in each group.

[0307] Solvent control group:

[0308] -G1 normal saline group;

[0309] Treatment Group:

[0310] -G2 TZT6 (10 mg / kg) group, and

[0311] -G3 TZT7 (10 mg / kg) group.

[0312] Intraperitoneal injection, twice a week, for 2 consecutive weeks, the experiment was terminated 4 days after the last dose. Tumor volume and body weight were measured twice a week, and the weight and tumor volume of the mice were recorded. At the end of the experiment, the mice were euthanized, and the tumor inhibition rate TGI (%) was calculated as [1-(Ti-T0) / (Vi-V0)]×100%. (Ti: mean tumor volume of the treatment group on the i-th day of administration, T0: mean tumor volume of the treatment group on the 0th day of administration; Vi: mean tumor volume of the solvent control group on the i-th day of administration, V0: mean tumor volume of the solvent control group on the 0th day of administration).

[0313] As shown in Figure 9, on day 14 after administration, the average tumor volume of the saline control group was 2170 mm 3 The average tumor volume in the TZT6 group was 1455 mm 3Compared with the saline control group, the tumor inhibition rate was 33.7%. The average tumor volume in the TZT7 group was 372 mm 3 Compared with the saline control group, the tumor inhibition rate was 84.9%, significantly inhibiting tumor volume growth. The results showed that in the hCD3e humanized mouse transplanted B16 OVA huCD20 1F11 model, both TZT6 and TZT7 exhibited tumor inhibitory effects at a dose level of 10 mg / kg.

[0314] Example 13: Inhibitory effect of TZT7 on B16 OVA huCD20 tumor growth

[0315] 1. Test Purpose

[0316] The anti-tumor effect of TZT7 of the present invention was evaluated in a mouse melanoma B16 OVA huCD20 subcutaneous transplant tumor model.

[0317] 2. Testing process

[0318] Female hCD3e humanized mice aged 6-8 weeks (purchased from Biocytogen Jiangsu Gene Biotechnology Co., Ltd.) were subcutaneously inoculated on the right side of the back with 2.5×10 5 B16 OVA huCD20 1F11 cells (transfected with human CD20 gene). The average tumor volume is about 88mm 3 When the tumor volume was quantified, appropriate animals were selected and randomly divided into three groups, with 6 animals in each group.

[0319] Solvent control group:

[0320] -G1 normal saline group;

[0321] Treatment Group:

[0322] -G2 TZT7 (10 mg / kg) group, and

[0323] -G3 RENG1979 (10 mg / kg) group.

[0324] Intraperitoneal injection, twice a week, continuous administration for 2 weeks, the experiment ended 4 days after the last administration. The tumor volume and body weight were measured twice a week, and the mouse body weight and tumor volume were recorded. At the end of the experiment, the mice were euthanized and the tumor inhibition rate TGI was calculated. TGI(%)=[1-(Ti-T0) / (Vi-V0)]×100%. (Ti: the mean tumor volume of the treatment group on the i-th day of dosing, T0: the mean tumor volume of the treatment group on the 0th day of dosing; Vi: the mean tumor volume of the solvent control group on the i-th day of dosing, V0: the mean tumor volume of the solvent control group on the 0th day of dosing).

[0325] As shown in Figure 10, on day 18 after administration, the average tumor volume of the saline control group was 2517 mm 3 The mean tumor volume in the REGN1979 (Regeneron Pharmaceuticals) group was 2646 mm 3 Compared with the saline control group, the tumor inhibition rate was -5.3%, and no tumor inhibition effect was observed. The average tumor volume in the TZT7 group was 371 mm 3 Compared with the saline control group, the tumor inhibition rate was 88.3%, significantly inhibiting tumor volume growth. The results showed that TZT7 at a dose level of 10 mg / kg can significantly inhibit the growth of B16 OVA huCD20 1F11 tumors transplanted into hCD3e humanized mice.

[0326] Example 14: Inhibitory effects of TZT6 and TZT7 on the Raji Mixeno model of human lymphoma

[0327] 1. Test Purpose

[0328] The anti-tumor effects of TZT6 and TZT7 of the present invention were evaluated in the human lymphoma Raji Mixeno model.

[0329] 2. Testing process

[0330] Female NDG mice aged 6-8 weeks (purchased from Biocytogen Jiangsu Gene Biotechnology Co., Ltd.) were subcutaneously inoculated on the right back with a premixed solution of 2.5×10 6 Raji cells and 5×10 6 A suspension of PBMC (purchased from Allcells) (0.2 ml / cell) was added to establish a subcutaneous transplant tumor model. When the average tumor volume was about 108 mm 3 When the tumor volume was 100 mg / kg, the appropriate animals were selected and randomly divided into 5 groups according to the tumor volume, with 6 animals in each group.

[0331] Solvent control group:

[0332] -G1 normal saline group;

[0333] Treatment Group:

[0334] -G2 TZT6 (1 mg / kg) group;

[0335] -G3 TZT6 (3 mg / kg) group;

[0336] -G4 TZT7 (1 mg / kg) group;

[0337] -G5 TZT7 (3 mg / kg) group;

[0338] Intraperitoneal injection, single administration, the experiment was ended after 18 days of administration. The tumor volume and body weight were measured twice a week, and the mouse body weight and tumor volume were recorded. At the end of the experiment, the mice were euthanized and the tumor inhibition rate TGI was calculated. TGI(%)=[1-(Ti-T0) / (Vi-V0)]×100%. (Ti: the mean tumor volume of the treatment group on the i-th day of dosing, T0: the mean tumor volume of the treatment group on the 0th day of dosing; Vi: the mean tumor volume of the solvent control group on the i-th day of dosing, V0: the mean tumor volume of the solvent control group on the 0th day of dosing).

[0339] As shown in Figure 11, on day 18 after the start of drug administration, the average tumor volume of the saline control group was 1610 mm 3 At the dose levels of 1 mg / kg and 3 mg / kg, the average tumor volume of TZT6 was 723 mm 3 and 233mm 3 Compared with the saline control group, the tumor inhibition rates were 58.7% and 91.5%, respectively, significantly inhibiting the growth of tumor volume. At the dose level of 1mg / kg and 3mg / kg, the average tumor volume was 264mm 3 and 99mm 3 The tumor inhibition rates were 89.6% and 100.6% respectively, significantly inhibiting the growth of tumor volume. The results showed that TZT6 and TZT7 at dose levels of 1 and 3 mg / kg could significantly inhibit the growth of subcutaneous transplanted tumors of human lymphoma Raji cells.

Claims

1. A bispecific antibody that specifically binds to CD20 and CD3, comprising: (1) an antigen-binding fragment having a CD20 binding domain, which comprises a first heavy chain variable region (VH1) and a first light chain variable region (VL1); and (2) An antigen-binding fragment having a CD3-binding domain, which comprises a second heavy chain variable region (VH2) and a second light chain variable region (VL2).

2. The bispecific antibody according to claim 1, comprising the following polypeptide chains: (1) A polypeptide chain represented by formula (I) and formula (II) having a CD20 binding domain: VH1-CH1-Fc1 Formula (I), VL1-CL Formula (II), and (2) A polypeptide chain represented by formula (III) and formula (II-2) having CD20 and CD3 binding domains: VH1-CH1-L-VL2-L-VH2-L-Fc2 formula (III); VL1-CL Formula (II-2); or A polypeptide chain represented by formula (IV) having a CD3 binding domain: VL2-L-VH2-L-Fc2 Formula (IV); Wherein VH represents the heavy chain variable region; VL represents the light chain variable region; Fc comprises CH2 and CH3; CH1, CH2 and CH3 represent domains 1, 2 and 3 of the heavy chain constant region, respectively; VL represents the light chain variable region; CL represents the light chain constant region; L represents a connecting peptide, each connecting peptide is the same or different; optionally, there is a hinge region between CH1 and Fc1; Formulas (Ⅰ), (Ⅱ), (Ⅱ-2), (Ⅲ) and (Ⅳ) are connected sequentially from the N-terminus to the C-terminus; The antigen binding site formed by VH1 and VL1 binds CD20, and the antigen binding site formed by VL2 and VH2 binds CD3.

3. The bispecific antibody of claim 1 or 2, wherein the connecting peptide comprises the amino acid sequence (GGGGS)n, in, n is independently selected from 1, 2, 3, 4, 5 and 6.

4. The bispecific antibody according to any one of claims 1 to 3, in: The VH1 comprises HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, and The VL1 comprises LCDR1, LCDR2 and LCDR3, the amino acid sequences of which are shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively.

5. The bispecific antibody according to claim 4, in: The VH1 comprises the amino acid sequence as shown in SEQ ID NO:7, or comprises a VH2 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence as shown in SEQ ID NO:

7. The amino acid sequence of The VL1 comprises the amino acid sequence as shown in SEQ ID NO:8; or comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:

8.

6. The bispecific antibody according to any one of claims 1 to 3, in: The VH2 comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11, respectively, and The VL2 comprises LCDR1, LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, respectively.

7. The bispecific antibody according to claim 6, in: The VH2 comprises the amino acid sequence as shown in SEQ ID NO: 15, or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence as shown in SEQ ID NO: 15; and The VL2 comprises the amino acid sequence as shown in SEQ ID NO:16; or comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:

16.

8. The bispecific antibody according to any one of claims 1 to 3, in: The polypeptide chain of formula (I) comprises the amino acid sequence as shown in SEQ ID NO: 17 or 23, or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence as shown in SEQ ID NO: 17 or 23; and The polypeptide chains of formula (II) and formula (II-2) contain the amino acid sequence as shown in SEQ ID NO:18, or contain an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:

18.

9. The bispecific antibody of any one of claims 1 to 3, wherein the polypeptide chain of formula (III) comprises the amino acid sequence as shown in SEQ ID NO: 19, or comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence as shown in SEQ ID NO:

19.

10. The bispecific antibody of any one of claims 1 to 3, wherein the polypeptide chain of formula (IV) comprises the amino acid sequence as shown in SEQ ID NO: 20, or comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence as shown in SEQ ID NO:

20.

11. The bispecific antibody according to any one of claims 1 to 3, comprising a polypeptide chain represented by formula (I) and formula (II), and a polypeptide chain represented by formula (III) and formula (II-2), wherein the amino acid sequence of the polypeptide chain of formula (I) is shown in SEQ ID NO: 23, the amino acid sequences of the polypeptide chains of formula (II) and formula (II-2) are shown in SEQ ID NO: 18, and the amino acid sequence of the polypeptide chain of formula (III) is shown in SEQ ID NO:

19.

12. The bispecific antibody of any one of claims 1 to 3, comprising a polypeptide chain represented by formula (I), formula (II), and a polypeptide chain represented by formula (IV), wherein the amino acid sequence of the polypeptide chain of formula (I) is shown in SEQ ID NO: 17, the amino acid sequence of the polypeptide chain of formula (II) is shown in SEQ ID NO: 18, and the amino acid sequence of the polypeptide chain of formula (IV) is shown in SEQ ID NO:

20.

13. The bispecific antibody according to any one of claims 1 to 12, wherein the formula (I) and the formula (II) and the formula (III) and the formula (II-2) are connected via a disulfide bond; and the formula (I) and the formula (III) or the formula (IV) are connected via a disulfide bond and a Knob into Hole structure of the CH3 domain; Preferably, Fc1 in formula (I) is knob-Fc, and Fc2 in formula (III) or (IV) is hole-Fc; or Fc1 in formula (I) is hole-Fc, and Fc2 in formula (III) or (IV) is knob-Fc.

14. The bispecific antibody according to any one of claims 1 to 13, in, The CH1-Fc1 in formula (I) and Fc2 in formula (III) or (IV) are in the form of IgG, such as IgG1, IgG2, IgG3 or IgG4, and / or the CL in formula (II) and (II-2) is derived from λ or κ chain.

15. An isolated nucleic acid encoding any one or more polypeptide chains of the bispecific antibody of any one of claims 1-14.

16. An expression vector comprising the nucleic acid according to claim 15, preferably, the expression vector is a eukaryotic expression vector.

17. A host cell comprising the nucleic acid according to claim 15 or the expression vector according to claim 16, preferably, the host cell is a eukaryotic cell, more preferably a mammalian cell.

18. A method for preparing the bispecific antibody according to any one of claims 1 to 14, comprising culturing the host cell according to claim 16 under conditions suitable for expressing the nucleic acid according to claim 15, and recovering the bispecific antibody from the host cell.

19. A pharmaceutical composition comprising the bispecific antibody of any one of claims 1 to 14, the nucleic acid of claim 15, the expression vector of claim 16 and / or the host cell of claim 17, and a pharmaceutically acceptable carrier or excipient.

20. Use of the bispecific antibody according to any one of claims 1 to 14, the nucleic acid according to claim 15, the expression vector according to claim 16, the host cell according to claim 17 and / or the pharmaceutical composition according to claim 19 in the preparation of a medicament for preventing or treating cancer, preferably, the cancer is selected from acute B lymphocytic leukemia, diffuse large B-cell lymphoma, chronic lymphocytic leukemia, follicular lymphoma, non-Hodgkin's lymphoma, chronic myeloid leukemia and Burkitt's lymphoma.

21. A method for preventing or treating cancer in a subject, comprising administering to a subject in need thereof the bispecific antibody of any one of claims 1 to 14, the nucleic acid of claim 15, the expression vector of claim 16, the host cell of claim 17 and / or the pharmaceutical composition of claim 19.

22. A method for detecting the presence of CD3 and / or CD20 in a sample using the bispecific antibody of any one of claims 1 to 14.

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