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

Abstract

The present invention provides bispecific antibodies targeting CD3 and CD20, and compositions comprising same. Further provided are nucleic acid molecules encoding the bispecific antibodies of the invention, vectors and host cells for expressing the bispecific antibodies of the invention, and therapeutic and diagnostic methods and uses of the antibodies or antigen-binding fragments thereof of the invention.

Description

[Technical field]

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

[0002] CD3 (T cell surface glycoprotein CD3, a signal transduction coreceptor of the T cell receptor containing subunits γ, δ, ε and ζ) is a differentiation antigen expressed on the surface of all T lymphocytes, which mainly mediates the transmission of T cell activation signals. It plays an important role in the anti-infection immunity of the body's immune system. The CD3 molecule forms a stable TCR-CD3 complex with the T cell antigen receptor, and 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 (T cell receptor, TCR) with MHC molecules, while the intracellular region enhances the activation signal of leukocyte CD3 transduction, thereby participating in and regulating the activation of the immune system. The index of the number of CD3 positive lymphocyte population is an important index to measure the immune status of somatic cells.

[0003] CD20 is a marker molecule specific to the surface of B lymphocytes, and is expressed in mature B cells and most malignant B lymphocytes, but not in early B cell precursors or late mature plasma cells. The molecule is composed of 297 amino acid residues, penetrates the cell membrane four times, and its antigen epitope is the only extracellularly exposed loop composed of 43 amino acid residues with no glycan chains in the third and fourth transmembrane regions. The exact function of CD20 is unknown, and CD20 may be involved in the activation and differentiation of B cells and function as a calcium channel. CD20 is not internalized into cells after binding with antibodies, and does not undergo obvious shedding on the cell surface, making it an ideal antigen for treating B lymphocyte-related diseases. For example, rituximab monoclonal antibody and obinutuzumab monoclonal antibody are both antibody drugs against CD20, and have good efficacy. Considering that the conventional antibodies against CD20 mediate the killing effect of immune cells against tumor cells, and there is still room for improvement and advantages of bispecific antibodies, a good anti-CD20 / CD3 bispecific antibody can well mediate the killing effect of immune cells against tumor cells, and has great prospects for clinical application.

[0004] Bispecific antibodies (BsAbs), also known as dual-function antibodies, can specifically bind to two different antigens or two different antigen epitopes simultaneously. Due to their specificity and dual functionality, they have good application effects and prospects in the fields of tumor immunotherapy and autoimmune diseases. There are many types of bispecific antibodies, and different bispecific antibody forms have different effects on different tumor-associated antigens. Due to their specific functions, they have broad application prospects in tumor immunotherapy.

[0005] There are currently no commercially available pharmaceuticals based on anti-CD3×CD20 bispecific antibodies, and the object of the present invention is to develop novel bispecific antibodies that are highly effective in terms of affinity, safety, stability, etc. Summary of the Invention

[0006] Disclosed herein are novel bispecific antibody molecules constructed by antibody engineering methods.

[0007] The present invention provides a bispecific antibody that specifically binds to CD20 and CD3, (1) an antigen-binding fragment having a CD20-binding domain, the antigen-binding fragment comprising a first heavy chain variable region (VH1) and a first light chain variable region (VL1); (2) an antigen-binding fragment having a CD3-binding domain, the antigen-binding fragment comprising a second heavy chain variable region (VH2) and a second light chain variable region (VL2).

[0008] In some embodiments, a bispecific antibody according to the invention comprises the following polypeptide chains: (1) A polypeptide chain having a CD20 binding domain as shown in formula (I) and formula (II): VH1-CH1-Fc1 formula (I), VL1-CL Formula (II), and (2) A polypeptide chain having a CD20 and CD3 binding domain as shown in formula (III) and formula (II-2): VH1-CH1-L-VL2-L-VH2-L-Fc2 formula (III), VL1-CL formula (II-2), or A polypeptide chain having a CD3 binding domain as shown in formula (IV): VL2-L-VH2-L-Fc2 formula (IV), wherein VH represents the heavy chain variable region, VL represents the light chain variable region, Fc includes 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, and L represents a connecting peptide, each connecting peptide being homologous or different, and optionally, there is a hinge region between CH1 and Fc1; Formulae (I), (II), (II-2), (III) and (IV) are linked in sequence 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.

[0009] In some embodiments, a connecting peptide according to the invention comprises the amino acid sequence (GGGGS) n , where n is independently selected from 1, 2, 3, 4, 5, or 6.

[0010] In some embodiments, the VH1 described in the present invention comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are set forth 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 set forth in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively.

[0011] In some embodiments, in the bispecific antibody according to the invention, The VH1 comprises an amino acid sequence set forth in SEQ ID NO:7 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO:7; and The VL1 comprises an amino acid sequence set forth in SEQ ID NO:8 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO:8.

[0012] In some embodiments, in the bispecific antibody described in the present invention, the VH2 comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are set forth 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 set forth in SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14, respectively.

[0013] In some embodiments, in the bispecific antibody according to the invention, The VH2 comprises an amino acid sequence set forth in SEQ ID NO:15 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO:15; and The VL2 comprises an amino acid sequence set forth in SEQ ID NO:16 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO:16.

[0014] In some embodiments, in the bispecific antibody according to the invention, The polypeptide chain of formula (I) comprises an amino acid sequence as set forth in SEQ ID NO: 17 or 23, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence as set forth in SEQ ID NO: 17 or 23, and The polypeptide chains of formulae (II) and (II-2) comprise an amino acid sequence as set forth in SEQ ID NO:18 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence as set forth in SEQ ID NO:18.

[0015] In some embodiments, in a bispecific antibody according to the invention, the polypeptide chain of formula (III) comprises the amino acid sequence set forth in SEQ ID NO:19 or comprises an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO:19.

[0016] In some embodiments, in a bispecific antibody according to the invention, the polypeptide chain of formula (IV) comprises the amino acid sequence set forth in SEQ ID NO:20 or comprises an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO:20.

[0017] In some embodiments, a bispecific antibody according to the invention comprises a polypeptide chain of Formula (I), Formula (II) and a polypeptide chain of Formula (III), Formula (II-2), wherein the amino acid sequence of the polypeptide chain of Formula (I) is set forth in SEQ ID NO:23, the amino acid sequence of the polypeptide chains of Formulas (II) and (II-2) is set forth in SEQ ID NO:18, and the amino acid sequence of the polypeptide chain of Formula (III) is set forth in SEQ ID NO:19.

[0018] In some embodiments, a bispecific antibody according to the invention comprises a polypeptide chain according to Formula (I), Formula (II) and a polypeptide chain according to Formula (IV), wherein the amino acid sequence of the polypeptide chain of Formula (I) is set forth in SEQ ID NO: 17, the amino acid sequence of the polypeptide chain of Formula (II) is set forth in SEQ ID NO: 18 and the amino acid sequence of the polypeptide chain of Formula (IV) is set forth in SEQ ID NO: 20.

[0019] In some embodiments, in the bispecific antibody according to the present invention, Formula (I) and Formula (II), and Formula (III) and Formula (II-2) are linked by a disulfide bond, and Formula (I) and Formula (III) or Formula (IV) are linked by 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 formula (IV) is hole-Fc, or Fc1 in formula (I) is hole-Fc and Fc2 in formula (III) or formula (IV) is knob-Fc.

[0020] In some embodiments, in the bispecific antibodies described in the present invention, CH1-Fc1 in formula (I) and Fc2 in formula (III) are in the form of IgG, such as IgG1, IgG2, IgG3 or IgG4, and / or CL in formula (II) and formula (II-2) is derived from a λ or κ chain.

[0021] In yet another aspect, the invention provides isolated nucleic acid encoding any one or more of the polypeptide chains in the bispecific antibodies described herein.

[0022] In yet another aspect, the present invention provides an expression vector comprising a nucleic acid described herein, preferably said expression vector being a eukaryotic expression vector.

[0023] In yet another aspect, the present invention provides a host cell comprising a nucleic acid as described herein or an expression vector as described in the present invention, said host cell being preferably a eukaryotic cell, more preferably a mammalian cell.

[0024] In yet another aspect, the invention provides a method of preparing a bispecific antibody as described herein comprising culturing a host cell as described herein under conditions suitable for expression of a nucleic acid as described herein and recovering said bispecific antibody from said host cell.

[0025] In yet another aspect, the present invention provides a pharmaceutical composition comprising a bispecific antibody as described herein, a polynucleotide as described herein, an expression vector as described herein, and / or a host cell as described herein, and a pharma- ceutically acceptable carrier or excipient.

[0026] In yet another aspect, the present invention provides the use of 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 in the preparation of a medicament for preventing or treating cancer, wherein said cancer is preferably 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's lymphoma.

[0027] In yet another aspect, the present invention provides a method of 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.

[0028] 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 treating cancer.

[0029] In some embodiments, the cancer according to the invention 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's lymphoma.

[0030] In yet another aspect, the 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.

[0031] In yet another aspect, the present invention provides a method of detecting the presence of CD3 and / or CD20 in a sample using the bispecific antibodies described herein.

[0032] In yet another aspect, the present invention provides a reagent kit comprising an antibody described herein or a pharmaceutical composition described herein. [Brief description of the drawings]

[0033] [Figure 1] Schematic diagrams of the molecular structures of anti-CD3×CD20 bispecific antibodies. 1a: Schematic diagram of TZT6 structure, 1b: Schematic diagram of TZT7 structure. [Diagram 2]FIG. 1 shows detection of binding of bispecific antibodies to human CD3ε by ELISA. [Diagram 3] FIG. 1 shows binding of bispecific antibodies to Raji cells. [Figure 4] FIG. 1 shows binding of bispecific antibodies to Jurkat cells. [Diagram 5] FIG. 1 shows binding of bispecific antibodies to overexpressing cynomolgus monkey CD3e cells. [Figure 6] FIG. 1 shows the activity of bispecific antibodies in a luciferase reporter gene system. [Figure 7] FIG. 1 shows the activation activity of bispecific antibodies against T lymphocytes. [Figure 8] FIG. 1 shows that bispecific antibodies enhance the killing activity of T cells against B lymphoma cells. [Figure 9] FIG. 1 shows inhibition of B16 OVA huCD20 tumor growth by bispecific antibodies TZT6 and TZT7. [Figure 10] FIG. 1 shows bispecific antibody TZT7 inhibition of B16 OVA huCD20 tumor growth. [Figure 11] FIG. 1 shows the inhibitory effect of bispecific antibodies on the human lymphoma Raji Mixeno model. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0034] definition Unless otherwise indicated, the practice of the present invention will employ conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, immunology and the like, which are within the skill of the art.

[0035] In order to make the present invention easier to understand, some technical terms are specifically defined as follows. Unless otherwise clearly defined in other parts of this specification, technical terms used in this specification have the meanings that are commonly understood by those skilled in the art of the present invention. For definitions and terms in the field, experts can specifically refer to Current Protocols in Molecular Biology (Ausubel). The abbreviations of amino acid residues are the standard three-letter and / or one-letter codes used in the field to represent one of the 20 commonly used L-amino acids. Unless otherwise clearly stated in this specification, the singular form used in this specification (including the claims) includes its corresponding plural form.

[0036] 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 of 5% less than the specified numerical value and an upper limit of 5% greater than the specified numerical value.

[0037] The term "and / or" should be understood to mean any one of the options or a combination of any two or more of the options.

[0038] The term "CD3" refers to the three distinct chains CD3ε, CD3δ and CD3γ as part of the T cell receptor complex. CD3 is concentrated on T cells, for example by the immobilizing effect of anti-CD3 antibodies on the T cell, resulting in activation of the T cell similar to T cell receptor-mediated activation, but independent of the specificity of the TCR clone. The majority of anti-CD3 antibodies recognize the CD3ε chain. The term refers to any native CD3 from any vertebrate, mammal, such as a primate (e.g., human), and rodent (e.g., mouse and rat), unless otherwise specified. The term includes "full-length" unprocessed CD3, and any form of CD3 or any fragment thereof produced by intracellular processing. The term further includes naturally occurring variants of CD3, such as splice variants or allelic variants. In a preferred embodiment, CD3 refers to full-length CD3 or a fragment thereof (e.g., a mature fragment thereof lacking the signal peptide) from human or cynomolgus monkey. In a preferred embodiment, CD3 refers to full length or a fragment thereof derived from mouse / rat (eg, a mature fragment thereof lacking the signal peptide).

[0039] The term "human CD20" or "CD20" refers to human CD20 (UniProtKB / Swiss-Prot No. P11836), and It includes any variant, isoform, and species homologue of CD20 that is naturally expressed by a cell (including a tumor cell) or expressed on a cell transfected with the CD20 gene or cDNA. Species homologues include rhesus monkey CD20 (macaca mulatta; UniProtKB / Swiss-Prot No H9YXP1) and cynomolgus monkey CD20.

[0040] The term "percentage (%) of amino acid sequence identity" or the abbreviation "identity" is defined as the percentage of amino acid residues in a candidate amino acid sequence that are the same as the amino acid residues in a reference amino acid sequence when the amino acid sequences are aligned (with gaps introduced if necessary) to obtain the maximum percentage of sequence identity, and any conservative substitutions are not considered part of the sequence identity. To measure the percentage of amino acid sequence identity, sequences can be aligned using various methods in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measurement and alignment, including any algorithms required to obtain maximum alignment over the entire length of the sequences being compared.

[0041] The term "immune response" refers to the action of, for example, lymphocytes, antigen-presenting cells, phagocytes, granulocytes and soluble macromolecules (including antibodies, cytokines and complement) produced by such cells or the liver, which results in the selective damage, destruction or removal from the human body of invading pathogens, pathogen-infected cells or tissues, cancer cells or normal human cells or tissues in the case of autoimmunity or pathological inflammation.

[0042] The term "signal transduction pathway" or "signal transduction activity" refers to a biochemical causal relationship, usually initiated by a protein-protein interaction, 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 part of the cell. Generally, transduction involves the specific phosphorylation of one or more tyrosine, serine, or threonine residues on one or more proteins in a series of reactions that triggers the signal transduction. The penultimate step usually involves a nuclear event, resulting in a change in gene expression.

[0043] The terms "activity" or "biological activity" or "biological properties" or "biological characteristics" 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, the 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, usually with non-human homologs of the target peptide, or with other proteins or tissues), and the ability to maintain high expression levels of the protein in mammalian cells. The aforementioned properties or characteristics are observed, measured, or assessed using techniques known in the art, including, but not limited to, ELISA, FACS, or BIACORE plasma resonance analysis, neutralization measurements in vivo or in vitro, receptor binding, production and / or secretion of cytokines or growth factors, signal transduction, and immunohistochemistry of tissue fragments of different origins (human, primate, or any other origin).

[0044] The term "antibody" refers to any form of antibody having the desired biological activity. Thus, when used in the broadest sense, it 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.

[0045] The term "isolated antibody" refers to the purified state of the binding compound, and in such cases means that the molecule is substantially free of other biological molecules, such as nucleic acids, proteins, lipids, sugars, cellular debris and other substances such as growth medium. The term "isolated" does not imply the complete absence of such substances, or the absence of water, buffers or salts, unless present in amounts that would clearly interfere with the experimental or therapeutic application of the binding compounds described herein.

[0046] The term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., each antibody from the population is the same except for minor variations that may occur naturally. Monoclonal antibodies are highly specific and are directed against a single antigenic epitope. In contrast, a conventional (polyclonal) antibody preparation will usually contain large numbers of antibodies directed against (or having specificities for) different epitopes. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.

[0047] The term "bispecific antibody" refers to an antibody molecule that can bind to two independent antigens or has binding specificities 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., a CD3 molecule), thus activating and initiating cellular immune-associated mechanisms in tumor cells.

[0048] The term "full-length antibody" refers to an immunoglobulin molecule that, when naturally occurring, contains four peptide chains, two heavy (H) chains (about 50-70 kDa for full-length) and two light (L) chains (about 25 kDa for full-length) linked together 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 divided into highly variable complementarity determining regions (CDRs) and more conserved regions between them, called framework regions (FRs). Each VH or VL region consists of three CDRs and four 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 a binding domain that interacts with an antigen. The constant regions of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including cells of the immune system (e.g., effector cells) and the first component (C1q) of the representative complement system.

[0049] The term "antigen-binding fragment" of an antibody ("parent antibody") includes fragments or derivatives of antibodies that typically contain at least a fragment of the antigen-binding or variable region (e.g., one or more CDRs) of the parent antibody and retain 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 formed from antibody fragments, and multispecific antibodies. When antigen-binding activity is expressed in molar concentrations, a binding fragment or derivative typically retains at least 10% of its antigen-binding activity. It is preferred that a binding fragment or derivative retains at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or more of the antigen-binding affinity of the parent antibody. Antigen-binding fragments of antibodies are expected to contain conservative or non-conservative amino acid substitutions (referred to as "conservative variants" or "function-conservative variants" of an antibody) that do not appreciably alter its biological activity. The term "binding compound" refers to both antibodies and binding fragments thereof.

[0050] 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 which enables the scFv to form the desired structure for antigen binding.

[0051] The terms "Fc" or "Fc region" or "Fc fragment" refer 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 via the hinge region. Although the resolution of the Fc fragment is variable, the heavy chain Fc fragment of human IgG usually refers to this polypeptide from A231 to the carboxy terminus.

[0052] The term "hinge region" refers to the polypeptide chain in an antibody that is located between CH1 and CH2, is proline-rich, and easily stretches and bends, whereas the recognized IgG hinge region is the polypeptide chain consisting of amino acid residues 216 to 230.

[0053] The term "domain antibody" refers to an immunologically functional immunoglobulin fragment containing only the heavy or light chain variable region. Optionally, 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.

[0054] The term "epitope" refers to a protein determinant that can specifically bind to an antibody. Epitopes usually consist of surface clustered molecules such as amino acids or sugar side chains, and generally have specific three-dimensional structural features, as well as specific charge characteristics. Conformational and non-conformational epitopes differ in that the binding to the former but not the latter is lost in the presence of denaturing solvents. Epitopes 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 residues are within the footprint of the specific antigen-binding peptide).

[0055] The term "bivalent antibody" comprises two antigen-binding sites. In some cases, the two binding sites have the same antigen specificity. However, a bivalent antibody may be bispecific.

[0056] The term "diabody" refers to a small antibody fragment with two antigen-binding sites, said fragment comprising a heavy-chain variable domain (VH) linked to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL or VL-VH), which is paired with the complementary domains of another chain by using a linker that is too short to allow pairing between the two domains in the same chain to produce two antigen-binding sites.

[0057] The term "chimeric antibody" refers to an antibody having the variable domains of a first antibody and the constant domains of a second antibody, where the first and second antibodies are from different species. Typically, the variable domains are obtained from an antibody such as a rodent (the "parent antibody") and 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 compared to the parent rodent antibody.

[0058] The term "humanized antibody" refers to forms of antibodies that contain sequences derived from human and non-human (e.g., mouse, rat) antibodies. Generally, a humanized antibody will contain substantially all of at least one, and usually two, variable domains, in which 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 those of a human immunoglobulin sequence. A humanized antibody optionally contains at least a portion of a human immunoglobulin constant region (Fc).

[0059] The term "fully human antibody" refers to an antibody that contains only human immunoglobulin protein sequences. For example, a fully human antibody may contain mouse glycosylation when produced in a mouse, a mouse cell, or a hybridoma derived from a mouse cell. Similarly, a "mouse antibody" refers to an antibody that contains only mouse immunoglobulin sequences. Alternatively, a fully human antibody may contain rat glycosylation when produced in a rat, a rat cell, or a hybridoma derived from a rat cell. Similarly, a "rat antibody" refers to an antibody that contains only rat immunoglobulin sequences.

[0060] An "isotype" of an antibody refers to the antibody class (e.g., IgM, IgE, IgG, such as IgG1, IgG2, or IgG4) that is provided by the heavy chain constant region genes. Isotypes further include modified forms of one of these classes, in which modifications have been made to alter Fc function, for example, to enhance or decrease effector function or binding to an Fc receptor.

[0061] The term "epitope" refers to the region of an antigen to which an antibody binds. Epitopes can be formed from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein.

[0062] "Affinity" or "binding affinity" refers to the intrinsic binding affinity that reflects the interaction between members of a binding pair. The affinity of a molecule X for its ligand Y is usually expressed as an equilibrium dissociation constant (KD), which is the ratio of the dissociation rate constant to the association rate constant (kdis and kkon, respectively). Affinity can be measured by common methods known in the art. One specific method for measuring affinity is the ForteBio kinetic binding assay herein.

[0063] The term "does not bind" to a protein or cell refers to not binding to a protein or cell or not binding to the same with high affinity, i.e., the KD for binding to a protein or cell is 1.0×10 ‐6 M or more, more preferably 1.0×10 ‐5 M or more, more preferably 1.0×10 ‐4 M or more, 1.0×10 ‐3 M or more, more preferably 1.0×10 ‐2 M or higher.

[0064] The term "high affinity" refers to an IgG antibody with 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 less, 5.0 x 10 ‐9 M or less, more preferably 1.0×10 ‐9 M or less. For other antibody isoforms, "high affinity" binding can vary. For example, "high affinity" binding for an IgM isoform is defined as a KD of 10 ‐6 M or less, preferably 10 ‐7M or less, more preferably 10 ‐8 It means M or less.

[0065] 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, whose cell membrane surface antigens are bound by an antibody, such as the Claudin 18.2 antibody.

[0066] The term "complement dependent cytotoxicity" or "CDC" is an effector function of IgG and IgM antibodies which, when binding to surface antigens, trigger the classical complement pathway involving the formation of a membrane attack complex and lysis of the target cell. The antibodies of the invention, when binding to CD20, trigger CDC against cancer cells.

[0067] The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single- or double-stranded form. Unless expressly limited, the term includes nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides (see U.S. Patent No. 8,278,036 to Kariko et al., which disclosed mRNA molecules in which uridine is substituted with pseudouridine, methods of synthesizing such mRNA molecules, and methods of using them to deliver therapeutic proteins in vivo). Unless otherwise indicated, a particular nucleic acid sequence also implicitly includes conservative and modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, complementary sequences, and the sequence explicitly indicated. Specifically, substitution of degenerate codons 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); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0068] "Construct" refers to any recombinant polynucleotide molecule (such as a plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, phage, or linear or circular single- or double-stranded DNA or RNA polynucleotide molecule) derived from any source, capable of genomic integration or autonomous replication, and constituting one or more polynucleotide molecules functionally linked (i.e., operably linked) to the following polynucleotide molecules. Recombinant constructs typically include a polynucleotide of the invention operably linked to a transcription initiation regulatory sequence that directs transcription of the polynucleotide in a host cell. Expression of the nucleic acid of the invention can be induced using both heterologous and non-heterologous (i.e., endogenous) promoters.

[0069] "Vector" refers to any recombinant polynucleotide construct that can be used for transformation purposes (i.e., to introduce heterologous DNA into a host cell). One type of vector is a "plasmid," which refers to a circular double-stranded DNA circle into which additional DNA segments can be ligated. Another type of vector is a viral vector, into which additional DNA segments can be ligated into the viral genome. Some vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Additionally, some vectors are capable of inducing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0070] As used herein, the term "expression vector" refers to a nucleic acid molecule capable of replicating and expressing a target gene 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, optionally, to provide amplification within the host.

[0071] Unless the context clearly dictates otherwise, "activation," "stimulation," and "treatment" as used with respect to a cell or receptor can have the same meaning, such as ligand activation, stimulation, or treatment as used with respect to a cell or receptor. "Ligand" includes natural and synthetic ligands, such as cytokines, cytokine variants, analogs, muteins, and antibody-derived binding compounds. "Ligand" also includes small molecules, such as peptide mimetics of cytokines and peptide mimetics of antibodies. "Activation" refers to the activation of a cell regulated by internal mechanisms and external or environmental factors. "Responses / reactions," e.g., responses of a cell, tissue, organ, or organism, include changes in biochemistry or physiological behavior (e.g., concentration, density, adhesion or migration within a biological compartment, gene expression rate, or differentiation state), where the changes are related to activation, stimulation, or treatment, or related to internal mechanisms, such as genetic programming.

[0072] As used herein, in one embodiment, the term "treatment" or "therapy" of any disease or condition refers to the amelioration of the disease or condition (i.e., alleviation, arrest, or reduction of the progression of the disease or at least one of its clinical symptoms). In another embodiment, "treatment" or "therapy" refers to the alleviation or improvement of at least one physical parameter, including those physical parameters that are not discernible by the patient. In another embodiment, "treatment" or "therapy" refers to modulating the disease or condition in the body (e.g., stabilization of discernible symptoms), physiology (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.

[0073] A "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, including mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. As used herein, the terms "cyno" or "cynomolgus monkey" refer to cynomolgus monkeys.

[0074] Administration "in combination with" one or more other therapeutic agents includes simultaneous (concomitant) and consecutive administration in any order.

[0075] "Therapeutically effective amount," "therapeutically effective dose," and "effective amount" refer to an amount of the CD20 antibody or antigen-binding fragment thereof of the present invention, when administered alone or in combination with other therapeutic agents to a cell, tissue, or subject, that effectively prevents or ameliorates one or more symptoms of a disease or condition or the progression of said disease or condition. A therapeutically effective dose also refers to an amount of the antibody or antigen-binding fragment thereof sufficient to bring about an improvement in symptoms, such as an amount that treats, cures, prevents, or ameliorates a relevant medical condition, or an amount that increases the rate of treatment, cure, prevention, or amelioration of said condition. When a single active ingredient is administered to an individual, the therapeutically effective dose refers to that ingredient alone. When administered in combination, the therapeutically effective dose refers to the total amount of active ingredients that elicits a therapeutic effect, regardless of combined, sequential, or simultaneous administration. An effective amount of a therapeutic agent improves a diagnostic standard or parameter by at least 10%, typically at least 20%, preferably at least about 30%, more preferably at least 40%, and most preferably at least 50%.

[0076] "Cancer" and "cancerous" refer to or describe a physiological disorder in mammals that is normally characterized by unregulated cell growth. This definition includes benign tumors, malignant tumors, and dormant tumors or tumor micrometastases. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specific examples of such cancers include squamous cell carcinoma, lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), peritoneal cancer, hepatocellular carcinoma, 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, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, and various head and neck cancers, 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, bulky lesions, and the like). These include chronic lymphocytic leukemia (CL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia, post-transplant lymphoproliferative disease (PTLD), and vascular abnormalities associated with phakomatoses, edema (e.g., associated with brain tumors), and Meigs syndrome.

[0077] antibody The present invention provides novel antibody molecules that can be used for the treatment, prevention and / or diagnosis of a wide variety of diseases.

[0078] A bispecific antibody of the invention comprises a first antigen-binding site that specifically binds CD3 and a second antigen-binding site that specifically binds CD20. Such antibodies may be referred to herein as, for example, "anti-CD3 / anti-CD20" or "anti-CD3xCD20" or "CD3xCD20" bispecific molecules, or other similar terms.

[0079] The terms "anti-CD3 antibody," "anti-CD3," "CD3 antibody," or "antibody that binds CD3" refer to an antibody that can bind to the 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.

[0080] The terms "anti-CD20 antibody," "anti-CD20," "CD20 antibody," or "antibody that binds to CD20" refer to an antibody that can bind to the 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.

[0081] In some embodiments, a bispecific antibody according to the invention comprises the following polypeptide chains: (1) A polypeptide chain as shown in formula (I) and formula (II) which binds to a domain of CD20: VH1-CH1-Fc1 formula (I), VL1-CL formula (II), (2) A polypeptide chain as shown in formula (III) and formula (II-2) which binds to the domains of CD20 and CD3: VH1-CH1-L-VL2-L-VH2-L-Fc2 formula (III), VL1-CL formula (II-2), wherein VH represents the heavy chain variable region, VL represents the light chain variable region, Fc includes 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, and L represents a connecting peptide, each connecting peptide being homologous or different, and optionally, there is a hinge region between CH1 and Fc1; Formulae (I), (II), (II-2) and (III) are linked in order from the N-terminus to the C-terminus, 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 according to the invention has the above structure.

[0082] In some embodiments, a bispecific antibody according to the invention comprises the following polypeptide chains: (1) A polypeptide chain as shown in formula (I) and formula (II) which binds to a domain of CD20: VH1-CH1-Fc1 formula (I), VL1-CL formula (II), (2) A polypeptide chain as shown in formula (IV) which binds to a domain of CD3: VL2-L-VH2-L-Fc2 Formula (IV).

[0083] wherein VH represents the heavy chain variable region, VL represents the light chain variable region, Fc includes 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, and L represents a connecting peptide, each connecting peptide being homologous or different, and optionally, there is a hinge region between CH1 and Fc1; Formulae (I), (II) and (IV) are linked in order from the N-terminus to the C-terminus, 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 according to the invention has the above structure.

[0084] In some embodiments, a connecting peptide according to the invention comprises the amino acid sequence (GGGGS)n, where n is independently selected from 1, 2, 3, 4, 5, or 6, preferably, n is independently selected from 2, 3, or 4.

[0085] In some embodiments, the VH1 described in the present invention comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are set forth 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 set forth in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively.

[0086] In some embodiments, in the bispecific antibody according to the invention, The VH1 comprises an amino acid sequence set forth in SEQ ID NO:7 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO:7; and The VL1 comprises an amino acid sequence set forth in SEQ ID NO:8 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO:8.

[0087] In some embodiments, in the bispecific antibody described in the present invention, the VH2 comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are set forth 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 set forth in SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14, respectively.

[0088] In some embodiments, in the bispecific antibody according to the invention, The VH2 comprises an amino acid sequence set forth in SEQ ID NO:15 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO:15; and The VL2 comprises an amino acid sequence set forth in SEQ ID NO:16 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO:16.

[0089] In some embodiments, in the bispecific antibody according to the invention, The polypeptide chain of formula (I) comprises an amino acid sequence as set forth in SEQ ID NO: 17 or 23, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence as set forth in SEQ ID NO: 17 or 23, and The polypeptide chains of formula (II) and formula (II-2) comprise an amino acid sequence as set forth in SEQ ID NO:18 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence as set forth in SEQ ID NO:18.

[0090] In some embodiments, in a bispecific antibody according to the invention, the polypeptide chain of formula (III) comprises the amino acid sequence set forth in SEQ ID NO:19 or comprises an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO:19.

[0091] In some embodiments, in a bispecific antibody according to the invention, the polypeptide chain of formula (IV) comprises the amino acid sequence set forth in SEQ ID NO:20 or comprises an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO:20.

[0092] In some embodiments, in a bispecific antibody according to the invention, the amino acid sequence of the polypeptide chain of Formula (I) is set forth in SEQ ID NO:23, the amino acid sequence of the polypeptide chain of Formula (II) and Formula (II-2) is set forth in SEQ ID NO:18, and the amino acid sequence of the polypeptide chain of Formula (III) is set forth in SEQ ID NO:19.

[0093] In some embodiments, in a bispecific antibody according to the invention, the amino acid sequence of the polypeptide chain of formula (I) is set forth in SEQ ID NO: 17, the amino acid sequence of the polypeptide chain of formula (II) is set forth in SEQ ID NO: 18, and the amino acid sequence of the polypeptide chain of formula (IV) is set forth in SEQ ID NO: 20.

[0094] In some embodiments, the antibody molecule of the present invention is humanized. Different methods for humanizing antibodies are known to those skilled in the art 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).

[0095] In some embodiments, the antibody molecule of the present invention is a human or humanized antibody. Human or humanized antibodies can be prepared using various techniques known in the art.

[0096] In some embodiments, the antibody molecule of the present invention is a chimeric antibody.

[0097] In some embodiments, at least a portion of the framework sequences of the antibody molecules of the invention are human consensus framework sequences. Further included are antigen-binding fragments thereof, such as Fab, Fab', F(ab')2, Fv, scFv or sdAb antibody fragments.

[0098] In the present invention, multiple strains of anti-human CD3 antibodies are analyzed and compared in detail, and the sequences are optimized and humanized to obtain the humanized antibody JSCD3.

[0099] The humanized anti-CD3 antibody described in the present invention shows stronger tumor killing activity when used to prepare bispecific antibodies. The humanized sequence disclosed in the present invention has more advantages and characteristics than other companies' humanized sequences, and shows higher tumor killing activity when used to prepare new antitumor drugs, and also has significantly improved physicochemical stability, which is more suitable for screening and developing antitumor drugs. This creative achievement constitutes the beneficial effect and extremely high medical application value of the present invention.

[0100] The precise amino acid sequence boundaries of the variable region CDRs of an antibody according to the invention can be determined by any of a number of known approaches, including those based on the three-dimensional structure of the antibody and the topology of the CDR rings as proposed by 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 variability of the antibody sequence as proposed by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, USDepartment of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), the international ImMunoGeneTics database (IMGT) (1999 Nucleic Acids Research, 27, 209-212), and a North CDR definition based on affinity propagation clustering in which a large number of crystal structures are utilized.

[0101] Unless otherwise specified, the CDRs of an antibody of the present invention can be delimited by one skilled in the art based on any solution in the art (eg, different allocation systems or combinations).

[0102] 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.

[0103] In a preferred embodiment, the amino acid changes according to the invention occur in regions outside the CDRs (e.g., FRs). More preferably, the amino acid changes according to the invention occur in regions outside the heavy chain variable region and / or outside the light chain variable region.

[0104] In some embodiments, the substitution is a conservative substitution, which refers to the replacement of one amino acid with another amino acid in the same class, for example, one acidic amino acid with another acidic amino acid, one basic amino acid with another basic amino acid, or one neutral amino acid with another neutral amino acid.

[0105] In some embodiments, bispecific antibodies or antigen-binding fragments thereof of the invention include those antibodies that have been mutated by amino acid deletion, insertion or substitution, but still have an amino acid sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the above antibodies (particularly in the CDR regions set forth in the sequences above). In some embodiments, the antibodies of the invention have no more than 1, 2, 3, 4 or 5 amino acid mutations resulting from amino acid deletion, insertion or substitution in the CDR regions when compared to the CDR regions set forth in the specific sequences.

[0106] In some embodiments, one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein to produce a variant Fc region. The variant Fc region may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) that includes an amino acid modification (e.g., a substitution, deletion, or insertion) at one or more amino acid positions.

[0107] In some embodiments, the antibody characteristics can be modified by altering the number of cysteine ​​residues in the antibody, for example by modifying the hinge region of CH1, where the modification alters (e.g., increases or decreases) the number of cysteine ​​residues in the hinge region.

[0108] The term "knob-into-hole structure" refers to mutating the hydrophobic amino acids of CH3 of antibody Fc. In order to enhance the hydrophobic action, the side chain amino acids of one chain CH3 are mutated to form a hydrophobic amino acid (knob) whose molecule is relatively large, and in order to reduce steric hindrance, the side chain amino acids of the other CH3 are mutated to form a small amino acid (hole). After mutation, CH3 with knob and CH3 with hole form a knob-into-hole structure (KiH) in the form of hydrophobic action, which is helpful for the formation of heavy chain heterodimers. KiH mutation mainly occurs in the internal hydrophobic amino acids of the spatial structure of the CH3 domain, and the amino acids exposed to the outside after mutation are almost unchanged, so that it does not affect the effector function of Fc or the immunogenicity caused. The term "knob-Fc" refers to a point mutation including T366W in the antibody Fc region to form a spatial structure similar to a knob. Accordingly, "hole-Fc" refers to point mutations including T366S, L368A, and Y407V in the antibody Fc region to form a hole-like spatial structure. To further promote the formation of heterodimers, the point mutations S354C and Y349C can be further introduced into knob-Fc and hole-Fc, respectively, to further promote the formation of heterodimers through disulfide bonds. At the same time, the point mutations H435R and Y436F can be further introduced into hole-Fc, respectively, to weaken the binding with Protein A.

[0109] The bispecific antibodies of the invention can comprise two Fc regions, each Fc region being part of a single antibody heavy chain. Fc1 in formula (I) and Fc2 in formula (III) or formula (IV) can have the same sequence, except for having mutations in the CH3 domains that are intended to facilitate or facilitate purification of the heterodimeric (i.e., bispecific) molecule.

[0110] In some embodiments, in the bispecific antibody described in the present invention, the linkage between the above formula (I) and formula (II), and the linkage between the above formula (III) and formula (II-2) are via a disulfide bond.

[0111] In some embodiments, the bispecific antibody of formula (I) and formula (III) may be directly or indirectly linked to each other. In some embodiments, the bispecific antibody of formula (I) and formula (III) may be linked to each other by a linker. In some embodiments, the linker is a peptide linker.

[0112] In some embodiments, the bispecific antibody of the present invention is linked to the antibody of formula (I) and the antibody of formula (III) or formula (IV) by a disulfide bond in the Fc region and a knob-into-hole structure in the CH3 domain. Preferably, Fc1 in formula (I) is knob-Fc and Fc2 in formula (III) or formula (IV) is hole-Fc, or Fc1 in formula (I) is hole-Fc and Fc2 in formula (III) or formula (IV) is knob-Fc.

[0113] In some embodiments, in the bispecific antibodies described in the present invention, CH1-Fc1 in formula (I) and Fc2 in formula (III) are in the form of IgG, such as IgG1, IgG2, IgG3 or IgG4, and / or CL in formula (II) and formula (II-2) is derived from a λ or κ chain.

[0114] The Fc region of the bispecific antibody of the present invention may be a human Fc region. The Fc region of the bispecific antibody of the present invention may be of any isotype, The Fc regions of the first and second antibodies are both of the IgG1 isotype, including but not limited to IgG1, IgG2, IgG3 or IgG4. In some embodiments, the Fc regions of the first and second antibodies are both of the IgG4 isotype. In some embodiments, one of the antibody Fc regions is of the IgG1 isotype and the other is of the IgG4 isotype. In the latter embodiment, the resulting bispecific antibody comprises an IgG1 Fc region and an IgG4 Fc region and may therefore have interesting intermediate properties with regard to activation of effector functions.

[0115] In some embodiments, the antibodies provided herein may be further modified to contain other non-proteinaceous moieties known in the art and readily available. Moieties suitable for derivatization of antibodies 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, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerin), polyvinyl alcohol, and mixtures thereof.

[0116] Antibody expression In yet another aspect, the present invention provides a nucleic acid encoding any of the above antibodies or fragments thereof, or any of the chains thereof. In one embodiment, the polynucleotide may comprise a polynucleotide encoding the amino acid sequence of the light chain variable region and / or the heavy chain variable region of the antibody, or may comprise a polynucleotide encoding the amino acid sequence of the light chain and / or the heavy chain of the antibody.

[0117] In some embodiments, the nucleic acid of the present invention includes a nucleic acid encoding an amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 1 to 20, or a nucleic acid encoding an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 20.

[0118] In yet another aspect, the present invention provides an expression vector comprising a nucleic acid as described herein, preferably, said vector is a eukaryotic expression vector. In some embodiments, the nucleic acid as described herein is included in one or more expression vectors. Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phages, or yeast artificial chromosomes (YACs).

[0119] In yet another aspect, the invention provides a host cell comprising a nucleic acid described herein or an expression vector described herein, preferably, said 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.

[0120] In one embodiment, the invention provides a method for preparing an anti-CD3xCD20 bispecific antibody of the invention, said method comprising introducing an expression vector into a mammalian host cell and culturing the host cell for a sufficient time to produce the antibody, such that the antibody is expressed in the host cell, or more preferably, the antibody is secreted into the host cell growth medium. The antibody can be recovered from the medium by standard protein purification methods.

[0121] 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), particularly Chinese hamster ovary (CHO) cells, NS0, 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 human, mouse, rat, dog, monkey, pig, goat, cow, horse, and hamster cells. Particularly preferred cell lines are selected by determining which cell lines have high expression levels.

[0122] Antibodies expressed in different cell lines or in transgenic animals may show different glycosylation. 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 some embodiments, nonfucosylated antibodies are advantageous because their glycostructure is a common component of natural human serum IgG and therefore they usually have a more potent effect in vitro and in vivo than their fucosylated counterparts and may not be immunogenic.

[0123] Pharmaceutical Compositions and Drug Formulations In yet another aspect, the present invention provides a pharmaceutical composition comprising an anti-CD3xCD20 bispecific antibody, or antigen-binding fragment thereof, described herein, and a pharma- ceutically acceptable carrier or excipient.

[0124] It should be understood that the anti-CD3×CD20 antibody or pharmaceutical composition thereof provided by the present invention can be administered in combination with suitable carriers, excipients and other reagents in the formulation to provide improved metastasis, delivery, tolerance, etc.

[0125] The term "pharmaceutical composition" refers to a formulation that allows the biological activity of the active ingredient contained therein to be present in an effective form and does not contain other ingredients that have unacceptable toxicity to the subject to which the formulation is administered.

[0126] Pharmaceutical formulations comprising the anti-CD3xCD20 bispecific antibodies described herein can be prepared by mixing the anti-CD3xCD20 bispecific antibodies of the 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 formulation.

[0127] The pharmaceutical composition or formulation of the present invention may further comprise one or more other active ingredients, which are as required for the particular indication being treated, and preferably have such active ingredients with 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 any known antitumor or anticancer agents, and the active ingredients are present in suitable combinations in amounts effective for the intended use.

[0128] In some embodiments, the pharmaceutical composition of the invention further comprises a composition of a nucleic acid encoding an anti-CD3xCD20 bispecific antibody.

[0129] In yet another aspect, the present invention provides a pharmaceutical combination comprising an anti-CD3xCD20 bispecific antibody or antigen-binding fragment thereof as described herein, or a pharmaceutical composition as described herein, and one or more additional therapeutic agents.

[0130] In yet another aspect, the present invention provides a reagent kit comprising an antibody or antigen-binding fragment thereof described herein, a polynucleotide described herein, an expression vector described herein, a host cell described herein, or a pharmaceutical composition described herein.

[0131] Medical Use In yet another aspect, the present invention provides the use of an anti-CD3xCD20 bispecific antibody or an antigen-binding fragment thereof as described herein, or a pharmaceutical composition as described herein, in the preparation of a medicament for the prevention and / or treatment of cancer.

[0132] In yet another aspect, the present invention provides an anti-CD3xCD20 bispecific antibody or an antigen-binding fragment thereof as described herein, or a pharmaceutical composition as described herein, for preventing and / or treating cancer.

[0133] In yet another aspect, the present invention provides a method for preventing and / or treating cancer comprising administering to a subject in need thereof an anti-CD3xCD20 bispecific antibody as described herein, or a pharmaceutical composition as described herein.

[0134] In some embodiments, the cancer according to the invention 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's lymphoma.

[0135] The subject may be a mammal, such as a primate, and is preferably an advanced primate, such as a human (e.g., a patient having or at risk of having a disease described herein). In one embodiment, the subject has or is at risk of having a disease described herein (e.g., a tumor described herein). In some embodiments, the subject is undergoing or has undergone other treatments, such as chemotherapy and / or radiation therapy.

[0136] In some embodiments, modes of administration of the present invention include, but are not limited to, oral administration, intravenous administration, subcutaneous administration, intramuscular administration, intraarterial administration, intra-articular administration (e.g., in an arthritic joint), inhalation, aerosol delivery, or intratumoral administration.

[0137] In some embodiments, the present invention provides one or more therapies (eg, therapeutics and / or other therapeutic agents) to be co-administered to a subject in therapeutically effective amounts.

[0138] In some embodiments, the methods or uses provided by the present invention further comprise administering to the individual one or more therapies (e.g., therapy and / or other therapeutic agents). The antibodies of the present invention may be used alone or in combination with other therapeutic agents of the therapy. For example, they may be administered in combination with at least one additional therapeutic agent.

[0139] In some embodiments, the therapeutic modality comprises surgery, radiation therapy, local or focused radiation, etc. In some embodiments, the therapeutic agent is selected from a chemotherapeutic agent, a cytotoxic agent, a vaccine, another antibody, an anti-infective agent, or an immunomodulatory agent.

[0140] Methods used in diagnosis and detection In yet another aspect, the present invention provides a method of detecting the presence of CD3 or CD20 in a sample utilizing any of the antibodies or antigen-binding fragments thereof described herein. The term "detection" as used herein includes quantitative detection or qualitative detection. Exemplary detection methods may relate to immunohistochemistry, immunocytochemistry, flow cytometry (e.g., FACS), antibody molecule complexed magnetic beads, ELISA assays, PCR-techniques (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 sample derived from an organism. In some embodiments, the biological sample comprises cells or tissues. In some embodiments, the biological sample is derived from a hyperproliferative or cancerous lesion.

[0141] In one aspect, the 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 the steps of 1) contacting the sample (and optionally a control sample) with an antibody molecule as described herein or administering said antibody molecule to a subject under conditions that allow the interaction to occur, and 2) detecting the formation of a complex between said antibody molecule and the sample (and optionally a control sample). The formation of a complex indicates the presence of the relevant antigen and may indicate the applicability or need for treatment and / or prevention as described herein.

[0142] In some embodiments, the invention provides a diagnostic reagent kit comprising an antibody molecule or antigen-binding fragment thereof described herein, or a pharmaceutical composition described herein and instructions for use.

[0143] The present invention includes all combinations of the specific embodiments described. Further embodiments of the present invention and its full scope of applicability will become apparent from the detailed description provided below. However, while preferred embodiments of the present invention have been set forth in the detailed description and specific examples, it should be understood that these descriptions and examples are provided by way of illustration only, since 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. For all purposes, all disclosures, patents, and patent applications cited herein, including the foregoing, are hereby incorporated by reference in their entirety.

[0144] The present invention employs the following abbreviations:

[0145] his-tag stands for histidine tag.

[0146] Fc tag stands for fragment crystallizable tag.

[0147] ECD stands for the extracellular domain.

[0148] PEI stands for polyethyleneimine.

[0149] BSA stands for bovine serum albumin.

[0150] PBS stands for phosphate buffered saline.

[0151] FBS stands for fetal bovine serum.

[0152] CFSE stands for carboxyfluorescein diacetate succinimidyl ester.

[0153] APC stands for allophycocyanin.

[0154] NA-PE stands for neutral avidin labeled with phycoerythrin.

[0155] PE stands for phycoerythrin.

[0156] TMB stands for 3,3',5,5'-tetramethylbenzidine.

[0157] PBST stands for phosphate Tween buffer.

[0158] Working Example The present invention will be described by the following examples, but is not intended to limit the present invention in any way. This specification has already described the present invention in detail, and specific embodiments thereof have been disclosed. It is obvious 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 present invention.

[0159] Example 1. Construction of expression vector for bispecific antibody molecules 1.1 Anti-CD3 antibody In the present invention, multiple strains of anti-human CD3 antibodies were analyzed, and one of them, the mouse antibody SP34, was humanized through sequence optimization to obtain the humanized anti-CD3 antibody JSCD3.

[0160] SP34 is a mouse monoclonal antibody against human CD3, whose heavy chain amino acid sequence is SEQ ID NO:24 and whose light chain amino acid sequence is SEQ ID NO:25.

[0161] The amino acid sequence of the humanized anti-CD3 antibody JSCD3 is as follows: 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. JSCD3 VH: SEQ ID NO: 15.

[0162] The amino acid sequences of its light chain complementarity determining regions LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs: 12, 13 and 14, respectively. JSCD3 VL: SEQ ID NO: 16.

[0163] The full length amino acid sequence of JSCD3 is as follows: JSCD3 HC: SEQ ID NO: 21 JSCD3 LC: SEQ ID NO:22

[0164] 1.2 Construction of expression vectors for bispecific antibody molecules Bispecific antibody molecule TZT6: The expression vectors were constructed by simultaneously expressing HXT2-JS CD20 LC-2, HXT4s-JS CD20 HC-2b and HX4-JSCD3ScFv NVL-Mut transiently, and the construction of the expression vectors was as follows: the synthesis of the gene encoding JSCD20 LC-2 was entrusted to GenScript Biotech Corporation, and the enzyme was digested with BSPQI and ligated to the HXT2 vector (a vector derived from pCDNA3.1 and autonomously modified by the primordial organism), to obtain the first expression vector HXT2-JS CD20 LC-2. The synthesis of the gene encoding JSCD20 LC-2 was entrusted to GenScript Biotech Corporation, and the enzyme was digested with HindIII and NheI and ligated to the HXT4s-Mut-b (a vector derived from pCDNA3.1 and autonomously modified by the primordial organism), to obtain the second expression vector HXT4s-JS CD20 HC-2b. The synthesis of the gene encoding JSCD3ScFv was entrusted to GenScript Biotech Corporation, and the gene was enzymatically digested with HindIII and NheI and ligated into the HX4-FC-mut-h vector (a vector derived from pCDNA3.1 and autonomously modified by the host organism) to obtain the third expression vector HX4-JSCD3ScFv NVL-Mut h.

[0165] Bispecific antibody molecule TZT7: HXT2-JS CD20 LC-2, HXT4s-JS CD20 HC-2 Mut h and HX4-JS CD20 HC-2 JS CD3ScFv NVL-G4 FC b V2 were simultaneously transiently expressed to obtain the expression vector, which was constructed in sequence as follows: the synthesis of the gene encoding JSCD20 LC-2 was entrusted to GenScript Biotech Corporation, and the enzyme digestion was performed by BSPQI and ligated to the HXT2 vector to obtain the first expression vector HXT2-JS CD20 LC-2. The synthesis of the gene encoding JSCD20 HC-2 was entrusted to GenScript Biotech Corporation, and the enzyme digestion was performed by HindIII and NheI and ligated to HXT4s-Mut-h to obtain the second expression vector HXT4s-JS CD20 HC-2 h. The synthesis of the gene encoding JSCD3ScFv was entrusted to GenScript Biotech Corporation, and the gene was enzymatically 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.

[0166] Example 2. Expression and purification of bispecific antibody molecules 2.1 Expression of bispecific antibody molecules Transient expression and purification of bispecific antibody molecules TZT6 and TZT7: The three plasmids constructed above were extracted using an endotoxin control reagent kit and used for subsequent mammalian cell expression. CHO-K1 cells in culture (modified at the genome level to be suitable for transient expression) were counted and the cell density was determined to be 2–6 × 10 6 When the cell density was 1.5–2.0 × 10 / mL, subculture was performed in CD CHO medium, and the cell density was adjusted to 1.5–2.0 × 10 6 / mL, and the next day the cell density was approximately 3.5 × 10 6Transfection was performed when the total volume of the medium reached 10 μg / mL. First, add 1 / 10 of the transfection volume of medium, then add 1-2 μg / mL of the transfection volume of plasmid, and finally add 3-14 μg / mL of PEI, mix evenly, and then incubate at room temperature, the time not exceeding 30 minutes, and finally add the transfection mixture slowly to the pre-treated cells, mixing evenly while adding. The transfection mixture was placed in a shaker and cultured under the culture conditions of 36.5 °C, 120 rpm, and 7% CO2. The culture cycle was 6-10 days after transfection, and replenished once every 2 days.

[0167] 2.2 Purification of bispecific antibody molecules After the above transfection mixture was cultured, it was centrifuged at 1000g for 10 minutes to discard the precipitate, and then centrifuged at 12000g for 30 minutes to collect the cell supernatant, which was then sterile filtered. It was purified using an AKTA Avant purification device, and first, a column packed with Praesto Jetted A50 affinity packing was CIPed with 0.1M NaOH for 15-20 minutes, then the sample was loaded after equilibration with PBS buffer for 3-5 CV, and after loading was completed, it was eluted with affinity chromatography elution buffer, and finally the target protein was eluted with acetic acid-sodium acetate buffer at pH 3.8, the sample was neutralized with 1M Tris buffer, and the pH was adjusted to 5.5, and the sample was left and sent to SEC-HPLC to detect the purity of the sample. The remaining sample was purified in the next step, using weak cationic packing EMD COOM packing, the equilibration solution was 50mM acetic acid-sodium acetate system at pH 5.5, the eluent was 50mM acetic acid-sodium acetate + 1M NaCl buffer system at pH 5.5, and the target protein was collected by linear elution, and the final SEC-HPLC monomer purity reached more than 95%, resulting in the bispecific antibody molecules TZT6 and TZT7, respectively. The structure of the bispecific antibody molecule TZT6 is shown in Figure 1a, and the structure of the bispecific antibody molecule TZT7 is shown in Figure 1b.

[0168] The amino acid sequence of the bispecific antibody molecule TZT6 is as follows: HC1 (HXT4s-JS CD20 HC-2 b): SEQ ID NO: 17 LC (HXT2-JS CD20 LC-2): SEQ ID NO: 18 HC2 (HX4-JSCD3ScFv NVL-Mut h): SEQ ID NO: 20 The amino acid sequence of the bispecific antibody molecule TZT7 is as follows: HC1 (HXT4s-JS CD20HC-2 Mut h): SEQ ID NO: 23 LC (HXT2-JS CD20 LC-2): SEQ ID NO: 18 HC2 (HX4-JS CD20 HC-2 JS CD3ScFv NVL-G4 FC b V2): SEQ ID NO: 19.

[0169] The CDR and variable region amino acid sequences of the bispecific antibody molecules TZT6 and TZT7 are shown in Table 1.

[0170] [Table 1]

[0171] Example 3. Stability of Bispecific Antibodies 3.1 Thermal stability of bispecific antibodies 1. Purpose of the test The thermal stability of the bispecific antibody of the present invention was detected. Differential scanning fluorescence (DSF) was used to study 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).

[0172] 2. Test process and results The antibody sample was added to the above buffer solution, the concentration of the antibody sample was controlled to about 4 mg / mL, and detection was performed by DSF. The results are shown in Table 2. In the above buffer solution, the bispecific antibodies TZT6 and TZT7 of the present invention both had thermal transition temperatures (Tm) of 60°C or higher, and showed good thermal stability.

[0173] [Table 2]

[0174] 3.2 High temperature stability of bispecific antibodies 1. Purpose of the test The stability of the bispecific antibody was examined under high temperature conditions.

[0175] 2. Test process and results The antibody sample was added to a pH 6.0 buffer system (20 mM citric acid-sodium citrate / 50 mM sodium chloride / 140 mM mannitol), the concentration of the sample was controlled to about 4 mg / mL, and 500 μL / bottle was dispensed into a vial. The samples were placed in a 40°C incubator and the stability was considered at 0W, 2W and 4W, and the samples were sent and detected according to Table 3. The stability was evaluated by the following parameters: (a) the content of antibody monomer, polymer, or fragment was measured by SEC-HPLC (size exclusion chromatography), (b) the molecular weight of the antibody was detected by two types of CE-SDS (sodium dodecyl sulfate capillary electrophoresis) methods, and (c) the biological activity of the antibody was detected by ELISA and reporter gene methods (see Examples 4 and 8 for the experimental methods).

[0176] The detection results showed that the purity (SEC-HPLC method, R-CE-SDS (reduced electrophoresis), NR-CE-SDS (non-reduced electrophoresis)) and biological activity of the bispecific antibodies TZT6 and TZT7 of the present invention did not change significantly even after being left at a high temperature of 40°C for 4 weeks, and they have good thermal stability. The specific results are shown in Table 3.

[0177] [Table 3]

[0178] Example 4: Detection of binding of bispecific antibodies to human CD3ε by ELISA 1. Purpose of the test The binding of the bispecific antibody of the present invention to human CD3ε was detected by ELISA.

[0179] 2. Testing process a. Recombinant human CD3ε (Novoprotein, C578) at a concentration of 1.0 μg / mL was used as an antigen and coated onto a 96-well plate at 100 μL / well, followed by incubation at 37° C. for 1.5 hours.

[0180] b. The plate was washed four times with 1x PBST at 300 μL / well, and 2% BSA was added at 200 μL / well, followed by blocking at 37° C. for 1.5 hours.

[0181] c. Wash 4 times with 1x PBST at 300 μL / well, add gradient diluted bispecific antibodies TZT6, TZT7 and negative control antibody (anti-KLH hIgG4) (initial concentration 10 μg / mL, 12 concentration points diluted 2.5-fold gradient, 100 μL / well) and incubate at 37°C for 1 hour.

[0182] d. Wash 4 times with 1x PBST at 300 μL / well.

[0183] e. 100 μL / well of mouse anti-human IgG4 Fc antibody coupled to horseradish peroxidase (HRP) (Southern Biotech, 9200-05) diluted 1:5000 was added and incubated at 37° C. for 1 hour.

[0184] f. Wash 4 times with 1x PBST at 300 μL / well.

[0185] g. 0.1 mg / mL TMB was added at 100 μL / well and incubated at 37° C. for 15 minutes, after which 2 M hydrochloric acid solution was added at 100 μL / well to stop the reaction.

[0186] h. Absorbance was measured at 450 nm / 620 nm using a plate reader and data was analyzed using Graphpad Prism7.

[0187] 3. Test results As shown in FIG. 2, the bispecific antibodies TZT6 and TZT7 of the present invention exhibited relatively strong binding to human CD3ε and EC 50 were 21.4ng / mL and 85.63ng / mL, respectively.

[0188] Example 5: Binding of bispecific antibodies to Raji cells Raji cells (human B lymphoma cells, CD20 positive) were incubated with different concentrations of bispecific antibodies TZT7 and TZT6 (initial concentration 100 μg / mL, diluted 3-fold, total 12 concentration gradients) at 4°C for 30 min, then washed and incubated with fluorescently labeled secondary antibodies at 4°C in the dark for 30 min. Finally, cells were collected using a flow cytometer (BD CantoII) to detect fluorescent antibodies binding to the cell surface. FlowJo was used to analyze the raw data to obtain MFI values, and GraphPad was used to fit the antibody dose-dependent binding curves (Figure 3) to obtain EC 50 was calculated, and the positive and negative controls were REGN1979 (Regeneron Pharmaceuticals) and anti-KLH hu-IgG4 antibody, respectively.

[0189] As shown in Figure 3, TZT7, TZT6 and REGN1979 could all bind to CD20 on the surface of Raji cells with high affinity and were expressed in EC 50 were 0.5152 μg / mL, 3.673 μg / mL, and 30.11 μg / mL, respectively, and the binding abilities of TZT7 and TZT6 to Raji cells were significantly superior to that of the positive control REGN1979.

[0190] Example 6: Binding of bispecific antibodies to Jurkat cells Jurkat cells (human T lymphoma cells, CD3 positive) were incubated with different concentrations of bispecific antibodies TZT6 and TZT7 (initial concentration 100 μg / mL, diluted 3-fold, total 12 concentration gradients) at 4 °C for 30 min, then washed and incubated with fluorescently labeled secondary antibodies at 4 °C in the dark for 30 min. Finally, cells were collected using a flow cytometer (BD CantoII) to detect fluorescent antibodies binding to the cell surface. FlowJo was used to analyze the raw data to obtain MFI values, and GraphPad was used to fit the antibody dose-dependent binding curves (Figure 4) to obtain EC 50 was calculated, and the positive and negative controls were REGN1979 (Regeneron Pharmaceuticals) and anti-KLH hu-IgG4 antibody, respectively.

[0191] As shown in FIG. 4, REGN1979, TZT6, and TZT7 were all able to bind to human CD3 on the surface of Jurkat cells and were able to mediate EC 50 were 4.698 μg / mL, 4.442 μg / mL, and 26.59 μg / mL, respectively.

[0192] Example 7: Binding of bispecific antibodies to overexpressing cynomolgus monkey CD3e cells 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 (initial concentration 100 μg / mL, diluted 3-fold, total 12 concentration gradients) for 30 min at 4 °C, then washed and incubated with fluorescently labeled secondary antibodies for 30 min in the dark at 4 °C. Finally, cells were collected using a flow cytometer (BD CantoII) to detect fluorescent antibodies binding to the cell surface. FlowJo was used to analyze the raw data to obtain MFI values, and GraphPad was used to fit the antibody dose-dependent binding curves (Figure 5) to obtain EC 50 was calculated, and the positive and negative controls were JSCD3 (anti-CD3 antibody) and anti-KLH hu-IgG4 antibody, respectively.

[0193] As shown in FIG. 5, both bispecific antibodies TZT7 and TZT6 can bind to cynomolgus monkey CD3e.

[0194] Example 8: Detection of bispecific antibody activity in a luciferase reporter gene system The target cells Raji (human B lymphoma cells, CD20 positive) and the effector cells Jurkat NFAT (stably expressing luc2P / NFAT-RE) were each cultured at 5 × 10 per well. 4 1 x 10 cells per well 5 The cells were added to a 96-well flat-bottom white plate (Corning, Cat#3917) at 100 μg / mL. Then, bispecific antibodies TZT7, TZT6 and REGN1979 (initial concentration 1 μg / mL, diluted 3-fold, total 12 concentration gradients) were added to the cell plate using experimental buffer (RPMI 1640 (1×) + 2% FBS) and co-incubated in a 37°C incubator for 4-6 h. Finally, ONE-Glo luciferase detection reagent (Promega) was added to the cell-antibody mixture, and the chemiluminescence signal was detected by a multifunction plate reader (TECAN M1000 pro). A four-parameter regression curve was fitted by GraphPad prism software to obtain the EC 50 The value was calculated.

[0195] As shown in FIG. 6, TZT7, TZT6 and REGN1979 (Regeneron Pharmaceuticals) have strong T cell activation activity and EC 50 were 0.2087 ng / mL, 0.6057 ng / mL, and 3.688 ng / mL, respectively, and TZT7 and TZT6 had significantly superior T cell activation activity to REGN1979.

[0196] Example 9: Study of the activation of bispecific antibodies against T lymphocytes Anti-CD3 / CD20 bispecific antibody can effectively promote the activation of T lymphocytes in peripheral blood monocytes. In general, upregulated expression of cell surface marker CD69 is an early marker of T cell activation, and upregulated expression of cell surface marker CD25 is a late marker of T cell activation. In this experiment, the activation effect of anti-CD3 / CD20 bispecific antibody on T cells in this study was evaluated by the increased proportion of the CD25 and CD69 double positive population.

[0197] Human total T cells were isolated and purified from commercially available PBMCs (Allcells, Cat#PB004F-C) using a human total T cell purification reagent kit (Miltenyi Biotec, Cat#130-096-535), and the purified human total T cells (1 × 10 per well) were then cultured in a 5% CO2-free medium. 5 cells) and Raji cells (2 × 10 per well 4 100 cells) were co-incubated with gradient diluted TZT7 and TZT6 or control samples (initial concentration 10 μg / mL, 5-fold diluted, total 12 concentration gradients) in 96-well plates at 37°C for 24 hours. Finally, cells were harvested and stained with APC anti-human CD8a (Biolegend, Cat#301049), PE anti-human CD25 (Biolegend, Cat#302606), BV421 anti-human CD69 (BD, Cat#562884), followed by mechanical detection with a flow cytometer (BD, CantoII). CD8 was detected using FlowJo software. + CD25 on T cells + CD69 + The percentage of double positive cell population was calculated and a four-parameter regression curve was fitted using GraphPad prism software to determine the EC 50 The value was calculated.

[0198] As shown in FIG. 7, the anti-CD3 / CD20 bispecific antibodies TZT7 and TZT6 of the present invention and the positive control REGN1979 (Regeneron Pharmaceuticals) all inhibited CD25 in CD8 T lymphocytes. + and CD69 +It can effectively promote the increase in the proportion of double positive cell population of EC 50 were 0.1476 ng / mL, 0.9190 ng / mL, and 5.632 ng / mL, respectively. TZT7 and TZT6 had significantly superior T cell activation activity to REGN1979.

[0199] Example 10: Study to enhance killing activity of T cells against B lymphoma cells by bispecific antibodies Flow cytometry was used to detect the ability of the anti-CD3 / CD20 bispecific antibody of the present invention to enhance the killing activity of T cells against B lymphoma cells (Raji cells).

[0200] First, human total T cells were isolated and purified from commercial PBMCs (Allcells, Cat#PB004F-C) using a Human pan T cell isolation kit (Miltenyi Biotec, Cat#130-096-535), and then Raji cells were labeled with a CFSE cell proliferation reagent kit (Invitrogen, Cat#C34554) and the purified human total T cells (1 × 10 per well) were cultured in 100% PBS-free medium. 5 cells) and CFSE-labeled Raji cells (2 × 10 per well 4 100 cells) were co-incubated with gradient diluted TZT7, TZT6 or control samples (initial concentration 10 μg / mL, diluted 6-fold, total 10 concentration gradient) in 96-well plates at 37°C for 72 hours. Finally, cells were harvested and stained with 7-AAD (BD, Cat#559925), followed by collection by flow cytometer (BD, Canto II). The percentage of the dead Raji cell population, i.e., CFSE, was determined using FlowJo software. + 7-AAD + (Percp-cy5.5 channel) The proportion of double positive populations was analyzed and a four-parameter regression curve was fitted using GraphPad prism software to obtain the EC 50 The value was calculated.

[0201] As shown in FIG. 8, the anti-CD3 / CD20 bispecific antibodies TZT7, TZT6 and REGN1979 (Regeneron Pharmaceuticals) of the present invention can effectively promote the killing of B lymphoma cells (i.e., Raji cells) by T cells, and can suppress the EC 50 were 0.9869 ng / mL, 41.40 ng / mL, and 62.95 ng / mL, respectively, indicating that the killing activity of TZT7 was significantly superior to that of REGN1979.

[0202] Example 11: Single-dose PK study in cynomolgus monkeys 1. Purpose of the test PK study of a bispecific antibody of the invention for a single dose in cynomolgus monkeys.

[0203] 2. Testing process a. Recombinant human CD3ε (Novoprotein, C578) at a concentration of 1.0 μg / mL was used as an antigen and coated onto a 96-well plate at 100 μL / well, followed by incubation at 37° C. for 1.5 hours.

[0204] b. The plate was washed four times with 1x PBST at 300 μL / well, and 2% BSA was added at 200 μL / well, followed by blocking at 37° C. for 1.5 hours.

[0205] c. TZT7 calibration curve samples were prepared with a range of 8μg / mL-125ng / mL using blank cynomolgus serum, TZT6 calibration curve samples were prepared with a range of 4μg / mL-62.5ng / mL using blank cynomolgus serum, and the specimens were diluted to the calibration curve range using blank cynomolgus serum. The calibration curve samples and specimens were then diluted 10-fold with 2% BSA. The specimens prepared above were added at 100μL / well, incubated at 37℃ for 1 hour, and the plate was washed.

[0206] d. Wash 4 times with 1x PBST at 300 μL / well.

[0207] e. 100 μL / well of mouse anti-human IgG4 Fc antibody coupled to horseradish peroxidase (HRP) (Southern Biotech, 9200-05) diluted 1:5000 was added and incubated at 37° C. for 1 hour.

[0208] f. Wash 4 times with 1x PBST at 300 μL / well.

[0209] g. 0.1 mg / mL TMB was added at 100 μL / well and incubated at 37° C. for 5 to 7 minutes, after which 2 M hydrochloric acid solution was added at 100 μL / well to stop the reaction.

[0210] h. Absorbance was measured at 450 nm / 620 nm using a plate reader, and data was analyzed using a four-parameter model in SoftMax Pro 5.4.1.

[0211] 3. Test results As shown in Table 4, the bispecific antibody TZT7 of the present invention in cynomolgus monkeys has a half-life of 19.1 hours when administered at a dose of 1 mg / kg in the body, and max is 24.1 mg / L and the dose is 10 mg / kg, the half-life is 138.3 hours, and C max The bispecific antibody TZT6 of the present invention had a half-life of 164.2 hours when administered at a dose of 1 mg / kg in the body, and a C max is 24.1 mg / L and the dose is 10 mg / kg, the half-life is 168.8 hours, and C max was 244.9 mg / L.

[0212] [Table 4]

[0213] Note: AUC (0-t) : Area under the drug concentration-time curve, t 1 / 2 :Terminal elimination half-life, T max : Time to peak, C max :Peak drug concentration.

[0214] Example 12: Inhibitory effect of bispecific antibodies on the growth of B16 OVA huCD20 tumors 1. Purpose of the test The antitumor effects of TZT6 and TZT7 of the present invention were evaluated in a mouse melanoma B16 OVA huCD20 subcutaneously transplanted tumor model.

[0215] 2. Testing process 2.5 × 10 5 B16 OVA huCD20 1F11 cells (transduced with human CD20 gene) were inoculated subcutaneously on the right dorsal side. The average tumor volume was approximately 52 mm 3 If so, suitable animals were selected and randomly divided into three groups with six animals per group according to tumor volume. Vehicle control group, - G1 saline group, treatment group, -G2 TZT6 (10 mg / kg) group, and - G3 TZT7 (10 mg / kg) group.

[0216] The mice were administered twice a week by intraperitoneal injection for two consecutive weeks, and the experiment was terminated four 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. After the experiment was completed, the mice were euthanized, and the tumor inhibition rate TGI (%) = [1-(Ti-T0) / (Vi-V0)] × 100% was calculated. (Ti: mean tumor volume of the treatment group on the ith 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 ith day of administration, and V0: mean tumor volume of the solvent control group on the 0th day of administration).

[0217] As shown in FIG. 9, on day 14 after administration, the saline control group had a mean tumor volume of 2170 mm 3 The mean tumor volume in the TZT6 group was 1455 mm 3The tumor inhibition rate was 33.7% compared to the saline control group. The mean tumor volume in the TZT7 group was 372 mm 3 The tumor inhibition rate was 84.9% compared to the saline control group, and the increase in tumor volume was significantly inhibited. The results showed that TZT6 and TZT7 both exhibited tumor inhibition effects at a dose level of 10 mg / kg in the hCD3e humanized mouse transplanted B16 OVA huCD20 1F11 model.

[0218] Example 13: Inhibitory effect of TZT7 on B16 OVA huCD20 tumor growth 1. Purpose of the test The antitumor effect of TZT7 of the present invention was evaluated in a mouse melanoma B16 OVA huCD20 subcutaneously transplanted tumor model.

[0219] 2. Testing process 2.5 × 10 5 B16 OVA huCD20 1F11 cells (transduced with human CD20 gene) were inoculated subcutaneously on the right dorsal side. The average tumor volume was approximately 88 mm 3 If so, suitable animals were selected and randomly divided into three groups with six animals per group according to tumor volume. Vehicle control group, - G1 saline group, treatment group, -G2 TZT7 (10 mg / kg) group, and - G3 RENG1979 (10 mg / kg) group.

[0220] The mice were administered twice a week by intraperitoneal injection for two consecutive weeks, and the experiment was terminated four 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. After the experiment was completed, the mice were euthanized, and the tumor inhibition rate TGI was calculated. TGI(%)=[1-(Ti-T0) / (Vi-V0)]×100%. (Ti: mean tumor volume of the treatment group on day i of administration, T0: mean tumor volume of the treatment group on day 0 of administration, Vi: mean tumor volume of the vehicle control group on day i of administration, and V0: mean tumor volume of the vehicle control group on day 0 of administration).

[0221] As shown in FIG. 10, on day 18 after administration, the saline control group had a mean tumor volume of 2517 mm 3 The mean tumor volume in the RENG1979 (Regeneron Pharmaceuticals) group was 2646 mm 3 The tumor inhibition rate was -5.3% compared to the saline control group, and no tumor inhibition effect was observed. The mean tumor volume in the TZT7 group was 371 mm 3 The tumor inhibition rate was 88.3% compared to the saline control group, and the increase in tumor volume was significantly inhibited. The results showed that TZT7 could significantly inhibit the growth of hCD3e humanized mouse transplanted B16 OVA huCD20 1F11 tumors at a dose level of 10 mg / kg.

[0222] Example 14: Inhibitory effect of TZT6 and TZT7 on human lymphoma Raji Mixeno model 1. Purpose of the test The antitumor effects of TZT6 and TZT7 of the present invention were evaluated in a human lymphoma Raji Mixeno model.

[0223] 2. Testing process Six to eight week old female NDG mice (purchased from Biocytogen JiangSu Co., Ltd.) were administered 2.5 × 10 6 Raji cells and 5 × 10 6A suspension (0.2 mL / mouse) of PBMCs (purchased from Allcells) was inoculated subcutaneously into the right dorsal region to establish a subcutaneous tumor model. The average tumor volume was approximately 108 mm. 3 If so, select appropriate animals and randomly divide them into 5 groups with 6 animals per group according to tumor volume, Vehicle control group, - G1 saline group, treatment group, -G2 TZT6 (1mg / kg) group, -G3 TZT6 (3mg / kg) group, -G4 TZT7 (1mg / kg) group, -G5 TZT7 (3 mg / kg) group, The mice were intraperitoneally injected and administered once, and the experiment was terminated after 18 days of administration. The tumor volume and body weight were measured twice a week, and the body weight and tumor volume of the mice were recorded. After the experiment was completed, the mice were euthanized, and the tumor inhibition rate TGI was calculated. TGI(%)=[1-(Ti-T0) / (Vi-V0)]×100%. (Ti: mean tumor volume of the treatment group on day i of administration, T0: mean tumor volume of the treatment group on day 0 of administration, Vi: mean tumor volume of the solvent control group on day i of administration, and V0: mean tumor volume of the solvent control group on day 0 of administration).

[0224] As shown in FIG. 11, on the 18th day after the start of administration, the saline control group had a mean tumor volume of 1610 mm 3 TZT6 had a mean tumor volume of 723 mm at dose levels of 1 mg / kg and 3 mg / kg, respectively. 3 and 233 mm 3 The tumor inhibition rates were 58.7% and 91.5%, respectively, compared with the saline control group, and the increase in tumor volume was significantly inhibited. TZT7 significantly inhibited the increase in tumor volume at the dose levels of 1 mg / kg and 3 mg / kg, respectively. 3 and 99mm 3 The tumor inhibition rates were 89.6% and 100.6%, respectively, and the increase in tumor volume was significantly inhibited. The results showed that TZT6 and TZT7 could significantly inhibit the growth of subcutaneously transplanted tumors of human lymphoma Raji cells at the dose levels of 1 mg / kg and 3 mg / kg.

Claims

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

2. The polypeptide chain comprises: (1) A polypeptide chain having a CD20-binding domain as shown in formula (I) and formula (II): VH1-CH1-Fc1 formula (I), VL1-CL Formula (II), and (2) A polypeptide chain having a CD20 and CD3 binding domain as shown in formula (III) and formula (II-2): VH1-CH1-L-VL2-L-VH2-L-Fc2 formula (III), VL1-CL Formula (II-2), or A polypeptide chain having a CD3 binding domain as shown in formula (IV): 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, and L represents a connecting peptide, each connecting peptide being homologous or different, and optionally, there is a hinge region between CH1 and Fc1; Formulae (I), (II), (II-2), (III) and (IV) are linked in order from the N-terminus to the C-terminus, 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. The bispecific antibody of claim 1.

3. the connecting peptide comprises an amino acid sequence (GGGGS)n, where n is independently selected from 1, 2, 3, 4, 5, and 6; A bispecific antibody according to claim 1 or 2.

4. The VH1 comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are set forth 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; The bispecific antibody of claim 1.

5. The VH1 comprises an amino acid sequence set forth in SEQ ID NO:7 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO:7; and The VL1 comprises an amino acid sequence as set forth in SEQ ID NO:8 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence as set forth in SEQ ID NO:8; The bispecific antibody of claim 4.

6. The VH2 comprises HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are set forth 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 set forth in SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14, respectively; The bispecific antibody of claim 1.

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

8. The polypeptide chain of formula (I) comprises an amino acid sequence as set forth in SEQ ID NO: 17 or 23, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence as set forth in SEQ ID NO: 17 or 23, and The polypeptide chain of formula (II) and formula (II-2) comprises an amino acid sequence as set forth in SEQ ID NO: 18 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence as set forth in SEQ ID NO: 18; The bispecific antibody of claim 1.

9. The polypeptide chain of formula (III) comprises an amino acid sequence as set forth in SEQ ID NO:19 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence as set forth in SEQ ID NO:19; The bispecific antibody of claim 1.

10. The polypeptide chain of formula (IV) comprises an amino acid sequence as set forth in SEQ ID NO:20 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence as set forth in SEQ ID NO:20; The bispecific antibody of claim 1.

11. The polypeptide chains include 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 represented by SEQ ID NO: 23, the amino acid sequences of the polypeptide chains of formula (II) and formula (II-2) are represented by SEQ ID NO: 18, and the amino acid sequence of the polypeptide chain of formula (III) is represented by SEQ ID NO:

19. The bispecific antibody of claim 1.

12. The polypeptide chain comprises a polypeptide chain represented by formula (I), a polypeptide chain represented by formula (II), and a polypeptide chain represented by formula (IV), wherein the amino acid sequence of the polypeptide chain of formula (I) is represented by SEQ ID NO: 17, the amino acid sequence of the polypeptide chain of formula (II) is represented by SEQ ID NO: 18, and the amino acid sequence of the polypeptide chain of formula (IV) is represented by SEQ ID NO:

20. The bispecific antibody of claim 1.

13. The formula (I) and the formula (II), and the formula (III) and the formula (II-2) are linked by a disulfide bond, and the formula (I) and the formula (III) or the formula (IV) are linked by a disulfide bond and a knob into hole structure of a CH3 domain; Preferably, Fc1 in formula (I) is a knob-Fc, and Fc2 in formula (III) or formula (IV) is a hole-Fc, or Fc1 in formula (I) is a hole-Fc, and Fc2 in formula (III) or formula (IV) is a knob-Fc. The bispecific antibody of claim 1.

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

15. An isolated nucleic acid encoding any one or more of the polypeptide chains in the bispecific antibody of claim 1. Isolated nucleic acid.

16. 16. An expression vector comprising the nucleic acid of claim 15, preferably said expression vector being a eukaryotic expression vector. Expression vector.

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

18. 17. A method for preparing a bispecific antibody according to claim 1, said method comprising culturing a host cell according to claim 16 under conditions suitable for expression of a nucleic acid according to claim 15, and recovering said bispecific antibody from said host cell. method.

19. 17. A method for the preparation of a ... Pharmaceutical compositions.

20. 17. Use of the bispecific antibody according to claim 1, the nucleic acid according to claim 15, the expression vector according to claim 16, a host cell comprising said nucleic acid or expression vector and / or a pharmaceutical composition comprising said bispecific antibody, said nucleic acid, said expression vector and / or said host cell and a pharma- ceutically acceptable carrier or excipient, in the preparation of a medicament for the prevention or treatment of cancer, preferably 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. use.

21. 19. A method for preventing or treating cancer in a subject, comprising administering to a subject in need thereof a bispecific antibody according to claim 1, a nucleic acid according to claim 15, an expression vector according to claim 16, a host cell comprising said nucleic acid or said expression vector, and / or a pharmaceutical composition comprising said bispecific antibody, said nucleic acid, said expression vector and / or said host cell, and a pharma- ceutical acceptable carrier or excipient. method.

22. Detecting the presence of CD3 and / or CD20 in a sample using the bispecific antibody of claim 1. method.

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