WO2022037582A1 - 抗cd3和抗cldn-18.2双特异性抗体及其用途 - Google Patents

抗cd3和抗cldn-18.2双特异性抗体及其用途 Download PDF

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WO2022037582A1
WO2022037582A1 PCT/CN2021/113033 CN2021113033W WO2022037582A1 WO 2022037582 A1 WO2022037582 A1 WO 2022037582A1 CN 2021113033 W CN2021113033 W CN 2021113033W WO 2022037582 A1 WO2022037582 A1 WO 2022037582A1
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seq
antibody
amino acid
antigen
heavy chain
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PCT/CN2021/113033
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French (fr)
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周岳华
李瑞胜
张静
刘洪川
姚盛
冯辉
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上海君实生物医药科技股份有限公司
苏州君盟生物医药科技有限公司
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Publication of WO2022037582A1 publication Critical patent/WO2022037582A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C12N15/867—Retroviral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer

Definitions

  • the present invention relates to anti-CD3 and anti-CLDN-18.2 bispecific antibodies and uses thereof.
  • CD3 T cell surface glycoprotein CD3, a signaling co-receptor of T cell receptors, which contains subunits ⁇ , ⁇ , ⁇ , and ⁇
  • CD3 is a differentiation antigen expressed on the surface of all T lymphocytes, mainly mediating Transduction of T cell activation signals. It plays an important role in the anti-infection immunity of the body's immune system.
  • CD3 molecule forms a stable TCR-CD3 complex with T cell antigen receptor (T cell receptor), and its extracellular domain recognizes and binds to major histocompatibility complex class II molecules, enhancing T cell antigen receptor (T cell receptor) , TCR) and the stability of MHC molecules; the intracellular region enhances the activation signal transduced by leukocytes CD3, thereby participating in and regulating the activation of the immune system.
  • T cell receptor T cell antigen receptor
  • TCR T cell antigen receptor
  • MHC molecules major histocompatibility complex class II molecules
  • the quantitative index of CD3-positive lymphocyte population is an important index to measure the cellular immunity of the body.
  • Claudin also known as CLDN
  • CLDN is a family of cell surface proteins that build paracellular barriers and control the flow of molecules between cells, and at least 26 species have been identified so far.
  • Members of the Claudin protein family are important structural components of tight junctions and play an important role in maintaining epithelial cell polarity, controlling paracellular spreading, and regulating cell growth and differentiation.
  • the Claudin molecule crosses the cell membrane four times and both the N- and C-termini land in the cytoplasm.
  • Claudin 18 (CLDN18) has two alternative splicing variants, CLDN-18.1 and CLDN-18.2.
  • Claudin 18.1 (CLDN-18.1) is selectively expressed in normal lung and gastric epithelium.
  • Claudin 18.2 (CLDN-18.2) is slightly expressed in normal gastric epithelial short-lived cells, but in tumor cells, Claudin 18.2 is strongly expressed in various cancer types.
  • Bispecific antibodies also known as bifunctional antibodies, can specifically bind to two different antigens or two different antigenic epitopes at the same time. Due to its specificity and dual function, it has good application effects and prospects in the fields of tumor immunotherapy and autoimmune diseases. There are various forms of bispecific antibodies, and for different tumor-related antigens, different forms of bispecific antibodies have different effects. Because of its special function, it has broad application prospects in tumor immunotherapy.
  • the present invention aims to provide an anti-CD3 antibody and an anti-CD3 ⁇ CLDN-18.2 bispecific antibody with better effects in terms of affinity, stability and the like.
  • the present invention provides antibodies or antigen-binding fragments thereof that bind to CD3, and bispecific antibodies targeting CD3 and CLDN-18.2.
  • the anti-CLDN-18.2 antibodies or antigen-binding fragments thereof provided by the present invention can be used as stand-alone therapy or in combination with other therapies/or other anti-cancer agents, such as in the treatment of cancer.
  • the invention provides an antibody or antigen-binding fragment thereof comprising an antigen-binding site that binds CD3.
  • the CD3-binding antigen binding site of the present invention comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, respectively, and amino acids
  • the sequences are LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, respectively.
  • the CD3-binding antigen binding site of the present invention comprises a heavy chain variable region (VH) whose amino acid sequence is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2 A light chain variable region (VL); or a heavy chain variable region (VH) comprising an amino acid sequence shown in SEQ ID NO:5, and a light chain variable region (VL) having an amino acid sequence shown in SEQ ID NO:6 ).
  • VH heavy chain variable region
  • VL light chain variable region having an amino acid sequence shown in SEQ ID NO:6
  • the antigen-binding fragment of the present invention comprises a heavy chain fragment whose amino acid sequence is shown in SEQ ID NO: 15, and a light chain fragment whose amino acid sequence is shown in SEQ ID NO: 4.
  • the antigen-binding fragments of the present invention are Fab fragments.
  • the antibody of the present invention comprises a heavy chain whose amino acid sequence is set forth in SEQ ID NO:3, and a light chain whose amino acid sequence is set forth in SEQ ID NO:4.
  • the antibodies of the invention are monoclonal, bispecific, or multispecific antibodies.
  • the antibody or antigen-binding fragment thereof of the invention further comprises an antigen-binding site that binds CLDN-18.2.
  • the antigen binding site of the present invention that binds CLDN-18.2 comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24, respectively, and LCDR1, LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NO: 25, SEQ ID NO: 26 and SEQ ID NO: 27, respectively.
  • the antigen binding site of the present invention that binds CLDN-18.2 comprises a heavy chain variable region whose amino acid sequence is shown in SEQ ID NO:9, and a light chain whose amino acid sequence is shown in SEQ ID NO:10. chain variable region.
  • the heavy chain variable region (VH) and light chain variable region (VL) of the CD3-binding antigen binding site of the present invention are linked by a linker peptide to form an scFv, the amino acid sequence of the linker peptide Each independently is (GGGGS)n, wherein n is 2, 3, 4, 5 or 6, preferably, the scFv further binds to the Fc region.
  • the Fc binding to the antigen binding site of CD3 and the antigen binding site of CLDN-18.2 of the present invention is an Fc from IgG, such as an Fc from IgG1, IgG2, IgG3, or IgG4, and/or
  • the CL of the light chain is the CL from a lambda or kappa light chain.
  • the CD3-binding antigen binding site of the present invention comprises the amino acid sequence shown in SEQ ID NO:7.
  • the antigen binding site of the present invention that binds CLDN-18.2 comprises a heavy chain whose amino acid sequence is shown in SEQ ID NO: 11, and a light chain whose amino acid sequence is shown in SEQ ID NO: 12.
  • the antigen-binding fragment of the present invention is a Fab, Fab', F(ab')2, Fv, scFv or sdAb.
  • the heavy chain of the CD3-binding antigen binding site of the invention has a hinge region-Fc-linking peptide-VL-linking peptide-VH from the N-terminus to the C-terminus.
  • the heavy chain of the antigen binding site of CLDN-18.2 of the present invention has VH-CH1-hinge region-Fc from N-terminus to C-terminus, and the light chain has VL-CL from N-terminus to C-terminus .
  • the Fc that binds the antigen binding site of CD3 and the Fc that binds the antigen binding site of CLDN-18.2 of the present invention are connected by a disulfide bond in the hinge region and a knob-hole structure in the CH3 domain.
  • the present invention provides a bispecific antibody having two heavy chains and one light chain, wherein the first heavy chain has VH-CH1-hinge region-Fc from the N-terminus to the C-terminus;
  • the double chain has hinge region-Fc-linking peptide-VL-linking peptide-VH from N-terminal to C-terminal;
  • the light chain has VL-CL from N-terminal to C-terminal; wherein, the VH of the first heavy chain -CH1 forms an antigen binding site with the VL-CL of the light chain to bind CLDN-18.2, and the antigen binding site of the VH-linking peptide-VL of the second heavy chain binds CD3.
  • the VH of the second heavy chain of the present invention comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are set forth in SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, respectively, and the VL comprises The amino acid sequences are LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, respectively.
  • the amino acid sequences of the linking peptides are each independently (GGGGS)n, wherein n is 2, 3, 4, 5 or 6.
  • the VH of the second heavy chain of the present invention has the amino acid sequence shown in SEQ ID NO: 1
  • the VL has the amino acid sequence shown in SEQ ID NO: 2
  • the amino acid of the linker peptide The sequences are independently (GGGGS)n, where n is 2, 3, 4, 5, or 6.
  • the VH of the second heavy chain of the present invention has the amino acid sequence shown in SEQ ID NO: 5, and the VL has the amino acid sequence shown in SEQ ID NO: 6, and the amino acid of the linker peptide
  • the sequences are independently (GGGGS)n, where n is 2, 3, 4, 5, or 6.
  • the second heavy chain of the present invention has the amino acid sequence shown in SEQ ID NO:7.
  • the VH of the first heavy chain of the present invention comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24, respectively.
  • the VH of the first heavy chain of the present invention has the amino acid sequence shown in SEQ ID NO:9.
  • the first heavy chain of the present invention has the amino acid sequence shown in SEQ ID NO: 11.
  • the VL of the light chain of the invention comprises LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO:25, SEQ ID NO:26 and SEQ ID NO:27, respectively.
  • the VL of the light chain of the present invention has a sequence of amino acids as shown in SEQ ID NO:10.
  • the light chain of the present invention has a sequence of amino acids as set forth in SEQ ID NO:12.
  • the VH of the first heavy chain comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24, respectively;
  • the VH of the second heavy chain comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18 respectively, and the VL comprises the amino acid sequences shown in SEQ ID NO: 19, SEQ ID NO: 18, respectively.
  • the VL of the light chain comprises LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO:25, SEQ ID NO:26 and SEQ ID NO:27, respectively.
  • the VH of the first heavy chain has the amino acid sequence shown in SEQ ID NO:9;
  • the VH of the second heavy chain has the amino acid sequence shown in SEQ ID NO:1, the VL has the amino acid sequence shown in SEQ ID NO:2; or the VH of the second heavy chain has the amino acid sequence shown in SEQ ID NO:5
  • the amino acid sequence, VL has the amino acid sequence shown in SEQ ID NO:6; and the amino acid sequence of the connecting peptide in the second heavy chain is each independently (GGGGS)n, wherein n is 2, 3, 4, 5 or 6 ;
  • the VL of the light chain has the sequence of amino acids shown in SEQ ID NO:10.
  • the first heavy chain has the amino acid sequence shown in SEQ ID NO: 11
  • the second heavy chain has the amino acid sequence shown in SEQ ID NO: 7
  • the light chain has the sequence of amino acids as set forth in SEQ ID NO:12.
  • the first heavy chain and the second heavy chain of the present invention are connected by a disulfide bond in the hinge region and a knob-hole structure in the CH3 domain.
  • the Fc of the first heavy chain and the second heavy chain of the invention is an Fc from IgG, such as an Fc from IgGl, IgG2, IgG3 or IgG4, and/or the CL of the light chain is from IgG CL of a lambda or kappa light chain.
  • the present invention provides a polynucleotide molecule whose nucleotide sequence is selected from:
  • nucleotide sequence encoding the first heavy chain, second heavy chain and/or light chain of an antibody or antigen-binding fragment thereof that binds to CD3 as described herein, or a bispecific antibody as described herein;
  • the present invention provides an expression vector comprising a polynucleotide molecule as described herein, preferably, the expression vector is a eukaryotic expression vector.
  • the present invention provides a host cell comprising a polynucleotide molecule or expression vector as described herein, or expressing an antibody as described in any of the embodiments herein; preferably, the host cell is a true host cell Nuclear cells, more preferably mammalian cells.
  • the present invention provides a method of making a bispecific antibody as described herein, the method comprising in a host cell as described herein under conditions suitable for expression of the bispecific antibody The bispecific antibody is expressed, and the expressed bispecific antibody is recovered from the host cell.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof as described herein, a bispecific antibody as described herein, a polynucleotide molecule as described herein, An expression vector as described herein, a host cell as described herein, and a pharmaceutically acceptable carrier or excipient.
  • the present invention provides an antibody or antigen-binding fragment thereof as described herein, a bispecific antibody as described herein, a polynucleotide molecule as described herein, an expression vector as described herein, Use of a host cell as described herein and a pharmaceutical composition as described herein in the manufacture of a medicament for the treatment of cancer.
  • the present invention provides an antibody or antigen-binding fragment thereof as described herein, a bispecific antibody as described herein, a polynucleotide molecule as described herein, an expression vector as described herein, Host cells as described herein and pharmaceutical compositions as described herein for use in the treatment of cancer.
  • the present invention provides a method of treating cancer comprising administering to a subject in need thereof an antibody or antigen-binding fragment thereof, a bispecific antibody as described herein, an antibody as described herein, or an antigen-binding fragment thereof as described herein
  • a method of treating cancer comprising administering to a subject in need thereof an antibody or antigen-binding fragment thereof, a bispecific antibody as described herein, an antibody as described herein, or an antigen-binding fragment thereof as described herein
  • the cancer of the present invention is selected from the group consisting of gastric cancer, esophageal cancer, gastroesophageal cancer, pancreatic cancer, bile duct cancer, lung cancer, ovarian cancer, colon cancer, liver cancer, head and neck cancer, gallbladder cancer, bowel cancer and bladder cancer. .
  • the present invention provides a pharmaceutical combination comprising an antibody or antigen-binding fragment thereof as described herein, a bispecific antibody as described herein, a polynucleotide molecule as described herein, a The expression vector, the host cell described herein, or the pharmaceutical composition described herein, and one or more additional therapeutic agents.
  • the present invention provides a kit comprising an antibody or antigen-binding fragment thereof as described herein, a bispecific antibody as described herein, a polynucleotide molecule as described herein, a The expression vector, the host cell described herein, or the pharmaceutical composition described herein.
  • the present invention provides a method of detecting the presence of CD3 and/or CLDN-18.2 in a sample using a bispecific antibody as described herein, the method comprising combining the bispecific antibody as described herein with the sample.
  • the conjugates formed by the bispecific antibody with CD3 and/or CLDN-18.2 are contacted and detected or quantified.
  • Figure 1 Schematic representation of the molecular structure of the anti-CD3 ⁇ CLDN-18.2 bispecific antibody.
  • Figure 2 Detection of the binding of anti-CD3 monoclonal antibody and anti-CD3 ⁇ CLDN-18.2 bispecific antibody to human CD3-expressing cells by FACS.
  • Figure 3 Detection of the binding of anti-CD3 monoclonal antibody and anti-CD3 ⁇ CLDN-18.2 bispecific antibody to cynomolgus monkey CD3-expressing cells by FACS.
  • Figure 4 The effect of anti-CD3 monoclonal antibody and anti-CD3 ⁇ CLDN-18.2 bispecific antibody on T cell activity detected by luciferase assay.
  • Figure 5 Inhibition of tumor growth in mice by anti-CD3xCLDN-18.2 bispecific antibodies.
  • Figure 6 Binding detection results of JSCD3 and SP34 to CD3E.
  • Figure 7 Binding assay results of JSCD3 and SP34 to CD3D&E.
  • CD3 refers to being part of the T cell receptor complex, consisting of three distinct chains CD3 ⁇ , CD3 ⁇ and CD3 ⁇ .
  • concentration of CD3 on T cells by, for example, their immobilization by anti-CD3 antibodies results in T cell activation, similar to T cell receptor-mediated activation, but independent of the specificity of the TCR clone.
  • the vast majority of anti-CD3 antibodies recognize the CD3 ⁇ chain.
  • the term refers to any native CD3 from any vertebrate (including mammals such as primates (eg, humans)) and rodents (eg, mice and rats), unless otherwise stated.
  • the term encompasses "full-length" unprocessed CD3 as well as any form of CD3 or any fragment thereof produced by intracellular processing.
  • CD3 refers to full-length CD3 from human and cynomolgus monkeys or a fragment thereof (such as a mature fragment thereof lacking a signal peptide). In a preferred embodiment, CD3 refers to full length from mouse/rat or a fragment thereof (such as a mature fragment thereof lacking a signal peptide).
  • CLDN-18.2 or "Claudin18.2” is one of two splice variants of Claudin 18.
  • the term refers to any native CLDN-18.2 from any vertebrate (including mammals such as primates (eg, humans)) and rodents (eg, mice and rats), unless otherwise stated.
  • the term encompasses "full-length” unprocessed CLDN-18.2 as well as any form of CLDN-18.2 or any fragment thereof produced by intracellular processing.
  • the term also includes naturally-occurring variants of CLDN-18.2, eg, splice variants or allelic variants.
  • CLDN-18.2 refers to the full length of CLDN-18.2 from human and cynomolgus monkeys or a fragment thereof (such as a mature fragment thereof lacking a signal peptide).
  • percent (%) amino acid sequence identity or simply “identity” is defined as the maximum percent sequence identity obtained when amino acid sequences are aligned (and where necessary gaps are introduced), and no conservative substitutions are considered to be Following the portion of sequence identity, the percentage of amino acid residues in the candidate amino acid sequence that are identical to those in the reference amino acid sequence.
  • Sequence alignments to determine percent amino acid sequence identity can be performed using various methods in the art, eg, 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 measuring alignment, including any algorithms needed to obtain maximal alignment over the full length of the sequences being compared.
  • immune response refers to the action by, for example, lymphocytes, antigen-presenting cells, phagocytes, granulocytes, and the production of soluble macromolecules (including antibodies, cytokines and complement) by these cells or by the liver, which results in selective Damage, destroy or eliminate invading pathogens, pathogen-infected cells or tissues, cancer cells, or normal human cells or tissues in the case of autoimmunity or pathological inflammation.
  • signal transduction pathway or “signal transduction activity” refers to a biochemical causal relationship, typically initiated by protein-protein interactions such as the binding of growth factors to receptors, that results in the transmission of signals from one part of a cell to another of the cell. part.
  • delivery involves specific phosphorylation of one or more tyrosine, serine, or threonine residues on one or more proteins in a series of reactions leading to signal transduction.
  • the penultimate process usually involves nuclear events that lead to changes in gene expression.
  • activity or “biological activity”, or the terms “biological property” or “biological signature” are used interchangeably herein and include, but are not limited to, epitope/antigen affinity and specificity, neutralization or antagonism of CLDN in vivo or in vitro -18.2 Capacity for activity, IC50, in vivo stability of the antibody and immunogenic properties of the antibody.
  • Other identifiable biological properties or characteristics of antibodies known in the art include, for example, cross-reactivity (ie, generally with non-human homologues of the targeting peptide, or with other proteins or tissues), and retention of The ability of proteins to be expressed at high levels in mammalian cells.
  • antibody refers to any form of antibody that possesses the desired biological activity. Accordingly, it is used in the broadest sense and specifically includes, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies), humanized antibodies, fully human antibodies, Chimeric and camelized single domain antibodies.
  • the basic antibody structural unit is known to comprise a tetramer. Each tetramer includes two identical pairs of polypeptide chains, each pair having a "light" chain (about 25 kDa) and a "heavy” chain (about 50-70 kDa).
  • the amino-terminal portion or fragment of each chain may include a variable region of about 100-110 or more amino acids primarily responsible for antigen recognition.
  • the carboxy-terminal portion or fragment of each chain may define a constant region primarily responsible for effector function.
  • Human light chains are generally classified as kappa and lambda light chains.
  • human heavy chains are generally classified as mu, delta, gamma, alpha, or epsilon, and the isotypes of antibodies are defined as IgM, IgD, IgG, IgA, and IgE, respectively.
  • the variable and constant regions are linked by a "J" region of about 12 or more amino acids, wherein the heavy chain also includes a "D” region of about 10 or more amino acids. See generally Chapter 7 of Fundamental Immunology (Paul, W. ed., 2nd ed. Raven Press, N.Y. (1989)).
  • isolated antibody refers to the purified state of the binding compound, and in this case means that the molecule is substantially free of other biomolecules such as nucleic acids, proteins, lipids, sugars or other substances such as cell debris and growth media .
  • isolated does not mean the complete absence of such materials or the absence of water, buffers or salts unless they are present in amounts that significantly interfere with the experimental or therapeutic use of the binding compound described herein.
  • the term "monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous antibodies, ie, the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, directed against a single epitope. In contrast, conventional (polyclonal) antibody preparations typically include large numbers of antibodies directed against (or specific for) different epitopes.
  • the modifier "monoclonal” indicates the characteristics of an antibody obtained from a substantially homogeneous population of antibodies and should not be construed as requiring the production of the antibody by any particular method.
  • bispecific antibody refers to an antibody that is artificially designed, which is composed of components from two different antigen-binding sites, and can bind to two different antigen-binding sites simultaneously.
  • full-length antibody refers to an immunoglobulin molecule comprising four peptide chains in nature: two heavy (H) chains (about 50-70 kDa in full length) and two light (L) chains (full length) 25kDa) are connected to each other by disulfide bonds.
  • Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH).
  • the heavy chain constant region consists of three domains, CH1, CH2 and CH3.
  • Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region.
  • the light chain constant region consists of one domain, CL.
  • VH and VL regions can be further subdivided into highly variable complementarity determining regions (CDRs) and spaced by more conserved regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • Each VH or VL region consists of 3 CDRs and 4 FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from amino terminus to carboxy terminus.
  • the variable regions of the heavy and light chains contain binding domains that interact with the antigen.
  • the constant regions of the antibodies mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.
  • antibody-binding fragment of an antibody (“parent antibody”) includes fragments or derivatives of an antibody, typically including at least one fragment of the antigen-binding or variable region (eg, one or more CDRs) of the parent antibody that retains the parental At least some binding specificity of an antibody.
  • 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.
  • a binding fragment or derivative typically retains at least 10% of its antigen-binding activity when the antigen-binding activity is expressed on a molar basis.
  • the 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 may include conservative or non-conservative amino acid substitutions that do not significantly alter their biological activity (referred to as “conservative variants” or “functionally conservative variants” of an antibody).
  • binding compound refers to both antibodies and binding fragments thereof.
  • 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.
  • Fv polypeptides typically also comprise a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding.
  • Fc or “Fc region” or “Fc fragment” refers to a polypeptide consisting of the CH2 and CH3 domains of IgA, IgD and IgG, or the CH2, CH3 and CH4 domains of IgE and IgM through the hinge region. Although the breakdown of Fc fragments is variable, the heavy chain Fc fragment of human IgG generally refers to the stretch of polypeptide from A231 to its carboxy terminus.
  • the term "hinge region” refers to a proline-rich polypeptide chain located between CH1 and CH2 in an antibody, which is easy to stretch and bend.
  • the recognized IgG hinge region is a polypeptide chain composed of amino acid residues from positions 216 to 230.
  • domain antibody is an immunologically functional immunoglobulin fragment containing only the heavy chain variable region or the light chain variable region.
  • two or more VH regions are covalently linked to 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.
  • bivalent antibody contains two antigen-binding sites. In some cases, the two binding sites have the same antigen specificity. However, bivalent antibodies can be bispecific.
  • diabody refers to a small antibody fragment with two antigen-binding sites comprising a heavy chain linked to a light chain variable domain (VL) in the same polypeptide chain (VH-VL or VL-VH) Variable domain (VH).
  • VL light chain variable domain
  • VH-VL or VL-VH Variable domain
  • chimeric antibody is an antibody having the variable domains of a first antibody and the constant domains of a second antibody, wherein the first antibody and the second antibody are from different species.
  • the variable domains are obtained from rodent or the like antibodies (“parental antibodies”), while the constant domain sequences are obtained from human antibodies, such that the resulting chimeric antibody induces induction in human subjects as compared to the parental rodent antibody The likelihood of an adverse immune response is low.
  • humanized antibody refers to a form of antibody that contains sequences from human and non-human (eg, mouse, rat) antibodies.
  • humanized antibodies comprise substantially all of at least one, usually two variable domains, wherein all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, and all or substantially all of the hypervariable loops Framework (FR) regions are the framework regions of human immunoglobulin sequences.
  • FR hypervariable loops Framework
  • a humanized antibody optionally may comprise at least a portion of a human immunoglobulin constant region (Fc).
  • Fully human antibody refers to an antibody comprising only human immunoglobulin protein sequences. Fully human antibodies may contain murine sugar chains if produced in mice, in mouse cells, or in hybridomas derived from mouse cells. Likewise, “mouse antibody” refers to an antibody comprising only mouse immunoglobulin sequences. Alternatively, fully human antibodies may contain rat sugar chains if produced in rats, in rat cells, or in hybridomas derived from rat cells. Likewise, “rat antibody” refers to an antibody comprising only rat immunoglobulin sequences.
  • an “isotype” antibody refers to the class of antibody provided by the heavy chain constant region genes (eg, IgM, IgE, IgG such as IgGl, IgG2, or IgG4). Isotypes also include modified forms of one of these species, wherein modifications have been made to alter Fc function, eg, to enhance or reduce effector function or binding to Fc receptors.
  • heavy chain constant region genes eg, IgM, IgE, IgG such as IgGl, IgG2, or IgG4
  • epitope refers to the region of an antigen to which an antibody binds. Epitopes can be formed from contiguous amino acids or non-contiguous amino acids juxtaposed by tertiary folding of the protein.
  • 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 partner Y can generally be represented by the equilibrium dissociation constant (KD), which is the ratio of the dissociation rate constant to the association rate constant (kdis and kon, respectively).
  • KD equilibrium dissociation constant
  • kdis and kon association rate constant
  • Affinity can be measured by common methods known in the art.
  • One specific method used to measure affinity is the ForteBio kinetic binding assay herein.
  • does not bind to a protein or cell means, does not bind to the protein or cell, or does not bind to it with high affinity, i.e. binds the protein or cell with a KD of 1.0 x 10 -6 M or higher, more preferably 1.0 x 10 - 5 M or higher, more preferably 1.0 ⁇ 10 -4 M or higher, 1.0 ⁇ 10 -3 M or higher, more preferably 1.0 ⁇ 10 -2 M or higher.
  • high affinity for IgG antibodies means a KD for antigen of 1.0x10-6 M or less, preferably 5.0x10-8 M or less, more preferably 1.0x10-8 M or lower, 5.0 ⁇ 10 ⁇ 9 M or lower, more preferably 1.0 ⁇ 10 ⁇ 9 M or lower.
  • high affinity binding may vary.
  • “high affinity” binding of an IgM subtype refers to a KD of 10-6 M or lower, preferably 10-7 M or lower, more preferably 10-8 M or lower.
  • antibody-dependent cytotoxicity refers to cell-mediated immune defense in which immune system effector cells actively associate cell membrane surface antigens with antibodies, such as Claudin18. 2 Antibodies that bind to target cells such as cancer cells are lysed.
  • CDC complement-dependent cytotoxicity
  • IgG and IgM antibodies that, when bound to surface antigens, initiate the canonical complement pathway, including formation of membrane attack complexes and lysis of target cells.
  • the antibodies of the present invention when bound to Claudin 18.2, trigger CDC against cancer cells.
  • 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 binding properties similar to the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides (see, in Kariko et al. Human US Patent No. 8,278,036, which discloses mRNA molecules in which uridine is replaced by pseudouridine, methods of synthesizing such mRNA molecules, and methods for delivering therapeutic proteins in vivo).
  • nucleic acid sequence also implicitly includes conservatively modified variants thereof (eg, degenerate codon substitutions), alleles, orthologs, SNPs, and complements thereof, as well as sequences explicitly indicated.
  • degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is replaced by mixed bases and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).
  • Construct refers to any recombinant polynucleotide molecule (such as a plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, bacteriophage, or linear or circular single- or double-stranded DNA or RNA polynucleotide molecule), derived from Any source, capable of integrating with the genome or replicating autonomously, constitutes a polynucleotide molecule in which one or more polynucleotide molecules have been functionally linked (ie, operably linked).
  • the recombinant construct will typically comprise a polynucleotide of the invention operably linked to transcription initiation regulatory sequences that direct transcription of the polynucleotide in a host cell. Expression of the nucleic acids of the invention can be directed using both heterologous and non-heterologous (ie, endogenous) promoters.
  • Vector refers to any recombinant polynucleotide construct that can be used for the purpose of transformation (ie, the introduction of heterologous DNA into a host cell).
  • plasmid refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated.
  • viral vector in which additional DNA segments can be ligated into the viral genome.
  • Certain vectors are capable of autonomous replication in the host cell into which they are introduced (eg, bacterial vectors with bacterial origins of replication and episomal mammalian vectors).
  • vectors After introduction into the host cell, other vectors (eg, non-episomal mammalian vectors) integrate into the genome of the host cell and thus replicate together with the host genome. In addition, certain vectors are capable of directing the expression of operably linked genes. Such vectors are referred to herein as "expression vectors".
  • expression vector refers to a nucleic acid molecule capable of replicating and expressing a gene of interest when transformed, transfected or transduced into a host cell.
  • Expression vectors contain one or more phenotypic selectable markers and origins of replication to ensure maintenance of the vector and to provide for amplification within the host if desired.
  • Activation can have the same meaning, eg, activation, stimulation, or treatment of a cell or receptor with a ligand, unless the context otherwise or clearly dictates.
  • Ligand includes natural and synthetic ligands, such as cytokines, cytokine variants, analogs, muteins, and binding compounds derived from antibodies.
  • Ligand also includes small molecules such as peptidomimetics of cytokines and peptidomimetics of antibodies.
  • Activation can refer to cellular activation regulated by internal mechanisms as well as external or environmental factors.
  • a “response/response”, eg, the response of a cell, tissue, organ, or organism, includes changes in biochemical or physiological behavior (eg, concentration, density, adhesion or migration, gene expression rate, or differentiation state within a biological compartment), wherein changes Related to activation, stimulation, or processing, or to internal mechanisms such as genetic programming.
  • the terms “treating” or “treating” of any disease or disorder refers, in one embodiment, to ameliorating the disease or disorder (ie, slowing or arresting or reducing the progression of the disease or at least one of its clinical symptoms). In another embodiment, “treating” or “treating” refers to alleviating or ameliorating at least one physical parameter, including those physical parameters that may not be discernible by a patient. In another embodiment, “treating” or “treating” refers to modulating a disease or disorder physically (eg, stabilization of discernible symptoms), physiologically (eg, stabilization of physical parameters), or both. Unless explicitly described herein, methods for assessing treatment and/or prevention of disease are generally known in the art.
  • Subject includes any human or non-human animal.
  • non-human animal includes all vertebrates, eg, mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cattle, chickens, amphibians, reptiles, and the like.
  • cyno or “cynomolgus monkey” refers to a cynomolgus monkey.
  • Administration "in combination with” one or more other therapeutic agents includes simultaneous (co) administration and sequential administration in any order.
  • “Therapeutically effective amount”, “therapeutically effective dose” and “effective amount” mean that the CLDN-18.2 antibody or antigen-binding fragment thereof of the present invention, when administered alone or in combination with other therapeutic agents, is effective in preventing Or an amount that ameliorates the symptoms of one or more diseases or conditions or the progression of that disease or condition.
  • a therapeutically effective dose also refers to an amount of the antibody or antigen-binding fragment thereof sufficient to cause amelioration of symptoms, eg, an amount that treats, cures, prevents or ameliorates a related medical condition or increases the rate of treatment, cure, prevention or amelioration of such a condition.
  • the therapeutically effective dose refers to that ingredient only.
  • a therapeutically effective dose refers to the combined amount of active ingredients that elicits a therapeutic effect, whether administered in combination, sequentially or simultaneously.
  • An effective amount of a therapeutic agent will result in an improvement in a diagnostic criterion or parameter of at least 10%; usually at least 20%; preferably at least about 30%; more preferably at least 40%, and most preferably at least 50%.
  • Cancer and “cancerous” refer to or describe a physiological disorder in mammals that is often characterized by unregulated cell growth. Benign and malignant cancers as well as dormant tumors or micrometastases are included in this definition. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia.
  • cancers include squamous cell carcinoma, lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung), peritoneal cancer, hepatocellular carcinoma, cancer of the stomach or gastric cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial cancer or uterine cancer, Cancer of salivary gland, kidney or kidney, liver, prostate, vulva, thyroid, liver, and various types of head and neck cancer, and B-cell lymphomas (including low-grade/follicular non-HoJ King's lymphoma (NHL), small lymphocytic (SL) NHL, intermediate/follicular NHL, intermediate diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small anucleate NHL, bulky disease (NHL), small lymph
  • anti-CD3 antibody refers to the ability to bind CD3 protein or fragment thereof with sufficient affinity such that the antibody can be used as a diagnostic agent targeting CD3 and/or or therapeutic agents.
  • anti-CLDN-18.2 antibody refers to the ability to bind CLDN-18.2 protein or fragment thereof with sufficient affinity such that the antibody can Use as a diagnostic and/or therapeutic agent targeting CLDN-18.2.
  • the present inventors conducted in-depth analysis and comparison of several anti-human CD3 antibodies, and sequenced one of the murine antibody SP34 (see Pessano, S. et al., EMBO J 4 (1985) 337-344) After optimization and humanization, the anti-CD3 antibody JSCD3 was obtained.
  • the humanized anti-CD3 antibodies of the present invention have many unique properties and advantages. These new properties make this new humanized antibody more suitable for the development of antibody drugs.
  • the optimized antibody sequence of the present invention has high expression of antibody protein, good thermal stability, higher affinity with human and cynomolgus monkey CD3 antigen, and can effectively activate the downstream signaling pathway of T cells; When the bispecific antibody was prepared, it showed stronger tumor-killing activity.
  • the humanized sequence disclosed in the present invention has more advantages and characteristics than the humanized sequences of other companies, and shows better tumoricidal activity when used for the preparation of new anti-tumor drugs, and the physical and chemical stability is also greatly improved, It is more suitable for the screening and development of antitumor drugs, and this creative achievement constitutes the beneficial effect and extremely high medical application value of the present invention.
  • the present invention relates to an antibody or antigen-binding fragment comprising an antigen-binding site that binds CD3, the CD3-binding antigen-binding site comprising a heavy chain variable having an amino acid sequence as shown in SEQ ID NO: 1 region (VH), and light chain variable region (VL) whose amino acid sequence is shown in SEQ ID NO:2.
  • the antibody or antigen-binding fragment of the invention is a monoclonal antibody or antigen-binding fragment thereof.
  • the antibodies or antigen-binding fragments of the invention may be chimeric, CDR-grafted, humanized, or fully human.
  • the antigen-binding fragment of the present invention is a Fab, Fab', F(ab')2, Fv, scFv, or sdAb.
  • the antibodies of the invention comprise the heavy chain amino acid sequence set forth in SEQ ID NO:3, and the light chain amino acid sequence set forth in SEQ ID NO:4.
  • the antibodies of the invention are bispecific or multispecific antibodies.
  • the antibody further comprises another antigen binding site that binds CLDN-18.2.
  • the invention provides a bispecific antibody comprising a first antigen binding site that specifically binds CD3 and a second antigen binding site that specifically binds Claudin 18.2 (CLDN-18.2).
  • Such antibodies may be referred to herein, for example, as “anti-CD3/anti-CLDN-18.2” or “anti-CD3 x CLDN-18.2” or “CD3 x CLDN-18.2” bispecific molecules, or other similar terms.
  • the first antigen-binding site that binds CD3 comprises HCDR1, HCDR2, and HCDR3 whose amino acid sequences are set forth in SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively, and amino acids
  • the sequences are LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, respectively.
  • the first antigen binding site of the present invention that binds to CD3 comprises a heavy chain variable region (VH) whose amino acid sequence is as shown in SEQ ID NO: 1, and the amino acid sequence is as shown in SEQ ID NO: 2 light chain variable region (VL) shown; or a heavy chain variable region (VH) comprising an amino acid sequence as shown in SEQ ID NO:5, and a light chain variable region with an amino acid sequence as shown in SEQ ID NO:6 (VL).
  • VH heavy chain variable region
  • the second antigen binding site of the present invention that binds to CLDN-18.2 comprises HCDR1, HCDR2 and HCDR3, and LCDR1, LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NO:25, SEQ ID NO:26 and SEQ ID NO:27, respectively.
  • the second antigen binding site of the present invention that binds to CLDN-18.2 comprises a heavy chain variable region whose amino acid sequence is shown in SEQ ID NO: 9, and the amino acid sequence is shown in SEQ ID NO: 10 light chain variable region.
  • the present invention provides a bispecific antibody having two heavy chains and one light chain, wherein the first heavy chain has VH-CH1-hinge region-Fc from the N-terminus to the C-terminus;
  • the double chain has hinge region-CH2-CH3-linking peptide-VL-linking peptide-VH from N-terminus to C-terminus;
  • the light chain has VL-CL from N-terminus to C-terminus, wherein the first heavy chain has The antigen binding site formed by VH-CH1 and the VL-CL of the light chain binds to CLDN-18.2, and the antigen binding site of the VH-linking peptide-VL of the second heavy chain binds CD3.
  • the amino acid sequences of the linking peptides are each independently (GGGGS)n, wherein n is 2, 3, 4, 5 or 6.
  • the VH of the second heavy chain of the present invention has the amino acid sequence shown in SEQ ID NO: 1
  • the VL has the amino acid sequence shown in SEQ ID NO: 2
  • the amino acid of the linker peptide The sequences are each independently (GGGGS)n, where n is 2, 3, 4, 5, or 6.
  • the second heavy chain of the present invention has the amino acid sequence shown in SEQ ID NO:7.
  • the VH of the first heavy chain of the present invention has the amino acid sequence shown in SEQ ID NO:9
  • the VL of the light chain has the amino acid sequence shown in SEQ ID NO:10.
  • the first heavy chain of the present invention has the amino acid sequence shown in SEQ ID NO: 11, and the light chain has the amino acid sequence shown in SEQ ID NO: 12.
  • the first heavy chain and the second heavy chain of the present invention are connected by a disulfide bond in the hinge region and a knob-hole structure in the CH3 domain.
  • the Fc of the first heavy chain and the second heavy chain of the invention is an Fc from IgG, such as an Fc from IgGl, IgG2, IgG3 or IgG4, and/or the CL of the light chain is from IgG CL of a lambda or kappa light chain.
  • variable region CDRs of the antibodies of the invention can be determined using any of a number of well-known protocols, including Chothia based on the three-dimensional structure of the antibody and topology of the CDR loops (Chothia et al.
  • the CDR boundaries of the variable region of the same antibody obtained based on different assignment systems may vary. That is, the CDR sequences of the variable regions of the same antibody defined under different assignment systems are different.
  • the scope of said antibodies also covers antibodies whose variable region sequences comprise said specific CDR sequences, but due to the application of different schemes (e.g. different assignment systems or combinations), resulting in their claimed CDR boundaries being different from the specific CDR boundaries defined by the present invention.
  • Antibodies with different specificities have different CDRs.
  • CDRs vary from antibody to antibody, only a limited number of amino acid positions within CDRs are directly involved in antigen binding.
  • the minimal binding unit can be a sub-portion of a CDR.
  • the residues of the remainder of the CDR sequence can be determined by the structure and protein folding of the antibody, as will be apparent to those skilled in the art. Accordingly, the present invention also contemplates variants of any of the CDRs presented herein. For example, in a variant of a CDR, the amino acid residues of the smallest binding unit may remain unchanged, while the remaining CDR residues as defined by Kabat or Chothia may be replaced by conservative amino acid residues.
  • murine CDR regions can be inserted into human germline framework regions using methods known in the art. See US Patent Nos. 5,225,539 to Winter et al. and US Patent Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen et al.
  • amino acid changes include amino acid deletions, insertions or substitutions.
  • the anti-CLDN-18.2 antibodies or antigen-binding fragments thereof of the invention include those that have been mutated by amino acid deletions, insertions, or substitutions, but which are still identical to the antibodies described above (particularly in the CDR regions depicted in the sequences above) Those antibodies that have amino acid sequences that are at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical.
  • the antibodies of the invention have no more than 1, 2, 3, 4, or 5 amino acid mutations in the CDR regions that have been mutated by amino acid deletions, insertions, or substitutions when compared to the CDR regions depicted in a particular sequence.
  • the polynucleotides encoding the antibodies of the invention include those that have been mutated by nucleotide deletions, insertions, or substitutions, but still have at least about 60, 70, or 60, 70 or more of the coding region corresponding to the CDRs depicted in the sequences described above. , 80, 90, 95 or 100% identical polynucleotides.
  • one or more amino acid modifications can be introduced into the Fc region of the antibodies provided herein, thereby generating Fc region variants.
  • An Fc region variant may comprise a human Fc region sequence (eg, a human IgGl, IgG2, IgG3, or IgG4 Fc region) comprising amino acid modifications (eg, substitutions) at one or more amino acid positions.
  • cysteine-engineered antibodies such as "thioMAbs,” in which one or more residues of the antibody are replaced with cysteine residues.
  • the antibodies provided herein can be further modified to contain other non-proteinaceous moieties known in the art and readily available.
  • Moieties suitable for antibody derivatization include, but are not limited to, water-soluble polymers.
  • Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, polyvinyl - 1,3-dioxane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), and dextran or poly(n-ethylene pyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (eg, glycerol), polyvinyl alcohol,
  • first antigen binding domain and the second antigen binding domain of the bispecific antibody may be linked to each other directly or indirectly.
  • the first antigen-binding domain and the first antigen-binding domain of the bispecific antibody may be linked to each other directly or indirectly.
  • the two antigen-binding domains can be linked to each other by a linker.
  • the linker is a peptide linker.
  • the first antigen-binding domain and the second antigen-binding domain of a bispecific antibody can be linked directly or indirectly to each other to form a bispecific antigen binding molecule (ie, a bispecific scFv), which further binds to Fc region.
  • a bispecific antigen binding molecule ie, a bispecific scFv
  • the first antigen binding domain and the second antigen binding domain may each be linked to separate Fc regions.
  • the bispecific antigen binding molecules of the invention may comprise two Fc regions, each Fc region being part of a separate antibody heavy chain.
  • the first and second Fc regions may have the same sequence, except for mutations in the CH3 domain intended to facilitate or facilitate purification of the heterodimeric (ie bispecific) molecule.
  • the Fc region of the bispecific antibody of the present invention may be a human Fc region.
  • the Fc region of the bispecific antibodies of the invention can be of any isotype, including but not limited to IgGl, IgG2, IgG3 or IgG4.
  • the Fc regions of both said first and said second antibodies are of the IgGl isotype.
  • the Fc regions of both said first and said second antibodies are of the IgG4 isotype.
  • one of the Fc regions of the antibody is of the IgGl isotype and the other is of the IgG4 isotype.
  • the resulting bispecific antibody comprises the Fc region of IgGl and the Fc region of IgG4, and thus may have intermediate properties of interest with respect to activation of effector functions.
  • knock hole structure refers to the mutation of the hydrophobic amino acid of CH3 of the antibody Fc.
  • the side chain amino acids of one CH3 chain are mutated to form larger hydrophobic amino acids (knob, knob) to strengthen the hydrophobic force; the other CH3 side chain amino acids are mutated to form small amino acids (hole, hole) to reduce steric positions
  • CH3 with Knob and with Hole form a knob-hole structure (Knob into Hole, KiH) in the form of hydrophobic interaction, which is conducive to the formation of heavy chain heterodimers
  • CH3 of knob is due to the space of amino acids after mutation The steric hindrance is large, and the hydrophobic force becomes weaker after hole mutation, both of which are not conducive to the formation of homodimers
  • KiH mutation mainly occurs in the internal hydrophobic amino acids of the spatial structure of the CH3 domain, and after mutation, it is exposed to The outer amino acids are almost unchanged, so the effector function of Fc and the resulting immunogenic
  • the Fc region may contain one or more amino acid changes (eg, insertions, deletions, or substitutions) compared to a designated chimeric form of the Fc region without altering the desired function sex.
  • the present invention includes bispecific antigen binding molecules comprising one or more modifications in the Fc region that result in a modified Fc region with modified binding interactions (eg, enhanced or reduced) between Fc and FcRn.
  • Fc modifications include, eg, a serine ("S") to proline (“P”) mutation at position 228 of the amino acid sequence of the human IgG4 Fc region.
  • the present invention provides bispecific CD3xCLDN-18.2 antibodies that effectively promote T cell-mediated killing of tumor cells expressing CLDN-18.2.
  • a particular antigen-binding region may be selected from the group of antibodies or antigen-binding regions provided herein, or share, for example, epitopes or cross-blocking regions with antibodies or antigen-binding regions provided herein. those antibody- or antigen-binding regions of the block.
  • Many different formats and uses of bispecific antibodies are known in the art and were recently reviewed by Chames and Baty (2009) Curr Opin Drug Disc Dev 12:276.
  • the antibody of the invention comprising an antigen binding site that binds CD3 and an antigen binding site that binds Claudin 18.2 (CLDN-18.2) may be a bispecific antibody or a multispecific antibody, preferably a bispecific antibody Antibodies, wherein bispecific antibodies can take a wide variety of configurations, such as bispecific antibodies containing Fc fragments (IgG-like bispecific antibodies) and bispecific antibodies without Fc fragments (non-IgG-like antibodies) bispecific antibodies).
  • IgG-like bispecific antibodies mainly include Triomabs/quadroma, DVD-Ig (dual variable domain Ig), CrossMAb, Two-in-one IgG, scFv2-Fc.
  • Non-IgG-like bispecific antibodies mainly include TandAb (tandem diabody), scFv-HSA-scFv, BiTE (bi-specific T-cell engager), DART (dual affinity retargeting), Nanobody.
  • Exemplary bispecific antibody molecules of the invention have two heavy chains and one light chain, wherein the first heavy chain has VH-CH1-hinge region-Fc from N-terminus to C-terminus; the second heavy chain is from N-terminus to C-terminus C-terminal has hinge region-Fc-linking peptide-VL-linking peptide-VH; the light chain has VL-CL from N-terminal to C-terminal; wherein, VH-CH1 of the first heavy chain and the light chain
  • the VL-CL forms an antigen-binding site that binds CLDN-18.2, and the VH-linked peptide of the second heavy chain-VL has an antigen-binding site that binds CD3;
  • this bispecific antibody format has many unique properties and advantages, such as Excellent stability, low toxicity, and excellent tumor-killing activity in the preparation of bispecific antibodies for multiple targets.
  • the linking peptide of the present invention may be a flexible polypeptide containing G (glycine) and S (serine) or composed of G and S, and its length may be 2-30 amino acid residues.
  • Exemplary linker peptides include, but are not limited to, (GS)n, (GGS)n, (GGGS)n, and (GGGGS)n, etc., where n can be an integer from 1-6.
  • the bispecific antibodies of the present invention are diabodies, cross-bodies, or bispecific antibodies obtained by those controlled Fab arm exchanges as described in the present invention.
  • bispecific antibodies examples include, but are not limited to:
  • IgG fusion molecules wherein a full-length IgG antibody is fused to another Fab fragment or part of a Fab fragment;
  • Fc fusion molecules wherein a single chain Fv molecule or a stabilized diabody is fused to a heavy chain constant domain, an Fc region or a portion thereof;
  • Antibodies based on scFv and diabodies and heavy chain antibodies eg, domain antibodies, Nanobodies
  • different single chain Fv molecules or different diabodies or different heavy chain antibodies eg, domain antibodies, Nanobodies
  • IgG-like molecules with complementary CH3 domain molecules include, but are not limited to, Triomab/Quadroma (Trion Pharma/Fresenius Biotech), Knobs-into-Holes (Genentech), CrossMAbs (Roche) and the electrostatically-matched ) (Amgen), LUZ-Y (Genentech), Strand Exchange Engineered Domain body (SEEDbody) (EMD Serono), Biclonic (Merus), and DuoBody (Genmab A/S).
  • IgG-like dual targeting molecules include, but are not limited to, Dual Targeting (DT)-Ig (GSK/Domantis), Two-in-one Antibody (Genentech), Cross-linked Mabs (Kamanos Cancer Center), mAb2 (F -Star), and CovX-body (CovX/Pfizer).
  • DT Dual Targeting
  • Genentech Two-in-one Antibody
  • Cross-linked Mabs Kamanos Cancer Center
  • mAb2 F -Star
  • CovX-body CovX/Pfizer
  • IgG fusion molecules include, but are not limited to, Dual Variable Domain (DVD)-Ig (Abbott), IgG-like Bispecific (ImClone/Eli Lilly), Ts2Ab (MedImmune/AZ) and BsAb (Zymogenetics), HERCULES (Biogen Idec), and TvAb (Roche).
  • DVD Dual Variable Domain
  • IgG-like Bispecific ImClone/Eli Lilly
  • Ts2Ab MedImmune/AZ
  • BsAb Zymogenetics
  • HERCULES Biogen Idec
  • TvAb Roche.
  • Fc fusion molecules include, but are not limited to, scFv/Fc Fusions (Academic Institution), SCORPION (Emergent BioSolutions/Trubion, Zymogenetics/BMS), DualAffinity Retargeting Technology (Fc-DART) (MacroGenics), and Dual(scFv)2-Fab (National Engineering Research Center for Antibody Drugs, China).
  • Fab fusion bispecific antibodies include, but are not limited to, F(ab)2 (Medarex/AMGEN), Dual-Action or Bis-Fab (Genentech), Dock-and-Lock (DNL) (ImmunoMedics), Bivalent Bispecific (Biotecnol) ), and Fab-Fv (UCB-Celltech).
  • scFv, diabody and domain antibodies based include, but are not limited to, Bispecific T Cell Engager (BiTE) (Micromet, Tandem Diabody (Tandab) (Affimed), Dual Affinity Retargeting Technology (DART) (MacroGenics), Single-chain Diabody (Academic) ), TCR-like Antibodies (AIT, ReceptorLogics), Human SerumAlbumin scFv Fusion (Merrimack) and COMBODY (Epigen Biotech), dual targeting nanobodies (Ablynx), dual targeting heavy chain domain only antibodies (dualtargeting heavy chain only domain antibodies).
  • the present invention provides a polynucleotide encoding an antibody or antigen-binding fragment thereof that binds to CD3 as described herein, or an anti-CD3xCLDN-18.2 bispecific antibody as described herein.
  • the polynucleotide may comprise a polynucleotide encoding the amino acid sequence of the light chain variable region and/or heavy chain variable region of an antibody, or a polynucleotide comprising the amino acid sequence encoding the light chain and/or heavy chain of an antibody .
  • polynucleotide sequence encoding the first heavy chain described herein is shown in SEQ ID NO: 13, and the polynucleotide sequence encoding the second heavy chain described herein is shown in SEQ ID NO: 8, encoding the double chain described herein.
  • the polynucleotide sequence of the light chain in the specific antibody is shown in SEQ ID NO:14.
  • the present invention provides an expression vector comprising the polynucleotide as described herein, preferably, the vector is a eukaryotic expression vector.
  • the polynucleotides as described herein are contained in one or more expression vectors.
  • the present invention provides a host cell comprising a polynucleotide as described herein or an expression vector as described herein, preferably the host cell is a eukaryotic cell, more preferably a mammalian cell .
  • the present invention provides a method for preparing an anti-CD3xCLDN-18.2 bispecific antibody as described herein, comprising the method described herein under conditions suitable for expression of said antibody
  • the antibody is expressed in a host cell, and the expressed antibody is recovered from the host cell.
  • the invention provides mammalian host cells for expressing the recombinant antibodies of the invention, including a number of immortalized cell lines available from the American Type Culture Collection (ATCC). These include, inter alia, Chinese Hamster Ovary (CHO) cells, NSO, SP2/0 cells, HeLa cells, Baby Hamster Kidney (BHK) cells, Monkey Kidney cells (COS), human hepatocellular carcinoma cells, A549 cells, 293T cells and many others cell line. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, bovine, horse and hamster cells. Particularly preferred cell lines are selected by determining which cell lines have high expression levels.
  • ATCC American Type Culture Collection
  • the present invention provides a method of preparing an anti-CD3xCLDN-18.2 bispecific antibody of the present invention, wherein the method comprises introducing an expression vector into a mammalian host cell, and culturing the host cell for a sufficient period of time to The antibody is produced by allowing the antibody to be expressed in the host cell or, more preferably, secreted into the medium in which the host cell is grown.
  • Antibodies can be recovered from the culture medium using standard protein purification methods.
  • afucosylated antibodies are advantageous because they generally have more potent potency than their fucosylated counterparts in vitro and in vivo, and are unlikely to be immunogenic , because their carbohydrate structure is a normal component of native human serum IgG.
  • compositions and pharmaceutical preparations are provided.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising an anti-CD3 antibody or antigen-binding fragment thereof as described herein, an anti-CD3 x CLDN-18.2 bispecific antibody as described herein, A polynucleotide, an expression vector described herein or a host cell described herein, and a pharmaceutically acceptable carrier or excipient.
  • anti-CD3 antibody or anti-CD3 ⁇ CLDN-18.2 antibody or pharmaceutical composition thereof provided by the present invention can be integrated with suitable carriers, excipients and other agents in the formulation for combined administration, thereby providing improved transfer, delivery , tolerance, etc.
  • composition refers to a formulation that allows the active ingredients contained therein to exist in a biologically effective form and does not contain additional ingredients that would be unacceptably toxic to the subject to whom the formulation is administered.
  • Anti-CD3xCLDN-18.2 bispecific antibodies of the invention of the desired purity can be prepared by mixing with one or more optional pharmaceutical excipients (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980) ) to prepare a pharmaceutical formulation comprising the anti-CD3xCLDN-18.2 bispecific antibody described herein, preferably in the form of an aqueous solution or a lyophilized formulation.
  • compositions or formulations of the present invention may also contain one or more other active ingredients required for the particular indication being treated, preferably those active ingredients having complementary activities that do not adversely affect each other .
  • the other active ingredient is a chemotherapeutic agent, an immune checkpoint inhibitor, a growth inhibitory agent, an antibiotic, or various anti-tumor or anti-cancer agents known, in a suitable amount effective for the intended use exist in combination.
  • compositions of the present invention further comprise compositions of polynucleotides encoding anti-CD3xCLDN-18.2 bispecific antibodies.
  • the present invention provides a pharmaceutical combination comprising an anti-CD3 antibody or antigen-binding fragment thereof as described herein, an anti-CD3 x CLDN-18.2 bispecific antibody as described herein, a multinucleate as described herein nucleotides, expression vectors described herein, host cells described herein, or pharmaceutical compositions described herein, and one or more additional therapeutic agents.
  • the present invention provides a kit 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, or the pharmaceutical compositions described herein.
  • the present invention provides an anti-CD3 antibody or antigen-binding fragment thereof as described herein, an anti-CD3 x CLDN-18.2 bispecific antibody as described herein, a polynucleotide as described herein, a Use of an expression vector, a host cell described herein, or a pharmaceutical composition described herein in the manufacture of a medicament for the treatment of cancer.
  • the present invention provides an anti-CD3 antibody or antigen-binding fragment thereof as described herein, an anti-CD3 x CLDN-18.2 bispecific antibody as described herein, a polynucleotide as described herein, a An expression vector, a host cell described herein, or a pharmaceutical composition described herein for use in the treatment of cancer.
  • the present invention provides a method of treating cancer comprising administering to a subject in need thereof an anti-CD3 antibody or antigen-binding fragment thereof as described herein, an anti-CD3 x CLDN-18.2 as described herein A bispecific antibody, a polynucleotide described herein, an expression vector described herein, a host cell described herein, or a pharmaceutical composition described herein.
  • the cancer is associated with CLDN18; more specifically, the cancer overexpresses Claudin 18.2. Therefore, in a preferred embodiment, the cancer is a cancer whose cancer cells highly express Claudin 18.2.
  • "Highly expressed" as used herein means that the expression level of Claudin 18.2 in cancer cells or cancer tissues is higher than that in normal cells or normal tissues. Conventional methods can be used to test the expression of Claudin 18.2 in normal cells or tissues in the normal population, and use this as a benchmark to determine whether Claudin 18.2 is highly expressed in the corresponding cells or tissues of the subject.
  • the cancer is selected from the group consisting of gastric cancer, esophageal cancer, gastroesophageal cancer, pancreatic cancer, bile duct cancer, lung cancer, ovarian cancer, colon cancer, liver cancer, head and neck cancer, gallbladder cancer, bowel cancer and bladder cancer.
  • modes of administration of the present invention include, but are not limited to, oral, intravenous, subcutaneous, intramuscular, intraarterial, intraarticular (eg, in arthritic joints), by inhalation, aerosol delivery, or intratumoral administration Wait.
  • the present invention provides for co-administration of a therapeutically effective amount of one or more therapies (eg, therapeutic modalities and/or other therapeutic agents) to a subject.
  • the therapy includes surgery and/or radiation therapy.
  • the methods or uses provided herein further comprise administering to an individual one or more therapies (eg, therapeutic modalities and/or other therapeutic agents).
  • therapies eg, therapeutic modalities and/or other therapeutic agents.
  • Antibodies of the invention can be used alone or in combination with other therapeutic agents in therapy. For example, it can be co-administered with at least one additional therapeutic agent.
  • PD-1 antibody, PD-L1 antibody, LAG-3 antibody and/or CTLA-4 antibody for example, PD-1 antibody, PD-L1 antibody, LAG-3 antibody and/or CTLA-4 antibody.
  • the cancer disease treatment method of the present invention further comprises administering a CD3xCLDN-18.2 expression stabilizing or increasing agent.
  • the expression of CD3xCLDN-18.2 is preferably on the cell surface of cancer cells.
  • the CD3xCLDN-18.2 expression stabilizing or increasing agent may be oxaliplatin and/or 5-FU.
  • the present invention provides a method of detecting the presence of CD3 or CLDN-18.2 in a sample using an antibody or antigen-binding fragment thereof as described herein.
  • detection includes quantitative or qualitative detection.
  • the sample is a biological sample.
  • the biological sample is blood, serum, or other fluid sample of biological origin.
  • the biological sample comprises cells or tissues.
  • the method comprises contacting an antibody or antigen-binding fragment thereof of the invention, particularly a bispecific antibody of the invention, with a sample, and detecting the association of the antibody or antigen-binding fragment thereof or the bispecific antibody with CD3 or CLDN -18.2 Presence and/or amount of conjugate formed.
  • the present invention analyzes multiple anti-human CD3 antibodies, optimizes the sequence of one of the mouse-derived antibodies SP34 and performs humanization transformation to obtain humanized anti-CD3 antibody JSCD3 and its antigen-binding fragment.
  • the specific steps of humanization transformation are as follows: A. Compare the gene sequence of each candidate antibody with the human embryonic antibody gene sequence to find out the sequence with high homology; B. Analyze and investigate HLA-DR affinity, and select The low-affinity human embryonic framework region sequence; C. Using computer simulation technology, the amino acid sequence of the variable region and its surrounding framework region is analyzed by molecular docking, and its spatial three-dimensional binding mode is investigated.
  • the gene sequences of each candidate antibody are analyzed for the key amino acids that can interact with CD3 and maintain the spatial framework region, and graft them into the selected human embryonic gene framework region. On this basis, the amino acid sites in the framework region that must be retained are marked to synthesize a humanized antibody or an antigen-binding fragment thereof.
  • SP34 is a murine monoclonal antibody against human CD3, its heavy chain amino acid sequence is SEQ ID NO: 28, and its light chain amino acid sequence is SEQ ID NO: 29.
  • VH and VL amino acid sequences of the humanized anti-CD3 antibodies are as follows (HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are indicated in bold and underlined):
  • HCDR1:TYAMN SEQ ID NO: 16
  • HCDR2 RIRSKYNNYATYYADSVKD (SEQ ID NO: 17);
  • HCDR3 HGNFGNSYVSWFAY (SEQ ID NO: 18).
  • LCDR1 RSSTGAVTTSNYAN (SEQ ID NO: 19);
  • LCDR2 GTNKRAP (SEQ ID NO: 20);
  • LCDR3 ALWYSNLWV (SEQ ID NO: 21).
  • amino acid sequences of the HC and LC of the antigen-binding fragment (Fab) of the anti-CD3 antibody are as follows:
  • JSCD3HC SEQ ID NO: 3:
  • JSCD3LC SEQ ID NO: 4:
  • Embodiment 2 the construction of bispecific antibody molecule expression vector
  • GenScript Biotechnology Co., Ltd. was entrusted to synthesize the gene encoding JS012-2B19LC-1, digested with BSPQI and ligated into the HXT2 vector to obtain the first expression vector HXT2-JS012-2B19-LC-1.
  • the plasmid HXT1S-JS012-2B19-HC-7-KDL was digested with HindIII and NheI to recover the gene fragment JS012-2B19-HC-7, and the plasmid HXT4S-JS012-1H17-HC-mut-h was digested with HindIII and NheI and The vector HXT4s-mut h was recovered and ligated with T4 DNA ligase to obtain the second expression vector HXT4S-JS012-2B19-HC-7-mut-h. GenScript Biotechnology Co., Ltd.
  • CHO-K1 cells modified at the genome level to make cells suitable for transient expression, Junshi Bio
  • CD CHO medium Gibco
  • 7% CO 2 when the cell density reached (2-6) ⁇ 10 6 /mL, use CD CHO medium for subculture expansion.
  • the CHO-K1 cell density was diluted to (1.5-2.0) ⁇ 10 6 /mL, placed on a shaker (culture conditions: 36.5°C, 120 rmp, 7% CO 2 ) to culture, the next day when the cell density reached about Transfection was performed at 3.5 ⁇ 10 6 /mL.
  • a shaker culture conditions: 36.5°C, 120 rmp, 7% CO 2
  • Transfection was performed at 3.5 ⁇ 10 6 /mL.
  • first add one-tenth of the transfection volume of CD CHO medium to the reactor then add 1-2ug/mL transfection volume of the above plasmid, 3-14ug/mL PEI (polyethyleneimine) , after mixing, incubate at room temperature for 20-30 minutes, then slowly add the above mixture to the pre-treated CHO-K1 cells to form a transfection mixture, and mix while adding.
  • the transfection mixture was cultured in a shaker at 36.5°C, 120 rmp, 7% CO 2 .
  • the first step is to purify with AKTA Avant purifier. First, sterilize the packed mabselect sure LX column with 0.1M NaOH for 15-20 minutes, then equilibrate with PBS buffer for 3-5 column volumes and load the sample.
  • the amino acid sequence (ie the second heavy chain) encoding the anti-CD3 full-length antibody is shown in SEQ ID NO: 7, and its coding sequence is shown in SEQ ID NO: 8; the antibody against CLDN-18.2
  • the amino acid sequence of the heavy chain (that is, the first heavy chain) is shown in SEQ ID NO: 11, and its coding sequence is shown in SEQ ID NO: 13;
  • the amino acid sequence of the light chain of the antibody against CLDN-18.2 is shown in SEQ ID NO: 12 shown, its coding sequence is shown in SEQ ID NO: 14;
  • the amino acid sequence of the heavy chain variable region of the anti-CLDN-18.2 antibody is shown in SEQ ID NO: 9
  • the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 9
  • the amino acid sequence of the heavy chain variable region of the anti-CD3 antibody is shown in SEQ ID NO: 5
  • the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 6;
  • SEQ ID NO: 7 (bold type indicates VL and VH, respectively, LCDR1-LCDR3 and HCDR1-HCDR3 are underlined)
  • thermostability of bispecific antibody JS009 was detected by nanoDSF (differential scanning fluorescence technique) in a buffer system of pH 6.0 (20 mM citric acid-sodium citrate/50 mM sodium chloride/150 mM mannitol).
  • the double antibody JS009 sample was replaced with the above buffer, and the concentration of the sample was controlled at about 10 mg/ml, and DSF was used for detection.
  • the results are shown in Table 1.
  • the bispecific antibody JS009 showed good thermal stability, with Tm1 and Tm2 being 63.3°C and 79.0°C, respectively.
  • Tm thermal transition temperature
  • the bispecific antibody JS009 was replaced with a buffer system of pH 6.0 (20mM citric acid-sodium citrate/50mM sodium chloride/150mM mannitol), and the concentration of the sample was controlled at about 10mg/ml. Tube) of the double antibody sample was placed in a 40°C incubator, the stability of 0W, 1W, 2W and 4W was investigated, and the sample purity and biological activity were detected according to time points. The test results show that the bispecific antibody JS009 has good stability and can withstand high temperature conditions, as shown in Table 2.
  • both JSCD3 and JS009 can effectively bind to human CD3-expressing cells, and the binding ability of JS009 to human CD3-expressing cells is reduced.
  • Cynomolgus monkey peripheral blood mononuclear cells (PBMC, Ocellus Biotechnology (Shanghai) Co., Ltd.) were diluted with serial concentrations of humanized CD3 mAb (JS202-JSCD3), SP34, bispecific antibody ( JS009) and negative control (anti-KLH monoclonal antibody, Anti KLH IgG4) were incubated at 4°C for 30 min, the residual antibody was washed away and then incubated with fluorescent secondary antibody (PE-anti-human IgG4), and finally the fluorescence was detected by BD Canto II flow cytometer The stronger the fluorescence signal, the higher the affinity of the antibody to the target. Antibody dose-dependent binding curves were fitted by GraphPad ( Figure 3).
  • SP34, JSCD3 and JS009 can effectively bind to cynomolgus monkey CD3 expressing cells, and the binding ability of JS009 to cynomolgus monkey CD3 expressing cells is reduced.
  • Anti-CD3 ⁇ Claudin18.2 double antibody can bind to Claudin18.2 on target cells and further bind to T cells expressing CD3, thereby activating the downstream signaling pathway of T cells.
  • JS202-JSCD3 monoclonal antibody SP34, JS009 double antibody were incubated with target cells NUGC4-CLDN-18.2 cells (Junshi Bio) and Jurkat NFAT effector cells (Junshi Bio), and the substrate one-glo was added. The signal was then detected with a microplate reader, and the data was finally analyzed with GraphPad to compare the cell viability in a dose-dependent manner (Fig. 4).
  • SP34, JS202-JSCD3 mAb and JS009 double antibody can effectively activate the downstream signaling pathway of T cells.
  • the activity of JS009 double antibody is better than that of mAb in the presence of target cells.
  • mice Twenty-five 6-8-week-old female B-hCD3e humanized mice (Biocyto Jiangsu Gene Biotechnology Co., Ltd.) were taken, and MC38 Claudin18.2 cells were inoculated into the right side of the mice at a concentration of 1 ⁇ 10 6 /0.1 mL. Subcutaneously on the back, when the tumor grows to about 96mm 3 , it is randomly divided into groups according to the tumor volume, 6 in each group, a total of 2 groups, namely: G1 normal saline (normal saline) negative control group and G2 JS009 (10 mg/kg) group.
  • G1 normal saline normal saline
  • G2 JS009 10 mg/kg
  • the drug was administered by intraperitoneal injection, twice a week, with a dose of 10 mg/kg and an administration concentration of 1 mg/ml, for 5 consecutive administrations, and the experiment was terminated 3 days after the last administration.
  • the tumor volume and body weight were measured twice a week, and the body weight and tumor volume of the mice were recorded.
  • T0 the mean tumor volume of the treatment group on the 0th day of administration
  • Vi the mean tumor volume of the negative control group on the ith day of administration
  • V0 the negative control Group mean tumor volume on dosing day 0.
  • the average tumor volume in the normal saline group was 2013 ⁇ 199 mm 3
  • the average tumor volume in the JS009 group was 1019 ⁇ 62 mm 3
  • the relative tumor inhibition rate was 51.9%, p ⁇ 0.001. It was shown that the bispecific antibody JS009 could inhibit the growth of B-hCD3e humanized mouse MC38 Claudin18.2 cells subcutaneously transplanted in vivo.

Abstract

本发明提供抗CD3和抗CLDN-18.2双特异性抗体及其用途。本发明的抗CD3抗体包含结合CD3的抗原结合位点，该结合CD3的抗原结合位点包含氨基酸序列如SEQ ID NO:16、SEQ ID NO:17和SEQ ID NO:18所示的HCDR1、HCDR2和HCDR3，和氨基酸序列分别如SEQ ID NO:19、SEQ ID NO:20和SEQ ID NO:21所示的LCDR1、LCDR2和LCDR3。本发明的双特异性抗体含有结合CD3的抗原结合位点和结合CLDN-18.2的结合位点。

Description

抗CD3和抗CLDN-18.2双特异性抗体及其用途 技术领域
本发明涉及抗CD3和抗CLDN-18.2双特异性抗体及其用途。
背景技术
CD3(T细胞表面糖蛋白CD3，T细胞受体的信号转导共受体，其包含亚基γ、δ、ε和ζ)是表达于所有T淋巴细胞表面的一种分化抗原，主要介导T细胞活化信号的转导。在机体免疫系统的抗感染免疫中起着重要作用。CD3分子与T细胞抗原受体(T cell receptor)形成稳定的TCR-CD3复合体，其胞外区识别、结合主要组织相容性复合体II类分子，增强T细胞抗原受体(T cell receptor，TCR)与MHC分子结合的稳定性；胞内区增强白细胞CD3转导的活化信号，从而参与并调节免疫系统的活化。CD3阳性淋巴细胞群的数量指标是衡量机体细胞免疫情况的重要指标。
Claudin，又称CLDN，是一类建立细胞旁屏障并控制细胞间分子流动的细胞表面蛋白家族，目前已发现至少有26种。Claudin蛋白家族成员是紧密连接的重要结构成分，在保持上皮细胞极性、控制细胞旁扩散、以及调控细胞生长分化方面起到重要作用。Claudin分子跨细胞膜四次，N端和C端均落在细胞质中。Claudin 18(CLDN18)有两种选择性剪接变异体CLDN-18.1和CLDN-18.2。Claudin 18.1(CLDN-18.1)选择性地在正常肺和胃上皮中表达。Claudin 18.2(CLDN-18.2)在正常胃上皮短寿细胞中呈现微量表达，但在肿瘤细胞中，Claudin 18.2在多种癌症类型中均呈现强烈表达。
双特异性抗体(bispecific antibody，BsAb)，又称为双功能抗体，可以同时特异性结合两个不同的抗原或两个不同的抗原表位。由于其特异性和双功能性，在肿瘤的免疫治疗及自身免疫病等领域具有良好的应用效果和前景。双特异抗体有多种形式，而且对于不同的肿瘤相关抗原，不同的双特异抗体的形式效果是不一样的，由于它的这种特殊的功能，在肿瘤的免疫治疗中具有广阔的应用前景。
本发明旨在提供亲和力、稳定性等方面均具有较好效果的抗CD3抗体和抗CD3×CLDN-18.2双特异性抗体。
发明内容
本发明提供了与CD3结合的抗体或其抗原结合片段，以及靶向CD3和CLDN-18.2的 双特异性抗体。本发明提供的抗CLDN-18.2抗体或其抗原结合片段可作为独立的疗法或与其它疗法/或其他抗癌药剂联合，用于诸如癌症的治疗。
在一个方面，本发明提供了一种抗体或其抗原结合片段，其包含结合CD3的抗原结合位点。
在一些实施方式中，本发明所述结合CD3的抗原结合位点包含氨基酸序列分别如SEQ ID NO:16、SEQ ID NO:17和SEQ ID NO:18所示的HCDR1、HCDR2和HCDR3，和氨基酸序列分别如SEQ ID NO:19、SEQ ID NO:20和SEQ ID NO:21所示的LCDR1、LCDR2和LCDR3。
在一些实施方式中，本发明所述结合CD3的抗原结合位点包含氨基酸序列如SEQ ID NO:1所示的重链可变区(VH)，和氨基酸序列如SEQ ID NO:2所示的轻链可变区(VL)；或包含氨基酸序列如SEQ ID NO:5所示的重链可变区(VH)，和氨基酸序列如SEQ ID NO:6所示的轻链可变区(VL)。
在一些实施方式中，本发明所述抗原结合片段包含氨基酸序列如SEQ ID NO:15所示的重链片段，和氨基酸序列如SEQ ID NO:4所示的轻链片段。在一些实施方式中，本发明所述抗原结合片段为Fab片段。
在一些实施方式中，本发明所述抗体包含氨基酸序列如SEQ ID NO:3所示的重链，和氨基酸序列如SEQ ID NO:4所示的轻链。
在一些实施方式中，本发明所述抗体是单克隆抗体、双特异性抗体或多特异性抗体。
在一些实施方式中，本发明所述抗体或其抗原结合片段还包含结合CLDN-18.2的抗原结合位点。
在一些实施方式中，本发明所述结合CLDN-18.2的抗原结合位点包含氨基酸序列分别如SEQ ID NO:22、SEQ ID NO:23和SEQ ID NO:24所示的HCDR1、HCDR2和HCDR3，和氨基酸序列分别如SEQ ID NO:25、SEQ ID NO:26和SEQ ID NO:27所示的LCDR1、LCDR2和LCDR3。
在一些实施方式中，本发明所述结合CLDN-18.2的抗原结合位点包含氨基酸序列如SEQ ID NO:9所示的重链可变区，和氨基酸序列如SEQ ID NO:10所示的轻链可变区。
在一些实施方式中，本发明所述结合CD3的抗原结合位点的重链可变区(VH)和轻链可变区(VL)通过连接肽连接形成scFv形式，所述连接肽的氨基酸序列各自独立为(GGGGS)n，其中，n为2、3、4、5或6，优选地，所述scFv进一步结合至Fc区。
在一些实施方式中，本发明所述结合CLDN-18.2的抗原结合位点的重链可变区(VH)、恒定区1(CH1)、轻链可变区(VL)和轻链恒定区(CL)形成Fab形式；优选地，所述Fab进一步结合至Fc区。
在一些实施方式中，本发明所述结合CD3的抗原结合位点和结合CLDN-18.2的抗原结合位点的Fc为来自IgG的Fc，例如来自IgG1、IgG2、IgG3或IgG4的Fc，和/或所述轻链的CL为来自λ或κ轻链的CL。
在一些实施方式中，本发明所述结合CD3的抗原结合位点包含氨基酸序列如SEQ ID NO:7所示的氨基酸序列。
在一些实施方式中，本发明所述结合CLDN-18.2的抗原结合位点包含氨基酸序列如SEQ ID NO:11所示的重链，和氨基酸序列如SEQ ID NO:12所示的轻链。
在一些实施方式中，本发明所述抗原结合片段为Fab、Fab'、F(ab')2、Fv、scFv或sdAb。
在一些实施方式中，本发明所述结合CD3的抗原结合位点的重链自N端到C端具有铰链区-Fc-连接肽-VL-连接肽-VH。
在一些实施方式中，本发明所述结合CLDN-18.2的抗原结合位点的重链自N端到C端具有VH-CH1-铰链区-Fc，轻链自N端到C端具有VL-CL。
在一些实施方式中，本发明所述结合CD3的抗原结合位点的Fc和结合CLDN-18.2的抗原结合位点的Fc之间通过铰链区的二硫键和CH3结构域的杵臼结构连接。
在又一个方面，本发明提供了一种双特异性抗体，其具有两条重链和一条轻链，其中，第一重链自N端到C端具有VH-CH1-铰链区-Fc；第二重链自N端到C端具有铰链区-Fc-连接肽-VL-连接肽-VH；所述轻链自N端到C端具有VL-CL；其中，所述第一重链的VH-CH1与所述轻链的VL-CL形成抗原结合位点结合CLDN-18.2，所述第二重链的VH-连接肽-VL的抗原结合位点结合CD3。
在一些实施方式中，本发明所述第二重链的VH包含氨基酸序列分别如SEQ ID NO:16、SEQ ID NO:17和SEQ ID NO:18所示的HCDR1、HCDR2和HCDR3，和VL包含氨基酸序列分别如SEQ ID NO:19、SEQ ID NO:20和SEQ ID NO:21所示的LCDR1、LCDR2和LCDR3。
在一些实施方式中，本发明所述第二重链中，所述连接肽的氨基酸序列各自独立为(GGGGS)n，其中，n为2、3、4、5或6。
在一些实施方式中，本发明所述第二重链的VH具有如SEQ ID NO:1所示的氨基酸序列，和VL具有如SEQ ID NO:2所示的氨基酸序列，所述连接肽的氨基酸序列独立为(GGGGS)n，其中，n为2、3、4、5或6。
在一些实施方式中，本发明所述第二重链的VH具有如SEQ ID NO:5所示的氨基酸序列，和VL具有如SEQ ID NO:6所示的氨基酸序列，所述连接肽的氨基酸序列独立为(GGGGS)n，其中，n为2、3、4、5或6。
在一些实施方式中，本发明所述第二重链具有如SEQ ID NO:7所示的氨基酸序列。
在一些实施方式中，本发明所述第一重链的VH包含氨基酸序列分别如SEQ ID NO:22、 SEQ ID NO:23和SEQ ID NO:24所示的HCDR1、HCDR2和HCDR3。
在一些实施方式中，本发明所述第一重链的VH具有如SEQ ID NO:9所示的氨基酸序列。
在一些实施方式中，本发明所述第一重链具有如SEQ ID NO:11所示的氨基酸序列。
在一些实施方式中，本发明所述轻链的VL包含分别如SEQ ID NO:25、SEQ ID NO:26和SEQ ID NO:27所示的LCDR1、LCDR2和LCDR3。
在一些实施方式中，本发明所述轻链的VL具有如SEQ ID NO:10所示的氨基酸的序列。
在一些实施方式中，本发明所述轻链具有如SEQ ID NO:12所示的氨基酸的序列。
在一些实施方式中，本发明所述双特异性抗体中：
第一重链的VH包含氨基酸序列分别如SEQ ID NO:22、SEQ ID NO:23和SEQ ID NO:24所示的HCDR1、HCDR2和HCDR3；
第二重链的VH包含氨基酸序列分别如SEQ ID NO:16、SEQ ID NO:17和SEQ ID NO:18所示的HCDR1、HCDR2和HCDR3，VL包含氨基酸序列分别如SEQ ID NO:19、SEQ ID NO:20和SEQ ID NO:21所示的LCDR1、LCDR2和LCDR3；
轻链的VL包含分别如SEQ ID NO:25、SEQ ID NO:26和SEQ ID NO:27所示的LCDR1、LCDR2和LCDR3。
在一些实施方式中，本发明所述双特异性抗体中：
第一重链的VH具有如SEQ ID NO:9所示的氨基酸序列；
第二重链的VH具有如SEQ ID NO:1所示的氨基酸序列，VL具有如SEQ ID NO:2所示的氨基酸序列；或第二重链的VH具有如SEQ ID NO:5所示的氨基酸序列，VL具有如SEQ ID NO:6所示的氨基酸序列；且第二重链中的连接肽的氨基酸序列各自独立为(GGGGS)n，其中，n为2、3、4、5或6；
轻链的VL具有如SEQ ID NO:10所示的氨基酸的序列。
在一些实施方式中，本发明所述双特异性抗体中，第一重链具有如SEQ ID NO:11所示的氨基酸序列，第二重链具有如SEQ ID NO:7所示的氨基酸序列，轻链具有如SEQ ID NO:12所示的氨基酸的序列。
在一些实施方式中，本发明所述第一重链与所述第二重链之间通过铰链区的二硫键和CH3结构域的杵臼结构连接。
在一些实施方式中，本发明所述第一重链和第二重链的Fc为来自IgG的Fc，例如来自IgG1、IgG2、IgG3或IgG4的Fc，和/或所述轻链的CL为来自λ或κ轻链的CL。
在又一个方面，本发明提供了一种多核苷酸分子，其核苷酸序列选自：
(1)编码如本文所述的与CD3结合的抗体或其抗原结合片段，或本文所示的双特异 性抗体的第一重链、第二重链和/或轻链的核苷酸序列；
(2)如(1)所述核苷酸序列的互补序列。
在又一个方面，本发明提供了一种表达载体，其包含如本文所述的多核苷酸分子，优选地，所述表达载体为真核表达载体。
在又一个方面，本发明提供了一种宿主细胞，其包含如本文所述的多核苷酸分子或表达载体，或表达本文任一实施方案所述的抗体；优选地，所述宿主细胞是真核细胞，更优选哺乳动物细胞。
在又一个方面，本发明提供了一种制备如本文所述的双特异性抗体的方法，所述方法包括在适合于所述双特异性抗体表达的条件下在如本文所述的宿主细胞中表达所述双特异性抗体，并从所述宿主细胞回收所表达的双特异性抗体。
在又一个方面，本发明提供了一种药物组合物，其包含如本文所述的抗体或其抗原结合片段、如本文所述的双特异性抗体、如本文所述的多核苷酸分子、如本文所述的表达载体、如本文所述的宿主细胞和药学上可接受的载体或赋形剂。
在又一个方面，本发明提供了如本文所述的抗体或其抗原结合片段、如本文所述的双特异性抗体、如本文所述的多核苷酸分子、如本文所述的表达载体、如本文所述的宿主细胞和如本文所述的药物组合物在制备用于治疗癌症的药物中的用途。
在又一个方面，本发明提供了如本文所述的抗体或其抗原结合片段、如本文所述的双特异性抗体、如本文所述的多核苷酸分子、如本文所述的表达载体、如本文所述的宿主细胞和如本文所述药物组合物，其用于治疗癌症。
在又一个方面，本发明提供了一种治疗癌症的方法，其包括向有需要的受试者施用如本文所述的抗体或其抗原结合片段、如本文所述的双特异性抗体、如本文所述的多核苷酸分子、如本文所述的表达载体、如本文所述的宿主细胞和如本文所述的药物组合物。
在一些实施方式中，本发明所述癌症选自胃癌、食道癌、胃食管癌、胰腺癌、胆管癌、肺癌、卵巢癌、结肠癌、肝癌、头颈癌、胆囊癌、肠癌和膀胱癌药物。
在又一个方面，本发明提供了一种药物组合，其包含如本文所述的抗体或其抗原结合片段、如本文所述的双特异性抗体、本文所述的多核苷酸分子、本文所述的表达载体、本文所述的宿主细胞、或本文所述的药物组合物，以及一种或多种另外的治疗剂。
在又一个方面，本发明提供了一种试剂盒，其包括如本文所述的抗体或其抗原结合片段、如本文所述的双特异性抗体、本文所述的多核苷酸分子、本文所述的表达载体、本文所述的宿主细胞、或本文所述的药物组合物。
在又一个方面，本发明提供了使用如本文所述的双特异性抗体检测CD3和/或CLDN-18.2在样品中的存在的方法，所述方法包括使本文所述的双特异性抗体与样品接触 并检测所述双特异性抗体与CD3和/或CLDN-18.2形成的结合物或对其进行定量测定。
附图说明
图1：抗CD3×CLDN-18.2双特异性抗体的分子结构示意图。
图2：FACS法检测抗CD3单抗和抗CD3×CLDN-18.2双特异性抗体与人CD3表达细胞的结合。
图3：FACS法检测抗CD3单抗和抗CD3×CLDN-18.2双特异性抗体与食蟹猴CD3表达细胞的结合。
图4：荧光素酶法检测抗CD3单抗和抗CD3×CLDN-18.2双特异性抗体的对T细胞活性的影响。
图5：抗CD3×CLDN-18.2双特异性抗体对小鼠肿瘤生长的抑制。
图6：JSCD3和SP34与CD3E的结合检测结果。
图7：JSCD3和SP34与CD3D&E的结合检测结果。
具体实施方式
定义
除非另有说明，本发明的实施将采用分子生物学(包括重组技术)、微生物学、细胞生物学、生物化学和免疫学的常规技术，这些都在本领域的技术范围内。
为了可以更容易地理解本发明，某些科技术语具体定义如下。除非本文其它部分另有明确定义，否则本文所用的科技术语都具有本发明所属领域普通技术人员通常理解的含义。关于本领域的定义及术语，专业人员具体可参考Current Protocolsin Molecular Biology(Ausubel)。氨基酸残基的缩写是本领域中所用的指代20个常用L-氨基酸之一的标准3字母和/或1字母代码。本文(包括权利要求书)所用单数形式包括其相应的复数形式，除非文中另有明确规定。
术语“约”在与数字数值联合使用时意为涵盖具有比指定数字数值小5％的下限和比指定数字数值大5％的上限的范围内的数字数值。
术语“和/或”应理解为意指可选项中的任一项或可选项中的任意两项或更多项的组合。
术语“CD3”指的是作为T细胞受体复合物的一部分，由三个不同的链CD3ε，CD3δ和CD3γ组成。CD3在T细胞上通过例如抗CD3抗体对其的固定作用而产生的集中，导致T细胞的活化，与T细胞受体介导的活化类似，但是不依赖于TCR克隆的特异性。绝大多数抗CD3抗体识别CD3ε链。该术语指来自任何脊椎动物(包括哺乳动物如灵长类动物(例如人))和啮齿类动物(例如，小鼠和大鼠)的任何天然CD3，除非另有说明。该术 语涵盖“全长”未加工的CD3以及由细胞内加工产生的任何形式的CD3或其任何片段。该术语还包括天然存在的CD3的变体，例如，剪接变体或等位变体。在一个优选实施方式中，CD3是指来自人和食蟹猴CD3全长或其片段(诸如其缺乏信号肽的成熟片段)。在一个优选实施方式中，CD3是指来自小鼠/大鼠全长或其片段(诸如其缺乏信号肽的成熟片段)。
术语“CLDN-18.2”或“Claudin18.2”，是Claudin 18的两个剪接变异体之一。该术语指来自任何脊椎动物(包括哺乳动物如灵长类动物(例如人))和啮齿类动物(例如，小鼠和大鼠)的任何天然CLDN-18.2，除非另有说明。该术语涵盖“全长”未加工的CLDN-18.2以及由细胞内加工产生的任何形式的CLDN-18.2或其任何片段。该术语还包括天然存在的CLDN-18.2的变体，例如，剪接变体或等位变体。在一个优选实施方式中，CLDN-18.2是指来自人和食蟹猴CLDN-18.2全长或其片段(诸如其缺乏信号肽的成熟片段)。
术语“百分比(％)氨基酸序列同一性”或简称“同一性”定义为在将氨基酸序列进行比对(并在必要时导入空位)以获取最大百分比序列同一性，且不将任何保守取代视为序列同一性的部分之后，候选氨基酸序列中的氨基酸残基与参比氨基酸序列中的相同氨基酸残基的百分比。可使用本领域各种方法进行序列比对以便测定百分比氨基酸序列同一性，例如，使用公众可得到的计算机软件如BLAST、BLAST-2、ALIGN或MEGALIGN(DNASTAR)软件。本领域技术人员可以决定测量比对的适宜参数，包括对所比较的序列全长获得最大比对所需的任何算法。
术语“免疫应答”是指由例如淋巴细胞、抗原呈递细胞、吞噬细胞、粒细胞和由上述细胞或肝产生可溶性大分子(包括抗体、细胞因子和补体)的作用，该作用导致从人体选择性损害、破坏或清除侵入的病原体、感染病原体的细胞或组织、癌细胞或者在自体免疫或病理性炎症的情况下的正常人细胞或组织。
术语“信号转导途径”或“信号转导活性”是指通常由蛋白质间相互作用诸如生长因子对受体的结合启动的生化因果关系，所述关系导致信号从细胞的一部分传递至细胞的另一部分。一般地，传递包括引起信号转导的系列反应中的一种或多种蛋白质上的一个或多个酪氨酸、丝氨酸或苏氨酸残基的特定磷酸化。倒数第二过程通常包括细胞核事件，从而导致基因表达的变化。
术语“活性”或“生物活性”，或术语“生物性质”或“生物特征”此处可互换使用，包括但不限于表位/抗原亲和力和特异性、在体内或体外中和或拮抗CLDN-18.2活性的能力、IC50、抗体的体内稳定性和抗体的免疫原性质。本领域公知的抗体的其它可鉴定的生物学性质或特征包括，例如，交叉反应性(即通常与靶定肽的非人同源物，或与其它蛋白质或组织的交叉反应性)，和保持哺乳动物细胞中蛋白质高表达水平的能力。使用本领域公知的技术观察、测定或评估前面提及的性质或特征，所述技术包括但不局限于ELISA、FACS或 BIACORE等离子体共振分析、不受限制的体外或体内中和测定、受体结合、细胞因子或生长因子的产生和/或分泌、信号转导和不同来源(包括人类、灵长类或任何其它来源)的组织切片的免疫组织化学。
术语“抗体”是指具有所需生物活性的任何形式的抗体。因此，其以最广义使用，具体包括但不限于单克隆抗体(包括全长单克隆抗体)、多克隆抗体、多特异性抗体(例如双特异性抗体)、人源化抗体、全人抗体、嵌合抗体和骆驼源化单结构域抗体。已知基本的抗体结构单位包含四聚体。每个四聚体包括两个相同的多肽链对，每对具有一条“轻”链(约25kDa)和一条“重”链(约50-70kDa)。每条链的氨基端部分或片段可包括主要负责抗原识别的约100-110个或更多个氨基酸的可变区。每条链的羧基端部分或片段可限定主要负责效应子功能的恒定区。通常将人轻链归类为κ和λ轻链。此外，通常将人重链归类为μ、δ、γ、α或ε，并将抗体的同种型分别定义为IgM、IgD、IgG、IgA和IgE。在轻链和重链内，可变区和恒定区通过约12个或更多个氨基酸的“J”区连接，其中重链还包括约10多个氨基酸的“D”区。一般参见Fundamental Immunology第7章(Paul，W.主编，第2版。Raven Press，N.Y.(1989))。
术语“分离的抗体”是指结合化合物的纯化状态，且在这种情况下意指该分子基本不含其它生物分子，例如核酸、蛋白质、脂质、糖或其它物质例如细胞碎片和生长培养基。术语“分离(的)”并非意指完全不存在这类物质或不存在水、缓冲液或盐，除非它们以明显干扰本文所述结合化合物的实验或治疗应用的量存在。
术语“单克隆抗体”是指获自基本均质抗体群的抗体，即组成该群的各个抗体除可少量存在的可能天然存在的突变之外是相同的。单克隆抗体是高度特异性的，针对单一抗原表位。相比之下，常规(多克隆)抗体制备物通常包括大量针对不同表位(或对不同表位有特异性)的抗体。修饰语“单克隆”表明获自基本均质抗体群的抗体的特征，且不得解释为需要通过任何特定方法产生抗体。
术语“双特异性抗体”是指是人工设计的抗体，其由两个不同抗原结合位点的组分构成，可同时与两种不同的抗原结合位点结合。
术语“全长抗体”，是指在天然存在时包含四条肽链的免疫球蛋白分子：两条重(H)链(全长时约50-70kDa)和两条轻(L)链(全长时约25kDa)通过二硫键互相连接。每一条重链由重链可变区(在本文中缩写为VH)和重链恒定区(在本文中缩写为CH)组成。重链恒定区由3个结构域CH1、CH2和CH3组成。每一条轻链由轻链可变区(在本文中缩写为VL)和轻链恒定区组成。轻链恒定区由一个结构域CL组成。VH和VL区可被进一步细分为具有高可变性的互补决定区(CDR)和其间隔以更保守的称为框架区(FR)的区域。每一个VH或VL区由按下列顺序：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4从氨基末端至羧基 末端排列的3个CDR和4个FR组成。重链和轻链的可变区含有与抗原相互作用的结合结构域。抗体的恒定区可介导免疫球蛋白对宿主组织或因子(包括免疫系统的各种细胞(例如，效应细胞)和经典补体系统的第一组分(Clq))的结合。
术语抗体(“亲代抗体”)的“抗原结合片段”包括抗体的片段或衍生物，通常包括亲代抗体的抗原结合区或可变区(例如一个或多个CDR)的至少一个片段，其保持亲代抗体的至少一些结合特异性。抗体结合片段的实例包括但不限于Fab，Fab'，F(ab')2和Fv片段；双抗体；线性抗体；单链抗体分子，例如sc-Fv；由抗体片段形成的纳米抗体(nanobody)和多特异性抗体。当抗原的结合活性在摩尔浓度基础上表示时，结合片段或衍生物通常保持其抗原结合活性的至少10％。优选结合片段或衍生物保持亲代抗体的抗原结合亲和力的至少20％、50％、70％、80％、90％、95％或100％或更高。还预期抗体的抗原结合片段可包括不明显改变其生物活性的保守或非保守氨基酸取代(称为抗体的“保守变体”或“功能保守变体”)。术语“结合化合物”是指抗体及其结合片段两者。
术语“单链Fv”或“scFv”抗体是指包含抗体的VH和VL结构域的抗体片段，其中这些结构域存在于单条多肽链中。Fv多肽一般还包含VH和VL结构域之间的多肽接头，其使scFv能够形成用于抗原结合的所需结构。
术语“Fc”或“Fc区”或“Fc片段”是指由IgA、IgD及IgG的CH2和CH3结构域，或者由IgE及IgM的CH2、CH3和CH4结构域通过铰链区的组成的多肽。尽管Fc片段的分解是可变的，但是人IgG的重链Fc片段通常指从A231到其羧基末端这一段多肽。
术语“铰链区”是指抗体中位于CH1与CH2之间，富含脯氨酸，易伸展弯曲的多肽链。而公认的IgG铰链区为第216位至230位的氨基酸残基构成的多肽链。
术语“结构域抗体”是只含有重链可变区或轻链可变区的免疫功能性免疫球蛋白片段。在某些情况下，两个或更多个VH区与肽接头共价连接形成二价结构域抗体。二价结构域抗体的2个VH区可靶向相同或不同的抗原。
术语“二价抗体”包含2个抗原结合部位。在某些情况下，2个结合部位具有相同的抗原特异性。然而，二价抗体可以是双特异性的。
术语“双抗体”是指具有两个抗原结合部位的小抗体片段，所述片段包含在同一多肽链(VH-VL或VL-VH)中与轻链可变结构域(VL)连接的重链可变结构域(VH)。通过使用短得不允许在同一链的两个结构域之间配对的接头，迫使该结构域与另一链的互补结构域配对并产生两个抗原结合部位。
术语“嵌合抗体”是具有第一抗体的可变结构域和第二抗体的恒定结构域的抗体，其中第一抗体和第二抗体来自不同物种。通常，可变结构域获自啮齿动物等的抗体(“亲代抗体”)，而恒定结构域序列获自人抗体，使得与亲代啮齿动物抗体相比，所得嵌合抗体 在人受试者中诱导不良免疫应答的可能性较低。
术语“人源化抗体”是指含有来自人和非人(例如小鼠、大鼠)抗体的序列的抗体形式。一般而言，人源化抗体包含基本所有的至少一个、通常两个可变结构域，其中所有或基本所有的超变环相当于非人免疫球蛋白的超变环，而所有或基本所有的构架(FR)区是人免疫球蛋白序列的构架区。人源化抗体任选可包含至少一部分的人免疫球蛋白恒定区(Fc)。
术语“全人抗体”是指只包含人免疫球蛋白蛋白质序列的抗体。如在小鼠中、在小鼠细胞中或在来源于小鼠细胞的杂交瘤中产生，则全人抗体可含有鼠糖链。同样，“小鼠抗体”是指仅包含小鼠免疫球蛋白序列的抗体。或者，如果在大鼠中、在大鼠细胞中或在来源于大鼠细胞的杂交瘤中产生，则全人抗体可含有大鼠糖链。同样，“大鼠抗体”是指仅包含大鼠免疫球蛋白序列的抗体。
“同种型”抗体是指由重链恒定区基因提供的抗体种类(例如，IgM、IgE、IgG诸如IgGl、IgG2或IgG4)。同种型还包括这些种类之一的修饰形式，其中修饰已被产生来改变Fc功能，例如以增强或减弱效应子功能或对Fc受体的结合。
术语“表位”是指抗体所结合的抗原区域。表位可以由连续的氨基酸形成或者通过蛋白的三级折叠而并置的非连续氨基酸形成。
“亲和力”或“结合亲和力”指反映结合对子的成员之间相互作用的固有结合亲和力。分子X对其配偶物Y的亲和力可以通常由平衡解离常数(KD)代表，平衡解离常数是解离速率常数和结合速率常数(分别是kdis和kon)的比值。亲和力可以由本领域已知的常见方法测量。用于测量亲和力的一个具体方法是本文中的ForteBio动力学结合测定法。
术语“不结合”蛋白或细胞是指，不与蛋白或细胞结合，或者不以高亲和力与其结合，即结合蛋白或细胞的KD为1.0×10 -6M或更高，更优选1.0×10 -5M或更高，更优选1.0×10 -4M或更高、1.0×10 -3M或更高，更优选1.0×10 -2M或更高。
术语“高亲和性”对于IgG抗体而言，是指对于抗原的KD为1.0×10 -6M或更低，优选5.0×10 -8M或更低，更优选1.0×10 -8M或更低、5.0×10 -9M或更低，更优选1.0×10 -9M或更低。对于其他抗体亚型，“高亲和性”结合可能会变化。例如，IgM亚型的“高亲和性”结合是指KD为10 -6M或更低，优选10 -7M或更低，更优选10 -8M或更低。
术语“抗体依赖的细胞毒性”、“抗体依赖的细胞介导的细胞毒性”或“ADCC”是指细胞介导的免疫防御，其中免疫系统效应细胞主动地将细胞膜表面抗原与抗体，例如Claudin18.2抗体，结合的靶细胞例如癌细胞裂解。
术语“补体依赖的细胞毒性”或“CDC”是指IgG和IgM抗体的效应功能，当与表面抗原结合时引发典型的补体途径，包括形成膜攻击复合体以及靶细胞裂解。本发明的抗体，与Claudin 18.2结合时，引发对癌细胞的CDC。
术语“核酸”或“多核苷酸”是指脱氧核糖核酸(DNA)或核糖核酸(RNA)及其呈单链或双链形式的聚合物。除非明确地限制，否则术语包括具有与参照核酸相似的结合性质并且以与天然存在的核苷酸相似的方式被代谢的含有已知的天然核苷酸的类似物的核酸(参见，属于Kariko等人的美国专利No.8,278,036，其公开了尿苷被假尿苷替代的mRNA分子，合成所述mRNA分子的方法以及用于在体内递送治疗性蛋白的方法)。除非另有所指，否则特定核酸序列还隐含地包括其保守修饰的变体(例如，简并密码子取代)、等位基因、直系同源物、SNP和互补序列以及明确指出的序列。具体地，简并密码子取代可通过生成其中一个或多个选择的(或全部)密码子的第三位被混合碱基和/或脱氧肌苷残基取代的序列来实现(Batzer等人，Nucleic Acid Res.19:5081(1991)；Ohtsuka等人，J.Biol.Chem.260:2605-2608(1985)；和Rossolini等人，Mol.Cell.Probes 8:91-98(1994))。
“构建体”是指任何重组多核苷酸分子(诸如质粒、粘粒、病毒、自主复制多核苷酸分子、噬菌体或线性或环状单链或双链DNA或RNA多核苷酸分子)，衍生自任何来源，能够与基因组整合或自主复制，构成如下多核苷酸分子，其中已经以功能操作的方式连接(即，可操作地连接)一或多个多核苷酸分子。重组构建体通常会包含可操作地连接至转录起始调节序列的本发明的多核苷酸，这些序列会导引多核苷酸在宿主细胞中的转录。可使用异源及非异源(即，内源)启动子两者导引本发明的核酸的表达。
“载体”是指任何重组多核苷酸构建体，该构建体可用于转化的目的(即将异源DNA引入到宿主细胞中)。一种类型的载体为“质粒”，是指环状双链DNA环，可将额外DNA区段连接至该环中。另一类型的载体为病毒载体，其中可将额外DNA区段连接至病毒基因组中。某些载体能够在被引入到的宿主细胞中自主复制(例如，具有细菌复制起点的细菌载体及游离型哺乳动物载体)。在引入到宿主细胞中后，其他载体(例如，非游离型哺乳动物载体)整合至宿主细胞的基因组中，且因此与宿主基因组一起复制。此外，某些载体能够导引被操作性连接的基因的表达。本文将此类载体称为“表达载体”。
本文所用术语“表达载体”是指能够在转化、转染或转导至宿主细胞中时复制及表达目的基因的核酸分子。表达载体包含一或多个表型选择标记及复制起点，以确保维护载体及以在需要的情况下于宿主内提供扩增。
用于细胞或受体的“活化”、“刺激”和“处理”可具有相同含义，例如细胞或受体用配体活化、刺激或处理，除非上下文另外或明确规定。“配体”包括天然和合成配体，例如细胞因子、细胞因子变体、类似物、突变蛋白和来源于抗体的结合化合物。“配体”还包括小分子，例如细胞因子的肽模拟物和抗体的肽模拟物。“活化”可指通过内部机制以及外部或环境因素调节的细胞活化。“应答/反应”，例如细胞、组织、器官或生物体的应答，包括生化或生理行为(例如生物区室内的浓度、密度、粘附或迁移、基因表达速率 或分化状态)的改变，其中改变与活化、刺激或处理有关，或者与例如遗传编程等内部机制有关。
如本文中所用，术语任何疾病或病症的“治疗”或“医治”在一个实施方式中是指改善疾病或病症(即，减缓或阻止或减少疾病的进展或其临床症状的至少一个)。在另一个实施方式中，“治疗”或“医治”是指缓解或改善至少一个身体参数，包括可能不能被患者辨别出的那些物理参数。在另一个实施方式中，“治疗”或“医治”是指在身体上(例如，可辨别的症状的稳定)、生理上(例如，身体参数的稳定)或在这两方面调节疾病或病症。除非在本文中明确描述，否则用于评估疾病的治疗和/或预防的方法在本领域中通常是已知的。
“受试者”包括任何人或非人动物。术语“非人动物”包括所有脊椎动物，例如哺乳动物和非哺乳动物，诸如非人灵长类动物、绵羊、狗、猫、马、牛、鸡、两栖动物、爬行动物等。如本文中所用，术语“cyno”或“食蟹猴”是指食蟹猴。
“联合”一种或多种其它治疗剂的施用包括同时(共同)施用和任意次序的连续施用。
“治疗有效量”、“治疗有效剂量”和“有效量”是指本发明的CLDN-18.2抗体或其抗原结合片段当单独或与其它治疗药物组合给予细胞、组织或受试者时，有效预防或改善一种或多种疾病或病况的症状或该疾病或病况的发展的量。治疗有效剂量还指足以导致症状改善的抗体或其抗原结合片段的量，例如治疗、治愈、预防或改善相关医学病况或者提高这类病况的治疗、治愈、预防或改善的速度的量。当对个体施用单独给予的活性成分时，治疗有效剂量仅是指该成分。当组合施用时，治疗有效剂量是指引起治疗效果的活性成分的综合量，不论是组合、依次给予还是同时给予。治疗剂的有效量将导致诊断标准或参数提高至少10％；通常至少20％；优选至少约30％；更优选至少40％，最优选至少50％。
“癌症”和“癌性”指或描述哺乳动物中特征通常为细胞生长不受调控的生理疾患。此定义中包括良性和恶性癌症以及休眠肿瘤或微转移。癌症的例子包括但不限于癌，淋巴瘤，母细胞瘤，肉瘤，和白血病。此类癌症的更具体例子包括鳞状细胞癌，肺癌(包括小细胞肺癌，非小细胞肺癌，肺的腺癌，和肺的鳞癌)，腹膜癌，肝细胞癌，胃的癌或胃癌(包括胃肠癌)，胰腺癌，成胶质细胞瘤，宫颈癌，卵巢癌，肝癌，膀胱癌，肝瘤(hepatoma)，乳腺癌，结肠癌，结肠直肠癌，子宫内膜癌或子宫癌，唾液腺癌，肾癌或肾的癌，肝癌，前列腺癌，外阴癌，甲状腺癌，肝的癌，及各种类型的头和颈癌，以及B细胞淋巴瘤(包括低级/滤泡性非何杰金氏淋巴瘤(NHL)，小淋巴细胞性(SL)NHL，中级/滤泡性NHL，中级弥漫性NHL，高级成免疫细胞性NHL，高级成淋巴细胞性NHL，高级小无核裂细胞性NHL，贮积病(bulky disease)NHL，套细胞淋巴瘤，AIDS相关淋巴瘤，和瓦尔登斯特伦氏(Waldenstrom)巨球蛋白血症)，慢性淋巴细胞性白血病(CLL)，急性成淋巴细胞性白血 病(ALL)，毛细胞性白血病，慢性成髓细胞性白血病，和移植后淋巴增殖性病症(PTLD)，以及与瘢痣病(phakomatoses)，水肿(诸如与脑瘤有关的)和梅格斯氏(Meigs)综合征有关的异常血管增殖。
抗体
术语“抗CD3抗体”、“抗CD3”、“CD3抗体”或“结合CD3”是指能够以足够的亲合力结合CD3蛋白或其片段以致所述抗体可以用作靶向CD3的诊断剂和/或治疗剂。
术语“抗CLDN-18.2抗体”、“抗CLDN-18.2”、“CLDN-18.2抗体”或“结合CLDN-18.2”是指能够以足够的亲合力结合CLDN-18.2蛋白或其片段以致所述抗体可以用作靶向CLDN-18.2的诊断剂和/或治疗剂。
本发明人对多株抗人CD3抗体进行了深入的分析和比较，并对其中一株鼠源抗体SP34(参见Pessano,S.et al.,EMBO J 4(1985)337-344)进行了序列优化及人源化改造，得到抗CD3抗体JSCD3。
本发明所述人源化的抗CD3抗体，具有许多独特的性能和优势。这些新的特性使这一新的人源化抗体更适合于抗体药物的开发。具体而言，本发明优化的抗体序列，抗体蛋白的表达量高，热稳定性好，与人和食蟹猴CD3抗原都有更高的亲和力，且能有效激活T细胞下游信号通路；在用于双特异性抗体制备时，显示出更强的肿瘤杀伤活性。本发明的公开的这一人源化序列比其他公司的人源化序列有更多的优势和特点，用于制备抗肿瘤新药显示出更好的杀肿瘤活性，而且理化稳定性也大幅度提高，更适合于抗肿瘤药物的筛选和开发，这一创造性的成就构成了本发明的有益的效果和极高的医疗应用价值。
在一个方面，本发明涉及一种抗体或抗原结合片段，其包含结合CD3的抗原结合位点，所述结合CD3的抗原结合位点包含氨基酸序列如SEQ ID NO:1所示的重链可变区(VH)，和氨基酸序列如SEQ ID NO:2所示的轻链可变区(VL)。
在一些实施方案中，本发明所述抗体或抗原结合片段是单克隆抗体或其抗原结合片段。
在一些实施方案中，本发明所述抗体或抗原结合片段可以是嵌合的、CDR移植的、人源化的或全人的。
在一些实施方案中，本发明所述抗原结合片段为Fab、Fab'、F(ab')2、Fv、scFv或sdAb。
在一些实施方案中，本发明所述抗体包含如SEQ ID NO:3所示的重链氨基酸序列，和如SEQ ID NO:4所示的轻链氨基酸序列。
在一些实施方案中，本发明所述抗体是双特异性抗体或多特异性抗体。
在一些实施方案中，所述抗体还包含结合CLDN-18.2的另一抗原结合位点。
在又一个方面，本发明提供了一种双特异性抗体，其包含特异性结合CD3的第一抗原结合位点和特异性结合Claudin18.2(CLDN-18.2)的第二抗原结合位点。这样的抗体在本文 中可被称为例如“抗CD3/抗CLDN-18.2”或“抗CD3×CLDN-18.2”或“CD3×CLDN-18.2”双特异性分子，或其他类似术语。在一些实施方案中，所述结合CD3的第一抗原结合位点包含氨基酸序列分别如SEQ ID NO:16、SEQ ID NO:17和SEQ ID NO:18所示的HCDR1、HCDR2和HCDR3，和氨基酸序列分别如SEQ ID NO:19、SEQ ID NO:20和SEQ ID NO:21所示的LCDR1、LCDR2和LCDR3。在一些实施方式中，本发明所述结合CD3的第一抗原结合位点包含氨基酸序列如SEQ ID NO:1所示的重链可变区(VH)，和氨基酸序列如SEQ ID NO:2所示的轻链可变区(VL)；或包含氨基酸序列如SEQ ID NO:5所示的重链可变区(VH)，和氨基酸序列如SEQ ID NO:6所示的轻链可变区(VL)。在一些实施方式中，本发明所述结合CLDN-18.2的第二抗原结合位点包含氨基酸序列分别如SEQ ID NO:22、SEQ ID NO:23和SEQ ID NO:24所示的HCDR1、HCDR2和HCDR3，和氨基酸序列分别如SEQ ID NO:25、SEQ ID NO:26和SEQ ID NO:27所示的LCDR1、LCDR2和LCDR3。在一些实施方式中，本发明所述结合CLDN-18.2的第二抗原结合位点包含氨基酸序列如SEQ ID NO:9所示的重链可变区，和氨基酸序列如SEQ ID NO:10所示的轻链可变区。
在又一个方面，本发明提供了一种双特异性抗体，其具有两条重链和一条轻链，其中，第一重链自N端到C端具有VH-CH1-铰链区-Fc；第二重链自N端到C端具有铰链区-CH2-CH3-连接肽-VL-连接肽-VH；所述轻链自N端到C端具有VL-CL，其中所述第一重链的VH-CH1与所述轻链的VL-CL形成的抗原结合位点结合CLDN-18.2，所述第二重链的VH-连接肽-VL的抗原结合位点结合CD3。
在一些实施方式中，本发明所述第二重链中，所述连接肽的氨基酸序列各自独立为(GGGGS)n，其中，n为2、3、4、5或6。
在一些实施方式中，本发明所述第二重链的VH具有如SEQ ID NO:1所示的氨基酸序列，和VL具有如SEQ ID NO:2所示的氨基酸序列，所述连接肽的氨基酸序列各自独立为(GGGGS)n，其中，n为2、3、4、5或6。
在一些实施方式中，本发明所述第二重链具有如SEQ ID NO:7所示的氨基酸序列。
在一些实施方式中，本发明所述第一重链的VH具有如SEQ ID NO:9所示的氨基酸序列，所述轻链的VL具有如SEQ ID NO:10所示的氨基酸的序列。
在一些实施方式中，本发明所述第一重链具有如SEQ ID NO:11所示的氨基酸序列，所述轻链具有如SEQ ID NO:12所示的氨基酸的序列。
在一些实施方式中，本发明所述第一重链与所述第二重链之间通过铰链区的二硫键和CH3结构域的杵臼结构连接。
在一些实施方式中，本发明所述第一重链和第二重链的Fc为来自IgG的Fc，例如来自IgG1、IgG2、IgG3或IgG4的Fc，和/或所述轻链的CL为来自λ或κ轻链的CL。
本发明的所述抗体的可变区CDR的精确氨基酸序列边界可使用许多公知的方案的任何方案来确定，包括基于抗体的三维结构和CDR环的拓扑学的Chothia(Chothia等人.(1989)Nature 342:877-883；Al-Lazikani等人,“Standard conformations for the canonical structures of immunoglobulins”,Journal of Molecular Biology,273,927-948(1997))基于抗体序列可变性的Kabat(Kabat等人,Sequences of Proteins of Immunological Interest，第4版，U.S.Department of Health and Human Services,National Institutes of Health(1987))，AbM(University of Bath)，Contact(University College London)，国际ImMunoGeneTics database(IMGT)(1999 Nucleic Acids Research,27,209-212)，以及基于利用大量晶体结构的近邻传播聚类(affinity propagation clustering)的North CDR定义。本发明抗体的CDR可以由本领域的技术人员根据本领域的任何方案(例如不同的指派系统或组合)确定边界。
应该注意，基于不同的指派系统获得的同一抗体的可变区的CDR的边界可能有所差异。即不同指派系统下定义的同一抗体可变区的CDR序列有所不同。因此，在涉及用本发明定义的具体CDR序列限定抗体时，所述抗体的范围还涵盖了这样的抗体，其可变区序列包含所述的具体CDR序列，但是由于应用了不同的方案(例如不同的指派系统或组合)而导致其所声称的CDR边界与本发明所定义的具体CDR边界不同。
具有不同特异性(即，针对不同抗原的不同结合位点)的抗体具有不同的CDR。然而，尽管CDR在抗体与抗体之间是不同的，但是CDR内只有有限数量的氨基酸位置直接参与抗原结合。使用Kabat、Chothia、AbM、Contact和North方法中的至少两种，可以确定最小重叠区域，从而提供用于抗原结合的“最小结合单位”。最小结合单位可以是CDR的一个子部分。正如本领域技术人员明了，通过抗体的结构和蛋白折叠，可以确定CDR序列其余部分的残基。因此，本发明也考虑本文所给出的任何CDR的变体。例如，在一个CDR的变体中，最小结合单位的氨基酸残基可以保持不变，而根据Kabat或Chothia定义的其余CDR残基可以被保守氨基酸残基替代。
本发明所述的人源化抗体，可以使用本领域已知的方法将鼠源CDR区插入人种系框架区。参见Winter等人的美国专利No.5,225,539及Queen等人的美国专利No.5,530,101；5,585,089；5,693,762和6,180,370。
在一些实施方式中，氨基酸变化包括氨基酸缺失、插入或置换。在一些实施方式中，本发明的抗CLDN-18.2抗体或其抗原结合片段包括具有已通过氨基酸缺失、插入或置换突变的，但仍与上述抗体(特别地在上述序列中描绘的CDR区中)有至少约90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列的那些抗体。在一些实施方式中，本发明的抗体与具体序列中描绘的CDR区相比较时，在CDR区中已通过氨基酸缺失、插入或置换的氨基酸突变不超过1、2、3、4或5个。
在一些实施方式中，编码本发明抗体的多核苷酸包括已通过核苷酸缺失、插入或置换突变的，但仍然与上文中所述的序列中描绘的CDR对应编码区具有至少约60、70、80、90、95或100％同一性的多核苷酸。
在一些实施方式中，可在本文中所提供抗体的Fc区中引入一个或多个氨基酸修饰，以此产生Fc区变体。Fc区变体可包含在一或多个氨基酸位置处包含氨基酸修饰(例如置换)的人Fc区序列(例如人IgG1、IgG2、IgG3或IgG4Fc区)。
在一些实施方式中，可能需要产生经半胱氨酸工程改造的抗体，例如“硫代MAb”，其中抗体的一或多个残基经半胱氨酸残基置换。
在一些实施方式中，本文中所提供的抗体可进一步经修饰为含有本领域中已知且轻易获得的其他非蛋白质部分。适合抗体衍生作用的部分包括，但不限于，水溶性聚合物。水溶性聚合物的非限制性实例包括，但不限于，聚乙二醇(PEG)、乙二醇/丙二醇共聚物、羧甲基纤维素、葡聚糖、聚乙烯醇、聚乙烯吡咯烷酮、聚-1，3-二烷、聚-1，3，6-三烷、乙烯/马来酸酐共聚物、聚氨基酸(均聚物或无规共聚物)、及葡聚糖或聚(n-乙烯基吡咯烷酮)聚乙二醇、丙二醇均聚物、聚环氧丙烷/氧化乙烯共聚物、聚氧乙基化多元醇(例如甘油)、聚乙烯醇、及其混合物。
在一些实施方案中，双特异性抗体的第一抗原结合结构域和第二抗原结合结构域可以直接或间接彼此连接。在一些实施方案中，双特异性抗体的第一抗原结合结构域和第
二抗原结合结构域可以通过接头彼此连接。在一些实施方案中，接头是肽接头。
在一些实施方案中，双特异性抗体的第一抗原结合结构域和第二抗原结合结构域可以彼此直接或间接连接以形成双特异性抗原结合分子(即双特异性scFv)，其进一步结合至Fc区。或者，第一抗原结合结构域和第二抗原结合结构域可以各自连接到单独的Fc区。本发明的双特异性抗原结合分子可包含两个Fc区，每个Fc区分别是单独的抗体重链的一部分。第一和第二Fc区可以具有相同的序列，除了在CH3结构域中具有旨在促进或易于纯化异二聚体(即双特异性)分子的突变之外。
本发明的双特异性抗体的Fc区可以是人Fc区。本发明的双特异性抗体的Fc区可以是任何同种型，包括但不限于IgG1，IgG2，IgG3或IgG4。在一些实施方案中，所述第一和所述第二抗体的Fc区都是IgG1同种型。在一些实施方案中，所述第一和所述第二抗体的Fc区都是IgG4同种型。在一些实施方案中，所述抗体的Fc区之一是IgG1同种型而另一个是IgG4同种型。在后一个实施方案中，所得到的双特异性抗体包含IgG1的Fc区和IgG4的Fc区，因此就效应子功能的激活而言可具有令人感兴趣的中间特性。
术语“杵臼结构”，是将抗体Fc的CH3的疏水氨基酸进行突变。将一条链CH3的侧链氨基酸突变形成分子比较大的疏水氨基酸(杵，knob)，以加强疏水作用力；另一个CH3 侧链氨基酸突变形成小的氨基酸(臼，hole)，用于减少空间位阻；突变后带有Knob的和带有Hole的CH3以疏水作用形式形成杵臼结构(Knob into Hole，KiH)，有利于重链异源二聚体的形成；杵的CH3由于突变后氨基酸的空间位阻大，而臼突变后由于疏水作用力变弱，这两者都不利于同源二聚体的形成；KiH突变主要是发生在CH3结构域的空间结构的内部疏水氨基酸，突变后暴露在外面的氨基酸几乎没有变化，所以不影响Fc的效应功能和引起的免疫原性。
在本发明的双特异性抗体的背景下，与指定的Fc区嵌合形式相比，Fc区可以包含一个或多个氨基酸改变(例如，插入，缺失或取代)，而不改变所需的功能性。例如，本发明包括在Fc区中包含一个或多个修饰的双特异性抗原结合分子，其导致在Fc和FcRn之间具有修饰的结合相互作用(例如增强或减弱)的修饰的Fc区。此类Fc修饰的非限制性实例包括例如人IgG4Fc区的氨基酸序列的位置228处的丝氨酸(“S”)至脯氨酸(“P”)的突变。
双特异性抗体形式
本发明提供双特异性CD3×CLDN-18.2抗体，其有效地促进T细胞介导的对表达CLDN-18.2的肿瘤细胞的杀灭。取决于具体用途的合意的功能性质，具体的抗原结合区可以选自本发明所提供的抗体或抗原结合区的组，或与本发明所提供的抗体或抗原结合区共享例如表位或交叉阻断区的那些抗体或抗原结合区。本领域已知双特异性抗体的许多不同的形式和用途，且其最近由Chames和Baty(2009)Curr Opin Drug Disc Dev12:276所综述。
在一些实施方案中，本发明包含结合CD3的抗原结合位点和结合Claudin18.2(CLDN-18.2)的抗原结合位点的抗体可以是双特异性抗体或多特异性抗体，优选为双特异性抗体，其中双特异性抗体可以采用广泛多种构形，例如为含Fc片段的双特异性抗体(IgG-like双特异性抗体)与不含Fc片段的双特异性抗体(non-IgG-like双特异性抗体)。IgG-like双特异性抗体主要包括Triomabs/quadroma、DVD-Ig(dual variable domain Ig)、CrossMAb、Two-in-one IgG、scFv2-Fc。non-IgG-like双特异性抗体主要包括TandAb(tandem diabody)、scFv-HSA-scFv、BiTE(bi-specific T-cell engager)、DART(dual affinity retargeting)、Nanobody。本发明的例示性双特异性抗体分子具有两条重链和一条轻链，其中，第一重链自N端到C端具有VH-CH1-铰链区-Fc；第二重链自N端到C端具有铰链区-Fc-连接肽-VL-连接肽-VH；所述轻链自N端到C端具有VL-CL；其中，所述第一重链的VH-CH1与所述轻链的VL-CL形成抗原结合位点结合CLDN-18.2，所述第二重链的VH-连接肽-VL的抗原结合位点结合CD3；该双特异性抗体形式具有许多独特的性能和优势，例如优异的稳定性，毒性低，在用于多个靶点的双特异性抗体制备时都显示出优异的肿瘤杀伤活性。
本发明的连接肽可以是含有G(甘氨酸)和S(丝氨酸)或由G和S组成的柔性多肽， 其长度可为2-30个氨基酸残基。示例性的连接肽包括但不限于(GS)n、(GGS)n、(GGGS)n和(GGGGS)n等，n可为1-6的整数。
在一个实施方案中，本发明的双特异性抗体是双抗体、交叉抗体(cross-body)、或如本发明中所描述的通过那些受控的Fab臂交换所获得的双特异性抗体。
不同种类的双特异性抗体的实例包括但不限于：
1)带有迫使异二聚化的互补性CH3域的IgG样分子；
2)重组IgG样双靶向性分子，其中所述分子的两面每个都包含至少两种不同抗体的Fab片段或Fab片段的部分；
3)IgG融合分子，其中将全长IgG抗体融合至另外的Fab片段或Fab片段的部分；
4)Fc融合分子，其中将单链Fv分子或稳定化(stabilized)的双抗体融合至重链恒定域、Fc区或其部分；
5)Fab融合分子，其中将不同Fab片段融合至一起；
6)基于scFv和双抗体和重链抗体(例如，域抗体、纳米抗体)的抗体，其中将不同的单链Fv分子或不同双抗体或不同重链抗体(例如，域抗体、纳米抗体)彼此融合或融合至另一个蛋白质或载体分子。
带有互补性CH3域分子的IgG样分子的实例包括但不限于Triomab/Quadroma(Trion Pharma/Fresenius Biotech)、Knobs-into-Holes(Genentech)、CrossMAbs(Roche)和静电配对的(the electrostatically-matched)(Amgen)、LUZ-Y(Genentech)、Strand Exchange Engineered Domain body(SEEDbody)(EMD Serono)、Biclonic(Merus)、和DuoBody(Genmab A/S)。
重组IgG样双靶向性分子的实例包括但不限于Dual Targeting(DT)-Ig(GSK/Domantis)、Two-in-one Antibody(Genentech)、Cross-linked Mabs(Karmanos Cancer Center)、mAb2(F-Star)、和CovX-body(CovX/Pfizer)。
IgG融合分子的实例包括但不限于Dual Variable Domain(DVD)-Ig(Abbott)、IgG样Bispecific(ImClone/Eli Lilly)、Ts2Ab(MedImmune/AZ)和BsAb(Zymogenetics)、HERCULES(Biogen Idec)、和TvAb(Roche)。
Fc融合分子的实例包括但不限于scFv/Fc Fusions(Academic Institution)、SCORPION(Emergent BioSolutions/Trubion,Zymogenetics/BMS)、DualAffinity Retargeting Technology(Fc-DART)(MacroGenics)、和Dual(scFv)2-Fab(抗体药物国家工程研究中心，中国)。
Fab融合双特异性抗体的实例包括但不限于F(ab)2(Medarex/AMGEN)、Dual-Action or Bis-Fab(Genentech)、Dock-and-Lock(DNL)(ImmunoMedics)、Bivalent Bispecific(Biotecnol)、和Fab-Fv(UCB-Celltech)。
基于scFv、双抗体和域抗体的实例包括但不限于Bispecific T Cell Engager(BiTE)(Micromet,Tandem Diabody(Tandab)(Affimed)、Dual Affinity Retargeting Technology(DART)(MacroGenics)、Single-chain Diabody(Academic)、TCR-like Antibodies(AIT,ReceptorLogics)、Human SerumAlbumin scFv Fusion(Merrimack)和COMBODY(Epigen Biotech)、双靶向性纳米抗体(dual targeting nanobodies)(Ablynx)、双靶向性仅重链域抗体(dualtargeting heavy chain only domain antibodies)。
抗体表达
在又一个方面，本发明提供了一种多核苷酸，其编码如本文所述的与CD3结合抗体或其抗原结合片段，或编码本文所述的抗CD3×CLDN-18.2双特异性抗体。所述多核苷酸可以包含编码抗体的轻链可变区和/或重链可变区的氨基酸序列的多核苷酸，或包含编码抗体的轻链和/或重链的氨基酸序列的多核苷酸。示例性的编码本文所述第一重链的多核苷酸序列如SEQ ID NO:13所示，编码本文所述第二重链的多核苷酸序列如SEQ ID NO:8所示，编码本文双特异性抗体中所述轻链的多核苷酸序列如SEQ ID NO:14所示。
在又一个方面，本发明提供了一种表达载体，其包含如本文所述的多核苷酸，优选地，所述载体为真核表达载体。在一些实施方式中，如本文所述的多核苷酸包含在一个或多个表达载体中。
在又一个方面，本发明提供了一种宿主细胞，其包含如本文所述的多核苷酸或如本文所述的表达载体，优选地，所述宿主细胞是真核细胞，更优选哺乳动物细胞。
在又一个方面，本发明提供了一种用于制备如本文所述的抗CD3×CLDN-18.2双特异性抗体的方法，所述方法包括在适合于所述抗体表达的条件下在本文所述的宿主细胞中表达所述抗体，并从所述宿主细胞回收所表达的抗体。
本发明提供用于表达本发明的重组抗体的哺乳动物宿主细胞，包括可获自美国典型培养物保藏中心(ATCC)的许多永生化细胞系。这些尤其包括中国仓鼠卵巢(CHO)细胞、NS0、SP2/0细胞、HeLa细胞、幼仓鼠肾(BHK)细胞、猴肾细胞(COS)、人肝细胞癌细胞、A549细胞、293T细胞和许多其它细胞系。哺乳动物宿主细胞包括人、小鼠、大鼠、狗、猴、猪、山羊、牛、马和仓鼠细胞。通过测定哪种细胞系具有高表达水平来选择特别优选的细胞系。
在一个实施方式中，本发明提供制备本发明抗CD3×CLDN-18.2双特异性抗体的方法，其中所述方法包括，将表达载体导入哺乳动物宿主细胞中，培养宿主细胞足够的一段时间，以允许抗体在宿主细胞中表达或者更优选使抗体分泌到宿主细胞生长的培养基中，以产生抗体。可采用标准蛋白质纯化方法从培养基中回收抗体。
很可能由不同细胞系表达或在转基因动物中表达的抗体彼此具有不同的糖基化。然而， 由本文提供的核酸分子编码的或包含本文提供的氨基酸序列的所有抗体是本发明的组成部分，而不论抗体的糖基化如何。同样，在某些实施方式中，非岩藻糖基化抗体是有利的，因为它们通常在体外和体内具有比其岩藻糖基化对应物更强力的功效，并且不可能是免疫原性的，因为它们的糖结构是天然人血清IgG的正常组分。
药物组合物和药物制剂
在又一个方面，本发明提供了一种药物组合物，其包含如本文所述的抗CD3抗体或其抗原结合片段、本文所述的抗CD3×CLDN-18.2双特异性抗体、本文所述的多核苷酸、本文所述的表达载体或本文所述的宿主细胞，和药学上可接受的载体或赋形剂。
应理解，本发明提供的抗CD3抗体或抗CD3×CLDN-18.2抗体或其药物组合物可以整合制剂中合适的运载体、赋形剂和其他试剂以联合给药，从而提供改善的转移、递送、耐受等。
术语“药物组合物”指这样的制剂，其允许包含在其中的活性成分的生物学活性有效的形式存在，并且不包含对施用所述制剂的受试者具有不可接受的毒性的另外的成分。
可以通过将具有所需纯度的本发明的抗CD3×CLDN-18.2双特异性抗体与一种或多种任选的药用辅料(Remington's Pharmaceutical Sciences，第16版，Osol,A.编辑(1980))混合来制备包含本文所述的抗CD3×CLDN-18.2双特异性抗体的药物制剂，优选地以水溶液或冻干制剂的形式。
本发明的药物组合物或制剂还可以包含一种或多种其它活性成分，所述活性成分是被治疗的特定适应证所需的，优选具有不会不利地影响彼此的互补活性的那些活性成分。在一些实施方式中，其它的活性成分为化疗剂、免疫检查点抑制剂、生长抑制剂、抗生素或已知的各种抗肿瘤或抗癌剂，所述活性成分以对于目的用途有效的量合适地组合存在。
在一些实施方式中，本发明的药物组合物还包含编码抗CD3×CLDN-18.2双特异性抗体的多核苷酸的组合物。
在又一个方面，本发明提供了一种药物组合，其包含如本文所述的抗CD3抗体或其抗原结合片段、本文所述的抗CD3×CLDN-18.2双特异性抗体、本文所述的多核苷酸、本文所述的表达载体、本文所述的宿主细胞、或本文所述的药物组合物，以及一种或多种另外的治疗剂。
在又一个方面，本发明提供了一种试剂盒，其包括如本文所述的抗体或其抗原结合片段、本文所述的多核苷酸、本文所述的表达载体、本文所述的宿主细胞、或本文所述的药物组合物。
医药用途
在又一个方面，本发明提供了如本文所述的抗CD3抗体或其抗原结合片段、本文所述的抗CD3×CLDN-18.2双特异性抗体、本文所述的多核苷酸、本文所述的表达载体、本文所述的宿主细胞、或本文所述的药物组合物在制备用于治疗癌症的药物中的用途。
在又一个方面，本发明提供了如本文所述的抗CD3抗体或其抗原结合片段、本文所述的抗CD3×CLDN-18.2双特异性抗体、本文所述的多核苷酸、本文所述的表达载体、本文所述的宿主细胞、或本文所述的药物组合物，其用于治疗癌症。
在又一个方面，本发明提供了一种治疗癌症的方法，其包括向有需要的受试者施用如本文所述的抗CD3抗体或其抗原结合片段、本文所述的抗CD3×CLDN-18.2双特异性抗体、本文所述的多核苷酸、本文所述的表达载体、本文所述的宿主细胞、或本文所述的药物组合物。
优选地，所述癌症与CLDN18相关；更具体而言，所述癌症高表达Claudin 18.2。因此，在优选的实施方案中，所述癌症为其癌细胞高表达Claudin 18.2的癌症。本文所述的“高表达”意指癌细胞或癌组织中的Claudin 18.2表达量高于正常细胞或正常组织中的Claudin 18.2表达量。可采用常规的方法测试正常人群中正常细胞或组织中的Claudin 18.2表达量，以此为基准判定对象相对应的细胞或组织中Claudin 18.2是否高表达。
在一些实施方式中，所述癌症选自胃癌、食道癌、胃食管癌、胰腺癌、胆管癌、肺癌、卵巢癌、结肠癌、肝癌、头颈癌、胆囊癌、肠癌和膀胱癌药物。
在一些实施方式中，本发明给药方式包括但不限于口服、静脉内、皮下、肌内、动脉内、关节内(例如在关节炎关节中)、通过吸入、气雾剂递送或肿瘤内给予等。
在一些实施方式中，本发明提供了向受试者联合施用治疗有效量的一种或多种疗法(例如治疗方式和/或其它治疗剂)。在一些实施方式中，所述疗法包括手术治疗和/或放射疗法。
在一些实施方式中，本发明提供的方法或用途还包括向个体施用一种或多种疗法(例如治疗方式和/或其它治疗剂)。可以单独或与疗法中的其它治疗剂组合使用本发明的抗体。例如，可以与至少一种另外的治疗剂共施用。例如，PD-1抗体、PD-L1抗体、LAG-3抗体和/或CTLA-4抗体。
在一个实施方式中，本发明的癌症疾病治疗方法还包括施用CD3×CLDN-18.2表达稳定或增加剂。CD3×CLDN-18.2的表达优选在癌细胞的细胞表面。CD3×CLDN-18.2表达稳定或增加剂可以是奥沙利铂和/或5-FU。
用于诊断和检测的方法
在又一个方面，本发明提供了一种使用如本文所述的抗体或其抗原结合片段检测CD3 或CLDN-18.2在样品中的存在的方法。术语“检测”用于本文中时，包括定量或定性检测。在一些实施方式中，所述样品是生物样品。在某些实施方式中，生物样品是血、血清或生物来源的其他液体样品。在某些实施方式中，生物样品包含细胞或组织。所述方法包括使本发明的抗体或其抗原结合片段，尤其是本发明的双特异性抗体，与样品接触，并检测所述抗体或其抗原结合片段或所述双特异性抗体与CD3或CLDN-18.2形成的结合物的存在和/或该结合物的量。
本发明包括所叙述特定实施方式的所有组合。本发明的进一步实施方式及可应用性的完整范畴将自下文所提供的详细描述变得显而易见。然而，应理解，尽管详细描述及特定实施例指示本发明的优选实施方式，但仅以说明的方式提供这些描述及实施例，因为本发明的精神及范畴内的各种改变及修改将自此详细描述对熟悉此项技术者变得显而易见。出于所有目的，包括引文在内的本文所引用的所有公开物、专利及专利申请将以引用的方式全部并入本文。
实施例
提供以下实施例以证明并进一步解释本发明的一些优选的实施方式和方面，不应被解释为限制其范围。
实施例1、抗CD3抗体或其抗原结合片段
本发明对多株抗人CD3抗体进行了分析，并对其中一株鼠源抗体SP34优化序列并进行人源化改造，得到人源化抗CD3抗体JSCD3及其抗原结合片段。人源化改造具体步骤如下：A、将各候选抗体的基因序列与人胚胎系抗体基因序列进行比对，找出同源性高的序列；B、分析考察HLA-DR亲和性，选出亲和力低的人胚胎系框架区序列；C、利用计算机模拟技术，应用分子对接分析可变区及其周边的框架区氨基酸序列，考察其空间立体结合方式。通过计算静电力，范德华力，亲疏水性和熵值，分析各候选的抗体的基因序列中可与CD3作用以及维护空间架构区的关键氨基酸个体，将其嫁接到经选择的人胚胎系基因框架区中，并在此基础上标示出必须保留的框架区氨基酸位点，合成人源化抗体或其抗原结合片段。
SP34是一株抗人CD3的鼠源单克隆抗体，其重链氨基酸序列为SEQ ID NO:28，轻链氨基酸序列为SEQ ID NO:29。
人源化抗CD3抗体的VH和VL氨基酸序列如下(黑体和下划线部分标出了HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3)：
JSCD3VH:
Figure PCTCN2021113033-appb-000001
HCDR1:TYAMN(SEQ ID NO:16)；
HCDR2:RIRSKYNNYATYYADSVKD(SEQ ID NO:17)；
HCDR3:HGNFGNSYVSWFAY(SEQ ID NO:18)。
JSCD3VL:
Figure PCTCN2021113033-appb-000002
LCDR1:RSSTGAVTTSNYAN(SEQ ID NO:19)；
LCDR2:GTNKRAP(SEQ ID NO:20)；
LCDR3:ALWYSNLWV(SEQ ID NO:21)。
抗CD3抗体的抗原结合片段(Fab)的HC和LC的氨基酸序列如下：
HC:SEQ ID NO:15：
Figure PCTCN2021113033-appb-000003
LC:SEQ ID NO:4。
抗CD3单抗(又称为JS202-JSCD3)的全长氨基酸序列(HC和LC)如下：JSCD3HC：SEQ ID NO:3：
Figure PCTCN2021113033-appb-000004
JSCD3LC：SEQ ID NO:4：
Figure PCTCN2021113033-appb-000005
实施例2、双特异抗体分子表达载体的构建
委托金斯瑞生物科技有限公司合成编码JS012-2B19LC-1的基因，通过BSPQI酶切并连接至HXT2载体，得到第一个表达载体HXT2-JS012-2B19-LC-1。将质粒HXT1S-JS012-2B19-HC-7-KDL用HindIII和NheI酶切回收获取基因片段JS012-2B19-HC-7，使用HindIII和NheI酶切质粒HXT4S-JS012-1H17-HC-mut-h并回收得到载体HXT4s-mut h，使用T4DNA连接酶连接，得到第二个表达载体HXT4S-JS012-2B19-HC-7-mut-h。委托金斯瑞生物科技有限公司合成编码JSCD3scFv的基因，通过BSPQI酶切并连接至HX4-FC-mut-b-BspQI载体，得到第三个表达载体HX4-FC-mut-b-JSCD3scFv。
实施例3、双特异性抗体分子的表达与纯化
3.1双特异性抗体分子的表达
将实施例2中构建的3个表达载体用无内毒素的试剂盒进行质粒大提，供后面哺乳动物细胞表达使用，具体操作按照常规的普通分子生物学方法进行。将CHO-K1细胞(经过基因组水平的改造使细胞适用于瞬转表达，君实生物)使用CD CHO培养基(Gibco)，在36.5℃，120rmp，7％CO 2条件下培养，当细胞密度达到(2-6)×10 6/mL时，用CD CHO培养基进行传代扩增。转染前一天，将CHO-K1细胞密度稀释至(1.5-2.0)×10 6/mL，放置摇床(培养条件为36.5℃，120rmp，7％CO 2)培养，次日当细胞密度达到约3.5×10 6/mL时进行转染。转染时，先向反应器中加入十分之一转染体积的CD CHO培养基，依次加入1-2ug/mL转染体积的上述质粒，3-14ug/mL的PEI(聚乙烯亚胺)，混匀后室温孵育20-30分钟，然后将上述混合物缓慢加入到预先处理好的上述CHO-K1细胞中，形成转染混合物，边加边混匀。将转染混合物放入摇床培养，培养条件为36.5℃，120rmp，7％CO 2。培养周期为转染后6-10天，每两天补料一次。
3.2双特异性抗体分子的纯化
上述转染混合物培养结束后，1000rmp离心10min弃去沉淀，然后12000rmp离心30min收集细胞上清并进行无菌过滤。第一步，用AKTA Avant纯化仪进行纯化，先用0.1M NaOH对装填mabselect sure LX柱子进行消毒15-20min，然后用PBS缓冲液平衡3-5个柱体积后上样，上样完成后，用pH 5.5醋酸钠缓冲液进行淋洗，最后用pH 3.6的醋酸- 醋酸钠缓冲液洗脱目的蛋白；第二步，用Eshmuno CPX进行精纯，平衡液为pH5.5，50mM醋酸-醋酸钠体系，洗脱液为pH5.5，50mM醋酸-醋酸钠+1M NaCl缓冲体系，采用线性洗脱方式，收集目的蛋白，经SEC-HPLC测定，单体纯度可达到95％以上，即为双特异性抗体JS009。
双特异性抗体JS009中，编码抗CD3全长抗体的氨基酸序列(即第二重链)如SEQ ID NO:7所示，其编码序列如SEQ ID NO:8所示；抗CLDN-18.2的抗体的重链氨基酸序列(即第一重链)如SEQ ID NO:11所示，其编码序列如SEQ ID NO:13所示；抗CLDN-18.2的抗体的轻链氨基酸序列如SEQ ID NO:12所示，其编码序列如SEQ ID NO:14所示；抗CLDN-18.2的抗体的重链可变区的氨基酸序列如SEQ ID NO:9所示，轻链可变区的氨基酸序列如SEQ ID NO:10所示，抗CD3抗体的重链可变区的氨基酸序列如SEQ ID NO:5所示，轻链可变区的氨基酸序列如SEQ ID NO:6所示；抗CLDN-18.2的抗体的重链可变区的HCDR1、HCDR2和HCDR3的氨基酸序列分别如SEQ ID NO:22、23和24所示，轻链可变区的LCDR1、LCDR2和LCDR3的氨基酸序列分别如SEQ ID NO:25、26和27所示；抗CD3的抗体的重链可变区的HCDR1、HCDR2和HCDR3的氨基酸序列分别如SEQ ID NO:16、17和18所示，轻链可变区的LCDR1、LCDR2和LCDR3的氨基酸序列分别如SEQ ID NO:19、20和21所示。
SEQ ID NO:7(黑体字分别表示VL和VH，下划线标出LCDR1-LCDR3和HCDR1-HCDR3)
Figure PCTCN2021113033-appb-000006
SEQ ID NO:11(黑体字表示VH，下划线标出HCDR1-HCDR3)
Figure PCTCN2021113033-appb-000007
Figure PCTCN2021113033-appb-000008
SEQ ID NO:12(黑体字表示VL，下划线标出LCDR1-LCDR3)
Figure PCTCN2021113033-appb-000009
双特异性抗体分子结构示意图如图1所示。
实施例4、双特异性抗体的稳定性
4.1双特异性抗体的热稳定性
1、测试目的
在pH6.0的缓冲体系(20mM柠檬酸-柠檬酸钠/50mM氯化钠/150mM甘露醇)中，利用nanoDSF(差示扫描荧光技术)检测双特异性抗体JS009的热稳定性。
2、测试过程及结果
将双抗JS009样品置换到上述的缓冲液中，控制样品浓度在10mg/ml左右，利用DSF进行检测。结果见表1，在上述缓冲液中，双特异性抗体JS009表现了良好的热稳定性，Tm1和Tm2分别为63.3℃和79.0℃。
表1：双特异性抗体的热稳定性
样品 Tm1(℃) Tm2(℃)
JS009 63.3 79.0
注：Tm:热转化温度。
4.2双特异性抗体的高温稳定性
1、测试目的
检测双特异性抗体JS009高温条件下稳定性。
2、测试过程及结果
将双特异性抗体JS009置换到pH6.0的缓冲体系(20mM柠檬酸-柠檬酸钠/50mM氯化钠/150mM甘露醇)中，控制样品浓度在10mg/ml左右，分装一定体积(200μl/管)的双抗样品放置于40℃恒温箱，考察0W、1W、2W和4W稳定性，根据时间点检测样品纯度和 生物学活性。检测结果显示双特异性抗体JS009具有良好的稳定性，可耐受高温条件，具体见表2。
表2：双特异性抗体的高温稳定性
Figure PCTCN2021113033-appb-000010
实施例5、抗CD3单抗和双特异性抗体与人和食蟹猴的CD3结合
5.1抗CD3单体和双特异性抗体与人CD3的结合
将内源性表达人CD3的细胞(Jurkat细胞，中国科学院典型培养物保藏委员会细胞库)分别与梯度稀释成系列浓度的CD3单抗(JS202-JSCD3)、双特异性抗体(JS009)及阴性对照(抗KLH单抗，Anti KLH IgG4)在4℃孵育30min，洗去残留抗体后与荧光二抗(PE-抗人IgG4)孵育，最后用BD Canto Ⅱ流式细胞仪检测荧光强度，荧光信号越强，抗体与靶点亲和力越高。通过GraphPad拟合抗体剂量依赖性的结合曲线(图2)。
如图2，JSCD3及JS009与人CD3表达细胞均能有效结合，并且，JS009与人CD3表达细胞的结合能力有所减低。
5.2抗CD3单抗和双特异性抗体与食蟹猴CD3的结合
将食蟹猴外周血单核细胞(PBMC，奥赛尔斯生物技术(上海)有限公司)分别与梯度稀释成系列浓度的人源化CD3单抗(JS202-JSCD3)、SP34、双特异性抗体(JS009)及阴性对照(抗KLH单抗，Anti KLH IgG4)在4℃孵育30min，洗去残留抗体后与荧光二抗(PE-抗人IgG4)孵育，最后用BD Canto Ⅱ流式细胞仪检测荧光强度，荧光信号越强，抗体与靶点亲和力越高。通过GraphPad拟合抗体剂量依赖性的结合曲线(图3)。
如图3，SP34、JSCD3及JS009与食蟹猴CD3表达细胞均能有效结合，并且，JS009与食蟹猴CD3表达细胞的结合能力有所减低。
实施例6、抗CD3单抗和双特异性抗体的对T细胞活性的影响
抗CD3×Claudin18.2双抗，能够与靶细胞上的Claudin18.2结合，进一步结合表达CD3的T细胞，从而激活T细胞下游信号通路。我们利用内源性表达人CD3的NFAT通路荧光素酶系统，以过表达Claudin18.2的NUGC4细胞为靶细胞，通过报告基因系统检测双抗活性。
将不同稀释浓度的JS202-JSCD3单抗、SP34、JS009双抗与靶细胞NUGC4-CLDN-18.2细胞(君实生物)以及Jurkat NFAT效应细胞(君实生物)共孵育，在加入底物one-glo后用酶标仪检测信号，最后用GraphPad分析数据，比较剂量依赖性的细胞活性(图4)。
如图4所示，SP34、JS202-JSCD3单抗与JS009双抗均能有效激活T细胞下游信号通路，与单抗相比，在靶细胞存在条件下，JS009双抗活性优于单抗活性。
实施例7、双特异性抗体对小鼠肿瘤生长的抑制
取25只6-8周龄雌性B-hCD3e人源化小鼠(百奥赛图江苏基因生物技术有限公司)，将MC38 Claudin18.2细胞以1×10 6个/0.1mL浓度接种小鼠的右后背皮下，待肿瘤生长到约96mm 3时按肿瘤体积随机分组，每组6只，共2组，分别为：G1生理盐水(生理盐水)阴性对照组和G2 JS009(10mg/kg)组。通过腹腔注射给药，每周给药2次，给药剂量10mg/kg，给药浓度1mg/ml，连续给药5次，末次给药3天后结束实验。每周测量肿瘤体积及体重2次，记录小鼠体重和肿瘤体积。实验结束时，动物安乐死，计算相对肿瘤抑制率TGI％＝(1-(Ti-T0)/(Vi-V0))×100％。Ti：治疗组在给药第i天的肿瘤体积均值；T0：治疗组在给药第0天的肿瘤体积均值；Vi：阴性对照组在给药第i天的肿瘤体积均值；V0：阴性对照组在给药第0天的肿瘤体积均值。
如图5所示，在动物接种肿瘤细胞后第21天，生理盐水组平均肿瘤体积为2013±199mm 3，JS009组平均肿瘤体积为1019±62mm 3，与生理盐水组相比，相对肿瘤抑制率为51.9％，p<0.001。表明双特异性抗体JS009能够体内抑制B-hCD3e人源化小鼠MC38 Claudin18.2细胞皮下移植瘤的生长。
实施例8、抗CD3单体JSCD3与人CD3的结合
先使用PBS(Hyclone，SH30256.01)稀释重组人CD3E(C-6His)(Novoprotein，C578)及重组人CD3D&E杂二聚体蛋白(Sino Biological，CT026-H0323H)至1.0μg/mL，以100μl/孔加入酶标板，37℃恒温培养箱中静置包被90min；洗板；加入200μl/孔2％BSA(Sigma，V900933)至板内，置37℃恒温培养箱内孵育90min；洗板；将样品JSCD3、SP34及阴性对照(抗KLH单抗，anti-KLH hIgG4)用稀释液(2％BSA)稀释至1000ng/mL，每次稀释倍数不高于10倍。然后，在样品稀释板上以3倍梯度稀释至0.017ng/mL及1个空白点，所有样品以100μl/孔加入酶标板中，置37℃恒温培养箱内孵育60min；洗板；将小鼠抗人IgG4 Fc-HRP(Southern Biotech,9200-05)用2％BSA稀释5000倍，以100μl/孔加入酶标板，置37℃恒温培养箱内孵育60min；洗板；加入0.1mg/mL TMB(Sigma,T2885)100μl/孔，37℃避光显色15min；最后2M HCl终止100μl/孔，避免气泡，10min内完成酶标仪读数(波长：450/620 nm)。
结果如图6和7所示，JSCD3和SP34与CD3E及CD3D&E均有特异性结合，结合活性基本相当。

Claims (20)

  1. 一种抗体或其抗原结合片段，其包含结合CD3的抗原结合位点，其中，所述结合CD3的抗原结合位点包含氨基酸序列分别如SEQ ID NO:16、SEQ ID NO:17和SEQ ID NO:18所示的HCDR1、HCDR2和HCDR3，和氨基酸序列分别如SEQ ID NO:19、SEQ ID NO:20和SEQ ID NO:21所示的LCDR1、LCDR2和LCDR3；优选地，所述结合CD3的抗原结合位点包含氨基酸序列如SEQ ID NO:1所示的重链可变区，和氨基酸序列如SEQ ID NO:2所示的轻链可变区；或包含氨基酸序列如SEQ ID NO:5所示的重链可变区，和氨基酸序列如SEQ ID NO:6所示的轻链可变区。
  2. 如权利要求1所述的抗体或其抗原结合片段，其中，
    所述抗原结合片段包含氨基酸序列如SEQ ID NO:15所示的重链片段，和氨基酸序列如SEQ ID NO:4所示的轻链片段；或
    所述抗体包含氨基酸序列如SEQ ID NO:3所示的重链，和氨基酸序列如SEQ ID NO:4所示的轻链。
  3. 如权利要求1所述的抗体或其抗原结合片段，其中，所述抗体是单克隆抗体、双特异性抗体或多特异性抗体。
  4. 如权利要求1所述的抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段还包含结合CLDN-18.2的抗原结合位点。
  5. 如权利要求4所述的抗体或其抗原结合片段，其中，所述结合CLDN-18.2的抗原结合位点包含氨基酸序列分别如SEQ ID NO:22、SEQ ID NO:23和SEQ ID NO:24所示的HCDR1、HCDR2和HCDR3，和氨基酸序列分别如SEQ ID NO:25、SEQ ID NO:26和SEQ ID NO:27所示的LCDR1、LCDR2和LCDR3；优选地，所述结合CLDN-18.2的抗原结合位点包含氨基酸序列如SEQ ID NO:9所示的重链可变区，和氨基酸序列如SEQ ID NO:10所示的轻链可变区；进一步优选地，所述结合CLDN-18.2的抗原结合位点包含氨基酸序列如SEQ ID NO:11所示的重链，和氨基酸序列如SEQ ID NO:12所示的轻链。
  6. 如权利要求1-5中任一项所述的抗体或其抗原结合片段，其中，所述抗原结合片段为Fab、Fab'、F(ab')2、Fv、scFv或sdAb。
  7. 一种双特异性抗体，其具有两条重链和一条轻链，其中，第一重链自N端到C端具有VH-CH1-铰链区-Fc；第二重链自N端到C端具有铰链区-Fc-连接肽-VL-连接肽-VH；所述轻链自N端到C端具有VL-CL，其中所述第一重链的VH-CH1与所述轻链的VL-CL形成抗原结合位点结合CLDN-18.2，所述第二重链的VH-连接肽-VL的抗原结合位点结合CD3。
  8. 如权利要求7所述的双特异性抗体，其中，所述第二重链的VH包含氨基酸序列分别如SEQ ID NO:16、SEQ ID NO:17和SEQ ID NO:18所示的HCDR1、HCDR2和HCDR3，和VL包含氨基酸序列分别如SEQ ID NO:19、SEQ ID NO:20和SEQ ID NO:21所示的LCDR1、LCDR2和LCDR3。
  9. 如权利要求8所述的双特异性抗体，其中，所述第二重链的VH具有如SEQ ID NO:1所示的氨基酸序列，和VL具有如SEQ ID NO:2所示的氨基酸序列；或所述第二重链的VH具有如SEQ ID NO:5所示的氨基酸序列，和VL具有如SEQ ID NO:6所示的氨基酸序列；
    优选地，所述连接肽的氨基酸序列含有甘氨酸残基和丝氨酸残基或由甘氨酸残基和丝氨酸残基组成，长度为2-30个氨基酸残基；优选各自独立为(GGGGS)n，其中，n为2、3、4、5或6；
    进一步优选地，所述第二重链具有如SEQ ID NO:7所示的氨基酸序列。
  10. 如权利要求7-9中任一项所述的双特异性抗体，其中，所述第一重链的VH包含氨基酸序列分别如SEQ ID NO:22、SEQ ID NO:23和SEQ ID NO:24所示的HCDR1、HCDR2和HCDR3，和所述轻链的VL包含氨基酸序列分别如SEQ ID NO:25、SEQ ID NO:26和SEQ ID NO:27所示的LCDR1、LCDR2和LCDR3。
  11. 如权利要求10所述的双特异性抗体，其中，所述第一重链的VH具有如SEQ ID NO:9所示的氨基酸序列，和所述轻链的VL具有如SEQ ID NO:10所示的氨基酸的序列；优选地，所述第一重链具有如SEQ ID NO:11所示的氨基酸序列，和所述轻链具有如SEQ ID NO:12所示的氨基酸的序列。
  12. 如权利要求7-11中任一项所述的双特异性抗体，其中，所述第一重链与所述第二重链之间通过铰链区的二硫键和CH3结构域的杵臼结构连接。
  13. 如权利要求7-12中任一项所述的双特异性抗体，其中，所述第一重链和第二重链的Fc为来自IgG的Fc，例如来自IgG1、IgG2、IgG3或IgG4的Fc，和/或所述轻链的CL为来自λ或κ轻链的CL。
  14. 一种多核苷酸分子，其核苷酸序列选自：
    (1)编码权利要求1-6中任一项所述的抗体或其抗原结合片段或权利要求7-13中任一项的双特异性抗体的第一重链、第二重链和/或轻链的核苷酸序列；和
    (2)如(1)所述核苷酸序列的互补序列；
    优选地，编码所述第一重链的多核苷酸序列如SEQ ID NO:13所示，编码所述第二重链的多核苷酸序列如SEQ ID NO:8所示，编码所述轻链的多核苷酸序列如SEQ ID NO:14所示。
  15. 一种表达载体，其包含如权利要求14所述的多核苷酸分子，优选地，所述表达载 体为真核表达载体。
  16. 一种宿主细胞，其包含如权利要求14所述的多核苷酸分子，或包含如权利要求15所述的表达载体，或表达权利要求1-6中任一项所述的抗体或其抗原结合片段，或表达权利要求7-13中任一项的双特异性抗体的第一重链、第二重链和/或轻链；优选地，所述宿主细胞是真核细胞，更优选哺乳动物细胞。
  17. 一种制备如权利要求1-6中任一项所述的抗体或其抗原结合片段或如权利要求7-13中任一项所述的双特异性抗体的方法，所述方法包括在适合于所述双特异性抗体表达的条件下培养权利要求16所述的宿主细胞，使其表达所述抗体或其抗原结合片段或所述双特异性抗体，并从所述宿主细胞回收所表达的抗体或其抗原结合片段或所述双特异性抗体。
  18. 一种药物组合物，其包含权利要求1-6中任一项所述的抗体或其抗原结合片段、权利要求7-13中任一项所述的双特异性抗体、权利要求14所述的多核苷酸分子、权利要求15所述的表达载体或权利要求16所述的宿主细胞，和药学上可接受的载体或赋形剂。
  19. 权利要求1-6中任一项所述的抗体或其抗原结合片段、权利要求7-13中任一项所述的双特异性抗体、权利要求14所述的多核苷酸分子、权利要求15所述的表达载体、权利要求16所述的宿主细胞或权利要求18所述的药物组合物在制备用于治疗癌症的药物中的用途；优选地，所述癌症与CLDN18相关；更优选地，所述癌症选自胃癌、食道癌、胃食管癌、胰腺癌、胆管癌、肺癌、卵巢癌、结肠癌、肝癌、头颈癌、胆囊癌、肠癌和膀胱癌。
  20. 使用如权利要求7-13中任一项所述的双特异性抗体检测CD3和/或CLDN-18.2在样品中的存在的方法，其特征在于，所述方法包括使所述的双特异性抗体与样品接触并检测所述双特异性抗体与CD3和/或CLDN-18.2形成的结合物或对其进行定量测定。
PCT/CN2021/113033 2020-08-18 2021-08-17 抗cd3和抗cldn-18.2双特异性抗体及其用途 WO2022037582A1 (zh)

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