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WO2023051727A1 - 结合cd3的抗体及其用途 - Google Patents

结合cd3的抗体及其用途 Download PDF

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Publication number
WO2023051727A1
WO2023051727A1 PCT/CN2022/122753 CN2022122753W WO2023051727A1 WO 2023051727 A1 WO2023051727 A1 WO 2023051727A1 CN 2022122753 W CN2022122753 W CN 2022122753W WO 2023051727 A1 WO2023051727 A1 WO 2023051727A1
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seq
amino acid
acid sequence
jscd3
polypeptide chain
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French (fr)
Inventor
周岳华
李瑞胜
张静
陈素芬
刘辉
刘丹丹
何志娟
姚盛
冯辉
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Shanghai Junshi Biosciences Co Ltd
Suzhou Junmeng Biosciences Co Ltd
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Shanghai Junshi Biosciences Co Ltd
Suzhou Junmeng Biosciences Co Ltd
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Publication of WO2023051727A1 publication Critical patent/WO2023051727A1/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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins

Definitions

  • the present invention relates to a CD3 antibody, and also relates to a bispecific antibody that simultaneously binds to CD3 and a tumor-associated antigen and uses thereof.
  • T lymphocytes are lymphatic stem cells derived from bone marrow. After differentiation and maturation in the thymus, they are distributed to immune organs and tissues throughout the body through lymphatic and blood circulation to exert immune functions. T cells are highly efficient killer cells that can rapidly destroy virus-infected cells and cancer cells. The killing effect of T cells requires the formation of immune synapses, and this process is highly dependent on the complex formed by TCR recognition of MHC molecules on the surface of antigen-presenting cells and the presented antigen peptides. Activated immune synapses will release cytotoxins and cytokines to play a killing role.
  • bispecific antibody-directed T cells In the process of immune synapse formation, limited by the distance between T cells and target cells, bispecific antibody-directed T cells must simulate the formation of immune synapses, and one end bridges T cell TCR receptors by targeting CD3 , and the other end bridging target cells is achieved by targeting target cell surface antigens.
  • CD3 T cell surface glycoprotein CD3, signal transduction co-receptor of T cell receptor, which includes 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 region recognizes and binds to major histocompatibility complex class II molecules to enhance T cell antigen receptor (T cell receptor) , TCR) and the stability of the combination of MHC molecules; the intracellular region enhances the activation signal transduced by leukocyte CD3, thereby participating in and regulating the activation of the immune system.
  • T cell receptor T cell antigen receptor
  • TCR T cell antigen receptor
  • the quantitative index of CD3 positive lymphocyte population is an important index to measure the cellular immunity of the body.
  • bispecific antibodies targeting cytotoxic T cells to cancer cells was first described in 1985. Bispecific antibody-mediated T cell redirection to kill tumor cells is considered to be the most promising cancer treatment strategy.
  • Removab the world's first bispecific antibody drug, was approved for marketing by the European Medicines Agency (EMA). The indication is malignant ascites caused by EpCAM-positive tumors that are ineffective or infeasible by standard treatment.
  • Catumaxomab (anti-CD3 and anti-EpCAM) is the first double antibody approved by the FDA.
  • the application goal it wants to achieve is "a three-function" bispecific antibody, which besides bridging tumor target cells and T cells , can also bind to Fc ⁇ receptor-positive effector cells, macrophages, etc.
  • catumaxomab As the first T cell recruiter double antibody approved by the FDA, catumaxomab has a milestone significance. In fact, catumaxomab did show good efficacy in the treatment of ovarian and other cancer patients with ascites. However, during this process, side effects such as fever, nausea, and vomiting caused by cytokine storm often appeared. % of patients developed human anti-mouse antibody/human anti-rat antibody (HAMA/HARA).
  • the present invention aims to provide antibodies with good effects in terms of affinity, stability and/or reducing cytokine release syndrome.
  • the present invention provides an antibody or an antigen-binding fragment thereof that binds to CD3.
  • the anti-CD3 antibody or an antigen-binding fragment thereof provided by the present invention can be used as an independent therapy or in combination with other therapies/or other anti-cancer agents for such as cancer It also provides a trivalent bispecific antibody that simultaneously binds to CD3 and tumor-associated antigens, which has good effects in terms of affinity, stability, and/or reduction of cytokine release syndrome, and can effectively inhibit tumors growth, which can be used in treatments such as cancer.
  • the present invention provides a bispecific antibody comprising a first binding domain, a second binding domain and a third binding domain, wherein the first binding domain binds to CD3, and the second binding domain
  • the second binding domain and the third binding domain bind to tumor-associated antigens
  • the first binding domain has a form of Fab molecule or Fv molecule or scFv molecule
  • the second binding domain has a Fab molecule or Fv molecule or scFv molecule
  • said third binding domain is in the form of a Fab molecule.
  • the first binding domain of the invention is in the form of a Fab molecule
  • the second binding domain is in the form of a Fv molecule or scFv molecule
  • the third binding domain is in the form of a Fab molecule .
  • the first binding domain of the invention is in the form of a Fab molecule
  • the second binding domain is in the form of a Fv molecule
  • the third binding domain is in the form of a Fab molecule.
  • the first binding domain of the invention is in the form of a Fab molecule
  • the second binding domain is in the form of a scFv molecule
  • the third binding domain is in the form of a Fab molecule.
  • the first binding domain of the invention is in the form of an Fv molecule
  • the second binding domain is in the form of a Fab molecule
  • the third binding domain is in the form of a Fab molecule.
  • the tumor-associated antigen of the present invention is selected from BCMA, CD19, CD20, CD22, CD30, CD33, CD38, CD40, CD123, CDK4, CEA, Claudin18.2, EGFR, ROR1, TGF- ⁇ and 5T4 ;
  • the tumor-associated antigen is Claudin 18.2, CD20 or BCMA.
  • the bispecific antibody of the present invention has four polypeptide chains, wherein the first polypeptide chain has VH1-CH1-connecting peptide-VL2-connecting peptide-hinge region from N-terminus to C-terminus - the first Fc region, the second polypeptide chain has VL1-CL-connecting peptide-VH2 from N-terminus to C-terminus; the third polypeptide chain has VH2-CH1-hinge region from N-terminus to C-terminus- In the second Fc region, the fourth polypeptide chain has VL2-CL from N-terminal to C-terminal.
  • the VH1 and VL1 form a structural domain
  • the VH2 and VL2 form a structural domain.
  • the VH1-CH1 of the first polypeptide chain of the present invention and the VL1-CL of the second polypeptide chain form the first binding domain
  • the VL2 of the first polypeptide chain and the VL1-CL of the second polypeptide chain form the first binding domain
  • VH2 of the second polypeptide chain forms the second binding domain
  • VH2-CH1 of the third polypeptide chain and VL2-CL of the fourth polypeptide chain form the third binding domain.
  • the bispecific antibody of the present invention has four polypeptide chains, wherein the first polypeptide chain has VH1-CH1-connecting peptide-VL2-connecting peptide-VH2- Connecting peptide-hinge region-first Fc region, the second polypeptide chain has VL1-CL from N-terminus to C-terminus; the third polypeptide chain has VH2-CH1-hinge region- from N-terminus to C-terminus In the second Fc region, the fourth polypeptide chain has VL2-CL from N-terminal to C-terminal.
  • the VH1 and VL1 form a structural domain
  • the VH2 and VL2 form a structural domain.
  • the VH1-CH1 of the first polypeptide chain of the present invention and the VL1-CL of the second polypeptide chain form the first binding domain
  • the VL2-CL of the first polypeptide chain The connecting peptide-VH2 forms the second binding domain
  • the VH2-CH1 of the third polypeptide chain and the VL2-CL of the fourth polypeptide chain form the third binding domain.
  • the bispecific antibody of the present invention has four polypeptide chains, wherein the first polypeptide chain has VH2-CH1-connecting peptide-VL1-connecting peptide-hinge region from N-terminus to C-terminus - the first Fc region, the second polypeptide chain has VL2-CL-connecting peptide-VH1 from N-terminus to C-terminus; the third polypeptide chain has VH2-CH1-hinge region from N-terminus to C-terminus- In the second Fc region, the fourth polypeptide chain has VL2-CL from N-terminal to C-terminal.
  • the VH1 and VL1 form a structural domain
  • the VH2 and VL2 form a structural domain.
  • the VH2-CH1 of the first polypeptide chain of the present invention and the VL2-CL of the second polypeptide chain form the second binding domain
  • the VL1 of the first polypeptide chain and the VL2-CL of the second polypeptide chain form the second binding domain
  • VH1 of the second polypeptide chain forms the first binding domain
  • VH2-CH1 of the third polypeptide chain and VL2-CL of the fourth polypeptide chain form the third binding domain.
  • the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 contained in the first binding domain of the present invention are respectively:
  • HCDR1 X1YAMN, where X1 is T or K;
  • HCDR2 RIRSKYNNYATYYAX2SVKX3, where X2 is D or A, X3 is D or G;
  • HCDR3 HGX3X4GX5X6X7X8X9X10X11AY, where X3 is N or A, X4 is F or A, X5 is N or A, X6 is S or A, X7 is Y or A, X8 is V or A, X9 is S or A, X10 is W or A, X11 is F or A;
  • LCDR1 X12SSTGAVTTX13NYX14N, where X12 is R or G, X13 is S or G, X14 is A or P;
  • LCDR2 GTNKRAP
  • LCDR3 AX15X16X17X18X19X20X21X22, among them, X15 is L or A, X16 is W or A, X17 is Y or A, X18 is S or A, X19 is N or A, X20 is L or A, X21 is W or A, X22 is V or A.
  • the first binding domain of the present invention comprises: HCDR1 with an amino acid sequence as shown in SEQ ID NO: 1 or 2, HCDR2 with an amino acid sequence as shown in SEQ ID NO: 3 or 4, and an amino acid sequence as shown in HCDR3 shown in any one of SEQ ID NO: 5-22, the amino acid sequence is LCDR1 shown in SEQ ID NO: 23 or 24, the amino acid sequence is LCDR2 shown in SEQ ID NO: 25, and the amino acid sequence is shown in SEQ ID NO: NO: LCDR3 shown in any one of 26-42.
  • the first binding domain of the present invention comprises HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3:
  • the HCDR1, HCDR2, and HCDR3 are:
  • the amino acid sequence is HCDR1 shown in SEQ ID NO: 1, the amino acid sequence is HCDR2 shown in SEQ ID NO: 3, and the amino acid sequence is HCDR3 shown in SEQ ID NO: 5; or
  • amino acid sequence is HCDR1 shown in SEQ ID NO: 1, the amino acid sequence is HCDR2 shown in SEQ ID NO: 3, and the amino acid sequence is HCDR3 shown in SEQ ID NO: 15; or
  • amino acid sequence is HCDR1 shown in SEQ ID NO: 2, the amino acid sequence is HCDR2 shown in SEQ ID NO: 4, and the amino acid sequence is HCDR3 shown in any one of SEQ ID NO: 5-22;
  • the LCDR1, LCDR2, LCDR3 are:
  • amino acid sequence is LCDR1 shown in SEQ ID NO: 23, the amino acid sequence is LCDR2 shown in SEQ ID NO: 25, and the amino acid sequence is LCDR3 shown in SEQ ID NO: 26; or
  • amino acid sequence is LCDR1 shown in SEQ ID NO: 23, the amino acid sequence is LCDR2 shown in SEQ ID NO: 25, and the amino acid sequence is LCDR3 shown in SEQ ID NO: 33; or
  • amino acid sequence is LCDR1 shown in SEQ ID NO: 24, the amino acid sequence is LCDR2 shown in SEQ ID NO: 25, and the amino acid sequence is LCDR3 shown in any one of SEQ ID NO: 26-42.
  • the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 contained in the first binding domain of the present invention are respectively:
  • HCDR1 X1YAMN, wherein X1 is T or K (preferably K);
  • HCDR2 RIRSKYNNYATYYAX2SVKX3, wherein X2 is D or A (preferably A), X3 is D or G (preferably G);
  • HCDR3 HGX3X4GX5X6X7X8X9X10X11AY, where X3 is N, X4 is F, X5 is N or A, X6 is S or A, X7 is Y or A, X8 is V or A, X9 is S or A, X10 is W, and X11 is F or A (preferably F);
  • LCDR1 X12SSTGAVTTX13NYX14N, where X12 is R or G, X13 is S or G, X14 is A or P;
  • LCDR2 GTNKRAP
  • LCDR3 AX15X16X17X18X19X20X21X22, where X15 is L or A, X16 is W, X17 is Y or A, X18 is S or A, X19 is N or A, X20 is L or A, X21 is W, and X22 is V or A.
  • the first binding domain of the present invention comprises: HCDR1 with an amino acid sequence as shown in SEQ ID NO: 1 or 2, HCDR2 with an amino acid sequence as shown in SEQ ID NO: 3 or 4, and an amino acid sequence as shown in HCDR3 shown in any one of SEQ ID NO: 5, 9, 10, 11, 15, 17, 18, 19 or 20, the amino acid sequence is shown in LCDR1 shown in SEQ ID NO: 23 or 24, the amino acid sequence is shown in SEQ ID LCDR2 shown in NO: 25, and LCDR3 shown in any one of SEQ ID NO: 26, 28, 30, 33, 35, 36, 37, 39, 40, 41 or 42 with amino acid sequence.
  • the first binding domain of the present invention comprises:
  • Amino acid sequence such as HCDR1 shown in SEQ ID NO: 1, amino acid sequence such as HCDR2 shown in SEQ ID NO: 3, amino acid sequence such as HCDR3 shown in SEQ ID NO: 15, amino acid sequence such as SEQ ID NO: 23
  • the LCDR1 shown, the amino acid sequence is LCDR2 shown in SEQ ID NO: 25, the amino acid sequence is LCDR3 shown in SEQ ID NO: 33; or
  • Amino acid sequence such as HCDR1 shown in SEQ ID NO: 2, amino acid sequence such as HCDR2 shown in SEQ ID NO: 4, amino acid sequence such as HCDR3 shown in SEQ ID NO: 5, amino acid sequence such as SEQ ID NO: 24
  • the LCDR1 shown, the amino acid sequence is LCDR2 shown in SEQ ID NO: 25, the amino acid sequence is LCDR3 shown in any one of SEQ ID NO: 26, 28, 33, 36 or 37; or
  • amino acid sequence such as HCDR1 shown in SEQ ID NO: 2, amino acid sequence such as HCDR2 shown in SEQ ID NO: 4, amino acid sequence such as HCDR3 shown in SEQ ID NO: 10, amino acid sequence such as SEQ ID NO: 24
  • the LCDR1 shown, the amino acid sequence is LCDR2 shown in SEQ ID NO: 25, the amino acid sequence is LCDR3 shown in any one of SEQ ID NO: 28, 33, 40, 41 or 42; or
  • the LCDR1 shown, the amino acid sequence is LCDR2 shown in SEQ ID NO: 25, the amino acid sequence is LCDR3 shown in any one of SEQ ID NO: 26, 28, 33, 35, 36, 37, 40, 41 or 42 ;
  • amino acid sequence such as HCDR1 shown in SEQ ID NO: 2, amino acid sequence such as HCDR2 shown in SEQ ID NO: 4, amino acid sequence such as HCDR3 shown in any one of SEQ ID NO: 9, 10 or 19,
  • the amino acid sequence is LCDR1 shown in SEQ ID NO: 23, the amino acid sequence is LCDR2 shown in SEQ ID NO: 25, and the amino acid sequence is LCDR3 shown in SEQ ID NO: 26; or
  • amino acid sequence such as HCDR1 shown in SEQ ID NO: 2, amino acid sequence such as HCDR2 shown in SEQ ID NO: 4, amino acid sequence such as HCDR3 shown in SEQ ID NO: 19, amino acid sequence such as SEQ ID NO: 24 As shown in LCDR1, the amino acid sequence is LCDR2 shown in SEQ ID NO: 25, and the amino acid sequence is LCDR3 shown in SEQ ID NO: 36.
  • the first binding domain of the present invention comprises: an amino acid sequence such as a VH shown in any one of SEQ ID NO: 43-62, and an amino acid sequence such as any one of SEQ ID NO: 63-81 VL shown in item.
  • the first binding domain of the present invention comprises:
  • VH with amino acid sequence as shown in SEQ ID NO: 44, and VL with amino acid sequence as shown in SEQ ID NO: 64; or
  • VH with amino acid sequence as shown in SEQ ID NO: 45
  • VL with amino acid sequence as shown in SEQ ID NO: 65, 67, 72, 75 or 76
  • VH with amino acid sequence as shown in SEQ ID NO: 50, and VL with amino acid sequence as shown in SEQ ID NO: 67, 72, 79, 80 or 81; or
  • VH with amino acid sequence as shown in SEQ ID NO: 57
  • VL with amino acid sequence as shown in SEQ ID NO: 72, 74, 75, 76 or 81; or
  • VIII VH with amino acid sequence as shown in SEQ ID NO: 58, and VL with amino acid sequence as shown in SEQ ID NO: 67, 74, 75, 76, 78, 80 or 81; or
  • the second binding domain and the third binding domain of the present invention bind to Claudin 18.2, which respectively comprise: HCDR1 with an amino acid sequence such as SEQ ID NO: 121, and an amino acid sequence such as SEQ ID NO: HCDR2 shown in 122, amino acid sequence such as HCDR3 shown in SEQ ID NO: 123, amino acid sequence such as LCDR1 shown in SEQ ID NO: 124, amino acid sequence such as LCDR2 shown in SEQ ID NO: 125, and amino acid sequence such as SEQ ID NO: ID NO: LCDR3 shown in 126.
  • Claudin 18.2 which respectively comprise: HCDR1 with an amino acid sequence such as SEQ ID NO: 121, and an amino acid sequence such as SEQ ID NO: HCDR2 shown in 122, amino acid sequence such as HCDR3 shown in SEQ ID NO: 123, amino acid sequence such as LCDR1 shown in SEQ ID NO: 124, amino acid sequence such as LCDR2 shown in SEQ ID NO: 125, and amino acid sequence such as
  • the second binding domain and the third binding domain of the present invention bind to CD20, which respectively comprise: HCDR1 with an amino acid sequence such as SEQ ID NO: 127, and an amino acid sequence such as SEQ ID NO: 128 As shown in HCDR2, the amino acid sequence is HCDR3 shown in SEQ ID NO: 129, the amino acid sequence is LCDR1 shown in SEQ ID NO: 130, the amino acid sequence is LCDR2 shown in SEQ ID NO: 131, and the amino acid sequence is shown in SEQ ID NO: LCDR3 shown in 132.
  • the second binding domain and the third binding domain of the present invention bind to Claudin 18.2, which respectively comprise: (a) a VH with an amino acid sequence as shown in SEQ ID NO: 133, and an amino acid sequence as shown in The VL shown in SEQ ID NO: 134; or (b) the VH with the amino acid sequence shown in SEQ ID NO: 166, and the VL with the amino acid sequence shown in SEQ ID NO: 167.
  • the second binding domain and the third binding domain of the present invention bind to CD20, which respectively comprise: a VH with an amino acid sequence such as SEQ ID NO: 135, and an amino acid sequence such as SEQ ID NO: 136 VL shown.
  • the first Fc region of the present invention is knob-Fc
  • the second Fc region is hole-Fc
  • the first Fc region of the present invention is connected to the second Fc region through a disulfide bond in the hinge region and a knob-and-hole structure.
  • the first Fc region and/or the second Fc region of the present invention is an Fc region from IgG, preferably an Fc region from IgG1, IgG2, IgG3 or IgG4.
  • the CH1 of the present invention is CH1 from IgG, preferably CH1 from IgG1, IgG2, IgG3 or IgG4.
  • the CL of the invention is a CL from a lambda or kappa light chain.
  • the connecting peptide of the present invention may be a flexible polypeptide comprising G (glycine), S (serine) and A (alanine) or composed of G and S, and its length may be 2-30 amino acid residues ; Further preferably, the connecting peptide includes but not limited to (GS)n, (GGS)n, (GGGS)n, (GGGGS)n and AS(GGGGS)n, etc., n can be an integer of 1-6, such as 1, 2, 3, 4, 5, 6.
  • the bispecific antibody of the present invention has four polypeptide chains, and the four polypeptide chains are:
  • the third polypeptide chain having the amino acid sequence of SEQ ID NO: 143 has the fourth polypeptide chain of the amino acid sequence; or
  • the third polypeptide chain having the amino acid sequence of SEQ ID NO: 143 has the fourth polypeptide chain of the amino acid sequence; or
  • the third polypeptide chain having the amino acid sequence of SEQ ID NO: 143 has the fourth polypeptide chain of the amino acid sequence; or
  • the third polypeptide chain having the amino acid sequence of SEQ ID NO: 153 has the fourth polypeptide chain of the amino acid sequence; or
  • the third polypeptide chain having the amino acid sequence of SEQ ID NO: 153 has the fourth polypeptide chain of the amino acid sequence; or
  • the third polypeptide chain having the amino acid sequence of SEQ ID NO: 153 has the fourth polypeptide chain of the amino acid sequence; or
  • the third polypeptide chain having the amino acid sequence shown in SEQ ID NO: 143 has the fourth polypeptide chain.
  • the present invention provides a polynucleotide molecule whose nucleotide sequence is selected from:
  • the present invention provides an expression vector comprising the polynucleotide molecule described herein, preferably, the expression vector is a eukaryotic expression vector.
  • the present invention provides a host cell comprising the polynucleotide molecule described herein, or comprising the expression vector described herein, or expressing any of the bispecific antibodies described herein; preferably , the host cell is a eukaryotic cell, more preferably a mammalian cell.
  • the present invention provides a method for preparing the bispecific antibody described in any one of this paper, the method comprising culturing the bispecific antibody described herein under conditions suitable for the expression of the bispecific antibody described in any one of this paper.
  • the host cell is made to express the bispecific antibody described in any one herein, and the expressed bispecific antibody described in any one herein is recovered from the host cell.
  • the present invention provides a pharmaceutical composition, which comprises any one of the bispecific antibody, polynucleotide molecule, expression vector or host cell described herein, and a pharmaceutically acceptable carrier or excipient agent.
  • the present invention provides the use of any one of the bispecific antibodies, polynucleotide molecules, expression vectors, host cells or pharmaceutical compositions described herein in the preparation of drugs for preventing or treating cancer.
  • the present invention provides any one of the bispecific antibodies, polynucleotide molecules, expression vectors, host cells or pharmaceutical compositions herein, which is used for preventing or treating cancer.
  • the present invention provides a method for preventing or treating cancer, comprising administering to a subject in need a bispecific antibody, polynucleotide molecule, expression vector, host cell as described herein or pharmaceutical compositions.
  • the cancer of the present invention is related to CLDN18 or CD20; more preferably, the cancer is selected from 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; or said cancer is selected from acute B-lymphoblastic leukemia, diffuse large B-cell lymphoma, chronic lymphocytic leukemia, follicular lymphoma, non-Hodgkin's lymphoma neoplasms, chronic myeloid leukemia, and Burkitt lymphoma.
  • the present invention provides a pharmaceutical combination comprising a bispecific antibody, a polynucleotide molecule, an expression vector, a host cell, or a pharmaceutical composition as described herein, and one or more additional therapeutic agent.
  • the present invention provides a kit comprising the bispecific antibody, polynucleotide molecule, expression vector, host cell or pharmaceutical composition as described in any one of the herein.
  • the present invention provides an antibody or antigen-binding fragment thereof comprising a CD3-binding binding domain, wherein the CD3-binding binding domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 They are:
  • HCDR1 X1YAMN, where X1 is T or K;
  • HCDR2 RIRSKYNNYATYYAX2SVKX3, where X2 is D or A, X3 is D or G;
  • HCDR3 HGX3X4GX5X6X7X8X9X10X11AY, where X3 is N or A, X4 is F or A, X5 is N or A, X6 is S or A, X7 is Y or A, X8 is V or A, X9 is S or A, X10 is W or A, X11 is F or A;
  • LCDR1 X12SSTGAVTTX13NYX14N, where X12 is R or G, X13 is S or G, X14 is A or P;
  • LCDR2 GTNKRAP
  • LCDR3 AX15X16X17X18X19X20X21X22, among them, X15 is L or A, X16 is W or A, X17 is Y or A, X18 is S or A, X19 is N or A, X20 is L or A, X21 is W or A, X22 is V or A;
  • the amino acid sequence is as shown in SEQ ID NO: HCDR1
  • the amino acid sequence is as shown in SEQ ID NO: 3 HCDR2 shown
  • amino acid sequence such as HCDR3 shown in SEQ ID NO: 5
  • amino acid sequence such as LCDR1 shown in SEQ ID NO: 23
  • amino acid sequence such as LCDR2 shown in SEQ ID NO: 25
  • amino acid sequence such as SEQ ID NO: LCDR3 shown in 26.
  • the CD3 binding domain of the present invention comprises: HCDR1 with an amino acid sequence as shown in SEQ ID NO: 1 or 2, HCDR2 with an amino acid sequence as shown in SEQ ID NO: 3 or 4, and an amino acid sequence HCDR3 as shown in any one of SEQ ID NOs: 5-22, the amino acid sequence as LCDR1 shown in SEQ ID NO: 23 or 24, the amino acid sequence as LCDR2 shown in SEQ ID NO: 25, and the amino acid sequence as SEQ ID NO: LCDR3 shown in any one of ID NO: 26-42.
  • the CD3 binding domains of the present invention comprise HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3:
  • the HCDR1, HCDR2, and HCDR3 are:
  • the amino acid sequence is HCDR1 shown in SEQ ID NO: 1, the amino acid sequence is HCDR2 shown in SEQ ID NO: 3, and the amino acid sequence is HCDR3 shown in SEQ ID NO: 5; or
  • amino acid sequence is HCDR1 shown in SEQ ID NO: 1, the amino acid sequence is HCDR2 shown in SEQ ID NO: 3, and the amino acid sequence is HCDR3 shown in SEQ ID NO: 15; or
  • amino acid sequence is HCDR1 shown in SEQ ID NO: 2, the amino acid sequence is HCDR2 shown in SEQ ID NO: 4, and the amino acid sequence is HCDR3 shown in any one of SEQ ID NO: 5-22;
  • the LCDR1, LCDR2, LCDR3 are:
  • amino acid sequence is LCDR1 shown in SEQ ID NO: 23, the amino acid sequence is LCDR2 shown in SEQ ID NO: 25, and the amino acid sequence is LCDR3 shown in SEQ ID NO: 26; or
  • amino acid sequence is LCDR1 shown in SEQ ID NO: 23, the amino acid sequence is LCDR2 shown in SEQ ID NO: 25, and the amino acid sequence is LCDR3 shown in SEQ ID NO: 33; or
  • amino acid sequence is LCDR1 shown in SEQ ID NO: 24, the amino acid sequence is LCDR2 shown in SEQ ID NO: 25, and the amino acid sequence is LCDR3 shown in any one of SEQ ID NO: 26-42.
  • the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 contained in the CD3-binding binding domain of the present invention are:
  • HCDR1 X1YAMN, wherein X1 is T or K (preferably K);
  • HCDR2 RIRSKYNNYATYYAX2SVKX3, wherein X2 is D or A (preferably A), X3 is D or G (preferably G);
  • HCDR3 HGX3X4GX5X6X7X8X9X10X11AY, where X3 is N, X4 is F, X5 is N or A, X6 is S or A, X7 is Y or A, X8 is V or A, X9 is S or A, X10 is W, and X11 is F or A (preferably F);
  • LCDR1 X12SSTGAVTTX13NYX14N, where X12 is R or G, X13 is S or G, X14 is A or P;
  • LCDR2 GTNKRAP
  • LCDR3 AX15X16X17X18X19X20X21X22, where X15 is L or A, X16 is W, X17 is Y or A, X18 is S or A, X19 is N or A, X20 is L or A, X21 is W, X22 is V or A;
  • the amino acid sequence is as shown in SEQ ID NO: HCDR1
  • the amino acid sequence is as shown in SEQ ID NO: 3 HCDR2 shown
  • amino acid sequence such as HCDR3 shown in SEQ ID NO: 5
  • amino acid sequence such as LCDR1 shown in SEQ ID NO: 23
  • amino acid sequence such as LCDR2 shown in SEQ ID NO: 25
  • amino acid sequence such as SEQ ID NO: LCDR3 shown in 26.
  • the CD3 binding domain of the present invention comprises: HCDR1 with an amino acid sequence as shown in SEQ ID NO: 1 or 2, HCDR2 with an amino acid sequence as shown in SEQ ID NO: 3 or 4, and an amino acid sequence HCDR3 as shown in any one of SEQ ID NO: 5, 9, 10, 11, 15, 17, 18, 19 or 20, the amino acid sequence is as shown in SEQ ID NO: 23 or 24 LCDR1, the amino acid sequence is as SEQ LCDR2 shown in ID NO: 25, and LCDR3 shown in any one of SEQ ID NO: 26, 28, 30, 33, 35, 36, 37, 39, 40, 41 or 42 with amino acid sequence.
  • the CD3-binding binding domain of the present invention comprises:
  • Amino acid sequence such as HCDR1 shown in SEQ ID NO: 1, amino acid sequence such as HCDR2 shown in SEQ ID NO: 3, amino acid sequence such as HCDR3 shown in SEQ ID NO: 15, amino acid sequence such as SEQ ID NO: 23
  • the LCDR1 shown, the amino acid sequence is LCDR2 shown in SEQ ID NO: 25, the amino acid sequence is LCDR3 shown in SEQ ID NO: 33; or
  • Amino acid sequence such as HCDR1 shown in SEQ ID NO: 2, amino acid sequence such as HCDR2 shown in SEQ ID NO: 4, amino acid sequence such as HCDR3 shown in SEQ ID NO: 5, amino acid sequence such as SEQ ID NO: 24 LCDR1 shown, the amino acid sequence of LCDR2 shown in SEQ ID NO: 25, the amino acid sequence of LCDR3 shown in SEQ ID NO: 26, 28, 33, 36 or 37; or
  • amino acid sequence such as HCDR1 shown in SEQ ID NO: 2, amino acid sequence such as HCDR2 shown in SEQ ID NO: 4, amino acid sequence such as HCDR3 shown in SEQ ID NO: 10, amino acid sequence such as SEQ ID NO: 24 LCDR1 shown, the amino acid sequence of LCDR2 shown in SEQ ID NO: 25, the amino acid sequence of LCDR3 shown in SEQ ID NO: 28, 33, 40, 41 or 42; or
  • Amino acid sequence such as HCDR1 shown in SEQ ID NO: 2, amino acid sequence such as HCDR2 shown in SEQ ID NO: 4, amino acid sequence such as HCDR3 shown in SEQ ID NO: 18, amino acid sequence such as SEQ ID NO: 24 the LCDR1 shown, the amino acid sequence of LCDR2 shown in SEQ ID NO: 25, the amino acid sequence of LCDR3 shown in SEQ ID NO: 28, 35, 36, 37, 39, 41 or 42; or
  • the CD3 binding domain of the present invention comprises: an amino acid sequence such as a VH shown in any one of SEQ ID NOs: 43-62, and an amino acid sequence such as any one of SEQ ID NOs: 63-81 One VL shown.
  • the CD3-binding binding domain of the present invention comprises:
  • VH with amino acid sequence as shown in SEQ ID NO: 44, and VL with amino acid sequence as shown in SEQ ID NO: 64; or
  • VH with amino acid sequence as shown in SEQ ID NO: 45
  • VL with amino acid sequence as shown in SEQ ID NO: 65, 67, 72, 75 or 76
  • VH with amino acid sequence as shown in SEQ ID NO: 50, and VL with amino acid sequence as shown in SEQ ID NO: 67, 72, 79, 80 or 81; or
  • VH with amino acid sequence as shown in SEQ ID NO: 57
  • VL with amino acid sequence as shown in SEQ ID NO: 72, 74, 75, 76 or 81; or
  • VIII VH with amino acid sequence as shown in SEQ ID NO: 58, and VL with amino acid sequence as shown in SEQ ID NO: 67, 74, 75, 76, 78, 80 or 81; or
  • the antibody of the present invention comprises a heavy chain with an amino acid sequence as shown in any one of SEQ ID NO: 82-101, and an amino acid sequence as shown in any one of SEQ ID NO: 102-120 light chain.
  • the antibodies of the invention comprise:
  • VII a heavy chain having an amino acid sequence as set forth in SEQ ID NO: 96, and a light chain having an amino acid sequence as set forth in SEQ ID NO: 111, 113, 114, 115 or 120; or
  • the antibody of the present invention is a monoclonal antibody, a bispecific antibody or a multispecific antibody, and the antigen-binding fragment is Fab, Fab', F(ab')2, Fv, dsFv, scFv or sdAb.
  • the antibody of the invention is a monoclonal antibody that specifically binds CD3.
  • the antibody or antigen-binding fragment thereof of the present invention further comprises one or two binding domains that bind tumor-associated antigens.
  • the tumor-associated antigen of the present invention is selected from BCMA, CD19, CD20, CD22, CD30, CD33, CD38, CD40, CD123, CDK4, CEA, Claudin18.2, EGFR, ROR1, TGF- ⁇ and 5T4 ;
  • the tumor-associated antigen is Claudin 18.2, CD20 or BCMA.
  • the tumor-associated antigen of the present invention is Claudin 18.2 or CD20.
  • the binding domain of the present invention that binds to Claudin 18.2 comprises: HCDR1 with an amino acid sequence such as SEQ ID NO: 121, HCDR2 with an amino acid sequence such as SEQ ID NO: 122, and an amino acid sequence such as SEQ ID HCDR3 shown in NO:123, LCDR1 shown in amino acid sequence as SEQ ID NO:124, LCDR2 shown in amino acid sequence as SEQ ID NO:125, and LCDR3 shown in amino acid sequence as SEQ ID NO:126.
  • the CD20-binding binding domain of the present invention comprises: HCDR1 with an amino acid sequence such as SEQ ID NO: 127, HCDR2 with an amino acid sequence such as SEQ ID NO: 128, and an amino acid sequence such as SEQ ID NO HCDR3 shown in: 129, LCDR1 shown in amino acid sequence as SEQ ID NO: 130, LCDR2 shown in amino acid sequence as SEQ ID NO: 131, and LCDR3 shown in amino acid sequence as SEQ ID NO: 132.
  • the binding domain of the present invention that binds to Claudin 18.2 comprises: (a) a VH with an amino acid sequence as shown in SEQ ID NO: 133, and a VL with an amino acid sequence as shown in SEQ ID NO: 134; or (b) VH whose amino acid sequence is shown in SEQ ID NO: 166, and VL whose amino acid sequence is shown in SEQ ID NO: 167.
  • the CD20-binding binding domain of the present invention comprises: a VH with an amino acid sequence as shown in SEQ ID NO: 135, and a VL with an amino acid sequence as shown in SEQ ID NO: 136.
  • the present invention provides an antibody comprising a binding domain that binds CD3 and a binding domain that binds CD20, wherein the binding domain that binds CD3 comprises: an amino acid sequence such as SEQ ID NO: 1 HCDR1 shown, amino acid sequence such as HCDR2 shown in SEQ ID NO: 3, amino acid sequence such as HCDR3 shown in SEQ ID NO: 5, amino acid sequence such as LCDR1 shown in SEQ ID NO: 23, amino acid sequence such as SEQ ID NO : LCDR2 shown in 25, the amino acid sequence is LCDR3 shown in SEQ ID NO: 26; the binding domain of the CD20 includes: amino acid sequence shown in HCDR1 as SEQ ID NO: 127, amino acid sequence is shown in SEQ ID NO: HCDR2 shown in 128, amino acid sequence such as HCDR3 shown in SEQ ID NO: 129, amino acid sequence such as LCDR1 shown in SEQ ID NO: 130, amino acid sequence such as LCDR2 shown in SEQ ID NO: 131, and amino acid sequence such
  • the present invention provides an antibody comprising a binding domain that binds CD3 and a binding domain that binds CD20, wherein the binding domain that binds CD3 comprises: an amino acid sequence such as SEQ ID NO: 43 The VH shown, and the VL with the amino acid sequence shown in SEQ ID NO: 63, the binding domain that binds CD20 includes: the VH with the amino acid sequence shown in SEQ ID NO: 135, and the amino acid sequence shown in SEQ ID NO: 136 VL shown.
  • the Fc region of the antibody of the present invention is Fc from IgG, such as Fc from IgG1, IgG2, IgG3 or IgG4, and/or the CL of the antibody is CL from ⁇ or ⁇ light chain;
  • the Fc region comprises a knob-and-hole structure.
  • the antibody of the present invention has four polypeptide chains, and the four polypeptide chains are:
  • the first polypeptide chain has an amino acid sequence as shown in SEQ ID NO: 144
  • the second polypeptide chain has an amino acid sequence as shown in SEQ ID NO: 145
  • the third polypeptide chain Having the amino acid sequence shown in SEQ ID NO: 142
  • the fourth polypeptide chain has the amino acid sequence shown in SEQ ID NO: 143; or
  • the first polypeptide chain has an amino acid sequence as shown in SEQ ID NO: 146
  • the second polypeptide chain has an amino acid sequence as shown in SEQ ID NO: 147
  • the third polypeptide chain Having the amino acid sequence shown in SEQ ID NO: 142
  • the fourth polypeptide chain has the amino acid sequence shown in SEQ ID NO: 143; or
  • the first polypeptide chain has an amino acid sequence as shown in SEQ ID NO: 148
  • the second polypeptide chain has an amino acid sequence as shown in SEQ ID NO: 149
  • the third polypeptide chain Having the amino acid sequence shown in SEQ ID NO: 142
  • the fourth polypeptide chain has the amino acid sequence shown in SEQ ID NO: 143; or
  • the first polypeptide chain has an amino acid sequence as shown in SEQ ID NO: 150
  • the second polypeptide chain has an amino acid sequence as shown in SEQ ID NO: 151
  • the third polypeptide chain Having the amino acid sequence shown in SEQ ID NO: 152
  • the fourth polypeptide chain has the amino acid sequence shown in SEQ ID NO: 153; or
  • the first polypeptide chain has the amino acid sequence shown in SEQ ID NO: 154
  • the second polypeptide chain has the amino acid sequence shown in SEQ ID NO: 155
  • the third polypeptide chain Having the amino acid sequence shown in SEQ ID NO: 152
  • the fourth polypeptide chain has the amino acid sequence shown in SEQ ID NO: 153; or
  • the first polypeptide chain has an amino acid sequence as shown in SEQ ID NO: 156
  • the second polypeptide chain has an amino acid sequence as shown in SEQ ID NO: 157
  • the third polypeptide chain Having the amino acid sequence shown in SEQ ID NO: 152
  • the fourth polypeptide chain has the amino acid sequence shown in SEQ ID NO: 153; or
  • the first polypeptide chain has the amino acid sequence shown in SEQ ID NO: 164
  • the second polypeptide chain has the amino acid sequence shown in SEQ ID NO: 165
  • the third polypeptide chain It has the amino acid sequence shown in SEQ ID NO: 142
  • the fourth polypeptide chain has the amino acid sequence shown in SEQ ID NO: 143.
  • the present invention provides a polynucleotide molecule whose nucleotide sequence is selected from:
  • the present invention provides an expression vector comprising the polynucleotide molecule described herein, preferably, the expression vector is a eukaryotic expression vector.
  • the present invention provides a host cell comprising the polynucleotide molecule described herein, or comprising the expression vector described herein, or expressing any antibody or antigen-binding fragment thereof herein; preferably,
  • the host cells are eukaryotic cells, more preferably mammalian cells.
  • the present invention provides a method of preparing any of the antibodies or antigen-binding fragments thereof herein, the method comprising culturing any of the antibodies or antigen-binding fragments thereof described herein under conditions suitable for expression of any of the antibodies or antigen-binding fragments thereof herein.
  • a host cell expressing any of the antibodies or antigen-binding fragments thereof herein, and recovering the expressed antibody or antigen-binding fragments of any of the herein from the host cells.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof, polynucleotide molecule, expression vector or host cell described in any embodiment herein, and a pharmaceutically acceptable carrier or excipients.
  • the present invention provides the use of the antibody or its antigen-binding fragment, polynucleotide molecule, expression vector, host cell or pharmaceutical composition described in any embodiment herein in the preparation of a drug for preventing or treating cancer use.
  • the present invention provides the antibody or antigen-binding fragment thereof, polynucleotide molecule, expression vector, host cell or pharmaceutical composition according to any one of the embodiments herein for use in the prevention or treatment of cancer.
  • the present invention provides a method of preventing or treating cancer, comprising administering to a subject in need the antibody or antigen-binding fragment thereof, polynucleotide molecule, expression Vector, host cell or pharmaceutical composition.
  • the cancer of the present invention is related to CLDN18 or CD20; more preferably, the cancer is selected from 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; or said cancer is selected from acute B-lymphoblastic leukemia, diffuse large B-cell lymphoma, chronic lymphocytic leukemia, follicular lymphoma, non-Hodgkin's lymphoma neoplasms, chronic myeloid leukemia, and Burkitt lymphoma.
  • the present invention provides a pharmaceutical combination comprising the antibody or antigen-binding fragment thereof, polynucleotide molecule, expression vector, host cell or pharmaceutical composition according to any embodiment herein, and a or more additional therapeutic agents.
  • the invention provides a kit comprising the antibody or antigen-binding fragment thereof, polynucleotide molecule, expression vector, host cell or pharmaceutical composition according to any embodiment herein.
  • the present invention provides the use of the monoclonal antibody specifically binding to CD3 according to any embodiment of the present invention in the preparation of molecules or drugs for reducing cytokine storm or for treating or preventing cytokine release syndrome application.
  • the molecule or drug is a bispecific antibody or a bispecific antibody-containing drug.
  • the bispecific antibody is the bispecific antibody described in any of the embodiments herein.
  • Figure 1 Detection results of the binding of the antibodies of Group 1 to human CD3.
  • Figure 2 The detection results of the binding of the antibodies of group 2 to human CD3.
  • Figure 3 Detection results of the binding of the antibodies of Group 3 to human CD3.
  • Figure 4 The detection results of the binding of the antibodies of Group 4 to human CD3.
  • Figure 5 The detection results of the binding of the antibodies of Group 5 to human CD3.
  • Figure 6 The detection results of the binding of the antibodies of Group 6 to human CD3.
  • Figure 7 Detection results of the binding of the antibodies of Group 7 to human CD3.
  • Figure 8 Detection results of the binding of the antibodies of Group 8 to human CD3.
  • Fig. 9 Detection results of the binding of the antibodies of Group 9 to human CD3.
  • Fig. 10 Detection results of the binding of the antibodies of Group 9 to human CD3.
  • Fig. 11 Detection results of the binding of the antibodies of Group 11 to human CD3.
  • Fig. 12 Detection results of the binding of the antibodies of Group 12 to human CD3.
  • Figure 13 Schematic diagram of the structure of the bispecific antibody JS202-7.
  • Figure 14 Schematic diagram of the structure of the bispecific antibody JS202-7 CD3 Fv.
  • Figure 15 Schematic diagram of the structure of bispecific antibody JS202-7 012 Fv and JS202-7 CD3 130 012 Fv.
  • Figure 16 Schematic diagram of the structure of bispecific antibodies JS203-7-JSCD3-CD20Fv and JS203-7-CD3 130-CD20Fv.
  • Figure 17 Schematic diagram of the structure of the bispecific antibody JS203-7-CD3-FV.
  • Figure 18 Schematic diagram of the structure of bispecific antibody JS202-7 CD3-130-012 scFv.
  • Figure 19 Detection results of bispecific antibodies JS202-7 CD3-Fv, JS202-7 012-Fv, JS202-7 CD3 130 012-Fv and JS202-7 binding to human CD3.
  • Figure 20 Detection results of the binding of bispecific antibodies JS202-7 CD3-Fv, JS202-7 012-Fv, JS202-7 CD3 130 012-Fv and JS202-7 to cynomolgus monkey CD3e.
  • Figure 21 The binding detection results of bispecific antibodies JS202-7 CD3-Fv, JS202-7 012-Fv, JS202-7 CD3 130 012-Fv and JS202-7 to CHO Claudin18.2.
  • Figure 22 Activation of T cell downstream signaling pathways by bispecific antibodies JS202-7 CD3-Fv, JS202-7 012-Fv, JS202-7 CD3 130 012-Fv and JS202-7.
  • Figure 23 Activation of T cells by bispecific antibodies JS202-7 CD3-Fv, JS202-7 012-Fv, JS202-7 CD3 130 012-Fv and JS202-7.
  • Figure 24 Research results of cytokine release syndrome induced by bispecific antibodies JS202-7 CD3-Fv, JS202-7 012-Fv, JS202-7 CD3 130 012-Fv and JS202-7.
  • Figure 25 Inhibition of bispecific antibody JS202-7 CD3 130 012-Fv and JS202-7 on the growth of mouse MC38hClaudin18.2 subcutaneously transplanted tumors.
  • Figure 26 Detection results of the binding of bispecific antibodies JS203-7-JSCD3-CD20-Fv and JS203-7-CD3 130-CD20-Fv to CHO-hCD3 cells.
  • Figure 27 Detection results of the binding of bispecific antibodies JS203-7-JSCD3-CD20-Fv and JS203-7-CD3 130-CD20-Fv to Raji cells.
  • Figure 28 The activity detection results of the bispecific antibodies JS203-7-JSCD3-CD20-Fv and JS203-7-CD3 130-CD20-Fv in the luciferase reporter gene system.
  • Figure 29 Detection results of the activation activity of bispecific antibodies JS203-7-JSCD3-CD20-Fv and JS203-7-CD3 130-CD20-Fv on T lymphocytes.
  • Figure 30 Research results of cytokine release syndrome induced by bispecific antibodies JS203-7-JSCD3-CD20-Fv and JS203-7-CD3 130-CD20-Fv.
  • antigen refers to a molecule or a part of a molecule capable of being bound by an antibody of the invention.
  • An antigen may have one or more than one epitope.
  • tumor-associated antigen refers to any molecule (eg, protein, peptide, lipid, carbohydrate, etc.) expressed alone or predominantly or overexpressed by tumor cells such that the antigen is associated with the tumor.
  • a tumor-associated antigen may be an antigen expressed by only one type of tumor such that the tumor antigen is associated with or unique to only one type of tumor.
  • the tumor antigen may be associated or specific tumor antigens of various types of tumors.
  • tumor-associated antigens can be expressed by both breast cancer cells and colon cancer cells, but not by normal, non-tumor, or non-cancerous cells.
  • Exemplary tumor-associated antigens are tumor cell surface antigens, which are more readily recognized by therapeutic and diagnostic antibodies.
  • tumor-associated antigens include, for example, BCMA, CD19, CD20, CD22, CD30, CD33, CD38, CD40, CD123, CDK4, CEA, Claudin 18.2, EGFR, ROR1, TGF-beta, and 5T4.
  • CD3 refers to being part of the T cell receptor complex consisting of three distinct chains CD3 ⁇ , CD38 and CD3 ⁇ . Concentration of CD3 on T cells by, for example, their immobilization by anti-CD3 antibodies results in activation of T cells similar to T cell receptor-mediated activation but independent of TCR clone specificity. 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 indicated.
  • the term encompasses "full length" unprocessed CD3 as well as any form of CD3 or any fragment thereof resulting from intracellular processing.
  • CD3 refers to CD3 full length or a fragment thereof (such as its mature fragment lacking a signal peptide) from human and cynomolgus monkey. In a preferred embodiment, CD3 refers to full length or a fragment thereof (such as its mature fragment lacking a signal peptide) from mouse/rat.
  • CLDN-18.2 or "Claudin 18.2” is one of the 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 indicated.
  • 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 or fragments of CLDN-18.2 from humans and cynomolgus monkeys (such as its mature fragment lacking a signal peptide).
  • CLDN18.2 protein is highly expressed in a variety of tumor tissues, such as gastric cancer (60-80%), pancreatic cancer (50%), esophageal cancer (30-50%) and lung cancer (40-60%), etc., but in normal Little to no expression in tissue.
  • human CD20 refers to human CD20 (UniProtKB/Swiss-Prot No. P11836) and CD20 including CD20 naturally expressed by cells (including tumor cells) or expressed on cells transfected with CD20 gene or cDNA Any variants, isoforms and species homologues.
  • Species homologs include rhesus monkey CD20 (macaca mulatta; UniProtKB/Swiss-Prot No H9YXP1) and cynomolgus monkey.
  • percent (%) amino acid sequence identity or simply “identity” is defined as aligning amino acid sequences (and introducing gaps where necessary) for maximum percent sequence identity and not considering any conservative substitutions as 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. Alignment of sequences to determine percent amino acid sequence identity can be performed using various methods in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • immune response refers to the action of, for example, lymphocytes, antigen-presenting cells, phagocytes, granulocytes, and the production of soluble macromolecules (including antibodies, cytokines, and complement) by such cells or the liver, which results in selective Damage, destroy, or eliminate invading pathogens, pathogen-infected cells or tissues, cancer cells, or in the case of autoimmunity or pathological inflammation, normal human cells or tissues.
  • signal transduction pathway or “signal transduction activity” refers to a biochemical causal relationship, usually initiated by protein-protein interactions, such as the binding of a growth factor to a receptor, that results in the transmission of a signal from one part of a cell to another part of the cell. part.
  • delivery involves specific phosphorylation of one or more tyrosine, serine, or threonine residues on one or more proteins in a cascade of reactions resulting in signal transduction.
  • the penultimate process often involves nuclear events, resulting in changes in gene expression.
  • activity or “biological activity”, or the terms “biological property” or “biological signature” are used interchangeably herein, including but not limited to epitope/antigen affinity and specificity, neutralization or antagonism of CLDN in vivo or in vitro - 18.2 Potency of activity, IC50, in vivo stability of antibody and immunogenic properties of antibody.
  • Other identifiable biological properties or characteristics of antibodies known in the art include, for example, cross-reactivity (i.e., often with non-human homologues of the targeting peptide, or with other proteins or tissues), and retention The ability to express proteins 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 (such as bispecific antibodies), humanized antibodies, fully human antibodies, Chimeric antibodies and camelized single domain antibodies.
  • the basic antibody structural unit is known to comprise tetramers. Each tetramer comprises two identical pairs of polypeptide chains, each pair having one "light” chain (about 25 kDa) and one "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.
  • 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 commonly classified as u, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.
  • the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D” region of about 10 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 a binding compound, and in this context 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 imply the complete absence of such substances or the absence of water, buffers or salts, unless they are present in amounts that significantly interfere with the experimental or therapeutic use of the binding compounds described herein.
  • the term "monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of 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 a large number of antibodies directed against (or specific for) different epitopes.
  • the modifier "monoclonal” characterizes an antibody obtained from a substantially homogeneous population of antibodies and should not be construed as requiring that the antibody be produced by any particular method.
  • multispecific antibody refers to an antibody comprising two or more antigen-binding domains capable of binding two or more different epitopes (e.g., two, three, four or more different epitope), the epitope can be on the same or a different antigen on the antibody.
  • multispecific antibodies include "bispecific antibodies” that bind two different antigens or two different epitopes.
  • full-length antibody refers to an immunoglobulin molecule that, as it occurs in nature, comprises four peptide chains: two heavy (H) chains (about 50-70 kDa in full length) and two light (L) chains (about 25 kDa) are interconnected by disulfide bonds.
  • Each heavy chain is composed 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 is composed 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 complementarity determining regions (CDRs) of high variability separated 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.
  • heavy chain constant region or "CH” are used interchangeably herein to refer to a region comprising at least three heavy chain constant domains (CH1, CH2 and CH3).
  • Non-limiting examples of human heavy chain constant regions include gamma, delta, and alpha.
  • Non-limiting examples of human heavy chain constant regions also include ⁇ and ⁇ .
  • Each constant region corresponds to an antibody isotype.
  • an antibody comprising a gamma constant region is an IgG antibody
  • an antibody comprising a delta constant region is an IgD antibody
  • an antibody comprising an alpha constant region is an IgA antibody.
  • antibodies comprising a mu constant region are IgM antibodies
  • antibodies comprising an epsilon constant region are IgE antibodies.
  • IgG antibodies include but are not limited to IgG1 (comprising ⁇ 1 constant region), IgG2 (comprising ⁇ 2 constant region), IgG3 (comprising ⁇ 3 constant region) and IgG4 (comprising ⁇ 4 constant region) antibodies;
  • IgA antibodies include but are not limited to IgA1 (comprising ⁇ 1 constant region) and IgA2 (comprising the ⁇ 2 constant region) antibodies;
  • IgM antibodies include, but are not limited to, IgM1 and IgM2.
  • CH1 domain or “CH1” or “heavy chain constant region 1” are used interchangeably herein to include the first (mostly amino-terminal) constant region domain of an immunoglobulin heavy chain, which is obtained, for example, from Kabat Approximately position 114-223 (EU position 118-215) in the numbering system extends.
  • the CH1 domain is adjacent to the VH domain and the amino terminus of the hinge region of an immunoglobulin heavy chain molecule and does not form part of the Fc region of an immunoglobulin heavy chain, e.g. human IgG1 CH1 domain, IgG2 CH1 domain, IgG3 CH1 structure domain and IgG4CH1 domain; and naturally occurring variants thereof.
  • CH2 domain or “CH2” or “heavy chain constant region 2" are used interchangeably herein to include that portion of a heavy chain immunoglobulin molecule e.g. from approximately positions 244-360 in the Kabat numbering system (EU position 231-340) extension.
  • the CH2 domain is unique in that it is not closely paired with another domain. Instead, two N-linked branched carbohydrate chains are inserted between the two CH2 domains of the intact native IgG molecule.
  • CH3 domain or “CH3” or “heavy chain constant region 3” are used interchangeably herein to include that portion of a heavy chain immunoglobulin molecule that extends from the terminus of the CH2 domain, e.g. from the Kabat numbering system. Approximately positions 361-476 (EU positions 341-445) extend for approximately 110 residues.
  • the CH3 domain usually forms the C-terminal portion of an antibody. In some immunoglobulins, however, additional domains may extend from the CH3 domain to form the C-terminal portion of the molecule (eg, the CH4 domain in the ⁇ chain of IgM and the ⁇ chain of IgE).
  • light chain constant region or "CL” are used interchangeably herein to refer to the region comprising the light chain constant domain (CL).
  • Non-limiting examples of light chain constant regions include lambda and kappa.
  • antigen-binding fragment of an antibody includes fragments or derivatives of the antibody, typically including at least a fragment of the antigen-binding region or variable region (e.g., one or more CDRs) of the parent antibody, which retains the parent antibody. At least some binding specificity of the antibody.
  • antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv and dsFv fragments; diabodies; linear antibodies; single-chain antibody molecules such as scFv; sdAb) and multispecific antibodies.
  • the binding fragment or derivative typically retains at least 10% of its antigen binding activity when the antigen binding activity is expressed on a molar concentration 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 appreciably alter their biological activity (referred to as “conservative variants” or “functionally conservative variants” of the antibody).
  • binding compound refers to both antibodies and binding fragments thereof.
  • Fab or "Fab molecule” generally refers to a fragment containing the variable domain of the heavy chain (VH) and the variable domain of the light chain (VL), and also contains the constant domain of the light chain ( CL) and the first constant domain (CH1) of the heavy chain.
  • Fab' generally refers to a fragment that differs from Fab by the addition of a few residues at the carboxy-terminus of the CH1 domain of the heavy chain, including one or more cysteines from the antibody hinge region.
  • F(ab')2 generally refers to a dimer of Fab', an antibody fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region.
  • Fv or "Fv molecule” generally refers to the smallest antibody fragment that contains the complete antigen recognition and binding site; in some cases, the fragment may consist of a heavy chain variable domain and a light chain variable domain in close Non-covalently bound dimers.
  • dsFv generally refers to a disulfide-stabilized Fv fragment in which the linkage between a single light chain variable domain and a single heavy chain variable domain is a disulfide bond.
  • sdAb fragment generally refers to an antibody fragment consisting of a VH domain.
  • single-chain Fv or "scFv” antibody refers to an antibody fragment comprising the VH and VL domains of the antibody, wherein these domains are present in a single polypeptide chain.
  • Fv polypeptides generally also comprise a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for antigen binding.
  • Fc or “Fc region” or “Fc fragment” or “Fc domain” are used interchangeably herein to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region.
  • the term includes native sequence Fc regions and variant Fc regions.
  • a native immunoglobulin "Fc domain” comprises two or three constant domains, a CH2 domain, a CH3 domain and optionally a CH4 domain.
  • the immunoglobulin Fc domain comprises the second and third constant domains (CH2 and CH3 domains) derived from the two heavy chains of antibodies of the IgG, IgA and IgD classes;
  • the Fc region herein may also comprise a hinge region.
  • hinge region refers to the proline-rich, easy-to-bend polypeptide chain located between CH1 and CH2 in an antibody.
  • domain antibody is an immunologically functional immunoglobulin fragment containing only the variable region of the heavy chain or the variable region of the light chain.
  • two or more VH regions are covalently linked with a peptide linker to form a bivalent domain antibody.
  • the two VH regions of a bivalent domain antibody can target the same or different antigens.
  • valency refers to the number of potential antigen-binding sites in a polypeptide. Each antigen binding site specifically binds to one antigen molecule or specific site on the antigen molecule. When a polypeptide comprises more than one antigen-binding site, each antigen-binding site can specifically bind the same or different molecules (for example, can bind different ligands or different antigens, or bind different epitopes on the same antigen). bits).
  • bivalent means that the antibody comprises two antigen combining sites.
  • trivalent means that the antibody comprises three antigen combining sites.
  • binding domain or "antigen-binding domain” or “antigen-binding site” refers to the portion of an antibody that includes a portion that specifically binds to, and is complementary to, part or all of an antigen. area. When an antigen is large, antibodies may only be able to bind to a specific part of the antigen, called an epitope. Binding domains may comprise heavy and light chain variable domains (VH and VL), each comprising four conserved framework regions (FRs) and three CDRs. The CDRs vary in sequence and determine specificity for a particular antigen.
  • VH and VL heavy and light chain variable domains
  • FRs conserved framework regions
  • 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 and second antibodies are from different species.
  • the variable domains are derived from an antibody of a rodent, etc. (the "parent antibody”), while the constant domain sequences are derived from a human antibody such that the resulting chimeric antibody induces greater The likelihood of an adverse immune response is low.
  • humanized antibody refers to forms of antibodies that contain sequences from both human and non-human (eg, mouse, rat) antibodies.
  • a humanized antibody will 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 correspond to those of a non-human immunoglobulin.
  • Framework (FR) regions are the framework regions of human immunoglobulin sequences.
  • a humanized antibody optionally can comprise at least a portion of a human immunoglobulin constant region (Fc).
  • Fully human antibody refers to an antibody that comprises only human immunoglobulin protein sequences. Fully human antibodies may contain murine sugar chains as 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 antibody class (eg, IgM, IgE, IgG such as IgG1, IgG2 or IgG4) that is contributed by the heavy chain constant region genes.
  • Isotypes also include modified forms of one of these classes, wherein modifications have been made to alter Fc function, for example to enhance or decrease effector function or binding to Fc receptors.
  • 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 the tertiary folding of the protein.
  • binding refers to the binding of an antibody to a predetermined epitope on an antigen. Specific binding can be measured, for example, by determining the binding of a molecule compared to the binding of a control molecule, typically a molecule of a similar structure that does not have binding activity. For example, specific binding can be determined by competition with a control molecule similar to the target.
  • 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 and 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 for measuring affinity is the ForteBio Kinetic Binding Assay herein.
  • not binding to proteins or cells refers to not binding to proteins or cells, or not binding to them with high affinity, that is, the KD of binding proteins or cells is 1.0 ⁇ 10 -6 M or higher, more preferably 1.0 ⁇ 10 -6 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 an IgG antibody means that the KD for the antigen is 1.0 ⁇ 10 -6 M or lower, preferably 5.0 ⁇ 10 -8 M or lower, more preferably 1.0 ⁇ 10 -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 for other antibody subtypes.
  • "high affinity" binding of IgM subtype means a KD of 10 -6 M or lower, preferably 10 -7 M or lower, more preferably 10 -8 M or lower.
  • antibody-dependent cellular 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, bound 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 the membrane attack complex and lysis of target cells.
  • the antibodies of the present invention when combined with Claudin 18.2, elicit CDC on 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 analogs of known natural nucleotides that have similar binding properties to the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides (see, in Kariko et al. Al's US Patent No. 8,278,036, which discloses mRNA molecules in which uridine is replaced by pseudouridine, methods of synthesizing said 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 complementary sequences, as well as the explicitly indicated sequence.
  • degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is replaced with 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 From any source, capable of integrating into the genome or replicating autonomously, constitutes a polynucleotide molecule in which one or more polynucleotide molecules have been functionally linked (ie, operably linked).
  • Recombinant constructs 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.
  • transcription initiation regulatory sequences that direct transcription of the polynucleotide in a host cell.
  • Both heterologous and non-heterologous (ie, endogenous) promoters can be used to direct expression of the nucleic acids of the invention.
  • 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 a bacterial origin of replication and episomal mammalian vectors).
  • vectors After introduction into a host cell, other vectors (eg, non-episomal mammalian vectors) integrate into the genome of the host cell and thus replicate along 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 upon transformation, transfection or transduction into a host cell.
  • Expression vectors contain one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to provide amplification in the host if desired.
  • Activation can have the same meaning, eg, the cell or receptor is activated, stimulated or treated with a ligand, unless the context dictates otherwise or clearly.
  • 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.
  • treatment refers in one embodiment to ameliorating the disease or condition (ie, slowing or arresting or reducing at least one of the progression of the disease or its clinical symptoms).
  • treating or “curing” refers to alleviating or improving at least one physical parameter, including those physical parameters that may not be discernible by the patient.
  • treating or “curing” refers to modulating a disease or disorder physically (eg, stabilization of discernible symptoms), physiological (eg, stabilization of physical parameters), or both.
  • methods for assessing treatment and/or prevention of disease are generally known in the art.
  • Subject or “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, cows, chickens, amphibians, reptiles, and the like.
  • cyno or “cynomolgus monkey” refers to cynomolgus monkeys.
  • Administration "in conjunction with” one or more other therapeutic agents includes both 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 is effective in preventing Or an amount that improves the symptoms of one or more diseases or conditions or the development of such diseases or conditions.
  • a therapeutically effective dose also refers to an amount of an antibody or antigen-binding fragment thereof sufficient to result in an amelioration of symptoms, eg, an amount that treats, cures, prevents or ameliorates an associated medical condition or increases the rate of treatment, cure, prevention or amelioration of such a condition.
  • a therapeutically effective dose refers to that ingredient only.
  • a therapeutically effective dose refers to the combined amount of the active ingredients which produces the therapeutic effect, whether in combination, sequentially or simultaneously.
  • An effective amount of a therapeutic agent will result in an increase in diagnostic criteria or parameters of at least 10%; usually at least 20%; preferably at least about 30%; more preferably at least 40%, most preferably at least 50%.
  • Cancer and “cancerous” refer to or describe the physiological condition in mammals that is often characterized by unregulated cell growth. Included in this definition are benign and malignant cancers as well as dormant tumors or micrometastases. Cancers can be solid tumors and hematological tumors, including but 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 carcinoma, hepatocellular carcinoma, carcinoma of the stomach or gastric cancer ( Including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma (hepatoma), breast cancer, colon cancer, colorectal cancer, endometrial cancer or uterine cancer, Salivary gland cancer, kidney cancer or cancer of the kidney, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, and various types of head and neck cancer, and B-cell lymphoma (including low-grade/follicular non-Hodgkin King's lymphoma (NHL), small lymphocytic (SL) NHL, intermediate/follicular NHL, intermediate diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblast
  • NHL low
  • Cytokine storm refers to a variety of cytokines in body fluids such as TNF- ⁇ , IL-1, IL-6, IL-12, IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , MCP-1
  • Cytokine release syndrome refers to a group of clinical syndromes caused by lymphocyte activation, lysis, and release of a large amount of cytokines after treatment with monoclonal antibodies, cytokines or infection.
  • anti-CD3 antibody refers to the ability to bind the CD3 protein or a fragment thereof with sufficient affinity such that the antibody can be used as a targeting A diagnostic and/or therapeutic agent for CD3.
  • anti-CLDN-18.2 antibody refers to the ability to bind the CLDN-18.2 protein with sufficient affinity or a fragment thereof such that the antibody can be used as a diagnostic and/or therapeutic agent targeting CLDN-18.2.
  • anti-CD20 antibody refers to the ability to bind the CD20 protein or a fragment thereof with sufficient affinity such that the antibody can be used as a targeting Diagnostic and/or therapeutic agents for CD20.
  • the humanized anti-CD3 antibody of the present invention is mutated starting from the antibody JSCD3, and has many unique properties and advantages.
  • the antibody JSCD3 refer to patent applications CN202110468621.2 and PCT/CN2021/113033. 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, good affinity with human and cynomolgus monkey CD3 antigens, and can effectively activate the downstream signaling pathway of T cells; When the bispecific antibody is prepared, it shows stronger tumor killing activity and can reduce the risk of cytokine release syndrome.
  • the humanized sequence disclosed in the present invention has more advantages and characteristics than the humanized sequence of other companies, and it shows better anti-tumor activity when used to prepare new anti-tumor drugs, and its 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 a bispecific antibody comprising a first binding domain, a second binding domain and a third binding domain, wherein the first binding domain binds to CD3, and the second The binding domain and the third binding domain each bind to a tumor-associated antigen, the first binding domain has a form of a Fab molecule or a Fv molecule or a scFv molecule, and the second binding domain has a Fab molecule or a Fv molecule or scFv molecule, and said third binding domain is in the form of a Fab molecule.
  • the first binding domain of the invention is in the form of a Fab molecule
  • the second binding domain is in the form of a Fv molecule or scFv molecule
  • the third binding domain is in the form of a Fab molecule .
  • the first binding domain of the invention is in the form of a Fab molecule
  • the second binding domain is in the form of a Fv molecule
  • the third binding domain is in the form of a Fab molecule.
  • the first binding domain of the invention is in the form of a Fab molecule
  • the second binding domain is in the form of a scFv molecule
  • the third binding domain is in the form of a Fab molecule.
  • the first binding domain of the invention is in the form of an Fv molecule
  • the second binding domain is in the form of a Fab molecule
  • the third binding domain is in the form of a Fab molecule.
  • the tumor-associated antigen of the present invention is selected from BCMA, CD19, CD20, CD22, CD30, CD33, CD38, CD40, CD123, CDK4, CEA, Claudin18.2, EGFR, ROR1, TGF- ⁇ and 5T4;
  • the tumor-associated antigen is Claudin 18.2, CD20 or BCMA.
  • the bispecific antibody of the present invention has four polypeptide chains, wherein the first polypeptide chain has VH1-CH1-connecting peptide-VL2-connecting peptide-hinge region-the first polypeptide chain from N-terminal to C-terminal An Fc region, the second polypeptide chain has VL1-CL-connecting peptide-VH2 from N-terminal to C-terminal; the third polypeptide chain has VH2-CH1-hinge region-second from N-terminal to C-terminal In the Fc region, the fourth polypeptide chain has VL2-CL from the N-terminal to the C-terminal.
  • the VH1 and VL1 form a structural domain
  • the VH2 and VL2 form a structural domain
  • the VH1-CH1 of the first polypeptide chain of the present invention and the VL1-CL of the second polypeptide chain form the first binding domain
  • the VL2 of the first polypeptide chain and the VL1-CL of the second polypeptide chain form the first binding domain
  • VH2 of the second polypeptide chain forms the second binding domain
  • VH2-CH1 of the third polypeptide chain and VL2-CL of the fourth polypeptide chain form the third binding domain.
  • the bispecific antibody of the present invention has four polypeptide chains, wherein the first polypeptide chain has VH1-CH1-connecting peptide-VL2-connecting peptide-VH2-connecting peptide from N-terminus to C-terminus -hinge region-first Fc region, the second polypeptide chain has VL1-CL from N-terminus to C-terminus; the third polypeptide chain has VH2-CH1-hinge region-second from N-terminus to C-terminus In the Fc region, the fourth polypeptide chain has VL2-CL from the N-terminal to the C-terminal.
  • the VH1 and VL1 form a structural domain
  • the VH2 and VL2 form a structural domain
  • the VH1-CH1 of the first polypeptide chain of the present invention and the VL1-CL of the second polypeptide chain form the first binding domain
  • the VL2-CL of the first polypeptide chain The connecting peptide-VH2 forms the second binding domain
  • the VH2-CH1 of the third polypeptide chain and the VL2-CL of the fourth polypeptide chain form the third binding domain.
  • the bispecific antibody of the present invention has four polypeptide chains, wherein the first polypeptide chain has VH2-CH1-connecting peptide-VL1-connecting peptide-hinge region-the first polypeptide chain from N-terminal to C-terminal An Fc region, the second polypeptide chain has VL2-CL-connecting peptide-VH1 from N-terminal to C-terminal; the third polypeptide chain has VH2-CH1-hinge region-second from N-terminal to C-terminal In the Fc region, the fourth polypeptide chain has VL2-CL from the N-terminal to the C-terminal.
  • the VH2-CH1 of the first polypeptide chain of the present invention and the VL2-CL of the second polypeptide chain form a second binding domain
  • the VL1 of the first polypeptide chain and the VH1 of the second polypeptide chain forms a first binding domain
  • VH2-CH1 of the third polypeptide chain forms a third binding domain with VL2-CL of the fourth polypeptide chain.
  • the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 contained in the first binding domain of the present invention are respectively:
  • HCDR1 X1YAMN (SEQ ID NO: 168), wherein X1 is T or K;
  • HCDR2 RIRSKYNNYATYYAX2SVKX3 (SEQ ID NO: 169), wherein X2 is D or A, X3 is D or G;
  • HCDR3 HGX3X4GX5X6X7X8X9X10X11AY (SEQ ID NO: 170), wherein, X3 is N or A, X4 is F or A, X5 is N or A, X6 is S or A, X7 is Y or A, X8 is V or A, X9 is S or A, X10 is W or A, X11 is F or A;
  • LCDR1 X12SSTGAVTTX13NYX14N (SEQ ID NO: 171), wherein X12 is R or G, X13 is S or G, and X14 is A or P;
  • LCDR2 GTNKRAP (SEQ ID NO: 172);
  • LCDR3 AX15X16X17X18X19X20X21X22 (SEQ ID NO: 173), wherein, X15 is L or A, X16 is W or A, X17 is Y or A, X18 is S or A, X19 is N or A, X20 is L or A, X21 It is W or A, and X22 is V or A.
  • the first binding domain of the present invention comprises: HCDR1 with an amino acid sequence as shown in SEQ ID NO: 1 or 2, HCDR2 with an amino acid sequence as shown in SEQ ID NO: 3 or 4, and an amino acid sequence as shown in HCDR3 shown in any one of SEQ ID NO: 5-22, the amino acid sequence is LCDR1 shown in SEQ ID NO: 23 or 24, the amino acid sequence is LCDR2 shown in SEQ ID NO: 25, and the amino acid sequence is shown in SEQ ID NO: NO: LCDR3 shown in any one of 26-42.
  • the first binding domain of the present invention comprises HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3; wherein, the HCDR1, HCDR2, HCDR3 are:
  • the amino acid sequence is HCDR1 shown in SEQ ID NO: 1, the amino acid sequence is HCDR2 shown in SEQ ID NO: 3, and the amino acid sequence is HCDR3 shown in SEQ ID NO: 5; or
  • amino acid sequence is HCDR1 shown in SEQ ID NO: 1, the amino acid sequence is HCDR2 shown in SEQ ID NO: 3, and the amino acid sequence is HCDR3 shown in SEQ ID NO: 15; or
  • amino acid sequence is HCDR1 shown in SEQ ID NO: 2, the amino acid sequence is HCDR2 shown in SEQ ID NO: 4, and the amino acid sequence is HCDR3 shown in any one of SEQ ID NO: 5-22;
  • the LCDR1, LCDR2, LCDR3 are:
  • amino acid sequence is LCDR1 shown in SEQ ID NO: 23, the amino acid sequence is LCDR2 shown in SEQ ID NO: 25, and the amino acid sequence is LCDR3 shown in SEQ ID NO: 26; or
  • amino acid sequence is LCDR1 shown in SEQ ID NO: 23, the amino acid sequence is LCDR2 shown in SEQ ID NO: 25, and the amino acid sequence is LCDR3 shown in SEQ ID NO: 33; or
  • amino acid sequence is LCDR1 shown in SEQ ID NO: 24, the amino acid sequence is LCDR2 shown in SEQ ID NO: 25, and the amino acid sequence is LCDR3 shown in any one of SEQ ID NO: 26-42.
  • the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 contained in the first binding domain of the present invention are respectively:
  • HCDR1 X1YAMN, wherein X1 is T or K (preferably K);
  • HCDR2 RIRSKYNNYATYYAX2SVKX3, wherein X2 is D or A (preferably A), X3 is D or G (preferably G);
  • HCDR3 HGX3X4GX5X6X7X8X9X10X11AY, where X3 is N, X4 is F, X5 is N or A, X6 is S or A, X7 is Y or A, X8 is V or A, X9 is S or A, X10 is W, and X11 is F or A (preferably F);
  • LCDR1 X12SSTGAVTTX13NYX14N, wherein X12 is R or G (preferably G), X13 is S or G (preferably G), X14 is A or P (preferably P);
  • LCDR2 GTNKRAP
  • LCDR3 AX15X16X17X18X19X20X21X22, wherein, X15 is L or A (preferably L), X16 is W, X17 is Y or A (preferably Y), X18 is S or A (preferably S), X19 is N or A (preferably N), X20 is L or A (preferably L), X21 is W, and X22 is V or A.
  • the first binding domain of the present invention comprises: HCDR1 with an amino acid sequence as shown in SEQ ID NO: 1 or 2, HCDR2 with an amino acid sequence as shown in SEQ ID NO: 3 or 4, and an amino acid sequence as shown in HCDR3 shown in SEQ ID NO: 5, 9, 10, 11, 15, 17, 18, 19 or 20, the amino acid sequence is shown in LCDR1 shown in SEQ ID NO: 23 or 24, the amino acid sequence is shown in SEQ ID NO: 25 The LCDR2 shown, and the LCDR3 shown in the amino acid sequence as SEQ ID NO: 26, 28, 30, 33, 35, 36, 37, 39, 40, 41 or 42.
  • the first binding domain of the present invention comprises:
  • Amino acid sequence such as HCDR1 shown in SEQ ID NO: 1, amino acid sequence such as HCDR2 shown in SEQ ID NO: 3, amino acid sequence such as HCDR3 shown in SEQ ID NO: 15, amino acid sequence such as SEQ ID NO: 23
  • the LCDR1 shown, the amino acid sequence is LCDR2 shown in SEQ ID NO: 25, the amino acid sequence is LCDR3 shown in SEQ ID NO: 33; or
  • Amino acid sequence such as HCDR1 shown in SEQ ID NO: 2, amino acid sequence such as HCDR2 shown in SEQ ID NO: 4, amino acid sequence such as HCDR3 shown in SEQ ID NO: 5, amino acid sequence such as SEQ ID NO: 24
  • the LCDR1 shown, the amino acid sequence is LCDR2 shown in SEQ ID NO: 25, the amino acid sequence is LCDR3 shown in any one of SEQ ID NO: 26, 28, 33, 36 or 37; or
  • amino acid sequence such as HCDR1 shown in SEQ ID NO: 2, amino acid sequence such as HCDR2 shown in SEQ ID NO: 4, amino acid sequence such as HCDR3 shown in SEQ ID NO: 10, amino acid sequence such as SEQ ID NO: 24
  • the LCDR1 shown, the amino acid sequence is LCDR2 shown in SEQ ID NO: 25, the amino acid sequence is LCDR3 shown in any one of SEQ ID NO: 28, 33, 40, 41 or 42; or
  • the LCDR1 shown, the amino acid sequence is LCDR2 shown in SEQ ID NO: 25, the amino acid sequence is LCDR3 shown in any one of SEQ ID NO: 26, 28, 33, 35, 36, 37, 40, 41 or 42 ;
  • amino acid sequence such as HCDR1 shown in SEQ ID NO: 2, amino acid sequence such as HCDR2 shown in SEQ ID NO: 4, amino acid sequence such as HCDR3 shown in any one of SEQ ID NO: 9, 10 or 19,
  • the amino acid sequence is LCDR1 shown in SEQ ID NO: 23, the amino acid sequence is LCDR2 shown in SEQ ID NO: 25, and the amino acid sequence is LCDR3 shown in SEQ ID NO: 26; or
  • amino acid sequence such as HCDR1 shown in SEQ ID NO: 2, amino acid sequence such as HCDR2 shown in SEQ ID NO: 4, amino acid sequence such as HCDR3 shown in SEQ ID NO: 19, amino acid sequence such as SEQ ID NO: 24 As shown in LCDR1, the amino acid sequence is LCDR2 shown in SEQ ID NO: 25, and the amino acid sequence is LCDR3 shown in SEQ ID NO: 36.
  • the first binding domain of the present invention comprises: a VH having an amino acid sequence such as any one of SEQ ID NO: 43-62, and an amino acid sequence such as any one of SEQ ID NO: 63-81 VL shown in item.
  • the first domain of the present invention comprises: an amino acid sequence such as the VH shown in any one of SEQ ID NO: 43-45, 49-51, 55 and 57-60, and an amino acid sequence such as SEQ ID NO: ID NO: VL shown in any one of 63-65, 67, 69, 72, 74-76 and 78-81.
  • the first binding domain of the present invention comprises:
  • VH with amino acid sequence as shown in SEQ ID NO: 44, and VL with amino acid sequence as shown in SEQ ID NO: 64; or
  • VH with amino acid sequence as shown in SEQ ID NO: 45
  • VL with amino acid sequence as shown in SEQ ID NO: 65, 67, 72, 75 or 76
  • VH with amino acid sequence as shown in SEQ ID NO: 50, and VL with amino acid sequence as shown in SEQ ID NO: 67, 72, 79, 80 or 81; or
  • VH with amino acid sequence as shown in SEQ ID NO: 57
  • VL with amino acid sequence as shown in SEQ ID NO: 72, 74, 75, 76 or 81; or
  • VIII a VH having an amino acid sequence as shown in SEQ ID NO: 58, and a VL having an amino acid sequence as shown in SEQ ID NO: 67, 74, 75, 76, 78, 80 or 81; or
  • the second binding domain and the third binding domain of the present invention bind to Claudin 18.2, which respectively comprise: HCDR1 with an amino acid sequence such as SEQ ID NO: 121, and an amino acid sequence such as SEQ ID NO: HCDR2 shown in 122, amino acid sequence such as HCDR3 shown in SEQ ID NO: 123, amino acid sequence such as LCDR1 shown in SEQ ID NO: 124, amino acid sequence such as LCDR2 shown in SEQ ID NO: 125, and amino acid sequence such as SEQ ID NO: ID NO: LCDR3 shown in 126.
  • Claudin 18.2 which respectively comprise: HCDR1 with an amino acid sequence such as SEQ ID NO: 121, and an amino acid sequence such as SEQ ID NO: HCDR2 shown in 122, amino acid sequence such as HCDR3 shown in SEQ ID NO: 123, amino acid sequence such as LCDR1 shown in SEQ ID NO: 124, amino acid sequence such as LCDR2 shown in SEQ ID NO: 125, and amino acid sequence such as
  • the second binding domain and the third binding domain of the present invention bind to CD20, which respectively comprise: HCDR1 with an amino acid sequence such as SEQ ID NO: 127, and an amino acid sequence such as SEQ ID NO: 128 As shown in HCDR2, the amino acid sequence is HCDR3 shown in SEQ ID NO: 129, the amino acid sequence is LCDR1 shown in SEQ ID NO: 130, the amino acid sequence is LCDR2 shown in SEQ ID NO: 131, and the amino acid sequence is shown in SEQ ID NO: LCDR3 shown in 132.
  • the second binding domain and the third binding domain of the present invention bind to Claudin 18.2, which respectively comprise: (a) a VH with an amino acid sequence as shown in SEQ ID NO: 133, and an amino acid sequence as shown in The VL shown in SEQ ID NO: 134; or (b) the VH with the amino acid sequence shown in SEQ ID NO: 166, and the VL with the amino acid sequence shown in SEQ ID NO: 167.
  • the second binding domain and the third binding domain of the present invention bind to CD20, which respectively comprise: a VH with an amino acid sequence such as SEQ ID NO: 135, and an amino acid sequence such as SEQ ID NO: 136 VL shown.
  • the first Fc region of the present invention is knob-Fc
  • the second Fc region is hole-Fc
  • the first Fc region of the present invention is connected to the second Fc region through a disulfide bond in the hinge region and a knob-and-hole structure.
  • the first Fc region and/or the second Fc region of the present invention is an Fc region from IgG, preferably an Fc region from IgG1, IgG2, IgG3 or IgG4.
  • the CH1 of the present invention is CH1 from IgG, preferably CH1 from IgG1, IgG2, IgG3 or IgG4.
  • the CL of the invention is a CL from a lambda or kappa light chain.
  • the connecting peptide of the present invention can be a flexible polypeptide comprising G (glycine), S (serine) and A (alanine) or composed of G and S, and its length can be 2-30 amino acid residues ; Further preferably, the connecting peptide includes but not limited to (GS)n, (GGS)n, (GGGS)n, (GGGGS)n and AS(GGGGS)n, etc., n can be an integer of 1-6, such as 1, 2, 3, 4, 5, 6.
  • HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of the first binding domain of the present invention are from any monoclonal antibody whose ratio of sample MFI to JSCD3MFI in Table 2-5 is between 0.30-0.80 HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3.
  • the bispecific antibody of the present invention has four polypeptide chains, and the four polypeptide chains are:
  • the first polypeptide chain has an amino acid sequence as shown in SEQ ID NO: 144
  • the second polypeptide chain has an amino acid sequence as shown in SEQ ID NO: 145
  • the third polypeptide chain Having the amino acid sequence shown in SEQ ID NO: 142
  • the fourth polypeptide chain has the amino acid sequence shown in SEQ ID NO: 143; or
  • the first polypeptide chain has an amino acid sequence as shown in SEQ ID NO: 146
  • the second polypeptide chain has an amino acid sequence as shown in SEQ ID NO: 147
  • the third polypeptide chain Having the amino acid sequence shown in SEQ ID NO: 142
  • the fourth polypeptide chain has the amino acid sequence shown in SEQ ID NO: 143; or
  • the first polypeptide chain has an amino acid sequence as shown in SEQ ID NO: 148
  • the second polypeptide chain has an amino acid sequence as shown in SEQ ID NO: 149
  • the third polypeptide chain Having the amino acid sequence shown in SEQ ID NO: 142
  • the fourth polypeptide chain has the amino acid sequence shown in SEQ ID NO: 143; or
  • the first polypeptide chain has an amino acid sequence as shown in SEQ ID NO: 150
  • the second polypeptide chain has an amino acid sequence as shown in SEQ ID NO: 151
  • the third polypeptide chain Having the amino acid sequence shown in SEQ ID NO: 152
  • the fourth polypeptide chain has the amino acid sequence shown in SEQ ID NO: 153; or
  • the first polypeptide chain has an amino acid sequence as shown in SEQ ID NO: 154
  • the second polypeptide chain has an amino acid sequence as shown in SEQ ID NO: 155
  • the third polypeptide chain Having the amino acid sequence shown in SEQ ID NO: 152
  • the fourth polypeptide chain has the amino acid sequence shown in SEQ ID NO: 153; or
  • the first polypeptide chain has an amino acid sequence as shown in SEQ ID NO: 156
  • the second polypeptide chain has an amino acid sequence as shown in SEQ ID NO: 157
  • the third polypeptide chain Having the amino acid sequence shown in SEQ ID NO: 152
  • the fourth polypeptide chain has the amino acid sequence shown in SEQ ID NO: 153; or
  • the first polypeptide chain has the amino acid sequence shown in SEQ ID NO: 164
  • the second polypeptide chain has the amino acid sequence shown in SEQ ID NO: 165
  • the third polypeptide chain It has the amino acid sequence shown in SEQ ID NO: 142
  • the fourth polypeptide chain has the amino acid sequence shown in SEQ ID NO: 143.
  • the present invention provides an antibody or antigen-binding fragment thereof comprising a CD3-binding binding domain, wherein the CD3-binding binding domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 They are:
  • HCDR1 X1YAMN, where X1 is T or K;
  • HCDR2 RIRSKYNNYATYYAX2SVKX3, where X2 is D or A, X3 is D or G;
  • HCDR3 HGX3X4GX5X6X7X8X9X10X11AY, where X3 is N or A, X4 is F or A, X5 is N or A, X6 is S or A, X7 is Y or A, X8 is V or A, X9 is S or A, X10 is W or A, X11 is F or A;
  • LCDR1 X12SSTGAVTTX13NYX14N, where X12 is R or G, X13 is S or G, X14 is A or P;
  • LCDR2 GTNKRAP
  • LCDR3 AX15X16X17X18X19X20X21X22, among them, X15 is L or A, X16 is W or A, X17 is Y or A, X18 is S or A, X19 is N or A, X20 is L or A, X21 is W or A, X22 is V or A;
  • the amino acid sequence is as shown in SEQ ID NO: HCDR1
  • the amino acid sequence is as shown in SEQ ID NO: 3 HCDR2 shown
  • amino acid sequence such as HCDR3 shown in SEQ ID NO: 5
  • amino acid sequence such as LCDR1 shown in SEQ ID NO: 23
  • amino acid sequence such as LCDR2 shown in SEQ ID NO: 25
  • amino acid sequence such as SEQ ID NO: LCDR3 shown in 26.
  • the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 included in the CD3-binding binding domain are:
  • HCDR1 X1YAMN, wherein X1 is T or K (preferably K);
  • HCDR2 RIRSKYNNYATYYAX2SVKX3, wherein X2 is D or A (preferably A), X3 is D or G (preferably G);
  • HCDR3 HGX3X4GX5X6X7X8X9X10X11AY, where X3 is N, X4 is F, X5 is N or A, X6 is S or A, X7 is Y or A, X8 is V or A, X9 is S or A, X10 is W, and X11 is F or A (preferably F);
  • LCDR1 X12SSTGAVTTX13NYX14N, wherein X12 is R or G (preferably G), X13 is S or G (preferably G), X14 is A or P (preferably P);
  • LCDR2 GTNKRAP
  • LCDR3 AX15X16X17X18X19X20X21X22, wherein, X15 is L or A (preferably L), X16 is W, X17 is Y or A (preferably Y), X18 is S or A (preferably S), X19 is N or A (preferably N), X20 is L or A (preferably L), X21 is W, X22 is V or A;
  • the amino acid sequence is as shown in SEQ ID NO: HCDR1
  • the amino acid sequence is as shown in SEQ ID NO: 3 HCDR2 shown
  • amino acid sequence such as HCDR3 shown in SEQ ID NO: 5
  • amino acid sequence such as LCDR1 shown in SEQ ID NO: 23
  • amino acid sequence such as LCDR2 shown in SEQ ID NO: 25
  • amino acid sequence such as SEQ ID NO: LCDR3 shown in 26.
  • the CD3 binding domain of the present invention comprises: HCDR1 with an amino acid sequence as shown in SEQ ID NO: 1 or 2, HCDR2 with an amino acid sequence as shown in SEQ ID NO: 3 or 4, and an amino acid sequence HCDR3 as shown in any one of SEQ ID NOs: 5-22, the amino acid sequence as LCDR1 shown in SEQ ID NO: 23 or 24, the amino acid sequence as LCDR2 shown in SEQ ID NO: 25, and the amino acid sequence as SEQ ID NO: LCDR3 shown in any one of ID NO: 26-42.
  • the CD3 binding domains of the present invention comprise HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3:
  • the HCDR1, HCDR2, and HCDR3 are:
  • the amino acid sequence is HCDR1 shown in SEQ ID NO: 1, the amino acid sequence is HCDR2 shown in SEQ ID NO: 3, and the amino acid sequence is HCDR3 shown in SEQ ID NO: 5; or
  • amino acid sequence is HCDR1 shown in SEQ ID NO: 1, the amino acid sequence is HCDR2 shown in SEQ ID NO: 3, and the amino acid sequence is HCDR3 shown in SEQ ID NO: 15; or
  • amino acid sequence is HCDR1 shown in SEQ ID NO: 2, the amino acid sequence is HCDR2 shown in SEQ ID NO: 4, and the amino acid sequence is HCDR3 shown in any one of SEQ ID NO: 5-22;
  • the LCDR1, LCDR2, LCDR3 are:
  • amino acid sequence is LCDR1 shown in SEQ ID NO: 23, the amino acid sequence is LCDR2 shown in SEQ ID NO: 25, and the amino acid sequence is LCDR3 shown in SEQ ID NO: 26; or
  • amino acid sequence is LCDR1 shown in SEQ ID NO: 23, the amino acid sequence is LCDR2 shown in SEQ ID NO: 25, and the amino acid sequence is LCDR3 shown in SEQ ID NO: 33; or
  • amino acid sequence is LCDR1 shown in SEQ ID NO: 24, the amino acid sequence is LCDR2 shown in SEQ ID NO: 25, and the amino acid sequence is LCDR3 shown in any one of SEQ ID NO: 26-42.
  • the CD3 binding domain of the present invention comprises: HCDR1 with an amino acid sequence as shown in SEQ ID NO: 1 or 2, HCDR2 with an amino acid sequence as shown in SEQ ID NO: 3 or 4, and an amino acid sequence HCDR3 as shown in SEQ ID NO: 5, 9, 10, 11, 15, 17, 18, 19 or 20, the amino acid sequence is as shown in SEQ ID NO: 23 or 24 LCDR1, the amino acid sequence is as SEQ ID NO: 25 LCDR2 as shown, and LCDR3 with amino acid sequence as shown in SEQ ID NO: 26, 28, 30, 33, 35, 36, 37, 39, 40, 41 or 42.
  • the CD3-binding binding domain of the present invention comprises:
  • Amino acid sequence such as HCDR1 shown in SEQ ID NO: 1, amino acid sequence such as HCDR2 shown in SEQ ID NO: 3, amino acid sequence such as HCDR3 shown in SEQ ID NO: 15, amino acid sequence such as SEQ ID NO: 23
  • the LCDR1 shown, the amino acid sequence is LCDR2 shown in SEQ ID NO: 25, the amino acid sequence is LCDR3 shown in SEQ ID NO: 33; or
  • Amino acid sequence such as HCDR1 shown in SEQ ID NO: 2, amino acid sequence such as HCDR2 shown in SEQ ID NO: 4, amino acid sequence such as HCDR3 shown in SEQ ID NO: 5, amino acid sequence such as SEQ ID NO: 24 LCDR1 shown, the amino acid sequence of LCDR2 shown in SEQ ID NO: 25, the amino acid sequence of LCDR3 shown in SEQ ID NO: 26, 28, 33, 36 or 37; or
  • amino acid sequence such as HCDR1 shown in SEQ ID NO: 2, amino acid sequence such as HCDR2 shown in SEQ ID NO: 4, amino acid sequence such as HCDR3 shown in SEQ ID NO: 10, amino acid sequence such as SEQ ID NO: 24 the LCDR1 shown, the amino acid sequence of LCDR2 shown in SEQ ID NO: 25, the amino acid sequence of LCDR3 shown in SEQ ID NO: 28, 33, 40, 41 or 42; or
  • Amino acid sequence such as HCDR1 shown in SEQ ID NO: 2, amino acid sequence such as HCDR2 shown in SEQ ID NO: 4, amino acid sequence such as HCDR3 shown in SEQ ID NO: 18, amino acid sequence such as SEQ ID NO: 24 the LCDR1 shown, the amino acid sequence of LCDR2 shown in SEQ ID NO: 25, the amino acid sequence of LCDR3 shown in SEQ ID NO: 28, 35, 36, 37, 39, 41 or 42; or
  • the CD3 binding domain of the present invention comprises: an amino acid sequence such as a VH shown in any one of SEQ ID NOs: 43-62, and an amino acid sequence such as any one of SEQ ID NOs: 63-81 One VL shown.
  • the CD3-binding binding domain of the present invention comprises: VH having an amino acid sequence as shown in any one of SEQ ID NO: 43-45, 49-51, 55, and 57-60, and an amino acid sequence VL as set forth in any one of SEQ ID NOs: 63-65, 67, 69, 72, 74-76 and 78-81.
  • the CD3-binding binding domain of the present invention comprises:
  • VH with amino acid sequence as shown in SEQ ID NO: 44, and VL with amino acid sequence as shown in SEQ ID NO: 64; or
  • VH with amino acid sequence as shown in SEQ ID NO: 45
  • VL with amino acid sequence as shown in SEQ ID NO: 65, 67, 72, 75 or 76
  • VH with amino acid sequence as shown in SEQ ID NO: 50, and VL with amino acid sequence as shown in SEQ ID NO: 67, 72, 79, 80 or 81; or
  • VH with amino acid sequence as shown in SEQ ID NO: 57
  • VL with amino acid sequence as shown in SEQ ID NO: 72, 74, 75, 76 or 81; or
  • VIII VH with amino acid sequence as shown in SEQ ID NO: 58, and VL with amino acid sequence as shown in SEQ ID NO: 67, 74, 75, 76, 78, 80 or 81; or
  • the antibody of the present invention comprises a heavy chain with an amino acid sequence as shown in any one of SEQ ID NO: 82-101, and an amino acid sequence as shown in any one of SEQ ID NO: 102-120 light chain.
  • the heavy chain amino acid sequence of the antibody of the present invention is shown in any one of SEQ ID NO: 82-84, 88-90, 94 and 96-99, and the light chain amino acid sequence is shown in SEQ ID NO: Any one of 102-104, 106, 108, 111, 113-115 and 117-120 is shown.
  • the antibodies of the invention comprise:
  • VII a heavy chain having an amino acid sequence as set forth in SEQ ID NO: 96, and a light chain having an amino acid sequence as set forth in SEQ ID NO: 111, 113, 114, 115 or 120; or
  • the antibody of the present invention is a monoclonal antibody, a bispecific antibody or a multispecific antibody, and the antigen-binding fragment is Fab, Fab', F(ab')2, Fv, dsFv, scFv or sdAb.
  • the antibody of the present invention is a monoclonal antibody specifically binding to CD3, and its HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are as described in any of the preceding embodiments; or its VH and VL are as described in any of the foregoing As described in one embodiment, or its CH and CL are as described in any of the preceding embodiments; the proviso is that the VH of the monoclonal antibody is not the amino acid sequence shown in SEQ ID NO: 43, and the VL is not SEQ ID NO: 63
  • the amino acid sequence shown, or the CH of the monoclonal antibody is not the amino acid sequence shown in SEQ ID NO:82, and the CL is not the amino acid sequence shown in SEQ ID NO:102.
  • the antibody of the present invention is a monoclonal antibody specifically binding to CD3, and its HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are from the samples in Table 2-5.
  • the ratio of MFI to JSCD3MFI is between 0.30-0.80 HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of any monoclonal antibody, or its VH and VL are from any monoclonal antibody whose ratio of sample MFI to JSCD3MFI in Table 2-5 is between 0.30-0.80, Or its CH and CL come from the CH and CL of any monoclonal antibody whose ratio of sample MFI to JSCD3MFI in Table 2-5 is between 0.30-0.80.
  • the antibody or antigen-binding fragment thereof of the present invention further comprises one or two binding domains that bind tumor-associated antigens.
  • the tumor-associated antigen of the present invention is selected from BCMA, CD19, CD20, CD22, CD30, CD33, CD38, CD40, CD123, CDK4, CEA, Claudin 18.2, EGFR, ROR1, TGF- ⁇ and 5T4;
  • the tumor-associated antigen is Claudin 18.2, CD20 or BCMA.
  • the tumor-associated antigen of the present invention is Claudin 18.2 or CD20.
  • the binding domain of the present invention that binds to Claudin 18.2 comprises: HCDR1 with an amino acid sequence such as SEQ ID NO: 121, HCDR2 with an amino acid sequence such as SEQ ID NO: 122, and an amino acid sequence such as SEQ ID HCDR3 shown in NO:123, LCDR1 shown in amino acid sequence as SEQ ID NO:124, LCDR2 shown in amino acid sequence as SEQ ID NO:125, and LCDR3 shown in amino acid sequence as SEQ ID NO:126.
  • the CD20-binding binding domain of the present invention comprises: HCDR1 with an amino acid sequence such as SEQ ID NO: 127, HCDR2 with an amino acid sequence such as SEQ ID NO: 128, and an amino acid sequence such as SEQ ID NO HCDR3 shown in: 129, LCDR1 shown in amino acid sequence as SEQ ID NO: 130, LCDR2 shown in amino acid sequence as SEQ ID NO: 131, and LCDR3 shown in amino acid sequence as SEQ ID NO: 132.
  • the binding domain of the present invention that binds to Claudin 18.2 comprises: (a) a VH with an amino acid sequence as shown in SEQ ID NO: 133, and a VL with an amino acid sequence as shown in SEQ ID NO: 134; or (b) VH whose amino acid sequence is shown in SEQ ID NO: 166, and VL whose amino acid sequence is shown in SEQ ID NO: 167.
  • the CD20-binding binding domain of the present invention comprises: a VH with an amino acid sequence as shown in SEQ ID NO: 135, and a VL with an amino acid sequence as shown in SEQ ID NO: 136.
  • the present invention provides an antibody (preferably a bispecific antibody) comprising a binding domain that binds CD3 and a binding domain that binds CD20, wherein the binding domain that binds CD3 comprises:
  • the amino acid sequence is HCDR1 shown in SEQ ID NO: 1, the amino acid sequence is HCDR2 shown in SEQ ID NO: 3, the amino acid sequence is HCDR3 shown in SEQ ID NO: 5, and the amino acid sequence is shown in SEQ ID NO: 23 LCDR1, the amino acid sequence is LCDR2 shown in SEQ ID NO: 25, and the amino acid sequence is LCDR3 shown in SEQ ID NO: 26;
  • the binding domain that binds CD20 includes: HCDR1 with an amino acid sequence shown in SEQ ID NO: 127 , the amino acid sequence is HCDR2 shown in SEQ ID NO: 128, the amino acid sequence is HCDR3 shown in SEQ ID NO: 129, the amino acid sequence is LCDR1 shown in SEQ ID NO: 130, the amino acid sequence is shown in SEQ ID NO:
  • the present invention provides an antibody (preferably a bispecific antibody) comprising a binding domain that binds CD3 and a binding domain that binds CD20, wherein the binding domain that binds CD3 comprises:
  • the amino acid sequence is VH as shown in SEQ ID NO: 43
  • the amino acid sequence is VL as shown in SEQ ID NO: 63
  • the binding domain for binding CD20 includes: the amino acid sequence is VH as shown in SEQ ID NO: 135, And amino acid sequence such as the VL shown in SEQ ID NO: 136.
  • the Fc region of the antibody of the present invention is Fc from IgG, such as Fc from IgG1, IgG2, IgG3 or IgG4, and/or the CL of the antibody is CL from ⁇ or ⁇ light chain;
  • the Fc region comprises a knob-and-hole structure.
  • the antibody of the present invention has four polypeptide chains, and the four polypeptide chains are:
  • the amino acid sequence has the amino acid sequence shown in SEQ ID NO: 143; or
  • the third polypeptide chain having the amino acid sequence of SEQ ID NO: 143 has the fourth polypeptide chain of the amino acid sequence; or
  • the third polypeptide chain having the amino acid sequence of SEQ ID NO: 143 has the fourth polypeptide chain of the amino acid sequence; or
  • the third polypeptide chain having the amino acid sequence of SEQ ID NO: 153 has the fourth polypeptide chain of the amino acid sequence; or
  • the third polypeptide chain having the amino acid sequence of SEQ ID NO: 153 has the fourth polypeptide chain of the amino acid sequence; or
  • the third polypeptide chain having the amino acid sequence of SEQ ID NO: 153 has the fourth polypeptide chain of the amino acid sequence; or
  • the third polypeptide chain having the amino acid sequence shown in SEQ ID NO: 143 has the fourth polypeptide chain.
  • variable region CDRs of the antibodies of the invention can be determined using any of a number of well-known schemes, including Chothia based on the three-dimensional structure of the antibody and the topology of the CDR loops (Chothia et al.
  • the boundaries of the CDRs of the variable region of the same antibody obtained based on different assignment systems may be different. That is, the CDR sequences of the variable region of the same antibody defined under different assignment systems are different.
  • the scope of said antibody also covers antibodies whose variable region sequences comprise said particular CDR sequence, but due to the application of a different protocol (e.g. Different assignment systems or combinations) cause the claimed CDR boundaries to be different from the specific CDR boundaries defined in the present invention.
  • Antibodies with different specificities have different binding sites for different antigens.
  • CDRs vary from antibody to antibody, only a limited number of amino acid positions within a CDR are directly involved in antigen binding.
  • a minimal binding unit may be a subsection of a CDR.
  • the residues of the remainder of the CDR sequences can be determined from the structure and protein folding of the antibody. Accordingly, the invention also contemplates variations of any of the CDRs presented herein. For example, in a variant of a CDR, the amino acid residues of the smallest binding unit can remain unchanged, while the remaining CDR residues defined according to Kabat or Chothia can be replaced by conserved amino acid residues.
  • the murine CDR regions can be inserted into the human germline framework regions using methods known in the art. See US Patent No. 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 present invention include mutations that have undergone amino acid deletions, insertions, or substitutions, but are still compatible with the above-mentioned antibodies (especially in the CDR regions depicted in the above-mentioned sequences) Those antibodies have amino acid sequences that are at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical.
  • an antibody of the invention has no more than 1, 2, 3, 4 or 5 amino acid mutations in the CDR region by amino acid deletion, insertion or substitution when compared to the CDR region depicted in the specific sequence.
  • polynucleotides encoding antibodies of the invention include coding regions that have been mutated by nucleotide deletions, insertions, or substitutions, but still have at least about 60, 70 , 80, 90, 95 or 100% identical polynucleotides.
  • Fc region variants may comprise human Fc region sequences (eg, human IgGl, IgG2, IgG3 or IgG4 Fc regions) 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-protein moieties known and readily available in the art.
  • 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, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, polyvinyl -1,3-dioxane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyamino acid (homopolymer or random copolymer), and dextran or poly(n-ethylene pyrrolidone) polyethylene glycol, propylene glycol homopolymer, polypropylene oxide/ethylene oxide copolymer, polyoxyethylated polyols (such as glycerin), polyvinyl alcohol, and mixtures thereof
  • the first binding domain and the second binding domain of the bispecific antibody can be connected to each other through a linker, and then further bind to the first Fc region; the third binding domain binds to the second Fc region.
  • the linker is a peptide linker.
  • the Fc region of the bispecific antibody of the present invention may be a human Fc region.
  • the Fc region of the bispecific antibody of the invention may be of any isotype, including but not limited to IgG1, IgG2, IgG3 or IgG4.
  • the Fc regions of said first and said second antibody are both IgGl isotypes.
  • the Fc regions of said first and said second antibody are both IgG4 isotypes.
  • 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 IgG1 and the Fc region of IgG4 and thus may have interesting intermediate properties with regard to the activation of effector functions.
  • Knob into Hole structure or "knob-hole structure” is to mutate the hydrophobic amino acid of CH3 of antibody Fc.
  • the side chain amino acid of one chain CH3 is mutated into a hydrophobic amino acid (knob) with a relatively large molecule to strengthen the hydrophobic force; the other CH3 side chain amino acid is mutated into a small amino acid (hole) to reduce steric hindrance; after mutation CH3 with Knob and CH3 with Hole form a Knob into Hole structure (KiH) in the form of hydrophobic interaction, which is conducive to the formation of heavy chain heterodimers; KiH mutations mainly occur in the internal hydrophobicity of the spatial structure of the CH3 domain Amino acids, the amino acids exposed to the outside after mutation have almost no change, so the effector function of Fc and the immunogenicity caused are not affected.
  • knob-Fc refers to a point mutation comprising T366W in the Fc region of an antibody to form a knob-like spatial structure.
  • hole-Fc refers to point mutations including T366S, L368A, and Y407V in the Fc region of the antibody to form a hole-like spatial structure.
  • point mutations of S354C and Y349C can be introduced into knob-Fc and hole-Fc, respectively, to further promote the formation of heterodimers through disulfide bonds.
  • point mutations of H435R and Y436F can also be introduced into hole-Fc respectively.
  • the Fc region may contain one or more amino acid changes (e.g. insertions, deletions or substitutions) compared to the specified chimeric form of the Fc region without altering the desired function sex.
  • the invention includes bispecific antigen binding molecules comprising one or more modifications in the Fc region that result in a modified Fc region with a modified binding interaction (eg, enhanced or reduced) between Fc and FcRn.
  • Fc modifications include, for example, 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 antibodies that effectively promote T cell-mediated killing of tumor cells.
  • a particular antigen-binding domain may be selected from the group of antibodies or antigen-binding domains provided herein, or share, for example, an epitope or cross-blocking domain with an antibody or antigen-binding domain provided herein. Those antibody or antigen-binding regions of the domain.
  • Many different formats and uses of bispecific antibodies are known in the art and are reviewed by Chames and Baty (2009) Curr Opin Drug Disc Dev 12:276.
  • an antibody of the invention comprising an antigen binding site that binds CD3 and an antigen binding site that binds a tumor-associated antigen may be a bispecific antibody, wherein the bispecific antibody may adopt 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 bispecific antibodies).
  • IgG-like bispecific antibodies mainly include Triomabs/quadroma, DVD-Ig (dual variable domain Ig), CrossMAb, Two-in-one IgG, and 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.
  • An exemplary bispecific antibody molecule of the present invention has four polypeptide chains, wherein (a) the first polypeptide chain has VH1-CH1-connecting peptide-VL2-connecting peptide-hinge region-first Fc region, the second polypeptide chain has VL1-CL-connecting peptide-VH2 from N-terminal to C-terminal; the third polypeptide chain has VH2-CH1-hinge region-second Fc from N-terminal to C-terminal region, said fourth polypeptide chain has VL2-CL from N-terminus to C-terminus; or (b) wherein said first polypeptide chain has VH2-CH1-connecting peptide-VL1-connecting peptide from N-terminus to C-terminus -hinge region-first Fc region, the second polypeptide chain has VL2-CL-connecting peptide-VH1 from N-terminus to C-terminus; the third polypeptide chain has VH2-CH1- from N
  • the connecting peptide comprises G (glycine), S (serine) and A (alanine) or a flexible polypeptide composed of G and S, and its length can be 2-30 amino acid residues;
  • Peptides include but are not limited to (GS)n, (GGS)n, (GGGS)n, (GGGGS)n and AS(GGGGS)n, etc., n can be an integer of 1-6.
  • This bispecific antibody format has many unique properties and advantages, such as excellent stability, low toxicity, and excellent tumor killing activity when used in the preparation of bispecific antibodies against multiple targets.
  • the bispecific antibody of the invention is a diabody, a cross-body, or a bispecific antibody obtained by controlled Fab arm exchange.
  • 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, Fc region or part thereof;
  • Antibodies based on scFv and diabodies and heavy chain antibodies e.g. domain antibodies, nanobodies
  • different single chain Fv molecules or different diabodies or different heavy chain antibodies e.g. 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 (Karmanos Cancer Center), mAb2 (F -Star), and CovX-body (CovX/Pfizer).
  • DT Dual Targeting
  • Genentech Two-in-one Antibody
  • Cross-linked Mabs Karmanos 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-based, diabodies, and domain antibodies 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 (EpigenBiotech), dual targeting nanobodies (Ablynx), dual targeting heavy chain domain only antibody (dualtargeting heavy chain only domain antibodies).
  • the invention provides a polynucleotide encoding a bispecific antibody as described herein.
  • the polynucleotide may comprise a polynucleotide encoding the amino acid sequences of the 4 polypeptide chains of the bispecific antibody.
  • the present invention provides an expression vector comprising the polynucleotide as described herein, preferably, the vector is a eukaryotic expression vector.
  • the vector is a eukaryotic expression vector.
  • a polynucleotide as described herein is 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 a bispecific antibody as described herein, said method comprising expressing said antibody in a host cell as described herein under conditions suitable for expression of said antibody. Said antibody is obtained, and the expressed antibody is recovered from said host cell.
  • the invention provides mammalian host cells for expressing the recombinant antibodies of the invention, including the many 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 for preparing the bispecific antibody described herein, wherein the method comprises introducing the expression vector into a mammalian host cell, and culturing the host cell for a period of time sufficient to allow the antibody to express itself in the host cell.
  • the antibody is produced by expressing it in the cell or, more preferably, secreting it into the medium in which the host cell is grown.
  • Antibodies can be recovered from the culture medium using standard protein purification methods.
  • non-fucosylated antibodies are advantageous because they generally have more potent potency than their fucosylated counterparts in vitro and in vivo and are less likely to be immunogenic , because their sugar structure is a normal component of natural human serum IgG.
  • the present invention provides a pharmaceutical composition, which comprises the bispecific antibody, polynucleotide, expression vector or host cell according to any embodiment herein, and a pharmaceutically acceptable carrier or excipient Forming agent.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof, polynucleotide, expression vector or host cell as described in any embodiment herein, and a pharmaceutically acceptable carrier or excipients.
  • compositions described herein can be administered in combination by incorporating suitable carriers, excipients and other agents in formulations to provide improved transfer, delivery, tolerance, and the like.
  • composition refers to a formulation that allows the active ingredients contained therein to be present in a biologically effective form and does not contain additional ingredients that would be unacceptably toxic to the subject to which the formulation is administered.
  • the bispecific antibody described herein may be mixed with one or more optional pharmaceutical excipients (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980)) having the desired purity.
  • Pharmaceutical formulations comprising the bispecific antibodies described herein are prepared, preferably in the form of aqueous solutions or lyophilized formulations.
  • compositions or formulations of the invention may also contain one or more other active ingredients as required for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other .
  • other active ingredients are chemotherapeutic agents, immune checkpoint inhibitors, growth inhibitors, antibiotics, or various known anti-tumor or anti-cancer agents, and the active ingredients are suitable in an effective amount for the intended use combination exists.
  • the pharmaceutical composition of the invention further comprises a composition of polynucleotides encoding the bispecific antibodies described herein.
  • the present invention provides a pharmaceutical combination comprising the bispecific antibody, polynucleotide, expression vector, host cell, or pharmaceutical composition as described in any embodiment herein, and one or more an additional therapeutic agent.
  • the present invention provides a kit comprising the bispecific antibody, polynucleotide, expression vector, host cell, or pharmaceutical composition according to any embodiment herein.
  • the pharmaceutical composition of the invention further comprises a composition of polynucleotides encoding the antibodies described herein.
  • the present invention provides a pharmaceutical combination comprising the antibody or antigen-binding fragment thereof, polynucleotide, expression vector, host cell, or pharmaceutical composition according to any embodiment herein, and a or more additional therapeutic agents.
  • the present invention provides a kit comprising the antibody or antigen-binding fragment thereof, polynucleotide, expression vector, host cell, or pharmaceutical composition according to any embodiment herein.
  • the present invention provides the use of the bispecific antibody, polynucleotide molecule, expression vector, host cell, or pharmaceutical composition as described in any embodiment herein in the preparation of a drug for preventing or treating cancer in a subject use.
  • the present invention provides a bispecific antibody, polynucleotide molecule, expression vector, host cell, or pharmaceutical composition according to any one of the embodiments herein for use in preventing or treating cancer in a subject.
  • the present invention provides a method of preventing or treating cancer in a subject, which comprises administering the bispecific antibody, polynucleotide molecule, expression vector as described in any embodiment herein to a subject in need , a host cell, or a pharmaceutical composition.
  • the present invention provides an antibody or an antigen-binding fragment thereof, a polynucleotide, an expression vector, a host cell, or a pharmaceutical composition as described in any embodiment herein in the preparation of a medicament for preventing or treating cancer the use of.
  • the present invention provides the antibody or antigen-binding fragment thereof, polynucleotide, expression vector, host cell, or pharmaceutical composition according to any one of the embodiments herein for use in the prevention or treatment of cancer.
  • the present invention provides a method for preventing or treating cancer, comprising administering the antibody or its antigen-binding fragment, polynucleotide, expression vector as described in any embodiment herein to a subject in need , a host cell, or a pharmaceutical composition.
  • the cancer is preferably a cancer expressing one or more tumor-associated antigens selected from the group consisting of BCMA, CD19, CD20, CD22, CD30, CD33, CD38, CD40, CD123, CDK4, CEA, Claudin 18.2, EGFR, ROR1, TGF- ⁇ and 5T4. More preferably, the tumor-associated antigen is selected from one or more of Claudin 18.2, CD20 and BCMA. Exemplary cancers include those described above.
  • the cancer is associated with CLDN18; more specifically, the cancer highly expresses Claudin 18.2. Therefore, in a preferred embodiment, the cancer is a cancer whose cancer cells highly express Claudin 18.2.
  • the "high expression” mentioned herein means that the expression level of Claudin 18.2 in cancer cells or cancer tissues is higher than the expression level of Claudin 18.2 in normal cells or normal tissues. Conventional methods can be used to test the expression level 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 associated with CLDN18 is selected from gastric cancer, esophageal cancer, gastroesophageal cancer, pancreatic cancer, cholangiocarcinoma, lung cancer, ovarian cancer, colon cancer, liver cancer, head and neck cancer, gallbladder cancer, bowel cancer, and bladder cancer. cancer.
  • the cancer is associated with CD20; in some embodiments, the cancer associated with CD20 is selected from acute B-lymphoblastic leukemia, diffuse large B-cell lymphoma, chronic lymphocytic leukemia, follicular lymphoma, Non-Hodgkin's lymphoma, chronic myeloid leukemia, and Burkitt's lymphoma.
  • modes of administration of the present invention include, but are not limited to, oral, intravenous, subcutaneous, intramuscular, intraarterial, intraarticular (eg, in arthritic joints), via inhalation, aerosol delivery, or intratumoral administration wait.
  • the invention also encompasses co-administering to a subject a therapeutically effective amount of one or more therapies (eg, treatment modalities and/or other therapeutic agents).
  • the therapy includes surgery and/or radiation therapy.
  • the cancer treatment method of the present invention further comprises administering the expression stabilizing or increasing agent of the bispecific antibody described herein.
  • Expression of the bispecific antibody is preferably on the cell surface of cancer cells.
  • the agent for stabilizing or increasing the expression of bispecific antibodies can be oxaliplatin and/or 5-FU.
  • the methods or uses provided herein further comprise administering one or more therapies (eg, treatment modalities and/or other therapeutic agents) to the individual.
  • therapies eg, treatment 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 invention provides a method of detecting the presence of CD3 or CLDN-18.2 or CD20 in a sample using a bispecific antibody as described herein.
  • detection includes quantitative or qualitative detection.
  • the sample is a biological sample.
  • the biological sample is blood, serum, or other liquid sample of biological origin.
  • a biological sample comprises cells or tissues. The method comprises contacting a bispecific antibody of the invention with a sample and detecting the presence and/or amount of a conjugate formed by said bispecific antibody with CD3 or CLDN-18.2 or CD20.
  • the CDR region of the antibody is mutated for the purpose of reducing the affinity of the antibody and reducing toxicity , screened out the following multiple amino acid sequences:
  • the CDR region of the light chain JSCD3-LC was mutated to produce CD3-LC1, CD3-LC2, CD3-LC3, CD3-LC4, CD3-LC5, CD3-LC6, CD3-LC7, CD3 -LC8, CD3-LC9, CD3-LC10, CD3-LC11, CD3-LC12, CD3-LC13, CD3-LC14, CD3-LC15, CD3-LC16, CD3-LC17, CD3-LC18 and JSCD3-LC-VtoA, total 18 sequences;
  • the CDR region of the heavy chain JSCD3-HC is mutated to generate CD3-HC1, CD3-HC2, CD3-HC
  • JSCD3-HC (italics represent VH, underline represents HCDR1, HCDR2 and HCDR3):
  • amino acid sequence of JSCD3-HC-FtoA is shown in SEQ ID NO: 83, and its variable region sequence is as follows (the underlines represent HCDR1, HCDR2 and HCDR3 in sequence):
  • CD3-HC1 The amino acid sequence of CD3-HC1 is shown in SEQ ID NO: 84, and its variable region sequence is as follows (the underlines represent HCDR1, HCDR2 and HCDR3 in sequence):
  • CD3-HC2 The amino acid sequence of CD3-HC2 is shown in SEQ ID NO: 85, and its variable region sequence is as follows (the underlines represent HCDR1, HCDR2 and HCDR3 in sequence):
  • CD3-HC3 The amino acid sequence of CD3-HC3 is shown in SEQ ID NO: 86, and its variable region sequence is as follows (the underlines represent HCDR1, HCDR2 and HCDR3 in sequence):
  • CD3-HC4 The amino acid sequence of CD3-HC4 is shown in SEQ ID NO: 87, and its variable region sequence is as follows (the underlines represent HCDR1, HCDR2 and HCDR3 in sequence):
  • CD3-HC5 The amino acid sequence of CD3-HC5 is shown in SEQ ID NO: 88, and its variable region sequence is as follows (the underlines represent HCDR1, HCDR2 and HCDR3 in sequence):
  • CD3-HC6 The amino acid sequence of CD3-HC6 is shown in SEQ ID NO: 89, and its variable region sequence is as follows (the underlines represent HCDR1, HCDR2 and HCDR3 in sequence):
  • CD3-HC7 The amino acid sequence of CD3-HC7 is as shown in SEQ ID NO: 90, and its variable region sequence is as follows (the underline represents HCDR1, HCDR2 and HCDR3 successively):
  • CD3-HC8 The amino acid sequence of CD3-HC8 is shown in SEQ ID NO: 91, and its variable region sequence is as follows (the underlines represent HCDR1, HCDR2 and HCDR3 in sequence):
  • CD3-HC9 The amino acid sequence of CD3-HC9 is shown in SEQ ID NO: 92, and its variable region sequence is as follows (the underlines represent HCDR1, HCDR2 and HCDR3 in sequence):
  • CD3-HC10 The amino acid sequence of CD3-HC10 is shown in SEQ ID NO: 93, and its variable region sequence is as follows (the underlines represent HCDR1, HCDR2 and HCDR3 in sequence):
  • CD3-HC11 The amino acid sequence of CD3-HC11 is shown in SEQ ID NO: 94, and its variable region sequence is as follows (the underlines represent HCDR1, HCDR2 and HCDR3 in sequence):
  • CD3-HC12 The amino acid sequence of CD3-HC12 is shown in SEQ ID NO: 95, and its variable region sequence is as follows (the underlines represent HCDR1, HCDR2 and HCDR3 in turn):
  • CD3-HC13 The amino acid sequence of CD3-HC13 is shown in SEQ ID NO: 96, and its variable region sequence is as follows (the underlines represent HCDR1, HCDR2 and HCDR3 in turn):
  • CD3-HC14 The amino acid sequence of CD3-HC14 is shown in SEQ ID NO: 97, and its variable region sequence is as follows (the underlines represent HCDR1, HCDR2 and HCDR3 in turn):
  • CD3-HC15 The amino acid sequence of CD3-HC15 is shown in SEQ ID NO: 98, and its variable region sequence is as follows (the underlines represent HCDR1, HCDR2 and HCDR3 in turn):
  • CD3-HC16 The amino acid sequence of CD3-HC16 is shown in SEQ ID NO: 99, and its variable region sequence is as follows (the underlines represent HCDR1, HCDR2 and HCDR3 in sequence):
  • CD3-HC17 The amino acid sequence of CD3-HC17 is shown in SEQ ID NO: 100, and its variable region sequence is as follows (the underlines represent HCDR1, HCDR2 and HCDR3 in sequence):
  • CD3-HC18 The amino acid sequence of CD3-HC18 is shown in SEQ ID NO: 101, and its variable region sequence is as follows (the underlines represent HCDR1, HCDR2 and HCDR3 in sequence):
  • amino acid sequence of JSCD3-HC-WtoA is shown in SEQ ID NO: 140, and its variable region sequence is as follows (the underlines represent HCDR1, HCDR2 and HCDR3 in turn):
  • JSCD3-LC (italics represent VL, underlines represent LCDR1, LCDR2 and LCDR3):
  • the amino acid sequence of JSCD3-LC-VtoA is shown in SEQ ID NO: 103, and its variable region sequence is as follows (the underlines represent LCDR1, LCDR2 and LCDR3 in sequence):
  • CD3-LC1 The amino acid sequence of CD3-LC1 is shown in SEQ ID NO: 104, and its variable region sequence is as follows (the underlines represent LCDR1, LCDR2 and LCDR3 in sequence):
  • CD3-LC2 The amino acid sequence of CD3-LC2 is shown in SEQ ID NO: 105, and its variable region sequence is as follows (the underline indicates LCDR1, LCDR2 and LCDR3 in sequence):
  • CD3-LC3 The amino acid sequence of CD3-LC3 is shown in SEQ ID NO: 106, and its variable region sequence is as follows (the underline indicates LCDR1, LCDR2 and LCDR3 in sequence):
  • CD3-LC4 The amino acid sequence of CD3-LC4 is shown in SEQ ID NO: 107, and its variable region sequence is as follows (the underline indicates LCDR1, LCDR2 and LCDR3 in sequence):
  • CD3-LC5 The amino acid sequence of CD3-LC5 is shown in SEQ ID NO: 108, and its variable region sequence is as follows (the underline indicates LCDR1, LCDR2 and LCDR3 in sequence):
  • CD3-LC6 The amino acid sequence of CD3-LC6 is shown in SEQ ID NO: 109, and its variable region sequence is as follows (the underline indicates LCDR1, LCDR2 and LCDR3 in sequence):
  • the amino acid sequence of CD3-LC7 is shown in SEQ ID NO: 110, and its variable region sequence is as follows (the underline indicates LCDR1, LCDR2 and LCDR3 in sequence):
  • CD3-LC8 The amino acid sequence of CD3-LC8 is shown in SEQ ID NO: 111, and its variable region sequence is as follows (the underline indicates LCDR1, LCDR2 and LCDR3 in sequence):
  • CD3-LC9 The amino acid sequence of CD3-LC9 is shown in SEQ ID NO: 112, and its variable region sequence is as follows (the underline indicates LCDR1, LCDR2 and LCDR3 in sequence):
  • CD3-LC10 The amino acid sequence of CD3-LC10 is shown in SEQ ID NO: 113, and its variable region sequence is as follows (the underlines represent LCDR1, LCDR2 and LCDR3 in sequence):
  • CD3-LC11 The amino acid sequence of CD3-LC11 is shown in SEQ ID NO: 114, and its variable region sequence is as follows (the underlines represent LCDR1, LCDR2 and LCDR3 in sequence):
  • CD3-LC12 The amino acid sequence of CD3-LC12 is shown in SEQ ID NO: 113, and its variable region sequence is as follows (the underline indicates LCDR1, LCDR2 and LCDR3 in sequence):
  • CD3-LC13 The amino acid sequence of CD3-LC13 is shown in SEQ ID NO: 115, and its variable region sequence is as follows (the underlines represent LCDR1, LCDR2 and LCDR3 in sequence):
  • CD3-LC14 The amino acid sequence of CD3-LC14 is shown in SEQ ID NO: 116, and its variable region sequence is as follows (the underlines represent LCDR1, LCDR2 and LCDR3 in sequence):
  • CD3-LC15 The amino acid sequence of CD3-LC15 is shown in SEQ ID NO: 117, and its variable region sequence is as follows (the underline indicates LCDR1, LCDR2 and LCDR3 in sequence):
  • CD3-LC16 The amino acid sequence of CD3-LC16 is shown in SEQ ID NO: 118, and its variable region sequence is as follows (the underline indicates LCDR1, LCDR2 and LCDR3 in sequence):
  • CD3-LC17 The amino acid sequence of CD3-LC17 is shown in SEQ ID NO: 119, and its variable region sequence is as follows (the underline indicates LCDR1, LCDR2 and LCDR3 in sequence):
  • the amino acid sequence of CD3-LC18 is shown in SEQ ID NO: 120, and its variable region sequence is as follows (the underlines indicate LCDR1, LCDR2 and LCDR3 in sequence):
  • the preparation method of anti-CD3 antibody is as follows:
  • GenScript was commissioned to synthesize the heavy chain of the antibody on puc57, which was digested with SapI and connected to the HXT4S vector to obtain the expression vector HXT4S-CD3-HC.
  • GenScript was commissioned to synthesize the light chain of the human antibody on puc57, which was digested with SapI and connected to the HXT2 vector to obtain the expression vector HXT2-CD3-LC.
  • the day before transfection dilute the CHO-K1 cell density to (1.5-2.0) ⁇ 10 6 /mL, and culture it on a shaker (the culture condition is 36.5°C, 120rmp, 7% CO 2 ). Transfection was carried out at 3.5 ⁇ 10 6 /mL.
  • transfecting 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 plasmids, 3-14ug/mL of PEI (polyethyleneimine) , mix well and incubate at room temperature for 20-30 minutes, then slowly add the above mixture into the pretreated CHO-K1 cells to form a transfection mixture, and mix well while adding.
  • the transfection mixture was cultured on a shaker at 36.5° C., 120 rpm, and 7% CO 2 . The culture period is 6-10 days after transfection, with feeding every two days.
  • the second step use Eshmuno CPX for purification, the balance solution is pH 5.5, 50mM acetic acid-sodium acetate System, the eluent is pH 5.5, 50mM acetic acid-sodium acetate + 1M NaCl buffer system, the target protein is collected by linear elution, and the purity of the monomer can reach more than 95% as determined by SEC-HPLC, that is, the anti-CD3 Antibody.
  • Table 1A Combination table of light and heavy chains of anti-CD3 antibodies
  • Table 1B Combination table of light and heavy chains of anti-CD3 antibodies
  • Table 1C Combination table of light and heavy chains of anti-CD3 antibodies
  • Embodiment 3 the SEC detection result of anti-CD3 antibody
  • the SEC-HPLC purity was obtained by installing an SEC chromatographic column (XBridge BEH SEC 7.8*300mm, 3.5 ⁇ m) HPLC (Agilent 1260) for detection.
  • the composition of the mobile phase was 50 mM phosphate, 300 mM sodium chloride, pH 6.8 ⁇ 0.2.
  • the results were quantified by peak area normalization. The peak area percentages for monomers, aggregates, and fragments were calculated separately. The specific test results are shown in Table 6.
  • the anti-CD3 antibody selected in Example 2 and JSCD3-A1, JSCD3-A2, JSCD3-Roche, JSCD3-AMG, REGN-CD3, and JS202-JSCD3 were divided into 12 groups for activity testing, and the specific groups were as follows:
  • Group 1 JSCD3-1, JSCD3-3, JSCD3-8, JSCD3-11, JSCD3-13, JSCD3-79, REGN-CD3, JS202-JSCD3;
  • Group 2 JSCD3-88, JSCD3-89, JSCD3-91, JSCD3-257, JSCD3-258, JSCD3-260, REGN-CD3, JS202-JSCD3;
  • Group 3 JSCD3-98, JSCD3-103, JSCD3-111, JSCD3-112, JSCD3-113, JSCD3-114, REGN-CD3, JS202-JSCD3;
  • Group 4 JSCD3-115, JSCD3-117, JSCD3-122, JSCD3-124, JSCD3-125, JSCD3-126, REGN-CD3, JS202-JSCD3;
  • Group 5 JSCD3-127, JSCD3-130, JSCD3-131, JSCD3-132, JSCD3-Roche, JSCD3-AMG, REGN-CD3, JS202-JSCD3;
  • Group 7 JSCD3-262, JSCD3-264, JSCD3-265, JSCD3-285, JSCD3-288, JSCD3-293, REGN-CD3, JS202-JSCD3;
  • Group 8 JSCD3-296, JSCD3-297, JSCD3-301, JSCD3-302, REGN-CD3, JS202-JSCD3;
  • Group 9 JSCD3-92, JSCD3-95, JSCD3-303, REGN-CD3, JS202-JSCD3;
  • Group 11 JSCD3-5, JSCD3-162, JSCD3-173, JSCD3-191, JSCD3-195, JSCD3-210, JSCD3-217, JSCD3-268, JSCD3-324, JSCD3-325, REGN-CD3, JS202- JSCD3;
  • the heavy chain amino acid sequence of JSCD3-Roche is shown in SEQ ID NO: 158
  • the light chain amino acid sequence is shown in SEQ ID NO: 159
  • the heavy chain amino acid sequence of JSCD3-AMG is shown in SEQ ID NO: 160
  • the light chain amino acid sequence is shown in SEQ ID NO: 160.
  • the chain amino acid sequence is shown in SEQ ID NO: 161
  • the heavy chain amino acid sequence of REGN-CD3 is shown in SEQ ID NO: 162
  • the light chain amino acid sequence is shown in SEQ ID NO: 163
  • the preparation of REGN-CD3 is as shown in the preparation method of anti-CD3 antibody in Example 1.
  • the above grouped anti-CD3 antibodies were diluted with different concentration gradients to fit the curve to further test the binding activity with human CD3.
  • Cells endogenously expressing human CD3 (Jurkat cells, Cell Bank of Type Culture Collection Committee, Chinese Academy of ) were incubated with anti-CD3 antibody at 4°C for 30 min at 4°C, washed away the residual antibody and then incubated with fluorescent secondary antibody (PE-anti-human IgG). Finally, the fluorescence intensity was detected by C6PLUS flow cytometer. Stronger, the higher the affinity of the antibody to the target.
  • TOP represents the fluorescence signal value (MFI-PE) when the antibody concentration is saturated
  • relative binding activity vs JSCD3(%) JSCD3 EC50 value/sample EC50 ⁇ 100%
  • relative binding activity vs REGN-CD3(%) REGN-CD3 EC50 Value/sample EC50 x 100%.
  • Table 8 Test results of binding activity of antibodies of group 2 to human CD3
  • Table 12 Test results of the binding activity of the antibodies of Group 6 to human CD3
  • Table 17 Test results of the binding activity of the antibodies of Group 12 to human CD3
  • the plasmids HXT4S-JS012-2B19-HC-7 and HXT4s-JS CD20HC-2Mut h-HY were respectively digested with NheI and NotI enzymes to obtain vectors and fragments, and connected with T4 DNA ligase to obtain the first expression vector JS012- 2B19HC- 7-IgG4-hole. Entrusted GenScript Biotechnology Co., Ltd.
  • GenScript Biotechnology Co., Ltd. was entrusted to synthesize the gene encoding JS012-2B19LC-1-Kappa, which was digested with SapI and connected to HXT2 to obtain the third expression vector JS012-2B19LC-1-kappa.
  • the first expression vector JS012-2B19HC-7-IgG4-hole was obtained by the method in Section 5.1.
  • HXT4S-JS012-2B19-HC-7-mutb HXT2-JS012-2B19LC-1 and HXT4S-JSCD3-HC7 as templates to amplify the vectors and fragments respectively
  • the second expression vector JSCD3-HC7-CH1-012ScFv was obtained by recombination -IgG4-knob. Sub-entrusted GenScript Biotechnology Co., Ltd.
  • the first expression vector JS012-2B19HC-7-IgG4-hole was obtained by the method in Section 5.1.
  • HXT4S-JS012-2B19-HC-7-mutb and HXT2-JSCD3-LC were respectively used as templates to amplify the vector and the fragment to obtain the second expression vector JS012-2B19HC-7-CH1-JSCD3 LC-IgG4-knob through recombination.
  • HXT2-JS012-2B19LC-1 and HXT4S-JSCD3-HC were used as template PCR to obtain the vector and fragment respectively, and the third expression vector JS012-2B19LC-1-Kappa-JSCD3-HC was obtained through recombination.
  • GenScript Biotechnology Co., Ltd. was entrusted to synthesize the gene encoding JS012-2B19LC-1-Kappa, which was digested with SapI and ligated to HXT2 to obtain the fourth expression vector JS012-2B19LC-1-kapp
  • the first expression vector JS012-2B19HC-7-IgG4-hole was obtained by the method in Section 5.1.
  • HXT4S-JSCD3-HC, HXT2-JS012-2B19LC-1 and HXT4S-1A6H2 QGQ-mutb were used as templates to obtain the vector and two fragments by PCR respectively, and the second expression vector JSCD3-VH-CH1-JS012-2B19LC was obtained by recombination -1-IgG4-knob.
  • HXT3-JSCD3-LC and HXT4S-JS012-2B19-HC-7 were used as templates to obtain vectors and fragments by PCR respectively, and the third expression vector JSCD3-LC-lambda-2B19HC-7 was obtained through recombination.
  • GenScript Biotechnology Co., Ltd. was entrusted to synthesize the gene encoding JS012-2B19LC-1-Kappa, which was digested with SapI and ligated to HXT2 to obtain the fourth expression vector JS012-2B19LC-1-kappa.
  • the first expression vector JS012-2B19HC-7-IgG4-hole was obtained by the method in Section 5.1.
  • HXT4S-JSCD3-HC7, HXT2-JS012-2B19LC-1 and HXT4S-1A6H2 QGQ-mutb were used as templates to obtain the vector and two fragments by PCR respectively, and the second expression vector JSCD3-HC7-CH1-JS012-2B19LC was obtained by recombination -1-IgG4-knob.
  • HXT3-JSCD3-LC16 and HXT4S-JS012-2B19-HC-7 were used as templates to obtain vectors and fragments by PCR respectively, and the third expression vector JSCD3-LC16-lambda-2B19HC-7 was obtained through recombination.
  • GenScript Biotechnology Co., Ltd. was entrusted to synthesize the gene encoding JS012-2B19LC-1-Kappa, which was digested with SapI and ligated to HXT2 to obtain the fourth expression vector JS012-2B19LC-1-kappa.
  • GenScript was entrusted to synthesize the heavy chain JSCD20-HC-2 of human CD20 in puc57, which was digested with BSPQI and connected to the HXT4S vector to obtain the expression vector HXT4S-JSCD20-HC-2.
  • the plasmid was digested with HindIII and NheI and recovered to obtain the gene fragment JSCD20-HC-2, and the plasmid HXT4S-JS012-1H17-HC-mut-h was digested with HindIII and NheI and recovered to obtain the vector HXT4s-mut h, and T4 DNA ligase was used Ligated to obtain the first expression vector JSCD20-HC-2-IgG4-hole.
  • the second expression vector JSCD3-HC-CH1-JSCD20-LC- 2-IgG4-knob was obtained by recombination.
  • GenScript entrusted GenScript to synthesize the light chain JSCD20-LC-2 of human CD20 in puc57, digest it with SapI and connect it to the HXT2 vector to obtain the fourth expression vector JSCD20-LC-2-kappa.
  • GenScript commissioned GenScript to synthesize the heavy chain JSCD20-HC-2 of human CD20 in puc57, digest it with SapI and connect it to the HXT4S vector to obtain the expression vector HXT4S-JSCD20-HC-2.
  • the plasmid was digested with HindIII and NheI and recovered to obtain the gene fragment JSCD20-HC-2, and the plasmid HXT4S-JS012-1H17-HC-mut-h was digested with HindIII and NheI and recovered to obtain the vector HXT4s-mut h, and T4 DNA ligase was used Ligated to obtain the first expression vector JSCD20-HC-2-IgG4-hole.
  • the second expression vector JSCD3-HC7-CH1-JSCD20-LC-2-IgG4 was obtained by recombination -knob.
  • HXT3-JSCD3-LC16-2B19 HC-7 and HXT4s-JSCD20HC-2Mut h as templates to amplify the vector and fragment respectively
  • the third expression vector JSCD3-LC16-lambda-JSCD20-HC-2 was obtained by recombination.
  • GenScript entrusted GenScript to synthesize the light chain JSCD20-LC-2 of human CD20 in puc57, digest it with SapI and connect it to the HXT2 vector to obtain the fourth expression vector JSCD20-LC-2-kappa.
  • GenScript commissioned GenScript to synthesize the heavy chain JSCD20-HC-2 of human CD20 in puc57, digest it with SapI and connect it to the HXT4S vector to obtain the expression vector HXT4S-JSCD20-HC-2.
  • the plasmid was digested with HindIII and NheI and recovered to obtain the gene fragment JSCD20-HC-2, and the plasmid HXT4S-JS012-1H17-HC-mut-h was digested with HindIII and NheI and recovered to obtain the vector HXT4s-mut h, and T4 DNA ligase was used Ligated to obtain the first expression vector JSCD20-HC-2-IgG4-hole.
  • the second expression vector JSCD20-VH-CH1-JSCD3-VL-IgG4-knob was obtained by recombination.
  • the third expression vector JSCD20-LC-2 Kappa-JSCD3-VH was obtained by recombination.
  • GenScript entrusted GenScript to synthesize the light chain JSCD20-LC-2 of human CD20 in puc57, digest it with SapI and connect it to the HXT2 vector to obtain the fourth expression vector JSCD20-LC-2-kappa.
  • CHO-K1 cells modified at the genome level to make the cells suitable for transient expression, Junshi Biotechnology
  • CD CHO medium Gibco
  • 7% CO 2 when the cell density reached (2-6) ⁇ 10 6 /mL, use CD CHO medium for passage expansion.
  • the day before transfection dilute the CHO-K1 cell density to (1.5-2.0) ⁇ 10 6 /mL, and culture it on a shaker (the culture condition is 36.5°C, 120rmp, 7% CO 2 ). Transfection was carried out at 3.5 ⁇ 10 6 /mL.
  • transfecting 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 plasmids, 3-14ug/mL of PEI (polyethyleneimine) , mix well and incubate at room temperature for 20-30 minutes, then slowly add the above mixture into the pretreated CHO-K1 cells to form a transfection mixture, and mix well while adding.
  • the transfection mixture was cultured on a shaker at 36.5° C., 120 rpm, and 7% CO 2 . The culture period is 6-10 days after transfection, with feeding every two days.
  • the first step is to purify with the AKTA Avant purification instrument. 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 second step use Eshmuno CPX for purification, the balance solution is pH 5.5, 50mM acetic acid-sodium acetate system, the eluent is pH 5.5, 50mM acetic acid-sodium acetate + 1M NaCl buffer system, the target protein is collected by linear elution, and the purity of the monomer can reach more than 95% as determined by SEC-HPLC, that is, the bispecific Sexual antibodies.
  • the amino acid sequence of the first polypeptide chain (expression vector JS012-2B19HC-7-CH1-JSCD3 scFv-IgG4-knob) is shown in SEQ ID NO: 141
  • the second polypeptide chain (expression The amino acid sequence of the vector JS012-2B19HC-7-IgG4-hole) is shown in SEQ ID NO: 142
  • the amino acid sequence of the third polypeptide chain (expression vector JS012-2B19LC-1-kappa) is shown in SEQ ID NO: 143.
  • the schematic structure of the bispecific antibody JS202-7 is shown in Figure 13.
  • SEQ ID NO: 141 (boldface indicates VL and VH respectively, and LCDR1-LCDR3 and HCDR1-HCDR3 are underlined)
  • VH amino acid sequence of JSCD3 scFv is shown in SEQ ID NO: 137
  • VL amino acid sequence is shown in SEQ ID NO: 138.
  • the amino acid sequence of the first polypeptide chain (expression vector JS012-2B19HC-7-CH1-JSCD3 LC-IgG4-knob) is shown in SEQ ID NO: 144
  • the second polypeptide chain Chain (expression vector JS012-2B19LC-1-Kappa-JSCD3-HC) amino acid sequence is shown in SEQ ID NO: 145
  • the third polypeptide chain (expression vector JS012-2B19HC-7-IgG4-hole) amino acid sequence is shown in SEQ ID
  • the amino acid sequence of the fourth polypeptide chain (expression vector JS012-2B19LC-1-kappa) is shown in SEQ ID NO: 143.
  • the schematic structure of the bispecific antibody JS202-7CD3-Fv is shown in Figure 14.
  • SEQ ID NO: 144 (boldface indicates VL and VH respectively, and LCDR1-LCDR3 and HCDR1-HCDR3 are underlined)
  • SEQ ID NO: 145 (Boldface indicates VL and VH respectively, underlined LCDR1-LCDR3 and HCDR1-HCDR3)
  • the amino acid sequence of the first polypeptide chain (expression vector JSCD3-VH-CH1-JS012-2B19LC-1-IgG4-knob) is shown in SEQ ID NO: 146, and the second most The amino acid sequence of the peptide chain (expression vector JSCD3-LC-lambda-2B19HC-7) is shown in SEQ ID NO: 147, and the amino acid sequence of the third polypeptide chain (expression vector JS012-2B19HC-7-IgG4-hole) is shown in SEQ ID NO Shown in: 142, the amino acid sequence of the fourth polypeptide chain (expression vector JS012-2B19LC-1-kappa) is shown in SEQ ID NO: 143.
  • the schematic structure of the bispecific antibody JS202-7 012-Fv is shown in Figure 15.
  • SEQ ID NO: 146 (Boldface indicates VL and VH respectively, and LCDR1-LCDR3 and HCDR1-HCDR3 are underlined)
  • SEQ ID NO: 147 (boldface indicates VL and VH respectively, underlined LCDR1-LCDR3 and HCDR1-HCDR3)
  • the amino acid sequence of the first polypeptide chain (expression vector JSCD3-HC7-CH1-JS012-2B19LC-1-IgG4-knob) is shown in SEQ ID NO: 148, the first The amino acid sequence of the second polypeptide chain (expression vector JSCD3-LC16-lambda-2B19HC-7) is shown in SEQ ID NO: 149, and the amino acid sequence of the third polypeptide chain (expression vector JS012-2B19HC-7-IgG4-hole) is as shown in SEQ ID NO: ID NO: 142, the amino acid sequence of the fourth polypeptide chain (expression vector JS012-2B19LC-1-kappa) is shown in SEQ ID NO: 143.
  • the schematic diagram of the structure of the bispecific antibody JS202-7CD3 130 012-Fv is shown in Figure 15.
  • SEQ ID NO: 148 (boldface indicates VL and VH respectively, underlined LCDR1-LCDR3 and HCDR1-HCDR3)
  • SEQ ID NO: 149 (boldface indicates VL and VH respectively, underlined LCDR1-LCDR3 and HCDR1-HCDR3)
  • the amino acid sequence of the first polypeptide chain (expression vector JSCD3-HC-CH1-JSCD20-LC-2-IgG4-knob) is shown in SEQ ID NO: 150
  • the amino acid sequence of the second polypeptide chain (expression vector JSCD3-LC-lambda-JSCD20-HC-2) is shown in SEQ ID NO: 151
  • the amino acid sequence of the third polypeptide chain (expression vector JSCD20-HC-2-IgG4-hole)
  • the sequence is shown in SEQ ID NO: 152
  • the amino acid sequence of the fourth polypeptide chain (expression vector JSCD20-LC-2-kappa) is shown in SEQ ID NO: 153.
  • the schematic diagram of the structure of the bispecific antibody JS203-7-JSCD3-CD20-Fv is shown in FIG. 16 .
  • SEQ ID NO: 150 (Boldface indicates VL and VH respectively, and LCDR1-LCDR3 and HCDR1-HCDR3 are underlined)
  • SEQ ID NO: 151 (boldface indicates VL and VH respectively, underlined LCDR1-LCDR3 and HCDR1-HCDR3)
  • SEQ ID NO: 153 (The boldface characters represent VL respectively, and the underline marks LCDR1-LCDR3)
  • the amino acid sequence of the first polypeptide chain (expression vector JSCD3-HC7-CH1-JSCD20-LC-2-IgG4-knob) is shown in SEQ ID NO: 154
  • the amino acid sequence of the second polypeptide chain (expression vector JSCD3-LC16-1ambda-JSCD20-HC-2) is shown in SEQ ID NO: 155
  • the third polypeptide chain (expression vector JSCD20-HC-2-IgG4-hole)
  • the amino acid sequence is shown in SEQ ID NO: 152
  • the amino acid sequence of the fourth polypeptide chain (expression vector JSCD20-LC-2-kappa) is shown in SEQ ID NO: 153.
  • the schematic diagram of the structure of the bispecific antibody JS203-7-CD3 130-CD20-Fv is shown in Figure 16.
  • SEQ ID NO: 154 (boldface indicates VL and VH respectively, and LCDR1-LCDR3 and HCDR1-HCDR3 are underlined)
  • SEQ ID NO: 155 (Boldface indicates VL and VH respectively, and LCDR1-LCDR3 and HCDR1-HCDR3 are underlined)
  • the amino acid sequence of the first polypeptide chain (expression vector JSCD20-VH-CH1-JSCD3-VL-IgG4-knob) is shown in SEQ ID NO: 156
  • the second polypeptide chain chain (expression vector JSCD20-LC-2Kappa-JSCD3-VH) amino acid sequence as shown in SEQ ID NO: 157
  • the third polypeptide chain (expression vector JSCD20-HC-2-IgG4-hole) amino acid sequence as shown in SEQ ID NO: Shown in 152
  • the amino acid sequence of the fourth polypeptide chain (expression vector JSCD20-LC-2-kappa) is shown in SEQ ID NO: 153.
  • the schematic structure of the bispecific antibody JS203-7-CD3-FV is shown in Figure 17.
  • SEQ ID NO: 156 (boldface indicates VL and VH respectively, and LCDR1-LCDR3 and HCDR1-HCDR3 are underlined)
  • SEQ ID NO: 157 (boldface represents VL and VH respectively, and the underline marks LCDR1-LCDR3 and HCDR1-HCDR3)
  • the amino acid sequence of the first polypeptide chain (expression vector JSCD3-HC7-CH1-012ScFv-IgG4-knob) is shown in SEQ ID NO: 164
  • the second polypeptide chain The amino acid sequence of the polypeptide chain (expression vector JSCD3-LC16-Lambda) is shown in SEQ ID NO: 165
  • the amino acid sequence of the third polypeptide chain (expression vector JS012-2B19HC-7-IgG4-hole) is shown in SEQ ID NO: 142.
  • the amino acid sequence of the fourth polypeptide chain (expression vector JS012-2B19LC-1-kappa) is shown in SEQ ID NO: 143.
  • the schematic diagram of the structure of the bispecific antibody JS202-7 CD3-130-012 scFv is shown in Figure 18.
  • SEQ ID NO: 164 (boldface indicates VL and VH respectively, and LCDR1-LCDR3 and HCDR1-HCDR3 are underlined)
  • SEQ ID NO: 165 (boldfaces represent VL respectively, LCDR1-LCDR3 and HCDR1-HCDR3 are underlined)
  • VH amino acid sequence of 012 scFv is shown in SEQ ID NO: 166
  • VL amino acid sequence is shown in SEQ ID NO: 167.
  • the cells endogenously expressing human CD3 (Jurkat cells, the cell bank of the Type Culture Collection Committee of the Chinese Academy of Sciences) were diluted with serial concentrations of anti-CD3 ⁇ Claudin18.2 bispecific antibodies (JS202-7 CD3-Fv, JS202 -7 012-Fv, JS202-7 CD3 130 012-Fv and JS202-7) were incubated at 4°C for 30 minutes, washed away residual antibodies and incubated with fluorescent secondary antibody (PE-anti-human IgG), and finally analyzed by C6PLUS flow cytometer Detect the fluorescence intensity, 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 19).
  • both JS202-7 012-Fv and JS202-7 CD3 130 012-Fv can effectively bind to human CD3 expressing cells, while the binding ability of JS202-7 CD3-Fv and JS202-7 to human CD3 expressing cells is weak.
  • CHO cynomolgus monkey CD3e (Junshi Bio) was diluted with serial concentrations of anti-CD3 ⁇ Claudin18.2 bispecific antibodies (JS202-7 CD3-Fv, JS202-7 012-Fv, JS202-7 CD3 130 012 -Fv and JS202-7) and negative control (anti-KLH monoclonal antibody, anti-KLHhIgG4) were incubated at 4°C for 30 min, washed away residual antibody, incubated with fluorescent secondary antibody (PE-anti-human IgG), and finally analyzed by C6 PLUS flow cytometry The cytometer detects the fluorescence intensity, and 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 20).
  • JS202-7 CD3-Fv, JS202-7 012-Fv, JS202-7 CD3 130 012-Fv and JS202-7 can effectively combine with CHO cynomolgus monkey CD3e cells, JS202-7 CD3-Fv and JS202 -7 binding TOP value is low, it is believed that the binding ability of JS202-7 CD3-Fv and JS202-7 to CHO cynomolgus monkey CD3e cells is reduced.
  • CHO Claudin18.2 (Junshi Biology) cells were respectively diluted with serial concentrations of anti-CD3 ⁇ Claudin18.2 bispecific antibodies (JS202-7 CD3-Fv, JS202-7 012-Fv, JS202-7 CD3 130 012-Fv and JS202-7) were incubated at 4°C for 30 min, washed away the residual antibody and then incubated with fluorescent secondary antibody (PE-anti-human IgG). Finally, the fluorescence intensity was detected by C6PLUS flow cytometer. The stronger the fluorescence signal, the stronger the antibody and The higher the target affinity. Antibody dose-dependent binding curves were fitted by GraphPad ( Figure 21).
  • JS202-7 CD3-Fv, JS202-7 012-Fv, JS202-7 CD3 130 012-Fv and JS202-7 can effectively bind to CHO Claudin18.2 cells, with EC50 of 160.6ng/mL and 169.8 ng/mL, 158.9 ng/mL and 138.7 ng/mL.
  • the anti-CD3 ⁇ Claudin18.2 double antibody can bind to Claudin18.2 on target cells and further bind to CD3-expressing T cells, thereby activating the downstream signaling pathway of T cells.
  • the anti-CD3 ⁇ Claudin18.2 bispecific antibody (JS202-7 CD3-Fv, JS202-7 012-Fv, JS202-7 CD3 130 012-Fv and JS202-7) which was serially diluted to a serial concentration was mixed with the target cell CHO Claudin18 .2 Cells (Junshi Bio) and Jurkat NFAT effector cells (Junshi Bio) were co-incubated, and the signal was detected with a microplate reader after adding the substrate One-Lite TM Luciferase Assay system (Vazyme, Cat#DD1203-04), and finally Data were analyzed with GraphPad to compare dose-dependent cell viability (Figure 22).
  • JS202-7 CD3-Fv, JS202-7 012-Fv, JS202-7 CD3 130 012-Fv and JS202-7 double antibodies can effectively activate T cell downstream signaling pathways, with EC50 of 0.7212ng/ mL, 0.1700 ng/mL, 1.551 ng/mL, and 2.850 ng/mL.
  • Anti-CD3 ⁇ Claudin18.2 bispecific antibody can effectively promote the activation of T lymphocytes in peripheral blood mononuclear cells.
  • the up-regulation of the expression of the cell surface marker CD69 is a sign of early T cell activation, while the expression of the cell surface marker CD25 Up-regulation is a sign of the later stage of T cell activation.
  • the up-regulation of the proportion of CD25 and CD69 double positive population was used to evaluate the activation effect of anti-CD3 ⁇ Claudin18.2 bispecific antibody on T cells in this study.
  • Human total T cells were isolated and purified from commercial PBMCs (Allcells, Cat#PB004F-C) using the human total T cell isolation kit (Miltenyi Biotec, Cat#130-096-535); Cells (1 ⁇ 105 cells per well) and NUGC4Claudin18.2 cells (2 ⁇ 104 cells per well, Junshi Biotechnology) and serially diluted JS202-7 CD3-Fv, JS202-7 012-Fv, JS202-7 CD3 130 012-Fv and JS202-7 and negative control sample anti-KLHhIgG4 were co-incubated in a 96-well plate at 37°C for 24 hours (24 hours).
  • CD25+ and CD69+ double-positive cell populations on CD8+ T cells were collected, stained with PerCP/Cy5.5 anti-human CD8a (Biolegend, Cat#301032), PE anti-human CD25 (Biolegend, Cat#302606), BV421 anti-human CD69 (BD, Cat#562884), and analyzed by flow cytometry Cytometer (BD, CantoII) on-machine detection.
  • the proportion of CD25+ and CD69+ double-positive cell populations on CD8+ T cells was derived by FlowJo software, and then the four-parameter regression curve was fitted by GraphPadprism software to calculate the EC50 value.
  • the anti-CD3 ⁇ Claudin18.2 bispecific antibodies JS202-7 CD3-Fv, JS202-7 012-Fv, JS202-7 CD3 130 012-Fv and JS202-7 of the present invention can all effectively promote CD8T
  • the increase in the proportion of CD25+ and CD69+ double-positive cell populations in lymphocytes promoted T cell activation, with EC50 of 1.323ng/mL, 1.664ng/mL, 127.5ng/mL and 3.300ng/mL, respectively.
  • Cytokine release syndrome is the release of massive inflammatory cytokines due to the cross-linking of immune effector cells and tumor cells.
  • Sources of cytokines include the target cells themselves and immune cells recruited to the tumor site. This chain reaction leads to hyperactivation of immune cells, leading to more activation of cytokines such as tumor necrosis factor (TNF) and interferon gamma (IFN ⁇ ), followed by interleukins IL-6, IL-10, IL -8 and IL-2 etc.
  • TNF tumor necrosis factor
  • IFN ⁇ interferon gamma
  • JS202-7 CD3-Fv, JS202-7 012-Fv, JS202-7 CD3 130 012-Fv and JS202-7 in the form of experimental detection solution positive control antibody OKT3 and negative control antibody anti-KLHhIgG4 were all tested in vitro at 10 ⁇ g/ml
  • the level was incubated with commercial PBMC (Allcells, Cat#PB004F-C), and the total amount of PBMC was 3.0 ⁇ 10 5 cells/well.
  • the experiment was carried out in PBMC of 2 different donors (ID: LP200810, PCH20210100021).
  • the cells were incubated with the antibody for 24 hours at 37°C and 5% CO 2 , and the supernatant was collected by centrifugation the next day and the release of cytokines was detected with BD CBA human Th1/Th2 cytokine kit II.
  • PBMC peripheral blood mononuclear cells
  • OKT3 obtainable from the hybridoma deposited in the American Type Culture Collection under the accession number ATCC CRL 8001.
  • the experimental results showed that JS202-7 012-Fv and JS202-7 CD3 130 012-Fv can significantly reduce the risk of cytokine release syndrome, and JS202-7 CD3 130 012-Fv will not cause cytokine release syndrome, both JS202-7 CD3-Fv and JS202-7 Can cause cytokine release syndrome.
  • the administration was administered by intraperitoneal injection, twice a week, with an administration volume of 10 ⁇ L/g, for five 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.
  • Ti mean tumor volume of the treatment group on day i of administration
  • T0 mean tumor volume of treatment group on day 0 of administration
  • Vi mean tumor volume of negative control group on day i of administration
  • V0 negative control The mean tumor volume of the group on day 0 of administration.
  • the average tumor volume of the normal saline group was 2066 ⁇ 422mm 3
  • the volumes were 1071 ⁇ 119mm 3 and 994 ⁇ 196mm 3
  • the TGIs were 51.0% and 55.0%, respectively, compared with the normal saline group, significantly inhibiting tumor growth.
  • G4 JS202-7 CD3 130 012-FV (0.1mg/kg) group, G5 JS202-7 CD3 130 012-FV (0.3mg/kg) group and G6 JS202-7 CD3 130 012-FV (3mg/kg) group mean The tumor volumes were 1098 ⁇ 195mm 3 , 854 ⁇ 83mm 3 and 783 ⁇ 179mm 3 , and the TGIs were 49.7%, 62.2% and 65.8% compared with the normal saline group, which significantly inhibited tumor growth.
  • JS202-7 CD3 130 012-FV could inhibit the growth of B-hCD3e humanized mouse MC38 hClaudin18.2 subcutaneously transplanted tumor in vivo.
  • test double antibodies are: JS202-7 CD3 130 012-Fv, JS202-7 CD3 132 012-Fv, JS202-7 CD3 238 012-Fv, JS202-7 CD3 285 012-Fv, of which JS202-7 CD3 132 012-Fv , JS202-7 CD3 238 012-Fv, JS202-7 CD3 285 012-Fv compared with JS202-7 CD3 130 012-Fv, only the CD3 sequence is different; the VH/VL of CD3 in JS202-7 CD3 130 012-Fv Replace the sequences with the VH/VL sequences of JSCD3-130, JSCD3-132, JSCD3-238, and JSCD3-285 respectively to obtain JS202-7 CD3 132 012-F
  • Tm is the thermal transition temperature
  • test double antibodies are: JS202-7 CD3 130 012-Fv, JS202-7 CD3 132 012-Fv, JS202-7 CD3 238 012-Fv, JS202-7 CD3 285 012-Fv, of which JS202-7 CD3 132 012-Fv , JS202-7 CD3 238 012-Fv, JS202-7 CD3 285 012-Fv compared with JS202-7 CD3 130 012-Fv, only the CD3 sequence is different; the VH/VL of CD3 in JS202-7 CD3 130 012-Fv Replace the sequences with the VH/VL sequences of JSCD3-130, JSCD3-132, JSCD3-238, and JSCD3-285 respectively to obtain JS202-7 CD3 132 012-Fv, JS202-7 CD3 238 012-Fv, and JS202-7 CD3 285 012-Fv.
  • the high-performance liquid chromatography of the SEC chromatographic column detects the SEC purity of each component sample.
  • the mobile phase is 50mM PB-300mM Na 2 SO 4 (pH 6.8 ⁇ 0.2), the flow rate is 0.5mL/min, the detection wavelength is 280nm, the test sample is injected 100 ⁇ g, and detected by high performance liquid chromatography. Quantitative analysis was carried out by area normalization, the peak area percentage of monomer was used as the purity of the sample, and the peak area percentage of aggregates and fragments was used as the content of aggregates and fragments.
  • the CE-SDS reduction purity of each component sample was detected by a capillary electrophoresis instrument (Maurice Instruments) equipped with a CE-SDS cartridge. Add 5 ⁇ L of 2-mercaptoethanol to 95 ⁇ l of the test solution (1 mg/mL), incubate at 70° C. for denaturation and heat treatment for 15 minutes, and detect using capillary electrophoresis (Maurice). Calculated by the area normalization method, the light chain, heavy chain 1 and heavy chain 2 purity are calculated respectively by the percentages of the corrected peak areas of the light chain and heavy chain to the sum of all corrected peak areas, and the sum of the three is the sample total purity.
  • CE-SDS non-reducing purity of each component sample was detected by a capillary electrophoresis apparatus (Maurice Instruments) equipped with a CE-SDS cartridge.
  • a capillary electrophoresis apparatus (Maurice Instruments) equipped with a CE-SDS cartridge.
  • Add 5 ⁇ L of 0.25M N-ethylmaleimide to 95 ⁇ L of the test solution (1 mg/mL) incubate at 70°C for denaturation and heat treatment for 5 min, and use capillary electrophoresis (Maurice) for detection.
  • the purity is calculated as the percentage of the corrected peak area of the main peak to the sum of all corrected peak areas.
  • a capillary electrophoresis instrument (Maurice Instruments) equipped with an icIEF cartridge was used to detect the isoelectric point of each component sample. After the test sample was diluted with iCIEF mixture, it was detected by capillary electrophoresis (Maurice). Different isoelectric point components are focused on different positions to achieve the effect of focusing and separation.
  • the experiments were all carried out at 37°C. Using a Thermo Scientific microplate reader, the plate was coated with a fixed concentration of His-tagged human CD3E (1.0 ⁇ g/mL) for 90 minutes. After washing, the plate was blocked with 2% BSA for 90 minutes.
  • JS202 samples (starting at 1 ⁇ g/mL, 3-fold serial dilution to 0.005645ng/mL, 12 concentrations in total) were incubated for 60 minutes, and after washing the plate, Mouse Anti-Human IgG4 Fc-HRP diluted 5000 times was added as a detection antibody and incubated for 60 minutes for detection , then use 0.1mg/mL TMB to develop color for 15min, and finally stop the reaction with 2M HCl, and read the plate at 450nm/620nm.
  • the four-parameter logarithmic regression (4PL) model in GraphPad Prism software was used to fit and obtain the corresponding EC50.
  • the anti-CD3 ⁇ Claudin18.2 double antibody can bind to Claudin18.2 on target cells and further bind to CD3-expressing T cells, thereby activating the downstream signaling pathway of T cells.
  • JS202-7 double antibody into the pH6.0 buffer system (20mM histidine hydrochloride histidine + 230mM sucrose), control the sample concentration at about 10mg/ml, and dispense a certain volume (1mL/tube) of JS202 -7 double antibody samples were placed in a medical refrigerator at 5 ⁇ 3°C, the stability of 0W, 2W and 4W was investigated, and the purity and biological activity of the samples were tested according to the time points.
  • the test results show that the JS202-7 double antibody has good long-term stability, see Table 19 for details.
  • CHO-hCD3 cells human CD3 protein overexpressed on CHO cells
  • CHO-hCD3 cells human CD3 protein overexpressed on CHO cells
  • control antibodies from The initial concentration was 100 ⁇ g/ml, diluted 3 times, and a total of 10 concentration gradients
  • JS203-7-JSCD3-CD20-Fv and JS203-7-CD3 130-CD20-Fv and control antibodies from The initial concentration was 100 ⁇ g/ml, diluted 3 times, and a total of 10 concentration gradients
  • fluorescently-labeled secondary antibodies for 30 minutes at 4°C in the dark.
  • the cells were collected by flow cytometry (BD CantoII), and the fluorescent antibodies bound to the cell surface were detected.
  • the MFI value was obtained by analyzing the raw data with FlowJo, and the antibody dose-dependent binding curve was fitted by GraphPad ( Figure 26) and the EC50 was calculated.
  • the positive control was REGN1979 (Regeneron Pharmaceuticals).
  • REGN1979, JS203-7-JSCD3-CD20-Fv and JS203-7-CD3 130-CD20-Fv can bind to human CD3 on the cell surface, with EC50 of 0.97 ⁇ g/mL, 0.09198 and 0.6511 ⁇ g/mL, respectively. mL.
  • Raji cells (a human B lymphoma cell, CD20 positive) were mixed with different concentrations of bispecific antibodies JS203-7-JSCD3-CD20-Fv and JS203-7-CD3 130-CD20-Fv and control antibodies (initial concentration 100 ⁇ g/ml, 3-fold dilution, a total of 10 concentration gradients) were incubated at 4°C for 30 minutes, then washed and incubated with fluorescently-labeled secondary antibodies for 30 minutes at 4°C in the dark. Finally, the cells were collected by flow cytometry (BD CantoII), and the fluorescent antibodies bound to the cell surface were detected. The MFI value was obtained by analyzing the raw data with FlowJo, and the antibody dose-dependent binding curve was fitted by GraphPad ( Figure 27) and the EC50 was calculated. The positive control was REGN1979 (Regeneron Pharmaceuticals)).
  • REGN1979, JS203-7-JSCD3-CD20-Fv and JS203-7-CD3 130-CD20-Fv can all bind to CD20 on the surface of Raji cells with high affinity, with EC50 of 2.251 ⁇ g/mL and 1.316 ⁇ g/mL, respectively. ⁇ g/mL and 1.939 ⁇ g/mL.
  • the target cell Raji (a human B lymphoma cell, CD20 positive) and the effector cell Jurkat NFAT (stable expression of luc2P/NFAT-RE) were divided into 5 ⁇ 104 cells per well and 1 ⁇ 105 cells per well Added to 96-well flat bottom white plate (Corning, Cat#3917). Afterwards, bispecific antibodies JS203-7-JSCD3-CD20-Fv and JS203-7-CD3 130-CD20-Fv and control antibody (initial concentration of 1 ⁇ g /ml, 3-fold dilution, a total of 12 concentration gradients) were added to the cell plate and co-incubated for 4-6 hours in a 37°C incubator.
  • ONE-Glo luciferase detection reagent Promega was added to the cell-antibody mixture system, and the chemiluminescent signal was detected with a multifunctional microplate reader (TECAN M1000pro). The four-parameter regression curve was fitted by GraphPad prism software, and the EC50 value was calculated.
  • JS203-7-JSCD3-CD20-Fv and JS203-7-CD3 130-CD20-Fv had strong expression in the luciferase reporter gene system composed of effector cells Jurkat NFAT and target cells Raji cells
  • the activated T cell activity, EC50 were 10.84ng/mL, 0.3967ng/mL and 2.780ng/mL.
  • Anti-CD3/CD20 bispecific antibody can effectively promote the activation of T lymphocytes in peripheral blood mononuclear cells.
  • the up-regulation of the expression of the cell surface marker CD69 is a sign of early T cell activation
  • the up-regulation of the expression of the cell surface marker CD25 is a sign of T lymphocyte activation.
  • this experiment uses the up-regulation of the proportion of CD25 and CD69 double positive population to evaluate the activation effect of anti-CD3/CD20 bispecific antibody on T cells in this study.
  • Human total T cells were isolated and purified from commercial PBMCs (Allcells, Cat#PB004F-C) using the human total T cell purification kit (Miltenyi Biotec, Cat#130-096-535); the purified human total T cells were then (1X105 cells per well) and Raji cells (2 ⁇ 104 cells per well), as well as serially diluted JS203-7-JSCD3-CD20-Fv and JS203-7-CD3 130-CD20-Fv and control antibody (initial Concentration of 10 ⁇ g/ml, 5-fold dilution, a total of 12 concentration gradients) were co-incubated in a 96-well plate at 37°C for 24 hours.
  • CD25+CD69+ double-positive cell population on CD8+T cells was derived by FlowJo software, and then the four-parameter regression curve was fitted by GraphPad prism software to calculate the EC50 value.
  • the anti-CD3/CD20 bispecific antibodies REGN1979, JS203-7-JSCD3-CD20-Fv and JS203-7-CD3 130-CD20-Fv of the present invention can effectively promote CD25+ and CD69+ in CD8 T lymphocytes.
  • bispecific antibodies in solution and control antibodies were incubated with resting PBMCs at in vitro levels, resulting in the layout of cytokine release.
  • the experiment was incubated under the condition of 1640 complete medium (RPMI 1640 Medium+10% FBS), and 50 ⁇ l of PBMC (3.0 ⁇ 10 5 cells/well) was added.
  • PBMCs of 4 different donors LP210817011, LP210825006, LP210821004 and PCH202100025
  • the antibody detection concentration was 10 ⁇ g/ml, 50 ⁇ l per well, and 3 replicate wells were set for each condition.
  • the cells were incubated with the antibody for 24 hours at 37°C and 5% CO 2 , and the supernatant was collected by centrifugation the next day and the release of cytokines was detected with BD CBA human Th1/Th2 cytokine kit II.
  • PBMCs were co-incubated with antibodies in solution in a resting state, and the positive control was OKT3.
  • the experimental results showed that double antibody molecules in solution could induce human PBMCs to release a large amount of cytokines, especially IL-6 and IFN- ⁇ . This may be due to the presence of target cells CD20+ B cells in the response system, which caused strong T cell activation and the release of multiple cytokines, so in subsequent clinical trials, additional attention should be paid to secondary pharmacological factors.
  • the CRS of JS203-7-JSCD3-CD20-Fv and JS203-7-CD3 130-CD20-Fv is lower than that of the positive control OKT3 and comparable to that of REGN1979 .

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Abstract

提供了一种结合CD3的抗体及其用途，以及能同时结合CD3与肿瘤相关抗原的双特异性抗体。所述双特异性抗体包含第一结合结构域、第二结合结构域和第三结合结构域，其中第一结合结构域与CD3结合、第二结合结构域和第三结合结构域与肿瘤相关抗原结合，第一结合结构域具有Fab分子的形式，第二结合结构域具有Fv分子或scFv分子的形式，第三结合结构域具有Fab分子的形式，所述双特异性抗体能够在结合肿瘤相关抗原表达细胞的同时，结合并激活CD3阳性的T细胞，进而促进T细胞对表达肿瘤相关抗原的肿瘤细胞特异性的杀伤。同时，还提供了包含所述双特异性抗体的药物组合物以及制备和使用所述双特异性抗体的方法。

Description

结合CD3的抗体及其用途 技术领域
本发明涉及CD3抗体，还涉及同时结合CD3和肿瘤相关抗原的双特异性抗体及其用途。
背景技术
T淋巴细胞(T lymphocyte)简称T细胞，是由来源于骨髓的淋巴干细胞，在胸腺中分化、发育成熟后，通过淋巴和血液循环而分布到全身的免疫器官和组织中发挥免疫功能。T细胞是一种高效的杀伤细胞，能迅速消灭病毒感染细胞和癌细胞。T细胞杀伤作用需要免疫突触的形成，而这一过程高度依赖于TCR识别抗原递呈细胞表面MHC分子与所递呈的抗原肽形成的复合物。激活的免疫突触将释放细胞毒素和细胞因子起到杀伤作用。在免疫突触形成过程中，受T细胞与靶细胞之间距离的限制，双特异性抗体定向T细胞就要模拟免疫突触的形成，一端通过靶向CD3来实现桥连T细胞TCR受体，另一端桥连靶细胞通过靶向靶细胞表面抗原来实现。
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。
双特异性抗体将细胞毒性T细胞导向癌细胞的基本原理在1985年首次被描述。双特异性抗体介导的T细胞重定向杀伤肿瘤细胞被认为是最有希望的癌症治疗策略。2009年，全球首个双特异性抗体药物Removab获得欧盟药品管理局(EMA)批准上市，适应症为标准治疗无效或不可行的因EpCAM阳性肿瘤所致的恶性腹水。Catumaxomab(anti-CD3and anti-EpCAM)做为第一例被FDA批准的双抗，想要达到的应用目标是“一个三功能”的双特异性抗体，它除了桥连的肿瘤靶细胞和T细胞，也可以结合Fcγ受体阳性效应细胞，巨噬细胞等。作为第一个被FDA批准的T cell recruiter类双抗，catumaxomab具有里程碑式的意义。事实上，在对有腹水的卵巢癌和其它癌患者的治疗中，catumaxomab的确显示了良 好的疗效。但在这一过程中，细胞因子风暴引起的发热、恶心、呕吐等副作用多有出现，同时，在第0，3，7和10天分别进行10，20，50，150μg四次注射后，70％的患者出现了人抗小鼠抗体/人抗大鼠抗体(HAMA/HARA)。这些副作用可能和以下因素相关：双抗对CD3较高的亲和力导致未结合肿瘤细胞时就低活化T细胞所引起的细胞因子风暴；双抗在结合肿瘤细胞后过度活化T细胞引起的细胞因子风暴；EpCAM在某些正常组织上也有相当量的表达引起的双抗对正常组织的潜在非特异性杀伤。
本发明旨在提供亲和力、稳定性和/或降低引起细胞因子释放综合征等方面均具有较好效果的抗体。
发明内容
本发明提供了一种与CD3结合的抗体或其抗原结合片段，本发明提供的抗CD3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；还提供了同时结合CD3和肿瘤相关抗原的三价双特异性抗体，其在亲和力、稳定性和/或降低引起细胞因子释放综合征等方面均具有较好效果，并能够有效抑制肿瘤生长，可用于诸如癌症的治疗。
在一个方面，本发明提供了一种双特异性抗体，其包含第一结合结构域、第二结合结构域和第三结合结构域，其中所述第一结合结构域与CD3结合，所述第二结合结构域和所述第三结合结构域与肿瘤相关抗原结合，所述第一结合结构域具有Fab分子或Fv分子或scFv分子的形式，所述第二结合结构域具有Fab分子或Fv分子或scFv分子的形式，和所述第三结合结构域具有Fab分子的形式。
在一些实施方式中，本发明所述第一结合结构域具有Fab分子的形式，所述第二结合结构域具有Fv分子或scFv分子的形式，和所述第三结合结构域具有Fab分子的形式。
在一些实施方式中，本发明所述第一结合结构域具有Fab分子的形式，所述第二结合结构域具有Fv分子的形式，和所述第三结合结构域具有Fab分子的形式。
在一些实施方式中，本发明所述第一结合结构域具有Fab分子的形式，所述第二结合结构域具有scFv分子的形式，和所述第三结合结构域具有Fab分子的形式。
在一些实施方式中，本发明所述第一结合结构域具有Fv分子的形式，所述第二结合结构域具有Fab分子的形式，和所述第三结合结构域具有Fab分子的形式。
在一些实施方式中，本发明所述肿瘤相关抗原选自BCMA、CD19、CD20、CD22、CD30、CD33、CD38、CD40、CD123、CDK4、CEA、Claudin18.2、EGFR、ROR1、TGF-β和5T4；优选地，所述肿瘤相关抗原是Claudin 18.2、CD20或BCMA。
在一些实施方式中，本发明所述双特异性抗体，其具有四条多肽链，其中所述第一多 肽链自N端到C端具有VH1-CH1-连接肽-VL2-连接肽-铰链区-第一Fc区，所述第二多肽链自N端到C端具有VL1-CL-连接肽-VH2；所述第三多肽链自N端到C端具有VH2-CH1-铰链区-第二Fc区，所述第四多肽链自N端到C端具有VL2-CL。其中，所述VH1和VL1形成一结构域，所述VH2和VL2形成一结构域。
在一些实施方式中，本发明所述第一多肽链的VH1-CH1与所述第二多肽链的VL1-CL形成所述第一结合结构域，所述第一多肽链的VL2与所述第二多肽链的VH2形成所述第二结合结构域，所述第三多肽链的VH2-CH1与所述第四多肽链的VL2-CL形成所述第三结合结构域。
在一些实施方式中，本发明所述双特异性抗体，其具有四条多肽链，其中所述第一多肽链自N端到C端具有VH1-CH1-连接肽-VL2-连接肽-VH2-连接肽-铰链区-第一Fc区，所述第二多肽链自N端到C端具有VL1-CL；所述第三多肽链自N端到C端具有VH2-CH1-铰链区-第二Fc区，所述第四多肽链自N端到C端具有VL2-CL。其中，所述VH1和VL1形成一结构域，所述VH2和VL2形成一结构域。
在一些实施方式中，本发明所述第一多肽链的VH1-CH1与所述第二多肽链的VL1-CL形成所述第一结合结构域，所述第一多肽链的VL2-连接肽-VH2形成所述第二结合结构域，所述第三多肽链的VH2-CH1与所述第四多肽链的VL2-CL形成所述第三结合结构域。
在一些实施方式中，本发明所述双特异性抗体，其具有四条多肽链，其中所述第一多肽链自N端到C端具有VH2-CH1-连接肽-VL1-连接肽-铰链区-第一Fc区，所述第二多肽链自N端到C端具有VL2-CL-连接肽-VH1；所述第三多肽链自N端到C端具有VH2-CH1-铰链区-第二Fc区，所述第四多肽链自N端到C端具有VL2-CL。其中，所述VH1和VL1形成一结构域，所述VH2和VL2形成一结构域。
在一些实施方式中，本发明所述第一多肽链的VH2-CH1与所述第二多肽链的VL2-CL形成所述第二结合结构域，所述第一多肽链的VL1与所述第二多肽链的VH1形成所述第一结合结构域，所述第三多肽链的VH2-CH1与所述第四多肽链的VL2-CL形成所述第三结合结构域。
在一些实施方式中，本发明所述第一结合结构域包含的HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3分别为：
HCDR1：X1YAMN，其中，X1为T或K；
HCDR2：RIRSKYNNYATYYAX2SVKX3，其中，X2为D或A，X3为D或G；
HCDR3：HGX3X4GX5X6X7X8X9X10X11AY，其中，X3为N或A，X4为F或A，X5为N或A，X6为S或A，X7为Y或A，X8为V或A，X9为S或A，X10为W或 A，X11为F或A；
LCDR1：X12SSTGAVTTX13NYX14N，其中，X12为R或G，X13为S或G，X14为A或P；
LCDR2：GTNKRAP；
LCDR3：AX15X16X17X18X19X20X21X22，其中，X15为L或A，X16为W或A，X17为Y或A，X18为S或A，X19为N或A，X20为L或A，X21为W或A，X22为V或A。
在一些实施方式中，本发明所述第一结合结构域包含：氨基酸序列如SEQ ID NO：1或2所示的HCDR1，氨基酸序列如SEQ ID NO：3或4所示的HCDR2，氨基酸序列如SEQ ID NO：5-22中任一项所示的HCDR3，氨基酸序列如SEQ ID NO：23或24所示的LCDR1，氨基酸序列如SEQ ID NO：25所示的LCDR2，和氨基酸序列如SEQ ID NO：26-42中任一项所示的LCDR3。
在一些实施方式中，本发明所述第一结合结构域包含HCDR1、HCDR2、HCDR3和LCDR1、LCDR2、LCDR3：
所述HCDR1、HCDR2、HCDR3为：
(1)氨基酸序列如SEQ ID NO：1所示的HCDR1，氨基酸序列如SEQ ID NO：3所示的HCDR2，氨基酸序列如SEQ ID NO：5所示的HCDR3；或
(2)氨基酸序列如SEQ ID NO：1所示的HCDR1，氨基酸序列如SEQ ID NO：3所示的HCDR2，氨基酸序列如SEQ ID NO：15所示的HCDR3；或
(3)氨基酸序列如SEQ ID NO：2所示的HCDR1，氨基酸序列如SEQ ID NO：4所示的HCDR2，氨基酸序列如SEQ ID NO：5-22中任一项所示的HCDR3；
所述LCDR1、LCDR2、LCDR3为：
(1)氨基酸序列如SEQ ID NO：23所示的LCDR1，氨基酸序列如SEQ ID NO：25所示的LCDR2，氨基酸序列如SEQ ID NO：26所示的LCDR3；或
(2)氨基酸序列如SEQ ID NO：23所示的LCDR1，氨基酸序列如SEQ ID NO：25所示的LCDR2，氨基酸序列如SEQ ID NO：33所示的LCDR3；或
(3)氨基酸序列如SEQ ID NO：24所示的LCDR1，氨基酸序列如SEQ ID NO：25所示的LCDR2，氨基酸序列如SEQ ID NO：26-42中任一项所示的LCDR3。
在一些实施方式中，本发明所述第一结合结构域包含的HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3分别为：
HCDR1：X1YAMN，其中，X1为T或K(优选为K)；
HCDR2：RIRSKYNNYATYYAX2SVKX3，其中，X2为D或A(优选为A)，X3为 D或G(优选为G)；
HCDR3：HGX3X4GX5X6X7X8X9X10X11AY，其中，X3为N，X4为F，X5为N或A，X6为S或A，X7为Y或A，X8为V或A，X9为S或A，X10为W，X11为F或A(优选为F)；
LCDR1：X12SSTGAVTTX13NYX14N，其中，X12为R或G，X13为S或G，X14为A或P；
LCDR2：GTNKRAP；
LCDR3：AX15X16X17X18X19X20X21X22，其中，X15为L或A，X16为W，X17为Y或A，X18为S或A，X19为N或A，X20为L或A，X21为W，X22为V或A。
在一些实施方式中，本发明所述第一结合结构域包含：氨基酸序列如SEQ ID NO：1或2所示的HCDR1，氨基酸序列如SEQ ID NO：3或4所示的HCDR2，氨基酸序列如SEQ ID NO：5、9、10、11、15、17、18、19或20中任一项所示的HCDR3，氨基酸序列如SEQ ID NO：23或24所示的LCDR1，氨基酸序列如SEQ ID NO：25所示的LCDR2，和氨基酸序列如SEQ ID NO：26、28、30、33、35、36、37、39、40、41或42中任一项所示的LCDR3。
在一些实施方式中，本发明所述第一结合结构域包含：
(I)氨基酸序列如SEQ ID NO：1所示的HCDR1，氨基酸序列如SEQ ID NO：3所示的HCDR2，氨基酸序列如SEQ ID NO：15所示的HCDR3，氨基酸序列如SEQ ID NO：23所示的LCDR1，氨基酸序列如SEQ ID NO：25所示的LCDR2，氨基酸序列如SEQ ID NO：33所示的LCDR3；或
(II)氨基酸序列如SEQ ID NO：2所示的HCDR1，氨基酸序列如SEQ ID NO：4所示的HCDR2，氨基酸序列如SEQ ID NO：5所示的HCDR3，氨基酸序列如SEQ ID NO：24所示的LCDR1，氨基酸序列如SEQ ID NO：25所示的LCDR2，氨基酸序列如SEQ ID NO：26、28、33、36或37中任一项所示的LCDR3；或
(III)氨基酸序列如SEQ ID NO：2所示的HCDR1，氨基酸序列如SEQ ID NO：4所示的HCDR2，氨基酸序列如SEQ ID NO：9所示的HCDR3，氨基酸序列如SEQ ID NO：24所示的LCDR1，氨基酸序列如SEQ ID NO：25所示的LCDR2，氨基酸序列如SEQ ID NO：28、35、37、39或40中任一项所示的LCDR3；或
(IV)氨基酸序列如SEQ ID NO：2所示的HCDR1，氨基酸序列如SEQ ID NO：4所示的HCDR2，氨基酸序列如SEQ ID NO：10所示的HCDR3，氨基酸序列如SEQ ID NO：24所示的LCDR1，氨基酸序列如SEQ ID NO：25所示的LCDR2，氨基酸序列如SEQ ID NO：28、33、40、41或42中任一项所示的LCDR3；或
(V)氨基酸序列如SEQ ID NO：2所示的HCDR1，氨基酸序列如SEQ ID NO：4所示 的HCDR2，氨基酸序列如SEQ ID NO：11所示的HCDR3，氨基酸序列如SEQ ID NO：24所示的LCDR1，氨基酸序列如SEQ ID NO：25所示的LCDR2，氨基酸序列如SEQ ID NO：26、28、33、35、36、37、40、41或42中任一项所示的LCDR3；或
(VI)氨基酸序列如SEQ ID NO：2所示的HCDR1，氨基酸序列如SEQ ID NO：4所示的HCDR2，氨基酸序列如SEQ ID NO：15所示的HCDR3，氨基酸序列如SEQ ID NO：24所示的LCDR1，氨基酸序列如SEQ ID NO：25所示的LCDR2，氨基酸序列如SEQ ID NO：26或30所示的LCDR3；或
(VII)氨基酸序列如SEQ ID NO：2所示的HCDR1，氨基酸序列如SEQ ID NO：4所示的HCDR2，氨基酸序列如SEQ ID NO：17所示的HCDR3，氨基酸序列如SEQ ID NO：24所示的LCDR1，氨基酸序列如SEQ ID NO：25所示的LCDR2，氨基酸序列如SEQ ID NO：33、35、36、37或42中任一项所示的LCDR3；或
(VIII)氨基酸序列如SEQ ID NO：2所示的HCDR1，氨基酸序列如SEQ ID NO：4所示的HCDR2，氨基酸序列如SEQ ID NO：18所示的HCDR3，氨基酸序列如SEQ ID NO：24所示的LCDR1，氨基酸序列如SEQ ID NO：25所示的LCDR2，氨基酸序列如SEQ ID NO：28、35、36、37、39、41或42中任一项所示的LCDR3；或
(IX)氨基酸序列如SEQ ID NO：2所示的HCDR1，氨基酸序列如SEQ ID NO：4所示的HCDR2，氨基酸序列如SEQ ID NO：20所示的HCDR3，氨基酸序列如SEQ ID NO：24所示的LCDR1，氨基酸序列如SEQ ID NO：25所示的LCDR2，氨基酸序列如SEQ ID NO：28、33、35、36、40、41或42中任一项所示的LCDR3；或
(X)氨基酸序列如SEQ ID NO：2所示的HCDR1，氨基酸序列如SEQ ID NO：4所示的HCDR2，氨基酸序列如SEQ ID NO：9、10或19中任一项所示的HCDR3，氨基酸序列如SEQ ID NO：23所示的LCDR1，氨基酸序列如SEQ ID NO：25所示的LCDR2，氨基酸序列如SEQ ID NO：26所示的LCDR3；或
(XI)氨基酸序列如SEQ ID NO：1所示的HCDR1、氨基酸序列如SEQ ID NO：3所示的HCDR2、氨基酸序列如SEQ ID NO：5所示的HCDR3、氨基酸序列如SEQ ID NO：23所示的LCDR1、氨基酸序列如SEQ ID NO：25所示的LCDR2、氨基酸序列如SEQ ID NO：26所示的LCDR3；或
(XII)氨基酸序列如SEQ ID NO：2所示的HCDR1，氨基酸序列如SEQ ID NO：4所示的HCDR2，氨基酸序列如SEQ ID NO：19所示的HCDR3，氨基酸序列如SEQ ID NO：24所示的LCDR1，氨基酸序列如SEQ ID NO：25所示的LCDR2，氨基酸序列如SEQ ID NO：36所示的LCDR3。
在一些实施方式中，本发明所述第一结合结构域包含：氨基酸序列如SEQ ID NO：43- 62中任一项所示的VH，和氨基酸序列如SEQ ID NO：63-81中任一项所示的VL。
在一些实施方式中，本发明所述第一结合结构域包含：
(I)氨基酸序列如SEQ ID NO：44所示的VH，和氨基酸序列如SEQ ID NO：64所示的VL；或
(II)氨基酸序列如SEQ ID NO：45所示的VH，和氨基酸序列如SEQ ID NO：65、67、72、75或76所示的VL；或
(III)氨基酸序列如SEQ ID NO：49所示的VH，和氨基酸序列如SEQ ID NO：67、74、76、78或79所示的VL；或
(IV)氨基酸序列如SEQ ID NO：50所示的VH，和氨基酸序列如SEQ ID NO：67、72、79、80或81所示的VL；或
(V)氨基酸序列如SEQ ID NO：51所示的VH，和氨基酸序列如SEQ ID NO：65、67、72、74、75、76、79、80或81所示的VL；或
(VI)氨基酸序列如SEQ ID NO：55所示的VH，和氨基酸序列如SEQ ID NO：65或69所示的VL；或
(VII)氨基酸序列如SEQ ID NO：57所示的VH，和氨基酸序列如SEQ ID NO：72、74、75、76或81所示的VL；或
(VIII)氨基酸序列如SEQ ID NO：58所示的VH，和氨基酸序列如SEQ ID NO：67、74、75、76、78、80或81所示的VL；或
(IX)氨基酸序列如SEQ ID NO：60所示的VH，和氨基酸序列如SEQ ID NO：67、72、74、75、79、80或81所示的VL；或
(X)氨基酸序列如SEQ ID NO：49、50或59中任一项所示的VH，和氨基酸序列如SEQ ID NO：63所示的VL；或
(XI)氨基酸序列如SEQ ID NO：43所示的VH，和氨基酸序列如SEQ ID NO：63所示的VL；或
(XII)氨基酸序列如SEQ ID NO：59所示的VH，和氨基酸序列如SEQ ID NO：75所示的VL。
在一些实施方式中，本发明所述第二结合结构域和第三结合结构域与Claudin 18.2结合，其分别包含：氨基酸序列如SEQ ID NO：121所示的HCDR1，氨基酸序列如SEQ ID NO：122所示的HCDR2，氨基酸序列如SEQ ID NO：123所示的HCDR3，氨基酸序列如SEQ ID NO：124所示的LCDR1，氨基酸序列如SEQ ID NO：125所示的LCDR2，和氨基酸序列如SEQ ID NO：126所示的LCDR3。
在一些实施方式中，本发明所述第二结合结构域和第三结合结构域与CD20结合，其 分别包含：氨基酸序列如SEQ ID NO：127所示的HCDR1，氨基酸序列如SEQ ID NO：128所示的HCDR2，氨基酸序列如SEQ ID NO：129所示的HCDR3，氨基酸序列如SEQ ID NO：130所示的LCDR1，氨基酸序列如SEQ ID NO：131所示的LCDR2，和氨基酸序列如SEQ ID NO：132所示的LCDR3。
在一些实施方式中，本发明所述第二结合结构域和第三结合结构域与Claudin 18.2结合，其分别包含：(a)氨基酸序列如SEQ ID NO：133所示的VH，和氨基酸序列如SEQ ID NO：134所示的VL；或(b)氨基酸序列如SEQ ID NO：166所示的VH，和氨基酸序列如SEQ ID NO：167所示的VL。
在一些实施方式中，本发明所述第二结合结构域和第三结合结构域与CD20结合，其分别包含：氨基酸序列如SEQ ID NO：135所示的VH，和氨基酸序列如SEQ ID NO：136所示的VL。
在一些实施方式中，本发明所述第一Fc区为knob-Fc，所述第二Fc区为hole-Fc。
在一些实施方式中，本发明所述第一Fc区与所述第二Fc区之间通过铰链区的二硫键和杵臼结构连接。
在一些实施方式中，本发明所述第一Fc区和/或第二Fc区为来自IgG的Fc区，优选为来自IgG1、IgG2、IgG3或IgG4的Fc区。
在一些实施方式中，本发明所述第CH1为来自IgG的CH1，优选为IgG1、IgG2、IgG3或IgG4的CH1。
在一些实施方式中，本发明所述CL为来自λ或κ轻链的CL。
在一些实施方式中，本发明连接肽可以是包含G(甘氨酸)、S(丝氨酸)和A(丙氨酸)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；进一步优选地，连接肽包括但不限于(GS)n、(GGS)n、(GGGS)n、(GGGGS)n和AS(GGGGS)n等，n可为1-6的整数，如1、2、3、4、5、6。
在一些实施方式中，本发明所述双特异性抗体具有四条多肽链，所述四条多肽链分别为：
(1)具有如SEQ ID NO：144所示的氨基酸序列的第一多肽链，具有如SEQ ID NO：145所示的氨基酸序列的第二多肽链，具有如SEQ ID NO：142所示的氨基酸序列的第三多肽链，具有如SEQ ID NO：143所示的氨基酸序列的第四多肽链；或
(2)具有如SEQ ID NO：146所示的氨基酸序列的第一多肽链，具有如SEQ ID NO：147所示的氨基酸序列的第二多肽链，具有如SEQ ID NO：142所示的氨基酸序列的第三多肽链，具有如SEQ ID NO：143所示的氨基酸序列的第四多肽链；或
(3)具有如SEQ ID NO：148所示的氨基酸序列的第一多肽链，具有如SEQ ID NO：149 所示的氨基酸序列的第二多肽链，具有如SEQ ID NO：142所示的氨基酸序列的第三多肽链，具有如SEQ ID NO：143所示的氨基酸序列的第四多肽链；或
(4)具有如SEQ ID NO：150所示的氨基酸序列的第一多肽链，具有如SEQ ID NO：151所示的氨基酸序列的第二多肽链，具有如SEQ ID NO：152所示的氨基酸序列的第三多肽链，具有如SEQ ID NO：153所示的氨基酸序列的第四多肽链；或
(5)具有如SEQ ID NO：154所示的氨基酸序列的第一多肽链，具有如SEQ ID NO：155所示的氨基酸序列的第二多肽链，具有如SEQ ID NO：152所示的氨基酸序列的第三多肽链，具有如SEQ ID NO：153所示的氨基酸序列的第四多肽链；或
(6)具有如SEQ ID NO：156所示的氨基酸序列的第一多肽链，具有如SEQ ID NO：157所示的氨基酸序列的第二多肽链，具有如SEQ ID NO：152所示的氨基酸序列的第三多肽链，具有如SEQ ID NO：153所示的氨基酸序列的第四多肽链；或
(7)具有如SEQ ID NO：164所示的氨基酸序列的第一多肽链，具有如SEQ ID NO：165所示的氨基酸序列的第二多肽链，具有如SEQ ID NO：142所示的氨基酸序列的第三多肽链，具有如SEQ ID NO：143所示的氨基酸序列的第四多肽链。
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)编码本文任一项所述的双特异性抗体的核苷酸序列；和
(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，其包含本文所述的多核苷酸分子，或包含本文所述的表达载体，或表达本文任一项所述的双特异性抗体；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本文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本文任一项所述的双特异性抗体表达的条件下培养本文所述的宿主细胞，使其表达所述本文任一项所述的双特异性抗体，并从所述宿主细胞回收所表达的本文任一项所述的双特异性抗体。
在又一个方面，本发明提供了一种药物组合物，其包含本文任一项所述的双特异性抗体、多核苷酸分子、表达载体或宿主细胞，和药学上可接受的载体或赋形剂。
在又一个方面，本发明提供了本文任一项所述双特异性抗体、多核苷酸分子、表达载体、宿主细胞或药物组合物在制备用于预防或治疗癌症的药物中的用途。
在又一个方面，本发明提供了本文任一项所述双特异性抗体、多核苷酸分子、表达载体、宿主细胞或药物组合物，其用于预防或治疗癌症。
在又一个方面，本发明提供了一种预防或治疗癌症的方法，其包括向有需要的受试者施用如本文任一项所述双特异性抗体、多核苷酸分子、表达载体、宿主细胞或药物组合物。
在一些实施方式中，本发明所述癌症与CLDN18或CD20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；或所述癌症选自急性B淋巴细胞性白血病、弥漫性大B细胞淋巴瘤、慢性淋巴细胞白血病、滤泡淋巴瘤，非霍奇金氏淋巴瘤、慢性骨髓细胞白血病和Burkitt淋巴瘤。
在又一个方面，本发明提供了一种药物组合，其包含如本文任一项所述双特异性抗体、多核苷酸分子、表达载体、宿主细胞或药物组合物，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，其包括如本文任一项所述双特异性抗体、多核苷酸分子、表达载体、宿主细胞或药物组合物。
在又一个方面，本发明提供了一种抗体或其抗原结合片段，其包含结合CD3的结合结构域，其中，所述结合CD3的结合结构域包含的HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3分别为：
HCDR1：X1YAMN，其中，X1为T或K；
HCDR2：RIRSKYNNYATYYAX2SVKX3，其中，X2为D或A，X3为D或G；
HCDR3：HGX3X4GX5X6X7X8X9X10X11AY，其中，X3为N或A，X4为F或A，X5为N或A，X6为S或A，X7为Y或A，X8为V或A，X9为S或A，X10为W或A，X11为F或A；
LCDR1：X12SSTGAVTTX13NYX14N，其中，X12为R或G，X13为S或G，X14为A或P；
LCDR2：GTNKRAP；
LCDR3：AX15X16X17X18X19X20X21X22，其中，X15为L或A，X16为W或A，X17为Y或A，X18为S或A，X19为N或A，X20为L或A，X21为W或A，X22为V或A；
条件是，所述结合CD3的结合结构域包含的HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3不同时为：氨基酸序列如SEQ ID NO：1所示的HCDR1、氨基酸序列如SEQ ID NO：3所示的HCDR2、氨基酸序列如SEQ ID NO：5所示的HCDR3、氨基酸序列如SEQ ID NO：23所示的LCDR1、氨基酸序列如SEQ ID NO：25所示的LCDR2、氨基酸序列如SEQ ID NO：26所示的LCDR3。
在一些实施方式中，本发明所述结合CD3的结合结构域包含：氨基酸序列如SEQ ID NO：1或2所示的HCDR1，氨基酸序列如SEQ ID NO：3或4所示的HCDR2，氨基酸序列 如SEQ ID NO：5-22中任一项所示的HCDR3，氨基酸序列如SEQ ID NO：23或24所示的LCDR1，氨基酸序列如SEQ ID NO：25所示的LCDR2，和氨基酸序列如SEQ ID NO：26-42中任一项所示的LCDR3。
在一些实施方式中，本发明所述结合CD3的结合结构域包含HCDR1、HCDR2、HCDR3和LCDR1、LCDR2、LCDR3：
所述HCDR1、HCDR2、HCDR3为：
(1)氨基酸序列如SEQ ID NO：1所示的HCDR1，氨基酸序列如SEQ ID NO：3所示的HCDR2，氨基酸序列如SEQ ID NO：5所示的HCDR3；或
(2)氨基酸序列如SEQ ID NO：1所示的HCDR1，氨基酸序列如SEQ ID NO：3所示的HCDR2，氨基酸序列如SEQ ID NO：15所示的HCDR3；或
(3)氨基酸序列如SEQ ID NO：2所示的HCDR1，氨基酸序列如SEQ ID NO：4所示的HCDR2，氨基酸序列如SEQ ID NO：5-22中任一项所示的HCDR3；
所述LCDR1、LCDR2、LCDR3为：
(1)氨基酸序列如SEQ ID NO：23所示的LCDR1，氨基酸序列如SEQ ID NO：25所示的LCDR2，氨基酸序列如SEQ ID NO：26所示的LCDR3；或
(2)氨基酸序列如SEQ ID NO：23所示的LCDR1，氨基酸序列如SEQ ID NO：25所示的LCDR2，氨基酸序列如SEQ ID NO：33所示的LCDR3；或
(3)氨基酸序列如SEQ ID NO：24所示的LCDR1，氨基酸序列如SEQ ID NO：25所示的LCDR2，氨基酸序列如SEQ ID NO：26-42中任一项所示的LCDR3。
在一些实施方式中，本发明所述结合CD3的结合结构域包含的HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3分别为：
HCDR1：X1YAMN，其中，X1为T或K(优选为K)；
HCDR2：RIRSKYNNYATYYAX2SVKX3，其中，X2为D或A(优选为A)，X3为D或G(优选为G)；
HCDR3：HGX3X4GX5X6X7X8X9X10X11AY，其中，X3为N，X4为F，X5为N或A，X6为S或A，X7为Y或A，X8为V或A，X9为S或A，X10为W，X11为F或A(优选为F)；
LCDR1：X12SSTGAVTTX13NYX14N，其中，X12为R或G，X13为S或G，X14为A或P；
LCDR2：GTNKRAP；
LCDR3：AX15X16X17X18X19X20X21X22，其中，X15为L或A，X16为W，X17为Y或A，X18为S或A，X19为N或A，X20为L或A，X21为W，X22为V或A；
条件是，所述结合CD3的结合结构域包含的HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3不同时为：氨基酸序列如SEQ ID NO：1所示的HCDR1、氨基酸序列如SEQ ID NO：3所示的HCDR2、氨基酸序列如SEQ ID NO：5所示的HCDR3、氨基酸序列如SEQ ID NO：23所示的LCDR1、氨基酸序列如SEQ ID NO：25所示的LCDR2、氨基酸序列如SEQ ID NO：26所示的LCDR3。
在一些实施方式中，本发明所述结合CD3的结合结构域包含：氨基酸序列如SEQ ID NO：1或2所示的HCDR1，氨基酸序列如SEQ ID NO：3或4所示的HCDR2，氨基酸序列如SEQ ID NO：5、9、10、11、15、17、18、19或20中任一项所示的HCDR3，氨基酸序列如SEQ ID NO：23或24所示的LCDR1，氨基酸序列如SEQ ID NO：25所示的LCDR2，和氨基酸序列如SEQ ID NO：26、28、30、33、35、36、37、39、40、41或42中任一项所示的LCDR3。
在一些实施方式中，本发明所述结合CD3的结合结构域包含：
(I)氨基酸序列如SEQ ID NO：1所示的HCDR1，氨基酸序列如SEQ ID NO：3所示的HCDR2，氨基酸序列如SEQ ID NO：15所示的HCDR3，氨基酸序列如SEQ ID NO：23所示的LCDR1，氨基酸序列如SEQ ID NO：25所示的LCDR2，氨基酸序列如SEQ ID NO：33所示的LCDR3；或
(II)氨基酸序列如SEQ ID NO：2所示的HCDR1，氨基酸序列如SEQ ID NO：4所示的HCDR2，氨基酸序列如SEQ ID NO：5所示的HCDR3，氨基酸序列如SEQ ID NO：24所示的LCDR1，氨基酸序列如SEQ ID NO：25所示的LCDR2，氨基酸序列如SEQ ID NO：26、28、33、36或37所示的LCDR3；或
(III)氨基酸序列如SEQ ID NO：2所示的HCDR1，氨基酸序列如SEQ ID NO：4所示的HCDR2，氨基酸序列如SEQ ID NO：9所示的HCDR3，氨基酸序列如SEQ ID NO：24所示的LCDR1，氨基酸序列如SEQ ID NO：25所示的LCDR2，氨基酸序列如SEQ ID NO：28、35、37、39或40所示的LCDR3；或
(IV)氨基酸序列如SEQ ID NO：2所示的HCDR1，氨基酸序列如SEQ ID NO：4所示的HCDR2，氨基酸序列如SEQ ID NO：10所示的HCDR3，氨基酸序列如SEQ ID NO：24所示的LCDR1，氨基酸序列如SEQ ID NO：25所示的LCDR2，氨基酸序列如SEQ ID NO：28、33、40、41或42所示的LCDR3；或
(V)氨基酸序列如SEQ ID NO：2所示的HCDR1，氨基酸序列如SEQ ID NO：4所示的HCDR2，氨基酸序列如SEQ ID NO：11所示的HCDR3，氨基酸序列如SEQ ID NO：24所示的LCDR1，氨基酸序列如SEQ ID NO：25所示的LCDR2，氨基酸序列如SEQ ID NO：26、28、33、35、36、37、40、41或42所示的LCDR3；或
(VI)氨基酸序列如SEQ ID NO：2所示的HCDR1，氨基酸序列如SEQ ID NO：4所示的HCDR2，氨基酸序列如SEQ ID NO：15所示的HCDR3，氨基酸序列如SEQ ID NO：24所示的LCDR1，氨基酸序列如SEQ ID NO：25所示的LCDR2，氨基酸序列如SEQ ID NO：26或30所示的LCDR3；或
(VII)氨基酸序列如SEQ ID NO：2所示的HCDR1，氨基酸序列如SEQ ID NO：4所示的HCDR2，氨基酸序列如SEQ ID NO：17所示的HCDR3，氨基酸序列如SEQ ID NO：24所示的LCDR1，氨基酸序列如SEQ ID NO：25所示的LCDR2，氨基酸序列如SEQ ID NO：33、35、36、37或42所示的LCDR3；或
(VIII)氨基酸序列如SEQ ID NO：2所示的HCDR1，氨基酸序列如SEQ ID NO：4所示的HCDR2，氨基酸序列如SEQ ID NO：18所示的HCDR3，氨基酸序列如SEQ ID NO：24所示的LCDR1，氨基酸序列如SEQ ID NO：25所示的LCDR2，氨基酸序列如SEQ ID NO：28、35、36、37、39、41或42所示的LCDR3；或
(IX)氨基酸序列如SEQ ID NO：2所示的HCDR1，氨基酸序列如SEQ ID NO：4所示的HCDR2，氨基酸序列如SEQ ID NO：20所示的HCDR3，氨基酸序列如SEQ ID NO：24所示的LCDR1，氨基酸序列如SEQ ID NO：25所示的LCDR2，氨基酸序列如SEQ ID NO：28、33、35、36、40、41或42所示的LCDR3；或
(X)氨基酸序列如SEQ ID NO：2所示的HCDR1，氨基酸序列如SEQ ID NO：4所示的HCDR2，氨基酸序列如SEQ ID NO：9、10或19所示的HCDR3，氨基酸序列如SEQ ID NO：23所示的LCDR1，氨基酸序列如SEQ ID NO：25所示的LCDR2，氨基酸序列如SEQ ID NO：26所示的LCDR3；或
(XI)氨基酸序列如SEQ ID NO：2所示的HCDR1，氨基酸序列如SEQ ID NO：4所示的HCDR2，氨基酸序列如SEQ ID NO：19所示的HCDR3，氨基酸序列如SEQ ID NO：24所示的LCDR1，氨基酸序列如SEQ ID NO：25所示的LCDR2，氨基酸序列如SEQ ID NO：36所示的LCDR3；或
(XII)氨基酸序列如SEQ ID NO：1所示的HCDR1、氨基酸序列如SEQ ID NO：3所示的HCDR2、氨基酸序列如SEQ ID NO：5所示的HCDR3、氨基酸序列如SEQ ID NO：23所示的LCDR1、氨基酸序列如SEQ ID NO：25所示的LCDR2、氨基酸序列如SEQ ID NO：26所示的LCDR3。
在一些实施方式中，本发明所述结合CD3的结合结构域包含：氨基酸序列如SEQ ID NO：43-62中任一项所示的VH，和氨基酸序列如SEQ ID NO：63-81中任一项所示的VL。
在一些实施方式中，本发明所述结合CD3的结合结构域包含：
(I)氨基酸序列如SEQ ID NO：44所示的VH，和氨基酸序列如SEQ ID NO：64所示 的VL；或
(II)氨基酸序列如SEQ ID NO：45所示的VH，和氨基酸序列如SEQ ID NO：65、67、72、75或76所示的VL；或
(III)氨基酸序列如SEQ ID NO：49所示的VH，和氨基酸序列如SEQ ID NO：67、74、76、78或79所示的VL；或
(IV)氨基酸序列如SEQ ID NO：50所示的VH，和氨基酸序列如SEQ ID NO：67、72、79、80或81所示的VL；或
(V)氨基酸序列如SEQ ID NO：51所示的VH，和氨基酸序列如SEQ ID NO：65、67、72、74、75、76、79、80或81所示的VL；或
(VI)氨基酸序列如SEQ ID NO：55所示的VH，和氨基酸序列如SEQ ID NO：65或69所示的VL；或
(VII)氨基酸序列如SEQ ID NO：57所示的VH，和氨基酸序列如SEQ ID NO：72、74、75、76或81所示的VL；或
(VIII)氨基酸序列如SEQ ID NO：58所示的VH，和氨基酸序列如SEQ ID NO：67、74、75、76、78、80或81所示的VL；或
(IX)氨基酸序列如SEQ ID NO：60所示的VH，和氨基酸序列如SEQ ID NO：67、72、74、75、79、80或81所示的VL；或
(X)氨基酸序列如SEQ ID NO：49、50或59所示的VH，和氨基酸序列如SEQ ID NO：63所示的VL；或
(XI)氨基酸序列如SEQ ID NO：59所示的VH，和氨基酸序列如SEQ ID NO：75所示的VL；或
(XII)氨基酸序列如SEQ ID NO：43所示的VH，和氨基酸序列如SEQ ID NO：63所示的VL。
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：82-101中任一项所示的重链，和氨基酸序列如SEQ ID NO：102-120中任一项所示的轻链。
在一些实施方式中，本发明所述抗体包含：
(I)氨基酸序列如SEQ ID NO：83所示的重链，和氨基酸序列如SEQ ID NO：103所示的轻链；或
(II)氨基酸序列如SEQ ID NO：84所示的重链，和氨基酸序列如SEQ ID NO：104、106、111、114或115所示的轻链；或
(III)氨基酸序列如SEQ ID NO：88所示的重链，和氨基酸序列如SEQ ID NO：106、113、115、117或118所示的轻链；或
(IV)氨基酸序列如SEQ ID NO：89所示的重链，和氨基酸序列如SEQ ID NO：106、111、118、119或120所示的轻链；或
(V)氨基酸序列如SEQ ID NO：90所示的重链，和氨基酸序列如SEQ ID NO：104、106、111、113、114、115、118、119或120所示的轻链；或
(VI)氨基酸序列如SEQ ID NO：94所示的重链，和氨基酸序列如SEQ ID NO：104或108所示的轻链；或
(VII)氨基酸序列如SEQ ID NO：96所示的重链，和氨基酸序列如SEQ ID NO：111、113、114、115或120所示的轻链；或
(VIII)氨基酸序列如SEQ ID NO：97所示的重链，和氨基酸序列如SEQ ID NO：106、113、114、115、117、119或120所示的轻链；或
(IX)氨基酸序列如SEQ ID NO：99所示的重链，和氨基酸序列如SEQ ID NO：106、111、113、114、118、119或120所示的轻链；或
(X)氨基酸序列如SEQ ID NO：88、89或98所示的重链，和氨基酸序列如SEQ ID NO：102所示的轻链；或
(XI)氨基酸序列如SEQ ID NO：98所示的重链，和氨基酸序列如SEQ ID NO：114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，所述抗原结合片段为Fab、Fab′、F(ab′)2、Fv、dsFv、scFv或sdAb。
在一些实施方式中，本发明所述抗体是特异性结合CD3的单克隆抗体。
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一个或两个结合肿瘤相关抗原的结合结构域。
在一些实施方式中，本发明所述肿瘤相关抗原选自BCMA、CD19、CD20、CD22、CD30、CD33、CD38、CD40、CD123、CDK4、CEA、Claudin18.2、EGFR、ROR1、TGF-β和5T4；优选地，所述肿瘤相关抗原是Claudin 18.2、CD20或BCMA。
在一些实施方式中，本发明所述肿瘤相关抗原是Claudin 18.2或CD20。在一些实施方式中，本发明所述结合Claudin 18.2的结合结构域包含：氨基酸序列如SEQ ID NO：121所示的HCDR1，氨基酸序列如SEQ ID NO：122所示的HCDR2，氨基酸序列如SEQ ID NO：123所示的HCDR3，氨基酸序列如SEQ ID NO：124所示的LCDR1，氨基酸序列如SEQ ID NO：125所示的LCDR2，和氨基酸序列如SEQ ID NO：126所示的LCDR3。在一些实施方式中，本发明所述结合CD20的结合结构域包含：氨基酸序列如SEQ ID NO：127所示的HCDR1，氨基酸序列如SEQ ID NO：128所示的HCDR2，氨基酸序列如SEQ ID NO：129所示的HCDR3，氨基酸序列如SEQ ID NO：130所示的LCDR1，氨基酸序列如SEQ ID  NO：131所示的LCDR2，和氨基酸序列如SEQ ID NO：132所示的LCDR3。
在一些实施方式中，本发明所述结合Claudin 18.2的结合结构域包含：(a)氨基酸序列如SEQ ID NO：133所示的VH，和氨基酸序列如SEQ ID NO：134所示的VL；或(b)氨基酸序列如SEQ ID NO：166所示的VH，和氨基酸序列如SEQ ID NO：167所示的VL。
在一些实施方式中，本发明所述结合CD20的结合结构域包含：氨基酸序列如SEQ ID NO：135所示的VH，和氨基酸序列如SEQ ID NO：136所示的VL。
在一些实施方式中，本发明提供了一种抗体，其包含结合CD3的结合结构域和结合CD20的结合结构域，其中，所述结合CD3的结合结构域包含：氨基酸序列如SEQ ID NO：1所示的HCDR1、氨基酸序列如SEQ ID NO：3所示的HCDR2、氨基酸序列如SEQ ID NO：5所示的HCDR3、氨基酸序列如SEQ ID NO：23所示的LCDR1、氨基酸序列如SEQ ID NO：25所示的LCDR2、氨基酸序列如SEQ ID NO：26所示的LCDR3；所述结合CD20的结合结构域包含：氨基酸序列如SEQ ID NO：127所示的HCDR1，氨基酸序列如SEQ ID NO：128所示的HCDR2，氨基酸序列如SEQ ID NO：129所示的HCDR3，氨基酸序列如SEQ ID NO：130所示的LCDR1，氨基酸序列如SEQ ID NO：131所示的LCDR2，和氨基酸序列如SEQ ID NO：132所示的LCDR3。
在一些实施方式中，本发明提供了一种抗体，其包含结合CD3的结合结构域和结合CD20的结合结构域，其中，所述结合CD3的结合结构域包含：氨基酸序列如SEQ ID NO：43所示的VH，和氨基酸序列如SEQ ID NO：63所示的VL，所述结合CD20的结合结构域包含：氨基酸序列如SEQ ID NO：135所示的VH，和氨基酸序列如SEQ ID NO：136所示的VL。
在一些实施方式中，本发明所述抗体的Fc区为来自IgG的Fc，例如来自IgG1、IgG2、IgG3或IgG4的Fc，和/或所述抗体的CL为来自λ或κ轻链的CL；优选地，所述Fc区包含杵臼结构。
在一些实施方式中，本发明所述抗体具有四条多肽链，所述四条多肽链分别为：
(1)所述第一多肽链具有如SEQ ID NO：144所示的氨基酸序列，所述第二多肽链具有如SEQ ID NO：145所示的氨基酸序列，所述第三多肽链具有如SEQ ID NO：142所示的氨基酸序列，所述第四多肽链具有如SEQ ID NO：143所示的氨基酸序列；或
(2)所述第一多肽链具有如SEQ ID NO：146所示的氨基酸序列，所述第二多肽链具有如SEQ ID NO：147所示的氨基酸序列，所述第三多肽链具有如SEQ ID NO：142所示的氨基酸序列，所述第四多肽链具有如SEQ ID NO：143所示的氨基酸序列；或
(3)所述第一多肽链具有如SEQ ID NO：148所示的氨基酸序列，所述第二多肽链具有如SEQ ID NO：149所示的氨基酸序列，所述第三多肽链具有如SEQ ID NO：142所示的氨 基酸序列，所述第四多肽链具有如SEQ ID NO：143所示的氨基酸序列；或
(4)所述第一多肽链具有如SEQ ID NO：150所示的氨基酸序列，所述第二多肽链具有如SEQ ID NO：151所示的氨基酸序列，所述第三多肽链具有如SEQ ID NO：152所示的氨基酸序列，所述第四多肽链具有如SEQ ID NO：153所示的氨基酸序列；或
(5)所述第一多肽链具有如SEQ ID NO：154所示的氨基酸序列，所述第二多肽链具有如SEQ ID NO：155所示的氨基酸序列，所述第三多肽链具有如SEQ ID NO：152所示的氨基酸序列，所述第四多肽链具有如SEQ ID NO：153所示的氨基酸序列；或
(6)所述第一多肽链具有如SEQ ID NO：156所示的氨基酸序列，所述第二多肽链具有如SEQ ID NO：157所示的氨基酸序列，所述第三多肽链具有如SEQ ID NO：152所示的氨基酸序列，所述第四多肽链具有如SEQ ID NO：153所示的氨基酸序列；或
(7)所述第一多肽链具有如SEQ ID NO：164所示的氨基酸序列，所述第二多肽链具有如SEQ ID NO：165所示的氨基酸序列，所述第三多肽链具有如SEQ ID NO：142所示的氨基酸序列，所述第四多肽链具有如SEQ ID NO：143所示的氨基酸序列。
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)编码本文任一项抗体或其抗原结合片段的核苷酸序列；和
(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，其包含本文所述的多核苷酸分子，或包含本文所述的表达载体，或表达本文任一项抗体或其抗原结合片段；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本文任一项抗体或其抗原结合片段的方法，所述方法包括在适合于本文任一项抗体或其抗原结合片段表达的条件下培养本文所述的宿主细胞，使其表达所述本文任一项抗体或其抗原结合片段，并从所述宿主细胞回收所表达的本文任一项抗体或其抗原结合片段。
在又一个方面，本发明提供了一种药物组合物，其包含本文任一实施方案所述的抗体或其抗原结合片段、多核苷酸分子、表达载体或宿主细胞，和药学上可接受的载体或赋形剂。
在又一个方面，本发明提供了本文任一实施方案所述的抗体或其抗原结合片段、多核苷酸分子、表达载体、宿主细胞或药物组合物在制备用于预防或治疗癌症的药物中的用途。
在又一个方面，本发明提供了本文任一实施方案所述的抗体或其抗原结合片段、多核苷酸分子、表达载体、宿主细胞或药物组合物，其用于预防或治疗癌症。
在又一个方面，本发明提供了一种预防或治疗癌症的方法，其包括向有需要的受试者施用如本文任一实施方案所述的抗体或其抗原结合片段、多核苷酸分子、表达载体、宿主细胞或药物组合物。
在一些实施方式中，本发明所述癌症与CLDN18或CD20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；或所述癌症选自急性B淋巴细胞性白血病、弥漫性大B细胞淋巴瘤、慢性淋巴细胞白血病、滤泡淋巴瘤，非霍奇金氏淋巴瘤、慢性骨髓细胞白血病和Burkitt淋巴瘤。
在又一个方面，本发明提供了一种药物组合，其包含如本文任一实施方案所述的抗体或其抗原结合片段、多核苷酸分子、表达载体、宿主细胞或药物组合物，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，其包括如本文任一实施方案所述的抗体或其抗原结合片段、多核苷酸分子、表达载体、宿主细胞或药物组合物。
在又一个方面，本发明提供本发明任一实施方案所述的特异性结合CD3的单克隆抗体在制备用于降低细胞因子风暴或用于治疗或预防细胞因子释放综合征的分子或药物中的应用。在一些实施方案中，所述分子或药物为双特异性抗体或含双特异性抗体的药物。在一些实施方案中，所述双特异性抗体为本文任一实施方案所述的双特异性抗体。
附图说明
图1：组一的抗体与人CD3的结合检测结果。
图2：组二的抗体与人CD3的结合检测结果。
图3：组三的抗体与人CD3的结合检测结果。
图4：组四的抗体与人CD3的结合检测结果。
图5：组五的抗体与人CD3的结合检测结果。
图6：组六的抗体与人CD3的结合检测结果。
图7：组七的抗体与人CD3的结合检测结果。
图8：组八的抗体与人CD3的结合检测结果。
图9：组九的抗体与人CD3的结合检测结果。
图10：组九的抗体与人CD3的结合检测结果。
图11：组十一的抗体与人CD3的结合检测结果。
图12：组十二的抗体与人CD3的结合检测结果。
图13：双特异性抗体JS202-7的结构示意图。
图14：双特异性抗体JS202-7 CD3 Fv的结构示意图。
图15：双特异性抗体JS202-7 012 Fv和JS202-7 CD3 130 012 Fv的结构示意图。
图16：双特异性抗体JS203-7-JSCD3-CD20Fv和JS203-7-CD3 130-CD20Fv的结构示意图。
图17：双特异性抗体JS203-7-CD3-FV的结构示意图。
图18：双特异性抗体JS202-7 CD3-130-012 scFv的结构示意图。
图19：双特异性抗体JS202-7 CD3-Fv、JS202-7 012-Fv、JS202-7 CD3 130 012-Fv和JS202-7与人CD3的结合检测结果。
图20：双特异性抗体JS202-7 CD3-Fv、JS202-7 012-Fv、JS202-7 CD3 130 012-Fv和JS202-7与食蟹猴CD3e的结合检测结果。
图21：双特异性抗体JS202-7 CD3-Fv、JS202-7 012-Fv、JS202-7 CD3 130 012-Fv和JS202-7与CHO Claudin18.2的结合检测结果。
图22：双特异性抗体JS202-7 CD3-Fv、JS202-7 012-Fv、JS202-7 CD3 130 012-Fv和JS202-7对T细胞下游信号通路的激活作用。
图23：双特异性抗体JS202-7 CD3-Fv、JS202-7 012-Fv、JS202-7 CD3 130 012-Fv和JS202-7对T细胞的激活作用。
图24：双特异性抗体JS202-7 CD3-Fv、JS202-7 012-Fv、JS202-7 CD3 130 012-Fv和JS202-7引起细胞因子释放综合征的研究结果。
图25：双特异性抗体JS202-7 CD3 130 012-Fv和JS202-7对小鼠MC38hClaudin18.2细胞皮下移植瘤生长的抑制。
图26：双特异性抗体JS203-7-JSCD3-CD20-Fv和JS203-7-CD3 130-CD20-Fv与CHO-hCD3细胞的结合检测结果。
图27：双特异性抗体JS203-7-JSCD3-CD20-Fv和JS203-7-CD3 130-CD20-Fv与Raji细胞的结合检测结果。
图28：双特异性抗体JS203-7-JSCD3-CD20-Fv和JS203-7-CD3 130-CD20-Fv在荧光素酶报告基因系统中的活性检测结果。
图29：双特异性抗体JS203-7-JSCD3-CD20-Fv和JS203-7-CD3 130-CD20-Fv对T淋巴细胞的激活活性检测结果。
图30：双特异性抗体JS203-7-JSCD3-CD20-Fv和JS203-7-CD3 130-CD20-Fv引起细胞因子释放综合征的研究结果。
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-氨基酸之一的标准三字母和/或单字母代码。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“抗原”系指能够被本发明的抗体所结合的分子或分子之一部分。抗原可有一个或多于一个表位。
术语“肿瘤相关抗原”指肿瘤细胞单独表达或主要表达或过表达的任何分子(例如蛋白、肽、脂质、碳水化合物等)，以使所述抗原与肿瘤相关。肿瘤相关抗原可以是仅一种类型的肿瘤表达的抗原，以使所述肿瘤抗原仅与一种类型的肿瘤相关或者仅是一种类型的肿瘤所特有的。可选地，肿瘤抗原可以是多种类型肿瘤相关或者特有的肿瘤抗原。例如，肿瘤相关抗原可以被乳腺癌细胞和结肠癌细胞都表达，但不被正常的、非肿瘤或非癌细胞所表达。示例性的肿瘤相关抗原为肿瘤细胞表面抗原，这类抗原更利于为治疗性和诊断性抗体所识别。“肿瘤相关抗原”的非限定示例包含，例如BCMA、CD19、CD20、CD22、CD30、CD33、CD38、CD40、CD123、CDK4、CEA、Claudin 18.2、EGFR、ROR1、TGF-β和5T4。
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，CD38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“Claudin 18.2”，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)。CLDN18.2蛋白在多种肿瘤组织中高度表达，比如胃癌(60-80％)、胰腺癌(50％)、食管癌(30-50％)和肺癌(40-60％)等，但是在正常组织中几乎没有表达。
术语“人CD20”或“CD20”是指人CD20(UniProtKB/Swiss-Prot No.P11836)和包括 由细胞(包括肿瘤细胞)天然表达或在用CD20基因或cDNA转染的细胞上表达的CD20的任何变体、同种型和物种同源物。物种同源物包括恒河猴CD20(macaca mulatta；UniProtKB/Swiss-Prot No H9YXP1)和食蟹猴。
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u、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“多特异性抗体”是指包含两个或更多个抗原结合结构域，能够结合两个或更多个不同的表位(例如，两个、三个、四个或更多个不同的表位)，表位可以在相同或不同的抗原上的抗体。多特异性抗体的示例包括结合两个不同抗原或两个不同表位的“双特异性抗体”。
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“重链恒定区”或“CH”此处可互换使用，是指包含至少三个重链恒定结构域(CH1、CH2和CH3)的区域。非限制性示例人重链恒定区包括γ、δ和α。非限制性示例人重链恒定区还包括ε和μ。各重恒定区对应于抗体同种型。例如，包含γ恒定区的抗体是IgG抗体，包含δ恒定区的抗体是IgD抗体，并且包含α恒定区的抗体是IgA抗体。此外，包含μ恒定区的抗体是IgM抗体，并且包含ε恒定区的抗体是IgE抗体。某些同种型可以进一步再分成亚类。例如，IgG抗体包括但不限于IgG1(包含γ1恒定区)、IgG2(包含γ2恒定区)、IgG3(包含γ3恒定区)和IgG4(包含γ4恒定区)抗体；IgA抗体包括但不限于IgA1(包含α1恒定区)和IgA2(包含α2恒定区)抗体；并且IgM抗体包括但不限于IgM1和IgM2。
术语“CH1结构域”或“CH1”或“重链恒定区1”此处可互换使用，包括免疫球蛋白重链的第一(大多数胺基末端)恒定区结构域，其例如从Kabat编号系统中的大约位置114-223(EU位置118-215)延伸。CH1结构域与VH结构域和免疫球蛋白重链分子的铰链区的胺基末端相邻，并且不构成免疫球蛋白重链的Fc区的一部分，例如人IgG1CH1结构域、IgG2CH1结构域、IgG3CH1结构域和IgG4CH1结构域；及其天然存在变体。
术语“CH2结构域”或“CH2”或“重链恒定区2”此处可互换使用，包括重链免疫球蛋白分子的这样的部分，其例如从Kabat编号系统中的大约位置244-360(EU位置231-340)延伸。CH2结构域是独特的，因为它不与另一个结构域紧密配对。而是将两个N连接的支链糖类链插入完整天然IgG分子的两个CH2结构域之间。
术语“CH3结构域”或“CH3”或“重链恒定区3”此处可互换使用，包括重链免疫球蛋白分子的这样的部分，其从CH2结构域的末端例如从Kabat编号系统的大约位置361-476(EU位置341-445)延伸约110个残基。CH3结构域通常形成抗体的C末端部分。然而，在一些免疫球蛋白中，附加的结构域可以从CH3结构域延伸以形成分子的C末端部分(例如，IgM的μ链和IgE的e链中的CH4结构域)。
术语“轻链恒定区”或“CL”此处可互换使用，是指包含轻链恒定结构域(CL)的区域。非限制性示例轻链恒定区包括λ和κ。
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。抗原结合片段的实例包括但不限于Fab，Fab′，F(ab′)2，Fv和dsFv片段；双抗体；线性抗体；单链抗体分子，例如scFv；由抗体片段形成的纳米抗体(nanobody、sdAb)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。在本申请中，术语“Fab”或“Fab分子”通常是指含有重链可变结构域(VH)和轻链可变结构域(VL)的片段，并且还含有轻链的恒定结构域(CL)和重链的第一恒定结构域(CH1)。术语“Fab′”通常是指在重链CH1结构域的羧基端添加少量残基(包括一个或多个来自抗体铰链区的半胱氨酸)而不同于Fab的片段。术语“F(ab′)2”通常是指Fab′的二聚体，包含通过铰链区上的二硫桥连接的两个Fab片段的抗体片段。术语“Fv”或“Fv分子”通常是指含有完整抗原识别与结合位点的最小抗体片段；在某些情形中，该片段可以由一个重链可变区和一个轻链可变区以紧密非共价结合的二聚体组成。术语“dsFv”通常是指二硫键稳定的Fv片段，其单个轻链可变区与单个重链可变区之间的键是二硫键。术语“sdAb片段”通常是指由VH结构域组成的抗体片段。
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”或“Fc结构域”此处可互换使用，在本文中用来定义免疫球蛋白重链的含有至少一部分恒定区的C端区域。该术语包括天然序列Fc区和变体Fc区。天然的免疫球蛋白“Fc结构域”包含两个或三个恒定结构域，即CH2结构域、CH3结构域和可选的CH4结构域。例如，在天然抗体中，免疫球蛋白Fc结构域包含源自IgG、IgA和IgD类抗体的两条重链的第二和第三恒定结构域(CH2结构域和CH3结构域)；或者包含源自IgM和IgE类抗体的两条重链的第二、第三和第四恒定结构域(CH2结构域、CH3结构域和CH4结构域)。本文中的Fc区也可包含铰链区。
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。
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“价”是指多肽中潜在的抗原结合位点的数目。每个抗原结合位点特异性地结合抗 原分子上的一个抗原分子或特异性位点。当多肽包含多于一个抗原结合位点时，每个抗原结合位点可特异性地结合相同或不同的分子(例如，可结合不同的配体或不同的抗原，或结合相同抗原上的不同表位)。术语“二价”表示抗体包含两个抗原结合位点。术语“三价”表示抗体包含三个抗原结合位点。
术语“结合结构域”或“抗原结合结构域”或“抗原结合位点”系指抗体中这样的部分，其包含特异地与抗原之部分或全部结合，并与抗原之部分或全部成互补的区域。当抗原很大时，抗体可能只能结合到抗原的一个特定的部分，该部分被称为表位。结合结构域可包含重链和轻链可变结构域(VH和VL)，其各自包括四个保守框架区(FR)和三个CDR。CDR在序列上变化并确定对特定抗原的特异性。
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。
术语“特异性结合”、“选择性结合”、“选择性地结合”和“特异性地结合”是指抗体对预 先确定的抗原上的表位的结合。特异性结合可以例如通过测定与对照分子的结合相比的分子的结合来测量，所述对照分子通常是具有不具有结合活性的类似结构的分子。例如，特异性结合可以通过与类似于靶的对照分子竞争来确定。
“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))。
术语“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。
术语“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”。
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”或“对象”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。癌症可以是实体瘤和血液肿瘤，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。
“细胞因子风暴(cytokine storm)”是指机体体液中多种细胞因子如TNF-α、IL-1、IL-6、IL-12、IFN-α、IFN-β、IFN-γ、MCP-1和IL-8等迅速大量产生的现象，是引起急性呼吸窘迫综合征和多脏器衰竭的重要原因。细胞因子释放综合征(CRS)指淋巴细胞在应用单克隆抗体、细胞因子等治疗或感染后出现活化、溶解，并释放出大量细胞因子所导致的一组临床综合征。
抗体
术语“抗CD3抗体”、“抗CD3”、“CD3抗体”、“与CD3结合”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”、“与CLDN-18.2 结合”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。
术语“抗CD20抗体”、“抗CD20”、“CD20抗体”、“与CD20结合”或“结合CD20”是指能够以足够的亲合力结合CD20蛋白或其片段以致所述抗体可以用作靶向CD20的诊断剂和/或治疗剂。
本发明所述人源化的抗CD3抗体是以抗体JSCD3为起始进行突变得到，具有许多独特的性能和优势，其中抗体JSCD3参见专利申请CN202110468621.2和PCT/CN2021/113033。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抗原都有较好的亲和力，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，并且能够降低引起细胞因子释放综合征的风险。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，本发明涉及一种双特异性抗体，其包含第一结合结构域、第二结合结构域和第三结合结构域，其中所述第一结合结构域与CD3结合，所述第二结合结构域和所述第三结合结构域各自与肿瘤相关抗原结合，所述第一结合结构域具有Fab分子或Fv分子或scFv分子的形式，所述第二结合结构域具有Fab分子或Fv分子或scFv分子的形式，和所述第三结合结构域具有Fab分子的形式。
在一些实施方式中，本发明所述第一结合结构域具有Fab分子的形式，所述第二结合结构域具有Fv分子或scFv分子的形式，和所述第三结合结构域具有Fab分子的形式。
在一些实施方式中，本发明所述第一结合结构域具有Fab分子的形式，所述第二结合结构域具有Fv分子的形式，和所述第三结合结构域具有Fab分子的形式。
在一些实施方式中，本发明所述第一结合结构域具有Fab分子的形式，所述第二结合结构域具有scFv分子的形式，和所述第三结合结构域具有Fab分子的形式。
在一些实施方式中，本发明所述第一结合结构域具有Fv分子的形式，所述第二结合结构域具有Fab分子的形式，和所述第三结合结构域具有Fab分子的形式。
在一些实施方式中，本发明所述的肿瘤相关抗原选自BCMA、CD19、CD20、CD22、CD30、CD33、CD38、CD40、CD123、CDK4、CEA、Claudin18.2、EGFR、ROR1、TGF-β和5T4；优选地，所述肿瘤相关抗原是Claudin 18.2、CD20或BCMA。
在一些实施方式中，本发明所述双特异性抗体具有四条多肽链，其中所述第一多肽链自N端到C端具有VH1-CH1-连接肽-VL2-连接肽-铰链区-第一Fc区，所述第二多肽链自 N端到C端具有VL1-CL-连接肽-VH2；所述第三多肽链自N端到C端具有VH2-CH1-铰链区-第二Fc区，所述第四多肽链自N端到C端具有VL2-CL。其中，所述VH1和VL1形成一结构域，所述VH2和VL2形成一结构域。在一些实施方式中，本发明所述第一多肽链的VH1-CH1与所述第二多肽链的VL1-CL形成所述第一结合结构域，所述第一多肽链的VL2与所述第二多肽链的VH2形成所述第二结合结构域，所述第三多肽链的VH2-CH1与所述第四多肽链的VL2-CL形成所述第三结合结构域。
在一些实施方式中，本发明所述双特异性抗体具有四条多肽链，其中所述第一多肽链自N端到C端具有VH1-CH1-连接肽-VL2-连接肽-VH2-连接肽-铰链区-第一Fc区，所述第二多肽链自N端到C端具有VL1-CL；所述第三多肽链自N端到C端具有VH2-CH1-铰链区-第二Fc区，所述第四多肽链自N端到C端具有VL2-CL。其中，所述VH1和VL1形成一结构域，所述VH2和VL2形成一结构域。在一些实施方式中，本发明所述第一多肽链的VH1-CH1与所述第二多肽链的VL1-CL形成所述第一结合结构域，所述第一多肽链的VL2-连接肽-VH2形成所述第二结合结构域，所述第三多肽链的VH2-CH1与所述第四多肽链的VL2-CL形成所述第三结合结构域。
在一些实施方式中，本发明所述双特异性抗体具有四条多肽链，其中所述第一多肽链自N端到C端具有VH2-CH1-连接肽-VL1-连接肽-铰链区-第一Fc区，所述第二多肽链自N端到C端具有VL2-CL-连接肽-VH1；所述第三多肽链自N端到C端具有VH2-CH1-铰链区-第二Fc区，所述第四多肽链自N端到C端具有VL2-CL。在一些实施方式中，本发明所述第一多肽链的VH2-CH1与所述第二多肽链的VL2-CL形成第二结合结构域，所述第一多肽链的VL1与所述第二多肽链的VH1形成第一结合结构域，所述第三多肽链的VH2-CH1与所述第四多肽链的VL2-CL形成第三结合结构域。
在一些实施方式中，本发明所述第一结合结构域包含的HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3分别为：
HCDR1：X1YAMN(SEQ ID NO：168)，其中，X1为T或K；
HCDR2：RIRSKYNNYATYYAX2SVKX3(SEQ ID NO：169)，其中，X2为D或A，X3为D或G；
HCDR3：HGX3X4GX5X6X7X8X9X10X11AY(SEQ ID NO：170)，其中，X3为N或A，X4为F或A，X5为N或A，X6为S或A，X7为Y或A，X8为V或A，X9为S或A，X10为W或A，X11为F或A；
LCDR1：X12SSTGAVTTX13NYX14N(SEQ ID NO：171)，其中，X12为R或G，X13为S或G，X14为A或P；
LCDR2：GTNKRAP(SEQ ID NO：172)；
LCDR3：AX15X16X17X18X19X20X21X22(SEQ ID NO：173)，其中，X15为L或A，X16为W或A，X17为Y或A，X18为S或A，X19为N或A，X20为L或A，X21为W或A，X22为V或A。
在一些实施方式中，本发明所述第一结合结构域包含：氨基酸序列如SEQ ID NO：1或2所示的HCDR1，氨基酸序列如SEQ ID NO：3或4所示的HCDR2，氨基酸序列如SEQ ID NO：5-22中任一项所示的HCDR3，氨基酸序列如SEQ ID NO：23或24所示的LCDR1，氨基酸序列如SEQ ID NO：25所示的LCDR2，和氨基酸序列如SEQ ID NO：26-42中任一项所示的LCDR3。
在一些实施方式中，本发明所述第一结合结构域包含HCDR1、HCDR2、HCDR3和LCDR1、LCDR2、LCDR3；其中，所述HCDR1、HCDR2、HCDR3为：
(1)氨基酸序列如SEQ ID NO：1所示的HCDR1，氨基酸序列如SEQ ID NO：3所示的HCDR2，氨基酸序列如SEQ ID NO：5所示的HCDR3；或
(2)氨基酸序列如SEQ ID NO：1所示的HCDR1，氨基酸序列如SEQ ID NO：3所示的HCDR2，氨基酸序列如SEQ ID NO：15所示的HCDR3；或
(3)氨基酸序列如SEQ ID NO：2所示的HCDR1，氨基酸序列如SEQ ID NO：4所示的HCDR2，氨基酸序列如SEQ ID NO：5-22中任一项所示的HCDR3；
所述LCDR1、LCDR2、LCDR3为：
(1)氨基酸序列如SEQ ID NO：23所示的LCDR1，氨基酸序列如SEQ ID NO：25所示的LCDR2，氨基酸序列如SEQ ID NO：26所示的LCDR3；或
(2)氨基酸序列如SEQ ID NO：23所示的LCDR1，氨基酸序列如SEQ ID NO：25所示的LCDR2，氨基酸序列如SEQ ID NO：33所示的LCDR3；或
(3)氨基酸序列如SEQ ID NO：24所示的LCDR1，氨基酸序列如SEQ ID NO：25所示的LCDR2，氨基酸序列如SEQ ID NO：26-42中任一项所示的LCDR3。
在一些实施方式中，本发明所述第一结合结构域包含的HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3分别为：
HCDR1：X1YAMN，其中，X1为T或K(优选为K)；
HCDR2：RIRSKYNNYATYYAX2SVKX3，其中，X2为D或A(优选为A)，X3为D或G(优选为G)；
HCDR3：HGX3X4GX5X6X7X8X9X10X11AY，其中，X3为N，X4为F，X5为N或A，X6为S或A，X7为Y或A，X8为V或A，X9为S或A，X10为W，X11为F或A(优选为F)；
LCDR1：X12SSTGAVTTX13NYX14N，其中，X12为R或G(优选G)，X13为S 或G(优选G)，X14为A或P(优选P)；
LCDR2：GTNKRAP；
LCDR3：AX15X16X17X18X19X20X21X22，其中，X15为L或A(优选L)，X16为W，X17为Y或A(优选Y)，X18为S或A(优选S)，X19为N或A(优选N)，X20为L或A(优选L)，X21为W，X22为V或A。
在一些实施方式中，本发明所述第一结合结构域包含：氨基酸序列如SEQ ID NO：1或2所示的HCDR1，氨基酸序列如SEQ ID NO：3或4所示的HCDR2，氨基酸序列如SEQ ID NO：5、9、10、11、15、17、18、19或20所示的HCDR3，氨基酸序列如SEQ ID NO：23或24所示的LCDR1，氨基酸序列如SEQ ID NO：25所示的LCDR2，和氨基酸序列如SEQ ID NO：26、28、30、33、35、36、37、39、40、41或42所示的LCDR3。
在一些实施方式中，本发明所述第一结合结构域包含：
(I)氨基酸序列如SEQ ID NO：1所示的HCDR1，氨基酸序列如SEQ ID NO：3所示的HCDR2，氨基酸序列如SEQ ID NO：15所示的HCDR3，氨基酸序列如SEQ ID NO：23所示的LCDR1，氨基酸序列如SEQ ID NO：25所示的LCDR2，氨基酸序列如SEQ ID NO：33所示的LCDR3；或
(II)氨基酸序列如SEQ ID NO：2所示的HCDR1，氨基酸序列如SEQ ID NO：4所示的HCDR2，氨基酸序列如SEQ ID NO：5所示的HCDR3，氨基酸序列如SEQ ID NO：24所示的LCDR1，氨基酸序列如SEQ ID NO：25所示的LCDR2，氨基酸序列如SEQ ID NO：26、28、33、36或37中任一项所示的LCDR3；或
(III)氨基酸序列如SEQ ID NO：2所示的HCDR1，氨基酸序列如SEQ ID NO：4所示的HCDR2，氨基酸序列如SEQ ID NO：9所示的HCDR3，氨基酸序列如SEQ ID NO：24所示的LCDR1，氨基酸序列如SEQ ID NO：25所示的LCDR2，氨基酸序列如SEQ ID NO：28、35、37、39或40中任一项所示的LCDR3；或
(IV)氨基酸序列如SEQ ID NO：2所示的HCDR1，氨基酸序列如SEQ ID NO：4所示的HCDR2，氨基酸序列如SEQ ID NO：10所示的HCDR3，氨基酸序列如SEQ ID NO：24所示的LCDR1，氨基酸序列如SEQ ID NO：25所示的LCDR2，氨基酸序列如SEQ ID NO：28、33、40、41或42中任一项所示的LCDR3；或
(V)氨基酸序列如SEQ ID NO：2所示的HCDR1，氨基酸序列如SEQ ID NO：4所示的HCDR2，氨基酸序列如SEQ ID NO：11所示的HCDR3，氨基酸序列如SEQ ID NO：24所示的LCDR1，氨基酸序列如SEQ ID NO：25所示的LCDR2，氨基酸序列如SEQ ID NO：26、28、33、35、36、37、40、41或42中任一项所示的LCDR3；或
(VI)氨基酸序列如SEQ ID NO：2所示的HCDR1，氨基酸序列如SEQ ID NO：4所 示的HCDR2，氨基酸序列如SEQ ID NO：15所示的HCDR3，氨基酸序列如SEQ ID NO：24所示的LCDR1，氨基酸序列如SEQ ID NO：25所示的LCDR2，氨基酸序列如SEQ ID NO：26或30所示的LCDR3；或
(VII)氨基酸序列如SEQ ID NO：2所示的HCDR1，氨基酸序列如SEQ ID NO：4所示的HCDR2，氨基酸序列如SEQ ID NO：17所示的HCDR3，氨基酸序列如SEQ ID NO：24所示的LCDR1，氨基酸序列如SEQ ID NO：25所示的LCDR2，氨基酸序列如SEQ ID NO：33、35、36、37或42中任一项所示的LCDR3；或
(VIII)氨基酸序列如SEQ ID NO：2所示的HCDR1，氨基酸序列如SEQ ID NO：4所示的HCDR2，氨基酸序列如SEQ ID NO：18所示的HCDR3，氨基酸序列如SEQ ID NO：24所示的LCDR1，氨基酸序列如SEQ ID NO：25所示的LCDR2，氨基酸序列如SEQ ID NO：28、35、36、37、39、41或42中任一项所示的LCDR3；或
(IX)氨基酸序列如SEQ ID NO：2所示的HCDR1，氨基酸序列如SEQ ID NO：4所示的HCDR2，氨基酸序列如SEQ ID NO：20所示的HCDR3，氨基酸序列如SEQ ID NO：24所示的LCDR1，氨基酸序列如SEQ ID NO：25所示的LCDR2，氨基酸序列如SEQ ID NO：28、33、35、36、40、41或42中任一项所示的LCDR3；或
(X)氨基酸序列如SEQ ID NO：2所示的HCDR1，氨基酸序列如SEQ ID NO：4所示的HCDR2，氨基酸序列如SEQ ID NO：9、10或19中任一项所示的HCDR3，氨基酸序列如SEQ ID NO：23所示的LCDR1，氨基酸序列如SEQ ID NO：25所示的LCDR2，氨基酸序列如SEQ ID NO：26所示的LCDR3；或
(XI)氨基酸序列如SEQ ID NO：1所示的HCDR1、氨基酸序列如SEQ ID NO：3所示的HCDR2、氨基酸序列如SEQ ID NO：5所示的HCDR3、氨基酸序列如SEQ ID NO：23所示的LCDR1、氨基酸序列如SEQ ID NO：25所示的LCDR2、氨基酸序列如SEQ ID NO：26所示的LCDR3；或
(XII)氨基酸序列如SEQ ID NO：2所示的HCDR1，氨基酸序列如SEQ ID NO：4所示的HCDR2，氨基酸序列如SEQ ID NO：19所示的HCDR3，氨基酸序列如SEQ ID NO：24所示的LCDR1，氨基酸序列如SEQ ID NO：25所示的LCDR2，氨基酸序列如SEQ ID NO：36所示的LCDR3。
在一些实施方式中，本发明所述第一结合结构域包含：氨基酸序列如SEQ ID NO：43-62中任一项所示的VH，和氨基酸序列如SEQ ID NO：63-81中任一项所示的VL。在一些实施方案中，本发明所述第一结构域包含：氨基酸序列如SEQ ID NO：43-45、49-51、55和57-60中任一项所示的VH，和氨基酸序列如SEQ ID NO：63-65、67、69、72、74-76和78-81中任一项所示的VL。
在一些实施方式中，本发明所述第一结合结构域包含：
(I)氨基酸序列如SEQ ID NO：44所示的VH，和氨基酸序列如SEQ ID NO：64所示的VL；或
(II)氨基酸序列如SEQ ID NO：45所示的VH，和氨基酸序列如SEQ ID NO：65、67、72、75或76所示的VL；或
(III)氨基酸序列如SEQ ID NO：49所示的VH，和氨基酸序列如SEQ ID NO：67、74、76、78或79所示的VL；或
(IV)氨基酸序列如SEQ ID NO：50所示的VH，和氨基酸序列如SEQ ID NO：67、72、79、80或81所示的VL；或
(V)氨基酸序列如SEQ ID NO：51所示的VH，和氨基酸序列如SEQ ID NO：65、67、72、74、75、76、79、80或81所示的VL；或
(VI)氨基酸序列如SEQ ID NO：55所示的VH，和氨基酸序列如SEQ ID NO：65或69所示的VL；或
(VII)氨基酸序列如SEQ ID NO：57所示的VH，和氨基酸序列如SEQ ID NO：72、74、75、76或81所示的VL；或
(VIII)氨基酸序列如SEQ ID NO：58所示的VH，和氨基酸序列如SEQ ID NO：67、74、75、76、78、80或81所示的VL；或
(IX)氨基酸序列如SEQ ID NO：60所示的VH，和氨基酸序列如SEQ ID NO：67、72、74、75、79、80或81所示的VL；或
(X)氨基酸序列如SEQ ID NO：49、50或59所示的VH，和氨基酸序列如SEQ ID NO：63所示的VL；或
(XI)氨基酸序列如SEQ ID NO：43所示的VH，和氨基酸序列如SEQ ID NO：63所示的VL；或
(XII)氨基酸序列如SEQ ID NO：59所示的VH，和氨基酸序列如SEQ ID NO：75所示的VL。
在一些实施方式中，本发明所述第二结合结构域和第三结合结构域与Claudin 18.2结合，其分别包含：氨基酸序列如SEQ ID NO：121所示的HCDR1，氨基酸序列如SEQ ID NO：122所示的HCDR2，氨基酸序列如SEQ ID NO：123所示的HCDR3，氨基酸序列如SEQ ID NO：124所示的LCDR1，氨基酸序列如SEQ ID NO：125所示的LCDR2，和氨基酸序列如SEQ ID NO：126所示的LCDR3。
在一些实施方式中，本发明所述第二结合结构域和第三结合结构域与CD20结合，其分别包含：氨基酸序列如SEQ ID NO：127所示的HCDR1，氨基酸序列如SEQ ID NO：128 所示的HCDR2，氨基酸序列如SEQ ID NO：129所示的HCDR3，氨基酸序列如SEQ ID NO：130所示的LCDR1，氨基酸序列如SEQ ID NO：131所示的LCDR2，和氨基酸序列如SEQ ID NO：132所示的LCDR3。
在一些实施方式中，本发明所述第二结合结构域和第三结合结构域与Claudin 18.2结合，其分别包含：(a)氨基酸序列如SEQ ID NO：133所示的VH，和氨基酸序列如SEQ ID NO：134所示的VL；或(b)氨基酸序列如SEQ ID NO：166所示的VH，和氨基酸序列如SEQ ID NO：167所示的VL。
在一些实施方式中，本发明所述第二结合结构域和第三结合结构域与CD20结合，其分别包含：氨基酸序列如SEQ ID NO：135所示的VH，和氨基酸序列如SEQ ID NO：136所示的VL。
在一些实施方式中，本发明所述第一Fc区为knob-Fc，所述第二Fc区为hole-Fc。
在一些实施方式中，本发明所述第一Fc区与所述第二Fc区之间通过铰链区的二硫键和杵臼结构连接。
在一些实施方式中，本发明所述第一Fc区和/或第二Fc区为来自IgG的Fc区，优选为来自IgG1、IgG2、IgG3或IgG4的Fc区。
在一些实施方式中，本发明所述第CH1为来自IgG的CH1，优选为IgG1、IgG2、IgG3或IgG4的CH1。
在一些实施方式中，本发明所述CL为来自λ或κ轻链的CL。
在一些实施方式中，本发明连接肽可以是包含G(甘氨酸)、S(丝氨酸)和A(丙氨酸)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；进一步优选地，连接肽包括但不限于(GS)n、(GGS)n、(GGGS)n、(GGGGS)n和AS(GGGGS)n等，n可为1-6的整数，如1、2、3、4、5、6。
在一些实施方案中，本发明所述第一结合结构域的HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3来自表2-5中样品MFI与JSCD3MFI比值在0.30-0.80之间的任意单克隆抗体的HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3。
在一些实施方式中，本发明所述双特异性抗体具有四条多肽链，所述四条多肽链分别为：
(1)所述第一多肽链具有如SEQ ID NO：144所示的氨基酸序列，所述第二多肽链具有如SEQ ID NO：145所示的氨基酸序列，所述第三多肽链具有如SEQ ID NO：142所示的氨基酸序列，所述第四多肽链具有如SEQ ID NO：143所示的氨基酸序列；或
(2)所述第一多肽链具有如SEQ ID NO：146所示的氨基酸序列，所述第二多肽链具有如SEQ ID NO：147所示的氨基酸序列，所述第三多肽链具有如SEQ ID NO：142所示的氨 基酸序列，所述第四多肽链具有如SEQ ID NO：143所示的氨基酸序列；或
(3)所述第一多肽链具有如SEQ ID NO：148所示的氨基酸序列，所述第二多肽链具有如SEQ ID NO：149所示的氨基酸序列，所述第三多肽链具有如SEQ ID NO：142所示的氨基酸序列，所述第四多肽链具有如SEQ ID NO：143所示的氨基酸序列；或
(4)所述第一多肽链具有如SEQ ID NO：150所示的氨基酸序列，所述第二多肽链具有如SEQ ID NO：151所示的氨基酸序列，所述第三多肽链具有如SEQ ID NO：152所示的氨基酸序列，所述第四多肽链具有如SEQ ID NO：153所示的氨基酸序列；或
(5)所述第一多肽链具有如SEQ ID NO：154所示的氨基酸序列，所述第二多肽链具有如SEQ ID NO：155所示的氨基酸序列，所述第三多肽链具有如SEQ ID NO：152所示的氨基酸序列，所述第四多肽链具有如SEQ ID NO：153所示的氨基酸序列；或
(6)所述第一多肽链具有如SEQ ID NO：156所示的氨基酸序列，所述第二多肽链具有如SEQ ID NO：157所示的氨基酸序列，所述第三多肽链具有如SEQ ID NO：152所示的氨基酸序列，所述第四多肽链具有如SEQ ID NO：153所示的氨基酸序列；或
(7)所述第一多肽链具有如SEQ ID NO：164所示的氨基酸序列，所述第二多肽链具有如SEQ ID NO：165所示的氨基酸序列，所述第三多肽链具有如SEQ ID NO：142所示的氨基酸序列，所述第四多肽链具有如SEQ ID NO：143所示的氨基酸序列。
在又一个方面，本发明提供了一种抗体或其抗原结合片段，其包含结合CD3的结合结构域，其中，所述结合CD3的结合结构域包含的HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3分别为：
HCDR1：X1YAMN，其中，X1为T或K；
HCDR2：RIRSKYNNYATYYAX2SVKX3，其中，X2为D或A，X3为D或G；
HCDR3：HGX3X4GX5X6X7X8X9X10X11AY，其中，X3为N或A，X4为F或A，X5为N或A，X6为S或A，X7为Y或A，X8为V或A，X9为S或A，X10为W或A，X11为F或A；
LCDR1：X12SSTGAVTTX13NYX14N，其中，X12为R或G，X13为S或G，X14为A或P；
LCDR2：GTNKRAP；
LCDR3：AX15X16X17X18X19X20X21X22，其中，X15为L或A，X16为W或A，X17为Y或A，X18为S或A，X19为N或A，X20为L或A，X21为W或A，X22为V或A；
条件是，所述结合CD3的结合结构域包含的HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3不同时为：氨基酸序列如SEQ ID NO：1所示的HCDR1、氨基酸序列如 SEQ ID NO：3所示的HCDR2、氨基酸序列如SEQ ID NO：5所示的HCDR3、氨基酸序列如SEQ ID NO：23所示的LCDR1、氨基酸序列如SEQ ID NO：25所示的LCDR2和氨基酸序列如SEQ ID NO：26所示的LCDR3。
在一些实施方式中，所述结合CD3的结合结构域包含的HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3分别为：
HCDR1：X1YAMN，其中，X1为T或K(优选为K)；
HCDR2：RIRSKYNNYATYYAX2SVKX3，其中，X2为D或A(优选为A)，X3为D或G(优选为G)；
HCDR3：HGX3X4GX5X6X7X8X9X10X11AY，其中，X3为N，X4为F，X5为N或A，X6为S或A，X7为Y或A，X8为V或A，X9为S或A，X10为W，X11为F或A(优选为F)；
LCDR1：X12SSTGAVTTX13NYX14N，其中，X12为R或G(优选G)，X13为S或G(优选G)，X14为A或P(优选P)；
LCDR2：GTNKRAP；
LCDR3：AX15X16X17X18X19X20X21X22，其中，X15为L或A(优选L)，X16为W，X17为Y或A(优选Y)，X18为S或A(优选S)，X19为N或A(优选N)，X20为L或A(优选L)，X21为W，X22为V或A；
条件是，所述结合CD3的结合结构域包含的HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3不同时为：氨基酸序列如SEQ ID NO：1所示的HCDR1、氨基酸序列如SEQ ID NO：3所示的HCDR2、氨基酸序列如SEQ ID NO：5所示的HCDR3、氨基酸序列如SEQ ID NO：23所示的LCDR1、氨基酸序列如SEQ ID NO：25所示的LCDR2和氨基酸序列如SEQ ID NO：26所示的LCDR3。
在一些实施方式中，本发明所述结合CD3的结合结构域包含：氨基酸序列如SEQ ID NO：1或2所示的HCDR1，氨基酸序列如SEQ ID NO：3或4所示的HCDR2，氨基酸序列如SEQ ID NO：5-22中任一项所示的HCDR3，氨基酸序列如SEQ ID NO：23或24所示的LCDR1，氨基酸序列如SEQ ID NO：25所示的LCDR2，和氨基酸序列如SEQ ID NO：26-42中任一项所示的LCDR3。
在一些实施方式中，本发明所述结合CD3的结合结构域包含HCDR1、HCDR2、HCDR3和LCDR1、LCDR2、LCDR3：
所述HCDR1、HCDR2、HCDR3为：
(1)氨基酸序列如SEQ ID NO：1所示的HCDR1，氨基酸序列如SEQ ID NO：3所示的HCDR2，氨基酸序列如SEQ ID NO：5所示的HCDR3；或
(2)氨基酸序列如SEQ ID NO：1所示的HCDR1，氨基酸序列如SEQ ID NO：3所示的HCDR2，氨基酸序列如SEQ ID NO：15所示的HCDR3；或
(3)氨基酸序列如SEQ ID NO：2所示的HCDR1，氨基酸序列如SEQ ID NO：4所示的HCDR2，氨基酸序列如SEQ ID NO：5-22中任一项所示的HCDR3；
所述LCDR1、LCDR2、LCDR3为：
(1)氨基酸序列如SEQ ID NO：23所示的LCDR1，氨基酸序列如SEQ ID NO：25所示的LCDR2，氨基酸序列如SEQ ID NO：26所示的LCDR3；或
(2)氨基酸序列如SEQ ID NO：23所示的LCDR1，氨基酸序列如SEQ ID NO：25所示的LCDR2，氨基酸序列如SEQ ID NO：33所示的LCDR3；或
(3)氨基酸序列如SEQ ID NO：24所示的LCDR1，氨基酸序列如SEQ ID NO：25所示的LCDR2，氨基酸序列如SEQ ID NO：26-42中任一项所示的LCDR3。
在一些实施方式中，本发明所述结合CD3的结合结构域包含：氨基酸序列如SEQ ID NO：1或2所示的HCDR1，氨基酸序列如SEQ ID NO：3或4所示的HCDR2，氨基酸序列如SEQ ID NO：5、9、10、11、15、17、18、19或20所示的HCDR3，氨基酸序列如SEQ ID NO：23或24所示的LCDR1，氨基酸序列如SEQ ID NO：25所示的LCDR2，和氨基酸序列如SEQ ID NO：26、28、30、33、35、36、37、39、40、41或42所示的LCDR3。
在一些实施方式中，本发明所述结合CD3的结合结构域包含：
(I)氨基酸序列如SEQ ID NO：1所示的HCDR1，氨基酸序列如SEQ ID NO：3所示的HCDR2，氨基酸序列如SEQ ID NO：15所示的HCDR3，氨基酸序列如SEQ ID NO：23所示的LCDR1，氨基酸序列如SEQ ID NO：25所示的LCDR2，氨基酸序列如SEQ ID NO：33所示的LCDR3；或
(II)氨基酸序列如SEQ ID NO：2所示的HCDR1，氨基酸序列如SEQ ID NO：4所示的HCDR2，氨基酸序列如SEQ ID NO：5所示的HCDR3，氨基酸序列如SEQ ID NO：24所示的LCDR1，氨基酸序列如SEQ ID NO：25所示的LCDR2，氨基酸序列如SEQ ID NO：26、28、33、36或37所示的LCDR3；或
(III)氨基酸序列如SEQ ID NO：2所示的HCDR1，氨基酸序列如SEQ ID NO：4所示的HCDR2，氨基酸序列如SEQ ID NO：9所示的HCDR3，氨基酸序列如SEQ ID NO：24所示的LCDR1，氨基酸序列如SEQ ID NO：25所示的LCDR2，氨基酸序列如SEQ ID NO：28、35、37、39或40所示的LCDR3；或
(IV)氨基酸序列如SEQ ID NO：2所示的HCDR1，氨基酸序列如SEQ ID NO：4所示的HCDR2，氨基酸序列如SEQ ID NO：10所示的HCDR3，氨基酸序列如SEQ ID NO：24所示的LCDR1，氨基酸序列如SEQ ID NO：25所示的LCDR2，氨基酸序列如SEQ ID  NO：28、33、40、41或42所示的LCDR3；或
(V)氨基酸序列如SEQ ID NO：2所示的HCDR1，氨基酸序列如SEQ ID NO：4所示的HCDR2，氨基酸序列如SEQ ID NO：11所示的HCDR3，氨基酸序列如SEQ ID NO：24所示的LCDR1，氨基酸序列如SEQ ID NO：25所示的LCDR2，氨基酸序列如SEQ ID NO：26、28、33、35、36、37、40、41或42所示的LCDR3；或
(VI)氨基酸序列如SEQ ID NO：2所示的HCDR1，氨基酸序列如SEQ ID NO：4所示的HCDR2，氨基酸序列如SEQ ID NO：15所示的HCDR3，氨基酸序列如SEQ ID NO：24所示的LCDR1，氨基酸序列如SEQ ID NO：25所示的LCDR2，氨基酸序列如SEQ IDNQ：26或30所示的LCDR3；或
(VII)氨基酸序列如SEQ ID NO：2所示的HCDR1，氨基酸序列如SEQ ID NO：4所示的HCDR2，氨基酸序列如SEQ ID NO：17所示的HCDR3，氨基酸序列如SEQ ID NO：24所示的LCDR1，氨基酸序列如SEQ ID NO：25所示的LCDR2，氨基酸序列如SEQ ID NO：33、35、36、37或42所示的LCDR3；或
(VIII)氨基酸序列如SEQ ID NO：2所示的HCDR1，氨基酸序列如SEQ ID NO：4所示的HCDR2，氨基酸序列如SEQ ID NO：18所示的HCDR3，氨基酸序列如SEQ ID NO：24所示的LCDR1，氨基酸序列如SEQ ID NO：25所示的LCDR2，氨基酸序列如SEQ ID NO：28、35、36、37、39、41或42所示的LCDR3；或
(IX)氨基酸序列如SEQ ID NO：2所示的HCDR1，氨基酸序列如SEQ ID NO：4所示的HCDR2，氨基酸序列如SEQ ID NO：20所示的HCDR3，氨基酸序列如SEQ ID NO：24所示的LCDR1，氨基酸序列如SEQ ID NO：25所示的LCDR2，氨基酸序列如SEQ ID NO：28、33、35、36、40、41或42所示的LCDR3；或
(X)氨基酸序列如SEQ ID NO：2所示的HCDR1，氨基酸序列如SEQ ID NO：4所示的HCDR2，氨基酸序列如SEQ ID NO：9、10或19所示的HCDR3，氨基酸序列如SEQ ID NO：23所示的LCDR1，氨基酸序列如SEQ ID NO：25所示的LCDR2，氨基酸序列如SEQ ID NO：26所示的LCDR3；或
(XI)氨基酸序列如SEQ ID NO：2所示的HCDR1，氨基酸序列如SEQ ID NO：4所示的HCDR2，氨基酸序列如SEQ ID NO：19所示的HCDR3，氨基酸序列如SEQ ID NO：24所示的LCDR1，氨基酸序列如SEQ ID NO：25所示的LCDR2，氨基酸序列如SEQ ID NO：36所示的LCDR3；或
(XII)氨基酸序列如SEQ ID NO：1所示的HCDR1、氨基酸序列如SEQ ID NO：3所示的HCDR2、氨基酸序列如SEQ ID NO：5所示的HCDR3、氨基酸序列如SEQ ID NO：23所示的LCDR1、氨基酸序列如SEQ ID NO：25所示的LCDR2、氨基酸序列如SEQ ID NO：26 所示的LCDR3。
在一些实施方式中，本发明所述结合CD3的结合结构域包含：氨基酸序列如SEQ ID NO：43-62中任一项所示的VH，和氨基酸序列如SEQ ID NO：63-81中任一项所示的VL。在一些实施方案中，本发明所述结合CD3的结合结构域包含：氨基酸序列如SEQ ID NO：43-45、49-51、55和57-60中任一项所示的VH，和氨基酸序列如SEQ ID NO：63-65、67、69、72、74-76和78-81中任一项所示的VL。
在一些实施方式中，本发明所述结合CD3的结合结构域包含：
(I)氨基酸序列如SEQ ID NO：44所示的VH，和氨基酸序列如SEQ ID NO：64所示的VL；或
(II)氨基酸序列如SEQ ID NO：45所示的VH，和氨基酸序列如SEQ ID NO：65、67、72、75或76所示的VL；或
(III)氨基酸序列如SEQ ID NO：49所示的VH，和氨基酸序列如SEQ ID NO：67、74、76、78或79所示的VL；或
(IV)氨基酸序列如SEQ ID NO：50所示的VH，和氨基酸序列如SEQ ID NO：67、72、79、80或81所示的VL；或
(V)氨基酸序列如SEQ ID NO：51所示的VH，和氨基酸序列如SEQ ID NO：65、67、72、74、75、76、79、80或81所示的VL；或
(VI)氨基酸序列如SEQ ID NO：55所示的VH，和氨基酸序列如SEQ ID NO：65或69所示的VL；或
(VII)氨基酸序列如SEQ ID NO：57所示的VH，和氨基酸序列如SEQ ID NO：72、74、75、76或81所示的VL；或
(VIII)氨基酸序列如SEQ ID NO：58所示的VH，和氨基酸序列如SEQ ID NO：67、74、75、76、78、80或81所示的VL；或
(IX)氨基酸序列如SEQ ID NO：60所示的VH，和氨基酸序列如SEQ ID NO：67、72、74、75、79、80或81所示的VL；或
(X)氨基酸序列如SEQ ID NO：49、50或59所示的VH，和氨基酸序列如SEQ ID NO：63所示的VL；或
(XI)氨基酸序列如SEQ ID NO：59所示的VH，和氨基酸序列如SEQ ID NO：75所示的VL；或
(XII)氨基酸序列如SEQ ID NO：43所示的VH，和氨基酸序列如SEQ ID NO：63所示的VL。
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：82-101中任一项 所示的重链，和氨基酸序列如SEQ ID NO：102-120中任一项所示的轻链。在优选的实施方案中，本发明所述抗体的重链氨基酸序列如SEQ ID NO：82-84、88-90、94和96-99中任一所示，轻链氨基酸序列如SEQ ID NO：102-104、106、108、111、113-115和117-120中任一所示。
在一些实施方式中，本发明所述抗体包含：
(I)氨基酸序列如SEQ ID NO：83所示的重链，和氨基酸序列如SEQ ID NO：103所示的轻链；或
(II)氨基酸序列如SEQ ID NO：84所示的重链，和氨基酸序列如SEQ ID NO：104、106、111、114或115所示的轻链；或
(III)氨基酸序列如SEQ ID NO：88所示的重链，和氨基酸序列如SEQ ID NO：106、113、115、117或118所示的轻链；或
(IV)氨基酸序列如SEQ ID NO：89所示的重链，和氨基酸序列如SEQ ID NO：106、111、118、119或120所示的轻链；或
(V)氨基酸序列如SEQ ID NO：90所示的重链，和氨基酸序列如SEQ ID NO：104、106、111、113、114、115、118、119或120所示的轻链；或
(VI)氨基酸序列如SEQ ID NO：94所示的重链，和氨基酸序列如SEQ ID NO：104或108所示的轻链；或
(VII)氨基酸序列如SEQ ID NO：96所示的重链，和氨基酸序列如SEQ ID NO：111、113、114、115或120所示的轻链；或
(VIII)氨基酸序列如SEQ ID NO：97所示的重链，和氨基酸序列如SEQ ID NO：106、113、114、115、117、119或120所示的轻链；或
(IX)氨基酸序列如SEQ ID NO：99所示的重链，和氨基酸序列如SEQ ID NO：106、111、113、114、118、119或120所示的轻链；或
(X)氨基酸序列如SEQ ID NO：88、89或98所示的重链，和氨基酸序列如SEQ ID NO：102所示的轻链；或
(XI)氨基酸序列如SEQ ID NO：98所示的重链，和氨基酸序列如SEQ ID NO：114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，所述抗原结合片段为Fab、Fab′、F(ab′)2、Fv、dsFv、scFv或sdAb。
在一些实施方案中，本发明所述抗体是特异性结合CD3的单克隆抗体，其HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3如前文任一实施方案所述；或其VH和VL如前文任一实施方案所述，或其CH和CL如前文任一实施方案所述；条件是，所述单克 隆抗体的VH不是SEQ ID NO：43所示的氨基酸序列，且VL不是SEQ ID NO：63所示的氨基酸序列，或所述单克隆抗体的CH不是SEQ ID NO：82所示的氨基酸序列，且CL不是SEQ ID NO：102所示的氨基酸序列。
在一些实施方案中，本发明所述抗体是特异性结合CD3的单克隆抗体，其HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3来自表2-5中样品MFI与JSCD3MFI比值在0.30-0.80之间的任意单克隆抗体的HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3，或其VH和VL来自表2-5中样品MFI与JSCD3MFI比值在0.30-0.80之间的任意单克隆抗体的VH和VL，或其CH和CL来自表2-5中样品MFI与JSCD3MFI比值在0.30-0.80之间的任意单克隆抗体的CH和CL。
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一个或两个结合肿瘤相关抗原的结合结构域。
在一些实施方式中，本发明所述肿瘤相关抗原选自BCMA、CD19、CD20、CD22、CD30、CD33、CD38、CD40、CD123、CDK4、CEA、Claudin 18.2、EGFR、ROR1、TGF-β和5T4；优选地，所述肿瘤相关抗原是Claudin 18.2、CD20或BCMA。
在一些实施方式中，本发明所述肿瘤相关抗原是Claudin 18.2或CD20。在一些实施方式中，本发明所述结合Claudin 18.2的结合结构域包含：氨基酸序列如SEQ ID NO：121所示的HCDR1，氨基酸序列如SEQ ID NO：122所示的HCDR2，氨基酸序列如SEQ ID NO：123所示的HCDR3，氨基酸序列如SEQ ID NO：124所示的LCDR1，氨基酸序列如SEQ ID NO：125所示的LCDR2，和氨基酸序列如SEQ ID NO：126所示的LCDR3。在一些实施方式中，本发明所述结合CD20的结合结构域包含：氨基酸序列如SEQ ID NO：127所示的HCDR1，氨基酸序列如SEQ ID NO：128所示的HCDR2，氨基酸序列如SEQ ID NO：129所示的HCDR3，氨基酸序列如SEQ ID NO：130所示的LCDR1，氨基酸序列如SEQ ID NO：131所示的LCDR2，和氨基酸序列如SEQ ID NO：132所示的LCDR3。
在一些实施方式中，本发明所述结合Claudin 18.2的结合结构域包含：(a)氨基酸序列如SEQ ID NO：133所示的VH，和氨基酸序列如SEQ ID NO：134所示的VL；或(b)氨基酸序列如SEQ ID NO：166所示的VH，和氨基酸序列如SEQ ID NO：167所示的VL。
在一些实施方式中，本发明所述结合CD20的结合结构域包含：氨基酸序列如SEQ ID NO：135所示的VH，和氨基酸序列如SEQ ID NO：136所示的VL。
在一些实施方式中，本发明提供了一种抗体(优选为双特异性抗体)，其包含结合CD3的结合结构域和结合CD20的结合结构域，其中，所述结合CD3的结合结构域包含：氨基酸序列如SEQ ID NO：1所示的HCDR1、氨基酸序列如SEQ ID NO：3所示的HCDR2、氨基酸序列如SEQ ID NO：5所示的HCDR3、氨基酸序列如SEQ ID NO：23所示的LCDR1、 氨基酸序列如SEQ ID NO：25所示的LCDR2、氨基酸序列如SEQ ID NO：26所示的LCDR3；所述结合CD20的结合结构域包含：氨基酸序列如SEQ ID NO：127所示的HCDR1，氨基酸序列如SEQ ID NO：128所示的HCDR2，氨基酸序列如SEQ ID NO：129所示的HCDR3，氨基酸序列如SEQ ID NO：130所示的LCDR1，氨基酸序列如SEQ ID NO：131所示的LCDR2，和氨基酸序列如SEQ ID NO：132所示的LCDR3。
在一些实施方式中，本发明提供了一种抗体(优选为双特异性抗体)，其包含结合CD3的结合结构域和结合CD20的结合结构域，其中，所述结合CD3的结合结构域包含：氨基酸序列如SEQ ID NO：43所示的VH，和氨基酸序列如SEQ ID NO：63所示的VL，所述结合CD20的结合结构域包含：氨基酸序列如SEQ ID NO：135所示的VH，和氨基酸序列如SEQ ID NO：136所示的VL。
在一些实施方式中，本发明所述抗体的Fc区为来自IgG的Fc，例如来自IgG1、IgG2、IgG3或IgG4的Fc，和/或所述抗体的CL为来自λ或κ轻链的CL；优选地，所述Fc区包含杵臼结构。
在一些实施方式中，本发明所述抗体具有四条多肽链，所述四条多肽链分别为：
(1)具有如SEQ ID NO：144所示的氨基酸序列的第一多肽链，具有如SEQ ID NO：145所示的氨基酸序列的第二多肽链，具有如SEQ ID NO：142所示的氨基酸序列，具有如SEQ ID NO：143所示的氨基酸序列；或
(2)具有如SEQ ID NO：146所示的氨基酸序列的第一多肽链，具有如SEQ ID NO：147所示的氨基酸序列的第二多肽链，具有如SEQ ID NO：142所示的氨基酸序列的第三多肽链，具有如SEQ ID NO：143所示的氨基酸序列的第四多肽链；或
(3)具有如SEQ ID NO：148所示的氨基酸序列的第一多肽链，具有如SEQ ID NO：149所示的氨基酸序列的第二多肽链，具有如SEQ ID NO：142所示的氨基酸序列的第三多肽链，具有如SEQ ID NO：143所示的氨基酸序列的第四多肽链；或
(4)具有如SEQ ID NO：150所示的氨基酸序列的第一多肽链，具有如SEQ ID NO：151所示的氨基酸序列的第二多肽链，具有如SEQ ID NO：152所示的氨基酸序列的第三多肽链，具有如SEQ ID NO：153所示的氨基酸序列的第四多肽链；或
(5)具有如SEQ ID NO：154所示的氨基酸序列的第一多肽链，具有如SEQ ID NO：155所示的氨基酸序列的第二多肽链，具有如SEQ ID NO：152所示的氨基酸序列的第三多肽链，具有如SEQ ID NO：153所示的氨基酸序列的第四多肽链；或
(6)具有如SEQ ID NO：156所示的氨基酸序列的第一多肽链，具有如SEQ ID NO：157所示的氨基酸序列的第二多肽链，具有如SEQ ID NO：152所示的氨基酸序列的第三多肽链，具有如SEQ ID NO：153所示的氨基酸序列的第四多肽链；或
(7)具有如SEQ ID NO：164所示的氨基酸序列的第一多肽链，具有如SEQ ID NO：165所示的氨基酸序列的第二多肽链，具有如SEQ ID NO：142所示的氨基酸序列的第三多肽链，具有如SEQ ID NO：143所示的氨基酸序列的第四多肽链。
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结合结构域和第二结合结构域可以通过接头彼此连接，再进一步结合至第一Fc区；第三结合结构域结合至第二Fc区。在一些实施方案中，接头是肽接头。
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“Knob into Hole结构”或“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(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(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Knob into Hole结构(KiH)，有利于重链异源二聚体的形成；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。术语“knob-Fc”是指在抗体Fc区包含T366W的点突变，以形成类似knob的空间结构。相对应地，“hole-Fc”是指在抗体Fc区包含T366S、L368A、Y407V的点突变，以形成类似hole的空间结构。为进一步地促进异二聚体的形成，还可在knob-Fc和hole-Fc分别引入S354C和Y349C的点突变，通过二硫键进一步促进异二聚体的形成。同时，为了减弱和蛋白A的结合，还可在hole-Fc分别引入H435R和Y436F的点突变。
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抗体，其有效地促进T细胞介导的对肿瘤细胞的杀灭。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肿瘤相关抗原的抗原结合位点的抗体可以是双特异性抗体，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四条多肽链，其中(a)所述第一多肽链自N端到C端具有VH1-CH1-连接肽-VL2-连接肽-铰链区-第一Fc区，所述第二多肽链自N端到C端具有VL1-CL-连接肽-VH2；所述第三多肽链自N端到C端具有VH2-CH1-铰链区-第二Fc区，所述第四多肽链自N端到C端具有VL2-CL；或(b)其中所述第一多肽链自N端到C端具有VH2-CH1-连接肽-VL1-连接肽-铰链区-第一Fc区，所述第二多肽链自N端到C端具有VL2-CL-连接肽-VH1；所述第三多肽链自N端到C端具有VH2-CH1-铰链区-第二Fc区，所述第四多肽链自N端到C端具有VL2-CL。其中，所述连接肽包含G(甘氨酸)、S(丝氨酸)和A(丙氨 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；进一步优选地，连接肽包括但不限于(GS)n、(GGS)n、(GGGS)n、(GGGGS)n和AS(GGGGS)n等，n可为1-6的整数。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。
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)、或通过受控的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(EpigenBiotech)、双靶向性纳米抗体(dualtargetingnanobodies)(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双特异性抗体。所述多核苷酸可以包含编码双特异性抗体的4条多肽链的氨基酸序列的多核苷酸。
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，本发明提供制备本文所述的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，其包含如本文任一实施方案所述的双特异性抗体、多核苷酸、表达载体或宿主细胞，和药学上可接受的载体或赋形剂。
在又一个方面，本发明提供了一种药物组合物，其包含如本文任一实施方案所述的抗体或其抗原结合片段、多核苷酸、表达载体或宿主细胞，和药学上可接受的载体或赋形剂。
应理解，本文所述的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文所述的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本文所述的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，其包含如本文任一实施方案所述的双特异性抗体、多核苷酸、表达载体、宿主细胞、或药物组合物，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，其包括如本文任一实施方案所述的双特异性抗体、多核苷酸、表达载体、宿主细胞、或药物组合物。
在一些实施方式中，本发明的药物组合物还包含编码本文所述的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，其包含如本文任一实施方案所述的抗体或其抗原结合片段、多核苷酸、表达载体、宿主细胞、或药物组合物，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，其包括如本文任一实施方案所述的抗体或其抗原结合片段、多核苷酸、表达载体、宿主细胞、或药物组合物。
医药用途
在又一个方面，本发明提供如本文任一实施方案所述的双特异性抗体、多核苷酸分子、表达载体、宿主细胞、或药物组合物在制备用于预防或治疗对象癌症的药物中的用途。
在又一个方面，本发明提供如本文任一实施方案所述的双特异性抗体、多核苷酸分子、表达载体、宿主细胞、或药物组合物，其用于预防或治疗对象的癌症。
在又一个方面，本发明提供一种预防或治疗对象的癌症的方法，其包括向有需要的受试者施用如本文任一实施方案所述的双特异性抗体、多核苷酸分子、表达载体、宿主细胞、或药物组合物。
在又一个方面，本发明提供了如本文任一实施方案所述的抗体或其抗原结合片段、多核苷酸、表达载体、宿主细胞、或药物组合物在制备用于预防或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任一实施方案所述的抗体或其抗原结合片段、多核苷酸、表达载体、宿主细胞、或药物组合物，其用于预防或治疗癌症。
在又一个方面，本发明提供了一种预防或治疗癌症的方法，其包括向有需要的受试者施用如本文任一实施方案所述的抗体或其抗原结合片段、多核苷酸、表达载体、宿主细胞、或药物组合物。
本文中，所述癌症优选是表达选自以下一种或多种肿瘤相关抗原的癌症：BCMA、CD19、CD20、CD22、CD30、CD33、CD38、CD40、CD123、CDK4、CEA、Claudin 18.2、EGFR、ROR1、TGF-β和5T4。更优选地，所述肿瘤相关抗原选自Claudin 18.2、CD20和BCMA中的一种或多种。示例性的癌症包括前文所述的癌症。
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，所述与CLDN18相关的癌症选自胃癌、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。
优选地，所述癌症与CD20相关；在一些实施方式中，所述与CD20相关的癌症选自急性B淋巴细胞性白血病、弥漫性大B细胞淋巴瘤、慢性淋巴细胞白血病、滤泡淋巴瘤，非霍奇金氏淋巴瘤、慢性骨髓细胞白血病和Burkitt淋巴瘤。
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，本发明还包括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。
在一个实施方式中，本发明的癌症治疗方法还包括施用本文所述的双特异性抗体的表达稳定或增加剂。双特异性抗体的表达优选在癌细胞的细胞表面。双特异性抗体表达稳定或增加剂可以是奥沙利铂和/或5-FU。
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。
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双特异性抗体检测CD3或CLDN-18.2或CD20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双特异性抗体与样品接触，并检测所述双特异性抗体与CD3或CLDN-18.2或CD20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
抗体序列设计：
以抗体JSCD3(又称为JS202-JSCD3，其重链序列为JSCD3-HC和轻链序列为JSCD3-LC)起始，出于降低抗体的亲和力、减少毒性等目的，对抗体的CDR区进行突变，筛选出以下多种氨基酸序列：轻链JSCD3-LC的CDR区进行突变，产生CD3-LC1、CD3-LC2、CD3-LC3、CD3-LC4、CD3-LC5、CD3-LC6、CD3-LC7、CD3-LC8、CD3-LC9、CD3-LC10、CD3-LC11、CD3-LC12、CD3-LC13、CD3-LC14、CD3-LC15、CD3-LC16、CD3-LC17、CD3-LC18和JSCD3-LC-VtoA，共18个序列；重链JSCD3-HC的CDR区进行突变，产生CD3-HC1、CD3-HC2、CD3-HC3、CD3-HC4、CD3-HC5、CD3-HC6、CD3-HC7、CD3-HC8、CD3-HC9、CD3-HC10、CD3-HC11、CD3-HC12、CD3-HC13、CD3-HC14、CD3-HC15、CD3-HC16、CD3-HC17、CD3-HC18、JSCD3-HC-WtoA和JSCD3-HC-FtoA共20个序列。序列如下：
JSCD3-HC(斜体字表示VH，下划线依次表示HCDR1、HCDR2和HCDR3)：
Figure PCTCN2022122753-appb-000001
JSCD3-HC-FtoA的氨基酸序列如SEQ ID NO：83所示，其可变区序列如下(下划线依次表示HCDR1、HCDR2和HCDR3)：
Figure PCTCN2022122753-appb-000002
CD3-HC1的氨基酸序列如SEQ ID NO：84所示，其可变区序列如下(下划线依次表示HCDR1、HCDR2和HCDR3)：
Figure PCTCN2022122753-appb-000003
CD3-HC2的氨基酸序列如SEQ ID NO：85所示，其可变区序列如下(下划线依次表示HCDR1、HCDR2和HCDR3)：
Figure PCTCN2022122753-appb-000004
CD3-HC3的氨基酸序列如SEQ ID NO：86所示，其可变区序列如下(下划线依次表示HCDR1、HCDR2和HCDR3)：
Figure PCTCN2022122753-appb-000005
CD3-HC4的氨基酸序列如SEQ ID NO：87所示，其可变区序列如下(下划线依次表示HCDR1、HCDR2和HCDR3)：
Figure PCTCN2022122753-appb-000006
CD3-HC5的氨基酸序列如SEQ ID NO：88所示，其可变区序列如下(下划线依次表示HCDR1、HCDR2和HCDR3)：
Figure PCTCN2022122753-appb-000007
CD3-HC6的氨基酸序列如SEQ ID NO：89所示，其可变区序列如下(下划线依次表示HCDR1、HCDR2和HCDR3)：
Figure PCTCN2022122753-appb-000008
CD3-HC7的氨基酸序列如SEQ ID NO：90所示，其可变区序列如下(下划线依次表 示HCDR1、HCDR2和HCDR3)：
Figure PCTCN2022122753-appb-000009
CD3-HC8的氨基酸序列如SEQ ID NO：91所示，其可变区序列如下(下划线依次表示HCDR1、HCDR2和HCDR3)：
Figure PCTCN2022122753-appb-000010
CD3-HC9的氨基酸序列如SEQ ID NO：92所示，其可变区序列如下(下划线依次表示HCDR1、HCDR2和HCDR3)：
Figure PCTCN2022122753-appb-000011
CD3-HC10的氨基酸序列如SEQ ID NO：93所示，其可变区序列如下(下划线依次表示HCDR1、HCDR2和HCDR3)：
Figure PCTCN2022122753-appb-000012
CD3-HC11的氨基酸序列如SEQ ID NO：94所示，其可变区序列如下(下划线依次表示HCDR1、HCDR2和HCDR3)：
Figure PCTCN2022122753-appb-000013
CD3-HC12的氨基酸序列如SEQ ID NO：95所示，其可变区序列如下(下划线依次表示HCDR1、HCDR2和HCDR3)：
Figure PCTCN2022122753-appb-000014
Figure PCTCN2022122753-appb-000015
CD3-HC13的氨基酸序列如SEQ ID NO：96所示，其可变区序列如下(下划线依次表示HCDR1、HCDR2和HCDR3)：
Figure PCTCN2022122753-appb-000016
CD3-HC14的氨基酸序列如SEQ ID NO：97所示，其可变区序列如下(下划线依次表示HCDR1、HCDR2和HCDR3)：
Figure PCTCN2022122753-appb-000017
CD3-HC15的氨基酸序列如SEQ ID NO：98所示，其可变区序列如下(下划线依次表示HCDR1、HCDR2和HCDR3)：
Figure PCTCN2022122753-appb-000018
CD3-HC16的氨基酸序列如SEQ ID NO：99所示，其可变区序列如下(下划线依次表示HCDR1、HCDR2和HCDR3)：
Figure PCTCN2022122753-appb-000019
CD3-HC17的氨基酸序列如SEQ ID NO：100所示，其可变区序列如下(下划线依次表示HCDR1、HCDR2和HCDR3)：
Figure PCTCN2022122753-appb-000020
CD3-HC18的氨基酸序列如SEQ ID NO：101所示，其可变区序列如下(下划线依次表示HCDR1、HCDR2和HCDR3)：
Figure PCTCN2022122753-appb-000021
JSCD3-HC-WtoA的氨基酸序列如SEQ ID NO：140所示，其可变区序列如下(下划线依次表示HCDR1、HCDR2和HCDR3)：
Figure PCTCN2022122753-appb-000022
JSCD3-LC(斜体字表示VL，下划线依次表示LCDR1、LCDR2和LCDR3)：
Figure PCTCN2022122753-appb-000023
JSCD3-LC-VtoA的氨基酸序列如SEQ ID NO：103所示，其可变区序列如下(下划线依次表示LCDR1、LCDR2和LCDR3)：
Figure PCTCN2022122753-appb-000024
CD3-LC1的氨基酸序列如SEQ ID NO：104所示，其可变区序列如下(下划线依次表示LCDR1、LCDR2和LCDR3)：
Figure PCTCN2022122753-appb-000025
CD3-LC2的氨基酸序列如SEQ ID NO：105所示，其可变区序列如下(下划线依次表示LCDR1、LCDR2和LCDR3)：
Figure PCTCN2022122753-appb-000026
CD3-LC3的氨基酸序列如SEQ ID NO：106所示，其可变区序列如下(下划线依次表示LCDR1、LCDR2和LCDR3)：
Figure PCTCN2022122753-appb-000027
CD3-LC4的氨基酸序列如SEQ ID NO：107所示，其可变区序列如下(下划线依次表示LCDR1、LCDR2和LCDR3)：
Figure PCTCN2022122753-appb-000028
CD3-LC5的氨基酸序列如SEQ ID NO：108所示，其可变区序列如下(下划线依次表示LCDR1、LCDR2和LCDR3)：
Figure PCTCN2022122753-appb-000029
CD3-LC6的氨基酸序列如SEQ ID NO：109所示，其可变区序列如下(下划线依次表示LCDR1、LCDR2和LCDR3)：
Figure PCTCN2022122753-appb-000030
CD3-LC7的氨基酸序列如SEQ ID NO：110所示，其可变区序列如下(下划线依次表示LCDR1、LCDR2和LCDR3)：
Figure PCTCN2022122753-appb-000031
CD3-LC8的氨基酸序列如SEQ ID NO：111所示，其可变区序列如下(下划线依次表示LCDR1、LCDR2和LCDR3)：
Figure PCTCN2022122753-appb-000032
CD3-LC9的氨基酸序列如SEQ ID NO：112所示，其可变区序列如下(下划线依次表示LCDR1、LCDR2和LCDR3)：
Figure PCTCN2022122753-appb-000033
CD3-LC10的氨基酸序列如SEQ ID NO：113所示，其可变区序列如下(下划线依次表示LCDR1、LCDR2和LCDR3)：
Figure PCTCN2022122753-appb-000034
CD3-LC11的氨基酸序列如SEQ ID NO：114所示，其可变区序列如下(下划线依次表示LCDR1、LCDR2和LCDR3)：
Figure PCTCN2022122753-appb-000035
CD3-LC12的氨基酸序列如SEQ ID NO：113所示，其可变区序列如下(下划线依次表示LCDR1、LCDR2和LCDR3)：
Figure PCTCN2022122753-appb-000036
CD3-LC13的氨基酸序列如SEQ ID NO：115所示，其可变区序列如下(下划线依次表示LCDR1、LCDR2和LCDR3)：
Figure PCTCN2022122753-appb-000037
CD3-LC14的氨基酸序列如SEQ ID NO：116所示，其可变区序列如下(下划线依次表示LCDR1、LCDR2和LCDR3)：
Figure PCTCN2022122753-appb-000038
CD3-LC15的氨基酸序列如SEQ ID NO：117所示，其可变区序列如下(下划线依次表示LCDR1、LCDR2和LCDR3)：
Figure PCTCN2022122753-appb-000039
CD3-LC16的氨基酸序列如SEQ ID NO：118所示，其可变区序列如下(下划线依次表示LCDR1、LCDR2和LCDR3)：
Figure PCTCN2022122753-appb-000040
CD3-LC17的氨基酸序列如SEQ ID NO：119所示，其可变区序列如下(下划线依次表示LCDR1、LCDR2和LCDR3)：
Figure PCTCN2022122753-appb-000041
CD3-LC18的氨基酸序列如SEQ ID NO：120所示，其可变区序列如下(下划线依次表示LCDR1、LCDR2和LCDR3)：
Figure PCTCN2022122753-appb-000042
上述重链、轻链、重链可变区、轻链可变区、重链CDR和轻链CDR序列的序列编号汇总如下：
Figure PCTCN2022122753-appb-000043
Figure PCTCN2022122753-appb-000044
将重链JSCD3-HC-WtoA和轻链JSCD3-LC-VtoA组合得到抗CD3体JSCD3-A1；将重链JSCD3-HC-FtoA和轻链JSCD3-LC-VtoA组合得到抗CD3体JSCD3-A2。
将上述重链JSCD3-HC、CD3-HC1～CD3-HC18，和轻链JSCD3-LC、CD3-LC1～CD3-LC17两两配对组合，配对组合得到的抗CD3抗体如下表1A、表1B和表1C所示。
抗CD3抗体的制备方法如下：
(1)抗CD3抗体分子的的质粒构建：委托金斯瑞合成抗体的重链于puc57，通过SapI酶切并连接至HXT4S载体，得到表达载体HXT4S-CD3-HC。委托金斯瑞合成人抗体的轻链于puc57，通过SapI酶切并连接至HXT2载体，得到表达载体HXT2-CD3-LC。
(2)抗CD3抗体分子的表达：将上述构建的每个抗CD3抗体各自的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)抗CD3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，即得到抗CD3抗体。
表1A：抗CD3抗体的轻重链组合表
  CD3-HC1 CD3-HC2 CD3-HC3 CD3-HC4 CD3-HC5 CD3-HC6
CD3-LC1 JSCD3-1 JSCD3-20 JSCD3-39 JSCD3-58 JSCD3-77 JSCD3-96
CD3-LC2 JSCD3-2 JSCD3-21 JSCD3-40 JSCD3-59 JSCD3-78 JSCD3-97
CD3-LC3 JSCD3-3 JSCD3-22 JSCD3-41 JSCD3-60 JSCD3-79 JSCD3-98
CD3-LC4 JSCD3-4 JSCD3-23 JSCD3-42 JSCD3-61 JSCD3-80 JSCD3-99
CD3-LC5 JSCD3-5 JSCD3-24 JSCD3-43 JSCD3-62 JSCD3-81 JSCD3-100
CD3-LC6 JSCD3-6 JSCD3-25 JSCD3-44 JSCD3-63 JSCD3-82 JSCD3-101
CD3-LC7 JSCD3-7 JSCD3-26 JSCD3-45 JSCD3-64 JSCD3-83 JSCD3-102
CD3-LC8 JSCD3-8 JSCD3-27 JSCD3-46 JSCD3-65 JSCD3-84 JSCD3-103
CD3-LC9 JSCD3-9 JSCD3-28 JSCD3-47 JSCD3-66 JSCD3-85 JSCD3-104
CD3-LC10 JSCD3-10 JSCD3-29 JSCD3-48 JSCD3-67 JSCD3-86 JSCD3-105
CD3-LC11 JSCD3-11 JSCD3-30 JSCD3-49 JSCD3-68 JSCD3-87 JSCD3-106
CD3-LC12 JSCD3-12 JSCD3-31 JSCD3-50 JSCD3-69 JSCD3-88 JSCD3-107
CD3-LC13 JSCD3-13 JSCD3-32 JSCD3-51 JSCD3-70 JSCD3-89 JSCD3-108
CD3-LC14 JSCD3-14 JSCD3-33 JSCD3-52 JSCD3-71 JSCD3-90 JSCD3-109
CD3-LC15 JSCD3-15 JSCD3-34 JSCD3-53 JSCD3-72 JSCD3-91 JSCD3-110
CD3-LC16 JSCD3-16 JSCD3-35 JSCD3-54 JSCD3-73 JSCD3-92 JSCD3-111
CD3-LC17 JSCD3-17 JSCD3-36 JSCD3-55 JSCD3-74 JSCD3-93 JSCD3-112
CD3-LC18 JSCD3-18 JSCD3-37 JSCD3-56 JSCD3-75 JSCD3-94 JSCD3-113
JSCD3-LC JSCD3-19 JSCD3-38 JSCD3-57 JSCD3-76 JSCD3-95 JSCD3-114
表1B：抗CD3抗体的轻重链组合表
  CD3-HC7 CD3-HC8 CD3-HC9 CD3-HC10 CD3-HC11 CD3-HC12
CD3-LC1 JSCD3-115 JSCD3-134 JSCD3-153 JSCD3-172 JSCD3-191 JSCD3-210
CD3-LC2 JSCD3-116 JSCD3-135 JSCD3-154 JSCD3-173 JSCD3-192 JSCD3-211
CD3-LC3 JSCD3-117 JSCD3-136 JSCD3-155 JSCD3-174 JSCD3-193 JSCD3-212
CD3-LC4 JSCD3-118 JSCD3-137 JSCD3-156 JSCD3-175 JSCD3-194 JSCD3-213
CD3-LC5 JSCD3-119 JSCD3-138 JSCD3-157 JSCD3-176 JSCD3-195 JSCD3-214
CD3-LC6 JSCD3-120 JSCD3-139 JSCD3-158 JSCD3-177 JSCD3-196 JSCD3-215
CD3-LC7 JSCD3-121 JSCD3-140 JSCD3-159 JSCD3-178 JSCD3-197 JSCD3-216
CD3-LC8 JSCD3-122 JSCD3-141 JSCD3-160 JSCD3-179 JSCD3-198 JSCD3-217
CD3-LC9 JSCD3-123 JSCD3-142 JSCD3-161 JSCD3-180 JSCD3-199 JSCD3-218
CD3-LC10 JSCD3-124 JSCD3-143 JSCD3-162 JSCD3-181 JSCD3-200 JSCD3-219
CD3-LC11 JSCD3-125 JSCD3-144 JSCD3-163 JSCD3-182 JSCD3-201 JSCD3-220
CD3-LC12 JSCD3-126 JSCD3-145 JSCD3-164 JSCD3-183 JSCD3-202 JSCD3-221
CD3-LC13 JSCD3-127 JSCD3-146 JSCD3-165 JSCD3-184 JSCD3-203 JSCD3-222
CD3-LC14 JSCD3-128 JSCD3-147 JSCD3-166 JSCD3-185 JSCD3-204 JSCD3-223
CD3-LC15 JSCD3-129 JSCD3-148 JSCD3-167 JSCD3-186 JSCD3-205 JSCD3-224
CD3-LC16 JSCD3-130 JSCD3-149 JSCD3-168 JSCD3-187 JSCD3-206 JSCD3-225
CD3-LC17 JSCD3-131 JSCD3-150 JSCD3-169 JSCD3-188 JSCD3-207 JSCD3-226
CD3-LC18 JSCD3-132 JSCD3-151 JSCD3-170 JSCD3-189 JSCD3-208 JSCD3-227
JSCD3-LC JSCD3-133 JSCD3-152 JSCD3-171 JSCD3-190 JSCD3-209 JSCD3-228
表1C：抗CD3抗体的轻重链组合表
  CD3-HC13 CD3-HC14 CD3-HC15 CD3-HC16 CD3-HC17 CD3-HC18 JSCD3-HC
CD3-LC1 JSCD3-229 JSCD3-248 JSCD3-267 JSCD3-286 JSCD3-305 JSCD3-324 JSCD3-343
CD3-LC2 JSCD3-230 JSCD3-249 JSCD3-268 JSCD3-287 JSCD3-306 JSCD3-325 JSCD3-344
CD3-LC3 JSCD3-231 JSCD3-250 JSCD3-269 JSCD3-288 JSCD3-307 JSCD3-326 JSCD3-345
CD3-LC4 JSCD3-232 JSCD3-251 JSCD3-270 JSCD3-289 JSCD3-308 JSCD3-327 JSCD3-346
CD3-LC5 JSCD3-233 JSCD3-252 JSCD3-271 JSCD3-290 JSCD3-309 JSCD3-328 JSCD3-347
CD3-LC6 JSCD3-234 JSCD3-253 JSCD3-272 JSCD3-291 JSCD3-310 JSCD3-329 JSCD3-348
CD3-LC7 JSCD3-235 JSCD3-254 JSCD3-273 JSCD3-292 JSCD3-311 JSCD3-330 JSCD3-349
CD3-LC8 JSCD3-236 JSCD3-255 JSCD3-274 JSCD3-293 JSCD3-312 JSCD3-331 JSCD3-350
CD3-LC9 JSCD3-237 JSCD3-256 JSCD3-275 JSCD3-294 JSCD3-313 JSCD3-332 JSCD3-351
CD3-LC10 JSCD3-238 JSCD3-257 JSCD3-276 JSCD3-295 JSCD3-314 JSCD3-333 JSCD3-352
CD3-LC11 JSCD3-239 JSCD3-258 JSCD3-277 JSCD3-296 JSCD3-315 JSCD3-334 JSCD3-353
CD3-LC12 JSCD3-240 JSCD3-259 JSCD3-278 JSCD3-297 JSCD3-316 JSCD3-335 JSCD3-354
CD3-LC13 JSCD3-241 JSCD3-260 JSCD3-279 JSCD3-298 JSCD3-317 JSCD3-336 JSCD3-355
CD3-LC14 JSCD3-242 JSCD3-261 JSCD3-280 JSCD3-299 JSCD3-318 JSCD3-337 JSCD3-356
CD3-LC15 JSCD3-243 JSCD3-262 JSCD3-281 JSCD3-300 JSCD3-319 JSCD3-338 JSCD3-357
CD3-LC16 JSCD3-244 JSCD3-263 JSCD3-282 JSCD3-301 JSCD3-320 JSCD3-339 JSCD3-358
CD3-LC17 JSCD3-245 JSCD3-264 JSCD3-283 JSCD3-302 JSCD3-321 JSCD3-340 JSCD3-359
CD3-LC18 JSCD3-246 JSCD3-265 JSCD3-284 JSCD3-303 JSCD3-322 JSCD3-341 JSCD3-360
JSCD3-LC JSCD3-247 JSCD3-266 JSCD3-285 JSCD3-304 JSCD3-323 JSCD3-342 JSCD3
实施例2、抗CD3抗体与人CD3的结合活性初步研究
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)分别与稀释成10μg/ml浓度的抗CD3抗体在4℃孵育30min，洗去残留抗体后与荧光二抗(PE-抗人IgG)孵育，最后用C6PLUS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。
根据表2-5的平均荧光强度(MFI)测试结果，CDR突变后的抗CD3抗体与人CD3的结合活性均有不同程度的降低，选择抗体JSCD3-1、JSCD3-3、JSCD3-5、JSCD3-8、JSCD3-11、JSCD3-13、JSCD3-79、JSCD3-88、JSCD3-89、JSCD3-91、JSCD3-92、JSCD3-93、JSCD3-94、JSCD3-95、JSCD3-98、JSCD3-103、JSCD3-111、JSCD3-112、JSCD3-113、JSCD3-114、JSCD3-115、JSCD3-117、JSCD3-122、JSCD3-124、JSCD3-125、JSCD3-126、JSCD3-127、JSCD3-130、JSCD3-131、JSCD3-132、JSCD3-162、JSCD3-173、JSCD3-191、JSCD3-195、JSCD3-210、JSCD3-217、JSCD3-236、JSCD3-238、JSCD3-239、JSCD3-241、JSCD3-246、JSCD3-250、JSCD3-255、JSCD3-257、JSCD3-258、JSCD3-259、JSCD3-260、JSCD3-262、JSCD3-264、JSCD3-265、JSCD3-268、JSCD3-277、JSCD3-285、JSCD3-288、JSCD3-293、JSCD3-296、JSCD3-297、JSCD3-301、JSCD3-302、JSCD3-303、JSCD3-324、JSCD3-325进行下一步研究。
表2：抗CD3抗体与人CD3结合活性结果I
Figure PCTCN2022122753-appb-000045
Figure PCTCN2022122753-appb-000046
表3：抗CD3抗体与人CD3结合活性结果II
Figure PCTCN2022122753-appb-000047
Figure PCTCN2022122753-appb-000048
表4：抗CD3抗体与人CD3结合活性结果III
Figure PCTCN2022122753-appb-000049
Figure PCTCN2022122753-appb-000050
表5：抗CD3抗体与人CD3结合活性结果IV
Figure PCTCN2022122753-appb-000051
Figure PCTCN2022122753-appb-000052
Figure PCTCN2022122753-appb-000053
实施例3、抗CD3抗体的SEC检测结果
对实施例2选择的抗CD3抗体进行SEC-HPLC纯度检测。
SEC-HPLC纯度采用安装了SEC色谱柱(XBridge BEH SEC
Figure PCTCN2022122753-appb-000054
7.8*300mm，3.5μm)的HPLC(Agilent 1260)进行检测。流动相组成为50mM磷酸盐，300mM氯化钠，pH 6.8±0.2。采用峰面积归一化对结果进行定量分析。分别计算单体、聚体和片段 的峰面积百分比。具体测试结果见表6。
由表6看出，抗体表达的质量较好，其中JSCD3-1、JSCD3-8、JSCD3-11、JSCD3-13、JSCD3-88、JSCD3-89、JSCD3-98、JSCD3-103、JSCD3-111、JSCD3-113、JSCD3-114、JSCD3-115、JSCD3-117、JSCD3-122、JSCD3-124、JSCD3-125、JSCD3-126、JSCD3-127、JSCD3-130、JSCD3-132、JSCD3-236、JSCD3-238、JSCD3-239、JSCD3-241、JSCD3-246、JSCD3-250、JSCD3-257、JSCD3-258、JSCD3-260、JSCD3-262、JSCD3-264、JSCD3-265、JSCD3-285、JSCD3-288、JSCD3-293、JSCD3-296、JSCD3-297、JSCD3-301、JSCD3-303表达纯化后的抗体质量优异。
表6：SEC纯度测试结果
样品名称 聚体(％) 单体(％) 片段(％)
JSCD3-1 6.9 75.8 17.2
JSCD3-3 9.3 66.8 23.8
JSCD3-8 3.1 94.5 2.4
JSCD3-11 2.9 95.1 2
JSCD3-13 5.4 74.8 19.8
JSCD3-79 7.3 69 23.7
JSCD3-88 2.2 78.8 19.1
JSCD3-89 2.9 75 22.1
JSCD3-91 5.4 64.3 30.2
JSCD3-98 4.5 74.1 21.4
JSCD3-103 2.3 87.1 10.5
JSCD3-111 2.4 85.1 12.5
JSCD3-112 15.2 48.2 36.6
JSCD3-113 3.2 84.4 12.4
JSCD3-114 3 87.9 9.1
JSCD3-115 4.5 80.7 14.8
JSCD3-117 3.8 72.4 23.8
JSCD3-122 4.2 86.9 8.9
JSCD3-124 1.9 89.1 9
JSCD3-125 1.4 90.9 7.7
JSCD3-126 4.9 79.2 15.9
JSCD3-127 4.2 79.2 16.6
JSCD3-130 1.9 85.9 12.1
JSCD3-131 16.1 47.6 36.2
JSCD3-132 2.3 83.3 14.4
JSCD3-236 2.1 86.3 11.6
JSCD3-238 3.5 84.4 12
JSCD3-239 2.6 87.7 9.7
JSCD3-241 5 75.4 19.6
JSCD3-246 3.7 83.1 13.2
JSCD3-250 2.2 85.5 12.2
JSCD3-257 1.3 87.2 11.5
JSCD3-258 0.9 87.7 11.5
JSCD3-260 1.5 88.1 10.4
JSCD3-262 1.5 80.5 18
JSCD3-264 3.7 70.3 25.9
JSCD3-265 0.2 85 14.9
JSCD3-285 1.6 97 1.4
JSCD3-288 3.6 81.5 14.9
JSCD3-293 3.5 87.2 9.3
JSCD3-296 1 90.4 8.6
JSCD3-297 1.9 86.2 11.9
JSCD3-301 2 82.9 15.2
JSCD3-302 9 45.5 45.5
JSCD3-303 1.3 82.3 16.4
实施例4、抗CD3抗体与人CD3的结合活性进一步研究
对实施例2选择的抗CD3抗体与JSCD3-A1、JSCD3-A2、JSCD3-Roche、JSCD3-AMG、REGN-CD3、JS202-JSCD3分为12组进行活性测试，具体分组如下：
组一：JSCD3-1、JSCD3-3、JSCD3-8、JSCD3-11、JSCD3-13、JSCD3-79、REGN-CD3、JS202-JSCD3；
组二：JSCD3-88、JSCD3-89、JSCD3-91、JSCD3-257、JSCD3-258、JSCD3-260、REGN-CD3、JS202-JSCD3；
组三：JSCD3-98、JSCD3-103、JSCD3-111、JSCD3-112、JSCD3-113、JSCD3-114、REGN-CD3、JS202-JSCD3；
组四：JSCD3-115、JSCD3-117、JSCD3-122、JSCD3-124、JSCD3-125、JSCD3-126、REGN-CD3、JS202-JSCD3；
组五：JSCD3-127、JSCD3-130、JSCD3-131、JSCD3-132、JSCD3-Roche、JSCD3-AMG、 REGN-CD3、JS202-JSCD3；
组六：JSCD3-236、JSCD3-238、JSCD3-239、JSCD3-241、JSCD3-246、JSCD3-250、REGN-CD3、JS202-JSCD3；
组七：JSCD3-262、JSCD3-264、JSCD3-265、JSCD3-285、JSCD3-288、JSCD3-293、REGN-CD3、JS202-JSCD3；
组八：JSCD3-296、JSCD3-297、JSCD3-301、JSCD3-302、REGN-CD3、JS202-JSCD3；
组九：JSCD3-92、JSCD3-95、JSCD3-303、REGN-CD3、JS202-JSCD3；
组十：JSCD3-A1、JSCD3-A2、JS202-JSCD3；
组十一：JSCD3-5、JSCD3-162、JSCD3-173、JSCD3-191、JSCD3-195、JSCD3-210、JSCD3-217、JSCD3-268、JSCD3-324、JSCD3-325、REGN-CD3、JS202-JSCD3；
组十二：JSCD3-93、JSCD3-94、JSCD3-255、JSCD3-259、JSCD3-277、REGN-CD3、JS202-JSCD3。
其中，JSCD3-Roche的重链氨基酸序列如SEQ ID NO：158所示，轻链氨基酸序列如SEQ ID NO：159所示；JSCD3-AMG的重链氨基酸序列如SEQ ID NO：160所示，轻链氨基酸序列如SEQ ID NO：161所示；REGN-CD3的重链氨基酸序列如SEQ ID NO：162所示，轻链氨基酸序列如SEQ ID NO：163所示；JSCD3-Roche、JSCD3-AMG和REGN-CD3的制备如实施例1中抗CD3抗体的制备方法所示。
将上述分组后的抗CD3抗体分别稀释不同的浓度梯度拟合曲线进一步的进行与人CD3的结合活性测试，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抗CD3抗体在4℃孵育30min，洗去残留抗体后与荧光二抗(PE-抗人IgG)孵育，最后用C6PLUS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。
具体测试结果见表7-17和图1-12，CDR突变后的抗CD3抗体与人CD3的结合活性均有不同程度的降低，其中JSCD3-130，JSCD3-132，JSCD3-238，JSCD3-285，JSCD3-191，JSCD3-195相比较于JSCD3亲和力下降约10倍，和REGN-CD3亲和力相当；JSCD3-A2相较于JSCD3亲和力下降约70倍；JSCD3-259相较于JSCD3亲和力下降约100倍；JSCD3-277相较于JSCD3亲和力下降约500倍。其中TOP代表抗体浓度饱和时荧光信号值(MFI-PE)，相对结合活性vs JSCD3(％)＝JSCD3 EC50值/样品EC50×100％，相对结合活性vs REGN-CD3(％)＝REGN-CD3 EC50值/样品EC50×100％。
表7：组一的抗体与人CD3的结合活性测试结果
Figure PCTCN2022122753-appb-000055
表8：组二的抗体与人CD3的结合活性测试结果
Figure PCTCN2022122753-appb-000056
表9：组三的抗体与人CD3的结合活性测试结果
Figure PCTCN2022122753-appb-000057
Figure PCTCN2022122753-appb-000058
表10：组四的抗体与人CD3的结合活性测试结果
Figure PCTCN2022122753-appb-000059
表11：组五的抗体与人CD3的结合活性测试结果
Figure PCTCN2022122753-appb-000060
表12：组六的抗体与人CD3的结合活性测试结果
Figure PCTCN2022122753-appb-000061
Figure PCTCN2022122753-appb-000062
表13：组七的抗体与人CD3的结合活性测试结果
Figure PCTCN2022122753-appb-000063
表14：组八的抗体与人CD3的结合活性测试结果
Figure PCTCN2022122753-appb-000064
表15：组九的抗体与人CD3的结合活性测试结果
Figure PCTCN2022122753-appb-000065
Figure PCTCN2022122753-appb-000066
表16：组十一的抗体与人CD3的结合活性测试结果
Figure PCTCN2022122753-appb-000067
表17：组十二的抗体与人CD3的结合活性测试结果
Figure PCTCN2022122753-appb-000068
实施例5、双特异抗体分子表达载体的构建
5.1 JS202-7(又称为JS202-7JSCD3 scFv)双特异抗体的质粒构建
用NheI和NotI酶分别酶切质粒HXT4S-JS012-2B19-HC-7和HXT4s-JS CD20HC-2Mut h-HY获取载体和片段，使用T4 DNA连接酶连接，得到第一个表达载体JS012- 2B19HC-7-IgG4-hole。委托金斯瑞生物科技有限公司合成编码JSCD3 scFv的基因用引物扩增后BSPQI酶切并连接至载体HXT4S-JS012-2B19-HC-mutb得到第二个表达载体JS012-2B19HC-7-CH1-JSCD3 scFv-IgG4-knob。委托金斯瑞生物科技有限公司合成编码JS012-2B19LC-1-Kappa的基因，通过SapI酶切并连接至HXT2得到第三个表达载体JS012-2B19LC-1-kappa。
5.2 JS202-7 CD3-130-012 scFv双特异抗体的质粒构建
通过5.1节中的方法得到第一个表达载体JS012-2B19HC-7-IgG4-hole。分别以HXT4S-JS012-2B19-HC-7-mutb，HXT2-JS012-2B19LC-1和HXT4S-JSCD3-HC7为模板扩增得到载体和片段通过重组得到第二个表达载体JSCD3-HC7-CH1-012ScFv-IgG4-knob。分委托金斯瑞生物科技有限公司合成编码JS012-2B19LC-1-Kappa的基因，通过SapI酶切并连接至HXT2得到第三个表达载体JS012-2B19LC-1-kappa。分委托金斯瑞生物科技有限公司合成编码JSCD3-LC16-Lambda的基因，通过SapI酶切并连接至HXT2得到第四个表达载体JSCD3-LC16-Lambda。
5.3 JS202-7 CD3-Fv双特异抗体的质粒构建
通过5.1节中的方法得到第一个表达载体JS012-2B19HC-7-IgG4-hole。分别以HXT4S-JS012-2B19-HC-7-mutb和HXT2-JSCD3-LC为模板扩增得到载体和片段通过重组得到第二个表达载体JS012-2B19HC-7-CH1-JSCD3 LC-IgG4-knob。分别以HXT2-JS012-2B19LC-1和HXT4S-JSCD3-HC为模板PCR得到载体和片段，通过重组得到第三个表达载体JS012-2B19LC-1-Kappa-JSCD3-HC。委托金斯瑞生物科技有限公司合成编码JS012-2B19LC-1-Kappa的基因，通过SapI酶切并连接至HXT2得到第四个表达载体JS012-2B19LC-1-kappa。
5.4 JS202-7 012-Fv双特异抗体的质粒构建
通过5.1节中的方法得到第一个表达载体JS012-2B19HC-7-IgG4-hole。分别以HXT4S-JSCD3-HC和HXT2-JS012-2B19LC-1以及HXT4S-1A6H2 QGQ-mutb为模板分别PCR得到载体和两个片段，通过重组得到第二个表达载体JSCD3-VH-CH1-JS012-2B19LC-1-IgG4-knob。分别以HXT3-JSCD3-LC和HXT4S-JS012-2B19-HC-7为模板PCR得到载体和片段，通过重组得到第三个表达载体JSCD3-LC-lambda-2B19HC-7。委托金斯瑞生物科技有限公司合成编码JS012-2B19LC-1-Kappa的基因，通过SapI酶切并连接至HXT2得到第四个表达载体JS012-2B19LC-1-kappa。
5.5 JS202-7 CD3 130 012-Fv双特异抗体的质粒构建
通过5.1节中的方法得到第一个表达载体JS012-2B19HC-7-IgG4-hole。分别以HXT4S-JSCD3-HC7和HXT2-JS012-2B19LC-1以及HXT4S-1A6H2 QGQ-mutb为模板分别PCR得 到载体和两个片段，通过重组得到第二个表达载体JSCD3-HC7-CH1-JS012-2B19LC-1-IgG4-knob。分别以HXT3-JSCD3-LC16和HXT4S-JS012-2B19-HC-7为模板PCR得到载体和片段，通过重组得到第三个表达载体JSCD3-LC16-lambda-2B19HC-7。委托金斯瑞生物科技有限公司合成编码JS012-2B19LC-1-Kappa的基因，通过SapI酶切并连接至HXT2得到第四个表达载体JS012-2B19LC-1-kappa。
5.6 JS203-7-JSCD3-CD20-Fv双特异抗体的质粒构建
委托金斯瑞合成人CD20的重链JSCD20-HC-2于puc57，通过BSPQI酶切并连接至HXT4S载体，得到表达载体HXT4S-JSCD20-HC-2。将质粒用HindIII和NheI酶切回收获取基因片段JSCD20-HC-2，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T4 DNA连接酶连接，得到第一个表达载体JSCD20-HC-2-IgG4-hole。分别以HX4-JSCD3 HC-7-2B19 LC-1-mutb和HXT2-JSCD20-LC-2为模板扩增载体和基因片段，通过重组得到第二个表达载体JSCD3-HC-CH1-JSCD20-LC-2-IgG4-knob。分别以HXT3-JSCD3-LC-2B19 HC-7和HXT4s-JS CD20HC-2Mut h为模板扩增载体和基因片段，通过重组得到第三个表达载体JSCD3-LC-lambda-JSCD20-HC-2。委托金斯瑞合成人CD20的轻链JSCD20-LC-2于puc57，通过SapI酶切并连接至HXT2载体，得到第四个表达载体JSCD20-LC-2-kappa。
5.7 JS203-7-CD3 130-CD20-Fv双特异抗体的质粒构建
委托金斯瑞合成人CD20的重链JSCD20-HC-2于puc57，通过SapI酶切并连接至HXT4S载体，得到表达载体HXT4S-JSCD20-HC-2。将质粒用HindIII和NheI酶切回收获取基因片段JSCD20-HC-2，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T4 DNA连接酶连接，得到第一个表达载体JSCD20-HC-2-IgG4-hole。分别以HX4-JSCD3 HC-2B19 LC-1-mutb和HXT2-JSCD20-LC-2为模板扩增载体和片段，通过重组得到第二个表达载体JSCD3-HC7-CH1-JSCD20-LC-2-IgG4-knob。分别以HXT3-JSCD3-LC16-2B19 HC-7和HXT4s-JSCD20HC-2Mut h为模板扩增载体和片段，通过重组得到第三个表达载体JSCD3-LC16-lambda-JSCD20-HC-2。委托金斯瑞合成人CD20的轻链JSCD20-LC-2于puc57，通过SapI酶切并连接至HXT2载体，得到第四个表达载体JSCD20-LC-2-kappa。
5.8 JS203-7-CD3-Fv双特异抗体的质粒构建
委托金斯瑞合成人CD20的重链JSCD20-HC-2于puc57，通过SapI酶切并连接至HXT4S载体，得到表达载体HXT4S-JSCD20-HC-2。将质粒用HindIII和NheI酶切回收获取基因片段JSCD20-HC-2，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T4 DNA连接酶连接，得到第一个表达载体JSCD20- HC-2-IgG4-hole。分别以HXT4s-JS CD20HC-2 mutb和HXT2-JSCD3-LC为模板扩增载体和片段，通过重组得到第二个表达载体JSCD20-VH-CH1-JSCD3-VL-IgG4-knob。分别以HXT2-JSCD20-LC-2和HXT2-JSCD3-HC为模板扩增载体和片段，通过重组得到第三个表达载体JSCD20-LC-2 Kappa-JSCD3-VH。委托金斯瑞合成人CD20的轻链JSCD20-LC-2于puc57，通过SapI酶切并连接至HXT2载体，得到第四个表达载体JSCD20-LC-2-kappa。
实施例6、双特异性抗体分子的表达与纯化
6.1双特异性抗体分子的表达
将实施例5中构建的每个双特异性抗体各自的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。
6.2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，即得到双特异性抗体。
双特异性抗体JS202-7中，第一多肽链(表达载体JS012-2B19HC-7-CH1-JSCD3 scFv-IgG4-knob)氨基酸序列如SEQ ID NO：141所示，第二多肽链(表达载体JS012-2B19HC-7-IgG4-hole)氨基酸序列如SEQ ID NO：142所示，第三多肽链(表达载体JS012-2B19LC-1-kappa)氨基酸序列如SEQ ID NO：143所示。双特异性抗体JS202-7的结构示意图如图 13所示。
SEQ ID NO：141(黑体字分别表示VL和VH，下划线标出LCDR1-LCDR3和HCDR1-HCDR3)
Figure PCTCN2022122753-appb-000069
其中JSCD3 scFv的VH氨基酸序列如SEQ ID NO：137所示，VL氨基酸序列如SEQ ID NO：138所示。
SEQ ID NO：142(黑体字分别表示VH，下划线标出HCDR1-HCDR3)
Figure PCTCN2022122753-appb-000070
SEQ ID NO：143(黑体字分别表示VL，下划线标出LCDR1-LCDR3)
Figure PCTCN2022122753-appb-000071
双特异性抗体JS202-7 CD3-Fv中，第一多肽链(表达载体JS012-2B19HC-7-CH1-JSCD3 LC-IgG4-knob)氨基酸序列如SEQ ID NO：144所示，第二多肽链(表达载体JS012-2B19LC-1-Kappa-JSCD3-HC)氨基酸序列如SEQ ID NO：145所示，第三多肽链(表达载体JS012-2B19HC-7-IgG4-hole)氨基酸序列如SEQ ID NO：142所示，第四多肽链(表达载体JS012-2B19LC-1-kappa)氨基酸序列如SEQ ID NO：143所示。双特异性抗体JS202-7CD3-Fv的结构示意图如图14所示。
SEQ ID NO：144(黑体字分别表示VL和VH，下划线标出LCDR1-LCDR3和HCDR1-HCDR3)
Figure PCTCN2022122753-appb-000072
SEQ ID NO：145(黑体字分别表示VL和VH，下划线标出LCDR1-LCDR3和HCDR1-HCDR3)
Figure PCTCN2022122753-appb-000073
双特异性抗体JS202-7 012-Fv中，第一多肽链(表达载体JSCD3-VH-CH1-JS012-2B19LC-1-IgG4-knob)氨基酸序列如SEQ ID NO：146所示，第二多肽链(表达载体JSCD3-LC-lambda-2B19HC-7)氨基酸序列如SEQ ID NO：147所示，第三多肽链(表达载体JS012-2B19HC-7-IgG4-hole)氨基酸序列如SEQ ID NO：142所示，第四多肽链(表达载体JS012- 2B19LC-1-kappa)氨基酸序列如SEQ ID NO：143所示。双特异性抗体JS202-7 012-Fv的结构示意图如图15所示。
SEQ ID NO：146(黑体字分别表示VL和VH，下划线标出LCDR1-LCDR3和HCDR1-HCDR3)
Figure PCTCN2022122753-appb-000074
SEQ ID NO：147(黑体字分别表示VL和VH，下划线标出LCDR1-LCDR3和HCDR1-HCDR3)
Figure PCTCN2022122753-appb-000075
双特异性抗体JS202-7 CD3 130 012-Fv中，第一多肽链(表达载体JSCD3-HC7-CH1-JS012-2B19LC-1-IgG4-knob)氨基酸序列如SEQ ID NO：148所示，第二多肽链(表达载体JSCD3-LC16-lambda-2B19HC-7)氨基酸序列如SEQ ID NO：149所示，第三多肽链(表达载体JS012-2B19HC-7-IgG4-hole)氨基酸序列如SEQ ID NO：142所示，第四多肽链(表达载体JS012-2B19LC-1-kappa)氨基酸序列如SEQ ID NO：143所示。双特异性抗体JS202-7CD3 130 012-Fv的结构示意图如图15所示。
SEQ ID NO：148(黑体字分别表示VL和VH，下划线标出LCDR1-LCDR3和HCDR1-HCDR3)
Figure PCTCN2022122753-appb-000076
SEQ ID NO：149(黑体字分别表示VL和VH，下划线标出LCDR1-LCDR3和HCDR1-HCDR3)
Figure PCTCN2022122753-appb-000077
双特异性抗体JS203-7-JSCD3-CD20-Fv中，第一多肽链(表达载体JSCD3-HC-CH1-JSCD20-LC-2-IgG4-knob)氨基酸序列如SEQ ID NO：150所示，第二多肽链(表达载体JSCD3-LC-lambda-JSCD20-HC-2)氨基酸序列如SEQ ID NO：151所示，第三多肽链(表达载体JSCD20-HC-2-IgG4-hole)氨基酸序列如SEQ ID NO：152所示，第四多肽链(表达载体JSCD20-LC-2-kappa)氨基酸序列如SEQ ID NO：153所示。双特异性抗体JS203-7-JSCD3-CD20-Fv的结构示意图如图16所示。
SEQ ID NO：150(黑体字分别表示VL和VH，下划线标出LCDR1-LCDR3和HCDR1-HCDR3)
Figure PCTCN2022122753-appb-000078
Figure PCTCN2022122753-appb-000079
SEQ ID NO：151(黑体字分别表示VL和VH，下划线标出LCDR1-LCDR3和HCDR1-HCDR3)
Figure PCTCN2022122753-appb-000080
SEQ ID NO：152(黑体字分别表示VH，下划线标出HCDR1-HCDR3)
Figure PCTCN2022122753-appb-000081
SEQ ID NO：153(黑体字分别表示VL，下划线标出LCDR1-LCDR3)
Figure PCTCN2022122753-appb-000082
双特异性抗体JS203-7-CD3 130-CD20-Fv中，第一多肽链(表达载体JSCD3-HC7-CH1-JSCD20-LC-2-IgG4-knob)氨基酸序列如SEQ ID NO：154所示，第二多肽链(表达载体 JSCD3-LC16-1ambda-JSCD20-HC-2)氨基酸序列如SEQ ID NO：155所示，第三多肽链(表达载体JSCD20-HC-2-IgG4-hole)氨基酸序列如SEQ ID NO：152所示，第四多肽链(表达载体JSCD20-LC-2-kappa)氨基酸序列如SEQ ID NO：153所示。双特异性抗体JS203-7-CD3 130-CD20-Fv的结构示意图如图16所示。
SEQ ID NO：154(黑体字分别表示VL和VH，下划线标出LCDR1-LCDR3和HCDR1-HCDR3)
Figure PCTCN2022122753-appb-000083
SEQ ID NO：155(黑体字分别表示VL和VH，下划线标出LCDR1-LCDR3和HCDR1-HCDR3)
Figure PCTCN2022122753-appb-000084
双特异性抗体JS203-7-CD3-FV中，第一多肽链(表达载体JSCD20-VH-CH1-JSCD3-VL-IgG4-knob)氨基酸序列如SEQ ID NO：156所示，第二多肽链(表达载体JSCD20-LC-2Kappa-JSCD3-VH)氨基酸序列如SEQ ID NO：157所示，第三多肽链(表达载体JSCD20-HC-2-IgG4-hole)氨基酸序列如SEQ ID NO：152所示，第四多肽链(表达载体JSCD20-LC-2-kappa)氨基酸序列如SEQ ID NO：153所示。双特异性抗体JS203-7-CD3-FV的结构示意图如图17所示。
SEQ ID NO：156(黑体字分别表示VL和VH，下划线标出LCDR1-LCDR3和HCDR1-HCDR3)
Figure PCTCN2022122753-appb-000085
SEQ ID NO：157(黑体字分别表示VL和VH，下划线标出LCDR1-LCDR3和HCDR1-HCDR3)
Figure PCTCN2022122753-appb-000086
双特异性抗体JS202-7 CD3-130-012 scFv中，第一多肽链(表达载体JSCD3-HC7-CH1-012ScFv-IgG4-knob)氨基酸序列如SEQ ID NO：164所示，第二多肽链(表达载体JSCD3-LC16-Lambda)氨基酸序列如SEQ ID NO：165所示，第三多肽链(表达载体JS012-2B19HC-7-IgG4-hole)氨基酸序列如SEQ ID NO：142所示，第四多肽链(表达载体JS012-2B19LC-1-kappa)氨基酸序列如SEQ ID NO：143所示。双特异性抗体JS202-7 CD3-130-012 scFv的结构示意图如图18所示。
SEQ ID NO：164(黑体字分别表示VL和VH，下划线标出LCDR1-LCDR3和HCDR1-HCDR3)
Figure PCTCN2022122753-appb-000087
Figure PCTCN2022122753-appb-000088
SEQ ID NO：165(黑体字分别表示VL，下划线标出LCDR1-LCDR3和HCDR1-HCDR3)
Figure PCTCN2022122753-appb-000089
其中012 scFv的VH氨基酸序列如SEQ ID NO：166所示，VL氨基酸序列如SEQ ID NO：167所示。
实施例7、双特异性抗体的结合活性研究
7.1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抗CD3×Claudin18.2双特异性抗体(JS202-7 CD3-Fv、JS202-7 012-Fv、JS202-7 CD3 130 012-Fv和JS202-7)在4℃孵育30min，洗去残留抗体后与荧光二抗(PE-抗人IgG)孵育，最后用C6PLUS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19)。
如图19，JS202-7 012-Fv和JS202-7 CD3 130 012-Fv与人CD3表达细胞均能有效结合，JS202-7 CD3-Fv和JS202-7与人CD3表达细胞的结合能力较弱。
7.2双特异性抗体与食蟹猴CD3e的结合
将CHO食蟹猴CD3e(君实生物)分别与梯度稀释成系列浓度的抗CD3×Claudin18.2双特异性抗体(JS202-7 CD3-Fv、JS202-7 012-Fv、JS202-7 CD3 130 012-Fv和JS202-7)及阴性对照(抗KLH单抗，anti-KLHhIgG4)在4℃孵育30min，洗去残留抗体后与荧光 二抗(PE-抗人IgG)孵育，最后用C6 PLUS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20)。
如图20，JS202-7 CD3-Fv、JS202-7 012-Fv、JS202-7 CD3 130 012-Fv及JS202-7与CHO食蟹猴CD3e细胞均能有效结合，JS202-7 CD3-Fv和JS202-7结合TOP值较低，固认为JS202-7 CD3-Fv和JS202-7与CHO食蟹猴CD3e细胞的结合能力有所减低。
7.3双特异性抗体与CHO Claudin18.2的结合
将CHO Claudin18.2(君实生物)细胞分别分别与梯度稀释成系列浓度的抗CD3×Claudin18.2双特异性抗体(JS202-7 CD3-Fv、JS202-7 012-Fv、JS202-7 CD3 130 012-Fv和JS202-7)在4℃孵育30min，洗去残留抗体后与荧光二抗(PE-抗人IgG)孵育，最后用C6PLUS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21)。
如图21，JS202-7 CD3-Fv、JS202-7 012-Fv、JS202-7 CD3 130 012-Fv及JS202-7与CHO Claudin18.2细胞均能有效结合，EC50分别为160.6ng/mL、169.8ng/mL、158.9ng/mL和138.7ng/mL。
实施例8、双特异性抗体的体外药效研究
8.1双特异性抗体对T细胞下游信号通路的激活作用
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CHO细胞为靶细胞，通过报告基因系统检测双抗活性。
将梯度稀释成系列浓度的抗CD3×Claudin18.2双特异性抗体(JS202-7 CD3-Fv、JS202-7 012-Fv、JS202-7 CD3 130 012-Fv和JS202-7)与靶细胞CHO Claudin18.2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-Lite TM Luciferase Assay system(Vazyme，Cat#DD1203-04)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22)。
如图22所示，JS202-7 CD3-Fv、JS202-7 012-Fv、JS202-7 CD3 130 012-Fv及JS202-7双抗均能有效激活T细胞下游信号通路，EC50分别为0.7212ng/mL、0.1700ng/mL、1.551ng/mL和2.850ng/mL。
8.2双特异性抗体对T细胞的激活作用
抗CD3×Claudin18.2双特异性抗体能够有效促进外周血单核细胞中T淋巴细胞的激活，一般细胞表面标志物CD69的表达上调是T细胞激活早期的标志，而细胞表面标志物 CD25的表达上调是T细胞激活后期的标志，本实验用CD25和CD69双阳性群体的比例上调来评估本研究中抗CD3×Claudin18.2双特异性抗体对T细胞的激活作用。
用人总T细胞分选试剂盒(Miltenyi Biotec，Cat#130-096-535)从商业化的PBMC(Allcells，Cat#PB004F-C)中分离纯化出人总T细胞；然后将纯化的人总T细胞(每孔1×105个细胞)和NUGC4Claudin18.2细胞(每孔2×104个细胞，君实生物)以及梯度稀释的JS202-7 CD3-Fv、JS202-7 012-Fv、JS202-7 CD3 130 012-Fv和JS202-7及阴性对照样品anti-KLHhIgG4，在96孔板中37℃共孵育24h(24小时)。最后收集细胞，用PerCP/Cy5.5抗人CD8a(Biolegend，Cat#301032)、PE抗人CD25(Biolegend，Cat#302606)、BV421抗人CD69(BD，Cat#562884)染色后，用流式细胞仪(BD，CantoII)上机检测。利用FlowJo软件导出CD8+T细胞上CD25+和CD69+双阳性细胞群体的比例，再通过GraphPadprism软件拟合四参数回归曲线，计算EC50值。
如图23所示，本发明的抗CD3×Claudin18.2双特异性抗体JS202-7 CD3-Fv、JS202-7 012-Fv、JS202-7 CD3 130 012-Fv和JS202-7都能够有效促进CD8T淋巴细胞中CD25+和CD69+双阳性细胞群体比例的增加，促进T细胞激活，EC50分别为1.323ng/mL、1.664ng/mL、127.5ng/mL和3.300ng/mL。
实施例9、双特异性抗体的CRS研究
细胞因子释放综合征(CRS)是由于免疫效应细胞和肿瘤细胞的交联导致大量的炎症因子释放。细胞因子的来源包括靶细胞本身和被募集至肿瘤部位的免疫细胞。这种连锁反应会导致免疫细胞的过度活化，导致更多的细胞因子活化，如肿瘤坏死因子(TNF)和干扰素γ(IFNγ)，其次还有白细胞介素IL-6、IL-10、IL-8和IL-2等。本实验分别在不同供体中检测了不同浓度的抗CD3×Claudin18.2双特异性抗体(溶液形式)与PBMC共孵育24小时之后，检测Th1和Th2细胞因子的释放水平，来评估抗CD3×Claudin18.2双特异性抗体是否能够引起细胞因子释放综合征。
实验检测溶液形式的JS202-7 CD3-Fv、JS202-7 012-Fv、JS202-7 CD3 130 012-Fv和JS202-7及阳性对照抗体OKT3以及阴性对照抗体anti-KLHhIgG4均以10μg/ml在体外水平与商业化的PBMC(Allcells，Cat#PB004F-C)孵育，PBMC的总量为3.0×10 5细胞/孔，该实验在2个不同donor(ID：LP200810、PCH20210100021)的PBMC中分别开展。细胞与抗体在37℃，5％CO 2条件下孵育24h，第二天离心收集上清用BD CBA human Th1/Th2 cytokine kit II检测细胞因子释放量。
如图24所示，PBMC与溶液形式的抗体共孵育，阳性对照为OKT3(可从登录号ATCC CRL 8001保藏于美国典型培养物保藏中心的杂交瘤获得)，实验结果表明JS202-7 012-Fv 和JS202-7 CD3 130 012-Fv能够明显降低引起细胞因子释放综合征的风险，且JS202-7 CD3 130 012-Fv不会引起细胞因子释放综合征，JS202-7 CD3-Fv和JS202-7均会引起细胞因子释放综合征。
实施例10、双特异性抗体对小鼠MC38hClaudin18.2细胞皮下移植瘤生长的抑制
取7-9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 hClaudin18.2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117mm 3时按肿瘤体积随机分组，每组6只，共6组，分别为：G1生理盐水(生理盐水)阴性对照组，G2 JS202-7(0.1mg/kg)组，G3 JS202-7(0.3mg/kg)组，G4 JS202-7 CD3 130 012-FV(0.1mg/kg)组，G5 JS202-7 CD3 130 012-FV(0.3mg/kg)组和G6 JS202-7 CD3 130 012-FV(3mg/kg)组。通过腹腔注射给药，每周给药2次，给药体积为10μL/g，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25所示，在动物给药后第17天，生理盐水组平均肿瘤体积为2066±422mm 3，JS202-7(0.1mg/kg)组和JS202-7(0.3mg/kg)组平均肿瘤体积分别为1071±119mm 3和994±196mm 3，与生理盐水组相比，TGI分别为51.0％和55.0％，显著抑制肿瘤生长。G4 JS202-7 CD3 130 012-FV(0.1mg/kg)组，G5 JS202-7 CD3 130 012-FV(0.3mg/kg)组和G6 JS202-7 CD3 130 012-FV(3mg/kg)组平均肿瘤体积分别为1098±195mm 3，854±83mm 3和783±179mm 3，与生理盐水组相比，TGI分别为49.7％，62.2％和65.8％，显著抑制肿瘤生长。
结果表明JS202-7 CD3 130 012-FV能够体内抑制B-hCD3e人源化小鼠MC38 hClaudin18.2细胞皮下移植瘤的生长。
实施例11、双特异性抗体的稳定性
11.1双抗结构的热稳定性
测试目的：
在pH6.0的缓冲体系(20mM组氨酸盐酸组氨酸+230mM蔗糖)中，利用nanoDSF(差示扫描荧光技术)检测JS202-7双抗结构的热稳定性。测试双抗为：JS202-7 CD3 130 012-Fv、JS202-7 CD3 132 012-Fv、JS202-7 CD3 238 012-Fv、JS202-7 CD3 285 012-Fv，其中 JS202-7 CD3 132 012-Fv、JS202-7 CD3 238 012-Fv、JS202-7 CD3 285 012-Fv与JS202-7 CD3 130 012-Fv相比，仅CD3序列不同；将JS202-7 CD3 130 012-Fv中CD3的VH/VL序列分别替换为JSCD3-130、JSCD3-132、JSCD3-238、JSCD3-285的VH/VL序列，即可得到JS202-7 CD3 132 012-Fv、JS202-7 CD3 238 012-Fv、JS202-7 CD3 285 012-Fv。
测试过程及结果：
将双抗JS202-7样品置换到上述的缓冲体系中，控制样品浓度在10mg/ml左右，利用DSF进行检测。结果见表18，JS202-7双抗结构表现了良好的热稳定性。
表18：双特异性抗体的热稳定性
样品 Tm1(℃) Tm2(℃)
JS202-7 CD3 130 012-Fv 63.7 79.1
JS202-7 CD3 132 012-Fv 63.8 79.4
JS202-7 CD3 238 012-Fv 68.1 79.5
JS202-7 CD3 285 012-Fv 67.8 79.1
注：Tm为热转化温度。
11.2双抗结构的长期稳定性
测试目的：
检测JS202-7双抗结构长期条件下稳定性。测试双抗为：JS202-7 CD3 130 012-Fv、JS202-7 CD3 132 012-Fv、JS202-7 CD3 238 012-Fv、JS202-7 CD3 285 012-Fv，其中JS202-7 CD3 132 012-Fv、JS202-7 CD3 238 012-Fv、JS202-7 CD3 285 012-Fv与JS202-7 CD3 130 012-Fv相比，仅CD3序列不同；将JS202-7 CD3 130 012-Fv中CD3的VH/VL序列分别替换为JSCD3-130、JSCD3-132、JSCD3-238、JSCD3-285的VH/VL序列，即可得到JS202-7 CD3 132 012-Fv、JS202-7 CD3 238 012-Fv、JS202-7 CD3 285 012-Fv。
SEC纯度检测方法：
采用安装了
Figure PCTCN2022122753-appb-000090
BEH
Figure PCTCN2022122753-appb-000091
SEC色谱柱的高效液相色谱仪检测各组分样品SEC纯度。流动相为50mM PB-300mM Na 2SO 4(pH 6.8±0.2)，流速为0.5mL/min，检测波长为280nm，供试品进样100μg，使用高效液相色谱仪检测。采用面积归一化进行定量分析，以单体的峰面积百分比作为样品的纯度，聚体和片段的峰面积百分比为聚体和片段的含量。
CE-SDS还原检测方法(r-CE-SDS)：
采用安装了CE-SDS卡盒的毛细管电泳仪(Maurice仪器)检测各组分样品CE-SDS 还原纯度。供试品溶液(1mg/mL)95μl加入2-巯基乙醇5μL，70℃孵育变性热处理15min，使用毛细管电泳仪(Maurice)检测。按面积归一化法计算，以轻链和重链的修正峰面积分别占所有修正峰面积之和的百分比分别计算轻链、重链1和重链2纯度，三者之和即为样品的总纯度。
CE-SDS非还原检测方法(nr-CE-SDS)：
采用安装了CE-SDS卡盒的毛细管电泳仪(Maurice仪器)检测各组分样品CE-SDS非还原纯度。供试品溶液(1mg/mL)95μL加入0.25M N-乙马来酰亚胺5μL，70℃孵育变性热处理5min，使用毛细管电泳仪(Maurice)检测。按面积归一化法计算，以主峰的修正峰面积占所有修正峰面积之和的百分比计算纯度。
icIEF检测方法：
采用安装了icIEF卡盒的毛细管电泳仪(Maurice仪器)检测各组分样品等电点。供试品经iCIEF混合液稀释处理后，使用毛细管电泳仪(Maurice)检测。不同等电点组分被聚焦在不同位置而达到聚焦和分离的效果。
结合ELISA实验(与Human CD3E结合)检测方法：
本实验均在37℃条件下进行，使用Thermo Scientific的酶标仪，用固定浓度的His标签human CD3E(1.0μg/mL)包板90min，洗板后用2％BSA封闭90min，加入梯度稀释的JS202样品(1μg/mL起始，3倍梯度稀释至0.005645ng/mL，共12个浓度)孵育60min，洗板后加入稀释5000倍的Mouse Anti-Human IgG4 Fc-HRP作为检测抗体孵育60min进行检测，然后用0.1mg/mL TMB显色15min，最后用2M HCl终止反应，在450nm/620nm下读板。使用GraphPad Prism软件中四参数对数回归(4PL)模型拟合及获取相应的EC50。
细胞活性检测方法：
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CHO细胞为靶细胞，通过报告基因系统检测双抗活性。
测试过程及结果：
将JS202-7双抗置换到pH6.0的缓冲体系(20mM组氨酸盐酸组氨酸+230mM蔗糖)中， 控制样品浓度在10mg/ml左右，分装一定体积(1mL/管)的JS202-7双抗样品放置于5±3℃医用冷藏箱，考察0W、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JS202-7双抗具有良好的长期稳定性，具体见表19。
表19：双特异性抗体的长期稳定性
Figure PCTCN2022122753-appb-000092
注：“/”表示未检测。
实施例12、双特异性抗体的活性研究
12.1 CD3×CD20双抗与CHO-hCD3细胞的结合研究
将CHO-hCD3细胞(在CHO细胞上面过表达了人CD3蛋白)与不同浓度的双特异性抗体JS203-7-JSCD3-CD20-Fv和JS203-7-CD3 130-CD20-Fv及对照抗体(起始浓度为100μg/ml，3倍稀释，共10个浓度梯度)在4℃孵育30min，然后洗涤并与荧光标记的二抗在4℃条件下避光孵育30分钟。最后用流式细胞仪(BD CantoII)收集细胞，检测细胞表面结合的荧光抗体。用FlowJo分析原始数据得到MFI值，并通过GraphPad拟合抗体剂量依赖性的结合曲线(图26)并计算EC50，阳性对照为REGN1979(Regeneron Pharmaceuticals)。
如图26所示，REGN1979、JS203-7-JSCD3-CD20-Fv和JS203-7-CD3 130-CD20-Fv能够与细胞表面的人CD3结合，EC50分别为0.97μg/mL、0.09198和0.6511μg/mL。
12.2双特异性抗体与Raji细胞的结合
将Raji细胞(一种人B淋巴瘤细胞，CD20阳性)与不同浓度的双特异性抗体JS203-7-JSCD3-CD20-Fv和JS203-7-CD3 130-CD20-Fv及对照抗体(起始浓度为100μg/ml，3倍稀释，共10个浓度梯度)在4℃孵育30min，然后洗涤并与荧光标记的二抗在4℃条件下避光孵育30分钟。最后用流式细胞仪(BD CantoII)收集细胞，检测细胞表面结合的荧光抗体。用FlowJo分析原始数据得到MFI值，并通过GraphPad拟合抗体剂量依赖性的结合曲线(图27)并计算EC50，阳性对照为REGN1979(Regeneron Pharmaceuticals))。
如图27所示，REGN1979、JS203-7-JSCD3-CD20-Fv和JS203-7-CD3 130-CD20-Fv均能够以高亲和力与Raji细胞表面的CD20结合，EC50分别为2.251μg/mL、1.316μg/mL和1.939μg/mL。
12.3双特异性抗体在荧光素酶报告基因系统中的活性检测
将靶细胞Raji(一种人B淋巴瘤细胞，CD20阳性)和效应细胞Jurkat NFAT(稳定表达luc2P/NFAT-RE)分别按照每孔5×10 4个细胞和每孔1×10 5个细胞数加入96孔平底白板中(Corning，Cat#3917)。之后用实验缓冲液(RPMI 1640(1X)+2％FBS)将双特异性抗体JS203-7-JSCD3-CD20-Fv和JS203-7-CD3 130-CD20-Fv及对照抗体(起始浓度为1μg/ml，3倍稀释，共12个浓度梯度)加入到细胞板中，并在37℃培养箱中共孵育4-6小时。最后在细胞抗体混合体系中加入ONE-Glo荧光素酶检测试剂(Promega)并用多功能酶标仪(TECAN M1000pro)检测化学发光信号。通过GraphPad prism软件拟合四参数回归曲线，计算EC50值。
如图28所示，REGN1979、JS203-7-JSCD3-CD20-Fv和JS203-7-CD3 130-CD20-Fv在效应细胞Jurkat NFAT和靶细胞Raji细胞构成的荧光素酶报告基因系统中有很强的的激活T细胞的活性，EC50分别为10.84ng/mL、0.3967ng/mL和2.780ng/mL。
12.4双特异性抗体对T淋巴细胞的激活活性研究
抗CD3/CD20双特异性抗体能够有效促进外周血单核细胞中T淋巴细胞的激活，一般细胞表面标志物CD69的表达上调是T细胞激活早期的标志，而细胞表面标志物CD25的表达上调是T细胞激活后期的标志，本实验用CD25和CD69双阳性群体的比例上调来评估本研究中抗CD3/CD20双特异性抗体对T细胞的激活作用。
用人总T细胞纯化试剂盒(Miltenyi Biotec，Cat#130-096-535)从商业化的PBMC(Allcells，Cat#PB004F-C)中分离纯化出人总T细胞；然后将纯化的人总T细胞(每孔1X105个细胞)和Raji细胞(每孔2×10 4个细胞)以及梯度稀释的JS203-7-JSCD3-CD20-Fv和JS203-7-CD3 130-CD20-Fv及对照抗体(起始浓度为10μg/ml，5倍稀释，共12个 浓度梯度)在96孔板中37℃共孵育24h。最后收集细胞，用APC抗人CD8a(Biolegend，Cat#301049)、PE抗人CD25(Biolegend，Cat#302606)、BV421抗人CD69(BD，Cat#562884)染色后，用流式细胞仪(BD，CantoII)上机检测。利用FlowJo软件导出CD8+T细胞上CD25+CD69+双阳性细胞群体的比例，再通过GraphPad prism软件拟合四参数回归曲线，计算EC50值。
如图29所示，本发明的抗CD3/CD20双特异性抗体REGN1979、JS203-7-JSCD3-CD20-Fv和JS203-7-CD3 130-CD20-Fv都能够有效促进CD8T淋巴细胞中CD25+和CD69+双阳性细胞群体比例的增加，促进T细胞激活，EC50分别为2.583ng/mL、0.2193ng/mL和5.499ng/mL。
12.5双特异性抗体CRS研究
本研究检测溶液形式的双特异性抗体和对照抗体在体外水平与静息状态下PBMC孵育，引起细胞因子释放的排版布局。该实验在1640完全培养基(RPMI 1640 Medium+10％FBS)条件下孵育，加入50μl的PBMC(3.0×10 5细胞/孔)。在4个不同donor(LP210817011，LP210825006，LP210821004和PCH202100025)的PBMC中分别开展，抗体检测浓度为10μg/ml，每孔50μl，每个条件设3个复孔。细胞与抗体在37℃，5％CO 2条件下孵育24h，第二天离心收集上清用BD CBA human Th1/Th2 cytokine kit II检测细胞因子释放量。
如图30所示，静息状态下PBMC与溶液形式的抗体共孵育，阳性对照为OKT3，实验结果表明，双抗分子以溶液的形式会诱导人PBMC大量释放细胞因子，特别是IL-6和IFN-γ。这可能是因为在反应体系中，有靶细胞CD20+B细胞的存在，因此引起了很强的T细胞激活和多个细胞因子的释放，故在后续临床试验中，需要额外关注继发于药理作用的细胞因子释放风险，做好细胞因子风暴的预防措施，另外JS203-7-JSCD3-CD20-Fv和JS203-7-CD3 130-CD20-Fv的CRS与阳性对照OKT3相比较低，与REGN1979相当。

Claims (20)

  1. 一种双特异性抗体，其包含第一结合结构域、第二结合结构域和第三结合结构域，其中所述第一结合结构域与CD3结合，所述第二结合结构域和所述第三结合结构域与肿瘤相关抗原结合，所述第一结合结构域具有Fab分子的形式，所述第二结合结构域具有Fv分子或scFv分子的形式，和所述第三结合结构域具有Fab分子的形式。
  2. 如权利要求1所述的双特异性抗体，其中，所述肿瘤相关抗原选自BCMA、CD19、CD20、CD22、CD30、CD33、CD38、CD40、CD123、CDK4、CEA、Claudin 18.2、EGFR、ROR1、TGF-β和5T4；优选地，所述肿瘤相关抗原是Claudin 18.2、CD20或BCMA。
  3. 如权利要求1-2中任一项所述的双特异性抗体，其具有四条多肽链，其中所述第一多肽链自N端到C端具有VH1-CH1-连接肽-VL2-连接肽-铰链区-第一Fc区，所述第二多肽链自N端到C端具有VL1-CL-连接肽-VH2；所述第三多肽链自N端到C端具有VH2-CH1-铰链区-第二Fc区，所述第四多肽链自N端到C端具有VL2-CL；
    优选地，所述第一多肽链的VH1-CH1与所述第二多肽链的VL1-CL形成所述第一结合结构域，所述第一多肽链的VL2与所述第二多肽链的VH2形成所述第二结合结构域，所述第三多肽链的VH2-CH1与所述第四多肽链的VL2-CL形成所述第三结合结构域。
  4. 如权利要求1-2中任一项所述的双特异性抗体，其具有四条多肽链，其中所述第一多肽链自N端到C端具有VH1-CH1-连接肽-VL2-连接肽-VH2-连接肽-铰链区-第一Fc区，所述第二多肽链自N端到C端具有VL1-CL；所述第三多肽链自N端到C端具有VH2-CH1-铰链区-第二Fc区，所述第四多肽链自N端到C端具有VL2-CL；
    优选地，所述第一多肽链的VH1-CH1与所述第二多肽链的VL1-CL形成所述第一结合结构域，所述第一多肽链的VL2-连接肽-VH2形成所述第二结合结构域，所述第三多肽链的VH2-CH1与所述第四多肽链的VL2-CL形成所述第三结合结构域。
  5. 如权利要求1-4中任一项所述的双特异性抗体，其中，所述第一结合结构域包含的HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3分别为：
    HCDR1：X1YAMN，其中，X1为T或K；
    HCDR2：RIRSKYNNYATYYAX2SVKX3，其中，X2为D或A，X3为D或G；
    HCDR3：HGX3X4GX5X6X7X8X9X10X11AY，其中，X3为N或A，X4为F或A，X5为N或A，X6为S或A，X7为Y或A，X8为V或A，X9为S或A，X10为W或A，X11为F或A；
    LCDR1：X12SSTGAVTTX13NYX14N，其中，X12为R或G，X13为S或G，X14为A或P；
    LCDR2：GTNKRAP；
    LCDR3：AX15X16X17X18X19X20X21X22，其中，X15为L或A，X16为W或A，X17为Y或A，X18为S或A，X19为N或A，X20为L或A，X21为W或A，X22为V或A；
    优选地，所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3分别为：
    HCDR1：X1YAMN，其中，X1为T或K；
    HCDR2：RIRSKYNNYATYYAX2SVKX3，其中，X2为D或A，X3为D或G；
    HCDR3：HGX3X4GX5X6X7X8X9X10X11AY，其中，X3为N，X4为F，X5为N或A，X6为S或A，X7为Y或A，X8为V或A，X9为S或A，X10为W，X11为F或A；
    LCDR1：X12SSTGAVTTX13NYX14N，其中，X12为R或G，X13为S或G，X14为A或P；
    LCDR2：GTNKRAP；
    LCDR3：AX15X16X17X18X19X20X21X22，其中，X15为L或A，X16为W，X17为Y或A，X18为S或A，X19为N或A，X20为L或A，X21为W，X22为V或A。
  6. 如权利要求5所述的双特异性抗体，其中，所述第一结合结构域包含：氨基酸序列如SEQ ID NO：1或2所示的HCDR1，氨基酸序列如SEQ ID NO：3或4所示的HCDR2，氨基酸序列如SEQ ID NO：5-22中任一项所示、优选如SEQ ID NO：5、9、10、11、15、17、18、19或20所示的HCDR3，氨基酸序列如SEQ ID NO：23或24所示的LCDR1，氨基酸序列如SEQ ID NO：25所示的LCDR2，和氨基酸序列如SEQ ID NO：26-42中任一项所示、优选如SEQ ID NO：26、28、30、33、35、36、37、39、40、41或42所示的LCDR3；
    优选地，所述HCDR1、HCDR2、HCDR3为：
    (1)氨基酸序列如SEQ ID NO：1所示的HCDR1，氨基酸序列如SEQ ID NO：3所示的HCDR2，氨基酸序列如SEQ ID NO：5所示的HCDR3；或
    (2)氨基酸序列如SEQ ID NO：1所示的HCDR1，氨基酸序列如SEQ ID NO：3所示的HCDR2，氨基酸序列如SEQ ID NO：15所示的HCDR3；或
    (3)氨基酸序列如SEQ ID NO：2所示的HCDR1，氨基酸序列如SEQ ID NO：4所示的HCDR2，氨基酸序列如SEQ ID NO：5-22中任一项所示的HCDR3；
    所述LCDR1、LCDR2、LCDR3为：
    (1)氨基酸序列如SEQ ID NO：23所示的LCDR1，氨基酸序列如SEQ ID NO：25所示的LCDR2，氨基酸序列如SEQ ID NO：26所示的LCDR3；或
    (2)氨基酸序列如SEQ ID NO：23所示的LCDR1，氨基酸序列如SEQ ID NO：25所 示的LCDR2，氨基酸序列如SEQ ID NO：33所示的LCDR3；或
    (3)氨基酸序列如SEQ ID NO：24所示的LCDR1，氨基酸序列如SEQ ID NO：25所示的LCDR2，氨基酸序列如SEQ ID NO：26-42中任一项所示的LCDR3。
  7. 如权利要求6所述的双特异性抗体，其中，所述第一结合结构域包含：
    (I)氨基酸序列如SEQ ID NO：1所示的HCDR1，氨基酸序列如SEQ ID NO：3所示的HCDR2，氨基酸序列如SEQ ID NO：15所示的HCDR3，氨基酸序列如SEQ ID NO：23所示的LCDR1，氨基酸序列如SEQ ID NO：25所示的LCDR2，氨基酸序列如SEQ ID NO：33所示的LCDR3；或
    (II)氨基酸序列如SEQ ID NO：2所示的HCDR1，氨基酸序列如SEQ ID NO：4所示的HCDR2，氨基酸序列如SEQ ID NO：5所示的HCDR3，氨基酸序列如SEQ ID NO：24所示的LCDR1，氨基酸序列如SEQ ID NO：25所示的LCDR2，氨基酸序列如SEQ ID NO：26、28、33、36或37中任一项所示的LCDR3；或
    (III)氨基酸序列如SEQ ID NO：2所示的HCDR1，氨基酸序列如SEQ ID NO：4所示的HCDR2，氨基酸序列如SEQ ID NO：9所示的HCDR3，氨基酸序列如SEQ ID NO：24所示的LCDR1，氨基酸序列如SEQ ID NO：25所示的LCDR2，氨基酸序列如SEQ ID NO：28、35、37、39或40中任一项所示的LCDR3；或
    (IV)氨基酸序列如SEQ ID NO：2所示的HCDR1，氨基酸序列如SEQ ID NO：4所示的HCDR2，氨基酸序列如SEQ ID NO：10所示的HCDR3，氨基酸序列如SEQ ID NO：24所示的LCDR1，氨基酸序列如SEQ ID NO：25所示的LCDR2，氨基酸序列如SEQ ID NO：28、33、40、41或42中任一项所示的LCDR3；或
    (V)氨基酸序列如SEQ ID NO：2所示的HCDR1，氨基酸序列如SEQ ID NO：4所示的HCDR2，氨基酸序列如SEQ ID NO：11所示的HCDR3，氨基酸序列如SEQ ID NO：24所示的LCDR1，氨基酸序列如SEQ ID NO：25所示的LCDR2，氨基酸序列如SEQ ID NO：26、28、33、35、36、37、40、41或42中任一项所示的LCDR3；或
    (VI)氨基酸序列如SEQ ID NO：2所示的HCDR1，氨基酸序列如SEQ ID NO：4所示的HCDR2，氨基酸序列如SEQ ID NO：15所示的HCDR3，氨基酸序列如SEQ ID NO：24所示的LCDR1，氨基酸序列如SEQ ID NO：25所示的LCDR2，氨基酸序列如SEQ ID NO：26或30所示的LCDR3；或
    (VII)氨基酸序列如SEQ ID NO：2所示的HCDR1，氨基酸序列如SEQ ID NO：4所示的HCDR2，氨基酸序列如SEQ ID NO：17所示的HCDR3，氨基酸序列如SEQ ID NO：24所示的LCDR1，氨基酸序列如SEQ ID NO：25所示的LCDR2，氨基酸序列如SEQ ID NO：33、35、36、37或42中任一项所示的LCDR3；或
    (VIII)氨基酸序列如SEQ ID NO：2所示的HCDR1，氨基酸序列如SEQ ID NO：4所示的HCDR2，氨基酸序列如SEQ ID NO：18所示的HCDR3，氨基酸序列如SEQ ID NO：24所示的LCDR1，氨基酸序列如SEQ ID NO：25所示的LCDR2，氨基酸序列如SEQ ID NO：28、35、36、37、39、41或42中任一项所示的LCDR3；或
    (IX)氨基酸序列如SEQ ID NO：2所示的HCDR1，氨基酸序列如SEQ ID NO：4所示的HCDR2，氨基酸序列如SEQ ID NO：20所示的HCDR3，氨基酸序列如SEQ ID NO：24所示的LCDR1，氨基酸序列如SEQ ID NO：25所示的LCDR2，氨基酸序列如SEQ ID NO：28、33、35、36、40、41或42中任一项所示的LCDR3；或
    (X)氨基酸序列如SEQ ID NO：2所示的HCDR1，氨基酸序列如SEQ ID NO：4所示的HCDR2，氨基酸序列如SEQ ID NO：9、10或19中任一项所示的HCDR3，氨基酸序列如SEQ ID NO：23所示的LCDR1，氨基酸序列如SEQ ID NO：25所示的LCDR2，氨基酸序列如SEQ ID NO：26所示的LCDR3；或
    (XI)氨基酸序列如SEQ ID NO：1所示的HCDR1、氨基酸序列如SEQ ID NO：3所示的HCDR2、氨基酸序列如SEQ ID NO：5所示的HCDR3、氨基酸序列如SEQ ID NO：23所示的LCDR1、氨基酸序列如SEQ ID NO：25所示的LCDR2、氨基酸序列如SEQ ID NO：26所示的LCDR3；或
    (XII)氨基酸序列如SEQ ID NO：2所示的HCDR1，氨基酸序列如SEQ ID NO：4所示的HCDR2，氨基酸序列如SEQ ID NO：19所示的HCDR3，氨基酸序列如SEQ ID NO：24所示的LCDR1，氨基酸序列如SEQ ID NO：25所示的LCDR2，氨基酸序列如SEQ ID NO：36所示的LCDR3。
  8. 如权利要求1所述的双特异性抗体，其中，所述第一结合结构域包含：氨基酸序列如SEQ ID NO：43-62中任一项所示的VH，和氨基酸序列如SEQ ID NO：63-81中任一项所示的VL；优选地，所述第一结合结构域包含：
    (I)氨基酸序列如SEQ ID NO：44所示的VH，和氨基酸序列如SEQ ID NO：64所示的VL；或
    (II)氨基酸序列如SEQ ID NO：45所示的VH，和氨基酸序列如SEQ ID NO：65、67、72、75或76所示的VL；或
    (III)氨基酸序列如SEQ ID NO：49所示的VH，和氨基酸序列如SEQ ID NO：67、74、76、78或79所示的VL；或
    (IV)氨基酸序列如SEQ ID NO：50所示的VH，和氨基酸序列如SEQ ID NO：67、72、79、80或81所示的VL；或
    (V)氨基酸序列如SEQ ID NO：51所示的VH，和氨基酸序列如SEQ ID NO：65、67、 72、74、75、76、79、80或81所示的VL；或
    (VI)氨基酸序列如SEQ ID NO：55所示的VH，和氨基酸序列如SEQ ID NO：65或69所示的VL；或
    (VII)氨基酸序列如SEQ ID NO：57所示的VH，和氨基酸序列如SEQ ID NO：72、74、75、76或81所示的VL；或
    (VIII)氨基酸序列如SEQ ID NO：58所示的VH，和氨基酸序列如SEQ ID NO：67、74、75、76、78、80或81所示的VL；或
    (IX)氨基酸序列如SEQ ID NO：60所示的VH，和氨基酸序列如SEQ ID NO：67、72、74、75、79、80或81所示的VL；或
    (X)氨基酸序列如SEQ ID NO：49、50或59中任一项所示的VH，和氨基酸序列如SEQ ID NO：63所示的VL；或
    (XI)氨基酸序列如SEQ ID NO：43所示的VH，和氨基酸序列如SEQ ID NO：63所示的VL；或
    (XII)氨基酸序列如SEQ ID NO：59所示的VH，和氨基酸序列如SEQ ID NO：75所示的VL。
  9. 如权利要求1-8中任一项所述的双特异性抗体，其中，所述第二结合结构域和第三结合结构域与Claudin 18.2结合，其分别包含：氨基酸序列如SEQ ID NO：121所示的HCDR1，氨基酸序列如SEQ ID NO：122所示的HCDR2，氨基酸序列如SEQ ID NO：123所示的HCDR3，氨基酸序列如SEQ ID NO：124所示的LCDR1，氨基酸序列如SEQ ID NO：125所示的LCDR2，和氨基酸序列如SEQ ID NO：126所示的LCDR3；或所述第二结合结构域和第三结合结构域与CD20结合，其分别包含：氨基酸序列如SEQ ID NO：127所示的HCDR1，氨基酸序列如SEQ ID NO：128所示的HCDR2，氨基酸序列如SEQ ID NO：129所示的HCDR3，氨基酸序列如SEQ ID NO：130所示的LCDR1，氨基酸序列如SEQ ID NO：131所示的LCDR2，和氨基酸序列如SEQ ID NO：132所示的LCDR3；
    优选地，所述第二结合结构域和第三结合结构域与Claudin 18.2结合，其分别包含：(a)氨基酸序列如SEQ ID NO：133所示的VH，和氨基酸序列如SEQ ID NO：134所示的VL；或(b)氨基酸序列如SEQ ID NO：166所示的VH，和氨基酸序列如SEQ ID NO：167所示的VL；或所述第二结合结构域和第三结合结构域与CD20结合，其分别包含：氨基酸序列如SEQ ID NO：135所示的VH，和氨基酸序列如SEQ ID NO：136所示的VL。
  10. 如权利要求3-9中任一项所述的双特异性抗体，其中，所述第一Fc区为knob-Fc，所述第二Fc区为hole-Fc；
    优选地，所述第一Fc区与所述第二Fc区之间通过铰链区的二硫键和杵臼结构连接；
    优选地，所述第一Fc区和/或第二Fc区为来自IgG的Fc区，优选为来自IgG1、IgG2、IgG3或IgG4的Fc区；
    优选地，所述第CH1为来自IgG的CH1，优选为IgG1、IgG2、IgG3或IgG4的CH1；
    优选地，所述CL为来自λ或κ轻链的CL；
    优选地，所述连接肽包含G(甘氨酸)、S(丝氨酸)和A(丙氨酸)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；进一步优选地，连接肽包括但不限于(GS)n、(GGS)n、(GGGS)n、(GGGGS)n和AS(GGGGS)n等，n为1、2、3、4、5或6。
  11. 如权利要求1所述的双特异性抗体，其具有四条多肽链，所述四条多肽链分别为：
    (1)所述第一多肽链具有如SEQ ID NO：144所示的氨基酸序列，所述第二多肽链具有如SEQ ID NO：145所示的氨基酸序列，所述第三多肽链具有如SEQ ID NO：142所示的氨基酸序列，所述第四多肽链具有如SEQ ID NO：143所示的氨基酸序列；或
    (2)所述第一多肽链具有如SEQ ID NO：146所示的氨基酸序列，所述第二多肽链具有如SEQ ID NO：147所示的氨基酸序列，所述第三多肽链具有如SEQ ID NO：142所示的氨基酸序列，所述第四多肽链具有如SEQ ID NO：143所示的氨基酸序列；或
    (3)所述第一多肽链具有如SEQ ID NO：148所示的氨基酸序列，所述第二多肽链具有如SEQ ID NO：149所示的氨基酸序列，所述第三多肽链具有如SEQ ID NO：142所示的氨基酸序列，所述第四多肽链具有如SEQ ID NO：143所示的氨基酸序列；或
    (4)所述第一多肽链具有如SEQ ID NO：150所示的氨基酸序列，所述第二多肽链具有如SEQ ID NO：151所示的氨基酸序列，所述第三多肽链具有如SEQ ID NO：152所示的氨基酸序列，所述第四多肽链具有如SEQ ID NO：153所示的氨基酸序列；或
    (5)所述第一多肽链具有如SEQ ID NO：154所示的氨基酸序列，所述第二多肽链具有如SEQ ID NO：155所示的氨基酸序列，所述第三多肽链具有如SEQ ID NO：152所示的氨基酸序列，所述第四多肽链具有如SEQ ID NO：153所示的氨基酸序列；或
    (6)所述第一多肽链具有如SEQ ID NO：156所示的氨基酸序列，所述第二多肽链具有如SEQ ID NO：157所示的氨基酸序列，所述第三多肽链具有如SEQ ID NO：152所示的氨基酸序列，所述第四多肽链具有如SEQ ID NO：153所示的氨基酸序列；或
    (7)所述第一多肽链具有如SEQ ID NO：164所示的氨基酸序列，所述第二多肽链具有如SEQ ID NO：165所示的氨基酸序列，所述第三多肽链具有如SEQ ID NO：142所示的氨基酸序列，所述第四多肽链具有如SEQ ID NO：143所示的氨基酸序列。
  12. 一种多核苷酸分子，其核苷酸序列选自：
    (1)编码权利要求1-11中任一项所述的双特异性抗体的核苷酸序列；和
    (2)如(1)所述核苷酸序列的互补序列。
  13. 一种表达载体，其包含如权利要求12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。
  14. 一种宿主细胞，其包含如权利要求12所述的多核苷酸分子，或包含如权利要求13所述的表达载体，或表达权利要求1-11中任一项所述的双特异性抗体；优选地，所述宿主细胞是真核细胞，更优选哺乳动物细胞。
  15. 一种制备如权利要求1-11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14所述的宿主细胞，使其表达所述权利要求1-11中任一项所述的双特异性抗体，并从所述宿主细胞回收所表达的双特异性抗体。
  16. 一种药物组合物，其包含权利要求1-11中任一项所述的双特异性抗体、权利要求12所述的多核苷酸分子、权利要求13所述的表达载体或权利要求14所述的宿主细胞，和药学上可接受的载体或赋形剂。
  17. 权利要求1-11中任一项所述的双特异性抗体、权利要求12所述的多核苷酸分子、权利要求13所述的表达载体、权利要求14所述的宿主细胞或权利要求16所述的药物组合物在制备用于预防或治疗癌症的药物中的用途；优选地，所述癌症与CLDN18或CD20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；或所述癌症选自急性B淋巴细胞性白血病、弥漫性大B细胞淋巴瘤、慢性淋巴细胞白血病、滤泡淋巴瘤，非霍奇金氏淋巴瘤、慢性骨髓细胞白血病和Burkitt淋巴瘤。
  18. 一种抗体，其包含结合CD3的结合结构域和结合CD20的结合结构域，其中，所述结合CD3的结合结构域包含：氨基酸序列如SEQ ID NO：1所示的HCDR1、氨基酸序列如SEQ ID NO：3所示的HCDR2、氨基酸序列如SEQ ID NO：5所示的HCDR3、氨基酸序列如SEQ ID NO：23所示的LCDR1、氨基酸序列如SEQ ID NO：25所示的LCDR2、氨基酸序列如SEQ ID NO：26所示的LCDR3；所述结合CD20的结合结构域包含：氨基酸序列如SEQ ID NO：127所示的HCDR1，氨基酸序列如SEQ ID NO：128所示的HCDR2，氨基酸序列如SEQ ID NO：129所示的HCDR3，氨基酸序列如SEQ ID NO：130所示的LCDR1，氨基酸序列如SEQ ID NO：131所示的LCDR2，和氨基酸序列如SEQ ID NO：132所示的LCDR3；
    优选地，所述结合CD3的结合结构域包含：氨基酸序列如SEQ ID NO：43所示的VH，和氨基酸序列如SEQ ID NO：63所示的VL，所述结合CD20的结合结构域包含：氨基酸 序列如SEQ ID NO：135所示的VH，和氨基酸序列如SEQ ID NO：136所示的VL。
  19. 如权利要求18所述的抗体，所述抗体的Fc区为来自IgG的Fc，例如来自IgG1、IgG2、IgG3或IgG4的Fc，和/或所述抗体的CL为来自λ或κ轻链的CL；优选地，所述Fc区包含杵臼结构。
  20. 如权利要求18或19所述的抗体，所述抗体具有四条多肽链，所述四条多肽链分别为：
    (1)具有如SEQ ID NO：150所示的氨基酸序列的第一多肽链，具有如SEQ ID NO：151所示的氨基酸序列的第二多肽链，具有如SEQ ID NO：152所示的氨基酸序列的第三多肽链，具有如SEQ ID NO：153所示的氨基酸序列的第四多肽链；或
    (2)具有如SEQ ID NO：154所示的氨基酸序列的第一多肽链，具有如SEQ ID NO：155所示的氨基酸序列的第二多肽链，具有如SEQ ID NO：152所示的氨基酸序列的第三多肽链，具有如SEQ ID NO：153所示的氨基酸序列的第四多肽链；或
    (3)具有如SEQ ID NO：156所示的氨基酸序列的第一多肽链，具有如SEQ ID NO：157所示的氨基酸序列的第二多肽链，具有如SEQ ID NO：152所示的氨基酸序列的第三多肽链，具有如SEQ ID NO：153所示的氨基酸序列的第四多肽链。
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