WO2022135536A1 - Cd3人源化抗体及其应用 - Google Patents

Cd3人源化抗体及其应用 Download PDF

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WO2022135536A1
WO2022135536A1 PCT/CN2021/140901 CN2021140901W WO2022135536A1 WO 2022135536 A1 WO2022135536 A1 WO 2022135536A1 CN 2021140901 W CN2021140901 W CN 2021140901W WO 2022135536 A1 WO2022135536 A1 WO 2022135536A1
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antigen
antibody
seq
cancer
binding
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PCT/CN2021/140901
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English (en)
French (fr)
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卫培培
葛虎
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江苏先声药业有限公司
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Publication of WO2022135536A1 publication Critical patent/WO2022135536A1/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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal 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

Definitions

  • the present disclosure relates to the field of antibodies, in particular, to CD3 humanized antibodies and applications thereof.
  • TCR-CD3 complex CD3 molecule and T cell (antigen) receptor (T cell receptor, TCR) form a TCR-CD3 complex, which plays an extremely important role in the process of T cell antigen recognition and immune response generation.
  • TCR has two polymorphic subunits (TCR ⁇ or TCR ⁇ ), the cytoplasmic region of TCR is very short, and its main function is to recognize antigens presented by binding MHC molecules.
  • CD3 molecule is an important differentiation antigen on the T cell membrane and is a characteristic marker of mature T cells. It consists of four chains of ⁇ or five chains of ⁇ .
  • CD3 ⁇ , ⁇ and ⁇ exist in two non-covalent bond forms, ⁇ and ⁇ heterodimers; the vast majority of ⁇ chains exist in the form of homodimers, namely ⁇ - ⁇ ; ⁇ and ⁇ chains It is two different spliced forms of the same gene, and only 10% exists in the form of heterodimer ( ⁇ - ⁇ ), but the expression level of human ⁇ is very low, and there is still lack of molecular level evidence in human cells to prove ⁇ Protein products and their presence in cells as transcripts.
  • Each chain of CD3 has an immunoreceptor tyrosine-based activation motif (ITAM) in the intracellular region.
  • ITAM immunoreceptor tyrosine-based activation motif
  • TCR can activate some PTKs after antigen binding, including TCR-linked p59fyn, ZAP-70 ( ⁇ associated protein-70), CD4/CD8-linked p56lck, and other src-related PTKs, which subsequently lead to tyrosine phosphorylation of various substrates , thereby transmitting downstream signals of the immune response.
  • the SP34 antibody is an antibody that specifically binds to the CD3 ⁇ subunit and is used in the development of antibody drugs and/or cell therapy.
  • SP34 antibody is a mouse-derived antibody, and the mouse antibody has strong immunogenicity. In clinical application, it will cause human anti-mouse antibody (HAMA) reaction and anti-antibody reaction (AAR), etc., resulting in a shortened half-life, easy to be cleared, treatment The effect is weakened, and it can even threaten the patient's life. Therefore, the humanization of murine CD3 ⁇ antibody SP34 is of great significance for the further development and drug development of this antibody.
  • HAMA human anti-mouse antibody
  • AAR anti-antibody reaction
  • the present disclosure provides a humanized antibody or antigen-binding fragment that specifically binds to the CD3 ⁇ subunit, a multispecific antigen-binding molecule, a nucleic acid fragment, a vector, a host cell, an immune effector cell, a preparation method, a pharmaceutical composition, Pharmaceutical uses and methods of treatment of cancer or tumors, infectious diseases or autoimmune diseases.
  • the present disclosure provides a humanized antibody or antigen-binding fragment that specifically binds to the CD3 ⁇ subunit, the antibody or antigen-binding fragment comprising:
  • (a) light chain variable region which sequentially comprises LFR1, LCDR1, LFR2, LCDR2, LFR3, LCDR3 and LFR4, and the amino acid sequences thereof are respectively as follows: QTVVTQEPSLTVSPGGTVTLTC (SEQ ID NO: 54), RSSTGAVTTSNYAN (SEQ ID NO: 55 ), WFQQKPGQAPRALIY (SEQ ID NO: 56), GTNKRAP (SEQ ID NO: 57), WTPARFSGSLLGGKAALTLSGVQPEDEAEYYC (SEQ ID NO: 58), ALWYSNLWV (SEQ ID NO: 59), and FGGGTKLTVL (SEQ ID NO: 60);
  • HFR1, HCDR1, HFR2, HCDR2, HFR3, HCDR3 and HFR4 in turn, the amino acid sequences of which are respectively as follows: EVQLVESGGGLVQPGGSLKLSCAASGFTFS (SEQ ID NO: 61), TYAMN (SEQ ID NO: 62), WVRQASGKGLEWVG (SEQ ID NO:63), RIRSKYNNYATYYADSVKD (SEQ ID NO:64), RFTISRDDSKNTAYLQMNSLKTEDTAVYYCTR (SEQ ID NO:65), HGNFGNSYVSWFAY (SEQ ID NO:66) and WGQGTLVTVSS (SEQ ID NO:67).
  • the LCDR1 is mutated by up to 6 amino acid residues, preferably, numbered according to the Kabat numbering system, and the mutation comprises at least one or more mutations selected from the group consisting of: R 24 S/G , T 29 S, S 30 G or A 33 P, more preferably, the mutation comprises at least R 24 S, T 29 S, S 30 G and A 33 P, or at least R 24 G, T 29 S, S 30 G and A 33 P; and/or,
  • Said LCDR2 is mutated by up to 6 amino acid residues; preferably, the mutation comprises at least one or more mutations selected from the group consisting of N 52 K, K 53 F, R 54 L or A, numbered according to the Kabat numbering system 55 S, more preferably, the mutation comprises at least N 52 K, K 53 F, R 54 L and A 55 S, or at least N 52 K, K 53 F and R 54 L; and/or,
  • Said LCDR3 is mutated in at most 6 amino acid residues, preferably, said mutations according to the Kabat numbering system at least comprise one or more mutations selected from the group consisting of A 89 S/V, L 90 I, Y 92 F, S 93 T, L 95 R or V 97 T, more preferably, the mutation comprises at least A 89 S and L 95 R, or at least A 89 V, L 90 I and L 95 R, or at least A 89 V , Y 92 F and L 95 R, or at least A 89 V, S 93 T and L 95 R, or at least A 89 V, L 95 R and V 97 T, or at least A 89 V and L 95 R; and / or,
  • Said HCDR1 is mutated by up to 6 amino acid residues, preferably, numbered according to the Kabat numbering system, said mutation comprising at least T 31 K; and/or,
  • Said HCDR2 is mutated by up to 6 amino acid residues, preferably, numbered according to the Kabat numbering system, said mutation comprising at least N54S; and/or,
  • Said HCDR3 is mutated at most 6 amino acid residues, preferably, numbered according to the Kabat numbering system, said mutation at least comprising one or more mutations selected from the group consisting of G 96 S, S 100A A, Y 100B F, V 100C I, W 100E Y, F 100F W/H or Y 102F /H, more preferably, the mutation comprises at least S 100A A, V 100C I, W 100E Y and F 100F W, or at least G 96 S, V 100C I, W 100E Y, and F 100F W, or at least the V 100C I, W 100E Y, F 100F W, and Y 102F , or at least the V 100C I, W 100E Y, F 100F W, and Y 102H mutations, Or at least V 100C I, W 100E Y and F 100F W, or at least Y 100B F, V 100C I, W 100E Y and F 100F W.
  • the framework region of the light chain variable region is mutated by up to 15 amino acid residues, preferably, numbered according to the Kabat numbering system, the mutation comprises at least one or more selected from the group consisting of Mutation: F36V , A46G , Y49G or W57G , more preferably, at least F36V, A46G , Y49G and W57G ; and/or, the framework region of the heavy chain variable region Up to 15 amino acid residues are mutated, preferably, the mutation comprises at least one or more mutations selected from the group consisting of S 30 N, S 41 P, G 49 A, S 82 BN or T 93 V, more preferably Typically, the mutation comprises at least S 30 N, S 41 P, G 49 A, S 82BN and T 93 V.
  • the number of amino acid residue mutations in the LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 or HCDR3 may be selected from 0, 1, 2, 3, 4, 5 or 6; the amino acids of the framework region
  • the number of residue mutations can be selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15; preferably, the mutations can be selected from insertion, Deletions or substitutions, preferably substitutions of conserved amino acid residues.
  • the heavy chain variable region has the sequence shown in any one of SEQ ID NOs: 9-18, and the light chain variable region has the sequence shown in any one of SEQ ID NOs: 3-8 an indicated sequence;
  • the heavy chain variable region has the sequence shown in SEQ ID NO: 9, and the light chain variable region has the sequence shown in any one of SEQ ID NOs: 3 to 7; or,
  • the heavy chain variable region has the sequence shown in SEQ ID NO: 10, and the light chain variable region has the sequence shown in any one of SEQ ID NOs: 3 to 7; or,
  • the heavy chain variable region has the sequence shown in SEQ ID NO: 11, and the light chain variable region has the sequence shown in any one of SEQ ID NOs: 3 to 7; or,
  • the heavy chain variable region has the sequence shown in SEQ ID NO: 12, and the light chain variable region has the sequence shown in any one of SEQ ID NOs: 3 to 7; or,
  • the heavy chain variable region has the sequence shown in any one of SEQ ID NOs: 13 to 18, and the light chain variable region has the sequence shown in SEQ ID NO:8.
  • the antibody or antigen-binding fragment comprises an scFv domain comprising the heavy chain variable region and the light chain variable region linked by a linker;
  • the connecting peptide can be selected from (G 4 S) n , optionally, the n is 3;
  • variable region of the heavy chain is located at the N-terminus of the connecting peptide
  • variable region of the light chain is located at the C-terminus of the connecting peptide
  • the scFv domain has the amino acid sequence shown in any one of SEQ ID NOs: 19-44.
  • the antibody or antigen-binding fragment comprises or does not comprise a heavy chain constant region and/or a light chain constant region;
  • the heavy chain constant region may be selected from a CH1, hinge region, Fc or CH3 domain, more preferably, the heavy chain constant region comprises a hinge region and an Fc, the Fc preferably having as SEQ ID NO:45
  • the sequence shown, the hinge region preferably has the sequence shown in SEQ ID NO: 46;
  • the heavy chain constant region and/or the light chain constant region are human heavy chain constant regions and/or human light chain constant regions;
  • the heavy chain constant region is an IgG heavy chain constant region, such as an IgG1 heavy chain constant region, an IgG2 heavy chain constant region, an IgG3 heavy chain constant region or an IgG4 heavy chain constant region;
  • the heavy chain constant region is a human IgG1 heavy chain constant region, a human IgG2 heavy chain constant region, a human IgG3 heavy chain constant region or a human IgG4 heavy chain constant region.
  • the antibody or antigen-binding fragment is selected from the group consisting of monoclonal antibodies, polyclonal antibodies, natural antibodies, engineered antibodies, monospecific antibodies, multispecific antibodies (eg, bispecific antibodies), monovalent antibodies Antibodies, Multivalent Antibodies, Whole Antibodies, Antibody Fragments, Naked Antibodies, Conjugated Antibodies, Fab, Fab', Fab'-SH, F(ab') 2 , Fd, Fv, scFv, diabody, bispecific Sexual T cell engager (BiTE) or single domain antibody.
  • monoclonal antibodies polyclonal antibodies, natural antibodies, engineered antibodies, monospecific antibodies, multispecific antibodies (eg, bispecific antibodies), monovalent antibodies Antibodies, Multivalent Antibodies, Whole Antibodies, Antibody Fragments, Naked Antibodies, Conjugated Antibodies, Fab, Fab', Fab'-SH, F(ab') 2 , Fd, Fv, scFv, diabody
  • the antibody or antigen-binding fragment specifically binds human CD3 ⁇ subunit and/or monkey CD3 ⁇ subunit, preferably, the antibody or antigen-binding fragment binds human CD3 ⁇ subunit with a KD of 1.00E -8M ⁇ 1.00E-11M, for example, 1.00E-9M, 2.00E-9M, 3.00E-9M, 4.00E-9M, 5.00E-9M, 6.00E-9M, 7.00E-9M, 8.00E-9M, 9.00E-9M, 1.00E-10M, 2.00E-10M, 3.00E-10M, 4.00E-10M, 5.00E-10M, 6.00E-10M, 7.00E-10M, 8.00E-10M or 9.00E-10M.
  • a KD of 1.00E -8M ⁇ 1.00E-11M for example, 1.00E-9M, 2.00E-9M, 3.00E-9M, 4.00E-9M, 5.00E-9M, 6.00E-9M, 7.00E-9M, 8.00E-9M, 9.00E-9
  • the antibody or antigen-binding fragment is further coupled with a therapeutic agent or a tracer; preferably, the therapeutic agent is selected from radioisotopes, chemotherapeutic agents or immunomodulatory agents, and the tracer Selected from radiographic contrast agents, paramagnetic ions, metals, fluorescent labels, chemiluminescent labels, ultrasound contrast agents and photosensitizers.
  • the therapeutic agent is selected from radioisotopes, chemotherapeutic agents or immunomodulatory agents
  • the tracer Selected from radiographic contrast agents, paramagnetic ions, metals, fluorescent labels, chemiluminescent labels, ultrasound contrast agents and photosensitizers.
  • the present disclosure also provides a multispecific antigen-binding molecule comprising at least a first antigen-binding moiety and other antigen-binding moieties, the first antigen-binding moiety comprising the aforementioned antibody or an antigen-binding fragment, the other antigen-binding moiety specifically binds to an antigen other than the CD3 ⁇ subunit or binds to a different CD3 ⁇ subunit epitope than the first antigen-binding moiety.
  • the multispecific antigen binding molecule can be a bispecific, trispecific or tetraspecific antigen binding molecule
  • the antigens other than the CD3 ⁇ subunit can be selected from tumor-associated antigens or infectious disease antigens, for example, can be selected from the following group: BCMA, CD19, CD20, CD33, CD38, CD70, CD123, CD138, CD276, CDH3, CEA, c-Met, CLDN18.2, CLL-1, DLL3, DLK1, ENPP3, EpCAM, GD2, GPC3, GPRC5d, gpA33, MSLN, MUC1, MUC16, MUC17, LRRC15, ROR1, EGFR, HER2, PSMA, PSCA, PD-1, PD-L1, TPBG, TYRP1, WT1, SSTR2, HIV, Hepatitis A, Hepatitis B, Hepatitis C, Herpes virus e.g.
  • the other antigen-binding moieties are antibodies or antigen-binding fragments
  • the multispecific antigen binding molecule is a bispecific T cell engager (BiTE).
  • the present disclosure also provides an isolated nucleic acid fragment encoding the aforementioned antibody, antigen-binding fragment or multispecific antigen-binding molecule.
  • the nucleic acid fragment further comprises a nucleic acid encoding a chimeric antigen receptor (CAR); preferably, the nucleic acid fragment encoding the aforementioned antibody, antigen binding fragment or multispecific antigen binding molecule is the same as the nucleic acid encoding the
  • the nucleic acid fragments of the chimeric antigen receptor are separated by nucleic acid fragments encoding a ribosome skipping moiety, which can be selected from P2A or T2A;
  • the antibody or antigen-binding fragment is a single-chain antibody, such as a scFv; preferably, the multispecific antigen-binding molecule is a bispecific T cell engager (BiTE).
  • BiTE bispecific T cell engager
  • the present disclosure also provides a vector comprising the aforementioned nucleic acid fragment.
  • the present disclosure also provides a host cell comprising the aforementioned vector; preferably, the cell is a prokaryotic cell or a eukaryotic cell, such as bacteria (E. coli), fungi (yeast), insect cells Or mammalian cells (CHO cell line or 293 cell line).
  • the cell is a prokaryotic cell or a eukaryotic cell, such as bacteria (E. coli), fungi (yeast), insect cells Or mammalian cells (CHO cell line or 293 cell line).
  • the present disclosure also provides an immune effector cell comprising the aforementioned nucleic acid fragment and/or expressing (a) the chimeric antigen receptor encoded by the aforementioned nucleic acid fragment and (b) the aforementioned nucleic acid fragment.
  • Antibodies, antigen-binding fragments or multispecific antigen-binding molecules are selected from T cells, NK cells (natural killer cells), NKT cells (natural killer T cells), DNT cells (double negative T cells) ), monocytes, macrophages, dendritic cells or mast cells, the T cells are preferably from cytotoxic T cells, regulatory T cells or helper T cells; the immune effector cells are preferably from autoimmune effector cells or allogeneic immune effector cells.
  • the present disclosure also provides a method for preparing the aforementioned antibody or antigen-binding fragment, or a multispecific antigen-binding molecule, the method comprising culturing the aforementioned cell, and isolating the antibody or antigen-binding fragment expressed by the cell, or Multispecific antigen binding molecules.
  • the present disclosure further provides a method for preparing the aforementioned immune effector cells, the method comprising introducing the aforementioned nucleic acid fragment into the immune effector cells, preferably, the method further comprising activating the chimeric antigen receptor expression, and initiating expression of the antibody, antigen-binding fragment or multispecific antigen-binding molecule.
  • the present disclosure also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the aforementioned antibody or antigen-binding fragment, multispecific antigen-binding molecule, nucleic acid fragment, vector, host cell, immune effector cell or according to the aforementioned method prepared product.
  • the present disclosure also provides the aforementioned antibodies or antigen-binding fragments, multispecific antigen-binding molecules, isolated nucleic acid fragments, vectors, host cells, immune effector cells, pharmaceutical compositions or products prepared according to the aforementioned methods in Use in the preparation of medicines for the treatment of cancer or tumors, infectious diseases or autoimmune diseases;
  • the cancer or tumor is selected from hematological tumors or solid tumors, which can be selected from B-cell lymphoma, mantle cell lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, Non-Hodgkin lymphoma, acute lymphoblastic leukemia, acute myeloid leukemia, multiple myeloma, stomach cancer, uterine cancer, ovarian cancer, breast cancer, liver cancer, prostate cancer, glioblastoma, lung cancer, pancreatic cancer, bile duct cancer, breast cancer, colorectal cancer, melanoma, renal cell carcinoma, or cancerous ascites;
  • B-cell lymphoma mantle cell lymphoma
  • follicular lymphoma diffuse large B-cell lymphoma
  • Non-Hodgkin lymphoma Non-Hodgkin lymphoma
  • acute lymphoblastic leukemia acute myeloid leukemia
  • multiple myeloma stomach cancer
  • the autoimmune disease is selected from the group consisting of rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, inflammatory bowel disease, myasthenia gravis, celiac disease, Sjogren's syndrome, Grave's disease, Crohn's disease Enn's disease, autoimmune hepatitis, psoriasis, psoriatic arthritis, asthma, allergic rhinitis, effects from organ transplantation or graft-versus-host disease (GVHD);
  • GVHD graft-versus-host disease
  • the infectious disease is selected from HIV virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, herpes virus eg, HSV-1, HSV-2, CMV, Chlamydia, Rickettsia, Mycobacterium, Staphylococcus , Streptococcus, Pneumococcus, Meningococcus, or Neisseria gonorrhoeae.
  • HIV virus hepatitis A virus
  • hepatitis B virus hepatitis C virus
  • herpes virus eg, HSV-1, HSV-2, CMV, Chlamydia, Rickettsia, Mycobacterium, Staphylococcus , Streptococcus, Pneumococcus, Meningococcus, or Neisseria gonorrhoeae.
  • the present disclosure also provides a method of treating cancer or tumor, infectious disease or autoimmune disease, the method comprising administering to a subject an effective amount of the aforementioned antibody or antigen-binding fragment, multispecific Antigen-binding molecules, nucleic acid fragments, vectors, host cells, immune effector cells, pharmaceutical compositions or products prepared according to the aforementioned methods;
  • the cancer or tumor is selected from hematological tumors or solid tumors, which can be selected from B-cell lymphoma, mantle cell lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, Non-Hodgkin lymphoma, acute lymphoblastic leukemia, acute myeloid leukemia, multiple myeloma, stomach cancer, uterine cancer, ovarian cancer, breast cancer, liver cancer, prostate cancer, glioblastoma, lung cancer, pancreatic cancer, bile duct cancer, breast cancer, colorectal cancer, melanoma, renal cell carcinoma, or cancerous ascites;
  • B-cell lymphoma mantle cell lymphoma
  • follicular lymphoma diffuse large B-cell lymphoma
  • Non-Hodgkin lymphoma Non-Hodgkin lymphoma
  • acute lymphoblastic leukemia acute myeloid leukemia
  • multiple myeloma stomach cancer
  • the autoimmune disease is selected from the group consisting of rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, inflammatory bowel disease, myasthenia gravis, celiac disease, Sjogren's syndrome, Grave's disease, Crohn's disease Enn's disease, autoimmune hepatitis, psoriasis, psoriatic arthritis, asthma, allergic rhinitis, effects from organ transplantation or graft-versus-host disease (GVHD);
  • GVHD graft-versus-host disease
  • the infectious disease is selected from HIV virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, herpes virus eg, HSV-1, HSV-2, CMV, Chlamydia, Rickettsia, Mycobacterium, Staphylococcus , Streptococcus, Pneumococcus, Meningococcus, or Neisseria gonorrhoeae.
  • HIV virus hepatitis A virus
  • hepatitis B virus hepatitis C virus
  • herpes virus eg, HSV-1, HSV-2, CMV, Chlamydia, Rickettsia, Mycobacterium, Staphylococcus , Streptococcus, Pneumococcus, Meningococcus, or Neisseria gonorrhoeae.
  • the present disclosure also provides the aforementioned antibodies or antigen-binding fragments, multispecific antigen-binding molecules, isolated nucleic acid fragments, vectors, host cells, immune effector cells, pharmaceutical compositions or products prepared according to the aforementioned methods , for the treatment of cancer or tumors, infectious diseases or autoimmune diseases;
  • the cancer or tumor is selected from hematological tumors or solid tumors, which can be selected from B-cell lymphoma, mantle cell lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, Non-Hodgkin lymphoma, acute lymphoblastic leukemia, acute myeloid leukemia, multiple myeloma, stomach cancer, uterine cancer, ovarian cancer, breast cancer, liver cancer, prostate cancer, glioblastoma, lung cancer, pancreatic cancer, bile duct cancer, breast cancer, colorectal cancer, melanoma, renal cell carcinoma, or cancerous ascites;
  • B-cell lymphoma mantle cell lymphoma
  • follicular lymphoma diffuse large B-cell lymphoma
  • Non-Hodgkin lymphoma Non-Hodgkin lymphoma
  • acute lymphoblastic leukemia acute myeloid leukemia
  • multiple myeloma stomach cancer
  • the autoimmune disease is selected from the group consisting of rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, inflammatory bowel disease, myasthenia gravis, celiac disease, Sjogren's syndrome, Grave's disease, Crohn's disease Enn's disease, autoimmune hepatitis, psoriasis, psoriatic arthritis, asthma, allergic rhinitis, effects from organ transplantation or graft-versus-host disease (GVHD);
  • GVHD graft-versus-host disease
  • the infectious disease is selected from HIV virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, herpes virus eg, HSV-1, HSV-2, CMV, Chlamydia, Rickettsia, Mycobacterium, Staphylococcus , Streptococcus, Pneumococcus, Meningococcus, or Neisseria gonorrhoeae.
  • HIV virus hepatitis A virus
  • hepatitis B virus hepatitis C virus
  • herpes virus eg, HSV-1, HSV-2, CMV, Chlamydia, Rickettsia, Mycobacterium, Staphylococcus , Streptococcus, Pneumococcus, Meningococcus, or Neisseria gonorrhoeae.
  • CD3 cluster of differentiation 3
  • the term "CD3" herein refers to a cluster of differentiation 3 protein derived from any vertebrate source, including mammals, such as primates (eg, humans, monkeys) and rodents (eg, mice and rats). mouse).
  • the CD3 molecule is a six-chain multiprotein complex, including a CD3 ⁇ chain, a CD3 ⁇ chain, two CD3 ⁇ chains, and a homodimer of CD3 ⁇ chains, where the CD3 ⁇ chain is the intracellular tail of the CD3 molecule, and CD3 ⁇ Chain, CD3 ⁇ chain and CD3 ⁇ chain all contain an extracellular domain (ECD) expressed on the surface of T cells.
  • Exemplary sequences of human CD3 include human CD3 ⁇ protein (NCBI Ref Seq No.
  • NCBI Ref Seq No. NP_000724 or NCBI: AAH49847.1 human CD3 ⁇ protein
  • NCBI Ref Seq No. NP_000723 human CD3 ⁇ protein
  • NCBI Ref Seq No. NP_000064 exemplary sequences of non-human CD3 include cynomolgus monkey (Macaca fascicularis) (monkey) CD3 epsilon protein (NCBI Ref Seq No. NP_001270544), cynomolgus monkey (Macaca fascicularis) (monkey) CD3 delta protein (NCBI Ref Seq No.
  • NP_001274617 Cynomolgus monkey (Macaca fascicularis) (monkey) CD3 ⁇ protein (NCBI Ref Seq No.NP_001270839); mouse CD3 ⁇ protein (NCBI Ref Seq No.NP_031674), mouse CD3 ⁇ protein (NCBI Ref Seq No.NP_038515), mouse CD3 ⁇ protein ( NCBI Ref Seq No.AAA37400); Rattus norvegicus (Rat) CD3 ⁇ protein (NCBI Ref Seq No.NP_001101610), Rattus norvegicus (Rat) CD3 ⁇ protein (NCBI Ref Seq No.NP_037301), Rattus norvegicus (Rat) ) CD3 ⁇ protein (NCBI Ref Seq No. NP_001071114).
  • CD3 ⁇ herein is intended to encompass any form of the CD3 ⁇ subunit, eg, 1) native unprocessed CD3 ⁇ molecules, "full-length” CD3 ⁇ chains or naturally occurring CD3 ⁇ variants, including, eg, splice variants or alleles Variants; 2) any form of CD3 ⁇ produced by processing in a cell; or 3) full-length, fragments (eg, truncated forms, extracellular/transmembrane domains) or modified CD3 ⁇ subunits produced by recombinant methods form (eg, mutated form, glycosylated/pegylated, histidine-tagged/immunofluorescent fusion form).
  • the term "specifically binds" herein refers to an antigen-binding molecule (eg, an antibody) that specifically binds an antigen and a substantially identical antigen, usually with high affinity, but does not bind with high affinity to an unrelated antigen. Affinity is usually reflected by the equilibrium dissociation constant (KD), where lower KD indicates higher affinity.
  • high affinity generally refers to having about 1.00E-7M or less, about 1.00E-8M or less, about 1.00E-9M or less, about 1.00E-10M or less, 1.00E-11M or lower or 1.00E-12M or lower KD.
  • the equilibrium dissociation constant KD can be measured using methods well known in the art, such as surface plasmon resonance (eg Biacore) or equilibrium dialysis.
  • antigen binding molecules include, but are not limited to, antibodies or antibody mimetics.
  • Antibody mimetic refers to an organic compound or binding domain that can specifically bind to an antigen, but is unrelated to the structure of an antibody.
  • antibody mimetics include, but are not limited to, affibody, affitin, affilin, designed ankyrin repeat proteins (DARPin), nucleic acid aptamer or Kunitz-type domain peptide.
  • antibody is used herein in the broadest sense to refer to a polypeptide comprising sufficient sequence from the variable region of an immunoglobulin heavy chain and/or sufficient sequence from the variable region of an immunoglobulin light chain to enable specific binding to an antigen or peptide combinations.
  • Antibody herein encompasses various forms and various structures so long as they exhibit the desired antigen-binding activity.
  • Antibody herein includes alternative protein scaffolds or artificial scaffolds with grafted complementarity determining regions (CDRs) or CDR derivatives. Such scaffolds include antibody-derived scaffolds comprising mutations introduced, eg, to stabilize the three-dimensional structure of the antibody, and fully synthetic scaffolds comprising, eg, biocompatible polymers.
  • Such scaffolds may also include non-antibody derived scaffolds, such as scaffold proteins known in the art to be useful for grafting CDRs, including but not limited to tenascin, fibronectin, peptide aptamers, and the like.
  • Antibody herein includes a typical "quad-chain antibody”, which is an immunoglobulin consisting of two heavy chains (HC) and two light chains (LC); heavy chain refers to a polypeptide chain that is The N-terminal to C-terminal direction consists of the heavy chain variable region (VH), the heavy chain constant region CH1 domain, the hinge region (HR), the heavy chain constant region CH2 domain, the heavy chain constant region CH3 domain; and, When the full-length antibody is of the IgE isotype, it optionally also includes a heavy chain constant region CH4 domain; the light chain is composed of a light chain variable region (VL) and a light chain constant in the N-terminal to C-terminal direction
  • the polypeptide chain composed of the region (CL); the heavy chain and the heavy chain, and the heavy chain and the light chain are connected by disulfide bonds to form a "Y"-shaped structure.
  • immunoglobulins Due to the different amino acid composition and arrangement sequence of the constant region of immunoglobulin heavy chain, its antigenicity is also different. Accordingly, the "immunoglobulins" herein can be divided into five classes, or isotypes called immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE, and their corresponding heavy chains are ⁇ and ⁇ chains, respectively. , ⁇ chain, ⁇ chain and ⁇ chain. The same type of Ig can be divided into different subclasses according to the difference in the amino acid composition of its hinge region and the number and position of disulfide bonds in the heavy chain.
  • IgG can be divided into IgG1, IgG2, IgG3, and IgG4, and IgA can be divided into IgA1 and IgA2.
  • Light chains are classified into kappa chains or lambda chains by the difference in the constant region.
  • Each of the five classes of Ig can have a kappa chain or a lambda chain.
  • Antibody herein also includes antibodies that do not contain a light chain, such as those produced by Camelus dromedarius, Camelus bactrianus, Lama glama, Lama guanicoe, and alpaca ( Vicugna pacos) and other camelids produced heavy-chain antibodies (heavy-chain antibodies, HCAbs) and sharks and other cartilaginous fish found in the new immunoglobulin receptor (Ig new antigen receptor, IgNAR).
  • HCAbs heavy-chain antibodies
  • an “antibody” herein can be derived from any animal, including, but not limited to, humans and non-human animals, which can be selected from primates, mammals, rodents, and vertebrates, such as camelid, llama , ostriches, alpacas, sheep, rabbits, mice, rats or cartilaginous fishes (eg sharks).
  • Antibody herein includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (eg, bispecific antibodies), monovalent antibodies, multivalent antibodies, intact antibodies, fragments of intact antibodies, naked antibodies , conjugated antibodies, chimeric antibodies, humanized antibodies or fully human antibodies.
  • the term "monoclonal antibody” herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., except for possible variants (e.g., containing naturally occurring mutations or produced during the manufacture of a preparation, such variants typically defined as except that the individual antibodies comprising the population are identical and/or bind the same epitope.
  • polyclonal antibody preparations typically include different antibodies directed against different determinants (epitopes)
  • each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant on an antigen.
  • the modifier "monoclonal” herein should not be construed as requiring the production of the antibody or antigen-binding molecule by any particular method.
  • monoclonal antibodies can be made by a variety of techniques including, but not limited to, hybridoma technology, recombinant DNA methods, phage library display technology, and the use of transgenic animals that contain all or part of the human immunoglobulin loci method and other methods known in the art.
  • natural antibody herein refers to an antibody that is produced and paired by the immune system of a multicellular organism.
  • the antibody of the term “engineered antibody” herein refers to a non-natural antibody obtained by genetic engineering, antibody engineering and other techniques.
  • engineered antibody includes humanized antibody, small molecule antibody (such as scFv, etc.), dual specific antibodies, etc.
  • the term "monospecific” herein refers to having one or more binding sites, wherein each binding site binds the same epitope of the same antigen.
  • multispecific antibody herein refers to having at least two antigen-binding sites, each of which is associated with a different epitope of the same antigen or a different epitope of a different antigen. Bit binding.
  • terms such as “bispecific”, “trispecific”, “tetraspecific” etc. refer to the number of different epitopes to which an antibody/antigen binding molecule can bind.
  • valency herein refers to the presence of a defined number of binding sites in an antibody/antigen binding molecule.
  • the terms “monovalent”, “bivalent”, “tetravalent” and “hexavalent” refer to one binding site, two binding sites, four binding sites and six binding sites, respectively, in an antibody/antigen binding molecule the existence of points.
  • full-length antibody “intact antibody,” and “intact antibody” are used interchangeably herein to mean having a structure that is substantially similar to that of a native antibody.
  • Antigen-binding fragment and “antibody fragment” are used interchangeably herein and do not possess the full structure of an intact antibody, but only include partial or partial variants of the intact antibody that have the ability to bind antigenic capacity.
  • Antigen-binding fragments” or “antibody fragments” herein include, but are not limited to, Fab, Fab', Fab'-SH, F(ab') 2 , Fd, Fv, scFv, diabodies, and single domain antibodies.
  • Papain digestion of intact antibodies produces two identical antigen-binding fragments, termed "Fab” fragments, each containing the heavy and light chain variable domains, as well as the light chain constant domain and the heavy chain first constant domain (CH1 ).
  • Fab fragment herein refers to a light chain fragment comprising the VL domain and constant domain (CL) of the light chain, and an antibody fragment comprising the VH domain and the first constant domain (CH1) of the heavy chain.
  • Fab' fragments differ from Fab fragments by adding a few residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region.
  • Fab'-SH is a Fab' fragment in which the cysteine residues of the constant domains carry free thiol groups. Pepsin treatment produces an F(ab') 2 fragment with two antigen binding sites (two Fab fragments) and a portion of the Fc region.
  • Fd refers to an antibody consisting of VH and CH1 domains.
  • Fv refers to antibody fragments consisting of one-armed VL and VH domains. Fv fragments are generally considered to be the smallest antibody fragments that can form a complete antigen-binding site. It is generally believed that the six CDRs confer antigen-binding specificity to an antibody. However, even a single variable region (eg, an Fd fragment, which contains only three CDRs specific for the antigen) is able to recognize and bind the antigen, albeit probably with lower affinity than the intact binding site.
  • scFv single-chain variable fragment
  • linker see, e.g., Bird et al., Science 242:423 -426 (1988); Huston et al, Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, eds. Roseburg and Moore, Springer-Verlag, New York, pp. 269-315 (1994)).
  • Such scFv molecules can have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH.
  • Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof.
  • GGGGS linker with the amino acid sequence
  • Other linkers useful in the present disclosure are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res.
  • a disulfide bond may also exist between the VH and VL of the scFv, forming a disulfide-linked Fv (dsFv).
  • the term herein is a "diabody” whose VH and VL domains are expressed on a single polypeptide chain, but use a linker that is too short to allow pairing between the two domains of the same chain, forcing the domains to interact with The complementary domains of the other chain pair and create two antigen-binding sites (see, e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993), and Poljak R.J. et al., Structure 2: 1121-1123 (1994)).
  • bispecific T cell engager (BiTE) is a bispecific antibody format based on a single chain antibody arranged in tandem, comprising two single chain variable regions (scFv), one targeting tumor cells and the other An scFv targets immune cells (eg, targeting CD3) and is capable of bridging tumor cells and immune cells (especially T cells).
  • scFv single chain variable regions
  • immune cells eg, targeting CD3
  • T cells especially T cells.
  • single domain antibody (sdAb), “VHH” and “nanobody” have the same meaning and are used interchangeably herein and refer to the variable region of a cloned antibody heavy chain, constructed from only one A single-domain antibody composed of the variable region of the heavy chain, which is the smallest fully functional antigen-binding fragment.
  • an antibody that naturally lacks the light chain and heavy chain constant region 1 (CH1) is obtained first, and then the variable region of the antibody heavy chain is cloned to construct a single-domain antibody consisting of only one heavy chain variable region.
  • Single domain antibodies can be derived from camelid heavy chain antibodies or from cartilaginous IgNARs.
  • naked antibody herein refers to an antibody that is not conjugated to a therapeutic agent or tracer; the term “conjugated antibody” refers to an antibody that is conjugated to a therapeutic agent or tracer.
  • chimeric antibody herein refers to an antibody in which a portion of the light or/and heavy chain is derived from an antibody (which may be derived from a particular species or belong to a particular antibody class or subclass). ), and the other part of the light chain or/and the heavy chain is derived from another antibody (which may be derived from the same or a different species or belong to the same or different antibody class or subclass), but in any case it retains its interest in the target Antigen binding activity (U.S.P 4,816,567 to Cabilly et al.; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851 6855 (1984)).
  • chimeric antibody can include antibodies (eg, human-mouse chimeric antibodies) in which the heavy and light chain variable regions of the antibody are derived from a primary antibody (eg, a murine antibody) and the heavy and The light chain constant region is derived from a second antibody (eg, a human antibody).
  • a primary antibody eg, a murine antibody
  • a second antibody eg, a human antibody
  • humanized antibody refers to a genetically engineered non-human antibody whose amino acid sequence has been modified to increase homology to the sequence of a human antibody.
  • CDR regions of a humanized antibody are derived from a non-human antibody (donor antibody), and all or part of the non-CDR regions (eg, variable FR and/or constant regions) are derived from human Immunoglobulins (receptor antibodies).
  • Humanized antibodies generally retain or partially retain the expected properties of the donor antibody, including, but not limited to, antigen specificity, affinity, reactivity, ability to increase immune cell activity, ability to enhance immune response, and the like.
  • Fully human antibody refers to an antibody having variable regions in which both the FRs and CDRs are derived from human germline immunoglobulin sequences. Furthermore, if the antibody comprises a constant region, the constant region is also derived from human germline immunoglobulin sequences.
  • Fully human antibodies herein may include amino acid residues not encoded by human germline immunoglobulin sequences (eg, mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, "fully human antibodies” herein do not include antibodies in which CDR sequences derived from the germline of another mammalian species (eg, mouse) have been grafted onto human framework sequences.
  • variable region herein refers to the region of an antibody heavy or light chain that is involved in binding an antibody to an antigen.
  • "Heavy chain variable region” is used interchangeably with “VH” and “HCVR”.
  • VL is used interchangeably with "VL”, “LCVR”.
  • the variable domains (VH and VL, respectively) of the heavy and light chains of native antibodies generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). See, eg, Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., p.91 (2007).
  • a single VH or VL domain may be sufficient to confer antigen binding specificity.
  • complementarity determining region and “CDR” are used interchangeably herein, and generally refer to the variable region of the heavy chain (VH) or the hypervariable region (HVR) of the light chain variable region (VL), which is spatially structured It can form precise complementarity with the antigenic epitope, so it is also called the complementarity determining region.
  • VH variable region of the heavy chain
  • HVR hypervariable region of the light chain variable region
  • HCDR variable region of the heavy chain
  • LCDR light chain variable region
  • frame region or "FR region” are used interchangeably and refer to those amino acid residues other than the CDRs in the variable region of the heavy or light chain of an antibody
  • HFR may refer to the framework of the variable region of the heavy chain region
  • LFR may refer to the framework region of the light chain variable region.
  • CDRs may be labeled and defined by means known in the art, including but not limited to the Kabat numbering system, the Chothia numbering system, or the IMGT numbering system, using tool websites including, but not limited to, the AbRSA website (http://cao.labshare.
  • CDRs herein include overlaps and subsets of amino acid residues differently defined.
  • Kabat numbering system herein generally refers to the immunoglobulin alignment and numbering system proposed by Elvin A. Kabat (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991).
  • Chothia numbering system generally refers to the immunoglobulin numbering system proposed by Chothia et al., which is a classical rule for identifying CDR region boundaries based on the position of structural loop regions (see, eg, Chothia & Lesk (1987) J. Mol. Biol 196:901-917; Chothia et al. (1989) Nature 342:878-883).
  • IMGT numbering system herein generally refers to the numbering system based on The International ImMunoGeneTics information system (IMGT) initiated by Lefranc et al., see Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003.
  • IMGT International ImMunoGeneTics information system
  • variable domains refers to the carboxy-terminal portion of an antibody heavy chain that is not directly involved in the binding of the antibody to an antigen, but exhibits effector functions, such as interaction with Fc receptors, relative to the availability of the antibody
  • the variable domains have more conserved amino acid sequences.
  • a “heavy chain constant region” comprises at least: a CH1 domain, a hinge region, a CH2 domain, a CH3 domain, or variants or fragments thereof.
  • “Heavy chain constant region” includes "full-length heavy chain constant region” and “heavy chain constant region fragment", the former has a substantially similar structure to that of natural antibody constant region, while the latter includes only "full-length heavy chain constant region” part".
  • a typical "full-length antibody heavy chain constant region” consists of a CH1 domain-hinge region-CH2 domain-CH3 domain; when the antibody is an IgE, it also includes a CH4 domain; when the antibody is a heavy chain In the case of an antibody, it does not include the CH1 domain.
  • a typical "heavy chain constant region fragment" can be selected from a CH1, Fc or CH3 domain.
  • light chain constant region refers to the carboxy-terminal portion of an antibody light chain that is not directly involved in binding the antibody to an antigen, which light chain constant region may be selected from a constant kappa domain or a constant lambda domain.
  • Fc refers to the papain hydrolyzed carboxy-terminal portion of an antibody from an intact antibody, which typically comprises the CH3 and CH2 domains of the antibody.
  • Fc regions include, for example, native sequence Fc regions, recombinant Fc regions, and variant Fc regions.
  • the boundaries of the Fc region of an immunoglobulin heavy chain can vary slightly, the Fc region of a human IgG heavy chain is generally defined as extending from the amino acid residue at position Cys226 or from Pro230 to its carboxy terminus.
  • the C-terminal lysine of the Fc region (residue 447 according to the Kabat numbering system) can be removed, for example, during production or purification of the antibody, or by recombinant engineering of nucleic acid encoding the antibody heavy chain, thus, the Fc region can include or excluding Lys447.
  • the numbering of amino acid residues in "antibodies” or “antigen-binding fragments” described herein is determined by the Kabat numbering system, as detailed in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991, for the numbering of amino acid residues in the variable region of antibodies, please refer to the following website: http://www.abysis.org/abysis/sequence_input/key_annotation/key_annotation.cgi .
  • a light chain variable region mutation R 24 S/G means that the light chain variable region is numbered at position 24 according to the Kabat numbering system
  • the amino acid residue is mutated from R to S, or from R to G, wherein, "/" means "or”, and the underline indicates the specific numbering
  • heavy chain variable region mutation S 100A A indicating that the heavy chain can be
  • the variable region is numbered according to the Kabat numbering system, and the amino acid residue at position 100A is mutated from S to A, and the underline indicates the specific numbering.
  • amino acids generally refers to amino acids that belong to the same class or have similar characteristics (eg, charge, side chain size, hydrophobicity, hydrophilicity, backbone conformation, and rigidity).
  • amino acids within each of the following groups belong to each other's conserved amino acid residues, and substitutions of amino acid residues within a group belong to conservative amino acid substitutions:
  • nucleic acid herein includes any compound and/or substance comprising a polymer of nucleotides.
  • Each nucleotide consists of a base, particularly a purine or pyrimidine base (i.e. cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), sugar (ie deoxyribose or ribose) and a phosphate group.
  • cytosine C
  • G guanine
  • A adenine
  • T thymine
  • U uracil
  • nucleic acid molecules are described by a sequence of bases, whereby the bases represent the primary structure (linear structure) of the nucleic acid molecule.
  • the sequence of bases is usually denoted 5' to 3'.
  • nucleic acid molecule encompasses deoxyribonucleic acid (DNA), including, for example, complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), especially messenger RNA (mRNA), synthetic forms of DNA or RNA, as well as synthetic forms of DNA or RNA. A mixed polymer of one or more of these molecules.
  • Nucleic acid molecules can be linear or circular.
  • nucleic acid molecule includes both sense and antisense strands, as well as single- and double-stranded forms.
  • nucleic acid molecules described herein may contain naturally occurring or non-naturally occurring nucleotides.
  • nucleic acid molecules also encompass DNA and RNA molecules suitable as vectors for the direct expression of the antibodies of the present disclosure in vitro and/or in vivo, eg, in a host or patient.
  • DNA eg, cDNA
  • RNA eg, mRNA
  • the mRNA can be chemically modified to enhance the stability of the RNA vector and/or the expression of the encoded molecule, so that the mRNA can be injected into a subject to generate antibodies in vivo (see, e.g., Stadler et al., Nature Medicine 2017, published online 12 June 2017, doi: 10.1038/nm.4356 or EP 2 101 823 B1).
  • An "isolated" nucleic acid herein refers to a nucleic acid molecule that has been separated from components of its natural environment.
  • An isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but which is present extrachromosomally or at a chromosomal location different from its natural chromosomal location.
  • chimeric antigen receptor herein refers to an artificial cell surface receptor engineered to be expressed on immune effector cells and to specifically bind an antigen, comprising at least (1) an extracellular antigen binding domain, eg, an antibody The variable heavy or light chain, (2) the transmembrane domain that anchors the CAR into immune effector cells, and (3) the intracellular signaling domain.
  • CARs can utilize extracellular antigen-binding domains to redirect T cells and other immune effector cells to selected targets, such as cancer cells, in a non-MHC-restricted manner.
  • vector refers to a nucleic acid molecule capable of amplifying another nucleic acid to which it is linked.
  • the term includes vectors that are self-replicating nucleic acid structures as well as vectors that integrate into the genome of the host cell into which the vector has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors”.
  • host cell herein refers to a cell into which exogenous nucleic acid has been introduced, including progeny of such cells.
  • Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages.
  • the progeny may not be identical in nucleic acid content to the parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected in the initially transformed cell are included herein.
  • pharmaceutical composition refers to a formulation that is in a form that permits the biological activity of the active ingredients contained therein to be effective and that does not contain unacceptable toxicity to the subject to whom the pharmaceutical composition is administered of additional ingredients.
  • treatment refers to surgical or therapeutic treatment for the purpose of preventing, slowing (reducing) undesired physiological changes or pathologies, such as cancer, autoimmune diseases and viral infections, in the subject being treated. progress.
  • Beneficial or desirable clinical outcomes include, but are not limited to, reduction of symptoms, reduction in disease severity, stable disease state (ie, no worsening), delayed or slowed disease progression, improvement or alleviation of disease state, and remission (whether partial remission or complete remission), whether detectable or undetectable.
  • Those in need of treatment include those already suffering from the disorder or disease as well as those prone to develop the disorder or disease or for whom the disorder or disease is to be prevented.
  • alleviation, alleviation, weakening, alleviation, alleviation, etc. the meanings also include elimination, disappearance, non-occurrence, etc.
  • subject herein refers to an organism receiving treatment for a particular disease or disorder as described in the present disclosure, and exemplarily includes mammals, such as humans, primates ( For example, monkeys) or non-primate mammals.
  • an effective amount herein refers to an amount of a therapeutic agent that, when administered alone or in combination with another therapeutic agent, to a cell, tissue, or subject, is effective to prevent or alleviate a disease condition or progression of the disease.
  • Effective amount also refers to an amount of the compound sufficient to relieve symptoms, eg, treat, cure, prevent or alleviate related medical conditions, or an increased rate of treatment, cure, prevention or alleviation of such conditions.
  • a therapeutically effective dose refers to that ingredient alone.
  • a therapeutically effective dose refers to the combined amount of active ingredients that produces a therapeutic effect, whether administered in combination, consecutively or simultaneously.
  • autoimmune disease refers to a disorder in which cells, tissues and/or organs are damaged by a subject's immune response to its own cells, tissues and/or organs.
  • cancer refers to or describes the physiological condition in mammals that is typically characterized by unregulated cell growth. Benign and malignant cancers are included in this definition.
  • tumor or “neoplastic” herein refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
  • cancer and “tumor” are not mutually exclusive when referred to herein.
  • EC50 refers to the half-maximal effective concentration, which includes the concentration of antibody that induces a half-way response between baseline and maximum after a specified exposure time. EC50 essentially represents the concentration of the antibody at which 50% of its maximal effect is observed and can be measured by methods known in the art.
  • Fig. 1 is the binding reaction of ELISA detection control antibody and human S004pep polypeptide
  • Figure 2 shows the CD3 expression of endogenous cell Jurkat detected by SP34 and OKT3 antibodies
  • Figure 3 shows the expression of CD3 in endogenous negative cells MOLT4 detected by SP34 and OKT3 antibodies
  • Figure 4 shows the binding reaction of SP34 antibody detection with CD3 on the surface of human T cells
  • Figure 5 shows the binding reaction of SP34 antibody detection with CD3 on the surface of monkey T cells
  • Figures 6A to 6F are ELISA detection of the binding reaction of SP34 humanized antibody and human CD3 ⁇ protein
  • Figures 7A to 7F are FACS detection of the binding reaction of SP34 humanized antibody to human Jurkat cells
  • 8A to 8F are FACS detection of the binding reaction of SP34 humanized antibody and human MOLT4 cells
  • Figures 9A to 9G are FACS detection of the binding reaction of SP34 humanized antibody and human T cells
  • FIGS 10A to 10C show the SPR detection of the affinity of SP34 humanized antibody to human CD3 ⁇ protein
  • Figures 11A to 11F show the binding reaction of SP34 humanized antibody and polypeptide detected by ELISA
  • Figures 12A-12G are FACS detection of the cross-binding reaction between SP34 humanized antibody and monkey T cells
  • Figures 13A to 13H are luciferase reporter gene experiments to detect the function of SP34 humanized antibody
  • IGLV7-43*01 and IGLJ2*01 were selected as the humanized light proteins selected by the mouse antibody SP34, respectively.
  • Chain templates, IGHV3-73*01 and IGHJ1*01 were used as humanized heavy chain templates.
  • the CDRs of murine antibody SP34 were grafted into the FRs of its human template, respectively, to form variable region sequences in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • the key amino acids in the FR region sequence of the humanized antibody are back-mutated to the corresponding amino acids of the mouse antibody to ensure the original affinity.
  • some amino acids in the CDR region sequence of the humanized antibody were mutated to homologous amino acids corresponding to the human template to improve the degree of humanization, or to other amino acids to optimize the physicochemical properties of the molecule to obtain a humanized anti-CD3 monoclonal antibody. cloned antibodies.
  • the numbering of amino acid residues and CDR regions of the antibody in this example are determined and annotated by the Kabat numbering system (see http://www.abysis.org/abysis/sequence_input/key_annotation/key_annotation.cgi for details) .
  • the humanized antibodies VL and VH designed by the above method are shown in Table 1 and Table 2, respectively.
  • SP34 antibody VL The specific sequence of SP34 antibody VL is shown below (SEQ ID NO: 1):
  • SP34 antibody VH sequence is shown below (SEQ ID NO: 2):
  • SP34 humanized VL is as follows, in which the underline is the CDR region, the rest are the framework region, and the bold indicates the amino acid residues mutated in the CDR region or the framework region.
  • SP34 humanized VH The specific sequence of SP34 humanized VH is as follows:
  • each humanized antibody corresponds to VL, VH (see Table 3- Table 4), wherein, S004-2-01 indicates that SP34 humanized antibody S004-2-01 has the variable region of light chain as described in SP34.VL1 and the variable region of heavy chain as described in SP34.VH1, and the others follow the same analogy.
  • the SP34 humanized antibody shown in Table 3-4 was used to construct a scFv antibody (format: VH-(G 4 S) 3 -VL), the specific sequence is as follows:
  • the SP34 humanized antibody is expressed in the form of scFv, and the variable region of the heavy chain and the variable region of the light chain are connected by a peptide segment consisting of 15 amino acids (GGGGGSGGGGSGGGGS) (see SEQ ID NO in Example 1 for details: 19-44).
  • Antibody expression and purification were carried out by Taizhou Baiying Biotechnology Co., Ltd.
  • the DNA sequence comprising the signal peptide and scFv-hinge region-hFc was directly synthesized and cloned into Expression vector BI3.4-huIgG1 (from Biointron), and prepare plasmids according to established standard molecular biology methods, see Sambrook, J., Fritsch, EF, and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, Second Edition (Plainview, New York: Cold Spring Harbor Laboratory Press).
  • the expression vector was transiently transfected into HEK293E cells (purchased from the Cell Bank of the Type Culture Collection, Chinese Academy of Sciences) according to the instructions of PEI (purchased from Polysciences, cat.
  • the cells were continuously cultured at °C for 5 days, and the cell components were removed by centrifugation to obtain a culture supernatant containing SP34 humanized antibody.
  • the culture supernatant was loaded onto a protein A chromatography column (Protein A packing AT Protein A Diamond and chromatography column BXK16/26 were purchased from Borglon, catalog number: AA0273 and B-1620), using PBS phosphate buffered saline (pH 7.4) and then washed with 20 mM PB, 1 M NaCl (pH 7.2), and finally eluted with pH 3.4 citrate buffer to collect the Fc tag eluted from the Protein A column.
  • the SP34 humanized antibody was neutralized with 1/10 volume of 1M Tris, pH 8.0, and dialyzed overnight at 4°C with PBS. The dialyzed protein was sterile filtered through a 0.22-micron filter membrane and aliquoted at -80°C. save.
  • EPKSADKTHTCPPCP SEQ ID NO: 46;
  • Positive control antibody (see SEQ ID NO: 1-2, SEQ ID NO: 47-52 for sequence information): SP34, OKT3, I2C, all of which recognize human CD3 antibodies, among which SP34 and I2C clones recognize human CD3 ⁇ protein, CD3 ⁇ / ⁇ heterodimer and CD3 ⁇ / ⁇ heterodimer; OKT3 clone recognizes only human CD3 ⁇ / ⁇ heterodimer.
  • the plasmid construction and antibody production and purification of the positive control antibody were completed by Taizhou Baiying Biotechnology Co., Ltd. The specific methods are as follows: I2C, referring to "Example 2.1", construct scFv(I2C)-hinge region-hFc antibody, the following Abbreviated as I2C.
  • SP34, OKT3 The light chain variable region sequences were cloned into the expression vector pcDNA3.4-B1HLK containing the signal peptide and the light chain constant region of human antibody IgG1, and the heavy chain variable region sequences were cloned into the expression vector pcDNA3.4-B1HLK containing the signal peptide (SEQ ID NO: 68) and the expression vector pcDNA3.4-B1HH1 of the heavy chain constant region of human antibody IgG1 to obtain the sequences of SP34-hIgG1, OKT3-hIgG1, hereinafter referred to as SP34, OKT3.
  • the expression and purification were carried out in HEK293E cells with reference to "Example 2.1", and the amino acid sequence information of SP34 and OKT3 is shown in Table 5 below.
  • the negative control antibody hIgG1 is the antibody anti-hel-hIgG1 against Hen Egg Lysozyme chicken egg lysozyme (purchased from Baiying, product number: B117901), hereinafter referred to as hIgG1.
  • the Gln22-Cys49 polypeptide S004pep of human CD3 ⁇ was commissioned by Gill Biochemical.
  • the prepared S004pep polypeptides were tested by ELISA with positive control antibodies that recognize different epitopes.
  • the test results are shown in Figure 1.
  • OKT3 cannot bind to S004pep polypeptides, but SP34 can bind to S004pep polypeptides.
  • the test results are consistent with those reported in the literature, indicating that they have been prepared.
  • the above-mentioned polypeptide having binding activity is obtained.
  • Amino acid sequence of S004pep polypeptide QDGNEEMGGITQTPYKVSISGTTVILTC (SEQ ID NO: 53).
  • Suspension cell Jurkat cells purchased from the cell bank of the Chinese Academy of Sciences
  • T-175 cell culture flask to the logarithmic growth phase
  • the medium supernatant was discarded by centrifugation
  • the cell pellet was washed twice with PBS.
  • the supernatant was removed by centrifugation, and the detection antibody and IgG negative control at an initial concentration of 100 nM, 1:5 serial dilution was added, 100 ⁇ l per well, the cells were mixed, and the cells were incubated on ice for 1 hour.
  • the cells were centrifuged and washed 3 times with PBS buffer, and 50 ⁇ l/well of Alexa647-labeled secondary antibody (purchased from Jackson, catalog number: 109-605-088) was added, and incubated on ice for 1 hour.
  • the cells were centrifuged and washed 5 times with PBS buffer, and the results were detected and analyzed by FACS (FACS CantoTM, purchased from BD Company).
  • MOLT4 cells purchased from the cell bank of the Chinese Academy of Sciences
  • MOLT4 cells were expanded and cultured in a T-175 cell culture flask to the logarithmic growth phase, the medium supernatant was discarded by centrifugation, and the cell pellet was washed twice with PBS.
  • FACS detection and data analysis were carried out according to the method of Example 3.1.
  • the detection antibody is SP34, OKT3 antibody, and the IgG control is hIgG1.
  • the analysis results are shown in Table 7 and FIG. 3 .
  • the results showed that MOLT4 cells did not bind to SP34 and OKT3, and MOLT4 cells could be used as negative cells for CD3 antibody screening.
  • Human PBMC cells purchased from Ocellus Biotechnology (Shanghai) Co., Ltd.
  • T Cell Activation/Expansion Kit Human (purchased from Miltenyi, Item No.: 130-091-441) to complete T cell activation.
  • Amplification experiments When the cells were cultured for 14 days, the medium supernatant was discarded by centrifugation, and the cell pellet was washed twice with PBS.
  • SP34 antibody as the primary antibody and Alexa647-labeled secondary antibody (purchased from Jackson, item number: 109-605-098)
  • FACS FACS CantoTM, purchased from BD Company
  • detection and data analysis were performed with reference to the method in Example 3.1. The analysis results are shown in Table 8 and FIG. 4 . The results showed that human PBMCs were expanded to obtain human T cells with higher CD3 protein expression.
  • Cynomolgus monkey PBMC cells (purchased from Medicilon) were completed according to the instructions of T Cell Activation/Expansion Kit, non-human primate (purchased from Miltenyi, item number: 130-092-919) to complete the cynomolgus monkey (hereinafter referred to as monkey) T cell expansion experiments.
  • monkey cynomolgus monkey
  • the medium supernatant was discarded by centrifugation, and the cell pellet was washed twice with PBS.
  • SP34 antibody as the primary antibody and Alexa647-labeled secondary antibody (purchased from Jackson, item number: 109-605-098)
  • FACS FACS CantoTM, purchased from BD Company
  • human CD3 ⁇ protein (the human CD3 ⁇ protein is CD3 ⁇ -His, purchased from Yiqiao Shenzhou, the product number is 10977-H08H) was diluted with PBS to a final concentration of 1 ⁇ g/ mL, and then added 50 ⁇ l/well to a 96-well ELISA plate, sealed with plastic film and incubated overnight at 4°C, washed the plate twice with PBST the next day, and added blocking solution [PBS+2% (w/w) BSA] at room temperature Closed for 2 hours.
  • OD450nm values were read with an ELISA plate reader (Multimode Plate Reader, EnSight, purchased from Perkin Elmer). The experimental results are shown in Figures 6A to 6F and Table 10. The IgG control was hIgG1; SP34 and I2C were positive controls. The data in the table are OD450nm values. The results indicated that all SP34 humanized antibodies had binding activity to human CD3 ⁇ protein in ELISA experiments.
  • Example 3.1 Collect Jurkat cells, MOLT4 cells and human T cells obtained after PBMC expansion, and perform FACS detection and data analysis according to the method of Example 3.1.
  • the analysis results are shown in Figures 7A-F, Figures 8A-F, Figures 9A-G and Tables 10-11, wherein the IgG control is hIgG1; SP34 and I2C are positive controls.
  • the results showed that all SP34 humanized antibodies had specific binding reaction with CD3 on the surface of Jurkat cells and human T cells.
  • SPR Surface plasmon resonance
  • the antibody to be tested was first captured with a Protein A chip, and then a single concentration of human CD3 ⁇ antigen protein was injected to record the binding and dissociation process of the antibody and the antigen protein. Finally, Glycine pH1.5 (GE Helthcare; BR-1003 -54) Complete chip regeneration. Binding was measured by injecting different concentrations of recombinant human CD3 ⁇ in solution for 240 seconds with a flow rate of 30 ⁇ L/min, starting at 200 nM (see detailed results for actual concentrations tested), diluted 1:1 for a total of 5 concentrations. The dissociation phase was monitored for up to 600 seconds and triggered by switching from sample solution to running buffer.
  • Glycine pH1.5 GE Helthcare
  • the polypeptide was diluted with PBS to a final concentration of 1 ⁇ g/mL, and then added to a 96-well ELISA plate at 50 ⁇ l/well.
  • the binding reaction between SP34 humanized antibody and CD3 ⁇ N-terminal polypeptide was detected according to the ELISA detection method in 3.1.
  • the results are shown in Figures 11A to 11F and Table 13, all the antibodies bound to the polypeptide S004pep at the ELISA level.
  • the monkey T cells obtained after PBMC expansion were collected and subjected to FACS detection and data analysis according to the method of Example 3.1.
  • the analysis results are shown in Figures 12A to 12G and Tables 14-15, wherein the IgG control is hIgG1, and SP34 and I2C are positive controls.
  • the results showed that all SP34 humanized antibodies had specific binding reactions to CD3 on the surface of monkey T cells.
  • the antibody to be tested was diluted with PBS, the initial concentration was 20 ⁇ g/mL, 1:1.5 gradient dilution, and then added to 96-well flat-bottom cell culture plate at 100 ⁇ l/well . Cover the cell culture plate, incubate at 37°C for 3 hours, and wash the plate three times with PBS.
  • Jurkat-Lucia NFAT purchased from InvivoGen, Cat. No. jktl-nfat
  • RPMI 1640 purchased from Gibco, Cat. No. 12633012

Abstract

涉及CD3人源化抗体及其应用，其公开特异性结合CD3ε亚基的人源化抗体或抗原结合片段、多特异性抗原结合分子、核酸片段、载体、宿主细胞、制备方法、药物组合物、制药用途以及，癌症或肿瘤，感染性疾病或自身免疫性疾病的治疗方法，对于开发CD3抗体药物和细胞治疗药物具有重要意义。

Description

CD3人源化抗体及其应用
本公开要求于2020年12月25日向中国专利局提交、发明名称为“CD3人源化抗体及其应用”，申请号为CN202011565227.2的中国专利申请的优先权，其全部内容通过引用结合在本公开中。
技术领域
本公开涉及抗体领域，具体而言，涉及CD3人源化抗体及其应用。
背景技术
CD3分子与T细胞(抗原)受体(T cell receptor，TCR)组成TCR-CD3复合体，在T细胞抗原识别和免疫应答产生过程中具有极其重要的作用。TCR/CD3复合体中，TCR有两个多态型亚单位(TCRαβ或TCRγδ)，TCR胞浆区非常短，主要功能是识别结合MHC分子提呈的抗原。CD3分子是T细胞膜上的重要分化抗原，是成熟T细胞的特征性标志。它由εδγζ四种链或εδγζη五种链组成。在复合体中，CD3γ、δ和ε以二种非共价键形式γε和δε异源二聚体存在；ζ链绝大多数以同源二聚体即ζ-ζ形式存在；ζ和η链是同一基因的两种不同的拼接形式，仅有10％以异源二聚体(ζ-η)形式存在，但人类η表达水平很低，人体细胞中至今还缺乏分子水平的证据来证明η蛋白产物及其在细胞中转录物的存在。CD3每条链在胞内区均有免疫受体酪氨酸活化基序(immunoreceptor tyrosine-based activation motif，简称ITAM)。经突变及嵌合分子等研究证实，ITAM是TCR信号转导的结构基础，单独分离的ITAM能够转TCR介导的信号。TCR与抗原结合后可激活一些PTKs，包括TCR相连的p59fyn、ZAP-70(ζassociated protein-70)、CD4/CD8相连的p56lck以及其它src相关的PTKs随后引起多种底物的酪氨酸磷酸化，从而传递免疫反应的下游信号。
SP34抗体为特异性结合CD3ε亚基的抗体，被用于开发抗体药物和/或细胞治疗。但SP34抗体为鼠源抗体，鼠抗具有较强的免疫原性，在临床应用中会引起人抗鼠抗体(HAMA)反应和抗抗体反应(AAR)等，导致半衰期缩短，易被清除，治疗效果减弱，严重的甚至威胁患者的生命。因此，鼠源CD3ε抗体SP34的人源化对该抗体进一步开发和成药具有重要意义。
发明内容
本公开提供一种特异性结合CD3ε亚基的人源化抗体或抗原结合片段、多特异性抗原结合分子、核酸片段、载体(vector)、宿主细胞、免疫效应细胞、制备方法、药物组合物、制药用途以及癌症或肿瘤、感染性疾病或自身免疫性疾病的治疗方法。
在第一方面，本公开提供一种特异性结合CD3ε亚基的人源化抗体或抗原结合片段，所述抗体或抗原结合片段包含：
(a)轻链可变区，其依次包含LFR1、LCDR1、LFR2、LCDR2、LFR3、LCDR3和 LFR4，其氨基酸序列分别如下所示：QTVVTQEPSLTVSPGGTVTLTC(SEQ ID NO：54)、RSSTGAVTTSNYAN(SEQ ID NO：55)、WFQQKPGQAPRALIY(SEQ ID NO：56)、GTNKRAP(SEQ ID NO：57)、WTPARFSGSLLGGKAALTLSGVQPEDEAEYYC(SEQ ID NO：58)、ALWYSNLWV(SEQ ID NO：59)和FGGGTKLTVL(SEQ ID NO：60)；
和(b)重链可变区，其依次包含HFR1、HCDR1、HFR2、HCDR2、HFR3、HCDR3和HFR4，其氨基酸序列分别如下所示：EVQLVESGGGLVQPGGSLKLSCAASGFTFS(SEQ ID NO：61)、TYAMN(SEQ ID NO：62)、WVRQASGKGLEWVG(SEQ ID NO：63)、RIRSKYNNYATYYADSVKD(SEQ ID NO：64)、RFTISRDDSKNTAYLQMNSLKTEDTAVYYCTR(SEQ ID NO：65)、HGNFGNSYVSWFAY(SEQ ID NO：66)和WGQGTLVTVSS(SEQ ID NO：67)。
在一些具体的实施方式中，所述LCDR1发生至多6个氨基酸残基突变，优选地，根据Kabat编号系统编号，所述突变至少包含选自下组的一个或多个突变：R 24S/G、T 29S、S 30G或A 33P，更优选地，所述突变至少包含R 24S、T 29S、S 30G和A 33P，或至少包含R 24G、T 29S、S 30G和A 33P；和/或，
所述LCDR2发生至多6个氨基酸残基突变；优选地，根据Kabat编号系统编号，所述突变至少包含选自下组的一个或多个突变：N 52K、K 53F、R 54L或A 55S，更优选地，所述突变至少包含N 52K、K 53F、R 54L和A 55S，或至少包含N 52K、K 53F和R 54L；和/或，
所述LCDR3发生至多6个氨基酸残基突变，优选地，根据Kabat编号系统编号所述突变至少包含选自下组的一个或多个突变：A 89S/V、L 90I、Y 92F、S 93T、L 95R或V 97T，更优选地，所述突变至少包含A 89S和L 95R，或至少包含A 89V、L 90I和L 95R，或至少包含A 89V、Y 92F和L 95R，或至少包含A 89V、S 93T和L 95R，或至少包含A 89V、L 95R和V 97T，或至少包含A 89V和L 95R；和/或，
所述HCDR1发生至多6个氨基酸残基突变，优选地，根据Kabat编号系统编号，所述突变至少包含T 31K；和/或，
所述HCDR2发生至多6个氨基酸残基突变，优选地，根据Kabat编号系统编号，所述突变至少包含N54S；和/或，
所述HCDR3发生至多6个氨基酸残基突变，优选地，根据Kabat编号系统编号，所述突变至少包含选自下组的一个或多个突变：G 96S、S 100AA、Y 100BF、V 100CI、W 100EY、F 100FW/H或Y 102F/H，更优选地，所述突变至少包含S 100AA、V 100CI、W 100EY和F 100FW，或至少包含G 96S、V 100CI、W 100EY和F 100FW，或至少包含V 100CI、W 100EY、F 100FW和Y 102F，或至少包含V 100CI、W 100EY、F 100FW和Y 102H突变，或至少包含V 100CI、W 100EY和F 100FW，或至少包含Y 100BF、V 100CI、W 100EY和F 100FW。
在一些具体的实施方式中，所述轻链可变区的框架区发生至多15个氨基酸残基突变，优选地，根据Kabat编号系统编号，所述突变至少包含选自下组的一个或多个突变：F 36V、A 46G、Y49G或W 57G，更优选地，至少包含F 36V、A 46G、Y49G和W 57G；和/或，所述重链可变区的框架区发生至多15个氨基酸残基突变，优选地，所述突变至少包含选自 下组的一个或多个突变：S 30N、S 41P、G 49A、S 82BN或T 93V，更优选地，所述突变至少包含S 30N、S 41P、G 49A、S 82BN和T 93V。
在一些具体的实施方式中，所述LCDR1、LCDR2、LCDR3、HCDR1、HCDR2或HCDR3的氨基酸残基突变数目可选自0,1,2,3,4,5或6；所述框架区的氨基酸残基突变数目可选自0,1,2,3,4,5,6,7,8,9,10,11,12,13,14或15；优选地，所述突变可选自插入、缺失或替换，所述替换优选为保守氨基酸残基的替换。
在一些具体的实施方式中，所述重链可变区具有如SEQ ID NO：9～18任一项所示的序列，和所述轻链可变区具有如SEQ ID NO：3～8任一项所示的序列；
优选地，所述重链可变区具有如SEQ ID NO：9所示的序列，和所述轻链可变区具有如SEQ ID NO：3～7任一项所示的序列；或，
所述重链可变区具有如SEQ ID NO：10所示的序列，和所述轻链可变区具有如SEQ ID NO：3～7任一项所示的序列；或，
所述重链可变区具有如SEQ ID NO：11所示的序列，和所述轻链可变区具有如SEQ ID NO：3～7任一项所示的序列；或，
所述重链可变区具有如SEQ ID NO：12所示的序列，和所述轻链可变区具有如SEQ ID NO：3～7任一项所示的序列；或，
所述重链可变区具有如SEQ ID NO：13～18任一项所示的序列，和所述轻链可变区具有如SEQ ID NO：8所示的序列。
在一些具体的实施方式中，所述抗体或抗原结合片段包括scFv结构域，所述scFv结构域包含通过连接肽(linker)连接的所述重链可变区和所述轻链可变区；
优选地，所述连接肽可选自(G 4S) n，可选地，所述n为3；
优选地，所述重链可变区位于所述连接肽N端，所述轻链可变区位于所述连接肽C端；
更优选地，所述scFv结构域具有如SEQ ID NO：19～44任一项所示的氨基酸序列。
在一些具体的实施方式中，所述抗体或抗原结合片段包含或不包含重链恒定区和/或轻链恒定区；
优选地，所述重链恒定区可选自CH1、铰链区、Fc或CH3结构域，更优选地，所述重链恒定区包含铰链区和Fc，所述Fc优选具有如SEQ ID NO：45所示序列，所述铰链区优选具有如SEQ ID NO：46所示序列；
优选地，所述重链恒定区和/或所述轻链恒定区为人重链恒定区和/或人轻链恒定区；
优选地，所述重链恒定区为IgG重链恒定区，例如IgG1重链恒定区、IgG2重链恒定区、IgG3重链恒定区或IgG4重链恒定区；
优选地，所述重链恒定区为人IgG1重链恒定区、人IgG2重链恒定区、人IgG3重链恒定区或人IgG4重链恒定区。
在一些具体的实施方式中，所述抗体或抗原结合片段选自单克隆抗体、多克隆抗体、天然抗体、工程化抗体、单特异性抗体、多特异性抗体(例如双特异性抗体)、单价抗体、多价抗体、完整抗体、抗体片段、裸抗体、缀合抗体、Fab、Fab'、Fab'-SH、F(ab') 2、Fd、 Fv、scFv、双抗体(diabody)、双特异性T细胞接合器(BiTE)或单域抗体。
在一些具体的实施方式中，所述抗体或抗原结合片段特异性结合人CD3ε亚基和/或猴CD3ε亚基，优选地，所述抗体或抗原结合片段结合人CD3ε亚基的KD为1.00E-8M～1.00E-11M，例如，1.00E-9M、2.00E-9M、3.00E-9M、4.00E-9M、5.00E-9M、6.00E-9M、7.00E-9M、8.00E-9M、9.00E-9M、1.00E-10M、2.00E-10M、3.00E-10M、4.00E-10M、5.00E-10M、6.00E-10M、7.00E-10M、8.00E-10M或9.00E-10M。
在一些具体的实施方式中，所述抗体或抗原结合片段还偶联有治疗剂或示踪剂；优选地，所述治疗剂选自放射性同位素、化疗药或免疫调节剂，所述示踪剂选自放射学造影剂、顺磁离子、金属、荧光标记、化学发光标记、超声造影剂和光敏剂。
在第二方面，本公开还提供一种多特异性抗原结合分子，所述多特异性抗原结合分子至少包括第一抗原结合模块和其他抗原结合模块，所述第一抗原结合模块包含前述的抗体或抗原结合片段，所述其他抗原结合模块特异性结合CD3ε亚基以外的抗原或结合与所述第一抗原结合模块不同的CD3ε亚基抗原表位。
在一些具体的实施方式中，所述多特异性抗原结合分子可为双特异性、三特异性或四特异性抗原结合分子；
优选地，所述CD3ε亚基以外的抗原可选自肿瘤相关抗原或传染性疾病抗原，例如可选自下组：BCMA、CD19、CD20、CD33、CD38、CD70、CD123、CD138、CD276、CDH3、CEA、c-Met、CLDN18.2、CLL-1、DLL3、DLK1、ENPP3、EpCAM、GD2、GPC3、GPRC5d、gpA33、MSLN、MUC1、MUC16、MUC17、LRRC15、ROR1、EGFR、HER2、PSMA、PSCA、PD-1、PD-L1、TPBG、TYRP1、WT1、SSTR2、HIV、甲肝病毒、乙肝病毒、丙肝病毒、疱疹病毒例如，HSV-1、HSV-2、CMV、衣原体、立克次氏体、分枝杆菌、葡萄球菌、链球菌、肺炎双球菌、脑膜炎双球菌或淋菌；
优选地，所述其他抗原结合模块为抗体或抗原结合片段；
优选地，所述多特异性抗原结合分子为双特异性T细胞结合器(BiTE)。
在第三方便，本公开还提供一种分离的核酸片段，所述核酸片段编码前述抗体、抗原结合片段或多特异性抗原结合分子。
在一些具体的实施方式中，所述核酸片段还包含编码嵌合抗原受体(CAR)的核酸；优选地，编码前述抗体、抗原结合片段或多特异性抗原结合分子的核酸片段与编码所述嵌合抗原受体的核酸片段被编码核糖体跳跃部分的核酸片段分开，所述核糖体跳跃部分可选自P2A或T2A；
优选地，所述抗体或抗原结合片段为单链抗体，例如scFv；优选地，所述多特异性抗原结合分子为双特异性T细胞结合器(BiTE)。
在第四方面，本公开还提供一种载体(vector)，所述载体包含前述核酸片段。
在第五方面，本公开还提供一种宿主细胞，所述宿主细胞包含前述载体；优选地，所述细胞为原核细胞或真核细胞，例如细菌(大肠杆菌)、真菌(酵母)、昆虫细胞或哺乳动物细胞(CHO细胞系或293细胞系)。
在第六方面，本公开还提供一种免疫效应细胞，所述免疫效应细胞包含前述核酸片段 和/或表达表达前述核酸片段编码的(a)所述嵌合抗原受体和(b)所述抗体、抗原结合片段或多特异性抗原结合分子；优选地，所述免疫效应细胞选自T细胞、NK细胞(natural killer cell)、NKT细胞(natural killer T cell)、DNT细胞(double negative T cell)、单核细胞、巨噬细胞、树突状细胞或肥大细胞，所述T细胞优选自细胞毒性T细胞、调节性T细胞或辅助性T细胞；所述免疫效应细胞优选自自体免疫效应细胞或同种异体免疫效应细胞。
在第七方面，本公开还提供一种制备前述抗体或抗原结合片段，或多特异性抗原结合分子的方法，所述方法包括培养前述细胞，以及分离所述细胞表达的抗体或抗原结合片段或多特异性抗原结合分子。
在第八方面，本公开还提供一种制备前述免疫效应细胞的方法，所述方法包括将前述核酸片段导入所述免疫效应细胞，优选地，所述方法还包括启动所述嵌合抗原受体表达、以及启动所述抗体、抗原结合片段或多特异性抗原结合分子表达。
在第九方面，本公开还提供一种药物组合物，所述药物组合物包含前述抗体或抗原结合片段、多特异性抗原结合分子、核酸片段、载体、宿主细胞、免疫效应细胞或根据前述方法制备得到的产品。
在第十方面，本公开还提供前述抗体或抗原结合片段、多特异性抗原结合分子、分离的的核酸片段、载体、宿主细胞、免疫效应细胞、药物组合物或根据前述方法制备得到的产品在制备治疗癌症或肿瘤、感染性疾病或自身免疫性疾病的药物中的应用；
优选地，所述癌症或肿瘤选自血液瘤或实体瘤，所述血液瘤或实体瘤可选自B细胞淋巴瘤、套细胞淋巴瘤、滤泡性淋巴瘤、弥漫性大B细胞淋巴瘤、非霍奇金淋巴瘤、急性淋巴细胞白血病、急性髓性白血病、多发性骨髓瘤、胃癌、子宫癌、卵巢癌、乳腺癌、肝癌、前列腺癌、胶质母细胞瘤、肺癌、胰腺癌、胆管癌、乳腺癌、结直肠癌、黑色素瘤、肾细胞癌或癌性腹水；
优选地，所述自身免疫性疾病选自类风湿性关节炎、全身性红斑狼疮、多发性硬化症、炎性肠病、重症肌无力、乳糜泻、Sjogren综合征、格雷弗氏病、克罗恩病、自身免疫性肝炎、银屑病、银屑病关节炎、哮喘、过敏性鼻炎、来自器官移植的影响或移植物抗宿主病(GVHD)；
优选地，所述感染性疾病选自HIV病毒、甲肝病毒、乙肝病毒、丙肝病毒、疱疹病毒例如，HSV-1、HSV-2、CMV、衣原体、立克次氏体、分枝杆菌、葡萄球菌、链球菌、肺炎双球菌、脑膜炎双球菌或淋菌的感染。
在第十一方面，本公开还提供一种治疗癌症或肿瘤、感染性疾病或自身免疫性疾病的方法，所述方法包括向受试者施用有效量的前述抗体或抗原结合片段、多特异性抗原结合分子、核酸片段、载体、宿主细胞、免疫效应细胞、药物组合物或根据前述方法制备得到的产品；
优选地，所述癌症或肿瘤选自血液瘤或实体瘤，所述血液瘤或实体瘤可选自B细胞淋巴瘤、套细胞淋巴瘤、滤泡性淋巴瘤、弥漫性大B细胞淋巴瘤、非霍奇金淋巴瘤、急性淋巴细胞白血病、急性髓性白血病、多发性骨髓瘤、胃癌、子宫癌、卵巢癌、乳腺癌、肝癌、前列腺癌、胶质母细胞瘤、肺癌、胰腺癌、胆管癌、乳腺癌、结直肠癌、黑色素瘤、 肾细胞癌或癌性腹水；
优选地，所述自身免疫性疾病选自类风湿性关节炎、全身性红斑狼疮、多发性硬化症、炎性肠病、重症肌无力、乳糜泻、Sjogren综合征、格雷弗氏病、克罗恩病、自身免疫性肝炎、银屑病、银屑病关节炎、哮喘、过敏性鼻炎、来自器官移植的影响或移植物抗宿主病(GVHD)；
优选地，所述感染性疾病选自HIV病毒、甲肝病毒、乙肝病毒、丙肝病毒、疱疹病毒例如，HSV-1、HSV-2、CMV、衣原体、立克次氏体、分枝杆菌、葡萄球菌、链球菌、肺炎双球菌、脑膜炎双球菌或淋菌的感染。
在第十二方面，本公开还提供前述抗体或抗原结合片段、多特异性抗原结合分子、分离的的核酸片段、载体、宿主细胞、免疫效应细胞、药物组合物或根据前述方法制备得到的产品，用于治疗癌症或肿瘤、感染性疾病或自身免疫性疾病；
优选地，所述癌症或肿瘤选自血液瘤或实体瘤，所述血液瘤或实体瘤可选自B细胞淋巴瘤、套细胞淋巴瘤、滤泡性淋巴瘤、弥漫性大B细胞淋巴瘤、非霍奇金淋巴瘤、急性淋巴细胞白血病、急性髓性白血病、多发性骨髓瘤、胃癌、子宫癌、卵巢癌、乳腺癌、肝癌、前列腺癌、胶质母细胞瘤、肺癌、胰腺癌、胆管癌、乳腺癌、结直肠癌、黑色素瘤、肾细胞癌或癌性腹水；
优选地，所述自身免疫性疾病选自类风湿性关节炎、全身性红斑狼疮、多发性硬化症、炎性肠病、重症肌无力、乳糜泻、Sjogren综合征、格雷弗氏病、克罗恩病、自身免疫性肝炎、银屑病、银屑病关节炎、哮喘、过敏性鼻炎、来自器官移植的影响或移植物抗宿主病(GVHD)；
优选地，所述感染性疾病选自HIV病毒、甲肝病毒、乙肝病毒、丙肝病毒、疱疹病毒例如，HSV-1、HSV-2、CMV、衣原体、立克次氏体、分枝杆菌、葡萄球菌、链球菌、肺炎双球菌、脑膜炎双球菌或淋菌的感染。
术语定义和说明
除非本文另外定义，与本公开相关的科学和技术术语应具有本领域普通技术人员所理解的含义。
此外，除非本文另有说明，本文单数形式的术语应包括复数形式，复数形式的术语应包括单数形式。更具体地，如在本说明书和所附权利要求中所使用的，除非另外明确指出，否则单数形式“一种”和“这种”包括复数指示物。
本文术语“包括”、“包含”和“具有”之间可互换使用，旨在表示方案的包含性，意味着所述方案可存在除所列出的元素之外的其他元素。同时应当理解，在本文中使用“包括”、“包含”和“具有”描述，也提供“由……组成”方案。
术语“和/或”在本文使用时，包括“和”、“或”和“由所属术语链接的要素的全部或任何其他组合”的含义。
本文术语“CD3”(cluster of differention 3)，是指来源于任何脊椎动物来源的分化簇3蛋白，包括哺乳动物，如灵长类动物(例如人、猴)和啮齿动物(例如小鼠和大鼠)。在哺乳动 物中，CD3分子是六条链的多蛋白质复合体，包括CD3γ链、CD3δ链、两条CD3ε链和CD3ζ链的同源二聚体，其中CD3ζ链是CD3分子的细胞内尾巴，并且CD3γ链、CD3δ链和CD3ε链全部含有表达在T细胞表面上的胞外域(ECD)。人CD3的示例性的序列包括人CD3ε蛋白(NCBI Ref Seq No.NP_000724或NCBI：AAH49847.1)、人CD3δ蛋白(NCBI Ref Seq No.NP_000723)和人CD3γ蛋白(NCBI Ref Seq No.NP_000064)。非人CD3的示例性的序列包括食蟹猴(Macaca fascicularis)(猴)CD3ε蛋白(NCBIRef Seq No.NP_001270544)、食蟹猴(Macaca fascicularis)(猴)CD3δ蛋白(NCBI Ref SeqNo.NP_001274617)、食蟹猴(Macaca fascicularis)(猴)CD3γ蛋白(NCBI Ref Seq No.NP_001270839)；小鼠CD3ε蛋白(NCBI Ref Seq No.NP_031674)、小鼠CD3δ蛋白(NCBI Ref SeqNo.NP_038515)、小鼠CD3γ蛋白(NCBI Ref Seq No.AAA37400)；褐家鼠(大鼠)CD3ε蛋白(NCBI Ref Seq No.NP_001101610)、褐家鼠(大鼠)CD3δ蛋白(NCBI Ref Seq No.NP_037301)、褐家鼠(大鼠)CD3γ蛋白(NCBI Ref Seq No.NP_001071114)。
本文术语“CD3ε”旨在包含任何形式的CD3ε亚基，例如，1)天然未经加工的CD3ε分子，“全长”CD3ε链或天然存在的CD3ε变体，包括例如剪接变体或等位基因变体；2)在细胞中的加工产生的任何形式的CD3ε；或3)通过重组方法产生的CD3ε亚单位的全长、片段(例如截短的形式、胞外域/跨膜域)或修饰的形式(例如突变的形式、糖基化/聚乙二醇化的、组氨酸-标记/免疫荧光融合的形式)。
本文术语“特异性结合”是指抗原结合分子(例如抗体)通常以高亲和力特异性结合抗原和实质上相同的抗原，但不以高亲和力结合不相关抗原。亲和力通常以平衡解离常数(equilibrium dissociation constant，KD)来反映，其中较低KD表示较高亲和力。以抗体为例，高亲和力通常指具有约1.00E-7M或更低、约1.00E-8M或更低、约1.00E-9M或更低、约1.00E-10M或更低、1.00E-11M或更低或1.00E-12M或更低的KD。KD计算方式如下：KD＝Kd/Ka，其中Kd表示解离速率，Ka表示结合速率。可采用本领域周知的方法测量平衡解离常数KD，如表面等离子共振(例如Biacore)或平衡透析法测定。
本文术语“抗原结合分子”按最广义使用，是指特异性结合抗原的分子。示例性地，抗原结合分子包括但不限于抗体或抗体模拟物。“抗体模拟物”是指能够与抗原特异性结合，但与抗体结构无关的有机化合物或结合域，示例性地，抗体模拟物包括但不限于affibody、affitin、affilin、经设计的锚蛋白重复蛋白(DARPin)、核酸适体或Kunitz型结构域肽。
本文术语“抗体”按最广义使用，是指包含来自免疫球蛋白重链可变区的足够序列和/或来自免疫球蛋白轻链可变区的足够序列，从而能够特异性结合至抗原的多肽或多肽组合。本文“抗体”涵盖各种形式和各种结构，只要它们展现出期望的抗原结合活性。本文“抗体”包括具有移植的互补决定区(CDR)或CDR衍生物的替代蛋白质支架或人工支架。此类支架包括抗体衍生的支架(其包含引入以例如稳定化抗体三维结构的突变)以及包含例如生物相容性聚合物的全合成支架。参见，例如Korndorfer et al.,2003,Proteins:Structure,Function,and Bioinformatics,53(1):121-129(2003)；Roque et al.,Biotechnol.Prog.20:639-654(2004)。此类支架还可以包括非抗体衍生的支架，例如本领域已知可用于移植CDR的支架蛋白，包括但不限于肌腱蛋白、纤连蛋白、肽适体等。
本文“抗体”包括一种典型的“四链抗体”，其属于由两条重链(HC)和两条轻链(LC)组成的免疫球蛋白；重链是指这样的多肽链，其在N端到C端的方向上由重链可变区(VH)、重链恒定区CH1结构域、铰链区(HR)、重链恒定区CH2结构域、重链恒定区CH3结构域组成；并且，当所述全长抗体为IgE同种型时，任选地还包括重链恒定区CH4结构域；轻链是在N端到C端方向上由轻链可变区(VL)和轻链恒定区(CL)组成的多肽链；重链与重链之间、重链与轻链之间通过二硫键连接，形成“Y”字型结构。由于免疫球蛋白重链恒定区的氨基酸组成和排列顺序不同，故其抗原性也不同。据此，可将本文“免疫球蛋白”分为五类，或称为免疫球蛋白的同种型，即IgM、IgD、IgG、IgA和IgE，其相应的重链分别为μ链、δ链、γ链、α链和ε链。同一类Ig根据其铰链区氨基酸组成和重链二硫键的数目和位置的差别，又可分为不同的亚类，如IgG可分为IgG1、IgG2、IgG3、IgG4，IgA可分为IgA1和IgA2。轻链通过恒定区的不同分为κ链或λ链。五类Ig中第每类Ig都可以有κ链或λ链。
本文“抗体”还包括不包含轻链的抗体，例如，由单峰驼(Camelus dromedarius)、双峰驼(Camelus bactrianus)、大羊驼(Lama glama)、原驼(Lama guanicoe)和羊驼(Vicugna pacos)等骆驼科动物产生的重链抗体(heavy-chain antibodies,HCAbs)以及在鲨等软骨鱼纲中发现的免疫球蛋白新抗原受体(Ig new antigen receptor,IgNAR)。
本文“抗体”可以来源于任何动物，包括但不限于人和非人动物，所述非人动物可选自灵长类动物、哺乳动物、啮齿动物和脊椎动物，例如骆驼科动物、大羊驼、原鸵、羊驼、羊、兔、小鼠、大鼠或软骨鱼纲(例如鲨)。
本文“抗体”包括但不限于单克隆抗体、多克隆抗体、单特异性抗体、多特异性抗体(例如双特异性抗体)、单价抗体、多价抗体、完整抗体、完整抗体的片段、裸抗体、缀合抗体、嵌合抗体、人源化抗体或全人抗体。
本文术语“单克隆抗体”是指从基本上同质的抗体群体获得的抗体，即，除了可能的变异体(例如含有天然存在的突变或在制剂的生产过程中产生，此类变体通常以少量存在)之外，包含所述群体的各个抗体是相同的和/或结合相同的表位。与通常包括针对不同决定簇(表位)的不同抗体的多克隆抗体制剂相反，单克隆抗体制剂中的每种单克隆抗体针对抗原上的单一决定簇。本文修饰语“单克隆”不应解释为需要通过任何特定方法产生所述抗体或抗原结合分子。举例来说，单克隆抗体可通过多种技术制得，包括(但不限于)杂交瘤技术、重组DNA方法、噬菌体库展示技术和利用含有全部或部分人免疫球蛋白基因座的转殖基因动物的方法和其它本领域已知的方法。
本文术语“天然抗体”是指通过多细胞生物体的免疫系统制造和配对的抗体。本文术语“工程化抗体”的抗体是指通过基因工程、抗体工程等技术获得的非天然抗体，示例性地，“工程化抗体”包括人源化抗体、小分子抗体(例如scFv等)、双特异性抗体等等。
本文术语“单特异性”是指表示具有一个或多个结合位点，其中每个结合位点结合相同抗原的相同表位。
本文术语“多特异性抗体”是指具有至少两个抗原结合位点，所述至少两个抗原结合位点中的每一个抗原结合位点与相同抗原的不同表位或与不同抗原的不同表位结合。因此，诸如“双特异性”、“三特异性”、“四特异性”等术语是指抗体/抗原结合分子可以结合 的不同表位的数目。
本文术语“价”表示抗体/抗原结合分子中规定数目的结合位点的存在。因此,术语“单价”、“二价”、“四价”和“六价”分别表示抗体/抗原结合分子中一个结合位点、两个结合位点、四个结合位点和六个结合位点的存在。
本文“全长抗体”、“完好抗体”和“完整抗体”在本文中可互换使用，是指具有基本上与天然抗体结构相似的结构。
本文“抗原结合片段”和“抗体片段”在本文中可互换使用，其不具备完整抗体的全部结构，仅包含完整抗体的局部或局部的变体，所述局部或局部的变体具备结合抗原的能力。本文“抗原结合片段”或“抗体片段”包括但不限于Fab、Fab'、Fab'-SH、F(ab') 2、Fd、Fv、scFv、双抗体(diabody)和单域抗体。
完整抗体的木瓜蛋白酶消化生成两个同一的抗原结合片段，称作“Fab”片段，每个含有重和轻链可变域，还有轻链的恒定域和重链的第一恒定域(CH1)。如此,本文术语“Fab片段”指包含轻链的VL域和恒定域(CL)的轻链片段,和重链的VH域和第一恒定域(CH1)的抗体片段。Fab’片段因在重链CH1域的羧基末端增加少数残基而与Fab片段不同,包括来自抗体铰链区的一个或多个半胱氨酸。Fab’-SH是其中恒定域的半胱氨酸残基携带游离硫醇基团的Fab’片段。胃蛋白酶处理产生具有两个抗原结合位点(两个Fab片段)和Fc区的一部分的F(ab’) 2片段。
本文术语“Fd”是指由VH和CH1结构域组成的抗体。本文术语“Fv”是指由单臂VL和VH结构域组成的抗体片段。Fv片段通常被认为是，能形成完整的抗原结合位点的最小抗体片段。一般认为，六个CDR赋予抗体的抗原结合特异性。然而，即便是一个可变区(例如Fd片段，其仅仅含有三个对抗原特异的CDR)也能够识别并结合抗原，尽管其亲和力可能低于完整的结合位点。
本文术语“scFv”(single-chain variable fragment)是指包含VL和VH结构域的单个多肽链，其中所述VL和VH通过接头(linker)相连(参见，例如,Bird等人,Science 242:423-426(1988)；Huston等人,Proc.Natl.Acad.Sci.USA 85:5879-5883(1988)；和Pluckthun，The Pharmacology of Monoclonal Antibodies，第113卷，Roseburg和Moore编，Springer-Verlag，纽约，第269-315页(1994))。此类scFv分子可具有一般结构：NH2-VL-接头-VH-COOH或NH2-VH-接头-VL-COOH。合适的现有技术接头由重复的GGGGS氨基酸序列或其变体组成。例如，可使用具有氨基酸序列(GGGGS) 4的接头，但也可使用其变体(Holliger等人(1993)，Proc.Natl.Acad.Sci.USA 90:6444-6448)。可用于本公开的其他接头由Alfthan等人(1995)，Protein Eng.8:725-731，Choi等人(2001)，Eur.J.Immunol.31:94-106，Hu等人(1996)，Cancer Res.56:3055-3061，Kipriyanov等人(1999)，J.Mol.Biol.293:41-56和Roovers等人(2001)，Cancer Immunol.描述。在一些情况下，scFv的VH与VL之间还可以存在二硫键，形成二硫键连接的Fv(dsFv)。
本文术语“双抗体(diabody)”，其VH和VL结构域在单个多肽链上表达，但使用太短的连接体以致不允许在相同链的两个结构域之间配对，从而迫使结构域与另一条链的互补结构域配对并且产生两个抗原结合部位(参见，例如,Holliger P.等人,Proc.Natl.Acad.Sci.USA 90:6444-6448(1993)，和Poljak R.J.等人,Structure  2:1121-1123(1994))。
本文术语“双特异性T细胞接合器(BiTE)”是基于串联排列的单链抗体的双特异性抗体形式，其包含两个单链可变区(scFv)，其一靶向肿瘤细胞，另一scFv靶向免疫细胞(例如，靶向CD3)，能够桥接肿瘤细胞和免疫细胞(特别是T细胞)。关于BiTE的进一步描述可参见WO 05/061547；Baeuerle,P等(2008)“BiTE:A New Class Of Antibodies That Recruit T Cells,”Drugs of the Future 33:137-147；Bargou,etal.2008)“Tumor Regression in Cancer Patients by Very Low Doses of a T Cell-Engaging Antibody,”Science 321:974-977。
本文术语“单域抗体”(single domain antibody,sdAb)、“VHH”和“纳米抗体(nanobody)”具有相同的含义并可互换使用，指克隆抗体重链的可变区，构建仅由一个重链可变区组成的单域抗体，它是具有完整功能的最小的抗原结合片段。通常先获得天然缺失轻链和重链恒定区1(CH1)的抗体后，再克隆抗体重链的可变区，构建仅由一个重链可变区组成的单域抗体。单域抗体可以衍生自骆驼科重链抗体或软骨纲鱼类IgNAR。
本文术语“裸抗体”是指不与治疗剂或示踪剂缀合的抗体；术语“缀合抗体”是指与治疗剂或示踪剂缀合的抗体。
本文术语“嵌合抗体(Chimeric antibody)”是指这样的抗体，其轻链或/和重链的一部分源自一个抗体(其可以源自某一特定物种或属于某一特定抗体类或亚类)，且轻链或/和重链的另一部分源自另一个抗体(其可以源自相同或不同的物种或属于相同或不同的抗体类或亚类)，但无论如何，其仍保留对目标抗原的结合活性(U.S.P 4,816,567to Cabilly et al.；Morrison et al.,Proc.Natl.Acad.Sci.USA,81:6851 6855(1984))。例如，术语“嵌合抗体”可包括这样的抗体(例如人鼠嵌合抗体)，其中抗体的重链和轻链可变区来自第一抗体(例如鼠源抗体)，而抗体的重链和轻链恒定区来自第二抗体(例如人抗体)。
本文术语“人源化抗体”是指，经基因工程改造的非人源抗体，其氨基酸序列经修饰以提高与人源抗体的序列的同源性。通常而言，人源化抗体的全部或部分CDR区来自于非人源抗体(供体抗体)，全部或部分的非CDR区(例如，可变区FR和/或恒定区)来自于人源免疫球蛋白(受体抗体)。人源化抗体通常保留或部分保留了供体抗体的预期性质，包括但不限于，抗原特异性、亲和性、反应性、提高免疫细胞活性的能力、增强免疫应答的能力等。
本文术语“全人抗体”是指具有其中FR和CDR二者都源自人种系免疫球蛋白序列的可变区的抗体。此外，如果抗体包含恒定区，则恒定区也源自人种系免疫球蛋白序列。本文全人抗体可以包括不由人种系免疫球蛋白序列编码的氨基酸残基(例如，通过体外随机或位点特异性诱变或通过体内体细胞突变引入的突变)。然而，本文“全人抗体”不包括其中来源于另一个哺乳动物物种(例如小鼠)的种系的CDR序列已被移植到人框架序列上的抗体。
本文术语“可变区”是指抗体重链或轻链中牵涉使抗体结合抗原的区域，“重链可变区”与“VH”、“HCVR”可互换使用，“轻链可变区”与“VL”、“LCVR”可互换使用。天然抗体的重链和轻链的可变域(分别是VH和VL)一般具有相似的结构,每个域包含四个保守的框架区(FR)和三个高变区(HVR)。参见例如Kindt et al.,Kuby Immunology,6th ed.,W.H.Freeman and Co.,p.91(2007)。单个VH或VL域可足以赋予抗原结合特异性。本文 术语“互补决定区”与“CDR”可互换使用，通常指重链可变区(VH)或轻链可变区(VL)的高变区(HVR)，该部位因在空间结构上可与抗原表位形成精密的互补，故又称为互补决定区，其中，重链可变区CDR可缩写为HCDR，轻链可变区CDR可缩写为LCDR。本术语“框架区”或“FR区”可互换，是指抗体重链可变区或轻链可变区中除CDR以外的那些氨基酸残基，HFR可指代重链可变区的框架区，LFR可指代轻链可变区的框架区。通常典型的抗体可变区由4个FR区和3个CDR区按以下顺序组成：FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4。
对于CDR的进一步描述，参考Kabat等人,J.Biol.Chem.,252:6609-6616(1977)；Kabat等人,美国卫生与公共服务部，“Sequences of proteins of immunological interest”(1991)；Chothia等人,J.Mol.Biol.196:901-917(1987)；Al-Lazikani B.等人,J.Mol.Biol.,273:927-948(1997)；MacCallum等人,J.Mol.Biol.262:732-745(1996)；Abhinandan和Martin,Mol.Immunol.,45:3832-3839(2008)；Lefranc M.P.等人,Dev.Comp.Immunol.,27:55-77(2003)；以及Honegger和Plückthun,J.Mol.Biol.,309:657-670(2001)。本文“CDR”可由本领域公知的方式加以标注和定义，包括但不限于Kabat编号系统、Chothia编号系统或IMGT编号系统，使用的工具网站包括但不限于AbRSA网站(http://cao.labshare.cn/AbRSA/cdrs.php)、abYsis网站(www.abysis.org/abysis/sequence_input/key_annotation/key_annotation.cgi)和IMGT网站(http://www.imgt.org/3Dstructure-DB/cgi/DomainGapAlign.cgi#results)。本文CDR包括不同定义方式的氨基酸残基的重叠(overlap)和子集。
本文术语“Kabat编号系统”通常是指由Elvin A.Kabat提出的免疫球蛋白比对及编号系统(参见，例如Kabat et al.,Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md.,1991)。
本文术语“Chothia编号系统”通常是指由Chothia等人提出的免疫球蛋白编号系统，其是基于结构环区的位置鉴定CDR区边界的经典规则(参见，例如Chothia&Lesk(1987)J.Mol.Biol.196:901-917；Chothia等人(1989)Nature 342:878-883)。
本文术语“IMGT编号系统”通常是指基于由Lefranc等人发起的国际免疫遗传学信息系统(The international ImMunoGeneTics information system(IMGT))的编号系统，可参阅Lefranc et al.,Dev.Comparat.Immunol.27:55-77,2003。
本文术语“重链恒定区”是指抗体重链的羧基端部分，其不直接参与抗体与抗原的结合，但是表现出效应子功能，诸如与Fc受体的相互作用，其相对于抗体的可变结构域具有更保守的氨基酸序列。“重链恒定区”至少包含：CH1结构域，铰链区，CH2结构域，CH3结构域，或其变体或片段。“重链恒定区”包括“全长重链恒定区”和“重链恒定区片段”，前者具有基本上与天然抗体恒定区基本相似的结构，而后者仅包括“全长重链恒定区的一部分”。示例性地，典型的“全长抗体重链恒定区”由CH1结构域-铰链区-CH2结构域-CH3结构域组成；当抗体为IgE时，其还包括CH4结构域；当抗体为重链抗体时，则其不包括CH1结构域。示例性地，典型的“重链恒定区片段”可选自CH1、Fc或CH3结构域。
本文术语“轻链恒定区”是指抗体轻链的羧基端部分，其不直接参与抗体与抗原的结合，所述轻链恒定区可选自恒定κ结构域或恒定λ结构域。
本文术语“Fc”是指完整抗体经木瓜蛋白酶水解而成的抗体羧基端部分，典型地，其包含抗体的CH3和CH2结构域。Fc区包括例如天然序列Fc区、重组Fc区和变体Fc区。尽管免疫球蛋白重链的Fc区的边界可以略微变化，但是人IgG重链的Fc区通常被定义为从Cys226位置的氨基酸残基或从Pro230延伸至其羧基末端。Fc区的C末端赖氨酸(根据Kabat编号系统的残基447)可以例如在抗体的产生或纯化过程中，或通过对编码抗体重链的核酸重组工程化而除去，因此，Fc区可包括或不包括Lys447。
如无其他说明，本文所述“抗体”或“抗原结合片段”氨基酸残基编号由Kabat编号系统确定，详见Kabat et al.,Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md.,1991，关于抗体可变区氨基酸残基的编号可参考以下网址： http://www.abysis.org/abysis/sequence_input/key_annotation/key_annotation.cgi。以下结合氨基酸残基突变的具体表示形式对氨基酸残基的编号方式加以说明，例如，轻链可变区突变R 24S/G，表示轻链可变区根据Kabat编号系统被编号为第24位的氨基酸残基由R突变为S，或由R突变为G，其中，“/”表示“或”的意思，下划线表示具体的编号；重链可变区突变S 100AA，表示，重链可变区根据Kabat编号系统被编号为第100A位的氨基酸残基由S突变为A，下划线表示具体编号。
本文术语“保守氨基酸”通常是指属于同一类或具有类似特征(例如电荷、侧链大小、疏水性、亲水性、主链构象和刚性)的氨基酸。示例性地，下述每组内的氨基酸属于彼此的保守氨基酸残基，组内氨基酸残基的替换属于保守氨基酸的替换：
(1)丙氨酸(A)、丝氨酸(S)、苏氨酸(T)；
(2)天冬氨酸(D)、谷氨酸(E)；
(3)天冬酰胺(N)、谷氨酰胺(Q)；
(4)精氨酸(R)、赖氨酸(K)、组氨酸(H)；
(5)异亮氨酸(I)、亮氨酸(L)、甲硫氨酸(M)、缬氨酸(V)；和
(6)苯丙氨酸(F)、酪氨酸(Y)、色氨酸(W)。
本文术语“核酸”包括包含核苷酸的聚合物的任何化合物和/或物质。每个核苷酸由碱基，特别是嘌呤或嘧啶碱基(即胞嘧啶(C)、鸟嘌呤(G)、腺嘌呤(A)、胸腺嘧啶(T)或尿嘧啶(U))、糖(即脱氧核糖或核糖)和磷酸基团组成。通常，核酸分子由碱基的序列描述，由此所述碱基代表核酸分子的一级结构(线性结构)。碱基的序列通常表示为5′至3′。在本文中，术语核酸分子涵盖脱氧核糖核酸(DNA)，包括例如互补DNA(cDNA)和基因组DNA、核糖核酸(RNA)，特别是信使RNA(mRNA)、DNA或RNA的合成形式，以及包含两种或更多种这些分子的混合的聚合物。核酸分子可以是线性的或环状的。此外，术语核酸分子包括有义链和反义链二者，以及单链和双链形式。而且，本文所述的核酸分子可含有天然存在的或非天然存在的核苷酸。非天然存在的核苷酸的例子包括具有衍生的糖或磷酸骨架键合或化学修饰的残基的修饰的核苷酸碱基。核酸分子还涵盖DNA和RNA分子，其适合作为载体用于在体外和/或体内，例如在宿主或患者中，直接表达本公开的抗体。此类DNA(例如cDNA)或RNA(例如mRNA)载体可以是未修饰的或修饰的。例如，可以对mRNA进行 化学修饰以增强RNA载体的稳定性和/或被编码分子的表达，从而可以将mRNA注入到受试者内以在体内产生抗体(参见例如Stadler等人，Nature Medicine 2017，published online 2017年6月12日，doi：10.1038/nm.4356或EP 2 101 823 B1)。本文“分离的”核酸指已经与其天然环境的组分分开的核酸分子。分离的核酸包括在下述细胞中含有的核酸分子，所述细胞通常含有该核酸分子，但该核酸分子存在于染色体外或存在于不同于其天然染色体位置的染色体位置处。
本文术语“嵌合抗原受体(CAR)”是指经改造以在免疫效应细胞上表达并且特异性结合抗原的人工细胞表面受体，其包含至少(1)细胞外抗原结合结构域，例如抗体的可变重链或轻链，(2)锚定CAR进入免疫效应细胞的跨膜结构域，和(3)胞内信号传导结构域。CAR能够利用细胞外抗原结合结构域以非MHC限制性的方式将T细胞和其它免疫效应细胞重定向至所选择的靶标，例如癌细胞。
本文术语“载体”是指能够扩增与其连接的另一个核酸的核酸分子。该术语包括作为自我复制型核酸结构的载体以及整合入已引入该载体的宿主细胞的基因组中的载体。某些载体能够指导与它们可操作连接的核酸的表达。这样的载体在本文中称为“表达载体”。
本文术语“宿主细胞”是指细胞中引入外源核酸的细胞，包括这种细胞的后代。宿主细胞包括“转化体”和“经转化的细胞”，其包括原代的经转化的细胞和来源于其的后代，而不考虑传代的次数。后代在核酸内容物上可能与亲本细胞不完全相同，而是可以包含突变。本文中包括具有与在初始转化的细胞中筛选或选择的相同功能或生物学活性的突变体后代。
本文术语“药物组合物”是指这样的制剂，其以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，并且不含有对施用所述药物组合物的受试者具有不可接受的毒性的另外的成分。
本文术语“治疗”是指外科手术或药物处理(surgical or therapeutic treatment)，其目的是预防、减缓(减少)治疗对象中不希望的生理变化或病变，如癌症、自身免疫性疾病和病毒感染的进展。有益的或所希望的临床结果包括但不限于症状的减轻、疾病程度减弱、疾病状态稳定(即，未恶化)、疾病进展的延迟或减慢、疾病状态的改善或缓和、以及缓解(无论是部分缓解或完全缓解)，无论是可检测的或不可检测的。需要治疗的对象包括已患有病症或疾病的对象以及易于患上病症或疾病的对象或打算预防病症或疾病的对象。当提到减缓、减轻、减弱、缓和、缓解等术语时，其含义也包括消除、消失、不发生等情况。
本文术语“受试者”是指接受对如本公开所述的特定疾病或病症的治疗的生物体，示例性地，其包括接受疾病或病症治疗的哺乳动物，如人、灵长类动物(例如，猴)或非灵长类哺乳动物。
本文术语“有效量”指单独给予或与另一治疗剂组合给予细胞、组织或对象时能有效防止或缓解疾病病症或该疾病进展的治疗剂用量。“有效量”还指足以缓解症状，例如治疗、治愈、防止或缓解相关医学病症，或治疗、治愈、防止或缓解这些病症的速度增加的化合物用量。当将活性成分单独给予个体时，治疗有效剂量单指该成分。当应用某一组合时，治疗有效剂量指产生治疗作用的活性成分的组合用量，而无论是组合、连续或同时给 予。
本文术语“自身免疫性疾病”是指对象对其自身的细胞、组织和/或器官产生免疫反应而引起的细胞、组织和/或器官损伤的病症。
本文术语“癌症”指向或描述哺乳动物中典型地以不受调节的细胞生长为特征的生理状况。此定义中包括良性和恶性癌症。本文术语“肿瘤”或“瘤”是指所有赘生性(neoplastic)细胞生长和增殖,无论是恶性的还是良性的,及所有癌前(pre-cancerous)和癌性细胞和组织。术语“癌症”和“肿瘤”在本文中提到时并不互相排斥。
本文术语“EC50”是指半最大有效浓度，其包括在指定暴露时间之后诱导基线与最大值之间的半途响应的抗体浓度。EC50本质上代表其中观察到其最大作用的50％的抗体浓度，可通过本领域已知方法测量。
附图说明
图1为ELISA检测对照抗体与人S004pep多肽的结合反应；
图2为SP34，OKT3抗体检测内源性细胞Jurkat的CD3表达情况；
图3为SP34，OKT3抗体检测内源性阴性细胞MOLT4的CD3表达情况；
图4为SP34抗体检测与人T细胞表面CD3的结合反应；
图5为SP34抗体检测与猴T细胞表面CD3的结合反应；
图6A～图6F为ELISA检测SP34人源化抗体与人CD3ε蛋白的结合反应；
图7A～图7F为FACS检测SP34人源化抗体与人Jurkat细胞的结合反应；
图8A～图8F为FACS检测SP34人源化抗体与人MOLT4细胞的结合反应；
图9A～图9G为FACS检测SP34人源化抗体与人T细胞的结合反应；
图10A～图10C为SPR检测SP34人源化抗体与人CD3ε蛋白的亲和力；
图11A～图11F为ELISA检测SP34人源化抗体与多肽的结合反应；
图12A～图12G为FACS检测SP34人源化抗体与猴T细胞的交叉结合反应；
图13A～图13H为荧光素酶报告基因实验检测SP34人源化抗体的功能；
具体实施方式
下面结合具体实施例来进一步描述本公开，本公开的优点和特点将会随着描述而更为清楚。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
本公开实施例仅是范例性的，并不对本公开的范围构成任何限制。本领域技术人员应该理解的是，在不偏离本公开的精神和范围下可以对本公开技术方案的细节和形式进行修改或替换，但这些修改和替换均落入本公开的保护范围内。
实施例1 SP34的人源化设计
通过比对IMGT(http://imgt.cines.fr)人类抗体重轻链可变区种系基因数据库，分别挑选IGLV7-43*01和IGLJ2*01作为鼠源抗体SP34选择的人源化轻链模板，IGHV3-73*01和IGHJ1*01作为人源化重链模板。首先，将鼠源抗体SP34的CDR分别移植到其人源模板的FR中，形成次序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4的可变区序列。根据抗体的三维结构模拟，将人源化抗体的FR区序列中关键氨基酸进行回复突变为鼠源抗体对应的氨基酸，以保证原有的亲和力。另外，将人源化抗体的CDR区序列中的部分氨基酸突变为人源模板对应的同源氨基酸以提高人源化程度，或者突变为其他氨基酸以优化分子的理化性质，得到人源化抗CD3单克隆抗体。 本实施例抗体氨基酸残基的编号和CDR区均 由Kabat编号系统确定并注释(详见 http://www.abysis.org/abysis/sequence_input/key_annotation/key_annotation.cgi)。通过上述方法设计得到的人源化抗体VL、VH分别见表1和表2。
表1 SP34的人源化VL的设计
Figure PCTCN2021140901-appb-000001
表2 SP34的人源化VH的设计
Figure PCTCN2021140901-appb-000002
Figure PCTCN2021140901-appb-000003
SP34抗体VL具体序列如下所示(SEQ ID NO：1)：
Figure PCTCN2021140901-appb-000004
SP34抗体VH序列如下所示(SEQ ID NO：2)：
Figure PCTCN2021140901-appb-000005
SP34人源化VL具体序列如下，其中下划线为CDR区域，其余为框架区，加粗表示在CDR区或框架区发生突变的氨基酸残基。
>SP34.VL1(SEQ ID NO：3)
Figure PCTCN2021140901-appb-000006
>SP34.VL2(SEQ ID NO：4)
Figure PCTCN2021140901-appb-000007
>SP34.VL3(SEQ ID NO：5)
Figure PCTCN2021140901-appb-000008
>SP34.VL4(SEQ ID NO：6)
Figure PCTCN2021140901-appb-000009
>SP34.VL5(SEQ ID NO：7)
Figure PCTCN2021140901-appb-000010
>SP34.VL6(SEQ ID NO：8)
Figure PCTCN2021140901-appb-000011
SP34人源化VH具体序列如下：
>SP34.VH1(SEQ ID NO:9)
Figure PCTCN2021140901-appb-000012
Figure PCTCN2021140901-appb-000013
>SP34.VH2(SEQ ID NO:10)
Figure PCTCN2021140901-appb-000014
>SP34.VH3(SEQ ID NO:11)
Figure PCTCN2021140901-appb-000015
>SP34.VH4(SEQ ID NO:12)
Figure PCTCN2021140901-appb-000016
>SP34.VH5(SEQ ID NO:13)
Figure PCTCN2021140901-appb-000017
>SP34.VH6(SEQ ID NO:14)
Figure PCTCN2021140901-appb-000018
>SP34.VH7(SEQ ID NO:15)
Figure PCTCN2021140901-appb-000019
>SP34.VH8(SEQ ID NO:16)
Figure PCTCN2021140901-appb-000020
>SP34.VH9(SEQ ID NO:17)
Figure PCTCN2021140901-appb-000021
>SP34.VH10(SEQ ID NO:18)
Figure PCTCN2021140901-appb-000022
对表1、表2的SP34人源化抗体轻、重链可变区的突变设计进行交叉组合，最终获得多种SP34人源化抗体，各人源化抗体对应VL、VH(参见表3-表4)，其中，S004-2-01表示SP34人源化抗体S004-2-01具有如SP34.VL1所述轻链可变区和如SP34.VH1所述重链可变区，其它依此类推。
表3 SP34人源化抗体对应VL、VH
Fv SP34.VH1 SP34.VH2 SP34.VH3 SP34.VH4
SP34.VL1 S004‐2‐01 S004‐2‐06 S004‐2‐11 S004‐2‐16
SP34.VL2 S004‐2‐02 S004‐2‐07 S004‐2‐12 S004‐2‐17
SP34.VL3 S004‐2‐03 S004‐2‐08 S004‐2‐13 S004‐2‐18
SP34.VL4 S004‐2‐04 S004‐2‐09 S004‐2‐14 S004‐2‐19
SP34.VL5 S004‐2‐05 S004‐2‐10 S004‐2‐15 S004‐2‐20
表4 SP34人源化抗体对应VL、VH
Fv SP34.VH5 SP34.VH6 SP34.VH7 SP34.VH8 SP34.VH9 SP34.VH10
SP34.VL6 S004‐2‐21 S004‐2‐22 S004‐2‐23 S004‐2‐24 S004‐2‐25 S004‐2‐26
将表3-4所示SP34人源化抗体构建scFv抗体(形式：VH-(G 4S) 3-VL)，具体序列如下：
>S004-2-01
Figure PCTCN2021140901-appb-000023
>S004-2-02
Figure PCTCN2021140901-appb-000024
>S004-2-03
Figure PCTCN2021140901-appb-000025
>S004-2-04
Figure PCTCN2021140901-appb-000026
>S004-2-05
Figure PCTCN2021140901-appb-000027
>S004-2-06
Figure PCTCN2021140901-appb-000028
>S004-2-07
Figure PCTCN2021140901-appb-000029
>S004-2-08
Figure PCTCN2021140901-appb-000030
Figure PCTCN2021140901-appb-000031
>S004-2-09
Figure PCTCN2021140901-appb-000032
>S004-2-10
Figure PCTCN2021140901-appb-000033
>S004-2-11
Figure PCTCN2021140901-appb-000034
>S004-2-12
Figure PCTCN2021140901-appb-000035
>S004-2-13
Figure PCTCN2021140901-appb-000036
>S004-2-14
Figure PCTCN2021140901-appb-000037
>S004-2-15
Figure PCTCN2021140901-appb-000038
>S004-2-16
Figure PCTCN2021140901-appb-000039
>S004-2-17
Figure PCTCN2021140901-appb-000040
>S004-2-18
Figure PCTCN2021140901-appb-000041
>S004-2-19
Figure PCTCN2021140901-appb-000042
>S004-2-20
Figure PCTCN2021140901-appb-000043
>S004-2-21
Figure PCTCN2021140901-appb-000044
>S004-2-22
Figure PCTCN2021140901-appb-000045
>S004-2-23
Figure PCTCN2021140901-appb-000046
>S004-2-24
Figure PCTCN2021140901-appb-000047
>S004-2-25
Figure PCTCN2021140901-appb-000048
Figure PCTCN2021140901-appb-000049
>S004-2-26
Figure PCTCN2021140901-appb-000050
实施例2抗体及蛋白的制备
2.1 SP34人源化抗体的制备
SP34人源化抗体以scFv的形式进行表达，重链可变区和轻链可变区之间通过由15个氨基酸(GGGGSGGGGSGGGGS)组成的肽段进行连接(详见实施例1的SEQ ID NO：19-44)。抗体表达纯化由泰州市百英生物科技有限公司负责。直接合成包含信号肽和scFv-铰链区-hFc的DNA序列(人IgG1Fc序列如SEQ ID NO：45，铰链区序列如SEQ ID NO：46，信号肽序列如SEQ ID NO:68)，然后克隆到表达载体BI3.4-huIgG1(来自Biointron)中，并按已建立的标准分子生物学方法制备质粒，具体方法参见Sambrook,J.,Fritsch,E.F.,and Maniatis,T.(1989).Molecular Cloning:A Laboratory Manual,Second Edition(Plainview,New York:Cold Spring Harbor Laboratory Press)。将表达载体按照PEI(购自Polysciences，货号：24765-1)说明书瞬时转染HEK293E细胞(购自中国科学院典型培养物保藏委员会细胞库)并使用FreeStyle  TM 293(Thermofisher scientific，货号：12338018)在37℃下连续培养5天，离心去除细胞成分，获得含SP34人源化抗体的培养上清液。将培养上清液上样到蛋白A层析柱(蛋白A填料AT Protein A Diamond和层析柱BXK16/26均购自博格隆，货号：AA0273和B-1620)，使用PBS磷酸盐缓冲液(pH7.4)清洗后再用20mM PB，1M NaCl(pH 7.2)进行清洗，最后使用pH3.4的柠檬酸缓冲液进行洗脱，收集从蛋白A层析柱上洗脱下来的带Fc标签的SP34人源化抗体，用1/10体积的pH8.0的1M Tris中和，用PBS在4℃条件透析过夜，透析后的蛋白经0.22微米滤膜无菌过滤后分装于-80℃保存。
人IgG1Fc的序列:
Figure PCTCN2021140901-appb-000051
铰链区序列:EPKSADKTHTCPPCP(SEQ ID NO：46)；
信号肽：MGWSWILLFLLSVTAGVHS(SEQ ID NO:68)。
2.2对照抗体的制备
阳性对照抗体(序列信息详见SEQ ID NO:1‐2,SEQ ID NO:47‐52)：SP34，OKT3，I2C，均识别人CD3的抗体，其中SP34和I2C克隆识别人CD3ε蛋白、CD3γ/ε异源二聚体和CD3ε/δ异源二聚体；OKT3克隆仅识别人CD3ε/δ异源二聚体。由泰州市百英生物科技有限公司完成阳性对照抗体的质粒构建及抗体生产纯化工作，具体方法如下：I2C，参照“实施例2.1”，构建scFv(I2C)‐铰链区‐hFc的形式抗体，以下简称I2C。SP34，OKT3：将其轻链可变区序列克隆到包含信号肽和人源抗体IgG1的轻链恒定区的表达载体 pcDNA3.4-B1HLK，重链可变区序列分别克隆到包含信号肽(SEQ ID NO:68)和人源抗体IgG1的重链恒定区的表达载体pcDNA3.4-B1HH1，获得SP34‐hIgG1，OKT3‐hIgG1的序列，以下简称SP34，OKT3。参照“实施例2.1”在HEK293E细胞中进行表达并纯化，SP34和OKT3的氨基酸序列信息如下表5所示。
阴性对照抗体hIgG1为针对Hen Egg Lysozyme鸡卵溶菌酶的抗体anti‐hel‐hIgG1(购自百英，货号：B117901)，以下简称hIgG1。
表5对照抗体序列
Figure PCTCN2021140901-appb-000052
2.3 S004多肽的制备
委托吉尔生化生产人源CD3ε(NCBI：AAH49847.1)的Gln22-Cys49多肽S004pep。对制备的上述S004pep多肽用识别不同表位的阳性对照抗体进行ELISA检测，检测结果如图1所示，OKT3不能结合S004pep多肽，SP34能结合S004pep多肽，检测结果与文献报道的一致，说明已经制备获得具有结合活性的上述多肽。S004pep多肽的氨基酸序列：QDGNEEMGGITQTPYKVSISGTTVILTC(SEQ ID NO：53)。
实施例3：内源性细胞株的鉴定
3.1内源性表达CD3细胞株的鉴定
将悬浮细胞Jurkat细胞(采购于中国科学院细胞库)在T-175细胞培养瓶中扩大培养至对数生长期，离心弃去培养基上清，细胞沉淀用PBS洗涤2次。对上一步的细胞进行细胞计数后将细胞沉淀用[PBS+2％(w/w)FBS]封闭液重悬至4x10 6个细胞/毫升，按50μl/孔加入到96孔FACS反应板中，离心去除上清，加入起始浓度100nM，1:5梯度稀释的检 测抗体及IgG阴性对照，每孔100μl，将细胞混匀，冰上孵育1小时。用PBS缓冲液离心洗涤3次，加入50μl/孔Alexa647标记的二抗(购自Jackson，货号：109-605-088)，冰上孵育1小时。用PBS缓冲液离心洗涤5次，用FACS(FACS CantoTM，购自BD公司)检测和分析结果。通过软件(CellQuest)进行数据分析，得到细胞的平均荧光强度(MFI)。再通过软件(GraphPad Prism8)分析，进行数据拟合，计算EC50值。其中检测抗体为SP34，OKT3抗体，对照为hIgG1。分析结果如表6以及图2所示。结果表明：Jurkat细胞可与SP34及OKT3结合，与hIgG1不结合，Jurkat细胞可作为CD3抗体筛选的阳性细胞。
表6内源性细胞系Jurkat的FACS检测结果
Figure PCTCN2021140901-appb-000053
3.2不表达CD3细胞株的鉴定
将MOLT4细胞(采购于中国科学院细胞库)在T-175细胞培养瓶中扩大培养至对数生长期，离心弃去培养基上清，细胞沉淀用PBS洗涤2次。按照实施例3.1的方法进行FACS检测与数据分析。其中检测抗体为SP34，OKT3抗体，IgG对照为hIgG1。分析结果如表7以及图3所示。结果表明：MOLT4细胞不与SP34及OKT3结合，MOLT4细胞可作为CD3抗体筛选的阴性细胞。
表7内源性细胞系MOLT4的FACS检测结果
Figure PCTCN2021140901-appb-000054
3.3人T细胞的扩增
将人PBMC细胞(采购于澳赛尔斯生物技术(上海)有限公司)按照T Cell Activation/Expansion Kit,human(购自美天旎，货号：130-091-441)的说明书描述完成T细胞的扩增实验。当细胞培养至14天时，离心弃去培养基上清，细胞沉淀用PBS洗涤2次。用SP34抗体作为一抗，Alexa647标记的二抗(购自Jackson，货号：109-605-098)，参照实施例3.1的方法进行FACS(FACS CantoTM，购自BD公司)检测和数据分析。分析结果如表8以及图4所示。结果表明人PBMC经扩增得到较高CD3蛋白表达的人T细胞。
表8人T细胞的FACS检测结果
Figure PCTCN2021140901-appb-000055
Figure PCTCN2021140901-appb-000056
3.4食蟹猴T细胞的扩增
将食蟹猴PBMC细胞(采购于美迪西)按照T Cell Activation/Expansion Kit,non-human primate(购自美天旎，货号：130-092-919)的说明书描述完成食蟹猴(以下简称猴)T细胞的扩增实验。当细胞培养至14天时，离心弃去培养基上清，细胞沉淀用PBS洗涤2次。用SP34抗体作为一抗，Alexa647标记的二抗(购自Jackson，货号：109-605-098)，参照实施例3.1的方法进行FACS(FACS CantoTM，购自BD公司)检测和数据分析。分析结果如表9以及图5所示。结果表明：猴PBMC经扩增后，得到有较高的CD3蛋白表达的猴T细胞。
表9猴T细胞的FACS检测结果
Figure PCTCN2021140901-appb-000057
实施例4 SP34人源化抗体与人CD3ε的结合能力鉴定
4.1酶联免疫吸附实验(ELISA)检测抗体与人CD3ε蛋白的结合
为检测SP34人源化抗体与人CD3ε蛋白的结合活性，将人CD3ε蛋白(所述人CD3ε蛋白为CD3ε-His，购自义翘神州，货号为10977-H08H)用PBS稀释到终浓度1μg/mL，然后以50μl/孔加到96孔ELISA板，用塑料膜封好4℃孵育过夜，第二天用PBST洗板2次，加入封闭液[PBS+2％(w/w)BSA]室温封闭2小时。倒掉封闭液，加入起始浓度100nM，1:10梯度稀释的抗体或阴性对照抗体50μl/孔。37℃孵育2小时后，用PBST洗板3次。加入HRP(辣根过氧化物酶)标记的二抗(购自Sigma，货号：A0170)，37℃孵育半小时后，用PBST洗板5次。加入TMB底物50μl/孔，室温孵育10～15分钟后，加入终止液(1.0N HCl)50μl/孔。用ELISA读板机(Multimode Plate Reader，EnSight，购自Perkin Elmer)读取OD450nm数值。实验结果如图6A～6F和表10所示。其中IgG对照为hIgG1；SP34、I2C为阳性对照。表中的数据为OD450nm值。结果说明，所有SP34人源化抗体与人CD3ε蛋白在ELISA实验中有结合活性。
表10 SP34人源化抗体与CD3ε蛋白结合ELISA水平的结合反应
Figure PCTCN2021140901-appb-000058
Figure PCTCN2021140901-appb-000059
4.2流式细胞实验(FACS)检测抗体与CD3ε表达细胞的结合
收集Jurkat细胞，MOLT4细胞和PBMC扩增后得到的人T细胞，按照实施例3.1的方法进行FACS检测与数据分析。分析结果如图7A-F、图8A-F、图9A-G以及表10-11所示，其中IgG对照为hIgG1；SP34、I2C为阳性对照。结果表明，所有SP34人源化抗体与Jurkat细胞及人T细胞表面的CD3有特异性结合反应。
表10 SP34人源化抗体与CD3ε表达细胞FACS水平的结合反应
Figure PCTCN2021140901-appb-000060
Figure PCTCN2021140901-appb-000061
表11 SP34人源化抗体与CD3ε表达细胞FACS水平的结合反应
Figure PCTCN2021140901-appb-000062
4.3表面等离子共振(SPR)检测抗体与人CD3ε蛋白的亲和力
使用表面等离子共振(SPR)方法来测定SP34人源化抗体与人CD3ε蛋白(所述人CD3ε蛋白为CD3ε-His，购自义翘神州，货号为10977-H08H)的亲和力，实验方法如下：采用Protein A芯片(GE Helthcare；29-127-558)来捕获抗人CD3抗体，运行缓冲液是HBS-EP+(10mM HEPES,150mM NaCl,3mM EDTA,0.05％surfactant P20)(GE Healthcare；BR-1006-69)。流经池设置为25℃。样品块设置为16℃。两者都用运行缓冲液预处理。在每一个循环中，首先用Protein A芯片捕获待测抗体，然后注入单一浓度的人CD3ε抗原蛋白，记录抗体和抗原蛋白的结合和解离过程，最后用Glycine pH1.5(GE Helthcare；BR-1003-54)完成芯片再生。通过注射溶液中不同浓度的重组人CD3ε持续240秒来测量结合，其中流速为30μL/分钟，从200nM起始(测试的实际浓度见详细结果)，以1：1稀释，总共5个浓度。监测解离相长达600秒，并通过从样品溶液切换到运行缓冲液触发。通过用10mM甘氨酸溶液(pH 1.5)以30μL/分钟的流速洗涤30秒，再生表面。通过减去从山羊抗人Fc表面获得的响应来校正本体折射率(Bulk refractive index)差异，同时减去空白注射(＝双重参照)。为了计算表观KD值和其他动力学参数，使用Langmuir 1：1模型。SP34人源化抗体与人CD3ε蛋白的结合速率(Kd)、解离速率(Ka)及结合亲和力(KD)如表12和图10A～图10C所示，所有抗体的亲和力都优于7.18E-09M。
表12 SPR检测SP34人源化抗体与人CD3ε蛋白结合的亲和力
抗体名称 ka(1/Ms) kd(1/s) KD(M)
S004‐2‐01 3.26E+05 1.38E‐04 4.23E‐10
S004‐2‐02 2.76E+05 1.86E‐04 6.74E‐10
S004‐2‐03 3.17E+05 1.89E‐04 5.96E‐10
S004‐2‐04 3.34E+05 1.87E‐04 5.60E‐10
S004‐2‐05 3.68E+05 1.92E‐04 5.22E‐10
S004‐2‐06 7.90E+05 2.35E‐04 2.97E‐10
S004‐2‐07 8.08E+05 2.33E‐04 2.88E‐10
S004‐2‐08 8.87E+05 2.08E‐04 2.34E‐10
S004‐2‐09 9.36E+05 2.25E‐04 2.41E‐10
S004‐2‐10 8.63E+05 1.76E‐04 2.04E‐10
S004‐2‐11 4.08E+05 1.86E‐04 4.56E‐10
S004‐2‐12 3.94E+05 2.08E‐04 5.28E‐10
S004‐2‐13 4.59E+05 2.16E‐04 4.71E‐10
S004‐2‐14 4.12E+05 1.97E‐04 4.79E‐10
S004‐2‐15 4.94E+05 1.93E‐04 3.91E‐10
S004‐2‐16 4.72E+05 1.89E‐04 4.00E‐10
S004‐2‐17 4.11E+05 2.08E‐04 5.06E‐10
S004‐2‐18 5.12E+05 2.21E‐04 4.31E‐10
S004‐2‐19 5.38E+05 2.21E‐04 4.11E‐10
S004‐2‐20 5.43E+05 1.87E‐04 3.44E‐10
S004‐2‐21 2.68E+05 3.70E‐04 1.38E‐09
抗体名称 ka(1/Ms) kd(1/s) KD(M)
S004‐2‐22 3.75E+05 3.64E‐04 9.70E‐10
S004‐2‐23 4.02E+05 4.02E‐04 9.99E‐10
S004‐2‐24 5.25E+05 4.01E‐04 7.63E‐10
S004‐2‐25 4.01E+05 3.97E‐04 9.91E‐10
S004‐2‐26 4.28E+04 3.07E‐04 7.18E‐09
SP34 1.13E+06 1.54E‐04 1.36E‐10
I2C 3.61E+05 3.80E‐04 1.05E‐09
实施例5 SP34人源化抗体结合位点的鉴定
5.1酶联免疫吸附实验(ELISA)检测SP34人源化抗体与S004pep的结合
为检测SP34人源化抗体与多肽S004pep的结合活性，将多肽用PBS稀释到终浓度1μg/mL，然后以50μl/孔加到96孔ELISA板。根据3.1的ELISA检测方法检测SP34人源化抗体与CD3εN端多肽的结合反应。结果如图11A～11F及表13所示，所有抗体与多肽S004pep在ELISA水平有结合。
表13 SP34人源化抗体与S004pep多肽结合ELISA水平的结合反应
Figure PCTCN2021140901-appb-000063
Figure PCTCN2021140901-appb-000064
实施例6 SP34人源化抗体交叉结合活性的鉴定
6.1流式细胞实验(FACS)检测抗体与猴T细胞的结合
收集PBMC扩增后得到的猴T细胞按照实施例3.1的方法进行FACS检测与数据分析。分析结果如图12A～图12G以及表14-15所示，其中IgG对照为hIgG1，SP34和I2C为阳性对照。结果表明，所有SP34人源化抗体与猴T细胞表面的CD3有特异性结合反应。
表14 SP34人源化抗体与猴T细胞的结合
Figure PCTCN2021140901-appb-000065
Figure PCTCN2021140901-appb-000066
表15 SP34人源化抗体与猴T细胞的结合
Figure PCTCN2021140901-appb-000067
实施例7 SP34人源化抗体的功能鉴定
7.1荧光素酶报告基因(report assay)检测SP34人源化抗体的功能
为检测SP34人源化抗体激活CD3细胞内的信号通路，将待检测的抗体用PBS稀释，起始浓度20μg/mL，1:1.5梯度稀释，然后以100μl/孔加到96孔平底细胞培养板。盖好细胞培养板的盖子，37℃孵育3小时后，用PBS洗板3次。收集处于对数生长期的Jurkat-Lucia NFAT(购自InvivoGen，货号jktl-nfat)，用含2％FBS的培养基(RPMI 1640，采购于Gibco，货号12633012)调节细胞浓度至2e6/mL，将细胞加入洗好的细胞培养板，200uL每孔。在37℃的培养箱中孵育24小时后400g离心5分钟，取上清20uL至黑色不透明96孔检测板中，加入50uL检测试剂(采购于QUANTI-Luc，品牌：Invivogen，货号：rep-qlc2)，通过PerkiElmer Ensightm酶标仪读取响应值，再通过软件(GraphPad Prism8)分析，进行数据拟合，计算EC50值。结果如表16及图13A～图13H所示，在report assay中，所有抗体能激活Jurkat细胞的下游信号通路。
表16荧光素酶报告基因检测SP34人源化抗体的功能
Figure PCTCN2021140901-appb-000068
Figure PCTCN2021140901-appb-000069
表17荧光素酶报告基因检测SP34人源化抗体的功能
Figure PCTCN2021140901-appb-000070

Claims (23)

  1. 一种特异性结合CD3ε亚基的人源化抗体或抗原结合片段，其中，所述抗体或抗原结合片段包含：
    (b)轻链可变区，其依次包含LFR1、LCDR1、LFR2、LCDR2、LFR3、LCDR3和LFR4，其氨基酸序列分别如下所示：QTVVTQEPSLTVSPGGTVTLTC(SEQ ID NO：54)、RSSTGAVTTSNYAN(SEQ ID NO：55)、WFQQKPGQAPRALIY(SEQ ID NO：56)、GTNKRAP(SEQ ID NO：57)、WTPARFSGSLLGGKAALTLSGVQPEDEAEYYC(SEQ ID NO：58)、ALWYSNLWV(SEQ ID NO：59)和FGGGTKLTVL(SEQ ID NO：60)；和，
    (c)重链可变区，其依次包含HFR1、HCDR1、HFR2、HCDR2、HFR3、HCDR3和HFR4，其氨基酸序列分别如下所示：EVQLVESGGGLVQPGGSLKLSCAASGFTFS(SEQ ID NO：61)、TYAMN(SEQ ID NO：62)、WVRQASGKGLEWVG(SEQ ID NO：63)、RIRSKYNNYATYYADSVKD(SEQ ID NO：64)、RFTISRDDSKNTAYLQMNSLKTEDTAVYYCTR(SEQ ID NO：65)、HGNFGNSYVSWFAY(SEQ ID NO：66)和WGQGTLVTVSS(SEQ ID NO：67)。
  2. 根据权利要求1所述的抗体或抗原结合片段，其中，所述LCDR1发生至多6个氨基酸残基突变，优选地，根据Kabat编号系统编号，所述突变至少包含选自下组的一个或多个突变：R 24S/G、T 29S、S 30G或A 33P，更优选地，所述突变至少包含R 24S、T 29S、S 30G和A 33P，或至少包含R 24G、T 29S、S 30G和A 33P；和/或，
    所述LCDR2发生至多6个氨基酸残基突变，优选地，根据Kabat编号系统编号，所述突变至少包含选自下组的一个或多个突变：N 52K、K 53F、R 54L或A 55S；更优选地，所述突变至少包含N 52K、K 53F、R 54L和A 55S，或至少包含N 52K、K 53F和R 54L；和/或，
    所述LCDR3发生至多6个氨基酸残基突变，优选地，根据Kabat编号系统编号，所述突变至少包含选自下组的一个或多个突变：A 89S/V、L 90I、Y 92F、S 93T、L 95R或V 97T，更优选地，所述突变至少包含A 89S和L 95R，或至少包含A 89V、L 90I和L 95R，或至少包含A 89V、Y 92F和L 95R，或至少包含A 89V、S 93T和L 95R，或至少包含A 89V、L 95R和V 97T，或至少包含A 89V和L 95R；和/或，
    所述HCDR1发生至多6个氨基酸残基突变或所述HCDR1不发生突变，优选地，根据Kabat编号系统编号，所述突变至少包含T 31K；和/或，
    所述HCDR2发生至多6个氨基酸残基突变或所述HCDR2不发生突变，优选地，根据Kabat编号系统编号，所述突变至少包含N 54S；和/或，
    根据Kabat编号系统编号，所述HCDR3发生至多6个氨基酸残基突变；优选地，所述突变至少包含选自下组的一个或多个突变：G 96S、S 100AA、Y 100BF、V 100CI、W 100EY、F 100FW/H或Y 102F/H，更优选地，所述突变至少包含S 100AA、V 100CI、W 100EY和F 100FW，或至少包含G 96S、V 100CI、W 100EY和F 100FW，或至少包含V 100CI、W 100EY、F 100FW和Y 102F，或至少包含V 100CI、W 100EY、F 100FW和Y 102H突变，或至少包含V 100CI、W 100EY和F 100FW，或至少包含Y 100BF、V 100CI、W 100EY和F 100FW，或 至少包含V 100CI、W 100EY和F 100FH。
  3. 根据权利要求1～2任一项所述的抗体或抗原结合片段，其中，所述轻链可变区的框架区发生至多15个氨基酸残基突变，优选地，根据Kabat编号系统编号，所述突变至少包含选自下组的一个或多个突变：F 36V、A 46G、Y49G或W 57G，更优选地，至少包含F 36V、A 46G、Y49G和W 57G；和/或，所述重链可变区的框架区发生至多15个氨基酸残基突变；优选地，根据Kabat编号系统编号，所述突变至少包含选自下组的一个或多个突变：S 30N、S 41P、G 49A、S 82BN或T 93V，更优选地，所述突变至少包含S 30N、S 41P、G 49A、S 82BN和T 93V。
  4. 根据权利要求2或3所述的抗体或抗原结合片段，其中，所述LCDR1、LCDR2、LCDR3、HCDR1、HCDR2或HCDR3的氨基酸残基突变数目可选自0,1,2,3,4,5或6；所述框架区的氨基酸残基突变数目可选自0,1,2,3,4,5,6,7,8,9,10,11,12,13,14或15；优选地，所述突变可选自插入、缺失或替换，所述替换优选为保守氨基酸残基的替换。
  5. 根据权利要求1～4任一项所述的抗体或抗原结合片段，其中，所述重链可变区具有如SEQ ID NO：9～18任一项所示的序列，和所述轻链可变区具有如SEQ ID NO：3～8任一项所示的序列；
    优选地，所述重链可变区具有如SEQ ID NO：9所示的序列，和所述轻链可变区具有如SEQ ID NO：3～7任一项所示的序列；或，
    所述重链可变区具有如SEQ ID NO：10所示的序列，和所述轻链可变区具有如SEQ ID NO：3～7任一项所示的序列；或，
    所述重链可变区具有如SEQ ID NO：11所示的序列，和所述轻链可变区具有如SEQ ID NO：3～7任一项所示的序列；或，
    所述重链可变区具有如SEQ ID NO：12所示的序列，和所述轻链可变区具有如SEQ ID NO：3～7任一项所示的序列；或，
    所述重链可变区具有如SEQ ID NO：13～18任一项所示的序列，和所述轻链可变区具有如SEQ ID NO：8所示的序列。
  6. 根据权利要求1～5任一项所述的抗体或抗原结合片段，其中，所述抗体或抗原结合片段包括scFv结构域，所述scFv结构域包含通过连接肽(linker)连接的所述重链可变区和所述轻链可变区；
    优选地，所述连接肽可选自(G 4S) n，可选地，所述n为3；
    优选地，所述重链可变区位于所述连接肽N端，所述轻链可变区位于所述连接肽C端；
    更优选地，所述scFv结构域具有如SEQ ID NO：19～44任一项所示的氨基酸序列。
  7. 根据权利要求1～6任一项所述的抗体或抗原结合片段，其中，所述抗体或抗原结合片段包含或不包含重链恒定区和/或轻链恒定区；
    优选地，所述重链恒定区可选自CH1、铰链区、Fc或CH3结构域，更优选地，所述重链恒定区包含铰链区和Fc，所述Fc优选具有如SEQ ID NO：45所示序列，所述铰链区优选具有如SEQ ID NO：46所示序列；
    优选地，所述重链恒定区和/或所述轻链恒定区为人重链恒定区和/或人轻链恒定区；
    优选地，所述重链恒定区为IgG重链恒定区，例如IgG1重链恒定区、IgG2重链恒定区、IgG3重链恒定区或IgG4重链恒定区；
    优选地，所述重链恒定区为人IgG1重链恒定区、人IgG2重链恒定区、人IgG3重链恒定区或人IgG4重链恒定区。
  8. 根据权利要求1～7任一项所述的抗体或抗原结合片段，其中，所述抗体或抗原结合片段选自单克隆抗体、多克隆抗体、天然抗体、工程化抗体、单特异性抗体、多特异性抗体(例如双特异性抗体)、单价抗体、多价抗体、完整抗体、抗体片段、裸抗体、缀合抗体、Fab、Fab'、Fab'-SH、F(ab') 2、Fd、Fv、scFv、双抗体(diabody)、双特异性T细胞接合器(BiTE)或单域抗体。
  9. 根据权利要求1～8任一项所述的抗体或抗原结合片段，其中，所述抗体或抗原结合片段特异性结合人CD3ε亚基和/或猴CD3ε亚基，优选地，所述抗体或抗原结合片段结合人CD3ε亚基的KD为1.00E-8M～1.00E-11M，例如，1.00E-9M、2.00E-9M、3.00E-9M、4.00E-9M、5.00E-9M、6.00E-9M、7.00E-9M、8.00E-9M、9.00E-9M、1.00E-10M、2.00E-10M、3.00E-10M、4.00E-10M、5.00E-10M、6.00E-10M、7.00E-10M、8.00E-10M或9.00E-10M。
  10. 根据权利要求1～9任一项所述的抗体或抗原结合片段，其中，所述抗体或抗原结合片段还偶联有治疗剂或示踪剂；优选地，所述治疗剂选自放射性同位素、化疗药或免疫调节剂，所述示踪剂选自放射学造影剂、顺磁离子、金属、荧光标记、化学发光标记、超声造影剂和光敏剂。
  11. 一种多特异性抗原结合分子，其中，所述多特异性抗原结合分子至少包括第一抗原结合模块和其他抗原结合模块，所述第一抗原结合模块包含权利要求1～10任一项所述的抗体或抗原结合片段，所述其他抗原结合模块特异性结合CD3ε亚基以外的抗原或结合与所述第一抗原结合模块不同的CD3ε亚基抗原表位。
  12. 根据权利要求11所述的多特异性抗原结合分子，其中，所述多特异性抗原结合分子可为双特异性、三特异性或四特异性抗原结合分子；
    优选地，所述CD3ε亚基以外的抗原可选自肿瘤相关抗原或传染性疾病抗原，例如可选自下组：BCMA、CD19、CD20、CD33、CD38、CD70、CD123、CD138、CD276、CDH3、CEA、c-Met、CLDN18.2、CLL-1、DLL3、DLK1、ENPP3、EpCAM、GD2、GPC3、GPRC5d、gpA33、MSLN、MUC1、MUC16、MUC17、LRRC15、ROR1、EGFR、HER2、PSMA、PSCA、PD-1、PD-L1、TPBG、TYRP1、WT1、SSTR2、HIV、甲肝病毒、乙肝病毒、丙肝病毒、疱疹病毒例如，HSV-1、HSV-2、CMV、衣原体、立克次氏体、分枝杆菌、葡萄球菌、链球菌、肺炎双球菌、脑膜炎双球菌或淋菌；
    优选地，所述其他抗原结合模块为抗体或抗原结合片段；
    优选地，所述多特异性抗原结合分子为双特异性T细胞结合器(BiTE)。
  13. 一种分离的核酸片段，其中，所述核酸片段编码权利要求1～10任一项所述抗体或抗原结合片段或权利要求11～12任一项所述多特异性抗原结合分子。
  14. 根据权利要求13所述的核酸片段，其中，所述核酸片段还包含编码嵌合抗原受体 (CAR)的核酸；优选地，编码所述抗体、抗原结合片段或多特异性抗原结合分子的核酸片段与编码所述嵌合抗原受体的核酸片段被编码核糖体跳跃部分的核酸片段分开，所述核糖体跳跃部分可选自P2A或T2A；
    优选地，所述抗体或抗原结合片段为单链抗体，例如scFv；优选地，所述多特异性抗原结合分子为双特异性T细胞结合器(BiTE)。
  15. 一种载体(vector)，其中，所述载体包含权利要求13或14所述核酸片段。
  16. 一种宿主细胞，其中，所述宿主细胞包含权利要求15所述载体；优选地，所述细胞为原核细胞或真核细胞，例如细菌(大肠杆菌)、真菌(酵母)、昆虫细胞或哺乳动物细胞(CHO细胞系或293细胞系)。
  17. 一种免疫效应细胞，其中，所述免疫效应细胞包含权利要求14所述核酸片段和/或表达权利要求14所述核酸片段编码的(a)所述嵌合抗原受体和(b)所述抗体、抗原结合片段或多特异性抗原结合分子；优选地，所述免疫效应细胞选自T细胞、NK细胞(natural killer cell)、NKT细胞(natural killer T cell)、DNT细胞(double negative T cell)、单核细胞、巨噬细胞、树突状细胞或肥大细胞，所述T细胞优选自细胞毒性T细胞、调节性T细胞或辅助性T细胞；所述免疫效应细胞优选自自体免疫效应细胞或同种异体免疫效应细胞。
  18. 一种制备权利要求1～10任一项所述抗体或抗原结合片段或权利要求11～12任一项所述多特异性抗原结合分子的方法，其中，所述方法包括培养权利要求16所述细胞，以及分离所述细胞表达的抗体、抗原结合片段或多特异性抗原结合分子。
  19. 一种制备权利要求17所述免疫效应细胞的方法，其中，所述方法包括将权利要求14所述核酸片段导入所述免疫效应细胞，优选地，所述方法还包括启动所述嵌合抗原受体表达，以及启动所述抗体、抗原结合片段或多特异性抗原结合分子表达。
  20. 一种药物组合物，其中，所述药物组合物包含权利要求1～10任一项所述抗体或抗原结合片段、权利要求11～12任一项所述多特异性抗原结合分子、权利要求13～14任一项所述核酸片段、权利要求15所述载体、权利要求16所述宿主细胞、权利要求17所述免疫效应细胞或根据权利要求18～19任一项所述方法制备得到的产品。
  21. 权利要求1～10任一项所述抗体或抗原结合片段、权利要求11～12任一项所述多特异性抗原结合分子、权利要求13～14任一项所述核酸片段、权利要求15所述载体、权利要求16所述宿主细胞、权利要求17所述免疫效应细胞、权利要求20所述药物组合物或根据权利要求18～19任一项所述方法制备得到的产品在制备治疗癌症或肿瘤、感染性疾病或自身免疫性疾病的药物中的应用；
    优选地，所述癌症或肿瘤选自血液瘤或实体瘤，所述血液瘤或实体瘤可选自B细胞淋巴瘤、套细胞淋巴瘤、滤泡性淋巴瘤、弥漫性大B细胞淋巴瘤、非霍奇金淋巴瘤、急性淋巴细胞白血病、急性髓性白血病、多发性骨髓瘤、胃癌、子宫癌、卵巢癌、乳腺癌、肝癌、前列腺癌、胶质母细胞瘤、肺癌、胰腺癌、胆管癌、乳腺癌、结直肠癌、黑色素瘤、肾细胞癌或癌性腹水；
    优选地，所述自身免疫性疾病选自类风湿性关节炎、全身性红斑狼疮、多发性硬化症、炎性肠病、重症肌无力、乳糜泻、Sjogren综合征、格雷弗氏病、克罗恩病、自身免疫性肝 炎、银屑病、银屑病关节炎、哮喘、过敏性鼻炎、来自器官移植的影响或移植物抗宿主病(GVHD)；
    优选地，所述感染性疾病选自HIV病毒、甲肝病毒、乙肝病毒、丙肝病毒、疱疹病毒例如，HSV-1、HSV-2、CMV、衣原体、立克次氏体、分枝杆菌、葡萄球菌、链球菌、肺炎双球菌、脑膜炎双球菌或淋菌的感染。
  22. 一种治疗癌症或肿瘤、感染性疾病或自身免疫性疾病的方法，其中，所述方法包括向受试者施用有效量的权利要求1～10任一项所述抗体或抗原结合片段、权利要求11～12任一项所述多特异性抗原结合分子、权利要求13～14任一项所述核酸片段、权利要求15所述载体、权利要求16所述宿主细胞、权利要求17所述免疫效应细胞、权利要求20所述药物组合物或根据权利要求18～19任一项所述方法制备得到的产品；
    优选地，所述癌症或肿瘤选自血液瘤或实体瘤，所述血液瘤或实体瘤可选自B细胞淋巴瘤、套细胞淋巴瘤、滤泡性淋巴瘤、弥漫性大B细胞淋巴瘤、非霍奇金淋巴瘤、急性淋巴细胞白血病、急性髓性白血病、多发性骨髓瘤、胃癌、子宫癌、卵巢癌、乳腺癌、肝癌、前列腺癌、胶质母细胞瘤、肺癌、胰腺癌、胆管癌、乳腺癌、结直肠癌、黑色素瘤、肾细胞癌或癌性腹水；
    优选地，所述自身免疫性疾病选自类风湿性关节炎、全身性红斑狼疮、多发性硬化症、炎性肠病、重症肌无力、乳糜泻、Sjogren综合征、格雷弗氏病、克罗恩病、自身免疫性肝炎、银屑病、银屑病关节炎、哮喘、过敏性鼻炎、来自器官移植的影响或移植物抗宿主病(GVHD)；
    优选地，所述感染性疾病选自HIV病毒、甲肝病毒、乙肝病毒、丙肝病毒、疱疹病毒例如，HSV-1、HSV-2、CMV、衣原体、立克次氏体、分枝杆菌、葡萄球菌、链球菌、肺炎双球菌、脑膜炎双球菌或淋菌的感染。
  23. 权利要求1～10任一项所述抗体或抗原结合片段、权利要求11～12任一项所述多特异性抗原结合分子、权利要求13～14任一项所述核酸片段、权利要求15所述载体、权利要求16所述宿主细胞、权利要求17所述免疫效应细胞、权利要求20所述药物组合物或根据权利要求18～19任一项所述方法制备得到的产品，其中，用于治疗癌症或肿瘤、感染性疾病或自身免疫性疾病；
    优选地，所述癌症或肿瘤选自血液瘤或实体瘤，所述血液瘤或实体瘤可选自B细胞淋巴瘤、套细胞淋巴瘤、滤泡性淋巴瘤、弥漫性大B细胞淋巴瘤、非霍奇金淋巴瘤、急性淋巴细胞白血病、急性髓性白血病、多发性骨髓瘤、胃癌、子宫癌、卵巢癌、乳腺癌、肝癌、前列腺癌、胶质母细胞瘤、肺癌、胰腺癌、胆管癌、乳腺癌、结直肠癌、黑色素瘤、肾细胞癌或癌性腹水；
    优选地，所述自身免疫性疾病选自类风湿性关节炎、全身性红斑狼疮、多发性硬化症、炎性肠病、重症肌无力、乳糜泻、Sjogren综合征、格雷弗氏病、克罗恩病、自身免疫性肝炎、银屑病、银屑病关节炎、哮喘、过敏性鼻炎、来自器官移植的影响或移植物抗宿主病(GVHD)；
    优选地，所述感染性疾病选自HIV病毒、甲肝病毒、乙肝病毒、丙肝病毒、疱疹病毒 例如，HSV-1、HSV-2、CMV、衣原体、立克次氏体、分枝杆菌、葡萄球菌、链球菌、肺炎双球菌、脑膜炎双球菌或淋菌的感染。
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