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WO2022105811A1 - Cd19人源化抗体及其应用 - Google Patents

Cd19人源化抗体及其应用 Download PDF

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Publication number
WO2022105811A1
WO2022105811A1 PCT/CN2021/131305 CN2021131305W WO2022105811A1 WO 2022105811 A1 WO2022105811 A1 WO 2022105811A1 CN 2021131305 W CN2021131305 W CN 2021131305W WO 2022105811 A1 WO2022105811 A1 WO 2022105811A1
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seq
antigen
antibody
cell
binding
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French (fr)
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葛虎
辛莲
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Jiangsu Simcere Pharmaceutical Co Ltd
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Jiangsu Simcere Pharmaceutical Co Ltd
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Priority to CN202180078166.3A priority Critical patent/CN116848147A/zh
Priority to US18/037,574 priority patent/US20230406922A1/en
Publication of WO2022105811A1 publication Critical patent/WO2022105811A1/zh
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • 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
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/567—Framework region [FR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates to the field of antibodies, in particular to CD19 humanized antibodies and applications thereof.
  • B cells include pre-B cells, early-developed B cells (ie, immature B cells), and mature B cells, which undergo terminal differentiation into plasma cells and malignant B cells.
  • Most pre-B acute lymphoblastic leukemia (Pre B ALL), non-Hodgkin's lymphoma, chronic lymphocytic leukemia (CLL), pre-lymphocytic leukemia, hairy cell leukemia, acute lymphoblastic leukemia (ALL) and some non-acute lymphoblastic leukemias highly express CD19 (Nadler et al., J. Immunol., 131:244-250 (1983); Loken et al., Blood, 70:1316-1324 (1987)).
  • CD19 on plasma cells further indicates that it can be expressed on different B cell tumors such as multiple myeloma, plasmacytoma, Waldenoma (Grossbard et al., Br. J. Haematol, 102:509-15 (1998) ) ; Treon et al., Semin. Oncol, 30:248-52 (2003)). Therefore CD19 is considered to be a target of various hematological tumors. At the same time, studies have shown that CD19 may play a role in regulating MHC class II expression and signaling in vivo, and CD19 may also serve as a potential immunotherapy target for autoimmune diseases.
  • the antibodies against CD19 are mainly mouse-derived antibodies (mouse anti), such as J Immunol. 1987 May 1; 138(9): 2793-9 discloses mouse anti-HD37, Amgen's listed drug Blinatumomab, etc.
  • the mouse-derived CD19 antibody FMC63 can recognize human CD19 protein. Many companies choose this antibody to develop drugs and cell therapy products.
  • the marketed CD19 CAR-T uses the mouse-derived CD19 antibody FMC63.
  • mouse antibody has strong immunogenicity, which can cause human anti-mouse antibody (HAMA) reaction and anti-antibody reaction (AAR) in clinical application, resulting in shortened half-life, easy to be cleared, weakened therapeutic effect, serious or even threatening patient's human life. Therefore, the successful humanization of the murine CD19 antibody FMC63 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 invention provides CD19 humanized antibodies or antigen-binding fragments, multispecific antigen-binding molecules, chimeric antigen receptors, immune effector cells, nucleic acid fragments, vectors, cells, compositions, preparation methods, pharmaceutical uses, and disease treatment methods.
  • the present invention provides a humanized antibody or antigen-binding fragment that specifically binds to CD19, the antibody or antigen-binding fragment comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region
  • the zone contains:
  • CDR1 which comprises HCDR1 of VH shown in any one of SEQ ID NO: 2 or 8-10;
  • B CDR2
  • it comprises the HCDR2 of VH shown in any one of SEQ ID NO:2 or 8-10;
  • a CDR3 comprising the HCDR3 of the VH shown in any one of SEQ ID NO: 2 or 8 to 10;
  • a framework region comprising the framework regions HFR1, HFR2 and HFR3 of IGHV2-26*01 shown in SEQ ID NO:3, and the framework region HFR4 of IGHJ6*01 shown in SEQ ID NO:4;
  • the light chain variable region comprises:
  • CDR1 it comprises LCDR1 of VL shown in SEQ ID NO: 1 or 7;
  • CDR2 it comprises the LCDR2 of VL shown in SEQ ID NO: 1 or 7;
  • a framework region comprising the framework regions LFR1, LFR2 and LFR3 of IGKV1-39*01 shown in SEQ ID NO: 5, and the framework region LFR4 of IGKJ4*01 shown in SEQ ID NO: 6.
  • the CDR regions and framework regions of the sequences shown in any one of SEQ ID NOs: 1 to 10 are determined according to the Kabat numbering system, the Chothia numbering system or the IMGT numbering system;
  • HCDR1 is DYGVS (SEQ ID NO:22);
  • the HCDR2 is VIWGSETTYYNSALKS (SEQ ID NO: 23);
  • the HCDR3 is HYYYGGSYAMDY (SEQ ID NO: 24);
  • HFR1 is QVTLKESGPVLVKPTETTLTLTCTVSGFSLS (SEQ ID NO:25);
  • the HFR2 is WIRQPPGKALEWLA (SEQ ID NO: 26);
  • the HFR3 is RLTISKDTSKSQVVLTMTNMDPVDTATYYCAR (SEQ ID NO: 27);
  • the HFR4 is WGQGTTVTVSS (SEQ ID NO: 4);
  • the LCDR1 is RASQDISKYLN (SEQ ID NO: 28);
  • the LCDR2 is HTSRLHS (SEQ ID NO: 29);
  • the LCDR3 is QQGNTLPYT (SEQ ID NO: 30);
  • LFR1 is DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO:31),
  • LFR2 is WYQQKPGKAPKLLIY (SEQ ID NO:32);
  • Described LFR3 is GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO:33);
  • the LFR4 is FGGGTKVEIK (SEQ ID NO: 6);
  • the HCDR1 is GVSLPDY (SEQ ID NO: 34), GFSLSDY (SEQ ID NO: 35) or GFSLPDY (SEQ ID NO: 36);
  • the HCDR2 is WGSET (SEQ ID NO: 37)
  • the HCDR3 is HYYYGGSYAMDY (SEQ ID NO: 24);
  • the HFR1 is QVTLKESGPVLVKPTETLTLTCTVS (SEQ ID NO: 38);
  • the HFR2 is GVSWIRQPPGKALEWLAHI (SEQ ID NO: 39);
  • the HFR3 is KSYSTSLKSRLTISKDTSKSQVVLTMTNMDPVDTATYYCAR (SEQ ID NO: 40);
  • the HFR4 is WGQGTTVTVSS (SEQ ID NO: 4);
  • Described LCDR1 is RASQDISKYLN (SEQ ID NO:28);
  • the LCDR2 is HTSRLHS (SEQ ID NO: 29);
  • Described LCDR3 is: QQGNTLPYT (SEQ ID NO:30);
  • LFR1 is DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO:31);
  • LFR2 is WYQQKPGKAPKLLIY (SEQ ID NO:32);
  • Described LFR3 is GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO:33);
  • LFR4 is FGGGTKVEIK (SEQ ID NO:6)
  • the HCDR1 is GVSLPDYG (SEQ ID NO: 41), GFSLSDYG (SEQ ID NO: 42) or GFSLPDYG (SEQ ID NO: 43);
  • the HCDR2 is IWGSETT (SEQ ID NO: 44)
  • the HCDR3 is AKHYYYGGSYAMDY (SEQ ID NO: 45);
  • the HFR1 is QVTLKESGPVLVKPTETLTLTCTVS (SEQ ID NO: 38);
  • the HFR2 is VSWIRQPPGKALEWLAH (SEQ ID NO: 46);
  • HFR3 SYSTSLKSRLTISKDTSKSQVVLTMTNMDPVDTATYY (SEQ ID NO: 47);
  • the HFR4 is WGQGTTVTVSS (SEQ ID NO: 4);
  • Described LCDR1 is QDISKY (SEQ ID NO:48);
  • the LCDR2 is HT (SEQ ID NO: 49) or HTS (SEQ ID NO: 50);
  • Described LCDR3 is QQGNTLPYT (SEQ ID NO:30);
  • LFR1 DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO51);
  • LFR2 is LNWYQQKPGKAPKLLIY (SEQ ID NO:52);
  • the LFR3 is SSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO:53) or SLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO:54);
  • the LFR4 is FGGGTKVEIK (SEQ ID NO:6).
  • the framework region of the heavy chain variable region further comprises one or more mutations selected from the group consisting of Q1E, F27V, S30P, T73N or R94K; Preferably includes Q1E and R94K; or preferably includes Q1E, S30P and R94K; or preferably includes Q1E, F27V, S30P, T73N and R94K.
  • the framework region of the light chain variable region further comprises one or more mutations selected from the group consisting of K42G, P44V or F71Y, preferably including K42G, P44V and F71Y.
  • the heavy chain variable region has the amino acid sequence set forth in any one of SEQ ID NOs: 8 to 10, and/or the light chain variable region has the amino acid sequence set forth in SEQ ID NO:7 the amino acid sequence described.
  • the CDR1 , CDR2 and/or CDR3 of the heavy chain variable region comprises at least 80%, 85%, 90%, 91%, 92% of the same as the HCDR1, HCDR2 and/or HCDR3 , 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical sequences;
  • the CDR1, CDR2 and/or CDR3 of the light chain variable region comprise at least 80%, 85%, 90%, 91%, 92%, 93%, Sequences of 94%, 95%, 96%, 97%, 98%, 99% or 100% identity.
  • the CDR1, CDR2 and/or CDR3 of the heavy chain variable region comprises a sequence with up to 6 amino acid mutations compared to the HCDR1, HCDR2 and/or HCDR3, the number of mutations may be selected from 0, 1, 2, 3, 4, 5 or 6;
  • the CDR1, CDR2 and/or CDR3 of the variable region of the light chain comprise sequences with at most 6 amino acid mutations compared to the LCDR1, LCDR2 and/or LCDR3, the number of which may be selected from 0 , 1, 2, 3, 4, 5, or 6.
  • the framework region of the heavy chain variable region comprises at least 80%, 85%, 90%, 91%, 92%, 93% compared to HFR1, HFR2, HFR3 and/or HFR4 , 94%, 95%, 96%, 97%, 98%, 99% or 100% identical sequences;
  • the framework region of the light chain variable region comprises at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, compared with LFR1, LFR2, LFR3 and/or LFR4. Sequences of 95%, 96%, 97%, 98%, 99% or 100% identity.
  • the framework region of the heavy chain variable region comprises a sequence with up to 15 amino acid mutations compared to the HFR1, HFR2, HFR3 and/or HFR4, the number of mutations can be selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15;
  • the light chain variable region comprises a sequence with at most 15 amino acid mutations compared to the LFR1, LFR2, LFR3 and/or LFR4, the number of mutations may be selected from 0, 1, 2, 3 , 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.
  • the mutations may be selected from insertions, deletions or substitutions, preferably, the substitutions are conservative amino acid substitutions.
  • 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 comprises a full-length heavy chain constant region or a fragment thereof, the fragment may be selected from the CH1 domain, the Fc domain or the CH3 domain;
  • the heavy chain constant region and/or light chain constant region is a human heavy chain constant region and/or a human light chain constant region;
  • the heavy chain constant region may be selected from 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 lacks fucosylation.
  • 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, Full Length Antibodies, Antibody Fragments, Naked Antibodies, Conjugated Antibodies, Humanized Antibodies, Fully Human Antibodies, Fab, Fab', Fab'-SH, F(ab') 2 , Fd, Fv, scFv, diabody or single domain antibody.
  • 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 or 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 or photosensitizers.
  • the antibody or antigen-binding fragment binds to human CD19 and/or monkey CD19; optionally, the antibody or antigen-binding fragment binds human CD19 with a KD value of less than 1.00E-8M, 1.00E -9M, 2.00E-09M, 3.00E-9M, 4.00E-09M, 5.00E-09M, 6.00E-09M, 7.00E-09M, 8.00E-09M, 9.00E-09M, 1.00E-10M, 2.00E -10M, 3.00E-10M, 4.00E-10M, 5.00E-10M, 6.00E-10M, 7.00E-10M, 8.00E-10M, 9.00E-10M, 1.00E-11M, 2.00E-11M, 3.00E -11M, 4.00E-11M, 5.00E-11M, 6.00E-11M, 7.00E-11M, 8.00E-11M, 9.00E-11M, 1.00E-12M, 2.00E-12M, 3.00E-12M, 4.00E
  • the present invention also discloses a multispecific antigen-binding molecule comprising a first antigen-binding moiety and a second antigen-binding moiety, the first antigen-binding moiety comprising the aforementioned antibody or an antigen-binding fragment, the second antigen-binding moiety specifically binds to other antigens other than CD19 or binds to a different CD19 epitope from the first antigen-binding moiety;
  • the other antigens are selected from CD3, CD16, CD16A, CD4, CD5, CD8, CD14, CD15, CD19, CD20, CD21, CD22, CD23, CD25, CD33, CD37, CD38, CD40, CD40L, CD46, CD52 , CD54, CD66 (a-d), CD74, CD80, CD126, CD138, B7, MUC, Ia, HLA-DR, tenascin, VEGF, P1GF, ED-B fibronectin, oncogene products, IL-2, IL -6, TRAIL-R1 or TRAIL-R2;
  • the multispecific antibody is bispecific, trispecific or tetraspecific.
  • the present invention also discloses a chimeric antigen receptor (CAR), the chimeric antigen receptor comprises an extracellular antigen binding domain, a transmembrane domain and an intracellular domain, the extracellular antigen
  • the binding domain comprises the aforementioned CD19 antibody or antigen-binding fragment.
  • the present invention further discloses an immune effector cell, the immune effector cell comprising the aforementioned chimeric antigen receptor or a nucleic acid fragment encoding the aforementioned chimeric antigen receptor;
  • the immune effector cells are selected from T cells, NK cells (natural killer cells), NKT cells (natural killer cells), monocytes, macrophages, dendritic cells or mast cells;
  • the T cells may be selected from cytotoxic T cells, regulatory T cells (Treg) or helper T cells;
  • the immune effector cells are allogeneic immune effector cells or autoimmune effector cells.
  • the present invention also discloses an isolated nucleic acid fragment encoding the aforementioned antibody or antigen-binding fragment or multispecific antigen-binding molecule or chimeric antigen receptor.
  • the present invention also discloses a vector, the vector comprising the aforementioned nucleic acid fragment.
  • the present invention also discloses a host cell, the host cell comprises 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 293T cell line); preferably, the cells lack a fucosyltransferase, more preferably, the fucosyltransferase is FUT8.
  • 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 293T cell line); preferably, the cells lack a fucosyltransferase, more preferably, the fucosyltransferase is FUT8.
  • the present invention also discloses a method for preparing the aforementioned antibody or antigen-binding fragment or the aforementioned 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 invention further discloses a method for preparing the aforementioned immune effector cells, the method comprising introducing a nucleic acid fragment encoding the aforementioned CAR into the immune effector cells, optionally, the method further comprising activating the immune effector cells
  • the effector cells express the aforementioned CAR.
  • the present invention also discloses a pharmaceutical composition
  • a pharmaceutical composition comprising the aforementioned antibody or antigen-binding fragment, multispecific antigen-binding molecule, chimeric antigen receptor, immune effector cell, nucleic acid fragment, vector or cells; preferably, the composition further comprises a pharmaceutically acceptable carrier, diluent or adjuvant.
  • the present invention also discloses that the aforementioned antibodies or antigen-binding fragments, multispecific antigen-binding molecules, chimeric antigen receptors, immune effector cells, nucleic acid fragments, vectors or cells are used in the preparation of a drug for the treatment of cancer or autoimmune diseases. application in medicine;
  • the cancer is selected from lymphoma or leukemia
  • the lymphoma or leukemia can be selected from B cell lymphoma, non-Hodgkin lymphoma, mantle cell lymphoma, follicular lymphoma, marginal zone lymphoma, Primary mediastinal B-cell lymphoma, diffuse large B-cell lymphoma, precursor B-cell acute lymphoblastic leukemia (pre-B ALL), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia, hairy cell leukemia, pre-lymphoid cellular leukemia, plasmacytoma, Walden's tumor, or multiple myeloma;
  • the autoimmune disease may be selected from rheumatoid arthritis, multiple sclerosis, systemic sclerosis, neuromyelitis optica spectrum disorders, systemic lupus erythematosus, myasthenia gravis or IgG4-related diseases.
  • the present invention also discloses a method of treating cancer or autoimmune disease, the method comprising administering to a subject an effective amount of the aforementioned antibody or antigen-binding fragment, multispecific antigen-binding molecule, chimeric antigen receptors, immune effector cells, nucleic acid fragments, vectors or cells;
  • the cancer is selected from lymphoma or leukemia
  • the lymphoma or leukemia can be selected from B cell lymphoma, non-Hodgkin lymphoma, mantle cell lymphoma, follicular lymphoma, marginal zone lymphoma, Primary mediastinal B-cell lymphoma, diffuse large B-cell lymphoma, precursor B-cell acute lymphoblastic leukemia (pre-B ALL), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia, hairy cell leukemia, pre-lymphoid cellular leukemia, plasmacytoma, Walden's tumor, or multiple myeloma;
  • the autoimmune disease may be selected from rheumatoid arthritis, multiple sclerosis, systemic sclerosis, neuromyelitis optica spectrum disorders, systemic lupus erythematosus, myasthenia gravis or IgG4-related diseases.
  • the present invention also discloses the aforementioned antibodies or antigen-binding fragments, multispecific antigen-binding molecules, chimeric antigen receptors, immune effector cells, nucleic acid fragments, vectors or cells for the treatment of cancer or autoimmunity disease;
  • the cancer is selected from lymphoma or leukemia
  • the lymphoma or leukemia can be selected from B cell lymphoma, non-Hodgkin lymphoma, mantle cell lymphoma, follicular lymphoma, marginal zone lymphoma, Primary mediastinal B-cell lymphoma, diffuse large B-cell lymphoma, precursor B-cell acute lymphoblastic leukemia (pre-B ALL), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia, hairy cell leukemia, pre-lymphoid cellular leukemia, plasmacytoma, Walden's tumor, or multiple myeloma;
  • the autoimmune disease may be selected from rheumatoid arthritis, multiple sclerosis, systemic sclerosis, neuromyelitis optica spectrum disorders, systemic lupus erythematosus, myasthenia gravis or IgG4-related diseases.
  • CD19 Cluster of Difference 19
  • CD19 Cluster of Difference 19
  • CD19 includes mature or immature full-length wild-type CD19 protein or mutants thereof (eg point mutations, insertion mutations or deletion mutations), splice variants, orthologs (Orthologs) and fragments thereof.
  • CD19 herein can be derived from humans, primates, such as monkeys (eg, rhesus monkeys, cynomolgus monkeys), and rodents, such as mice and rats.
  • the amino acid sequence of human CD19 protein can be found in NCBI: NP_001761.3
  • the amino acid sequence of monkey CD19 protein can be found in NCBI: XM_005591542.1.
  • binding refers to an antigen-binding molecule (eg, an antibody) that specifically binds an antigen and a substantially identical antigen, usually with high affinity, but not an unrelated antigen with high affinity. Affinity is usually reflected in the equilibrium dissociation constant (KD), where lower KD indicates higher affinity.
  • KD equilibrium dissociation constant
  • high affinity generally refers to having about 10-7 M or less, about 10-8 M or less, about 1 ⁇ 10-9 M or less, about 1 ⁇ 10-10 M or less, 1 ⁇ 10 -11 M or lower, 1 ⁇ 10 -12 M 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, and for example, KD can be obtained by referring to the method shown in Example 6 herein.
  • 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 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 region ( CL) composed of polypeptide chains; heavy chains and heavy chains, heavy chains and light chains 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 heavy chain antibodies (heavy-chain antibodies, HCAbs) and in sharks and other cartilaginous fish found in the new immunoglobulin antigen receptors (Ig new antigen receptor, IgNAR).
  • a light chain such as those produced by Camelus dromedarius, Camelus bactrianus, Lama glama, Lama guanicoe, and alpaca ( Vicugna pacos) and other heavy chain antibodies (heavy-chain antibodies, HCAbs) and in sharks and other cartilaginous fish found in the new immunoglobulin antigen receptors (Ig new antigen receptor, IgNAR).
  • 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, whole antibodies, antigen-binding fragments, naked antibodies, Conjugated, humanized 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/or 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 herein refers to having at least two antigen-binding sites, each of which is associated with a different epitope of the same antigen or with a different epitope of a different antigen combine.
  • 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.
  • Fully-length antibody “intact antibody,” and “intact antibody” are used interchangeably herein to mean that they have 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 antigen .
  • Antigen-binding fragments” or “antibody fragments” herein include, but are not limited to, Fab, F(ab') 2 , Fab', Fab'-SH, Fd, Fv, scFv, diabodies, and single domain antibodies.
  • Intact antibodies are papain-digested to produce two identical antigen-binding fragments, termed "Fab" fragments, each Fab fragment containing the variable region of the heavy chain and the variable region of the light chain, as well as the constant region of the light chain and the variable region of the heavy chain.
  • Fab fragment herein refers to a light chain fragment comprising the VL region and constant region (CL) of the light chain, and an antibody fragment comprising the VH region and the first constant region (CH1) of the heavy chain.
  • Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 region, including one or more cysteines from the antibody hinge region.
  • Fab'-SH is a Fab' fragment whose cysteine residue in the constant region of the heavy chain carries a free thiol group.
  • F(ab') 2 is an antibody fragment with two antigen binding sites (two Fab fragments) and a portion of the Fc region produced by pepsin treatment of an intact antibody.
  • 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
  • scFv single-chain variable fragment
  • linker see, eg, 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 invention 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 linkers that are too short to allow pairing between the two domains of the same chain, forcing the structure The domains pair with the complementary domains of the other chain 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. Man, Structure 2:1121-1123 (1994)).
  • 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 An antibody composed of the variable region of the heavy chain, which is the smallest fully functional antigen-binding fragment.
  • an antibody that naturally lacks 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 (VHH) 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.
  • 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 Antibodies (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 are not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species (eg, mouse) have been grafted onto human framework sequences.
  • another mammalian species eg, mouse
  • 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 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 an antibody heavy or light chain, where the framework region of the variable heavy chain may be abbreviated As HFR, the framework region of the light chain variable region can be abbreviated as LFR.
  • HFR framework region of the variable heavy chain
  • LFR framework region of the light chain variable region
  • CDRs may be labeled and defined by numbering systems 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 .cn/AbRSA/cdrs.php), abYsis website (www.abysis.org/abysis/sequence_input/key_annotation/key_annotation.cgi) and IMGT website (http://www.imgt.org/3Dstructure-DB/cgi/DomainGapAlign .cgi#results).
  • the 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
  • the chothia numbering system or the IMGT numbering system for CDR definition and FR region definition are as follows (www.abysis.org/abysis/sequence_input/key_annotation/key_annotation.cgi, http://www.imgt.org/3Dstructure-DB/ cgi/DomainGapAlign.cgi#results):
  • HCDR1 is DYGVS (SEQ ID NO: 22);
  • HCDR2 is VIWGSETTYYNSALKS (SEQ ID NO: 23);
  • HCDR3 is HYYYGGSYAMDY (SEQ ID NO: 24).
  • HFR1 is QVTLKESGPVLVKPTETTLTLTCTVSGFSLS (SEQ ID NO: 25);
  • HFR2 is WIRQPPGKALEWLA (SEQ ID NO: 26);
  • HFR3 is RLTISKDTSKSQVVLTMTNMDPVDTATYYCAR (SEQ ID NO: 27);
  • HFR4 is WGQGTTVTVSS (SEQ ID NO: 4).
  • LCDR1 is RASQDISKYLN (SEQ ID NO: 28);
  • LCDR2 is HTSRLHS (SEQ ID NO: 29);
  • LCDR3 is QQGNTLPYT (SEQ ID NO: 30).
  • LFR1 is DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO:31);
  • LFR2 is WYQQKPGKAPKLLIY (SEQ ID NO:32);
  • LFR3 is GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 33);
  • LFR4 is FGGGTKVEIK (SEQ ID NO:6).
  • HCDR1 is GVSLPDY (SEQ ID NO:34), GFSLSDY (SEQ ID NO:35) or GFSLPDY (SEQ ID NO:36);
  • HCDR2 is WGSET (SEQ ID NO: 37)
  • HCDR3 is HYYYGGSYAMDY (SEQ ID NO: 24);
  • HFR1 is QVTLKESGPVLVKPTETLTLTCTVS (SEQ ID NO: 38);
  • HFR2 is GVSWIRQPPGKALEWLAHI (SEQ ID NO:39);
  • HFR3 is KSYSTSLKSRLTISKDTSKSQVVLTMTNMDPVDTATYYCAR (SEQ ID NO: 40);
  • HFR4 is WGQGTTVTVSS (SEQ ID NO: 4);
  • LCDR1 is RASQDISKYLN (SEQ ID NO: 28);
  • LCDR2 is HTSRLHS (SEQ ID NO: 29);
  • LCDR3 is: QQGNTLPYT (SEQ ID NO:30);
  • LFR1 is DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO:31);
  • LFR2 is WYQQKPGKAPKLLIY (SEQ ID NO:32);
  • LFR3 is GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 33);
  • LFR4 is FGGGTKVEIK (SEQ ID NO:6).
  • HCDR1 is GVSLPDYG (SEQ ID NO:41), GFSLSDYG (SEQ ID NO:42) or GFSLPDYG (SEQ ID NO:43);
  • HCDR2 is IWGSETT (SEQ ID NO: 44);
  • HCDR3 is AKHYYYGGSYAMDY (SEQ ID NO: 45);
  • HFR1 is QVTLKESGPVLVKPTETLTLTCTVS (SEQ ID NO: 38);
  • HFR2 is VSWIRQPPGKALEWLAH (SEQ ID NO:46);
  • HFR3 is SYSTSLKSRLTISKDTSKSQVVLTMTNMDPVDTATYYC (SEQ ID NO: 47);
  • HFR4 is WGQGTTVTVSS (SEQ ID NO: 4);
  • LCDR1 is QDISKY (SEQ ID NO: 48);
  • LCDR2 is HT (SEQ ID NO:49) or HTS (SEQ ID NO:50);
  • LCDR3 is QQGNTLPYT (SEQ ID NO: 30);
  • LFR1 is DIQMTQSPSSLSASVGDRVTITCRAS (SEQ ID NO:51);
  • LFR2 is LNWYQQKPGKAPKLLIY (SEQ ID NO: 52);
  • LFR3 is SSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO:53) or SLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO:54);
  • LFR4 is FGGGTKVEIK (SEQ ID NO:6).
  • 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 one of the following: 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 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, see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991). The description will be given below in conjunction with amino acid residue mutations.
  • the heavy chain variable region F27V mutation means that the amino acid residue at position 27 of the heavy chain determined according to the Kabat numbering system is mutated from F to V.
  • 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:
  • 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:
  • identity herein can be calculated by aligning the sequences for optimal comparison purposes in order to determine the percent "identity" of two amino acid sequences or two nucleic acid sequences (eg, it may be optimal). The alignment may introduce gaps in either or both of the first and second amino acid sequences or nucleic acid sequences or non-homologous sequences may be discarded for comparison purposes). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, then the molecules are identical at that position.
  • the percent identity between the two sequences varies with the identical positions shared by the sequences.
  • Sequence comparisons and calculation of percent identity between two sequences can be accomplished using mathematical algorithms. For example, using the Needlema and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm (available at www.gcg.com), which has been integrated into the GAP program of the GCG software package, using the Blossum 62 matrix or The PAM250 matrix and gap weights 16, 14, 12, 10, 8, 6 or 4 and length weights 1, 2, 3, 4, 5 or 6 determine the percent identity between two amino acid sequences.
  • the GAP program in the GCG software package (available at www.gcg.com) using the NWSgapdna.CMP matrix and gap weights 40, 50, 60, 70 or 80 and length weights 1, 2, 3, 4, 5 or 6, determine the percent identity between the two nucleotide sequences.
  • a particularly preferred set of parameters is the Blossum62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.
  • gapped BLAST can be used as described in Altschul et al., (1997) Nucleic Acids Res. 25:3389-3402.
  • the default parameters of the corresponding programs eg, XBLAST and NBLAST
  • XBLAST and NBLAST the default parameters of the corresponding programs. See www.ncbi.nlm.nih.gov.
  • antigen chimeric 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.
  • nucleic acid herein includes any compound and/or substance comprising a polymer of nucleotides.
  • Each nucleotide consists of a base, especially 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 generally represented as 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 invention 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 823B1).
  • 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.
  • 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, the purpose of which is to prevent, slow (reduce) the progression of undesired physiological changes or pathologies, such as cancer, autoimmune diseases, in the subject being treated.
  • 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 refers to an organism receiving treatment for a particular disease or disorder as described herein.
  • subjects and patients include mammals, such as humans, primates (eg, monkeys) or non-primate mammals, receiving treatment for a disease or disorder.
  • 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.
  • the technical solution of the present invention has at least one of the following beneficial effects:
  • the humanized antibody of the present invention not only has the ability to bind CD19, but also reduces the immunogenicity, which helps to reduce the risk of immune rejection when used by human subjects.
  • the humanized antibody of the present invention is equivalent to or better than its parent murine antibody FMC63, and is superior to the conventional humanized antibody (conventional humanized antibody).
  • the binding capacity is 2 to 3 times lower than that of the parental murine antibody).
  • the humanized antibody of the present invention shows good binding ability with human CD19 and/or monkey CD19, which is beneficial to improve its therapeutic effect and/or carry out preclinical animal experiments.
  • Figure 1 shows the detection results of human CD19 exon1-3-his protein samples on SDS-PAGE reducing gel and non-reducing gel, lane M is protein marker, lane 1 is non-reducing condition, and lane 2 is reducing condition;
  • Figure 2A is the FACS result of FMC63 antibody detecting the expression of CD19 in Raji cells
  • Figure 2B is the FACS result of 9G8 antibody detecting the expression of CD19 in Raji cells
  • Figure 3 shows the results of FACS screening of CHO-K1 cells transfected with human CD19 protein
  • Figure 4 is the FACS result of FMC63 antibody detecting HEK293T cells transfected with monkey CD19 protein
  • Figure 5 shows the binding reaction of FMC63 humanized antibody and human CD19-His protein detected by ELISA
  • Fig. 6A is FACS to detect the binding reaction of FMC63 humanized antibody and Raji;
  • Figure 6B is FACS detection of the binding reaction of FMC63 humanized antibody and CHO-K1-human CD19;
  • Fig. 6C is FACS detection of the binding reaction of FMC63 humanized antibody and MOLT-4;
  • Figure 6D shows the FACS detection of the binding reaction of FMC63 humanized antibody and CHO-K1
  • Fig. 7 is ELISA to detect the binding reaction of FMC63 humanized antibody and mouse CD19-his protein
  • Fig. 8 is ELISA to detect the binding reaction of FMC63 humanized antibody and monkey CD19-his protein
  • Figure 9A is FACS detection of the binding reaction of FMC63 humanized antibody and HEK293T-monkey CD19;
  • Fig. 9B is FACS to detect the binding reaction of FMC63 humanized antibody and HEK293T;
  • Figure 10 shows the FACS detection of the binding reaction between FMC63 humanized antibody and cynomolgus monkey B cells, wherein FMC63-L2H5 is FMC63.25, FMC63-L2H6 is FMC63.26, and FMC63-L2H7 is FMC63.27;
  • Figure 11A shows the affinity of FMC63 and human CD19 protein detected by SPR
  • Figure 11B shows the affinity of FMC63.25 humanized antibody to human CD19 protein detected by SPR;
  • Figure 11C shows the affinity of FMC63.26 humanized antibody to human CD19 protein detected by SPR
  • Figure 11D shows the affinity of FMC63.27 humanized antibody to human CD19 protein detected by SPR;
  • Figure 12 shows the binding reaction of FMC63 humanized antibody and human CD19 exon1-3-his protein for ELISA detection.
  • IGKV1-39*01 and IGKJ4*01 were selected as the humanized light chain templates of FMC63, and IGHV2- 26*01 and IGHJ6*01 were used as humanized heavy chain templates for FMC63.
  • the CDRs of the murine antibody were transplanted into the corresponding human templates to form variable region sequences in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and the individual amino acid residues in the framework sequence were restored Mutations and hotspot mutations (see Table 1 for details).
  • the amino acid residue sequence numbering, CDR regions and FR regions of the antibodies of this example are all determined by the Kabat numbering system.
  • Grafted represents the insertion of mouse antibody CDRs into the human germline FR region sequence
  • K42G represents the mutation of K at position 42 of Grafted back to G, and so on.
  • FMC63.25 means that the FMC63 humanized antibody FMC63.25 has a light chain variable region as described in FMC63.VL2 and a heavy chain variable region as described in FMC63.VH5, and so on.
  • FMC63 light chain variable region (SEQ ID NO: 1):
  • FMC63 heavy chain variable region (SEQ ID NO: 2):
  • IGHJ6*01 (SEQ ID NO: 4): WGQGTTVTVSS.
  • IGKJ4*01 (SEQ ID NO: 6): FGGGTKVEIK.
  • FMC63.VL2 amino acid sequence (SEQ ID NO: 7):
  • FMC63.VH5 amino acid sequence (SEQ ID NO: 8):
  • FMC63.VH6 amino acid sequence (SEQ ID NO: 9):
  • FMC63.VH7 amino acid sequence (SEQ ID NO: 10):
  • the heavy chain variable region sequences of FMC63 humanized antibodies FMC63.25, FMC63.26 and FMC63.27 were cloned by Taizhou Baiying Biotechnology Co., Ltd. into the heavy chain constant region containing the signal peptide and murine antibody IgG1 expression
  • the vector pcDNA3.4-B1HH1 (the heavy chain constant region sequence is shown in SEQ ID NO: 11)
  • the light chain variable region sequence was cloned into the expression vector pcDNA3.4- containing the signal peptide and the light chain constant region of the murine antibody IgG1 B1HLK (the light chain constant region sequence is shown in SEQ ID NO: 12)
  • the sequences of FMC63.25-mlgG1, FMC63.26-mlgG1 and FMC63.27-mlgG1 were obtained, and plasmids were prepared according to established standard molecular biology methods , see Sambrook, J., Fritsch, E.F., and Maniatis, T.
  • HEK293E cells purchased from Suzhou Yiyan Biotechnology Co., Ltd.
  • FreeStyleTM 293 Thermofisher scientific, Cat. No. 12338018
  • the culture supernatant was loaded onto a protein A chromatography column (the protein A filler AT Protein A Diamond and the chromatography column BXK16/26 were purchased from Borglon, the product numbers were respectively For: AA0273 and B-1620), washed with PBS phosphate buffer (pH 7.4), then washed with 20mM PB, 1M NaCl (pH 7.2), and finally eluted with pH 3.4 citrate buffer, The Fc-tagged antibody eluted from the protein A column was collected, neutralized with 1/10 volume of 1M Tris pH8.0, dialyzed overnight at 4°C with PBS, and the dialyzed protein was filtered through 0.22 micron. The membranes were sterile filtered and stored in aliquots at -80°C.
  • Murine antibody IgG1 heavy chain constant region SEQ ID NO: 11:
  • Murine antibody IgG1 light chain constant region SEQ ID NO: 12:
  • the nucleotide sequence containing the amino acid sequence (SEQ ID NO: 13) encoding human CD19 exon1-3 Pro 20-Gln186 was cloned into the pTT5 vector (completed by General Biosystems Anhui Co., Ltd.) and followed established standard molecular biology methods Plasmids were prepared, and the corresponding amino acid sequence information was shown in Table 3 below. For specific 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).
  • HEK293E cells purchased from Suzhou Yiyan Biotechnology Co., Ltd.
  • PEI Polysciences, Cat. No. 24765-1
  • FreeStyleTM 293 Thermofisher scientific, Cat. No. 12338018
  • the cell culture medium was collected, and the cell components were removed by centrifugation to obtain a culture supernatant containing human CD19 exon1-3.
  • the culture supernatant was loaded onto a nickel ion affinity chromatography column HisTrap TM Excel (GE Healthcare, Cat. No.: GE17-3712-06), and at the same time an ultraviolet (UV) detector was used to monitor the change of the UV absorbance value (A280nm).
  • UV ultraviolet
  • the nickel ion affinity chromatography column was washed with 20mM PB, 0.5M NaCl (pH7.4) until the UV absorption value returned to the baseline, and then buffer A: 20mM PB, 0.5M NaCl (pH7.4) and buffer B : 20 mM PB, 0.5 M NaCl, 500 mM imidazole for gradient elution (2%, 4%, 8%, 16%, 50%, 100%), and the His-bands eluted from the nickel ion affinity chromatography column were collected.
  • the tagged human CD19 exon1-3 protein was dialyzed against PBS phosphate buffered saline (pH 7.4) overnight in a refrigerator at 4°C. The dialyzed protein was sterile filtered through a 0.22-micron filter membrane and then stored at -80 °C to obtain purified human CD19 exon1-3 protein. Show.
  • the FMC63 and 9G8 clones are antibodies that recognize human CD19, and their antigen-binding epitopes are located at the proximal end of the membrane.
  • the heavy chain variable region and light chain variable region sequences of FMC63 clone were obtained according to patent WO2016033570A1, and the heavy chain variable region and light chain variable region sequences of 9G8 clone were obtained according to patent WO2018083535.
  • the light chain variable region sequences cloned from FMC63 and 9G8 were cloned by Taizhou Baiying Biotechnology Co., Ltd.
  • FMC63-mIgG1 and 9G8-mIgG1 The sequence was cloned into the expression vector pcDNA3.4-B1HLK containing the signal peptide and the constant region of the heavy chain of the mouse antibody IgG1 to obtain the sequences of FMC63-mIgG1 and 9G8-mIgG1. If there is no special description, FMC63 and 9G8 in the following refer to FMC63 respectively. -mlgG1 and 9G8-mlgG1. And prepare plasmids according to established standard molecular biology methods.
  • the culture supernatant was loaded onto a protein A chromatography column (protein A filler AT Protein A Diamond and a chromatography column).
  • BXK16/26 were purchased from Borglon, the product numbers are: AA0273 and B-1620), washed with PBS phosphate buffer (pH 7.4) and then washed with 20mM PB, 1M NaCl (pH 7.2), and finally used Elution was performed with citrate buffer pH 3.4, and the Fc-tagged antibody eluted from the Protein A column was collected, neutralized with 1/10 volume of 1 M Tris, pH 8.0, and PBS at 4°C. Conditional dialysis was performed overnight, and the dialyzed proteins were sterile filtered through a 0.22-micron filter membrane and stored in aliquots at -80°C.
  • nucleotide sequence encoding the full-length amino acid sequence of human CD19 (NCBI: NP_001761.3, SEQ ID NO: 20) was cloned into pcDNA3.1 vector and a plasmid was prepared (completed by General Biosystems (Anhui) Co., Ltd.).
  • CHO-K1 cell line (purchased from Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) was transfected with plasmids ( 3000Transfection Kit, purchased from Invitrogen, Cat. No.
  • the specific selection results are shown in Table 6 and Figure 3.
  • the IgG subtype control is the mouse IgG1 control.
  • the abscissa is the cell fluorescence intensity, and the ordinate is the number of cells.
  • the results in Table 6 and Figure 3 show that CHO-K1 monoclonal cell lines expressing high levels of human CD19 have been prepared: CHO-K1-human CD19-2C8, CHO-K1-human CD19-1C4, CHO-K1-human CD19 -2G4, and CHO-K1-human CD22 1C9.
  • the CHO-K1-human CD19-2C8 cell line was selected for subsequent antibody binding experiments.
  • the nucleotide sequence encoding the monkey CD19 full-length amino acid sequence (NCBI: XM_005591542.1, SEQ ID NO: 21) was cloned into pcDNA3.1 vector (purchased from Thermofisher scientific) and plasmids were prepared.
  • pcDNA3.1 vector purchased from Thermofisher scientific
  • Human CD19-his protein (purchased from ACROBiosystems, Cat. No.: CD9-H52H2) was diluted with PBS to a final concentration of 4 ⁇ g/mL, and then added to a 96-well ELISA plate at 100 ⁇ l per well. Cover with plastic film and incubate at 4°C overnight, wash the plate twice with PBS the next day, add blocking solution [PBS+2% (w/w) BSA] and block for 2 hours at room temperature. Pour off the blocking solution and add 50 ⁇ l of 100 nM serially diluted FMC63 humanized antibody, FMC63 or negative control antibody to each well. After incubation at 37°C for 2 hours, the plate was washed 3 times with PBS.
  • Table 8 illustrates that FMC63 humanized All the antibodies could bind to human CD19 at the ELISA level.
  • the negative control antibody mIgG1 is the antibody anti-hel-mIgG1 against chicken egg lysozyme (purchased from Baiying, article number: B118301), and the data in the table is the OD450nm value.
  • the desired cells were expanded to the logarithmic growth phase in T-75 cell culture flasks.
  • adherent cells CHO-K1 the medium was aspirated, washed twice with PBS buffer, and then the cells were trypsinized. After terminating the digestion Cells were washed twice with PBS buffer; for suspension cells Raji was directly centrifuged to discard the medium supernatant, and the cell pellet was washed twice with PBS.
  • the IgG control is mIgG1
  • the Alpaca serum is the human CD19-ECD-His immunized alpaca serum as a positive control (Alpaca serum has been verified to be able to bind to mouse CD19).
  • the data in the table are OD450nm values.
  • Alpaca serum serum was serially diluted 10 times from 1:100.
  • FIG. 8 and Table 11 illustrate that FMC63 humanized antibody and monkey CD19 did not bind at the ELISA level.
  • the IgG control is mIgG1
  • S003-NB151-89 is human CD19-His immunized alpacas
  • the clones that can bind to monkey CD19-His protein are screened out from the peripheral blood library as positive control.
  • the data in the table are OD450nm values.
  • Table 11 ELISA detects the binding reaction of FMC63 humanized antibody and monkey CD19 protein
  • the HEK293T-monkey CD19 cells were subjected to FACS detection and data analysis according to the method of Example 4.2.
  • the analysis results are shown in Table 12 and Figure 9A, FMC63.25 has good binding activity to 293T cells overexpressing monkey CD19, FMC63.26 has weak binding activity to 293T cells overexpressing monkey CD19, FMC63.27 and FMC63 had weak binding activity to 293T cells overexpressing monkey CD19 only at the highest concentration.
  • the binding of FMC63 humanized antibodies to HEK293T cells was simultaneously detected.
  • Figure 9B all FMC63 humanized antibodies did not bind to HEK293T cells and had good specificity.
  • Monkey peripheral blood mononuclear cells were extracted from fresh cynomolgus monkey peripheral blood (purchased from Shanghai Medicilon Biopharmaceutical Co., Ltd.) according to the instructions of Ficoll-Paque Plus (purchased from GE Healthcare, Cat. No.: 171440-02). Cell suspension After centrifugation, the cells were resuspended in PBS containing 1% BSA and counted. At the same time, the mouse antibody Brilliant Violet 605anti-human CD20 (Cat. No.: 302334, purchased from Biolegend) and the FMC63 humanized antibody to be tested (1nM, 10nM and 100nM) were added. . Incubate for 1 hour at room temperature.
  • the proportion of the positive cell population treated with FMC63 humanized antibody to the B cell population at the concentrations of 100 nM, 10 nM and 1 nM, respectively, is calculated as shown in Table 13.
  • Figure 10 shows the double-stained cell scatter plot of Brilliant Violet 605-labeled CD20 and Alexa Fluor 647 secondary antibody indirectly labeled FMC63 humanized antibody. It can be seen from the results that FMC63.25 has a higher proportion of binding to cynomolgus monkey B cells at a concentration of 100 nM, and has comparable or better binding activity than the positive antibody 9G8; other antibodies have no binding to cynomolgus monkey CD19. or relatively weak binding.
  • Anti-human CD19 antibodies were captured using a Protein A chip (GE Helthcare; 29-127-558).
  • Sample and running buffer were HBS-EP+ (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20) (GE Healthcare; BR-1006-69).
  • the flow-through cell was set to 25 °C.
  • the sample block was set to 16°C. Both were pretreated with running buffer.
  • the antibody to be tested was first captured with a Protein A chip, then a single concentration of CD19 antigen protein was injected to record the binding and dissociation process of the antibody and antigen protein, and finally Glycine pH1.5 (GE Helthcare; BR-1003- 54) Complete chip regeneration.
  • Binding was measured by injecting different concentrations of recombinant human CD19-His in solution for 240 sec 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.
  • the binding rate (Kon), dissociation rate (Koff) and binding affinity (KD) of FMC63 humanized antibody to human CD19-His protein are shown in Table 14, wherein antibody FMC63 was used as a control.
  • the KD of FMC63 humanized antibody binding to human CD19 is better than that of 2E-09M, for example, 1.81E-09, the better is 3.46E-10, and the best is 1.95E- 11M, showing comparable or better affinity to FMC63.
  • the antigen-binding epitope of murine FMC63 is the membrane-proximal end (exon4-7) of the outer membrane region of CD19 protein.
  • human CD19exon 1-3-His distal membrane end
  • the FMC63 humanized antibody has no binding activity to human CD19exon 1-3-His.
  • the antigen-binding epitope of the FMC63 humanized antibody is the same as that of the parent antibody FMC63, and both are located at the membrane-proximal end (exon4-7) of the outer region of the CD19 protein.

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Abstract

提供一种CD19人源化抗体及其应用，具体提供能够结合CD19的抗体或抗原结合片段、多特异性抗原结合分子、嵌合抗原受体、免疫效应细胞、核酸片段、载体、细胞、组合物、制备方法、制药用途以及癌症和自身免疫性疾病的治疗方法。

Description

CD19人源化抗体及其应用
本发明要求2020年11月20日向中国国家知识产权局提交的，专利申请号为202011305760.5，发明名称为“CD19人源化抗体及其应用”的在先申请的优先权。上述在先申请的全文通过引用的方式结合于本发明中。
技术领域
本发明涉及抗体领域，具体而言，涉及CD19人源化抗体及其应用。
背景技术
B细胞包括前B细胞(pre-B cell)、早期发育的B细胞(即，不成熟B细胞)和成熟B细胞等，成熟B细胞通过末端分化成为浆细胞和恶性B细胞。大多数的前B急性淋巴细胞性白血病(Pre B ALL)、非霍奇金恶性淋巴瘤、慢性淋巴细胞性白血病(CLL)、前淋巴细胞性白血病、毛细胞白血病、急性淋巴细胞白血病(ALL)和一些非急性淋巴母细胞性白血病都高表达CD19(Nadler等人，J.Immunol.,131:244-250(1983)；Loken等人，Blood,70：1316-1324(1987))。在浆细胞上的CD19的表达进一步说明其可在不同的B细胞瘤例如多发性骨髓瘤、浆细胞瘤、华氏瘤上表达(Grossbard等，Br.J.Haematol,102:509-15(1998)；Treon等，Semin.Oncol，30:248-52(2003))。因此CD19被认为是多种血液肿瘤的靶标。同时，有研究表明，CD19可能在体内调节MHCⅡ类表达和信号传导中起作用，CD19还可作为自身免疫性疾病的潜在免疫治疗靶点。
目前针对CD19的抗体主要是鼠源抗体(鼠抗)，如J Immunol.1987May 1；138(9):2793-9公开了鼠抗HD37、安进公司己上市的药物Blinatumomab等。鼠源CD19抗体FMC63，能够识别人CD19蛋白，多家公司选择该抗体开发药物以及细胞治疗类产品。如已经上市的CD19CAR-T采用鼠源CD19抗体FMC63。但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人生命。因此，鼠源CD19抗体FMC63的成功人源化对该抗体的进一步开发和成药具有重要意义。
发明内容
本发明提供CD19人源化抗体或抗原结合片段、多特异性抗原结合分子、嵌合抗原受体、免疫效应细胞、核酸片段、载体、细胞、组合物、制备方法、制药用途以及疾病治疗方法。
在第一方面，本发明提供一种特异性结合CD19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重链可变区和轻链可变区，所述重链可变区包含：
a、CDR1，其包含SEQ ID NO：2或8～10任一项所示VH的HCDR1；
b、CDR2，其包含SEQ ID NO：2或8～10任一项所示VH的HCDR2；
c、CDR3，其包含SEQ ID NO：2或8～10任一项所示VH的HCDR3；和，
d、框架区，其包含SEQ ID NO：3所示IGHV2-26*01的框架区HFR1、HFR2和HFR3，以及SEQ ID NO：4所示IGHJ6*01的框架区HFR4；
所述轻链可变区包含：
a、CDR1，其包含SEQ ID NO：1或7所示VL的LCDR1；
b、CDR2，其包含SEQ ID NO：1或7所示VL的LCDR2；
c、CDR3，其包含SEQ ID NO：1或7所示VL的LCDR3；和，
d、框架区，其包含SEQ ID NO：5所示IGKV1-39*01的框架区LFR1、LFR2和LFR3，以及SEQ ID NO：6所示IGKJ4*01的框架区LFR4。
在一些具体的实施方案中，SEQ ID NO：1～10任一项所示序列的CDR区和框架区根据Kabat编号系统、Chothia编号系统或IMGT编号系统确定；
可选地，根据Kabat编号系统：
所述HCDR1为DYGVS(SEQ ID NO:22)；
所述HCDR2为VIWGSETTYYNSALKS(SEQ ID NO：23)；
所述HCDR3为HYYYGGSYAMDY(SEQ ID NO：24)；
所述HFR1为QVTLKESGPVLVKPTETLTLTCTVSGFSLS(SEQ ID NO：25)；
所述HFR2为WIRQPPGKALEWLA(SEQ ID NO：26)；
所述HFR3为RLTISKDTSKSQVVLTMTNMDPVDTATYYCAR(SEQ ID NO：27)；
所述HFR4为WGQGTTVTVSS(SEQ ID NO:4)；
所述LCDR1为RASQDISKYLN(SEQ ID NO：28)；
所述LCDR2为HTSRLHS(SEQ ID NO：29)；
所述LCDR3为QQGNTLPYT(SEQ ID NO：30)；
所述LFR1为DIQMTQSPSSLSASVGDRVTITC(SEQ ID NO:31)，
所述LFR2为WYQQKPGKAPKLLIY(SEQ ID NO:32)；
所述LFR3为GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC(SEQ ID NO：33)；
所述LFR4为FGGGTKVEIK(SEQ ID NO:6)；
可选地，根据Chothia编号系统：
所述HCDR1为GVSLPDY(SEQ ID NO:34)、GFSLSDY(SEQ ID NO：35)或GFSLPDY(SEQ ID NO：36)；
所述HCDR2为WGSET(SEQ ID NO：37)
所述HCDR3为HYYYGGSYAMDY(SEQ ID NO：24)；
所述HFR1为QVTLKESGPVLVKPTETLTLTCTVS(SEQ ID NO：38)；
所述HFR2为GVSWIRQPPGKALEWLAHI(SEQ ID NO：39)；
所述HFR3为KSYSTSLKSRLTISKDTSKSQVVLTMTNMDPVDTATYYCAR(SEQ ID NO：40)；
所述HFR4为WGQGTTVTVSS(SEQ ID NO:4)；
所述LCDR1为RASQDISKYLN(SEQ ID NO:28)；
所述LCDR2为HTSRLHS(SEQ ID NO:29)；
所述LCDR3为：QQGNTLPYT(SEQ ID NO:30)；
所述LFR1为DIQMTQSPSSLSASVGDRVTITC(SEQ ID NO:31)；
所述LFR2为WYQQKPGKAPKLLIY(SEQ ID NO:32)；
所述LFR3为GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC(SEQ ID NO：33)；
所述LFR4为FGGGTKVEIK(SEQ ID NO:6)
可选地，根据IMGT编号系统：
所述HCDR1为GVSLPDYG(SEQ ID NO:41)、GFSLSDYG(SEQ ID NO：42)或GFSLPDYG(SEQ ID NO：43)；
所述HCDR2为IWGSETT(SEQ ID NO：44)
所述HCDR3为AKHYYYGGSYAMDY(SEQ ID NO：45)；
所述HFR1为QVTLKESGPVLVKPTETLTLTCTVS(SEQ ID NO：38)；
所述HFR2为VSWIRQPPGKALEWLAH(SEQ ID NO：46)；
所述HFR3为SYSTSLKSRLTISKDTSKSQVVLTMTNMDPVDTATYY(SEQ ID NO：47)；
所述HFR4为WGQGTTVTVSS(SEQ ID NO:4)；
所述LCDR1为QDISKY(SEQ ID NO:48)；
所述LCDR2为HT(SEQ ID NO:49)或HTS(SEQ ID NO：50)；
所述LCDR3为QQGNTLPYT(SEQ ID NO:30)；
所述LFR1为DIQMTQSPSSLSASVGDRVTITC(SEQ ID NO51)；
所述LFR2为LNWYQQKPGKAPKLLIY(SEQ ID NO:52)；
所述LFR3为SSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC(SEQ ID NO：53)或者SLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC(SEQ ID NO：54)；
所述LFR4为FGGGTKVEIK(SEQ ID NO:6)。
在一些具体的实施方案中，根据Kabat编号系统确定编号，所述重链可变区的框架区还包括选自下组的一种或多种的突变：Q1E、F27V、S30P、T73N或R94K；优选包括Q1E和R94K；或优选包括Q1E、S30P和R94K；或优选包括Q1E、F27V、S30P、T73N和R94K。
在一些具体的实施方案中，根据Kabat编号系统编号，所述轻链可变区的框架区还包括选自下组的一种或多种突变：K42G、P44V或F71Y，优选包括K42G、P44V和F71Y。
在一些具体的实施方案中，所述重链可变区具有SEQ ID NO:8～10任一项所述的氨基酸序列，和/或所述轻链可变区具有如SEQ ID NO:7所述的氨基酸序列。
在一些具体的实施方案中，所述重链可变区的CDR1、CDR2和/或CDR3包含与所述HCDR1、HCDR2和/或HCDR3具有至少80％、85％、90％、91％、92％、93％、94％、95％、 96％、97％、98％、99％或100％同一性的序列；
和/或，所述轻链可变区的CDR1、CDR2和/或CDR3包含与所述LCDR1、LCDR2和/或LCDR3具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的序列。
在一些具体的实施方案中，所述重链可变区的CDR1、CDR2和/或CDR3包含与所述HCDR1、HCDR2和/或HCDR3相比发生至多6个氨基酸突变的序列，所述突变的数目可选自0、1、2、3、4、5或6；
和/或，所述轻链可变区的CDR1、CDR2和/或CDR3包含与所述LCDR1、LCDR2和/或LCDR3相比发生至多6个氨基酸突变的序列，所述突变的数目可选自0、1、2、3、4、5或6。
在一些具体的实施方案中，所述重链可变区的框架区包含与HFR1、HFR2、HFR3和/或HFR4相比具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的序列；
和/或，所述轻链可变区的框架区包含与LFR1、LFR2、LFR3和/或LFR4相比具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的序列。
在一些具体的实施方案中，所述重链可变区的框架区包含与所述HFR1、HFR2、HFR3和/或HFR4相比发生至多15个氨基酸突变的序列，所述突变的数目可选自0、1、2、3、4、5、6、7、8、9、10、11、12、13、14或15；
和/或，所述轻链可变区包含与所述LFR1、LFR2、LFR3和/或LFR4相比发生至多15个氨基酸突变的序列，所述突变的数目可选自0、1、2、3、4、5、6、7、8、9、10、11、12、13、14或15。
在一些具体的实施方式中，所述突变可选自插入、缺失或替换，优选地，所述替换为保守氨基酸的替换。
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，所述重链恒定区包含全长重链恒定区或其片段，所述片段可选自CH1结构域、Fc结构域或CH3结构域；
优选地，所述重链恒定区和/或轻链恒定区为人重链恒定区和/或人轻链恒定区；
优选地，所述重链恒定区可选自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，所述重链恒定区是人Ig G1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；
优选地，所述抗体或抗原结合片段缺乏岩藻糖基化。
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、全长抗体、抗体片段、裸抗体、缀合抗体、人源化抗体、全人抗体、Fab、Fab’、Fab’-SH、F(ab’) 2、Fd、Fv、scFv、双抗体(diabody)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、超声造影剂或光敏剂。
在一些具体的实施方式中，所述抗体或抗原结合片段与人CD19和/或猴CD19结合；可选地，所述抗体或抗原结合片段结合人CD19的KD值小于1.00E-8M、1.00E-9M、2.00E-09M、3.00E-9M、4.00E-09M、5.00E-09M、6.00E-09M、7.00E-09M、8.00E-09M、9.00E-09M、1.00E-10M、2.00E-10M、3.00E-10M、4.00E-10M、5.00E-10M、6.00E-10M、7.00E-10M、8.00E-10M、9.00E-10M、1.00E-11M、2.00E-11M、3.00E-11M、4.00E-11M、5.00E-11M、6.00E-11M、7.00E-11M、8.00E-11M、9.00E-11M、1.00E-12M、2.00E-12M、3.00E-12M、4.00E-12M、5.00E-12M、6.00E-12M、7.00E-12M、8.00E-12M或9.00E-12M。
在第二方面，本发明还公开一种多特异性抗原结合分子，所述多特异性抗原结合分子包含第一抗原结合模块和第二抗原结合模块，所述第一抗原结合模块包含前述的抗体或抗原结合片段，所述第二抗原结合模块特异性结合CD19以外的其他抗原或结合与第一抗原结合模块不同的CD19抗原表位；
优选地，所述其他抗原选自CD3、CD16、CD16A、CD4、CD5、CD8、CD14、CD15、CD19、CD20、CD21、CD22、CD23、CD25、CD33、CD37、CD38、CD40、CD40L、CD46、CD52、CD54、CD66(a-d)、CD74、CD80、CD126、CD138、B7、MUC、Ia、HLA-DR、腱生蛋白、VEGF、P1GF、ED-B纤连蛋白、癌基因产物、IL-2、IL-6、TRAIL-R1或TRAIL-R2；
优选地，所述多特异性抗体为双特异性、三特异性或四特异性。
在第三方面，本发明还公开一种嵌合抗原受体(CAR)，所述嵌合抗原受体包含细胞外抗原结合结构域、跨膜结构域和胞内结构域，所述细胞外抗原结合结构域包含前述CD19抗体或抗原结合片段。
在第四方面，本发明还公开一种免疫效应细胞，所述免疫效应细胞包含前述嵌合抗原受体或包含编码前述嵌合抗原受体的核酸片段；
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 cell)、单核细胞、巨噬细胞、树突状细胞或肥大细胞；所述T细胞可选自细胞毒性T细胞、调节性T细胞(Treg)或辅助性T细胞；
优选地，所述免疫效应细胞为同种异体免疫效应细胞或自体免疫效应细胞。
在第五方面，本发明还公开一种分离的核酸片段，所述核酸片段编码前述的抗体或抗原结合片段或多特异性抗原结合分子或嵌合抗原受体。
在第六方面，本发还公开一种载体(vector)，所述载体包含前述核酸片段。
在第七方面，本发明还公开一种宿主细胞，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293T细胞系)；优选地，所述细胞缺乏岩藻糖基转移酶，更优选地， 所述岩藻糖基转移酶是FUT8。
在第八方面，本发明还公开一种制备前述抗体或抗原结合片段或前述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。
在第九方面，本发明还公开一种制备前述免疫效应细胞的方法，所述方法包括将编码前述CAR的核酸片段导入所述免疫效应细胞，可选地，所述方法还包括启动所述免疫效应细胞表达前述CAR。
在第十方面，本发明还公开一种药物组合物，所述组合物包含前述的抗体或抗原结合片段、多特异性抗原结合分子、嵌合抗原受体、免疫效应细胞、核酸片段、载体或细胞；优选地，所述组合物还包含药学上可接受的运载体(carrier)、稀释剂或助剂。
在第十一方面，本发明还公开前述抗体或抗原结合片段、多特异性抗原结合分子、嵌合抗原受体、免疫效应细胞、核酸片段、载体或细胞在制备治疗癌症或自身免疫性疾病的药物中的应用；
优选地，所述癌症选自淋巴瘤或白血病，所述淋巴瘤或白血病可选自B细胞淋巴瘤、非霍奇金淋巴瘤、套细胞淋巴瘤、滤泡性淋巴瘤、边缘区淋巴瘤、原发纵隔B细胞淋巴瘤、弥漫性大B细胞淋巴瘤、前体B细胞急性淋巴细胞白血病(pre-B ALL)、急性淋巴细胞白血病(ALL)、慢性淋巴细胞白血病、毛细胞白血病、前淋巴细胞性白血病、浆细胞瘤、华氏瘤或多发性骨髓瘤；
优先地，所述自身免疫性疾病可选自类风湿性关节炎、多发性硬化症、系统性硬化症、视神经脊髓炎谱系病、系统性红斑狼疮、重症肌无力或IgG4相关性疾病。
在第十二方面，本发明还公开一种治疗癌症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、嵌合抗原受体、免疫效应细胞、核酸片段、载体或细胞；
优选地，所述癌症选自淋巴瘤或白血病，所述淋巴瘤或白血病可选自B细胞淋巴瘤、非霍奇金淋巴瘤、套细胞淋巴瘤、滤泡性淋巴瘤、边缘区淋巴瘤、原发纵隔B细胞淋巴瘤、弥漫性大B细胞淋巴瘤、前体B细胞急性淋巴细胞白血病(pre-B ALL)、急性淋巴细胞白血病(ALL)、慢性淋巴细胞白血病、毛细胞白血病、前淋巴细胞性白血病、浆细胞瘤、华氏瘤或多发性骨髓瘤；
优选地，所述自身免疫性疾病可选自类风湿性关节炎、多发性硬化症、系统性硬化症、视神经脊髓炎谱系病、系统性红斑狼疮、重症肌无力或IgG4相关性疾病。
在第十三方面，本发明还公开前述的抗体或抗原结合片段、多特异性抗原结合分子、嵌合抗原受体、免疫效应细胞、核酸片段、载体或细胞，用于治疗癌症或自身免疫性疾病；
优选地，所述癌症选自淋巴瘤或白血病，所述淋巴瘤或白血病可选自B细胞淋巴瘤、非霍奇金淋巴瘤、套细胞淋巴瘤、滤泡性淋巴瘤、边缘区淋巴瘤、原发纵隔B细胞淋巴瘤、弥漫性大B细胞淋巴瘤、前体B细胞急性淋巴细胞白血病(pre-B ALL)、急性淋巴细胞白血病(ALL)、慢性淋巴细胞白血病、毛细胞白血病、前淋巴细胞性白血病、浆细胞瘤、华氏瘤或多发性骨髓瘤；
优选地，所述自身免疫性疾病可选自类风湿性关节炎、多发性硬化症、系统性硬化症、视神经脊髓炎谱系病、系统性红斑狼疮、重症肌无力或IgG4相关性疾病。
术语定义和说明
除非本文另外定义，本文所有术语均具有本领域普通技术人员通常理解的含义。
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“CD19”(Cluster of Differention 19)是表达于B淋巴细胞及滤泡树突状细胞的表面蛋白，属于免疫球蛋白超家族成员，本文“CD19”包括成熟或未成熟的全长野生型CD19蛋白或其突变体(例如点突变、插入突变或缺失突变)、剪切变体(splice variant)、直系同源物(Orthologs)及其片段。本文“CD19”可以来源于人、灵长类动物，如猴(例如恒河猴、食蟹猴)和啮齿类动物，例如小鼠和大鼠。示例性地，人CD19蛋白的氨基酸序列可参见NCBI：NP_001761.3，猴CD19蛋白的氨基酸序列可参见NCBI：XM_005591542.1。
本文术语“结合”或“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10 -7M或更低、约10 -8M或更低、约1×10 -9M或更低、约1×10 -10M或更低、1×10 -11M或更低、1×10 -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，示例性地，可参见本文实施例6所示方法获得KD。
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、抗原结合片段、裸抗体、缀合抗体、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/或配对的抗体。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”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”可互换使用，是指其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”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、F(ab’) 2、Fab’、Fab’-SH、Fd、Fv、scFv、双抗体(diabody)和单域抗体。
完整抗体经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Fab片段含有重链可变区和轻链可变区，还有轻链的恒定区和重链的第一恒定区(CH1)。如此，本文术语“Fab片段”指包含轻链的VL区和恒定区(CL)的轻链片段，和重链的VH区和第一恒定区(CH1)的抗体片段。Fab’片段因在重链CH1区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是其重链恒定区的半胱氨酸残基携带游离硫醇基团的Fab’片段。F(ab’) 2是完整抗体经胃蛋白酶处理产生的具有两个抗原结合位点(两个Fab片段)和Fc区的一部分的抗体片段。
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,Science242:423-426(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)。可用于本发明的其他接头由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，但使用太短的接头(linker)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“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(VHH)。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“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)来自于人源抗体(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”不意图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“构架区”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，其中重链可变区的框架区可缩写为HFR，轻链可变区的框架区可缩写为LFR。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”可由本领域公知的编号系统加以标注和定义，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。
示例性地，FMC63及其人源化抗体重链可变区(SEQ ID NO:2、8～10)和轻链可变区(SEQ ID NO:1,7)以及人源化模板IGHV2-26*01(SEQ ID NO：3)和IGHJ6*01(SEQ ID NO：4)、IGHV2-26*01(SEQ ID NO：3)和IGHJ6*01(SEQ ID NO：4)，按kabat编号系统、chothia编号系统或IMGT编号系统进行CDR定义和FR区定义，具体如下所示(www.abysis.org/abysis/sequence_input/key_annotation/key_annotation.cgi，http://www.imgt.org/3Dstructure-DB/cgi/DomainGapAlign.cgi#results)：
根据Kabat编号系统
1.SEQ ID NO：2、8～10所示VH的HCDR1、HCDR2和HCDR3：
HCDR1为DYGVS(SEQ ID NO:22)；
HCDR2为VIWGSETTYYNSALKS(SEQ ID NO：23)；
HCDR3为HYYYGGSYAMDY(SEQ ID NO：24)。
2.根据SEQ ID NO：3所示IGHV2-26*01和SEQ ID NO：4所示IGHJ6*01的框架区FR1、FR2、FR3和FR4：
HFR1为QVTLKESGPVLVKPTETLTLTCTVSGFSLS(SEQ ID NO：25)；
HFR2为WIRQPPGKALEWLA(SEQ ID NO：26)；
HFR3为RLTISKDTSKSQVVLTMTNMDPVDTATYYCAR(SEQ ID NO：27)；
HFR4为WGQGTTVTVSS(SEQ ID NO:4)。
3.SEQ ID NO：1或7所示VL的LCDR1、LCDR2和LCDR3：
LCDR1为RASQDISKYLN(SEQ ID NO：28)；
LCDR2为HTSRLHS(SEQ ID NO：29)；
LCDR3为QQGNTLPYT(SEQ ID NO：30)。
4.根据SEQ ID NO：5所示IGKV1-39*01和SEQ ID NO：6所示IGKJ4*01的框架区FR1、FR2、FR3和FR4：
LFR1为DIQMTQSPSSLSASVGDRVTITC(SEQ ID NO:31)；
LFR2为WYQQKPGKAPKLLIY(SEQ ID NO:32)；
LFR3为GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC(SEQ ID NO：33)；
LFR4为FGGGTKVEIK(SEQ ID NO:6)。
根据Chothia编号系统
1.SEQ ID NO：2、8～10所示VH的HCDR1、HCDR2和HCDR3
HCDR1为GVSLPDY(SEQ ID NO:34)、GFSLSDY(SEQ ID NO：35)或GFSLPDY(SEQ ID NO：36)；
HCDR2为WGSET(SEQ ID NO：37)
HCDR3为HYYYGGSYAMDY(SEQ ID NO：24)；
2.根据SEQ ID NO：3所示IGHV2-26*01和SEQ ID NO：4所示IGHJ6*01的框架区FR1、FR2、FR3和FR4
HFR1为QVTLKESGPVLVKPTETLTLTCTVS(SEQ ID NO：38)；
HFR2为GVSWIRQPPGKALEWLAHI(SEQ ID NO：39)；
HFR3为KSYSTSLKSRLTISKDTSKSQVVLTMTNMDPVDTATYYCAR(SEQ ID NO：40)；
HFR4为WGQGTTVTVSS(SEQ ID NO:4)；
3.SEQ ID NO：1或7所示VL的LCDR1、LCDR2和LCDR3
LCDR1为RASQDISKYLN(SEQ ID NO:28)；
LCDR2为HTSRLHS(SEQ ID NO:29)；
LCDR3为：QQGNTLPYT(SEQ ID NO:30)；
4.根据SEQ ID NO：5所示IGKV1-39*01和SEQ ID NO：6所示IGKJ4*01的框架区FR1、FR2、FR3和FR4
LFR1为DIQMTQSPSSLSASVGDRVTITC(SEQ ID NO:31)；
LFR2为WYQQKPGKAPKLLIY(SEQ ID NO:32)；
LFR3为GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC(SEQ ID NO：33)；
LFR4为FGGGTKVEIK(SEQ ID NO:6)。
根据IMGT编号系统
1.SEQ ID NO：2、8～10所示VH的HCDR1、HCDR2和HCDR3
HCDR1为GVSLPDYG(SEQ ID NO:41)、GFSLSDYG(SEQ ID NO：42)或GFSLPDYG(SEQ ID NO：43)；
HCDR2为IWGSETT(SEQ ID NO：44)；
HCDR3为AKHYYYGGSYAMDY(SEQ ID NO：45)；
2.根据SEQ ID NO：3所示IGHV2-26*01和SEQ ID NO：4所示IGHJ6*01的框架区FR1、FR2、FR3和FR4
HFR1为QVTLKESGPVLVKPTETLTLTCTVS(SEQ ID NO：38)；
HFR2为VSWIRQPPGKALEWLAH(SEQ ID NO：46)；
HFR3为SYSTSLKSRLTISKDTSKSQVVLTMTNMDPVDTATYYC(SEQ ID NO：47)；
HFR4为WGQGTTVTVSS(SEQ ID NO:4)；
3.SEQ ID NO：1或7所示VL的LCDR1、LCDR2和LCDR3
LCDR1为QDISKY(SEQ ID NO:48)；
LCDR2为HT(SEQ ID NO:49)或HTS(SEQ ID NO：50)；
LCDR3为QQGNTLPYT(SEQ ID NO:30)；
4.根据SEQ ID NO：5所示IGKV1-39*01和SEQ ID NO：6所示IGKJ4*01的框架区FR1、FR2、FR3和FR4
LFR1为DIQMTQSPSSLSASVGDRVTITCRAS(SEQ ID NO:51)；
LFR2为LNWYQQKPGKAPKLLIY(SEQ ID NO:52)；
LFR3为SSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC(SEQ ID NO：53)或者SLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC(SEQ ID NO：54)；
LFR4为FGGGTKVEIK(SEQ ID NO:6)。
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”至少包含以下之一：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)。以下结合氨基酸残基突变加以说明，例如，重链可变区F27V突变是指根据前述Kabat编号系统确定的重链第27位氨基酸残基由F突变为V。
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：
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“同一性”可通过以下方式计算获得：为确定两个氨基酸序列或两个核酸序列的“同一性”百分数，将所述序列出于最佳比较目的比对(例如，可以为最佳比对而在第一和第二氨基酸序列或核酸序列之一或二者中引入空位或可以为比较目的而抛弃非同源序列)。随后比较在对应氨基酸位置或核苷酸位置处的氨基酸残基或核苷酸。当第一序列中的位置由第二序列中对应位置处的相同氨基酸残基或核苷酸占据时，则所述分子在这个位置处是相同的。
考虑到为最佳比对这两个序列而需要引入的空位的数目和每个空位的长度，两个序列之间的同一性百分数随所述序列共有的相同位置变化而变化。
可以利用数学算法实现两个序列间的序列比较和同一性百分数的计算。例如，使用已经集成至GCG软件包的GAP程序中的Needlema和Wunsch((1970)J.Mol.Biol.48:444-453)算法(在www.gcg.com可获得)，使用Blossum 62矩阵或PAM250矩阵和空位权重16、14、12、10、8、6或4和长度权重1、2、3、4、5或6，确定两个氨基酸序列之间的同一性百分数。又例如，使用GCG软件包中的GAP程序(在www.gcg.com可获得)，使用NWSgapdna.CMP矩阵和空位权重40、50、60、70或80和长度权重1、2、3、4、5或6，确定两个核苷酸序列之间的同一性百分数。特别优选的参数集合(和除非另外说明否则应当使用的一个参数集合)是采用空位罚分12、空位延伸罚分4和移码空位罚分5的Blossum62评分矩阵。
还可以使用PAM120加权余数表、空位长度罚分12，空位罚分4，利用已经并入ALIGN程序(2.0版)的E.Meyers和W.Miller算法,((1989)CABIOS,4:11-17)确定两个氨基酸序列或核苷酸序列之间的同一性百分数。
额外地或备选地，可以进一步使用本发明所述的核酸序列和蛋白质序列作为“查询序列”以针对公共数据库执行检索，以例如鉴定其他家族成员序列或相关序列。例如，可以使用Altschul等人,(1990)J.Mol.Biol.215:403-10的NBLAST及XBLAST程序(版本2.0)执行此类检索。BLAST核苷酸检索可以用NBLAST程序，评分＝100、字长度＝12执行，以获得与本发明核酸(SEQ ID NO:1)分子同源的核苷酸序列。BLAST蛋白质检索可以用XBLAST程序、评 分＝50、字长度＝3执行，以获得与本发明蛋白质分子同源的氨基酸序列。为了出于比较目的获得带空位的比对结果，可以如Altschul等人,(1997)Nucleic Acids Res.25:3389-3402中所述那样使用空位BLAST。当使用BLAST和空位BLAST程序时，可以使用相应程序(例如，XBLAST和NBLAST)的默认参数。参见www.ncbi.nlm.nih.gov。
本文术语“抗原嵌合受体(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“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，直接表达本发明的抗体。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 823B1)。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“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”是指接受对如本发明所述的特定疾病或病症的治疗的生物体。对象和患者的实例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。
有益效果
与现有技术相比，本发明的技术方案至少具有以下之一的有益效果：
1.与鼠源FMC63抗体相比，本发明所述人源化抗体不但具有结合CD19的能力，同时还降低了免疫原性，有助于降低人受试者使用时的免疫排斥风险。
2.在与人CD19和/或猴CD19结合方面，意外发现本发明所述人源化抗体与其亲本鼠源抗体FMC63相当或更优，优于常规的人源化抗体(常规人源化抗体的结合能力较之亲本鼠源抗体降低2～3倍)。
3.本发明所述人源化抗体显示与人CD19和/或猴CD19的良好结合能力，有利于提高其治疗效果和/或开展临床前的动物实验。
附图说明
图1为人CD19 exon1-3-his蛋白样品SDS-PAGE还原胶和非还原胶检测结果，泳道M为蛋白marker，泳道1为非还原条件，泳道2为还原条件；
图2A为FMC63抗体检测Raji细胞CD19表达量的FACS结果；
图2B为9G8抗体检测Raji细胞CD19表达量的FACS结果；
图3为人CD19蛋白转染的CHO-K1细胞FACS筛选检测结果；
图4为FMC63抗体检测猴CD19蛋白转染的HEK293T细胞的FACS结果；
图5为ELISA检测FMC63人源化抗体与人CD19-His蛋白的结合反应；
图6A为FACS检测FMC63人源化抗体与Raji的结合反应；
图6B为FACS检测FMC63人源化抗体与CHO-K1-人CD19的结合反应；
图6C为FACS检测FMC63人源化抗体与MOLT-4的结合反应；
图6D为FACS检测FMC63人源化抗体与CHO-K1的结合反应；
图7为ELISA检测FMC63人源化抗体与鼠CD19-his蛋白的结合反应；
图8为ELISA检测FMC63人源化抗体与猴CD19-his蛋白的结合反应；
图9A为FACS检测FMC63人源化抗体与HEK293T-猴CD19的结合反应；；
图9B为FACS检测FMC63人源化抗体与HEK293T的结合反应；
图10为FACS检测FMC63人源化抗体与食蟹猴B细胞的结合反应，其中FMC63-L2H5即FMC63.25，FMC63-L2H6即FMC63.26，FMC63-L2H7即FMC63.27；
图11A为SPR检测FMC63与人CD19蛋白的亲和力；
图11B为SPR检测FMC63.25人源化抗体与人CD19蛋白的亲和力；
图11C为SPR检测FMC63.26人源化抗体与人CD19蛋白的亲和力；
图11D为SPR检测FMC63.27人源化抗体与人CD19蛋白的亲和力；
图12为为ELISA检测FMC63人源化抗体与人CD19 exon1-3-his蛋白的结合反应。
具体实施方式
下面结合具体实施例来进一步描述本发明，本发明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。
本发明实施例仅是范例性的，并不对本发明的范围构成任何限制。本领域技术人员应该理解的是，在不偏离本发明的精神和范围下可以对本发明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发明的保护范围内。
实施例1 FMC63抗体的人源化
1.1 鼠源抗体FMC63的人源化设计
比对IMGT(http://imgt.cines.fr)人类抗体重轻链可变区种系基因数据库，挑选 IGKV1-39*01和IGKJ4*01作为FMC63的人源化轻链模板，挑选IGHV2-26*01和IGHJ6*01作为FMC63的人源化重链模板。将鼠源抗体的CDR分别移植到相应的人源模板中，形成次序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4的可变区序列，并对框架序列中的个别氨基酸残基进行回复突变和热点突变(具体参见表1)。本实施例抗体的氨基酸残基序列编号、CDR区和FR区均由Kabat编号系统确定。
表1 FMC63的人源化抗体回复突变和热点突变设计
Figure PCTCN2021131305-appb-000001
注：Grafted代表将鼠抗体CDR植入人种系FR区序列；K42G表示将Grafted第42位K突变回G，其它依此类推。
对上述表1的FMC63的人源化抗体轻、重链可变区的突变设计进行交叉组合，最终获得多种FMC63人源化抗体(参见表2)。
表2 FMC63人源化抗体可变区对应氨基酸序列
  FMC63.VH5 FMC63.VH6 FMC63.VH7
FMC63.VL2 FMC63.25 FMC63.26 FMC63.27
注：FMC63.25表示FMC63人源化抗体FMC63.25具有如FMC63.VL2所述轻链可变区和如FMC63.VH5所述重链可变区，其它依此类推。
1.2 鼠源FMC63抗体、人源化模板及FMC63人源化抗体可变区具体序列
FMC63轻链可变区(SEQ ID NO:1)：
Figure PCTCN2021131305-appb-000002
FMC63重链可变区(SEQ ID NO:2)：
Figure PCTCN2021131305-appb-000003
IGHV2-26*01(SEQ ID NO：3)：
Figure PCTCN2021131305-appb-000004
IGHJ6*01(SEQ ID NO：4)：WGQGTTVTVSS。
IGKV1-39*01(SEQ ID NO:5)：
Figure PCTCN2021131305-appb-000005
IGKJ4*01(SEQ ID NO：6)：FGGGTKVEIK。
FMC63.VL2氨基酸序列(SEQ ID NO：7)：
Figure PCTCN2021131305-appb-000006
FMC63.VH5氨基酸序列(SEQ ID NO：8)：
Figure PCTCN2021131305-appb-000007
FMC63.VH6氨基酸序列(SEQ ID NO：9)：
Figure PCTCN2021131305-appb-000008
FMC63.VH7氨基酸序列(SEQ ID NO：10)：
Figure PCTCN2021131305-appb-000009
1.3 FMC63人源化抗体制备
由泰州市百英生物科技有限公司将FMC63人源化抗体FMC63.25、FMC63.26和FMC63.27的重链可变区序列克隆到包含信号肽和鼠源抗体IgG1的重链恒定区的表达载体pcDNA3.4-B1HH1(重链恒定区序列如SEQ ID NO：11所示)，轻链可变区序列克隆到包含信号肽和鼠源抗体IgG1的轻链恒定区的表达载体pcDNA3.4-B1HLK(轻链恒定区序列如SEQ ID NO：12所示)，获得FMC63.25-mIgG1、FMC63.26-mIgG1和FMC63.27-mIgG1的序列，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(购自苏州益研生物科技有限公司)，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，货号分别为：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。
鼠源抗体IgG1重链恒定区(SEQ ID NO:11)：
Figure PCTCN2021131305-appb-000010
鼠源抗体IgG1轻链恒定区(SEQ ID NO：12)：
Figure PCTCN2021131305-appb-000011
实施例2 CD19抗原以及阳性抗体的制备
2.1 人CD19 exon1-3-His标签蛋白的制备
将含有编码人CD19 exon1-3 Pro 20-Gln186氨基酸序列(SEQ ID NO：13)的核苷酸序列克隆到pTT5载体(由通用生物系统安徽有限公司完成)并按已建立的标准分子生物学方法制备质粒，对应的氨基酸序列信息如下表3所示。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)。对HEK293E细胞(购自苏州益研生物科技有限公司)进行瞬时转染(PEI，Polysciences，货号：24765-1)并使用FreeStyle TM 293(Thermofisher scientific，货号：12338018)在37℃下进行扩大培养。6天后收集细胞培养液，离心去除细胞成分，获得含人CD19 exon1-3的培养上清液。将培养上清液上样到镍离子亲和层析柱HisTrap TM Excel(GE Healthcare，货号：GE17-3712-06)，同时用紫外(UV)检测仪监测紫外吸收值(A280nm)的变化。上样后用20mM PB，0.5M NaCl(pH7.4)清洗镍离子亲和层析柱直到紫外吸收值回到基线，然后用buffer A：20mM PB，0.5M NaCl(pH7.4)和buffer B：20mM PB，0.5M NaCl，500mM咪唑进行梯度洗脱(2％,4％,8％,16％,50％,100％)，收集从镍离子亲和层析柱上洗脱下来的带His标签的人CD19 exon1-3蛋白，用PBS磷酸盐缓冲液(pH7.4)在4℃冰箱透析过夜。透析后的蛋白经0.22微米滤膜无菌过滤后分装于-80℃保存，即获得纯化的人CD19 exon1-3蛋白，SDS-PAGE还原胶和非还原胶检测样品目的条带如图1所示。
表3 人CD19 exon1-23蛋白氨基酸序列
Figure PCTCN2021131305-appb-000012
2.2 人CD19对照抗体的制备
FMC63和9G8克隆是识别人CD19的抗体，两者的抗原结合表位位于近膜端。FMC63克隆的重链可变区和轻链可变区序列根据专利WO2016033570A1获得，9G8克隆的重链可变区和轻链可变区序列根据专利WO2018083535获得。由泰州市百英生物科技有限公司将FMC63和9G8克隆的轻链可变区序列克隆到包含信号肽和鼠源抗体IgG1的轻链恒定区的表达载体pcDNA3.4-B1HH1，重链可变区序列克隆到包含信号肽和鼠源抗体IgG1的重链恒定区的表达载体pcDNA3.4-B1HLK，获得FMC63-mIgG1和9G8-mIgG1的序列，如无特殊说明则后文中FMC63和9G8均分别指FMC63-mIgG1和9G8-mIgG1。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(购自苏州益研生物科技有限公司)，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，货号分别为：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。
表4 抗人CD19的抗体FMC63-mIgG1和9G8-mIgG1的重链和轻链序列信息
Figure PCTCN2021131305-appb-000013
Figure PCTCN2021131305-appb-000014
实施例3 内源表达细胞株的鉴定、过表达人CD19以及猴CD19的细胞株构建和鉴定
3.1 内源性表达CD19细胞株的鉴定
将Raji细胞(购自武汉大学中国典型培养物保藏中心)在T-25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。用FMC63和9G8抗体作为一抗，APC标记的二抗(购自Biolegend，货号：409306)，用FACS(FACS CantoTM，购自BD公司)检测和分析结果。分析结果如表5以及图2A-2B所示，Raji细胞均可与FMC63和9G8结合。
表5 内源细胞系Raji细胞的FACS检测结果
Figure PCTCN2021131305-appb-000015
3.2 CHO-K1稳转人CD19单克隆细胞株的制备
编码人CD19全长氨基酸序列(NCBI：NP_001761.3，SEQ ID NO：20)的核苷酸序列被克隆到pcDNA3.1载体并制备质粒(由通用生物系统(安徽)有限公司完成)。对CHO-K1细胞系(购自中国科学院上海生命科学研究院)进行质粒转染(
Figure PCTCN2021131305-appb-000016
3000Transfection Kit，购自Invitrogen，货号：L3000-015)后，在含10μg/ml嘌呤霉素和含10％(w/w)胎牛血清的DMEM/F12培养基中选择性培养2周，用FMC63抗体作为一抗，Alexa Fluor 647标记的二抗(Jackson，货号：109605088)在流式细胞仪FACS AriaII(BD Biosciences)上分选阳性单克隆细胞到96孔板，并置于37℃，5％(v/v)CO 2细胞培养箱中培养，大约2周后选择部分单克隆孔进行扩增。对扩增后的克隆经流式细胞分析法进行筛选。选择长势较 好、荧光强度较高的单克隆细胞系继续扩大培养并液氮冻存。
具体选择结果如表6和图3所示，IgG亚型对照为鼠IgG1对照，图3中，横坐标为细胞荧光强度，纵坐标为细胞数。表6和图3的结果说明，已制得人CD19高水平表达的CHO-K1单克隆细胞株：CHO-K1-人CD19-2C8、CHO-K1-人CD19-1C4、CHO-K1-人CD19-2G4、以及CHO-K1-人CD22 1C9。选择CHO-K1-人CD19-2C8细胞株用于后续抗体结合实验。
人CD19全长氨基酸序列(SEQ ID NO：20)：
Figure PCTCN2021131305-appb-000017
表6 人CD19蛋白的CHO-K1稳转细胞系FACS检测结果
Figure PCTCN2021131305-appb-000018
3.3 HEK293T稳转猴CD19细胞株的制备
编码猴CD19全长氨基酸序列(NCBI：XM_005591542.1,SEQ ID NO:21)的核苷酸序列被克隆到pcDNA3.1载体(购自Thermofisher scientific)并制备质粒。对HEK293T细胞系用
Figure PCTCN2021131305-appb-000019
HD(Promega，货号：#E2311)进行质粒转染后，在含10μg/ml puromycin的含10％(w/w)胎牛血清的DMEM培养基中选择性培养2周，用有限稀释法在96孔培养板中进行亚克隆，并置于37℃，5％(v/v)CO 2培养，大约2周后选择部分多克隆孔扩增到6孔板中。对扩增后的克隆用具有猴交叉活性的CD19抗体9G8经流式细胞分析法进行筛选，选择长势较好、荧光强度较高的细胞系继续扩大培养并液氮冻存。结果如表7和图4所示。9G8抗体检测HEK293T细胞株经流式细胞分析显示经过puromycin筛选后过表达猴CD19的阳性细胞峰，可用于检测FMC63人源化抗体的交叉活性。
猴CD19全长氨基酸序列(SEQ ID NO：21)：
Figure PCTCN2021131305-appb-000020
Figure PCTCN2021131305-appb-000021
表7.猴CD19蛋白的HEK293T稳转细胞系FACS检测结果
Figure PCTCN2021131305-appb-000022
实施例4 FMC63人源化抗体的鉴定
4.1 酶联免疫吸附实验(ELISA)检测抗体与CD19蛋白的结合
将人CD19-his蛋白(购自ACROBiosystems，货号：CD9-H52H2)用PBS稀释到终浓度4μg/mL，然后以100μl每孔加到96孔ELISA板。用塑料膜封好4℃孵育过夜，第二天用PBS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100nM梯度稀释的FMC63人源化抗体、FMC63或阴性对照抗体50μl每孔。37℃孵育2小时后，用PBS洗板3次。加入HRP(辣根过氧化物酶)标记的二抗(购自Jackson Immuno，货号：115-035-003)，37℃孵育1小时后，用PBS洗板5次。加入TMB底物50μl每孔。室温孵育10分钟后，加入终止液(1.0N HCl)50μl每孔。用ELISA读板机(Multimode Plate Reader，EnSight，购自Perkin Elmer)读取OD450nm数值，FMC63人源化抗体与人CD19的ELISA结果如图5和表8所示，表8说明，FMC63人源化抗体均能与人CD19在ELISA水平结合。其中阴性对照抗体mIgG1为针对鸡卵溶菌酶的抗体anti-hel-mIgG1(购自百英，货号：B118301)，表中的数据为OD450nm值。
表8.ELISA检测FMC人源化抗体与人CD19蛋白的结合反应
Figure PCTCN2021131305-appb-000023
4.2 流式细胞实验(FACS)检测FMC63人源化抗体与不同CD19表达细胞的结合
将所需细胞在T-75细胞培养瓶中扩大培养至对数生长期，对于贴壁细胞CHO-K1吸除培养基，用PBS缓冲液洗涤2次，然后用胰酶消化细胞，终止消化后用PBS缓冲液洗涤细胞2次；对于悬浮细胞Raji直接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)BSA]封闭液重悬至2×10 6个细胞/毫升， 按50μl/孔加入到96孔FACS反应板中，加入待测样品(FMC63人源化抗体、FMC63或阴性对照抗体)50μl/孔，冰上孵育2小时。用PBS缓冲液离心洗涤3次，加入50μl/孔Alexa Fluor647标记的二抗(购自Jackson Immuno，货号：115-605-003)，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。分析结果如表9以及图6A-6D所示，FMC63人源化抗体均可结合Raji细胞和CHO-K1-人CD19细胞表面的人CD19蛋白(图6A-6B)。使用同样的方法同时检测了FMC63人源化抗体与内源CD19阴性的细胞MOLT-4细胞(购自ATCC，CRL-1582)以及CHO-K1细胞的结合，结果如图6C-6D所示，FMC63人源化抗体均不结合MoLT4细胞以及CHO-K1细胞，具有很好的特异性。
表9.FACS检测FMC63人源化抗体与Raji和CHO-K1-人CD19细胞的结合反应
Figure PCTCN2021131305-appb-000024
实施例5 FMC63人源化抗体的种属交叉结合活性检测
5.1 ELISA检测FMC63人源化抗体与不同种属CD19蛋白的结合
为检测FMC63人源化抗体的种属交叉活性，将商品化的鼠CD19(ACROBiosystems，货号：50510-M08H)和猴CD19(ACROBiosystems，货号：90051-C08H)分别包被ELISA板，按照实施例4.1的方法进行ELISA检测。FMC63人源化抗体与鼠CD19的ELISA结果如图7和表10所示，图7和表10说明，FMC63人源化抗体与鼠CD19在ELISA水平均无结合。其中IgG对照为mIgG1，Alpaca serum为人CD19-ECD-His免疫羊驼后的血清作为阳性对照(Alpaca serum已验证能与鼠CD19结合)，表中的数据为OD450nm值。
表10.ELISA检测FMC63人源化抗体与鼠CD19蛋白的结合反应
Figure PCTCN2021131305-appb-000025
注：Alpaca serum血清从1：100开始进行10倍浓度梯度稀释。
FMC63人源化抗体与猴CD19的ELISA结果如图8和表11所示，图8和表11说明，FMC63人源化抗体与猴CD19在ELISA水平均无结合。其中IgG对照为mIgG1， S003-NB151-89为人CD19-His免疫羊驼后取外周血建库筛选出来的能结合猴CD19-His蛋白的克隆作为阳性对照，表中的数据为OD450nm值。
表11 ELISA检测FMC63人源化抗体与猴CD19蛋白的结合反应
Figure PCTCN2021131305-appb-000026
5.2 FACS检测FMC63人源化抗体与猴CD19表达细胞的结合
将HEK293T-猴CD19细胞按照实施例4.2的方法进行FACS检测与数据分析。分析结果如表12以及图9A所示，FMC63.25与过表达猴CD19的293T细胞有很好的结合活性，FMC63.26与过表达猴CD19的293T细胞有较弱的结合活性，FMC63.27和FMC63与过表达猴CD19的293T细胞仅最高浓度有较弱的结合活性。使用同样的方法同时检测了FMC63人源化抗体与HEK293T细胞的结合，结果如图9B所示，所有FMC63人源化抗体均不结合HEK293T细胞，具有很好的特异性。
表12 FACS检测FMC63人源化抗体与293T-猴CD19细胞的结合反应
Figure PCTCN2021131305-appb-000027
5.3 FACS检测FMC63人源化抗体与食蟹猴(拉丁名：Macaca fascicularis)外周血B细胞的结合
从新鲜食蟹猴外周血(购自上海美迪西生物医药股份有限公司)中按照Ficoll-Paque Plus(购自GE Healthcae，货号:171440-02)说明书提取猴外周血单核细胞，细胞悬液离心后以含1％BSA的PBS重悬细胞后计数，同时加入鼠源抗体Brilliant Violet 605anti-human CD20(货号:302334，购自Biolegend)和待测FMC63人源化抗体(1nM、10nM和100nM)。室温孵育1小时。清洗细胞三次后加入Alexa Fluor 647标记的二抗anti-mouse IgG H+L(货号：115-605-003，购自Jackson Immuno)，避光室温孵育30分钟后清洗细胞5次，用PBS轻轻重悬细胞，用FACS(FACS CantoTM，购自BD公司)检测和分析，其中CD20作为B细胞的标记物，对CD20阳性的B细胞群进行圈门，分析其中FMC63人源化抗体阳性细胞所占比例，分别计算100nM，10nM和1nM浓度下FMC63人源化抗体处理的呈阳性细胞群占B 细胞群的比例如表13所示。Brilliant Violet 605标记CD20和Alexa Fluor 647二抗间接标记FMC63人源化抗体的双染细胞散点图见图10。由结果可知，FMC63.25在100nM浓度条件下与食蟹猴B细胞有较高比例的结合，且相比于阳性抗体9G8有相当或者更好的结合活性；其它抗体与食蟹猴CD19无结合或者相对较弱结合。
表13.FACS检测FMC63人源化抗体与食蟹猴B细胞的结合反应
Figure PCTCN2021131305-appb-000028
实施例6 CD19抗体亲和力测定
6.1 人源化抗体与人CD19-His蛋白亲和力测定
使用Protein A芯片(GE Helthcare；29-127-558)捕获抗人CD19抗体。样品和运行缓冲液是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CD19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CD19-His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。也减去空白注射(＝双重参照)。为了计算表观KD和其他动力学参数，使用Langmuir 1：1模型。FMC63人源化抗体与人CD19-His蛋白的结合速率(Kon)、解离速率(Koff)及结合亲和力(KD)如表14所示，其中抗体FMC63作为对照。如图11和表14所示，FMC63人源化抗体与人CD19结合的KD都优于2E-09M，例如，1.81E-09，更优可至3.46E-10，最优可达到1.95E-11M，表现出与FMC63相当或更优的亲和力。
表14.SPR(biacore)检测FMC63人源化抗体与人CD19的亲和力
抗体名称 ka(1/Ms) kd(1/s) KD(M)
FMC63.25 9.24E+04 1.80E-06 1.95E-11
FMC63.26 9.72E+04 3.36E-05 3.46E-10
FMC63.27 8.42E+04 1.52E-04 1.81E-09
FMC63 9.53E+04 1.17E-04 1.23E-09
实施例7 FMC63人源化抗体与抗原结合区域的鉴定
鼠源FMC63的抗原结合表位是CD19蛋白膜外区的近膜端(exon4-7)。为了进一步确认人源化后的抗体的抗原结合表位仍然保持不变，按照实施例4.1中的ELISA方法，将人CD19exon 1-3-His(远膜端)以2μg/mL包被，如图12和表15所示，FMC63人源化抗体与人CD19exon 1-3-His均无结合活性。因此可判断FMC63人源化抗体的抗原结合表位与母抗体FMC63相同，均位于CD19蛋白膜外区的近膜端(exon4-7)。
表15 FMC63人源化抗体表位分类
Figure PCTCN2021131305-appb-000029

Claims (26)

  1. 一种特异性结合CD19的人源化抗体或抗原结合片段，其特征在于，所述抗体或抗原结合片段包含重链可变区和轻链可变区，所述重链可变区包含：
    a、CDR1，其包含SEQ ID NO：2或8～10任一项所示VH的HCDR1；
    b、CDR2，其包含SEQ ID NO：2或8～10任一项所示VH的HCDR2；
    c、CDR3，其包含SEQ ID NO：2或8～10任一项所示VH的HCDR3；和，
    d、框架区，其包含SEQ ID NO：3所示IGHV2-26*01的框架区HFR1、HFR2和HFR3，以及SEQ ID NO：4所示IGHJ6*01的框架区HFR4；
    所述轻链可变区包含：
    a、CDR1，其包含SEQ ID NO：1或7所示VL的LCDR1；
    b、CDR2，其包含SEQ ID NO：1或7所示VL的LCDR2；
    c、CDR3，其包含SEQ ID NO：1或7所示VL的LCDR3；和，
    d、框架区，其包含SEQ ID NO：5所示IGKV1-39*01的框架区LFR1、LFR2和LFR3，以及SEQ ID NO：6所示IGKJ4*01的框架区LFR4。
  2. 根据权利要求1所述的抗体或抗原结合片段，其特征在于，SEQ ID NO：1～10任一项所示序列的CDR区和框架区根据Kabat编号系统、Chothia编号系统或IMGT编号系统确定；
    可选地，根据Kabat编号系统：
    所述HCDR1为DYGVS(SEQ ID NO:22)；
    所述HCDR2为VIWGSETTYYNSALKS(SEQ ID NO：23)；
    所述HCDR3为HYYYGGSYAMDY(SEQ ID NO：24)；
    所述HFR1为QVTLKESGPVLVKPTETLTLTCTVSGFSLS(SEQ ID NO：25)；
    所述HFR2为WIRQPPGKALEWLA(SEQ ID NO：26)；
    所述HFR3为RLTISKDTSKSQVVLTMTNMDPVDTATYYCAR(SEQ ID NO：27)；
    所述HFR4为WGQGTTVTVSS(SEQ ID NO:4)；
    所述LCDR1为RASQDISKYLN(SEQ ID NO：28)；
    所述LCDR2为HTSRLHS(SEQ ID NO：29)；
    所述LCDR3为QQGNTLPYT(SEQ ID NO：30)；
    所述LFR1为DIQMTQSPSSLSASVGDRVTITC(SEQ ID NO:31)；
    所述LFR2为WYQQKPGKAPKLLIY(SEQ ID NO:32)；
    所述LFR3为GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC(SEQ ID NO：33)；
    所述LFR4为FGGGTKVEIK(SEQ ID NO:6)；
    可选地，根据Chothia编号系统：
    所述HCDR1为GVSLPDY(SEQ ID NO:34)、GFSLSDY(SEQ ID NO：35)或GFSLPDY(SEQ ID NO：36)；
    所述HCDR2为WGSET(SEQ ID NO：37)
    所述HCDR3为HYYYGGSYAMDY(SEQ ID NO：24)；
    所述HFR1为QVTLKESGPVLVKPTETLTLTCTVS(SEQ ID NO：38)；
    所述HFR2为GVSWIRQPPGKALEWLAHI(SEQ ID NO：39)；
    所述HFR3为KSYSTSLKSRLTISKDTSKSQVVLTMTNMDPVDTATYYCAR(SEQ ID NO：40)；
    所述HFR4为WGQGTTVTVSS(SEQ ID NO:4)；
    所述LCDR1为RASQDISKYLN(SEQ ID NO:28)；
    所述LCDR2为HTSRLHS(SEQ ID NO:29)；
    所述LCDR3为：QQGNTLPYT(SEQ ID NO:30)；
    所述LFR1为DIQMTQSPSSLSASVGDRVTITC(SEQ ID NO:31)；
    所述LFR2为WYQQKPGKAPKLLIY(SEQ ID NO:32)；
    所述LFR3为GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC(SEQ ID NO：33)；
    所述LFR4为FGGGTKVEIK(SEQ ID NO:6)
    可选地，根据IMGT编号系统：
    所述HCDR1为GVSLPDYG(SEQ ID NO:41)、GFSLSDYG(SEQ ID NO：42)或GFSLPDYG(SEQ ID NO：43)；
    所述HCDR2为IWGSETT(SEQ ID NO：44)；
    所述HCDR3为AKHYYYGGSYAMDY(SEQ ID NO：45)；
    所述HFR1为QVTLKESGPVLVKPTETLTLTCTVS(SEQ ID NO：38)；
    所述HFR2为VSWIRQPPGKALEWLAH(SEQ ID NO：46)；
    所述HFR3为SYSTSLKSRLTISKDTSKSQVVLTMTNMDPVDTATYYC(SEQ ID NO：47)；
    所述HFR4为WGQGTTVTVSS(SEQ ID NO:4)；
    所述LCDR1为QDISKY(SEQ ID NO:48)；
    所述LCDR2为HT(SEQ ID NO:49)或HTS(SEQ ID NO：50)；
    所述LCDR3为QQGNTLPYT(SEQ ID NO:30)；
    所述LFR1为DIQMTQSPSSLSASVGDRVTITCRAS(SEQ ID NO:51)；
    所述LFR2为LNWYQQKPGKAPKLLIY(SEQ ID NO:52)；
    所述LFR3为SSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC(SEQ ID NO：53)或者SLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC(SEQ ID NO：54)；
    所述LFR4为FGGGTKVEIK(SEQ ID NO:6)。
  3. 根据权利要求1或2所述的抗体或抗原结合片段，其特征在于，根据Kabat编号系统编号，所述重链可变区的框架区还包括选自下组的一种或多种的突变：Q1E、F27V、S30P、T73N或R94K；优选包括Q1E和R94K；或优选包括Q1E、S30P和R94K；或优选包括Q1E、 F27V、S30P、T73N和R94K。
  4. 根据权利要求1～3任一项所述的抗体或抗原结合片段，其特征在于，根据Kabat编号系统编号，所述轻链可变区的框架区还包括选自下组的一种或多种突变：K42G、P44V或F71Y，优选包括K42G、P44V和F71Y。
  5. 根据权利要求1～4任一项所述的抗体或抗原结合片段，其特征在于，所述重链可变区具有SEQ ID NO:8～10任一项所述的氨基酸序列，和/或所述轻链可变区具有SEQ ID NO:7所述的氨基酸序列。
  6. 根据权利要求1～5任一项所述的抗体或抗原结合片段，其特征在于，所述重链可变区的CDR1、CDR2和/或CDR3包含与所述HCDR1、HCDR2和/或HCDR3具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的序列；
    和/或，所述轻链可变区的CDR1、CDR2和/或CDR3包含与所述LCDR1、LCDR2和/或LCDR3具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的序列。
  7. 根据权利要求1～5任一项所述的抗体或抗原结合片段，其特征在于，所述重链可变区的CDR1、CDR2和/或CDR3包含与所述HCDR1、HCDR2和/或HCDR3相比发生至多6个氨基酸突变的序列，所述突变的数目可选自0、1、2、3、4、5或6；
    和/或，所述轻链可变区的CDR1、CDR2和/或CDR3包含与所述LCDR1、LCDR2和/或LCDR3相比发生至多6个氨基酸突变的序列，所述突变的数目可选自0、1、2、3、4、5或6。
  8. 根据权利要求1～7任一项所述的抗体或抗原结合片段，其特征在于，所述重链可变区的框架区包含与所述HFR1、HFR2、HFR3和/或HFR4相比具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的序列；
    和/或，所述轻链可变区的框架区包含与LFR1、LFR2、LFR3和/或LFR4相比具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的序列。
  9. 根据权利要求1～7任一项所述的抗体或抗原结合片段，其特征在于，所述重链可变区的框架区包含与所述HFR1、HFR2、HFR3和/或HFR4相比发生至多15个氨基酸突变的序列，所述突变的数目可选自0、1、2、3、4、5、6、7、8、9、10、11、12、13、14或15；
    和/或，所述轻链可变区包含与所述LFR1、LFR2、LFR3和/或LFR4相比发生至多15个氨基酸突变的序列，所述突变的数目可选自0、1、2、3、4、5、6、7、8、9、10、11、12、13、14或15。
  10. 根据权利要求7或9所述的抗体或抗原结合片段，其特征在于，所述突变可选自插入、缺失或替换，优选地，所述替换为保守氨基酸的替换。
  11. 根据权利要求1～10任一项所述的抗体或抗原结合片段，其特征在于，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，所述重链恒定区包含全长重链恒定区或其片段，所述片段可选自CH1结构域、 Fc结构域或CH3结构域；
    优选地，所述重链恒定区和/或轻链恒定区为人重链恒定区和/或人轻链恒定区；
    优选地，所述重链恒定区可选自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，所述重链恒定区是人Ig G1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；
    优选地，所述抗体或抗原结合片段缺乏岩藻糖基化。
  12. 根据权利要求1～11任一项所述的抗体或抗原结合片段，其特征在于，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、全长抗体、抗体片段、裸抗体、缀合抗体、人源化抗体、全人抗体、Fab、Fab’、Fab’-SH、F(ab’) 2、Fd、Fv、scFv、双抗体(diabody)或单域抗体。
  13. 根据权利要求1～12任一项所述的抗体或抗原结合片段，其特征在于，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、超声造影剂或光敏剂。
  14. 根据权利要求1～13任一项所述的抗体或抗原结合片段，其特征在于，所述抗体或抗原结合片段与人CD19和/或猴CD19结合；可选地，所述抗体或抗原结合片段结合人CD19的KD值小于1.00E-8M、1.00E-9M、2.00E-09M、3.00E-9M、4.00E-09M、5.00E-09M、6.00E-09M、7.00E-09M、8.00E-09M、9.00E-09M、1.00E-10M、2.00E-10M、3.00E-10M、4.00E-10M、5.00E-10M、6.00E-10M、7.00E-10M、8.00E-10M、9.00E-10M、1.00E-11M、2.00E-11M、3.00E-11M、4.00E-11M、5.00E-11M、6.00E-11M、7.00E-11M、8.00E-11M、9.00E-11M、1.00E-12M、2.00E-12M、3.00E-12M、4.00E-12M、5.00E-12M、6.00E-12M、7.00E-12M、8.00E-12M或9.00E-12M。
  15. 一种多特异性抗原结合分子，其特征在于，所述多特异性抗原结合分子包含第一抗原结合模块和第二抗原结合模块，所述第一抗原结合模块包含权利要求1～14任一项所述的抗体或抗原结合片段，所述第二抗原结合模块特异性结合CD19以外的其他抗原或结合与第一抗原结合模块不同的CD19抗原表位；
    优选地，所述其他抗原选自CD3、CD16、CD16A、CD4、CD5、CD8、CD14、CD15、CD19、CD20、CD21、CD22、CD23、CD25、CD33、CD37、CD38、CD40、CD40L、CD46、CD52、CD54、CD66(a-d)、CD74、CD80、CD126、CD138、B7、MUC、Ia、HLA-DR、腱生蛋白、VEGF、P1GF、ED-B纤连蛋白、癌基因产物、IL-2、IL-6、TRAIL-R1或TRAIL-R2；
    优选地，所述多特异性抗体为双特异性、三特异性或四特异性。
  16. 一种嵌合抗原受体(CAR)，其特征在于，所述嵌合抗原受体包含细胞外抗原结合结构域、跨膜结构域和胞内信号传导结构域，所述细胞外抗原结合结构域包含权利要求1～14任一项所述CD19抗体或抗原结合片段。
  17. 一种免疫效应细胞，其特征在于，所述免疫效应细胞包含权利要求16所述嵌合抗原 受体或包含编码权利要求16所述嵌合抗原受体的核酸片段；
    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 cell)、单核细胞、巨噬细胞、树突状细胞或肥大细胞；所述T细胞可选自细胞毒性T细胞、调节性T细胞(Treg)或辅助性T细胞；
    优选地，所述免疫效应细胞为同种异体免疫效应细胞或自体免疫效应细胞。
  18. 一种分离的核酸片段，其特征在于，所述核酸片段编码权利要求1～14任一项所述的抗体或抗原结合片段、权利要求15所述的多特异性抗原结合分子或权利要求16所述的嵌合抗原受体。
  19. 一种载体(vector)，其特征在于，所述载体包含权利要求18所述核酸片段。
  20. 一种宿主细胞，其特征在于，所述宿主细胞包含权利要求19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293T细胞系)；优选地，所述细胞缺乏岩藻糖基转移酶，更优选地，所述岩藻糖基转移酶是FUT8。
  21. 一种制备权利要求1～14任一项所述抗体或抗原结合片段或权利要求15所述多特异性抗原结合分子的方法，其特征在于，所述方法包括培养权利要求20所述细胞，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。
  22. 一种制备权利要求17所述免疫效应细胞的方法，其特征在于，所述方法包括将编码权利要求16所述CAR的核酸片段导入所述免疫效应细胞，可选地，所述方法还包括启动所述免疫效应细胞表达权利要求16所述CAR。
  23. 一种药物组合物，其特征在于，所述组合物包含权利要求1～14任一项所述的抗体或抗原结合片段、权利要求15所述的多特异性抗原结合分子、权利要求16所述的嵌合抗原受体、权利要求17所述的免疫效应细胞、权利要求18所述的核酸片段、权利要求19所述的载体或权利要求20所述的细胞；优选地，所述组合物还包含药学上可接受的运载体(carrier)、稀释剂或助剂。
  24. 权利要求1～14任一项所述抗体或抗原结合片段、权利要求15所述多特异性抗原结合分子、权利要求16所述的嵌合抗原受体、权利要求17所述的免疫效应细胞、权利要求18所述的核酸片段、权利要求19所述的载体或权利要求20所述的细胞在制备治疗癌症或自身免疫性疾病的药物中的应用；
    优选地，所述癌症选自淋巴瘤或白血病，所述淋巴瘤或白血病可选自B细胞淋巴瘤、非霍奇金淋巴瘤、套细胞淋巴瘤、滤泡性淋巴瘤、边缘区淋巴瘤、原发纵隔B细胞淋巴瘤、弥漫性大B细胞淋巴瘤、前体B细胞急性淋巴细胞白血病(pre-B ALL)、急性淋巴细胞白血病(ALL)、慢性淋巴细胞白血病、毛细胞白血病、前淋巴细胞性白血病、浆细胞瘤、华氏瘤或多发性骨髓瘤；
    优选地，所述自身免疫性疾病可选自类风湿性关节炎、多发性硬化症、系统性硬化症、视神经脊髓炎谱系病、系统性红斑狼疮、重症肌无力或IgG4相关性疾病。
  25. 一种治疗癌症或自身免疫性疾病的方法，其特征在于，所述方法包括向受试者施用有效量的权利要求1～14任一项所述的抗体、权利要求15所述多特异性抗原结合分子、权利要 求16所述的嵌合抗原受体、权利要求17所述的免疫效应细胞、权利要求18所述的核酸片段、权利要求19所述的载体或权利要求20所述的细胞；
    优选地，所述癌症选自淋巴瘤或白血病，所述淋巴瘤或白血病可选自B细胞淋巴瘤、非霍奇金淋巴瘤、套细胞淋巴瘤、滤泡性淋巴瘤、边缘区淋巴瘤、原发纵隔B细胞淋巴瘤、弥漫性大B细胞淋巴瘤、前体B细胞急性淋巴细胞白血病(pre-B ALL)、急性淋巴细胞白血病(ALL)、慢性淋巴细胞白血病、毛细胞白血病、前淋巴细胞性白血病、浆细胞瘤、华氏瘤或多发性骨髓瘤；
    优先地，所述自身免疫性疾病可选自类风湿性关节炎、多发性硬化症、系统性硬化症、视神经脊髓炎谱系病、系统性红斑狼疮、重症肌无力或IgG4相关性疾病。
  26. 权利要求1～14任一项所述的抗体或抗原结合片段、权利要求15所述多特异性抗原结合分子、权利要求16所述的嵌合抗原受体、权利要求17所述的免疫效应细胞、权利要求18所述的核酸片段、权利要求19所述的载体或权利要求20所述的细胞，其特征在于，用于治疗癌症或自身免疫性疾病；
    优选地，所述癌症选自淋巴瘤或白血病，所述淋巴瘤或白血病可选自B细胞淋巴瘤、非霍奇金淋巴瘤、套细胞淋巴瘤、滤泡性淋巴瘤、边缘区淋巴瘤、原发纵隔B细胞淋巴瘤、弥漫性大B细胞淋巴瘤、前体B细胞急性淋巴细胞白血病(pre-B ALL)、急性淋巴细胞白血病(ALL)、慢性淋巴细胞白血病、毛细胞白血病、前淋巴细胞性白血病、浆细胞瘤、华氏瘤或多发性骨髓瘤；
    优选地，所述自身免疫性疾病可选自类风湿性关节炎、多发性硬化症、系统性硬化症、视神经脊髓炎谱系病、系统性红斑狼疮、重症肌无力或IgG4相关性疾病。
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WO2024055928A1 (zh) * 2022-09-16 2024-03-21 菲鹏生物股份有限公司 抗猴痘病毒抗体或其抗原结合片段、检测猴痘病毒的试剂和试剂盒
WO2024251263A1 (en) 2023-06-09 2024-12-12 Jw Therapeutics R & D (Shanghai) Co., Ltd. Fusion protein and medical use thereof

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