WO2022007543A1 - Anticorps anti-fgl1 et son utilisation - Google Patents

Anticorps anti-fgl1 et son utilisation Download PDF

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WO2022007543A1
WO2022007543A1 PCT/CN2021/097569 CN2021097569W WO2022007543A1 WO 2022007543 A1 WO2022007543 A1 WO 2022007543A1 CN 2021097569 W CN2021097569 W CN 2021097569W WO 2022007543 A1 WO2022007543 A1 WO 2022007543A1
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seq
amino acid
antibody
variable region
acid sequence
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PCT/CN2021/097569
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Chinese (zh)
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周冲
姜晓玲
王艺臻
黄真真
吴崇兵
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盛禾(中国)生物制药有限公司
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Publication of WO2022007543A1 publication Critical patent/WO2022007543A1/fr

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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
    • C07K16/36—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against blood coagulation factors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • 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]

Definitions

  • the invention belongs to the field of tumor immunotherapy and molecular immunology, in particular to an anti-FGL1 antibody and its application.
  • LAG-3 lymphocyte activation gene 3
  • NK natural killer cells
  • LAG3 protein is a type I membrane protein composed of 498 amino acids, including signal peptide, extracellular domain, transmembrane domain and cytoplasmic domain, and is selectively expressed on activated T lymphocytes, NK cells and dendritic cells.
  • LAG-3 protein is composed of four highly homologous extracellular immunoglobulin superfamily-like (IgSF-like) domains (D1-D4), and its regulatory function on T cells is similar to that of PD-1. Receptors that deliver inhibitory signals function.
  • the extracellular region has four Ig domains, and the entire protein shares sequence similarity with CD4.
  • LAG-3 is an immunosuppressive receptor, and major histocompatibility complex class II (MHC-II) is a typical ligand; there are two other predicted ligands, LSECtin (expressed in melanoma cells) and Galectin-3 (Expressed on stromal cells and CD8+ T cells in the tumor microenvironment).
  • MHC-II major histocompatibility complex class II
  • LAG-3 works with other immune checkpoints to inhibit T cell activation, especially PD-1, and antagonist antibodies targeting inhibitory immune checkpoints can restore T cell function.
  • LAG-3 transmits inhibitory signals through the KIEELE motif in the cytoplasmic tail. Therefore, the development of monoclonal antibodies only against LAG-3 is not very effective.
  • FGL-1 Fibrinogen-like protein 1, fibrinogen-like protein 1, also known as HPS
  • HPS fibrinogen-like protein 1
  • FGL1 is an inhibitory functional ligand of LAG-3, which is independent of MHC-II
  • FGL1-LAG-3 signaling pathway is independent of the PD1 pathway.
  • FGL1 inhibits antigen-specific T cell activation, and elimination of FGL1 in mice promotes T cell immunity.
  • Blockade of FGL1-LAG-3 interaction by monoclonal antibody stimulates tumor immunity and treats established mouse tumors in a receptor-ligand interdependent manner.
  • FGL1 is a highly expressed product of human cancer cells, and the increase of FGL1 in the plasma of tumor patients is associated with poor prognosis and resistance to anti-PD1/PDL1 therapy.
  • the present invention takes FGL1 as the target of immunotherapy, and develops a new anti-FGL1 antibody.
  • an anti-FGL1 antibody with good performance which can specifically recognize/bind FGL1, block the binding of FGL1 to MHC II or LAG-3, and can in vitro / Enhance immune cell activity in vivo and stimulate immune response.
  • the anti-FGL1 antibody has the potential for preventing and/or treating tumors, infections or autoimmune diseases.
  • the first object of the present invention is to provide an anti-FGL1 antibody or an antigen-binding fragment thereof and its application.
  • the second object of the present invention is to provide a murine antibody, nanobody, chimeric antibody, fully human antibody or humanized antibody of the above-mentioned anti-FGL1 antibody or its antigen-binding fragment.
  • the third object of the present invention is to provide a monospecific, bispecific, multispecific antibody, antibody conjugate or cell therapy of the above-mentioned anti-FGL1 antibody or antigen-binding fragment thereof.
  • the fourth object of the present invention is to provide a gene encoding the above-mentioned anti-FGL1 antibody or antigen-binding fragment thereof.
  • the fifth object of the present invention is to provide a method for preparing the above-mentioned anti-FGL1 antibody or antigen-binding fragment thereof.
  • the sixth object of the present invention is to provide a pharmaceutical composition.
  • An anti-FGL1 antibody or an antigen-binding fragment thereof the whole or part of the binding region of the antibody or the antigen-binding fragment thereof is contained in the amino acid sequence described in SEQ ID NO: 163.
  • the antibody or antigen binding fragment 1nM smaller K D or binding FGL1.
  • the antibody or antigen binding fragment 1pM smaller K D or binding FGL1.
  • the antibody is a murine antibody, nanobody, chimeric antibody, fully human antibody or humanized antibody.
  • the antibody is a monospecific, bispecific, multispecific antibody, antibody conjugate or cell therapy.
  • the antibody conjugate is a cytotoxic moiety, a radioisotope, a drug or a cytokine.
  • the bispecific antibody includes any of the above-mentioned antibodies or antigen-binding fragments thereof, as well as antibodies or antigen-binding fragments directed against other antigens and/or other antigenic epitopes.
  • the bispecific antibody is an anti-FGL1/CD3 bispecific antibody, an anti-FGL1/PD-1 bispecific antibody, an anti-FGL1/PD-L1 bispecific antibody or an anti-FGL1/CD16A bispecific antibody .
  • the antibody is a monoclonal antibody.
  • an anti-FGL1 antibody or an antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising the heavy chain complementarity determining regions CDR1, CDR2 and CDR3, the light chain variable region Comprising light chain complementarity determining regions CDR1, CDR2 and CDR3, wherein,
  • CDR1 of the heavy chain variable region selected from any amino acid sequence of SEQ ID NO: 37-44, or having at least 50%, 60%, 70% with any amino acid sequence of SEQ ID NO: 37-44 , 80%, 85%, 90%, 95% or more identical sequences, or have one or more (preferably 2 or 3) conserved amino acids compared to any of the amino acid sequences of SEQ ID NOs: 37-44 A mutated (preferably substitution, insertion or deletion) amino acid sequence;
  • CDR2 of the heavy chain variable region selected from any amino acid sequence of SEQ ID NO: 47-54, or having at least 50%, 60%, 70% with any amino acid sequence of SEQ ID NO: 47-54 , 80%, 85%, 90%, 95% or more identical sequences, or have one or more (preferably 2 or 3) conserved amino acids compared to any of the amino acid sequences of SEQ ID NOs: 47-54 A mutated (preferably substitution, insertion or deletion) amino acid sequence;
  • CDR3 of the heavy chain variable region selected from any amino acid sequence of SEQ ID NOs: 57-64, or having at least 50%, 60%, 70% with any amino acid sequence of SEQ ID NOs: 57-64 , 80%, 85%, 90%, 95% or more identical sequences, or have one or more (preferably 2 or 3) conserved amino acids compared to any of the amino acid sequences of SEQ ID NOs: 57-64 A mutated (preferably substitution, insertion or deletion) amino acid sequence;
  • CDR1 of the light chain variable region selected from any amino acid sequence of SEQ ID NO: 67-74, or having at least 50%, 60%, 70% of the amino acid sequence of any of SEQ ID NO: 67-74 , 80%, 85%, 90%, 95% or more identical sequences, or have one or more (preferably 2 or 3) conserved amino acids compared to any of the amino acid sequences of SEQ ID NOs: 67-74 A mutated (preferably substitution, insertion or deletion) amino acid sequence;
  • CDR2 of the light chain variable region selected from any amino acid sequence of SEQ ID NO: 75-82, or having at least 50%, 60%, 70% with any amino acid sequence of SEQ ID NO: 75-82 , 80%, 85%, 90%, 95% or more identical sequences, or have one or more (preferably 2 or 3) conserved amino acids compared to any of the amino acid sequences of SEQ ID NOs: 75-82 A mutated (preferably substitution, insertion or deletion) amino acid sequence;
  • CDR3 of the light chain variable region selected from any amino acid sequence of SEQ ID NO: 83-90, or having at least 50%, 60%, 70% with any amino acid sequence of SEQ ID NO: 83-90 , 80%, 85%, 90%, 95% or more identical sequences, or have one or more (preferably 2 or 3) conserved amino acids compared to any of the amino acid sequences of SEQ ID NOs: 83-90 A mutated (preferably substitution, insertion or deletion) amino acid sequence.
  • the heavy chain variable region also includes a framework region FR, and the framework region FR includes:
  • (a) is selected from any amino acid sequence of SEQ ID NO:91-98,
  • (b) is selected from any amino acid sequence of SEQ ID NO: 101-108,
  • (c) is selected from any amino acid sequence of SEQ ID NO: 111-118,
  • (d) is selected from any amino acid sequence of SEQ ID NO: 121-128,
  • the light chain variable region also includes a framework region FR, and the framework region FR includes:
  • (e) is selected from any amino acid sequence of SEQ ID NO: 131-138,
  • (f) is selected from any amino acid sequence of SEQ ID NO: 139-146,
  • (g) is selected from any amino acid sequence of SEQ ID NO: 147-154,
  • (h) is selected from any amino acid sequence of SEQ ID NO: 155-162,
  • CDR1, CDR2 and CDR3 of the heavy chain variable region are respectively composed of amino acid sequences SEQ ID NO:37, SEQ ID NO:47 and SEQ ID NO:57, and CDR1, CDR2 and CDR3 of the light chain variable region CDR2 and CDR3 consist of SEQ ID NO: 67, SEQ ID NO: 75 and SEQ ID NO: 83, respectively;
  • the heavy chain variable region includes framework region FR and CDR1, CDR2 and CDR3 of the above-mentioned heavy chain variable region; more preferably, the framework region FR includes: FR1 shown in SEQ ID NO: 91 , FR2 shown in SEQ ID NO: 101, FR3 shown in SEQ ID NO: 111, and FR4 shown in SEQ ID NO: 121.
  • the light chain variable region includes framework region FR and CDR1, CDR2 and CDR3 of the above-mentioned light chain variable region; more preferably, the framework region FR includes: FR1 shown in SEQ ID NO: 131 , FR2 shown in SEQ ID NO: 139, FR3 shown in SEQ ID NO: 147, and FR4 shown in SEQ ID NO: 155.
  • CDR1, CDR2 and CDR3 of the heavy chain variable region are respectively composed of amino acid sequences SEQ ID NO:38, SEQ ID NO:48 and SEQ ID NO:58, and CDR1, CDR2 and CDR3 of the light chain variable region CDR2 and CDR3 consist of SEQ ID NO: 68, SEQ ID NO: 76 and SEQ ID NO: 84, respectively;
  • the heavy chain variable region includes framework region FR and CDR1, CDR2 and CDR3 of the above-mentioned heavy chain variable region; more preferably, the framework region FR includes: FR1 shown in SEQ ID NO: 92 , FR2 shown in SEQ ID NO: 102, FR3 shown in SEQ ID NO: 112, and FR4 shown in SEQ ID NO: 122.
  • the light chain variable region includes framework region FR and CDR1, CDR2 and CDR3 of the above-mentioned light chain variable region; more preferably, the framework region FR includes: FR1 shown in SEQ ID NO: 132 , FR2 shown in SEQ ID NO: 140, FR3 shown in SEQ ID NO: 148, and FR4 shown in SEQ ID NO: 156.
  • the CDR1, CDR2 and CDR3 of the heavy chain variable region are respectively composed of amino acid sequences SEQ ID NO:39, SEQ ID NO:49 and SEQ ID NO:59, and the CDR1, CDR2 and CDR3 of the light chain variable region CDR2 and CDR3 consist of SEQ ID NO: 69, SEQ ID NO: 77 and SEQ ID NO: 85, respectively;
  • the heavy chain variable region includes framework region FR and CDR1, CDR2 and CDR3 of the heavy chain variable region; more preferably, the framework region FR includes: FR1 shown in SEQ ID NO: 93 , FR2 shown in SEQ ID NO: 103, FR3 shown in SEQ ID NO: 113, and FR4 shown in SEQ ID NO: 123.
  • the light chain variable region includes framework region FR and CDR1, CDR2 and CDR3 of the above-mentioned light chain variable region; more preferably, the framework region FR includes: FR1 shown in SEQ ID NO: 133 , FR2 shown in SEQ ID NO: 141, FR3 shown in SEQ ID NO: 149, and FR4 shown in SEQ ID NO: 157.
  • CDR1, CDR2 and CDR3 of the heavy chain variable region are respectively composed of amino acid sequences SEQ ID NO: 40, SEQ ID NO: 50 and SEQ ID NO: 60, and CDR1, CDR2 and CDR3 of the light chain variable region CDR2 and CDR3 consist of SEQ ID NO: 70, SEQ ID NO: 78 and SEQ ID NO: 86, respectively;
  • the light chain variable region includes framework region FR and CDR1, CDR2 and CDR3 of the above-mentioned light chain variable region; more preferably, the framework region FR includes: FR1 shown in SEQ ID NO: 134 , FR2 shown in SEQ ID NO: 142, FR3 shown in SEQ ID NO: 150, and FR4 shown in SEQ ID NO: 158.
  • CDR1, CDR2 and CDR3 of the heavy chain variable region are respectively composed of amino acid sequences SEQ ID NO:41, SEQ ID NO:51 and SEQ ID NO:61, and CDR1, CDR2 and CDR3 of the light chain variable region CDR2 and CDR3 consist of SEQ ID NO: 71, SEQ ID NO: 79 and SEQ ID NO: 87, respectively;
  • the heavy chain variable region includes framework region FR and CDR1, CDR2 and CDR3 of the above-mentioned heavy chain variable region; more preferably, the framework region FR includes: FR1 shown in SEQ ID NO: 95 , FR2 shown in SEQ ID NO: 105, FR3 shown in SEQ ID NO: 115, and FR4 shown in SEQ ID NO: 125.
  • the light chain variable region includes framework region FR and CDR1, CDR2 and CDR3 of the above-mentioned light chain variable region; more preferably, the framework region FR includes: FR1 shown in SEQ ID NO: 135 , FR2 shown in SEQ ID NO: 143, FR3 shown in SEQ ID NO: 151, and FR4 shown in SEQ ID NO: 159.
  • the CDR1, CDR2 and CDR3 of the heavy chain variable region are respectively composed of amino acid sequences SEQ ID NO: 42, SEQ ID NO: 52 and SEQ ID NO: 62, and the CDR1, CDR2 and CDR3 of the light chain variable region CDR2 and CDR3 consist of SEQ ID NO: 72, SEQ ID NO: 80 and SEQ ID NO: 88, respectively;
  • the heavy chain variable region includes framework region FR and CDR1, CDR2 and CDR3 of the above-mentioned heavy chain variable region; more preferably, the framework region FR includes: FR1 shown in SEQ ID NO: 96 , FR2 shown in SEQ ID NO: 106, FR3 shown in SEQ ID NO: 116, and FR4 shown in SEQ ID NO: 126.
  • the light chain variable region includes framework region FR and CDR1, CDR2 and CDR3 of the above-mentioned light chain variable region; more preferably, the framework region FR includes: FR1 shown in SEQ ID NO: 136 , FR2 shown in SEQ ID NO: 144, FR3 shown in SEQ ID NO: 152, and FR4 shown in SEQ ID NO: 160.
  • the CDR1, CDR2 and CDR3 of the heavy chain variable region are respectively composed of amino acid sequences SEQ ID NO:43, SEQ ID NO:53 and SEQ ID NO:63, and the CDR1, CDR2 and CDR3 of the light chain variable region CDR2 and CDR3 consist of SEQ ID NO: 73, SEQ ID NO: 81 and SEQ ID NO: 89, respectively;
  • the heavy chain variable region includes framework region FR and CDR1, CDR2 and CDR3 of the above-mentioned heavy chain variable region; more preferably, the framework region FR includes: FR1 shown in SEQ ID NO: 97 , FR2 shown in SEQ ID NO: 107, FR3 shown in SEQ ID NO: 117, and FR4 shown in SEQ ID NO: 127.
  • the light chain variable region includes framework region FR and CDR1, CDR2 and CDR3 of the above-mentioned light chain variable region; more preferably, the framework region FR includes: FR1 shown in SEQ ID NO: 137 , FR2 shown in SEQ ID NO: 145, FR3 shown in SEQ ID NO: 153, and FR4 shown in SEQ ID NO: 161.
  • the CDR1, CDR2 and CDR3 of the heavy chain variable region are respectively composed of amino acid sequences SEQ ID NO:44, SEQ ID NO:54 and SEQ ID NO:64, and the CDR1, CDR2 and CDR3 of the light chain variable region CDR2 and CDR3 consist of SEQ ID NO:74, SEQ ID NO:82 and SEQ ID NO:90, respectively.
  • the heavy chain variable region includes framework region FR and CDR1, CDR2 and CDR3 of the above-mentioned heavy chain variable region; more preferably, the framework region FR includes: FR1 shown in SEQ ID NO: 98 , FR2 shown in SEQ ID NO: 108, FR3 shown in SEQ ID NO: 118, and FR4 shown in SEQ ID NO: 128.
  • the light chain variable region includes framework region FR and CDR1, CDR2 and CDR3 of the above-mentioned light chain variable region; more preferably, the framework region FR includes: FR1 shown in SEQ ID NO: 138 , FR2 shown in SEQ ID NO: 146, FR3 shown in SEQ ID NO: 154, and FR4 shown in SEQ ID NO: 162.
  • An anti-FGL1 antibody or an antigen-binding fragment thereof comprising a heavy chain variable region comprising the heavy chain complementarity determining regions CDR1, CDR2 and CDR3, wherein,
  • CDR1 of heavy chain variable region selected from any amino acid sequence of SEQ ID NO: 45-46, or having at least 50%, 60%, 70% with any amino acid sequence of SEQ ID NO: 45-46 , 80%, 85%, 90%, 95% or more identical sequences, or have one or more (preferably 2 or 3) conserved amino acids compared to any of the amino acid sequences of SEQ ID NOs: 45-46 A mutated (preferably substitution, insertion or deletion) amino acid sequence;
  • CDR2 of the heavy chain variable region selected from any amino acid sequence of SEQ ID NOs: 55-56, or having at least 50%, 60%, 70% with any amino acid sequence of SEQ ID NOs: 55-56 , 80%, 85%, 90%, 95% or more identical sequences, or have one or more (preferably 2 or 3) conserved amino acids compared to any of the amino acid sequences of SEQ ID NOs: 55-56 A mutated (preferably substitution, insertion or deletion) amino acid sequence;
  • CDR3 of the heavy chain variable region selected from the amino acid sequence of any of SEQ ID NOs: 65-66, or having at least 50%, 60%, 70% of the amino acid sequence of any of SEQ ID NOs: 65-66 , 80%, 85%, 90%, 95% or more identical sequences, or have one or more (preferably 2 or 3) conserved amino acids compared to any of the amino acid sequences of SEQ ID NOs: 65-66 a mutated (preferably substitution, insertion or deletion) amino acid sequence;
  • the heavy chain variable region also includes a framework region FR, and the framework region FR includes:
  • (b) is selected from any amino acid sequence of SEQ ID NO: 109-110,
  • (c) is selected from any amino acid sequence of SEQ ID NO: 119-120,
  • (d) is selected from any amino acid sequence of SEQ ID NO: 129-130,
  • CDR1, CDR2 and CDR3 of the heavy chain variable region are respectively composed of amino acid sequences SEQ ID NO: 45, SEQ ID NO: 55 and SEQ ID NO: 65.
  • the heavy chain variable region includes framework region FR and CDR1, CDR2 and CDR3 of the above-mentioned heavy chain variable region; more preferably, the framework region FR includes: FR1 shown in SEQ ID NO: 99 , FR2 shown in SEQ ID NO: 109, FR3 shown in SEQ ID NO: 119, and FR4 shown in SEQ ID NO: 129.
  • CDR1, CDR2 and CDR3 of the heavy chain variable region are respectively composed of amino acid sequences SEQ ID NO: 46, SEQ ID NO: 56 and SEQ ID NO: 66.
  • the heavy chain variable region includes the framework region FR and the CDR1, CDR2 and CDR3 of the above-mentioned heavy chain variable region; more preferably, the framework region FR includes: FR1 shown in SEQ ID NO: 100 , FR2 shown in SEQ ID NO: 110, FR3 shown in SEQ ID NO: 120, and FR4 shown in SEQ ID NO: 130.
  • An anti-FGL1 antibody or an antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region; it is characterized in that, wherein:
  • the heavy chain variable region has any of the amino acid sequences given in SEQ ID Nos: 1-8,
  • the light chain variable region has any of the amino acid sequences given in SEQ ID Nos: 21-28;
  • the heavy chain variable region and the light chain variable region are selected from any one of the following amino acid sequences (1)-(8):
  • An anti-FGL1 antibody or antigen-binding fragment thereof comprising a heavy chain variable region; wherein:
  • the heavy chain variable region has any of the amino acid sequences given in SEQ ID NOs: 9-10,
  • the heavy chain variable region is selected from the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10.
  • the present invention discloses a fusion protein comprising the antibody or antigen-binding fragment thereof described in any one of the above.
  • the present invention discloses a gene encoding any of the above-mentioned anti-FGL1 antibodies.
  • the gene is selected from any one of the following (1)-(10):
  • the present invention discloses recombinant vectors, expression cassettes, transgenic cell lines or recombinant bacteria of the above-mentioned genes.
  • the invention discloses the application of the above-mentioned recombinant vector, expression cassette, transgenic cell line or recombinant bacteria in preparing anti-FGL1 antibody.
  • the present invention discloses the application of any of the above-mentioned antibodies or antigen-binding fragments thereof in the following (a) and/or (b) and/or (c):
  • the drug may be administered simultaneously, separately or sequentially with one or more drugs.
  • the present invention discloses a pharmaceutical composition
  • a pharmaceutical composition comprising the above-mentioned antibody or its antigen-binding fragment and a pharmaceutically acceptable carrier.
  • the present invention discloses the application of the above-mentioned antibody or its antigen-binding fragment in preparing a medicine for treating cancer.
  • the present invention discloses the application of the above-mentioned antibody or its antigen-binding fragment in the preparation of medicine for treating cancer, the cancer is lung cancer, liver cancer, melanoma, malignant glioma, head and neck cancer, colorectal cancer, gastric cancer, prostate cancer, ovarian cancer cancer, bladder cancer, pancreatic cancer, stomach cancer, colon cancer, cervical cancer or related tumors.
  • FR antibody framework region, i.e. amino acid residues other than CDR residues in the variable region of an antibody
  • VH antibody heavy chain variable region
  • VL antibody light chain variable region
  • IgG Immunoglobulin G
  • FACS Fluorescence Activated Cell Sorting
  • IL-2 Interleukin 2
  • antibody may refer to a natural immunoglobulin or an immunoglobulin prepared by partial or complete synthesis.
  • Antibodies can be obtained from natural resources such as plasma or serum in which the antibodies naturally occur, or from culture supernatants of hybridoma cells that produce antibodies, from animal immune serum, from phage libraries, from phage-displayed mouse immune antibody libraries, and from phage-displayed alpaca immunizations.
  • the library, human natural phage display antibody library, etc. were screened for reconstruction and isolation. Alternatively, it may be partially or completely synthesized by techniques using genetic recombination or the like.
  • Preferred antibodies include, for example, antibodies of immunoglobulin isotypes or subclasses of these isotypes.
  • Human immunoglobulins are known to include 9 classes (isotypes) of IgG1, IgG2, IgG3, IgG4, IgAl, IgA2, IgD, IgE, and IgM.
  • the antibodies of the invention may include IgGl, IgG2, IgG3, IgG4.
  • an "antibody” as used in this specification is an immunoglobulin molecule composed of two pairs of polypeptide chains, each pair having one light chain (LC) and one heavy chain (HC).
  • Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH).
  • the heavy chain constant region consists of 3 domains (CH1, CH2 and CH3).
  • Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL), or only the light chain constant region (CL).
  • the light chain constant region consists of one domain, CL.
  • Constant domains are not directly involved in the binding of antibodies to antigens, but exhibit a variety of effector functions, such as mediating immunoglobulins with host tissues or factors, including various cells of the immune system (eg, effector cells) and classical complement Binding of the first component (C1q) of the system.
  • the VH and VL regions can also be subdivided into regions of high variability called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • Each VH and VL consists of 3 CDRs and 4 FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from amino terminus to carboxy terminus.
  • the term “antibody” herein refers to single domain antibodies comprising only heavy chains, including full-length antibodies, individual chains, and all parts, domains or fragments thereof (including but not limited to antigen binding domains or fragments, such as VHH domains or VH/VL domains, respectively).
  • sequence eg, in terms of "immunoglobulin sequence", “antibody sequence”, “single variable domain sequence”, “VHH sequence” or “protein sequence”, etc.
  • sequences should generally be understood to include both Relevant amino acid sequences, in turn, include nucleic acid sequences or nucleotide sequences encoding said sequences, unless a more limited interpretation is required herein.
  • murine antibody in the present invention is a monoclonal antibody prepared according to knowledge and skill in the art. In preparation, the test subject is injected with the antigen, and hybridomas expressing antibodies with the desired sequence or functional properties are isolated.
  • chimeric immunoglobulin refers to an immunoglobulin or antibody whose variable regions are derived from a first species and whose constant regions are derived from a second species. Chimeric immunoglobulins or antibodies can be constructed, eg, by genetic engineering, from immunoglobulin gene segments belonging to different species.
  • the term "screening of human natural phage-displayed antibody library” refers to the selection of human natural phage-displayed antibody library by using the target protein or overexpressing cell line as an antigen. According to the results of the audition, a sufficient number of monoclones were selected for primary screening, positive clones were selected for sequencing, and sequence diversity analysis was performed to select sequence-specific antibody clones, and then prepare antibody samples.
  • “human natural phage display antibody library” and “human natural library” are used interchangeably.
  • the term “Phage Displaying Alpaca Immune Library Screening” refers to using the target protein or overexpressing cell line as an antigen to immunize alpaca, preferably alpaca PBMC cells with high immune titer to construct an immune library.
  • the alpaca nanobody phage-displayed antibody library was selected, and a sufficient number of monoclones were selected for primary screening, positive clones were selected for sequencing, and sequence diversity analysis was performed to select the sequence Specific antibody cloning followed by antibody sample preparation.
  • phage display alpaca immune library and “alpaca phage library” are used interchangeably.
  • antigen-binding fragment of an antibody refers to a polypeptide fragment of an antibody, such as a polypeptide fragment of a full-length antibody, that retains the ability to specifically bind to the same antigen to which the full-length antibody binds, and/or compete with the full-length antibody for binding to the antigen.
  • specific binding which is also referred to as an "antigen binding moiety”.
  • Antigen-binding fragments of antibodies can be generated by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies.
  • Non-limiting examples of antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, dAb and complementarity determining region (CDR) fragments, single chain antibodies (eg, scFv), chimeric antibodies, diabodies (diabody), linear antibody (linear antibody), nanobody (eg technology from Ablynx), domain antibody (eg technology from Domantis), and polypeptides comprising at least a portion of the antibody sufficient to confer specific antigen-binding ability to the polypeptide .
  • CDR complementarity determining region
  • vector refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked.
  • the term includes vectors that are self-replicating nucleic acid structures and vectors that are incorporated into the genome of the host cell into which they are 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”.
  • pharmaceutically acceptable carrier includes any standard pharmaceutical carrier, such as phosphate buffered saline solutions, water and emulsions, such as oil/water or water/oil emulsions, and wetting agents of various types.
  • polypeptide refers to an amino acid chain of any length, regardless of modification (eg, phosphorylation or glycosylation).
  • polypeptide includes proteins and fragments thereof.
  • Polypeptides may be "foreign”, meaning that they are “heterologous”, ie foreign to the host cell being utilized, eg, human polypeptides produced by bacterial cells.
  • Polypeptides are disclosed herein as sequences of amino acid residues. Those sequences are written left to right in amino-terminal to carboxy-terminal direction.
  • amino acid residue sequences are named with three-letter or one-letter codes as follows: alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), Partic acid (Asp, D), cysteine (Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), Isoleucine (Ile, I), Leucine (Leu, L), Lysine (Lys, K), Methionine (Met, M), Phenylalanine (Phe, F) , proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y) and valine (Val, V).
  • the numbering of amino acid positions in the antibodies described herein eg, amino acid residues of an Fc region
  • regions of interest eg, amino acid
  • Percent (%) amino acid sequence identity with respect to a reference polypeptide sequence is defined as the amino acid residues in a candidate sequence that are identical to those in the reference polypeptide sequence after aligning the sequences and introducing gaps where necessary to obtain maximum percent sequence identity percentage of residues. Alignment for purposes of determining percent amino acid sequence identity can be performed in a variety of ways that are within the skill in the art, for example using publicly available computer software such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software or the FASTA program Bag.
  • domain comprising an antibody variable region having FGL1 binding activity refers to an antibody portion comprising a region that specifically binds to and is also complementary to all or part of the above-mentioned FGL1 protein or a partial peptide of the FGL1 protein.
  • Domains comprising antibody variable regions can be provided from the variable domains of one or more antibodies.
  • the antibody variable region-containing domains comprise antibody light and heavy chain variable regions (VL and VH).
  • Suitable examples of such domains comprising antibody variable regions include "single chain Fv (scFv)", “single chain antibody”, “Fv”, “single chain Fv 2 (scFv2)", “Fab”, "Fv” (ab)2", “VHH", etc.
  • Specific means that one of the molecules involved in specific binding does not exhibit any significant binding to molecules other than one or more of the binding partner molecules.
  • the term is also used when the antibody variable region-containing domain is specific for a particular epitope of multiple epitopes in an antigen.
  • an antigen-binding molecule comprising the antibody variable region-containing domain can bind to various antigens having the epitope.
  • Epitope means an antigenic determinant in an antigen, and refers to an antigenic site to which a domain of an antigen-binding molecule comprising an antibody variable region disclosed in the present specification binds.
  • epitopes can be defined in terms of their structure.
  • the epitope can also be defined based on the antigen-binding activity in the antigen-binding molecule that recognizes the epitope.
  • the antigen is a peptide or polypeptide
  • the epitope can be specified by the amino acid residues that form the epitope.
  • the epitope is a sugar chain
  • the epitope can be identified by its specific sugar chain structure.
  • a linear epitope present in a FGL1 molecule by a test antigen-binding molecule comprising a domain comprising an antibody variable region having FGL1 binding activity can be confirmed as follows.
  • a linear peptide comprising the amino acid sequence constituting the extracellular domain of FGL1 was synthesized.
  • the peptides may be chemically synthesized.
  • it can be obtained by genetic engineering methods using a region in the cDNA encoding FGL1 corresponding to the amino acid sequence of the extracellular domain.
  • the binding activity between the linear peptide comprising the amino acid sequence constituting the extracellular domain and the test antigen-binding molecule comprising the domain comprising the variable region of the antibody having FGL1 binding activity was evaluated.
  • ELISA using immobilized linear peptides as antigens can assess the binding activity of antigen-binding molecules to peptides.
  • the binding activity to linear peptides can be elucidated based on the level of inhibition caused by the linear peptides in the binding of antigen-binding molecules to cells expressing FGL1. These tests can elucidate the binding activity of antigen-binding molecules to linear peptides.
  • test antigen-binding molecule comprising the domain of the antibody variable region having FGL1-binding activity
  • cells expressing FGL1 were prepared for the above purposes.
  • a test antigen-binding molecule comprising a domain of an antibody variable region having FGL1-binding activity contacts a cell expressing FGL1, it binds tightly to the cell, but on the other hand, there are cases in which the antigen-binding molecule
  • the immobilized linear peptide comprising the amino acid sequence constituting the extracellular domain of FGL1 is substantially not bound.
  • substantially no binding means that the binding activity is 80% or less, usually 50% or less, preferably 30% or less, and particularly preferably 15%, relative to the binding activity to cells expressing human FGL1 %the following.
  • BR can be evaluated based on the principles of ELISA or fluorescence-activated cell sorting (FACS) using FGL1-expressing cells as antigens.
  • the antibody variable region-containing domains in the antigen-binding molecules of the present invention can bind to the same epitope.
  • the same epitope may be present in a protein comprising the amino acid sequence of SEQ ID NO: 163.
  • the antibody variable region-containing domains in the antigen-binding molecules of the present invention may bind to different epitopes, respectively.
  • different epitopes may be present in the protein comprising the amino acid sequence of SEQ ID NO: 163.
  • the present application discloses an anti-FGL1 antibody or an antigen-binding fragment thereof, the antibody binds to an epitope, and the epitope is wholly or partially contained in the amino acid sequence described in SEQ ID NO: 163.
  • Monoclonal antibody-producing hybridomas can be produced using known techniques, for example, as follows. Specifically, mammals were immunized according to conventional immunization methods using FGL1 protein as a sensitizing antigen. The obtained immune cells are fused with known parental cells by a conventional cell fusion method. Then, by screening for monoclonal antibody-producing cells using conventional screening methods, hybridomas producing anti-FGL1 antibodies can be selected.
  • FGL1 protein was purchased from a conventional biochemical reagent store.
  • the FGL1 protein is used as a sensitizing antigen for immunization in mammals.
  • Partial peptides of FGL1 can also be used as sensitizing antigens.
  • the partial peptide can also be obtained from the human FGL1 amino acid sequence by chemical synthesis.
  • it can also be obtained by integrating a part of the FGL1 gene into an expression vector and expressing it.
  • it can also be obtained by degrading FGL1 protein using a protease, but the region and size of the FGL1 peptide used as a partial peptide are not particularly limited to specific embodiments.
  • the number of amino acids constituting the peptide used as the sensitizing antigen is at least five or more, or preferably, for example, six or more, or seven or more. More specifically, a peptide consisting of 8 to 50 residues or preferably 10 to 30 residues can be used as the sensitizing antigen.
  • the mammal to be immunized with the sensitizing antigen is not limited to a specific animal. However, the selection is preferably made in consideration of suitability with the parent cell for cell fusion. Generally rodents such as mice, rats and hamsters, alpacas, rabbits and monkeys are preferably used.
  • the above-mentioned animals are immunized with a sensitizing antigen according to a known method.
  • immunization is carried out by intraperitoneal, plantar or subcutaneous injection of a sensitizing antigen into mammals.
  • the sensitizing antigen is appropriately diluted with PBS (phosphate buffered saline), physiological saline, or the like.
  • a conventional adjuvant such as Freund's complete adjuvant is mixed with the antigen and the mixture is emulsified.
  • the sensitizing antigen is then administered to the mammal multiple times at intervals of 4 to 21 days.
  • Appropriate carriers can be used in the immunization of sensitizing antigens, especially in the case of using partial peptides with smaller molecular weights as sensitizing antigens, which are combined with albumin, keyhole limpet hemocyanin (KIH), etc.
  • the sensitizing antigenic peptide in which the carrier protein is bound is sometimes desirable for immunization.
  • hybridomas producing desired antibodies can also be produced using DNA immunization as follows.
  • DNA immunization refers to the expression of a sensitizing antigen in vivo of the immunized animal to which the immunized animal has been administered with a vector DNA constructed so as to enable expression of a gene encoding an antigenic protein to confer Immune methods of immune stimulation.
  • DNA immunization is expected to have the following advantages: maintaining the structure of proteins such as FGL1 to impart immune stimulation; and requiring no purification of immunizing antigens.
  • DNA expressing FGL1 protein is administered to the immunized animal.
  • DNA encoding FGL1 can be synthesized by a known method such as PCR.
  • the resulting FGL1 was inserted into an appropriate expression vector and administered to immunized animals.
  • the expression vector a commercially available expression vector such as PCDNA3.1 can be suitably used.
  • a method of administering the carrier into a living body a commonly used method can be used.
  • DNA immunization is carried out by introducing a gold particle to which an expression vector is adsorbed into the cells of an immunized animal individual with a gene gun.
  • the production of an antibody that recognizes FGL1 can also be produced by the method described in International Publication WO 2003/104453.
  • splenocytes can be used in particular.
  • Mammalian myeloma cells can be used as the cells to be fused with the above-mentioned immune cells.
  • Myeloma cells are preferably provided with appropriate selectable markers for screening.
  • a selectable marker refers to a property that enables (or does not) survive under specific culture conditions.
  • Well-known selectable markers include hypoxanthine-guanine-phosphoribosyltransferase deficiency (hereinafter abbreviated as HGPRT deficiency) or thymidine kinase deficiency (hereinafter abbreviated as TK deficiency).
  • HAT sensitivity hypoxanthine-aminopterin-thymidine sensitivity
  • Cells lacking HGPRT or lacking TK can be selected in a medium containing 6-thioguanine, 8-azaguanine (hereinafter abbreviated as 8AG), or 5'-bromodeoxyuridine, respectively. Normal cells that incorporate these pyrimidine analogs into their DNA will die. At the same time, cells lacking these enzymes that do not take up these pyrimidine analogs can survive in selective culture.
  • a selectable marker called G418 resistance confers resistance to 2-deoxystreptamine antibiotics (a gentamicin analog) through a neomycin resistance gene.
  • Various myeloma cells suitable for cell fusion are known.
  • myeloma cells including the following cells can be preferably used: P3 (P3x63Ag8.653) (J. Immunol. (1979) 123(4), 1548-1550);
  • Cell fusion of the above-mentioned immune cells and myeloma cells is basically carried out according to known methods, for example, the method of Kohler and Milstein et al. (Methods Enzymol. (1981) 73, 3-46).
  • the above-mentioned cell fusion can be carried out in a conventional nutrient medium in the presence of a cell fusion promoter.
  • Fusion promoters include, for example, polyethylene glycol (PEG) and Sendai virus (HVJ).
  • Adjuvants such as dimethyl sulfoxide can be added as needed to further improve the fusion efficiency.
  • the use ratio of immune cells to myeloma cells can be arbitrarily set. For example, it is preferable to use 1 to 10 times more immune cells than myeloma cells.
  • the culture medium for the above-mentioned cell fusion for example, RPMI1640 medium suitable for the growth of the above-mentioned myeloma cell line, MEM medium, and conventional culture medium used for cell culture of this species can be used.
  • serum supplements such as fetal calf serum (FCS) can be suitably added.
  • predetermined amounts of the above-mentioned immune cells and myeloma cells can be sufficiently mixed in the above-mentioned culture medium.
  • a PEG solution for example, an average molecular weight of about 1,000 to 6,000 in average molecular weight
  • the desired fused cells can be formed by slowly mixing the mixture.
  • the appropriate medium exemplified above was gradually added to the cells, and centrifugation was repeated to remove the supernatant.
  • Cell fusion agents, etc. which are not conducive to the growth of hybridomas, can be removed.
  • the hybridoma thus obtained can be selected by culturing with a conventional selection medium, for example, a HAT medium (a medium containing hypoxanthine, aminopterin, and thymidine).
  • a HAT medium a medium containing hypoxanthine, aminopterin, and thymidine.
  • the culture using the above-mentioned HAT medium is continued for a sufficient time for the cells other than the desired hybridomas (non-fused cells) to die. Typically, the sufficient time is from several days to several weeks.
  • screening and single cloning of hybridomas producing the desired antibody can be performed using conventional limiting dilution methods.
  • the hybridoma thus obtained can be selected by using a selection medium based on the selectable marker possessed by the myeloma used for cell fusion.
  • a selection medium based on the selectable marker possessed by the myeloma used for cell fusion.
  • cells lacking HGPRT or TK can be selected by culturing with HAT medium (a medium containing hypoxanthine, aminopterin, and thymidine). That is, when HAT-sensitive myeloma cells are used for cell fusion, cells successfully fused with normal cells can selectively proliferate in the HAT medium.
  • the culture using the above-mentioned HAT medium is continued for a time sufficient to kill cells other than the desired hybridomas (non-fused cells).
  • a desired hybridoma can usually be selected by culturing for several days to several weeks. Next, screening and single cloning of hybridomas producing the desired antibody can be performed using conventional limiting dilution methods.
  • FACS fluorescence-activated cell sorting
  • hybridomas producing the monoclonal antibodies of the invention were first prepared.
  • Preferred cells for screening are mammalian cells that are forced to express FGL1.
  • untransformed mammalian cells can be used as host cells to selectively detect the binding activity of an antibody to FGL1 on the cell surface. That is, hybridomas that produce anti-FGL1 antibodies can be obtained by selecting hybridomas that produce antibodies that do not bind to host cells but that bind to cells forcibly expressing FGL1.
  • the binding activity of the antibody to immobilized FGL1-expressing cells can be assessed based on the principles of ELISA.
  • cells expressing FGL1 are immobilized in the wells of an ELISA plate.
  • the antibody bound to the immobilized cells is detected by contacting the culture supernatant of the hybridoma with the immobilized cells in the well.
  • the monoclonal antibody is of mouse origin, the antibody bound to the cells can be detected by an anti-mouse immunoglobulin antibody.
  • Hybridomas having antigen-binding ability and producing the desired antibody selected by the above screening can be cloned by a limiting dilution method or the like.
  • the monoclonal antibody-producing hybridoma thus produced can be subcultured in a conventional culture medium.
  • the hybridoma can be stored in liquid nitrogen for a long time.
  • the hybridoma is cultured according to a conventional method, and the desired monoclonal antibody can be obtained from the culture supernatant.
  • monoclonal antibodies can be obtained from the ascites fluid of the hybridoma by administering it to an adapted mammal and multiplying it.
  • the former method is suitable for obtaining high-purity antibodies.
  • Antibodies encoded by antibody genes cloned from antibody-producing cells such as hybridomas can also be suitably used. By inserting the cloned antibody gene into an appropriate vector and introducing it into a host, the antibody encoded by the gene is expressed. Methods for isolation of antibody genes, insertion of genes into vectors, and transformation of host cells have been established, for example, by Vandamme et al. (Eur. J. Biochem. (1990) 192(3), 767-775). As described below, methods for producing recombinant antibodies are also known.
  • cDNA encoding the variable region (V region) of the anti-FGL1 antibody can be prepared from hybridoma cells expressing the anti-FGL1 antibody.
  • total RNA is usually first extracted from the hybridoma.
  • a method for extracting mRNA from cells for example, the following methods can be used:
  • the guanidine ultracentrifugation method (Biochemistry (1979) 18(24), 5294-5299); and the AGPC method (Anal. Biochem. (1987) 162(1), 156-159).
  • the extracted mRNA can be purified using an mRNA purification kit (GE Healthcare Bioscience) or the like. Alternatively, kits for the direct extraction of total mRNA from cells such as the QuickPrep mRNA purification kit (GE Healthcare Bioscience) are also commercially available. mRNA can be obtained from hybridomas using such kits.
  • the cDNA encoding the antibody V region can be synthesized from the prepared mRNA using reverse transcriptase.
  • cDNA can be synthesized using AMV Reverse Transcriptase First-strand cDNA Synthesis Kit (Biochemical Industry Co., Ltd.) or the like.
  • AMV Reverse Transcriptase First-strand cDNA Synthesis Kit Biochemical Industry Co., Ltd.
  • SMART RACE cDNA Amplification Kit (Clontech) and PCR-based 5'-RACE method (Proc. Natl. Acad. Sci. USA (1988) 85(23), 8998) can be appropriately used -9002; Nucleic Acids Res. (1989) 17(8), 2919-2932).
  • appropriate restriction enzyme sites described later may be introduced into both ends of the cDNA.
  • the target cDNA fragment was purified from the obtained PCR product, followed by ligation with vector DNA.
  • a recombinant vector is produced and introduced into Escherichia coli or the like.
  • the desired recombinant vector can be prepared from the E. coli forming the colony.
  • whether or not the recombinant vector has the target cDNA nucleotide sequence can be confirmed by a known method, for example, the dideoxynucleotide chain termination method or the like.
  • cDNA was synthesized using RNA extracted from hybridoma cells as template to obtain 5'-RACE cDNA library.
  • the 5'-RACE cDNA library can be synthesized using a commercially available kit such as the SMART RACE cDNA Amplification Kit as appropriate.
  • the antibody gene was amplified by PCR method.
  • Primers for amplifying mouse antibody genes can be designed based on known antibody gene sequences.
  • the nucleotide sequences of these primers vary depending on the immunoglobulin subclass. Therefore, the subclass is preferably determined in advance using a commercially available kit such as the Iso Strip Mouse Monoclonal Antibody Isotype Kit (Roche Diagnostics).
  • the gene encoding mouse IgG can be isolated using primers capable of amplifying genes encoding Y1, Y2a, Y2b, Y3 as the heavy chain, the kappa chain and the Y chain as the light chain.
  • primers capable of amplifying genes encoding Y1, Y2a, Y2b, Y3 as the heavy chain, the kappa chain and the Y chain as the light chain In order to amplify the variable region gene of IgG, a primer annealing to a constant region site close to the variable region can usually be used as a 3'-side primer.
  • the primers attached to the 5'RACE cDNA library construction kit were used as 5' side primers.
  • immunoglobulins composed of combinations of heavy and light chains can be reconstituted. Desired antibodies can be screened using the FGL1-binding activity of the reconstituted immunoglobulin as an indicator. For example, for the purpose of isolating an antibody against FGL1, it is more preferable that the binding of the antibody to FGL1 is specific. Antibodies that bind to FGL1 can be screened, for example, by the following steps:
  • Methods for detecting antibody binding to cells expressing FGL1 are well known. Specifically, the binding of the antibody to cells expressing FGL1 can be detected by techniques such as FACS as described above. In order to evaluate the binding activity of the antibody, a fixed sample of FGL1-expressing cells can be appropriately used.
  • Screening methods for antibodies using binding activity as an index also include panning methods using phage vectors. Screening methods utilizing phage vectors are advantageous in cases where antibody genes are isolated from heavy and light chain subclass libraries of cell populations expressing polyclonal antibodies.
  • the genes encoding the variable regions of the heavy and light chains can be ligated with appropriate linker sequences to form single-chain Fvs (scFvs).
  • Phage with scFv presented on the surface can be obtained by inserting a gene encoding scFv into a phage vector.
  • the phage is contacted with the antigen of interest. By collecting phages that bind to the antigen, the DNA encoding the scFv with target-binding activity can be isolated. Repeating this process as needed, the scFv with the desired binding activity can be concentrated.
  • the cDNA is digested by restriction enzymes that recognize restriction enzyme sites inserted at both ends of the cDNA.
  • Preferred restriction enzymes recognize and cleave nucleotide sequences that occur with low frequency in the nucleotide sequence of antibody genes. And it is preferred to introduce restriction sites conferring sticky ends into the vector to insert a single copy of the digested fragment in the correct orientation.
  • the cDNA encoding the V region of the anti-FGL1 antibody was digested as described above and inserted into a suitable expression vector to construct an antibody expression vector.
  • chimeric antibody means that the origin of the constant region is different from the origin of the variable region. Therefore, human-human allochimeric antibodies are also included in the chimeric antibodies of the present invention in addition to mouse-human heterozygous chimeric antibodies.
  • a chimeric antibody expression vector can be constructed by inserting the above V region gene into an expression vector already having a constant region.
  • a recognition sequence of a restriction enzyme that excises the above-mentioned V region gene can be appropriately placed on the 5' side of an expression vector carrying DNA encoding a desired antibody constant region (C region).
  • Chimeric antibody expression vectors were constructed by in-frame fusion of two genes digested with the same combination of restriction enzymes.
  • an anti-FGL1 antibody the antibody gene is inserted into an expression vector such that the gene is expressed under the control of an expression control region.
  • Expression control regions used to express antibodies include, for example, enhancers and promoters.
  • an appropriate signal sequence can be appended to the amino terminus to allow secretion of the expressed antibody outside the cell.
  • other suitable signal sequences are attached.
  • the expressed polypeptide is cleaved at the carboxy-terminal portion of the above sequence, and the resulting polypeptide can be secreted out of the cell as a mature polypeptide.
  • recombinant cells expressing DNA encoding anti-FGL1 antibody can be obtained.
  • DNAs encoding the antibody heavy chain (H chain) and light chain (L chain) are inserted into different expression vectors, respectively.
  • Antibody molecules having H and L chains can be expressed by co-transfecting the same host cell with a vector into which H and L chains are inserted.
  • host cells can be transformed with a single expression vector into which DNAs encoding the H and L chains are inserted (refer to International Publication WO 94/11523).
  • Suitable eukaryotic cells for use as host cells include animal cells, plant cells, or fungal cells.
  • animal cells include, for example, the following cells:
  • Mammalian cells CHO, COS, myeloma, baby hamster kidney (BHK), Hela, Vero, etc.;
  • Amphibian cells Xenopus oocytes, etc.
  • Insect cells sf9, sf21, Tn5, etc.
  • Nicotiana genus such as Nicotiana tabacum
  • Callus-cultured cells can suitably be used to transform plant cells.
  • Saccharomyces such as Saccharomyces serevisiae, Pichia such as Pichia pastoris;
  • Filamentous fungi Aspergillus such as Aspergillus niger.
  • antibody gene expression systems using prokaryotic cells are also known.
  • bacterial cells such as Escherichia coli (E.coli) and Bacillus subtilis can be appropriately used.
  • An expression vector containing the antibody gene of interest is introduced into these cells by transformation.
  • the desired antibody can be obtained from the culture of transformed cells by culturing the transformed cells in vitro.
  • transgenic animals can also be used to produce recombinant antibodies. That is, the antibody can be obtained from an animal into which a gene encoding the desired antibody has been introduced.
  • an antibody gene can be constructed by in-frame insertion into a gene encoding a protein inherently produced in milk to construct a fusion gene.
  • a protein secreted into milk for example, goat B casein and the like can be used.
  • a DNA fragment containing the fusion gene into which the antibody gene is inserted is injected into a goat embryo, and the embryo is then introduced into a female goat.
  • the milk produced by the transgenic goat from the goat that received the embryo (or its progeny) can obtain the desired antibody as a fusion protein with the milk protein.
  • hormones can be administered to the transgenic goats in order to increase the amount of milk containing the desired antibodies produced by the transgenic goats (Ebert, K.M. et al., Bio/Technology (1994), 12(7), 699-702).
  • domains derived from genetic recombinant antibodies that have been artificially modified to reduce heterologous antigenicity to humans, etc. can be suitably used as containing antibody variable regions domain of an antigen-binding molecule.
  • Such genetically recombinant antibodies include, for example, humanized antibodies. These modified antibodies are suitably prepared by known methods.
  • a humanized antibody is also called a reshaped human antibody, and specifically, a humanized antibody obtained by grafting the CDRs of a non-human animal antibody, eg, a mouse antibody, to a human antibody, and the like are known.
  • Conventional genetic recombination methods for obtaining humanized antibodies are also known.
  • a method of grafting mouse antibody CDRs to human FRs for example, overlap extension PCR (overlap extension PCR) is known.
  • overlap extension PCR overlap extension PCR
  • nucleotide sequences encoding the CDRs of the mouse antibody to be transplanted are appended to primers for synthesizing FRs of human antibodies. Primers were prepared separately for the 4 FRs.
  • mouse CDRs when mouse CDRs are transplanted into human FRs, it is beneficial to select human FRs with high identity to mouse FRs to maintain the function of CDRs. That is, it is generally preferable to use a human FR comprising an amino acid sequence having a high amino acid sequence identity with the FR adjacent to the mouse CDR to be transplanted.
  • V regions of human antibodies are expressed as single-chain antibodies (scFv) on the surface of phage by phage display methods.
  • Phages can be selected that express scFvs that bind to the antigen.
  • the DNA sequence encoding the V region of a human antibody that binds an antigen can be determined by analyzing the genes of selected phages.
  • the DNA sequence of the scFv bound to the antigen was determined.
  • An expression vector can be produced by fusing the V region sequence with the desired human antibody C region sequence in frame and inserting it into an appropriate expression vector.
  • the expression vector is introduced into cells suitable for expression, such as those described above.
  • Human antibodies can be produced by expressing a gene encoding a human antibody in a cell. These methods are already known (cf. International Patent Publication Nos. WO1992/001047, WO1992/02079, WO1993/006213, WO1993/011236, WO1993/019172, WO1995/001438, WO1995/015388).
  • the anti-FGL1 antibody provided by the present invention can specifically bind to FGL1, block the combination of FGL1 and LAG-3, reverse the inhibitory effect of FGL1 on T cells, and activate T cells to secrete cytokines; the above functions are close to or surpass the current anti-FGL1 antibodies. level.
  • Figure 1 is a schematic diagram of the ELISA results of the binding of anti-FGL1 antibody to human FGL1;
  • Figure 2 is a schematic diagram of the ELISA results of the binding of anti-FGL1 antibody to mouse FGL1;
  • Figure 3 is a schematic diagram of the FACS results of anti-FGL1 antibody blocking the binding of FGL1 to human LAG3;
  • FIG. 4a and Figure 4b are schematic diagrams showing that anti-FGL1 antibody reverses the inhibitory effect of human FGL1 on T cells;
  • Figure 5 is a graph of tumor volume after administration in mice.
  • Mouse immunization Using recombinant human FGL1-his protein (Sinobio, Cat: 13484-H08B) and monkey FGL1-huFC (Acro, Cat: FG1-C5269) as immunogens, immunize mice, in order to avoid poor mouse response or If they die during the immunization process, 3 to 4 animals can be immunized at the same time. Negative serum was taken 3 days in advance, the first immunization, subcutaneous immunization, intraperitoneal immunization and plantar immunization were used to inject 60 ⁇ g of recombinant human FGL1-his protein fully emulsified with Freund's complete adjuvant at multiple points.
  • the immunization method was multi-point injection of 30 ⁇ g of recombinant human FGL1-his protein fully emulsified with Freund's complete adjuvant for secondary immunization, and 30 ⁇ g of monkey FGL1-huFc immunogen was injected in the same way as the second immunization after two weeks and two weeks later.
  • Fourth immunization After 6 days, antiserum titers were assessed by assaying sera collected from tail bleeds in recombinant human FGL1-huFc protein-coated ELISA plates at various dilutions from 1:100 to 1:1,000,000. When the titer results meet the requirements and anti-human FGL1 antibody is detected at a dilution of >1:1,000,000, the mouse spleen and lymph nodes can be harvested for cell fusion and phage library construction.
  • Alpaca immunization Recombinant human FGL1-his protein (Sinobio, Cat: 13484-H08B) and monkey FGL1-huFc (Acro, Cat: FG1-C5269) were prepared as immunogens, and the total antigen weight for each immunization was kept at 1-2 mg Between, the volume is less than 2mL, the antigen and adjuvant are emulsified 1:1 to form a homogeneous mixture and stored at 4°C. After recording the alpaca ear number, the immunization experiment was started. Each time, the mice were injected into the left and right sides near the neck lymph nodes of the alpaca.
  • Each side was injected at 2 points, and 0.4 mL of the mixed antigen was injected into each point.
  • the blood was centrifuged at 400 ⁇ g for 30 minutes in a pre-cooled 25°C centrifuge, and the upper layer was separated and stored. serum. Then, the lymphocytes were separated, that is, 3 mL of cell separation solution was firstly added to a 15 mL centrifuge tube, and then 3 mL of blood was slowly added. When adding blood, be careful and slow to prevent mixing of blood and separation solution. After that, the centrifuge was pre-cooled to room temperature. After centrifugation at 400 x g for 30 minutes, the blood separation in the centrifuge tube was observed, and the intermediate cotton-like upper immune cells were carefully drawn out with a 200 ⁇ L pipette.
  • Myeloma cells SP2/0 (ATCC) were passaged one day before fusion, so that the cells were in logarithmic growth phase during the experiment. Before fusion, the cells were collected in a centrifuge tube, centrifuged at 100 rpm for 8 minutes, and the supernatant was discarded. 10 mL of serum-free 1640 medium was added to mix the cells for use. The B lymphocytes and lymph node cells used in the experiment were obtained from mice immunized alternately with recombinant human FGL1-his and monkey FGL1-huFc proteins. Before fusion, the mice were sacrificed to remove the spleen and lymph nodes, and 10 mL of serum-free 1640 medium was added to grind the cells.
  • the obtained cell suspension was centrifuged at 1000 rpm for 8 minutes, the supernatant was discarded, and serum-free 1640 medium was added to mix the cells for use.
  • the feeder cells used in the fusion selection culture were obtained from macrophages in the peritoneal cavity of unimmunized animals; before fusion, the collected macrophage suspension was centrifuged at 100 rpm for 8 minutes, the supernatant was discarded and 25 mL of HAT was added.
  • the selection medium was mixed and distributed into two 24-well culture plates to assist the growth of new hybrid-B lymphocyte hybridomas.
  • B lymphocytes and myeloma cells were mixed at a ratio of 1:1, the suspension was centrifuged at 1000 rpm for 8 minutes, the pellet was collected, and washed twice with PM solution. Take the pellet and add PM solution to 1.2 mL, and inject 0.4 mL of cell suspension into each of the three multi-electrode cells.
  • the alternating electric field of dielectric electrophoresis the frequency of the sinusoidal signal was 1MHz, the amplitude was 250V/cm, and applied for 30 seconds; then the perforated RC electric pulse was immediately added—the amplitude was 5kV/cm, The pulse width is 20 ⁇ s, the number of pulses is 3, and the time interval is 1 second.
  • the fusion was washed out with a total of 5 mL of PFM solution, and incubated at 37°C for 30 minutes.
  • centrifuge at 100 rpm for 8 minutes, take the pellet, add 250 mL of HAT, mix well and dispense into a 24-well culture plate (there are 10 4 feeder cells/well), put it into a CO 2 incubator and culture at 37°C.
  • Positive cell clones were screened by ELISA.
  • the phalanx assay determines the coating concentration of the detection antigen human FGL1-his protein.
  • the detection antigen coating buffer was serially diluted horizontally, and 50 ⁇ L per well was used to coat the ELISA plate, overnight at 4°C; PBST was washed 3 times, 200 ⁇ L of blocking solution was added to each well, overnight at 4°C; the immunized mouse serum was longitudinally diluted, 50 ⁇ L per well, Normal mouse serum was diluted in the same fold as a negative control, incubated at 37°C for 2h; washed with PBST for the third time, added the enzyme-labeled secondary antibody at the working concentration, 50 ⁇ L per well, incubated at 37°C for 1.5h, after washing with PBST, TMB developed color.
  • the OD450 value was determined by an enzyme-linked detector to determine the optimal coating concentration for detecting the antigen.
  • the established ELISA method was used to detect the antibody secreted by the hybridoma cells.
  • the specific method is as follows: add the culture supernatant of hybridoma cells to the pre-coated ELISA plate, 50 ⁇ L/well, use the SP2/0 cell supernatant as the negative control and the immune polyanti-serum as the positive control, water bath at 37°C for 2 hours; PBST Wash 3 times; add working concentration of HRP-labeled goat anti-mouse IgG and IgM antibodies, 50 ⁇ L/well, water bath at 37 °C for 1.5 h; The OD450 reading of the tested wells was more than two times higher than that of the negative control, and it was judged as positive.
  • monoclonal antibodies are mainly obtained by in vitro culture method.
  • the cell line was expanded into a T75 culture flask, and cultured until the cell coverage was 80-90%, the cell supernatant was discarded, and 30 ml of hybridoma-SFM (Gibco) was added to culture at 37°C, 5% CO 2 . After 2-3 days of culture, add 30 mL of hybridoma-SFM, if the cell viability is less than 30%, fresh viable cells can be added. After culturing for 6-7 days, when the cell viability was lower than 20%, the culture supernatant was collected after low-speed centrifugation, and stored at 4°C for later use.
  • the method of phage library construction is universal.
  • a total of three phage libraries are constructed, which are derived from different peripheral blood lymphocytes.
  • the alpaca phage library is derived from peripheral blood lymphocytes derived from alpaca immunization
  • the mouse phage library is derived from mouse immunization.
  • the human natural pool (commercial general platform) is derived from the mixed human natural peripheral blood lymphocytes.
  • the phage library construction process is as follows: extract RNA, transfer the peripheral blood lymphocytes preserved with Trizol to a 1.5 mL centrifuge tube, add 1/5 volume of chloroform and mix well; stand at room temperature for 5 minutes and then centrifuge at 12000g at 4°C for 15 minutes; be careful Transfer the centrifuged supernatant to a new centrifuge tube; add 0.5-1 volume of isopropanol to the new centrifuge tube; stand at room temperature for 10 minutes, then centrifuge at 12,000g at 4°C for 10 minutes; use the peripheral blood stored with Trizol
  • the lymphocytes were washed with an equal volume of 75% ethanol, centrifuged at 7500g for 5 minutes at 4°C, and dissolved in an appropriate amount of RNase-free water.
  • the cDNA was then reverse transcribed, and the RNA obtained in the previous step was reverse transcribed into cDNA according to the instructions of the reverse transcription kit.
  • Antibody fragments are then amplified, and specific antibody fragments are amplified from reverse transcribed cNDA by PCR using Taq DNA Polymerase Hot Start enzyme.
  • the PCR reaction system was: 2 ⁇ L of cDNA template, 2 ⁇ L of forward primer, 2 ⁇ L of reverse primer, 5 ⁇ L of 10X Taq Buffer, 4 ⁇ L of dNTP, 0.25 ⁇ L of Taq(HS), and 50 ⁇ L of sterile water.
  • the PCR reaction conditions were: 98°C for 3 minutes; 95°C for 30 seconds, 57°C for 30 seconds, 68°C for 40 seconds, each cycle increased by 2 seconds, and repeated 22 cycles; 68°C for 5 minutes.
  • the PCR reaction system is: DNA template 2 ⁇ L, forward primer 2 ⁇ L, reverse primer 2 ⁇ L, 10X Taq Buffer 5 ⁇ L, dNTP 4 ⁇ L, Taq(HS) 0.25 ⁇ L, and sterile water to make up to 50 ⁇ L.
  • the PCR reaction conditions were: 98°C for 3 minutes; 95°C for 50 seconds, 55°C for 30 seconds, 72°C for 40 seconds, repeating 12 cycles; 72°C for 10 minutes.
  • the obtained PCR amplification product was recovered using a DNA purification and recovery kit according to the instructions.
  • the antibody gene sequence and phage vector amplified in the previous step were digested with BglI, and the digestion system was: amplified gene 12 ⁇ g or vector 3 ⁇ g, 10X BglI Buffer 3 ⁇ L, BglI 4.5 ⁇ L, make up water to 30 ⁇ L .
  • the digestion was carried out at 37°C for 3-4 hours. After enzyme digestion, use a DNA purification and recovery kit to recover the vector and amplify the gene according to the instructions, and then carry out the ligation reaction.
  • the ligation reaction system is: 200ng of vector, 80ng of amplified gene, 2 ⁇ L of T4 ligase, 5 ⁇ L of 10X ligation buffer, add water to 50 ⁇ L, react overnight at 4°C, and recover the ligation product using a DNA purification and recovery kit according to the instructions, and use ultra Pure water dissolves. Then transform into SS320 competent cells, put the electroporation cup on ice to pre-cool, add 1 ⁇ L of the recovered ligation product after the SS320 competent cells are thawed, and transfer the mixed competent cells and ligation products to the pre-cooled electroporation In the cup, use the preset Bacteria transformation program of the electroporator for electroporation transformation.
  • helper phage According to the ratio of helper phage: bacterial cell number of 20:1, the helper phage was added and incubated at 37°C for 30 minutes. The final concentration of kanamycin and 0.2 mM IPTG was added, and the cells were incubated at 30°C overnight. The overnight cultured cells were centrifuged at 13000rpm at 4°C for 5 minutes, the supernatant was transferred to a new centrifuge tube, 1/4 volume of pre-cooled 5X PEG8000/NaCl was added, and the cells were incubated on ice for 30 minutes. After centrifugation at 13000rpm for 10 minutes at 4°C, the supernatant was removed, and 1 mL of PBS buffer was added to dissolve the precipitate.
  • the immunotubes incubated with antigen and phage were washed 20 times with PBS containing 0.01% Tween for 5 minutes each time. Then 1 mL of 100 mM tricarboximide was added to the immunotube, incubated at room temperature for 10 minutes, 1 M Tris-HCl was added to neutralize the tricarboximide, and the last 1.5 mL of the eluted phage was transferred to a new centrifuge tube. Then, the eluted phage was amplified according to the amplification and purification phage library, and then the screening process was repeated twice, and the amount of antibody coated on the immune tube was successively reduced by half to obtain the eluted phage after three screenings.
  • the cell fluid after overnight culture was centrifuged to obtain a supernatant.
  • the phage supernatant obtained in the previous step was added to the 96-well ELISA plate coated with antigen overnight and blocked with 3% BSA, and incubated at room temperature for 1 h. After washing 3 times with PBS containing 0.05% Tween, human FGL1-his was used as the primary antibody, and the corresponding secondary antibody anti-VHH-HRP or anti-mouse Fab-HRP was used to read each well at wavelength 450 after TMB color development. absorbance value. The SS320 colony with the highest absorbance reading was selected and sent for sequencing to obtain the gene sequence of the antibody.
  • amino acid sequences of CDR1, CDR2 and CDR3 of the SH-01 heavy chain variable region are: SEQ ID NO: 37, SEQ ID NO: 47 and SEQ ID NO: 57;
  • amino acid sequence of SH-01 heavy chain variable region is SEQ ID NO: 1;
  • the nucleotide sequence of SH-01 heavy chain variable region is SEQ ID NO: 11;
  • amino acid sequences of CDR1, CDR2 and CDR3 of the SH-01 light chain variable region are: SEQ ID NO: 67, SEQ ID NO: 75 and SEQ ID NO: 83;
  • amino acid sequence of the SH-01 light chain variable region is SEQ ID NO: 21;
  • the nucleotide sequence of the variable region of the SH-01 light chain is SEQ ID NO: 29;
  • amino acid sequences of CDR1, CDR2 and CDR3 of the SH-02 heavy chain variable region are: SEQ ID NO: 38, SEQ ID NO: 48 and SEQ ID NO: 58;
  • amino acid sequence of SH-02 heavy chain variable region is SEQ ID NO: 2;
  • the nucleotide sequence of SH-02 heavy chain variable region is SEQ ID NO: 12;
  • amino acid sequences of CDR1, CDR2 and CDR3 of the SH-02 light chain variable region are: SEQ ID NO: 68, SEQ ID NO: 76 and SEQ ID NO: 84;
  • amino acid sequence of the SH-02 light chain variable region is SEQ ID NO: 22;
  • the nucleotide sequence of the SH-02 light chain variable region is SEQ ID NO: 30;
  • amino acid sequences of CDR1, CDR2 and CDR3 of SH-03 heavy chain variable region are: SEQ ID NO: 39, SEQ ID NO: 49 and SEQ ID NO: 59;
  • amino acid sequence of the SH-03 heavy chain variable region is SEQ ID NO: 3;
  • the nucleotide sequence of the SH-03 heavy chain variable region is SEQ ID NO: 13;
  • amino acid sequences of CDR1, CDR2 and CDR3 of the SH-03 light chain variable region are: SEQ ID NO: 69, SEQ ID NO: 77 and SEQ ID NO: 85;
  • amino acid sequence of the SH-03 light chain variable region is SEQ ID NO: 23;
  • the nucleotide sequence of the SH-03 light chain variable region is SEQ ID NO: 31;
  • amino acid sequences of CDR1, CDR2 and CDR3 of the SH-04 heavy chain variable region are: SEQ ID NO: 40, SEQ ID NO: 50 and SEQ ID NO: 60;
  • amino acid sequence of the SH-04 heavy chain variable region is SEQ ID NO: 4.
  • the nucleotide sequence of the SH-04 heavy chain variable region is SEQ ID NO: 14;
  • amino acid sequences of CDR1, CDR2 and CDR3 of the SH-04 light chain variable region are: SEQ ID NO: 70, SEQ ID NO: 78 and SEQ ID NO: 86;
  • amino acid sequence of the SH-04 light chain variable region is SEQ ID NO: 24;
  • the nucleotide sequence of the SH-04 light chain variable region is SEQ ID NO:32.
  • sequences screened by the human natural phage display antibody library were sequenced for antibody gene, and finally the variable regions of 4 antibodies SH-05, SH-06, SH-07 and SH-08 were obtained, their heavy chain variable regions and light
  • the amino acid/nucleotide sequences of the chain variable regions are shown below, respectively:
  • amino acid sequences of CDR1, CDR2 and CDR3 of the SH-05 heavy chain variable region are: SEQ ID NO: 41, SEQ ID NO: 51 and SEQ ID NO: 61;
  • amino acid sequence of the SH-05 heavy chain variable region is SEQ ID NO: 5;
  • the nucleotide sequence of the SH-05 heavy chain variable region is SEQ ID NO: 15;
  • amino acid sequences of CDR1, CDR2 and CDR3 of SH-05 light chain variable region are: SEQ ID NO: 71, SEQ ID NO: 79 and SEQ ID NO: 87;
  • amino acid sequence of the SH-05 light chain variable region is SEQ ID NO: 25;
  • the nucleotide sequence of the SH-05 light chain variable region is SEQ ID NO: 33;
  • amino acid sequences of CDR1, CDR2 and CDR3 of the SH-06 heavy chain variable region are: SEQ ID NO: 42, SEQ ID NO: 52 and SEQ ID NO: 62;
  • amino acid sequence of the SH-06 heavy chain variable region is SEQ ID NO: 6;
  • the nucleotide sequence of the SH-06 heavy chain variable region is SEQ ID NO: 16;
  • amino acid sequences of CDR1, CDR2 and CDR3 of the SH-06 light chain variable region are: SEQ ID NO: 72, SEQ ID NO: 80 and SEQ ID NO: 88;
  • amino acid sequence of the SH-06 light chain variable region is SEQ ID NO: 26;
  • the nucleotide sequence of the SH-06 light chain variable region is SEQ ID NO: 34;
  • amino acid sequences of CDR1, CDR2 and CDR3 of the SH-07 heavy chain variable region are: SEQ ID NO: 43, SEQ ID NO: 53 and SEQ ID NO: 63;
  • amino acid sequence of the SH-07 heavy chain variable region is SEQ ID NO: 7;
  • the nucleotide sequence of the SH-07 heavy chain variable region is SEQ ID NO: 17;
  • amino acid sequences of CDR1, CDR2 and CDR3 of the SH-07 light chain variable region are: SEQ ID NO: 73, SEQ ID NO: 81 and SEQ ID NO: 89;
  • amino acid sequence of the SH-07 light chain variable region is SEQ ID NO: 27;
  • the nucleotide sequence of the SH-07 light chain variable region is SEQ ID NO: 35;
  • amino acid sequences of CDR1, CDR2 and CDR3 of the SH-08 heavy chain variable region are: SEQ ID NO: 44, SEQ ID NO: 54 and SEQ ID NO: 64;
  • amino acid sequence of the SH-08 heavy chain variable region is SEQ ID NO: 8;
  • the nucleotide sequence of SH-08 heavy chain variable region is SEQ ID NO: 18;
  • amino acid sequences of CDR1, CDR2 and CDR3 of the SH-08 light chain variable region are: SEQ ID NO: 74, SEQ ID NO: 82 and SEQ ID NO: 90;
  • amino acid sequence of the SH-08 light chain variable region is SEQ ID NO: 28;
  • the nucleotide sequence of the SH-08 light chain variable region is SEQ ID NO:36.
  • variable regions are as follows:
  • amino acid sequences of CDR1, CDR2 and CDR3 of SH-09 variable region are: SEQ ID NO: 45, SEQ ID NO: 55 and SEQ ID NO: 65;
  • amino acid sequence of the variable region of SH-09 is SEQ ID NO: 9;
  • the nucleotide sequence of the variable region of SH-09 is SEQ ID NO: 19;
  • amino acid sequences of CDR1, CDR2 and CDR3 of the SH-10 variable region are: SEQ ID NO: 46, SEQ ID NO: 56 and SEQ ID NO: 66;
  • amino acid sequence of the SH-10 variable region is SEQ ID NO: 10;
  • the nucleotide sequence of the SH-10 variable region is SEQ ID NO:20.
  • the antibody fragments obtained by screening and sequencing of hybridoma and phage libraries were synthesized into human IgG frameworks, and then the antibody fragments were inserted into the PCDNA3.1 vector by molecular cloning technology to construct mammalian cell expression plasmids.
  • the plastid transfection method is introduced into the host cell line CHO cell, and the fermentation supernatant is obtained by using the cell fed-batch.
  • the supernatant of the fermentation liquid is purified by a series of steps such as affinity chromatography and ion exchange chromatography, and finally the construction is obtained. of monoclonal antibodies.
  • the purified monoclonal antibody was tested for expression level, purity, endotoxin, etc.
  • amino acid sequence of the heavy chain constant region of antibodies SH-01, SH-02, SH-03, and SH-04 is SEQ ID NO: 164, and the amino acid sequence of the light chain constant region is SEQ ID NO: 165.
  • amino acid sequence of the heavy chain constant region of antibodies SH-05, SH-06, SH-07 and SH-08 is SEQ ID NO: 164, and the amino acid sequence of the light chain constant region is SEQ ID NO: 165.
  • amino acid sequence of antibody SH-09, SH-10 constant region is SEQ ID NO:164.
  • Example 8 Species cross detection by ELISA method
  • the affinity of the antibody to human and mouse FGL1 protein was determined by ELISA detection method.
  • the plates coated with human FGL1-his or mouse FGL1-his were incubated at 4°C overnight, the antibodies to be detected were added to the 96-well ELISA plates blocked with 5% PBS-milk, and incubated at room temperature for 1 h. After washing three times with PBS containing 0.05% Tween, the detection antibody was used as the primary antibody, and human-IgG-FC-HRP was used as the secondary antibody. After TMB color development, the absorbance value of each well was read at a wavelength of 450. Elisa method screened antibodies that specifically bind to human FGL1 and cross-react with mouse species, and finally determined the affinity of FGL1 antibodies to human and mouse FGL1 proteins.
  • the binding of the anti-FGL1 antibody to human FGL1 was detected, and the results were shown in Figure 1, and the binding of the anti-FGL1 antibody to mouse FGL1 was detected, and the results were shown in Figure 2.
  • the results showed that the EC 50 of the anti-FGL1 antibody binding activity to human FGL1 was between 0.06-2.3 ⁇ g/mL.
  • the binding activity of anti-FGL1 antibody to murine FGL1 protein has an EC 50 between 0.06-2.3 ⁇ g/mL. It can be seen that the anti-FGL1 antibody has good binding activity.
  • Example 9 FACS determination of blocking activity of anti-FGL1 antibody on huLAG-3-CHO-K1
  • the CHO-K1 cell line was transfected with a lentiviral vector carrying human LAG3 (Ref accession number NP_002277.4) to obtain a huLAG3-CHO-K1 stably transfected cell line.
  • Anti-FGL1 was incubated with human FGL1-mFc (Sinobio, Cat: 13484-H38H) (final concentration 0.6 ⁇ g/ml) for 1 h at 4°C.
  • the cells were added to the flat bottom plate, the supernatant was removed by centrifugation, 100 ⁇ L of antibody and huFGL1-mFc dilution were added to the cells, incubated at 4°C for 1 h, the cells were washed three times with FACS buffer, and mixed with PE anti-mouse IgG Fc (Abeam, 98742) The antibody was incubated at 4°C for 30 minutes, and after washing the cells with FACS buffer, the blocking effect of anti-FGL1 antibody on the binding of FGL1 to LAG-3 was analyzed by flow cytometry (FACS).
  • FACS flow cytometry
  • 1 ⁇ g/mL anti-human CD3 antibody (OKT3) and 0.5 ⁇ g/mL anti-human CD28 antibody were coated on a 96-well flat-bottom tissue culture plate and incubated overnight at 4°C. Aspirate the coating solution, wash the wells three times with 1 ⁇ PBS, immediately add 10 ⁇ g/ml of FGL1-his or control substance (huLAG3-huFc) to a 96-well flat-bottom tissue culture plate, incubate at 37°C for 3h, and then use 1 ⁇ plates were washed three times with PBS, and added after from whole PBMC 1.5E5 / hole healthy human donors to the wells, flat bottom 96-well tissue culture plate was placed in CO 2 incubator, 37 °C, 5% CO 2 72 hours incubation .
  • huLAG3-huFc FGL1-his or control substance
  • Activator Preparation Prepare by mixing 400 mM EDC and 100 mM NHS(GE) immediately before use.
  • the CM5 sensor chip was activated for 420 s at a flow rate of 10 ⁇ L/min.
  • the channel was then injected with 30 ⁇ g/mL of anti-human Fc IgG in 10 mM NaAc (pH 4.5) at a flow rate of 10 ⁇ L/min.
  • the chips were inactivated with 1 M ethanolamine-hydrochloric acid (GE) at a flow rate of 10 ⁇ L/min for 420 s.
  • GE ethanolamine-hydrochloric acid
  • Samples in running buffer 1 ⁇ HBS-EP+ (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% Tween 20, pH 7.4) were captured onto Fc2 with anti-human Fc IgG at a flow rate of 10 ⁇ L/min.
  • 10 nm of human FGL1-his protein (Sinobio; Cat: 13484-H08B) and running buffer were sequentially injected into Fc1-Fc2 at a flow rate of 30 min, bound for 180 s, and then dissociated for 3600 s.
  • 10 mM glycine (pH 1.5) was injected as regeneration buffer after each dissociation.
  • Chips were regenerated with 10 mM glycine (pH 1.5).
  • Example 12 Evaluation of antitumor efficacy in vivo
  • the anti-tumor efficacy of antibody SH-07 with human-mouse cross was evaluated in vivo.
  • the C57BL/MC38 mouse model was established by the intestinal cancer cell line MC38, and the effect of antibody SH-07 administration on tumor growth was evaluated.
  • Mouse colon cancer MC38 cells were cultured in monolayer in vitro, and 0.1 mL (1.5x10 7 cells) of MC38 cells were subcutaneously inoculated into the right back of each mouse. When the average tumor volume reached 100 mm 3 , group administration was started.
  • the tumor-bearing mice were given alternating injections of antibody SH-07 (5mpk), PDL1 antibody Avelumab (1mpk), and Comb, namely Avelumab (1mpk) + SH-07 (5mpk), in the abdominal cavity and tail vein, once every 3 days, weekly
  • the drug was administered twice, and the same volume of PBS was administered as a control, for a total of 8 administrations.
  • the health status and death of the animals were monitored daily. Routine examinations included observation of tumor growth and the effects of drug treatment on the animals' daily behaviors such as behavioral activities, food and water intake (visual inspection only), physical signs or other abnormal conditions. Animal deaths and side effects within each group were recorded based on the number of animals in each group.
  • the antitumor efficacy of the drug was evaluated by TGI (%) or relative tumor proliferation rate T/C (%). TGI (%), reflecting tumor growth inhibition rate.
  • TGI(%) [1-(average tumor volume at the end of administration of a certain treatment group-average tumor volume at the beginning of administration of this treatment group)/(average tumor volume at the end of treatment in the solvent control group- The average tumor volume at the start of treatment in the solvent control group)] ⁇ 100%.
  • the mouse colon cancer cell line MC38 xenograft model was given to the tumor-bearing mice as a PBS control, respectively.
  • the tumor growth curves of antibodies SH-07 (5mpk), Avelumab (1mpk) and Comb are shown in Figure 5, where the abscissa represents after inoculation with MC38 cells The number of days, the ordinate represents the tumor volume, and the tumor inhibition rate TGI (%) is shown in Table 2.
  • the cells After 7 days of cell inoculation, the cells reached 100 mm 3 to be divided into cages and administered. 28 days after administration, the tumor-bearing volume of the model control group grew to 678 ⁇ 115 mm 3 , which was 8 times higher than that of the grouping on the 7th day. At 28 days of administration, the tumor-bearing volume of the mice in the Avelumab-1mpk control antibody group was 449 ⁇ 189 mm 3 , and the tumor growth inhibition rate was 33.8% compared with the control group; the candidate molecule SH-07 test antibody was 30.5% compared with the control group.

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Abstract

L'invention concerne un anticorps anti-FGL1 ou un fragment de liaison à l'antigène de celui-ci, et des utilisations et des compositions pharmaceutiques correspondantes. L'anticorps anti-FGL1 de la présente invention peut se lier spécifiquement à FGL1, bloquer la liaison de FGL1 à LAG-3, inverser l'effet inhibiteur de FGL1 sur les lymphocytes T et activer les lymphocytes T pour provoquer la sécrétion de cytokines.
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