WO2023051683A1 - 抗lag3双特异性抗体、药物组合物及用途 - Google Patents

抗lag3双特异性抗体、药物组合物及用途 Download PDF

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WO2023051683A1
WO2023051683A1 PCT/CN2022/122556 CN2022122556W WO2023051683A1 WO 2023051683 A1 WO2023051683 A1 WO 2023051683A1 CN 2022122556 W CN2022122556 W CN 2022122556W WO 2023051683 A1 WO2023051683 A1 WO 2023051683A1
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antibody
seq
amino acid
variable region
lag3
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French (fr)
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张鹏
李百勇
夏瑜
王忠民
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中山康方生物医药有限公司
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Priority to IL311692A priority Critical patent/IL311692A/en
Priority to AU2022355986A priority patent/AU2022355986A1/en
Priority to CA3233192A priority patent/CA3233192A1/en
Publication of WO2023051683A1 publication Critical patent/WO2023051683A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/06—Antianaemics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells

Definitions

  • the invention belongs to the field of biomedicine, and relates to a bispecific antibody against LAG3 antibody, its pharmaceutical composition and application.
  • the bispecific antibody is an anti-LAG3-anti-PD-1 bispecific antibody.
  • Tumors are diseases that seriously endanger human health in the world today, ranking second among the deaths caused by various diseases. And in recent years, its incidence has shown an obvious upward trend.
  • the curative effect of malignant tumors is poor, the rate of advanced metastasis is high, and the prognosis is often poor.
  • conventional treatment methods such as radiotherapy, chemotherapy and surgery are currently used clinically, although the pain is relieved to a large extent and the survival time is prolonged, these methods have great limitations, and the curative effect is difficult to further improve.
  • Lymphocyte-activation gene 3 (LAG3), or CD223, is a type I transmembrane protein composed of 498 amino acids, which belongs to the immunoglobulin superfamily (IgSF) member.
  • LAG3 is mainly expressed in activated CD4 + T cells and CD8 + T cells, in addition to natural killer (natural killer, NK) cells, B cells, regulatory T cells (regulatory T cells, Treg) and plasmacytoid dendritic cells ( Plasmacytoid dendritic cells, pDC) and other cells also express LAG3.
  • NK natural killer
  • Treg regulatory T cells
  • Plasmacytoid dendritic cells Plasmacytoid dendritic cells, pDC
  • the LAG3 molecular gene is located on human chromosome 12 (20p13.3), adjacent to the CD4 molecular gene, and both have the same exons and introns. Although the amino acid sequence homology between the two is only about 20%, the LAG3 molecule and the CD4 molecule have a high similarity in structure.
  • Major histocompatibility complex class II molecules MHC II
  • LSECtin liver sinusoidal endothelial cell agglutinin
  • galectin-3 galectin-3
  • MHC class II molecules are the main ligands of LAG3, and their affinity (Kd: 60nmol L-1) with MHC class II molecules is 100 times that of CD4 molecules, indicating that LAG3 molecules can effectively compete for the binding of CD4 and MHC class II molecules. Inhibits T cell activation.
  • the expression of the immunosuppressive molecule LAG3 can be detected 24 hours after T cell activation, leading to T cell incapacity or apoptosis.
  • the LAG3 molecule forms a dimer molecule through its D1 domain (containing a proline-rich ring structure) and is specific to the MHC II molecule in the first signaling axis "CD3-TCR-MHC II" of CD4 + T cell activation.
  • the combination blocks the signal transduction pathway of T cell activation, and on the other hand, the intracellular segment of LAG3 molecule (KIEELE motif) produces an immunosuppressive signal to down-regulate the activity of CD4 + T cells.
  • LAG3 molecules can promote the differentiation of Treg cells, participate in signal transduction and transcription activator 5 downstream signaling, thereby enhancing the inhibitory effect of Treg cells, which is one of the mechanisms for tumors to escape the killing of the immune system (Andrews Lawrence P, Marciscano Ariel E, Drake Charles G et al.LAG3(CD223) as a cancer immunotherapy target.[J].Immunol Rev,2017,276:80-96.).
  • LAG3 is overexpressed in tumor-infiltrating CD8 + T cells in various malignancies.
  • tumor-infiltrating New York esophageal squamous cell carcinoma 1 (NY-ESO-1)-specific CD8 + T cells express high levels of PD-1 and LAG3 and have reduced ability to produce IFN- ⁇ and TNF- ⁇ , resulting in inactivation of lymphocytes.
  • Galectin-3 and LSECtin mainly interact with LAG3 to regulate the activation and function of CD8 + T cells.
  • melanoma antigen-specific T cells isolated from patients with melanoma metastasis were significantly upregulated in the expression of LAG3 and other immune checkpoint molecules CTLA-4 and TIM-3.
  • LAG3 antibody drugs have entered the clinical research stage.
  • Bristol-Myers Squibb's Relatlimab has made the fastest progress.
  • 10 clinical studies have been carried out, and most of them are combined drugs of Relatlimab and Nivolumab. It is used to treat tumors such as hematoma, melanoma, glioblastoma, renal cell carcinoma and non-small cell lung cancer.
  • the transmembrane receptor PD-1 (programmed cell death-1) is a member of the CD28 family and is expressed in activated T cells, B cells and myeloid cells. Both PDL1 and PDL2, the receptors of PD-1, belong to the B7 superfamily, in which PDL1 is expressed in many cells, including T cells, B cells, endothelial cells and epithelial cells, while PDL2 is only expressed in antigen-presenting cells such as dendritic cells and macrophages.
  • PD-1 plays a very important role in the process of negatively regulating the activation of T cells.
  • the negative regulation of T cells mediated by PD-1 is one of the important mechanisms for tumor immune evasion.
  • PD-L1 expressed on the surface of tumors can interact with PD-1 on the surface of immune cells binds, and then inhibits immune cells from killing tumor tissue through the PD-1/PD-L1 signaling pathway.
  • Tumors with high expression of PD-L1 are accompanied by cancers that are difficult to detect (Hamanishi et al. USA 2007; 104:3360-5).
  • An effective approach to antagonize PD-1, thereby inhibiting the PD-1/PD-L1 signaling pathway, is the in vivo injection of anti-PD-1 antibodies.
  • Anti-PD-1 antibodies have broad-spectrum anti-tumor prospects and amazing drug efficacy. Antibodies targeting the PD-1 pathway will bring breakthroughs in the treatment of various tumors: for the treatment of non-small cell lung cancer, renal cell carcinoma, Ovarian cancer, melanoma (Homet M.B., Parisi G., et al., Anti-PD-1 Therapy in Melanoma.Semin Oncol.2015Jun; 42(3):466-473), hematological tumors and anemia (Held SA, Heine A , et al., Advances in immunotherapy of chronic myeloid leukemia CML. Curr Cancer Drug Targets. 2013 Sep; 13(7):768-74).
  • Bifunctional antibodies also known as bispecific antibodies (Bispecific Antibody) are specific antibody drugs that simultaneously target and bind two different antigens. They can be produced by immuno-sorting and purification, and can also be obtained by genetic engineering. Genetic engineering has corresponding flexibility in terms of optimization of binding sites, consideration of synthetic form, and yield, so it has certain advantages. At present, more than 45 forms of bispecific antibodies have been proven (Müller D, Kontermann RE. Bispecific antibodies for cancer immunotherapy: Current perspectives. BioDrugs 2010; 24:89-98).
  • the IgG-ScFv form is the Morrison model (Coloma MJ, Morrison SL. Design and production of novel tetravalent bispecific antibodies. Nat Biotechnol.
  • the inventors After in-depth research and creative work, the inventors obtained an anti-LAG3 antibody, and based on this, prepared an anti-LAG3-anti-PD-1 bispecific antibody.
  • the present inventors have surprisingly found that the anti-LAG3 antibody of the present invention (also referred to simply as the antibody or the antibody of the present invention) and the anti-LAG3-anti-PD-1 bispecific antibody (also referred to as the bispecific antibody of the present invention or the bispecific antibody of the present invention) Sexual antibodies) have superior affinity and/or specificity, and are even superior to positive control antibodies (such as Nivolumab, Pembrolizumab, Relatlimab, etc.) in one or more aspects.
  • positive control antibodies such as Nivolumab, Pembrolizumab, Relatlimab, etc.
  • One aspect of the present invention relates to an anti-LAG3 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein,
  • the heavy chain variable region comprises: amino acid sequences as shown in SEQ ID NOs: 5-7 HCDR1-HCDR3; and the light chain variable region comprises: amino acid sequences as shown in SEQ ID NOs: 8-10 LCDR1-LCDR3;
  • the heavy chain variable region comprises: amino acid sequences such as HCDR1-HCDR3 shown in SEQ ID NOs: 5-7; and the light chain variable region comprises: amino acid sequences such as SEQ ID NO: 8, SEQ ID NO LCDR1-LCDR3 shown in :46 and SEQ ID NO:47;
  • the heavy chain variable region comprises: amino acid sequences such as HCDR1-HCDR3 shown in SEQ ID NOs: 5-7; and the light chain variable region comprises: amino acid sequences such as SEQ ID NO: 48, SEQ ID NO :46 and LCDR1-LCDR3 shown in SEQ ID NO:10.
  • the antibody or antigen-binding fragment thereof wherein,
  • amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:2, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO:4;
  • amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:2, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO:42;
  • amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:2
  • amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO:44.
  • the antibody or antigen-binding fragment thereof wherein the antibody or antigen-binding fragment thereof is selected from Fab, Fab', F(ab')2, Fd, Fv, dAb, complementary Determining region fragments, single chain antibodies, humanized antibodies, chimeric antibodies or diabodies.
  • the antibody or antigen-binding fragment thereof wherein, the antibody is less than 0.2nM, such as less than 0.15nM, less than 0.1nM, less than 0.08nM, 0.06nM or less than 0.05nM or A smaller EC50 binding to human LAG3-mFc; preferably, said EC50 is determined by an indirect ELISA method.
  • the antibody or antigen-binding fragment thereof wherein,
  • the antibody includes non-CDR regions, and the non-CDR regions are from a species other than murine, eg, from a human antibody.
  • the antibody or antigen-binding fragment thereof wherein,
  • the antibody whose constant region is derived from a human antibody
  • the constant region of the antibody is selected from the constant region of human IgG1, IgG2, IgG3 or IgG4.
  • the antibody or antigen-binding fragment thereof wherein,
  • the heavy chain constant region of the anti-LAG3 antibody is Ig gamma-1 chain C region (for example, as shown in SEQ ID NO:39) or Ig gamma-4 chain C region (for example, as shown in SEQ ID NO:45) the light chain constant region is an Ig kappa chain C region (eg, as shown in SEQ ID NO: 40).
  • the antibody or antigen-binding fragment thereof wherein,
  • the antibody is of human IgG1 subtype
  • the heavy chain constant region of the antibody has the following mutations:
  • amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO: 11
  • amino acid sequence of the light chain is shown in SEQ ID NO: 12.
  • the antibody or antigen-binding fragment thereof wherein,
  • the antibody is of human IgG4 subtype
  • the heavy chain constant region of the antibody has the following mutations:
  • amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO: 13
  • amino acid sequence of the light chain is shown in SEQ ID NO: 12.
  • the anti-LAG3 antibody is a monoclonal antibody.
  • the anti-LAG3 antibody is in the form of immunoglobulin.
  • the anti-LAG3 antibody is a single chain antibody.
  • an antibody-drug conjugate which includes an antibody or its antigen-binding fragment and a small molecule drug, wherein the antibody or its antigen-binding fragment is any one of the present invention
  • ADC antibody-drug conjugate
  • the small-molecule drug is a small-molecule cytotoxic drug; more preferably, the small-molecule drug is a tumor chemotherapy drug.
  • the chemotherapeutic drugs can be conventional tumor chemotherapeutic drugs, such as alkylating agents, antimetabolites, antitumor antibiotics, plant anticancer drugs, hormones, immune preparations and the like.
  • the antibody-drug conjugate wherein the antibody or its antigen-binding fragment is connected to the small molecule drug through a linker;
  • the linker can be a person skilled in the art Known linkers are, for example, hydrazone bonds, disulfide bonds or peptide bonds.
  • the antibody-drug conjugate wherein the molar ratio of the antibody or its antigen-binding fragment to the small-molecule drug is 1: (2-4), for example, 1: 2, 1:3 or 1:4.
  • Yet another aspect of the present invention relates to a bispecific antibody comprising a first protein domain and a second protein domain, wherein:
  • the first protein domain targets LAG3,
  • said second protein domain targets a target other than LAG3 (e.g., PD-1),
  • the first protein functional domain is the antibody or antigen-binding fragment according to any one of the present invention.
  • the bispecific antibody is in IgG-scFv format
  • the first protein functional region is an antibody according to any one of the present invention, and the second protein functional region is a single-chain antibody; or
  • the first protein functional domain is a single-chain antibody
  • the second protein functional domain is an antibody according to any one of the present invention.
  • the bispecific antibody of the present invention is an anti-LAG3-anti-PD-1 bispecific antibody.
  • the bispecific antibody wherein the first protein functional domain and the second protein functional domain are directly connected or connected through a linking fragment
  • the connecting segment is (GGGGS)m, where m is a positive integer, such as 1, 2, 3, 4, 5 or 6;
  • the connecting segment is (GGGGS)nG, n is a positive integer, such as 1, 2, 3, 4, 5 or 6.
  • the first protein functional domain and the second protein functional domain are independently 1, 2 or more than 2.
  • the bispecific antibody wherein the single-chain antibody is linked to the C-terminus of the heavy chain of the antibody.
  • the bispecific antibody includes:
  • the first protein functional region is the anti-LAG3 antibody according to any one of the present invention, and the anti-LAG3 antibody is in the form of immunoglobulin,
  • the second protein functional region is an anti-PD-1 single-chain antibody.
  • the bispecific antibody wherein the anti-PD-1 single-chain antibody comprises a heavy chain variable region and a light chain variable region, wherein,
  • the heavy chain variable region comprises: HCDR1-HCDR3 whose amino acid sequences are shown in SEQ ID NOs: 26-28, respectively;
  • the light chain variable region comprises: LCDR1-LCDR3 with amino acid sequences shown in SEQ ID NOs: 29-31, respectively.
  • the bispecific antibody wherein the anti-PD-1 single chain antibody
  • amino acid sequence of its heavy chain variable region is set forth in SEQ ID NO: 15, and the amino acid sequence of its light chain variable region is set forth in SEQ ID NO: 17; or
  • amino acid sequence of its heavy chain variable region is shown in SEQ ID NO: 19, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO: 21 or SEQ ID NO: 38.
  • the bispecific antibody wherein the heavy chain variable region and the light chain variable region in the anti-PD-1 single-chain antibody are connected directly or through a connecting fragment;
  • the connecting segment is (GGGGS)m, where m is a positive integer, such as 1, 2, 3, 4, 5 or 6;
  • the connecting segment is (GGGGS)nG, n is a positive integer, such as 1, 2, 3, 4, 5 or 6.
  • the bispecific antibody wherein,
  • the bispecific antibodies include:
  • the functional region of the first protein is immunoglobulin, and the functional region of the second protein is a single-chain antibody;
  • amino acid sequence of the heavy chain of the immunoglobulin is shown in SEQ ID NO: 11 or SEQ ID NO: 13, and the amino acid sequence of its light chain is shown in SEQ ID NO: 12;
  • amino acid sequence of the heavy chain variable region of the single-chain antibody is as shown in SEQ ID NO:19, and the amino acid sequence of the light chain variable region of the single-chain antibody is as SEQ ID NO:21 or SEQ ID NO:38 shown;
  • the single-chain antibody is connected to the C-termini of the two heavy chains of the immunoglobulin;
  • the first protein functional region is connected to the second protein functional region through a first connecting segment; and the heavy chain variable region of the single-chain antibody is connected to the light chain variable region of the single-chain antibody through a second Segment concatenation; said first concatenated fragment and said second concatenated fragment are the same or different;
  • amino acid sequences of the first connecting segment and the second connecting segment are independently selected from SEQ ID NOs: 35-37;
  • amino acid sequences of the first connecting fragment and the second connecting fragment are shown in SEQ ID NO:36.
  • the bispecific antibody includes:
  • the first protein functional region is an anti-LAG3 single-chain antibody
  • the second protein functional region is an anti-PD-1 antibody
  • the anti-PD-1 antibody is in the form of immunoglobulin
  • the anti-LAG3 single-chain antibody comprises a heavy chain variable region and a light chain variable region, wherein,
  • the heavy chain variable region comprises: HCDR1-HCDR3 whose amino acid sequences are shown in SEQ ID NOs: 5-7; and
  • the light chain variable region comprises: LCDR1-LCDR3 with amino acid sequences shown in SEQ ID NOs: 8-10, respectively.
  • the bispecific antibody wherein the anti-LAG3 single chain antibody
  • amino acid sequence of its heavy chain variable region is shown in SEQ ID NO: 2, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO: 4;
  • amino acid sequence of its heavy chain variable region is set forth in SEQ ID NO: 2
  • amino acid sequence of its light chain variable region is set forth in SEQ ID NO: 42; or
  • amino acid sequence of its heavy chain variable region is shown in SEQ ID NO:2
  • amino acid sequence of its light chain variable region is shown in SEQ ID NO:44.
  • the bispecific antibody wherein the heavy chain variable region and the light chain variable region in the anti-LAG3 single-chain antibody are connected directly or through a linking fragment;
  • the connecting segment is (GGGGS)m, where m is a positive integer, such as 1, 2, 3, 4, 5 or 6;
  • the connecting segment is (GGGGS)nG, n is a positive integer, such as 1, 2, 3, 4, 5 or 6.
  • the bispecific antibody wherein the anti-PD-1 antibody comprises a heavy chain variable region and a light chain variable region, wherein,
  • the heavy chain variable region comprises: HCDR1-HCDR3 whose amino acid sequences are shown in SEQ ID NOs: 26-28, respectively;
  • the light chain variable region comprises: LCDR1-LCDR3 with amino acid sequences shown in SEQ ID NOs: 29-31, respectively.
  • the bispecific antibody wherein the anti-PD-1 antibody,
  • amino acid sequence of its heavy chain variable region is set forth in SEQ ID NO: 15, and the amino acid sequence of its light chain variable region is set forth in SEQ ID NO: 17; or
  • amino acid sequence of its heavy chain variable region is shown in SEQ ID NO: 19, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO: 21 or SEQ ID NO: 38.
  • the bispecific antibody wherein, the heavy chain constant region of the anti-PD-1 antibody is Ig gamma-1 chain C region (for example, as SEQ ID NO: 39 shown) or Ig gamma-4 chain C region (for example, as shown in SEQ ID NO:45); the light chain constant region is Ig kappa chain C region (for example, as shown in SEQ ID NO:40).
  • the bispecific antibody wherein,
  • the anti-PD-1 antibody is of human IgG1 subtype
  • the anti-PD-1 antibody has the following mutations:
  • amino acid sequence of the heavy chain of the anti-PD-1 antibody is shown in SEQ ID NO:34, and the amino acid sequence of the light chain is shown in SEQ ID NO:25.
  • the bispecific antibody wherein,
  • the anti-PD-1 antibody is of human IgG4 subtype
  • the anti-PD-1 antibody has the following mutations:
  • amino acid sequence of the heavy chain of the anti-PD-1 antibody is shown in SEQ ID NO:32, and the amino acid sequence of the light chain is shown in SEQ ID NO:25.
  • the bispecific antibody wherein,
  • the bispecific antibodies include:
  • the first protein functional region is a single-chain antibody
  • the second protein functional region is an immunoglobulin
  • amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 4;
  • amino acid sequence of the heavy chain of the immunoglobulin is shown in SEQ ID NO:34 or SEQ ID NO:32, and the amino acid sequence of its light chain is shown in SEQ ID NO:25;
  • the single-chain antibody is connected to the C-termini of the two heavy chains of the immunoglobulin;
  • the first protein functional region is connected to the second protein functional region through a first connecting segment; and the heavy chain variable region of the single-chain antibody is connected to the light chain variable region of the single-chain antibody through a second Segment concatenation; said first concatenated fragment and said second concatenated fragment are the same or different;
  • amino acid sequences of the first connecting segment and the second connecting segment are independently selected from SEQ ID NOs: 35-37;
  • amino acid sequences of the first connecting fragment and the second connecting fragment are shown in SEQ ID NO:36.
  • one immunoglobulin molecule is linked with two single-chain antibody molecules; preferably, the two single-chain antibody molecules are the same.
  • Another aspect of the present invention relates to an isolated nucleic acid molecule, which encodes the anti-LAG3 antibody according to any one of the present invention, or encodes the bispecific antibody according to any one of the present invention.
  • Yet another aspect of the present invention relates to a recombinant vector comprising the isolated nucleic acid molecule of the present invention.
  • Yet another aspect of the present invention relates to a host cell comprising an isolated nucleic acid molecule of the present invention, or a recombinant vector of the present invention.
  • Another aspect of the present invention relates to a method for preparing the antibody or antigen-binding fragment thereof according to any one of the present invention or the bispecific antibody according to any one of the present invention, which comprises culturing under suitable conditions The host cell of the present invention, and the step of recovering the antibody or its antigen-binding fragment or bispecific antibody from cell culture.
  • Another aspect of the present invention relates to a pharmaceutical composition, which comprises any of the antibodies or antigen-binding fragments of the present invention, any of the antibody drug conjugates of the present invention or any of the present invention
  • Another aspect of the present invention relates to the antibody or antigen-binding fragment thereof according to any one of the present invention, the antibody drug conjugate according to any one of the present invention or the bispecific antibody according to any one of the present invention Use of antibodies in the preparation of drugs for treating and/or preventing tumors or anemia;
  • the tumor is selected from ovarian cancer, esophageal cancer, melanoma, hematoma, glioblastoma, renal cell carcinoma, lung cancer, prostate cancer, bladder cancer, colon cancer, rectal cancer, liver cancer, gastrointestinal One or more of cancers of the tract, breast, brain, pancreas, thyroid, head and neck, and kidney;
  • the lung cancer is non-small cell lung cancer
  • the hematological tumor is leukemia
  • the esophageal cancer is esophageal squamous cell carcinoma.
  • the tumor is selected from ovarian cancer, esophageal cancer, melanoma, hematoma, glioblastoma, renal cell carcinoma, lung cancer, prostate cancer, bladder cancer, colon cancer, rectal cancer, liver cancer, gastrointestinal One or more of cancers of the tract, breast, brain, pancreas, thyroid, head and neck, and kidney;
  • the lung cancer is non-small cell lung cancer
  • the hematological tumor is leukemia
  • the esophageal cancer is esophageal squamous cell carcinoma.
  • Another aspect of the present invention relates to a method for treating and/or preventing tumor or anemia, comprising administering to a subject in need an effective amount of the antibody or antigen-binding fragment thereof described in any one of the present invention, the present invention.
  • the step of the antibody drug conjugate described in any one of the inventions or the bispecific antibody described in any one of the present invention comprising administering to a subject in need an effective amount of the antibody or antigen-binding fragment thereof described in any one of the present invention, the present invention.
  • the tumor is selected from ovarian cancer, esophageal cancer, melanoma, hematoma, glioblastoma, renal cell carcinoma, lung cancer, prostate cancer, bladder cancer, colon cancer, rectal cancer, liver cancer, gastrointestinal One or more of cancers of the tract, breast, brain, pancreas, thyroid, head and neck, and kidney;
  • the lung cancer is non-small cell lung cancer
  • the hematological tumor is leukemia
  • the esophageal cancer is esophageal squamous cell carcinoma.
  • EC 50 refers to the concentration for 50% of maximal effect, which refers to the concentration that can cause 50% of the maximal effect.
  • antibody refers to an immunoglobulin molecule generally composed of two pairs of polypeptide chains, each pair having a "light” (L) chain and a “heavy” (H) chain.
  • Antibody light chains can be classified as kappa and lambda light chains.
  • Heavy chains can be classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.
  • the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also comprising a "D" region of about 3 or more amino acids.
  • Each heavy chain is composed 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 is composed of a light chain variable region (VL) and a light chain constant region (CL).
  • the light chain constant region consists of one domain, CL.
  • the constant regions of the antibodies mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.
  • 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
  • Each VH and VL consists of 3 CDRs and 4 FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4, from amino-terminus to carboxy-terminus.
  • the variable regions (VH and VL) of each heavy chain/light chain pair form the antibody binding site, respectively. Assignment of amino acids to regions or domains follows Bethesda M.d., Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, (1987 and 1991)), or Chothia & Lesk J. Mol. Biol. 1987; 196:901-917; Chothia et al.
  • the heavy chain may also comprise more than 3 CDRs, such as 6, 9, or 12.
  • the heavy chain in the bispecific antibody of the present invention, can be a ScFv connected to the C-terminus of the heavy chain of an IgG antibody. In this case, the heavy chain contains 9 CDRs.
  • antibody is not limited to any particular method of producing antibodies. For example, it includes recombinant antibodies, monoclonal antibodies and polyclonal antibodies. Antibodies can be of different isotypes, eg, IgG (eg, IgGl, IgG2, IgG3, or IgG4 subtype), IgAl, IgA2, IgD, IgE, or IgM antibodies.
  • IgG eg, IgGl, IgG2, IgG3, or IgG4 subtype
  • IgAl IgA2, IgD, IgE, or IgM antibodies.
  • the terms “monoclonal antibody” and “monoclonal antibody” refer to an antibody or a fragment of an antibody from a group of highly homologous antibody molecules, that is, except for natural mutations that may occur spontaneously, A population of identical antibody molecules.
  • mAbs are highly specific for a single epitope on an antigen.
  • polyclonal antibodies usually contain at least two or more different antibodies, and these different antibodies usually recognize different epitopes on antigens.
  • Monoclonal antibodies can usually be obtained using hybridoma technology first reported by Kohler et al. ( G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity [J]. Nature, 1975; 256(5517): 495), but it can also be obtained by recombinant DNA technology (see US Patent 4,816,567).
  • humanized antibody refers to the replacement of all or part of the CDR regions of a human immunoglobulin (recipient antibody) with the CDR regions of a non-human antibody (donor antibody).
  • Antibodies or antibody fragments, wherein the donor antibody can be a non-human (eg, mouse, rat or rabbit) antibody with the desired specificity, affinity or reactivity.
  • some amino acid residues in the framework region (FR) of the acceptor antibody can also be replaced by amino acid residues of corresponding non-human antibodies, or by amino acid residues of other antibodies, so as to further improve or optimize the performance of the antibody.
  • the antigen-binding fragments of antibodies are diabodies, in which the VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for two domains on the same chain pairing, thereby forcing the domain to pair with the complementary domain of another chain and create two antigen-binding sites (see, e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA 1993; 90:6444 -6448 and Poljak RJ et al., Structure 1994; 2:1121-1123).
  • single chain fragment variable refers to an antibody comprising a heavy chain variable region (V H ) and an antibody light chain variable region (V L ) connected by a linker. ) molecules. where the VL and VH domains pair to form a monovalent molecule via a linker that enables production as a single polypeptide chain (see, e.g., Bird et al, Science 1988; 242:423-426 and Huston et al, Proc. Natl. Acad. Sci. USA 1988; 85:5879-5883).
  • Such scFv molecules may have the general structure: NH2- VL -Linker- VH -COOH or NH2- VH -Linker- VL -COOH.
  • Suitable prior art linkers consist of the repeated GGGGS amino acid sequence or variants thereof.
  • a linker having the amino acid sequence (GGGGS)4 can be used, but variants thereof can also be used (Holliger et al, Proc. Natl. Acad. Sci. USA 1993; 90:6444-6448).
  • Other linkers that can be used in the present invention are described by Alfthan et al, Protein Eng. 1995; 8:725-731, Choi et al, Eur.J.
  • isolated means obtained from the natural state by artificial means. If an "isolated" substance or component occurs in nature, it may be that its natural environment has been altered, the substance has been isolated from its natural environment, or both. For example, an unisolated polynucleotide or polypeptide naturally exists in a living animal, and the same polynucleotide or polypeptide with high purity isolated from this natural state is called isolation. of.
  • isolated or “isolated” do not exclude the admixture of artificial or synthetic substances, nor the presence of other impurities which do not affect the activity of the substance.
  • vector refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted.
  • the vector is called an expression vector.
  • a vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements it carries can be expressed in the host cell.
  • Vectors are well known to those skilled in the art, including but not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC) ; Phage such as lambda phage or M13 phage and animal viruses.
  • artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC)
  • Phage such as lambda phage or M13 phage and animal viruses.
  • Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses, papillomaviruses, Polyoma vacuolar virus (eg SV40).
  • retroviruses including lentiviruses
  • adenoviruses such as herpes simplex virus
  • poxviruses such as herpes simplex virus
  • baculoviruses such as herpes simplex virus
  • baculoviruses such as herpes simplex virus
  • papillomaviruses papillomaviruses
  • papillomaviruses papillomaviruses
  • Polyoma vacuolar virus eg
  • the term "host cell” refers to cells that can be used to introduce vectors, including, but not limited to, prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, Insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, GS cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.
  • prokaryotic cells such as Escherichia coli or Bacillus subtilis
  • fungal cells such as yeast cells or Aspergillus
  • Insect cells such as S2 Drosophila cells or Sf9
  • animal cells such as fibroblasts, CHO cells, GS cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.
  • an antibody that specifically binds to an antigen refers to an antibody that is less than about 10 -5 M, such as less than about 10 -6 M, 10 -7 M, Binds the antigen with an affinity (K D ) of 10 ⁇ 8 M, 10 ⁇ 9 M or 10 ⁇ 10 M or less.
  • KD refers to the dissociation equilibrium constant for a particular antibody-antigen interaction, which is used to describe the binding affinity between an antibody and antigen.
  • the antibody has a dissociation equilibrium constant (K D ) of less than about 10 ⁇ 5 M, such as less than about 10 ⁇ 6 M, 10 ⁇ 7 M, 10 ⁇ 8 M, 10 ⁇ 9 M, or 10 ⁇ 10 M or less.
  • Binds antigen eg, PD-1 protein). KD can be determined using methods known to those skilled in the art, for example using a Fortebio Molecular Interaction Instrument.
  • amino acids are generally represented by single-letter and three-letter abbreviations known in the art.
  • alanine can be represented by A or Ala.
  • the term "pharmaceutically acceptable carrier and/or excipient” refers to a carrier and/or excipient compatible with the subject and the active ingredient pharmacologically and/or physiologically, These are well known in the art (see e.g. Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and include, but are not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers agent.
  • pH regulators include but not limited to phosphate buffer
  • surfactants include but not limited to cationic, anionic or nonionic surfactants such as Tween-80
  • ionic strength enhancers include but not limited to sodium chloride.
  • an effective amount refers to an amount sufficient to achieve, or at least partially achieve, the desired effect.
  • an effective amount for preventing a disease refers to an amount sufficient to prevent, arrest, or delay the occurrence of a disease (such as a tumor);
  • an effective amount for treating a disease refers to an amount sufficient to cure or at least partially prevent the occurrence of a disease in a patient Amount of disease and its complications. Determining such an effective amount is well within the capability of those skilled in the art.
  • amounts effective for therapeutic use will depend on the severity of the disease to be treated, the general state of the patient's own immune system, the general condition of the patient such as age, weight and sex, the mode of administration of the drug, and other treatments administered concomitantly etc.
  • the amino acid sequence of the PD-1 protein when referring to the amino acid sequence of the PD-1 protein (NCBI GenBank: NM_005018), it includes the full length of the PD-1 protein, or the extracellular fragment of PD-1, PD-1 ECD, or includes PD -1 Fragments of ECD; also include full-length fusion proteins of PD-1 protein or fusion proteins of PD-1 ECD, such as fragments fused with Fc protein fragments (mFc or hFc) of mouse or human IgG.
  • Fc protein fragments mFc or hFc
  • those skilled in the art understand that in the amino acid sequence of the PD-1 protein, mutations or variations (including but not limited to substitutions, deletions and/or additions) can be naturally produced or artificially introduced without affecting its biological function. Therefore, in the present invention, the term "additional protein" shall include all such sequences, including their natural or artificial variants. And, when describing the sequence fragment of PD-1 protein, it also includes the
  • lymphocyte-activation gene 3 when referring to the amino acid sequence of lymphocyte activation gene 3 (lymphocyte-activation gene 3, LAG3), it includes the full length of the LAG3 protein, or the extracellular fragment LAG3 ECD of LAG3 or a protein comprising LAG3 ECD Fragments; also include full-length fusion proteins of LAG3 protein or fusion proteins of LAG3 ECD, for example, fragments fused with Fc protein fragments (mFc or hFc) of mouse or human IgG.
  • Fc protein fragments mFc or hFc
  • first and second are for distinction in reference or clarity of expression, Does not have typical sequential implications.
  • the anti-LAG3 antibody of the present invention has superior affinity and specificity
  • bispecific antibodies of the present invention can specifically bind to LAG3 well, and can effectively block the binding of LAG3 to MHC II, and specifically release LAG3 Immunosuppression of the body;
  • the bispecific antibodies of the present invention can specifically bind to PD-1 well, and can effectively block the binding of PD-1 to PDL1. It can effectively relieve the immune suppression of the body by PD-1 and activate the immune response.
  • the bispecific antibody of the present invention completely eliminates its binding activity to Fc receptors Fc ⁇ RI, Fc ⁇ RIIb, Fc ⁇ RIIa_H131, Fc ⁇ RIIIa_V158 and/or Fc ⁇ RIIIa_F158, thereby eliminating its ADCC activity or ADCP activity.
  • the bispecific antibody of the present invention especially BS-PL022B, completely eliminates its binding activity to complement C1q, thereby eliminating its CDC activity.
  • Figure 1 The detection results of binding activity between BS-PL021A, BS-PL022B, BS-PL023C, Bs-PLV02, 14C12H1L1(hG1TM) and antigen PD-1-mFc by indirect ELISA method.
  • Figure 2 The detection results of binding activity between BS-PL021A, BS-PL022B, BS-PL023C, BS-PLV02, Relatlimab, H7L8(hG1WT) and antigen LAG3-mFc by indirect ELISA method.
  • Figure 3 The competition ELISA method to determine the activity detection results of the anti-LAG3-anti-PD-1 bispecific antibody competing with human PDL1-mFc to bind to human PD-1-mFc-Biotin.
  • Figure 4 FACS detection results of anti-LAG3-anti-PD-1 bispecific antibody binding to PD-1 on the surface of 293T-PD1 membrane.
  • Figure 5 FACS detection results of the binding activity of anti-LAG3-anti-PD-1 bispecific antibody to LAG3 on the surface of 293T-LAG3 membrane.
  • Figure 6 The detection results of the competition flow cytometry assay of the anti-LAG3-anti-PD-1 bispecific antibody and PDL1 in binding to the cell membrane surface antigen PD-1.
  • Figure 7 The detection results of competition flow cytometry of anti-LAG3-anti-PD-1 bispecific antibody and LAG3 in binding to cell membrane surface antigen MHC II.
  • Figure 8A Detection results of anti-LAG3-anti-PD-1 bispecific antibody blocking LAG3 binding to MHCII.
  • Figure 8B Detection results of anti-LAG3-anti-PD-1 bispecific antibody blocking LAG3 binding to MHCII.
  • Figure 9A Detection results of anti-LAG3-anti-PD-1 bispecific antibody blocking PD-1 binding to PD-L1.
  • Figure 9B Detection results of anti-LAG3-anti-PD-1 bispecific antibody blocking PD-1 binding to PD-L1.
  • Figure 10A Anti-LAG3-anti-PD-1 bispecific antibody simultaneously blocked the binding detection results of LAG3 and MHCII and PD-1 and PD-L1.
  • Figure 10B Anti-LAG3-anti-PD-1 bispecific antibody simultaneously blocked the binding detection results of LAG3 and MHCII and PD-1 and PD-L1.
  • Figure 11 Detection results of anti-LAG3-anti-PD-1 bispecific antibody bridging experiment.
  • Fig. 12A The biological activity detection results of anti-LAG3-anti-PD-1 bispecific antibody promoting IFN- ⁇ secretion by mixed lymphocyte reaction MLR.
  • Fig. 12B The biological activity detection results of anti-LAG3-anti-PD-1 bispecific antibody promoting IL-2 secretion by mixed lymphocyte reaction MLR.
  • Fig. 16 A graph of the detection results of the affinity constant between H7L8(hG1WT) and Fc ⁇ RIIIa_V158.
  • Fig. 20 Detection results of affinity constants between H7L8(hG1WT) and Fc ⁇ RIIa_H131.
  • Figure 26 Drug effect of anti-LAG3-anti-PD-1 bispecific antibody on BALB/c-hPD1/hLAG3 mouse CT26 xenograft tumor model. *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001, VS isotype control group (two-way ANOVA)
  • Figure 27 Effect of anti-LAG3-anti-PD-1 bispecific antibody on body weight of BALB/c-hPD1/hLAG3 mouse CT26 xenograft tumor model.
  • mice were purchased from Guangdong Medical Experimental Animal Center.
  • Nivolumab was purchased from BMS Company, batch number: ABA0330. Nivolumab is an anti-PD-1 antibody.
  • Pembrolizumab was purchased from MSD Ireland (Carlow), product number: S023942. Pembrolizumab is an anti-PD-1 antibody.
  • Relatlimab refers to the US Patent Publication: US20160326248A1 for its sequence.
  • amino acid sequence of the heavy chain refers to SEQ ID NO:1 in the patent disclosure
  • amino acid sequence of the light chain refers to SEQ ID NO:2 in the patent disclosure.
  • Relatlimab is an anti-LAG-3 antibody.
  • the 293T-PD1 cell line was constructed by Zhongshan Akefang Biomedical Co., Ltd.
  • the 293T-PD1 cell line was obtained by virus infection of HEK293T cells.
  • the virus was prepared using 3rd Generation Lentiviral Systems, see, for example, A Third Generation Lentivirus Vector with a Conditional Packaging System. Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, and Naldini L.J Virol.1998.72(11):8463-8471.
  • the lentiviral expression vector used in it is plenti6.3/V5-PD1FL-BSD (wherein PD1, Genebank ID: NM_005018; vector plenti6. 3/V5-BSD, available from Invitrogen, product number: K5315-20).
  • the 293T-LAG3 cell line was constructed by Zhongshan Akefang Biopharmaceutical Co., Ltd.
  • the 293T-LAG3 cell line was obtained by virus infection of HEK293T cells.
  • the virus was prepared using 3rd Generation Lentiviral Systems, see, for example, A Third Generation Lentivirus Vector with a Conditional Packaging System. Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, and Naldini L.J Virol.1998.72(11):8463-8471.
  • the lentiviral expression vector used in it is plenti6.3/V5-huLAG3FL-BSD (where LAG3, Genebank ID: NM_002277.4; vector plenti6.3/V5-BSD, purchased from Invitrogen, product number: K5315-20).
  • the Raji-PDL1 cell line was constructed by Zhongshan Akefang Biopharmaceutical Co., Ltd.
  • the Raji-PDL1 cell line was obtained by virus infection of Raji cells using 3rd Generation Lentiviral Systems, see, for example, A Third Generation Lentivirus Vector with a Conditional Packaging System. Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, and Naldini L.J Virol.1998.72(11):8463-8471.
  • the lentiviral expression vector used in it is plenti6.3/V5-PDL1 (wherein PDL1, Genebank ID: NP_054862.1; vector plenti6. 3/V5, purchased from Invitrogen, catalog number: K5315-20).
  • the Jurkat-NFAT-PD1-LAG3 cell line was constructed by Zhongshan Akeso Biopharmaceutical Co., Ltd.
  • the Jurkat-NFAT-PD1-LAG3 cell line is obtained from PD-1 Effector cells (CPM), manufacturer: Promega, product number: J112A) cells infected by virus, the virus is prepared using 3rd Generation Lentiviral Systems, See, for example A Third Generation Lentivirus Vector with a Conditional Packaging System. Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, and Naldini L.J Virol.1998.72(11):8463-8471.
  • the lentiviral expression vector is pCDH-huLAG3FL-RFP-NEO (wherein LAG3, Genebank ID: NM_002277.4; vector pCDH-CMV-MCS-EF1-RFP+Neo, purchased from Youbao Biology, product number: VT9005).
  • the CHO-K1-PD1 cell line was constructed by Zhongshan Akefang Biopharmaceutical Co., Ltd.
  • the CHO-K1-PD1 cell line was produced by virus infection of CHO-K1 cells using 3rd Generation Lentiviral Systems, see, for example, A Third Generation Lentivirus Vector with a Conditional Packaging System. Dull T, Zufferey R, Kelly M , Mandel RJ, Nguyen M, Trono D, and Naldini L.J Virol.1998.72(11):8463-8471.
  • the lentiviral expression vector used therein is pCDH-CMV-PD-1FL-Puro (where PD1, Genebank ID: NM_005018 ; Vector pCDH-CMV-Puro, purchased from Youbao Biology, product number: VT1480).
  • the CHO-K1-LAG3 cell line was constructed by Zhongshan Akeso Biopharmaceutical Co., Ltd.
  • the CHO-K1-LAG3 cell line was obtained by virus infection of CHO-K1 cells using 3rd Generation Lentiviral Systems, see, for example, A Third Generation Lentivirus Vector with a Conditional Packaging System. Dull T, Zufferey R, Kelly M , Mandel RJ, Nguyen M, Trono D, and Naldini L.J Virol.1998.72(11):8463-8471.
  • the lentiviral expression vector used therein is plenti6.3/V5-huLAG3FL-BSD (where LAG3, Genebank ID: NM_002277 .4; vector plenti6.3/V5-BSD, purchased from Invitrogen, product number: K5315-20).
  • the Jurkat-NFAT-CD64-CD32R cell line was constructed by Zhongshan Akefang Biopharmaceutical Co., Ltd.
  • the Jurkat-NFAT-CD64-CD32R cell line was produced by virus infection of Jurkat cells using 3rd Generation Lentiviral Systems, see, for example, A Third Generation Lentivirus Vector with a Conditional Packaging System. Dull T, Zufferey R, Kelly M , Mandel RJ, Nguyen M, Trono D, and Naldini L.J Virol.1998.72 (11): 8463-8471.
  • the lentiviral expression vector used therein is pCDH-NFAT-Hygro (wherein the carrier pCDH-Hygro is based on pCDH-CMV-MCS -EF1-Puro (purchased from Youbao Biology, article number: VT1480) based on transformation in our laboratory), pcDH-hFCGR1AFL-Neo (carrier pCDH-Neo based on pCDH-CMV-MCS-EF1-Puro (purchased from Youbao Biology, Cat.
  • VT1480 based on transformation in our laboratory) and pCDH-hFCGR2A(H167)-puro (wherein hFCGR2A(H167), Genebank ID: P12318; vector pCDH-CMV-MCS-EF1-Puro was purchased from Youbao Biology, Cat. No.: VT1480).
  • the CHO-K1-PD1-LAG3 cell line was constructed by Zhongshan Kangfang Biomedical Co., Ltd.
  • the CHO-K1-PD1-LAG3 cell line was obtained by virus infection of CHO-K1 cells.
  • the virus was prepared using 3rd Generation Lentiviral Systems, see, for example, A Third Generation Lentivirus Vector with a Conditional Packaging System. Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, and Naldini L.J Virol.1998.72(11):8463-8471.
  • the lentiviral expression vector used in it is pCDH-hPD1-FL-puro (where PD-1, Genebank ID : NM_005018; its vector pCDH-CMV-MCS-EF1-Puro was purchased from Youbao Biology, article number: VT1480) and plenti6.3/V5-huLAG3FL-BSD (where LAG3, Genebank ID: NM_002277.4; vector plenti6.3/ V5-BSD, purchased from Invitrogen, catalog number: K5315-20)
  • the nucleotide sequence of the heavy chain variable region H7v of H7L9 is identical to the nucleotide sequence of the heavy chain variable region H7v of H7L8, namely SEQ ID NO:1.
  • the amino acid sequence of the heavy chain variable region H7v of H7L9 is identical to the amino acid sequence of the heavy chain variable region H7v of H7L8, namely SEQ ID NO:2.
  • the nucleotide sequence of the heavy chain variable region H7v of H7L10 is identical to the nucleotide sequence of the heavy chain variable region H7v of H7L8, namely SEQ ID NO:1.
  • the amino acid sequence of the heavy chain variable region H7v of H7L10 is identical to the amino acid sequence of the heavy chain variable region H7v of H7L8, namely SEQ ID NO:2.
  • amino acid sequence of the CDR of antibody H7L8 is as follows (according to the IMGT numbering system):
  • HCDR1 GGSISDYY (SEQ ID NO: 5);
  • HCDR2 INHRGTT (SEQ ID NO: 6);
  • HCDR3 AFGYSDYEYDWFDP (SEQ ID NO: 7);
  • LCDR1 QTISSY (SEQ ID NO: 8);
  • LCDR2 DAS (SEQ ID NO:9);
  • LCDR3 QQRSNWPIT (SEQ ID NO: 10).
  • amino acid sequence of the CDR of antibody H7L9 is as follows (according to the IMGT numbering system):
  • HCDR1 GGSISDYY (SEQ ID NO: 5);
  • HCDR2 INHRGTT (SEQ ID NO: 6);
  • HCDR3 AFGYSDYEYDWFDP (SEQ ID NO: 7);
  • LCDR1 QTISSY (SEQ ID NO: 8);
  • LCDR2 DGS (SEQ ID NO: 46);
  • LCDR3 QQRSNWPLT (SEQ ID NO: 47).
  • amino acid sequence of the CDR of antibody H7L10 is as follows (according to the IMGT numbering system):
  • HCDR1 GGSISDYY (SEQ ID NO: 5);
  • HCDR2 INHRGTT (SEQ ID NO: 6);
  • HCDR3 AFGYSDYEYDWFDP (SEQ ID NO: 7);
  • LCDR1 QSISSY (SEQ ID NO: 48);
  • LCDR2 DGS (SEQ ID NO: 46);
  • LCDR3 QQRSNWPIT (SEQ ID NO: 10).
  • the heavy chain cDNA sequence of H7L8 (hG1WT) (the variable region coding sequence is shown in SEQ ID NO: 1; the constant region is Ig gamma-1 chain C region, SEQ ID NO: 39) and the cDNA sequence of the light chain (can be
  • the variable region coding sequence is shown in SEQ ID NO: 3; the constant region is P01834.1 (human Ig kappa chain C region, SEQ ID NO: 40) respectively cloned into pUC57simple (provided by GenScript) vector, respectively to obtain pUC57simple -H7 and pUC57simple-L8 plasmids.
  • Plasmids pUC57simple-H7 and pUC57simple-L8 were respectively digested (HindIII & EcoRI), and the heavy and light chains recovered by electrophoresis were subcloned into pcDNA3.1 vectors, and the recombinant plasmids were extracted and co-transfected into 293F Cells. After 7 days of cell culture, the culture medium was centrifuged at high speed, the supernatant was concentrated, and then loaded onto the HiTrap MabSelect SuRe column. The protein was eluted with Elution Buffer in one step, and the target sample was recovered and replaced with PBS.
  • H7L8 H7L8 (hG1WT)
  • the present inventor introduced a point mutation (L234A) from leucine to alanine (L234A) at the No. 234 position of its heavy chain (according to the EU numbering system, the same below), No. 235 A point mutation from leucine to alanine (L235A) was introduced at site No. 237, and a point mutation from glycine to alanine (G237A) was introduced at site 237 to obtain the humanized antibody H7L8 (hG1TM ).
  • the amino acid sequence of the heavy chain H7 (hG1TM) of H7L8 (hG1TM) is shown in SEQ ID NO: 11; the amino acid sequence of the light chain L8 is shown in SEQ ID NO: 12.
  • the preparation of the humanized antibody H7L8 can refer to the method in step 2 above.
  • H7L8 On the basis of H7L8 (hG1WT), the inventors used the Ig gamma-4 chain C region as the heavy chain constant region on the basis of H7L8 (hG1WT), and introduced the benzene A point mutation from alanine to alanine (F234A), and a point mutation from leucine to alanine at site 235 (L235A) were used to obtain the humanized antibody H7L8 (hG4DM) with mutations in the constant region.
  • the heavy chain amino acid sequence of H7L8 (hG4DM) is shown in SEQ ID NO: 13; the light chain amino acid sequence is shown in SEQ ID NO: 12.
  • the amino acid sequence of the light chain L8 of H7L8 (hG4DM) is identical to the amino acid sequence of the light chain of H7L8 (hG1TM), which is SEQ ID NO: 12.
  • the heavy chain cDNA sequences of H7L8 (hG4WT), H7L9 (hG4WT) and H7L10 (hG4WT) (the variable region coding sequence is shown in SEQ ID NO: 1; the constant region is Ig gamma-4 chain C region, as shown in SEQ ID NO :45) and the cDNA sequence of H7L8 (hG4WT) light chain (variable region coding sequence is shown in SEQ ID NO:3; constant region is human Ig kappa chain C region, as shown in SEQ ID NO:40),
  • the cDNA sequence of H7L9 (hG4WT) light chain (variable region coding sequence is shown in SEQ ID NO: 42; the constant region is human Ig kappa chain C region, as shown in SEQ ID NO: 40), H7L10 (hG4WT) light chain
  • the cDNA sequence (the variable region coding sequence is shown in SEQ ID NO: 44; the constant region is human Ig
  • Plasmids pUC57simple-H7 and pUC57simple-L8, pUC57simple-L9, pUC57simple-L10 were digested with restriction enzymes (HindIII&EcoRI), and the heavy and light chains recovered by electrophoresis were respectively subcloned into pcDNA3.1 vectors, and the recombinant plasmids were extracted and co-transfected into 293F cell. After the cells were cultured for 7 days, the culture medium was concentrated by high-speed centrifugation and the supernatant was concentrated and loaded onto the HiTrap MabSelect SuRe column. The protein was eluted in one step with Elution Buffer, and the target sample was recovered and replaced with PBS.
  • restriction enzymes HindIII&EcoRI
  • H7L8 H7L9
  • H7L10 H7L10
  • H7L8 H7L9 (hG4WT) or H7L10 (hG4WT) light chain constant region amino acid sequence:
  • amino acid sequences of the heavy chain and light chain of the anti-PD-1 antibody 14C12 and its humanized antibody 14C12H1L1, as well as the coding nucleic acid sequence are completely identical to 14C12 and 14C12H1L1 in Chinese Patent Publication CN 106967172A (or CN 106977602A).
  • the CDRs of antibodies 14C12 and 14C12H1L1 are identical, as follows (according to the IMGT numbering system):
  • LCDR1 QDINTY (SEQ ID NO: 29)
  • LCDR2 RAN (SEQ ID NO:30)
  • the inventors kept the antibody variable region unchanged, adopted Ig gamma-4 chain C region as the heavy chain constant region, and introduced phenylalanine at the No. 234 site of the heavy chain constant region A point mutation to alanine (F234A), and a point mutation from leucine to alanine at position 235 (L235A) were used to obtain the humanized antibody 14C12H1L1 (hG4DM) with mutations in the constant region.
  • the amino acid sequence of the heavy chain 14C12H1 (hG4DM) of 14C12H1L1 (hG4DM) is shown in SEQ ID NO:32; the amino acid sequence of the light chain is shown in SEQ ID NO:25.
  • the amino acid sequence of the light chain of 14C12H1L1 is identical to the amino acid sequence of the light chain 14C12L1 of 14C12H1L1, which is SEQ ID NO:25.
  • L234A point mutation from leucine to alanine at the 234th position in the hinge region of the heavy chain according to the EU numbering system.
  • L235A leucine to alanine
  • G237A glycine to alanine
  • hG1TM mutant humanized 14C12H1L1
  • the nucleotide sequence of the light chain of 14C12H1L1 is the same as SEQ ID NO: 24.
  • the amino acid sequence of the light chain of 14C12H1L1 (hG1TM) is the same as the amino acid sequence of the light chain 14C12L1 of 14C12H1L1, namely SEQ ID NO:25.
  • the structural patterns of the bifunctional antibodies BS-PL021A, Bs-PL022B, BS-PL023C, and Bs-PLV02 in the present invention belong to the Morrison pattern (IgG-scFv), that is, the C-terminals of the two heavy chains of an IgG antibody are connected to the other A scFv fragment of an antibody, the main composition of its heavy chain and light chain is designed as shown in Table 1 below.
  • variable region of the corresponding heavy chain or the variable region of the corresponding light chain is the full length including the constant region.
  • the heavy chain cDNA sequences and light chain cDNA sequences of Bs-PL021A, Bs-PL022B, Bs-PL023C, and Bs-PLV02 were respectively cloned into pUC57simple (provided by GenScript) vectors to obtain pUC57simple-Bs-PL021AH/pUC57simple -Bs-PL021AL, pUC57simple-Bs-PL022BH/pUC57simple-Bs-PL022BL, pUC57simple-Bs-PL023CH/pUC57simple-Bs-PL023CL, pUC57simple-Bs-PLV02H/pUC57simple-Bs-PLV02L, pUC57simple/Bi-PGV02 - PGV02 plasmid.
  • pUC57simple provided by GenScript
  • the culture medium was concentrated by high-speed centrifugation and the supernatant was concentrated and loaded onto the HiTrap MabSelect SuRe column.
  • the protein was eluted in one step with Elution Buffer, and the target sample was recovered and replaced with PBS.
  • amino acid sequences of the fusion proteins PD-1-mFc, PD-1-hFc, and PDL1-hFc in this preparation example and their encoding nucleic acid sequences are respectively the same as those of PD-1-mFc, PD-1-mFc, PD-1-hFc and PDL1-hFc are the same.
  • Fusion proteins PD-1-mFc, PD-1-hFc and PDL1-hFc were prepared.
  • Human anti-Hen Egg Lysozyme IgG human anti-Hen Egg Lysozyme IgG, anti-HEL, namely human IgG, hIgG for short
  • its sequence comes from Affinity maturation increases the stability and plasticity of the Fv domain of anti published by Acierno et al. - Variable region sequence of Fab F10.6.6 sequence in protein antibodies study (Acierno et al. J Mol Biol. 2007; 374(1):130-46.).
  • the preparation method is as follows:
  • Experimental example 1 Determination of the binding activity of anti-LAG3-anti-PD-1 bispecific antibody to antigen by ELISA method
  • the indirect ELISA method was used to measure the binding activity of BS-PL021A, BS-PL022B, BS-PL023C, Bs-PLV02, 14C12H1L1(hG1TM) and antigen PD-1-mFc respectively.
  • the specific method is as follows:
  • Table 2 ELISA detection of binding of BS-PL021A, BS-PL022B, BS-PL023C, Bs-PLV02, 14C12H1L1(hG1TM) to human PD-1-mFc
  • BS-PL021A, BS-PL022B, BS-PL023C, Bs-PLV02, 14C12H1L1(hG1TM) can effectively bind to the antigen human PD-1-mFc, and the binding efficiency is dose-dependent.
  • the binding efficiencies EC 50 of antibodies BS-PL021A, BS-PL022B, BS-PL023C, Bs-PLV02, 14C12H1L1 (hG1TM) (as a control) (as a control) were 0.066nM, 0.074 nM, 0.046nM, 0.103nM, 0.02nM.
  • the indirect ELISA method was used to measure the binding activities of BS-PL021A, BS-PL022B, BS-PL023C, Bs-PLV02, Relatlimab, H7L8(hG1WT) and antigen LAG3-mFc respectively.
  • Table 3 Binding of BS-PL021A, BS-PL022B, BS-PL023C, Bs-PLV02, Relatlimab, H7L8(hG1WT) to LAG3-mFc detected by ELISA
  • the curve simulation calculation obtained the antibodies BS-PL021A, BS-PL022B, BS-PL023C, Bs-PLV02, Relatlimab (as a positive control), H7L8 (hG1WT) (as a positive control)
  • the binding efficiencies EC 50 of control were 0.073nM, 0.081nM, 0.377nM, 0.685nM, 0.106nM, 0.045nM, respectively.
  • Human PDL1-mFc (PD-L1 Genbank ID: NP_054862.1, mFc SEQ ID NO:) was coated on the microtiter plate at 2 ⁇ g/mL and incubated overnight at 4°C. After the incubation, the ELISA plate was blocked with 1% BSA in PBS at 37°C for 2 hours. After the blocking, the plate was washed three times and patted dry.
  • the test results are shown in Figure 3.
  • the OD values of each dose are shown in Table 4.
  • the curve simulates the binding efficiency of the antibody blocking human PD-1-mFc-Biotin and its ligand human PDL1-mFc to obtain the competitive binding EC 50 (Table 4).
  • Table 4 Activity detection results of BS-PL021A, BS-PL022B, BS-PL023C, Bs-PLV02, 14C12H1L1(hG1TM) competing with human PDL1-mFc to bind to human PD-1-mFc-Biotin
  • the efficiency of BS-PL021A, BS-PL022B, and BS-PL023C in blocking the binding of human PD-1-mFc-Biotin to its ligand human PDL1-mFc is basically equivalent to that of 14C12H1L1 (hG1TM).
  • Collect 293T-PD1 cells in the logarithmic growth phase transfer the cells to a conical 96-well plate at 3 ⁇ 10 5 cells/well, add 100 ⁇ L of 1% PBSA, centrifuge at 350 ⁇ g for 5 minutes, and remove the supernatant.
  • Add 100 ⁇ L of antibodies diluted in PBSA final concentrations are 300, 100, 33.3, 11.1, 3.7, 1.23, 0.123 and 0.0123 nM), mix gently and incubate on ice for 1 h.
  • Table 5 FACS detection of PD-1 binding activity on the cell surface of 14C12H1L1(hG1TM), BS-PL021A, BS-PL022B, Bs-PLV02, Bi-PGV02, Nivolumab, Pembrolizumab 293T-PD1
  • the EC 50 of 14C12H1L1(hG1TM), BS-PL021A, BS-PL022B, Bs-PLV02, Bi-PGV02, Nivolumab, and Pembrolizumab binding to 293T-PD1 cells were 5.351nM, 6.851nM, 6.066nM, 6.866nM, 7.206nM, 3.073nM, 3.970nM.
  • the 293T-LAG3 cells in the logarithmic phase were routinely trypsinized, and the cells were transferred to a conical 96-well plate at 3 ⁇ 10 5 cells/well, and 100 ⁇ L of 1% PBSA was added, centrifuged at 350 ⁇ g for 5 minutes, and the supernatant was discarded. Add 100 ⁇ L of antibodies diluted in 1% PBSA (final concentrations are 300, 100, 33.3, 11.1, 3.7, 1.23, 0.123, 0.0123 and 0.00123 nM), mix well and incubate on ice for 1 h.
  • Table 6 FACS detection of the binding activity of BS-PL022B, Relatlimab and LAG33 on the surface of 293T-LAG3 cells.
  • the 293T-PD1 cells were routinely digested, transferred to a conical bottom 96-well plate at 3 ⁇ 10 5 cells/well, and washed by centrifugation by adding 100 ⁇ L of 1% PBSA.
  • Add corresponding serially diluted antibodies (final concentrations are 300, 100, 33.3, 11.1, 3.7, 1.23, 0.123 and 0.0123 nM, respectively), 100 ⁇ L per sample, and incubate on ice for 30 min.
  • Table 7 Fluorescence intensity analysis of FACS detection of Nivolumab, Pembrolizumab, 14C12H1L1(hG1TM), BS-PL022B competitively binding to 293T-PD-1 surface antigen
  • Table 8 Fluorescence intensity analysis of the competitive binding of Relalimab and BS-PL022B to Raji cell surface antigen detected by FACS
  • Anti-LAG3-anti-PD-1 bispecific antibody blocks the binding of LAG3 and MHCII
  • Place SEE Staphylococcal enterotoxin E (final concentration 0.05ng/mL, Toxin Technology, product number: ET404) and Raji cells in a 37°C, 5% CO 2 incubator and incubate for 30min; 10 4 /well, 25 ⁇ L/well was added to a 96-well plate, and the final volume of the system was 80 ⁇ L; after mixing, place it in a 37°C, 5% CO 2 incubator and incubate for 16h. Take out the culture plate, equilibrate to room temperature, add 80 ⁇ L/well of Bright-Glo TM Luciferase Assay System (Promega, product number: E2650), incubate in the dark for 2 minutes, and read the RLU value.
  • Bright-Glo TM Luciferase Assay System Promega, product number: E2650
  • Anti-LAG3-anti-PD-1 bispecific antibody blocks the binding of PD-1 and PD-L1
  • PDL1aAPC/CHO-K1 cells (Promega, product number: J1081A) 4 ⁇ 104 cells/well were seeded in a 96-well flat-bottomed black plate (Corning, model: 3916) and cultured overnight, 100 ⁇ L per well (the medium was the growth medium of the cells) : Ham F-12+10% FBS).
  • Anti-LAG3-anti-PD-1 bispecific antibody simultaneously blocks the combination of LAG-3 and MHCII as well as PD-1 and PD-L1
  • Pembrolizumab, Relatlimab, 14C12H1L1(hG1TM), 14C12H1L1(hG1TM)+Relatlimab, and Bs-PL022B can simultaneously block the binding of PD-1 and PD-L1 and the binding of LAG3 and MHCII, and the blocking ability of Bs-PL022B is stronger than that of other antibodies .
  • CHO-K1-PD1 Cell Resource Center, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, article number: 3111C0001CCC000004
  • CHO-K1-PD1 constructed by Akeso Bio
  • CHO-K1-LAG3 constructed by Akeso Bio
  • CFSE CFSE Cell Division Tracker Kit, Biolegend, Cat. No. 423801
  • treatment concentration 1 ⁇ M, 1 mL/10 ⁇ 10 6 cells CHO-K1-LAG3 cells were stained with Far red (Thermofisher, Cat. No.
  • CHO-K1 cells were stained with Far red or CFSE, and stained in the incubator for 20 minutes; complete medium was used to stop staining, centrifuged at 170 ⁇ g for 5 minutes, and the supernatant was discarded. Then add the complete medium to the incubator and incubate for 10 minutes, centrifuge at 170 ⁇ g for 5 minutes to discard the supernatant, repeat washing once, and resuspend the complete medium for counting.
  • CHO-K1-PD1, CHO-K1-LAG3 and CHO-K1 cells were transferred to conical 96-well plates at 1.5 ⁇ 105 cells/well, and buffer (PBS+1% human serum) was added ( Human serum, Zhongke Chenyu, Cat.
  • the anti-LAG3-anti-PD-1 bispecific antibody can simultaneously bind to CHO-K1-PD1 and CHO-K1-LAG3 cells, and bridge the two cells, while 14C12H1L1(hG1TM), Relatlimab even Combination doesn't work either.
  • Experimental example 7 Mixed lymphocyte reaction MLR detection anti-LAG3-anti-PD-1 bispecific antibody promotes IFN- ⁇ and the biological activity of IL-2 secretion
  • Raji-PDL1 cells were conventionally subcultured; revived PBMCs were cultured with 10 mL of 1640 complete medium, and stimulated with 0.5 ⁇ g/mL SEB (Staphylococcal enterotoxin B) (Denotec, product number: S010201) for two days.
  • SEB Staphylococcal enterotoxin B
  • Raji-PDL1 cells were treated with MMC (Stressmarq, product number: SIH-246-10MG) at a final concentration of 2 ⁇ g/mL, and placed in a 37°C, 5% CO2 incubator for 1 hour; PBMC and Raji-PDL1 cells treated with MMC for 1 hour were washed twice with PBS; after resuspended in complete medium and counted, each 10 ⁇ 104 cells/well were added to a U-shaped 96-well plate (Corning, model: 3799) for co-cultivation .
  • MMC Stressmarq, product number: SIH-246-10MG
  • Antibodies were added according to the experimental design (final concentrations of 300nM, 30nM, 3nM), and co-cultivated in the incubator for 3 days; after 3 days, centrifuged at 1200rpm for 5min, the cell culture supernatant was collected, and IFN- ⁇ was detected by ELISA.
  • the mixed culture of human PBMC and Raji-PDL1 cells can significantly promote the secretion of IFN- ⁇ of PBMC, and the addition of antibodies in the mixed culture system can significantly induce PBMC to further secrete IFN- ⁇ , and promote IFN- ⁇ secretion.
  • the activity of BS-PL022B antibody was better than that of PD-1 single-target antibody 14C12H1L1 (hG1TM) and LAG-3 single-target control antibody Relatlimab.
  • BS-PL022B has better IFN- ⁇ secretion-promoting potential at two different antibody concentration levels (30nM and 300nM).
  • Raji-PDL1 cells were routinely subcultured; revived PBMCs, cultured with 10 mL of 1640 complete medium, and added 0.5 ⁇ g/mL of SEB (Staphylococcal enterotoxin B) (Denotec, product number: S010201) to stimulate for two days.
  • Raji-PDL1 cells were treated with MMC (Stressmarq, product number: SIH-246-10MG) at a final concentration of 2 ⁇ g/mL, and placed in a 37° C., 5% CO 2 carbon dioxide incubator for 1 hour.
  • Antibodies were added according to the experimental design (final concentrations of 300nM, 30nM, 3nM), and co-cultured for 3 days; after 3 days, centrifuged at 1200rpm for 5min, the cell culture supernatant was collected, and IL-2 was detected by ELISA.
  • the mixed culture of human PBMC and Raji-PDL1 cells can promote the secretion of IL-2 in PBMC to a certain extent, and adding antibodies to the mixed culture system can significantly induce PBMC to further secrete IL-2, which has a significant effect.
  • BS-PL022B compared with the PD-1 single-target antibody 14C12H1L1(hG1TM) and the LAG-3 single-target control antibody Relatlimab, BS-PL022B has the same effect at three different antibody concentration levels. Better IL-2 secretion-promoting potential. Even compared with the combination of 14C12H1L1(hG1TM) and Relatlimab, BS-PL022B has better potential to promote IL-2 secretion at three different antibody concentration levels.
  • the Fc receptor Fc ⁇ RI (also known as CD64) can bind to the Fc end of an IgG antibody and participate in antibody-dependent cell-mediated cytotoxicity (ADCC).
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • the ability of a therapeutic antibody to bind to Fc receptors affects the safety and efficacy of that antibody.
  • the Fortebio Octet molecular interaction instrument was used to detect the affinity constant between BS-PL022B and Fc ⁇ RI to evaluate the ADCC activity of the antibody.
  • the sample dilution buffer is a solution of PBS, 0.02% Tween-20, 0.1% BSA, and pH 7.4.
  • a solution of Fc ⁇ RI (purchased from Sinobio) at a concentration of 1 ⁇ g/mL was added to the HIS1K sensor for a fixation time of 50 s, so that Fc ⁇ RI was immobilized on the surface of the sensor.
  • the determination of the binding and dissociation parameters of the antibody to Fc ⁇ RI was carried out in buffer, and the antibody concentration was 3.12-50 nM (two-fold serial dilution).
  • the vibration rate of the sample plate is 1000 rpm
  • the detection temperature is 30° C.
  • the frequency is 5.0 Hz.
  • the data were analyzed with a 1:1 model fit to obtain affinity constants.
  • N/A means that the antibody did not bind to the antigen or the binding signal was extremely low, and the results were not analyzed, so the corresponding data could not be obtained.
  • H7L8 (hG1WT) can bind to Fc ⁇ RI with an affinity constant of 6.59E-09M.
  • BS-PL022B did not bind to Fc ⁇ RI or the binding signal was extremely low, and the results were not analyzed, so corresponding data could not be obtained.
  • the Fc receptor Fc ⁇ RIIIa_V158 (also known as CD16a_V158) can bind to the Fc end of an IgG antibody to mediate the ADCC effect.
  • the Fortebio Octet molecular interaction instrument was used to detect the affinity constant between BS-PL022B and Fc ⁇ RIIIa_V158 to evaluate the ADCC activity of the antibody.
  • the sample dilution buffer is PBS, 0.02% Tween-20, 0.1% BSA, pH7.4.
  • 5 ⁇ g/mL Fc ⁇ RIIIa_V158 is fixed on the HIS1K sensor, and the time 60s, the sensor is equilibrated in the buffer for 60s, Fc ⁇ RIIIa_V158 immobilized on the sensor binds to each antibody, the antibody concentration is 31.25-500nM (two-fold dilution), the time is 60s, the antibody is dissociated in the buffer, the time is 60s.
  • the vibration rate of the sample plate is 1000 rpm, the detection temperature is 30° C., and the frequency is 5.0 Hz.
  • the data were analyzed with a 1:1 model fit to obtain affinity constants.
  • Antibody K D (M) kon(1/Ms) SE(kon) kdis(1/s) SE(kdis) Rmax(nm) BS-PL022B N/A N/A N/A N/A N/A N/A H7L8(hG1WT) 8.77E-08 4.95E+05 2.36E+04 4.34E-02 7.00E-04 0.18-0.59
  • N/A means that the antibody did not bind to the antigen or the binding signal was extremely low, and the results were not analyzed, so the corresponding data could not be obtained.
  • H7L8 (hG1WT) could bind to Fc ⁇ RIIIa_V158 with an affinity constant of 8.77E-08M.
  • BS-PL022B did not analyze the results because it did not bind to Fc ⁇ RIIIa_V158 or the binding signal was extremely low.
  • Fc receptor Fc ⁇ RIIIa_F158 (also known as CD16a_F158) can bind to the Fc end of IgG antibody to mediate ADCC.
  • CD16a_F158 the Fortebio Octet molecular interaction instrument was used to detect the affinity constant between BS-PL022B and Fc ⁇ RIIIa_F158 to evaluate the ADCC activity of each antibody.
  • the sample dilution buffer is PBS, 0.02% Tween-20, 0.1% BSA, pH7.4.
  • 5 ⁇ g/mL Fc ⁇ RIIIa_F158 is immobilized on the HIS1K sensor , the time is 120s, the sensor is equilibrated in the buffer for 60s, the Fc ⁇ RIIIa_F158 immobilized on the sensor is bound to each antibody, the antibody concentration is 31.25-500nM (two-fold dilution), the time is 60s, the antibody is dissociated in the buffer, the time is 60s.
  • the vibration rate of the sample plate is 1000 rpm, the detection temperature is 30° C., and the frequency is 5.0 Hz.
  • the data were analyzed with a 1:1 model fit to obtain affinity constants.
  • Antibody K D (M) kon(1/Ms) SE(kon) kdis(1/s) SE(kdis) Rmax(nm) BS-PL022B N/A N/A N/A N/A N/A N/A N/A N/A N/A
  • N/A means that the antibody did not bind to the antigen or the binding signal was extremely low, and the results were not analyzed, so the corresponding data could not be obtained.
  • the Fc receptor Fc ⁇ RIIa_H131 (also known as CD32a_H131) can bind to the Fc end of an IgG antibody and participate in antibody-dependent cell-mediated phagocytosis (ADCP) or antibody-dependent cell-mediated cytotoxicity (ADCC).
  • ADCP antibody-dependent cell-mediated phagocytosis
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • the ability of a therapeutic antibody to bind to Fc receptors affects the safety and efficacy of that antibody.
  • the Fortebio Octet molecular interaction instrument was used to detect the affinity constant between BS-PL022B and Fc ⁇ RIIa_H131, so as to evaluate the binding ability of each antibody to be tested to the Fc receptor.
  • the sample dilution buffer is PBS, 0.02% Tween-20, 0.1% BSA, pH7.4. 5 ⁇ g/mL of Fc ⁇ RIIa_H131 is fixed on NTA
  • the immobilized height is about 1.0nm, and the sensor is equilibrated in the buffer for 60s.
  • the Fc ⁇ RIIa_H131 immobilized on the sensor binds to each antibody, the antibody concentration is 12.5-200nM (two-fold serial dilution), and the time is 60s.
  • the antibody is in the buffer Dissociation, time 60s.
  • the vibration rate of the sample plate is 1000 rpm, the detection temperature is 30° C., and the frequency is 5.0 Hz.
  • the data were analyzed with a 1:1 model fit to obtain affinity constants.
  • N/A means that the antibody did not bind to the antigen or the binding signal was extremely low, and the results were not analyzed, so the corresponding data could not be obtained.
  • Fc receptor Fc ⁇ RIIb (also known as CD32b) can bind to the Fc end of IgG antibodies.
  • the Fortebio Octet molecular interaction instrument was used to detect the affinity constant of each antibody to be tested and Fc ⁇ RIIb to evaluate the binding ability of BS-PL022B to Fc receptors.
  • the sample dilution buffer is PBS, 0.02% Tween-20, 0.1% BSA, pH7.4. 5 ⁇ g/mL Fc ⁇ RIIb is immobilized on NTA On the sensor, the fixed height is about 1.0nm. The sensor is equilibrated in the buffer for 60s. The hFCGR2B-his immobilized on the sensor is bound to each antibody. The antibody concentration is 12.5-200nM (two-fold serial dilution) for 60s. Dissociation in the liquid, time 60s. The vibration rate of the sample plate is 1000 rpm, the detection temperature is 30° C., and the frequency is 5.0 Hz. The data were analyzed with a 1:1 model fit to obtain affinity constants.
  • Antibody K D (M) kon(1/Ms) SE(kon) kdis(1/s) SE(kdis) Rmax(nm) BS-PL022B N/A N/A N/A N/A N/A H7L8(hG1WT) 1.21E-07 3.74E+05 3.48E+04 4.53E-02 1.28E-03 0.12-0.34
  • N/A means that the antibody did not bind to the antigen or the binding signal was extremely low, and the results were not analyzed, so the corresponding data could not be obtained.
  • H7L8 (hG1WT) can bind to Fc ⁇ RIIb with an affinity constant of 1.21E-07M.
  • BS-PL022B did not bind to Fc ⁇ RIIb or had a very low binding signal, so the results were not analyzed, so corresponding data could not be obtained.
  • Serum complement C1q can bind to the Fc end of an IgG antibody to mediate the CDC effect, and the ability of a therapeutic antibody to bind to C1q affects the safety and effectiveness of the antibody.
  • the Fortebio Octet molecular interaction instrument was used to detect the affinity constant between BS-PL022B and C1q to evaluate the CDC activity of the antibody.
  • the Fortebio Octet molecular interaction instrument detects the affinity constant of the corresponding antibody and C1q.
  • the experimental method is briefly described as follows: the sample dilution buffer is PBS, 0.02% Tween-20, 0.1% BSA, pH7.4. 50 ⁇ g/mL of antibody is immobilized on the FAB2G sensor Above, the fixed height is about 2.0nm, the sensor is equilibrated in the buffer for 60s, the antibody immobilized on the sensor is combined with the antigen C1q, the antigen concentration is 0.625-10nM (two-fold serial dilution), the time is 60s, the antigen and antibody are in the buffer Dissociation, time 60s.
  • the vibration rate of the sample plate is 1000 rpm
  • the detection temperature is 30 degrees
  • the detection frequency is 5.0 Hz.
  • the data were analyzed with a 1:1 model fit to obtain affinity constants.
  • the data acquisition software is Fortebio Data Acquisition 7.0
  • the data analysis software is Fortebio Data Analysis 7.0.
  • Antibody K D (M) kon(1/Ms) SE(kon) kdis(1/s) SE(kdis) Rmax(nm) BS-PL022B N/A N/A N/A N/A N/A H7L8(hG1WT) 1.75E-09 2.05E+06 3.27E+04 3.58E-03 5.24E-05 0.56-0.71
  • N/A means that the antibody did not bind to the antigen or the binding signal was extremely low, and the results were not analyzed, so the corresponding data could not be obtained.
  • CT26 colon cancer cells purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.
  • hPD1/hLAG3 mice purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.
  • the grouping day was defined as D0 day.
  • the administration method was intraperitoneal injection (ip), administered twice a week (BIW), and administered 6 times in total. See Table 18 for modeling and specific administration methods. After administration, the length and width of the tumors in each group were measured, and the tumor volume was calculated.
  • Table 18 Dosing regimen of anti-LAG3-anti-PD-1 bispecific antibody in the treatment of BALB/c-hPD1/hLAG3 mouse CT26 colon cancer xenograft model
  • the doses of isotype control 15mg/kg, Relatlimab 15mg/kg and BS-PL022B 20mg/kg are equimolar concentrations; the doses of Relatlimab 3mg/kg and BS-PL022B 4mg/kg are equimolar concentrations.

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Abstract

提供了一种抗LAG3抗体的双特异性抗体、其药物组合物及用途，属于生物医药领域。具体地，所述双特异性抗体，其包括第一蛋白功能区和第二蛋白功能区，其中：所述第一蛋白功能区靶向LAG3，所述第二蛋白功能区靶向不同于LAG3的靶点，其中，所述第一蛋白功能区为抗LAG3抗体或其抗原结合片段，包含重链可变区和轻链可变区，其中，所述重链可变区包含：氨基酸序列分别如SEQ ID NOs:5-7所示的HCDR1-HCDR3；并且所述轻链可变区包含：氨基酸序列分别如SEQ ID NOs:8-10所示的LCDR1-LCDR3。所述双特异性抗体具有优越的亲和力和特异性，具有良好的应用前景。

Description

抗LAG3双特异性抗体、药物组合物及用途 技术领域
本发明属于生物医药领域，涉及一种抗LAG3抗体的双特异性抗体、其药物组合物及用途。具体地，所述双特异性抗体为一种抗LAG3-抗PD-1双特异性抗体。
背景技术
肿瘤尤其是恶性肿瘤是当今世界严重危害人类健康的疾病，在各种疾病所致死亡中高居第二位。而且近年来，其发病率呈明显上升趋势。恶性肿瘤治疗效果差，晚期转移率高，预后多不佳。目前临床上所采用的常规治疗方法如放、化疗和手术治疗虽然在很大程度上缓解了病痛，延长了生存时间，但这些方法均存在很大的局限性，其疗效难以进一步提高。
淋巴细胞活化基因3(lymphocyte-activation gene 3，LAG3)，即CD223，是一种由498个氨基酸组成的Ⅰ型跨膜蛋白，属于免疫球蛋白超家族(immunoglobulin superfamily，IgSF)成员。LAG3主要表达于活化的CD4 +T细胞和CD8 +T细胞，另外自然杀伤(natural killer，NK)细胞、B细胞、调节性T细胞(regulatory T cell，Treg)和浆细胞样树突状细胞(plasmacytoid dendritic cells，pDC)等细胞也表达LAG3。(Ruffo Elisa,Wu Richard C,Bruno Tullia C et al.Lymphocyte-activation gene 3(LAG3):The next immune checkpoint receptor.[J].Semin Immunol,2019,42:101305.)。
LAG3分子基因定位于人12号染色体(20p13.3)上，与CD4分子基因相邻，二者具有相同的外显子和内含子。虽然二者的氨基酸序列同源性只有约20％，但LAG3分子与CD4分子在结构上具有较高的相似性。主要组织相容性复合体Ⅱ类分子(MHC Ⅱ)、肝窦内皮细胞凝集素(LSECtin)分子和半乳糖凝集素3(galectin-3)分子是LAG3分子的相关配体。MHC Ⅱ类分子是LAG3的主要配体，其与MHC Ⅱ类分子的亲和力(Kd：60nmol·L-1)是CD4分子的100倍，表明LAG3分子能够有效竞争CD4与MHC Ⅱ类分子的结合，抑制T细胞活化。
肿瘤微环境中，T细胞活化24小时后便能检测到免疫抑制分子LAG3分子的表达，进而导致T细胞失能或者凋亡。LAG3分子通过其D1结构域(含有1个富含脯氨酸的环状结构)形成二聚体分子与CD4 +T细胞活化第一信号轴“CD3-TCR-MHC  Ⅱ”中MHC Ⅱ分子特异性结合，一方面阻断T细胞活化的信号转导通路，另一方面LAG3分子胞内段(KIEELE基序)产生免疫抑制信号下调CD4 +T细胞活性。LAG3分子能促进Treg细胞分化，参与信号转导及转录激活因子5下游信号，从而增强Treg细胞的抑制作用，这是肿瘤逃逸免疫系统杀伤的机制之一(Andrews Lawrence P,Marciscano Ariel E,Drake Charles G et al.LAG3(CD223)as a cancer immunotherapy target.[J].Immunol Rev,2017,276:80-96.)。
多项研究表明，LAG3在各种恶性肿瘤的肿瘤浸润性CD8 +T细胞中过表达。例如卵巢癌中，肿瘤浸润的纽约食管鳞状细胞癌1抗原(NY-ESO-1)特异性CD8 +T细胞表达高水平的PD-1和LAG3，产生IFN-γ和TNF-α的能力降低，进而导致淋巴细胞失活。Galectin-3和LSECtin主要与LAG3相互作用来调节CD8 +T细胞的活化与功能。另外从黑色素瘤转移患者中分离的黑色素瘤抗原特异性T细胞在LAG3及其他免疫检查点分子CTLA-4和TIM-3表达上调显著。(刘昊,李新颖,罗龙龙,等.淋巴细胞活化基因3分子生物学功能及其抗体药物临床应用研究进展[J].中国药理学与毒理学杂志,2019,33(01):70-78.)。
目前已有多种LAG3抗体药物进入临床研究阶段，其中，百时美施贵宝的Relatlimab进展最快，目前已开展了10个临床研究，绝大多数是以Relatlimab与纳武单抗进行联合用药，用于治疗血液瘤、黑色素瘤、神经胶质母细胞瘤、肾细胞癌和非小细胞肺癌等肿瘤。
跨膜受体PD-1(程序性细胞死亡-1)是CD28家族成员之一，在活化的T细胞，B细胞以及骨髓系细胞都有表达。PD-1的受体PDL1和PDL2均属于B7超家族，其中PDL1多种细胞都有表达，包括T细胞，B细胞以及内皮细胞和上皮细胞，PDL2则仅表达于抗原呈递细胞如树突状细胞和巨噬细胞。
PD-1在负调节T细胞的活化过程中起着非常重要的作用，PD-1介导的对T细胞负调节作用是肿瘤免疫逃避的重要机制之一，肿瘤表面表达的PD-L1可与免疫细胞表面的PD-1结合，进而通过PD-1/PD-L1信号通路抑制免疫细胞对肿瘤组织的杀伤，高表达PD-L1的肿瘤伴随着很难被检测到的癌症(Hamanishi et al.,Proc.Natl.Acad.Sci.USA 2007；104:3360-5)。拮抗PD-1，从而抑制PD-1/PD-L1信号通路的一种有效方法是体内注射抗PD-1抗体。
PD-1抗体具有广谱抗肿瘤前景和惊人的药效，针对PD-1通路的抗体将带来治疗多种肿瘤治疗的突破性的进展：用于治疗非小细胞性肺癌，肾细胞癌，卵巢癌，黑色 素瘤(Homet M.B.,Parisi G.,et al.,Anti-PD-1Therapy in Melanoma.Semin Oncol.2015Jun；42(3):466-473)，血液肿瘤以及贫血(Held SA,Heine A,et al.,Advances in immunotherapy of chronic myeloid leukemia CML.Curr Cancer Drug Targets.2013 Sep；13(7):768-74)。
双功能抗体亦称为双特异性抗体(Bispecific Antibody)，是同时靶向结合两种不同抗原的特异性抗体药物，其可通过免疫分选纯化生产，也可通过基因工程获得。基因工程在结合位点优化，合成形式的考量以及产量等方面都具有相应的灵活性，所以具有一定的优势。目前，双特异性抗体存在形式已被证明有超过45种(Müller D,Kontermann RE.Bispecific antibodies for cancer immunotherapy:Current perspectives.BioDrugs 2010；24:89-98)。IgG-ScFv形式即Morrison模式(Coloma MJ,Morrison SL.Design and production of novel tetravalent bispecific antibodies.Nat Biotechnol.Nature Biotechnology,1997；15:159-163)由于类似于天然存在的IgG的形式，在抗体工程、表达和纯化上具有优势，已被证明是双功能抗体的一种理想存在形式(Miller BR,Demarest SJ,et al.,Stability engineering of scFvs for the development of bispecific and multivalent antibodies.Protein Eng Des Sel 2010；23:549-57；Fitzgerald J,Lugovskoy A.Rational engineering of antibody therapeutics targeting multiple oncogene pathways.MAbs 2011；3:299-309)。
目前，尚需要开发新的抗LAG3抗体以及同时靶向PD-1和LAG3的双功能抗体药物。
发明内容
本发明人经过深入的研究和创造性的劳动，得到了一种抗LAG3抗体，并在此基础上制得了抗LAG3-抗PD-1双特异性抗体。本发明人惊奇地发现，本发明的抗LAG3抗体(也简称为抗体或本发明的抗体)和抗LAG3-抗PD-1双特异性抗体(也简称为双特异性抗体或本发明的双特异性抗体)具有优越的亲和力和/或特异性，甚至在一个或多个方面优于阳性对照抗体(例如Nivolumab、Pembrolizumab、Relatlimab等)。由此提供了下述发明：
本发明的一个方面涉及一种抗LAG3抗体或其抗原结合片段，包含重链可变区和轻链可变区，其中，
所述重链可变区包含：氨基酸序列分别如SEQ ID NOs:5-7所示的HCDR1-HCDR3；并且所述轻链可变区包含：氨基酸序列分别如SEQ ID NOs:8-10所示的LCDR1-LCDR3；
所述重链可变区包含：氨基酸序列分别如SEQ ID NOs:5-7所示的HCDR1-HCDR3；并且所述轻链可变区包含：氨基酸序列分别如SEQ ID NO:8、SEQ ID NO:46和SEQ ID NO:47所示的LCDR1-LCDR3；
或者
所述重链可变区包含：氨基酸序列分别如SEQ ID NOs:5-7所示的HCDR1-HCDR3；并且所述轻链可变区包含：氨基酸序列分别如SEQ ID NO:48、SEQ ID NO:46和SEQ ID NO:10所示的LCDR1-LCDR3。
在本发明的一些实施方式中，所述的抗体或其抗原结合片段，其中，
所述抗体的重链可变区的氨基酸序列如SEQ ID NO:2所示，并且所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:4所示；
所述抗体的重链可变区的氨基酸序列如SEQ ID NO:2所示，并且所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:42所示；
或者
所述抗体的重链可变区的氨基酸序列如SEQ ID NO:2所示，并且所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:44所示。
在本发明的一些实施方式中，所述的抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段选自Fab、Fab'、F(ab')2、Fd、Fv、dAb、互补决定区片段、单链抗体、人源化抗体、嵌合抗体或双抗体。
在本发明的一些实施方式中，所述的抗体或其抗原结合片段，其中，所述的抗体以小于0.2nM，例如小于0.15nM、小于0.1nM、小于0.08nM、0.06nM或小于0.05nM或更小的EC 50结合人LAG3-mFc；优选地，所述EC 50通过间接ELISA方法测得。
在本发明的一些实施方式中，所述的抗体或其抗原结合片段，其中，
所述的抗体包括非-CDR区，且所述非-CDR区来自不是鼠类的物种，例如来自人抗体。
在本发明的一些实施方式中，所述的抗体或其抗原结合片段，其中，
所述的抗体，其恒定区来自人抗体；
优选地，所述抗体的恒定区选自人IgG1、IgG2、IgG3或IgG4的恒定区。
在本发明的一些实施方式中，所述的抗体或其抗原结合片段，其中，
所述抗LAG3抗体的重链恒定区为Ig gamma-1 chain C region(例如，如SEQ ID NO:39所示)或Ig gamma-4 chain C region(例如，如SEQ ID NO:45所示)；轻链恒定区为Ig kappa chain C region(例如，如SEQ ID NO:40所示)。
在本发明的一些实施方式中，所述的抗体或其抗原结合片段，其中，
所述抗体为人IgG1亚型，
其中，按照EU编号系统，所述抗体的重链恒定区具有如下突变：
L234A和L235A，
L234A和G237A，
L235A和G237A，
或者
L234A、L235A和G237A；
优选地，所述抗体的重链的氨基酸序列如SEQ ID NO:11所示，并且轻链的氨基酸序列如SEQ ID NO:12所示。
在本发明的一些实施方式中，所述的抗体或其抗原结合片段，其中，
所述抗体为人IgG4亚型，
其中，按照EU编号系统，所述抗体的重链恒定区具有如下突变：
F234A和L235A，
F234A和G237A，
L235A和G237A，
或者
F234A、L235A和G237A；
优选地，所述抗体的重链的氨基酸序列如SEQ ID NO:13所示，并且轻链的氨基酸序列如SEQ ID NO:12所示。
在本发明的一些实施方式中，所述的抗LAG3抗体为单克隆抗体。
在本发明的一些实施方式中，所述的抗LAG3抗体为免疫球蛋白形式。
在本发明的一些实施方式中，所述的抗LAG3抗体为单链抗体。
本发明的另一方面涉及抗体药物偶联物(antibody-drug conjugate，ADC)，其包括抗体或其抗原结合片段以及小分子药物，其中，所述抗体或其抗原结合片段为本发明中任一项所述的抗LAG3抗体或其抗原结合片段；优选地，所述小分子药物为小分子细胞毒药 物；更优选地，所述小分子药物为肿瘤化疗药物。
所述化疗药物可以是常规的肿瘤化疗药物，例如烷化剂、抗代谢药、抗肿瘤抗生素、植物类抗癌药、激素、免疫制剂等。
在本发明的一个或多个实施方案中，所述的抗体药物偶联物，其中，所述抗体或其抗原结合片段通过连接子与小分子药物连接；所述连接子可以是本领域技术人员知悉的连接子，例如，所述连接子为腙键、二硫键或肽键。
在本发明的一个或多个实施方案中，所述的抗体药物偶联物，其中，所述抗体或其抗原结合片段与小分子药物的摩尔比为1：(2－4)，例如1：2，1：3或1：4。
本发明的再一方面涉及一种双特异性抗体，其包括第一蛋白功能区和第二蛋白功能区，其中：
所述第一蛋白功能区靶向LAG3，
所述第二蛋白功能区靶向不同于LAG3的靶点(例如，PD-1)，
其中，所述第一蛋白功能区为本发明中任一项所述的抗体或抗原结合片段；
优选地，所述双特异性抗体为IgG-scFv模式；
优选地，所述第一蛋白功能区为本发明中任一项所述的抗体，并且所述第二蛋白功能区为单链抗体；或者
优选地，所述第一蛋白功能区为单链抗体，并且所述第二蛋白功能区为本发明中任一项所述的抗体。
本发明的双特异性抗体为抗LAG3-抗PD-1双特异性抗体。
在本发明的一些实施方式中，所述的双特异性抗体，其中，所述第一蛋白功能区和第二蛋白功能区直接连接或者通过连接片段连接；
优选地，所述连接片段为(GGGGS)m，m为正整数，例如1、2、3、4、5或6；
优选地，所述连接片段为(GGGGS)nG，n为正整数，例如1、2、3、4、5或6。
在本发明的一些实施方式中，所述的双特异性抗体，其中，所述第一蛋白功能区和第二蛋白功能区独立地为1个、2个或者2个以上。
在本发明的一些实施方式中，所述的双特异性抗体，其中，所述单链抗体连接在抗体的重链的C末端。
在本发明的一些实施方式中，所述的双特异性抗体，其包括：
靶向LAG3的第一蛋白功能区，和
靶向PD-1的第二蛋白功能区；
其中，
所述第一蛋白功能区为本发明中任一项所述的抗LAG3抗体，并且所述抗LAG3抗体为免疫球蛋白形式，
所述第二蛋白功能区为抗PD-1的单链抗体。
在本发明的一些实施方式中，所述的双特异性抗体，其中，所述抗PD-1的单链抗体包含重链可变区和轻链可变区，其中，
所述重链可变区包含：氨基酸序列分别如SEQ ID NOs:26-28所示的HCDR1-HCDR3；和
所述轻链可变区包含：氨基酸序列分别如SEQ ID NOs:29-31所示的LCDR1-LCDR3。
在本发明的一些实施方式中，所述的双特异性抗体，其中，所述抗PD-1的单链抗体，
其重链可变区的氨基酸序列如SEQ ID NO:15所示，并且其轻链可变区的氨基酸序列如SEQ ID NO:17所示；或者
其重链可变区的氨基酸序列如SEQ ID NO:19所示，并且其轻链可变区的氨基酸序列如SEQ ID NO:21或SEQ ID NO:38所示。
在本发明的一些实施方式中，所述的双特异性抗体，其中，所述抗PD-1的单链抗体中的重链可变区和轻链可变区直接连接或者通过连接片段连接；
优选地，所述连接片段为(GGGGS)m，m为正整数，例如1、2、3、4、5或6；
优选地，所述连接片段为(GGGGS)nG，n为正整数，例如1、2、3、4、5或6。
在本发明的一些实施方式中，所述的双特异性抗体，其中，
所述双特异性抗体包括：
靶向LAG3的第一蛋白功能区，和
靶向PD-1的第二蛋白功能区；
所述第一蛋白功能区为1个，所述第二蛋白功能区为2个；
其中，所述第一蛋白功能区为免疫球蛋白，所述第二蛋白功能区为单链抗体；
所述免疫球蛋白的重链的氨基酸序列如SEQ ID NO:11或SEQ ID NO:13所示，并且其轻链的氨基酸序列如SEQ ID NO:12所示；
所述单链抗体的重链可变区的氨基酸序列如SEQ ID NO:19所示，并且所述单链抗体的轻链可变区的氨基酸序列如SEQ ID NO:21或SEQ ID NO:38所示；
所述单链抗体连接在免疫球蛋白的两条重链的C末端；
所述第一蛋白功能区与所述第二蛋白功能区通过第一连接片段连接；并且所述单链抗体的重链可变区与所述单链抗体的轻链可变区通过第二连接片段连接；所述第一连接片段和所述第二连接片段相同或不同；
优选地，所述第一连接片段和所述第二连接片段的氨基酸序列独立地选自SEQ ID NOs:35-37；
优选地，所述第一连接片段和所述第二连接片段的氨基酸序列均如SEQ ID NO:36所示。
在本发明的一些实施方式中，所述的双特异性抗体，其包括：
靶向LAG3的第一蛋白功能区，和
靶向PD-1的第二蛋白功能区；
其中，所述第一蛋白功能区为抗LAG3的单链抗体，所述第二蛋白功能区为抗PD-1的抗体，并且所述抗PD-1的抗体为免疫球蛋白形式；
其中，所述抗LAG3的单链抗体包含重链可变区和轻链可变区，其中，
所述重链可变区包含：氨基酸序列分别如SEQ ID NOs:5-7所示的HCDR1-HCDR3；和
所述轻链可变区包含：氨基酸序列分别如SEQ ID NOs:8-10所示的LCDR1-LCDR3。
在本发明的一些实施方式中，所述的双特异性抗体，其中，所述抗LAG3的单链抗体，
其重链可变区的氨基酸序列如SEQ ID NO:2所示，并且其轻链可变区的氨基酸序列如SEQ ID NO:4所示；
其重链可变区的氨基酸序列如SEQ ID NO:2所示，并且其轻链可变区的氨基酸序列如SEQ ID NO:42所示；或者
其重链可变区的氨基酸序列如SEQ ID NO:2所示，并且其轻链可变区的氨基酸序列如SEQ ID NO:44所示。
在本发明的一些实施方式中，所述的双特异性抗体，其中，所述抗LAG3的单链抗体中的重链可变区和轻链可变区直接连接或者通过连接片段连接；
优选地，所述连接片段为(GGGGS)m，m为正整数，例如1、2、3、4、5或6；
优选地，所述连接片段为(GGGGS)nG，n为正整数，例如1、2、3、4、5或6。
在本发明的一些实施方式中，所述的双特异性抗体，其中，所述抗PD-1的抗体包含重链可变区和轻链可变区，其中，
所述重链可变区包含：氨基酸序列分别如SEQ ID NOs:26-28所示的HCDR1-HCDR3；和
所述轻链可变区包含：氨基酸序列分别如SEQ ID NOs:29-31所示的LCDR1-LCDR3。
在本发明的一些实施方式中，所述的双特异性抗体，其中，所述抗PD-1的抗体，
其重链可变区的氨基酸序列如SEQ ID NO:15所示，并且其轻链可变区的氨基酸序列如SEQ ID NO:17所示；或者
其重链可变区的氨基酸序列如SEQ ID NO:19所示，并且其轻链可变区的氨基酸序列如SEQ ID NO:21或SEQ ID NO:38所示。
在本发明的一些实施方式中，所述的双特异性抗体，其中，所述抗PD-1的抗体，其重链恒定区为Ig gamma-1 chain C region(例如，如SEQ ID NO:39所示)或Ig gamma-4 chain C region(例如，如SEQ ID NO:45所示)；轻链恒定区为Ig kappa chain C region(例如，如SEQ ID NO:40所示)。
在本发明的一些实施方式中，所述的双特异性抗体，其中，
所述抗PD-1的抗体为人IgG1亚型，
其中，按照EU编号系统，所述抗PD-1的抗体具有如下突变：
L234A和L235A，
L234A和G237A，
L235A和G237A，
或者
L234A、L235A和G237A；
优选地，所述抗PD-1的抗体的重链的氨基酸序列如SEQ ID NO:34所示，并且轻链的氨基酸序列如SEQ ID NO:25所示。
在本发明的一些实施方式中，所述的双特异性抗体，其中，
所述抗PD-1的抗体为人IgG4亚型，
其中，按照EU编号系统，所述抗PD-1的抗体具有如下突变：
F234A和L235A，
F234A和G237A，
L235A和G237A，
或者
F234A、L235A和G237A；
优选地，所述抗PD-1的抗体的重链的氨基酸序列如SEQ ID NO:32所示，并且轻链的氨基酸序列如SEQ ID NO:25所示。
在本发明的一些实施方式中，所述的双特异性抗体，其中，
所述双特异性抗体包括：
靶向LAG3的第一蛋白功能区，和
靶向PD-1的第二蛋白功能区；
所述第一蛋白功能区为1个，所述第二蛋白功能区为2个；
其中，所述第一蛋白功能区为单链抗体，所述第二蛋白功能区为免疫球蛋白；
所述单链抗体的重链可变区的氨基酸序列如SEQ ID NO:2所示，并且所述单链抗体的轻链可变区的氨基酸序列如SEQ ID NO:4所示；
所述免疫球蛋白的重链的氨基酸序列如SEQ ID NO:34或SEQ ID NO:32所示，并且其轻链的氨基酸序列如SEQ ID NO:25所示；
所述单链抗体连接在免疫球蛋白的两条重链的C末端；
所述第一蛋白功能区与所述第二蛋白功能区通过第一连接片段连接；并且所述单链抗体的重链可变区与所述单链抗体的轻链可变区通过第二连接片段连接；所述第一连接片段和所述第二连接片段相同或不同；
优选地，所述第一连接片段和所述第二连接片段的氨基酸序列独立地选自SEQ ID NOs:35-37；
优选地，所述第一连接片段和所述第二连接片段的氨基酸序列均如SEQ ID NO:36所示。
在本发明的一些实施方式中，所述的双特异性抗体，其中，一个免疫球蛋白分子连接有两个单链抗体分子；优选地，两个单链抗体分子相同。
本发明的再一方面涉及一种分离的核酸分子，其编码本发明中任一项所述的抗LAG3抗体，或者编码本发明中任一项所述的双特异性抗体。
本发明的再一方面涉及一种重组载体，其包含本发明的分离的核酸分子。
本发明的再一方面涉及一种宿主细胞，其包含本发明的分离的核酸分子，或者包含本 发明的重组载体。
本发明的再一方面涉及一种制备本发明中任一项所述的抗体或其抗原结合片段或者本发明中任一项所述的双特异性抗体的方法，其包括在合适的条件下培养本发明的宿主细胞，以及从细胞培养物中回收所述抗体或其抗原结合片段或者双特异性抗体的步骤。
本发明的再一方面涉及一种药物组合物，其包含本发明中任一项所述的抗体或其抗原结合片段、本发明中任一项所述的抗体药物偶联物或者本发明中任一项所述的双特异性抗体；可选地，其还包括药学上可接受的辅料。
本发明的再一方面涉及本发明中任一项所述的抗体或其抗原结合片段、本发明中任一项所述的抗体药物偶联物或者本发明中任一项所述的双特异性抗体在制备治疗和/或预防肿瘤或贫血病的药物中的用途；
优选地，所述肿瘤为选自卵巢癌、食管癌、黑色素瘤、血液瘤、神经胶质母细胞瘤、肾细胞癌、肺癌、前列腺癌、膀胱癌、结肠癌、直肠癌、肝癌、胃肠道癌、乳腺癌、脑癌、胰腺癌、甲状腺癌、头颈癌和肾癌中的一种或多种；
优选地，所述肺癌为非小细胞肺癌；
优选地，所述血液瘤为白血病；
优选地，所述食管癌为食管鳞癌。
根据本发明中任一项所述的抗体或其抗原结合片段、本发明中任一项所述的抗体药物偶联物或者本发明中任一项所述的双特异性抗体，其用于治疗和/或预防肿瘤或贫血病；
优选地，所述肿瘤为选自卵巢癌、食管癌、黑色素瘤、血液瘤、神经胶质母细胞瘤、肾细胞癌、肺癌、前列腺癌、膀胱癌、结肠癌、直肠癌、肝癌、胃肠道癌、乳腺癌、脑癌、胰腺癌、甲状腺癌、头颈癌和肾癌中的一种或多种；
优选地，所述肺癌为非小细胞肺癌；
优选地，所述血液瘤为白血病；
优选地，所述食管癌为食管鳞癌。
本发明的再一方面涉及一种治疗和/或预防肿瘤或贫血病的方法，包括给予有需求的受试者以有效量的本发明中任一项所述的抗体或其抗原结合片段、本发明中任一项所述的抗体药物偶联物或者本发明中任一项所述的双特异性抗体的步骤；
优选地，所述肿瘤为选自卵巢癌、食管癌、黑色素瘤、血液瘤、神经胶质母细胞瘤、 肾细胞癌、肺癌、前列腺癌、膀胱癌、结肠癌、直肠癌、肝癌、胃肠道癌、乳腺癌、脑癌、胰腺癌、甲状腺癌、头颈癌和肾癌中的一种或多种；
优选地，所述肺癌为非小细胞肺癌；
优选地，所述血液瘤为白血病；
优选地，所述食管癌为食管鳞癌。
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的细胞培养、分子遗传学、核酸化学、免疫学实验室操作步骤均为相应领域内广泛使用的常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
如本文中所使用的，术语EC 50是指半最大效应浓度(concentration for 50％of maximal effect)，是指能引起50％最大效应的浓度。
如本文中所使用的，术语“抗体”是指通常由两对多肽链(每对具有一条“轻”(L)链和一条“重”(H)链)组成的免疫球蛋白分子。抗体轻链可分类为κ和λ轻链。重链可分类为μ、δ、γ、α或ε，并且分别将抗体的同种型定义为IgM、IgD、IgG、IgA和IgE。在轻链和重链内，可变区和恒定区通过大约12或更多个氨基酸的“J”区连接，重链还包含大约3个或更多个氨基酸的“D”区。各重链由重链可变区(VH)和重链恒定区(CH)组成。重链恒定区由3个结构域(CH1、CH2和CH3)组成。各轻链由轻链可变区(VL)和轻链恒定区(CL)组成。轻链恒定区由一个结构域CL组成。抗体的恒定区可介导免疫球蛋白与宿主组织或因子，包括免疫系统的各种细胞(例如，效应细胞)和经典补体系统的第一组分(C1q)的结合。VH和VL区还可被细分为具有高变性的区域(称为互补决定区(CDR))，其间散布有较保守的称为构架区(FR)的区域。各VH和VL由按下列顺序：FR1、CDR1、FR2、CDR2、FR3、CDR3和FR4从氨基末端至羧基末端排列的3个CDR和4个FR组成。各重链/轻链对的可变区(VH和VL)分别形成抗体结合部位。氨基酸至各区域或结构域的分配遵循Bethesda M.d.,Kabat Sequences of Proteins of Immunological Interest(National Institutes of Health,(1987 and 1991))，或Chothia&Lesk J.Mol.Biol.1987；196:901-917；Chothia等人Nature 1989；342:878-883，或者IMGT编号系统定义，见Ehrenmann F,Kaas Q,Lefranc M P.IMGT/3Dstructure-DB and IMGT/DomainGapAlign:a database and a tool for immunoglobulins or antibodies,T cell receptors,MHC,IgSF and MhcSF[J].Nucleic  acids research,2009；38(suppl_1):D301-D307的定义。
特别地，重链还可以包含3个以上CDR，例如6、9、或12个。例如在本发明的双特异性抗体中，重链可以是IgG抗体的重链的C端连接一个ScFv，这种情况下重链含有9个CDR。
术语“抗体”不受任何特定的产生抗体的方法限制。例如，其包括，重组抗体、单克隆抗体和多克隆抗体。抗体可以是不同同种型的抗体，例如，IgG(例如，IgG1，IgG2，IgG3或IgG4亚型)，IgA1，IgA2，IgD，IgE或IgM抗体。
如本文中所使用的，术语“单抗”和“单克隆抗体”是指，来自一群高度同源的抗体分子中的一个抗体或抗体的一个片段，也即除可能自发出现的自然突变外，一群完全相同的抗体分子。单抗对抗原上的单一表位具有高特异性。多克隆抗体是相对于单克隆抗体而言的，其通常包含至少2种或更多种的不同抗体，这些不同的抗体通常识别抗原上的不同表位。单克隆抗体通常可采用Kohler等首次报道的杂交瘤技术获得(
Figure PCTCN2022122556-appb-000001
G,Milstein C.Continuous cultures of fused cells secreting antibody of predefined specificity[J].nature,1975；256(5517):495)，但也可采用重组DNA技术获得(如参见U.S.Patent 4,816,567)。
如本文中所使用的，术语“人源化抗体”是指，人源免疫球蛋白(受体抗体)的全部或部分CDR区被一非人源抗体(供体抗体)的CDR区替换后得到的抗体或抗体片段，其中的供体抗体可以是具有预期特异性、亲和性或反应性的非人源(例如，小鼠、大鼠或兔)抗体。此外，受体抗体的构架区(FR)的一些氨基酸残基也可被相应的非人源抗体的氨基酸残基替换，或被其他抗体的氨基酸残基替换，以进一步完善或优化抗体的性能。关于人源化抗体的更多详细内容，可参见例如，Jones et al.,Nature 1986；321:522 525；Reichmann et al.,Nature,1988；332:323 329；Presta,Curr.Op.Struct.Biol.1992；2:593-596；和Clark,Immunol.Today 2000；21:397 402。在一些情况下，抗体的抗原结合片段是双抗体(Diabodies)，其中V H和V L结构域在单个多肽链上表达，但使用太短的连接体以致不允许在相同链的两个结构域之间配对，从而迫使结构域与另一条链的互补结构域配对并且产生两个抗原结合部位(参见，例如,Holliger P.et al.,Proc.Natl.Acad.Sci.USA 1993；90:6444-6448和Poljak R.J.et al.,Structure 1994；2:1121-1123)。
如本文中所使用的，术语“单链抗体(single chain fragment variable，ScFv)”是指，包含通过连接体连接的抗体重链可变区(V H)和抗体轻链可变区(V L)的分子。其中V L 和V H结构域通过使其能够产生为单个多肽链的连接体配对形成单价分子(参见，例如,Bird et al,Science 1988；242:423-426和Huston et al,Proc.Natl.Acad.Sci.USA 1988；85:5879-5883)。此类scFv分子可具有一般结构：NH2-V L-连接片段-V H-COOH或NH2-V H-连接片段-V L-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 et al，Proc.Natl.Acad.Sci.USA 1993；90:6444-6448)。可用于本发明的其他接头由Alfthan et al，Protein Eng.1995；8:725-731，Choi et al，Eur.J.Immunol.2001；31:94-106，Hu et al，Cancer Res.1996；56:3055-3061，Kipriyanov et al，J.Mol.Biol.1999；293:41-56和Roovers et al,Cancer Immunology,Immunotherapy,2001,50(1):51-59.描述。
如本文中所使用的，术语“分离的”或“被分离的”指的是，从天然状态下经人工手段获得的。如果自然界中出现某一种“分离”的物质或成分，那么可能是其所处的天然环境发生了改变，或从天然环境下分离出该物质，或二者情况均有发生。例如，某一活体动物体内天然存在某种未被分离的多聚核苷酸或多肽，而从这种天然状态下分离出来的高纯度的相同的多聚核苷酸或多肽即称之为分离的。术语“分离的”或“被分离的”不排除混有人工或合成的物质，也不排除存在不影响物质活性的其它不纯物质。
如本文中所使用的，术语“载体(vector)”是指，可将多聚核苷酸插入其中的一种核酸运载工具。当载体能使插入的多核苷酸编码的蛋白获得表达时，载体称为表达载体。载体可以通过转化，转导或者转染导入宿主细胞，使其携带的遗传物质元件在宿主细胞中获得表达。载体是本领域技术人员公知的，包括但不限于：质粒；噬菌粒；柯斯质粒；人工染色体，例如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。可用作载体的动物病毒包括但不限于，逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。一种载体可以含有多种控制表达的元件，包括但不限于，启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。
如本文中所使用的，术语“宿主细胞”是指，可用于导入载体的细胞，其包括但不限于，如大肠杆菌或枯草杆菌等的原核细胞，如酵母细胞或曲霉菌等的真菌细胞，如S2果蝇细胞或Sf9等的昆虫细胞，或者如纤维原细胞，CHO细胞，GS细胞，COS 细胞，NSO细胞，HeLa细胞，BHK细胞，HEK 293细胞或人细胞等的动物细胞。
如本文中使用的，术语“特异性结合”是指，两分子间的非随机的结合反应，如抗体和其所针对的抗原之间的反应。在某些实施方式中，特异性结合某抗原的抗体(或对某抗原具有特异性的抗体)是指，抗体以小于大约10 -5M，例如小于大约10 -6M、10 -7M、10 -8M、10 -9M或10 -10M或更小的亲和力(K D)结合该抗原。
如本文中所使用的，术语“K D”是指特定抗体-抗原相互作用的解离平衡常数，其用于描述抗体与抗原之间的结合亲和力。平衡解离常数越小，抗体-抗原结合越紧密，抗体与抗原之间的亲和力越高。通常，抗体以小于大约10 -5M，例如小于大约10 -6M、10 -7M、10 -8M、10 -9M或10 -10M或更小的解离平衡常数(K D)结合抗原(例如，PD-1蛋白)。可以使用本领域技术人员知悉的方法测定K D，例如使用Fortebio分子相互作用仪测定。
如本文中所使用的，术语“单克隆抗体”和“单抗”具有相同的含义且可互换使用；术语“多克隆抗体”和“多抗”具有相同的含义且可互换使用。并且在本发明中，氨基酸通常用本领域公知的单字母和三字母缩写来表示。例如，丙氨酸可用A或Ala表示。
如本文中所使用的，术语“药学上可接受的载体和/或赋形剂”是指在药理学和/或生理学上与受试者和活性成分相容的载体和/或赋形剂，其是本领域公知的(参见例如Remington's Pharmaceutical Sciences.Edited by Gennaro AR,19th ed.Pennsylvania:Mack Publishing Company,1995)，并且包括但不限于：pH调节剂，表面活性剂，佐剂，离子强度增强剂。例如，pH调节剂包括但不限于磷酸盐缓冲液；表面活性剂包括但不限于阳离子，阴离子或者非离子型表面活性剂，例如Tween-80；离子强度增强剂包括但不限于氯化钠。
如本文中所使用的，术语“有效量”是指足以获得或至少部分获得期望的效果的量。例如，预防疾病(例如肿瘤)有效量是指，足以预防，阻止，或延迟疾病(例如肿瘤)的发生的量；治疗疾病有效量是指，足以治愈或至少部分阻止已患有疾病的患者的疾病和其并发症的量。测定这样的有效量完全在本领域技术人员的能力范围之内。例如，对于治疗用途有效的量将取决于待治疗的疾病的严重度、患者自己的免疫系统的总体状态、患者的一般情况例如年龄，体重和性别，药物的施用方式，以及同时施用的其他治疗等等。
如本文中所使用的，当提及PD-1蛋白(NCBI GenBank:NM_005018)的氨基酸序列时，其包括PD-1蛋白的全长，或者PD-1的胞外片段PD-1 ECD或者包含PD-1  ECD的片段；还包括PD-1蛋白的全长的融合蛋白或PD-1 ECD的融合蛋白，例如与小鼠或人IgG的Fc蛋白片段(mFc或hFc)进行融合的片段。然而，本领域技术人员理解，在PD-1蛋白的氨基酸序列中，可天然产生或人工引入突变或变异(包括但不限于置换，缺失和/或添加)，而不影响其生物学功能。因此，在本发明中，术语“添加)，蛋白”应包括所有此类序列，包括其天然或人工的变体。并且，当描述PD-1蛋白的序列片段时，其还包括其天然或人工变体中的相应序列片段。
如本文中所使用的，当提及淋巴细胞活化基因3(lymphocyte-activation gene 3，LAG3)的氨基酸序列时，其包括LAG3蛋白的全长，或者LAG3的胞外片段LAG3 ECD或者包含LAG3 ECD的片段；还包括LAG3蛋白的全长的融合蛋白或LAG3 ECD的融合蛋白，例如与小鼠或人IgG的Fc蛋白片段(mFc或hFc)进行融合的片段。然而，本领域技术人员理解，在LAG3蛋白的氨基酸序列中，可天然产生或人工引入突变或变异(包括但不限于置换，缺失和/或添加)，而不影响其生物学功能。因此，在本发明中，术语“添加)，蛋白”应包括所有此类序列，包括其天然或人工的变体。并且，当描述LAG3蛋白的序列片段时，其还包括其天然或人工变体中的相应序列片段。
在本发明中，如果没有特别说明，所述“第一”(例如第一蛋白功能区)和“第二”(例如第二蛋白功能区)是为了指代上的区分或表述上的清楚，并不具有典型的次序上的含义。
发明的有益效果
本发明取得了如下效果中的一项或多项：
(1)本发明的抗LAG3抗体具有优越的亲和力和特异性；
(2)本发明的双特异性抗体例如BS-PL021A、BS-PL022B、BS-PL023C能够很好地特异性与LAG3结合，并且能够十分有效地阻断LAG3与MHC Ⅱ的结合，特异地解除LAG3对机体免疫抑制；
(3)本发明的双特异性抗体例如BS-PL021A、BS-PL022B、BS-PL023C能够很好地特异性与PD-1结合，并且能够十分有效地阻断PD-1与PDL1的结合，特异地解除PD-1对机体免疫抑制，激活免疫反应。
(4)本发明的双特异性抗体中的第一蛋白功能区和第二蛋白功能区之间具有协同作用。
(5)本发明的双特异性抗体特别是BS-PL022B，完全消除了其与Fc受体FcγRI、 FcγRIIb、FcγRIIa_H131、FcγRIIIa_V158和/或FcγRIIIa_F158的结合活性，进而消除了其ADCC活性或ADCP活性。
(6)本发明的双特异性抗体特别是BS-PL022B，完全消除了其与补体C1q的结合活性，进而消除了其CDC活性。
附图说明
图1：间接ELISA方法测定BS-PL021A、BS-PL022B、BS-PL023C、Bs-PLV02、14C12H1L1(hG1TM)与抗原PD-1-mFc结合活性检测结果。
图2：间接ELISA方法测定BS-PL021A、BS-PL022B、BS-PL023C、BS-PLV02、Relatlimab、H7L8(hG1WT)与抗原LAG3-mFc结合活性检测结果。
图3：竞争ELISA方法测定抗体抗LAG3-抗PD-1双特异性抗体与人PDL1-mFc竞争结合人PD-1-mFc-Biotin活性检测结果。
图4：FACS检测抗LAG3-抗PD-1双特异性抗体与293T-PD1膜表面PD-1结合活性检测结果。
图5：FACS检测抗LAG3-抗PD-1双特异性抗体与293T-LAG3膜表面LAG3结合活性检测结果。
图6：竞争流式细胞法测定抗LAG3-抗PD-1双特异性抗体与PDL1竞争结合细胞膜表面抗原PD-1检测结果。
图7：竞争流式细胞法测定抗LAG3-抗PD-1双特异性抗体与LAG3竞争结合细胞膜表面抗原MHC II检测结果。
图8A：抗LAG3-抗PD-1双特异性抗体阻断LAG3与MHCII结合检测结果。
图8B：抗LAG3-抗PD-1双特异性抗体阻断LAG3与MHCII结合检测结果。
图9A：抗LAG3-抗PD-1双特异性抗体阻断PD-1与PD-L1结合检测结果。
图9B：抗LAG3-抗PD-1双特异性抗体阻断PD-1与PD-L1结合检测结果。
图10A：抗LAG3-抗PD-1双特异性抗体同时阻断LAG3与MHCII以及PD-1与PD-L1结合检测结果。
图10B：抗LAG3-抗PD-1双特异性抗体同时阻断LAG3与MHCII以及PD-1与PD-L1结合检测结果。
图11：抗LAG3-抗PD-1双特异性抗体桥连实验检测结果。
图12A：混合淋巴细胞反应MLR检测抗LAG3-抗PD-1双特异性抗体促IFN-γ 分泌的生物活性检测结果。
图12B：混合淋巴细胞反应MLR检测抗LAG3-抗PD-1双特异性抗体促IL-2分泌的生物活性检测结果。
图13：BS-PL022B与FcγRI的亲和力常数检测结果。
图14：H7L8(hG1WT)与FcγRI的亲和力常数检测结果。
图15：BS-PL022B与FcγRIIIa_V158的亲和力常数检测结果。
图16：H7L8(hG1WT)与FcγRIIIa_V158的亲和力常数检测结果图。
图17：BS-PL022B与FcγRIIIa_F158的亲和力常数检测结果。
图18：H7L8(hG1WT)与FcγRIIIa_F158的亲和力常数检测结果。
图19：BS-PL022B与FcγRIIa_H131的亲和力常数检测结果。
图20：H7L8(hG1WT)与FcγRIIa_H131的亲和力常数检测结果。
图21：BS-PL022B与FcγRIIb的亲和力常数检测结果。
图22：H7L8(hG1WT)与FcγRIIb的亲和力常数检测结果。
图23：BS-PL022B与C1q的亲和力常数检测结果。
图24：H7L8(hG1WT)与C1q的亲和力常数检测结果。
图25：BS-PL022B的ADCP效应结果。
图26：抗LAG3-抗PD-1双特异性抗体对BALB/c-hPD1/hLAG3小鼠CT26移植瘤模型药效。*P<0.05，**P<0.01，***P<0.001，VS同型对照组(two-way ANOVA)
图27：抗LAG3-抗PD-1双特异性抗体对BALB/c-hPD1/hLAG3小鼠CT26移植瘤模型体重影响。
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述。本领域技术人员将会理解，下面的实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件(例如参考J.萨姆布鲁克等著，黄培堂等译的《分子克隆实验指南》，第三版，科学出版社)或按照产品说明书进行。所用试剂或仪器未注明生产厂商者，为可以通过市场购买获得的常规产品。例如MDA-MB-231细胞和U87-MG细胞可以购自ATCC。
BALB/c小鼠购自广东省医学实验动物中心。
Nivolumab购自BMS公司，批号：ABA0330。Nivolumab是抗PD-1抗体。
Pembrolizumab购自MSD Ireland(Carlow)公司，货号：S023942。Pembrolizumab是抗PD-1抗体。
阳性对照抗体Relatlimab，其序列参考美国专利公开：US20160326248A1。其中重链氨基酸序列参照该专利公开中的SEQ ID NO:1，轻链氨基酸序列参照该专利公开中的SEQ ID NO:2。Relatlimab是抗LAG-3抗体。
293T-PD1细胞系由中山康方生物医药有限公司构建。293T-PD1细胞系由HEK293T细胞经病毒感染制得，病毒制备使用的是3rd Generation Lentiviral Systems，参见，例如A Third Generation Lentivirus Vector with a Conditional Packaging System.Dull T,Zufferey R,Kelly M,Mandel RJ,Nguyen M,Trono D,and Naldini L.J Virol.1998.72(11):8463-8471.其中所使用的慢病毒表达载体为plenti6.3/V5-PD1FL-BSD(其中PD1，Genebank ID:NM_005018；载体plenti6.3/V5-BSD，购自Invitrogen，产品编号：K5315-20)。
293T-LAG3细胞系由中山康方生物医药有限公司构建。293T-LAG3细胞系由HEK293T细胞经病毒感染制得，病毒制备使用的是3rd Generation Lentiviral Systems，参见，例如A Third Generation Lentivirus Vector with a Conditional Packaging System.Dull T,Zufferey R,Kelly M,Mandel RJ,Nguyen M,Trono D,and Naldini L.J Virol.1998.72(11):8463-8471.其中所使用的慢病毒表达载体为plenti6.3/V5-huLAG3FL-BSD(其中LAG3，Genebank ID:NM_002277.4；载体plenti6.3/V5-BSD，购自Invitrogen，产品编号：K5315-20)。
Raji-PDL1细胞系由中山康方生物医药有限公司构建。Raji-PDL1细胞系由Raji细胞经病毒感染制得，病毒制备使用的是3rd Generation Lentiviral Systems，参见，例如A Third Generation Lentivirus Vector with a Conditional Packaging System.Dull T,Zufferey R,Kelly M,Mandel RJ,Nguyen M,Trono D,and Naldini L.J Virol.1998.72(11):8463-8471.其中所使用的慢病毒表达载体为plenti6.3/V5-PDL1(其中PDL1，Genebank ID:NP_054862.1；载体plenti6.3/V5，购自Invitrogen，货号：K5315-20)。
Jurkat-NFAT-PD1-LAG3细胞系由中山康方生物医药有限公司构建。Jurkat-NFAT-PD1-LAG3细胞系由PD-1效应细胞(PD-1 Effector cells(CPM)，厂家：Promega，货号：J112A)细胞经病毒感染制得，病毒制备使用的是3rd Generation Lentiviral Systems，参见，例如A Third Generation Lentivirus Vector with a  Conditional Packaging System.Dull T,Zufferey R,Kelly M,Mandel RJ,Nguyen M,Trono D,and Naldini L.J Virol.1998.72(11):8463-8471.其中所使用的慢病毒表达载体为pCDH-huLAG3FL-RFP-NEO(其中LAG3，Genebank ID:NM_002277.4；载体pCDH-CMV-MCS-EF1-RFP+Neo，购自优宝生物，产品编号：VT9005)。
CHO-K1-PD1细胞系由中山康方生物医药有限公司构建。CHO-K1-PD1细胞系由CHO-K1细胞经病毒感染制得，病毒制备使用的是3rd Generation Lentiviral Systems，参见，例如A Third Generation Lentivirus Vector with a Conditional Packaging System.Dull T,Zufferey R,Kelly M,Mandel RJ,Nguyen M,Trono D,and Naldini L.J Virol.1998.72(11):8463-8471.其中所使用的慢病毒表达载体为pCDH-CMV-PD-1FL-Puro(其中PD1，Genebank ID:NM_005018；载体pCDH-CMV-Puro，购自优宝生物，产品编号：VT1480)。
CHO-K1-LAG3细胞系由中山康方生物医药有限公司构建。CHO-K1-LAG3细胞系由CHO-K1细胞经病毒感染制得，病毒制备使用的是3rd Generation Lentiviral Systems，参见，例如A Third Generation Lentivirus Vector with a Conditional Packaging System.Dull T,Zufferey R,Kelly M,Mandel RJ,Nguyen M,Trono D,and Naldini L.J Virol.1998.72(11):8463-8471.其中所使用的慢病毒表达载体为plenti6.3/V5-huLAG3FL-BSD(其中LAG3，Genebank ID:NM_002277.4；载体plenti6.3/V5-BSD，购自Invitrogen，产品编号：K5315-20)。
Jurkat-NFAT-CD64-CD32R细胞系由中山康方生物医药有限公司构建。Jurkat-NFAT-CD64-CD32R细胞系由Jurkat细胞经病毒感染制得，病毒制备使用的是3rd Generation Lentiviral Systems，参见，例如A Third Generation Lentivirus Vector with a Conditional Packaging System.Dull T,Zufferey R,Kelly M,Mandel RJ,Nguyen M,Trono D,and Naldini L.J Virol.1998.72(11):8463-8471.其中所使用的慢病毒表达载体为pCDH-NFAT-Hygro(其中载体pCDH-Hygro基于pCDH-CMV-MCS-EF1-Puro(购自优宝生物，货号：VT1480)基础上，本实验室改造获得)、pcDH-hFCGR1AFL-Neo(载体pCDH-Neo基于pCDH-CMV-MCS-EF1-Puro(购自优宝生物，货号：VT1480)基础上，本实验室改造获得)和pCDH-hFCGR2A(H167)-puro(其中hFCGR2A(H167)，Genebank ID:P12318；载体pCDH-CMV-MCS-EF1-Puro购自优宝生物，货号：VT1480)。
CHO-K1-PD1-LAG3细胞系由中山康方生物医药有限公司构建。 CHO-K1-PD1-LAG3细胞系由CHO-K1细胞经病毒感染制得，病毒制备使用的是3rd Generation Lentiviral Systems，参见，例如A Third Generation Lentivirus Vector with a Conditional Packaging System.Dull T,Zufferey R,Kelly M,Mandel RJ,Nguyen M,Trono D,and Naldini L.J Virol.1998.72(11):8463-8471.其中所使用的慢病毒表达载体为pCDH-hPD1-FL-puro(其中PD-1，Genebank ID:NM_005018；其载体pCDH-CMV-MCS-EF1-Puro购自优宝生物，货号：VT1480)和plenti6.3/V5-huLAG3FL-BSD(其中LAG3，Genebank ID:NM_002277.4；载体plenti6.3/V5-BSD，购自Invitrogen，货号：K5315-20)
制备例1：抗LAG3抗体的设计和制备
1.抗体的设计
本发明人根据已有的LAG3蛋白序列(NCBI Reference Sequence:NP_002277.4)及其蛋白三维晶体结构等，创造性地人工设计了一系列的抗体序列。通过大量的筛选和检测，最终得到了与LAG3特异性结合的人源化单克隆抗体，分别命名为H7L8、H7L9、H7L10。该单克隆抗体重链和轻链可变区的氨基酸序列及其编码序列如下。
H7L8的重链可变区H7v的核酸序列(360bp)：
Figure PCTCN2022122556-appb-000002
H7L8的重链可变区H7v的氨基酸序列(120aa)：
Figure PCTCN2022122556-appb-000003
H7L8的轻链可变区L8v的核酸序列(321bp)：
Figure PCTCN2022122556-appb-000004
Figure PCTCN2022122556-appb-000005
H7L8的轻链可变区L8v的氨基酸序列(107aa)：
Figure PCTCN2022122556-appb-000006
H7L9的重链可变区H7v的核酸序列与H7L8的重链可变区H7v的核酸序列相同，即SEQ ID NO:1。
H7L9的重链可变区H7v的氨基酸序列与H7L8的重链可变区H7v的氨基酸序列相同，即SEQ ID NO:2。
H7L9的轻链可变区L9v的核酸序列(321bp)：
Figure PCTCN2022122556-appb-000007
H7L9的轻链可变区L9v的氨基酸序列(107bp)：
Figure PCTCN2022122556-appb-000008
H7L10的重链可变区H7v的核酸序列与H7L8的重链可变区H7v的核酸序列相同，即SEQ ID NO:1。
H7L10的重链可变区H7v的氨基酸序列与H7L8的重链可变区H7v的氨基酸序列相同，即SEQ ID NO:2。
H7L10的轻链可变区L10v的核酸序列(321bp)：
Figure PCTCN2022122556-appb-000009
H7L10的轻链可变区L10v的氨基酸序列(107bp)：
Figure PCTCN2022122556-appb-000010
抗体H7L8的CDR的氨基酸序列如下(按照IMGT编号系统)：
HCDR1：GGSISDYY(SEQ ID NO:5)；
HCDR2：INHRGTT(SEQ ID NO:6)；
HCDR3：AFGYSDYEYDWFDP(SEQ ID NO:7)；
LCDR1：QTISSY(SEQ ID NO:8)；
LCDR2：DAS(SEQ ID NO:9)；
LCDR3：QQRSNWPIT(SEQ ID NO:10)。
抗体H7L9的CDR的氨基酸序列如下(按照IMGT编号系统)：
HCDR1：GGSISDYY(SEQ ID NO:5)；
HCDR2：INHRGTT(SEQ ID NO:6)；
HCDR3：AFGYSDYEYDWFDP(SEQ ID NO:7)；
LCDR1：QTISSY(SEQ ID NO:8)；
LCDR2：DGS(SEQ ID NO:46)；
LCDR3：QQRSNWPLT(SEQ ID NO:47)。
抗体H7L10的CDR的氨基酸序列如下(按照IMGT编号系统)：
HCDR1：GGSISDYY(SEQ ID NO:5)；
HCDR2：INHRGTT(SEQ ID NO:6)；
HCDR3：AFGYSDYEYDWFDP(SEQ ID NO:7)；
LCDR1：QSISSY(SEQ ID NO:48)；
LCDR2：DGS(SEQ ID NO:46)；
LCDR3：QQRSNWPIT(SEQ ID NO:10)。
2.人源化抗体抗体H7L8(hG1WT)的表达和纯化
将H7L8(hG1WT)的重链cDNA序列(可变区编码序列如SEQ ID NO:1所示；恒定区是Ig gamma-1 chain C region,SEQ ID NO:39)和轻链的cDNA序列(可变区编码序列如SEQ ID NO:3所示；恒定区为P01834.1(human Ig kappa chain C region，SEQ ID NO:40)分别克隆到pUC57simple(金斯瑞公司提供)载体中，分别获得pUC57simple-H7和pUC57simple-L8质粒。分别将质粒pUC57simple-H7和pUC57simple-L8进行酶切(HindIII&EcoRI)，电泳回收得到的重链轻链分别亚克隆到pcDNA3.1载体中，提取重组质粒共转染293F细胞。细胞培养7天后，将培养液通过高速离心、上清浓缩后上样至HiTrap MabSelect SuRe柱，用Elution Buffer一步洗脱蛋白并回收目标样品并换液至PBS。
H7L8(hG1WT)重链恒定区氨基酸序列
Figure PCTCN2022122556-appb-000011
H7L8(hG1WT)轻链恒定区氨基酸序列
Figure PCTCN2022122556-appb-000012
Figure PCTCN2022122556-appb-000013
3、人源化抗体H7L8(hG1TM)的设计
本发明人在H7L8(hG1WT)的基础上，通过在其重链的第234号位点(按照EU编号系统，下同)引进了亮氨酸到丙氨酸的点突变(L234A)，第235号位点引进了亮氨酸到丙氨酸的点突变(L235A)，第237号位点引进了甘氨酸到丙氨酸的点突变(G237A)获得了恒定区突变的人源化抗体H7L8(hG1TM)。H7L8(hG1TM)的重链H7(hG1TM)的氨基酸序列如SEQ ID NO:11所示；轻链L8的氨基酸序列如SEQ ID NO:12所示。
人源化抗体H7L8(hG1TM)的制备可以参照上面步骤2中的方法。
H7L8(hG1TM)的重链H7(hG1TM)的氨基酸序列
Figure PCTCN2022122556-appb-000014
H7L8(hG1TM)的轻链L8的氨基酸序列
Figure PCTCN2022122556-appb-000015
4、人源化抗体H7L8(hG4DM)的设计
本发明人在H7L8(hG1WT)的基础上，抗体可变区不变，采用Ig gamma-4 chain C  region作为重链恒定区，并通过在其重链恒定区的第234号位点引进了苯丙氨酸到丙氨酸的点突变(F234A)，和第235号位点引进了亮氨酸到丙氨酸的点突变(L235A)获得了恒定区突变的人源化抗体H7L8(hG4DM)。H7L8(hG4DM)的重链氨基酸序列如SEQ ID NO:13所示；轻链氨基酸序列如SEQ ID NO:12所示。
H7L8(hG4DM)的重链H7(hG4DM)的氨基酸序列：
Figure PCTCN2022122556-appb-000016
H7L8(hG4DM)的轻链L8的氨基酸序列与H7L8(hG1TM)轻链氨基酸序列相同，即为SEQ ID NO:12。
5.人源化抗体H7L8(hG4WT)、H7L9(hG4WT)以及H7L10(hG4WT)的表达和纯化
将H7L8(hG4WT)、H7L9(hG4WT)以及H7L10(hG4WT)的重链cDNA序列(可变区编码序列如SEQ ID NO:1所示；恒定区是Ig gamma-4 chain C region,如SEQ ID NO:45所示)和H7L8(hG4WT)轻链的cDNA序列(可变区编码序列如SEQ ID NO:3所示；恒定区为human Ig kappa chain C region，如SEQ ID NO:40所示)、H7L9(hG4WT)轻链的cDNA序列(可变区编码序列如SEQ ID NO:42所示；恒定区为human Ig kappa chain C region，如SEQ ID NO:40所示)、H7L10(hG4WT)轻链的cDNA序列(可变区编码序列如SEQ ID NO:44所示；恒定区为human Ig kappa chain C region，如SEQ ID NO:40所示)分别克隆到pUC57simple(金斯瑞公司提供)载体中，分别获得pUC57simple-H7和pUC57simple-L8、pUC57simple-L9、pUC57simple-L10质粒。分别将质粒pUC57simple-H7和pUC57simple-L8、 pUC57simple-L9、pUC57simple-L10进行酶切(HindIII&EcoRI)，电泳回收得到的重链轻链分别亚克隆到pcDNA3.1载体中，提取重组质粒共转染293F细胞。细胞培养7天后，将培养液通过高速离心、上清浓缩后上样至HiTrap MabSelect SuRe柱，用Elution Buffer一步洗脱蛋白并回收目标样品并换液至PBS。
H7L8(hG4WT)、H7L9(hG4WT)或H7L10(hG4WT)重链恒定区氨基酸序列：
Figure PCTCN2022122556-appb-000017
H7L8(hG4WT)、H7L9(hG4WT)或H7L10(hG4WT)轻链恒定区氨基酸序列：
Figure PCTCN2022122556-appb-000018
制备例2：抗PD-1的抗体14C12及其人源化抗体14C12H1L1的设计和制备
抗PD-1的抗体14C12及其人源化抗体14C12H1L1的重链和轻链的氨基酸序列、以及编码核酸序列分别与中国专利公开CN 106967172A(或者CN 106977602A)中的14C12、14C12H1L1完全相同。
(1)14C12的重链可变区序列和轻链可变区序列
14C12的重链可变区的核酸序列：(354bp)
Figure PCTCN2022122556-appb-000019
Figure PCTCN2022122556-appb-000020
14C12的重链可变区的氨基酸序列：(118aa)
Figure PCTCN2022122556-appb-000021
14C12的轻链可变区的核酸序列：(321bp)
Figure PCTCN2022122556-appb-000022
14C12的轻链可变区的氨基酸序列：(107aa)
Figure PCTCN2022122556-appb-000023
(2)人源化单克隆抗体14C12H1L1的重链可变区序列和轻链可变区序列、重链序列和轻链序列
14C12H1L1的重链可变区14C12H1v的核酸序列：(354bp)
Figure PCTCN2022122556-appb-000024
Figure PCTCN2022122556-appb-000025
14C12H1L1的重链可变区14C12H1v的氨基酸序列：(118aa)
Figure PCTCN2022122556-appb-000026
14C12H1L1的轻链可变区14C12L1v的核酸序列：(321bp)
Figure PCTCN2022122556-appb-000027
14C12H1L1的轻链可变区14C12L1v的氨基酸序列：(107aa)
Figure PCTCN2022122556-appb-000028
14C12H1L1的重链14C12H1的核酸序列：(1344bp)
Figure PCTCN2022122556-appb-000029
Figure PCTCN2022122556-appb-000030
14C12H1L1的重链14C12H1的氨基酸序列：(448aa)
Figure PCTCN2022122556-appb-000031
14C12H1L1的轻链14C12L1的核酸序列：(642bp)
Figure PCTCN2022122556-appb-000032
Figure PCTCN2022122556-appb-000033
14C12H1L1的轻链14C12L1的氨基酸序列：(214aa)
Figure PCTCN2022122556-appb-000034
抗体14C12和14C12H1L1的CDR相同，如下(按照IMGT编号系统)：
HCDR1:GFAFSSYD(SEQ ID NO:26)
HCDR2:ISGGGRYT(SEQ ID NO:27)
HCDR3:ANRYGEAWFAY(SEQ ID NO:28)
LCDR1:QDINTY(SEQ ID NO:29)
LCDR2:RAN(SEQ ID NO:30)
LCDR3:LQYDEFPLT(SEQ ID NO:31)
14C12H1L1(M)的重链可变区序列和轻链可变区序列
以14C12H1L1为基础对其骨架区(轻链)中个别氨基酸进行了突变得到14C12H1L1(M)。
14C12H1L1(M)的重链可变区14C12H1(M)：
与14C12H1L1的重链可变区14C12H1相同，即氨基酸序列如SEQ ID NO:19所示。
14C12H1L1(M)的轻链可变区14C12L1(M)：
Figure PCTCN2022122556-appb-000035
制备例3：人源化抗体14C12H1L1(hG4DM)的设计
本发明人在14C12H1L1的基础上，抗体可变区不变，采用Ig gamma-4 chain C region作为重链恒定区，并通过在其重链恒定区的第234号位点引进了苯丙氨酸到丙氨酸的点突变(F234A)，和第235号位点引进了亮氨酸到丙氨酸的点突变(L235A)获得了恒定区突变的人源化抗体14C12H1L1(hG4DM)。14C12H1L1(hG4DM)的重链14C12H1(hG4DM)的氨基酸序列如SEQ ID NO:32所示；轻链氨基酸序列如SEQ ID NO:25所示。
14C12H1L1(hG4DM)重链氨基酸序列
Figure PCTCN2022122556-appb-000036
14C12H1L1(hG4DM)轻链氨基酸序列与14C12H1L1的轻链14C12L1的氨基酸序列相同，即为SEQ ID NO:25。
制备例4：人源化抗体14C12H1L1(hG1TM)的序列设计
本发明人在人源化抗体14C12H1L1的基础上，按照EU编号系统(EU numbering  system)，通过在其重链铰链区域第234号位点引进了亮氨酸到丙氨酸的点突变(L234A)，第235号位点引进了亮氨酸到丙氨酸的点突变(L235A)，第237号位点引进了甘氨酸到丙氨酸的点突变(G237A)，获得了突变的人源化14C12H1L1(hG1TM)。
14C12H1L1(hG1TM)的重链14C12H1(hG1TM)的核酸序列：(1344bp)
Figure PCTCN2022122556-appb-000037
Figure PCTCN2022122556-appb-000038
14C12H1L1(hG1TM)的重链14C12H1(hG1TM)的氨基酸序列：(448aa)
Figure PCTCN2022122556-appb-000039
14C12H1L1(hG1TM)的轻链的核酸序列同SEQ ID NO:24。
14C12H1L1(hG1TM)的轻链的氨基酸序列同14C12H1L1的轻链14C12L1的氨基酸序列，即SEQ ID NO:25。
制备例5：抗LAG3/PD-1双功能抗体的设计和制备
1.序列设计
本发明中的双功能抗体BS-PL021A、Bs-PL022B、BS-PL023C、Bs-PLV02的结构模式属于Morrison模式(IgG-scFv)，即在一个IgG抗体的两条重链的C端均连接另一个抗体的scFv片段，其重链和轻链的主要组成设计如下面的表1。
Figure PCTCN2022122556-appb-000040
上面的表1中:
(1)连接片段(GGGGS)3氨基酸序列：
Figure PCTCN2022122556-appb-000041
连接片段(GGGGS)4氨基酸序列：
Figure PCTCN2022122556-appb-000042
连接片段(GGGGS)4G氨基酸序列：
Figure PCTCN2022122556-appb-000043
(2)右下角标注“v”的，是指相应重链的可变区或者相应轻链的可变区。没有标注“v”的，相应重链或者轻链为包含恒定区的全长。这些可变区或者全长的氨基酸序列及其编码核酸序列均参照上面的制备例中记载的相应序列。
2.抗体的表达和纯化
分别将Bs-PL021A、Bs-PL022B、Bs-PL023C、Bs-PLV02的重链cDNA序列和轻链的cDNA序列克隆到pUC57simple(金斯瑞公司提供)载体中，分别获得pUC57simple-Bs-PL021AH/pUC57simple-Bs-PL021AL、pUC57simple-Bs-PL022BH/pUC57simple-Bs-PL022BL、pUC57simple-Bs-PL023CH/pUC57simple-Bs-PL023CL、pUC57simple-Bs-PLV02H/pUC57simple-Bs-PLV02L、pUC57simple-Bi-PGV02/pUC57simple-Bi-PGV02质粒。
分别将质粒pUC57simple-Bs-PL021AH/pUC57simple-Bs-PL021AL、pUC57simple-Bs-PL022BH/pUC57simple-Bs-PL022BL、pUC57simple-Bs-PL023CH/pUC57simple-Bs-PL023CL、pUC57simple-Bs-PLV02H/pUC57simple-Bs-PLV02LpUC57simple-Bi-PGV02/pUC57simple-Bi-PGV02进行酶切(HindIII&EcoRI)，电泳回收得到的重链轻链分别亚克隆到pcDNA3.1载体中，提取重组质粒共转染293F细胞。细胞培养7天后，将培养液通过高速离心、上清浓缩后上样至HiTrap MabSelect SuRe柱，用Elution Buffer一步洗脱蛋白并回收目标样品并换液至PBS。
制备例6：融合蛋白PD-1-mFc、PD-1-hFc和PDL1-hFc的制备
融合蛋白PD-1-mFc、PD-1-hFc和PDL1-hFc的制备以及SDS-PAGE电泳检测完全参照中国专利公开CN106632674A的制备例1进行。
其中，本制备例中的融合蛋白PD-1-mFc、PD-1-hFc和PDL1-hFc的氨基酸序列及其编码核酸序列分别与中国专利公开CN106632674A的制备例1中的PD-1-mFc、PD-1-hFc和PDL1-hFc相同。
制得了融合蛋白PD-1-mFc、PD-1-hFc和PDL1-hFc。
制备例7：人抗鸡蛋溶酶体抗体的制备
人抗鸡蛋溶酶体抗体(human anti-Hen Egg Lysozyme IgG，anti-HEL，即human IgG，简称hIgG)，其序列来自于Acierno等人发表的Affinity maturation increases the stability and plasticity of the Fv domain of anti-protein antibodies研究中Fab F10.6.6序列的可变区序列(Acierno等人.J Mol Biol.2007；374(1):130-46.)。制备方法如下：
human IgG委托南京金斯瑞生物对抗体的重轻链(全序列或可变区)基因进行氨基酸的密码子优化和基因合成，参照《分子克隆实验指南(第三版)》介绍的标准技术，采用PCR、酶切、DNA胶回收、连接转化、菌落PCR或酶切鉴定等标准的分子克隆技术将重轻链基因分别亚克隆到哺乳动物表达系统的抗体重链表达载体和抗体轻链表达载体，并进一步对重组表达载体的重轻链基因进行测序分析。测序验证正确后，中大量制备去内毒素级别的表达质粒并将重轻链表达质粒瞬时共转染HEK293细胞进行重组抗体的表达。培养7天后收集细胞培养液，进行rProtein A柱(GE)亲和纯化，收获的抗体样品用SDS-PAGE和SEC-HPLC标准分析技术对其进行质量鉴定。
实验例1：ELISA方法测定抗LAG3-抗PD-1双特异性抗体与抗原的结合活性
1.间接ELISA方法分别测定BS-PL021A、BS-PL022B、BS-PL023C、Bs-PLV02、14C12H1L1(hG1TM)与抗原PD-1-mFc的结合活性。具体方法如下：
将人PD-1-mFc，0.5μg/mL包被酶标板置于4℃孵育过夜，然后使用PBST洗板包被了抗原的酶标板1次，再使用1％BSA的PBS溶液作为封闭液在37℃下对酶标板进行封闭2小时。酶标板封闭结束后用PBST洗板3次，加入PBST溶液梯度稀释的抗体(抗体稀释梯度详见表2)，加入待测抗体的酶标板置于37℃条件下孵育30分钟，孵育完成后用PBST洗板3次。洗板后加入1:5000比例稀释的HRP标记羊抗人IgG FC(H+L)(Jackson,货号：109-035-098)二抗工作液，然后置于37℃条件下孵育30分钟。孵育完成后使用PBST洗板4次，后加入TMB(Neogen，308177)避光 显色5min，加入终止液终止显色反应。立即把酶标板放入酶标仪中，选择450nm光波长读取酶标板各孔的OD数值。用SoftMax Pro 6.2.1软件对数据进行分析处理。
检测结果如表2和图1所示。
表2：ELISA检测BS-PL021A、BS-PL022B、BS-PL023C、Bs-PLV02、14C12H1L1(hG1TM)与人PD-1-mFc的结合
Figure PCTCN2022122556-appb-000044
由图1可知，BS-PL021A、BS-PL022B、BS-PL023C、Bs-PLV02、14C12H1L1(hG1TM)与抗原人PD-1-mFc能够有效地结合，并且其结合效率呈剂量依赖关系。通过对结合的抗体进行吸光度定量分析，曲线模拟计算获得抗体BS-PL021A、BS-PL022B、BS-PL023C、Bs-PLV02、14C12H1L1(hG1TM)(作为对照)的结合效率EC 50分别为0.066nM、0.074nM、0.046nM、0.103nM、0.02nM。
结果表明，在相同实验条件下，BS-PL021A、BS-PL022B、BS-PL023C结合PD-1-mFc的活性与同靶点阳性对照14C12H1L1(hG1TM)基本相当，提示BS-PL021A、BS-PL022B、BS-PL023C、Bs-PLV02具有有效结合PD-1-mFc的活性。
2.间接ELISA方法分别测定BS-PL021A、BS-PL022B、BS-PL023C、Bs-PLV02、Relatlimab、H7L8(hG1WT)与抗原LAG3-mFc的结合活性。
具体方法如下：
将人LAG3-mFc(康方生物，批号：20200417)，2μg/mL包被酶标板置于4℃ 孵育过夜，然后使用PBST洗板包被了抗原的酶标板1次，再使用1％BSA的PBS溶液作为封闭液在37℃下对酶标板进行封闭2小时。酶标板封闭结束后用PBST洗板3次，加入PBST溶液梯度稀释的抗体(抗体稀释梯度详见表3)，加入待测抗体的酶标板置于37℃条件下孵育30分钟，孵育完成后用PBST洗板3次。洗板后加入1:5000比例稀释的HRP标记羊抗人IgG FC(H+L)(Jackson,货号：109-035-098)二抗工作液，然后置于37℃条件下孵育30分钟。孵育完成后使用PBST洗板4次，后加入TMB(Neogen，308177)避光显色5min，加入终止液终止显色反应。立即把酶标板放入酶标仪中，选择450nm光波长读取酶标板各孔的OD数值。用SoftMax Pro 6.2.1软件对数据进行分析处理。
检测结果如表3和图2所示。
表3：ELISA检测BS-PL021A、BS-PL022B、BS-PL023C、Bs-PLV02、Relatlimab、H7L8(hG1WT)与LAG3-mFc的结合
Figure PCTCN2022122556-appb-000045
由图2可知，BS-PL021A、BS-PL022B、BS-PL023C、Bs-PLV02、Relatlimab、H7L8(hG1WT)与抗原人LAG3-mFc能够有效的结合，并且其结合效率呈剂量依赖关系，各剂量的吸光度强度见表3，通过对结合的抗体进行吸光度定量分析，曲线模拟计算获得抗体BS-PL021A、BS-PL022B、BS-PL023C、Bs-PLV02、Relatlimab(作为阳性对照)、H7L8(hG1WT)(作为对照)的结合效率EC 50分别为0.073nM、0.081nM、0.377nM、0.685nM、0.106nM、0.045nM。
以上实验结果表明，在相同实验条件下，BS-PL021A、BS-PL022B、H7L8(hG1WT)具有有效结合LAG3-mFc的活性，且BS-PL021A、BS-PL022B、H7L8(hG1WT)结合人LAG3-mFc的活性强于靶点阳性药Relatlimab。特别是H7L8(hG1WT)，其结合人LAG3-mFc的活性显著强于靶点阳性药Relatlimab。
实验例2：竞争ELISA方法分别测定抗体抗LAG3-抗PD-1双特异性抗体与人 PDL1-mFc竞争结合人PD-1-mFc-Biotin的活性
将人PDL1-mFc(PD-L1Genbank ID:NP_054862.1，mFc SEQ ID NO:)以2μg/mL包被酶标板后，4℃孵育过夜。孵育结束后用1％BSA的PBS溶液在37℃条件下对酶标板进行封闭2小时，封闭结束后洗板三次并拍干。在稀释板上以80nM(终浓度40nM)作为起始浓度进行一系列3倍稀释比例将抗体进行梯度稀释至7个浓度，并设空白对照，然后加入等体积的1.2μg/mL(终浓度0.6μg/mL)的人PD-1-mFc-Biotin溶液，和抗体混匀后在室温孵育10分钟。然后将反应后的混合液加入到包被好的酶标板，在37℃条件下孵育30分钟。孵育结束用PBST洗板三次并拍干，加入SA-HRP(KPL，14-30-00)工作液，在37℃条件下孵育30分钟。孵育完成后洗板四次拍干，后加入TMB(Neogen，308177)避光显色5min，加入终止液终止显色反应。立即把酶标板放入酶标仪中，选择450nm光波长读取酶标板各孔的OD数值，用SoftMax Pro 6.2.1软件对数据进行分析处理。
检测结果如图3所示。各剂量的OD值见表4。通过对结合的抗体进行吸光强度定量分析，曲线模拟抗体阻断人PD-1-mFc-Biotin与其配体人PDL1-mFc的结合效率获得竞争结合EC 50(表4)。
表4：BS-PL021A、BS-PL022B、BS-PL023C、Bs-PLV02、14C12H1L1(hG1TM)与人PDL1-mFc竞争结合人PD-1-mFc-Biotin的活性检测结果
Figure PCTCN2022122556-appb-000046
Figure PCTCN2022122556-appb-000047
结果表明，BS-PL021A、BS-PL022B、BS-PL023C、Bs-PLV02、14C12H1L1(hG1TM)(作为对照)能有效地阻断抗原人PD-1-mFc-Biotin与其配体人PDL1-mFc的结合，且阻断效率呈现剂量依赖关系，BS-PL021A、BS-PL022B、BS-PL023C、Bs-PLV02、14C12H1L1(hG1TM)阻断人PD-1-mFc-Biotin与其配体人PDL1-mFc结合的EC 50分别为3.031nM、3.462nM、2.982nM、5.045nM、2.606nM。BS-PL021A、BS-PL022B、BS-PL023C阻断人PD-1-mFc-Biotin与其配体人PDL1-mFc结合的效率与14C12H1L1(hG1TM)基本相当。
实验例3：FACS检测抗LAG3-抗PD-1双特异性抗体的结合活性
1.FACS检测抗LAG3-抗PD-1双特异性抗体与293T-PD1膜表面PD-1的结合活性
收集对数生长期293T-PD1细胞，按3×10 5个细胞/孔将细胞转移至尖底96孔板中，加入100μL 1％PBSA，350×g离心5min，去上清。加入PBSA稀释的抗体100μL(终浓度分别为300、100、33.3、11.1、3.7、1.23、0.123和0.0123nM)，轻柔混匀后于冰上孵育1h。加入100μL 1％PBSA,350×g离心5min，去上清，并用200μL 1％PBSA洗涤两次。加入400倍稀释的FITC标记羊抗人IgG二抗(Jackson，货号：109-095-098)重悬混匀，冰上避光孵育0.5h。加入100μL 1％PBSA,350xg离心5min，去上清，并用200μL 1％PBSA洗涤两次。加入400μL 1％PBSA重悬细胞沉淀，并转移至流式管，FACSCalibur检测。
实验结果如表5和图4所示，14C12H1L1(hG1TM)、BS-PL021A、BS-PL022B、Bs-PLV02、Bi-PGV02、Nivolumab、Pembrolizumab均可特异性与293T-PD1细胞膜表面PD-1受体结合。
表5：FACS检测14C12H1L1(hG1TM)、BS-PL021A、BS-PL022B、Bs-PLV02、Bi-PGV02、Nivolumab、Pembrolizumab 293T-PD1细胞表面PD-1的结合活性
Figure PCTCN2022122556-appb-000048
Figure PCTCN2022122556-appb-000049
在相同实验条件下，14C12H1L1(hG1TM)、BS-PL021A、BS-PL022B、Bs-PLV02、Bi-PGV02、Nivolumab、Pembrolizumab与293T-PD1细胞结合的EC 50分别为5.351nM、6.851nM、6.066nM、6.866nM、7.206nM、3.073nM、3.970nM。以上实验结果表明，在相同实验条件下14C12H1L1(hG1TM)、BS-PL021A、BS-PL022B、Bs-PLV02、Bi-PGV02与293T-PD1细胞结合活性与对照抗体Nivolumab、Pembrolizumab相当，提示14C12H1L1(hG1TM)、BS-PL021A、BS-PL022B、Bs-PLV02、Bi-PGV02具有有效结合293T-PD1细胞膜表面PD-1的活性。
2.FACS检测抗LAG3-抗PD-1双特异性抗体与293T-LAG3细胞膜表面LAG3的结合活性
常规胰酶消化对数期293T-LAG3细胞，按3×10 5个细胞/孔将细胞转移至尖底96孔板中，加入100μL 1％PBSA，350×g离心5min，去上清。加入1％PBSA稀释的抗体100μL(终浓度分别为300、100、33.3、11.1、3.7、1.23、0.123、0.0123和0.00123nM)，混匀后于冰上孵育1h。加入100μL 1％PBSA，350×g离心5min，去上清，并用200μL1％PBSA洗涤两次。加入300倍稀释的FITC标记羊抗人IgG二抗(Jackson,货号：109-095-098)重悬混匀，冰上避光孵育0.5h。加入500μLPBSA,350×g离心5min，去上清，并用200μL 1％PBSA洗涤两次。加入300μL 1％PBSA重悬细胞沉淀，并转移至流式管，FACSCalibur检测。
实验结果如表6和图5所示。
表6：FACS检测BS-PL022B、Relatlimab与293T-LAG3细胞表面LAG33的结合活性。
  Relatlimab BS-PL022B
EC 50(nM) 4.113 3.213
结果显示，在相同实验条件下，BS-PL022B、Relatlimab与293T-LAG3细胞膜表面LAG3结合的EC 50分别为3.213nM、4.113nM，可见BS-PL022B与293T-LAG3细胞膜表面LAG3的结合活性高于Relatlimab。
以上实验结果表明，BS-PL022B和同靶点阳性药Relatlimab均可呈剂量依赖性与293T-LAG3细胞膜表面LAG-3特异性结合。提示BS-PL022B具有有效结合 293T-LAG3膜表面LAG3的活性，且其结合能力强于Relatlimab。
实验例4：抗LAG3-抗PD-1双特异性抗体对细胞膜表面抗原的竞争性结合
1、竞争流式细胞法测定抗LAG3-抗PD-1双特异性抗体与PDL1竞争结合细胞膜表面抗原PD-1。
取293T-PD1细胞常规消化，按3×10 5个细胞/孔将细胞转移至尖底96孔板中，加入100μL 1％PBSA离心洗涤。加入相应梯度稀释的抗体(终浓度分别为300、100、33.3、11.1、3.7、1.23、0.123和0.0123nM)，每样本100μL，冰上孵育30min。每管加入100μL PDL-1-mFc，混匀，使得最终浓度为20nM，冰上孵育1h。350×g离心5min，去上清；用200μL 1％PBSA洗涤两次。加入100μL 400倍稀释的FITC羊抗鼠IgG/IgM抗体(BD，货号：555988)，空白样本则加入100μL 1％PBSA，混匀后冰上避光孵育30min。洗涤离心后重悬，转移至流式上样管，上机测试。
结果如图6所示，各样品的EC 50值见表7。通过荧光定量分析和曲线拟合，计算出抗体Nivolumab、Pembrolizumab、14C12H1L1(hG1TM)、BS-PL022B的竞争结合EC 50分别为3.608nM、2.769nM、2.511nM、5.123nM。
表7：FACS检测Nivolumab、Pembrolizumab、14C12H1L1(hG1TM)、BS-PL022B竞争结合293T-PD-1表面抗原的荧光强度分析
Figure PCTCN2022122556-appb-000050
结果显示，BS-PL022B抗体能有效地阻断PD-L1对293T-PD1宿主细胞表面的PD-1的结合，且呈剂量依赖关系。
2、竞争流式细胞法测定抗LAG3-抗PD-1双特异性抗体与LAG3-mG1Fc竞争结合细胞膜表面抗原MHC II。
按实验设计稀释抗体和LAG3-mG1Fc，1:1混合均匀，使得LAG3-mG1Fc(康方生产，批号20190508)终浓度为3nM，抗体终浓度为300、100、33.3、11.1、3.7、1.23、0.123、0.0123、0.00123nM，冰上孵育30min；收集Raji细胞(中国科学院上海生命科学研究院细胞资源中心，货号：TCHu 44)，每样本300000细胞接种至96孔尖底板，补加1％PBSA，500xg离心5min弃上清；100μL抗体与蛋白预孵液重悬细胞，设计空白对照(细胞+PBSA+PBSA)、阴性对照(细胞+PBSA+二抗)及同型对照， 冰上避光孵育1h；加入100μL 1％PBSA，500xg离心5min，去上清；200μL 1％PBSA重悬细胞，500xg离心5min，去上清，重复洗涤一次；100μL APC goat anti mouse IgG二抗(BioLegend，货号：405308)(1:300稀释)重悬细胞，空白对照则用100μL 1％PBSA重悬，冰上避光孵育30min；加入100μL 1％PBSA，500xg离心5min，去上清；200μL 1％PBSA重悬细胞，500xg离心5min，去上清，重复洗涤一次；200μL 1％PBSA重悬细胞，转移至流式上样管，上机测试。
结果如图7、表8所示，各样品的EC 50值见表。通过荧光定量分析和曲线拟合，计算出抗体Relatlimab、BS-PL022B的竞争结合EC 50分别为0.9689nM、1.306nM。
表8：FACS检测Relalimab、BS-PL022B竞争结合Raji细胞表面抗原的荧光强度分析
  Relatlimab BS-PL022B
EC 50(nM) 0.9689 1.306
R2 0.9964 0.9948
结果显示，BS-PL022B抗体能有效地阻断LAG-3对Raji宿主细胞表面的MHC II的结合，且呈剂量依赖关系。
实验例5：抗LAG3-抗PD-1双特异性抗体阻断实验
1、抗LAG3-抗PD-1双特异性抗体阻断LAG3与MHCII结合
收集Jurkat-NFAT-PD1-LAG3(康方生物构建，P9，活率：97.75％)细胞和Raji细胞(中国科学院上海生命科学研究院细胞资源中心，货号：TCHu 44)，110×g离心5min，去上清，用1640培养基(含10％FBS)重悬细胞，计数。将Jurkat-NFAT-PD1-LAG3细胞按10×10 4个细胞/孔接种至黑底96孔板(Corning，型号：3916)，25μL/孔；按实验设计加入抗体，30μL/孔(终浓度分别为900nM、300nM、100nM、33.3nM、3.3nM、0.3nM、0.03nM和0.003nM)，置于37℃，5％CO 2培养箱中预先孵育30min。将SEE(Staphylococcal enterotoxin E)(终浓度0.05ng/mL，Toxin Technology，货号：ET404)和Raji细胞置于37℃，5％CO 2培养箱中孵育30min；孵育30min后，将Raji细胞按2×10 4/孔、25μL/孔加入到96孔板中，体系终体积为80μL；混匀后置于37℃，5％CO 2培养箱中孵育16h。取出培养板，平衡至室温，加 入Bright-Glo  TM Luciferase Assay System(Promega，货号：E2650)80μL/孔，避光孵育2min后读取RLU值。
结果如图8A、图8B和表9所示。
表9：抗LAG3-抗PD-1双特异性抗体阻断LAG3与MHCII结合
  Relatlimab Bs-PLV02 Bi-PGV02 BS-PL022B
EC 50(nM) 8.563 1.210 1.483 0.9762
结果显示，Bs-PLV02、Bi-PGV02、BS-PL022B和Relatlimab阻断LAG3与MHCII结合的EC 50(nM)分别为1.21nM、1.483nM、0.9762nM、8.563nM。Bs-PLV02、Bi-PGV02、BS-PL022B阻断LAG3与MHCII结合的能力强于阳性对照抗体Relatlimab。
2、抗LAG3-抗PD-1双特异性抗体阻断PD-1与PD-L1结合
PDL1aAPC/CHO-K1细胞(Promega，货号：J1081A)4×10 4个细胞/孔接种于96孔平底黑板(Corning，型号：3916)培养过夜，每孔100μL(培养基为该细胞的生长培养基：Ham F-12+10％FBS)。次日，去板内培养基，加入PD1效应细胞(Promega，货号：J1121A)5×10 4个细胞/孔，40μL/孔(培养基为：1640+10％FBS)；加入抗体，40μL/孔(终浓度分别为1000nM、300nM、100nM、33.3nM、11.1nM、3.7nM、1.23nM、0.123nM和0.0123nM)，与此同时设置同型对照组和阴性对照组，终体积为80μL/孔；置于培养箱孵育6h。取出培养板，平衡至室温，加入Bright-GloTM Luciferase Assay System(Promega，货号：E2650)80μL/孔，避光孵育2min后读取RLU值。
结果如图9A、图9B和表10所示。
表10：抗LAG3-抗PD-1双特异性抗体阻断PD-1与PD-L1结合
Figure PCTCN2022122556-appb-000051
结果显示，Nivolumab、Pembrolizumab、14C12H1L1(hG1TM)、BS-PL022B阻断PD-1和PD-L1结合的EC 50(nM)分别为4.089nM、1.281nM、5.219nM、20.01nM。结果说明Nivolumab、Pembrolizumab、14C12H1L1(hG1TM)、BS-PL022B均能阻断PD-1和PD-L1结合。
3、抗LAG3-抗PD-1双特异性抗体同时阻断LAG-3与MHCII以及PD-1与PD-L1 结合
收集Jurkat-NFAT-PD1-LAG3细胞和Raji-PDL1细胞，110×g离心5min去上清，用1640培养基(含10％FBS)重悬细胞，计数。
Jurkat-NFAT-PD1-LAG3细胞10×10 4个细胞/孔接种至黑底96孔板(Corning，型号：3916)，25μL/孔，按实验设计加入抗体，30μL/孔(终浓度分别为3000、1000、300、30、3、0.3、0.03和0.003nM)，置于37℃，5％CO 2培养箱中预先孵育30min；向Raji-PDL1细胞加入SEE(Staphylococcal enterotoxin E)(终浓度0.1ng/mL)后置于37℃，5％CO 2培养箱中孵育30min。孵育30min后，将Raji-PDL1细胞按3×10 4/孔、25μL/孔加入到96孔板中，体系终体积为80μL；混匀后置于37℃，5％CO 2培养箱中孵育15h。取出培养板，平衡至室温，加入Bright-Glo  TMLuciferase Assay System(Promega，货号：E2650)80μL/孔，避光孵育5min后读取RLU值。
结果如图10A、图10B和表11所示。
表11：抗LAG3-抗PD-1双特异性抗体同时阻断LAG-3与MHCII以及PD-1与PD-L1结合
Figure PCTCN2022122556-appb-000052
结果显示，Pembrolizumab、Relatlimab、14C12H1L1(hG1TM)、14C12H1L1(hG1TM)+Relatlimab、BS-PL022B同时阻断PD1和PD-L1结合以及LAG3与MHCII结合的EC 50(nM)分别为24.01nM、5.525nM、44.86nM、29.75nM、16.21nM。Pembrolizumab、Relatlimab、14C12H1L1(hG1TM)、14C12H1L1(hG1TM)+Relatlimab、Bs-PL022B均能同时阻断PD-1和PD-L1结合以及LAG3与MHCII结合，且Bs-PL022B的阻断能力要强于其他抗体。
实验例6：抗LAG3-抗PD-1双特异性抗体桥连实验
常规消化CHO-K1(中国医学科学院基础医学研究所细胞资源中心，货号：3111C0001CCC000004)，CHO-K1-PD1(康方生物构建)，CHO-K1-LAG3(康方生物构建)细胞，170×g离心5min，弃上清；用完全培养基重悬细胞后，计数及活率。 对CHO-K1-PD1细胞染CFSE(CFSE Cell Division Tracker Kit，Biolegend，货号：423801)(处理浓度1μM，1mL/10×10 6个细胞)，CHO-K1-LAG3细胞染Far red(Thermofisher，货号：C34564)(处理浓度0.3μM，1mL/10×10 6个细胞)，CHO-K1细胞染Far red或CFSE，培养箱中染色20min；完全培养基终止染色，170×g离心5min弃上清，再加入完全培养基培养箱孵育10min，170×g离心5min弃上清，重复洗涤1次，完全培养基重悬计数。染色后的CHO-K1-PD1、CHO-K1-LAG3及CHO-K1细胞按1.5×10 5个细胞/孔分别转移至尖底96孔板中，补加buffer(PBS+1％人血清)(人血清，中科晨宇，货号：168014-100mL)，离心弃上清；按实验设计向CHO-K1-PD1加入抗体(终浓度为30、3、1、0.3、0.1nM)，向CHO-K1-LAG3补加buffer或加入抗体(终浓度为30nM、3nM、1nM、0.3nM、0.1nM)，向CHO-K1补加buffer，100μL/孔，冰上孵育60min。
加100μL buffer，350×g离心5min，弃上清，200μL buffer重复洗涤2次。用100μL buffer分别重悬CHO-K1-LAG3及CHO-K1细胞，并转移至相应的CHO-K1-PD1样品孔，混匀，1.5×10 5个细胞/孔冰上避光孵育40min。加200μL buffer重悬细胞，转至流式上样管，上机测试。
结果如图11所示。与同型对照相比，抗LAG3-抗PD-1双特异性抗体可同时结合CHO-K1-PD1和CHO-K1-LAG3细胞，将两个细胞桥连起来，而14C12H1L1(hG1TM)、Relatlimab即便是联用也无此作用。
实验例7：混合淋巴细胞反应MLR检测抗LAG3-抗PD-1双特异性抗体促IFN-γ 和IL-2分泌的生物活性
1.抗LAG3-抗PD-1双特异性抗体促Raji-PDL1混合淋巴反应体系分泌IFN-γ的生物活性检测
Raji-PDL1细胞常规传代培养；复苏PBMC，用10mL 1640完全培养基培养，0.5μg/mL的SEB(Staphylococcal enterotoxin B)(德诺泰克，货号：S010201)刺激两天。Raji-PDL1细胞用终浓度为2μg/mL的MMC(Stressmarq，货号：SIH-246-10MG)处理，置于37℃、5％CO 2二氧化碳培养箱中1小时；收集经SEB刺激2天后PBMC和经MMC处理1小时的Raji-PDL1细胞，用PBS洗涤两次；完全培养基重悬计数后，各10×10 4个细胞/孔加入到U型96孔板(Corning，型号：3799)中共培养。按实验设计加入抗体(终浓度为300nM、30nM、3nM)，于培养箱共培 养3天；3天后，1200rpm离心5min，收集细胞培养上清，ELISA法进行IFN-γ检测。
如图12A所示，人PBMC和Raji-PDL1细胞混合培养后对PBMC的IFN-γ的分泌具有显著促进作用，在混合培养体系中同时加入抗体能显著诱导PBMC进一步分泌IFN-γ，促IFN-γ分泌活性水平方面，BS-PL022B抗体的活性优于PD-1单靶点抗体14C12H1L1(hG1TM)以及LAG-3单靶点对照抗体Relatlimab。甚至与14C12H1L1(hG1TM)和体Relatlimab联用相比，BS-PL022B在两个不同抗体浓度水平(30nM和300nM)都具更佳的促IFN-γ分泌潜能。
2.抗LAG-3-抗PD-1双特异性抗体促Raji-PDL1混合淋巴反应体系分泌IL-2的生物活性检测
Raji-PDL1细胞常规传代培养；复苏PBMC，用10mL 1640完全培养基培养，添加0.5μg/mL的SEB(Staphylococcal enterotoxin B)(德诺泰克，货号：S010201)刺激两天。Raji-PDL1细胞用终浓度为2μg/mL的MMC(Stressmarq，货号：SIH-246-10MG)处理，置于37℃、5％CO 2二氧化碳培养箱中1小时。收集经SEB刺激2天后的PBMC和经MMC处理1小时的Raji-PDL1细胞，用PBS洗两次；完全培养基重悬计数后，各10×10 4个细胞/孔加入U型96孔板(Corning，型号：3799)中共培养。按实验设计加入抗体(终浓度为300nM、30nM、3nM)，共培养3天；3天后，1200rpm离心5min，收集细胞培养上清，ELISA法进行IL-2检测。
如图12B所示，人PBMC和Raji-PDL1细胞混合培养后对PBMC的IL-2的分泌有一定的促进作用，在混合培养体系中同时加入抗体能显著诱导PBMC进一步分泌IL-2，具有显著剂量依赖关系，促IL-2分泌活性水平方面，与PD-1单靶点抗体14C12H1L1(hG1TM)以及LAG-3单靶点对照抗体Relatlimab相比，BS-PL022B在三个不同抗体浓度水平都具更佳的促IL-2分泌潜能。甚至与14C12H1L1(hG1TM)和体Relatlimab联用相比，BS-PL022B在三个不同抗体浓度水平都具更佳的促IL-2分泌潜能。
实验例8：BS-PL022B与Fc受体FcγRI亲和力检测
Fc受体FcγRI(又名CD64)可与IgG抗体的Fc端结合，参与抗体依赖细胞介导的细胞毒作用(ADCC)。治疗性抗体与Fc受体结合的能力影响到该抗体的安全性和有效性。本实验使用Fortebio Octet分子相互作用仪检测BS-PL022B与FcγRI的亲和力常数，以评价抗体的ADCC活性。
Fortebio Octet分子相互作用仪检测相应抗体与FcγRI的亲和力常数实验方法简述如下：样品稀释缓冲液为PBS，0.02％Tween-20，0.1％BSA，pH7.4的溶液。HIS1K传感器上加入浓度为1μg/mL的FcγRI(购自Sinobio)溶液，固定时间50s，以使得FcγRI固定于传感器表面。抗体与FcγRI的结合和解离参数的测定均在缓冲液中进行，抗体浓度为3.12-50nM(两倍梯度稀释)。样品板震动速率为1000rpm，检测温度为30℃，频率为5.0Hz。数据以1:1模型拟合分析，得到亲和力常数。
FcγRI与BS-PL022B的亲和力常数测定结果如表12和图13－图14所示。
表12：BS-PL022B与FcγRI结合的动力学参数
Figure PCTCN2022122556-appb-000053
N/A表示抗体与抗原没有结合或结合信号极低，没有对结果进行分析，故未能获得相应数据。
结果显示，H7L8(hG1WT)能够与FcγRI结合，亲和力常数为6.59E-09M，BS-PL022B由于与FcγRI没有结合或结合信号极低，没有对结果进行分析，故而未能获得相应数据。
结果表明，BS-PL022B与FcγRI的结合活性被有效地消除。
实验例9：BS-PL022B与Fc受体FcγRIIIa及其亚型的亲和力测定
(1)FcγRIIIa_V158与BS-PL022B的亲和力常数测定
Fc受体FcγRIIIa_V158(又名CD16a_V158)，可与IgG抗体的Fc端结合，介导ADCC效应。实验中使用Fortebio Octet分子相互作用仪检测BS-PL022B与FcγRIIIa_V158的亲和力常数，以评价抗体的ADCC活性。
Fortebio Octet分子相互作用仪检测相应抗体亲和力常数实验方法简述如下：样品稀释缓冲液为PBS，0.02％Tween-20，0.1％BSA，pH7.4。5μg/mL的FcγRIIIa_V158固定在HIS1K传感器上，时间60s，传感器在缓冲液中平衡60s，固定在传感器上的 FcγRIIIa_V158与各抗体结合，抗体浓度为31.25-500nM(两倍稀释)，时间60s，抗体在缓冲液中解离，时间60s。样品板震动速率为1000rpm，检测温度为30℃，频率为5.0Hz。数据以1:1模型拟合分析，得到亲和力常数。
FcγRIIIa_V158与BS-PL022B的亲和力常数测定结果如表13和图15－图16所示。
表13：BS-PL022B与FcγRIIIa_V158结合的动力学参数
抗体 K D(M) kon(1/Ms) SE(kon) kdis(1/s) SE(kdis) Rmax(nm)
BS-PL022B N/A N/A N/A N/A N/A N/A
H7L8(hG1WT) 8.77E-08 4.95E+05 2.36E+04 4.34E-02 7.00E-04 0.18-0.59
N/A表示抗体与抗原没有结合或结合信号极低，没有对结果进行分析，故未能获得相应数据。
结果显示，H7L8(hG1WT)能够与FcγRIIIa_V158结合，亲和力常数为8.77E-08M，BS-PL022B由于与FcγRIIIa_V158没有结合或结合信号极低，没有对结果进行分析。
结果表明，BS-PL022B与FcγR IIIa_V158的结合活性被有效地消除。
(2)FcγRIIIa_F158与BS-PL022B的亲和力常数测定
Fc受体FcγRIIIa_F158(又名CD16a_F158)可与IgG抗体的Fc端结合，介导ADCC。本实验使用Fortebio Octet分子相互作用仪检测BS-PL022B与FcγRIIIa_F158的亲和力常数，以评价各抗体的ADCC活性。
Fortebio Octet分子相互作用仪检测TF01与FcγRIIIa_F158的亲和力常数实验方法简述如下：样品稀释缓冲液为PBS，0.02％Tween-20，0.1％BSA，pH7.4。5μg/mL的FcγRIIIa_F158固定在HIS1K传感器上，时间120s，传感器在缓冲液中平衡60s，固定在传感器上的FcγRIIIa_F158与各抗体结合，抗体浓度为31.25-500nM(两倍稀释)，时间60s，抗体在缓冲液中解离，时间60s。样品板震动速率为1000rpm，检测温度为30℃，频率为5.0Hz。数据以1:1模型拟合分析，得到亲和力常数。
FcγRIIIa_F158与BS-PL022B的亲和力常数测定结果如表14和图17－图18所示。
表14：TF01FcγRIIIa_F158结合的动力学参数
抗体 K D(M) kon(1/Ms) SE(kon) kdis(1/s) SE(kdis) Rmax(nm)
BS-PL022B N/A N/A N/A N/A N/A N/A
H7L8(hG1WT) 3.64E-07 4.15E+05 3.03E+04 1.51E-01 3.46E-03 0.06-0.25
N/A表示抗体与抗原没有结合或结合信号极低，没有对结果进行分析，故未能获得相应数据。
结果显示，H7L8(hG1WT)能够与FcγRIIIa_F158结合，亲和力常数为3.64E-07M，BS-PL022B与FcγRIIIa_F158没有结合或结合信号极低，没有对结果进行分析，故而未能获得相应数据。
结果表明，BS-PL022B与FcγRIIIa_F158的结合活性被有效地消除。
实验例10：BS-PL022B与Fc受体FcγRIIa及其亚型的亲和力测定
(1)FcγRIIa_H131与BS-PL022B的亲和力常数测定
Fc受体FcγRIIa_H131(又名CD32a_H131)可与IgG抗体的Fc端结合，参与抗体依赖性细胞介导的细胞吞噬作用(ADCP)或者抗体依赖细胞介导的细胞毒作用(ADCC)。治疗性抗体与Fc受体结合的能力影响到该抗体的安全性和有效性。本实验使用Fortebio Octet分子相互作用仪检测BS-PL022B与FcγRIIa_H131的亲和力常数，以评价各待测抗体与Fc受体的结合能力。
Fortebio Octet分子相互作用仪检测BS-PL022B与FcγRIIa_H131的亲和力常数实验方法简述如下：样品稀释缓冲液为PBS，0.02％Tween-20，0.1％BSA，pH7.4。5μg/mL的FcγRIIa_H131固定在NTA传感器上，固定高度约为1.0nm，传感器在缓冲液中平衡60s，固定在传感器上的FcγRIIa_H131与各抗体结合，抗体浓度为12.5-200nM(两倍梯度稀释)，时间60s，抗体在缓冲液中解离，时间60s。样品板震动速率为1000rpm，检测温度为30℃，频率为5.0Hz。数据以1:1模型拟合分析，得到亲和力常数。
FcγRIIa_H131与BS-PL022B的亲和力常数测定结果如表15和图19－图20所示。
表15：BS-PL022B与FcγRIIa_H131结合的动力学参数
Sample ID K D(M) kon(1/Ms) SE(kon) kdis(1/s) SE(kdis) Rmax(nm)
BS-PL022B N/A N/A N/A N/A N/A N/A
H7L8(hG1WT) 1.78E-07 4.19E+05 3.55E+04 7.44E-02 1.66E-03 1.14-1.46
N/A表示抗体与抗原没有结合或结合信号极低，没有对结果进行分析，故未能获得相应数据。
结果显示，H7L8(hG1WT)能够与FcγRIIa_H131结合，亲和力常数为1.78E-07M，BS-PL022B与FcγRIIa_H131没有结合或结合信号极低，没有对结果进行分析，故而未能获得相应数据。
结果表明，BS-PL022B与FcγRIIa_H131的结合活性被有效地消除。
实验例11：FcγRIIb与BS-PL022B的亲和力常数测定
Fc受体FcγRIIb(又名CD32b)可与IgG抗体的Fc端结合。本实验使用Fortebio Octet分子相互作用仪检测各待测抗体与FcγRIIb的亲和力常数，以评价BS-PL022B与Fc受体的结合能力。
Fortebio Octet分子相互作用仪检测BS-PL022B与FcγRIIb的亲和力常数实验方法简述如下：样品稀释缓冲液为PBS，0.02％Tween-20，0.1％BSA，pH7.4。5μg/mL的FcγRIIb固定在NTA传感器上，固定高度约为1.0nm，传感器在缓冲液中平衡60s，固定在传感器上的hFCGR2B-his与各抗体结合，抗体浓度为12.5-200nM(两倍梯度稀释)，时间60s，抗体在缓冲液中解离，时间60s。样品板震动速率为1000rpm，检测温度为30℃，频率为5.0Hz。数据以1:1模型拟合分析，得到亲和力常数。
FcγRIIb与BS-PL022B的亲和力常数测定结果如表17和图21－图22所示。
表16：BS-PL022B与FcγRIIb结合的动力学参数
抗体 K D(M) kon(1/Ms) SE(kon) kdis(1/s) SE(kdis) Rmax(nm)
BS-PL022B N/A N/A N/A N/A N/A N/A
H7L8(hG1WT) 1.21E-07 3.74E+05 3.48E+04 4.53E-02 1.28E-03 0.12-0.34
N/A表示抗体与抗原没有结合或结合信号极低，没有对结果进行分析，故未能获得相应数据。
结果显示，H7L8(hG1WT)能够与FcγRIIb结合，亲和力常数为1.21E-07M，BS-PL022B由于与FcγRIIb没有结合或结合信号极低，没有对结果进行分析，故而未能获得相应数据。
结果表明，BS-PL022B与FcγRIIb的结合活性被有效地消除。
实验例12：BS-PL022B与C1q的亲和力测定
血清补体C1q可与IgG抗体的Fc端结合，介导CDC效应，治疗性抗体与C1q 结合的能力影响到该抗体的安全性和有效性。本实验使用Fortebio Octet分子相互作用仪检测BS-PL022B与C1q的亲和力常数，以评价抗体的CDC活性。
Fortebio Octet分子相互作用仪检测相应抗体与C1q的亲和力常数实验方法简述如下：样品稀释缓冲液为PBS，0.02％Tween-20，0.1％BSA，pH7.4。50μg/mL的抗体固定在FAB2G传感器上，固定高度约为2.0nm，传感器在缓冲液中平衡60s，固定在传感器上的抗体与抗原C1q结合,抗原浓度为0.625-10nM(两倍梯度稀释)，时间60s，抗原抗体在缓冲液中解离，时间60s。样品板震动速率为1000rpm，检测温度为30度，检测频率为5.0Hz。数据以1:1模型拟合分析，得到亲和力常数。数据采集软件为Fortebio Data Acquisition 7.0，数据分析软件为Fortebio Data Analysis 7.0。
BS-PL022B与C1q的亲和力常数测定结果如表18和图23－图24所示。
表17：BS-PL022B与C1q结合的动力学参数
抗体 K D(M) kon(1/Ms) SE(kon) kdis(1/s) SE(kdis) Rmax(nm)
BS-PL022B N/A N/A N/A N/A N/A N/A
H7L8(hG1WT) 1.75E-09 2.05E+06 3.27E+04 3.58E-03 5.24E-05 0.56-0.71
N/A表示抗体与抗原没有结合或结合信号极低，没有对结果进行分析，故未能获得相应数据。
结果显示，H7L8(hG1WT)能够与C1q结合，亲和力常数为1.75E-09M，BS-PL022B由于与C1q没有结合或结合信号极低，没有对结果进行分析，故而未能获得相应数据。
结果表明，BS-PL022B与C1q的结合活性被有效地消除。
实验例13：BS-PL022B对CHO-K1-PD1-LAG3的抗体介导的细胞吞噬的活性
常规收集Jurkat-NFAT-CD64-CD32R(康方生物构建)，CHO-K1-PD1-LAG3(康方生物构建)细胞，110xg离心5min，弃上清；用1640+4％FBS重悬后计数并测定细胞活率，调整细胞浓度；按实验设计用1640+4％FBS将抗体稀释至50nM、5nM、0.5nM(工作浓度为10nM、1nM、0.1nM或5nM、0.5nM、0.05nM)，对照抗体稀释至50nM(工作浓度为10nM)；按40μL/样本将Jurkat-NFAT-CD64-CD32R细胞悬液加至96孔黑板中(4万细胞/孔)；按40μL/样本将靶细胞CHO-K1-PD1-LAG3细胞(4万细胞/孔)加至已含Jurkat-NFAT-CD64-CD32R的样本中；按20μL/孔将抗体加至相应样本，混匀；同时设置空白对照和同型性对照，置于培养箱孵育5h；按50μL/孔将Bright-GloTM Luciferase Assay System(Promega，货号：E2650)加至样 本，混匀，读板。
结果如图25所示。
结果显示，同浓度时14C12H1L1(G1WT)+H7L8(hG1WT)、Nivolumab+Relatlimab具有ADCP效应；而BS-PL022B不具有ADCP效应。
实验例14：抗LAG3-抗PD-1双特异性抗体在小鼠肿瘤细胞皮下移植模型中的药 效学评价
为检测抗LAG3-抗PD-1双特异性抗体体内抑瘤活性，首先用CT26结肠癌细胞(购自江苏集萃药康生物科技股份有限公司)，接种于7.1-7.3周龄的雌性BALB/c-hPD1/hLAG3小鼠(购自江苏集萃药康生物科技股份有限公司)小鼠右侧背部大腿上方皮下。分组当天定义为D0天。给药方式为腹腔注射(ip)，每周给药2次(BIW)，共给药6次。造模与具体给药方式见表18。给药后测量各组肿瘤的长宽，计算肿瘤体积。
表18：抗LAG3-抗PD-1双特异性抗体治疗BALB/c-hPD1/hLAG3小鼠CT26结肠癌细胞移植瘤模型的给药方案
Figure PCTCN2022122556-appb-000054
注：同型对照15mg/kg、Relatlimab 15mg/kg与BS-PL022B 20mg/kg的剂量为等摩尔浓度；Relatlimab 3mg/kg与BS-PL022B 4mg/kg的剂量为等摩尔浓度。
结果如图26所示。结果显示，相比同型对照抗体，抗LAG3-抗PD-1双特异性抗体BS-PL022B以及阳性对照抗体Relatlimab均能有效地抑制小鼠肿瘤的生长。结果表明，抗LAG3-抗PD-1双特异性抗体BS-PL022B抑瘤效果显著优于阳性对照抗体Relatimab。
此外，如图27所示，荷瘤鼠对受试药BS-PL022B的耐受性均良好，各组对荷瘤小鼠体重无影响。
尽管本发明的具体实施方式已经得到详细的描述，本领域技术人员将会理解。根据已经公开的所有教导，可以对那些细节进行各种修改和替换，这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。

Claims (36)

  1. 一种双特异性抗体，其包括第一蛋白功能区和第二蛋白功能区，其中：
    所述第一蛋白功能区靶向LAG3，
    所述第二蛋白功能区靶向不同于LAG3的靶点(例如，PD-1)，
    其中，所述第一蛋白功能区为抗LAG3抗体或其抗原结合片段，包含重链可变区和轻链可变区，其中，
    所述重链可变区包含：氨基酸序列分别如SEQ ID NOs:5-7所示的HCDR1-HCDR3；并且所述轻链可变区包含：氨基酸序列分别如SEQ ID NOs:8-10所示的LCDR1-LCDR3。
  2. 根据权利要求1所述的双特异性抗体，其中，
    所述抗LAG3抗体的重链可变区的氨基酸序列如SEQ ID NO:2所示，并且所述抗LAG3抗体的轻链可变区的氨基酸序列如SEQ ID NO:4所示。
  3. 根据权利要求1至2中任一权利要求所述的双特异性抗体，其中，所述抗LAG3抗体或其抗原结合片段选自Fab、Fab'、F(ab')2、Fd、Fv、dAb、互补决定区片段、单链抗体、人源化抗体、嵌合抗体或双抗体。
  4. 根据权利要求1至3中任一权利要求所述的双特异性抗体，其中，所述抗LAG3抗体以小于0.2nM，例如小于0.15nM、小于0.1nM、小于0.08nM、0.06nM或小于0.05nM或更小的EC 50结合人LAG3-mFc；优选地，所述EC 50通过间接ELISA方法测得。
  5. 根据权利要求1至4中任一权利要求所述的双特异性抗体，其中，
    所述抗LAG3抗体包括非-CDR区，且所述非-CDR区来自不是鼠类的物种，例如来自人抗体。
  6. 根据权利要求1至5中任一权利要求所述的双特异性抗体，其中，
    所述抗LAG3抗体，其恒定区来自人抗体；
    优选地，所述抗体的恒定区选自人IgG1、IgG2、IgG3或IgG4的恒定区。
  7. 根据权利要求1至6中任一权利要求所述的双特异性抗体，其中，
    所述抗LAG3抗体的重链恒定区为Ig gamma-1 chain C region(例如，如SEQ ID NO:39所示)或Ig gamma-4 chain C region(例如，如SEQ ID NO:45所示)；轻链恒定区为Ig kappa chain C region(例如，如SEQ ID NO:40所示)。
  8. 根据权利要求1至7中任一权利要求所述的双特异性抗体，其中，
    所述抗LAG3抗体为人IgG1亚型，
    其中，按照EU编号系统，所述抗体的重链恒定区具有如下突变：
    L234A和L235A，
    L234A和G237A，
    L235A和G237A，
    或者
    L234A、L235A和G237A；
    优选地，所述抗LAG3抗体的重链的氨基酸序列如SEQ ID NO:11所示，并且轻链的氨基酸序列如SEQ ID NO:12所示。
  9. 根据权利要求1至8中任一权利要求所述的双特异性抗体，其中，
    所述抗LAG3抗体为人IgG4亚型，
    其中，按照EU编号系统，所述抗体的重链恒定区具有如下突变：
    F234A和L235A，
    F234A和G237A，
    L235A和G237A，
    或者
    F234A、L235A和G237A；
    优选地，所述抗LAG3抗体的重链的氨基酸序列如SEQ ID NO:13所示，并且轻链的氨基酸序列如SEQ ID NO:12所示。
  10. 根据权利要求1至9中任一权利要求所述的双特异性抗体，其中，
    所述双特异性抗体为IgG-scFv模式。
  11. 根据权利要求1至10中任一权利要求所述的双特异性抗体，其中，
    所述第一蛋白功能区为抗LAG3抗体的抗体，并且所述第二蛋白功能区为单链抗体；或者
    所述第一蛋白功能区为单链抗体，并且所述第二蛋白功能区为靶向不同于LAG3的靶点的抗体。
  12. 根据权利要求1至11中任一权利要求所述的双特异性抗体，其中，所述第一蛋白功能区和第二蛋白功能区直接连接或者通过连接片段连接；
    优选地，所述连接片段为(GGGGS)m，m为正整数，例如1、2、3、4、5或6；
    优选地，所述连接片段为(GGGGS)nG，n为正整数，例如1、2、3、4、5或6。
  13. 根据权利要求1至12中任一权利要求所述的双特异性抗体，其中，所述第一蛋白功能区和第二蛋白功能区独立地为1个、2个或者2个以上。
  14. 根据权利要求1至13中任一权利要求所述的双特异性抗体，其中，所述单链抗体连接在抗体的重链的C末端。
  15. 根据权利要求1至14中任一权利要求所述的双特异性抗体，其包括：
    靶向LAG3的第一蛋白功能区，和
    靶向PD-1的第二蛋白功能区；
    其中，
    所述第一蛋白功能区为抗LAG3抗体，并且所述抗LAG3抗体为免疫球蛋白形式，
    所述第二蛋白功能区为抗PD-1的单链抗体。
  16. 根据权利要求15所述的双特异性抗体，其中，所述抗PD-1的单链抗体包含重链可变区和轻链可变区，其中，
    所述重链可变区包含：氨基酸序列分别如SEQ ID NOs:26-28所示的HCDR1-HCDR3；和
    所述轻链可变区包含：氨基酸序列分别如SEQ ID NOs:29-31所示的LCDR1-LCDR3。
  17. 根据权利要求15至16中任一权利要求所述的双特异性抗体，其中，所述抗PD-1的单链抗体，
    其重链可变区的氨基酸序列如SEQ ID NO:15所示，并且其轻链可变区的氨基酸序列如SEQ ID NO:17所示；或者
    其重链可变区的氨基酸序列如SEQ ID NO:19所示，并且其轻链可变区的氨基酸序列如SEQ ID NO:21或SEQ ID NO:38所示。
  18. 根据权利要求15至17任一权利要求所述的双特异性抗体，其中，所述抗PD-1的单链抗体中的重链可变区和轻链可变区直接连接或者通过连接片段连接；
    优选地，所述连接片段为(GGGGS)m，m为正整数，例如1、2、3、4、5或6；
    优选地，所述连接片段为(GGGGS)nG，n为正整数，例如1、2、3、4、5或6。
  19. 根据权利要求15至18中任一权利要求所述的双特异性抗体，其中，
    所述双特异性抗体包括：
    靶向LAG3的第一蛋白功能区，和
    靶向PD-1的第二蛋白功能区；
    所述第一蛋白功能区为1个，所述第二蛋白功能区为2个；
    其中，所述第一蛋白功能区为免疫球蛋白，所述第二蛋白功能区为单链抗体；
    所述免疫球蛋白的重链的氨基酸序列如SEQ ID NO:11或SEQ ID NO:13所示，并且其轻链的氨基酸序列如SEQ ID NO:12所示；
    所述单链抗体的重链可变区的氨基酸序列如SEQ ID NO:19所示，并且所述单链抗体的轻链可变区的氨基酸序列如SEQ ID NO:21或SEQ ID NO:38所示；
    所述单链抗体连接在免疫球蛋白的两条重链的C末端；
    所述第一蛋白功能区与所述第二蛋白功能区通过第一连接片段连接；并且所述单链抗体的重链可变区与所述单链抗体的轻链可变区通过第二连接片段连接；所述第一连接片段和所述第二连接片段相同或不同；
    优选地，所述第一连接片段和所述第二连接片段的氨基酸序列独立地选自SEQ ID NOs:35-37；
    优选地，所述第一连接片段和所述第二连接片段的氨基酸序列均如SEQ ID NO:36所示。
  20. 根据权利要求1至14中任一权利要求所述的双特异性抗体，其包括：
    靶向LAG3的第一蛋白功能区，和
    靶向PD-1的第二蛋白功能区；
    其中，所述第一蛋白功能区为抗LAG3的单链抗体，所述第二蛋白功能区为抗PD-1的抗体，并且所述抗PD-1的抗体为免疫球蛋白形式；
    其中，所述抗LAG3的单链抗体包含重链可变区和轻链可变区，其中，
    所述重链可变区包含：氨基酸序列分别如SEQ ID NOs:5-7所示的HCDR1-HCDR3；和
    所述轻链可变区包含：氨基酸序列分别如SEQ ID NOs:8-10所示的LCDR1-LCDR3。
  21. 根据权利要求20所述的双特异性抗体，其中，所述抗LAG3的单链抗体，
    其重链可变区的氨基酸序列如SEQ ID NO:2所示，并且其轻链可变区的氨基酸序列如SEQ ID NO:4所示。
  22. 根据权利要求20至21中任一权利要求所述的双特异性抗体，其中，所述抗LAG3的单链抗体中的重链可变区和轻链可变区直接连接或者通过连接片段连接；
    优选地，所述连接片段为(GGGGS)m，m为正整数，例如1、2、3、4、5或6；
    优选地，所述连接片段为(GGGGS)nG，n为正整数，例如1、2、3、4、5或6。
  23. 根据权利要求20至22中任一权利要求所述的双特异性抗体，其中，所述抗PD-1的抗体包含重链可变区和轻链可变区，其中，
    所述重链可变区包含：氨基酸序列分别如SEQ ID NOs:26-28所示的HCDR1-HCDR3；和
    所述轻链可变区包含：氨基酸序列分别如SEQ ID NOs:29-31所示的LCDR1-LCDR3。
  24. 根据权利要求20至23中任一权利要求所述的双特异性抗体，其中，所述抗PD-1的抗体，
    其重链可变区的氨基酸序列如SEQ ID NO:15所示，并且其轻链可变区的氨基酸序列如SEQ ID NO:17所示；或者
    其重链可变区的氨基酸序列如SEQ ID NO:19所示，并且其轻链可变区的氨基酸序列如SEQ ID NO:21或SEQ ID NO:38所示。
  25. 根据权利要求22至26中任一权利要求所述的双特异性抗体，其中，所述抗PD-1的抗体，其重链恒定区为Ig gamma-1 chain C region(例如，如SEQID:39所示)或Ig gamma-4 chain C region(例如，如SEQ ID NO:45所示)；轻链恒定区为Ig kappa chain C region(例如，如SEQID:40所示)。
  26. 根据权利要求20至25中任一权利要求所述的双特异性抗体，其中，
    所述抗PD-1的抗体为人IgG1亚型，
    其中，按照EU编号系统，所述抗PD-1的抗体具有如下突变：
    L234A和L235A，
    L234A和G237A，
    L235A和G237A，
    或者
    L234A、L235A和G237A；
    优选地，所述抗PD-1的抗体的重链的氨基酸序列如SEQ ID NO:34所示，并且轻链的氨基酸序列如SEQ ID NO:25所示。
  27. 根据权利要求20至25中任一权利要求所述的双特异性抗体，其中，
    所述抗PD-1的抗体为人IgG4亚型，
    其中，按照EU编号系统，所述抗PD-1的抗体具有如下突变：
    F234A和L235A，
    F234A和G237A，
    L235A和G237A，
    或者
    F234A、L235A和G237A；
    优选地，所述抗PD-1的抗体的重链的氨基酸序列如SEQ ID NO:32所示，并且轻链的氨基酸序列如SEQ ID NO:25所示。
  28. 根据权利要求20至27中任一权利要求所述的双特异性抗体，其中，
    所述双特异性抗体包括：
    靶向LAG3的第一蛋白功能区，和
    靶向PD-1的第二蛋白功能区；
    所述第一蛋白功能区为1个，所述第二蛋白功能区为2个；
    其中，所述第一蛋白功能区为单链抗体，所述第二蛋白功能区为免疫球蛋白；
    所述单链抗体的重链可变区的氨基酸序列如SEQ ID NO:2所示，并且所述单链抗体的轻链可变区的氨基酸序列如SEQ ID NO:4所示；
    所述免疫球蛋白的重链的氨基酸序列如SEQ ID NO:34或SEQ ID NO:32所示，并且其轻链的氨基酸序列如SEQ ID NO:25所示；
    所述单链抗体连接在免疫球蛋白的两条重链的C末端；
    所述第一蛋白功能区与所述第二蛋白功能区通过第一连接片段连接；并且所述单链抗体的重链可变区与所述单链抗体的轻链可变区通过第二连接片段连接；所述第一连接片段和所述第二连接片段相同或不同；
    优选地，所述第一连接片段和所述第二连接片段的氨基酸序列独立地选自SEQ ID NOs:35-37；
    优选地，所述第一连接片段和所述第二连接片段的氨基酸序列均如SEQ ID NOs:36所示；
    优选地，所述第一连接片段和所述第二连接片段的氨基酸序列均如SEQ ID NO:37所示。
  29. 分离的核酸分子，其编码权利要求1至28中任一权利要求所述的双特异性抗体。
  30. 一种重组载体，其包含权利要求29所述的分离的核酸分子。
  31. 一种宿主细胞，其包含权利要求29所述的分离的核酸分子，或者包含权利要求30所述的重组载体。
  32. 制备权利要求13至30中任一权利要求所述的双特异性抗体的方法，其包括在合适的条件下培养权利要求31的宿主细胞，以及从细胞培养物中回收双特异性抗体的步骤。
  33. 一种药物组合物，其包含权利要求1至28中任一权利要求所述的双特异性抗体；可选地，其还包括药学上可接受的辅料。
  34. 权利要求1至28中任一权利要求所述的双特异性抗体在制备治疗和/或预防肿瘤或贫血病的药物中的用途；
    优选地，所述肿瘤为选自卵巢癌、食管癌、黑色素瘤、血液瘤、神经胶质母细胞瘤、肾细胞癌、肺癌、前列腺癌、膀胱癌、结肠癌、直肠癌、肝癌、胃肠道癌、乳腺癌、脑癌、胰腺癌、甲状腺癌、头颈癌和肾癌中的一种或多种；
    优选地，所述肺癌为非小细胞肺癌；
    优选地，所述血液瘤为白血病；
    优选地，所述食管癌为食管鳞癌。
  35. 根据权利要求1至28中任一权利要求所述的双特异性抗体，其用于治疗和/或预防肿瘤或贫血病；
    优选地，所述肿瘤为选自卵巢癌、食管癌、黑色素瘤、血液瘤、神经胶质母细胞瘤、肾细胞癌、肺癌、前列腺癌、膀胱癌、结肠癌、直肠癌、肝癌、胃肠道癌、乳腺癌、脑癌、胰腺癌、甲状腺癌、头颈癌和肾癌中的一种或多种；
    优选地，所述肺癌为非小细胞肺癌；
    优选地，所述血液瘤为白血病；
    优选地，所述食管癌为食管鳞癌。
  36. 一种治疗和/或预防肿瘤或贫血病的方法，包括给予有需求的受试者以有效量的权利要求1至28中任一权利要求所述的双特异性抗体的步骤；
    优选地，所述肿瘤为选自卵巢癌、食管癌、黑色素瘤、血液瘤、神经胶质母细胞瘤、肾细胞癌、肺癌、前列腺癌、膀胱癌、结肠癌、直肠癌、肝癌、胃肠道癌、乳腺癌、脑癌、胰腺癌、甲状腺癌、头颈癌和肾癌中的一种或多种；
    优选地，所述肺癌为非小细胞肺癌；
    优选地，所述血液瘤为白血病；
    优选地，所述食管癌为食管鳞癌。
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