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WO2020173378A1 - 结合人lag-3的抗体、其制备方法和用途 - Google Patents

结合人lag-3的抗体、其制备方法和用途 Download PDF

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Publication number
WO2020173378A1
WO2020173378A1 PCT/CN2020/076023 CN2020076023W WO2020173378A1 WO 2020173378 A1 WO2020173378 A1 WO 2020173378A1 CN 2020076023 W CN2020076023 W CN 2020076023W WO 2020173378 A1 WO2020173378 A1 WO 2020173378A1
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seq
antibody
amino acid
acid sequence
antigen
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PCT/CN2020/076023
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English (en)
French (fr)
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赵杰
黄浩旻
朱祯平
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三生国健药业（上海）股份有限公司
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Priority to JP2021549338A priority Critical patent/JP2022523929A/ja
Priority to EP20762071.7A priority patent/EP3932948A4/en
Priority to CN202080015287.9A priority patent/CN113508139B/zh
Publication of WO2020173378A1 publication Critical patent/WO2020173378A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/75—Agonist effect on antigen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates to the field of antibodies. More specifically, the present invention discloses an antibody that binds to human LAG-3, a preparation method and use thereof. Background technique
  • Tumor immunotherapy is the application of immunological principles and methods to activate the immune system, break the immune tolerance of the immune system to tumors, enhance the recognition of tumor antigens by immune cells, stimulate and enhance the body's anti-tumor immune response, thereby suppressing the body's immune system And kill tumor cells, and finally achieve the goal of treating tumors.
  • Tumor immunotherapy has attracted much attention recently and is the focus of the field of tumor treatment. In recent years, great news of tumor immunotherapy has continued to emerge.
  • programmed death receptor 1 (PD-1) is undoubtedly the most dazzling star target in cancer immunotherapy, and immune checkpoint inhibitors such as nivolumab and pembrolizumab are used as targets.
  • Anti-tumor drugs have shown strong anti-tumor activity in the clinical treatment of some tumor types such as melanoma, non-small cell lung cancer, etc., and have been approved by the US FD A (Food and Dmg Administration, FDA) for clinical application.
  • Tumor immunotherapy was named the most important scientific breakthrough of the year by Science magazine in 2013 due to its excellent efficacy and innovation. Tumor immunotherapy is expected to become a revolution in the field of tumor treatment following surgery, chemotherapy, radiotherapy and targeted therapy.
  • T cells play an important role in the immune system, but the activation of T cells depends on antigen presenting cells to digest harmful foreign antigens and re-present them into an antigen type that T cells can recognize.
  • antigen presenting cells There are a group of proteins involved in regulation on T cells and antigen-presenting cells, which have the function of assisting in regulating the signal transduction of T cell receptors.
  • helper receptors are divided into two categories, one of which is costimulatory which is responsible for transmitting activation signals.
  • Receptors the other is a co-inhibitory receptor that transmits inhibitory signals, and these inhibitory molecules are now called immune checkpoint receptors and ligands.
  • the aforementioned star molecule PD-1 is a typical receptor for immune checkpoints, and belongs to the CD28 superfamily.
  • PD-L1 programmed death ligand 1
  • PD-1 and PD-L1 mediates the co-inhibitory signal of T cell activation, and negatively regulates T cell activation and proliferation.
  • PD-L1 is highly expressed in tumor tissues and regulates the function of tumor infiltrating CD8+ T cells.
  • immunomodulatory therapy targeting PD-1/PD-L1 has achieved great success in clinical anti-tumor therapy.
  • Lymphocyte activation gene-3 (LAG-3) is mainly expressed on activated T lymphocytes and is a negative immunoregulatory molecule similar to PD-1.
  • the main ligand of LAG-3 is MHC II A family of molecules that can compete with CD4 molecules for binding to MHC II ligands with higher affinity, and transduce inhibitory signals to the inside of the cell, thus negative
  • LAG-3 can help T cells maintain a state of immune tolerance, or keep continuously activated T cells in a state of exhaustion. It has the functions of maintaining internal environment stability and participating in negative immune regulation, and is closely related to the occurrence and development of tumors.
  • some preliminary studies have shown that antibodies that block LAG-3 can enhance the immune response, and the combination of PD-1 antibodies can play a significant synergistic activation effect, and has great potential in the field of cancer immunotherapy.
  • BMS Bristol-Myers Squibb
  • Novartis Novartis
  • BMS-986016 anti-LAG-3 monoclonal antibody BMS-986016 is in the first phase of clinical trials.
  • the main research goal is the safety and effectiveness of the PD-1 antibody nivolumab in combination.
  • the research results show that BMS-986016 is The combined application of nivolumab has shown good efficacy in the clinical treatment of melanoma.
  • anti-LAG-3 antibodies can also be used to treat autoimmune diseases.
  • the basic mechanism is to kill activated T cells through antibody-dependent cell-mediated cytotoxicity (ADCC), thereby inhibiting The purpose of immune response and treatment of autoimmune diseases.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • the inventors of the present invention conducted a large number of experiments, from antigen immunization, hybridoma screening, antibody expression and purification to biological activity identification, and screened and obtained two mouse sources 134 and 2 that specifically bind to human LAG-3.
  • -34 (ie 5E7) antibody and on this basis, further construct and obtain its chimeric antibodies 134-Chimeric, 5E7-Chimeric and humanized antibodies 134-Hu-IgG4-C91S, 5E7-Hu-IgG4.
  • the study of the present invention shows that the mouse 134 and 2-34 (ie, 5E7) antibodies have novel epitopes that bind to human LAG-3.
  • the first object of the present invention is to provide an antibody or antigen-binding fragment thereof that binds to human LAG-3.
  • the second object of the present invention is to provide another antibody or antigen-binding fragment thereof that binds to human LAG-3.
  • the third object of the present invention is to provide a nucleotide sequence encoding the antibody or antigen-binding fragment thereof that binds to human LAG-3.
  • the fourth object of the present invention is to provide an expression vector containing the nucleotide sequence.
  • the fifth object of the present invention is to provide a host cell containing the expression vector.
  • the sixth object of the present invention is to provide a method for preparing the antibody or antigen-binding fragment thereof that binds to human LAG-3.
  • the seventh object of the present invention is to provide a pharmaceutical composition containing the antibody or antigen-binding fragment thereof that binds to human LAG-3.
  • the eighth object of the present invention is to provide the use of the antibody or antigen-binding fragment thereof that binds to human LAG-3, or the pharmaceutical composition.
  • the first aspect of the present invention provides an antibody or antigen-binding fragment thereof that binds human LAG-3, which binds human
  • the LAG-3 epitope includes the following amino acid sequence: AAAPGHPLA (SEQ ID NO: 50).
  • the human LAG-3 epitope to which it binds includes the following amino acid sequence: GPPAAAPGHPLA (SEQ ID NO: 48) or AAAPGHPLAPGPHPAAPSS (SEQ ID NO: 49).
  • the second aspect of the present invention provides an antibody or antigen-binding fragment thereof that binds to human LAG-3, comprising:
  • the antibody is a monoclonal antibody or a polyclonal antibody.
  • the antibody is a monoclonal antibody.
  • the antibody is a murine antibody, a chimeric antibody, or a humanized antibody.
  • the antibody is a humanized antibody.
  • the antigen-binding fragments include Fab fragments, F(ab , )2 fragments, Fv fragments and the like.
  • the antibody or antigen-binding fragment thereof that binds to human LAG-3 includes a heavy chain variable region and a light chain variable region, selected from:
  • the antibody or antigen-binding fragment thereof that binds to human LAG-3 includes a heavy chain constant region and a light chain constant region, and the heavy chain constant region is selected from the group consisting of IgGl, IgG2, IgG3, and IgG4 heavy chains Constant region, the light chain constant region is selected from K or X light chain constant regions.
  • the heavy chain constant region is an IgG4 heavy chain constant region, and the light chain constant region is a K light chain constant region. More preferably, the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO: 30, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO: 34.
  • the antibody or antigen-binding fragment thereof that binds to human LAG-3 includes a heavy chain and a light chain, Selected from:
  • the third aspect of the present invention provides a nucleotide sequence that encodes the antibody or antigen-binding fragment thereof that binds to human LAG-3 as described in any one of the above.
  • the nucleotide sequence includes:
  • the nucleotide sequence includes the nucleotide sequence encoding the heavy chain constant region as shown in SEQ ID NO: 29, and the nucleus encoding the light chain constant region as shown in SEQ ID NO: 33 Nucleotide sequence.
  • the nucleotide sequence includes:
  • the fourth aspect of the present invention provides an expression vector containing the nucleotide sequence described in any one of the above.
  • the fifth aspect of the present invention provides a host cell containing the expression vector as described above.
  • the sixth aspect of the present invention provides a method for preparing an antibody or antigen-binding fragment thereof that binds to human LAG-3 as described above, and the method includes the following steps:
  • the seventh aspect of the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof that binds to human LAG-3 as described in any one of the above, and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition further includes a PD-1 inhibitor.
  • the PD-1 inhibitor is an antibody or an antigen-binding fragment thereof that binds PD-1.
  • the eighth aspect of the present invention provides the use of the antibody or antigen-binding fragment thereof that binds to human LAG-3 as described in any one of the above, or the pharmaceutical composition according to any one of the above, in the preparation of a medicament for the treatment of cancer.
  • the cancer is selected from melanoma, renal cell carcinoma, non-small cell lung cancer, classic Hodgkin's lymphoma, urothelial cancer, colorectal cancer, liver cancer, and the like.
  • the present invention discloses an antibody or antigen-binding fragment thereof capable of specifically binding to human LAG-3, which has a novel epitope that binds to human LAG-3 and a good biological activity of enhancing mixed lymphocyte reaction, and can be applied to preparation Drugs used for cancer immunotherapy have good clinical application prospects.
  • Figure 1 shows the results of the effect of murine anti-human LAG-3 antibody on the mixed lymphocyte reaction, where Figure 1A is the first round of screening, and Figure 1B is the second round of screening.
  • Figure 2 shows the comparison of the relative affinity between the humanized antibody and the chimeric antibody of the anti-human LAG-3 antibody.
  • Fig. 3 shows the result of the enhancement effect of humanized anti-human LAG-3 antibody on PBMC secreting IL-2, wherein the PBMC in Figs. 3A and 3B are from different donors.
  • Figure 4 shows the result of the binding of humanized anti-human LAG-3 antibody to LAG-3 on the cell surface.
  • Figure 5 is a schematic diagram of the epitope of the anti-human LAG-3 antibody.
  • Figure 6 shows the result of the blocking effect of humanized anti-human LAG-3 antibody on LAG-3 binding to Raji cells.
  • Figure 7 is the result of flow cytometry detecting the blocking effect of anti-human LAG-3 antibody on FGL1 binding to LAG-3.
  • Figure 8 shows the inhibitory effect of anti-human LAG-3 antibody on the MC38 transplanted tumor model in transgenic mice.
  • LAG-3 refers to lymphocyte activation gene 3, also known as CD223.
  • the terms "antibody (Ab)” and “immunoglobulin G (IgG)” are heterotetrameric glycoproteins with the same structural characteristics of about 150,000 daltons, which consist of two identical light chains (L) And two identical heavy chains (H). Each light chain is connected to the heavy chain through a covalent disulfide bond, and the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes is different. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. There is a variable region (VH) at one end of each heavy chain, followed by a constant region. The constant region of the heavy chain is composed of three structural domains CHI, CH2, and CH3.
  • Each light chain has a variable region (VL) at one end and a constant region at the other end.
  • the light chain constant region includes a structural domain CL; the constant region of the light chain is opposite to the first constant region of the heavy chain.
  • the variable region is opposite to the variable region of the heavy chain.
  • Constant regions do not directly participate in the binding of antibodies and antigens, but they exhibit different effector functions, such as participating in antibody-dependent cell-mediated cytotoxicity (ADCC).
  • the heavy chain constant region includes IgG1, IgG2, IgG3, IgG4, IgM, IgAl, IgA2, IgD, and IgE; the light chain constant region includes K (Kappa) or X (Lambda).
  • the antibodies of the present invention include monoclonal antibodies, polyclonal antibodies, multispecific antibodies (such as bispecific antibodies) formed from at least two antibodies, and the like.
  • monoclonal antibody monoclonal antibody
  • monoclonal antibody refers to an antibody obtained from a substantially homogeneous population, that is, the single antibodies contained in the population are the same except for a few naturally occurring mutations that may exist.
  • Monoclonal antibodies are highly specific to a single antigen site. Moreover, unlike conventional polyclonal antibody preparations (usually with different antibodies directed against different determinants), each monoclonal antibody is directed against a single determinant on the antigen.
  • monoclonal antibodies are synthesized by hybridoma culture and will not be contaminated by other immunoglobulins.
  • the modifier "monoclonal” indicates the characteristics of the antibody, which is obtained from a substantially uniform antibody population, which should not be interpreted as requiring any special method to produce antibodies.
  • antigen-binding fragment refers to a fragment of an antibody capable of specifically binding to an antigen (for example, human LAG-3).
  • antigen-binding fragments of the present invention include Fab fragments, F(ab , )2 fragments, Fv fragments, and the like.
  • Fab fragments are fragments produced by digesting antibodies with papain.
  • the F(ab')2 fragment is a fragment produced by digesting an antibody with pepsin.
  • Fv fragments are composed of dimers in which the variable regions of the heavy and light chains of an antibody are closely and non-covalently related.
  • Fc segment means that papain can cleave an antibody into two identical Fab segments and an Fc segment.
  • the Fc segment can be a fragment crystallizable (Fc), which is composed of the CH2 and CH3 structures of the antibody. Domain composition.
  • the Fc segment has no antigen binding activity and is the site where the antibody interacts with effector molecules or cells.
  • variable means that some parts of the variable region of the antibody are different in sequence, which forms the binding and specificity of various specific antibodies to their specific antigens.
  • variability is not evenly distributed throughout the variable regions of antibodies. It is concentrated in three fragments called the complementarity-determining region (CDR) or hypervariable region in the variable region of the heavy chain and the variable region of the light chain.
  • CDR complementarity-determining region
  • FR frame region
  • the variable regions of the natural heavy chain and light chain each contain four FR regions, which are roughly in a folded configuration, connected by three CDRs forming a connecting loop, and in some cases can form a partial 0-fold structure.
  • the CDRs in each chain are closely placed together through the FR region and form the antigen binding site of the antibody together with the CDRs of the other chain (see Kabat et al., NIH Publ. No. 91-3242, Volume I, pages 647-669 (1991)).
  • the antibodies of the present invention include murine antibodies, chimeric antibodies, humanized antibodies and the like.
  • murine antibody refers to an antibody derived from rat or mouse, preferably mouse.
  • the murine antibody of the present invention is obtained by immunizing mice with the extracellular domain of human LAG-3 as an antigen and screening hybridoma cells.
  • the murine antibodies of the present invention are antibodies No. 134 and No. 2-34 (ie 5E7).
  • the term "chimeric antibody” refers to an antibody comprising heavy chain variable region and light chain variable region sequences derived from one species and constant region sequences derived from another species, for example, having a human constant region
  • the mouse heavy chain variable region and light chain variable region antibodies Preferably, the chimeric antibody of the present invention is obtained by splicing the heavy chain variable region and light chain variable region sequences of mouse No. 134 and No. 2-34 (SP5E7) antibodies with a human constant region, respectively. More preferably, the heavy chain of the chimeric antibody of the present invention is obtained by splicing the heavy chain variable region sequence of the mouse No.
  • the chimeric antibody of the present invention includes 134-Chimeric and 5E7-Chimeric.
  • the term "humanized antibody” means that its CDRs are derived from non-human species (preferably mouse) antibodies, and the remaining parts of the antibody molecule (including framework regions and constant regions) are derived from human antibodies. In addition, the framework residues can be changed to maintain Combine affinity.
  • the humanized antibody of the present invention is recombined from the CDR regions of mouse No. 134 and No. 2-34 (ie 5E7) antibodies and non-CDR regions derived from human antibodies, and has embedded residues and CDR regions. Directly interacting residues and residues that have an important influence on the conformation of VL and VH of antibodies No. 134 and 2-34 (ie 5E7) were obtained by back mutation. More preferably, the humanized antibody of the present invention includes 134-Hu-IgG4-C91 S and 5E7-Hu-IgG4.
  • the terms “epitope” and “human LAG-3 epitope” refer to the region located on human LAG-3 and specifically binding to an antibody.
  • the human LAG-3 epitope of the present invention is located in the extracellular domain of human LAG-3, and the extracellular domain of human LAG-3 includes the amino acid sequence shown in SEQ ID NO: 41.
  • the human LAG-3 epitope of the present invention includes the following amino acid sequence: AAAPGHPLA (SEQ ID NO: 50).
  • the term "antibody that binds to human LAG-3" or "anti-human LAG-3 antibody” refers to an antibody that specifically binds to an epitope of human LAG-3.
  • the term “specifically binds” refers to an antibody with a concentration of less than 1 X 10 '7 M or less, preferably 1 X 10_ 8 M or less, more preferably 1 X 10_ 9 M or less, 1 X 10_ 1 () M or less, 1 X 10_ U M or less equilibrium dissociation constant (KD) binding.
  • KD refers to the equilibrium dissociation constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between the antibody and the antigen.
  • SPR Surface Plasmon Resonance
  • ELISA ELISA
  • expression vector can be pTT5, pSECtag series, pCGS3 series, pCDNA series vectors, etc., as well as other vectors used in mammalian expression systems, etc.
  • the expression vector includes those connected with appropriate transcription and translation regulatory sequences. Fusion DNA sequence.
  • the term "host cell” refers to a cell suitable for expressing the above expression vector. It can be a eukaryotic cell, such as mammalian or insect host cell culture systems, which can be used to express the fusion protein of the present invention. CHO (Chinese hamster) Ovary, Chinese Hamster Ovary), HEK293, COS, BHK and derived cells of the above-mentioned cells are all suitable for the present invention.
  • the term "pharmaceutical composition” means that the antibody or antigen-binding fragment thereof that binds to human LAG-3 of the present invention can be combined with a pharmaceutically acceptable carrier to form a pharmaceutical preparation composition to more stably exert a therapeutic effect.
  • a pharmaceutically acceptable carrier to form a pharmaceutical preparation composition to more stably exert a therapeutic effect.
  • These preparations It can ensure the conformational integrity of the amino acid core sequence of the antibody or antigen-binding fragment thereof that binds to human LAG-3 disclosed in the present invention, while also protecting the protein's multifunctional groups from degradation (including but not limited to aggregation, deamination or oxidation).
  • the following examples and experimental examples further illustrate the present invention, and should not be construed as limiting the present invention.
  • the amino acid sequence of the extracellular segment of human LAG-3 is from https:/7www. uniprot. ore/uniprot/P 18627, the amino acid sequence is shown in SEQ ID NO: 41, and the codon optimized by Shenggong Bioengineering (Shanghai) Co., Ltd. And synthesize the gene, and then clone the synthesized gene fragment into the pUC57 vector.
  • coli TOP 10 competent cells and coated Cultivate overnight in LB (Amp resistant) plate medium; pick a single colony, culture and amplify, extract the plasmid, cut the plasmid with restriction enzymes to identify whether the target gene fragment is inserted; select qualified positive clones Sequencing; select clones with completely correct sequences and extract plasmids after amplification.
  • PEI Polyethyleneimine
  • amino acid sequences of the heavy chain variable region and the light chain variable region of the anti-LAG-3 monoclonal antibody used as a positive control are derived from SEQ ID NOs: 12 and 14 in the US patent application US20140093511A1, and were provided by Shenggong Bioengineering (Shanghai) Co., Ltd. Co., Ltd. conducts codon optimization and synthesizes genes.
  • the heavy chain variable region and light chain variable region genes are respectively associated with the human IgG4 heavy chain constant region (the nucleotide sequence is shown in SEQ ID NO: 29, and the amino acid sequence is shown in : 30) and human Kappa light chain constant region (the nucleotide sequence is shown in SEQ ID NO: 33, the amino acid sequence is shown in SEQ ID NO: 34), and the signal peptide sequence (SEQ ID NO: 42 )
  • To construct complete heavy chain and light chain genes and then clone the complete heavy chain and light chain genes into the pTT5 expression vector according to the similar method described above.
  • the pTT5 expression vector containing the heavy and light chain genes was combined in the same way, and then transferred into HEK293E cells for expression.
  • HEK293E cells are cultured in the serum-free medium FreeStyle 293 Expression Medium (purchased from Thermo Fisher Scientific) with restricted chemical composition. Protein quantification is determined by ultraviolet spectrophotometry.
  • Example 2 Using human LAG-3-ECD-hFc as antigen to immunize mice and the preparation and screening of hybridomas
  • the LAG-3-ECD-hFc prepared in Example 1 was diluted with normal saline to a suitable concentration, and mixed with an equal volume of Freund’s complete adjuvant (purchased from Sigma). After phacoemulsification, the 4-5 weeks old Balb/c Mice (purchased from Shanghai Lingchang Biological Technology Co., Ltd., animal production license number: SCXK (Shanghai) 2013-0018) were injected with multiple subcutaneous injections; three weeks later, physiological saline LAG-3-ECD-hFc was diluted with water and mixed with an equal volume of Freund's incomplete adjuvant (purchased from Sigma).
  • mice were injected with multiple subcutaneous injections; this step was repeated again two weeks later; all mice On the seventh day after the third immunization, a small amount of blood was taken to separate the serum, and the serum titer was detected by conventional ELISA; mice with serum antibody titer> 10000 were selected, and each mouse was injected with 10
  • mice On the third day after intravenous injection of antigen protein, a certain number of mice were selected, sacrificed and dissected to remove the spleen. After grinding, the spleen cells were collected and counted to prepare hybridomas.
  • myeloma Sp2/0 cells from the cell bank of the Type Culture Collection Committee of the Chinese Academy of Sciences) are selected as the cell fusion ligand.
  • Sp2/0 cells are cultured in a cell incubator (37 ° C, 5% CO 2 ), and the medium used is RPMI-1640 complete medium (the preparation method is as follows: Add 10% fetal cattle to RPMI-1640 basic medium Serum and 1% Penicillin-Streptomycin, the above materials were purchased from Thermo Fisher Scientific), the medium was changed one day before the cell fusion, so that the cells were in a good growth state.
  • the electrofusion method is used to prepare hybridoma cells.
  • LAG-3-ECD-hFc antigen coated ELISA plate the coating amount is 100ng/well; After incubating for 2 hours at room temperature, wash with PBST (PBS containing 0.05% Tween-20); add 200[jl containing 1% to each well The PBST solution of bovine serum albumin (Bovine Serum Albumin, BSA) was sealed and incubated at room temperature for 1 hour; washed with PBST, drained, and stored in the refrigerator for later use.
  • PBST Bovine Serum Albumin
  • the selected positive clones must not be single clones, and some clones may be unstable, so subcloning needs to be performed in time, and a single clone is finally obtained.
  • the subcloning is completed by the limiting dilution method.
  • the process is as follows: Prepare a positive clone of hybridoma cell suspension and use HT medium (the preparation method of HT medium is as follows: Use RPMI-1640 complete medium to dilute the 100xHT solution to the required concentration, 100xHT (Purchased from Thermo Fisher Scientific) Adjust the cell density to a dilution of 10 or 20 cells per milliliter; inoculate a 96-well plate for each dilution, and the volume per well is 0.2ml, and the average number of hybridoma cells per well is 2 or 4; Place in a CO 2 cell incubator for 7-10 days. When there are macroscopically visible clones, observe under an inverted microscope to mark the wells where only a single clone grows.
  • the stable monoclonal cell line was expanded and cultured in the serum-free medium HybriGRO SF (purchased from Corning) for about 5-7 days.
  • the cell culture supernatant was centrifuged and filtered to remove the precipitate, and then the protein G (purchased from GE Healthcare) ) Purified mouse antibody.
  • the present invention successfully established more than 200 stable hybridoma monoclonal cell strains, and purified corresponding murine antibodies for further experiments.
  • Example 3 The effect of murine anti-human LAG-3 antibody on mixed lymphocyte reaction
  • the mixed lymphocyte reaction (Mixed Lymphocyte Reaction, MLR) is used to evaluate the activity of the mouse-derived anti-human LAG-3 antibody, and Anti-LAG3.5 is added as a positive control.
  • the MLR experiment method is described as follows: Use Histopaque (purchased from Sigma) to isolate Peripheral Blood Mononuclear Cell (PBMC) from human blood, and separate the monocyte subpopulations in PBMC by the adhesion method. Then use IL-4 and GM-CSF to induce monocytes to differentiate into induced dendritic cells (p38 MAPK-inhibited dendritic cells induce superior antitumour immune responses and overcome regulatory T-cell-mediated immunosuppression. Nat Commun. 2014 Jun 24 ;5:4229.
  • PBMC Peripheral Blood Mononuclear Cell
  • monoclonal antibodies No. 134 and No. 5E7 were finally selected as lead antibodies.
  • the light chain variable region and the heavy chain variable region of the murine anti-human LAG-3 antibody were amplified by PCR Base Therefore, the PCR product was then cloned into the pMD18-T vector, and the variable region gene sequence was sequenced and analyzed.
  • the sequence information of clone No. 134 is as follows: the heavy chain variable region gene sequence is 360 bp in length, encoding 120 amino acid residues, the nucleotide sequence is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2; The light chain variable region gene sequence is 321 bp in length and encodes 107 amino acid residues. The nucleotide sequence is shown in SEQ ID NO: 3, and the amino acid sequence is shown in SEQ ID NO: 4.
  • sequence information of clone No. 5E7 is as follows: the full length of the heavy chain variable region gene sequence is 348 bp, encoding 116 amino acid residues, the nucleotide sequence is shown in SEQ ID NO: 5, and the amino acid sequence is shown in SEQ ID NO: 6; The light chain variable region gene sequence is 321 bp in length and encodes 107 amino acid residues. The nucleotide sequence is shown in SEQ ID NO: 7, and the amino acid sequence is shown in SEQ ID NO: 8.
  • the amino acid sequences of the heavy chain variable region and the light chain variable region in Example 4 were analyzed, and the 3 antigen complementarity determining regions (CDR) and the light chain of the heavy chain and light chain of antibody No. 134 and 5E7 were determined according to the Rabat coding rules. 4 frame areas (FR).
  • the amino acid sequence of the heavy chain complementarity determining region of clone No. 134 is HCDR1: AYYMNCSEQ ID NO: 9), HCDR2: VINPYNGDSSYNQKFKG (SEQ ID NO: 10) and HCDR3: DDGYYRWYFDV (SEQ ID NO: 11), the light chain complementarity determining region
  • the amino acid sequence is LCDR1: RASQDIGSRLN (SEQ ID NO: 12), LCDR2: ATSSLES (SEQ ID NO: 13) and LCDR3: LQCGSSPPT (SEQ ID NO: 14).
  • the amino acid sequence of the heavy chain complementarity determining region of clone No. 5E7 is HCDR1: DDYMA (SEQ ID NO: 15), HCDR2: SISHGGDYIYYADNLKG (SEQ ID NO: 16) and HCDR3: DRRSIDY (SEQ ID NO: 17).
  • the amino acid sequence is LCDR1: RASQDISNYLS (SEQ ID NO: 18), LCDR2: YTSRLHS (SEQ ID NO: 19) and LCDR3: QQGKTLPYT (SEQ ID NO: 20).
  • Back mutation is to mutate certain amino acids in the framework region of the CDR grafted variable region (amino acids that are important for maintaining the structure and affinity of the antibody) into the amino acids at the corresponding positions in the mouse framework region.
  • amino acids in the framework region of the CDR grafted variable region amino acids that are important for maintaining the structure and affinity of the antibody
  • the amino acid sequence is coded according to the Rabat rules, and the position of each amino acid is indicated by the Rabat code.
  • the G at position 44 of the variable region of heavy chain CDR grafting is backmutated to S
  • M at position 48 is mutated to I
  • V at position 67 is mutated to A
  • M at position 69 is mutated to L
  • R at position 71 is mutated to V
  • the T mutation at position 73 is K
  • the V mutation at position 78 is A.
  • the Y at position 36 of the light chain CDR grafted variable region is backmutated to L
  • the A at position 43 is mutated to S
  • the P at position 44 is mutated to I
  • the L at position 46 is mutated to R
  • the position at 66 is The G mutation at position 69 is R
  • the T mutation at position 69 is S
  • the F mutation at position 71 is Y.
  • the CDR3 (SEQ ID NO: 14) of the light chain of antibody No. 134 contains an extra cysteine (Rabat coded at position 91, C91), which may cause abnormal antibody structure, so we will make it here. Become serine (C91 S, SEQ ID NO: 43).
  • the amino acid sequences of WGQGTKVEIK (SEQ ID NO: 44) and FGQGTKVEIK (SEQ ID NO: 45) were added to the ends of the heavy chain and light chain CDR grafted variable regions carrying the above-mentioned back mutations to construct a complete humanized
  • the heavy chain and light chain variable regions, and related gene sequences are codon optimized and gene synthesized by Sangong Bioengineering (Shanghai) Co., Ltd.
  • the finally obtained 134 humanized heavy chain variable region (134-Hu-VH) gene sequence is 354 bp in length and encodes 118 amino acid residues.
  • the nucleotide sequence is shown in SEQ ID NO: 21, and the amino acid sequence is shown in SEQ ID. NO: 22;
  • the 134 humanized light chain variable region (134-HU-VL-C91 S) gene sequence is 321 bp in length, encoding 107 amino acid residues, and the nucleotide sequence is shown in SEQ ID NO: 23
  • the amino acid sequence is shown in SEQ ID NO: 24.
  • Amino acid sequence of 134 humanized light chain variable region (134-HU-VL-C91 S)
  • DIOMTOSPSSLSASVGDRVTITCRASODIGSRLNWLOOKPGKSIKRLIYATSSLESGVPSRFS GSRSGSDYTLTISSLOPEDFATYYCLOSGSSPPTFGOGTKVEIK (SEQ ID NO: 24, where the underlined part is the light chain complementarity determining region)
  • variable regions of the heavy chain and light chain CDR grafted some amino acid positions in the framework region were backmutated.
  • the amino acid sequence is coded according to the Rabat rule, and the position of each amino acid is indicated by the Kabat code.
  • the V at position 37 of the heavy chain CDR grafted variable region is backmutated to F
  • the G at position 44 is mutated to R
  • the S at position 49 is mutated to A
  • the Y at position 91 is mutated to F
  • a at position 93 is mutated to S.
  • a at position 43 is mutated to T
  • P at position 44 is mutated to I
  • F at position 71 is mutated to Y
  • Y at position 87 is mutated to F.
  • the amino acid sequences of WGQGTKVEIK (SEQ ID NO: 44) and FGQGTKVEIK (SEQ ID NO: 45) were added to the ends of the heavy chain and light chain CDR grafted variable regions carrying the above-mentioned back mutations respectively to construct a complete humanized
  • the heavy chain and light chain variable regions and related gene sequences are codon optimized and gene synthesized by Sangong Bioengineering (Shanghai) Co., Ltd.
  • the finally obtained 5E7 humanized heavy chain variable region (5E7-HU-VH) gene sequence is 354 bp in length, encoding 118 amino acid residues, the nucleotide sequence is shown in SEQ ID NO: 25, and the amino acid sequence is shown in SEQ ID Shown at NO: 26; the 5E7 humanized light chain variable region (5E7-HU-VL) gene sequence is 321 bp in length, encoding 107 amino acid residues, and the nucleotide sequence is as shown in SEQ ID NO: 27.
  • the amino acid sequence As shown in SEQ ID NO: 28.
  • DIOMTOSPSSLSASVGDRVTITCRASODISNYLSWYOOKPGKTIKLLIYYTSRLHSGVPSRFS GSGSGTDYTFTISSLOPEDIATYFCOOGKTLPYTFGOGTKVEIK (SEP ID NO: 28, where the underlined part is the light chain complementarity determining region)
  • 134 humanized heavy chain variable region and human immunoglobulin IgG4 constant region are connected to construct Into a complete 134 humanized heavy chain, named 134-Hu-IgG4-HC (the nucleotide sequence is shown in SEQ ID NO: 31, and the amino acid sequence is shown in SEQ ID NO: 32); 134 humanized light
  • the chain variable region is connected with the constant region of the human immunoglobulin Kappa chain (the nucleotide sequence is shown in SEQ ID NO: 33, and the amino acid sequence is shown in SEQ ID NO: 34) to construct a complete 134 humanized light chain , Named 134-HU-LC-C91 S (the nucleotide sequence is shown in SEQ ID NO: 35, and the amino acid sequence is shown in SEQ ID NO: 36).
  • HEALHNHYTQKSLSLSLGK SEQ ID NO: 30
  • 5E7 humanized heavy chain variable region with the constant region of human immunoglobulin IgG4 (the nucleotide sequence is shown in SEQ ID NO: 29, and the amino acid sequence is shown in SEQ ID NO: 30) to construct Into a complete 5E7 humanized heavy chain, named 5E7-Hu-IgG4-HC (nucleotide sequence is shown in SEQ ID NO: 37, amino acid sequence is shown in SEQ ID NO: 38); 5E7 humanized light chain The chain variable region is connected with the constant region of the human immunoglobulin Kappa chain (the nucleotide sequence is shown in SEQ ID NO: 33, and the amino acid sequence is shown in SEQ ID NO: 34) to construct a complete 5E7 humanized light chain , Named as 5E7-HU-LC (the nucleotide sequence is shown in SEQ ID NO: 39, and the amino acid sequence is shown in SEQ ID NO: 40).
  • HNHYTQKSLSLSLGK SEQ ID NO: 38, where the underlined part is the heavy chain complementarity determining region
  • the above 134-Hu-IgG4-HC and 134-Hu-LC-C91S genes were respectively constructed into pTT5 expression vector, and the constructed expression vector was co-transfected into HEK293E cells with PEI for transient expression.
  • HEK293E cells were cultured in FreeStyle 293 Expression Medium (purchased from Thermo Fisher Scientific) in serum-free medium. After 5 days, the cells were centrifuged and the cell supernatant was collected. The antibody was purified by Protein A affinity chromatography.
  • the final prepared antibody was named 134-Hu- IgG4-C91S o uses the same method to construct 5E7 humanized heavy chain and light chain expression vectors, and uses the same method to express and purify the antibody.
  • the final prepared antibody is named 5E7-Hu-IgG4.
  • variable region of the heavy chain and the variable region of the light chain of the murine antibody No. 134 were respectively combined with the constant region of the human immunoglobulin IgG4 heavy chain by recombinant PCR (the nucleotide sequence is shown in SEQ ID NO: 29, The amino acid sequence is shown in SEQ ID NO: 30) and the Kappa light chain constant region (the nucleotide sequence is shown in SEQ ID NO: 33, and the amino acid sequence is shown in SEQ ID NO: 34) are assembled into pTT5 expression vector.
  • the antibody expressed and purified by the above method, preparing a chimeric antibody designated as 134-chimeric o chimeric 5E7 antibody is the antibody 5E7-chimeric same way.
  • the ELISA method was used to detect the influence of humanized modification on the affinity between antibody and antigen.
  • PBST solution containing 1% BSA Block with Tween-20 in PBS
  • PBST solution containing 1% BSA to each well for blocking
  • Wash the plate with PBST and pat dry Add gradiently diluted antibodies in the blocking solution to the plate Human LAG-3 antibody, incubate at room temperature for about 1 hour, then wash the plate
  • Goat-Anti-Human IgG (Fc specific) secondary antibody purchased from Sigma
  • diluted with blocking solution incubate at room temperature for about 1 hour, then wash ELISA plate
  • Staphylococcal Enterotoxin A is a superantigen that can cross-link MHC class II molecules of Antigen Presenting Cell (APC) and T cell receptors on the surface of T cells.
  • APC Antigen Presenting Cell
  • TCR T cell receptors on the surface of T cells.
  • APC Antigen Presenting Cell
  • LAG-3 is expressed, and LAG-3 can bind to MHC class II molecules on the surface of APC, thereby exerting an immunosuppressive effect.
  • SEA is used After stimulating the PBMC, an anti-human LAG-3 antibody is added, and the functional activity of the humanized anti-human LAG-3 antibody is evaluated by detecting the secretion of IL-2.
  • the experimental method is described as follows: The SEA gene with histidine tag was cloned into the pET28a vector, the protein was expressed in the E. coli system, and then the SEA was purified by a nickel affinity chromatography column; PBMC was separated from human blood by Histopaque , Washed with PBS and centrifuged; resuspended cells in RPMI-1640 medium (containing 10% fetal bovine serum), added the above-mentioned self-made SEA (SEQ ID NO: 52) to a final concentration of 1ng/ml, and then inoculated PBMC to a circle
  • each well has 2 ⁇ 10 5 cells, and each well has a volume of 150 ⁇ 1; then add 50M serially diluted anti-human LAG-3 antibody to each well, and place the 96-well plate in the cell incubator (37 ° C, 5% CO 2 ) incubate for 2 days; take appropriate amount of culture supernatant, use double antibody sandwich
  • the PBMCs in Figures 3A and 3B are from different donors. Experimental results show that Anti-LAG3.5, 134-Hu-IgG4-C91S and 5E7-Hu-IgG4 can effectively enhance the secretion of IL-2 by SEA-stimulated PBMC.
  • the EC50 of Anti-LAG3.5, 134-Hu-IgG4-C91S and 5E7-Hu-IgG4 in the experiment shown in Figure 3A were 98.09 ng/ml, 50.71 ng/ml and 78.12 ng/ml, respectively; in the experiment shown in Figure 3B
  • the EC50 of the above three antibodies were 16.21 ng/ml, 10.85 ng/ml and 3.068 ng/ml.
  • the superantigen SEA can activate T cell clones in PBMC, and the activated T cells will express LAG-3.
  • the binding effect of the humanized anti-human LAG-3 antibody of the present invention on the cell surface LAG-3 was measured by flow cytometry.
  • the experimental procedure is described as follows: Separate PBMC from human blood with Histopaque, wash twice with PBS, and collect the cells by centrifugation; Resuspend the cells in RPMI-1640 medium (containing 10% fetal bovine serum), and add SEA to a final concentration of 1ng/ ml, then inoculate PBMC into a round-bottomed 96-well cell culture plate with 2 ⁇ 10 5 cells per well; place the 96-well plate in a cell incubator (37 ° C, 5% CO 2 ) and incubate for 2 days; use PBS Wash the cells in the 96-well plate, centrifuge and discard the supernatant; Use Biotin N-hydroxysuccinimide ester (Cat.
  • Anti-LAG3.5, 134-Hu-IgG4-C91S and 5E7-Hu-IgG4 can effectively bind to LAG-3 on the cell surface, with EC50 of 71.68ng/ml and 80.45ng/ml, respectively And 53.13ng/ml, the binding ability of the three is basically the same.
  • the isotype control is an IgG4 antibody that does not bind to LAG-3, and the ordinate is the average fluorescence intensity.
  • Example 8 Epitope analysis of anti-human LAG-3 antibody
  • the first immunoglobulin-like domain of the extracellular segment of LAG-3 contains an exposed “extra loop” whose amino acid sequence is: GPPAAAPGHPLAPGPHPAAPSSWGPRPRRY (SEQ ID NO: 46). According to literature reports, this peptide plays an important role in the binding of LAG-3 to MHC-II molecules (Baixeras E, Huard B, Miossec C, et al. Characterization of the lymphocyte activation gene 3 -encoded protein. A new ligand for human leukocyte antigen class II antigens. [J] Journal of Experimental Medicine, 1992, 176(2): 327-337. ).
  • biotin tags to the N-terminus of these peptides; coat the microplate with Streptavidin (200ng/well), wash the microplate with blocking solution (containing 1%) BSA PBST solution) blocking; Dilute the biotinylated peptides with blocking solution to lpg/ml and add them to the ELISA plate, incubate at room temperature for 1 hour to be captured by Streptavidin, and then wash the ELISA plate; The anti-human LAG-3 antibody diluted in the blocking solution is incubated at room temperature for about 1 hour, then the ELISA plate is washed; Goat-Anti-Human IgG (Fc specific) secondary antibody (purchased from Sigma) appropriately diluted in the blocking solution is added for detection For the binding of anti-human LAG-3 antibody to the peptide, incubate at room temperature for about 1 hour; add 100M color developing solution (TMB substrate) to each well for color development, and then add 50( ⁇ 1 2M H 2 SO 4 stop solution to each well to stop the reaction
  • Raji cells are a type of Burkitt lymphoma cells that originate from B lymphocytes and highly express MHC-II molecules.
  • flow cytometry was used to determine the blocking effect of the humanized anti-human LAG-3 antibody of the present invention on LAG-3 binding to MHC-II molecules on the surface of Raji cells.
  • the experimental procedure is as follows: Raji cells (purchased from American Type Culture Collection, abbreviated as ATCC; ATCC ® CCL-86ä) were washed twice with PBS, and then seeded into a round bottom 96-well cell culture plate, 2> ⁇ 10 5 per well After centrifugation, the supernatant was discarded; the biotinylated LAG-3 (purchased from ACROBiosy stems) was diluted to 0.5 ⁇ g/ml with PBS containing 1% BSA, and then the anti-human LAG- 3 Antibody, then add the mixed solution containing biotinylated LAG-3 and anti-human LAG-3 antibody to the above cells, incubate for about 1 hour at room temperature, wash the cells twice with PBS; use PBS containing 1% BSA Appropriately dilute PE-Streptavidin (purchased from BD Biosciences), add it to the above cells, incubate for about 1 hour, wash the cells twice with PBS; add 4% paraformalde
  • Anti-LAG3.5 and 134-Hu-IgG4-C91 S can effectively block the binding of LAG-3 to Raji cells, with IC50 of 256.9ng/ml and 288.8ng/ml, respectively.
  • the blocking ability of the participants is basically the same.
  • the effect of 5E7-Hu-IgG4 on LAG-3 binding to Raji cells is different from Anti-LAG3.5 and 134-Hu-IgG4-C91 S: At high concentrations, 5E7-Hu-IgG4 can only partially block LAG-3 Binding of Raji cells; At low concentrations, 5E7-Hu-IgG4 can instead promote the binding of LAG-3 to Raji cells.
  • the isotype control is an IgG4 antibody that does not bind to human LAG-3, and the negative control represents the background fluorescence value of Raji cells without LAG-3.
  • the molecular interaction instrument Biacore 8K (GE healthcare) was used to detect the affinity between Anti-LAG3.5, 134-Hu-IgG4-C91 S and 5E7-Hu-IgG4 and LAG-3.
  • the anti-LAG-3 antibody sample was diluted to a concentration of 0.5ng/mL, and recombinant LAG-3 (purchased from SinoBiological, catalog number: 16498-H08H) was diluted to 1.024nM, 2.56nM, 6.4nM, 16nM, 40nM and 100nM, with 0 concentration added; 6M guanidine hydrochloride solution as regeneration buffer;
  • Biacore 8K use Protein A chip to capture anti-LAG-3 antibody, and then inject recombinant LAG-3 to obtain Binding-dissociation curve, repeat the next cycle after regeneration buffer elution; use Biacore 8K Insight Evaluation Software to analyze the data. The results are shown in Table 2.
  • Example 11 Flow cytometry to detect the blocking effect of anti-human LAG-3 antibody on FGL1 binding to LAG-3
  • FGL1 Fibrinogen-like protein 1
  • FGL1 Fibrinogen-like protein 1
  • LAG-3 LAG-3
  • This example uses flow cytometry to detect Anti-LAG3.5 , 134-Hu-IgG4-C91S and 5E7-Hu-IgG4 block the ability of FGL1 to interact with LAG-3.
  • the method of preparing recombinant human FGL1 protein is as follows:
  • the human FGL1 sequence is from http:/7www.uniprot.ore (Entry: Q08830), and the amino acid sequence is as follows:
  • the gene encoding the above amino acid sequence was synthesized by Shenggong Bioengineering (Shanghai) Co., Ltd.; the FGL1 gene and the Fc segment gene of human IgG1 were fused together by the method of recombinant PCR, and the above gene with Fc tag was constructed into In a mammalian expression vector; transiently transfected and expressed in HEK293E cells; five days later, the recombinant protein in the cell culture supernatant was purified by Protein A affinity chromatography, and the protein concentration was determined by ultraviolet spectrophotometry, and the resulting protein was named FGL1 -hFc o
  • the biotin labeling method of FGLl-hFc is as follows: Biotin N-hydroxysuccinimide ester (Sigma/Cat.
  • H1759-100MG is formulated into 100mM mother liquor with anhydrous DMSO; the corresponding substance is calculated according to the molecular weight and concentration of FGLl-hFc Take an appropriate volume of Biotin N-hydroxysuccinimide ester solution, mix it with FGLl-hFc at a ratio of 20:1, and incubate at room temperature for 1 hour; after dialysis, the protein concentration is determined by ultraviolet spectrophotometry, and the resulting organism The vegetarian protein was named Biotin-FGLl-hFc.
  • the method for constructing a CHO-S cell line stably expressing LAG-3 is as follows: CHO-S cells were purchased from Thermo Fisher Scientific, and the full-length human LAG-3 gene was constructed into pCHO 1.0 expression vector, using PEI (polyethyleneimine) The expression vector was transfected into CHO-S cells, and selected with methotrexate and puromycin under pressure, and then a single clone was selected by the limiting dilution method to obtain a stable expressing human LAG- 3, the CHO-S monoclonal cell line was named CHO-S-LAG-3.
  • the flow cytometry method for detecting antibody blocking the interaction between FGL1 and LAG-3 is described as follows: Inoculate CHO-S-LAG-3 cells into a 96-well plate (round bottom with lid) with 200,000 cells per well; after centrifugation at 300g for 5 min, aspirate the cell culture supernatant with a row gun, and add 200nlPBS+1 to each well %BSA (1% bovine serum albumin in PBS) was used to resuspend the cells, then 300g centrifugation, aspirate the supernatant; add serially diluted anti-LAG-3 antibody and a constant concentration of 2 /ml Biotin-FGLl-hFc , Incubate at room temperature for 1 hour; centrifuge at 300g, aspirate the supernatant, wash the cells with PBS+1%BSA twice; centrifuge at 300g, aspirate the supernatant, add PBS+1%BSA diluted 1:2000 to each well PE Streptavidin, add 200M
  • the animal experiment procedures are as follows: Human LAG-3 transgenic mice (germline background is C57BL/6) and MC38 mouse colorectal cell lines are provided by Shanghai Southern Model Biology Research Center. In the transgenic mouse, the extracellular segment of mouse LAG-3 has been replaced by the homologous portion of the human LAG-3 gene. Therefore, the humanized anti-LAG-3 antibody of the present invention can recognize LAG- in the transgenic mouse. 3 molecules.
  • the specific implementation steps are as follows: expand MC38 in vitro, the medium is DMEM containing 10% fetal bovine serum (fetal bovine serum and DMEM medium purchased from Thermo Fisher Scientific); inoculate the expanded MC38 cells into human LAG-3
  • DMEM fetal bovine serum
  • DMEM medium purchased from Thermo Fisher Scientific
  • each mouse was inoculated subcutaneously with 1x10 6 cells; when the tumor cells to be inoculated grew to a volume close to 100mm 3 , the animals were randomly divided into five groups, 5 in each group: Isotype control antibody group (only injected isotype Control antibody);
  • Anti-mPD- 1 (purchased from Bio X Cell, catalog number BP0146) 10 mg/kg; Anti-mPD- 1 10mg/kg+Anti-LAG3.5 20 mg/kg; Anti-mPD- 1 10 mg/kg+134 -Hu-IgG4-C91 S 20 mg/kg; Anti-mPD-1 10 mg/kg+5E7-Hu-IgG4 20 mg/kg. It is administered by intraperitoneal injection and the frequency of administration is twice a week. Subsequently, the drug was administered according to the above-designed plan for three weeks, and the tumor volume was measured and calculated twice a week. Finally, the tumor growth curve of each group is shown in Figure 8. The anti-tumor effect of anti-LAG-3 monoclonal antibody when used as a single agent is relatively weak (Reference: Woo S R, Turnis

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Abstract

提供了能够特异性结合人LAG-3的抗体或其抗原结合片段，所述抗体或其抗原结合片段具有增强混合淋巴细胞反应的生物学活性，可用于制备癌症免疫疗法的药物。

Description

结合人 LAG-3的抗体、 其制备方法和用途 技术领域
本发明涉及抗体领域， 更具体地， 本发明公开了结合人 LAG-3 的抗体、 其制备方法和 用途。 背景技术
各种类型的恶性肿瘤目前已经成为人类的主要杀手，并且发病率年年攀升。常规的手术、 化疗和放疗虽有一定疗效， 但是往往带给病人身体带来巨大损害和毒副作用。 现阶段的靶向 性单克隆抗体类药物 (例如， 革 E1向 HER2的 trastuzumab ) 以及革 El向性小分子激酶抑制剂 (例 如， 靶向某些酪氨酸激酶的 imatinib )虽然在肿瘤治疗中取得了可喜的临床效果， 但这类药物 只对表达特定靶点的肿瘤有效， 能治疗的癌症类型有限， 往往客观反应率较低， 应用这类抗 体之后的肿瘤易产生耐药性从而复发和重新进展。 肿瘤免疫治疗是应用免疫学原理和方法， 激活免疫系统， 打破免疫系统对肿瘤的免疫耐受状态， 增强免疫细胞对肿瘤抗原的识别， 激 发和增强机体抗肿瘤免疫应答， 从而使机体免疫系统抑制和杀伤肿瘤细胞， 最终达到治疗肿 瘤的目的。 肿瘤免疫治疗近来备受关注， 是肿瘤治疗领域的焦点。 近几年， 肿瘤免疫治疗的 重大好消息不断涌现。目前，在癌症免疫治疗中程序性死亡受体 1 ( programmed death 1，PD-1 ) 无疑是最耀眼的明星革 G点， 以其为革 G点的免疫检查点抑制剂如 nivolumab 和 pembrolizumab 等单抗类药物已在一些肿瘤类型如黑色素瘤、 非小细胞肺癌等的临床治疗中展示出了强大的 抗肿瘤活性， 已获得美国 FD A (Food and Dmg Administration, FDA) 批准临床应用。 肿瘤免 疫治疗由于其卓越的疗效和创新性， 在 2013年被 《科学》 杂志评为年度最重要的科学突破。 肿瘤免疫治疗有望成为继手术、 化疗、 放疗和靶向治疗后肿瘤治疗领域的一场革命。
T细胞在免疫系统中扮演重要角色， 不过 T细胞的活化需依赖抗原递呈细胞将外来有害 的抗原消化并重新呈现为 T细胞可以识别的抗原型态。 T细胞和抗原递呈细胞上有一群参与 调节的蛋白质， 具有辅助调节 T细胞受体信号转导的功能， 这些辅助性受体分为 2大类， 有 一类是负责传递激活信号的共刺激性受体， 另一类是传递抑制信号的共抑制性受体， 如今这 类抑制性分子被称为免疫检查点的受体和配体。上述提到的明星分子 PD-1就是典型的免疫检 查点的受体， 属于 CD28 超家族成员， 其重要配体为程序性死亡配体 -1 ( programmed death ligand 1， PD-L1 ) 。 PD-1与 PD-L1的结合介导 T细胞活化的共抑制信号， 负向调节 T细胞 活化和增殖。 另外， 华裔科学家陈列平教授首先发现 PD-L1在肿瘤组织高表达， 而且调节肿 瘤浸润 CD8+T细胞的功能。 目前， 以 PD-1/PD-L1为靶点的免疫调节疗法已在临床抗肿瘤治 疗中取得了巨大成功。
淋巴细胞活化基因 -3 ( lymphocyte activation gene-3 , LAG-3 ) 主要表达于活化的 T淋巴 细胞， 是一种类似于 PD-1的免疫负调节分子， LAG-3的主要配体是 MHC II分子家族， 它能 够以更高的亲和力与 CD4分子竞争结合 MHC II配体， 并向细胞内部转导抑制信号， 从而负 向调节细胞的激活、增殖和动态平衡， 其分子机制非常类似于 PD-1。 LAG-3能够帮助 T细胞 维持在免疫耐受状态， 或使持续激活的 T细胞处于耗竭的状态， 具有维持内环境稳定和参与 免疫负向调节的功能，与肿瘤的发生发展密切相关。 目前，一些初步的研究表明， 阻断 LAG-3 的抗体能够增强免疫反应，与 PD-1抗体联用能够起到显著的协同激活作用，在癌症免疫治疗 领域中潜力巨大。
目前国际上以 LAG-3为靶点的单抗类药物的研发大部分尚处于初期阶段，主要研发公司 都是百时美施贵宝 (Bristol-Myers Squibb， BMS) 、 诺华 (Novartis) 等国际医药巨头， 其中 百时美施贵宝的抗 LAG-3单抗 BMS-986016正处于临床一期阶段， 主要研究目标是与 PD-1 抗体 nivolumab联合使用的安全性和有效性， 研究结果显示， BMS-986016与 nivolumab联合 应用在黑色素瘤的临床治疗中展现出良好疗效。 另外， 抗 LAG-3抗体还可以用来治疗自身免 疫病， 基本机制是通过抗体依赖细胞介导的细胞毒效应 ( antibody-dependent cell-mediated cytotoxicity， ADCC)杀伤激活的 T细胞， 从而起到抑制免疫反应和治疗自身免疫病的目的。 然而，仍亟待开发更多新型、特异、高效的以 LAG-3为靶点的药物用于免疫疗法的临床应用。 发明内容
为了解决上述技术问题， 本发明的发明人进行了大量试验， 从抗原免疫、 杂交瘤筛选、 抗体表达纯化到生物活性鉴定， 筛选获得了两个特异性结合人 LAG-3的鼠源 134和 2-34 (即 5E7) 号抗体， 并在此基础上， 进一步构建获得其嵌合抗体 134-Chimeric、 5E7-Chimeric以及 人源化抗体 134-Hu-IgG4-C91S、 5E7-Hu-IgG4。本发明的研究表明鼠源 134和 2-34 (即 5E7) 号抗体具有全新的与人 LAG-3 结合的表位。 细胞水平实验结果表明人源化抗体 134-Hu-IgG4-C91S、 5E7-Hu-IgG4能够有效地阻断 LAG-3对 Raji细胞的结合和增强 SEA刺 激的 PBMC分泌 IL-2。 因此， 本发明开发的结合人 LAG-3的抗体或其抗原结合片段可应用 于制备用于癌症免疫疗法的药物。
因此， 本发明的第一个目的在于提供一种结合人 LAG-3的抗体或其抗原结合片段。
本发明的第二个目的在于提供另一种结合人 LAG-3的抗体或其抗原结合片段。
本发明的第三个目的在于提供编码所述的结合人 LAG-3的抗体或其抗原结合片段的核苷 酸序列。
本发明的第四个目的在于提供含有所述的核苷酸序列的表达载体。
本发明的第五个目的在于提供含有所述的表达载体的宿主细胞。
本发明的第六个目的在于提供所述的结合人 LAG-3的抗体或其抗原结合片段的制备方法。 本发明的第七个目的在于提供含有所述的结合人 LAG-3的抗体或其抗原结合片段的药物 组合物。
本发明的第八个目的在于提供所述的结合人 LAG-3的抗体或其抗原结合片段或所述的药 物组合物的用途。
为了实现上述目的， 本发明采用了如下技术方案： 本发明的第一个方面提供了一种结合人 LAG-3 的抗体或其抗原结合片段， 其结合的人
LAG-3表位包括以下氨基酸序列： AAAPGHPLA (SEQIDNO: 50) 。
根据本发明的优选实施例， 其结合的人 LAG-3 表位包括以下氨基酸序列： GPPAAAPGHPLA (SEQIDNO： 48) 或 AAAPGHPLAPGPHPAAPSS (SEQIDNO： 49) 。
本发明的第二个方面提供了一种结合人 LAG-3的抗体或其抗原结合片段， 包括：
(a)重链互补决定区 HCDR1、 HCDR2、 HCDR3, 所述 HCDR1的氨基酸序列如 SEQ ID NO： 9所示， 所述 HCDR2的氨基酸序列如 SEQ ID NO： 10所示， 所述 HCDR3的氨基酸序 列如 SEQ ID NO: 11所示， 和轻链互补决定区 LCDR1、 LCDR2、 LCDR3， 所述 LCDR1的 氨基酸序列如 SEQIDNO: 12所示， 所述 LCDR2的氨基酸序列如 SEQIDNO: 13所示， 所 述 LCDR3的氨基酸序列如 SEQID NO: 14或 SEQ ID NO： 43所示； 或
(b)重链互补决定区 HCDR1、 HCDR2、 HCDR3, 所述 HCDR1的氨基酸序列如 SEQ ID NO： 15所示， 所述 HCDR2的氨基酸序列如 SEQIDNO: 16所示， 所述 HCDR3的氨基酸序 列如 SEQ ID NO: 17所示， 和轻链互补决定区 LCDR1、 LCDR2、 LCDR3， 所述 LCDR1的 氨基酸序列如 SEQIDNO: 18所示， 所述 LCDR2的氨基酸序列如 SEQIDNO: 19所示， 所 述 LCDR3的氨基酸序列如 SEQ ID NO： 20所示。
根据本发明， 所述抗体为单克隆抗体或多克隆抗体。 优选的， 所述抗体为单克隆抗体。 根据本发明， 所述抗体为鼠源抗体、 嵌合抗体或人源化抗体等。 优选的， 所述抗体为人 源化抗体。
根据本发明， 所述抗原结合片段包括 Fab片段、 F(ab, )2片段、 Fv片段等。
根据本发明的优选实施例，所述结合人 LAG-3的抗体或其抗原结合片段包括重链可变区 和轻链可变区， 选自：
(a)所述重链可变区的氨基酸序列如 SEQIDNO: 2所示， 所述轻链可变区的氨基酸序 列如 SEQIDNO: 4所示；
(b)所述重链可变区的氨基酸序列如 SEQIDNO: 6所示， 所述轻链可变区的氨基酸序 列如 SEQIDNO: 8所不；
(c) 所述重链可变区的氨基酸序列如 SEQ ID NO: 22所示， 所述轻链可变区的氨基酸 序列如 SEQ ID NO： 24所示； 和
(d) 所述重链可变区的氨基酸序列如 SEQ IDNO: 26所示， 所述轻链可变区的氨基酸 序列如 SEQ ID NO： 28所示。
根据本发明的优选实施例，所述结合人 LAG-3的抗体或其抗原结合片段包括重链恒定区 和轻链恒定区， 所述重链恒定区选自 IgGl、 IgG2、 IgG3和 IgG4重链恒定区， 所述轻链恒定 区选自 K或 X轻链恒定区。 优选地， 所述重链恒定区为 IgG4重链恒定区， 所述轻链恒定区 为 K轻链恒定区。 更优选的， 所述重链恒定区的氨基酸序列如 SEQIDNO: 30所示， 所述轻 链恒定区的氨基酸序列如 SEQID NO： 34所示。
根据本发明的优选实施例，所述结合人 LAG-3的抗体或其抗原结合片段包括重链和轻链， 选自:
(a)所述重链的氨基酸序列如 SEQ ID NO: 32所示， 所述轻链的氨基酸序列如 SEQ ID NO： 36所示； 和
(b)所述重链的氨基酸序列如 SEQ ID NO: 38所示， 所述轻链的氨基酸序列如 SEQ ID NO： 40所示。
本发明的第三个方面提供了一种核苷酸序列， 所述核苷酸序列编码如上任一项所述的结 合人 LAG-3的抗体或其抗原结合片段。
根据本发明的优选实施例， 所述核苷酸序列包括：
(a) 如 SEQ ID NO: 1所示编码重链可变区的核苷酸序列， 如 SEQIDNO: 3所示编码 轻链可变区的核苷酸序列；
(b) 如 SEQ ID NO: 5所示编码重链可变区的核苷酸序列， 如 SEQIDNO: 7所示编码 轻链可变区的核苷酸序列；
(c) 如 SEQIDNO: 21所示编码重链可变区的核苷酸序列， 如 SEQIDNO: 23所示编 码轻链可变区的核苷酸序列； 或
(d) 如 SEQ ID NO: 25所示编码重链可变区的核苷酸序列， 如 SEQIDNO: 27所示编 码轻链可变区的核苷酸序列。
根据本发明的优选实施例， 所述核苷酸序列包括如 SEQ ID NO: 29所示的编码重链恒定 区的核苷酸序列， 和如 SEQIDNO: 33所示的编码轻链恒定区的核苷酸序列。
根据本发明的优选实施例， 所述核苷酸序列包括：
(a) 如 SEQ ID NO: 31所示编码重链的核苷酸序列， 如 SEQIDNO: 35所示编码轻链 的核苷酸序列； 或
(b) 如 SEQ ID NO: 37所示编码重链的核苷酸序列， 如 SEQIDNO: 39所示编码轻链 的核苷酸序列。
本发明的第四个方面提供了一种表达载体， 所述表达载体含有如上任一项所述的核苷酸 序列。
本发明的第五个方面提供了一种宿主细胞， 所述宿主细胞含有如上所述的表达载体。 本发明的第六个方面提供了如上所述的结合人 LAG-3的抗体或其抗原结合片段的制备方 法， 所述方法包括以下步骤：
(a) 在表达条件下， 培养如上所述的宿主细胞， 从而表达所述的结合人 LAG-3 的抗体 或其抗原结合片段；
(b) 分离并纯化 (a) 所述的结合人 LAG-3的抗体或其抗原结合片段。
本发明的第七个方面提供了一种药物组合物， 所述药物组合物含有如上任一项所述的结 合人 LAG-3的抗体或其抗原结合片段和药学上可接受的载体。
根据本发明的优选实施例， 所述药物组合物还包括 PD-1抑制剂。 优选的， 所述 PD-1抑 制剂为结合 PD-1的抗体或其抗原结合片段。 本发明的第八个方面提供了如上任一项所述的结合人 LAG-3的抗体或其抗原结合片段或 如上任一项所述的药物组合物在制备治疗癌症的药物中的用途。
根据本发明， 所述癌症选自黑色素瘤、 肾细胞癌、 非小细胞肺癌、 经典霍奇金淋巴瘤、 尿路上皮癌、 结直肠癌以及肝癌等。
有益效果：
本发明公开了能够特异性结合人 LAG-3 的抗体或其抗原结合片段， 其具有全新的与人 LAG-3结合的表位以及良好的增强混合淋巴细胞反应的生物学活性， 可应用于制备用于癌症 免疫疗法的药物， 具有良好的临床应用前景。 附图说明
图 1为鼠源抗人 LAG-3抗体对混合淋巴细胞反应的影响的结果， 其中， 图 1A为第一轮 筛选， 图 1B为第二轮筛选。
图 2为抗人 LAG-3抗体的人源化抗体和嵌合抗体之间的相对亲和力比较的结果。
图 3为人源化抗人 LAG-3抗体对 PBMC分泌 IL-2的增强作用的结果， 其中， 图 3A和 3B中的 PBMC分别来自不同的捐献者。
图 4为人源化抗人 LAG-3抗体对细胞表面 LAG-3的结合作用的结果。
图 5为抗人 LAG-3抗体表位的示意图。
图 6为人源化抗人 LAG-3抗体对 LAG-3结合 Raji细胞的阻断作用的结果。
图 7为流式细胞法检测抗人 LAG-3抗体对 FGL1结合 LAG-3的阻断作用的结果。
图 8为抗人 LAG-3抗体对转基因小鼠 MC38移植瘤模型的抑制作用。 具体实施方式
本发明中， 术语“LAG-3”是指淋巴细胞活化基因 3， 也称作 CD223。
本发明中， 术语“抗体（Ab）”和“免疫球蛋白 G（IgG）”是有相同结构特征的约 150000 道尔顿的异四聚糖蛋白， 其由两个相同的轻链 （L） 和两个相同的重链 （H） 组成。 每条轻链 通过一个共价二硫键与重链相连， 而不同免疫球蛋白同种型的重链间的二硫键数目不同。 每 条重链和轻链也有规则间隔的链内二硫键。每条重链的一端有可变区（VH），其后是恒定区， 重链恒定区由三个结构域 CHI、 CH2、 以及 CH3构成。 每条轻链的一端有可变区 （VL）， 另 一端有恒定区， 轻链恒定区包括一个结构域 CL; 轻链的恒定区与重链的第一个恒定区相对， 轻链的可变区与重链的可变区相对。 恒定区不直接参与抗体与抗原的结合， 但是它们表现出 不同的效应功能， 例如参与抗体依赖的细胞介导的细胞毒性作用（ADCC， antibody-dependent cell-mediated cytotoxicity）等。重链恒定区包括 IgGl、 IgG2、 IgG3、 IgG4、 IgM、 IgAl、 IgA2、 IgD和 IgE; 轻链恒定区包括 K （Kappa） 或 X （Lambda）。
本发明的抗体包括单克隆抗体、 多克隆抗体、 由至少两种抗体形成的多特异性抗体 （例 如双特异性抗体） 等。 本发明中， 术语“单克隆抗体 (单抗)”指从一类基本均一的群体获得的抗体， 即该群体 中包含的单个抗体是相同的， 除少数可能存在的天然发生的突变外。 单克隆抗体高特异性地 针对单个抗原位点。而且，与常规多克隆抗体制剂 (通常是具有针对不同决定簇的不同抗体) 不同， 各单克隆抗体是针对抗原上的单个决定簇。 除了它们的特异性外， 单克隆抗体的好处 还在于它们是通过杂交瘤培养来合成的， 不会被其它免疫球蛋白污染。 修饰语“单克隆”表 示了抗体的特性， 是从基本均一的抗体群中获得的， 这不应被解释成需要用任何特殊方法来 生产抗体。
本发明中， 术语“抗原结合片段”是指能够与抗原 (例如人 LAG-3 ) 特异性结合的抗体 的片段。 本发明的抗原结合片段的例子包括 Fab片段、 F(ab, )2片段、 Fv片段等。 Fab片段 是用木瓜蛋白酶消化抗体产生的片段。 F(ab’ )2片段是用胃蛋白酶消化抗体产生的片段。 Fv 片段是由抗体的重链可变区和轻链可变区紧密非共价关联的二聚物组成。
本发明中， 术语“Fc段”是指木瓜蛋白酶可将抗体裂解为两个完全相同的 Fab段和一个 Fc段， Fc段即可结晶片段 ( fragment crystallizable， Fc)， 由抗体的 CH2和 CH3结构域组成。 Fc段无抗原结合活性， 是抗体与效应分子或细胞相互作用的部位。
本发明中， 术语“可变”表示抗体中可变区的某些部分在序列上有所不同， 它形成各种 特定抗体对其特定抗原的结合和特异性。然而，可变性并不均匀地分布在整个抗体可变区中。 它集中于重链可变区和轻链可变区中称为互补决定区 ( complementarity-determining region, CDR) 或超变区中的三个片段中。 可变区中较保守的部分称为框架区 (frame region, FR)。 天然重链和轻链的可变区中各自包含四个 FR区， 它们大致上呈 折叠构型， 由形成连接环 的三个 CDR相连， 在某些情况下可形成部分 0折叠结构。 每条链中的 CDR通过 FR区紧密 地靠在一起并与另一链的 CDR 一起形成了抗体的抗原结合部位 (参见 Kabat 等， NIH Publ.No.91-3242， 卷 I， 647-669页 ( 1991 ) )。
本发明的抗体包括鼠源抗体、 嵌合抗体、 人源化抗体等。
本发明中， 术语“鼠源抗体”是指来源于大鼠或小鼠的抗体， 优选小鼠。 本发明的鼠源 抗体为使用人 LAG-3的胞外域为抗原免疫小鼠并进行杂交瘤细胞筛选获得。 优选的， 本发明 的鼠源抗体为 134号和 2-34 (即 5E7) 号抗体。
本发明中， 术语“嵌合抗体”是指包含来源于一个物种的重链可变区和轻链可变区序列 以及来源于另一个物种的恒定区序列的抗体， 例如具有与人恒定区连接的鼠重链可变区和轻 链可变区的抗体。 优选的， 本发明的嵌合抗体是由鼠源 134号和 2-34 ( SP5E7) 号抗体的重链 可变区和轻链可变区序列分别与人的恒定区拼接获得。 更优选的， 本发明的嵌合抗体的重链 是由鼠源 134号和 2-34 ( SP5E7 ) 号抗体的重链可变区序列分别与人的 IgG4重链恒定区拼接获 得， 轻链是由鼠源 134号和 2-34 ( BP5E7)号抗体的轻链可变区序列分别与人的 Kappa轻链恒定 区拼接获得。 最优选的， 本发明的嵌合抗体包括 134-Chimeric和 5E7-Chimeric。
本发明中， 术语“人源化抗体”是指其 CDR来源于非人物种 (优选小鼠)抗体， 抗体分 子中残余的部分 (包括框架区和恒定区) 来源于人抗体。 此外， 框架区残基可被改变以维持 结合亲和性。 优选的， 本发明的人源化抗体由鼠源 134号和 2-34 (即 5E7)号抗体的 CDR区 和来源自人抗体的非 CDR区重组， 并对包埋残基、 与 CDR区有直接相互作用的残基， 以及 对 134号和 2-34 (即 5E7)号抗体的 VL和 VH的构象有重要影响的残基进行回复突变获得。 更优选的， 本发明的人源化抗体包括 134-Hu-IgG4-C91 S和 5E7-Hu-IgG4。
本发明中， 术语“表位”和“人 LAG-3表位”是指位于人 LAG-3上并与抗体特异性结 合的区域。 优选的， 本发明的人 LAG-3表位位于人 LAG-3的胞外域内， 所述的人 LAG-3的 胞外域包括如 SEQ ID NO: 41所示的氨基酸序列。 更优选的， 本发明的人 LAG-3表位包括 以下氨基酸序列： AAAPGHPLA ( SEQ ID NO： 50)。 最优选的， 本发明的人 LAG-3表位包 括以下氨基酸序列： GPPAAAPGHPLA C SEQ ID NO: 48 )或 AAAPGHPLAPGPHPAAPSS ( SEQ ID NO： 49) 0
本发明中， 术语“结合人 LAG-3的抗体”或“抗人 LAG-3抗体”是指与人 LAG-3表位 特异性结合的抗体。 术语“特异性结合”是指抗体以小于 1 X 10'7M或更小、 优选为 1 X 10_8M 或更小、更优选为 1 X 10_9M或更小、 1 X 10_1()M或更小、 1 X 10_UM或更小的平衡解离常数 (KD) 结合。 本发明中， 术语“KD”是指特定抗体 -抗原相互作用的平衡解离常数， 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。 例如， 使用表面等离子体共振术 ( Surface Plasmon Resonance， 缩写 SPR) 在 BIACORE仪中测定抗体与抗原的结合亲和力或使用 ELISA测定抗体与抗原结合的相对亲和 力。
本发明中， 术语“表达载体”可以为 pTT5， pSECtag系列， pCGS3系列， pCDNA系列 载体等， 以及其它用于哺乳动物表达系统的载体等， 表达载体中包括连接有合适的转录和翻 译调节序列的融合 DNA序列。
本发明中， 术语“宿主细胞”是指适用于表达上述表达载体的细胞， 可以是真核细胞， 如哺乳动物或昆虫宿主细胞培养系统均可用于本发明的融合蛋白的表达， CHO (中国仓鼠卵 巢， Chinese Hamster Ovary), HEK293， COS， BHK以及上述细胞的衍生细胞均可适用于本 发明。
本发明中， 术语“药物组合物”是指本发明的结合人 LAG-3的抗体或其抗原结合片段可 以和药学上可以接受的载体一起组成药物制剂组合物从而更稳定地发挥疗效， 这些制剂可以 保证本发明公开的结合人 LAG-3 的抗体或其抗原结合片段的氨基酸核心序列的构象完整性， 同时还保护蛋白质的多官能团防止其降解 (包括但不限于凝聚、 脱氨或氧化)。 以下实施例、 实验例是对本发明进行进一步的说明， 不应理解为对本发明的限制。 实施 例不包括对传统方法的详细描述， 如那些用于构建载体和质粒的方法， 将编码蛋白的基因插 入到这样的载体和质粒的方法或将质粒引入宿主细胞的方法。 这样的方法对于本领域中具有 普通技术的人员是众所周知的， 并且在许多出版物中都有所描述， 包括 Sambrook, I, Fritsch, E. F . and Maniais, T. ( 1989) Molecular Cloning： A Laboratory Manual, 2nd edition, Cold spring Harbor Laboratory Press o 实施例 1. 人 LAG-3蛋白以及抗 LAG-3阳性对照抗体的制备
人 LAG-3胞外段氨基酸序列来自 https :/7www. uniprot. ore/uniprot/P 18627, 氨基酸序列如 SEQ ID NO： 41所示， 由生工生物工程 (上海) 股份有限公司进行密码子优化并合成基因， 然后将合成的基因片段克隆到 pUC57 载体中。设计引物，用 PCR( Polymerase Chain Reaction) 法扩增 LAG-3胞外段 (Extracellular Domain， ECD) 编码区 ( SEQ ID NO: 41 ) 和人免疫球 蛋白 IgGl的 Fc段编码区 ( SEQ ID NO： 51 )； 将上述扩增出的片段回收， 通过重组 PCR将 上述片段拼接在一起， 同时引入信号肽 (MGVKVLFALICIAVAEA， SEQ ID NO： 42) 编码 区， 并在两端引入相应的酶切位点； 用限制性内切酶切割 pTT5 表达载体 (购自 NRC Biotechnology Research Institute) 和前述重组 PCR片段； 两组酶切产物纯化后， 用连接酶连 接后转化到大肠杆菌 TOP 10感受态细胞中，涂布于 LB (Amp抗性)平板培养基中过夜培养； 挑取单克隆菌落， 培养扩增之后抽提质粒， 用限制性内切酶切割质粒以鉴定是否有目的基因 片段插入； 挑选合格的阳性克隆测序； 选取序列完全正确的克隆扩增之后抽提质粒。 利用聚 乙烯亚胺 (Polyethyleneimine, PEI)将上述构建好的 LAG-3表达载体转入 HEK293E细胞 (购 自 NRC Biotechnology Research Institute) 中进行表达， 5天后收取细胞上清， 利用 Protein A 亲和层析纯化， 所得蛋白命名为 LAG-3-ECD-hFc， 用于免疫小鼠及后续筛选过程。 HEK293E 细胞在化学成分限定的无血清培养基 FreeStyle 293 Expression Medium (购自 Thermo Fisher Scientific) 中培养。 蛋白定量通过紫外分光光度法进行。
作为阳性对照的抗 LAG-3单克隆抗体的重链可变区和轻链可变区的氨基酸序列来自美国 专利申请 US20140093511A1中的 SEQ ID NO: 12和 14， 由生工生物工程 (上海) 股份有限 公司进行密码子优化并合成基因， 重链可变区和轻链可变区基因分别与人 IgG4 重链恒定区 (核苷酸序列如 SEQ ID NO： 29所示， 氨基酸序列如 SEQ ID NO： 30所示) 和人 Kappa轻 链恒定区 (核苷酸序列如 SEQ ID NO: 33所示， 氨基酸序列如 SEQ ID NO: 34所示)连接， 并引入信号肽序列 ( SEQ ID NO： 42)， 构建成完整的重链和轻链基因， 然后按照上述类似的 方法将完整的重链和轻链基因分别克隆到 pTT5 表达载体中。 用同样的方法将含有重链和轻 链基因的 pTT5表达载体组合后， 转入 HEK293E细胞中进行表达， 5天后收取细胞上清， 利 用 Protein A亲和层析纯化抗体， 所得抗体命名为 Anti-LAG3.5。 HEK293E细胞在化学成分限 定的无血清培养基 FreeStyle 293 Expression Medium (购自 Thermo Fisher Scientific) 中培养。 蛋白定量通过紫外分光光度法测定。 实施例 2. 用人 LAG-3-ECD-hFc作为抗原免疫小鼠以及杂交瘤的制备和筛选
将实施例 1中制备 LAG-3-ECD-hFc用生理盐水稀释至合适浓度后，与等体积弗氏完全佐 剂 (购自 Sigma) 混合， 超声乳化后对 4-5周龄的 Balb/c小鼠 (购自上海灵畅生物科技有限 公司， 动物生产许可证号： SCXK (沪) 2013-0018 ) 进行多点皮下注射； 三周后， 用生理盐 水稀释 LAG-3-ECD-hFc并与等体积弗氏不完全佐剂（购自 Sigma）混合， 超声乳化完全后对 小鼠进行多点皮下注射； 两周后再次重复此步骤； 所有小鼠在第三次免疫后的第七天取少量 血样分离血清， 利用常规 ELISA法检测血清滴度； 选取血清抗体效价＞10000的小鼠， 七天后 每只小鼠通过尾静脉注射 10|ig抗原蛋白。
静脉注射抗原蛋白后的第三天， 选取一定数量的小鼠， 处死并解剖取出脾脏， 研磨后收 集脾脏细胞并计数， 用于制备杂交瘤。 在此选取骨髓瘤 Sp2/0细胞 （来源自中国科学院典型 培养物保藏委员会细胞库） 作为细胞融合配体。 Sp2/0细胞在细胞培养箱 （37°C， 5% C02） 中培养， 所用培养基为 RPMI- 1640完全培养基（配制方法如下： 在 RPMI-1640基础培养基中 添加 10%的胎牛血清和 1%的 Penicillin-Streptomycin， 上述材料均购自 Thermo Fisher Scientific）, 细胞融合前一天更换培养基， 使细胞处于较好的生长状态。 在此采取电融合法制 备杂交瘤细胞。 电融合法与杂交瘤筛选过程如下： 按照脾脏细胞： Sp2/0细胞 =2: 1的比例混 合两种细胞， 离心后弃上清液； 然后用细胞融合缓冲液 （购自 BTX） 洗涤细胞三次； 细胞沉 淀以 l x lO7 /ml的密度悬浮于细胞融合缓冲液中； 加 2ml细胞悬液于细胞融合池中，置于电融 合仪 ECM2001 （购自 BTX）上， 按一定条件 （AC 60V， 30S； DC 1700V， 40pS， 3X； POST AC 60V， 3S） 进行处理； 然后轻轻地将处理后的细胞转移至 37°C预热的 RPMI-1640完全培 养基中， 室温放置 1小时； 将处理后的细胞按 104/孔的密度接种入 96孔板， 每孔 1000; 次 日每孔补加 100M含有 2xHAT （50xHAT购自 Thermo Fisher Scientific, 使用时按照需要用培 养基适当稀释） 的 RPMI-1640完全培养基； 分别在第四天和第七天用新鲜的含有 l xHAT的 RPMI-1640完全培养基置换旧培养液;于融合后第九天取样进行 ELISA检测，鉴定阳性克隆。
阳性克隆 （能够分泌抗 LAG-3 抗体的克隆） 的检测通过 ELISA 方法进行： 利用
LAG-3-ECD-hFc抗原包被酶标板，包被量为 100ng/孔； 室温孵育 2小时后用 PBST（含 0.05% Tween-20的 PBS）清洗；每孔加入 200[jl含 1%牛血清白蛋白（Bovine Serum Albumin, BSA） 的 PBST溶液进行封闭， 室温下孵育 1小时； 用 PBST洗涤后排干， 放置冰箱中保存待用。 检测时在酶标板中加入杂交瘤培养上清 1000/孔， 孵育 1小时左右； 用 PBST洗涤 3遍； 用 含有 1% BSA的 PBST适当稀释 HRP-Goat- Anti-Mouse IgG抗体 （购自 KPL）， 室温孵育 1小 时左右; PBST洗涤 3遍;每孔加 100M显色液（以 TMB为底物）显色；随后每孔加入 2M H2S04 终止液终止反应； 立即用酶标仪（Molecular Device）在 450nm波长处测量各孔 OD值； 分析 数据， 确定阳性克隆。
筛选出的阳性克隆并一定是单克隆， 并且有些克隆可能不稳定， 因此需要及时进行亚克 隆， 并最终获得单克隆。 亚克隆采用有限稀释法完成， 过程如下： 制备阳性克隆的杂交瘤细 胞悬液， 用 HT培养基（HT培养基配制方法如下： 用 RPMI-1640完全培养基将 100xHT溶液 稀释至所需浓度， 100xHT购自 Thermo Fisher Scientific） 将细胞密度调整至 10或 20个细胞 每毫升的稀释度； 每个稀释度接种 96孔板一块， 每孔量为 0.2ml， 每孔的杂交瘤细胞数平均 为 2或 4; 置于 C02细胞培养箱中培养 7-10天， 出现肉眼可见的克隆时， 在倒置显微镜下观 察， 标出只有单个克隆生长的孔， 取上清作 ELISA检测 （检测方法如上所述）； 取 ELISA检 测为阳性的孔在 24孔板中进行扩大培养， 并用有限稀释法继续进行新一轮亚克隆， 挑选不少 于 24个克隆孔， 进行 ELISA检测， 当检测呈 100%阳性时， 即为稳定表达目的抗体的单克隆 杂交瘤细胞株； 将单克隆杂交瘤细胞株进行扩大培养， 并进行冻存保种。
将稳定的单克隆细胞株扩大培养， 在无血清培养基 HybriGRO SF（购自 Corning）中培养 5-7天左右， 细胞培养上清经过离心和过滤除去沉淀后， 用 Protein G（购自 GE Healthcare） 纯化鼠源抗体。 经过若干次融合和筛选， 本发明成功建立了 200多株稳定的杂交瘤单克隆细 胞株， 并且纯化得到了相应的鼠源抗体， 用于进一步的实验。 实施例 3. 鼠源抗人 LAG-3抗体对混合淋巴细胞反应的影响
在此采用混合淋巴细胞反应（Mixed Lymphocyte Reaction， MLR） 对鼠源抗人 LAG-3抗 体的活性进行评估， 并加入 Anti-LAG3.5作为阳性对照。
MLR实验方法描述如下： 用 Histopaque （购自 Sigma） 从人血液中分离出外周血单个核 细胞 （Peripheral Blood Mononuclear Cell， 缩写 PBMC）， 将 PBMC中的单核细胞亚群通过贴 壁法分离出来， 然后用 IL-4 和 GM-CSF 联合诱导单核细胞分化成诱导的树突状细胞 （p38 MAPK-inhibited dendritic cells induce superior antitumour immune responses and overcome regulatory T-cell-mediated immunosuppression. Nat Commun. 2014 Jun 24;5:4229. doi: 10.1038/ncomms5229.）； 七天之后， 消化收集上述诱导的树突状细胞； 从另外供体的血液中 用上述方法分离出 PBMC， 然后用 MACS磁铁和 CD4 MicroBeads （购自 MiltenyiBiotec） 从 中分离 CD4阳性 T细胞；将诱导的树突状细胞（ 104个 /孔）和分离出的 CD4阳性 T细胞（ 105 个 /孔）按 10: 1的比例混匀后接种到 96孔板中， 每孔 150M; 2小时后， 在上述 96孔板中加 入 50M用 AIM-V培养基稀释好的鼠源抗 LAG-3抗体；同时设置阳性对照抗体 Anti-LAG3.5， 以及不结合 LAG-3的无关同型对照抗体； 将 96孔板置于细胞培养箱 （37°C， 5% C02） 中孵 育 3-4天， 取适量培养上清， 用双抗夹心 ELISA检测 IL-2的分泌量 （相关检测试剂购自 BD Biosciences）； 用多功能酶标仪（Molecular Device） 读取 OD450， 用 GraphPad Prism6进行数 据分析和作图。 本实施例中使用无血清培养基 AIM-V（购自 Thermo Fisher Scientific）培养细 胞。
实验结果如图 1A和 1B所示， 通过两轮筛选 （图 1A和 1B）， 获得两个能够明显增强 MLR分泌 IL-2的鼠源抗人 LAG-3抗体， 分别是 134号和 2-34 （即 5E7） 号单抗。 实施例 4. 优选鼠源抗人 LAG-3抗体的核苷酸和氨基酸序列的测定
根据实施例 3的筛选结果， 最终挑取了 134号和 5E7号单抗作为先导抗体。 使用 TRIzol （购自 Thermo Fisher Scientific） 从 134和 5E7对应的杂交瘤单克隆细胞株中提取总 RNA， 用逆转录试剂盒 （购自 Takara公司） 将 mRNA逆转录成 cDNA， 通过文献报道的引物组合 （《Antibody Engineering》 Volume 1， Edited by Roland Kontermann and Stefan Diibel； 引物组 合的序列来自第 323页） 用 PCR扩增鼠源的抗人 LAG-3抗体的轻链可变区和重链可变区基 因， 然后将 PCR产物克隆入 pMD18-T载体， 测序并分析可变区基因序列。
134号克隆的序列信息如下： 重链可变区基因序列全长 360bp， 编码 120个氨基酸残基， 核苷酸序列如 SEQ ID NO: 1所示， 氨基酸序列如 SEQ ID NO: 2所示； 轻链可变区基因序 列全长 321bp， 编码 107个氨基酸残基， 核苷酸序列如 SEQ ID NO: 3所示， 氨基酸序列如 SEQIDNO： 4所示。
134号克隆的重链可变区的氨基酸序列
EVQLQQSGPVLVKPGASVKMSCKASGYTLTAYYMNWVKQSRGKSLEWIGVINPYNGDSSY NQKFKGKATLTVDKSSSTAYMELNSLTSEDSAVYYCARDDGYYRWYFDVWGTGTTVTVSS
(SEQIDNO： 2)
134号克隆的轻链可变区的氨基酸序列
DIQMTQSPSSLSASLGERVSLTCRASQDIGSRLNWLQQGPDGSIKRLIYATSSLESGVPKRFS GSRSGSDYFLTISSLESEDFVDYYCLQCGSSPPTFGGGTKLEIK (SEQIDNO： 4)
5E7号克隆的序列信息如下： 重链可变区基因序列全长 348bp， 编码 116个氨基酸残基， 核苷酸序列如 SEQ ID NO: 5所示， 氨基酸序列如 SEQ ID NO: 6所示； 轻链可变区基因序 列全长 321bp， 编码 107个氨基酸残基， 核苷酸序列如 SEQ ID NO: 7所示， 氨基酸序列如 SEQIDNO： 8所示。
5E7号克隆的重链可变区的氨基酸序列
EVKLVESGGGLVKPGGSLKLSCAASGFTFSDDYMAWFRQTPEKRLEWVASISHGGDYIYYA DNLKGRFTISRDNAKNTLYLQMSSLKSEDTAIYFCSRDRRSIDYWGQGTTLTVSS ( SEQ ID
NO： 6)
5E7号克隆的轻链可变区的氨基酸序列
DIQMTQITSSLSASLGDRVTITCRASQDISNYLSWYQQKPDGTIKLLIYYTSRLHSGVPSRFS GSGSGTDYSLTISDLEQEDIATYFCQQGKTLPYTFGGGTKLERK ( SEQ ID NO： 8) 实施例 5. 鼠源抗人 LAG-3抗体的人源化
对实施例 4中的重链可变区和轻链可变区的氨基酸序列进行分析， 依据 Rabat编码规则 分别确定 134和 5E7号抗体重链和轻链的 3个抗原互补决定区 (CDR)和 4个框架区 (FR)。
其中， 134号克隆的重链互补决定区的氨基酸序列为 HCDR1： AYYMNCSEQ ID NO: 9)、 HCDR2： VINPYNGDSSYNQKFKG (SEQIDNO： 10)和 HCDR3: DDGYYRWYFDV (SEQ ID NO： 11)， 轻链互补决定区的氨基酸序列为 LCDR1: RASQDIGSRLN ( SEQ ID NO： 12)、 LCDR2： ATSSLES (SEQIDNO： 13) 和 LCDR3: LQCGSSPPT (SEQIDNO： 14)。
其中， 5E7号克隆的重链互补决定区的氨基酸序列为 HCDR1: DDYMA (SEQIDNO： 15)、 HCDR2： SISHGGDYIYYADNLKG (SEQIDNO： 16)和 HCDR3: DRRSIDY (SEQ ID NO： 17)， 轻链互补决定区的氨基酸序列为 LCDR1: RASQDISNYLS (SEQ ID NO： 18)、 LCDR2： YTSRLHS (SEQIDNO： 19) 和 LCDR3: QQGKTLPYT (SEQIDNO： 20)。 在 https://www.ncbi. nlm.nih.gov/igblast/中，将 134号抗体重链可变区氨基酸序列与人免疫 球蛋白可变区胚系 ( Germline)序列进行同源性比较，选择与 134号同源性最高的 IGHV1 -46*01 为重链可变区人源化模板， 用鼠源 134号重链 CDR替换 IGHV1-46*01中对应的 CDR， 构建 成重链 CDR移植可变区。 同样地， 经过同源性比较， 选择 IGKV1-39*01胚系序列为轻链可 变区人源化模板，将鼠源 134号轻链 CDR替换 IGKV1-39*01中对应的 CDR，构建成轻链 CDR 移植可变区。然后在重链和轻链 CDR移植可变区的基础上，对一些框架区内的氨基酸位点进 行回复突变。 回复突变就是将 CDR移植可变区的框架区内的某些氨基酸 (对维持抗体结构和 亲和力比较重要的氨基酸) 突变成鼠源框架区对应位置上的氨基酸。 在进行回复突变时， 依 据 Rabat规则将氨基酸序列编码，每个氨基酸的位置由 Rabat编码指示。优选的，将重链 CDR 移植可变区 44位的 G回复突变为 S， 48位的 M突变为 I， 67位的 V突变为 A， 69位的 M 突变为 L， 71位的 R突变为 V， 73位的 T突变为 K， 78位的 V突变为 A。 优选的， 将轻链 CDR移植可变区 36位的 Y回复突变为 L， 第 43位的 A突变为 S， 第 44位的 P突变为 I， 第 46位的 L突变为 R，第 66位的 G突变为 R，第 69位的 T突变为 S，第 71位的 F突变为 Y。 需要注意的是， 134号抗体轻链 CDR3 ( SEQ ID NO： 14 )中含有一个多余的半胱氨酸 (Rabat 编码的 91位， C91 )，其可能导致抗体结构异常，故在此将其突变成丝氨酸 ( C91 S， SEQ ID NO： 43 )。 在携带上述回复突变的重链和轻链 CDR移植可变区的末端分别加上氨基酸序列： WGQGTKVEIK ( SEQ ID NO： 44) 和 FGQGTKVEIK ( SEQ ID NO: 45 )， 构建成完整的人 源化的重链和轻链可变区， 相关基因序列由生工生物工程 (上海) 股份有限公司进行密码子 优化和基因合成。
最终获得的 134人源化重链可变区 ( 134-Hu-VH)基因序列全长 354bp， 编码 118个氨基 酸残基， 核苷酸序列如 SEQ ID NO: 21所示， 氨基酸序列如 SEQ ID NO: 22所示； 134人源 化轻链可变区 ( 134-HU-VL-C91 S ) 基因序列全长 321bp， 编码 107个氨基酸残基， 核苷酸序 列如 SEQ ID NO： 23所示， 氨基酸序列如 SEQ ID NO： 24所示。
134人源化重链可变区 ( 134-Hu-VH) 的氨基酸序列
OVOLVOSGAEVKKPGASVKVSCKASGYTLTAYYMNWVROAPGOSLEWIGVINPYNGDSS YNOKFKGRATLTVDKSTSTAYMELSSLRSEDTAVYYCARDDGYYRWYFDVWGOGTLVTV SS ( SEQ ID NO: 22， 其中下划线部分为重链互补决定区)
134人源化轻链可变区 ( 134-HU-VL-C91 S) 的氨基酸序列
DIOMTOSPSSLSASVGDRVTITCRASODIGSRLNWLOOKPGKSIKRLIYATSSLESGVPSRFS GSRSGSDYTLTISSLOPEDFATYYCLOSGSSPPTFGOGTKVEIK ( SEQ ID NO: 24， 其中下划 线部分为轻链互补决定区)
用同样的方法对 5E7号抗体进行人源化。选择与 5E7号同源性最高的 IGHV3-21 *01为重 链可变区人源化模板， 用鼠源 5E7号重链 CDR替换 IGHV3-21 *01中对应的 CDR， 构建成重 链 CDR移植可变区。 同样地， 经过同源性比较， 选择 IGKV1-33*01胚系序列为轻链可变区 人源化模板， 将鼠源 5E7号轻链 CDR替换 IGKV1-33*01 中对应的 CDR， 构建成轻链 CDR 移植可变区。然后在重链和轻链 CDR移植可变区的基础上，对一些框架区内的氨基酸位点进 行回复突变。 在进行回复突变时， 依据 Rabat规则将氨基酸序列编码， 每个氨基酸的位置由 Kabat编码指示。 优选的， 将重链 CDR移植可变区 37位的 V回复突变为 F， 44位的 G突变 为 R， 49位的 S突变为 A， 91位的 Y突变为 F， 93位的 A突变为 S。 优选的， 将轻链 CDR 移植可变区 43位的 A突变为 T，第 44位的 P突变为 I，第 71位的 F突变为 Y，第 87位的 Y 突变为 F。 在携带上述回复突变的重链和轻链 CDR移植可变区的末端分别加上氨基酸序列： WGQGTKVEIK ( SEQ ID NO： 44) 和 FGQGTKVEIK ( SEQ ID NO： 45 )， 构建成完整的人 源化的重链和轻链可变区， 相关基因序列由生工生物工程 (上海) 股份有限公司进行密码子 优化和基因合成。
最终获得的 5E7人源化重链可变区 ( 5E7-HU-VH) 基因序列全长 354bp， 编码 118个氨 基酸残基， 核苷酸序列如 SEQ ID NO： 25所示， 氨基酸序列如 SEQ ID NO： 26所示； 5E7 人源化轻链可变区 ( 5E7-HU-VL)基因序列全长 321bp，编码 107个氨基酸残基， 核苷酸序列 如 SEQ ID NO： 27所示， 氨基酸序列如 SEQ ID NO： 28所示。
5E7人源化重链可变区 ( 5E7-HU-VH) 的氨基酸序列
EVOLVESGGGLVKPGGSLRLSCAASGFTFSDDYMAWFROAPGKRLEWVASISHGGDYIYY ADNLKGRFTISRDNAKNSLYLOMNSLRAEDTAVYFCSRDRRSIDYWGOGTLVTVSS ( SEQ
ID NO: 26， 其中下划线部分为重链互补决定区)
5E7人源化轻链可变区 ( 5E7-HU-VL) 的氨基酸序列
DIOMTOSPSSLSASVGDRVTITCRASODISNYLSWYOOKPGKTIKLLIYYTSRLHSGVPSRFS GSGSGTDYTFTISSLOPEDIATYFCOOGKTLPYTFGOGTKVEIK ( SEP ID NO： 28, 其中下划 线部分为轻链互补决定区)
将上述人工合成的 134人源化重链可变区与人免疫球蛋白 IgG4恒定区 (核苷酸序列如 SEQ ID NO： 29所示， 氨基酸序列如 SEQ ID NO: 30所示)相连， 构建成完整的 134人源化 重链， 命名为 134-Hu-IgG4-HC (核苷酸序列如 SEQ ID NO: 31所示， 氨基酸序列如 SEQ ID NO： 32所示)； 134人源化轻链可变区与人免疫球蛋白 Kappa链恒定区 (核苷酸序列如 SEQ ID NO： 33所示，氨基酸序列如 SEQ ID NO： 34所示)相连，构建成完整的 134人源化轻链， 命名为 134-HU-LC-C91 S (核苷酸序列如 SEQ ID NO： 35所示， 氨基酸序列如 SEQ ID NO： 36所示)。
人免疫球蛋白 IgG4恒定区的氨基酸序列
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY
SLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPK
PKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVL
TVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTC
LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVM
HEALHNHYTQKSLSLSLGK ( SEQ ID NO： 30) 134人源化重链 134-Hu-IgG4-HC的氨基酸序列
OVOLVOSGAEVKKPGASVKVSCKASGYTLTAYYMNWVROAPGOSLEWIGVINPYNGDSS
YNOKFKGRATLTVDKSTSTAYMELSSLRSEDTAVYYCARDDGYYRWYFDVWGOGTLVTV
SSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFP
PKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSL
TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSV
MHEALHNHYTQKSLSLSLGK （SEQ ID NO： 32, 其中下划线部分为重链互补决定区） 人免疫球蛋白 Kappa链恒定区的氨基酸序列
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDS KD ST YSLS STLTLSKAD YEKHKVYACEVTHQGLS SP VTKSFNRGEC （SEQ ID NO： 34）
134人源化轻链 134-HU-LC-C91 S的氨基酸序列
DIOMTOSPSSLSASVGDRVTITCRASODIGSRLNWLOOKPGKSIKRLIYATSSLESGVPSRFS GSRSGSDYTLTISSLOPEDFATYYCLOSGSSPPTFGOGTKVEIKRTVAAPSVFIFPPSDEOLKS GTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE KHKVYACEVTHQGLS SP VTKSFNRGEC （SEQ ID NO: 36， 其中下划线部分为轻链互补决 定区）
将上述人工合成的 5E7人源化重链可变区与人免疫球蛋白 IgG4恒定区 （核苷酸序列如 SEQ ID NO： 29所示， 氨基酸序列如 SEQ ID NO: 30所示）相连， 构建成完整的 5E7人源化 重链， 命名为 5E7-Hu-IgG4-HC （核苷酸序列如 SEQ ID NO: 37所示， 氨基酸序列如 SEQ ID NO： 38所示）； 5E7人源化轻链可变区与人免疫球蛋白 Kappa链恒定区 （核苷酸序列如 SEQ ID NO： 33所示，氨基酸序列如 SEQ ID NO： 34所示）相连，构建成完整的 5E7人源化轻链， 命名为 5E7-HU-LC （核苷酸序列如 SEQ ID NO： 39所示， 氨基酸序列如 SEQ ID NO: 40所 示）。
5E7人源化重链 5E7-Hu-IgG4-HC的氨基酸序列
EVOLVESGGGLVKPGGSLRLSCAASGFTFSDDYMAWFROAPGKRLEWVASISHGGDYIYY
ADNLKGRFTISRDNAKNSLYLOMNSLRAEDTAVYFCSRDRRSIDYWGOGTLVTVSSASTKG
PSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV
VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDT
LMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLH
QDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKG
FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEAL
HNHYTQKSLSLSLGK （SEQ ID NO： 38, 其中下划线部分为重链互补决定区）
5E7人源化轻链 5E7-HU-LC的氨基酸序列 DIOMTOSPSSLSASVGDRVTITCRASODISNYLSWYOOKPGKTIKLLIYYTSRLHSGVPSRFS GSGSGTDYTFTISSLOPEDIATYFCOOGKTLPYTFGOGTKVEIKRTYAAPSVFIFPPSDEOLKS GTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE KHK VYACEVTHQGLS SP VTKSFNRGEC （SEQ ID NO: 40， 其中下划线部分为轻链互补决 定区）
将上述 134-Hu-IgG4-HC和 134-Hu-LC-C91S基因分别构建到 pTT5表达载体中， 用 PEI 将构建好的表达载体共同转染至 HEK293E细胞中进行瞬时表达。 HEK293E细胞在 FreeStyle 293 Expression Medium （购自 Thermo Fisher Scientific）无血清培养基中培养， 5天后离心并 收取细胞上清， 利用 Protein A 亲和层析纯化抗体， 最终制备所得抗体命名为 134-Hu-IgG4-C91So 用同样的方法构建 5E7人源化重链和轻链的表达载体， 用同样的方法表 达和纯化抗体， 最终制备所得抗体命名为 5E7-Hu-IgG4。
另外，分别将鼠源 134号抗体的重链可变区和轻链可变区利用重组 PCR法分别与人免疫 球蛋白 IgG4重链恒定区 （核苷酸序列如 SEQ ID NO: 29所示， 氨基酸序列如 SEQ ID NO: 30所示） 和 Kappa轻链恒定区 （核苷酸序列如 SEQ ID NO: 33所示， 氨基酸序列如 SEQ ID NO： 34所示）拼接， 分别构建到 pTT5表达载体中， 用上述方法表达并纯化抗体， 制备的嵌 合抗体命名为 134-Chimeric o 用同样方法获得 5E7抗体的嵌合抗体 5E7-Chimeric。
采用 ELISA 法检测人源化改造对抗体和抗原之间的亲和力产生的影响。 用 LAG-3-ECD-His（带有 His标签的 LAG-3胞外段，购自 Sino Biological Inc.）包被酶标板（ 10ng/ 孔）， 室温孵育 2小时后用 PBST（含 0.05% Tween-20的 PBS）清洗； 每孔加入 200M封闭液 （含 1% BSA的 PBST溶液） 进行封闭； 用 PBST洗涤酶标板后拍干； 在酶标板中加入在封 闭液中梯度稀释的抗人 LAG-3抗体， 室温孵育 1小时左右， 然后洗涤酶标板； 加入用封闭液 稀释的 Goat- Anti -Human IgG（Fc specific） 二抗 （购自 Sigma）， 室温孵育 1小时左右， 然后 洗涤酶标板； 每孔加 1000显色液 （TMB底物） 显色； 随后每孔加入 50M 2M H2S04终止显 色反应；用 SpectraMax 190（Molecular Device）在 450nm波长处测量各孔 OD值；用 GraphPad Prism6进行数据分析和作图， 并计算 EC50。
实验结果如图 2 所示， Anti-LAG3.5、 134-Chimeric、 134-Hu-IgG4-C91S、 5E7-Chimeric 和 5E7-Hu-IgG4均能有效结合 LAG-3 , EC50分别为 27.63ng/ml、 8.685ng/ml、 8.782ng/ml、 9.741ng/ml和 9.078 ng/ml， 对应的人源化抗体与嵌合抗体的 EC50基本一致， 表明人源化改 造没有降低抗体抗原间的亲和力。 其中， 同种型对照为不结合 LAG-3的 IgG4抗体。 实施例 6. 检验人源化抗人 LAG-3抗体对 PBMC分泌 IL-2的增强作用
葡萄球菌肠毒素 （ Staphylococcal Enterotoxin A, SEA） 是一种超抗原， 能够交联抗原递 呈细胞（Antigen Presenting Cell, APC）的 MHC II类分子和 T细胞表面的 T细胞受体（T cell receptor, TCR），从而激活大量的 T细胞克隆，强烈刺激免疫反应。 T细胞激活后表达 LAG-3， LAG-3可与 APC表面的 MHC II类分子结合， 从而发挥免疫抑制作用。 本实施例中， 用 SEA 刺激 PBMC之后， 加入抗人 LAG-3抗体， 通过检测 IL-2的分泌， 评估人源化抗人 LAG-3抗 体的功能活性。
实验方法描述如下： 将带有组氨酸标签的 SEA基因克隆到 pET28a载体中， 用大肠杆菌 系统表达此蛋白，然后用镍亲和层析柱对 SEA进行纯化;用 Histopaque从人血液中分离 PBMC， 用 PBS洗涤后离心； 用 RPMI-1640培养基 （含 10%胎牛血清） 重悬细胞， 加入上述自制的 SEA（SEQ IDNO： 52） 至终浓度为 lng/ml， 然后将 PBMC接种至圆底 96孔细胞培养板中， 每孔 2x l05个细胞，每孔体积为 150^1；然后在每孔中加入 50M梯度稀释的抗人 LAG-3抗体， 将 96孔板置于细胞培养箱（37°C， 5% C02）中孵育 2天；取适量培养上清，用双抗夹心 ELISA 检测 IL-2的分泌量（相关检测试剂购自 BD Biosciences）； 用酶标仪（Molecular Device）读取 OD450， 用 GraphPad Prism6进行数据分析和作图。
图 3A 和 3B 中的 PBMC 分别来自不同的捐献者。 实验结果显示， Anti-LAG3.5、 134-Hu-IgG4-C91S和 5E7-Hu-IgG4均能有效增强 SEA刺激的 PBMC分泌 IL-2。 图 3A所示 实验中 Anti-LAG3.5、 134-Hu-IgG4-C91S和 5E7-Hu-IgG4的 EC50分别为 98.09 ng/ml、 50.71 ng/ml和 78.12 ng/ml;图 3B所示实验中上述三个抗体的 EC50分别为 16.21 ng/ml、 10.85 ng/ml 和 3.068 ng/ml 两次实验结果均显示， 134-Hu-IgG4-C91S和 5E7-Hu-IgG4刺激活化 PBMC 分泌 IL-2的功能活性均强于 Anti-LAG3.5 o其中，同种型对照为不结合 LAG-3的 IgG4抗体。 实施例 7. 人源化抗人 LAG-3抗体对细胞表面 LAG-3的结合作用
超抗原 SEA能够激活 PBMC中的 T细胞克隆， 激活的 T细胞会表达 LAG-3。 本实施例 用流式细胞法测定本发明的人源化抗人 LAG-3抗体对细胞表面 LAG-3的结合作用。
实验步骤描述如下： 用 Histopaque从人血液中分离 PBMC， 用 PBS洗涤 2遍， 离心收集 细胞； 用 RPMI-1640培养基 （含 10%胎牛血清） 重悬细胞， 加入 SEA至终浓度为 lng/ml， 然后将 PBMC接种至圆底 96孔细胞培养板中， 每孔 2x l05个细胞； 将 96孔板置于细胞培养 箱（37°C， 5% C02） 中孵育 2天； 用 PBS洗涤 96孔板中的细胞， 离心后弃掉上清； 用 Biotin N-hydroxysuccinimide ester （货号 /规格： H1759-100MG， 购自 Sigma） 对抗体进行生物素化， 具体操作按照生产商提供的说明书进行； 加入梯度稀释的用生物素标记的抗人 LAG-3抗体， 孵育 1小时左右，然后用 PBS洗涤细胞 2遍；用含 1%BSA的 PBS适当稀释的 PE-Streptavidin （购自 BD Biosciences）， 加入上述细胞中， 孵育 1小时左右， 然后用 PBS洗涤细胞 2遍； 加 入 4%的多聚甲醛固定细胞； 在流式细胞仪上 （ CytoFLEX Cytometer System， 购自 Beckman Coulter） 检测细胞的 PE 荧光强度； 用流式细胞仪分析软件处理实验数据并计算平均荧光强 度； 用 GraphPad Prism6进行数据分析和作图， 计算 EC50。
实验结果如图 4所示， Anti-LAG3.5、 134-Hu-IgG4-C91S和 5E7-Hu-IgG4均能有效结合 细胞表面的 LAG-3， EC50分别为 71.68ng/ml、 80.45ng/ml和 53.13ng/ml， 三者的结合能力基 本一致。 其中， 同种型对照为不结合 LAG-3的 IgG4抗体， 纵坐标为平均荧光强度。 实施例 8. 抗人 LAG-3抗体的表位分析
LAG-3 胞外段的第一个免疫球蛋白样结构域含有一段暴露的“extra loop”， 其氨基酸序 列为： GPPAAAPGHPLAPGPHPAAPSSWGPRPRRY ( SEQ ID NO： 46)。 据文献报道， 该肽 段对 LAG-3 与 MHC-II类分子的结合起重要作用 (Baixeras E, Huard B, Miossec C, et al. Characterization of the lymphocyte activation gene 3 -encoded protein. A new ligand for human leukocyte antigen class II antigens. [J] Journal of Experimental Medicine, 1992, 176(2): 327-337. )。 为检验本发明的抗人 LAG-3抗体与该肽段的结合情况， 并确定每个抗体的结合表位， 实施了 如下肽段扫描实验： 通过化学方法合成一系列部分重叠的肽段， 这些部分重叠的肽段覆盖整 个“extra loop”区， 在这些肽段的 N端连接生物素标签； 用 Streptavidin包被酶标板 (200ng/ 孔)， 洗涤酶标板后用封闭液 (含 1%BSA 的 PBST溶液)封闭； 用封闭液将生物素化的肽段 稀释至 lpg/ml后加入酶标板中， 室温孵育 1小时， 使之被 Streptavidin捕获， 然后洗涤酶标 板； 在其中加入用封闭液梯度稀释的抗人 LAG-3抗体， 室温孵育 1小时左右， 然后洗涤酶标 板； 加入在封闭液中适当稀释的 Goat-Anti-Human IgG (Fc specific)二抗 (购自 Sigma)检测 抗人 LAG-3抗体对肽段的结合， 室温孵育 1小时左右； 每孔加 100M显色液 ( TMB底物)显 色，随后每孔加入 50(^1 2M H2SO4终止液终止反应;用 SpectraMax 190( Molecular Devices, Inc. ) 在 450nm波长处测量各孔 OD值； 用 GraphPad Prism6进行数据分析和作图， 并计算 EC50。 肽段扫描实验的结果总结在表 1中。
表 1. 肽段扫描实验的结果汇总
Figure imgf000018_0001
备注： 表示无明显结合， “+”表示有明显结合， “+”越多表示结合越强； EC50越小“+”越多。 如果抗体与多条肽段均明显结合， 那么重叠的最短的肽段就是该抗体的表位。 表 1实验 结果显示， Anti-LAG3.5抗体的表位是 PHPAAPSSW ( SEQ ID NO: 47)， 134号抗体的表位 是 GPPAAAPGHPLA ( SEQ ID NO： 48 );而 5E7号抗体的表位是 AAAPGHPLAPGPHPAAPSS ( SEQ ID NO： 49)。 相关结果展示在图 5中。 实施例 9. 人源化抗人 LAG-3抗体对 LAG-3结合 Raji细胞的阻断作用
Raji细胞是一种 Burkitt淋巴瘤细胞， 起源于 B淋巴细胞， 高表达 MHC-II类分子。 本实 施例用流式细胞法测定本发明的人源化抗人 LAG-3抗体对 LAG-3结合 Raji细胞表面 MHC-II 类分子的阻断作用。
实验步骤如下：用 PBS将 Raji细胞 (购自 American Type Culture Collection,缩写为 ATCC; ATCC® CCL-86ä) 洗涤 2遍， 然后接种至圆底 96孔细胞培养板中， 每孔 2>< 105个细胞， 离 心后弃掉上清； 用含 1%BSA 的 PBS将生物素化 LAG-3 (购自 ACROBiosy stems) 稀释至 0.5^g/ml, 然后再在此溶液中梯度稀释抗人 LAG-3抗体， 然后将配好的含有生物素化 LAG-3 和抗人 LAG-3抗体的混合溶液加入上述细胞中， 室温孵育 1小时左右， 用 PBS洗涤细胞 2 遍； 用含 1%BSA的 PBS适当稀释 PE-Streptavidin (购自 BD Biosciences),加入上述细胞中， 孵育 1 小时左右， 用 PBS洗涤细胞 2遍； 加入 4%的多聚甲醛固定细胞； 在流式细胞仪上 ( CytoFLEX Cytometer System, 购自 Beckman Coulter) 检测细胞的 PE焚光强度； 用流式细 胞仪分析软件处理实验数据并计算平均荧光强度； 用 GraphPad Prism6进行数据分析和作图， 计算 EC50。
实验结果如图 6所示， Anti-LAG3.5和 134-Hu-IgG4-C91 S均能有效阻断 LAG-3对 Raji 细胞的结合， IC50分别为 256.9ng/ml和 288.8ng/ml，两者的阻断能力基本一致。 5E7-Hu-IgG4 对 LAG-3 结合 Raji细胞的影响不同于 Anti-LAG3.5 和 134-Hu-IgG4-C91 S： 在高浓度时， 5E7-Hu-IgG4只能部分阻断 LAG-3对 Raji细胞的结合； 在低浓度时， 5E7-Hu-IgG4反而能促 进 LAG-3对 Raji细胞的结合。 其中， 同种型对照为不结合人 LAG-3的 IgG4抗体， 阴性对照 表示没有 LAG-3存在时 Raji细胞的背景荧光值。
实施例 10. Biacore检测亲和力
本实施例采用分子相互作用仪 Biacore 8K ( GE healthcare ) 检测 Anti-LAG3.5、 134-Hu-IgG4-C91 S和 5E7-Hu-IgG4与 LAG-3之间的亲和力。
用 HBS-EP+ pH7.4作为稀释缓冲液，抗 LAG-3抗体样品稀释至浓度 0.5ng/mL，重组 LAG-3 (购自 SinoBiological， 货号： 16498-H08H)稀释成 1.024nM、 2.56nM、 6.4nM、 16nM、 40nM 和 100nM， 并增设 0浓度； 采用 6M盐酸胍溶液作为再生缓冲液； 在 Biacore 8K上， 使用 Protein A芯片捕获抗 LAG-3抗体， 再将重组 LAG-3进样， 得到结合 -解离曲线， 再生缓冲液 洗脱后重复下一个循环； 利用 Biacore 8K Insight Evaluation Software对数据进行分析。结果如 表 2所示。
Figure imgf000019_0001
Figure imgf000020_0001
实验结果显示， Anti-LAG3.5、 134-Hu-IgG4-C91S和 5E7-Hu-IgG4对 LAG-3的平衡解离 常数 (KD ) 分别是 5.68E-10M、 4.78E-10M 和 4.17E-12M， 这表明 134-Hu-IgG4-C91S 和 5E7-Hu-IgG4对 LAG-3的亲和力高于 Anti-LAG3.5。 5E7-Hu-IgG4的解离常数 (Koff) 最小， 其 KD最小， 因此其亲和力也最高。
实施例 11. 流式细胞法检测抗人 LAG-3抗体对 FGL1结合 LAG-3的阻断作用
最近， 陈列平团队发现 FGL1 (Fibrinogen-like protein 1 ) 是 LAG-3的主要配体， 并阐明 了这一肿瘤抑制通路的基本原理 (参考文献： Wang J, Sanmamed M F, Datar I, et al. Fibrinogen-like protein 1 is a major immune inhibitory ligand of LAG-3 [J] Cell, 2019, 176(1-2): 334-34T el2. )o本实施例用流式细胞术检测 Anti-LAG3.5、 134-Hu-IgG4-C91S和 5E7-Hu-IgG4 阻断 FGL1与 LAG-3相互作用的能力。
制备重组人 FGL1蛋白的方法如下： 人 FGL1序列来自于 http :/7www.uniprot. ore (Entry： Q08830 ) ， 氨 基 酸 序 列 如 下 所 示 ：
LEDCAQEQMRLRAQVRLLETRVKQQQVKIKQLLQENEVQFLDKGDENTVIDLGSKRQYAD CSEIFNDGYKLSGFYKIKPLQSPAEFSVYCDMSDGGGWTVIQRRSDGSENFNRGWKDYEN GFGNFVQKHGEYWLGNKNLHFLTTQEDYTLKIDLADFEKNSRYAQYKNFKVGDEKNFYEL NIGEYSGTAGDSLAGNFHPEVQWWASHQRMKFSTWDRDHDNYEGNCAEEDQSGWWFNR CHSANLNGVYYSGPYTAKTDNGIVWYTWHGWWYSLKSVVMKIRPNDFIPNVI ( SEQIDNO： 53 ) 。 然后通过生工生物工程 (上海) 股份有限公司合成上述氨基酸序列的编码基因； 通过 重组 PCR的方法将 FGL1的基因与人 IgGl的 Fc段基因融合在一起， 将上述带有 Fc标签的 基因构建到哺乳动物表达载体中；在 HEK293E细胞中瞬时转染并表达；五天后通过 Protein A 亲和层析法纯化细胞培养上清中的重组蛋白， 通过紫外分光光度法测定蛋白浓度， 所得蛋白 命名为 FGLl-hFc o FGLl-hFc 的生物素标记方法如下： 用无水 DMSO 将 Biotin N-hydroxysuccinimide ester( Sigma/货号: H1759-100MG)配制成 100mM的母液;根据 FGLl-hFc 的分子量和浓度计算相应的物质的量浓度； 取适当体积的 Biotin N-hydroxysuccinimide ester 溶液， 与 FGLl-hFc按照 20: 1的物质的量之比混匀， 室温孵育 1小时； 透析之后用紫外分 光光度法测定蛋白浓度， 所得生物素化蛋白命名为 Biotin-FGLl-hFc。
稳定表达 LAG-3 的 CHO-S 细胞株的构建方法如下： CHO-S 细胞购自 Thermo Fisher Scientific， 将全长人 LAG-3基因构建到 pCHO 1.0表达载体中， 用 PEI (聚乙烯亚胺)将该表 达载体转染至 CHO-S细胞中， 用甲氨蝶呤 (methotrexate) 和嘌呤霉素 (puromycin) 加压筛 选，然后经过有限稀释法挑取单克隆，获得一株稳定表达人 LAG-3的 CHO-S单克隆细胞株， 将该细胞命名为 CHO-S-LAG-3。
流式细胞术检测抗体阻断 FGL1与 LAG-3相互作用的方法描述如下： 将 CHO-S-LAG-3细胞接种到 96孔板（圆底带盖）中，每孔 20万个细胞； 300g离心 5min 后， 用排枪将细胞培养上清吸掉， 每孔加入 200nlPBS+l%BSA （含 1%牛血清白蛋白的 PBS 溶液） 以重悬细胞， 然后 300g离心， 吸掉上清； 加入梯度稀释的抗 LAG-3抗体和浓度恒定 为 2 /ml的 Biotin-FGLl-hFc， 室温孵育 1小时； 300g离心， 吸掉上清， 用 PBS+1%BSA洗 涤细胞 2 遍； 300g 离心， 吸掉上清， 在每孔中加入用 PBS+1%BSA 以 1 :2000 稀释的 PE Streptavidin， 每孔加入 200M， 室温孵育 0.5小时； 300g离心， 吸掉上清， 用 PBS+1%BSA洗 涤细胞 2遍； 最终用 200^1 PBS重悬细胞； 在流式细胞仪上检测各孔细胞在 PE通道的荧光强 度； 用 GraphPad Prism6进行数据分析和作图， 并计算 IC50。
实验结果如图 7所示， Anti-LAG3.5、 134-Hu-IgG4-C91 S和 5E7-Hu-IgG4阻断 FGL1与 LAG-3相互作用的 IC50分别是 785.6ng/mK 97.57ng/ml和 97.71ng/ml,这表明 134-Hu-IgG4-C91 S 和 5E7-Hu-IgG4的阻断能力强于 Anti-LAG3.5。
实施例 12.抗人 LAG-3抗体对转基因小鼠 MC38移植瘤模型的抑制作用
动物实验步骤如下： 人 LAG-3转基因小鼠 （种系背景为 C57BL/6）和 MC38小鼠结直肠 细胞株由上海南方模式生物研究中心提供。 该转基因小鼠中， 小鼠 LAG-3胞外段部分已被人 LAG-3基因的同源部分取代，因此本发明的人源化抗 LAG-3抗体能够识别该转基因小鼠中的 LAG-3分子。
具体实施步骤如下：将 MC38在体外扩大培养，培养基为含有 10%胎牛血清的 DMEM（胎 牛血清和 DMEM培养基购自 Thermo Fisher Scientific）； 将扩大培养的 MC38细胞接种于人 LAG-3转基因小鼠中， 每只小鼠皮下接种 l x lO6个细胞； 待接种的肿瘤细胞生长至体积接近 100mm3时， 随机将动物分成五组， 每组 5 只： 同型对照抗体组 （仅注射同型对照抗体）；
Anti-mPD- 1（购自 Bio X Cell，货号 BP0146） 10 mg/kg； Anti-mPD- 1 10mg/kg+Anti-LAG3.5 20 mg/kg ； Anti-mPD- 1 10 mg/kg+134-Hu-IgG4-C91 S 20 mg/kg ； Anti-mPD- 1 10 mg/kg+5E7-Hu-IgG4 20 mg/kg。 通过腹腔注射给药， 给药频率为每周两次。 随后， 按照上述 设计好的方案给药， 持续时间三周， 每周测量并计算肿瘤体积 2次。 最终， 各组肿瘤生长曲 线图 8所示。抗 LAG-3单抗作为单药使用时的抗肿瘤作用比较弱（参考文献： Woo S R, Turnis
M E, Goldberg M V, et al. Immune inhibitory molecules LAG-3 and PD-1 synergistically regulate T-cell function to promote tumoral immune escape[J] Cancer research, 2012, 72（4）: 917-927.），考 虑到动物福利， 此处未设置抗 LAG-3单抗的单药组。
实验结果显示， 与对照组相比， Anti-mPD- 1能够明显地抑制小鼠体内 MC38肿瘤的生长 （尸 =0.0249） ; 与 Anti-mPD- 1单药相比， Anti-LAG3.5与 Anti-mPD- 1联合应用能够进一步 抑制 MC38肿瘤的生长， 但无统计学显著性差异 （尸 =0.1174， 尸＜0.05认为有显著性差异） ； 与 Anti-mPD- 1单药相比， 134-Hu-IgG4-C91 S与 Anti-mPD- 1联合应用能够进一步抑制 MC38 肿瘤的生长 （尸 =0.0159） ; 与 Anti-mPD- 1单药相比， 5E7-Hu-IgG4与 Anti-mPD- 1联合应用 也能进一步抑制 MC38肿瘤的生长 （尸 =0.0003） 。

Claims

权利要求书
1、 结合人 LAG-3的抗体或其抗原结合片段， 其特征在于， 其结合的人 LAG-3表位包括 以下氨基酸序列： AAAPGHPLA ( SEQ ID NO: 50) 。
2、 如权利要求 1所述的结合人 LAG-3的抗体或其抗原结合片段， 其特征在于， 其结合 的人 LAG-3 表位包括以下氨基酸序列： GPPAAAPGHPLA ( SEQ ID NO： 48 ) 或 AAAPGHPLAPGPHPAAPSS ( SEQ ID NO： 49) 。
3、 结合人 LAG-3的抗体或其抗原结合片段， 其特征在于， 包括：
( a)重链互补决定区 HCDR1、 HCDR2、 HCDR3 , 所述 HCDR1的氨基酸序列如 SEQ ID NO： 9所示， 所述 HCDR2的氨基酸序列如 SEQ ID NO： 10所示， 所述 HCDR3的氨基酸序 列如 SEQ ID NO: 11所示， 和轻链互补决定区 LCDR1、 LCDR2、 LCDR3， 所述 LCDR1的 氨基酸序列如 SEQ ID NO: 12所示， 所述 LCDR2的氨基酸序列如 SEQ ID NO: 13所示， 所 述 LCDR3的氨基酸序列如 SEQ ID NO: 14或 SEQ ID NO： 43所示； 或
(b )重链互补决定区 HCDR1、 HCDR2、 HCDR3 , 所述 HCDR1的氨基酸序列如 SEQ ID NO： 15所示， 所述 HCDR2的氨基酸序列如 SEQ ID NO: 16所示， 所述 HCDR3的氨基酸序 列如 SEQ ID NO: 17所示， 和轻链互补决定区 LCDR1、 LCDR2、 LCDR3， 所述 LCDR1的 氨基酸序列如 SEQ ID NO： 18所示， 所述 L-CDR2的氨基酸序列如 SEQ ID NO： 19所示， 所述 LCDR3的氨基酸序列如 SEQ ID NO： 20所示。
4、如权利要求 1-3任一项所述的结合人 LAG-3的抗体或其抗原结合片段，其特征在于， 所述抗体为单克隆抗体或多克隆抗体。
5、 如权利要求 4所述的结合人 LAG-3的抗体或其抗原结合片段， 其特征在于， 所述抗 体为单克隆抗体。
6、如权利要求 1-3任一项所述的结合人 LAG-3的抗体或其抗原结合片段，其特征在于， 所述抗体为鼠源抗体、 嵌合抗体或人源化抗体。
7、 如权利要求 6所述的结合人 LAG-3的抗体或其抗原结合片段， 其特征在于， 所述抗 体为人源化抗体。
8、如权利要求 1-3任一项所述的结合人 LAG-3的抗体或其抗原结合片段，其特征在于， 所述抗原结合片段包括 Fab片段、 F(ab , )2片段、 Fv片段。
9、 如权利要求 1-3 中任一项所述的结合人 LAG-3 的抗体或其抗原结合片段， 其特征在 于， 所述结合人 LAG-3的抗体或其抗原结合片段包括重链可变区和轻链可变区， 选自：
( a)所述重链可变区的氨基酸序列如 SEQ ID NO: 2所示， 所述轻链可变区的氨基酸序 列如 SEQ ID NO: 4所示；
(b )所述重链可变区的氨基酸序列如 SEQ ID NO: 6所示， 所述轻链可变区的氨基酸序 列如 SEQ ID NO: 8所示；
( c) 所述重链可变区的氨基酸序列如 SEQ ID NO: 22所示， 所述轻链可变区的氨基酸 序列如 SEQ ID NO： 24所示； 和 ( d) 所述重链可变区的氨基酸序列如 SEQ ID NO: 26所示， 所述轻链可变区的氨基酸 序列如 SEQ ID NO： 28所示。
10、 如权利要求 9所述的结合人 LAG-3的抗体或其抗原结合片段， 其特征在于， 所述结 合人 LAG-3 的抗体或其抗原结合片段包括重链恒定区和轻链恒定区， 所述重链恒定区选自 IgGl、 IgG2、 IgG3和 IgG4重链恒定区， 所述轻链恒定区选自 K或人轻链恒定区。
11、 如权利要求 10所述的结合人 LAG-3的抗体或其抗原结合片段， 其特征在于， 所述 重链恒定区为 IgG4重链恒定区， 所述轻链恒定区为 K轻链恒定区。
12、 如权利要求 11所述的结合人 LAG-3的抗体或其抗原结合片段， 其特征在于， 所述 重链恒定区的氨基酸序列如 SEQ ID NO： 30所示，所述轻链恒定区的氨基酸序列如 SEQ ID NO： 34所示。
13、 如权利要求 9-12任一项所述的结合人 LAG-3 的抗体或其抗原结合片段， 其特征在 于， 所述结合人 LAG-3的抗体或其抗原结合片段包括重链和轻链， 选自：
( a)所述重链的氨基酸序列如 SEQ ID NO: 32所示， 所述轻链的氨基酸序列如 SEQ ID NO： 36所示； 和
(b )所述重链的氨基酸序列如 SEQ ID NO: 38所示， 所述轻链的氨基酸序列如 SEQ ID NO： 40所示。
14、 核苷酸序列， 其特征在于， 所述核苷酸序列编码如权利要求 1-13任一项所述的结合 人 LAG-3的抗体或其抗原结合片段。
15、 如权利要求 14所述的核苷酸序列， 其特征在于， 所述核苷酸序列包括：
( a) 如 SEQ ID NO: 1所示编码重链可变区的核苷酸序列， 如 SEQ ID NO: 3所示编码 轻链可变区的核苷酸序列；
(b ) 如 SEQ ID NO: 5所示编码重链可变区的核苷酸序列， 如 SEQ ID NO: 7所示编码 轻链可变区的核苷酸序列；
( c) 如 SEQ ID NO: 21所示编码重链可变区的核苷酸序列， 如 SEQ ID NO: 23所示编 码轻链可变区的核苷酸序列； 或
( d) 如 SEQ ID NO: 25所示编码重链可变区的核苷酸序列， 如 SEQ ID NO: 27所示编 码轻链可变区的核苷酸序列。
16、 如权利要求 15所述的核苷酸序列， 所述核苷酸序列包括如 SEQ ID NO： 29所示的 编码重链恒定区的核苷酸序列， 和如 SEQ ID NO： 33所示的编码轻链恒定区的核苷酸序列。
17、 如权利要求 14-16任一项所述的核苷酸序列， 所述核苷酸序列包括：
( a) 如 SEQ ID NO: 31所示编码重链的核苷酸序列， 如 SEQ ID NO: 35所示编码轻链 的核苷酸序列； 或
(b ) 如 SEQ ID NO: 37所示编码重链的核苷酸序列， 如 SEQ ID NO: 39所示编码轻链 的核苷酸序列。
18、 表达载体， 其特征在于， 所述表达载体含有如权利要求 14-17任一项所述的核苷酸 序列。
19、 宿主细胞， 其特征在于， 所述宿主细胞含有如权利要求 18所述的表达载体。
20、如权利要求 1-13任一项所述的结合人 LAG-3的抗体或其抗原结合片段的制备方法， 其特征在于， 所述方法包括以下步骤：
( a) 在表达条件下， 培养权利要求 19所述的宿主细胞， 从而表达所述的结合人 LAG-3 的抗体或其抗原结合片段；
(b ) 分离并纯化 ( a) 所述的结合人 LAG-3的抗体或其抗原结合片段。
21、 药物组合物， 其特征在于， 所述药物组合物含有如权利要求 1-13任一项所述的结合 人 LAG-3的抗体或其抗原结合片段和药学上可接受的载体。
22、 如权利要求 21所述的药物组合物， 其特征在于， 所述药物组合物还包括 PD-1抑制 剂。
23、 如权利要求 22所述的药物组合物， 其特征在于， 所述 PD-1抑制剂为结合 PD-1的 抗体或其抗原结合片段。
24、 如权利要求 1-13任一项所述的结合人 LAG-3 的抗体或其抗原结合片段或如权利要 求 21-23任一项所述的药物组合物在制备治疗癌症的药物中的用途。
25、 如权利要求 24所述的用途， 其特征在于， 所述癌症选自黑色素瘤、 肾细胞癌、 非小 细胞肺癌、 经典霍奇金淋巴瘤、 尿路上皮癌、 结直肠癌和肝癌。
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114456267A (zh) * 2020-11-03 2022-05-10 浙江大学医学院附属第二医院 一种抗cd73人源化单克隆抗体及其应用
CN114901697A (zh) * 2020-12-10 2022-08-12 北京东方百泰生物科技股份有限公司 一种抗lag-3的单克隆抗体、其抗原结合片段及其应用
WO2023273958A1 (zh) 2021-06-29 2023-01-05 三生国健药业（上海）股份有限公司 三特异性抗体、其制备方法和用途

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112759648B (zh) * 2020-09-30 2024-01-16 白先宏 Lag-3结合分子及其应用
CN117159703B (zh) * 2023-11-02 2024-04-02 正大天晴(广州)医药有限公司 含抗lag-3抗体的药物组合物及其用途

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102176921A (zh) * 2008-08-11 2011-09-07 梅达雷克斯股份有限公司 结合淋巴细胞活化基因3(lag-3)之人类抗体及其用途
US20140093511A1 (en) 2012-07-02 2014-04-03 Bristol-Myers Squibb Company Optimization of antibodies that bind lymphocyte activation gene-3 (lag-3), and uses thereof
WO2017219995A1 (zh) * 2016-06-23 2017-12-28 江苏恒瑞医药股份有限公司 Lag-3抗体、其抗原结合片段及其医药用途
WO2019011306A1 (en) * 2017-07-13 2019-01-17 Nanjing Leads Biolabs Co., Ltd. LAG-3 BINDING ANTIBODIES AND USES THEREOF

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2016316730B2 (en) * 2015-09-02 2023-05-25 Immutep S.A.S. Anti-LAG-3 Antibodies
AU2017343621B2 (en) * 2016-10-11 2021-12-02 Agenus Inc. Anti-LAG-3 antibodies and methods of use thereof
KR102583190B1 (ko) * 2016-10-13 2023-09-26 치아타이 티안큉 파마수티컬 그룹 주식회사 항-lag-3 항체 및 조성물
CN111050792B (zh) * 2017-08-30 2024-05-31 凡恩世制药(北京)有限公司 抗lag-3抗体及其用途

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102176921A (zh) * 2008-08-11 2011-09-07 梅达雷克斯股份有限公司 结合淋巴细胞活化基因3(lag-3)之人类抗体及其用途
US20140093511A1 (en) 2012-07-02 2014-04-03 Bristol-Myers Squibb Company Optimization of antibodies that bind lymphocyte activation gene-3 (lag-3), and uses thereof
WO2017219995A1 (zh) * 2016-06-23 2017-12-28 江苏恒瑞医药股份有限公司 Lag-3抗体、其抗原结合片段及其医药用途
WO2019011306A1 (en) * 2017-07-13 2019-01-17 Nanjing Leads Biolabs Co., Ltd. LAG-3 BINDING ANTIBODIES AND USES THEREOF

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
BAIXERAS EHUARD BMIOSSEC C ET AL.: "Characterization of the lymphocyte activation gene 3-encoded protein. A new ligand for human leukocyte antigen class II antigens.[J", JOURNAL OF EXPERIMENTAL MEDICINE, vol. 176, no. 2, 1992, pages 327 - 337, XP000672285, DOI: 10.1084/jem.176.2.327
KABAT ET AL., NIH PUBL, vol. I, no. 91-3242, 1991, pages 647 - 669
NAT COMMUN., vol. 5, 24 June 2014 (2014-06-24), pages 4229
SAMBROOK, J.FRITSCH, E.F.MANIAIS, T.: "Molecular Cloning: A Laboratory Manual", 1989, COLD SPRING HARBOR LABORATORY PRESS
See also references of EP3932948A4
WANG JSANMAMED MFDATAR I ET AL.: "Fibrinogen-like protein 1 is a major immune inhibitory ligand of LAG-3[J", CELL, vol. 176, no. 1-2, 2019, pages 334 - 347, XP055629972, DOI: 10.1016/j.cell.2018.11.010

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114456267A (zh) * 2020-11-03 2022-05-10 浙江大学医学院附属第二医院 一种抗cd73人源化单克隆抗体及其应用
CN114456267B (zh) * 2020-11-03 2023-12-01 浙江大学医学院附属第二医院 一种抗cd73人源化单克隆抗体及其应用
CN114901697A (zh) * 2020-12-10 2022-08-12 北京东方百泰生物科技股份有限公司 一种抗lag-3的单克隆抗体、其抗原结合片段及其应用
CN114901697B (zh) * 2020-12-10 2022-12-13 北京东方百泰生物科技股份有限公司 一种抗lag-3的单克隆抗体、其抗原结合片段及其应用
CN116514972A (zh) * 2020-12-10 2023-08-01 北京东方百泰生物科技股份有限公司 一种抗lag-3的单克隆抗体、其抗原结合片段及其应用
CN116514971A (zh) * 2020-12-10 2023-08-01 北京东方百泰生物科技股份有限公司 一种抗lag-3的单克隆抗体、其抗原结合片段及其应用
CN116514972B (zh) * 2020-12-10 2023-10-27 北京东方百泰生物科技股份有限公司 一种抗lag-3的单克隆抗体、其抗原结合片段及其应用
CN116514971B (zh) * 2020-12-10 2023-10-27 北京东方百泰生物科技股份有限公司 一种抗lag-3的单克隆抗体、其抗原结合片段及其应用
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