WO2022121720A1 - 一种抗lag-3的单克隆抗体、其抗原结合片段及其应用 - Google Patents

一种抗lag-3的单克隆抗体、其抗原结合片段及其应用 Download PDF

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WO2022121720A1
WO2022121720A1 PCT/CN2021/133941 CN2021133941W WO2022121720A1 WO 2022121720 A1 WO2022121720 A1 WO 2022121720A1 CN 2021133941 W CN2021133941 W CN 2021133941W WO 2022121720 A1 WO2022121720 A1 WO 2022121720A1
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amino acid
acid sequence
heavy chain
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PCT/CN2021/133941
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French (fr)
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白义
周俊杰
刘思
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北京东方百泰生物科技股份有限公司
北京精益泰翔技术发展有限公司
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Priority to KR1020237023286A priority Critical patent/KR20230118922A/ko
Priority to IL303443A priority patent/IL303443A/en
Priority to EP21902427.0A priority patent/EP4242234A4/en
Priority to AU2021398061A priority patent/AU2021398061A1/en
Priority to CN202180007833.9A priority patent/CN114901697B/zh
Priority to US18/039,720 priority patent/US20240025994A1/en
Priority to CA3204858A priority patent/CA3204858A1/en
Priority to JP2023535502A priority patent/JP2023552621A/ja
Publication of WO2022121720A1 publication Critical patent/WO2022121720A1/zh

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Definitions

  • the invention relates to the technical field of biomedicine, in particular to an anti-LAG-3 monoclonal antibody, an antigen-binding fragment thereof and applications thereof.
  • Immunotherapy is the hottest cancer treatment method today, and is known as the third revolution in cancer treatment.
  • the so-called “cancer immunotherapy” is a treatment method that uses the body's own immune system to attack cancer cells.
  • the battle between the immune system and cancer cells is a dynamic process of a long-term game that is both head-to-head and intertwined.
  • the immune cells of a healthy body can find and kill cancer cells, but under the induction of various innate and acquired factors, the immune system will lose its absolute advantage, and even be “insured” by cancer cells, promoting the occurrence and development of cancer.
  • Targeted drugs had high hopes for their precision, but due to the fickleness and complexity of cancer cells, cancer cells are prone to drug resistance, and precision is easily useless. For this reason, since the discovery of CTLA-4 and PD-1, immune checkpoint inhibition in cancer treatment has gradually become a research hotspot.
  • LAG-3 is gradually recognized as a potential immune checkpoint receptor, and it has been reported that LAG-3 plays an important role in promoting regulatory T cell activity and downregulating T cell activation and proliferation effect (Workman CJ et al., J. Immunol. 2005; 174:688-695).
  • LAG-3 (Lymphocyte-activation gene 3), also known as CD223, this gene includes 8 exons, located on human chromosome 12 (mouse chromosome 6), belonging to the immunoglobulin superfamily, which consists of extracellular It consists of three parts: the transmembrane region, the transmembrane region and the cytoplasmic region, encoding a type I transmembrane protein composed of 498 amino acids.
  • LAG-3 is widely expressed on the surface of activated NK cells, T cells and other immune cells, and can negatively regulate lymphocyte function, and some studies have confirmed that it plays an important role in tumors and autoimmune diseases.
  • LAG-3 is an immune checkpoint receptor capable of regulating T cell function, and inhibition of LAG-3 may benefit patients more, especially those whose tumors contain immune cells expressing LAG-3.
  • LAG- 3 is considered a more attractive target than other immune checkpoint proteins.
  • LAG-3 is a target with more clinical data and relatively certain druggability.
  • the present invention obtains an anti-LAG-3 monoclonal antibody or antigen-binding fragment that can specifically bind to LAG-3 and has higher biological activity by screening the immune library.
  • the present invention provides an anti-LAG-3 monoclonal antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes 3 heavy chain complementarity determining regions, 3
  • the heavy chain complementarity determining regions are represented by HCDR1, HCDR2 and HCDR3 respectively
  • the light chain variable region includes three light chain complementarity determining regions
  • the three light chain complementarity determining regions are respectively represented by LCDR1, LCDR2 and LCDR3
  • the monoclonal antibody or its antigen-binding fragment is selected from any of the following:
  • the heavy chain complementarity determining region HCDR1 comprises the amino acid sequence shown in SEQ ID No: 1
  • the heavy chain complementarity determining region HCDR2 comprises the amino acid sequence shown in SEQ ID No: 2
  • the heavy chain The complementarity determining region HCDR3 comprises the amino acid sequence shown in SEQ ID No: 3
  • the light chain complementarity determining region LCDR1 comprises the amino acid sequence shown in SEQ ID No: 4
  • the light chain complementarity determining region LCDR2 comprises the amino acid sequence shown in SEQ ID No: 4
  • the light chain complementarity determining region LCDR3 comprises the amino acid sequence shown in SEQ ID No: 6;
  • the heavy chain complementarity determining region HCDR1 comprises the amino acid sequence shown in SEQ ID No: 1
  • the heavy chain complementarity determining region HCDR2 comprises the amino acid sequence shown in SEQ ID No: 2
  • the heavy chain The complementarity determining region HCDR3 comprises the amino acid sequence shown in SEQ ID No: 3
  • the light chain complementarity determining region LCDR1 comprises the amino acid sequence shown in SEQ ID No: 4
  • the light chain complementarity determining region LCDR2 comprises the amino acid sequence shown in SEQ ID No: 4
  • the light chain complementarity determining region LCDR3 comprises the amino acid sequence shown in SEQ ID No: 8;
  • the heavy chain complementarity determining region HCDR1 comprises the amino acid sequence shown in SEQ ID No: 9
  • the heavy chain complementarity determining region HCDR2 comprises the amino acid sequence shown in SEQ ID No: 10
  • the heavy chain The complementarity determining region HCDR3 comprises the amino acid sequence shown in SEQ ID No: 11
  • the light chain complementarity determining region LCDR1 comprises the amino acid sequence shown in SEQ ID No: 12
  • the light chain complementarity determining region LCDR2 comprises the amino acid sequence shown in SEQ ID No: 12
  • the light chain complementarity determining region LCDR3 comprises the amino acid sequence shown in SEQ ID No: 14;
  • the heavy chain complementarity determining region HCDR1 comprises the amino acid sequence shown in SEQ ID No: 15
  • the heavy chain complementarity determining region HCDR2 comprises the amino acid sequence shown in SEQ ID No: 16
  • the heavy chain The complementarity determining region HCDR3 comprises the amino acid sequence shown in SEQ ID No: 17
  • the light chain complementarity determining region LCDR1 comprises the amino acid sequence shown in SEQ ID No: 18,
  • the light chain complementarity determining region LCDR2 comprises the amino acid sequence shown in SEQ ID No: 18
  • the light chain complementarity determining region LCDR3 comprises the amino acid sequence shown in SEQ ID No: 20.
  • the above-mentioned anti-LAG-3 monoclonal antibody or its antigen-binding fragment provided by the present invention can specifically bind to LAG-3, and has better binding activity.
  • Blocking LAG-3 can reverse the inhibitory effect of LAG-3 on T cells.
  • Boosting T cell activity and reducing the number of regulatory T cells can also increase the sensitivity of T cell immune responses for the treatment of immune or cancer diseases.
  • the monoclonal antibody or its antigen-binding fragment is a mouse-derived antibody molecule, and the mouse-derived antibody molecule is selected from any of the following:
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID No: 21, and the light chain variable region comprises the amino acid sequence shown in SEQ ID No: 22;
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID No: 21, and the light chain variable region comprises the amino acid sequence shown in SEQ ID No: 23;
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID No: 24, and the light chain variable region comprises the amino acid sequence shown in SEQ ID No: 25;
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID No: 26 and the light chain variable region comprises the amino acid sequence shown in SEQ ID No: 27;
  • the murine antibody molecule is MA-1.
  • the present invention uses LAG-3 antigen for mouse immunization to immunize mice, optimizes the immunization method, creates a phage display library, and screens out the above-mentioned mouse-derived antibody molecules with higher affinity, better activity and more stability, through a large number of cell-level experiments After verification, it is found that MA-1 has higher biological activity than the other three mouse-derived antibody molecules. Therefore, MA-1 is preferably selected in the present invention.
  • the murine antibody molecule also includes a heavy chain constant region and a light chain constant region, and the heavy chain constant region is one of mouse IgG1 type, IgG2a type, IgG2b type, and IgG3 type, and the IgG1 type
  • the heavy chain constant region amino acid sequence of the IgG2a type is shown in SEQ ID No: 29
  • the heavy chain constant region amino acid sequence of the IgG2a type is shown in SEQ ID No: 30
  • the heavy chain constant region amino acid sequence of the IgG2b type is shown in SEQ ID No.
  • the heavy chain constant region amino acid sequence of the IgG3 type is shown in SEQ ID No: 32
  • the light chain constant region is a murine C kappa chain whose amino acid sequence is shown in SEQ ID No: 28;
  • the heavy chain constant region is of murine IgG1 type.
  • the monoclonal antibody or its antigen-binding fragment is a chimeric antibody molecule, and the chimeric antibody molecule includes the heavy chain variable region of the murine antibody molecule, the light chain variable region of the murine antibody molecule Region and human antibody constant region;
  • the human antibody constant region includes human antibody heavy chain constant region and human antibody light chain constant region, the human antibody heavy chain constant region is human IgG1 type, IgG2 type or IgG4
  • the heavy chain constant region amino acid sequence of the IgG1 type is shown in SEQ ID No: 39
  • the heavy chain constant region amino acid sequence of the IgG2 type is shown in SEQ ID No: 40
  • the IgG4 type The heavy chain constant region amino acid sequence is shown in SEQ ID No:41;
  • the human antibody light chain constant region is the human C kappa chain whose amino acid sequence is shown in SEQ ID No:42;
  • the human antibody heavy chain constant region is of human IgG4 type.
  • the chimeric antibody molecule includes the variable region sequence of the murine antibody molecule and the human antibody constant region.
  • the chimeric antibody molecule is designed to verify that the function of the CDR is not changed after the humanization of the constant region of the present invention, and it is a humanized antibody molecule.
  • the research provides the basis for further research and development.
  • the monoclonal antibody or its antigen-binding fragment is a humanized antibody molecule, and the humanized antibody molecule is selected from any of the following:
  • HA-1 the heavy chain variable region comprises the amino acid sequence shown in SEQ ID No: 33, and the light chain variable region comprises the amino acid sequence shown in SEQ ID No: 34;
  • HA-2 the heavy chain variable region comprises the amino acid sequence shown in SEQ ID No: 33, and the light chain variable region comprises the amino acid sequence shown in SEQ ID No: 35;
  • HA-3 the heavy chain variable region comprises the amino acid sequence shown in SEQ ID No: 36, and the light chain variable region comprises the amino acid sequence shown in SEQ ID No: 35;
  • HA-4 the heavy chain variable region comprises the amino acid sequence shown in SEQ ID No: 36, and the light chain variable region comprises the amino acid sequence shown in SEQ ID No: 34;
  • HA-5 the heavy chain variable region comprises the amino acid sequence shown in SEQ ID No: 37, and the light chain variable region comprises the amino acid sequence shown in SEQ ID No: 38;
  • the humanized antibody molecule is HA-1.
  • humanized antibody molecules are obtained after humanization screening. Through in vitro and in vivo experiments, it is found that among the five humanized antibody molecules provided in the present invention, HA-1 has higher activity and has the most significant medicinal effect. For this reason HA-1 is preferred in the present invention.
  • the humanized antibody molecule also includes a heavy chain constant region and a light chain constant region, the heavy chain constant region is one of human IgG1 type, IgG2 type or IgG4 type, and the heavy chain of the IgG1 type
  • the amino acid sequence of the constant region is shown in SEQ ID No: 39
  • the amino acid sequence of the heavy chain constant region of the IgG2 type is shown in SEQ ID No: 40
  • the amino acid sequence of the heavy chain constant region of the IgG4 type is shown in SEQ ID No: 41
  • the light chain constant region is a human C kappa chain whose amino acid sequence is shown in SEQ ID No: 42;
  • the heavy chain constant region is of human IgG4 type.
  • the humanized antibody molecule is a full-length antibody or an antibody fragment, and the humanized antibody molecule includes one or a combination of Fab, F(ab)2, Fv or ScFv.
  • the present invention also provides a polypeptide or protein comprising the anti-LAG-3 monoclonal antibody or an antigen-binding fragment thereof.
  • the present invention also provides a polynucleotide sequence or combination, the polynucleotide sequence or combination encoding the amino acid sequence of the anti-LAG-3 monoclonal antibody or its antigen-binding fragment.
  • the present invention also provides a recombinant DNA expression vector, the recombinant DNA expression vector comprising the polynucleotide sequence or combination.
  • the present invention also provides a host cell transfected with the recombinant DNA expression vector, the host cell includes prokaryotic cells, yeast cells, insect cells or mammalian cells;
  • the host cells are mammalian cells, and the mammalian cells are HEK293E cells, CHO cells or NSO cells.
  • the present invention also provides a medicine or a pharmaceutical composition comprising the anti-LAG-3 monoclonal antibody or an antigen-binding fragment thereof.
  • the present invention also provides the application of the anti-LAG-3 monoclonal antibody or its antigen-binding fragment in the preparation of medicines for treating cancer or immune diseases;
  • the cancer includes leukemia, lung cancer, gastric cancer, esophageal cancer, ovarian cancer, head and neck cancer, melanoma, kidney cancer, breast cancer, colorectal cancer, liver cancer, pancreatic cancer or bladder cancer;
  • Such immune diseases include psoriasis, Crohn's disease, rheumatoid arthritis, primary biliary cirrhosis, systemic lupus erythematosus, multiple sclerosis, ulcerative colitis, and autoimmune hepatitis.
  • the present invention also provides the application of the anti-LAG-3 monoclonal antibody or its antigen-binding fragment in combination with the anti-PD-1 monoclonal antibody for preparing a medicine for treating cancer or immune diseases.
  • the anti-PD-1 monoclonal antibodies include DFPD1-9, DFPD1-10, DFPD1-11, DFPD1-12, DFPD1-13, Nivolumab, Pembrolizumab, toripalizumab, sintilimab, Tislelizumab, camrelizumab, pianpilimumab, or sapalimumab.
  • the anti-PD-1 monoclonal antibody is DFPD1-10
  • the DFPD1-10 comprises a light chain variable region shown in SEQ ID No:45 and a heavy chain variable region shown in SEQ ID No:43 variable area.
  • the anti-PD-1 monoclonal antibody is Nivolumab.
  • the cancer is selected from leukemia, lung cancer, gastric cancer, esophageal cancer, ovarian cancer, head and neck cancer, melanoma, kidney cancer, breast cancer, colorectal cancer, liver cancer, pancreatic cancer or bladder cancer;
  • the immune disease includes silver Psoriasis, Crohn's disease, rheumatoid arthritis, primary biliary cirrhosis, systemic lupus erythematosus, multiple sclerosis, ulcerative colitis and autoimmune hepatitis.
  • the anti-LAG-3 monoclonal antibody or its antigen-binding fragment provided by the present invention can specifically bind to LAG-3, has high affinity, and has good biological activity, and can be used for the treatment of multiple cancers or immune diseases
  • cancers include but are not limited to leukemia, lung cancer, stomach cancer, esophageal cancer, ovarian cancer, head and neck cancer, melanoma, kidney cancer, breast cancer, colorectal cancer, liver cancer, pancreatic cancer or bladder cancer
  • immune diseases include But not limited to psoriasis, Crohn's disease, rheumatoid arthritis, primary biliary cirrhosis, systemic lupus erythematosus, multiple sclerosis, ulcerative colitis and autoimmune hepatitis.
  • Fig. 1 is the plasmid map of pScFv-Disb-HS vector in Example 2 of the present invention
  • Fig. 2 is the comparative graph of the relative affinity of the anti-LAG-3 phage monoclonal antibody of gradient dilution ELISA in Example 3 of the present invention
  • Figure 3 is a map of the vector pTSE in Example 5 of the present invention.
  • Figure 4 is a denaturing polyacrylamide gel electrophoresis image of the mouse-derived antibody molecule in Example 5 of the present invention.
  • Fig. 5 is a graph comparing the binding ability of murine antibody and LAG-3 in Example 6 of the present invention.
  • Example 6 is a graph showing the secretion of the cytokine IL-2 by murine antibody in the mixed lymphocyte reaction in Example 7 of the present invention.
  • Example 7 is a denaturing polyacrylamide gel electrophoresis image of the humanized antibody molecule in Example 12 of the present invention.
  • FIG. 8 is a comparison diagram of the binding ability of humanized antibody molecule and LAG-3 in Example 15 of the present invention.
  • Figure 9 shows the activity of the anti-LAG-3 humanized antibody molecule tested by the mixed lymphocyte reaction (MLR) in Example 16 of the present invention.
  • Figure 10 is a graph showing the effect of the inhibition test of anti-LAG-3 monoclonal antibody on MC38 colorectal cancer in mice in Example 17 of the present invention.
  • FIG. 11 is a graph showing the thermal stability evaluation of the anti-LAG-3 monoclonal antibody HA-1 protein molecule in Example 18 of the present invention.
  • Figure 12 is a graph showing the growth of tumor volume in the MC38 colorectal cancer model in combination with anti-LAG-3 monoclonal antibody and anti-PD-1 monoclonal antibody in Example 22 of the present invention
  • Example 13 is a bar graph of tumor weight in the MC38 colorectal cancer model in which the anti-LAG-3 monoclonal antibody and the anti-PD-1 monoclonal antibody are combined in Example 22 of the present invention.
  • LAG-3 refers to lymphocyte activating factor 3, where LAG-3 includes, but is not limited to, dimer-expressed LAG-3 on the surface of activated T cells, NK cells, and B cells (eg, A soluble form of LAG-3 found in the art, known in the art as CD223) and human serum, is referred to herein as LAG-3.
  • antibody as used herein includes whole antibodies and any antigen-binding fragments thereof.
  • Antibodies include murine antibodies, humanized antibodies, bispecific antibodies or chimeric antibodies.
  • Antibodies can also be Fab, F(ab)2 , Fv or ScFv (single chain antibody), the antibody may be a naturally occurring antibody or an antibody that has been altered (eg, mutation, deletion, substitution, etc.).
  • variable region and “constant region” as used herein mean that the sequence region of the heavy chain and light chain of the antibody near the N segment is the variable region (V region), and the remaining amino acid sequences near the C segment are relatively stable and are Constant region (C region), variable region includes 3 complementarity determining regions (CDR) and 4 framework regions (FR), each light chain variable region and heavy chain variable region have 3 CDR regions and 4
  • CDR1, HCDR2 and HCDR3 3 complementarity determining regions
  • FR framework regions
  • each light chain variable region and heavy chain variable region have 3 CDR regions and 4
  • the three CDR regions of the heavy chain are respectively represented by HCDR1, HCDR2 and HCDR3
  • the three CDR regions of the light chain are respectively represented by LCDR1, LCDR2 and LCDR3.
  • antibody molecule of murine origin is derived from antibodies obtained after immunizing mice with LAG-3 antigen.
  • chimeric antibody molecule is an antibody obtained by fusing the variable region of a murine antibody with the constant region of a human antibody, which can alleviate the immune response induced by the murine antibody in the human body.
  • Chimeric antibodies use DNA recombination technology to insert the light and heavy chain variable region genes of mouse monoclonal antibodies into an expression vector containing human antibody constant regions.
  • the variable regions of the light and heavy chains in the antibody molecule thus expressed are of mouse origin.
  • the constant region is of human origin, and nearly 2/3 of the entire antibody molecule is of human origin. The resulting antibody reduces the immunogenicity of the murine antibody while retaining the ability of the parent antibody to specifically bind to the antigen.
  • humanized antibody molecule refers to grafting the CDRs of a murine monoclonal antibody to the variable region of a human antibody, replacing the CDRs of the human antibody, so that the human antibody obtains the antigen-binding specificity of the murine monoclonal antibody. sex, while reducing its heterogeneity.
  • CHO cell is a Chinese hamster ovary cell
  • HEK293E cell is a human embryonic kidney 293E cell (human embryonic kidney 293E cell)
  • NS0 cell is a mouse NSO thymoma cell.
  • Embodiment 1 of the present invention provides an anti-LAG-3 monoclonal antibody or an antigen-binding fragment thereof, which specifically includes a heavy chain variable region and a light chain variable region, and the heavy chain variable region includes three heavy chain complementarity determining regions , the three heavy chain complementarity determining regions are represented by HCDR1, HCDR2 and HCDR3 respectively, the light chain variable region includes three light chain complementarity determining regions, and the three light chain complementarity determining regions are respectively represented by LCDR1, LCDR2 and LCDR3, monoclonal antibody or an antigen-binding fragment thereof is selected from any one of the following.
  • the present invention uses LAG-3 antigen to immunize mice, optimizes the immunization method, and creates a phage display library.
  • the construction, screening and identification of the specific phage display library are as follows:
  • Step 1 Immune mice with LAG-3 antigen
  • Step 2 Construction of phage antibody library
  • mice spleen cells with higher titers were taken, and Trizol reagent (purchased from Ambion, article number: 15596026) was used to extract the total RNA in the mouse spleen cells, and cDNA was obtained by RT-PCR.
  • the cDNA was used as a template, and degenerate primers ( References for degenerate primers used: Journal of Immunological Methods 233 (2000) 167-177) for PCR amplification to obtain immunized mouse antibody heavy chain variable region gene library (VH) and light chain variable region gene library (VL ), the light and heavy chains were respectively double digested, connected to the same step-by-step digested carrier, and the pScFv-Disb-HS-VH-VL gene library was constructed.
  • VH immunized mouse antibody heavy chain variable region gene library
  • VL light chain variable region gene library
  • the PscFv-DisB-HS carrier adopts a series of gene cloning methods to
  • the vector pComb3 vector purchased from the China Plasmid Vector Strain Cell Line Gene Collection Center
  • the modified vector was named pScFv-Disb-HS vector, and its plasmid map was obtained as shown in Figure 1. Based on this vector, a mouse immune phage antibody library was constructed.
  • Step 3 Coat the immune tube with LAG-3 as antigen, the amount of antigen coating is 5 ⁇ g/500 ⁇ L/tube, coat overnight at 4°C, and then block the immune tube and the immune phage antibody library with 4% nonfat milk powder/PBST respectively, at room temperature closed for 1h.
  • the blocked immune phage antibody library was added to the immune tube for antigen-antibody binding.
  • the amount of phage input was about 10 9 to 10 12 .
  • PBST-PBS was used to wash away the unbound phage, and the unbound phage was washed away by 0.1M pH2.2 Glycine-HCl was eluted, and finally the eluted phage antibody solution was neutralized to pH 7.0 with 1.5M Tris-HCl pH 8.8.
  • Step 4 Infect 10 ml of the TG1 bacterial liquid that has grown to log phase with the neutralized phage, let stand for 30 min in a 37°C incubator, take out part of the bacterial liquid for gradient dilution, and spread it on a 2YTAG plate for calculating the phage output. The remaining bacterial liquid was centrifuged and the supernatant was discarded, and the bacterial pellet was resuspended in a small amount of medium, aspirated and spread on a large 2YTAG plate to prepare for the next round of screening.
  • Step 5 Scrape the plated bacteria from the large plate, inoculate the bacteria into 2YTAG liquid medium, shake to log phase, add M13KO7 helper phage for superinfection, and cultivate overnight at 28°C at 220rpm to prepare Phage, PEG/NaCl sedimentation-purified phage was used for the next round of screening, and a total of one round of phage library enrichment screening was performed.
  • Step 6 Screening of LAG-3 phage single-chain antibody positive clones: After one round of screening, pick well-separated monoclonal colonies and inoculate them in 96-well deep-well plates supplemented with 2YTAG liquid medium at 37°C , cultivated to its logarithmic growth phase under the condition of 220rpm, added about 10 10 helper phage M13KO7 to each well, and infected statically for 30min at 37°C. Centrifuge at 4000 rpm for 15 min, discard the supernatant, resuspend the pellet with 2YTAK, and cultivate overnight at 28° C. and 220 rpm.
  • the amplified phage supernatant was drawn for ELISA identification, and finally four anti-LAG-3 mouse-derived antibody molecules with higher affinity were screened and named as MA-1, MA -2, MA-3 and MA-4, the monoclonal antibody obtained above is determined by gene sequencing to be the correct antibody sequence, after sequencing, the sequences of the four monoclonal antibodies screened above are as follows:
  • mouse antibody molecule heavy chain variable region sequence light chain variable region sequence MA-1 SEQ ID No: 21 SEQ ID No: 22 MA-2 SEQ ID No: 21 SEQ ID No: 23 MA-3 SEQ ID No: 24 SEQ ID No: 25 MA-4 SEQ ID No: 26 SEQ ID No: 27
  • SEQ ID No: 21 amino acid sequences of heavy chain variable regions of MA-1 and MA-2):
  • SEQ ID No: 22 amino acid sequence of light chain variable region of MA-1):
  • SEQ ID No: 23 amino acid sequence of the light chain variable region of MA-2):
  • SEQ ID No: 24 amino acid sequence of heavy chain variable region of MA-3):
  • SEQ ID No: 25 amino acid sequence of the light chain variable region of MA-3):
  • SEQ ID No: 26 amino acid sequence of heavy chain variable region of MA-4):
  • SEQ ID No: 27 amino acid sequence of the light chain variable region of MA-4):
  • the 4 mouse-derived antibody molecules (MA-1, MA-2, MA-3 and MA-4) obtained in Example 2 were displayed and purified by monoclonal phage, and then the phage gradient dilution ELISA experiment was performed to identify the affinity, and the control Antibody selection patent CN105992595A provides anti-LAG-3 monoclonal antibody, the specific method is as follows:
  • LAG-3 was coated with carbonate buffer pH 9.6, 100ng/well/100 ⁇ l, overnight at 4°C, washed three times with PBST, and the 4 phage monoclones screened in Example 2 were Antibodies were serially diluted three times with PBST, 100 ⁇ l of the diluted samples were added to each well, and they were allowed to stand at room temperature for 1 hour.
  • the ELISA plate was washed with PBST, and the HRP-anti-M13 monoclonal antibody diluted with PBST was added to the ELISA plate and placed at room temperature for 1 h.
  • TMB color development kit developed color, developed color at room temperature for 10 minutes, terminated with 2M H 2 SO 4 , read at 450nm/630nm, and calculated the corresponding EC50 value.
  • the specific data are as follows:
  • the four different mouse-derived antibody molecules screened in Example 2 can all bind to LAG-3, and the monoclonal antibodies provided by the present invention all have high affinity for LAG-3 .
  • Example 4 of the present invention further defines that the mouse-derived antibody molecule also includes a heavy chain constant region and a light chain constant region, and the heavy chain constant region is one of mouse IgG1, IgG2a, IgG2b, and IgG3 types.
  • the heavy chain constant region amino acid sequence of IgG1 type is shown in SEQ ID No: 29
  • the heavy chain constant region amino acid sequence of IgG2a type is shown in SEQ ID No: 30
  • the heavy chain constant region amino acid sequence of IgG2b type is shown in SEQ ID No: 30 ID No: 31
  • the heavy chain constant region amino acid sequence of IgG3 type is shown in SEQ ID No: 32
  • the light chain constant region is a murine C kappa chain whose amino acid sequence is shown in SEQ ID No: 28
  • the specific sequence is as follows :
  • SEQ ID No: 28 (light chain constant region sequence of murine C kappa chain):
  • SEQ ID No: 29 (heavy chain constant region sequence of murine IgG1 type):
  • SEQ ID No: 30 (heavy chain constant region sequence of murine IgG2a type):
  • SEQ ID No: 31 (heavy chain constant region sequence of murine IgG2b type):
  • SEQ ID No: 32 (heavy chain constant region sequence of murine IgG3 type):
  • Example 5 of the present invention preferably defines that the heavy chain constant region of the mouse-derived antibody molecule is the mouse IgG1 type, and the IgG1 type includes the amino acid sequence shown in SEQ ID No: 29; the light chain constant region Being a murine C k chain, the murine C k chain comprises the amino acid sequence shown in SEQ ID No:28.
  • the antibody preparation method is as follows:
  • the coding genes of the heavy chain VH and light chain VL of the 4 monoclonal antibodies screened in Example 2 were cloned into the vector pTSE (as shown in Figure 3) containing the heavy chain and light chain constant region genes, respectively,
  • the preferred heavy chain constant region is mouse IgG1 type constant region (amino acid sequence shown in SEQ ID No: 29)
  • the light chain constant region is murine C k chain (amino acid sequence shown in SEQ ID No: 28)
  • pTSE The structure of the carrier is shown in Figure 3 (for the preparation process of the pTSE carrier, see paragraph [0019] on page 3 of the specification of CN103525868A).
  • LAG-3 was coated with carbonate buffer pH 9.6, 100 ng/well/100 ⁇ l, overnight at 4°C. Wash five times with 300 ⁇ l/well PBST, then add 1% BSA-PBST to block for 1 h at 37 °C, add MA-1, MA-2, MA-3 and MA-4 murine antibodies at different dilution concentrations, 4 The initial maximum concentration of all kinds of whole antibodies was 5 ⁇ g/ml. After 3-fold dilution, 12 gradients were made for each antibody and incubated at 37 °C for 1 h. Wash five times with 300 ⁇ l/well PBST, then add Anti-Mouse Fc-HRP diluted 1:10000 with 1% BSA-PBST, and incubate at 37° C. for 1 h.
  • the four different mouse-derived antibodies screened can bind to LAG-3.
  • MA-1 has the lowest EC50 value, indicating that it is closely related to LAG-3.
  • LAG-3 has the best binding ability and the highest affinity.
  • Example 7 The effect of murine antibody on the secretion of cytokine IL-2 in mixed lymphocyte reaction
  • Fresh peripheral blood PBMCs were isolated by density gradient centrifugation, and CD14 + T cells were separated by magnetic beads; CD14 + T cells were cultured with a medium of 20ng/mL GM-CSF and 10ng/mL IL-4, and the medium was changed every 2 days for 7 days. -10 days induction into dendritic DC cells. Two days before the DCs were used, 25 ng/mL of TNF- ⁇ was added to induce the DCs to become mature DC cells, and the mature DC cells were collected and prepared into a cell suspension with a cell density of 1 ⁇ 10 5 cells/mL.
  • CD4 + T cells were sorted by magnetic beads from fresh peripheral blood PBMC, counted, and made into a cell suspension with a cell density of 1 ⁇ 10 6 cells/mL. Take 100 ⁇ L of CD4 + T cells and DC cells each and add them to a 96-well plate at a ratio of 10:1.
  • the MA-1, MA-2, MA-3 and MA-4 murine antibodies prepared in Example 5 were diluted 4-fold, each antibody was set up with 6 gradients, and 50 ⁇ l of each was added to a 96-well plate. , after culturing for 5 days, detect the concentration of IL-2, read at 450nm, and calculate the corresponding EC50 value.
  • the specific data are as follows:
  • the four mouse-derived antibody molecules screened by the present invention all have good activities.
  • the EC50 value of MA-1 is the lowest, so its activity is the best. Therefore, the present invention can carry out humanization treatment for the mouse antibody molecule MA-1.
  • Example 8 of the present invention further defines that the monoclonal antibody or its antigen-binding fragment is a chimeric antibody molecule, and the chimeric antibody molecule includes the variable region of the heavy chain of the murine antibody molecule and the light chain of the murine antibody molecule in Example 2 Variable region and human antibody constant region; human antibody constant region includes human antibody heavy chain constant region and human antibody light chain constant region, human antibody heavy chain constant region is human IgG1 type, IgG2 type or IgG4 type A kind of, the heavy chain constant region amino acid sequence of IgG1 type is shown as SEQ ID No:39, the heavy chain constant region amino acid sequence of IgG2 type is shown as SEQ ID No:40, the heavy chain constant region amino acid sequence of IgG4 type is shown as SEQ ID No: 41; the human antibody light chain constant region is a human C kappa chain whose amino acid sequence is shown in SEQ ID No: 42.
  • SEQ ID No: 39 (human IgG1 type heavy chain constant region amino acid sequence):
  • SEQ ID No: 40 human IgG2 type heavy chain constant region amino acid sequence
  • SEQ ID No: 41 human IgG4 heavy chain constant region amino acid sequence
  • SEQ ID No: 42 amino acid sequence of light chain constant region of human C kappa chain
  • Example 9 of the present invention further defines that the heavy chain constant region of the chimeric antibody molecule is of human IgG4 type, and the amino acid sequence of the heavy chain constant region of IgG4 type is shown in SEQ ID No: 41; the chimeric antibody The light chain constant region is a human C kappa chain whose amino acid sequence is shown in SEQ ID No:42.
  • the heavy chain variable region VH (SEQ ID No: 21) and light chain variable region VL gene (SEQ ID No: 22) of the antibody molecule MA-1 screened by the immunophage antibody library in Example 2 were kept murine sequences unchanged, respectively cloned into the vector pTSE (as shown in Figure 3) equipped with heavy chain constant region and light chain constant region genes, the heavy chain constant region is human IgG4 type (amino acid sequence shown in SEQ ID NO: 41), The light chain constant region is the human C kappa chain (amino acid sequence shown in SEQ ID NO: 42).
  • HEK293E cells purchased from: Institute of Basic Medicine, Chinese Academy of Medical Sciences, item number: GNHu43 were transiently transfected, and the antibody was expressed to obtain the chimeric antibody CA-1.
  • Monoclonal antibodies heavy chain variable region light chain variable region HA-1 SEQ ID No: 33 SEQ ID No: 34 HA-2 SEQ ID No: 33 SEQ ID No: 35 HA-3 SEQ ID No: 36 SEQ ID No: 35 HA-4 SEQ ID No: 36 SEQ ID No: 34 HA-5 SEQ ID No: 37 SEQ ID No: 38
  • SEQ ID No: 33 (the amino acid sequences of the heavy chain variable regions of HA-1 and HA-2):
  • SEQ ID No: 34 amino acid sequences of light chain variable regions of HA-1 and HA-4.
  • SEQ ID No: 35 amino acid sequences of light chain variable regions of HA-2 and HA-3):
  • SEQ ID No: 36 amino acid sequences of heavy chain variable regions of HA-3 and HA-4.
  • SEQ ID No: 37 amino acid sequence of heavy chain variable region of HA-5):
  • SEQ ID No: 38 amino acid sequence of light chain variable region of HA-5):
  • Example 11 of the present invention further defines that the humanized antibody molecule further includes a heavy chain constant region and a light chain constant region, and the heavy chain constant region is one of human IgG1 type, IgG2 type or IgG4 type.
  • the heavy chain constant region amino acid sequence of IgG1 type is shown in SEQ ID No: 39
  • the heavy chain constant region amino acid sequence of IgG2 type is shown in SEQ ID No: 40
  • the heavy chain constant region amino acid sequence of IgG4 type is shown in SEQ ID No: 40.
  • the light chain constant region is a human C kappa chain whose amino acid sequence is shown in SEQ ID No: 42.
  • Example 12 of the present invention further defines that the heavy chain constant region of the humanized antibody molecule is of human IgG4 type, and the amino acid sequence of the heavy chain constant region of IgG4 type is shown in SEQ ID No: 41;
  • the chain constant region is a human C kappa chain whose amino acid sequence is shown in SEQ ID No:42.
  • the coding genes of the heavy chain VH and light chain VL of the five humanized antibody molecules obtained by humanization in the above Example 10 were cloned into the vector pTSE containing the heavy chain constant region and light chain constant region genes respectively (as shown in Figure 3).
  • the constant region of the heavy chain is of human IgG4 type (the amino acid sequence is shown in SEQ ID NO: 41), and the constant region of the light chain is the C k chain (the amino acid sequence is shown in SEQ ID NO: 42).
  • the chimeric antibody CA-1 and the humanized antibody were transiently transfected into HEK293E cells (purchased from the Institute of Basic Medicine, Chinese Academy of Medical Sciences, Cat. No. GNHu43) for antibody expression, and monoclonal was obtained by purification using an AKTA instrument through a protein A affinity column
  • the BCA kit purchased from: Beijing Huitian Oriental Technology Co., Ltd., product number: BCA0020
  • the sequence is the non-reduced protein molecular weight Marker1, HA-1, HA-2, HA-3, HA-4, HA-5, the chimeric antibody CA-1 prepared in Example 9, and the anti-LAG-3 provided by the core patent CN105992595A Monoclonal antibody and reduced protein molecular weight Marker2, HA-1, HA-2, HA-3, HA-4, HA-5, chimeric antibody CA-1 and anti-LAG-3 monoclonal antibody provided by core patent CN105992595A, each The molecular weight of the band is consistent with the theory.
  • Embodiment 13 of the present invention further defines the humanized antibody molecule as a full-length antibody or antibody fragment on the basis of the above-mentioned embodiment, and the humanized antibody molecule includes one of Fab, F(ab)2, Fv or ScFv or several combinations.
  • Embodiment 14 of the present invention further defines the following scheme on the basis of the above-mentioned embodiment:
  • a polypeptide or protein is further defined, and the polypeptide or the protein comprises the anti-LAG-3 monoclonal antibody or antigen-binding fragment thereof as defined in any one of the above embodiments.
  • a polynucleotide sequence or combination is further defined, and the polynucleotide sequence or combination encodes the amino acid sequence of the anti-LAG-3 monoclonal antibody or antigen-binding fragment thereof as defined in any one of the above embodiments.
  • a recombinant DNA expression vector is further defined, and the recombinant DNA expression vector comprises the above-mentioned polynucleotide sequence or combination.
  • the host cell includes prokaryotic cells, yeast cells, insect cells or mammalian cells;
  • the host cells are mammalian cells, and the mammalian cells are HEK293E cells, CHO cells or NSO cells.
  • a medicament or a pharmaceutical composition is further defined, and the medicament or pharmaceutical composition comprises the anti-LAG-3 monoclonal antibody or its antigen-binding fragment as defined in any one of the above embodiments.
  • the present invention further provides the application of the anti-LAG-3 monoclonal antibody or its antigen-binding fragment in the preparation of a medicine for treating cancer or immune diseases;
  • cancer includes but is not limited to leukemia, lung cancer, stomach cancer, esophageal cancer, ovarian cancer, head and neck cancer, melanoma, kidney cancer, breast cancer, colorectal cancer, liver cancer, pancreatic cancer or bladder cancer; immune diseases include but are not limited to Psoriasis, Crohn's disease, rheumatoid arthritis, primary biliary cirrhosis, systemic lupus erythematosus, multiple sclerosis, ulcerative colitis and autoimmune hepatitis.
  • immune diseases include but are not limited to Psoriasis, Crohn's disease, rheumatoid arthritis, primary biliary cirrhosis, systemic lupus erythematosus, multiple sclerosis, ulcerative colitis and autoimmune hepatitis.
  • LAG-3 was coated with carbonate buffer pH 9.6, 100 ng/well/100 ⁇ l, overnight at 4°C. Wash five times with 300 ⁇ l/well PBST, then add 1% BSA-PBS to block for 2 h at 37 °C, add humanized antibodies HA-1, HA-2, HA-3, HA-4 at different dilution concentrations, HA-5 and the chimeric antibody CA-1 prepared in Example 9 and the anti-LAG-3 antibody in the patent CN105992595A, the initial maximum concentration of the seven antibodies was 5 ⁇ g/ml, and each antibody was diluted 5 times. Eight gradients were made and incubated at 37°C for 1 h.
  • Fresh peripheral blood PBMCs were isolated by density gradient centrifugation, and CD14 + T cells were separated by magnetic beads; CD14 + T cells were cultured with a medium of 20ng/ml GM-CSF and 10ng/ml IL-4, and the medium was changed every two days, 7 -10 days induction into dendritic DC cells. Two days before the DCs were used, 25ng/ml of TNF- ⁇ was added to induce the DCs to become mature DC cells, and the mature DC cells were collected to prepare a cell suspension with a cell density of 1 ⁇ 10 5 cells/ml. CD14 + T cells were sorted by magnetic beads from fresh PMBC, counted, and made into a cell suspension with a cell density of 1 ⁇ 10 6 cells/ml. 100 ⁇ L of CD14 + T cells and DC cells were taken and added to a 96-well plate at a ratio of 10:1.
  • Example 12 The five anti-LAG-3 humanized antibody molecules prepared in Example 12, the chimeric antibody CA-1 prepared in Example 9, and the anti-LAG-3 antibody provided by patent CN105992595A were used as positive controls, and were respectively diluted 4-fold. Eight gradients were set for each antibody, and 50 ⁇ L of each was added to a 96-well plate. After 5 days, CCK8 tested the proliferation of CD14 + T cells, read at 450nm/630nm, and calculated the corresponding EC50 value. The specific data are as follows:
  • the EC50 values of the five different anti-LAG-3 antibodies screened in the present invention are significantly lower than the anti-LAG-3 antibodies provided in the patent CN105992595A, indicating that the anti-LAG-3 antibodies provided by the present invention The activity of the -3 antibody is relatively high.
  • the EC50 value of the anti-LAG-3 whole antibody HA-1 is the lowest, indicating that its activity is the highest.
  • the affinity of the chimeric antibody CA-1 is close to the EC50 value of the anti-LAG-3 antibody HA-1, indicating that the humanized antibody HA-1 retains the biological activity of the mouse parent antibody MA-1, and its biological activity does not decrease .
  • Mus Musculus Musculus, NCG, mouse;
  • MC38 tumor cells purchased from: Biovector NTCC Inc., item number: NTCC-MC38).
  • DMEM medium purchased from Thermo Fisher Scientific
  • FND500 10% fetal bovine serum
  • Gibco, Cat. No. 10566-016 culture tumor cells in an incubator at 37°C, 5% CO 2 , and divide the cells into flasks every 3 to 4 days after they are fully grown. Tumor cells in growth phase were used for in vivo tumor seeding.
  • Inoculation and grouping of tumor cells MC38 tumor cells resuspended in PBS at a concentration of 1.0 ⁇ 10 7 /ml were inoculated subcutaneously on the right flank of the experimental animals, 100 ⁇ L per animal, when the tumor grew to about 61 mm 3
  • Grouped administration a total of 3 groups, each group of 8, respectively, vehicle control group, HA-1 (10mg/kg, ip, biw ⁇ 3w), HA-1 (30mg/kg, ip, biw ⁇ 3w).
  • the data shown in Figure 10 shows that the anti-LAG-3 monoclonal antibody HA-1 can inhibit the growth of the tumor, and shows a dose-dependent response.
  • the anti-LAG-3 monoclonal antibody HA-1 protein molecule was ultrafiltered and exchanged into the PBS buffer system, 12000rpm, centrifuged at 4°C for 5min, and the multifunctional protein thermal stability analysis system (purchased from Unchained Labs) was used to resist The thermal stability of the LAG-3 monoclonal antibody HA-1 protein molecule was evaluated.
  • the thermal stability of the LAG-3 monoclonal antibody HA-1 protein molecule was evaluated.
  • temperature starting from 25°C, heating up to 95°C at a heating rate of 0.3°C/min
  • Tm protein melting temperature
  • the colloidal stability of the protein (characterized by Tagg) is determined by static light scattering. The results are shown in the following table and Figure 11.
  • the temperature of the anti-LAG-3 monoclonal antibody HA-1 protein molecule was 66.6°C, and the average Tagg was 68.0°C, showing good conformational stability and colloidal stability.
  • Embodiment 19 of the present invention further defines the following scheme on the basis of the above-mentioned embodiments:
  • the present invention also provides the application of the anti-LAG-3 monoclonal antibody or its antigen-binding fragment in combination with the anti-PD-1 monoclonal antibody for preparing a medicine for treating cancer or immune diseases.
  • the anti-PD-1 monoclonal antibody is selected from DFPD1-9, DFPD1-10, DFPD1-11, DFPD1-12, DFPD1-13, Nivolumab, Pembrolizumab, toripalizumab, sintilimab, Lelizumab, camrelizumab, pianpilimumab, or sapalizumab.
  • Nivolumab Nivolumab, Pembrolizumab, Toripalizumab, Sintilimab, Tislelizumab, Camrelizumab, Pembrolizumab or Sepalizumab are all marketed products .
  • DFPD1-9, DFPD1-10, DFPD1-11, DFPD1-12, and DFPD1-13 are anti-PD-1 monoclonal antibodies provided by the application number CN201510312910.8, and DFPD1-9 includes as shown in SEQ ID No:44 DFPD1-10 includes the light chain variable region shown in SEQ ID No:45 and the heavy chain variable region shown in SEQ ID No:43; The variable region of the heavy chain; DFPD1-11 includes the variable region of the light chain as shown in SEQ ID No: 44 and the variable region of the heavy chain as shown in SEQ ID No: 46; DFPD1-12 includes as shown in SEQ ID No: 46 The light chain variable region shown in 44 and the heavy chain variable region shown in SEQ ID No:47; DFPD1-13 include the light chain variable region shown in SEQ ID No:45 and the light chain variable region shown in SEQ ID No:46 The heavy chain variable region shown, the specific sequence is as follows:
  • SEQ ID No:45 the sequence is as follows:
  • the anti-PD-1 monoclonal antibody also includes a heavy chain constant region and a light chain constant region.
  • the heavy chain constant region and the light chain constant region are the same.
  • the cancer is selected from leukemia, lung cancer, gastric cancer, esophageal cancer, ovarian cancer, head and neck cancer, melanoma, kidney cancer, breast cancer, colorectal cancer, liver cancer, pancreatic cancer or bladder cancer; immune diseases include psoriasis, Roan's disease, rheumatoid arthritis, primary biliary cirrhosis, systemic lupus erythematosus, multiple sclerosis, ulcerative colitis and autoimmune hepatitis.
  • immune diseases include psoriasis, Roan's disease, rheumatoid arthritis, primary biliary cirrhosis, systemic lupus erythematosus, multiple sclerosis, ulcerative colitis and autoimmune hepatitis.
  • Example 20 of the present invention further defines the anti-PD-1 monoclonal antibody as DFPD1-10, and DFPD1-10 includes the light chain variable region shown in SEQ ID No: 45 and the light chain variable region shown in SEQ ID No: 45.
  • Example 21 of the present invention further defines the anti-PD-1 monoclonal antibody as Nivolumab.
  • Example 22 In vivo efficacy experiment of anti-LAG-3 monoclonal antibody and anti-PD-1 monoclonal antibody combined in MC38 colorectal cancer model
  • Anti-LAG-3 monoclonal antibody HA-1 provided in Example 10 above, numbered JY03;
  • Quantity 11mL/piece, 4.9mg/mL, 7 pieces in total;
  • Example 20 The anti-PD-1 monoclonal antibody provided in Example 20 is DFPD1-10, and the number in the experiment is JY034;
  • Quantity 10ml, 10mg/ml, 3 bottles, a total of 300mg;
  • MusMusculus B6/JGpt-Pdcd1em1Cin(hPDCD1)Lag3em1Cin(hLAG3)/Gpt
  • MC38 tumor cells were cultured in DMEM medium containing inactivated 10% fetal bovine serum, 100 U/mL penicillin and 100 ⁇ g/mL streptomycin, and 2 mM glutamine in a 37°C, 5% CO2 incubator. After 3 to 4 days, the cells were subcultured, and the tumor cells in the logarithmic growth phase were used for inoculation of tumors in vivo.
  • the MC38 tumor cells resuspended in PBS at a concentration of 1 ⁇ 10 7 /mL were inoculated subcutaneously on the right flank of experimental animals, 1 ⁇ 10 6 /100 ⁇ L per animal, and administered in groups when the tumor grew to about 50 mm 3 (The day was recorded as PG-D0), a total of 8 groups, 10 animals in each group.
  • the tumor volume was measured 2-3 times with a vernier caliper every week, and the mice were weighed with an electronic balance to measure the long and short diameters of the tumors.
  • T is the mean value of the relative tumor volume (RTV) of each test substance-treated group
  • C is the mean value of the relative tumor volume (RTV) of the control group
  • RTV is the mean value of the relative tumor volume (RTV) after administration and administration. previous tumor volume ratios.
  • Tumor growth inhibition rate TGITV (%) (1-T/C) ⁇ 100%.
  • the evaluation criteria are: T/C(%)>40% is invalid; T/C(%) ⁇ 40%, and P ⁇ 0.05 is effective after statistical processing.
  • the administration volume of each group is 10 ⁇ L/g according to the animal’s body weight, and the dosage can be adjusted when the body weight decreases by 15-20%; i.p.: intraperitoneal injection; biw x 3wks: twice a week for three weeks , a total of 6 times; #: It is the second dose of JY03, and the actual dose is 5 times in three weeks.
  • test drugs JY03 group, JY034 group, Opdivo group, JY03+JY034 group and JY03+Opdivo group were effective against PD-1 and LAG3 dual-target humanized colorectal cancer.
  • the treatment of MC38 model produced a clear anti-tumor effect.
  • the inhibitory effect of JY03+JY034 group on tumors was significantly stronger than that of single-agent JY03 group and single-agent JY034 group, and the inhibitory effect of JY03+Opdivo group was significantly stronger than that of single-agent JY03 group.
  • Drug JY03 group and single drug Opdivo group it can be shown that anti-LAG-3 monoclonal antibody can improve the inhibitory effect of anti-PD-1 monoclonal antibody on tumor; in addition, animals in each group tolerated well, and no obvious adverse effects were found reaction.
  • the present invention is not limited to the above-mentioned best embodiment, and anyone can draw other various forms of products under the inspiration of the present invention, but no matter if any changes are made in its shape or structure, all products with the same or similar characteristics as those of the present application can be obtained. Similar technical solutions all fall within the protection scope of the present invention.

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Abstract

涉及生物医药领域，具体提供了一种抗LAG-3的单克隆抗体、其抗原结合片段及其应用，其包括重链可变区和轻链可变区，单克隆抗体或其抗原结合片段选自A-1、A-2、A-3、A-4中任意一种。提供的单克隆抗体能够特异性结合LAG-3，亲和力较高，且具有很好的生物学活性，能够用于治疗多种癌症或者免疫疾病，癌症包括白血病、肺癌、胃癌、食道癌、卵巢癌、头颈癌、黑色素瘤、肾癌、乳腺癌、结直肠癌、肝癌、胰腺癌或膀胱癌，免疫疾病包括银屑病、克罗恩病、类风湿性关节炎、原发性胆汁性肝硬化、系统性红斑狼疮、多发性硬化症、溃疡性结肠炎和自身免疫性肝炎。

Description

一种抗LAG-3的单克隆抗体、其抗原结合片段及其应用
相关申请的交叉引用
本申请要求2020年12月10日提交的中国专利申请202011436581.5的权益，该申请的内容通过引用被合并于本文。
技术领域
本发明涉及生物医药技术领域，特别涉及一种抗LAG-3的单克隆抗体、其抗原结合片段及其应用。
背景技术
免疫疗法是当今最火热的癌症治疗方式，被誉为癌症治疗的第三次革命。所谓的“癌症免疫治疗”是借助人体自身的免疫系统，去攻击癌细胞的治疗方法。免疫系统和癌细胞之间的抗衡是一个长期博弈的动态过程，既正面交锋也相互交织。健康机体的免疫细胞能发现和杀灭癌细胞，但在各种先天和后天因素的诱导下，免疫系统会失去绝对优势，甚至被癌细胞“策反”，助长癌症的发生和发展。靶向药物曾以其精准性被寄予厚望，但由于癌细胞的善变和复杂，癌细胞很容易产生耐药性，精准性也很容易没有了用武之地。为此，自CTLA-4和PD-1被发现后，免疫检查点抑制治疗癌症逐渐成为研究热点。
随着免疫抗癌疗法的兴起，LAG-3逐渐被认为是一个颇有潜力的免疫检查点受体，据报道，LAG-3在促进调节性T细胞活性和下调T细胞活化和增殖中起重要作用(Workman CJ等人，J.Immunol.2005；174:688-695)。LAG-3(Lymphocyte-activation gene 3)又称为CD223，该基因包括8个外显子，位于人的12号染色体(小鼠的6号染色体)，属于免疫球蛋白超家族，其由胞外区、跨膜区和胞质区3个部分组成，编码由498个氨基酸构成的I型跨膜蛋白。LAG-3在激活的NK细胞、T细胞等免疫细胞表面广泛表达，可负向调控淋巴细胞功能，并且已有部分研究证实其在肿瘤以及自身免疫性疾病方面均发挥着重要作用。
临床前研究显示，抑制LAG-3能够让T细胞重新恢复细胞毒性，从而限制肿瘤的生长，进而增强对肿瘤的杀伤效果，同时抑制LAG-3还能够降低调节T细胞抑制免疫反应的功能，所以LAG-3是能够调节T细胞功能的免疫检查点受体，抑制LAG-3有助于使患者受益更多，特别是那些肿瘤中含有表达LAG-3的免疫细胞的患者，为此，LAG-3被认为是一个比其它免疫检查点蛋白更吸引人的靶点。目前免疫检查点二代靶点中，LAG-3是临床数据较多、成药 性相对确定的靶点。鉴于LAG-3存在的上述重要性，为了满足国内外肿瘤或其他免疫疾病患者的需求，急需开发具有较高生物学活性的抗LAG-3的单克隆抗体。
发明内容
为了满足国内市场的需求，本发明通过对免疫文库的筛选，得到了可以与LAG-3特异性结合且具有较高生物学活性的抗LAG-3的单克隆抗体或抗原结合片段。
本发明具体技术方案如下：
本发明提供了一种抗LAG-3的单克隆抗体或其抗原结合片段，包括重链可变区和轻链可变区，所述重链可变区包括3个重链互补决定区，3个所述重链互补决定区分别用HCDR1、HCDR2和HCDR3表示，所述轻链可变区包括3个轻链互补决定区，3个所述轻链互补决定区分别用LCDR1、LCDR2和LCDR3表示，所述单克隆抗体或其抗原结合片段选自以下任意一种：
A-1:所述重链互补决定区HCDR1包含如SEQ ID No:1所示的氨基酸序列，所述重链互补决定区HCDR2包含如SEQ ID No:2所示的氨基酸序列，所述重链互补决定区HCDR3包含如SEQ ID No:3所示的氨基酸序列，所述轻链互补决定区LCDR1包含如SEQ ID No:4所示的氨基酸序列，所述轻链互补决定区LCDR2包含如SEQ ID No:5所示的氨基酸序列，所述轻链互补决定区LCDR3包含如SEQ ID No:6所示的氨基酸序列；
A-2：所述重链互补决定区HCDR1包含如SEQ ID No:1所示的氨基酸序列，所述重链互补决定区HCDR2包含如SEQ ID No:2所示的氨基酸序列，所述重链互补决定区HCDR3包含如SEQ ID No:3所示的氨基酸序列，所述轻链互补决定区LCDR1包含如SEQ ID No:4所示的氨基酸序列，所述轻链互补决定区LCDR2包含如SEQ ID No:7所示的氨基酸序列，所述轻链互补决定区LCDR3包含如SEQ ID No:8所示的氨基酸序列；
A-3：所述重链互补决定区HCDR1包含如SEQ ID No:9所示的氨基酸序列，所述重链互补决定区HCDR2包含如SEQ ID No:10所示的氨基酸序列，所述重链互补决定区HCDR3包含如SEQ ID No:11所示的氨基酸序列，所述轻链互补决定区LCDR1包含如SEQ ID No:12所示的氨基酸序列，所述轻链互补决定区LCDR2包含如SEQ ID No:13所示的氨基酸序列，所述轻链互补决定区LCDR3包含如SEQ ID No:14所示的氨基酸序列；
A-4：所述重链互补决定区HCDR1包含如SEQ ID No:15所示的氨基酸序列，所述重链互补决定区HCDR2包含如SEQ ID No:16所示的氨基酸序列，所述重链互补决定区HCDR3包含如SEQ ID No:17所示的氨基酸序列，所述轻链互补决定区LCDR1包含如SEQ ID No:18 所示的氨基酸序列，所述轻链互补决定区LCDR2包含如SEQ ID No:19所示的氨基酸序列，所述轻链互补决定区LCDR3包含如SEQ ID No:20所示的氨基酸序列。
本发明提供的上述抗LAG-3的单克隆抗体或其抗原结合片段能够与LAG-3特异性结合，结合活性更好，阻断LAG-3则可以逆转LAG-3对T细胞的抑制作用，增强T细胞活性，并减少调节性T细胞数量，还可以提高T细胞免疫应答的敏感度，从而用于治疗免疫或癌症疾病。
进一步的，所述单克隆抗体或其抗原结合片段为鼠源抗体分子，所述鼠源抗体分子选自以下任意一种：
MA-1：所述重链可变区包含如SEQ ID No:21所示的氨基酸序列，所述轻链可变区包含如SEQ ID No:22所示的氨基酸序列；
MA-2：所述重链可变区包含如SEQ ID No:21所示的氨基酸序列，所述轻链可变区包含如SEQ ID No:23所示的氨基酸序列；
MA-3：所述重链可变区包含如SEQ ID No:24所示的氨基酸序列，所述轻链可变区包含如SEQ ID No:25所示的氨基酸序列；
MA-4：所述重链可变区包含如SEQ ID No:26所示的氨基酸序列，所述轻链可变区包含如SEQ ID No:27所示的氨基酸序列；
优选的，所述鼠源抗体分子为MA-1。
本发明通过小鼠免疫用LAG-3抗原免疫小鼠，优化免疫方法，创建噬菌体展示库，并筛选出上述亲和力较高，活性较好且较为稳定的鼠源抗体分子，通过大量的细胞水平实验验证，发现相对其他3个鼠源抗体分子，MA-1具有更高的生物学活性，为此，本发明优选的选择MA-1。
进一步的，所述鼠源抗体分子还包括重链恒定区和轻链恒定区，所述重链恒定区为鼠的IgG1型、IgG2a型、IgG2b型、IgG3型中的一种，所述IgG1型的重链恒定区氨基酸序列如SEQ ID No:29所示，所述IgG2a型的重链恒定区氨基酸序列如SEQ ID No:30所示，所述IgG2b型的重链恒定区氨基酸序列如SEQ ID No:31所示，所述IgG3型的重链恒定区氨基酸序列如SEQ ID No:32所示；所述轻链恒定区为氨基酸序列如SEQ ID No:28所示的鼠源C k链；
优选的，所述重链恒定区为鼠的IgG1型。
进一步的，所述单克隆抗体或其抗原结合片段为嵌合抗体分子，所述嵌合抗体分子包括所述鼠源抗体分子的重链可变区、所述鼠源抗体分子的轻链可变区和人源抗体恒定区；所述人源抗体恒定区包括人源抗体重链恒定区和人源抗体轻链恒定区，所述人源抗体重链恒定区 为人的IgG1型、IgG2型或IgG4型中的一种，所述IgG1型的重链恒定区氨基酸序列如SEQ ID No:39所示，所述IgG2型的重链恒定区氨基酸序列如SEQ ID No:40所示，所述IgG4型的重链恒定区氨基酸序列如SEQ ID No:41所示；所述人源抗体轻链恒定区为氨基酸序列如SEQ ID No:42所示的人的C k链；
优选的，所述人源抗体重链恒定区为人的IgG4型。
嵌合抗体分子包括鼠源抗体分子的可变区序列和人源抗体恒定区，嵌合抗体分子的设计用于验证本发明恒定区人源化后没有改变CDR的功能，为人源化抗体分子的研究提供了进一步的研发基础。
进一步的，所述单克隆抗体或其抗原结合片段为人源化抗体分子，所述人源化抗体分子选自以下任意一种：
HA-1：所述重链可变区包含如SEQ ID No:33所示的氨基酸序列，所述轻链可变区包含如SEQ ID No:34所示的氨基酸序列；
HA-2：所述重链可变区包含如SEQ ID No:33所示的氨基酸序列，所述轻链可变区包含如SEQ ID No:35所示的氨基酸序列；
HA-3：所述重链可变区包含如SEQ ID No:36所示的氨基酸序列，所述轻链可变区包含如SEQ ID No:35所示的氨基酸序列；
HA-4：所述重链可变区包含如SEQ ID No:36所示的氨基酸序列，所述轻链可变区包含如SEQ ID No:34所示的氨基酸序列；
HA-5：所述重链可变区包含如SEQ ID No:37所示的氨基酸序列，所述轻链可变区包含如SEQ ID No:38所示的氨基酸序列；
优选的，所述人源化抗体分子为HA-1。
本发明通过人源化筛选后得到了人源化抗体分子，通过体内外的实验验证发现本发明中提供的5种人源化抗体分子中，HA-1的活性较高，药效最为显著，为此本发明优选HA-1。
进一步的，所述人源化抗体分子还包括重链恒定区和轻链恒定区，所述重链恒定区为人的IgG1型、IgG2型或IgG4型中的一种，所述IgG1型的重链恒定区氨基酸序列如SEQ ID No:39所示，所述IgG2型的重链恒定区氨基酸序列如SEQ ID No:40所示，所述IgG4型的重链恒定区氨基酸序列如SEQ ID No:41所示，所述轻链恒定区为氨基酸序列如SEQ ID No:42所示的人的C k链；
优选的，所述重链恒定区为人的IgG4型。
进一步的，所述人源化抗体分子为全长抗体或抗体片段，所述人源化抗体分子包括Fab、 F(ab)2、Fv或ScFv中的一种或几种组合。
本发明还提供了一种多肽或蛋白，所述多肽或所述蛋白包含所述的抗LAG-3的单克隆抗体或其抗原结合片段。
本发明还提供了一种多核苷酸序列或组合，所述多核苷酸序列或组合编码所述的抗LAG-3的单克隆抗体或其抗原结合片段的氨基酸序列。
本发明还提供了一种重组DNA表达载体，所述重组DNA表达载体包含所述的多核苷酸序列或组合。
本发明还提供了一种转染所述的重组DNA表达载体的宿主细胞，所述宿主细胞包括原核细胞、酵母细胞、昆虫细胞或哺乳动物细胞；
优选的，所述宿主细胞为哺乳动物细胞，所述哺乳动物细胞为HEK293E细胞、CHO细胞或NS0细胞。
本发明还提供了一种药物或药物组合物，所述药物或所述药物组合物包含所述的抗LAG-3的单克隆抗体或其抗原结合片段。
本发明还提供了所述的抗LAG-3的单克隆抗体或其抗原结合片段在制备治疗癌症或免疫疾病药物中的应用；
优选的，所述癌症包括白血病、肺癌、胃癌、食道癌、卵巢癌、头颈癌、黑色素瘤、肾癌、乳腺癌、结直肠癌、肝癌、胰腺癌或膀胱癌；
所述免疫疾病包括银屑病、克罗恩病、类风湿性关节炎、原发性胆汁性肝硬化、系统性红斑狼疮、多发性硬化症、溃疡性结肠炎和自身免疫性肝炎。
本发明还提供了所述的抗LAG-3的单克隆抗体或其抗原结合片段与抗PD-1单克隆抗体联合用于制备治疗癌症或免疫疾病药物中的应用。
进一步的，所述抗PD-1单克隆抗体包括DFPD1-9、DFPD1-10、DFPD1-11、DFPD1-12、DFPD1-13、Nivolumab、Pembrolizumab、特瑞普利单抗、信迪利单抗、替雷利珠单抗、卡瑞利珠单抗、派安普利单抗或赛帕利单抗。
优选的，所述抗PD-1单克隆抗体为DFPD1-10，所述DFPD1-10包括如SEQ ID No:45所示的轻链可变区和如SEQ ID No:43所示的重链可变区。
优选的，所述抗PD-1单克隆抗体为Nivolumab。
优选的，所述癌症选自白血病、肺癌、胃癌、食道癌、卵巢癌、头颈癌、黑色素瘤、肾癌、乳腺癌、结直肠癌、肝癌、胰腺癌或膀胱癌；所述免疫疾病包括银屑病、克罗恩病、类风湿性关节炎、原发性胆汁性肝硬化、系统性红斑狼疮、多发性硬化症、溃疡性结肠炎和自 身免疫性肝炎。
本发明的有益效果如下：本发明提供的抗LAG-3的单克隆抗体或其抗原结合片段能够特异性结合LAG-3，亲和力较高，且具有很好的生物学活性，能够用于治疗多种癌症或者免疫疾病，癌症包括但不限于白血病、肺癌、胃癌、食道癌、卵巢癌、头颈癌、黑色素瘤、肾癌、乳腺癌、结直肠癌、肝癌、胰腺癌或膀胱癌，免疫疾病包括但不限于银屑病、克罗恩病、类风湿性关节炎、原发性胆汁性肝硬化、系统性红斑狼疮、多发性硬化症、溃疡性结肠炎和自身免疫性肝炎。
附图说明
图1为本发明实施例2中pScFv-Disb-HS载体的质粒图谱；
图2为本发明实施例3中梯度稀释ELISA抗LAG-3噬菌体单克隆抗体相对亲和力的比较图；
图3为本发明实施例5中载体pTSE的图谱；
图4为本发明实施例5中鼠源抗体分子的变性聚丙烯酰胺凝胶电泳图；
图5为本发明实施例6中鼠源抗体与LAG-3的结合能力比较图；
图6为本发明实施例7中鼠源抗体在混合淋巴细胞反应中对细胞因子IL-2分泌情况图；
图7为本发明实施例12中人源化抗体分子的变性聚丙烯酰胺凝胶电泳图；
图8为本发明实施例15中人源化抗体分子与LAG-3的结合能力比较图；
图9为本发明实施例16中混合淋巴细胞反应(MLR)测试抗LAG-3人源化抗体分子活性。
图10为本发明实施例17中抗LAG-3单克隆抗体对小鼠体内MC38结直肠癌的抑制试验效果图；
图11为本发明实施例18中抗LAG-3单克隆抗体HA-1蛋白分子热稳定性评价图。
图12为本发明实施例22中抗LAG-3单克隆抗体与抗PD-1单克隆抗体联合用于MC38结直肠癌模型中肿瘤体积生长曲线图；
图13为本发明实施例22中抗LAG-3单克隆抗体与抗PD-1单克隆抗体联合用于MC38结直肠癌模型中肿瘤重量柱形图。
具体实施方式
为了更加容易理解本发明，描述实施例之前，先对本发明某些技术和科学术语作以下说 明：
本文所使用的术语“LAG-3”是指淋巴细胞活化因子3，这里的LAG-3包括但不限于活化的T细胞、NK细胞和B细胞表面上的二聚物表达的LAG-3(例如本领域公知的CD223)和人血清中发现的LAG-3的可溶形式，在本文中均称为LAG-3。
本文所使用的术语“抗体”，包含全抗体及其任一抗原结合片段，抗体包括鼠源抗体、人源化抗体、双特异抗体或嵌合抗体，抗体也可以是Fab、F(ab)2、Fv或ScFv(单链抗体)，抗体可以是天然存在的抗体也可以是通过改变(例如突变、缺失、置换等)的抗体。
本文所使用的术语“可变区”和“恒定区”，即为抗体重链和轻链靠近N段的序列区为可变区(V区)，靠近C段的其余氨基酸序列相对稳定，为恒定区(C区)，可变区包括3个互补性决定区(CDR)和4个框架区(FR)，每条轻链可变区和重链可变区均有3个CDR区和4个FR区组成，重链的3个CDR区分别通过HCDR1、HCDR2和HCDR3表示，轻链的3个CDR区分别通过LCDR1、LCDR2和LCDR3表示。
本文所使用的术语“鼠源抗体分子”，其来源是用LAG-3抗原免疫注射小鼠后得到的抗体。
本文所使用的术语“嵌合抗体分子”，是将鼠源抗体的可变区与人源抗体的恒定区融合而成的抗体，可以减轻鼠源抗体在人体内诱发的免疫应答反应。嵌合抗体是利用DNA重组技术，将鼠源单抗的轻、重链可变区基因插入含有人抗体恒定区的表达载体中，这样表达的抗体分子中轻重链的可变区是鼠源的，而恒定区是人源的，整个抗体分子的近2/3部分都是人源的。这样产生的抗体，减少了鼠源抗体的免疫原性，同时保留了亲本抗体特异性结合抗原的能力。
本文所使用的术语“人源化抗体分子”，其是将鼠源单抗的CDR移植至人源抗体可变区，替代人源抗体CDR，使人源抗体获得鼠源单抗的抗原结合特异性，同时减少其异源性。
术语“CHO细胞”为中国仓鼠卵巢细胞(chinese hamster ovary cell)；术语“HEK293E细胞”为人胚肾293E细胞(human embryonic kidney 293E cell)，术语“NS0细胞”为小鼠NS0胸腺瘤细胞。
下面结合以下实施例对本发明作进一步详细说明。
实施例1
本发明实施例1提供了一种抗LAG-3的单克隆抗体或其抗原结合片段，具体包括重链可变区和轻链可变区，重链可变区包括3个重链互补决定区，3个重链互补决定区分别用HCDR1、HCDR2和HCDR3表示，轻链可变区包括3个轻链互补决定区，3个轻链互补决定区分别用LCDR1、LCDR2和LCDR3表示，单克隆抗体或其抗原结合片段选自以下任意一种。
Figure PCTCN2021133941-appb-000001
实施例2鼠源抗体分子筛选
本发明通过用LAG-3抗原免疫小鼠，优化免疫方法，创建噬菌体展示库，具体噬菌体展示库的构建与筛选鉴定如下：
步骤一：LAG-3抗原免疫小鼠
1、实验动物：
种属品系：BALB/c，雌性，小鼠；
体重：18-20g；
实验动物提供商：北京华阜康生物科技股份有限公司。
2、免疫：对小鼠进行免疫，免疫抗原为人LAG-3(南京金斯瑞生物科技有限公司合成基因，本公司构建载体并表达纯化)。
步骤二：噬菌体抗体库的构建
取效价较高的小鼠脾细胞，利用Trizol试剂(购买自Ambion，货号：15596026)，提取小鼠脾细胞中的总RNA，RT-PCR获得cDNA，以cDNA为模板，采用简并引物(所用简并引物参考文献：Journal of Immunological Methods 233(2000)167-177)进行PCR扩增，从而获得免疫小鼠抗体重链可变区基因库(VH)及轻链可变区基因库(VL)，轻重链分别双酶切，连接至同样分步骤酶切处理过的载体上，构建pScFv-Disb-HS-VH-VL基因库，PscFv-DisB-HS载体是采用一系列基因克隆的方法对载体pComb3载体(购自中国质粒载体菌株细胞株基因保藏中心)进行改造，使之用于噬菌体单链抗体库的构建和表达。改造后的载体命名pScFv-Disb-HS载体，获得其质粒图谱如图1所示，并以此载体为基础，构建小鼠免疫噬菌体抗体库。
步骤三：以LAG-3为抗原包被免疫管，抗原包被量为5μg/500μL/管，4℃包被过夜，再用4％脱脂奶粉/PBST分别封闭免疫管和免疫噬菌体抗体库，室温封闭1h。封闭后的免疫噬菌体抗体库加入免疫管中进行抗原抗体结合，噬菌体投入量约为10 9～10 12个，室温反应1h后，使用PBST-PBS洗去未结合的噬菌体，通过0.1MpH2.2的Glycine-HCl洗脱，最后使用1.5M  pH 8.8的Tris-HCl中和洗脱下来的噬菌体抗体溶液至pH7.0左右。
步骤四：将上述中和后的噬菌体感染10ml生长至对数期的TG1菌液，37℃培养箱中静置30min，取出部分菌液进行梯度稀释，涂布于2YTAG平板上，用于计算噬菌体产出量。剩余的菌液离心弃上清，将菌体沉淀重悬于少量培养基，吸出后涂布于2YTAG大平板，为下一轮筛选做准备。
步骤五：将上述感染后涂板的菌体从大平板上刮下，接菌至2YTAG液体培养基，摇至对数期后加入M13KO7辅助噬菌体超感染，在28℃条件下，220rpm培养过夜制备噬菌体，PEG/NaCl沉降纯化噬菌体用于下一轮筛选，共进行一轮噬菌体库富集筛选。
步骤六：LAG-3噬菌体单链抗体阳性克隆的筛选：经过一轮筛选后，挑取分隔良好的单克隆菌落，接种于加有2YTAG液体培养基的96孔深孔板，在37℃条件下，220rpm的条件下培养至其对数生长期，每孔加入约10 10的辅助噬菌体M13KO7，在37℃的温度条件下静止感染30min。4000rpm，离心15min，弃去上清，菌体用2YTAK重悬沉淀，在28℃且220rpm的条件下培养过夜。4000rpm，4℃的条件下离心15min后，吸取扩增后的噬菌体上清进行ELISA鉴定，最终筛选得到四个亲和力较高的抗LAG-3的鼠源抗体分子，分别命名为MA-1，MA-2，MA-3和MA-4，将上述得到的单克隆抗体进行基因测序确定为正确的抗体序列，经过测序，上述筛选到的4个单克隆抗体序列如下：
鼠源抗体分子 重链可变区序列 轻链可变区序列
MA-1 SEQ ID No:21 SEQ ID No:22
MA-2 SEQ ID No:21 SEQ ID No:23
MA-3 SEQ ID No:24 SEQ ID No:25
MA-4 SEQ ID No:26 SEQ ID No:27
具体的，SEQ ID No:21(MA-1和MA-2的重链可变区的氨基酸序列)：
Figure PCTCN2021133941-appb-000002
SEQ ID No:22(MA-1的轻链可变区的氨基酸序列)：
Figure PCTCN2021133941-appb-000003
SEQ ID No:23(MA-2的轻链可变区的氨基酸序列)：
Figure PCTCN2021133941-appb-000004
SEQ ID No:24(MA-3的重链可变区的氨基酸序列)：
Figure PCTCN2021133941-appb-000005
SEQ ID No:25(MA-3的轻链可变区的氨基酸序列)：
Figure PCTCN2021133941-appb-000006
SEQ ID No:26(MA-4的重链可变区的氨基酸序列)：
Figure PCTCN2021133941-appb-000007
SEQ ID No:27(MA-4的轻链可变区的氨基酸序列)：
Figure PCTCN2021133941-appb-000008
实施例3梯度稀释ELISA比较抗LAG-3噬菌体单克隆抗体的亲和力
将实施例2中获得的4个鼠源抗体分子(MA-1，MA-2，MA-3和MA-4)进行单克隆噬菌体的展示和纯化，然后进行噬菌体梯度稀释ELISA实验鉴定亲和力，对照抗体选择专利CN105992595A中的中提供的抗LAG-3的单克隆抗体，具体方法如下：
用pH9.6的碳酸盐缓冲液包被LAG-3，100ng/孔/100μl，在4℃温度条件下包被过夜，使用PBST洗涤三次，将实施例2中筛选得到的4个噬菌体单克隆抗体分别用PBST三倍梯度稀释，每孔加入100μl稀释后的样品，在室温下静置1小时。用PBST洗涤ELISA板，将PBST稀释后的HRP-anti-M13单克隆抗体加入ELISA板中，在室温放置1h。TMB显色试剂盒显色，室温显色10分钟，用2M H 2SO 4终止后，在450nm/630nm下读数，并计算对应的EC50值，具体数据如下：
克隆 MA-1 MA-2 MA-3 MA-4 对照抗体
EC50 0.010 0.058 0.024 0.117 0.115
通过上述数据及如图2所示，实施例2筛选出的4个不同的鼠源抗体分子均能够与LAG-3进行结合，本发明提供的单克隆抗体与LAG-3均具有较高的亲和力。
实施例4
本发明实施例4在实施例2基础上进一步限定了鼠源抗体分子还包括重链恒定区和轻链恒定区，重链恒定区为鼠的IgG1型、IgG2a型、IgG2b型、IgG3型中的一种，IgG1型的重 链恒定区氨基酸序列如SEQ ID No:29所示，IgG2a型的重链恒定区氨基酸序列如SEQ ID No:30所示，IgG2b型的重链恒定区氨基酸序列如SEQ ID No:31所示，IgG3型的重链恒定区氨基酸序列如SEQ ID No:32所示；轻链恒定区为氨基酸序列如SEQ ID No:28所示的鼠源C k链；具体序列如下：
SEQ ID No:28(鼠源C k链的轻链恒定区序列)：
Figure PCTCN2021133941-appb-000009
SEQ ID No:29(鼠的IgG1型的重链恒定区序列)：
Figure PCTCN2021133941-appb-000010
SEQ ID No:30(鼠的IgG2a型的重链恒定区序列)：
Figure PCTCN2021133941-appb-000011
SEQ ID No:31(鼠的IgG2b型的重链恒定区序列)：
Figure PCTCN2021133941-appb-000012
SEQ ID No:32(鼠的IgG3型的重链恒定区序列)：
Figure PCTCN2021133941-appb-000013
Figure PCTCN2021133941-appb-000014
实施例5抗LAG-3鼠源抗体分子制备
本发明实施例5在实施例4的基础上优选的限定了鼠源抗体分子的重链恒定区为鼠的IgG1型，IgG1型包含如SEQ ID No:29所示的氨基酸序列；轻链恒定区为鼠源C k链，鼠源C k链包含如SEQ ID No:28所示的氨基酸序列。抗体制备方法具体如下：
1、在将实施例2筛选出来的4个单克隆抗体的重链VH和轻链VL的编码基因分别克隆至装有重链和轻链恒定区基因的载体pTSE(如图3所示)，优选的重链恒定区为鼠的IgG1型恒定区(氨基酸序列如SEQ ID No:29所示)，轻链恒定区为鼠源C k链(氨基酸序列如SEQ ID No:28所示)，pTSE载体结构如图3所示(pTSE载体制备过程参见CN103525868A说明书第3页第[0019]段)。
2、瞬时转染HEK293E细胞(购自中国医学科学院基础医学研究所，货号为GNHu43)，进行抗体表达，使用AKTA仪器通过protein A亲和柱纯化获得4个单克隆抗体，同时使用BCA试剂盒(购买自：北京汇天东方科技有限公司，货号：BCA0020)进行蛋白浓度测定，之后通过SDS-PAGE鉴定蛋白大小，结果如图4所示，从左侧到右侧依次为非还原MA-1，MA-2，MA-3和MA-4和蛋白质分子量Marker1、Marker2及还原MA-1，MA-2，MA-3和MA-4鼠源抗LAG-3单克隆抗体，每条带的分子量大小与理论一致。
实施例6鼠源抗体与LAG-3的结合实验
用pH9.6的碳酸盐缓冲液包被LAG-3，100ng/孔/100μl，在4℃的温度条件下过夜包被。用300μl/孔PBST洗涤五次，再加入1％BSA-PBST在37℃温度条件下封闭1h，加入不同稀释浓度的MA-1，MA-2，MA-3和MA-4鼠源抗体，4种全抗体的起始最高浓度均是5μg/ml，分别经过3倍稀释后每个抗体均做12个梯度，在37℃温度条件下孵育1h。用300μl/孔PBST洗涤五次，再加入用1％BSA-PBST 1:10000稀释的Anti-Mouse Fc-HRP，在37℃温度条件下孵育1h。TMB显色试剂盒显色，100μl/孔，室温显色8min，然后用2MH 2SO 4终止显色。在450nm/630nm下读数，并计算对应的EC50值，具体数据如下：
克隆 MA-1 MA-2 MA-3 MA-4
EC50(ng/ml) 10.08 25.99 255.9 22.12
通过上述数据及如图5所示，筛选出的4个不同的鼠源抗体均能与LAG-3进行结合，此外，这4个鼠源抗体分子中MA-1的EC50值最低，说明其与LAG-3结合能力最好，亲和力最高。
实施例7鼠源抗体在混合淋巴细胞反应中对细胞因子IL-2分泌量的影响
按密度梯度离心法分离新鲜外周血PBMC，磁珠分选CD14 +T细胞；用20ng/mL GM-CSF与10ng/mL IL-4的培养基培养CD14 +T细胞，每2天换液，7-10天诱导成为树突状DC细胞。在DC使用前两天，加入25ng/mL的TNF-α诱导DC为成熟的DC细胞，收集成熟的DC细胞，配制成细胞密度为1x10 5个/mL细胞悬液。从新鲜外周血PBMC中磁珠分选出CD4 +T细胞，计数，制成细胞密度为1x10 6个/mL细胞悬液。将CD4 +T细胞与DC细胞各取100μL，按比例10：1加入96孔板。
将实施例5中制备的MA-1，MA-2，MA-3和MA-4鼠源抗体分别进行4倍梯度稀释，每个抗体均设置6个梯度，各取50μl加入到96孔板中，培养5天后检测IL-2的浓度，在450nm下读数，并计算对应的EC50值，具体数据如下：
克隆 MA-1 MA-2 MA-3 MA-4
EC50(ug/ml) 0.024 0.030 0.076 0.121
通过上述数据及如图6所示，本发明筛选出来的4个鼠源抗体分子，均具有较好的活性，此外，通过上述数据还可以得出，本发明筛选出的4个单克隆抗体中MA-1的EC50值最低，所以其活性最好，为此，本发明可以针对鼠源抗体分子MA-1进行人源化处理。
实施例8
本发明实施例8进一步的限定了单克隆抗体或其抗原结合片段为嵌合抗体分子，嵌合抗体分子包括实施例2中鼠源抗体分子的重链可变区、鼠源抗体分子的轻链可变区和人源抗体恒定区；人源抗体恒定区包括人源抗体重链恒定区和人源抗体轻链恒定区，人源抗体重链恒定区为人的IgG1型、IgG2型或IgG4型中的一种，IgG1型的重链恒定区氨基酸序列如SEQ ID No:39所示，IgG2型的重链恒定区氨基酸序列如SEQ ID No:40所示，IgG4型的重链恒定区氨基酸序列如SEQ ID No:41所示；人源抗体轻链恒定区为氨基酸序列如SEQ ID No:42所示的人的C k链。
SEQ ID No:39(人的IgG1型的重链恒定区氨基酸序列)：
Figure PCTCN2021133941-appb-000015
Figure PCTCN2021133941-appb-000016
SEQ ID No:40(人的IgG2型的重链恒定区氨基酸序列)：
Figure PCTCN2021133941-appb-000017
SEQ ID No:41(人的IgG4型的重链恒定区氨基酸序列)：
Figure PCTCN2021133941-appb-000018
SEQ ID No:42(人的C k链的轻链恒定区氨基酸序列)：
Figure PCTCN2021133941-appb-000019
实施例9嵌合抗体分子抗体的制备
本发明实施例9在实施例8的基础上进一步限定了嵌合抗体分子的重链恒定区为人的IgG4型，IgG4型的重链恒定区氨基酸序列如SEQ ID No:41所示；嵌合抗体轻链恒定区为氨基酸序列如SEQ ID No:42所示的人的C k链。
具体的制备方法：
将实施例2中免疫噬菌体抗体库筛选得到的抗体分子MA-1的重链可变区VH(SEQ ID No:21)和轻链可变区VL基因(SEQ ID No:22)保持鼠源序列不变，分别克隆至装有重链恒定区和轻链恒定区基因的载体pTSE(如图3所示)，重链恒定区为人的IgG4型(氨基酸序 列如SEQ ID NO:41所示)，轻链恒定区为人的C k链(氨基酸序列如SEQ ID NO:42所示)。瞬时转染HEK293E细胞(购买自：中国医学科学院基础医学研究所，货号为：GNHu43)，进行抗体表达，得到嵌合抗体CA-1。
实施例10鼠源抗体分子MA-1进行人源化
首先使用实施例2中鼠源抗体分子MA-1的序列和人抗体种系数据库(v-base)比较，寻找同源性较高的人抗体轻、重链种系作为候选序列，然后将鼠源抗体分子MA-1的CDR的序列移植到人源候选序列上进行同源建模。然后通过三维结构模拟计算可能对于维持CDR环状结构起重要作用的关键框架氨基酸残基，从而设计人源化抗体的回复突变。将设计好的包含回复突变的人源化抗体的轻、重链可变区分别由南京金斯瑞生物科技有限公司优化合成，然后再连接到瞬时表达载体上，对人源化得到的轻重链组合分析，得到如下人源化抗体分子：HA-1，HA-2，HA-3，HA-4，HA-5，上述筛选到的5个单克隆抗体序列如下：
单克隆抗体 重链可变区 轻链可变区
HA-1 SEQ ID No:33 SEQ ID No:34
HA-2 SEQ ID No:33 SEQ ID No:35
HA-3 SEQ ID No:36 SEQ ID No:35
HA-4 SEQ ID No:36 SEQ ID No:34
HA-5 SEQ ID No:37 SEQ ID No:38
具体的，SEQ ID No:33(HA-1和HA-2的重链可变区的氨基酸序列)：
Figure PCTCN2021133941-appb-000020
SEQ ID No:34(HA-1和HA-4的轻链可变区的氨基酸序列)：
Figure PCTCN2021133941-appb-000021
SEQ ID No:35(HA-2和HA-3的轻链可变区的氨基酸序列)：
Figure PCTCN2021133941-appb-000022
SEQ ID No:36(HA-3和HA-4的重链可变区的氨基酸序列)：
Figure PCTCN2021133941-appb-000023
SEQ ID No:37(HA-5的重链可变区的氨基酸序列)：
Figure PCTCN2021133941-appb-000024
SEQ ID No:38(HA-5的轻链可变区的氨基酸序列)：
Figure PCTCN2021133941-appb-000025
实施例11
本发明实施例11在实施例10的基础上进一步的限定了人源化抗体分子还包括重链恒定区和轻链恒定区，重链恒定区为人的IgG1型、IgG2型或IgG4型中的一种，IgG1型的重链恒定区氨基酸序列如SEQ ID No:39所示，IgG2型的重链恒定区氨基酸序列如SEQ ID No:40所示，IgG4型的重链恒定区氨基酸序列如SEQ ID No:41所示，轻链恒定区为氨基酸序列如SEQ ID No:42所示的人的C k链。
实施例12人源化抗体分子的制备
本发明实施例12在实施例11的基础上进一步的限定了人源化抗体分子的重链恒定区为人的IgG4型，IgG4型的重链恒定区氨基酸序列如SEQ ID No:41所示；轻链恒定区为氨基酸序列如SEQ ID No:42所示的人的C k链。
将上述实施例10人源化得到的5个人源化抗体分子的重链VH和轻链VL的编码基因分别克隆至装有重链恒定区和轻链恒定区基因的载体pTSE(如图3所示)，重链恒定区为人的IgG4型(氨基酸序列如SEQ ID NO:41所示)，轻链恒定区为C k链(氨基酸序列如SEQ ID NO:42所示)。
将嵌合抗体CA-1和人源化抗体瞬时转染HEK293E细胞(购自中国医学科学院基础医学研究所，货号为GNHu43)，进行抗体表达，使用AKTA仪器通过protein A亲和柱纯化获得单克隆抗体，同时使用BCA试剂盒(购买自：北京汇天东方科技有限公司，货号：BCA0020)进行蛋白浓度测定，之后通过SDS-PAGE鉴定蛋白大小，结果如图7所示，从左侧到右侧依次为非还原蛋白质分子量Marker1、HA-1、HA-2、HA-3、HA-4、HA-5、实施例9中制备的嵌合抗体CA-1及核心专利CN105992595A提供的抗LAG-3单克隆抗体和还原蛋白质分子量Marker2、HA-1、HA-2、HA-3、HA-4、HA-5、嵌合抗体CA-1及核心专利CN105992595A提供的抗LAG-3单克隆抗体，每条带的分子量大小与理论一致。
实施例13
本发明实施例13在上述实施例的基础上进一步的限定了人源化抗体分子为全长抗体或抗体片段，人源化抗体分子包括Fab、F(ab)2、Fv或ScFv中的一种或几种组合。
实施例14
本发明实施例14在上述实施例的基础上进一步的限定了如下方案：
进一步限定了一种多肽或蛋白，多肽或所述蛋白包含上述任意一个实施例限定的抗LAG-3的单克隆抗体或其抗原结合片段。
进一步的限定了一种多核苷酸序列或组合，多核苷酸序列或组合编码上述任意一个实施例限定的抗LAG-3的单克隆抗体或其抗原结合片段的氨基酸序列。
进一步的限定了一种重组DNA表达载体，重组DNA表达载体包含上述的多核苷酸序列或组合。
进一步的限定了一种转染上述限定的重组DNA表达载体的宿主细胞，宿主细胞包括原核细胞、酵母细胞、昆虫细胞或哺乳动物细胞；
优选的，宿主细胞为哺乳动物细胞，哺乳动物细胞为HEK293E细胞、CHO细胞或NS0细胞。
进一步的限定了一种药物或药物组合物，药物或药物组合物包含上述任意一个实施例限定的抗LAG-3的单克隆抗体或其抗原结合片段。
本发明进一步的提供了抗LAG-3的单克隆抗体或其抗原结合片段在制备治疗癌症或免疫疾病药物中的应用；
优选的，癌症包括但不限于白血病、肺癌、胃癌、食道癌、卵巢癌、头颈癌、黑色素瘤、肾癌、乳腺癌、结直肠癌、肝癌、胰腺癌或膀胱癌；免疫疾病包括但不限于银屑病、克罗恩病、类风湿性关节炎、原发性胆汁性肝硬化、系统性红斑狼疮、多发性硬化症、溃疡性结肠炎和自身免疫性肝炎。
实施例15人源化抗体分子与LAG-3结合实验
用pH9.6的碳酸盐缓冲液包被LAG-3，100ng/孔/100μl，在4℃的温度条件下过夜包被。用300μl/孔PBST洗涤五次，再加入1％BSA-PBS在37℃温度条件下封闭2h，加入不同稀释浓度的人源化抗体HA-1，HA-2，HA-3，HA-4，HA-5和实施例9中制备的嵌合抗体CA-1及专利CN105992595A中的抗LAG-3抗体，7个抗体的起始最高浓度均是5μg/ml，分别经过5倍稀释后每个抗体均做8个梯度，在37℃温度条件下孵育1h。用300μl/孔PBST洗涤五次，再加入用1％BSA-PBS 1:10000稀释的Anti-Human Fc-HRP，在37℃温度条件下孵育1h。TMB显色试剂盒显色，100μl/孔，室温显色8min，然后用2M H 2SO 4终止显色。在450nm/630nm 下读数，并计算对应的EC50值，具体数据如下：
克隆 HA-1 HA-2 HA-3 HA-4 HA-5 嵌合抗体CA-1 CN1059925A
EC50(ng/ml) 46.25 150.7 102.5 538.8 350.3 52.16 747.2
通过上述数据及实验结果如图8所示，5个不同的人源化抗体分子均能与LAG-3进行结合，本发明提供的5个不同的单克隆抗体的EC50值均明显低于专利CN105992595A中的抗LAG-3抗体，说明本发明提供的单克隆抗体与LAG-3的结合能力强，亲和力高，此外，从图8及上述数据中还可以得出，5个不同的单克隆抗体中HA-1的EC50值最低，说明其与LAG-3结合能力最好，亲和力最高；同时HA-1的EC50值与嵌合抗体CA-1相似，说明人源化后的HA-1保留了鼠源亲本抗体MA-1与LAG-3的高亲和力，亲和力没有下降。
实施例16混合淋巴细胞反应(MLR)测试抗LAG-3人源化抗体分子活性
按密度梯度离心法分离新鲜外周血PBMC，磁珠分选CD14 +T细胞；用20ng/ml GM-CSF与10ng/ml IL-4的培养基培养CD14 +T细胞，每两天换液，7-10天诱导成为树突状DC细胞。在DC使用前两天，加入25ng/ml的TNF-α诱导DC为成熟的DC细胞，收集成熟的DC细胞，配制成细胞密度为1×10 5个/ml细胞悬液。从新鲜PMBC中磁珠分选出CD14 +T细胞，计数，制成细胞密度为1×10 6个/ml细胞悬液。将CD14 +T细胞与DC细胞各取100μL，按比例10:1加入96孔板。
将实施例12制备的5个抗LAG-3人源化抗体分子、实施例9制备的嵌合抗体CA-1及专利CN105992595A提供的抗LAG-3抗体作为阳性对照，分别进行4倍梯度稀释，每个抗体均设置8个梯度，各取50μL加入到96孔板中，5天后，CCK8测试CD14 +T细胞增殖，在450nm/630nm下读数，并计算对应的EC50值，具体数据如下：
克隆 HA-1 HA-2 HA-3 HA-4 HA-5 嵌合抗体CA-1 CN105992595A
EC50(ng/ml) 6.875 27.09 15.01 33.8 31.84 7.226 39.59
通过上述数据及如图9所示，本发明筛选出的5个不同的抗LAG-3抗体的EC50值均明显低于专利CN105992595A中提供的抗LAG-3的抗体，说明本发明提供的抗LAG-3抗体的活性均比较高，同时本发明筛选出的5个单克隆抗体中，抗LAG-3全抗体HA-1的EC50值最低，说明其活性最高，此外，从图9还可以看出，嵌合抗体CA-1的亲和力与抗LAG-3抗体HA-1的EC50值比较接近，说明人源化抗体HA-1保留了鼠源亲本抗体MA-1的生物活性，其生物活性没有下降。
实施例17抗LAG-3单克隆抗体HA-1对小鼠体内MC38结直肠癌的抑制试验
1、实验动物：
种属品系：Mus Musculus，NCG，小鼠；
周龄：6-8周；
实验动物提供商:江苏集萃药康生物科技有限公司。
2、细胞培养，MC38肿瘤细胞(购自：普如汀生物技术(北京)有限公司(Biovector NTCC Inc.)，货号：NTCC-MC38)。用含有灭活的10％胎牛血清(ExCell Bio,货号：FND500)，100U/ml的青霉素和100μg/ml的链霉素以及2mM谷氨酰胺的DMEM培养基(购自：赛默飞世尔科技(中国)有限公司(Gibco)，货号：10566-016)在37℃、5％CO 2的培养箱中培养肿瘤细胞，每隔3至4天待细胞长满后分瓶传代，将处于对数生长期的肿瘤细胞用于体内肿瘤的接种。
3、肿瘤细胞的接种与分组：PBS重悬的MC38肿瘤细胞，浓度为1.0×10 7/ml，接种于实验动物的右侧胁肋部皮下，100μL/只，在肿瘤生长至61mm 3左右时分组给药，共3组，每组8只，分别为溶媒对照组、HA-1(10mg/kg，i.p.，biw×3w)、HA-1(30mg/kg，i.p.，biw×3w)。
4、检测指标：每周使用游标卡尺对肿瘤体积进行2次的测量，测量肿瘤的长径和短径，其体积计算公式为：体积＝0.5×长径×短径 2；记录肿瘤体积的变化与给药时间的关系，实验结果如图10所示。
通过图10数据显示，抗LAG-3单克隆抗体HA-1能够抑制肿瘤的生长，且呈现剂量依赖性反应。
实施例18抗LAG-3单克隆抗体HA-1蛋白分子热稳定性评估
将抗LAG-3单克隆抗体HA-1蛋白分子超滤换液到PBS缓冲体系中，12000rpm，在4℃条件下，离心5min，使用多功能蛋白热稳定性分析系统(购买自Unchained Labs)对抗LAG-3单克隆抗体HA-1蛋白分子的热稳定性进行评估。通过监测蛋白内源性荧光随温度变化(从25℃开始，以0.3℃/min的升温速度升温至95℃)检测蛋白构象的变化，从而确定蛋白熔解温度Tm，评估蛋白构象稳定性。样品发生聚集时，会导致散射光波发生干涉，散射光信号增加，通过静态光散射测定蛋白的胶体稳定性(以Tagg进行表征)，结果参加如下表和附图11所示。
Figure PCTCN2021133941-appb-000026
如上表和图11显示，抗LAG-3单克隆抗体HA-1蛋白分子的温度为66.6℃，平均Tagg 为68.0℃，显示出较好的构象稳定性和胶体稳定性。
实施例19
本发明实施例19在上述实施例的基础上进一步的限定了如下方案：
本发明还提供了抗LAG-3的单克隆抗体或其抗原结合片段与抗PD-1单克隆抗体联合用于制备治疗癌症或免疫疾病药物中的应用。
进一步的，抗PD-1单克隆抗体选自DFPD1-9、DFPD1-10、DFPD1-11、DFPD1-12、DFPD1-13、Nivolumab、Pembrolizumab、特瑞普利单抗、信迪利单抗、替雷利珠单抗、卡瑞利珠单抗、派安普利单抗或赛帕利单抗。
其中，Nivolumab、Pembrolizumab、特瑞普利单抗、信迪利单抗、替雷利珠单抗、卡瑞利珠单抗、派安普利单抗或赛帕利单抗均为已上市产品。
其中，DFPD1-9、DFPD1-10、DFPD1-11、DFPD1-12、DFPD1-13为申请号为CN201510312910.8提供的抗PD-1的单克隆抗体，DFPD1-9包括如SEQ ID No:44所示的轻链可变区和如SEQ ID No:43所示的重链可变区；DFPD1-10包括如SEQ ID No:45所示的轻链可变区和如SEQ ID No:43所示的重链可变区；DFPD1-11包括如SEQ ID No:44所示的轻链可变区和如SEQ ID No:46所示的重链可变区；DFPD1-12包括如SEQ ID No:44所示的轻链可变区和如SEQ ID No:47所示的重链可变区；DFPD1-13包括如SEQ ID No:45所示的轻链可变区和如SEQ ID No:46所示的重链可变区，具体序列如下：
SEQ ID No:43，序列如下：
Figure PCTCN2021133941-appb-000027
SEQ ID No:44，序列如下：
Figure PCTCN2021133941-appb-000028
SEQ ID No:45，序列如下：
Figure PCTCN2021133941-appb-000029
SEQ ID No:46，序列如下：
Figure PCTCN2021133941-appb-000030
SEQ ID No:47，序列如下：
Figure PCTCN2021133941-appb-000031
此外，抗PD-1单克隆抗体还包括重链恒定区和轻链恒定区，重链恒定区和轻链恒定区序列与专利申请号为CN201510312910.8提供的抗PD-1的单克隆抗体的重链恒定区和轻链恒定区相同。
进一步的，癌症选自白血病、肺癌、胃癌、食道癌、卵巢癌、头颈癌、黑色素瘤、肾癌、乳腺癌、结直肠癌、肝癌、胰腺癌或膀胱癌；免疫疾病包括银屑病、克罗恩病、类风湿性关节炎、原发性胆汁性肝硬化、系统性红斑狼疮、多发性硬化症、溃疡性结肠炎和自身免疫性肝炎。
实施例20
本发明实施例20在实施例19的基础上，进一步的限定了抗PD-1单克隆抗体为DFPD1-10，DFPD1-10包括如SEQ ID No:45所示的轻链可变区和如SEQ ID No:43所示的重链可变区。
实施例21
本发明实施例21在实施例19的基础上，进一步的限定了抗PD-1单克隆抗体为Nivolumab。
实施例22抗LAG-3单克隆抗体与抗PD-1单克隆抗体联合用于MC38结直肠癌模型的体内药效实验
1、供试药物
(1)上述实施例10提供的抗LAG-3的单克隆抗体HA-1，编号为JY03；
提供单位：北京东方百泰生物科技股份有限公司；
数量：11mL/支，4.9mg/mL，共7支；
(2)实施例20提供的抗PD-1单克隆抗体为DFPD1-10，实验中编号为JY034；
提供单位：北京东方百泰生物科技股份有限公司；
数量：10ml，10mg/ml，3瓶，共300mg；
(3)Opdivo(Nivolumab)
厂家：百时美施贵宝
数量：100mg/10ml，1支
2、实验动物
种属品系：MusMusculus，B6/JGpt-Pdcd1em1Cin(hPDCD1)Lag3em1Cin(hLAG3)/Gpt
性别：雌性
周龄：6-8周
体重：18-22g
数量：75只
实验动物提供商：江苏集萃药康生物科技股份有限公司
生产许可证号：SCXK(苏)2018-0008
3、实验方法
(1)细胞培养
MC38肿瘤细胞用含有灭活的10％胎牛血清，100U/mL的青霉素和100μg/mL的链霉素以及2mM谷氨酰胺的DMEM培养基在37℃、5％CO2的培养箱中培养，每隔3至4天待细胞长满后分瓶传代，将处于对数生长期的肿瘤细胞用于体内肿瘤的接种。
(2)肿瘤细胞接种与分组
将PBS重悬的MC38肿瘤细胞，浓度为1×10 7/mL，接种于实验动物的右侧胁肋部皮下，1×10 6/100μL/只，在肿瘤生长至50mm 3左右时分组给药(当天记为PG-D0)，共8组，每组10只。
(3)小鼠体重的测量及实验指标
每周使用游标卡尺对肿瘤体积进行2-3次测量并用电子天平称量小鼠体重，测量肿瘤的长径和短径，其体积计算公式为：体积＝0.5×长径×短径 2。根据肿瘤体积计算T/C值，其中T为各受试物处理组相对肿瘤体积(RTV)的平均值，C为对照组相对肿瘤体积(RTV)的平均值，RTV为给药后与给药前的肿瘤体积比值。肿瘤生长抑制率TGITV(％)＝(1－T/C)×100％。
实验结束时，动物安乐死，剥离肿瘤称重、摆放整齐拍照，并计算瘤重抑制率TGITW(％)，公式为(1－T/C)×100％，其中T/C＝治疗组TW平均值/对照组TW平均值。
原则上，评价标准为：T/C(％)>40％为无效；T/C(％)≤40％，并经统计学处理P<0.05为有效。
4、给药方案
Figure PCTCN2021133941-appb-000032
Figure PCTCN2021133941-appb-000033
注：*各组给药容积依动物体重按10μL/g，出现体重下降15-20％时可调节给药量；i.p.：腹腔注射；biw x 3wks：每周给药两次，给药三周，共6次；#：为JY03第二次给药时开始给药，实际给药次数为三周共5次。
5、统计学分析
应用IBM SPSS Statistics 22.0统计学软件，应用One-Way ANOVA检验对肿瘤体积、肿瘤重量进行组间统计学分析，p<0.05认为有显著性差异。
6、数据结果如下：
(1)肿瘤生长抑制结果
Figure PCTCN2021133941-appb-000034
注：各组进行One-Way ANOVA单因素方差分析并采用LSD(least significant difference)进行事后多重比较； a .均数±标准误； b .与Vehicle组比较； c .与Opdivo组比较； d .与JY03组比较； e .与JY03+JY034组比较； f .与JY03+Opdivo组比较。JY034治疗组间比较均无统计学差异(p>0.05)。其中JY034组、Opdivo组、JY03+JY034组和JY03+Opdivo组分别有3只、5只、1只、4只小鼠肿瘤完全消退。
(2)肿瘤重量
Figure PCTCN2021133941-appb-000035
注：各组进行One-Way ANOVA单因素方差分析并采用LSD(least significant difference)进行事后多重比较； a .均数±标准误； b .与Vehicle组比较； c .与Opdivo组比较； d .与JY03组比较； e .与JY03+JY034组比较； f .与JY03+Opdivo组比较。
综上，通过上述数据及图12和13所示，受试药物JY03组、JY034组、Opdivo组、JY03+JY034组和JY03+Opdivo组对PD-1和LAG3双靶点人源化结直肠癌MC38模型的治疗均产生了明确的抗肿瘤作用，其中，JY03+JY034组对肿瘤的抑制效果明显强于单药JY03组和单药JY034组，JY03+Opdivo组对肿瘤的抑制效果明显强于单药JY03组和单药Opdivo组，为此可以说明，抗LAG-3单克隆抗体能够提高抗PD-1单克隆抗体对肿瘤的抑制作用；此外各组中动物耐受性良好，未见明显不良反应。
本发明不局限于上述最佳实施方式，任何人在本发明的启示下都可得出其他各种形式的产品，但不论在其形状或结构上作任何变化，凡是具有与本申请相同或相近似的技术方案，均落在本发明的保护范围之内。

Claims (18)

  1. 一种抗LAG-3的单克隆抗体或其抗原结合片段，其特征在于，包括重链可变区和轻链可变区，所述重链可变区包括3个重链互补决定区，3个所述重链互补决定区分别用HCDR1、HCDR2和HCDR3表示，所述轻链可变区包括3个轻链互补决定区，3个所述轻链互补决定区分别用LCDR1、LCDR2和LCDR3表示，所述单克隆抗体或其抗原结合片段选自以下任意一种：
    A-1:所述重链互补决定区HCDR1包含如SEQ ID No:1所示的氨基酸序列，所述重链互补决定区HCDR2包含如SEQ ID No:2所示的氨基酸序列，所述重链互补决定区HCDR3包含如SEQ ID No:3所示的氨基酸序列，所述轻链互补决定区LCDR1包含如SEQ ID No:4所示的氨基酸序列，所述轻链互补决定区LCDR2包含如SEQ ID No:5所示的氨基酸序列，所述轻链互补决定区LCDR3包含如SEQ ID No:6所示的氨基酸序列；
    A-2：所述重链互补决定区HCDR1包含如SEQ ID No:1所示的氨基酸序列，所述重链互补决定区HCDR2包含如SEQ ID No:2所示的氨基酸序列，所述重链互补决定区HCDR3包含如SEQ ID No:3所示的氨基酸序列，所述轻链互补决定区LCDR1包含如SEQ ID No:4所示的氨基酸序列，所述轻链互补决定区LCDR2包含如SEQ ID No:7所示的氨基酸序列，所述轻链互补决定区LCDR3包含如SEQ ID No:8所示的氨基酸序列；
    A-3：所述重链互补决定区HCDR1包含如SEQ ID No:9所示的氨基酸序列，所述重链互补决定区HCDR2包含如SEQ ID No:10所示的氨基酸序列，所述重链互补决定区HCDR3包含如SEQ ID No:11所示的氨基酸序列，所述轻链互补决定区LCDR1包含如SEQ ID No:12所示的氨基酸序列，所述轻链互补决定区LCDR2包含如SEQ ID No:13所示的氨基酸序列，所述轻链互补决定区LCDR3包含如SEQ ID No:14所示的氨基酸序列；
    A-4：所述重链互补决定区HCDR1包含如SEQ ID No:15所示的氨基酸序列，所述重链互补决定区HCDR2包含如SEQ ID No:16所示的氨基酸序列，所述重链互补决定区HCDR3包含如SEQ ID No:17所示的氨基酸序列，所述轻链互补决定区LCDR1包含如SEQ ID No:18所示的氨基酸序列，所述轻链互补决定区LCDR2包含如SEQ ID No:19所示的氨基酸序列，所述轻链互补决定区LCDR3包含如SEQ ID No:20所示的氨基酸序列。
  2. 如权利要求1所述的抗LAG-3的单克隆抗体或其抗原结合片段，其特征在于，所述单克隆抗体或其抗原结合片段为鼠源抗体分子，所述鼠源抗体分子选自以下任意一种：
    MA-1：所述重链可变区包含如SEQ ID No:21所示的氨基酸序列，所述轻链可变区包含如SEQ ID No:22所示的氨基酸序列；
    MA-2：所述重链可变区包含如SEQ ID No:21所示的氨基酸序列，所述轻链可变区包含如SEQ ID No:23所示的氨基酸序列；
    MA-3：所述重链可变区包含如SEQ ID No:24所示的氨基酸序列，所述轻链可变区包含如SEQ ID No:25所示的氨基酸序列；
    MA-4：所述重链可变区包含如SEQ ID No:26所示的氨基酸序列，所述轻链可变区包含如SEQ ID No:27所示的氨基酸序列；
    优选的，所述鼠源抗体分子为MA-1。
  3. 如权利要求2所述的抗LAG-3的单克隆抗体或其抗原结合片段，其特征在于，所述鼠源抗体分子还包括重链恒定区和轻链恒定区，所述重链恒定区为鼠的IgG1型、IgG2a型、IgG2b型、IgG3型中的一种，所述IgG1型的重链恒定区氨基酸序列如SEQ ID No:29所示，所述IgG2a型的重链恒定区氨基酸序列如SEQ ID No:30所示，所述IgG2b型的重链恒定区氨基酸序列如SEQ ID No:31所示，所述IgG3型的重链恒定区氨基酸序列如SEQ ID No:32所示；所述轻链恒定区为氨基酸序列如SEQ ID No:28所示的鼠源C k链；
    优选的，所述重链恒定区为鼠的IgG1型。
  4. 如权利要求2所述的抗LAG-3的单克隆抗体或其抗原结合片段，其特征在于，所述单克隆抗体或其抗原结合片段为嵌合抗体分子，所述嵌合抗体分子包括所述鼠源抗体分子的重链可变区、所述鼠源抗体分子的轻链可变区和人源抗体恒定区；所述人源抗体恒定区包括人源抗体重链恒定区和人源抗体轻链恒定区，所述人源抗体重链恒定区为人的IgG1型、IgG2型或IgG4型中的一种，所述IgG1型的重链恒定区氨基酸序列如SEQ ID No:39所示，所述IgG2型的重链恒定区氨基酸序列如SEQ ID No:40所示，所述IgG4型的重链恒定区氨基酸序列如SEQ ID No:41所示；所述人源抗体轻链恒定区为氨基酸序列如SEQ ID No:42所示的人的C k链；
    优选的，所述人源抗体重链恒定区为人的IgG4型。
  5. 如权利要求1所述的抗LAG-3的单克隆抗体或其抗原结合片段，其特征在于，所述单克隆抗体或其抗原结合片段为人源化抗体分子，所述人源化抗体分子选自以下任意一种：
    HA-1：所述重链可变区包含如SEQ ID No:33所示的氨基酸序列，所述轻链可变区包含如SEQ ID No:34所示的氨基酸序列；
    HA-2：所述重链可变区包含如SEQ ID No:33所示的氨基酸序列，所述轻链可变区包含如SEQ ID No:35所示的氨基酸序列；
    HA-3：所述重链可变区包含如SEQ ID No:36所示的氨基酸序列，所述轻链可变区包含如SEQ ID No:35所示的氨基酸序列；
    HA-4：所述重链可变区包含如SEQ ID No:36所示的氨基酸序列，所述轻链可变区包含如SEQ ID No:34所示的氨基酸序列；
    HA-5：所述重链可变区包含如SEQ ID No:37所示的氨基酸序列，所述轻链可变区包含如SEQ ID No:38所示的氨基酸序列；
    优选的，所述人源化抗体分子为HA-1。
  6. 如权利要求5所述的抗LAG-3的单克隆抗体或其抗原结合片段，其特征在于，所述人源化抗体分子还包括重链恒定区和轻链恒定区，所述重链恒定区为人的IgG1型、IgG2型或IgG4型中的一种，所述IgG1型的重链恒定区氨基酸序列如SEQ ID No:39所示，所述IgG2型的重链恒定区氨基酸序列如SEQ ID No:40所示，所述IgG4型的重链恒定区氨基酸序列如SEQ ID No:41所示，所述轻链恒定区为氨基酸序列如SEQ ID No:42所示的人的C k链；
    优选的，所述重链恒定区为人的IgG4型。
  7. 如权利要求5所述的抗LAG-3的单克隆抗体或其抗原结合片段，其特征在于，所述人源化抗体分子为全长抗体或抗体片段，所述人源化抗体分子包括Fab、F(ab)2、Fv或ScFv中的一种或几种组合。
  8. 一种多肽或蛋白，其特征在于，所述多肽或所述蛋白包含权利要求1-7任一项所述的抗LAG-3的单克隆抗体或其抗原结合片段。
  9. 一种多核苷酸序列或组合，其特征在于，所述多核苷酸序列或组合编码权利要求1-7任一项所述的抗LAG-3的单克隆抗体或其抗原结合片段的氨基酸序列。
  10. 一种重组DNA表达载体，其特征在于，所述重组DNA表达载体包含权利要求9所述的多核苷酸序列或组合。
  11. 一种转染如权利要求10所述的重组DNA表达载体的宿主细胞，其特征在于，所述宿主细胞包括原核细胞、酵母细胞、昆虫细胞或哺乳动物细胞；
    优选的，所述宿主细胞为哺乳动物细胞，所述哺乳动物细胞为HEK293E细胞、CHO细胞或NS0细胞。
  12. 一种药物或药物组合物，其特征在于，所述药物或所述药物组合物包含权利要求1-7任一项所述的抗LAG-3的单克隆抗体或其抗原结合片段。
  13. 权利要求1-7任一项所述的抗LAG-3的单克隆抗体或其抗原结合片段在制备治疗癌症或免疫疾病药物中的应用；
    优选的，所述癌症包括白血病、肺癌、胃癌、食道癌、卵巢癌、头颈癌、黑色素瘤、肾癌、乳腺癌、结直肠癌、肝癌、胰腺癌或膀胱癌；
    所述免疫疾病包括银屑病、克罗恩病、类风湿性关节炎、原发性胆汁性肝硬化、系统性红斑狼疮、多发性硬化症、溃疡性结肠炎和自身免疫性肝炎。
  14. 如权利要求1-7任一项所述的抗LAG-3的单克隆抗体或其抗原结合片段与抗PD-1单克隆抗体联合用于制备治疗癌症或免疫疾病药物中的应用。
  15. 根据权利要求14所述的应用，其特征在于，所述抗PD-1单克隆抗体选自DFPD1-9、DFPD1-10、DFPD1-11、DFPD1-12、DFPD1-13、Nivolumab、Pembrolizumab、特瑞普利单抗、信迪利单抗、替雷利珠单抗、卡瑞利珠单抗、派安普利单抗或赛帕利单抗。
  16. 根据权利要求15所述的应用，其特征在于，所述抗PD-1单克隆抗体为DFPD1-10，所述DFPD1-10包括如SEQ ID No:45所示的轻链可变区和如SEQ ID No:43所示的重链可变区。
  17. 根据权利要求15所述的应用，其特征在于，所述抗PD-1单克隆抗体为Nivolumab。
  18. 根据权利要求14所述的应用，其特征在于，所述癌症选自白血病、肺癌、胃癌、食道癌、卵巢癌、头颈癌、黑色素瘤、肾癌、乳腺癌、结直肠癌、肝癌、胰腺癌或膀胱癌；所述免疫疾病包括银屑病、克罗恩病、类风湿性关节炎、原发性胆汁性肝硬化、系统性红斑狼疮、多发性硬化症、溃疡性结肠炎和自身免疫性肝炎。
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