Lag-3 binding molecules and uses thereof

Abstract

The application discloses a LAG-3 binding molecule, which comprises a LAG-3 antibody, or an antigen binding fragment of the LAG-3 antibody, or a fusion protein containing the LAG-3 antibody, or a fusion protein containing the antigen binding fragment, or an antibody drug conjugate containing the LAG-3 antibody, or an antibody drug conjugate containing the antigen binding fragment, or a bispecific antibody containing the LAG-3 antibody or a bispecific antibody containing the antigen binding fragment. The LAG-3 binding molecule provided by the application can bind to human and monkey LAG-3, has high affinity to human LAG-3, effectively enhances T cell response, can be used for regulating T cell and antibody-mediated immune response, and has wide therapeutic uses as an immunomodulator, such as cancer, autoimmune diseases, inflammatory diseases, and infectious diseases.

Description

Technical Field

[0001] This invention relates to the field of antibody engineering, and particularly to LAG-3 binding molecules and their applications. Background Technology

[0002] Lymphocyte activation gene 3 (LAG-3), also known as CD223, is a transmembrane protein composed of three parts: an extracellular region, a transmembrane region, and an intracellular region. The extracellular region consists of four parts: Domain1, Domain2, Domain3, and Domain4. Human LAG-3 has 498 amino acids, a relative molecular weight of 70 kDa, and is located on human chromosome 12 (20p13.3). LAG-3 was initially identified as being expressed on the surface of T cells (especially activated T cells), natural killer cells, B cells, and plasmacytoid dendritic cells. LAG-3 is an inhibitory receptor that forms a dimer molecule through its D1 domain and specifically binds to MHC class II molecules on the surface of antigen-presenting cells (APCs). Through signal transduction of the highly conserved KIEELE sequence in its cytoplasmic region, it negatively regulates T cell proliferation and controls the memory T cell pool. LAG-3 also has a direct regulatory effect on the inhibitory function of CD4+ and CD25+ regulatory T cells (Treg cells) and is an essential molecule for Treg cells to perform their functions.

[0003] In addition to MHC class II molecules, LAG-3 has also been reported to bind to three other ligands: fibrinogen 1 (FGL-1), hepatic sinusoidal endothelial cell lectin (LSECtin), and galactose-binding lectin-3 (Galectin-3). FGL-1 is secreted by hepatocytes, and studies have shown that when FGL-1 binds to LAG-3 on the surface of T cells, T cell proliferation is inhibited and immune activity is affected.

[0004] Furthermore, LAG-3 plays a crucial role in maintaining immune homeostasis, and its expression level has significant marker effects in both autoimmune diseases and cancer. LAG-3 has a protective effect in autoimmune diseases, and blocking or deletion of the LAG-3 gene accelerates the progression of these diseases. In cancer and chronic viral infections, upregulated LAG-3 expression suppresses T-cell immune function; in cancer, LAG-3 typically marks dysfunctional or exhausted T cells. Moreover, LAG-3 is highly expressed in animal T cells resistant to PD-1 antibodies. Studies have shown that in preclinical solid tumor and hematologic malignancy models, simultaneously blocking both the LAG-3 and PD-1 pathways improves antitumor effector function and inhibits tumor growth more effectively than blocking PD-1 alone. Therefore, the combined use of LAG-3 antibodies and PD-1 antibodies can mitigate the resistance issues arising from PD-1 antibody use.

[0005] Currently, there are no LAG-3 inhibitor drugs on the market. The most advanced LAG-3 inhibitors are in clinical trials or recruiting participants as anti-cancer treatments. Examples include BMS-986016, a fully human anti-LAG-3 IgG4 monoclonal antibody developed by Bristol-Myers Squibb, and MK-4280, an anti-LAG-3 fully human IgG4 monoclonal antibody developed by Merck Sharp & Dohme. These are primarily used in combination with anti-PD-1 / PD-L1 antibodies to treat various solid tumors and hematological malignancies. While LAG-3 plays an important clinical role in tumor immunotherapy as a novel immunotherapy target, there is still no clear evidence of immunotherapy benefits for many malignant tumors. The application scope and efficacy of anti-LAG-3 antibody immunotherapy still require significant research. Summary of the Invention

[0006] The purpose of this invention is to provide a LAG-3 binding molecule so that more cancer patients can benefit from treatment.

[0007] In a first aspect, the present invention provides a LAG-3 binding molecule, said LAG-3 binding molecule comprising a LAG-3 antibody, or an antigen-binding fragment of said LAG-3 antibody, or a fusion protein containing said LAG-3 antibody, or a fusion protein containing said antigen-binding fragment, or an antibody-drug conjugate containing said LAG-3 antibody, or an antibody-drug conjugate containing said antigen-binding fragment, or a bispecific antibody containing said LAG-3 antibody, or a bispecific antibody containing said antigen-binding fragment, wherein said LAG-3 binding molecule contains a heavy chain variable region and a light chain variable region, said heavy chain variable region and light chain variable region comprising the CDR region sequence described below (13H4) or (3F6).

[0008] The amino acid sequences of HCDR1 in the heavy chain variable region (13H4) are shown as positions 26-35 of SEQ ID No. 1, the amino acid sequences of HCDR2 are shown as positions 50-66 of SEQ ID No. 1, and the amino acid sequences of HCDR3 are shown as positions 99-109 of SEQ ID No. 1 or SEQ ID No. 6; the amino acid sequences of LCDR1 in the light chain variable region are shown as positions 24-34 of SEQ ID No. 3, the amino acid sequences of LCDR2 are shown as positions 50-56 of SEQ ID No. 3, and the amino acid sequences of LCDR3 are shown as positions 89-96 of SEQ ID No. 3, SEQ ID No. 5, or SEQ ID No. 7.

[0009] (3F6) The amino acid sequence of HCDR1 in the heavy chain variable region is shown as positions 26-35 of SEQ ID No. 8, the amino acid sequence of HCDR2 is shown as positions 50-66 of SEQ ID No. 8 or positions 50-66 of SEQ ID No. 12, and the amino acid sequence of HCDR3 is shown as positions 97-105 of SEQ ID No. 8; the amino acid sequence of LCDR1 in the light chain variable region is shown as positions 24-34 of SEQ ID No. 10, the amino acid sequence of LCDR2 is shown as positions 50-56 of SEQ ID No. 10, and the amino acid sequence of LCDR3 is shown as positions 89-97 of SEQ ID No. 10.

[0010] The CDR described in this invention stands for "complementarity-determining region," which is a region within the antibody variable domain that is highly variable in sequence and forms a structurally defined "hypervariant loop" and / or contains antigen contact residues, or "antigen contact sites." The CDR is primarily responsible for binding to antigen epitopes. The CDRs of the heavy and light chains are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus. CDRs located within the antibody heavy chain variable domain are referred to as HCDR1, HCDR2, and HCDR3, while CDRs located within the antibody light chain variable domain are referred to as LCDR1, LCDR2, and LCDR3.

[0011] Further, the amino acid sequence of the heavy chain variable region of the aforementioned LAG-3 binding molecule (13H4) is as shown in positions 1-120 of SEQ ID No. 1 or SEQ ID No. 6, or has at least 80% identity with the amino acid sequence shown in positions 1-120 of SEQ ID No. 1 or SEQ ID No. 6 (the inconsistency is preferably in the FR region); the amino acid sequence of the light chain variable region is as shown in positions 1-106 of SEQ ID No. 3 or SEQ ID No. 5 or SEQ ID No. 7, or has at least 80% identity with the amino acid sequence shown in positions 1-106 of SEQ ID No. 3 or SEQ ID No. 5 or SEQ ID No. 7 (the inconsistency is preferably in the FR region);

[0012] The amino acid sequence of the heavy chain variable region is as shown in positions 1-116 of SEQ ID No. 8 or SEQ ID No. 12, or has at least 80% identity with the amino acid sequence shown in positions 1-116 of SEQ ID No. 8 or SEQ ID No. 12 (the inconsistency is preferably in the FR region); the amino acid sequence of the light chain variable region is as shown in positions 1-107 of SEQ ID No. 10, or has at least 80% identity with the amino acid sequence shown in positions 1-107 of SEQ ID No. 10 (the inconsistency is preferably in the FR region).

[0013] Furthermore, in the aforementioned LAG-3 binding molecule, the LAG-3 antibody further includes a heavy chain constant region and a light chain constant region. The heavy chain constant region can be any one of IgG, IgM, IgE, IgA, or IgD; the light chain type of the antibody can be a κ chain or a λ chain.

[0014] The IgG may be any one of IgG1, IgG2, IgG3 and IgG4 or a mutant thereof.

[0015] The IgG1 may be mouse IgG1, human IgG1, or a mutant thereof.

[0016] The mutant of human IgG1 may be an IgG1 mutant with mutation type L234A and / or L235A (Kabat count) mutation.

[0017] Specifically, the amino acid sequence of the heavy chain constant region is shown as positions 121-450 of SEQ ID No. 1.

[0018] The light chain type of the antibody described in this invention may be a κ chain.

[0019] Specifically, the amino acid sequence of the light chain constant region is shown in positions 107-213 of SEQ ID No. 3.

[0020] Further, in the aforementioned LAG-3 binding molecule, the amino acid sequence of the heavy chain of the LAG-3 antibody is as shown in SEQ ID No. 1 or has at least 80% identity with the amino acid sequence shown in SEQ ID No. 1, and the amino acid sequence of the light chain is as shown in SEQ ID No. 3 or has at least 80% identity with the amino acid sequence shown in SEQ ID No. 3; or,

[0021] The amino acid sequence of the heavy chain is as shown in SEQ ID No. 8 or has at least 80% identity with the amino acid sequence shown in SEQ ID No. 8, and the amino acid sequence of the light chain is as shown in SEQ ID No. 10 or has at least 80% identity with the amino acid sequence shown in SEQ ID No. 10.

[0022] In the above applications, identity refers to the similarity of amino acid sequences or nucleotide sequences. The identity of amino acid sequences can be determined using homology search sites on the internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, using blastp as the program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, setting the Gap existence cost, Per residue gap cost, and Lambdaratio to 11, 1, and 0.85 (default values) respectively, and performing an identity search on a pair of amino acid sequences, the identity value (%) can then be obtained.

[0023] In the above applications, the 80% or more of identity can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%.

[0024] The antibody provided by this invention has one or more of the following properties or characteristics:

[0025] 1) Specifically binds to human LAG-3;

[0026] 2) Can be combined with cynomolgus monkey LAG-3;

[0027] 3) It binds weakly to mouse LAG-3;

[0028] 4) To a certain extent, it can block the binding of ligands such as MHC II molecules and FGL1 to human LAG-3;

[0029] 5) It is IgG, such as IgG1, IgG2, IgG3 or IgG4, or a mutant of the above IgG;

[0030] 6) It is a humanized antibody, chimeric antibody, or murine antibody.

[0031] 7) It mainly binds to Domain1 in the extracellular region of human LAG-3.

[0032] The antigen-binding fragments in the LAG-3 binding molecule mentioned above include one or more combinations of Fab, Fab', F(ab')2, Fab'-SH, Fv, and ScFv.

[0033] The Fab fragment is obtained by digesting the antibody with papain. Fab includes a variable domain of the heavy chain and a variable domain of the light chain, as well as a constant domain of the light chain and a first constant domain (CH1) of the heavy chain.

[0034] The Fab' fragment differs from the Fab fragment by adding some residues (including one or more cysteine ​​residues from the antibody hinge region) to the carboxyl terminus of the CH1 domain of the heavy chain.

[0035] F(ab')2 is obtained by digesting the entire IgG antibody with pepsin (removing most of the Fc region while retaining some hinge regions intact). The F(ab')2 fragment has two antigen-binding F(ab) parts linked together by disulfide bonds, so the F(ab')2 fragment is bivalent.

[0036] Fab'-SH is the designation for a Fab' whose constant domain contains a cysteine ​​residue carrying a free thiol group. The F(ab')2 antibody fragment was initially generated as a pair of Fab' fragments with a hinge cysteine ​​between them.

[0037] Fv is the smallest antibody fragment containing a complete antigen-binding site. Double-chain Fvs consist of a heavy-chain variable domain and a light-chain variable domain in a tightly bound, non-covalently associated dimer. In single-chain Fvs (scFvs), a heavy-chain variable domain and a light-chain variable domain are covalently linked by a flexible peptide linker, allowing the light and heavy chains to associate in a “dimeric” structure similar to that of double-chain Fvs. In this configuration, it is the three CDRs of each variable domain that define the antigen-binding site on the surface of the VH-VL dimer.

[0038] scFv, or single-chain Fv, refers to an antibody fragment containing both the VH and VL domains of an antibody, where these domains exist as a single polypeptide chain. Typically, Fv polypeptides also include a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for antigen binding.

[0039] "Bispecific antibody" refers to an antibody that has binding sites that bind to two different antigens, or an antibody that has binding sites that bind to different epitopes of the same antigen.

[0040] Antibody-drug conjugates (ADCs) are drugs formed by linking a target-specific antibody with a highly toxic small molecule drug via a linker peptide.

[0041] A second aspect of the present invention is to provide a biomaterial associated with the aforementioned LAG-3 binding molecule, said biomaterial being any of the following:

[0042] B1) Nucleic acid molecules that encode the aforementioned LAG-3 binding molecules;

[0043] B2) An expression cassette containing the nucleic acid molecule described in B1);

[0044] B3) A recombinant vector containing the nucleic acid molecule described in B1) or the expression cassette described in B2);

[0045] B4) Recombinant microorganisms containing the nucleic acid molecule described in B1), or the expression cassette described in B2), or the recombinant vector described in B3);

[0046] B5) An animal cell line containing the nucleic acid molecule described in B1), or containing the expression cassette described in B2), or containing the recombinant vector described in B3);

[0047] B6) A plant cell line containing the nucleic acid molecule described in B1), or containing the expression cassette described in B2), or containing the recombinant vector described in B3);

[0048] B7) Host cells that produce the aforementioned LAG-3 binding molecules.

[0049] Furthermore, in the aforementioned biological materials, the nucleic acid molecules described in B1) include:

[0050] g1) A DNA molecule whose coding sequence is shown as positions 76-105 of SEQ ID No. 2;

[0051] g2) The coding sequence of the coding strand is shown in positions 148-198 of SEQ ID No. 2 for the DNA molecule;

[0052] g3) The coding sequence of the coding strand is as shown in positions 295-327 of SEQ ID No. 2 for the DNA molecule;

[0053] g4) The coding sequence of the coding strand is shown in positions 69-102 of SEQ ID No. 4 for the DNA molecule;

[0054] g5) The coding sequence of the coding strand is as shown in positions 148-168 of SEQ ID No. 4 for a DNA molecule;

[0055] g6) The coding sequence of the coding strand is as shown in positions 265-288 of SEQ ID No. 4 for the DNA molecule;

[0056] g7) The coding sequence of the coding strand is as shown in nucleotides 1-360 of SEQ ID No. 2 for the DNA molecule;

[0057] g8) The coding sequence of the coding strand is as shown in nucleotides 1-318 of SEQ ID No. 4 for a DNA molecule;

[0058] g9) The coding sequence of the coding strand is as shown in SEQ ID No. 2 of the DNA molecule;

[0059] The coding sequence of the g10) coding strand is as shown in SEQ ID No. 4 of the DNA molecule;

[0060] g11) The coding sequence of the coding strand is shown in positions 76-105 of SEQ ID No. 9 for the DNA molecule;

[0061] g12) The coding sequence of the coding strand is shown in positions 148-198 of SEQ ID No. 9 for the DNA molecule;

[0062] g13) The coding sequence of the coding strand is as shown in positions 277-315 of SEQ ID No. 9 for the DNA molecule;

[0063] g14) The coding sequence of the coding strand is shown in positions 69-102 of SEQ ID No. 11 for the DNA molecule;

[0064] g15) The coding sequence of the coding strand is shown in positions 148-168 of SEQ ID No. 11 for the DNA molecule;

[0065] The coding sequence of the g16) coding strand is shown in positions 265-291 of SEQ ID No. 11 for the DNA molecule;

[0066] g17) The coding sequence of the coding strand is shown in nucleotides 1-348 of SEQ ID No. 9;

[0067] The coding sequence of the g18) coding strand is shown in nucleotides 1-321 of SEQ ID No. 11;

[0068] The coding sequence of the g19) coding strand is as shown in SEQ ID No. 9 of the DNA molecule;

[0069] The coding sequence of the g20 coding strand is shown in SEQ ID No. 11 of the DNA molecule.

[0070] A third aspect of the invention is to provide a medicament or pharmaceutical composition containing the aforementioned LAG-3 binding molecule.

[0071] Furthermore, the drug or drug composition further includes a PD-1 antibody, including but not limited to: Pembrolizumab, Nivolumab, Toripalimab, Sintilimab, Camrelizumab, Tislelizumab, Cemiplimab and / or Prolgolimab.

[0072] The drug or drug composition may be a LAG-3 inhibitor (a substance that inhibits the activity of LAG-3).

[0073] A fourth aspect of the present invention is to provide the use of the above-described LAG-3 binding molecule or biomaterials related to the above-described LAG-3 binding molecule in the preparation of LAG-3 inhibitors.

[0074] The LAG-3 inhibitors mentioned above can be used to treat diseases including cancer.

[0075] The cancers mentioned include, but are not limited to: B-cell lymphoma, melanoma, non-small cell lung cancer, soft tissue cell carcinoma, head and neck cancer, squamous cell carcinoma of the head and neck, gastric cancer, esophageal cancer, MSS colorectal cancer, chordoma, hematologic malignancies, non-Hodgkin lymphoma, Hodgkin lymphoma, cervical cancer, endometrial cancer, pancreatic cancer, breast cancer, peritoneal cancer, and / or renal cell carcinoma.

[0076] The LAG-3 binding molecule described in this invention has inhibitory activity, which can inhibit the negative regulatory effect of LAG-3 on immune cells, thereby stimulating CD4. + T and CD8 + T cell proliferation was significantly increased, and IFN-γ expression was also significantly elevated. In vivo experiments demonstrated that the combined use of LAG-3 antibody and PD-1 antibody significantly inhibited tumor growth. These experimental results indicate that the LAG-3 binding molecule can regulate the immune system by modulating immune cell activity, and thus can be used as an immune enhancer for anti-tumor or antiviral immune responses, or as an immunomodulator for T cell-mediated autoimmune diseases. Attached Figure Description

[0077] Figure 1A The affinity of the first part of the mouse antibody for human LAG-3 was determined by FACS.

[0078] Figure 1B The second part of the FACS test was conducted to determine the affinity of the murine antibody for human LAG-3.

[0079] Figure 2 Experiment to stimulate T cells to release IFN-γ using chimeric antibodies.

[0080] Figure 3The study investigated tumor volume changes in an in vivo efficacy experiment of chimeric antibodies.

[0081] Figure 4A The affinity of the first part of the 13H4 humanized antibody for human LAG-3 was determined by FACS.

[0082] Figure 4B The affinity of the second part of the 13H4 humanized antibody for human LAG-3 was determined by FACS.

[0083] Figure 4C The affinity of the humanized 13H4 antibody for human LAG-3 was determined by FACS testing in Part III.

[0084] Figure 5A The affinity of the 3F6 humanized antibody for human LAG-3 was determined by FACS testing in Part 1.

[0085] Figure 5B The affinity of the second part of the FACS test for the humanized antibody 3F6 to human LAG-3 was determined.

[0086] Figure 6 The affinity of the 13H4-affinity-matured single-chain antibody to human LAG-3 was determined.

[0087] Figure 7 The affinity of the 13H4-affinity mature antibody to human LAG-3 was determined.

[0088] Figure 8A The affinity of the humanized antibody IgG1 subtype for binding to human LAG-3.

[0089] Figure 8B The affinity of the humanized antibody IgG1 subtype for binding to monkey LAG-3.

[0090] Figure 8C The affinity of the humanized antibody IgG1 subtype for binding to mouse LAG-3.

[0091] Figure 9 It demonstrates the binding blocking activity of humanized antibodies against MHC II.

[0092] Figure 10 The binding blocking activity of the humanized antibody against FGL1 was observed.

[0093] Figure 11A Epitope competition between the humanized antibody and the control antibody Ab1.

[0094] Figure 11B Epitope competition between the humanized antibody and the control antibody Ab2.

[0095] Figure 12 Changes in tumor volume were observed in the in vivo efficacy experiment of humanized antibodies. Detailed Implementation

[0096] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0097] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0098] In the quantitative experiments described below, three replicate experiments were conducted, and the average value of the results was taken.

[0099] In the following examples, the positive control antibody Ab1 was BMS-986016 (Relatlimab, patent number: US9505839 B2) from Bristol Myers Squibb, and the positive control antibody Ab2 was MK4280 (patent number: US20170097333A1) from Merck Sharp & Dohme. The negative control antibody was human IgG (a product of Nanjing Genscript).

[0100] The vector pcDNA3.4 in the following examples is Invitrogen's pcDNA™ 3.4 TOPO® vector of Cat:A14697.

[0101] The following examples use ExpiCHO-S cells (purchased from Thermo Fisher Scientific, catalog number 29127).

[0102] The HEK293F cells (purchased from Thermo Fisher Scientific, catalog number A14527) used in the following examples were equipped with the following standard equipment and reagents:

[0103] 1. 96-well microplate (Nunc);

[0104] 2. Plating buffer: 0.05M NaHCO3 solution;

[0105] 3. Washing buffer (PBST): Phosphate buffer with a pH of 7.0 containing only 0.05% (v / v) Tween 20;

[0106] 4. Sealing solution: Washing solution containing only 10g / L BSA.

[0107] 5. Horseradish peroxidase-labeled avidin;

[0108] 6. Chromogenic substrate: Tetramethylbenzidine;

[0109] 7. Termination solution: 1M sulfuric acid.

[0110] Example 1: Mouse Immunization and Hybridoma Screening

[0111] 1.1 Mouse Immunization

[0112] Three Balb / c mice and three C57b1 / 6 mice aged 4-6 weeks were selected and simultaneously immunized with human LAG-3 extracellular fragment and monkey LAG-3 extracellular fragment as antigens. Immunization was repeated every 2 weeks for a total of 4 immunizations. After the third immunization, blood was collected from the tail vein, and antibody titers were detected by ELISA. Spleen cells from mice with high titers were fused with myeloma cells SP2 / 0, and the resulting hybridoma cells were used for further screening.

[0113] 1.2 ELISA screening for positive clones

[0114] ELISA plates were coated with 1 μg / ml recombinant human LAG-3 (23L-450L), incubated overnight at 4 ℃, and then blocked. After washing the plates three times with PBST, the supernatant of hybridoma cell culture collected in step 1.1 was added, and the plates were incubated at 37 ℃ for 1 hour. The plates were then washed three more times with PBST, and 4000-fold diluted goat anti-mouse IgG-HRP (Thermo Fisher Scientific) was added and incubated at 37 ℃ for 45 min. After washing three more times with PBST, TMB chromogenic solution was added and the plates were developed for 15 minutes. The absorbance was measured at 450 nm. Positive clones were further subcloned until all subclones showed 100% positivity in the cell culture supernatant. Single-clone cell lines with positive clones and good growth were expanded to obtain LAG-3 monoclonal antibody hybridoma cell lines, which were then cryopreserved for later use.

[0115] 1.3 Purification and Concentration of Mouse Antibodies

[0116] The hybridoma cell lines obtained from screening in section 1.2 were progressively expanded. The cell culture medium was centrifuged at 10,000 rpm for 10 minutes, and the supernatant was collected. The supernatant was purified using a Protein G affinity chromatography column. The specific procedure was as follows: First, the Protein G column (GE) was equilibrated with PBS. Then, the culture supernatant was passed through the column. Five column volumes were pre-eluted with solution A (solvent: water; solute and concentration: 20 mM sodium phosphate, 500 mM NaCl, pH 5.0). Then, five column volumes were eluted with solution B (solvent: water; solute and concentration: 20 mM sodium acetate, 150 mM NaCl, pH 3.5). The elution peaks were collected, and then concentrated using a 30 kDa centrifuge tube to obtain a concentrated solution containing the antibody.

[0117] 1.4 Subtype Identification

[0118] Preliminary screening yielded a total of eight hybridoma cell lines: HKL3F6, HKL15A10, HKL5E10, HKL11B6, HKL13H4, HKL9A1, HKL7E5, and HKL1G8. All antibody subtypes were identified as IgG1 and κ. Sequencing of the obtained anti-LAG-3 antibodies revealed a molecular weight of 150 kb, containing both heavy and light chains, indicating they are typical intact antibodies.

[0119] 1.5. FACS determination of the affinity between murine anti-LAG-3 antibody and human LAG-3

[0120] The pcDNA3.4-HuLAG3 plasmid was transfected into ExpiCHO-S cells. Cells were collected 24 hours later for analysis. The resulting cells expressing human LAG-3 were named CHO-S / pcDNA3.4-HuLAG3. CHO-S / pcDNA3.4-HuLAG3 cells were digested with trypsin, and the cells were collected, centrifuged, and the supernatant was discarded. The cells were resuspended in 1xPBS for counting, and 200 μL of PBS was added for a total of 2x10⁻⁶ cells / mL. 5 The cells were transferred to EP tubes and centrifuged at 1000 rpm for 2 min. The cells were washed once with 1% BSA (dissolved in PBS) and the supernatant was discarded. The murine anti-LAG-3 antibody secreted by the hybridoma was used as the test antibody. The test antibody was diluted to 20 μg / ml with 1% BSA as the starting concentration. This was serially diluted 4-fold to obtain 6 different antibody concentrations. 100 μl of each antibody dilution was added to EP tubes containing the CHO-S / pcDNA3.4-HuLAG3 cells. The cells were resuspended and mixed thoroughly. The tubes were incubated at 4°C in the dark for 1 hour. After incubation, 400 μl of 1xPBS containing 2% BSA was added, and the tubes were centrifuged at 2000 rpm for 5 min. The supernatant was discarded. This process was repeated once. Add 100 μl of goat anti-mouse IgG-FITC (Jackson) diluted 200-fold to each EP tube, incubate at room temperature for 0.5 hours, add 400 μl of 1xPBS containing 2% BSA, centrifuge at 2000 rpm for 5 minutes and discard the supernatant. Repeat this operation once. After washing, resuspend the cells in 400 μl of 1xPBS and analyze by flow cytometry. GraphPad Prism statistical software was used to process the data. The data processing results are shown in Table 1 and... Figure 1A , Figure 1B As shown.

[0121] The results showed that all hybridoma-secreted antibodies could specifically bind to CHO-S cells expressing human LAG-3, with HKL3F6, HKL13H4, and HKL9A1 exhibiting higher affinity.

[0122] Table 1: Affinity of hybridoma-secreted antibodies as determined by FACS

[0123]

[0124] 1.6. ELISA method for determining the affinity of mouse antibodies for cynomolgus monkey LAG-3.

[0125] The affinity of hybridoma-secreted antibodies for recognizing LAG-3 in monkeys was determined using the ELISA method. The specific steps are as follows: Dilute monkey LAG-3 (Jackson) to 2 μg / mL with NaHCO3, add 100 μL to each well of the ELISA plate, and incubate overnight at 4 ℃; wash the plate 3 times with PBST; add 350 μL of 1% BSA (Sangon Biotech) to each well of the ELISA plate, and incubate at 37 ℃ for 2 hours after blocking; wash the plate 3 times with PBST; dilute the LAG-3 antibody to be tested to 1000 ng / mL with 1% BSA, perform 6 4-fold serial dilutions to obtain 7 test sample concentrations, add 100 μL of the LAG-3 antibody to each well of the ELISA plate, and incubate at room temperature for 1 hour; wash the plate 3 times with PBST; dilute goat anti-mouse IgG-HRP 4000 times with 1% BSA, add 100 μL to each well of the ELISA plate, and incubate at room temperature for 0.5 hours; wash the plate 3 times with PBST; add 50 μL of TMB to each well. Incubate the microplate with 50 μL of the sample at room temperature in the dark for 15 minutes; stop the color development reaction by adding 2 mol / L H2SO4 to each well; place the microplate in a SeptraMax Versa microplate reader and measure the OD. 450nm The values ​​were statistically analyzed using GraphPad Prism, and the EC50 values ​​were calculated. The results are shown in Table 2. The results indicate that the murine antibodies secreted by the hybridoma can all recognize monkey LAG-3.

[0126] Table 2: Mouse antibodies recognize LAG-3 in monkeys

[0127]

[0128] Based on the results of affinity tests for human LAG-3 and monkey LAG-3, HKL13H4, HKL3F6, and HKL9A1 showed the highest affinity and were used for the construction of chimeric antibodies.

[0129] Example 2: Construction and Screening of Chimeric Antibodies

[0130] 2.1 Construction of chimeric antibodies

[0131] HKL3F6, HKL13H4, and HKL9A1 hybridoma cells were collected and sent to Nanjing Genscript Biotech Co., Ltd. for sequencing. The amino acid sequence of the mouse antibody obtained from sequencing is as follows:

[0132] Table 3: Amino acid sequences of the variable regions of HKL13H4, HKL3F6 and HKL9A1

[0133]

[0134] The amino acid sequences of the light and heavy chain variable regions of HKL3F6, HKL13H4, and HKL9A1 were grafted into the constant regions of human κappa and IgG1. After reverse translation into DNA, the amino acid sequences were synthesized. These were then constructed into the pcDNA3.4 vector using genetic engineering techniques and transiently transfected into HEK293F cells for protein expression, yielding chimeric antibodies CHL3F6, CHL13H4, and CHL9A1. The harvested chimeric antibodies were purified and concentrated using the method described in section 1.3 and stored for later use.

[0135] 2.2 Detection of the PBMC cell activation effect of chimeric antibodies

[0136] (1) Staphylococcus aureus enterotoxin B (SEB, purchased from Sigma-Aldrich) was diluted to 0.05 ng / ml with PBS to obtain an SEB solution. Chimeric antibodies CHL3F6, CHL9A1, and CHL13H4 were diluted with PBS to obtain chimeric antibody solutions with concentrations of 10 μg / ml, 0.33 μg / ml, and 0.01 μg / ml, respectively. The SEB solution was mixed with the above antibody solutions at a 1:1 volume ratio and then added to a 96-well plate at 50 μL / well, with 3 replicates. 50 μL / well of SEB solution was used as a control. The 96-well plate was incubated at 37 ℃ for 1 hour. Before use, the plate was washed three times with PBS.

[0137] (2) Human PBMC cell isolation: Whole blood was drawn from healthy human subjects, and PBMC cells were isolated and counted using lymphocyte separation fluid (Sigma).

[0138] (3) According to 5×10 4 The amount of PBMC cells added per well was determined by adding the cells to the 96-well cell culture plate from step (1), and then incubating the 96-well plate at 37 ℃ in a 5% CO2 incubator for 3 days. Cell supernatant was collected, and the secretion of the cytokine IFN-γ was measured. The experimental results are shown in [Figure number missing]. Figure 2 .

[0139] The results showed that the chimeric antibodies CHL3F6, CHL9A1, and CHL13H4 could all stimulate PBMC cells to activate and secrete the cytokine IFN-γ, with CHL3F6 and CHL9A1 showing the best effects.

[0140] 2.3. In vivo efficacy

[0141] The experiment selected B-hPD-1 / hLAG3 mice as test mice to detect the in vivo antitumor efficacy of LAG-3 antibody. The B-hPD-1 / hLAG3 mouse model is a genetically engineered mouse, which is a chimeric human HuLAG-3 gene and HuPD-1 gene in the genome of a C57BL / 6 mouse with a genetic background, and was purchased from Biocytogen.

[0142] MC38 cells (mouse colon cancer cells, purchased from Nanjing Kebai) were cultured to 80% confluence, digested with trypsin, centrifuged at 1000 rpm for 5 min, and the cells were collected, washed, centrifuged again, and resuspended to obtain a cell suspension. The cell viability was ensured to be >95%, and the cells were counted for later use.

[0143] MC38 cells were subcutaneously injected into the back (shaved side) of test mice (6-8 weeks old, weighing 20±0.3 g) at a dose of 100 μL per mouse, for a total of 2×10⁻⁶ cells. 6 (cells). When the average tumor volume in tumor-bearing mice reaches 100-150 mm. 3 Mice were randomly divided into 6 groups, with 6 mice in each group. Five experimental groups were set up: CHL3F6+ anti PD-1, CHL9A1+ anti PD-1, CHL13H4+ anti PD-1, Ab2+ anti PD-1, and anti-PD-1, with physiological saline as a control. The anti-LAG-3 concentration was 5 mg / kg, and the anti-PD-1 concentration was 0.1 mg / kg. The anti-PD-1 antibody was provided by Anhui Anke Biotechnology (Group) Co., Ltd.

[0144] The antibodies used in the above experiments were diluted with physiological saline to the corresponding concentration according to the injection concentration and stored in a refrigerator at 4°C for later use.

[0145] The CHL3F6+ anti PD-1 group received 100 μL of CHL3F6 and anti PD-1 solution (solvent was physiological saline, solute was CHL3F6 and anti PD-1) per injection, with each CHL3F6 dose being 5 mg / kg body weight and each anti PD-1 dose being 0.1 mg / kg body weight, administered twice a week for a total of 7 weeks;

[0146] The CHL9A1+ anti PD-1 group received 100 μL of CHL9A1 and anti PD-1 solution (the solvent was physiological saline, and the solutes were CHL9A1 and anti PD-1) per injection, with each injection making the CHL9A1 dose 5 mg / kg body weight and the anti PD-1 dose 0.1 mg / kg body weight per injection, twice a week for a total of 7 injections;

[0147] The CHL13H4+ anti PD-1 group received 100 μL of CHL13H4 and anti PD-1 solution (solvent was physiological saline, solute was CHL13H4 and anti PD-1) per injection, with each injection making the CHL13H4 dose 5 mg / kg body weight and the anti PD-1 dose 0.1 mg / kg body weight per injection, twice a week for a total of 7 injections;

[0148] The Ab2+ anti PD-1 group received 100 μL of Ab2 and anti PD-1 solution (solvent was physiological saline, solute was Ab2 and anti PD-1) per injection, with an Ab2 dose of 5 mg / kg body weight and an anti PD-1 dose of 0.1 mg / kg body weight per injection, administered twice a week for a total of 7 weeks.

[0149] The antiPD-1 group received 100 μL of antiPD-1 solution (solvent is physiological saline, solute is antiPD-1) per injection, making the antiPD-1 dose 0.1 mg / kg body weight per administration, twice a week for a total of 7 administrations;

[0150] The saline group served as the control group. Each mouse was subcutaneously injected with 100 μL of saline, and the administration was repeated twice a week for a total of 7 weeks.

[0151] After tumor inoculation, the animals' survival and activity levels were checked twice a week, including tumor growth, weight, activity level, and diet, and the results were recorded.

[0152] Tumor growth results are shown Figure 3 The results showed that, compared with saline, the antibodies in each experimental group could inhibit the growth of MC38 tumors; among them, ChL13H4 and ChL3F6 combined with PD-1 showed the best tumor inhibition effect.

[0153] Example 3: Construction and Expression of Humanized Antibodies

[0154] 3.1 Construction of humanized antibodies

[0155] Humanization of chimeric antibodies CHL13H4 and CHL3F6 was carried out using homologous sequence modeling, antibody complementary determining region (CDR) transplantation, and framework region (FR) key amino acid back mutation techniques.

[0156] The CHL13H4 antibody gene sequence was analyzed, and then, through homology modeling and optimization using antibody Fab, surface scanning was used to determine humanized mutation sites. Virtual mutation and molecular dynamics simulations were conducted to identify key amino acids, among other analyses, to design a rationally designed humanized antibody. Ultimately, four heavy chain candidate sequences and three light chain candidate sequences were obtained, which were combined to yield 12 candidate antibodies for further identification as described in this embodiment. The 12 candidate antibodies are HuL13H4-H1L1, HuL13H4-H1L2, HuL13H4-H1L3, HuL13H4-H2L1, HuL13H4-H2L2, HuL13H4-H2L3, HuL13H4-H3L1, HuL13H4-H3L2, HuL13H4-H3L3, HuL13H4-H4L1, HuL13H4-H4L2, and HuL13H4-H4L3.

[0157] The CHL3F6 antibody gene sequence was analyzed, followed by homology modeling and optimization using antibody Fab, surface scanning to identify humanized mutation sites, virtual mutation and molecular dynamics simulations to determine key amino acids, and a series of other analyses to design a rationally designed humanized antibody. Ultimately, three candidate sequences for the heavy chain variable region (VH) and three candidate sequences for the light chain variable region (VL) were obtained. Combinations of the VH and VL candidate sequences yielded nine candidate antibodies for further identification. The nine candidate antibodies are: HuL3F6-H1L1, HuL3F6-H1L2, HuL3F6-H1L3, HuL3F6-H2L1, HuL3F6-H2L2, HuL3F6-H2L3, HuL3F6-H3L1, HuL3F6-H3L2, and HuL3F6-H3L3.

[0158] The amino acid sequences of the candidate variable region sequences are shown in Table 4.

[0159] Table 4: List of amino acid sequences of candidate variable region sequences

[0160]

[0161]

[0162] 3.2. FACS determination of the affinity between humanized anti-LAG-3 antibody and human LAG-3

[0163] CHO-S cells were transfected with the pcDNA3.4-HuLAG3 plasmid. Cells were collected 24 hours later for analysis. The resulting cells expressing human LAG-3 were named CHO-S / pcDNA3.4-HuLAG3. CHO-S / pcDNA3.4-HuLAG3 cells were digested with trypsin, and the cells were collected, centrifuged, and the supernatant was discarded. The cells were resuspended in 1xPBS and counted, adjusting the cell count to 1.5 x 10-1 cells per EP tube.5 Centrifuge cells at 1000 rpm for 2 min, wash once with 1% BSA (dissolved in PBS), and discard the supernatant. Humanized anti-LAG-3 antibody was used as the test antibody. The test antibody was diluted to 30 μg / ml with 1% BSA as the starting concentration. Six different concentrations of antibody were obtained by serial dilution five times (3-fold) to obtain six different antibody concentrations. 100 μl of each concentration of antibody dilution was added to EP tubes containing the above-mentioned CHO-S / pcDNA3.4-HuLAG3 cells. The cells were resuspended and mixed well. The cells were incubated at 4 °C in the dark for 1 hour. 1% BSA dilution was used as a negative control. After incubation, 400 μl of 1xPBS containing 2% BSA was added, centrifuged at 2000 rpm for 5 min, and the supernatant was discarded. This operation was repeated once. Add 100 μl of goat anti-human IgG-FITC (Jackson) diluted 200 times to each EP tube, incubate at room temperature for 0.5 hours, add 400 μl of 1xPBS containing 2% BSA, centrifuge at 2000 rpm for 5 minutes and discard the supernatant. Repeat this operation once. After washing, resuspend the cells in 400 μl of 1xPBS and analyze them by flow cytometry. Process the data using GraphPad Prism statistical software.

[0164] The affinity results for HuL13H4 are shown in Table 5.1 and Figure 4A , Figure 4B , Figure 4C As shown.

[0165] The affinity results for HuL3F6 are shown in Table 5.2 and Figure 5A , Figure 5B As shown.

[0166] Table 5.1: HuL13H4 antibody recognizes human LAG-3

[0167]

[0168] Table 5.2: HuL3F6 antibody molecular recognition of human LAG-3

[0169]

[0170] Example 4: Affinity maturation and potential deamidation site mutations

[0171] 4.1 Mutations at HuL13H4-H1L3 Affinity Maturation and Potential Deamidation Sites

[0172] 4.1.1 Obtaining HuL13H4-H1L3 affinity maturation candidate molecules

[0173] The humanized HuL13H4-H1L3 molecule was constructed into a single-chain antibody, namely VH-(G4S)3-VL-Fc. The amino acid sequence of the heavy chain variable region of HuL13H4-H1L3 is shown in SEQ ID No. 6, and the amino acid sequence of the light chain variable region is shown in SEQ ID No. 7. Affinity maturation was performed by Abstudio to modify the CDR regions of the light and heavy chains, resulting in five candidate molecules: scF-HuL13H4-2B8-Fc, scFv-HuL13H4-F4-Fc, scFv-HuL13H4-2E8, scFv-HuL13H4-2B4-Fc, and scFv-HuL13H4-2A6-Fc.

[0174] 4.1.2. FACS determination of the affinity of mature candidate molecules for HuL13H4-H1L3

[0175] The pcDNA3.4-HuLAG3 plasmid was transfected into ExpiCHO-S cells. After 24 h, the cells were collected for testing. The cells expressing the antigen human LAG-3 were named ExpiCHO-S / pcDNA3.4-HuLAG3.

[0176] ExpiCHO-S / pcDNA3.4-HuLAG-3 cells were digested with trypsin, and the cells were collected, centrifuged, the supernatant was discarded, and the cells were resuspended in 1xPBS for counting. The cell count was adjusted to 1.5 x 10^6 cells per EP tube. 5 Centrifuge cells at 1000 rpm for 2 min, wash once with 1% BSA (dissolved in PBS), and discard the supernatant. The single-chain antibody obtained from affinity maturation is the test antibody. Dilute the test antibody to 30 μg / ml with 1% BSA as the starting concentration, and serially dilute 5 times (3-fold) to obtain 6 different antibody concentrations. Take 100 μl of each antibody dilution and add it to an EP tube containing the above CHO-S / pcDNA3.4-HuLAG3 cells. Resuspend the cells and mix well. Incubate at 4°C in the dark for 1 hour. Use 1% BSA dilution as a negative control. After incubation, add 400 μl of 1xPBS containing 2% BSA, centrifuge at 2000 rpm for 5 min, discard the supernatant, and repeat this operation once. Add 100 μl of goat anti-human IgG-FITC (Jackson) diluted 200-fold to each EP tube, incubate at room temperature for 0.5 hours, add 400 μl of 1xPBS containing 2% BSA, centrifuge at 2000 rpm for 5 minutes and discard the supernatant. Repeat this operation once. After washing, resuspend the cells in 400 μl of 1xPBS and analyze by flow cytometry. GraphPad Prism statistical software was used to process the data. The data processing results are shown in Table 6. Figure 6As shown in the figure. The results indicate that scFv-L13H4-F4-Fc and scFv-L13H4-2B4-Fc have higher affinity.

[0177] Table 6: Affinity Maturation Candidate Affinities-ScFv of HKL13H4

[0178]

[0179] scFv-L13H4-F4-Fc and scFv-L13H4-2B4-Fc are preferred candidates. The amino acid sequence of the light chain variable region of scFv-L13H4-F4-Fc is shown in positions 1-106 of SEQ ID No. 3, and its heavy chain variable region sequence is consistent with that of the humanized HuL13-H1L3 molecule, as shown in SEQ ID No. 6. The amino acid sequence of the light chain variable region of scFv-L13H4-2B4-Fc is shown in SEQ ID No. 5, and its heavy chain variable region sequence is consistent with that of the humanized HuL13-H1L3 molecule, as shown in SEQ ID No. 6. scFv-L13H4-F4-Fc and scFv-L13H4-2B4-Fc were reduced to complete antibodies by grafting the heavy chain variable region onto the heavy chain constant region (IgG1 subtype with L234A and L235A mutations, its amino acid sequence is shown as positions 121-450 of SEQ ID No. 1), and grafting the light chain variable region onto the light chain constant region (i.e., the Kappa subtype, its amino acid sequence is shown as positions 107-213 of SEQ ID No. 3), yielding antibodies HuL13H4-2B4 and HuL13H4-F4. Their binding affinity to human LAG-3 was further detected by FACS, following the same experimental procedure as in 4.1.2, and the results are as follows. Figure 7 As shown.

[0180] 4.1.3, Potential deamidation site mutations in HuL13H4-2B4 and HuL13H4-F4

[0181] The antibody HankeL13 is obtained by mutating the affinity maturation antibody HuL13H4-F4. The asparagine at position 104 or 105 of the heavy chain variable region is mutated to avoid potential deamidation sites. This mutation can be to replace the asparagine at position 104 with glutamic acid or alanine, or to replace the asparagine at position 105 with glutamic acid or alanine, or to mutate both positions simultaneously. Preferably, the asparagine at position 104 is mutated to glutamic acid, while keeping the other sequences of HuL13H4-F4 unchanged. The amino acid sequence of the mutated heavy chain variable region is shown in positions 1-120 of SEQ ID.1; preferably, the heavy chain constant region is the IgG1 isotype with L234A and L235A mutations. The full-length amino acid sequence of the heavy chain of HankeL3 is shown in SEQ ID No. 1, and the coding sequence of its coding strand is shown in SEQ ID No. 2; the full-length amino acid sequence of the light chain of HankeL3 is shown in SEQ ID. 3, and the coding sequence of its coding strand is shown in SEQ ID. 4.

[0182] 4.2 Potential deamidation site mutation in HuL3F6-H2L2

[0183] The antibody HankeL3 was obtained by mutating the humanized antibody HuL3F6-H2L2. The heavy chain variable region underwent a G56A mutation to avoid potential deamide sites; the other sequences of HuL3F6-H2L2 remained unchanged. The sequence of the mutated heavy chain variable region is shown in positions 1-116 of SEQ ID.8. The preferred heavy chain constant region is the IgG1 isotype, with L234A and L235A mutations. The full-length amino acid sequence of the heavy chain of HankeL3 is shown in SEQ ID.8, and the coding sequence of its coding strand is shown in SEQ ID.9. The full-length amino acid sequence of the light chain of HankeL3 is shown in SEQ ID.10, and the coding sequence of its coding strand is shown in SEQ ID.11.

[0184] Example 5: Expression and functional verification of antibodies HankeL13 and HankeL3

[0185] 5.1 Expression and purification of antibodies HankeL13 and HankeL3

[0186] Based on the gene sequences obtained from sequencing, the full-length gene sequences were synthesized. The heavy chain coding gene of the HankeL13 antibody is shown in SEQ ID No. 2, and the light chain coding gene of the HankeL13 antibody is shown in SEQ ID No. 4. Positions 1-360 of SEQ ID No. 2 are the coding genes for the variable region VH of the HankeL13 antibody heavy chain, where the coding sequences for CDR1, CDR2, and CDR3 are shown in positions 76-105, 148-198, and 295-327 of SEQ ID No. 2, respectively. Positions 1-318 of SEQ ID No. 4 are the coding genes for the variable region VL of the HankeL13 antibody light chain, where the coding sequences for CDR1, CDR2, and CDR3 are shown in positions 69-102, 148-168, and 265-288 of SEQ ID No. 4, respectively.

[0187] Based on the gene sequences obtained from sequencing, the full-length genes were synthesized. The coding gene for the HankeL3 antibody heavy chain is shown in SEQ ID No. 9, and the coding gene for the HankeL3 antibody light chain is shown in SEQ ID No. 11. Positions 1-348 of SEQ ID No. 9 encode the variable region VH of the HankeL3 antibody heavy chain, with the coding sequences for CDR1, CDR2, and CDR3 shown in positions 76-105, 148-198, and 277-315 of SEQ ID No. 9, respectively. Positions 1-321 of SEQ ID No. 11 encode the variable region VL of the HankeL3 antibody light chain, with the coding sequences for CDR1, CDR2, and CDR3 shown in positions 69-102, 148-168, and 265-291 of SEQ ID No. 11, respectively.

[0188] The DNA fragment (encoding gene of the antibody heavy chain) shown in SEQ ID No. 2 or SEQ ID No. 9 was cloned into the restriction sites XbaI and Hind III of the vector pcDNA3.4 to obtain recombinant expression vectors expressing the heavy chain of the antibody, named pcDNA3.4-HankeL13-H and pcDNA3.4-HankeL3-H, respectively. To further facilitate protein expression, when constructing the recombinant expression vector of the antibody heavy chain, an XbaI restriction site, a kozak co-recognition sequence (5'-GCCACC-3'), and a single peptide coding sequence (5'-ATGGAGTTTGGACTGTCTTGGGTGTTCCTGGTGGCTATCCTGAAAGGAGTCCAGTGC-3') were sequentially introduced upstream of the DNA fragment (encoding gene of the antibody heavy chain) shown in SEQ ID No. 2 or SEQ ID No. 9. The stop codon TGA and HindIII restriction site were introduced downstream of the DNA fragment shown in No. 9 (antibody heavy chain encoding gene). The above gene sequence was sent to Nanjing Genscript Biotech Co., Ltd. for synthesis, and then digested with XbaI and HindIII restriction sites, and ligated into the XbaI and HindIII digested pcDNA3.4 vector.

[0189] The DNA fragment (encoding gene of the antibody light chain) shown in SEQ ID No. 4 or SEQ ID No. 11 was cloned into the restriction sites Xba I and Hind III of the pcDNA3.4 vector to obtain recombinant expression vectors expressing the light chain of the antibody (named pcDNA3.4-HankeL3-L and pcDNA3.4-HankeL13-L, respectively). To facilitate protein expression, when constructing the recombinant expression vector for the antibody light chain, an XbaI restriction site, a kozak co-recognition sequence (5'-GCCACC-3'), and a single peptide coding sequence (5'-ATGGAAACAGATACACTCCTCCTCTGGGTGCTGCTCCTCTGGGTGCCAGGATCTACAGGA-3') were sequentially introduced upstream of the 5' end of the DNA fragment shown in SEQ ID No. 4 or SEQ ID No. 11 (the antibody light chain coding gene). A stop codon TGA and a HindIII restriction site were introduced downstream of the 3' end of the DNA fragment shown in SEQ ID No. 4 or SEQ ID No. 11 (the antibody light chain coding gene). The above gene sequences were synthesized at Nanjing Genscript Biotech Co., Ltd., and subsequently digested with XbaI and HindIII restriction sites, then ligated into the XbaI and HindIII-digested pcDNA3.4 vector.

[0190] pcDNA3.4-HankeL13-H and pcDNA3.4-HankeL13-L were introduced into the human embryonic kidney cell line HEK293F to obtain recombinant cells HEK293F / pcDNA3.4-HankeL13. The recombinant cells HEK293F / pcDNA3.4-HankeL13 can express the antibody HankeL13.

[0191] pcDNA3.4-HankeL3-H and pcDNA3.4-HankeL3-L were introduced into the human embryonic kidney cell line HEK293F to obtain recombinant cells HEK293F / pcDNA3.4-HankeL3. The recombinant cells HEK293F / pcDNA3.4-HankeL3 can express the antibody HankeL3.

[0192] The recombinant cells HEK293F / pcDNA3.4-HankeL13 and HEK293F / pcDNA3.4-HankeL3 were then cultured in a 37 ℃, 5% CO2 shaking incubator at 120 rpm.

[0193] Antibody proteins were purified from culture supernatant using a Protein A affinity chromatography column. The specific procedure was as follows: First, the Protein A column (GE) was equilibrated with PBS. Then, the culture supernatant was passed through the column. Pre-eluting was performed with solution A (solvent: water; solute and concentration: 20 mM sodium phosphate, 500 mM NaCl, pH 5.0) for 5 column volumes. Next, elution was performed with solution B (solvent: water; solute and concentration: 20 mM sodium acetate, 150 mM NaCl, pH 3.5) for another 5 column volumes. The elution peaks were collected, and then concentrated in a 30 kDa centrifuge tube to obtain the antibodies, thus yielding anti-human LAG-3 antibodies (Hanke L3 and Hanke L13).

[0194] Sequencing of the antibody HankeL13 revealed that the obtained anti-human LAG-3 antibody HankeL13 consists of a heavy chain and a light chain. The amino acid sequence of the heavy chain is shown in SEQ ID No. 1, with CDR1, CDR2, and CDR3 sequences shown at positions 26-35, 50-66, and 99-109 from the N-terminus of SEQ ID No. 1, respectively. The amino acid sequence of the light chain is shown in SEQ ID No. 3, with CDR1, CDR2, and CDR3 sequences shown at positions 24-34, 50-56, and 89-96 from the N-terminus of SEQ ID No. 3, respectively.

[0195] The obtained anti-human LAG-3 antibody HankeL3 is a complete antibody, composed of a heavy chain and a light chain. The amino acid sequence of the heavy chain is shown in SEQ ID No. 8, and the sequences of its CDR1, CDR2 and CDR3 are shown at positions 26-35, 50-66 and 97-105 from the N-terminus of SEQ ID No. 8, respectively. The amino acid sequence of the light chain is shown in SEQ ID No. 10, and the sequences of its CDR1, CDR2 and CDR3 are shown at positions 24-34, 50-56 and 89-97 from the N-terminus of SEQ ID No. 10, respectively.

[0196] 5.2. Identification of the affinity of antibodies HankeL13 and HankeL3 for human, monkey, and mouse LAG-3.

[0197] 5.2.1 Antigen Preparation

[0198] XbaI restriction site and kozak co-recognition sequence (5'-GCCACC-3') were sequentially added to the 5' end of the extracellular region gene sequences of human, monkey, and mouse LAG3 antigens, respectively. The mouse Fc gene sequence, stop codon TGA, and HindIII restriction site were sequentially added to the 3' end. These gene sequences were sent to Nanjing Genscript Biotech Co., Ltd. for synthesis, followed by XbaI and HindIII restriction enzyme digestion, and ligation into the similarly digested pcDNA3.4 vector to obtain pcDNA3.4 / HuLAG3-Fc, pcDNA3.4 / CyLAG3-Fc, and pcDNA3.4 / MuLAG3-Fc vectors, respectively. These vectors were then introduced into HEK293F cells and cultured at 37 ℃ in a 5% CO2 shaking incubator at 120 rpm. Three days later, the cell supernatant was collected, and the antibody protein was purified from the culture supernatant using a Protein A affinity chromatography column. The specific procedure was the same as in 5.1. HuLAG-3-Fc, CyLAG-3-Fc, and MuLAG-3-Fc antigen proteins were obtained, and their amino acid sequences are shown in Table 7.

[0199] Table 7: Amino acid sequences of expressed human / mouse / monkey proteins

[0200]

[0201] 5.2.2 Determination of the affinity of antibodies HankeL13 and HankeL3 for human LAG-3

[0202] The method for determining the epitope competition between the test antibody and Ab1 was as follows: Goat anti-mouse Fc (Jackson Biotech) was diluted with NaHCO3 to the working concentration, and 100 μl was added to each well of the ELISA plate and incubated overnight at 4°C; the plate was washed three times with PBST; 200 μl of 5% NON-Fat Milk (Sangon Biotech) was added to each well for blocking at 37°C for 2 hours; the plate was washed three times with PBST; HuLAG-3-Fc antigen (prepared in 5.2.1) was diluted with 1% NON-Fat Milk to 1 μg / ml, and 100 μl was added to each well of the ELISA plate and incubated overnight at 4°C; the plate was washed three times with PBST; HankeL13 and HankeL3 antibodies were used as test antibodies, and the test samples were serially diluted 4-fold six times with 1% NON-Fat Milk to obtain seven gradient concentrations, with the highest concentration being 50 nM, and 100 μl was added to each well. Add μl of the solution to the microplate, using Ab1 as a positive control; incubate at room temperature for 1 hour; wash the plate 3 times with PBST; dilute goat anti-human-HRP with 1% NON-Fat Milk, add 100 μl to each well of the microplate, and incubate at room temperature for 0.5 hours; wash the plate 3 times with PBST; add TMB to each well and incubate at room temperature in the dark; stop the color development reaction by adding H2SO4; place the microplate in a SeptraMax Versa microplate reader and measure the absorbance (OD value) at 450 nm, statistically analyze the results using GraphPad Prism and calculate the EC50 value.

[0203] The affinity assays for antibodies HankeL13 and HankeL3 against monkey and mouse LAG-3 were performed using the same method as above, except that human LAG-3 was replaced with monkey LAG-3 (prepared in 5.2.1) and mouse LAG-3 (prepared in 5.2.1), with maximum concentrations of 10 nM and 2 ug / ml, respectively. The remaining steps were the same as above.

[0204] The experimental results are shown in Table 8 and Figure 8A , Figure 8B , Figure 8C The results showed that antibodies HankeL13 and HankeL3 could recognize both human LAG-3 and monkey LAG-3, but had a weaker binding to mice.

[0205] Table 8: ELISA assay for binding of monoclonal antibodies to human / monkey / mouse LAG-3 antigen (EC50 (nM))

[0206]

[0207] 5.3 Ligand Competition

[0208] 5.3.1 Determination of the competitive binding activity of Hanke L13 and Hanke L3 with ligand MHCII

[0209] Using A375 cells (expressing MHCII, purchased from Nanjing Kebai Biotechnology Co., Ltd.) to detect the competitive binding activity of antibodies HankeL3 and HankeL13 with MHCII ligands to LAG-3 antigen, Ab1 and Ab2 were used as positive controls, and IgG1 was used as a negative control. (1) A375 cells cultured for 24 h were digested with trypsin, the cells were collected, centrifuged to remove the supernatant, resuspended in 1xPBS for counting, and 200 μL of PBS was added for a total of 2x10 5 (1) Add cells to a 1.5 mL EP tube. (2) HankeL13 and HankeL3 are the test antibodies. Dilute the test antibodies to 25 uM with PBS. 5-fold serial dilution 6 times to obtain 7 concentrations of antibody test solutions. Dilute the HuLAG-3-Fc antigen (self-made in 5.2.1) to 250 nM with PBS. Mix equal volumes of antigen and antibody dilution solution to a total of 100 μL and add to the EP tube described in step (1). Incubate at room temperature for 1 hour, centrifuge at 2000 rpm for 3 minutes, add PBS to resuspend the cells, centrifuge at 2000 rpm for 3 minutes, and discard the supernatant. (3) Add 200-fold diluted goat anti-mouse-FITC secondary antibody, incubate at room temperature for 30 minutes, centrifuge at 2000 rpm for 3 minutes, add PBS to resuspend the cells, centrifuge at 2000 rpm for 3 minutes, discard the supernatant, add 400 μL PBS to resuspend and perform detection. Use GraphPadPrism to statistically analyze the results and calculate the IC50 value.

[0210] The results are shown in Table 9 and Figure 10 As shown in the figure. The results indicate that antibodies HankeL13 and HankeL3 can block the binding of the antigen to the ligand MHCII.

[0211] Table 9: Blockade of LAG-3 antigen and its ligand MHCII molecule by FACS detection

[0212]

[0213] 5.3.2 Determination of the competitive binding activity of HankeL13 and HankeL3 with ligand FGL1

[0214] The activity of anti-LAG-3 antibody in competitive binding to LAG-3 antigen with human FGL1 was detected using ELISA, with Ab1 and Ab2 as positive controls and IgG1 as a negative control. Dilute FGL1-hFc (Acro, catalog number FG1-H5258) with NaHCO3 to 1 μg / ml, add 100 μl to each well of the ELISA plate, and incubate overnight at 4°C; wash the plate 3 times with PBST; add 200 μl of 1% BSA (Sangon Biotech) to each well of the ELISA plate, and incubate at 37°C for 2 hours to block; wash the plate 3 times with PBST; dilute the HuLAG-3-Fc antigen (prepared in 5.2.1) with 1% BSA, dilute the LAG-3 antibody to 200 nM with 1% BSA, perform 6 2-fold serial dilutions to obtain 7 concentration gradients, mix equal volumes of antigen and antibody, add 100 μl to each well of the ELISA plate, and incubate at room temperature for 1 hour; wash the plate 3 times with PBST; dilute goat anti-mouse IgG-HRP with 1% BSA, add 100 μl to each well. Add μl to the microplate and incubate at room temperature for 0.5 hours; wash the plate 3 times with PBST; add TMB to each well and incubate at room temperature in the dark; add H2SO4 to stop the color development reaction; place the microplate in a SeptraMax Versa microplate reader and measure the absorbance (OD value) at 450 nm, and use GraphPad Prism to analyze the results and calculate the IC50 value.

[0215] The experimental results are shown in Table 10 and Figure 10 As shown in the figure. The results indicate that antibodies HankeL13 and HankeL3 can effectively block the binding of LAG-3 antigen to ligand FGL1.

[0216] Table 10: Blocking effect of LAG-3 antigen on ligand FGL-1 molecule detected by ELISA

[0217]

[0218] 5.4 Antigen Epitope Binding Analysis

[0219] 5.4.1 Analysis of antigen-binding domain epitopes

[0220] Human LAG-3 antigen extracellular domains 1 (37G-167G), 2 (168Q-252S), 3 (265P-343N), and 4 (348L-419R), or combinations thereof, were replaced with the corresponding extracellular regions of mouse TIM-3 to synthesize the corresponding target genes. These genes were then inserted into the pcDNA3.4 vector to obtain different human-mouse chimeric LAG-3 antigen DNA plasmids. The plasmids were introduced into HEK293F cells, and cells were collected after 24 hours for FACS analysis (see Table 11 for details). The cell count was adjusted to 2 x 10⁵ cells per EP tube using PBS, centrifuged at 1000 rpm for 2 min, washed once with 1% BSA (dissolved in PBS), and the supernatant was discarded. Antibodies Ab1, HankeL13, and HankeL3 were diluted to 30 μg / ml with 1% BSA and added to HEK293F cells expressing different human-mouse chimeric antigens. The mixture was incubated at 4°C in the dark for 1 hour. After incubation, 400 μL of 1xPBS containing 2% BSA was added, and the cells were centrifuged at 2000 rpm for 5 minutes, discarding the supernatant. This process was repeated once. 100 μL of goat anti-human IgG-FITC (Jackson) diluted 200-fold was added to each EP tube, and the cells were incubated at room temperature for 0.5 hours. Then, 400 μL of 1xPBS containing 2% BSA was added, and the cells were centrifuged at 2000 rpm for 5 minutes, discarding the supernatant. This process was repeated once. After washing, the cells were resuspended in 400 μL of 1xPBS and analyzed by flow cytometry.

[0221] Through the above operations, it can be seen that the fluorescence intensity of Ab1 decreases in cell groups C, D, E, and F, indicating that Ab1 mainly binds to Doamin1 and Domain2 in the extracellular region of human LAG-3; the fluorescence intensity of HankeL13 and HankeL3 decreases in cell groups C, E, and F, indicating that HankeL13 and HankeL3 mainly bind to Domain1 in the extracellular region of human LAG-3. Specific results are shown in Table 11.

[0222] Table 11: Analysis of mean fluorescence intensity of antibodies binding to different human-mouse chimeric LAG-3 antigen epitopes

[0223]

[0224] 5.4.2 Competition with positive control antibody epitopes

[0225] The epitope competition between HankeL3 and HankeL13 antibodies and control antibodies Ab1 and Ab2 was compared using ELISA.

[0226] The method for determining the epitope competition between the antibody and Ab1 was as follows: Goat anti-mouse Fc (Jackson) was diluted with NaHCO3 to the working concentration, and 100 μl was added to each well of the ELISA plate and incubated overnight at 4°C; the plate was washed 3 times with PBST; 200 μl of 5% NON-FatMilk (Sangon Biotech) was added to each well of the ELISA plate and incubated at 37°C for 2 hours to block; the plate was washed 3 times with PBST; HuLAG-3-Fc antigen (prepared in 5.2.1) was diluted with 1% NON-Fat Milk to 1 μg / ml, and 100 μl was added to each well of the ELISA plate and incubated at room temperature for 1 hour; the plate was washed 3 times with PBST. Positive control antibody Ab1 was labeled with streptavidin-biotin (SA-Biotin, SA). The biotin-labeled Ab1 was diluted to 2 nM with non-Fat Milk and labeled as solution A. Simultaneously, unlabeled Ab1, Hanke L3, Hanke L13, and IgG were prepared to 2 μM with non-Fat Milk, diluted 5-fold for one spot, followed by 10-fold serial dilutions for four spots, labeled as solution B. 50 μL of solution A and each concentration of solution B were mixed and added to a 96-well plate coated with LAG-3 antigen. The plate was incubated at room temperature for 1 hour and washed three times with PBST. Add 100 μL of 8000-fold diluted SA-HRP (Biolegend) to each well and incubate at room temperature for 0.5 hours. Wash the plate three times with PBST, add TMB to each well, and incubate at room temperature in the dark. Terminate the colorimetric reaction with H2SO4. Place the plate in a SeptraMax Versa microplate reader and measure the absorbance at 450 nm. Analyze the results using GraphPad Prism and calculate the EC50 value.

[0227] The method for determining the epitope competition between the antibody to be tested and Ab2 is the same as above, except that solution A is replaced with biotin-labeled Ab2 at a concentration of 2 nM.

[0228] Specific results are shown in Table 12 and... Figure 11A and Figure 11B As shown in the figure. Experiments show that HankeL13 and HankeL3 have different antigen-binding epitopes from the positive control antibodies Ab1 and Ab2.

[0229] Table 12: Epitope Competition Detected by ELISA (OD) 450nm )

[0230]

[0231] 5.5. In vivo efficacy

[0232] A20 tumor model

[0233] The A20 mouse model reconstructed by PBMCs was used in the experiment to detect the in vivo antitumor efficacy of LAG-3 antibody. The B-NDG B2M KOplus mouse was created by simultaneously knocking out the B2m gene in the gene knockout mouse and expressing the B2m gene fused to the FcRn gene. This mouse combines the B-NDG mouse background with MHC class I deletion. The genetically engineered B-NDG B2M KOplus mouse was purchased from Biocytogen.

[0234] A20 cells (mouse B-cell lymphoma, purchased from Nanjing Cobioer, catalog number: Cobioer / cbp60279) were cultured to 80% confluence, digested with trypsin, centrifuged at 1000 rpm for 5 min, and the cells were collected, washed, centrifuged again, and resuspended in a resuspension solution to obtain a cell suspension. The cell viability was guaranteed to be >95%, and the cells were counted for later use.

[0235] A20 cell line was subcutaneously injected into the back (shaved side) of the test B-NDG B2M KO plus mice (6-8 weeks old, weighing 20±0.3 g) at a dose of 2×10⁻⁶ cells per mouse. 6 100 μl of cells). Five days later, human PBMCs were inoculated (5 × 10⁶ cells per mouse). 6 (cells). When the average tumor volume in tumor-bearing mice reaches approximately 50 mm... 3 Mice were randomly divided into 6 groups, with 6 mice in each group. A total of 5 experimental groups were designed: Ab1+Keytruda group, Ab2+Keytruda group, HankelL3+Keytruda group, HankelL13+Keytruda group, and Keytruda group, with physiological saline as the control.

[0236] The Ab1+Keytruda group received 100 μL of Ab1 and Keytruda solution (the solvent was physiological saline, and the solutes were Ab1 and Keytruda) per injection, with each Ab1 dose being 10 mg / kg body weight and each Keytruda dose being 10 mg / kg body weight, administered twice a week for a total of 2 weeks.

[0237] The Ab2+Keytruda group received 100 μL of Ab2 and Keytruda solution (solvent was physiological saline, solute was Ab2 and Keytruda) per injection, with each Ab2 dose being 10 mg / kg body weight and each Keytruda dose being 10 mg / kg body weight, administered twice a week for a total of 2 weeks;

[0238] The HankelL3+Keytruda group received 100 μL of HankelL3 and Keytruda solution (solvent was physiological saline, solute was HankelL3 and Keytruda) per injection, with each injection bringing the HankelL3 dose to 10 mg / kg body weight and the Keytruda dose to 10 mg / kg body weight per injection, twice a week for a total of 2 weeks;

[0239] The HankelL13+Keytruda group received 100 μL of HankelL13 and Keytruda solution (the solvent was physiological saline, and the solutes were HankelL13 and Keytruda) per injection, with each injection making the HankelL13 dose 10 mg / kg body weight and the Keytruda dose 10 mg / kg body weight per injection, twice a week for a total of 2 weeks;

[0240] The Keytruda group received 100 μL of Keytruda solution (physiological saline as solvent and Keytruda as solute) per injection, making the Keytruda dosage 10 mg / kg body weight per administration, twice a week for a total of 2 weeks;

[0241] The saline group served as the control group. Each mouse was subcutaneously injected with 100 μL of saline, and the administration was carried out twice a week for a total of 2 weeks.

[0242] Pembrolizumab injection (Keytruda, purchased from MSD Ireland, import drug registration certificate number: S20180019) should be diluted with physiological saline according to the required concentration and stored at 4°C.

[0243] After tumor inoculation, the animals' survival and activity levels were checked twice a week, including tumor growth, weight, activity level, and diet, and the results were recorded.

[0244] Tumor volume seen Figure 12 (Day 0 is the day of tumor inoculation). The results showed that, compared with saline, the antibodies in all experimental groups could inhibit the growth of A20 tumors without affecting the activity and weight of mice; among them, the HankelL13+Keytruda experimental group had the best tumor inhibition effect among all experimental groups.

[0245] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims. sequence list <110> Hefei Hanke Maibo Biotechnology Co., Ltd. <120> LAG-3-bound molecules and their applications <160> 12 <170> SIPOSequenceListing 1.0 <210> 1 <211> 450 <212> PRT <213> Artificial Sequence <400> 1 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Asn Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Tyr Pro Tyr Thr Gly Gly Thr Gly Tyr Asn Gln Lys Phe 50 55 60 Lys Asn Arg Val Thr Leu Thr Val Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Gly Asp Arg Tyr Glu Asp Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Lys 450 <210> 2 <211> 1350 <212> DNA <213> Artificial Sequence <400> 2 gaagtgcagc tggtgcagtc tggagcagaa gtgaagaagc caggagcttc cgtgaaggtg 60 tcttgtaagg cttccggcta tacctttacc gactacaaca tccattgggt gagacaggct 120 ccaggaaagg gcctcgagtg gatcggatac atctaccctt acacaggcgg cacaggctat 180 aatcagaagt tcaagaacag ggtgaccctg acagtggata catctatctc caccgcctac 240 atggaactgt ctagactgag atccgaggac acagcagtgt actattgcgc tagatccgga 300 gataggtacg aagacgctat ggactattgg ggacagggaa catcagtgac agtgtcttcc 360 gcttctacaa aggggccctc cgtgtttcct ctggctcctt cttctaagtc tacaagcgga 420 ggaacagcag ctctgggttg tctggtgaag gattacttcc cagagccagt gacagtgtct 480 tggaactccg gagctctgac ctcaggagtg catacatttc cagcagtgct gcagagttca 540 ggactgtatt ctctgtcttc cgtggtgaca gtgccttctt cttctctggg aacacagacc 600 tacatttgca acgtgaacca caagccctcc aacacaaagg tggacaagag agtggagcct 660 aagtcttgcg acaagaccca cacttgtcct ccttgtccag ctccagaagc agcaggagga 720 ccttccgtgt ttctgtttcc tcctaagcct aaggacaccc tgatgatctc cagaacacca 780 gaagtgactt gcgtggtggt ggacgtgtct cacgaggacc ccgaggtgaa gttcaattgg 840 tacgtggacg gagtggaagt gcataacgct aaaaccaagc ctagagagga gcagtacaac 900 tctacctaca gagtggtgtc agtgctgaca gtgctgcatc aggattggct gaacggaaag 960 gagtacaagt gcaaggtgtc caacaaggct ctgccagctc ctattgaaaa gaccatctct 1020 aaggctaagg gacagcctag agaacctcag gtgtacaccc tgcctccttc ccgggaggag 1080 atgaccaaga accaggtgtc tctgacttgt ctggtgaagg gattctaccc ttccgacatc 1140 gccgtcgagt gggaatctaa cggacagcca gagaacaact ataagaccac ccctcctgtg 1200 ctggattcag acggctcctt cttcctgtac tccaagctga ccgtggataa gtctaggtgg 1260 cagcagggaa acgtgttctc ttgtagcgtg atgcacgaag ctctgcataa ccactacaca 1320 cagaagtctc tgtctctgtc tccaggaaag 1350 <210> 3 <211> 213 <212> PRT <213> Artificial Sequence <400> 3 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Ser Leu Lys Leu Leu Ile 35 40 45 Ser Phe Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Gly Ile Gly His Trp Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro 100 105 110 Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr 115 120 125 Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys 130 135 140 Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu [[ID=第十八条]]145 150 155 160 Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser 165 170 175 Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala 180 185 190 Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe 195 200 205 Asn Arg Gly Glu Cys 210 <210> 4 <211> 639 <212> DNA <213> Artificial Sequence <400> 4 It should be noted that in the original text, "第十八条" seems to be an incorrect or mislabeled item. It has been left as it is in the translation for the purpose of maintaining the integrity of the original content. If this is an error, it may need to be corrected in the source material.gacatccaga tgacccagtc tccttcttct ctgtctgctt cagtgggaga tagagtgacc 60 atcacctgta gagcttctca ggacatctcc aactacctca actggtacca gcagaagcca 120 gacggatctc tgaagctgct gatctctttc acctccagac tgcattccgg agtgccttct 180 agattctctg gctccggctc tagaaccgac tttacactga caatctctag tctgcagcca 240 gaggacgtgg ctacatatta ttgccagcag ggaatcggcc attggacatt tggcggagga 300 acaaaggtgg agatcaagag aaccgtggct gctccttccg tgtttatttt ccctccttct 360 gacgaacagc tgaaatccgg aacagcttca gtcgtctgcc tgctgaacaa cttctaccct 420 agagaggcca aagtccagtg gaaagtggat aacgctctgc agtccggaaa ttctcaggaa 480 tccgtgaccg agcaggattc taaggattct acctactccc tgtcttctac cctgacactg 540 tctaaggccg attacgagaa gcacaaggtg tacgcttgcg aagtgacaca tcagggactg 600 tcttctccag tgaccaagtc cttcaacaga ggcgagtgt 639 <210> 5 <211> 106 <212> PRT <213> Artificial Sequence <400> 5 Asp Ile Gln Met Thr Gln Ser Thr Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Ser Leu Lys Leu Leu Ile 35 40 45 Ser Phe Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Arg Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Val Ala Thr Tyr Phe Cys Gln Gln Gly Ile Arg Gln Trp Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 6 <211> 120 <212> PRT <213> Artificial Sequence <400> 6 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Asn Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Tyr Pro Tyr Thr Gly Gly Thr Gly Tyr Asn Gln Lys Phe 50 55 60 Lys Asn Arg Val Thr Leu Thr Val Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Gly Asp Arg Tyr Asp Asp Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Ser Val Thr Val Ser Ser 115 120 <210> 7 <211> 106 <212> PRT <213> Artificial Sequence <400> 7 Asp Ile Gln Met Thr Gln Ser Thr Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Ser Leu Lys Leu Leu Ile 35 40 45 Ser Phe Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Arg Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Val Ala Thr Tyr Phe Cys Gln Gln Gly Ile Thr Leu Trp Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 8 <211> 446 <212> PRT <213> Artificial Sequence <400> 8 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Tyr 20 25 30 Phe Ile His Trp Val Arg Gln Arg Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asp Pro Glu Asn Ala Asp Thr Glu Tyr Asp Pro Lys Phe 50 55 60 Gln Gly Arg Ala Thr Met Thr Val Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Leu Glu Leu Ser Arg Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Asn Ala Arg Glu Pro Gly Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 260 265 270 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350 Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 9 <211> 1,338 <212> DNA <213> Artificial Sequence <400> 9 caggttcagc tggtgcagag cggagctgaa gtgaagaagc ccggagcttc cgtgaagctg 60 tcctgtacag cttccggctt caatatcaag gactacttca tccactgggt gcggcagaga 120 cctggacagg gactggagtg gatgggatgg atcgacccag agaacgctga caccgagtac 180 gatcccaagt tccagggcag ggctaccatg acagtggata ccagcatctc caccgcctac 240 ctggagttgt ccaggttgag aagcgaggac accgccgttt actattgcaa cgccagggag 300 cctggcctgg attattgggg acagggtacc ctggtgacag tgagctctgc ctctaccaag 360 gggcccagcg tgttcccact ggccccctct agcaagtcta ccagcggagg cacagccgcc 420 ctgggatgcc tggtgaagga ctacttccca gagccagtga ccgtgagctg gaactccggc 480 gccctgacca gcggagtgca cacatttcca gccgtgctgc agtcctctgg cctgtactcc 540 ctgagctccg tggtgaccgt gccctctagc tccctgggca cccagacata tatctgcaac 600 gtgaatcaca agccatctaa tacaaaggtg gacaagaagg tggagcccaa gagctgtgat 660 aagacccaca catgcccccc ttgtcctgca ccagaggccg ccggcggccc tagcgtgttc 720 ctgtttccac ccaagcctaa ggacaccctg atgatctccc ggaccccaga ggtgacatgc 780 gtggtggtgg acgtgtctca cgaggacccc gaggtgaagt ttaactggta cgtggatggc 840 gtggaggtgc acaatgccaa gaccaagcct cgggaggagc agtacaacag cacctataga 900 gtggtgtccg tgctgacagt gctgcaccag gactggctga acggcaagga gtataagtgc 960 aaggtgagca ataaggccct gcccgcccct atcgagaaga ccatctccaa ggccaagggc 1020 cagcctaggg agccacaggt ctatacactg cctccaagcc gcgacgagct gaccaagaac 1080 caggtgtccc tgacatgtct ggtgaagggc ttctatcctt ccgatatcgc cgtggagtgg 1140 gagtctaatg gccagccaga gaacaattac aagaccacac cccctgtgct ggactctgat 1200 ggcagcttct ttctgtattc taagctgacc gtggataaga gcaggtggca gcagggcaac 1260 gtgttttcct gctctgtgat gcacgaggcc ctgcacaatc actatacaca gaagagcctg 1320 tccctgtctc ccggcaag 1338 <210> 10 <211> 214 <212> PRT <213> Artificial Sequence <400> 10 Asp Val Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ser Ser Gln Asp Ile Gly Ser Tyr 20 25 30 Leu Asn Trp Phe Gln Gln Lys Pro Asp Gly Thr Ile Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Thr Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Gly Tyr Thr Leu Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 11 <211> 642<​​​​​​gatgtgcaga tgacccagag cccatcctcc ttgagcgctt ctctgggaga cagagtgacc 60 gatgtgcaga tgacccagag cccatcctcc ttgagcgctt ctctgggaga cagagtgacc 60 atcacctgca gatcctccca ggacatcggc tcttatctga actggttcca acagaagcct 120 atcacctgca gatcctccca ggacatcggc tcttatctga actggttcca acagaagcct 120 gacggcacca tcaagctgct gatctattac acctccaccc tgcactccgg cgtgccttcc 180 gacggcacca tcaagctgct gatctattac acctccaccc tgcactccgg cgtgccttcc 180 aggttctccg gatccggatc tggaaccgat ttcaccctga ccatcagcag cctgcagcca 240 aggttctccg gatccggatc tggaaccgat ttcaccctga ccatcagcag cctgcagcca 240 gaagactttg ccacctactt ttgccagcag ggctacaccc tgccttatac ctttggccag 300 gaagactttg ccacctactt ttgccagcag ggctacaccc tgccttatac ctttggccag 300 ggtaccaagc tggagatcaa gaggaccgtg gccgctccat ccgtgttcat ctttccccct 360 ggtaccaagc tggagatcaa gaggaccgtg gccgctccat ccgtgttcat ctttccccct 360 agcgacgagc agctgaagag cggcacagct tctgtggtgt gcctgctgaa caatttctac 420 agcgacgagc agctgaagag cggcacagct tctgtggtgt gcctgctgaa caatttctac 420 cccagggagg ccaaggtgca gtggaaggtg gataacgctc tgcagagcgg caattctcag 480 cccagggagg ccaaggtgca gtggaaggtg gataacgctc tgcagagcgg caattctcag 480 gagtccgtga ccgagcagga cagcaaggat tctacatatt ccctgagctc taccctgaca 540 gagtccgtga ccgagcagga cagcaaggat tctacatatt ccctgagctc taccctgaca 540 ctgagcaagg ccgactacga gaagcacaag gtgtatgctt gcgaggtgac ccatcagggc 600 ctgagcaagg ccgactacga gaagcacaag gtgtatgctt gcgaggtgac ccatcagggc 600 ctgtccagcc ccgtgacaaa gtcttttaac aggggcgagt gt 642 ctgtccagcc ccgtgacaaa gtcttttaac aggggcgagt gt 642 <210> 12 <210> 12 <211> 116 <211> 116 <212> PRT <212> PRT <213> 人工序列(Artificial Sequence) <213> Artificial Sequence <400> 12 <400> 12 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Tyr 20 25 30 Phe Ile His Trp Val Arg Gln Arg Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Asp Pro Lys Phe 50 55 60 Gln Gly Arg Ala Thr Met Thr Val Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Leu Glu Leu Ser Arg Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Asn Ala Arg Glu Pro Gly Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115

Claims

1. A LAG-3 binding molecule, characterized by: The LAG-3 binding molecule comprises a LAG-3 antibody, or an antigen-binding fragment of the LAG-3 antibody, or an antibody-drug conjugate containing the LAG-3 antibody, or an antibody-drug conjugate containing the antigen-binding fragment, or a bispecific antibody containing the LAG-3 antibody, or a bispecific antibody containing the antigen-binding fragment. The LAG-3 binding molecule contains a heavy chain variable region and a light chain variable region, and the heavy chain variable region and light chain variable region contain the CDR sequence described below (13H4). The amino acid sequences of HCDR1 in the heavy chain variable region (13H4) are shown as positions 26-35 of SEQ ID No. 1, positions 50-66 of SEQ ID No. 1, and positions 99-109 of SEQ ID No. 1 or SEQ ID No. 6; the amino acid sequences of LCDR1 in the light chain variable region are shown as positions 24-34 of SEQ ID No. 3, positions 50-56 of SEQ ID No. 3, and positions 89-96 of SEQ ID No. 3, SEQ ID No. 5, or SEQ ID No.

7.

2. The LAG-3 binding molecule as described in claim 1, characterized in that: The amino acid sequence of the heavy chain variable region (13H4) is shown in positions 1-120 of SEQ ID No. 1 or SEQ ID No. 6; the amino acid sequence of the light chain variable region is shown in positions 1-106 of SEQ ID No. 3 or SEQ ID No. 5 or SEQ ID No.

7.

3. The LAG-3 binding molecule as described in claim 1 or 2, characterized in that: The LAG-3 antibody further includes a heavy chain constant region and a light chain constant region. The heavy chain constant region is of the type IgG, IgM, IgE, IgA or IgD; the light chain constant region is of the type κ chain or λ chain.

4. The LAG-3 binding molecule as described in claim 3, characterized in that: The amino acid sequence of the heavy chain of the LAG-3 antibody is shown in SEQ ID No. 1, and the amino acid sequence of the light chain is shown in SEQ ID No.

3.

5. The LAG-3 binding molecule as described in claim 1 or 2, characterized in that: The antigen-binding fragments include one or more combinations of Fab, Fab', F(ab')2, Fab'-SH, Fv, and ScFv.

6. A nucleic acid molecule, characterized by: The nucleic acid molecule is a nucleic acid molecule encoding the LAG-3 binding molecule as described in any one of claims 1 to 5.

7. An expression box, characterized in that: The expression cassette contains the nucleic acid molecule as described in claim 6.

8. A recombinant vector, characterized in that: The recombinant vector contains the nucleic acid molecule of claim 6 or the expression cassette of claim 7.

9. Recombinant microorganisms, characterized by: The recombinant microorganism contains the nucleic acid molecule of claim 6, the expression cassette of claim 7, or the recombinant vector of claim 8.

10. Recombinant cells, characterized by: The recombinant cells contain the nucleic acid molecule of claim 6, the expression cassette of claim 7, or the recombinant vector of claim 8.

11. The nucleic acid molecule according to claim 6, characterized in that: The nucleic acid molecule includes at least one of the following: g1) A DNA molecule whose coding sequence is shown as positions 76-105 of SEQ ID No. 2; g2) The coding sequence of the coding strand is shown in positions 148-198 of SEQ ID No. 2 for the DNA molecule; g3) The coding sequence of the coding strand is as shown in positions 295-327 of SEQ ID No. 2 for the DNA molecule; g4) The coding sequence of the coding strand is shown in positions 69-102 of SEQ ID No. 4 for the DNA molecule; g5) The coding sequence of the coding strand is as shown in positions 148-168 of SEQ ID No. 4 for a DNA molecule; g6) The coding sequence of the coding strand is as shown in positions 265-288 of SEQ ID No. 4 for the DNA molecule; g7) The coding sequence of the coding strand is as shown in nucleotides 1-360 of SEQ ID No. 2 for the DNA molecule; g8) The coding sequence of the coding strand is as shown in nucleotides 1-318 of SEQ ID No. 4 for a DNA molecule; g9) The coding sequence of the coding strand is as shown in SEQ ID No. 2 of the DNA molecule; The coding sequence of the g10 coding strand is shown in SEQ ID No. 4 of the DNA molecule.

12. A drug or drug composition, characterized in that: The drug or drug composition contains the LAG-3 binding molecule as described in any one of claims 1-5.

13. The medicament or pharmaceutical composition according to claim 12, characterized in that: The drug or drug composition also includes an anti-PD-1 antibody.

14. The use of the LAG-3 binding molecule according to any one of claims 1-5 in the preparation of LAG-3 inhibitors.

15. The use of the nucleic acid molecule of claim 6 in the preparation of LAG-3 inhibitors.

16. The use of the expression cassette according to claim 7 in the preparation of LAG-3 inhibitors.

17. The use of the recombinant vector according to claim 8 in the preparation of LAG-3 inhibitors.

18. The use of the recombinant microorganism according to claim 9 in the preparation of LAG-3 inhibitors.

19. The use of the recombinant cells of claim 10 in the preparation of LAG-3 inhibitors.

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