CN115724969A - LAG-3 binding molecules and uses thereof - Google Patents

Abstract

The present invention discloses LAG-3 binding molecules comprising a LAG-3 antibody, or an antigen-binding fragment of said LAG-3 antibody, or a fusion protein comprising said antigen-binding fragment, or an antibody drug conjugate comprising said LAG-3 antibody, or an antibody drug conjugate comprising said antigen-binding fragment, or a bispecific antibody comprising said LAG-3 antibody, or a bispecific antibody comprising said antigen-binding fragment. The LAG-3 binding molecules provided by the present invention bind human and monkey LAG-3, exhibit high affinity for human LAG-3 and effectively enhance T cell responses, can be used to modulate T cell and antibody-mediated immune responses, and have a wide range of therapeutic uses as immunomodulators, such as cancer, autoimmune diseases, inflammatory diseases, and infectious diseases.

Description

LAG-3 binding molecules and uses thereof

Technical Field

The invention relates to the field of antibody engineering, in particular to LAG-3 binding molecules and application thereof.

Background

Lymphocyte activation gene 3 (LAG-3), also known as CD223, is a transmembrane protein composed of 3 parts of extracellular, transmembrane and intracellular regions. The extracellular region consists of 4 parts including Domain1, domain2, domain3 and Domain 4. Human LAG-3 has 498 amino acids with a relative molecular weight of 70kDa and is located on human chromosome 12 (20p13.3). LAG-3 was originally identified as expressed on the surface of T cells (particularly activated T cells), natural killer cells, B cells, and plasmacytoid dendritic cells. LAG-3 is an inhibitory receptor that specifically binds to MHC class II molecules on the surface of APC by forming dimeric molecules through the D1 domain, negatively regulates T cell expansion and controls the memory T cell pool by signaling through the highly conserved KIEELE sequence of the cytoplasmic domain. LAG-3 also has a direct regulatory effect on the suppressive function of CD4+, CD25+ regulatory T cells (Treg cells), and is an essential molecule for Treg cell function.

In addition to MHC class II molecules, LAG-3 has been reported to bind three other ligands, fibrinogen protein 1 (FGL-1), antral endothelial 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 also affected.

In addition, LAG-3 plays an important role in maintaining immune homeostasis, and has obvious marker effect on the expression level of the LAG in autoimmunity and cancer. LAG-3 has a protective effect in autoimmunity, and the blocking and deletion of LAG-3 gene can accelerate the progress of autoimmune diseases. In cancer, where LAG-3 is often indicative of incapacitated or depleted T cells, and chronic viral infection, LAG-3 is up-regulated in expression and inhibits immune function of T cells. In addition, LAG-3 is highly expressed in animal T cells with resistance to PD-1, and studies show that the blocking of LAG-3 and PD1 pathways in preclinical solid tumor and blood tumor models can better improve the function of anti-tumor effect and inhibit tumor growth than the blocking of PD1 pathway alone. Therefore, the LAG-3 antibody and the PD-1 antibody are combined to improve the drug resistance problem generated when the PD-1 antibody is used.

No LAG-3 inhibitor drugs are currently on the market, and the leading LAG-3 inhibitors are in clinical trials as anti-cancer therapeutics or are being recruited as anti-cancer therapeutics, such as BMS-986016, a fully human IgG4 monoclonal antibody against LAG-3, developed by Bristol-Myers Squibb, inc., MK-4280, a fully human IgG4 monoclonal antibody against LAG-3, developed by Merck Sharp and Dohme, inc., used primarily in combination with anti-PD-1/PD-L1 antibodies to treat a variety of solid and hematological malignancies. LAG-3 plays an important clinical significance in tumor immunotherapy as a novel immunotherapy target, but at present, many malignant tumors have evidence that no clear immunotherapy benefit is obtained, and the application range and the treatment effectiveness of anti-LAG-3 antibody immunotherapy still have a great research space.

Disclosure of Invention

It is an object of the present invention to provide LAG-3 binding molecules that enable more cancer patients to benefit from treatment.

In a first aspect, the invention provides LAG-3 binding molecules, the LAG-3 binding molecules comprising a LAG-3 antibody, or an antigen-binding fragment of the LAG-3 antibody, or a fusion protein comprising the antigen-binding fragment, or an antibody drug conjugate comprising the LAG-3 antibody, or an antibody drug conjugate comprising the antigen-binding fragment, or a bispecific antibody comprising the LAG-3 antibody, or a bispecific antibody comprising the antigen-binding fragment, the LAG-3 binding molecules comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region and the light chain variable region comprising CDR region sequences of 13H 4) or 3F 6) below,

13H4) The amino acid sequence of HCDR1 in the heavy chain variable region is shown as 26 th-35 th sites of SEQ ID No.1, the amino acid sequence of HCDR2 is shown as 50 th-66 th sites of SEQ ID No.1, and the amino acid sequence of HCDR3 is shown as 99 th-109 th sites of SEQ ID No.1 or 99 th-109 th sites of SEQ ID No. 6; the amino acid sequence of LCDR1 in the light chain variable region is shown as SEQ ID No.3, the amino acid sequence of LCDR2 is shown as SEQ ID No.3, the amino acid sequence of LCDR3 is shown as SEQ ID No.3, the positions 89-96 or SEQ ID No.5, the positions 89-96 or SEQ ID No.7, the positions 89-96;

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

The CDRs of the invention are "complementarity determining regions," which are regions of antibody variable domains that are mutated in sequence and form structurally defined "hypervariable loops" and/or contain antigen-contacting residues "antigen-contacting points. The CDRs are primarily responsible for binding to an epitope of the antigen. The CDRs of the heavy and light chains are commonly referred to as CDR1, CDR2 and CDR3, numbered sequentially from the N-terminus. The CDRs located within the antibody heavy chain variable domain are referred to as HCDR1, HCDR2 and HCDR3, while the CDRs located within the antibody light chain variable domain are referred to as LCDR1, LCDR2 and LCDR3.

Further, the LAG-3 binding molecule as described above, 13H 4) wherein the heavy chain variable region has an amino acid sequence as set forth in positions 1 to 120 of SEQ ID No.1 or SEQ ID No.6, or has at least 80% identity (where non-uniformity is preferably in the FR region) with the amino acid sequence set forth in positions 1 to 120 of SEQ ID No.1 or SEQ ID No. 6; the light chain variable region has an amino acid sequence as set forth 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 (where the inconsistency is preferably in the FR region) with an amino acid sequence as set forth in positions 1-106 of SEQ ID No.3 or SEQ ID No.5 or SEQ ID No. 7;

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

Further, in the above LAG-3 binding molecules, the LAG-3 antibody further comprises a heavy chain constant region and a light chain constant region, and the heavy chain constant region can be any one of IgG, igM, igE, igA, or IgD; the light chain type of the antibody may be a kappa chain or a lambda chain.

The IgG may be any one of IgG1, igG2, igG3 and IgG4, or a mutant thereof.

The IgG1 can be murine IgG1, human IgG1, or a mutant thereof.

The human IgG1 mutant can be an IgG1 mutant with mutations of the L234A and/or L235A (Kabat counting).

Specifically, the amino acid sequence of the heavy chain constant region is shown as the 121 th to 450 th positions of SEQ ID No. 1.

The light chain type of the antibody of the present invention may be a kappa chain.

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

Further, in the above LAG-3 binding molecule, the amino acid sequence of the heavy chain of the LAG-3 antibody is represented by SEQ ID No.1 or has at least 80% identity with the amino acid sequence represented by SEQ ID No.1, and the amino acid sequence of the light chain is represented by SEQ ID No.3 or has at least 80% identity with the amino acid sequence represented by SEQ ID No. 3; or the like, or a combination thereof,

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

In the above applications, identity refers to the identity of amino acid sequences or nucleotide sequences. The identity of the amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST web pages of the NCBI home website. For example, in advanced BLAST2.1, by using blastp as a program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as Matrix, setting Gap existence cost, per response Gap cost, and Lambda ratio to 11,1 and 0.85 (default values), respectively, and searching for identity of a pair of amino acid sequences to calculate, then, a value (%) of identity can be obtained.

In the above applications, the 80% or more identity may 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%, 99.9% identity.

The antibodies provided by the invention have one or more of the following properties or characteristics:

1) Specifically binds human LAG-3;

2) Can bind to cynomolgus monkey LAG-3;

3) Weak binding to murine LAG-3;

4) To some extent, can block the combination of ligands such as MHCII molecules and FGL1 and human LAG-3;

5) Is IgG, such as IgG1, igG2, igG3 or IgG4, or a mutant of said IgG;

6) Is a humanized or chimeric or murine antibody.

7) Domain1, which binds predominantly to the extracellular Domain of human LAG-3.

The antigen-binding fragment of the LAG-3 binding molecule described above comprises: fab, fab ', F (ab') ₂ One or a plurality of combinations of Fab' -SH, fv and ScFv.

Fab fragments are obtained by papain digestion of antibodies, and comprise a heavy chain variable domain and a light chain variable domain, and also a constant domain of the light chain and a first constant domain of the heavy chain (CH 1).

Fab' fragments differ from Fab fragments by the addition of residues (including one or more cysteines from the antibody hinge region) at the carboxy terminus of the heavy chain CH1 domain on a Fab basis.

F (ab ') 2 is obtained by pepsin digestion of whole IgG antibodies (after removal of most of the Fc region while leaving some hinge region intact, F (ab') ₂ Fragments have two antigen-binding F (ab) moieties linked together by a disulfide bond, thus F (ab') ₂ The fragment is bivalent.

Fab '-SH is the designation for Fab' in which the cysteine residues of the constant domains carry a free thiol group. F (ab ') 2 antibody fragments were originally generated as pairs of Fab ' fragments with hinge cysteines between the Fab ' fragments.

Fv is the smallest antibody fragment that contains the entire antigen binding site. A two-chain Fv species consists of dimers of one heavy chain variable domain and one light chain variable domain in tight, non-covalent association. In single chain Fv (scFv) species, one heavy chain variable domain and one light chain variable domain may be covalently linked by a flexible peptide linker so that the light and heavy chains may associate in a "dimeric" structure similar to that of a two-chain Fv species. In this configuration, it is the three CDRs of each variable domain that act to define the antigen binding site on the surface of the VH-VL dimer.

scFv or single chain Fv, refers to antibody fragments comprising the VH domain and the VL domain of an antibody, wherein these domains are present as a single polypeptide chain. Typically, the Fv polypeptide further comprises a polypeptide linker between the VH domain and the VL domain that enables the scFv to form the desired structure for antigen binding.

"bispecific antibody" refers to an antibody having a binding site that binds to two different antigens, or an antibody having a binding site that binds to different epitopes of the same antigen.

An Antibody Drug Conjugate (ADC) is a drug formed by coupling an antibody with targeting specificity and a small molecule drug with high toxicity through a connecting peptide.

In a second aspect of the present invention, there is provided a biomaterial related to the LAG-3 binding molecule as described above, which is any one of:

b1 Nucleic acid molecules encoding the LAG-3 binding molecules described above;

b2 An expression cassette comprising the nucleic acid molecule according to B1);

b3 A recombinant vector containing the nucleic acid molecule according to B1) or containing the expression cassette according to B2);

b4 A recombinant microorganism containing the nucleic acid molecule according to B1) or containing the expression cassette according to B2) or containing the recombinant vector according to B3);

b5 An animal cell line containing the nucleic acid molecule according to B1) or containing the expression cassette according to B2) or containing the recombinant vector according to B3);

b6 A plant cell line containing the nucleic acid molecule according to B1) or containing the expression cassette according to B2) or containing the recombinant vector according to B3);

b7 Host cells producing the LAG-3 binding molecules described above.

Further, in the above-mentioned biomaterial, the nucleic acid molecule according to B1) comprises:

g1 A DNA molecule having the coding sequence of the coding strand shown in positions 76-105 of SEQ ID No. 2;

g2 A DNA molecule having the coding sequence of the coding strand as shown in SEQ ID No.2 at positions 148-198;

g3 A DNA molecule whose coding sequence of the coding strand is shown in the 295-327 th position of SEQ ID No. 2;

g4 A DNA molecule having the coding sequence of the coding strand shown in positions 69 to 102 of SEQ ID No. 4;

g5 A DNA molecule having the coding sequence of the coding strand as shown in SEQ ID No.4 at positions 148-168;

g6 A DNA molecule whose coding sequence of the coding strand is shown in the 265 th to 288 th positions of SEQ ID No. 4;

g7 A DNA molecule with the coding sequence of the coding chain as shown in the 1 st to 360 th nucleotides of SEQ ID No. 2;

g8 A DNA molecule with the coding sequence of the coding chain as shown in the 1 st to 318 th nucleotides of SEQ ID No. 4;

g9 A DNA molecule with the coding sequence of the coding chain as shown in SEQ ID No. 2;

g10 A DNA molecule with the coding sequence of the coding chain as shown in SEQ ID No. 4;

g11 A DNA molecule having the coding sequence of the coding strand shown in positions 76-105 of SEQ ID No. 9;

g12 A DNA molecule having the coding sequence of the coding strand as shown in SEQ ID No.9 at positions 148-198;

g13 A DNA molecule having the coding sequence of the coding strand as shown in SEQ ID No.9 at position 277-315;

g14 A DNA molecule having the coding sequence of the coding strand shown in positions 69-102 of SEQ ID No. 11;

g15 A DNA molecule having the coding sequence of the coding strand as shown in SEQ ID No.11 at positions 148-168;

g16 A DNA molecule whose coding sequence of the coding strand is shown as 265 th to 291 th positions of SEQ ID No. 11;

g17 A DNA molecule with the coding sequence of the coding chain as shown in the 1 st to 348 th nucleotides of SEQ ID No. 9;

g18 A DNA molecule with the coding sequence of the coding chain as shown in the 1 st to 321 st nucleotides of SEQ ID No. 11;

g19 A DNA molecule with the coding sequence of the coding chain as shown in SEQ ID No. 9;

g20 A DNA molecule shown as SEQ ID No. 11) encoding the coding sequence of the coding strand.

In a third aspect of the invention, there is provided a medicament or pharmaceutical composition comprising the LAG-3 binding molecule as defined above.

Further, the medicament or pharmaceutical composition further comprises PD-1 antibodies, including but not limited to: pembrolizumab, nivolumab, torpilimab, sintillomab, camrelizumab, tislelizumab, cemiplimab and/or Prolgolimab.

The medicament or pharmaceutical composition may be a LAG-3 inhibitor (a substance that inhibits the activity of LAG-3).

In a fourth aspect, the present invention provides the use of the above-described LAG-3 binding molecule or a biological material associated with the above-described LAG-3 binding molecule in the preparation of a LAG-3 inhibitor.

Hereinbefore, the LAG-3 inhibitor may be used for the treatment of diseases including cancer.

Such cancers include, but are not limited to: b-lymphocytic lymphoma, melanoma, non-small cell lung cancer, soft tissue cell carcinoma, head and neck cancer, head and neck squamous cell carcinoma, gastric cancer, esophageal cancer, MSS colorectal cancer, chordoma, hematologic tumor, non-hodgkin lymphoma, cervical cancer, endometrial cancer, pancreatic cancer, breast cancer, peritoneal cancer, and/or renal cell carcinoma.

The LAG-3 binding molecules of the present invention have inhibitory activity and inhibit the down-regulation of immune cells by LAG-3, thereby stimulating CD4 ⁺ T and CD8 ⁺ T cells are proliferated, the expression quantity of IFN-gamma is obviously increased, and in vivo experiments prove that the combined administration of the LAG-3 antibody and the PD-1 antibody can obviously inhibit the growth of tumors. The above experimental results show that the LAG-3 binding molecules can regulate and control the immune system by regulating the activity of immune cells, and can be applied to immune enhancers of immune enhancer antitumor or antiviral immune response, or immune modulators of T cell mediated autoimmune diseases.

Drawings

FIG. 1A shows FACS detection of the affinity of the first murine antibody to human LAG-3.

FIG. 1B shows FACS detection of the affinity of a second portion of murine antibody to human LAG-3.

FIG. 2 is an experiment showing that chimeric antibody stimulates T cells to release IFN-. Gamma..

FIG. 3 shows the change in tumor volume in the in vivo potency test of the chimeric antibody.

FIG. 4A shows FACS detection of the affinity of the first 13H4 humanized antibody to human LAG-3.

FIG. 4B shows FACS detection of the affinity of the second portion of the 13H4 humanized antibody to human LAG-3.

FIG. 4C shows FACS detection of the affinity of the third portion of the 13H4 humanized antibody to human LAG-3.

FIG. 5A shows FACS detection of the affinity of the first 3F6 humanized antibody to human LAG-3.

FIG. 5B shows FACS detection of the affinity of the second portion of 3F6 humanized antibody to human LAG-3.

FIG. 6 is an affinity assay of 13H4 affinity matured single chain antibody with human LAG-3.

FIG. 7 is an affinity assay for human LAG-3 for the 13H4 affinity matured antibody.

FIG. 8A is the affinity of the IgG1 subtype of humanized antibodies for binding to human LAG-3.

FIG. 8B is the affinity of IgG1 subtype of humanized antibodies for binding to monkey LAG-3.

FIG. 8C is the affinity of the IgG1 subtype of humanized antibodies for binding to murine LAG-3.

FIG. 9 shows the binding blocking activity of the humanized antibody against MHC II.

Fig. 10 shows binding blocking activity of the humanized antibody to FGL 1.

Figure 11A is a competition of the epitope of the humanized antibody with the control antibody Ab 1.

Figure 11B is a competition of the epitope of the humanized antibody with the control antibody Ab2.

FIG. 12 shows the in vivo efficacy of humanized antibodies in tumor volume changes.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

In the quantitative tests in the following examples, three replicates were set up and the results averaged.

In the following examples, positive control antibody Ab1 was BMS-986016 (Relatimab, U.S. Pat. No.: US 9505839B 2) from Bristol Myers Squibb, and positive control antibody Ab2 was MK4280 from Merck Sharp & Dohme (U.S. Pat. No.: US20170097333A 1). The negative control antibody was human IgG (product of jinsburg).

In the following examplesThe vector pcDNA3.4 is Invitrogen Cat: A14697 pcDNA ^TM 3.4

And (3) a carrier.

ExpicCHO-S cells (purchased from Saimer Fei, cat # 29127) in the examples below

HEK293F cells (purchased from seimer fly, cat # a 14527) conventional equipment and reagents are as follows in the following examples:

1. a 96-well microplate (Nunc Corp.);

2. plating buffer solution: naHCO3 solution with concentration of 0.05M;

3. wash solution (PBST): phosphate buffer at pH 7.0 containing only 0.05% by volume of Tween 20;

4. sealing liquid: washing containing only 10g/L BSA.

5. Horse radish peroxidase labeled avidin;

6. chromogenic substrate: tetramethyl benzidine;

7. stopping liquid: 1M sulfuric acid.

Example 1 mouse immunization and hybridoma screening

1.1 immunization of mice

Selecting 3 Balb/C mice and C57b1/6 mice with the age of 4-6 weeks, simultaneously immunizing the mice by taking human LAG-3 extracellular domain and monkey LAG-3 extracellular domain as antigens, and immunizing for 1 time every 2 weeks and 4 times in total. And (3) collecting blood from tail veins after 3 rd immunization, detecting antibody titer by ELISA, fusing high-titer mouse spleen cells with myeloma cells SP2/0, and using the obtained hybridoma cells for further screening.

1.2 screening of Positive clones by ELISA

ELISA plates were coated with 1. Mu.g/ml recombinant human LAG-3 (23L-450L), overnight at 4 ℃ and blocked. Washing the plate with PBST for 3 times, sequentially adding 1.1 of hybridoma cell culture supernatant, incubating at 37 deg.C for 1 hr, washing the plate with PBST for 3 times, adding 4000-fold diluted goat anti-mouse IgG-HRP (Thermo Fisher Co.) and incubating at 37 deg.C for 45min, washing the plate with PBST for 3 times, adding color development liquid TMB for color development for 15 min, and measuring absorbance at 450 nm. The primary screened positive clones continue to be subcloned until all subcloned cell culture supernatants are detected as 100% positive. And (3) carrying out amplification culture on the monoclonal cell strain with positive clone and good growth vigor to obtain the LAG-3 monoclonal antibody hybridoma cell strain, and freezing for later use.

1.3 purification and concentration of murine antibody

And (3) carrying out step-by-step expansion culture on the hybridoma cell strain obtained by screening the 1.2, centrifuging cell culture solution at 10000rpm for 10 minutes, and taking supernatant. Purifying the obtained supernatant with Protein G affinity chromatography column, and the specific operation method is as follows: the Protein G column (GE company) was equilibrated with PBS, and then the culture supernatant was passed through the column, 5 column volumes were pre-eluted with solution A (formulation: solvent: water, solute and concentration: 20mM sodium phosphate, 500mM NaCl, pH 5.0), 5 column volumes were eluted with solution B (formulation: solvent: water, solute and concentration: 20mM sodium acetate, 150mM NaCl, pH 3.5), elution peaks were collected, and then concentrated with a 30KDa concentration centrifuge tube to obtain a concentrated solution containing the antibody.

1.4 subtype identification

Primarily screening to obtain 8 hybridoma cell strains in total, wherein the hybridoma cell strains are HKL3F6, HKL15A10, HKL5E10, HKL11B6, HKL13H4, HKL9A1, HKL7E5 and HKL1G8; the antibody subtypes are identified as IgG1, kappa type. The anti-LAG-3 antibody obtained was sequenced and showed a molecular weight of 150Kb, comprising a heavy chain and a light chain, typical of intact antibodies.

1.5 FACS determination of the affinity of murine anti-LAG-3 antibodies to human LAG-3

The pcDNA3.4-HuLAG3 plasmid is transfected into ExpicHO-S cells, the cells are collected for detection after 24h, and the obtained cells expressing human LAG-3 are named as CHO-S/pcDNA3.4-HuLAG3. Trypsinizing CHO-S/pcDNA3.4-HuLAG3 cells, collecting cells, centrifuging to remove supernatant, counting by resuspension at 1xPBS, adding 200. Mu.L of 2x10 ⁵ The cells were centrifuged at 1000rpm for 2min in an EP tube, washed once with 1% BSA (in PBS), and the supernatant was removed. The murine anti-LAG-3 antibody secreted by hybridoma was used as the test antibody, the test antibody was diluted to 20. Mu.g/ml with 1% BSA as the starting concentration, diluted 4-fold to obtain 6 antibodies with different concentrations, and 100. Mu.l of each antibody dilution was added to the sample containing CHO-S/pcDNIn EP tubes containing A3.4-HuLAG3 cells, the cells were resuspended, mixed well, and incubated at 4 ℃ for 1 hour in the dark. After completion of the incubation, 400. Mu.l of 2% BSA in 1xPBS 2000rpm was added and centrifuged for 5 minutes to discard the supernatant, and this operation was repeated once. 100 μ l of goat anti-mouse IgG-FITC (Jackson) diluted 200-fold was added to each EP tube, incubated at room temperature for 0.5 hour, 400. Mu.l of 2-BSA-containing 1xPBS 2000rpm was added, centrifuged for 5 minutes, the supernatant was discarded, this operation was repeated once, after washing, 400. Mu.l of 1xPBS was added to resuspend the cells, followed by detection by an up-flow cytometer, and the data was processed using GraphPad Prism statistical software, as shown in Table 1, and in FIGS. 1A and 1B.

The results show that: the hybridoma secreting antibodies were all able to specifically bind to CHO-S cells expressing human LAG-3, with HKL3F6 and HKL13H4, HKL9A1 having higher affinity.

Table 1: FACS determination of the affinity of the antibody secreted by hybridomas

Numbering	HKL3F6	HKL15A10	HKL5E10	HKL11B6	HKL13H4	HKL9A1	HKL7E5	HKL1G8
									EC50(ug/ml)	0.982	0.861	4.417	0.937	0.505	0.395	1.939	3.545
R2	0.999	0.998	1	0.998	0.999	0.997	0.999	0.998

1.6, determining affinity of murine antibody and cynomolgus monkey LAG-3 by ELISA method

The affinity of hybridoma-secreted antibodies for recognition of monkey LAG-3 was determined by ELISA. The method comprises the following specific steps: with NaHCO ₃ Monkey LAG-3 (Jackson) was diluted to 2. Mu.g/mL, 100. Mu.L per well was added to the microplate and incubated overnight at 4 ℃; PBST plate washing 3 times; 350ul 1% BSA per well (Sangon Biotech) was added to the microplate and incubated at 37 ℃ for 2 hours after blocking; PBST plate washing 3 times; PBST plate washing 3 times; 1, diluting the to-be-detected LAG-3 antibody to 1000ng/mL by BSA (bovine serum albumin), diluting for 6 times by 4 times to obtain the concentration of 7 to-be-detected samples, adding 100 mu L of to-be-detected LAG-3 antibody per hole into an enzyme label plate, and incubating for 1 hour at room temperature; PBST plate washing 3 times; 1% BSA diluted goat anti-mouse IgG-HRP 4000 times, 100. Mu.l per well was added to the ELISA plate and incubated at room temperature for 0.5 hours; PBST plate washing 3 times; adding 50 mu L of TMB into each hole, and developing for 15 minutes at room temperature in a dark place; adding 2mol/LH to 50 mu L of each hole ₂ SO ₄ TerminateCarrying out color development reaction; placing the enzyme label plate into a SepctraMax Versa enzyme label instrument, and determining OD _450nm Values, the results were counted using GraphPad Prism and EC50 values were calculated, and the results are shown in table 2. The results show that: the murine antibody secreted by the hybridoma recognizes monkey LAG-3.

Table 2: mouse antibody identification of monkey LAG-3

Number of	HKL1G8	HKL3F6	HKL5E10	HKL7E5	HKL9A1	HKL11B6	HKL13H4	HKL15A10
									EC50(ng/ml)	4.746	5.798	0.035	2.50	0.109	43.450	5.518	0.532
R 2	0.9520	0.9382	0.9904	0.9548	0.9800	0.9548	0.9586	0.9504

HKL13H4 and HKL3F6 and HKL9A1 showed the highest affinity for the chimeric antibody construction, as determined by affinity for human LAG-3 and affinity for monkey LAG-3.

Example 2 chimeric antibody construction and screening

2.1 construction of chimeric antibodies

HKL3F6, HKL13H4 and HKL9A1 hybridoma cells were collected and sent to Nanjing Kinsry Biotech, inc., limited Biotech for sequencing. The amino acid sequence of the murine antibody obtained by sequencing is as follows:

table 3: HKL13H4, HKL3F6 and HKL9A1 variable region amino acid sequences

The amino acid sequences of the light and heavy chain variable regions of HKL3F6, HKL13H4, HKL9A1 were grafted to the constant regions of human kappa appa and IgG1, and gene synthesis was performed after reverse translation of the amino acid sequences into DNA. Further, the chimeric antibody is constructed on a pcDNA3.4 vector by a genetic engineering technology, and a transient transfection HEK293F cell expression system is carried out for protein expression to obtain chimeric antibodies CHL3F6, CHL13H4 and CHL9A1. The harvested chimeric antibody was purified and concentrated by the method of 1.3 and stored for further use.

2.2 detection of PBMC activation Effect of chimeric antibodies

(1) Staphylococcus aureus enterotoxin B (SEB, available from sigma-aldrich) was diluted to 0.05ng/ml with PBS to obtain SEB solution chimeric antibodies CHL3F6, CHL9A1, CHL13H4 were diluted with PBS to obtain chimeric antibody solutions with concentrations of 10. Mu.g/ml, 0.33. Mu.g/ml, and 0.01. Mu.g/ml, respectively. The SEB solution and the antibody solution are respectively and uniformly mixed according to the volume ratio of 1:1, and then added into a 96-well plate package plate, 50 mu L/well and 3 compound wells are arranged, and 50 mu L/well SEB solution is used as a control. The 96-well plate was incubated at 37 ℃ for 1 hour. Before use, the cells were washed three times with PBS.

(2) Human PBMC cell isolation: whole blood was collected from healthy humans, and PBMC cell count was obtained by isolation using lymphocyte separation medium (Sigma).

(3) According to 5X10 ⁴ Addition amount per well PBMC cells were added to the 96-well cell culture plate of step (1), and the 96-well plate was placed at 37 ℃ 5% ₂ Culturing in an incubator for 3 days, collecting cell supernatant, and detecting the secretion of the cell factor IFN-gamma, wherein the experimental result is shown in figure 2.

The results show that: the chimeric antibodies CHL3F6, CHL9A1 and CHL13H4 can stimulate PBMC cells to activate and secrete cytokines IFN-gamma, wherein the CHL3F6 and CHL9A1 have the best effect.

2.3 in vivo drug action

Experimental selection B-hPD-1/hLAG3 mice were used as test mice to test the in vivo antitumor effect of LAG-3 antibody, and B-hPD-1/hLAG3 mouse model was a genetically engineered mouse in which a human HuLAG-3 gene and a HuPD-1 gene were chimeric in the genome of a C57BL/6 mouse in genetic background and purchased from Poiosael.

Culturing MC38 cells (mouse colon cancer cells, purchased from Nanjing Kebai) to 80% full, digesting with pancreatin, centrifuging at 1000rpm for 5min, collecting cells, cleaning, centrifuging, and resuspending cells to obtain cell suspension, ensuring cell viability >95%, and counting for use.

After the test mice (week-old 6-8 weeks, body weight 20 + -0.3 g) were shaved) One side was inoculated subcutaneously with MC38 cells (100. Mu.L per mouse for a total of 2X 10) ⁶ Individual cells). When the average tumor volume of the tumor-bearing mice reaches 100-150mm ³ At this time, the mice were randomly divided into 6 groups of 6 mice each. 5 experimental groups, namely CHL3F6+ anti PD-1, CHL9A1+ anti PD-1, CHL13H4+ anti PD-1, ab2+ anti PD-1 and anti-PD-1 are set, and normal saline is used as a control. The administration concentration of anti LAG-3 is 5mg/kg, and the administration concentration of anti PD-1 is 0.1mg/kg. Among them, the anti PD-1 antibody is provided by Anhui biological engineering (group) Inc., anhui.

The antibodies for the experiments are diluted to corresponding concentrations by physiological saline according to injection concentrations and stored in a refrigerator at 4 ℃ for later use.

In the CHL3F6+ anti PD-1 group, 100ul CHL3F6 and anti PD-1 solution (solvent is normal saline, and solute is CHL3F6 and anti PD-1) are injected each time, so that the dosage of CHL3F6 administered each time is 5mg/kg body weight, and the dosage of anti PD-1 administered each time is 0.1mg/kg body weight, and the administration is performed 2 times per week and 7 times in total;

in the CHL9A1+ anti PD-1 group, 100ul CHL9A1 and anti PD-1 solutions (the solvent is normal saline, and the solutes are CHL9A1 and anti PD-1) are injected into each time, so that the dosage of CHL9A1 administered each time is 5mg/kg body weight, and the dosage of anti PD-1 administered each time is 0.1mg/kg body weight, and the administration is performed 2 times per week for 7 times;

in the CHL13H4+ anti PD-1 group, 100ul CHL13H4 and anti PD-1 solution (solvent is normal saline, solute is CHL13H4 and anti PD-1) are injected each time, so that the dosage of CHL13H4 administered each time is 5mg/kg body weight, and the dosage of anti PD-1 administered each time is 0.1mg/kg body weight, and the administration is performed 2 times per week and 7 times in total;

in the Ab2+ anti PD-1 group, 100ul of Ab2 and anti PD-1 solution (solvent is normal saline, solute is Ab2 and anti PD-1) are injected each time, so that Ab2 is administrated at a dose of 5mg/kg body weight each time, and anti PD-1 is administrated at a dose of 0.1mg/kg body weight each time, 2 times are administrated each week, and 7 times are administrated in total;

the anti PD-1 group is injected with 100ul of anti PD-1 solution (solvent is normal saline and solute is anti PD-1) each time, so that the dosage of the anti PD-1 is 0.1mg/kg body weight each time, and the administration is carried out 2 times per week for 7 times;

the normal saline group is a control group, 100ul of normal saline is injected into each mouse subcutaneously, and the administration is carried out 2 times per week for 7 times;

animal survival and activity was examined twice weekly after tumor inoculation and included: tumor growth, body weight, activity, diet and recording.

The tumor growth results are shown in fig. 3, which shows that: compared with normal saline, the antibodies of each experimental group can inhibit the growth of the MC38 tumor; among them, chL13H4 and ChL3F6 had the best effect on tumor suppression when administered in combination with PD-1.

Example 3 construction and expression of humanized antibody

3.1 construction of humanized antibodies

The chimeric antibodies CHL13H4 and CHL3F6 were each humanized by using homologous sequence modeling-antibody complementary region (CDRs) transplantation-Framework Region (FR) key amino acid back mutation (back mutation) technique.

Analyzing the gene sequence of the antibody CHL13H4, then determining a humanized mutation site through homologous modeling and optimization of an antibody Fab, performing surface scanning, simulating virtual mutation and molecular dynamics, determining key amino acids and the like. Finally, 4 heavy chain candidate sequences and 3 light chain candidate sequences were obtained, and 12 candidate antibodies were obtained by combination for further characterization as described in this example. 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.

Analyzing the gene sequence of the antibody CHL3F6, then determining a humanized mutation site through homologous modeling and optimization of an antibody Fab, performing surface scanning, simulating virtual mutation and molecular dynamics, determining key amino acids and the like. Finally, 3 heavy chain variable region (VH) candidate sequences and 3 light chain variable region (VL) candidate sequences are obtained. VH candidate sequences and VL candidate sequences were combined to give 9 candidate antibodies for further identification. The 9 candidate antibodies were: huL3F6-H1L1, huL3F6-H1L2, huL3F6-H1L3, huL3F6-H2L1, huL3F6-H2L2, huL3F6-H2L3, huL3F6-H3L1, huL3F6-H3L2, and HuL3F6-H3L3.

The amino acid sequences of the variable region candidate sequences are shown in Table 4

Table 4: amino acid sequence listing of candidate sequences for variable regions

3.2 FACS determination of the affinity of the humanized anti-LAG-3 antibody to human LAG-3

The pcDNA3.4-HuLAG3 plasmid is transfected into CHO-S cells, the cells are collected for detection after 24h, and the obtained cells expressing human LAG-3 are named as CHO-S/pcDNA3.4-HuLAG3. Trypsinizing CHO-S/pcDNA3.4-HuLAG3 cells, collecting cells, centrifuging to remove supernatant, counting by resuspension of 1xPBS, adjusting the cell number to 1.5x10 per EP tube ⁵ The cells were centrifuged at 1000rpm for 2min, washed once with 1% BSA (in PBS), and the supernatant was removed. Humanized anti-LAG-3 antibody was used as a test antibody, which was diluted to 30. Mu.g/ml with 1% BSA as an initial concentration, and diluted 5 times at a 3-fold ratio to obtain 6 antibodies with different concentrations, 100. Mu.l of each concentration of the antibody dilution was added to each EP tube to which the CHO-S/pcDNA3.4-HuLAG3 cells were added, and the resuspended cells were mixed well, incubated at 4 ℃ in the dark for 1 hour, and 1% BSA dilution was used as a negative control. After completion of the incubation, 400. Mu.l of 2% BSA in 1xPBS 2000rpm was added and centrifuged for 5 minutes to discard the supernatant, and this operation was repeated once. 100. Mu.l of goat anti-human IgG-FITC (Jackson) diluted 200-fold was added per EP tube, incubated at room temperature for 0.5 hour, 400. Mu.l of 2 BSA in 1xPBS 2000rpm was added, centrifuged at 5 minutes to discard the supernatant, this operation was repeated once, after washing, 400ul of 1xPBS was added to resuspend the cells, and the data were processed by GraphPad Prism statistical software by up-flow cytometry.

The affinity results for HuL13H4 are shown in table 5.1 and fig. 4A, 4B, 4C.

The affinity results for HuL3F6 are shown in table 5.2 and fig. 5A, 5B.

Table 5.1: human LAG-3 recognition by the HuL13H4 antibody

Table 5.2: human LAG-3 recognized by HuL3F6 antibody molecule

Name (R)	HuL3F6-H1L1	HuL3F6-H1L2	HuL3F6-H1L3	HuL3F6-H2L1	HuL3F6-H2L2
						EC50(μg/ml)	1.542	1.453	1.402	1.112	1.038
Name (R)	HuL3F6-H2L3	HuL3F6-H3L1	HuL3F6-H3L2	HuL3F6-H3L3
						EC50(μg/ml)	2.450	2.857	3.950	4.906

Example 4 affinity maturation and potential deamidation site mutation

4.1, huL13H4-H1L3 affinity maturation and potential deamidation site mutation

4.1.1 obtaining of affinity maturation candidate molecules of HuL13H4-H1L3

HuL13H4-H1L3 humanized molecule is constructed into a single chain antibody form, namely VH- (G4S) 3-VL-Fc, the amino acid sequence of the heavy chain variable region of HuL13H4-H1L3 is shown as SEQ ID No.6, and the amino acid sequence of the light chain variable region is shown as SEQ ID No. 7. The affinity maturation was carried out by Abstudio corporation to transform the CDR regions of the light chain and the heavy chain to obtain 5 candidate molecules, scF-HuL13H4-2B8-Fc, scFv-HuL13H4-F4-Fc, scFv-HuL13H4-2E8, scFv-HuL13H4-2B4-Fc, and scFv-HuL13H4-2A6-Fc.

4.1.2 FACS determination of the affinity of the HuL13H4-H1L3 affinity maturation candidate molecules

The pcDNA3.4-HuLAG3 plasmid is transfected into ExpicHO-S cells, the cells are collected for detection after 24h, and the obtained cells expressing the human LAG-3 are named as ExpicHO-S/pcDNA3.4-HuLAG3.

Pancreatin digestion of ExpicHO-S/pcDNA3.4-HuLAG-3 cells, collection of cells, centrifugation to remove supernatant, 1xPBS heavy suspension counting, cell number adjusted to 1.5x10 per EP tube ⁵ The cells were centrifuged at 1000rpm for 2min, washed once with 1% BSA (in PBS), and the supernatant was removed. The single-chain antibody obtained by affinity maturation was used as an antibody to be tested, which was diluted to 30. Mu.g/ml with 1% BSA as the starting concentration, and 3-fold diluted 5 times to obtain 6 antibodies of different concentrations, and 100. Mu.l of each antibody dilution was added to each EP tube to which the above-mentioned CHO-S/pcDNA3.4-HuLAG3 cells were addedResuspended cells were mixed, incubated at 4 ℃ in the dark for 1 hour, and 1% BSA dilution was used as a negative control. After completion of the incubation, 400. Mu.l of 2% BSA in 1xPBS 2000rpm was added and centrifuged for 5 minutes to discard the supernatant, and this operation was repeated once. 100. Mu.l of goat anti-human IgG-FITC (Jackson) diluted 200-fold was added per EP tube, incubated at room temperature for 0.5 hour, 400. Mu.l of 2 BSA-containing 1xPBS 2000rpm was added, centrifuged at 5 minutes to discard the supernatant, this operation was repeated once, after washing, 400ul of 1xPBS-resuspended cells were added, detection was performed by up-flow cytometry, and data was processed by GraphPad Prism statistical software, with the data processing results shown in Table 6 and FIG. 6. The results show that: scFv-L13H4-F4-Fc and scFv-L13H4-2B4-Fc have higher affinity.

Table 6: HKL13H4 affinity maturation candidate affinity-ScFv

scFv-L13H4-F4-Fc and scFv-L13H4-2B4-Fc are preferred as candidates, wherein the amino acid sequence of the light chain variable region of scFv-L13H4-F4-Fc is shown in SEQ ID No.3, positions 1-106, and the sequence of the heavy chain variable region remains identical to the HuL13-H1L3 humanized molecule, shown in SEQ ID No. 6; the amino acid sequence of the variable region of the light chain of the scFv-L13H4-2B4-Fc is shown as SEQ ID No.5, and the sequence of the variable region of the heavy chain thereof keeps consistent with HuL13-H1L3 humanized molecule, which is shown as SEQ ID No. 6. scFv-L13H4-F4-Fc and scFv-L13H4-2B4-Fc were reduced to whole antibodies, i.e., heavy chain variable region was grafted onto heavy chain constant region (IgG 1 subtype, with L234A and L235A mutations, whose amino acid sequence is shown in SEQ ID No.1 at positions 121-450), and light chain variable region was grafted onto light chain constant region (Kappa subtype, whose amino acid sequence is shown in SEQ ID No.3 at positions 107-213), to obtain antibodies HuL13H4-2B4 and HuL13H4-F4. The binding ability to human LAG-3 was further tested by FACS, the procedure was as described in 4.1.2, and the results are shown in FIG. 7.

4.1.3 potential deamidation site mutations of HuL13H4-2B4 and HuL13H4-F4

The antibody Hankel13 is obtained by mutation of an affinity maturation antibody HuL13H4-F4, asparagine at position 104 or 105 in a heavy chain variable region is mutated to avoid potential deamidation sites, and the mutation can be to mutate asparagine at position 104 into glutamic acid or alanine, or mutate asparagine at position 105 into glutamic acid or alanine, or mutate asparagine at positions 104 and 105 simultaneously, preferably to mutate asparagine at position 104 into glutamic acid, and keep other sequences of HuL13H4-F4 unchanged. The amino acid sequence of the mutated heavy chain variable region is shown as the 1 st to 120 th positions of SEQ ID.1; heavy chain constant region IgG1 subtypes are preferred and L234A and L235A mutations are performed. The full-length amino acid sequence of the heavy chain of the Hankel3 is shown as SEQ ID No.1, and the coding sequence of the coding chain is shown as SEQ ID No. 2; the Hankel3 light chain full-length amino acid sequence is shown as SEQ ID.3, and the coding sequence of the coding chain is shown as SEQ ID.4.

4.2 potential deamidation site mutation of HuL3F6-H2L2

The antibody Hankel3 is obtained by mutation of a humanized antibody HuL3F6-H2L2, and a heavy chain variable region is subjected to G56A mutation so as to avoid potential deamidation sites; the other sequences of HuL3F6-H2L2 were kept unchanged. The sequence of the mutated heavy chain variable region is shown in SEQ ID.8, 1 st to 116 th; preferably selecting a heavy chain constant region IgG1 subtype, carrying out L234A and L235A mutation, wherein the full-length amino acid sequence of the heavy chain of Hankel3 is shown as SEQ ID.8, and the coding sequence of the coding chain is shown as SEQ ID.9; the Hankel3 light chain full-length amino acid sequence is shown as SEQ ID.10, and the coding sequence of the coding chain is shown as SEQ ID.11.

Example 5 expression and functional verification of antibodies Hankel13 and Hankel3

5.1 expression and purification of antibodies Hankel13 and Hankel3

According to the gene sequence obtained by sequencing, a full-length gene sequence is synthesized, the heavy chain coding gene of the Hankel13 antibody is shown as SEQ ID No.2, and the light chain coding gene of the Hankel13 antibody is shown as SEQ ID No. 4. The 1 st to 360 th sites of SEQ ID No.2 are encoding genes of a Hankel13 antibody heavy chain variable region VH, wherein the encoding sequences of CDR1, CDR2 and CDR3 are respectively shown as the 76 th to 105 th sites, the 148 th to 198 th sites and the 295 th to 327 th sites of SEQ ID No. 2. The 1 st to 318 th sites of SEQ ID No.4 are encoding genes of a Hankel13 antibody light chain variable region VL, wherein the encoding sequences of CDR1, CDR2 and CDR3 are respectively shown as 69 th to 102 th sites, 148 th to 168 th sites and 265 th to 288 th sites of SEQ ID No. 4.

According to the gene sequence obtained by sequencing, a full-length gene is synthesized, the coding gene of the Hankel3 antibody heavy chain is shown as SEQ ID No.9, and the coding gene of the Hankel3 antibody light chain is shown as SEQ ID No. 11. The 1 st to 348 th positions of SEQ ID No.9 are encoding genes of a Hankel3 antibody heavy chain variable region VH, wherein the encoding sequences of CDR1, CDR2 and CDR3 are respectively shown as the 76 th to 105 th positions, 148 th to 198 th positions and 277 th to 315 th positions of SEQ ID No. 9. The 1 st to 321 th sites of SEQ ID No.11 are encoding genes of a Hankel3 antibody light chain variable region VL, wherein the encoding sequences of CDR1, CDR2 and CDR3 are respectively shown as 69 th to 102 th sites, 148 th to 168 th sites and 265 th to 291 th sites of SEQ ID No. 11.

Cloning the DNA fragment (encoding gene of antibody heavy chain) shown in SEQ ID No.2 or SEQ ID No.9 to the enzyme cutting site Xba I and Hind III of the vector pcDNA3.4 to obtain recombinant expression vectors for expressing the heavy chain of the antibody, wherein the recombinant expression vectors are named pcDNA3.4-Hankel13-H and pcDNA3.4-Hankel3-H respectively; in order to be more beneficial to protein expression, when a recombinant expression vector of the heavy chain of the antibody is constructed, an XbaI enzyme digestion site, a kozak consensus sequence (5 '-GCCACC-3') and a coding sequence (5'-ATGGAGTTTGGACTGTCTTGGGTGTTCCTGGTGGCTATCCTGAAAGGAGTCCAGTGC-3') of single peptide are sequentially introduced into the 5 'upstream of a DNA fragment (antibody heavy chain coding gene) shown in SEQ ID No.2 or SEQ ID No.9, and a stop codon TGA and a HindIII enzyme digestion site are introduced into the 3' downstream of the DNA fragment (antibody heavy chain coding gene) shown in SEQ ID No.2 or SEQ ID No. 9. The above gene sequence was sent to Nanjing King Shirui Biotech Co., ltd for synthesis, and then cleaved with XbaI and HindIII cleavage sites, and ligated to XbaI and HindIII cleaved pcDNA3.4 vector.

The DNA fragment shown in SEQ ID No.4 or SEQ ID No.11 (coding gene of antibody light chain) is cloned between enzyme cutting sites Xba I and Hind III of pcDNA3.4 vector to obtain recombinant expression vector for expressing the light chain of the antibody (named pcDNA3.4-Hankel3-L and pcDNA3.4-Hankel13-L respectively). In order to be more beneficial to protein expression, when a recombinant expression vector of the light chain of the antibody is constructed, an XbaI enzyme cutting site, a kozak consensus sequence (5 '-GCCACC-3') and a coding sequence (5'-ATGGAAACAGATACACTCCTCCTCTGGGTGCTGCTCCTCTGGGTGCCAGGATCTACAGGA-3') of single peptide are sequentially introduced into the 5 'upstream of the DNA fragment (coding gene of the antibody light chain) shown in SEQ ID No.4 or SEQ ID No.11, and a stop codon TGA and a HindIII enzyme cutting site are introduced into the 3' downstream of the DNA fragment (coding gene of the antibody heavy chain) shown in SEQ ID No.4 or SEQ ID No. 11. The above gene sequence was synthesized by the Nanjing King Shirui Biotechnology Co., ltd, and then digested with XbaI and HindIII restriction sites, and ligated to XbaI and HindIII-digested pcDNA3.4 vector.

The pcDNA3.4-Hankel13-H and pcDNA3.4-Hankel13-L were introduced into the human embryonic kidney cell line HEK293F to obtain recombinant cell HEK293F/pcDNA3.4-Hankel13. The recombinant cell HEK293F/pcDNA3.4-Hankel13 can express the antibody Hankel13.

The pcDNA3.4-Hankel3-H and pcDNA3.4-Hankel3-L were introduced into the human embryonic kidney cell line HEK293F to obtain the recombinant cell HEK293F/pcDNA3.4-Hankel3. The recombinant cell HEK293F/pcDNA3.4-Hankel3 can express the antibody Hankel3.

The recombinant cells HEK293F/pcDNA3.4-Hankel13 and HEK293F/pcDNA3.4-Hankel3 were then each treated at 37 ℃ with 5% CO ₂ The culture was carried out in a shaking incubator at 120rpm.

The antibody Protein was purified from the culture supernatant using Protein A affinity chromatography column. The specific operation is as follows: the Protein A column (GE company) was equilibrated with PBS, and the supernatant was cultured and passed through the column, and 5 column volumes were pre-eluted with solution A (formulation: solvent: water, solute and concentration: 20mM sodium phosphate, 500mM NaCl, pH 5.0), and then 5 column volumes were eluted with solution B (formulation: solvent: water, solute and concentration: 20mM sodium acetate, 150mM NaCl, pH 3.5), and the eluted peaks were collected, and then the antibody was obtained by concentrating a 30kDa concentration centrifuge tube to obtain anti-human LAG-3 antibodies (Hankel 3 and Hankel 13).

Sequencing the antibody Hankel13, and displaying that the obtained anti-human LAG-3 antibody Hankel13 consists of a heavy chain and a light chain, wherein the amino acid sequence of the heavy chain is shown as SEQ ID No.1, and the sequences of CDR1, CDR2 and CDR3 are respectively shown as 26 th to 35 th, 50 th to 66 th and 99 th to 109 th from the N end of SEQ ID No. 1; the amino acid sequence of the light chain is shown as SEQ ID No.3, and the sequences of CDR1, CDR2 and CDR3 are respectively shown as 24 th to 34 th, 50 th to 56 th and 89 th to 96 th from the N end of the SEQ ID No. 3.

The obtained anti-human LAG-3 antibody Hankel3 is a complete antibody and consists of a heavy chain and a light chain, the amino acid sequence of the heavy chain is shown as SEQ ID No.8, and the sequences of CDR1, CDR2 and CDR3 are respectively shown as 26 th to 35 th, 50 th to 66 th and 97 th to 105 th from the N end of the SEQ ID No. 8; the amino acid sequence of the light chain is shown as SEQ ID No.10, and the sequences of CDR1, CDR2 and CDR3 are respectively shown as 24 th to 34 th, 50 th to 56 th and 89 th to 97 th from the N end of the SEQ ID No. 10.

5.2 identification of the affinity of the antibodies Hankel13 and Hankel3 for human, monkey, murine LAG-3

5.2.1 antigen preparation

XbaI enzyme cutting sites and kozak consensus sequences (5 '-GCCACC-3') are sequentially added to the 5 'end of the gene sequence of the extracellular region of the human LAG3 antigen, monkey and mouse, the 3' end of the gene sequence is sequentially added with the gene sequence of mouse Fc, stop codons TGA and HindIII enzyme cutting sites, the gene sequences are sent to Nanjing King Shirui biotechnology GmbH for synthesis, then the XbaI enzyme cutting sites and the HindIII enzyme cutting sites are used for enzyme cutting, and the gene sequences are connected to a pcDNA3.4 vector which is cut by the same enzyme, so that pcDNA3.4/HuLAG3-Fc, pcDNA3.4/CyLAG3-Fc and pcDNA3.4/Mu 3-Fc vectors are respectively obtained. The pcDNA3.4/HuLAG3-Fc, pcDNA3.4/CyLAG3-Fc and pcDNA3.4/MuLAG3-Fc vectors were each introduced into HEK293F cells and subjected to 5% CO at 37 ℃% ₂ The culture was performed in a shaking incubator at 120rpm. After 3 days, cell supernatants were collected, and antibody proteins were purified from the culture supernatants using Protein A affinity chromatography columns. The specific operation is the same as 5.1. HuLAG-3-Fc, cyLAG-3-Fc and MuLAG-3-Fc antigen proteins are obtained respectively, and the amino acid sequences of the proteins are shown in Table 7.

Table 7: expressed human/murine/monkey protein amino acid sequences

5.2.2 affinity assay for human LAG-3 with antibodies Hankel13 and Hankel3

The epitope competition assay method of the antibody to be tested and Ab1 comprises the following steps: dilution of goat anti-mouse Fc with NaHCO3 (Jacks)on company), adding 100 microlitres of the enzyme label plate into each hole to reach the working concentration, and incubating overnight at 4 ℃; PBST plate washing 3 times; 200ul 5% NON-Fat Milk per well (Sangon Biotech) was added to the microplate and incubated at 37 ℃ for 2 hours after blocking; PBST plate washing 3 times; further diluting HuLAG-3-Fc antigen (homemade in 5.2.1) to 1ug/ml by 1% of NON-Fat Milk, adding 100. Mu.l per well to the enzyme plate, incubating overnight at 4 ℃ and washing the plate 3 times with PBST; using antibodies Hankel13 and Hankel3 as antibodies to be detected, diluting a sample to be detected by 1% NON-Fat Milk for 6 times at a 4-fold ratio to obtain 7 gradient concentrations, wherein the highest concentration is 50nM, 100 mul of each well is added into an enzyme label plate, and Ab1 is used as a positive control; incubating for 1 hour at room temperature; PBST plate washing 3 times; 1% NON-Fat Milk diluted goat anti-human-HRP, 100. Mu.l per well was added to the microplate and incubated for 0.5 hours at room temperature; PBST plate washing 3 times; adding TMB into each hole, and developing at room temperature in a dark place; adding H ₂ SO ₄ Terminating the color development reaction; and (3) placing the enzyme label plate into a SepctraMax Versa enzyme label instrument, measuring the light absorption value (OD value) at 450nm, counting the result by using GraphPad Prism and calculating the EC50 value.

The affinity of the antibodies Hankel13 and Hankel3 for monkey and murine LAG-3 was determined as above, with only the human LAG-3 being exchanged for monkey LAG-3 (homemade in 5.2.1) and murine LAG-3 (homemade in 5.2.1), with maximum concentrations of 10nM and 2ug/ml, respectively, and the rest of the procedures being as above.

The results of the experiment are shown in Table 8 and FIGS. 8A, 8B and 8C. The results show that the antibodies Hankel13 and Hankel3 can be recognized by human LAG-3 and monkey LAG-3, and have weak binding with mice.

Table 8: ELISA determination of binding of monoclonal antibodies to human/monkey/mouse LAG-3 antigen (EC 50 (nM))

Antibodies	HankeL3	HankeL13	Ab1	Ab2	Anti-Mouse-LAG-3
						Human LAG-3	0.2395	0.7137	1.236	NA	NA
Monkey LAG-3	0.196	1.466	NA	0.145	NA
						Mouse LAG-3	25.42	19228	NA	NA	0.0467

5.3 ligand Competition

5.3.1, hankel13 and Hankel3 assays for competitive binding Activity with ligand MHCII

A375 cells (expressing MHCII, purchased from Bai Biotech Co., ltd., nanjing) were used to detect the activity of antibodies Hankel3 and Hankel13 competing with ligand MHCII molecules for binding to LAG-3 antigen, with Ab1 and Ab2 as positive controls and IgG1 as negative controls. (1) Trypsinizing cultured A375 cells for 24h, collecting cells, centrifuging to remove supernatant, counting by resuspension at 1xPBS, adding 200. Mu.L of 2X10 ⁵ Cells were plated into 1.5mL EP tubes. (2) Hankel13 and Hankel3 are antibodies to be detected, the antibodies to be detected are diluted by PBS to 25uM,5 times of the ratio to obtain antibody test solutions with 7 concentrations, the HuLAG-3-Fc antigen (self-made in 5.2.1) is diluted by PBS to 250nM, the antigen-antibody dilution solution is added into the EP tube in the step (1) in an equal volume mixing way, the mixture is incubated for 1 hour at room temperature and centrifuged for 3 minutes at 2000rpm, after PBS is added to resuspend the cells, the mixture is centrifuged for 3 minutes at 2000rpm, and the supernatant is discarded. (3) Adding 200 times diluted goat anti-mouse-FITC secondary antibody, incubating for 30 minutes at room temperature, centrifuging for 3 minutes at 2000rpm, adding PBS to resuspend cells, centrifuging for 3 minutes at 2000rpm, discarding supernatant, adding 400ul PBS to resuspend on the machine for detection. The results were counted using GraphPad Prism and the IC50 values were calculated.

The results are shown in table 9 and fig. 10. The results show that: the antibodies Hankel13 and Hankel3 can block the binding of antigen to the ligand MHCII.

Table 9: FACS detection of blockade of LAG-3 antigen with ligand MHCII molecules

	HankeL3	HankeL13	Ab1	Ab2	IgG
						IC50(uM)	0.047	0.045	0.031	0.034	NA

5.3.2, hankel13 and Hankel3 assays for competitive binding Activity with ligand FGL1

The activity of anti LAG-3 antibody competing with human FGL1 for binding to LAG-3 antigen was detected by ELISA using Ab1 and Ab2 as positive controls and IgG1 as negative control. FGL1-hFc (Acro company, product number FG 1-H5258) is diluted by NaHCO3 to 1ug/ml, 100ul of each well is added into an enzyme label plate, and the mixture is incubated overnight at 4 ℃; PBST plate washing 3 times; 200ul 1% BSA per well (Sangon Biotech) was added to the microplate and incubated at 37 ℃ for 2 hours after blocking; PBST plate washing 3 times; diluting the antigen HuLAG-3-Fc antigen with 1% BSA (made by the manufacturer 5.2.1), diluting the LAG-3 antibody with 1% BSA to 200nM, 6-fold dilutions to obtain 7 concentration gradients, mixing the antigen and antibody in equal volumes, adding 100. Mu.l of each well to the ELISA plate, and incubating at room temperature for 1 hour; PBST plate washing 3 times; 1% BSA dilution goat anti-mouse IgG-HRP, 100. Mu.l per well, was added to the ELISA plate and incubated for 0.5 hours at room temperature; PBST plate washing 3 times; adding TMB into each hole, and developing at room temperature in a dark place; adding H ₂ SO ₄ Terminating the color development reaction; and (3) placing the enzyme label plate into a SepctraMax Versa enzyme label instrument, measuring the light absorption value (OD value) at 450nm, counting the result by using GraphPad Prism and calculating the IC50 value.

The experimental results are shown in table 10 and fig. 10. The results show that: the antibodies Hankel13 and Hankel3 can effectively block the binding of the LAG-3 antigen and the ligand FGL 1.

Table 10: ELISA detection of blocking of LAG-3 antigen and ligand FGL-1 molecule

	HankeL3	HankeL13	Ab1	Ab2	IgG
						IC50(nM)	0.579	1.625	0.998	1.525	NA

5.4 epitope binding assays

5.4.1 antigen binding Domain epitope analysis

Human LAG-3 antigens extracellular Domain1 (37G-167G), domain2 (168Q-252S), domain3 (265P-343N) and Domain4 (348L-419R) or the combination thereof are respectively replaced by corresponding extracellular regions of mouse TIM-3, corresponding target genes are synthesized, the corresponding target genes are respectively inserted into pcDNA3.4 vectors to obtain DNA plasmids of different human and mouse chimeric LAG-3 antigens, the plasmids are introduced into HEK293F cells, and the cells are collected after 24h for FACS detection, which is specifically shown in Table 11. The cell number was adjusted to 2X105 cells per EP tube using PBS solution, centrifuged at 1000rpm for 2min, washed once with 1% BSA (in PBS) solution, and the supernatant removed. The test antibodies Ab1, hankel13 and Hankel3 were diluted to 30. Mu.g/ml with 1% BSA, added to the above HEK293F cells expressing the different human and mouse chimeric antigens, mixed, and incubated at 4 ℃ for 1 hour in the dark. After completion of the incubation, 400. Mu.l of 2% BSA in 1xPBS 2000rpm was added and centrifuged for 5 minutes to discard the supernatant, and this operation was repeated once. 100. Mu.l of goat anti-human IgG-FITC (Jackson) diluted 200-fold was added to each EP tube, incubated at room temperature for 0.5 hour, 400. Mu.l of 2-BSA-containing 1xPBS 2000rpm was added, centrifuged at 5 minutes to discard the supernatant, this operation was repeated once, and after washing, 400ul of 1xPBS was added to resuspend the cells, and detection was carried out by an up-flow cytometer.

Through the above operation, the trend of the fluorescence intensity of Ab1 is found to be reduced in cells C, D, E and group F, which indicates that Ab1 mainly binds to Doamin1 and Domain2 in the extracellular region of human LAG-3; the trend of the fluorescence intensity of Hankel13 and Hankel3 is reduced in cells C, E and F group, which indicates that Hankel13 and Hankel3 mainly bind to Domain1 in human LAG-3 extracellular region. The specific results are shown in Table 11.

Table 11: antibody binding to different human murine chimeric LAG-3 epitopes mean fluorescence intensity analysis

Cell number	Specific combined structure of antigen	Ab1	HankeL13	HankeL3
					A	Human LAG-3 full Length	14440.9	13141.1	6296.5
B	Human LAG-3 Domain1-2+ mouse LAG-3 Domain 3-4	13543.1	11802.4	6101.2
					C	Mouse LAG-3 Domain1-2+ human LAG-3 Domain 3-4	675.5	6464.6	734
D	Human LAG-3 Domain1+ mouse LAG-3 Domain 2-4	8787.1	12338.4	7721.4
					E	Human LAG-3 Domain2+ mouse LAG-3 Domain1, 3-4	709.9	8103.8	558.2
F	Murine LAG-3 full Length	590.6	8889.5	619.4

5.4.2 Competition with Positive control antibody epitopes

The competition relationship of the Hankel3 and Hankel13 antibodies with the epitopes of the control antibodies Ab1 and Ab2 was compared using an ELISA assay.

The epitope competition assay method of the antibody to be tested and Ab1 comprises the following steps: with NaHCO ₃ Diluting goat anti-mouse Fc (Jackson) to working concentration, adding 100 μ l per well into an enzyme label plate, and incubating overnight at 4 ℃; PBST plate washing 3 times; 200ul 5% NON-Fat Milk per well (Sangon Biotech Co.) was added to the microplate, and incubated at 37 ℃ for 2 hours after blocking; PBST plate washing 3 times; then, the HuLAG-3-Fc antigen (manufactured by 5.2.1) was diluted to 1ug/ml by 1% of NON-Fat Milk, and 100. Mu.l of the diluted solution was added to the microplate, and the plate was incubated at room temperature1 hour; PBST plate washing 3 times. Positive control antibody Ab1 was labeled with streptavidin-Biotin (SA-Biotin Biotin, SA), biotin-labeled Ab1 was diluted with NON-Fat Mill to a concentration of 2nM as solution A, while unlabeled Ab1, hankel3, hankel13, and IgG were prepared with NON-Fat Mill to 2. Mu.M, diluted 5-fold for 1 point, followed by 10-fold gradient dilution for 4 points as solution B. 50 μ L of each solution A and B at each concentration were mixed and added to LAG-3 antigen-coated 96-well plates, incubated at room temperature for 1 hour, and washed 3 times with PBST. Add 100. Mu.L of SA-HRP (Biolegend) diluted 8000 times to each well, incubate for 0.5 hours at room temperature, wash the plate 3 times with PBST, add TMB to each well, color development in the dark at room temperature; adding H ₂ SO ₄ Terminating the color development reaction; and (3) placing the enzyme label plate into a SepctraMax Versa enzyme label instrument, measuring the light absorption value at 450nm, counting the result by GraphPad Prism and calculating the EC50 value.

Epitope competition assay method for the antibody to be tested and Ab2 was performed as above, and solution A was replaced with biotin-labeled Ab2 at a concentration of 2 nM.

Specific results are shown in table 12 and fig. 11A and 11B. Experiments showed that HankeL13 and HankeL3 differ from the antigen binding epitopes of the positive control antibodies Ab1 and Ab2.

Table 12: ELISA for epitope Competition detection (OD) _450nm )

5.5 in vivo efficacy

A20 tumor model

Experimental selection of PBMC reconstructed mouse A20 model for testing the in vivo antitumor effect of LAG-3 antibody. The B-NDG B2M KO plus mouse expresses the B2M gene fused in the FcRn gene while knocking off the mouse B2M gene. This mouse binds to the background of B-NDG mice with MHC class I deletions. Genetically engineered mouse B-NDG B2M KO plus mice, purchased from Poiosael.

Culturing A20 cells (mouse B cell lymphoma, purchased from Nanjing Kebai, cat number: cobioer/cbp 60279) to 80% full density, trypsinizing, centrifuging at 1000rpm for 5min, collecting cells, cleaning, centrifuging, resuspending the cells with resuspension solution to obtain cell suspension, ensuring cell viability rate to be more than 95%, and counting for use.

A20 cell line (2X 10 cells/mouse) was inoculated subcutaneously on one side of the back (shaved hair) of test B-NDG B2M KO plus mice (week-old 6-8 weeks, body weight 20. + -.0.3 g) ⁶ Cell, 100 μ l). Human PBMC were inoculated 5 days later (5X 10 for each mouse) ⁶ Individual cells). When the average tumor volume of the tumor-bearing mice reaches about 50mm ³ At the time, the mice were randomly divided into 6 groups of 6 mice each. A total of 5 experimental groups were designed, which were: ab1+ Keytruda group, ab2+ Keytruda group, hankelL3+ Keytruda group, hankelL13+ Keytruda group and Keytruda group, with physiological saline as control.

The Ab1+ Keytruda group was injected with 100ul Ab1 and Keytruda solutions (solvent is physiological saline, solute is Ab1 and Keytruda) each time, so that Ab1 was administered at a dose of 10mg/kg body weight each time, keytruda was administered at a dose of 10mg/kg body weight each time, 2 times per week, for 2 weeks;

the Ab2+ Keytruda group was injected with 100ul Ab2 and Keytruda solutions (solvent is physiological saline, solute is Ab2 and Keytruda) at a time to give Ab2 at a dose of 10mg/kg body weight per administration and Keytruda at a dose of 10mg/kg body weight per administration, 2 times per week for 2 weeks;

the HankelL3+ Keytruda group was injected with 100ul HankelL3 and Keytruda solutions (solvent is normal saline, solute is HankelL3 and Keytruda) each time, so that HankelL3 was administered at a dose of 10mg/kg body weight and Keytruda was administered at a dose of 10mg/kg body weight each time, 2 times per week for 2 weeks;

the HankelL13+ Keytruda group was injected with 100ul of HankelL13 and Keytruda solutions (solvent physiological saline, solute HankelL13 and Keytruda) at a time to give HankelL13 at a dose of 10mg/kg body weight per administration and Keytruda at a dose of 10mg/kg body weight per administration for 2 times per week for 2 weeks;

keytruda group was injected with 100ul Keytruda solution (solvent normal saline, solute Keytruda) per dose of 10mg/kg body weight per administration, 2 times per week for 2 weeks;

the normal saline group is a control group, 100ul of normal saline is injected into each mouse subcutaneously, and the administration is carried out 2 times per week for 2 weeks;

pabolizumab injection (Keytruda, from MSD ireland, import medicine registration No.: S20180019), the above antibody was diluted with physiological saline as required for concentration, and stored in a refrigerator at 4 ℃.

Animal survival and activity was examined twice weekly after tumor inoculation and included: tumor growth, body weight, activity, diet and recording.

The tumor volume is shown in figure 12 (day 0 is the day of tumor inoculation), and the results show that compared with normal saline, the antibodies of each experimental group can inhibit the growth of the A20 tumor and do not influence the mobility and the body weight of the mouse; of these, the HankelL13+ Keytruda experimental group had the best tumor suppression among all experimental groups.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is made possible within the scope of the claims attached below.

Sequence listing

<110> Sakuaimaibo Biotechnology Limited

<120> LAG-3 binding molecules and uses thereof

<160> 12

<170> SIPOSequenceListing 1.0

<210> 1

<211> 450

<212> PRT

<213> Artificial Sequence (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 (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 (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

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 (Artificial Sequence)

<400> 4

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 (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 (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 (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 (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> 1338

<212> DNA

<213> Artificial Sequence (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 (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

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

gatgtgcaga tgacccagag cccatcctcc ttgagcgctt ctctgggaga cagagtgacc 60

atcacctgca gatcctccca ggacatcggc tcttatctga actggttcca acagaagcct 120

gacggcacca tcaagctgct gatctattac acctccaccc tgcactccgg cgtgccttcc 180

aggttctccg gatccggatc tggaaccgat ttcaccctga ccatcagcag cctgcagcca 240

gaagactttg ccacctactt ttgccagcag ggctacaccc tgccttatac ctttggccag 300

ggtaccaagc tggagatcaa gaggaccgtg gccgctccat ccgtgttcat ctttccccct 360

agcgacgagc agctgaagag cggcacagct tctgtggtgt gcctgctgaa caatttctac 420

cccagggagg ccaaggtgca gtggaaggtg gataacgctc tgcagagcgg caattctcag 480

gagtccgtga ccgagcagga cagcaaggat tctacatatt ccctgagctc taccctgaca 540

ctgagcaagg ccgactacga gaagcacaag gtgtatgctt gcgaggtgac ccatcagggc 600

ctgtccagcc ccgtgacaaa gtcttttaac aggggcgagt gt 642

<210> 12

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<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 (10)

1. LAG-3 binding molecule, wherein the LAG-3 binding molecule comprises a LAG-3 antibody, or an antigen-binding fragment of the LAG-3 antibody, or a fusion protein comprising the antigen-binding fragment, or an antibody drug conjugate comprising the LAG-3 antibody, or an antibody drug conjugate comprising the antigen-binding fragment, or a bispecific antibody comprising the LAG-3 antibody, or a bispecific antibody comprising the antigen-binding fragment, wherein the LAG-3 binding molecule comprises a heavy chain variable region and a light chain variable region comprising the CDR sequences of 13H 4) or 3F 6) below,

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

   3F6) The amino acid sequence of HCDR1 in the heavy chain variable region is shown as 26 th-35 th sites of SEQ ID No.8, the amino acid sequence of HCDR2 is shown as 50 th-66 th sites of SEQ ID No.8 or 50 th-66 th sites of SEQ ID No.12, and the amino acid sequence of HCDR3 is shown as 97 th-105 th sites of SEQ ID No. 8; the amino acid sequence of LCDR1 in the light chain variable region is shown as SEQ ID No.10, the amino acid sequence of LCDR2 is shown as SEQ ID No.10, the amino acid sequence of LCDR3 is shown as SEQ ID No.10, the amino acid sequence of LCDR is shown as SEQ ID No. 89-97.
2. 2. The LAG-3 binding molecule of claim 1, wherein:

   13H4) The amino acid sequence of the heavy chain variable region is shown as SEQ ID No.1, 1 to 120 th position or SEQ ID No.6, or has at least 80% of identity with the amino acid sequence shown as SEQ ID No.1, 1 to 120 th position or SEQ ID No. 6; the amino acid sequence of the light chain variable region is shown as SEQ ID No.3 sites 1-106 or SEQ ID No.5 or SEQ ID No.7, or has at least 80% of identity with the amino acid sequence shown as SEQ ID No.3 sites 1-106 or SEQ ID No.5 or SEQ ID No. 7;

   3F6) The amino acid sequence of the heavy chain variable region is shown in1 st to 116 th positions of SEQ ID No.8 or SEQ ID No.12, or has at least 80 percent of identity with the amino acid sequence shown in1 st to 116 th positions of SEQ ID No.8 or SEQ ID No. 12; the variable region of the light chain has an amino acid sequence as shown in SEQ ID No.10, positions 1-107, or has at least 80% identity with the amino acid sequence shown in SEQ ID No.10, positions 1-107.
3. 3. The LAG-3 binding molecule of claim 1 or 2, wherein: the LAG-3 antibody further comprises a heavy chain constant region and a light chain constant region, the heavy chain constant region being of the class IgG, igM, igE, igA, or IgD; the light chain constant region is classified as a kappa chain or a lambda chain.
4. 4. A LAG-3 binding molecule according to any one of claims 1 to 3, wherein the amino acid sequence of the heavy chain of the LAG-3 antibody is as set forth in SEQ ID No.1, or has at least 80% identity with the amino acid sequence set forth in SEQ ID No.1, and the amino acid sequence of the light chain is as set forth in SEQ ID No.3, or has at least 80% identity with the amino acid sequence set forth in SEQ ID No. 3; or the amino acid sequence of the heavy chain is shown as SEQ ID No.8, or has at least 80% of identity with the amino acid sequence shown as SEQ ID No.8, and the amino acid sequence of the light chain is shown as SEQ ID No.10, or has at least 80% of identity with the amino acid sequence shown as SEQ ID No. 10.
5. 5. The LAG-3 binding molecule of claim 1 or 2, wherein: the antigen-binding fragment comprises: fab, fab ', F (ab') ₂ One or a plurality of combinations of Fab' -SH, fv and ScFv.
6. 6. Biomaterial associated with the LAG-3 binding molecule of any one of claims 1-5, characterized in that: the biological material is any one of the following materials:

   b1 A nucleic acid molecule encoding a LAG-3 binding molecule according to any one of claims 1 to 5;

   b2 An expression cassette containing the nucleic acid molecule according to B1);

   b3 A recombinant vector containing the nucleic acid molecule according to B1) or containing the expression cassette according to B2);

   b4 A recombinant microorganism containing the nucleic acid molecule according to B1) or containing the expression cassette according to B2) or containing the recombinant vector according to B3);

   b5 An animal cell line containing the nucleic acid molecule according to B1) or containing the expression cassette according to B2) or containing the recombinant vector according to B3);

   b6 A plant cell line containing the nucleic acid molecule according to B1) or containing the expression cassette according to B2) or containing the recombinant vector according to B3);

   b7 Host cell for producing a LAG-3 binding molecule according to any one of claims 1 to 5.
7. 7. The biomaterial of claim 5, wherein the nucleic acid molecules of B1) comprise:

   g1 A DNA molecule having the coding sequence of the coding strand shown in positions 76-105 of SEQ ID No. 2;

   g2 A DNA molecule having the coding sequence of the coding strand as shown in SEQ ID No.2 at positions 148-198;

   g3 A DNA molecule whose coding sequence of the coding strand is shown in positions 295-327 of SEQ ID No. 2;

   g4 A DNA molecule having the coding sequence of the coding strand shown in positions 69 to 102 of SEQ ID No. 4;

   g5 A DNA molecule having the coding sequence of the coding strand as shown in SEQ ID No.4 at positions 148-168;

   g6 A DNA molecule whose coding sequence of the coding strand is shown in the 265 th to 288 th positions of SEQ ID No. 4;

   g7 A DNA molecule with the coding sequence of the coding chain as shown in the 1 st to 360 th nucleotides of SEQ ID No. 2;

   g8 A DNA molecule with the coding sequence of the coding chain as shown in the 1 st to 318 th nucleotides of SEQ ID No. 4;

   g9 A DNA molecule with the coding sequence of the coding chain as shown in SEQ ID No. 2;

   g10 A DNA molecule with the coding sequence of the coding chain as shown in SEQ ID No. 4;

   g11 A DNA molecule having the coding sequence of the coding strand shown in positions 76-105 of SEQ ID No. 9;

   g12 A DNA molecule having the coding sequence of the coding strand as shown in SEQ ID No.9 at positions 148-198;

   g13 A DNA molecule having the coding sequence of the coding strand as shown in SEQ ID No.9 at position 277-315;

   g14 A DNA molecule whose coding sequence of the coding strand is shown in the 69-102 th position of SEQ ID No. 11;

   g15 A DNA molecule having the coding sequence of the coding strand as shown in SEQ ID No.11 at positions 148-168;

   g16 A DNA molecule whose coding sequence of the coding strand is shown as 265 th to 291 th positions of SEQ ID No. 11;

   g17 A DNA molecule with the coding sequence of the coding chain shown as the 1 st to 348 nd nucleotides in SEQ ID No. 9;

   g18 A DNA molecule with the coding sequence of the coding chain as shown in the 1 st to 321 st nucleotides of SEQ ID No. 11;

   g19 A DNA molecule with the coding sequence of the coding chain as shown in SEQ ID No. 9;

   g20 A DNA molecule whose coding sequence of the coding strand is shown in SEQ ID No. 11.
8. 8. A medicament or pharmaceutical composition characterized by: the medicament or pharmaceutical composition comprising the LAG-3 binding molecule of any one of claims 1-5.
9. 9. The drug or pharmaceutical composition of claim 8, further comprising an anti-PD-1 antibody.
10. 10. Use of the LAG-3 binding molecule of any one of claims 1-5 or the biomaterial of claim 6 or 7 for the preparation of a LAG-3 inhibitor.

Images