CN112094348A

CN112094348A - Anti-human Tim3 antibody or functional fragment thereof and application thereof

Info

Publication number: CN112094348A
Application number: CN202011000703.6A
Authority: CN
Inventors: 闫卓红; 张洪涛; 王小珏; 易玲
Original assignee: Beijing Chest Hospital; Beijing Tuberculosis and Thoracic Tumor Research Institute
Current assignee: Beijing Chest Hospital; Beijing Tuberculosis and Thoracic Tumor Research Institute
Priority date: 2020-09-22
Filing date: 2020-09-22
Publication date: 2020-12-18
Anticipated expiration: 2040-09-22
Also published as: CN112094348B

Abstract

The invention provides an anti-human Tim3 antibody or a functional fragment thereof and application thereof. The invention obtains the anti-human Tim3 antibody with good specificity, high affinity and sensitivity by screening from a phage antibody library, and the anti-human Tim3 antibody obtained by the invention can specifically recognize and combine soluble Tim3 and cell membrane surface Tim3 protein.

Description

Anti-human Tim3 antibody or functional fragment thereof and application thereof

Technical Field

The invention relates to the technical field of biomedicine and antibody engineering, in particular to an anti-human Tim3 antibody or a functional fragment thereof and application thereof.

Background

Immunotherapy offers a new approach for tumor therapy, and antibody drugs, represented by PD1/PDL1 and CTLA4 immune checkpoint inhibitors, have benefited some tumor patients, but most patients show drug-initial unresponsiveness or partial response patients still develop tumor progression, which may be associated with the presence of other immunosuppressive molecules and incomplete activation of the immune system. Screening and combining with new immunotherapy targets, optimizing treatment strategies, is a great development trend of current tumor treatment.

T-cell immunoglobulin mucin 3(T-cell immunoglobulin and mucin domain-3, Tim 3; also known as HAVCR2) is one of the hottest immunosuppressive molecules following PD1/PDL1 and CTLA 4.Tim3 is a type I transmembrane glycoprotein originally associated with IFN-gamma producing Th1 and cytotoxic CD8⁺T (Tc1) cell surface. It was subsequently discovered that Tim3 can be expressed on a variety of tumor cells and other immune cells, such as Th17 cells, Tumor Infiltrating Lymphocytes (TILs), regulatory T cells (tregs), and natural immune cells. The binding of Tim3 to its ligand negatively modulates the immune response. There are 4 Tim3 ligands reported so far, including Galectin-9 (Galetin-9, Gal9), carcinoembryonic antigen-related cell adhesion molecule 1(carcino-embryonic antigen related cellular adhesion molecule 1, CEACAM1), high mobility group protein B1(high mobility group protein B1, HMGB1), and phosphatidylserine (PtdSer). Tim3 binds to its ligand to mediate immunosuppression. Specifically, Tim3 in combination with PtdSer mediates clearance of apoptotic cells; tim3 binds to HMGB1 to inhibit dendritic cell-mediated innate immune responses in the tumor microenvironment; tim3 binding to CEACAM1 was associated with Tim 3-mediated immune tolerance and T cell depletion; tim3 and Gal9 combine to regulate T cell apoptosis in the tumor microenvironment, and mediate T cell immunosuppression.

The Tim3 mediates the immune tolerance of autoimmunity and alloimmunity, the expression disorder of Tim3 is related to autoimmune diseases, and the expression level of Tim3 on T cells is inversely related to the progression of the autoimmune diseases. However, during chronic viral infections (e.g., HIV, HCV) and tumor progression, high levels of expression of Tim3 are associated with T cell suppression and functional depletion. Elevated expression of Tim3 can be detected in various types of tumors, such as non-small cell lung cancer, hepatocellular carcinoma, cervical cancer, colorectal cancer, ovarian cancer, head and neck cancer, renal cell carcinoma, gastric cancer, esophageal cancer, prostate cancer, bladder urothelial cancer, non-hodgkin's lymphoma, and the like. Elevated Tim3 expression has been reported to be often associated with shorter survival in patients. Tim3 is a potential prognostic marker for solid tumors. Tim3 can be cleaved from the cell surface to become soluble Tim3 (sttim 3). The level of sTim3 reflects the expression level of Tim3 on the surface of the cell membrane to a certain extent.

The main effector cell of anti-tumor immunity, CD8, in the tumor microenvironment⁺Once T cells express or co-express immunosuppressive molecules such as Tim3, PD1, and CTLA4, they are marked with CD8⁺Depletion of T cell function. The expression level of Tim3 in tumor infiltrating lymphocytes is positively correlated with the level of PD1/PDL1, and the up-regulation of Tim3 expression is probably an important mechanism for inducing the treatment resistance of PD1 antibodies. The Tim3 is an effective target point of tumor immunotherapy, and the anti-Tim 3 monoclonal antibody can play a synergistic effect with CTLA-4, PD-1, PD-L1 antibodies and the like, and can treat patients with PD-1 and PD-L1 drug resistance. Anti-tumor treatment with the Tim3 antibody is currently in clinical trials. The Tim3 antibody has good clinical application prospect in tumor treatment. Most of the existing Tim3 monoclonal antibodies are prepared by a mouse hybridoma technology. The progress of antibody engineering technology promotes the preparation of antibodies and improves the detection performance thereof. Phage antibody library technology can be used to screen for antibodies of any antigen specificity. The high-performance human Tim3 monoclonal antibody is screened by using the immune mouse phage antibody library, and compared with a mouse hybridoma antibody screening technology, the high-affinity antibody is obtained more organically.

Disclosure of Invention

The invention aims to provide an anti-human Tim3 antibody or a functional fragment thereof and application thereof. The antibody provided by the invention has the characteristics of high sensitivity, high specificity, high affinity and combination with different antigen epitopes, can be used for detecting soluble Tim3 protein and Tim3 protein on the surface of a cell membrane, and is favorable for improving the accuracy of a detection result.

In order to realize the purpose of the invention, the invention provides the following technical scheme:

an anti-human Tim3 antibody or functional fragment thereof comprising the following CDR regions:

the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain are respectively shown in SEQ ID NO.1-3, or respectively shown in SEQ ID NO.4-5 and SEQ ID NO.3, or respectively shown in SEQ ID NO. 6-8;

the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain are respectively shown in SEQ ID NO. 14-16; or respectively shown as SEQ ID NO. 17-19; or respectively shown as SEQ ID NO. 20-22; or as shown in SEQ ID NO.23-25, respectively.

In some embodiments, the amino acid sequences of the heavy chain variable region sequence and the light chain variable region sequence of the antibody or functional fragment thereof are set forth in SEQ ID No.9 and SEQ ID No.26, respectively, or SEQ ID No.10 and SEQ ID No.27, respectively, or SEQ ID No.11 and SEQ ID No.28, respectively, or SEQ ID No.12 and SEQ ID No.29, respectively.

In some embodiments, the antibody or functional fragment thereof further comprises an antibody constant region Fc; preferably, the constant region Fc comprises a heavy chain constant region and a light chain constant region;

preferably, the heavy chain constant region is selected from: any one of IgD, IgE, IgM, IgA, IgG1, IgG2a, IgG2b, IgG3 and IgG 4;

preferably, the light chain constant region is a kappa-type or lambda-type light chain constant region;

more preferably, the heavy chain constant region is selected from IgG1, and the amino acid sequence is shown in SEQ ID NO. 13;

more preferably, the light chain constant region is of the kappa type and has the amino acid sequence shown in SEQ ID NO. 30.

In some embodiments, the functional fragment is selected from the group consisting of Fab, Fab ', F (ab')₂One or more of Fv and scFv。

In some embodiments, the antibody is a mouse monoclonal antibody.

A nucleic acid molecule encoding said antibody or functional fragment thereof.

A vector comprising said nucleic acid molecule.

A host cell comprising said nucleic acid molecule or said vector.

Preferably, the host cell is a mammalian cell, and the mammalian cell is a HEK293E (human embryo kidney 293E cell), a cho (Chinese hamster over cell) cell or an NSO thymoma cell.

An antibody conjugate comprising the antibody or a functional fragment thereof.

The antibody or the functional fragment thereof is applied to the preparation of a reagent or a kit for detecting the Tim3 protein.

A kit comprising one or more of the antibody or functional fragment thereof, the nucleic acid molecule, the carrier and the antibody conjugate.

Has the advantages that:

the anti-Tim 3 antibody provided by the invention has stronger affinity and higher specificity, can identify and combine different epitopes of Tim3, can be used for detecting soluble Tim3 protein and Tim3 protein on the surface of a cell membrane, and is beneficial to improving the accuracy of a detection result, and the antibody provided by the invention has wide development prospect.

The invention provides a brand-new Tim3 antibody, which is a mouse anti-human Tim3 monoclonal antibody, has high sensitivity, high specificity and high affinity for the detection of soluble Tim3 protein and Tim3 protein on the surface of cell membranes, has the characteristics of being capable of combining different epitopes of Tim3, can detect Tim3 protein on the surfaces of tumor cells and immune cell membranes and sTim3 in body fluid by various immunological methods, has higher sensitivity and specificity when being used for detecting cells or body fluid taking Tim3 as targets, provides a new antibody selection for the detection of Tim3 protein, and provides a reference basis for the diagnosis and treatment of tumors, autoimmune diseases and infectious diseases.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is an SDS-PAGE identification of the purity of Tim3 antibody; in the figure, A: non-reducing conditions; b: reducing conditions; m: marker; 1: MsT001, MsT; 2: MsT 065; 3: MsT229, 229; 4: MsT286, 286;

FIG. 2 shows the ELISA identification of the Tim3 antibody detection Tim3 protein specificity;

FIG. 3 is Western blotting identification of Tim3 protein specificity in Tim3 antibody detection; in the figure, A: representative results of ponceau staining; b: western blotting detection results; m: marker; 1: tim3 recombinant protein under non-reducing conditions; 2: tim3 recombinant protein under reducing conditions;

FIG. 4 is a flow cytometry identification of Tim3 antibody recognizing Tim3-GFP recombinant plasmid transfected 293FT cell surface Tim3 protein;

FIG. 5 shows the flow cytometry identification of Tim3 antibody recognizing the Tim3 protein naturally expressed on the surface of U937 cells;

FIG. 6 shows the ELISA identification of Tim3 antibody for detecting Tim3 protein sensitivity;

FIG. 7 is a magnified ELISA identification of the sensitivity of the antibody labeled with biotin to Tim3 to detect Tim3 protein;

FIG. 8 is an identification of the affinity of the Tim3 antibody for Tim3 protein binding;

FIG. 9 is a sandwich ELISA identification of the binding of Tim3 antibody to different epitopes of Tim3 protein;

FIG. 10 is a forteBio Octet protein interaction identification of the Tim3 antibody binding to different epitopes of the Tim3 protein;

FIG. 11 is a sandwich ELISA assay for detection of Tim3 protein sensitivity by Tim3 paired antibodies recognizing different epitopes of Tim 3.

Detailed Description

In order that the invention may be more readily understood, it is further described as follows:

the invention provides a Tim3 antibody or a functional fragment thereof and application thereof.

The invention relates to an anti-T cell immunoglobulin mucin 3(Tim3) mouse monoclonal antibody, in particular to an antibody which specifically binds to a Tim3 protein and a soluble Tim3 protein on the surface of a cell membrane by using a specific nucleic acid sequence of the monoclonal antibody.

The present invention provides an antibody or a functional fragment thereof that specifically binds to Tim3, wherein the complementarity determining regions of the light and heavy chains have the sequences shown in any one of (1) to (4) below:

(1): the amino acid sequences of the CDR1-3 of the heavy chain are respectively shown as SEQ ID NO. 1-3; the amino acid sequences of CDR1-3 of the light chain are respectively shown in SEQ ID NO. 14-16;

(2): the amino acid sequences of the CDR1-3 of the heavy chain are respectively shown as SEQ ID NO.4-5 and SEQ ID NO. 3; the amino acid sequences of CDR1-3 of the light chain are respectively shown in SEQ ID NO. 17-19;

(3): the amino acid sequences of the CDR1-3 of the heavy chain are respectively shown as SEQ ID NO. 6-8; the amino acid sequences of CDR1-3 of the light chain are shown in SEQ ID NO.20-22, respectively.

(4) The amino acid sequences of the CDR1-3 of the heavy chain are respectively shown as SEQ ID NO. 6-8; the amino acid sequences of CDR1-3 of the light chain are shown in SEQ ID NO.23-25, respectively.

In some embodiments of the present invention, the antibody or functional fragment thereof has the CDR sequence shown in item (1) above, the amino acid sequence of the heavy chain variable region of the antibody or functional fragment thereof is shown in SEQ ID NO.9, and the amino acid sequence of the light chain variable region of the antibody or functional fragment thereof is shown in SEQ ID NO. 26.

In some embodiments of the present invention, the antibody or functional fragment thereof has the CDR sequence shown in item (2) above, the amino acid sequence of the heavy chain variable region of the antibody or functional fragment thereof is shown in SEQ ID NO.10, and the amino acid sequence of the light chain variable region of the antibody or functional fragment thereof is shown in SEQ ID NO. 27.

In some embodiments of the present invention, the antibody or functional fragment thereof has the CDRs sequence as set forth in item (3) above, the amino acid sequence of the heavy chain variable region of the antibody or functional fragment thereof is set forth in SEQ ID NO.11, and the amino acid sequence of the light chain variable region of the antibody or functional fragment thereof is set forth in SEQ ID NO. 28.

In some embodiments of the present invention, the antibody or functional fragment thereof has the CDRs sequence as set forth in item (4) above, the amino acid sequence of the light chain variable region of the antibody or functional fragment thereof is set forth in SEQ ID NO.12, and the amino acid sequence of the heavy chain variable region of the antibody or functional fragment thereof is set forth in SEQ ID NO. 29.

The term "antibody" refers to a protein comprising at least one and preferably two heavy chain (H) variable regions (abbreviated herein as VH), at least one and preferably two light chain (L) variable regions (abbreviated herein as VL), and at least one and preferably two heavy chain constant regions. The VH and VL regions may be further subdivided into hypervariable regions, termed "complementarity determining regions" (CDRs), interspersed with more conserved regions termed "framework regions" (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR 4.

It is well known in the art that both the binding specificity and avidity of an antibody are determined primarily by the CDR sequences, and that variants with similar biological activity can be obtained by readily altering the amino acid sequence of the non-CDR regions according to well-established, well-known techniques of the art. The monoclonal antibody variant of the present invention having the CDR sequences identical to those described above has similar biological activity because it has the CDR sequences identical to those of the antibody of the present invention.

The term "functional fragment" as used herein refers in particular to an antibody fragment having the same specificity as the parent antibody. These functional fragments typically have the same binding specificity as the antibody from which they are derived. The functional fragment of the invention is selected from Fab, Fab ', F (ab')₂In Fv, scFvOne or more of (a). Antibody functional fragments can also be obtained by peptide synthesis by recombinant genetic techniques also known to those skilled in the art or by, for example, automated peptide synthesizers, such as those sold by Applied BioSystems and the like.

In the present invention, the amino acid sequence of the above-mentioned monoclonal antibody of Tim3 or functional fragment thereof is disclosed, and those skilled in the art can easily obtain the above-mentioned monoclonal antibody of Tim3 or functional fragment thereof by using the conventional techniques in the art such as genetic engineering, and it is within the scope of the present invention to obtain the above-mentioned monoclonal antibody of Tim3 or functional fragment thereof by whatever means.

The invention also provides a paired antibody for quantitative detection of Tim3, wherein the first antibody comprises a heavy chain variable region with the amino acid sequence of SEQ ID NO.9 and a light chain variable region with the amino acid sequence of SEQ ID NO. 26; the second antibody comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO.11 and a light chain variable region having an amino acid sequence of SEQ ID NO.28, or the second antibody comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO.12 and a light chain variable region having an amino acid sequence of SEQ ID NO. 29.

The first antibody is a capture antibody and the second antibody is a detection antibody.

The paired antibody provided by the invention is used for detecting Tim3, and can be used for establishing a double-antibody sandwich enzyme-linked immunosorbent assay system, a colloidal gold chromatography detection system, a chemiluminescence quantitative detection system or a fluorescence immune layer plate detection system for detection.

A double-antibody sandwich ELISA kit for detecting human Tim3, which comprises the first antibody and the second antibody.

It is also another aspect of the present invention to provide a nucleic acid molecule which is DNA or RNA encoding the antibody or functional fragment thereof as described above.

Herein, nucleic acids include conservatively substituted variants thereof (e.g., substitution of degenerate codons) and complementary sequences, including genes, cDNA molecules, mRNA molecules, and fragments thereof such as oligonucleotides.

Based on the amino acid sequences disclosed in the present invention, those skilled in the art can easily obtain the nucleotide sequences encoding these antibodies, and no matter how the nucleotide molecule sequences are changed, the monoclonal antibodies encoding the anti-Tim 3 or functional fragments thereof are within the protection scope of the present invention.

In another aspect, the present invention provides a vector comprising the nucleic acid molecule described above. The vector can be a cloning vector and an expression vector, and comprises a plasmid vector, a phage vector, a virus vector and the like.

It is also a further aspect of the present invention to provide a host cell comprising a nucleic acid molecule as described above or a vector as described above.

The host cell mainly relates to eukaryotic cells, and the eukaryotic cells comprise mammalian cells, yeast cells and insect cells. Especially, the whole antibody or the full-length antibody is prepared, is commonly used for mammalian cells, and can be CHO, 293 and NSO cells. In some embodiments, the host cell is a mammalian cell 293.

A method of producing antibodies and functional fragments thereof capable of specifically binding to Tim3 comprising the steps of:

culturing the host cell as described above in a culture medium and under suitable culture conditions;

the antibodies and functional fragments thereof thus produced are recovered from the culture medium or from the cultured host cells.

The invention also provides application of the antibody or the functional fragment thereof in a reagent or a kit for detecting the Tim3 protein.

On the basis of the antibody disclosed in the present invention, those skilled in the art can easily use the above monoclonal antibody or functional fragment thereof for preparing a reagent or kit for detecting Tim3 protein, regardless of the detection principle, such as immunoprecipitation, ELISA, etc., on which the reagent or kit is based, as long as it utilizes the antibody or functional fragment thereof of the present invention, and thus it is within the scope of the present invention.

It is also another aspect of the present invention to provide an antibody conjugate or an antibody composition comprising the antibody or a functional fragment thereof.

The invention also provides a kit comprising the aforementioned antibody or functional fragment thereof, a nucleic acid molecule, a vector or an antibody conjugate.

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

Example 1: preparation of mouse anti-human Tim3 monoclonal antibody

(1) Animal immunization

The experimental animals were 6-8 week old female Balb/c mice purchased from Beijing Wittingle laboratory animals technologies, Inc. After raising for one week without abnormality, numbering. About 0.1ml of blood is collected through the retroorbital venous plexus. Standing at room temperature for 1 hr, standing at 4 deg.C overnight, centrifuging at 1500g, collecting upper layer serum, and storing at-20 deg.C for inspection. The basic immunization was carried out by emulsifying and mixing 100. mu.g/100. mu.l of Tim3-His recombinant protein (ACRO biosystems) with equal volume of Freund's complete adjuvant (Sigma; cat. No. F5881), and injecting subcutaneously at multiple sites of the abdomen and back. The booster immunizations were performed at 3-week intervals, and the same amount of Tim3-His recombinant protein was emulsified and mixed with Freund's incomplete adjuvant (Sigma Co., cat No. F5506) and injected subcutaneously at multiple points for immunization. After three times of immunization, the mice have a rest for six months, the boosting immunization is carried out once, blood is collected through retroorbital veins 7 days after the last immunization, serum is separated, and the serum titer is detected. And injecting 100 mu g of Tim3-His recombinant protein into the abdominal cavity of a mouse with higher titer after one month for impact immunization, killing the mouse 3 days after immunization, aseptically collecting the spleen, and quickly transferring to liquid nitrogen for freezing and storing for later use.

(2) Valence detection process

Diluting the Tim3-His recombinant protein to 5 mu g/ml by using a coating buffer solution (0.1M sodium carbonate with pH9.5), adding the diluted protein into an enzyme label plate of a 96-pore plate, slightly shaking and uniformly mixing the protein and the enzyme label plate, sealing the enzyme label plate by using a preservative film, and standing overnight at 4 ℃; washing the plate for three times by a plate washing machine, patting the enzyme-labeled plate dry, adding 200 mul/hole blocking liquid to block the non-specific binding sites, and carrying out 37 ℃ for 2 h; washing with a plate washing machine for three times, adding 100 mu l/hole of serum diluted in a gradient manner, and reacting for 2h at 37 ℃; washing with a plate washing machine for five times, adding 1:5000 diluted goat anti-mouse IgG (H + L) marked by horseradish peroxidase, 100 mu L/hole, and reacting for 1H at 37 ℃; washing with plate washing machine for five times, adding 100 μ l/hole color development liquid, standing at 37 deg.C for 30 min; adding 50 mul/hole of stop solution; OD450nm values were determined using a microplate reader. The dilution of the mouse serum is 1:16000, the antibody detection is positive, and the mouse with higher OD450 value is selected for impact immunization.

(3) Extraction and separation of antibody-secreting lymphocytes and synthesis of cDNA

The spleen of the immunized mouse is extracted with total RNA by Trizol method, and cDNA is synthesized by reverse transcription process.

(4) scFv phage antibody library construction and antibody screening

After reverse transcription, PCR amplifying antibody variable region (V) gene with specific primer pair, splicing heavy chain variable region (VH) and light chain variable region (VL) fragments to obtain fused scFv fragment, inserting it into pCOMB3X phage vector, and electrically transferring XL1-Blue competent cell to obtain phage antibody library with library capacity not less than 10⁸. The phage library is screened by coating Tim3-His recombinant protein by ELISA method, and positive clone is obtained by adopting a 'panning-amplification-enrichment' circulation mode for more than 2 rounds generally. And carrying out PCR amplification on ELISA detection positive clones, carrying out enzyme digestion and typing, then sequencing, and selecting a suitable variable region sequence according to a sequencing result.

(5) Antibody expression vector construction

The variable region gene is amplified by taking phage bacterium liquid or plasmid as a template, and the variable region gene fragment is inserted into an expression vector containing an antibody constant region to construct an IgG antibody heavy chain (pCMV3-H) and light chain (pCMV3-L) expression vector. The plasmid is amplified and then sent to downstream for expression.

Example 2: tim3 antibody sequence analysis

Analysis of amino acid sequences of antibody variable regions phage antibody library screening by scFv confirmed 4 Tim3 monoclonal antibodies, designated MsT001, MsT065, MsT229 and MsT 286.

Positive clones were sequenced to obtain light and heavy chain variable region sequences.

Analysis by IGBLAST software (https:// www.ncbi.nlm.nih.gov/IGBLAST /) revealed that the scFv antibodies belong to the VH class and the V κ type; ABodyBuilder software (http:// sight. stats. ox. ac. uk/webpps/newsbab/sabpred/ABodyBuilder /) the three CDR region positions for each antibody VH (heavy chain variable region) and VL (light chain variable region) were further analyzed in the Kabat model.

The CDR sequences of the heavy chain variable regions of the antibodies are shown in table 1 below.

TABLE 1

The heavy chain variable region amino acid sequence is as follows (underlined is the signal peptide amino acid sequence):

MsT001-VH amino acid sequence (SEQ ID NO. 9):

MGWSLILLFLVAVATRVLSEVQLQQSGAEVVMPGASVKMSCKTSGYTFTDYLIHWVKQRPGQGLVWIGAIDTSDSYASYNQKFKGKATLTLDESSRTAYMQLSSLTSEDSAVYFCTGEGLDYWGQGTSVTVSS；

MsT065-VH amino acid sequence SEQ ID NO. 10):

MGWSLILLFLVAVATRVLSQVQIQQSGAELVMPGASVKMSCKASGYTFTDHLMHWVKQRPGQGLEWIGAIDTSDSYASYNRKFKGKATLTVDESSRTAYMQLSSLTSEDSAVYYCAGEGLDYWGQGTSVTVSS；

MsT229-VH amino acid sequence SEQ ID NO. 11):

MGWSLILLFLVAVATRVLSEIQLVQSGPELVKPGASMKMSCKASGYSFTGYTMNWVKQSHGQNLEWIGLINPQTGGTSYNQNFRGKATLTVDKSSNTAYMELLSLTSEDSAVYYCARERGFDSPGFPYWGQGTLVTVSA；

MsT286-VH amino acid sequence SEQ ID NO. 12):

MGWSCIILFLVATATGVHSQVQLQQSGPELVKPGASMKMSCKASGYSFTGYTMNWVKQSHGQNLEWIGLINPQTGGTSYNQNFRGKATLTVDKSSNTAYMELLSLTSEDSAVYYCARERGFDSPGFPYWGQGTLVTVSA；

the amino acid sequences of the four antibody heavy chain constant regions are identical (SEQ ID NO. 13):

AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK；

the light chain variable region CDR amino acid sequences are shown in table 2.

TABLE 2

The light chain variable region amino acid sequence is as follows (underlined is the signal peptide amino acid sequence):

MsT001-VL amino acid sequence (SEQ ID NO. 26):

MGWSCIILFLVATATGVHSDIQMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLNWYLQKPGQSPKLLIYKVSYRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPPLTFGAGTKLELK；

MsT065-VL amino acid sequence (SEQ ID NO. 27):

MGWSCIILFLVATATGVHSDVVMTQTPLSLPVSLGDQASISCRSSQNLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPLTFGAGTKLEMK；

MsT229-VL amino acid sequence (SEQ ID NO. 28):

MGWSCIILFLVATATGVHSDIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPLTFGAGTKLELK；

MsT286-VL amino acid sequence (SEQ ID NO. 29):

MGWSCIILFLVATATGVHSDIVLTQSPASLAVSLGQRATISCKASQSVDYDGDSYMNWYQQKPGQPPKLLIYAASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQSNEDPYTFGGGTKLEIK；

the four antibody light chain constant region amino acid sequences are identical (SEQ ID No. 30):

RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC；

in addition, the nucleotide sequence encoding the antibody is shown below:

nucleotide sequence encoding the variable region of the antibody heavy chain (underlined signal peptide coding sequence):

MsT001-VH nucleotide sequence (SEQ ID NO. 31):

ATGGGCTGGTCCCTGATTCTGCTGTTCCTGGTGGCTGTGGCTACCAGGGTGCTGAGTGAGGTTCAGCTGCAGCAGTCTGGGGCTGAGGTTGTGATGCCTGGGGCTTCAGTGAAGATGTCCTGCAAGACTTCTGGCTACACATTCACTGACTACTTGATACACTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGTATGGATCGGAGCGATTGATACTTCTGATAGTTATGCTAGCTACAATCAAAAGTTCAAGGGCAAGGCCACATTGACTTTAGACGAATCCTCCAGGACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTTCTGTACAGGAGAGGGTCTGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA；

MsT065-VH nucleotide sequence (SEQ ID NO. 32):

ATGGGCTGGTCCCTGATTCTGCTGTTCCTGGTGGCTGTGGCTACCAGGGTGCTGAGTCAGGTCCAGATACAGCAGTCTGGGGCTGAGCTTGTGATGCCTGGGGCTTCAGTGAAGATGTCCTGCAAGGCTTCTGGCTACACATTCACTGACCACTTGATGCACTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATCGGAGCGATTGATACTTCTGATAGTTATGCTAGCTACAATCGAAAGTTCAAGGGCAAGGCCACATTGACTGTAGACGAGTCCTCCAGGACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCAGGAGAGGGTCTGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA；

MsT229-VH nucleotide sequence (SEQ ID NO. 33):

ATGGGCTGGTCCCTGATTCTGCTGTTCCTGGTGGCTGTGGCTACCAGGGTGCTGAGTGAGATTCAGTTGGTGCAGTCTGGACCTGAACTGGTGAAGCCTGGAGCTTCAATGAAGATGTCCTGCAAGGCTTCTGGTTACTCATTCACTGGCTACACCATGAACTGGGTGAAGCAGAGCCATGGACAGAACCTTGAGTGGATTGGACTTATTAATCCTCAGACTGGAGGCACTTCCTACAACCAGAATTTCAGGGGCAAGGCCACATTAACTGTAGACAAGTCATCCAACACAGCCTACATGGAGCTCCTCAGTCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGAGAAAGGGGGTTTGATTCACCCGGGTTTCCTTACTGGGGCCAAGGAACTCTGGTCACTGTCTCTGCA；

MsT286-VH nucleotide sequence (SEQ ID NO. 34):

ATGGGCTGGTCCCTGATTCTGCTGTTCCTGGTGGCTGTGGCTACCAGGGTGCTGAGTCAGGTTCAGCTGCAACAGTCTGGACCTGAACTGGTGAAGCCTGGAGCTTCAATGAAGATGTCCTGCAAGGCTTCTGGTTACTCATTCACTGGCTACACCATGAACTGGGTGAAGCAGAGCCATGGACAGAACCTTGAGTGGATTGGACTTATTAATCCTCAGACTGGAGGCACTTCCTACAACCAGAATTTCAGGGGCAAGGCCACATTAACTGTAGACAAGTCATCCAACACAGCCTACATGGAGCTCCTCAGTCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGAGAAAGGGGGTTTGATTCACCCGGGTTTCCTTACTGGGGCCAAGGAACTCTGGTCACTGTCTCTGCA；

the nucleotide sequences of the four antibody-encoding heavy chain constant regions are identical (SEQ ID NO. 35):

GCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCTGGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTCACCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGGGATTGTGGTTGTAAGCCTTGCATATGTACAGTCCCAGAAGTATCATCTGTCTTCATCTTCCCCCCAAAGCCCAAGGATGTGCTCACCATTACTCTGACTCCTAAGGTCACGTGTGTTGTGGTAGACATCAGCAAGGATGATCCCGAGGTCCAGTTCAGCTGGTTTGTAGATGATGTGGAGGTGCACACAGCTCAGACGCAACCCCGGGAGGAGCAGTTCAACAGCACTTTCCGCTCAGTCAGTGAACTTCCCATCATGCACCAGGACTGGCTCAATGGCAAGGAGTTCAAATGCAGGGTCAACAGTGCAGCTTTCCCTGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGCAGACCGAAGGCTCCACAGGTGTACACCATTCCACCTCCCAAGGAGCAGATGGCCAAGGATAAAGTCAGTCTGACCTGCATGATAACAGACTTCTTCCCTGAAGACATTACTGTGGAGTGGCAGTGGAATGGGCAGCCAGCGGAGAACTACAAGAACACTCAGCCCATCATGGACACAGATGGCTCTTACTTCGTCTACAGCAAGCTCAATGTGCAGAAGAGCAACTGGGAGGCAGGAAATACTTTCACCTGCTCTGTGTTACATGAGGGCCTGCACAACCACCATACTGAGAAGAGCCTCTCCCACTCTCCTGGTAAATAA；

the amino acid sequence encoding the variable region of the antibody light chain is as follows (underlined is the signal peptide coding sequence):

MsT001-VL nucleotide sequence (SEQ ID NO. 36):

ATGGGCTGGTCCTGTATCATCCTGTTCCTGGTGGCTACAGCCACAGGAGTGCATAGTGATATCCAGATGACTCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTATTTAAATTGGTACCTGCAGAAGCCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCTACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTTCTGCTCTCAAAGTACACATGTTCCTCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA；

MsT065-VL nucleotide sequence (SEQ ID NO. 37):

ATGGGCTGGTCCTGTATCATCCTGTTCCTGGTGGCTACAGCCACAGGAGTGCATAGTGATGTTGTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAACCTTGTACACAGTAATGGAAACACCTATTTACATTGGTACCTGCAGAAGCCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTTCTGCTCTCAAAGTACACATGTTCCGCTCACGTTCGGTGCTGGCACCAAGCTGGAAATGAAA；

MsT229-VL nucleotide sequence (SEQ ID NO. 38):

ATGGGCTGGTCCTGTATCATCCTGTTCCTGGTGGCTACAGCCACAGGAGTGCATAGTGATATCCAGATGACTCAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGCAATTATTTAAACTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACTACACATCAAGATTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGAGCAAGAAGATATTGCCACTTACTTTTGCCAACAGGGTAATACGCTTCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA；

MsT286-VL nucleotide sequence (SEQ ID NO. 39):

ATGGGCTGGTCCTGTATCATCCTGTTCCTGGTGGCTACAGCCACAGGAGTGCATAGTGATATTGTGCTAACCCAATCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAGGGCCACCATCTCCTGCAAGGCCAGCCAAAGTGTTGATTATGATGGTGATAGTTATATGAACTGGTACCAACAGAAACCAGGACAGCCACCCAAACTCCTCATCTATGCTGCATCCAATCTAGAATCTGGGATCCCAGCCAGGTTTAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCAGCAAAGTAATGAGGATCCGTACACGTTCGGAGGGGGGACAAAGCTGGAAATAAAA；

the nucleotide sequences of the four antibodies encoding the light chain constant region are identical (SEQ ID No. 40):

CGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGTTAA。

example 3 antibody expression and purification characterization

The light chain and heavy chain expression vector of the same antibody are simultaneously transfected into 293 cells transiently, culture supernatant is taken 6-7 days after transfection, and affinity purification is carried out through a protein A purification column to obtain a purified antibody. The molecular weight of the antibody is predicted according to a protein molecular weight calculator, and the purity and the molecular weight of the antibody are identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

SDS-PAGE electrophoresis detection flow: separately, 5. mu.g of purified antibody was subjected to electrophoretic separation with 10% SDS-PAGE under non-reducing conditions (no addition of beta mercaptoethanol to the buffer for protein electrophoresis, no boiling of the sample) and reducing conditions (addition of beta mercaptoethanol to the buffer for protein electrophoresis, boiling of the sample for 5min), and subjected to electrophoresis at 150V for about 1 hour. After the electrophoresis, the electrophoresis was carried out overnight at 4 ℃ by staining with Coomassie brilliant blue. And (4) decolorizing the decolorizing solution on the second day until the strips are clear, and taking a picture by a camera to record the dyeing result after the background is clean.

SDS-PAG assay results (FIG. 1): the molecular weight of the four antibody heavy chains is predicted to be about 48kDa, the molecular weight of the light chains is predicted to be about 24kDa, and the molecular weight of the whole IgG antibodies is predicted to be about 145 kDa. The molecular weight of the protein is similar to the predicted molecular weight through SDS-PAGE electrophoretic identification, and the purity of the antibody is good and about 95%. In the figure, A: detecting results under non-reduction conditions; b: detecting the result under a reduction condition; m: marker; 1: MsT001, MsT; 2: MsT 065; 3: MsT229, 229; 4: MsT286, 286.

Example 4 identification of antibody specificity and sensitivity

(1) Identification of antibody specificity

Detecting specificity of Tim3 antibody by ELISA

The specificity of the Tim3 antibody was identified using human Tim3, murine Tim3, human CD137, human PD-L1, and human OX40 recombinant proteins.

ELISA detection process: the coating liquid coats human Tim3, murine Tim3, human CD137, human PD-L1 and human OX40 recombinant protein (all His tags), 1 μ g/ml, 4 deg.CAnd (4) at night. The next day, the plate washer was washed with Wash Buffer working solution 3 times, and then 5% skim milk powder was added to block the non-specific binding sites, 200. mu.l/well, and the reaction was carried out at 37 ℃ for 2 hours. After 3 times of washing with a plate washer, 100. mu.l/well of Tim3 antibody (Tim3 antibody control F38-2E2 and MsT001, MsT065, MsT229 and MsT286) diluted with 5% skim milk powder at 1. mu.g/ml was added, and reacted at 37 ℃ for 2 hours. After washing for 5 times, adding 1:5000 diluted horse radish peroxidase-labeled goat anti-rabbit IgG (H + L) (Beijing Zhonghua Jinqiao Biotech Co., Ltd.) to react for 1H at 37 ℃. After washing 5 times, TMB substrate solution was added at 100. mu.l/well and reacted at 37 ℃ for 30 min. Stop solution was added at 50. mu.l/well. OD determination by means of enzyme-linked immunosorbent assay₄₅₀The value is obtained. And analyzing the detection result by Graphpad software.

Injecting main reagent sources and preparation:

coating solution (BD OptEIA Coating Buffer, 51-2713KC) 0.1M sodium carbonate pH 9.5;

washing solution (BD OptEIA Wash Buffer, 51-9003739)20 × concentrated washing solution, which should be diluted to 1 × working solution with deionized water or distilled water;

color developing solution A (BD OptEIA Substrate reagent A, 51-2606KZ) contains hydrogen peroxide buffer;

the color developing solution B (BD OptEIA Substrate reagent B, 51-2607KZ) contains an organic solvent of 3,3',5,5' Tetramethylbenzidine (TMB);

stop Solution (BD OptEIA Stop Solution, 51-2608KZ)1 sulfuric acid;

5% of milk: 5 g of milk are added to 100ml of 1 Xwash (1 Xwash buffer).

Test results (fig. 2): the Tim3 antibodies F38-2E2, MsT001, MsT065, MsT229 and MsT286 can well identify human Tim3 recombinant protein, only MsT286 has weak cross-binding to mouse Tim3 recombinant protein, the prepared four antibodies are not combined with human CD137, PDL1 and OX40 recombinant protein, and the four antibodies have good specificity to human Tim3 protein.

② Western blotting detection of specificity of Tim3 antibody

The binding property of the Tim3 antibody to the Tim3 recombinant protein under non-reducing conditions and reducing conditions is identified by a western blotting method. A commercially available Tim3 antibody F38-2E2 was used as a control.

Western blotting detection flow: 1 mu g of Tim3 recombinant protein is respectively taken and subjected to electrophoretic separation by using 10% SDS-PAGE gel under non-reducing conditions (beta mercaptoethanol is not added into protein electrophoresis loading buffer, samples are not boiled) and reducing conditions (beta mercaptoethanol is added into protein electrophoresis loading buffer, samples are boiled for 5min), and the electrophoresis is carried out for about 1h at 150V. After the electrophoresis is finished, the model is transferred, and the protein separated by the SDS-PAGE gel is transferred to a nitrocellulose membrane at 300mA for 1 h. The film was stained with ponceau red stain for 10min and the staining was recorded by photography (a in fig. 3). After washing with deionized water, sealing with 5% skimmed milk powder at room temperature for 2 h. Antibodies (F38-2E2, MsT001, MsT065, MsT229 and MsT286) at 0.5. mu.g/ml were each bound overnight at 4 ℃. PBST solution washing membrane 3 times, 5 min/time. And adding horseradish peroxidase-labeled goat anti-mouse IgG (H + L) diluted by 1:10000 into the secondary antibody, and reacting for 1H at room temperature. PBST washing removes unbound antibody. Adding a chemiluminescence substrate, and recording the detection result by a gel imager.

Test results (fig. 3): the control antibodies F38-2E2, MsT229 and MsT286 antibodies mainly recognize Tim3 recombinant protein in non-reducing conditions (only the band of interest in non-reducing conditions is visible in short exposure), considering that the epitope recognized by them is mainly a conformational epitope; MsT001 and MsT065 antibodies can well recognize Tim3 recombinant proteins under non-reducing conditions and reducing conditions, and the recognized antigen epitope is mainly sequential epitope (also called linear epitope). In the figure, A: representative results of ponceau staining; b: western blotting detection results; m is Marker; 1, Tim3 recombinant protein under non-reducing condition; 2 Tim3 recombinant protein under reducing conditions.

③ detecting the Tim3 protein on the surface of the cell membrane by flow cytometry

The Tim3 protein expressed on the surface of a cell membrane after a 293FT cell is transfected by a Tim3-GFP recombinant plasmid and the naturally expressed Tim3 protein on the surface of a U937 cell membrane are identified by flow cytometry. A commercially available mouse anti-human Tim3 antibody (F38-2E2) was used as a positive control.

Flow cytometry detection procedure: after 293FT cells are transfected by the Tim3-GFP recombinant plasmid for 24h, single cell suspension is prepared by trypsinizing the cells. U937 cells were suspension cells. The cells to be examined were washed with PBSAfter one time, 1. mu.g of anti-Tim 3 antibody (F38-2E2, MsT001, MsT065, mAb and MsT286) was added to react at room temperature for 30min, washed with PBS 1 time, added with 1:2000 diluted AF 647-labeled goat anti-mouse IgG (H + L) antibody, and reacted at room temperature for 30min in the dark. PBS wash 2 times, cell heavy suspension in 200 u l PBS solution. Using BD LSRFortessa^TMAnd (4) detecting by using a flow cytometer, and analyzing the result by using BDFACSDiva software.

Flow cytometry results for detecting Tim3-GFP recombinant plasmid transfected 293FT cell surface Tim3 protein (FIG. 4): the Tim3 antibody can be combined with Tim3 protein expressed on the surface of 293FT cell membrane transfected by Tim3 recombinant plasmid. When the test is carried out, certain 293FT cells are positive in GFP label, and the Tim3 antibody recognizes the Tim3 negative, which is considered to be caused by interference of the GFP label on recognition of the Tim3 protein by the Tim3 antibody.

Flow cytometry results for detection of Tim3 protein on the U937 cell surface (fig. 5): the Tim3 antibody can be combined with Tim3 recombinant protein naturally expressed on the surface of the U937 cell, and the detection positive rate is about 20 percent.

(2) Determination of antibody sensitivity

Indirect ELISA detection of Tim3 antibody detection Tim3 protein sensitivity

And (3) detection flow: the Tim3 recombinant protein was diluted in 10-fold gradient (1000-0.001ng/ml) to coat 96-well microtiter plates at 100. mu.l/well overnight at 4 ℃. Primary antibodies were added at 1 μ g/ml of MsT001, MsT065, MsT229, MsT286 and control antibody (F38-2E 2). The rest steps are the same as the ELISA detection process.

ELISA sensitivity assay results (fig. 6): MsT001 and MsT286 can detect that the sensitivity of Tim3-His recombinant protein is 1ng/ml, the sensitivity of MsT065 and MsT229 is 10ng/ml and the sensitivity of F38-2E2 is 100ng/ml by indirect ELISA. The sensitivity of the 4 screened antibodies is higher than that of the commercial Tim3 antibody F38-2E 2.

② the biotin-streptavidin amplified ELISA for detecting the sensitivity of Tim3 antibody recognition Tim3 protein

The Tim3 antibody entrusts the relevant company to mark biotin, and the sensitivity of the biotin-marked Tim3 antibody for recognizing Tim3-His recombinant protein is identified by biotin-streptavidin amplification ELISA.

And (3) detection flow: the Tim3-His recombinant protein was diluted in 10-fold gradient (1000-0.001ng/ml) to coat 96-well microtiter plates at 100. mu.l/well overnight at 4 ℃. Primary antibodies were added with 1. mu.g/ml biotin-labeled MsT001, MsT065, MsT229, MsT 286. The secondary antibody was added with horseradish peroxidase-labeled streptavidin diluted 1: 5000. The rest steps are the same as the ELISA detection process.

Amplification ELISA sensitivity assay results (fig. 7): the sensitivity of the Tim3 antibody for recognizing the Tim3-His recombinant protein is detected by an amplification ELISA method, the detection sensitivity of MsT001-Bio and MsT286-Bio antibodies is 100pg/ml, the detection sensitivity of MsT065-Bio antibody is 10ng/ml, and the detection sensitivity of MsT229-Bio antibody is (1 ng/ml).

Comparison of detection sensitivity of unlabeled antibody and biotin-labeled antibody (fig. 6 and 7): the detection sensitivity (100pg/ml) of MsT001-Bio and MsT286-Bio is 10 times higher than that (1ng/ml) of MsT001 and MsT 286-Bio. MsT065-Bio and MsT065 showed the same sensitivity (10ng/ml), and MsT065 labeled biotin did not improve the sensitivity. MsT229-Bio test sensitivity (1ng/ml) was 10-fold higher than MsT229 test sensitivity (10 ng/ml).

Example 5: tim3 antibody affinity identification

Antibody affinity was detected by ForteBio Octet molecular interaction instrument. Tim3-hFc recombinant protein (Sino Biological) as the detection antigen, FORTEBIO anti-human IgG Fc capture (AHC) biosensor, and Tim3 antibody F38-2E2 as the control antibody.

And (3) affinity detection flow: prewetting a FORTEBIO anti-human IgG Fc capture (AHC) biosensor in a balance solution (0.1% BSA + 0.02% Tween20 in PBS) for 10 minutes, diluting the Tim3-hFc recombinant protein to 5 mu g/mL by using the balance solution, adding the diluted protein into a second row of a light-proof 96-well plate, and keeping the concentration at 200 mu l/well; the Tim3 antibody was diluted from 250nM to 15.6nM starting fold ratio, added to the fourth column of a light-protected 96-well plate at 200. mu.l/well, antibody 0nM blank was set and 200. mu.l of the equilibration buffer was added. The equilibration solution was added to the first and third columns at 200. mu.l/well. ForteBio Octet molecular interaction meter test, the sensor was equilibrated in the first column for 60s to obtain a base equilibrium curve, and then antigen immobilization was performed in the second column for 100 s. A wash is performed 120s in the third column, binding antibody 180s in the fourth column to obtain a binding curve, and dissociation 300s in the first column to obtain a dissociation curve. The ForteBio Octet analysis software performs fitting analysis on the curve to obtain an affinity value.

Affinity assay results (fig. 8): the affinity of the antibody was determined by ForteBio Octet system using a buffered saline solution without Tim3 protein as a blank and the commercial Tim3 antibody F38-2E2 as a detection control. Affinity K of the antibody after data analysis_DThe values are respectively 8.40X 10^-10M(F38-2E2)、4.61×10^-11M(MsT001)、1.59×10^-9M(MsT065)、3.70×10^-9M (MsT229) and 7.73X 10^-10M (MsT 286). Goodness of curve fit R²0.9956(F38-2E2), 0.9929(MsT001), 0.9917(MsT065), 0.9702(MsT229) and 0.9965(MsT286), respectively.

Example 6: tim3 paired antibody screening and detection sensitivity identification

(1) Sandwich ELISA screening for paired antibodies to Tim3

And (3) detection flow: capture antibody immobilized 2 μ g/ml of Tim3 monoclonal antibody (MsT001, MsT065, MsT229, or MsT286) overnight at 4 ℃; the detection time of the Tim3-His recombinant protein is 1 mu g/ml, 100 mu l/hole, 37 ℃ and 2 h; the detection was carried out with biotin-labeled Tim3 antibody (MsT001-Bio, MsT065-Bio, MsT229-Bio or MsT286-Bio) at a concentration of 1. mu.g/ml, 37 ℃ for 2 h; biotin recognition add 1:5000 peroxidase-labeled streptavidin, 37 ℃, 1 h. The rest steps are the same as the ELISA detection process.

Sandwich ELISA screening for Tim3 antibody epitope pairing results (FIG. 9; Table 3): MsT001 can be paired with MsT001-Bio, MsT229-Bio and MsT286-Bio antibodies, and MsT001 can be specifically distinguished from MsT229 and MsT286 by considering that MsT001 antibody might bind to a repeated epitope of Tim3 protein; MsT065 can be paired with MsT001-Bio, MsT229-Bio and MsT286-Bio antibodies, taking into account MsT065 and MsT229 and MsT286 specific different antigen binding epitopes. The results of MsT001 and MsT065 antibodies were inconsistent, considering that the antigen binding sites of the two antibodies partially overlap or due to steric hindrance after antibody binding, the Tim3 protein bound to MsT001 first, which hinders MsT065 binding, and MsT065 first, which has less effect on MsT001 binding again. MsT229 can be paired with MsT001-Bio antibody; the detection of OD450 values with the pair MsT065-Bio was low, considering that there was interference with binding of MsT065-Bio antibody; no pairing with MsT286-Bio was possible, and both were considered to bind the same epitope. MsT286 can be paired with MsT001-Bio, MsT065-Bio antibodies; no pairing with MsT229-Bio antibody was achieved, again indicating that both binding epitopes are identical.

TABLE 3 Tim3 antibody pairing

+++(OD450>2.0)；+(0.12<OD450<0.5)；-(OD450<0.1)

(2) ForteBio Octet molecular interaction instrument for identifying Tim3 antibody binding with Tim3 protein epitope

The epitope characteristic that the Tim3 antibody binds to the Tim3 protein was identified by a ForteBio Octet molecular interaction instrument. The Tim3-hFc recombinant protein is used as an antigen for detection, and a FORTEBIO anti-human IgG Fc capture (AHC) biosensor is applied.

Antibody binding epitope identification procedure: FORTEBIO anti-human IgG Fc capture (AHC) biosensor was prewetted in equilibration solution (0.1% BSA + 0.02% Tween20 in PBS) for 10 minutes. One set of AHC biosensors (4) reacted in the equilibrium for 60s, bound with 5. mu.g/ml Tim3-hFc recombinant protein for 100s, equilibrated in the equilibrium for 120s, bound with 5. mu.g/ml MsT001 antibody (or MsT065, MsT229, MsT286) for 300s, saturated, and reacted with four antibodies (5. mu.g/ml) for 180s, respectively. Antibody binding epitope characteristics were analyzed by ForteBio Octet analysis software.

Antibody epitope characterization results (fig. 10): binding detection of Tim3-hFc protein immobilized by AHC biosensor Tim3 antibody (MsT001, MsT065, MsT229 or MsT286) reaches saturation, then respectively reacting with 4 Tim3 antibody junctions, and analyzing the epitope characteristics of antibody binding. The results partially show the characteristics of the subsequent reaction curve that marks the detection antibody as zero point when saturated. MsT001 and MsT065 antibodies are not completely same when combined with the Tim3 epitope, the antigen-combined epitopes of the two antibodies may be partially overlapped or steric interference exists after the two antibodies are combined with the antigen, and after MsT001 is combined with the Tim3 protein, MsT065 cannot be combined with the protein; after MsT065 is combined with Tim3, MsT001 can still be combined with Tim3 protein; both differ from the binding epitopes of MsT229 and MsT286 antibodies. MsT229 and MsT286 antibodies bind to the same epitope of the Tim3 protein, and one antibody binds to the Tim3 protein, while the other antibody is no longer able to bind.

(3) Sandwich ELISA determination Tim3 paired antibody detection Tim3 protein sensitivity

And (3) detection flow: the sensitivity of the paired antibody detection was identified by sandwich ELISA. Detection was performed using a 10-fold gradient dilution (1000-0.001ng/ml) of Tim-His recombinant protein. The remaining steps were the same as in the sandwich ELISA protocol described above.

Sandwich ELISA assay Tim3 paired antibody detection Tim3 protein sensitivity (fig. 11, table 4): MsT001 has low detection sensitivity (10ng/ml) when paired with self MsT-Bio and has highest detection sensitivity (0.01ng/ml) when paired with MsT229-Bio and MsT-Bio. MsT065 were all 1.0ng/ml sensitive to pairing with other antibodies. MsT229 and MsT286 were paired with MsT001-Bio, respectively, with a sensitivity of 0.1 ng/ml. MsT229 and MsT286 were paired with MsT065-Bio, respectively, with a sensitivity of 10 ng/ml. In summary, MsT001 was paired with MsT229-Bio or MsT286-Bio for the highest detection sensitivity.

TABLE 4 Tim3 paired antibody detection sensitivity (ng/ml)

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

SEQUENCE LISTING

<110> the university of capital medical department affiliated to the Beijing thoracic hospital; research institute of tuberculosis and breast tumor in Beijing

<120> anti-human Tim3 antibody or functional fragment thereof and application thereof

<130> PA20023125

<160> 40

<170> PatentIn version 3.3

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Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys

85 90 95

Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Pro

100 105 110

Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu

115 120 125

Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser

130 135 140

Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu

145 150 155 160

Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr

165 170 175

Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn

180 185 190

Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro

195 200 205

Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln

210 215 220

Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val

225 230 235 240

Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val

245 250 255

Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln

260 265 270

Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn

275 280 285

Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val

290 295 300

Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu Ser His

305 310 315 320

Ser Pro Gly Lys

<210> 14

<211> 16

<212> PRT

<213> Artificial sequence

<400> 14

Arg Ser Ser Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu Asn

1 5 10 15

<210> 15

<211> 7

<212> PRT

<213> Artificial sequence

<400> 15

Lys Val Ser Tyr Arg Phe Ser

1 5

<210> 16

<211> 10

<212> PRT

<213> Artificial sequence

<400> 16

Ser Gln Ser Thr His Val Pro Pro Leu Thr

1 5 10

<210> 17

<211> 16

<212> PRT

<213> Artificial sequence

<400> 17

Arg Ser Ser Gln Asn Leu Val His Ser Asn Gly Asn Thr Tyr Leu His

1 5 10 15

<210> 18

<211> 7

<212> PRT

<213> Artificial sequence

<400> 18

Lys Val Ser Asn Arg Phe Ser

1 5

<210> 19

<211> 9

<212> PRT

<213> Artificial sequence

<400> 19

Ser Gln Ser Thr His Val Pro Leu Thr

1 5

<210> 20

<211> 11

<212> PRT

<213> Artificial sequence

<400> 20

Arg Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn

1 5 10

<210> 21

<211> 7

<212> PRT

<213> Artificial sequence

<400> 21

Tyr Thr Ser Arg Leu His Ser

1 5

<210> 22

<211> 9

<212> PRT

<213> Artificial sequence

<400> 22

Gln Gln Gly Asn Thr Leu Pro Leu Thr

1 5

<210> 23

<211> 15

<212> PRT

<213> Artificial sequence

<400> 23

Lys Ala Ser Gln Ser Val Asp Tyr Asp Gly Asp Ser Tyr Met Asn

1 5 10 15

<210> 24

<211> 7

<212> PRT

<213> Artificial sequence

<400> 24

Ala Ala Ser Asn Leu Glu Ser

1 5

<210> 25

<211> 9

<212> PRT

<213> Artificial sequence

<400> 25

Gln Gln Ser Asn Glu Asp Pro Tyr Thr

1 5

<210> 26

<211> 132

<212> PRT

<213> Artificial sequence

<400> 26

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Asp Ile Gln Met Thr Gln Thr Pro Leu Ser Leu Pro Val

20 25 30

Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu

35 40 45

Val His Ser Asn Gly Asn Thr Tyr Leu Asn Trp Tyr Leu Gln Lys Pro

50 55 60

Gly Gln Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser Tyr Arg Phe Ser

65 70 75 80

Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

85 90 95

Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys

100 105 110

Ser Gln Ser Thr His Val Pro Pro Leu Thr Phe Gly Ala Gly Thr Lys

115 120 125

Leu Glu Leu Lys

130

<210> 27

<211> 131

<212> PRT

<213> Artificial sequence

<400> 27

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val

20 25 30

Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Asn Leu

35 40 45

Val His Ser Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro

50 55 60

Gly Gln Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser

65 70 75 80

Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

85 90 95

Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys

100 105 110

Ser Gln Ser Thr His Val Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu

115 120 125

Glu Met Lys

130

<210> 28

<211> 126

<212> PRT

<213> Artificial sequence

<400> 28

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala

20 25 30

Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile

35 40 45

Ser Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys

50 55 60

Leu Leu Ile Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg

65 70 75 80

Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn

85 90 95

Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr

100 105 110

Leu Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

115 120 125

<210> 29

<211> 130

<212> PRT

<213> Artificial sequence

<400> 29

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val

20 25 30

Ser Leu Gly Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser Val

35 40 45

Asp Tyr Asp Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly

50 55 60

Gln Pro Pro Lys Leu Leu Ile Tyr Ala Ala Ser Asn Leu Glu Ser Gly

65 70 75 80

Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu

85 90 95

Asn Ile His Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln

100 105 110

Gln Ser Asn Glu Asp Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu

115 120 125

Ile Lys

130

<210> 30

<211> 107

<212> PRT

<213> Artificial sequence

<400> 30

Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu

1 5 10 15

Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe

20 25 30

Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg

35 40 45

Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu

65 70 75 80

Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser

85 90 95

Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys

100 105

<210> 31

<211> 399

<212> DNA

<213> Artificial sequence

<400> 31

atgggctggt ccctgattct gctgttcctg gtggctgtgg ctaccagggt gctgagtgag 60

gttcagctgc agcagtctgg ggctgaggtt gtgatgcctg gggcttcagt gaagatgtcc 120

tgcaagactt ctggctacac attcactgac tacttgatac actgggtgaa gcagaggcct 180

ggacaaggcc ttgtatggat cggagcgatt gatacttctg atagttatgc tagctacaat 240

caaaagttca agggcaaggc cacattgact ttagacgaat cctccaggac agcctacatg 300

cagctcagca gcctgacatc tgaggactct gcggtctatt tctgtacagg agagggtctg 360

gactactggg gtcaaggaac ctcagtcacc gtctcctca 399

<210> 32

<211> 399

<212> DNA

<213> Artificial sequence

<400> 32

atgggctggt ccctgattct gctgttcctg gtggctgtgg ctaccagggt gctgagtcag 60

gtccagatac agcagtctgg ggctgagctt gtgatgcctg gggcttcagt gaagatgtcc 120

tgcaaggctt ctggctacac attcactgac cacttgatgc actgggtgaa gcagaggcct 180

ggacaaggcc ttgagtggat cggagcgatt gatacttctg atagttatgc tagctacaat 240

cgaaagttca agggcaaggc cacattgact gtagacgagt cctccaggac agcctacatg 300

cagctcagca gcctgacatc tgaggactct gcggtctatt actgtgcagg agagggtctg 360

gactactggg gtcaaggaac ctcagtcacc gtctcctca 399

<210> 33

<211> 417

<212> DNA

<213> Artificial sequence

<400> 33

atgggctggt ccctgattct gctgttcctg gtggctgtgg ctaccagggt gctgagtgag 60

attcagttgg tgcagtctgg acctgaactg gtgaagcctg gagcttcaat gaagatgtcc 120

tgcaaggctt ctggttactc attcactggc tacaccatga actgggtgaa gcagagccat 180

ggacagaacc ttgagtggat tggacttatt aatcctcaga ctggaggcac ttcctacaac 240

cagaatttca ggggcaaggc cacattaact gtagacaagt catccaacac agcctacatg 300

gagctcctca gtctgacatc tgaggactct gcagtctatt actgtgcaag agaaaggggg 360

tttgattcac ccgggtttcc ttactggggc caaggaactc tggtcactgt ctctgca 417

<210> 34

<211> 417

<212> DNA

<213> Artificial sequence

<400> 34

atgggctggt ccctgattct gctgttcctg gtggctgtgg ctaccagggt gctgagtcag 60

gttcagctgc aacagtctgg acctgaactg gtgaagcctg gagcttcaat gaagatgtcc 120

tgcaaggctt ctggttactc attcactggc tacaccatga actgggtgaa gcagagccat 180

ggacagaacc ttgagtggat tggacttatt aatcctcaga ctggaggcac ttcctacaac 240

cagaatttca ggggcaaggc cacattaact gtagacaagt catccaacac agcctacatg 300

gagctcctca gtctgacatc tgaggactct gcagtctatt actgtgcaag agaaaggggg 360

tttgattcac ccgggtttcc ttactggggc caaggaactc tggtcactgt ctctgca 417

<210> 35

<211> 975

<212> DNA

<213> Artificial sequence

<400> 35

gccaaaacga cacccccatc tgtctatcca ctggcccctg gatctgctgc ccaaactaac 60

tccatggtga ccctgggatg cctggtcaag ggctatttcc ctgagccagt gacagtgacc 120

tggaactctg gatccctgtc cagcggtgtg cacaccttcc cagctgtcct gcagtctgac 180

ctctacactc tgagcagctc agtgactgtc ccctccagca cctggcccag cgagaccgtc 240

acctgcaacg ttgcccaccc ggccagcagc accaaggtgg acaagaaaat tgtgcccagg 300

gattgtggtt gtaagccttg catatgtaca gtcccagaag tatcatctgt cttcatcttc 360

cccccaaagc ccaaggatgt gctcaccatt actctgactc ctaaggtcac gtgtgttgtg 420

gtagacatca gcaaggatga tcccgaggtc cagttcagct ggtttgtaga tgatgtggag 480

gtgcacacag ctcagacgca accccgggag gagcagttca acagcacttt ccgctcagtc 540

agtgaacttc ccatcatgca ccaggactgg ctcaatggca aggagttcaa atgcagggtc 600

aacagtgcag ctttccctgc ccccatcgag aaaaccatct ccaaaaccaa aggcagaccg 660

aaggctccac aggtgtacac cattccacct cccaaggagc agatggccaa ggataaagtc 720

agtctgacct gcatgataac agacttcttc cctgaagaca ttactgtgga gtggcagtgg 780

aatgggcagc cagcggagaa ctacaagaac actcagccca tcatggacac agatggctct 840

tacttcgtct acagcaagct caatgtgcag aagagcaact gggaggcagg aaatactttc 900

acctgctctg tgttacatga gggcctgcac aaccaccata ctgagaagag cctctcccac 960

tctcctggta aataa 975

<210> 36

<211> 396

<212> DNA

<213> Artificial sequence

<400> 36

atgggctggt cctgtatcat cctgttcctg gtggctacag ccacaggagt gcatagtgat 60

atccagatga ctcaaactcc actctccctg cctgtcagtc ttggagatca agcctccatc 120

tcttgcagat ctagtcagag ccttgtacac agtaatggaa acacctattt aaattggtac 180

ctgcagaagc caggccagtc tccaaagctc ctgatctaca aagtttccta ccgattttct 240

ggggtcccag acaggttcag tggcagtgga tcagggacag atttcacact caagatcagc 300

agagtggagg ctgaggatct gggagtttat ttctgctctc aaagtacaca tgttcctccg 360

ctcacgttcg gtgctgggac caagctggag ctgaaa 396

<210> 37

<211> 393

<212> DNA

<213> Artificial sequence

<400> 37

atgggctggt cctgtatcat cctgttcctg gtggctacag ccacaggagt gcatagtgat 60

gttgtgatga cccaaactcc actctccctg cctgtcagtc ttggagatca agcctccatc 120

tcttgcagat ctagtcagaa ccttgtacac agtaatggaa acacctattt acattggtac 180

ctgcagaagc caggccagtc tccaaagctc ctgatctaca aagtttccaa ccgattttct 240

ggggtcccag acaggttcag tggcagtgga tcagggacag atttcacact caagatcagc 300

agagtggagg ctgaggatct gggagtttat ttctgctctc aaagtacaca tgttccgctc 360

acgttcggtg ctggcaccaa gctggaaatg aaa 393

<210> 38

<211> 378

<212> DNA

<213> Artificial sequence

<400> 38

atgggctggt cctgtatcat cctgttcctg gtggctacag ccacaggagt gcatagtgat 60

atccagatga ctcagactac atcctccctg tctgcctctc tgggagacag agtcaccatc 120

agttgcaggg caagtcagga cattagcaat tatttaaact ggtatcagca gaaaccagat 180

ggaactgtta aactcctgat ctactacaca tcaagattac actcaggagt cccatcaagg 240

ttcagtggca gtgggtctgg aacagattat tctctcacca ttagcaacct ggagcaagaa 300

gatattgcca cttacttttg ccaacagggt aatacgcttc cgctcacgtt cggtgctggg 360

accaagctgg agctgaaa 378

<210> 39

<211> 390

<212> DNA

<213> Artificial sequence

<400> 39

atgggctggt cctgtatcat cctgttcctg gtggctacag ccacaggagt gcatagtgat 60

attgtgctaa cccaatctcc agcttctttg gctgtgtctc tagggcagag ggccaccatc 120

tcctgcaagg ccagccaaag tgttgattat gatggtgata gttatatgaa ctggtaccaa 180

cagaaaccag gacagccacc caaactcctc atctatgctg catccaatct agaatctggg 240

atcccagcca ggtttagtgg cagtgggtct gggacagact tcaccctcaa catccatcct 300

gtggaggagg aggatgctgc aacctattac tgtcagcaaa gtaatgagga tccgtacacg 360

ttcggagggg ggacaaagct ggaaataaaa 390

<210> 40

<211> 324

<212> DNA

<213> Artificial sequence

<400> 40

cgggctgatg ctgcaccaac tgtatccatc ttcccaccat ccagtgagca gttaacatct 60

ggaggtgcct cagtcgtgtg cttcttgaac aacttctacc ccaaagacat caatgtcaag 120

tggaagattg atggcagtga acgacaaaat ggcgtcctga acagttggac tgatcaggac 180

agcaaagaca gcacctacag catgagcagc accctcacgt tgaccaagga cgagtatgaa 240

cgacataaca gctatacctg tgaggccact cacaagacat caacttcacc cattgtcaag 300

agcttcaaca ggaatgagtg ttaa 324

Claims

1. An anti-human Tim3 antibody or functional fragment thereof, wherein said antibody or functional fragment thereof comprises the following CDR regions:

2. The antibody or functional fragment thereof according to claim 1, wherein the amino acid sequences of the heavy chain variable region sequence and the light chain variable region sequence of the antibody or functional fragment thereof are shown as SEQ ID No.9 and SEQ ID No.26, respectively, or as SEQ ID No.10 and SEQ ID No.27, respectively, or as SEQ ID No.11 and SEQ ID No.28, respectively, or as SEQ ID No.12 and SEQ ID No.29, respectively.

3. The antibody or functional fragment thereof according to claim 1, wherein the antibody or functional fragment thereof further comprises an antibody constant region Fc; preferably, the constant region Fc comprises a heavy chain constant region and a light chain constant region;

4. The antibody or functional fragment thereof according to claim 3, wherein the functional fragment is selected from the group consisting of Fab, Fab ', F (ab')₂Fv, scFv.

5. A nucleic acid molecule encoding the antibody or functional fragment thereof according to any one of claims 1 to 4.

6. A vector comprising the nucleic acid molecule of claim 5.

7. A host cell comprising the nucleic acid molecule of claim 5 or the vector of claim 6.

8. A paired antibody for use in the quantitative detection of Tim3, wherein the first antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID No.9 and a light chain variable region having the amino acid sequence of SEQ ID No. 26; the second antibody comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO.11 and a light chain variable region having an amino acid sequence of SEQ ID NO.28, or the second antibody comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO.12 and a light chain variable region having an amino acid sequence of SEQ ID NO. 29.

9. An antibody conjugate comprising the antibody or functional fragment thereof of any one of claims 1-4.

10. A kit comprising one or more of the antibody or functional fragment thereof of any one of claims 1 to 4, the nucleic acid molecule of claim 5, the vector of claim 6 and the antibody conjugate of claim 9.