A TIM-3 affinity peptide and its application

Abstract

The present invention belongs to the field of biopharmaceutical technology, and specifically discloses a TIM-3 affinity peptide and its application. The present invention obtains TIM-3 protein affinity peptide TBS-22 (as shown in SEQ ID NO.1) by screening through phage display seven peptide library high-throughput screening technology, and obtains mutant peptide TBSM-3 (isoleucine at position 6 is mutated to histidine, as shown in SEQ ID NO.2) through later optimization and transformation. The present invention shows through in vitro cell level experiments and mouse tumor-bearing experiments that TIM-3 affinity peptide can affinity TIM-3 protein, block the interaction between TIM-3 and its ligand Galectin-9, thereby exerting anti-tumor efficacy, can be used to prepare anti-tumor drugs, has good medical application prospects, and provides a new option for tumor immunotherapy.

Description

Technical Field

[0001] The present invention belongs to the field of biopharmaceutical technology, and specifically relates to a TIM-3 affinity peptide and its application in preventing and treating tumors and other related diseases. Background Art

[0002] Currently, cancer immunotherapy has become a major cancer treatment modality alongside surgery, radiotherapy, and chemotherapy. Cancer immunotherapy works by reactivating and rejuvenating the immune system, restoring the body's anti-tumor immunity, and ultimately controlling and eliminating tumors. Immune checkpoints are inhibitory pathways crucial for controlling the duration and magnitude of immune responses, and tumors can exploit these pathways to resist immune responses. Targeting immune checkpoints in cancer therapy has made significant progress in the past decade, and immune checkpoint blockade has proven to be a promising approach for cancer treatment in clinical trials. In the body, the immune system uses immune checkpoint molecules to recognize tumor cell surface antigens, distinguishing tumor cells from normal cells, and regulates these responses through co-stimulatory and inhibitory signals. Under normal physiological conditions, immune checkpoints are crucial for maintaining self-tolerance and protecting tissues from damage when the immune system responds to pathogen infection. However, within the tumor microenvironment, tumor cells exploit negative immune checkpoint molecules expressed on immune cells to prevent the immune system from recognizing and eliminating tumor cells, a key factor in tumor development and progression.

[0003] Immune checkpoint inhibitors are a class of anti-tumor drugs developed for corresponding immune checkpoints. Their main function is to block negative immune checkpoint signaling pathways, activate immune cell function, and restore the body's anti-tumor immunity, such as programmed cell death protein 1 (PD-1), programmed cell death ligand 1 (PD-L1), and cytotoxic T lymphocyte antigen 4 (CTLA-4). By blocking immunosuppressive signals and enabling patients to produce effective signals, they can achieve good anti-tumor responses in various cancers.

[0004] TIM-3 (HAVCR2) belongs to the TIM gene family and is a type of T cell surface inhibitory molecule that exists in different types of immune cells. Like PD-1, it is a CD + 4 and CD +TIM-3 is a marker of T cell exhaustion and is expressed on a variety of immune cells, including T cells, regulatory T cells (Tregs), dendritic cells (DCs), B cells, macrophages, natural killer (NK) cells, and mast cells. TIM-3 ligands include phosphatidylserine (Ptdser), Galectin-9, HMGB1, and CEACAM-1, which bind to the extracellular IgV domain of TIM-3, respectively. TIM-3 activity in the TME is important for anti-tumor immunity. In various cancers, the interaction between TIM-3 and Galectin-9 inhibits anti-tumor immunity mediated by innate and adaptive immune cells. Therefore, blocking the TIM3 / Gal-9 interaction is a promising cancer treatment method.

[0005] Currently, antibodies and small molecule drugs targeting TIM-3 have achieved good results in preclinical models. For example, Chinese patent CN109983032B (Hengrui Medicine) discloses an anti-human TIM-3 monoclonal antibody or antigen-binding fragment thereof that can specifically recognize human TIM-3 and bind to the amino acid sequence or three-dimensional structure of the extracellular region, thereby preparing reagents for immunodetection or measurement of TIM-3, or for diagnosing diseases associated with TIM-3-positive cells. Chinese patent application CN113896791A (Novartis) discloses an antibody that binds to TIM-3 with high affinity and specificity and can be used to treat, prevent, or diagnose immune disorders, cancer, infectious diseases, Crohn's disease, sepsis, SIRS, glomerulonephritis, etc. However, peptide drugs also have similar specificity and affinity as antibodies, while peptides have smaller molecular weight, higher stability, lower immunogenicity, better tissue penetration, and lower toxicity and side effects. In addition, artificially synthesized and modified peptides have the advantages of low cost and high production efficiency. Therefore, the development of safer and more effective peptide blockers has good development value and application prospects. Summary of the Invention

[0006] The present invention primarily addresses the technical problem of providing a TIM-3 affinity peptide that can bind to the TIM-3 protein, block the interaction between TIM-3 and its ligand, Galectin-9, and exhibit anti-tumor activity. The present invention also provides a use of the TIM-3 affinity peptide in preventing and treating tumors and other related diseases.

[0007] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0008] A TIM-3 affinity peptide, the amino acid sequence of the TIM-3 affinity peptide is shown as SEQ ID NO.1 or SEQ ID NO.2.

[0009] The amino acid sequence shown in SEQ ID NO. 1 is the parent peptide TBS-22 (Leu-Pro-Ser-Ile-Trp-Ile-Thr). The amino acid sequence shown in SEQ ID NO. 2 is the mutant peptide TBSM-3 (Leu-Pro-Ser-Ile-Trp-His-Thr) in which the isoleucine at position 6 of the parent peptide is mutated to histidine.

[0010] As a preferred embodiment of the present invention, the configuration of each amino acid in the TIM-3 affinity peptide is independently selected from D-type or L-type. For example, all amino acids in the TIM-3 affinity peptide are D-type or L-type. When a certain amino acid is specified as D-type without specifying the configuration of other amino acids, it is assumed that the other amino acids are L-type. For example, the amino acid sequence of the mutant peptide TBSM-3 is Leu D -Pro D -Ser D -Ile-Trp-His-Thr D , when lowercase single letters are used to represent the D configuration of the corresponding amino acids, the above sequence can be abbreviated as lpsIWHt.

[0011] Specifically, the TIM-3 affinity peptide can be prepared by solid phase synthesis, for example, using Fmoc solid phase synthesis.

[0012] A use of a TIM-3 affinity peptide in the preparation of a drug or a detection reagent.

[0013] A drug or detection reagent comprising a TIM-3 affinity peptide, wherein the amino acid sequence of the TIM-3 affinity peptide in the drug or detection reagent is shown as SEQ ID NO.1 or SEQ ID NO.2.

[0014] As a preferred embodiment of the present invention, the TIM-3 affinity peptide in the drug or detection reagent exists in a free form or in the form of a pharmaceutically acceptable salt thereof.

[0015] As a preferred embodiment of the present invention, the drug has one or more of the following uses:

[0016] (1) Anti-tumor;

[0017] (2) blocking the binding of TIM-3 protein to its ligand Galectin-9;

[0018] (3) Affinity (human / mouse) TIM-3 protein.

[0019] Specifically, the tumor includes colorectal cancer, etc. The anti-tumor effect is mainly to inhibit tumor growth or eliminate tumor.

[0020] As a preferred embodiment of the present invention, the drug may contain, in addition to the TIM-3 affinity peptide, other pharmacological ingredients to achieve combined therapy through combined use.

[0021] Specifically, the contents of the TIM-3 affinity peptide and / or other pharmacological ingredients in the drug are all clinically effective doses.

[0022] As a preferred embodiment of the present invention, the drug may further comprise a pharmaceutically acceptable adjuvant or excipient, the type and amount of which may be selected and adjusted according to the different dosage forms of the drug.

[0023] As a preferred embodiment of the present invention, the detection reagent can be used to detect the affinity and / or blocking ability of the test substance for the TIM-3 protein (for example, blocking the binding between TIM-3 and Galectin-9), or for qualitatively, quantitatively or positionally detecting whether the TIM-3 protein is expressed in a biological sample, the expression amount or the expression location.

[0024] Specifically, the TIM-3 protein can be a human TIM-3 protein or a mouse TIM-3 protein, or a TIM-3 protein from other sources.

[0025] Beneficial effects of the invention:

[0026] The present invention screened the TIM-3 protein affinity peptide TBS-22 (as shown in SEQ ID NO.1) through phage display heptapeptide library high-throughput screening technology, and obtained the mutant peptide TBSM-3 (isoleucine at position 6 was mutated to histidine, as shown in SEQ ID NO.2) after subsequent optimization and transformation. In vitro cell level experiments and mouse tumor-bearing experiments showed that the TIM-3 affinity peptide can affinity with TIM-3 protein, blocking the interaction between TIM-3 and its ligand Galectin-9, thereby exerting anti-tumor efficacy. Given the significant tumor inhibition effect of the TIM-3 affinity peptide and the lack of obvious toxic side effects, it can be used to prepare anti-tumor drugs (including anti-tumor immunotherapy drugs or anti-tumor-related drugs) or detection reagents targeting TIM-3, has good medical application prospects, and provides a new option for tumor immunotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The results of the experiment in which the parent peptide TBS-22 blocked the interaction between human TIM-3 and Galectin-9 proteins;

[0028] Figure 2 The experimental results show that the mutant peptide TBSM-3 blocks the interaction between human TIM-3 and Galectin-9 proteins.

[0029] Figure 3 Modified peptide TBSM-3 aaa-a Experimental results on blocking the interaction between human TIM-3 and Galectin-9 proteins;

[0030] Figure 4 Modified peptide TBSM-3 aaa-a Results of affinity experiments on hTIM-3 protein;

[0031] Figure 5 Modified peptide TBSM-3 aaa-a Results of affinity experiments on mTIM-3 protein;

[0032] Figure 6 Modified peptide TBSM-3 aaa-a Effects on tumor volume in C57BL / 6 mice with MC38 transplanted tumor model;

[0033] In the figure, the significance analysis symbol * indicates P < 0.05, and ** indicates P < 0.01;

[0034] Figure 7 Modified peptide TBSM-3 aaa-a Effect on body weight changes in C57BL / 6 mice with MC38 transplanted tumor model.

[0035] Figure 8 Modified peptide TBSM-3 aaa-a Effects of 2 mg / kg combined with OPBP-1 peptide (0.5 mg / kg) on ​​the tumor volume of C57BL / 6 mice with B16 xenograft tumor model;

[0036] In the figures, the significance analysis symbol * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001. DETAILED DESCRIPTION

[0037] The technical solutions of the present invention will be clearly and completely described below in conjunction with specific embodiments. However, it should be understood by those skilled in the art that the embodiments are only used to illustrate the technical solutions of the present invention and should not be regarded as limiting the scope of protection of the present invention. Based on the following embodiments, all other embodiments obtained by those of ordinary skill in the art without making creative work, such as modifications or simple replacements, fall within the scope of protection of the present invention.

[0038] Unless otherwise specified, the experimental methods used in the following examples or experimental examples are conventional methods, and the raw materials, reagents, instruments, etc. used are all commercially available unless otherwise specified.

[0039] Main reagents and kits:

[0040] Bovine serum albumin (BSA), Solebao Biotechnology Co., Ltd.

[0041] Fetal bovine serum (FBS) was from BI Company, Israel.

[0042] Monolith NT TM His-Tag Labeling Kit RED-Tris-NTA protein labeling kit, Notamp Technology (Beijing) Co., Ltd.

[0043] Media and solutions:

[0044] LB medium, top agar, LB / IPTG / X-gal plates, RMPI 1640 medium (containing 10% FBS, 100 U / mL penicillin and 100 μg / mL streptomycin), Protein A / GMix Magnetic Beads, TBS buffer (50 mM Tris-HCl (pH 7.5), containing 150 mM NaCl), Tris-HCl (pH 9.1) neutralizing solution, PEG-8000 / NaCl precipitation solution, Tris-T buffer, elution solution (0.2 M Glycine-HCl (pH 2.2), 1 mg / mL BSA), neutralizing solution (1 M Tris-HCl (pH 9.1)), PBS buffer (pH 7.2), PBST, BSA, Tween-20, TBST washing buffer, etc. were all prepared according to conventional techniques and are not described here.

[0045] Main instruments:

[0046] MST instruments, Nano Temper Technology GmbH, Germany.

[0047] Flow cytometer, BD Biosciences, USA.

[0048] Biomaterials:

[0049] MC38 cell line was purchased commercially and maintained in our laboratory.

[0050] The CHO-K1-hTIM-3 cell line (cells overexpressing hTIM-3) was constructed using conventional techniques and maintained in our laboratory.

[0051] C57BL / 6 mice (6-8 weeks old, female) were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. and housed in an SPF animal room.

[0052] Experimental example

[0053] 1. Screening and synthesis of TIM-3 affinity peptides

[0054] A phage-displayed seven-peptide library was used to screen affinity peptides for human TIM-3. The steps are briefly as follows:

[0055] (1) Screening of phage-displayed heptapeptide libraries using a cell-based screening method;

[0056] (2) After several rounds of screening, phage monoclones with affinity for the extracellular segment of the target protein human TIM-3 were enriched in each round;

[0057] (3) Positive clones were selected for sequencing, and multiple inserted heptapeptide sequences were obtained, namely human TIM-3 affinity heptapeptide sequences. Among them, one with repeated clones was affinity peptide TBS-22, with the sequence of Leu-Pro-Ser-Ile-Trp-Ile-Thr, and the amino acid configuration was all L-type.

[0058] The affinity peptide TBS-22 obtained based on the above screening is subjected to standard Fmoc solid phase synthesis. After purification by high performance liquid chromatography and identification by mass spectrometry, a blocking experiment can be performed to detect the blocking ability of the peptide.

[0059] Optimization and modification of human TIM-3 affinity peptide: The TBS-22 peptide was subjected to a single-point mutation to obtain the mutant peptide TBSM-3 (isoleucine at position 6 was mutated to histidine). The affinity and blocking abilities of the mutant peptide can then be tested through affinity and blocking experiments.

[0060] Modification of mutant peptide TBSM-3: The TBSM-3 peptide was modified by replacing amino acids with D configuration to obtain modified peptide TBSM-3 aaa-a The single-letter abbreviation sequence is lpsIWHt, where lowercase letters represent amino acids in the D configuration. Affinity and blocking experiments can then be performed to investigate the blocking ability of the modified peptide and test its stability, further confirming its anti-tumor effect.

[0061] 2. In vitro blocking experiment

[0062] (1) CHO-K1 and CHO-K1-hTIM-3 cells were cultured in RMPI 1640 medium (containing 10% FBS, 100 U / mL penicillin and 100 μg / mL streptomycin). Cells in the logarithmic growth phase were collected and counted, and then aliquoted into 3×10 5 Pre-chilled PBS (pH 7.2) buffer was added to the cells / tube, centrifuged at 3500 rpm for 5 min at 4°C, washed and placed on ice.

[0063] (2) The peptides (parent peptide TBS-22, mutant peptide TBSM-3 and modified peptide TBSM-3) were synthesized. aaa-a) were dissolved in PBS buffer (pH 7.2) and serially diluted to obtain six samples with a concentration gradient (200 μM, 100 μM, 50 μM, 25 μM, 12.5 μM, and 6.25 μM). 50 μL of each sample was transferred to a microcentrifuge tube, and an equal volume of PBS buffer (pH 7.2) was added to the control tube. Then, 10 μL of 150 ng of recombinant hGalectin-9-Fc protein was added to each sample tube and the control tube, respectively. The samples were thoroughly mixed and incubated in an ice-water bath for 30 min.

[0064] (3) Add an equal amount of the incubated mixture to the cells, resuspend the cells, add the flow cytometry detection antibody anti-human Fc PE, mix well, and incubate in an ice water bath for 30 minutes.

[0065] (4) Add pre-cooled FACS Buffer (1 mL / tube) to the incubated mixture and centrifuge for washing. Then add 200 μL FACS Buffer to resuspend the cells, filter, and use flow cytometry to detect the mean fluorescence intensity of the cells. The results are as follows: Figure 1-3 shown.

[0066] from Figure 1-3 It can be seen that the parent peptide TBS-22, the mutant peptide TBSM-3 and the modified peptide TBSM-3 aaa-a Both can block the binding between hTIM-3 and hGalectin-9, with the IC50 of the mutant peptide and modified peptide being 25.35±0.14μM and 48.07±2.07μM, respectively.

[0067] 3. Affinity experiment

[0068] Detection of modified peptide TBSM-3 by microthermophoresis (MST) aaa-a The affinity for hTIM-3 (human) and mTIM-3 (mouse) is determined as follows:

[0069] (1) Labeling protein: Monolith NT TMDilute the 5×PBST in the His-Tag Labeling Kit RED-Tris-NTA protein labeling kit to 1×PBST, dissolve the dye in 50μL of 1×PBST to obtain a dye concentration of 5μM, then mix 2μL of dye with 98μL of 1×PBST to make a 100nM dye solution; adjust the protein concentration to 200nM (volume of 100μL), then mix the dye and protein in a volume ratio of 1:1 (i.e., 100μL of 200nM protein, 100μL of 100nM dye), incubate at room temperature in the dark for 30min, and then centrifuge the labeled protein at 4°C, 15000g for 10min, retain the supernatant, discard the precipitate, and protein labeling is complete.

[0070] (2) Sample preparation: Dissolve the polypeptide to an appropriate concentration and perform 15 serial dilutions to obtain 16 samples with a concentration gradient of 5 μL. Then, add 5 μL of labeled protein sample to each tube, mix well, and centrifuge to remove bubbles. After incubation on ice for 5 min, use an MST-specific capillary to absorb the incubated liquid and place it on the instrument holder.

[0071] (3) On-computer testing: Turn on the computer and start the MO.Control software, and select the Red channel Binding Affinity mode for testing.

[0072] (4) Analysis results: The binding dissociation constant (K) was calculated using Nano Temper analysis software MO.Affinity Analysis v2.2.4. D value), the result is as follows Figure 4-5 shown.

[0073] from Figure 4-5 It can be seen that the modified peptide TBSM-3 aaa-a Ability to bind to hTIM-3 and mTIM-3, K D The values ​​were 1.68±7.85μM and 7.51±4.96μM, respectively.

[0074] 4. Enzyme degradation stability test

[0075] (1) Weigh the mutant peptide TBSM-3 and modified peptide TBSM-3 aaa-a The dry powder was dissolved to 200 μM with physiological saline, and human serum was added to prepare a mixed solution containing 10% (V / V) serum. After rapid mixing, the solution was placed in a metal bath and incubated at 37°C for 48 hours. Some samples were taken out at 0 hours, 0.5 hours, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 24 hours, 36 hours, 48 ​​hours, and 72 hours for subsequent testing.

[0076] (2) Add the samples taken at different time points to a mixture of acetonitrile and glacial acetic acid with a final concentration of 90% (v / v), quickly shake and mix to terminate proteolysis, centrifuge at 12,000 g for 15 min at 4°C, collect the supernatant, and place in an ice-water bath.

[0077] (3) RP-HPLC analysis of peptide-serum mixture samples and statistical analysis of experimental results.

[0078] The results of enzyme degradation stability experiments showed that the modified peptide TBSM-3 aaa-a Compared with the mutant peptide, the serum stability of TBSM-3 was significantly improved, and the concentration remained almost the same as the initial concentration at 72 h.

[0079] 5. Anti-tumor animal experiments

[0080] To detect the modified peptide TBSM-3 aaa-a The following experiments were specially designed to investigate the anti-tumor activity of

[0081] (1) Inoculate 1×10 6 MC38 tumor cells or 2×10 5 B16 tumor cells, and wait until the tumor volume of the mouse reaches 40-80mm 3 The MC38 tumor model was divided into S-type groups according to tumor size. 2 mg / kg TBSM-3 was injected daily. aaa-a peptide (low dose group), 6mg / kg TBSM-3 aaa-a Peptide (high dose group) or saline was used as negative control. For B16 tumor model, 2 mg / kg TBSM-3 was injected daily. aaa-a peptide, 0.5mg / kg OPBP-1 peptide, 2mg / kg TBSM-3 aaa-a Peptide + 0.5 mg / kg OPBP-1 peptide or saline was used as a negative control and administered daily for 14 consecutive days.

[0082] (2) Measure the length (a), width (b), diameter, and height (c) of the tumor every other day, and calculate the tumor volume according to the formula (calculation formula: V = 1 / 2 × a × b × c), and draw a tumor growth curve. The results are as follows Figure 6 As shown; The weight of mice was measured and recorded every other day, and a curve was drawn to test the modified peptide TBSM-3 aaa-a The side effects of Figure 7 shown.

[0083] from Figure 6 It can be seen that in the MC38 tumor model, compared with the saline group, the modified peptide TBSM-3 aaa-aThe tumor volume of mice in the high-dose and low-dose groups was significantly reduced, and the tumor volume of mice in the high-dose group was the smallest.

[0084] from Figure 7 It can be seen that in the MC38 tumor model, the modified peptide TBSM-3 aaa-a The weight change trend of mice in the high and low dose groups was almost the same as that in the normal saline group, indicating that the injection of modified peptide TBSM-3 aaa-a It has no toxic side effects on the growth of mice.

[0085] from Figure 8 It can be seen that in the B16 tumor model, compared with the saline group, the modified peptide TBSM-3 aaa-a The tumor volume was the smallest when the drug (2 mg / kg) was combined with OPBP-1 peptide (0.5 mg / kg).

[0086] Example 1

[0087] This example provides a TIM-3 affinity peptide TBS-22, whose amino acid sequence is shown in SEQ ID NO.1.

[0088] This embodiment also provides a use of a TIM-3 affinity peptide TBS-22 in the preparation of anti-tumor drugs or detection reagents.

[0089] This embodiment also provides an anti-tumor drug comprising a clinically effective dose of the TIM-3 affinity peptide TBS-22.

[0090] Example 2

[0091] This example provides a TIM-3 affinity peptide TBSM-3, which is a mutant peptide in which the isoleucine at position 6 of the parent peptide TBS-22 in Example 1 is mutated to histidine, and its amino acid sequence is shown in SEQ ID NO.2.

[0092] This embodiment also provides a use of a TIM-3 affinity peptide TBSM-3 in the preparation of anti-tumor drugs or detection reagents.

[0093] This embodiment also provides an anti-tumor drug comprising a clinically effective dose of the TIM-3 affinity peptide TBSM-3.

[0094] Example 3

[0095] This example provides a TIM-3 affinity peptide TBSM-3 aaa-a , is a modified peptide of the mutant peptide TBSM-3 in Example 2, and its single-letter abbreviation sequence is lpsIWHt, where lowercase single letters represent amino acids in the D configuration.

[0096] This example also provides a TIM-3 affinity peptide TBSM-3aaa-a Application in the preparation of anti-tumor drugs or detection reagents.

[0097] This embodiment also provides an anti-tumor drug comprising a clinically effective dose of a TIM-3 affinity peptide TBSM-3 aaa -a .

[0098] Example 4

[0099] This embodiment provides a detection reagent comprising a detection effective amount of the TIM-3 affinity peptide TBSM-3 in Example 3. aaa-a The reagent is mainly used to detect the affinity and / or blocking ability of the test substance to the TIM-3 protein, or to qualitatively, quantitatively or positionally detect whether the TIM-3 protein is expressed, the expression amount or the expression location in the biological sample.

[0100] The TIM-3 affinity peptides TBS-22, TBSM-3, and TBSM-3 in the above examples aaa-a All were prepared by standard Fmoc solid-phase synthesis method.

[0101] The TIM-3 affinity peptide provided by the present invention can bind to the TIM-3 protein and block the interaction between TIM-3 and its ligand Galectin-9, thereby exerting anti-tumor efficacy.

[0102] The present invention has shown through in vitro cell level experiments and mouse tumor-bearing experiments that the affinity peptide has obvious tumor inhibition effect and no obvious toxic side effects, and has good medical application prospects.

[0103] The affinity peptide of the present invention can be used to prepare anti-tumor drugs (including anti-tumor immunotherapy drugs or anti-tumor-related drugs) or detection reagents targeting TIM-3, providing a new option for tumor immunotherapy.

[0104] Although the technical solution of the present invention has been described in detail above using general descriptions, specific implementation methods, and experimental examples, it should be noted that the embodiments and experimental examples are only intended to illustrate the technical solution and technical effects of the present invention and should not be construed as limiting the scope of protection of the present invention. Simple variations, modifications, or improvements based on the technical concept of the present invention are all within the scope of protection claimed by the present invention.

Claims

1. A TIM-3 affinity peptide, characterized in that: The amino acid sequence of the TIM-3 affinity peptide is as shown in SEQ ID NO.1 or SEQ ID NO.2, and the amino acid configuration is L-type; or the amino acid sequence of the TIM-3 affinity peptide is Leu D -Pro D -Ser D -Ile-Trp-His-Thr D .

2. Use of a TIM-3 affinity peptide in the preparation of an anti-tumor drug, characterized in that: The tumor is colorectal cancer or melanoma, and the amino acid sequence of the TIM-3 affinity peptide is Leu D -Pro D -Ser D -Ile-Trp-His-Thr D .

3. Use of the TIM-3 affinity peptide according to claim 1 in the preparation of a detection reagent for blocking the binding of TIM-3 protein to its ligand Galectin-9.

4. A drug comprising a TIM-3 affinity peptide, characterized in that: The drug is an anti-tumor drug, the tumor is colorectal cancer or melanoma, and the amino acid sequence of the TIM-3 affinity peptide is Leu D -Pro D -Ser D -Ile-Trp-His-Thr D .

5. The drug according to claim 4, characterized in that: The TIM-3 affinity peptide in the drug exists in a free form or in the form of a pharmaceutically acceptable salt thereof.

6. The drug according to claim 4, characterized in that: The content of TIM-3 affinity peptide in the drug is the clinical efficacy dose; And / or, the drug contains, in addition to the TIM-3 affinity peptide, other pharmacological ingredients, and the content of the other pharmacological ingredients is a clinical pharmacological dose; And / or, the medicine further comprises pharmaceutically acceptable excipients or excipients.

7. A detection reagent comprising the TIM-3 affinity peptide according to claim 1.

8. The detection reagent according to claim 7, characterized in that: The detection reagent is used to detect the affinity and / or blocking ability of the analyte for the TIM-3 protein, or to qualitatively, quantitatively or positionally detect whether the TIM-3 protein is expressed, the expression amount or the expression location in a biological sample.

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