CN116253774A - TIM-3 affinity peptide and application thereof - Google Patents

Abstract

The invention belongs to the technical field of biological pharmacy, and particularly discloses a TIM-3 affinity peptide and application thereof. The TIM-3 protein affinity peptide TBS-22 (shown as SEQ ID NO. 1) is obtained by screening by a phage display heptapeptide library high-throughput screening technology, and mutant peptide TBSM-3 (the 6 th isoleucine is mutated into histidine and shown as SEQ ID NO. 2) is obtained by post optimization transformation. According to the invention, through in vitro cell level experiments and mouse tumor-bearing experiments, the TIM-3 affinity peptide can be used for affinity of TIM-3 protein and blocking interaction between TIM-3 and ligand Galectin-9 thereof, so that the anti-tumor effect is exerted, the anti-tumor peptide can be used for preparing anti-tumor medicines, has a good medical application prospect, and provides a new choice for tumor immunotherapy.

Description

TIM-3 affinity peptide and application thereof

Technical Field

The invention belongs to the technical field of biological pharmacy, and particularly relates to a TIM-3 affinity peptide and application thereof in the aspect of preventing and treating related diseases such as tumors.

Background

Currently, tumor immunotherapy has become another large tumor treatment modality in addition to surgery, radiation therapy, chemotherapy. Tumor immunotherapy achieves the purposes of controlling and eliminating tumors by restarting and activating the immune system, recovering the anti-tumor immunity of the organism. Immune checkpoints are inhibitory pathways critical to control the duration and magnitude of immune responses that tumors can use to combat immune effects. Tumor therapy targeting immune checkpoints has progressed tremendously over the last 10 years, and immune checkpoint blockade has proven to be an ideal method for cancer therapy in clinical trials. In the body, the immune system can distinguish tumor cells from normal cells by recognizing tumor cell surface antigens through immune checkpoint molecules, and is regulated by co-stimulatory and inhibitory signals. Under normal physiological conditions, immune checkpoints are critical for maintaining self-tolerance and can protect self-tissues from damage when the immune system reacts to pathogen infection, whereas in tumor microenvironments, tumor cells can utilize negative immune checkpoint molecules expressed on immune cells, which prevent the immune system from recognizing and eliminating tumor cells, which is also an important factor leading to tumorigenesis and development.

Immune checkpoint inhibitors are a class of anti-tumor drugs developed for corresponding immune checkpoints, and have the main functions of blocking the signal path of negative immune checkpoints, activating immune cell functions, recovering the anti-tumor immunity of organisms, such as programmed cell death protein 1 (PD-1), programmed cell death ligand 1 (PD-L1) and cytotoxic T lymphocyte antigen 4 (CTLA-4), and obtaining good anti-tumor response in various cancers by blocking immune inhibition signals and enabling patients to generate effective signals.

TIM-3 (HAVCR 2), which belongs to the family of TIM genes, is a class of T cell surface inhibitory molecules that are present in different types of immune cells, and as with PD-1, is CD ⁺ 4 and CD ⁺ Markers of 8T cell failure are expressed on a variety of immune cells, including T cells, regulatory T cells (Tregs), dendritic Cells (DCs), B cells, macrophages, natural killer cells (NK), and mast cells. The ligand of TIM-3 is phosphatidylserine (Ptdser), galectin-9,HMGB1 and CEACAM-1, which bind to the TIM-3 extracellular IgV domain, respectively. The activity of TIM-3 in TME is important against tumor immunity. In various cancers, the interaction of TIM-3 and Galectin-9 inhibits innate and adaptive immune cell-mediated anti-tumor immunity. Thus, blocking the interaction of TIM3/Gal-9 is a promising approach to cancer treatment.

At present, antibodies and small molecule drugs targeting TIM-3 have achieved good effects in preclinical models. For example, chinese patent CN109983032B (constant Rayleigh) discloses an anti-human TIM-3 monoclonal antibody or antigen-binding fragment thereof, which can specifically recognize human TIM-3 and bind to the amino acid sequence of the extracellular region or its three-dimensional structure, and can be used for preparing reagents for immunodetection or assay of TIM-3, or reagents for diagnosis of diseases associated with TIM-3 positive cells, etc. Chinese patent application CN113896791a (nohua) discloses an antibody that binds TIM-3 with high affinity and specificity, useful for the treatment, prevention or diagnosis of immune disorders, cancer, infectious diseases, crohn's disease, sepsis, SIRS, glomerulonephritis, etc. However, polypeptide drugs also have similar specificity and affinity as antibodies, while polypeptides have smaller molecular weight, higher stability, lower immunogenicity, better tissue penetration, and lower toxicity and side effects. In addition, the synthetic and modified polypeptide has the advantages of low cost, high production efficiency and the like. Therefore, the development of the safer and more effective polypeptide blocker has better development value and application prospect.

Disclosure of Invention

The invention mainly solves the technical problem of providing a TIM-3 affinity peptide which can be used for affinity of TIM-3 protein, blocking the interaction between TIM-3 and ligand Galectin-9 thereof and has anti-tumor activity. Meanwhile, the invention also provides application of the TIM-3 affinity peptide in preventing and treating related diseases such as tumors.

In order to solve the technical problems, the invention provides the following technical scheme:

a TIM-3 affinity peptide has an amino acid sequence shown in SEQ ID NO.1 or SEQ ID NO. 2.

Wherein the amino acid sequence shown in SEQ ID NO.1 is parent peptide TBS-22 (Leu-Pro-Ser-Ile-Trp-Ile-Thr). The amino acid sequence shown in SEQ ID NO.2 is a mutant peptide TBSM-3 (Leu-Pro-Ser-Ile-Trp-His-Thr) with isoleucine at the 6 th position of the parent peptide mutated into histidine.

As a preferred embodiment of the present invention, the configuration of each amino acid in the TIM-3 affinity peptide is independently selected from the D-form or the L-form. For example, all amino acids in the TIM-3 affinity peptide are D-or L-forms. When an amino acid is defined as D-form without specifying the configuration of the other amino acid, the other amino acid is L-form by default. 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 the D configuration of the corresponding amino acid is represented by a lower case single letter, the above sequence may be abbreviated as l-p-s-I-W-H-t.

In particular, the TIM-3 affinity peptide may be prepared by solid phase synthesis. For example, fmoc solid phase synthesis is used.

Use of TIM-3 affinity peptide in the preparation of a medicament or detection reagent.

A drug or test agent comprising a TIM-3 affinity peptide, wherein the amino acid sequence of the TIM-3 affinity peptide in the drug or test agent is shown as SEQ ID No.1 or SEQ ID No. 2.

As a preferred embodiment of the present invention, the TIM-3 affinity peptide in the drug or test agent is present in free form or in the form of a pharmaceutically acceptable salt thereof.

As a preferred embodiment of the invention, the medicament has one or more of the following uses:

(1) An anti-tumor;

(2) Blocking the binding of the TIM-3 protein to its ligand Galectin-9;

(3) Affinity (human/murine) TIM-3 proteins.

In particular, the tumors include colorectal cancer tumors and the like. The anti-tumor is mainly used for inhibiting tumor growth or eliminating tumor.

As a preferred embodiment of the present invention, the medicament may contain other pharmaceutically effective components in addition to the TIM-3 affinity peptide, and the combination therapy is achieved by combination.

Specifically, the content of TIM-3 affinity peptide and/or other medicinal components in the medicament is clinical medicinal dose.

As a preferred embodiment of the present invention, the medicament may further comprise pharmaceutically acceptable auxiliary materials or excipients. The types and the dosage of the auxiliary materials or the excipients can be selected and adjusted according to different dosage forms of the medicines.

As a preferred embodiment of the present invention, the detection reagent may be used to detect the affinity and/or blocking ability of a test substance to the TIM-3 protein (e.g., blocking the binding between TIM-3 and Galectin-9), or to qualitatively, quantitatively or positionally detect the presence or absence of TIM-3 protein expression, the amount of expression or the location of expression in a biological sample.

Specifically, the TIM-3 protein may be human TIM-3 protein or mouse TIM-3 protein, or TIM-3 protein of other sources.

The invention has the beneficial effects that:

the TIM-3 protein affinity peptide TBS-22 (shown as SEQ ID NO. 1) is obtained by screening by a phage display heptapeptide library high-throughput screening technology, and mutant peptide TBSM-3 (the 6 th isoleucine is mutated into histidine and shown as SEQ ID NO. 2) is obtained by post optimization transformation. In vitro cell level experiments and mouse tumor-bearing experiments show that the TIM-3 affinity peptide can be used for affinity of TIM-3 protein and blocking interaction between TIM-3 and ligand Galectin-9 thereof, thereby exerting anti-tumor effect. In view of the remarkable tumor inhibiting effect of the TIM-3 affinity peptide and no obvious toxic or side effect, the TIM-3 affinity peptide can be used for preparing antitumor drugs (including antitumor immunotherapeutic drugs or antitumor related drugs) or detection reagents taking TIM-3 as targets, has better medical application prospect and provides a new choice for tumor immunotherapy.

Drawings

FIG. 1 shows experimental results of the parent peptide TBS-22 blocking human TIM-3/Galectin-9 protein interaction;

FIG. 2 shows the experimental results of blocking human TIM-3/Galectin-9 protein interaction by mutant peptide TBSM-3;

FIG. 3 is a repairDecorative peptide TBSM-3 ^aaa-a Blocking experimental results of human TIM-3/Galectin-9 protein interaction;

FIG. 4 is a modified peptide TBSM-3 ^aaa-a Affinity assay results for hTIM-3 protein;

FIG. 5 is a modified peptide TBSM-3 ^aaa-a Experimental results of affinity for mTIM-3 protein;

FIG. 6 is a modified peptide TBSM-3 ^aaa-a Effect on tumor volume of MC38 engrafted tumor model C57BL/6 mice;

in the figure, significance analysis marks are represented by P < 0.05, and P < 0.01;

FIG. 7 is a modified peptide TBSM-3 ^aaa-a Effect on weight change in MC38 engrafted tumor model C57BL/6 mice.

FIG. 8 is a modified peptide TBSM-3 ^aaa-a (2 mg/kg) effect of the combined OPBP-1 peptide (0.5 mg/kg) on tumor volume of B16-engrafted tumor model C57BL/6 mice;

in the figure, the significance analysis mark represents that P is less than 0.05, P is less than 0.01, and P is less than 0.001.

Detailed Description

The technical scheme of the present invention will be clearly and completely described in the following in connection with specific embodiments. It should be understood by those skilled in the art that the examples are only for illustrating the technical scheme of the present invention and should not be construed as limiting the scope of the present invention. All other examples, such as modified, simple substituted embodiments, which are obtained by a person of ordinary skill in the art without making any inventive effort, are within the scope of the present invention based on the following examples.

The experimental methods used in the following examples or experimental examples are conventional methods unless otherwise specified, and the raw materials, reagents, instruments and the like used are commercially available unless otherwise specified.

Major reagents and kits:

bovine Serum Albumin (BSA), soribao biotechnology limited.

Fetal Bovine Serum (FBS), israel BI company.

Monolith NT ^TM His-Tag LabelingKit RED-Tris-NTA protein labeling Kit, nortanpu technologies (Beijing) Co., ltd.

Culture medium and solution:

LB medium, top agar, LB/IPTG/X-gal plates, RMPI 1640 medium (containing 10% FBS,100U/mL penicillin and 100. Mu.g/mL streptomycin), protein A/GMix Magnetic Beads, TBS buffer (50 mM Tris-HCl (pH 7.5), containing 150mM NaCl), tris-HCl (pH 9.1) neutralizer, PEG-8000/NaCl pellet, tris-T buffer, eluent (0.2M Glycine-HCl (pH 2.2), 1mg/mL BSA), neutralizer (1M Tris-HCl (pH 9.1)), PBS buffer (pH 7.2), PBST, BSA, tween-20, TBST wash buffer, etc. were prepared according to conventional techniques and will not be described here.

The main instrument is as follows:

MST instrument, nano Temper technology Co., ltd.

Flow cytometry, BD company, usa.

Biological material:

MC38 cell lines, commercially available, were maintained by the present laboratory.

CHO-K1-hTIM-3 cell line (hTIM-3 overexpressing cell) was constructed and maintained in the laboratory according to conventional techniques.

C57BL/6 mice (6-8 weeks old, female), beijing Vitolihua laboratory animal technology Co., ltd., purchased and then fed to SPF-class animal houses.

Experimental example

1. TIM-3 affinity peptide screening and synthesis

The phage display heptapeptide library was used to screen for the affinity peptide for human TIM-3, briefly as follows:

(1) Screening phage display heptapeptide library by adopting a cell screening method;

(2) After several rounds of screening, the phage monoclonal with affinity with the extracellular segment of the target protein human TIM-3 is enriched round by round;

(3) And selecting positive clones for sequencing to obtain a plurality of inserted heptapeptide sequences, namely human TIM-3 affinity heptapeptide sequences, wherein 1 of the inserted heptapeptide sequences has repeated cloning as affinity peptide TBS-22, the sequence is Leu-Pro-Ser-Ile-Trp-Ile-Thr, and the amino acid configuration is L-shaped.

And carrying out standard Fmoc solid-phase synthesis on the affinity peptide TBS-22 obtained by screening, and carrying out blocking experiments to detect the blocking capability of the polypeptide after high performance liquid chromatography purification and mass spectrum identification in sequence.

Optimization of human TIM-3 affinity peptide: the TBS-22 peptide is subjected to single-point mutation to obtain mutant peptide TBSM-3 (isoleucine at position 6 is mutated into histidine), and then affinity and blocking abilities of the mutant peptide can be detected through an affinity experiment and a blocking experiment.

Modification of mutant peptide TBSM-3: modification of TBSM-3 peptide by D-configuration amino acid substitution scheme to obtain modified peptide TBSM-3 ^aaa-a The single letter abbreviation sequence is l-p-s-I-W-H-t, and the lower case single letter represents the amino acid in the D configuration. And then, the blocking capacity of the modified peptide can be researched through an affinity experiment and a blocking experiment, and the stability of the peptide is detected, so that the anti-tumor effect of the modified peptide is further confirmed.

2. In vitro blocking experiments

(1) Culturing CHO-K1 and CHO-K1-hTIM-3 cells in RMPI 1640 medium (containing 10% FBS,100U/mL penicillin and 100 μg/mL streptomycin), collecting logarithmic phase cells, counting, and packaging into 3×10 ⁵ Cells/tubes were centrifuged at 3500rpm at 4℃for 5min in a pre-chilled PBS buffer (pH 7.2), washed and placed on ice.

(2) The polypeptide (parent peptide TBS-22, mutant peptide TBSM-3 and modified peptide TBSM-3) ^aaa-a ) Respectively dissolving in PBS buffer solution (pH 7.2), diluting by multiple ratio to obtain 6 concentration gradient samples (200 mu M,100 mu M,50 mu M,25 mu M,12.5 mu M and 6.25 mu M), respectively taking 50 mu L into a microcentrifuge tube, adding an equal volume of PBS buffer solution (pH 7.2) into a control tube, adding 10 mu L of 150ng recombinant hGALIECin-9-Fc protein into each sample tube and the control tube, fully mixing, and placing in an ice-water bath for incubation for 30min.

(3) And adding the incubated mixture into cells in equal quantity, re-suspending the cells, adding the detection flow antibody anti-human Fc PE, uniformly mixing, and placing in an ice-water bath for incubation for 30min.

(4) After the incubation, the mixture was washed by adding pre-cooled FACS Buffer (1 mL/tube) and then 200. Mu.L of FACS Buffer was added to resuspend the cells, and the cells were filtered and the average fluorescence intensity of the cells was measured by flow cytometry, and the results are shown in FIGS. 1-3.

As can be seen from FIGS. 1-3, the parent peptide TBS-22, the mutant peptide TBSM-3 and the modified peptide TBSM-3 ^aaa-a Can block the combination of hTIM-3/hGALICTIN-9, and the IC50 of the mutant peptide and the modified peptide are 25.35+ -0.14 μm and 48.07+ -2.07 μm respectively.

3. Affinity experiments

Detection of modified peptide TBSM-3 using micro-thermophoresis (MST) ^aaa-a Affinity with hTIM-3 (human source) and mTIM-3 (murine source), the detection process is as follows:

(1) Labeling proteins: monolith NT with pure water ^TM 5 XPBS in His-Tag Labeling Kit RED-Tris-NTA protein labelling kit was diluted to 1 XPBS, 50. Mu.L of 1 XPBS was used to dissolve the dye to give a dye at a concentration of 5. Mu.M, and then 2. Mu.L of dye was mixed with 98. Mu.L of 1 XPBS to give a 100nM dye solution; the protein concentration was adjusted to 200nM (volume 100. Mu.L), after which the dye was mixed with protein in a volume ratio of 1:1 (i.e. 100. Mu.L of 200nM protein, 100. Mu.L of 100nM dye), incubating at room temperature in the absence of light for 30min, centrifuging the labeled protein at 4deg.C and 15000g for 10min, retaining the supernatant, discarding the precipitate, and labeling the protein.

(2) Sample preparation: dissolving the polypeptide to proper concentration, carrying out 15 times of ratio dilution to obtain 16 concentration gradient samples with the volume of 5 mu L, adding 5 mu L of marked protein sample into each tube, mixing uniformly, centrifuging to remove bubbles, incubating on ice for 5min, absorbing the incubated liquid by using a special MST capillary, and placing on an instrument holder.

(3) And (3) detecting: and starting the MO.control software by opening the computer, and selecting a Red channel Binding Affinity mode for detection.

(4) Analysis results: calculation of binding dissociation constant (K) using Nano template Analysis software MO.affinity Analysis v2.2.4 _D Values), the results are shown in fig. 4-5.

As can be seen from FIGS. 4-5, the modified peptide TBSM-3 ^aaa-a Can be compatible with hTIM-3 and mTIM-3, K _D The values are 1.68+ -7.85 μm,7.51±4.96μM。

4. Enzyme degradation stability test

(1) Weighing mutant peptide TBSM-3 and modified peptide TBSM-3 ^aaa-a The dry powder is dissolved to 200 mu M by normal saline, human serum is added to prepare a mixed solution containing 10% (V/V) serum, the mixed solution is placed in a metal bath after being quickly and evenly mixed, incubated for 48 hours at 37 ℃, and partial samples are 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 respectively for subsequent detection.

(2) Samples taken at different time points were added to an acetonitrile-glacial acetic acid mixture having a final concentration of 90% (V/V), mixed by rapid shaking to terminate proteolysis, centrifuged at 12000g at 4℃for 15min, the supernatant was collected, and placed in an ice-water bath.

(3) RP-HPLC analysis of peptide-serum mixture samples, statistical test results.

The results of the enzyme degradation stability experiments show that the modified peptide TBSM-3 ^aaa-a The serum stability was significantly improved compared to the mutant peptide TBSM-3, and remained at almost the same concentration as initially at 72 h.

5. Anti-tumor animal experiments

For detection of the modified peptide TBSM-3 ^aaa-a Specifically designed are the following experiments:

(1) Inoculating 1×10 on the right back of each C57BL/6 mouse ⁶ Individual MC38 tumor cells or 2X 10 ⁵ B16 tumor cells, when the tumor volume of the mice reaches 40-80mm ³ At the same time, the tumor cells are grouped according to the size of tumor volume and S type, and 2mg/kg TBSM-3 is injected into MC38 tumor model every day ^aaa-a Peptide (Low dose group), 6mg/kg TBSM-3 ^aaa-a Peptide (high dose group) or physiological saline served as negative control. For the B16 tumor model, 2mg/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 physiological saline was used as a negative control. Daily administration was continued for 14 days.

(2) Measuring the length (a), width (b) diameter and height (c) of the tumor every other day, calculating the tumor volume according to a formula (calculation formula: v=1/2×a×b×c), and drawing a tumor growth curve, wherein the result is shown in fig. 6; daily mouse body weight measurements and notesRecording and plotting to examine the modified peptide TBSM-3 ^aaa-a The results of the toxic and side effects of (2) are shown in FIG. 7.

As can be seen from FIG. 6, in the MC38 tumor model, the modified peptide TBSM-3 was compared to the saline group ^aaa-a The tumor volume of the mice in the high and low dose groups is significantly reduced, and the tumor volume of the mice in the high dose group is minimal.

As can be seen from FIG. 7, in the MC38 tumor model, the modified peptide TBSM-3 ^aaa-a The body weight change trend of the mice in the high and low dose groups is almost the same as that of the mice in the normal saline group, which indicates that the modified peptide TBSM-3 is injected ^aaa-a Has no toxic or side effect on the growth of mice.

As can be seen from FIG. 8, in the B16 tumor model, the modified peptide TBSM-3 was compared to the saline group ^aaa-a The tumor volume of the (2 mg/kg) combined OPBP-1 peptide (0.5 mg/kg) was minimal.

Example 1

The present example provides a TIM-3 affinity peptide TBS-22, the amino acid sequence of which is shown as SEQ ID NO. 1.

The embodiment also provides application of the TIM-3 affinity peptide TBS-22 in preparing antitumor drugs or detection reagents.

The embodiment also provides an antitumor drug comprising a clinically effective dose of TIM-3 affinity peptide TBS-22.

Example 2

The present example provides a TIM-3 affinity peptide TBSM-3, which is a mutant peptide obtained by mutating isoleucine at 6 th position of parent peptide TBS-22 in example 1 into histidine, and the amino acid sequence of the mutant peptide is shown as SEQ ID NO. 2.

The embodiment also provides an application of the TIM-3 affinity peptide TBSM-3 in preparing antitumor drugs or detection reagents.

The embodiment also provides an antitumor drug comprising a clinically effective dose of TIM-3 affinity peptide TBSM-3.

Example 3

This example provides a TIM-3 affinity peptide TBSM-3 ^aaa-a The modified peptide of the mutant peptide TBSM-3 of example 2, whose single letter abbreviation sequence is l-p-s-I-W-H-t, and whose lower case single letter represents the D configurationIs an amino acid of (a).

This example also provides a TIM-3 affinity peptide TBSM-3 ^aaa-a The application in preparing antitumor drugs or detection reagents.

The embodiment also provides an antitumor drug comprising a clinically effective dose of TIM-3 affinity peptide TBSM-3 ^{aaa -a} 。

Example 4

This example provides a detection reagent comprising a detection effective amount of TIM-3 affinity peptide TBSM-3 of example 3 ^aaa-a The reagent is mainly used for detecting the affinity and/or blocking capacity of an analyte to the TIM-3 protein, or is used for qualitatively, quantitatively or positionally detecting whether the TIM-3 protein is expressed or not, the expression quantity or the expression position in a biological sample.

TIM-3 affinity peptides TBS-22, TBSM-3 in the above examples ^aaa-a All were prepared using standard Fmoc solid phase synthesis.

The TIM-3 affinity peptide provided by the invention can be used for affinity of TIM-3 protein and blocking interaction between TIM-3 and ligand Galectin-9 thereof, thereby exerting anti-tumor effect.

The in vitro cell level experiment and the mouse tumor-bearing experiment show that the affinity peptide has obvious tumor-inhibiting effect, no obvious toxic or side effect and good medical application prospect.

The affinity peptide of the invention can be used for preparing antitumor drugs (including antitumor immunotherapeutic drugs or antitumor related drugs) or detection reagents with TIM-3 as a target spot, and provides a new choice for tumor immunotherapy.

Although the technical solutions of the present invention have been described in detail in the foregoing general description, the specific embodiments and the experimental examples, it should be noted that the examples and the experimental examples are only for illustrating the technical solutions and the technical effects of the present invention, and should not be construed as limiting the scope of the present invention. Simple variations, modifications or improvements made on the basis of the technical idea of the invention fall within the scope of the invention as claimed.

Claims (10)

1. A TIM-3 affinity peptide, characterized by: the amino acid sequence of the TIM-3 affinity peptide is shown as SEQ ID NO.1 or SEQ ID NO. 2.

2. The TIM-3 affinity peptide of claim 1, wherein: the configuration of each amino acid in the TIM-3 affinity peptide is independently selected from D or L;

preferably, all amino acids in the TIM-3 affinity peptide are in D-form or L-form.

3. The TIM-3 affinity peptide according to claim 1 or 2, characterized in that: the amino acid sequence of the TIM-3 affinity peptide is Leu ^D -Pro ^D -Ser ^D -Ile-Trp-His-Thr ^D 。

4. Use of a TIM-3 affinity peptide according to any one of claims 1-3 in the manufacture of a medicament or a detection reagent.

5. A pharmaceutical or detection reagent comprising a TIM-3 affinity peptide according to any one of claims 1-3.

6. The drug or test agent of claim 5 wherein: the TIM-3 affinity peptide in the drug or test agent is present in free form or in the form of a pharmaceutically acceptable salt thereof.

7. The drug or test agent of claim 5 wherein: the medicament has one or more of the following uses:

(1) An anti-tumor;

(2) Blocking the binding of the TIM-3 protein to its ligand Galectin-9;

(3) Affinity TIM-3 protein.

8. The drug or test agent of claim 7 wherein: the tumor includes colorectal cancer tumor.

9. The drug or test agent of claim 5 wherein: the content of TIM-3 affinity peptide in the medicine is clinical efficacy dose;

and/or, the medicine contains other medicinal components besides TIM-3 affinity peptide, and the content of the other medicinal components is clinical medicinal dose;

and/or the medicine also comprises pharmaceutically acceptable auxiliary materials or excipients.

10. The drug or test agent of claim 5 wherein: the detection reagent can be used for detecting the affinity and/or blocking capacity of an analyte on the TIM-3 protein, or can be used for qualitatively, quantitatively or positionally detecting whether the TIM-3 protein is expressed or not, the expression quantity or the expression position in a biological sample.

Images