CN112409450A

CN112409450A - Affinity agent of TIGIT-IgV and application thereof

Info

Publication number: CN112409450A
Application number: CN202010233379.6A
Authority: CN
Inventors: 高艳锋; 周秀曼; 李琬琼; 翟文杰; 吴亚红; 赵文珊; 祁元明
Original assignee: Zhengzhou University
Current assignee: Zhengzhou University
Priority date: 2020-03-29
Filing date: 2020-03-29
Publication date: 2021-02-26
Anticipated expiration: 2040-03-29
Also published as: CN112409450B

Abstract

The invention relates to a affinity agent of TIGIT-IgV and application thereof, wherein the affinity agent is selected from amino acids shown in SEQ ID NOs:1, 3 or 4, or the structure of the affinity agent is shown in formula I or formula II, X1-Tyr-X2-His-X3-Arg-X4-X5(I) X1-Tyr-X2-His-X3-Arg-X4-X5- (L) n-X6 (II). The invention develops a new method, and the affinity agent obtained by repeated screening and optimization can better block the interaction between TIGIT/PVR, thereby treating tumors or other types of diseases.

Description

Affinity agent of TIGIT-IgV and application thereof

The technical field is as follows:

the invention belongs to the technical field of biological pharmacy, and particularly relates to a TIGIT-IgV affinity agent and application thereof in tumor and other related diseases.

Background art:

tumor immunotherapy, especially based on the blockade of immune checkpoint molecules, is the most promising direction of research in the field of tumor therapy. Immune checkpoint molecules refer to a series of functionally non-redundant negative co-stimulatory molecules expressed by T cells or NK cells, such as PD-1, TIM-3, LAG-3, TIGIT, and the like. When the immune system is activated by external stimulation, the expression of immune check point molecules is up-regulated, and the immune check point molecules are combined with ligand molecules on antigen presenting cells to play a 'brake' function to prevent over-activation of T cells. Over-expression or over-strong function of immune checkpoint molecules can mediate immune tolerance to cause diseases such as tumors and the like; conversely, the body may be in a state of sustained immune activation leading to hyperimmunity or autoimmune disease.

Currently, the best studied immune checkpoint molecule is PD-1, blocking its mediated negative signaling pathway of PD-1/PD-L1 can break immune tolerance, activate T cell killing in the tumor microenvironment and clear tumors. The PD-1/PD-L1 antibody has made a breakthrough in the clinical treatment of melanoma, non-small cell lung cancer and Hodgkin's lymphoma. However, the low response rate of the PD-1/PD-L1 antibody and the adaptive resistance due to the up-regulation of the expression of other immune checkpoint molecules, there is an urgent need to find new therapeutic targets and develop corresponding drugs.

TIGIT is an immune checkpoint molecule that is expressed primarily in NK cells, activated T cells and regulatory T cells, and binds to a ligand molecule PVR expressed on tumor cells or DC cells to exert an inhibitory function. TIGIT direct inhibitionCan inhibit the cell toxicity, granzyme polarization and cytokine secretion of NK cells, and can also inhibit the proliferation of T cells and the secretion of cytokines. The TIGIT is blocked to break a TIGIT/PVR mediated negative signal path, the functions of NK cells and T cells exhausted in a tumor-bearing mouse body can be recovered simultaneously, and the tumor growth is obviously inhibited. The antibody combined blocking TIGIT, PD-1 and PD-L1 can obviously inhibit the tumor growth (even regress) of tumor-bearing mice and can also promote CD8 of tumor patients⁺TIL cell proliferation and cytokine secretion. TIGIT has become a new star in immune checkpoint molecules, blocking TIGIT alone or in combination with other immune checkpoint molecules or will become a new trend in immunotherapy. In addition, TIGIT plays an important role in the immune system of the body, playing an important regulatory role in the immune pathogenesis of various autoimmune diseases, in addition to the above-mentioned widespread applications in the oncological field, as well as in bacterial and viral infections, such as chronic infection with Human Immunodeficiency Virus (HIV), infection with human T-lymphoblastic leukemia virus type I and infection with lymphocytic choriomeningitis virus. Therefore, based on the TIGIT/PVR pathway regulation, the problem that scientists are facing and urgently need to solve is to find a reasonable and effective treatment strategy.

TIGIT blockers are still studied at an early stage and are mostly antibody drugs. Some characteristics of the antibody drug such as large molecular weight and difficult infiltration to tumor sites; the Fc segment of the antibody is easy to cause immune-related adverse reactions; the half life is too long, and once side effects appear in the treatment process, the side effects are difficult to treat in time; the antibody has strong immunogenicity, and is easy to generate anti-antibody to cause drug resistance and the like, so that the antibody drug is limited to play a full role. Polypeptide drugs have the advantages of strong permeability, weak immunogenicity, and easy synthesis and modification, and research reports have shown that polypeptides aiming at immune checkpoints can be used for diagnosis and in vivo tracing, and can also be designed into nanoparticles for drug delivery, drug sustained release, and the like.

Most of the existing polypeptide drugs targeting immune checkpoint molecules have natural L configuration, and are easily degraded by enzymes in vivo and difficult to exert the corresponding effect. Because the enzyme for degrading the D-configuration polypeptide does not exist in vivo, the D-configuration polypeptide drug is more stable and has better application prospect.

At present, polypeptide drugs targeting TIGIT, in particular D-configuration polypeptide drugs with stable activity, are in urgent need of development and are applied to TIGIT-related scientific research and disease treatment.

Disclosure of Invention

The invention develops a new method, obtains the affinity agent of TIGIT-IgV by repeated screening, and proves that the affinity agent has affinity TIGIT and activity of blocking TIGIT/PVR protein combination, and can be used for targeting in vivo and treating tumors or other types of diseases.

In a first aspect, the invention provides an affinity agent of TIGIT-IgV or a drug-forming modified form thereof, wherein the affinity agent is selected from amino acids shown in SEQ ID NOs:1, 3 or 4, or the structure of the affinity agent is shown in a formula I or a formula II,

X1-Tyr-X2-His-X3-Arg-X4-X5 (I)

X1-Tyr-X2-His-X3-Arg-X4-X5-(L)n-X6 (II)

formula I or formula II:

x1, X2, X3 or X5 are independently selected from peptide fragments consisting of 0-2 amino acids (for conciseness of the text, the structure of <2 amino acids is also referred to as peptide fragment),

x4 is absent or is leucine,

(L) n represents a flexible linker, n is a positive integer, and X6 is a peptide fragment consisting of 1-12 amino acids;

the affinity agent is an IgV-like structural domain which can be compatible with TIGIT protein and can block TIGIT/PVR protein from combining with each other, and the TIGIT protein and a PVR ligand can be wild type or mutant protein still retaining the activity of the TIGIT protein and the PVR ligand; the drug-forming modification mode means that the drug-forming modification mode is a drug-forming modification in the field, and the affinity agent is subjected to chemical modification to prolong the half-life period on the premise of keeping the activity, such as cyclization modification, acetylation modification, PAS modification, PEG modification, fatty acid modification, albumin affinity peptide coupling, tumor homing peptide coupling, membrane-penetrating peptide coupling and nano-carrier coupling.

Optionally, said X1 is absent or selected from Gly-Gly-, Ala-Gly-, Gly-Ala-or Gly-.

Optionally, the X2 is absent or selected from peptidyl Thr-Phe, Ala-Phe, or Thr-Ala.

Optionally, the X3 is absent or selected from peptidyl Trp-His, Ala-His or Trp-Ala.

Optionally, X5 is absent or selected from peptidyl Asn-Pro, Ala-Pro or Asn-Ala.

Optionally, the L is peptidyl-Ala-Ala-.

Optionally, n-1-6, preferably n-1.

Optionally, said X6 is the peptide fragment-Asn-Tyr-Ser-Lys-Pro-Thr-Asp-Arg-Gln-Tyr-His-Phe.

Optionally, the sequence of the affinity peptide is selected from SEQ ID NOs: 1.2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18(NOs represent a side-by-side listing of sequence numbers in the art, i.e., the amino acid sequences are SEQ ID No.1, SEQ ID No.2 … …, respectively).

Optionally, the configuration of the amino acids of the affinity peptide is independently selected from D-or L-form (glycine, which is not classified into D-or L-form, so as to make the context concise), and both D-or L-form, e.g., the amino acids are in D-form.

It is further noted that the affinity and neutralization agents of this patent, either in free form or in the form of their pharmaceutically acceptable salts, constitute an equivalent infringement of this patent based on a simple modification of the patent's idea.

In a second aspect, the present invention provides a pharmaceutical composition or kit comprising an affinity agent as described in the first aspect above, or a pharmaceutically modified form thereof.

In a third aspect, the present invention provides the use of an affinity agent according to the first aspect as hereinbefore described in the manufacture of a medicament (or a pharmaceutical composition comprising the same) or a diagnostic kit.

The pharmaceutical composition of the second aspect or the third aspect, or the medicament of the third aspect, may be used for at least one of the following uses:

1) against tumors, e.g. colon, melanoma or breast cancer

2) Treating infections caused by bacteria, viruses or fungi

3) Treatment of autoimmune diseases

4) Blocking the combination of TIGIT protein and PVR ligand; the TIGIT and PVR may be wild-type of human (hTIGIT) or mouse origin or a mutant protein that still retains its activity.

The diagnostic kit of the second aspect or the third aspect can be used for detecting the affinity or blocking ability of a test object to the TIGIT protein, or detecting the expression, expression position or expression content of the TIGIT protein in a sample.

The polypeptide fragment can be prepared by solid phase synthesis, such as preparation of I, X6 (L) n by Fmoc scheme, and splicing to obtain II.

The invention has the beneficial effects that:

the invention develops a new method, obtains the affinity agents TIGIT-IgV through repeated screening and optimization, and the affinity blocking activity experiment proves that the affinity agents can block the combination of TIGIT/PVR. Further in vitro affinity experiments and in vivo anti-tumor experiments of mice prove that the high blocking rate peptide can obviously inhibit the growth of CT26 colon cancer and B16-OVA melanoma of the mice, inhibit the growth of 4T1 breast cancer tumor and the lung metastasis of the tumor of the mice, and has no obvious toxic or side effect.

Description of the drawings:

FIG. 1 is a graph of the results of experiments in which the parent peptide of the present invention blocks TIGIT/PVR protein binding;

FIG. 2 is a graph showing the results of an experiment in which the affinity peptide derivative of the present invention blocks TIGIT/PVR protein binding;

FIG. 3 is a graph showing the results of experiments in which the alanine scanning peptide of the present invention blocks TIGIT/PVR protein binding;

FIG. 4 is a graph showing the results of experiments in which a truncated peptide of the present invention blocks TIGIT/PVR protein binding;

FIG. 5 shows the result of the bioinformatic analysis of experiment 7 according to the present invention;

FIG. 6 is a graph showing the effect of exemplary peptides of the present invention on the body weight change of BALB/c mice inoculated with CT 26;

FIG. 7 is a graph of the effect of exemplary peptides of the invention on the change in the volume of transplanted tumors in BALB/c mice inoculated with CT 26;

FIG. 8 is a graph showing the effect of exemplary peptides of the present invention on the weight change of C56BL/6 mice inoculated with B16-OVA;

FIG. 9 is a graph of the effect of exemplary peptides of the invention on the change in the volume of transplanted tumors in C56BL/6 mice inoculated with B16-OVA;

FIG. 10 is a graph showing the effect of exemplary peptides of the present invention on the body weight change of BALB/c mice inoculated with 4T 1;

FIG. 11 is a graph of the effect of exemplary peptides of the invention on the change in the volume of BALB/c mouse transplants inoculated with 4T 1;

FIG. 12 is a graph of the effect of exemplary peptides of the invention on the change in the number of pulmonary metastases in BALB/c mice vaccinated with 4T 1; FIG. 13 is a graph of the results of a tissue infiltration experiment for exemplary peptides of the present invention;

the significance analysis signatures involved in each figure represent P <0.05,. indicates 0.01.

The specific implementation mode is as follows:

embodiments of the present invention will be described in detail below with reference to examples, but the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention.

Unless otherwise specified, the reagents, biological materials, culture media and solutions used below are all commonly used, publicly available or commercially available in the art.

1. Phage image display peptide library liquid phase screening is carried out to obtain parent peptides such as affinity peptide TBP-1 of TIGIT-IgV, and the general screening process is as follows:

a) D-TIGIT-IgV-biotin is synthesized by complete chemistry, Streptavidin (SA) magnetic beads are adopted to capture the D-configuration protein, and a differential liquid phase screening method is adopted to carry out screening work of a phage display dodecapeptide library;

b) after multiple rounds of screening, the phage monoclonals with affinity to the target protein D-TIGIT-IgV are enriched one by one;

c) selecting positive clones from the phage clones, sequencing to obtain a plurality of inserted dodecapeptide sequences, determining a plurality of enriched phage clones according to the sequencing result, wherein the displayed polypeptides can be combined with TIGIT-IgV, and the obtained parent peptides are named as TBP-1, TBP-3, TBP-5 and TBP-17 (the sequences are sequentially shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, and the configurations of all amino acids are synthesized into D type).

2. The affinity of the polypeptide and human TIGIT is detected by Surface Plasmon Resonance (SPR), and the detection process is as follows:

a) preparation of the instrument: SPR experiments were performed using a Biacore T200 instrument (GE Healthcare) at 25 ℃ with vacuum filtered and degassed running buffer (10mM Na2HPO4, 2.7mM KCl, 137mM NaCl, pH 7.4, GE Healthcare) containing 0.05% surfactant P20.

b) Preparation of an SA chip: the biotin-tagged lyophilized protein hIgVTIGIT (TIT-H82E5, ACRO Biosystems) was dissolved at a protein concentration of 5.7. mu.M, and the resulting aqueous protein buffer was diluted in 10mM sodium acetate buffer (pH 4.5, GE Healthcare) and immediately immobilized on Series S sensor SA chips at a fixed level of 1800 according to standard protocols.

c) And (3) sample testing: to confirm affinity, a concentration gradient (2-fold serial dilution) of the selected peptide or hPVR ectodomain was freshly prepared in running buffer. Using running buffer, the analyte was flowed through the sensor chip at a flow rate of 30L/min and a contact and dissociation time of 60 seconds. Untreated channels without hIgVTIGIT protein were used as blanks to avoid non-specific binding. The sensor chip surface was prepared for the next analyte by regeneration with 30mL/min 10mM glycine 3.0 for 20 seconds to completely remove the remaining analyte.

The result shows that TBP-1, TBP-3, TBP-5 and TBP-17 can be well compatible with TIGIT protein, K_dThe value (mu M) is 2.79 plus or minus 0.51, 5.60 plus or minus 2.56, 6.75 plus or minus 1.07 and 48.3 plus or minus 11.2 in sequence.

3. Blocking experiments:

a) culturing CHO-K1-hTIGIT cells in RMPI 1640 medium containing 10% FBS until the cells grow to the logarithmic phase and collecting the cells in 1.5mL EP tubes, each tube having a cell size of 3X 10⁵Washing with 1mL of PBS7.2 twice, and placing on ice for later use;

b) polypeptide and CHO-K1-hTIGIT cell were incubated: a certain amount of polypeptide was weighed, PBS7.2 was dissolved to 1000. mu.M, 50. mu.L of the dissolved polypeptide was resuspended in cells, both tubes of which were resuspended in 50. mu.L of PBS, used as negative and positive controls, and incubated together on vortex-mixed ice for 30 min.

c) 10ng of hPVR-Fc protein was added to the cell mixture, an equal volume of PBS was added to the negative control tube, and the cells were incubated for 30min on vortex-mixed ice.

d) Add 0.3. mu.L of antibody (Human Fc. gamma. specific PE) to all the above cell mixtures, vortex to mix well and incubate on ice for 30min in the dark.

f) Antibody washing: after washing with 1mL of ice-cold PBS once at 7.2, centrifuging at 1800rpm for 5min, adding 200. mu.L of PBS to resuspend the cells, and transferring the cells into a flow tube to detect the binding of CHO-K1-hTIGIT cells and hPVR-Fc protein in a flow mode.

The blocking results show that: the parent peptides (TBP-1, TBP-3, TBP-5 and TBP-17) of the invention can block the binding of hPVR protein and CHO-K1-hTIGIT cells to different degrees, and the blocking rate is shown in figure 1.

4. Blocking rates of affinity peptide derivatives at different concentrations:

the blocking rate of TBP-3 derivatives was determined at different peptide concentrations as described in experiment 3 above and the results are shown in FIG. 2:

we synthesized derivatives, coupling TBP-3(Gly-Gly-Tyr-Thr-Phe-His-Trp-His-Arg-Leu-Asn-Pro) with the peptide Asn-Tyr-Ser-Lys-Pro-Thr-Asp-Arg-Gln-Tyr-His-Phe (intermediate linker-Ala-Ala-) to obtain derivatives of the affinity peptide TBP-3, and tested the blocking efficiency of the affinity peptide TBP-3 on TIGIT and PVR, and the results show that the derivatives of TBP-3 also have the capability of blocking TIGIT/PVR interaction, and the results are shown in FIG. 2.

5. Peptide TBP-3 is subjected to alanine scanning, and blocking experiments are carried out to study the blocking capacity of the peptide in the alanine scanning, and the specific implementation method is basically the same as that in experiment 3.

Replacing any one amino acid of the peptide TBP-3 with alanine to obtain a series of single mutation peptides (the amino acid configuration keeps D type), which are called alanine scanning peptides, for example, the sequence of alanine scanning peptide A9 is Gly-Gly-Tyr-Thr-Phe-His-Trp-His-Ala-Leu-Asn-Pro, the sequence of alanine scanning peptide A9 is shown as Gly-Gly-Tyr-Thr-Phe-His-Trp-His-Ala-Leu-Asn-Pro, the sequence of alanine scanning peptide A1, A2, A4, A5, A7, A8, A11 and A12 is shown as SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12 and SEQ ID NO.13, the blocking rate of TBP-3 to hTIGIIT is taken as 100%, and the blocking rate of alanine scanning peptides A1, A2, A4, A5, A7, A8, A9 and A11 and A12 of TBP 3 are shown as the relative blocking: alanine scanning peptides A1, A2, A4, A5, A7, A8, A11 and A12 of TBP-3 all blocked TIGIT/PVR protein binding well at a concentration of 500. mu.M.

6. The peptide TBP-3 is truncated, and the blocking experiment is used for researching the blocking capability of the truncated peptide, and the specific implementation method is basically the same as that of the experiment 3.

A series of shorter peptides (the amino acid configuration is kept in a D type) obtained by selectively cutting 1 to more amino acids from the N end or the C end of the peptide TBP-3 are called as cut-off peptides, the sequence of the cut-off peptide P4 is Gly-Gly-Tyr-Thr-Phe-His-Trp-His, the sequence of the cut-off peptide P7 is Thr-Phe-His-Trp-His-Arg-Leu-Asn-Pro, the sequences of the cut-off peptides P1, P2, P3, P5 and P6 are sequentially shown as SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17 and SEQ ID NO.18, the relative blocking rate of TBP-3 to hTIGIT is calculated by taking the value of the blocking rate of the TBP-3 to the peptide as 100%, and the result is shown as FIG. 4.

7. Biological information analysis and verification

a) PEPstMOD online prediction of the 3D structure of TBP-3 peptide: opening a PEPstMOD webpage, inputting a polypeptide sequence, selecting and submitting a D-configuration amino acid module, and storing a polypeptide structure to a mailbox after prediction is completed.

b) Carrying out Z-DOCK molecular docking: and (3) performing molecular docking on the predicted TBP-3 peptide structure and hTIGIT (PDB ID:3UDW), performing Z-DOCK online docking on the molecular docking, analyzing the docking result by MOE software, and selecting a docking mode (comprehensive bond energy, distance and interaction site for consideration).

c) Analyzing a docking mode: analyzing the interaction of TIGIT protein and TBP-3 peptide in MOE software, selecting a binding pattern consistent with the key amino acids in the above alanine scanning peptide results, which shows that the interaction sites of TBP-3 peptide and TIGIT protein are H6-N58, R9-E60, R9-D63, Y3-N70 and L10-S80;

d) verification of key amino acids: mutating amino acid positions Q56, N58, E60, D63, N70, L73 and S80 of the TIGIT protein into alanine respectively, expressing the mutant protein on the cell surface, synthesizing a TBP-3 peptide (TBP-3-biotin) marked by biotin, detecting the affinity of the TBP-3-biotin with cells expressing the TIGIT protein and the mutant protein through flow type, and calculating the relative affinity of each group relative to the affinity of the TBP-3-biotin to the TIGIT wild protein.

The results shown in FIG. 5 show that the TIGIT protein (WT) has amino acid positions N58, E60, D63, N70, and S80 mutated to alanine, and that the affinity of TBP-3-biotin peptide to TIGIT protein is decreased, and important positions Q56 and L73 other than the predicted results have no interference with the affinity of TBP-3-biotin peptide to TIGIT protein, and that the key amino acid positions of TBP-3 peptide in the binding pattern are Y3, H6, R9, and the like.

8. Affinity peptidase degradation stability experiment:

a) dissolving peptide TBP-3 to 100 mu M mother liquor by PBS7.2, rapidly and uniformly mixing 190 mu L of 10% human serum solution with 10 mu L of the mother liquor of the TBP-3 peptide, taking out 20 mu L of the mixed liquor, timing to be 0h, putting the rest in a 37 ℃ incubator for enzymolysis reaction, and respectively taking out 20 mu L of reaction products in a 1.5mL EP tube for detection of enzymolysis conditions at each time point in the following 0.5h, 1h, 2h, 4h, 8h, 16h, 24h and 48 h;

b) carefully adding 90 mu L of acetonitrile into the samples taken out at different time points by using a pipette, immediately shaking and uniformly mixing, placing on ice for 10min, and adding 90 mu L of 0.5% glacial acetic acid solution into the pipette to stop the enzymolysis reaction;

c) precooling a centrifuge at 4 ℃, centrifuging for 20min at 10000g, collecting supernatant, putting the supernatant into a new EP tube, and using the supernatant in a subsequent RP-HPLC (reverse phase high performance liquid chromatography) analysis enzyme degradation stability experiment to show that the affinity peptide TBP-3 still stably exists within 48h and has obvious enzymolysis resistance.

9. The anti-tumor effect of TBP-3 is researched in a CT26 colorectal cancer transplantation tumor model, a B16-OVA melanoma transplantation tumor model and a 4T1 breast cancer model, and the specific implementation method is as follows:

a) carrying out tumor loading: collecting good-growth-state CT26 colorectal cancer cells, B16-OVA melanoma cells and 4T1 breast cancer cells, and respectively carrying tumor BALB/c mice (2X 10)⁵Cell/cell), C57BL/6 mice (5X 10)⁵Cell/cell) and BALB/c mice (1X 10)⁴Cell/cell).

b) For CThe tumor volume of the T26 or B16-OVA tumor-bearing mice is about one week after the mice have the tumor volume of about 40-80mm³The method comprises the following steps of carrying out S-shaped grouping according to the size of a mouse tumor, carrying out intraperitoneal administration every day for 2 weeks, weighing the weight of the mouse by using an electronic balance every other day during administration, measuring the mouse tumor by using a digital vernier caliper, and calculating and recording the volume change of the mouse tumor according to a formula V which is 1/2 × a (length) × b (width) × c (height);

c) for 4T1 tumor-bearing mice, after 12 days of tumor bearing, the mice are divided into S-shaped groups according to the tumor sizes of the mice, the mice are subjected to intraperitoneal administration every day for 2 weeks, the weight and the tumor volume changes of the mice are recorded, after 34 days of tumor bearing, the tumor-bearing mice are dissected, and the number of the lung metastases of each group of mice is observed and recorded.

The mental status of mice was good during the administration of the three transplanted tumor models, and the results of each experiment are shown in FIGS. 6-12.

10. In vivo imaging experiments prove the tissue infiltration of the affinity peptide TBP-3, and the specific implementation method is as follows:

a) selecting CT26 tumor-bearing mice with uniform tumor size, and injecting 200 mug Cy5.5 fluorescence-labeled affinity peptide TBP-3-Cy5.5 and equimolar fluorescent dye Cy5.5 into tail vein;

b) after tail vein injection is carried out for 0h and 24h, a living body imaging system of a small animal is utilized to detect the distribution of fluorescence in a mouse body, the tumor-bearing mouse is dissected after 24h, and the tumor tissue and organs (heart, liver, spleen, lung, kidney and brain) of the mouse are taken to carry out fluorescence detection;

the result shows that the fluorescent-labeled affinity peptide TBP-3-Cy5.5 is mainly distributed at the tumor tissue part of the tumor-bearing mouse, which indicates that the affinity peptide has stronger tumor tissue infiltration capacity, the fluorescent dye is mainly distributed at the kidney, and the fluorescence intensity statistical result is shown in figure 13.

While particular embodiments of the present invention have been illustrated and described, it would be obvious that various other changes and modifications can be made without departing from the spirit and scope of the invention, and it is intended to cover in the appended claims all such changes and modifications that are within the scope of the invention.

Sequence listing

<110> Zhengzhou university

Affinity agent of <120> TIGIT-IgV and application thereof

<160> 18

<170> SIPOSequenceListing 1.0

<210> 1

<211> 12

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Leu Thr Pro His Lys His His Lys His Leu His Ala

1 5 10

<210> 2

<211> 12

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Gly Gly Tyr Thr Phe His Trp His Arg Leu Asn Pro

1 5 10

<210> 3

<211> 12

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Gly Asn Leu Thr Leu His Met His Arg Ser Pro Ser

1 5 10

<210> 4

<211> 12

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Ser Ala Ile His Phe His His Pro Arg Trp Lys Pro

1 5 10

<210> 5

<211> 26

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 5

Gly Gly Tyr Thr Phe His Trp His Arg Leu Asn Pro Ala Ala Asn Tyr

1 5 10 15

Ser Lys Pro Thr Asp Arg Gln Tyr His Phe

20 25

<210> 6

<211> 12

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

Ala Gly Tyr Thr Phe His Trp His Arg Leu Asn Pro

1 5 10

<210> 7

<211> 12

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 7

Gly Ala Tyr Thr Phe His Trp His Arg Leu Asn Pro

1 5 10

<210> 8

<211> 12

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 8

Gly Gly Tyr Ala Phe His Trp His Arg Leu Asn Pro

1 5 10

<210> 9

<211> 12

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 9

Gly Gly Tyr Thr Ala His Trp His Arg Leu Asn Pro

1 5 10

<210> 10

<211> 12

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 10

Gly Gly Tyr Thr Phe His Ala His Arg Leu Asn Pro

1 5 10

<210> 11

<211> 12

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 11

Gly Gly Tyr Thr Phe His Trp Ala Arg Leu Asn Pro

1 5 10

<210> 12

<211> 12

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 12

Gly Gly Tyr Thr Phe His Trp His Arg Leu Ala Pro

1 5 10

<210> 13

<211> 12

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 13

Gly Gly Tyr Thr Phe His Trp His Arg Leu Asn Ala

1 5 10

<210> 14

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 14

Gly Gly Tyr Thr Phe His Trp His Arg Leu Asn

1 5 10

<210> 15

<211> 10

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 15

Gly Gly Tyr Thr Phe His Trp His Arg Leu

1 5 10

<210> 16

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 16

Gly Gly Tyr Thr Phe His Trp His Arg

1 5

<210> 17

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 17

Gly Tyr Thr Phe His Trp His Arg Leu Asn Pro

1 5 10

<210> 18

<211> 10

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 18

Tyr Thr Phe His Trp His Arg Leu Asn Pro

1 5 10

Claims

An affinity agent of TIGIT-IgV or a drug-modified form thereof, wherein the affinity agent can block TIGIT/PVR protein combination, the affinity agent is selected from amino acids shown in SEQ ID NOs:1, 3 or 4, or the structure of the affinity agent is shown in a formula I or a formula II,

X1-Tyr-X2-His-X3-Arg-X4-X5 (I)

X1-Tyr-X2-His-X3-Arg-X4-X5-(L)n-X6 (II)

in the formula I or the formula II,

x1, X2, X3 or X5 are independently selected from peptide fragments consisting of 0-2 amino acids,

x4 is absent or is leucine,

(L) n represents a flexible linker, n is a positive integer, and X6 is a peptide fragment consisting of 1-12 amino acids.
2. The affinity agent according to claim 1, wherein X1 is absent or selected from Gly-, Ala-Gly-, Gly-Ala-or Gly-; independently optionally, the X2 is absent or selected from peptidyl Thr-Phe, Ala-Phe, or Thr-Ala; independently optionally, said X3 is absent or selected from peptidyl Trp-His, Ala-His or Trp-Ala; independently optionally, X5 is absent or selected from peptidyl Asn-Pro, Ala-Pro or Asn-Ala.
3. An affinity agent according to any one of the preceding claims wherein L is peptidyl-Ala-.
4. An affinity agent according to any one of the preceding claims, wherein n-1-6, preferably n-1.
5. An affinity agent according to any one of the preceding claims wherein X6 is the peptide fragment-Asn-Tyr-Ser-Lys-Pro-Thr-Asp-Arg-Gln-Tyr-His-Phe.
6. The affinity agent of claim 1, wherein the sequence of said affinity peptide is selected from the group consisting of SEQ ID NOs: 1.2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18.
7. An affinity agent according to any preceding claim, wherein the configuration of each amino acid of the affinity agent is independently selected from D or L, and each amino acid may be in D or L form, e.g. each amino acid is in D configuration.
8. A pharmaceutical composition or diagnostic kit comprising an affinity agent according to any preceding claim or a pharmaceutically modified form thereof.
9. Use of an affinity agent according to any preceding claim in the manufacture of a medicament or diagnostic kit.
10. Use according to claim 9, wherein the medicament is for at least one of the following uses:

1) resisting tumor, wherein the tumor can include colon cancer, melanoma or breast cancer

2) Treating infections caused by bacteria, viruses or fungi

3) Treating autoimmune diseases.