CN116514913B

CN116514913B - TNFR2 targeting peptide with anti-tumor activity and its application

Info

Publication number: CN116514913B
Application number: CN202310667572.4A
Authority: CN
Inventors: 陈新; 廖萍; 陈忠豪
Original assignee: University of Macau
Current assignee: University of Macau
Priority date: 2023-06-07
Filing date: 2023-06-07
Publication date: 2025-03-21
Anticipated expiration: 2043-06-07
Also published as: CN116514913A

Abstract

The invention discloses a TNFR2 targeting peptide with anti-tumor activity and application thereof. The targeting peptide is a targeting TNFR2 affinity peptide with anti-tumor activity, can inhibit the combination of TNF and TNFR2, and can inhibit the increase of the proportion of Treg cells induced by the TNF. The inventor further proves that the targeted TNFR2 polypeptide provided by the invention has better anti-tumor activity through a mouse subcutaneous tumor-bearing experiment, and can obviously inhibit the growth of mouse tumors. Meanwhile, the combination of the anti-tumor peptide and the PD-L1 antibody can enhance the curative effect of cancer immunotherapy, reduce the proportion of tregs in tumor tissues and improve the proportion of CD8+IFNgamma+.

Description

TNFR2 targeting peptide with anti-tumor activity and application thereof

Technical Field

The invention relates to the technical field of biomedicine, in particular to a TNFR2 targeting peptide with anti-tumor activity and application thereof.

Background

In recent years, the burden of cancer has been continuously rising. Malignant tumor morbidity and mortality both show a trend of increasing year by year. Finding new therapeutic targets and developing new therapeutic drugs has been the subject of global research. Compared with the traditional methods such as surgical excision, chemotherapy, radiotherapy and the like, the tumor immunotherapy can activate or induce the autoimmune reaction of a patient, and can more safely and effectively inhibit the development and metastasis of tumors.

Tumor necrosis factor receptor 2 (TNFR 2) is one of the receptors mediating TNF biological functions, TNFR2 has a determining role in activation, function, proliferation and phenotypic stabilization of cd4+foxp3+ regulatory T cells (regulatory T cell, treg). Treg cells play a critical role in maintaining the stability of the immune system in vivo and in suppressing autoimmune responses. The Treg cells gather in a large amount in the tumor to inhibit the activities of effector T cells and cytotoxic T cells, so that the immune escape of the tumor is caused, and the growth and the metastasis of the tumor are further promoted. But systemic killer tregs may impair their normal biological function, leading to severe systemic inflammation. Therefore, the targeted TNFR2 selectively inhibits the activity of tumor-infiltrating Treg cells, can achieve the effects of enhancing anti-tumor immune response and inhibiting tumor growth, and is a safe and effective strategy for tumor immunotherapy.

The advent of tumor immunotherapy is one of the most important scientific breakthroughs in recent years, and there is currently a lot of immunotherapeutic drugs (such as immune checkpoint inhibitors, anti-PD-1, anti-PD-L1, anti-CTLA-4, etc. antibody drugs). However, anti-PD-1/PD-L1 therapy is generally effective in only 20% of patients, some of whom also develop resistance and even disease progression. While TNFR2 is a novel target for tumor immunotherapy, a large number of pharmaceutical enterprises have begun to put into development of anti-TNFR 2 antibody drugs. The targeted TNFR2 antibody medicines developed by a plurality of pharmaceutical enterprises including Bioinvent, baiji Shenzhou, pioneer pharmaceutical industry, arno medicine and HIFIBIO have greatly progressed in anti-tumor treatment and gradually enter clinical researches.

Although antibody drugs have advantages in tumor immunotherapy, searching for active peptides targeting TNFR2 has not been reported yet. The targeting TNFR2 active peptide has good application prospect and safety when used as a tumor immunotherapy medicament, and the polypeptide medicament has the advantages of low immunogenicity, easy synthesis and transformation, difficult accumulation in vivo and the like. At the same time, the drug effect can be provided by coupling the ligand or by carrying the nano-delivery system. However, in the prior art, polypeptide medicines for better targeting TNFR2 are not available.

In view of this, the present invention has been made.

Disclosure of Invention

The invention aims to provide a TNFR2 targeting peptide with anti-tumor activity and application thereof, and the targeting peptide can reduce tumor infiltration Treg cells by blocking the combination of TNF/TNFR2, thereby achieving the effects of enhancing anti-tumor immune response and inhibiting tumor growth.

The invention is realized in the following way:

In a first aspect, the invention provides a TNFR2 targeting peptide having anti-tumor activity as follows:

(1) P1 has an amino acid sequence shown in SEQ ID NO.1, or

(2) P19, the amino acid sequence of which is shown as SEQ ID NO.2, or

(3) P20, the amino acid sequence of which is shown as SEQ ID NO.3, or

(4) An amino acid sequence in which one or more amino acids are deleted, added and/or substituted from the amino acid sequence of any one of (1) to (3), but the function of the targeting peptide is unchanged.

In a second aspect, the invention provides a method for preparing the TNFR2 targeting peptide, which comprises the steps of performing high-throughput screening by taking TNFR2 molecules as targets through phage display peptide library screening technology, and then synthesizing the targeting TNFR2 antitumor active peptide through artificial solid phase.

In a third aspect, the present invention provides a gene encoding the above TNFR2 targeting peptide.

In a fourth aspect, the present invention provides a recombinant vector comprising the above gene.

In a fifth aspect, the present invention provides a recombinant host cell comprising the recombinant vector described above.

In a sixth aspect, the invention provides the use of a TNFR2 targeting peptide in the preparation of a fusion protein, a targeting drug or a targeted delivery system.

In a seventh aspect, the invention provides an application of a TNFR2 targeting peptide in preparing a targeting medicament for treating tumors, wherein the tumors comprise colon cancer, colorectal cancer, gastric cancer, esophageal cancer, pancreatic cancer and liver cancer, and preferably the tumors are colon cancer.

In an eighth aspect, the invention provides an application of a TNFR2 targeting peptide and a PD-L1 antibody in combination in preparing a targeting medicament for treating tumors, wherein the tumors comprise colon cancer, colorectal cancer, gastric cancer, esophageal cancer, pancreatic cancer and liver cancer, and preferably the tumors are colon cancer.

The invention has the following beneficial effects:

The targeting peptide provided by the invention is a targeting TNFR2 affinity peptide with anti-tumor activity, can inhibit the combination of TNF and TNFR2, and can inhibit the increase of the proportion of Treg cells induced by the TNF. The inventor further proves that the targeted TNFR2 polypeptide provided by the invention has better anti-tumor activity through a mouse subcutaneous tumor-bearing experiment, and can obviously inhibit the growth of mouse tumors. The combination of the anti-tumor antigen and the PD-L1 antibody can enhance the curative effect of cancer immunotherapy, reduce the proportion of tregs in tumor tissues and improve the proportion of CD8+IFNgamma+cells.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a graph showing the results of a binding assay for TNF-TNFR2 by Jurkat cells that overexpress TNFR2 by the polypeptides of example 2;

FIG. 2 is a graph showing the experimental results of the effect of the polypeptide of example 3 on FOXP3 expression on CD4 cells in vitro;

FIG. 3 is a graph showing the inhibition of tumor growth in CT26 subcutaneous tumor-bearing mice by targeting peptides;

Fig. 4 is a graph of the targeting peptide reducing the extent of Treg infiltration in the tumor microenvironment;

FIG. 5 is a graph showing that targeting peptide in combination with anti-PD-L1 antibody can significantly inhibit the growth of MC38 tumor-bearing mice tumors.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

The invention provides a TNFR2 targeting peptide with anti-tumor activity, which comprises the following components:

(1) P1 has an amino acid sequence shown in SEQ ID NO.1, or

(2) P19, the amino acid sequence of which is shown as SEQ ID NO.2, or

(3) P20, the amino acid sequence of which is shown as SEQ ID NO.3, or

As is well known to those skilled in the art, the 20 amino acid residues that make up a protein can be divided into four classes according to the polarity of the side chain:

(1) Non-polar amino acids alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), methionine (Met), phenylalanine (Phe), tryptophan (Trp) and proline (Pro);

(2) Polar uncharged amino acids glycine (Gly), serine (Ser), threonine (Thr), cysteine (Cys), asparagine (Asn), glutamine (Gln) and tyrosine (Tyr);

(3) Positively charged amino acids arginine (Arg), lysine (Lys) and histidine (His);

(4) Negatively charged amino acids aspartic acid (Asp) and glutamic acid (Glu) (see "biochemistry" (second edition) upper books, shen Tong, wang Jingyan, pages 82-83, higher education Press, 12 months 1990). If amino acid residue substitution of the same class occurs in the protein, for example Arg for Lys or Leu for Ile, the function of the residue in the protein domain (such as the function of providing positive charge or forming a hydrophobic pocket structure) is not changed, so that the steric structure of the protein is not affected, and thus the function of the protein can still be achieved. For example, it is well known to those skilled in the art that Ala and Ser, val and Ile, asp and Glu, ser and Thr, ala and Gly, ala and Thr, ser and Asn, ala and Val, ser and Gly, tyr and Phe, ala and Pro, lys and Arg, asp and Asn, leu and Ile, leu and Val, ala and Glu, and Asp and Gly are substituted for each other one by one without affecting the steric structure and function of the protein. Amino acid residue substitutions belonging to one class can occur at any one amino acid residue position on the targeting peptide of the invention. In contrast, amino acid residues of different classes are substituted, or amino acid substitutions do not meet the substitution rules listed above, and are likely to change the structure of the protein and cause differences in function.

The targeting peptides of the invention may also be modified or mutated to provide derived polypeptides or proteins. The term "derivative polypeptide or protein" as used herein refers to a polypeptide which differs in amino acid sequence from a target peptide having the amino acid sequence described above, or in modified form which does not affect the sequence, or both. These proteins include natural or induced genetic variants. The above-described induced variants may be obtained by various techniques such as random mutagenesis by irradiation or mutagens and the like, or by techniques such as site-directed mutagenesis or other known molecular biology. The "derived polypeptides or proteins" described above also include analogs having residues of the natural L-form amino acid (e.g., D-amino acids), as well as analogs having non-naturally occurring or synthetic amino acids (e.g., beta-amino acids, gamma-amino acids, etc.).

Modified (typically without altering the primary structure) forms include chemically derivatized forms of the protein, such as acetylated or carboxylated, in vivo or in vitro. Modifications also include glycosylation, such as those resulting from glycosylation modifications during synthesis and processing of the protein or during further processing steps. Such modification may be accomplished by exposing the protein to an enzyme that performs glycosylation (e.g., mammalian glycosylase or deglycosylase). Modified forms also include sequences having phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). Proteins modified to increase their proteolytic resistance or to optimize their solubility properties are also included.

In the invention, the amino acid sequence of the targeting peptide P1 is GLCWVSPCSTK, which is shown as SEQ ID NO. 1;

The amino acid sequence of P19 is SGKHCSKVYDCH as shown in SEQ ID NO. 2;

the amino acid sequence of P20 is SHWGGNVRGTQT, shown as SEQ ID NO. 3.

In some embodiments, the targeting peptide is P19 or P20, more preferably, the targeting peptide is P20.

The invention also provides a preparation method of the targeting peptide, which comprises the steps of utilizing phage display peptide library screening technology, taking TNFR2 molecules as targets for high-throughput screening, and synthesizing the targeting TNFR2 anti-tumor active peptide by artificial solid phase.

Specifically, the phage display peptide library screening technology is to fuse the exogenous DNA fragment encoding the polypeptide with the encoding gene of the phage surface protein (insert between the signal peptide and the capsid protein gene), then present the fusion protein on the phage surface, each phage contains only 1 exogenous gene, the displayed polypeptide or protein can maintain relative spatial structure and biological activity, and display on the phage surface. A group of phages into which various exogenous genes have been introduced constitutes a phage display library displaying various exogenous peptides. When a protein is used to screen a phage display library, it is selectively bound to a foreign peptide that interacts with it, thereby isolating a particular phage in the display library.

The invention adopts molecular level in-situ screening to coat specific target protein on a 96-well plate, and the polypeptide screening process is not easy to be interfered by nonspecific factors because only a single target is contacted.

The invention relates to an artificial solid phase synthesis, which uses Fmoc as a protecting group of amino acid alpha-amino, connects a first amino acid C end of target peptide with a solid phase carrier through a covalent bond, uses an amino acid N end as a synthesis starting point, carries out reaction through deamination protecting group and excessive activated second amino acid, stretches a peptide chain, repeats operation to reach ideal synthetic peptide chain length, and finally, cracks the peptide chain from resin, separates and purifies to obtain the target polypeptide.

The invention also provides a nucleotide sequence for encoding the targeting peptide.

It is well known in the art that of the 20 different amino acids that make up a protein, other than Met (ATG) or Trp (TGG) are each encoded by a single codon, the 18 other amino acids are each encoded by 2-6 codons (Sambrook et al, molecular cloning, cold spring harbor laboratory Press, new York, U.S. second edition, 1989, see page 950 appendix D). That is, due to the degeneracy of the genetic code, the nucleotide sequence of the gene encoding the same protein may differ, since the substitution of the third nucleotide in the triplet codon, which determines most of the codons of one amino acid, does not change the composition of the amino acid. The nucleotide sequences of genes encoding them can be deduced entirely from the amino acid sequences disclosed in the present invention and the amino acid sequences whose functions of the targeting peptide obtained from the above amino acid sequences according to well known codon tables, and the above nucleotide sequences can be obtained by biological methods (e.g., PCR method, mutation method) or chemical synthesis method, and thus, the partial nucleotide sequences should be included in the scope of the present invention. In contrast, by using the DNA sequences disclosed in the present invention, amino acid sequences functionally equivalent to the targeting peptides described above can also be obtained by modifying the nucleic acid sequences provided in the present invention by methods known in the art, such as the method of Sambrook et al (molecular cloning, cold spring harbor laboratory Press, new York, U.S. A., second edition, 1989).

According to the targeting peptides disclosed herein, fusion proteins comprising the above-described targeting peptides can also be obtained. The fusion protein may also include other protein sequences or polypeptide sequences linked at the N-or C-terminus of the fusion protein.

According to the targeting peptide disclosed by the invention, a pharmaceutical composition comprising the targeting peptide can be obtained, and the pharmaceutical composition further comprises pharmaceutically acceptable components. Such ingredients include, but are not limited to, pharmaceutically acceptable carriers.

According to the targeting peptide disclosed by the invention, a targeting delivery system which comprises the targeting peptide and is packed in a nano carrier or is modified on the surface of the nano carrier and is constructed by the physicochemical properties of the nano carrier can be also obtained.

Proved by experiments, the TNFR2 targeting peptide provided by the invention has better anti-tumor activity and can obviously inhibit the growth of mice tumors. In particular to a TNFR 2-targeted anti-tumor active peptide P20 which plays a role in inhibiting the growth of colon cancer (CT 26, MC 38) as an active ingredient.

Meanwhile, the inventor finds that the combination of the P20 and the PD-L1 antibody can enhance the curative effect of cancer immunotherapy, reduce the proportion of tregs in tumor tissues and improve the proportion of CD8+IFNgamma+ cells.

The features and capabilities of the present invention are described in further detail below in connection with the examples.

Example 1

This example is the preparation of a TNFR2 targeting peptide comprising:

1. phage display peptide library screening polypeptides

1.1 Phage titer detection

1) ER2738 strain is inoculated into LB culture medium (containing Tet antibiotics with the concentration of 30 mug/mL) for activation, and then inoculated into 5mL of fresh LB culture medium (containing Tet antibiotics with the concentration of 30 mug/mL) according to the proportion of 1:100 for culture until OD 600.4-0.6;

2) Preparing an agar plate, namely weighing 1.25g of IPTG and 1g of Xgal, dissolving in 25mL of DMF, and storing at-20 ℃ for later use;

3) 15g/L agar is added into 1L LB culture medium, 1mL IPTG/Xgal stock solution is added at the same time to prepare a flat plate, and the flat plate is stored at 4 ℃ for light-shielding standby;

4) Diluting phage by LB culture according to 10-fold gradient dilution method, equally dividing host bacteria subjected to expansion culture according to requirements, adding 10 mu L of LB diluted gradient phage into each tube, slightly mixing, and incubating at room temperature for 30min;

5) Plating the infected host bacteria in the step 4) on the flat plate prepared in the step 3), and culturing at 37 ℃ overnight;

6) Plaque count.

1.2 Phage expansion culture

2) 10 mu L of phage are inoculated to the host strain subjected to the expansion culture, and shake culture is carried out at 200rpm and 37 ℃ for 3-6 hours. Centrifuging at 8000rpm to collect supernatant to obtain phage culture.

1.3 Host bacteria verification

4) Diluting phage by LB culture according to 10-fold gradient dilution method, equally dividing host bacteria subjected to expansion culture according to requirements, adding 1 μl of LB diluted gradient phage into each tube, slightly mixing, and incubating at room temperature for 30min;

5) The infected host bacteria of step 4) were plated on the plates prepared in step 3) and incubated overnight at 37℃to see whether there was blue-white spots.

1.4 Panning of recombinant phages

1) Antigen coating, namely diluting purified TNFRSF1B recombinant protein to 4 mug/mL by using PBS buffer solution, taking a 96-well ELISA plate, selecting 3 wells, adding 100 mug (400 ng/well) into each well, coating overnight at 4 ℃, and taking PBS as a negative control;

2) Sealing, namely removing the coating liquid, adding 150 mu L of 2% concentration skimmed milk powder into each hole, and sealing for 1h at room temperature;

3) Incubating phage, namely washing 4 times by using PBST, taking phage solution prepared by 1.2, diluting to 5X 10 ¹¹ PFU/mL by using 2% skimmed milk powder, adding an ELISA plate, and incubating for 2 hours at room temperature;

4) Eluting by discarding phage sample, washing with PBST for 10 times, washing with PBS for 5 times, adding 100 μl of freshly prepared 0.1M triethylamine into each well, standing at room temperature for 10min, and rapidly neutralizing the eluate with equal volume of 1M Tris-HCl (pH 7.4);

5) Determining the titer of the recombinant phage in the eluent by referring to the method 1.1;

6) Concentrating and purifying phage particles, namely concentrating and purifying phage particles according to the operation method of the step 1.2, and using the phage particles for the next round of screening;

7) Repeating the steps 1) to 6) twice to finish the second round and the third round of panning.

1.5 Picking up monoclonal from bacterial plates after the third round of screening, extracting plasmids for sequencing after amplification culture.

Fomc solid-phase polypeptide synthesis targeting peptides

The C-terminal first Fmoc-amino acid carboxyl of the candidate peptide is connected in a covalent bond mode, and then the N-terminal of the amino acid is used as a starting point of candidate polypeptide synthesis to be dehydrated and condensed with the carboxyl terminal of the second amino acid in the sequence to form a peptide bond. Then deprotecting Fmoc-amino acid protecting group at N terminal, then reacting second amino acid N terminal with subsequent amino acid carboxyl, repeating the process until the synthesis of polypeptide is completed. Finally, precipitating and washing to obtain the polypeptide. And (3) identifying that the molecular weight of the polypeptide accords with the theoretical molecular weight by mass spectrum, and storing at-20 ℃.

Example 2

This example is a Jurkat cell TNF-TNFR2 binding assay in which the polypeptide inhibits the expression of TNFR2, and comprises the following steps:

1) Collecting Jurkat cells which over express TNFR2, centrifuging 300g for 5 minutes to remove supernatant, and re-suspending the cells in a serum-free culture medium to dilute the cells so as to adjust the cell concentration to 1X 10 ⁶ cells/mL;

2) Plating 100 μl of the cell suspension to a 96-well plate, adding polypeptide/positive control/negative control to incubate cells at 37 ℃ for 1 hour, adding biotin-TNF and continuing incubation for 1 hour;

3) Cells were collected and centrifuged at 300g for 5 min to remove supernatant, and after 30min of blocking, APC-strepitavidin was added for 30min, and after two PBS washes, the cells were checked on-machine.

The APC-strepitavidins fluorescence peak is shown in FIG. 1C, D. ETA is Etanercept as positive control.

As shown in FIG. 1, 1-A is a Jurkat cell over-expressing TNFR2 constructed by lentiviral transfection, 1-B is a change in fluorescence intensity of a Jurkat cell over-expressing TNFR2 after incubation with Biotin-TNF for 1 hour with a Jurkat cell over-expressing TNFR2, and then, with the addition of APC-labeled streptavidins, the binding of different concentrations of TNF to TNFR2 on the Jurkat cell membrane was detected by flow cytometry, 1-C and 1-D are the fluorescence intensities of NC (negative control, WT Jurkat cells) and Jurkat cell over-expressing TNFR2 (JK-R2), 1-E is a positive control of Jurkat cell over-expressing TNFR2 after P20 treatment, and ETA (etanercept, fusion protein of soluble TNFR. LINKED IGG 1) is a positive control, and 1-F is the signal intensity on the cells after treatment with different polypeptides.

It can be seen from FIG. 1 that targeting peptide P20 inhibits the binding effect of TNF-TNFR 2.

Example 3

The example is the effect of polypeptide in vitro culture on FOXP3 expression, and the steps are as follows:

1) CD4+ T cells were purified from lymph nodes, spleens, of wild type C57BL/6 mice by magnetic bead sorting techniques, labeled with CELL TRACE ^TM Violet staining of primary cells, and cultured in vitro with IL-2, TNF, 10ng/mL each.

2) Cells were collected 72 hours after treatment with different polypeptides, stained with LIVE/DEAD ^TM dye, analyzed by flow cytometry, and labeled with FACS antibodies for staining for CD4, FOXP3, etc.

3) The effect of polypeptide in vitro culture on FOXP3 expression was analyzed. The experimental results are shown in FIG. 2.

As can be seen from FIG. 2, cells cultured in IL-2 (10 ng/mL) containing culture medium, TNF (10 ng/mL) can specifically stimulate proliferation of Treg cells, and flow cytometry analyzed the proportion of Foxp3+ cells (FIG. 2-A). Positive drug ETA (Etanercept) and polypeptide 10 μg/mL both significantly inhibited TNF-induced Treg cell elevation (fig. 2-B, 2-C).

Example 4

The embodiment is an in vivo experiment of the targeting peptide in tumor-bearing mice, and specifically comprises the following steps:

In this example, CT26 colon cancer cells were selected and the cell concentration was adjusted to 5X 10 ⁶ cells/mL with PBS, 100. Mu.L of the cell suspension was inoculated subcutaneously into BALB/C mice after conventional sterilization, and the mice were respectively intraperitoneally injected with PBS,10mg/kg P1, P19, P20 after 7 days of inoculation. The administration is continued for 12 days, and the growth of subcutaneous tumor is continuously observed. The experimental results are shown in FIG. 3.

The BALB/C mice were inoculated subcutaneously with CT26 cells on the right back and when the tumor diameter was 5-7 mm, intraperitoneal injection of polypeptides P01, P19 and P20 (10 mg/kg) was started, and the specific experimental procedure was shown in FIG. 3-A. As can be seen from fig. 3-B, P01, P19 and P20 each inhibited tumor growth, wherein P19 and P20 significantly inhibited tumor growth (P <0.05, P < 0.01), whereas as can be seen from fig. 3-C, there was no significant change in mouse body weight.

In this example, MC38 colon cancer cells were selected and the cell concentration was adjusted to 5X 10 ⁶ cells/mL with PBS, after conventional sterilization, 100. Mu.L of the cell suspension was inoculated subcutaneously into C57 mice, and after 7 days of inoculation, the mice were given intraperitoneal injections of PBS, PD-L1 antibody, P20, PD-L1 antibody in combination with P20, respectively. The subcutaneous tumor growth was observed continuously for 9 days of administration. The experimental results are shown in FIG. 5.

C57BL/6 mice are inoculated with MC38 cells subcutaneously, and when tumors grow to 5-7 mm, intraperitoneal injection of the polypeptide P20 and anti-PD-L1 is started. The experimental design is shown in FIG. 5-A. The method has the advantages that both P20 and anti-PD-L1 can inhibit tumor growth, the effect of inhibiting tumor growth by combining P20 with anti-PD-L1 is more obvious, the proportion of tumor-infiltrating Treg cells in a combined treatment group is obviously reduced by combining 5-C, the expression of IFN gamma of tumor-infiltrating CD8 cells can be improved by combining P20, anti-PD-L1 and P20 with anti-PD-L1 by combining 5-D, and the improvement effect of P20 with anti-PD-L1 is more obvious.

Example 5

The embodiment is an immune cell infiltration detection in a mouse tumor microenvironment, and specifically comprises the following steps:

After the end of the administration, the mice were euthanized, the tumor tissue was exfoliated, and the tumor tissue was digested with GENTLEMACS ^TM and filtered through a 70 μm sieve to obtain a single cell suspension. After incubation with LIVE/DEAD ^TM Fixable Near-IR DEAD CELL STAIN KIT dye for 30 min after two PBS washes, the PBS resuspended cells were washed two times. After fixing cells for 12-18 hours with fixative, resuspended cells were washed twice with membrane-disrupting buffer, after 30 minutes of blocking, CD45, CD3, CD4, CD8, FOXP3 antibodies were added and after 30 minutes of staining, washed twice with membrane-disrupting buffer and then on-machine tested, cd4+ foxp3+ cells ratios such as shown in fig. 4.

It can be seen from FIGS. 4-A and 4-B that P20 significantly increases the proportion of draining lymph node CD8 cells, increases the proportion of CD8/CD4, that P20 significantly decreases the proportion of CD4 cells in the tumor environment, significantly increases the proportion of CD8/CD4, as can be seen from FIGS. 4-C and 4-D, and that P19, P20 significantly decreases the proportion of tumor-infiltrating Treg cells, as can be seen from FIGS. 4-E and 4-F.

The single cell suspension was used to detect the cd8+ifnγ+ ratio by taking 5×10 ⁶ cells, and after 6 hours of stimulation with Cell Stimulation Cocktail (plus protein transport inhibitors) (500×), the staining procedure was performed using the staining protocol described above, and the cd8+ifnγ+ cell ratios are shown in fig. 5, for example.

The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A TNFR2 targeting peptide having anti-tumor activity, wherein the targeting peptide is:

(1) P1 has an amino acid sequence shown in SEQ ID NO.1, or

(2) P19, the amino acid sequence of which is shown as SEQ ID NO.2, or

(3) And P20 has an amino acid sequence shown in SEQ ID NO. 3.

2. A gene encoding the TNFR2 targeting peptide of claim 1.

3.A recombinant vector comprising the gene of claim 2.

4. A recombinant host cell comprising the recombinant vector of claim 3.

5. Use of the TNFR2 targeting peptide of claim 1 in the preparation of a fusion protein, a targeting drug, or a targeted delivery system.

6. The use according to claim 5, wherein the fusion protein, targeted drug or targeted delivery system targets cells expressing TNFR2 protein;

the cells expressing the TNFR2 protein comprise tumor cells and immune cells.

7. Use of the TNFR2 targeting peptide of claim 1 in the preparation of a targeting drug for the treatment of colon cancer.

8. Use of the TNFR2 targeting peptide of claim 1 in combination with a PD-L1 antibody in the manufacture of a targeted medicament for the treatment of colon cancer.