TNFR2 targeting peptide with anti-tumor activity and its application

Abstract

The present invention discloses a TNFR2 targeting peptide with anti-tumor activity and its application. The targeting peptide is an affinity peptide targeting TNFR2 with anti-tumor activity, which can inhibit the binding of TNF to TNFR2 and inhibit the increase in the proportion of Treg cells induced by TNF. The inventors further proved through a mouse subcutaneous tumor-bearing experiment that the targeted TNFR2 polypeptide provided by the present invention has good anti-tumor activity and can significantly inhibit the growth of mouse tumors. At the same time, its combination with PD-L1 antibody can enhance the efficacy of cancer immunotherapy, reduce the proportion of Treg in tumor tissue, and increase the proportion of CD8+IFNγ+.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, in particular to a TNFR2 targeting peptide with anti-tumor activity and application thereof. Background Art

[0002] In recent years, the burden of cancer has continued to rise. The incidence and mortality of malignant tumors have both increased year by year. Finding new therapeutic targets and developing new therapeutic drugs have always been a hot topic in global research. Compared with traditional methods such as surgical resection, chemotherapy and radiotherapy, tumor immunotherapy can activate or induce the patient's autoimmune response and can more safely and effectively inhibit the development and metastasis of tumors.

[0003] Tumor necrosis factor receptor 2 (TNFR2) is one of the receptors that mediate the biological functions of TNF. TNFR2 plays a decisive role in the activation, function, proliferation and phenotypic stability of CD4+Foxp3+ regulatory T cells (Treg). Treg cells play a vital role in maintaining the stability of the body's immune system and inhibiting autoimmune responses. Treg cells accumulate in large numbers in tumors to inhibit the activity of effector T cells and cytotoxic T cells, leading to tumor immune escape and promoting tumor growth and metastasis. However, systemic killing of Treg may damage its normal biological functions and lead to severe systemic inflammation. Therefore, targeting TNFR2 to selectively inhibit the activity of tumor-infiltrating Treg cells can achieve the effect of enhancing anti-tumor immune responses and inhibiting tumor growth, which is a safe and effective strategy for tumor immunotherapy.

[0004] The emergence of tumor immunotherapy is one of the most important scientific breakthroughs in recent years. At present, there are a number of immunotherapy drugs (such as immune checkpoint inhibitors, anti-PD-1, anti-PD-L1, anti-CTLA-4 and other antibody drugs). However, anti-PD-1 / PD-L1 therapy is generally effective for only 20% of patients, and some patients have developed drug resistance or even super-progression of the disease. As a new target for tumor immunotherapy, TNFR2 has been invested in the research and development of antibody drugs for anti-TNFR2. Antibody drugs targeting TNFR2 developed by many pharmaceutical companies including Bioinvent, BeiGene, Innovent, Arno Pharmaceuticals, and HIFIBIO have made great progress in anti-tumor treatment and are gradually entering clinical research.

[0005] Although antibody drugs have advantages in tumor immunotherapy, there are still no reports on the search for active peptides targeting TNFR2. The use of active peptides targeting TNFR2 as tumor immunotherapy drugs has good application prospects and safety, and peptide drugs have the advantages of low immunogenicity, easy synthesis and modification, and not easy to accumulate in the body. At the same time, the efficacy can also be provided by coupling ligands or by carrying nano-delivery systems. However, in the prior art, there is still a lack of good peptide drugs targeting TNFR2.

[0006] In view of this, the present invention is proposed. Summary of the invention

[0007] The purpose of the present invention is to provide a TNFR2 targeting peptide with anti-tumor activity and its application, which can reduce tumor-infiltrating Treg cells by blocking the binding of TNF / TNFR2, thereby enhancing the anti-tumor immune response and inhibiting tumor growth.

[0008] The present invention is achieved in that:

[0009] In a first aspect, the present invention provides the following TNFR2 targeting peptides having anti-tumor activity:

[0010] (1) P1, the amino acid sequence of which is shown in SEQ ID NO. 1, or

[0011] (2) P19, the amino acid sequence of which is shown in SEQ ID NO. 2, or

[0012] (3) P20, the amino acid sequence of which is shown in SEQ ID NO. 3, or

[0013] (4) An amino acid sequence in which one or more amino acids are deleted, added and / or substituted in any one of (1) to (3), but the targeting peptide function remains unchanged.

[0014] In a second aspect, the present invention provides a method for preparing the above-mentioned TNFR2 targeting peptide, which comprises firstly performing high-throughput screening using phage display peptide library screening technology with TNFR2 molecules as targets, and then synthesizing anti-tumor active peptides targeting TNFR2 by artificial solid phase synthesis.

[0015] In a third aspect, the present invention provides a gene encoding the above-mentioned TNFR2 targeting peptide.

[0016] In a fourth aspect, the present invention provides a recombinant vector comprising the above gene.

[0017] In a fifth aspect, the present invention provides a recombinant host cell comprising the above-mentioned recombinant vector.

[0018] In a sixth aspect, the present invention provides the use of TNFR2 targeting peptides in the preparation of fusion proteins, targeted drugs or targeted delivery systems.

[0019] In a seventh aspect, the present invention provides the use of TNFR2 targeting peptides in the preparation of targeted drugs for treating tumors, wherein the tumors include colon cancer, large intestinal cancer, gastric cancer, esophageal cancer, pancreatic cancer and liver cancer; preferably, the above tumor is colon cancer.

[0020] In an eighth aspect, the present invention provides the use of a TNFR2 targeting peptide in combination with a PD-L1 antibody in the preparation of a targeted drug for treating tumors, wherein the tumors include colon cancer, large intestinal cancer, gastric cancer, esophageal cancer, pancreatic cancer and liver cancer; preferably, the above tumor is colon cancer.

[0021] The present invention has the following beneficial effects:

[0022] The targeting peptide of the present invention is an affinity peptide targeting TNFR2 with anti-tumor activity, which can inhibit the binding of TNF to TNFR2 and inhibit the increase in the proportion of Treg cells induced by TNF. The inventors further proved through subcutaneous tumor-bearing experiments in mice that the targeting TNFR2 polypeptide provided by the present invention has good anti-tumor activity and can significantly inhibit the growth of mouse tumors. Its combination with PD-L1 antibodies can enhance the efficacy of cancer immunotherapy, reduce the proportion of Treg in tumor tissues, and increase the proportion of CD8+IFNγ+ cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0024] Figure 1 The results of the TNF-TNFR2 binding experiment in Example 2 in which the polypeptide inhibited the TNFR2-overexpressing Jurkat cells;

[0025] Figure 2 The experimental results of Example 3 on the effect of the polypeptide on the expression of FOXP3 on CD4 cells in vitro culture;

[0026] Figure 3 The inhibition of tumor growth by targeting peptide in CT26 subcutaneous tumor-bearing mice;

[0027] Figure 4 The targeted peptide reduces the level of Treg infiltration in the tumor microenvironment;

[0028] Figure 5The targeted peptide combined with anti-PD-L1 antibody can significantly inhibit the growth of tumors in MC38 tumor-bearing mice. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0030] The TNFR2 targeting peptide with anti-tumor activity provided by the present invention comprises:

[0031] (1) P1, the amino acid sequence of which is shown in SEQ ID NO. 1, or

[0032] (2) P19, the amino acid sequence of which is shown in SEQ ID NO. 2, or

[0033] (3) P20, the amino acid sequence of which is shown in SEQ ID NO. 3, or

[0034] (4) An amino acid sequence in which one or more amino acids are deleted, added and / or substituted in any one of (1) to (3), but the targeting peptide function remains unchanged.

[0035] It is well known in the art that the 20 amino acid residues that make up proteins can be divided into four categories according to the polarity of their side chains:

[0036] (1) Non-polar amino acids: alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), methionine (Met), phenylalanine (Phe), tryptophan (Trp) and proline (Pro);

[0037] (2) Polar uncharged amino acids: glycine (Gly), serine (Ser), threonine (Thr), cysteine ​​(Cys), asparagine (Asn), glutamine (Gln) and tyrosine (Tyr);

[0038] (3) Positively charged amino acids: arginine (Arg), lysine (Lys), and histidine (His);

[0039] (4) Negatively charged amino acids: aspartic acid (Asp) and glutamic acid (Glu) (see "Biochemistry" (Second Edition), Volume 1, Shen Tong, Wang Jingyan, pages 82-83, Higher Education Press, December 1990). If amino acid residues belonging to the same category are substituted in a protein, such as Arg replacing Lys or Leu replacing Ile, the role of the residue in the protein domain (such as providing positive charge or forming a hydrophobic pocket structure) does not change, so it does not affect the three-dimensional structure of the protein, and the protein's function 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, replace each other in pairs, and can not affect the three-dimensional structure and function of protein. The amino acid residue substitution belonging to the same category can occur in any amino acid residue position on the targeting peptide of the present invention. On the contrary, the amino acid residues of different classes replace, or the amino acid whose substitution does not conform to the substitution rule listed above, may cause the structure of protein to change, and the function may differ.

[0040] The targeting peptide of the present invention can also be modified or mutated to obtain a derived polypeptide or protein. The "derived polypeptide or protein" of the present invention refers to a polypeptide having a difference in amino acid sequence with the targeting peptide having the above amino acid sequence, or a difference in the form of modification that does not affect the sequence, or both. These proteins include natural or induced genetic variants. The above-mentioned induced variants can be obtained by various techniques, such as random mutations produced by radiation or mutagens, or by techniques such as site-directed mutagenesis or other known molecular biology. The above-mentioned "derived polypeptide or protein" also includes analogs with residues of natural L-type amino acids (such as D-type amino acids), and analogs with non-naturally occurring or synthetic amino acids (such as β-amino acids, γ-amino acids, etc.).

[0041] Modifications (usually without changing the primary structure) include: chemical derivatization of proteins in vivo or in vitro, such as acetylation or carboxylation. Modifications also include glycosylation, such as those produced by glycosylation modification during protein synthesis and processing or in further processing steps. This modification can be accomplished by exposing the protein to a glycosylation enzyme (such as a mammalian glycosylase or deglycosylase). Modified forms also include sequences with phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, phosphothreonine). Also included are proteins that have been modified to increase their resistance to proteolysis or optimize their solubility.

[0042] In the present invention, the amino acid sequence of the targeting peptide P1 is: GLCWVSPCSTK, as shown in SEQ ID NO.1;

[0043] The amino acid sequence of P19 is: SGKHCSKVYDCH, as shown in SEQ ID NO.2;

[0044] The amino acid sequence of P20 is: SHWGGNVRGTQT, as shown in SEQ ID NO.3.

[0045] In some embodiments, the targeting peptide is P19 or P20; more preferably, the targeting peptide is P20.

[0046] As a general technical concept, the present invention also provides a method for preparing the above-mentioned targeting peptide, which comprises utilizing phage display peptide library screening technology, taking TNFR2 molecules as targets for high-throughput screening, and artificially solid-phase synthesizing anti-tumor active peptides targeting TNFR2.

[0047] Specifically, the phage display peptide library screening technology is to fuse the exogenous DNA fragment encoding the polypeptide with the coding gene of the phage surface protein (inserted between the signal peptide and the capsid protein gene), and then present it on the surface of the phage in the form of a fusion protein. Each phage contains only one exogenous gene, and the displayed polypeptide or protein can maintain a relative spatial structure and biological activity and be displayed on the surface of the phage. A group of phages introduced with various exogenous genes constitutes a phage display library that displays various exogenous peptides. When a protein is used to screen a phage display library, it will selectively combine with a certain exogenous peptide that interacts with it, thereby isolating a specific phage in the display library.

[0048] Among them, the present invention adopts in situ screening at the molecular level, and the specific target protein is coated on a 96-well plate. Since only a single target is contacted, the polypeptide screening process is not easily interfered by non-specific factors.

[0049] The artificial solid phase synthesis in the present invention uses Fmoc as the protecting group of the α-amino group of the amino acid, connects the C-terminus of the first amino acid of the target peptide to the solid phase carrier through a covalent bond, and then uses the N-terminus of the amino acid as the starting point of synthesis, removes the amino protecting group and reacts with an excess of the activated second amino acid, connects the peptide chain, repeats the operation to achieve the ideal synthetic peptide chain length, and finally cleaves the peptide chain from the resin, separates and purifies it to obtain the target polypeptide.

[0050] The present invention also provides a nucleotide sequence encoding the above-mentioned targeting peptide.

[0051] It is well known in the art that among the 20 different amino acids constituting proteins, except for Met (ATG) or Trp (TGG) which are respectively encoded by a single codon, the other 18 amino acids are respectively encoded by 2-6 codons (Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory Press, New York, USA, second edition, 1989, see Appendix D on page 950). That is, due to the degeneracy of the genetic code, there are mostly more than one codons that determine an amino acid, and the substitution of the third nucleotide in the triplet codon often does not change the composition of the amino acid, so the nucleotide sequence of the gene encoding the same protein can be different. According to the known codon table, the person skilled in the art can fully deduce the nucleotide sequence of the gene that can encode them from the amino acid sequence disclosed in the present invention, and the amino acid sequence with unchanged targeting peptide function obtained from the above amino acid sequence, and obtain the above nucleotide sequence by biological methods (such as PCR method, mutation method) or chemical synthesis method, so the partial nucleotide sequence should be included in the scope of the present invention. On the contrary, using the DNA sequence disclosed in the present invention, it is also possible to obtain an amino acid sequence consistent with the above-mentioned targeting peptide function of the present invention by modifying the nucleic acid sequence provided by the present invention through methods known in the art, such as the method of Sambrook et al. (Molecular Cloning, Cold Spring Harbor Laboratory Press, New York, USA, Second Edition, 1989).

[0052] According to the targeting peptide disclosed in the present invention, a fusion protein comprising the targeting peptide can also be obtained. The fusion protein can also include other protein sequences or polypeptide sequences, which are connected to the N-terminus or C-terminus of the fusion protein.

[0053] According to the targeting peptide disclosed in the present invention, a pharmaceutical composition comprising the targeting peptide can also be obtained, and the pharmaceutical composition also includes pharmaceutically acceptable ingredients, including but not limited to pharmaceutically acceptable carriers.

[0054] According to the targeting peptide disclosed in the present invention, a targeted delivery system can also be obtained by encapsulating the targeting peptide inside a nanocarrier or modifying the targeting peptide on the surface of the nanocarrier and constructing the targeting delivery system through the physicochemical properties of the nanocarrier.

[0055] Experiments have shown that the TNFR2 targeting peptide provided by the present invention has good anti-tumor activity and can significantly inhibit the growth of mouse tumors. In particular, the TNFR2 targeting anti-tumor active peptide P20, as an active ingredient, plays an inhibitory role in the growth of colon cancer (CT26, MC38).

[0056] At the same time, the inventors found that the combination of P20 and PD-L1 antibodies can enhance the efficacy of cancer immunotherapy, reduce the proportion of Treg in tumor tissue, and increase the proportion of CD8+IFNγ+ cells.

[0057] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0058] Example 1

[0059] This example is the preparation of TNFR2 targeting peptide, which includes:

[0060] 1. Screening of peptides using phage display peptide library

[0061] 1.1 Phage titer detection

[0062] 1) After the ER2738 strain was inoculated into LB medium (containing 30 μg / mL Tet antibiotics) for activation, it was inoculated into 5 mL of fresh LB medium (containing 30 μg / mL Tet antibiotics) at a ratio of 1:100 and cultured until OD600 was 0.4-0.6;

[0063] 2) Prepare agar plates: weigh 1.25 g IPTG and 1 g Xgal and dissolve in 25 mL DMF and store at -20°C until use;

[0064] 3) Add 15 g / L agar to 1L LB medium and 1 mL IPTG / Xgal stock solution to prepare plates and store at 4°C in the dark until use;

[0065] 4) Dilute the phage using LB culture according to the 10-fold gradient dilution method; divide the expanded host bacteria equally according to the needs, add 10 μL of the gradient phage diluted by LB to each tube, mix gently and incubate at room temperature for 30 minutes;

[0066] 5) Spread the infected host bacteria in step 4) on the plate prepared in step 3); culture overnight at 37°C;

[0067] 6) Plaque counting.

[0068] 1.2 Phage expansion culture

[0069] 1) After the ER2738 strain was inoculated into LB medium (containing 30 μg / mL Tet antibiotics) for activation, it was inoculated into 5 mL of fresh LB medium (containing 30 μg / mL Tet antibiotics) at a ratio of 1:100 and cultured until OD600 was 0.4-0.6;

[0070] 2) Take 10 μL of phage and inoculate it into the above-mentioned expanded host strain, shake and culture at 200 rpm and 37°C for 3-6 hours. Centrifuge at 8000 rpm to collect the supernatant, which is the phage culture.

[0071] 1.3 Host bacteria verification

[0072] 1) After the ER2738 strain was inoculated into LB medium (containing 30 μg / mL Tet antibiotics) for activation, it was inoculated into 5 mL of fresh LB medium (containing 30 μg / mL Tet antibiotics) at a ratio of 1:100 and cultured until OD600 was 0.4-0.6;

[0073] 2) Prepare agar plates: weigh 1.25 g IPTG and 1 g Xgal and dissolve in 25 mL DMF and store at -20°C until use;

[0074] 3) Add 15 g / L agar to 1L LB medium and 1 mL IPTG / Xgal stock solution to prepare plates and store at 4°C in the dark until use;

[0075] 4) Dilute the phage using LB culture according to the 10-fold gradient dilution method; divide the expanded host bacteria equally according to the needs, add 1 μL of the gradient phage diluted by LB to each tube, mix gently and incubate at room temperature for 30 minutes;

[0076] 5) Spread the infected host bacteria in step 4) on the plate prepared in step 3); culture overnight at 37°C and observe whether there are blue-white spots.

[0077] 1.4 Selection of recombinant phage

[0078] 1) Antigen coating: Dilute the purified TNFRSF1B recombinant protein to 4 μg / mL with PBS buffer, select 3 replicate wells in a 96-well ELISA plate, add 100 μL (400 ng / well) to each well, and coat overnight at 4°C. PBS is used as a negative control.

[0079] 2) Blocking: discard the coating solution, add 150 μL of 2% skim milk powder to each well, and block at room temperature for 1 hour;

[0080] 3) Incubate phages: Wash 4 times with PBST, take the phage solution prepared in 1.2, dilute to 5×10 11 PFU / mL, add to ELISA plate, 100 μL / well, incubate at room temperature for 2 h;

[0081] 4) Elution: discard the phage sample, wash with PBST 10 times, then wash with PBS 5 times, add 100 μL of freshly prepared 0.1 M triethylamine to each well, let stand at room temperature for 10 min, aspirate the eluate and quickly neutralize with an equal volume of 1 M Tris-HCl (pH 7.4);

[0082] 5) Determination of phage titer in the eluate: Determine the titer of the recombinant phage in the eluate according to the method in 1.1;

[0083] 6) Concentration and purification of phage particles: Follow the operation method in step 1.2 to concentrate and purify phage particles for the next round of screening;

[0084] 7) Repeat steps 1) to 6) twice to complete the second and third rounds of panning.

[0085] 1.5 Pick a single clone from the bacterial plate after the third round of screening, extract the plasmid after amplification and culture, and then sequence it.

[0086] 2. Fomc solid phase peptide synthesis targeting peptide

[0087] The first Fmoc-amino acid carboxyl group at the C-terminus of the candidate peptide is connected in the form of a covalent bond, and then the N-terminus of this amino acid is used as the starting point for the synthesis of the candidate peptide to dehydrate and condense with the carboxyl group of the second amino acid in the sequence to form a peptide bond. Then the Fmoc-amino acid protecting group at the N-terminus is deprotected, and then the N-terminus of the second amino acid is reacted with the carboxyl group of the following amino acid, and this process is repeated until the synthesis of the peptide is completed. Finally, the peptide is precipitated and washed to obtain the peptide. The molecular weight of the peptide identified by mass spectrometry is consistent with the theoretical molecular weight, and it is stored at -20°C.

[0088] Example 2

[0089] This example is a polypeptide-inhibited TNF-TNFR2 binding experiment in Jurkat cells that overexpress TNFR2, and the steps are as follows:

[0090] 1) Jurkat cells overexpressing TNFR2 were collected, centrifuged at 300 g for 5 minutes, the supernatant was removed, and the cells were resuspended in serum-free medium and diluted to adjust the cell concentration to 1×10 6 cells / mL;

[0091] 2) Plate 100 μL of cell suspension into a 96-well plate, add peptide / positive control / negative control and incubate the cells at 37°C for 1 hour, add biotin-TNF and continue incubation for 1 hour;

[0092] 3) Collect cells into flow cytometry tubes and centrifuge at 300g for 5 minutes to remove supernatant. After blocking for 30 minutes, add APC-Streptavidin for staining for 30 minutes, wash twice with PBS and then detect on the flow cytometer.

[0093] APC-Streptavidin fluorescence peak diagram Figure 1 As shown in C and D. ETA is Etanercept, which is used as a positive control.

[0094] like Figure 1As shown, 1-A is a Jurkat cell overexpressing TNFR2 constructed by lentiviral transfection; 1-B is a Jurkat cell overexpressing TNFR2 incubated with Biotin-TNF for 1 hour, and then APC-labeled streptavidin was added, and 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 intensity of the NC (negative control, WT Jurkat cells) group and the Jurkat cell group overexpressing TNFR2 (JK-R2); 1-E is the change in fluorescence intensity of Jurkat cells overexpressing TNFR2 after P20 treatment, and ETA (etanercept, fusion protein of soluble TNFR2 linked IgG1) is the positive control; 1-F is the observation of APC signal intensity on cells after treatment with different peptides.

[0095] pass Figure 1 It can be seen that the targeting peptide P20 can inhibit the binding effect of TNF-TNFR2.

[0096] Example 3

[0097] This example is about the effect of polypeptide on FOXP3 expression in vitro, and the steps are as follows:

[0098] 1) CD4+ T cells were purified from lymph nodes and spleen of wild-type C57BL / 6 mice by magnetic bead sorting technology and used Cell Trace TM The primary cells were stained with Violet, and 10 ng / mL of IL-2 and TNF were added to culture CD4+T cells in vitro.

[0099] 2) After treatment with different peptides for 72 hours, cells were collected and analyzed using LIVE / DEAD TM The cells are stained with dyes to distinguish between live and dead cells, analyzed by flow cytometry, and FACS antibodies are used to stain CD4, FOXP3 and other markers.

[0100] 3) Analyze the effect of peptide on FOXP3 expression in vitro. Figure 2 .

[0101] pass Figure 2 It can be concluded that cells cultured in the medium containing IL-2 (10 ng / mL) and TNF (10 ng / mL) can specifically stimulate the proliferation of Treg cells. Flow cytometry analysis showed that the proportion of Foxp3+ cells ( Figure 2-A). The positive drug ETA (Etanercept) and peptide 10μg / mL can significantly inhibit the increase of Treg cells induced by TNF ( Figure 2 -B, 2-C).

[0102] Example 4

[0103] This example is an in vivo experiment of targeting peptides in tumor-bearing mice, as follows:

[0104] In this example, CT26 colon cancer cells were selected and the cell concentration was adjusted to 5×10 6 After routine disinfection, 100 μL of cell suspension was inoculated subcutaneously in BALB / C mice. Seven days after inoculation, mice were intraperitoneally injected with PBS, 10 mg / kg P1, P19, and P20. The drugs were administered for a total of 12 days, and the growth of subcutaneous tumors was continuously observed. The experimental results are shown in Figure 3 .

[0105] BALB / C mice were subcutaneously inoculated with CT26 cells on the right back. When the tumor grew to 5-7 mm in diameter, peptides P01, P19 and P20 (10 mg / kg) were injected intraperitoneally. The specific experimental steps are shown in Figure 3 -A. From Figure 3 -B shows that P01, P19 and P20 can inhibit tumor growth, among which P19 and P20 can significantly inhibit tumor growth (*, P<0.05, **, P<0.01). Figure 3 -C As can be seen, there was no significant change in the body weight of mice.

[0106] In this example, MC38 colon cancer cells were selected and the cell concentration was adjusted to 5×10 6 After routine disinfection, 100 μL of cell suspension was inoculated subcutaneously in C57 mice. Seven days after inoculation, mice were intraperitoneally injected with PBS, PD-L1 antibody, P20, and PD-L1 antibody combined with P20. The drugs were administered for a total of 9 days, and the growth of subcutaneous tumors was continuously observed. The experimental results are shown in Figure 5 .

[0107] C57BL / 6 mice were subcutaneously inoculated with MC38 cells, and when the tumor grew to 5-7 mm, peptide P20 and anti-PD-L1 were injected intraperitoneally. Figure 5-A. As shown in Figure 5-B, both P20 and anti-PD-L1 can inhibit tumor growth, and the inhibitory effect of P20 combined with anti-PD-L1 is more significant; as shown in Figure 5-C, the combined treatment group significantly reduced the proportion of tumor-infiltrating Treg cells; as shown in Figure 5-D, P20, anti-PD-L1 and P20 combined with anti-PD-L1 can increase the expression of IFNγ in tumor-infiltrating CD8 cells, and the improvement effect of P20 combined with anti-PD-L1 is more significant.

[0108] Example 5

[0109] This example is the detection of immune cell infiltration in the mouse tumor microenvironment, as follows:

[0110] After the administration, the mice were euthanized, and the tumor tissues were removed and analyzed using gentleMACS TM The tumor tissue was digested and filtered through a 70 μm filter to obtain a single cell suspension. After washing twice with PBS, LIVE / DEAD TM After incubation with Fixable Near-IR Dead CellStain Kit dye for 30 minutes, resuspend the cells in PBS and wash twice. After fixing the cells with fixative for 12-18 hours, resuspend the cells in permeabilization buffer and wash twice. After blocking for 30 minutes, add CD45, CD3, CD4, CD8, and FOXP3 antibodies for staining for 30 minutes, wash twice with permeabilization buffer and then test on the machine. The ratio of CD4+FOXP3+ cells is as follows. Figure 4 shown.

[0111] from Figure 4 -A and 4-B, P20 significantly increased the proportion of CD8 cells in draining lymph nodes and increased the CD8 / CD4 ratio; Figure 4 -C and 4-D, it can be seen that P20 significantly reduced the proportion of CD4 cells in the tumor environment and significantly increased the CD8 / CD4 ratio; Figure 4 As can be seen from 4-E and 4-F, P19 and P20 significantly reduced the proportion of tumor-infiltrating Treg cells.

[0112] Take 5×10 6 The cells were used to detect the CD8+IFNγ+ ratio. After stimulation with Cell Stimulation Cocktail (plus protein transport inhibitors) (500X) for 6 hours, the staining was performed using the above staining method. The CD8+IFNγ+ cell ratio was, for example, Figure 5 shown.

[0113] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A TNFR2 targeting peptide with anti-tumor activity, characterized in that: The targeting peptide is: (1) P1, the amino acid sequence of which is shown in SEQ ID NO.1, or (2) P19, the amino acid sequence of which is shown in SEQ ID NO. 2, or (3) P20, the amino acid sequence of which is shown in SEQ ID NO.

3.

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

3. A recombinant vector comprising the gene according to claim 2.

4. A recombinant host cell comprising the recombinant vector according to claim 3.

5. Use of the TNFR2 targeting peptide as claimed in claim 1 in preparing fusion proteins, targeted drugs or targeted delivery systems.

6. The use according to claim 5, characterized in that: The fusion protein, targeted drug or targeted delivery system targets cells expressing TNFR2 protein; The cells expressing TNFR2 protein include tumor cells and immune cells.

7. Use of the TNFR2 targeting peptide as claimed in claim 1 in the preparation of a targeted drug for treating colon cancer.

8. Use of the TNFR2 targeting peptide as claimed in claim 1 in combination with a PD-L1 antibody in the preparation of a targeted drug for treating colon cancer.

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