CN113769063A

CN113769063A - Application of polypeptide PTPR in preparation of tumor immunotherapy medicine

Info

Publication number: CN113769063A
Application number: CN202111156751.9A
Authority: CN
Inventors: 林爱福; 石成瑜
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2021-09-30
Filing date: 2021-09-30
Publication date: 2021-12-10
Anticipated expiration: 2041-09-30
Also published as: CN113769063B

Abstract

The invention discloses an application of polypeptide PTPR in preparing a tumor immunotherapy medicament, which is a novel polypeptide designed by utilizing the principle of competitive inhibition on the basis of regulating and controlling an amino acid sequence of a PD-L1 stability functional segment by TMUB 1. The polypeptide can enhance the ubiquitination of PD-L1 in cells and promote the degradation and elimination of PD-L1; the invention inhibits the tumor cells from generating drug resistance to the antibody by inhibiting the stability regulation of TMUB1 on PD-L1, avoids the problems of prolonged administration time, reduced curative effect and the like, induces the PD-L1 protein to be degraded by proteasome so as to reduce the abundance of PD-L1 on the surface and in the tumor cells and fundamentally inhibits the function of the PD-L1 for promoting the escape of tumor immunity.

Description

Application of polypeptide PTPR in preparation of tumor immunotherapy medicine

Technical Field

The invention relates to the technical field of biological medicines, in particular to a PTPR polypeptide medicine for enhancing anti-tumor immunity of an organism to realize tumor treatment.

Background

The cancer treatment method comprises the following steps of traditional surgical excision, radiotherapy and chemotherapy, and also comprises the targeted treatment of drugs and immunotherapy. Although anti-tumor immunotherapy based on the binding of relevant molecules such as immunosuppressive agents to the human immune system to treat cancer has achieved a good effect in many cancers in recent years, the binding of tumor cells themselves to various immunosuppressive molecule receptors (e.g., PD1, TIM3, LAG3, etc.) on the surface of T cells by highly expressing inhibitory molecules of the anti-tumor immune response, such as PDL1, IDO, TGF β, etc., leads to the inhibition of tumor immune microenvironment and the proliferation and spread of cancer cells due to T cell depletion. This is a bottleneck in anti-cancer immunotherapy. The key to current anti-immunotherapy of cancer is therefore to find new inhibitors or antibodies of inhibitory molecules that block tumor immune responses efficiently and specifically. Thereby helping to recover the recognition of the tumor cells by immune cells and reducing the immune escape process of the tumor cells.

PD1(Programmed Death-1) is a type I transmembrane protein of CD28 family, is expressed on the surface of T cells, B cells and macrophages of human beings, and provides an inhibition signal for activated T cells; PD-L1(Programmed Death 1ligand1) is a ligand of PD1 and is a cell surface type I transmembrane protein of B7 family. As an important immunosuppressive molecule, PD-L1 is widely expressed in tumor cells, dendritic cells, macrophages, fibroblasts, and T cells in the tumor microenvironment. High expression PD-L1 in tumor tissue is combined with PD1 on the surface of immunocyte, so that the tumor immune reaction is inactivated, and the effect of immunotherapy is reduced. The PD-L1 related inhibitor can be used as a synergistic drug target of immune checkpoint blockade therapy, improve the activity of T cells in immune microenvironment and enhance anti-tumor immune response.

TMUB1(Transmembrane and Ubiquitin-Like Domain binding 1) is a Transmembrane and Ubiquitin-Like Domain protein, first reported in 2015, whose gene is located on human chromosome seven and encodes 245 amino acids. The TMUB1 protein contains a Ubiquitin (UBL) -like domain and three transmembrane domains, can shuttle between the nucleus and cytoplasm, and is involved in assembly formation of centrosomes and degradation of endoplasmic reticulum-associated proteins. The invention discovers that TMUB1 can be competitively combined with protein promoting PD-L1 ubiquitination, thereby stabilizing PD-L1 at the protein level and continuously inhibiting tumor immune response reaction. Based on the theory, the invention develops a polypeptide molecule targeting TMUB1, which can be competitively combined with PD-L1, weaken the stability of PD-L1, enable immune cells to be normally activated, specifically recognize and kill tumor cells.

Disclosure of Invention

The invention aims to provide application of a competitive polypeptide PTPR of TMUB1 protein in preparing a tumor immunotherapy medicament, and aims to reduce accumulation of immune checkpoint protein molecules PD-L1 in tumor cells and enhance anti-tumor immunity of an organism by competitively combining the polypeptide PTPR and the TMUB1 with the PD-L1 on the basis that the TMUB1 can competitively combine with the protein promoting ubiquitination of PD-L1.

The technical scheme adopted by the invention is as follows:

the invention provides an application of a competitive polypeptide PTPR of TMUB1 protein in preparing a tumor immunotherapy medicament based on an interaction domain of TMUB1 and PD-L1, wherein the polypeptide PTPR comprises one or more of the following components:

(a) and the amino acid sequence is SEQ ID NO. 1: GFTATPPAPDSPQEP;

(b) a polypeptide having 90% or more identity to the amino acid sequence shown in (a);

(c) the polypeptide comprises an amino acid sequence obtained by one or more of substitution, deletion and insertion of one or more amino acids of the amino acid sequence shown in the (a);

(d) and (b) a polypeptide comprising an amino acid sequence which is modified by one or more of acetylation, phosphorylation, glycosylation, succinylation and ubiquitination of the amino acid sequence shown in (a).

The TMUB1 protein has the Gene ID: 83590 or its homologous sequence, the nucleotide sequence is shown in SEQ ID NO.2, and the amino acid sequence is shown in SEQ ID NO. 3.

The polypeptide PTPR is a chemically modified polypeptide PTPR and is used for enhancing the stability of the polypeptide and realizing intracellular tracing, and the polypeptide PTPR comprises but is not limited to: cyclization, N-methylation, phosphorylation, myristoylation, palmitoylation, glycosylation, prenylation, polyethylene glycol modification, fluorescent labeling, biotinylation, and the like. FITC fluorescent labels are preferred.

The tumor is selected from: lung cancer (such as non-small cell lung cancer), melanoma, breast cancer, ovarian cancer, prostate cancer, liver cancer, renal cancer, intestinal cancer, head and neck cancer, skin cancer, bladder cancer, and pancreatic cancer.

The medicament is a PD-L1 inhibitor, and the preparation form of the medicament comprises: solutions, emulsions, suspensions or injections.

The medicament may be used in combination with medicaments or therapies known in the art for preventing and/or treating tumor immune tolerance in a subject. The combination comprises: simultaneous, sequential, separate or separate administration of the substances or products of the invention and other known drugs or therapies

In view of the present disclosure, those skilled in the art can freely combine the foregoing technical solutions and features without departing from the spirit and scope of the present invention.

Compared with the prior art, the invention has the advantages that:

(1) the invention relates to a novel polypeptide designed by utilizing the principle of competitive inhibition on the basis of the amino acid sequence of a PD-L1 stability functional segment regulated and controlled by TMUB 1. The polypeptide can enhance the ubiquitination of PD-L1 in cells and promote the degradation and elimination of PD-L1;

(2) the invention inhibits the tumor cells from generating drug resistance to the antibody by inhibiting the stability regulation of TMUB1 on PD-L1, avoids the problems of prolonged administration time, reduced curative effect and the like, induces the PD-L1 protein to be degraded by proteasome so as to reduce the abundance of PD-L1 on the surface and in the tumor cells and fundamentally inhibits the function of the PD-L1 for promoting the escape of tumor immunity.

Drawings

FIG. 1 is the mRNA expression level of TMUB1 in 2 breast cancer cells after targeted knockdown of TMUB1 in example 1.

FIG. 2 is a gel diagram of the protein content of PD-L1, TMUB1, VCL after targeted knockdown of TMUB1 in 2 breast cancer cells of example 1.

FIG. 3 is the amino acid sequence diagram (A) and gel diagram (B, C) of the functional segment of PD-L1 regulated by TMUB1 in example 2; WT in A refers to Wild Type TMUB1 protein, Δ MU1 refers to TMUB1 protein with truncated MU1 segment, Δ UBL refers to TMUB1 protein with truncated UBL segment, Δ MU2 refers to TMUB1 protein with truncated MU2 segment, and Δ TM1 refers to TMUB1 protein with truncated TM1 segment; TM1 refers to position 11 to position 31 in the amino acid sequence of TMUB1, MU1 refers to position 32 to position 102 in the amino acid sequence of TMUB1, UBL refers to position 103 to position 176 in the amino acid sequence of TMUB1, TM2 refers to position 195 to position 215 in the amino acid sequence of TMUB1, and TM3 refers to position 222 to position 241 in the amino acid sequence of TMUB 1; MU2 refers to amino acid sequence from 177 to 194, 216 to 221, 242 to 246 of TMUB 1; the plasmid vector in B is a PCDNA3.1 plasmid vector without an insert, TMUB1-Flag is TMUB1 full-length protein with a Flag tag at the C end, MU1-Flag truncation is that TMUB1 truncates an MU1 region, UBL-Flag truncation is that TMUB1 truncates a BUL region, MU2-Flag truncation is that TMUB1 truncates an MU2 region, and TM2-Flag truncation is that TMUB1 truncates a TM2 region; the plasmid vector in C is a PCDNA3.1 plasmid vector without an insert, TMUB1-Flag is TMUB1 full-length protein with a Flag tag at the C end, and the truncations 32-45, 46-59, 60-73, 74-87 and 88-102 are TMUB1 truncated protein expression plasmids which respectively truncate the corresponding positions of an MU1 region.

FIG. 4 is a schematic amino acid sequence of PTPR-FITC in example 2.

FIG. 5 is a confocal fluorescence micrograph of PTPR-FITC entering tumor cells in example 2.

FIG. 6 is a gel image of PTPR-FITC incubated with MDA-MB-231 human breast cancer cells in example 2.

FIG. 7 is a photograph (A) of PTPR-FITC co-cultured with T cells and a bar graph (B) of tumor cell survival rate in example 3.

FIG. 8 is a statistical graph of the effect of PTPR-FITC on breast tumor volume growth in mice in example 4.

FIG. 9 is a bar graph of the PD-L1 protein level (A) in tumor cells of each treatment group, the CD8 positive cell fraction in tumor infiltrating lymphocytes at CD3 positive cells (B) and the GzmB positive cell fraction in tumor infiltrating lymphocytes at CD3, CD8 double positive cells (C) in the PTPR in vivo test in example 4.

FIG. 10 is a statistical picture of the prolongation of survival of tumor-bearing mice in the PTPR in vivo assay of example 4.

FIG. 11 is a graphical representation of the status of mice observed as endpoints in the toxicity assessment assay for PTPR in example 5.

Detailed Description

The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:

the conception, the specific structural steps and the technical effects produced by the present invention will be further described with reference to the accompanying drawings so as to fully understand the objects, the features and the effects of the present invention.

Example 1 cellular level functional validation of PTPR peptides

1. TMUB1 small molecule RNA inhibitors

According to a TMUB1 Gene nucleotide sequence (Gene ID: 83590, http:// siderect2. rnai. jp/, a nucleotide sequence is shown as SEQ ID NO.2, and an amino acid sequence is shown as SEQ ID NO.3), two cholesterol-modified small-molecule RNA nucleotide sequences are obtained according to a base complementary pairing principle and an energy minimum principle, wherein the two cholesterol-modified small-molecule RNA nucleotide sequences are respectively a TMUB1 sequence 1 and a TMUB1 sequence 2, and the two cholesterol-modified small-molecule RNA nucleotide sequences are the TMUB1 small-molecule RNA inhibitor.

TMUB1 gene sequence (SEQ ID NO. 2): atga ccctgattgaaggggtgggt gatgaggtga ccgtcctttt ctcggtgctt gcctgccttc tggtgctggcccttgcctgg gtctcaacgc acaccgctga gggcggggac ccactgcccc agccgtcagggaccccaacg ccatcccagc ccagcgcagc catggcagct accgacagca tgagaggggaggccccaggg gcagagaccc ccagcctgag acacagaggt caagctgcac agccagagcccagcacgggg ttcacagcaa caccgccagc cccggactcc ccgcaggagc ccctcgtgctacggctgaaa ttcctcaatg attcagagca ggtggccagg gcctggcccc acgacaccattggctccttg aaaaggaccc agtttcccgg ccgggaacag caggtgcgac tcatctaccaagggcagctg ctaggcgacg acacccagac cctgggcagc cttcacctcc ctcccaactgcgttctccac tgccacgtgt ccacgagagt cggtccccca aatcccccct gcccgccggggtccgagccc ggcccctccg ggctggaaat cggcagcctg ctgctgcccc tgctgctcctgctgttgctg ctgctctggt actgccagat ccagtaccgg cccttctttc ccctgaccgccactctgggc ctggccggct tcaccctgct cctcagtctc ctggcctttg ccatgtaccgcccgtag are provided.

TMUB1 amino acid sequence (SEQ ID NO. 3):

MTLIEGVGDEVTVLFSVLACLLVLALAWVSTHTAEGGDPLPQPSGTPTPSQPS AAMAATDSMRGEAPGAETPSLRHRGQAAQPEPSTGFTATPPAPDSPQEPLVLRLKFL NDSEQVARAWPHDTIGSLKRTQFPGREQQVRLIYQGQLLGDDTQTLGSLHLPPNCV LHCHVSTRVGPPNPPCPPGSEPGPSGLEIGSLLLPLLLLLLLLLWYCQIQYRPFFPLTA TLGLAGFTLLLSLLAFAMYRP。

TMUB1 sequence 1(SEQ ID NO. 4): 5'-GGCUGAAAUUCCUCAAUGA-3',

TMUB1 sequence 2(SEQ ID NO. 5): 5'-CUACGGCUGAAAUUCCUCA-3' are provided.

Nonsense targeting sequence: 5'-UUCUCCGAACGUGUCACGU-3' are provided.

2. Tumor cells

Human breast cancer cell lines MDA-MB-231 and MDA-MB-468 were purchased from American Standard cell culture center (ATCC) using DMEM/F12 medium (Gibco) containing 10% FBS (Bovogen), 37 ℃, 5% CO₂Culturing in an incubator, and detecting cells by a MycoAlert kit (Lonza) without mycoplasma pollution and cross contamination.

3. Construction of cell lines with a specifically knockdown of TMUB1

Plasmid transfection was performed using the TMUB1 small RNA inhibitor prepared above, according to Lipofectamine2000(ThermoScientific) reagent instructions, specifically: according to the ViraPowerTMLentiviral expression System (Thermo Scientific) method, the TMUB1 sequence 1, the TMUB1 sequence 2 and the nonsense targeting sequence are respectively transfected into a human breast cancer cell line MDA-MB-231, two strains of TMUB1 specifically-knocked-down cell strains are respectively marked as targeting knocking-down #1 and targeting knocking-down #2, and one cell strain with the nonsense targeting sequence is marked as nonsense targeting knocking-down contrast.

Under the same conditions, the human breast cancer cell line MDA-MB-231 was replaced with the human breast cancer cell line MDA-MB-468.

4. QPCR detection

The expression condition of TMUB1 mRNA in cells is detected by QPCR, and figure 1 shows that the TMUB1 small-molecule RNA inhibitor has good targeting effect and effectively reduces the mRNA expression in the cells.

5. Western blot detection

The levels of PD-L1 protein and TMUB1 protein, as well as the reference protein VCL, were examined in control as well as targeted knockdown 1# and targeted knockdown 2# cells by western blots.

The results in fig. 2 show that the targeted knockdown of TMUB1 in MDA-MB-231 and MDA-MB-468 cell lines, PD-L1 protein levels were significantly reduced compared to controls. This example demonstrates that knock-down of TMUB1 can significantly inhibit PD-L1 protein stability at the cellular level.

Example 2 design of the TMUB1 competitive polypeptide PTPR and its entry into cells and effect on PD-L1 expression.

1. Design of TMUB1 competitive polypeptide PTPR by utilizing competitive inhibition principle

(1) Construction of Flag-tagged TMUB1 truncated protein

TMUB1 protein (Wild Type) amino acid sequence composition (a in fig. 3): the 11 th to 31 th bits are TM1, the 32 th to 102 th bits are MU1, the 103 th to 176 th bits are UBL, the 177 th to 194 th bits, the 216 th to 221 th bits, the 242 th to 246 th bits are MU2, the 195 th to 215 th bits are TM2, and the 222 th to 241 th bits are TM 3.

In view of the important regulatory role of TMUB1 protein (Wild Type) on PD-L1 stability and amino acid sequence characteristics, we truncated a portion of the amino acid sequence in TMUB1 protein (Wild Type) as follows:

the nucleotide sequences of translated MU1, UBL, MU2 and TM1 in TMUB1 are respectively truncated to obtain a TMUB1 nucleotide truncated sequence delta MU1, delta UBL, delta MU2 and delta TM1, and a Flag tag is added to the C end of each TMUB1 truncated nucleotide sequence to construct a Flag tag-carrying TMUB1 truncated protein PCDNA3.1 vector expression plasmid.

Meanwhile, the C end of TMUB1 is added with a Flag tag and transferred into a PCDNA3.1 plasmid vector to construct TMUB 1-Flag. The PCDNA3.1 plasmid vector was used as a control.

(2) TMUB1 truncated protein functional segment screening

And (2) respectively carrying out the steps of (1) and mixing the Flag-tagged TMUB1 truncated protein expression plasmid and the HA-tagged PD-L1 protein PCDNA3.1 vector expression plasmid in a ratio of 1: 1, and after incubation for 36 hours, using Anti-Flag magnetic beads for co-immunoprecipitation, the binding and status of the different TMUB1 truncated proteins to PD-L1 were examined to determine the functional segment of TMUB1 (B in fig. 3) that regulates the stability of PD-L1, and as shown in B in fig. 3, the truncated protein of TMUB1 that truncates the MU1 segment lost the binding and regulating functions to PD-L1.

(3) MU1 region truncation selection of TMUB1

Truncating the MU1 region of TMUB1 by using the method in the step (1) and (2) to generate TMUB1 truncated protein expression plasmids of delta 32-45, delta 46-59, delta 60-73, delta 74-87 and delta 88-102 with C-terminal Flag tags, and combining the TMUB1 truncated protein expression plasmid with a PD-L1-HA expression plasmid in a ratio of 1: 1, and after incubation for 36 hours and immunoprecipitation using Anti-Flag magnetic beads, as shown by C in fig. 3, the truncated protein of TMUB1 truncated at amino acids 88-102 (sequence glyphe thralathrapropro pro alapro pro sper gln glupro, amino acid sequence shown in SEQ ID No. 1) failed to bind to PD-L1 and regulated PD-L1 concentration, indicating that 88-102 is the binding and functional segment of TMUB1 regulating PD-L1.

Therefore, the 88 th-102 th short peptide of TMUB1 is polypeptide PTPR, which is used as a "bait" for competitive binding with PD-L1 and is modified by a fluorescent group FITC for intracellular tracing, and is named polypeptide FITC-PTPR (FIG. 4). The design utilizes the principle of competitive inhibition to weaken the combination of TMUB1 and PD-L1 in cells and reduce the expression of endogenous PD-L1.

2.2 demonstration that the polypeptide FITC-PTPR can enter tumor cells to attenuate the abundance of intracellular PD-L1

A human breast cancer cell line MDA-MB-231 was inoculated at 2 x10 ^5 into a 24-well plate containing 1ml of DMEM/F12 medium containing 10% FBS (Bovogen) by volume, after the cells were attached, FITC-PTPR solution (solvent phosphate buffered saline, PBS) synthesized in vitro was added thereto at 10. mu.M concentration, incubated at 37 ℃ for 12 hours, and then PTPR peptides which failed to enter the cells were eliminated by proteinase K to prepare a fluorescent slide. The entry of the polypeptide FITC-PTPR into the cells was detected by confocal fluorescence microscopy. As shown in fig. 5, PTPR can enter cells directly.

Three identical human breast cancer cell lines MDA-MB-231 were inoculated in amounts of 5X 10^6 into 10cm dishes supplemented with 10mL of DMEM/F12 medium containing 10% FBS (Bovogen) by volume, and the following treatments were performed after the cells adhered: one was not treated at all (FIG. 6, column 1), one was added with 1. mu.L of dimethyl sulfoxide as a control (FIG. 6, column 2), and one was added with a 10. mu.M final concentration of the polypeptide FITC-PTPR in FBS (FIG. 6, column 3), and after incubation at 37 ℃ for 12 hours, the expression of PD-L1 protein was detected in a gel electrophoresis immunoblot using a PD-L1-specific antibody. As shown in FIG. 6, the addition of the polypeptide FITC-PTPR obviously reduced the expression of PD-L1 in human breast cancer cells, while dimethyl sulfoxide serving as a solvent control did not have the function, which indicates that the polypeptide FITC-PTPR polypeptide inhibits PD-L1 through competition, so that the protein expression level of PD-L1 is reduced.

Example 3 attenuation of tumor cell immune escape Using FITC-PTPR to enhance T cell killing of tumor cells

In view of the inhibitory effect that FITC-PTPR exhibits on the stability of PD-L1 at the cellular level, FITC-PTPR should have a significant inhibitory effect on tumor immune escape.

Human peripheral blood mononuclear cells PBMC were purchased from American Standard cell culture center (ATCC) using RMPI-1640 medium (Gibco) containing 10% FBS (Bovogen), 37 ℃, 5% CO₂Culturing in an incubator, and detecting cells by a MycoAlert kit (Lonza) without mycoplasma pollution and cross contamination.

PBMCs were activated as cytotoxic T cells according to human CD3/CD28/CD 2T cell activators (STEMCELL Technologies) using instructions. Human breast cancer cell line MDA-MB-231 was inoculated in an amount of 2 x10 ^5 into 6-well plates supplemented with 3ml of 10% by volume FBS (Bovogen) DMEM/F12 medium at 37 ℃ with 5% CO₂The incubator was 12h, divided into 4 groups, group 1 and group 2 were not treated, and 3x10 ^6 amounts of cytotoxic T cells were added to each of group 3 and group 4, and each incubation was continued for 24 h. Groups 1 and 3 were added with FITC-PTPR in PBS at a final concentration of 10. mu.M, and incubation was continued for 12 hours, and tumor cell survival was examined by crystal violet staining. As shown in FIG. 7, FITC-PTPR treatment did not affect tumor cell survival in the control group (group 1 vs group 3), whereas the survival number of tumor cells in the PBMC co-cultured groups (group 3 and group 4) was significantly less than that in the control group (group 1, group 2 vs group 3, group 4), and that in the FITC-PTPR-treated group (group 3 vs group 4), indicating that FITC-PTPR enhanced the immune escape of tumor cells by attenuating the expression level of PD-L1, enhancing T cell-to-tumor cell enhancement by attenuating the immune escape of tumor cellsKilling of the cells.

Example 4 verification of the function of FITC-PTPR mouse tumor model

4.1 establishment of mouse tumor model

12 Balb/c mice were purchased, and the Balb/c mice were injected subcutaneously with 4T1 mouse breast cancer cells 3x10 using a 4T1 mouse breast cancer cell line⁶On the third day after the injection of the breast cancer cell 4T1, a tumor visible to the touch appeared subcutaneously, and a mouse tumor model was established to verify the therapeutic effect of the above FITC-PTPR on breast cancer.

The major diameter (mm) and the minor diameter (mm) of the tumor generated in each mouse were measured by a vernier caliper, and the tumor volume (mm) were calculated by the following formula³) Long diameter (mm) x short diameter (mm) 0.5x²The size of the tumor to be detected is about 50mm³The treatment of medication is carried out.

4.2 grouping and administration methods

The size of the tumor is about 50mm³The mice were randomly divided into 2 groups of 6 mice each, and then were individually labeled and photographed, and the tumor size before and after the drug administration was recorded. Each group of mice was treated in the manner and amount shown in Table 1, in which the mice numbered 1 were treated with the drug using physiological saline, and the mice numbered 2 were treated with the drug using FITC-PTPR dissolved in physiological saline, and administered once a day.

TABLE 1

Numbering	Mode of administration	Dosage of medicine
			1	Intratumoral injection	0.9% of normal saline per doseAdministered 10 times with a dose of 100 μ l
2	Intratumoral injection	10mg/ml FITC-PTPR solution 100 μ l each time, 10 times

4.3 Observation

Each group of mice was photographed 3 days after drug treatment, and the tumor size of each mouse after drug treatment was compared. 18 days after tumor loading, mice in groups 1 and 2 were sacrificed and the status of CD3+ CD8+ Gzmb + positive T cells and tumor PD-L1 expression in tumor infiltrating lymphocytes were examined. Mice in groups numbered 3, 4 were housed until 42 days post tumor-bearing or dead, and the time to death of each mouse in each group was recorded.

4.4 results of the experiment

Fig. 8 and 9 show experimental results of the drug solution treatment of the mouse PTPR polypeptides numbered 1 and 2, respectively, and it can be seen from fig. 8 that the use of the PTPR peptides significantly delays the growth of tumors; as can be seen in FIG. 9, the use of PTPR reduced PD-L1 expression in tumor cells and increased CD3 in mouse tumor-infiltrating lymphocytes⁺CD8⁺GzmB⁺Cell number, indicating that the use of PTPR peptides enhances the body's anti-tumor immunity. Fig. 10 shows the survival of the drug solution treated PTPR polypeptides of mice nos. 3 and 4, and it can be seen from fig. 10 that the use of PTPR peptides significantly increases the survival time of tumor-bearing mice.

Example 5 evaluation of in vivo toxicity of PTPR peptides

5.1 grouping and methods of administration

12 Balb/c mice aged 4 weeks were purchased and randomly divided into 4 groups of 3 mice each treated according to the administration method and the amount shown in Table 2, wherein the mice numbered 1 were treated with physiological saline, the mice numbered 2 were treated with 100mg/kg of FITC-PTPR physiological saline, the mice numbered 3 were treated with 200mg/kg of FITC-PTPR physiological saline, and the mice numbered 4 were treated with 500mg/kg of FITC-PTPR physiological saline.

TABLE 2 PTPR in vivo toxicity assessment dosing regimen and dosage

Numbering	Mode of administration	Dosage of medicine
			1	Abdominal injection	0.9% physiological saline, administered on the first and seventh days
2	Abdominal injection	The dosage is 100mg/kg, and the dosage is administered on the first day and the seventh day
			3	Abdominal injection	The dosage is 200mg/kg, and the dosage is administered on the first day and the seventh day
4	Abdominal injection	The drug is administered at a dose of 500mg/kg, the drug is administered on the first day and the seventh day

5.2 Observation

Taking the first administration as an observation starting point and taking 14 days after the first administration as an observation end point, photographing and recording the activity and the appearance state of the mouse, and recording the survival condition of the mouse.

5.3 results of the experiment

Figure 11 shows the appearance and survival of representative mice in each group at the observation endpoint. As can be seen in fig. 11, all mice survived the high dose PTPR treatment and were normal in appearance and activity, indicating that PTPR was not significantly toxic.

The foregoing detailed description of the preferred embodiments of the invention has been presented. It should be understood that numerous modifications and variations could be devised by those skilled in the art in light of the present teachings without departing from the inventive concepts. Therefore, the technical solutions available to those skilled in the art through logic analysis, reasoning and limited experiments based on the prior art according to the concept of the present invention should be within the scope of protection defined by the claims.

Sequence listing

<110> Zhejiang university

<120> application of polypeptide PTPR in preparation of tumor immunotherapy drug

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<210> 1

<211> 15

<212> PRT

<213> Unknown (Unknown)

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Gly Phe Thr Ala Thr Pro Pro Ala Pro Asp Ser Pro Gln Glu Pro

1 5 10 15

<210> 2

<211> 741

<212> DNA

<213> Unknown (Unknown)

<400> 2

atgaccctga ttgaaggggt gggtgatgag gtgaccgtcc ttttctcggt gcttgcctgc 60

cttctggtgc tggcccttgc ctgggtctca acgcacaccg ctgagggcgg ggacccactg 120

ccccagccgt cagggacccc aacgccatcc cagcccagcg cagccatggc agctaccgac 180

agcatgagag gggaggcccc aggggcagag acccccagcc tgagacacag aggtcaagct 240

gcacagccag agcccagcac ggggttcaca gcaacaccgc cagccccgga ctccccgcag 300

gagcccctcg tgctacggct gaaattcctc aatgattcag agcaggtggc cagggcctgg 360

ccccacgaca ccattggctc cttgaaaagg acccagtttc ccggccggga acagcaggtg 420

cgactcatct accaagggca gctgctaggc gacgacaccc agaccctggg cagccttcac 480

ctccctccca actgcgttct ccactgccac gtgtccacga gagtcggtcc cccaaatccc 540

ccctgcccgc cggggtccga gcccggcccc tccgggctgg aaatcggcag cctgctgctg 600

cccctgctgc tcctgctgtt gctgctgctc tggtactgcc agatccagta ccggcccttc 660

tttcccctga ccgccactct gggcctggcc ggcttcaccc tgctcctcag tctcctggcc 720

tttgccatgt accgcccgta g 741

<210> 3

<211> 246

<212> PRT

<213> Unknown (Unknown)

<400> 3

Met Thr Leu Ile Glu Gly Val Gly Asp Glu Val Thr Val Leu Phe Ser

1 5 10 15

Val Leu Ala Cys Leu Leu Val Leu Ala Leu Ala Trp Val Ser Thr His

20 25 30

Thr Ala Glu Gly Gly Asp Pro Leu Pro Gln Pro Ser Gly Thr Pro Thr

35 40 45

Pro Ser Gln Pro Ser Ala Ala Met Ala Ala Thr Asp Ser Met Arg Gly

50 55 60

Glu Ala Pro Gly Ala Glu Thr Pro Ser Leu Arg His Arg Gly Gln Ala

65 70 75 80

Ala Gln Pro Glu Pro Ser Thr Gly Phe Thr Ala Thr Pro Pro Ala Pro

85 90 95

Asp Ser Pro Gln Glu Pro Leu Val Leu Arg Leu Lys Phe Leu Asn Asp

100 105 110

Ser Glu Gln Val Ala Arg Ala Trp Pro His Asp Thr Ile Gly Ser Leu

115 120 125

Lys Arg Thr Gln Phe Pro Gly Arg Glu Gln Gln Val Arg Leu Ile Tyr

130 135 140

Gln Gly Gln Leu Leu Gly Asp Asp Thr Gln Thr Leu Gly Ser Leu His

145 150 155 160

Leu Pro Pro Asn Cys Val Leu His Cys His Val Ser Thr Arg Val Gly

165 170 175

Pro Pro Asn Pro Pro Cys Pro Pro Gly Ser Glu Pro Gly Pro Ser Gly

180 185 190

Leu Glu Ile Gly Ser Leu Leu Leu Pro Leu Leu Leu Leu Leu Leu Leu

195 200 205

Leu Leu Trp Tyr Cys Gln Ile Gln Tyr Arg Pro Phe Phe Pro Leu Thr

210 215 220

Ala Thr Leu Gly Leu Ala Gly Phe Thr Leu Leu Leu Ser Leu Leu Ala

225 230 235 240

Phe Ala Met Tyr Arg Pro

245

<210> 4

<211> 19

<212> RNA

<213> Unknown (Unknown)

<400> 4

ggcugaaauu ccucaauga 19

<210> 5

<211> 19

<212> RNA

<213> Unknown (Unknown)

<400> 5

cuacggcuga aauuccuca 19

<210> 6

<211> 19

<212> RNA

<213> Unknown (Unknown)

<400> 6

uucuccgaac gugucacgu 19

Claims

1. The application of the polypeptide PTPR in preparing the tumor immunotherapy medicine is characterized in that the polypeptide PTPR comprises one or more of the following components:

(a) and the amino acid sequence is SEQ ID NO. 1: GFTATPPAPDSPQEP;

2. Use according to claim 1, characterized in that the polypeptide PTPR amino acid sequence is as shown in SEQ ID No. 1.

3. Use according to claim 1, characterized in that the polypeptide PTPR is a chemically modified polypeptide PTPR.

4. The use according to claim 3, wherein said chemical modification comprises: cyclization, N-methylation, phosphorylation, myristoylation, palmitoylation, glycosylation, prenylation, polyethylene glycol modification, fluorescent labeling, biotinylation.

5. The use according to claim 1, wherein said tumor is selected from the group consisting of: lung cancer, melanoma, breast cancer, ovarian cancer, prostate cancer, liver cancer, renal cancer, intestinal cancer, head and neck cancer, skin cancer, bladder cancer, and pancreatic cancer.

6. Use according to claim 1, characterized in that the medicament is a PD-L1 inhibitor.

7. The use according to claim 1, wherein the medicament is in a formulation comprising: solutions, emulsions, suspensions or injections.

8. Use according to claim 1, characterized in that the medicament is used in combination with an immune-tolerizing medicament or therapy for the prevention and/or treatment of tumors.