CN115925815A

CN115925815A - PAK 4-targeting polypeptide, application thereof and medicine

Info

Publication number: CN115925815A
Application number: CN202211410760.0A
Authority: CN
Inventors: 徐珊; 马伯涵
Original assignee: First Affiliated Hospital of Medical College of Xian Jiaotong University
Current assignee: First Affiliated Hospital of Medical College of Xian Jiaotong University
Priority date: 2022-11-11
Filing date: 2022-11-11
Publication date: 2023-04-07

Abstract

The invention provides a PAK 4-targeted polypeptide, and application and a medicament thereof, and belongs to the technical field of biological medicines. The amino acid sequence of the polypeptide is shown as SEQ ID No.1, the polypeptide has stronger binding capacity with p21 activated kinase 4 protein, the polypeptide is delivered to tumors, the proliferation of cancer cells can be effectively inhibited, and meanwhile, the polypeptide has the capacity of targeted degradation of p21 activated kinase 4. Compared with the existing p21 activated kinase 4 inhibitor, the PAK 4-targeted polypeptide drug can provide a new treatment strategy for treating the middle-high risk tumor, namely, the combination of the PD-1 antibody, and particularly can overcome the tumor patients with drug resistance of the PD-1 antibody.

Description

PAK 4-targeting polypeptide, application thereof and medicine

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a PAK 4-targeted polypeptide, and application and a medicament thereof.

Background

Tumors, especially malignant tumors, are the first major diseases threatening human health and life, and are the troublesome problems facing modern medicine. Early stage tumors can be removed by surgery, and high-risk metastatic tumors lose the chance of surgery. Compared with a single targeting medicament, the immunosuppressive agent and the targeting medicament improve the overall survival time, the objective effective rate and the progression-free survival time of the patients with the high-risk metastatic tumors. Unfortunately, up to 70% of patients still do not respond to immunosuppressant therapy.

p 21-activated kinase 4 (PAK 4) is a conserved serine/threonine protein kinase, an effector protein of the small gtpases CDC42 and Rac1 in the Rho family, that mediates transduction of its downstream signaling pathway. PAK4 is a member of PAKs, and when PAK4 is abnormally expressed, cells become cancerous. Wang C and other researches show that the expression content of PAK4 in lung cancer, colon cancer, prostatic cancer, pancreatic cancer and breast cancer cells is far higher than that of normal cells, and the PAK4 has important influence on the occurrence, development, invasion and migration of tumors. Cabriel et al found that in patients who did not respond to immunotherapy, the oncogene PAK4 was highly expressed with a concomitant lack of infiltration of immune cells (T cells and dendritic cells). Gene knockout or inhibition of PAK4 can increase the proportion of tumor-specific T cells infiltrating in tumor tissue, thereby making tumors more sensitive to PD-1 blocking therapy. Therefore, the development of PAK4 inhibitors is one of the effective strategies for the treatment of various tumors. KPT-9274 entering phase I clinical trial at present is also directed against PAK family, and no medicine targeting PAK4 degradation is used.

Disclosure of Invention

In view of this, the present invention aims to provide a PAK 4-targeting polypeptide, and applications and drugs thereof, wherein the polypeptide has high binding capacity with p 21-activated kinase 4 protein, effectively degrades p 21-activated kinase 4, significantly inhibits cancer cell proliferation, and can be used for preparing drugs for treating cancer.

In order to achieve the above purpose, the invention provides the following technical scheme:

the invention provides a polypeptide of a targeted p21 activated kinase 4, and the amino acid sequence of the polypeptide is shown as SEQ ID No. 1.

The invention also provides an application of the polypeptide in preparing a medicament for preventing and/or treating tumors.

Preferably, the polypeptide has a binding constant of 456.6nM to the p 21-activated kinase 4 protein.

Preferably, the tumor comprises one or more of renal cancer, lung cancer, colon cancer, prostate cancer, pancreatic cancer, and breast cancer.

The invention also provides application of the polypeptide in preparing a reagent for degrading p21 activated kinase 4 protein.

The invention also provides a medicament for preventing and/or treating tumors, which comprises the polypeptide.

The invention also provides an application of the combined use of the polypeptide and the PD-1 antibody in the preparation of drugs for preventing and/or treating tumors.

The invention also provides a pharmaceutical composition for preventing and/or treating tumors, which comprises the polypeptide and the PD-1 antibody.

Preferably, the above drug or pharmaceutical composition further comprises a delivery system, wherein the delivery system comprises nanogold, liposome or nano selenium.

Compared with the prior art, the invention has the following beneficial effects:

the amino acid sequence of the polypeptide of the invention is shown in SEQ ID No.1, and the polypeptide targets p21 activated kinase 4. According to the invention, isothermal calorimetry titration experiments are adopted to respectively detect the binding capacity of the polypeptide and the p21 activated kinase 4 protein, and the binding constant of the polypeptide and the p21 activated kinase 4 protein is 456.6nM, which shows that the polypeptide and the p21 activated kinase 4 protein have stronger binding capacity. The polypeptide is delivered to cancer cells, and the proliferation of the cancer cells can be effectively inhibited. Therefore, the polypeptide can be applied to the preparation of medicaments for preventing and/or treating tumors or the preparation of reagents for degrading p21 activated kinase 4 protein.

The invention provides a drug or a drug composition for preventing and/or treating tumors, wherein the polypeptide is combined with a PD-1 antibody for use, so that the survival cycle of a cancer mouse and the infiltration proportion of immune T cells in tumor tissues are obviously improved, and the synergistic effect is realized. Compared with the existing p21 activated kinase 4 targeted drug, the drug or the drug composition can provide a new treatment strategy for drug resistance and treatment of middle and late stage tumors, particularly can solve the problem of PD-1 insensitive or PD-1 drug resistant cancer patients, and fills the blank of the drug for targeted degradation of the polypeptide of the targeted p21 activated kinase 4 in the global scope.

Drawings

FIG. 1 shows the result of designing a polypeptide targeting p21 activated kinase 4 provided by the present invention, wherein A is the structure of a complex of a polypeptide targeting p21 activated kinase 4 and a protein targeting p21 activated kinase 4; b and C are the result of protein-aided design of the PpD drug polypeptide; d is the detection result of the affinity of the PpD drug polypeptide with p21 activated kinase 4 and MDM2 protein respectively; e is a trimer detection result formed by inducing p21 to activate kinase 4 and MDM2 protein by PpD drug polypeptide;

FIG. 2 shows the results of the effect of PpD drugs on the proliferation potency of 786-O, ACHN and Caki-1 cancer cells; D. e and F are results of detecting that PpD drugs with different concentrations respectively degrade p21 activated kinase 4 in 786-O, ACHN and Caki-1 cancer cells by adopting a protein immunoblotting method; G. h and I are results of detecting that p21 activates kinase 4 in 786-O, ACHN and Caki-1 cancer cells degraded by the PpD medicament at different time points by adopting a protein immunoblotting method; J. k and L respectively represent the results calculated according to the degradation rate of the PpD drug to the p21 activated kinase 4 in 786-O, ACHN and Caki-1 cancer cells at different times;

in fig. 3, a is an experimental flow chart for verifying the influence of the PpD drug on the tumor at an animal level, and B is the influence result of the control group, the nano-selenium, the PAK4 inhibitor and the PpD drug treatment group on the tumor size; c is the effect result of the control group, the nano-selenium, the PAK4 inhibitor and the PpD drug treatment group on the tumor weight; d is the growth curve of 786-O xenograft tumor in BALB/c nude mouse after the control group, the nano-selenium, the PAK4 inhibitor and the PpD drug treatment group are treated; e is the result of histopathological analysis of the tumor by a control group, a nano-selenium, PAK4 inhibitor and PpD drug treatment group; f and G are the results of Ki-67 and p21 activated kinase 4 immunohistochemical statistical analysis respectively carried out after the control group, the nano-selenium, the PAK4 inhibitor and the PpD are treated;

FIG. 4A is a flowchart of an experiment for verifying the effect of PpD drug and PD-1 monoclonal antibody on tumors at animal level; b is a survival curve of the mouse after being treated by the control group, the nano selenium, the mAb PD-1, the PpD and the PpD combined with the mAb PD-1; c is the combination of control group, nano-selenium, mAb PD-1, ppD and PpDmonitoring the tumor size and survival state of the mouse after the mAb PD-1 treatment; d is animal level verification that PpD medicine and PD-1 monoclonal antibody are used alone or in combination to detect CD8 in BALB/c tumor-bearing mouse tumor tissue ⁺ T cells and CD4 ⁺ Schematic representation of T cell experiments; e is CD45 in tumor tissues treated by combination of control group, nano-selenium, mAb PD-1, ppD and mAb PD-1 ⁺ CD3 ⁺ CD8 in Positive lymphocytes ⁺ T cells and CD4 ⁺ Representative dot plots of T cells; f is CD45 in tumor tissues treated by the combination of the control group, nano selenium, mAb PD-1, ppD and mAb PD-1 ⁺ CD3 ⁺ CD8 in Positive lymphocytes ⁺ Statistical analysis of T cell infiltration proportion;

FIG. 5 is a schematic diagram of the effect of a polypeptide targeting p21 activated kinase 4.

Detailed Description

The invention provides a polypeptide of a targeted p21 activated kinase 4, and the amino acid sequence of the polypeptide is CRQLRKGKFFSEGGSGGTSFEQFWAWWLWP (SEQ ID No. 1).

The source of the above-mentioned polypeptide is not particularly limited in the present invention, and any polypeptide source known in the art may be used. As in the present example, the polypeptides are synthesized using a polypeptide solid phase synthesis method. The solid phase synthesis method of the polypeptide is not particularly limited in the present invention, and a polypeptide synthesis method known in the art, such as Fmoc polypeptide synthesis, may be used. The Fmoc protected amino acids were purchased from gill biochemical, HBTU and HIBT condensation agents from suzhou haohima.

In view of the functions of the polypeptide in targeting combination with p21 activated kinase 4 and inhibiting tumor cell proliferation, the invention provides application of the polypeptide in preparing a medicament for preventing and/or treating tumors.

In the present invention, the polypeptides are delivered into cancer cells using nanoselenium, and are found to inhibit cell proliferation. IC of polypeptide drug on 786-O renal carcinoma cells ₅₀ 230.8nM, IC on ACHN renal carcinoma cells ₅₀ 248.1nM, IC for Caki-1 renal carcinoma cells ₅₀ It was 126.9nM.

In the present invention, the tumor preferably includes one or more of kidney cancer, lung cancer, colon cancer, prostate cancer, pancreatic cancer and breast cancer, and further preferably includes kidney cancer. The renal cancer includes but is not limited to one or more of clear cell carcinoma, papillary cell carcinoma (also called chromophobe cell carcinoma), chromophobe cell carcinoma, multiple atrial cystic renal cell carcinoma, renal medullary carcinoma, and the like. Still further preferably, the renal cancer cells include one or more of 786-O, caki-1, ACHN, and OS-RC-2.

In view of the function of the polypeptide for degrading the p21 activated kinase 4 protein in cells, the invention provides application of the polypeptide in preparing a reagent for degrading the p21 activated kinase 4 protein.

In the invention, isothermal calorimetry titration experiment is adopted to detect the binding capacity of the polypeptide and the p21 activated kinase 4 protein, and the binding constant of the polypeptide and the p21 activated kinase 4 protein is preferably 456.6nM. The polypeptide is combined with PAK4 protein and MDM2 protein to induce the MDM2 protein to ubiquitinate PAK4, so that the result of mediating the degradation of PAK4 in tumors is realized.

The invention also provides application of the combined use of the polypeptide and the PD-1 antibody in preparing a medicament for preventing and/or treating tumors.

In the present invention, the above-mentioned drug or pharmaceutical composition further preferably comprises a delivery system, preferably nanogold, liposome or nanoselenium. In the embodiment of the present invention, nano-selenium is taken as an example of a delivery system to illustrate the preparation method and the efficacy of the drug. Experiments prove that the medicine has no toxicity at a cellular level and an animal level and has higher safety.

In the present invention, in addition to the delivery system, the medicament or pharmaceutical composition of the present invention preferably comprises pharmaceutically other acceptable excipients, such as one or more of diluents, flavoring agents, binders, suspending agents. The diluent can be one or more of water, glycerol, polyethylene glycol, ethanol, propanol, starch slurry, dextrin, glucose solution, acacia slurry, gelatin slurry, sodium carboxymethylcellulose, potassium phosphate, and polyvinylpyrrolidone. The correctant may be one or more of saccharin sodium, aspartame, sucrose, syrup, glycyrrhetinic acid, and essence. The binder can be one or more of acacia, gelatin, ethanol, honey, liquid sugar, rice paste and batter. The suspending agent can be one or more of polyvinylpyrrolidone, microcrystalline cellulose, sucrose, agar, and hydroxypropyl methylcellulose. The medicament dosage form is preferably a solution, a suspension, an injection, a capsule, a freeze-dried powder injection or an emulsion. The routes of administration of the drug of the present invention include oral, intravenous, intramuscular, subcutaneous, intraperitoneal, intranasal or rectal administration. The dosage of the drug of the present invention can be determined by the type of the disease to be treated, the severity of the disease, the administration route, the age, sex, health condition of the patient, and the like, and for example, the dosage of the drug of the present invention can be 0.01. Mu.g to 1000mg per day per patient.

The PAK 4-targeting polypeptide provided by the present invention, its use and pharmaceutical composition are described in detail in the following examples, which should not be construed as limiting the scope of the present invention.

Description of the Experimental materials

The test materials were divided into the synthesis part: fmoc amino acids were purchased from Gell, shanghai, DIEA, HOBT, HBTU, sigma-Aldrich (Merck Life sciences, inc.). The preparation part of the nano gold comprises: branched PEI, tetrachloroauric acid, was purchased from alatin reagent.

Noun description

Se represents a single nanoselenium delivery system;

PpD represents a finished product coupled with a polypeptide drug and a nano-selenium delivery system;

control represents a finished product coupled with a polypeptide negative Control and a nano-selenium delivery system;

PAK4 represents the target protein p21 activated kinase 4;

beta-actin is a representative actin, and serves as an internal reference in western immunoblotting;

chx represents cycloheximide, and is protein production inhibitor.

Example 1

1.1 ProTAC polypeptides targeting the p 21-activated kinase 4DNA domain were obtained using protein-aided design.

Amino acids by solid phase synthesis using polypeptides

Sequence synthesis of polypeptide drugs (for self laboratory synthesis). The synthesis method is general Fmoc polypeptide synthesis. Fmoc protected amino acids were purchased from Gill Biochemical, HBTU, HIBT condensation reagent from Sazhou Haima Sativa. The synthesis method comprises the following steps:

1) Deprotection: fmoc protected columns and monomers must be freed of the amino protecting group with a basic solvent (piperidine).

2) Activation and crosslinking: the carboxyl group of the next amino acid is activated by an activator. The activated monomer reacts with the free amino group to crosslink, forming a peptide bond. The reaction is driven to completion at this step using a large excess of reagent. And (3) circulation: these two reactions are repeatedly cycled until the synthesis is complete.

3) Elution and deprotection: the polypeptide is eluted from the column and its protecting group is eluted with a deprotecting agent (TFA) and deprotected to yield the crude product.

The amino acid sequence of the PROTAC polypeptide is CRQLRKGKFFSEGGSGGTSFEQFWAWWLWP (SEQ ID NO: 1).

The complex structure of the aforementioned PROTAC polypeptide targeting p21 activated kinase 4 is shown in a of fig. 1.

Example 2

2.1 preparation of Se-PAK4 PROTAC (Nano selenium-polypeptide) medicine

The coupling method comprises the following steps: 2mg of the polypeptide obtained is dissolved in 1mL of pure water, 0.2mL of 50mM sodium selenite, 0.6mL of 5% chitosan and 1.6mL of 50mM ascorbic acid are added, the mixture is heated to 50 ℃, reacted for 20 minutes and cooled to the normal temperature, and the polypeptide drug PpD drug coupled with nano-selenium is obtained.

As can be seen from B and C in FIG. 1, the binding site of the p21 activated kinase 4 and the polypeptide targeting p21 activated kinase 4 is designed by computer assistance, and the optimized polypeptide drug sequence can be obtained according to the result.

As shown in D in FIG. 1, the binding constant of the PROTAC polypeptide targeting p21 activated kinase 4 to p21 activated kinase 4 protein is 456.6nM, and the binding constant of the PROTAC polypeptide targeting p21 activated kinase 4 to MDM2 protein is 82.4nM, which indicates that the PROTAC polypeptide targeting p21 activated kinase 4 has strong binding ability to p21 activated kinase 4 protein.

From E in fig. 1, it can be seen that the PROTAC polypeptide targeting p21 activated kinase 4 can bind to PAK4 protein and MDM2 protein, and PAK4 PROTAC can effectively induce the formation of trimer complex, and the result of trimer formation shows that the PROTAC polypeptide of the present invention can promote ubiquitination and degradation of PAK4 protein.

FIG. 1 conclusion: the PAK4 PROTAC designed by the invention is a PAK4 targeting peptide antagonist with high selectivity and good binding affinity, and can form a trimer with E3 ligase MDM2 to achieve the aim of targeting and degrading PAK4 protein.

Example 3

3.1 cell level assay of the nanoselenium-polypeptide drug prepared in example 2

The cell culture method comprises the following steps: the culture conditions of ACHN, caki-1, OS-RC-2 and 786-O cell lines were 1640 medium, 10% fetal bovine serum, 5% CO ₂ The saturation humidity of the culture medium is that the culture density of the cells is up to 90 percent of fusion degree by adherent culture at 37 ℃.

Experiment of cancer cell proliferation inhibition capacity by drugs:

the Cell Counting Kit-8 detection method analyzes the cancer Cell proliferation inhibition capacity of the medicine.

The Cell Counting Kit-8 is CCK-8 for short, and is a rapid high-sensitivity detection Kit widely applied to Cell proliferation and cytotoxicity based on WST-8. WST-8 is a compound similar to MTT and can be reduced by some dehydrogenases in mitochondria to generate a compound with orange yellow color in the presence of an electron coupling reagent. The more rapid the cell proliferation, the darker the color; the more cytotoxic, the lighter the color. The shade of color and the number of cells were linear for the same cells.

In performing experiments to detect cell activity, ACHN, caki-1 and 786-O cells were plated at 3X 10 ⁴ The density of cells/mL is determined,the cells were plated in 96-well cell culture plates, and 100. Mu.L of cell fluid was added to each well. After 24h of adherent culture of cells, the cells were treated by adding different concentrations of the PpD drug prepared in example 2 (0, 31.3, 62.5, 125, 250, 500, 1000, 2000 nM), and negative controls were set as naked selenium nanoparticles and selenium-loaded nanoparticles of reversed-phase peptide (excluding causes of nonspecific killing). After 48h of treatment, 10. Mu.L of CCK8 reagent was added to each well and incubated for 2 hours in an incubator at 37 ℃. After the development was completed, absorbance values at a wavelength of 450nm and a wavelength of 690nm were measured for each well using a microplate reader for detection. After measurement, the absorbance of each well was calibrated according to formula I. Finally, cell viability was calculated according to formula II.

A＝OD ₄₅₀ -OD ₆₉₀ Formula I

Cell viability (%) = (a (medicated) -a (background))/(a (control) -a (blank)) × 100 formula II

After the drug treatment, the inhibition proliferation effect of the drug which represents the coupling of the polypeptide drug and the nano-selenium delivery system on the kidney cancer cells is obtained through detection and calculation.

3.2 to investigate the ability of the PpD drug prepared in example 2 to cleave PAK4, the PAK4 was analyzed by Immunoblotting (IB). The specific experimental process comprises the following steps:

1) Effect of different concentrations of PpD drug on PAK4 degradation capacity assay: 786-O/ACHN/Caki-1 cells at 3X 10 ⁴ cells/mL were plated into 12-well cell culture plates, and 1mL of cell broth was added to each well. After the cells were cultured for 24h, the cells were treated with 10ng/mL protease inhibitor or different concentrations of PpD drug (0, 31.3, 62.5, 125, 250, 500, 1000, 2000 nM) with 0nM as control. After 48h of treatment, RIPA lysate containing 50 μ L protease inhibitor was added to each well and the lysate was collected.

2) The total protein content of each group of samples was quantified by means of the BCA quantification kit, and the protein concentration of each group of samples was made uniform by means of adjusting the sample volume. After the amount of protein was adjusted, a loading buffer was added and incubated at 100 ℃ for 10 minutes to completely denature the protein.

3) Samples from different groups were separated by polyacrylamide gel electrophoresis. 12% polyacrylamide gel with sodium lauryl sulfate and 5% polyacrylamide gel concentrate were prepared. And then adding the prepared sample and a prestained protein sample with the same volume into the loading hole for carrying out an electrophoretic separation experiment. The electrophoresis conditions are as follows: the voltage was set at 70v and the separation was about 15min until bromophenol blue reached the separation gel. Then, the voltage is adjusted to 120v, the separation is carried out for about 60min until bromophenol blue reaches the position of about 1cm at the end of the separation gel, and the electrophoresis is stopped.

4) And (3) carrying out membrane conversion treatment on the protein sample. All IB experiments of this example used polyvinylidene fluoride membranes, sequentially discharged on a rotary film meter: three layers of filter paper, a polyvinylidene fluoride membrane, glue and three layers of filter paper. The membrane rotating current is set to be 1mA, and the membrane rotating time is set to be 1h.

5) And (5) sealing. The polyvinylidene fluoride membrane after membrane transfer is immersed in a sealing solution containing 5% bovine serum albumin, and incubated for 1h at room temperature to remove the influence of nonspecific adsorption.

6) Primary antibody incubation. And (3) preparing diluents of different antibodies according to requirements, and then incubating overnight at 4 ℃ so as to achieve the purpose that the antibodies recognize specific antigens.

7) And (5) incubating a secondary antibody. A horseradish peroxidase-labeled secondary antibody (anti-mouse or anti-rabbit) with species specificity was prepared according to the source species of the different antibodies, and diluted 1. After which incubation was carried out for 1h at room temperature.

8) And (4) developing color. Preparing a color developing solution, infiltrating the polyvinylidene fluoride membrane with the second antibody for complete incubation, and performing color development analysis by using a chemiluminescence instrument.

As can be seen from FIG. 2, the PpD drug can effectively inhibit the growth of renal cancer cells, the PpD drug shows a dose-dependent growth inhibition effect in 786-O, ACHN and Caki-1 cells, and the nano-selenium particles and the polypeptide control group have no effect on the cancer cells. Half maximal inhibitory concentration (IC 50) values for the PpD drugs on 786-O, ACHN and Caki-1 cells were 305.9nM, 340.4nM and 532.2nM, respectively (A-C in FIG. 2). The PpD drug induces the degradation of PAK4 in a dose-dependent manner, and when the PpD drug concentration reaches 250nM, the content of the PAK4 protein in cells is remarkably reduced (D-F in figure 2). The experimental result of the half-life period of the PAK4 protein shows that: the PpD drug effectively improves the degradation of the PAK4 protein of the renal cancer cells (shown as G-L in figure 2).

From the results of fig. 2 it can be derived: the PpD medicament obviously inhibits the proliferation of renal cancer cells by effectively targeting and reducing the content of the PAK4 protein in the cells.

Example 4

Verification of PpD drug effect on inhibiting tumor growth on animal level

To evaluate the therapeutic efficacy of PpD drugs in vivo, 786-O cells (2X 10) were used ⁶ ) A subcutaneous transplantation kidney cancer mouse model is established, 20 mice are randomly divided into 4 groups, and the mice are treated by PBS, nano selenium particles, ppD drugs and KPT-9274 (PAK 4 inhibitor) with the same dose (2.5 mg/kg) every 3 days for 3 weeks. Continuously observing, and recording the tumor volume when the tumor volume is increased to 50-100 mm ³ The medicine is injected. Tumor volume calculation was done according to formula IV. All the medicines are injected every other day, the injection mode is intraperitoneal injection, the injection volume is 100 mu L, the final injection concentration is 2.5mg/kg, and the change condition of the tumor size is recorded while injection is carried out. After 21 days of drug treatment, dissecting a mouse, taking out a tumor part and other main organs, and performing treatments such as HE staining and immunohistochemical Ki67 and PAK4 staining experiments, wherein the HE staining is used for detecting the pathological form of tumor tissues; ki67 is a tumor cell growth marker, and the expression level of the Ki-67 often indicates the proliferation activity degree of cells; PAK4 staining was performed to detect PAK4 protein levels in tumor cells.

Tumor volume (V) = length × width ² Formula IV,/2.

HE staining method was as follows:

1) Tissue fixation

The removed fresh tissue is put into a prepared 10% formalin fixing solution to denature and coagulate the proteins of the tissue and cells, so as to prevent autolysis or bacterial decomposition after cell death, thereby maintaining the original morphological structure of the cells.

2) Tissue dehydration

The tissue after fixation was trimmed to 25px × 25px × 5px and the tissue was washed with pure water to remove the fixative. Then gradually replacing water in the tissue with alcohol according to the low concentration to the high concentration of the tissue. Then the tissue block is placed in a clearing agent xylene which is dissolved in alcohol and paraffin, and the xylene is used for replacing the alcohol in the tissue block, so that the tissue block can be embedded by paraffin.

3) Tissue embedding

Placing the transparent tissue block in melted paraffin, and placing the tissue block in a paraffin dissolving box for heat preservation. And standing, cooling and solidifying into blocks after the paraffin is completely immersed into the tissue blocks.

4) Tissue section

The embedded wax blocks are fixed on a microtome and cut into thin sections, typically 5 to 8 μm thick.

5) Staining of the sections

Before tissue staining, paraffin in the sections is removed again by xylene, then alcohol is removed by high-concentration to low-concentration alcohol, and finally the sections are immersed in pure water. Staining was then started and the sections were stained in aqueous hematoxylin for 10min. Then, the sliced acid water and ammonia water are subjected to color separation for several seconds. After rinsing with pure water, the slices were dehydrated in 70% and 90% alcohol for 10min each, and then stained with an alcohol eosin staining solution for 2min.

6) Slicing and re-dehydrating

The stained sections were re-dehydrated as described above for tissue dehydration.

7) Sealing sheet

The transparent sections were dropped with gum and covered with coverslips. After which it was observed under a microscope and photographed.

Immunohistochemical Ki67 and PAK4 staining experiments were as follows:

1) Immunohistochemical PBS reagent formula and preparation method

The following formulation is exemplified by the reagents required to formulate 1000mL of potassium-free immunohistostaining PBS buffer 0.01m, ph = 7.4:

2) Formula and preparation method of immunohistochemical antigen repairing liquid

The following formula takes the reagents required for preparing 1000mL of citric acid buffer solution with 0.01M and pH =6.0 of immune tissue staining antigen repairing solution as an example:

trisodium citrate 2H ₂ O 3g

Citric acid H ₂ O 0.4g

ddH ₂ O 1000mL

3) Gradient alcohol preparation (100 mL)

4) Tissue fixation, slicing and baking

The tissue fixing and slicing method is the same as the HE staining fixing and slicing method, and then the tissue slices are placed in an oven and baked for 2h at the temperature of 60 ℃.

5) Dewaxing

Taking out the slices from the oven, and immediately and sequentially putting the slices into dimethylbenzene for dewaxing treatment:

xylene I8 min

Xylene II 8min

Xylene III 8min

3) Alcohol gradient hydration

Taking out the slices from the xylene III, and immediately and sequentially putting the slices into gradient alcohol for hydration treatment:

6) PBS Wash

The sections were removed from 50% ethanol and immediately placed in PBS for 3 washes, 3min each.

7) Antigen retrieval

Taking out the slices from PBS, putting the slices into a tissue slice box containing citric acid buffer solution with the pH of 0.01M and 6.0, putting the tissue slice box into a numerical control heat antigen restoration instrument, and performing antigen restoration at the temperature of 121 ℃ for 5 min. After the antigen is repaired, naturally reducing the pressure, and then taking out the assembling film box to naturally cool to the room temperature.

8) PBS Wash

And taking out the slices from the citric acid buffer solution, immediately putting the slices into PBS for washing treatment by the citric acid buffer solution, and washing for 3 times, wherein each time lasts for 3min.

9) Cell punching

The sections were put into 2% TritonX-100 for cell punching treatment. The PBS buffer around the tissue chip was wiped dry with filter paper and the outside of the tissue section was sketched with a brush pen. Pipette up 1mL of tissue covered by TritonX-100 on the entire tissue chip, and stand still at room temperature for 10min.

10 ) PBS Wash

The sections were washed 3 times for 3min each time with endogenous peroxidase in PBS.

11 Endogenous peroxidase blockade

The sections were removed from the PBS, the PBS buffer surrounding the tissue sections was wiped dry with filter paper, and the outside of the tissue chip was outlined with a brush pen. Pipette 1mL of nonspecific blocking agent to cover the tissue on the entire tissue chip, and allow to stand at room temperature for 30min.

12 ) PBS Wash

The sections were washed 3 times for 3min each time in PBS for non-specific blocking agent.

13 Primary antibody incubation

Primary antibody was diluted to the appropriate concentration with a primary antibody diluent according to the antibody specification. The sections were removed from the PBS, wiped dry with filter paper to remove PBS buffer around the sections, and the outside of the sections were outlined with a brush pen. Pipette 1mL of the prepared primary anti-solution to cover the tissue on the entire tissue chip and set the primary anti-diluent as a negative control (i.e., no primary antibody is added to the primary anti-diluent). The sections added with the primary antibody are placed into a tissue-dissolving wet box, placed in a medical refrigerator at 4 ℃ and kept still overnight.

14 ) rewarming

The combined wet box is taken out from a medical refrigerator at 4 ℃ and is placed for 1h at room temperature.

15 ) PBS Wash

The sections were washed in PBS for 3 times for 3min each time.

16 Incubation with secondary antibody

The sections were removed from the PBS, the PBS buffer around the tissue chip was wiped dry with filter paper, and the outside of the tissue chip was outlined with a brush pen. The tissue on the whole section is covered by the secondary antibody solution sucked by a pipette, and the section added with the secondary antibody is placed in a combined wet box and is placed for 1h at room temperature.

17 ) PBS Wash

The sections were washed in PBS for 3 times for 3min each with a secondary antibody solution.

18 ) DAB color development

The sections were removed from the PBS, the PBS buffer surrounding the sections was wiped dry with filter paper, and the outside of the sections was delineated with a set of paintbrush. 1mL of DAB dye solution (DAB: substrate buffer = 26. Mu.L: 1000. Mu.L) was pipetted to cover the whole section, the time for development of primary antibody was varied, the time for termination was judged under a microscope, and the section was placed in deionized water to terminate the staining.

19 ) PBS Wash

And (3) putting the slices into PBS for DAB staining solution washing treatment, wherein the washing is carried out for 3 times and 3min each time.

20 Double dyeing)

The sections were removed from the PBS, the PBS buffer surrounding the sections was wiped dry with filter paper, and the outside of the sections was delineated with a set of paintbrush. Sucking 1mL hematoxylin staining solution by a pipette, standing at room temperature for 5min, and placing into deionized water to stop staining.

21 ) PBS Wash

The renal clear cell carcinoma tissue chip is placed into PBS for hematoxylin staining and washing treatment, and washing is carried out for 3 times, 3min each time.

22 Differentiation with hydrochloric acid

The sections were removed from the PBS, the PBS buffer surrounding the sections was wiped dry with filter paper, and the outside of the sections was delineated with a set of paintbrush. 1mL of hydrochloric acid solution was pipetted, allowed to stand at room temperature for 5s, and then placed in deionized water to stop staining.

23 Ammonia bluing

After the section differentiation is finished, 1% ammonia water solution is immediately added, the section is kept still for 10s at room temperature, and the section is added into deionized water to stop the reaction.

24 Gradient alcohol dehydration and xylene clarification

Putting the renal clear cell carcinoma tissue chip into a gradient alcohol and xylene solution in sequence for dehydration treatment:

25 Sealing sheet

And taking the section out of the xylene III, dripping neutral gum into the area where the tissue is located after the xylene is volatilized, covering the tissue with 8 cm-8 cm cover glass, placing the tissue in a ventilation cabinet for air drying, and then observing and taking a picture by using a microscope.

As shown in fig. 3, compared with the control group, both the PAK4 inhibitor KPT-9274 and the PpD drug group significantly inhibited tumor growth in vivo, as shown in B in fig. 3. Compared with the PAK4 inhibitor KPT-9274 group, the PpD drug group has better inhibition effect on the tumor volume and weight, which are shown as C and D in figure 3. The immunohistochemical staining result of the tumor cell proliferation index Ki67 shows that: compared with the control group, the ratios of the tumor cells Ki67 positive cells in the PAK4 inhibitor KPT-9274 and the PpD drug group are obviously reduced, and are shown as E and F in figure 3. In the PpD group and the PAK4 inhibitor KPT-9274 group, the PAK4 positive cell ratio of the tumor cells is also obviously reduced, and is shown as E and G in figure 3.

From the results of fig. 3, it can be concluded that: the PpD drug group shows that the experimental data in vivo are superior to those in the PAK4 inhibitor KPT-9274 group in four aspects of inhibiting the tumor volume and the tumor weight of a mouse and reducing the proliferation Ki67 and the PAK4 protein expression of tumor cells.

Example 5

The effect of combined use of the PpD medicament and the PD-1 monoclonal antibody on tumor immunotherapy is verified on an animal level, and in order to evaluate the in-vivo immunotherapy effect of the PpD medicament, a subcutaneous transplantation mouse source renal carcinoma mouse model is established. BALB/c mice were injected subcutaneously with 40X 10 ⁴ Renca cells. After 2 weeks, 50 mice were randomly divided into 5 groups and given controls, respectivelyGroup (PBS, n =10, injected once every 2 days), nano-selenium (Se mutant peptide nanoparticles, n =10, injected once every 2 days), ppD (2.5 mg/kg, n =10, injected once every 2 days), PD-1 (2.5 mg/kg, n =10, injected once every 3 days), ppD in combination with PD-1 (2.5 mg/kg, n = 10), wherein PpD is treated in combination with PD-1 by injecting PpD once 3 days after PD-1 mab treatment in mice, once PD-1 mab every 2 days, (see a in fig. 4). BALB/c nude mice were recorded for body weight and tumor volume every 3 days. In order to simultaneously explore a molecular mechanism of the PpD medicament for increasing the immunotherapy sensitivity in vivo, a mouse model of the mouse-derived renal carcinoma transplanted subcutaneously is established. BALB/c mice were injected subcutaneously with 100X 10 ⁴ Renca cells. When the tumors grew to about soybean size, 30 mice were randomly divided into 5 groups, and each group was administered with control group (PBS, n =6, injected once every 2 days), nano-selenium (Se mutant peptide nanoparticles, n =6, injected once every 2 days), ppD (2.5 mg/kg, n =6, injected once every 2 days), PD-1 (2.5 mg/kg, n =6, injected once every 3 days), and PpD in combination with PD-1 (2.5 mg/kg, n = 6), wherein the PpD in combination with PD-1 was administered once 3 days after the PD-1 mab-treated mice, and once PD-1 mab was administered at 2 days intervals (see D in fig. 4). After 10 days, separating tumor tissue, extracting tumor immunocyte, and directly killing immunocyte CD8 on tumor ⁺ T cell and immunocyte CD4 ⁺ And (4) staining the T cells, and detecting the infiltration ratio by a flow cytometer.

The injection mode of the administration of the mice is intraperitoneal injection, the injection volume is 100 mu L, and the injection is carried out while the change condition of tumors is observed. Tumors greater than 2cm in length, width, height or ulceration greater than 1cm on the surface of the tumor and poor mental status of the mice were considered dead. The mice were dissected and tumor tissue was obtained.

Tumor volume (V) = length × width ² Formula IV,/2.

As can be seen from FIG. 4, the polypeptides targeting PAK4 can block the PD-I/PD-L1 axis to enhance the immunotherapeutic effect. Of these, the PpD group, the PD-1 mAb group, and the PpD in combination with the PD-1 group all showed better survival rates, with the PpD in combination with the PD-1 group mice having prolonged survival times (see B in FIG. 4). Mice survived at the experimental endpoint in both the PpD group, the PD-1 monoclonal antibody group, and the PpD combined PD-1 group, with the largest surviving individuals in the PpD combined PD-1 group and the smallest in the PpD groupMice survived one more than the PD-1 mab group (see C in FIG. 4). From E in FIG. 4, it can be seen that the tumor tissues of the mice in the PpD group, the PD-1 monoclonal antibody group, and the PpD-combined PD-1 group were CD8 in comparison with the control group ⁺ The proportion of T cell infiltration is significantly increased. PpD group, PD-1 monoclonal antibody group, ppD in combination with PD-1 group CD8 ⁺ The infiltration ratio of T cells is obviously increased, wherein the PpD group and the PpD combined PD-1 group have statistical significance compared with a control group, and the PpD combined PD-1 group has more than the PD-1 monoclonal antibody group mouse tumor tissue CD8 ⁺ T cell infiltration accounted for more (see F in fig. 4).

From the results of fig. 4, it can be concluded that: the PpD, the PD-1 monoclonal antibody group and the PpD and PD-1 combined group can effectively prolong the service life of mice and obviously improve the CD8 of tumor direct killer cells ⁺ Infiltration of T cells. And (4) prompting: the PpD drug and the immune checkpoint drug can increase the curative effect of clinical immunotherapy.

FIG. 5 is a schematic view of the PpD drug designed by the present invention degrading PAK4 protein in tumor body to kill tumor cells.

In the invention, the PpD drug can effectively induce the degradation of the PAK4 protein, and the PpD drug can inhibit the growth of tumor cells and increase the infiltration of T cells of immune cells while inducing the degradation of the PAK4 protein, thereby achieving the clinical effect of enhancing the sensitivity of immunotherapy.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

1. A polypeptide targeting p21 activated kinase 4 is characterized in that the amino acid sequence of the polypeptide is shown as SEQ ID No. 1.

2. Use of the polypeptide of claim 1 for the preparation of a medicament for the prevention and/or treatment of tumors.

3. The use of claim 2, wherein the polypeptide has a binding constant for p 21-activated kinase 4 protein of 456.6nM.

4. The use of claim 3, wherein the tumor comprises one or more of renal cancer, lung cancer, colon cancer, prostate cancer, pancreatic cancer, and breast cancer.

5. Use of a polypeptide according to claim 1 for the preparation of a reagent for degrading the p 21-activated kinase 4 protein.

6. A medicament for the prevention and/or treatment of tumors comprising the polypeptide of claim 1.

7. Use of a polypeptide according to claim 1 in combination with a PD-1 antibody for the preparation of a medicament for the prevention and/or treatment of a tumor.

8. A pharmaceutical composition for preventing and/or treating tumor, comprising the polypeptide of claim 1 and a PD-1 antibody.

9. The drug of claim 6 or the pharmaceutical composition of claim 8, further comprising a delivery system comprising nanogold, liposomes, or nanoselenium.

10. The medicament of claim 6 or the pharmaceutical composition of claim 8, wherein the tumor comprises one or more of kidney cancer, lung cancer, colon cancer, prostate cancer, pancreatic cancer, and breast cancer.