A pd-1 and αvβ3 bifunctional polypeptide and applications thereof

Abstract

This invention relates to polypeptides, specifically to a PD-1 and αvβ3 bifunctional polypeptide and its applications, the amino acid sequence of which is shown in SEQ ID NO.1. The PD-1 / αvβ3 bifunctional polypeptide can simultaneously inhibit the PD-1 / PD-L1 pathway and the αvβ3 pathway, not only inhibiting tumor growth and invasion and suppressing abnormal tumor angiogenesis, but also promoting immune cell infiltration into tumors by blocking the PD-1 pathway, inhibiting angiogenesis, and regulating PD-L1 expression, thereby modulating the tumor microenvironment and exerting a synergistic immune effect.

Description

Technical Field

[0001] The invention relates to polypeptides, specifically to a bifunctional polypeptide of PD-1 and αvβ3 and its applications.

[0002] Research Background

[0003] Overexpression of immune checkpoint proteins by tumor cells can suppress the activity of immune cells, thereby evading the surveillance of the immune system. PD-1, first discovered in 1992, is expressed on the surface of T cells, B cells, monocytes, dendritic cells, and natural killer T cells (NK cells). PD-L1 and PD-L2 are mainly expressed on tumor cells and antigen-presenting cells (APCs). The interaction between PD-1 and PD-L1 induces tumor immune escape. When PD-1 is activated, it induces intracellular signaling pathways, thereby inhibiting T cell proliferation, cytokine secretion, and cytotoxic function. PD-1 and PD-L1 play an important role in the negative regulation of the immune response. By blocking the binding of PD-1 and PD-L1, T cell function can be restored, T cells can be reactivated, and endogenous anti-tumor immune responses can be activated, thereby exerting a therapeutic effect on tumors. However, although PD-1 antagonist peptides have good immunomodulatory and anti-tumor activities, they have a short half-life and poor water solubility. In animal models, they are administered intratumorally in the form of nanoparticles. Meanwhile, PD-1 is also expressed in normal tissues and is an important component in maintaining the homeostasis of the immune system. Single-target blocking of PD-1 may lead to some adverse immune reactions. Therefore, enhancing its targeting and improving its water solubility are urgent problems to be solved.

[0004] Integrins are transmembrane cell surface heterodimer proteins composed of α and β subunits. They mainly mediate signal transduction between cells and the extracellular environment (such as the extracellular matrix (ECM), regulate cell proliferation and survival, and promote angiogenesis in endothelial cells. Currently, 18 α subunits and 8 β subunits have been identified in mammals, forming 24 different integrin dimers, each with its own function and tissue specificity. Based on the type of ligand they recognize, integrins can be divided into four categories: (1) receptors that recognize the tripeptide RGD (Arg-Gly-Asp) sequence; (2) receptors that have... (2) Leukocyte-specific receptors that bind to the LDV (L / ID / EV / S / TP / S) sequence; (3) receptors that selectively bind to laminin; (4) collagen-binding receptors (including four β1 integrins (α1, α2, α10, α11) that recognize the GFOGER motif. Among them, αvβ3 belongs to the first class of integrins that recognize the RGD motif. It is highly expressed in endothelial cells and tumor tissues and plays an important role in tumor growth. αvβ3 is considered a therapeutic target for various types of cancer, but there are currently no αvβ3 antagonists approved for treatment.

[0005] Previous studies on integrin αvβ3 have often focused on tumorigenesis and development, epithelial-mesenchymal transition (EMT), tumor angiogenesis, and drug resistance. Recent research indicates that integrin αvβ3 can participate in tumor immune escape by regulating PD-L1 expression on tumor cell surfaces. Silencing the β3 subunit or administering the specific inhibitor silengilide can reduce PD-L1 expression in tumor cells stimulated by interferon. Furthermore, it was found that integrin αvβ3 exerts its effects by activating downstream IFN-γ pathways. In a mouse melanin model, silencing the β3 subunit or administering silengilide reduced PD-L1 expression in tumor tissue and enhanced CD8+ T cell infiltration and IFN-γ levels in tumor tissue, with even higher efficacy observed after combining with an anti-PD-1 antibody. However, this experiment did not extend the results to more tumor types. Moreover, blocking integrins and inhibiting PD-1 are achieved through two separate mechanisms, suggesting potential drug interactions.

[0006] Based on the above research background, this invention combines the inhibition of integrin αvβ3 with the principle of immune checkpoint blockade to design a PD-1 / αvβ3 dual-targeting peptide to address the problem of PD-1 presence. Summary of the Invention

[0007] Purpose of the invention

[0008] This invention links the sequence of the integrin αvβ3 that specifically targets PD-1 to the PD-1 blocking peptide P-F4 via a linker, forming a novel bifunctional polypeptide.

[0009] Technical solution

[0010] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0011] A bifunctional polypeptide of PD-1 and αvβ3, characterized in that the amino acid sequence of the polypeptide is: GRGDSPKGGGGFSGTVTTAGLLF, as shown in SEQ ID NO.1.

[0012] The application of the polypeptide in the preparation of therapeutic antitumor drugs.

[0013] The tumor in question is colorectal cancer.

[0014] Specifically as follows:

[0015] (1) Peptide Design: To preserve the activity of the PD-1 antagonist peptide P-F4 and the targeting integrin sequence peptide as much as possible, different amounts of Gly were selected as linkers to connect the "RGD" sequence to the N-terminus and C-terminus of P-F4, respectively. The relative blocking activity of the peptides relative to P-F4 was detected by molecular docking and HTRF assay. The molecular docking model showed that the linker affected the activity, with PF4-RGD5 and PF4-RGD11 showing the best docking results. Further blocking experiments verified that PF4-RGD5 completely preserved the blocking ability of the P-F4 peptide, while the blocking ability of PF4-RGD1 without the linker and PF4-RGD11 coupled to the C-terminus decreased significantly. This indicates that the modification method of coupling the RGD sequence to the N-terminus of the P-F4 peptide with a linker can maximize the preservation of the original peptide's blocking ability against PD-1.

[0016] However, since RGD-PF4 could not effectively inhibit the migration of vascular endothelial cells, the "RGD" tripeptide sequence was replaced with the specific targeting peptide sequence "GRGDSPK". This was then coupled with the precursor peptide P-F4 via a linker to form the bifunctional peptide SPK-PF4. The blocking ability of the bifunctional peptide against PD-1 / PD-L1 relative to P-F4 was verified using HTRF, with experimental procedures as described in Example 2; the results are as follows. Figure 2 As shown, the relative blocking rate of SPK-PF4 is 74.06%. This indicates that the bifunctional peptide has the ability to block PD-1 / PD-L1 binding at the molecular level.

[0017] (2) Cellular activity study: The effects of SPK-PF4 on the proliferation, migration and adhesion of vascular endothelial cells (HUVECs) were detected; the effects of SPK-PF4 on the migration and apoptosis of tumor cells were detected; the regulatory effect of SPK-PF4 on the PD-L1 level of tumor cells was detected; and the restorative effect of SPK-PF4 on the killing activity of spleen cells was detected.

[0018] (3) Animal-level activity study: A mouse CT-26 colon cancer model was established, and in vivo pharmacodynamic experiments were conducted to explore the anti-tumor effect and in vivo immune regulation effect of SPK-PF4.

[0019] Beneficial effects

[0020] The PD-1 / αvβ3 dual-targeting peptide can simultaneously inhibit the PD-1 / PD-L1 pathway and the αvβ3 pathway. It can not only inhibit tumor growth and invasion and suppress abnormal tumor angiogenesis, but also promote the infiltration of immune cells into tumors by blocking the PD-1 pathway, inhibiting angiogenesis, regulating PD-L1 expression, regulating the tumor microenvironment, and exerting a synergistic immune effect.

[0021] Specifically, this invention designs a polypeptide that can inhibit the migration and adhesion of vascular endothelial cells at the cellular level, while also inhibiting tumor cell migration and PD-L1 expression, promoting tumor cell apoptosis, and enhancing the killing effect of spleen cells on tumor cells. In a mouse colorectal cancer model, this polypeptide can effectively inhibit tumor growth and angiogenesis within the tumor, while overcoming the disadvantage of poor water solubility and difficulty in administration of precursor polypeptides.

[0022] This polypeptide has the following advantages compared to the original PD-1 blocking peptide P-F4:

[0023] 1. Due to the high expression of αvβ3 at the tumor site, the dual-targeting peptide can specifically target the tumor site;

[0024] 2. By blocking the αvβ3 pathway, it regulates the expression of PD-L1, which has a synergistic effect with the blocking of the PD-1 pathway.

[0025] 3. Blocking αvβ3 can inhibit tumor angiogenesis and regulate the tumor microenvironment by inhibiting the VEGFR pathway, breaking the negative cycle between tumor angiogenesis and immunosuppression;

[0026] 4. Blocking αvβ3 can inhibit the proliferation, adhesion, and invasion of tumor cells;

[0027] 5. P-F4 acts on the hydrophobic interface of PD-1 / PD-L1 and contains a large number of hydrophobic amino acids, resulting in poor water solubility. However, αvβ3-targeting peptide modification can effectively improve the water solubility of P-F4. Attached image description:

[0028] Figure 1 The blocking rates of PF4-RGD1, PF4-RGD5, and PF4-RGD11 relative to P-F4 for PD-1 / PD-L1 in this invention;

[0029] Figure 2 This refers to the blocking rate of SPK-PF4 against PD-1 / PD-L1 relative to P-F4 in this invention;

[0030] Figure 3 This invention describes the cytotoxic effects of SPK-PF4 at different concentrations on vascular endothelial cells (HUVECs): Figure 3 A represents the effect at a concentration of 50 μM; Figure 3 B represents the effect at a concentration of 100 μM.

[0031] Figure 4 This invention relates to the effect of SPK-PF4 at different concentrations on HUVEC cell migration: Figure 4 A represents statistical data on the migration area of ​​HUVEC cells after treatment with 50 μM peptide. Figure 4B represents statistical data on the migration area of ​​HUVEC cells after treatment with 100 μM peptide. Figure 4 C is a comparison of HUVEC cell scratches after treatment with 100 μM peptide;

[0032] Figure 5 This invention relates to the effect of SPK-PF4 peptide on HUVEC cell adhesion.

[0033] Figure 6 The effect of the SPK-PF4 peptide on the migration of SKOV3 tumor cells in this invention: Figure 6 A represents statistical data on the migration area of ​​SKOV3 cells after treatment with 100 μM peptide. Figure 6 B is a comparison of SKOV3 cell scratches after treatment with 100μM peptide;

[0034] Figure 7 The effect of SPK-PF4 peptide on tumor cell surface and total PD-L1 expression in this invention: Figure 7 A represents the PD-L1 expression level on the surface of SKOV3 cells after treatment with 100 μM peptide; Figure 7 B represents the statistical results of PD-L1 expression on the surface of SKOV3 cells after treatment with 100 μM peptide; Figure 7 C represents the total PD-L1 expression level in SKOV3 and A549 cells after treatment with 100 μM peptide;

[0035] Figure 8 The effect of SPK-PF4 peptide on SKOV3 cell apoptosis in this invention: Figure 8 A represents the apoptosis level of SKOV3 cells after treatment with 100 μM peptide; Figure 8 B represents the statistical results of the early apoptosis rate of SKOV3 cells after treatment with 100 μM peptide. Figure 8 C represents the statistical results of the proportion of late apoptosis and necrosis in SKOV3 cells after treatment with 100 μM peptide.

[0036] Figure 9 The effect of SPK-PF4 peptide on the recovery of spleen cell activity in this invention: Figure 9 A represents the apoptosis level of SKOV3 cells after co-incubation with SKOV3 cells and spleen cells followed by treatment with 100 μM peptide. Figure 9 B represents the statistical results of the early apoptosis rate of SKOV3 cells after co-incubation and treatment with 100 μM peptide; Figure 9 C represents the statistical results of the late apoptosis and necrosis ratios of SKOV3 cells after co-incubation and treatment with 100 μM peptide;

[0037] Figure 10 These are the in vivo pharmacodynamic results of the SPK-PF4 peptide in this invention: Figure 10 A is a tumor growth curve; Figure 10B is a graph showing the tumor weight. Figure 10 C is the mouse weight gain curve during the drug administration period; Figure 10 D is a statistical chart of the weight of the mouse's core organs;

[0038] Figure 10 E represents the detection and statistical analysis of the killing ability of splenic lymphocytes in each group of mice at different effector-to-target ratios;

[0039] Figure 11 The effect of the SPK-PF4 peptide on the tumor microenvironment in this invention is as follows: Figure 11 A shows the expression and statistical analysis of CD31+ vascular endothelial cells in immunohistochemistry. Figure 11 B shows the infiltration of CD8+ T cells in immunohistochemistry and statistical analysis. Figure 11 C represents the infiltration of CD4+ T cells in immunohistochemistry and statistical analysis; Figure 11 D represents the infiltration of Foxp3+ T cells in immunohistochemistry and statistical analysis. Detailed implementation method:

[0040] The present invention will be described in further detail below with reference to the embodiments; however, it should be understood that these embodiments are only for illustrative purposes and are not intended to limit the present invention; any modification of the linking of the precursor polypeptide P-F4 with other targeting integrin sequences under the premise of the present invention is within the scope of protection of the present invention.

[0041] The polypeptides SPK-PF4, PF4-RGD1, PF4-RGD5, PF4-RGD11 and P-F4 involved in this invention were synthesized by Shanghai Sangon Biotech.

[0042] Example 1: Preliminary design of the RGD-PF4 bifunctional peptide and determination of its relative activity in blocking the PD-1 / PD-L1 pathway. Activity verification: 1. In order to preserve the activity of the PD-1 antagonist peptide P-F4 and the peptide targeting the integrin sequence as much as possible, different numbers of Gly were selected as linkers to connect the RGD sequence to the N-terminus and C-terminus of P-F4, respectively, and 12 peptides were designed, named PF4-RGD1, PF4-RGD2, PF4-RGD3, PF4-RGD4, PF4-RGD5, PF4-RGD6, PF4-RGD7, PF4-RGD8, PF4-RGD9, PF4-RGD10, PF4-RGD11, and PF4-RGD12.

[0043] 2. The PD-1 protein crystal structure was retrieved from the PDB (protein database bank) database. The PDB ID was 3RRQ, and the H2O molecule was removed. Based on previous research on the P-F4 peptide in our laboratory, the MDockPeP docking software platform was selected to simulate docking of the P-F4 peptide with the PD-1 protein crystal structure.

[0044] 3. Select 12 peptide docking poses around the PD-L1 binding hotspot residues on the PD-1 protein and have the highest absolute value score of ITScorePeP (MDockPeP's built-in scoring system, the higher the absolute value, the better) and record their scores.

[0045] result:

[0046] As shown in Table 1, PF4-RGD5 and PF4-RGD11 showed the best docking results.

[0047] Table 1. Scoring of PF4-RGD1 to PF4-RGD12 docking with PD-1 protein molecules.

[0048]

[0049] 4. As shown in Table 1, the score of N-terminal coupling was significantly higher than that of C-terminal coupling. Therefore, PF4-RGD1 was selected as a candidate peptide, and its blocking activity relative to P-F4 was tested. Prepare the PD-1 / PD-L1 HTRF kit (Tag1-PD-L1, Tag2-PD-1, AntiTag1-Eu3+, Anti-Tag2-XL665) and thaw it on ice.

[0050] 5. Use the buffer provided with the kit to dilute Tag1-PD-L1 and Tag2-PD-1 100 times to obtain working solutions for later use. Use the buffer to dilute Anti-Tag1-Eu3+ and Anti-Tag2-XL665 100 times and premix them at a 1:1 ratio to prepare working solutions for later use.

[0051] 6. Prepare a 600 μM peptide solution using PBS, maintaining a DMSO concentration of 5%. Add 2 μL of the peptide solution to each well of a low-volume 96-well plate, with three replicates per peptide. Add 4 μL of Tag1-PD-L1 and 4 μL of Tag2-PD-1, mix thoroughly, and incubate at room temperature for 15 min. Use the experimental group without Tag1-PD-L1 as a positive control, PBS as a negative control instead of peptide solution, and P-F4 as an experimental control. For groups with less than 10 μL, supplement with buffer.

[0052] 7. Add 10 μL of premixed Anti-Tag1-Eu3+ and Anti-Tag2-XL665 to each well, seal with sealing film, mix thoroughly, centrifuge, and incubate at room temperature for 2 hours.

[0053] 8. After incubation, use an EnVision multi-functional microplate reader to excite at 320nm and emit at 665nm and 620nm respectively, and measure the emission signal value of each well.

[0054] 9. Calculate the transmit signal ratio, suppression ratio, and relative suppression ratio for each aperture according to the following formulas, and perform data analysis using Graphpad 9.5 software.

[0055] Ratio = (Signal 665nm / Signal 620nm )×10 4

[0056] Inhibition Rate = (Ratio 阴性 -Ratio 实验 ) / (Ratio 阴性 -Ratio 阳性) ×100%

[0057] Relative Inhibition Rate=Inhibition Rate 实验 / Inhibition Rate 实验对照

[0058] result:

[0059] like Figure 1 As shown, PF4-RGD5 completely retains the blocking ability of the P-F4 peptide, while the blocking ability of PF4-RGD1 without a linker and PF4-RGD11 coupled to the C-terminus is significantly reduced. This indicates that the modification method of coupling the P-F4 peptide to the RGD sequence via a linker at the N-terminus can maximize the preservation of the original peptide's blocking ability against PD-1.

[0060] Example 2: Design of SPK-PF4 bifunctional peptide and determination of relative activity against PD-1 / PD-L1 pathway

[0061] To further enhance the ability of the peptide to target integrin αvβ3, the "RGD" tripeptide sequence in PF4-RGD5 was replaced with the peptide sequence "GRGDSPK" which has a specific targeting effect. The bifunctional peptide SPK-PF4 was formed by coupling it with the precursor peptide P-F4 through a linker. The blocking ability of the bifunctional peptide against PD-1 / PD-L1 relative to P-F4 was verified by HTRF. The experimental steps were the same as in Example 1.

[0062] result:

[0063] like Figure 2 As shown, the relative blocking rate of SPK-PF4 is 74.06%. This indicates that the bifunctional peptide retains the ability to block PD-1 / PD-L1 binding at the molecular level.

[0064] Example 3: Cytotoxicity of SPK-PF4 bifunctional peptide against HUVEC cells

[0065] 1. Collect and count HUVEC cells by digestion, then resuspend and adjust the cell concentration to 4 × 10⁻⁶. 4 per ml.

[0066] 2. Use a 96-well plate, add 100 μl of cell suspension to each well, and let it adhere overnight.

[0067] 3. Dilute the DMSO-prepared peptide stock solution to 50 μM and 100 μM with complete culture medium, and dilute the positive control drug Cilengitide to 0.5 μM and 1 μM. Prepare 600 μl of each peptide-containing culture medium in 1 ml EP tubes for later use. Set up the DMSO group as a solvent control.

[0068] 4. Discard the culture medium in the well plate, add 100 μl of the mixed drug-containing culture medium to each well, with 5 parallel wells per group, and place in a cell culture incubator at 37℃ and 5% CO2 for 24 h.

[0069] 5. Remove the 96-well plate, add 10 μl of CCK-8 reagent to each well, and continue incubation in the incubator for 2 hours.

[0070] 6. Detection of OD using an enzyme-linked immunosorbent assay (ELISA) reader 450 Values ​​and OD 650 Cell viability was calculated using the following formula, and experimental data were analyzed using GraphPad Prism 9.5 software.

[0071] Cell Viability (%) = (OD) 450 -OD 650 ) 实验组 / (OD 450 -OD 650 Negative control

[0072] result:

[0073] like Figure 3 As shown in the CCK8 data, both concentrations of the bifunctional peptide and the positive control drug showed no cytotoxicity to HUVEC cells, indicating that the bifunctional peptide has good targeting properties.

[0074] Example 4: Effects of SPK-PF4 bifunctional peptide on HUVEC cell migration

[0075] 1. Collect and count HUVEC cells by digestion, then resuspend and adjust the cell concentration to 1.5 × 10⁻⁶. 7 per ml.

[0076] 2. Use a 6-well plate. Draw 6 parallel straight lines on the bottom of the plate with a marker beforehand. Add 100 μl of cell suspension to each well, add 900 μl of culture medium, mix well and culture until the cells cover the bottom of the entire well.

[0077] 3. Replace DMEM+10% FBS with serum-free DMEM medium and starve the medium for 12 hours.

[0078] 4. Dilute the DMSO-prepared peptide stock solution to 50 μM and 100 μM with serum-free culture medium, and dilute silengitide to 0.5 μM and 1 μM. Set up the culture medium group as a negative control and the 0.1% DMSO group as a solvent control.

[0079] 5. Using a 20μL pipette tip, draw three lines on the straight line drawn before each well is perpendicular to the target. Quickly aspirate the serum-free culture medium and wash three times with PBS to remove cell debris.

[0080] 6. Add 1 ml of drug-containing culture medium to each well and incubate at 37°C and 5% CO2 for 24 h.

[0081] 7. Take photos using CellSens at 0h and 24h, analyze the scratch area using ImageJ, and analyze the scratch area data using GraphPad Prism 9.5 software.

[0082] result:

[0083] like Figure 4 As shown, compared with the precursor peptide P-F4, the bifunctional peptide effectively restricted the migration of HUVEC cells, while there was no significant difference between the precursor peptide group and the control group. The inhibitory effect was significantly stronger at a concentration of 100 μM in the experimental group than at 50 μM; therefore, subsequent experiments controlled the peptide concentration in the experimental group at 100 μM and the positive control drug concentration at 1 μM. Notably, the SPK-PF4 peptide group exhibited similar cell contact inhibition activity to the positive control drug, indicating that SPK-PF4 possesses a stronger targeted blocking function.

[0084] Experimental Example 5: Effect of SPK-PF4 bifunctional peptide on HUVEC cell adhesion

[0085] 1. Dilute the DMSO-prepared peptide stock solution to 200 μM with serum-free culture medium, and dilute C-silagetide.

[0086] Prepare 300 μl of each peptide-containing culture medium at 2 μM in a 1 ml EP tube for later use. Set up the DMSO group as a solvent control.

[0087] 2. Collect and count HUVEC cells after digestion. Wash three times with PBS to remove serum. Resuspend the cells in serum-free medium and adjust the cell concentration to 8 × 10⁻⁶. 4 per ml.

[0088] 3. Add 300 μl of cell suspension to each tube of drug-containing culture medium. HUVEC cells are very easy to clump together after digestion. During the addition process, continuously pipette the cells to make them uniform. After adding, pipette the cells and drugs to mix them evenly.

[0089] 4. Using a 96-well plate, add 100 μl of the mixed drug-containing cell suspension to each well of the plate, with 5 parallel wells per group.

[0090] 5. After culturing in the incubator for 2 hours, the original culture medium was aspirated and the cells were washed three times with PBS to remove non-adherent cells.

[0091] 7. Add 90 μL of PBS and 10 μL of CCK-8 reagent to each well and incubate in the dark for 2 hours.

[0092] 8. Detection of OD using an ELISA reader 450 The experimental data were analyzed using GraphPad Prism 9.5 software.

[0093] result:

[0094] like Figure 5 As shown, the precursor P-F4 peptide group did not inhibit adhesion, while the SPK-PF4 peptide group had the strongest inhibitory effect among the experimental groups, indicating that this peptide can effectively block the function of HUVEC cells in integrin-mediated cell adhesion.

[0095] Example 6: Effects of SPK-PF4 bifunctional peptide on tumor cell migration

[0096] The steps regarding the effect on cell migration are as described in Example 4.

[0097] result:

[0098] like Figure 6 As shown, the P-F4 group did not inhibit cell migration, while the SPK-PF4 group showed significant inhibitory effects and also inhibited cell-to-cell contact, indicating that the bifunctional peptide SPK-PF4 can inhibit tumor cell migration.

[0099] Example 7: Effects of SPK-PF4 bifunctional peptide on the expression of PD-L1 on the surface of tumor cells and in total PD-L1

[0100] Flow cytometry detection of PD-L1 expression on cell surface

[0101] 1. Digest and centrifuge to collect SKOV3 cells, resuspend them, and adjust the cell density to 1×10⁻⁶. 5 per ml.

[0102] 2. Using a 24-well plate, add 100 μl of cell suspension to each well, add 400 μl of culture medium, and gently blow the cells to mix them overnight for adhesion.

[0103] 3. Dilute the polypeptide stock solution prepared in DMSO to 100 μM with medium containing 1000 U / ml hIFN-γ, and dilute silengitide to 1 μM.

[0104] 4. Discard the culture medium in the well plate, add 500 μl of culture medium containing peptide and hIFN-γ to each well, set up a blank control with only blank culture medium, a negative control with only hIFN-γ, and a solvent control with hIFN-γ and DMSO. Set up 3 replicates for each group and incubate for 24 h.

[0105] 5. Digest and collect cells from each well separately, wash three times with pre-cooled PBS, and resuspend in 200 μL of 2% BSA / PBS for the last wash.

[0106] 6. Add 2 μl of APC-labeled anti-hPD-L1 antibody to each tube of cells and incubate in the dark for 30 min.

[0107] 7. Wash three times with pre-cooled 2% BSA / PBS, and resuspend the cells in 200 μL of PBS for the last wash. Analyze the cells using flow cytometry and use Flowjo 10 for analysis.

[0108] Western blot analysis of changes in total PD-L1 expression in cells before and after treatment

[0109] 1. Simultaneously, tumor cell lines SKOV3 and A549, which highly express the β3 subunit, were cultured in 6cm cell plates.

[0110] 2. Digest and centrifuge the cells separately to collect 2 × 10⁻⁶ cells per cell. 6 Each cell was washed twice with pre-cooled PBS.

[0111] 3. Remove as much supernatant as possible, add 200 μl of pre-chilled RIPA (20 μl of 100 PMSF) lysis buffer to the cell pellet, and mix well by pipetting. Incubate on ice for 30 min, vortexing once every five minutes.

[0112] 4. Centrifuge at 14000g, 4℃ for 15 minutes, and collect the supernatant.

[0113] 5. Take the supernatant of the lysis buffer and quantify it using the BCA kit. Dilute the sample to the same concentration with PBS, then mix it thoroughly with Loading buffer and β-mercaptoethanol at a ratio of 40:10:1. Boil for 10 minutes, then centrifuge briefly before use.

[0114] 6. SDS-PAGE: Prepare a 10% separating gel and a 5% stacking gel according to the gel preparation kit instructions. Add 700 ml of electrophoresis buffer (for both gels). Add different volumes of sample and 5 μl of tri-color pre-stained marker to the sample wells according to the fixed total protein content. Initially, maintain a constant voltage of 80V. After the samples reach the separating gel layer, switch to a constant voltage of 120V. After approximately 80 minutes, the samples will reach the bottom of the gel.

[0115] 7. Carefully peel off the separating gel. Cut the PVDF membrane and hybridization filter paper to appropriate sizes. Activate the PVDF membrane in methanol solution for 15 seconds. Then, from cathode to anode, sandwich the membrane into the transfer plate in the following order: three layers of filter paper - gel - PVDF membrane - three layers of filter paper. Next, place the prepared transfer plate into the transfer tank and add the pre-prepared transfer buffer (containing 20% ​​methanol). Turn on the power and maintain a constant current of 200mA at 4°C. Determine the specific transfer time based on the protein molecular weight.

[0116] 8. Sealing: After the transfer, PVDF is immersed in 5% skim milk powder and shaken at low speed at 37°C for 2 hours in a shaker.

[0117] 9. Antibody incubation: After blocking, cut the target band from the PVDF membrane according to the pre-stained marker and immerse it in diluted primary antibody solution for overnight incubation at 4°C. Immerse the overnight incubated band in TBST and wash three times on a low-speed shaker for 5 minutes each time. Then immerse the band in diluted HRP-tagged secondary antibody solution and incubate at room temperature for 2 hours.

[0118] 10. Exposure and color development: Immerse the incubated bands in TBST and wash three times on a low-speed shaker for 10 minutes each time. Finally, prepare the color development solution according to the instructions, add it evenly to the surface of the membrane protein, and observe and photograph it under a chemiluminescence detector.

[0119] result:

[0120] like Figure 7 As shown, Figure 7 Ablation showed that IFNγ stimulation significantly increased PD-L1 expression on the surface of SKOV3 cells. The SPK-PF4 peptide group effectively inhibited PD-L1 expression, while the P-F4 group did not. To further verify the peptide activity, Western blotting was used to detect changes in overall PD-L1 expression in cells. Figure 8C showed that SPK-PF4 could reduce the expression of total PD-L1 in multiple cell lines.

[0121] Example 8: Effect of SPK-PF4 bifunctional peptide on tumor apoptosis

[0122] 1. Collect SKOV3 cells by digestion, resuspend the cells, and adjust the cell density to 2×10⁻⁶. 6 per ml.

[0123] 2. Use a 24-well plate, add 1×10 to each well. 5 Cells. Dilute the DMSO-prepared peptide stock solution to 200 μM with culture medium, and dilute silengitide to 2 μM, then store in 1 ml EP tubes for later use.

[0124] 3. Add 250 μl of drug-containing culture medium to the cells and bring the volume up to 500 μl. Set up a blank culture medium as a negative control and DMSO of the same concentration as a solvent control. Incubate at 37°C and 5% CO2 for 24 h.

[0125] 4. After incubation, collect the culture medium from each well into a centrifuge tube. After digesting the SKOV3 cells, stop the digestion with the original culture medium and collect the cells by centrifugation.

[0126] 6. Wash three times with pre-chilled PBS, centrifuge at 1500 rpm for 3 min. Resuspend the cells in pre-chilled PBS / 2% BSA, add 100 μL of 1×Binding Buffer to each tube, and gently mix until a single-cell suspension is formed.

[0127] 7. Add 5 μL Annexin V-FITC and PI Staining Solution to each tube, mix well, and incubate at room temperature for 15 min in the dark.

[0128] 8. Add 400 μL of 1×Binding Buffer to each tube, mix well, and all samples must be detected by flow cytometry within 1 hour after staining.

[0129] 9. Use Flowjo 10 software to analyze streaming data.

[0130] result:

[0131] like Figure 8 As shown, Figure 8 B showed that the experimental group could slightly increase the early apoptosis rate of SKOV3 cells, although the difference was not statistically significant. Figure 8C showed that the SPK-PF4 experimental group also increased the proportion of late apoptosis or necrosis of tumor cells, while the P-F4 group did not show a promoting effect on tumor apoptosis, indicating that the bifunctional peptide can affect integrin-mediated apoptosis. Example 9: Study on the restoration of spleen cell killing activity by the SPK-PF4 bifunctional peptide.

[0132] 1. The mice, which had not undergone any treatment, were euthanized by dislocation of their necks and then immersed in 75% ethanol.

[0133] 2. In a clean bench, dissect the mouse and remove the complete spleen. Rinse it thoroughly in a pre-prepared petri dish containing PBS.

[0134] 3. Using a 1mL sterile syringe, draw up clean PBS, insert the needle into the spleen (be careful not to puncture the spleen), inject PBS into the spleen to wash out the spleen cells, place them in a new petri dish, and repeat several times.

[0135] 4. Centrifuge at 4℃ and 2000 rpm for 5 min, discard the supernatant, resuspend in red blood cell lysis buffer, lyse for 1-2 min, repeat centrifugation and discard the supernatant until no red blood cell precipitate is found at the bottom after centrifugation.

[0136] 5. Resuspend in 1640+10% FBS medium, add PHA to the medium to a final concentration of 10μM / mL, and stimulate culture for 24h before use.

[0137] 6. SKOV3 cells were selected as target cells and cultured under the following conditions: 1640 + 10% FBS, 37℃, 5% CO2, and passaged at a ratio of 1:2.

[0138] 7. Digest and collect cells, resuspend the cells, and adjust the cell density to 2×10⁻⁶. 6 per ml.

[0139] 8. Use a 24-well plate, add 1×10⁻⁶ ppm to each well. 5 Cells were stimulated with hIFN-γ to a final concentration of 500 U / ml for 24 hours before use.

[0140] 9. Centrifuge at 2000 rpm for 5 min, discard the supernatant, remove PHA, collect and count spleen cells, and adjust the cell concentration to 2 × 10⁻⁶. 7 per ml.

[0141] 10. After stimulation and adhesion of SKOV3 cells in a 24-well plate, the supernatant was aspirated, and 2 × 10⁶ cells were added to each well. 6 One mouse spleen cell.

[0142] 11. Dilute the DMSO-prepared polypeptide stock solution to 200 μM with culture medium, dilute silengitide to 2 μM, and store in 1 ml EP tubes for later use.

[0143] 12. Add 250 μl of drug-containing culture medium to the co-incubated cells and bring the volume up to 500 μl. Set up a blank control group without spleen cells, use blank culture medium as a negative control, and use DMSO of the same concentration as a solvent control. Incubate at 37°C and 5% CO2 for 24 h.

[0144] 13. After incubation, collect the culture medium from the wells into centrifuge tubes. After digesting the SKOV3 cells, stop the digestion with the original culture medium and collect the cells by centrifugation.

[0145] 14. Wash three times with pre-cooled PBS, then centrifuge at 1500 rpm for 3 min.

[0146] 15. The cells were finally resuspended in pre-cooled PBS / 2% BSA. 100 μL of 1×Binding Buffer was added to each tube and gently mixed until a single-cell suspension was formed.

[0147] 16. Add 5 μL Annexin V-FITC and PI Staining Solution to each tube, mix well, and incubate at room temperature for 15 min in the dark.

[0148] 17. Add 400 μL of 1×Binding Buffer to each tube, mix well, and all samples must be detected by flow cytometry within 1 hour after staining.

[0149] Flowjo 10 software was used to analyze streaming data.

[0150] result:

[0151] like Figure 9 As shown, after the addition of immune cells, the proportion of tumor cells undergoing either early or late apoptosis was increased. Compared to the single-drug administration experiment, the experimental group of peptides enhanced the apoptosis-promoting effect on tumor cells in the co-incubation experiment, and the effect was superior to P-F4. In summary, these experiments indicate that SPK-PF4 has the best anti-tumor effect.

[0152] Example 10: Effects of SPK-PF4 bifunctional peptide on a mouse colorectal cancer model

[0153] 1. The precursor peptide P-F4 has extremely poor water solubility, making it difficult to conduct direct animal experiments. SPK-PF4, however, exhibits significantly improved water solubility compared to the precursor peptide. Female Balb / c mice aged 4-6 weeks were randomly divided into six groups of five mice each. The groups included: ① negative control group ② positive drug group ③ SPK-PF4 group. The negative control group received intratumoral administration with physiological saline, the positive control group received 3 mg / kg of commercially available DOX·HCl via tail vein, and the SPK-PF4 group received 10 mg / kg via intratumoral administration. The peptide group received the drug once daily, and the positive control group received the drug every two days. All administration was via tail vein (iv). The drug concentration was diluted, and each administration volume was approximately 100 μL (the actual dosage was slightly adjusted according to the mouse's actual body weight). During the administration period, tumor volume was measured daily using the formula (V = L*W² / 2, where L represents the longest diameter of the tumor and W represents the shortest diameter perpendicular to the longest diameter), and mouse body weight was monitored.

[0154] 2. Digest and centrifuge the pre-cultured cells to remove the culture medium. Tap the bottom of the tube with your fingers to prevent cell clumping. Wash the cells 2-3 times with sterile PBS to remove trypsin and culture medium. Resuspend the cells in PBS, count the cells, and maintain a cell density of 1×10⁻⁶ cells using PBS. 6 100 μL of single-cell suspension (1 × 10⁶ cells / mL) was subcutaneously injected into mice (near the mammary pad, an area rich in blood vessels and prone to tumor formation). 5 (The cells are resuspended in the EP tube every three to five minutes to ensure uniform cell number.) After inoculation, the cells are fed in an SPF-grade environment, and tumor formation is expected in about 10 days.

[0155] 3. After administering the final dose of medication to the mice, and measuring their tumor volume and body weight, the mice were euthanized. Their organs (heart, liver, spleen, and kidneys) were removed in a clean bench and immersed in physiological saline. Before weighing, the organs were first dried with filter paper. The ratio of the weight of the mouse's organs (heart, liver, spleen, and kidneys) to its body weight was used as the organ (heart, liver, spleen, and kidney) index to determine the drug's toxicity to the organs.

[0156] 4. Place the removed spleen on a 200-mesh sterile sieve in a laminar flow hood, grind the spleen with the rubber stopper of a sterile 5mL syringe, and then sieve it with physiological saline. Repeat this process several times until there are no large pieces of tissue on the sieve.

[0157] 5. Pre-cool the centrifuge to 4°C, collect the mouse spleen lymphocyte suspension and centrifuge at 1000 rpm for 5 min. Discard the supernatant and turbid liquid, tap the bottom of the tube with your finger to prevent the cells from clumping, and wash once with sterile PBS. The operation is the same as above.

[0158] After centrifugation, a blood-red precipitate appeared at the bottom of the centrifuge tube. 1 ml of erythrocyte lysis buffer was added to the tube for erythrocyte lysis treatment, and the tube was incubated at room temperature for 10 min. Then, the cell supernatant was removed by centrifugation at 1000 rpm for 5 min. The tube was then washed 1-2 times with sterile PBS, sealed with sealing film, and transferred to the cell culture chamber for further processing.

[0159] 6. Culture CT-26 cells. After their growth stabilizes, digest them with trypsin, wash them once with sterile PBS, and then resuspend the CT-26 cells in complete culture medium for cell counting.

[0160] 7. Count the previously collected spleen cells and resuspend them in complete culture medium. Co-incubate effector cells and target cells at E:T ratios of 10:1, 50:1, and 100:1 (the total number of effector cells and target cells in this co-incubation system is 2 × 10⁻⁶ cells). 5 The total volume was set to 200 μL, with 3-5 replicates per group. PBS was added to the outermost ring of the 96-well plate to avoid edge effects. Blank background wells were filled with culture medium but no cells. Individual CT-26 cell wells were used as spontaneous release wells without cell lysis buffer. Individual CT-26 cell wells were used as maximum enzyme activity wells after adding cell lysis buffer. Maximum release wells (CT-26 + LDH release reagent) and spontaneous release wells (CT-26 only) and background correction blank control wells (complete culture medium) were set up under different effector-to-target ratios. The well plates were placed in a cell culture incubator and cultured for 6 hours.

[0161] 8. One hour before the end of the incubation period, add LDH release reagent (10% by volume) to the largest enzyme well, repeatedly pipette, and return to the incubator.

[0162] 9. After 6 hours of incubation, place the culture plate in a horizontal centrifuge and centrifuge at 1000 rpm for 5 minutes to collect all cells. Use a pipette tip to transfer 60 μL of the supernatant culture medium to a new cell culture plate (to avoid cell interference with the final reading).

[0163] 10. Prepare the LDH detection working solution (lactic acid: 1×INT: enzyme = 1:1:1) according to the sample volume. Add 30 μL to each well and mix thoroughly, avoiding air bubbles. Wrap the solution in aluminum foil and gently shake on a shaker. Incubate at room temperature for 30 min. Then perform dual-wavelength measurement at 490 / 630 nm, using OD... 490 -OD 630 Perform calibration.

[0164] The LDH release rate (i.e., target cell killing rate) was calculated using the following formula, and statistical analysis was performed using the t-test to compare the differences between groups (n=3-5 mice per group).

[0165] LDH release rate = (sample wells - spontaneous release wells - blank background) / (maximum release wells - spontaneous release wells - blank background) × 100%

[0166] result:

[0167] Figure 10 Tumor growth curves showed that the bifunctional peptide SPK-PF4 effectively inhibited tumor growth until the end of administration; the tumor volume in both the positive control DOX group and the experimental group did not exceed 500 mmHg. 3 The tumor-suppressing effect in the experimental group was consistent with that of the positive control drug DOX. Figure 10 B also showed that the tumor weight in the experimental group was significantly smaller than that in the blank control group. Figure 10 The body weight curves and organ weight diagrams of mice C and D show that neither the experimental group nor the blank control group experienced significant fluctuations in body weight throughout the experiment, indicating that neither had any effect on the survival of the mice. The positive experimental group experienced a significant decrease in body weight after administration, which further demonstrates that DOX has severe drug toxicity, while the bifunctional peptide has good safety. Figure 10 The E-splenic cell killing experiment showed that the killing effect of the experimental group was much higher than that of the other two groups. It is worth noting that the killing rate of the DOX group was always the lowest, indicating that DOX seriously damaged the killing function of splenic lymphocytes. The bifunctional peptide can effectively improve the immunosuppressive microenvironment in vivo and activate lymphocytes to further kill tumor cells.

[0168] Example 11: Immunohistochemical study on the regulation of the tumor immune microenvironment by SPK-PF4 bifunctional peptide

[0169] 1. The excised tumor tissue was soaked in paraformaldehyde for up to 24 hours, and then delivered to the company for paraffin embedding and sectioning, with a section thickness of 4 μM.

[0170] 2. Bake the sections at 60℃ for 30 minutes to melt the wax layer. In a fume hood, immerse the sections in xylene I and xylene II for 10 minutes each to dewax them. Then, immerse them in alcohol solutions of 100%, 90%, 80%, 70%, 60%, 50%, and 30% concentrations for 5 minutes each, followed by rinsing with tap water for 10 minutes.

[0171] 3. Permeabilize the sections with 0.3% Triton X-100 to enhance the staining effect of intracellular antigens. Rinse three times with PBS, 5 min each time.

[0172] 4. Immerse the slides completely in EDTA antigen retrieval solution (pH 9.0) and place them in a microwave oven for antigen retrieval. Microwave at 92-98℃ in a gentle boiling state for 15-20 minutes. Then remove the slides and allow them to return to room temperature. Rinse three times with PBS for 5 minutes each time.

[0173] 5. Treat the slides with 3% H2O2 for 15 min to reduce endogenous peroxidase activity and lower background signal. Rinse with PBS three times with shaking, 5 min each time.

[0174] 6. Draw a closed hydrophobic circle around the tissue using a histochemical pen. Add 5% BSA to cover the tissue and block at room temperature for 2 hours. Then dilute the primary antibody (CD41: 500, CD81: 500, Foxp31: 500) with 5% BSA, cover the tissue, place the slide in a humidified chamber, add an appropriate amount of water to the bottom, and incubate overnight at 4°C.

[0175] 7. First, rinse the sections with PBS with shaking for 5 minutes each time. Then, dilute the secondary antibody containing HRP according to the dilution ratio in the instructions, add it to the histochemistry zone, and cover the tissue. Incubate at room temperature for 1 hour, and then repeat the washing steps.

[0176] 8. Remove excess water from the tissue sections, add freshly prepared DAB solution to the tissue surface, and develop the stain in the dark for 3-5 minutes. Discard the staining solution and rinse the sections with double-distilled water to stop the development process.

[0177] 9. Counterstain sections with hematoxylin for 3-5 minutes, then rinse with running water. Differentiate with 1% hydrochloric acid ethanol for a few seconds, then rinse slightly with tap water to stop the differentiation. Use 0.6% ammonia water to make the hematoxylin turn blue, then rinse several times with running water.

[0178] 10. Immerse the slides sequentially in 30%, 50%, 60%, 70%, 80%, 90%, and anhydrous ethanol to dehydrate them, air dry, add a drop of mounting medium, cover with a coverslip, and examine under a microscope.

[0179] Images (7–9 fields of view) were selected from various parts of the panoramic scan, with a scale bar of 200 μM. ImageJ was used for positive image analysis, and One-Way ANOVA was used for statistical analysis to compare differences between groups.

[0180] result

[0181] like Figure 11 As shown: CD31 is a marker of angiogenesis. Staining with CD31 revealed a statistically significant reduction in angiogenesis in the SPK-PF4 group of tumor tissue compared to both the positive control and the blank control groups, indicating that the peptide can inhibit angiogenesis by suppressing integrin αvβ3 in vascular endothelial cells; CD4 is a marker of T helper cells. Figure 11 B showed that the infiltration of CD4+ T cells in the experimental group was significantly higher than that in the two control groups. CD8 is a marker of cytotoxic T cells (CTLs). Figure 11C showed that CD8+ T cell infiltration in the experimental group was also significantly higher than in the two control groups. Foxp3 is a marker for regulating T cells (Tregs). Figure 11 The D-scan showed that the Foxp3 content in the experimental group was lower than that in the control group, indicating that the infiltration rate of regulatory T cells was lower in the experimental group, CD4+ and CD8+ T cell infiltration increased, and Foxp3+ T cell infiltration was less, indicating that the bifunctional peptide can activate the immune system and create an immune microenvironment that is conducive to killing tumors.

Claims

1. A PD-1 and ανβ3 bifunctional polypeptide, characterized in that, The amino acid sequence of the polypeptide is: GRGDSPKGGGGFSGTVTTAGLLF, as shown in SEQ ID NO.

1.

2. Use of the polypeptide according to claim 1 for the preparation of a medicament for the treatment of tumors, characterized in that, The tumor is colorectal cancer.

Images