PD-1 and alpha v beta 3 bifunctional polypeptide and application thereof

Abstract

The invention relates to a polypeptide, in particular to a PD-1 and alpha v beta 3 bifunctional polypeptide and application thereof, and the amino acid sequence of the polypeptide is shown as SEQ ID NO.1. The PD-1 / alpha v beta 3 dual-targeting polypeptide can simultaneously inhibit a PD-1 / PD-L1 pathway and an alpha v beta 3 pathway, not only can inhibit the growth and invasion of tumors and inhibit the abnormal generation of tumor blood vessels, but also can promote the infiltration of immune cells to tumors and regulate the tumor microenvironment by blocking the PD-1 pathway, inhibiting the generation of the blood vessels and regulating the expression of PD-L1 so as to play an immune synergistic effect.

Description

Technical Field

[0001] The invention relates to a polypeptide, specifically to a PD-1 and αvβ3 bifunctional polypeptide and its application

[0002] Background

[0003] Overexpression of immune checkpoint proteins by tumor cells can inhibit the activity of immune cells, thereby escaping the surveillance of the immune system. PD-1 was first discovered in 1992 and is expressed on the cell 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 can induce tumor immune escape. When PD-1 is activated, it can induce intracellular signaling pathways, thereby inhibiting T cell proliferation, cytokine secretion, and cell killing function. PD-1 and PD-L1 play an important role in the negative regulation of immune responses. By blocking the binding of PD-1 and PD-L1, the function of T cells 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, nanoparticles are used for intratumoral administration. At the same time, PD-1 is also expressed to a certain extent in normal tissues and is an important component for maintaining the homeostasis of the immune system. Single targeted blocking of PD-1 may lead to some adverse immune reactions. Therefore, strengthening its targeting and improving its water solubility are issues that need to be urgently addressed.

[0004] Integrins are transmembrane cell surface heterodimeric proteins composed of two subunits, α and β. They mainly mediate signal transduction between cells and the extracellular environment (such as the extracellular matrix (ECM), regulate the proliferation and survival of tissue cells and the angiogenesis function of endothelial cells. Currently, 18 α subunits and 8 β subunits have been found in mammals, which together form 24 different integrin dimers, each of which has functional and tissue specificity. Integrins can be divided into four categories based on the type of ligand recognized: (1) receptors that recognize the tripeptide RGD (Arg-Gly-Asp) sequence; (2) receptors with (1) Receptors that are specific to leukocytes and bind to the LDV (L / ID / EV / S / TP / S) sequence; (2) Receptors that selectively bind to laminin; (3) Collagen binding receptors (including four β1 integrins (α1, α2, α10, α11) that recognize the GFOGER motif. Among them, αvβ3 belongs to the first type of integrin that recognizes 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 currently no αvβ3 antagonists have been approved for treatment.

[0005] In previous studies, integrin αvβ3 often focused on tumor development, epithelial-mesenchymal transition (EMT), tumor angiogenesis, and drug resistance. Recent studies have shown that integrin αvβ3 can participate in tumor immune escape by regulating the expression of PD-L1 on the surface of tumor cells. Silencing the β3 subunit or administering the specific inhibitor cilengitide can reduce the expression of PD-L1 by tumor cells stimulated by interferon. At the same time, it was found that integrin αvβ3 plays a role by activating the IFNR downstream pathway. In the mouse melanoma model, by silencing the β3 subunit or administering cilengitide, it was found that the expression of PD-L1 in tumor tissues was reduced and the infiltration of CD8+T cells and the level of IFN-γ in tumor tissues were enhanced, and a higher level of efficacy was shown after combining with Anti-PD-1 antibodies. However, this experiment did not extend the experimental results to more types. Blocking integrins and inhibiting PD-1 are achieved in two ways, and there are certain drug interactions.

[0006] Based on the above research background, the present invention combines the inhibition of integrin αvβ3 with the immune checkpoint blocking principle to design a PD-1 / αvβ3 dual-targeting peptide to solve the problem of the existence of PD-1. Summary of the invention

[0007] Purpose of the Invention

[0008] The present invention connects the sequence specifically targeting integrin αvβ3 with the targeted PD-1 blocking peptide P-F4 through a linker to form a new bifunctional polypeptide.

[0009] Technical Solution

[0010] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows:

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

[0012] The polypeptide is used in the preparation of anti-tumor drugs.

[0013] The tumor is colorectal cancer

[0014] The details are as follows:

[0015] (1) Peptide design: In order to retain the activity of the PD-1 antagonist peptide P-F4 and the integrin targeting peptide, different numbers of Gly were selected as linkers, and the "RGD" sequence was connected to the N-terminus and C-terminus of P-F4, respectively. The relative blocking activity of the peptide relative to P-F4 was detected by molecular docking and HTRF kit. The molecular docking model showed that the linker had an effect on the activity, among which PF4-RGD5 and PF4-RGD11 had the best docking results. Further blocking experiments verified that PF4-RGD5 completely retained 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 was greatly reduced, indicating that the modification method of coupling the N-terminus of the P-F4 peptide with the RGD sequence through a linker can retain the blocking ability of the original peptide against PD-1 to the greatest extent.

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

[0017] (2) Activity studies at the cell level: Detect the effect of SPK-PF4 on the proliferation, migration and adhesion of vascular endothelial cells (HUVEC); detect the effect of SPK-PF4 on the migration and apoptosis of tumor cells; detect the regulatory effect of SPK-PF4 on the PD-L1 level of tumor cells, and detect the restorative effect of SPK-PF4 on the killing activity of spleen cells.

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

[0019] Beneficial Effects

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

[0021] Specifically: The present invention designs a polypeptide that can inhibit the migration and adhesion of vascular endothelial cells at the cellular level, and can also inhibit the migration of tumor cells and the expression of PD-L1, promote the apoptosis of tumor cells, and enhance the killing effect of spleen cells on tumor cells. In the mouse colorectal cancer model, the polypeptide can effectively inhibit the growth of tumors and the formation of new blood vessels inside tumors, while overcoming the disadvantage of poor water solubility of the precursor polypeptide that is difficult to administer.

[0022] Compared with the original PD-1 blocking peptide P-F4, this peptide has the following advantages:

[0023] 1. Due to the high expression of αvβ3 in tumor sites, dual-targeting peptides can specifically target tumor sites;

[0024] 2. Regulate the expression of PD-L1 by blocking the αvβ3 pathway, and play an immune synergistic role with the PD-1 pathway blockade;

[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, contains a large number of hydrophobic amino acids, and has poor water solubility. αvβ3-targeted peptide modification can effectively improve the water solubility of P-F4. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 is the blocking rate of PF4-RGD1, PF4-RGD5, and PF4-RGD11 on PD-1 / PD-L1 relative to P-F4 in the present invention;

[0029] Figure 2 is the blocking rate of SPK-PF4 on PD-1 / PD-L1 relative to P-F4 in the present invention;

[0030] Figure 3 The cytotoxic effect of SPK-PF4 on HUVEC at different concentrations in the present invention is: Figure 3 A is the effect at 50 μM concentration; Figure 3 B is the effect at a concentration of 100 μM;

[0031] Figure 4 The effect of SPK-PF4 at different concentrations on HUVEC cell migration in the present invention is: Figure 4 A is the statistical data of HUVEC cell migration area after 50 μM peptide treatment; Figure 4B is the statistical data of HUVEC cell migration area after 100 μM peptide treatment; Figure 4 C is the comparison of HUVEC cell scratches after 100 μM peptide treatment;

[0032] Figure 5 It is the effect of the SPK-PF4 polypeptide of the present invention on HUVEC cell adhesion;

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

[0034] Figure 7 The effect of the SPK-PF4 polypeptide of the present invention on the expression of PD-L1 on the surface of tumor cells and total PD-L1: Figure 7 A is the expression level of PD-L1 on the surface of SKOV3 cells after treatment with 100 μM peptide; Figure 7 B is the statistical result of PD-L1 expression on the surface of SKOV3 cells after treatment with 100 μM peptide; Figure 7 C is the total PD-L1 expression level of SKOV3 and A549 cells after 100 μM peptide treatment; Figure 8 The effect of the SPK-PF4 polypeptide of the present invention on apoptosis of SKOV3 cells: Figure 8 A is the apoptosis level of SKOV3 cells after treatment with 100 μM peptide; Figure 8 B is the statistical result of the early apoptosis ratio of SKOV3 cells after treatment with 100 μM peptide; Figure 8 C is the statistical result of the proportion of late apoptosis and necrosis of SKOV3 cells after treatment with 100 μM peptide;

[0035] Fig. 9 The effect of the SPK-PF4 polypeptide of the present invention on the recovery of splenocyte activity: Fig. 9 A is the apoptosis level of SKOV3 cells after co-incubation of SKOV3 cells and spleen cells and treatment with 100 μM peptide; Fig. 9 B is the statistical result of the early apoptosis ratio of SKOV3 cells after co-incubation and treatment with 100 μM peptide; Fig. 9 C is the statistical result of the proportion of late apoptosis and necrosis of SKOV3 cells after co-incubation and treatment with 100 μM peptide;

[0036] Fig.10 The results of the in vivo pharmacodynamics experiment of the SPK-PF4 polypeptide of the present invention are as follows: Fig.10 A is a graph of tumor growth; Fig.10 B is a statistical graph of tumor weight; Fig.10 C is a graph showing the body weight of mice during the drug administration period; Fig.10 D is a statistical graph of the weight of the core organs of mice;

[0037] Fig.10 E is the detection and statistical analysis of the killing ability of spleen lymphocytes of each group of mice at different effector-target ratios;

[0038] Fig.11 The effect of the SPK-PF4 polypeptide of the present invention on the tumor microenvironment: Fig.11 A is the expression of CD31+ vascular endothelial cells in immunohistochemistry and statistical analysis; Fig.11 B is the infiltration of CD8+T cells in immunohistochemistry and statistical analysis; Fig.11 C is the infiltration of CD4+T cells in immunohistochemistry and statistical analysis; Fig.11 D is the infiltration of Foxp3+T cells in immunohistochemistry and statistical analysis. DETAILED DESCRIPTION

[0039] The present invention will be further described in detail below in conjunction with the examples; however, it should be understood that these examples are only for illustrative purposes and are not intended to limit the present invention; under the premise of the concept of the present invention, the connection modification of the precursor polypeptide P-F4 with other targeting integrin sequences all fall within the scope of protection claimed by the present invention.

[0040] The polypeptides SPK-PF4, PF4-RGD1, PF4-RGD5, PF4-RGD11 and P-F4 involved in the present invention are synthesized by Shanghai Bioengineering.

[0041] Example 1 Preliminary design of RGD-PF4 bifunctional polypeptide and relative activity determination of blocking PD-1 / PD-L1 pathway Activity verification 1. In order to retain the activity of PD-1 antagonist peptide P-F4 and targeting integrin sequence polypeptide as much as possible, different numbers of Gly were selected as linkers, and the RGD sequence was connected to the N-terminus and C-terminus of P-F4, respectively. 12 polypeptides were designed and 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.

[0042] 2. Retrieve the PD-1 protein crystal structure from the PDB (protein database bank) database, PDB ID: 3RRQ, delete the H2O molecule. Based on the laboratory's previous research on the P-F4 peptide, the MDockPeP docking software platform was selected to simulate the docking of the P-F4 peptide with the PD-1 protein crystal structure.

[0043] 3. Select 12 peptide docking postures around the hotspot residues binding to PD-L1 on the PD-1 protein and the model with the highest absolute score of ITScorePeP (MDockPeP has its own scoring system, the higher the absolute value, the better), and record its score.

[0044] result:

[0045] As shown in Table 1, PF4-RGD5 and PF4-RGD11 had the best docking results.

[0046] Table 1 Scoring of docking of PF4-RGD1~PF4-RGD12 with PD-1 protein molecules

[0047]

[0048] 4. As shown in Table 1, the score after N-terminal coupling is significantly higher than that after C-terminal coupling, so PF4-RGD1 was selected as a candidate peptide and its blocking activity relative to P-F4 was tested. Prepare PD-1 / PD-L1 HTRF kit (Tag1-PD-L1, Tag2-PD-1, AntiTag1-Eu3+, Anti-Tag2-XL665) and thaw on ice.

[0049] 5. Use the buffer provided by the kit to dilute Tag1-PD-L1 and Tag2-PD-1 100 times to obtain working solutions for use. Use the buffer to dilute Anti-Tag1-Eu3+ and Anti-Tag2-XL665 100 times and pre-mix them at a ratio of 1:1 to prepare working solutions for use.

[0050] 6. Prepare the peptide to be tested into a 600μM peptide solution with PBS, and keep the DMSO concentration at 5%. Add 2μL of the peptide solution to be tested to each well of a low-volume 96-well plate, set up 3 replicate wells for each peptide, 4μL Tag1-PD-L1 and 4μLTag2-PD-1, mix well and incubate at room temperature for 15min. The experimental group without Tag1-PD-L1 was used as the positive control, PBS was used as the negative control instead of the peptide solution, P-F4 was used as the experimental control, and the group with less than 10μL was supplemented with Buffer.

[0051] 7. Add 10 μL of pre-mixed Anti-Tag1-Eu3+ and Anti-Tag2-XL665 to each well, seal with a sealing film, mix evenly, centrifuge, and incubate at room temperature for 2 hours.

[0052] 8. After incubation, use EnVision multi-function microplate reader to measure the emission signal value of each well at 320nm excitation, 665nm and 620nm emission.

[0053] 9. Calculate the emission signal ratio, inhibition rate and relative inhibition rate of each well according to the following formula, and use Graphpad 9.5 software for data analysis.

[0054] Ratio=(Signal 665nm / Signal 620nm )×10 4

[0055] Inhibition Rate=(Ratio 阴性 -Ratio 实验 ) / (Ratio 阴性 -Ratio 阳性) ×100%

[0056] Relative Inhibition Rate=Inhibition Rate 实验 / Inhibition Rate 实验对照

[0057] result:

[0058] like Figure 1 As shown, PF4-RGD5 completely retained the blocking ability of the P-F4 polypeptide, while the blocking ability of PF4-RGD1 without a linker and PF4-RGD11 coupled to the C-terminus was greatly reduced, indicating that the modification method of coupling the N-terminus of the P-F4 polypeptide with the RGD sequence through a linker can retain the blocking ability of the original polypeptide against PD-1 to the greatest extent.

[0059] Example 2: Design of SPK-PF4 bifunctional peptide and determination of relative activity of blocking PD-1 / PD-L1 pathway

[0060] In order to further enhance the ability of the peptide to target integrin αvβ3, the "RGD" tripeptide sequence in PF4-RGD5 was replaced with a peptide sequence "GRGDSPK" with specific targeting, and coupled with the precursor peptide P-F4 through a linker to form a bifunctional peptide SPK-PF4. The blocking ability of the bifunctional peptide relative to P-F4 on PD-1 / PD-L1 was verified by HTRF, and the experimental steps were the same as those in Example 1;

[0061] result:

[0062] like Figure 2 As shown, the relative blocking rate of SPK-PF4 is 74.06%, indicating that the bifunctional peptide retains the ability to block PD-1 / PD-L1 binding at the molecular level.

[0063] Example 3: Cytotoxicity of SPK-PF4 bifunctional polypeptide to HUVEC cells

[0064] 1. Digest and collect HUVEC cells and count them. After resuspending, adjust the cell concentration to 4×10 4 Pieces / ml.

[0065] 2. Use a 96-well plate, add 100 μl of cell suspension to each well, and allow cells to adhere overnight.

[0066] 3. Dilute the DMSO-prepared peptide stock solution to 50μM and 100μM with complete culture medium, dilute the positive drug Cilengitide to 0.5μM and 1μM, prepare 600μl of each peptide-containing culture medium in a 1ml EP tube for later use. Set the DMSO group as the solvent control.

[0067] 4. Aspirate the culture medium in the well plate, add 100 μl of the mixed drug-containing culture medium to each well of the well plate, and place 5 parallel wells in each group in a cell culture incubator at 37°C and 5% CO2 for 24 hours.

[0068] 5. Take out the 96-well plate, add 10 μl of CCK-8 reagent to each well, and continue to culture in the incubator for 2 hours.

[0069] 6. Detect OD with ELISA 450 Value and OD 650 The cell viability was calculated according to the following formula, and the experimental data were analyzed by GraphPad Prism9.5 software.

[0070] Cell Viability (%) = (OD 450 -OD 650 ) 实验组 / (OD 450 -OD 650 ) Negative control

[0071] result:

[0072] like Figure 3 As shown, the CCK8 data showed that the two concentrations of bifunctional peptides and positive drugs had no cytotoxicity to HUVEC cells, indicating that the bifunctional peptides had good targeting properties.

[0073] Example 4: Effect of SPK-PF4 bifunctional polypeptide on HUVEC cell migration

[0074] 1. Digest and collect HUVEC cells and count them. After resuspending, adjust the cell concentration to 1.5×10 7 Pieces / ml.

[0075] 2. Use a 6-well plate and draw 6 parallel straight lines on the bottom of the well plate with a marker. Add 100 μl of cell suspension to each well, add 900 μl of culture medium, pipet evenly and culture until the cells cover the entire bottom of the well plate.

[0076] 3. Replace DMEM+10% FBS with serum-free DMEM medium and starve the cells for 12 hours.

[0077] 4. Dilute the DMSO-prepared peptide stock solution to 50 μM and 100 μM with serum-free culture medium, dilute silengitide to 0.5 μM and 1 μM, set the culture medium group as the negative control, and the 0.1% DMSO group as the solvent control.

[0078] 5. Use a 20 μL pipette tip to make three scratches in each well perpendicular to the straight line drawn before, quickly aspirate the serum-free medium and wash three times with PBS to remove cell debris.

[0079] 6. Add 1 ml of drug-containing culture medium to each well and culture at 37°C, 5% CO2 for 24 hours.

[0080] 7. Use CellSens to take pictures at 0h and 24h, and use ImageJ to analyze the scratch area. GraphPad Prism9.5 software is used to analyze the scratch area data.

[0081] result:

[0082] like Figure 4 As shown in the figure, compared with the precursor peptide P-F4, the bifunctional peptide can effectively restrict the migration of HUVEC cells, while there is no significant difference between the precursor peptide group and the control group. When the concentration of the experimental group reaches 100μM, the inhibitory effect is much stronger than that of 50μM. Therefore, in subsequent experiments, the peptide concentration of the experimental group is controlled to 100μM and the positive drug concentration is controlled to 1μM. It is worth noting that the SPK-PF4 peptide group can show similar activity in inhibiting cell contact as the positive drug, indicating that SPK-PF4 has a stronger targeted blocking function.

[0083] Experimental Example 5: Effect of SPK-PF4 bifunctional peptide on HUVEC cell adhesion

[0084] 1. Dilute the DMSO-prepared peptide stock solution to 200 μM with serum-free medium.

[0085] To 2μM, 300μl of each peptide-containing medium was prepared in a 1ml EP tube for later use. The DMSO group was set as the solvent control.

[0086] 2. HUVEC cells were digested, collected and counted, washed three times with PBS to remove serum, and resuspended in serum-free medium to adjust the cell concentration to 8×10 4 Pieces / ml.

[0087] 3. Add 300 μl of cell suspension to each tube of drug-containing culture medium. HUVEC cells are very easy to clump after digestion. During the addition process, continue to blow the cells to make the cells uniform. After adding, blow and mix the cells and drugs.

[0088] 4. Use a 96-well plate and add 100 μl of the mixed drug-containing cell suspension into each well of the plate, with 5 parallel wells in each group.

[0089] 5. After culturing in the incubator for 2 hours, discard the original culture medium and wash three times with PBS to remove non-adherent cells.

[0090] 7. Add 90ul PBS and 10μl CCK-8 reagent to each well and incubate in the incubator in the dark for 2h.

[0091] 8. Detect OD with ELISA 450 The experimental data were analyzed using GraphPad Prism 9.5 software.

[0092] result:

[0093] like Figure 5 As shown, the precursor P-F4 polypeptide group had no effect on inhibiting adhesion, and the SPK-PF4 polypeptide had the strongest inhibitory ability among the experimental groups, indicating that the polypeptide can effectively block the function of HUVEC cells mediating cell adhesion through integrin.

[0094] Example 6: Effect of SPK-PF4 bifunctional polypeptide on tumor cell migration

[0095] The steps for influencing cell migration refer to Example 4

[0096] result:

[0097] like Figure 6 As shown, the P-F4 group had no inhibitory effect on cell migration, while the SPK-PF4 group had a significant inhibitory effect and could also inhibit cell-to-cell contact, indicating that the bifunctional peptide SPK-PF4 can inhibit the migration of tumor cells.

[0098] Example 7: Effect of SPK-PF4 bifunctional polypeptide on PD-L1 expression on tumor cell surface and overall PD-L1 expression

[0099] Flow cytometry was used to detect the expression of PD-L1 on the cell surface

[0100] 1. Digest and centrifuge SKOV3 cells, resuspend, and adjust the cell density to 1×10 5 Pieces / ml.

[0101] 2. Use a 24-well plate, add 100 μl of cell suspension to each well, add 400 μl of culture medium, and gently blow the cells evenly overnight to allow them to adhere.

[0102] 3. Dilute the DMSO-prepared peptide stock solution to 100 μM with culture medium containing 1000 U / ml hIFN-γ, and dilute cilengitide to 1 μM.

[0103] 4. Aspirate the culture medium in the well plate, add 500 μl of culture medium containing polypeptide and hIFN-γ to each well, set a blank control containing only blank culture medium, a hIFN-γ group as a negative control, and a hIFN-γ and DMSO group as a solvent control. Set up 3 replicate wells for each group and culture for 24 hours.

[0104] 5. Digest and collect cells from each well, wash with pre-cooled PBS for 3 times, and resuspend with 200ul 2% BSA / PBS for the last time.

[0105] 6. Add 2 μl of APC-labeled anti-hPD-L1 antibody to each tube of cells and incubate in the dark for 30 minutes.

[0106] 7. Wash three times with pre-cooled 2% BSA / PBS, resuspend the cells in 200ul PBS for the last time, and detect by flow cytometry. The results are analyzed using Flowjo 10.

[0107] Western Blot detection of PD-L1 expression in cells before and after changes

[0108] 1. Use 6 cm cell dishes at the same time to culture the tumor cell lines SKOV3 and A549 that highly express the β3 subunit.

[0109] 2. Digest and centrifuge the cells separately, collect 2×10 6 Cells were washed twice with pre-cooled PBS.

[0110] 3. Remove the supernatant as much as possible, add 200 μl of pre-cooled RIPA (20 μl 100 PMSF) lysis buffer to the cell pellet, and pipette evenly. Lyse on ice for 30 minutes, vortexing every five minutes.

[0111] 4. Centrifuge at 14000g, 4°C for 15 min and collect the supernatant.

[0112] 5. Take the supernatant of the lysate and use the BCA kit for quantification. Add PBS to dilute the sample to the same concentration, mix it thoroughly with the loading buffer and β-mercaptoethanol in a ratio of 40:10:1, boil it for 10 minutes, centrifuge it instantly and set it aside.

[0113] 6. SDS-PAGE: Prepare 10% separation gel and 5% concentration gel according to the instructions of the gel preparation kit, add 700ml electrophoresis buffer (two gels), add different volumes of samples and 5μl three-color pre-stained markers to the sample loading wells according to the fixed total protein amount. First, keep the voltage at 80V, and change to 120V when the sample runs to the separation gel layer. After about 80 minutes, the sample runs to the bottom of the gel.

[0114] 7. Carefully peel off the separation gel, cut the PVDF membrane and hybridization filter paper to the appropriate size, activate the PVDF membrane in methanol solution for 15 seconds, and then clamp it into the transfer plate in the order of three layers of filter paper-gel-PVDF membrane-three layers of filter paper from cathode to anode. Then put the treated transfer plate into the transfer tank and add the pre-prepared transfer buffer (containing 20% ​​methanol). Turn on the power supply, constant current 200mA at 4℃, and determine the specific transfer time according to the protein molecular weight.

[0115] 8. Blocking: After transfer, immerse the PVDF in 5% skimmed milk powder and shake at low speed at 37°C in a shaker for 2 hours.

[0116] 9. Antibody incubation: After blocking, cut the target band from the PVDF membrane according to the pre-stained marker and immerse it in the diluted primary antibody solution and incubate it overnight at 4°C. Immerse the overnight incubated band in TBST and wash it on a low-speed shaker 3 times, 5 minutes each time. Then immerse the band in the diluted HRP-labeled secondary antibody solution and incubate it at room temperature for 2 hours.

[0117] 10. Exposure and color development: Immerse the incubated strips in TBST and wash them on a low-speed shaker for 3 times, 10 minutes each time. Finally, prepare the color development solution according to the instructions and evenly add it to the membrane protein surface, and place it under a chemiluminescence detector for observation and photography.

[0118] result:

[0119] like Figure 7 As shown, Figure 7 AB showed that after the SKOV3 cell line was stimulated by IFNγ, the expression of PD-L1 on the cell surface increased significantly. The SPK-PF4 peptide group could effectively inhibit the expression of PD-L1, while the P-F4 group could not inhibit the expression of PD-L1 on the tumor cell surface. In order to further verify the activity of the peptide, Western Blot was used to detect the changes in the overall PD-L1 expression of the cells. Figure 8C shows that SPK-PF4 can reduce the expression of total PD-L1 in multiple cell lines.

[0120] Example 8: Effect of SPK-PF4 bifunctional polypeptide on tumor apoptosis

[0121] 1. Digest and collect SKOV3 cells, resuspend the cells, and adjust the cell density to 2×10 6 Pieces / ml.

[0122] 2. Use a 24-well plate and add 1×10 5 The peptide stock solution prepared with DMSO was diluted to 200 μM with culture medium, and cilengitide was diluted to 2 μM, and stored in a 1 ml EP tube for later use.

[0123] 3. Add 250 μl of drug-containing culture medium to the cells and make up the volume to 500 μl. Set up blank culture medium as negative control and DMSO of the same concentration as solvent control. Incubate at 37°C, 5% CO2 for 24 h.

[0124] 4. After the incubation, collect the culture medium in each well into a centrifuge tube. After the SKOV3 cells are digested, use the original culture medium to terminate the digestion and collect the cells by centrifugation.

[0125] 6. Wash with pre-cooled PBS for 3 times, centrifuge at 1500 rpm for 3 min. Resuspend the cells in pre-cooled PBS / 2% BSA, add 100 μL 1× Binding Buffer to each tube, and gently blow to a single cell suspension.

[0126] 7. Add 5 μL Annexin V-FITC and PI Staining Solution to each tube, mix well, protect from light, and incubate at room temperature for 15 minutes.

[0127] 8. Add 400 μL 1×Binding Buffer to each tube and mix well. All samples must be detected by flow cytometry within 1 hour after staining.

[0128] 9. Use Flowjo 10 software to analyze flow cytometry data.

[0129] result:

[0130] like Figure 8 As shown, Figure 8 B shows that the experimental group can slightly increase the early apoptosis rate of SKOV3 cells, although there is no significant difference. Figure 8C shows that the SPK-PF4 experimental group can also increase the proportion of late apoptosis or necrosis of tumor cells, while the P-F4 group did not show an effect of promoting tumor apoptosis, indicating that the bifunctional polypeptide can affect integrin-mediated cell apoptosis. Example 9: Study on the restoration of spleen cell killing activity by SPK-PF4 bifunctional polypeptide

[0131] 1. The untreated mice were killed by cervical dislocation and immersed in 75% ethanol.

[0132] 2. In a clean bench, dissect the mouse, remove the complete spleen, and rinse it in a prepared dish containing PBS.

[0133] 3. Use a 1 mL sterile syringe to draw up clean PBS, insert the needle into the spleen (be careful not to puncture the spleen), inject PBS into the spleen, flush out the spleen cells, place them in a new dish, and repeat several times.

[0134] 4. Centrifuge at 4°C, 2000 rpm for 5 min, discard the supernatant, resuspend with red blood cell lysis buffer, lyse for 1-2 min, repeat centrifugation and discard the supernatant until there is no red blood cell sediment at the bottom after centrifugation.

[0135] 5. Resuspend with 1640+10% FBS medium, add PHA to the medium to a final concentration of 10 μM / mL, and use after 24 hours of stimulation and culture.

[0136] 6. Select SKOV3 cells as target cells, and culture conditions are 1640 + 10% FBS, 37°C, 5% CO2, and 1:2 passaging.

[0137] 7. Digest and collect cells, resuspend the cells, and adjust the cell density to 2×10 6 Pieces / ml.

[0138] 8. Use a 24-well plate and add 1×10 5 cells, and added hIFN-γ at a final concentration of 500 U / ml, and used after stimulation for 24 hours.

[0139] 9. Centrifuge at 2000 rpm for 5 min, remove the supernatant, remove PHA, collect and count spleen cells, and adjust the cell concentration to 2×10 7 Pieces / ml.

[0140] 10. The stimulated and attached SKOV3 cells in the 24-well plate were discarded, and 2×10 6 Mouse spleen cells.

[0141] 11. Dilute the DMSO-prepared peptide stock solution to 200 μM with culture medium and dilute cilengitide to 2 μM and place in a 1 ml EP tube for later use.

[0142] 12. Add 250 μl of drug-containing culture medium to the co-incubated cells and make up the volume to 500 μl. Set up a blank control group without spleen cells, with blank culture medium as a negative control and DMSO of the same concentration as a solvent control. Incubate at 37°C, 5% CO2 for 24 h.

[0143] 13. After the incubation, collect the culture medium in each well into a centrifuge tube. After the SKOV3 cells are digested, use the original culture medium to terminate the digestion and collect the cells by centrifugation.

[0144] 14. Wash three times with pre-cooled PBS and centrifuge at 1500 rpm for 3 min.

[0145] 15. The cells were finally resuspended in pre-cooled PBS / 2% BSA, 100 μL 1× Binding Buffer was added to each tube, and the suspension was gently blown to form a single cell suspension.

[0146] 16. Add 5 μL Annexin V-FITC and PI Staining Solution to each tube, mix well, protect from light, and incubate at room temperature for 15 minutes.

[0147] 17. Add 400 μL 1×Binding Buffer to each tube and mix well. All samples must be detected by flow cytometry within 1 hour after staining.

[0148] Flow cytometry data were analyzed using Flowjo 10 software.

[0149] result:

[0150] like Fig. 9 As shown in the figure, after adding immune cells, the proportion of tumor cells in both early and late apoptosis has been increased. Compared with the single-dose experiment, the experimental group peptides enhanced the apoptosis-promoting effect on tumor cells in the co-incubation experiment, and the effect was better than P-F4. In summary, we know that SPK-PF4 has the best anti-tumor effect.

[0151] Example 10: Effect of SPK-PF4 bifunctional polypeptide on mouse colorectal cancer model

[0152] 1. The precursor peptide P-F4 is difficult to directly conduct animal experiments due to its extremely poor water solubility, while SPK-PF4 has greatly improved water solubility compared to the precursor peptide. Female Balb / c mice aged 4-6 weeks were randomly divided into six groups. There were 5 mice in each group, including: ① negative control group ② positive drug group ③ SPK-PF4 group; the negative control group was intratumorally administered with normal saline, the positive control group was intravenously administered with 3 mg / kg commercially available DOX·HCl, the SPK-PF4 group was intratumorally administered with 10 mg / kg, the peptide group was administered once a day, and the positive control was administered once every two days. The administration method was intravenous administration (intravenous, iv), the drug concentration was diluted, and the volume of each administration was about 100 μL (the actual dosage was slightly adjusted according to the actual weight of the mouse). During the administration period, the tumor volume of the mouse was measured every day according to (V=L*W2 / 2, L represents the longest diameter of the tumor, and W represents the short diameter perpendicular to the longest diameter) and the weight of the mouse was monitored.

[0153] 2. Digest the cells in good pre-culture state, centrifuge, remove the culture medium, tap the bottom of the tube with your fingers to prevent the cells from clumping, wash the cells 2 to 3 times with sterile PBS, wash the trypsin and culture medium, and then resuspend the cells with PBS, count the cells, and use PBS to control the cell density to 1×10 6 100 μL of single cell suspension (1×10 5 cells, and resuspend the EP tube every three to five minutes to ensure a uniform number of cells). After inoculation, the cells are raised in an SPF environment and are expected to form tumors in about 10 days.

[0154] 3. After the last dose of the drug, the mice were killed after their tumor volume and body weight were measured. The organs (heart, liver, spleen, and kidney) were removed in a clean bench and soaked in saline. When weighing, the moisture outside the organs was first absorbed with filter paper before measurement. The weight of the mouse organs (heart, liver, spleen, and kidney) was compared with the weight of the mouse itself as the organ (heart, liver, spleen, and kidney) index, so as to judge the toxicity of the drug to the organ.

[0155] 4. Place the removed spleen on a 200-mesh sterile sieve in a clean bench, grind the spleen with the rubber stopper of a sterile 5mL syringe, and then sieve it with physiological saline, repeating the process several times until there are no large pieces of tissue on the sieve.

[0156] 5. Pre-cool the centrifuge at 4°C, collect the mouse spleen lymphocyte suspension and centrifuge at 1000rpm for 5min, discard the supernatant, tap the bottom of the tube with your fingers to prevent cells from clumping, and add sterile PBS to wash again. Repeat the above steps.

[0157] After centrifugation, a blood-red precipitate appears at the bottom of the centrifuge tube. Add 1 ml of red blood cell lysis solution to the tube for red blood cell lysis and place it at room temperature for 10 minutes. Then, remove the cell supernatant at 1000 rpm for 5 minutes, wash it with sterile PBS 1 to 2 times, wrap it with sealing film and bring it into the cell room for subsequent processing.

[0158] 6. Culture CT-26 cells, digest them with trypsin after they grow stably, wash them with sterile PBS, and then resuspend the CT-26 cells with complete culture medium, count the cells, and set aside.

[0159] 7. Count the spleen cells and resuspend them in complete culture medium. Co-culture the 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 the co-culture system is 2×10 5 ), the total volume was set to 200 μL, 3-5 duplicate wells were set for each group, and PBS was added to the outermost circle of the 96-well plate to avoid edge effects. The blank background wells were only laid with culture medium without cells, and the single CT-26 cell wells were used as spontaneous release wells without adding cell lysate. The single CT-26 cell wells were then added with cell lysate as the maximum enzyme activity wells. The maximum release wells (CT-26+LDH release reagent) and spontaneous release wells (CT-26 only) under different effect-target ratios were set, as well as the background correction blank control wells (complete culture medium), and the wells were placed in a cell culture incubator for 6 hours.

[0160] 8. 1 h before the end of the culture time, add LDH releasing reagent (10% volume) to the well with maximum enzyme activity, pipette and vortex repeatedly, and return to culture.

[0161] 9. After 6 hours of culture, place the culture plate in a horizontal centrifuge at 1000 rpm for 5 minutes to precipitate all the cells. Use a pipette to transfer 60 μL of the upper culture medium to a new cell culture plate (to avoid cells interfering with the final reading).

[0162] 10. Prepare LDH detection working solution (lactic acid: 1×INT: enzyme = 1:1:1) according to the sample volume, add 30μL to each well, mix thoroughly to avoid bubbles. Wrap with tin foil and shake slowly on a shaker, incubate at room temperature for 30 minutes. Then perform dual wavelength measurement at 490 / 630nm, and use OD 490 -OD 630 Perform calibration.

[0163] The LDH release rate (ie, target cell killing rate) was calculated according to the following formula, and the t test was used for statistical analysis to compare the differences among the groups (n=3-5 mouse samples in each group).

[0164] LDH release rate = (sample well - spontaneous release well - blank background) / (maximum release well - spontaneous release well - blank background) × 100%

[0165] result:

[0166] Fig.10 A The tumor volume growth curve shows that the bifunctional peptide SPK-PF4 can effectively inhibit tumor growth. Until the end of administration, the tumor volume of the positive control DOX group and the experimental group did not exceed 500mm 3 The tumor inhibition effect of the experimental group was consistent with that of the positive drug DOX. Fig.10 B also shows that the tumor weight in the experimental group is significantly smaller than that in the blank control group. Fig.10 C and D mouse weight curves and organ weight graphs show that the experimental group and the blank control group did not experience significant fluctuations in body weight during the entire experimental process, indicating that the two had no effect on the survival of the mice. The positive experimental group experienced a significant weight loss after administration, which further illustrates that DOX has severe drug toxicity, while the bifunctional peptide has good safety. Fig.10 E spleen 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 spleen lymphocytes, while the bifunctional peptide could well improve the immunosuppressive microenvironment in the body and activate lymphocytes to further kill tumor cells.

[0167] Example 11 Immunohistochemical study of SPK-PF4 bifunctional polypeptide regulating tumor immune microenvironment

[0168] 1. The above-dissected tumor tissue was immersed in paraformaldehyde (immersion for up to 24 hours), and then delivered to the company for paraffin embedding and sectioning, with a section thickness of 4μM.

[0169] 2. Bake the slices at 60℃ for 30 minutes to melt the wax layer. Dewax the slices in xylene I and xylene II in a fume hood for 10 minutes each, then rinse them in 100%, 90%, 80%, 70%, 60%, 50%, and 30% alcohol in order for 5 minutes, then rinse them in tap water for 10 minutes.

[0170] 3. Permeabilize the slices in 0.3% Triton X-100 to enhance the intracellular antigen staining effect. Rinse with PBS for three times with shaking for 5 minutes each time.

[0171] 4. Immerse the slices completely in EDTA antigen repair solution (pH 9.0) and place them in a microwave oven for antigen repair. Heat the microwave oven to a slightly boiling state of 92-98°C for 15-20 minutes, then take out the slices and return them to room temperature, and rinse them with PBS three times, 5 minutes each time.

[0172] 5. Treat the slices with 3% H2O2 for 15 minutes to reduce endogenous peroxidase activity and background signal. Rinse with PBS for three times, 5 minutes each time.

[0173] 6. Use a histochemical pen to draw a closed hydrophobic circle around the tissue. 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, put the slices into a wet box, add appropriate amount of water at the bottom, and incubate at 4°C overnight.

[0174] 7. The slices were first washed with PBS for 3 times with shaking, 5 minutes each time, and then the secondary antibody with HRP was diluted according to the dilution ratio in the instructions, and dropped into the histochemical circle to cover the tissue. Incubate at room temperature for 1 hour, and then repeat the washing steps.

[0175] 8. Shake off the moisture on the surface of the slices, add freshly prepared DAB solution to the surface of the tissue, and color for 3-5 minutes in the dark. Aspirate and discard the dye, and rinse the slices with double distilled water to stop color development.

[0176] 9. Re-stain the sections with hematoxylin for 3-5 minutes, rinse with running water. Differentiate with 1% hydrochloric acid ethanol for a few seconds, rinse with tap water to terminate the differentiation, turn hematoxylin blue with 0.6% ammonia water, and rinse with running water several times.

[0177] 10. Immerse the slices in 30%, 50%, 60%, 70%, 80%, 90% anhydrous ethanol in sequence for gradient dehydration, dry them, add a drop of mounting medium, cover with coverslip, complete the mounting, and examine under a microscope.

[0178] Images (7 to 9 fields of view) were selected from various parts of the panoramic scan, with a scale of 200 μM. ImageJ was used for image positivity analysis, and One Way ANOVA was used for statistical analysis to compare the differences among the groups.

[0179] result

[0180] like Fig.11 As shown: CD31 is a marker of new blood vessels. By staining CD31, it was found that the new blood vessels in the tumor tissue of the SPK-PF4 group were statistically reduced compared with the positive control and blank groups, indicating that the polypeptide can inhibit angiogenesis by inhibiting the integrin αvβ3 of vascular endothelial cells; CD4 is a marker of helper T cells (T helper), Fig.11 B shows 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 (CTL). Fig.11C shows that the infiltration of CD8+T in the experimental group was also significantly higher than that in the two control groups. Foxp3 is a marker of regulatory T cells (Treg). Fig.11 D shows that the content of Foxp3 in the experimental group is lower than that in the control group, indicating that the infiltration rate of regulatory T cells in the experimental group is lower, the infiltration of CD4+ and CD8+ T cells is increased, and the infiltration of Foxp3+ T cells is less, indicating that the bifunctional polypeptide can activate the immune system and create an immune microenvironment that is conducive to killing tumors.

Claims

1. A PD-1 and αvβ3 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 in the preparation of anti-tumor drugs.

3. The use according to claim 2, characterized in that: The tumor is colorectal cancer.

Images