Cancer immunotherapy polypeptide and application

Abstract

The present application relates to a kind of cancer immunotherapy polypeptide and application, disclose a kind of targeting PD-L1 lactylation modified treatment peptide, its preparation method and application in tumor immunotherapy drug, the treatment peptide amino acid sequence as shown in SEQ ID NO.3.The present application is based on the lactylation modification mechanism of PD-L1, according to the interaction region of lactoyltransferase HAT1 and PD-L1 design the treatment peptide, it can significantly reduce the interaction of HAT1 and PD-L1, reduce the lactylation level of PD-L1;The treatment peptide is combined with PD-1 antibody can further inhibit tumor growth, improve the immunotherapy effect of PD-1 antibody, suitable for the immunotherapy of ovarian cancer, lung cancer and other tumors.The present application provides new ideas and effective means for the immunological checkpoint therapy with PD-L1 as target, solves the technical problem of low objective response rate of existing PD-1 / PD-L1 inhibitor.

Description

Technical Field

[0001] This invention belongs to the field of tumor biomedicine, specifically relating to a cancer immunotherapy polypeptide and its application. Background Technology

[0002] Tumor immunotherapy is an important approach in cancer treatment, aiming to eliminate malignant tumor cells by mobilizing and restoring the host's immune system to fight tumors. Among these therapies, immune checkpoint inhibitors (ICIs) are the most rapidly advancing and clinically impactful. Antibody drugs targeting programmed death-1 (PD-1), programmed death-1 ligand (PD-L1), and cytotoxic T lymphocyte antigen 4 (CTLA4) have shown durable clinical benefits in various cancers, including lung cancer, melanoma, kidney cancer, head and neck cancer, and gastric cancer.

[0003] However, as the clinical use of ICIs has increased, their inherent clinical limitations have gradually become apparent, resulting in an objective response rate of only 19.56% for PD-1 / PD-L1 inhibitors in various cancer patients, thus limiting their clinical efficacy. To address the technical problems of ineffective treatment and low response rates with existing ICIs, there is an urgent need to develop novel therapeutic approaches that regulate immune checkpoints, providing new strategies and technical solutions to enhance the efficacy of tumor immunotherapy.

[0004] Lactation modification of PD-L1 is an important post-translational modification mechanism regulating its function. The specific interaction between the lactyltransferase HAT1 and PD-L1 is a key step mediating PD-L1 lactation modification. Abnormal activation of this interaction leads to increased PD-L1 lactation levels, thereby inhibiting the body's anti-tumor immune response. Based on this mechanism, designing targeting molecules that can interfere with the interaction between HAT1 and PD-L1 is a potential research direction for improving the therapeutic effects of PD-1 / PD-L1 inhibitors. Summary of the Invention

[0005] To address the shortcomings of existing PD-1 / PD-L1 inhibitors, such as low objective response rates and limited therapeutic effects, this invention provides the design and application of cancer immunotherapy peptides. By designing therapeutic peptides that target PD-L1 lactation modification, the interaction between HAT1 and PD-L1 is interfered with, reducing PD-L1 lactation levels and enhancing the immunotherapy effect of PD-1 antibodies, thus providing new targeted molecules and drug compositions for tumor immunotherapy.

[0006] To achieve the above-mentioned objectives, the present invention adopts the following technical solution: A therapeutic peptide for cancer immunotherapy, the amino acid sequence of which is shown in SEQ ID NO.3, is composed of a HAT1-PD-L1 interaction region sequence (SEQ ID NO.1: HTYSVLSPTGGENFT) and a transmembrane peptide sequence (SEQ ID NO.2: YGRKKRRQRRR), which can effectively enter tumor cells and interfere with the interaction between HAT1 and PD-L1.

[0007] The above-mentioned therapeutic peptide is used in the preparation of tumor immunotherapy drugs. This therapeutic peptide reduces the lactation level of PD-L1 by inhibiting the interaction between HAT1 and PD-L1, thereby achieving the effect of tumor immunotherapy. It is suitable for the treatment of tumors such as ovarian cancer and lung cancer.

[0008] A tumor immunotherapy drug comprising the above-mentioned therapeutic peptide, preferably, the drug further comprising a PD-1 antibody, more preferably, the PD-1 antibody being nivolumab; the dosage form of the drug is preferably an injection, wherein the injection dose of the therapeutic peptide is 10 mg / kg.

[0009] Application of a reagent capable of inhibiting the interaction between HAT1 and PD-L1 in the preparation of immunotherapeutic drugs for ovarian cancer and lung cancer.

[0010] The therapeutic peptide was prepared using the Fmoc solid-phase peptide synthesis method, with Rink amide resin as the solid-phase carrier. The purified product was obtained through steps such as deprotection, amino acid coupling, cleavage and deprotection, purification and freeze-drying. The specific synthesis process is a conventional technique in the relevant technical field and can be industrialized.

[0011] Compared with the prior art, the present invention has the following significant advantages: 1. This invention is based on the lactation modification mechanism of PD-L1 and designs therapeutic peptides that precisely target the interaction region between HAT1 and PD-L1. These therapeutic peptides can significantly reduce the interaction between HAT1 and PD-L1, effectively inhibit the lactation modification of PD-L1, regulate the function of PD-L1 at the molecular level, thereby restoring the body's anti-tumor immune response and improving the immunotherapy effect of PD-1 antibodies. This provides a new approach and target molecule for immune checkpoint therapy targeting PD-L1.

[0012] 2. The therapeutic peptide of the present invention can work synergistically with PD-1 antibodies. When the two are used together, they can further inhibit tumor growth, significantly reduce tumor volume, and slow down the tumor growth rate. Compared with single PD-1 antibody treatment, the therapeutic effect is greatly improved, effectively solving the technical problem of low response rate of existing PD-1 / PD-L1 inhibitors.

[0013] 3. The therapeutic peptides of this invention can effectively enter tumor cells such as ovarian cancer cells and lung cancer cells to exert their effects, and animal experiments have shown that they have no significant liver and kidney toxicity, demonstrating high drug safety and providing a good safety basis for clinical translation and application.

[0014] 4. The therapeutic peptide of the present invention is prepared by solid-phase peptide synthesis, which has a mature synthesis process and controllable steps, enabling large-scale and industrialized production. Moreover, the drug dosage form is an injection, which is convenient for administration and suitable for clinical application. Attached Figure Description

[0015] Figure 1 The distribution of FITC-labeled therapeutic peptides in human ovarian cancer cells HEY-A8 and lung cancer cells A549 is shown, demonstrating that the therapeutic peptides can effectively enter tumor cells. Figure 2 The figure shows the effect of the therapeutic peptide of the present invention on the interaction between HAT1 and PD-L1 and the lactation modification of PD-L1. It shows that the therapeutic peptide can reduce the binding of HAT1 and PD-L1 and reduce the lactation level of PD-L1. Figure 3 The figure shows the effect of the therapeutic peptide of the present invention on the growth of tumors in humanized mice. A is a picture of the tumor tissue and B is a tumor growth curve, showing that the therapeutic peptide can inhibit tumor growth and the effect is more significant when used in combination with PD-1 antibody. Figure 4 This is a graph showing the effect of the therapeutic peptide of the present invention on tumor-infiltrating lymphocytes in humanized mice, where A represents TNF-α. + CD8 + T cell percentage, B represents IFN-γ + CD8 + The percentage of T cells indicates that the therapeutic peptide can promote tumor infiltration of effector T cells and enhance the anti-tumor killing effect; Figure 5 The image shows the H&E staining of liver and kidney tissues from humanized mice, indicating that the therapeutic peptides did not cause significant damage to the liver and kidney tissues of mice and had no hepatotoxicity. Detailed Implementation

[0016] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments and accompanying drawings. The described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the protection scope of the present invention.

[0017] Example 1: Design and synthesis of a cancer immunotherapy peptide; Based on the interaction region between lactyltransferase HAT1 and PD-L1, a therapeutic peptide was designed to interfere with their interaction. The sequence located in the interaction region of the HAT1 protein (as shown in SEQ ID NO.1) is: HTYSVLSPTGGENFT. Adding the membrane-penetrating peptide sequence (as shown in SEQ ID NO.2) is: YGRKKRRQRRR, the therapeutic peptide sequence of the present invention (as shown in SEQ ID NO.3) is: YGRKKRRQRRRHTYSVLSPTGGENFT.

[0018] Specifically, the synthesis process of this therapeutic peptide is as follows: The peptides were prepared using the Fmoc solid-phase synthesis method, and the synthesis was carried out at room temperature. Rink amide resin was used as the solid-phase support. The Fmoc protecting groups of amino acids on the resin were removed using a 20% piperidine / dimethylformamide (DMF) solution. After deprotection, the resin was washed with DMF.

[0019] Subsequently, amino acid monomers were sequentially coupled to the resin from the C-terminus to the N-terminus. The amino acid coupling reaction used HBTU and N,N-diisopropylethylamine (DIEA) as the condensation system in DMF solvent. After each coupling step, the completeness of coupling was checked using standard colorimetric reagents. After coupling was completed, the next round of Fmoc deprotection was performed. The above deprotection and coupling steps were repeated until the entire target polypeptide sequence was synthesized.

[0020] The side chains of the amino acids used are protected as needed. Arginine is protected with a Pbf protecting group, lysine with a Boc protecting group, glutamic acid with a tert-butyl ester (OtBu) protecting group, serine, threonine and tyrosine with a tert-butyl (tBu) protecting group, and histidine, asparagine and glutamine with a triphenylmethyl (Trt) protecting group.

[0021] After peptide chain assembly and removal of the terminal Fmoc protecting group, the peptide was cleaved from the resin using a trifluoroacetic acid-based cleavage system, simultaneously removing side chain protecting groups to obtain crude peptide. The crude peptide was then purified by reversed-phase high-performance liquid chromatography, and the target fraction was collected and lyophilized to obtain the purified target peptide product.

[0022] Example 2: In this example, experiments demonstrate that the therapeutic peptide synthesized in Example 1 can effectively enter tumor cells (ovarian cancer cells HEY-A8 and lung cancer cells A549); The experimental procedure includes: 1) On the first day, HEY-A8 and A549 cells that were in good condition, tested negative for mycoplasma, and were in the logarithmic growth phase were collected, digested, centrifuged, and then 3×10⁻⁶ cells were added. 4 One cell was seeded in a confocal cell culture dish.

[0023] 2) On the second day, 10 µM of FITC-labeled therapeutic peptide was added to the target tumor cells, while the control group was given an equal volume of phosphate buffered saline (PBS), and the cells were cultured for another 8 hours.

[0024] 3) Discard the culture medium, wash the cells with PBS, add 1 µM Hoechst 33258 and incubate in an incubator for 30 minutes.

[0025] 4) Discard the dye and add PBS; observe the uptake of FITC-labeled therapeutic peptides by live cells under a confocal microscope within 30 minutes.

[0026] The distribution of FITC-labeled therapeutic peptides in human ovarian cancer cells HEY-A8 and lung cancer cells A549 was observed using confocal microscopy. Figure 1 As shown in the figure, punctate green fluorescence is observed within the cells, indicating that FITC-labeled therapeutic peptides can effectively enter the target tumor cells.

[0027] Example 3: In this example, Flag-IP and WB experiments showed that treatment with the therapeutic peptide synthesized in Example 1 reduced the interaction between HAT1 and PD-L1 and inhibited the lactation modification of PD-L1. The experimental procedure is as follows: 1) On the first day, healthy human embryonic kidney cells HEK293T in the logarithmic growth phase were collected, digested, and centrifuged. 3×10⁻⁶ cells were then separately... 6 One cell was seeded into three 10 cm cell culture dishes.

[0028] 2) On the second day, the plenti-PD-L1-Flag plasmid was transfected into HEK293T cells using jetPRIME DNA transfection reagent. 1 mL of jetPRIME buffer was added to an RNase-free centrifuge tube, followed by 10 µg of plasmid and vortexing for 10 seconds. Then, 20 µL of jetPRIME transfection reagent was added, vortexed for 10 seconds, and incubated at room temperature for 10 minutes. Finally, the transfection complex was added dropwise to the culture medium of HEK293T cells.

[0029] 3) 24 hours after transfection, change the culture medium of the cells and add 10 µM therapeutic peptide, and continue to culture for another 24 hours.

[0030] 4) Extract cell proteins using RIPA lysis buffer and perform Flag-IP.

[0031] 5) The Flag-tagged immunoprecipitation (Flag-IP) eluent was analyzed by Western blotting to detect the expression of HAT1, PD-L1 and their lactation in the eluent.

[0032] Test results are as follows Figure 2 As shown, the Flag band indicates enrichment of PD-L1-Flag protein via Flag-IP, the HAT1 band indicates that the therapeutic peptide reduces the amount of HAT1 protein interacting with PD-L1-Flag, and the K-lac band indicates that the therapeutic peptide reduces the degree of lactation modification of PD-L1-Flag protein; thus, it is demonstrated that the therapeutic peptide can effectively inhibit the interaction between HAT1 and PD-L1 and the lactation of PD-L1.

[0033] Example 4: In this example, the therapeutic peptide synthesized in Example 1 was applied to humanized mice for relevant experiments and tests; 1) Therapeutic peptides inhibit tumor growth in humanized mice The experimental procedure is as follows: In a humanized mouse model, 4×10 6 HEY-A8 cells were injected into the flank of NOG mice. Tumor volume was measured every 2–3 days, and tumor size was calculated using the formula "volume = width² × length / 2". On day 3, 5 × 10⁵ cells were transplanted via the tail vein. 6 Peripheral blood mononuclear cells (PBMCs) were collected from individuals. Therapeutic peptides (10 mg / kg) and PD-1 antibody (nivolumab, Selleck, A2002) (100 µg per mouse) were administered intraperitoneally on days 5, 9, 13, 17, and 21, for a total of five injections. Tumor tissue, liver, and kidney tissue were collected at the end of the experiment on day 23.

[0034] Test results are as follows Figure 3 As shown, Figure 3 In this context, A represents the condition of the tumor tissue. Figure 3 In the figure, B represents the tumor growth curve; from Figure 3 The results show that the application of therapeutic peptides and PD-1 antibodies can reduce tumor volume and delay tumor growth; thus, it can be seen that therapeutic peptides significantly inhibit the growth of humanized mouse tumors, and when combined with PD-1 antibodies, the tumor volume is further reduced.

[0035] 2) Effects of therapeutic peptides on tumor-infiltrating lymphocytes in humanized mice The experimental procedure is as follows: Digestion: Tumor tissue was placed in a 6-well cell culture plate, and 2 mL of enzyme digestion solution (HBSS solution containing 2 mg / mL collagenase IV, 0.2 mg / mL DNase I, and 0.25 mg / mL hyaluronidase) was added. The tumor was cut into pieces of approximately 1 mm³ using ophthalmic scissors. The culture plate was placed in a 37°C incubator and shaken for 1 hour. Subsequently, the digestion solution was filtered through a 70 μm cell sieve into 50 mL centrifuge tubes, and the filter was rinsed with 1640 complete culture medium. The tubes were centrifuged at 1500 rpm at 4°C for 5 minutes, and the supernatant was discarded. 3 mL of FACS was added to each tube to wash away any remaining digestion solution, and the tubes were then transferred to 15 mL centrifuge tubes and centrifuged at 1500 rpm at 4°C for 5 minutes, and the supernatant was discarded completely.

[0036] Lymphocyte isolation: Resuspend the centrifuged cell pellet in 3 mL of 30% Percoll separation buffer, and centrifuge at 1500 rpm density gradient for 20 minutes. The centrifuge speed should be slowed down during this step. (Preparation of Percoll separation buffer: 30% Percoll, 40 mL: 1.2 mL 10× PBS, 10.8 mL Percoll and 28 mL 1x PBS) Lyse red blood cells: Collect the pellet and resuspend it in a 15 mL centrifuge tube containing 6 mL of FACS. Centrifuge at 1500 rpm, 4°C for 5 minutes, and discard the supernatant. If the cell pellet is very red, dissolve the red blood cells with red blood cell lysis buffer. Add an appropriate volume of red blood cell lysis buffer (usually 3 mL) according to the amount of cell pellet, vortex thoroughly, and incubate at room temperature for 4 minutes. Centrifuge at 1500 rpm, 4°C for 5 minutes, and discard the supernatant. Wash the cells once more with FACS.

[0037] Cell activation: Cells were resuspended in 24-well cell culture plates containing 1640 complete medium, and 500× Cell Activation Cocktail stock solution containing phorbol-12-myristate 13-acetate (40.5 µM), ionomycin (669.3 µM), and Brefeldin A (2.5 mg / ml) was added. The plates were then incubated at 37°C in a 5% CO2 incubator for 6 hours.

[0038] Staining and blocking: Collect cells into 1.5 mL EP tubes, centrifuge at 1500 rpm for 5 minutes at 4°C, and discard the supernatant. Wash once with 1 mL PBS, centrifuge again, and discard the supernatant. Add 100 μL of Viability Dye (diluted 1:1000 with PBS) to each tube to kill cells, and incubate at room temperature in the dark for 10-15 minutes. Add 1 mL of FACS directly, mix well, wash away residual dye, centrifuge at 1500 rpm for 5 minutes at 4°C, and discard the supernatant. Add 100 μL of Fc blocking buffer to each centrifuge tube, incubate at 4°C in the dark for 15 minutes, then add 1 mL of FACS directly, centrifuge at 1500 rpm for 5 minutes at 4°C, and discard the supernatant.

[0039] Cell membrane surface marker staining: Add 100 μL of T cell surface marker antibody mixture to each sample and incubate at 4°C in the dark for 30 minutes. After incubation, add 1 mL of FACS and mix by inverting. Centrifuge at 1500 rpm at 4°C for 5 minutes and discard the supernatant.

[0040] Cell fixation and permeabilization: Add 500 μL of 1× fixative to each sample tube, vortex to mix, and incubate at room temperature for 45-60 minutes. Then add 500 μL of 1×Perm buffer, centrifuge at 1500 rpm at room temperature for 5 minutes, and discard the supernatant. Resuspend the cells in 1 mL of 1×Perm buffer, centrifuge at 1500 rpm at room temperature for 5 minutes, and discard the supernatant.

[0041] Intracellular marker staining: Add 100 μL of T cell intracellular antibody mixture prepared with 1×Perm buffer to each sample and incubate at room temperature in the dark for 30 minutes. Then add 1 mL of 1×Perm buffer, centrifuge at 1500 rpm at room temperature for 5 minutes, and discard the supernatant.

[0042] Flow cytometry: After adding 1 mL of FACS, centrifuge at 1500 rpm for 5 minutes at room temperature and discard the supernatant. Then, resuspend the sample in 400 μL of FACS, pass it through a 200-mesh sieve, transfer it to a flow cytometer tube, and perform flow cytometry analysis to determine the presence of tumor-infiltrating lymphocytes.

[0043] The results show that... Figure 4 As shown; Figure 4 In the diagram, A and B represent TNF-α in each treatment group, respectively. + CD8 + T cells, IFN-γ + CD8 + T cells account for a significant portion of CD8. + The proportion of T cells indicates the presence of TNF-α. + IFN-γ+ CD8 + Increased T cell infiltration suggests that the therapeutic peptide promotes the killing of tumor cells by T cells, thereby delaying tumor growth.

[0044] 3) The therapeutic peptides showed no significant toxicity to the liver and kidney tissues of humanized mice. The experimental procedure is as follows: In the above mouse model, the liver and kidney tissues of mice were stained with H&E. The results are as follows Figure 5 As shown, the morphology of each organ and tissue is normal, and the therapeutic peptide has no obvious liver and kidney toxicity.

[0045] In the description of this specification, references to terms such as "an embodiment," "example," "specific example," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0046] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claimed invention.

Claims

1. A therapeutic peptide for cancer immunotherapy, characterized in that, The amino acid sequence of the therapeutic peptide is shown in SEQ ID NO.

3.

2. The use of the therapeutic peptide as described in claim 1 in the preparation of tumor immunotherapy drugs.

3. The application according to claim 2, characterized in that, The application involves achieving tumor immunotherapy by inhibiting the interaction between lactyltransferase HAT1 and PD-L1.

4. The application according to claim 2, characterized in that, The tumor is at least one of ovarian cancer or lung cancer.

5. A tumor immunotherapy drug, characterized in that, It contains the therapeutic peptide as described in claim 1.

6. The tumor immunotherapy drug according to claim 5, characterized in that, It also contains PD-1 antibodies.

7. The tumor immunotherapy drug according to claim 6, characterized in that, The PD-1 antibody is nivolumab.

8. The tumor immunotherapy drug according to claim 5, characterized in that, The drug is in the form of an injection.

9. The tumor immunotherapy drug according to claim 8, characterized in that, The injection dose of the therapeutic peptide in the injection is 10 mg / kg.

10. The application of a reagent capable of inhibiting the interaction between HAT1 and PD-L1 in the preparation of tumor immunotherapy drugs.

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