A polypeptide and a drug product for specifically blocking the interaction of PD-L1 with endogenous double-stranded RNA

Abstract

The present application relates to the technical field of biological medicines, and particularly relates to a polypeptide for specifically blocking the interaction between PD-L1 and endogenous double-stranded RNA and a medicine. The present application provides a polypeptide for specifically binding to endogenous double-stranded RNA, which has a strong effect on the activation of innate immune genes. The polypeptide fused with a cell-penetrating peptide can competitively bind to endogenous double-stranded RNA in cells and inhibit the A-to-I editing of endogenous double-stranded RNA, thereby improving the sensitivity of tumor cells to drugs. The present application provides a new strategy for the immunotherapy of tumor patients and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical technology, and more particularly to a polypeptide and drug that specifically blocks the interaction between PD-L1 and endogenous double-stranded RNA. Background Technology

[0002] The immune system is crucial for the body to recognize and kill tumor cells. Simultaneously, tumors possess a series of immune escape mechanisms to evade the immune system's surveillance and killing. Among these, tumor immune checkpoint molecules help tumor cells achieve immune escape by inhibiting the function of immune cells such as T cells. PD-L1 molecules on the tumor surface interact with PD-1 molecules on the surface of T cells, inhibiting T cell proliferation and cytotoxic capabilities. These immune checkpoints have become targets for tumor immunotherapy.

[0003] Immune checkpoint blockade, as an emerging immunotherapy approach, offers a novel treatment option for cancer. The most widely used cancer immunotherapy is anti-PD therapy, which involves antibodies targeting and binding to PD-1 or PD-L1 on the cell surface, blocking the interaction between PD-1 and PD-L1, thereby reactivating the immune system to kill tumors. Triple-negative breast cancer (TNBC) is a subtype of breast cancer with a very low clinical cure rate. Due to its high level of tumor immune infiltration, immune checkpoint inhibitors offer hope for the treatment of triple-negative breast cancer.

[0004] Besides antibody drugs, cell-penetrating peptides can deliver various molecules into cells, including nucleic acids, peptides, and nanoparticles. Specifically designed peptides can target and bind to protein or nucleic acid molecules, and peptide drugs have proven to be an effective method for regulating protein-protein and protein-nucleic acid interactions.

[0005] Currently, tumor immunotherapy primarily utilizes monoclonal antibodies to block the interaction between PD-1 and PD-L1. However, in clinical treatment, only a small percentage of patients respond to anti-PD therapy, with most patients either not responding or developing resistance after a period of treatment. A 2020 phase III clinical trial (IMpassion031) of neoadjuvant PD-L1 antibody (Atezolizumab) combined with chemotherapy in triple-negative breast cancer patients showed a complete response rate of 58% in the Atezolizumab plus chemotherapy group, compared to 41% in the placebo plus chemotherapy group. Furthermore, the efficacy of Atezolizumab was not significantly correlated with PD-L1 expression. This indicates that there is significant room for improvement in the clinical treatment of triple-negative breast cancer with PD-L1 antibodies, particularly in patients with high PD-L1 expression, where the use of other PD-L1-targeting drugs may further improve response rates.

[0006] Therefore, developing cell-delivered peptide drugs that target the intracellular function of PD-L1 and improve the response rate of patients to PD-L1-targeted immunotherapy drugs has become a technical challenge that needs to be solved in this field. Summary of the Invention

[0007] To address the problem of poor therapeutic effects of antibody-targeted PD-L1 treatment on cell surfaces in existing technologies, this invention first provides a polypeptide that specifically blocks the interaction between PD-L1 and endogenous double-stranded RNA, with the amino acid sequence IQDTNSKKQSDT (SEQ ID No. 1).

[0008] The inventors discovered that in triple-negative breast cancer, intracellular PD-L1 promotes ADAR1-mediated A-to-I editing by binding to endogenous double-stranded RNA, thereby inhibiting the innate immune response and promoting tumor immune escape. Therefore, targeting the interaction between PD-L1 and endogenous double-stranded RNA may be an effective strategy to overcome resistance to tumor immunotherapy. This invention, through the design and screening of different peptide sequences, found that the aforementioned peptides have a strong activation effect on innate immune genes, can competitively bind to cellular endogenous double-stranded RNA with PD-L1, and inhibit A-to-I editing of endogenous double-stranded RNA, thereby improving the sensitivity of tumor cells to drugs (such as interferon, DNMT inhibitors, PD-1 antibodies, etc.).

[0009] Furthermore, the present invention provides a nucleic acid encoding the polypeptide.

[0010] Furthermore, the present invention provides a biomaterial containing the aforementioned polypeptide or nucleic acid.

[0011] In some implementations, the biomaterial is a carrier, expression cassette, transposon, cell, or engineered bacteria.

[0012] Furthermore, the present invention provides a fusion polypeptide (cell delivery polypeptide) containing the aforementioned polypeptide and a cell-penetrating peptide.

[0013] In specific implementation schemes, appropriate cell-penetrating peptides can be selected to improve cell delivery efficiency or tissue specificity.

[0014] In some implementations, the cell-penetrating peptide has no effect on the expression of innate immune genes.

[0015] Preferably, the cell-penetrating peptide is HLYVSPWGG (SEQ ID No. 2).

[0016] Preferably, the amino acid sequence of the fusion polypeptide (PD-Compep) is HLYVSPWGGIQDTNSKKQSDT (shown in SEQ ID No. 3).

[0017] The combination of the above-mentioned fusion peptides with DNMT inhibitors significantly promoted the infiltration of immune cells into the tumor microenvironment and inhibited tumor growth.

[0018] Furthermore, the present invention provides the application of the aforementioned polypeptide, nucleic acid, biomaterial, and fusion polypeptide in the preparation of pharmaceuticals.

[0019] Preferably, the drug is used to treat tumors; more preferably, the tumor is cancer; more preferably, the cancer is triple-negative breast cancer.

[0020] Furthermore, the present invention provides a pharmaceutical product containing the aforementioned polypeptide, nucleic acid, biomaterial, or fusion polypeptide.

[0021] Preferably, the drug is used to treat tumors; more preferably, the tumor is cancer; more preferably, the cancer is triple-negative breast cancer.

[0022] In some implementations, the pharmaceutical product also includes pharmaceutically acceptable excipients.

[0023] In some implementation schemes, pharmaceutically acceptable excipients include fillers, excipients, lubricants, wetting agents, diluents, etc.

[0024] In some embodiments, the formulation type of the drug may be a solid dosage form (e.g., powder, granules, capsules, tablets, etc.) or a liquid dosage form (e.g., oral liquid, etc.).

[0025] In some implementations, the medicine also includes other drugs for treating tumors.

[0026] Preferably, the other drugs for treating tumors include DNMT inhibitors, interferons, or PD-1 antibodies.

[0027] More preferably, the DNMT inhibitor is 5-AZA-CdR; or the interferon is IFNβ.

[0028] When the peptides of the present invention are used in combination with interferon, DNMT inhibitors, or PD-1 antibodies, they help overcome tumor resistance to immunotherapy and provide a new strategy for immunotherapy in cancer patients.

[0029] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention provides a polypeptide that specifically binds to endogenous double-stranded RNA, exhibiting a strong activation effect on innate immune genes. The polypeptide fused with a cell-penetrating peptide can competitively bind to endogenous double-stranded RNA and inhibit A-to-I editing of endogenous double-stranded RNA, thereby enhancing the sensitivity of tumor cells to drugs. This invention provides a novel strategy for immunotherapy in cancer patients and has broad application prospects. Attached Figure Description

[0030] Figure 1 This demonstrates that PD-L1 interacts with endogenous double-stranded RNA through its ICD (260aa-290aa).

[0031] Figure 2 This demonstrates PD-L1 regulation of A-to-I editing of endogenous double-stranded RNA.

[0032] Figure 3 Stable PD-L1 knockdown was observed, leading to innate immune activation.

[0033] Figure 4 The study showed that the combination of the competitively binding peptide PD-Compep and the immunotherapy drug IFNβ increased the expression of innate immune genes.

[0034] Figure 5 The study demonstrated the regulatory role of the peptide PD-Compep in A-to-I editing of endogenous double-stranded RNA.

[0035] Figure 6 The study showed that the combination of the peptide PD-Compep and the immunotherapy drug 5-AZA-CdR increased the expression of innate immune genes.

[0036] Figure 7 The study demonstrated that the combination of the peptide PD-Compep and the immunotherapy drug 5-AZA-CdR inhibited tumor growth in a 4T1 mouse triple-negative breast cancer model.

[0037] Figure 8The study showed that the combination of the peptide PD-Compep and the immunotherapy drug 5-AZA-CdR increased the infiltration of immune cells in a 4T1 mouse triple-negative breast cancer model.

[0038] Figure 9 The effect of peptide PD-Compep treatment on mouse body weight was shown.

[0039] Figure 10 The study demonstrated that the combination of the peptide PD-Compep and the immunotherapy drug PD-1 antibody inhibited tumor growth in a 4T1 mouse triple-negative breast cancer model.

[0040] Figure 11 The study showed that the combination of the peptide PD-Compep and the immunotherapy drug PD-1 antibody increased the infiltration of immune cells in a 4T1 mouse triple-negative breast cancer model. Detailed Implementation

[0041] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention. In the embodiments provided in this specification, where specific techniques or conditions are not specified, they are performed according to the techniques or conditions described in the literature in this field, or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0042] This invention relates to molecular biology experiments. Unless otherwise specified, reference can be made to the book *Molecular Cloning* (J. Sambrook, E.F. Fritsch, and T. Maniatis, Science Press, 1994). This book and its subsequent editions are the most commonly used and guiding reference books for those skilled in the art when performing experiments related to molecular biology. In addition, depending on the experimental purpose, those skilled in the art may conduct corresponding experiments under the guidance of the operating manuals accompanying various commercially available kits or entrust them to specialized companies, such as gene sequencing.

[0043] Example 1: PD-L1 interacts with endogenous double-stranded RNA This embodiment uses gel electrophoresis migration (EMSA) experiments to demonstrate the interaction between PD-L1 and endogenous double-stranded RNA. The specific steps are as follows: 1) The forward Alu (SEQ ID No. 4) and reverse Alu (SEQ ID No. 5) located in the 3'UTR region of the NICN1 gene were transcribed using the T7 in vitro transcription system (purchased from New England Biolabs), and the sequences are shown in Table 1 below.

[0044] Table 1 Alu sequences

[0045] 2) Add 20 pmol γP32-ATP to the transcribed Alu RNA for RNA labeling, and make a 20 μL system with 1 μL polynucleotide kinase and 2 μL 10x reaction buffer A.

[0046] 3) Incubate at 37℃ for 30 min.

[0047] 4) Add 1 μL of 0.5 M EDTA and heat at 75℃ for 10 min.

[0048] 5) Mix 20 μL each of the sense and antisense strands, add 10 μL of 5x Annealing Buffer (50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 5 mM EDTA), heat at 90°C for 2 min, and then place on hot water to cool naturally to room temperature for annealing.

[0049] 6) Dilute PD-L1 protein (Flag-PD-L1, 20-290aa, purchased from OriGene) and purified expressed mutant PD-L1 protein (GST-PD-L1 ΔC, 20aa-238aa) with binding buffer (10 mM HEPES, 50 mM KCl, 1 mM EDTA, 0.05% TritonX-100, 5% Glycerol, 0.01 mg / ml BSA, 1 mM DTT, 40 U / ml RNase inhibitor) to 8 μM, 4 μM, 1 μM, and 0.1 μM.

[0050] 7) Add 1 μL of diluted protein and 1 μL of annealed double-stranded RNA to 8 μL of binding buffer and incubate at room temperature for 30 min.

[0051] 8) Add 4 μL of 5 x TBE high density loading buffer and centrifuge briefly.

[0052] 9) Electrophoresis at 25 mA in an 8% Native PAGE gel until the loading buffer is 1 / 3 of the gel length from the bottom edge.

[0053] 10) Dry the adhesive at 75°C for 1 hour, then transfer the signal to the phosphor screen and develop it.

[0054] The results showed that the PD-L1 protein (Flag-PD-L1) could bind double-stranded RNA in vitro, while the PD-L1 protein mutant lacking the ICD and transmembrane region (GST-PD-L1 ΔC, 20aa-238aa) could not bind double-stranded RNA. Figure 1 ).

[0055] Example 2: PD-L1 regulates A-to-I editing of endogenous double-stranded RNA This embodiment uses RNA-seq experiments to demonstrate that PD-L1 regulates A-to-I editing of endogenous double-stranded RNA. The specific steps are as follows: 1) Wild-type and PD-L1 knockdown MDA-MB-231 cells were treated with 2.5 ng / mL IFNβ (purchased from Proteintech) for 48 h.

[0056] 2) Collect 10 6 One MDA-MB-231 cell was centrifuged, the culture medium was discarded, and the cells were directly lysed with 1000 μL TRIzol reagent.

[0057] 3) After incubating for 5 min, add 200 μL of chloroform and gently mix by turning the container several times.

[0058] 4) Incubate the sample at room temperature for 3 minutes, then centrifuge at 12,000 g for 15 minutes at 4°C.

[0059] 5) Collect the aqueous phase containing RNA and mix it with an equal volume of freshly prepared 70% ethanol. Centrifuge at 12,000 g for 15 minutes to obtain the precipitate, which is RNA.

[0060] 6) Total RNA was quantitatively determined using a NanoDrop 2000 spectrophotometer.

[0061] 7) Sequencing was performed using a standard eukaryotic transcriptome library to obtain 150 bp end-paired reads.

[0062] 8) To analyze the RNA-seq data, adapter sequences were removed and quality filtered using Trimmomatic. All files were aligned to the reference human genome (hg38) and mapped using STAR, and sorted and indexed using samtools.

[0063] 9) Calculate the A-to-I editing index using RNAEditingIndexer.

[0064] The results showed that, under IFNβ treatment, PD-L1 knockdown MDA-MB-231 cells had a lower A-to-I editing index ( Figure 2 ).

[0065] Example 3: Stable knockdown of PD-L1, activation of innate immunity This embodiment uses qRT-PCR experiments to demonstrate stable PD-L1 knockdown and activation of the innate immune system. The specific steps are as follows: 1) Wild-type and PD-L1 knockdown MDA-MB-231 cells were treated with 2.5 ng / mL IFNβ (purchased from Proteintech) for 48 h.

[0066] 2) Collect 10 6 One MDA-MB-231 cell was centrifuged, the culture medium was discarded, and the cells were directly lysed with 1000 μL TRIzol reagent.

[0067] 3) After incubating for 5 min, add 200 μL of chloroform and gently mix by turning the container several times.

[0068] 4) Incubate the sample at room temperature for 3 minutes, then centrifuge at 12,000 g for 15 minutes at 4°C.

[0069] 5) Collect the aqueous phase containing RNA and mix it with an equal volume of freshly prepared 70% ethanol. Centrifuge at 12,000 g for 15 minutes to obtain the precipitate, which is RNA.

[0070] 6) Total RNA was quantitatively determined using a NanoDrop 2000 spectrophotometer.

[0071] 7) Use a reverse transcription kit to convert the extracted RNA into complementary DNA (cDNA).

[0072] 8) Perform qPCR using SYBR Green premixed reagent and specific primers (sequences are shown in Table 2 below).

[0073] Table 2 qRT-PCR primer sequences

[0074] In this embodiment, the expression levels of innate immune genes in wild-type and PD-L1 knockdown MDA-MB-231 cells were analyzed by normalizing GAPDH expression. The results showed that the expression levels of innate immune genes in PD-L1 knockdown MDA-MB-231 cells were higher under IFNβ treatment. Figure 3 ).

[0075] Example 4 Synthesis of Polypeptides In this embodiment, peptides (as shown in Table 4) were designed based on the sequence of the ICD region of PD-L1 (as shown in Table 3 below). S1, S2, and S3 were fused with the membrane-penetrating peptide Pep2 (sequence: HLYVSPWGG) to form cell delivery peptides (HLYVSPWGG+S1, HLYVSPWGG+S2, HLYVSPWGG+S3). The peptides were synthesized and purified by Nanjing Jietai Biotechnology Co., Ltd. The synthesis of the above peptides is a prior art and will not be described in detail here.

[0076] Table 3. Sequences of ICD segments of PD-L1

[0077] Table 4. Design of PD-L1 peptide sequences

[0078] This embodiment further examines the effect of the combined use of cell-delivery peptides and the immunotherapy drug IFNβ on the expression of innate immune genes. The specific steps are as follows: 1) The experiment was divided into six groups, including: Wild-type MDA-MB-231 cells were treated with 2.5 ng / mL IFNβ for 72 h; PD-L1 knockdown MDA-MB-231 cells were treated with 2.5 ng / mL IFNβ for 72 h; wild-type MDA-MB-231 cells were treated with 20 μM membrane-penetrating peptide fused with S1 peptide plus 2.5 ng / mL IFNβ for 72 h; wild-type MDA-MB-231 cells were treated with 20 μM membrane-penetrating peptide fused with S2 peptide plus 2.5 ng / mL IFNβ for 72 h; wild-type MDA-MB-231 cells were treated with 20 μM membrane-penetrating peptide fused with S3 peptide plus 2.5 ng / mL IFNβ for 72 h; wild-type MDA-MB-231 cells were treated with 20 μM membrane-penetrating peptide plus 2.5 ng / mL IFNβ for 72 h.

[0079] 2) Collect 10 6 RNA was extracted from the above-treated MDA-MB-231 cells, reverse transcribed, and the expression of innate immune genes was detected by qRT-PCR (the method and primers were the same as in Example 3).

[0080] The results are as follows Figure 4 The polypeptide formed by fusing the transmembrane peptide with S1 showed the strongest activation effect on innate immune genes. This polypeptide is referred to as PD-Compep, shown in SEQ ID No. 3. The transmembrane peptide itself has no effect on the expression of innate immune genes. The transmembrane peptide is used as a control peptide and is called Ctrpep, shown in SEQ ID No. 2.

[0081] Example 5: The peptide PD-Compep inhibits A-to-I editing of endogenous double-stranded RNA. 1) The experiment was divided into two groups, including: Wild-type MDA-MB-231 cells were treated with 20 μM of the control peptide Ctrpep plus 2.5 ng / mL IFNβ for 72 h, and wild-type MDA-MB-231 cells were treated with 20 μM of the competitive binding peptide PD-Compep plus 2.5 ng / mL IFNβ for 72 h.

[0082] 2) Collect 10 6 RNA was extracted from the above-treated MDA-MB-231 cells, and strand-specific RNA-seq was performed. The A-to-I editing index was calculated using RNAEditingIndexer (same method as in Example 2).

[0083] The results are as follows Figure 5 Compared to the control group peptide Ctrpep, the PD-Compep peptide treatment group showed lower levels of A-to-I editing of endogenous double-stranded RNA.

[0084] Example 6: Combination of peptide PD-Compep and immunotherapy drug 5-AZA-CdR increases the expression of innate immune genes. 1) The experiment was divided into six groups, including: Wild-type MDA-MB-231 cells were treated with 300 nM 5-AZA-CdR for 72 h; PD-L1 knockdown MDA-MB-231 cells were treated with 300 nM 5-AZA-CdR for 72 h; wild-type MDA-MB-231 cells were treated with 20 μM membrane-penetrating peptide fused with S1 peptide plus 300 nM 5-AZA-CdR for 72 h; wild-type MDA-MB-231 cells were treated with 20 μM membrane-penetrating peptide fused with S2 peptide plus 300 nM 5-AZA-CdR for 72 h; wild-type MDA-MB-231 cells were treated with 20 μM membrane-penetrating peptide fused with S3 peptide plus 300 nM 5-AZA-CdR for 72 h; wild-type MDA-MB-231 cells were treated with 20 μM membrane-penetrating peptide plus 300 nM 5-AZA-CdR for 72 h.

[0085] 2) Collect 10 6 RNA was extracted from the above-treated MDA-MB-231 cells, reverse transcribed, and the expression of innate immune genes was detected by qRT-PCR (the method and primers were the same as in Example 3).

[0086] The results are as follows Figure 6 The peptide formed by fusing the transmembrane peptide with S1 (PD-Compep) has the strongest activation effect on innate immune genes, and the transmembrane peptide (Ctrpep) itself has no effect on the expression of innate immune genes.

[0087] Example 7: Inhibition of tumor growth in a 4T1 mouse triple-negative breast cancer model by combination of peptide PD-Compep and immunotherapy drug 5-AZA-CdR. 1) Animal grouping: Six-week-old female BALB / c mice were divided into four groups of six each. The experiment consisted of four groups, including: Control group; 5-AZA-CdR group; PD-Compep group; 5-AZA-CdR plus PD-Compep group.

[0088] 2) Tumor cell preparation and inoculation: Adherent mouse triple-negative breast cancer 4T1 cells were digested with trypsin and washed three times with PBS. 5 × 10⁶ tumor cells were then inoculated. 5 Three-negative breast cancer 4T1 cells were injected into the right axilla of mice to induce tumor formation.

[0089] 3) Mouse administration: When the tumor volume reaches 100 mm 3 Treatment began. 5-AZA-CdR was administered intraperitoneally at a dose of 0.5 mg / kg daily, and peptide PD-Compep was administered intraperitoneally at a dose of 5 mg / kg daily. Two cycles of administration were performed, with a 5-day rest period followed by a 1-day rest period. Tumor size was measured with calipers every two days. The volume was calculated using the formula: Volume = Length × Width. 2 / 2.

[0090] The results are as follows Figure 7 As shown, the combined use of 5-AZA-CdR and the peptide PD-Compep inhibited the growth of triple-negative breast cancer in mice.

[0091] Example 8: Combination of peptide PD-Compep and immunotherapy drug 5-AZA-CdR increases the infiltration of immune cells in a 4T1 mouse triple-negative breast cancer model. In this embodiment, the infiltration of immune cells in the tumor obtained in Example 7 was analyzed using the cell loss technique. The specific experimental steps are as follows: 1) Surgically remove the mouse tumor and place it in a sterile culture dish, then soak it in 3 ml of ice-cold RPMI. Take care not to remove the lymph nodes within the mammary gland / tumor tissue.

[0092] 2) Place one tumor into a 15 mL centrifuge tube and add 2 mL of preheated dissociation buffer (Collagenase IV (final concentration 2 mg / mL, purchased from MedChemExpress), DNase I (final concentration 4 U / mL, purchased from TAKARA) in RPMI).

[0093] 3) Incubate at 37°C in a shaker incubator for 30 minutes. Pipette the liquid vigorously up and down, disrupting the tissue every 10 minutes.

[0094] 4) Transfer the cell suspension up and down for 2 minutes, then centrifuge at 300 g for 10 minutes at room temperature.

[0095] 5) Aspirate the supernatant and gently suspend the microspheres in 1 mL of DPBS containing 0.5% BSA.

[0096] 6) Filter the resuspension through a 100 μm cell filter.

[0097] 7) Centrifuge at 300 g for 10 minutes at room temperature, then aspirate and discard the supernatant.

[0098] 8) If the tumor cell suspension is not bloody, skip this step: Resuspend the particles in 2 mL of erythrocyte lysis buffer (RBCL). Gently stir for 1 minute at room temperature. Add 20 mL of DPBS supplemented with 0.5% BSA and centrifuge at 300 g for 7 minutes at room temperature. Discard the supernatant. Resuspend the cells in 20 mL of DPBS supplemented with 0.5% BSA. Centrifuge at 300 g for 7 minutes at room temperature and discard the supernatant.

[0099] 9) Gently suspend the microspheres in 1 mL of DPBS containing 0.5% BSA.

[0100] 10) Filter the resuspension through a 70 μm cell filter.

[0101] 11) Centrifuge the cell suspension at 300 g for 4 minutes at 4°C.

[0102] 12) Take 1 × 10 6 One cell was resuspended in 50 µL of DPBS containing 2% BSA and pre-incubated on ice for 5-10 minutes to block the Fc receptor.

[0103] 13) Prepare antibody solutions using 50 µL FACS buffer (DPBS containing 2% BSA).

[0104] CD8 + T cell marker: CD45 + CD3 + CD8 + NK cell marker: CD45 + CD3 - NKp46 + 14) Add 50 µL of antibody solution to the corresponding well and mix by pipetting. Incubate the cells in the dark on ice for 30 minutes.

[0105] 15) Add 150 µL of ice-cold FACS buffer to each well to wash the cells. Centrifuge at 300 g for 4 minutes at 4°C.

[0106] 16) Discard the supernatant. Wash twice with 200 µL of ice-cold FACS buffer.

[0107] 17) Transfer the cells to a 1.5 mL EP tube and add FACS buffer to bring the final volume to 500 µL. Acquire the sample on a flow cytometer as quickly as possible.

[0108] The results are as follows Figure 8 As shown, the combination of the peptide PD-Compep and the immunotherapy drug 5-AZA-CdR increased CD8 levels in a 4T1 mouse triple-negative breast cancer model. + Infiltration of T cells and NK cells.

[0109] Example 9: Peptide PD-Compep treatment had no effect on mouse body weight. 1) Animal grouping: Six-week-old female BALB / c mice were divided into two groups of six each. The experiment consisted of two groups: a control group and a group using the peptide PD-Compep.

[0110] 2) Tumor cell preparation and inoculation: Adherent mouse triple-negative breast cancer 4T1 cells were digested with trypsin and washed three times with PBS. 5 × 10⁶ tumor cells were then inoculated. 5 Three-negative breast cancer 4T1 cells were injected into the right axilla of mice to induce tumor formation.

[0111] 3) Mouse administration: When the tumor volume reaches 100 mm 3 Treatment began. The peptide PD-Compep was administered intraperitoneally at a dose of 5 mg / kg daily, with a 5-day administration followed by a 1-day rest period, for two rounds. Mouse weight was measured every two days.

[0112] The results are as follows Figure 9 As shown, treatment with the peptide PD-Compep had no effect on mouse body weight.

[0113] Example 10: Inhibition of tumor growth in a 4T1 mouse triple-negative breast cancer model by combination of peptide PD-Compep and immunotherapy drug PD-1 antibody. 1) Animal grouping: Six-week-old female BALB / c mice were divided into four groups of six each. The experiment consisted of four groups, including: The control group used IgG (catalog number HY-P990679, MedChemExpress); the group using PD-1 antibody (clone RMP1-14, catalog number HY-P99144, MedChemExpress); the group using peptide PD-Compep; and the group using PD-1 antibody plus peptide PD-Compep.

[0114] 2) Tumor cell preparation and inoculation: Adherent mouse triple-negative breast cancer 4T1 cells were digested with trypsin and washed three times with PBS. 5 × 10⁶ tumor cells were then inoculated. 5 Three-negative breast cancer 4T1 cells were injected into the right axilla of mice to induce tumor formation.

[0115] 3) Mouse administration: When the tumor volume reaches 100 mm 3 Treatment begins. IgG or PD-1 antibodies are administered intraperitoneally at a dose of 200 μg / dose four times, and PD-Compep peptide is administered intraperitoneally at a dose of 100 μg / dose eight times. Tumor size is measured with calipers every two days. Volume is calculated using the formula: Volume = Length × Width. 2 / 2.

[0116] The results are as follows Figure 10 As shown, the combined use of PD-1 antibody and peptide PD-Compep inhibited the growth of triple-negative breast cancer in mice.

[0117] Example 11: Combination of peptide PD-Compep and immunotherapy drug PD-1 antibody increases the infiltration of immune cells in a 4T1 mouse triple-negative breast cancer model. In this embodiment, the infiltration of immune cells in the tumor obtained in Example 10 was analyzed using the cell loss technique, and the specific experimental steps were the same as in Example 8.

[0118] The results are as follows Figure 11 As shown, the combination of the peptide PD-Compep and the immunotherapy drug PD-1 antibody increased CD8 levels in a 4T1 mouse triple-negative breast cancer model. + Infiltration of T cells and NK cells.

[0119] In summary, this invention targets intracellular PD-L1 with a PD-L1-specific peptide, blocking the inhibitory function of PD-L1 in innate immunity. When used in combination with traditional immunotherapy, it can inhibit the immune escape process of tumors, providing a promising treatment strategy for improving the response rate of patients with high PD-L1 expression to immunotherapy.

[0120] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A polypeptide that specifically blocks the interaction between PD-L1 and endogenous double-stranded RNA, characterized in that, Its amino acid sequence is IQDTNSKKQSDT.

2. A nucleic acid encoding the polypeptide of claim 1.

3. A biomaterial, characterized in that, It contains the polypeptide of claim 1 or the nucleic acid of claim 2.

4. The biomaterial according to claim 3, characterized in that, The biomaterials are vectors, expression cassettes, transposons, cells, or engineered bacteria.

5. A fusion polypeptide, characterized in that, It contains the polypeptide described in claim 1 and cell-penetrating peptides.

6. The fusion polypeptide according to claim 5, characterized in that, The cell-penetrating peptide is HLYVSPWGG; preferably, the amino acid sequence of the fusion polypeptide is HLYVSPWGGIQDTNSKKQSDT.

7. The use of the polypeptide of claim 1, the nucleic acid of claim 2, the biomaterial of claim 3 or 4, or the fusion polypeptide of claim 5 or 6 in the preparation of a pharmaceutical product; preferably, the pharmaceutical product is used to treat tumors; more preferably, the tumor is cancer; more preferably, the cancer is triple-negative breast cancer.

8. A medicine, characterized in that, It contains the polypeptide of claim 1, the nucleic acid of claim 2, the biomaterial of claim 3 or 4, or the fusion polypeptide of claim 5 or 6.

9. The pharmaceutical product according to claim 8, characterized in that, The drug may also include pharmaceutically acceptable excipients, or may include other drugs for treating tumors.

10. The pharmaceutical product according to claim 9, characterized in that, Other drugs used to treat tumors include DNMT inhibitors, interferons, or PD-1 antibodies; Preferably, the DNMT inhibitor is 5-AZA-CdR; or the interferon is IFNβ.

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