Immunomodulatory peptides and uses thereof

Abstract

The application firstly discovers polypeptides of several HLA A11:01 ligands, which are derived from immune regulatory proteins ARG-1, ARG-2, FoxP3, IDO, PDL-1, PDL-2, PD-L2, TGF-beta. Specifically, the application screens 14 polypeptides by the prediction score of proteasome cleavage, TAP transport and MHC binding, wherein 9 polypeptides are newly discovered immune regulatory polypeptides. The 9 polypeptides can effectively activate the immune response of T cells and can stimulate the production of specific T cells with high killing efficiency. The activated T cells have the potential to resist cancers such as pancreatic cancer and myeloid cell cancer by releasing cytokines. The immune regulatory polypeptides of the application have the efficacy of preventing / treating cancers such as pancreatic cancer, breast cancer and sarcoma.

Description

Technical Field

[0001] This invention relates to the fields of immunology, peptide screening, and cancer treatment. Specifically, this invention provides immunomodulatory peptides and their use in the preparation of medicaments or pharmaceutical compositions for treating cancer. Background Technology

[0002] A core objective of anticancer immunotherapy is to identify and characterize appropriate tumor antigens. Generally, tumor-associated antigens (TAAs) are highly expressed specifically in tumor cells, while tumor-specific antigens (TSAs) are entirely unique to tumor cells. Recent research has focused on identifying patient-specific neoantigens, which are not expressed in healthy tissues and possess high immunogenicity. Recently, a class of tumor suppressor antigens with immunomodulatory properties has been identified, capable of recognizing and attacking many cell types in the tumor microenvironment, such as regulatory immune cells and tumor cells. These antigens can originate from metabolic enzymes (e.g., IDO, arginase 1, arginase 2), checkpoint inhibitors (e.g., PD-L1, PD-L2), inhibitory cytokines (e.g., transforming growth factor-β), or transcription factors (e.g., FoxP3).

[0003] Immunomodulatory peptide-based vaccination aims to reshape the immune cell repertoire and transform the immunosuppressive microenvironment into a pro-inflammatory one. During treatment, pro-inflammatory cytokines released by immunomodulatory peptide-specific CD4+ T cells may synergistically interact with target cell killing effects mediated by immunomodulatory peptide-specific CD8+ T cells. Studies have shown that melanoma patients receiving anti-PD-1 therapy may experience synergistic therapeutic effects through vaccination with peptide vaccines containing IDO and PD-L1 epitopes (covering both CD8+ and CD4+ T cell epitopes).

[0004] Targeted immunosuppressive antigens can not only directly attack tumor cells, but also modulate the tumor microenvironment, imbuing it with immune capabilities and enabling it to attack the tumor. Different immunomodulatory peptides are expressed by different cells and can be used in combination in anticancer immunotherapy to directly attack tumor cells and modulate local immune cells, thereby creating a tumor-hostile microenvironment and inhibiting tumor angiogenesis.

[0005] Therefore, there is an urgent need in this field to discover new immunomodulatory peptides, thereby providing a new treatment approach for cancer immunotherapy. Summary of the Invention

[0006] The purpose of this invention is to provide immunomodulatory peptides and their use in the preparation of medicaments or pharmaceutical compositions for treating cancer.

[0007] In a first aspect, the present invention provides a method for screening immunomodulatory peptides, the method comprising:

[0008] (1) Provide candidate peptides;

[0009] (2) Calculate the total score, which is the sum of the proteasome cleavage score, the TAP transport score, and the MHC binding score; the proteasome cleavage score, the TAP transport score, and the MHC binding score are predicted using the MHC-I prediction tool;

[0010] (3) Select the polypeptide with the highest total score from the candidate polypeptides as the immunomodulatory polypeptide.

[0011] In another preferred embodiment, step (2) further includes: calculating a processing fraction, which is the sum of the proteasome cleavage fraction and the TAP transport fraction.

[0012] In another preferred embodiment, the candidate polypeptide is derived from arginase 1, FOXP-3, IDO1, PD-L1, PD-L2, TGF-β, or a combination thereof.

[0013] In another preferred embodiment, the MHC-I prediction tool includes: IEDB MHC-I prediction tool, Anthem, DeepHLApan, Evaxion AI-Immunology, and NetMHCpan.

[0014] In another preferred embodiment, the MHC-I prediction tool is the IEDB MHC-I prediction tool.

[0015] In another preferred embodiment, the proteasome cleavage fraction is the logarithm of the total amount of cleavage sites used at the C-terminus of the released polypeptide.

[0016] In another preferred embodiment, the TAP transport fraction is the effective value of the binding of the polypeptide or its N-terminal extended precursor to TAP.

[0017] In another preferred embodiment, the effective value is -log(IC50).

[0018] In another preferred embodiment, the MHC binding score is a score obtained through MHC-I class prediction.

[0019] In another preferred embodiment, the MHC binding fraction is -log(IC50).

[0020] In a second aspect, the present invention provides an immunomodulatory polypeptide, which is obtained by screening using the method described in the first aspect of the present invention.

[0021] In another preferred embodiment, the polypeptide has an amino acid sequence as shown in any one of SEQ ID NO:1-14.

[0022] In another preferred embodiment, the polypeptide has an amino acid sequence as shown in any one of SEQ ID NO:1-9.

[0023] In another preferred embodiment, the immunomodulatory polypeptide has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence similarity to the amino acid sequence shown in any one of SEQ ID NO:1-9.

[0024] In another preferred embodiment, the immunomodulatory polypeptide has a similar function to the amino acid sequence shown in any one of SEQ ID NO:1-9.

[0025] A third aspect of the present invention provides the use of the immunomodulatory polypeptide described in the second aspect of the present invention for the preparation of a medicament or combination of medicaments for the prevention and / or treatment of cancer.

[0026] In another preferred embodiment, the cancer includes pancreatic cancer, breast cancer, and sarcoma.

[0027] In another preferred embodiment, the immunomodulatory polypeptide used to prepare a drug or combination of drugs for the prevention and / or treatment of pancreatic cancer is selected from the group consisting of: SEQ ID NO:1-9, or combinations thereof; preferably SEQ ID NO:1, 2, 5, 6, or combinations thereof; more preferably SEQ ID NO:1, 2, or combinations thereof; and most preferably SEQ ID NO:1.

[0028] In another preferred embodiment, the immunomodulatory peptide used to prepare a medicament or combination of medicaments for the prevention and / or treatment of breast cancer is selected from the group consisting of SEQ ID NO:1, 2, 5, 6, or combinations thereof; preferably SEQ ID NO:1, 2, or combinations thereof.

[0029] In another preferred embodiment, the immunomodulatory polypeptide used to prepare a drug or combination of drugs for the prevention and / or treatment of sarcoma is selected from the group consisting of: SEQ ID NO:1, 2, 5, 6, or combinations thereof; preferably SEQ ID NO:1, 5, or combinations thereof; more preferably SEQ ID NO:5.

[0030] In another preferred embodiment, the drug or pharmaceutical composition further includes a pharmaceutically acceptable carrier, diluent, or excipient.

[0031] A fourth aspect of the present invention provides a medicament or pharmaceutical composition comprising:

[0032] (I) A polypeptide having an amino acid sequence as shown in any one of SEQ ID NO:1-9;

[0033] (II) Pharmaceutically acceptable carriers.

[0034] In another preferred embodiment, the drug or drug composition further includes: (III) other drugs for treating cancer.

[0035] In another preferred embodiment, the cancer includes pancreatic cancer, breast cancer, and sarcoma.

[0036] In another preferred embodiment, the dosage form of the drug or drug composition is selected from the group consisting of lyophilized preparations, liquid preparations, or combinations thereof.

[0037] In another preferred embodiment, the drug or drug composition is a liquid formulation.

[0038] A fifth aspect of the present invention provides a medicine box, comprising:

[0039] (A) A first container, and an immunomodulatory polypeptide located in the first container, the immunomodulatory polypeptide comprising a polypeptide with an amino acid sequence as shown in any one of SEQ ID NO:1-9;

[0040] (B) The second container, and other cancer treatment drugs located in the second container.

[0041] In another preferred embodiment, the other drugs for treating cancer include: immunotherapy drugs.

[0042] In another preferred embodiment, the immunotherapy drug includes a PD-1 inhibitor.

[0043] A sixth aspect of the present invention provides a method for activating T cells in vitro, comprising the steps of:

[0044] (a) Provide a T cell to be activated;

[0045] (b) The immunomodulatory polypeptide described in the second aspect of the present invention is incubated with the T cells to be activated to obtain activated T cells.

[0046] In another preferred embodiment, the T cells to be activated are derived from PBMC cells.

[0047] In another preferred embodiment, the PBMC cells are derived from healthy individuals or cancer patients with HLA A11:01 subtype.

[0048] In another preferred embodiment, the tumor patient includes: pancreatic cancer patient, breast cancer patient, and sarcoma patient.

[0049] In another preferred embodiment, in step (b), the immunomodulatory peptides and cytokines are incubated with the T cells to be activated to obtain activated T cells.

[0050] In another preferred embodiment, the immunomodulatory polypeptide is selected from the group consisting of SEQ ID NO:1-9 or combinations thereof.

[0051] In another preferred embodiment, the cytokines include IL-7 and IL-2.

[0052] In another preferred embodiment, step (b) includes the step:

[0053] (b1) The immunomodulatory peptide and IL-7 were incubated with the T cells to be activated;

[0054] (b2) The immunomodulatory peptide, IL-7, and IL-2 are incubated with the T cells to be activated to obtain activated T cells.

[0055] In another preferred embodiment, the concentration of the immunomodulatory peptide is 10 ± 1 μg / mL.

[0056] In another preferred embodiment, the concentration of IL-7 is 20 ± 2 ng / mL.

[0057] In another preferred embodiment, the concentration of IL-2 is 20 ± 2 U / mL.

[0058] In another preferred embodiment, the incubation period is 12-14 days, preferably 14 days.

[0059] In another preferred embodiment, the activated T cells are selected through cell sorting.

[0060] In another preferred embodiment, the cells used for cell sorting are PBMCs incubated in step (b).

[0061] In another preferred embodiment, the cell sorting is performed using CD8+ (APC) antibody labeling.

[0062] A seventh aspect of the present invention provides a method for killing tumor cells in vitro, comprising the steps of:

[0063] (i) Provide a tumor cell line;

[0064] (ii) Co-culturing activated T cells with the tumor cell line to kill tumor cells;

[0065] The activated T cells are prepared by the method described in the sixth aspect of this invention.

[0066] In another preferred embodiment, the tumor cells are tumor cells expressing HLA-A11:01.

[0067] In another preferred embodiment, the tumor cell line includes: human pancreatic cancer cell line and human chronic myeloid leukemia cell line.

[0068] In another preferred embodiment, a presenting polypeptide is added to the tumor cells, thereby allowing the tumor cells to take up the presenting polypeptide.

[0069] In another preferred embodiment, the presented polypeptide is selected from the group consisting of any one of the amino acid sequences shown in SEQ ID NO:1-9, or combinations thereof.

[0070] In another preferred embodiment, the cytotoxicity T1 of activated T cells against human pancreatic cancer cell line (PANC1) with added polypeptide, compared to the cytotoxicity T0 of activated T cells against the untreated human pancreatic cancer cell line (PANC1), satisfies T1 / T0 ≥ 150%, preferably ≥ 4000%, wherein the activated T cells are selected from the group consisting of: T cells activated by the amino acid sequence shown in SEQ ID NO: 8, T cells activated by the amino acid sequence shown in SEQ ID NO: 1, T cells activated by the amino acid sequence shown in SEQ ID NO: 7, T cells activated by the amino acid sequence shown in SEQ ID NO: 5, or combinations thereof.

[0071] In another preferred embodiment, the cytotoxicity U1 of activated T cells against human chronic myeloid leukemia cell lines (K562-A11) with added peptides, compared to the cytotoxicity U0 of activated T cells against such cell lines, satisfies U1 / U0 ≥ 150%, preferably ≥ 340%, wherein the activated T cells are selected from the group consisting of: T cells activated by the amino acid sequence shown in SEQ ID NO:1, T cells activated by the amino acid sequence shown in SEQ ID NO:2, T cells activated by the amino acid sequence shown in SEQ ID NO:4, T cells activated by the amino acid sequence shown in SEQ ID NO:6, T cells activated by the amino acid sequence shown in SEQ ID NO:7, T cells activated by the amino acid sequence shown in SEQ ID NO:9, or combinations thereof.

[0072] In another preferred embodiment, in step (ii), activated T cells are co-cultured with a human pancreatic cancer cell line, the activated T cells having high TNF-α secretion levels.

[0073] In another preferred embodiment, in step (ii), activated T cells are co-cultured with a human chronic myeloid leukemia cell line, wherein the activated T cells have high levels of TNF-α and IFN-γ secretion.

[0074] An eighth aspect of the present invention provides a screening system for immunomodulatory peptides, comprising:

[0075] An input unit configured to input candidate immunomodulatory protein sequences;

[0076] A prediction unit, configured to predict the candidate immunomodulatory protein sequence, includes:

[0077] (S1) Predict the proteasome cleavage fraction and calculate the logarithm of the total number of cleavage sites used to release the C-terminal cleavage sites of the candidate polypeptides in the candidate immunomodulatory protein sequence; and / or

[0078] (S2) Predict the TAP transport fraction and calculate the effective binding value of the candidate polypeptide or its N-terminal elongated precursor in the candidate immunomodulatory protein sequence to TAP; and / or

[0079] (S3) Predict the MHC binding score and calculate the score obtained by the prediction of MHC-I class;

[0080] (S4) Calculate the processing fraction, which is the sum of the proteasome cleavage fraction and the TAP transport fraction; and / or

[0081] (S5) Calculate the total score, which is the sum of the proteasome cleavage fraction, the TAP transport fraction, and the MHC binding fraction;

[0082] Select the polypeptide with the highest total score from the immunomodulatory protein sequence as the immunomodulatory polypeptide;

[0083] An output unit is configured to output the result of the prediction unit.

[0084] In another preferred embodiment, the candidate immunomodulatory protein sequence is selected from the group consisting of arginase 1, FOXP-3, IDO1, PD-L1, PD-L2, TGF-β, or combinations thereof.

[0085] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0086] Figure 1 The results of IFN-γELISpot in PBMCs of healthy individuals are shown.

[0087] Figure 2 The results of PBMC IFN-γELISpot in patients with different tumor types are shown.

[0088] Figure 3 The results for IFN-γELISpot are summarized.

[0089] Figure 4 The results of the screening of the peptide IFN-γELISpot are shown.

[0090] Figure 5 The heatmap results of the screened peptide IFN-γELISpot are shown.

[0091] Figure 6 The results of ICS analysis are shown after IDO 05 specific T cells were co-incubated with PANC-1 for 24 hours.

[0092] Figure 7 The results of ICS analysis are shown after IDO 05 specific T cells were co-incubated with K562-A11 for 24 hours. Detailed Implementation

[0093] Through extensive and in-depth experiments, the inventors have for the first time discovered several HLA A11:01 ligand peptides derived from immunomodulatory proteins ARG-1, ARG-2, FoxP3, IDO, PDL-1, PDL-2, PD-L2, and TGF-β. Specifically, this invention screened 14 peptides based on predicted scores for proteasome cleavage, TAP transport, and MHC binding, as well as processing scores based on proteasome cleavage and TAP transport, and the total scores based on proteasome cleavage, TAP transport, and MHC binding. Nine of these are newly discovered immunomodulatory peptides. These nine peptides are non-toxic, can effectively activate T cell immune responses, and can stimulate the production of highly efficient, specific T cells. Activated T cells, through the release of cytokines, have the potential to fight cancers such as pancreatic cancer and myeloid cell carcinoma. Based on this, this invention was completed.

[0094] Therefore, this invention relates to an anticancer drug or pharmaceutical composition that can be selectively used in combination with other immunotherapies and to serve as a cancer treatment method by adoptive transfer or induction of antigen-specific T cells in vivo via vaccination. One aspect of the invention is that the drug provided herein can be used in combination with cancer chemotherapy. Another aspect relates to the prevention and treatment of infections using the same methods described above.

[0095] It should be understood that the specific methods and experimental conditions of the invention described below in varying degrees of detail are intended to provide a substantive understanding of the invention. Definitions of certain terms used in this specification are provided below. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0096] the term

[0097] Where a numerical range is provided, unless the context clearly indicates otherwise, it should be understood that every intermediate integer of the value, every tenth of every intermediate integer of the value, any other intermediate value between the upper and lower limits of the range, and any other intermediate value within the specified range are included within the scope of this invention. The upper and lower limits of these smaller ranges may be independently included within the smaller range and also covered within the scope of this invention, but are subject to any express exclusions within the specified range. For example, "1 to 50" includes "2 to 25", "5 to 20", "25 to 50", "1 to 10", etc.

[0098] As used herein, the terms “containing” or “including (comprise)” can be open-ended, semi-closed, or closed-ended. In other words, the terms also include “consistently made of” or “made of”.

[0099] As used herein, the term “and / or” refers to and covers any and all possible combinations of one or more of the related listed items.

[0100] The immunomodulatory polypeptide of the present invention

[0101] As used herein, the terms “immunomodulatory polypeptide,” “IMDP,” and “IMDP polypeptide” are used interchangeably to refer to several polypeptide sequences derived from immunomodulatory proteins discovered in this invention.

[0102] In this invention, the MHC-I screening tool is used to screen the sequences of immunomodulatory proteins (including arginase 1, FOXP-3, IDO1, PD-L1, PD-L2 and TGF-β) to find candidate HLA-A11:01 ligands.

[0103] Ultimately, this invention screened out 14 polypeptide sequences, of which 9 sequences had not been reported before, as shown in SEQ ID NO:1-9.

[0104] The present invention is protected by any amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence similarity to any of the amino acid sequences shown in SEQ ID NO:1-9, and / or precursor or source proteins of any of the amino acid sequences shown in SEQ ID NO:1-9, and / or amino acid sequences having similar functions to any of the amino acid sequences shown in SEQ ID NO:1-9. Similar sequences include sequences in which one or more amino acids are added to both ends of any of the amino acid sequences shown in SEQ ID NO:1-9 without affecting the function of the sequence itself; the one or more amino acids may be tag sequences, detection sequences, etc.

[0105] The present invention provides a method and system for screening immunomodulatory peptides.

[0106] This invention utilizes an MHC-I screening tool to screen for immunomodulatory protein sequences, thereby obtaining immunomodulatory peptides. Therefore, this invention provides a method and system for screening immunomodulatory peptides.

[0107] (1) Provide candidate peptides;

[0108] (2) Calculate the total score, which is the sum of the proteasome cleavage score, the TAP transport score, and the MHC binding score; the proteasome cleavage score, the TAP transport score, and the MHC binding score are predicted using the MHC-I prediction tool;

[0109] (3) Select the polypeptide with the highest total score from the candidate polypeptides as the immunomodulatory polypeptide.

[0110] Preferably, step (2) further includes: calculating the processing fraction, which is the sum of the proteasome cleavage fraction and the TAP transport fraction.

[0111] Wherein, the proteasome cleavage fraction is the logarithm of the total amount of cleavage sites used at the C-terminus of the released polypeptide; the TAP transport fraction is the effective value of TAP binding to the polypeptide or its N-terminal elongation precursor; and the MHC binding fraction is a score obtained through MHC-I class prediction.

[0112] It is understood that any MHC-I prediction tool, software, or platform capable of predicting proteasome cleavage fraction, TAP transport fraction, and MHC binding fraction can be used in the immunomodulatory peptide screening method of the present invention. Preferably, the MHC-I prediction tool used is the IEDB MHC-I prediction tool.

[0113] The system includes:

[0114] An input unit configured to input candidate immunomodulatory protein sequences;

[0115] A prediction unit, configured to predict the candidate immunomodulatory protein sequence, includes:

[0116] (S1) Predict the proteasome cleavage fraction and calculate the logarithm of the total number of cleavage sites used to release the C-terminal cleavage sites of the candidate polypeptides in the candidate immunomodulatory protein sequence; and / or

[0117] (S2) Predict the TAP transport fraction and calculate the effective binding value of the candidate polypeptide or its N-terminal elongated precursor in the candidate immunomodulatory protein sequence to TAP; and / or

[0118] (S3) Predict the MHC binding score and calculate the score obtained by the prediction of MHC-I class;

[0119] (S4) Calculate the processing fraction, which is the sum of the proteasome cleavage fraction and the TAP transport fraction; and / or

[0120] (S5) Calculate the total score, which is the sum of the proteasome cleavage fraction, the TAP transport fraction, and the MHC binding fraction;

[0121] Select the polypeptide with the highest total score from the immunomodulatory protein sequence as the immunomodulatory polypeptide;

[0122] An output unit is configured to output the result of the prediction unit.

[0123] The main advantages of this invention include:

[0124] (1) The present invention provides a method for screening immunomodulatory peptides. The method obtains scores by predicting proteasome cleavage, TAP transport and MHC binding, selects peptides with high scores as potential HLAA11:01 ligands, and finally screens out 14 peptides, of which 9 are newly discovered immunomodulatory peptides.

[0125] (2) The polypeptide of the present invention can effectively activate the immune response of T cells derived from patients with pancreatic cancer, breast cancer, and sarcoma.

[0126] (3) The polypeptide of the present invention can stimulate the production of highly efficient specific T cells, which have higher cell killing efficacy against pancreatic cancer cells.

[0127] (4) The polypeptide of the present invention can effectively induce T cells co-cultured with cancer cell lines to release cytokines. In pancreatic cancer cell lines, it induces T cells to secrete more TNF-α; in chronic myeloid leukemia cell lines, it induces T cells to secrete more IFN-γ and TNF-α.

[0128] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0129] Example 1: Prediction of Immunogenicity of Peptides

[0130] This embodiment relates to the prediction of peptides with binding restriction to the HLA-A11:01 allele. Specifically, the IEDB MHC-I processing prediction tool was used to screen sequences of different immunomodulatory proteins (Arginase 1, FOXP-3, IDO 1, PD-L1, PD-L2, TGF-Beta) to identify potential HLA-A11:01 ligands (i.e., peptides naturally processed from their source proteins and presented by MHC-I class molecules). Three main prediction scores were evaluated:

[0131] 1. Proteasome cleavage: This is defined as the logarithm of the total amount of cleavage sites at the C-terminus of the polypeptide.

[0132] 2. TAP transport: Estimating the effective -log(IC50) value of TAP binding of the peptide or its N-terminal elongated precursor. It has been shown that high affinity of the peptide translates to high transport rate.

[0133] 3. MHC binding: MHC binding prediction is the same as for Class I. To maintain consistency, output the -log(IC50) value.

[0134] All scores indicate that higher values ​​are associated with higher predictive efficiency.

[0135] In addition to the scores mentioned above, two composite scores were also calculated:

[0136] 1. Processing Fraction: This fraction combines proteasome cleavage and TAP transport prediction. It predicts an amount of peptide in proportion to the number of peptides in the endoplasmic reticulum (ER), where peptides can bind multiple MHC molecules. This allows for the prediction of T cell epitope candidates independently of MHC restriction.

[0137] 2. Total Score: This score combines proteasome cleavage, TAP transport, and MHC binding predictions. It predicts the amount of peptide presented in proportion to the number of MHC molecules on the cell surface.

[0138] Peptides with high total scores were selected as potential HLA-A11:01 ligands. The sequences of the screened peptides are shown in Table 1; the predicted presentation ability and affinity are shown in Table 2. In addition, their physicochemical properties and toxicity predictions were further evaluated using Toxinpred (Table 3).

[0139] Table 1. Peptide sequences

[0140]

[0141] Table 2. Predicted scores of presentation ability and affinity of peptide sequence to HLA-A11:01 allele.

[0142]

[0143]

[0144] Table 3. Physicochemical properties and toxicity predicted using the Toxinpred algorithm

[0145]

[0146] Example 2: PBMC cell polypeptide-induced stimulation culture

[0147] This embodiment relates to the in vitro induction and stimulation of PBMC cells using the polypeptide of the present invention.

[0148] PBMCs of healthy volunteers with HLA-A11:01 subtype were harvested and cultured in X-VIVO15 complete medium at a ratio of 2 × 10⁻⁶ cells / mL. 5 Add PBMCs to 96-well plates, adding IMDP peptide (10 μg / mL) and IL-7 (20 ng / mL IL-7) to each well for stimulation. Incubate at 37°C, marking day 1. On day 4, change the medium and add IMDP peptide (10 μg / mL), IL-7 (20 ng / mL IL-7), and IL-2 (20 U / mL). Thereafter, change the medium and add the corresponding volumes of cytokines and peptides every one day. Co-stimulate and culture for 12-14 days, maintaining a concentration of 3 × 10⁻⁶ cells / well. 6 Within.

[0149] Example 3: ELISPOTs method to verify peptide-activated T cell immune response

[0150] This embodiment relates to the verification of the activation of T cell immune responses by the polypeptide of the present invention.

[0151] On day 13, cells cultured in Example 2 were collected and added to human IFN gamma interferon ELISPOTs plates for incubation at 37°C for 24 hours for color development. Spots generated in the ELISPOT assay were counted according to the ELISPOTs assay instructions (see kit manual). The immunogenicity requirement for the experimental peptide is as follows: the ratio of experimental peptide spots to irrelevant peptide spots > 1, meaning the number of spots caused by the experimental peptide is more than twice the number of irrelevant peptide spots.

[0152] This embodiment further selected 5 healthy individuals ( Figure 1 ) and 7 patients with different types of tumors ( Figure 2 PBMCs were cultured in vitro with experimental peptides, and IFN-γ ELISpot was detected. The results were then summarized and analyzed. Figure 3 ), from which peptides with a positive rate ≥33% were selected for display. Figure 4-5 ). Figures 1-4The vertical axis represents the number of spots in the peptide experimental group minus the blank control group, and the horizontal axis represents different experimental peptides. Different shapes represent different PBMC samples.

[0153] Example 4: Sorting and rapid expansion of antigen-specific T cells

[0154] This embodiment involves the sorting and rapid expansion culture of T cells using flow cytometry and a suitable culture medium.

[0155] Using PBMCs cultured for 14 days as described in Example 2, flow cytometry was used to sort IMDP antigen-specific activated T cells that expressed the 4-1BB surface marker. The enriched antigen-specific T cells were then expanded from the sorted cells using a rapid expansion protocol. The initial cell count before expansion was approximately 1-2 × 10⁻⁶. 5 In an X-VIVO15 complete culture medium system, 0.03 μg / mL of anti-CD3 (OKT3) reagent and 6000 U / mL of IL2 were added to stimulate the rapid expansion of specific T cells. During this period, the expanding T cells were counted every 2 days using a cell counter. On the 4th-5th day of expansion, the number of sorted T cells was observed to have doubled, and the culture medium was replaced to replenish anti-CD3 (OKT3) reagent and IL2 for continued stimulation. Thereafter, the culture medium was changed every 3 days, and the concentration of T cells in the culture system was controlled at 3 × 10⁻⁶. 6 Within.

[0156] Example 5: LDH release assay demonstrates the cytotoxic activity of peptide-specific T cells.

[0157] This embodiment involves the verification of peptide-specific T-cell killing activity.

[0158] The rapidly expanded T cells from Example 4 were co-cultured with HLA-A11:01-expressing tumor cells. The tumor cells were divided into two groups: one group was pre-cultured with peptides to allow cells to take up and present the peptides, and the other group was not pre-cultured with peptides. These two groups served as control groups when co-cultured with IMDP-specific T cells. In the LDH experiment, wells for maximum target cell release, volume correction, culture medium control, effector cell spontaneous release, and target cell spontaneous release were set up as controls, with three replicates for each group. After co-culturing for 4 hours, 50 μL of the co-cultured cell supernatant was added to 50 μL of LDH substrate mixture to catalyze the LDH substrate reaction. The final wavelength was read at 490 nm, and the killing activity of IMDP-specific T cells against the two different treated tumor cells was calculated using the following formula.

[0159] The formula for calculating the killing activity is: Killing efficiency (%) = (Experimental wells - spontaneous release from effector cells - spontaneous release from target cells + culture medium wells) / (maximum release from target cells - volume correction wells - spontaneous release from target cells + culture medium wells) × 100%.

[0160] In the co-culture experiment of PANC-1 cells and specific T cells, the cytotoxicity of T cells against PANC-1 cells cultured with pre-added peptides was significantly higher (Table 4). Similarly, in the co-culture experiment of K562-A11 cells and specific T cells, the cytotoxicity of T cells against K562-A11 cells cultured with pre-added peptides was also higher than that against the group without peptides, indicating that the peptides have the ability to stimulate the production of highly efficient specific T cells and can exhibit higher killing activity against tumor cells expressing HLA-A11:01 and presenting peptides (Table 5).

[0161] Table 4. Summary of LDH release experiment results (PANC-1 cells)

[0162]

[0163] Table 5. Summary of LDH release assay results (K562-A11 cells)

[0164]

[0165]

[0166] Example 6: Flow cytometry staining to verify the amount of cytokines released.

[0167] This embodiment involves verifying the release of cytokines and investigating the secretion of cytokines by T cells co-cultured with different cancer cells.

[0168] After the peptide-specific T cells and target cells co-cultured in Example 4 and the effector cell spontaneous release group were cultured for another 24 hours, flow cytometry staining was performed. Extracellular flow cytometry antibodies for CD3 and CD8 were added at a ratio of 1:200 and incubated for 30 minutes. After the incubation, the cells were perforated overnight with a perforation reagent. After resuspending the cells in 50 μL of perforation buffer, intracellular flow cytometry antibodies for IFN-γ and TNF-α were added at a ratio of 1:50 and incubated for 30 minutes. The cells were then detected by flow cytometry.

[0169] See results Figure 6-7After co-culturing for 24 hours, in the experiment of co-culturing PANC-1 cells with IMDP-specific T cells, T cells co-cultured with PANC-1 cells that had been pre-cultured with peptides showed higher TNF-α secretion. In the experiment of co-culturing K562-A11 cells with peptide-specific T cells, T cells co-cultured with K562-A11 cells that had been pre-cultured with peptides showed higher IFN-γ and TNF-α secretion.

[0170] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A method for screening immunomodulatory peptides, characterized in that, The method includes: (1) Provide candidate peptides; (2) Calculate the total score, which is the sum of the proteasome cleavage score, the TAP transport score, and the MHC binding score; the proteasome cleavage score, the TAP transport score, and the MHC binding score are predicted using the MHC-I prediction tool; (3) Select the polypeptide with the highest total score from the candidate polypeptides as the immunomodulatory polypeptide.

2. The method as described in claim 1, characterized in that, The MHC-I prediction tool is the IEDB MHC-I prediction tool.

3. An immunomodulatory polypeptide, characterized in that, The polypeptide was obtained by screening using the method described in claim 1.

4. The immunomodulatory polypeptide according to claim 3, characterized in that, The polypeptide has an amino acid sequence as shown in any one of SEQ ID NO:1-9.

5. The use of the immunomodulatory polypeptide according to claim 3, characterized in that, Used to prepare medicines or combinations of medicines for the prevention and / or treatment of cancer; said cancers include: pancreatic cancer, breast cancer, sarcoma.

6. A drug or drug composition, characterized in that, The drug or drug composition includes: (I) A polypeptide having an amino acid sequence as shown in any one of SEQ ID NO:1-9; (II) Pharmaceutically acceptable carriers.

7. A medicine box, characterized in that, include: (A) A first container, and an immunomodulatory polypeptide located in the first container, the immunomodulatory polypeptide comprising a polypeptide with an amino acid sequence as shown in any one of SEQ ID NO: 1-9; (B) The second container, and other cancer treatment drugs located in the second container.

8. A method for activating T cells in vitro, characterized in that, Including the following steps: (a) Provide a T cell to be activated; (b) The immunomodulatory polypeptide of claim 3 is incubated with the T cells to be activated to obtain activated T cells.

9. A method for killing tumor cells in vitro, characterized in that, Including the following steps: (i) Provide a tumor cell line; (ii) Co-culturing activated T cells with the tumor cell line to kill tumor cells; The activated T cells are prepared by the method described in claim 8.

10. A screening system for immunomodulatory peptides, characterized in that, include: An input unit configured to input candidate immunomodulatory protein sequences; A prediction unit, configured to predict the candidate immunomodulatory protein sequence, includes: (S1) Predict the proteasome cleavage fraction and calculate the logarithm of the total number of cleavage sites used to release the C-terminal cleavage sites of the candidate polypeptides in the candidate immunomodulatory protein sequence; and / or (S2) Predict the TAP transport fraction and calculate the effective binding value of the candidate polypeptide or its N-terminal elongated precursor in the candidate immunomodulatory protein sequence to TAP; and / or (S3) Predict the MHC binding score and calculate the score obtained by the prediction of MHC-I class; (S4) Calculate the processing fraction, which is the sum of the proteasome cleavage fraction and the TAP transport fraction; (S5) Calculate the total score, which is the sum of the proteasome cleavage fraction, the TAP transport fraction, and the MHC binding fraction; Select the polypeptide with the highest total score from the immunomodulatory protein sequence as the immunomodulatory polypeptide; An output unit is configured to output the result of the prediction unit.

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