TSSK4 antigen HLA-A2 restrictive CTL epitope peptide and application thereof
By screening and identifying the TSSK4 antigen HLA-A2 restricted CTL epitope peptide, the problem of limited effectiveness of existing tumor treatment methods in some types of tumors is solved, effective killing of tumor cells is achieved, and new tumor immunotherapy targets are provided.
Patent Information
- Application Number
- CN202510270540.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-07
- Publication Date
- 2025-06-13
AI Technical Summary
Existing tumor treatment methods are limited in some types of tumors, and tumor cells are prone to drug resistance, resulting in treatment failure.
TSSK4 antigen HLA-A2 restriction CTL epitope peptide was screened and identified, its immune activity was verified by in vitro experiments, and a polypeptide vaccine was designed to activate specific CTL responses.
Multiple HLA-A2 restriction epitope peptides with potential immunoactivity were successfully screened, providing a new candidate target for TSSK4-related tumor immunotherapy, which can effectively activate CTL response and have a killing effect on tumor cells.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biomedical technologies, and relates to a TSSK4 antigen HLA-A2 restricted CTL epitope peptide and its application in tumor immunotherapy. Background Art
[0002] With the changes in lifestyle and the aging of the population, the incidence and mortality of tumors continue to rise. Traditional treatment methods (such as surgery, radiotherapy, and chemotherapy) have limited effects in certain types of tumors. Many tumors may develop drug resistance after initial treatment, resulting in treatment failure. However, immunotherapy can combat drug resistance through different mechanisms. Especially for some recurrent and refractory tumors, immunotherapy not only helps to improve the survival rate and quality of life of tumor patients, but also provides an important scientific basis for future cancer treatment innovation. Therefore, increasing investment in tumor immunotherapy research is an important task in the current medical field.
[0003] Since mutations (such as point mutations, insertions, or deletions) occurring in the genome of tumor cells can lead to the production of abnormal proteins, these mutations enable tumor cells to generate new peptide segments, which are neoantigens. Identifying these antigens is the key to immunotherapy. The TSSK4 (Testis specific serine / threonine protein kinase 4) gene is a member of the family encoding testis-specific serine / threonine kinases. The encoded protein is thought to participate in spermatogenesis by phosphorylating the cAMP response element binding protein transcription factor to stimulate the CREB / CRE response pathway. Studies have found that in normal tissues, TSSK4 is almost only expressed in the testes of males, but in tumors such as Acute Myeloid Leukemia (abbreviated as LAML), TSSK4 is abnormally expressed. In previous studies, the functional research on TSSK4 and its family mainly focused on the process of sperm occurrence and development. However, it is worth noting that in mouse testes, it was found that TSSK4 maintains its kinase activity through autophosphorylation at the Thr-197 site, thereby inducing apoptosis. In addition, in the study of TSSK4 and pulmonary fibrosis, it was also found that TSSK4 can promote the apoptosis of alveolar type II cells through the mitochondrial death mechanism.
[0004] Since the testis is an immune-privileged organ, this makes us speculate that TSSK4, which is hardly expressed in other normal tissues but is expressed in the testis, may be a cancer-testis antigen (abbreviated as CTA). Its expression in tumors can make it a potential target for immunotherapy. Summary of the Invention
[0005] The present invention aims to screen and identify HLA-A2 (human leukocyte antigen type A2)-restricted cytotoxic T lymphocyte (CTL) epitope peptides of the TSSK4 antigen, with a view to providing new targets or drug options for tumor immunotherapy.
[0006] To achieve the above object, first, four epitope prediction softwares, namely NetMHC4.0, NetCTL1.2, SYFPEITHI and IEDB, were used to screen potential HLA-A2-restricted epitope peptides from the amino acid sequence of TSSK4. Subsequently, in vitro experiments were carried out using the T2A2 cell line to verify the immunological activities of these epitope peptides. The experimental results showed that the epitope peptides T5, T6, T7, and T9 could significantly induce specific CD8 + T cell responses in HLA-A2 donor-derived PBMCs, that is, CD8 + T cells secreted IFN-γ and Granzyme B, and showed significant killing effects on peptide-loaded T2A2 cells, confirming that these epitope peptides could effectively activate the CTL response and have a killing effect on tumor cells. This study successfully screened multiple HLA-A2-restricted epitope peptides with potential immunological activities, providing new candidate targets for TSSK4-related tumor immunotherapy.
[0007] The amino acid sequences of the above-mentioned epitope peptides are as follows:
[0008] Equivalent variants having at least 80% homology with any of the sequences of SEQ ID NO: 1-4 and retaining the functions of inducing HLA-A2-restricted CTL responses and killing TSSK4-positive tumor cells are also within the protection scope of the present invention.
[0009] The above-mentioned HLA-A2-restricted CTL epitope peptides of the TSSK4 antigen can be synthesized by the well-known solid-phase synthesis method in the art.
[0010] The above-mentioned HLA-A2-restricted CTL epitope peptides of the TSSK4 antigen have been proven to be able to effectively induce specific CTLs and have a killing effect on tumor cells expressing TSSK4. Therefore, the epitope peptides or their coding nucleic acids can be applied to the preparation of tumor immunotherapeutic agents, and targeted polypeptide vaccines can be designed to induce specific immune responses in the body, thereby enhancing the anti-tumor immune effect and providing new treatment strategies and options for tumor patients.
[0011] The present invention also provides a tumor immunotherapeutic agent, which comprises the above-mentioned epitope peptides or their coding nucleic acids, and the therapeutic agent is a pharmaceutical composition or a vaccine.
[0012] Among them, the vaccine is a polypeptide vaccine or a nucleic acid vaccine, and further comprises an immune adjuvant.
[0013] Furthermore, the immunoadjuvant is selected from cytokines, Toll-like receptor agonists, aluminum salt adjuvants or liposome delivery systems and is not limited thereto.
[0014] The beneficial effects of the present invention are as follows: The present invention screened and obtained TSSK4 antigenic epitope peptides and identified them through in vitro immune activity detection, providing a basis for the subsequent development of vaccines and diagnostic agents based on tumor-specific antigens, and providing more options for the design of TCR-T (T cell receptor-T lymphocyte) and tumor multi-epitope vaccines based on the epitope. The peptide vaccine designed based on specific tumor-associated antigens in the present invention has strong targeting ability, can highly specifically recognize tumor cells, reduce the immune response to normal cells, thereby improving the clinical efficacy and the quality of life of patients, and promoting the development of tumor immunotherapy. Description of the Drawings
[0015] Figure 1 : Intracellular cytokine staining was used to detect the level of IFN-γ secreted after the interaction between CTLs induced by epitope peptides from PBMCs derived from healthy donors and T2A2 cells loaded with epitope peptides.
[0016] Figure 2 : Intracellular cytokine staining was used to detect the level of Granzyme B secreted after the interaction between CTLs induced by epitope peptides from PBMCs derived from healthy donors and T2A2 cells loaded with epitope peptides.
[0017] Figure 3 : Intracellular cytokine staining was used to detect the level of IFN-γ secreted after the interaction between CTLs induced by epitope peptides from PBMCs derived from colorectal cancer patients and T2A2 cells loaded with epitope peptides.
[0018] Figure 4 : Intracellular cytokine staining was used to detect the level of Granzyme B secreted after the interaction between CTLs induced by epitope peptides from PBMCs derived from colorectal cancer patients and T2A2 cells loaded with epitope peptides.
[0019] Figure 5 : Cytotoxicity experiments were used to verify the killing effect of CTLs induced by epitope peptides from PBMCs derived from healthy donors on T2A2 cells loaded with epitope peptides.
[0020] In the significance analysis markers involved in each figure, * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001. Detailed Embodiments
[0021] The present invention will be described in detail below in conjunction with specific embodiments. It should be noted that the specific embodiments are only used to explain the present invention and are not used to limit the present invention. In addition, for the experimental operations involved in the embodiments, the parts not specifically described in the text are all conventional operations in the art, and those of ordinary skill in the art can refer to various commonly used reference books, scientific and technological literature, or relevant specifications, manuals, etc. before the filing date of the present invention application for implementation.
[0022] I. Experimental Materials 1. Experimental cell lines T2A2 cell line: The T2 cell line is generated by modifying the human acute lymphoblastic leukemia cell line (CEM). It expresses specific HLA molecules through genetic engineering modification, usually HLA-A2. A significant feature of T2A2 cells is their lack of TAP (antigen peptide transporter) function, resulting in the HLA-A2 molecules on the cell surface mainly existing in the "empty" form. Only after exogenous addition of antigen peptides can the HLA-A2 molecules be stably expressed. This characteristic makes T2A2 cells an ideal model for studying HLA-A2-restricted antigen epitopes, immune responses, tumor immune escape mechanisms, and developing immunotherapies (such as peptide vaccines).
[0023] 2. Peripheral blood: Peripheral blood from HLA-A2 healthy donors comes from healthy volunteers of HLA-A2 type; peripheral blood from HLA-A2 colorectal cancer patients comes from the oncology ward of our hospital. The collection of all peripheral blood samples has been approved by the ethics committee of our hospital and the consent of the patients or their family members has been obtained.
[0024] 3. Experimental reagents
[0025] All the peptide segments involved in the experiment were synthesized by Nanjing Jiepeptide Biotechnology Co., Ltd.
[0026] II. Experimental Methods 1. Screening of potential new epitopes binding to HLA-A2 molecules HLA-A2 molecule is a subtype of the human major histocompatibility complex (MHC) class I molecule, located on chromosome 6, and mainly participates in the T cell-mediated immune recognition and response process. The functional characteristics of HLA-A2 molecules make them an important target for studying antigen epitopes.
[0027] NetMHC-4.0 predicts the binding affinity of peptides to MHC-I through an artificial neural network, which is trained using 81 different human MHC alleles, including HLA-A, HLA-B, HLA-C, and HLA-E; the SYFPEITHI epitope prediction software calculates the affinity score based on the binding of the primary and secondary anchor residues of the epitope peptide to MHC, and the result of summing the scores of each amino acid residue is the score of the epitope peptide; NetCTL is a comprehensive scoring that combines the antigen peptide presentation efficiency and the cleavage site; IEDB is a sequence-based linear epitope prediction tool. Therefore, by combining the prediction results of SYFPEITHI, NetMHC-4.0, and IEDB and then comprehensively analyzing the scoring results of the NetCTL epitope prediction software, the use of four different online prediction software can improve the accuracy of epitope prediction. Enter the amino acid sequence of the candidate antigen and set the corresponding experimental parameters: A2 Supertype, predicted antigen peptide length (nonamers 9aa), and the remaining parameters are the default parameter values. Potential HLA class I-restricted CTL epitopes are defined as epitopes with a comprehensive prediction score greater than or equal to the default threshold (0.75), and the target subtype is HLA-A*02 (A*0201, A*0203, A*6802, and A*0206, etc.). Use four prediction databases for comprehensive scoring, rank the results obtained in the epitope prediction analysis, and select candidate dominant epitopes. See Table 1 for details.
[0028] 2. In vitro immunological activity study of antigen epitope peptides 1) Isolation of peripheral blood mononuclear cells (PBMCs) Identify the HLA type of healthy human peripheral blood, and then draw 40 mL of peripheral blood from HLA-A2 type volunteers. Dilute the peripheral blood 2-fold, that is, mix 1 part of PBS and 1 part of peripheral blood. Isolate PBMCs. Take a 50 mL centrifuge tube and add 15 mL of the separation solution and the diluted peripheral blood each. Slowly add the diluted peripheral blood to the separation solution so that peripheral blood: PBS: lymphocyte separation solution = 1:1:2. Centrifuge at 2000 rpm for 30 min. It can be seen that the centrifuge tube is divided into 4 layers from top to bottom: plasma layer, lymphocyte layer, separation solution layer, and red blood cell layer. Carefully aspirate the lymphocyte layer in the centrifuge tube and transfer it to a new 50 mL centrifuge tube. Add 10 - 15 mL of PBS to the tube to mix the cells and centrifuge and wash 2 times: 2000 rpm, 20 min. Resuspend PBMC with the medium and adjust the cell density to 1 - 1.5×10 6 / mL.
[0029] Approved by the hospital's ethics committee and with the consent of the patient or the patient's family, approximately 5 mL of peripheral blood was collected from colorectal cancer patients. The HLA type of the peripheral blood was identified, and the peripheral blood identified as HLA-A2 type was used to isolate peripheral blood mononuclear cells according to the above steps. The PBMCs were resuspended in the medium, and the cell density was adjusted to 1 - 1.5×10 6 / mL.
[0030] 2) Inducing epitope peptide-specific T lymphocytes The principle of the experiment for inducing epitope peptide-specific T lymphocytes (usually referred to as the T cell epitope stimulation experiment or the T cell response experiment) is based on the recognition and response of T cells to specific antigenic epitopes (peptide segments). The purpose of the experiment is to verify a specific immune response by inducing T cells to specifically recognize the target epitope peptide. The process includes: antigen presentation, T cell recognition, T cell activation and proliferation, and detection of the immune response.
[0031] The experimental steps are as follows: The previously synthesized epitope peptide was dissolved in PBS to prepare a solution with a concentration of 1 mg / mL. Using GT-T551 medium, 100 U / mL rhIL-2 was added. The cell density of PBMCs from healthy donors was adjusted to 1 - 1.5×10 6 cells / mL, and 4 ml / well was plated in a 6-well plate. 50 μg / mL of the epitope peptide was added, and the cells were cultured at 37°C for 5 days. On the 5th day, half of the medium (2 ml) was replaced and fresh medium containing rhIL-2 was added, and 50 μg / mL of the epitope peptide was added and the culture continued. On the 8th and 11th days, the operation on the 5th day was repeated. On the 14th day, half of the medium (2 ml) was replaced and fresh medium containing rhIL-2 was used instead (without adding the epitope peptide). On the 15th day, the induced cytotoxic T lymphocytes (CTLs) were collected for the next functional analysis. In addition, due to the small number of PBMCs from colorectal cancer patients, the whole cell density was 1 - 1.5×10 6 cells / mL, and 1 ml / well was plated in a 24-well plate. 50 μg / mL of the epitope peptide was added, and the cells were cultured at 37°C for 5 days. On the 5th day, half of the medium (500 μL) was replaced and fresh medium containing rhIL-2 was added, and 50 μg / mL of the epitope peptide was added and the culture continued. On the 8th and 11th days, the operation on the 5th day was repeated. On the 14th day, half of the medium (500 μL) was replaced and fresh medium containing rhIL-2 was used instead (without adding the epitope peptide). On the 15th day, the induced cytotoxic T lymphocytes (CTLs) were collected for the next functional analysis.
[0032] 3) Detection of IFN-γ level released by T cells by intracellular cytokine staining method Intracellular Cytokine Staining (ICS) is a flow cytometry-based technique used to detect the production of specific cytokines (such as IFN-γ, TNF-α, IL-2, etc.) inside activated T cells. Its principle is to stain intracellular cytokines with specific antibodies and analyze them using a flow cytometer, thereby quantitatively or qualitatively evaluating T cell function. As mentioned above, there are two sources of epitope peptide-induced cytotoxic T lymphocytes (CTLs), namely CTLs induced from PBMCs of healthy donors by epitope peptides and CTLs induced from PBMCs of colorectal cancer patients by epitope peptides. In this experiment, CTLs refer to CTLs induced from the above two different sources of PBMCs by epitope peptides, corresponding to Figure 1-2 and Figure 3-4 .
[0033] A. Target cell preparation: Collect T2A2 cells and adjust the cell density to 2×10 6 cells / mL, seed them into 24-well plates, add β 2 -microglobulin (β 2 -M) at 3 μg / mL, add epitope peptide at 50 μg / mL, and incubate at 37°C and 5% CO 2 for 4 h to obtain peptide-loaded target cells.
[0034] B. Co-incubate target cells with induced CTLs: Collect induced CTLs and adjust the cell density to 2×10 6 cells / mL. Add 500 μL of peptide-loaded target cells and 500 μL of induced CTLs into 1.5 mL EPPendorf tubes respectively, and co-incubate at 37°C and 5% CO 2 for 4 h. After incubating for 4 h, add 1 μL of BFA to each tube and incubate for 1 h. Set up control groups, PBS group, CD3 single-positive group, CD8 single-positive group, CD3CD8 double-positive group, IFN-γ single-positive group, granzyme B single-positive group, and experimental group.
[0035] C. Incubate with CD3 and CD8 antibodies, mix well and incubate at 4°C in the dark for 30 min. Add 200 μL of fixative to resuspend the cells, let them stand at room temperature in the dark for 30 min, then add 200 μL of 1× permeabilizing agent, centrifuge at 1500 rpm for 3 min, and repeat the operation once.
[0036] D. Incubate intracellular factor antibodies: Add 100 μL of 1× permeabilization agent (to resuspend the cells) and appropriate amounts of Human IFN-γ and granzyme B antibodies, and incubate at 4°C in the dark for 30 min. After the antibody incubation, add FACS buffer and centrifuge at 1500 rpm for 3 min for one wash. Then add buffer salt solution (FACS buffer) to resuspend the cells, and detect by flow cytometry.
[0037] 4) Detection of cytotoxicity experiment by LDH method The cytotoxicity experiment detected by the LDH method is an experimental method based on the release of lactate dehydrogenase (LDH), which is used to evaluate cytotoxicity or cell lysis. LDH is an enzyme commonly present in the cytoplasm. When the cell membrane is damaged or the cell lyses, LDH is released into the culture medium. By detecting the activity of LDH in the culture medium, the degree of cell death and toxic effects can be indirectly evaluated. The CTLs in this experiment are specific T lymphocytes induced by epitope peptides from PBMCs of healthy donors, corresponding to the experimental results Figure 5 。
[0038] A. In this experiment, peptide-loaded T2A2 cells were used as target cells, and the cell concentration was adjusted to 2×10 5 cells / mL, seeded into the well plates, and epitope peptides were added at a final concentration of 10 μg / mL, and then placed in the cell culture incubator for 4 h to obtain peptide-loaded T2A2 cells; Collect the CTLs induced by epitope peptides, and adjust the cell density to 2×10 6 cells / mL; The CTLs and peptide-loaded T2A2 cells were seeded into 96-well U-bottom plates according to different effector-to-target ratios. The number of target cells in each well was 5×10 3 cells, and CTLs induced by epitope peptides were added to each well according to different effector-to-target ratios (E:T = 12.5:1, 25:1, 50:1). After mixing, incubate in a 37°C, 5% CO 2 incubator for 4 h; And supplement serum-free IMDM medium to a total volume of 100 μL, and set 3 replicates for each group. At the same time, set the following controls: target cell spontaneous release group, target cell maximum release group, effector cell spontaneous release group, background control group, and total volume correction control group. Each control group also has 3 replicates, and the total volume of each well is 100 μL.
[0039] B. Place the 96-well cell culture plate at 37°C, 5% CO 2Cultivate for 4 h in an incubator. 45 min before the end of incubation, add 10 μL of 10× lysis buffer to the maximum release wells and volume correction wells of the target cells. After the cultivation ends, centrifuge the culture plate at 1000 rpm for 10 min; take out the 96-well plate, gently aspirate 50 μL of the supernatant from each well and place it into the corresponding well of another 96-well plate. After all the transfers are completed, quickly add 50 μL of lactate dehydrogenase substrate mixture to each well, and incubate in the dark at room temperature for 30 min; after the incubation ends, add 50 μL of stop solution to each well; detect the absorbance OD value at 490 nm on an enzyme-linked immunosorbent assay (ELISA) reader, and calculate the killing rate. Killing rate = (experimental group release - effector cell spontaneous release - target cell spontaneous release) / (target cell maximum release - target cell spontaneous release) × 100%.
[0040] III. Experimental Results 1. Prediction Results of HLA-A2 Restricted Epitope Peptides Derived from TSSK4 Based on the amino acid sequence of the target TSSK4, four epitope prediction software, NetMHC4.0, NetCTL1.2, SYFPEITHI, and IEDB, were used for comprehensive scoring to predict potential candidate epitopes. As shown in Table 1, a total of 10 HLA-A2 restricted antigen epitope peptides were screened (Table 1). The candidate epitope peptides were synthesized using the Fmoc solid-phase synthesis method, and the actual molecular weight of the epitope peptides was confirmed to be consistent with the theoretical molecular weight by mass spectrometry.
[0041] Table 1. Prediction Results of HLA-A2 Restricted Epitope Peptides Derived from TSSK4
[0042] 2. Results of Intracellular Cytokine Staining Experiment In the intracellular cytokine staining experiment, specific cell markers (such as CD8 to label T cells) and intracellular cytokine markers (such as IFN-γ and Granzyme B) were selected, and scatter plots were used to present the expression of cytokines. The horizontal and vertical axes represent the expression of different cytokines, and the four quadrants of the scatter plot show different populations of cells. In this experiment, the upper right quadrant shows CD8 + T cells secreting IFN-γ / Granzyme B, and the lower left quadrant shows CD8 + T cells that do not secrete any cytokines.
[0043] The intracellular cytokine staining experiment showed that compared with the PBS group, the proportion of CD8 + T lymphocytes secreting IFN-γ and Granzyme B increased significantly after the CTLs induced by epitope peptides T5, T6, T7, and T9 from healthy donor-derived PBMCs interacted with T2A2 cells loaded with epitope peptides ( Figure 1 、 2 ).
[0044] Intracellular cytokine staining experiments showed that, compared with the PBS group, the proportion of CD8 T lymphocytes secreting IFN-γ and Granzyme B after the interaction of CTLs induced by epitope peptides T5, T6, T7, and T9 from PBMCs of colorectal cancer patients with T2A2 cells loaded with epitope peptides was significantly increased ( + . Figure 3 and 4 ).
[0045] 3. Results of cytotoxicity assay In the cytotoxicity assay detected by the LDH method, LDH (lactate dehydrogenase) is an enzyme inside cells. When the cell membrane ruptures, LDH will be released into the external medium. Therefore, by detecting the LDH concentration in the culture medium, the killing degree of immune cells against target cells can be indirectly reflected.
[0046] Cytotoxicity experiments showed that CTLs induced in vitro by epitope peptides T5, T6, T7, and T9 from PBMCs of healthy donors had a significant killing function against peptide-loaded T2A2 ( Figure 5 ).
Claims
1. A TSSK4 antigen HLA-A2 restricted CTL epitope peptide, characterized in that: The amino acid sequence of the epitope peptide is selected from any one of the following sequences: a) LLLDKWENV (SEQ ID NO:1); b) KMVPSNQPV (SEQ ID NO:2); c) FAYACPEIL (SEQ ID NO:3); d) GVILYTLVV (SEQ ID NO:4); or a functionally equivalent variant thereof, wherein the functionally equivalent variant has at least 80% homology with any one of SEQ ID NOs: 1-4 and retains the function of inducing HLA-A2 restricted CTL response and killing TSSK4 positive tumor cells.
2. An isolated nucleic acid molecule, characterized in that Encodes the TSSK4 antigen HLA-A2 restricted CTL epitope peptide as described in claim 1.
3. Use of the TSSK4 antigen HLA-A2 restricted CTL epitope peptide or its encoding nucleic acid according to claim 1 in the preparation of a tumor immunotherapeutic agent, wherein the tumor immunotherapeutic agent can activate CD8+ T cells and induce them to secrete IFN-γ and Granzyme B, thereby producing a specific killing effect on tumor cells expressing TSSK4.
4. The use according to claim 3, wherein the tumor is a malignant tumor derived from a patient with an HLA-A2 phenotype.
5. A tumor immunotherapeutic agent, comprising the TSSK4 antigen HLA-A2 restricted CTL epitope peptide or its encoding nucleic acid according to claim 1, wherein the therapeutic agent is a pharmaceutical composition or a vaccine.
6. The tumor immunotherapeutic agent according to claim 5, characterized in that The vaccine is a polypeptide vaccine or a nucleic acid vaccine, and further comprises an immune adjuvant.
7. The tumor immunotherapeutic agent according to claim 6, characterized in that The immune adjuvant is selected from cytokines, Toll-like receptor agonists, aluminum salt adjuvants or liposome delivery systems.