CKAP4-targeted tumor antigen peptide, vaccine and application of CKAP4-targeted tumor antigen peptide

Abstract

The invention relates to the technical field of biological medicines, in particular to a CKAP4-targeted tumor antigen peptide, a vaccine and application of the CKAP4-targeted tumor antigen peptide. The invention provides a high-immunogenicity tumor antigen peptide RLTELTKSI targeting human and mouse homologous CKAP4 protein, and the tumor antigen peptide and a vaccine thereof can realize efficient killing of CKAP4 positive tumor cells and remarkable inhibition of CT26 subcutaneous tumor by activating specific CD8 + T cell immune response. The traditional single-target limitation is broken through, the co-expression characteristic of CKAP4 in tumor cells and immunosuppressive cells (TAM / TAN) is utilized, a double-target and double-channel mechanism is initiated, and the immunosuppressive state of cold tumors is effectively reversed by inducing T cells to synchronously kill tumor cells and remodel an immune microenvironment. According to the technology, CKAP4 is expanded from an antibody target to a T cell vaccine target, lasting specific CTL response can be stimulated, the off-target risk of antibody treatment is avoided, a universal treatment scheme can be provided for solid tumors, and the clinical transformation potential and the treatment broad spectrum are remarkably improved.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a tumor antigen peptide targeting CKAP4, a vaccine, and its applications. Background Technology

[0002] Malignant tumors remain a major global health challenge, with their high heterogeneity and immune escape mechanisms leading to limitations in current treatments. While targeted therapies and immune checkpoint inhibitors have achieved clinical breakthroughs in some cancer types, overall response rates remain unsatisfactory, particularly for tumor types lacking clearly defined driver mutations. In recent years, tumor-specific antigen immunotherapy (such as neoantigen vaccines) has shown revolutionary therapeutic potential, but its application is limited by individual variability in tumor mutational burden and the complexity of its preparation.

[0003] Currently, major technologies in the field of cancer treatment all have significant limitations. Targeted drugs are only applicable to patients with specific gene mutations; the efficacy of immune checkpoint inhibitors is constrained by the tumor microenvironment and is prone to drug resistance; while neoantigen vaccines face problems such as complex individualized preparation and high costs. Traditional tumor-associated antigen vaccines have failed to achieve breakthroughs in clinical efficacy due to a lack of sufficient specificity and immunogenicity. These technological bottlenecks severely restrict the widespread application of cancer immunotherapy.

[0004] CKAP4, a key regulatory molecule of the endoplasmic reticulum-cytoskeleton network, is not only highly expressed in various malignant tumors but also participates in regulating key processes such as tumor proliferation, metastasis, and immune escape. Therefore, this invention employs a peptide vaccine strategy based on tumor microenvironment antigens (TMAs). By selecting CKAP4 (Cytoskeleton-Associated Protein 4), which is co-expressed highly on the surface of tumor cells and suppressive immune cells in the tumor microenvironment (TME) (such as M2 macrophages, MDSCs, and Tregs), as a target, a specific peptide vaccine is designed and screened to achieve "one target, two effects," aiming to overcome the limitations of single-pathway therapy. Summary of the Invention

[0005] The first technical problem to be solved by the present invention is to provide a tumor antigen peptide that targets CKAP4, in order to address the shortcomings of the prior art.

[0006] The second technical problem to be solved by the present invention is to provide a nucleic acid molecule.

[0007] The third technical problem to be solved by the present invention is to provide an expression carrier.

[0008] The fourth technical problem to be solved by the present invention is to provide a tumor antigen peptide vaccine that targets CKAP4.

[0009] The fifth technical problem to be solved by the present invention is to provide the application of the tumor antigen peptide vaccine in the preparation of drugs for targeted therapy or immunotherapy of solid tumors.

[0010] The sixth technical problem to be solved by the present invention is to provide a detection kit.

[0011] The overall concept of this invention is as follows: By integrating and analyzing multi-omics tumor databases, it was discovered that the CKAP4 gene exhibits significantly high expression in various solid tumor tissues, including gastric cancer, colorectal cancer, and breast cancer, and its expression level is significantly correlated with poor patient prognosis. Further single-cell sequencing analysis showed that CKAP4 is not only highly expressed in tumor cells but also specifically expressed in immunosuppressive cell populations such as Treg cells, M2 macrophages, and myeloid-derived suppressor cells (MDSCs) within the tumor microenvironment. Based on this unique expression characteristic, this invention uses bioinformatics prediction combined with in vitro experimental verification to screen and obtain CKAP4-specific antigenic epitopes with strong immunogenicity, providing a novel strategy for overcoming drug resistance in solid tumor immunotherapy. Simultaneously, CKAP4 is innovatively selected as a dual-effect target to design and develop a peptide vaccine that can simultaneously target tumor cells and the immunosuppressive microenvironment. This vaccine, by inducing a specific T-cell immune response, can achieve a dual therapeutic effect of direct killing of tumor cells and remodeling of the immune microenvironment, demonstrating broad-spectrum anti-tumor application potential.

[0012] To solve the above-mentioned technical problems, the present invention discloses the following technical solution:

[0013] In a first aspect, the present invention provides a tumor antigen peptide targeting CKAP4, the amino acid sequence of which is shown in SEQ ID NO.1.

[0014] The tumor antigen peptide is obtained by MHC typing screening based on the amino acid sequence of the CKAP4 protein.

[0015] Specifically, the tumor antigen peptide is a human-mouse homologous antigen peptide.

[0016] Specifically, the tumor antigen peptide is an HLA-A*0201 molecule-restricted antigen peptide.

[0017] Furthermore, the tumor antigen peptide targeting CKAP4 was obtained through screening using the following technical process: First, based on the complete amino acid sequence of the CKAP4 protein, MHC molecular typing technology was used, and an immune epitope prediction system was employed to perform CD8 typing on the protein sequence. + T cell epitope analysis; then, candidate polypeptide fragments were chemically synthesized based on the predicted results; finally, the ability of each polypeptide fragment to activate specific T cell immune responses was verified through in vitro functional experiments, thereby screening out targeted polypeptides with significant immunogenicity.

[0018] The immune epitope prediction system is preferably any one or a combination of several bioinformatics analysis tools such as SYFPEITHI, RANKPEP, or NetMHCpan4.1.

[0019] Secondly, the present invention provides a nucleic acid molecule.

[0020] The nucleic acid molecule encodes the tumor antigen peptide.

[0021] Thirdly, the present invention provides an expression carrier.

[0022] The expression vector contains the nucleic acid molecule.

[0023] Fourthly, the present invention provides a tumor antigen peptide vaccine targeting CKAP4.

[0024] The effective active ingredient of the tumor antigen peptide vaccine is the tumor antigen peptide.

[0025] Furthermore, the tumor antigen peptide vaccine contains the adjuvant CpG ODN.

[0026] Furthermore, the present invention also provides a method for preparing the tumor antigen peptide vaccine, wherein an adjuvant CpG ODN is added to the tumor antigen peptide targeting CKAP4 to prepare a tumor peptide vaccine targeting CKAP4.

[0027] In some embodiments of the present invention, the mass ratio of the tumor antigen peptide targeting CKAP4 to the adjuvant CpG ODN is 5:1.

[0028] Fifthly, the present invention provides the application of the tumor antigen peptide vaccine in the preparation of drugs for targeted therapy or immunotherapy of solid tumors.

[0029] The solid tumors include, but are not limited to, at least one of gastric cancer, colorectal cancer, breast cancer, and lung cancer.

[0030] The drugs mentioned are antigen-reactive T-cell drugs and engineered T-cell drugs.

[0031] Sixthly, the present invention provides a detection kit.

[0032] The effective component of the detection kit is the tumor antigen peptide.

[0033] Beneficial effects:

[0034] (1) This invention successfully identified a highly immunogenic antigenic epitope peptide, RLTELTKSI, targeting the human and mouse homologous CKAP4 protein by integrating T cell epitope prediction tools such as SYFPEITHI, RANKPEP, and NetMHCpan4.1, thus expanding the target selection range for tumor vaccines. Flow cytometry, cytokine detection array (CBA), and in vitro killing experiments confirmed that this peptide can effectively activate T cell immune responses and significantly enhance the killing efficacy of T cells against CKAP4-positive tumor cells. In in vitro experiments, it showed a killing efficiency of more than 25% against CKAP4-overexpressing tumor cells (effect-to-target ratio 10:1). Further validation in the CT26 subcutaneous xenograft model showed that the CKAP4-targeting peptide vaccine not only significantly inhibited tumor growth (tumor volume reduced by 86%, p<0.001) but also increased CD8+. + T-cell infiltration and reduction of suppressor myeloid cells TAM and TAN demonstrate its dual therapeutic efficacy in reshaping the immune microenvironment.

[0035] (2) Compared with traditional peptide vaccines that only target tumor cells, this invention makes full use of the co-expression characteristics of CKAP4 in inhibitory immune cells (TAN, TAM) in the TME to design a vaccine that can activate specific CD8 + Vaccine components that simultaneously recognize and kill both tumor cells and immunosuppressive cells exhibit a dual anti-tumor effect, representing a functional advancement from single-target killing to microenvironment regulation. Specifically, vaccination induces an endogenous specific T-cell response, simultaneously killing tumor cells and suppressive immune cells in the tumor microenvironment (TME), thereby achieving a dual anti-tumor effect of directly inhibiting tumors and reshaping the immune microenvironment. This dual targeting of "tumor cells + immunosuppressive cells" effectively alleviates local immunosuppression in tumors, activates and expands the infiltration and effector functions of anti-tumor T-cell clones, thereby enhancing the transformation of "cold tumors" into "hot tumors" and improving the therapeutic response rate of vaccines in immune-tolerant or advanced tumors. This "dual-target, dual-pathway" mechanism overcomes the limitations of traditional vaccines that only target tumor cells.

[0036] (3) This invention provides experimental evidence and translational prospects for tumor immunotherapy based on CKAP4. By simultaneously targeting tumor cells and the immunosuppressive microenvironment, it achieves a dual anti-tumor effect. This technology is applicable to adoptive T-cell therapy, engineered T-cell preparation and combined immunotherapy for patients with solid tumors, and provides new treatment options for various solid tumors such as gastric cancer and breast cancer.

[0037] (4) This invention expands CKAP4 from existing antibody-based immunomodulatory interventions to a T-cell-mediated peptide vaccine target, achieving for the first time the active induction of a durable cytotoxic T lymphocyte (CTL) population in vivo by stimulating a specific T-cell response through the CKAP4 epitope peptide. This response has stronger specificity, more durable memory, and broader antitumor activity, avoiding the dose dependence and tumor antigen off-target problems existing in antibody therapy, and can be further synergized with other antitumor therapies to improve clinical translatability and therapeutic breadth. Attached Figure Description

[0038] The present invention will be further described in detail below with reference to the accompanying drawings, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0039] Figure 1 The activation of T cells in peripheral blood mononuclear cells from gastric cancer patients after stimulation with CKAP4 antigen peptide 160 was analyzed by flow cytometry; negative control: no peptide was added. A is a schematic diagram of the experimental workflow for studying immune cells in gastric cancer patients; B is CD8... + T cell phenotype analysis results; C represents IFN-γ detection results.

[0040] Figure 2 The activation of T cells in peripheral blood mononuclear cells from healthy individuals after stimulation with CKAP4 antigen peptide 160 was analyzed by flow cytometry; negative control: no peptide was added. A is a schematic diagram of the experimental procedure for studying immune cells in healthy individuals; B is CD8... + T cell phenotype analysis results; C represents IFN-γ detection results.

[0041] Figure 3 The activation of T cells in Balb / c mouse lymph node cells after stimulation with CKAP4 antigen peptide 160 was analyzed by flow cytometry; negative control: no peptide was added. A and D are schematic diagrams of the experimental workflow for immune cell studies in 4T1 tumor-bearing Balb / c mice and CT26 tumor-bearing Balb / c mice, respectively; B and E are CD8+ cells in 4T1 tumor-bearing Balb / c mice and CT26 tumor-bearing Balb / c mice, respectively. + T cell phenotype analysis results; C and F are the IFN-γ detection results of 4T1 tumor-bearing Balb / c mice and CT26 tumor-bearing Balb / c mice, respectively.

[0042] Figure 4This study aimed to determine and analyze the specific killing ability of Balb / c mouse lymph node cells against homologous tumor cells after stimulation with CKAP4 antigen peptide 160 in vitro. The negative control was no peptide added. Figures A and C show the flowcharts of the in vitro killing experiments in 4T1-bearing Balb / c mice and CT26-bearing Balb / c mice, respectively. Figures B and D show the tumor cell killing results in 4T1-bearing Balb / c mice and CT26-bearing Balb / c mice, respectively. Specifically, Figures A and B show the killing experiments of Balb / c mouse lymph node cells activated by CKAP4 peptide in vitro, comparing lymph node cells with 4T1 tumor cells at different effector-target ratios; the negative control was mouse lymph node cells without peptide induction. Figures C and D show the killing experiments of Balb / c mouse lymph node cells activated by CKAP4 peptide in vitro, comparing lymph node cells with CT26 tumor cells at different effector-target ratios; the negative control was mouse lymph node cells without peptide induction.

[0043] Figure 5 This study analyzed the inhibitory effect of CKAP4 antigen peptide 160 on tumor growth in Balb / c tumor-bearing mice using in vivo tumor inhibition experiments; negative control: no peptide added. A and D are schematic diagrams of the in vivo experimental procedures for 4T1 and CT26 tumor-bearing Balb / c mice, respectively; B and E are photographs of the tumors in 4T1 and CT26 tumor-bearing Balb / c mice, respectively; C and F are statistical results of tumor weight in 4T1 and CT26 tumor-bearing Balb / c mice, respectively. Specifically, A-C are schematic diagrams of the treatment mode of CKAP peptide vaccine administered to 4T1 allograft tumor-bearing mice with breast cancer, images of tumor size at the experimental endpoint, and tumor weight at the experimental endpoint; negative control: saline. D-F are schematic diagrams of the treatment mode of P2 and P3 vaccines administered to CT26 allograft tumor-bearing mice with colorectal cancer, images of tumor size at the experimental endpoint, and tumor weight at the experimental endpoint; negative control: saline. Detailed Implementation

[0044] The present invention will be further described in detail below with reference to specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0045] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0046] In the following examples, the CKAP4 antigenic peptide (i.e., P160) was synthesized and prepared by GenScript.

[0047] In the following examples, the 4T1 tumor-bearing Balb / c mice and the CT26 tumor-bearing Balb / c mice were respectively fed 100 μL of a solution containing 1×10⁻⁶ mol / L of ... 4 ~2×10 6 Cell suspensions of 4T1 mouse breast cancer cells and CT26 mouse colorectal cancer cells were prepared by inoculating SPF-grade Balb / c female mice. The 4T1 mouse breast cancer cells and CT26 mouse colorectal cancer cells were purchased from the Cell Bank of the Chinese Academy of Sciences, and the SPF-grade Balb / c female mice were purchased from Nanjing Cavens Biotechnology Co., Ltd.

[0048] Example 1: Prediction and Synthesis of CKAP4 Antigen Peptide

[0049] Based on the amino acid sequence of the human and mouse homologous CKAP4 protein, combined with the human leukocyte antigen (HLA) typing HLA-A*0201 (which accounts for 15-25% of the Chinese population) and the Balb / c mouse major histocompatibility complex (MHC) typing H2d, three T cell epitope prediction tools, SYFPEITHI, RANKPEP, and NetMHCpan4.1, were used for comprehensive analysis to select epitope peptides with a length of 9-11 amino acids for synthesis.

[0050] Specifically, the higher the score of the epitope prediction software SYFPEITHI and RANKPEP, the stronger the peptide is considered to bind to MHC molecules; the positive interpretation of the prediction results of MHCpan4.1 software is: IC50 < 500 nM or %Rank < 2.0 is considered to be able to bind to MHC molecules; IC50 < 50 nM or %Rank < 0.5 is considered to be able to bind strongly to MHC molecules.

[0051] Based on the prediction results of the above tools, peptide sequences with high SYFPEITHI and RANKPEP scores and low MHCpan4.1 %Rank scores were selected for synthesis. This invention preferentially selected a 9-amino acid CKAP4 antigen peptide that is homologous to both humans and mice and has the highest predicted score for chemical synthesis, named P160 (corresponding to amino acids 160-168 of the human CKAP4 protein). Detailed results for this CKAP4 antigen peptide are shown in Table 1. The data indicate that P160 has high affinity for both HLA-A*0201 and H-2d molecules, especially with a %Rank value of NetMHCpan4.1 much less than 0.5, classifying it as a strong-binding peptide suitable for subsequent immunological function studies.

[0052] Table 1. Prediction results of highly immunogenic CKAP4 antigenic peptides

[0053]

[0054] Example 2: Patient HLA typing and cell sample preparation

[0055] 2.1 HLA typing

[0056] Two mL of peripheral blood was drawn from gastric cancer patients and anticoagulated using EDTA or sodium citrate. High-resolution genotyping (four-digit) of the HLA-A locus was performed using PCR-SBT (polymerase chain reaction-sequencing genotyping) technology to screen for HLA-A*0201-positive gastric cancer patients for subsequent experiments.

[0057] 2.2 Obtaining human peripheral blood mononuclear cells (PBMCs)

[0058] (1) After sterilizing the cell bags (or heparinized blood drawn by syringe) obtained by the COBE Spectra™ MNC system from gastric cancer patients and healthy individuals, place them in a clean bench, transfer the cells to a 50 mL centrifuge tube using a 20 mL syringe, and dilute them with physiological saline at a 1:1 ratio.

[0059] (2) Add 20 mL of human lymphocyte separation medium (purchased from Tianjin Haoyang Biological Products Technology Co., Ltd., product number LTS1077) to the centrifuge tube.

[0060] (3) The cell solution diluted with physiological saline was slowly poured onto the human lymphocyte separation solution along the tube wall using a Pasteur pipette;

[0061] (4) Centrifuge horizontally at 800 g for 20 min;

[0062] (5) After centrifugation, the tube is divided into three layers: the upper layer is plasma, the lower layer is mainly red blood cells and granulocytes, the middle layer is lymphocyte separation fluid, and there is a white membrane layer mainly composed of mononuclear cells between the plasma layer and the lymphocyte separation fluid, which is the peripheral blood mononuclear cell layer.

[0063] (6) Insert a pipette into the white membrane layer, carefully aspirate the mononuclear cells, transfer them to a new 50 mL centrifuge tube, add physiological saline, and centrifuge horizontally at 300 g for 10 min;

[0064] (7) Discard the supernatant, add physiological saline, mix well, and centrifuge horizontally at 300 g for 10 min;

[0065] (8) Discard the supernatant, and finally dilute with AIM-V medium (Gibico, USA) to count cells. Take an appropriate amount of cells for cryopreservation.

[0066] 2.3 Preparation of single-cell suspension from mouse draining lymph nodes

[0067] (1) Freshly dissected and intact draining lymph nodes of 4T1 tumor-bearing Balb / c mice and CT26 tumor-bearing Balb / c mice were soaked in ice-c physiological saline.

[0068] (2) After wetting the 70 μm cell filter with physiological saline, put the lymph node in, gently grind it, rinse it with physiological saline, and collect the lymph node grinding fluid.

[0069] (3) Centrifuge the lymph node homogenate at 1200 rpm for 5 min, discard the supernatant, and resuspend in physiological saline to a concentration of 1×10⁻⁶. 6 Cells were collected at a density of 100 cells / mL to prepare a single-cell suspension, which was then transferred to a flow cytometry tube, labeled with grouping information, and ready for use.

[0070] Example 3: In vitro induction of PBMCs and detection of mouse lymph node cell activation and immune response

[0071] 3.1 Cell activation culture

[0072] (1) The human PBMCs or mouse lymph node single cells isolated in Example 2 were suspended in AIM-V complete medium (Gibico, USA) and counted.

[0073] (2) Using a 96-well plate with a U-shaped bottom designed to promote better cell-cell contact, fill each well with 1 × 10⁻⁶ cells. 5 Cells were seeded into 200 μL of culture medium and co-incubated with 10 μM of CKAP4 antigen peptide P160. The culture medium consisted of AIM-V medium (Gibco) containing a small amount of interleukin-2 (IL-2, 100 U / mL, Peprotech) and 10% fetal bovine serum (FCS, Gibco).

[0074] (3) Each round of stimulation culture lasts 3 days, and the culture medium is replaced once with half the volume. At the same time, fresh antigen peptide (10 μM) and IL-2 (100 U / mL) are added.

[0075] (4) After 2-3 rounds of in vitro stimulation with peptides, the culture medium was replaced with fresh medium, and 10 μM of fresh CKAP4 antigen peptide P160 was added again for overnight stimulation. Cells were collected within 24 hours of the last stimulation (i.e., on day 7 and / or day 10 of culture), and the expression level of the T cell activation marker 4-1BB (CD137) was evaluated by flow cytometry to detect the immunogenicity of the antigen peptide. PBMCs stimulated with peptide-free (culture medium) or irrelevant peptides served as negative controls (NS).

[0076] 3.2 Detection of IFN-γ

[0077] The secretion level of interferon-γ (IFN-γ) in cell culture supernatant was detected using flow cytometry microsphere (CBA) technology. The specific procedure is as follows.

[0078] (1) Prepare human and mouse soluble protein free assortment reagent standards respectively.

[0079] 1) Transfer a bottle of lyophilized cytokine standard beads to a 15 mL centrifuge tube and label it as high concentration standard (2500 pg / mL).

[0080] 2) Dilute the standard with 4 mL of Assay Diluent and let it stand at room temperature for at least 15 minutes;

[0081] 3) Gently pipette the standard to mix it; do not vortex or shake vigorously.

[0082] 4) Mark the gradient dilution ratios of 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, and 1:256 on the 9 branch pipettes respectively;

[0083] 5) Add 500 µL of Assay Diluent to each tube;

[0084] 6) Starting with the highest concentration standard, add 500 µL of diluent one by one to serially dilute the standards;

[0085] 7) Add 500 µL of Assay Diluent to a new flow cytometry tube as a control tube (0 pg / mL).

[0086] (2) Microspheres were captured by a mixture of human and mouse soluble protein free-association reagents:

[0087] 1) Determine the number of BD CBA human soluble protein free-association reagents (i.e., the types of free-association cytokines).

[0088] 2) Determine the number of test samples. Note: To avoid insufficient reagent due to liquid loss during sample addition, the amount of 3 more samples can be mixed when mixing the microspheres.

[0089] 3) Before mixing the capture microspheres, each type of capture microsphere needs to be vortexed for at least 15 seconds to ensure thorough mixing;

[0090] 4) Determine the total amount of capture microspheres required for dilution in the experiment based on a volume of 50 µL per sample;

[0091] 5) Determine the volume of each type of capture microsphere based on a quantity of 1 µL per sample; for example, if the number of samples is 40, then the volume of the capture microsphere is 40 µL.

[0092] 6) Determine the volume of the microsphere dilution solution;

[0093] 7) Take a new tube, label it "Mixed Capture Microspheres", and mix the capture microspheres with the capture microsphere dilution solution in the tube.

[0094] (3) Prepare detection antibodies labeled with PE reagent for the free assortment of human and mouse soluble proteins respectively.

[0095] 1) Determine the number of BD CBA human soluble protein free-association reagents (i.e., the types of free-association cytokines).

[0096] 2) Determine the number of test samples;

[0097] 3) Determine the total amount of PE-labeled detection antibody to be diluted in the experiment based on a volume of 50 µL per sample;

[0098] 4) Determine the amount of PE-labeled detection antibody reagent according to 1 µL per sample;

[0099] 5) Determine the volume of the PE-labeled antibody dilution buffer;

[0100] 6) Dilute the PE detection antibody reagent with the detection antibody diluent, mix well in a tube, and store at 4°C protected from light before use.

[0101] (4) Incubation of human and mouse soluble protein free combination reagent with samples

[0102] 1) Standards and test samples: Add 50 µL of serially diluted standard to each standard tube, and add 50 µL of test sample to each sample tube;

[0103] 2) Vortex mix the microspheres for at least 5 seconds, adding 50 µL of microspheres to each tube and gently mixing.

[0104] 3) Incubate at room temperature for 1 hour;

[0105] 4) Add 50 µL of PE-labeled detection antibody to each tube and mix gently;

[0106] 5) Incubate at room temperature for 2 hours.

[0107] (5) On-machine testing

[0108] 1) Add 1 mL of PBS solution to each tube to wash the sample, and centrifuge at 200 g for 5 minutes;

[0109] 2) Carefully aspirate or gently discard the supernatant, and resuspend the cells in 300 µL of PBS solution in each tube;

[0110] 3) Test the instrument as soon as possible after calibration.

[0111] (6) Data Analysis

[0112] After the sample data collection was completed (FCS2.0 format), the standard curve was plotted and the data was analyzed using the CBA-specific analysis software FCAP Array V3.0 to calculate the concentration of IFN-γ in the sample.

[0113] 3.3 Detection of T cell activation markers

[0114] The surface markers of activated T cells were detected by flow cytometry, and the specific procedure is as follows.

[0115] (1) Adjust the concentration of activated PBMCs or mouse lymph node cells to 1×10 6 Approximately 10 cells / mL. After mixing the cell suspension, centrifuge at 1500 rpm for 5 min, discard the supernatant, add PBS, centrifuge at 1500 rpm for 5 min, discard the supernatant, and resuspend the cells in 100 μL PBS into flow cytometry tubes. Label the cells of each group on the tube wall.

[0116] (2) Add fluorescent antibodies against human CD3, CD8, CD25, CD69, CD107a, CD137 and other antibodies to human PBMCs, and set up negative control group, single label group and isotype control group; add fluorescent antibodies against mouse CD3, CD8, CD25, CD69, CD137 and other antibodies to mouse lymph node cells, and set up negative control group, single label group and isotype control group;

[0117] (3) Stain at 4°C in the dark for 30 min;

[0118] (4) Add 1 mL of PBS and gently pipette to mix.

[0119] (5) Centrifuge at 1500 rpm for 5 min and discard the supernatant;

[0120] (6) Resuspend the cells in 200 μL PBS and perform flow cytometry analysis. Collect data using Beckman software and analyze the results using Flowjo software.

[0121] 3.4 Experimental Results

[0122] (1) Results of IFN-γ secretion levels and T cell activation markers in gastric cancer patients and tumor-bearing mice

[0123] Activation of PBMCs from HLA-A*0201 gastric cancer patients and lymph node cells from tumor-bearing mice was induced in vitro using the CKAP4 antigen peptide P160. The IFN-γ secretion level in the supernatant of activated cells was detected by CBA assay. Results are as follows: Figure 1 C in Figure 3As shown in C and F, P160-stimulated human PBMCs and mouse lymph node cells secreted IFN-γ levels were 3-4 times higher than those in the negative control group.

[0124] After stimulating and activating T cells from HLA-A*0201 gastric cancer patients and tumor-bearing mice with CKAP4 antigen peptide P160, flow cytometry analysis revealed the following results after CKAP4 antigen peptide P160 stimulation: Figure 1 B in Figure 3 As shown in B and E, human CD8 + T cells showed a 10-fold increase in CD25 and CD137 compared to the control group, and varying degrees of increase in CD69 and CD107a; mouse CD8 + T cell CD25 and CD137 levels were 2-4 times higher than in the control group. This indicates that the CKAP4 antigen peptide P160 can significantly activate T cells in gastric cancer patients and various tumor-bearing mice.

[0125] (2) Results of IFN-γ secretion level and T cell activation marker detection in healthy individuals

[0126] Similarly, activation of HLA-A*0201-positive healthy human PBMCs was induced in vitro using the CKAP4 antigen peptide P160, and the levels of IFN-γ secretion and CD8+ in the supernatant of activated cells were detected. + The expression of surface markers on T cells, experimental procedure as follows: Figure 2 As shown in A, the results are similar to those above: the IFN-γ level secreted by PBMCs stimulated by the CKAP4 antigen peptide P160 was 3 times higher than that in the negative control group; CD8 + T cells showed a 10-fold increase in CD137 compared to the control group, and a 2-3 fold increase in CD25, CD69, and CD107a. This indicates that the CKAP4 antigen peptide P160 can also significantly activate T cells in healthy individuals.

[0127] The above results indicate that the CKAP4 antigenic peptide P160 can effectively activate T cells derived from HLA-A*0201 positive individuals and tumor-bearing mice, inducing a strong antigen-specific immune response.

[0128] Example 4: In vitro tumor cell killing experiment using antigen-specific T cells

[0129] Using the CFSE / PI labeling method, lymph node T cells were extracted from 4T1 and CT26 tumor-bearing mice, respectively. The specific T cells induced by CKAP4 antigen peptide P160 for one week were used as effector cells (E), and the mouse 4T1 breast cancer cell line and mouse CT26 colorectal cancer cell line were used as target cells (T), respectively, to evaluate the antigen-specific killing ability of the specific T cells.

[0130] (1) Marking of target cells (T)

[0131] Log-phase tumor-bearing mouse 4T1 (breast cancer) and CT26 (colorectal cancer) cells were incubated in serum-free RPMI 1640 medium at 37°C for 2–4 h. Then, 4T1 and CT26 cells were collected in centrifuge tubes, centrifuged at 1000 rpm for 5 min to pellet the cells, washed once with PBS, and then the target cell concentration was adjusted to 10-1 with PBS. 6 Cells / mL were added, CFSE dye (final concentration 4 nM) was added, and the mixture was incubated at 37°C in the dark for 10 min. Then, at least 10 volumes of PBS were added, and the mixture was centrifuged again at 1000 rpm for 5 min, the supernatant was discarded, and the cells were washed twice. The target cells were resuspended in complete cell culture medium to a cell concentration of 10n. 5 Cells / mL; incubate at 37°C in a CO2 incubator for later use.

[0132] (2) Preparation of effector cells (E)

[0133] T cells (effective cells) from tumor-bearing mice (4T1 and CT26) induced and stimulated with CKAP4 antigen peptide P160 in vitro for one week were collected into centrifuge tubes. The cells were centrifuged at 1000 rpm for 5 min to collect the cell pellet, and the supernatant was discarded. The cells were resuspended in complete cell culture medium at a concentration of 5 × 10⁻⁶ cells / mL. 6 Cells / mL.

[0134] (3) Co-incubation and kill detection

[0135] Effector cells and CFSE-labeled target cells were mixed at different effector-target ratios (1:1, 5:1, 10:1) and incubated together at 37°C in a 5% CO2 incubator for 4–16 hours.

[0136] The cell mixture was centrifuged to precipitate the cells, and the supernatant was discarded. Each group of cells was resuspended in 100 μL PBS, and propidium iodide (PI, final concentration 50 μg / mL) was added. The cells were incubated at 4°C in the dark for 15 min. The cell mixture was centrifuged again to precipitate the cells, and then the cells were resuspended in 200 μL PBS. The results were detected and analyzed by flow cytometry. Specific killing efficiency was calculated by analyzing the proportion of PI-positive (dead cells) cells in the CFSE-positive (target cell) population.

[0137] Results of the effective target cell killing experiment are as follows Figure 4As shown, CT26 and 4T1 tumor-bearing mouse T cells specifically stimulated with CKAP4 antigen peptide P160 significantly increased their killing ability against corresponding CKAP4-positive tumor cells at various effector-to-target ratios (1:1, 5:1, and 10:1), and the killing efficiency increased synchronously with the gradient increase. At an effector-to-target ratio of 10:1, CKAP4 antigen peptide P160 showed a killing efficiency of more than 25% against CKAP4-highly expressed tumor cells. This indicates that CKAP4 antigen peptide P160 can effectively activate the T cell immune response and significantly enhance the killing efficacy of T cells against CKAP4-positive tumor cells.

[0138] Example 5: Establishment of mouse 4T1 and CT26 subcutaneous tumor models

[0139] (1) Select mouse 4T1 and CT26 cell lines in the logarithmic proliferation phase, collect cells after trypsin digestion and washing, and adjust the final cell concentration with physiological saline to 2×10⁻⁶. 6 cells / mL and 2×10 7 Tumor cell suspension was administered to 4-5 week old female Balb / c mice using a 1 mL sterile syringe (pre-cooled on ice for 2 hours). The suspension was then subcutaneously implanted into the left groin area. Each mouse received 100 μL of the suspension; for the 4T1 model, the injection volume was approximately 2 × 10⁻⁶ cells / mL. 5 Cells / animal, CT26 model approximately 2 × 10 6 Cells / cell. A small subcutaneous bulge is observed at the implantation site; monitor tumor formation after 5-7 days.

[0140] (2) Evaluation of antitumor efficacy in animal models of 4T1 and CT26 subcutaneous tumors

[0141] Subcutaneous tumor models of 4T1 and CT26 were established using the method in step (1). The day of tumor implantation was designated as the start time, marked as Day 0 (D0). The tumor-forming mice were randomly divided into three groups of five mice each, including a blank control group (Ctrl) and an experimental group (P160) that received subcutaneous injection of P160 vaccine. The P160 vaccine contained 100 μg of P160 per mouse and 20 μg of CpG adjuvant per mouse.

[0142] The 4T1 model was administered the drug on days 6, 18, and 30. Mice were weighed every other day, and the long diameter (a) and short diameter (b) of the tumor were measured. The tumor volume was calculated using the formula a × b. 2 / 2; Mice were sacrificed on day 32, and the tumors were completely removed and their weight recorded. The CT26 model was administered the drug on days 3, 9, and 15; mice were weighed every other day, and the long axis (a) and short axis (b) of the tumor were measured. The tumor volume was calculated using the formula a × b. 2 / 2; On day 17, mice were sacrificed and the tumors were completely removed and their weight was recorded.

[0143] Experimental results are as follows Figure 5 As shown, compared with the blank control group, tumor growth was significantly inhibited in mice in the subcutaneous injection group of P160 vaccine (P160 antigen peptide + CpG adjuvant). At the end of the experiment, the tumor volume of the P160 group mice was reduced by an average of 86% (p<0.001), and the tumor weight was also significantly reduced. Simultaneously, flow cytometry analysis showed that CD8+ was present in the tumor tissue of the subcutaneously injected P160 vaccine experimental group. + The number of infiltrating T cells increased significantly, while the proportion of immunosuppressive cell populations (such as TAM and TAN) decreased significantly. This indicates that the P160 vaccine can not only directly kill tumors but also reshape the immune microenvironment.

[0144] In summary, the short peptide P160 (RLTELTKSI) targeting the human and mouse homologous CKAP4 protein developed in this invention exhibits significant dual antitumor properties. This antigenic peptide can effectively activate specific CD8 cells through an HLA-restricted antigen presentation mechanism. + T-cell immune responses, as demonstrated in vitro, have shown that the induced T cells possess potent killing activity against CKAP4-positive tumor cells. Animal models further validated its therapeutic potential; this peptide not only directly inhibits tumor progression but also reduces the proportion of immunosuppressive cell populations by regulating the immune microenvironment. Based on its unique biological effects, P160 can be used as a core functional component in: 1) adoptive T-cell therapy for CKAP4-positive patients; 2) antigen component formulation design for tumor vaccines; and 3) the development of engineered T-cell receptors, providing innovative solutions for solid tumor immunotherapy systems.

[0145] This invention provides a concept and method for targeting CKAP4 tumor antigen peptides, vaccines, and their applications. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A tumor antigen peptide targeting CKAP4, characterized in that, The amino acid sequence of the tumor antigen peptide is shown as SEQ ID NO.

1.

2. The tumor antigen peptide according to claim 1, characterized in that, The tumor antigen peptide is a human, murine homologous antigen peptide.

3. The tumor antigen peptide of claim 1, wherein, The tumor antigen peptide is an HLA-A*0201 molecule restricted antigen peptide.

4. A nucleic acid molecule, characterized in that, It encodes the tumor antigen peptide according to any one of claims 1-3.

5. An expression vector, characterized by, It comprises the nucleic acid molecule according to claim 4.

6. A tumor antigen peptide vaccine targeting CKAP4, characterized in that, The effective active ingredient of the tumor antigen peptide vaccine is the tumor antigen peptide according to any one of claims 1-3.

7. The tumor antigen peptide vaccine according to claim 6, characterized by, The tumor antigen peptide vaccine comprises an adjuvant CpG ODN.

8. The tumor antigen peptide vaccine according to claim 6 or 7 in the preparation of a solid tumor targeted therapy or immunotherapy drug.

9. Use according to claim 8, characterized in that, The drug is an antigen reactive T cell drug and an engineered T cell drug.

10. A test kit comprising, The effective constituent element of the detection kit is the tumor antigen peptide according to any one of claims 1-3.

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