A universal assay protocol for lung cancer and gastrointestinal tumor antigen-specific thymus-dependent lymphocytes for east asian populations

Abstract

This invention discloses a universal detection method for tumor antigen-specific thymus-dependent lymphocytes in a broad population of East Asia, applicable to lung cancer and gastrointestinal cancers. The broad-spectrum T-cell epitope peptide library used in this detection method contains specific antigen T-cell epitope peptides presented by 13 HLA-A molecules, 15 HLA-B molecules, and 14 HLA-C molecules with an allele frequency greater than 1% in the East Asian population. The total gene frequencies of these dominant HLA-A, B, and C molecules account for approximately 95%, 71%, and 93% of the Chinese population, respectively, and are similar in total gene frequencies in Northeast and Southeast Asian populations. Therefore, this detection method is applicable to the vast majority of individuals in this region, providing a universal method for evaluating specific tumor antigen-specific T-cell immune function in patients with lung cancer or gastrointestinal tumors.

Description

Technical Field

[0001] This invention relates to the fields of medical immunology and immunological testing technology, specifically to a universal detection scheme and its application for detecting tumor antigen-specific thymus-dependent lymphocytes for lung cancer and gastrointestinal cancers in a wide population in Northeast Asia and Southeast Asia, including China. Background Technology

[0002] 1. Antitumor effects of tumor antigen-specific T cells:

[0003] Tumor cells process intracellular tumor antigens into T-cell epitope peptides, which bind to HLA molecules to form antigen peptide / HLA complexes, and then bind to CD8. + The specific binding of T cell receptor (TCR) molecules on the T cell membrane promotes the activation of tumor antigen-specific T cell clones, which differentiate into cytotoxic T cells (CTLs), ultimately becoming the main effector cells in the human body capable of killing tumor cells. Therefore, the reactivity and quantity of tumor antigen-specific T cells are key factors affecting tumor progression and prognosis, exhibiting dynamic changes at different stages of tumor disease and having significant clinicopathological and prognostic implications. For example, in lung cancer, the reactivity of tumor antigen-specific T cells such as MAGE-A3, NY-ESO-1, WT1, and Survivin gradually decreases from early to late stages. Similarly, in the progression of gastrointestinal tumors such as gastrointestinal and esophageal cancer, the reactivity of tumor antigen-specific T cells such as MAGE-A1 and SALL4 gradually decreases, suggesting that these tumor antigens may play an immune protective role in the early stages. Conversely, the reactivity of PD-L1 and KRAS mutant antigen-specific T cells is weak in the early stages but gradually increases in the late stages, potentially marking tumor progression and malignancy.

[0004] 2. Clinical value of tumor antigen-specific T cell detection:

[0005] Tumor patients in early stage receive surgical resection, radiofrequency ablation, in the middle and late stage receive radiotherapy and chemotherapy, due to the change of tumor load in the body, the amount of tumor antigen released by tumor cells is different, and the individual differences of the body's immune response, etc., so the reactivity and number of tumor antigen-specific T cells will appear dynamic changes and individual differences, which affect the final efficacy, survival and recurrence. At present, immunotherapy is widely implemented in various cancers, its main purpose is to expand the number of tumor antigen-specific T cells or prevent their functional exhaustion, and promote their tumor killing activity. Therefore, the detection of tumor antigen-specific T cells can evaluate the strength of the specific T cell immune function of patients after traditional treatment or immunotherapy. Some studies show that the dynamic changes of tumor antigen-specific T cell reactivity have a significant correlation with disease progression and recurrence after treatment. For example: in lung cancer, gastrointestinal cancer and esophageal cancer, after immunotherapy such as Anti-PD-1 / PD-L1 / CTLA-4 immune checkpoint inhibitors, MUC1\NY-ESO-1\WT1, CEA / CA724 / CA19-9 polypeptide vaccine, antigen peptide modified DC or adoptive T cells, etc., the enhancement degree of tumor antigen-specific CD8 + T cell activity is significantly positively correlated with the prolongation of patient's recurrence-free\progression-free survival and even overall survival and the reduction of recurrence rate. However, there is no systematic study to confirm that it can directly guide the treatment plan and drug selection of clinicians. However, the immediate detection of the specific T cell immune function of patients against specific antigens can provide precise immunological indicators for the study of the immune interaction mechanism between tumor and body, so as to ultimately provide theoretical support for the development of new methods for disease treatment and prevention, and provide new immunological indicators for clinicians to evaluate the specific cellular immune function state of tumor patients.

[0006] 3. Difficulties and current situation of antigen-specific T cell detection technology:

[0007] 3.1 Technical difficulties:

[0008] Compared with the quantitative detection of antigens and antibodies, the detection of antigen-specific T cells is much more difficult, mainly restricted by two factors. One is the high polymorphism of HLA in the population, which seriously restricts the population universality of the detection method: human major histocompatibility antigen (HLA) is responsible for presenting antigen peptides (T cell epitope peptides) to T cells, initiating the adaptive immune response of the body. Among them, HLA-A, B, C and other class I molecules present antigen peptides to activate CD8 + T cells, which differentiate into cytotoxic T cells (CTLs), which are the main effector cells targeting tumor cells; HLA-DR, DQ, DP and other class II molecules present antigen peptides to activate CD4 + T cells, which differentiate into effector Th cells, which secrete a large amount of pro-inflammatory cytokines, and at the same time assist CD8 +T cell or B cell activation. However, the HLA gene system is a group of closely linked genes. There are a large number of alleles at each HLA locus in the human population. The HLA alleles are not completely identical between different individuals, and are highly polymorphic in the human population. Each HLA molecule can bind and present a large number of antigenic peptide sequences, and the resulting T cell immune response is also different in strength. Therefore, for a specific tumor antigen or other antigen such as a pathogen antigen, a broad-spectrum T cell epitope peptide library covering the HLA allele polymorphism of a wide range of population is needed to establish a population-universal detection method for the antigen-specific T cells.

[0009] The second limitation is that the T cell epitope spectrum in most tumor antigens has not been clearly defined, which limits the establishment of a broad-spectrum T cell epitope peptide library: although there have been many studies on tumor antigens, there have been few studies on T cell epitopes (antigenic peptides) presented by various HLA molecules in these antigens. For example: although there are many tumor antigens related to lung cancer, gastrointestinal cancer and esophageal cancer, such as tumor-associated antigens CYFRA 21-1, CEA, CA724, SCC, CA19-9, NSE, hTERT, HER2, MUC1 and STEAP1, etc.; cancer-testis antigens MAGE-A1, MAGE-A3, NY-ESO-1 and SSX2, etc. However, there are very few experimentally verified and reported T cell epitopes, such as CD8 + Among the T cell epitopes, NSE has only 4, CA125 has 7, CEA has only 12, and other antigens have even fewer, and these reported epitope peptides are only presented by a few HLA-A molecules (such as HLA-A0201, A2402 or A1101), which cannot cover the HLA allele polymorphism of the population in a specific region.

[0010] 3.2 Current technology:

[0011] There are two types of antigen-specific T cell detection techniques currently in use: one is peptide-HLA multimer fluorescent staining and flow analysis, which is the most specific because the specificity of T cells is reflected in the specific binding of TCR to peptide-HLA complexes. However, it has three disadvantages: 1) the preparation of peptide-HLA complexes and their multimers is difficult, the cost is high, the stable period is short, and it is very difficult to prepare hundreds of pHLA multimer libraries covering a large number of different HLA molecules and a large number of T cell epitope peptides to detect a wide range of clinical patients, so it is difficult to promote in clinical laboratories; 2) this method can only detect the number of antigen-specific T cells, and cannot evaluate the function, and exhausted, apoptotic or non-antigen-reactive specific T cells can also be detected; 3) the sensitivity is low, and it is difficult to distinguish individual differences or dynamic changes in frequency for antigen-specific T cells with very low frequency.

[0012] The second type of technology utilizes CD8. + T-cell epitope peptides (9-10 aa) or CD4 + T-cell epitope peptides (16-19aa) are co-cultured with patient lymphocytes, followed by detection of cytokine secretion using enzyme-linked immunospot / fluorescent immunospot (ELISpot / FluoroSpot) or intracellular cytokine fluorescence staining (ICS). While this technique has slightly lower specificity than peptide-HLA multimer fluorescence staining and flow cytometry, it is the most widely used and accepted method by scholars both domestically and internationally due to three advantages: 1) Reagent preparation is easy and inexpensive; 2) The sensitivity of ELISpot or FluoroSpot methods is far higher than that of peptide-HLA multimer fluorescence staining; even a single activated cell secreting cytokines out of a million cells can form a spot and be detected; 3) It allows for counting and is also a cellular function assay. Only T cells that are reactive after stimulation by the antigen peptide can be detected and counted. The drawback of this technique is the lack of an experimentally validated, real T-cell epitope peptide library that covers a broad range of HLA gene polymorphisms. Currently, most researchers can only use a few known epitope peptides to test a small number of patients carrying specific HLA alleles, which cannot fully reflect the functional status of the patient's tumor antigen-specific T-cell clone library. Some researchers use overlapping peptide libraries covering the full length of tumor antigens or computer-simulated predictive peptide libraries to test a wide range of patients. Overlapping peptides are generally designed as overlapping long peptides (15-19 amino acids / peptide) spaced 5-8 amino acids apart, and predictive peptides are mostly CD8. + Short peptides (9-10 amino acids / peptide) are found for T cell epitopes. However, overlapping peptide libraries or predicted peptide libraries have serious limitations: 1) They are not experimentally validated true epitope peptides; recent studies have further confirmed that the vast majority are pseudoepitopes; 2) The HLA restriction of these overlapping peptides or predicted peptides is unclear, making it unknown which HLA molecules they can cross-bind to or which HLA allele populations they can cover. Therefore, using overlapping peptide libraries or predicted peptide libraries to test a wide range of patients is currently a last resort.

[0013] The latest detection technologies include single-cell transcriptome sequencing and TCR gene sequencing, which are used to analyze the clonal characteristics of antigen-specific T cell repositories in vivo. However, these technologies are difficult to popularize due to both cost and technical limitations, and the analysis and interpretation of the results are difficult to concretize and simplify.

[0014] Due to the aforementioned limitations, although some laboratories have begun to assess patients' immune function status by detecting changes in the frequency of functional subtypes such as activated / proliferating T cells, regulatory T cells, and depleted T cells in the overall T cell pool of patients, there are still no universal detection technologies and reagents for tumor antigen-specific T cells applicable to a wide range of patient populations, both domestically and internationally. Furthermore, there are no reagents and reference values ​​that conform to the HLA gene polymorphism of the Chinese population. Therefore, this seriously hinders clinicians from evaluating patients' specific T cell immune function status. Summary of the Invention

[0015] To address the problems mentioned in the background section, this invention provides a universal detection method and kit for tumor antigen-specific thymus-dependent lymphocytes in a broad population of East Asia, applicable to lung cancer or gastrointestinal cancers, for non-diagnostic purposes.

[0016] To achieve the above objectives, the present invention provides the following technical solution:

[0017] One object of the present invention is to provide a universal detection method and kit for tumor antigen-specific thymus-dependent lymphocytes in a broad population of East Asian regions, specifically including the following steps:

[0018] (1) Screening and verification of a series of antigenic peptides against specific viral antigens or tumor antigens presented by all HLA-A, B, and C molecules with allele frequencies greater than 1% in East Asian populations: Using multiple online epitope prediction databases, candidate epitope peptides against lung cancer-related antigens (Cyfra21-1, CA125, NSE), colorectal cancer-related antigens (CEA, CA724, CA242), or esophageal cancer-related antigens (SCC-Ag, CA199) were virtually predicted from 13 HLA-A molecules, 15 HLA-B molecules, and 14 HLA-C molecules with allele frequencies greater than 1%. Then, using peripheral blood mononuclear cells (PBMCs) from cancer patients for lymphocyte functional experiments and using stable transfected cell lines of dominant HLA-A, B, and C molecules in the Chinese population for peptide competitive binding experiments, the immunogenicity of each fei'a candidate epitope peptide was verified, and a positive epitope peptide library was established for each dominant HLA molecule.

[0019] The 13 HLA-A molecules (with a total gene frequency greater than 95% in the Chinese population) are as follows: HLA-A1101, A2402, A0201, A3101, A0206, A0207, A3303, A3001, A0203, A1102, A0301, A0101, A2601, etc.

[0020] The following are 15 HLA-B molecules (with a total gene frequency greater than 70% in the Chinese population): HLA-B4001, B4601,

[0021] B5801, B5101, B1302, B1501, B1301, B4006, B1502, B3501, B5401, B5201, B4403, B4801, B0702, etc.

[0022] The 14 HLA-C molecules (with a total gene frequency greater than 90% in the Chinese population) are as follows: HLA-Cw0702, Cw0102, Cw0304, Cw0801, Cw0602, Cw0303, Cw0302, Cw0401, Cw1402, Cw1502, Cw0701, Cw1202, Cw1403, and Cw1203. In subsequent descriptions, the "w" in HLA-C molecules is usually omitted.

[0023] (2) Construct a broad-spectrum T-cell epitope peptide library to achieve universal detection. Dominant T-cell epitope peptides have multiple meanings: strong immunogenicity, cross-binding with multiple dominant HLA molecules, and the ability to detect specific T-cell clones in most patients. Based on the amino acid sequence length of each viral antigen or tumor antigen, 30-100 epitope peptides are screened to form a broad-spectrum T-cell epitope peptide library. These broad-spectrum T-cell epitope peptides are respectively presented by the epitope peptides of the aforementioned 42 HLA molecules, and each HLA-A / B / C molecule cross-binds with multiple epitope peptides. Thus, each patient's 6 HLA-A / B / C molecules can cross-bind with dozens of epitope peptides. This broad-spectrum epitope peptide library not only conforms to the HLA polymorphism of Chinese and East Asian populations but also fully reflects the richness of T-cell epitopes in antigen molecules, thus enabling universal detection and fully reflecting the immune function status of the antigen-specific T-cell clone library within the individual patient.

[0024] (3) Purchase commercial reagents and use the peptide pool array of the broad-spectrum T cell epitope peptide library of the above-mentioned specific antigens to co-culture with patient PBMCs in microwell reaction plates to establish enzyme-linked immunospot (ELISpot) or fluorescent immunospot (FluroSpot) assays to quantitatively detect the number of cells secreting IFN-γ, TNF-α or IL-2.

[0025] Further, in step (2), a broad-spectrum T-cell epitope peptide library is constructed: based on the sequence length of each tumor antigen, 30-100 epitope peptides are screened from the above-mentioned positive epitope peptide library to form a broad-spectrum T-cell epitope peptide library; these broad-spectrum T-cell epitope peptides are respectively epitope peptides presented by the above-mentioned 42 HLA molecules, and each HLA-A / B / C molecule cross-binds 5-15 of the epitope peptides; therefore, the 6 HLA-A / B / C molecules of each patient can cross-bind 30-90 epitope peptides in the broad-spectrum T-cell epitope peptide library, that is, for each tumor antigen, each patient can be tested for 30-90 specific CD8. + T-cell clones.

[0026] Furthermore, in step (3) of this invention, enzyme-linked immunosorbent assay (ELISA) or fluorescent immunospot assay can be replaced with magnetic microsphere chemiluminescence assay: Purchase streptavidin-labeled magnetic microspheres, biotinylated IFN-γ capture, enzyme-labeled IFN-γ detection antibody, chemiluminescent substrate, and IFN-γ standard, etc., and use the peptide pool array of the broad-spectrum T-cell epitope peptide library of the specific antigen from step (2) above to co-culture with patient PBMCs in a 96-well cell culture plate, collecting the culture supernatant from each well as the sample to be tested. Establish a magnetic microsphere chemiluminescence assay in another microplate to detect the levels of IFN-γ, TNF-α, or IL-2 in the culture supernatant.

[0027] Furthermore, in step (3) of this invention, enzyme-linked immunosorbent assay (ELISA) or fluorescent immunospot assay can be replaced with enzyme-linked immunosorbent assay (ELISA): IFN-γ capture antibody, biotinylated IFN-γ detection antibody, streptavidin-labeled horseradish peroxidase or alkaline phosphatase, enzyme substrate, and IFN-γ standard are procured. Using the peptide pool array of the broad-spectrum T-cell epitope peptide library of the specific antigen from step (2) above, the peptides are co-cultured with patient PBMCs in a 96-well cell culture plate, and the culture supernatant from each well is collected as the sample to be tested. An ELISA method is established in another microplate to detect the levels of IFN-γ, TNF-α, or IL-2 in the culture supernatant.

[0028] Furthermore, in step (3) of this invention, the enzyme-linked immunospot assay can be replaced by a combination of cytometric beads array (CBA) and T cell multicolor fluorescence staining to analyze the functional subtypes of antigen-specific T cells: a kit reagent for detecting multiple molecules is assembled using commercially available CBA individual reagents. The peptide pool array of the specific antigen from step (2) above is co-cultured with patient PBMCs in a 96-well cell culture plate for 20 hours, and then the supernatant is collected. The secretion levels of multiple soluble molecules secreted by patient PBMCs are detected using CBA reagents and flow cytometry. At the same time, cells are collected for multicolor fluorescence monoclonal antibody staining and flow cytometry analysis to quantitatively analyze the frequency of multiple functional subpopulations of antigen-specific T cells.

[0029] Furthermore, the soluble molecules secreted by the patient's BMCs are any one or more of human interferon-γ, human interleukin-17, granzyme A, granzyme B, perforin, tumor necrosis factor-α, and tumor necrosis factor-β.

[0030] The invention's method and kit can be used to universally detect the immune function status of specific antigen-specific T cells in lung cancer, gastrointestinal cancer, or esophageal cancer patients in East Asia. The detection results will provide precise immunological indicators for studying the immune interaction mechanisms between tumors and patients, thus providing fundamental theoretical support for the subsequent development of new treatment and prevention methods.

[0031] Another objective of this invention is to provide the application of the above-mentioned antigen-specific thymus-dependent lymphocyte detection method applicable to a wide range of populations in East Asia in the preparation of specific T-cell detection kits for detecting lung cancer-related tumor antigens, gastrointestinal cancer-related tumor antigens, and esophageal cancer-related tumor antigens.

[0032] The method described in this invention can be used to prepare specific T-cell detection kits for lung cancer-related tumor antigens, gastrointestinal cancer-related tumor antigens, and esophageal cancer-related tumor antigens.

[0033] Preferably, the kit is an enzyme-linked immunospot assay kit, but it can also be a fluorescent immunospot assay kit, an enzyme-linked immunosorbent assay (ELISA) kit, a chemiluminescence assay kit, a cytometric beads array kit, or a kit for T cell membrane fluorescence staining flow cytometry analysis.

[0034] Compared with the prior art, the beneficial effects of the present invention are:

[0035] 1. This invention screens and verifies authentic T-cell epitope peptides from specific tumor antigens that can be presented by 42 dominant HLA-A, -B, and -C molecules in Chinese and East Asian populations, thereby enabling the construction of a broad-spectrum authentic T-cell epitope peptide library that conforms to HLA gene polymorphisms in Chinese and East Asian populations. Existing detection technologies use overlapping peptide libraries or predicted peptide libraries of specific tumor antigens, which cannot reflect the actual functional state of tumor antigen-specific T cells in patients.

[0036] 2. This invention utilizes the aforementioned broad-spectrum, real T-cell epitope peptide library to establish a specific T-cell detection kit for specific tumor antigens applicable to Chinese and East Asian populations. It eliminates the need for HLA allele typing of the tested subjects and can simultaneously detect the responses of numerous specific T-cell clones presented by HLA-A, -B, and -C molecules. Existing detection technologies use only a few known T-cell epitope peptides, limiting detection to a small number of patients carrying specific HLA-A, -B, or -C alleles, and failing to fully reflect the functional status of the overall specific T-cell clone library against tumor antigens within the patient's body.

[0037] 3. When screening and verifying T-cell epitope peptides in specific tumor antigens, this invention, in addition to using an online epitope prediction database to virtually predict candidate epitope peptides, also uses PBMCs from a tumor patient population to conduct co-culture experiments of candidate peptides and PBMCs to verify the real immunogenicity of candidate epitope peptides in the real tumor world (this experiment is an improved method based on existing technology). Furthermore, this invention utilizes a cell line array with dominant HLA-A, B, and C alleles in the Chinese population to conduct peptide competitive binding experiments, analyzing the binding affinity of each epitope peptide with multiple related HLA molecules, thereby determining the types of HLA alleles that each epitope peptide can cover and the breadth of the applicable population. In existing technologies, only a very small number of T-cell epitopes have been experimentally verified, and most of them use online epitope prediction databases to virtually predict epitope peptides. Very few use lymphocyte samples from patient populations to conduct peptide-PBMC co-stimulation experiments to verify the immunogenicity of epitope peptides in the real world of tumors. Furthermore, no one has used cell line arrays with dominant HLA-A, B, and C alleles in the Chinese population to conduct peptide competitive binding experiments to clarify the HLA allele coverage of each epitope peptide.

[0038] 4. The technical solution of this invention is similar to the invention previously filed by the applicant. However, the technical solution of the previous invention was not used to screen T-cell epitope peptides of lung cancer-associated antigens (Cyfra21-1, CA125, NSE), colorectal cancer-associated antigens (CEA, CA724, CA242), or esophageal cancer-associated antigens (SCC-Ag, CA199), nor was a specific T-cell detection protocol for these antigens established accordingly. This invention, however, seeks protection for the technical solution of the aforementioned tumor-associated antigens and provides specific implementation schemes for some of these antigens, such as Cyfra21-1, CEA, and SCC-Ag, as well as experimental data supporting this invention, such as T-cell epitope screening data, specific T-cell detection data of clinical specimens, and clinical significance analysis data. There is no relevant data regarding the aforementioned tumor-associated antigens in the prior art. Attached Figure Description

[0039] Figure 1 This is a technical solution diagram of Embodiment 1 of the present invention;

[0040] Figure 2 A statistical chart showing the results of screening positive epitope peptides for Cyfra21-1 antigen in blood samples from each lung cancer patient.

[0041] Figure 3 Flow cytometry analysis of positive epitope peptides of Cyfra21-1 antigen in blood samples from some lung cancer patients;

[0042] Figure 4 Flow cytometry analysis of peptide competitive binding assays of some Cyfra21-1 antigenic epitope peptides;

[0043] Figure 5 ELISPOT dot plots and clinical significance statistical analysis of Cyfra21-1 antigen-specific T cell detection in blood samples of lung cancer patients;

[0044] Figure 6 A statistical chart showing the results of screening positive epitope peptides for CEA antigen in blood samples from colorectal cancer patients.

[0045] Figure 7 Flow cytometry analysis of positive epitope peptides for CEA antigen in blood samples from some colorectal cancer patients;

[0046] Figure 8 Flow cytometry analysis of peptide competitive binding assays for some CEA antigen epitope peptides;

[0047] Figure 9 This is an ELISPOT dot pattern of four blood samples for repeatable CEA-specific T cell assays.

[0048] Figure 10Statistical analysis of the clinical significance of CEA-specific T cell detection results in blood samples of colorectal cancer patients;

[0049] Figure 11 Flow cytometry analysis of the peptide competitive binding assay for the SCC-Ag antigen epitope peptide;

[0050] Figure 12 An example of SCC-Ag-specific T-cell detection in blood samples from esophageal cancer patients (ELISPOT speckle image). Detailed Implementation

[0051] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0052] Example 1

[0053] This method was used to screen T-cell epitope peptides of Cyfra21-1, a lung cancer-associated tumor antigen presented by 42 dominant HLA-A, B, and C molecules, to construct a broad-spectrum T-cell epitope peptide library. A universally applicable detection method and kit for Cyfra21-1 tumor antigen-specific T-cell antigens was then established for a wide range of lung cancer patients in China. The main procedures are as follows: Figure 1 .

[0054] 1) Virtual prediction of candidate T-cell epitope peptides of Cyfra21-1, a lung cancer-associated antigen presented by HLA-A, B, and C molecules:

[0055] ① The full-length amino acid sequence of Cyfra21-1 was obtained from databases such as UniProt / NCBI and COSMIC. Multiple sequence alignment was performed using the ClustalW method in Molecular Evolutionary Genetics Analysis (MEGA7, version 1.0.0.0). The alignment results were then analyzed using GeneDoc (version 2.7.0) to determine the conserved regions of the sequence. The conservation properties of each amino acid were assessed using 100%, 95%, and 80% as cutoff points.

[0056] ② Thirteen HLA-A molecules, 15 HLA-B molecules, and 14 HLA-C molecules with gene frequencies greater than 1% in the Chinese population were identified. Their total allele frequencies covered over 90%, 70%, and 90% of the Chinese and East Asian populations, respectively. Epitope peptides presented by the aforementioned 42 dominant HLA-A, B, and C molecules were virtually predicted using the epitope prediction algorithms and tools listed in Table 1. Epitope peptide lengths were set to 9-mer and 10-mer, and they had to meet the evaluation criteria of at least three prediction algorithms. The top 5-10 peptides in terms of score were selected as candidate epitopes. A total of 132 candidate epitopes were ultimately screened, and all 59 epitopes with different sequences were synthesized into candidate epitope peptides for subsequent validation experiments.

[0057] Table 1. Epitope Peptide Prediction Database Websites

[0058]

[0059] The amino acid sequence of Cyfra21-1 (a soluble antigen fragment 21-1 of cytokeratin 19) is as follows:

[0060] Scientifically named genera and species: Phylum Vertebrata, Subclass Eutheria, Humans

[0061] Cyfra21-1 (Cytokeratin Fragment Antigen 21-1) (ID: P08727)

[0062] MTSYSYRQSSATSSFGGLGGGSVRFGPGVAFRAPSIHGGSGGRGVSVSSA 50

[0063] RFVSSSSSGAYGGGYGGVLTASDGLLAGNEKLTMQNLNDRLASYLDKVRA 100

[0064] LEAANGELEVKIRDWYQKQGPGPSRDYSHYYTTIQDLRDKILGATIENSR 150

[0065] IVLQIDNARLAADDFRTKFETEQALRMSVEADINGLRRVLDELTLARTDL 200

[0066] EMQIEGLKEELAYLKKNHEEEISTLRGQVGGQVSVEVDSAPGTDLAKILS 250

[0067] DMRSQYEVMAEQNRKDAEAAWFTSRTEELNREVAGHTEQLQMSRSEVTDLR 300

[0068] RTLQGLEIELQSQLSMKAALEDTLAETEARFGAQLAHIQALISGIEAQLG 350

[0069] DVRADSERQNQEYQRLMDIKSRLEQEIATYRSLLEGQEDHYNNLSASKVL 400

[0070] 2) Using PBMCs from lung cancer patients, the immunogenicity of candidate epitope peptides was verified through a peptide-PBMC co-stimulation assay:

[0071] ① Peripheral blood was collected from lung cancer patients by the Department of Thoracic Surgery, First Affiliated Hospital of Bengbu Medical University. PBMCs were routinely separated and kept warm for later use. A portion of cells were also taken and HLA-A, B, and C allele typing was performed according to the following steps.

[0072] 200 µL of anticoagulated blood was collected, and genomic DNA was extracted using a human whole blood genomic DNA extraction kit (Tiangen Biotech, Beijing). PCR was performed using HLA-A site-specific primers A1 and A3 (Table 2) to amplify the DNA sequences of exon 2, intron 2, exon 3, and some introns 1 and 3 at site A, with a product size of 985 bp. PCR was performed using HLA-B site-specific primers B1 and B3 (Table 2) to amplify the DNA sequences of exon 2, intron 2, exon 3, and some introns 1 and 3 at site B, with a product size of 985 bp. PCR was performed using HLA-C site-specific primers CF and CR (Table 2) to amplify the DNA sequences of exon 2, intron 2, exon 3, and some introns 1 and 3 at site C, with a product size of 985 bp. The amplification conditions were: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s; 62℃ annealing for 15 s; 72℃ extension for 90 s; 35 cycles; 72℃ extension for 5 min. The amplified products were identified by 1% agarose gel electrophoresis and sent to Shanghai Sunny Biotechnology Co., Ltd. for purification and bidirectional sequencing. PCR reagents were purchased from Nanjing Novizan Biotechnology Co., Ltd.

[0073] Table 2. HLA-A, B, and C site-specific PCR amplification primers

[0074]

[0075] ② Immunogenicity verification of single peptides:

[0076] Based on the identification results of the patient's HLA-A, B, and C alleles, candidate epitope peptides corresponding to specific HLA molecules of the patient were selected and co-cultured with the patient's PBMCs for 6 hours. Intracellular IFN-γ fluorescence staining was then performed to detect CD8+ secreting IFN-γ.+ T cells and CD4 + T cell frequency compared to the peptide-free culture group. This resulted in IFN-γ... + / CD8 + Epitopes with significantly elevated T cell frequency (more than twice that of the group without peptide culture) are immunogenic and are positive epitopes, meaning that the patient's PBMCs contain memory CD8 specific to that epitope peptide. + T cells. Each candidate epitope peptide was tested with at least 5 PBMCs from patients with relatively HLA genes, and epitope peptides with strong immunogenicity and positive reactions in a majority of patients (3 / 5) were selected for inclusion in the dominant epitope peptide library. Results: Using blood samples from 150 lung cancer patients, 37 positive epitope peptides were obtained. Figure 2 A statistical graph showing the results of screening Cyfra21-1 antigen for positive epitope peptides in blood samples from lung cancer patients. Immunogenic positive epitope peptides can increase IFN-γ levels in co-cultured PBMCs. + / CD8 + The T cell frequency was significantly increased (more than twice that of the group without peptide culture). Figure 3 Flow cytometry analysis of positive epitope peptides for Cyfra21-1 antigen in blood samples from selected patients, and IFN-γ in PBMCs. + / CD8 + The frequency of T cells was analyzed.

[0077] 3) Using an array of HMy2.CIR cell lines expressing 12 specific HLA-A molecules, 15 specific HLA-B molecules, and 14 specific HLA-C molecules, competitive binding experiments were conducted between HLA molecules and peptides to verify the cross-binding affinity of positive epitope peptides with various related HLA molecules:

[0078] ①HMy2.CIR is a human B lymphoblast cell line lacking HLA class I antigens. It does not express HLA-A and B molecules, but only a trace amount of HLA-Cw4 molecules. Our laboratory constructed an array of HMy2.CIR cell lines expressing 12 specific HLA-A molecules, 15 specific HLA-B molecules, and 14 specific HLA-C molecules. The main steps were: extracting mRNA from healthy PBMCs, amplifying each HLA-A and cDNA, constructing the pcDNATM3.1 / myc-His(-)A recombinant plasmid using standard methods, electroporating HMy2.CIR cell lines, selecting stable cell lines using G418, staining with the HLA-ABC fluorescent monoclonal antibody W6 / 32, and then sorting CIR cell lines highly expressing the HLA molecule using flow cytometry for pure culture, followed by sequencing identification.

[0079] ② Positive epitope peptides validated by the peptide-PBMC co-stimulation experiment were classified. Based on the predicted HLA molecule restriction and the actual HLA-A, B, and C alleles of blood donors in the validation experiment, a list of positive epitope peptides that could bind to each HLA molecule was compiled. HMy2.CIR cell lines stably expressing a specific HLA-A molecule were co-incubated with each positive epitope peptide associated with that HLA molecule and a fluorescein-labeled reference peptide to perform a peptide competition binding experiment: First, the HLA-bound peptides on the CIR cell membrane were eluted with an acid solution (0.131 mol / L citric acid and 0.061 mol / L disodium hydrogen phosphate, pH 3.0), then neutralized with cell culture medium, centrifuged and washed, and seeded into 96-well cell culture plates (1×10⁻⁶). 5 Cells / well / 100μL) were cultured, β2-microglobulin (1μg / mL) was added, followed by a fluorescein-labeled reference peptide (200nM), and then unlabeled positive epitope peptides (5μM, 15μM) were added respectively. After 24h of incubation, flow cytometry analysis was performed, and the results were analyzed by IC50. 50 The affinity of each positive epitope peptide to a specific HLA molecule was determined by the value and competitive inhibition percentage. The results clarified the binding affinity of each positive epitope peptide to multiple related HLA-A, -B, or -C molecules. Table 3 summarizes the affinity ranking of each epitope peptide to related HLA molecules; Figure 4 This is a flow cytometry chromatogram of peptide competitive binding assays for some Cyfra21-1 antigenic epitope peptides.

[0080] 4) Construct a broad-spectrum T-cell epitope peptide library of the lung cancer-associated antigen Cyfra21-1, and detect Cyfra21-1-specific T cells in a wide range of PBMCs from lung cancer patients:

[0081] Through the above-mentioned single peptide immunogenicity verification and HLA molecule-peptide competitive binding experiments, 22 dominant epitope peptides cross-linked with 13 dominant HLA-A molecules, 23 dominant epitope peptides cross-linked with 15 dominant HLA-B molecules, and 13 dominant epitope peptides cross-linked with 14 dominant HLA-C molecules were screened. These peptides can specifically target 73, 111, and 70 peptide / HLA complex CD8, respectively. + T cell clones were used. These 37 epitope peptides with different sequences were used to construct a broad-spectrum T cell epitope peptide library, Cyfra21-1, and further divided into two mixed peptide libraries (18 and 19 peptides) based on antigen origin and pH. An IFN-γ ELISpot detection method was then established. Theoretically, these 37 T cell epitopes can cross-detect approximately 254 peptide / HLA complex-specific CD8+. + T cell clones. The number of Cyfra21-1 dominant T cell epitope peptides that cross-bind with each dominant HLA-A, B, and C molecule is shown in Table 4.

[0082] Table 3. Peptide competitive binding assays using 41 HMy2.CIR cell lines to detect the binding affinity of each HLA-A, -B, and -C molecule to the corresponding Cyfra21-1 antigen T cell epitope peptide.

[0083]

[0084]

[0085] Table 4. Number of Cyfra21-1 dominant T cell epitope peptides cross-linked with dominant HLA-A, B, and C molecules

[0086]

[0087] 5) Assemble the ELISPOT kit to perform universal detection of lung cancer antigen Cyfra21-1 specific T cells:

[0088] ①PBMC isolation: Peripheral anticoagulated blood samples were collected from the patients to be tested. PBMCs were isolated using the same routine method as above, counted, and the cell concentration was adjusted to 4 × 10⁻⁶ cells using serum-free cell culture medium (Dakeway Biotechnology, Shenzhen). 6 / ml.

[0089] ② Add cell suspension: Remove the ELISPOT strips pre-coated with human IFN-γ capture antibody, preheat at room temperature for 30 minutes, and add 4 × 10⁻⁶ PBMCs from each patient. 6 Add 100 μL / mL to 4 wells, with 100 μL added to wells 1 through 3 (i.e., 4 × 10⁻⁶). 5 50 μL / well was added to the 4th well (positive control well) (i.e., 2 × 10⁻⁶ cells / well). 5 (cells).

[0090] ③ Add antigen peptides: Each blood sample is tested in 4 wells. Add antigen peptide library 1 and peptide library 2 to wells 1 and 2, respectively, at 26 μL / well, for 2 experimental wells; add 26 μL of negative control replenishment to well 3, for negative control; well 4 is the positive control, add 5 μL of PHA working solution and 71 μL of serum-free cell culture medium. The final volume of each well is 126 μL.

[0091] ④ Culture and incubation: Cover the plate and place it in a 37℃, 5% CO2 cell culture incubator for 20-22 hours.

[0092] ⑤ Cell lysis and plate washing: Remove the ELISPOT strips, shake off the cell suspension, add 200 μL of deionized water to each well, incubate at 4°C for 10 minutes, shake off the deionized water, and pat dry on absorbent paper; add 200 μL of ordinary washing buffer (1×PBS) to each well, let stand for 1 minute, shake off the liquid, and repeat this washing process 5 times. After the last shake, pat dry on absorbent paper.

[0093] ⑥ Add detection antibody and wash plate: Add 100 μL of biotin-IFN-γ detection antibody working solution (BDBioscience) to each well and incubate at room temperature in the dark (20-25℃) for 2 hours; shake off the liquid, add 200 μL of ordinary washing buffer (1×PBS) to each well, let stand for 1 minute, shake off the liquid vigorously, repeat this washing process 5 times, and after the last shake off the liquid, pat dry on absorbent paper.

[0094] ⑦ Add HRP-streptavidin and wash the plate: Add 100 μL of HRP-streptavidin working solution (BDBioscience) to each well and incubate at room temperature (20-25℃) in the dark for 1 hour; remove the liquid, add 200 μL of ordinary washing buffer (1×PBS) to each well, let stand for 1 minute, and then shake off the liquid vigorously. Repeat this washing process 5 times. After the last time, shake off the liquid and pat dry on absorbent paper.

[0095] ⑧ Developing and terminating developing: Add 100 μL of AEC developing solution (prepared on the spot) to each well and let it stand at room temperature (20-25℃) in the dark for 25 minutes; shake off the liquid forcefully, remove the plate base, wash the front and back sides of the PVDF membrane and the base 5 times with deionized water to terminate developing, and then place the strip in a dark place at room temperature to air dry naturally before reinstalling the plate base.

[0096] 9. Spot Counting: Count the spots manually under a low-power microscope in an upright position. If there are many spots, the experimenter can also use an enzyme-linked immunospot analyzer to take pictures and count the spots.

[0097] ⑩ Result Interpretation: Subtract the number of spots in the negative control well from the number of spots in each experimental well to obtain the actual number of spots in each experimental well. Then, add the actual number of spots in the two experimental wells to obtain the total number of spots in the specimen (SFUs / 4×10⁻⁶). 5 PBMCs); when the number of spots in the experimental well is less than the number of spots in the negative control well, the actual number of spots in the experimental well is zero.

[0098] 6) Statistical analysis of examples and clinical significance of blood sample test results in lung cancer patients:

[0099] Below are the Cyfra21-1 specific T cell detection results and their ELISPOT spectra of three blood samples from lung cancer patients before and after anti-PD-1 antibody treatment at the First Affiliated Hospital of Bengbu Medical University.Figure 5 A. Pretreatment testing was also performed on 70 non-small cell lung cancer (NSCLC) patients scheduled for PD-1 / PD-L1 antibody therapy. The baseline number of Cyfra21-1 specific T cells in responder PBMCs was significantly higher than in non-responders (see A). Figure 5 Multivariate regression analysis showed that the baseline number of Cyfra21-1 specific T cells in PBMCs was an independent predictor of patient response to the antibody treatment. ROC analysis showed an area under the curve (AUC) of 0.757, based on 25.5 SFUs / 4×10⁻⁶ cells. 5 PBMCs are threshold values ​​used to distinguish between responders and non-responders. See Figure 5 C in the middle. See Figure 5 B. In the patient cohort with ≥ 25.5 SFUs before treatment, 83.4% of patients responded to antibody therapy, see [link to relevant documentation]. Figure 5 D in the middle.

[0100] Example 2

[0101] This method was used to screen and validate T-cell epitope peptides of CEA (carcinoma-associated gastrointestinal cancer) presented by 42 dominant HLA-A, B, and C molecules, construct a broad-spectrum T-cell epitope peptide library, and establish a universally applicable method and kit for CEA tumor antigen-specific T-cell detection in a wide range of gastrointestinal cancer patients in China. The main procedure is as described in Example 1. Figure 1 .

[0102] 1) Virtual prediction of candidate T-cell epitope peptides for colorectal cancer-associated antigen CEA presented by HLA-A, B, and C molecules:

[0103] ① The full-length amino acid sequence of CEA was obtained from databases such as UniProt / NCBI and COSMIC. Multiple sequence alignment was performed using the ClustalW method in Molecular Evolutionary Genetics Analysis (MEGA7, version 1.0.0.0). The alignment results were then analyzed using GeneDoc (version 2.7.0) software to identify conserved regions. The conservation of each amino acid was assessed using 100%, 95%, and 80% as cutoff points.

[0104] ② Thirteen HLA-A molecules, 15 HLA-B molecules, and 14 HLA-C molecules with gene frequencies greater than 1% in the Chinese population were identified. Their total allele frequencies covered over 90%, 70%, and 90% of the Chinese and East Asian populations, respectively. Epitope peptides presented by the aforementioned 42 dominant HLA-A, B, and C molecules were virtually predicted using the epitope prediction algorithms and tools listed in Table 1 of Example 1. Epitope peptide lengths were set to 9-mer and 10-mer, and they had to meet the evaluation criteria of at least three prediction algorithms. The top 5-10 peptides in terms of score were selected as candidate epitopes. Finally, 215 candidate epitopes were screened, and all 85 epitopes with different sequences were synthesized into candidate epitope peptides for subsequent validation experiments.

[0105] The amino acid sequence of colorectal cancer-associated tumor antigen CEA (carcinoembryonic antigen) is as follows:

[0106] Scientifically named genera and species: Phylum Vertebrata, Subclass Eutheria, Humans

[0107] CEA (Carcinoembryonic antigen) (ID: P06731)

[0108] MESPSAPPHRWCIPWQRLLLTASLLTFWNPPTTAKLTIESTPFNVAEGKEVLLLVHNLPQ 60

[0109] HLFGYSWYKGERVDGNRQIIGYVIGTQQATPGPAYSGREIIYPNASLLIQNIIQNDTGFY 120

[0110] TLHVIKSDLVNEEATGQFRVYPELPKPSISSNNSKPVEDKDAVAFTCEPETQDATYLWWV 180

[0111] NNQSLPVSPRLQLSNGNRTLTLFNVTRNDTASYKCETQNPVSARRSDSVILNVLYGPDAP 240

[0112] TISPLNTSYRSGENLNLSCHAASNPPAQYSWFVNGTFQQSTQELFIPNITVNNSGSYTCQ 300

[0113] AHNSDTGLNRTTVTTITVYAEPPKPFITSNNSNPVEDEDAVALTCEPEIQNTTYLWWVNN 360

[0114] QSLPVSPRLQLSNDNRTLTLLSVTRNDVGPYECGIQNELSVDHSDPVILNVLYGPDDPTI 420

[0115] SPSYTYYRPGVNLSLSCHAASNPPAQYSWLIDGNIQQHTQELFISNITEKNSGLYTCQAN 480

[0116] NSASGHSRTTVKTITVSAELPKPSISSNNSKPVEDKDAAVAFTCEPEAQNTTYLWWVNGQS 540

[0117] LPVSPRLQLSNGNRTLTLFNVTRNDARAYVCGIQNSVSANRSDPVTLDVLYGPDTPIISP 600

[0118] PDSSYLSGANLNLSCHSASNPSPQYSWRINGIPQQHTQVLFIAKITPNNNGTYACFVSNL 660

[0119] ATGRNNSIVKSITVSASGTSPGLSAGATVGIMIGVLVGVALI 702

[0120] 2) Using PBMCs from colorectal cancer patients, the immunogenicity of candidate epitope peptides was verified through a peptide-PBMC co-stimulation assay:

[0121] ① Peripheral blood was collected from patients with colorectal cancer from the Department of General Surgery and Gastrointestinal Surgery of the Affiliated Hospital of Xuzhou Medical University. PBMCs were routinely separated and kept warm for later use. A portion of cells were also taken and HLA-A, B, and C allele typing was performed according to the following steps.

[0122] 200 µL of anticoagulated blood was collected, and genomic DNA was extracted using a human whole blood genomic DNA extraction kit (Tiangen Biotech, Beijing). PCR was performed using HLA-A site-specific primers A1 and A3 (same as Table 2 in Example 1) to amplify the DNA sequences of exon 2, intron 2, exon 3, and some introns 1 and 3 at site A, with a product size of 985 bp. PCR was performed using HLA-B site-specific primers B1 and B3 (same as Table 2 in Example 1) to amplify the DNA sequences of exon 2, intron 2, exon 3, and some introns 1 and 3 at site B, with a product size of 985 bp. PCR was performed using HLA-C site-specific primers CF and CR (same as Table 2 in Example 1) to amplify the DNA sequences of exon 2, intron 2, exon 3, and some introns 1 and 3 at site C, with a product size of 985 bp. The amplification conditions were: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s; 62℃ annealing for 15 s; 72℃ extension for 90 s; 35 cycles; 72℃ extension for 5 min. The amplified products were identified by 1% agarose gel electrophoresis and sent to Shanghai Sunny Biotechnology Co., Ltd. for purification and bidirectional sequencing. PCR reagents were purchased from Nanjing Novizan Biotechnology Co., Ltd.

[0123] ② Immunogenicity verification of single peptides:

[0124] Based on the identification results of the patient's HLA-A, B, and C alleles, candidate epitope peptides corresponding to specific HLA molecules of the patient were selected and co-cultured with the patient's PBMCs for 6 hours. Intracellular IFN-γ fluorescence staining was then performed to detect CD8+ secreting IFN-γ. + T cells and CD4 + T cell frequency compared to the peptide-free culture group. This resulted in IFN-γ... + / CD8 + Epitopes with significantly elevated T cell frequency (more than twice that of the group without peptide culture) are immunogenic and are positive epitopes, meaning that the patient's PBMCs contain memory CD8 specific to that epitope peptide. + T cells. Each candidate epitope peptide was tested against PBMCs from at least 5 patients with relatively HLA genes, and epitope peptides with strong immunogenicity and positive reactions in a majority of patients (3 / 5) were selected for inclusion in the dominant epitope peptide library. Results: 59 positive epitope peptides were obtained using blood samples from 230 colorectal cancer patients. Figure 6 A statistical chart showing the results of screening positive epitope peptides for CEA antigen in blood samples from colorectal cancer patients. Figure 7 Flow cytometry analysis of positive epitope peptides for CEA antigen in blood samples from a subset of patients.

[0125] 3) Using an array of HMy2.CIR cell lines expressing 12 specific HLA-A molecules, 15 specific HLA-B molecules, and 14 specific HLA-C molecules, competitive binding experiments were conducted between HLA molecules and peptides to verify the cross-binding affinity of positive epitope peptides with various related HLA molecules:

[0126] ①HMy2.CIR is a human B lymphoblast cell line lacking HLA class I antigens. It does not express HLA-A and B molecules, but only a trace amount of HLA-Cw4 molecules. Our laboratory constructed an array of HMy2.CIR cell lines expressing 12 specific HLA-A molecules, 15 specific HLA-B molecules, and 14 specific HLA-C molecules. The main steps were: extracting mRNA from healthy PBMCs, amplifying each HLA-A and cDNA, constructing the pcDNATM3.1 / myc-His(-)A recombinant plasmid using standard methods, electroporating HMy2.CIR cell lines, selecting stable cell lines using G418, staining with the HLA-ABC fluorescent monoclonal antibody W6 / 32, and then sorting CIR cell lines highly expressing the HLA molecule using flow cytometry for pure culture, followed by sequencing identification.

[0127] ② Positive epitope peptides validated by the peptide-PBMC co-stimulation experiment were classified. Based on the predicted HLA molecule restriction and the actual HLA-A, B, and C alleles of blood donors in the validation experiment, a list of positive epitope peptides that could bind to each HLA molecule was compiled. HMy2.CIR cell lines stably expressing a specific HLA-A molecule were co-incubated with each positive epitope peptide associated with that HLA molecule and a fluorescein-labeled reference peptide to perform a peptide competition binding experiment: First, the HLA-bound peptides on the CIR cell membrane were eluted with an acid solution (0.131 mol / L citric acid and 0.061 mol / L disodium hydrogen phosphate, pH 3.0), then neutralized with cell culture medium, centrifuged and washed, and seeded into 96-well cell culture plates (1×10⁻⁶). 5 Cells / well / 100μL) were cultured, β2-microglobulin (1μg / mL) was added, followed by a fluorescein-labeled reference peptide (200nM), and then unlabeled positive epitope peptides (5μM, 15μM) were added respectively. After 24h of incubation, flow cytometry analysis was performed, and the results were analyzed by IC50. 50 The affinity of each positive epitope peptide for a specific HLA molecule was determined by the value and competitive inhibition percentage. The results clarified the binding affinity of each positive epitope peptide for multiple related HLA-A, -B, or -C molecules. Table 5 summarizes the affinity ranking of each epitope peptide for its associated HLA molecules; Figure 8 This is a flow cytometry chromatogram of a peptide competitive binding assay for some CEA antigen epitope peptides.

[0128] Table 5. Peptide competitive binding assays using 41 HMy2.CIR cell lines to detect the binding affinity of each HLA-A, -B, and -C molecule to the corresponding CEA antigen T cell epitope peptide.

[0129]

[0130]

[0131] 4) Construct a broad-spectrum T-cell epitope peptide library of colorectal cancer-associated antigen CEA, and detect CEA-specific T cells in a wide range of PBMCs from colorectal cancer patients:

[0132] Through the above-mentioned single peptide immunogenicity verification and HLA molecule-peptide competitive binding experiments, 34 dominant epitope peptides cross-linked with 13 dominant HLA-A molecules, 28 dominant epitope peptides cross-linked with 15 dominant HLA-B molecules, and 23 dominant epitope peptides cross-linked with 14 dominant HLA-C molecules were screened. These peptides can specifically target 101, 84, and 72 peptide / HLA complex CD8 molecules, respectively. + T-cell clones were used. These 59 epitope peptides with different sequences were used to form a broad-spectrum T-cell epitope peptide library for CEA. Based on antigen origin and pH, these 59 epitope peptides were further divided into three mixed peptide libraries (20, 20, and 19 peptides each) to establish an IFN-γ ELISpot detection method. Theoretically, these 59 T-cell epitopes can cross-detect approximately 257 peptide / HLA complex-specific CD8+. + T cell clones. The number of CEA-dominant T cell epitope peptides that cross-bind with each dominant HLA-A, B, and C molecule is shown in Table 6.

[0133] Table 6. Number of CEA-dominant T cell epitope peptides cross-linked with dominant HLA-A, B, and C molecules.

[0134]

[0135] 5) Assemble the ELISPOT kit and perform universal detection of colorectal cancer antigen CEA-specific T cells:

[0136] ①PBMC isolation: Peripheral anticoagulated blood samples were collected from the patients to be tested. PBMCs were isolated using the same routine method as above, counted, and the cell concentration was adjusted to 4 × 10⁻⁶ cells using serum-free cell culture medium (Dakeway Biotechnology, Shenzhen). 6 / ml.

[0137] ② Add cell suspension: Remove the ELISPOT strips pre-coated with human IFN-γ capture antibody, preheat at room temperature for 30 minutes, and add 4 × 10⁻⁶ PBMCs from each patient. 6Add 100 μL / well to 5 wells, with 100 μL added to wells 1 through 4 (i.e., 4 × 10⁻⁶). 5 50 μL / well (i.e., 2 × 10⁶ cells / well) was added to well 5 (positive control well). 5 (cells).

[0138] ③ Add antigen peptides: Each blood sample is tested in 5 wells. Add antigen peptide library 1, antigen peptide library 2, and peptide library 3 to wells 1 to 3, respectively, at 33 μL / well, for 3 experimental wells; add 33 μL of negative control replenishment to well 4, which is a negative control well; well 5 is a positive control well, with 5 μL of PHA working solution added, and 78 μL of serum-free cell culture medium added. The final volume of each well is 133 μL.

[0139] ④ Culture and incubation: Cover the plate and place it in a 37℃, 5% CO2 cell culture incubator for 20-22 hours.

[0140] ⑤ Cell lysis and plate washing: Remove the ELISPOT strips, shake off the cell suspension, add 200 μL of deionized water to each well, incubate at 4°C for 10 minutes, shake off the deionized water, and pat dry on absorbent paper; add 200 μL of ordinary washing buffer (1×PBS) to each well, let stand for 1 minute, shake off the liquid, and repeat this washing process 5 times. After the last shake, pat dry on absorbent paper.

[0141] ⑥ Add detection antibody and wash plate: Add 100 μL of biotin-IFN-γ detection antibody working solution (BDBioscience) to each well and incubate at room temperature in the dark (20-25℃) for 2 hours; shake off the liquid, add 200 μL of ordinary washing buffer (1×PBS) to each well, let stand for 1 minute, shake off the liquid vigorously, repeat this washing process 5 times, and after the last shake off the liquid, pat dry on absorbent paper.

[0142] ⑦ Add HRP-streptavidin and wash the plate: Add 100 μL of HRP-streptavidin working solution (BDBioscience) to each well and incubate at room temperature (20-25℃) in the dark for 1 hour; remove the liquid, add 200 μL of ordinary washing buffer (1×PBS) to each well, let stand for 1 minute, and then shake off the liquid vigorously. Repeat this washing process 5 times. After the last time, shake off the liquid and pat dry on absorbent paper.

[0143] ⑧ Developing and terminating developing: Add 100 μL of AEC developing solution (prepared on the spot) to each well and let it stand at room temperature (20-25℃) in the dark for 25 minutes; shake off the liquid forcefully, remove the plate base, wash the front and back sides of the PVDF membrane and the base 5 times with deionized water to terminate developing, and then place the strip in a dark place at room temperature to air dry naturally before reinstalling the plate base.

[0144] 9. Spot Counting: Count the spots manually under a low-power microscope in an upright position. If there are many spots, the experimenter can also use an enzyme-linked immunospot analyzer to take pictures and count the spots.

[0145] ⑩ Result Interpretation: Subtract the number of spots in the negative control well from the number of spots in each experimental well to obtain the actual number of spots in each experimental well. Then, add the actual number of spots in the three experimental wells to obtain the total number of spots in the specimen (SFUs / 4×10⁻⁶). 5 PBMCs); when the number of spots in the experimental well is less than the number of spots in the negative control well, the actual number of spots in the experimental well is zero.

[0146] 6) Statistical analysis of examples and clinical significance of blood sample test results in colorectal cancer patients:

[0147] The following describes the reproducibility of CEA-specific T-cell detection using blood samples from patients in the Department of General Surgery and Gastrointestinal Surgery at the First Affiliated Hospital of Xuzhou Medical University. The intra-assay and inter-assay coefficients of variation (CV) were 6.43% and 6.61%, respectively. Figure 9 This is an ELISPOT dot pattern of four blood samples for repeatable CEA-specific T cell testing.

[0148] In addition, CEA-specific T-cell counts were performed on 64 colorectal cancer patients scheduled for surgical resection and 68 colorectal cancer patients scheduled for anti-PD-1 antibody therapy. Results showed that in the surgical cohort, patients with disease-free survival (DFS) > 12 months (n=45) had significantly higher CEA-specific T-cell counts (spot-forming units, SFUs) after treatment than patients with DFS ≤ 12 months (n=19), with median counts of 92 SFUs and 26 SFUs, respectively (p=0.003). Figure 10 (E in the text). Receiver operating characteristic (ROC) curve analysis showed that the AUC for differentiating DFS > 12 months from ≤ 12 months was 0.837; with > 86.5 SFUs / 4 × 10 5 PBMCs are Cut-off values ​​( Figure 10 (F in the text). ≥86.5 SFUs / 4×10 after treatment. 5 In the PBMCs group, the 12-month disease-free survival rate was significantly higher than that of patients with a count <86.5, with 82.7% of patients having a DFS >12 months. Figure 10 In the anti-PD-1 antibody treatment cohort, patients with progression-free survival (PFS) > 6 months (n=29) had significantly higher CEA-specific T cell counts after treatment than patients with PFS ≤ 6 months (n=39), with median values ​​of 123 SFUs and 36 SFUs, respectively (P=0.002, G). Figure 10(E in the text). Similarly, ROC analysis showed an AUC value of 0.865; at 116.5 SFUs / 4×10⁻⁶. 5 PBMCs are cut-off values ​​that can predict PFS > 6 months and ≤ 6 months in patients ( Figure 10 In the group of patients with ≥116.5 SFUs after treatment, 82.4% of patients had a PFS > 6 months (F in the original text). Figure 10 (H in the text).

[0149] Example 3:

[0150] This method was used to prepare a broad-spectrum T-cell epitope peptide library of esophageal cancer-associated tumor antigen (SCC-Ag) presented by HLA-A, B, and C molecules, and to establish a universally applicable method and kit for the detection of SCC-Ag tumor antigen-specific T cells in a wide range of esophageal cancers in China. The main procedure is as described in Example 1. Figure 1 .

[0151] 1) Virtual prediction of candidate T-cell epitope peptides for esophageal cancer-associated antigen SCC-Ag presented by HLA-A, B, and C molecules:

[0152] ① The full-length amino acid sequence of SCC-Ag was obtained from databases such as UniProt / NCBI and COSMIC. Multiple sequence alignment was performed using the ClustalW method in Molecular Evolutionary Genetics Analysis (MEGA7, version 1.0.0.0). The alignment results were then analyzed using GeneDoc (version 2.7.0) to determine the conserved regions of the sequence. The conservation properties of each amino acid were assessed using 100%, 95%, and 80% as cutoff points.

[0153] ② Thirteen HLA-A molecules, 15 HLA-B molecules, and 14 HLA-C molecules with gene frequencies greater than 1% in the Chinese population were identified. Their total allele frequencies covered over 90%, 70%, and 90% of the Chinese and East Asian populations, respectively. Epitope peptides presented by the aforementioned 42 dominant HLA-A, B, and C molecules were virtually predicted using the epitope prediction algorithms and tools listed in Table 1 of Example 1. Epitope peptide lengths were set to 9-mer and 10-mer, and they had to meet the evaluation criteria of at least three prediction algorithms. The top 5-10 peptides in terms of score were selected as candidate epitope peptides.

[0154] The amino acid sequence of esophageal cancer-associated tumor antigen SCC-Ag is as follows:

[0155] Scientifically named genera and species: Phylum Vertebrata, Subclass Eutheria, Humans

[0156] SCC-Ag (Squamous cell carcinoma antigen) (ID: P29508)

[0157] MNSLSEANTKFMFDLFQQFRKSKENNIFYSPISITSALGMVLLGAKDNTAQQIKKVLHFD 60

[0158] QVTENTTGKAATYHVDRSGNVHHQFQKLLTEFNKSTDAYELKIANKLFGEKTYLFLQEYL 120

[0159] DAIKKFYQTSVESVDFANAPEESRKKINSWVESQTNEKIKNLIPEGNIGSNTTLVLVNAI 180

[0160] YFKGQWEKKFNKEDTKEEKFWPNKNTYKSIQMMRQYTSFHFASLEDVQAKVLEIPYKGKD 240

[0161] LSMIVLLPNEIDGLQKLEEKLTAEKLMEWTSLQNMRETRVDLHLPRFKVEESYDLKDTLR 300

[0162] TMGMVDIFNGDADLSGMTGSRGLVLSGVLHKAFVEVTEEGAEAAAATAVVGFGSSPTSTN 360

[0163] EEFHCNHPFLFFIRQNKTNSILFYGRFSSP 390

[0164] 2) Using PBMCs from esophageal cancer patients, the immunogenicity of candidate epitope peptides was verified through a peptide-PBMC co-stimulation assay:

[0165] ① Peripheral blood was collected from esophageal cancer patients from the Department of Gastroenterology, Affiliated Hospital of Xuzhou Medical University. PBMCs were routinely separated and kept warm for later use. A portion of cells were also taken and HLA-A, B, and C allele typing was performed according to the following steps.

[0166] 200 µL of anticoagulated blood was collected, and genomic DNA was extracted using a human whole blood genomic DNA extraction kit (Tiangen Biotech, Beijing). PCR was performed using HLA-A site-specific primers A1 and A3 (same as Table 2 in Example 1) to amplify the DNA sequences of exon 2, intron 2, exon 3, and some introns 1 and 3 at site A, with a product size of 985 bp. PCR was performed using HLA-B site-specific primers B1 and B3 (same as Table 2 in Example 1) to amplify the DNA sequences of exon 2, intron 2, exon 3, and some introns 1 and 3 at site B, with a product size of 985 bp. PCR was performed using HLA-C site-specific primers CF and CR (same as Table 2 in Example 1) to amplify the DNA sequences of exon 2, intron 2, exon 3, and some introns 1 and 3 at site C, with a product size of 985 bp. The amplification conditions were: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s; 62℃ annealing for 15 s; 72℃ extension for 90 s; 35 cycles; 72℃ extension for 5 min. The amplified products were identified by 1% agarose gel electrophoresis and sent to Shanghai Sunny Biotechnology Co., Ltd. for purification and bidirectional sequencing. PCR reagents were purchased from Nanjing Novizan Biotechnology Co., Ltd.

[0167] ② Immunogenicity verification of single peptides:

[0168] Based on the identification results of the patient's HLA-A, B, and C alleles, candidate epitope peptides corresponding to specific HLA molecules of the patient were selected and co-cultured with the patient's PBMCs for 6 hours. Intracellular IFN-γ fluorescence staining was then performed to detect CD8+ secreting IFN-γ. + T cells and CD4 + T cell frequency compared to the peptide-free culture group. This resulted in IFN-γ... + / CD8 + Epitopes with significantly elevated T cell frequency (more than twice that of the group without peptide culture) are immunogenic and are positive epitopes, meaning that the patient's PBMCs contain memory CD8 specific to that epitope peptide. + T cells. Each candidate epitope peptide was tested against at least 5 PBMCs from patients with relatively HLA genes, and epitope peptides with strong immunogenicity and positive reactions in a majority of patients (3 / 5) were selected for inclusion in the dominant epitope peptide library. Results validation yielded 51 positive epitope peptides with distinct sequences.

[0169] 3) Using an array of HMy2.CIR cell lines expressing 12 specific HLA-A molecules, 15 specific HLA-B molecules, and 14 specific HLA-C molecules, competitive binding experiments were conducted between HLA molecules and peptides to verify the cross-binding affinity of positive epitope peptides with various related HLA molecules:

[0170] ①HMy2.CIR is a human B lymphoblast cell line lacking HLA class I antigens. It does not express HLA-A and B molecules, but only a trace amount of HLA-Cw4 molecules. Our laboratory constructed an array of HMy2.CIR cell lines expressing 12 specific HLA-A molecules, 15 specific HLA-B molecules, and 14 specific HLA-C molecules. The main steps were: extracting mRNA from healthy PBMCs, amplifying each HLA-A and cDNA, constructing the pcDNATM3.1 / myc-His(-)A recombinant plasmid using standard methods, electroporating HMy2.CIR cell lines, selecting stable cell lines using G418, staining with the HLA-ABC fluorescent monoclonal antibody W6 / 32, and then sorting CIR cell lines highly expressing the HLA molecule using flow cytometry for pure culture, followed by sequencing identification.

[0171] ② Positive epitope peptides validated by the peptide-PBMC co-stimulation experiment were classified. Based on the predicted HLA molecule restriction and the actual HLA-A, B, and C alleles of blood donors in the validation experiment, a list of positive epitope peptides that could bind to each HLA molecule was compiled. HMy2.CIR cell lines stably expressing a specific HLA-A molecule were co-incubated with each positive epitope peptide associated with that HLA molecule and a fluorescein-labeled reference peptide to perform a peptide competition binding experiment: First, the HLA-bound peptides on the CIR cell membrane were eluted with an acid solution (0.131 mol / L citric acid and 0.061 mol / L disodium hydrogen phosphate, pH 3.0), then neutralized with cell culture medium, centrifuged and washed, and seeded into 96-well cell culture plates (1×10⁻⁶). 5 Cells / well / 100μL) were cultured, β2-microglobulin (1μg / mL) was added, followed by a fluorescein-labeled reference peptide (200nM), and then unlabeled positive epitope peptides (5μM, 15μM) were added respectively. After 24h of incubation, flow cytometry analysis was performed, and the results were analyzed by IC50. 50 The affinity of each positive epitope peptide for a specific HLA molecule was determined by the value and the percentage of competitive inhibition.

[0172] 4) Construct a broad-spectrum T-cell epitope peptide library of esophageal cancer-associated antigen SCC-Ag, and detect SCC-Ag-specific T cells in a wide range of PBMCs from esophageal cancer patients:

[0173] Through the above-mentioned single peptide immunogenicity verification and HLA molecule-peptide competitive binding experiments, 31 dominant epitope peptides cross-linked with 13 dominant HLA-A molecules, 25 dominant epitope peptides cross-linked with 15 dominant HLA-B molecules, and 16 dominant epitope peptides cross-linked with 14 dominant HLA-C molecules were screened. A total of 51 epitope peptides with different sequences were identified, forming the SCC-Ag broad-spectrum T cell epitope peptide library. Based on antigen source and pH, the 51 epitope peptides were divided into three mixed peptide libraries (16, 17, and 18 types), theoretically allowing for cross-detection of 388 CEA-specific T cell clones. The number of SCC-Ag dominant T cell epitope peptides cross-linked with each dominant HLA-A, B, and C molecule is shown in Table 7. Figure 11 This is a flow cytometry chromatogram of a peptide competitive binding assay for SCC-Ag antigenic epitope peptides. For each HLA molecule, some candidate epitope peptides can compete with the fluorescently labeled reference peptide for binding to HLA molecules on the cell line, thus weakening cell fluorescence. Other candidate epitope peptides are less competitive and therefore cannot change the fluorescence intensity of the cell line.

[0174] Table 7. Number of SCC-Ag dominant T cell epitope peptides cross-linked with dominant HLA-A, B, and C molecules.

[0175]

[0176] 5) Assemble the ELISPOT kit for universal detection of esophageal cancer antigen SCC-Ag-specific T cells:

[0177] ①PBMC isolation: Peripheral anticoagulated blood samples were collected from the patients to be tested. PBMCs were isolated using the same routine method as above, counted, and the cell concentration was adjusted to 4 × 10⁻⁶ cells using serum-free cell culture medium (Dakeway Biotechnology, Shenzhen). 6 / ml.

[0178] ② Add cell suspension: Remove the ELISPOT strips pre-coated with human IFN-γ capture antibody, preheat at room temperature for 30 minutes, and add 4 × 10⁻⁶ PBMCs from each patient. 6 Add 100 μL / well to 5 wells, with 100 μL added to wells 1 through 4 (i.e., 4 × 10⁻⁶). 5 50 μL / well (i.e., 2 × 10⁶ cells / well) was added to well 5 (positive control well). 5 (cells).

[0179] ③ Add antigen peptides: Each blood sample is tested in 5 wells. Add antigen peptide library 1, antigen peptide library 2, and peptide library 3 to wells 1 to 3, respectively, at 33 μL / well, for 3 experimental wells; add 33 μL of negative control replenishment to well 4, which is a negative control well; well 5 is a positive control well, with 5 μL of PHA working solution added, and 78 μL of serum-free cell culture medium added. The final volume of each well is 133 μL.

[0180] ④ Culture and incubation: Cover the plate and place it in a 37℃, 5% CO2 cell culture incubator for 20-22 hours.

[0181] ⑤ Cell lysis and plate washing: Remove the ELISPOT strips, shake off the cell suspension, add 200 μL of deionized water to each well, incubate at 4°C for 10 minutes, shake off the deionized water, and pat dry on absorbent paper; add 200 μL of ordinary washing buffer (1×PBS) to each well, let stand for 1 minute, shake off the liquid, and repeat this washing process 5 times. After the last shake, pat dry on absorbent paper.

[0182] ⑥ Add detection antibody and wash plate: Add 100 μL of biotin-IFN-γ detection antibody working solution (BDBioscience) to each well and incubate at room temperature in the dark (20-25℃) for 2 hours; shake off the liquid, add 200 μL of ordinary washing buffer (1×PBS) to each well, let stand for 1 minute, shake off the liquid vigorously, repeat this washing process 5 times, and after the last shake off the liquid, pat dry on absorbent paper.

[0183] ⑦ Add HRP-streptavidin and wash the plate: Add 100 μL of HRP-streptavidin working solution (BDBioscience) to each well and incubate at room temperature (20-25℃) in the dark for 1 hour; remove the liquid, add 200 μL of ordinary washing buffer (1×PBS) to each well, let stand for 1 minute, and then shake off the liquid vigorously. Repeat this washing process 5 times. After the last time, shake off the liquid and pat dry on absorbent paper.

[0184] ⑧ Developing and terminating developing: Add 100 μL of AEC developing solution (prepared on the spot) to each well and let it stand at room temperature (20-25℃) in the dark for 25 minutes; shake off the liquid forcefully, remove the plate base, wash the front and back sides of the PVDF membrane and the base 5 times with deionized water to terminate developing, and then place the strip in a dark place at room temperature to air dry naturally before reinstalling the plate base.

[0185] 9. Spot Counting: Count the spots manually under a low-power microscope in an upright position. If there are many spots, the experimenter can also use an enzyme-linked immunospot analyzer to take pictures and count the spots.

[0186] ⑩ Result Interpretation: Subtract the number of spots in the negative control well from the number of spots in each experimental well to obtain the actual number of spots in each experimental well. Then add the actual number of spots in the two experimental wells to obtain the number of spots in the specimen. When the number of spots in an experimental well is less than the number of spots in the negative control well, the actual number of spots in that experimental well is zero.

[0187] 6) Example of blood sample test results from esophageal cancer patients:

[0188] Below is an example of SCC-Ag-specific T cell detection using three blood samples from patients in the Department of Gastroenterology, First Affiliated Hospital of Xuzhou Medical University. The ELISPOT dot plot shows how each epitope peptide library stimulates specific T cell activation in the patient's PBMCs to secrete IFN-γ, forming a dot. Each dot represents a T cell specific to one epitope peptide. NC represents the negative control well, and PHA represents the positive control well. Figure 12 Further testing is underway on more patients receiving immunotherapy, and the clinical value of SCC-Ag antigen-specific T cell counts requires further investigation.

[0189] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0190] It should be noted that the above content merely illustrates the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. For those skilled in the art, various improvements and modifications can be made without departing from the principle of the present invention, and all such improvements and modifications fall within the scope of protection of the claims of the present invention.

Claims

1. A non-diagnostic, tumor antigen-specific thymus-dependent lymphocyte detection protocol for lung or gastrointestinal cancers applicable to a broad population in East Asia, comprising the following steps: (1) Screening and verification of a series of antigenic peptides against specific tumor antigens presented by HLA-A, B, and C molecules with an allele frequency greater than 1% in East Asian populations: Using multiple online epitope prediction databases, candidate epitope peptides against tumor antigens presented by HLA-A, HLA-B, and HLA-C molecules with an allele frequency greater than 1% were virtually predicted; then, lymphocyte functional experiments were performed using peripheral blood mononuclear cells from cancer patients to verify the immunogenicity of candidate epitope peptides in real tumor patients, and positive epitope peptides presented by each dominant HLA molecule were screened; then, HLA-A, B, or C molecules were used on HLA-stable transfected cell line arrays to perform peptide competitive binding experiments on each immunogenic positive epitope peptide to clarify the HLA cross-restriction and population coverage of each positive epitope peptide; (2) Construct a broad-spectrum T-cell epitope peptide library: Based on the length of the amino acid sequence of each tumor antigen, 30-100 epitope peptides are screened from the above positive epitope peptide library to form a broad-spectrum T-cell epitope peptide library; these broad-spectrum T-cell epitope peptides are epitope peptides that are respectively presented by the above HLA molecules. (3) The broad-spectrum T cell epitope peptide library of the above-mentioned specific antigens is packaged into a peptide pool array and co-cultured with patient PBMCs in a microwell reaction plate to establish an enzyme-linked immunospot method or a fluorescent immunospot method to quantitatively detect the number of cells secreting IFN-γ, TNF-α or IL-2. The feature is that, in step (1), the tumor antigens include: lung cancer-related tumor antigens Cyfra21-1, CA125 and NSE, gastrointestinal cancer tumor antigens CEA, CA724 and CA242, and esophageal cancer-related tumor antigens SCC-Ag and CA199. The HLA-A molecules include: HLA-A1101, A2402, A0201, A3101, A0206, A0207, A3303, A3001, A0203, A1102, A0301, A0101, A2601; HLA-B molecules include: HLA-B4001, B4601, B5801, B5101, B1302, B1501, B1301, B4006, B1502, B3501, B5401, B5201, B4403, B4801, and B0702; HLA-C molecules include: HLA-C0702, C0102, C0304, C0801, C0602, C0303, C0302, C0401, C1402, C1502, C0701, C1202, C1403, and C1203.

2. The tumor antigen-specific thymus-dependent lymphocyte detection protocol for lung or digestive tract cancers applicable to a broad population in East Asia for non-diagnostic purposes, as described in claim 1, is characterized in that... In step (3), the enzyme-linked immunosorbent assay (ELISA) or fluorescent immunospot assay is replaced by magnetic microsphere chemiluminescence assay.

3. The tumor antigen-specific thymus-dependent lymphocyte detection protocol for lung or digestive tract cancers applicable to a broad population in East Asia for non-diagnostic purposes, as described in claim 1, is characterized in that... In step (3), the enzyme-linked immunosorbent assay (ELISA) or fluorescent immunospot assay is replaced by the enzyme-linked immunosorbent assay (ELISA).

4. The tumor antigen-specific thymus-dependent lymphocyte detection protocol for lung or digestive tract cancers applicable to a broad population in East Asia for non-diagnostic purposes, as described in claim 1, is characterized in that... In step (3), the enzyme-linked immunospot assay is replaced by a combination of cytometric beads array and T cell multicolor fluorescence staining to analyze the functional subtypes of antigen-specific T cells.

5. A non-diagnostic thymus-dependent lymphocyte detection protocol for lung or digestive tract cancers applicable to a broad population in East Asia, as described in claims 1-4, characterized in that... The soluble molecules detected by the patient's T cells are any one or more of human interferon-γ, human interleukin-17, granzyme A, granzyme B, perforin, tumor necrosis factor-α, and tumor necrosis factor-β.

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