Thymus-dependent lymphocyte antigen epitope peptide of lung cancer-related antigen Cyfra21-1 and its application

Abstract

The present invention discloses a thymus-dependent lymphocyte antigen epitope peptide of lung cancer-associated antigen Cyfra21-1 and its application, belonging to the fields of medical immunology and oncology. A thymus-dependent lymphocyte antigen epitope peptide of lung cancer-associated antigen Cyfra21-1, the amino acid sequence of the epitope peptide includes any one or more of the following: RQSSATSSF; VRFGPGVAF; VSVSSARFV; the amino acid sequence after the removal or replacement of a single amino acid of the above amino acid sequence. The epitope peptide of the present application can specifically bind to cytotoxic thymus-dependent lymphocytes, stimulate the latter to activate, proliferate and differentiate, thereby exerting an anti-tumor immune effector effect; these antigen peptides can be used to prepare therapeutic and preventive vaccines for lung cancer, and can also be used to prepare a detection reagent for quantitatively detecting specific T cells of lung cancer-associated antigen Cyfra21-1, which has potential application value in the prevention, treatment and diagnosis of lung cancer.

Description

Technical Field

[0001] The present invention relates to the fields of medical immunology and oncology, and in particular to a thymus-dependent lymphocyte antigen epitope peptide of lung cancer-related antigen Cyfra21-1 and application thereof. Background Art

[0002] The high incidence and mortality of lung cancer seriously threaten the lives and health of the Chinese people. Non-small cell lung cancer (NSCLC) is the most common subtype, accounting for about 85% of newly diagnosed lung cancer cases. Most NSCLC patients are diagnosed at an advanced stage and have a poor prognosis, with a 5-year survival rate of <5%.

[0003] In my country, the high-risk groups for NSCLC mainly include long-term smokers, those exposed to secondhand smoke, environmental pollution, occupational carcinogens (such as asbestos and radon gas), and those with a family history of lung cancer. In addition, chronic lung disease, tuberculosis, and certain genetic susceptibility may also increase the risk of disease. Non-small cell lung cancer is usually divided into three main subtypes: adenocarcinoma, squamous cell carcinoma, and large cell carcinoma. Adenocarcinoma is more common in women, while squamous cell carcinoma is more common in male smokers. Clinical symptoms include persistent cough, chest pain, shortness of breath, and weight loss.

[0004] Keratin 19 fragment (Cyfra21-1) is a fragment of keratin 19. Under normal circumstances, it exists in the form of oligomers and its content in serum is extremely low. When cells become cancerous, the content of keratin 19 in the cells increases. After the cancer cells necrotize and fall off, they are released into the blood and body fluids in the form of dissolved fragments. Cyfra21-1 is one of the most valuable tumor-associated antigens of NSCLC and is also a commonly used tumor marker for clinical diagnosis of NSCLC and monitoring and evaluation of prognosis and recurrence. This patent selects lung cancer-associated tumor antigen Cyfra21-1 for research.

[0005] Cytotoxic T lymphocytes (CTLs) are core cells that mediate adaptive immune responses and play a vital role in anti-infection, anti-tumor, hypersensitivity reactions and autoimmune diseases. The T cell receptors (TCRs) on their cell membranes can specifically recognize and bind to the complex of MHC class I molecules and antigenic peptides on the surface of antigen-presenting cells, i.e., MHC / antigen peptide complex molecules. CTL epitopes refer to antigenic peptides bound to MHC class I molecules. They are linear fragments or spatial conformational structures in antigen molecules that can be specifically recognized by TCRs. They are the basic antigenic units that cause immune responses and play a key role in the CTL activation process.

[0006] The MHC system refers to the major histocompatibility complex (MHC), which is a group of tightly linked genes in the vertebrate genome that encode and express MHC class I and class II protein molecules. HLA (human leukocyte antigen) is the human MHC system, responsible for presenting antigenic peptides (T cell epitope peptides) to T cells and initiating the body's specific immune response. Among them, HLA-A, B, C and other class I molecules present antigenic peptides to activate CD8 + T cells, which differentiate into CTLs, are the main effector cells that target and kill tumor cells. However, HLA alleles are highly polymorphic in the population. HLA molecules are not exactly the same between different individuals, and the antigen peptide sequences that each HLA molecule can present are also very different, causing different strengths of T cell immune responses. Therefore, the same tumor antigen can cause different strengths of specific immune responses in different individuals. For human lung cancer, HLA class I molecules are mainly responsible for presenting endogenous lung cancer-related antigens to CD8 + CTL, activated CTLs secrete perforin and granzyme to cause apoptosis of tumor cells expressing tumor-associated antigens. + The number and function of T cells can accurately reflect the specific immune function status of lung cancer patients against lung cancer-related antigens. Due to the different HLA molecular types of different people, their processing, handling and presentation capabilities for different lung cancer antigens are also different, thus causing lung cancer-related antigen-specific T cell immune responses of different intensities. According to the different HLA molecular types of lung cancer patients, the antigen peptides of lung cancer-related antigens presented by them are selected to dynamically monitor the CD8 specificity of the lung cancer-related antigen. + The number and reactivity of T cells are of great significance for monitoring the disease progression, formulating diagnosis and treatment plans, observing efficacy, and judging the prognosis of lung cancer patients, and are important technical means to achieve precision medical treatment of lung cancer. At the same time, peptide vaccines or gene vaccines can be prepared by using the antigenic peptides of lung cancer-related antigens that these HLA molecules bind with high affinity to prevent and treat lung cancer.

[0007] However, there are still very few Cyfra21-1-specific T cell epitopes that have been identified and presented by various HLA molecules and can stimulate the body to induce T cell responses. This limits the detection of Cyfra21-1-specific T cells in lung cancer patients carrying different HLA alleles, the role of lung cancer Cyfra21-1-specific T cells in the occurrence and development of lung cancer, and the personalized detection and precision immunotherapy based on individual differences in HLA genes and the differences in the lung cancer Cyfra21-1 antigen peptides they present. Summary of the invention

[0008] In view of the deficiencies of the prior art, the present invention proposes a thymus-dependent lymphocyte antigen epitope peptide of lung cancer-related antigen Cyfra21-1 and its application.

[0009] The purpose of the present invention can be achieved through the following technical solutions:

[0010] The first aspect of the present invention relates to a thymus-dependent lymphocyte antigen epitope peptide of the lung cancer-associated antigen Cyfra21-1, wherein the amino acid sequence of the epitope peptide comprises any one or more of the following:

[0011] RQSSATSSF;

[0012] VRFGPGVAF;

[0013] VSVSSARFV;

[0014] The amino acid sequence above is an amino acid sequence after a single amino acid is removed or replaced.

[0015] The second aspect of the present invention relates to the use of the thymus-dependent lymphocyte antigen epitope peptide of the lung cancer-associated antigen Cyfra21-1 in the preparation of lung cancer vaccines.

[0016] The third aspect of the present invention relates to a nucleic acid molecule encoding the thymus-dependent lymphocyte antigen epitope peptide of the lung cancer-associated antigen Cyfra21-1.

[0017] The fourth aspect of the present invention relates to a vector containing the above-mentioned nucleic acid molecule.

[0018] The fifth aspect of the present invention relates to a vaccine for treating or preventing lung cancer, comprising the above-mentioned nucleic acid molecule or the above-mentioned vector.

[0019] The sixth aspect of the present invention relates to the use of the thymus-dependent lymphocyte antigen epitope peptide of the lung cancer-associated antigen Cyfra21-1 in the preparation of a drug for treating or preventing lung cancer.

[0020] The seventh aspect of the present invention relates to the use of the above-mentioned thymus-dependent lymphocyte antigen epitope peptide of lung cancer-associated antigen Cyfra21-1 in a kit for preparing lung cancer-associated antigen Cyfra21-1 T cells.

[0021] Optionally, the kit includes: the detection reagent is an enzyme-linked immunosorbent assay reagent, an intracellular cytokine fluorescent staining reagent, an enzyme-linked immunosorbent assay reagent, a human leukocyte antigen polymer fluorescent staining or a flow cytometry reagent.

[0022] The eighth aspect of the present invention relates to a monoclonal antibody capable of specifically binding to the above-mentioned epitope peptide.

[0023] The ninth aspect of the present invention relates to a drug for treating lung cancer, comprising the above-mentioned monoclonal antibody.

[0024] More specifically, the present invention provides a novel thymus-dependent lymphocyte antigen epitope peptide of lung cancer-associated antigen Cyfra21-1, the amino acid sequence of which is any one of the epitope peptide sequences shown below:

[0025]

[0026]

[0027] As can be seen from the above table, the above sequence is the antigen peptide sequence of keratin 19 fragment (Cyfra21-1), and can bind with high affinity to A1101, A2402, A0201, A3001, B4001, B1502, B5101, B1301, B1501, B3501, B0702, C0602, C0401, C1402, C1403, C1203, C0304, C0303, C1502 molecules, and form "HLA / antigen peptide" complex molecules on the surface of antigen presenting cells, and bind to Cyfra21-1 antigen peptide-specific CD8 + T cell clones bind to stimulate their activation, proliferation and differentiation, and exert anti-lung cancer immune effector effects.

[0028] The thymus-dependent lymphocyte antigen epitope peptide sequence of lung cancer-related antigen Cyfra21-1 can be used to prepare lung cancer polypeptide vaccine or gene vaccine: polypeptide vaccine preparation: one or more polypeptides are artificially synthesized according to the polypeptide sequence of the present invention, mixed with adjuvant to form a soluble preparation, or loaded with biological nanomaterials to form a nano polypeptide vaccine, injected into lung cancer patients, stimulate the activation and proliferation of Cyfra21-1-specific T cells of lung cancer patients, and enhance their tumor-killing cell activity, thereby preparing lung cancer polypeptide vaccine. Gene vaccine preparation: according to the polypeptide sequence of the present invention, a recombinant DNA gene fragment, recombinant plasmid or recombinant virus vector of one or more polypeptides is constructed, injected into lung cancer patients, so that the recombinant gene expresses one or more polypeptides in vivo, stimulates the activation and proliferation of Cyfra21-1-specific T cells of lung cancer patients, and enhances their tumor-killing cell activity, thereby preparing lung cancer gene vaccine.

[0029] The thymus-dependent lymphocyte antigen epitope peptide sequence of lung cancer-associated antigen Cyfra21-1 can be used to prepare a detection preparation or kit for detecting lung cancer-associated antigen Cyfra21-1-specific T cells: one or more polypeptides are artificially synthesized according to the polypeptide sequence of the present invention, and used as antigen preparations in enzyme-linked immunospot method, intracellular cytokine fluorescence staining method and enzyme-linked immunosorbent assay, and mixed cultured with patient peripheral blood mononuclear cells (PBMCs) to stimulate Cyfra21-1-specific T cells to activate, proliferate and secrete cytokines, and then the synthesis amount of the cytokine is detected by other combined reagents, thereby reflecting the number and reaction activity of Cyfra21-1-specific T cells; these polypeptides can also be used to prepare human leukocyte antigen and polypeptide complexes (peptide-HLA complex) and polymers thereof through genetic engineering technology and protein engineering technology, and then further prepared into fluorescently labeled preparations, and the number of Cyfra21-1-specific T cells in the patient's peripheral blood mononuclear cell population is detected by flow cytometry. The relevant kit refers to a lung cancer-related antigen Cyfra21-1 specific T cell detection kit assembled from the above-mentioned preparation and other reagents commonly used in different detection methods.

[0030] The thymus-dependent lymphocyte antigen epitope peptide sequence of the lung cancer-associated antigen Cyfra21-1 can be used to prepare drugs for treating lung cancer: the above-mentioned polypeptide vaccine or gene vaccine based on the polypeptide sequence of the present invention is combined with other immunotherapy preparations or chemotherapeutic preparations to prepare clinical drugs for treating lung cancer.

[0031] The present invention uses six online epitope prediction databases to virtually predict 13 HLA-A (A0201, A1101, A2402, A3101, A0206, A0207, A3303, A3001, A0203, A1102, A0301, A0101, A2601), 15 HLA-B (B4601, B4001, B5801, B1502, B5101, B1301, B1302, B1501, B4006, B5401, B3501, B5201, B0702, B4403, B4801 ) and 14 HLA-C (C0102, C0602, C0702, C0801, C0304, C0302, C0303, C0401, C1402, C1202, C1502, C1403, C1203, C0701) molecule restricted lung cancer associated antigen Cyfra21-1 specific antigen epitope peptide sequence, and then verified its immunogenicity through intracellular cytokine staining (ICS) functional experiment, providing specific antigen peptides for the preparation of lung cancer treatment and preventive vaccines and the development of lung cancer Cyfra21-1 specific T cell detection reagents.

[0032] 1. Select the keratin 19 (Cyfra21-1) amino acid sequence as the targeting sequence;

[0033] 2. Select the prediction results to obtain six epitope prediction databases commonly used in the art with high accuracy: SYFPEITHI, BIMAS, SVMHC, IEDB, NETMHC and EPIJEN to predict the above-mentioned 13 HLA-A molecules, 15 HLA-B molecules and 14 HLA-C molecules restricted Cyfra21-1 antigen epitope peptide sequences;

[0034] 3. According to certain prediction criteria, the prediction results of six online epitope prediction websites were integrated and analyzed to obtain candidate antigen peptide sequences with relatively consistent prediction results from the six websites.

[0035] 4. The immunogenicity of lung cancer-related antigen peptide Cyfra21-1 was verified through IFN-γICS cell functional experiments.

[0036] 5. The peptide competition binding experiment of HLA molecules was carried out by using the HMy2.CIR cell line expressing specific HLA molecules to analyze the binding force between positive epitope peptides and specific HLA molecules.

[0037] 6. The immunogenicity of positive epitope peptides in stimulating specific T cell activation and proliferation in vivo was verified by immunizing HLA transgenic mice.

[0038] The present invention is an antigenic peptide sequence in a keratin 19 fragment (Cyfra21-1) that can bind to A1101, A2402, A0201, A3001, B4001, B1502, B5101, B1301, B1501, B3501, B0702, C0602, C0401, C1402, C1403, C1203, C03:04, C03:03, and C15:02 molecules in HLA molecules with high affinity and has immunogenicity; in addition, the present application also relates to a lung cancer polypeptide vaccine and a gene vaccine based on the above-mentioned antigenic peptide, as well as a reagent and method for detecting lung cancer Cyfra21-1-specific T cells based on the above-mentioned antigenic peptide.

[0039] Compared with the prior art, the advantages of the present invention are as follows:

[0040] The present invention obtains A1101, A2402, A0201, A3001, B4001, B1502, B5101, B1301, B1501, B3501, B0702, C0602, C0401, C1402, C1403, C1203, C0304, C0303, C1502 molecules restricted lung cancer associated antigen Cyfra21-1 specific antigen epitope peptides which have not been reported before. These HLA molecules have not been reported to have restricted lung cancer associated antigen peptides before. The experimental data reflect that these new antigen epitope peptide sequences have good affinity with specific HLA molecules, and the vaccine prepared based on the epitope peptide sequence of the present invention can cause the body to produce a strong immune response. Therefore, the epitope peptides provided by the present invention will provide the required key antigen components, i.e., antigen epitope peptide sequences, for the development of therapeutic and preventive polypeptide vaccines and gene vaccines for lung cancer, and the design of reagents and methods for detecting Cyfra21-1-specific T cells for lung cancer; at the same time, these antigen epitope peptides also provide key antigen components for individualized detection and precision medicine for lung cancer patients with these specific HLA alleles. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 Epitope peptides stimulate CD8 in peptide-PBMCs co-culture experiments + Flow cytometry detection of T cell activation; Figure 1 A is the CD3 + / CD8 + The flow cytometry analysis scatter plot of IFN-γ secretion in T cell population after being stimulated by specific epitope peptides; B is the CD3 + / CD8 + Statistical graph of the proportion of cells in the T cell population that secrete IFN-γ after being stimulated by specific epitope peptides. NC: negative control; No. 1: epitope peptide No. 1 (SEQ ID NO. 1); No. 2: epitope peptide No. 2 (SEQ ID NO. 2); No. 3: epitope peptide No. 3 (SEQ ID NO. 3).

[0042] Figure 2 Peptide competition binding assay to analyze the binding affinity of three Cyfra21-1 positive T cell epitopes to specific HLA molecules. NC, negative control; 1: epitope peptide No. 1 (SEQ ID NO.1); 2: epitope peptide No. 2 (SEQ ID NO.2); 3: epitope peptide No. 3 (SEQ ID NO.3); The blue solid line and red solid line show the cell fluorescence peaks at 5μM and 15μM concentrations of the tested peptide, respectively, while the gray filled line represents the maximum fluorescence intensity of the cells when there is only FITC-reference peptide. The light green solid line in the NC graph represents the negative control background fluorescence of the cells without peptide wells.

[0043] Figure 3 It provides a technical roadmap for epitope peptide prediction and screening, immunogenicity verification, and peptide competition binding experiments.

[0044] Figure 4 Epitope peptides induced specific CD8 in HLA-A2 / DR1 transgenic mice + T cell response

[0045] A mixed polypeptide vaccine was prepared by using three HLA-A0201-restricted positive epitope peptides and combined with adjuvant poly(I:C) to immunize HLA-A2 / DR1 transgenic mice on days 0, 7, and 21, respectively. Seven days after the last immunization, the mouse spleen cells were taken and restimulated with PBS and each positive epitope peptide for 10 h, and then BFA / Monesion (to block cytokine release) was added to continue culturing for 6 h. Then, IFN-γ intracellular staining and flow cytometry analysis were performed to detect IFN-γ. + / CD3 + / CD8 + The frequency of T cells. Figure 4 A is a representative flow cytometry analysis; B is the IFN-γ + / CD3 + / CD8 + T cell frequency statistics. DETAILED DESCRIPTION

[0046] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0047] In some embodiments of the present invention, disclosed are A1101, A2402, A0201, A3001, B4001, B1502, B5101, B1301, B1501, B3501, B0702, C0602, C0401, C1402, C1403, C1203, C0304, C03:03, C15:02 molecularly restricted lung cancer-associated antigen Cyfra21-1 T cell epitopes, the sequences are screened and identified by the following steps:

[0048] 1Online virtual prediction of dominant T cell epitope peptides of lung cancer-associated antigen Cyfra21-1 restricted by 13 HLA-A, 15 HLA-B and 14 HLA-C molecules

[0049] The lung cancer-related antigen protein: keratin 19 fragment (Cyfra21-1) was selected, and its amino acid sequence was obtained by searching the UniProt global protein resource database, and the most studied standard sequence was selected; then six commonly used epitope peptide prediction databases such as SYFPEITHI, BIMAS, SVMHC, IEDB, NETMHC, and EPIJEN were used to predict the epitope peptides A0201, A1101, A2402, A3101, A0206, A0207, A3303, A3001, A0203, A1102, A0301, A0101, A2601, B4601, and B40 01, B5801, B1502, B5101, B1301, B1302, B1501, B4006, B5401, B3501, B5201, B0702, B4403, B4801, C0102, C0602, C0702, C0801, C0304, C0302, C0303, C0401, C1402, C1202, C1502, C1403, C1203, C0701 molecularly restricted T cell epitope peptides for Cyfra21-1 antigen protein, among which the SVMHC database includes two prediction algorithms: MHCPEP model and SYFEPITHI model;

[0050] The IEDB database includes three methods: ANN, SMM and ARB. This example uses the ANN and SMM methods with better statistical significance. The websites of these epitope peptide prediction databases are shown in Table 1. The amino acid sequence of the lung cancer-related antigen Cyfra21-1 used to predict antigen epitopes is as follows:

[0051] Cyfra21-1(P08727): Human protein

[0052] MTSYSYRQSSATSSFGGLGGGSVRFGPGVAFRAPSIHGGSGGRGVSVSSA 50

[0053] RFVSSSSSGAYGGGYGGVLTASDGLLAGNEKLTMQNLNDRLASYLDKVRA 100

[0054] LEAANGELEVKIRDWYQKQGPGPSRDYSHYYTTIQDLRDKILGATIENSR 150

[0055] IVLQIDNARLAADDFRTKFETEQALRMSVEADINGLRRVLDELTLARTDL 200

[0056] EMQIEGLKEELAYLKNHEEEISTLRGQVGGQVSVEVDSAPGTDLAKILS 250

[0057] DMRSQYEVMAEQNRKDAEAAWFTSRTEELNREVAGHTEQLQMSRSEVTDLR 300

[0058] RTLQGLEIELQSQLSMKAALEDTLAETEARFGAQLAHIQALISGIEAQLG 350

[0059] DVRADSERQNQEYQRLMDIKSRLEQEIATYRSLLEGQEDHYNNLSASKVL 400

[0060] The antigen binding groove of HLA class I molecules is closed at both ends, and the length of the antigen peptide accepted is 8-11 amino acid residues, among which 9 and 10 amino acids are the most common. Therefore, this embodiment mainly selects polypeptides with a length of 9 and 10 amino acids as the research object. The amino acid sequence of Cyfra21-1 is input into the corresponding amino acid sequence input box of the prediction database website, and then the length of the epitope peptide is selected as 9 and 10 amino acids respectively, and then a specific HLA molecule is selected to perform online virtual prediction of the T cell epitope peptide of the lung cancer-related antigen Cyfra21-1.

[0061] Table 1 Epitope peptide prediction database website

[0062]

[0063]

[0064] For each HLA molecule, the peptides predicted by different databases are arranged from high to low according to the scores, and then the epitope peptides that meet the scoring criteria of at least 4 prediction methods are selected as candidate epitope peptides. For each HLA molecule, 1-5 peptides with the highest scores (highest affinity) are selected from the candidate epitope peptides as epitope peptides to be identified. For the above 13 HLA-A, 15 HLA-B and 14 HLA-C molecules, a total of 59 dominant T cell epitope peptides to be identified were screened and predicted.

[0065] 2. Isolation of PBMC from peripheral blood of lung cancer patients

[0066] Take fresh anticoagulated whole blood stored at room temperature and dilute it appropriately with sterile PBS; add 1 times the volume of original blood human lymphocyte separation solution (Dakoway Biotechnology, Shenzhen) to a 15 mL centrifuge tube; slowly spread the diluted blood on the separation solution and centrifuge at room temperature for 20 min at 2000 rpm; aspirate the mononuclear cell (PBMCs) layer and wash it twice by centrifugation; resuspend it with serum-free medium (Dakoway Biotechnology, Shenzhen), count the cells, and adjust the cell concentration to 3×10 6 / mL for future use.

[0067] 3. Identification of the immunogenicity of lung cancer-associated antigen Cyfra21-1 T cell epitope peptide using IFN-γICS method

[0068] Technical route such as Figure 3 As shown:

[0069] 4HLA allele typing

[0070] 200 μL of anticoagulated blood was collected from the patient, and genomic DNA was extracted using a human whole blood genomic DNA extraction kit (Tiangen Bio, Beijing). HLA locus-specific primers were used for PCR to amplify the DNA sequences of exon 2, intron 2, exon 3, and part of introns 1 and 3 at locus A, B, or C, respectively, with a product size of 985 bp. The amplification conditions were: 95°C pre-denaturation for 3 min; 95°C denaturation for 15 s; 62°C annealing for 15 s; 72°C extension for 90 s; 35 cycles; 72°C extension for 5 min. The amplified product was 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 Novozyme Biotechnology Co., Ltd.

[0071] Table 2 HLA site-specific PCR amplification primers and sequencing primers (sequences are SEQ ID NO. 5 to 22, respectively)

[0072]

[0073]

[0074] The sequencing results of exon 2 and exon 3 were assembled into a complete HLA overlapping sequence (Contig) by using the Seqman software of the Lasergene program. The bases of the bidirectional sequencing were carefully checked to see if they were complete and consistent. The bases of the heterozygotes were found and replaced with annexed bases, such as M for A and C, R for A and G, W for A and T, S for C and G, Y for C and T, and K for G and T. Finally, the sequence fragments of the amplified HLA alleles were determined. The Nucleotide BLAST tool was used to compare the assembled HLA base sequence with the exon 2 and exon 3 sequences of all HLA-A alleles in the database until a completely matched gene combination was obtained, thereby determining the HLA alleles.

[0075] 5. Peptide-PBMCs co-culture and IFN-γ intracellular staining experiment

[0076] PBMCs were separated by density gradient centrifugation and the concentration of PBMCs was adjusted to 3 × 10 6 / mL, inoculated into a U-bottom 96-well plate, 100 μL / well, and placed at 37°C, 5% CO 2 The constant temperature incubator was set aside. According to the patient's HLA-A allele typing results, the corresponding candidate epitope peptides were co-cultured with the PBMCs in the above 96-well plate for 6 hours, and the Brefeldin A Solution / Monensin Solution (1000×) blocking agent was added for co-culture, and then IFN-γ intracellular fluorescence staining was performed, and flow cytometry was used to detect the CD8 secretion of IFN-γ. + T cell frequency; at the same time, a single culture well of PBMCs without peptide stimulation was set up as a negative control group. Compared with the negative control, IFN-γ + / CD8 + The candidate epitope peptides with a T cell frequency increased by more than 2 times were set as positive epitope peptides, that is, the epitope peptides could stimulate the memory CD8 + T cells become activated and secrete cytokines.

[0077] Through the IFN-γICS experiment, PBMCs from 150 lung cancer patients were collected and co-cultured with related peptides from 42 candidate epitope peptides. Among them, three candidate epitope peptides (sequences of RQSSATSSF, VRFGPGVAF, and VSVSSARFV) induced strong CD8 + T cell response caused IFN-γ to appear in PBMCs of at least 4 lung cancer patients + / CD8 +The increase in T cell frequency determined that the three candidate epitope peptides were positive epitopes and dominant epitope peptides with strong immunogenicity ( Figure 1 ).

[0078] 6. Peptide competition binding assay to analyze the affinity of candidate epitope peptides to corresponding HLA-A molecules

[0079] 59 candidate epitopes (HMy2.CIR cell lines expressing HLA-A2601 are not available, so 10 candidate epitopes cannot participate in this experiment), including the three positive epitope peptides No. 1, No. 2, and No. 3 screened in the previous step, which were verified by the peptide-PBMCs costimulation experiment, were assigned to the relevant epitope peptide queues of each HLA molecule according to the predicted HLA restriction and the HLA allele type of the patient with positive T cell response. As the peptides to be tested, they were subjected to peptide competition binding experiments with the fluorescent reference peptides of the HLA molecule. Finally, the three positive peptides No. 1, 2, and 3 all shifted the cell fluorescence peak to the left ( Figure 2 ), indicating that these positive epitope peptides can compete with the fluorescent reference peptides for binding to the relevant HLA molecules on the HMy2.CIR cell membrane. These three positive epitope peptides all showed high or medium affinity binding to the corresponding HLA molecules (Table 3).

[0080] Table 3 Peptide competition binding assay to analyze the binding affinity of three Cyfra21-1 positive T cell epitopes to 16 dominant HLA molecules

[0081]

[0082]

[0083] Immunization of HLA transgenic mice with 7 epitope peptides to verify their immunogenicity in inducing specific T cell responses in vivo

[0084] Three epitope peptides restricted by HLA-A0201 molecules (including epitope peptides No. 1, No. 2 and No. 3 of the present application) were made into mixed polypeptides and then mixed with adjuvant poly I: C. HLA-A0201 / DR0101 transgenic mice were randomly divided into 2 groups, 3 mice in each group. The experimental groups were inoculated with peptide cocktail / poly(I: C) (epitope peptide: 10 μg / single peptide, poly(I: C): 100 μg /

[0085] The adjuvant group was also vaccinated with poly(I:C) (100 μg /

[0086] Subcutaneous injections were given at four points (neck back, tail base, and bilateral groins).

[0087] On the 28th day, mice were killed and spleen cell suspension was routinely prepared. The cell concentration was adjusted to 1×10 7 / mL, inoculated into a 48-well cell culture plate (0.5mL / well), each mouse spleen cell inoculation well was divided into 4 groups, and PBS (negative control) and each positive epitope peptide (including the three epitope peptides SEQ ID NO.1-3 of the present application, 20μg / mL / peptide) were added respectively; after culturing for 10 hours, BFA and Monensin were added to each well and incubated for another 6 hours; then the cells in each well were collected, blocked with FcR Blocking Reagent mouse for 15 minutes, and then co-stained with FITC-anti-mouse CD3 and PE-anti-mouse CD8a for 30 minutes; after washing, each tube was treated with a membrane permeabilizing agent, and finally APC-anti-mouse IFN-γ monoclonal antibody was added for intracellular staining for 30 minutes; after washing, CD3 was analyzed by flow cytometry + / CD8 + / IFN-γ + T cell frequency, observation of specific CD8 induced by epitope peptide + The strength of the T cell response.

[0088] The results showed that the three positive epitope peptides (peptide cocktail / polyI:C group) of the present application all induced strong epitope peptide-specific CD8 + T cell response. Compared with the adjuvant group, the IFN-γ induced by these three epitope peptides + / CD8 + The frequency of T cells was 3-5 times that of the adjuvant group.

[0089] In the description of this specification, the description with reference to the terms "one embodiment", "example", "specific example", etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0090] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and these changes and improvements all fall within the scope of the present invention to be protected.

Claims

1. A thymus-dependent lymphocyte antigen epitope peptide of lung cancer-associated antigen Cyfra21-1, characterized in that: The amino acid sequence of the epitope peptide includes any one or more of the following: RQSSATSSF; VRFGPGVAF; VSVSSARFV; The amino acid sequence above is an amino acid sequence after a single amino acid is removed or replaced.

2. Use of the thymus-dependent lymphocyte antigen epitope peptide of the lung cancer-associated antigen Cyfra21-1 according to claim 1 in the preparation of lung cancer vaccine.

3. A nucleic acid molecule encoding the thymus-dependent lymphocyte antigen epitope peptide of the lung cancer-associated antigen Cyfra21-1 according to claim 1. A vector comprising the nucleic acid molecule according to claim 3.

5. A vaccine for treating or preventing lung cancer, characterized in that: Containing the nucleic acid molecule according to claim 3 or the vector according to claim 4.

6. Use of the thymus-dependent lymphocyte antigen epitope peptide of the lung cancer-associated antigen Cyfra21-1 according to claim 1 in the preparation of a drug for treating or preventing lung cancer.

7. Use of the thymus-dependent lymphocyte antigen epitope peptide of lung cancer-associated antigen Cyfra21-1 according to claim 1 in a kit for preparing lung cancer-associated antigen Cyfra21-1 T cells.

8. The use of the thymus-dependent lymphocyte antigen epitope peptide of lung cancer-associated antigen Cyfra21-1 according to claim 1 in a kit for preparing lung cancer-associated antigen Cyfra21-1 T cells according to claim 7, characterized in that: The kit comprises: the detection reagent is an enzyme-linked immunosorbent assay reagent, an intracellular cytokine fluorescent staining reagent, an enzyme-linked immunosorbent assay reagent, a human leukocyte antigen polymer fluorescent staining or a flow cytometric analysis reagent.

9. A monoclonal antibody, characterized in that Capable of specifically binding to the epitope peptide according to claim 1.

10. A drug for treating lung cancer, characterized in that: Comprising the monoclonal antibody according to claim 9.

Images