Thymus dependent lymphocyte antigen epitope peptide of lung cancer associated antigen Cyfra21-1 and application of thymus dependent lymphocyte antigen epitope peptide

Abstract

The invention discloses a thymus dependent lymphocyte antigen epitope peptide of a lung cancer associated antigen Cyfra21-1 and application, and belongs to the field of medical immunology and oncology. The invention relates to a thymus dependent lymphocyte antigen epitope peptide of a lung cancer associated antigen Cyfra21-1. The amino acid sequence of the epitope peptide comprises any one or more of the following: EVKIRDWYQK; a YQKQGPGPSR (YQKQGPGPSR) A DYSHYYTTI (Transmission Time Interval); the amino acid sequence is an amino acid sequence obtained by removing or replacing a single amino acid of the amino acid sequence. The epitope peptide disclosed by the invention can be specifically combined with cytotoxic thymus dependent lymphocytes to stimulate the cytotoxic thymus dependent lymphocytes to activate, proliferate and differentiate, so that an anti-tumor immune effect is exerted; the antigen peptides can be used for preparing therapeutic and preventive vaccines for lung cancer, can also be used for preparing quantitative detection reagents for detecting specific T cells of the lung cancer related antigen Cyfra21-1, and have potential application value in 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-associated antigen Cyfra21-1 and its application. Background Art

[0002] According to the 2022 China Malignant Tumor Burden Report released by the National Cancer Center, lung cancer ranked first in both new cases and deaths in my country in 2022. The high incidence and mortality of lung cancer pose a serious threat to the lives and health of the Chinese people. Non-small cell lung cancer (NSCLC) is the most common subtype, accounting for approximately 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 less than 5%.

[0003] In my country, high-risk groups for NSCLC mainly include long-term smokers, those exposed to secondhand smoke, environmental pollution, occupational carcinogens (such as asbestos and radon), and those with a family history of lung cancer. In addition, chronic lung disease, tuberculosis, and certain genetic susceptibilities may also increase the risk of disease. Non-small cell lung cancer is generally divided into three major 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] Cyfra21-1 is a fragment of keratin 19. Normally, it exists as oligomers and is present at very low levels in serum. When cells become cancerous, intracellular keratin 19 levels increase, and after necrosis and shedding of cancer cells, the fragments are released into the blood and body fluids as dissolved fragments. Cyfra21-1 is one of the most valuable tumor-associated antigens in NSCLC and a commonly used tumor marker for clinical diagnosis of NSCLC and for monitoring and assessing prognosis and recurrence. This patent uses Cyfra21-1, a lung cancer-associated tumor antigen, for research.

[0005] Cytotoxic T lymphocytes (CTLs) are core cells that mediate adaptive immune responses and play a crucial role in anti-infection, anti-tumor, hypersensitivity reactions, and autoimmune diseases. The T cell receptor (TCR) on their cell membrane can specifically recognize and bind to the complex of MHC class I molecules and antigenic peptides on the surface of antigen-presenting cells, known as the MHC / antigen-peptide complex. CTL epitopes are antigenic peptides bound to MHC class I molecules. They are linear segments or spatial conformational structures within the antigen molecule that are specifically recognized by the TCR. They are the basic antigenic units that trigger immune responses and play a key role in CTL activation.

[0006] The MHC system refers to the major histocompatibility complex (MHC), which is a group of tightly linked genes in the genome of vertebrates, encoding MHC class I and class II protein molecules. HLA (human leukocyte antigen) is the MHC system of humans, responsible for presenting antigen peptides (T cell epitope peptides) to T cells to initiate specific immune responses 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 CTLs, are the main effector cells targeting tumor cells. However, HLA alleles are highly polymorphic in the human population. Between different individuals, HLA molecules are not completely the same, and each HLA molecule can present a lot of antigen peptide sequences, causing different T cell immune responses. Therefore, the same tumor antigen causes different 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 + CTLs, and activating CTLs to make tumor cells expressing tumor-related antigens apoptotic by secreting perforin and granzyme. Therefore, dynamic monitoring of the number and function of lung cancer-related antigen-specific CD8 + T cells can accurately reflect the specific immune function status of lung cancer patients against lung cancer-related antigens. Because different people have different HLA molecule types, their ability to process, process and present different lung cancer antigens is also different, causing different intensities of lung cancer-related antigen-specific T cell immune response reactions. According to the different HLA molecule types of lung cancer patients, select the antigen peptides of the lung cancer-related antigens presented by the HLA molecules, and dynamically monitor the number and reactivity of the lung cancer-related antigen-specific CD8 + T cells, which are of great significance to the monitoring of disease progression, diagnosis, treatment plan development, efficacy observation and prognosis of lung cancer patients, and are an important technical means to achieve precise medical treatment of lung cancer. At the same time, using these lung cancer-related antigen peptides with high affinity binding to HLA molecules, polypeptide vaccines or genetic vaccines can also be prepared to prevent and treat lung cancer.

[0007] However, there are still very few Cyfra21-1-specific T cell epitopes that have been clearly presented by various HLA molecules and can stimulate the body to cause T cell response reactions, thereby limiting the detection of Cyfra21-1-specific T cells in lung cancer patients carrying different HLA alleles, and limiting the role of lung cancer Cyfra21-1-specific T cells in the development of lung cancer, and further limiting personalized detection and precise immunotherapy based on individual differences in HLA genes and differences in lung cancer Cyfra21-1 antigen peptides presented by HLA genes. SUMMARY

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

[0009] The object of the present application can be achieved by the following technical solutions.

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

[0011] EVKIRDWYQK;

[0012] YQKQGPGPSR;

[0013] DYSHYYTTI;

[0014] an amino acid sequence obtained by removing or replacing a single amino acid of the above amino acid sequence.

[0015] In a second aspect, the present application relates to the use of the thymus-dependent lymphocyte antigen epitope peptide of lung cancer related antigen Cyfra21-1 described above in the preparation of a lung cancer vaccine.

[0016] In a third aspect, the present application relates to a nucleic acid molecule encoding the thymus-dependent lymphocyte antigen epitope peptide of lung cancer related antigen Cyfra21-1 described above.

[0017] In a fourth aspect, the present application relates to a vector containing the nucleic acid molecule described above.

[0018] In a fifth aspect, the present application relates to a vaccine for treating or preventing lung cancer, containing the nucleic acid molecule described above or the vector described above.

[0019] In a sixth aspect, the present application relates to the use of the thymus-dependent lymphocyte antigen epitope peptide of lung cancer related antigen Cyfra21-1 described above in the preparation of a medicament for treating or preventing lung cancer.

[0020] In a seventh aspect, the present application relates to the use of the thymus-dependent lymphocyte antigen epitope peptide of lung cancer related antigen Cyfra21-1 described above in the preparation of a lung cancer related antigen Cyfra21-1 T cell kit.

[0021] Optionally, the kit comprises: the detection reagent is an enzyme-linked immunospot method reagent, an intracellular cytokine fluorescence staining method reagent, an enzyme-linked immunosorbent assay reagent, a human leukocyte antigen multimer fluorescence staining or flow cytometry analysis method reagent.

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

[0023] The ninth aspect of the present application relates to a medicament for treating lung cancer, comprising the monoclonal antibody described above.

[0024] More specifically, the present application provides a new thymus-dependent T cell epitope peptide of lung cancer-associated antigen Cyfra21-1, which has any one of the amino acid sequences shown in the following epitope peptide sequences:

[0025]

[0026] As can be seen from the above table, the above sequences are antigen peptide sequences of keratin 19 fragment (Cyfra21-1), and are capable of high-affinity binding to A2402, A0201, A3303, and A1101 molecules, respectively, to form "HLA / antigen peptide" complex molecules on the surface of antigen-presenting cells, which specifically bind to Cyfra21-1 antigen peptide-specific CD8 + T cell clones, stimulate their activation, proliferation, and differentiation, and exert anti-lung cancer immune effector functions.

[0027] The thymus-dependent T cell epitope peptide sequence of lung cancer-associated antigen Cyfra21-1 can be used to prepare a lung cancer polypeptide vaccine or a gene vaccine: polypeptide vaccine preparation: one or more polypeptides are artificially synthesized according to the polypeptide sequences of the present application, mixed with an adjuvant to form a soluble preparation, or loaded with a biological nanomaterial to form a nanopolypeptide vaccine, which is injected into a lung cancer patient to stimulate Cyfra21-1-specific T cell activation, proliferation, and enhancement of their tumor cell killing activity, thereby preparing a lung cancer polypeptide vaccine. Gene vaccine preparation: a recombinant DNA gene fragment, a recombinant plasmid, or a recombinant viral vector of one or more polypeptides is constructed according to the polypeptide sequences of the present application, which is injected into a lung cancer patient to allow the recombinant gene to express one or more polypeptides in vivo, stimulate Cyfra21-1-specific T cell activation, proliferation, and enhancement of their tumor cell killing activity, thereby preparing a lung cancer gene vaccine.

[0028] The thymus-dependent lymphocyte antigen epitope peptide sequence of lung cancer related antigen Cyfra21-1 can be used to prepare a detection preparation or kit for detecting lung cancer related antigen Cyfra21-1 specific T cells: according to the polypeptide sequence of the present application, one or more polypeptides are artificially synthesized, and in enzyme-linked immunospot assay, intracellular cytokine fluorescence staining and enzyme-linked immunosorbent assay, the polypeptides are mixed and cultured with peripheral blood mononuclear cells (PBMCs) of a patient as an antigen preparation to stimulate Cyfra21-1 specific T cells to activate, proliferate and secrete cytokines, and then the amount of the synthesized cytokines is detected by other combined reagents to reflect the number and reactivity of Cyfra21-1 specific T cells; the polypeptides can also be used to prepare a peptide-HLA complex and a multimer thereof by genetic engineering technology and protein engineering technology, and then the complex or the multimer is further prepared into a fluorescein-labeled preparation to detect the number of Cyfra21-1 specific T cells in the peripheral blood mononuclear cell group of the patient by flow cytometry. The related kit refers to a lung cancer related antigen Cyfra21-1 specific T cell detection kit assembled by the above preparation and other reagents commonly used in different detection methods.

[0029] The thymus-dependent lymphocyte antigen epitope peptide sequence of lung cancer related antigen Cyfra21-1 can be used to prepare a drug for treating lung cancer: the polypeptide vaccine or the genetic vaccine based on the polypeptide sequence of the present application is combined with other immunotherapy preparations or chemotherapy preparations to prepare a clinical drug for treating lung cancer.

[0030] The present application virtually predicts 13 kinds of HLA-A (A0201, A1101, A2402, A3101, A0206, A0207, A3303, A3001, A0203, A1102, A0301, A0101, A2601) molecule restricted lung cancer related antigen Cyfra21-1 specific antigen epitope peptide sequences by using six online epitope prediction databases, and then verifies the immunogenicity thereof by intracellular cytokine staining (ICS) functional experiment, thereby providing specific antigen peptides for preparing a lung cancer treatment and prevention vaccine and developing a lung cancer Cyfra21-1 specific T cell detection reagent.

[0031] 1. Selecting keratin 19 (Cyfra21-1) amino acid sequence as a target sequence;

[0032] 2. Selecting six kinds of epitope prediction databases with high accuracy and commonly used by researchers, i.e. SYFPEITHI, BIMAS, SVMHC, IEDB, NETMHC and EPIJEN, to predict the 13 kinds of HLA-A molecule restricted Cyfra21-1 antigen epitope peptide sequences;

[0033] 3. According to certain prediction criteria, the prediction results of six online epitope prediction websites are integrated and analyzed to obtain candidate antigen peptide sequences with consistent prediction results of the six websites.

[0034] 4. The immunogenicity of lung cancer related antigen peptide Cyfra21-1 is verified by IFN-γ ICS cell functional experiment.

[0035] 5. The polypeptide competition binding experiment of HLA molecules is carried out by HMy2.CIR cell strain expressing specific HLA molecules, and the binding force of positive epitope peptide and specific HLA molecules is analyzed.

[0036] 6. The immunogenicity of positive epitope peptide in stimulating specific T cell activation and proliferation in vivo is verified by immunizing C57BL / 6J mice.

[0037] The application is an antigen peptide sequence in keratin 19 fragment (Cyfra21-1) which can be combined with A2402, A0201, A3303 and A1101 molecules with high affinity and has immunogenicity; and also relates to lung cancer polypeptide vaccine, gene vaccine based on the above antigen peptide, and reagent and method for detecting lung cancer Cyfra21-1 specific T cells based on the above antigen peptide.

[0038] Compared with the prior art, the application has the following advantages:

[0039] The A2402, A0201, A3303 and A1101 molecule restricted lung cancer related antigen Cyfra21-1 specific antigen epitope peptide obtained by online virtual prediction and functional experiment verification has not been reported before. These HLA molecules have not been reported to be restricted by lung cancer related antigen peptides before. Therefore, these new antigen epitope peptide sequences will provide the required key antigen components, i.e. antigen epitope peptide sequences, for developing therapeutic and preventive polypeptide vaccines and gene vaccines for lung cancer, designing reagents and methods for detecting lung cancer Cyfra21-1 specific T cells, etc. Meanwhile, 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 DRAWINGS

[0040] Figure 1 Flow cytometry detection of epitope peptide stimulated CD8 + T cell activation in polypeptide-PBMC co-culture experiment; Figure 1 A is the flow cytometry analysis scatter plot of IFN-γ secreting cells in CD3 + / CD8 + T cell population of each lung cancer patient stimulated by specific epitope peptide; B is the flow cytometry analysis scatter plot of IFN-γ secreting cells in CD3 + / CD8+ The proportion of cells secreting IFN-γ in T cell population after stimulation by specific epitope peptides. NC: negative control; No. 1: No. 1 epitope peptide; No. 2: No. 2 epitope peptide; No. 3: No. 3 epitope peptide.

[0041] Figure 2 The polypeptide competition binding experiment analyzes the binding affinity of three Cyfra21-1 positive T cell epitopes to specific HLA molecules. NC, negative control; blue solid line and red solid line show the cell fluorescence peaks of the tested peptides at 5 μM and 15 μM concentrations, respectively, and the gray filled line represents the maximum fluorescence intensity of the cells with only FITC-reference peptide. The cyan solid line in the NC graph represents the negative control background fluorescence of the cells without peptide wells.

[0042] Figure 3 The technical roadmap for epitope peptide prediction and screening, immunogenicity verification, and polypeptide competition binding experiment.

[0043] Figure 4 Epitope peptides induced specific CD8 + T cell responses in C57BL / 6J mice.

[0044] Three positive epitope peptides were prepared into a mixed polypeptide vaccine, combined with adjuvant poly(IC), and used to immunize C57BL / 6J mice on days 0, 7, and 21. Seven days after the last immunization, the mouse spleen cells were taken and cultured with PBS, each positive epitope peptide for 10 h, then BFA / Monesion (blocking cytokine release) was added for 6 h of further culture, followed by IFN-γ intracellular staining and flow analysis to detect the frequency of IFN-γ + / CD3 + / CD8 + T cells. Figure 4 Among them, A is a representative flow analysis graph; B is a frequency histogram of IFN-γ + / CD3 + / CD8 + T cells. DETAILED DESCRIPTION

[0045] The technical solutions in the embodiments of the present application will be described clearly and completely below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the scope of the present application.

[0046] In some embodiments of the present application, A2402, A0201, A3303, A1101 molecule restrictive lung cancer related antigen Cyfra21-1 T cell epitope, and its exploration and immunogenicity verification method are disclosed, such as Figure 3 As shown, comprising the following steps:

[0047] 1 Online virtual prediction of 13 HLA-A molecule restricted lung cancer related antigen Cyfra21-1 dominant T cell epitope peptides

[0048] Select lung cancer related antigen protein: keratin 19 fragment (Cyfra21-1), obtain its amino acid sequence by UniProt global protein resource database retrieval, select the most studied standard sequence; Then, through six commonly used epitope peptide prediction databases such as SYFPEITHI, BIMAS, SVMHC, IEDB, NETMHC, EPIJEN, T cell epitope peptides restricted by A0201, A1101, A2402, A3101, A0206, A0207, A3303, A3001, A0203, A1102, A0301, A0101, A2601 molecules against Cyfra21-1 antigen protein are virtually predicted, wherein the SVMHC database includes two prediction algorithms of MHCPEP model and SYFEPITHI model; The IEDB database includes ANN, SMM and ARB three methods, and the ANN and SMM methods with better statistical significance are used in this embodiment. The epitope peptide prediction database websites are shown in Table 1.

[0049] The antigen binding groove of HLA class I molecule is closed at both ends, and the length of the accepted antigen peptide is 8-11 amino acid residues, of which 9 and 10 amino acids are most common, therefore, polypeptides with 9 and 10 amino acids are mainly selected as research objects. 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, and the T cell epitope peptide of lung cancer related antigen Cyfra21-1 is virtually predicted online. In this embodiment, the amino acid sequence of the lung cancer related antigen Cyfra21-1 used to predict the antigen epitope is SED ID NO:4:

[0050] Cyfra21-1 (P08727): Human protein

[0051]

[0052] Table 1 Epitope peptide prediction database website

[0053]

[0054]

[0055] For each HLA molecule, the polypeptides predicted by different databases were ranked from high to low according to the scores, and at least 4 or more predicted method score standard epitope peptides were selected as candidate epitope peptides. For each HLA molecule, 1-5 polypeptides with the highest score (highest affinity) were selected from the candidate epitope peptides as the epitope peptides to be identified. For the above 13 HLA-A molecules, a total of 59 predicted dominant T cell epitope peptides to be identified were screened.

[0056] 2 Isolation of peripheral blood PBMCs of lung cancer patients

[0057] The fresh anticoagulant whole blood stored at room temperature was diluted with sterile PBS; 1 times the volume of human lymphocyte separation medium (Da Ke Bio, Shenzhen) was added to a 15 mL centrifuge tube; the diluted blood was slowly laid on the separation medium, centrifuged at room temperature for 20 min at 2000 rpm; the mononuclear cell (PBMC) layer was aspirated and washed twice by centrifugation; resuspended with serum-free medium (Da Ke Bio, Shenzhen), counted the cells, and adjusted the cell concentration to 3x10 6 / mL for standby.

[0058] 3 HLA allele typing

[0059] 200 μL of anticoagulant blood of the patient was collected, and genomic DNA was extracted using a human whole blood genomic DNA extraction kit (Tiangen Bio, Beijing); HLA site-specific primers were used for PCR to amplify the DNA sequences of exon 2, intron 2, exon 3, and part of intron 1 and 3 of A site, and the product size was 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 amplification product was identified by 1% agarose gel electrophoresis, and was sent to Shanghai Sunny Biotech Company for purification and bidirectional sequencing. PCR reagents were purchased from Nanjing Novozyme Biotech Company.

[0060] Table 2 HLA site-specific PCR amplification primers and sequencing primers

[0061]

[0062]

[0063] The sequencing results of exon 2 and exon 3 were assembled into a complete HLA contig using the Lasergene Seqman software. The bidirectional sequencing was carefully checked for base integrity and consistency. Heterozygous bases were identified and replaced with degenerate bases (e.g., 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). The resulting sequence fragments were then identified as the amplified HLA alleles. The Nucleotide BLAST tool was then used to align the assembled HLA base sequence with the exon 2 and exon 3 sequences of all HLA alleles in the database until a perfect match was obtained, thereby confirming the HLA allele.

[0064] 4. Peptide-PBMCs co-culture and IFN-γ intracellular staining experiment

[0065] PBMCs were isolated 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 in a 37°C, 5% CO2 constant temperature incubator for use. According to the patient's HLA allele typing results, the corresponding candidate epitope peptides were co-cultured with PBMCs in the above 96-well plate for 6 hours, and Brefeldin ASolution / Monensin Solution (1000×) blocking agent was added for co-culture. IFN-γ intracellular fluorescence staining was then performed, and IFN-γ secretion of CD8 + T cell frequency; at the same time, PBMCs culture wells without peptide stimulation were set up as negative control group. Compared with the negative control, IFN-γ + / CD8 + The candidate epitope peptides whose T cell frequency increased by more than 2 times were defined as positive epitope peptides, that is, the epitope peptides could stimulate the memory CD8 + T cells are activated and secrete cytokines.

[0066] Through IFN-γICS experiments, PBMCs from 150 lung cancer patients were collected and co-cultured with related peptides from 59 candidate epitope peptides. Among them, three candidate epitope peptides (sequences were EVKIRDWYQK; YQKQGPGPSR; DYSHYYTTI) induced strong CD8 + T cell response caused IFN-γ to appear in PBMCs of at least 3 or more lung cancer patients + / CD8 + The T cell frequency increased more than 10 times, so the three candidate epitope peptides were determined to be positive epitopes and dominant epitope peptides with strong immunogenicity (Figure 1 ).

[0067] 5 Polypeptide competition binding assay to analyze the affinity of candidate epitope peptides to the corresponding HLA molecules

[0068] Fifty-nine candidate epitopes (HMy2.CIR cell line expressing HLA-A2601 was temporarily unavailable, so 10 candidate epitopes could not participate in this experiment) including the three positive epitope peptides No. 1 (EVKIRDWYQK), No. 2 (YQKQGPGPSR), and No. 3 (DYSHYYTTI) screened in the previous step and verified by peptide-PBMCs co-stimulation experiment were assigned to the relevant epitope peptide queue 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 test peptides, and were subjected to polypeptide competition binding assay with the fluorescent reference peptides of the HLA molecules. The fluorescent peptide sequences of A2402 and A0201 were EYLVSK(FITC)GVW (SEQ ID NO: 5) and FLPSDK(FITC)FPSV (SEQ ID NO: 6), respectively. The fluorescent peptide sequences of A3303 and A1101 were YVNVNK(FITC)GLK (SEQ ID NO: 7).

[0069] Finally, the three positive peptides No. 1, No. 2, and No. 3 all caused the cell fluorescence peak to shift to the left ( Figure 2 ), indicating that these positive epitope peptides could compete with the fluorescent reference peptides for binding to the relevant HLA molecules on the cell membrane of HMy2.CIR. The three positive epitope peptides all showed high or medium affinity for the corresponding HLA molecules (Table 3).

[0070] Table 3 Polypeptide competition binding assay to analyze the binding affinity of three Cyfra21-1 positive T cell epitopes to four dominant HLA molecules

[0071]

[0072] 6 Immunization of C57BL / 6J mice with epitope peptides to verify their immunogenicity in inducing specific T cell responses in vivo

[0073] Three positive epitope peptides (including No. 1, No. 2 and No. 3 epitope peptides of this example) were prepared into a mixed polypeptide, and then mixed with the adjuvant poly I:C. C57BL / 6J mice were randomly divided into two groups, 3 mice in each group. The experimental group was inoculated with peptide cocktail / poly(I:C) (epitope peptide: 10 μg / monopeptide, poly(I:C): 100 μg / mouse) on day 0, day 7 and day 21, respectively; the adjuvant group was also inoculated with poly(I:C) (100 μg / mouse) on day 0, day 7 and day 21, respectively. Subcutaneous injection was performed at four points (neck, back, tail root, bilateral groin).

[0074] On day 28, the mice were killed, and the spleen cell suspension was prepared conventionally. The cell concentration was adjusted to 1×10 7 / mL with 10% FBS-1640 culture solution, and inoculated into a 48-well cell culture plate (0.5 mL / well). The spleen cells of each mouse were inoculated into 4 wells, and PBS (negative control) and each positive epitope peptide (including the three epitope peptides screened in this example, 20 μg / mL / peptide) were added, respectively. After 10 hours of culture, BFA and Monensin were added to each well, and incubated for 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, the cells were treated with a membrane breaker, and then APC-anti-mouse IFN-γ monoclonal antibody was added for intracellular staining for 30 minutes. After washing, the CD3 + / CD8 + / IFN-γ + T cell frequency was analyzed by flow cytometry. The strength of the epitope peptide-induced specific CD8 + T cell response was observed.

[0075] The results showed that the three positive epitope peptides (peptide cocktail / poly I:C group) of this example all induced strong epitope peptide-specific CD8 + T cell response in mice. Compared with the adjuvant group, the IFN-γ + / CD8 + T cell frequency induced by the three epitope peptides was 3-4 times that of the adjuvant group.

[0076] In the description of the specification, the description of the terms "one embodiment", "an example", "a specific example" and the like means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In the specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any appropriate way in one or more embodiments or examples.

[0077] The basic principles, main features and advantages of the present application are shown and described above. Those skilled in the art should understand that the present application is not limited by the above embodiments, and the above embodiments and descriptions in the specification are only to illustrate the principles of the present application. Without departing from the spirit and scope of the present application, various changes and improvements can be made to the present application, and these changes and improvements all fall within the scope of the claimed present application.

Claims

1. A thymus dependent T cell epitope peptide of a lung cancer associated antigen Cyfra 21-1, characterized in that, The amino acid sequence of the epitope peptide includes any one or more of the following: EVKIRDWYQK; YQKQGPGPSR; DYSHYYTTI; an amino acid sequence after removal or replacement of a single amino acid of the above-mentioned amino acid sequence.

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

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

4. A vector containing the nucleic acid molecule according to claim 3.

5. A vaccine for treating or preventing lung cancer, characterized by, containing the nucleic acid molecule according to claim 3 or the vector according to claim 4.

6. Use of the thymus-dependent T-lymphocyte epitope peptide of the lung cancer-related antigen Cyfra21-1 according to claim 1 in the preparation of a medicament for the treatment or prevention of lung cancer.

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

8. Use of the thymus-dependent T cell epitope peptide of the lung cancer-related antigen Cyfra 21-1 according to claim 7 for the preparation of a kit for lung cancer-related antigen Cyfra 21-1 T cells according to claim 1, characterized in that, The kit includes: the detection reagent is an enzyme-linked immunospot reagent, an intracellular cytokine fluorescence staining reagent, an enzyme-linked immunosorbent assay reagent, a human leukocyte antigen multimer fluorescence staining or flow cytometry reagent.

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

10. A medicament for treating lung cancer, characterized by, including the monoclonal antibody according to claim 9.

Images