CN119751570A - Thymus-dependent lymphocyte epitope peptide of lung cancer related antigen Cyfra21-1 and application - Google Patents

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: LAGNEKLTM; KLTMQNLNDR; SYLDKVRAL; 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

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

Technical Field

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

Background

According to the recently released burden of malignant tumor diseases in 2022 China by the cancer center of China, the new occurrence of lung cancer in 2022 China is 1 st, the death case is 1 st, and the high occurrence and high mortality rate of the lung cancer seriously threatens the life and health of 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 with advanced stage, with a poor prognosis, with a 5-year survival rate of <5%.

In our country, the high risk group of NSCLC mainly includes those who smoke for a long period of time, are exposed to secondhand smoke, environmental pollution, occupational carcinogens (such as asbestos and radon gas) exposure, and have a history of familial 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 generally divided into three major subtypes, adenocarcinoma, squamous cell carcinoma, and large cell carcinoma. Adenocarcinomas are more common in women, while squamous cell carcinomas are common in men who smoke. Clinical symptoms include persistent cough, chest pain, shortness of breath, weight loss, and the like.

The keratin 19 fragment (Cyfra 21-1) is a fragment of keratin 19, and is normally present in the form of an oligomer, and the content of serum is extremely low, and when cells become cancerous, the content of keratin 19 in the cells is increased, and after necrosis and shedding of cancer cells, the keratin 19 is released in the form of a dissolved fragment in blood and body fluid. Cyfra21-1 is one of the most valuable tumor-associated antigens of NSCLC and is also a common tumor marker for clinical diagnosis of NSCLC and for monitoring and assessing prognosis recurrence. The patent selects the lung cancer related tumor antigen Cyfra21-1 for research.

Cytotoxic T cells (Cytotoxic T lymphocyte, CTLs) are core cells that mediate adaptive immune responses, play a vital role in the development of anti-infective, anti-neoplastic and hypersensitive responses and autoimmune diseases, and their cell membrane-associated T Cell Receptors (TCRs) are capable of specifically recognizing and binding to complexes of antigen presenting cell surface MHC class I molecules with antigenic peptides, i.e. MHC/antigenic peptide complex molecules. CTL epitopes refer to antigenic peptides bound to MHC class I molecules, which are linear fragments or spatially conformational structures of the antigenic molecules that can be specifically recognized by TCRs, are the basic antigenic units that elicit an immune response and play a critical role in CTL activation.

The MHC system refers to the major histocompatibility complex (major histocompatibility complex, MHC), a group of closely linked gene groups in the vertebrate genome, encoding molecules that express MHC class I and class II proteins. HLA (human leukocyte antigen) is the human MHC system responsible for presenting antigenic peptides (T cell epitope peptides) to T cells, initiating specific immune responses in the body. The antigen peptide presented by HLA-A, B, C and other I molecules activates CD8 ⁺ T cells, and the antigen peptide is differentiated into CTLs, so that the antigen peptide is a main effector cell for targeted killing of tumor cells. However, HLA alleles are highly polymorphic in the population. The HLA molecules are not completely identical among different individuals, and the antigen peptide sequences presented by each HLA molecule are different, so that the T cell immune response is different. Thus, the specific immune response elicited by the same tumor antigen varies from individual to individual. For human lung cancer, HLA class I molecules are mainly responsible for presentation of endogenous lung cancer-associated antigens to CD8 ⁺ CTLs, and activated CTLs apoptosis tumor cells expressing tumor-associated antigens by secretion of perforin and granzyme, etc. Therefore, the number and the function of the lung cancer related antigen specific CD8 ⁺ T cells can be dynamically monitored to accurately reflect the specific immune function state of a lung cancer patient aiming at the lung cancer related antigen. Because of different HLA molecular types of different people, the processing, treatment and presenting capability of different antigens of lung cancer are different, so that the lung cancer related antigen specific T cell immune response reactions with different intensities are caused. According to different HLA molecular types of a lung cancer patient, the antigen peptide of the presented lung cancer related antigen is selected, the number and the reactivity of the specific CD8 ⁺ T cells of the lung cancer related antigen are dynamically monitored, and the method has important significance for monitoring the disease process of the lung cancer patient, diagnosing and formulating a treatment scheme, observing the curative effect, judging the prognosis and the like, and is an important technical means for realizing accurate medical treatment of the lung cancer. Meanwhile, the antigen peptide of the lung cancer related antigen combined by the HLA molecules with high affinity can be used for preparing polypeptide vaccine or gene vaccine for preventing and treating lung cancer.

However, currently, the specific T cell epitope of Cyfra21-1 which is clearly presented by various HLA molecules and can stimulate an organism to cause T cell response is still very few, so that the detection of the specific T cell of the Cyfra21-1 on lung cancer patients carrying different HLA alleles is limited, the research on the action of the specific T cell of the lung cancer Cyfra21-1 in the occurrence and development of lung cancer is also limited, and the personalized detection and accurate immunotherapy of the peptide difference of the antigen peptide of the lung cancer Cyfra21-1 based on the individual difference of HLA genes and the presentation thereof are further limited.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a thymus-dependent lymphocyte epitope peptide of a lung cancer related antigen Cyfra21-1 and application thereof.

The aim of the invention can be achieved by the following technical scheme:

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

LAGNEKLTM;

KLTMQNLNDR;

SYLDKVRAL;

The amino acid sequence after removal or replacement of a single amino acid of the above amino acid sequence.

The second aspect of the invention relates to application of the thymus-dependent lymphocyte epitope peptide of the lung cancer related antigen Cyfra21-1 in preparing lung cancer vaccines.

In a third aspect, the invention relates to a nucleic acid molecule encoding the thymus-dependent lymphocyte epitope peptide of the lung cancer-associated antigen Cyfra 21-1.

In a fourth aspect, the invention relates to a vector comprising a nucleic acid molecule as described above.

In a fifth aspect, the invention relates to a vaccine for the treatment or prophylaxis of lung cancer, comprising a nucleic acid molecule as described above or a vector as described above.

The sixth aspect of the invention relates to an application of the thymus-dependent lymphocyte epitope peptide of the lung cancer related antigen Cyfra21-1 in preparing a medicament for treating or preventing lung cancer.

The seventh aspect of the invention relates to application of the thymus-dependent lymphocyte epitope peptide of the lung cancer related antigen Cyfra21-1 in preparation of a kit of lung cancer related antigen Cyfra 21-1T cells.

Optionally, the kit comprises an ELISA reagent, an intracellular cytokine fluorescent staining reagent, an ELISA reagent, a human leukocyte antigen multimer fluorescent staining or a flow cytometry reagent.

In an eighth aspect, the present invention relates to a monoclonal antibody capable of specifically binding to the above epitope peptide.

In a ninth aspect, the invention relates to a medicament for treating lung cancer, comprising a monoclonal antibody as described above.

More specifically, the invention provides a novel thymus-dependent lymphocyte antigen epitope peptide of a lung cancer related antigen Cyfra21-1, wherein the amino acid sequence of the novel thymus-dependent lymphocyte antigen epitope peptide is any one of epitope peptide sequences shown in the following specification:

from the above table, the above sequence is the antigenic peptide sequence of keratin 19 fragment (Cyfra 21-1) and can be combined with A2402, A1101, B5101, B3501, B4601, B4001, B1302, B5201, C0702, C0701, C0602, C0302 and C1203 molecules with high affinity, and forms an HLA/antigenic peptide complex molecule on the surface of antigen presenting cells, and can be combined with the clone of Cyfra21-1 antigenic peptide specific CD8 ⁺ T cell to stimulate activation, proliferation and differentiation and exert the effect of anti-lung cancer immunity.

The thymus-dependent lymphocyte antigen epitope peptide sequence of the lung cancer related antigen Cyfra21-1 can be used for preparing lung cancer polypeptide vaccine or gene vaccine, wherein the polypeptide vaccine is prepared by artificially synthesizing one or more polypeptides according to the polypeptide sequence, mixing with an adjuvant to prepare a soluble preparation, or loading biological nano materials to prepare a nano polypeptide vaccine, injecting the nano polypeptide vaccine into a lung cancer patient, activating and proliferating Cyfra21-1 specific T cells of the lung cancer patient, and enhancing the tumor killing activity of the T cells of the lung cancer patient, thereby preparing the lung cancer polypeptide vaccine. The preparation of gene vaccine includes constructing one kind of polypeptide or several kinds of polypeptide recombinant DNA gene segment, recombinant plasmid or recombinant virus vector, injecting into lung cancer patient to express one or several kinds of polypeptide, activating and proliferation specific T cell of Cyfra21-1 of lung cancer patient and enhancing tumor killing activity.

The thymic dependent lymphocyte antigen epitope peptide sequence of lung cancer related antigen Cyfra21-1 can be used for preparing detection preparation or kit for detecting lung cancer related antigen Cyfra21-1 specific T cells, one or more polypeptides can be artificially synthesized according to the polypeptide sequence, and can be further prepared into fluorescein-labeled preparation by using a genetic engineering technology and protein engineering technology to detect the number and reactivity of Cyfra21-1 specific T cells in a patient peripheral blood mononuclear cell population by using a flow cytometry method as antigen preparation by mixing and culturing with the patient Peripheral Blood Mononuclear Cells (PBMCs) to stimulate the activation, proliferation and secretion of cytokines of the Cyfra21-1 specific T cells and detecting the synthesis amount of the cytokines by other combined reagents in an ELISA method, wherein the polypeptide sequence can be used for preparing a human leukocyte antigen-polypeptide complex (peptide-HLA complex) and a polymer thereof by using a genetic engineering technology and a protein engineering technology. The related kit is a lung cancer related antigen Cyfra21-1 specific T cell detection kit assembled by the preparation and other reagents commonly used in different detection methods.

The thymus-dependent lymphocyte antigen epitope peptide sequence of the lung cancer related antigen Cyfra21-1 can be used for preparing medicaments for treating lung cancer, wherein the polypeptide vaccine or gene vaccine based on the polypeptide sequence is combined with other immunotherapeutic preparations or chemotherapeutic preparations to prepare clinical medicaments for treating lung cancer.

The invention virtually predicts the specific antigen epitope peptide sequences of lung cancer related antigens Cyfra21-1 limited by 13 HLA-A (A0201, A1101, A2402, A3101, A0206, A0207, A3303, A3001, A0203, A1102, A0301, A0101 and 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 and C0701) molecules by utilizing six online epitope prediction databases, and then verifies the immunogenicity of lung cancer related antigens Cyfra21-1 through an Intracellular Cytokine Staining (ICS) experiment, thereby providing specific antigen peptide sequences for preparing and detecting lung cancer specific antigen peptides of lung cancer cells of cell tumor cells.

1. Selecting the keratin 19 (Cyfra 21-1) amino acid sequence as a targeting sequence;

2. the predicted result is selected to obtain six commonly used epitope prediction databases which are commonly used in the field and have higher accuracy, wherein SYFPEITHI, BIMAS, SVMHC, IEDB, NETMHC and EPIJEN are used for predicting the restricted Cyfra21-1 epitope peptide sequences of the 13 HLA-A molecules, 15 HLA-B molecules and 14 HLA-C molecules;

3. and carrying out integration analysis on the predicted results of the six online epitope predicted websites according to a certain prediction standard to obtain candidate antigen peptide sequences with more consistent predicted results of the six websites.

4. The immunogenicity of the lung cancer related antigen peptide Cyfra21-1 is verified by IFN-gamma ICS cell functional experiments.

5. And (3) carrying out polypeptide competition binding experiments of HLA molecules by using HMy2.CIR cell lines expressing the specific HLA molecules, and analyzing the binding force of the positive epitope peptide and the specific HLA molecules.

6. The immunogenicity of positive epitope peptides to stimulate proliferation of specific T cell activation in vivo was verified by immunizing C57BL/6J mice.

The invention relates to an antigen peptide sequence which can be combined with A2402, A1101, B5101, B3501, B4601, B4001, B1302, B5201, C0702, C0701, C0602, C0302 and C1203 molecules in HLA molecules in a keratin 19 fragment (Cyfra 21-1) in a high affinity way and has immunogenicity, a lung cancer polypeptide vaccine and a gene vaccine based on the antigen peptide, and a reagent and a method for detecting lung cancer Cyfra21-1 specific T cells based on the antigen peptide.

Compared with the prior art, the invention has the following advantages:

The specific epitope peptide of the lung cancer related antigen Cyfra21-1 with the restriction of A2402, A1101, B5101, B3501, B4601, B4001, B1302, B5201, C0702, C0701, C0602, C0302 and C1203 molecules obtained through on-line virtual prediction and functional experiment verification has not been reported before. These HLA molecules have not previously been reported to have a restricted lung cancer associated antigenic peptide. The experimental data show that the novel epitope peptide sequences have better affinity with specific HLA molecules, and the vaccine prepared based on the epitope peptide sequences can enable organisms to generate strong immune response. Therefore, the epitope peptide provided by the invention provides a key antigen component, namely an epitope peptide sequence, for developing a polypeptide vaccine and a gene vaccine for treating and preventing lung cancer, designing a reagent and a method for detecting lung cancer Cyfra21-1 specific T cells, and the like, and simultaneously provides a key antigen component for individual detection and accurate medical treatment of lung cancer patients aiming at specific HLA alleles.

Drawings

FIG. 1 shows the flow assay of the activation of epitope peptide-stimulated CD8 ⁺ T cells in a polypeptide-PBMCs co-culture experiment, FIG. 1 shows a flow assay scatter plot of IFN-gamma secretion after stimulation with a specific epitope peptide in CD3 ⁺/CD8⁺ T cell populations of each lung cancer patient, and FIG. 1 shows a statistical plot of the proportion of IFN-gamma secretion after stimulation with a specific epitope peptide in CD3 ⁺/CD8⁺ T cell populations of each lung cancer patient. NC is negative control, no.1 is epitope peptide No.1 (SEQ ID NO. 1), no.2 is epitope peptide No.2 (SEQ ID NO. 2), and No.3 is epitope peptide No.3 (SEQ ID NO. 3).

FIG. 2 polypeptide competition binding experiments 3 Cyfra21-1 positive T cell epitopes were analyzed for binding affinity to specific HLA molecules. NC, negative control, epitope peptide No. 1:1 (SEQ ID NO. 1), epitope peptide No. 2:2 (SEQ ID NO. 2), epitope peptide No. 3:3 (SEQ ID NO. 3), blue and red solid lines show the fluorescence peaks of the test peptide at 5. Mu.M and 15. Mu.M concentrations, respectively, while gray filled lines represent the maximum fluorescence intensity of the cells with FITC-reference peptide alone. The light green solid line in the NC plot represents the negative control background fluorescence of the peptide-free cells.

FIG. 3 is a technical scheme of epitope peptide prediction and screening, immunogenicity validation, and polypeptide competition binding experiments.

FIG. 4 epitope peptide induced a specific CD8 ⁺ T cell response in C57BL/6J mice

The 3 positive epitope peptides are prepared into a mixed polypeptide vaccine, and are combined with an adjuvant poly (I: C), C57BL/6J mice are immunized on 0,7,21 days respectively, spleen cells of the mice are taken after 7 days of last immunization, the spleen cells are respectively stimulated and cultured for 10 hours with PBS and each positive epitope peptide, BFA/Monesion (blocking cytokine release) is added for continuous culture for 6 hours, and then IFN-gamma intracellular staining and flow analysis are carried out to detect the frequency of IFN-gamma ⁺/CD3⁺/CD8⁺ T cells. In FIG. 4, A is a representative flow chart and B is a statistical plot of the frequency of IFN-. Gamma. ⁺/CD3⁺/CD8⁺ T cells per group of mice.

Detailed Description

The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

In some embodiments of the invention, a2402, a1101, B5101, B3501, B4601, B4001, B1302, B5201, C0702, C0701, C0602, C0302, C1203 molecule restricted lung cancer associated antigen Cyfra 21-1T cell epitopes are disclosed, the sequence is screened and identified by:

1 on-line virtual prediction of dominant T cell epitope peptide of lung cancer related antigen Cyfra21-1 limited by 13 HLA-A, 15 HLA-B and 14 HLA-C molecules

The lung cancer related antigen protein is selected, keratin 19 fragment (Cyfra 21-1, SEQ ID NO. 4) is searched through UniProt global protein resource database to obtain the amino acid sequence, the most studied standard sequence is selected, then, A0201、A1101、A2402、A3101、A0206、A0207、A3303、A3001、A0203、A1102、A0301、A0101、A2601、B4601、B4001、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 molecule limited T cell epitope peptide aiming at Cyfra21-1 antigen protein is virtually predicted through SYFPEITHI, BIMAS, SVMHC, IEDB, NETMHC, EPIJEN and other six common epitope peptide prediction databases, wherein SVMHC database comprises MHCPEP model and SYFEPITHI MODEL prediction algorithms, IEDB database comprises ANN, SMM and ARB methods, and the embodiment adopts the ANN and the SMM methods with better statistical significance. These epitope peptide predictive database websites are shown in table 1. The amino acid sequence of the lung cancer related antigen Cyfra21-1 used for predicting the epitope is as follows:

The antigen binding groove of HLA class I molecules is closed at both ends, and the received antigenic peptide is 8-11 amino acid residues in length, with 9 and 10 amino acids being the most common, so in this example, polypeptides of 9 and 10 amino acids are mainly selected as subjects. The amino acid sequence of Cyfra21-1 is input into a corresponding amino acid sequence input frame of a forecast database website, the length of epitope peptide is respectively selected to be 9 and 10 amino acids, then a specific HLA molecule is selected, and on-line virtual forecast is carried out on the T cell epitope peptide of the lung cancer related antigen Cyfra 21-1.

Table 1 epitope peptide predictive database website

For each HLA molecule, the polypeptides predicted by different databases are respectively arranged from high score to low score, and then epitope peptides meeting at least more than 4 prediction method scoring standards are selected as candidate epitope peptides. For each HLA molecule, 1-5 polypeptides with highest scores (highest affinities) 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, 59 dominant T cell epitope peptides to be identified were predicted by total screening.

2 Separation of peripheral blood PBMC of lung cancer patient

Taking fresh anticoagulated whole blood stored at room temperature, properly diluting with sterile PBS, adding a human lymphocyte separation solution (Daidae biology, shenzhen) with a volume of 1 time of the whole blood into a 15mL centrifuge tube, slowly spreading the diluted blood on the separation solution, centrifuging at room temperature for 20min at 2000rpm, sucking a mononuclear cell (PBMCs) layer, centrifuging and washing for 2 times, re-suspending with a serum-free culture medium (Daidae biology, shenzhen) and adjusting the cell concentration to 3X 10 ⁶/mL for standby.

3 Identification of immunogenicity of lung cancer related antigen Cyfra 21-1T cell epitope peptide by IFN-gamma ICS method

The technical route is shown in fig. 3:

4HLA allele typing

200 Mu L of anticoagulation is collected from a patient, genomic DNA is extracted by using a human whole blood genomic DNA extraction kit (Tian Gen organism, beijing), PCR is performed by using HLA site-specific primers, and DNA sequences of exon 2, intron 2, exon 3 and partial introns L and 3 of the A site, the B site or the C site are amplified respectively, and the size of the product is 985bp. The amplification conditions were 95℃pre-denaturation for 3min, 95℃denaturation for 15s, 62℃annealing for 15s, 72℃extension for 90s, 35 cycles, 72℃extension for 5min. The amplified products were sized by 1% agarose gel electrophoresis and sent to Shanghai Sanny Biotech for purification and two-way sequencing. PCR reagents were purchased from Nanjinouzan Biotech.

TABLE 2HLA site-specific PCR amplification primers and sequencing primers (SEQ ID NO. 5-22, respectively)

Sequencing results of exon 2 and exon 3 are spliced into a complete HLA overlapping sequence (Contig) by Seqman software of a Lasergene program, whether bases sequenced in two directions are completely consistent or not is carefully checked, bases of heterozygotes are found and replaced by facultative bases, for example M represents A and C, R represents A and G, W represents A and T, S represents C and G, Y represents C and T, K represents G and T, and sequence fragments of amplified HLA alleles are finally determined. And (3) comparing the spliced HLA base sequences with the exon 2 and exon 3 sequences of all HLA alleles in the database by using a nucleotides BLAST tool until a completely matched gene combination is obtained, so as to determine the HLA alleles.

5 Polypeptide-PBMCs Co-culture and IFN-gamma intracellular staining experiments

PBMCs were isolated by density gradient centrifugation, adjusted to a concentration of 3X 10 ⁶/mL in ELISPOT serum-free cell culture medium, seeded in U-bottom 96-well plates at 100. Mu.L/well and placed in a 37℃incubator at 5% CO ₂ for use. According to the HLA allele typing result of the patient, co-culturing the corresponding candidate epitope peptide with the PBMCs in the 96-well plate for 6 hours, synchronously adding Brefeldin ASolution/Monensin Solution (1000X) blocking agent for co-culturing, then carrying out IFN-gamma intracellular fluorescent staining, detecting the frequency of CD8 ⁺ T cells secreting IFN-gamma by a flow cytometer, and simultaneously setting a single culture hole of the PBMCs without polypeptide stimulation as a negative control group. The candidate epitope peptide that increased the frequency of IFN-. Gamma. ⁺/CD8⁺ T cells by more than 2-fold compared to the negative control was set as a positive epitope peptide, i.e., the epitope peptide stimulated activation and cytokine secretion of memory CD8 ⁺ T cells in the patient's PBMCs.

Through IFN-gamma ICS experiments, total collection of 150 lung cancer patients of PBMCs and 59 candidate epitope peptides of related polypeptides for co-culture experiments. Wherein, the 3 candidate epitope peptides (the sequences are LAGNEKLTM, KLTMQNLNDR and SYLDKVRAL respectively) induce strong CD8 ⁺ T cell reaction, and at least the frequency of IFN-gamma ⁺/CD8⁺ T cells of more than 3 cases of lung cancer patients is increased by 2-10 times, so that the 3 candidate epitope peptides are positive epitopes and dominant epitope peptides, and have stronger immunogenicity (figure 1).

6 Polypeptide competitive binding assay for the affinity of candidate epitope peptides to corresponding HLA molecules

59 Candidate epitopes (HMy 2.CIR cell lines which temporarily lack expression of HLA-A2601, so 10 candidate epitopes cannot participate in the experiment) including three positive epitope peptides screened in the previous step, namely No.1, no.2 and No. 3, which are verified by the peptide-PBMCs co-stimulation experiment, are distributed into relevant epitope peptide queues of each HLA molecule according to the predicted HLA restriction and the HLA allele type of a patient with positive T cell reaction, and are used as peptides to be tested, and polypeptide competitive binding experiments are respectively carried out with fluorescent reference peptides of the HLA molecules. Finally, these three positive peptides 1,2, 3 all shifted the cell fluorescence peak to the left (fig. 2), suggesting that these positive epitope peptides could compete with the fluorescent reference peptide for binding to the relevant HLA molecule on hmy2.Cir cell membrane, and these 3 positive epitope peptides all exhibited high or medium affinity binding to the corresponding HLA molecule (table 3).

Table 3 polypeptide competitive binding assay 3 Cyfra21-1 positive T cell epitopes were analyzed for binding affinity to 13 dominant HLA molecules

C57BL/6J mice immunized with the 7 epitope peptide were validated for immunogenicity inducing specific T cell responses in vivo

3 Positive epitope peptides (including epitope peptides No. 1, no. 2 and No. 3 of the present application) were made into mixed polypeptides, which were then mixed with the adjuvant polyI: C. The C57BL/6J mice were randomly divided into 2 groups of 3 mice each. The experimental groups were inoculated with peptide cocktail/poly (I: C) (epitope peptide: 10. Mu.g/mono peptide, poly (I: C): 100. Mu.g/C) on day 0, day 7, and day 21, respectively, and the adjuvant groups were also inoculated with poly (I: C) (100. Mu.g/C) on day 0, day 7, respectively. Four subcutaneous injections (dorsal cervical, caudal, bilateral inguinal) were performed.

On day 28, splenocyte suspensions were routinely prepared, cell concentrations were adjusted to 1X 10 ⁷/mL with 10% FBS-1640 medium, 48-well cell culture plates (0.5 mL/well) were inoculated, each rat splenocyte inoculation well was divided into 4 groups, PBS (negative control) and each positive epitope peptide (including the three epitope peptides of the application SEQ ID NO. 1-3, 20. Mu.g/mL/peptide) were added separately, BFA and Monensin were added to each well after 10 hours of incubation, cells were collected and blocked with FcR Blocking Reagent mouse for 15 minutes, stained with FITC-anti-mouse CD3 and PE-anti-mouse CD8a for 30 minutes, membrane breaker treatment was added to each tube after washing, intracellular staining was performed with APC-anti-mouse IFN-gamma monoclonal antibodies for 30 minutes, CD3 ⁺/CD8⁺/IFN-γ⁺ T cell frequencies were analyzed with a flow cytometer after washing, and epitope peptide-induced specific CD8 ⁺ T cell reaction was observed to be weak.

The results show that all three positive epitope peptides (peptide cocktail/polyI: group C) of the application induced a strong epitope peptide specific CD8 ⁺ T cell response in mice. These three epitope peptides induced IFN-. Gamma. ⁺/CD8⁺ T cells at 20-30 times the frequency of the adjuvant group compared to the adjuvant group.

In the description of the present specification, the descriptions of the terms "one embodiment," "example," "specific example," and the like, mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiments or examples. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

The foregoing has shown and described the basic principles, principal features and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, and that the above embodiments and descriptions are merely illustrative of the principles of the present invention, and various changes and modifications may be made without departing from the spirit and scope of the invention, which is defined in the appended claims.

Claims (10)

1. A thymus-dependent lymphocyte antigen epitope peptide of a lung cancer-associated antigen Cyfra21-1, wherein the amino acid sequence of said epitope peptide comprises any one or more of the following:

LAGNEKLTM;

KLTMQNLNDR;

SYLDKVRAL;

The amino acid sequence after removal or replacement of a single amino acid of the above amino acid sequence.

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

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

4. A vector comprising the nucleic acid molecule of claim 3.

5. A vaccine for the treatment or prophylaxis of lung cancer, comprising the nucleic acid molecule of claim 3 or the vector of claim 4.

6. An application comprising any one of the following:

use of the thymus-dependent lymphocyte epitope peptide of the lung cancer-associated antigen Cyfra21-1 according to claim 1, in the manufacture of a medicament for treating or preventing lung cancer;

Use of the thymus-dependent lymphocyte epitope peptide of the lung cancer-associated antigen Cyfra21-1, the nucleic acid molecule of claim 3 or the vector of claim 4 in the preparation of a kit for lung cancer-associated antigen Cyfra 21-1T cells.

7. The use of the thymus-dependent lymphocyte epitope peptide of the lung cancer-associated antigen Cyfra21-1 according to claim 6, in the preparation of a kit for preparing the lung cancer-associated antigen Cyfra 21-1T cells, wherein the kit comprises an enzyme-linked immunosorbent assay reagent, an intracellular cytokine fluorescent staining method reagent, an enzyme-linked immunosorbent assay reagent, a human leukocyte antigen multimer fluorescent staining or a flow cytometry assay reagent.

8. A kit for detecting a lung cancer associated antigen Cyfra 21-1T cell, comprising the epitope peptide of claim 1, or further comprising a recombinant HLA molecule that binds to said epitope peptide.

9. A monoclonal antibody capable of specifically binding to the epitope peptide of claim 1.

10. A medicament for treating lung cancer comprising the monoclonal antibody of claim 9.