Tumor-specific peptide sequences and their applications

Abstract

Provided are tumor-specific polypeptide sequences and applications thereof. The polypeptide includes at least one polypeptide in the first peptide group, and may also selectively include at least one polypeptide in the second peptide group; the first peptide group includes polypeptides having sequences shown in SEQ ID NO:1 to SEQ ID NO:5; the second peptide group includes derivative peptides of sequences shown in SEQ ID NO:1 to SEQ ID NO:5, the derivative peptides including sequentially connected front, middle and back peptide segments, the middle peptide segment having at least 80% homology with the sequences shown in SEQ ID NO:1 to SEQ ID NO:5, and the sum of the lengths of the front and back peptide segments being 14 to 16 amino acids. Also provided are isolated nucleic acids, constructs, expression vectors, pharmaceutical compositions, antigen presenting cells, immune effector cells, tumor vaccines, and uses of polypeptides in preparing drugs for preventing or treating tumors and methods for treating tumor patients.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a tumor-specific polypeptide sequence and its application, specifically to a group of isolated polypeptides, isolated nucleic acids, constructs, expression vectors, host cells, pharmaceutical compositions, antigen presenting cells, immune effector cells, tumor vaccines, and the use of polypeptides in preparing drugs for preventing or treating tumors and methods for treating tumors in patients. Background Art

[0002] Cancer, a disease caused by uncontrolled cell proliferation due to gene mutations in cells, has become a major threat to human health and a major cause of death. The "2015 Analysis of the Prevalence of Malignant Tumors in China" released by the National Cancer Center pointed out that in 2015, there were about 3.929 million cases of malignant tumors in China and about 2.338 million deaths. The burden of cancer continues to rise. In the past 10 years, the incidence of malignant tumors has maintained an annual increase of about 3.9%, and the mortality rate has maintained an annual increase of 2.5%. Among them, the main high-incidence malignant tumors are lung cancer, gastric cancer, colorectal cancer, liver cancer, breast cancer and esophageal cancer. Therefore, finding efficient and specific cancer treatment methods has great clinical value.

[0003] Immunotherapy mobilizes the body's immune system and enhances the anti-tumor immunity of the tumor microenvironment, thereby achieving the purpose of controlling and killing tumor cells. It has the advantages of high efficiency, strong specificity, and good tolerance, and has broad prospects in tumor treatment. Immunotherapy mainly includes cytokine therapy, immune checkpoint monoclonal antibodies, adoptive cell transfer, tumor immunotherapy vaccines, etc. Among them, tumor immunotherapy vaccines mainly include tumor cell vaccines, dendritic cell vaccines, protein & peptide vaccines, nucleic acid vaccines, genetic engineering vaccines and anti-idiotypic antibody vaccines. The main mechanism by which these vaccines can kill tumors is to induce the patient's immune response against the tumor, allowing T cells to recognize tumor cells and then kill tumor cells.

[0004] Tumor antigens targeted by tumor immunotherapy vaccines include tumor-associated antigens and tumor neoantigens (neoantigens). Tumor-associated antigens are derived from proteins that are highly expressed in tumor tissues but lowly expressed or not expressed in normal tissues; while tumor neoantigens are derived from mutant proteins produced by mutations in the tumor genome. Because tumor neoantigens only exist in tumor cells and not in normal cells, they can bypass central immune tolerance and induce strong T cell immune responses, and have the characteristics of good effects; at the same time, the tumor-specific characteristics make tumor neoantigens have the advantages of good safety and few side effects. However, suitable tumor neoantigens targeted by tumor immunotherapy vaccines need to be further improved. Summary of the invention

[0005] The present invention aims to solve one of the technical problems in the related art at least to a certain extent. To this end, one object of the present invention is to provide a tumor-specific polypeptide sequence and its application, specifically a group of isolated polypeptides, isolated nucleic acids, constructs, expression vectors, host cells, pharmaceutical compositions, antigen presenting cells, immune effector cells, tumor vaccines, and the use of polypeptides in the preparation of drugs for preventing or treating tumors and methods for treating tumor patients.

[0006] When treating patients with tumor immunotherapy, several schemes are often used: 1. Treating patients with tumor-associated antigens that are highly expressed in the patient's tumor. This treatment method may be poor because the tumor-associated antigens are also expressed in some normal tissues and have low immunogenicity. 2. Treating with tumor-associated antigens or tumor neoantigens that have been experimentally identified in some patients. However, tumor mutations are patient-specific, and most tumor mutations will not recur in multiple patients. Therefore, if the tumor-specific antigens identified in some patients have not been verified by the frequency of occurrence in a large number of tumor patients, the probability of being reused in new patients is low, resulting in a small number of patients who can be treated with these tumor neoantigens. 3. Individualized tumor neoantigen screening for each patient, for example, the patient's tumor-specific mutations and the variant peptides that may be generated by these mutations can be obtained by analyzing the sequencing data of their genome and transcriptome, and then predicting which variant peptides may be presented as antigens by MHC molecules through machine learning algorithms, and then using these predicted tumor neoantigens for patient treatment. The personalized tumor neoantigen screening program based on sequencing can screen out tumor neoantigens for treating a patient by sequencing the genome and transcriptome of a patient, analyzing the sequencing data and predicting antigens through algorithms, but the whole process is costly and time-consuming. In addition, due to the low accuracy of the current antigen prediction algorithm, the false positive rate of the screened antigens is high, and some predicted antigens cannot effectively induce an immune response in the patient's body, resulting in poor efficacy for the patient. 4. Combining the above schemes, that is, using the identified tumor-related antigens and tumor neoantigen sets, combined with personalized tumor neoantigen screening schemes. For example, the known antigens in Scheme 1 or Scheme 2 are used to treat the patient in the first stage, and the patient is screened for personalized antigens according to Scheme 3, and then the antigens obtained by the screening are used for the second stage of treatment of the patient. Although this scheme can make up for the problem of long time for personalized tumor neoantigen screening, the cost of treatment cannot be reduced because it also involves personalized tumor neoantigen screening schemes.

[0007] Through a large amount of data analysis and experimental screening, the present invention discovered a high-frequency mutation gene MUC3A (the wild-type MUC3A gene encodes mucin 3A, providing lubrication, cell signaling pathways and chemical barrier functions) that repeatedly appears in a variety of cancers. The high-frequency mutation gene causes the amino acid at the 326th position encoded by it to change from serine (S) to threonine (T). The mutant polypeptide can be specifically and highly expressed in tumor tissues. The present invention experimentally verified the high affinity of the mutant polypeptide to the molecules of HLA-A11:01 typing and the presentation in tumor cells. Further, the mutant polypeptide is sequenced and a large number of experiments are performed to screen out a deformed polypeptide that can be recognized by the same T cells as the original mutant polypeptide, but has a stronger ability to activate T cells and induce antigen-specific T cells to kill tumors.

[0008] Specifically, the present invention provides the following technical solutions:

[0009] In the first aspect of the present invention, the present invention proposes a group of separated polypeptides. According to an embodiment of the present invention, the polypeptide includes at least any one polypeptide in the first peptide group, and may also selectively include at least any one polypeptide in the second peptide group; the first peptide group includes a polypeptide having an amino acid sequence shown in SEQ ID NO:1 to SEQ ID NO:5; the second peptide group includes a derivative peptide of the amino acid sequence shown in SEQ ID NO:1 to SEQ ID NO:5, the derivative peptide includes a sequentially connected pre-peptide segment, a mid-peptide segment and a post-peptide segment, the mid-peptide segment has at least 80% homology with the amino acid sequence shown in SEQ ID NO:1 to SEQ ID NO:5, and the sum of the lengths of the pre-peptide segment and the post-peptide segment is 14 to 16 amino acids.

[0010] The polypeptide sequence proposed in the present invention is a tumor-specific antigen and its variants generated by tumor gene mutations. It will not be expressed and presented in normal tissues, thus overcoming the problem of low safety of using tumor-associated antigens for treatment. At the same time, the proposed polypeptide sequence comes from genes with high frequency mutations in various cancers and can be presented by HLA molecules that appear frequently in the population. Therefore, it can appear repeatedly in tumors of patients with various cancers, and can cover patients that are not covered by currently known tumor neoantigen sequences.

[0011] According to an embodiment of the present invention, the isolated polypeptide described above may further include the following technical features:

[0012] In some embodiments of the present invention, the mid-peptide segment has at least 90% homology with the amino acid sequence shown in SEQ ID NO: 1 to SEQ ID NO: 5.

[0013] In some embodiments of the present invention, the mid-peptide segment is identical to the amino acid sequence shown in SEQ ID NO:1 to SEQ ID NO:5.

[0014] In some embodiments of the present invention, the derivative peptide has an amino acid sequence shown in SEQ ID NO:6 to SEQ ID NO:10.

[0015] In some embodiments of the present invention, the polypeptide is selected from at least one of the following groups:

[0016] (1) at least two polypeptides having the amino acid sequences shown in SEQ ID NO: 1 to SEQ ID NO: 5;

[0017] (2) at least one polypeptide having an amino acid sequence as shown in SEQ ID NO: 1 to SEQ ID NO: 5, and at least one polypeptide having an amino acid sequence as shown in SEQ ID NO: 6 to SEQ ID NO: 10.

[0018] In the second aspect of the present invention, the present invention provides an isolated nucleic acid, according to an embodiment of the present invention, the nucleic acid encodes the above polypeptide or its complementary sequence. As mentioned above, the above polypeptide can be presented as an antigen on the surface of tumor cells by HLA molecules with affinity, and has the ability to activate T cells and guide these T cells to kill tumors, so the nucleic acid sequence encoding the above polypeptide or the complementary sequence of the nucleic acid sequence encoding the above polypeptide can be used to prevent or treat tumors.

[0019] In the third aspect of the present invention, the present invention proposes a construct. According to an embodiment of the present invention, the construct comprises the nucleic acid and control sequence described in the second aspect of the present invention, and the control sequence is operably connected to the nucleic acid. The construct proposed in the embodiment of the present invention can efficiently express the above-mentioned polypeptide in a suitable host cell under suitable conditions, and can be effectively used for the treatment or prevention of tumors. Among them, the control sequence can guide the nucleic acid to express the above-mentioned polypeptide in the host, and these control sequences can be one or more. These control sequences can be promoters, terminators, SD sequences, regulatory genes for regulating gene expression, etc. as needed.

[0020] In the fourth aspect of the present invention, the present invention provides an expression vector. According to an embodiment of the present invention, the expression vector comprises the construct described in the third aspect of the present invention. The expression vector provided by the present invention can efficiently express the above polypeptide in a host under appropriate conditions, and the expression vector can be effectively used for the treatment or prevention of tumors.

[0021] In the fifth aspect of the present invention, the present invention provides a host cell. According to an embodiment of the present invention, the host cell carries the construct described in the third aspect of the present invention or the expression vector described in the fourth aspect of the present invention, and the host cell can be obtained by transfecting or transforming the aforementioned nucleic acid construct or expression vector. The host cell can efficiently express the above polypeptide under appropriate conditions, and the host cell can be effectively used for the treatment or prevention of tumors.

[0022] In the sixth aspect of the present invention, the present invention proposes the use of a polypeptide in the preparation of a drug for preventing or treating tumors or in the preparation of a kit for diagnosing tumors. If the tumor expresses the above-mentioned mutant gene MUC3A, the high-frequency mutant gene causes the amino acid at the 326th position encoded by it to change from serine (S) to threonine (T), and expresses an HLA molecule of the HLA-A11:01 type with affinity to the polypeptide, then the above-mentioned polypeptide has the ability to be presented as an antigen on the surface of tumor cells by the HLA molecule of the HLA-A11:01 type with affinity to it, activate T cells and guide these T cells to kill tumors. Therefore, the proposed polypeptide can be used to prevent and control tumors. At the same time, as mentioned above, since the polypeptide proposed by the present invention is specifically expressed in tumor cells, it is used to treat or prevent tumors with good safety. It can also be used in the preparation of a kit for diagnosing tumors.

[0023] In the seventh aspect of the present invention, the present invention proposes a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition comprises the aforementioned polypeptide and a pharmaceutically acceptable excipient. The pharmaceutical composition comprising the aforementioned polypeptide and excipient can significantly stimulate the proliferation of tumor-specific T cells and the cytokine secretion of these T cells, thereby killing tumor cells expressing corresponding mutant genes, and can be used for the treatment or prevention of tumors. Of course, the provided pharmaceutical composition may also include some pharmaceutically acceptable adjuvants, which act as non-specific immunopotentiators. When injected with the aforementioned polypeptide or pre-injected into the body, the body's immune response to the polypeptide antigen can be enhanced or the type of immune response can be changed. Available adjuvants include, but are not limited to, PD-1 inhibitors.

[0024] In the eighth aspect of the present invention, the present invention proposes an antigen presenting cell. According to an embodiment of the present invention, the antigen presenting cell can present the aforementioned polypeptide. Antigen presenting cells can be obtained by loading the polypeptide, transfecting or transforming the aforementioned nucleic acid construct or expression vector, or engulfing the aforementioned host cells. According to an embodiment of the present invention, the antigen presenting cells presenting the aforementioned polypeptide significantly stimulate the proliferation of tumor-specific T cells and the cytokine secretion of these T cells, thereby killing tumor cells expressing corresponding mutant genes, and can be used for the treatment or prevention of tumors. These available antigen presenting cells can be dendritic cells, macrophages, B cells, etc.

[0025] In the ninth aspect of the present invention, the present invention proposes an immune effector cell. According to an embodiment of the present invention, the immune effector cell can recognize the aforementioned polypeptide or recognize the antigen presenting cell described in the eighth aspect of the present invention. The immune effector cell can be induced by the aforementioned polypeptide or the aforementioned antigen presenting cell. The immune effector cell can specifically kill tumor cells expressing corresponding mutant genes for the treatment or prevention of tumors. These available immune effector cells can be T cells, effector T cells, NK cells, etc.

[0026] In the tenth aspect of the present invention, the present invention provides a tumor vaccine. According to an embodiment of the present invention, the tumor vaccine comprises the aforementioned nucleic acid, or the aforementioned nucleic acid construct, or the aforementioned expression vector, or the aforementioned host cell, or the aforementioned antigen presenting cell, or the aforementioned immune effector cell.

[0027] In the eleventh aspect of the present invention, the present invention provides a method for treating tumor patients, the method comprising administering to the patient an effective amount of a pharmaceutical composition or an effective amount of a tumor vaccine, wherein the pharmaceutical composition is the pharmaceutical composition described in the seventh aspect of the present invention, and the tumor vaccine is the tumor vaccine described in the tenth aspect of the present invention. Among them, an "effective amount" of a pharmaceutical composition refers to any amount that can achieve the purpose of inhibiting tumor growth or intervening in tumor proliferation. An "effective amount" of a tumor vaccine refers to the introduction of these tumor vaccines into the patient's body, which can overcome the immunosuppressive state caused by the tumor and activate the patient's own immune system, thereby achieving the purpose of controlling or eliminating the tumor. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is a diagram of the mass spectrometry identification result of the mutant polypeptide provided in an embodiment of the present invention.

[0029] Figure 2 It is a flow cytometry verification result diagram of the affinity between the polypeptide and T2 provided in an embodiment of the present invention.

[0030] Figure 3 It is a diagram of the results of in vitro immunogenicity ELISPOTs detection of polypeptides provided according to an embodiment of the present invention.

[0031] Figure 4 This is a graph showing the results of controlling mouse tumor growth with a polypeptide vaccine provided in an embodiment of the present invention.

[0032] Figure 5 This is a graph showing the results of controlling mouse tumor growth with the polypeptide DC vaccine provided in an embodiment of the present invention.

[0033] Figure 6This is a graph showing the results of controlling mouse tumor growth with the DC-CTL vaccine provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0034] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0035] At the same time, in order to facilitate the understanding of those skilled in the art, certain terms of the present invention are explained and illustrated. It should be noted that these explanations and illustrations are only used to help the understanding of the technical solution of the present invention, and should not be regarded as limiting the scope of protection of the present invention.

[0036] Herein, the term "first peptide group" or "second peptide group" refers to polypeptides comprising different amino acid sequences, respectively.

[0037] Herein, the term "derivative peptide" is used to refer to a polypeptide sequence derived from a polypeptide having an amino acid sequence as shown in SEQ ID NO:1 to SEQ ID NO:5, wherein these derived sequences include, from the N-terminus to the C-terminus, a connected pre-peptide segment, a mid-peptide segment and a post-peptide segment, wherein the mid-peptide segment has at least 80% homology with the amino acid sequence as shown in SEQ ID NO:1 to SEQ ID NO:5, preferably at least 90% homology, and the sum of the lengths of the pre-peptide segment and the post-peptide segment is 14 to 16 amino acids. There is no particular limitation on the specific types of amino acids in the pre-peptide segment and the post-peptide segment. In at least some embodiments, these derived peptides may be long peptide sequences with a total length of 23mer to 25mer obtained by extending the amino acid sequence as shown in SEQ ID NO:1 to SEQ ID NO:5 to both sides. In some preferred embodiments, these derived peptides may be polypeptides having an amino acid sequence as shown in SEQ ID NO:6 to SEQ ID NO:10.

[0038] In at least some embodiments of the present invention, the isolated polypeptide provided by the present invention is selected from at least one group of the following: 1) a polypeptide having an amino acid sequence shown in SEQ ID NO:1-SEQ ID NO:5; (2) at least any one polypeptide having an amino acid sequence shown in SEQ ID NO:1-SEQ ID NO:5, and at least any one polypeptide having an amino acid sequence shown in SEQ ID NO:6 to SEQ ID NO:10.

[0039] The polypeptides with amino acid sequences shown in SEQ ID NO: 1 to SEQ ID NO: 10 are shown in Table 1 below.

[0040] Table 1 Peptide and its variant sequences

[0041] SEQ ID Peptide sequence Mutation information SEQ ID Derived peptide sequences NO:1 TILPTTITK MUC3A_S326T NO:6 PLSTLVTILPTTITKSTPTSETT NO:2 TSLPTTITK MUC3A_S326T NO:7 PLSTLVTSLPTTITKSTPTSETT NO:3 TTLPTTITR MUC3A_S326T NO:8 PLSTLVTTLPTTITRSTPTSETT NO:4 TTLPTTITK MUC3A_S326T NO:9 PLSTLVTTLPTTITKSTPTSETT NO:5 TVLPTTITK MUC3A_S326T NO:10 PLSTLVTVLPTTITKSTPTSETT

[0042] These polypeptide sequences are derived from tumor-specific antigens generated by tumor gene mutations and will not be expressed or presented in normal tissues. Therefore, they have higher specificity and induce higher specific immune responses. They are safe for treatment and have few side effects. They are simple in structure and easy to synthesize artificially. At the same time, these polypeptide sequences have the characteristics of affinity with HLA molecules, ability to stimulate T cell proliferation and secretion of cytokines, and strong ability to induce antigen-specific T cells to kill target cells. They do not change their specificity with T cells and have better tumor control effects.

[0043] The scheme of the present invention will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used are not indicated by the manufacturer and are all conventional products that can be obtained commercially.

[0044] Embodiment 1

[0045] Based on a large amount of tumor mutation data in public databases such as TCGA / ICGC, the high-frequency mutations were counted, and the high-frequency typing of the Chinese population was predicted, screened, and experimentally verified to obtain polypeptide sequences, which are shown in SEQID NO:1 to SEQ ID NO:5. At the same time, tumor mutation data were also used to obtain derived peptides, which are polypeptide sequences formed by extending the polypeptide sequence to a length of 23 amino acids on both sides, and the derived peptide sequences are shown in SEQ ID NO:6 to SEQID NO:10.

[0046] The following examples study various polypeptide sequences and derived peptide sequences. For the sake of convenience, the sequence shown in SEQ ID NO: 3 can be referred to as a mutant polypeptide, and the sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 5 can be referred to as deformed polypeptides.

[0047] Example 2 Mass spectrometry experiments verify that peptides are presented by HLA molecules on the surface of tumor cells

[0048] In the present invention, the polypeptide sequence obtained in Example 1 and the coding gene of the derived peptide are transfected into tumor cells by lentivirus, and then the polypeptide-MHC complex on the cell surface is enriched by immunoprecipitation-mass spectrometry, and whether the MHC molecules on the surface of the tumor cells present the mutant polypeptide is identified. The specific method is as follows:

[0049] 1. Isolation and purification of MHC-I restricted T cell epitope peptides:

[0050] Use pan-MHC-I A / B / C antibody (clone number: w6 / 32) to bind to sepharose CL-4B beads with protein A molecules on the surface at 4℃ for 1 hour. Use NanoDrop to detect the residual antibody content in the supernatant. The antibody binding rate >90% is considered qualified, and pan-MHC-I A / B / C binding sepharose is prepared and kept at 4℃ for use. Add 40ml RIPA lysis buffer to the cell sample, incubate at 4℃ for 1 hour, centrifuge at 12000rpm for 30min, add sepharose CL-4B beads to the supernatant for pre-hybridization, incubate at 4℃ for 1 hour; remove beads by centrifugation, add pan-MHC-A / B / C binding sepharose CL-4B beads to the supernatant, and incubate at 4℃ overnight (16-18 hours). The beads were washed with PBS precooled at 4°C, and repeated three times; the beads were washed with ultrapure water; the washing solution was removed by centrifugation, and the antibody-MHC-I protein complex on the surface of the beads was eluted with 0.1N acetic acid. The antibody-MHC-I protein complex was dissociated under acidic conditions. Further, the proteins and peptides in the eluted product were separated and purified using a 3kDa ultrafiltration tube or a c18 solid phase extraction column (25 mg, waters), and the purified product was concentrated using a refrigerated vacuum centrifuge, and the concentrated product was stored at -20°C until it was loaded on the mass spectrometer.

[0051] 2. Mass spectrometry identification of MHC-I restricted T cell epitope peptides:

[0052] The concentrated MHC-I restricted epitope peptide solution was analyzed by a Q Exactive mass spectrometer (Thermo Fisher Scientific) connected online to a nanoflow HPLC (Thermo Fisher Scientific). The separation was performed using a 15 cm long, 75 um inner diameter separation column manually packed with ReproSil-Pur C18-AQ 1.9 um filler. The peptides were eluted using a linear gradient of 2-30% buffer B (80% ACN / 0.5% acetic acid), with a flow rate of 250 nl / min and an elution time of 90 min. The secondary mass spectrometry was fragmented using HCD, and the data collection selected the data-dependent "Top 20" method. The MS spectrum was collected at a resolution of 70,000, 200 m / z, and a target value of 3E6 ions; the top 10 ions in ion intensity were usually separated and accumulated using a maximum injection time of 120 ms until the automatic gain controller displayed a value of 1E5. The peptide matching option was set to “disable” and the MS / MS resolution was set to 17,500 (200 m / z).

[0053] 3. Mass spectrometry data analysis of MHC-I restricted T cell epitope peptides:

[0054] Data analysis was performed using MaxQuant (version 1.3.10.15) to compare mass spectra with a human protein library (Uniprot, 86,749 proteins), tumor-associated antigens, tumor-specific mutant peptides, and a list of spectra generated from a data set containing 247 common contaminants (keratin, bovine serum albumin, and proteases). Variable modification detection settings: N-terminal acetylation and methionine oxidation. Second-position peptide identification settings: enable; specific enzyme cleavage settings: unspecific; peptide identification FDR (false discovery rate) set to 1%, protein identification FDR not set; sequence match length limit set to 8-15aa, maximum peptide mass set to 1500Da, and maximum charge state set to 3. The initial allowed mass deviation of the leading ion was set to 6ppm, and the maximum fragment mass deviation was set to 20ppm. The "match between runs" setting was turned on. The identification results were output and saved in the "peptide.txt" file, and the peptides matched to the counter-library and the contamination library were removed, and the rest were the identification results of MHC-I restricted epitopes.

[0055] The experimental results show that mutant polypeptides, deformed polypeptides and derivative peptide sequences can be expressed and presented on HLA molecules on the cell surface. Taking the mutant polypeptide SEQ ID NO:3 as an example, the mass spectrometry of the polypeptide is shown in Figure 1The results showed that the above polypeptides can be expressed and presented on the HLA molecules on the cell surface.

[0056] Example 3 Verification of the affinity of polypeptide T2

[0057] T2 cells are cell lines deficient in antigen polypeptide transporters essential for the endogenous antigen presentation pathway and can be used to study the antigen presentation process and the strength of the mutual recognition of MHC molecules.

[0058] In order to test the affinity of exogenous peptides to T2 cells, peptides that have been proven to have strong affinity to T2 cells are used as positive controls, and T2 cells without peptides are used as background controls. The binding of exogenous peptides to MHC I molecules on the surface of T2 cells can increase the expression of MHC I molecules on their surface. The more stable the binding, the more MHC I molecules can be detected. Finally, the average fluorescence intensity is used as the detection index, and the fluorescence coefficient (FI) is used as the measurement index. In this way, the affinity of the peptide to T2 cells is judged. The higher the FI value, the stronger the affinity of the peptide to T2 cells, which is beneficial to the subsequent specific CD8 + T cell recognition.

[0059] The synthesized peptide was added to 2*10 5 T2 cells were added with human β2 microglobulin (final concentration, 3ug / ml), cultured in 24-well plates, and cultured overnight in an incubator (37°C, 5% CO2). T2 cells without peptides were used as background controls, and CMV peptides (whose sequence is NLVPMVATV, a viral peptide that is known to have a strong affinity with T2 cells) were used as positive controls. Two replicate wells were set up in the experiment and the average value was taken.

[0060] The cultured cells were centrifuged at 200 g for 5 minutes to collect the cells. After washing the cells twice with PBS, the cells were directly incubated with FITC-labeled monoclonal antibodies against the corresponding HLA typing (HLA-A*11:01) at 4°C for 30 minutes. Then, the cells were analyzed by flow cytometry (BD FACSJazz TM ) and its software to detect and analyze its average fluorescence intensity, see Figure 2 The T2 affinity results are shown in Table 2 below.

[0061] Table 2 T2 affinity results

[0062] Peptide sequence FI in conclusion Positive Control 1.63 TTLPTTITR 4.09 High affinity

[0063] As can be seen from Table 2, compared with the positive control, the provided polypeptide TTLPTTITR exhibits very high affinity.

[0064] Example 4 Peptide stimulates CD8+ T cells in vitro

[0065] Take PBMC cells from volunteers who are positive for the corresponding subtype of the peptide, 2×10 7 PBMC cells were isolated by the adherence method (attachment for 3 hours), and CD8+ T cells were isolated by the CD8 magnetic bead method. GM-CSF (1000U / ml) and IL-4 (1000U / ml) were used to induce adherent monocytes into immature DCs, and then IFN-gamma (100U / ml), LPS (10ng / ml), and various peptides were used to induce mature DC cells into peptide-specific mature DC cells. The obtained peptide-specific mature DC cells were compared with the volunteers' CD8 + T cells were co-cultured and IL-21 was added. After 3 days, IL-2 and IL-7 were added. IL-2 and IL-7 were added on days 5 and 7. On day 10, the co-cultured cells were counted and subsequently tested by ELISPOTs and LDH.

[0066] Example 5 ELISPOTs method verifies the peptide-activated CD8+ T cell immune response

[0067] The ELISPOT method, or Enzyme linked immunospot assay, can detect the cytokines secreted by a single cell. In the experiment, specific monoclonal antibodies are coated on the culture plate, and then the cells to be tested and antigen stimulants are added for culture. Under the stimulation of the stimulant, the T cells secrete the corresponding cytokines, and the secreted cytokines are captured by the antibodies coated on the culture plate. After washing the cells, the captured cytokines can be combined with the fluorescently labeled second antibody to form spots. That is, the coated antibodies can be used to capture the cytokines secreted by the cells in culture and presented in the form of enzyme-linked spot color development, so as to detect and verify the strength of the immune response of the peptide to activate CD8+T cells.

[0068] Referring to the description in the ELISPOTs kit manual, the cultured cells in Experimental Example 4 and T2 cells loaded with the experimental polypeptide (i.e., TTLPTTITR) and an irrelevant polypeptide (a polypeptide that does not stimulate T cells to secrete IFN-gamma interferon, the specific sequence is LSYRNKPSI, and the irrelevant polypeptide used in the following examples is also this sequence) were added to the ELISPOTs plate for culture, and ELISPOTs detection was performed after 20 hours (see the kit manual). ELISPOTs results are shown in Figure 3 The results are summarized in Table 3 below:

[0069] Table 3 Peptides stimulate specific CD8+ T cells to secrete IFN-gamma interferon

[0070]

[0071] Among them, the second and third columns in Table 3 represent the number of spots detected using the experimental peptide as a stimulus or using an irrelevant peptide as a stimulus, respectively, and the fourth column multiple represents the ratio of the number of spots generated by using the experimental peptide as a stimulus to using an irrelevant peptide as a stimulus. Generally speaking, when the ratio exceeds a certain multiple (>=2), it is considered to be immunogenic, and the higher the ratio, the stronger the immunogenicity of the peptide.

[0072] Example 6 LDH release experiment proves CD8 + T cell peptide-specific killing activity

[0073] LDH (Lactate Dehydrogenase) is an enzyme present in the cytoplasm. When the cell membrane is damaged, LDH will be released into the culture medium. Since the released LDH is stable, the amount of LDH in the detection culture medium can be used as an indicator to determine the number of dead and damaged cells.

[0074] The cells cultured in Experimental Example 4 were co-cultured with T2 cells loaded with experimental polypeptides or irrelevant polypeptides or unloaded polypeptides. The maximum release well, volume correction well, culture medium control well, spontaneous release well, different effect-target ratios (ratio of the number of T cells to T2 cells) and other controls were set in the experiment. Three duplicate wells were set for each group. After 4 hours, 50 μl of the co-cultured cell supernatant was taken out and added to 50 ul of LDH substrate mixture to catalyze the LDH substrate reaction by the cell supernatant. Finally, the wavelength of 490 nm and the reference wavelength of 680 nm were read, and the killing activity of the target cell killing T2 was calculated according to the control well. The results are shown in Table 4 below. The larger the value shown in Table 4, the stronger the killing effect.

[0075] The results showed that these peptides stimulated the production of CD8 + T cells have peptide-specific killing activity.

[0076] Table 4 T cells specifically recognize and kill target cells presenting experimental peptides

[0077]

[0078] The results showed that CD8 + T cells have peptide-specific killing activity.

[0079] Example 7 Establishment of subcutaneous transplanted tumor model in mice

[0080] This example constructs a mouse subcutaneous transplant tumor model, which is used to verify the control effect of the polypeptide drug combination, antigen presenting cells, and vaccine proposed in the present invention on tumors.

[0081] 1. The coding genes of each polypeptide are introduced by lentiviral transfection to construct and package a recombinant lentivirus expressing the aforementioned mutant polypeptide or its variant.

[0082] 2. Establishment of human lung cancer cell lines expressing peptides

[0083] The human lung cancer cell line HCC827 was purchased from ATCC (No.: CRL-2868), and its HLA subtype was HLA-A*1101 positive. The cells were cultured in DMEM medium containing 10% fetal bovine serum, 100U / mL penicillin and streptomycin. The cells were cultured in an incubator at 37°C and 5% CO2. The packaged lentivirus was transfected into the HCC827 cell line, and Puromycin antibiotics were used to continuously screen the surviving HCC827 cell line, and finally the HCC827 cell line expressing the polypeptide was established.

[0084] 3. Human immune reconstitution in NOD SCID mice

[0085] Collect 600 to 900 ml of anticoagulated peripheral blood from healthy volunteers. Separate peripheral blood mononuclear cells (PBMC) with Ficoll and collect the cells for later use. Take 300 NODSCID mice that have been excluded from immune leakage and inject 2*10 PBMC into each mouse intraperitoneally. 7 / 0.5ml, human immune reconstitution was performed on NOD SCID mice. After 4 weeks, mice were selected to prepare for inoculation of human lung cancer cell line model.

[0086] 4. Construction of human lung cancer tumor model

[0087] The established human lung cancer cell line was cultured in DMEM medium containing 10% fetal bovine serum, 100U / mL penicillin and streptomycin. The cells were cultured in an incubator at 37°C and 5% CO2. The tumor cells were collected and centrifuged at 3000 rpm. The tumor cells were washed three times with sterile saline. Appropriate dilution was made, and 40 μl of the cell suspension was added with 10 μl of 0.4% trypan blue for staining and microscopic counting to make a concentration of 1*10 8 The NOD / SCID mice after immune reconstruction were selected and 100 ml of tumor cell suspension was subcutaneously inoculated into each mouse. After the inoculation, the inoculation site was observed for infection and whether the tumor naturally subsided after growth. After 7 days, a tumor about the size of a grain of rice could be felt in the subcutaneous tumor of the mouse. The NOD / SCID mice with subcutaneous tumor model after 4 weeks of immune reconstruction were treated with DC vaccine, and the volume of the tumor was recorded every 3-4 days.

[0088] Example 8 Preparation of polypeptide vaccine and treatment regimen

[0089] The HCC827 subcutaneous tumor model NOD / SCID mice with immune reconstruction for 4 weeks were randomly divided into 4 groups: adjuvant + wild-type peptide group (the wild-type peptide is TTLPTTISR), adjuvant + blank peptide group (i.e., only containing adjuvant), adjuvant + mutant peptide group (the mutant peptide is TTLPTITR), adjuvant + deformed peptide group (which can be divided into four groups according to the deformed peptides used, and the deformed peptides used are TILPTTITK, TSLPTTITK, TTLPTTITK, and TVLPTTITK), each group has 6 mice. The adjuvant used is Freund's adjuvant.

[0090] The initial immunization dose of each group of polypeptides is 100 ml / mouse. After the above polypeptides are resuspended with PBS, mixed with 150 ml / mouse Freund's complete adjuvant, adjusted to 300 ml / mouse with PBS, and injected subcutaneously at two points on the back. Two weeks later, the same dose is used for booster immunization (complete Freund's adjuvant is used for the first time, and incomplete Freund's adjuvant is used thereafter), for a total of 4 immunizations. After the injection, the vital signs of the mice are observed, and the vertical and horizontal sizes of the tumors are measured with a vernier caliper every 3-4 days. Tumor volume is calculated as: tumor volume = 1 / 2*length*width 2 At the same time, the weight changes of mice were recorded. Figure 4 .

[0091] The results showed that compared with the peptide vaccine group loaded with adjuvant + wild-type peptide and the adjuvant + blank peptide group, the peptide vaccine group loaded with adjuvant + mutant peptide or adjuvant + deformed peptide could significantly slow down the growth of mouse tumors.

[0092] Example 9 Preparation and treatment plan of DC polypeptide vaccine

[0093] 100 to 150 ml of anticoagulated peripheral blood was collected from healthy volunteers. Peripheral blood mononuclear cells (PBMC) were separated by Ficoll, and PBMC cells were collected and counted at 2-3*10 6 / ml was resuspended in RPMI 1640 medium and incubated at 37℃ for 2h. The adherent cells were DCs, and the non-adherent cells were peripheral blood lymphocytes (PBL) for later use. GM-CSF (1000U / ml) and IL-4 (1000U / ml) were used to induce adherent monocytes into immature DCs, and then IFN-gamma (100U / ml) and CD40L (10ng / ml) were added. Finally, wild-type peptide combination and mutant peptide combination (at a concentration of 10 micrograms / ml) were added to induce adherent cells into mature DC cells. Mature DCs were harvested and washed 3 times with saline. The DC loaded with peptides was adjusted to (4.0±0.5)*10 with saline.7 / ml for subsequent experiments. The mice were randomly divided into 4 groups: DC-loaded wild-type polypeptide group (the wild-type polypeptide is TTLPTTISR), DC-loaded mutant polypeptide group (the mutant polypeptide is TTLPTITR), DC-loaded deformed polypeptide group (according to the different denatured polypeptides used, it can be divided into four groups, and the denatured polypeptides used are TILPTTITK, TSLPTTITK, TTLPTTITK, TVLPTTITK), blank polypeptide group (i.e., no polypeptide is loaded), with 6 mice in each group. Prepare polypeptide cell suspensions of DC-loaded wild-type polypeptide, DC-loaded mutant polypeptide, DC-loaded deformed polypeptide and DC-loaded blank polypeptide. Intradermal injections were performed on the inner thigh near the groin of the mice, with 0.1 ml injected on each side, once a week. The dose is (4.0±0.5)*10 6 cells / time, injected 2 times in total. After the injection, observe the vital signs of the mice, and measure the vertical and horizontal size of the tumor with a vernier caliper every 3-4 days. Tumor volume is calculated as: tumor volume = 1 / 2*length*width 2 At the same time, the weight changes of mice were recorded. Figure 5 .

[0094] The results showed that compared with the DC vaccine group loaded with wild-type peptide and the DC vaccine group loaded with blank peptide, the DC vaccine group loaded with mutant peptide or deformed peptide could significantly slow down the growth of mouse tumors.

[0095] Example 10 Preparation of polypeptide-specific DC-CTL vaccine and treatment regimen

[0096] Example 9 PBL collected by magnetic bead sorting to obtain CD8 + T cells were co-cultured with DC loaded with blank peptide, DC loaded with wild-type peptide, DC loaded with mutant peptide, and DC loaded with deformed peptide. The cell ratio was DC:CD8 +T cells = 1:4. 500 IU / ml IL-2 and 50 ng / ml IL-7 were added to the culture medium, and the cells were incubated in a 37V 5% CO2 incubator. The cells were counted after 1 week of culture. In the second week, DC loaded with blank peptide, DC loaded with wild-type peptide, DC loaded with mutant peptide, and DC loaded with deformed peptide were used for the second round of stimulation. Three rounds of stimulation were performed in total, and culture medium was added appropriately during the culture period. The number of lymphocytes was counted on the 0th, 7th, 14th and 21st days of culture, and the cell proliferation index (PI) was calculated. Among them, PI = number of cells after amplification / number of cells inoculated. Cytotoxic T lymphocytes (CTL) were harvested after 21 days of culture. The cells were resuspended in physiological saline, the resuspension volume was 0.2 ml, and the cells were reinfused through the tail vein. The number of cells reinfused into each tumor model mouse was approximately 1*10 8 After the injection, the vital signs of the mice were observed, and the vertical and horizontal dimensions of the tumor were measured with a vernier caliper every 3-4 days. The tumor volume was calculated as: tumor volume = 1 / 2 * length * width 2 At the same time, the weight changes of mice were recorded. Figure 6 .

[0097] The results showed that compared with the blank peptide control group and the wild-type peptide group, the DC-CTL vaccine activated by the mutant peptide or deformed peptide could significantly slow down the growth of mouse tumors.

[0098] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" 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 representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0099] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention. SEQUENCE LISTING <110> Wuhan BGI Genentech Biotechnology Co., Ltd. <120> Tumor-specific peptide sequences and their applications <130> PII220478PCN <160> 10 <170> PatentIn version 3.5 <210> 1 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Deformed Peptide <400> 1 Thr Ile Leu Pro Thr Thr Ile Thr Lys 1 5 <210> 2 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Deformed Peptide <400> 2 Thr Ser Leu Pro Thr Thr Ile Thr Lys 1 5 <210> 3 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Mutant Peptide <400> 3 Thr Thr Leu Pro Thr Thr Ile Thr Arg 1 5 <210> 4 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Deformed Peptide <400> 4 Thr Thr Leu Pro Thr Thr Ile Thr Lys 1 5 <210> 5 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Deformed Peptide <400> 5 Thr Val Leu Pro Thr Thr Ile Thr Lys 1 5 <210> 6 <211> twenty three <212> PRT <213> Artificial Sequence <220> <223> Derivatized Peptides <400> 6 Pro Leu Ser Thr Leu Val Thr Ile Leu Pro Thr Thr Ile Thr Lys Ser 1 5 10 15 Thr Pro Thr Ser Glu Thr Thr 20 <210> 7 <211> twenty three <212> PRT <213> Artificial Sequence <220> <223> Derivatized peptides <400> 7 Pro Leu Ser Thr Leu Val Thr Ser Leu Pro Thr Thr Ile Thr Lys Ser 1 5 10 15 Thr Pro Thr Ser Glu Thr Thr 20 <210> 8 <211> twenty three <212> PRT <213> Artificial Sequence <220> <223> Derivatized peptides <400> 8 Pro Leu Ser Thr Leu Val Thr Thr Leu Pro Thr Thr Ile Thr Arg Ser 1 5 10 15 Thr Pro Thr Ser Glu Thr Thr 20 <210> 9 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> Derived peptide <400> 9 Pro Leu Ser Thr Leu Val Thr Thr Leu Pro Thr Thr Ile Thr Lys Ser 1 5 10 15 Thr Pro Thr Ser Glu Thr Thr 20 <210> 10 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> Derived peptide <400> 10 Pro Leu Ser Thr Leu Val Thr Val Leu Pro Thr Thr Ile Thr Lys Ser 1 5 10 15 Thr Pro Thr Ser Glu Thr Thr 20

Claims

1. A group of isolated polypeptides, characterized in that The polypeptide includes at least any one polypeptide in the first peptide group, and the first peptide group includes polypeptides with amino acid sequences as shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:

5.

2. The polypeptide according to claim 1, characterized in that The polypeptide is selected from at least two polypeptides having amino acid sequences shown by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO:

5.

3. An isolated nucleic acid, characterized in that The nucleic acid encodes the polypeptide of claim 1 or 2.

4. A construct, characterized in that The construct comprises the nucleic acid of claim 3 and a control sequence operably linked to the nucleic acid.

5. An expression vector, characterized in that: The expression vector comprises the construct of claim 4.

6. A host cell, characterized in that The host cell carries the construct according to claim 4 or the expression vector according to claim 5.

7. Use of the polypeptide according to claim 1 or 2 in the preparation of a drug for preventing or treating lung cancer.

8. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the polypeptide according to claim 1 or 2 and pharmaceutically acceptable excipients.

9. An antigen presenting cell, characterized in that The antigen-presenting cells present the polypeptide of claim 1 or 2.

10. A tumor vaccine, characterized in that: The tumor vaccine comprises the polypeptide of claim 1 or 2, or the nucleic acid of claim 3, or the construct of claim 4, or the expression vector of claim 5, or the host cell of claim 6, or the antigen presenting cell of claim 9.

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