Optimized antigen peptide aiming at L858R point mutation of EGFR exon 21 region and application thereof

Abstract

The invention belongs to the field of biological medicines, and particularly relates to an optimized antigen peptide aiming at L858R point mutation of an EGFR exon 21 region and application of the optimized antigen peptide. Compared with a mutant peptide, the optimized antigen peptide has stronger immune cross reaction, can improve the immunoreactivity or curative effect of related tumor vaccines, and can effectively cover more patient crowds.

Description

Technical Field

[0001] The present application belongs to the field of biomedicine, and specifically relates to an optimized antigen peptide targeting the L858R point mutation in the exon 21 region of EGFR and its application. Background Art

[0002] Lung cancer is the most common malignant tumor in China, with approximately 900,000 cases in 2022, of which non-small cell lung cancer (NSCLC) accounts for about 85%. Among typical non-small cell lung cancers, squamous cell carcinoma and adenocarcinoma account for the vast majority. The occurrence of lung adenocarcinoma is closely related to some gene mutations, such as KRAS, EGFR, ROS, ALK, etc.; these mutations show racial differences.

[0003] Taking EGFR (epithelial growth factor receptor) mutation as an example, the proportion of lung adenocarcinoma patients with EGFR mutation is about 15% in European and American Caucasian patients, while it reaches 60% in patients in the Far East. This leads to regional differences in the pathological process, treatment methods, and prognosis recovery of EGFR mutation-positive lung adenocarcinoma. Different types of mutations can occur in exons 18, 19, 20, and 21 of the EGFR gene, including single-base mutations, deletions, and deletion insertions, which are closely related to the efficacy and resistance of targeted drugs such as EGFR-related tyrosine kinase inhibitors (EGFR-TKI).

[0004] The main mutations that are sensitive to EGFR-TKI are EGFR exon 19 deletion and EGFR exon 21 L858R point mutation, also known as EGFR-TKI drug-sensitive mutations, which account for 45% and 40% of all EGFR mutations, respectively. Among patients with drug-sensitive EGFR mutations, about 70-80% of patients are sensitive to the first and second generation EGFR targeted drugs, and their tumors can be better controlled; tumor cells of insensitive patients may have more complex natural drug resistance mechanisms.

[0005] In the use of targeted drugs, most patients will sooner or later develop secondary EGFR T790M mutations, leading to failure of targeted drugs and tumor progression. The improved third-generation targeted drugs, such as osimertinib, are effective for resistant patients with EGFR double mutations (exon 19 deletion + T790M and L858R + T790M), but resistance will eventually occur. Therefore, although EGFR-TKI can effectively inhibit tumor cells with related mutations and prolong patient survival. However, there are no other recognized effective medical treatments after targeted drug resistance, leaving a large medical demand space. The fourth-generation EGFR-TKI is about to be launched, but clinical data show that the fourth-generation targeted drugs are only effective for some patients with EGFR triple mutations (accounting for 36% of patients with EGFR double mutations).

[0006] Based on the analysis and prediction of EGFR-related mutations, it was found that EGFR mutations have different affinity in patients expressing different human leukocyte antigen (HLA) alleles; this means that some mutations have certain immunogenicity in some patient groups, called tumor neoantigens, which may trigger the body's protective immune response. Foreign studies have shown that patients with both EGFR drug-sensitive mutations and certain HLA alleles with affinity for mutations have milder conditions due to the protective effect of active T cells; the survival of patients using EGFR exon 19 deletion or EGFR exon 21 L858R mutation peptide vaccines is significantly prolonged. These antigen peptides or sequences can therefore be used in the development of new tumor vaccine drugs as an adjuvant treatment for tumor immunotherapy or targeted drug resistance in some patient populations.

[0007] However, existing antigen peptides have technical problems such as small coverage of patient population and weak immune cross-reaction. Summary of the invention

[0008] Based on this, the present application provides an optimized antigen peptide targeting the L858R point mutation in the EGFR exon 21 region to solve the technical problems in traditional technologies that antigen peptides cover a small patient population and have weak immune cross-reactions.

[0009] On the one hand, the present application provides an optimized antigen peptide for the L858R point mutation in the EGFR exon 21 region, which comprises at least one of the following four polypeptides:

[0010] MVKITRFGR, HVKITRFGR, RAYLLGAFK and RMYLLGAFK.

[0011] In one embodiment, it is a composition comprising 2, 3 or 4 of the four polypeptides.

[0012] In one embodiment, it is a fusion polypeptide comprising 2, 3 or 4 of the four polypeptides.

[0013] In one embodiment, the fusion polypeptide further includes one or more connecting peptides.

[0014] On the other hand, the present application provides a nucleic acid that expresses the above-mentioned optimized antigen peptide.

[0015] The present application also provides a vector, which contains the above-mentioned nucleic acid.

[0016] On the other hand, the present application provides a vaccine, which contains the above-mentioned optimized antigen peptide, or the above-mentioned nucleic acid, or the above-mentioned vector.

[0017] In one embodiment, it further comprises an adjuvant.

[0018] The present application also provides a kit, which comprises the above-mentioned optimized antigen peptide, or the above-mentioned nucleic acid, or the above-mentioned vector, or the above-mentioned vaccine.

[0019] On the other hand, the present application provides a use of the above-mentioned optimized antigen peptide, or the above-mentioned nucleic acid, or the above-mentioned vector, or the above-mentioned vaccine in the preparation of a drug for sensitizing and stimulating immune cells of the body.

[0020] In one embodiment, the immune cells are of HLA-A11 phenotype, HLA-A31 phenotype, HLA-A30 phenotype, and / or HLA-A3 phenotype.

[0021] The present application also provides the use of the above-mentioned optimized antigen peptide, or the above-mentioned nucleic acid, or the above-mentioned vector, or the above-mentioned vaccine in the preparation of a drug for treating tumors; the tumor has an L858R point mutation in the EGFR exon 21 region.

[0022] In one embodiment, the tumor is lung adenocarcinoma.

[0023] Compared with the traditional technology, the beneficial effects of this application include:

[0024] An optimized antigen peptide targeting the L858R point mutation in the exon 21 region of EGFR is provided. Compared with the mutant peptide, the optimized antigen peptide has a stronger immune cross-reaction, can improve the immune reactivity or efficacy of related tumor vaccines, and can effectively cover a wider patient population. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the technical solutions in the embodiments of the present application and to more completely understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present application, and those skilled in the art can obtain other drawings based on these drawings without creative work.

[0026] Figure 1 The immune reactivity of DC+T cells (HLA-A33) stimulated by EGFR L858R mutation and optimized structural peptides;

[0027] Figure 2 The immune reactivity of DC+T cells (HLA-A3) stimulated by EGFR L858R mutation and optimized structure peptide. DETAILED DESCRIPTION

[0028] Below in conjunction with embodiment and example, the application is further described in detail.It should be understood that these embodiments and examples are only used to illustrate the application and are not used to limit the scope of the application, and the purpose of providing these embodiments and examples is to make the understanding of the disclosure of the application more thorough and comprehensive.It should also be understood that the application can be implemented in many different forms, is not limited to the embodiment and example described herein, and those skilled in the art can make various changes or modifications without violating the connotation of the application, and the equivalent form obtained also falls within the protection scope of the application.In addition, in the description below, a large number of specific details are given in order to provide a more comprehensive understanding of the application, and it should be understood that the application can be implemented without one or more of these details.

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0030] the term

[0031] All documents mentioned in this application are cited as references in this application, just as each document is cited as reference separately. Unless they conflict with the application purpose and / or technical solution of this application, the cited documents involved in this application are cited with all contents and all purposes. When the cited documents are involved in this application, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When the cited documents are involved in this application, the examples and preferred methods of the cited relevant technical features can also be incorporated into this application as references, but are limited to the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, the present application shall prevail or be modified adaptively according to the description of this application.

[0032] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0033] The terms "and / or", "or / and", and "and / or" used in this article include any one of two or more related listed items, and also include any and all combinations of related listed items, and the arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions that are all connected by "logical and", and undoubtedly includes technical solutions that are all connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution that is all connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the combination of four of A, B, C, and D (that is, the technical solution that is all connected by "logical AND").

[0034] In the present application, when it comes to numerical intervals (i.e., numerical ranges), unless otherwise specified, the optional numerical distribution is considered continuous within the above numerical interval, and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical range, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to the integers within the numerical interval, it includes the two endpoint integers of the numerical range, and each integer between the two endpoints. In this article, it is equivalent to directly listing each integer, such as t is an integer selected from 1 to 10, indicating that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges included therein.

[0035] On the one hand, the present application provides an optimized antigen peptide for the L858R point mutation in the exon 21 region of EGFR, which comprises at least one of the following four polypeptides: MVKITRFGR, HVKITRFGR, RAYLLGAFK and RMYLLGAFK.

[0036] In a specific example, it is a composition comprising 2, 3 or 4 of the four polypeptides.

[0037] Optionally, it is a fusion polypeptide containing 2, 3 or 4 of the four polypeptides.

[0038] In a specific example, the fusion polypeptide further includes one or more connecting peptides.

[0039] On the other hand, the present application provides a nucleic acid that expresses the above-mentioned optimized antigen peptide.

[0040] The present application also provides a vector, which contains the above-mentioned nucleic acid.

[0041] The term "vector" refers to a nucleic acid carrier into which a polynucleotide can be inserted. When a vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements it carries are expressed in the host cell. Vectors are well known to those skilled in the art, and include but are not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); bacteriophages such as lambda phage or M13 phage and animal viruses. Animal viruses that can be used as vectors include but are not limited to retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomas (such as SV40). In some embodiments, the vector of the present invention contains regulatory elements commonly used in genetic engineering, such as enhancers, promoters, internal ribosome entry sites (IRES) and other expression control elements (such as transcription termination signals, or polyadenylation signals and poly-U sequences, etc.).

[0042] On the other hand, the present application provides a vaccine, which contains the above-mentioned optimized antigen peptide, or the above-mentioned nucleic acid, or the above-mentioned vector.

[0043] Optionally, it further comprises an adjuvant.

[0044] Adjuvants suitable for the vaccine of the present invention include adjuvants that can enhance the immune response to the above-mentioned antigenic peptides, preferably adjuvants that can activate the activity of dendritic cells (DC) or T cells (especially CTL cells) to make them more likely to produce immune responses. The adjuvants are well known in the art.

[0045] The present application also provides a kit, which comprises the above-mentioned optimized antigen peptide, or the above-mentioned nucleic acid, or the above-mentioned vector, or the above-mentioned vaccine.

[0046] Optionally, the vaccine is a water-in-oil emulsion having an aqueous phase and an oily phase.

[0047] Further optionally, the vaccine is an oil-in-water emulsion having an aqueous phase and an oily phase.

[0048] Vaccines are typically formulated for administration by injection. Typical immunizations are vaccinations by intravenous (IV) injection routes, but the invention also contemplates oral and subcutaneous (SC), intramuscular (IM), intraperitoneal (IP) or intradermal (ID) injections.

[0049] Vaccines can also be delivered to the subject by a delivery system that delivers antigenic peptides, nucleic acids or vectors. Non-viral methods of delivering nucleic acids include lipofection, nucleofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycations or lipid: nucleic acid conjugates, naked DNA, artificial virus particles and agents that enhance the uptake of DNA.

[0050] Viral delivery of nucleic acid methods can be administered directly to the subject or can be used to treat cells (particularly immune cells) in vitro, and the treated cells can optionally be administered to the subject.

[0051] On the other hand, the present application provides a use of the above-mentioned optimized antigen peptide, or the above-mentioned nucleic acid, or the above-mentioned vector, or the above-mentioned vaccine in the preparation of a drug for sensitizing and stimulating immune cells of the body.

[0052] In a specific example, the immune cell is of HLA-A11 phenotype, HLA-A31 phenotype, HLA-A30 phenotype, and / or HLA-A3 phenotype.

[0053] The present application also provides the use of the above-mentioned optimized antigen peptide, or the above-mentioned nucleic acid, or the above-mentioned vector, or the above-mentioned vaccine in the preparation of a drug for treating tumors; the tumor has an L858R point mutation in the EGFR exon 21 region.

[0054] In one specific example, the tumor is lung adenocarcinoma. DETAILED DESCRIPTION

[0056] Example 1: Optimization of new antigenic peptides associated with EGFR L858R mutation

[0057] Taking the L858R point mutation in the exon 21 region of EGFR as an example, its immunogenic (HLA-A affinity) sequence is located at wild-type amino acid positions 850-867: the mutant sequence is HVKITDFGRAKLLGAEEK (SEQ ID NO.12), and the wild sequence is HVKITDFGLAKLLGAEEK (SEQ ID NO.13). According to NETMHCpan v4.0, the L858R mutant peptide sequence HVKITDFGR (SEQ ID NO.2) has a high affinity (strong binder) to HLA-A*31:01 (IC50=18.9nM), HLA-A*33:03 (IC50=22.9nM) and HLA-A*68:01 (IC50=19.6nM), while the L858R mutant peptide sequence RAKLLGAEEK (SEQ ID NO.6) has a high affinity to HLA-A*30:01 (IC50 MT=47.5nM). This means that tumor vaccines using the above sequences may produce immunotherapy methods or prevent tumor recurrence for patients with normal expression of the relevant HLA alleles.

[0058] Using NETMHC 4.0, a new prediction was made for the above-mentioned related peptide sequences, and it was found that the data given by different models were somewhat different (see Table 1-Table 6). In the following Tables 1-2, the affinity differences of the EGFR L858R mutant peptide HVKITDFGR (SEQ ID NO.2) (wild type HVKITDFGL (SEQ ID NO.1)) to HLA-A*31:01 (Table 1) and HLA-A*33:01 were compared.

[0059] Table 1. Comparison of affinity predictions for EGFR L858R mutation-associated peptides and allele HLA-A31

[0060]

[0061] Table 2. Comparison of affinity predictions for EGFR L858R mutation-associated peptides and allele HLA-A33

[0062]

[0063] Similarly, the EGFR L858R mutant peptide RAKLLGAEEK (SEQ ID NO.6) (wild type LAKLLGAEEK (SEQ ID NO.5)) was analyzed and predicted, and the results are shown in Table 3. If the optimized peptide RAKLLGAFK (SEQ ID NO.8) obtained by changing the 865th and 866th positions E to amino acid F can enhance the immune cross-reaction and improve the immune reactivity or efficacy of related tumor vaccines, but replacing the amino acids at other positions in the sequence, A859M or K860Y, cannot further improve the affinity for HLA-A30.

[0064] Table 3. Comparison of affinity predictions for EGFR L858R mutation-associated peptides and allele HLA-A30

[0065]

[0066]

[0067] Except for HLA-A30 and -A33, which are distributed in small amounts in the northern population of my country, HLA--A31 is mainly distributed in the American population. We further analyzed and predicted the affinity of the EGFR L858R mutant peptide RAKLLGAEEK (SEQ ID NO.6) (wild-type LAKLLGAEEK (SEQ ID NO.5)) for other HLA alleles, and the results are shown in Tables 4-6. The results showed that if the two amino acids E at position 865 and E at position 866 were replaced by amino acid F to obtain the optimized peptide RAKLLGAFK (SEQ ID NO.8), the immune cross-reaction could be enhanced; if K860Y and A859M were further changed, the obtained optimized structural peptide RMYLLGAFK (SEQ ID NO.11) would maximize the immunoreactivity or efficacy of the relevant tumor vaccine in HLA-A3, HLA-A11, and HLA-A31 (Tables 4-6). HLA-A11 is mainly distributed in the Far East and central, southern and coastal areas of my country. Optimizing the structural peptide RMYLLGAFK (SEQ ID NO.11) can greatly increase the applicable population for related tumor vaccines.

[0068] Table 4. Comparison of affinity predictions for EGFR L858R mutation-associated peptides and allele HLA-A11

[0069]

[0070] Table 5. Comparison of affinity predictions for EGFR L858R mutation-associated peptides and allele HLA-A3

[0071]

[0072] Table 6. Comparison of affinity predictions for EGFR L858R mutation-associated peptides and allele HLA-A31

[0073]

[0074] Example 2: Effect of optimized structural peptides on immune response to EGFR exon 21 L858R mutant peptide HVKITDFGR (SEQ ID NO. 2)

[0075] The HLA-A*33:03 phenotype of healthy blood donors and the target cell human lung adenocarcinoma HCC 4006 (HLA-A*33:03: HLA-A*33:03) cell line were tested and confirmed by PCR-SSCP method.

[0076] Collect venous blood from healthy people with HLA-A*33:03 phenotype, separate peripheral blood mononuclear cells (PBMC), count; use appropriate amount of adherent culture medium to adhere for 90 minutes; culture DCs with serum-free culture medium containing 1000 units / ml GM-CSF and 500 units / ml IL-4 or 150 units / ml IL-15, and change the medium every two days. Count the suspended T cells and freeze them.

[0077] On the 5th day, the cells were infected with DC adjuvant recombinant adenovirus HBAD-DCML; on the 6th day, the neoantigen peptide HVKITDFGR (SEQ ID NO.2) or the optimized antigen peptide HVKITRFGR (SEQ ID NO.3) and MVKITRFGR (SEQ ID NO.4) were added for stimulation and sensitization (60 μg / ml).

[0078] On day 7, 50 ng / ml TNFα was added to mature DCs; frozen suspension T cells and target cells HCC 4006 (HLA-A*33:03: HLA-A*33:03) were thawed and prepared.

[0079] On day 8, DCs were collected and mixed and cultured at a ratio of DC:T=1:10 to activate effector T cells.

[0080] On the 9th day, 15 μg / ml of mutant peptide or wild-type control peptide was added to the culture medium of target cells and co-cultured for 16 hours to form type I histocompatibility complex MHC I (HLA-A*33:03+mutant peptide+β2 microglobulin).

[0081] On the 10th day, activated effector T cells were mixed with target cells at an effector-target ratio of 2:1 or 3:1, and then inoculated into a 96-well microplate with a polyvinylidene fluoride membrane (PVDF) coated with γ-interferon antibody at the bottom and co-cultured for 24 hours; the plate was washed and the peroxidase color reaction was performed according to the requirements of the γ-interferon Elispot kit.

[0082] The results of Example 2 are shown in Figure 1 After stimulating sensitized DCs and activating T cells in vitro with mutant peptide HVKITDFGR (SEQ ID NO.2) (MT), optimized antigen peptide HVKITRFGR (SEQ ID NO.3), and optimized antigen peptide MVKITRFGR (SEQ ID NO.4), no interferon-γ signal released by active T cells was detected in the group without effector T cells (-T) or without antigen peptide (-P) complex; while in the wild peptide (HVKITDFGL (SEQ ID NO.1), WT) group and mutant peptide HVKITDFGR (SEQ ID NO.2) (MT) group, interferon-γ signal of active effector T cells after specific stimulation appeared. The strength of the effector T cell immune reactivity produced by different stimulation DC allergens is the optimized structural peptide MVKITRFGR (SEQ ID NO.4) group > optimized structural peptide HVKITRFGR (SEQ ID NO.3) > mutant peptide HVKITDFGR (SEQ ID NO.2). If the optimized structural peptide MVKITRFGR (SEQID NO.4) or HVKITRFGR (SEQ ID NO.3) is used for tumor vaccine, it will provide stronger protection and clinical efficacy for relevant patients than the mutant peptide.

[0083] Example 3 Effect of optimized structural peptides on immune response to EGFR exon 21 L858R mutant peptide RAKLLGAEEK (SEQ ID NO.6)

[0084] Healthy blood donors with HLA-A*03:01 phenotype and target cell human Burkitt's lymphoma RAJI (HLA-A*03:01:HLA-A*03:01) cell line were tested and confirmed by PCR-SSCP method. Venous blood of healthy people with HLA-A*03:01 phenotype was collected, peripheral blood mononuclear cells (PBMC) were separated and counted; adhered to the wall for 90 minutes with appropriate amount of adherent culture medium; adherent cells were cultured with serum-free culture medium containing 1000units / mL GM-CSF and 500units / mL IL-4 or 150units / mL IL-15, and the medium was changed every two days. Suspended T cells were counted and frozen. On the 5th day, DC adjuvant recombinant adenovirus HBAD-DCML was added for infection; on the 6th day, the neoantigen peptide RAKLLGAEEK (SEQ ID NO.6) or the optimized antigen peptide RAYLLGAFK (SEQ ID NO.9) and RMYLLGAFK (SEQ ID NO.11) were added for stimulation and sensitization (60μg / ml); on the 7th day, 50ng / ml TNFɑ was added to make DC mature; frozen suspension T cells and target cells RAJI (HLA-A*03:01:HLA-A*03:01) were resuscitated and prepared. On the 8th day, DCs were collected and mixed cultured at a ratio of DC:T=1:10 to activate effector T cells; on the 9th day, mutant peptides or wild control peptides 15ug / ml were added to the culture medium of target cells for co-culture for 16 hours to form type I histocompatibility complex MHC I (HLA-A*03:01+mutant peptide+β2 microglobulin). On the 10th day, activated effector T cells were mixed with target cells at an effector-target ratio of 2:1 or 3:1, and then inoculated into a 96-well microplate with a polyvinylidene fluoride membrane (PVDF) coated with γ-interferon antibody at the bottom and co-cultured for 24 hours; the plate was washed and the peroxidase color reaction was performed according to the requirements of the γ-interferon Elispot kit.

[0085] The results of Example 3 are shown in Figure 2After stimulating sensitized DCs and activating T cells in vitro with mutant peptide RAKLLGAEEK (SEQ ID NO.6) (MT), optimized antigen peptide RAYLLGAFK (SEQ ID NO.9), and optimized antigen peptide RMYLLGAFK (SEQ ID NO.11), no interferon-γ signal released by active T cells was detected in the group without effector T cells (-T), and a small amount of non-specific interferon-γ signal background of activated T cells was detected in the group without antigen peptide (-P) complex; while in the wild peptide (LAKLLGAEEK (SEQ ID NO.5), WT) group and mutant peptide RAKLLGAEEK (SEQ ID NO.6) (MT) group, strong interferon-γ signal of active effector T cells after specific stimulation appeared. The strength of the effector T cell immune reactivity generated by different DC sensitizers was optimized structure peptide RMYLLGAFK (SEQ ID NO.11) group> optimized structure peptide RAYLLGAFK (SEQ ID NO.9)> mutant peptide RAKLLGAEEK (SEQ ID NO.6). Theoretically, if the optimized structural nonapeptide RMYLLGAFK (SEQ ID NO.11) or RAYLLGAFK (SEQ ID NO.9) is used for tumor vaccine, it can not only provide stronger protection or therapeutic effect for patients (HLA-A30 positive), but also may effectively cover more patient populations (HLA-A3, HLA-A11 or HLA-A31 positive).

[0086] Tumor mutation-related peptides are used in personalized neoantigen vaccines with relevant HLA affinity, which can not only prevent tumor occurrence and tumor recurrence, but can also be used directly for tumor treatment in vitro in the form of DC+T. After DCs are stimulated with neoantigens, T cells activated in vitro express polyclonal ɑβTCRs with high and low affinities, which provides the possibility for further development of monoclonal therapeutic recombinant TCRs.

[0087] The above-described embodiments only express several implementation methods of the present application, which is convenient for understanding the technical solution of the present application in detail, but it cannot be understood as a limitation on the scope of protection of the patent application. It should be pointed out that for ordinary technicians in this field, without departing from the concept of the present application, several deformations and improvements can be made, which all belong to the protection scope of the present application. In addition, it should be understood that after reading the above-mentioned teaching content of the present application, the technicians in this field can make various changes or modifications to the present application, and the equivalent forms obtained also fall within the protection scope of the present application. It should also be understood that the technical solutions obtained by the technicians in this field through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided in the present application are all within the protection scope of the claims attached to the present application. Therefore, the protection scope of the patent of the present application shall be based on the content of the attached claims, and the description and drawings can be used to explain the content of the claims.

Claims

1. An optimized antigen peptide targeting the L858R point mutation in the EGFR exon 21 region, comprising at least one of the following four polypeptides: MVKITRFGR, HVKITRFGR, RAYLLGAFK and RMYLLGAFK.

2. The optimized antigen peptide according to claim 1, which is a composition comprising 2, 3 or 4 of the four polypeptides.

3. The optimized antigen peptide according to claim 1, which is a fusion polypeptide, wherein the fusion polypeptide contains 2, 3 or 4 of the four polypeptides.

4. The optimized antigen peptide according to claim 3, wherein the fusion polypeptide further comprises one or more connecting peptides.

5. A nucleic acid expressing the optimized antigen peptide according to any one of claims 1 to 4. A vector comprising the nucleic acid according to claim 5.

7. A vaccine comprising the optimized antigen peptide according to any one of claims 1 to 4, or the nucleic acid according to claim 5, or the vector according to claim 6.

8. The vaccine according to claim 7, further comprising an adjuvant.

9. A kit comprising the optimized antigen peptide according to any one of claims 1 to 4, or the nucleic acid according to claim 5, or the vector according to claim 6, or the vaccine according to claim 7 or 8.

10. Use of the optimized antigen peptide according to any one of claims 1 to 4, or the nucleic acid according to claim 5, or the vector according to claim 6, or the vaccine according to claim 7 or 8 in the preparation of a drug for sensitizing and stimulating immune cells of the body.

11. The use according to claim 10, wherein the immune cells are of HLA-A11 phenotype, HLA-A31 phenotype, HLA-A30 phenotype, and / or HLA-A3 phenotype.

12. Use of the optimized antigen peptide according to any one of claims 1 to 4, or the nucleic acid according to claim 5, or the vector according to claim 6, or the vaccine according to claim 7 or 8 in the preparation of a medicament for treating a tumor; the tumor has a L858R point mutation in the exon 21 region of EGFR.

13. The use according to claim 12, wherein the tumor is lung adenocarcinoma.

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