CN114790235A - Neoantigen aiming at EGFR exon 19 deletion mutation and application thereof - Google Patents

Abstract

The invention relates to the field of biomedicine, in particular to an antigenic peptide aiming at EGFR exon 19 deletion mutation and application thereof. The antigenic peptide comprises at least one of the following four polypeptides: AIXXPKANK, AIXXXKANK, (V) AIXXXTSPK and VAIKXXSPK; each occurrence of X is independently selected from any one of amino acids A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, V. The antigenic peptide can effectively cover common EGFR exon 19 deletion mutation antigenic peptides through the matching of a plurality of limited polypeptides.

Description

Neoantigen aiming at EGFR exon 19 deletion mutation and application thereof

The priority of the chinese patent application entitled "neoantigen against EGFR exon 19 deletion mutation and use thereof" filed by the chinese patent office on 26/01/2021, application No. 2021101044685, the entire contents of which are incorporated herein by reference, is claimed in the present application.

Technical Field

The invention relates to the field of biomedicine, in particular to an antigenic peptide aiming at EGFR exon 19 deletion mutation and application thereof.

Background

Lung cancer is classified into small cell lung cancer and non-small cell lung cancer according to cell type; Non-Small Cell Lung Cancer (NSCLC) can be further classified into squamous Cell carcinoma, adenocarcinoma, and large Cell Lung carcinoma.

The annual incidence of lung adenocarcinoma accounts for 40% of lung cancers, with approximately 50-60% of EGFR (Epidermal growth factor receptor) mutations, and in the majority of new annual cases, there are exon 19 deletion mutations (45%), exon 20 insertion mutations (10%), or exon 21 point mutations L858R (37-40%) in EGFR. The exon 19 deletion mutation and the L858R are also called drug sensitive mutation, and can be effectively treated by an EGFR tyrosine kinase inhibitor (EGFR-TKI) to become a targeted drug treatment method. The targeted drug solves the drug resistance problem of T790M mutant oxitinib from first generation Iressa, second generation Afatinib, third generation AZD9291 to fourth generation EGFR-TKI targeted drug.

Not only EGFR-TKI targeted drugs are used for treating NSCLC, but monoclonal antibody drugs aiming at EGFR are also continuously marketed, such as cetuximab, panitumumab, cetuximab and nimotuzumab, and the like, which indicates that EGFR is one of important targets for treating NSCLC.

Disclosure of Invention

The present invention relates to antigenic peptides directed against EGFR exon 19 deletion mutations, comprising at least one of the following four polypeptides:

AIXXPKANK, AIXXXKANK, (V) AIXXXTSPK and VAIKXXSPK;

each occurrence of X is independently selected from any one of amino acids A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, V.

Alternatively, the antigenic peptide as described above, which is a composition, comprises 2, 3 or 4 of the four polypeptide sequences.

Optionally, the antigenic peptide is a fusion polypeptide comprising 2, 3 or 4 of the four polypeptide sequences.

Optionally, the antigenic peptide as described above, further comprising one or more linker peptides in the fusion polypeptide.

Optionally, the antigenic peptide as described above, X is independently selected at each occurrence from any one of F, M, A, S, V.

The present invention relates to a vaccine comprising the antigenic peptide as described above.

Optionally, the vaccine as described above, further comprising an adjuvant.

The present invention relates to a kit comprising an antigenic peptide as described above; or a vaccine as described above.

The invention also relates to the application of the antigenic peptide in the preparation of the medicine for sensitizing and stimulating the immune cells of the body.

Alternatively, for use as described above, the immune cell is an HLA-A11 phenotype and/or an HLA-A3 phenotype.

The invention also relates to the application of the antigenic peptide in the preparation of the medicine for treating tumors; the tumor has an EGFR exon 19 deletion mutation.

Alternatively, for use as described above, the tumour is lung adenocarcinoma.

The beneficial effects of the invention are as follows:

provides a universal antigenic peptide aiming at EGFR exon 19 deletion mutation, and can effectively cover common EGFR exon 19 deletion mutation by matching a plurality of limited polypeptides.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments or the prior art descriptions will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is a chromatogram of mutant peptide VAIKATSPK;

FIG. 2 is a chromatogram of mutant peptide AIKTSPKANK;

FIG. 3 is a chromatogram of mutant peptide IPVAIKTSPK;

FIG. 4 is a chromatogram of mutant peptide AIKASPKANK;

FIG. 5 is a chromatogram of mutant peptide AIKISPKANK;

FIG. 6 is a chromatogram of mutant peptide IPVAIKISPK;

FIG. 7 is a chromatogram of mutant peptide AIKSPKANK;

FIG. 8 is a chromatogram of mutant peptide AIKAPKANK;

FIG. 9 is a chromatogram of mutant peptide AIKDPKANK;

FIG. 10 is a chromatogram of mutant peptide AIKVPKANK;

FIG. 11 is a chromatogram of mutant peptide AIKEATSPK;

FIG. 12 is a chromatogram of mutant peptide AIKEPTSPK;

FIG. 13 is a chromatogram of mutant peptide AIKESTSPK;

FIG. 14 is a chromatogram of mutant peptide VAIKETSPK;

FIG. 15 is a chromatogram of mutant peptide VAIKEPSPK;

FIG. 16 is a chromatogram of mutant peptide VAIKESSPK;

FIG. 17 is a chromatogram of mutant peptide AIKESPKANK;

FIG. 18 is a chromatogram of mutant peptide AIKEPKANK;

FIG. 19 is a chromatogram of mutant peptide AIKEQKANK;

FIG. 20 is a chromatogram of mutant peptide AIKESKANK;

FIG. 21 shows the results of experiments on the immune response of representative mutant peptide combinations to EGFR exon 19 deletion mutations in one embodiment of the present invention;

fig. 22 is a chromatogram of a representative optimized peptide combination 19DA1103T 1;

figure 23 is a mass spectrum of a representative optimized peptide combination 19DA1103T 1;

FIG. 24 shows the results of a representative mutant peptide combination 19DA1103T versus representative optimized peptide combination in an example of the present invention comparing the results of an immune response experiment in the HLA-A11: 01 phenotype DC-T model;

figure 25 shows the results of a representative mutant peptide combination 19DA1103T versus representative optimized peptide combination 19DA1103T1 comparing HLA-a 03:01 phenotype DC-T model immune response experiments in one embodiment of the present invention;

fig. 26 is a chromatogram of stimulating peptide VAIMFPKANK;

fig. 27 is a chromatogram of wild-type peptide C2;

FIG. 28 is a comparison of the immune response reactivity of representative mutant peptide AIKASPKANK and representative optimized peptide VAIMFPKANK against known mutant peptides according to one embodiment of the present invention;

FIG. 29 is a chromatogram of AIKMFSTSPK;

FIG. 30 is a chromatogram of VAIKMFTSPK;

FIG. 31 is a chromatogram of KTPREATSPK;

FIG. 32 is a chromatogram of wild-type peptide C1;

FIG. 33 is a comparison of the immune response reactivity of representative optimized peptides AIKMFSTSPK and VAIKMFTSPK to known mutant peptides in one embodiment of the invention.

Detailed Description

Reference will now be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the invention. Indeed, it will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. For instance, features illustrated or described as part of one embodiment, can be used on another embodiment to yield a still further embodiment.

Unless defined otherwise, 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 invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

The present invention relates to antigenic peptides directed against exon 19 deletion mutations of EGFR comprising at least one (e.g., 2, 3, 4) of the following four polypeptides:

AIXXPKANK, AIXXXKANK, (V) AIXXXTSPK and VAIKXXSPK;

each occurrence of X is independently selected from any one of amino acids A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, V.

(V) AIXXXTSPK stands for V are optional.

In some embodiments, the antigenic peptide is a composition comprising 2, 3, or 4 of the four polypeptide sequences.

In some embodiments, the antigenic peptide is a fusion polypeptide comprising 2, 3, or 4 of the four polypeptide sequences.

The fusion polypeptide may be artificially synthesized.

In some embodiments, the fusion polypeptide further comprises one or more linker peptides.

In some embodiments, the number of amino acids of the linker peptide is 1 to 30; there may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30.

In some embodiments, the amino acids of the linker peptide are nonsense polypeptides that do not have additional functions (e.g., protein localization, cleavage sites, etc.) other than linking.

In some embodiments, the linker peptide is a flexible linker peptide;

in some embodiments, the amino acid sequence of the linking peptide is selected from one or more of Gly, Ser, Pro, Ala, and Glu.

In some embodiments, the amino acid sequence of the linker peptide is selected from (GGGGS) n, (GGGS) n, (GGS) n, (GS) n, or (G) n, wherein n represents the number of repeats selected from 1, 2, 3, 4, 5, or 6.

In many embodiments, the reverse transcriptase is linked to the thermostable fusion protein described above via a linker peptide. However, the reverse transcriptase and the thermostable fusion protein may also be fused directly to each other. The polypeptides comprising the fusion protein are preferably linked from N-terminus to C-terminus. However, the reverse transcriptase and thermostable fusion protein can be linked together in either order. For example, the two peptide sequences may be linked from C-terminus to N-terminus or from N-terminus to C-terminus. In some embodiments, a linker peptide is included between the linking C-terminus and N-terminus of the reverse transcriptase and thermostable fusion protein.

The linker peptide is generally flexible and can reduce steric hindrance between the fusion protein and the protein of interest, thereby facilitating proper folding of the protein.

In further embodiments, the linker peptide is a rigid linker peptide; i.e. a relatively inflexible peptide linker. Rigid linker peptides do not require a complete lack of flexibility, but are less flexible than flexible linker peptides such as glycine-rich peptide linkers. Due to its relative lack of flexibility, the rigid linker peptide reduces the movement of two protein domains (in the present case a stabilizer protein and a thermostable reverse transcriptase) linked together by the rigid linker peptide. A linker peptide providing an ordered chain (e.g., an alpha helical structure) can provide a rigid linker peptide. For example, arginine, leucine, glutamic acid, glutamine, and methionine all exhibit a relatively high propensity for helical linker structure. However, non-helical linkers containing many proline residues may also exhibit significant rigidity. Examples of rigid linking peptides include polylysine and poly-DL-alanine polylysine. Further description of rigid peptide linkers is provided by Wriggers et al, Biopolymers, 80, pages 736-46 (2005). In addition, rigid linker peptides are described in the linker database described by George et al, Protein Engineering, 15, pp 871-79 (2003). Preferably, the rigid linking peptide is also a non-cleavable linker peptide, i.e. a non-cleavable rigid linking peptide.

In some embodiments, each occurrence of X is independently selected from any one of F, M, A, S, V, L.

In some embodiments, each occurrence of X is independently selected from F or M.

In some embodiments, when the polypeptide is AIXXPKANK and VAIKXXSPK, X is independently selected at each occurrence from F, M.

In some embodiments, when the polypeptide is aixxxxank and vaixxxxtspk, X is independently selected at each occurrence from any one of F, M, A, S, V.

In some embodiments, the AIXXPKANK can be any one of the following polypeptides: AIMFPKANK, AIFFPKANK, AIMMPKANK, AIFMPKANK, AIASPKANK, respectively;

in some embodiments, the aixxxxank can be any one of AIMFPKANK, AIMFSKANK, AIMFVKANK polypeptides.

In some embodiments, (V) the aixxxtpk can be any one of AIMFSTSPK polypeptides.

Since (V) AIXXXTSPK cross-reacts with (V) AIXXTSPK, it may also be used interchangeably with (V) AIXXTSPK, which may be VAIFFTSPK in some embodiments.

In some embodiments, VAIKXXSPK may be VAIKFMSPK.

In some embodiments, the antigenic peptide comprises any one of IPVAIKETSPK, IPVAIFFSPK, IPVAIFFSSPK.

The present invention also provides nucleic acids that express the antigenic peptides described above.

The nucleic acid may be RNA or DNA.

The invention also provides a vector comprising a nucleic acid as described above.

The term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing a protein encoded by an inserted polynucleotide, the vector is referred to as an expression vector. The vector may be introduced into a host cell by transformation, transduction, or transfection, and the genetic material elements carried thereby are expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to: a plasmid; phagemid; a cosmid; artificial chromosomes such as Yeast Artificial Chromosomes (YACs), Bacterial Artificial Chromosomes (BACs), or artificial chromosomes (PACs) derived from P1; bacteriophage such as lambda phage or M13 phage, animal virus, etc. Animal viruses that may be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), pox viruses, baculoviruses, papilloma viruses, papova viruses (e.g., SV 40). In some embodiments, regulatory elements commonly used in genetic engineering, such as enhancers, promoters, Internal Ribosome Entry Sites (IRES), and other expression control elements (e.g., transcription termination signals, or polyadenylation signals and poly-U sequences, etc.) are included in the vectors of the present invention.

The invention also relates to a vaccine comprising an antigenic peptide as described above, or a nucleic acid as described above, or a vector as described above.

In some embodiments, the vaccine further comprises an adjuvant.

Adjuvants suitable for use in the vaccine of the present invention include adjuvants that enhance the immune response against the above-mentioned antigenic peptides, preferably those capable of activating the activity of Dendritic Cells (DCs) or T cells (particularly CTL cells) to make it easier to generate an immune response, which adjuvants are well known in the art.

In some embodiments, the vaccine is a water-in-oil emulsion having an aqueous phase and an oil phase.

In some embodiments, the vaccine is an oil-in-water emulsion having an aqueous phase and an oil phase.

Vaccines are typically formulated for administration by injection. Typical immunizations are by Intravenous (IV) routes of vaccination, but oral and Subcutaneous (SC), Intramuscular (IM), Intraperitoneal (IP), or Intradermal (ID) injection are also contemplated by the present invention.

Vaccines may also deliver antigenic peptides, nucleic acids or vectors to a subject via a delivery system. Methods for non-viral delivery of nucleic acids include lipofection, nucleofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycations or lipids nucleic acid conjugates, naked DNA, artificial virions and agent-enhanced uptake of DNA.

The methods of viral delivery of nucleic acids 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.

The invention also relates to a kit comprising an antigenic peptide as described above, or a nucleic acid as described above, or a vector as described above, or a vaccine as described above.

The kit may further comprise a container for vaccination of the vaccine. The inoculation container is preferably a medical syringe.

The invention also relates to the application of the antigen peptide, the nucleic acid, the carrier or the vaccine in preparing medicine for sensitizing and stimulating immune cells of body.

In some embodiments, the immune cell is an HLA-A11 phenotype and/or an HLA-A3 phenotype.

In some embodiments, the HLA-a11 is HLA-a x 11: 01.

In some embodiments, the HLA-a3 is HLA-a 03: 01.

In some embodiments, the immune cell is a dendritic cell.

The invention also relates to the use of an antigenic peptide as described above, or a nucleic acid as described above, or a vector as described above, or a vaccine as described above, for the preparation of a medicament for the treatment of a tumour.

In some embodiments, the tumor has an EGFR exon 19 deletion mutation.

In some embodiments, the tumor comprises: bone, bone junction, muscle, lung, trachea, heart, spleen, artery, vein, blood, capillary vessel, lymph node, lymphatic vessel, lymph fluid, oral cavity, pharynx, esophagus, stomach, duodenum, small intestine, colon, rectum, anus, appendix, liver, gallbladder, pancreas, parotid gland, sublingual gland, urinary kidney, ureter, bladder, urethra, ovary, fallopian tube, uterus, vagina, vulva, scrotum, testis, vas deferens, penis, eye, ear, nose, tongue, skin, brain stem, medulla oblongata, spinal cord, cerebrospinal fluid, nerve, thyroid, parathyroid gland, adrenal gland, pituitary, pineal gland, pancreatic islet, thymus, gonadal gland, and parotid gland.

In some embodiments, the tumor is a solid tumor.

In some embodiments, the tumor is selected from advanced or metastatic malignancies.

The term "advanced or metastatic malignancy" refers to a histologically or cytologically confirmed diagnosed advanced, unresectable and/or metastatic relapsed or refractory malignancy that is ineffective or absent for standard therapy to which it has been proven effective. According to the present invention, malignant tumors include, but are not limited to, carcinomas, sarcomas, melanomas, and lymphomas.

In some embodiments, the tumor is lung cancer, further non-small cell lung cancer, further lung adenocarcinoma.

The invention further provides a method of sensitising an immune cell in a subject and/or treating a tumour, which comprises administering to the animal an effective amount of an antigenic peptide, nucleic acid, vector or vaccine according to the invention.

In some embodiments, the subject is an animal, preferably a mammal, such as a cow, pig, dog, cat, sheep, rat, mouse, rabbit, horse. The subject is further preferably a primate, more preferably a human.

The term "effective amount" as used herein refers to a dose of a component to which the term corresponds that achieves treatment, prevention, alleviation and/or alleviation of a disease or condition described herein in a subject.

Embodiments of the present invention will be described in detail with reference to examples.

Example 1

EGFR exon 19 deletion mutation there are nearly 20 different deletion mutations reported in the literature, see Table 1. The frequencies listed in the table are the frequencies reported in the literature for mutations occurring in a large 354 NSCLC sample (EGFR mutation 48.02%).

EGFR exon Deletion mutation (Deletion) and reported Frequency (Frequency)

*Quan X，et al.Oncol Lett 2018 15:2131-2138

Through NetMHC4.0 messenger software and database comparison analysis, we found that the mutations of EGFR exon 19(EGFR1, NM _005228.3, P00533-1) listed in the table have different strong and weak binding force with HLA-A03: 01 and HLA-A11: 01, respectively (Table 2); ranking (RANK value) <0.5 was considered Strong binding and force (Strong binder), ranking values between 0.5 and 2.0 were considered Weak binding and force (Weak binder), and most mutant peptides appeared to have the potential to generate immune responses against both HLA phenotypes or to develop therapeutic vaccines.

TABLE 2 binding force ranking and theoretical affinity (EC) of EGFR exon 19 deletion mutant peptides and HLA phenotypes ₅₀ )

N.a. means that no binding force was found.The mutation sites are underlined in the tables.

Through further analysis, we found that the exon 19 deletion mutant peptides can be divided into 3-4 types according to the basic structure, and the main type is AIXXPKANK (9/18), the primary antigenic structure may be "AIXXPKANK "nucleus, shown as" AI "in Table 3XXPKANK' the amino acid composition of the first two sites affects the binding force of the mutant peptide and the corresponding sites, and for HLA-A03: 01 and HLA-A11: 01, the hydrophilicity and steric hindrance of methionine (M) and phenylalanine (F) become the first-choice amino acids for optimizing the structure of the polypeptide. Optimizing peptide AIFFPKANK and representative peptide AIASPKANK can represent the two extremes of this optimized peptide, AIASPKANK represents a characteristic representative structure of various EGFR exon 19 natural deletion mutant peptides, while in AIFFThe optimized selection of FF site in PKANK can make the mutant peptide of the same type reach the maximumHigh affinity overall coverage.

TABLE 3 optimization of core nonapeptide AIXXPKANK and mutant peptide AIASPKANK affinity

And "AIXXPKANK "is similar but different to" AIKXXKANK's core structure, currently collected as L747_ G753>Q and L747_ G753>S has two mutations, and the binding force is weaker. Through amino acid adjustment and optimization, the theoretical binding force can be enhanced by 5-10 times (see table 4.).

TABLE 4 optimization of core nonapeptide AIKXXKANK affinity

Further optimization is made theoretically, "AIXXPKANK "and" AIKXXKANK can be combined into the same optimized structure to cover all AIXXXKANK' structure. The results in Table 5 show that the peptide "AI" is optimized by analytical comparisonMFPKANK "is probably the most balanced choice to cover the deletion mutant peptides in the table completely.

TABLE 5 optimization of core nonapeptides AIXXPKANK and AIKXXKANK affinity

The third type of EGFR exon 19 appears to be "VAIXXXTSPK "hybrid, see Table 6, where the results obtained are L747_ A750, which is the strongest>S deletion mutant AIKEOptimized peptide "AI" for STSPKMFSTSPK”。

TABLE 6 optimization of core nonapeptide "VAIXXXTSPK”

Another group of optimized peptides with carboxyl terminal "SPK" is shown in Table 7, and the representative optimized peptide structure is "VAIKFMSPK ". In comparison with the results of tables 4 and 5, "AI" in Table 6 cannot be confirmedMFSTSPK "can cover the deletion mutant peptide in Table 7, substituting for" VAIKFMSPK "optimizes the peptide.

TABLE 7 optimization of core nonapeptide "VAIKXXSPK”

According to the above analysis, there are different structural types of the mutant peptide sequence in which exon 19 of EGFR is deleted (Table 8), and representative mutant peptide and optimized peptide sequences are present in each structure. To simplify the use and validation of representative mutant and optimized peptides, we designed combinatorial mutant peptides or synthetic combinatorial long peptides based on different structural types of EGFR exon 19 deletion mutant peptides to test whether they can stimulate Dendritic Cells (DCs) to generate an immune response in vitro and validated reactivity across various antigens using the gamma interferon Elispot assay active T cell method and corresponding tumor target cell models.

TABLE 8 EGFR exon 19 mutant core structure types

The first combined long peptide was synthesized from three structural type deletion mutations selecting representative mutant peptides as follows, named 19DA 1103T:

AIKASPKANK-IPVAIKETSPK-AIKESKANK

the second combination, optimized peptide, was selected for combination and was named 19DA1103T1:

AIFFPKANK-VAIFFSTSPK-IPVAIFFSPK

and the third combination is formed by selecting optimized peptides, and is named as 19DA1103T2:

AIMFVKANK-GGS-VAIMFSTSPK-IPVAIFFSSPK

adjusting the order of arrangement of the combinations may form other combinations.

Based on the obtained experimental results, the invention confirms that EGFR exon 19 deletion mutation is mainly divided into four structural types (Table 8), and the representative mutant peptide or representative optimized peptide with four structures can generate immune response on an in vitro cell model in a single, simple combination or synthesized long peptide neoantigen form, so that the invention has the prospect of developing various therapeutic vaccine products (polypeptide, DNA, RNA or DC vaccine) and can be used for clinically treating patients with HLA-A11: 01 phenotype, HLA-A03: 01 phenotype and corresponding EGFR exon 19 deletion mutation NSCLC.

Example 2 immune response of representative mutant peptide combinations to EGFR exon 19 deletion mutation

By the PCR-SSCP method, we tested and confirmed healthy blood donors with HLA-a 11:01 phenotype and the target cell K562 cell line. Collecting HLA-A11: 01 phenotype healthy human venous blood, separating Peripheral Blood Mononuclear Cells (PBMC), and counting; using a proper amount of adherent culture solution to adhere to the wall for 90 minutes; adherent cells were cultured with DCs in serum-free medium containing 1000units/ml GM-CSF and 500units/ml IL-4, with changes every two days. And counting suspended T cells and freezing. Adding DC adjuvant to the recombinant adenovirus HBAD-DCML on the 5 th day for infection; on day 6, the neoantigen combined peptide 19DA1103T (AIKASPKANKIPVAIKETSPKAIKESKANK) was added for sensitization (60 ug/ml); on day 7, adding 50ng/ml TN F alpha to mature the DC; the frozen suspension of T cells was thawed and the target cells K562 (HLA-A11: 01 phenotype). DC were collected on day 8, as DC: t-1: 10 mixed culture to activate T cells; on day 9, 15ug/ml of the mutant or control peptide was added to the target cell culture medium and co-cultured for 16 hours to bind to MHC I (HLA-A11: 01+ mutant peptide + beta 2 microglobulin) which is a type I histocompatibility complex. On the 10 th day, co-culturing the activated T cells and the target cells in a 96-well micro-porous plate with a polyvinylidene fluoride (PVDF) film coated with a gamma-interferon antibody fixed at the bottom for 24 hours according to the target-to-effect ratio of 0.5:1, 1:1, 2:1 and 3:1 respectively; washing the plate, and carrying out the color development reaction of peroxidase according to the requirements of a gamma-interferon Elispot kit.

The results of the experiment are shown in FIG. 21. Neo-antigen combination peptide 19DA1103T following priming of DCs and activation of T cells in vitro, reactivity of T cells overlaid various deletion mutant peptides in the map, with respect to an unrelated control peptide, wild-type nonapeptide (EATS)PKANK) Or wild type nonapeptide (ELREA)TSPK) The reactivity of the panel was comparable to the background (no peptide added panel). 19DA1103T the combination peptide is E746_ T751>A (AIKASPKANK), L747_ A750del (IPVAIKETSPK) and L747_ G753>S (AIKESKANK) three representative mutant peptides with deletion mutant structures are combined, and the immunoreactivity generated by the combined peptide can generate cross-reactivity with similar intensity on other mutant peptides with small structural difference.

Example 3 comparison of representative mutant peptide combination 19DA1103T with representative optimized peptide combination HLA-A11: 01 phenotype DC-T model immune response

The experimental procedure was similar to that of example 2, using a model of K562 (DC-T-K562) of DC, T cells and target cells prepared from peripheral blood of healthy humans of HLA-A11: 01 phenotype. After adding the DC adjuvant HBAD-DCML, dividing the DC into two groups, respectively adding 60ug/ml of a representative mutant peptide combination 19DA1103T or a representative optimized peptide combination 19DA1103T1(AIFFPKANKVAIFFSTSPKIPVAIFFSPK, see FIGS. 22 and 23) into the culture solution to stimulate sensitization, and adding TNF alpha to mature the DC after solution change, thereby activating T cells. Mixing the activated T cells with target cells K562 which are respectively combined with various polypeptides in advance in a ratio of 1:1, and culturing for 24 hours on a microporous plate of a PVDF membrane with a gamma-interferon antibody fixed and coated at the bottom; the plate is then washed and the peroxidase development is carried out as required by the interferon gamma Elispot kit.

The results are shown in fig. 24, and the optimized peptide combination 19DA1103T1 and representative mutant peptide combination 19DA1103 x 1103T of different structural types (table 8) were also able to generate an immune response covering most of the exon 19 deletion mutant peptides on the DC-T-K562 model, although this immune response cytology was only qualitatively tested, but also reflected the prospect of developing a vaccine product using a panel of polypeptides or one synthetic polypeptide suitable for immunotherapy of exon 19 deletion mutations in most of HLA-a 11:01 phenotype patients.

Example 4 comparison of representative mutant peptide combination 19DA1103T with representative optimized peptide combination 19DA1103T1 HLA-A03: 01 phenotypic DC-T model immune response

The model of HLA-A11: 01 phenotype DC-T-K562 cells was used in examples 2 and 3.

To validate the immune response of representative mutant peptide combination 19DA1103T and representative optimized peptide combination 19DA1103T1 on the DC-T model of HLA-a 03:01 phenotype, we tested and confirmed healthy donors and target cell Raji cell lines of HLA-a 03:01 phenotype using high-precision PCR-SSCP-based HLA allele typing.

The experimental procedure was similar to example 2 and example 3. 20-30ml of peripheral blood of healthy human with HLA-A03: 01 phenotype was collected, PBMC was prepared, and DC and suspension T cells were prepared as described above. Unlike K562 cells, Raji cells are anchorage-growing cells; counting one day in advance is required and seeded into the bottom of the microplate for Elispot testing. Similarly, the DC were divided into two groups, and 60ug/ml of a representative mutant peptide combination 19DA1103T or a representative optimized peptide combination 19DA1103T1 was added to the culture solution f to perform sensitization stimulation, and the mixture was matured with TNF α, and then mixed with autologous suspension T cells to culture the cells, thereby activating T cells, and detecting the reactivity to different EGFR exon deletion mutant peptides (Elispot kit).

The results are shown in fig. 25, and the optimized peptide combination 19DA1103T1 and the representative mutant peptide combination 19DA1103T of different structural types (table 8) are also able to generate immune responses covering most exon 19 deletion mutant peptides on the DC-T-Raji model, although this immune response cytology was only qualitatively tested, but also reflects the prospect of developing vaccine products using a panel of polypeptides or one synthetic polypeptide suitable for immunotherapy of exon 19 deletion mutations in most HLA-a 03:01 phenotype patients.

Example 5 comparison of the immune response reactivity of representative mutant peptide AIKASPKANK and representative optimized peptide VAIMFPKANK to known mutant peptides

From the above example, the reactivity of activated T cells was not as high as expected for representative mutant peptide AIKASPKANK, perhaps due to AIMFPKANK membrane permeability. This example was compared with AIKASPKANK using decapeptide VAIMFPKANK as the stimulatory peptide (fig. 26). The experimental procedure was similar to that of examples 2 and 3, using a model of K562 (DC-T-K562) of DC, T cells and target cells prepared from peripheral blood of healthy humans of HLA-A11: 01 phenotype. After adding DC adjuvant HBAD-DCML, dividing DC into two groups, respectively adding 20ug/ml representative mutant peptide AIKASPKANK or representative optimized peptide VAIMFPKANK into the culture solution to stimulate sensitization, adding TNF alpha after changing the solution to mature DC, and then activating T cells. Mixing the activated T cells with target cells K562 which are respectively combined with various polypeptides in advance in a ratio of 1:1, and culturing for 24 hours on a microporous plate of a PVDF membrane with a gamma-interferon antibody fixed and coated at the bottom; then the plate is washed, and the color reaction of peroxidase is carried out according to the requirements of a gamma-interferon Elispot kit. The mutated peptide sequence binding to K-562 used in this example is as follows:

E746_A750del AIKTSPKANK；E746_T751>A AIKASPKANK；E746_T751>I AIISPKANK；E746_T751del AIKSPKANK；E746_S752>A AIKAPKANK；E746_S752>D AIKDPKANK；E746_S752>V AIKVPKANK；L747_T751del AIKESPKANK；L747_S752del AIKEPKANK；L747_G753>S AIKESKANK；L747_G753>Q AIKEQKANK；

controls were provided with cell-free (-T), non-mutated (-P), wild-type peptide C2(EATSPKANK, FIG. 27), and irrelevant peptide C3 (LPGLFSLPA).

The results are shown in FIGS. 28a and 28 b. Optimized decapeptides VAIMFPKANK and AIKASPKANK as well, activated T cells after stimulation of DCs had higher reactivity to mutant peptides of all "KANK" structures (AIXXPKANK and AIKXXKANK).

Example 6 comparison of the reactivity of the immune responses of representative optimized peptides AIKMFSTSPK with VAIKMFTSPK to known mutant peptides

From the results of example 5, this example compares the immune response and T cell activity of two different classes of "SPK" (table 6) as stimulatory peptides using representative optimized peptide AIKMFSTSPK (fig. 29) and VAIKMFTSPK (fig. 30). The experimental procedure was similar to that of example 5, and a model of DC, T cells and target cell K562 (DC-T-K562) was prepared using peripheral blood of healthy human with HLA-A11: 01 phenotype. Adding DC adjuvant HBAD-DCML, dividing DC into two groups, adding 20ug/ml representative optimized peptide AIKMFSTSPK or VAIKMFTSPK into the culture solution to stimulate sensitization, changing the solution, adding TNF alpha to mature DC, and activating T cells. Mixing the activated T cells with target cells K562 which are respectively combined with various polypeptides in advance in a ratio of 1:1, and culturing for 24 hours on a microporous plate of a PVDF membrane with a gamma-interferon antibody fixed and coated at the bottom; then the plate is washed, and the color reaction of peroxidase is carried out according to the requirements of a gamma-interferon Elispot kit. The mutated peptide sequence binding to K-562 used in this example is as follows:

E746_E749del VAIKATSPK；E746_A750del IPVAIKTSPK；E746_T751>I IPVAIKISPK；

l747_ E749del AIKEATSPK; l747_ a750del VAIKETSPK; l747_ A750> P AIKEPTSPK; l747_ a750> SAIKESTSPK; l747_ T751> S VAIKESSPK; l747_ T751> P VAIKEPSPK; e746_ L747 delinstTP KTPREATSPK (FIG. 31).

Controls included cell-free (-T), non-mutated (-P), wild-type peptide C1(ELREATSPK, FIG. 32), and irrelevant peptide C3 (LPGLFSLPA).

Wherein the mutant peptide E746_ A750del and E746_ T751> I have both "KANK" and "SPK" high affinity structural sequences.

The results are shown in FIGS. 33a and 33 b. The results show that optimized peptide VAIKMFTSPK is significantly better than AIKMFSTSPK. AIKMFSTSPK the peptide core is 9 peptide AIMFSTSPK, which has high theoretical affinity in the "SPK" mutant peptide, and may be too hydrophilic to affect the uptake of the polypeptide by DC.

Combining the results of example 5 and example 6, a representative combination peptide can be a minimum of 20 peptides.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is specific and detailed, but not to be understood as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent should be subject to the appended claims.

Claims (14)

1. An antigenic peptide directed against an EGFR exon 19 deletion mutation, comprising at least one of the following four polypeptides:

AIXXPKANK, AIXXXKANK, (V) AIXXXTSPK and VAIKXXSPK;

each occurrence of X is independently selected from any one of amino acids A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, V.

2. The antigenic peptide of claim 1, which is a composition comprising 2, 3 or 4 of the four polypeptide sequences.

3. The antigenic peptide of claim 1, which is a fusion polypeptide comprising 2, 3 or 4 of the four polypeptide sequences.

4. The antigenic peptide of claim 3, further comprising one or more linker peptides in said fusion polypeptide.

5. The antigenic peptide of any one of claims 1 to 4, wherein X, at each occurrence, is independently selected from F, M, A, S, V or L.

6. A nucleic acid which expresses the antigenic peptide of any one of claims 1 to 5.

7. A vector comprising the nucleic acid of claim 6.

8. A vaccine comprising the antigenic peptide of any one of claims 1 to 5, or the nucleic acid of claim 6, or the vector of claim 7.

9. The vaccine of claim 8, further comprising an adjuvant.

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

11. Use of the antigenic peptide of any one of claims 1 to 5, or the nucleic acid of claim 6, or the vector of claim 7, or the vaccine of claim 8 or 9, for the manufacture of a medicament for the sensitizing stimulation of immune cells in a body.

12. The use of claim 11, wherein the immune cell is an HLA-a11 phenotype and/or an HLA-A3 phenotype.

13. Use of the antigenic peptide of any one of claims 1 to 5, or the nucleic acid of claim 6, or the vector of claim 7, or the vaccine of claim 8 or 9 for the manufacture of a medicament for the treatment of a tumour; the tumor has an EGFR exon 19 deletion mutation.

14. The use of claim 13, wherein the tumor is lung adenocarcinoma.

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