Antigenic peptides and peptide compositions for triple negative breast cancer immunotherapy

Abstract

The invention belongs to the technical field of medicaments related to cancer immunotherapy, relates to peptides, proteins, nucleic acids and cells for immunotherapy, and particularly relates to antigenic peptides and peptide compositions for triple negative breast cancer immunotherapy. In particular to a polypeptide which is selected from polypeptides with sequences as shown in SEQ ID NO: 1 to SEQ ID NO: 89. Or a variant sequence thereof. The invention also relates to independent use of the main component of the polypeptide vaccine or combined use of the main component of the polypeptide vaccine and other tumor-related drug active components for stimulating anti-tumor immune response or stimulating DC cells, T cells or vaccine compounds in vitro. Peptides bound to the major histocompatibility complex (MHC), or peptides as such, can be used as antibodies to recognize soluble T cell receptors and other binding molecular targets.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology related to cancer immunotherapy, and relates to peptides, proteins, nucleic acids, and cells for immunotherapy. This invention also relates to the use, alone or in combination with other tumor-associated active pharmaceutical ingredients that stimulate antitumor immune responses or in vitro stimulate dendritic cells (DCs), T cells, or vaccine complexes. Peptides that bind to the major histocompatibility complex (MHC), or peptides of the same type, may also be antibodies, soluble T-cell receptors, and other binding molecules. This invention also relates to several novel peptide sequences and their variants, derived from polypeptides that bind to HLA-I class molecules in human tumor cells, which can be used in vaccine compositions to induce antitumor immune responses or as components for the development of drugs or immunomodulatory compounds and cells. Background Technology

[0002] Existing technologies reveal that breast cancer is the most common malignant tumor among women worldwide and a leading cause of cancer death. A 2018 report on national cancer incidence and mortality rates released by the National Cancer Center shows that breast cancer ranks first among cancers affecting women in China, with nearly 279,000 women diagnosed annually and accounting for 15% of all cancer deaths. With the increasing population and aging trend, the disease burden from breast cancer is becoming increasingly severe, making the active and effective prevention and treatment of breast cancer a crucial issue in global public health.

[0003] With the gradual advancement of molecular biology techniques and the increasing sophistication of diagnostic and treatment methods, the diagnosis and treatment of breast cancer has entered the era of molecular subtyping. Based on the status of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor (HER2) with Ki-67, breast cancer can be classified into Luminal A, Luminal B, HER2-positive, and triple-negative breast cancer. 12 Triple-negative breast cancer is a type of breast cancer characterized by negative status for estrogen receptor, progesterone receptor, and HER2 receptor, accounting for approximately 12%-17% of all invasive breast cancers.

[0004] Unlike other molecular subtypes, triple-negative breast cancer (TNBC) predominantly affects young women and is characterized by rapid progression and high invasiveness. Due to the lack of specific therapeutic targets, current treatment for TNBC primarily relies on systemic chemotherapy. Therefore, compared to other subtypes, TNBC patients have a worse prognosis and a higher recurrence rate. TNBC is considered the most aggressive type of breast cancer, as it is unresponsive to conventional endocrine therapy and molecular targeted therapy. Researchers have been continuously seeking new and effective treatment options. For many years, chemotherapy has been the standard treatment, but its effectiveness has been unsatisfactory, with a median overall survival (OS) of only 12 to 18 months, and a high risk of drug resistance and recurrence, resulting in a poor prognosis. Therefore, there is an urgent need for improved therapies. Immunotherapy, represented by PD-1 / PD-L1 immune checkpoint inhibitors, has made significant progress in many tumor types in recent years, including breakthroughs in mTNBC.

[0005] Several characteristics of triple-negative breast cancer (TNBC) make it more sensitive to PD-1 / PD-1 inhibitor therapy. TNBC has numerous gene mutations, and immune checkpoint inhibitors (ICIs) can enhance the activation of specific T cells by tumor-specific neoantigens, exerting an anti-tumor effect. Based on the results of a Phase III clinical trial called IMpassion130, in March 2019, the FDA granted accelerated approval to atezolizumab in combination with nab-paclitaxel (abraxane) for first-line treatment of unresectable locally advanced or metastatic PD-L1-positive triple-negative breast cancer. Atezolizumab is the first PD-L1 inhibitor approved by the FDA and also the first immunotherapy regimen for breast cancer. The study enrolled 902 patients from 41 countries, of whom 369 were PD-L1 positive. The primary endpoints of the study were progression-free survival (PFS) and overall survival (OS). The results showed that in PD-L1-positive patients, median PFS increased from 5.0 months to 7.5 months, and median OS increased from 15.5 months to 25.0 months, an improvement of nearly 10 months.

[0006] However, more clinical trials have shown that monotherapy with immune checkpoint inhibitors, including atezolizumab, has a low efficacy rate in treating mTNBC, indicating the limitations of single-agent immune checkpoint inhibitor therapy. Future research should focus on combination therapies such as immunotherapy with chemotherapy / targeted therapy / radiotherapy and others.

[0007] Studies show that gene mutations are the initiation of tumorigenesis, and triple-negative breast cancer is no exception, being one of the tumors with a high mutation burden. Mutations typically produce variant proteins not found in normal cells. These variant proteins are hydrolyzed into short peptides by intracellular proteases, which bind with high affinity to MHC class I or MHC class II molecules and are presented on the cell surface. They are then recognized as "non-self" by T cells, triggering T cell activation, which in turn attacks and eliminates tumor cells.3 These abnormal proteins that trigger T cell activation are called "neoantigens." The more significant the difference between the mutation and the original coding sequence, the more easily it is recognized by T cells. Insertion / deletion and frameshift mutations result in significant changes to the amino acid sequence and spatial structure, leading to a stronger affinity for MHC molecules and a greater likelihood of being recognized as neoantigens by T cells.

[0008] A growing body of research indicates that personalized cancer vaccines based on mutated neoantigens hold great promise. In 2017, research teams from the United States and Germany independently conducted clinical trials using neoantigen-based technology for cancer treatment, achieving promising results in melanoma treatment. These were the first two successful clinical trials of neoantigen-based personalized cancer vaccines. Both studies suggest that personalized vaccines targeting multiple neoantigen epitopes may be an ideal strategy for overcoming tumor heterogeneity.

[0009] Alternative splicing of pre-mRNA is a key step in the regulation of post-transcriptional gene expression. This makes it possible for a single gene to be transcribed into different messenger RNAs, greatly enriching the diversity and complexity of proteome structure and function, and meeting the needs of different tissues and organs for protein diversity.

[0010] Alternative splicing (AS) of RNA refers to the way different splice variants of mRNA are produced by splicing at different splice sites in the precursor mRNA. The five most common forms of alternative splicing in mRNA are: exon skipping, intron retention, mutually exclusive exon splicing, alternative 3' splice site, and alternative 5' splice site. Analysis has shown that at least 15,000 cancer-specific splicing events have been identified in 27 types of cancer.

[0011] The application of NGS technology provides further evidence supporting the diverse sources of tumor mutations. Currently discovered sources of various types of neoantigens can be categorized as follows: genomic-level mutations, including SNVs and small fragment In Dels; genomic-level gene fusions; transcriptomic-level alternative splicing; transcriptomic-level RNA editing; transcriptomic-level mutations in the non-coding regions of translated proteins; and proteomic-level alternative splicing.

[0012] Since 99% of tumor mutations occur in non-coding regions, the exome covers only 2% of the human genome. However, up to 75% of the human genome can be transcribed and potentially translated into proteins. Therefore, focusing solely on the exome as a source of tumor-specific neoantigens is limiting.

[0013] Recent studies have shown that many so-called non-coding regions are actually protein-coding regions that can produce numerous peptides presented by MHC-I, some of which have been identified as targets of TILs. However, no MHC-II-restricted neoantigens that may initiate CD4+ T cell activation have been reported to originate from non-coding regions. Compared to mutations at other genomic and transcriptomic levels, neoantigens originating from non-coding regions are rarely mentioned. Effective computational tools need to be developed to identify such neoantigens and validate their immunogenicity.

[0014] Alternative splicing is a process regulated by multiple factors. Systemic mutations in splicing factors (such as U2AF1 and SF3B1) can lead to a wide range of landscape changes in the splicing process in cancer cells. Kahles et al. conducted a pan-cancer analysis of alternative splicing mutations in 8705 patients in TCGA. This study revealed that tumor-specific neo-links generated by alternative splicing are an important source of neoantigens. In particular, intron-preserving splicing (an RNA splicing error that results in the inclusion of introns in the mRNA transcript) is a source of tumor neoantigens. Some studies have also confirmed that novel epitopes generated by this type can be presented by MHC-I or MHC-II molecules and trigger an immune response from CD4+ or CD8+ lymphocytes. Epitopes are present in the amino acid sequence of the antigen to ensure that this "immunogenic peptide" derived from the tumor antigen can elicit an in vitro or in vivo T cell response.

[0015] Structural differences between different HLA loci, or between different alleles at the same locus of different genes, can create different HLA molecule groove structures. This results in the selective binding of different HLA allele-encoded molecules to various antigenic peptides. Furthermore, antigenic peptides that can bind to the same class of HLA molecules often share the same or similar anchoring sites and residues. This indicates that specific HLA molecules can selectively bind antigenic peptides based on desired common motifs; in this sense, the binding is selective. In fact, different HLA molecules can selectively bind peptides with different anchoring sites and residues. Therefore, different HLA allele products may present different epitopes of the same antigen molecule, leading to differences in the intensity of responses to the same antigen in different individuals (carrying different MHC alleles). This is an important mechanism by which HLA participates in and regulates immune responses through polymorphism.

[0016] Further research has revealed that the recognition of antigenic peptides by HLA molecules is not a strict one-to-one relationship. This flexibility manifests in several ways: First, the sequence and structure of antigenic peptides that form a common basic sequence can vary. Second, the anchoring residues required by the same HLA molecule (such as HLA-II molecules) often consist of more than one amino acid, resulting in a considerable number of peptide chains corresponding to a specific common basic sequence. This allows one HLA molecule to bind to multiple antigenic peptides, activating multiple specific T cell clones. Third, antigenic peptides accepted by different HLA molecules can possess similar common motifs. For example, at least four families—A2, A3, B4, and B44—are known in HLA-I molecules. Members of these families (various allele products) can selectively recognize antigenic peptides with the same or similar anchoring residues. This means that an antigenic peptide that can be recognized and presented by one HLA molecule may also be presented by other molecules in its family. This facilitates the application of peptide vaccines, in vitro sensitized dendritic cell vaccines, or T-cell vaccines for immunoprophylaxis and immunotherapy. This also provides a basis for identifying and modifying the peptide sequences of tumor vaccines. Not only can each HLA-I molecule bind to a certain number of different peptides, but T cells also exhibit cross-reactivity, meaning a single T cell can recognize two or more different peptide antigens and MHC protein complexes. This, in turn, provides another basis for identifying and modifying the peptide sequences of tumor vaccines.

[0017] For MHC-I peptides, which trigger cellular immune responses, they must also bind to MHC molecules. This process depends on the alleles of the MHC molecule and the specificity of the peptide's amino acid sequence. MHC-I molecule-binding peptides are typically 8-12 amino acid residues long. Furthermore, their interaction sequences with the corresponding binding grooves of MHC molecules usually include two conserved residues called "anchors," thus each MHC allele possesses a "binding motif," which determines which peptides can specifically bind to the binding groove.

[0018] Essentially, any peptide that can bind to MHC molecules can potentially act as a T cell epitope. The prerequisite for inducing an in vitro or in vivo T cell response is the presence of T cells with the corresponding TCR and the absence of immune tolerance to that specific epitope. Therefore, in a highly preferred embodiment of this invention, it is crucial to select only antigenic epitope peptides formed by RNA splicing abnormalities, as these tumor-specific antigens can induce specific anti-tumor immune responses.

[0019] Statistics show that HLA-A2 (including A*0201, A*0202, A*0204, A*0205, A*0206, A*0207, and A*0208) alone has an average distribution frequency exceeding 40% in the Chinese population. The peptide sequences identified within the same large group are highly similar.

[0020] Based on the current state of the technology, the inventors of this application intend to provide cancer immunotherapy-related pharmaceuticals, involving peptides, proteins, nucleic acids, and cells for immunotherapy. In particular, antigenic peptides and peptide compositions for the immunotherapy of triple-negative breast cancer.

[0021] Reference List

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[0039] Jiang T, Shi T, Zhang H, et al. Tumor neoantigens: from basic research to clinical applications. J Hematol Oncol. 2019; 12(1):93.

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[0041] Laumont CM, Vincent K, Hesnard L et al. Noncoding regions are the main source of targetable tumor-specific antigens. Sci Transl Med. 2018; 10(470). Summary of the Invention

[0042] Based on the current state of the technology, the inventors of this application provide peptides, proteins, nucleic acids, and cells for immunotherapy. This invention also relates to the use alone or in combination with other tumor-associated active pharmaceutical ingredients that stimulate antitumor immune responses or in vitro stimulate DC cells, T cells, or vaccine complexes. Peptides that bind to major histocompatibility complexes (MHC) or similar peptides may also be antibodies, soluble T-cell receptors, and other binding molecules. More specifically, this invention also provides antigenic peptides and peptide compositions for immunotherapy of triple-negative breast cancer.

[0043] Specifically,

[0044] In a first aspect, the present invention relates to a peptide comprising an amino acid sequence selected from SEQ ID NO: 1 to SEQ ID NO: 84, or a variant sequence having at least 80%, preferably at least 90%, homology to SEQ ID NO: 1 to SEQ ID NO: 84 ( wherein the variant binds to MHC and / or induces cross-reactivity between T cells and the peptide). The peptide is capable of binding to HLA-I or HLA-II molecules, and when bound to MHC, it is recognized by CD8+ T cells and / or CD4+ T cells.

[0045] A second aspect of the invention relates to a peptide comprising an amino acid sequence selected from SEQ ID NO: 1 to SEQ ID NO: 89, preferably SEQ ID NO: 1 to SEQ ID NO: 9, or a variant having at least 80%, preferably at least 90%, homology to SEQ ID NO: 1 to SEQ ID NO: 9, wherein the total length of the peptide or its variant is 8 to 50, preferably 8 to 30, and most preferably 8 to 16 amino acids. The following table shows the peptides according to the invention, their respective SEQ IDs, and the probable (potential) gene sources of these peptides. All peptides in Tables 1 and 2 are associated with HLA-A*02.

[0046] Table 1: Preferred Examples of the Polypeptides of the Present Invention

[0047] Serial Number ID sequence Gene symbol HLA-A2 affinity score 1(SEQ ID NO3) FLFCFVLFL ZC3H11A 36 2(SEQ ID NO 9) TLFRYPTPI ZC3H11A 29 3(SEQ ID NO 4) FLWSCCQPL SERPINA3 36 4(SEQ ID NO 89) FLSSEAANV TNFAIP2 21 5(SEQ ID NO 85) ALASQIAGI FIP1L1 24 6(SEQ ID NO 86) FLYFVPFFV SREK1 24 7(SEQ ID NO 5) FMWCYMKVL WWC1 35 8(SEQ ID NO 87) ILLTWLSLL WWC1 24 9(SEQ ID NO 88) FLLEFWQQV CDK13 24

[0048] Table 2: List of peptides screened by this invention

[0049]

[0050]

[0051]

[0052] Table 3: The HLA-A*02 peptide of the present invention and its use in other oncological diseases

[0053]

[0054]

[0055]

[0056]

[0057] The table above shows that other tumor types share epitopes derived from the same gene antigen. This invention also relates to peptides of the invention that have the ability to bind to major histocompatibility complex (MHC) I or to peptide-MHC II molecules in an elongated form, such as those with varying lengths.

[0058] In this invention, the term "variant" or "variant of a peptide" refers to a variant that is equivalent to or has better activity than the peptide in terms of protein activity, such as antigenicity, epitope activity, or immunological activity. In some embodiments, those skilled in the art can obtain the variant by altering portions of the peptide that do not impair its activity, such as by substituting, deleting, inserting, or adding one or more amino acids in the peptide's amino acid sequence. In some embodiments, the variant can be obtained by substituting relevant amino acids into the peptide by identifying conserved molecule residues and portions among similar peptides. Those skilled in the art can also analyze the three-dimensional structure and amino acid sequence associated with similar peptide structures. Based on such information, those skilled in the art can predict amino acid sequence alignments of the antigen's three-dimensional structure, thereby preparing experimental variants containing single amino acid substitutions at each desired amino acid residue. These variants can then be screened using activity assays known to those skilled in the art.

[0059] In this invention, the word "comprising" in the expression "a peptide contains a certain amino acid sequence" includes the meaning of "having".

[0060] In one aspect of the invention, a peptide fragment (immunogenic fragment) having the amino acid sequence of SEQ ID NO:1 to SEQ ID NO:89 is provided, i.e., having a continuous portion of the amino acid sequence of SEQ ID NO:1 to SEQ ID NO:89, which generates an immune response recognizing the amino acid sequence peptide shown in SEQ ID NO:1 to SEQ ID NO:89. Preferred fragments include, for example, truncated peptides having a portion of a continuous amino acid sequence of SEQ ID NO:1.

[0061] In another aspect of the invention, the aforementioned peptide can bind to HLA-I molecules and be recognized by CD8+ T cells. CD8+ T cells are also known as cytotoxic T cells (CTLs). In yet another aspect of the invention, the HLA-I is of type HLA-A2. In this document, "substantially identical amino acid sequence" means that one or more amino acids (e.g., 2, 3, 4, or 5) in the amino acid sequence are substituted, deleted, added, or inserted, resulting in the same or similar or better activity compared to that amino acid sequence. In this invention, the activity can refer to, for example, recognition by HLA I and HLA II and subsequent activation of tumor-associated T cell activity.

[0062] The peptides of the present invention can be synthesized using methods employed in conventional peptide chemistry. The polypeptides of the present invention can also be prepared using conventional genetic engineering. For example, the polypeptides can be prepared using nucleotides encoding the polypeptides prepared using conventional DNA synthesis and genetic engineering methods. The present invention also provides isolated nucleic acids consisting of a base sequence encoding the aforementioned peptides of the present invention. The nucleic acids of the present invention can be cDNA, mRNA, or a DNA / RNA chimera, preferably DNA. When the nucleic acid is double-stranded, it can be double-stranded DNA, double-stranded RNA, or a DNA:RNA hybrid. When the polynucleotides of the present invention are double-stranded, they can be inserted into an expression vector to produce a recombinant expression vector for expressing the peptides of the present invention. Therefore, the nucleic acids of the present invention comprise recombinant expression vectors obtained by inserting the double-stranded polynucleotides of the present invention into an expression vector.

[0063] The base sequence encoding the signal peptide of the present invention is not particularly limited, as long as it produces any of the amino acid sequences of the polypeptides of the present invention after translation. The base sequence encoding the amino acid sequence can be obtained by PCR or other methods using genomic DNA or RNA containing the peptide sequence as a template. On the other hand, considering expression efficiency in host cells, codons that are frequently used in the host cells are usually preferred. Codon usage frequency data in various biological species can be obtained from genetic code usage frequency databases. The nucleic acid of the present invention can also be chemically synthesized using an automated DNA / RNA synthesizer.

[0064] In one aspect of the invention, vaccine and pharmaceutical compositions comprising the peptides or nucleic acids of the present invention are provided. The vaccine and pharmaceutical compositions provided by the present invention can be used to treat or prevent cancer. The peptides of the present invention can be used as vaccine and pharmaceutical compositions for treating or preventing cancer. The nucleic acids of the present invention can also be used as vaccine and pharmaceutical compositions for treating or preventing cancer. The nucleic acids of the present invention can be expressed in a conventional manner to obtain the peptides of the present invention, which can then be used for the aforementioned purposes. The peptides of the present invention can be presented to HLA antigens on antigen-presenting cells, thus treating or preventing tumors in patients. The polypeptides of the present invention can be presented to HLA antigen-presenting cells, specifically stimulating T cells, particularly CD8+ T cells, capable of recognizing the complex of the binding of HLA antigens and the presented polypeptides, which can proliferate and kill tumor cells, thus being used to treat or prevent tumors in patients.

[0065] The reagents for inducing T cells, particularly CD8+ T cells, containing the peptides of the present invention as active ingredients, can be mixed or combined with pharmaceutically acceptable carriers, such as suitable adjuvants, to effectively establish cellular immunity.

[0066] The peptides of the present invention are particularly effective in presenting and inducing specific cytotoxic T cells (CTLs) in the HLA-A2 type. The HLA-A2 type includes A*0201, A*0202, A*0204, A*0205, A*0206, A*0207, and A*0208. Therefore, the peptides of the present invention and vaccine or pharmaceutical compositions based on the peptides of the present invention for the prevention and treatment of tumors can effectively cover a wider population. Preferably, the peptides of the present invention are effective in presenting and inducing specific cytotoxic T cells (CTLs) in the HLA-A2 type. More preferably, the peptides of the present invention are effective in presenting and inducing specific cytotoxic T cells (CTLs) in the A*0201 and A*0202 types.

[0067] Vaccines containing peptides or derivatives of the present invention as active ingredients for inducing preventive or therapeutic immune responses can be mixed or administered in combination with pharmaceutically acceptable carriers, such as suitable adjuvants, to more effectively establish an immune response. Examples of adjuvants that can be used include, for example, microbial-derived components or derivatives thereof, cytokines (GM-CSF), plant-derived components or derivatives thereof, marine-derived components or derivatives thereof, mineral gels such as aluminum hydroxide, lysophosphatidylcholine, surfactants such as polyols, polyanionic peptides, oily emulsifiers (emulsifier formulations), etc. Liposome formulations, nanoparticle formulations linked to beads having a diameter of a few micrometers, formulations with linked lipids, microsphere formulations, microcapsule formulations, etc., are also contemplated.

[0068] The methods of using the above-described peptides or pharmaceutical compositions of the present invention include intradermal, subcutaneous, intramuscular, intravenous administration, etc. The dosage of the peptides of the present invention in the formulation can be appropriately adjusted according to the disease to be treated, the patient's age and weight, etc. Typically, the dosage of the peptides of the present invention in the formulation is 0.0001 to 1000 mg, preferably 0.001 to 1000 mg, more preferably 0.1 to 10 mg, and preferably once every few days or once every one to several months.

[0069] The peptides and pharmaceutical compositions of the present invention described above can also be used in vitro to treat cancer patients. In other words, the peptides or nucleic acids of the present invention can be contacted in vitro with antigen-presenting cells and / or immune effector cells to generate antigen-presenting cells capable of recognizing the antigens or antigen complexes of the present invention, thereby inducing specific T cells, particularly CD8+ T cells, which can then be returned to the patient for the prevention or treatment of cancer.

[0070] The present invention provides T cells, particularly CD8+ T cells, induced in vitro by contacting peripheral blood lymphocytes derived from tumor patients with the peptides or nucleic acids of the present invention, and a method for generating the above-mentioned cells.

[0071] In another aspect of the invention, a method for generating antigen-presenting cells is provided. In one aspect of the invention, the method includes the step of contacting the peptides of the present invention described above with cells capable of antigen presentation. In one aspect of the invention, the method includes the step of contacting the nucleic acids of the present invention described above with cells capable of antigen presentation. The peptides and nucleic acids of the present invention described above can be contacted with cells capable of antigen presentation to generate antigen-presenting cells. Contacting the peptides or nucleic acids of the present invention with cells capable of antigen presentation can generate antigen-presenting cells. The peptides and nucleic acids of the present invention can be used in vitro for the prevention or treatment of tumors. For example, antigen-presenting cells can be generated by contacting the peptides or nucleic acids of the present invention with cells capable of antigen presentation in vitro. The peptides and nucleic acids of the present invention can also be used in vivo for the prevention or treatment of tumors.

[0072] In this invention, the cells with antigen-presenting capacity are cells that express HLA antigens with presenting peptides on their cell surface. One type of cell with antigen-presenting capacity is a dendritic cell.

[0073] This invention provides an antigen-presenting cell, wherein the cell surface expresses an HLA antigen that presents the peptide of this invention. One type of cell with antigen-presenting capacity is a dendritic cell. The antigen-presenting cell of this invention can be prepared by adding the peptide or nucleic acid of this invention to the aforementioned cells with antigen-presenting capacity.

[0074] The antigen-presenting cells of the present invention can be obtained by isolating antigen-presenting cells from tumor patients, stimulating the cells in vitro with the peptides of the present invention, and allowing the antigen-presenting cells to present a complex of HLA antigen and polypeptide. When using dendritic cells, lymphocytes can be isolated from the peripheral blood of tumor patients, cells that cannot adhere to the culture dish can be removed, adhering cells can be cultured in the presence of GM-CSF and IL-4 to induce dendritic cells, and the antigen-presenting cells of the present invention can be prepared by culturing and stimulating the dendritic cells with the peptides of the present invention.

[0075] The antigen-presenting cells of the present invention are prepared by adding the nucleic acid of the present invention into cells capable of antigen presentation. The nucleic acid may be in the form of DNA or RNA. The nucleic acid can express the peptide of the present invention in the antigen-presenting cells.

[0076] This invention provides an immune effector cell capable of specifically recognizing and being activated by presenting cells that express and present the peptide and HLA antigen complex of this invention, thus proliferating and differentiating into effector cells. Effector cells include various T cells, such as CD8+ T cells and CD4+ T cells. In one aspect of this invention, an immune effector cell, specifically a CD8+ T cell, is provided that is cytotoxic to cancer cells, causing them to rupture and die, and also serves as a memory cell, which, upon re-encountering the same antigen stimulation, will proliferate and differentiate into effector cells more rapidly.

[0077] The immune effector cells of the present invention can be prepared by adding the peptides or nucleic acids of the present invention to cells with antigen-presenting capabilities, and then contacting the presenting cells, which present the peptides and HLA antigen complex of the present invention, with cells with immune effector capabilities.

[0078] The antigen-presenting cells of the present invention described above can be used as vaccines or pharmaceutical compositions for the treatment or prevention of cancer. The antigen-presenting cells of the present invention possess immunoinducing activity and can be used to prepare reagents for inducing antigen-specific effector cells. The induced CD8+ T cells can exert anti-tumor effects through cytotoxicity and the production of lymphokines. Therefore, the antigen-presenting cells of the present invention can be an active ingredient in vaccine or pharmaceutical compositions for the treatment or prevention of tumors. Vaccines for inducing CTLs containing antigen-presenting cells as an active ingredient may contain saline, phosphate-buffered saline (PBS), culture medium, etc., to stably maintain the antigen-presenting cells. Administration methods include intravenous administration. The reagents for inducing CD8+ T cells containing antigen-presenting cells as an active ingredient can be returned to the patient, thus effectively inducing specific CD8+ T cells in patients who respond to the peptides of the present invention, resulting in the treatment or prevention of tumors.

[0079] The immune effector cells of the present invention can be used as vaccine or pharmaceutical compositions for the treatment or prevention of cancer. The effector cells of the present invention include various T cells, such as CD8+ T cells. The CD8+ T cells of the present invention can exert anti-tumor effects through cytotoxicity and the production of lymphokines. Therefore, the immune effector cells of the present invention can be active ingredients in vaccine or pharmaceutical compositions for the treatment or prevention of tumors. Vaccines containing immune effector cells as active ingredients may contain saline, phosphate-buffered saline (PBS), culture medium, etc., to stably maintain the immune effector cells. Administration methods include intravenous administration. The reagent containing immune effector cells as active ingredients can be returned to the patient, resulting in the treatment or prevention of tumors.

[0080] In one aspect of the invention, a vaccine and pharmaceutical composition comprising the antigen-presenting cells or immune effector cells of the present invention is provided. The vaccine and pharmaceutical composition provided by the present invention can be used to treat or prevent cancer. The antigen-presenting cells or immune effector cells of the present invention can be used as a vaccine and pharmaceutical composition for treating or preventing cancer.

[0081] The vaccine and pharmaceutical compositions containing antigen-presenting cells or immune effector cells of the above invention can be used to treat cancer patients via in vitro methods. The peptides or nucleic acids of the present invention can be contacted in vitro with antigen-presenting cells and / or immune effector cells to generate antigen-presenting cells capable of recognizing the antigens or antigen complexes of the present invention, thereby inducing specific effector cells, such as T cells (especially CD8+ T cells), and then the antigen-presenting cells and / or immune effector cells are returned to the patient for the prevention or treatment of cancer. In one aspect of the invention, the patient is HLA-I type, preferably HLA-A2 or HLA-A3 type, more preferably HLA-A2 type, and most preferably A*0201 or A*0202 type.

[0082] The immune effector cells of the present invention, such as T cells (especially CD8+ T cells), can be used as active ingredients in vaccine or pharmaceutical compositions for the treatment or prevention of tumors.

[0083] The present invention also provides the use of the polypeptides or variants thereof as described above, or the nucleic acids of the present invention as described above, or the antigen-presenting cells of the present invention as described above, or the immune effector cells of the present invention as described above, in the preparation of vaccine or pharmaceutical compositions for treating or preventing cancer. The cancers include leukemia, solid tumors, etc., and more specifically, breast cancer, lung cancer, malignant lymphoma, cancers of digestive organs (e.g., stomach cancer, pancreatic cancer, liver cancer, colon cancer, rectal cancer, etc.), ovarian cancer, bladder cancer, leukemia (e.g., acute leukemia, including acute exacerbations of chronic myeloid leukemia), kidney cancer, prostate cancer, etc., preferably breast cancer.

[0084] In the description of this invention, the terms "comprising," "including," or any other variations thereof are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising a reference structure" does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element. It should be noted that, herein, relational terms such as "first," "second," etc., are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Attached Figure Description

[0085] To more clearly illustrate the technical solutions of this application, the drawings used in the embodiments will be briefly introduced below. Obviously, each drawing described below is for a part of the embodiments of this application. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0086] Figure 1 Flowchart of screening and identification technology for antigen epitope peptides in triple-negative breast cancer immunotherapy.

[0087] Figure 2 Binding rates of antigenic peptides of different lengths to HLA-A*01:01 / HLA-A*02:01 molecules.

[0088] Figure 3 Construction of the prediction model of the anchor positioning integral algorithm.

[0089] Figure 4 The neoantigen peptides predicted by the anchor positioning integral algorithm prediction model were validated as having high affinity in NetMHCpan.

[0090] Figure 5 Flowchart for ELISOT identification of the immunogenicity of antigenic epitope peptides.

[0091] Figure 6 ELISPOT identified immunogenic epitope peptides.

[0092] Figure 7 P1 antigen-specific T cells kill tumor cells that stably express neoantigens HLA-A2+.

[0093] Figure 8 P2 antigen-specific T cells kill tumor cells that stably express HLA-A2+ neoantigens.

[0094] Figure 9 P3 antigen-specific T cells kill tumor cells that stably express neoantigens HLA-A2+.

[0095] Figure 10 P4 antigen-specific T cells kill tumor cells that stably express neoantigens HLA-A2+.

[0096] Figure 11 P5 antigen-specific T cells kill tumor cells that stably express neoantigens HLA-A2+.

[0097] Figure 12 P6 antigen-specific T cells kill tumor cells that stably express neoantigens HLA-A2+.

[0098] Figure 13 P7 antigen-specific T cells kill tumor cells that stably express neoantigens HLA-A2+.

[0099] Figure 14 P8 antigen-specific T cells kill tumor cells that stably express HLA-A2+ neoantigens.

[0100] Figure 15 P9 antigen-specific T cells kill tumor cells that stably express neoantigens HLA-A2+. Detailed Implementation

[0101] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0102] Example 1: Flowchart of Antigen Epitope Peptide Screening and Identification Technology for Triple-Negative Breast Cancer Immunotherapy

[0103] Based on the TNBC gene sequence database, an RNA splicing regulation model was used to read RNA alternative splicing patterns, including exon skipping, intron retention, mutually exclusive variable exons, variable 3' splice sites, and variable 5' splice sites. Next-generation sequencing (NGS) high-throughput sequencing technology was used to process the gene sequences. Due to the large required data sample size and the short sequence length of the mutated fragments, the high throughput and short read length of NGS are well-suited for this situation. Simultaneously, NGS-derived targeted resequencing technology was used to identify target RNA splicing aberrations by binding complementary oligonucleotide probes to specific sequence regions. These aberration fragments were then copied and stored to construct a triple-negative breast cancer RNA aberration splice body library. Using the UCSC gene database and Python as the text processing platform, the relevant data transformations (splice reconnection, FASTA format conversion, amino acid translation, and gene identification) were completed, ultimately obtaining 14,880 valid polypeptide sequence format triple-negative breast cancer RNA aberration splice bodies. A novel neoantigen prediction model was constructed by optimizing the original prediction model using a two-stage anchor residue screening method and an integral algorithm, which is beneficial for screening higher-quality neoantigens. Subsequently, the immunogenicity of the screened peptides was identified using ELIST. (See appendix) Figure 1 .

[0104] Example 2: Construction of the Anchor Positioning Integral Algorithm Prediction Model

[0105] First, peptides satisfying the amino acid requirements at positions 2 and 9 were screened using secondary anchor residues. Based on the known affinity correlations of fixed amino acids for MHC molecules in the anchoring model, an integral algorithm was introduced to score them: all peptides started with 20 points, and the additional score was the square of the number of good binding peptides minus the square of the number of no binding peptides. This model optimization can pre-simulate the differences in binding rates between strongly binding neoantigen peptides, reflecting the possible performance of antigen peptides in the T cell activation pathway, which is beneficial for screening higher-quality neoantigen peptides for experimental validation with limited resources. Peptides were prioritized according to the following criteria: ① High affinity for MHC molecules: IC50 < 50 nM or % Rank; ② Mutant (high VAF); ③ A peptide can bind to two or more HLA molecules; ④ Different algorithms can be used to predict peptides and further identify the specificity of pre-existing T cell responses to preferentially mutated peptides. See Appendix. Figure 2 and 3 .

[0106] Example 3: The neoantigen peptide predicted by the anchor localization integral algorithm prediction model was validated as having high affinity in NetMHCpan. The NetMHCpan-4.1 web server was used to predict the binding between peptides and MHC-I. This system utilizes tailored machine learning strategies to integrate different training data types, thereby achieving performance superior to traditional prediction models. NetMHCpan-4.1 covers over 11,000 MHC molecules, including human (HLA-A, HLA-B, HLA-C, HLA-E, HLA-G), mouse (H-2), bovine (BoLA), primate (Patr, Mamu, Gogo), pig (SLA), horse (EQCA), and dog (DLA) MHC alleles, and determines whether a sequence is a strong MHC binder (SB) or a weak MHC binder (WB) based on a %Rank score.

[0107] The high-scoring neoantigen peptides obtained in this invention have been certified using the NetMHCpan-4.1 system, and all selected high-scoring antigen peptides were evaluated as SB (strong binding) in this system. See appendix. Figure 4 .

[0108] Example 4 Synthesis of antigenic epitope peptides

[0109] Synthetic peptides (SEQ ID NO:1 to SEQ ID NO:9), hereinafter referred to as antigenic peptides, are abbreviated as P1, P2, P3, P4, P5, P6, P7, P8, and P9, respectively. The synthesized peptides were purified to 95%. After passing mass spectrometry identification, the lyophilized antigenic peptides were dissolved in dimethyl sulfoxide (25 mM) and stored at -80°C.

[0110] Example 4: Preparation of Dendritic Cells (DCs)

[0111] Anticoagulated blood was obtained from healthy volunteers, diluted twice with PBS, and then added to the surface of Ficoll-Hypaque separation solution. The mixture was centrifuged at 18-20°C and 800g density gradient for 30 minutes. After centrifugation, the contents of the centrifuge tube were separated into three layers: the upper layer was plasma (containing platelets), the middle layer was the separation solution, and the bottom layer was red blood cells and granulocytes. A milky white turbid PBMC layer was visible at the interface between the upper and middle layers. Remove the PBMC layer, resuspend it in PBS, centrifuge at 400g for 10 min, repeat the above step, resuspend the cell pellet in RPMI1640 containing 10% fetal bovine serum, and culture at 37°C for 2 hours. Remove the non-adherent cells for T cell isolation. Add RPMI1640 medium containing 1000 U / ml hGM-CSF and 500 U / ml hIL-4 to the adherent cells and culture at 37°C for 1 week to induce monocyte differentiation into DCs. Change the medium and cytokines every two days. Harvest the non-adherent and loosely adhering cells, which are the DCs.

[0112] Example 5: Preparation of Antigen-Specific T Cells

[0113] PBMCs were isolated from peripheral blood and resuspended in PBS. The cells were centrifuged at 400g for 10 min. This step was repeated, and the cell pellet was resuspended in RPMI 1640 containing 10% fetal bovine serum. The cells were incubated at 37°C for 2 hours. Non-adherent cells were then removed and resuspended in an appropriate volume of RPMI 1640 to achieve a cell concentration of 0.5–1.0 x 10⁻⁶ cells / mL. 7 ml, collect T cell suspension by immunomagnetic beads. Centrifuge at 400g for 5 min, resuspend the cell pellet in cell cryopreservation solution, and store for later use.

[0114] Add appropriate concentrations of antigens, such as the antigenic peptides used in this experiment, to the culture medium of DCs. Incubate at 37°C for 4 hours, harvest DCs, and wash once with PBS. Add DCs and T cells from the same volunteer to a 96-well flat-bottom culture plate according to a certain cell ratio. Co-culture at 37°C for 5 days, then add 3H-TdR, IuCi / well, and incubate at 37°C for 18 hours. Use a multi-head cell collector to collect the cells onto glass fiber filter paper, and wash three times each with PBS, 5% trichloroacetic acid, and anhydrous ethanol. Dry the filter paper, place it in scintillation solution, and measure the cpm value on a β-scintillation counter.

[0115] Example 6: Identification of antigen-specific CTL cells using ELISA_spot (ELISPOT) method

[0116] The ELISPOT assay for cytokines is used to determine the number of cytokine-secreting cells in a single-cell suspension. It offers rapid detection, high sensitivity, and ease of operation. The principle involves coating an ELISPOT plate with a high-affinity anti-cytokine antibody. When test cells are added to the ELISPOT plate, the secreted cytokines are captured by the coated antibody. After removing the test cell suspension, another labeled anti-cytokine antibody is added, followed by the appropriate chromogenic reagent. This produces spots on the ELISPOT plate indicating the number and location of cytokine secretion.

[0117] Add appropriate concentrations of antigens, such as the antigenic peptides used in this experiment, to the culture medium of DCs. Incubate at 37°C for 4 hours. Harvest DCs and wash once with PBS. Add DCs and T cells from the same volunteer to 24-well culture plates at a certain ratio and co-culture at 37°C for 7-10 days, adding 20 U / ml hIL-2 to the culture medium. Resuspend effector cells and target cells in a certain volume of culture medium and add them to 96-well ELISP0T reaction plates pre-coated with IFN-γ antibody at a certain ratio. Incubate at 37°C for 18 hours. Remove the cell suspension, wash 10 times with PBST, add biotin-labeled IFN-γ antibody, incubate at 37°C for 2 hours, wash 10 times with PBST, add enzyme-labeled anti-biotin antibody, incubate at 37°C for 2 hours, wash 10 times with PBST, add substrate solution, and react in the dark at room temperature for 15-30 minutes. Count the cells using an ELISP0T automatic reader. See Appendix. Figure 5 , attached Figure 6 .

[0118] Example 7: DCs loaded with antigenic peptides can activate tumor cell-specific CTLs and effectively attack tumor cells. In this experiment, a tandem DNA sequence encoding peptides SEQ NO 1 to SEQ NO 9 was synthesized, with EcoRI and XhoI restriction sites at the 5' and 3' ends, respectively. The sequence was subcloned into the expression vector pCDNA 3.0, amplified, recovered, and stored at -20°C.

[0119] The cytotoxicity of T lymphocytes was measured in the following experiments: Target cells (tumor cells SMMC-7721 and SMMC-7721-020, where SMMC-7721 was HLA-A2 negative and SMMC-7721-020 was HLA-A2 positive) were digested with 0.25% trypsin and washed once with 1640 complete medium (containing 10% FBS and 1% penicillin-streptomycin); the cells were then resuspended in 1640 complete medium and the cell number was adjusted to 1 × 10⁻⁶ cells / cells. 6cells / ml. SMMC-7721 and SMMC-7721-020 were transfected with plasmid

[0084] . 48 hours after transfection, cell lines that stably expressed tandem DNA sequences encoding peptides SEQ NO 1 to SEQ NO 9 were screened in selective medium containing G418 (1000 μg / ml).

[0120] Appropriate concentrations of antigens, including antigenic peptides, were added to the culture medium of DCs as described in Example 6 to stimulate the DCs, and then the resulting DCs loaded with the antigenic peptides were used to stimulate the body's own T cells.

[0121] Collect stimulated CD8+ T cells, centrifuge at 1000 rpm, discard the supernatant, resuspend the CD8+ T cells in 1640 complete culture medium, and adjust the cell number to 1×10⁻⁶. 6 cells / ml.

[0122] 100 μl of CD8+ T cells and 100 μl of tumor cells were added to 96-well plates. A blank control group (culture medium control) was also set up. Each experimental group and control group had 3 replicate wells. 50 μl of target cells (8000 cells / well) were added to each experimental group. The effector-target ratio was 25:1, 12:1, 6:1 and 3:1, with the corresponding number of effector cells added to each experimental group, and the volume added was 50 μl. After adding target cells and effector cells to the experimental wells, the following control groups need to be added to the corresponding cells or culture media: 1. Effector cell spontaneous release well group: According to the corresponding effector-to-target ratio, add effector cells corresponding to each effector-to-target ratio to 3 replicates to obtain the effector cell spontaneous release value, with a final volume of 100 μl for each well; 2. Target cell spontaneous LDH release group: Add target cells (8000 cells / well) to 3 replicates, with a final volume of 100 μl for each well; 3. Target cell maximum LDH release group: Add target cells (8000 cells / well) to 3 replicates, with a final volume of 100 μl for each well; 4. Culture medium background group: Add 100 μl of culture medium to 3 replicates. This control is used to correct the background caused by LDH activity in phenol red and serum-containing culture media; 5. Volume correction control group: Add 10 μl of Lysis Solution (10X) to 3 replicates containing 100 μl of 5% FBS RPMI 1640 culture medium (cell-free). This control was used to correct for volume changes caused by the addition of Lysis Solution (10X). After the test plate was prepared, it was centrifuged at 250g for 4 min and incubated at 37°C for 6 h; Lysis of cells and harvesting of supernatant: 45 minutes before collecting the supernatant, 10 μl of Lysis Solution (10X) was added to the target cell maximum release well. After incubation for 6 h, it was centrifuged at 250g for 4 min; (5) Measurement of LDH: 50 μl of supernatant was transferred from each well to a new 96-well using a pipette, and then 50 μl of the prepared substrate was added to each well. The well was incubated at room temperature in the dark for 30 min. After incubation, 50 μl of stop solution was added to each well, and large air bubbles were punctured with a syringe needle. The absorbance was measured on a 490nm microplate reader within 1 h; (6) Calculation of experimental results: The absorbance of all experimental wells, target cell spontaneous LDH release wells, target cell maximum LDH release wells and effector cell spontaneous LDH release wells was subtracted from the average absorbance of the culture medium background. Substitute the corrected values ​​obtained above into the formula below to calculate the percentage of cytotoxicity produced for each effector-to-target ratio. See Figure 7 - Figure 15 .

[0123] The calculation formula is as follows:

[0124]

[0125] This invention belongs to the fields of molecular biology and immunology. The amino acid sequence of the tumor antigen epitope peptide of this invention is as shown in any one of SEQ ID NO. 1-SEQ ID NO. 89; or the amino acid sequence of the tumor antigen epitope peptide has more than 90% similarity to any one of the sequences shown in SEQ ID NO. 1-SEQ ID NO. 89 through amino acid substitution, deletion, or addition; or the amino acid sequence of the tumor antigen epitope peptide includes any one of the sequences shown in SEQ ID NO. 1-SEQ ID NO. 89 or includes sequences that have more than 90% similarity to any one of the sequences shown in SEQ ID NO. 1-SEQ ID NO. 89 through amino acid substitution, deletion, or addition. The multimeric antigen peptide of this invention is the aforementioned tumor antigen epitope peptide. This invention provides insights for clinical immunotherapy of cancer, better serving the development of cancer vaccine combination drugs and benefiting cancer patients.

Claims

1. A polypeptide, characterized in that, The polypeptide is selected from one or more of the following polypeptides: (1) Polypeptides with sequences as shown in SEQ ID NO: 1 to SEQ ID NO: 89; (2) Variant sequences of which have at least 88% homology with SEQ ID NO: 1 to SEQ ID NO: 89; (3) A continuous amino acid extension region in any one of the sequences from SEQ ID NO: 1 to SEQ ID NO: 89; or, The variant binds to the major histocompatibility complex and / or induces T-cell cross-reactivity with the variant peptide.

2. The polypeptide according to claim 1, characterized in that, The polypeptide is capable of binding to HLA-I or HLA-II molecules; wherein the polypeptide can be recognized by CD8+ T cells and / or CD4+ T cells when it binds to MHC. The total length of the polypeptide or its variants is 8-50 amino acids, preferably 8-30 amino acids, more preferably 8-16 amino acids, and most preferably the polypeptide, which is composed of or substantially composed of any amino acid sequence according to SEQ ID NO: 1 to SEQ ID NO: 89; The polypeptide is modified and / or contains non-peptide bonds.

3. The polypeptide according to claim 2, characterized in that, The polypeptide is part of a fusion protein, and the fragment can bind to HLA-I molecules and be recognized by CD8+ T cells; wherein the HLA-I is HLA-A2 or HLA-A3, preferably HLA-A2, and more preferably A*0201.

4. A nucleic acid, characterized in that, The nucleic acid is the nucleic acid encoding the polypeptide of any one of claims 1 to 3, and / or optionally linked to a heterologous promoter sequence.

5. An expression carrier, characterized in that, It contains the nucleic acid as described in claim 4.

6. A host cell, characterized in that, The host cell comprises: (A) The polypeptide according to claims 1 to 3; (B) The nucleic acid according to claim 4; or, (C) The expression vector according to claim 5; The host cells mentioned are preferably antigen-presenting cells, such as dendritic cells.

7. A method for preparing the polypeptide according to any one of claims 1 to 3, characterized in that, The method includes culturing a host cell of claim 6, which presents the polypeptide of claims 1 to 3 and / or expresses the nucleic acid of claim 4 or carries the expression vector of claim 5, and isolating the polypeptide or a variant thereof from the host cell or its culture medium.

8. A method for preparing in vitro activated T lymphocytes, characterized in that, The method involves in vitro linking T cells to human MHC class I or MHC class II molecules carrying antigens, the molecules being expressed on the surface of antigen-presenting cells or on the surface of artificially simulated antigen-presenting cell structures, thereby activating T cells in an antigen-specific manner. The antigen described therein is a polypeptide as described in any one of claims 1 to 3.

9. The method according to claim 8, characterized in that, The generated activating T lymphocytes recognize specific cells, said specific cells presenting a polypeptide according to any one of claims 1 to 3.

10. A method for killing target cells in vitro, characterized in that, The targeted cells present a polypeptide, the polypeptide comprising the polypeptide described in any one of claims 1 to 3; The method for killing target cells further includes administering the effective amount of activating T lymphocytes as described in claim 9.

11. An antibody, characterized in that, The antibody is a soluble or membrane-bound antibody that specifically recognizes the polypeptide or variant thereof as described in claims 1 to 3, and is preferably the polypeptide or variant thereof as described in any one of claims 1 to 3 when binding to an MHC molecule.

12. A medicine box, characterized in that, include: (1) A first container comprising a pharmaceutical composition containing a polypeptide or a variant thereof as claimed in any one of claims 1 to 3, a nucleic acid as claimed in claim 4, an expression vector as claimed in claim 5, a host cell as claimed in claim 6, a T-lymphocyte initiator as claimed in claim 9, or an antibody as claimed in claim 11; (2) Optionally, the second container contains a diluent or reconstituted solution for the lyophilized powder formulation; (3) Optionally, at least one polypeptide is selected from polypeptides with sequences as shown in SEQ ID NO: 1 to SEQ ID NO: 89; (4) Optionally, use the instructions for use with a diluent or reconstituted solution and / or with a lyophilized powder formulation; or The kit includes one or more (i) buffers, (ii) diluents, (iii) filtrates, and (iv) needles or syringes.

13. A pharmaceutical composition, characterized in that, It includes the polypeptide or a variant thereof as described in any one of claims 1 to 3, the nucleic acid as described in claim 4, the expression vector as described in claim 5, the host cell as described in claim 6, the T-lymphocyte initiation method as described in claim 9, or the antibody as described in claim 11.

14. A vaccine, characterized in that, The vaccine is a personalized treatment or prevention vaccine for cancer, comprising the polypeptide or a variant thereof as described in any one of claims 1 to 3, the nucleic acid as described in claim 4, the expression vector as described in claim 5, the host cell as described in claim 6, the initiating T lymphocyte as described in claim 9, or the antibody as described in claim 11.

15. Use of a drug in the preparation of a medicament for treating or alleviating cancer; The drug is selected from: the nucleic acid of claim 4, the expression vector of claim 5, the host cell of claim 6, the T lymphocyte initiation method of claim 9, or the antibody of claim 11; The cancer is selected from breast cancer, lung cancer, colorectal cancer, esophageal cancer, kidney cancer, liver cancer, ovarian cancer, gastric cancer, melanoma, and leukemia. The cancer tumor overexpresses proteins derived from polypeptides from SEQ ID NO: 1 to SEQ ID NO:

89. The cells of the targeted patient are of HLA type HLA A1, preferably HLA A2 or HLA A3, preferably HLA A2, and more preferably A*0201 or A*0202.

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