TWI701262B - Immunogenic peptide and use thereof - Google Patents

Abstract

The present invention relates to an immunogenic peptide comprising a T cell epitope peptide derived from tumor necrosis factor receptor-associated protein 1 (TRAP1), and the immunogenic peptide is able to induce cytotoxic T lymphocytes. In addition, the present invention also relates to a drug comprising the above immunogenic peptide for treating a cancer associated with the overexpression of TRAP1.

Description

Immunogenic peptides and their uses

The present invention relates to an immunogenic peptide and its use in the treatment of cancer, characterized in that the immunogenic peptide is a T cell epitope peptide derived from tumor necrosis factor receptor related protein 1 (TRAP1) , Which can trigger a cytotoxic T cell response against cancer cells in cancer patients to treat cancer.

Tumor necrosis factor receptor-associated protein 1 (TRAP1) is a homolog of heat shock protein 90 (Hsp90), and it has been confirmed that it binds and hydrolyzes in mitochondria in a similar manner to Hsp90 ATP. Recently, accumulated evidence has pointed out that TRAP1 will be present in cancer cells in large quantities and play an important role in the mitochondrial transformation associated with tumorigenesis. Conversely, down-regulation of TRAP1 was found to accelerate the apoptosis of cancer cells and exhibit the ability to inhibit neoplastic transformation, indicating that targeting TRAP1 can be evaluated as a potential strategy for cancer treatment. Based on sequence similarity, Hsp90 inhibitors and its derivatives are theoretically ideal drug candidates for inactivating TRAP1; however, their inhibitory mechanism is controversial because these molecules may lack specificity, so It is impossible to distinguish TRAP1 from other chaperone homologs; because of this, the focus on the role of client protein in normal cells and homeostasis has been increased. Therefore, a new TRAP 1 targeting strategy is necessary.

In terms of targeting intracellular tumor-related proteins, inducing cytotoxic T lymphocyte (CTL) can be used as an alternative, which involves the immunity of peptides related to human leukocyte antigen (HLA) Monitoring role. HLA-A, HLA-B, and HLA-C are responsible for presenting peptide fragments of intracellular proteins for CD8 ⁺ T cells to recognize. HLA molecules present abnormal peptides of cancer-related proteins to T cells, leading to activation of T cells, and then destroying target cancer cells through specific cytolysis responses. To this day, the induction of antigen-specific CTL is still considered an effective strategy to eliminate tumors. In order to achieve this goal, the discovery of potential epitopes required for inducing CTL immune response is a key step. Therefore, it is necessary to identify the specific peptides of TRAP1 on cancer cells that can bind to HLA molecules.

Mass spectrometry (MS) can be used as an effective tool for analyzing peptide sequences presented on HLA. Complex peptide mixtures can be separated and then ionized in sequence in a mass spectrometer. Then the peptide sequence and possible post-translational modifications (post-translation) can be analyzed according to the corresponding tandem MS (MS/MS) spectrum. translational modifications, PTMs). Therefore, it was discovered that the specifically shared phosphorylation site on TRAP1 is involved in the blocking mechanism of the tyrosine-protein kinase Src-mediated signaling pathway, which is an alternate signal for cell transformation. Generally speaking, these dysregulated phosphorylated proteins will bind to ubiquitin for proteasomal degradation to maintain cell stability, which will increase the appearance of phosphorylated peptides in HLA molecules. Therefore, the phosphorylated peptides displayed on HLA molecules are closely related to the dysregulated phosphorylated proteins, so these phosphorylated peptides are potential CTL peptide candidates.

Therefore, the cancer vaccine developed based on the CTL epitope is an anti-cancer drug with future prospects. This type of cancer vaccine induces a cytotoxic response to tumor cells that express the target antigen, thereby destroying the tumor cells. Therefore, the identification of CTL epitopes derived from tumor target antigens is necessary for the development of cancer vaccines based on CTL epitopes.

The present invention uses AAD transgenic mice with genes encoding the α1 and α2 domains of the HLA-A*02:01 gene and the α3 transmembrane domain of the mouse H-2Db gene to simulate the expression of HLA-A2 restriction Human cell lines with sex epitopes. In the present invention, AAD-transfected mouse lung epithelial cancer cell line TC1/AAD is selected as a model to discover the potential HLA-A2-restricted cytotoxic T lymphocyte (CTL) epitope of TRAP1. In addition, single AAD gene transfection can avoid the laborious process required to confirm the HLA type of the identified peptide. Sequence analysis showed that the TRAP1 sequence is highly conserved in human and mouse cancer cell lines. Based on these characteristics, it is assumed that mouse TC1/AAD cells and human lung cancer cells exhibit similar HLA-A2 restricted peptides of TRAP1. With the aid of MS-based phosphorylation proteomics analysis; a new HLA-A2-restricted TRAP1 phosphorylation peptide was identified from TC1/AAD cells. This peptide has a new phosphorylation site, which is the first time it has been observed in TRAP1 protein. The present invention proves that the HLA-A2 restricted phosphorylation peptide derived from TRAP1 has immunogenicity and can induce CTL response, and the phosphorylation peptide can specifically lyse cancer cells and effectively inhibit AAD transgenic mice. Tumor growth. These anti-tumor results indicate the therapeutic efficacy of TRAP1-derived HLA-A2-restricted phosphorylated peptides in cancer treatment.

In addition, the present invention has also developed a lipidated peptide, which is composed of the above-mentioned phosphorylated peptide, a lipid, and a helper T cell epitope, wherein the above-mentioned three are all combined with an amine Base acid to connect. The lipidated peptide can also induce a cytotoxic reaction against tumor cells expressing the target antigen, and achieve the effect of inhibiting tumors. At the same time, since the lipids and helper T cell epitopes in the lipidated peptide can be used as internal adjuvants, when the lipidated peptide is made into a vaccine, the vaccine does not require additional adjuvants, which can cause intense Immune response.

The term "a" or "an" as used herein describes the elements and ingredients of the present invention. This term is only used for convenience and to provide the basic concept of the invention. This description should be understood to include one or at least one, and unless the context clearly dictates otherwise, singular terms shall include pluralities, and plural terms shall include the singular. When used with the word "including" in the scope of the patent application, the term "a" or "an" can mean one or more than one.

The term "or" used herein can mean "and/or".

The present invention provides a peptide comprising an amino acid sequence consisting of Lys-XX-Ser-Val-Glu-Thr-Asp-X (SEQ ID NO: 9), wherein X is leucine (Leu) or iso Leucine (Ile), and the serine (Ser) is phosphorylated.

In a specific embodiment, the peptide is a phosphopeptide. In another specific embodiment, the amino acid sequence is SEQ ID NO: 6, wherein the amino acid (Ser) at the fourth position in the sequence of SEQ ID NO: 6 (the fourth residue at the N terminal) is Phosphorylation.

The peptide of the present invention is a human leukocyte antigen (HLA) restricted peptide. HLA is a human-specific major histocompatibility complex (Major Histocompatibility Complex Class I, MHC) antigen. MHC includes Class 1 MHC (MHC class I), Class 2 MHC (MHC class II), and Class 3 MHC (MHC class III). When a peptide has suitable binding motifs, the Class 1 MHC molecule can bind to the peptide and present the peptide to cytotoxic T lymphocytes. In a specific embodiment, the HLA of the MHC class 1 includes HLA-A, HLA-B, and HLA-C. In a preferred embodiment, the HLA of the MHC type 1 is HLA-A.

In another specific embodiment, the HLA-A comprises HLA-A1, HLA-A2, HLA-A3, HLA-A9, HLA-A10, HLA-A11, HLA-Aw19, HLA-A23, HLA-A24, HLA -A25, HLA-A26, HLA-A28, HLA-A29, HLA-A30, HLA-A31, HLA-A32, HLA-Aw33, HLA-Aw34, HLA-Aw36, HLA-Aw43, HLA-Aw66, HLA-Aw6 And HLA-A69. In a preferred embodiment, the HLA is HLA-A1, HLA-A2, HLA-A3, HLA-A11 or HLA-A24. In a more preferred embodiment, the HLA is HLA-A2. In another specific embodiment, the peptide is an HLA-restricted peptide. In a preferred embodiment, the peptide is an HLA-A restricted peptide. In a more preferred embodiment, the peptide is an HLA-A2 restricted peptide.

Therefore, the peptide will combine with HLA (such as HLA-A2) to form an HLA/peptide complex, and immune cells will recognize the HLA/peptide complex and induce an immune response. In a specific embodiment, the peptide is an immunogenic peptide.

It is currently known that tumor necrosis factor receptor related protein 1 (TRAP1) is highly expressed on cancer cells, especially in lung cancer. Therefore, TRAP1 is a tumor specific antigen. Therefore, the peptide is designed based on the amino acid sequence of the T cell epitope derived from tumor necrosis factor receptor-related protein 1 (TRAP1). In a specific embodiment, the peptide is a T cell epitope peptide derived from TRAP1 (TRAP1-derived T cell epitope peptide). In a preferred embodiment, the peptide is a TRAP1-derived T cell phosphorylated epitope peptide (TRAP1-derived T cell phosphorylated epitope peptide). In another specific embodiment, the peptide is a cytotoxic T cell epitope peptide. In a preferred embodiment, the peptide is a cytotoxic T cell phosphorylated epitope peptide. Therefore, the peptide of the present invention can induce an effective and specific immune response to cells expressing TRAP1. In another embodiment, the peptide induces an immune response against cells expressing TRAP1. Further, the peptide induces the production of T cells (such as CTL) that secrete interferon-γ (IFN-γ) to combat cells expressing TRAP1, such as cancer cells. Therefore, the peptide can induce a CTL response against tumor cells expressing TRAP1. In a preferred embodiment, the peptide induces a CTL response to treat cancer caused by cancer cells expressing TRAP1.

The present invention also provides a composition comprising a peptide, wherein the peptide comprises an amino acid sequence consisting of Lys-XX-Ser-Val-Glu-Thr-Asp-X (SEQ ID NO: 9), wherein X is leucine (Leu) or isoleucine (Ile), and the serine (Ser) is phosphorylated.

In a specific embodiment, the peptide is a phosphopeptide. In another specific embodiment, the amino acid sequence is SEQ ID NO: 6, wherein the amino acid (Ser) at the fourth position in the sequence of SEQ ID NO: 6 (the fourth residue at the N terminal) is Phosphorylation.

In a specific embodiment, the composition is a pharmaceutical composition. The pharmaceutical composition may optionally include a carrier, such as a pharmaceutically acceptable carrier. In a preferred embodiment, the pharmaceutical composition includes a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is determined by the specific composition to be administered and the specific method used to administer the composition. Therefore, there are many suitable formulations of pharmaceutical compositions. Formulations suitable for parenteral administration can be formulated, for example, for intravenous, intramuscular, intradermal, intraperitoneal and subcutaneous routes. The carrier may contain an aqueous isotonic sterile injection solution, which may contain antioxidants, buffers, bacteriostatic agents and solutes, so that the preparation is isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions, which may Including suspending agents, solubilizers, thickening agents, pharmaceuticals, stabilizers, preservatives, liposomes, microspheres and emulsions. The term "pharmaceutically acceptable" refers to compounds and compositions that can be administered to mammals without excessive toxicity. The term "pharmaceutically acceptable carrier" does not include tissue culture medium.

In a specific embodiment, the composition may be an immunogenic composition or vaccine capable of eliciting an immune response to cancer cells. In a preferred embodiment, the composition is a vaccine. As used herein, the term "immunogenic composition or vaccine" refers to a composition that elicits at least one type of immune response against cancer cells. Therefore, this immune response can be of any type, such as a cytotoxic T lymphocyte (CTL) response. CTL can recognize the HLA/peptide complex presented on the surface of antigen presenting cells (APC), which leads to Cell lysis, that is, the vaccine can trigger the production of effector T cells (effector T cells) in the vaccinated subject, which has a cytotoxic effect on cancer cells.

Since the peptides of the present invention are relatively small molecules, it may be necessary to combine the peptides with various materials (such as adjuvants) in such compositions to produce vaccines or immunogenic compositions, etc. Adjuvants are broadly defined as substances that can promote an immune response. Generally, the selected adjuvant is selected from complete Freund's adjuvant, incomplete Freund's adjuvant, alum, vector virus, high molecular weight polysaccharide, glycoprotein, microparticle, liposome, lipid, and combinations thereof. When the antigen of interest is of low molecular weight or poor immunogenicity, it is recommended to couple with an immunogenic carrier. Specific examples of such immunogenic carrier molecules include keyhole limpet haemocyanin, bovine serum albumin, ovalbumin and fowl immunoglobulin. Various saponin extracts have also been proposed as adjuvants in immunogenic compositions. Recently, it has been proposed to use granulocyte-macrophage colony stimulating factor (GM-CSF), a well-known cytokine, as an adjuvant (WO 97/28816). In a specific embodiment, the composition includes an adjuvant.

In a specific embodiment, the peptide is an HLA restricted peptide. In a preferred embodiment, the peptide is an HLA-A restricted peptide. In a more preferred embodiment, the peptide is an HLA-A2 restricted peptide.

Since the peptide is a T cell epitope peptide derived from TRAP1, after the peptide binds to an HLA molecule on an antigen-presenting cell, it allows T cells to recognize and induce a CTL response to enable cells expressing TRAP1 (such as cancer Cell) cell lysis occurs. In another embodiment, the peptide induces an immune response against cells expressing TRAP1. In a preferred embodiment, the immune response includes a CTL response. Therefore, the composition containing the peptide has the ability to induce the response of cytotoxic T lymphocytes, and can be used to treat cancer cells expressing TRAP1. In a more preferred embodiment, the peptide induces a CTL response against cancer cells expressing TRAP1.

The present invention further provides a composition for the preparation of a medicament for the treatment of cancer in a body, wherein the composition comprises an effective dose of peptide, wherein the peptide comprises Lys-XX-Ser-Val-Glu-Thr -An amino acid sequence composed of Asp-X (SEQ ID NO: 9), where X is leucine (Leu) or isoleucine (Ile), and the serine (Ser) is phosphorylated.

In a specific embodiment, the peptide is a phosphopeptide. In another specific embodiment, the amino acid sequence is SEQ ID NO: 6, wherein the amino acid (Ser) at the fourth position in the sequence of SEQ ID NO: 6 (the fourth residue at the N terminal) is Phosphorylation.

In another embodiment, the peptide is an HLA restricted peptide. In a preferred embodiment, the peptide is an HLA-A restricted peptide. In a more preferred embodiment, the peptide is an HLA-A2 restricted peptide. Therefore, the peptide will combine with HLA-A molecules on an antigen presenting cell (APC) (such as B lymphocytes, macrophages, and dendritic cells) to form an HLA-A/peptide complex for T cells to recognize , And activate T cells. Therefore, the individual is an individual with HLA-A2 in one body, so that after the drug containing the peptide is administered into the individual, the peptide can bind to the HLA-A2 molecule to activate T cells. In another specific embodiment, the individual is an individual with an HLA molecule of HLA-A. In a preferred embodiment, the individual is an individual with an HLA molecule HLA-A2.

In a specific embodiment, the individual is an animal, preferably a mammal, and more preferably a human.

The peptide is designed based on the amino acid sequence of the T cell epitope derived from tumor necrosis factor receptor related protein 1 (TRAP1). Therefore, the peptide can induce immune cells to produce an immune response against cells expressing TRAP1. In a specific embodiment, the immune cells include type IT helper cells (Th1), cytotoxic T lymphocytes (CTL), antigen presenting cells (APC) capable of inducing Th1 cells, and APC capable of inducing CTL. In a preferred embodiment, the immune cells include type 1 helper T cells (Th1) and cytotoxic T lymphocytes (CTL). In a more preferred embodiment, the cytotoxic T lymphocytes are CD8 ⁺ T cells. Therefore, in another embodiment, the peptide is a cytotoxic T cell epitope peptide. In a preferred embodiment, the peptide is a cytotoxic T cell phosphorylated epitope peptide.

The term "cancer" as used herein refers to a condition in which cell populations do not respond to varying degrees of control mechanisms that normally control cell proliferation and differentiation. Cancer refers to various types of malignant tumors and tumors, including primary tumors and tumor metastases. In a specific embodiment, the cancer is cancer caused by cancer cells expressing TRAP1. In another specific embodiment, the cancer includes lung cancer, head and neck cancer, breast cancer, pancreatic cancer, lymphoma, myeloma, leukemia, kidney cancer, ovarian cancer, Bone cancer, liver cancer, prostate cancer, skin cancer, colon cancer and thyroid cancer. In a preferred embodiment, the cancer is lung cancer, breast cancer, ovarian cancer, colorectal cancer or prostate cancer. In a more preferred embodiment, the cancer is lung cancer.

As used herein, the term "treatment" refers to a therapeutic treatment that can help the individual reverse, reduce, ameliorate, inhibit, slow down, or stop the course or severity of the disease or condition associated with the disease, such as cancer. The term "treatment" includes reducing or alleviating at least one adverse effect or symptom of a condition, disease or condition associated with cancer. If one or more symptoms or clinical markers are reduced, the treatment is usually effective. Alternatively, if the course of the disease is reduced or stopped, the treatment is effective. In other words, "treatment" includes not only the improvement of symptoms or markers, but also the cessation or at least slowing down of the progression or deterioration of symptoms compared to expectations without treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptoms, reduction of disease severity, stabilization (i.e. not worsening) of disease state, delay or slowing down of disease progression, improvement or alleviation of disease state, alleviation (whether partial or full), Reduce interventions, shorten hospital stays and/or reduce mortality, whether detectable or undetectable. The term "treatment" of a disease also includes alleviation of symptoms or side effects of the disease (including palliative treatment).

The term "effective dose" as used herein refers to a therapeutic dose that can prevent, reduce, stop, or reverse the symptoms that have developed in the individual under certain conditions; or partially or completely relieve the individual when the individual begins to receive treatment Symptoms that already exist under certain conditions.

In a specific embodiment, the effective dose of the peptide ranges from 0.1 mg/kg body weight to 10 mg/kg body weight. In a preferred embodiment, the effective dose of the peptide ranges from 0.5 mg/kg body weight to 4 mg/kg body weight. In a more preferred embodiment, the effective dose of the peptide ranges from 1.5 mg/kg body weight to 2 mg/kg body weight.

Therefore, after the drug containing the peptide enters the body of the individual, it will combine with the HLA-A molecule on an antigen-presenting cell to form an HLA-A/peptide complex to activate T cells that can secrete IFN-γ (such as Th1 and CTL) to inhibit the growth of tumor cells expressing TRAP1, thereby treating cancer. In a specific embodiment, the peptide has the ability to induce the immune response of T cells to inhibit cancer cells. In a preferred embodiment, the peptide has the ability to induce the immune response of T cells to treat cancer. In a more preferred embodiment, the peptide has the ability to induce the immune response of T cells secreting IFN-γ to treat cancer.

In another specific embodiment, the T cell or the IFN-γ-secreting T cell includes type 1 helper T cells (Th1) and cytotoxic T lymphocytes (CTL). In a preferred embodiment, the cytotoxic T lymphocytes are CD8 ⁺ T cells.

Therefore, the dosage form of the drug can be a vaccine; the vaccine includes the peptide and an adjuvant, wherein the adjuvant can improve immunogenicity or promote immune response. The vaccine can induce CTL responses to cells expressing TRAP1 to treat diseases or cancers caused by cells expressing TRAP1.

The present invention further provides a lipidated peptide (lipopeptide), comprising a lipid, a T helper cell epitope and a peptide, wherein the lipid, the T helper cell epitope and the The three peptides are not connected to each other, but all three are connected to an amino acid, wherein the peptide contains Lys-XX-Ser-Val-Glu-Thr-Asp-X (SEQ ID NO: 9) The composition of the amino acid sequence, where X is leucine (Leu) or isoleucine (Ile), and the serine (Ser) is phosphorylated.

In a specific embodiment, the structure of the lipidated peptide is as follows:

Wherein W is the helper T cell epitope; Y is the peptide; Z is the lipid; and A is the amino acid. W, Y, and Z are not connected to each other. In addition, the positions of W, Y and Z in the structure can be interchanged, which does not affect the efficacy of the lipidated peptide.

In a specific embodiment, the peptide is a phosphorylated peptide. In a preferred embodiment, the amino acid sequence of the peptide is SEQ ID NO: 6, wherein the amino acid (Ser) at the fourth position in the sequence of SEQ ID NO: 6 (the fourth at the N-terminus) Residues) are phosphorylated.

The function of this lipid is to act as a ligand for toll-like receptor (TLR) to trigger the toll-like receptor pathway, thereby activating the response of immune cells, such as promoting dendritic cell (DC) ) Maturity, and enhance Th1-biased immune response (Th1-biased immune response) to promote CTL. In a specific embodiment, the lipid has the function of triggering the toll-like receptor pathway. In a preferred embodiment, the toll-like receptor (TLR) includes TLR2, TLR4, TLR5 and TLR9. In a more preferred embodiment, the toll-like receptor is toll-like receptor 2 (TLR2).

In another specific embodiment, the lipid comprises a fatty acid. In a preferred embodiment, the fatty acid comprises an unsaturated fatty acid and a saturated fatty acid. In a more preferred embodiment, the saturated fatty acid includes palmitic acid. Therefore, the side chain of the amino acid can be joined with two or three palmitic acids. In another specific embodiment, the palmitic acid is Cys((RS)-2,3-(palmitoyloxy)-propyl)-OH(Cys((RS)-2,3-di(palmitoyloxy) -propyl)-OH); or N-α-palmitoyl-S-[2,3-bis(palmitoyloxy)-2RS]-propyl]-L-cysteine (N-α- Palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-L-cysteine).

The function of the T helper cell epitope is to induce the immune response of T helper cells. In a specific embodiment, the helper T cell epitope has the function of inducing the immune response of the helper T cell. In a preferred embodiment, the sequence of the helper T cell epitope includes an amino acid sequence of SEQ ID NO:5.

The helper T cell epitope is a class 2 MHC restricted peptide. Therefore, the helper T cell epitope can be related to the second MHC molecule. When a peptide has suitable binding motifs, the second type of MHC molecule can bind to the peptide and present the peptide to helper T cells. In a specific embodiment, the HLA of the Type 2 MHC includes HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, and HLA-DR. In a preferred embodiment, the HLA of the second MHC is HLA-DR. In a more preferred embodiment, the helper T cell epitope is an HLA-DR-restricted peptide. Therefore, the helper T cell epitope can combine with the HLA-DR molecule on the antigen presenting cell to form an HLA-DR/helper T cell epitope complex, allowing the helper T cell to recognize and activate the helper Sex T cells.

"Major histocompatibility complex (MHC)", "MHC molecule", "HLA" or "HLA molecule" should be understood as particularly a protein capable of binding to a peptide, wherein the peptide is a protein antigen. The peptide produced by hydrolytic cleavage, and the peptide will exhibit potential T cell epitopes, the peptide will be transported to the cell surface and the peptide will be presented to specific cells, especially cytotoxic T lymphocytes or helper Sex T cells.

The amino acid is connected to the lipid, the helper T cell epitope and the peptide. Since the side chain of the amino acid has an active functional group (for example, OH or COOH), it can be connected to lipids. In a specific embodiment, the amino acid is an amino acid with a reactive functional group on one side chain. In a preferred embodiment, the amino acid is lysine. In another specific embodiment, the active functional group is an OH group or a COOH group.

In another embodiment, the peptide is an HLA restricted peptide. In a preferred embodiment, the peptide is an HLA-A restricted peptide. In a more preferred embodiment, the peptide is an HLA-A2 restricted peptide. Therefore, the lipidated peptide containing the peptide will combine with the HLA-A molecule (such as HLA-A2) on an antigen presenting cell (APC) to form an HLA-A/peptide complex, thereby activating T cells. In a specific embodiment, the lipidated peptide is an immunogenic peptide.

The peptide is designed based on the amino acid sequence of the T cell epitope derived from tumor necrosis factor receptor related protein 1 (TRAP1). In a specific embodiment, the peptide is a T cell epitope peptide derived from TRAP1. In a preferred embodiment, the peptide is a T cell phosphorylated epitope peptide derived from TRAP1. In another specific embodiment, the peptide is a cytotoxic T cell epitope peptide. In a preferred embodiment, the peptide is a cytotoxic T cell phosphorylated epitope peptide. Therefore, the peptide can induce immune cells to produce an immune response against cells expressing TRAP1. Therefore, the lipidated peptide will combine with the HLA molecules on the antigen-presenting cell to form an HLA/lipidated peptide complex, allowing T cells to recognize it, and then activate the cells that secrete IFN-γ to fight against cells expressing TRAP1 (such as cancer). cell). Therefore, the lipidated peptide can be used as a therapeutic agent for treating cancer. In a specific embodiment, the IFN-γ secreting cells include type 1 helper T cells (Th1) and cytotoxic T lymphocytes (CTL).

As used herein, “T cell epitope peptide”, “T cell phosphorylation epitope peptide” or “helper T cell epitope” should be understood to mean that it can be used in conjunction with type 1 or type 2. The peptide sequence bound by the MHC molecule of the MHC molecule is formed in the form of a peptide-based MHC molecule or an MHC complex, and then in this form, it is recognized and bound by cytotoxic T lymphocytes or helper T cells, respectively.

The present invention also provides a composition comprising a lipidated peptide, which comprises a lipid, a helper T cell epitope and a peptide, wherein the lipid, the helper T cell epitope and the peptide are three Those are not connected to each other, but the three are connected to an amino acid, wherein the peptide contains an amine composed of Lys-XX-Ser-Val-Glu-Thr-Asp-X (SEQ ID NO: 9) Base acid sequence, where X is leucine (Leu) or isoleucine (Ile), and the serine (Ser) is phosphorylated.

In a specific embodiment, the peptide is a phosphorylated peptide. In a preferred embodiment, the amino acid sequence of the peptide is SEQ ID NO: 6, wherein the amino acid (Ser) at the fourth position in the sequence of SEQ ID NO: 6 (the fourth residue at the N terminal) Base) is phosphorylated.

In another embodiment, the peptide is an HLA restricted peptide. In a preferred embodiment, the peptide is an HLA-A restricted peptide. In a more preferred embodiment, the peptide is an HLA-A2 restricted peptide.

The peptide is designed based on the amino acid sequence of the T cell epitope derived from tumor necrosis factor receptor related protein 1 (TRAP1). Therefore, the peptide can induce immune cells to produce an immune response against cells expressing TRAP1.

In a specific embodiment, the lipid has the function of triggering the toll-like receptor pathway. In a preferred embodiment, the toll-like receptor is toll-like receptor 2 (TLR2).

In another specific embodiment, the lipid comprises a fatty acid. In a preferred embodiment, the fatty acid includes palmitic acid. In a more preferred embodiment, the palmitic acid is Cys((RS)-2,3-(palmitoyloxy)-propyl)-OH(Cys((RS)-2,3-di(palmitoyloxy )-propyl)-OH); or N-α-palmitoyl-S-[2,3-bis(palmitoyloxy)-2RS]-propyl]-L-cysteine (N-α -Palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-L-cysteine).

In a specific embodiment, the helper T cell epitope has the function of inducing the immune response of the helper T cell. In a preferred embodiment, the sequence of the helper T cell epitope includes an amino acid sequence of SEQ ID NO:5. In another specific embodiment, the helper T cell epitope sequence is an HLA-DR-restricted peptide. Therefore, the helper T cell epitope can combine with the HLA-DR molecule on the antigen presenting cell to form an HLA-DR/helper T cell epitope complex, allowing the helper T cell to recognize and activate the helper Sex T cells.

In another embodiment, the composition is a pharmaceutical composition. In a preferred embodiment, the pharmaceutical composition includes a pharmaceutically acceptable carrier.

In a specific embodiment, the composition is an immunogenic composition or a vaccine. In a preferred embodiment, the composition is a vaccine. Since the lipid and the helper T cell epitope can be used as the internal adjuvant of the lipidated peptide, when the composition is prepared into a vaccine, no additional adjuvant is needed, and the vaccine can cause the individual's immune response. Therefore, the lipid and the helper T cell epitope have the effect of improving the immunogenicity of the vaccine.

After the vaccine containing the lipidated peptide of the present invention is injected into an individual, the vaccine can induce an immune response in the individual to inhibit cancer cells expressing TRAP1. In a specific embodiment, the vaccine containing the lipidated peptide has the ability to induce an immune response. In a preferred embodiment, the immune response includes T cell immune response. In a more preferred embodiment, the T cells include helper T cells and cytotoxic T lymphocytes. In another specific embodiment, the vaccine is used to treat cancer. In a preferred embodiment, the vaccine is used to treat cancers caused by cancer cells expressing TRAP1. In a more preferred embodiment, the vaccine inhibits the growth of cancer cells expressing TRAP1.

The present invention further provides a composition for the preparation of a medicament for the treatment of cancer in a body, wherein the composition comprises an effective dose of lipidated peptide, which comprises a lipid, a helper T cell epitope and a A peptide, wherein the lipid, the helper T cell epitope, and the peptide are not connected to each other, but all three are connected to an amino acid, wherein the peptide contains Lys-XX-Ser- An amino acid sequence composed of Val-Glu-Thr-Asp-X (SEQ ID NO: 9), where X is leucine (Leu) or isoleucine (Ile), and the serine (Ser) Is phosphorylated.

In a specific embodiment, the peptide is a phosphorylated peptide. In a preferred embodiment, the amino acid sequence of the peptide is SEQ ID NO: 6, wherein the amino acid (Ser) at the fourth position in the sequence of SEQ ID NO: 6 (the fourth residue at the N terminal) Base) is phosphorylated.

In a specific embodiment, the lipid has the function of triggering the toll-like receptor pathway. In a preferred embodiment, the toll-like receptor is toll-like receptor 2 (TLR2).

In another specific embodiment, the lipid comprises a fatty acid. In a preferred embodiment, the fatty acid comprises palmitic acid. In a more preferred embodiment, the palmitic acid is Cys((RS)-2,3-(palmitoyloxy)-propyl)-OH(Cys((RS)-2,3-di(palmitoyloxy )-propyl)-OH); or N-α-palmitoyl-S-[2,3-bis(palmitoyloxy)-2RS]-propyl]-L-cysteine (N-α -Palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-L-cysteine).

In a specific embodiment, the helper T cell epitope has the function of inducing the immune response of the helper T cell. In a preferred embodiment, the sequence of the helper T cell epitope includes an amino acid sequence of SEQ ID NO:5. In another specific embodiment, the helper T cell epitope is an HLA-DR-restricted peptide. Therefore, the helper T cell epitope can combine with the HLA-DR molecule on the antigen presenting cell to form an HLA-DR/helper T cell epitope complex, allowing the helper T cell to recognize and activate the helper Sex T cells.

In another embodiment, the peptide is an HLA restricted peptide. In a preferred embodiment, the peptide is an HLA-A restricted peptide. In a more preferred embodiment, the peptide is an HLA-A2 restricted peptide. Therefore, the individual is an individual with HLA-A2 in one body, so that the drug containing the lipidated peptide can be administered into the individual's body and can bind with HLA-A2 molecules to induce an immune response. In another specific embodiment, the individual is an individual with an HLA molecule of HLA-A. In a preferred embodiment, the individual is an individual with an HLA molecule HLA-A2

The peptide is designed based on the amino acid sequence of the T cell epitope derived from tumor necrosis factor receptor related protein 1 (TRAP1). In a specific embodiment, the peptide is a T cell epitope peptide derived from TRAP1. In a preferred embodiment, the peptide is a T cell phosphorylated epitope peptide derived from TRAP1. Therefore, the peptide can induce immune cells to produce an immune response against cells expressing TRAP1. In a specific embodiment, the immune cells include type 1 helper T cells (Th1), cytotoxic T lymphocytes (CTL), antigen presenting cells (APC) capable of inducing Th1 cells, and those capable of inducing CTL APC. In a preferred embodiment, the immune cells include type 1 helper T cells (Th1) and cytotoxic T lymphocytes (CTL). In a more preferred embodiment, the cytotoxic T lymphocytes are CD8 ⁺ T cells. In another specific embodiment, the peptide is a cytotoxic T cell epitope peptide. In a preferred embodiment, the peptide is a cytotoxic T cell phosphorylated epitope peptide.

In another embodiment, the cancer is a cancer caused by cancer cells expressing TRAP1. In a specific embodiment, the cancer includes lung cancer, head and neck cancer, breast cancer, pancreatic cancer, lymphoma, myeloma, leukemia, kidney cancer, ovarian cancer, bone cancer, liver cancer, prostate cancer, skin cancer, colorectal cancer, and thyroid cancer. cancer. In a preferred embodiment, the cancer is lung cancer, breast cancer, ovarian cancer, colorectal cancer or prostate cancer. In a more preferred embodiment, the cancer is lung cancer.

In a specific embodiment, the effective dose of the lipidated peptide ranges from 0.1 mg/kg body weight to 10 mg/kg body weight. In a preferred embodiment, the effective dose of the lipidated peptide ranges from 0.5 mg/kg body weight to 4 mg/kg body weight. In a more preferred embodiment, the effective dose of the lipidated peptide ranges from 1.5 mg/kg body weight to 2 mg/kg body weight.

Because the lipid and the helper T cell epitope can be used as the internal adjuvant of the lipidated peptide, it can improve immunogenicity or promote immune response. In a specific embodiment, the lipid and the helper T cell epitope have the function of improving immunogenicity or promoting immune response. Therefore, after the drug containing the lipidated peptide enters the body of the individual, it will combine with HLA molecules on an antigen-presenting cell to form an HLA/lipidated peptide complex to activate T cells that can secrete IFN-γ (such as Th1 and CTL) to inhibit the growth of tumor cells expressing TRAP1, thereby treating cancer. In a specific embodiment, the lipidated peptide has the ability to induce the immune response of immune cells to treat cancer. In a preferred embodiment, the lipidated peptide has the ability to induce the immune response of T cells to treat cancer. In a preferred embodiment, the lipidated peptide has the ability to induce the immune response of IFN-γ-secreting T cells to treat cancer.

In another specific embodiment, the T cell or the IFN-γ-secreting T cell includes type 1 helper T cells (Th1) and cytotoxic T lymphocytes (CTL). In a preferred embodiment, the cytotoxic T lymphocytes are CD8 ⁺ T cells.

Therefore, the dosage form of the drug containing the lipidated peptide can be a vaccine, and the vaccine does not need to add an adjuvant. The vaccine can induce T cell response to cells expressing TRAP1 to treat diseases or cancers caused by cells expressing TRAP1.

The present invention proves that the tumor-reactive T cell epitope of TRAP1 can be used for cancer treatment. In the present invention, the new phosphorylation site on the HLA-A2-restricted phosphorylation peptide KLIpS (ie SEQ ID NO: 6) derived from TRAP provides a unique feature for peptide-specific T cell induction. First, phosphorylation enhances the binding affinity of KLIpS to HLA-A2 molecules (a stable HLA-peptide complex is necessary for T cell receptor (TCR) recognition). Second, phosphoric acid binds to the fourth amino acid from the N-terminus on the phosphorylated peptide sequence, which is the main residue recognized by TCR. It is known that TCR can distinguish changes in the HLA/peptide complex and then trigger a cytokine response to target cells. As a good HLA-A2 conjugate with site-specific phosphorylation ligand, the immune effect of KLIpS has been shown to trigger an antigen-specific T cell response. In addition, KLIpS-induced T cells can be stimulated by phosphopeptides to secrete IFN-γ, so as to induce sufficient IFN-γ-secreting T cells to effectively eliminate tumors. Among them, IFN-γ can up-regulate MHC expression on target cells, and Mobilization of cytotoxic lymphocytes to tumor-associated antigens.

In addition, the present invention also proves that lipids, helper T cell epitopes and lipidated peptides constructed with the above phosphorylated peptides (KLIpS) can also trigger antigen-specific T cell responses. Since the lipid and the helper T cell epitope can be used as the internal adjuvant of the lipidated peptide, it can improve immunogenicity or promote immune response. The lipidated peptide can stimulate T cells that secrete IFN-γ to inhibit the growth of tumors expressing TRAP1.

In view of the above, the present invention identifies HLA-A2-restricted phosphorylated peptides (KLIpS) derived from tumor-associated TRAP1 protein. KLIpS seems to be an immunogenic CTL epitope, which can trigger antigen-specific CTLs against cancer cells and inhibit tumor growth. This highlights KLIpS as a vaccine candidate for developing cancer therapy with TRAP1. Until today, accumulated studies have shown that TRAP1 is highly expressed in cancer cell lines; the present invention proves that KLIpS is an ideal target for triggering CTL activation to control TRAP1 high-performance tumors. The principle of peptide-based cancer immunotherapy is based on the matching between antigen-specific CTLs and targets on human cancer cell lines. Therefore, the anti-tumor effect of KLIpS can be used for different cancer cell lines.

Figure 1 shows that TRAP1 protein is highly expressed in lung cancer cell lines and patients with malignant lung tumor tissues. Figure 1(A) shows the abundance of TRAP1 protein in TC1/AAD (containing the α1+α2 domain of HLA-A2.1 and the α3 domain of H-2Dd), TC1 and H2981 tumor tissue and normal mouse lung tissue (abundance) of Western ink dot analysis. β-actin (β-actin) served as the loading internal control in this analysis. Figure 1(B) shows the results of immunohistochemical staining of the expression level of TRAP1 protein in the patient's malignant lung tumor tissue and adjacent normal tissues.

Figure 2 shows a comparative tandem MS mass spectrometry analysis of immunoprecipitated KLIpS and a reference synthetic peptide. The significant ions (b and y, m/z) of the peptide fragment are marked on the peak of the matched m/z ion.

Figure 3 shows a comparative tandem MS analysis of immunoprecipitated RQLpS and a reference synthetic peptide. The significant ions (b and y, m/z) of the peptide fragment are marked on the peak of the matched m/z ion.

Figure 4 shows T2 cell stability analysis for selected peptide candidates. Figure 4(A) shows that after 10 μM synthetic peptide solution is mixed with T2 cells, the number of stable HLA-A *02:01 complexes is then quantified to analyze the affinity bonding between HLA molecules and each peptide. Experimental group: KLIpS (phosphorylated peptide, black solid line) and KLIS (phosphate-free peptide, black dashed line); and control group: VYC (HLA-A11 peptide, light gray thick line) and YML (HLA) -A2 restricted peptide, light gray thin line). Cells stained with isotype antibody are marked with dark gray solid lines. Figure 4(B) shows a bar graph comparing the binding affinity of HLA-A*02:01 and various peptides. The relative average fluorescence intensity (MFI) is calculated as: (MFI of peptide/MFI of VYC)×100% (KLIS vs. KLIpS, [p]<0.01 **). The data shown is representative of three experiments.

Figure 5 shows that the immune effect of KLIpS causes the secretion of IFN-γ antigen-specific CD8 ⁺ T cell population. Figure 5(A) shows that AAD transgenic mice and wild-type (WT) mice were immunized with KLIpS, and then the secretion of IFN-γ was detected by ELISPOT analysis. KLIpS immunized AAD transgenic mice induced enough IFN-γ-secreting cells, but wild-type mice did not (p<0.01 **). Figure 5(B) shows the immune induction effect of peptides on T cells in transgenic mice detected by ELISPOT analysis. The peptide candidates were used to immunize AAD transgenic mice (the number is 5), and then the lymph nodes in the groin were collected for detection of IFN-γ-secreting cell populations. Under antigen restimulation, sufficient IFN-γ-secreting cells were detected in the lymph nodes of mice immunized with KLIpS and YML (positive control group), but almost in mice immunized with KLIS and VYC (negative control group) can not see. (KLIS vs. KLIpS, p<0.01 **). Figure 5(C) shows cross-antigen restimulation to determine the specificity of peptide-induced IFN-γ secreting cells in AAD transgenic mice. The cells secreting IFN-γ were detected by ELISPOT analysis (in the KLIpS re-stimulation group: KLIS is immune to KLIpS; for KLIpS immunity: KLIS is re-stimulated to KLIpS; p<0.01 **). Figure 5(D) shows the detection of effector T cell population in the spleen cells of peptide-immunized mice. After the antigen was administered, flow cytometry showed that there were significantly stimulated IFN-γ-secreting CD8 ⁺ T cell populations in the lymphocytes of KLIpS-immunized AAD transgenic mice. Figure 5(E) shows the percentage of IFN-γ secreting cells in CD8 ⁺ T cells (KLIS vs. KLIpS, p<0.01 **). The data shown are representative of three experiments.

Figure 6 shows that KLIpS immunization can induce the antigen-specific cytotoxic activity of CTL. Figure 6(A) shows that AAD transgenic mice were immunized with 50 μl of KLIS or KLIpS formulated with IFA on day 0 and day 7; PBS was used as a control group. On the 14th day, the cytotoxic activity of CTL against peptide-pulsed specific target cells was detected by in vivo killing test (KLIS vs. KLIpS, p<0.01 **). Figure 6(B) shows AAD transgenic mice immunized with 50 μg KLIpS/IFA on day 0 and day 7. Purify CD8 ⁺ T cells from the spleen and draining lymph nodes with the MACS isolation kit. 5×10 ⁵ purified CD8 ⁺ T cells were co-cultured with 5×10 ^{3 51} Cr-labeled H2981 for 18 hours. For inhibition experiments, first incubate ⁵¹ Cr-labeled H2981 cells with 20μg/ml W6/32 antibody or incubate purified CD8 ⁺ T cells with 10μg/ml 53-6.7 antibody for 15 minutes, and then co-culture. The specific lysis in each group was determined by ⁵¹ Cr-release assay (w/o antibody anti-HLA antibody, p<0.05*; w/o antibody anti-CD8 antibody, p<0.01 **).

Figure 7 shows that the immune effect of KLIpS can delay tumor growth. On the 7th and 14th days, 50μg of KLIS, KLIpS or YML (control group) formulated with IFA was used to immunize TC1/AAD tumor-bearing mice. Figure 7(A) shows the average tumor size (mm ³ ). Data are expressed as mean ± standard error (SEM) (KLIS vs. KLIpS, p<0.01 **). Figure 7(B) shows the survival rate. Figure 7(C) shows that the CTL response induced by KLIpS immunity can inhibit the growth of human lung cancer tumors in a xenograft model.

Figure 8 shows the monitoring of T cell induction under the immune effect of palmitate peptides in transgenic mice by ELISPOT analysis.

Figure 9 shows that the immune effect of palmitate peptides can trigger an anti-tumor response to inhibit tumor growth.

The present invention can be implemented in different forms and is not limited to the embodiments mentioned below. The following embodiments only show the various aspects and features of the present invention.

(A) Materials and methods

1. Animals and cell lines

The present invention uses C57BL/6 wild-type mice from the National Animal Center. The transgenic mouse line was purchased from The Jackson Laboratory (Sacramento, CA). All animal studies were approved by the Laboratory Animal Care and Use Committee of the National Health Research Institutes (NHRI). Mouse lung cancer epithelial cells, TC1/AAD, are derived from AAD-expressing parental cell TC1, which is transgenic from AAD (containing the α1+α2 domain of HLA-A2.1 and the α3 domain of H-2Dd) Gene obtained from mice. In brief, a pair of primers 5'-CCCAAGCTTATGGCCGTCATGGCGCCCCGA-3' (SEQ ID NO: 1) and 5'-GCTCTAGATCACACTTTACAATCTGGGAG-3' (SEQ ID NO: 2) were used to amplify the spleens of HLA-AAD transgenic mice Cells (splenocytes) can encode DNA fragments of AAD chimeric protein. The amplified DNA fragment was further cloned into pcDNA4/TO/myc-His plastid (Thermo Fisher Scientific Inc., USA) to generate pcDNA4/TO/myc-His/AAD. PcDNA4/TO/myc-His/AAD was transfected into TC1 cells to produce a stable TC1/AAD cell line. The H2981 (HLA-A*02:01) human lung cancer cell line was cultured in complete RPMI medium (RPMI 1640, GIBCO), which contains 10% heat-inactivated fetal bovine serum (HyClone), 100 units/mL penicillin and 100μg/mL streptomycin (GIBCO), 1mM sodium pyruvate and 5mM HEPES, and the medium is placed at 37℃ and 5% CO ₂ Incubate in the box.

2. Analysis of western ink dot method

Wash the collected cells with cold phosphate buffered saline (PBS) buffer, and then use RIPA buffer containing protease inhibitor tablets (cOmplete, Roche) (containing 0.1% RIPA in 100mM PBS buffer, pH 7.8) Lyse cells. The lung tissues of AAD transgenic mice were used as a control group in western blot analysis. The lung tissue was crushed with ceramic beads (100 μm, EE-TEC Ltd., Taiwan) in an ice bath, and then the cells were lysed with 10 mL of RIPA buffer at 4°C. Subsequently, BCA reagent (BCA Protein Assay Kit, Pierce) was used to quantify the protein concentration of cell lysates and control tissues. BLUeye prestained protein ladder (BLUeye prestained protein ladder, GeneDireX, Canada) standard reagents were used for sodium dodecyl sulfonate-polyacrylamide gel electrophoresis (SDS-PAGE). A total of 50μg of protein was separated by 4-12% SDS PAGE (NuPAGE, ThermoFisher Scientific), and then transferred to PVDF membrane. In order to reduce the occurrence of non-specific binding, PBST (1×PBS buffer containing 0.05% Tween 20) buffer was used as the washing buffer in each washing step. The ink dot film was blocked with PBST containing 5% skim milk and reacted overnight at 4°C. Subsequently, the ink dot film was cut and combined with diluted anti-TRAP1 antibody (1:1000 in PBST, rabbit polyclonal antibody, ab8227, Abcam) and anti-actin antibody (1:10000 in PBST, Rabbit polyclonal antibody, ab151239, Abcam) were incubated together. Then use horseradish peroxidase (HRP)-conjugated detection antibody (1:10000 in PBST, goat polyclonal antibody, ab6721, Abcam) in enhanced chemiluminescence solution (Millipore) to test the ink dot membrane Perform dyeing.

3. Immunohistochemical staining

The lung non-small cell carcinoma tissue array was purchased from a US company (US Biomax, LC10012). As described in the previous literature (Lin CY et al., 2014, cancer research 74: 5229-5243), immunohistochemical staining was used to evaluate the tumor necrosis factor receptor-related protein 1 (TRAP1) in clinical samples from lung cancer patients. Degree of performance. Paired tumor tissues and adjacent tissues are used to compare the staining intensity; and because one core is missing from the tissue array, 44 paired samples from a total of 45 cases will be included in this analysis. Tissue sections will be combined with rabbit anti-human TRAP1 protein (LS-C31383; LifeSpan BioSciences) and HRP conjugated avidin-biotin complex (ABC) (from Vectastain Elite ABC reagent (Vector Laboratories) and AEC chromogen Chromogen (Vector Laboratories) interacts with each other. The intensity of TRAP1 staining in all samples was scored by a pathologist.

4. Separation of HLA-AAD peptides

According to the method described in the previous literature (Meyer VS et al., 2009, journal of proteome research 8: 3666-3674), the HLA-AAD peptide was obtained by immunoprecipitation with slight modification. First use two spinner flasks (1×10 ⁹ cells/liter) to collect TC1/AAD cells, then use CHAPS solution (containing 1% CHAPS in ice PBS buffer, pH 7.8) to lyse the cells, and then Centrifuge at 2000 rpm for 10 minutes to remove cell debris. The HLA-AAD-peptide complex was immunoprecipitated by anti-HLA-A2 antibody (BB7.2)-immobilized column, and an ultracentrifuge column (10kDa retention membrane, Millipore, Billerica, MA) and a reverse phase spin column (C18, Invitrogen) were used to extract related peptides in sequence. The desalted analyte is lyophilized and then resuspended in acetonitrile (ACN) solution (a deionized water solution containing 5% ACN) for liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis .

5. Identify HLA peptides by LC-MS/MS

A system using a Q Exactive mass spectrometer (Thermo Scientific, USA) with an Ultimate 3000 RSLC system (Dionex) was used for sequencing HLA peptides. A reverse phase column (C18, Acclaim pepmap RSLC, 75 μm×150 mm, pore size 2 μm, Dionex) was used to separate the composite peptide mixture. The mobile phase was prepared as follows: A: 0.1% formic acid (FA) in 5% acetonitrile (ACN) (V/V); and B: 0.1% FA in 95% ACN (V/V). The present invention designs a linear gradient, which starts from 1% B to 25% B within 40 minutes, and then increases to 80% B within 18 minutes to elute all analytes in the column. Mass spectrometry scans were performed in the range of m/z 300-2000, and the 10 strongest ions from the mass spectrometry scans were fragmented to obtain MS/MS (tandem MS) mass spectra. Use the software (Proteome Discoverer 1.3) to convert the MS raw data into a peak list. Use the MASCOT search engine with a self-built database (mouse species, UniProt) for sequence analysis. The parameters used include: coenzyme, oxidation (M) and phosphorylation (ST, Y) variable modification ( variable modification of oxidization), intensity ratio cutoff 10%, tolerance ±0.002Da, mass tolerance ±10ppm. The MS mass spectrum of the identified sequence will be further checked and confirmed manually.

6. MHC binding prediction

Use the SYFPEITHI algorithm and the IEDB analysis resource NetMHC (IEDB analysis resource NetMHC) (4.0 version) tool (31-33) to carry out the combined prediction of MHC class I (MHC class I). The parameters used include the following: HLA-A*02:01 Human alleles of H2-Db, mouse alleles of H2-Db, and peptides of 8-12 amino acids in length.

7. Stability analysis of T2 cells

As described in previous documents (Schweitizer S et al., 2000, Cytometry 41:271-278), the binding affinity between peptides and HLA-A2 molecules can be assessed by T2 cell stability analysis. To put it simply, HLA-A*02:01-positive, TAP-deficient T2 cells (ATCC) and 10μM candidate peptides are incubated in DMEM medium containing 0.1% FBS, 5×10 ^-5 M β-sulfhydryl Ethanol (β-mercaptoethanol), 5×10 ^-7 M β2-microglobulin (β2-microglobulin), and incubate overnight in a 25°C, 5% CO ₂ incubator. The synthetic peptide is obtained from the core facility of the National Institutes of Health (purity>95%). The peptides and T2 cells were incubated at 37°C for 3 hours, and then stained with PE-conjugated anti-HLA-A*02:01 antibody (BB7.2, BD Bioscience, USA). The amount of stable peptide-HLA-A*02:01 and β2-microglobulin complex (pHLA-A2) was monitored by flow cytometry (FACSCalibur, BD Bioscience, USA). The affinity is determined based on the average fluorescence intensity (Mean Fluorescence Intensity, MFI) of peptide-pulsed T2 cells. In this analysis, VYCKQQLLR (SEQ ID NO: 3, VYC) (HLA-A11 epitope) and YMLDLQPETT are used respectively (SEQ ID NO: 4, YML) (HLA-A2 epitope) synthetic peptides were used as negative and positive controls.

8. IFN-γ secretion analysis

The present invention uses the method taught in the previous literature (Tu SH et al., 2012, Journal of immunotherapy 35: 235-244) with some modifications, by using enzyme-linked immunospot assay (ELISPOT) to analyze peptide-specific T cell responses . In order to enhance peptide immunity (immunogenicity), the same amount of CD4 helper T cell (CD4 helper T cell, Th) epitope (AKFVAAWTLKAAA (SEQ ID NO: 5), PADRE) and incomplete Freund’s adjuvant ( The target peptide in incomplete Freund's adjuvant (IFA) solution (containing 50% incomplete Freund's adjuvant in PBS buffer) was mixed, and the final peptide concentration was 1 mg/mL. The synthetic peptides of VYCKQQLLR (SEQ ID NO: 3) (VYC) and YMLDLQPETT (SEQ ID NO: 4) (YML) were used as the negative and positive control groups in the analysis, respectively. Then 50 μL of the prepared immunogen was injected into the mouse paw on day 0 and day 7. A week after the last immunization, a total of 5×10 ⁵ lymph node cells were collected, and the cells and peptides (final concentration: 5μg/ml) were incubated in complete RPMI medium at 37°C and 5% CO ₂ for 48 hours . After incubation, the cells were washed with PBST buffer, and then 50 μL of biotin-conjugated anti-IFN-γ antibody (R46A2, eBioscience, CA) solution and streptomycin-conjugated HRP (eBioscience) were sequentially added to the well. Use 3-amine-9-ethyl carbazole solution (ACE, Sigma) to develop ink dots, and then use ELISOT reader (Cellular Technology Ltd., Shaker Heights, OH) to calculate The number of ink dots.

9. Intracellular staining

Spleen cells (5×10 ⁶ ) were collected from the peptide-immunized AAD transgenic mice, and incubated with the corresponding immunogen (final concentration: 10 μg/ml) at 37° C. and 5% CO ₂ overnight. After incubation, the cells were washed once with ice PBS, and stained with FITC-conjugated monoclonal anti-mouse CD8 antibody (53-6.7, BD). FITC-labeled cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% saponin/PBS for 5 minutes, and then the supernatant was removed. PECy7-conjugated anti-mouse IFN-γ (R3-34, BD) was used to detect intracellular cytokine staining, and then the number of stained cells was determined by flow cytometry (BD Bioscience). Use FCS Express software (De Novo Software) for data analysis.

10. Bloodthirsty analysis in vivo

Spleen cells (5×10 ⁷ ) were collected from AAD transgenic mice, and then combined with KLIpSVETDI (where p represents the position of S (serine, Serine) is phosphorylated) (SEQ ID NO: 6) (KLIpS, pS551 -TRAP1548-556) (5μg/ml) or PBS (untreated control group) incubated together at 37°C for 30 minutes. After incubation, the peptide-pulsed spleen cells and the untreated control group were labeled with CFSE (Molecular Probes, Eugene, OR) at a concentration of 10 μM and 1 μM at 37° C. for 15 minutes. Then add ice-cold complete RPMI medium to stop the CFSE labeling reaction. Mix non-peptide-pulsed cells and peptide-pulsed spleen cells at a ratio of 1:1, and inject a total of 2×10 ⁷ CFSE-labeled cells through the tail vein into AAD-transgenic mice immunized with peptides Inside. 18 hours after adoptive transfer, spleen cells were collected and analyzed by flow cytometry (BD Bioscience). Use FCS Express software (De Novo Software) for data analysis.

11. Tumor attack analysis

(1) Inoculate 2×10 ⁵ TC1/AAD cells into AAD transgenic mice 6-8 weeks old to establish tumor development. One week after tumor injection, 50μg peptide prepared with immuno-enhanced solution IFA containing PADRE will be injected into the tumor-bearing mice through the mouse paw on the 7th and 14th days. Twice to induce an immune response. In addition, PBS buffer was used to induce the immune response of tumor-bearing mice (the number is 5) and served as an untreated control group in the analysis. The tumor size was monitored by palpation with calipers twice a week; the tumor volume was calculated by the formula: length×width×width/2. When the tumor size exceeds 2000 mm ³ , the mice bearing the tumor are sacrificed.

(2) On day 0 and day 7, 50 μg of KLIpS and PADRE prepared with IFA were used to make AAD transgenic mice produce immune responses. SCID mice were subcutaneously inoculated with 1×10 ⁷ H2981 human lung cancer cell lines on the 7th day. On the 14th day, spleen cells were collected from the bodies of AAD mice injected with PBS or immunized AAD mice, and CD8 ⁺ T cells were sorted by a cell sorting device (Magnetic-activated cell sorting, MACS). CD8 ⁺ T cells were adoptively transferred to SCID mice with H2981 tumor by intravenous injection. The size of the tumor was measured by a vernier and monitored 3 times a week until the tumor size grew to 2000mm ³ .

12. ⁵¹ Cr release analysis

CD8 ⁺ T cell isolation kit (Miltenyl Biotech) was used to isolate CD8 ⁺ T cells from the spleen of mice immunized with KLIpS. When targeting cells, H2981 cells (5×10 ⁵ /ml) were labeled with 100 μCi of ⁵¹ Cr (Na251CrO4, PerkinElmer, MA) at 37°C for 1 hour to serve as target cells. Then CD8 ⁺ T cells (5×10 ⁵ ) and ⁵¹ Cr-labeled target cells (5×10 ³ ) were mixed at a ratio of 100:1 (effector: target, E: T), and incubated at 37°C 18 hours. After the incubation, the supernatant was harvested and the radioactivity was measured using a gamma counter. The following formula is used to calculate the percentage of specific lysis: 100×[(experimental release-spontaneous release)/(maximum release-spontaneous release)].

13. Preparation of palmitated peptides

In order to synthesize a branched palmitoylated peptide (palmitoylated peptide), which contains pan HLA-DR binding epitope (pan HLA-DR binding epitope, PADRE) and CTL epitope (SEQ ID NO: 6, KLIpS), and PADRE and KLIpS are connected by a lysine (K) residue, and the side chain of the lysine residue is joined to Pam2 or Pam3. The pan HLA-DR binding epitope is also a pan-Th epitope peptide (SEQ ID NO: 5). The chemical formula of Pam2 is Cys((RS)-2,3-(palmitoyloxy)-propyl)-OH(Cys((RS)-2,3-di(palmitoyloxy)-propyl)-OH); and The chemical formula of Pam3 is N-α-palmitoyl-S-[2,3-bis(palmitoyloxy)-2RS]-propyl]-L-cysteine (N-α-Palmitoyl-S- [2,3-bis(palmitoyloxy)-(2RS)-propyl]-L-cysteine). Therefore, the structure of the palmitated peptide is as follows:

In addition, the positions of PADRE, KLIpS and Pam2 (or Pam3) in the palmitate esterified peptide can all be interchanged, and Pam2 (or Pam3) can also be interchanged with other fatty acids. The above behavior of swapping positions does not affect the effect of palmitated peptides (lipidated peptides).

Use standard F-moc chemistry to synthesize the peptide that connects PADRE to the CTL epitope; after this step is performed, it is cleaved to produce a non-lipidated peptide; finally, connect Pam2 or Pam3 to the inserted lysine Acid residues on the amine group to construct the palmitated peptide. Therefore, the present invention uses dipalmitate peptide (Pam2KLIpS) and tripalmitate peptide (Pam3KLIpS) for subsequent experiments.

14. Induction assay of antigen-specific cells

On day 0 and day 7, 50μg Pam2KLIpS, 50μg Pam3KLIpS and a mixture (KLIpS/IFA) containing IFA, PADRE and 50μg KLIpS were used to induce the immune response of AAD transgenic mice. On day 14, lymphocytes were collected and stimulated with 5 μg/ml KLIpS for another 48 hours. Antigen-specific cells are cells that secrete IFN-γ were stimulated by KLIpS and further detected by ELISPOT.

15. Tumor suppression analysis

In order to evaluate the tumor growth inhibitory effect of Pam2KLIpS or Pam3KLIpS, 50μg of Pam2KLIpS and Pam3KLIpS were used to induce immune response in AAD mice 14 and 7 days before tumor inoculation. On day 0, immunized mice were inoculated with 1×10 ⁵ TC1-AAD tumor cells. Measure the tumor size with a vernier, and monitor 3 times a week until the tumor size reaches 2000mm ³ .

16. Statistical analysis

The statistical significance was evaluated by Student's t-test (two-tailed) and a one-way analysis of variance (ANOVA) with a significance level of 5%.

B. Results

1. The degree of TRAP1 expression in lung cancer cells and tumors

In order to evaluate the expression level of TRAP1 in mouse and human lung cancer cell lines, the present invention uses the Western blot method to quantify the target protein and compares its abundance with the TRAP1 abundance in normal mouse lung cells. As shown in Figure 1(A), TRAP has a significant high degree of expression in the tested lung cancer cells, but it is almost invisible in the lung tissues of normal mice. This result is consistent with previous studies (Agorreta J et al. , 2014, Mol Cancer Res 12: 660-669) consistent. Subsequently, the anti-TRAP1 antibody was stained with immunohistochemistry (IHC) to analyze the abundance of TRAP1 in human lung tumor tissues and adjacent normal corresponding tissues. Among 44 lung tumor tissues, high-level TRAP1 staining was detected in 17 samples (17/44, 38.6%). In contrast, TRAP1 was almost undetectable in adjacent normal human tissues, and only 7 samples showed positive staining (7/44, 15.9%), as shown in Table 1 and Figure 1(B). The intensity of TRAP1 staining in all samples was scored by a pathologist and summarized in Table 1. Score <=1 means low staining; score>1 means high staining.

2. Identification of HLA-A2 binding peptides

In order to identify the HLA-A2 restricted epitope of TRAP1, liquid chromatography mass spectrometry (LCMS) was used to analyze immunoprecipitated peptides of TC1/AAD cells. According to the MASCOT search, a total of 11 significant HLA-A2-restricted peptides were identified in 1x10 ⁹ TC1/AAD cells, and they were further confirmed by manual inspection of their corresponding tandem MS mass spectra the sequence of. By entering the sequence into the IEDB (www.IEDB.org) database funded by the National Institute of Allergy and Infectious Diseases America (National Institute of Allergy and Infectious Diseases America) and SYFPEITHI (www.syfpeithi.de) developed by the Rammensee team. ) In the database, all peptides identified by MS conform to the binding motif of the HLA-A*02:01 conjugate, but do not conform to the binding motif of the mouse molecule H2-Db. KLIpSVETDI (SEQ ID NO: 6) (KLIpS, pS551-TRAP1548-556, where p represents the position of S (serine, Serine) is phosphorylated) and RQLpSSGVSEI (SEQ ID NO: 7) (RQLpS, pS82-HSP2779 -88, where p represents the position of S (serine, Serine) is phosphorylated) are the phosphorylated peptides of cancer-related proteins derived from TRAP1_mouse (Q9CQN1, UniProt) and HSP27_mouse (P14602, UniProt), respectively. In addition to manual inspection, the sequence of these phosphorylated peptides was further confirmed by comparison with reference synthetic peptide tandem MS mass spectrometry, as shown in Figures 2 and 3. Therefore, HLA-phosphorylated peptides are considered to induce immunogenic epitopes of antigen-specific T cells. Therefore, TRAP1-derived phosphorylated peptides can cause immune responses, which will have high appeal in therapeutic applications.

Previous study (Proc Natl Acad Sci US A. 2006 Oct 3; 103(40): 14889-94. Identification of class I MHC-associated phosphopeptides as targets for cancer immunotherapy. Zarling AL, Polefrone JM, Evans AM, Mikesh LM, Shabanowitz J, Lewis ST, Engelhard VH, Hunt DF) pointed out that Hsp27-derived phosphorylated peptides were identified as known HLA-in human ovarian carcinoma (cov413) and melanoma cells (DM331). A2-restricted CTL epitope (Figure 3). It is worth noting that RQLpS was first observed in lung cancer cell lines, which further supports the sequencing results of MS in this previous study.

In addition, isoleucine (Isoleucine, IlE, I) and leucine (Leucine, Leu, L) are structural isomers. Therefore, IL or Leu in SEQ ID NO: 6 can be interchanged. The present invention uses the SYFPEITHI online simulation software (http://www.syfpeithi.de/index.html) developed by the Rammensee team to test the binding effect of phosphorylated peptide peptides, and SYFPEITHI is the MHC ligand (ligand) and peptide sequence database. As shown in Table 2, the simulation results confirm that the exchange of amino acid residues in SEQ ID NO: 6 does not affect the binding affinity between the phosphorylated peptide and the HLA molecule.

3. HLA-peptide stability analysis

A stable HLA-peptide complex is necessary for T cell identification. In the present invention, T2 cells are used for stability testing. The T2 cell line is a cell with a TAP defect but an empty HLA-A2 molecule on the cell membrane, which can be stabilized by placing an allele-restricted peptide. The present invention quantifies stable HLA-A2-peptide complexes using pan-HLA antibodies related to the non-binding negative control group. As shown in Figure 4(A) and 4(B), the KLIpS peptide and the positive control YMLDLQPET peptide (YML) (a known HLA-A*02:01 conjugate) can be used in T2 cells with TAP deficiency It effectively forms a stable complex with HLA-A2 molecules. In contrast, when the non-phosphorylated peptide (KLIS, SEQ ID NO: 8) or the VYC peptide (HLA-A*11) of the negative control group was used for implantation in the test, only a few or less were detected. The stable HLA-peptide complex is shown in Figures 4(A) and 4(B). In contrast, binding to phosphate is essential for enhancing the binding affinity of the peptide to HLA-A*02:01 molecules.

4. Antigen-specific CTL immune response induced by TRAP1-derived HLA-A2 restricted phosphorylation peptide

Based on the test results of T2 cells, the present invention evaluates the ability of KLIpS to induce T cells. This experiment started with C57BL/6 wild-type (WT) and AAD transgenic mice immunized with peptides. After peptide re-stimulation, only high amounts of IFN-γ secreting cells were detected in the lymph nodes of peptide-immunized AAD transgenic mice, but not in the wild-type control group; this result reflects cytokines The reaction is mediated through AAD molecules, as shown in Figure 5(A). Next, the AAD transgenic mice were immunized with peptides KLIpS, KLIS, YML, and VYC to compare the efficacy of inducing cytotoxic T cells. During the peptide restimulation, ELISPOT analysis showed that the lymph nodes of the AAD transgenic mice immunized with the peptide KLIpS or YML (positive control group) had a greater number of IFN-γ-secreting cells (KLIpS: ink dots: 230 ±45; YML: Ink dot: 161±81) (Figure 5(B)). In addition, further analysis of cross-restimulation was performed, and the results are shown in Figure 5(C). As shown in Figure 5(C), the strong cytokine response is limited to the peptide KLIpS, but no immune response is induced for other non-phosphorylated peptides.

In addition, intracellular staining analysis is used to assess the performance of cytokines within a heterogeneous population of cells. After fixing the cell membrane and Gogi's body, accumulated IFN-γ (KLIpS: 3.7%) was detected in the CD8 ⁺ T cell population of the spleen cells of the KLIpS-immunized AAD transgenic mice, which was used KLIS or VYC The immunized AAD transgenic mice constituted a relatively small group (KLIS: 0.5%; VYC: 0.7%), as shown in Figures 5(D) and 5(E). This cytokine secretion analysis confirmed that KLIpS is an immunogenic peptide, which can induce CD8 ⁺ T cell responses through peptide immunity.

5. Cytolysis of specific CTL response induced by KLIpS

In addition to the cytokine response to in vitro peptide re-stimulation, the present invention further evaluates the cytolytic effect of the specific CTL response induced by KLIpS. For this experiment, a mixture of equal amounts of epitope pulsed (CFSE ^high ) and untreated (CFSE ^low ) spleen cells was injected into peptide-immunized AAD transgenic mice through the tail vein. The spleen cells of the recipient were then collected to determine the percentage of CFSE ^high and CFSE ^low in the total CFSE ⁺ cells. CFSE ^high is used as a specific target cell in this analysis. When the cytotoxic T lymphocytes induced by KLIpS/IFA immunity perform a killer response, specific target cells will be lysed specifically. As shown in Figure 6(A), in mice immunized with KLIpS peptide, specific target cells (CFSE ^high ) were reduced by nearly half (KLIpS: 48.11±18.36%).

Subsequently, the present invention uses the chromium ( ⁵¹ Cr) release assay to examine the cytolytic effect of the specific CTL response induced by KLIpS on the H2981 human lung cancer cell line. This cell line is HLA-A2 positive and is similar to the TRAP1 protein that TC1/AAD cells express in large quantities (Figure 1). When ⁵¹ Cr labeled H2981 is incubated with mouse effector CD8 ⁺ T cells, up to 20% of the target cells are lysed at an effector: target ratio of 100:1 (E/T). As shown in Figure 6(B). It is worth noting that in the presence of anti-CD8 or anti-HLA antibodies, this cytolysis is significantly inhibited, which means that effector T cell cytolysis is mediated through the interaction of HLA and TCR.

6. Anti-tumor effect of specific CTL response induced by KLIpS

Based on the killing effect on cancer cells, a tumor attack model was established to test the cytolytic effect of specific CTL responses induced by KLIpS in tumor tissues. In the tumor attack analysis, mice with tumors (number of 5) immunized with KLIS or PBS buffer were sacrificed at 7 weeks; in contrast, as shown in Figures 7(A) and 7(B), The immune function of KLIpS can significantly reduce the growth of tumors and prolong the survival period of mice suffering from tumors for more than 7 weeks, which proves the efficacy of KLIpS as a therapeutic vaccine for cancer treatment.

In addition, in a xenograft model, KLIpS immunization-induced CTL can also inhibit the growth of human lung cancer tumors, as shown in Figure 7(C). This result showed that compared with H2981 tumor-bearing mice without CD8 ⁺ T cell adoptive transfer, the tumor growth of recipients with CTL response induced by KLIpS was very slow.

7. Antigen-specific CTL response induced by palmitate peptide

Lipids (Pam2 or Pam3) can trigger the toll-like receptor 2 (TLR2) pathway to promote the maturation of dendritic cells (DC) and enhance the Th1-biased immune response (Th1-biased immune response). Promote CTL. In addition, palmitate peptides can induce anti-tumor CTL responses by promoting cross-presentation of CTL epitopes by DC. Therefore, the present invention uses palmitic acid or tripalmitic acid conjugate as TLR2 agonist and PADRE to induce the activation of DC and helper T cells (Th) to promote CTL. In order to enhance the immunogenicity of KLIpS, the present invention synthesizes a palmitoylated peptide, which contains PADRE and KLIpS, and the two are connected to a lysine (K) residue, and the lysine ( K) The side chain of the residue is joined to dipalmitic acid or tripalmitic acid. The lipid (Pam2 or Pam3) moiety and PADRE are intrinsic adjuvants of dipalmitate peptide (Pam2KLIpS) or tripalmitate peptide (Pam3KLIpS). The lipidated peptide can induce antigen-specific CTL responses without conventional adjuvants, like incomplete Freund's adjuvant (IFA). To assess the immunogenicity of the self-adjuvanted lipidated peptide, AAD mice were immunized with 50 μg palmitated peptide on day 0 and day 7. The cells secreting IFN-γ were stimulated again with 5μg/ml KLIpS for 48 hours, and then confirmed by ELISPOT analysis. The cells that secrete IFN-γ represent specific CTLs induced by KLIpS. As shown in Figure 8, this result indicates that the immune effects of Pam2KLIpS and Pam3KLIpS can induce KLIpS-specific T cells and the frequency of KLIpS/IFA immunity.

In addition, a transgenic mouse model was used to evaluate the anti-tumor efficacy of lipidated peptides. Figure 9 demonstrates that compared with mice injected with PBS buffer (404.25±47.8mm ³ ), mice immunized with Pam2KLIpS or Pam3KLIpS can effectively inhibit tumor growth (Pam2KLIpS: 198±33.4mm ³ ; Pam3KLIpS: 46.9±24.8mm ³ ). The results of this tumor attack indicate that the immune effect of the palmitate peptide is effective in inhibiting tumor growth, and it may be a potential vaccine candidate for tumor therapy.

The proper description of the present invention can be implemented under elements or limitations not specifically disclosed herein. The terms that have been used for description are not limiting. There is no difference between the performance and description of these terms and any other equivalents, but it should be recognized that the rights in the present invention may be modified. Therefore, although the present invention has described embodiments and other situations, the content disclosed herein can be modified and changed by those skilled in the art, and such modifications and changes are considered to be within the scope of the rights of the present invention.

<110> National Institutes of Health

<120> Immunogenic peptides and their uses

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<170> PatentIn version 3.5

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<223> Introduction

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<223> A peptide designed based on human HLA-A11

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<223> A peptide designed based on human HLA-A2

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<223> Synthetic peptide

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<223> A peptide designed based on human TRAP1

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<223> Phosphorylation

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<223> A peptide designed based on human HSP27

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<223> A peptide designed based on human TRAP1

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<223> A peptide designed based on human TRAP1

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<223> Xaa is leucine or isoleucine

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Claims (18)

A peptide comprising an amino acid sequence composed of Lys-XX-Ser-Val-Glu-Thr-Asp-X (SEQ ID NO: 9), where X is leucine (Leu) or isoleucine (Ile), and the serine (Ser) is phosphorylated. The peptide described in item 1 of the scope of patent application, wherein the amino acid sequence is SEQ ID NO:6. Use of a composition for preparing a medicine for treating cancer in a body, wherein the composition comprises an effective dose of a peptide, wherein the peptide comprises Lys-XX-Ser-Val-Glu-Thr-Asp-X (SEQ ID NO: 9) An amino acid sequence composed of X is leucine (Leu) or isoleucine (Ile), and the serine (Ser) is phosphorylated. The use described in item 3 of the scope of patent application, wherein the individual is an individual with an HLA molecule of HLA-A2. The use described in item 3 of the scope of patent application, wherein the cancer is caused by cancer cells expressing TRAP1. The use described in item 5 of the scope of patent application, wherein the cancer is lung cancer, breast cancer, ovarian cancer, colorectal cancer or prostate cancer. The use described in item 3 of the scope of patent application, wherein the peptide has the ability to induce T cell immune response to treat cancer. The use as described in item 7 of the scope of patent application, wherein the T cells include type 1 helper T cells (Th1) and cytotoxic T lymphocytes (CTL). A lipidated peptide comprising a lipid, a helper T cell epitope and a peptide, wherein the lipid, the helper T cell epitope and the peptide are all linked to an amino acid, wherein the The peptide contains an amino acid sequence consisting of Lys-XX-Ser-Val-Glu-Thr-Asp-X (SEQ ID NO: 9), where X is leucine (Leu) or isoleucine (Ile ), and the serine (Ser) is phosphorylated. The lipidated peptide according to claim 9, wherein the lipid contains a fatty acid. The lipidated peptide according to item 9 of the scope of patent application, wherein the sequence of the helper T cell epitope includes an amino acid sequence of SEQ ID NO: 5. The lipidated peptide according to item 9 of the scope of patent application, wherein the amino acid is an amino acid with an active functional group on one side chain, wherein the active functional group can be connected to the lipid. Use of a composition for preparing a medicine for treating cancer in a body, wherein the composition comprises an effective dose of lipidated peptide, wherein the lipidated peptide comprises a lipid, a helper T cell epitope, and A peptide, wherein the lipid, the helper T cell epitope and the peptide are all linked to an amino acid, wherein the peptide comprises Lys-XX-Ser-Val-Glu-Thr-Asp-X (SEQ ID NO: 9) An amino acid sequence composed of X is leucine (Leu) or isoleucine (Ile), and the serine (Ser) is phosphorylated. The use as described in item 13 of the scope of patent application, wherein the individual is an individual with an HLA molecule of HLA-A2. The use described in item 13 of the scope of patent application, wherein the cancer is caused by cancer cells expressing TRAP1. The use described in item 15 of the scope of patent application, wherein the cancer is lung cancer, breast cancer, ovarian cancer, colorectal cancer or prostate cancer. The use as described in item 13 of the scope of patent application, wherein the lipidated peptide has the ability to induce the immune response of T cells to treat cancer. The use described in item 17 of the scope of the patent application, wherein the T cells include type 1 helper T cells (Th1) and cytotoxic T lymphocytes (CTL).

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