Polypeptide with tumor cell killing activity

Abstract

The invention relates to a polypeptide and a derivative thereof, in particular to a polypeptide with immunocompetence, the core structure of the polypeptide is K-M-X1-E-L-X2-H-F-L. X1 is selected from tyrosine (Y), valine (V) and proline (P); x2 is selected from valine (V) and lysine (K). The polypeptide can form a complex with an HLA molecule, and can be effectively loaded and presented on the surface of an antigen presenting cell (APC), thereby inducing a specific cytotoxic T cell (CTL) response. The invention also relates to a method for stimulating and activating APC cells by the polypeptide or the complex thereof, APC cells, CTL cells or cell compositions thereof obtained thereby, and pharmaceutical compositions and cancer vaccines comprising the polypeptide, the nucleic acid, the complex or the cells. The polypeptide and the application thereof have wide application prospects in anti-tumor immunotherapy, especially targeted therapy of lung cancer.

Description

Technical Field

[0001] This invention relates to the field of immunology, and in particular to a polypeptide with tumor cell killing activity and its uses. Background Technology

[0002] Cytotoxic T lymphocytes (CTLs) play a central role in anti-tumor immunity. Specific peptides, by binding to human leukocyte antigen (HLA) molecules, can be processed and presented by antigen-presenting cells (APCs), further activating CTL responses and inducing tumor cell killing. This process forms the basis of current peptide vaccines and cell immunotherapies.

[0003] However, existing tumor-associated peptides face challenges such as low presentation efficiency, poor immunogenicity, or insufficient specificity, especially in highly heterogeneous solid tumors like lung cancer. There is an urgent need to develop new targeted peptides with stronger T-cell response activation capabilities. Furthermore, using these peptides to prepare cell compositions that activate APCs, induce CTLs or CIK cells, and develop novel cancer vaccines is also a promising direction.

[0004] Therefore, developing a new class of peptides that can activate immune responses and constructing related cell therapy and vaccine products is of great significance for improving the treatment of lung cancer and other tumors. Summary of the Invention

[0005] This invention relates to the field of tumor immunotherapy technology, specifically a novel immune-related polypeptide and its applications. The polypeptide can be used to activate antigen-presenting cells (APCs), induce cytotoxic T lymphocytes (CTLs) or cytokine-induced killer cells (CIKs) to specifically recognize and kill cancer cells, thus making it widely applicable to preventative or therapeutic immune interventions for solid tumors such as lung cancer.

[0006] To address the issues of insufficient efficacy and poor HLA binding affinity of existing peptide antigens in immune responses, this invention provides a short peptide with an optimized structure. Its amino acid sequence is optimized through structural biology and immunoprediction calculations, which enhances its affinity for human leukocyte antigen (HLA) while exhibiting good immunogenicity and processing and presentation capabilities.

[0007] This invention first provides a polypeptide having the following general formula: KM-X1-EL-X2-HFL.

[0008] in:

[0009] X1 is selected from tyrosine (Y), valine (V), and proline (P);

[0010] X2 is selected from valine (V) and lysine (K);

[0011] Or its active variants or active fragments.

[0012] In further optimization, the present invention preferably incorporates the following combinations of active variants:

[0013] X1 is selected from tyrosine (Y) and valine (V);

[0014] X2 is selected from valine (V) and lysine (K).

[0015] In specific embodiments, preferred representative polypeptides of the present invention include, but are not limited to, the following sequences:

[0016] KMVELVHFL (SEQ ID NO:1);

[0017] KMYELVHFL(SEQ ID NO:2);

[0018] KMYELKHFL (SEQ ID NO:3);

[0019] KMVELKHFL(SEQ ID NO:4);

[0020] KMPELVHFL (SEQ ID NO:5).

[0021] The aforementioned peptide can efficiently bind to HLA class I molecules (such as HLA-A*02:01) to form a stable complex, which is then recognized by APCs and presented to T cells, inducing the production of CTL cells with anti-tumor activity. This invention also provides products or use systems based on this peptide, including but not limited to:

[0022] A peptide-HLA complex for activating T-cell immunity in vitro or in vivo;

[0023] APC cells activated in vitro were obtained by co-incubating the above-mentioned peptides or complexes with immature DCs or other APCs.

[0024] Specific CTL cells are generated by co-culturing peptide-loaded APC cells with autologous or allogeneic lymphocytes.

[0025] Cell composition comprising the above-described APC cells and a combination of CTL cells or CIK cells;

[0026] Therapeutic or preventive vaccines containing the aforementioned polypeptides, complexes, or cellular components;

[0027] The pharmaceutical composition, including any of the above-mentioned immune components, can be used as an individualized treatment plan for patients with tumors such as lung cancer.

[0028] The nucleotide sequences encoding the above-mentioned polypeptides are used to develop mRNA vaccines, gene therapies, or vector vaccines.

[0029] In this disclosure, for therapeutic or preventive vaccine compositions and pharmaceutical preparations, the therapeutic or preventive vaccine compositions may be delivered to the body alone or in combination with adjuvants; the pharmaceutical preparations include injections, lyophilized preparations, etc.; the polypeptides of the present invention and their encoded nucleic acids can be used to prepare drugs for the prevention or treatment of cancer, especially suitable for patients with solid tumors expressing corresponding antigens such as lung cancer, gastric cancer, and pancreatic cancer.

[0030] This invention provides a peptide-based immunotherapy technology system through systematic structural optimization, functional verification, and application expansion. It not only improves antigen presentation efficiency but also significantly enhances CTL cell response intensity, demonstrating promising application prospects and clinical translation potential. Attached Figure Description

[0031] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below.

[0032] Figure 1 This is a flow cytometry result image: CD8 + TCR + The percentage of cells was significantly higher than that of the negative control group (19.792% vs 0.470%). Detailed Implementation

[0033] To make the objectives, technical solutions and advantages of the present invention more clear, the embodiments of the present invention will be described in further detail below with reference to the accompanying drawings.

[0034] It should be understood that oligopeptides and polypeptides are interchangeable in this invention, as there is no strict distinction between oligopeptides and polypeptides for those skilled in the art.

[0035] It will be readily understood that the present invention covers variants of polypeptides, such as amino acid sequences having 1, 2, or 3 amino acid substitutions, deletions, or insertions in any of the sequences in SEQ ID NO:1-5.

[0036] For example, substitution refers to the replacement of an amino acid residue at the same position by another amino acid residue, preferably a substitution between amino acids of the same nature, such as a substitution between hydrophobic amino acids. The inserted amino acid residue can be inserted at any position, and the inserted amino acid residues can be all or partially adjacent, or none of the inserted amino acids can be adjacent to each other. Amino acid deletion can be the deletion of 1, 2, or 3 amino acid residues at any position, preferably the deletion of 1 or 2 amino acid residues.

[0037] Further exemplarily, substitution can occur between any amino acids. Conservative amino acid substitution is preferred. The term "conservative amino acid substitution" refers to the substitution of an amino acid residue by another amino acid residue with a side chain having similar properties. Amino acid residues are classified into several families based on their side chains. Examples of side chains include: basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., asparagine, glutamine, serine, threonine, tyrosine, and cysteine), nonpolar side chains (e.g., glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan), β-branched side chains (e.g., threonine, valine, and isoleucine), aliphatic side chains (e.g., glycine, alanine, valine, leucine, isoleucine, serine, and threonine), aromatic side chains (e.g., tyrosine, phenylalanine, and tryptophan), amide side chains (e.g., asparagine and glutamine), and sulfur-containing side chains (e.g., cysteine ​​and methionine). Conservative amino acid substitutions are preferably substitutions between amino acid residues within the same family. Examples of conserved amino acid substitutions include glutamic acid residues replaced by aspartic acid residues, phenylalanine residues replaced by tyrosine residues, leucine residues replaced by isoleucine residues, isoleucine residues replaced by valine residues, alanine residues replaced by serine residues, and histidine residues replaced by arginine residues.

[0038] For peptide variants, it is possible to verify whether the variant is an active variant. For example, the variant peptide can be contacted with APCs (e.g., DC cells), and then the APC cells can be tested for target monoclonal antibodies. If CD80 and CD86 are highly expressed on the APC cells, then the variant is an active variant.

[0039] This invention also covers tandem peptides, such as those comprising two or more of SEQ ID NO:1 or its active variants, or at least two repeating units thereof. It should be noted that, in order to present tumor-specific antigens on APCs, the peptides of this invention can be contacted with APCs in the form of tandem peptides.

[0040] Those skilled in the art will know that the polypeptides described in this invention can be post-translational modified at one or more positions between amino acid sequences, such as acetylation, phosphorylation, etc. Further, the polypeptides can be artificially modified, for example, by replacing their amino acid residues with amino acid analogs or mimics. Even further, one or more substances, such as amino acids, peptides, and their analogs, can be added to the N-terminus and / or C-terminus of the polypeptide. For example, a histidine tag can be added, or a fusion polypeptide can be formed with the protein. Detectable tags can also be bound to the polypeptide. When such a substance binds to the polypeptide, the substance can be processed, for example, using a biological enzyme or through intracellular processing, to produce the polypeptide. Such substances can modulate the solubility of the polypeptide, improve the stability of the peptide (e.g., protease resistance), allow the specific delivery of the polypeptide to a desired tissue or organ, or enhance the uptake of the polypeptide by antigen-presenting cells. Such substances can also be substances that increase the ability of a peptide to induce CTLs, for example, another peptide that activates T cells.

[0041] As is known to those skilled in the art, tumor antigens are enzymatically cleaved into polypeptide fragments by proteases within antigen-presenting cells. These fragments then bind to the major histocompatibility complex (MHC) to form a polypeptide-MHC complex, which is presented to the surface of APC cells. The polypeptide-MHC complex further binds to the surface of lymphocytes, stimulating and activating these lymphocytes to become cytotoxic T cells (CTLs).

[0042] Therefore, the present invention provides a polypeptide-MHC complex comprising the polypeptide or a variant thereof described herein. The MHC molecule may be an MHC class I molecule or an MHC class II molecule. In a preferred embodiment, the MHC molecule is HLA-A, HLA-B, and HLA-C, or HLA-DR, HLA-DQ, and HLA-DP. Further, the MHC molecule may also be an MHC class III molecule.

[0043] The present invention also provides molecules and cells that bind to the above-mentioned polypeptides, tandem polypeptides or complexes.

[0044] The methods for generating the polypeptide-MHC complex of the present invention are known to those skilled in the art, such as binding the polypeptide to an HLA molecule.

[0045] The polypeptide-MHC complex of the present invention can be used to screen or detect molecules that bind to it, such as T cell receptors (TCRs).

[0046] In cellular immunity, antigens are typically presented to the cell surface along with MHC complexes. Therefore, this invention also provides isolated cells capable of presenting the polypeptides or polypeptide-MHC complexes of this invention to their surface. Exemplarily, the cells are immune cells, such as APCs, DCs (dendritic cells), B cells, or T2 cells. Preferably, the cells presenting the polypeptides or polypeptide-MHC complexes of this invention are isolated. The cells may not naturally present the complexes of this invention. Cells presenting the polypeptide-MHC complexes of this invention can be used to isolate T cells and T cell receptors, the T cells being activated by the polypeptides or complexes and further sorted, proliferated in vitro, and then reinfused into a subject.

[0047] In one specific embodiment, the method for obtaining isolated T cells includes contacting the T cells with the polypeptide of the present invention or the polypeptide-MHC complex or cells presenting thereon. Using labeled antibodies, the activated T cells can be sorted by flow cytometry (FACS), and the sorted cells can be proliferated and cultured in vitro.

[0048] The present invention also provides nucleic acid molecules comprising polypeptides, polypeptide variants, tandem polypeptides, such as cDNA, encoding the present invention. The nucleic acids can be synthesized using artificial synthesis methods known in the art. Due to the degeneracy of the genetic code, those skilled in the art will understand that different nucleic acid sequences can encode the same amino acid sequence.

[0049] Based on the aforementioned nucleic acid, the present invention also provides a vector. This vector includes the nucleic acid sequence described in this invention. Exemplarily, the vector is an expression vector, such as a plasmid, bacteriophage, or virus. Suitable bacteriophage and viral vectors include λ-phage, EMBL-phage, simian virus, bovine wart virus, Epstein-Barr virus, oncolytic virus, mouse sarcoma virus, murine mammary carcinoma virus, lentivirus, etc.

[0050] The present invention also provides a host cell comprising the vector or nucleic acid described in the present invention, wherein such cell may be a mammalian cell expressing the peptide of the present invention.

[0051] The present invention also provides a molecule (e.g., a TCR and an antibody) that can be used as an immunotherapeutic agent or a diagnostic reagent. This molecule can bind to a peptide or to a complex formed by a peptide and an MHC molecule.

[0052] The TCR of the present invention can be any form known in the art, for example, a heterodimer.

[0053] In this invention, "antibody" refers to immunoglobulin molecules and the immunoactive portion of immunoglobulin molecules, i.e., molecules containing specific binding sites, which can be all-natural, partially synthetic, or fully synthetic. The term "antibody" includes antibody fragments, their derivatives, functional equivalents, and homologous antibodies and humanized antibodies. The antibody fragments include an immunoglobulin-binding region, which is an antibody-binding region or homologous to an antibody-binding region. They can be all-natural, partially synthetic, or fully synthetic. Humanized antibodies can be modified antibodies containing a variable region of a non-human antibody (e.g., mouse) and a constant region of a human antibody.

[0054] Examples of antibodies can be isotype immunoglobulins (e.g., IgG, IgE, IgM, IgD, and IgA) and their isotype subclasses; fragments include antigen-binding regions such as Fab, scFv, Fv, dAb, and Fd; and double-chain antibodies. Antibodies can be polyclonal or monoclonal, preferably monoclonal antibodies.

[0055] In this invention, the TCR and antibody can be present on the cell surface, such as the surface of T cells. Therefore, this invention also provides an isolated T cell that binds to the complex of this invention.

[0056] The present invention also provides the use of the aforementioned polypeptides and their variants, fusion polypeptides, polypeptide-MHC complexes, tandem polypeptides, cells, and binding molecules, for example, for the preparation of drugs for the prevention or treatment of cancer.

[0057] The present invention also provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and the peptide, fusion polypeptide, polypeptide-MHC complex, tandem polypeptide, cell, binding molecule or cell.

[0058] The dosage form of the pharmaceutical composition of the present invention can be suitable for any appropriate route of administration, such as injection (including subcutaneous, intramuscular, intraperitoneal, or intravenous injection), inhalation or oral administration, or via the nose or anus. The composition can be prepared by any method known in the pharmaceutical field, for example, under aseptic conditions by mixing the active ingredient with a carrier or excipient.

[0059] The polypeptides, fusion polypeptides, tandem polypeptides, complexes, or cells of the present invention can be provided in the form of vaccine compositions. These vaccine compositions can be used to treat or prevent cancer, and may further contain adjuvants, such as inactivated vaccines, live attenuated vaccines, viral vector vaccines, RNA vaccines, etc.

[0060] In this invention, antigen-presenting cells (APCs) are selected from, for example, monocytes, monocyte-derived cells, macrophages, dendritic cells (DCs), and preferably DC cells.

[0061] The antigen-presenting cells are preferably dendritic cells (DCs).

[0062] The major histocompatibility complex (MHC) is a gene complex encoding the human leukocyte antigen (HLA) gene. The HLA gene is expressed as a protein heterodimer displayed on the surface of human cells to circulating T cells. The HLA gene is highly polymorphic, allowing for fine-tuning of the adaptive immune system.

[0063] There are three classes of MHC molecules: MHC I, MHC II, and MHC III. MHC class I molecules consist of an α heavy chain and a β-2-microglobulin; MHC class II molecules consist of an α chain and a β chain. The MHC molecule structure contains a binding groove for non-covalent interactions with peptides; MHC III mainly encodes complement components, such as tumor necrosis factor (TNF) and heat shock protein 70 (HSP70).

[0064] Most nucleated cells express MHC class I molecules, primarily presenting endogenous proteins, defective ribosome products (DRIPs), and peptides derived from the cleavage of larger peptides. However, exogenous peptides are also frequently found on MHC class I molecules. MHC class II molecules are primarily found on antigen-presenting cells (APCs) and are mainly presented, for example, peptides of exogenous or transmembrane proteins that are occupied by APCs during endocytosis and subsequently processed.

[0065] Complexes of peptides and MHC class I molecules are recognized by CD8-positive T cells loaded with the corresponding T cell receptor (TCR), while complexes of peptides and MHC class II molecules are recognized by CD4-positive helper T cells loaded with the corresponding TCR.

[0066] CD4 + T helper cells play a crucial role in inducing and maintaining an effective response to CD8-positive cytotoxic T cells. At the tumor site, T helper cells maintain a cytokine environment favorable to cytotoxic T cells (CTLs) and attract effector cells such as CTLs, natural killer (NK) cells, macrophages, and granulocytes.

[0067] In the absence of inflammation, the expression of MHC class II molecules is primarily confined to immune system cells, especially antigen-presenting cells (APCs). The expression of MHC class II molecules has been found in tumor cells of cancer patients.

[0068] The peptides or variants of the present invention may be further modified to improve stability and / or bind to MHC molecules, thereby triggering a stronger immune response. Methods for improving peptide sequences are well known in the art, such as the introduction of trans peptide bonds and non-peptide bonds.

[0069] The term "isolation" means the removal of a substance from its original environment (e.g., the natural environment if it occurs naturally). For example, naturally occurring nucleotides or polypeptides in a living animal are not isolated; however, nucleotides or polypeptides isolated from some or all of the coexisting substances in a natural system are isolated. Such polynucleotides may be part of a carrier and / or such polynucleotides and polypeptides may be part of a composition, and because the carrier or composition is not part of its natural environment, it is still isolated.

[0070] This invention screens and identifies positive molecular targets, namely tumor-specific antigens, in lung cancer subjects from peripheral blood. These positive molecular targets are then co-cultured with the subject's immune cells in vitro and reinfused into the subject, thereby killing tumor cells in the body.

[0071] Example 1: Screening for tumor-specific molecular targets from lung cancer tissue

[0072] To screen for specific molecular targets for lung cancer, the following procedures were performed.

[0073] (1) Collect samples containing HLA-peptide complexes, perform tissue homogenization, primary purification, enrichment and purification, and collection:

[0074] Tissue homogenization: If the sample is tumor tissue or tumor cell line, it is necessary to homogenize the sample and centrifuge to collect the supernatant.

[0075] Primary purification: Add an equal volume of enrichment solution to the supernatant or serum, mix well, centrifuge and collect the supernatant;

[0076] Enrichment and purification: The supernatant was added to an Oasis Prime HLB SPE column for enrichment and purification;

[0077] Elution and collection: After the filtrate has completely passed through, the target peptide adsorbed on the elution column is used to collect approximately 20 μL of eluent.

[0078] (2) The obtained peptide solution was subjected to liquid chromatography-mass spectrometry (LC-MS): After the sample entered the high-performance liquid chromatography (HPLC) column, it underwent effective separation, allowing different substances to enter the ion source of the mass spectrometer in chronological order. The various charged molecules formed after ionization and fragmentation of the detected substances in the ion source reached the TOF mass spectrometer detector sequentially according to their mass-to-charge ratios. The detector signal was analyzed to obtain specific data such as the molecular weight of each molecule. Through comparison with a target database, specific molecular targets for lung cancer, SEQ ID NOs: 1-5, were finally identified and screened using multi-site point mutations.

[0079] (3) The target SEQ ID NOs: 1-5 were synthesized and purified using a peptide solid-phase synthesis platform to obtain lyophilized peptide powder with a purity of ≥95%.

[0080] Example 2: CTL cell preparation

[0081] Collect 80-120 ml of peripheral blood and separate mononuclear cells (PBMCs) from the blood using density gradient centrifugation with lymphocyte separation medium. Based on the PBMC count, adjust the cell count to approximately 1 × 10⁻⁶ cells / mL using culture medium. 7 Cells / ml were seeded into culture flasks, with 10-15 ml of culture medium per flask. The flasks were then incubated in a carbon dioxide incubator at 37°C with 5% CO2 for 30 min.

[0082] Dendritic cells were prepared by collecting adherent cells from PBMC culture flasks. Simultaneously, T cells were isolated from PBMCs of the same subject using Ficol-Paque density gradient centrifugation or magnetic cell sorting. After treating the dendritic cells in peptide medium, they were co-cultured with T cells, and the cultured T cells were then recovered. The activity of target peptide-specific T cells could be detected by measuring TCR. + The proportion of T cells and the killing ability of T cells against target cells expressing the target peptide were comprehensively evaluated.

[0083] Specifically, the collected dendritic cells were added to DC culture medium containing the target peptide (concentration of 5 μg / mL) and cultured in an incubator at 37°C and 5% CO2. Subsequently, the activated DC cells were harvested and co-cultured with suspended T cells to obtain cytotoxic T cells (CTL cells). The CTL cells were then further cultured and proliferated, and on day 5 of culture, the proportion of TCR+ T cells and their cytotoxic effect on target cells were detected to evaluate the functional activity of CTL cells.

[0084] Example 3: Detection of TCR+ T cell ratio in CTL

[0085] Prepare DSNS16 polypeptide tetramer according to the kit instructions.

[0086] The CTL cells prepared in Example 2 were collected, washed, counted, and grouped as follows:

[0087] (1) Blank group: normal samples, without the addition of any antibodies, used to adjust the voltage;

[0088] (2) Negative control group: Negative tetramer was added to determine the location of the TCR+ positive gate;

[0089] (3) Test sample group: Add tetramer corresponding to DSNS16 peptide.

[0090] Subsequently, 5-10 μL of tetramer was added to each group, with the negative control group receiving an equal volume of tetramer replaced by the negative peptide. After mixing, the mixture was incubated at 4°C for 30 minutes. Except for the Blank group, 2-5 μL of each of the four antibodies (CD8, etc.) were added to the other groups, and the mixture was incubated at room temperature for 15-30 minutes. After incubation, the mixture was centrifuged (1500 rpm, 5 min), the supernatant was discarded, and the cells were resuspended in physiological saline for flow cytometry analysis. The results are as follows: Figure 1 (SEQ ID NO: 1).

[0091] from Figure 1 As can be seen from CD8 + TCR + The percentage of cells was significantly higher in the control group than in the negative control group (19.782% vs 0.470%). This indicates that the peptide can effectively activate the corresponding TCR. + T cells.

[0092] Example 4: Analysis of the activating effect of peptides on human dendritic cells (DCs)

[0093] Human peripheral blood mononuclear cells (PBMCs) from healthy donors were used to isolate dendritic cells (DCs) using the adherent method. PBMCs were cultured in RPMI-1640 medium (containing 10% autologous plasma) at 37°C and 5% CO2 for 30 minutes. Non-adherent cells were then removed, and adherent cells were cultured further to obtain immature DCs.

[0094] The experimental groups were treated with each target peptide (SEQ ID NO: 1–5) at a final concentration of 5 μg / mL and cultured in CellGenix dendritic cell culture medium at 37°C and 5% CO2 for 5 days. The control group received no peptides. After culture, the expression of the activation markers CD80 and CD86 on the surface of dendritic cells was detected by flow cytometry.

[0095] The results are shown in the table below:

[0096] polypeptide number polypeptide sequence CD80 (%) CD86 (%) SEQ ID NO:1 KMVELVHFL 95.5 96.0 SEQ ID NO:2 KMYELVHFL 96.2 95.3 SEQ ID NO:3 KMYELKHFL 94.7 94.8 SEQ ID NO:4 KMVELKHFL 93.5 95.0 SEQ ID NO:5 KMPELVHFL 93.9 96.5 negative control Peptide-free 52.8 53.5

[0097] As shown above, all peptides significantly upregulated CD80 / CD86 expression on the DC surface (experimental group: 93.5%–96.5%; negative control: ≤75.5%), indicating that the optimized structure retained high APC activation capacity. The introduction of Y at the N-terminus (enhanced hydrophobicity) in SEQ ID NO:2 and SEQ ID NO:3 increased CD80 expression to 96.2%, but after replacing V with K (lysine, positively charged) in the center (NO:3), CD86 slightly decreased to 94.8%. The M→P substitution (change in flexibility) in SEQ ID NO:5 resulted in a peak CD86 expression of 96.5%, possibly related to HLA binding conformation optimization. The expression rates of activation markers in DC cells without peptide loading were significantly lower than in the experimental group, confirming the peptide-specific activation effect.

[0098] Example 5: Induction of Cytotoxic T Cells (CTLs) by Peptide-Loaded DC Cells and Functional Detection. The DC cells obtained in Example 4 were further cultured and each peptide (SEQ ID NO: 1–5) was added to a final concentration of 25 μg / mL. The culture medium was changed every 2–3 days, and IL-4, TNF-α, and GM-CSF were supplemented. After 5 days of culture, mature DC cells were harvested and co-cultured with T cells isolated from PBMCs at a DC:T ratio of 1:8 for ≥3 days to obtain primary CTL cells.

[0099] Set up a control group:

[0100] • Control 1: Co-culture of DC cells and T cells without peptide stimulation;

[0101] • Control 2: T cell culture alone.

[0102] The proliferation of T cells on days 0, 2, 4, and 6 was detected using the CCK-8 assay. Simultaneously, the cytotoxic effect of CTLs on NCI-H2228 cells was evaluated (target-efficacy ratio 1:30, cultured for 18h and 24h, 3 replicates). Results showed:

[0103] AT cell proliferation analysis (mean OD 450nm)

[0104] Group Day 0 Day 2 Day 4 Day 6 SEQ ID NO:1 0.21 0.32 0.76 1.12 SEQ ID NO:2 0.22 0.34 0.79 1.15 SEQ ID NO:3 0.20 0.30 0.71 1.06 SEQ ID NO:4 0.23 0.31 0.73 1.09 SEQ ID NO:5 0.21 0.32 0.75 1.11 Control group 1 0.19 0.27 0.53 0.74 Control group 2 0.18 0.25 0.50 0.70

[0105] B. CTL killing rate of NCI-H2228 cells (%)

[0106]

[0107]

[0108] The data above indicate that all five peptides can significantly promote T cell proliferation and induce highly efficient CTL cells, demonstrating good potential for immunostimulation.

[0109] Example 6: Killing activity of peptide-induced CTL cells against various lung cancer cell lines

[0110] Five human lung adenocarcinoma cell lines (NCI-H2228, NCI-H522, NCI-H1650, NCI-H2347, and NCI-H1703) were selected as target cells and co-cultured in vitro with CTL cells obtained in Example 5. Effector-to-target ratios of 40:1, 20:1, and 10:1 were set. After 24 hours of culture, cytotoxicity was detected using the LDH release assay. Using SEQ ID NO:1 as an example, the killing rates against the five lung cancer cell lines are as follows (%):

[0111] target cell lines 40:1 20:1 10:1 NCI-H2228 83.7 76.5 66.2 NCI-H522 80.1 72.0 61.3 NCI-H1650 77.9 70.8 59.1 NCI-H2347 76.5 70.2 58.0 NCI-H1703 75.3 68.1 56.4

[0112] Other peptides also exhibited similar broad-spectrum antitumor activity, with SEQ ID NO:2 and SEQ ID NO:5 showing the most significant CTL killing.

[0113] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A polypeptide, characterized in that, Its general structure is: KM-X1-EL-X2-HFL, in: X1 is selected from tyrosine (Y), valine (V), and proline (P); X2 is selected from valine (V) and lysine (K); Or its active variants or active fragments.

2. The polypeptide according to claim 1, characterized in that, The amino acids of the polypeptide are replaced with the following combinations: X1 is selected from tyrosine (Y) and valine (V); X2 is selected from valine (V) and lysine (K).

3. The polypeptide according to claim 1, characterized in that, The polypeptide is any of the following sequences: KMVELVHFL (SEQ ID NO:1); KMYELVHFL(SEQ ID NO:2); KMYELKHFL (SEQ ID NO:3); KMVELKHFL(SEQ ID NO:4); KMPELVHFL (SEQ ID NO:5).

4. A complex comprising HLA and the polypeptide of any one of claims 1-3.

5. A method for stimulating and activating APC cells in vitro, comprising the step of contacting a polypeptide of any one of claims 1-3 or a complex of claim 4 with APC cells to be activated, thereby loading the APC cells with the polypeptide.

6. An activated APC cell, obtained by contacting and culturing an APC cell to be activated with a polypeptide of any one of claims 1-3 or a complex of claim 4.

7. A cytotoxic T cell (CTL) comprising co-culturing the APC cells of claim 6 with lymphocytes to obtain cytotoxic T cells (CTL) specifically targeting lung cancer.

8. A cell composition comprising the APC cells and CTL cells of claim 6, or comprising the APC cells and CIK cells of claim 6.

9. A therapeutic or preventive vaccine against cancer, comprising the polypeptide of any one of claims 1-3 or the complex of claim 4, or the cell or cell composition of any one of claims 6-8.

10. Use of the polypeptide or its encoded nucleotide as described in any one of claims 1-3 in the preparation of a medicament for the prevention or treatment of cancer.

11. A drug comprising the polypeptide or its encoded nucleotide of any one of claims 1-3, the complex of claim 4, and the cell or cell composition of any one of claims 6-8.

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