Polypeptide with tumor cell killing activity

Abstract

The invention discloses a polypeptide with tumor cell killing activity. The specific molecular target or the fusion polypeptide thereof is co-cultured with autoimmune cells of a subject, and then the autoimmune cells are transfused back into the body of the subject, so that targeted killing of tumor cells is realized, and the purpose of treating tumors is achieved. The dendritic cell (DC) load positive rate activated by the molecular target is greater than 90%. Furthermore, the activated DC cells highly express CD80 and CD86 on the surface, and can effectively present tumor antigens to T lymphocytes, thereby inducing to generate CTL cells with the capability of specifically recognizing cancer cells.

Description

Technical Field

[0001] The present invention relates to the field of immunology, and in particular to a polypeptide with tumor cell killing activity and uses thereof. Background Art

[0002] Cancer is a serious threat to human health and one of the leading causes of death worldwide. According to the World Health Organization, approximately 19.29 million new cancer cases and 9.96 million deaths were reported worldwide in 2020. While advances in medical technology have led to significant progress in cancer diagnosis and treatment, treatment remains a significant challenge due to the heterogeneity and high recurrence rate of cancer. Currently, cancer treatment remains primarily based on traditional methods: surgery, radiotherapy, and chemotherapy.

[0003] All three traditional treatments present significant disadvantages when faced with tumor heterogeneity, high metastatic potential, and high recurrence rates. Surgery is a cornerstone of cancer treatment, particularly for early-stage, localized tumors. By removing the tumor and surrounding affected tissue, surgery can achieve local control and potentially cure. However, surgery is ineffective for micrometastatic lesions, and approximately 70% of patients are incurable by surgery at the time of diagnosis. Furthermore, the high recurrence rate after surgery necessitates additional treatment options to prolong survival. Radiotherapy, which uses high-energy radiation to kill tumor cells, is an important method for local tumor control. Modern radiotherapy techniques, such as intensity-modulated radiation therapy (IMRT) and stereotactic body body therapy (SBRT), have improved treatment precision. However, adverse effects of radiotherapy, including damage to normal tissue, limit its application. Furthermore, the insensitivity of some tumor types to radiotherapy, such as melanoma, reduces its efficacy. Chemotherapy, a drug-based approach to killing rapidly dividing cells, is widely used in the treatment of various cancers. Although chemotherapy has a certain effect in prolonging survival and alleviating symptoms, its nonspecific toxicity leads to serious side effects (such as bone marrow suppression, nausea and vomiting), which limits further dose increases. In addition, the problem of chemotherapy resistance is particularly prominent in patients with advanced disease.

[0004] In recent years, biotherapeutic approaches for cancer have shown promising promise as an emerging cancer treatment option, becoming a new treatment option. Immune checkpoint inhibitors, including PD-1 (PD-L1) and CTLA-4 antibodies, have been widely used. These checkpoint inhibitors can relieve T cell suppression, restore T cell cytotoxicity, and enhance the body's immune response to tumors. These inhibitors are suitable for a variety of cancers (such as melanoma and non-small cell lung cancer), producing sustained anti-tumor responses and achieving long-term remission in some patients. Furthermore, another category of biotherapeutic technologies is cell therapy, primarily including CAR-T, TCR-T, and TIL.

[0005] CAR (chimeric antigen receptor) is composed of a single-chain antibody fragment (scFv) that can recognize tumor cell surface antigens, combined with a T cell signaling domain (such as CD3ζ) and a co-stimulatory signaling domain (such as CD28, 4-1BB). T cells carrying CAR can specifically recognize and kill tumor cells. CAR-T directly recognizes tumor surface antigens (without the need for MHC restriction), such as CD19, CD22, and other antigens. It is suitable for tumors that highly express surface antigens and is primarily used for hematological malignancies (such as B-cell acute lymphoblastic leukemia and diffuse large B-cell lymphoma), where it has shown significant therapeutic effects. T cells prepared by this technology are not dependent on MHC, are applicable to a wide range of populations, and have strong tumor-killing ability and rapid expansion capabilities.

[0006] TCR-T cell therapy (T-Cell Receptor Engineered T-Cell Therapy) is an immunotherapy that uses genetic engineering to enhance the ability of T cells to recognize and kill tumors. It utilizes T cells from a patient or donor, genetically modified to express specific T cell receptors (TCRs). These receptors recognize antigenic peptides presented on the surface of tumor cells and can target intracellular antigens (such as the cancer / testis antigen NY-ESO-1 or the mutant antigen KRAS). It can be used to treat both solid tumors and hematologic malignancies, and is particularly suitable for cancers with clearly defined specific antigens.

[0007] TIL therapy (Tumor-Infiltrating Lymphocyte Therapy) extracts tumor-infiltrating lymphocytes (TILs) from a patient's tumor tissue, expands them in vitro, and then reinfuses them into the patient to enhance the anti-tumor immune response. TILs are T cells from the patient's own immune system that have already responded to tumor antigens. They do not require genetic engineering and have the ability to target multiple antigens. This technology has currently demonstrated promising results in the treatment of specific solid tumors, such as melanoma and cervical cancer.

[0008] There are many technologies for treating cancer at present. Three traditional treatment methods are the most widely used technologies. However, they have not been able to continuously improve the five-year survival rate of cancer patients over the years.

[0009] Surgery, the oldest cancer treatment, can only treat localized tumors and is difficult to treat for metastatic cancer or small lesions. It may not be able to completely remove all cancer cells, leading to recurrence. Surgery is usually not suitable for advanced or widely metastatic cancers.

[0010] As one of the traditional treatment methods, radiotherapy is powerless against certain cancers that are insensitive to or tolerant to radiotherapy. Moreover, long-term use not only damages normal tissues around the lesions, but may also lead to secondary cancers induced by radiation.

[0011] Chemotherapy has poor non-specificity and is difficult to accurately target tumor cells. While killing normal cells, its efficacy is limited, so it is highly toxic and often has toxic effects on cells throughout the body (including normal rapidly dividing cells), leading to serious side effects such as hair loss, nausea, and bone marrow suppression. In addition, high drug resistance is also a problem that cannot be ignored, resulting in limited efficacy and high recurrence rate. Similar to chemotherapy is targeted therapy, which is also a very widely used treatment method. In addition to having the same drug resistance as chemical drugs, the drug targets are very limited and the coverage population is limited. A large number of gene mutation sites still do not have corresponding targeted drugs. Furthermore, the cost is high, and it is difficult for ordinary families to adhere to long-term treatment.

[0012] Currently, immune checkpoint inhibitors and cell therapy technologies are widely used in biological treatments.

[0013] Although immune checkpoint inhibitors are increasingly used and PD-1 antibodies have become first-line clinical drugs, their low efficacy still reminds people that they need to continue to develop new treatment methods. Since tumor cells have dynamic regulatory capabilities, the use of immune checkpoints can also cause tumor cells to mutate to resist immune checkpoint inhibitors, thereby losing the therapeutic effect.

[0014] Cell therapy technologies primarily include CAR-T, TCR-T, and TIL. CAR-T has limited therapeutic targets, primarily applicable to hematologic malignancies, with limited efficacy against solid tumors. Furthermore, the potential for serious adverse reactions such as cytokine storm (CRS) or neurotoxicity cannot be ignored. The high cost of treatment has deterred many, and cases of relapse have recently been reported. TCR-T, another cancer therapy utilizing genetic engineering, can overexpress TCRs with high affinity for specific HLA peptides. However, because each cell has only one or two HLA peptide binding sites, it is unable to cope with tumor heterogeneity and high mutation rates. TIL, which expands a patient's own infiltrating T cells and then infuses them into the body to kill tumors, is typically the safest approach. However, the limited efficacy caused by cell depletion cannot be ignored, and the tumor microenvironment must also suppress the infused T cells, resulting in less than satisfactory results.

[0015] Both traditional treatments and emerging engineered cell technologies exhibit significant limitations when faced with the high heterogeneity and high mutational potential of tumors. These challenges have driven researchers to continuously explore novel therapeutic approaches. The emergence of DC-T therapy has responded to this need, injecting new vitality into the field of cancer treatment. Compared with technologies such as CAR-T and TCR-T, DC-T technology does not require genetic engineering of cells, offering significant safety advantages. Furthermore, the T cells activated by this technology can recognize a wide range of cell types expressing corresponding HLA peptides on their cell surfaces, encompassing a wide range of cells in different tissues and organs. This means that T cells generated using DC-T technology can broadly target cells expressing corresponding HLA peptides, regardless of their location. More importantly, by creating mixed T cells with a mixture of multiple HLA peptides, this technology can simultaneously target multiple cell types within tumor tissues, effectively addressing the therapeutic challenges presented by tumor heterogeneity and mutation. In summary, DC-T technology not only offers superior safety but also possesses broad targeting capabilities across tumors in multiple tissues and organs, providing a more comprehensive and effective solution for cancer treatment. Summary of the Invention

[0016] To address the problems of the prior art, the present invention provides active peptides and combinations thereof that can kill lung cancer cells. These peptides activate antigen-presenting cells (APCs), causing them to present specific antigens targeting lung cancer. This process stimulates the body to produce specific immune cells, achieving targeted immunotherapy for lung cancer cells.

[0017] To obtain lung cancer-specific peptides, the researchers of this invention conducted continuous screening, identification, and validation. First, the serum of lung cancer patients was enriched and purified. Subsequently, through mass spectrometry detection, specific peptides for lung cancer were screened and identified. In addition, the researchers used ultra-high performance liquid chromatography and mass spectrometry to hydrolyze the serum of tumor patients to obtain the amino acid sequences of the peptides. By comparing these amino acid sequences with a peptide library, peptides that were expressed only in lung cancer tissue and not in negative tissues were selected, and molecular targets with excellent performance were further screened. Negative tissues here include non-lung cancer tissues, such as normal tissue.

[0018] Through this series of screening and validation processes, the present invention successfully obtained excellent peptides and fusion polypeptides containing them that are specific for tumors (e.g., lung cancer). These peptides or fusion polypeptides not only provide new markers for lung cancer diagnosis but also lay an important foundation for lung cancer immunotherapy. This invention can provide lung cancer patients with more effective and personalized treatment options, opening up new avenues for the treatment and management of lung cancer.

[0019] Therefore, the present invention provides a polypeptide comprising the following amino acid sequence

[0020] ALSELTQGV (SEQ ID NO: 1, DSNS16 polypeptide) or an active variant thereof.

[0021] The present invention also provides a fusion polypeptide comprising a DSNS16 polypeptide or an active variant thereof.

[0022] Illustratively, the fusion polypeptide is as follows: DSNS16-linker-DSNS16 or DSNS16-linker-DSNS16-linker-DSNS16.

[0023] Furthermore, the fusion polypeptide is as follows: DSNS16-GGGS-DSNS16 or DSNS16-GGGS-DSNS16-GGGS-DSNS16.

[0024] Preferably, the polypeptide of the present invention or its fusion polypeptide is capable of binding to the major histocompatibility complex (MHC) to form a polypeptide-MHC complex. For example, the polypeptide of the present invention is capable of binding to an HLA molecule to form a polypeptide-HLA complex. Furthermore, the complex is capable of being recognized by T cells. Preferably, the HLA is present on an antigen-presenting cell (APC).

[0025] For example, ALSELTQGV (DSNS16 polypeptide) has 9 amino acid residues. Therefore, it should be understood that the polypeptides of the present invention should be allowed to have a length that binds to MHC I or MHC II molecules, such as 9 to 45, 9 to 40, 9 to 35, 9 to 30, or 9 to 25 amino acids in length. For example, the polypeptide can be composed of 9 to 18, 9 to 19, 9 to 20, 9 to 18, 9 to 17, 9 to 16, 9 to 15, 9 to 14, 9 to 13, 9 to 12, 9 to 11, 9 to 10, or 9 amino acid residues. In theory, the antigenic peptide that binds to the MHC molecule can be longer because the longer polypeptide contains the motif portion that binds to the MHC, and the other amino acid segments outside the motif are located on both sides or one side of the binding region.

[0026] The present invention also provides a fusion polypeptide comprising the polypeptide of the present invention. Since one of the objectives of the present invention is to present the polypeptide of the present invention on APCs and further stimulate or induce the production of CTLs, the fusion polypeptide should not be too long, as excessively long fusion polypeptides will require enzymatic cleavage before presentation to APCs. Therefore, illustratively, the fusion polypeptide may be no longer than 100 Aa, for example, 45-100, 45-80, 45-60, 45-55 Aa, and so on.

[0027] One or more (e.g., two or three) amino acid residues can be added, deleted, or substituted at any position in the parent sequence (the aforementioned polypeptide or its fusion polypeptide). For example, one to three amino acid substitutions can be made to enhance solubility or cell penetration; or some amino acid deletions can be made to reduce molecular size or eliminate potential undesirable immunogenicity. These modifications can optimize the properties of the parent peptide without significantly affecting its function.

[0028] The present invention also provides a complex comprising HLA and the polypeptide or fusion polypeptide of the present invention.

[0029] The present invention also provides a method for stimulating and activating APC cells, comprising the step of contacting the polypeptide or fusion polypeptide of the present invention with APC cells to be activated, thereby allowing the APC cells to be loaded with the polypeptide or fusion polypeptide.

[0030] The present invention also provides an isolated activated APC cell, wherein the cell surface presents a polypeptide, fusion polypeptide, or complex thereof (e.g., a polypeptide-HLA complex or a polypeptide-MHC complex) of the present invention. Exemplarily, the isolated APC is obtained by contacting and culturing the polypeptide of the present invention with an APC cell to be activated.

[0031] Since the polypeptide of the present invention is a tumor (lung cancer) specific antigen, the present invention also relates to molecules (such as antibodies) that can specifically bind to the polypeptide of the present invention, so as to be used for detecting tumor (lung cancer) specific antigens in the blood of a subject for diagnostic purposes, or for therapeutic purposes.

[0032] Therefore, the present invention also provides a method for detecting the risk of lung cancer in a subject, wherein the method comprises the step of contacting the subject's blood or serum with the molecule (eg, antibody) that specifically binds to the polypeptide of the present invention, for example, by ELISA.

[0033] The present invention also provides a detection reagent for detecting the risk of a subject suffering from lung cancer, which comprises a molecule (eg, an antibody) that specifically binds to the polypeptide of the present invention.

[0034] The present invention also provides a T cell receptor (TCR) capable of binding to the polypeptide of the present invention or a complex thereof.

[0035] The present invention also provides uses of the polypeptide, fusion polypeptide, complex or tandem polypeptide of the present invention for activating immune cells, such as APC cells or T cells.

[0036] The present invention also provides uses of the polypeptides, fusion polypeptides, complexes, and tandem polypeptides of the present invention, such as for preparing drugs for preventing or treating cancer (eg, lung cancer).

[0037] The present invention also provides a pharmaceutical composition or drug, which contains a pharmaceutically acceptable carrier and the peptide, complex, fusion polypeptide, tandem polypeptide, or isolated cell (eg, APC or T cell) of the present invention.

[0038] Exemplarily, the drug or the pharmaceutical composition is a vaccine.

[0039] The present invention also provides a method for preventing or treating a disease (eg, lung cancer), comprising administering an effective amount of the polypeptide, complex, fusion polypeptide, tandem polypeptide, or cell of the present invention to a subject in need thereof.

[0040] The present invention also provides a cytotoxic T cell (CTL) specifically targeting lung cancer, which comprises co-culturing the APC cells loaded with molecular targets of the present invention with T cells to obtain cytotoxic T cells (CTL) specifically targeting lung cancer.

[0041] The present invention also provides a cell composition comprising the activated APC cells and T cells or CTL cells of the present invention.

[0042] The present invention also provides another cell composition, which comprises the activated APC cells and CIK cells of the present invention.

[0043] Because the activated APC cells of the present invention can present lung cancer-specific molecular targets, they can be used as therapeutic or preventive vaccines against lung cancer. Therefore, the present invention also provides a therapeutic or preventive vaccine against lung cancer, which comprises the peptide, complex, fusion polypeptide, tandem polypeptide, isolated cells (e.g., activated APC cells) of the present invention, and an optional adjuvant, for example, coupled to KLH and / or combined with the immune adjuvant GM-CSF.

[0044] Furthermore, the present invention also provides a method for preventing or treating lung cancer, which comprises administering the vaccine of the present invention into a subject.

[0045] To evade the immune system, cancer cells often undergo genetic mutations and evolve new functions, leading to drug resistance. To prevent cancer cells from "escaping" a single molecular target, it is possible to increase the number of molecular targets to improve efficacy. Based on this, the present invention provides a multi-target solution that combines the present invention's molecular target for lung cancer with other molecular targets for lung cancer.

[0046] Therefore, the present invention provides a fusion polypeptide comprising:

[0047] Contains a DSNS16 polypeptide or an active variant thereof.

[0048] The present invention also provides a tandem polypeptide, illustratively comprising at least two repeating units of a DSNS16 polypeptide or an active variant thereof, for example, 2, 3, 4, 5, 6, 7, 8, or 9 repeating units of a DSNS16 polypeptide or an active variant thereof, connected in series. Adjacent polypeptides in the tandem polypeptide can be directly linked or connected via a linker.

[0049] Furthermore, the present invention also provides a method for stimulating and activating APC cells, which comprises the step of contacting the polypeptide, fusion polypeptide or tandem polypeptide of the present invention with APC cells to be activated, thereby allowing the APC cells to be loaded with the polypeptide or fusion polypeptide.

[0050] The present invention also provides an activated APC cell, which is obtained by contacting and culturing the fusion polypeptide or tandem polypeptide of the present invention with the APC cell to be activated.

[0051] The present invention also provides a cytotoxic T cell (CTL) specifically targeting lung cancer, which comprises co-culturing APC cells loaded with the polypeptide or its fusion polypeptide of the present invention with lymphocytes to obtain multi-target cytotoxic T cells (CTL) specifically targeting lung cancer.

[0052] The present invention also provides a cell composition comprising the activated APC cells and immune cells of the present invention.

[0053] The present invention also provides another cell composition, which comprises the activated APC cells and CIK cells of the present invention.

[0054] Similarly, because the activated APC cells of the present invention can present lung cancer-specific molecular targets, they can be used as therapeutic or preventive vaccines against lung cancer. Therefore, the present invention also provides a therapeutic or preventive vaccine against lung cancer, which comprises multiple molecular targets of the present invention, or comprises activated APC cells.

[0055] Furthermore, the present invention also provides a method for preventing or treating lung cancer, which comprises administering the activated APC cells of the present invention or the cell composition of the present invention into a subject.

[0056] In fact, based on the polypeptides of the present invention and their related complexes, fusion polypeptides, tandem proteins, cells, etc., those skilled in the art can apply them to various aspects. For example, the complexes of the present invention can be used to screen TCRs that bind thereto, and the steps include:

[0057] (i) contacting the candidate TCR molecule with the polypeptide-MHC complex of the present invention;

[0058] (ii) Screening out TCR molecules that bind to the peptide-MHC complex in (i).

[0059] Because the molecular targets for lung cancer in this invention were obtained through extensive screening and statistical analysis, they are highly representative for lung cancer. Therefore, this invention offers significant advantages for the treatment or prevention of lung cancer, including ease of use, a short treatment cycle, and significant therapeutic efficacy.

[0060] Compared to CIK technology, this method boasts high specificity and strong targeting. CTLs or CTLs utilize small peptide antigens screened from a target peptide library to precisely localize tumor cells. These small peptides also meet the antigen presentation requirements of APCs, significantly improving presentation efficiency and enhancing the immune cell's ability to kill tumor cells.

[0061] The present invention successfully screens, identifies and verifies tumor-specific antigens (molecular targets) in the subject's body, synthesizes therapeutic targets in vitro, and co-cultures them with the subject's autologous immune cells. After being reinfused into the subject's body, the tumor cells are killed in a targeted manner, thereby achieving the purpose of treating tumors.

[0062] In a specific embodiment of the present invention, DC cells activated by the molecular target of the present invention have a positive loading rate greater than 90% when detected by the target monoclonal antibody. Furthermore, the activated DC cells highly express CD80 and CD86 and present T lymphocytes, becoming CTLs or CTL cells that specifically recognize lung cancer cells.

[0063] The therapeutic vaccine described in the present invention is co-cultured with the subject's autologous immune cells, directly killing tumor cells through precise positioning and multiple targets, and by acting on the immune system, it enhances the body's immune response, ultimately prolonging the patient's survival and improving the quality of life.

[0064] definition:

[0065] Therapeutic vaccines: biological products that are natural, artificially synthesized, or expressed using genetic recombination technology, which treat or prevent the worsening of diseases by inducing specific immune responses in organisms infected with pathogenic microorganisms or suffering from certain diseases.

[0066] CTL (Cytotoxic T Lymphocyte): Cytotoxic T cells

[0067] DC-SCT (Specific Cluster Target of Dendritic Cell): Specific dendritic cell cluster target

[0068] DC (Dendritic Cell): Dendritic Cell

[0069] CIK (Cytokine-Induced Killer): Cytokine-induced killer cells

[0070] PBMC (Peripheral blood mononuclear cell): peripheral blood mononuclear cells

[0071] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions, and will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments.

[0073] Figure 1 Flow cytometry results: CD8 + TCR + The proportion of cells was significantly higher than that in the negative control group (1.399% vs 0.464%). DETAILED DESCRIPTION

[0074] 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.

[0075] It should be understood that oligopeptides and polypeptides are interchangeable in the present invention, because for those skilled in the art, there is no strict distinction between oligopeptides and polypeptides.

[0076] It is obvious that the present invention encompasses variants of the polypeptide, such as amino acid sequences having 1, 2 or 3 amino acid substitutions, deletions or insertions in any one of SEQ ID NO: 1 or 2.

[0077] Illustratively, the substitution refers to the replacement of an amino acid residue by another amino acid residue at the same position, preferably a replacement between amino acids of the same nature, such as a replacement between hydrophobic amino acids. The inserted amino acid residues can be inserted at any position, and the inserted amino acid residues can also be all or partially adjacent, or the inserted amino acids are not adjacent to each other. Amino acid deletions 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.

[0078] Further illustratively, substitution can occur between any amino acid. Conservative amino acid substitution is preferred. The term "conservative amino acid substitution" refers to that an amino acid residue is substituted by another amino acid residue with a side chain of similar properties. According to the side chain, amino acid residues are divided into multiple families. Examples of side chains include: basic side chains (such as lysine, arginine and histidine), acidic side chains (such as aspartic acid and glutamic acid), uncharged polar side chains (such as asparagine, glutamine, serine, threonine, tyrosine and cysteine), non-polar side chains (such as glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine and tryptophan), β-branched side chains (such as threonine, valine and isoleucine), aliphatic side chains (such as glycine, alanine, valine, leucine, isoleucine, serine and threonine), aromatic side chains (such as tyrosine, phenylalanine and tryptophan), amide side chains (such as asparagine and glutamine) and sulfur-containing side chains (such as cysteine ​​and methionine). Conservative amino acid substitution is preferably a substitution between amino acid residues within the same family. Examples of conservative amino acid substitutions include that glutamic acid residues are substituted for aspartic acid residues, phenylalanine residues are substituted for tyrosine residues, leucine residues are substituted for isoleucine residues, isoleucine residues are substituted for valine residues, alanine residues are substituted for serine residues, and histidine residues are substituted for arginine residues.

[0079] For polypeptide variants, it is possible to verify whether the variant is an active variant. For example, the variant polypeptide can be contacted with APCs (e.g., DCs), and then the APCs are tested for target monoclonal antibodies. If CD80 and CD86 are highly expressed on the APCs, the variant is an active variant.

[0080] The present invention also encompasses tandem polypeptides, for example, comprising two or more repeating units of SEQ ID NO: 1 or an active variant thereof, or at least two repeating units thereof. It should be noted that, in order to present tumor-specific antigens on APCs, the polypeptides of the present invention can be contacted with APCs in the form of tandem polypeptides.

[0081] It is known to those skilled in the art that the polypeptides of the present invention may be post-translationally modified at one or more positions between the amino acid sequences, such as acetylation, phosphorylation, etc. Further, the polypeptides may be artificially modified, for example, by replacing their amino acid residues with amino acid analogs or mimetics. Furthermore, one or more substances, such as amino acids, peptides, and their analogs, may be added to the N-terminus and / or C-terminus of the polypeptide. For example, a histidine tag may be added, or a fusion polypeptide may be formed with a protein. A detectable label may also be bound to the polypeptide. When such a substance is bound to the polypeptide, the substance may be processed, for example, with an enzyme or by intracellular processing, to produce the polypeptide. Such substances may regulate the solubility of the polypeptide, improve the stability of the peptide (e.g., protease resistance), allow the polypeptide to be specifically delivered to the desired tissue or organ, or enhance the uptake of the polypeptide by antigen-presenting cells. Such substances may also be substances that increase the ability of the peptide to induce CTLs, for example, another peptide that activates T cells.

[0082] As is well known to those skilled in the art, tumor antigens are enzymatically broken down into peptide fragments by proteases within antigen-presenting cells. These peptide fragments then bind to major histocompatibility complex (MHC) molecules to form peptide-MHC complexes, which are then presented on the surface of APCs. The peptide-MHC complexes further bind to the surface of lymphocytes, stimulating and activating them to become cytotoxic T cells (CTLs).

[0083] Therefore, the present invention provides a polypeptide-MHC complex comprising a polypeptide or 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. Furthermore, the MHC molecule may be an MHC class III molecule.

[0084] The present invention also provides molecules and cells that bind to the above polypeptide, tandem polypeptide or complex.

[0085] Methods for producing a polypeptide-MHC complex of the present invention are known to those skilled in the art, for example, by binding the polypeptide to an HLA molecule.

[0086] The polypeptide-MHC complex of the present invention can be used to screen or detect molecules bound thereto, such as T cell receptors (TCR).

[0087] In the process of cellular immunity, antigens are usually presented to the cell surface together with MHC complexes. Therefore, the present invention also provides a separated cell, which is capable of presenting the polypeptide or polypeptide-MHC complex of the present invention to its surface. Exemplarily, the cell is an immune cell, such as an APC, such as a DC (dendritic cell), or a B cell, or a T2 cell. Preferably, the cell presenting the polypeptide or polypeptide-MHC complex of the present invention is separated. The cell may not naturally present the complex of the present invention. The cell presenting the polypeptide-MHC complex of the present invention can be used to separate T cells and T cell receptors, and the T cells are activated by the polypeptide or complex and further sorted out, and used for reinfusion into the subject after in vitro proliferation.

[0088] In a specific embodiment, the method for obtaining isolated T cells comprises contacting T cells with a polypeptide or polypeptide-MHC complex of the present invention or a cell presenting the same. Using a labeled antibody, the activated T cells can be sorted by flow cytometry (FACS), and the sorted cells can be proliferated and cultured in vitro.

[0089] The present invention also provides nucleic acid molecules comprising nucleic acid molecules encoding polypeptides, polypeptide variants, and tandem polypeptides of the present invention, such as cDNAs. The nucleic acids can be synthesized by synthetic methods known in the art. Due to the degeneracy of the genetic code, it will be understood by those skilled in the art that different nucleic acid sequences can encode the same amino acid sequence.

[0090] Based on the above nucleic acid, the present invention also provides a vector. The vector comprises the nucleic acid sequence of the present invention. Exemplarily, the vector is an expression vector, such as a plasmid, a phage, a virus, etc. Suitable phage and viral vectors include lambda phage, EMBL phage, simian virus, bovine wart virus, Epstein-Barr virus, oncolytic virus, mouse sarcoma virus, murine mammary cancer virus, lentivirus, etc.

[0091] The present invention also provides a host cell comprising the vector or nucleic acid of the present invention, such a cell may be a mammalian cell, and expressing the peptide of the present invention.

[0092] The present invention also provides a molecule (e.g., TCR and antibody) that can be used as an immunotherapeutic agent or a diagnostic agent. The molecule can bind to a peptide or a complex formed by a peptide and an MHC molecule.

[0093] The TCR of the present invention can be in any form known in the art, for example, a heterodimer.

[0094] In the present invention, "antibody" refers to immunoglobulin molecules and immunologically active parts of immunoglobulin molecules, i.e., molecules containing specific binding sites, which can be all-natural, or partially artificially synthesized, or all artificially synthesized. The term "antibody" includes antibody fragments, derivatives thereof, functional equivalents, and homologous antibodies, humanized antibodies, and the antibody fragment includes an immunoglobulin binding region, which is an antibody binding region or is homologous to the antibody binding region. It can be all-natural, or partially artificially synthesized, or all artificially synthesized. A humanized antibody can be a modified antibody that contains the variable region of a non-human antibody (e.g., mouse) and the constant region of a human antibody.

[0095] Examples of antibodies include immunoglobulin isotypes (e.g., IgG, IgE, IgM, IgD, and IgA) and their subtypes; fragments including antigen-binding regions, such as Fab, scFv, Fv, dAb, Fd; and double-chain antibodies. Antibodies can be polyclonal or monoclonal, preferably monoclonal.

[0096] In the present invention, TCR and antibodies may be present on the surface of a cell, such as a T cell. Therefore, the present invention also provides an isolated T cell, which binds to the complex of the present invention.

[0097] The present invention also provides uses of the polypeptide and its variants, fusion polypeptides, polypeptide-MHC complexes, tandem polypeptides, cells, and binding molecules, for example, for preparing drugs for preventing or treating cancer.

[0098] 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.

[0099] The pharmaceutical composition of the present invention may be in a dosage form suitable for any appropriate route of administration, such as injection (including subcutaneous, intramuscular, intraperitoneal, or intravenous injection), inhalation, oral administration, nasal administration, or anal administration. The composition may be prepared by any method known in the pharmaceutical art, for example, by mixing the active ingredient with a carrier or excipient under sterile conditions.

[0100] The polypeptides, fusion polypeptides, tandem polypeptides, complexes, or cells of the present invention can be provided in the form of a vaccine composition. The vaccine composition can be used to treat or prevent cancer, wherein the vaccine composition can further contain an adjuvant, such as an inactivated vaccine, an attenuated vaccine, a viral vector vaccine, an RNA vaccine, or the like.

[0101] In the present invention, antigen presenting cells (APCs) are, for example, selected from monocytes, monocyte-derived cells, macrophages, and dendritic cells (DCs), preferably DCs.

[0102] The antigen presenting cells are preferably dendritic cells (DCs).

[0103] The major histocompatibility complex (MHC) is a gene complex that encodes human leukocyte antigen (HLA) genes. HLA genes are expressed as protein heterodimers on the surface of human cells that are displayed to circulating T cells. HLA genes are highly polymorphic, allowing them to fine-tune the adaptive immune system.

[0104] There are three classes of MHC molecules: MHC I, MHC II, and MHC III. MHC class I molecules are composed of an alpha heavy chain and beta-2-microglobulin; MHC class II molecules are composed of one alpha and one beta chain. MHC molecules contain a binding groove for non-covalent interactions with peptides; MHC III primarily encodes complement components, such as tumor necrosis factor (TNF) and heat shock protein 70 (HSP70).

[0105] MHC class I molecules are expressed on most nucleated cells and primarily present endogenous proteins, defective ribosomal products (DRIPs), and peptides generated by cleavage of larger peptides. However, peptides of exogenous origin are also frequently found on MHC class I molecules. MHC class II molecules are primarily found on antigen-presenting cells (APCs) and primarily present peptides of exogenous or transmembrane proteins that are taken up by APCs during endocytosis and subsequently processed.

[0106] The complex of peptide and MHC class I is recognized by CD8-positive T cells bearing the corresponding T cell receptor (TCR), while the complex of peptide and MHC class II molecule is recognized by CD4-positive helper T cells bearing the corresponding TCR.

[0107] CD4 + T helper cells play an important role in inducing and maintaining effective CD8-positive cytotoxic T cell responses. At the tumor site, T helper cells maintain a cytokine environment that is favorable to cytotoxic T cells (CTLs) and attract effector cells such as CTLs, natural killer (NK) cells, macrophages, and granulocytes.

[0108] In the absence of inflammation, the expression of MHC class II molecules is primarily restricted to cells of the immune system, particularly antigen-presenting cells (APCs). However, MHC class II molecules have been found to be expressed in tumor cells from cancer patients.

[0109] The peptides or variants of the present invention may be further modified to improve stability and / or binding to MHC molecules, thereby eliciting a stronger immune response. Methods for improving peptide sequences are well known in the art, for example, the introduction of trans-peptide bonds and non-peptide bonds.

[0110] The term "isolated" means that a material is removed from its original environment (e.g., its natural environment if naturally occurring). For example, a naturally occurring nucleotide or polypeptide in a living animal is not isolated, but a nucleotide or polypeptide separated from some or all coexisting materials in the natural system is isolated. Such polynucleotides may be part of a vector and / or such polynucleotides and polypeptides may be part of a composition and still be isolated because the vector or composition is not part of its natural environment.

[0111] The present invention screens and identifies positive molecular targets, namely tumor-specific antigens, in lung cancer subjects from peripheral blood. These positive molecular targets are co-cultured with the subject's immune cells in vitro and then infused back into the subject, thereby correspondingly killing tumor cells in the body.

[0112] Example 1: Screening for tumor-specific molecular targets from lung cancer tissue

[0113] To screen specific molecular targets for lung cancer, the following operations were performed.

[0114] (1) Collect samples containing HLA-peptide complexes, perform tissue homogenization, primary purification, enrichment, purification, and collection:

[0115] Tissue homogenate: If the sample is tumor tissue or tumor cell line, the sample needs to be homogenized and centrifuged to collect the supernatant.

[0116] Initial purification: add an equal volume of enrichment solution to the supernatant or serum, mix well, and collect the supernatant by centrifugation;

[0117] Enrichment and purification: The supernatant was added to the Oasis Prime HLB SPE column for enrichment and purification;

[0118] Elution collection: After the filtrate has completely passed through, the target peptide adsorbed on the elution column is eluted and about 20 μL of the eluate is collected.

[0119] (2) The treated peptide solution is subjected to liquid phase mass spectrometry detection: After the sample enters the high performance liquid chromatography, it is effectively separated by the chromatographic column, so that different substances enter the ion source of the mass spectrometer in chronological order. After the detected substances enter the ion source, they are ionized and fragmented to form various charged molecules that will reach the mass spectrometer TOF detector in sequence according to their mass-to-charge ratios. The detector signal is analyzed to obtain specific data such as the molecular weight of each molecule. By comparing with the target database, the specific molecular target ALSELTQGV (DSNS16 peptide) for lung cancer was finally identified.

[0120] (3) The target ALSELTQGV was synthesized and purified using a peptide solid phase synthesis platform to obtain a peptide freeze-dried powder with a purity greater than or equal to 95%.

[0121] Example 2: CTL cell preparation

[0122] Collect 80-120 ml of peripheral blood and separate the mononuclear cells (PBMC) from the blood by density gradient centrifugation using lymphocyte separation medium. Based on the PBMC count results, adjust the cell count to about 1×10 7 cells / ml, inoculated into culture flasks, inoculating 10-15 ml of culture medium per flask, and incubated in a carbon dioxide incubator at 37°C; 5% CO2; incubated for 30 minutes.

[0123] Dendritic cells are prepared by collecting adherent cells from PBMC culture flasks. At the same time, T cells are separated from PBMC of the same subject using Ficoll-Paque density gradient centrifugation or magnetic cell sorting. Dendritic cells are treated in peptide culture medium, mixed with T cells, and the cultured T cells are recovered. The activity of target peptide-specific T cells can be detected by TCR + The proportion of T cells and the ability of T cells to kill target cells expressing the target peptide are comprehensively evaluated.

[0124] Specifically, the collected dendritic cells were added to a DC culture medium containing the target polypeptide (at a concentration of 5 μg / mL) and cultured in an incubator at 37°C and 5% CO2. Subsequently, the activated DC cells were harvested and mixed with suspended T cells to obtain cytotoxic T cells (CTL cells). The CTL cells were then further cultured and proliferated, and the proportion of TCR+ T cells and their killing effect on target cells were detected on the fifth day of culture to evaluate the functional activity of the CTL cells.

[0125] Example 3: Detection of the proportion of TCR+T cells in CTL

[0126] According to the kit instructions, DSNS16 polypeptide tetramer was prepared for use.

[0127] The CTL cells prepared in Example 2 were collected, washed, counted, and grouped as follows:

[0128] (1) Blank group: normal sample, no antibody added, used for voltage adjustment;

[0129] (2) Negative control group: negative tetramers were added to determine the position of the TCR+ positive gate;

[0130] (3) Test sample group: tetramer corresponding to DSNS16 polypeptide was added.

[0131] Subsequently, 5-10 μL of tetramer was added to each group, and the negative control group was added with an equal volume of tetramer replaced by 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 including CD8 was added to the other groups and incubated at room temperature for 15-30 minutes. After incubation, the mixture was centrifuged (1500 rpm, 5 min), the supernatant was discarded, and the mixture was resuspended with physiological saline and then detected by flow cytometry. The results are shown in Figure 2. Figure 1 .

[0132] from Figure 1 As can be seen, CD8 + TCR + The percentage of cells was significantly higher than that of the negative control group (1.399% vs 0.464%). This indicates that the peptide can effectively activate the corresponding TCR + T cells.

[0133] Example 4: Activation of DC cells by molecular targets

[0134] Peripheral blood was collected from humans, and PBMCs were isolated. The cells were cultured in RPM1640 medium containing autologous plasma at 37°C and 5% CO2 for 30 minutes. Human peripheral blood dendritic cells (DCs) were isolated by adherence isolation, and non-adherent cells were removed. In the experimental group, DCs were cultured in fresh CellGenix dendritic cell culture medium containing the target peptide at 37°C and 5% CO2. After 5 days of culture, the harvested cells were analyzed by flow cytometry.

[0135] The results were validated against single peptides (DSNS16 peptide, 5 μg / mL) and fusion peptides (DSNS16-GGGS-DSNS16 and DSNS16-GGGS-DSNS16-GGGS-DSNS16, 5 μg / mL). A control group was also established. DCs were cultured in fresh CellGenix dendritic cell culture medium without the target peptide at 37°C and 5% CO2. After 5 days of culture, the cells were harvested and analyzed by flow cytometry. GGGS is the tetrapeptide linker. DSNS16-GGGS-DSNS16 was designated as fusion peptide 1, and DSNS16-GGGS-DSNS16-GGGS-DSNS16 was designated as fusion peptide 2.

[0136] The results showed that after adding the target, the activation rate of DC cells loaded increased, and they highly expressed CD80 and CD86. DC cells without target (control) expressed low CD80 and CD86 (see Table 1 below).

[0137] Table 1: Effects of each molecular target on DC activation

[0138] name CD80 CD86 DSNS16 95.6 97.4 DSNS16-GGGS-DSNS16 96.6 94.9 DSNS16-GGGS-DSNS16-GGGS-DSNS16 96.4 96.7 Negative control 52.8 75.5

[0139] Example 5

[0140] Human peripheral blood DC cells were isolated and cultured using the adherence method. PBMCs were cultured statically in RPM1640 culture medium containing autologous plasma at 37°C and 5% CO2. Non-adherent cells were removed (for subsequent CTL cell preparation) and adherent cells were cultured in fresh CellGenix dendritic cell culture medium containing TNF-a, GM-CSF, and IL-4 at 37°C and 5% CO2. Fluids and cytokine supplementation were performed every 2-3 days.

[0141] DSNS16 peptide, fusion peptide 1, and fusion peptide 2 were added to the DC cell culture system of the experimental group, with each peptide concentration being 25 μg / mL. The cells were cultured in a 37°C, 5% CO2 incubator. Subsequently, the activated DC cells were harvested and mixed with suspended T cells to obtain cytotoxic T cells (CTL cells).

[0142] After culturing peripheral blood DCs for 5 days, they were mixed at a ratio of DC:T = 1:8 and cultured for ≥ 3 days before performing lymphocyte function tests, including:

[0143] Control group 1: DC cells (without peptide stimulation) + T cells co-cultured (DC-T);

[0144] Control group 2: simple T cells,

[0145] Among them, (1) the cell proliferation was detected by CCK-8 assay at 0 days, 2 days, 4 days, and 6 days respectively; (2) the killing effect of different groups of cells on tumor cell line (NCI-H2228) was detected by CCK8 assay at 18 hours and 24 hours respectively (target-effect ratio 1:30). At the same time, single effector cell group and single target cell group were set up, and 3 replicates were set up in each group.

[0146] The experimental results are as follows:

[0147] 1. The CCK-8 assay was used to measure the effects of DSNS16 polypeptide, fusion polypeptide 1, and fusion polypeptide 2 on lymphocyte proliferation in vitro, with measurements taken on days 0, 2, 4, and 6. The results showed significant proliferation in both the experimental and control groups. There was no significant difference on days 0 and 2, but on days 4 and 6, the proliferation of the DSNS16 polypeptide, fusion polypeptide 1, and fusion polypeptide 2 groups was significantly greater (p<0.001) than that of control groups 1 and 2, indicating that the DSNS16 polypeptide, fusion polypeptide 1, and fusion polypeptide 2 groups can effectively stimulate lymphocyte proliferation.

[0148] 2. At a target / effect ratio of 1:30, the killing efficiency results of different groups showed that the experimental group, control group 1 and control group 2 all showed killing effects at 30 hours, among which the DSNS16 polypeptide group (78.5%), fusion polypeptide group 1 (79.4%) and fusion polypeptide group 2 (77.3%) showed higher efficiency than the other two control groups (45.2-50.0%).

[0149] Example 6: Killing of tumor cells by fusion polypeptide

[0150] Five lung adenocarcinoma cell lines (NCI-H2228, NCI-H522, NCI-H1650, NCI-H2347, and NCI-H1703) were selected as target cells to evaluate the in vitro cytotoxic activity of the CTL prepared in Example 5 over a 24-h period, with effector-target ratios of 40:1, 20:1, and 10:1.

[0151] The results showed that CTL cells from the DSNS16 peptide group, fusion peptide group 1, and fusion peptide group 2 exhibited cytotoxicity against all five lung adenocarcinoma cell lines. Specifically, at effector-target ratios of 40:1, 20:1, and 10:1, the effector cells exhibited cytotoxicity against all five target cell lines, with the most significant cytotoxicity against NCI-H2228.

[0152] 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 in the scope of protection of the present invention.

Claims

1. A polypeptide or a fusion polypeptide thereof comprising the amino acid sequence of ALSELTQGV (SEQ ID NO: 1) or an active variant or active fragment thereof: Preferably, the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 1 or an active variant thereof, more preferably, the fusion polypeptide comprises the amino acid sequence of SEQ ID NO:

1.

2. A complex comprising HLA and the polypeptide according to claim 1 or a fusion polypeptide thereof.

3. A method for stimulating and activating APC cells in vitro, comprising the step of contacting the polypeptide or fusion polypeptide thereof according to claim 1 or the complex according to claim 2 with APC cells to be activated, thereby causing the APC cells to be loaded with the polypeptide.

4. An activated APC cell, which is obtained by contacting and culturing the APC cell to be activated with the polypeptide or fusion polypeptide thereof according to claim 1 or the complex according to claim 2.

5. A cytotoxic T cell (CTL) specifically targeting lung cancer, comprising co-culturing the APC cell of claim 4 with lymphocytes to obtain the cytotoxic T cell (CTL) specifically targeting lung cancer. A cell composition comprising the APC cells and CTL cells according to claim 4. A cell composition comprising the APC cells and CIK cells according to claim 4 .

8. A therapeutic or preventive vaccine against cancer, comprising the polypeptide or fusion polypeptide thereof according to claim 1 or the complex according to claim 4, or the cell or cell composition according to any one of claims 6 to 7.

9. A molecule that specifically binds to the polypeptide of claim 1 or its fusion polypeptide or active variant thereof, wherein the binding molecule is an antibody.

10. Use of the cell or cell composition according to any one of claims 6 to 7 in the preparation of a medicament for preventing or treating lung cancer.

11. Use of the polypeptide or fusion polypeptide thereof according to claim 1 or the complex according to claim 2 in preparing CTLs targeting lung cancer.

12. A medicament comprising the polypeptide or fusion polypeptide thereof according to claim 1, the complex according to claim 2, or the cell or cell composition according to any one of claims 6 to 7.

13. Use of the specific binding antibody of claim 9 in the preparation of a detection reagent for detecting the risk of a subject suffering from lung cancer, wherein the detection comprises the step of contacting the subject's blood or serum with the specific binding antibody of claim 9.

14. A detection reagent for detecting a subject's risk of having lung cancer, comprising the specific binding antibody according to claim 9.

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