WO2023184861A1 - Hpv epitope and identification method therefor, and application thereof - Google Patents

Hpv epitope and identification method therefor, and application thereof Download PDF

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WO2023184861A1
WO2023184861A1 PCT/CN2022/116872 CN2022116872W WO2023184861A1 WO 2023184861 A1 WO2023184861 A1 WO 2023184861A1 CN 2022116872 W CN2022116872 W CN 2022116872W WO 2023184861 A1 WO2023184861 A1 WO 2023184861A1
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cancer
hpv
cells
mutant
antigen
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Chinese (zh)
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李波
周璀
张攀
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深圳吉诺因生物科技有限公司
武汉华大吉诺因生物科技有限公司
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
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    • C12N5/0645—Macrophages, e.g. Kuepfer cells in the liver; Monocytes
    • G—PHYSICS
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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
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    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5154—Antigen presenting cells [APCs], e.g. dendritic cells or macrophages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
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    • A61K2039/5156—Animal cells expressing foreign proteins
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    • C12N2510/00—Genetically modified cells
    • C12N2510/02—Cells for production

Definitions

  • the present invention relates to the field of biotechnology. Specifically, the present invention relates to HPV antigenic epitopes and their identification methods and applications. More specifically, the present invention relates to a method for identifying HPV antigenic epitopes, isolated peptides, mutants, and expression vectors. , the use of antigen-presenting cells, immune effector cells, reagents in the preparation of kits, the use of kits, the isolated peptides or expression vectors or antigen-presenting cells or immune cells in the preparation of drugs, treatment and/or prevention of diseases, Drugs, vaccines and diagnostic systems, methods of diagnosis, treatment and/or prevention of disease.
  • Human papillomavirus (HPV) infection is the cause of almost all invasive cervical cancers and some anogenital malignancies and oral cancers. Taking cervical cancer as an example, persistent infection with human papillomavirus is the main causative factor of cervical cancer. It has now become the fourth most common female cancer in the world, with a very serious disease burden.
  • HPV tumor immunotherapy is mainly based on preventive HPV vaccines.
  • the preventive vaccines that have been launched are mainly HPV 2-valent, 4-valent and 9-valent vaccines, which can be used to target specific types of HPV high-risk and low-risk types. immune protection.
  • preventive vaccines are mainly for prevention and cannot clear existing infections. They cannot cure infected HPV patients.
  • the development and clinical application of HPV therapeutic vaccines have become urgent.
  • HPV therapeutic vaccines stimulate cell-mediated immune responses by generating neutralizing antibodies against HPV virus particles, thereby targeting specific targets to kill HPV-infected cells.
  • HPV virus is a double-stranded spherical DNA virus of the genus Human Papillomavirus A, with a simple structure and no envelope. Its genome encodes six early regulatory proteins (E1, E2, E4, E5, E6, E7) and two late structural proteins (L1 and L2). Early gene-encoded proteins are responsible for viral DNA replication, transcription, carcinogenesis, and transformation.
  • the target antigen genes that are usually targeted when designing HPV therapeutic vaccines are the viral early proteins E6 and E7. E6 and E7 proteins have long been considered the most critical oncogenic proteins in HPV-related research.
  • the E6 and E7 expression proteins derived from virus proliferation can promote the rapid occurrence and development of late-stage tumors by affecting the p53 tumor suppressor gene and PRB retinoblastoma protein.
  • both protein sequences are well protected across a wide range of HPV subtypes and can be used as very attractive and applicable targets in exploratory research on HPV therapeutic vaccines.
  • HLA human leukocyte antigen
  • HLA antigens depends on the ⁇ heavy chain of the HLA molecule, which is encoded by the HLA-A, B, and C loci, and different genotypes of HLA-A, B, and C are derived from this.
  • the main function of the HLA class I complex is to process the gene products of HLA class I derived from the host or foreign substances such as viral expressed proteins through intracellular endogenous mechanisms and then translate and cleave them to form key therapeutic antigen peptides.
  • MHC complexes present antigenic peptides to CD8 + cytotoxic T cells to initiate and regulate specific immune responses.
  • the antigenic determinants related to the antigenic polypeptide sequence are called HLA class I restricted antigenic epitopes.
  • HLA-A0201 genotype Due to the high degree of polymorphism of HLA genes, the different sources of HLA-A, B, and C genes correspond to the different types of HLA-A, B, and C-restricted antigen epitopes. In the study of patients with HPV-related viral infections, HLA -A0201 genotype has the highest proportion. At the same time, there are more than 200 known types of HPV viruses. Based on their biological characteristics and carcinogenic potential, they are usually divided into high risk and low risk. Low-risk HPV types such as 6, 11, 42, 43, and 44 often cause benign lesions such as external genital warts. High-risk HPV types include 16, 18, 31, 33, 35, 39, 45, 51, 52, and 56. , 58, 59, 68, etc.
  • HPV16 is closely related to cervical cancer and intraepithelial lesions.
  • HPV16 accounts for the highest proportion among all types of patients, and treatment is more urgent. Therefore, identification of HLA-I class-restricted polypeptide epitopes of HPV16 viruses is of great significance for the preparation of therapeutic vaccines.
  • HPV therapeutic vaccines are a research hotspot in the field of immunotherapy, in which obtaining effective HPV viral epitopes is extremely critical.
  • the complex is subjected to immunoaffinity capture, and the antigen peptides in the complex are solid-phase extracted, and the obtained antigen peptides are analyzed using ultra-high performance liquid chromatography-mass spectrometry technology to identify the HPV virus epitope.
  • the present invention proposes a method for identifying HPV antigenic epitopes.
  • the following steps are included: 1) Combine HLA-I class antibodies and carriers at a volume mass ratio of 1mL:5mg-1mL:2mg to obtain an immunoaffinity column; 2) Conduct HPV-positive cells Lysis treatment; pass the cell lysate obtained in step 2) through a column, and the column is the immunoaffinity column to obtain an HLA complex containing HPV antigen peptides; 3) pass the HLA complex containing HPV antigen peptides HLA complexes were subjected to solid phase extraction to obtain peptide fragments.
  • the inventors screened and optimized the method for identifying HPV epitopes.
  • the HLA-I class antibody is combined with the carrier at a volume-to-mass ratio of 1mL:5mg-1mL:2mg, the binding rate of the antibody is higher, and the antibody has a higher binding rate.
  • the obtained HLA complex containing HPV antigen peptides has a high identification throughput when subjected to solid phase extraction.
  • the method of the embodiment of the present invention significantly improves the probability of obtaining HPV antigen peptides that effectively bind to the HLA class I molecules and significantly shortens the identification time. identification time.
  • the invention provides an isolated peptide. According to embodiments of the present invention, it includes at least one of the following: 1) amino acids 65-72 of HPV E6 protein; 2) amino acids 4-11 of HPV E7 protein; 3) amino acids 75-82 of HPV E7 protein ; 4) Amino acids 75-83 of HPV E7 protein; 5) Amino acids 77-86 of HPV E7 protein; 6) Amino acids 77-90 of HPV E7 protein; and 7) Amino acids 79-88 of HPV E7 protein.
  • the isolated peptide according to the embodiment of the present invention is obtained by expression and translation of the HPV E6 or E7 early coding region.
  • the isolated peptide can be presented by HLA class I molecules, recognized by CTL cells or TIL cells, and can then be expressed by HLA - Presenting cells of class I molecules are presented to CTL or TIL cells to activate specific T cell immunity, constituting the physiological target of the immune response of HPV-positive tumors, performing high-sensitivity and specific detection for the prevention and treatment of HPV-related diseases is of great value.
  • the present invention proposes an HPV antigenic epitope.
  • it includes at least one of the following: 1) amino acids 65-72 of HPV E6 protein; 2) amino acids 4-11 of HPV E7 protein; 3) amino acids 75-82 of HPV E7 protein ; 4) Amino acids 75-83 of HPV E7 protein; 5) Amino acids 77-86 of HPV E7 protein; 6) Amino acids 77-90 of HPV E7 protein; and 7) Amino acids 79-88 of HPV E7 protein.
  • the HPV antigenic epitope according to the embodiment of the present invention is obtained by expression and translation of the HPV E6 or E7 early coding region.
  • HPV antigenic epitope can be presented by HLA-I class molecules, recognized by CTL or TIL cells, and then can be expressed Presenting cells of HLA-I molecules are presented to CTL or TIL cells to activate specific T cell immunity, which constitutes the physiological target of the immune response of HPV-positive tumors. Highly sensitive and specific detection is required for the prevention and treatment of HPV-related diseases. It is of great value for treatment.
  • the invention provides a mutant.
  • the mutant compared to wild-type HPV E6 protein, the mutant has at least one mutation site except for the anchor position.
  • the amino acids at positions 2 and 9 (for 8 peptides, position 8) of the obtained HPV antigenic epitope are the anchor positions of HLA, and other amino acids in the epitope are substituted.
  • CTL or TIL cells activate specific T cell immunity, which constitutes the physiological target of the immune response of HPV-positive tumors. Highly sensitive and specific detection is of great value for the prevention and treatment of HPV-related diseases.
  • the invention provides a mutant.
  • the mutant compared to wild-type HPV E7 protein, the mutant has at least one mutation site except for the anchor position.
  • the amino acids at positions 2 and 9 (for 8 peptides, position 8) of the obtained HPV antigenic epitope are the anchor positions of HLA, and other amino acids in the epitope are substituted.
  • CTL or TIL cells activate specific T cell immunity, which constitutes the physiological target of the immune response of HPV-positive tumors. Highly sensitive and specific detection is of great value for the prevention and treatment of HPV-related diseases.
  • the invention provides a nucleic acid molecule.
  • the nucleic acid molecule encodes the isolated peptide described in the second aspect, the HPV epitope described in the third aspect, or the mutant described in the fourth or fifth aspect.
  • the isolated peptides, HPV epitopes or mutants encoded by the nucleic acid molecules according to the embodiments of the present invention can be presented by HLA class I molecules and recognized by CTL or TIL cells, that is, presented by presentation cells expressing HLA class I molecules.
  • CTL or TIL cells to activate specific T cell immunity, which constitutes the physiological target of the immune response of HPV-positive tumors. Highly sensitive and specific detection is of great value for the prevention and treatment of HPV-related diseases.
  • the present invention provides an expression vector.
  • nucleic acids carrying the isolated peptides described in the second aspect or the HPV epitopes described in the third aspect or the mutants described in the fourth or fifth aspects or the sixth aspect are nucleic acid molecules.
  • the expression vector may include optional control sequences operably linked to the nucleic acid molecule. Wherein, the control sequence is one or more control sequences that can direct the expression of the polypeptide in the host.
  • the expression vector proposed in the embodiment of the present invention can efficiently express the isolated peptide, HPV epitope or mutant in a suitable host cell, and can be effectively used to treat tumors, especially the simultaneous expression of HLA-I class molecules and Specific treatment or prevention of tumors with the above isolated peptides or HPV epitopes.
  • the present invention provides a recombinant cell.
  • the aforementioned nucleic acid molecules, expression vectors, isolated peptides, HPV epitopes or mutants are carried.
  • the recombinant cells are obtained by transfection or transformation of the expression vector.
  • the host cells can efficiently express the above-mentioned isolated peptides, HPV epitopes or mutants under appropriate conditions, and the recombinant cells can be effectively used to treat tumors, especially those that simultaneously express HLA-I class. Molecules and the above isolated peptides or HPV epitopes for specific treatment or prevention of tumors.
  • the present invention provides an antigen-presenting cell.
  • the cells may present isolated peptides, HPV epitopes or mutants as described above.
  • antigen-presenting cells presenting the aforementioned isolated peptides, HPV epitopes or mutants can effectively induce immunity in patients against tumor-specific antigens-the aforementioned isolated peptides/HPV epitopes/mutants. reaction, thereby activating the specific killing function of CTL.
  • the antigen-presenting cells proposed in the embodiments of the present invention have significant efficacy in treating tumors expressing the above-mentioned isolated peptides, HPV epitopes or mutants, and their therapeutic effects are significant and safe. high.
  • the present invention provides an immune effector cell.
  • the immune effector cells can recognize the polypeptides, HPV epitopes, and mutants described above, or recognize the polypeptides or HPV epitopes or mutants that are presented on the cell surface.
  • Antigen presenting cells According to embodiments of the present invention, the immune effector cells can specifically kill tumor cells that co-express HLA class I molecules and the above-mentioned isolated peptides, HPV epitopes or mutants.
  • the present invention proposes the use of reagents for detecting the previously described isolated peptides, HPV epitopes or mutants in the preparation of kits.
  • the kit is used to diagnose HPV or detect the therapeutic effect of HPV.
  • the reagent can accurately detect the isolated peptide, HPV epitope or mutant, for example, detect whether a biological sample contains the isolated peptide, HPV epitope or mutant, because the isolated peptide , HPV antigenic epitopes or mutants are highly expressed in HPV-infected tissues.
  • a kit containing the reagent can accurately diagnose whether the individual from whom the biological sample is derived is infected by HPV, and further, whether it is an individual at high risk for HPV.
  • the test kit can be used to detect the changes of HPV during the treatment process, such as aggravation, slowing down or cure, etc.
  • the present invention provides a kit.
  • reagents suitable for detecting isolated peptides, HPV epitopes or mutants as described above are included.
  • the reagent can accurately detect the isolated peptide, HPV epitope or mutant, for example, detect whether a biological sample contains the isolated peptide, HPV epitope or mutant, because the isolated peptide , HPV antigenic epitopes or mutants are highly expressed in HPV-infected tissues. Therefore, a kit containing the reagent can accurately diagnose whether the individual from whom the biological sample is derived is infected by HPV, and further, whether it is an individual at high risk for HPV. In the same way, during the treatment process of an individual infected by HPV, the test kit can be used to detect the changes of HPV during the treatment process, such as aggravation, slowing down or cure, etc.
  • the present invention proposes the use of the previously described isolated peptides, HPV epitopes, mutants, nucleic acid molecules, expression vectors, recombinant cells, antigen-presenting cells or immune effector cells in the preparation of medicines. use.
  • the medicine is used to treat or prevent HPV-related diseases.
  • the isolated peptide, HPV epitope, mutant, nucleic acid molecule encoding the isolated peptide or HPV epitope or mutant, expression vector, recombinant cell, antigen presenting cell or immune effector cell All can be effectively used for the specific treatment or prevention of tumors, especially tumors that express both HLA class I molecules and the above-mentioned isolated peptides, HPV epitopes or mutants. Therefore, drugs containing some or all of the above substances are also It has a significant effect of treating or preventing tumors expressing HLA class I molecules and the isolated peptide, HPV antigen epitope or mutant, and has higher safety and smaller side effects.
  • the invention provides a medicament.
  • the aforementioned isolated peptides, HPV epitopes, mutants, nucleic acid molecules, expression vectors, recombinant cells, antigen-presenting cells or immune effector cells are included.
  • the isolated peptide, HPV epitope, mutant, nucleic acid molecule encoding the isolated peptide or HPV epitope or mutant, expression vector, recombinant cell, antigen presenting cell or immune effector cell All can be effectively used for the specific treatment or prevention of tumors, especially tumors that express both HLA class I molecules and the above-mentioned isolated peptides, HPV epitopes or mutants. Therefore, drugs containing some or all of the above substances are also It has a significant effect of treating or preventing tumors expressing HLA class I molecules and the isolated peptide, HPV antigen epitope or mutant, and has higher safety and smaller side effects.
  • the invention provides a vaccine.
  • the aforementioned isolated peptides, HPV epitopes, mutants, nucleic acid molecules, expression vectors or antigen-presenting cells are included.
  • the nucleic acid molecules, expression vectors or recombinant cells of the embodiments of the present invention express the aforementioned isolated peptides, HPV epitopes or mutants under appropriate conditions, and the antigen-presenting cells can express the isolated peptides.
  • HPV antigenic epitopes or mutants when combined with HLA-I class molecules, are presented by antigen-presenting cells, allowing them to be recognized by CTL or TIL cells, that is, they are presented to antigen-presenting cells expressing HLA-I class molecules.
  • CTL or TIL cells activate specific T cell immunity. Therefore, the vaccine proposed in the embodiments of the present invention has a significant effect in treating or preventing tumors expressing HLA class I molecules and the isolated peptides, HPV epitopes or mutants, and is safer and has fewer side effects. .
  • the present invention provides a method for treating and/or preventing HPV-related diseases.
  • the subject is administered the aforementioned isolated peptides, HPV epitopes, mutants, nucleic acid molecules, expression vectors, recombinant cells, antigen-presenting cells, immune effector cells, drugs or vaccines.
  • the treatment and/or prevention methods proposed in the embodiments of the present invention including administering any effective amount of the aforementioned isolated peptides, mutants and other related substances, can effectively treat or prevent the expression of HLA- Class I molecules and the isolated peptides or HPV epitopes of tumors.
  • the present invention proposes the use of the previously described isolated peptides, HPV epitopes, mutants, nucleic acid molecules, expression vectors, and recombinant cells in the preparation of kits.
  • the kit is used to detect HLA.
  • the isolated peptides, HPV epitopes, mutants and materials that can indirectly obtain the isolated peptides, HPV epitopes or mutants according to the embodiments of the present invention can be combined with HLA. Therefore, the above materials can be used to prepare A kit for effectively detecting HLA.
  • the kit can accurately qualitatively or quantitatively detect HLA in biological samples. Furthermore, it can also detect the HLA level in an individual to determine the status of the individual, such as the individual's status. HLA levels are significantly lower or higher than normal levels.
  • the present invention provides a kit for detecting HLA.
  • the above-mentioned isolated peptides, HPV epitopes, mutants, nucleic acid molecules, expression vectors, and recombinant cells are included.
  • isolated peptides, HPV epitopes, mutants, and substances that can indirectly obtain the isolated peptides, HPV epitopes, or mutants can bind to HLA. Therefore, a kit containing the above substances
  • the HLA of biological samples can be accurately detected qualitatively or quantitatively.
  • the HLA level in an individual can also be detected to determine the status of the individual. For example, the HLA level of the individual is significantly lower or higher than the normal level.
  • the present invention provides a method for diagnosing whether a subject suffers from HPV-related diseases. According to an embodiment of the present invention, it includes the step of detecting whether the biological sample derived from the subject carries the aforementioned isolated peptide, HPV epitope, mutant, or nucleic acid molecule. As mentioned above, the isolated peptides, HPV epitopes, mutants and nucleic acid molecules are present in individuals infected with HPV. Therefore, by detecting whether biological samples derived from subjects carry the substances, it is possible to Effectively diagnose whether subjects suffer from or are susceptible to HPV-related diseases.
  • a diagnostic system in a twentieth aspect of the invention, it includes: a peptide detection device, the peptide detection device is used to detect whether a biological sample derived from a subject carries the previously described isolated peptide, HPV epitope or mutant; a diagnostic result determination device , the diagnostic result determination device is connected to the peptide detection device, and is used to determine whether the patient suffers from a tumor based on whether the biological sample carries the isolated peptide, HPV epitope or mutant.
  • the isolated peptide, HPV epitope or mutant exists in a subject infected by HPV, and the diagnostic system according to the embodiment of the present invention can detect whether the biological sample carries the isolated peptide. , HPV antigen epitope or mutant, therefore, the diagnostic system can accurately determine whether the subject from which the biological sample is derived is a tumor patient.
  • the present invention proposes the use of the previously described isolated peptides, mutants, expression vectors, antigen-presenting cells, immune effector cells, drugs or vaccines in the treatment and/or prevention of HPV-related diseases. use.
  • the isolated peptide and its mutants can effectively inhibit tumor growth. Therefore, the isolated peptide or mutant can express or contain the isolated peptide or mutant. Nucleic acids, expression vectors, cells, drugs, and vaccines can effectively treat and/or prevent HPV-related diseases.
  • Figure 1 shows a structural diagram of the diagnostic system of the present invention
  • Figure 2 shows the flow chart of the research technical solution of the present invention
  • Figures 3-A and 3-B show the epitope E7 77-86 RTLEDLLMGT (divalent precursor ion 574.8.26++) verification positive data analysis chart, where Retention Time represents retention time, Intensity represents density, and Replicate represents the number of repeats. , Peak Area percentage represents the proportion of peak area;
  • Figures 4-A and 4-B show the epitope E7 75-83 DIRTLEDLL (divalent precursor ion-544.3033++) verification analysis spectrum;
  • Figures 5-A and 5-B show the epitope E7 4-11 DTPTLHEY (divalent precursor ion-488.2245++) verification analysis spectrum;
  • Figures 6-A and 6-B show the epitope E7 75-82 DIRTLEDL (divalent precursor ion-487.7613++) verification analysis spectrum;
  • Figures 7-A and 7-B show epitope E7 79-88 LEDLLMGTG (divalent precursor ion-531.2810++) verification analysis spectrum;
  • Figures 8-A and 8-B show the epitope E6 65-72 NPYAVCDK (divalent precursor ion-488.7211++) verification analysis spectrum;
  • Figures 9-A and 9-B show the epitope E7 77-90 RTLEDLLMGTLGIV (trivalent precursor ion-510.9568+++) verification analysis spectrum;
  • Figure 10 shows an example analysis diagram of ions carried by polypeptides in the mass spectrometry detection system
  • FIG. 11 shows the process flow diagram for preparation of HPV-specific CTL cells.
  • first and second are used for descriptive purposes only and cannot be understood as indicating or implying relative importance or implicitly indicating the quantity of indicated technical features. Therefore, features defined as “first” and “second” may explicitly or implicitly include at least one of these features.
  • “plurality” means at least two, such as two, three, etc., unless otherwise expressly and specifically limited.
  • epitope also known as antigenic determinant, refers to the specific structural site of the antigen molecule recognized by specific effector molecules or T lymphocytes and B lymphocytes in the immune response, thereby inducing cellular immunity and humoral immunity. produce an immune effect.
  • the HPV antigenic epitope in this application exists in HPV and can bind to HLA class I molecules.
  • anchoring refers to the structure of the HLA-I molecule that accepts the HPV epitope when the HPV epitope binds to the HLA class I molecule. It is the antigen-binding groove located at the far membrane end of the molecule. Analysis of natural HPV antigens The primary structure of epitopes is found to have two or more specific sites that bind to the antigen-binding groove of HLA class I molecules, called anchor sites. The amino acid residue at this position is called the anchor residue.
  • antigen-presenting cells refers to a type of immune cells that can take in and process antigens, and present the processed antigens to T cells.
  • APC mainly includes monocytes-phagocytic cells, dendritic cells, B cells, Langerhans cells, and virus-infected target cells of tumor cells.
  • immune effector cells refer to immune cells that participate in the elimination of foreign antigens and perform effector functions in immune responses.
  • the present invention proposes a method for identifying HPV epitopes, which includes the following steps: 1) Combine HLA-I class antibodies and carriers at a volume-to-mass ratio of 1 mL: 5 mg to obtain an immunoaffinity column; 2 ) Lyse HPV-positive cells; pass the cell lysate obtained in step 2) through a column, and the column is the immunoaffinity column to obtain an HLA complex containing HPV antigen peptides; 3) pass the cell lysate obtained in step 2)
  • the HLA complex containing HPV antigen peptides is subjected to solid phase extraction to obtain peptide fragments.
  • the method according to the embodiment of the present invention significantly improves the probability of obtaining HPV antigen epitopes that effectively bind to the HLA class I molecule, and significantly improves the identification efficiency and identification accuracy.
  • the lysis treatment is performed in a lysis solution, and the final concentration of the HPV-positive cells in the lysis solution is 0.5e 8 -1e 8 /mL.
  • the HLA-I class is anti-HLA-I class A, B, C antibodies (W6/32, ATCC HB-95).
  • the carrier is Protein A Sepharose.
  • the HPV-positive cells are HPV 16-positive cells.
  • the HPV 16-positive cells are Caski cells and/or Siha cells.
  • the method further includes the following steps: 5) detecting the peptide fragment obtained in step 4) to obtain the HPV antigen epitope.
  • the detection includes PRM targeted mass spectrometry analysis to identify the sequence of the obtained peptide fragment.
  • HPV mass spectrometry sample processing methods mainly use immunoaffinity purification methods to facilitate HLA immune complex antibodies to capture the HLA complex molecules that interact with them in biological samples, and then extract and re-extract the peptide fragments in the complex molecules. High-resolution mass spectrometry identification was achieved.
  • the present invention systematically optimizes the sample preparation and instrument analysis at key points in the process, thereby obtaining high-quality analysis data to increase the probability of successful identification of HPV immune polypeptide epitopes.
  • the optimization part includes the following content: HPV-positive biological samples used the HPV16-positive cell line Caski (human cervical cancer epithelial) cell line and Siha (human cervical squamous cell carcinoma) cell line (human cervical squamous cell carcinoma). Cancer) cell line (constructed by HLA-A0201 transfection).
  • HPV-positive biological samples used the HPV16-positive cell line Caski (human cervical cancer epithelial) cell line and Siha (human cervical squamous cell carcinoma) cell line (human cervical squamous cell carcinoma). Cancer) cell line (constructed by HLA-A0201 transfection).
  • each affinity purification experiment used a cell sample with an initial cell count of more than 8e 8 for membrane protein lysis.
  • Immunoaffinity purification utilizes the highly specific affinity between antigens and antibodies existing in organisms to separate and purify. Antigens and antibodies can be bound to the molecular surface through non-covalent bonds, and can be separated through subsequent changes in experimental strips.
  • the key factors that affect the immunoaffinity purification experiment are the reaction form and reaction system for the antigen-antibody affinity during the experiment, the matrix influence of the antigen sample, the optimization of incubation and elution conditions, and the separation of subsequent samples. Purification, etc. will all affect the final efficiency of the experiment.
  • Instrumental analysis uses the EASY-nLC 1000 ultra-high performance liquid chromatography system to perform liquid phase gradient separation on the immunoaffinity-purified peptide detection samples and then enters the Orbitrap Fusion Lumos Tribrid mass spectrometer for PRM-based targeted mass spectrometry analysis.
  • the sample first enters the trap column for enrichment and desalination, and is then connected in series with a self-assembled C18 column (150 ⁇ m inner diameter, 1.8 ⁇ m column particle size, approximately 35 cm column length), and separated through the following effective gradient at a flow rate of 500 nL/min: 0 to 5 minutes , 5% mobile phase B (98% ACN, 0.1% FA); 5 to 45 minutes, mobile phase B rises linearly from 5% to 25%; 45 to 50 minutes, mobile phase B rises from 25% to 35%; 50 to 52 minutes , mobile phase B increased from 35% to 80%; 52-54min, 80% mobile phase B; 54-54.5min, mobile phase B dropped from 80% to 5%; 54.5-65min, 5% mobile phase B.
  • a self-assembled C18 column 150 ⁇ m inner diameter, 1.8 ⁇ m column particle size, approximately 35 cm column length
  • the end of the nanoliter liquid phase separation is directly connected to the mass spectrometer and detected according to the following parameters.
  • the peptide fragments were ionized by the nanoESI source and entered into the tandem mass spectrometer Orbitrap Fusion Lumos (Thermo Fisher Scientific, San Jose, CA) for MSOT+tMS2OT mode detection.
  • ion source voltage is set to 2kV; primary mass spectrometry scanning range is 350 ⁇ 1,400m/z; resolution is set to 60,000, maximum ion injection time (MIT) is 50ms; secondary mass spectrometry fragmentation mode is HCD, fragmentation The energy is set to 30; the resolution is set to 30,000, the maximum ion implantation time (MIT) is 50ms, and the AGC is set to: 4E5 for the first level and 5E4 for the second level.
  • the present invention proposes an isolated peptide, including at least one of the following: 1) amino acids 65-72 of HPV E6 protein; 2) amino acids 4-11 of HPV E7 protein; 3) HPV E7 protein 75-82 amino acids of HPV E7 protein; 4) 75-83 amino acids of HPV E7 protein; 5) 77-86 amino acids of HPV E7 protein; 6) 77-90 amino acids of HPV E7 protein; and 7) HPV E7 protein of Amino acids 79-88.
  • the isolated peptides according to some specific embodiments of the present invention are obtained by expression and translation of the HPV E6 or E7 early coding region.
  • the isolated peptides can be presented by HLA-I class molecules, recognized by CTL cells or TIL cells, and then can be Presenting cells expressing HLA class I molecules are presented to CTL or TIL cells to activate specific T cell immunity, which constitutes the physiological target of the immune response of HPV-positive tumors. Highly sensitive and specific detection is performed for the prevention of HPV-related diseases. It is of great value for treatment.
  • the isolated peptide is no more than 15 consecutive amino acids in length.
  • the isolated peptide is more than 6 consecutive amino acids in length.
  • the HPV E6 protein is HPV16 E6 protein.
  • the HPV E7 protein is HPV16 E7 protein.
  • the isolated peptide comprises an amino acid fragment that binds to an HLA-A, HLA-B or HLA-C molecule.
  • the isolated peptide includes at least one of the amino acid sequences shown in SEQ ID NO: 1-7.
  • the isolated peptide is obtained by expression and translation of the HPV E6 or E7 early coding region.
  • the isolated peptide can be presented by HLA-I class molecules and recognized by CTL cells or TIL cells, and then It can be presented to CTL or TIL cells by presentation cells expressing HLA-I molecules to activate specific T cell immunity. It constitutes the physiological target of the immune response of HPV-positive tumors and performs highly sensitive and specific detection for HPV-related diseases. of great value for prevention and treatment.
  • the present invention proposes an HPV antigenic epitope, including at least one of the following: 1) amino acids 65-72 of HPV E6 protein; 2) amino acids 4-11 of HPV E7 protein; 3) HPV Amino acids 75-82 of the E7 protein; 4) Amino acids 75-83 of the HPV E7 protein; 5) Amino acids 77-86 of the HPV E7 protein; 6) Amino acids 77-90 of the HPV E7 protein; and 7) HPV E7 Amino acids 79-88 of the protein.
  • the HPV antigenic epitope according to the embodiment of the present invention is obtained by expression and translation of the HPV E6 or E7 early coding region.
  • HPV antigenic epitope can be presented by HLA-I class molecules, recognized by CTL or TIL cells, and then can be expressed Presenting cells of HLA-I molecules are presented to CTL or TIL cells to activate specific T cell immunity, which constitutes the physiological target of the immune response of HPV-positive tumors. Highly sensitive and specific detection is required for the prevention and treatment of HPV-related diseases. It is of great value for treatment.
  • the HPV antigenic epitope includes at least one of the amino acid sequences shown in SEQ ID NO: 1-7.
  • the present invention provides a mutant having at least one mutation site in addition to the anchor position.
  • the second amino acid of the obtained HPV epitope is usually the anchor position of HLA.
  • the E6HPV epitope is Mutation at the 1st, 3rd, 5th, 6th and 7th positions can still have the same or related immunogenicity and potential therapeutic effect, that is, it can be presented by HLA-I class molecules and be Recognized by CTL cells or TIL cells, they can then be presented to CTL or TIL cells by presentation cells expressing HLA-I molecules to activate specific T cell immunity. This constitutes the physiological target of the immune response of HPV-positive tumors and can be used for high-sensitivity and Specific detection is of great value for the prevention and treatment of HPV-related diseases.
  • the mutant compared to wild-type HPV E6 protein, has at least one of the following mutation sites: position 65, position 67, position 69, position 70 and position 70. 71 bits.
  • the wild-type HPV 16E6 protein has the amino acid sequence shown in SEQ ID NO: 25.
  • the mutant compared to the previously described isolated peptide (HPV E6, SEQ ID NO: 1), has at least one of the following mutations: 1) N at position 1 Mutation to F; 2) Y at position 3 mutated to L; 3) V at position 5 mutated to E; 4) C at position 6 mutated to L; and D at position 7 mutated to V.
  • the mutant has the amino acid sequence shown in SEQ ID NO: 8-12.
  • mutants with the amino acid sequences shown can be presented by HLA class I molecules, recognized by CTL cells or TIL cells, and can then be presented to CTL or TIL cells by presentation cells expressing HLA class I molecules.
  • TIL cells activate specific T cell immunity and constitute the physiological target of the immune response to HPV-positive tumors. Highly sensitive and specific detection is of great value for the prevention and treatment of HPV-related diseases.
  • NPLAVCDK (SEQ ID NO: 8).
  • NPYAVCVK SEQ ID NO: 9
  • NPYAVLDK SEQ ID NO: 11
  • NPYAECDK SEQ ID NO: 12
  • the present invention provides a mutant having at least one mutation site in addition to the anchor position.
  • the 2nd and 9th amino acids of the obtained HPV epitope are usually the anchor positions of HLA. After the amino acids at other positions in the epitope are substituted, they can still have the same or related immunogenicity. and its potential therapeutic effect, that is, it can be presented by HLA class I molecules, recognized by CTL cells or TIL cells, and then presented to CTL or TIL cells by presentation cells expressing HLA class I molecules to activate specific T cell immunity. , constitutes the physiological target of the immune response of HPV-positive tumors, and high-sensitivity and specific detection is of great value for the prevention and treatment of HPV-related diseases.
  • the mutant compared to wild-type HPV E7 protein, has at least one of the following mutation sites: position 75, position 77, position 79, position 80 and position 80. 82 bits.
  • the wild-type HPV 16 E7 protein has the amino acid sequence shown in SEQ ID NO: 26:
  • the mutant has at least one of the following mutations compared to the wild-type HPV E7 protein: 1) D at position 75 is mutated to F or Y; 2) R at position 77 Mutated to L or P; 3) L at position 79 mutated to Y or deleted; 4) E at position 80 mutated to Y; 5) L at position 82 mutated to T.
  • the mutant compared to the previously described isolated peptide (HPV E7 75-83 , SEQ ID NO: 4), the mutant has at least one of the following mutations: position 1, 3rd, 5th, 6th, 8th.
  • the mutant compared to the previously described isolated peptide (HPV E7 75-83 , SEQ ID NO: 4), the mutant has at least one of the following mutations: 1) 1 D at position mutates to F or Y; 2) R at position 3 mutates to L or P; 3) L at position 5 mutates to Y or is deleted; 4) E at position 6 mutates to Y; 5) E at position 8 The L bit is mutated to T.
  • the mutant has the amino acid sequence shown in SEQ ID NO: 13-17.
  • mutants with the amino acid sequences shown can be presented by HLA class I molecules, recognized by CTL cells or TIL cells, and can then be presented to CTL or TIL cells by presentation cells expressing HLA class I molecules.
  • TIL cells activate specific T cell immunity and constitute the physiological target of the immune response to HPV-positive tumors. Highly sensitive and specific detection is of great value for the prevention and treatment of HPV-related diseases.
  • DILTLEDLL SEQ ID NO: 17
  • the present invention provides a nucleic acid molecule encoding the aforementioned isolated peptide, the HPV epitope or mutant.
  • the isolated peptides, HPV epitopes or mutants encoded by the nucleic acid molecules according to some specific embodiments of the present invention can be presented by HLA class I molecules, recognized by CTL or TIL cells, and can then be expressed by HLA class I molecules.
  • the nucleic acid molecule has at least one of the nucleotide sequences shown in SEQ ID NO: 18-24.
  • the nucleic acid encoding NPYAVCDK includes:
  • Nucleic acids encoding DTPTLHEY include:
  • Nucleic acids encoding DIRTLEDL include:
  • Nucleic acids encoding DIRTLEDLL include:
  • Nucleic acids encoding RTLEDLLMGT include:
  • Nucleic acids encoding RTLEDLLMGTLGIV include:
  • Nucleic acids encoding LEDLLMGTGG include:
  • nucleic acids mentioned in the description and claims of the present invention actually include either or both complementary double strands.
  • the gene sequence in this application includes DNA form or RNA form. Disclosing one of them means that the other one is also disclosed.
  • the present invention provides an expression vector carrying nucleic acid expressing the aforementioned isolated peptide, HPV epitope or mutant.
  • the type of expression vector is not particularly limited, as long as it can achieve efficient expression of the nucleic acid construct described above in recipient cells.
  • Expression vectors include but are not limited to retroviral vectors, lentiviral vectors and/or adenoviral vectors. Virus-related viral vectors.
  • the expression vector may include optional control sequences operably linked to the nucleic acid molecule. Wherein, the control sequence is one or more control sequences that can direct the expression of the polypeptide in the host.
  • the expression vectors proposed in some specific embodiments of the present invention can efficiently express the isolated peptide, HPV epitope or mutant in a suitable host cell, and thus can be effectively used to treat tumors, especially while expressing HLA-I. Specific treatment or prevention of tumors using class molecules and the above isolated peptides or HPV epitopes.
  • the present invention provides a recombinant cell carrying the aforementioned nucleic acid molecule, expression vector, isolated peptide, HPV epitope or mutant.
  • the recombinant cells are obtained by transfection or transformation of the expression vector. Transformation or transfection can be carried out by electroporation, viral transfection or transformation of competent cells.
  • the method of transfection or transformation used is determined by the nature of the host cell and the nature of the nucleic acid construct or expression vector to be transferred, as long as the high-efficiency expression of the polypeptide described above can be achieved in the host cell and the host cell The good cell condition does not have a major impact.
  • the host cells can efficiently express the above-mentioned isolated peptides, HPV epitopes or mutants under appropriate conditions, and the recombinant cells can be effectively used to treat tumors, especially while expressing HLA- Specific treatment or prevention of tumors with class I molecules and the above isolated peptides, HPV epitopes or mutants.
  • suitable conditions refer to conditions suitable for the expression of the isolated peptides, HPV epitopes or mutants described in this application. It is easily understood by those skilled in the art that conditions suitable for the expression of isolated peptides, HPV epitopes or mutants include, but are not limited to, suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy host cell status, suitable The host cell density, suitable cell culture environment, and suitable cell culture time. "Suitable conditions” are not particularly limited, and those skilled in the art can optimize the optimal conditions for the expression of the polypeptide according to the specific environment of the laboratory.
  • the present invention provides an antigen-presenting cell that can present the isolated peptide, HPV epitope or mutant as described above.
  • antigen-presenting cells presenting the aforementioned isolated peptides, HPV epitopes or mutants can effectively induce immunity in patients against tumor-specific antigens-the aforementioned isolated peptides/HPV epitopes/mutants. reaction, thereby activating the specific killing function of CTL.
  • the antigen-presenting cells proposed in the embodiments of the present invention have significant efficacy in treating tumors expressing the above-mentioned isolated peptides, HPV epitopes or mutants, and their therapeutic effects are significant and safe. high.
  • the antigen-presenting cells are obtained by at least one of the following: contacting cells with antigen-presenting ability with the polypeptide; introducing the aforementioned nucleic acid or expression vector into the antigen-presenting cells. Presenting competent cells.
  • the cells with antigen-presenting ability are dendritic cells, B cells or monocyte-phagocytic cells.
  • the present invention provides an immune effector cell.
  • the immune effector cells can recognize the polypeptides, HPV epitopes, and mutants described above, or recognize the polypeptides or HPV epitopes or mutants that are presented on the cell surface.
  • Antigen presenting cells According to embodiments of the present invention, the immune effector cells can specifically kill tumor cells that co-express HLA class I molecules and the above-mentioned isolated peptides, HPV epitopes or mutants.
  • the immune effector cells are obtained by contacting the aforementioned antigen-presenting cells with cells having immune effector capabilities.
  • the cells with immune effector capabilities are T cells, preferably CD8 + T cells.
  • the inventors found that by contacting antigen-presenting cells presenting the previously described isolated peptides, HPV epitopes or mutants with cells having immune effector capabilities, the antigen-presenting cells can activate unactivated cells with immune effector capabilities, presenting The antigen - the polypeptide mentioned above, then activates cells with immune effector ability, producing a large number of immune effector cells.
  • the immune effector cells have the function of specifically killing the target cells presenting the antigen - the polypeptide.
  • CD8 + T cells have a stronger ability to accept the activation effect of antigen-presenting cells, and the obtained CD8 + T cells have a stronger specific killing effect on target cells presenting antigen-the isolated peptide/HPV epitope.
  • the invention provides a medicament.
  • the aforementioned isolated peptides, HPV epitopes, mutants, nucleic acid molecules, expression vectors, recombinant cells, antigen-presenting cells or immune effector cells are included.
  • the isolated peptide, HPV epitope, mutant, nucleic acid molecule encoding the isolated peptide or HPV epitope or mutant, expression vector, recombinant cell, antigen presenting cell or immune effector cell All can be effectively used for the specific treatment or prevention of tumors, especially tumors that express both HLA class I molecules and the above-mentioned isolated peptides, HPV epitopes or mutants. Therefore, drugs containing some or all of the above substances are also It has a significant effect of treating or preventing tumors expressing HLA class I molecules and the isolated peptide, HPV antigen epitope or mutant, and has higher safety and smaller side effects.
  • Medicaments provided according to some specific embodiments of the present invention include pharmaceutically acceptable carriers and an effective amount of active ingredients of the above substances.
  • the term "effective amount” or “effective dose” refers to an amount that produces a function or activity in humans and/or animals and is acceptable to humans and/or animals.
  • a "pharmaceutically acceptable” ingredient is one that is suitable for use by humans and/or mammals without undue adverse side effects (e.g., toxicity, irritation, and allergic reactions), i.e., a substance that has a reasonable benefit/risk ratio.
  • pharmaceutically acceptable carrier refers to a carrier used for the administration of a therapeutic agent, including various excipients and diluents.
  • the pharmaceutical composition of the present invention contains a safe and effective amount of the active ingredient of the present invention and a pharmaceutically acceptable carrier.
  • Such carriers include, but are not limited to: saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof.
  • pharmaceutical preparations should match the mode of administration.
  • the dosage forms of the pharmaceutical composition of the present invention are injections, oral preparations (tablets, capsules, oral liquids), transdermal agents, and sustained-release agents. For example, it is prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants.
  • the pharmaceutical composition is preferably manufactured under sterile conditions.
  • the effective amount of the active ingredients of the present invention may vary depending on the mode of administration and the severity of the disease to be treated.
  • the selection of a preferred effective amount can be determined by one of ordinary skill in the art based on various factors (eg, through clinical trials).
  • the factors include but are not limited to: the pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the patient's weight, the patient's immune status, drug administration ways, etc. For example, several divided doses may be administered daily, or the dosage may be proportionally reduced as dictated by the exigencies of the treatment situation.
  • Pharmaceutically acceptable carriers of the present invention include (but are not limited to): water, saline, liposomes, lipids, proteins, protein-antibody conjugates, peptides, cellulose, nanogels, or its combination.
  • the choice of carrier should be compatible with the mode of administration, which are well known to those of ordinary skill in the art.
  • cervical cancer vulvar cancer, vaginal cancer, anal cancer, penile cancer, head and neck cancer, cervical intraepithelial neoplasia, vulvar intraepithelial neoplasia, vaginal intraepithelial neoplasia, anal intraepithelial neoplasia, and penile intraepithelial neoplasia.
  • Neoplasia oral cancer, laryngeal cancer, esophageal cancer, intranasal cancer or tonsil cancer, especially cervical cancer tissue specifically expresses the above-mentioned isolated polypeptide or HPV antigen epitope, and when the tumor is the above-mentioned tumor, the drug can treat Effectiveness is further improved.
  • the present invention provides a vaccine.
  • the aforementioned isolated peptides, HPV epitopes, mutants, nucleic acid molecules, expression vectors or antigen-presenting cells are included.
  • the nucleic acid molecules, expression vectors or recombinant cells of the embodiments of the present invention express the aforementioned isolated peptides, HPV epitopes or mutants under appropriate conditions, and the antigen-presenting cells can express the isolated peptides.
  • HPV antigenic epitopes or mutants when combined with HLA-I class molecules, are presented by antigen-presenting cells, allowing them to be recognized by CTL or TIL cells, that is, they are presented to antigen-presenting cells expressing HLA-I class molecules.
  • CTL or TIL cells activate specific T cell immunity. Therefore, the vaccine proposed in the embodiments of the present invention has a significant effect in treating or preventing tumors expressing HLA class I molecules and the isolated peptides, HPV epitopes or mutants, and is safer and has fewer side effects. .
  • the vaccine can effectively treat or prevent cervical cancer, vulvar cancer, vaginal cancer, anal cancer, penile cancer, head and neck cancer, cervical intraepithelial neoplasia, vulvar intraepithelial neoplasia, vaginal intraepithelial neoplasia, anus Intraepithelial neoplasia, penile intraepithelial neoplasia, oral cancer, laryngeal cancer, esophageal cancer, intranasal cancer or tonsil cancer, especially cervical cancer, tissue-specific high expression of the above isolated polypeptides, HPV epitopes or mutants, and then The effectiveness of this drug treatment is further enhanced when the tumor is cervical cancer as described above.
  • the vaccine is in a form suitable for administration by inhalation or injection.
  • the vaccine further comprises at least one adjuvant.
  • the present invention proposes the use of reagents for detecting the aforementioned isolated peptides, HPV epitopes or mutants in the preparation of kits for diagnosing HPV or detecting HPV. treatment effect.
  • the reagent can accurately detect the isolated peptide, HPV epitope or mutant, for example, detect whether a biological sample contains the isolated peptide, HPV epitope or mutant, because the isolated peptide , HPV antigenic epitopes or mutants are highly expressed in HPV-infected tissues. Therefore, a kit containing the reagent can accurately diagnose whether the individual from whom the biological sample is derived is infected by HPV, and further, whether it is an individual at high risk for HPV. In the same way, during the treatment process of an individual infected by HPV, the test kit can be used to detect the changes of HPV during the treatment process, such as aggravation, slowing down or cure, etc.
  • the present invention proposes the use of the aforementioned isolated peptides, HPV epitopes, mutants, nucleic acid molecules, expression vectors, recombinant cells, antigen-presenting cells or immune effector cells in the preparation of medicines.
  • the medicine is used to treat or prevent HPV-related diseases.
  • the isolated peptide, HPV epitope, mutant, nucleic acid molecule encoding the isolated peptide or HPV epitope or mutant, expression vector, recombinant cell, antigen presenting cell or immune effector cell All can be effectively used for the specific treatment or prevention of tumors, especially tumors that express both HLA class I molecules and the above-mentioned isolated peptides, HPV epitopes or mutants. Therefore, drugs containing some or all of the above substances are also It has a significant effect of treating or preventing tumors expressing HLA class I molecules and the isolated peptide, HPV antigen epitope or mutant, and has higher safety and smaller side effects.
  • the HPV-related diseases include at least one of the following: cervical cancer, vulvar cancer, vaginal cancer, anal cancer, penile cancer, head and neck cancer, cervical intraepithelial neoplasia, vulvar intraepithelial neoplasia vaginal intraepithelial neoplasia, anal intraepithelial neoplasia, penile intraepithelial neoplasia, oral cancer, laryngeal cancer, esophageal cancer, intranasal cancer and tonsil cancer.
  • the drug can effectively treat or prevent the above-mentioned diseases, especially cervical cancer tissue that specifically expresses the above-mentioned isolated polypeptide, HPV epitope or mutant. Furthermore, when the disease is the above-mentioned cervical cancer, the drug treatment is effective. Sexuality is further improved.
  • the present invention proposes the use of the previously described isolated peptides, HPV epitopes, mutants, nucleic acid molecules, expression vectors or recombinant cells in the preparation of kits for detecting HLA.
  • the isolated peptides, HPV epitopes, mutants and corresponding substances according to some specific embodiments of the present invention can be combined with HLA. Therefore, the above substances can be used to prepare kits for effectively detecting HLA.
  • the present invention provides a kit comprising reagents suitable for detecting the isolated peptide, HPV epitope or mutant as described above.
  • the reagent can accurately detect the isolated peptide, HPV epitope or mutant, for example, detect whether a biological sample contains the isolated peptide, HPV epitope or mutant, because the isolated peptide , HPV antigenic epitopes or mutants are highly expressed in HPV-infected tissues. Therefore, a kit containing the reagent can accurately diagnose whether the individual from whom the biological sample is derived is infected by HPV, and further, whether it is an individual at high risk for HPV. In the same way, during the treatment process of an individual infected by HPV, the test kit can be used to detect the changes of HPV during the treatment process, such as aggravation, slowing down or cure, etc.
  • kits for detecting HLA including the aforementioned isolated peptide, HPV epitope, mutant, nucleic acid molecule, expression vector or recombinant cell.
  • isolated peptides, HPV epitopes, mutants and their corresponding substances can bind to HLA. Therefore, kits containing the above substances can be used to effectively detect HLA qualitatively or quantitatively.
  • the present invention proposes a method for preventing or treating HPV-related diseases by administering to the subject the previously described isolated peptides, HPV epitopes, mutants, nucleic acid molecules, expression vectors, recombinant cells, and antigen-presenting cells. , immune effector cells, vaccines or drugs.
  • the preventive methods proposed by some specific embodiments of the present invention include administering any effective amount of the previously described isolated peptides and other substances, which can effectively treat or prevent the expression of HLA-I class molecules and the tumors with isolated peptides, HPV epitopes or mutants.
  • the HPV-related diseases include at least one of the following: cervical cancer, vulvar cancer, vaginal cancer, anal cancer, penile cancer, head and neck cancer, cervical intraepithelial neoplasia, vulvar intraepithelial neoplasia vaginal intraepithelial neoplasia, anal intraepithelial neoplasia, penile intraepithelial neoplasia, oral cancer, laryngeal cancer, esophageal cancer, intranasal cancer and tonsil cancer.
  • compositions of the present invention may be administered in an injectable formulation.
  • pharmaceutical compositions of the present invention may be administered using specific devices that deliver the active ingredients to target cells.
  • the administration frequency and dosage of isolated peptides, HPV epitopes, mutants, nucleic acids, expression vectors, recombinant cells, antigen-presenting cells, immune effector cells, drugs, and vaccines in the embodiments of the present invention can be determined by multiple related factors. To be sure, factors include the type of disease being treated, the route of administration, patient age, gender, weight and severity of the disease and the type of drug that is the active ingredient. According to some embodiments of the present invention, the daily dose may be divided into 1 dose, 2 doses, or multiple doses in a suitable form to be administered once, twice, or multiple times throughout the time period, as long as a therapeutically effective amount is achieved .
  • terapéuticaally effective amount refers to an amount sufficient to significantly ameliorate certain symptoms associated with a disease or condition, ie, an amount that provides a therapeutic effect for a given condition and dosage regimen.
  • treatment is used to refer to obtaining a desired pharmacological and/or physiological effect.
  • Treatment encompasses the administration of isolated peptides, HPV epitopes, mutants, nucleic acids, expression vectors, recombinant cells, vaccines, antigen-presenting cells, immune effector cells or drugs in the embodiments of the invention to an individual for treatment, including However, it is not limited to administration to an individual in need containing what is described herein.
  • the present invention proposes the use of the aforementioned isolated peptides, mutants, expression vectors, antigen-presenting cells, immune effector cells, drugs or vaccines in the treatment and/or prevention of HPV-related diseases.
  • the isolated peptide and its mutants can effectively inhibit tumor growth. Therefore, the isolated peptide or mutant, the nucleic acid capable of expressing or containing the isolated peptide or mutant , expression vectors, cells, drugs, and vaccines can all effectively treat and/or prevent HPV-related diseases.
  • the HPV-related diseases include at least one of the following: cervical cancer, vulvar cancer, vaginal cancer, anal cancer, penile cancer, head and neck cancer, cervical intraepithelial neoplasia, vulvar intraepithelial neoplasia vaginal intraepithelial neoplasia, anal intraepithelial neoplasia, penile intraepithelial neoplasia, oral cancer, laryngeal cancer, esophageal cancer, intranasal cancer and tonsil cancer.
  • the present invention proposes a method for diagnosing whether a subject suffers from HPV-related diseases, including detecting whether a biological sample derived from the subject carries the previously described isolated peptide, HPV epitope, mutant or Nucleic acid molecule steps.
  • the isolated peptide, HPV epitope, mutant or nucleic acid molecule is present in an individual infected by HPV. Therefore, by detecting whether the biological sample derived from the subject carries the substance, it is possible to Effectively diagnose whether subjects suffer from or are susceptible to HPV-related diseases.
  • the HPV-related diseases include at least one of the following: cervical cancer, vulvar cancer, vaginal cancer, anal cancer, penile cancer, head and neck cancer, cervical intraepithelial neoplasia, vulvar intraepithelial neoplasia vaginal intraepithelial neoplasia, anal intraepithelial neoplasia, penile intraepithelial neoplasia, oral cancer, laryngeal cancer, esophageal cancer, intranasal cancer and tonsil cancer.
  • the present invention proposes a diagnostic system.
  • the diagnostic system includes: a peptide detection device 100; a diagnostic result determination device 200.
  • the peptide detection device 100 is used to detect whether the biological sample derived from the subject carries the aforementioned isolated peptide, HPV epitope or mutant, and the diagnostic result determination device 200 is connected to the peptide detection device 100 for It is determined whether the patient has a tumor based on whether the biological sample carries the isolated peptide, HPV epitope or mutant.
  • a mass spectrometer can be used to detect whether the isolated peptide, HPV antigen epitope or mutant is present in the subject's serum, and then a mass spectrometry data analysis device can be used to determine whether the isolated peptide, HPV antigen is present in the subject's serum. epitopes or mutants to determine whether the patient has a tumor.
  • the inventor found that the isolated peptides, HPV antigen epitopes or mutants are specifically highly expressed in tumor tissues.
  • the diagnostic system proposed in the embodiment of the present invention can be used to effectively determine the specific highly expressed polypeptides, HPV antigen epitopes. or mutant tumor patients.
  • cervical cancer vulvar cancer, vaginal cancer, anal cancer, penile cancer, head and neck cancer
  • cervical intraepithelial neoplasia vulvar intraepithelial neoplasia
  • vaginal intraepithelial neoplasia anal intraepithelial neoplasia
  • penile epithelial neoplasia Intra-neoplasia, oral cancer, laryngeal cancer, esophageal cancer, intranasal cancer or tonsil cancer specifically express the polypeptide at a high level
  • the diagnostic system proposed in the embodiment of the present invention further improves the diagnostic accuracy of the above-mentioned tumors.
  • HLA-I class molecules have a strong affinity with the isolated peptide, HPV epitope or mutant, and the isolated peptide, HPV epitope or mutant interacts with HLA-I on the cell surface.
  • the molecules bind and trigger a series of immune responses. Therefore, the diagnostic system proposed by the embodiment of the present invention has a higher probability of diagnosing tumor patients who express both HLA class I molecules and the isolated peptide, HPV epitope or mutant.
  • isolated peptides, HPV epitopes, mutants and their uses nucleic acids encoding the isolated peptides or HPV epitopes or mutants, expression vectors, recombinant cells, and drugs , antigen-presenting cells, immune effector cells, vaccines, kits, methods and systems for treating and diagnosing HPV were discovered and completed by the inventor of the present application after painstaking creative labor and optimization work.
  • This embodiment uses an immunoaffinity method to obtain immune polypeptides.
  • the specific operation process is shown in Figure 2.
  • the specific operation steps are as follows:
  • HLA protein complexes major histocompatibility complex (MHC) class I complex molecules
  • HPV16-positive cell lines Caski (human cervical cancer epithelial) cell line and Siha (human cervical squamous cell carcinoma) cell line to prepare immune peptide samples.
  • the above cell lines are all constructed by HLA-A0201 transfection, using 8e HPV-positive cell lines above 8 are lysed and extracted.
  • the membrane protein lysate is composed of 0.5% sodium deoxycholate and 2% glucopyranoside PBS buffer;
  • step 8) Centrifuge the product obtained in step 7) at 4°C and 14000g for 30 min at low temperature and high speed. The supernatant after centrifugation is filtered with a 0.8 ⁇ m filter membrane and the filtrate is stored at 4°C for later use.
  • Each eluate is collected as one component. A total of 5 components are collected to obtain components 1-5. At the same time, 5 components are collected. The components are mixed to obtain component 6 to obtain the HLA protein complex containing immune polypeptides.
  • This example uses solid phase extraction method to separate the HLA protein complex obtained in step 1.1.
  • the specific experimental operations are as follows:
  • the immune peptides obtained in experiment 1.2.1 were subjected to secondary separation and mass spectrometry detection, and EASY-nLC 1000 ultra-high performance liquid chromatography tandem Orbitrap Fusion Lumos Tribrid mass spectrometer system was used to conduct DDA high-throughput mass spectrometry identification and analysis of immune peptide samples. Specific experiments Here's how to do it:
  • HPV immune polypeptide first enters the trap column for enrichment and desalination, and is then connected in series with a self-assembled C18 column (150 ⁇ m inner diameter, 1.8 ⁇ m column material particle size, approximately 35 cm column length), and passes through the following effective gradient at a flow rate of 500 nL/min Separation: 0 ⁇ 5min, 5% mobile B (98% ACN, 0.1% FA); 5 ⁇ 45min, mobile phase B rises linearly from 5% to 25%; 45 ⁇ 50min, mobile phase B rises from 25% to 35% %; 50 ⁇ 52min, mobile phase B rises from 35% to 80%; 52 ⁇ 54min, 80% mobile phase B; 54 ⁇ 54.5min, mobile phase B drops from 80% to 5%; 54.5 ⁇ 65min, 5% mobile phase B.
  • the end of the nanoliquid phase separation is directly connected to the mass spectrometer and detected according to the following parameters: the peptides after liquid phase separation are ionized by the nanoESI source and then enter the tandem mass spectrometer Orbitrap Fusion Lumos (Thermo Fisher Scientific, San Jose, CA) for MSOT.
  • Orbitrap Fusion Lumos Thermo Fisher Scientific, San Jose, CA
  • the main parameter settings are: ion source voltage is set to 2kV; primary mass spectrometry scanning range is 350 ⁇ 1,400m/z; resolution is set to 60,000, maximum ion injection time (MIT) is 50ms; secondary mass spectrometry fragmentation
  • the mode is HCD, the fragmentation energy is set to 30; the resolution is set to 30,000, the maximum ion implantation time (MIT) is 50ms, and the AGC is set to: 4E5 for the first level and 5E4 for the second level. Targeted acquisition of candidate target ions.
  • the offline sample files and synthetic peptide sample files in the result chart are as follows: Figure 3-A and 3-B: Test samples: (1) caski-1; (2) caski-2; (3) caski-3; (4) Synthetic peptide sample caski-mix; Figure 4-A and 4-B: Test samples: (1) caski-1; (2) caski-2; (3) caski-3; (4) Synthetic peptide sample: caski-mix ; Figure 5-A and 5-B: Detection samples: (1) caski-1; (2) caski-2; (3) caski-3; (4) Synthetic peptide sample: caski-mix; Figure 6-A and 6-B: Test samples: (1) caski-1; (2) caski-2; (3) caski-3; (4) siha-1; (5) siha-2; (6) Synthetic peptide sample: caski -siha-mix; Figure 7-A and 7-B: Test samples: (1) siha-1; (2) siha-2; (3) siha-3; (4) Synthetic peptide sample: siha-mix; Figure 8-A and 8-B: Test samples: (1) caski-1; (2)
  • peptides are protonated in positive ion mode to form charged precursor ions.
  • the charged precursor ion is initially located on the N-terminal or basic residue side chain, but due to internal cleavage, it can move along the main chain and generate product ion fragments after being broken at different sites.
  • backbone bonds that can be broken into peptide fragments: alkyl carbonyl (CHR-CO), peptide amide bond (CO-NH) and aminoalkyl bond (NH-CHR).
  • sequence of the HPV-specific immune polypeptide epitope newly identified in the immune polypeptide sample prepared by the present invention is as follows:
  • Example 2 Enzyme-linked immunospot assay (ELISPOT) to evaluate the immunogenicity of HPV-specific tumor antigen peptides
  • the experiment uses T2 cells to load the newly identified 7 HPV16 type-specific mixed antigen polypeptides described in Example 1, and presents them to cytotoxic T cells CTL through the antigen presentation function of T2 cells and the TAP (antigen presentation transport) of T2 cells.
  • the molecular defect activates the immune response after the experimental antigen is presented through the payload of exogenous peptides.
  • the experiment consists of a positive control group, a negative control group and a test product group. Collect viable T2 (ATCC: CRL-1992) cells in logarithmic growth phase after cell culture treatment, dilute them with culture medium and resuspend them to a cell density of 5e 5 cells/mL for later use.
  • T2 cells Take 1mL of T2 cells from each group, add phytohemagglutinin PHA (Sigma, Cat. No. L8902-25MG) to the positive control group, and the final concentration of PHA is 4 ⁇ g/mL; no treatment is added to the negative control group; newly identified HPV antigen is added to the test group
  • the final concentration of the antigen peptide mixture is approximately 5 ⁇ g/mL, and cultured in a CO 2 incubator for 3.5 h.
  • the experimental data of ELISPOT detection of the number of IFN- ⁇ secretion spots are shown in Table 1. Among them, the number of IFN- ⁇ spots in the test group was significantly increased compared with the negative control group. This shows that the HPV-specific tumor antigen polypeptide identified has good immunogenicity.
  • Example 3 Enzyme-linked immunospot assay (ELISPOT) to evaluate the immunogenicity of HPV-specific tumor antigen peptide mutants
  • the inventor replaced the non-weighted amino acids of the effective antigenic epitope obtained in Example 1 or 2 to explore whether this method can enhance the immunogenicity and potential efficacy of the CTL epitope.
  • the inventors performed amino acid substitutions at other sites except amino acids 2 and 9, and performed immunogenicity testing on the obtained mutant extracts.
  • the specific experimental operations are as follows:
  • the inventor modified E7 75-83 DIRTLEDLL and E6 65-72 NPYAVCDK among the HPV epitopes identified above, and used them in subsequent experiments after synthesis.
  • the amino acid sequences of the obtained mutants are shown in Tables 2 and 3.
  • An ELISPOT detection experiment is used to verify whether the variant of the HPV epitope obtained in step 3.1 can activate the immune response of CD8+ T cells.
  • the experiment consists of a positive control group, a negative control group and a test product group.
  • the final concentration of PHA is 4 ⁇ g/mL; no treatment is added to the negative control group; new identifications are added to the test group separately.
  • the two HPV antigen peptides and their corresponding mutants were processed.
  • the final concentration of the antigen peptide DIRTLEDLL and its mutant peptide was set to about 5 ⁇ g/mL. They were cultured using HIPP-T009 lymphocyte serum-free medium and cultured in a CO 2 incubator. 3.5h. Take the prepared T2 cells and CTL cells for ELISPOT plating, and add 50 ⁇ L T cells and 50 ⁇ L T2 cells to each well. After culturing for 18 hours in a 37°C, 5% CO2 incubator, use a kit (Human IFN-gamma ELISpotPRO (ALP), Cat. No.: 3420-2AST-10) for antibody incubation, color development, and use an EliSpot plate reader to secrete IFN- gamma spot count.
  • ALP Human IFN-gamma ELISpotPRO
  • test polypeptide The requirements for the test polypeptide to be immunogenic are as follows: number of spots (test polypeptide)/number of spots (unrelated polypeptide) >2; that is, the number of spots caused by the test polypeptide exceeds the number of spots caused by the irrelevant polypeptide by more than twice, indicating that the test polypeptide is immunogenic.
  • ELISPOT detection results of the above-mentioned HPV epitope E7 75-83 DIRTLEDLL and its mutants are shown in Table 2.
  • the ELISPOT detection results of the above-mentioned HPV antigenic epitope E6 65-72 NPYAVCDK and its mutants are shown in Table 3.
  • the number of spots caused by the preferred variant polypeptides tested is better than that of the target epitope before mutation, indicating that the target epitope in the present invention
  • the polypeptide and its preferred variant forms are immunogenic and can specifically activate CD8 + T cell immune responses.
  • Polypeptides and variants thereof Number of spots in experimental group Number of spots in negative control group Multiples (experimental group/negative control group) NPYAVCDK(SEQ ID NO:1) 235 29 8.1 FPYAVCDK(SEQ ID NO:10) 316 31 10.19 NPYAECDK(SEQ ID NO:12) 281 35 8.03 NPYAVLDK(SEQ ID NO:11) 299 34 8.79 NPLAVCDK(SEQ ID NO:8) 336 26 12.92 NPYAVCVK(SEQ ID NO:9) 385 32 12.03
  • HPV-specific CTL are prepared from peripheral blood collected from HPV-positive infected patients through an in vitro preparation process.
  • the specific preparation process flow is shown in Figure 11, in which the HPV-specific antigen polypeptide mix refers to the one obtained in Example 1
  • a mixture of 7 antigenic peptides the specific experimental procedures are as follows:
  • Cervical cancer positive cells caski were inoculated subcutaneously into 6-8 week old NOG dKO mice with severe combined immune deficiencies.
  • the HPV-specific CTL high-dose group was intravenously injected with 2*10e 7 CTL cells/time, and administered twice within 24 hours; the HPV-specific CTL low-dose group was intravenously injected with 2*10e 6 CTL cells/time , administered twice within 24 hours; human interleukin 2 (IL-2) was administered at 10,000 U/animal/day, administered intraperitoneally for 14 consecutive days. Repeated administration of HPV-specific CTL on the 14th day maintained the retention ratio of CTL cells in the body and the therapeutic effect. The mice were cultured for 38 days, and the tumors were harvested and weighed to calculate the tumor (volume) inhibition rate.
  • IL-2 human interleukin 2
  • iv means intravenous injection and ip means intraperitoneal injection.
  • first and second are used for descriptive purposes only and cannot be understood as indicating or implying relative importance or implicitly indicating the quantity of indicated technical features. Therefore, features defined as “first” and “second” may explicitly or implicitly include at least one of these features.
  • “plurality” means at least two, such as two, three, etc., unless otherwise expressly and specifically limited.
  • references to the terms “one embodiment,” “some embodiments,” “an example,” “specific examples,” or “some examples” or the like means that specific features are described in connection with the embodiment or example. , structures, materials or features are included in at least one embodiment or example of the invention. In this specification, the schematic expressions of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the specific features, structures, materials or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, those skilled in the art may combine and combine different embodiments or examples and features of different embodiments or examples described in this specification unless they are inconsistent with each other.

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Abstract

An HPV epitope and an identification method therefor, and an application thereof. The HPV epitope comprises amino acids at positions 65-72 of an HPV E6 protein. The screened and optimized HPV epitope identification method is used to obtain an HPV epitope, the obtained HPV epitope is applied, and a plurality of dominant mutants are further obtained; the drug or the vaccine prepared from the HPV epitope or the mutant can effectively treat various cancers, the safety of the HPV epitope or the mutant is higher, and the side effect thereof are fewer.

Description

HPV抗原表位及其鉴定方法、应用HPV antigenic epitopes and their identification methods and applications
优先权信息priority information
本申请请求2022年03月29日向中国国家知识产权局提交的、专利申请号为202210320031.X的专利申请的优先权和权益，并且通过参照将其全文并入此处。This application requests the priority and rights of the patent application with patent application number 202210320031.X submitted to the State Intellectual Property Office of China on March 29, 2022, and its full text is incorporated herein by reference.
技术领域Technical field
本发明涉及生物技术领域，具体地，本发明涉及HPV抗原表位及其鉴定方法、应用，更具体地，本发明涉及一种鉴定HPV抗原表位的方法、分离的肽、突变体、表达载体、抗原呈递细胞、免疫效应细胞、试剂在制备试剂盒中的用途、试剂盒、所述分离的肽或表达载体或抗原呈递细胞或免疫细胞在制备药物、治疗和/或预防疾病中的用途、药物、疫苗和诊断***、诊断方法、治疗和/或预防疾病的方法。The present invention relates to the field of biotechnology. Specifically, the present invention relates to HPV antigenic epitopes and their identification methods and applications. More specifically, the present invention relates to a method for identifying HPV antigenic epitopes, isolated peptides, mutants, and expression vectors. , the use of antigen-presenting cells, immune effector cells, reagents in the preparation of kits, the use of kits, the isolated peptides or expression vectors or antigen-presenting cells or immune cells in the preparation of drugs, treatment and/or prevention of diseases, Drugs, vaccines and diagnostic systems, methods of diagnosis, treatment and/or prevention of disease.
背景技术Background technique
人***瘤病毒(HPV)感染是几乎所有浸润性***和部分***生殖器恶性肿瘤和口腔癌的致病原因。以***为例，人***瘤病毒的持续感染是导致子***的主要致病因素，当前已发展为世界上第四大常见女性癌症，疾病负担十分严重。Human papillomavirus (HPV) infection is the cause of almost all invasive cervical cancers and some anogenital malignancies and oral cancers. Taking cervical cancer as an example, persistent infection with human papillomavirus is the main causative factor of cervical cancer. It has now become the fourth most common female cancer in the world, with a very serious disease burden.
随着精准医疗号角的吹起，各种免疫治疗的肿瘤靶向药踊跃出现。其中肿瘤疫苗因其高度特异性，疗效强，不良反应发生率低等特性，获得了极高的研发关注。已有的HPV肿瘤免疫疗法以预防性HPV疫苗为主，已上市的预防性疫苗主要为HPV2价、4价及9价疫苗，可以针对特定的几种HPV高危分型及低危分型病毒进行免疫保护。但预防性疫苗以预防为主，并不能清除已存在的感染，对已感染的HPV患者来说并不能起到治愈的效果，HPV治疗性疫苗的研发及临床应用变得刻不容缓。With the clarion call of precision medicine, various immunotherapy tumor-targeted drugs are emerging. Among them, tumor vaccines have received great research and development attention because of their high specificity, strong efficacy, and low incidence of adverse reactions. Existing HPV tumor immunotherapy is mainly based on preventive HPV vaccines. The preventive vaccines that have been launched are mainly HPV 2-valent, 4-valent and 9-valent vaccines, which can be used to target specific types of HPV high-risk and low-risk types. immune protection. However, preventive vaccines are mainly for prevention and cannot clear existing infections. They cannot cure infected HPV patients. The development and clinical application of HPV therapeutic vaccines have become urgent.
HPV治疗性疫苗通过产生针对HPV病毒颗粒的中和抗体来刺激细胞介导的免疫反应，从而针对特异性靶标杀死受HPV感染的细胞。HPV病毒属人***瘤空泡病毒A属的双链球形DNA病毒，结构简单、无包膜。其基因组编码了6种早期调控蛋白(E1、E2、E4、E5、E6、E7)和两种晚期结构蛋白(L1和L2)。早期基因编码蛋白负责病毒DNA的复制、转录、癌变及转化。设计HPV治疗性疫苗通常针对的靶抗原基因为病毒早期蛋白E6和E7。E6和E7蛋白一直以来被认为是HPV相关研究中最关键的致癌蛋白。HPV病毒感染发生后，由病毒增殖衍生的E6、E7表达蛋白可以通过影响p53抑癌基因和PRB视网膜母细胞瘤蛋白来促进肿瘤后期的快速发生及发展。同时，两种蛋白序列在广泛的HPV亚型中均得到了良好的保护，可以作为HPV治疗性疫苗探索性研究中极具吸引力和应用性的靶标。HPV therapeutic vaccines stimulate cell-mediated immune responses by generating neutralizing antibodies against HPV virus particles, thereby targeting specific targets to kill HPV-infected cells. HPV virus is a double-stranded spherical DNA virus of the genus Human Papillomavirus A, with a simple structure and no envelope. Its genome encodes six early regulatory proteins (E1, E2, E4, E5, E6, E7) and two late structural proteins (L1 and L2). Early gene-encoded proteins are responsible for viral DNA replication, transcription, carcinogenesis, and transformation. The target antigen genes that are usually targeted when designing HPV therapeutic vaccines are the viral early proteins E6 and E7. E6 and E7 proteins have long been considered the most critical oncogenic proteins in HPV-related research. After HPV virus infection occurs, the E6 and E7 expression proteins derived from virus proliferation can promote the rapid occurrence and development of late-stage tumors by affecting the p53 tumor suppressor gene and PRB retinoblastoma protein. At the same time, both protein sequences are well protected across a wide range of HPV subtypes and can be used as very attractive and applicable targets in exploratory research on HPV therapeutic vaccines.
通常治疗性疫苗会将来源于靶标具有潜在治疗效力的靶抗原以多种形式传递给抗原呈递细胞(Antigen-Presenting Cell,APC)，从而递送至主要组织相容性复合物(Major histocompatibility complex，MHC)分子上，激活抗原特异性的CD8+毒性T细胞或CD4+辅助T细胞产生特异性免疫反应，以达到治疗作用。主要组织相容性复合物是一种存在于脊椎动物的庞大基因家族编码的细胞表面分子，其与机体免疫反应关系十分密切。在人类中，其又被称为人类白细胞抗原(human leukocyte antigen,HLA)。HLA具有高度多态性，化学本质是一类糖蛋白，在所有有核细胞中均有表达。Ⅰ类HLA抗原的特异性取决于HLA分子的α重链，由HLA-A、B、C位点编码，并由此衍生出HLA-A、B、C的不同基因分型。HLAⅠ类复合物的主要功能是将HLAⅠ类来源于宿主或外来物如病毒表达蛋白等的基因产物，通过胞内内源机制加工后翻译剪切形成关键的治疗性抗原肽，近而通过形成的MHC复合物将抗原肽呈递给CD8 +细胞毒性T细胞启动和调节特异性免疫应答。抗原多肽序列相关的抗原决定簇，即被称之为HLAⅠ类限制性抗原表位。因HLA基因的高度多态性，HLA-A、B、C基因来源的不同对应了HLA-A、B、C不同分型限制性抗原表位，在HPV相关的病毒感染病患研究中，HLA-A0201基因分型占比最高。同时，已知的HPV病毒分型，约有200种以上。综合生物学特性及致癌潜能，通常被分为高危性和低危性。低危型HPV如6、11、42、43、44等，常引起外生殖器疣等良性病变，高危型HPV分型如16、18、31、33、35、39、45、51、52、56、58、59、68等与***及上皮内病变关系密切，其中高危致病分型又以HPV16型在各类病患中占比最高，治疗性更为迫切。因此，鉴定HPV16型病毒的HLA-Ⅰ类限制性多肽表位对于治疗性疫苗的制备具有重要意义。 Usually therapeutic vaccines deliver target antigens with potential therapeutic efficacy from the target to antigen-presenting cells (APCs) in various forms, thereby delivering them to the major histocompatibility complex (MHC). ) Molecularly, it activates antigen-specific CD8+ toxic T cells or CD4+ helper T cells to produce a specific immune response to achieve a therapeutic effect. The major histocompatibility complex is a cell surface molecule encoded by a large gene family that exists in vertebrates and is closely related to the body's immune response. In humans, it is also called human leukocyte antigen (HLA). HLA is highly polymorphic, chemically a type of glycoprotein, and is expressed in all nucleated cells. The specificity of class I HLA antigens depends on the α heavy chain of the HLA molecule, which is encoded by the HLA-A, B, and C loci, and different genotypes of HLA-A, B, and C are derived from this. The main function of the HLA class I complex is to process the gene products of HLA class I derived from the host or foreign substances such as viral expressed proteins through intracellular endogenous mechanisms and then translate and cleave them to form key therapeutic antigen peptides. MHC complexes present antigenic peptides to CD8 + cytotoxic T cells to initiate and regulate specific immune responses. The antigenic determinants related to the antigenic polypeptide sequence are called HLA class I restricted antigenic epitopes. Due to the high degree of polymorphism of HLA genes, the different sources of HLA-A, B, and C genes correspond to the different types of HLA-A, B, and C-restricted antigen epitopes. In the study of patients with HPV-related viral infections, HLA -A0201 genotype has the highest proportion. At the same time, there are more than 200 known types of HPV viruses. Based on their biological characteristics and carcinogenic potential, they are usually divided into high risk and low risk. Low-risk HPV types such as 6, 11, 42, 43, and 44 often cause benign lesions such as external genital warts. High-risk HPV types include 16, 18, 31, 33, 35, 39, 45, 51, 52, and 56. , 58, 59, 68, etc. are closely related to cervical cancer and intraepithelial lesions. Among the high-risk pathogenic types, HPV16 accounts for the highest proportion among all types of patients, and treatment is more urgent. Therefore, identification of HLA-Ⅰ class-restricted polypeptide epitopes of HPV16 viruses is of great significance for the preparation of therapeutic vaccines.
发明内容Contents of the invention
本发明是基于发明人的下列发现而完成的：HPV治疗疫苗是免疫治疗领域的研究热点，其中，获得有效的HPV病毒表位是极其关键的，发明人通过对HLA-I类MHC-抗原肽复合物进行免疫亲和捕获，固相萃取复合物中的抗原肽，并采用超高效液相色谱-质谱技术对获得的抗原肽进行分析，从而鉴定出HPV病毒表位。The present invention is completed based on the following findings of the inventor: HPV therapeutic vaccines are a research hotspot in the field of immunotherapy, in which obtaining effective HPV viral epitopes is extremely critical. The complex is subjected to immunoaffinity capture, and the antigen peptides in the complex are solid-phase extracted, and the obtained antigen peptides are analyzed using ultra-high performance liquid chromatography-mass spectrometry technology to identify the HPV virus epitope.
由此，在本发明的第一方面，本发明提出了一种鉴定HPV抗原表位的方法。根据本发明的实施例，包括以下步骤：1)将HLA-I类抗体与载体以1mL：5mg-1mL：2mg的体积质量比进行结合，以获得免疫亲和柱；2)将HPV阳性细胞进行裂解处理；将步骤2)所获得的细胞裂解液进行过柱处理，所述柱为所述免疫亲和柱，以获得包含HPV抗原肽的HLA复合物；3)将所述包含HPV抗原肽的HLA复合物进行固相萃取，以获得肽段。发明人对鉴定HPV 抗原表位的方法进行了筛选和优化，当所述HLA-I类抗体与载体以1mL：5mg-1mL：2mg的体积质量比进行结合时，抗体的结合率较高，将获得的包含HPV抗原肽的HLA复合物进行固相萃取时鉴定通量较高，本发明实施例的方法显著提高了获得与所述HLA-I类分子有效结合的HPV抗原肽的概率，显著缩短了鉴定时间。Therefore, in the first aspect of the present invention, the present invention proposes a method for identifying HPV antigenic epitopes. According to an embodiment of the present invention, the following steps are included: 1) Combine HLA-I class antibodies and carriers at a volume mass ratio of 1mL:5mg-1mL:2mg to obtain an immunoaffinity column; 2) Conduct HPV-positive cells Lysis treatment; pass the cell lysate obtained in step 2) through a column, and the column is the immunoaffinity column to obtain an HLA complex containing HPV antigen peptides; 3) pass the HLA complex containing HPV antigen peptides HLA complexes were subjected to solid phase extraction to obtain peptide fragments. The inventors screened and optimized the method for identifying HPV epitopes. When the HLA-I class antibody is combined with the carrier at a volume-to-mass ratio of 1mL:5mg-1mL:2mg, the binding rate of the antibody is higher, and the antibody has a higher binding rate. The obtained HLA complex containing HPV antigen peptides has a high identification throughput when subjected to solid phase extraction. The method of the embodiment of the present invention significantly improves the probability of obtaining HPV antigen peptides that effectively bind to the HLA class I molecules and significantly shortens the identification time. identification time.
在本发明的第二方面，本发明提出了一种分离的肽。根据本发明的实施例，包括下列中的至少之一：1)HPV E6蛋白的65-72位氨基酸；2)HPV E7蛋白的4-11位氨基酸；3)HPV E7蛋白的75-82位氨基酸；4)HPV E7蛋白的75-83位氨基酸；5)HPV E7蛋白的77-86位氨基酸；6)HPV E7蛋白的77-90位氨基酸；以及7)HPV E7蛋白的79-88位氨基酸。根据本发明实施例的分离的肽是由HPV E6或E7早期编码区表达翻译获得的，所述分离的肽可以由HLA-I类分子呈递，被CTL细胞或TIL细胞识别，进而可被表达HLA-I类分子的呈递细胞递呈给CTL或TIL细胞而激活特异性T细胞免疫，构成了HPV阳性肿瘤的免疫应答的生理靶标，进行高灵敏度及特异性检测，对于HPV相关疾病的预防和治疗来说具有重要价值。In a second aspect of the invention, the invention provides an isolated peptide. According to embodiments of the present invention, it includes at least one of the following: 1) amino acids 65-72 of HPV E6 protein; 2) amino acids 4-11 of HPV E7 protein; 3) amino acids 75-82 of HPV E7 protein ; 4) Amino acids 75-83 of HPV E7 protein; 5) Amino acids 77-86 of HPV E7 protein; 6) Amino acids 77-90 of HPV E7 protein; and 7) Amino acids 79-88 of HPV E7 protein. The isolated peptide according to the embodiment of the present invention is obtained by expression and translation of the HPV E6 or E7 early coding region. The isolated peptide can be presented by HLA class I molecules, recognized by CTL cells or TIL cells, and can then be expressed by HLA - Presenting cells of class I molecules are presented to CTL or TIL cells to activate specific T cell immunity, constituting the physiological target of the immune response of HPV-positive tumors, performing high-sensitivity and specific detection for the prevention and treatment of HPV-related diseases is of great value.
在本发明的第三方面，本发明提出了一种HPV抗原表位。根据本发明的实施例，包括下列中的至少之一：1)HPV E6蛋白的65-72位氨基酸；2)HPV E7蛋白的4-11位氨基酸；3)HPV E7蛋白的75-82位氨基酸；4)HPV E7蛋白的75-83位氨基酸；5)HPV E7蛋白的77-86位氨基酸；6)HPV E7蛋白的77-90位氨基酸；以及7)HPV E7蛋白的79-88位氨基酸。根据本发明实施例的HPV抗原表位是由HPV E6或E7早期编码区表达翻译获得的，所述HPV抗原表位可以由HLA-I类分子呈递，被CTL或TIL细胞识别，进而可被表达HLA-I类分子的呈递细胞递呈给CTL或TIL细胞而激活特异性T细胞免疫，构成了HPV阳性肿瘤的免疫应答的生理靶标，进行高灵敏度及特异性检测，对于HPV相关疾病的预防和治疗来说具有重要价值。In the third aspect of the present invention, the present invention proposes an HPV antigenic epitope. According to embodiments of the present invention, it includes at least one of the following: 1) amino acids 65-72 of HPV E6 protein; 2) amino acids 4-11 of HPV E7 protein; 3) amino acids 75-82 of HPV E7 protein ; 4) Amino acids 75-83 of HPV E7 protein; 5) Amino acids 77-86 of HPV E7 protein; 6) Amino acids 77-90 of HPV E7 protein; and 7) Amino acids 79-88 of HPV E7 protein. The HPV antigenic epitope according to the embodiment of the present invention is obtained by expression and translation of the HPV E6 or E7 early coding region. The HPV antigenic epitope can be presented by HLA-I class molecules, recognized by CTL or TIL cells, and then can be expressed Presenting cells of HLA-I molecules are presented to CTL or TIL cells to activate specific T cell immunity, which constitutes the physiological target of the immune response of HPV-positive tumors. Highly sensitive and specific detection is required for the prevention and treatment of HPV-related diseases. It is of great value for treatment.
在本发明的第四方面，本发明提出了一种突变体。根据本发明的实施例，相较于野生型HPV E6蛋白，所述突变体在除锚定位外具有至少一个突变位点。根据本发明的具体实施例，获得的HPV抗原表位的第2和9位(对于8肽来说，为第8位)氨基酸为HLA的锚定位，表位中的其他位点氨基酸进行取代后，依然可以具备相同或相关的免疫原性及其潜在治疗效果，即可以由HLA-I类分子呈递，被CTL细胞或TIL细胞识别，进而可被表达HLA-I类分子的呈递细胞递呈给CTL或TIL细胞而激活特异性T细胞免疫，构成了HPV阳性肿瘤的免疫应答的生理靶标，进行高灵敏度及特异性检测，对于HPV相关疾病的预防和治疗来说具有重要价值。In a fourth aspect of the invention, the invention provides a mutant. According to embodiments of the present invention, compared to wild-type HPV E6 protein, the mutant has at least one mutation site except for the anchor position. According to specific embodiments of the present invention, the amino acids at positions 2 and 9 (for 8 peptides, position 8) of the obtained HPV antigenic epitope are the anchor positions of HLA, and other amino acids in the epitope are substituted. , can still have the same or related immunogenicity and potential therapeutic effect, that is, it can be presented by HLA class I molecules, recognized by CTL cells or TIL cells, and then can be presented by presentation cells expressing HLA class I molecules. CTL or TIL cells activate specific T cell immunity, which constitutes the physiological target of the immune response of HPV-positive tumors. Highly sensitive and specific detection is of great value for the prevention and treatment of HPV-related diseases.
在本发明的第五方面，本发明提出了一种突变体。根据本发明的实施例，相较于野生型HPV E7蛋白，所述突变体在除锚定位外具有至少一个突变位点。根据本发明的具体实施例，获得的HPV抗原表位的第2和9位(对于8肽来说，为第8位)氨基酸为HLA的锚定位，表位中的其他位点氨基酸进行取代后，依然可以具备相同或相关的免疫原性及其潜在治疗效果，即可以由HLA-I类分子呈递，被CTL细胞或TIL细胞识别，进而可被表达HLA-I类分子的呈递细胞递呈给CTL或TIL细胞而激活特异性T细胞免疫，构成了HPV阳性肿瘤的免疫应答的生理靶标，进行高灵敏度及特异性检测，对于HPV相关疾病的预防和治疗来说具有重要价值。In a fifth aspect of the invention, the invention provides a mutant. According to embodiments of the present invention, compared to wild-type HPV E7 protein, the mutant has at least one mutation site except for the anchor position. According to specific embodiments of the present invention, the amino acids at positions 2 and 9 (for 8 peptides, position 8) of the obtained HPV antigenic epitope are the anchor positions of HLA, and other amino acids in the epitope are substituted. , can still have the same or related immunogenicity and potential therapeutic effect, that is, it can be presented by HLA class I molecules, recognized by CTL cells or TIL cells, and then can be presented by presentation cells expressing HLA class I molecules. CTL or TIL cells activate specific T cell immunity, which constitutes the physiological target of the immune response of HPV-positive tumors. Highly sensitive and specific detection is of great value for the prevention and treatment of HPV-related diseases.
在本发明的第六方面，本发明提出了一种核酸分子。根据本发明的实施例，所述核酸分子编码第二方面所述的分离的肽、第三方面所述的HPV抗原表位、或第四方面或第五方面所述的突变体。根据本发明实施例的核酸分子编码获得的分离的肽、HPV抗原表位或突变体可以由HLA-I类分子呈递，被CTL或TIL细胞识别，即被表达HLA-I类分子的呈递细胞递呈给CTL或TIL细胞而激活特异性T细胞免疫，构成了HPV阳性肿瘤的免疫应答的生理靶标，进行高灵敏度及特异性检测，对于HPV相关疾病的预防和治疗来说具有重要价值。In a sixth aspect of the invention, the invention provides a nucleic acid molecule. According to an embodiment of the present invention, the nucleic acid molecule encodes the isolated peptide described in the second aspect, the HPV epitope described in the third aspect, or the mutant described in the fourth or fifth aspect. The isolated peptides, HPV epitopes or mutants encoded by the nucleic acid molecules according to the embodiments of the present invention can be presented by HLA class I molecules and recognized by CTL or TIL cells, that is, presented by presentation cells expressing HLA class I molecules. Presented to CTL or TIL cells to activate specific T cell immunity, which constitutes the physiological target of the immune response of HPV-positive tumors. Highly sensitive and specific detection is of great value for the prevention and treatment of HPV-related diseases.
在本发明的第七方面，本发明提出了一种表达载体。根据本发明的实施例，携带表达第二方面所述的分离的肽或第三方面所述的HPV抗原表位或第四方面或第五方面所述的突变体的核酸或第六方面所述的核酸分子。所述表达载体可包括可选的控制序列，所述控制序列与所述核酸分子可操作地连接。其中，所述控制序列为可指导多肽在宿主中表达的一个或多个控制序列。本发明实施例所提出的表达载体可在适合的宿主细胞中高效表达所述分离的肽、HPV抗原表位或突变体，进而可有效用于对肿瘤，特别是同时表达HLA-I类分子和上述分离的肽或HPV抗原表位的肿瘤的特异性治疗或预防。In the seventh aspect of the present invention, the present invention provides an expression vector. According to embodiments of the present invention, nucleic acids carrying the isolated peptides described in the second aspect or the HPV epitopes described in the third aspect or the mutants described in the fourth or fifth aspects or the sixth aspect are nucleic acid molecules. The expression vector may include optional control sequences operably linked to the nucleic acid molecule. Wherein, the control sequence is one or more control sequences that can direct the expression of the polypeptide in the host. The expression vector proposed in the embodiment of the present invention can efficiently express the isolated peptide, HPV epitope or mutant in a suitable host cell, and can be effectively used to treat tumors, especially the simultaneous expression of HLA-I class molecules and Specific treatment or prevention of tumors with the above isolated peptides or HPV epitopes.
在本发明的第八方面，本发明提出了一种重组细胞。根据本发明的实施例，携带前面所述的核酸分子、表达载体、分离的肽、HPV抗原表位或突变体。所述重组细胞是通过转染或者转化所述表达载体获得的。根据本发明的实施例，所述宿主细胞在合适条件下可高效表达上述分离的肽、HPV抗原表位或突变体，所述重组细胞可有效用于对肿瘤，特别是同时表达HLA-I类分子和上述分离的肽或HPV抗原表位的肿瘤的特异性治疗或预防。In an eighth aspect of the present invention, the present invention provides a recombinant cell. According to embodiments of the present invention, the aforementioned nucleic acid molecules, expression vectors, isolated peptides, HPV epitopes or mutants are carried. The recombinant cells are obtained by transfection or transformation of the expression vector. According to embodiments of the present invention, the host cells can efficiently express the above-mentioned isolated peptides, HPV epitopes or mutants under appropriate conditions, and the recombinant cells can be effectively used to treat tumors, especially those that simultaneously express HLA-I class. Molecules and the above isolated peptides or HPV epitopes for specific treatment or prevention of tumors.
在本发明的第九方面，本发明提出了一种抗原呈递细胞。根据本发明的实施例，所述细胞可呈递前面所述的分离的肽、HPV抗原表位或突变体。根据本发明的实施例，呈递前面所述分离的肽、HPV抗原表位或突变体的抗原呈递细胞可有效引起患者针对肿瘤特异性抗原-上述分离的肽/HPV抗原表位/突变体的免疫反应，进而激活CTL特异性杀伤功能，本发明实施例所提出的抗原呈递细胞具有显著的治疗表达上述分离的肽、HPV抗原表位或突变体的肿瘤的功效，其治疗的效果显著，安全性高。In a ninth aspect of the present invention, the present invention provides an antigen-presenting cell. According to embodiments of the invention, the cells may present isolated peptides, HPV epitopes or mutants as described above. According to embodiments of the present invention, antigen-presenting cells presenting the aforementioned isolated peptides, HPV epitopes or mutants can effectively induce immunity in patients against tumor-specific antigens-the aforementioned isolated peptides/HPV epitopes/mutants. reaction, thereby activating the specific killing function of CTL. The antigen-presenting cells proposed in the embodiments of the present invention have significant efficacy in treating tumors expressing the above-mentioned isolated peptides, HPV epitopes or mutants, and their therapeutic effects are significant and safe. high.
在本发明的第十方面，本发明提出了一种免疫效应细胞。根据本发明的实施例，所述免疫效应细胞可识别前面所述的多肽、HPV抗原表位、突变体或识别在细胞表面呈递前面所述的多肽或所述的HPV抗原表位或突变体的抗原呈递细胞。根据本发明的实施例，所述免疫效应细胞可特异性杀伤共表达HLA-I类分子和上述分离的肽、HPV抗原表位或突变体的肿瘤细胞。In a tenth aspect of the present invention, the present invention provides an immune effector cell. According to embodiments of the present invention, the immune effector cells can recognize the polypeptides, HPV epitopes, and mutants described above, or recognize the polypeptides or HPV epitopes or mutants that are presented on the cell surface. Antigen presenting cells. According to embodiments of the present invention, the immune effector cells can specifically kill tumor cells that co-express HLA class I molecules and the above-mentioned isolated peptides, HPV epitopes or mutants.
在本发明的第十一方面，本发明提出了试剂在制备试剂盒中的用途，所述试剂用于检测前面所述的分离的肽、HPV抗原表位或突变体。根据本发明的实施例，所述试剂盒用于诊断HPV或者检测HPV的治疗效果。所述试剂可以对所述分离的肽、HPV抗原表位或突变体进行准确检测，如，检测生物样品中是否含有所述分离的肽、HPV抗原表位或突变体， 由于所述分离的肽、HPV抗原表位或突变体在被HPV感染的组织中高表达，因此，包含所述试剂的试剂盒可以准确诊断生物样品来源的个体是否被HPV感染，进一步地，是否为HPV高风险个体。同理，被HPV感染的个体在治疗过程中，利用所述试剂盒进行检测，可以检测治疗过程中HPV的变化，如加重、减缓或治愈等。In an eleventh aspect of the present invention, the present invention proposes the use of reagents for detecting the previously described isolated peptides, HPV epitopes or mutants in the preparation of kits. According to an embodiment of the present invention, the kit is used to diagnose HPV or detect the therapeutic effect of HPV. The reagent can accurately detect the isolated peptide, HPV epitope or mutant, for example, detect whether a biological sample contains the isolated peptide, HPV epitope or mutant, because the isolated peptide , HPV antigenic epitopes or mutants are highly expressed in HPV-infected tissues. Therefore, a kit containing the reagent can accurately diagnose whether the individual from whom the biological sample is derived is infected by HPV, and further, whether it is an individual at high risk for HPV. In the same way, during the treatment process of an individual infected by HPV, the test kit can be used to detect the changes of HPV during the treatment process, such as aggravation, slowing down or cure, etc.
在本发明的第十二方面，本发明提出了一种试剂盒。根据本发明的实施例，包括适于检测前面所述的分离的肽、HPV抗原表位或突变体的试剂。所述试剂可以对所述分离的肽、HPV抗原表位或突变体进行准确检测，如，检测生物样品中是否含有所述分离的肽、HPV抗原表位或突变体，由于所述分离的肽、HPV抗原表位或突变体在被HPV感染的组织中高表达，因此，包含所述试剂的试剂盒可以准确诊断生物样品来源的个体是否被HPV感染，进一步地，是否为HPV高风险个体。同理，被HPV感染的个体在治疗过程中，利用所述试剂盒进行检测，可以检测治疗过程中HPV的变化，如加重、减缓或治愈等。In a twelfth aspect of the present invention, the present invention provides a kit. According to embodiments of the present invention, reagents suitable for detecting isolated peptides, HPV epitopes or mutants as described above are included. The reagent can accurately detect the isolated peptide, HPV epitope or mutant, for example, detect whether a biological sample contains the isolated peptide, HPV epitope or mutant, because the isolated peptide , HPV antigenic epitopes or mutants are highly expressed in HPV-infected tissues. Therefore, a kit containing the reagent can accurately diagnose whether the individual from whom the biological sample is derived is infected by HPV, and further, whether it is an individual at high risk for HPV. In the same way, during the treatment process of an individual infected by HPV, the test kit can be used to detect the changes of HPV during the treatment process, such as aggravation, slowing down or cure, etc.
在本发明的第十三方面，本发明提出了前面所述的分离的肽、HPV抗原表位、突变体、核酸分子、表达载体、重组细胞、抗原呈递细胞或免疫效应细胞在制备药物中的用途。根据本发明的实施例，所述药物用于治疗或者预防HPV相关疾病。如前所述，所述分离的肽、HPV抗原表位、突变体、编码所述分离的肽或HPV抗原表位或突变体的核酸分子、表达载体、重组细胞、抗原呈递细胞或免疫效应细胞均可有效用于对肿瘤，特别是同时表达HLA-I类分子和上述分离的肽、HPV抗原表位或突变体的肿瘤的特异性治疗或预防，因此，包含部分或全部上述物质的药物同样具有显著的治疗或预防表达HLA-I类分子和所述分离的肽、HPV抗原表位或突变体的肿瘤的作用，其安全性更高、副作用更小。In the thirteenth aspect of the present invention, the present invention proposes the use of the previously described isolated peptides, HPV epitopes, mutants, nucleic acid molecules, expression vectors, recombinant cells, antigen-presenting cells or immune effector cells in the preparation of medicines. use. According to embodiments of the present invention, the medicine is used to treat or prevent HPV-related diseases. As mentioned above, the isolated peptide, HPV epitope, mutant, nucleic acid molecule encoding the isolated peptide or HPV epitope or mutant, expression vector, recombinant cell, antigen presenting cell or immune effector cell All can be effectively used for the specific treatment or prevention of tumors, especially tumors that express both HLA class I molecules and the above-mentioned isolated peptides, HPV epitopes or mutants. Therefore, drugs containing some or all of the above substances are also It has a significant effect of treating or preventing tumors expressing HLA class I molecules and the isolated peptide, HPV antigen epitope or mutant, and has higher safety and smaller side effects.
在本发明的第十四方面，本发明提出了一种药物。根据本发明的实施例，包含前面所述的分离的肽、HPV抗原表位、突变体、核酸分子、表达载体、重组细胞、抗原呈递细胞或免疫效应细胞。如前所述，所述分离的肽、HPV抗原表位、突变体、编码所述分离的肽或HPV抗原表位或突变体的核酸分子、表达载体、重组细胞、抗原呈递细胞或免疫效应细胞均可有效用于对肿瘤，特别是同时表达HLA-I类分子和上述分离的肽、HPV抗原表位或突变体的肿瘤的特异性治疗或预防，因此，包含部分或全部上述物质的药物同样具有显著的治疗或预防表达HLA-I类分子和所述分离的肽、HPV抗原表位或突变体的肿瘤的作用，其安全性更高、副作用更小。In a fourteenth aspect of the invention, the invention provides a medicament. According to embodiments of the present invention, the aforementioned isolated peptides, HPV epitopes, mutants, nucleic acid molecules, expression vectors, recombinant cells, antigen-presenting cells or immune effector cells are included. As mentioned above, the isolated peptide, HPV epitope, mutant, nucleic acid molecule encoding the isolated peptide or HPV epitope or mutant, expression vector, recombinant cell, antigen presenting cell or immune effector cell All can be effectively used for the specific treatment or prevention of tumors, especially tumors that express both HLA class I molecules and the above-mentioned isolated peptides, HPV epitopes or mutants. Therefore, drugs containing some or all of the above substances are also It has a significant effect of treating or preventing tumors expressing HLA class I molecules and the isolated peptide, HPV antigen epitope or mutant, and has higher safety and smaller side effects.
在本发明的第十五方面，本发明提出了一种疫苗。根据本发明的实施例，包含前面所述的分离的肽、HPV抗原表位、突变体、核酸分子、表达载体或抗原呈递细胞。如前所述，本发明实施例的核酸分子、表达载体或重组细胞在合适的条件下表达前面所述的分离的肽、HPV抗原表位或突变体，抗原呈递细胞可以表达所述分离的肽、HPV抗原表位或突变体，当其与HLA-I类分子结合后，被抗原呈递细胞呈递，使其被CTL或TIL细胞识别，即被表达HLA-I类分子的抗原呈递细胞递呈给CTL或TIL细胞而激活特异性T细胞免疫。因此，本发明实施例所提出的疫苗具有显著的治疗或预防表达HLA-I类分子和所述分离的肽、HPV抗原表位或突变体的肿瘤的作用，其安全性更高、副作用更小。In a fifteenth aspect of the invention, the invention provides a vaccine. According to embodiments of the present invention, the aforementioned isolated peptides, HPV epitopes, mutants, nucleic acid molecules, expression vectors or antigen-presenting cells are included. As mentioned above, the nucleic acid molecules, expression vectors or recombinant cells of the embodiments of the present invention express the aforementioned isolated peptides, HPV epitopes or mutants under appropriate conditions, and the antigen-presenting cells can express the isolated peptides. , HPV antigenic epitopes or mutants, when combined with HLA-I class molecules, are presented by antigen-presenting cells, allowing them to be recognized by CTL or TIL cells, that is, they are presented to antigen-presenting cells expressing HLA-I class molecules. CTL or TIL cells activate specific T cell immunity. Therefore, the vaccine proposed in the embodiments of the present invention has a significant effect in treating or preventing tumors expressing HLA class I molecules and the isolated peptides, HPV epitopes or mutants, and is safer and has fewer side effects. .
在本发明的第十六方面，本发明提出了一种治疗和/或预防HPV相关疾病的方法。根据本发明的实施例，给与受试者前面所述的分离的肽、HPV抗原表位、突变体、核酸分子、表达载体、重组细胞、抗原呈递细胞、免疫效应细胞、药物或疫苗。如前所述，本发明实施例所提出的治疗和/或预防方法，包括给予任一种有效量的前面所述的分离的肽、突变体等相关物质，均可有效治疗或预防表达HLA-I类分子和所述分离的肽或HPV抗原表位的肿瘤。In a sixteenth aspect of the present invention, the present invention provides a method for treating and/or preventing HPV-related diseases. According to embodiments of the present invention, the subject is administered the aforementioned isolated peptides, HPV epitopes, mutants, nucleic acid molecules, expression vectors, recombinant cells, antigen-presenting cells, immune effector cells, drugs or vaccines. As mentioned above, the treatment and/or prevention methods proposed in the embodiments of the present invention, including administering any effective amount of the aforementioned isolated peptides, mutants and other related substances, can effectively treat or prevent the expression of HLA- Class I molecules and the isolated peptides or HPV epitopes of tumors.
在本发明的第十七方面，本发明提出了前面所述的分离的肽、HPV抗原表位、突变体、核酸分子、表达载体、重组细胞在制备试剂盒中的用途。根据本发明的实施例，所述试剂盒用于检测HLA。根据本发明实施例的分离的肽、HPV抗原表位、突变体及能够间接获得所述分离的肽、HPV抗原表位或突变体的物质均可与HLA进行结合，因此，上述物质可用于制备有效检测HLA的试剂盒，所述试剂盒可以准确的定性或定量检测生物样本的HLA，更进一步地，也可对个体中HLA水平进行检测，以判断所述个体的状态，如所述个体的HLA水平显著低于或高于正常水平。In the seventeenth aspect of the present invention, the present invention proposes the use of the previously described isolated peptides, HPV epitopes, mutants, nucleic acid molecules, expression vectors, and recombinant cells in the preparation of kits. According to an embodiment of the present invention, the kit is used to detect HLA. The isolated peptides, HPV epitopes, mutants and materials that can indirectly obtain the isolated peptides, HPV epitopes or mutants according to the embodiments of the present invention can be combined with HLA. Therefore, the above materials can be used to prepare A kit for effectively detecting HLA. The kit can accurately qualitatively or quantitatively detect HLA in biological samples. Furthermore, it can also detect the HLA level in an individual to determine the status of the individual, such as the individual's status. HLA levels are significantly lower or higher than normal levels.
在本发明的第十八方面，本发明提出了一种检测HLA的试剂盒。根据本发明的实施例，包括第前面所述的分离的肽、HPV抗原表位、突变体、核酸分子、表达载体、重组细胞。如前所述，分离的肽、HPV抗原表位、突变体及能够间接获得所述分离的肽、HPV抗原表位或突变体的物质均可与HLA进行结合，因此，包含上述物质的试剂盒可以准确的定性或定量检测生物样本的HLA，更进一步地，也可对个体中HLA水平进行检测，以判断所述个体的状态，如所述个体的HLA水平显著低于或高于正常水平。In the eighteenth aspect of the present invention, the present invention provides a kit for detecting HLA. According to embodiments of the present invention, the above-mentioned isolated peptides, HPV epitopes, mutants, nucleic acid molecules, expression vectors, and recombinant cells are included. As mentioned above, isolated peptides, HPV epitopes, mutants, and substances that can indirectly obtain the isolated peptides, HPV epitopes, or mutants can bind to HLA. Therefore, a kit containing the above substances The HLA of biological samples can be accurately detected qualitatively or quantitatively. Furthermore, the HLA level in an individual can also be detected to determine the status of the individual. For example, the HLA level of the individual is significantly lower or higher than the normal level.
在本发明的第十九方面，本发明提出了一种诊断受试者是否患有HPV相关疾病的方法。根据本发明的实施例，包括检测受试者来源的生物样品是否携带前面所述的分离的肽、HPV抗原表位、突变体、核酸分子的步骤。如前所述，所述的分离的肽、HPV抗原表位、突变体及核酸分子存在于被HPV感染的个体内，因此，通过检测来源于受试者的生物样品是否携带所述物质，可以有效诊断受试者是否患有或易患HPV相关疾病。In a nineteenth aspect of the present invention, the present invention provides a method for diagnosing whether a subject suffers from HPV-related diseases. According to an embodiment of the present invention, it includes the step of detecting whether the biological sample derived from the subject carries the aforementioned isolated peptide, HPV epitope, mutant, or nucleic acid molecule. As mentioned above, the isolated peptides, HPV epitopes, mutants and nucleic acid molecules are present in individuals infected with HPV. Therefore, by detecting whether biological samples derived from subjects carry the substances, it is possible to Effectively diagnose whether subjects suffer from or are susceptible to HPV-related diseases.
在本发明的第二十方面，本发明提出了一种诊断***。根据本发明的实施例，包括：肽检测装置，所述肽检测装置用于检测受试者来源的生物样品是否携带前面所述的分离的肽、HPV抗原表位或突变体；诊断结果确定装置，所述诊断结果确定装置与所述肽检测装置相连，用于基于所述生物样品是否携带所述分离的肽、HPV抗原表位或突变体，确定所述患者是否患肿瘤。如前所述，所述分离的肽、HPV抗原表位或突变体存在于被HPV感染的受试者体内，根据本发明实施例的诊断***能够检测所述生物样品是否携带所述分离的肽、HPV抗原表位或突变体，因此，所述诊断***可以准确判断所述生物样品来源的受试者是否为肿瘤患者。In a twentieth aspect of the invention, a diagnostic system is provided. According to an embodiment of the present invention, it includes: a peptide detection device, the peptide detection device is used to detect whether a biological sample derived from a subject carries the previously described isolated peptide, HPV epitope or mutant; a diagnostic result determination device , the diagnostic result determination device is connected to the peptide detection device, and is used to determine whether the patient suffers from a tumor based on whether the biological sample carries the isolated peptide, HPV epitope or mutant. As mentioned above, the isolated peptide, HPV epitope or mutant exists in a subject infected by HPV, and the diagnostic system according to the embodiment of the present invention can detect whether the biological sample carries the isolated peptide. , HPV antigen epitope or mutant, therefore, the diagnostic system can accurately determine whether the subject from which the biological sample is derived is a tumor patient.
在本发明的第二十一方面，本发明提出了前面所述的分离的肽、突变体、表达载体、抗原呈递细胞、免疫效应细胞、药物或疫苗在治疗和/或预防HPV相关疾病中的用途。根据本发明实施例的一些具体实施例，所述分离的肽及其突变体能够有效的抑制肿瘤生长，因此，所述分离的肽或突变体、能够表达或包含所述分离的肽或突变体的核酸、表达载体、细胞、药物、疫苗均能够有效治疗和/或预防HPV相关疾病。In the twenty-first aspect of the present invention, the present invention proposes the use of the previously described isolated peptides, mutants, expression vectors, antigen-presenting cells, immune effector cells, drugs or vaccines in the treatment and/or prevention of HPV-related diseases. use. According to some specific embodiments of the present invention, the isolated peptide and its mutants can effectively inhibit tumor growth. Therefore, the isolated peptide or mutant can express or contain the isolated peptide or mutant. Nucleic acids, expression vectors, cells, drugs, and vaccines can effectively treat and/or prevent HPV-related diseases.
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。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 embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described one by one here.
附图说明Description of drawings
图1显示了本发明的诊断***的结构图；Figure 1 shows a structural diagram of the diagnostic system of the present invention;
图2显示了本发明的研究技术方案流程图；Figure 2 shows the flow chart of the research technical solution of the present invention;
图3-A和3-B显示了表位E7 77-86RTLEDLLMGT(二价前离子574.8.26++)验证阳性数据分析图谱，其中，Retention Time表示保留时间，Intensity表示密度，Replicate表示重复数，Peak Area percentage表示峰值面积的占比； Figures 3-A and 3-B show the epitope E7 77-86 RTLEDLLMGT (divalent precursor ion 574.8.26++) verification positive data analysis chart, where Retention Time represents retention time, Intensity represents density, and Replicate represents the number of repeats. , Peak Area percentage represents the proportion of peak area;
图4-A和4-B显示了表位E7 75-83DIRTLEDLL(二价前体离子-544.3033++)验证分析图谱； Figures 4-A and 4-B show the epitope E7 75-83 DIRTLEDLL (divalent precursor ion-544.3033++) verification analysis spectrum;
图5-A和5-B显示了表位E7 4-11DTPTLHEY(二价前体离子-488.2245++)验证分析图谱； Figures 5-A and 5-B show the epitope E7 4-11 DTPTLHEY (divalent precursor ion-488.2245++) verification analysis spectrum;
图6-A和6-B显示了表位E7 75-82DIRTLEDL(二价前体离子-487.7613++)验证分析图谱； Figures 6-A and 6-B show the epitope E7 75-82 DIRTLEDL (divalent precursor ion-487.7613++) verification analysis spectrum;
图7-A和7-B显示了表位E7 79-88LEDLLMGTLG(二价前体离子-531.2810++)验证分析图谱； Figures 7-A and 7-B show epitope E7 79-88 LEDLLMGTG (divalent precursor ion-531.2810++) verification analysis spectrum;
图8-A和8-B显示了表位E6 65-72NPYAVCDK(二价前体离子-488.7211++)验证分析图谱； Figures 8-A and 8-B show the epitope E6 65-72 NPYAVCDK (divalent precursor ion-488.7211++) verification analysis spectrum;
图9-A和9-B显示了表位E7 77-90RTLEDLLMGTLGIV(三价前体离子-510.9568+++)验证分析图谱； Figures 9-A and 9-B show the epitope E7 77-90 RTLEDLLMGTLGIV (trivalent precursor ion-510.9568+++) verification analysis spectrum;
图10显示了质谱检测***中多肽携带离子情况的举例分析图；Figure 10 shows an example analysis diagram of ions carried by polypeptides in the mass spectrometry detection system;
图11显示了HPV特异性CTL细胞制备的工艺流程图。Figure 11 shows the process flow diagram for preparation of HPV-specific CTL cells.
具体实施方式Detailed ways
下面详细描述本发明的实施例，所述实施例的示例在附图中示出。下面通过参考附图描述的实施例是示例性的，旨在用于解释本发明，而不能理解为对本发明的限制。Embodiments of the invention are described in detail below, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the drawings are exemplary and are intended to explain the present invention and are not to be construed as limiting the present invention.
此外，术语“第一”、“第二”仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此，限定有“第一”、“第二”的特征可以明示或者隐含地包括至少一个该特征。在本发明的描述中，“多个”的含义是至少两个，例如两个，三个等，除非另有明确具体的限定。In addition, the terms “first” and “second” are used for descriptive purposes only and cannot be understood as indicating or implying relative importance or implicitly indicating the quantity of indicated technical features. Therefore, features defined as "first" and "second" may explicitly or implicitly include at least one of these features. In the description of the present invention, "plurality" means at least two, such as two, three, etc., unless otherwise expressly and specifically limited.
在本文中所披露的范围的端点和任何值都不限于该精确的范围或值，这些范围或值应当理解为包含接近这些范围或值的值。对于数值范围来说，各个范围的端点值之间、各个范围的端点值和单独的点值之间，以及单独的点值之间可以彼此组合而得到一个或多个新的数值范围，这些数值范围应被视为在本文中具体公开。The endpoints of ranges and any values disclosed herein are not limited to the precise range or value, but these ranges or values are to be understood to include values approaching such ranges or values. For numerical ranges, the endpoint values of each range, the endpoint values of each range and individual point values, and the individual point values can be combined with each other to obtain one or more new numerical ranges. These values The scope shall be deemed to be specifically disclosed herein.
为了更容易理解本发明，以下具体定义了某些技术和科学术语。除显而易见在本文件中的它处另有明确定义，否则本文中使用的所有其它技术和科学术语都具有本发明所属领域的一般技术人员通常理解的含义。氨基酸残基的缩写是本领域中所用的指代20个常用L-氨基酸之一的标准3字母和/或1字母代码。In order to make the present invention easier to understand, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Abbreviations for amino acid residues are the standard 3-letter and/or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.
本文中，“表位”，又称抗原决定簇，是指免疫应答中被特异的效应分子或T淋巴细胞、B淋巴细胞识别的抗原分子的特定结构位点，从而诱导细胞免疫和体液免疫，产生免疫效应。如，本申请中的HPV抗原表位存在于HPV中，能够与HLA-I类分子结合。In this article, "epitope", also known as antigenic determinant, refers to the specific structural site of the antigen molecule recognized by specific effector molecules or T lymphocytes and B lymphocytes in the immune response, thereby inducing cellular immunity and humoral immunity. produce an immune effect. For example, the HPV antigenic epitope in this application exists in HPV and can bind to HLA class I molecules.
本文中，“锚定位”是指HPV抗原表位与HLA-I类分子结合时，HLA-I类分子接纳HPV抗原表位的结构，是位于该分子远膜端的抗原结合槽，分析天然HPV抗原表位的一级结构，发现都带有两个或两个以上与HLA-I类分子抗原结合槽相结合的特定部位，称锚定位。该位置的氨基酸残基称为锚定残基(anchor residue)。In this article, "anchoring" refers to the structure of the HLA-I molecule that accepts the HPV epitope when the HPV epitope binds to the HLA class I molecule. It is the antigen-binding groove located at the far membrane end of the molecule. Analysis of natural HPV antigens The primary structure of epitopes is found to have two or more specific sites that bind to the antigen-binding groove of HLA class I molecules, called anchor sites. The amino acid residue at this position is called the anchor residue.
本文中，“抗原呈递细胞”是指能够摄取、加工处理抗原，并将处理过的抗原呈递给T细胞的一类免疫细胞。APC主要包括单核-吞噬细胞、树突状细胞、B细胞、朗格汉斯细胞以及肿瘤细胞的病毒感染的靶细胞等。In this article, “antigen-presenting cells” refers to a type of immune cells that can take in and process antigens, and present the processed antigens to T cells. APC mainly includes monocytes-phagocytic cells, dendritic cells, B cells, Langerhans cells, and virus-infected target cells of tumor cells.
本文中，“免疫效应细胞”是指在免疫应答中参与清除异物抗原和行使效应功能的免疫细胞。As used herein, “immune effector cells” refer to immune cells that participate in the elimination of foreign antigens and perform effector functions in immune responses.
鉴定方法Identification method
一方面，本发明提出了一种鉴定HPV抗原表位的方法，包括以下步骤：1)将HLA-I类抗体与载体以1mL：5mg的体积质量比进行结合，以获得免疫亲和柱；2)将HPV阳性细胞进行裂解处理；将步骤2)所获得的细胞裂解液进行过柱处理，所述柱为所述免疫亲和柱，以获得包含HPV抗原肽的HLA复合物；3)将所述包含HPV抗原肽的HLA复合物进行固相萃取，以获得肽段。根据本发明实施例的方法显著提高了获得与所述HLA-I类分子有效结合的HPV抗原表位的概率，显著提高了鉴定效率和鉴定的准确性。On the one hand, the present invention proposes a method for identifying HPV epitopes, which includes the following steps: 1) Combine HLA-I class antibodies and carriers at a volume-to-mass ratio of 1 mL: 5 mg to obtain an immunoaffinity column; 2 ) Lyse HPV-positive cells; pass the cell lysate obtained in step 2) through a column, and the column is the immunoaffinity column to obtain an HLA complex containing HPV antigen peptides; 3) pass the cell lysate obtained in step 2) The HLA complex containing HPV antigen peptides is subjected to solid phase extraction to obtain peptide fragments. The method according to the embodiment of the present invention significantly improves the probability of obtaining HPV antigen epitopes that effectively bind to the HLA class I molecule, and significantly improves the identification efficiency and identification accuracy.
根据本发明的一些具体实施方案，所述裂解处理是在裂解液中进行，所述HPV阳性细胞在所述裂解液中的终浓度为0.5e 8-1e 8/mL。 According to some specific embodiments of the present invention, the lysis treatment is performed in a lysis solution, and the final concentration of the HPV-positive cells in the lysis solution is 0.5e 8 -1e 8 /mL.
根据本发明的一些具体实施方案，所述HLA-I类为anti-HLA-Ⅰ类A、B、C抗体(W6/32，ATCC HB-95)。According to some specific embodiments of the invention, the HLA-I class is anti-HLA-I class A, B, C antibodies (W6/32, ATCC HB-95).
根据本发明的一些具体实施方案，所述载体为Protein A琼脂糖凝胶。According to some specific embodiments of the invention, the carrier is Protein A Sepharose.
根据本发明的一些具体实施方案，所述HPV阳性细胞为HPV 16阳性细胞。According to some specific embodiments of the invention, the HPV-positive cells are HPV 16-positive cells.
根据本发明的一些具体实施方案，所述HPV 16阳性细胞为Caski细胞和/或Siha细胞。According to some specific embodiments of the invention, the HPV 16-positive cells are Caski cells and/or Siha cells.
根据本发明的一些具体实施方案，进一步包括以下步骤：5)对步骤4)获得的所述肽段进行检测，以便获得所述HPV抗原表位。根据本发明的一些具体实施方案，所述检测包括PRM靶向质谱分析，以对获得的所述肽段的序列进 行鉴定。According to some specific embodiments of the present invention, the method further includes the following steps: 5) detecting the peptide fragment obtained in step 4) to obtain the HPV antigen epitope. According to some specific embodiments of the present invention, the detection includes PRM targeted mass spectrometry analysis to identify the sequence of the obtained peptide fragment.
由于HPV基因调控等免疫逃逸效应的影响，免疫多肽表位的暴露往往会被降低至相对较低的呈递丰度水平，直接鉴定免疫多肽表位难度较大。已有的HPV质谱鉴定样品处理方法主要为通过免疫亲和纯化方法利于HLA免疫复合物抗体捕获生物样品中与之相互作用的HLA复合物分子，后通过萃取复取复合物分子中的肽段进行高分辨质谱鉴定而得以实现。本发明对流程中关键点的样品制备及仪器分析均做了***性优化，由此获得高质量的分析数据，以提高HPV免疫多肽表位鉴定成功的概率。Due to the influence of immune evasion effects such as HPV gene regulation, the exposure of immune peptide epitopes is often reduced to a relatively low presentation abundance level, making it difficult to directly identify immune peptide epitopes. Existing HPV mass spectrometry sample processing methods mainly use immunoaffinity purification methods to facilitate HLA immune complex antibodies to capture the HLA complex molecules that interact with them in biological samples, and then extract and re-extract the peptide fragments in the complex molecules. High-resolution mass spectrometry identification was achieved. The present invention systematically optimizes the sample preparation and instrument analysis at key points in the process, thereby obtaining high-quality analysis data to increase the probability of successful identification of HPV immune polypeptide epitopes.
其中，优化部分包括如下内容：HPV阳性生物样本使用了HPV16型阳性细胞株Caski(人子***上皮)细胞系系及Siha(人子宫颈鳞状细胞癌)细胞系(人子宫颈鳞状细胞癌)细胞(经HLA-A0201转染构建)细胞系。在免疫亲和实验过程中，根据裂解液的优化配比，每次亲和纯化实验采用8e 8以上细胞计数起始量的细胞样本进行膜蛋白裂解。免疫亲和纯化利用生物体内存在的抗原、抗体间高度特异性的亲和力作用而进行分离、纯化，抗原抗体可通过非共价键结合在分子表面，并通过后续实验条体的改变而得以分离。影响免疫亲和纯化实验关键的除了抗体的高效及特异性外，实验过程中针对抗原抗体亲和的反应形式及反应体系、抗原样品的基质影响、孵育及洗脱条件的优化及后续样品的分离纯化等等都会影响实验的最终效率。仪器分析使用EASY-nLC 1000超高效液相色谱***对免疫亲和纯化后的多肽检测样品进行液相梯度分离后进入Orbitrap Fusion Lumos Tribrid质谱仪进行依PRM靶向质谱分析。样品首先进入trap柱富集并除盐，随后与自装C18柱(150μm内径，1.8μm柱料粒径，约35cm柱长)串联，以500nL/min流速通过如下有效梯度进行分离：0～5min，5％流动相B(98％ACN，0.1％FA)；5～45min，流动相B从5％线性升至25％；45～50min，流动相B从25％升至35％；50～52min，流动相B从35％升至80％；52～54min，80％流动相B；54～54.5min，流动相B从80％降至5％；54.5～65min，5％流动相B。纳升液相分离末端直接连接质谱仪并按如下参数进行检测。经过液相分离的肽段通过nanoESI源离子化后进入到串联质谱仪Orbitrap Fusion Lumos(Thermo Fisher Scientific，San Jose，CA)进行MSOT+tMS2OT模式检测。主要参数设置：离子源电压设置为2kV；一级质谱扫描范围350～1,400m/z；分辨率设置为60,000，最大离子注入时间(MIT)为50ms；二级质谱碎裂模式为HCD，碎裂能量设置为30；分辨率设置为30,000，最大离子注入时间(MIT)为50ms，AGC设置为：一级4E5,二级5E4。 Among them, the optimization part includes the following content: HPV-positive biological samples used the HPV16-positive cell line Caski (human cervical cancer epithelial) cell line and Siha (human cervical squamous cell carcinoma) cell line (human cervical squamous cell carcinoma). Cancer) cell line (constructed by HLA-A0201 transfection). During the immunoaffinity experiment, according to the optimized ratio of the lysate, each affinity purification experiment used a cell sample with an initial cell count of more than 8e 8 for membrane protein lysis. Immunoaffinity purification utilizes the highly specific affinity between antigens and antibodies existing in organisms to separate and purify. Antigens and antibodies can be bound to the molecular surface through non-covalent bonds, and can be separated through subsequent changes in experimental strips. In addition to the high efficiency and specificity of the antibody, the key factors that affect the immunoaffinity purification experiment are the reaction form and reaction system for the antigen-antibody affinity during the experiment, the matrix influence of the antigen sample, the optimization of incubation and elution conditions, and the separation of subsequent samples. Purification, etc. will all affect the final efficiency of the experiment. Instrumental analysis uses the EASY-nLC 1000 ultra-high performance liquid chromatography system to perform liquid phase gradient separation on the immunoaffinity-purified peptide detection samples and then enters the Orbitrap Fusion Lumos Tribrid mass spectrometer for PRM-based targeted mass spectrometry analysis. The sample first enters the trap column for enrichment and desalination, and is then connected in series with a self-assembled C18 column (150 μm inner diameter, 1.8 μm column particle size, approximately 35 cm column length), and separated through the following effective gradient at a flow rate of 500 nL/min: 0 to 5 minutes , 5% mobile phase B (98% ACN, 0.1% FA); 5 to 45 minutes, mobile phase B rises linearly from 5% to 25%; 45 to 50 minutes, mobile phase B rises from 25% to 35%; 50 to 52 minutes , mobile phase B increased from 35% to 80%; 52-54min, 80% mobile phase B; 54-54.5min, mobile phase B dropped from 80% to 5%; 54.5-65min, 5% mobile phase B. The end of the nanoliter liquid phase separation is directly connected to the mass spectrometer and detected according to the following parameters. After liquid phase separation, the peptide fragments were ionized by the nanoESI source and entered into the tandem mass spectrometer Orbitrap Fusion Lumos (Thermo Fisher Scientific, San Jose, CA) for MSOT+tMS2OT mode detection. Main parameter settings: ion source voltage is set to 2kV; primary mass spectrometry scanning range is 350~1,400m/z; resolution is set to 60,000, maximum ion injection time (MIT) is 50ms; secondary mass spectrometry fragmentation mode is HCD, fragmentation The energy is set to 30; the resolution is set to 30,000, the maximum ion implantation time (MIT) is 50ms, and the AGC is set to: 4E5 for the first level and 5E4 for the second level.
分离的肽isolated peptide
一方面，本发明提出了一种分离的肽，包括下列中的至少之一：1)HPV E6蛋白的65-72位氨基酸；2)HPV E7蛋白的4-11位氨基酸；3)HPV E7蛋白的75-82位氨基酸；4)HPV E7蛋白的75-83位氨基酸；5)HPV E7蛋白的77-86位氨基酸；6)HPV E7蛋白的77-90位氨基酸；以及7)HPV E7蛋白的79-88位氨基酸。根据本发明一些具体实施例的分离的肽是由HPV E6或E7早期编码区表达翻译获得的，所述分离的肽可以由HLA-I类分子呈递，被CTL细胞或TIL细胞识别，进而可被表达HLA-I类分子的呈递细胞递呈给CTL或TIL细胞而激活特异性T细胞免疫，构成了HPV阳性肿瘤的免疫应答的生理靶标，进行高灵敏度及特异性检测，对于HPV相关疾病的预防和治疗来说具有重要价值。On the one hand, the present invention proposes an isolated peptide, including at least one of the following: 1) amino acids 65-72 of HPV E6 protein; 2) amino acids 4-11 of HPV E7 protein; 3) HPV E7 protein 75-82 amino acids of HPV E7 protein; 4) 75-83 amino acids of HPV E7 protein; 5) 77-86 amino acids of HPV E7 protein; 6) 77-90 amino acids of HPV E7 protein; and 7) HPV E7 protein of Amino acids 79-88. The isolated peptides according to some specific embodiments of the present invention are obtained by expression and translation of the HPV E6 or E7 early coding region. The isolated peptides can be presented by HLA-I class molecules, recognized by CTL cells or TIL cells, and then can be Presenting cells expressing HLA class I molecules are presented to CTL or TIL cells to activate specific T cell immunity, which constitutes the physiological target of the immune response of HPV-positive tumors. Highly sensitive and specific detection is performed for the prevention of HPV-related diseases. It is of great value for treatment.
根据本发明的一些具体实施方案，所述分离的肽的长度不超过15个连续的氨基酸。According to some embodiments of the invention, the isolated peptide is no more than 15 consecutive amino acids in length.
根据本发明的一些具体实施方案，所述分离的肽的长度超过6个连续的氨基酸。According to some specific embodiments of the invention, the isolated peptide is more than 6 consecutive amino acids in length.
根据本发明的一些具体实施方案，所述HPV E6蛋白为HPV16 E6蛋白。According to some specific embodiments of the invention, the HPV E6 protein is HPV16 E6 protein.
根据本发明的一些具体实施方案，所述HPV E7蛋白为HPV16 E7蛋白。According to some specific embodiments of the invention, the HPV E7 protein is HPV16 E7 protein.
根据本发明的一些具体实施方案，所述分离的肽包含与HLA-A、HLA-B或HLA-C分子结合的氨基酸片段。According to some specific embodiments of the invention, the isolated peptide comprises an amino acid fragment that binds to an HLA-A, HLA-B or HLA-C molecule.
根据本发明的一些具体实施方案，所述分离的肽包括SEQ ID NO:1-7所示氨基酸序列中的至少之一。根据本发明的一些具体实施方案中的分离的肽是由HPV E6或E7早期编码区表达翻译获得的，所述分离的肽可以由HLA-I类分子呈递，被CTL细胞或TIL细胞识别，进而可被表达HLA-I类分子的呈递细胞递呈给CTL或TIL细胞而激活特异性T细胞免疫，构成了HPV阳性肿瘤的免疫应答的生理靶标，进行高灵敏度及特异性检测，对于HPV相关疾病的预防和治疗来说具有重要价值。According to some specific embodiments of the invention, the isolated peptide includes at least one of the amino acid sequences shown in SEQ ID NO: 1-7. According to some specific embodiments of the present invention, the isolated peptide is obtained by expression and translation of the HPV E6 or E7 early coding region. The isolated peptide can be presented by HLA-I class molecules and recognized by CTL cells or TIL cells, and then It can be presented to CTL or TIL cells by presentation cells expressing HLA-I molecules to activate specific T cell immunity. It constitutes the physiological target of the immune response of HPV-positive tumors and performs highly sensitive and specific detection for HPV-related diseases. of great value for prevention and treatment.
NPYAVCDK(SEQ ID NO:1)。NPYAVCDK(SEQ ID NO:1).
DTPTLHEY(SEQ ID NO:2)。DTPTLHEY(SEQ ID NO:2).
DIRTLEDL(SEQ ID NO:3)。DIRTLEDL(SEQ ID NO:3).
DIRTLEDLL(SEQ ID NO:4)。DIRTLEDLL(SEQ ID NO:4).
RTLEDLLMGT(SEQ ID NO:5)。RTLEDLLMGT(SEQ ID NO:5).
RTLEDLLMGTLGIV(SEQ ID NO:6)。RTLEDLLMGTLGIV(SEQ ID NO:6).
LEDLLMGTLG(SEQ ID NO:7)。LEDLLMGTLG(SEQ ID NO:7).
另一方面，本发明提出了一种HPV抗原表位，包括下列中的至少之一：1)HPV E6蛋白的65-72位氨基酸；2)HPV E7蛋白的4-11位氨基酸；3)HPV E7蛋白的75-82位氨基酸；4)HPV E7蛋白的75-83位氨基酸；5)HPV E7蛋白的77-86位氨基酸；6)HPV E7蛋白的77-90位氨基酸；以及7)HPV E7蛋白的79-88位氨基酸。根据本发明实施例的HPV抗原表位是由HPV E6或E7早期编码区表达翻译获得的，所述HPV抗原表位可以由HLA-I类分子呈递，被CTL或TIL细胞识别，进而可被表达HLA-I类分子的呈递细胞递呈给CTL或TIL细胞而激活特异性T细胞免疫，构成了HPV阳性肿瘤的免疫应答的生理靶标，进行高灵敏度及特异性检测，对于HPV相关疾病的预防和治疗来说具有重要价值。On the other hand, the present invention proposes an HPV antigenic epitope, including at least one of the following: 1) amino acids 65-72 of HPV E6 protein; 2) amino acids 4-11 of HPV E7 protein; 3) HPV Amino acids 75-82 of the E7 protein; 4) Amino acids 75-83 of the HPV E7 protein; 5) Amino acids 77-86 of the HPV E7 protein; 6) Amino acids 77-90 of the HPV E7 protein; and 7) HPV E7 Amino acids 79-88 of the protein. The HPV antigenic epitope according to the embodiment of the present invention is obtained by expression and translation of the HPV E6 or E7 early coding region. The HPV antigenic epitope can be presented by HLA-I class molecules, recognized by CTL or TIL cells, and then can be expressed Presenting cells of HLA-I molecules are presented to CTL or TIL cells to activate specific T cell immunity, which constitutes the physiological target of the immune response of HPV-positive tumors. Highly sensitive and specific detection is required for the prevention and treatment of HPV-related diseases. It is of great value for treatment.
根据本发明的一些具体实施方案，所述HPV抗原表位包括SEQ ID NO:1-7所示氨基酸序列中的至少之一。According to some specific embodiments of the present invention, the HPV antigenic epitope includes at least one of the amino acid sequences shown in SEQ ID NO: 1-7.
再一方面，本发明提出了一种突变体，所述突变体在除锚定位外具有至少一个突变位点。根据本发明的具体实施例，获得的HPV抗原表位的第2位氨基酸通常为HLA的锚定位，表位中的其他位点氨基酸进行取代后，例如，本申请中将所述E6HPV抗原表位的第1位、第3位、第5位、第6位、第7位进行突变，依然可以具备相同或相关的免疫原性及 其潜在治疗效果，即可以由HLA-I类分子呈递，被CTL细胞或TIL细胞识别，进而可被表达HLA-I类分子的呈递细胞递呈给CTL或TIL细胞而激活特异性T细胞免疫，构成了HPV阳性肿瘤的免疫应答的生理靶标，进行高灵敏度及特异性检测，对于HPV相关疾病的预防和治疗来说具有重要价值。In yet another aspect, the present invention provides a mutant having at least one mutation site in addition to the anchor position. According to specific embodiments of the present invention, the second amino acid of the obtained HPV epitope is usually the anchor position of HLA. After the other amino acids in the epitope are substituted, for example, in this application, the E6HPV epitope is Mutation at the 1st, 3rd, 5th, 6th and 7th positions can still have the same or related immunogenicity and potential therapeutic effect, that is, it can be presented by HLA-I class molecules and be Recognized by CTL cells or TIL cells, they can then be presented to CTL or TIL cells by presentation cells expressing HLA-I molecules to activate specific T cell immunity. This constitutes the physiological target of the immune response of HPV-positive tumors and can be used for high-sensitivity and Specific detection is of great value for the prevention and treatment of HPV-related diseases.
根据本发明的一些具体实施方案，相较于野生型HPV E6蛋白，所述突变体具有如下突变位点中的至少之一：第65位、第67位、第69位、第70位和第71位。According to some specific embodiments of the invention, compared to wild-type HPV E6 protein, the mutant has at least one of the following mutation sites: position 65, position 67, position 69, position 70 and position 70. 71 bits.
根据本发明的一些具体实施方案，所述野生型HPV 16E6蛋白具有SEQ ID NO：25所示的氨基酸序列。According to some specific embodiments of the invention, the wild-type HPV 16E6 protein has the amino acid sequence shown in SEQ ID NO: 25.
Figure PCTCN2022116872-appb-000001
Figure PCTCN2022116872-appb-000001
根据本发明的一些具体实施方案，相较于前面所述的分离的肽(HPV E6，SEQ ID NO:1)，所述突变体具有如下突变中的至少之一：1)第1位的N突变为F；2)第3位的Y突变为L；3)第5位的V突变为E；4)第6位的C突变为L；和第7位的D突变为V。According to some specific embodiments of the invention, compared to the previously described isolated peptide (HPV E6, SEQ ID NO: 1), the mutant has at least one of the following mutations: 1) N at position 1 Mutation to F; 2) Y at position 3 mutated to L; 3) V at position 5 mutated to E; 4) C at position 6 mutated to L; and D at position 7 mutated to V.
根据本发明的一些具体实施方案，所述突变体具有SEQ ID NO：8-12所示的氨基酸序列。根据本发明的具体实施例，具有所示氨基酸序列的突变体可以由HLA-I类分子呈递，被CTL细胞或TIL细胞识别，进而可被表达HLA-I类分子的呈递细胞递呈给CTL或TIL细胞而激活特异性T细胞免疫，构成了HPV阳性肿瘤的免疫应答的生理靶标，进行高灵敏度及特异性检测，对于HPV相关疾病的预防和治疗来说具有重要价值。According to some specific embodiments of the invention, the mutant has the amino acid sequence shown in SEQ ID NO: 8-12. According to specific embodiments of the present invention, mutants with the amino acid sequences shown can be presented by HLA class I molecules, recognized by CTL cells or TIL cells, and can then be presented to CTL or TIL cells by presentation cells expressing HLA class I molecules. TIL cells activate specific T cell immunity and constitute the physiological target of the immune response to HPV-positive tumors. Highly sensitive and specific detection is of great value for the prevention and treatment of HPV-related diseases.
NPLAVCDK(SEQ ID NO：8)。NPLAVCDK (SEQ ID NO: 8).
NPYAVCVK(SEQ ID NO：9)。NPYAVCVK (SEQ ID NO: 9).
FPYAVCDK(SEQ ID NO：10)。FPYAVCDK (SEQ ID NO: 10).
NPYAVLDK(SEQ ID NO：11)。NPYAVLDK (SEQ ID NO: 11).
NPYAECDK(SEQ ID NO：12)。NPYAECDK (SEQ ID NO: 12).
又一方面，本发明提出了一种突变体，所述突变体在除锚定位外具有至少一个突变位点。根据本发明的具体实施例，获得的HPV抗原表位的第2和9位氨基酸通常为HLA的锚定位，表位中的其他位点氨基酸进行取代后，依然可以具备相同或相关的免疫原性及其潜在治疗效果，即可以由HLA-I类分子呈递，被CTL细胞或TIL细胞识别，进而可被表达HLA-I类分子的呈递细胞递呈给CTL或TIL细胞而激活特异性T细胞免疫，构成了HPV阳性肿瘤的免疫应答的生理靶标，进行高灵敏度及特异性检测，对于HPV相关疾病的预防和治疗来说具有重要价值。In yet another aspect, the present invention provides a mutant having at least one mutation site in addition to the anchor position. According to specific embodiments of the present invention, the 2nd and 9th amino acids of the obtained HPV epitope are usually the anchor positions of HLA. After the amino acids at other positions in the epitope are substituted, they can still have the same or related immunogenicity. and its potential therapeutic effect, that is, it can be presented by HLA class I molecules, recognized by CTL cells or TIL cells, and then presented to CTL or TIL cells by presentation cells expressing HLA class I molecules to activate specific T cell immunity. , constitutes the physiological target of the immune response of HPV-positive tumors, and high-sensitivity and specific detection is of great value for the prevention and treatment of HPV-related diseases.
根据本发明的一些具体实施方案，相较于野生型HPV E7蛋白，所述突变体具有如下突变位点中的至少之一：第75位、第77位、第79位、第80位和第82位。According to some specific embodiments of the present invention, compared to wild-type HPV E7 protein, the mutant has at least one of the following mutation sites: position 75, position 77, position 79, position 80 and position 80. 82 bits.
根据本发明的一些具体实施方案，所述野生型HPV 16 E7蛋白具有SEQ ID NO：26所示的氨基酸序列：According to some specific embodiments of the invention, the wild-type HPV 16 E7 protein has the amino acid sequence shown in SEQ ID NO: 26:
Figure PCTCN2022116872-appb-000002
Figure PCTCN2022116872-appb-000002
根据本发明的一些具体实施方案，所述突变体相较于野生型HPV E7蛋白具有如下突变中的至少之一：1)第75位的D突变为F或Y；2)第77位的R突变为L或P；3)第79位的L突变为Y或缺失；4)第80位的E突变为Y；5)第82位的L突变为T。According to some specific embodiments of the present invention, the mutant has at least one of the following mutations compared to the wild-type HPV E7 protein: 1) D at position 75 is mutated to F or Y; 2) R at position 77 Mutated to L or P; 3) L at position 79 mutated to Y or deleted; 4) E at position 80 mutated to Y; 5) L at position 82 mutated to T.
根据本发明的一些具体实施方案，相较于前面所述的分离的肽(HPV E7 75-83，SEQ ID NO:4)，所述突变体具有如下突变中的至少之一：第1位、第3位、第5位、第6位、第8位。 According to some specific embodiments of the invention, compared to the previously described isolated peptide (HPV E7 75-83 , SEQ ID NO: 4), the mutant has at least one of the following mutations: position 1, 3rd, 5th, 6th, 8th.
根据本发明的一些具体实施方案，相较于前面所述的分离的肽(HPV E7 75-83，SEQ ID NO:4)，所述突变体具有如下突变中的至少之一：1)第1位的D突变为F或Y；2)第3位的R突变为L或P；3)第5位的L突变为Y或缺失；4)第6位的E突变为Y；5)第8位的L突变为T。 According to some specific embodiments of the invention, compared to the previously described isolated peptide (HPV E7 75-83 , SEQ ID NO: 4), the mutant has at least one of the following mutations: 1) 1 D at position mutates to F or Y; 2) R at position 3 mutates to L or P; 3) L at position 5 mutates to Y or is deleted; 4) E at position 6 mutates to Y; 5) E at position 8 The L bit is mutated to T.
根据本发明的一些具体实施方案，所述突变体具有SEQ ID NO：13-17所示的氨基酸序列。根据本发明的具体实施例，具有所示氨基酸序列的突变体可以由HLA-I类分子呈递，被CTL细胞或TIL细胞识别，进而可被表达HLA-I类分子的呈递细胞递呈给CTL或TIL细胞而激活特异性T细胞免疫，构成了HPV阳性肿瘤的免疫应答的生理靶标，进行高灵敏度及特异性检测，对于HPV相关疾病的预防和治疗来说具有重要价值。According to some specific embodiments of the invention, the mutant has the amino acid sequence shown in SEQ ID NO: 13-17. According to specific embodiments of the present invention, mutants with the amino acid sequences shown can be presented by HLA class I molecules, recognized by CTL cells or TIL cells, and can then be presented to CTL or TIL cells by presentation cells expressing HLA class I molecules. TIL cells activate specific T cell immunity and constitute the physiological target of the immune response to HPV-positive tumors. Highly sensitive and specific detection is of great value for the prevention and treatment of HPV-related diseases.
FIRTLYDTL(SEQ ID NO：13)。FIRTLYDTL (SEQ ID NO: 13).
YIRTLEDLL(SEQ ID NO：14)。YIRTLEDLL (SEQ ID NO: 14).
DIPTEDLL(SEQ ID NO：15)。DIPTEDLL (SEQ ID NO: 15).
DIRTYEDLL(SEQ ID NO：16)。DIRTYEDLL (SEQ ID NO: 16).
DILTLEDLL(SEQ ID NO：17)。DILTLEDLL (SEQ ID NO: 17).
预防或治疗组合物Preventive or therapeutic compositions
另一方面，本发明提出了一种核酸分子，所述核酸分子编码前面所述的分离的肽、所述的HPV抗原表位或突变体。根据本发明的一些具体实施方案的核酸分子编码获得的分离的肽、HPV抗原表位或突变体可以由HLA-I类分子呈递，被CTL或TIL细胞识别，进而可被表达HLA-I类分子的呈递细胞递呈给CTL或TIL细胞而激活特异性T细胞免疫，构成了HPV阳性肿瘤的免疫应答的生理靶标，进行高灵敏度及特异性检测，对于HPV相关疾病的预防和治疗来说具有重要价值。On the other hand, the present invention provides a nucleic acid molecule encoding the aforementioned isolated peptide, the HPV epitope or mutant. The isolated peptides, HPV epitopes or mutants encoded by the nucleic acid molecules according to some specific embodiments of the present invention can be presented by HLA class I molecules, recognized by CTL or TIL cells, and can then be expressed by HLA class I molecules. Presenting cells present to CTL or TIL cells to activate specific T cell immunity, which constitutes the physiological target of the immune response of HPV-positive tumors. Highly sensitive and specific detection is important for the prevention and treatment of HPV-related diseases. value.
根据本发明的一些具体实施方案，所述核酸分子具有SEQ ID NO:18-24所示核苷酸序列中的至少之一。According to some specific embodiments of the invention, the nucleic acid molecule has at least one of the nucleotide sequences shown in SEQ ID NO: 18-24.
编码NPYAVCDK(SEQ ID NO:1)的核酸包括：The nucleic acid encoding NPYAVCDK (SEQ ID NO:1) includes:
AAUCCAUAUGCUGUAUGUGAUAAA(SEQ ID NO:18)AAUCCAUAUGCUGUAUGUGAUAAA(SEQ ID NO:18)
编码DTPTLHEY(SEQ ID NO:2)的核酸包括：Nucleic acids encoding DTPTLHEY (SEQ ID NO:2) include:
GAUACACCUACAUUGCAUGAAUAU(SEQ ID NO:19)GAUACACCUACAUUGCAUGAAUAU(SEQ ID NO:19)
编码DIRTLEDL(SEQ ID NO:3)的核酸包括：Nucleic acids encoding DIRTLEDL (SEQ ID NO:3) include:
GACAUUCGUACUUUGGAAGACCUG(SEQ ID NO:20)GACAUUCGUACUUUGGAAGACCUG(SEQ ID NO:20)
编码DIRTLEDLL(SEQ ID NO:4)的核酸包括：Nucleic acids encoding DIRTLEDLL (SEQ ID NO:4) include:
GACAUUCGUACUUUGGAAGACCUGUUA(SEQ ID NO:21)GACAUUCGUACUUUGGAAGACCUGUUA(SEQ ID NO:21)
编码RTLEDLLMGT(SEQ ID NO:5)的核酸包括：Nucleic acids encoding RTLEDLLMGT (SEQ ID NO:5) include:
CGUACUUUGGAAGACCUGUUAAUGGGCACA(SEQ ID NO:22)CGUACUUUGGAAGACCUGUUAAUGGGCACA(SEQ ID NO:22)
编码RTLEDLLMGTLGIV(SEQ ID NO:6)的核酸包括：Nucleic acids encoding RTLEDLLMGTLGIV (SEQ ID NO:6) include:
CGUACUUUGGAAGACCUGUUAAUGGGCACACUAGGAAUUGUG(SEQ ID NO:23)CGUACUUUGGAAGACCUGUUAAUGGGCACACUAGGAAUUGUG(SEQ ID NO:23)
编码LEDLLMGTLG(SEQ ID NO:7)的核酸包括：Nucleic acids encoding LEDLLMGTGG (SEQ ID NO:7) include:
UUGGAAGACCUGUUAAUGGGCACACUAGGA(SEQ ID NO:24)。UUGGAAGACCUGUUAAUGGGCACACUAGGA(SEQ ID NO:24).
需要说明的是，对于本发明说明书和权利要求书中所提及的核酸，本领域技术人员应当理解，实际包括互补双链的任意一条，或者两条。为了方便，在本说明书和权利要求书中，虽然多数情况下只给出了一条链，但实际上也公开了与之互补的另一条链。另外，本申请中的基因序列包括DNA形式或RNA形式，公开其中一种，意味着另一种也被公开。It should be noted that those skilled in the art will understand that the nucleic acids mentioned in the description and claims of the present invention actually include either or both complementary double strands. For convenience, in this description and claims, although in most cases only one strand is given, another strand complementary to it is actually disclosed. In addition, the gene sequence in this application includes DNA form or RNA form. Disclosing one of them means that the other one is also disclosed.
再一方面，本发明提出了一种表达载体，携带表达前面所述的分离的肽、HPV抗原表位或突变体的核酸。所述表达载体的类型不受特别限制，只要能够实现前面所述的核酸构建体在受体细胞中高效表达即可，表达载体包括但不限于反转录病毒载体、慢病毒载体和/或腺病毒相关病毒载体。所述表达载体可包括可选的控制序列，所述控制序列与所述核酸分子可操作地连接。其中，所述控制序列为可指导多肽在宿主中表达的一个或多个控制序列。本发明的一些具体实施方案所提出的表达载体可在适合的宿主细胞中高效表达所述分离的肽、HPV抗原表位或突变体，进而可有效用于对肿瘤，特别是同时表达HLA-I类分子和上述分离的肽或HPV抗原表位的肿瘤的特异性治疗或预防。In yet another aspect, the present invention provides an expression vector carrying nucleic acid expressing the aforementioned isolated peptide, HPV epitope or mutant. The type of expression vector is not particularly limited, as long as it can achieve efficient expression of the nucleic acid construct described above in recipient cells. Expression vectors include but are not limited to retroviral vectors, lentiviral vectors and/or adenoviral vectors. Virus-related viral vectors. The expression vector may include optional control sequences operably linked to the nucleic acid molecule. Wherein, the control sequence is one or more control sequences that can direct the expression of the polypeptide in the host. The expression vectors proposed in some specific embodiments of the present invention can efficiently express the isolated peptide, HPV epitope or mutant in a suitable host cell, and thus can be effectively used to treat tumors, especially while expressing HLA-I. Specific treatment or prevention of tumors using class molecules and the above isolated peptides or HPV epitopes.
又一方面，本发明提出了一种重组细胞，携带前面所述的核酸分子、表达载体、分离的肽、HPV抗原表位或突变体。所述重组细胞是通过转染或者转化所述表达载体获得的。转化或转染可采用电转、病毒转染或感受态细胞转化的方式进行。采用何种转染或转化的方式是根据宿主细胞的性质以及待转核酸构建体或表达载体的性质所决定的，只要能够在所述宿主细胞中实现前面所述多肽的高效表达并对宿主细胞的良好的细胞状态不产生较大影响即可。根据本发明的一些具体实施方案，所述宿主细胞在合适条件下可高效表达上述分离的肽、HPV抗原表位或突变体，所述重组细胞可有效用于对肿瘤，特别是同时表达HLA-I类分子和上述分离的肽、HPV抗原表位或突变体的肿瘤的特异性治疗或预防。In another aspect, the present invention provides a recombinant cell carrying the aforementioned nucleic acid molecule, expression vector, isolated peptide, HPV epitope or mutant. The recombinant cells are obtained by transfection or transformation of the expression vector. Transformation or transfection can be carried out by electroporation, viral transfection or transformation of competent cells. The method of transfection or transformation used is determined by the nature of the host cell and the nature of the nucleic acid construct or expression vector to be transferred, as long as the high-efficiency expression of the polypeptide described above can be achieved in the host cell and the host cell The good cell condition does not have a major impact. According to some specific embodiments of the present invention, the host cells can efficiently express the above-mentioned isolated peptides, HPV epitopes or mutants under appropriate conditions, and the recombinant cells can be effectively used to treat tumors, especially while expressing HLA- Specific treatment or prevention of tumors with class I molecules and the above isolated peptides, HPV epitopes or mutants.
需要说明的是，本申请说明书中所述的“适合条件”，是指适合本申请所述分离的肽、HPV抗原表位或突变体表达的条件。本领域技术人员容易理解的是，适合分离的肽、HPV抗原表位或突变体表达的条件包括但不限于合适的转化或转染方式、合适的转化或转条件、健康的宿主细胞状态、合适的宿主细胞密度、适宜的细胞培养环境、适宜的细胞培养时间。“适合条件”不受特别限制，本领域技术人员可根据实验室的具体环境，优化最适的所述多肽表达的条件。It should be noted that the "suitable conditions" mentioned in the specification of this application refer to conditions suitable for the expression of the isolated peptides, HPV epitopes or mutants described in this application. It is easily understood by those skilled in the art that conditions suitable for the expression of isolated peptides, HPV epitopes or mutants include, but are not limited to, suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy host cell status, suitable The host cell density, suitable cell culture environment, and suitable cell culture time. "Suitable conditions" are not particularly limited, and those skilled in the art can optimize the optimal conditions for the expression of the polypeptide according to the specific environment of the laboratory.
一个方面，本发明提出了一种抗原呈递细胞，所述细胞可呈递前面所述的分离的肽、HPV抗原表位或突变体。根据本发明的实施例，呈递前面所述分离的肽、HPV抗原表位或突变体的抗原呈递细胞可有效引起患者针对肿瘤特异性抗原-上述分离的肽/HPV抗原表位/突变体的免疫反应，进而激活CTL特异性杀伤功能，本发明实施例所提出的抗原呈递细胞具有显著的治疗表达上述分离的肽、HPV抗原表位或突变体的肿瘤的功效，其治疗的效果显著，安全性高。In one aspect, the present invention provides an antigen-presenting cell that can present the isolated peptide, HPV epitope or mutant as described above. According to embodiments of the present invention, antigen-presenting cells presenting the aforementioned isolated peptides, HPV epitopes or mutants can effectively induce immunity in patients against tumor-specific antigens-the aforementioned isolated peptides/HPV epitopes/mutants. reaction, thereby activating the specific killing function of CTL. The antigen-presenting cells proposed in the embodiments of the present invention have significant efficacy in treating tumors expressing the above-mentioned isolated peptides, HPV epitopes or mutants, and their therapeutic effects are significant and safe. high.
根据本发明的一些具体实施方案，所述抗原呈递细胞是通过下列至少之一获得的：将具有抗原呈递能力的细胞与所述多肽接触；将前面所述的核酸或表达载体导入所述具有抗原呈递能力的细胞。According to some specific embodiments of the present invention, the antigen-presenting cells are obtained by at least one of the following: contacting cells with antigen-presenting ability with the polypeptide; introducing the aforementioned nucleic acid or expression vector into the antigen-presenting cells. Presenting competent cells.
根据本发明的一些具体实施方案，所述具有抗原呈递能力的细胞为树突状细胞、B细胞或单核-吞噬细胞。According to some specific embodiments of the present invention, the cells with antigen-presenting ability are dendritic cells, B cells or monocyte-phagocytic cells.
另一方面，本发明提出了一种免疫效应细胞。根据本发明的实施例，所述免疫效应细胞可识别前面所述的多肽、HPV抗原表位、突变体或识别在细胞表面呈递前面所述的多肽或所述的HPV抗原表位或突变体的抗原呈递细胞。根据本发明的实施例，所述免疫效应细胞可特异性杀伤共表达HLA-I类分子和上述分离的肽、HPV抗原表位或突变体的肿瘤细胞。On the other hand, the present invention provides an immune effector cell. According to embodiments of the present invention, the immune effector cells can recognize the polypeptides, HPV epitopes, and mutants described above, or recognize the polypeptides or HPV epitopes or mutants that are presented on the cell surface. Antigen presenting cells. According to embodiments of the present invention, the immune effector cells can specifically kill tumor cells that co-express HLA class I molecules and the above-mentioned isolated peptides, HPV epitopes or mutants.
根据本发明的一些具体实施方案，所述免疫效应细胞是通过下列方式获得的：将前面所述的抗原呈递细胞与具有免疫效应能力的细胞接触。According to some specific embodiments of the present invention, the immune effector cells are obtained by contacting the aforementioned antigen-presenting cells with cells having immune effector capabilities.
根据本发明的一些具体实施方案，所述具有免疫效应能力的细胞为T细胞，优选为CD8 +T细胞。发明人发现，通过呈递前面所述的分离的肽、HPV抗原表位或突变体的抗原呈递细胞与具有免疫效应能力的细胞接触，抗原呈递细胞可活化具有免疫效应能力的未激活细胞，递呈抗原-前面所述的多肽，进而激活具有免疫效应能力的细胞，大量产生免疫效应细胞，该免疫效应细胞具有特异性杀伤呈递抗原-所述多肽的靶细胞的作用。CD8 +T细胞接受抗原呈递细胞激活作用的能力更强，获得的CD8 +T细胞的特异性杀伤呈递抗原-所述分离的肽/HPV抗原表位的靶细胞的作用更强。 According to some specific embodiments of the present invention, the cells with immune effector capabilities are T cells, preferably CD8 + T cells. The inventors found that by contacting antigen-presenting cells presenting the previously described isolated peptides, HPV epitopes or mutants with cells having immune effector capabilities, the antigen-presenting cells can activate unactivated cells with immune effector capabilities, presenting The antigen - the polypeptide mentioned above, then activates cells with immune effector ability, producing a large number of immune effector cells. The immune effector cells have the function of specifically killing the target cells presenting the antigen - the polypeptide. CD8 + T cells have a stronger ability to accept the activation effect of antigen-presenting cells, and the obtained CD8 + T cells have a stronger specific killing effect on target cells presenting antigen-the isolated peptide/HPV epitope.
在再一方面，本发明提出了一种药物。根据本发明的实施例，包含前面所述的分离的肽、HPV抗原表位、突变体、核酸分子、表达载体、重组细胞、抗原呈递细胞或免疫效应细胞。如前所述，所述分离的肽、HPV抗原表位、突变体、编码所述分离的肽或HPV抗原表位或突变体的核酸分子、表达载体、重组细胞、抗原呈递细胞或免疫效应细胞均可有效用于对肿瘤，特别是同时表达HLA-I类分子和上述分离的肽、HPV抗原表位或突变体的肿瘤的特异性治疗或预防，因此，包含部分或全部上述物质的药物同样具有显著的治疗或预防表达HLA-I类分子和所述分离的肽、HPV抗原表位或突变体的肿瘤的作用，其安全性更高、副作用更小。In yet another aspect, the invention provides a medicament. According to embodiments of the present invention, the aforementioned isolated peptides, HPV epitopes, mutants, nucleic acid molecules, expression vectors, recombinant cells, antigen-presenting cells or immune effector cells are included. As mentioned above, the isolated peptide, HPV epitope, mutant, nucleic acid molecule encoding the isolated peptide or HPV epitope or mutant, expression vector, recombinant cell, antigen presenting cell or immune effector cell All can be effectively used for the specific treatment or prevention of tumors, especially tumors that express both HLA class I molecules and the above-mentioned isolated peptides, HPV epitopes or mutants. Therefore, drugs containing some or all of the above substances are also It has a significant effect of treating or preventing tumors expressing HLA class I molecules and the isolated peptide, HPV antigen epitope or mutant, and has higher safety and smaller side effects.
根据本发明的一些具体实施方案提供的药物，包括药学上可接受的载体和有效量的上述物质的活性成分。Medicaments provided according to some specific embodiments of the present invention include pharmaceutically acceptable carriers and an effective amount of active ingredients of the above substances.
如本文所用，术语“有效量”或“有效剂量”是指可对人和/或动物产生功能或活性的且可被人和/或动物所接受的量。As used herein, the term "effective amount" or "effective dose" refers to an amount that produces a function or activity in humans and/or animals and is acceptable to humans and/or animals.
如本文所用，“药学上可接受的”的成分是适用于人和/或哺乳动物而无过度不良副反应(如毒性、刺激和*** 反应)的，即具有合理的效益/风险比的物质。术语“药学上可接受的载体”指用于治疗剂给药的载体，包括各种赋形剂和稀释剂。As used herein, a "pharmaceutically acceptable" ingredient is one that is suitable for use by humans and/or mammals without undue adverse side effects (e.g., toxicity, irritation, and allergic reactions), i.e., a substance that has a reasonable benefit/risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier used for the administration of a therapeutic agent, including various excipients and diluents.
本发明的药物组合物含有安全有效量的本发明的活性成分以及药学上可接受的载体。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、及其组合。通常药物制剂应与给药方式相匹配，本发明的药物组合物的剂型为注射剂、口服制剂(片剂、胶囊、口服液)、透皮剂、缓释剂。例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。所述的药物组合物宜在无菌条件下制造。The pharmaceutical composition of the present invention contains a safe and effective amount of the active ingredient of the present invention and a pharmaceutically acceptable carrier. Such carriers include, but are not limited to: saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. Generally, pharmaceutical preparations should match the mode of administration. The dosage forms of the pharmaceutical composition of the present invention are injections, oral preparations (tablets, capsules, oral liquids), transdermal agents, and sustained-release agents. For example, it is prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. The pharmaceutical composition is preferably manufactured under sterile conditions.
本发明所述的活性成分的有效量可随给药的模式和待治疗的疾病的严重程度等而变化。优选的有效量的选择可以由本领域普通技术人员根据各种因素来确定(例如通过临床试验)。所述的因素包括但不限于：所述的活性成分的药代动力学参数例如生物利用率、代谢、半衰期等；患者所要治疗的疾病的严重程度、患者的体重、患者的免疫状况、给药的途径等。例如，由治疗状况的迫切要求，可每天给予若干次分开的剂量，或将剂量按比例地减少。The effective amount of the active ingredients of the present invention may vary depending on the mode of administration and the severity of the disease to be treated. The selection of a preferred effective amount can be determined by one of ordinary skill in the art based on various factors (eg, through clinical trials). The factors include but are not limited to: the pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the patient's weight, the patient's immune status, drug administration ways, etc. For example, several divided doses may be administered daily, or the dosage may be proportionally reduced as dictated by the exigencies of the treatment situation.
本发明所述的药学上可接受的载体包括(但不限于)：水、盐水、脂质体、脂质、蛋白、蛋白-抗体缀合物、肽类物质、纤维素、纳米凝胶、或其组合。载体的选择应与给药方式相匹配，这些都是本领域的普通技术人员所熟知的。Pharmaceutically acceptable carriers of the present invention include (but are not limited to): water, saline, liposomes, lipids, proteins, protein-antibody conjugates, peptides, cellulose, nanogels, or its combination. The choice of carrier should be compatible with the mode of administration, which are well known to those of ordinary skill in the art.
同时，发明人发现***、外阴癌、***癌、***癌、***癌、头颈癌、宫颈上皮内瘤变、外阴上皮内瘤变、***上皮内瘤变、***上皮内瘤变、***上皮内瘤变、口腔癌、喉癌、食道癌、鼻腔内癌或扁桃体癌，尤其是***组织特异性高表达上述分离的多肽或HPV抗原表位，进而当肿瘤为上述肿瘤时，该药物治疗的有效性得到进一步提高。At the same time, the inventor discovered cervical cancer, vulvar cancer, vaginal cancer, anal cancer, penile cancer, head and neck cancer, cervical intraepithelial neoplasia, vulvar intraepithelial neoplasia, vaginal intraepithelial neoplasia, anal intraepithelial neoplasia, and penile intraepithelial neoplasia. Neoplasia, oral cancer, laryngeal cancer, esophageal cancer, intranasal cancer or tonsil cancer, especially cervical cancer tissue specifically expresses the above-mentioned isolated polypeptide or HPV antigen epitope, and when the tumor is the above-mentioned tumor, the drug can treat Effectiveness is further improved.
又一方面，本发明提出了一种疫苗。根据本发明的实施例，包含前面所述的分离的肽、HPV抗原表位、突变体、核酸分子、表达载体或抗原呈递细胞。如前所述，本发明实施例的核酸分子、表达载体或重组细胞在合适的条件下表达前面所述的分离的肽、HPV抗原表位或突变体，抗原呈递细胞可以表达所述分离的肽、HPV抗原表位或突变体，当其与HLA-I类分子结合后，被抗原呈递细胞呈递，使其被CTL或TIL细胞识别，即被表达HLA-I类分子的抗原呈递细胞递呈给CTL或TIL细胞而激活特异性T细胞免疫。因此，本发明实施例所提出的疫苗具有显著的治疗或预防表达HLA-I类分子和所述分离的肽、HPV抗原表位或突变体的肿瘤的作用，其安全性更高、副作用更小。同时，发明人发现所述疫苗可以有效治疗或预防***、外阴癌、***癌、***癌、***癌、头颈癌、宫颈上皮内瘤变、外阴上皮内瘤变、***上皮内瘤变、***上皮内瘤变、***上皮内瘤变、口腔癌、喉癌、食道癌、鼻腔内癌或扁桃体癌，尤其是***组织特异性高表达上述分离的多肽、HPV抗原表位或突变体，进而当肿瘤为上述***时，该药物治疗的有效性得到进一步提高。In yet another aspect, the present invention provides a vaccine. According to embodiments of the present invention, the aforementioned isolated peptides, HPV epitopes, mutants, nucleic acid molecules, expression vectors or antigen-presenting cells are included. As mentioned above, the nucleic acid molecules, expression vectors or recombinant cells of the embodiments of the present invention express the aforementioned isolated peptides, HPV epitopes or mutants under appropriate conditions, and the antigen-presenting cells can express the isolated peptides. , HPV antigenic epitopes or mutants, when combined with HLA-I class molecules, are presented by antigen-presenting cells, allowing them to be recognized by CTL or TIL cells, that is, they are presented to antigen-presenting cells expressing HLA-I class molecules. CTL or TIL cells activate specific T cell immunity. Therefore, the vaccine proposed in the embodiments of the present invention has a significant effect in treating or preventing tumors expressing HLA class I molecules and the isolated peptides, HPV epitopes or mutants, and is safer and has fewer side effects. . At the same time, the inventor found that the vaccine can effectively treat or prevent cervical cancer, vulvar cancer, vaginal cancer, anal cancer, penile cancer, head and neck cancer, cervical intraepithelial neoplasia, vulvar intraepithelial neoplasia, vaginal intraepithelial neoplasia, anus Intraepithelial neoplasia, penile intraepithelial neoplasia, oral cancer, laryngeal cancer, esophageal cancer, intranasal cancer or tonsil cancer, especially cervical cancer, tissue-specific high expression of the above isolated polypeptides, HPV epitopes or mutants, and then The effectiveness of this drug treatment is further enhanced when the tumor is cervical cancer as described above.
根据本发明的一些具体实施方案，所述疫苗呈适于吸入或者注射方式给药的形式。According to some embodiments of the invention, the vaccine is in a form suitable for administration by inhalation or injection.
根据本发明的一些具体实施方案，所述疫苗还包含至少一种佐剂。According to some specific embodiments of the invention, the vaccine further comprises at least one adjuvant.
用途use
一方面，本发明提出了试剂在制备试剂盒中的用途，所述试剂用于检测前面所述的分离的肽、HPV抗原表位或突变体，所述试剂盒用于诊断HPV或者检测HPV的治疗效果。所述试剂可以对所述分离的肽、HPV抗原表位或突变体进行准确检测，如，检测生物样品中是否含有所述分离的肽、HPV抗原表位或突变体，由于所述分离的肽、HPV抗原表位或突变体在被HPV感染的组织中高表达，因此，包含所述试剂的试剂盒可以准确诊断生物样品来源的个体是否被HPV感染，进一步地，是否为HPV高风险个体。同理，被HPV感染的个体在治疗过程中，利用所述试剂盒进行检测，可以检测治疗过程中HPV的变化，如加重、减缓或治愈等。On the one hand, the present invention proposes the use of reagents for detecting the aforementioned isolated peptides, HPV epitopes or mutants in the preparation of kits for diagnosing HPV or detecting HPV. treatment effect. The reagent can accurately detect the isolated peptide, HPV epitope or mutant, for example, detect whether a biological sample contains the isolated peptide, HPV epitope or mutant, because the isolated peptide , HPV antigenic epitopes or mutants are highly expressed in HPV-infected tissues. Therefore, a kit containing the reagent can accurately diagnose whether the individual from whom the biological sample is derived is infected by HPV, and further, whether it is an individual at high risk for HPV. In the same way, during the treatment process of an individual infected by HPV, the test kit can be used to detect the changes of HPV during the treatment process, such as aggravation, slowing down or cure, etc.
另一方面，本发明提出了前面所述的分离的肽、HPV抗原表位、突变体、核酸分子、表达载体、重组细胞、抗原呈递细胞或免疫效应细胞在制备药物中的用途。根据本发明的实施例，所述药物用于治疗或者预防HPV相关疾病。如前所述，所述分离的肽、HPV抗原表位、突变体、编码所述分离的肽或HPV抗原表位或突变体的核酸分子、表达载体、重组细胞、抗原呈递细胞或免疫效应细胞均可有效用于对肿瘤，特别是同时表达HLA-I类分子和上述分离的肽、HPV抗原表位或突变体的肿瘤的特异性治疗或预防，因此，包含部分或全部上述物质的药物同样具有显著的治疗或预防表达HLA-I类分子和所述分离的肽、HPV抗原表位或突变体的肿瘤的作用，其安全性更高、副作用更小。On the other hand, the present invention proposes the use of the aforementioned isolated peptides, HPV epitopes, mutants, nucleic acid molecules, expression vectors, recombinant cells, antigen-presenting cells or immune effector cells in the preparation of medicines. According to embodiments of the present invention, the medicine is used to treat or prevent HPV-related diseases. As mentioned above, the isolated peptide, HPV epitope, mutant, nucleic acid molecule encoding the isolated peptide or HPV epitope or mutant, expression vector, recombinant cell, antigen presenting cell or immune effector cell All can be effectively used for the specific treatment or prevention of tumors, especially tumors that express both HLA class I molecules and the above-mentioned isolated peptides, HPV epitopes or mutants. Therefore, drugs containing some or all of the above substances are also It has a significant effect of treating or preventing tumors expressing HLA class I molecules and the isolated peptide, HPV antigen epitope or mutant, and has higher safety and smaller side effects.
根据本发明的一些具体实施方案，所述HPV相关疾病包括下列中的至少之一：***、外阴癌、***癌、***癌、***癌、头颈癌、宫颈上皮内瘤变、外阴上皮内瘤变、***上皮内瘤变、***上皮内瘤变、***上皮内瘤变、口腔癌、喉癌、食道癌、鼻腔内癌和扁桃体癌。发明人发现所述药物可以有效治疗或者预防上述疾病，尤其是***组织特异性高表达上述分离的多肽、HPV抗原表位或突变体，进而当疾病为上述***时，该药物治疗的有效性得到进一步提高。According to some specific embodiments of the present invention, the HPV-related diseases include at least one of the following: cervical cancer, vulvar cancer, vaginal cancer, anal cancer, penile cancer, head and neck cancer, cervical intraepithelial neoplasia, vulvar intraepithelial neoplasia vaginal intraepithelial neoplasia, anal intraepithelial neoplasia, penile intraepithelial neoplasia, oral cancer, laryngeal cancer, esophageal cancer, intranasal cancer and tonsil cancer. The inventor found that the drug can effectively treat or prevent the above-mentioned diseases, especially cervical cancer tissue that specifically expresses the above-mentioned isolated polypeptide, HPV epitope or mutant. Furthermore, when the disease is the above-mentioned cervical cancer, the drug treatment is effective. Sexuality is further improved.
再一方面，本发明提出了前面所述的分离的肽、HPV抗原表位、突变体、核酸分子、表达载体或重组细胞在制备试剂盒中的用途，所述试剂盒用于检测HLA。根据本发明的一些具体实施方案的分离的肽、HPV抗原表位、突变体及其相应物质可与HLA结合，因此，上述物质可用于制备有效检测HLA的试剂盒。In another aspect, the present invention proposes the use of the previously described isolated peptides, HPV epitopes, mutants, nucleic acid molecules, expression vectors or recombinant cells in the preparation of kits for detecting HLA. The isolated peptides, HPV epitopes, mutants and corresponding substances according to some specific embodiments of the present invention can be combined with HLA. Therefore, the above substances can be used to prepare kits for effectively detecting HLA.
试剂盒Reagent test kit
一个方面，本发明提出了一种试剂盒，包括适于检测前面所述的分离的肽、HPV抗原表位或突变体的试剂。所述试剂可以对所述分离的肽、HPV抗原表位或突变体进行准确检测，如，检测生物样品中是否含有所述分离的肽、HPV抗原 表位或突变体，由于所述分离的肽、HPV抗原表位或突变体在被HPV感染的组织中高表达，因此，包含所述试剂的试剂盒可以准确诊断生物样品来源的个体是否被HPV感染，进一步地，是否为HPV高风险个体。同理，被HPV感染的个体在治疗过程中，利用所述试剂盒进行检测，可以检测治疗过程中HPV的变化，如加重、减缓或治愈等。In one aspect, the present invention provides a kit comprising reagents suitable for detecting the isolated peptide, HPV epitope or mutant as described above. The reagent can accurately detect the isolated peptide, HPV epitope or mutant, for example, detect whether a biological sample contains the isolated peptide, HPV epitope or mutant, because the isolated peptide , HPV antigenic epitopes or mutants are highly expressed in HPV-infected tissues. Therefore, a kit containing the reagent can accurately diagnose whether the individual from whom the biological sample is derived is infected by HPV, and further, whether it is an individual at high risk for HPV. In the same way, during the treatment process of an individual infected by HPV, the test kit can be used to detect the changes of HPV during the treatment process, such as aggravation, slowing down or cure, etc.
又一方面，本发明提出了一种检测HLA的试剂盒，包括前面所述的分离的肽、HPV抗原表位、突变体、核酸分子、表达载体或重组细胞。如前所述，分离的肽、HPV抗原表位、突变体及其相应物质可与HLA结合，因此，包含上述物质的试剂盒可用于有效定性或定量检测HLA。In another aspect, the present invention proposes a kit for detecting HLA, including the aforementioned isolated peptide, HPV epitope, mutant, nucleic acid molecule, expression vector or recombinant cell. As mentioned before, isolated peptides, HPV epitopes, mutants and their corresponding substances can bind to HLA. Therefore, kits containing the above substances can be used to effectively detect HLA qualitatively or quantitatively.
预防或治疗方法及用途Prevention or treatment methods and uses
一方面，本发明提出了一种预防或治疗HPV相关疾病的方法，给予受试者前面所述的分离的肽、HPV抗原表位、突变体、核酸分子、表达载体、重组细胞、抗原呈递细胞、免疫效应细胞、疫苗或药物。如前所述，本发明的一些具体实施方案所提出的预防方法，包括给予任一种有效量的前面所述的分离的肽等物质，均可有效治疗或预防表达HLA-I类分子和所述分离的肽、HPV抗原表位或突变体的肿瘤。On the one hand, the present invention proposes a method for preventing or treating HPV-related diseases by administering to the subject the previously described isolated peptides, HPV epitopes, mutants, nucleic acid molecules, expression vectors, recombinant cells, and antigen-presenting cells. , immune effector cells, vaccines or drugs. As mentioned above, the preventive methods proposed by some specific embodiments of the present invention include administering any effective amount of the previously described isolated peptides and other substances, which can effectively treat or prevent the expression of HLA-I class molecules and the tumors with isolated peptides, HPV epitopes or mutants.
根据本发明的一些具体实施方案，所述HPV相关疾病包括下列中的至少之一：***、外阴癌、***癌、***癌、***癌、头颈癌、宫颈上皮内瘤变、外阴上皮内瘤变、***上皮内瘤变、***上皮内瘤变、***上皮内瘤变、口腔癌、喉癌、食道癌、鼻腔内癌和扁桃体癌。According to some specific embodiments of the present invention, the HPV-related diseases include at least one of the following: cervical cancer, vulvar cancer, vaginal cancer, anal cancer, penile cancer, head and neck cancer, cervical intraepithelial neoplasia, vulvar intraepithelial neoplasia vaginal intraepithelial neoplasia, anal intraepithelial neoplasia, penile intraepithelial neoplasia, oral cancer, laryngeal cancer, esophageal cancer, intranasal cancer and tonsil cancer.
给药的各种方式是可以预期的，包括腹膜，静脉，肌肉，皮下，皮层，口服，局部，鼻腔，肺部和直肠，但是本发明不限于这些已举例的给药方式。然而，由于口服给药时，口服给药的组合物的活性成分应该被包被或被配制以防止其在胃部被降解。优选地，本发明的组合物可以注射制剂给药。此外，本发明的药物组合物可以使用将活性成分传送到靶细胞的特定器械来给药。Various modes of administration are contemplated, including peritoneal, intravenous, intramuscular, subcutaneous, cortical, oral, topical, nasal, pulmonary and rectal, but the invention is not limited to these exemplified modes of administration. However, since it is to be administered orally, the active ingredient of the composition for oral administration should be coated or formulated to prevent degradation in the stomach. Preferably, the compositions of the present invention may be administered in an injectable formulation. Furthermore, the pharmaceutical compositions of the present invention may be administered using specific devices that deliver the active ingredients to target cells.
本发明实施例中的分离的肽、HPV抗原表位、突变体、核酸、表达载体、重组细胞、抗原呈递细胞、免疫效应细胞、药物、疫苗的给药频率和剂量可以通过多个相关因素被确定，该因素包括要被治疗的疾病类型，给药途径，病人年龄，性别，体重和疾病的严重程度以及作为活性成分的药物类型。根据本发明的一些实施例，日剂量可分为适宜形式的1剂、2剂或多剂，以在整个时间段内以1次、2次或多次给药，只要达到治疗有效量即可。The administration frequency and dosage of isolated peptides, HPV epitopes, mutants, nucleic acids, expression vectors, recombinant cells, antigen-presenting cells, immune effector cells, drugs, and vaccines in the embodiments of the present invention can be determined by multiple related factors. To be sure, factors include the type of disease being treated, the route of administration, patient age, gender, weight and severity of the disease and the type of drug that is the active ingredient. According to some embodiments of the present invention, the daily dose may be divided into 1 dose, 2 doses, or multiple doses in a suitable form to be administered once, twice, or multiple times throughout the time period, as long as a therapeutically effective amount is achieved .
术语“治疗有效量”是指足以显著改善某些与疾病或病症相关的症状的量，也即为给定病症和给药方案提供治疗效果的量。术语“治疗”用于指获得期望的药理学和/或生理学效果。本文使用的“治疗”涵盖将发明实施例中的分离的肽、HPV抗原表位、突变体、核酸、表达载体、重组细胞、疫苗、抗原呈递细胞、免疫效应细胞或药物给予个体以治疗，包括但不限于将含本文所述的给予有需要的个体。The term "therapeutically effective amount" refers to an amount sufficient to significantly ameliorate certain symptoms associated with a disease or condition, ie, an amount that provides a therapeutic effect for a given condition and dosage regimen. The term "treatment" is used to refer to obtaining a desired pharmacological and/or physiological effect. "Treatment" as used herein encompasses the administration of isolated peptides, HPV epitopes, mutants, nucleic acids, expression vectors, recombinant cells, vaccines, antigen-presenting cells, immune effector cells or drugs in the embodiments of the invention to an individual for treatment, including However, it is not limited to administration to an individual in need containing what is described herein.
另一方面，本发明提出了前面所述的分离的肽、突变体、表达载体、抗原呈递细胞、免疫效应细胞、药物或疫苗在治疗和/或预防HPV相关疾病中的用途。根据本发明的一些具体实施例，所述分离的肽及其突变体能够有效的抑制肿瘤生长，因此，所述分离的肽或突变体、能够表达或包含所述分离的肽或突变体的核酸、表达载体、细胞、药物、疫苗均能够有效治疗和/或预防HPV相关疾病。On the other hand, the present invention proposes the use of the aforementioned isolated peptides, mutants, expression vectors, antigen-presenting cells, immune effector cells, drugs or vaccines in the treatment and/or prevention of HPV-related diseases. According to some specific embodiments of the present invention, the isolated peptide and its mutants can effectively inhibit tumor growth. Therefore, the isolated peptide or mutant, the nucleic acid capable of expressing or containing the isolated peptide or mutant , expression vectors, cells, drugs, and vaccines can all effectively treat and/or prevent HPV-related diseases.
根据本发明的一些具体实施方案，所述HPV相关疾病包括下列中的至少之一：***、外阴癌、***癌、***癌、***癌、头颈癌、宫颈上皮内瘤变、外阴上皮内瘤变、***上皮内瘤变、***上皮内瘤变、***上皮内瘤变、口腔癌、喉癌、食道癌、鼻腔内癌和扁桃体癌。According to some specific embodiments of the present invention, the HPV-related diseases include at least one of the following: cervical cancer, vulvar cancer, vaginal cancer, anal cancer, penile cancer, head and neck cancer, cervical intraepithelial neoplasia, vulvar intraepithelial neoplasia vaginal intraepithelial neoplasia, anal intraepithelial neoplasia, penile intraepithelial neoplasia, oral cancer, laryngeal cancer, esophageal cancer, intranasal cancer and tonsil cancer.
诊断方法diagnosis method
一方面，本发明提出了一种诊断受试者体内是否患有HPV相关疾病的方法，包括检测受试者来源的生物样品是否携带前面所述的分离的肽、HPV抗原表位、突变体或核酸分子的步骤。如前所述，所述的分离的肽、HPV抗原表位、突变体或核酸分子存在于被HPV感染的个体内，因此，通过检测来源于受试者的生物样品是否携带所述物质，可以有效诊断受试者是否患有或易患HPV相关疾病。On the one hand, the present invention proposes a method for diagnosing whether a subject suffers from HPV-related diseases, including detecting whether a biological sample derived from the subject carries the previously described isolated peptide, HPV epitope, mutant or Nucleic acid molecule steps. As mentioned above, the isolated peptide, HPV epitope, mutant or nucleic acid molecule is present in an individual infected by HPV. Therefore, by detecting whether the biological sample derived from the subject carries the substance, it is possible to Effectively diagnose whether subjects suffer from or are susceptible to HPV-related diseases.
根据本发明的一些具体实施方案，所述HPV相关疾病包括下列中的至少之一：***、外阴癌、***癌、***癌、***癌、头颈癌、宫颈上皮内瘤变、外阴上皮内瘤变、***上皮内瘤变、***上皮内瘤变、***上皮内瘤变、口腔癌、喉癌、食道癌、鼻腔内癌和扁桃体癌。According to some specific embodiments of the present invention, the HPV-related diseases include at least one of the following: cervical cancer, vulvar cancer, vaginal cancer, anal cancer, penile cancer, head and neck cancer, cervical intraepithelial neoplasia, vulvar intraepithelial neoplasia vaginal intraepithelial neoplasia, anal intraepithelial neoplasia, penile intraepithelial neoplasia, oral cancer, laryngeal cancer, esophageal cancer, intranasal cancer and tonsil cancer.
诊断***diagnostic system
最后，本发明提出了一种诊断***。根据本发明的实施例，参考图1，该诊断***包括：肽检测装置100；诊断结果确定装置200。其中，肽检测装置100用于检测受试者来源的生物样品是否携带前面所述的分离的肽、HPV抗原表位或突变体，诊断结果确定装置200与所述肽检测装置100相连，用于基于生物样品是否携带所述分离的肽、HPV抗原表位或突变体，确定所述患者是否患有肿瘤。如：可以采用质谱仪检测受试者血清中是否存在所述的分离的肽、HPV抗原表位或突变体，进而通过质谱数据分析装置确定受试者血清中是否存在该分离的肽、HPV抗原表位或突变体，确定所述患者是否患有肿瘤。发明人发现，所述分离的肽、HPV抗原表位或突变体在肿瘤组织中特异性高表达，本发明实施例所提出的诊断***可用于有效确定特异性高表达所述多肽、HPV抗原表位或突变体的肿瘤患者。Finally, the present invention proposes a diagnostic system. According to an embodiment of the present invention, referring to Figure 1, the diagnostic system includes: a peptide detection device 100; a diagnostic result determination device 200. Among them, the peptide detection device 100 is used to detect whether the biological sample derived from the subject carries the aforementioned isolated peptide, HPV epitope or mutant, and the diagnostic result determination device 200 is connected to the peptide detection device 100 for It is determined whether the patient has a tumor based on whether the biological sample carries the isolated peptide, HPV epitope or mutant. For example, a mass spectrometer can be used to detect whether the isolated peptide, HPV antigen epitope or mutant is present in the subject's serum, and then a mass spectrometry data analysis device can be used to determine whether the isolated peptide, HPV antigen is present in the subject's serum. epitopes or mutants to determine whether the patient has a tumor. The inventor found that the isolated peptides, HPV antigen epitopes or mutants are specifically highly expressed in tumor tissues. The diagnostic system proposed in the embodiment of the present invention can be used to effectively determine the specific highly expressed polypeptides, HPV antigen epitopes. or mutant tumor patients.
另外，发明人发现，***、外阴癌、***癌、***癌、***癌、头颈癌、宫颈上皮内瘤变、外阴上皮内瘤变、***上皮内瘤变、***上皮内瘤变、***上皮内瘤变、口腔癌、喉癌、食道癌、鼻腔内癌或扁桃体癌特异性高表达所述多肽，本发明实施例所提出的诊断***对上述肿瘤的诊断准确性进一步提高。In addition, the inventor found that cervical cancer, vulvar cancer, vaginal cancer, anal cancer, penile cancer, head and neck cancer, cervical intraepithelial neoplasia, vulvar intraepithelial neoplasia, vaginal intraepithelial neoplasia, anal intraepithelial neoplasia, penile epithelial neoplasia Intra-neoplasia, oral cancer, laryngeal cancer, esophageal cancer, intranasal cancer or tonsil cancer specifically express the polypeptide at a high level, and the diagnostic system proposed in the embodiment of the present invention further improves the diagnostic accuracy of the above-mentioned tumors.
同时，发明人发现HLA-I类分子与所述分离的肽、HPV抗原表位或突变体有着较强的亲和力，所述分离的肽、HPV抗原表位或突变体通过与细胞表面HLA-I类分子结合进而激发一系列的免疫反应。因而，本发明实施例所提出的诊断***诊断出同时表达HLA-I类分子和所述分离的肽、HPV抗原表位或突变体的肿瘤患者的几率更高。At the same time, the inventor found that HLA-I class molecules have a strong affinity with the isolated peptide, HPV epitope or mutant, and the isolated peptide, HPV epitope or mutant interacts with HLA-I on the cell surface. The molecules bind and trigger a series of immune responses. Therefore, the diagnostic system proposed by the embodiment of the present invention has a higher probability of diagnosing tumor patients who express both HLA class I molecules and the isolated peptide, HPV epitope or mutant.
需要说明的是，根据本发明实施例的分离的肽、HPV抗原表位、突变体及其用途、编码所述分离的肽或HPV抗原表位或突变体的核酸、表达载体、重组细胞、药物、抗原呈递细胞、免疫效应细胞、疫苗、试剂盒、治疗和诊断HPV的方法和***是本申请的发明人经过艰苦的创造性劳动和优化工作才发现和完成的。It should be noted that according to the embodiments of the present invention, isolated peptides, HPV epitopes, mutants and their uses, nucleic acids encoding the isolated peptides or HPV epitopes or mutants, expression vectors, recombinant cells, and drugs , antigen-presenting cells, immune effector cells, vaccines, kits, methods and systems for treating and diagnosing HPV were discovered and completed by the inventor of the present application after painstaking creative labor and optimization work.
下面参考具体实施例，对本发明进行描述，需要说明的是，这些实施例仅仅是描述性的，而不以任何方式限制本发明。实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件(例如参考J.萨姆布鲁克等著，黄培堂等译的《分子克隆实验指南》，第三版，科学出版社)或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。The present invention will be described below with reference to specific embodiments. It should be noted that these embodiments are only illustrative and do not limit the present invention in any way. If no specific techniques or conditions are specified in the examples, the techniques or conditions described in literature in the field shall be followed (for example, refer to "Molecular Cloning Experimental Guide" translated by J. Sambrook et al., Huang Peitang et al., third edition, Science Press) or follow the product instructions. If the manufacturer of the reagents or instruments used is not indicated, they are all conventional products that can be purchased commercially.
实施例1 HPV抗原表位的鉴定Example 1 Identification of HPV epitopes
本实施例采用免疫亲和的方法获得免疫多肽，具体操作流程如图2所示，具体操作步骤如下：This embodiment uses an immunoaffinity method to obtain immune polypeptides. The specific operation process is shown in Figure 2. The specific operation steps are as follows:
1.1 HLA蛋白复合物(主要组织相容性复合物(MHC)I类复合物分子)的获得1.1 Obtaining HLA protein complexes (major histocompatibility complex (MHC) class I complex molecules)
本实验的具体实验操作如下：The specific experimental operations of this experiment are as follows:
1)取1mL protein A Sepharose CL-4B(GE healthcare)50％resin至Poly-Prep Chromatography Columns中，流穿超纯水及PBS清洗后，流穿4mg anti-HLA-Ⅰ类A、B、C抗体(W6/32，ATCC HB-95)；1) Take 1mL protein A Sepharose CL-4B (GE healthcare) 50% resin into Poly-Prep Chromatography Columns, flow through ultrapure water and PBS after washing, and flow through 4mg anti-HLA-Ⅰ class A, B, C antibodies (W6/32, ATCC HB-95);
2)流穿PBS后，流穿交联平衡液(200mM三乙醇胺)；2) After flowing through the PBS, flow through the cross-linking balance solution (200mM triethanolamine);
3)流穿交联反应液，容留1mL交联反应液(50mM DMP)后关闭下通路，室温(25℃)静置1小时；3) Flow through the cross-linking reaction solution, hold 1 mL of cross-linking reaction solution (50mM DMP), close the lower channel, and let it stand at room temperature (25°C) for 1 hour;
4)流穿交联终止液(100mM乙醇胺)；4) Flow through the cross-linking stop solution (100mM ethanolamine);
5)流穿PBS，容留1mLPBS，制备的抗体亲和柱成品4℃冰箱保存备用；5) Flow through PBS, retain 1mL of PBS, and store the prepared antibody affinity column in a refrigerator at 4°C for later use;
6)使用HPV16型阳性细胞株Caski(人子***上皮)细胞系、Siha(人子宫颈鳞状细胞癌)细胞系进行免疫多肽样品制备，上述细胞系均经HLA-A0201转染构建，采用8e 8以上HPV阳性细胞系裂解提取，膜蛋白裂解液由0.5％脱氧胆酸钠，2％吡喃葡萄糖苷PBS缓冲液组成； 6) Use HPV16-positive cell lines Caski (human cervical cancer epithelial) cell line and Siha (human cervical squamous cell carcinoma) cell line to prepare immune peptide samples. The above cell lines are all constructed by HLA-A0201 transfection, using 8e HPV-positive cell lines above 8 are lysed and extracted. The membrane protein lysate is composed of 0.5% sodium deoxycholate and 2% glucopyranoside PBS buffer;
7)在收集后的HPV细胞样品中加入1x细胞裂解液，使细胞终浓度为0.5e 8-1e 8个/mL，瞬时涡旋振荡混匀后于4℃、20rpm上下颠倒混匀1小时； 7) Add 1x cell lysis solution to the collected HPV cell samples to make the final cell concentration 0.5e 8 -1e 8 cells/mL. Vortex briefly to mix and then mix upside down at 4°C and 20 rpm for 1 hour;
8)将步骤7)获得的产物经4℃、14000g低温高速离心30min，离心后的上清用0.8μm滤膜过滤，滤液4℃保存备用。8) Centrifuge the product obtained in step 7) at 4°C and 14000g for 30 min at low temperature and high speed. The supernatant after centrifugation is filtered with a 0.8 μm filter membrane and the filtrate is stored at 4°C for later use.
9)将步骤8)获得的滤液流穿经抗体亲和柱，使用缓冲液1(150mM NaCl,20mM Tris(pH＝8)水溶液)、2(400mM NaCl,20mM Tris(pH＝8)水溶液)、3(20mM Tris(pH＝8)水溶液)清洗，并使用每次1mL醋酸进行洗脱，每次洗脱液收为一个组分，共收集5组分，获得组分1-5，同时将5个组分混合，获得组分6，以获得含有免疫多肽的所述HLA蛋白复合物。9) Pass the filtrate obtained in step 8) through the antibody affinity column, using buffers 1 (150mM NaCl, 20mM Tris (pH=8) aqueous solution), 2 (400mM NaCl, 20mM Tris (pH=8) aqueous solution), 3 (20mM Tris (pH=8) aqueous solution), and use 1mL acetic acid each time for elution. Each eluate is collected as one component. A total of 5 components are collected to obtain components 1-5. At the same time, 5 components are collected. The components are mixed to obtain component 6 to obtain the HLA protein complex containing immune polypeptides.
1.2 HLA蛋白复合物及免疫多肽分离及检测1.2 Separation and detection of HLA protein complexes and immune peptides
1.2.1固相萃取分离HLA蛋白复合物1.2.1 Solid phase extraction to separate HLA protein complexes
本实施例采用固相萃取方法对步骤1.1中获得的HLA蛋白复合物进行分离。具体的实验操作如下：This example uses solid phase extraction method to separate the HLA protein complex obtained in step 1.1. The specific experimental operations are as follows:
将实验1.1收集的合并后的6种组分分别流穿经30％ACN/0.1％FA水溶液流穿活化及.1％FA水溶液流穿酸化平衡后的Waters Sep-pak tC18固相萃取小柱(WAT036790)，并使用30％ACN/0.1％FA水溶液进行固相萃取洗脱；然后将洗脱液收集后使用冻干机或浓缩仪进行浓缩，-20℃保存待质谱检测。The combined 6 components collected in Experiment 1.1 were flowed through the Waters Sep-pak tC18 solid phase extraction cartridge that was activated by 30% ACN/0.1% FA aqueous solution and .1% FA aqueous solution was flowed through acidification and equilibrium ( WAT036790), and use 30% ACN/0.1% FA aqueous solution for solid phase extraction and elution; then collect the eluate and concentrate it using a freeze dryer or concentrator, and store it at -20°C for mass spectrometry detection.
1.2.2免疫多肽的质谱检测1.2.2 Mass spectrometry detection of immune peptides
将实验1.2.1获得的免疫多肽进行二级分离及质谱检测，使用EASY-nLC 1000超高效液相色谱串联Orbitrap Fusion Lumos Tribrid质谱仪***对免疫多肽样品进行DDA高通量质谱鉴定分析，具体实验操作如下：The immune peptides obtained in experiment 1.2.1 were subjected to secondary separation and mass spectrometry detection, and EASY-nLC 1000 ultra-high performance liquid chromatography tandem Orbitrap Fusion Lumos Tribrid mass spectrometer system was used to conduct DDA high-throughput mass spectrometry identification and analysis of immune peptide samples. Specific experiments Here's how to do it:
获得的所述HPV免疫多肽首先进入trap柱富集并除盐，随后与自装C18柱(150μm内径，1.8μm柱料粒径，约35cm柱长)串联，以500nL/min流速通过如下有效梯度进行分离：0～5min，5％流动B(98％ACN，0.1％FA)；5～45min，流动相B从5％线性升至25％；45～50min，流动相B从25％升至35％；50～52min，流动相B从35％升至80％；52～54min，80％流动相B 54～54.5min，流动相B从80％降至5％；54.5～65min，5％流动相B。纳升液相分离末端直接连接质谱仪并按如下参数进行检测：经过液相分离的肽段通过nanoESI源离子化后进入到串联质谱仪Orbitrap Fusion Lumos(Thermo Fisher Scientific，San Jose，CA)进行MSOT+tMS2OT模式检测，主要参数设置为：离子源电压设置为2kV；一级质谱扫描范围350～1,400m/z；分辨率设置为60,000，最大离子注入时间(MIT)为50ms；二级质谱碎裂模式为HCD，碎裂能量设置为30； 分辨率设置为30,000，最大离子注入时间(MIT)为50ms，AGC设置为：一级4E5,二级5E4。对候选目标离子进行靶向采集。The obtained HPV immune polypeptide first enters the trap column for enrichment and desalination, and is then connected in series with a self-assembled C18 column (150 μm inner diameter, 1.8 μm column material particle size, approximately 35 cm column length), and passes through the following effective gradient at a flow rate of 500 nL/min Separation: 0~5min, 5% mobile B (98% ACN, 0.1% FA); 5~45min, mobile phase B rises linearly from 5% to 25%; 45~50min, mobile phase B rises from 25% to 35% %; 50～52min, mobile phase B rises from 35% to 80%; 52～54min, 80% mobile phase B; 54～54.5min, mobile phase B drops from 80% to 5%; 54.5～65min, 5% mobile phase B. The end of the nanoliquid phase separation is directly connected to the mass spectrometer and detected according to the following parameters: the peptides after liquid phase separation are ionized by the nanoESI source and then enter the tandem mass spectrometer Orbitrap Fusion Lumos (Thermo Fisher Scientific, San Jose, CA) for MSOT. +tMS2OT mode detection, the main parameter settings are: ion source voltage is set to 2kV; primary mass spectrometry scanning range is 350~1,400m/z; resolution is set to 60,000, maximum ion injection time (MIT) is 50ms; secondary mass spectrometry fragmentation The mode is HCD, the fragmentation energy is set to 30; the resolution is set to 30,000, the maximum ion implantation time (MIT) is 50ms, and the AGC is set to: 4E5 for the first level and 5E4 for the second level. Targeted acquisition of candidate target ions.
1.2.3免疫多肽靶向数据分析1.2.3 Immune peptide targeting data analysis
使用skyline靶向数据分析方法针对上述1.2.2步骤获得的12051条多肽样品分析是否具备合成肽相同的靶向捕获迹象。Use the skyline targeted data analysis method to analyze whether the 12051 peptide samples obtained in step 1.2.2 above have the same target capture signs as the synthetic peptides.
将经质谱靶向采样后不同批次的下机检测样品文件及合成多肽采样后下机检测样品文件原始数据导入skyline分析软件，用于检查离子在样本及合成肽样本中的鉴定及匹配。Import the raw data of different batches of off-machine detection sample files after mass spectrometry targeted sampling and off-machine detection sample files after synthetic peptide sampling into the skyline analysis software to check the identification and matching of ions in the samples and synthetic peptide samples.
结果图中下机样品文件及合成肽样品文件如下：图3-A和3-B：检测样品：(1)caski-1；(2)caski-2；(3)caski-3；(4)合成肽样品caski-mix；图4-A和4-B：检测样品：(1)caski-1；(2)caski-2；(3)caski-3；(4)合成肽样品：caski-mix；图5-A和5-B：检测样品：(1)caski-1；(2)caski-2；(3)caski-3；(4)合成肽样品：caski-mix；图6-A和6-B：检测样品：(1)caski-1；(2)caski-2；(3)caski-3；(4)siha-1；(5)siha-2；(6)合成肽样品：caski-siha-mix；图7-A和7-B：检测样品：(1)siha-1；(2)siha-2；(3)siha-3；(4)合成肽样品：siha-mix；图8-A和8-B：检测样品：(1)caski-1；(2)caski-2；(3)caski-3；(4)合成肽样品：caski-mix；图9-A和9-B：检测样品：(1)siha-1；(2)siha-2；(3)siha-3；(4)合成肽样品：siha-mix。The offline sample files and synthetic peptide sample files in the result chart are as follows: Figure 3-A and 3-B: Test samples: (1) caski-1; (2) caski-2; (3) caski-3; (4) Synthetic peptide sample caski-mix; Figure 4-A and 4-B: Test samples: (1) caski-1; (2) caski-2; (3) caski-3; (4) Synthetic peptide sample: caski-mix ; Figure 5-A and 5-B: Detection samples: (1) caski-1; (2) caski-2; (3) caski-3; (4) Synthetic peptide sample: caski-mix; Figure 6-A and 6-B: Test samples: (1) caski-1; (2) caski-2; (3) caski-3; (4) siha-1; (5) siha-2; (6) Synthetic peptide sample: caski -siha-mix; Figure 7-A and 7-B: Test samples: (1) siha-1; (2) siha-2; (3) siha-3; (4) Synthetic peptide sample: siha-mix; Figure 8-A and 8-B: Test samples: (1) caski-1; (2) caski-2; (3) caski-3; (4) Synthetic peptide sample: caski-mix; Figure 9-A and 9- B: Test samples: (1) siha-1; (2) siha-2; (3) siha-3; (4) Synthetic peptide sample: siha-mix.
本实验中，成功验证到的肽段在样本及合成肽样本中的鉴定及匹配满足以下条件：XIC出峰的液相保留时间一致，误差不超过3min；匹配的质量精度不超过10ppm；匹配子离子数超过5个以上。具体的实验结果如图3-A、3-B、4-A、4-B、5-A、5-B、6-A、6-B、7-A、7-B、8-A、8-B、9-A、9-B所示，其中，图中每例提取谱图展示的左上方已标明合成肽及样品的信息，提取出的结果具备数据分析中标出的匹配标准阳性。In this experiment, the identification and matching of successfully verified peptides in samples and synthetic peptide samples met the following conditions: the liquid phase retention time of the XIC peak was consistent, and the error did not exceed 3 minutes; the mass accuracy of the match did not exceed 10 ppm; the matching The number of ions exceeds 5 or more. The specific experimental results are shown in Figures 3-A, 3-B, 4-A, 4-B, 5-A, 5-B, 6-A, 6-B, 7-A, 7-B, 8-A, 8-B, 9-A, and 9-B, in which the information of the synthetic peptide and sample is marked on the upper left side of the extracted spectrum of each example in the figure, and the extracted results have the matching standard positive marked in the data analysis.
检测分析中，多肽在正离子模式下质子化形成带电前体离子。带电前体离子最初位于N端或碱性残基侧链上，但由于内部裂解，可沿主链移动，在不同的位点断裂后生成子离子片段。其中：有三种不同类型的主链键可以断裂成肽片段：烷基羰基(CHR-CO)，肽酰胺键(CO-NH)和氨基烷基键(NH-CHR)。In detection analysis, peptides are protonated in positive ion mode to form charged precursor ions. The charged precursor ion is initially located on the N-terminal or basic residue side chain, but due to internal cleavage, it can move along the main chain and generate product ion fragments after being broken at different sites. Among them: There are three different types of backbone bonds that can be broken into peptide fragments: alkyl carbonyl (CHR-CO), peptide amide bond (CO-NH) and aminoalkyl bond (NH-CHR).
在经HCD及CID碰撞生成二级离子检测的质谱***中，因为肽酰胺键(CO-NH)最脆弱的，b和y离子是最常见的离子类型，举例说明，如图10所示，其中，b、y离子右下角标注数字代表氨基酸残基的数目。In the mass spectrometry system that detects secondary ions generated by HCD and CID collisions, because the peptide amide bond (CO-NH) is the most fragile, b and y ions are the most common ion types. For example, as shown in Figure 10, where , The numbers marked in the lower right corner of b and y ions represent the number of amino acid residues.
因此，本发明制备的免疫多肽样品新鉴定的HPV特异性免疫多肽表位的序列如下所示：Therefore, the sequence of the HPV-specific immune polypeptide epitope newly identified in the immune polypeptide sample prepared by the present invention is as follows:
E6 65-72NPYAVCDK(SEQ ID NO:1)。 E6 65-72 NPYAVCDK (SEQ ID NO: 1).
E7 4-11DTPTLHEY(SEQ ID NO:2)。 E7 4-11 DTPTLHEY (SEQ ID NO: 2).
E7 75-82DIRTLEDL(SEQ ID NO:3)。 E7 75-82 DIRTLEDL (SEQ ID NO: 3).
E7 75-83DIRTLEDLL(SEQ ID NO:4)。 E7 75-83 DIRTLEDLL (SEQ ID NO: 4).
E7 77-86RTLEDLLMGT(SEQ ID NO:5)。 E7 77-86 RTLEDLLMGT (SEQ ID NO: 5).
E7 77-90RTLEDLLMGTLGIV(SEQ ID NO:6)。 E7 77-90 RTLEDLLMGTGIV (SEQ ID NO: 6).
E7 79-88LEDLLMGTLG(SEQ ID NO:7)。 E7 79-88 LEDLLMGTTLG (SEQ ID NO: 7).
实施例2酶联免疫斑点法(ELISPOT)评估HPV特异性肿瘤抗原肽免疫原性Example 2 Enzyme-linked immunospot assay (ELISPOT) to evaluate the immunogenicity of HPV-specific tumor antigen peptides
实验采用T2细胞负载新鉴定的实施例1所述的7种HPV16型特异性混合抗原多肽，通过T2细胞的抗原提呈功能呈递给细胞毒性T细胞CTL，T2细胞的TAP(抗原提呈转运)分子缺陷通过有效负载外源多肽，实验抗原提呈后而激活免疫应答。实验设置阳性对照组、阴性对组及供试品组。将经细胞培养处理对数生长期的T2(ATCC：CRL-1992)活细胞收集后，经培养基稀释重悬至5e 5个/mL细胞密度备用。每组取1mL T2细胞组，阳性对照组加入植物血凝素PHA(Sigma,货号L8902-25MG)，PHA终浓度为4μg/mL；阴性对照组不添加处理；供试品组添加新鉴定HPV抗原肽混合物处理，抗原多肽混合物终浓度约为5μg/mL，CO 2培养箱中培养3.5h。取制备好的T2细胞和CTL细胞进行ELISPOT铺板，每孔分别加入50μL CTL细胞及50μLT2细胞。于37℃、5％CO 2培养箱培养18h后使用试剂盒(Human IFN-gamma ELISpotPRO(ALP)，货号：3420-2AST-10)进行抗体孵育、显色后使用EliSpot读板仪进行分泌IFN-γ斑点计数。 The experiment uses T2 cells to load the newly identified 7 HPV16 type-specific mixed antigen polypeptides described in Example 1, and presents them to cytotoxic T cells CTL through the antigen presentation function of T2 cells and the TAP (antigen presentation transport) of T2 cells. The molecular defect activates the immune response after the experimental antigen is presented through the payload of exogenous peptides. The experiment consists of a positive control group, a negative control group and a test product group. Collect viable T2 (ATCC: CRL-1992) cells in logarithmic growth phase after cell culture treatment, dilute them with culture medium and resuspend them to a cell density of 5e 5 cells/mL for later use. Take 1mL of T2 cells from each group, add phytohemagglutinin PHA (Sigma, Cat. No. L8902-25MG) to the positive control group, and the final concentration of PHA is 4 μg/mL; no treatment is added to the negative control group; newly identified HPV antigen is added to the test group For peptide mixture treatment, the final concentration of the antigen peptide mixture is approximately 5 μg/mL, and cultured in a CO 2 incubator for 3.5 h. Take the prepared T2 cells and CTL cells for ELISPOT plating, and add 50 μL of CTL cells and 50 μL of T2 cells to each well. After culturing for 18 hours in a 37°C, 5% CO2 incubator, use a kit (Human IFN-gamma ELISpotPRO (ALP), Cat. No.: 3420-2AST-10) for antibody incubation, color development, and use an EliSpot plate reader to secrete IFN- gamma spot count.
ELISPOT检测IFN-γ分泌光斑数实验数据如表1所示，其中，供试品组的IFN-γ斑点数量相对阴性对照组显著增多。说明鉴定HPV特异性肿瘤抗原多肽具有良好的免疫原性。The experimental data of ELISPOT detection of the number of IFN-γ secretion spots are shown in Table 1. Among them, the number of IFN-γ spots in the test group was significantly increased compared with the negative control group. This shows that the HPV-specific tumor antigen polypeptide identified has good immunogenicity.
表1：Table 1:
试验组别test group 阴性对照组negative control group 阳性对照组positive control group 供试品组(鉴定表位混合处理组)Test product group (identification epitope mixed treatment group)
IFN-γ斑点数IFN-γ spot number 2525 289289 377377
实施例3酶联免疫斑点法(ELISPOT)评估HPV特异性肿瘤抗原肽突变体的免疫原性Example 3 Enzyme-linked immunospot assay (ELISPOT) to evaluate the immunogenicity of HPV-specific tumor antigen peptide mutants
本实施例中，发明人替换了实施例1或2获得的有效抗原表位的非权重位点氨基酸，探索该方式是否可以增强CTL表位的免疫原性及潜在药效。具体地，针对HLA-A0201分型，发明人对除2和9位氨基酸外的其他位点进行氨基酸取代，并对获得的突变体提进行免疫原性检测。具体的实验操作如下：In this example, the inventor replaced the non-weighted amino acids of the effective antigenic epitope obtained in Example 1 or 2 to explore whether this method can enhance the immunogenicity and potential efficacy of the CTL epitope. Specifically, for HLA-A0201 typing, the inventors performed amino acid substitutions at other sites except amino acids 2 and 9, and performed immunogenicity testing on the obtained mutant extracts. The specific experimental operations are as follows:
3.1 HPV特异性肿瘤抗原肽突变体的制备3.1 Preparation of HPV-specific tumor antigen peptide mutants
本实施例中，发明人对上述鉴定到的HPV抗原表位中的E7 75-83DIRTLEDLL及E6 65-72NPYAVCDK进行改造，经合成后用于后续实验。获得的突变体的氨基酸序列如表2和表3所示。 In this example, the inventor modified E7 75-83 DIRTLEDLL and E6 65-72 NPYAVCDK among the HPV epitopes identified above, and used them in subsequent experiments after synthesis. The amino acid sequences of the obtained mutants are shown in Tables 2 and 3.
3.2 HPV特异性肿瘤抗原肽突变体免疫原性检测3.2 Immunogenicity detection of HPV-specific tumor antigen peptide mutants
采用ELISPOT检测实验，验证步骤3.1获得的HPV抗原表位的所述变体是否能够激活CD8+T细胞的免疫反应。An ELISPOT detection experiment is used to verify whether the variant of the HPV epitope obtained in step 3.1 can activate the immune response of CD8+ T cells.
实验设置阳性对照组、阴性对组及供试品组。将经细胞培养处理对数生长期的T2(ATCC：CRL-1992)活细胞收集后，经培养基稀释重悬至5e 5个/mL细胞密度备用。每组取1ml T2细胞组，阳性对照组加入植物血凝素PHA(Sigma,货号L8902-25MG)，PHA终浓度为4μg/mL；阴性对照组不添加处理；供试品组分别单独添加新鉴定的2种HPV抗原肽及其对应的突变体处理，抗原多肽DIRTLEDLL及其突变形式多肽设置终浓度约为5μg/mL，采用HIPP-T009淋巴细胞无血清培养基进行培养，CO 2培养箱中培养3.5h。取制备好的T2细胞和CTL细胞进行ELISPOT铺板，每孔分别加入50μL T细胞及50μL T2细胞。于37℃、5％CO 2培养箱培养18h后使用试剂盒(Human IFN-gamma ELISpotPRO(ALP)，货号：3420-2AST-10)进行抗体孵育、显色后使用EliSpot读板仪进行分泌IFN-γ斑点计数。 The experiment consists of a positive control group, a negative control group and a test product group. Collect viable T2 (ATCC: CRL-1992) cells in logarithmic growth phase after cell culture treatment, dilute them with culture medium and resuspend them to a cell density of 5e 5 cells/mL for later use. Take 1ml of T2 cells from each group, and add phytohemagglutinin PHA (Sigma, Cat. No. L8902-25MG) to the positive control group. The final concentration of PHA is 4 μg/mL; no treatment is added to the negative control group; new identifications are added to the test group separately. The two HPV antigen peptides and their corresponding mutants were processed. The final concentration of the antigen peptide DIRTLEDLL and its mutant peptide was set to about 5 μg/mL. They were cultured using HIPP-T009 lymphocyte serum-free medium and cultured in a CO 2 incubator. 3.5h. Take the prepared T2 cells and CTL cells for ELISPOT plating, and add 50 μL T cells and 50 μL T2 cells to each well. After culturing for 18 hours in a 37°C, 5% CO2 incubator, use a kit (Human IFN-gamma ELISpotPRO (ALP), Cat. No.: 3420-2AST-10) for antibody incubation, color development, and use an EliSpot plate reader to secrete IFN- gamma spot count.
测试多肽具有免疫原性的要求如下：斑点数(测试多肽)/斑点数(无关多肽)＞2；即，测试多肽引起的斑点数超过无关多肽斑点数目的两倍以上，表明测试多肽具有免疫原性，关于上述HPV抗原表位E7 75-83DIRTLEDLL及其突变体的ELISPOT检测结果见如表2所示。 The requirements for the test polypeptide to be immunogenic are as follows: number of spots (test polypeptide)/number of spots (unrelated polypeptide) >2; that is, the number of spots caused by the test polypeptide exceeds the number of spots caused by the irrelevant polypeptide by more than twice, indicating that the test polypeptide is immunogenic. Sexually, the ELISPOT detection results of the above-mentioned HPV epitope E7 75-83 DIRTLEDLL and its mutants are shown in Table 2.
表2：Table 2:
验证多肽及其变体Verify peptides and their variants 实验斑点数Number of experimental spots 阴性斑点数Number of negative spots 倍数(实验组/阴性对照组)Multiples (experimental group/negative control group)
DIRTLEDLL(SEQ ID NO:4)DIRTLEDLL(SEQ ID NO:4) 245245 3333 7.427.42
DIRTYEDLL(SEQ ID NO：16)DIRTYEDLL(SEQ ID NO：16) 209209 2828 7.467.46
DILTLEDLL(SEQ ID NO：17)DILTLEDLL(SEQ ID NO：17) 256256 3737 6.916.91
FIRTLYDTL(SEQ ID NO：13)FIRTLYDTL(SEQ ID NO：13) 312312 24twenty four 1313
YIRTLEDLL(SEQ ID NO：14)YIRTLEDLL(SEQ ID NO：14) 328328 3232 10.2510.25
DIPTEDLL(SEQ ID NO：15)DIPTEDLL(SEQ ID NO：15) 279279 3333 8.458.45
由表2结果可知，制备的HPV抗原肽的突变体引起的斑点数均优于突变前的目标表位(DIRTLEDLL)，表明本发明中的多肽及其突变体均具有良好的免疫原性，可以特异性地激活CD8 +T细胞免疫反应。 It can be seen from the results in Table 2 that the number of spots caused by the prepared mutants of the HPV antigen peptide is better than that of the pre-mutation target epitope (DIRTLEDLL), indicating that the polypeptides of the present invention and their mutants have good immunogenicity and can Specifically activates CD8 + T cell immune responses.
关于上述HPV抗原表位E6 65-72NPYAVCDK及其突变体的ELISPOT检测结果见如表3所示，测试优选变体多肽引起的斑点数均优于突变前的目标表位，表明本发明中的多肽及其优选变体形式均具有免疫原性，可以特异性地激活CD8 +T细胞免疫反应。 The ELISPOT detection results of the above-mentioned HPV antigenic epitope E6 65-72 NPYAVCDK and its mutants are shown in Table 3. The number of spots caused by the preferred variant polypeptides tested is better than that of the target epitope before mutation, indicating that the target epitope in the present invention The polypeptide and its preferred variant forms are immunogenic and can specifically activate CD8 + T cell immune responses.
表3：table 3:
多肽及其变体Polypeptides and variants thereof 实验组斑点数Number of spots in experimental group 阴性对照组斑点数Number of spots in negative control group 倍数(实验组/阴性对照组)Multiples (experimental group/negative control group)
NPYAVCDK(SEQ ID NO:1)NPYAVCDK(SEQ ID NO:1) 235235 2929 8.18.1
FPYAVCDK(SEQ ID NO：10)FPYAVCDK(SEQ ID NO：10) 316316 3131 10.1910.19
NPYAECDK(SEQ ID NO：12)NPYAECDK(SEQ ID NO：12) 281281 3535 8.038.03
NPYAVLDK(SEQ ID NO：11)NPYAVLDK(SEQ ID NO：11) 299299 3434 8.798.79
NPLAVCDK(SEQ ID NO：8)NPLAVCDK(SEQ ID NO：8) 336336 2626 12.9212.92
NPYAVCVK(SEQ ID NO：9)NPYAVCVK(SEQ ID NO：9) 385385 3232 12.0312.03
实施例4 HPV特异性CTL小鼠模型体内药效检测Example 4 In vivo drug efficacy testing of HPV-specific CTL mouse model
HPV特异性CTL使用来源于HPV阳性的感染患者采集的外周血，通过体外制备工艺进行制备，具体制备的工艺流 程如图11所示，其中，HPV特异性抗原多肽mix是指实施例1获得的7种抗原多肽的混合物，具体实验操作如下：HPV-specific CTL are prepared from peripheral blood collected from HPV-positive infected patients through an in vitro preparation process. The specific preparation process flow is shown in Figure 11, in which the HPV-specific antigen polypeptide mix refers to the one obtained in Example 1 A mixture of 7 antigenic peptides, the specific experimental procedures are as follows:
将***阳性细胞caski接种至6-8周龄的重度免疫联合缺陷小鼠NOG dKO小鼠皮下，待成瘤生长至平均体积约50-75mm 3时随机分为PBS(对照)组，HPV特异性CTL高剂量组静脉注射组、HPV特异性CTL高剂量联用人白介素2(IL-2)腹腔给药组，HPV特异性CTL低剂量联用人白介素2(IL-2)腹腔给药组(n＝3)，进行HPV特异性CTL小鼠模型初步体内药效实验。HPV特异性CTL高剂量组采用进行静脉注射CTL细胞2*10e 7/只/次，24h内给药2次；HPV特异性CTL低剂量组采用进行静脉注射CTL细胞2*10e 6/只/次，24h内给药2次；人白介素2(IL-2)采用10000U/只/天，腹腔连续给药14天。HPV特异性CTL在第14天重复给药，维持CTL细胞在体内的存留比例及治疗效果。培养至38天对小鼠进行处理，获取肿瘤，并称重，计算肿瘤(体积)抑制率。皮下移植Caski小鼠模型(NOG dKO)中HPV特异性CTL药效实验设计及肿瘤抑制数据如表3所示，不同给药组给药对肿瘤生长抑制程度表现不同，小鼠培养至38天，高剂量HPV特异性CTL单药组(TGI＝31％)，高剂量HPV特异性CTL联用IL-2组(TGI＝63％)、低剂量HPV特异性CTL联用IL-2组(TGI＝52％)，表现出显著的肿瘤抑制效果。 Cervical cancer positive cells caski were inoculated subcutaneously into 6-8 week old NOG dKO mice with severe combined immune deficiencies. When the tumors grew to an average volume of about 50-75 mm, they were randomly divided into PBS (control) group and HPV-specific HPV-specific CTL high-dose intravenous injection group, HPV-specific CTL high-dose combined with intraperitoneal administration of human interleukin-2 (IL-2) group, HPV-specific CTL low-dose combined with intraperitoneal administration of human interleukin-2 (IL-2) group (n=3), preliminary in vivo efficacy experiments on HPV-specific CTL mouse models were conducted. The HPV-specific CTL high-dose group was intravenously injected with 2*10e 7 CTL cells/time, and administered twice within 24 hours; the HPV-specific CTL low-dose group was intravenously injected with 2*10e 6 CTL cells/time , administered twice within 24 hours; human interleukin 2 (IL-2) was administered at 10,000 U/animal/day, administered intraperitoneally for 14 consecutive days. Repeated administration of HPV-specific CTL on the 14th day maintained the retention ratio of CTL cells in the body and the therapeutic effect. The mice were cultured for 38 days, and the tumors were harvested and weighed to calculate the tumor (volume) inhibition rate. The experimental design and tumor inhibition data of HPV-specific CTL efficacy in the subcutaneously transplanted Caski mouse model (NOG dKO) are shown in Table 3. Different administration groups have different inhibitory effects on tumor growth. The mice were cultured for 38 days. High-dose HPV-specific CTL alone group (TGI=31%), high-dose HPV-specific CTL combined with IL-2 group (TGI=63%), low-dose HPV-specific CTL combined with IL-2 group (TGI= 52%), showing significant tumor inhibitory effect.
表4：Table 4:
Figure PCTCN2022116872-appb-000003
Figure PCTCN2022116872-appb-000003
其中，iv表示静脉注射，ip表示腹腔注射。Among them, iv means intravenous injection and ip means intraperitoneal injection.
此外，术语“第一”、“第二”仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此，限定有“第一”、“第二”的特征可以明示或者隐含地包括至少一个该特征。在本发明的描述中，“多个”的含义是至少两个，例如两个，三个等，除非另有明确具体的限定。In addition, the terms “first” and “second” are used for descriptive purposes only and cannot be understood as indicating or implying relative importance or implicitly indicating the quantity of indicated technical features. Therefore, features defined as "first" and "second" may explicitly or implicitly include at least one of these features. In the description of the present invention, "plurality" means at least two, such as two, three, etc., unless otherwise expressly and specifically limited.
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。In the description of this specification, reference to the terms "one embodiment," "some embodiments," "an example," "specific examples," or "some examples" or the like means that specific features are described in connection with the embodiment or example. , structures, materials or features are included in at least one embodiment or example of the invention. In this specification, the schematic expressions of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the specific features, structures, materials or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, those skilled in the art may combine and combine different embodiments or examples and features of different embodiments or examples described in this specification unless they are inconsistent with each other.
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。Although the embodiments of the present invention have been shown and described above, it can be understood that the above-mentioned embodiments are illustrative and should not be construed as limitations of the present invention. Those of ordinary skill in the art can make modifications to the above-mentioned embodiments within the scope of the present invention. The embodiments are subject to changes, modifications, substitutions and variations.

Claims (31)

  1. 一种分离的肽，其特征在于，包括HPV E6蛋白的65-72位氨基酸。An isolated peptide is characterized by comprising amino acids 65-72 of HPV E6 protein.
  2. 根据权利要求1所述的分离的肽，其特征在于，所述HPV E6蛋白为HPV16 E6蛋白。The isolated peptide of claim 1, wherein the HPV E6 protein is HPV16 E6 protein.
  3. 根据权利要求1或2所述的分离的肽，其特征在于，所述分离的肽包含与HLA-A、HLA-B或HLA-C分子结合的氨基酸片段。The isolated peptide according to claim 1 or 2, characterized in that the isolated peptide comprises an amino acid fragment that binds to an HLA-A, HLA-B or HLA-C molecule.
  4. 根据权利要求1-3任一项所述的分离的肽，其特征在于，包括SEQ ID NO:1所示氨基酸序列。The isolated peptide according to any one of claims 1-3, characterized in that it includes the amino acid sequence shown in SEQ ID NO: 1.
  5. 一种突变体，其特征在于，相较于权利要求1-4任一项所述的分离的肽，所述突变体在除锚定位外具有至少一个突变位点。A mutant, characterized in that, compared to the isolated peptide according to any one of claims 1 to 4, the mutant has at least one mutation site except for the anchor position.
  6. 根据权利要求5所述的突变体，其特征在于，相较于权利要求1-4任一项所述的分离的肽，所述突变体具有如下突变中的至少之一：第1位、第3位、第5位、第6位、第7位。The mutant according to claim 5, characterized in that, compared to the isolated peptide according to any one of claims 1 to 4, the mutant has at least one of the following mutations: position 1, position 1 3rd, 5th, 6th, 7th.
  7. 根据权利要求5所述的突变体，其特征在于，相较于权利要求1-4任一项所述的分离的肽，所述突变体具有如下突变中的至少之一：The mutant according to claim 5, characterized in that, compared to the isolated peptide according to any one of claims 1-4, the mutant has at least one of the following mutations:
    1)第1位的N突变为F；1) The N at position 1 is mutated to F;
    2)第3位的Y突变为L；2) The Y at position 3 is mutated to L;
    3)第5位的V突变为E；3) The V at position 5 is mutated to E;
    4)第6位的C突变为L；和4) C at position 6 is mutated to L; and
    5)第7位的D突变为V。5) D at position 7 is mutated to V.
  8. 根据权利要求5-7任一项所述的突变体，其特征在于，所述突变体具有SEQ ID NO：8～12任一所示的氨基酸序列。The mutant according to any one of claims 5-7, characterized in that the mutant has an amino acid sequence shown in any one of SEQ ID NO: 8 to 12.
  9. 一种表达载体，其特征在于，携带表达权利要求1-4任一项所述的分离的肽或权利要求5-8任一项所述的突变体的核酸。An expression vector, characterized by carrying a nucleic acid for expressing the isolated peptide described in any one of claims 1-4 or the mutant described in any one of claims 5-8.
  10. 一种抗原呈递细胞，其特征在于，所述细胞可呈递权利要求1-4任一项所述的分离的肽或权利要求5-8任一项所述的突变体。An antigen-presenting cell, characterized in that the cell can present the isolated peptide described in any one of claims 1-4 or the mutant described in any one of claims 5-8.
  11. 根据权利要求10所述的抗原呈递细胞，其特征在于，所述抗原呈递细胞是通过下列至少之一获得的：The antigen-presenting cell according to claim 10, characterized in that the antigen-presenting cell is obtained by at least one of the following:
    将具有抗原呈递能力的细胞与所述分离的肽或HPV抗原表位进行接触；contacting cells with antigen-presenting ability with the isolated peptide or HPV epitope;
    将权利要求9所述的表达载体导入所述具有抗原呈递能力的细胞。The expression vector of claim 9 is introduced into the cell with antigen presentation ability.
  12. 根据权利要求11所述的抗原呈递细胞，其特征在于，所述具有抗原呈递能力的细胞为树突状细胞、B细胞或单核-吞噬细胞。The antigen-presenting cell according to claim 11, wherein the cells with antigen-presenting ability are dendritic cells, B cells or mononuclear phagocytes.
  13. 一种免疫效应细胞，其特征在于，所述免疫效应细胞可识别权利要求1-4任一项所述分离的肽、权利要求5-8任一项所述的突变体或识别在细胞表面呈递权利要求1-4任一项所述的分离的肽或权利要求5-8任一项所述的突变体的抗原呈递细胞。An immune effector cell, characterized in that the immune effector cell can recognize the isolated peptide described in any one of claims 1-4, the mutant described in any one of claims 5-8, or recognize the peptide presented on the cell surface. Antigen-presenting cells of the isolated peptide according to any one of claims 1-4 or the mutant according to any one of claims 5-8.
  14. 根据权利要求13所述的免疫效应细胞，其特征在于，所述免疫效应细胞是通过下列方式获得的：The immune effector cell according to claim 13, characterized in that the immune effector cell is obtained in the following manner:
    将权利要求10-12任一项所述的抗原呈递细胞与具有免疫效应能力的细胞接触。The antigen-presenting cell according to any one of claims 10-12 is contacted with a cell having immune effector ability.
  15. 根据权利要求14所述的免疫效应细胞，其特征在于，所述具有免疫效应能力的细胞为T细胞。The immune effector cell according to claim 14, wherein the cells with immune effector ability are T cells.
  16. 根据权利要求14所述的免疫效应细胞，其特征在于，所述具有免疫效应能力的细胞为CD8 +T细胞。 The immune effector cell according to claim 14, wherein the cells with immune effector ability are CD8 + T cells.
  17. 试剂在制备试剂盒中的用途，所述试剂用于检测权利要求1-4中任一项所述的分离的肽或权利要求5-8任一项所述的突变体，所述试剂盒用于诊断HPV或者检测HPV的治疗效果。The use of reagents in preparing kits for detecting the isolated peptides described in any one of claims 1-4 or the mutants described in any one of claims 5-8, wherein the kits are used For diagnosing HPV or testing the effectiveness of HPV treatment.
  18. 一种试剂盒，其特征在于，包括适于检测权利要求1-4中任一项所述的分离的肽或权利要求5-8任一项所述的突变体的试剂。A kit comprising reagents suitable for detecting the isolated peptide according to any one of claims 1-4 or the mutant according to any one of claims 5-8.
  19. 权利要求1-4任一项所述的分离的肽、权利要求5-8任一项所述的突变体、权利要求9所述的表达载体、权利要求10-12任一项所述的抗原呈递细胞或权利要求13-16任一项所述的免疫效应细胞在制备药物中的用途，所述药物用于预防或治疗HPV相关疾病。The isolated peptide according to any one of claims 1 to 4, the mutant according to any one of claims 5 to 8, the expression vector according to claim 9, and the antigen according to any one of claims 10 to 12. The use of presenting cells or immune effector cells according to any one of claims 13 to 16 in the preparation of medicines for preventing or treating HPV-related diseases.
  20. 根据权利要求19所述的用途，其特征在于，所述HPV相关疾病包括下列中的至少之一：***、外阴癌、***癌、***癌、***癌、头颈癌、宫颈上皮内瘤变、外阴上皮内瘤变、***上皮内瘤变、***上皮内瘤变、***上皮内瘤变、口腔癌、喉癌、食道癌、鼻腔内癌和扁桃体癌。The use according to claim 19, wherein the HPV-related diseases include at least one of the following: cervical cancer, vulvar cancer, vaginal cancer, anal cancer, penile cancer, head and neck cancer, cervical intraepithelial neoplasia, Vulvar intraepithelial neoplasia, vaginal intraepithelial neoplasia, anal intraepithelial neoplasia, penile intraepithelial neoplasia, oral cancer, laryngeal cancer, esophageal cancer, intranasal cancer and tonsil cancer.
  21. 一种药物，其特征在于，包含权利要求1-4任一项所述的分离的肽、权利要求5-8任一项所述的突变体、权利要求9所述的表达载体、权利要求10-12任一项所述的抗原呈递细胞或权利要求13-16任一项所述的免疫效应细胞。A medicine, characterized by comprising the isolated peptide according to any one of claims 1-4, the mutant according to any one of claims 5-8, the expression vector according to claim 9, and the expression vector according to claim 10 The antigen-presenting cell according to any one of -12 or the immune effector cell according to any one of claims 13-16.
  22. 一种疫苗，其特征在于，包含权利要求10-12任一项所述的抗原呈递细胞。A vaccine, characterized by comprising the antigen-presenting cell according to any one of claims 10-12.
  23. 根据权利要求22所述的疫苗，其特征在于，进一步包括至少一种佐剂。The vaccine of claim 22, further comprising at least one adjuvant.
  24. 一种诊断***，其特征在于，包括：A diagnostic system, characterized by including:
    肽检测装置，所述肽检测装置用于检测受试者来源的生物样品是否携带权利要求1-4任一项所述的分离的肽或权利要求5-8任一项所述的突变体；A peptide detection device, which is used to detect whether a biological sample derived from a subject carries the isolated peptide described in any one of claims 1-4 or the mutant described in any one of claims 5-8;
    诊断结果确定装置，所述诊断结果确定装置与所述肽检测装置相连，用于基于所述生物样品是否携带所述分离的肽或突变体，确定所述受试者是否患肿瘤。A diagnostic result determination device, the diagnostic result determination device is connected to the peptide detection device, and is used to determine whether the subject suffers from a tumor based on whether the biological sample carries the isolated peptide or mutant.
  25. 根据权利要求24所述的诊断***，其特征在于，所述肿瘤为***、外阴癌、***癌、***癌、***癌、头颈癌、宫颈上皮内瘤变、外阴上皮内瘤变、***上皮内瘤变、***上皮内瘤变、***上皮内瘤变、口腔癌、喉癌、食道癌、鼻腔内癌或扁桃体癌。The diagnostic system according to claim 24, wherein the tumor is cervical cancer, vulvar cancer, vaginal cancer, anal cancer, penile cancer, head and neck cancer, cervical intraepithelial neoplasia, vulvar intraepithelial neoplasia, vaginal epithelium Intraepithelial neoplasia, anal intraepithelial neoplasia, penile intraepithelial neoplasia, oral cancer, laryngeal cancer, esophageal cancer, intranasal cancer or tonsil cancer.
  26. 权利要求1-4任一项所述的分离的肽、权利要求5-8任一项所述的突变体、权利要求9所述的表达载体、权利要求10-12任一项所述的抗原呈递细胞、权利要求13-16任一项所述的免疫效应细胞、权利要求21所述的药物或权利要求22或23所述的疫苗在治疗和/或预防HPV相关疾病中的用途。The isolated peptide according to any one of claims 1 to 4, the mutant according to any one of claims 5 to 8, the expression vector according to claim 9, and the antigen according to any one of claims 10 to 12. Use of the presenting cells, the immune effector cells of any one of claims 13-16, the medicine of claim 21 or the vaccine of claims 22 or 23 in the treatment and/or prevention of HPV-related diseases.
  27. 根据权利要求26所述的用途，其特征在于，所述HPV相关疾病包括选自下列中的至少之一：***、外阴癌、***癌、***癌、***癌、头颈癌、宫颈上皮内瘤变、外阴上皮内瘤变、***上皮内瘤变、***上皮内瘤变、***上皮内瘤变、口腔癌、喉癌、食道癌、鼻腔内癌和扁桃体癌。The use according to claim 26, wherein the HPV-related diseases include at least one selected from the following: cervical cancer, vulvar cancer, vaginal cancer, anal cancer, penile cancer, head and neck cancer, cervical intraepithelial neoplasia Vulvar intraepithelial neoplasia, vaginal intraepithelial neoplasia, anal intraepithelial neoplasia, penile intraepithelial neoplasia, oral cancer, laryngeal cancer, esophageal cancer, intranasal cancer and tonsil cancer.
  28. 一种治疗和/或预防HPV相关疾病的方法，其特征在于，包括向受试者施用以下中的至少之一：A method for treating and/or preventing HPV-related diseases, comprising administering to a subject at least one of the following:
    1)权利要求1-4任一项所述的分离的肽；1) The isolated peptide of any one of claims 1-4;
    2)权利要求5-8任一项所述的突变体；2) The mutant described in any one of claims 5-8;
    3)权利要求9所述的表达载体；3) The expression vector according to claim 9;
    4)权利要求10-12任一项所述的抗原呈递细胞；4) The antigen-presenting cell according to any one of claims 10-12;
    5)权利要求13-16任一项所述的免疫效应细胞；5) The immune effector cell according to any one of claims 13-16;
    6)权利要求21所述的药物；和6) The medicine of claim 21; and
    7)权利要求22或23所述的疫苗。7) The vaccine according to claim 22 or 23.
  29. 根据权利要求28所述的方法，其特征在于，所述HPV相关疾病包括选自下列中的至少之一：***、外阴癌、***癌、***癌、***癌、头颈癌、宫颈上皮内瘤变、外阴上皮内瘤变、***上皮内瘤变、***上皮内瘤变、***上皮内瘤变、口腔癌、喉癌、食道癌、鼻腔内癌和扁桃体癌。The method according to claim 28, wherein the HPV-related diseases include at least one selected from the following: cervical cancer, vulvar cancer, vaginal cancer, anal cancer, penile cancer, head and neck cancer, cervical intraepithelial neoplasia Vulvar intraepithelial neoplasia, vaginal intraepithelial neoplasia, anal intraepithelial neoplasia, penile intraepithelial neoplasia, oral cancer, laryngeal cancer, esophageal cancer, intranasal cancer and tonsil cancer.
  30. 一种诊断受试者是否患有HPV相关疾病的方法，其特征在于，包括检测受试者来源的生物样品是否携带权利要求1-4任一项所述分离的肽、权利要求5-8任一项所述的突变体、或编码权利要求1-4任一项所述分离的肽或权利要求5-8任一项所述的突变体的核酸分子的步骤。A method for diagnosing whether a subject suffers from HPV-related diseases, which includes detecting whether a biological sample derived from the subject carries the isolated peptide described in any one of claims 1-4, any of claims 5-8 The step of encoding the mutant described in one of the claims, or the nucleic acid molecule encoding the isolated peptide described in any one of claims 1-4, or the mutant described in any one of claims 5-8.
  31. 根据权利要求30所述的方法，其特征在于，所述HPV相关疾病包括选自下列中的至少之一：***、外阴癌、***癌、***癌、***癌、头颈癌、宫颈上皮内瘤变、外阴上皮内瘤变、***上皮内瘤变、***上皮内瘤变、***上皮内瘤变、口腔癌、喉癌、食道癌、鼻腔内癌或扁桃体癌。The method according to claim 30, wherein the HPV-related diseases include at least one selected from the following: cervical cancer, vulvar cancer, vaginal cancer, anal cancer, penile cancer, head and neck cancer, cervical intraepithelial neoplasia Vulvar intraepithelial neoplasia, vaginal intraepithelial neoplasia, anal intraepithelial neoplasia, penile intraepithelial neoplasia, oral cancer, laryngeal cancer, esophageal cancer, intranasal cancer or tonsil cancer.
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