Hepatitis B virus antigen peptide suitable for individuals with leukocyte antigen haplotype HLA-A2 and its application

Abstract

The present invention relates to the technical field of immunotherapy drugs, and in particular to antigenic peptides for hepatitis B virus and their applications. The purpose of the present invention is to provide a personalized treatment plan for hepatitis B virus that is suitable for individuals with human leukocyte antigen haplotype HLA-A2 haplotype in view of the deficiencies in the prior art. The antigenic polypeptide of the present invention comprises at least one of the polypeptides having an amino acid sequence as shown in SEQID No.1-32. The antigenic peptide of the present invention can significantly activate human T cells specific to HBV, increase the killing ability of T cells to HBV-infected liver cancer cells, and large-scale synthesis can be used for standardized and personalized HBV-related liver cancer immunotherapy, filling the gap in personalized antigenic peptides in the treatment of HBV-related diseases, especially liver cancer.

Description

Technical Field

[0001] The present invention relates to the technical field of immunotherapy drugs, and in particular to antigenic peptides targeting hepatitis B virus and applications thereof. Background Art

[0002] Hepatocellular carcinoma (HCC) ranks fifth among the most common cancers, with wide regional variation. Approximately 80% of liver cancer cases are linked to the hepatitis B virus (HBV). Approximately 15%-40% of untreated HCC patients will develop cirrhosis, liver failure, or liver cancer. Generally, many HCC patients present with advanced disease by the time symptoms appear, making resection impossible and resulting in a mid-term survival rate of less than six months. For a small number of patients who undergo liver cancer resection, the five-year survival rate is approximately 40%. Cancer immunotherapy is considered a new generation of cancer treatment, following surgery, radiotherapy, chemotherapy, and small molecule targeted therapy. It differs from traditional treatments in that it shifts its approach from directly targeting tumor cells with drugs to enhancing immune cells, targeting the patient's own anti-tumor immunity. Compared to traditional treatments, immunotherapy offers advantages such as precise targeting, minimal side effects, long-lasting efficacy, and a high degree of personalized treatment. Furthermore, the body's immune system possesses immune memory, so immunotherapy can help patients develop a memory immune response, offering significant advantages in preventing tumor recurrence and metastasis.

[0003] The HBV genome consists of 3200 bases and contains four open reading frames (ORFs), all located on the negative strand: the S region, the C region, the P region, and the X region. The S region is further divided into three coding regions: pre-S1, pre-S2, and S, encoding the pre-S1 and pre-S2 proteins on the envelope and HBsAg, respectively. The pre-S protein is highly immunogenic, and HBV's hepatotropism is primarily mediated by recognition between the pre-S protein and hepatocyte receptors. The C region is further divided into the pre-C gene and the C gene, encoding HBeAg and HBcAg. The protein encoded by the pre-C gene is processed and secreted extracellularly to become HBeAg; the protein encoded by the C gene is HBcAg. The P region is the longest open reading frame and encodes a large basic polypeptide with a molecular weight of approximately 90 kDa, containing multiple functional proteins. The X gene encodes the X protein, also known as HBxAg. HBxAg has a transactivating effect, activating various regulatory genes within HBV itself, other viruses, or cells, promoting HBV replication.

[0004] To identify personalized antigenic peptides targeting HBV, it is necessary to predict the antigenicity of HBV-expressed proteins and peptides, synthesize potential antigenic peptides in vitro, and finally activate immune cells using high-concentration and high-intensity tumor vaccines. Research on T cell antigen recognition has made significant progress. Studies have found that certain mutant proteins in tumors, after degradation, form peptides (also known as antigen epitopes) that can be specifically recognized by MHC molecules, forming MHC-antigen peptide complexes. These complexes are then presented on the surface of antigen-presenting cells and recognized as non-self by T cells, leading to their activation into effector T cells that attack and eliminate tumor cells. The most critical step in this process is the binding of MHC molecules to peptide molecules. MHC molecules in the human body, also known as human leukocyte antigens (HLA), are primarily divided into MHC class I and MHC class II molecules. The former are expressed by most cells and primarily participate in the presentation of endogenous antigens, while the latter are primarily expressed by antigen-presenting cells and primarily participate in the presentation of exogenous antigens. The human genome contains three genes encoding MHC I (HLA-A, HLA-B, and HLA-C) and three MHC II genes (HLA-DR, HLA-DQ, and HLA-DP). These genes vary greatly, and tens of thousands of different genotypes have been discovered, known as HLA haplotypes. Each haplotype is named according to international statistics, with the gene name separated from the number that follows by an asterisk (*), and alleles of different levels are separated by a comma (:). The more accurate classification is currently three-level, such as HLA-A*02:03:01, which represents the HLA-A gene's first classification of 02, the second classification of 03, and finally the third classification of 01. Different haplotypes generally have different affinities for the same polypeptide, that is, the ability to recognize it as an antigen. The stronger the affinity, the stronger the recognition ability. In addition, the same peptide has more similar affinities under the same haplotype. For example, the difference in affinity between the same peptide and different haplotypes under the HLA-A*02 (HLA-A2) classification is much smaller than the difference in affinity between the peptide and HLA-A11. To date, scientists in related fields have constructed multiple epitope databases and developed multiple epitope prediction software based on personalized HLA haplotypes (such as NetMHCpan4.0, NetMHCpan-BA, NetMHCpan-EL, MHCFlurry, ANN, SMM or CombLib). The more different software predicts an antigen peptide at the same time, the higher the accuracy of the antigen. Based on current research and analysis methods, it is possible to predict and design targeted antigen peptides for different personalized targets for personalized treatment.

[0005] HLA-A*02 is a specific class I major histocompatibility complex (MHC) allele at the HLA-A locus. Over 400 different HLA-A*02 alleles have been reported, making HLA-A2 the most polymorphic haplotype among the different HLA class I alleles. Among them, HLA-A*02:01 is the most frequent allele and has been widely used in previous studies of HLA-A2-restricted CTL response models. Aside from A*02:01, the frequency of HLA-A2 alleles in Asians is the highest among all MHC class I molecules. The affinity of the same peptide for different HLA-A2 haplotypes is highly similar. Therefore, designing antigenic peptides tailored to HLA-A2 haplotypes can maximize the number of patients who benefit.

[0006] Viral vaccines primarily include live attenuated vaccines, inactivated vaccines, subunit vaccines (recombinant protein or peptide vaccines), nucleic acid vaccines, and viral vectors. Vaccines have successfully prevented numerous diseases worldwide: smallpox has been eradicated, and the incidence of hepatitis B, polio, measles, and other childhood illnesses has been significantly reduced worldwide. However, these vaccines have various development and application shortcomings. For example, the development cycle for live attenuated or inactivated vaccines is relatively long. Because they contain pathogens, they carry the risk of host infection. To ensure safety, routine testing during the R&D process is complex and time-consuming. Summary of the Invention

[0007] The purpose of the present invention is to provide a personalized treatment plan for hepatitis B virus in response to the deficiencies in the prior art.

[0008] In order to solve the technical problem of the present invention, the first aspect of the present invention is an antigenic polypeptide targeting hepatitis B virus.

[0009] The antigen polypeptide provided by the present invention is suitable for antigen polypeptides of individuals with human leukocyte antigen haplotype HLA-A2 haplotype, including at least one of the polypeptides with amino acid sequences as shown in SEQ ID No. 1-32;

[0010] Alternatively, a polypeptide with the same or similar function obtained by replacing and / or deleting and / or adding at least one amino acid in the amino acid sequence of each of the above polypeptides.

[0011] The present invention further provides an antigen polypeptide combination comprising the antigen polypeptides shown in SEQ ID No. 1-32.

[0012] The present invention also provides a fusion polypeptide comprising the above antigen polypeptide.

[0013] The fusion polypeptides described above are fused with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 of the aforementioned antigenic polypeptides. Furthermore, the fusion polypeptides described above are a combination of fusion polypeptides, each of which contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 of the aforementioned antigenic polypeptides. Furthermore, all fusion polypeptides in the combination together contain at least all of the polypeptides set forth in SEQ ID NOs: 1-32. Furthermore, the antigenic polypeptides described above may be connected by linkers. These linkers can be selected from various connecting peptides commonly used in the art. Obviously, if a large number of antigenic polypeptides are required, multiple fusion polypeptides can be designed and used separately or together.

[0014] The present invention also provides antibodies against the above polypeptides. Further, the above antibodies are polyclonal antibodies or monoclonal antibodies.

[0015] Furthermore, the above-mentioned antibody can specifically bind to the above-mentioned polypeptide. Furthermore, the above-mentioned antibody also forms a conjugate with a coupling moiety; the coupling moiety is one or more selected from radionuclides, drugs, toxins, cytokines, enzymes, fluorescein, carrier proteins or biotin.

[0016] On this basis, the present invention provides the use of the above-mentioned antigenic polypeptide, fusion polypeptide, derivative of the above-mentioned antigenic polypeptide or fusion polypeptide, chemically modified product of the above-mentioned antigenic polypeptide or fusion polypeptide, or the above-mentioned antibody in any of the following:

[0017] a. Preparation of anti-HBV products, or anti-HBV products;

[0018] b. Preparation of products for treating and / or preventing diseases caused by hepatitis B virus infection; or treatment or prevention of diseases caused by hepatitis B virus infection;

[0019] c. Preparation of products that can improve symptoms caused by hepatitis B virus infection; or improvement of symptoms caused by hepatitis B virus infection;

[0020] In the above application, the disease caused by hepatitis B virus infection is at least one of hepatitis B, severe hepatitis, cirrhosis, ascites, or liver cancer. Furthermore, the disease caused by hepatitis B virus infection is especially hepatitis B virus-positive liver cancer.

[0021] The present invention also provides a method for preparing personalized hepatitis B virus antigen peptides. The method comprises the following steps:

[0022] a. Detect the human leukocyte antigen haplotype of the individual being tested; confirm that the individual has the HLA-A2 typing of the following human leukocyte antigen haplotypes associated with hepatitis B virus;

[0023] b. After confirming, based on the result of step a, that the individual has a hepatitis B virus-related human leukocyte antigen haplotype of HLA-A2, using the above-mentioned antigen polypeptide or the above-mentioned fusion polypeptide as a candidate polypeptide;

[0024] c. Prepare the candidate polypeptide.

[0025] Wherein, the detection of the human leukocyte antigen haplotype of the individual in step a of the above method is performed by detecting the individual's blood sample in vitro.

[0026] Furthermore, in step c of the above method, when selecting polypeptides, all polypeptides corresponding to HLA-A2 are selected from the candidate polypeptides confirmed in step b according to the type of hepatitis B virus-related human leukocyte antigen haplotype possessed by the individual.

[0027] The individual described in the above method is an HBV-positive individual. Further, the individual is an HBV-positive liver cancer patient.

[0028] On this basis, the present invention also provides personalized hepatitis B virus antigen peptides prepared by the above method.

[0029] The present invention also provides a protein or polypeptide vaccine for preventing and / or treating diseases caused by hepatitis B virus infection. The vaccine contains the aforementioned various antigenic polypeptides, fusion polypeptides, or personalized hepatitis B virus antigen peptides as the main antigenic component, as well as pharmaceutically acceptable excipients or auxiliary ingredients.

[0030] Furthermore, the above-mentioned vaccine also contains an immune adjuvant, wherein the immune adjuvant is Freund's incomplete adjuvant, complete Freund's adjuvant, aluminum hydroxide adjuvant, aluminum phosphate adjuvant, emulsified adjuvant, liposome adjuvant, microbial adjuvant, etc.

[0031] The above-mentioned disease caused by hepatitis B virus infection is at least one of hepatitis B, severe hepatitis, cirrhosis, ascites or liver cancer.

[0032] Furthermore, the above-mentioned vaccine for preventing and / or treating HBV-positive related diseases also contains other components that have the activity of preventing and / or treating HBV-positive related diseases.

[0033] Furthermore, the above-mentioned medicine also contains pharmaceutically acceptable excipients or auxiliary ingredients.

[0034] Among them, the pharmaceutically acceptable excipients or auxiliary ingredients are at least one of cell culture medium, solvent, solubilizer, cosolvent, emulsifier, colorant, adhesive, osmotic pressure regulator, stabilizer, flow aid, preservative, suspending agent, penetration enhancer, pH regulator, buffer, surfactant, absorbent, diluent or flocculant and deflocculant.

[0035] The present invention also provides a method for preventing and / or treating diseases caused by hepatitis B virus infection, comprising the step of administering an effective amount of the above-mentioned antigen polypeptide, fusion polypeptide, hepatitis B virus antigen peptide or vaccine to a hepatitis B virus-positive individual.

[0036] Furthermore, the administration method is injection. Furthermore, the injection is at least one of intravenous injection, intramuscular injection, intraperitoneal injection or intratumoral injection.

[0037] In the above method of the present invention, when administering, what is administered is an antigen polypeptide combination containing the antigen polypeptides shown in SEQ ID No. 1-32; or what is administered is a fusion polypeptide or fusion polypeptide combination containing the antigen polypeptides shown in SEQ ID No. 1-32.

[0038] The present invention also encompasses genes encoding the aforementioned antigenic polypeptides, fusion polypeptides, or antibodies. It also provides vectors containing these encoding genes. Furthermore, the vectors are expression vectors. These expression vectors can be selected from commonly used vectors such as plasmid vectors, adenoviral vectors, lentiviral vectors, or adeno-associated viral vectors. When using an adenoviral vector, a replication-defective adenoviral vector is generally employed.

[0039] The present invention provides a set of antigenic peptides whose personalized effectiveness has been verified using immunoassays in clinical trials, thus filling the gap in personalized antigenic peptides for HLA-A2 haplotypes in the treatment of HBV-related diseases, particularly liver cancer. Since HLA-A2 haplotypes are highly prevalent in Asian populations, with over 40% of people carrying at least one HLA-A2 haplotype, antigenic peptides targeting HLA-A2 haplotypes as HLA-restricted epitope vaccines have the potential to maximize the patient population benefiting and thus achieve wider application. Furthermore, centralized pre-production of antigenic peptides can significantly reduce the time and labor costs of new antigenic immunotherapies and improve treatment efficiency. The high polymorphism of HLA-A2 haplotypes means that the antigenic peptide ligands that bind to them may vary significantly in both physicochemical properties and spatial conformation. Therefore, the probability of antigenic peptides presented by HLA-A2 molecules being recognized by the surface-specific receptors (TCRs) of CD8+ T cells in patients is greatly increased, thereby activating an adaptive anti-tumor immune response. The antigen peptide combination of the present invention can significantly activate the human body's T cells specific to HBV and increase the killing ability of T cells against HBV-infected liver cancer cells. Large-scale synthesis can be used in standardized and individualized HBV-related liver cancer immunotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 Dendritic cells were loaded with antigen peptides to activate T cells. The in vitro reactivity of T cells from liver cancer patient 1 (haplotype HLA-A*02:01) to mixed antigen peptides was analyzed by IFN-γ ELISPOT assay. Positive control: phytohemagglutinin (PHA); negative control: no peptide added.

[0041] Figure 2 Dendritic cells were loaded with antigen peptides. After stimulating activated T cells, the in vitro reactivity of T cells from HCC patient 2 (haplotype HLA-A*02:01 and HLA-A*02:03) to mixed antigen peptides was analyzed by IFN-γ ELISPOT assay. Positive control: phytohemagglutinin (PHA); negative control: no peptide added.

[0042] Figure 3 The in vitro reactivity of T cells from HCC patient 3 (haplotype HLA-A*02:01) to the mixed antigen peptides was analyzed by IFN-γ ELISPOT assay after stimulation of activated T cells using a dendritic cell antigen mixed peptide panel. Positive control: phytohemagglutinin (PHA); negative control: no peptide added.

[0043] Figure 4Dendritic cells were loaded with antigen peptides to activate T cells. The in vitro reactivity of T cells from HCC patient 4 (haplotype HLA-A*02:06) to mixed antigen peptides was analyzed by IFN-γ ELISPOT assay. Positive control: phytohemagglutinin (PHA); negative control: no peptide added. DETAILED DESCRIPTION

[0044] The present invention is based on a clinical prediction method for personalized tumor neoantigen sequencing. The method mainly includes the following steps

[0045] Step 1: Compare the sequencing data of the patient's tumor cells with the preset corresponding reference genome to obtain DNA comparison results and RNA comparison results;

[0046] Step 2: pre-processing the DNA comparison results and the RNA comparison results;

[0047] Step 3: Analyze somatic cell variation, clone type, tumor purity, gene expression in tumor cells, and somatic cell variation allele expression abundance in tumor cells using the DNA comparison results and RNA comparison results to obtain analysis results;

[0048] Step 4: Predicting the MHC class I genotype of the patient's tumor cells, and analyzing the affinity of potential neoantigens and calculating the peptide presentation efficacy based on the DNA analysis results and the genotype of the MHC class I molecules of the patient's tumor cells;

[0049] Step 5: Scoring and ranking the corresponding neoantigens based on the analysis results, affinity of potential neoantigens, and peptide presentation efficacy, and then presenting them;

[0050] Step 6: Based on the experimental results, use machine learning methods to optimize the affinity analysis method of potential neoantigens in step 5 to improve the accuracy of prediction.

[0051] For specific steps, please refer to Chinese patent document CN111415707A, a clinical method for predicting individualized tumor neoantigens.

[0052] Using this method, the inventors sequenced and HLA-typed samples from multiple HBV-positive liver cancer patients. Combining different human leukocyte antigen (HLA) haplotypes and using the aforementioned bioinformatics prediction method, they predicted and initially validated antigenic peptides targeting proteins encoded by the hepatitis B virus. Ultimately, they generated a set of high-affinity antigenic peptides targeting the HLA-A2 haplotype, a common type found in HBV-positive liver cancer patients.

[0053] The sequences of the group of antigenic peptides, applicable haplotypes, and targets of HBV-encoded proteins are shown in Table 1.

[0054] Table 1

[0055]

[0056]

[0057] In the present invention, the expression "proteins having the same or similar functions as the above-mentioned proteins, which are obtained by replacing and / or deleting and / or adding at least one amino acid in the amino acid sequence of each polypeptide" includes but is not limited to the deletion, insertion and / or substitution of several (usually 1-10, preferably 1-5, more preferably 1-3) amino acids, and the addition of one or several (usually within 20, preferably within 10, more preferably within 5, and most preferably within 3) amino acids at the C-terminus and / or N-terminus. For example, in the protein, when amino acids with similar or similar properties are substituted, the function of the protein is generally not changed. For another example, adding one or several amino acids to the C-terminus and / or N-terminus generally does not change the function of the protein. The term also includes active fragments and active derivatives of the protein.

[0058] The expression "peptide segments obtained by substitution and / or deletion and / or addition of at least one amino acid in the amino acid sequence of each polypeptide" also includes, but is not limited to, polypeptides formed by substitution of up to 10 (i.e., one or more), preferably up to 8, and more preferably up to 5 (5, 4, 3, 2, or 1) amino acids with amino acids having similar or similar properties, i.e., conservative variant polypeptides. Furthermore, these conservative variant polypeptides can be generated by substitution according to Table 3.

[0059] Table 2 Amino acid substitution table

[0060] Initial residue Representative replacement Preferred substitutions Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe Leu Leu(L) Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; Ala; Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile;Leu;Met;Phe;Ala Leu

[0061] The aforementioned polypeptides can be used as active ingredients in the preparation of drugs for preventing and / or treating hepatitis B virus infection. Generally speaking, those skilled in the art can use the aforementioned polypeptides as antigenic active ingredients to design and prepare protein or polypeptide vaccines for preventing and / or treating diseases caused by hepatitis B virus infection. Such vaccines comprise the aforementioned polypeptides as antigenic components, along with pharmaceutically acceptable excipients or auxiliary ingredients.

[0062] When preparing vaccines, immune adjuvants are often added to enhance the body's immune response to the vaccine. Examples of immune adjuvants include incomplete Freund's adjuvant, complete Freund's adjuvant, aluminum hydroxide adjuvant, aluminum phosphate adjuvant, emulsified adjuvant, liposome adjuvant, and microbial adjuvant.

[0063] Naturally, based on the proteins described in the present invention, it is easy to obtain antibodies against the above proteins in the art. The above antibodies are polyclonal antibodies or monoclonal antibodies; preferably monoclonal antibodies. The above antibodies also form conjugates with coupling moieties. Furthermore, the coupling moieties are one or more selected from radionuclides, drugs, toxins, cytokines, enzymes, fluorescein, carrier proteins or biotin. Antibodies that can specifically bind to the aforementioned proteins can be used, on the one hand, to prepare drugs for preventing and / or treating hepatitis B virus infection to resist hepatitis B virus infection, and on the other hand, can be used for hepatitis B virus-related immune detection.

[0064] Furthermore, the present invention also encompasses genes encoding the aforementioned proteins. These genes can be used to express and produce the aforementioned proteins or antibodies; they can also be operably incorporated into expression vectors for formulation into vector vaccines or pharmaceuticals. Expression can be achieved using commonly available vectors, including plasmids, adenoviral vectors, lentiviral vectors, and adeno-associated viral vectors. When using an adenoviral vector, a replication-defective adenoviral vector is generally employed.

[0065] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts shall fall within the scope of protection of the present invention.

[0066] In the present invention, unless otherwise specified, the amino acid sequence from left to right is from nitrogen end to carbon end.

[0067] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features therein may be combined with each other.

[0068] The present invention will be further described below with reference to the accompanying drawings and specific examples, but they are not intended to limit the present invention.

[0069] 1. Patient HLA typing test

[0070] 2 mL of peripheral blood was drawn from each of the four patients, anticoagulated with EDTA or sodium citrate, and the four-digit high-resolution typing of each HLA locus was detected using PCR-SBT technology.

[0071] 2.4 Collection of Peripheral Blood PBMCs from Patients

[0072] (1) COBE Spectra will be used TMAfter disinfection, place the cell bag obtained by the MNC system (or heparin anticoagulated blood drawn with a syringe) in a clean bench. Use a 20mL syringe to transfer the cells to a 50mL centrifuge tube and dilute them 1:1 with normal saline.

[0073] (2) Add 20 mL of human lymphocyte separation medium into the centrifuge tube;

[0074] (3) Using a Pasteur pipette, slowly add the cell solution diluted with physiological saline to the human lymphocyte separation solution along the tube wall;

[0075] (4) Horizontal centrifugation at 800 g for 20 minutes;

[0076] (5) After centrifugation, the tube is divided into three layers: the upper layer is plasma, the lower layer is mainly red blood cells and granulocytes, and the middle layer is lymphocyte separation fluid. At the interface between the upper and middle layers, there is a white film layer mainly composed of monocytes, which is the peripheral blood mononuclear cell layer;

[0077] (6) Insert a pipette into the buffy coat layer, aspirate the mononuclear cells, place them into another 50 mL centrifuge tube, add physiological saline, and centrifuge horizontally at 300 g for 10 min.

[0078] (7) Discard the supernatant, add physiological saline, mix well, and centrifuge horizontally at 300g for 10 min;

[0079] (8) Discard the supernatant, add cell culture medium, mix thoroughly, and centrifuge horizontally at 300 g for 5 min;

[0080] (9) Discard the supernatant, dilute with AIM-V culture medium, count the cells, and take an appropriate amount of cells to freeze as a specimen.

[0081] 3. Dendritic cell (DC) culture:

[0082] (1) According to the above-mentioned PBMC acquisition method, PBMCs obtained from four patients were placed in multiple six-well culture flasks and cultured in AIM-V medium;

[0083] (2) Incubate in a 37°C, 5% CO2 incubator for 2 h to allow the monocytes to adhere;

[0084] (3) Isolate adherent cells, add 20 mL of AIM-V medium, supplemented with recombinant human GM-CSF (800 U / mL) and recombinant human IL-4 (1000 U / mL), and culture in a 37°C, 5% CO2 incubator to induce monocyte differentiation into DCs;

[0085] (4) On the third day, the medium should be changed halfway depending on the cell count. If the medium is changed, GM-CSF and IL-4 should be supplemented.

[0086] (5) On the sixth day, DC maturation-promoting factors LPS (10 ng / mL) and IFN-γ (100 IU / mL) were added to induce DC maturation for 16-48 h;

[0087] (6) On the seventh day, mature dendritic cells were obtained.

[0088] (7) The mature DCs harvested above were cultured in 32 groups for each patient. 10 μg / mL of an antigen peptide was added to each DC culture medium and incubated in a 37°C, 5% CO2 incubator for 4-6 hours. The cells were centrifuged and resuspended in preheated PBS for later use.

[0089] The mixed antigen peptide used in the present embodiment is composed of 32 polypeptides shown in SEQ ID No. 1 to 32. In this embodiment, these polypeptides are synthesized separately, and each antigen peptide is stored separately on a daily basis to form an antigen peptide combination.

[0090] 4. Enzyme-linked immunosorbent assay (ELISPOT)

[0091] (1) Activate the pre-coated plate by adding 200 μL of AIM-V serum-free medium, letting it stand at room temperature for 10 minutes, and then pouring it;

[0092] (2) According to the designed grouping, T cells and antigen peptides isolated from PBMC were added at 10-50 μg / mL, with three replicates in each group;

[0093] (3) After adding all samples, cover the plate, mark it, and place it in a 37°C, 5% CO2 incubator for 20 hours.

[0094] (4) Pour the cells and culture medium from the well;

[0095] (5) Cell lysis: Add 200 μL of ice-cold deionized water to each well and place in a 4°C refrigerator for 10 min (hypotonic cell lysis);

[0096] (6) Washing: Wash each well six times with 260 μL 1× Washing buffer for 60 seconds each time. Dry on absorbent paper after each wash.

[0097] (7) Detection antibody incubation: Add 100 μL of diluted biotin-labeled antibody to each well and incubate at 37°C in a 5% CO2 incubator for 1 hour;

[0098] (8) Washing: Wash each well six times with 260 μL 1× Washing buffer for 60 seconds each time. Dry on absorbent paper after each wash.

[0099] (9) Avidin incubation: Add 100 μL of diluted enzyme-labeled avidin to each well and incubate at 37°C for 1 hour;

[0100] (10) Washing: Wash each well six times with 260 μL 1× Washing buffer for 60 seconds each time. Dry on absorbent paper after each wash.

[0101] (11) Color development: Prepare AEC color development solution according to the reagent preparation instructions, add 100 μL of color development solution to each well, and incubate at 37°C, 5% CO2 incubator for color development, checking every 5 minutes;

[0102] (12) After the spots have grown to a suitable size, wash them twice with deionized water to terminate the color development process. Place the plate upside down on absorbent paper and pat dry the small water droplets. Then remove the protective layer and place it in a ventilated place at room temperature to allow the membrane to dry naturally.

[0103] (13) Count the spots on the ELISPOT plate and record various parameters of the spots for analysis.

[0104] 5. Flow cytometry to measure markers of T cell activation

[0105] (1) Adjust the concentration of activated PBMCs or T lymphocytes to 1×10 6 After the cell suspension is evenly dispersed, centrifuge at 1500 rpm for 5 minutes, discard the supernatant, add PBS, centrifuge at 1500 rpm for 5 minutes, discard the supernatant, and resuspend the cells in 100 μL PBS and transfer them to the flow cytometry tube. Label the tube wall with cells of each group.

[0106] (2) Add fluorescent antibodies such as anti-human CD8 and CD137 to label cells, and set up negative control group, single-label group, and isotype control group;

[0107] (3) Stain at 4°C in the dark for 30 min;

[0108] (4) Add 1 mL of PBS and mix gently by pipetting;

[0109] (5) Centrifugation, 1500 rpm, 5 min, discard the supernatant;

[0110] (6) Resuspend the cells in 100 μL of PBS and analyze the results using flow cytometry. BD software was used to collect data and FlowJo software was used to analyze the results.

[0111] 6. In vitro PBMC activation

[0112] (1) Heparinized anticoagulated blood was used to separate PBMCs by the above method and suspended in AIM-V medium (Gibico, USA) and counted.

[0113] (2) Use a 96-well plate with a U-shaped bottom and add 1×10 5Each PBMC was diluted in 200 μL of culture medium and incubated with 10 μM of one of the antigenic peptides from the combination. The U-shaped bottom of the culture medium was used to promote better cell-cell contact. The culture medium consisted of AIM-V medium (Gibco) supplemented with a small amount of interleukin-2 (IL-2, 100 U / mL, Peprotech) and 10% fetal calf serum (FCS, Gibco).

[0114] (3) PBMCs were cultured for 3 days per stimulation cycle, with half of the culture medium replaced once, and the same antigen peptide (10 μM) and IL-2 (100 U / mL) were supplemented.

[0115] (4) After 2-3 cycles of peptide stimulation in vitro, fresh culture medium was replaced and the same antigenic peptide was added again at 10 μM for overnight stimulation. Within 24 hours of the last round of stimulation (i.e., on the 7th and / or 10th day of culture), the T cell immune response level was assessed by ELISPOT assay for IFN-γ expression to identify the immunogenicity of different antigenic peptides. PBMCs stimulated with no peptide (culture medium) or irrelevant peptides served as negative controls (NS), and stimulation with plant lectin (PHA) served as a positive control.

[0116] 7. Use dendritic cells (DC) loaded with antigen peptides to stimulate and activate T cells

[0117] Mature DC (1×10 4 The cells were incubated with 10 μM of the antigen peptide combination (cells / 100 μL / well) in a 37 ° C, 5% CO2 incubator for 4-6 hours, washed with preheated PBS, and then incubated with T cells overnight at a stimulator to effector ratio of 1:10 in complete AIM-V medium. The activation level of T cells was evaluated the next day. The number and intensity of activated T cells after stimulation culture were detected by INF-γ ELISPOT, and the expression level of T cell activation marker 4-1BB (CD137) was evaluated by flow cytometry.

[0118] 8. Preparation of Neoantigen Peptide-Reactive T Cells (NRT)

[0119] (1) Transfer the suspended cells to the F225 culture flask and adjust the cell concentration to 1×10 7 / mL, supplemented with IL-2 (100 U / mL), IL-7 (10 ng / mL), and IL-15 (10 ng / mL), and cultured in a 37°C, 5% CO2 incubator for 7 days;

[0120] (2) Mature DCs were harvested on day 7. 32 groups of mature DCs were cultured for each patient. 25 μg / mL of an antigen peptide was added to the DC culture medium and incubated in a 37°C, 5% CO2 incubator for 4–6 h.

[0121] (3) Centrifuge at 1500 rpm for 5 min, discard the supernatant, mix the DC loaded with antigen peptides with the suspension cells, and add some fresh culture medium containing (IL-2, IL-7, and IL-15). Incubate at 37°C, 5% CO2 for 10 days.

[0122] (4) Depending on the cell growth and culture medium color, fresh complete culture medium and corresponding cytokines IL-7, IL-15, and IL-2 were added at half volume every 2 to 3 days and the culture was passaged. Depending on the T cell expansion, OKT3 monoclonal antibody was added in appropriate amounts, and irradiated ECCE cells or APC-loaded peptide prepared from revived frozen PBMC were added for a second round of stimulation.

[0123] (5) About 10 days after T cells are stimulated with antigenic peptides, the T cells of each patient are harvested and mixed for testing and functional experiments.

[0124] Experimental results:

[0125] The results of HLA typing and T cell activation experiments on the four patients using mixed antigen peptides are shown in Figure 2. Figures 1 to 4 As shown, the results showed that all four patients carried one or two HLA-A2 haplotypes. The mixed antigenic peptides of the present invention (a mixture of polypeptides shown in SEQ ID No. 1 to 32) activated the T cells of these patients against these antigenic peptides, that is, generated a strong immune response, which has the potential to kill HBV-positive liver cancer cells. In summary, the antigenic peptides of the present invention have been verified in clinical trials using immunoassays to be effective for individuals with HLA-A2 haplotypes, thus filling the gap in personalized antigenic peptides in the treatment of HBV-positive liver cancer. The antigenic peptides of the present invention can significantly activate the human body's T cells specific for HBV and increase the killing ability of T cells against HBV-infected liver cancer cells. Large-scale synthesis can be used in standardized personalized liver cancer immunotherapy.

[0126] The above description is only a preferred embodiment of the present invention and does not limit the implementation mode and protection scope of the present invention. For those skilled in the art, it should be aware that all solutions obtained by equivalent substitutions and obvious changes made using the description and illustrations of the present invention should be included in the protection scope of the present invention. Sequence Listing <110> Chengdu Langgu Biotechnology Co., Ltd. <120> Hepatitis B virus antigen peptide suitable for individuals with leukocyte antigen haplotype HLA-A2 and its application <160> 32 <170> SIPOSequenceListing 1.0 <210> 1 <211> 9 <212> PRT <213> Artificial Sequence <400> 1 Phe Leu Pro Asp Phe Phe Pro Ser Ile 1 5 <210> 2 <211> 9 <212> PRT <213> Artificial Sequence <400> 2 Tyr Met Asp Asp Val Val Leu Gly Ala 1 5 <210> 3 <211> 9 <212> PRT <213> Artificial Sequence <400> 3 Phe Leu Leu Thr Lys Ile Leu Thr Ile 1 5 <210> 4 <211> 9 <212> PRT <213> Artificial Sequence <400> 4 His Leu Tyr Ser His Pro Ile Ile Leu 1 5 <210> 5 <211> 9 <212> PRT <213> Artificial Sequence <400> 5 Ile Leu Ser Pro Phe Met Pro Leu Leu 1 5 <210> 6 <211> 9 <212> PRT <213> Artificial Sequence <400> 6 Ser Leu Tyr Ala Asp Ser Pro Ser Val 1 5 <210> 7 <211> 9 <212> PRT <213> Artificial Sequence <400> 7 Phe Leu Ser Lys Gln Tyr Leu Asn Leu 1 5 <210> 8 <211> 9 <212> PRT <213> Artificial Sequence <400> 8 Gly Leu Leu Gly Pro Leu Leu Val Leu 1 5 <210> 9 <211> 9 <212> PRT <213> Artificial Sequence <400> 9 Phe Leu Leu Ser Leu Gly Ile His Leu 1 5 <210> 10 <211> 9 <212> PRT <213> Artificial Sequence <400> 10 Lys Leu Ile Gly Thr Asp Asn Ser Val 1 5 <210> 11 <211> 9 <212> PRT <213> Artificial Sequence <400> 11 His Leu Pro Asp Arg Val His Phe Ala 1 5 <210> 12 <211> 9 <212> PRT <213> Artificial Sequence <400> 12 Phe Leu Leu Ala Gln Phe Thr Ser Ala 1 5 <210> 13 <211> 9 <212> PRT <213> Artificial Sequence <400> 13 Arg Val Thr Gly Gly Val Phe Leu Val 1 5 <210> 14 <211> 9 <212> PRT <213> Artificial Sequence <400> 14 Ala Leu Pro Pro Ala Ser Pro Pro Val 1 5 <210> 15 <211> 9 <212> PRT <213> Artificial Sequence <400> 15 Gly Leu Ser Arg Tyr Val Ala Arg Leu 1 5 <210> 16 <211> 9 <212> PRT <213> Artificial Sequence <400> 16 Leu Leu Ala Gln Phe Thr Ser Ala Ile 1 5 <210> 17 <211> 9 <212> PRT <213> Artificial Sequence <400> 17 Leu Leu Ser Ser Asn Leu Ser Trp Leu 1 5 <210> 18 <211> 9 <212> PRT <213> Artificial Sequence <400> 18 Val Leu Gln Ala Gly Phe Phe Leu Leu 1 5 <210> 19 <211> 10 <212> PRT <213> Artificial Sequence <400> 19 Gly Leu Ser Pro Thr Val Trp Leu Ser Val 1 5 10 <210> 20 <211> 9 <212> PRT <213> Artificial Sequence <400> 20 Val Leu His Lys Arg Thr Leu Gly Leu 1 5 <210> twenty one <211> 9 <212> PRT <213> Artificial Sequence <400> twenty one Val Ser Ile Pro Trp Thr His Lys Val 1 5 <210> twenty two <211> 9 <212> PRT <213> Artificial Sequence <400> twenty two Phe Ala Val Pro Asn Leu Gln Ser Leu 1 5 <210> twenty three <211> 9 <212> PRT <213> Artificial Sequence <400> twenty three Phe Thr Phe Ser Pro Thr Tyr Lys Ala 1 5 <210> twenty four <211> 9 <212> PRT <213> Artificial Sequence <400> twenty four Ala Val Thr Asn Phe Leu Leu Ser Leu 1 5 <210> 25 <211> 10 <212> PRT <213> Artificial Sequence <400> 25 Leu Leu Ala Pro Phe Val Gln Trp Phe Val 1 5 10 <210> 26 <211> 9 <212> PRT <213> Artificial Sequence <400> 26 Lys Ile Leu Thr Ile Pro Gln Ser Leu 1 5 <210> 27 <211> 9 <212> PRT <213> Artificial Sequence <400> 27 Met Met Trp Tyr Trp Gly Pro Ser Leu 1 5 <210> 28 <211> 9 <212> PRT <213> Artificial Sequence <400> 28 Tyr Leu Val Ser Phe Gly Val Trp Ile 1 5 <210> 29 <211> 9 <212> PRT <213> Artificial Sequence <400> 29 Trp Leu Ser Leu Leu Ala Pro Phe Val 1 5 <210> 30 <211> 9 <212> PRT <213> Artificial Sequence <400> 30 Leu Leu Trp Phe His Ile Ser Cys Leu 1 5 <210> 31 <211> 9 <212> PRT <213> Artificial Sequence <400> 31 Lys Gln Tyr Leu Asn Leu Tyr Pro Val 1 5 <210> 32 <211> 9 <212> PRT <213> Artificial Sequence <400> 32 Ser Leu Asp Ser Trp Trp Thr Ser Leu 1 5

Claims

1. An antigenic polypeptide against hepatitis B virus, characterized in that: It is a polypeptide combination with amino acid sequences as shown in SEQ ID NOs: 1 to 32.

2. Use of the antigen polypeptide according to claim 1 in the preparation of a product for treating and / or preventing liver cancer caused by hepatitis B virus infection; the liver cancer patient contains the human leukocyte antigen HLA-A2 haplotype.

3. A protein or polypeptide vaccine for preventing and / or treating diseases caused by hepatitis B virus infection, characterized in that: The method comprises the antigen polypeptide according to claim 1 as an antigen component, and pharmaceutically acceptable adjuvants or auxiliary components; the disease caused by hepatitis B virus infection is liver cancer; and the patient with liver cancer contains the human leukocyte antigen HLA-A2 haplotype.

4. The vaccine according to claim 3, characterized in that: It also contains an immune adjuvant.

5. The vaccine according to claim 4, characterized in that: The immune adjuvant is at least one of Freund's incomplete adjuvant, complete Freund's adjuvant, aluminum hydroxide adjuvant, aluminum phosphate adjuvant, emulsion adjuvant, liposome adjuvant or microbial adjuvant.

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