CN114591404A - Hepatitis B virus antigen peptide suitable for leukocyte antigen haplotype as HLA-A2 individual and application thereof - Google Patents

Hepatitis B virus antigen peptide suitable for leukocyte antigen haplotype as HLA-A2 individual and application thereof Download PDF

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CN114591404A
CN114591404A CN202210289717.7A CN202210289717A CN114591404A CN 114591404 A CN114591404 A CN 114591404A CN 202210289717 A CN202210289717 A CN 202210289717A CN 114591404 A CN114591404 A CN 114591404A
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polypeptide
hepatitis
antigen
virus
hla
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许恒
舒洋
蒿青
杨莉
丁振宇
魏于全
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Chengdu Langgu Biotechnology Co ltd
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Abstract

The invention relates to the technical field of immunotherapy drugs, in particular to an antigenic peptide aiming at hepatitis B virus and application thereof. The invention aims to overcome the defects in the prior art and provide a personalized treatment scheme for individual with human leukocyte antigen haplotype as HLA-A2 haplotype aiming at hepatitis B virus. The antigen polypeptide comprises at least one polypeptide with an amino acid sequence shown as SEQ ID No. 1-32. The antigen peptide can obviously activate the human body specificity to the T cell of HBV, increase the killing capacity of the T cell to the liver cancer cell infected by HBV, can be synthesized in a large scale and can be used in standardized individualized HBV related liver cancer immunotherapy, and fills the blank of individualized antigen peptide in HBV related diseases, particularly liver cancer therapy.

Description

Hepatitis B virus antigen peptide suitable for leukocyte antigen haplotype as HLA-A2 individual and application thereof
Technical Field
The invention relates to the technical field of immunotherapy drugs, in particular to an antigenic peptide aiming at hepatitis B virus and application thereof.
Background
Hepatocellular carcinoma (HCC) is the fifth most common tumor with quite wide regional differences, the annual incidence rate around 100 ten thousand worldwide and mainly concentrated in China. About 80% of liver cancers are associated with Hepatitis B Virus (HBV). About 15% -40% of untreated infected persons develop cirrhosis, liver failure or liver cancer. In general, liver cancer is less than twenty one hundred thousand in Chinese each year, but unfortunately many patients are already advanced in symptoms and lose the timing of resection, resulting in a mid-term survival rate of less than half a year. A small number of patients have a 5-year survival rate of about 40% after resection of cancerous tissue from the liver. Immunotherapy of tumors is considered as a new generation of tumor treatment following surgery, radiotherapy, chemotherapy and small molecule targeted therapy, which differs from the previous traditional treatment methods in that: immunotherapy transfers the treatment means from direct killing of tumor cells by drugs to enhancement of immune cells, treats tumors by improving the anti-tumor immunity of patients, and has the advantages of accurate killing, small side effect, lasting curative effect, high degree of individuation and the like compared with the traditional treatment. In addition, the immune system of the body has the characteristic of immunological memory, so that immunotherapy can help patients to form memory type immunity, and has remarkable advantages in preventing tumor recurrence and metastasis.
The HBV genome contains 3200 bases and comprises 4 Open Reading Frames (ORF) which are all positioned on a negative strand and are respectively an S region, a C region, a P region and an X region. The S region is divided into three coding regions of former S1, former S2 and S, which respectively code former S1 protein, former S2 protein and HBsAg on the envelope. The pre-S protein is highly immunogenic and the hepatotropic properties of HBV are mainly recognized and mediated by the pre-S protein and hepatocyte receptors. The C region is divided into pre-C gene and C gene, coding HBeAg and HBcAg. The protein coded by the pre-C gene is processed and then secreted out of the cell to obtain HBeAg; the protein encoded from the C gene was HBcAg. The P region is the longest reading frame, encodes a macromolecular basic polypeptide with a molecular weight of about 90KD, and contains multiple functional proteins. The X gene encodes the X protein, HBxAg. HBxAg has transactivation, and can activate various regulatory genes of HBV itself, other viruses or cells, and promote HBV replication.
In order to search for individual antigenic peptides against HBV, it is necessary to perform antigenic prediction on proteins and polypeptides expressed by HBV, synthesize potential antigenic peptides in vitro, and finally activate immune cells using tumor vaccines with high concentration and high strength, and research on T cell antigen recognition has been advanced greatly. It has been found that polypeptides (also called epitopes) formed by degradation of certain variant proteins in tumors can be specifically recognized by MHC molecules and form MHC-antigenic peptide complexes, and then the complexes are presented on the surface of antigen presenting cells and recognized as non-self components by T cells, so that the T cells are activated into effector T cells to attack and eliminate the tumor cells. The most critical step is the binding of MHC molecules to polypeptide molecules. MHC molecules in the human body, also known as Human Leukocyte Antigens (HLA), are mainly classified into MHC class I molecules, which are expressed by most cells and mainly involved in the presentation of endogenous antigens, and MHC class II molecules; the latter is mainly expressed by antigen presenting cells and mainly participates in the presentation of foreign antigens. In which three genes encoding MHC I (HLA-A, HLA-B and HLA-C) and three MHC II (HLA-DR, HLA-DQ, HLA-DP) are present in the human genome, and the variation of these genes is very large, and several tens of thousands of different genotypes are currently found, called HLA haplotypes, and each haplotype is internationally and statistically named, wherein the gene name is separated from the following number by one, and alleles of different grades are separated by (: and it is currently more accurate to divide into three grades, such as HLA-A02: 03:01, representing the second class 03 under the first class 02 of HLA-A genes, and finally, the third class 01. Different haplotypes generally have different affinities for the same polypeptide, i.e., the ability to recognize it as an antigen, with the stronger the affinity the stronger the recognition. In addition, the same polypeptide has a more similar affinity for the same class of haplotypes, as a polypeptide differs in affinity from different haplotypes under the HLA-A02 (abbreviated as HLA-A2) classification by a much smaller amount than for HLA-A11. Until now, scientists in the related art have constructed a plurality of epitope databases and developed a plurality of epitope prediction related software (such as netmhcpan4.0, netmhcpan-ba, netmhcpan-el, mhcflurry, ann, smm or combblib) based on individualized HLA haplotypes, and the more different software predict antigen peptides at the same time, the more accurate the antigen is. Based on current research and analytical approaches, it is possible to predict and design targeted antigenic peptides for personalized therapy for different personalized targets.
HLA-A02 is a specific class I Major Histocompatibility Complex (MHC) allele at the HLA-A locus. Over 400 different HLA-A x 02 alleles have been reported, making HLA-A2 the most polymorphic group of haplotypes in the specificity of different HLA class alleles. Among them, HLA-A02: 01 is the highest-frequency allele, and has been widely used in the study of HLA-A2-restricted CTL response models. With the exception of a × 02:01, the HLA-a2 allele was most frequently found in asia among all MHCI molecules. The affinity of the same polypeptide to a haplotype of a different HLA-A2 is more similar. Therefore, antigen peptides are designed aiming at HLA-A2 haplotype, and the beneficial population can be maximized.
The virus vaccines mainly comprise attenuated live vaccines, inactivated vaccines, subunit vaccines (recombinant protein vaccines or polypeptide vaccines), nucleic acid vaccines, virus vectors and the like. The use of vaccines has successfully prevented a variety of diseases worldwide: smallpox virus has been eradicated and the incidence of hepatitis b, poliomyelitis, measles and other childhood diseases has been greatly reduced worldwide. However, these vaccines have different development and application short boards, such as live attenuated vaccines or inactivated vaccines, which have relatively long development period, risk of host infection due to pathogen, and complicated and time-consuming routine test items in the development process for safety.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provides a personalized treatment scheme for hepatitis B virus.
In order to solve the technical problems of the present invention, the first aspect of the present invention is directed to an antigenic polypeptide of hepatitis B virus.
The antigen polypeptide provided by the invention is suitable for antigen polypeptides of individuals with human leukocyte antigen haplotypes of HLA-A2 haplotypes, and comprises at least one of polypeptides with amino acid sequences shown as SEQ ID Nos. 1-32;
or functionally identical or similar polypeptides obtained by substituting and/or deleting and/or adding at least one amino acid in the amino acid sequence of each polypeptide.
The invention further provides an antigen polypeptide combination comprising the antigen polypeptides shown in SEQ ID No. 1-32.
The invention also provides a fusion polypeptide comprising the antigen polypeptide.
Wherein the above fusion polypeptide is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 of the above antigen polypeptides fused thereto. Further, the above-mentioned fusion polypeptides are combinations which form fusion polypeptides, each of the combinations comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 of the above-mentioned antigenic polypeptides. Furthermore, all the fusion polypeptides in the combination at least contain all the polypeptides shown in SEQ ID NO 1-32. Furthermore, the antigen polypeptides can be connected with one another by a linker. These linkers may be selected from a variety of linking peptides commonly used in the art. Obviously, a large number of antigenic polypeptides are required, and multiple fusion polypeptides can be designed separately and used separately or together.
The invention also provides an antibody against the polypeptide. Further, the antibody is a polyclonal antibody or a monoclonal antibody.
Further, the above-mentioned antibody can specifically bind to the above-mentioned polypeptide. Further, the above antibody may form a conjugate with a conjugate moiety; the coupling moiety is one or more selected from the group consisting of a radionuclide, a drug, a toxin, a cytokine, an enzyme, a fluorescein, a carrier protein, or a biotin.
On the basis, the invention provides the application of the antigen polypeptide, the fusion polypeptide, the derivative of the antigen polypeptide or the fusion polypeptide, the chemical modification product of the antigen polypeptide or the fusion polypeptide or the antibody in any one of the following items:
a. preparing anti-hepatitis B virus products, or anti-hepatitis B virus products;
b. preparing a product for treating and/or preventing diseases caused by hepatitis B virus infection; or treating or preventing diseases caused by hepatitis B virus infection;
c. preparing a product capable of improving symptoms caused by hepatitis B virus infection; or improving symptoms due to hepatitis B virus infection;
in the above application, the disease caused by hepatitis B virus infection is at least one of hepatitis B, severe hepatitis, liver cirrhosis, hepatic ascites or liver cancer. Further, the disease caused by hepatitis B virus infection is especially hepatitis B virus positive liver cancer.
The invention also provides a method for preparing the personalized hepatitis B virus antigen peptide. The method comprises the following steps:
a. detecting a human leukocyte antigen haplotype of the subject to which it is directed; confirming that it contains HLA-A2 typing among the following human leukocyte antigen haplotypes related to hepatitis B virus;
b. according to the result of the step a, after the individual is confirmed to have the human leucocyte antigen haplotype related to the hepatitis B virus as HLA-A2; the antigen polypeptide or the fusion polypeptide is used as a candidate polypeptide;
c. preparing the candidate polypeptide to obtain the polypeptide.
Wherein, the step a of the method is to detect the individual human leukocyte antigen haplotype by detecting an ex vivo blood sample thereof.
Further, in step c of the method, when selecting the polypeptides, all polypeptides corresponding to HLA-A2 are selected from the candidate polypeptides confirmed in step b, depending on the type of the human leukocyte antigen haplotype associated with hepatitis B virus possessed by the individual.
Wherein the individual in the above method is an HBV positive individual. Further, the individual is a liver cancer patient positive for HBV.
The invention also provides the personalized hepatitis B virus antigen peptide prepared by the method.
The invention also provides a protein or polypeptide vaccine for preventing and/or treating diseases caused by hepatitis B virus infection. The vaccine contains various antigen polypeptides, fusion polypeptides or the personalized hepatitis B virus antigen peptide as main antigen components and pharmaceutically acceptable auxiliary materials or auxiliary components.
Furthermore, the vaccine also contains an immunologic adjuvant. Wherein the immunological adjuvant is Freund's incomplete adjuvant, Freund's complete adjuvant, aluminum hydroxide adjuvant, aluminum phosphate adjuvant, milk adjuvant, liposome adjuvant, microbial adjuvant, etc.
Wherein the disease caused by hepatitis B virus infection is at least one of hepatitis B, severe hepatitis, liver cirrhosis, hepatic ascites or liver cancer.
Furthermore, the vaccine for preventing and/or treating HBV positive related diseases also contains other components with the activity of preventing and/or treating HBV positive related diseases.
Furthermore, the medicine also contains pharmaceutically acceptable auxiliary materials or auxiliary components.
Wherein the pharmaceutically acceptable auxiliary materials or auxiliary components are at least one of cell culture medium, solvent, solubilizer, cosolvent, emulsifier, colorant, adhesive, osmotic pressure regulator, stabilizer, glidant, preservative, suspending agent, osmotic promoter, pH value regulator, buffering agent, surfactant, absorbent, diluent or flocculant and deflocculant.
The invention also provides a method for preventing and/or treating diseases caused by hepatitis B virus infection. The method comprises the step of administering an effective amount of the above-described antigenic polypeptide, fusion polypeptide, hepatitis b virus antigenic peptide, or vaccine to an individual positive for hepatitis b virus.
Further, the above administration mode is injection administration. Furthermore, the injection is at least one of intravenous injection, intramuscular injection, intraperitoneal injection or intratumoral injection.
The above method of the present invention, wherein, at the time of administration, an antigenic polypeptide combination comprising the antigenic polypeptides represented by SEQ ID Nos. 1 to 32 is administered; or a fusion polypeptide or a combination of fusion polypeptides comprising the antigenic polypeptides shown in SEQ ID Nos. 1-32 as described above.
The present invention also encompasses a gene encoding the above-mentioned antigen polypeptide, fusion polypeptide or antibody. And provides a vector containing the coding gene. Furthermore, the vector is an expression vector. The expression vector can be selected from common vectors such as plasmid vectors, adenovirus vectors, lentivirus vectors or adeno-associated virus vectors. When an adenovirus vector is used, a replication-defective adenovirus vector is generally used.
The invention has the beneficial effects that: the invention provides a group of antigen peptides, and the individual effectiveness of the antigen peptides is verified by utilizing an immunity experiment in a human body experiment, so that the blank of the individual antigen peptides of HLA-A2 haplotype in the treatment of HBV related diseases, particularly liver cancer is filled. Because HLA-A2 haplotype appears in Asian people at high frequency, more than 40% of people carry at least one HLA-A2 haplotype, the antigen peptide aiming at HLA-A2 haplotype has the maximized beneficial patient population as one HLA restricted epitope vaccine, thereby obtaining wider application. In addition, the antigen peptide is prepared in a centralized scale, so that the time cost and the labor cost of the new antigen immunotherapy can be greatly reduced, and the treatment efficiency is improved. The high polymorphism of HLA-A2 haplotype suggests that the antigenic peptide ligands to which they bind may differ significantly, both in physicochemical and spatial conformations. Therefore, the probability that the antigen peptide presented by the HLA-A2 molecule is recognized by the CD8+ T cell surface specific receptor (TCR) in the body of a patient is greatly increased, and the adaptive immune response of the anti-tumor can be activated. The antigen peptide combination can obviously activate the human body specificity to the T cells of HBV, increases the killing capacity of the T cells to the liver cancer cells infected by HBV, and can be synthesized on a large scale and used for standardized and individualized HBV related liver cancer immunotherapy.
Drawings
FIG. 1 is a graph showing the in vitro reactivity of T cells derived from liver cancer patient 1 (haplotype HLA-A02: 01) to mixed antigenic peptides by IFN-. gamma.ELISPOT assay after stimulation of activated T cells using dendritic cells loaded with antigen polypeptide groups; positive control: phytohemagglutinin PHA; negative control: no peptide was added.
FIG. 2 is a graph showing the in vitro reactivity of T cells derived from liver cancer patient 2 (haplotypes HLA-A02: 01 and HLA-A02: 03) to mixed antigen peptides by IFN-. gamma.ELISPOT assay after stimulation of activated T cells using dendritic cells loaded with antigen polypeptide groups; positive control: phytohemagglutinin PHA; negative control: no peptide was added.
FIG. 3 is a graph showing in vitro reactivity of T cells derived from liver cancer patient 3 (haplotype HLA-A02: 01) to mixed antigen peptides by IFN-. gamma.ELISPOT assay after stimulation of activated T cells using a dendritic cell antigen mixed peptide group; positive control: phytohemagglutinin PHA; negative control: no peptide was added.
FIG. 4 shows the in vitro reactivity of T cells from hepatocarcinoma patient 4 (haplotype HLA-A02: 06) to mixed antigenic peptides by IFN- γ ELISPOT assay after stimulation of activated T cells using dendritic cell loaded antigen-polypeptide set; positive control: phytohemagglutinin PHA; negative control: no peptide was added.
Detailed Description
The early stage of the invention is based on a prediction method for clinical individualized tumor neoantigen sequencing. The method mainly comprises the following steps
Step 1, comparing sequencing data of tumor cells of a patient with a preset corresponding reference genome to obtain a DNA comparison result and an RNA comparison result;
step 2, preprocessing a DNA comparison result and an RNA comparison result;
step 3, analyzing somatic variation, clone type, tumor purity, gene expression quantity in tumor cells and expression abundance of somatic variation allele by using the DNA comparison result and the RNA comparison result to obtain an analysis result;
step 4, predicting the genotype of MHC class I molecules of the tumor cells of the patient, analyzing the affinity of potential new antigens and calculating the presenting effect of the polypeptide according to the DNA analysis result and the genotype of the MHC class I molecules of the tumor cells of the patient;
step 5, scoring and sequencing each corresponding new antigen according to the analysis result, the affinity of the potential new antigen and the presenting effect of the polypeptide, and presenting the new antigen;
and 6, optimizing the analysis method of the affinity of the potential new antigen in the step 5 by using a machine learning method based on the experimental result. For improving the accuracy of the prediction.
The specific steps are shown in Chinese patent document CN111415707A, and the clinical individualized tumor neoantigen prediction method.
According to the method, a plurality of HBV positive liver cancer patient samples are sequenced and HLA is typed. Combining different human leucocyte antigen haplotypes, using the biological information prediction method, prediction and preliminary verification of an antigen peptide aiming at hepatitis B virus coding protein are carried out, and finally a group of high-affinity antigen peptides aiming at the common human leucocyte antigen haplotype HLA-A2 type in HBV positive liver cancer patients are obtained.
The sequences of this group of antigenic peptides, the applicable haplotypes, and the targets of the HBV-encoded proteins are shown in Table 1.
TABLE 1
Figure BDA0003561245290000061
Figure BDA0003561245290000071
In the present invention, the expression "a protein having the same or similar function as that of the above-mentioned protein, which is a peptide fragment obtained by substituting 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, deletion, insertion and/or substitution of several (usually 1 to 10, more preferably 1 to 5, and preferably 1 to 3) amino acids, and addition of one or several (usually 20 or less, preferably 10 or less, more preferably 5 or less, and most preferably 3 or less) amino acids at the C-terminus and/or N-terminus. For example, substitutions in the protein with amino acids of similar or similar properties will not generally alter the function of the protein. Also, for example, the addition of one or several amino acids at the C-terminus and/or N-terminus does not generally alter the function of the protein. The term also includes active fragments and active derivatives of the protein.
The expression "a peptide fragment obtained by substituting and/or deleting and/or adding at least one amino acid in the amino acid sequence of each polypeptide" also includes, but is not limited to, polypeptides formed by replacing at most 10 (i.e., one or several), preferably at most 8, and more preferably at most 5 (5, 4, 3, 2, or 1) amino acids with amino acids having similar or similar properties, i.e., conservative variant polypeptides. Further, these conservative variant polypeptides may be generated by making substitutions according to table 3.
TABLE 2 amino acid substitution Table
Initial residue(s) Representative substitutions 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
The polypeptide can be used as active ingredient for preparing medicine for preventing and/or treating hepatitis B virus infection. Generally, those skilled in the art can design and prepare protein or polypeptide vaccines for preventing and/or treating diseases caused by hepatitis B virus infection by using the polypeptides as antigen active ingredients. The vaccine takes the polypeptide as an antigen component and pharmaceutically acceptable auxiliary materials or auxiliary components.
In the preparation of vaccines, immunological adjuvants are often added to enhance the immune response of the body to the vaccine. Wherein the immunological adjuvant is Freund's incomplete adjuvant, Freund's complete adjuvant, aluminum hydroxide adjuvant, aluminum phosphate adjuvant, milk adjuvant, liposome adjuvant, microbial adjuvant, etc.
Naturally, in the art, antibodies against the proteins described herein are readily available in addition to the proteins described herein. The antibody is a polyclonal antibody or a monoclonal antibody; preferably a monoclonal antibody. The above antibodies also form conjugates with a conjugate moiety. Further, the coupling moiety is one or more selected from the group consisting of a radionuclide, a drug, a toxin, a cytokine, an enzyme, a fluorescein, a carrier protein, and a biotin. The antibody capable of being specifically combined with the protein can be used for preparing a medicament for preventing and/or treating hepatitis B virus infection so as to resist the hepatitis B virus infection on one hand, and can be used for immunoassay related to the hepatitis B virus on the other hand.
In addition, the present invention also encompasses a gene encoding the above protein. The coding gene of the protein can be used for expressing and preparing the protein or the antibody; on the other hand, the recombinant expression vector can be loaded in an expression vector in an operable way, and further can be prepared into vector vaccines or vector medicaments. Expression can be selected from commonly used vectors such as plasmid vectors, adenovirus vectors, lentiviral vectors, or adeno-associated virus vectors. When an adenovirus vector is used, a replication-defective adenovirus vector is generally used.
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
In the present invention, the amino acid sequence is from left to right from the nitrogen terminus to the carbon terminus unless otherwise specified.
It should be noted that the embodiments and features of the embodiments may be combined with each other without conflict.
The invention is further described with reference to the following drawings and specific examples, which are not intended to be limiting. EXAMPLES functional experiments on the antigenic peptides of the present invention
1. Patient HLA typing detection
2mL of peripheral blood of 4 patients is respectively extracted, EDTA or sodium citrate is adopted for anticoagulation, and PCR-SBT technology is used for detecting four-digit high-resolution typing of each HLA site.
Acquisition of peripheral blood PBMC of 2.4 patients
(1) Will use COBE SpectraTMDisinfecting a cell bag (or heparin anticoagulation blood extracted by an injector) collected by an MNC system machine, placing the cell bag into a super clean bench, transferring cells into a 50mL centrifugal tube by using a 20mL injector, and diluting the cells by using normal saline 1: 1;
(2) 20mL of human lymphocyte separation solution is added into a centrifugal tube;
(3) slowly superposing the cell sap diluted by the normal saline on the human lymphocyte separation fluid along the tube wall by using a Pasteur pipette;
(4) horizontally centrifuging at 800g for 20 minutes;
(5) the tube is divided into three layers after centrifugation, the upper layer is plasma, the lower layer is mainly red blood cells and granulocytes, the middle layer is lymphocyte separation liquid, and a leucocyte layer which mainly comprises mononuclear cells is arranged at the interface of the upper layer and the middle layer, namely a peripheral blood mononuclear cell layer;
(6) inserting the mononuclear cells into the tunica albuginea layer by using a suction tube, sucking the mononuclear cells, putting the mononuclear cells into another 50mL centrifuge tube, supplementing physiological saline, horizontally centrifuging the mononuclear cells for 300g, and performing centrifugation for 10 min;
(7) discarding the supernatant, adding normal saline, mixing uniformly, horizontally centrifuging for 300g, and 10 min;
(8) discarding the supernatant, supplementing a cell culture medium, horizontally centrifuging for 5min after uniformly mixing the supernatant and the cell culture medium;
(9) the supernatant was discarded, diluted with AIM-V medium, the cells were counted, and a suitable amount of cells was frozen and the specimen was retained.
3. Dendritic Cell (DC) culture:
(1) according to the PBMC obtaining method, the obtained 4 patients PBMCs are respectively placed in a plurality of six-hole plate culture flasks and cultured by AIM-V culture medium;
(2)37℃，5％CO2incubating for 2h in the incubator to allow the monocytes to adhere to the walls;
(3) adherent cells were isolated and 20mL AIM-V medium was added while supplementing recombinant human GM-CSF (800U/mL) and recombinant human IL-4(1000U/mL), 37 ℃, 5% CO2Culturing in an incubator, and inducing the monocyte to differentiate to DC;
(4) determining whether half amount of liquid is changed according to the cell amount on the third day, and if the liquid is changed, complementing GM-CSF and IL-4;
(5) adding DC maturation promoting factors LPS (10ng/mL) and IFN-gamma (100IU/mL) on the sixth day to induce DC maturation for 16-48 h;
(6) on day seven, mature dendritic cells were obtained.
(7) Harvesting mature DCs, culturing 32 groups of mature DCs of each patient, adding an antigen peptide 10 μ g/mL into the culture solution of each DC, standing at 37 deg.C and 5% CO2Incubate in incubator for 4-6 hours, centrifuge cells and resuspend using preheated PBS for use.
The mixed antigen peptide used in the embodiment of the invention is composed of 32 polypeptides shown in SEQ ID No. 1-32. In this example, these polypeptides were synthesized separately and each antigenic peptide was stored separately daily to form a combination of antigenic peptides.
4. Enzyme-linked immunosorbent assay (ELISPOT method)
(1) Activating the pre-coated plate, adding 200 mu L of AIM-V serum-free culture medium, standing at room temperature for 10 minutes, and pouring;
(2) adding 10-50 mu g/mL of T cells and antigen peptides separated from PBMC according to the designed groups, and 3 multiple holes for each group;
(3) after all samples were added, the plate was covered, marked, and placed at 37 ℃ with 5% CO2The incubator was incubated for 20 hours.
(4) Pouring cells and culture medium in the holes;
(5) cell lysis: add 200. mu.L ice-cold deionized water to each well, ice-cool for 10min in a refrigerator at 4 ℃ (cell lysis by hypotonic method);
(6) washing the plate: washing each well with 260 μ L of 1 × Washing buffer for 6 times, each for 60 seconds, and drying on absorbent paper after each Washing;
(7) and (3) incubation of the detection antibody: mu.L of diluted biotin-labeled antibody was added to each well at 37 ℃ with 5% CO2Incubating in an incubator for 1 hour;
(8) washing the plate: washing each well with 260 μ L of 1 × Washing buffer for 6 times, each for 60 seconds, and drying on absorbent paper after each Washing;
(9) and (3) avidin incubation: adding 100 mu L of diluted enzyme-labeled avidin into each hole, and incubating for 1 hour at 37 ℃;
(10) washing the plate: washing each well with 260 μ L of 1 × Washing buffer for 6 times, each for 60 seconds, and drying on absorbent paper after each Washing;
(11) color development: according to the reagent configuration, AEC color developing solution is prepared, 100 μ L of color developing solution is added into each well, the temperature is 37 ℃, and 5% CO is added2Developing color in the incubator, and checking once every 5 minutes;
(12) after the spots have grown to a suitable size, they are washed 2 times with deionized water and the color development is terminated. Reversely buckling the plate on absorbent paper, patting to dry fine water drops, taking down the protective layer, placing the protective layer in a ventilated place, standing at room temperature, and naturally drying the film;
(13) the ELISPOT plate spots were counted and various parameters of the spots were recorded and analyzed.
5. Flow cytometry to determine markers of T cell activation
(1) Modulating the concentration of activated PBMCs or T lymphocytes to 1X 106about/mL. After the cell suspension is evenly blown to the air,centrifuging at 1500rpm for 5min, discarding the supernatant, adding PBS, centrifuging at 1500rpm for 5min, discarding the supernatant, resuspending the cells with 100 μ L PBS to flow tube, and labeling each group of cells on the tube wall;
(2) adding fluorescent antibody labeled cells such as anti-human CD8 and CD137, and setting a negative control group, a single-standard group and an isotype control group;
(3) dyeing at 4 ℃ in dark for 30 min;
(4) adding 1mL of PBS, and gently blowing and beating by using a pipette gun to uniformly mix;
(5) centrifuging at 1500rpm for 5min, and discarding the supernatant;
(6) add 100. mu.L PBS to resuspend the cells and test on the machine. BD software collected the data and analyzed the streaming results with Flowjo software.
6. Induction of PBMC activation in vitro
(1) Heparinized anticoagulated PBMC were isolated by the method described above and suspended in AIM-V medium (Gibico, USA) and counted.
(2) 96-well plate with U-shaped bottom, 1X 10 per well5Each PBMC was diluted in 200. mu.L of medium and incubated with 10. mu.M of each antigenic peptide in the combination, the U-shaped bottom being used to promote better cell-to-cell contact. The medium consisted of AIM-V medium (Gibco) containing small amounts of interleukin-2 (IL-2, 100U/mL, Peprotech) and 10% fetal calf serum (FCS, Gibco).
(3) PBMC were cultured for 3 days per round of stimulation, with medium changed half-way while being supplemented with the same antigenic peptide (10uM) and IL-2 (100U/mL).
(4) After 2-3 cycles of peptide in vitro stimulation culture, the fresh culture medium is replaced, and the same antigen peptide 10. mu.M is added again for stimulation culture overnight. Within 24h of the last round of stimulation (i.e. at day 7 and/or day 10 of culture), the level of T cell immune response was assessed by ELISPOT detection of IFN- γ expression, identifying the immunogenicity of the different antigenic peptides. PBMC were stimulated as negative control (NS) without peptide (medium) or with irrelevant peptide, and Phytohemagglutinin (PHA) was stimulated as positive control.
7. Stimulation of activated T cells using Dendritic Cell (DC) loaded antigenic peptides
Mature DCs (1X 10)4Per 100. mu.L/well) at 37℃，5％CO2In the incubator, one of the antigen peptide combinations was pulsed at 10 μ M for 4-6 hours, washed with pre-warmed PBS, and then incubated with T cells overnight in complete AIM-V medium at a ratio of stimulator to effector of 1: 10. The level of activation of T cells was assessed the following day. The number and intensity of activated T cells after stimulated culture were measured by INF- γ ELISPOT and the expression level of the T cell activation marker 4-1BB (CD137) was evaluated by flow cytometry.
8. Preparation of novel antigenic peptide reactive T cells (NRT)
(1) Suspension cells transferred to F225 culture flask to adjust cell concentration to 1X 107/mL, supplemented with IL-2(100U/mL), IL-7(10ng/mL), IL-15(10ng/mL), 37 ℃, 5% CO2Culturing in an incubator for 7 days;
(2) harvesting mature DCs on day 7, culturing 32 groups of mature DCs of each patient, adding an antigen peptide 25 μ g/mL into the culture solution, placing at 37 deg.C and 5% CO2Incubating for 4-6 hours in an incubator;
(3) centrifuging at 1500rpm for 5min, discarding supernatant, mixing and culturing antigen peptide-loaded DC and suspension cells, and supplementing part of fresh culture medium containing (IL-2, IL-7, IL-15). 37 ℃ and 5% CO2Incubating for 10 days;
(4) supplementing a fresh complete culture medium and corresponding cytokines IL-7, IL-15 and IL-2 by half every 2 to 3 days according to the growth condition of the cells and the color of the culture medium, and then bottling; adding OKT3 monoclonal antibody in proper amount according to T cell expansion condition, adding irradiated ECCE cells or APC loading peptide prepared by resuscitating cryopreserved PBMC for second round stimulation;
(5) the T cells are stimulated by the antigen peptide for about 10 days, and the respective T cells of the patients are harvested and mixed for detection and function experiments.
The experimental results are as follows:
HLA typing results of four patients and results of T cell activation experiments on four patients using mixed antigen peptides are shown in FIGS. 1-4, and the results show that four patients all carry one or two HLA-A2 haplotypes, and the mixed antigen peptides (mixture of polypeptides shown in SEQ ID Nos. 1-32) of the present invention activate T cells of these patients against the antigen peptides, i.e., generate strong immune response, and are likely to kill liver cancer cells positive to HBV infection. In conclusion, the antigen peptide of the invention has verified the effectiveness of the antigen peptide on individuals containing haplotype of HLA-A2 in human body experiments by utilizing immune experiments, thereby making up the blank of individual antigen peptide in the treatment of liver cancer positive to HBV infection; the antigen peptide can obviously activate the human body specificity to the HBV T cells, increase the killing capacity of the T cells to HBV-infected liver cancer cells, and can be synthesized in a large scale and used for standardized and individualized liver cancer immunotherapy.
While the invention has been described with reference to a preferred embodiment, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention.
Sequence listing
<110> Chengdu Langgu Biotechnology Ltd
<120> hepatitis B virus antigen peptide suitable for leukocyte antigen haplotype of individual HLA-A2 and use thereof
<160> 32
<170> SIPOSequenceListing 1.0
<210> 1
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Phe Leu Pro Asp Phe Phe Pro Ser Ile
1 5
<210> 2
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 2
Tyr Met Asp Asp Val Val Leu Gly Ala
1 5
<210> 3
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 3
Phe Leu Leu Thr Lys Ile Leu Thr Ile
1 5
<210> 4
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 4
His Leu Tyr Ser His Pro Ile Ile Leu
1 5
<210> 5
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 5
Ile Leu Ser Pro Phe Met Pro Leu Leu
1 5
<210> 6
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 6
Ser Leu Tyr Ala Asp Ser Pro Ser Val
1 5
<210> 7
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 7
Phe Leu Ser Lys Gln Tyr Leu Asn Leu
1 5
<210> 8
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 8
Gly Leu Leu Gly Pro Leu Leu Val Leu
1 5
<210> 9
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 9
Phe Leu Leu Ser Leu Gly Ile His Leu
1 5
<210> 10
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 10
Lys Leu Ile Gly Thr Asp Asn Ser Val
1 5
<210> 11
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 11
His Leu Pro Asp Arg Val His Phe Ala
1 5
<210> 12
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 12
Phe Leu Leu Ala Gln Phe Thr Ser Ala
1 5
<210> 13
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 13
Arg Val Thr Gly Gly Val Phe Leu Val
1 5
<210> 14
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 14
Ala Leu Pro Pro Ala Ser Pro Pro Val
1 5
<210> 15
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 15
Gly Leu Ser Arg Tyr Val Ala Arg Leu
1 5
<210> 16
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 16
Leu Leu Ala Gln Phe Thr Ser Ala Ile
1 5
<210> 17
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 17
Leu Leu Ser Ser Asn Leu Ser Trp Leu
1 5
<210> 18
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 18
Val Leu Gln Ala Gly Phe Phe Leu Leu
1 5
<210> 19
<211> 10
<212> PRT
<213> Artificial Sequence (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 (Artificial Sequence)
<400> 20
Val Leu His Lys Arg Thr Leu Gly Leu
1 5
<210> 21
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 21
Val Ser Ile Pro Trp Thr His Lys Val
1 5
<210> 22
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 22
Phe Ala Val Pro Asn Leu Gln Ser Leu
1 5
<210> 23
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 23
Phe Thr Phe Ser Pro Thr Tyr Lys Ala
1 5
<210> 24
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 24
Ala Val Thr Asn Phe Leu Leu Ser Leu
1 5
<210> 25
<211> 10
<212> PRT
<213> Artificial Sequence (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 (Artificial Sequence)
<400> 26
Lys Ile Leu Thr Ile Pro Gln Ser Leu
1 5
<210> 27
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 27
Met Met Trp Tyr Trp Gly Pro Ser Leu
1 5
<210> 28
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 28
Tyr Leu Val Ser Phe Gly Val Trp Ile
1 5
<210> 29
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 29
Trp Leu Ser Leu Leu Ala Pro Phe Val
1 5
<210> 30
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 30
Leu Leu Trp Phe His Ile Ser Cys Leu
1 5
<210> 31
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 31
Lys Gln Tyr Leu Asn Leu Tyr Pro Val
1 5
<210> 32
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 32
Ser Leu Asp Ser Trp Trp Thr Ser Leu
1 5

Claims (25)

1. An antigenic polypeptide directed against hepatitis b virus, characterized in that: is suitable for individuals with haplotype of HLA-A2, and comprises at least one polypeptide with amino acid sequence shown in SEQ ID No. 1-32;
or functionally identical or similar polypeptides obtained by substituting and/or deleting and/or adding at least one amino acid in the amino acid sequence of each polypeptide.
2. The antigenic polypeptide of claim 1, wherein: is a polypeptide combination containing polypeptides with amino acid sequences shown as SEQ ID NO 1-32.
3. A fusion polypeptide comprising the antigenic polypeptide of any one of claims 1 or 2.
4. The fusion polypeptide of claim 3, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 of the antigenic polypeptides set forth in any one of SEQ ID NOs 1-32 are fused.
5. The fusion polypeptide of claim 3 or 4, characterized in that: is a combination of fusion polypeptides, which contains polypeptides shown in SEQ ID NO. 1-32.
6. The fusion polypeptide of any one of claims 3 to 5, characterized in that: the polypeptides are connected with each other at intervals by a linker.
7. An antibody against the polypeptide of any one of claims 1 to 6.
8. The antibody of claim 7, wherein: is polyclonal antibody or monoclonal antibody.
9. The antibody of claim 7 or 8, wherein: specifically binding to the polypeptide of any one of claims 1 to 6.
10. The antibody of any one of claims 7 to 9, wherein: also forming a conjugate with a coupling moiety; the coupling moiety is one or more selected from the group consisting of a radionuclide, a drug, a toxin, a cytokine, an enzyme, a fluorescein, a carrier protein, or a biotin.
11. Use of the antigenic polypeptide of any one of claims 1 to 2, the fusion polypeptide of any one of claims 3 to 6, a derivative of said antigenic polypeptide or fusion polypeptide, or a chemically modified product of said antigenic polypeptide or fusion polypeptide, or the antibody of any one of claims 7 to 10, in any one of:
a. preparing anti-hepatitis B virus products, or anti-hepatitis B virus products;
b. preparing a product for treating and/or preventing diseases caused by hepatitis B virus infection; or treating or preventing diseases caused by hepatitis B virus infection;
c. preparing a product capable of improving symptoms caused by hepatitis B virus infection; or improving symptoms caused by hepatitis B virus infection.
12. Use according to claim 11, characterized in that: the disease caused by hepatitis B virus infection is at least one of hepatitis B, severe hepatitis, liver cirrhosis, hepatic ascites or liver cancer; further, the disease caused by hepatitis B virus infection is especially hepatitis B virus positive liver cancer.
13. The method for preparing the personalized hepatitis B virus antigen peptide comprises the following steps: the method is characterized by comprising the following steps:
a. detecting a human leukocyte antigen haplotype of the individual; confirming that the human leukocyte antigen haplotype related to hepatitis B virus is contained in HLA-A2;
b. according to the result of the step a, after the individual is confirmed to have the human leucocyte antigen haplotype related to the hepatitis B virus as HLA-A2; the antigen polypeptide of claim 1 or 2, the fusion polypeptide of any one of claims 3 to 7 as a candidate polypeptide;
c. and c, preparing the alternative polypeptide confirmed in the step b to obtain the polypeptide.
14. The method of claim 13: the method is characterized in that: the step a is carried out by detecting the individual's human leucocyte antigen haplotype in an ex vivo blood sample.
15. The method of claim 13 or 14: the method is characterized in that: the individual is an HBV positive individual.
16. The method of any one of claims 13 to 15: the method is characterized in that: the individual is a liver cancer patient with HBV positive.
17. A personalized hepatitis b virus antigen peptide produced by the method of any one of claims 13 to 16.
18. The protein or polypeptide vaccine for preventing and/or treating diseases caused by hepatitis B virus infection is characterized in that: the antigen peptide of any one of claims 1 to 2, the fusion polypeptide of any one of claims 3 to 6, a derivative of the antigen polypeptide or the fusion polypeptide, or a chemically modified product of the antigen polypeptide or the fusion polypeptide, or the personalized hepatitis B virus antigen peptide of claim 17 as an antigen component, and a pharmaceutically acceptable adjuvant or auxiliary component.
19. The vaccine of claim 18, wherein: also contains immunological adjuvant.
20. The vaccine of claim 19, wherein: the immunological adjuvant is Freund incomplete adjuvant, complete Freund adjuvant, aluminum hydroxide adjuvant, aluminum phosphate adjuvant, milk adjuvant, liposome adjuvant, microbial adjuvant, etc.
21. The vaccine of any one of claims 17-20, characterized in that: the disease caused by hepatitis B virus infection is at least one of hepatitis B, severe hepatitis, liver cirrhosis, hepatic ascites or liver cancer.
22. A gene encoding the antigenic polypeptide of any one of claims 1 to 2, the fusion polypeptide of any one of claims 3 to 6, a derivative of said antigenic polypeptide or fusion polypeptide, or a chemically modified product of said antigenic polypeptide or fusion polypeptide, or the antibody of any one of claims 6 to 9.
23. A vector comprising the encoding gene of claim 22.
24. The vector according to claim 23, characterized in that said vector is an expression vector.
25. The vector of claim 24, wherein the expression vector is a plasmid vector, an adenoviral vector, a lentiviral vector or an adeno-associated viral vector.
CN202210289717.7A 2022-03-23 2022-03-23 Hepatitis B virus antigen peptide suitable for leukocyte antigen haplotype as HLA-A2 individual and application thereof Pending CN114591404A (en)

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Publication number Priority date Publication date Assignee Title
CN107022006A (en) * 2017-03-28 2017-08-08 东南大学 The t lymphocyte epitope peptide sequence of hepatitis B virus antigen
CN111116719A (en) * 2019-12-13 2020-05-08 南京大户生物科技有限公司 Thymus-dependent lymphocyte antigen epitope peptide of hepatitis B virus antigen and application thereof
CN114478711A (en) * 2022-01-05 2022-05-13 成都朗谷生物科技股份有限公司 Antigenic peptide aiming at hepatitis B virus and application thereof

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Publication number Priority date Publication date Assignee Title
CN107022006A (en) * 2017-03-28 2017-08-08 东南大学 The t lymphocyte epitope peptide sequence of hepatitis B virus antigen
CN111116719A (en) * 2019-12-13 2020-05-08 南京大户生物科技有限公司 Thymus-dependent lymphocyte antigen epitope peptide of hepatitis B virus antigen and application thereof
CN114478711A (en) * 2022-01-05 2022-05-13 成都朗谷生物科技股份有限公司 Antigenic peptide aiming at hepatitis B virus and application thereof

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