CN118580321A

CN118580321A - Antigenic peptide for hepatitis B virus and application thereof

Info

Publication number: CN118580321A
Application number: CN202410863906.XA
Authority: CN
Inventors: 许恒; 舒洋; 杨莉; 魏于全; 丁振宇
Original assignee: Chengdu Langgu Biotechnology Co ltd
Current assignee: Chengdu Langgu Biotechnology Co ltd
Priority date: 2022-01-05
Filing date: 2022-01-05
Publication date: 2024-09-03
Also published as: CN118580322A; CN114478711A; CN114478711B; CN118620040A

Abstract

The invention relates to an antigenic peptide aiming at hepatitis B virus, which is synthesized based on the amino acid sequence of hepatitis B virus encoding protein and by combining human leukocyte antigen haplotypes in different major histocompatibility complex I class molecules and using a biological information prediction method, wherein the antigenic peptide comprises the following components: at least one of the amino acid sequences shown in SEQ ID NO 1-124. The antigen peptide of the invention has verified the individuation effectiveness by using immune experiment in human body experiment, thereby making up the blank of individuation antigen peptide in liver cancer treatment; the antigen peptide can obviously activate the T cells of HBV specific to human body, increase the killing capacity of the T cells to HBV infected liver cancer cells, and can be used for standardized and individualized HBV related liver cancer immunotherapy by large-scale synthesis.

Description

Antigenic peptide for hepatitis B virus and application thereof

The invention relates to a split application of Chinese invention patent application with the application number of 202210011045.3 and the application name of antigenic peptide aiming at hepatitis B virus and application thereof and with the application date of 2022, 1 and 5.

Technical Field

The invention relates to the technical field of immunotherapeutic medicines, in particular to an antigenic peptide aiming at hepatitis B virus and application thereof.

Background

Immunotherapy of tumors is considered to be a new generation of tumor treatment following surgery, radiotherapy, chemotherapy and small molecule targeted therapies, which differs from the previous traditional treatments in that: the immunotherapy changes the treatment means from directly killing tumor cells by the medicine to enhancing immune cells, treats tumor by improving the anti-tumor immunity of the patient, and has the advantages of accurate killing, small side effect, durable curative effect and the like compared with the traditional treatment. In addition, the immune system of the organism has the characteristic of immune memory, so that the immune therapy can help a patient to form memory type immunity, and has remarkable advantages in preventing tumor recurrence and metastasis.

Hepatocellular carcinoma (HCC) is the fifth most common tumor, with quite extensive regional differences, with annual worldwide incidence of about 100 tens of thousands, mainly in china. About 80% of liver cancers are associated with hepatitis b virus infection (HBV). About 15% -40% of untreated infected persons develop cirrhosis, liver failure or liver cancer. In general, liver cancer is less than twenty parts per million per year in Chinese, but many patients are advanced when symptomatic, losing timing of excision, and thus median survival is less than half a year. Few patients have a 5-year survival rate of about 40% after removal of cancerous tissue from the liver.

The HBV genome contains 3200 bases and comprises 4 Open Reading Frames (ORFs) which are all located in the 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 pre-S1, pre-S2 and S, and the pre-S1 protein, the pre-S2 protein and the HBsAg on the envelope are respectively coded. The pre-S protein has strong immunogenicity, and the hepadnavigability of HBV is mainly identified and mediated by the pre-S protein and hepatocyte receptor. The C region is divided into a pre-C gene and a C gene, and HBeAg and HBcAg are encoded. The protein coded from the front C gene is secreted outside the cell after being processed, namely HBeAg; the protein encoded from the C gene is HBcAg. The P region is the longest 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 trans-activating effect, and can activate HBV own, other viral or cellular regulatory genes, and promote HBV replication.

In order to find individual antigenic peptides against HBV, antigenic prediction of proteins and polypeptides expressed by HBV is required, potential antigenic peptides are synthesized in vitro, and finally immune cells are activated by using tumor vaccine with high concentration and high intensity. Scientists have long been on the basis of basic research in immunology, where studies on T cell antigen recognition have made significant progress. It has been found that polypeptides (also referred to as epitopes) formed upon degradation of certain variant proteins in tumors can be specifically recognized by Major Histocompatibility Complex (MHC) molecules and form MHC-antigen peptide complexes, which are then presented to the surface of cells and recognized by T cells as non-hexon components, allowing T cells to activate into effector T cells to attack and clear the tumor cells. The most critical step is the binding of MHC molecules to polypeptide molecules. MHC molecules in humans, also known as Human Leukocyte Antigens (HLA), are largely divided into MHC class I molecules, which are expressed by most cells and are largely involved in the presentation of endogenous antigens, and MHC class II molecules; the latter is mainly expressed by antigen presenting cells, mainly involved in the presentation of foreign antigens. Wherein three genes encoding MHC I (HLA-A, HLA-B and HLA-C) and three MHC II (HLA-DR, HLA-DQ, HLA-DP) exist in the human genome, these genes vary very much, and several tens of thousands of different genotypes, called HLA haplotypes, each haplotype being designated by international statistics, have been found. Different haplotypes generally have different affinities for the same polypeptide, i.e., the ability to recognize it as an antigen, the stronger the affinity the stronger the recognition ability. Because the antigen peptides recognized by the major histocompatibility complex class I molecules are relatively short, related studies and analyses of major histocompatibility complex-antigen peptide complexes are also more complete. To date, scientists in the relevant field have constructed a number of antigen epitope databases and developed a number of antigen epitope prediction related software based on individual HLA haplotypes for major histocompatibility complex class I molecules (e.g., netMHCpan4.0, netmhcpan-ba, netmhcpan-el, mhcflurry, ann, smm or comblib). The more different software is predicted to be antigen peptide at the same time, the higher the accuracy of the antigen. Based on current research and analytical approaches, targeted antigenic peptides have been predicted and designed for different personalized targets for personalized therapies.

Disclosure of Invention

The invention aims at overcoming the defects in the prior art and provides an antigen peptide aiming at hepatitis B virus and application thereof.

In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:

In a first aspect, the present invention provides an antigenic peptide for hepatitis b virus, based on the amino acid sequence of hepatitis b virus encoded protein, combined with human leukocyte antigen haplotypes in different major histocompatibility complex class I molecules, using a bioinformatic prediction method, to synthesize an antigenic peptide comprising at least one of the amino acid sequences shown in the following table.

Preferably, the hepatitis b virus encoded protein comprises: at least one of hbv_env, hbv_ polymerase, HBV _c or hbv_x.

Preferably, the human leukocyte antigen typing comprises at least one of ：HLa-a*02:01、HLa-a*02:03、HLa-a*02:06、HLa-a*03:01、HLa-a*11:01、HLa-a*24:02、HLa-a*30:01、HLa-a*31:01、HLa-a*32:01、HLa-a*68:01、HLA-B*07:02、HLA-B*40:01、HLA-B*40:02、HLA-B*54:01、HLA-B*58:01、HLA-C*12:03、HLA-C*14:02 or HLA-C15:02.

The second aspect of the invention provides an application of the antigen peptide in preparing an immunotherapeutic medicine for liver cancer infected by hepatitis B virus.

Preferably, the immunotherapeutic agent comprises: at least one of an antigenic peptide cell therapeutic drug or vaccine.

Compared with the prior art, the invention has the following technical effects:

The antigen peptide of the invention has verified the individuation effectiveness by using immune experiment in human body experiment, thereby making up the blank of individuation antigen peptide in liver cancer treatment; the antigen peptide can obviously activate the T cells of HBV specific to human body, increase the killing capacity of the T cells to HBV infected liver cancer cells, and can be used for standardized and individualized HBV related liver cancer immunotherapy by large-scale synthesis.

Drawings

FIG. 1 is an in vitro assay of T cells from liver cancer patient 1 for reactivity to antigen peptide 6 and antigen peptide 115 by IFN-. Gamma.ELISPOT assay after stimulation of activated T cells using dendritic cell-loaded antigen peptide 6 and antigen peptide 115; positive control: phytohemagglutinin PHA; negative control: no peptide was added.

FIG. 2 is an in vitro assay of T cells from liver cancer patient 2 for reactivity to antigen peptide 6 and antigen peptide 23 by IFN-. Gamma.ELISPOT assay after stimulation of activated T cells using dendritic cell-loaded antigen peptide 6 and antigen peptide 23; positive control: phytohemagglutinin PHA; negative control: no peptide was added.

FIG. 3 is an in vitro assay of T cells from liver cancer patient 3 for reactivity to antigen peptide 3 and antigen peptide 84 by IFN-. Gamma.ELISPOT assay after stimulation of activated T cells using dendritic cells loaded with antigen peptide 3 and antigen peptide 84; positive control: phytohemagglutinin PHA; negative control: no peptide was added.

FIG. 4 is an in vitro assay of T cells from liver cancer patient 4 for reactivity to antigen peptide 79 and antigen peptide 115 by IFN-. Gamma.ELISPOT assay after stimulation of activated T cells using dendritic cell-loaded antigen peptide 79 and antigen peptide 115; positive control: phytohemagglutinin PHA; negative control: no peptide was added.

FIG. 5 is an in vitro assay of T cells from liver cancer patient 5 for reactivity to antigen peptide 6 and antigen peptide 124 by IFN-. Gamma.ELISPOT assay after stimulation of activated T cells using dendritic cells loaded with antigen peptide 6 and antigen peptide 124; positive control: phytohemagglutinin PHA; negative control: no peptide was added.

Detailed Description

The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

It should be noted that, without conflict, the embodiments of the present invention and features of the embodiments may be combined with each other.

The invention is further described below with reference to the drawings and specific examples, which are not intended to be limiting.

Examples

1. Patient HLA typing assay

Extracting 2mL of peripheral blood of a patient, anticoagulating by EDTA or sodium citrate, and detecting four-digit high-resolution typing of HLA-A, HLA-B and HLA-C sites of the major histocompatibility complex class I molecules by using PCR-SBT, sanger sequencing or second generation sequencing technology.

2. Peripheral blood PBMC acquisition

(1) Sterilizing a cell bag (or heparin anticoagulation extracted by a syringe) mechanically extracted by using a COBE Spectra ^TM MNC system, placing the sterilized cell bag into a super clean bench, transferring cells into a 50mL centrifuge tube by using a 20mL syringe, and diluting the cell bag with physiological saline in a ratio of 1:1;

(2) Adding 20mL of human lymphocyte separation liquid into the centrifuge tube;

(3) Slowly superposing the cell liquid diluted by the normal saline on the human lymphocyte separation liquid along the pipe wall by using a Pasteur pipette;

(4) Horizontally centrifuging 800g for 20 minutes;

(5) The inside of the tube is divided into three layers after centrifugation, the upper layer is blood plasma, the lower layer is mainly red blood cells and granulocytes, the middle layer is lymphocyte separating liquid, and a white membrane layer mainly containing 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 a white membrane layer by using a suction tube, sucking the mononuclear cells, placing the mononuclear cells into another 50mL centrifuge tube, supplementing physiological saline, and horizontally centrifuging for 300g and 10min;

(7) Discarding the supernatant, adding physiological saline, mixing, and horizontally centrifuging for 300g,10min;

(8) Discarding the supernatant, adding a cell culture medium, uniformly mixing, and horizontally centrifuging for 300g and 5min;

(9) The supernatant was discarded, diluted with AIM-V medium, counted, and a suitable amount of frozen stock was taken.

3. Dendritic Cell (DC) culture:

(1) According to the method for obtaining the PBMC, placing the obtained PBMC in a plurality of six-hole plate culture flasks and culturing AIM-V culture medium;

(2) Incubation for 2h at 37℃in a 5% CO ₂ incubator to allow adherence of monocytes;

(3) Adherent cells were isolated, 20mL AIM-V medium was added, recombinant human GM-CSF (800U/mL) and recombinant human IL-4 (1000U/mL) were supplemented simultaneously, cultured in a 5% CO ₂ incubator at 37℃and monocytes induced to differentiate toward DC;

(4) The third day of visual cell quantity determines whether half liquid exchange is carried out, if so, GM-CSF and IL-4 are supplemented;

(5) Adding DC maturation promoting factor LPS (10 ng/mL) and IFN-gamma (100 IU/mL) to induce DC maturation for 16-48h;

(6) Mature dendritic cells were obtained on day seven.

(7) The mature DC is harvested, 10 mug/mL of antigen peptide is added into the DC culture solution, the mixture is placed in a 37 ℃ and 5% CO ₂ incubator for incubation for 4 to 6 hours, cells are centrifuged and resuspended by using preheated PBS, and the cells are frozen for standby after being irradiated by radioactive rays of 30 Gy.

4. ELISA (ELISPOT method)

(1) Activating the pre-coated plate, adding 200 mu L of AIM-V serum-free medium, standing at room temperature for 10 minutes, and pouring;

(2) Adding T cells and polypeptides according to the designed groups, wherein the concentration of the T cells and the polypeptides is 10-50 mug/mL, and 3 compound holes are formed in each group;

(3) After all samples are added, the plate cover is covered, marks are made, and the samples are put into a 37 ℃ and 5% CO ₂ incubator for 20 hours.

(4) Pouring the cells and medium in the wells;

(5) Lysing the cells: 200 mu L ice-cold deionized water is added into each hole, and the ice bath is carried out for 10min at 4 ℃ in a refrigerator (cell lysis by hypotonic method);

(6) Washing the plate: washing each hole with 260 μl of 1×washing buffer for 6 times, each for 60 seconds, and drying on absorbent paper after each Washing;

(7) Incubation of detection antibody: 100. Mu.L of diluted biotin-labeled antibody was added to each well, and incubated at 37℃in a 5% CO ₂ incubator for 1 hour;

(8) Washing the plate: washing each hole with 260 μl of 1×washing buffer for 6 times, each for 60 seconds, and drying on absorbent paper after each Washing;

(9) Avidin incubation: 100 mu L of diluted enzyme-labeled avidin is added to each hole, and the mixture is incubated for 1 hour at 37 ℃;

(10) Washing the plate: washing each hole with 260 μl 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, preparing AEC color development liquid, adding 100 mu L of color development liquid into each hole, developing color by using a 5% CO ₂ incubator at 37 ℃, and checking every 5 minutes;

(12) After the spots grew to the appropriate size, the development process was terminated by washing with deionized water for 2 times. Reversely buckling the plate on water-absorbing paper, beating up tiny water drops, then taking down the protective layer, placing the protective layer in a ventilated place, standing at room temperature, and naturally airing the film;

(13) ELISPOT plate spot counts and various parameters of the spots were recorded for analysis.

5. Markers for determining T cell activation by flow cytometry

(1) The concentration of activated PBMCs or T lymphocytes was adjusted to about 1X 10 ⁶/mL. After the cell suspension was homogenized, the cells were centrifuged at 1500rpm for 5min, the supernatant was discarded, PBS was added, centrifugation was performed at 1500rpm for 5min, the supernatant was discarded, and 100. Mu.L of PBS was used to resuspend the cells into the flow tube, and the wall of the tube marked each group of cells;

(2) Adding fluorescent antibody labeled cells such as anti-human CD8, CD137 and the like, and setting a negative control group, a single standard group and a isotype control group;

(3) Dyeing at 4 ℃ for 30min in dark;

(4) Adding 1mL of PBS, and gently blowing and uniformly mixing by a pipetting gun;

(5) Centrifuging at 1500rpm for 5min, and discarding the supernatant;

(6) Cells were resuspended by adding 100. Mu.L PBS and detected on-press. BD software collects data and analyzes the streaming results with Flowjo software.

6. Induction of activation of PBMC in vitro

(1) Heparinized anticoagulants PBMCs were isolated by the method described above and suspended in AIM-V medium (Gibico, USA) and counted.

(2) 96-Well plates with U-shaped bottoms were used to promote better cell-to-cell contact by diluting 1X 10 ⁵ PBMC per well in 200. Mu.L of medium and adding 10. Mu.M of antigen peptide for incubation. The medium consisted of AIM-V medium (Gibco) containing small amounts of interleukin-2 (IL-2, 100U/mL, peprotech), 10% fetal calf serum (FCS, gibco).

(3) PBMC were cultured for 3 days with half-replacement of medium, with supplementation of the antigenic peptide (10 uM) and IL-2 (100U/mL) at the same time.

(4) After 2-3 cycles of in vitro stimulation culture of the peptides, the fresh medium was changed and 10. Mu.M of antigen peptide was added again for stimulation culture overnight. Within 24h of the last round of stimulation (i.e. on day 7 and/or day 10 of culture), the level of T cell immune response was assessed by detection of IFN- γ expression by ELISPOT, identifying the immunogenicity of the different antigenic peptides. Peptide-free (medium) or unrelated peptide stimulated PBMCs as negative control (NS), stimulated Phytohemagglutinin (PHA) was used as positive control.

7. Dendritic Cells (DCs) were used to load antigen peptide, DCs that stimulated maturation of activated T cells (1 x 10 ⁴/100 μl/well) were pulsed with 10 μΜ of peptide in a 5% co ₂ incubator at 37 ℃ for 4-6 hours, washed with pre-warmed PBS, and then incubated overnight with T cells in complete AIM-V medium at a stimulus to effector ratio of 1:10. The next day the level of activation of T cells was assessed. The number and intensity of activated T cells after stimulation of the culture were examined by INF-. Gamma.ELISPOT, and the expression level of T cell activation marker 4-1BB (CD 137) was evaluated by flow cytometry.

8. Preparation of neoantigen-reactive T cells (NRT)

(1) The suspension cells transferred to an F225 flask were cultured in a 5% CO ₂ incubator at 37℃for 7 days with the cell concentration adjusted to 1X 10 ⁷/mL, IL-2 (100U/mL), IL-7 (10 ng/mL) and IL-15 (10 ng/mL) being supplemented;

(2) Harvesting mature DC on day 7, adding 25 mug/mL of antigen peptide into the DC culture solution, placing the mixture in a 37 ℃ and 5% CO ₂ incubator for incubation for 4-6 hours;

(3) The supernatant was discarded after centrifugation at 1500rpm for 5min, and the fresh antigen-loaded DCs were mixed with suspension cells and supplemented with a portion of fresh medium containing (IL-2, IL-7, IL-15). Incubation at 37 ℃,5% co ₂, for 10 days;

(4) Half-supplementing fresh complete culture medium and corresponding cytokines IL-7, IL-15 and IL-2 every 2-3 days according to the growth condition of cells and the color of the culture medium, and transferring the culture medium into a bottle; adding a proper amount of OKT3 monoclonal antibody according to the expansion condition of T cells, and adding the irradiated ECCE cells or APC load peptide prepared by resuscitating frozen PBMC for second-round stimulation;

(5) T cells are subjected to antigen stimulation for about 10 days, and the T cells are harvested for detection and function experiments.

Analysis of experimental results:

As shown in fig. 1-5, patient 1, patient 2 and patient 5 have the same HLA-A x 02:01 haplotype and therefore also produce strong immunoreactivity when using the same polypeptide 6; whereas polypeptide 23 is a haplotype for a specific HLA-A 02:03 in patient 2, also generating an individualized immune response; similarly, polypeptide 115 can generate an immune response against HLA-C14:02 in both patient 1 and patient 4; polypeptide 84 and polypeptide 79 produce an immune response against HLA-C30:01 of patient 3 and HLA-a 11:01 of patient 4, respectively; in addition, both patient 1 and patient 3 carry haplotypes of HLA-A 02:01, and both polypeptide 6 and polypeptide 3 are capable of generating an immune response. This illustrates that different patients with the same HLA haplotype can produce an immune response to the same polypeptide or polypeptides, and that different patients have individualized antigenic peptides suitable for the respective HLA haplotype.

In conclusion, the antigen peptide provided by the invention has the advantages that the individuation effectiveness of the antigen peptide is verified by utilizing an immune experiment in a human body experiment, so that the blank of the individuation antigen peptide in liver cancer treatment is filled up; the antigen peptide can obviously activate the T cells of HBV specific to human body, increase the killing capacity of the T cells to HBV infected liver cancer cells, and can be used for standardized individual liver cancer immunotherapy by large-scale synthesis.

The foregoing description is only illustrative of the preferred embodiments of the present invention and is not to be construed as limiting the scope of the invention, and it will be appreciated by those skilled in the art that equivalent substitutions and obvious variations may be made using the description and illustrations of the present invention, and are intended to be included within the scope of the present invention.

Claims

1. An antigenic peptide directed against hepatitis b virus, characterized by: the amino acid sequence is shown as SEQ ID NO. 115.

2. The antigenic peptide of claim 1, further comprising an antigenic peptide as set forth in SEQ ID NO. 3, SEQ ID NO. 6, SEQ ID NO. 84, SEQ ID NO. 124.

3. The antigenic peptide of claim 1, wherein: is a polypeptide combination comprising at least two of the antigen peptides shown in the amino acid sequences SEQ ID NO. 1-124 and containing the antigen peptide shown in the amino acid sequence SEQ ID NO. 115;

and the polypeptide combination also needs to satisfy: 1) A polypeptide combination which does not comprise the polypeptide combination of claim 2, and 2) a polypeptide combination which does not comprise the antigenic peptide of the amino acid sequence shown as SEQ ID No. 23 or SEQ ID No. 79.

4. Use of the antigenic peptide of any one of claims 1 to 3 for the preparation of an immunotherapeutic agent for liver cancer infected with hepatitis b virus.

5. The use according to claim 4, wherein the immunotherapeutic agent comprises: at least one of an antigenic peptide cell therapeutic drug or vaccine.

6. The use according to claim 4, wherein the hepatitis b virus infected liver cancer is a liver cancer carrying HLA-C14:02 haplotypes.