CN117886919A - TCR for recognizing HLA-A2 restrictive hepatitis B virus HBs346-354 epitope and application thereof - Google Patents

Abstract

The invention belongs to the technical field of cellular immunotherapy, and particularly discloses a TCR for recognizing HLA-A2 restrictive hepatitis B virus HBs346-354 epitope and application thereof. The invention discloses a TCR capable of specifically recognizing and combining HLA-A2-HBs346-354 antigen complex, which can enable T cells which originally do not have antigen specific recognition capability to recognize and kill cells expressing HBV surface antigen, thereby achieving the purpose of treating HBV chronic infection or liver cancer. The invention also discloses a slow virus carrier containing TCR, a TCR-T cell capable of expressing TCR, a pharmaceutical composition taking the TCR, the slow virus carrier and the TCR-T cell as active ingredients, and application of the TCR, the slow virus carrier, the TCR-T cell and the pharmaceutical composition in preparing medicines for treating chronic hepatitis B or liver cell liver cancer caused by HBV infection.

Description

TCR for recognizing HLA-A2 restrictive hepatitis B virus HBs346-354 epitope and application thereof

Technical Field

The invention relates to the technical field of cellular immunotherapy, in particular to a TCR for recognizing HLA-A2 restrictive hepatitis B virus HBs346-354 epitope and application thereof.

Background

Hepatocellular carcinoma (Hepatocellular Carcinoma, HCC) lacks typical symptoms early, has hidden clinical manifestations, is often diagnosed in the late stages of the disease, and has a high mortality rate in the second place of cancer-related death. In asia, at least 80% of liver cancer is caused by hepatitis b virus (Hepatitis B Virus, HBV) as a major pathogenic substance, and 90% of liver cancer patients in our country are positive for HBsAg. In acute infections and disease pathologies, HBV infection can stimulate the body to produce a series of humoral and cellular immune responses for viral clearance; if the immune response of the organism is low, the immune response of the clearing virus is insufficient, HBV infection becomes a chronic process. Chronic infections can trigger immune pathologies, leading to chronic liver inflammation and fibrosis, ultimately leading to cirrhosis and/or hepatocellular carcinoma.

Currently, interferon (IFNs) and nucleotide (nucleotide) drugs are mainly used for treating HBV infection in clinic, and antagonism of viral replication is achieved through immunoregulation or interference. However, these treatments cannot completely eliminate the virus, and the use of interferon may cause various adverse reactions, and the nucleoside (nucleotide) drugs are prone to drug resistance, resulting in repeated disease. Surgical resection and liver transplantation are the most effective treatments for liver cancer, but fewer than 30% of patients who meet these surgical criteria and the long waiting time of the donor liver result in approximately 25% of patients failing to undergo liver transplantation due to tumor development. Chemotherapy and molecular targeted therapy have a certain effect on prognosis of liver cancer patients, but the postoperative recurrence rate is still up to 80% within five years. Therefore, the existing treatment means can only inhibit but not clear HBV, and the recurrence rate of patients is high, so that drug resistance is easy to generate.

Existing studies have found that naturally occurring HBV epitope-specific T cells play a decisive role in inhibiting viral replication and clearing viral infection in some subjects. However, persistent infection of chronic hepatitis B can create an intrahepatic immune tolerance environment, which in turn can lead to tolerance or depletion of HBV-specific CD8+ T cells, while leading to immune-mediated inflammatory liver injury. Reconstitution of the immune system of a patient suffering from chronic HBV infection can result in the resolution or even elimination of HBV infection. HBV-specific CD8+ T cells have been shown to play a decisive role in inhibiting viral replication and clearing viral infection. Adoptive transfer of HBV-specific TCR gene modified T cells (TCR-T) has been shown to eliminate HBV infection and reconstitute the immune system of HBV infected patients.

TCRs are T cell antigen receptors, consisting of two peptide chains, α and β, each of which is in turn divided into a variable region (V region) and a constant region (C region), which are important components of T cell immune responses, by recognizing antigenic peptides that rely on presentation of histocompatibility complex (Major histocompatibility complex, MHC) molecules, causing intracellular cell signaling and other physiological responses, thereby allowing T cells of different antigen specificities to exert immune effects on their target cells.

It has been shown that upon HBV infection, DNA can integrate into the host genome and integration frequently occurs at the early stages of infection, and this integration can trigger not only oncogenic turnover of normal hepatocytes but also the expression of HBV antigen in HCC cells. These viral proteins can be processed into viral short peptides, assembled from MHC class I molecules onto the cell surface, forming the ideal target for T cells, and are not affected by the presence of large amounts of circulating hepatitis B virus surface antigen (HBsAg) in the serum of HBV infected subjects, or circulating monocytes and B cells that can accumulate HBsAg.

Thus, HBV TCR-T cells targeting HBV-associated proteins (e.g., HBsAg, HBcAg, etc.) will more specifically attack HCC cells, and are now new ideal targets for the treatment of HCC. How to isolate, screen and identify T Cell antigen receptors (TCRs) that recognize and bind specific antigens is a key issue to be addressed in the application of such immunotherapies.

Disclosure of Invention

To solve the above problems, it is an object of the present invention to provide a reactive TCR capable of recognizing and binding to the HLA-A2-HBs346-354 antigen complex presented by MHC class I molecules. The TCR gene capable of recognizing HBV antigen is stably integrated into T cells of a patient by redirecting lymphocytes of the HBV infected patient by using an exogenous T cell receptor, so that the T cells which originally have no antigen specificity recognition capability can recognize and kill target cells expressing corresponding HBV antigen, and a personalized TCR-T cell treatment technology for recognizing HBV antigen is established, thereby achieving the aim of treating hepatitis and liver cancer.

Specifically, the TCR recognizing the epitope of HLA-A 2-restricted hepatitis B virus HBs346-354 comprises an alpha chain variable region and a beta chain variable region; the coding genes of the alpha chain variable region are shown in the 1 st-272 th positions of SEQ ID No.1, and in the SEQ ID No.1, the 76 th-96 th, 148 th-162 th and 265 th-303 th positions are respectively the coding genes of three complementarity determining regions CDR1, CDR2 and CDR 3; the coding genes of the beta chain variable region are shown in the 1 st-282 th positions of SEQ ID No.3, and in the SEQ ID No.3, the 79 th-93 th positions, the 145 th-162 th positions and the 274 th-303 th positions are respectively the coding genes of three complementarity determining regions of CDR1, CDR2 and CDR 3.

Preferably, the amino acid sequence of the alpha chain variable region is shown as SEQ ID No.2, wherein positions 26 to 32, 50 to 54 and 89 to 101 are the amino acid sequences of three complementarity determining regions CDR1, CDR2 and CDR3, respectively; the amino acid sequence of the beta chain variable region is shown as SEQ ID No.4, wherein 27-31, 49-54 and 92-101 are the amino acid sequences of three complementarity determining regions CDR1, CDR2 and CDR3 respectively.

Preferably, the TCR recognizing the epitope of HLA-A 2-restricted hepatitis B virus HBs346-354 further comprises an alpha chain constant region and a beta chain constant region, wherein the sequence of the alpha chain constant region is shown as SEQ ID No.5, and the sequence of the beta chain constant region is shown as SEQ ID No. 6.

Preferably, the screening method of the TCR for recognizing HLA-A2 restrictive hepatitis B virus HBs346-354 epitope is as follows:

step 1: target selection: screening out HBs346-354 epitope which has immunogenicity and predicts binding affinity with HLA-A 0201 to be less than 50nm through calculation biological simulation after enzyme cutting of all peptide fragments of HBV;

step 2: in vitro synthesizing HBs346-354 antigen peptide;

step 3: peripheral blood lymphocytes (PBMC) were isolated from HBV infected rehabilitation volunteer blood and PBMC HLA-a2 were screened;

step 4: in vitro stimulation and culture of HBs346-354 epitope specific T cells: incubating the isolated PBMCs with HBs346-354 polypeptide;

step 5: antigen peptide specific CTL (cytotoxic T lymphocytes) flow screen: the culture in the step 4 is centrifugally resuspended, added HBs346-354 tetramer (tetramer) and anti-CD8-BV421 antibodies are uniformly mixed and incubated, and CD8+ and tetramer+ double-positive T cells are separated and collected by using a flow cytometry, namely the CTL with the antigen peptide specificity;

step 6: targeting HBs346-354 antigen peptide TCR sequencing: sequencing single isolated antigen peptide specific CTL is realized through primer design, cDNA template preparation, nested PCR amplification and electrophoresis recovery amplification product sequencing, and antigen specific CTL alpha chains and beta chains matched with each other are screened out according to sequencing results;

step 7: affinity prediction: the binding energy of the screened TCR alpha and beta chains to the HLA-A2-HBs346-354 complex was calculated and the alpha chain and beta chain group with the lower binding energy (the smaller the binding energy, the better the affinity) was selected.

It is a second object of the present invention to provide a lentiviral vector, which is a reactive HLA-A2 restricted hepatitis B virus HBs346-354 specific TCR receptor vector, comprising an alpha chain and a beta chain of TCR, the coding gene sequence of which is shown as SEQ ID No.7, wherein positions 7-66 are SP sequences, positions 73-405 are beta chain variable region sequences, positions 415-944 are beta chain constant region sequences, positions 945-1077 are Furin and P2A sequences, positions 1084-1419 are alpha chain variable region sequences, and positions 1438-1845 are alpha chain constant region sequences.

It is a further object of the present invention to provide a TCR-T cell comprising a TCR or lentiviral vector recognizing an epitope of HLA-A 2-restricted hepatitis b virus HBs346-354 as described above, which is a receptor cell introduced by suitable means such as electrotransfection, transfection or transduction, CRISPR/Cas9 gene editing techniques, capable of expressing a TCR specifically binding to the HLA-A2-HBs346-354 complex.

It is a fourth object of the present invention to provide a pharmaceutical composition comprising an active ingredient which is the above-mentioned TCR, lentiviral vector or TCR-T cell recognizing epitope of HLA-A 2-restricted hepatitis B virus HBs 346-354.

The fifth object of the invention is to provide the application of TCR, lentiviral vector, TCR-T cell and pharmaceutical composition for recognizing HLA-A2 restrictive hepatitis B virus HBs346-354 epitope in preparing medicines for treating chronic hepatitis B or liver cell liver cancer caused by HBV infection.

The invention has the following beneficial effects:

the HBV-reactive TCRs of the present invention recognize HLA-A 2-restricted HBV epitopes, whereas about 50% of the general population expresses HLA-A2 molecules, and therefore HLA-A 2-restricted TCRs may have general therapeutic uses. The TCR screened by the invention can be used for targeting the hepatitis B virus surface antigen, has good affinity, and ensures that the T cells which originally have no antigen specificity recognition capability can recognize and kill cells expressing HBV surface antigen, thereby achieving the aim of treating HBV chronic infection or liver cancer.

The method for screening TCR can obtain a plurality of TCRs at a time, has the advantages of high screening efficiency, low screening cost and the like, and the obtained TCR can specifically identify the epitope peptide, especially when a plurality of epitope peptides are derived from the same virus or pathogenic cell, the plurality of TCRs obtained by the method can specifically identify the virus or pathogenic cell, and has the advantages of high screening efficiency and the like.

Drawings

Fig. 1: flow cytometry identified HLA-A 2-typed PBMC results in example 1.

Fig. 2: cd8+, tetramer+ double positive T cell results were collected by flow cytometry sorting in example 1.

Fig. 3: electrophoretogram of the PCR amplification products of example 1; the upper panel shows the TCR-alpha chain and the lower panel shows the TCR-beta chain.

Fig. 4: the desired fragment of the TCR lentiviral vector was mapped in example 2.

Fig. 5: fig. 2 shows lentiviral packaging effect of 293T cell transfection for 48h in the adherent state; in the figure, the left is a white light image under a microscope, and the right is a fluorescence image under the microscope.

Fig. 6: flow cytometry detection of CD8/TCR biscationic cell results in example 3; in the figure, i is a microscopic view, wherein Phase is a view without excitation light, GFP is a view under excitation light, and Merge is a combined view of Phase and GFP; ii and iii are the results of the double positive assay.

Fig. 7: the results of the cytokine secretion experiment in example 4 are shown.

Fig. 8: results of killing rates of transfected T cells in HepAD38, huh7, huh7+2 cell lines in example 4.

Fig. 9: microscopic white light image of the effect of transfected T cells in example 4 on HepAD38, huh7, huh7+2 cell lines.

Detailed Description

The invention is further described below with reference to the drawings and specific examples.

Example 1: cloning of the TCR Gene of HBs346-354 epitope-specific T cells

Step 1: target selection: all peptide fragments of HBV are subjected to enzyme digestion and then are subjected to calculation biological simulation, meanwhile, compared with results verified by experiments in the literature, the HBV peptide fragments are screened to have immunogenicity, and 8-11 peptides with binding affinity smaller than 50nm with HLA-A x 0201 are predicted to be high-risk high-reactivity antigen peptide fragments, and HBs346-354 antigen epitope (WLSLLVPFV) is finally selected through screening in the research.

Step 2: in vitro synthesis of HBs346-354 antigen peptide: 2mg of specific HBs346-354 antigen peptide was synthesized, and the purity after HPLC purification was > 95% (Nanjing Style biotechnology Co., ltd.).

Step 3: peripheral blood lymphocytes (PBMCs) were isolated from fresh blood obtained from HBV infected rehabilitation volunteers and PBMCs HLA-typed as HLA-a2 were screened: the fresh blood obtained was mixed with PBS at a ratio of 1:1, and then slowly added dropwise to the same volume of lymphocyte separation solution (Ficoll-Hypaque; GE, # 17-1440-02), PBMC were isolated by density gradient centrifugation, and phenotypes were identified by flow cytometry (as shown in FIG. 1).

Step 4: in vitro stimulation and culture of HBs346-354 epitope specific T cells: the isolated PBMC were resuspended in lymphocyte medium (stock, #T110 KJ) and 2% immune cell serum replacement (Gibco) ^TM , # A2596101), while adding HBs346-354 polypeptide at a final concentration of 1uM, stimulating at 37deg.C for 1h, washing with PBS, incubating with four times the amount of PBMC, and adding 20IU/ml interleukin 2 (IL-2; peproTech, # 200-02), 20IU/ml interleukin 15 (IL-15; peproTech, # 200-15) and CD3/CD28 beads (20 ul/106PBMC; gibco ^TM # 11132D), two rounds of stimulation were performed after 7 days of incubation in 24-well plates.

Step 5: antigen peptide specific CTL (cytotoxic T lymphocytes) flow screen: centrifugal washing at 1000rpm for 5min, discarding supernatant, re-suspending cells in PBS (containing 1% FBS), and adjusting cell concentration to 2×10 ⁷ /mL, add Flex-T ^TM HLA-A.times.02:01 Monomer UVX (BioLegend, # 280004) assembled HBs346-354 tetramer, anti-CD8-BV421 antibody (BD, # 740078), incubated at 4deg.C for 30min in the absence of light after mixing, while non-antigenic peptide stimulated cells were established as controls; flow cytometry sorting and collecting cd8+, tetramer+ double positive T cells into 96-well plates (as shown in fig. 2), collecting one cell per well, and harvesting the cells as antigen peptide specific CTLs for subsequent TCR sequencing.

Step 6: targeting HBs346-354 antigen peptide TCR sequencing: prior studies have been performed to amplify TCR a and β chains from individual cd8+ T cells (Ouyang Lichen, university of science and technology, 2013), and this experiment was intended to be used to sequence CTLs specific for individual isolated antigenic peptides by: according to the sequences of V Gene (TRAV and TRBV) and C Gene (TRAC and TRBC) fragments of all TCR alpha and beta chains provided by IMGT/Gene-DB (http:// IMGT. Cines. Fr /), 59 upstream primers are designed by using primer premier 5 software according to the primer design principle through Gene scanning and comparison, and can be paired with all known V Gene initiation sequences of TCR alpha and beta chains with a coding function, and four downstream primers are designed according to the sequences of C Gene fragments of relatively single TCR alpha and beta chains, and can be paired with the inner side or the outer side parts of C genes of the TCR alpha and beta chains respectively. Primer sequences (Shanghai Jieli Biotechnology Co., ltd.) are shown in the following tables 1-2:

table 1: primer group for amplifying TCR alpha chain

Table 2: primer group for amplifying TCR beta chain

RNA was extracted from the isolated single specific CD8+ T cells by adding reverse transcription system Mix1 (0.5. Mu.L Random primer+4.5. Mu.L DEPC water) to the isolated single CD8+ T cells (96 well plate-well), ice-bathing at 70℃for 5min, and then adding reverse transcription system Mix2 (5. Mu.L 2 Xbuffer+0.5. Mu.L RT enzyme), 5min at 25℃30min at 42℃for 5min at 85℃for reverse transcription to cDNA. Using the primer group as a template to carry out nested PCR amplification by using the designed primer group, wherein the first round of reaction procedure is carried out for 5min at 95 ℃;95℃20s,56℃20s,72℃45s,34 cycles; 7min at 72 ℃. The second round of reaction procedure was: 95 ℃ for 5min;95℃20s,56℃20s,72℃45s,34 cycles; 7min at 72 ℃. The amplification systems are respectively as follows

Tables 3-4 show:

paired TCR-alpha and TCR-beta variable region sequences can be obtained after two rounds of PCR amplification (as shown in FIG. 3, the bands of the electrophoresis are single cell amplified bands respectively). And cutting gel to recover PCR amplified product and sequencing cloned gene. In the study, 45 antigen-specific CTLs are sorted totally, wherein 14 antigen-specific CTL alpha chains and 2 antigen-specific CTL beta chains are detected, and 2 pairs of alpha chains and beta chains are paired with each other, so that 2 pairs of TCR receptor sequences are obtained. And analyzed, the 2 antigen-specific CTLs were more abundant in the α and β chain. The TCR receptor sequence screened by the method has strong tumor antigenicity and greatly saves the cost.

Step 7: affinity prediction of specific TCR α and β chains with HLA-A2-HBs346-354 complexes: the affinity of the 2 pairs of TCR alpha and beta chains screened above for the (HLA-A 2) - (HBs 346-354) complex was predicted using computational biological modeling, mainly as follows: firstly converting into protein FASTA data by using an ORF finder tool based on base sequence information; secondly, based on the FASTA sequence, predicting a composite structure by using Alpha Fold2, and selecting a structure with the highest scoring as an initial structure of molecular dynamics simulation; thirdly, using a ff14SB force field TIP3P water model to simulate 10ns, and analyzing the generated track; finally, the binding energy was calculated using MM-PBSA. The smaller the binding energy, the better the affinity. The predicted results are shown in Table 5 below, group 1 affinity is better and group 2 demonstrates that there is substantially no potential for affinity. Thus, the study selected group 1 for vector construction.

TABLE 5 calculation of affinity of biologically mimicked TCR alpha and beta chains to HLA-A2-HBs346-354 complexes

The TCR sequence composition of group 1 was identified by online alignment with the Human TCR database (IMGT/V-Quest, http:// www.imgt.org): the coding genes of the TCR alpha chain variable region are shown in the 1 st to 272 th positions of SEQ ID No.1, and in the SEQ ID No.1, the 76 th to 96 th positions, the 148 th to 162 th positions and the 265 th to 303 th positions are respectively the coding genes of three CDR1, CDR2 and CDR 3. The amino acid sequence of the alpha chain variable region is shown as SEQ ID No.2, and the 26 th to 32 th, 50 th to 54 th and 89 th positions are the amino acid sequences of three complementarity determining regions CDR1, CDR2 and CDR3 respectively. The coding genes of the beta chain variable region are shown in the 1 st-282 th positions of SEQ ID No.3, and in the SEQ ID No.3, the 79 th-93 rd, 145 th-162 th and 274 th-303 th positions are respectively the coding genes of three CDRs. The amino acid sequence of the beta-chain variable region is shown as SEQ ID No.4, wherein the 27-31 th, 49-54 th and 92-101 th positions are the amino acid sequences of three complementarity determining regions CDR1, CDR2 and CDR3, respectively.

Example 2: preparation of lentiviruses containing TCR chains

Step 8: construction of TCR viral vectors and lentiviral packaging: based on the sequences of the α chain variable region and the β chain variable region obtained as described above, the sequences of the constant regions of the α chain and the β chain of the human genome T cell receptor were obtained with reference to NCBI, the obtained sequences of the α chain constant region are shown in SEQ ID No.5 (GenBank: EU 863816.1), and the obtained sequences of the β chain constant region are shown in SEQ ID No.6 (GenBank: EU 885174.1). The α and β chain sequences of the above reactive TCRs were codon optimized using the wild type sequence and assembled into TCRs onto lentiviral vectors, each consisting of 1 α chain and 1 β chain linked at their constant domains by disulfide bonds. Each chain comprises 2 extracellular domains: the variable (V) region and the constant (C) region, and the fragment of interest construction map is shown in FIG. 4. The construction of the reactive HLA-A2 restrictive hepatitis B virus HBs346-354 specific TCR receptor vector has the target fragment complete coding gene shown in SEQ ID No.7, wherein, the 7 th to 66 th positions are SP sequences, the 73 rd to 405 th positions are beta chain variable region sequences, the 415 th to 944 th positions are beta chain constant region sequences, the 945 th to 1077 th positions are Furin and P2A sequences, the 1084 th to 1419 th positions are alpha chain variable region sequences, and the 1438 th to 1845 th positions are alpha chain constant region sequences.

Respectively carrying out slow virus packaging on the slow virus vectors which are successfully constructed, and counting 3 multiplied by 10 ⁶ 293T cells are suspended in 10ml serum-free DMEM culture medium, inoculated on a 10cm culture dish, transfected after the cell wall is more than 60% of the total area of the culture dish, and TCR lentiviral vector and lentiviral packaging plasmids PMD2.G and psPAX2 are used for 2:0.5:1.5, while adding 36. Mu.L of transfection reagent (EZ Trans, lissajous, oriental, # AC04L 092) to the 293T cells, and placing in a 5% CO2 cell incubator at 37 ℃After 6-8 hours of internal incubation, fresh DMEM medium (Hitachi Biotechnology, #41401ES76, inc. (Shanghai) containing 10% FBS) was replaced once, the virus-containing culture supernatants were collected at 48h and 72h, respectively, filtered through a 0.45 μm filter (Hitachi Biotechnology, #84310ES 03), and lentiviral concentrate (Lissajous, # AC04L 091) was concentrated and stored in aliquots at-80℃for a long period of time, and the titre was measured slightly as described above for TCR lentiviral control vector fluorescent expression, as shown in FIG. 5.

Example 3: pentameric binding ability of transfected T cells and T cells after transfection

Step 9: transfection and culture of TCR-T cells: resuscitating PBMCs of HLA-A2 phenotype, counting, and adjusting cell concentration to 5X 10 ⁵ In/ml lymphocyte culture medium (containing 10% human serum substitute +25μl CD3/28Beads +IL-15 10 ng/ml), spread in 24-well plate, 1ml lymphocyte culture medium per well, and placed at 37deg.C under 5% CO ₂ After 48h incubation in the cell incubator, the culture medium was replaced with fresh medium, transfected and recounting, concentrated viral supernatant was added at MOI=100, polybrene (Henry Biotechnology (Shanghai) Co., #HB-PB-500) was added at the same time to a final concentration of 6ug/ml, centrifuged at 1000g for 90min at room temperature, and the culture was performed in an incubator for amplification culture with half a liquid change every three days.

Step 10: amplification and identification of HBsAg-targeted TCR-T cells: culturing for the seventh day by 1×10 ⁶ Individual cells were stained for CD8 and TCR (HLA-A 2-tetramer), the proportion of CD8/TCR biscationic cells was examined by flow cytometry, the infection efficiency was identified, and the remaining cells continued to be grown in expansion. As shown in FIG. 6 (i, ii and iii), the CD8/TCR double-positive ratio was as high as 54.4% in the T-cell receptor gene pair cloned from HLA-A 2-restricted HBs346-354 epitope-reactive T cells isolated from peripheral blood derived from HBV infected rehabilitation volunteers. As can be seen, HBs346-354 can effectively activate TCR-T cells and can effectively stimulate the proliferation of TCR-T cells.

Example 4: cell killing ability of transfected T cells

Step 11: functional identification of HBsAg-targeted TCR-T cells: t2 cells are a speciesLymphoblastic, HLA-A2 positive, is commonly used to study antigen presentation processes and the mutual recognition of T cells and MHC-molecules. Culturing T2 cells to logarithmic phase, taking 1×10 ⁶ The individual cells were loaded with antigen peptide by adding HBs346-354 antigen peptide to a final concentration of 1. Mu.g/ml, incubating in an incubator for 1h, washing 3 times with HBSS, adding the same number of HBs346-354 TCR-T cells as described above, resuspending with lymphocyte medium (containing 2. Mu.g/ml Brefeldin A; division of Saint Biotech (Shanghai) #50502ES 03), and incubating overnight. After 24h, cells were collected and stained for flow (membrane rupture) as follows: anti-CD3-APC (BD, # 555335), anti-CD107a-Percp (Biolegend, # 328642), anti-IFNr-PECY7 (BD, # 557643), anti-TNFa-APC-Cy7 (Biolegend, # MAb 11).

Cytokine secretion experiments showed (fig. 7) that the secretion of CD107a (marker of TCR-T cell activation) and INF-gamma cytokines (key cytokines associated with anti-tumor immunity) was significantly up-regulated in the T cell receptor gene pairs cloned from HLA-A2 restricted HBs346-354 epitope-reactive T cells isolated from peripheral blood of HBV infected rehabilitation volunteers compared to the control group.

The TCR-T cells were co-cultured with three cell lines, hep-AD38, huh7+2, respectively, for an effective target ratio experiment, wherein Hep-AD38 was an HLA-A2 positive and HBV positive liver cancer cell line, huh7 was a liver cancer cell line, huh7+2 cells were an HLA-A2 positive liver cancer cell line. The results of the effect target ratio experiments are shown in figures 8-9, and the killing effect of the transfected T cells on the HepAD38 cell line is obviously better than that of other two control cell lines from the viewpoints of tumor killing rate and cell morphology, and meanwhile, HLA-A2 and HBV positive HepAD38 cell lines are killed by TCR-T targeting, so that the T cell receptor has good activity of killing target cells, and can also effectively clear chronic infection of HBV in vivo.

In summary, the present invention isolates and identifies a pair of HBV-specific TCR sequences, successfully constructs a vector for the pair of TCRs, and verifies in vitro that TCR transgenic positive CD8 cells (i.e., TCR-T cells) have HBV polypeptide epitope-dependent activation and proliferation capabilities; the in vitro target cell killing experiment proves that the TCR has good target cell killing activity; in vitro validation of the ability of Human TCR-T to specifically kill HLA-A2+ target cells as well; suggesting that this pair of TCR sequences may be one of the effective methods for clearing HBV infected cells.

The invention has important application value, identifies and separates the T cell for identifying HBs346-354 antigen peptide from the peripheral blood of the patient, obtains the TCR sequence carried by the T cell, redirects the T cell into the peripheral blood T cell of the patient, can establish the HBV antigen identification TCR-T cell which is derived from the patient, namely individuation, and is used for individuation hepatitis or liver cancer treatment.

The present embodiments are merely illustrative of the invention and not limiting of the invention, and any changes made by those skilled in the art after reading the specification of the invention will be protected by the patent laws within the scope of the appended claims.

Claims (8)

1. A TCR which recognizes the epitope of HLA-A 2-restricted hepatitis b virus HBs346-354, characterized in that: comprising an alpha chain variable region and a beta chain variable region;

the coding genes of the alpha chain variable region are shown in the 1 st-272 th positions of SEQ ID No.1, and in the SEQ ID No.1, the 76 th-96 th, 148 th-162 th and 265 th-303 th positions are respectively the coding genes of three complementarity determining regions CDR1, CDR2 and CDR 3;

the coding genes of the beta chain variable region are shown in the 1 st-282 th positions of SEQ ID No.3, and in the SEQ ID No.3, the 79 th-93 th positions, the 145 th-162 th positions and the 274 th-303 th positions are respectively the coding genes of three complementarity determining regions of CDR1, CDR2 and CDR 3.

2. The TCR which recognizes HLA-A 2-restricted hepatitis b virus HBs346-354 epitope according to claim 1, characterized in that: the amino acid sequence of the alpha chain variable region is shown as SEQ ID No.2, wherein the 26 th to 32 th positions, the 50 th to 54 th positions and the 89 th to 101 th positions are the amino acid sequences of three complementarity determining regions CDR1, CDR2 and CDR3 respectively;

the amino acid sequence of the beta chain variable region is shown as SEQ ID No.4, wherein 27-31, 49-54 and 92-101 are the amino acid sequences of three complementarity determining regions CDR1, CDR2 and CDR3 respectively.

3. The TCR which recognizes HLA-A 2-restricted hepatitis b virus HBs346-354 epitope according to claim 1, characterized in that: also comprises an alpha chain constant region and a beta chain constant region, wherein the sequence of the alpha chain constant region is shown as SEQ ID No.5, and the sequence of the beta chain constant region is shown as SEQ ID No. 6.

4. The TCR recognizing the epitope of HLA-A 2-restricted hepatitis B virus HBs346-354 of claim 1,

the method is characterized in that: the screening method comprises the following steps:

step 1: target selection: screening out HBs346-354 epitope which has immunogenicity and predicts binding affinity with HLA-A 0201 to be less than 50nm through calculation biological simulation after enzyme cutting of all peptide fragments of HBV;

step 2: in vitro synthesizing HBs346-354 antigen peptide;

step 3: peripheral blood lymphocytes were isolated from HBV infected rehabilitation volunteer blood and PBMCs HLA-typed as HLA-a2 were screened;

step 4: in vitro stimulation and culture of HBs346-354 epitope specific T cells: incubating the isolated PBMCs with HBs346-354 polypeptide;

step 5: antigen peptide specific CTL flow screen: the culture in the step 4 is centrifugally resuspended, HBs346-354 tetramer and anti-CD8-BV421 antibody are added, evenly mixed and incubated, and CD8+ and tetramer+ double-positive T cells are separated and collected by utilizing a flow cytometry, so that antigen peptide specific CTL is obtained;

step 6: targeting HBs346-354 antigen peptide TCR sequencing: sequencing single isolated antigen peptide specific CTL is realized through primer design, cDNA template preparation, nested PCR amplification and electrophoresis recovery amplification product sequencing, and antigen specific CTL alpha chains and beta chains matched with each other are screened out according to sequencing results;

step 7: affinity prediction: the binding energy of the screened TCR alpha and beta chains to the HLA-A2-HBs346-354 complex was calculated and the alpha chain and beta chain group with the small binding energy was selected.

5. A lentiviral vector, characterized in that: the specific TCR receptor vector for the reactive HLA-A2 restrictive hepatitis B virus HBs346-354 has the coding gene sequence shown in SEQ ID No.7, wherein the 7 th to 66 th positions are SP sequences, the 73 rd to 405 th positions are beta-chain variable region sequences, the 415 th to 944 th positions are beta-chain constant region sequences, the 945 th to 1077 th positions are Furin and P2A sequences, the 1084 th to 1419 th positions are alpha-chain variable region sequences, and the 1438 th to 1845 th positions are alpha-chain constant region sequences.

6. A TCR-T cell, comprising: a TCR comprising an epitope recognizing HLA-A 2-restricted hepatitis b virus HBs346-354 according to any one of claims 1 to 4 or a lentiviral vector according to claim 5.

7. A pharmaceutical composition characterized by: comprising an active ingredient which is a TCR which recognizes the epitope of HLA-A 2-restricted hepatitis b virus HBs346-354 as defined in any one of claims 1 to 4, a lentiviral vector as defined in claim 5 or a TCR-T cell as defined in claim 6.

8. Use of a TCR which recognizes an epitope of HLA-A 2-restricted hepatitis b virus HBs346-354 according to any one of claims 1-4, a lentiviral vector according to claim 5, a TCR-T cell according to claim 6 or a pharmaceutical composition according to claim 7 in the manufacture of a medicament for the treatment of chronic hepatitis b or hepatocellular carcinoma resulting from HBV infection.