Modified immune cell targeting hepatitis B surface antigen and medical application thereof

Abstract

The invention provides a modified immune cell which comprises a T cell receptor (TCR) capable of specifically targeting hepatitis B surface antigen (HBsAg) and a chimeric transformation receptor, the chimeric transformation receptor adopts an intracellular activation signal domain of a co-stimulatory molecule to replace an intracellular suppression signal domain of an immunosuppression receptor, and the chimeric transformation receptor can specifically target the hepatitis B surface antigen (HBsAg). An inhibitory signal induced by binding of an immunosuppressive receptor and a ligand is converted into an activation signal. The invention also provides a nucleic acid molecule for coding the TCR and / or coding the chimeric transformation receptor, a carrier containing the nucleic acid molecule, and application of the nucleic acid molecule in drugs for preventing and / or treating HBV infection and related diseases thereof.

Description

[0001] Modified immune cells targeting hepatitis B surface antigen and their medical uses Technical field This application relates to the field of immunotherapy technology, and more specifically to a modified immune cell targeting hepatitis B surface antigen (HBsAg) and its medical uses.

[0002] 1.1 Global prevalence of HBV

[0003] HBV is a non-cytopathic, double-stranded DNA virus primarily transmitted through blood, mother-to-child transmission, and sexual transmission. Despite the availability of vaccines and highly effective direct-acting antiviral drugs, HBV continues to pose a significant burden on the global population. In 2015, the number of people living with HBV worldwide reached 257 million, with over 884,000 deaths resulting from complications of chronic HBV infection, equivalent to one death every 36 seconds. Epidemiological studies estimate that HBV infection rates are higher in Asian and African countries compared to those in the Americas or Europe.

[0004] 1.2 The most common HBV genotypes infecting Asians

[0005] HBV is prone to mutation, producing different genotypes. Ten different HBV genotypes are reported worldwide. In Asian countries, HBV genotypes are primarily subtypes B and C. HBV genotypes are significantly associated with prognostic factors such as liver disease progression, risk of cirrhosis, and development of hepatocellular carcinoma (HCC). Researchers are focusing on the prevalence of different genotypes in different regions. For example, genotypes C and D are more likely to develop cirrhosis and HCC than other genotypes. Human leukocyte antigen (HLA) is the most polymorphic gene complex in the human genome. Genotypes include HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1. Studies have found that HLA-A alleles play a more important role in the Han Chinese population than other HLA genes. The most common HLA-A*02 subtypes in Asia are *02:01, *02:06, and *02:07.

[0006] 1.3 HCC and Its Carcinogenic Pathways Hepatocellular carcinoma (HCC) is a fatal, long-term complication of chronic hepatitis infection, most commonly occurring in Asians. In 2017, HCC caused approximately 470,000 deaths. The molecular pathology of HCC is a complex process involving multiple molecular aberrations and genetic mutations, ultimately leading to malignancy and HCC progression.

[0007] HBV-DNA integration is found in 80-90% of HBV-related hepatocellular carcinomas. HBV-DNA integration plays a crucial role in the development and progression of HBV-related HCC. First, HBV-DNA contains genes associated with liver cancer development, such as those encoding telomerase reverse transcriptase, lysine methyltransferase 2B, and cyclin A2. Second, when HBV integrates into the human genome, it increases genomic instability by affecting gene expression at the insertion site, thereby activating certain oncogenic pathways, such as phosphoinositide 3-kinase / Akt, myc, Wnt / R-catenin, c-Met, and hedgehog. Studies have shown that activation of Akt signaling inhibits transforming growth factor (TGF)-induced apoptosis. Molecular changes caused by HBV-DNA integration can also damage DNA checkpoints and contribute to tumor formation in cirrhotic livers. These changes include loss of function of the p53 tumor suppressor gene, inactivation of the p27 cell cycle regulator, loss of heterozygosity at the insulin-like growth inhibitor 2 receptor locus, and loss of expression of the p16 cell cycle inhibitor protein. In summary, HBV triggers changes in gene expression in the liver, conferring neoplastic characteristics on cells. These changes involve alterations in DNA methylation, changes in mRNA expression profiles, and the combined activation of numerous signal transduction pathways. The primary mechanism for inducing liver cancer is mediated by the immune response. The virus exploits the oxidative environment created by the immune response in the liver to constitutively activate signaling pathways, blocking apoptosis, promoting cell survival, and supporting HBV replication, thereby promoting viral persistence. Simultaneously, the virus may suppress or block innate immunity, thereby affecting the development of adaptive immune responses. This chronic, persistent immune response manifests pathologically as hepatitis and long-term fibrosis, ultimately leading to cirrhosis and liver cancer. Integrated HBV DNA contains an envelope protein with a complete open reading frame (ORF), which serves as a template for the production of HBs protein. Transcription levels of integrated HBV DNA in the HCC cell genome are positively correlated with HBs protein expression levels. HBs protein is also expressed in HBV-related HCC metastases. Studies have shown that when the complete HBs protein ORF is integrated into the host genome, HBs protein can be detected in plasma samples and HCC tumor tissue, but not in adjacent liver tissue. This suggests that these HBs proteins are not caused by infection, but rather originate from HBV genes that become embedded in the host cell genome during the development and progression of liver cancer. In other words, HCC cells continue to express HBs protein even in the absence of HBV infection. Antiviral drugs have no inhibitory or downregulatory effect on proteins expressed by these integrated HBV genes. Consequently, HCC patients can still express positive HBs protein even under antiviral treatment.

[0008] 1.4 Current Treatment of HCC: Currently, surgical resection and liver transplantation are the most effective treatments for HCC. However, less than 30% of HCC patients meet these surgical criteria, and the 5-year postoperative recurrence rate for eligible patients is as high as 80%. Furthermore, the waiting time for donor livers is long, and the number of patients waiting for transplantation continues to increase, resulting in nearly 25% of patients ultimately being unable to undergo transplantation due to tumor progression. The most commonly used conservative treatment is transcatheter arterial chemoembolization (TACE), but this therapy is contraindicated in patients with advanced cirrhosis and decompensated liver function. Furthermore, embolization-related ischemic injury can lead to increased ascites and potentially death. Currently, the FDA-approved targeted drugs for the treatment of HCC are sorafenib and regorafenib, both tyrosine kinase inhibitors. Sorafenib has a median overall survival (mOS) of 10.7 months and a median time to disease progression of 5.5 months in previously untreated patients with advanced HCC. In a clinical study of regorafenib in second-line advanced HCC patients who had failed first-line sorafenib, the median OS (median progression-free survival) was 10.6 months, the median PFS (median progression-free survival) was 3.1 months, and the objective response rate (ORR) was 11%. Furthermore, clinical trial data in patients with advanced HCC suggest that sorafenib and regorafenib are associated with an increased risk of bleeding and arterial and venous thrombosis, and their survival advantage in the indicated population is only 2-3 months. Over time, patients are more likely to develop disease progression. Furthermore, a phase III clinical trial of apatinib in patients with advanced HCC who had failed or were intolerant to at least one previous line of systemic therapy showed a median OS (median progression-free survival) of 8.7 months, a median PFS of 4.5 months, and an ORR of 10.7%. These survival data demonstrate the limited efficacy of current targeted drugs. In recent years, significant breakthroughs have been made in immunotherapy research for HCC. The role of immunotherapy in the first-line treatment of HCC is supported by relevant clinical studies. Results from the Phase III IMbravel50 study of first-line immunotherapy combination therapy showed that compared with the standard treatment of sorafenib alone, the combination of atezolizumab and bevacizumab (T+A) significantly reduced the risk of death and disease progression. However, the ORR (RECIST v1.1) was only 27.3%, and the mean progression-free survival (MPFS) was only 6.8 months. Among Chinese patients with advanced HCC, who have a worse prognosis, the mPFS was only 5.7 months.Furthermore, the evidence for PD-1 in second-line HCC treatment was confirmed in the CheckMate-040 study. PD-1 monotherapy in patients with advanced HCC without HCV or HBV infection resulted in an ORR of 15-20%, with a median OS of 28.6 months in subjects who had not received sorafenib, and 15.6 months in those who had received sorafenib. These data indicate that immunotherapy for HCC has made significant progress compared to previous treatments, but its efficacy remains unsatisfactory and warrants further study. Chemotherapy generally does not offer a significant survival advantage for HCC patients. Although several chemotherapy combination therapies are currently under development, such as PIAF (cisplatin, interferon, doxorubicin, and 5-fluorouracil), GEMOX (gemcitabine and oxaliplatin), and FOLFOX4 (fluorouracil, leucovorin, and oxaliplatin infusion), clinical trials have yet to be successful, and the results are incomplete.

[0009] Patients with HBV-related HCC require antiviral therapy to suppress viral replication, reduce viral load, and improve cirrhosis prognosis. Furthermore, because anticancer therapy can reactivate HBV, the 2020 CSCO Guidelines for the Diagnosis and Treatment of Primary Liver Cancer emphasize the importance of prioritizing underlying liver disease and recommend the use of antiviral therapy throughout HCC diagnosis, treatment, and clinical research. Entecavir and tenofovir are recommended for prophylactic antiviral therapy. According to the CSCO 2020 Guidelines for the Second-Line Treatment of Advanced HCC, Level I Experts recommend regorafenib and apatinib (with Class IA evidence) and PD-1 monoclonal antibodies (with Class 2A evidence), but these have not demonstrated significant advantages in current clinical trials. In summary, given the limitations and risks of currently available treatments and drugs, new therapeutic strategies are needed to provide more treatment options for patients with HBV-related HCC.

[0010] 1.5 Role of T cells in immunotherapy of HBV-related HCC and the rationale for using TCR T cells

[0011] T cells are immune cells that originate from the bone marrow and lymph nodes and mature in the thymus. They express T cell antigen receptors (TCRs) on their surface and play a crucial role in cell-mediated immunity, clearing infections and cancer cells. TCRs recognize and specifically bind to target antigen epitopes presented by major histocompatibility complex (MHC) molecules. Once a T cell recognizes its target, it kills the target cell through massive proliferation, cytokine release, and cytotoxicity. Adoptive T cell immunotherapy is being explored to treat human malignancies (such as leukemia) and viral diseases (such as cytomegalovirus (CMV) and Epstein-Barr virus (EBV)). However, isolating and amplifying virus- or tumor-specific T cells from patient blood is difficult and time-consuming. Therefore, researchers have adopted novel therapeutic strategies. By introducing antigen-specific T cell receptors (TCRs) or TCRα / β heterodimers, these gene-edited T lymphocytes are primed to target specific viral or tumor antigens, conferring precise antigenic specificity. The application of TCR-T cells to solid tumors, particularly HBV-related HCC, has high clinical value and significance. However, understanding the process and mechanism of HBV HBs protein expression is crucial to clarifying the key to TCR-T action.

[0012] TCR-T cell therapy relies on the presentation of MHC molecules (major histocompatibility complex) to recognize targets and activate T cells. These cells can recognize intracellular antigen fragments presented by MHC molecules. This characteristic allows TCR-T cell therapy to target a wider range of targets, including intracellular antigens, cell surface antigens, and neoantigens produced by tumor cell mutations. This overcomes the differences in antigen expression on malignant tumor cells caused by tumor heterogeneity, demonstrating greater therapeutic value. Furthermore, clinical evidence demonstrates that adoptive immunotherapy with HBV-specific T cells can control HBV replication or tumor growth. Leukemia patients can even acquire HBV immunity after receiving bone marrow transplantation from donors with specific HBV immunity (either from HBV vaccination or through autoimmunity). Similarly, transplanting an HBV-positive liver into a recipient with specific HBV immunity can eliminate HBV infection in the transplanted liver. Studies have shown that gene-edited T cells expressing HBV-specific T cell receptors (TCRs) can recognize HBV-related HCC cells in vivo, become activated, and expand. This reduces HBs protein levels without exacerbating liver inflammation or other toxicities. Therefore, HBs protein is a promising potential therapeutic target for HBV-related HCC. Furthermore, T cell exhaustion contributes to the poor efficacy of cell therapy in solid tumors. T cell exhaustion is common in malignant tumors and is a result of T cell dysfunction caused by long-term exposure to persistent antigenic stimulation. This condition, characterized by defective production of tumor necrosis factor (TNF) and interferon-γ (IFN-γ), is also a mechanism for malignant tumor cells to evade immune responses. Furthermore, in patients with HBV-related HCC, HBV-specific T cell immunity is often severely suppressed. In these patients, HBV-specific T cells are functionally deficient, scarce, and prone to exhaustion. Due to these functional defects, ex vivo analysis of patient blood rarely detects HBV-specific T cells. Therefore, developing specific TCRs targeting HBV surface antigens through genetic engineering and other biological approaches, transfecting these TCRs into isolated patient T cells in vitro, and then constructing high-affinity TCR-T cells for transfusion therapy has become a new option.However, currently, there is no proven safe and effective TCR-T cell for treating HBV-related diseases, particularly HBV-induced liver cancer. Furthermore, in solid tumor treatment trials, TCR-T cells are highly susceptible to TCR-T cell exhaustion due to immunosuppression in the tumor microenvironment, resulting in poor anti-tumor efficacy. SUMMARY OF THE INVENTION In response to existing technical issues, the present application provides a modified immune cell that specifically targets hepatitis B surface antigen (HBsAg) and is capable of overcoming immune cell exhaustion caused by immunosuppression in the tumor microenvironment. It is capable of specifically killing liver cancer cells induced by HBV infection, thereby treating HBV-induced liver cancer. In a first aspect, the present application provides a modified immune cell comprising a T cell receptor (HBV TCR) targeting hepatitis B surface antigen and a chimeric switch receptor; the chimeric switch receptor comprises the extracellular domain (ECD) of an immunosuppressive receptor and the intracellular domain (ICD) of a costimulatory molecule that mediates immune cell activation signals. Binding of the ECD to its ligand produces immune cell activation signals, rather than immune cell inactivation signals, in the modified immune cell. In some embodiments, the TCR comprises a TCR alpha chain variable region and a TCR rr chain variable region; wherein the amino acid sequence of aCDR3 in the TCR alpha chain variable region is as shown in SEQ ID NO:3, or a variant thereof, in which one or two amino acids are replaced by other amino acids; and the amino acid sequence of RCDR3 in the TCR rr chain variable region is as shown in SEQ ID NO:6, or a variant thereof, in which one or two amino acids are replaced by other amino acids. In some embodiments, the TCR alpha chain variable region comprises complementarity determining regions aCDR1, aCDR2, and aCDR3, and the TCR β chain variable region comprises complementarity determining regions pCDRK, RCDR2, and pCDR3, wherein: aCDR1, aCDR2, and aCDR3 are as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; pCDRK, RCDR2, and pCDR3 are as shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively; or CDR variants in which one or two amino acids in one or more CDRs are replaced by other amino acids. In some embodiments, the TCR α chain variable region comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 7; in some embodiments, the TCR β chain variable region comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 8.In some embodiments, the immunosuppressive receptor is selected from any one of PD1, CTLA4, BTLA, TIM3, TIGIT, TGFβ receptor, and any other protein with immunosuppressive function or associated with an immunosuppressive signaling pathway, and combinations thereof. The ECD of the immunosuppressive receptor may have at least one amino acid mutation. In some embodiments, the ECD of the present application is PD1 ECD; in some embodiments, the PD1 ECD sequence may have at least one amino acid mutation; in some embodiments, the PD1 ECD has an amino acid mutation, where alanine at position 132 is mutated to leucine, which increases the affinity of the PD1 ECD for PDL1. The ECD amino acid sequence is set forth in SEQ ID NO:9. In some embodiments, the costimulatory molecule comprises CD28, 4-1BB, ICOS, CD27, IL-12R, CD3, OX40, and combinations thereof. The ICD of the above-mentioned costimulatory molecule may have at least one amino acid mutation. In some embodiments, the ICD of the present application is CD28 ICD; in some embodiments, the ICD of the present application is 4-1BB ICD; in some embodiments, the CD28 ICD sequence may have at least one amino acid mutation; in some embodiments, the 4-1BB ICD sequence may have at least one amino acid mutation; in some embodiments, the CD28 ICD amino acid sequence is as shown in SEQ ID NO: 12; in some embodiments, the 4-1BB ICD amino acid sequence is as shown in SEQ ID NO: 13. In some embodiments, the ECD and the ICD are connected by a transmembrane region (TM) sequence; in some embodiments, the transmembrane region comprises a transmembrane domain of a protein selected from the group consisting of: α, R, or β chains of T cell receptors, CD28, CD3s, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154, and combinations thereof, wherein the transmembrane domains of the foregoing proteins may have at least one amino acid mutation. In some embodiments, the transmembrane region sequence is a CD8 transmembrane region sequence or a CD28 transmembrane region sequence; in some embodiments, the CD8 transmembrane region sequence is as shown in SEQ ID NO: 10; in some embodiments, the CD28 transmembrane region sequence is as shown in SEQ ID NO: 11.In some embodiments, the chimeric switch receptor is PD1 (ECD)-CD8 (TM)-4-1BB (ICD) (PD1-BB); in some embodiments, the PD1-BB amino acid sequence is as shown in SEQ ID NO: 15; in some embodiments, the chimeric switch receptor is PD1 (ECD)-CD28 (TM)-CD28 (ICD) (PD1-28); in some embodiments, the PD1-28 amino acid sequence is as shown in SEQ ID NO: 16. In some embodiments, the immune cell is selected from lymphocytes, dendritic cells, monocytes, macrophages, granulocytes, and mast cells; in some embodiments, the cell is a T cell. The second aspect of this application provides a nucleic acid molecule comprising a nucleic acid sequence encoding the TCR molecule described in the first aspect of this application; and / or comprising a nucleic acid sequence encoding the chimeric switch receptor described in the first aspect of this application. The third aspect of this application provides a vector, wherein the vector comprises the nucleic acid molecule described in the second aspect of this application. The fourth aspect of this application provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and the modified immune cells described in the first aspect of this application, the nucleic acid molecules described in the second aspect of this application, and the vector described in the third aspect of this application. The fourth aspect of this application provides the use of the modified immune cells described in the first aspect of this application, the nucleic acid molecules described in the second aspect of this application, the vector described in the third aspect of this application, or the pharmaceutical composition described in the fourth aspect of this application in the preparation of a medicament for preventing or treating diseases related to HBV infection; the HBV infection-related diseases include one or more of hepatitis, liver fibrosis, cirrhosis, and liver cancer. Advantageous effects of this application: In one aspect, this application isolates HBV-specific T lymphocytes from donors who have cleared HBV infection through autoimmunity, and further establishes a hepatitis B surface antigen-specific TCR library based on these T lymphocytes. The construction of HBV TCR T cells is based on the results of screening TCR molecules in this library. The modified immune cells constructed can specifically recognize and lyse HBsAg-positive HCC cells.On the other hand, the modified immune cells of the present application express not only a TCR-T targeting HBsAg but also a chimeric switch receptor that highly competitively binds to the PD-L1 molecule on tumor cells, converting the PD-1 / PD-L1 inhibitory signal into an immune cell activation signal. This effectively blocks cell exhaustion induced by native PD-1 molecules on the T cell surface, reduces TCR-T cell dysfunction in an immunosuppressive microenvironment, and increases cytokine release, cell proliferation, and tumor-killing capacity. Furthermore, these cells are expected to overcome a series of difficult challenges currently faced by adoptive T cell immunotherapy for liver cancer, such as tumor heterogeneity, difficulty penetrating the tumor barrier, short duration of action, and a local immunosuppressive microenvironment. These cells hold great promise for application in the field of cellular immunotherapy for solid tumors. On the other hand, in vitro and in vivo experiments have shown that the modified immune cells of the present application enhance the therapeutic efficacy of TCR-T cells while not producing non-specific effects on antigen-negative, PD-L1-positive cells. Their side effects and safety are superior to the combination of PD1 / PD-L1 antibodies and TCR-T cells. BRIEF DESCRIPTION OF THE DRAWINGS To more clearly illustrate the technical solutions in the embodiments of the present application, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below represent only some embodiments of the present application. Those skilled in the art can derive other drawings based on these drawings without inventive effort.Figure 1 is a schematic diagram of the mechanism of simultaneous expression of HBV TCR and chimeric conversion receptor; Figure 2 is a diagram of the structural sequence of simultaneous expression of HBV TCR and chimeric conversion receptor; Figure 3 is the titer of HBV TCR, HBV TCR-PD1-BB, HBV TCR-PD1-28 lentivirus and T cell MOI; Figure 4 is the expression of HBV TCR, HBV TCR-PD1-BB, HBV TCR-PD1-28 on T cells; Figure 5 is the killing of target cells and cytokines by HBV TCR, HBV TCR-PD1-BB, HBV TCR-PD1-28; Figure 6 is a multi-round tumor stimulation killing experiment of HBV TCR, HBV TCR-PD1-BB, HBV TCR-PD1-28; Figure 7 is a multi-round tumor stimulation cytokine expression of HBV TCR, HBV TCR-PD1-BB, HBV TCR-PD1-28; Figure 8 is a multi-round tumor stimulation cytokine expression of HBV TCR-PD1-BB and HBV TCR killing of various target cells and cytokine function; Figure 9 shows the proliferation capacity of HBV TCR-PD1-BB and HBV TCR target cells stimulated over multiple rounds; Figure 10 shows the in vivo efficacy, body weight, and body distribution of HBV TCR-PD1-BB in an immunodeficient mouse HBsAg+PDL1+ xenograft tumor model; Figure 11 shows a comparison of the long-term in vivo efficacy of HBV TCR-PD1-BB and HBV TCR-T in an immunodeficient mouse HBsAg+PDL1+ xenograft tumor model. DETAILED DESCRIPTION To facilitate understanding of the present invention, certain technical and scientific terms are explained before describing the examples. Unless otherwise clearly defined elsewhere in the present application, all other technical and scientific terms used herein have the meanings commonly understood by persons of ordinary skill in the art to which this invention belongs. The term "hepatitis B surface antigen T cell receptor (TCR)" is defined herein as a TCR that binds to hepatitis B surface antigen in the context of major histocompatibility complex (MHC) molecules, thereby inducing a helper or cytotoxic response in cells expressing the recombinant TCR. The TCR or fragment thereof of the present application is capable of binding to a hepatitis B surface antigen polypeptide presented by HLA-A*02. Specifically, the hepatitis B surface antigen may be HBs20-28, which are used interchangeably herein as HBs20, HBs20-28, S20-28, and S20. Unless otherwise specified, these refer to the S20-28 antigen of genotypes A and D, i.e., the FLLTRILTI polypeptide. In some embodiments, the hepatitis B surface antigen is HBV genotypes A and D; in some embodiments, the hepatitis B surface antigen is HBV genotypes B and C.In some embodiments, the hepatitis B surface antigen comprises the amino acid sequence of FLLTRILTI (SEQ ID NO: 19); in some embodiments, the hepatitis B surface antigen comprises the amino acid sequence of FLLTKILTI (SEQ ID NO: 20). In some embodiments, the hepatitis B surface antigen is the amino acid sequence of FLLTRILTI (SEQ ID NO: 19); in some embodiments, the hepatitis B surface antigen is the amino acid sequence of FLLTKILTI (SEQ ID NO: 20). The term "MHC molecule" refers to a protein of the immunoglobulin superfamily, which can be either class I or class II MHC molecules. Therefore, it is specific for antigen presentation. Different individuals have different MHCs, capable of presenting different short peptides from a protein antigen on the surface of their respective APCs. The human MHC is commonly referred to as the HLA gene or HLA complex.

[0013] TCRs are glycoproteins found on the cell membrane, consisting of either α-chains (small chains) or γ-chains (small chains) as heterodimers. In 95% of T cells, TCR heterodimers consist of α and β chains, while 5% have TCRs composed of γ and β chains. Natural β heterodimeric TCRs have α chains and β chains, which together constitute the subunits of the β heterodimeric TCR. The α and β chains each contain a variable region and a constant region. Each variable region comprises three complementarity-determining regions (CDRs): CDR1, CDR2, and CDR3, embedded within a framework. The CDR regions of the α and β chains of the TCRs in this application are delineated using the IMGT numbering convention. The CDR regions determine TCR binding to the pMHC complex. The sequence of the TCR constant domain can be found in the public database of the International Immunogenetics Information System (IMGT). For example, the constant domain sequence of the TCR molecule α chain is "TRAC*01," and the constant domain sequence of the TCR molecule R chain is "TRBC1*01" or "TRBC2*01." In this application, the terms "T cell receptor," "TCR," and "TCR molecule" are used interchangeably.

[0014] TCR Molecules: The TCRs or fragments of the present application recognize HLA-A2-restricted hepatitis B surface antigen. Approximately 50% of the population express the MHC class I molecule HLA-A*02. Therefore, HLA-A*02-restricted TCRs may have widespread therapeutic applications. Specifically, the TCRs of the present application can recognize products of many HLA-A*02 alleles, including HLA-A*0201, *0202, *0203, *0206, and *0207. Although HLA subtypes can differ significantly between Caucasians and Asians, over 95% of HLA-A2-positive Caucasians are HLA-A0201, while HLA-A2-positive Chinese individuals are composed of the following HLA-A2 subtypes: 23% HLA-A0201; 45% HLA-A0207; 8% HLA-A0206; and 23% HLA-A0203. In some embodiments, the TCR comprises a TCR α chain variable region and a TCR β chain variable region, and the TCR α and β chains each have three complementarity determining regions (CDRs). In some embodiments, the amino acid sequence of the aCDR3 of the TCR α chain variable region is as shown in SEQ ID NO: 3, or a variant thereof, in which one or two amino acids are replaced by other amino acids; and / or the amino acid sequence of the RCDR3 of the TCR β chain variable region is as shown in SEQ ID NO: 6, or a variant thereof, in which one or two amino acids are replaced by other amino acids. In some embodiments, the aCDR1, aCDR2, and aCDR3 are as shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively; and / or the pCDR1, RCDR2, and RCDR3 are as shown in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively. The CDR region amino acid sequences described herein can be embedded into any suitable framework structure to create a chimeric TCR. As long as the framework structure is compatible with the CDR regions of the TCR described herein, those skilled in the art can design or synthesize TCR molecules with corresponding functions based on the CDR regions disclosed herein. Therefore, the TCR molecules described herein refer to TCR molecules comprising the aforementioned alpha and / or alpha-linked CDR region sequences and any suitable framework structure.The variable region of the TCR α chain of the present application is an amino acid sequence having at least 90%, preferably 95%, and more preferably 98% sequence identity with SEQ ID NO: 7; and / or the variable region of the TCR RR chain of the present application is an amino acid sequence having at least 90%, preferably 95%, and more preferably 98% sequence identity with SEQ ID NO: 8. In some embodiments, the TCR is a β heterodimer comprising a TCR α chain constant domain and a TCR RR chain constant domain. In some embodiments, the constant domains of the TCR molecules of the present application are human constant domains. Those skilled in the art are aware of or can obtain human constant domain amino acid sequences by consulting relevant books or the public database of IMGT (International Immunogenetics Information System). For example, the constant region sequence of the α chain of the TCR molecule of the present invention can be "TRAC*01", and the constant region sequence of the R chain of the TCR molecule can be "TRBC1*01" or "TRBC2*01". ,,In some embodiments, an additional disulfide bond is introduced into the constant region to improve stability and reduce mispairing between exogenously transferred TCR molecules and endogenous TCR molecules. The constant region of the TCR molecules of the present application can also be mouse constant region. Simultaneously replacing TRAC and TRBC with mouse constant regions can prevent mispairing between exogenously transferred TCR molecules and endogenous TCR molecules, which can lead to TCR targeting errors. This effect is similar to the purpose of exogenously introducing artificial disulfide bonds. Chimeric switch receptors of the present application comprise: an extracellular domain (ECD) of an immunosuppressive receptor, wherein the ECD is fused to the intracellular domain (ICD) of a costimulatory molecule that mediates immune cell activation signals; wherein the extracellular domain of the immunosuppressive receptor binds to its ligand to generate an immune cell activation signal rather than an immune cell inactivation signal in the modified immune cell. The chimeric switch receptor converts the inactivation signal mediated by the immunosuppressive receptor into an immune cell activation signal, thereby further enhancing the immune activity of the modified immune cell. Immunosuppressive receptors include any one of PD1, CTLA4, BTLA, TIM3, TIGIT, TGβ receptors, and any other proteins with immunosuppressive functions or associated with immunosuppressive signaling pathways, and combinations thereof, and may contain at least one amino acid mutation. In some embodiments, the ECD of the present application is the PD1 ECD; in some embodiments, the PD1 ECD sequence may contain at least one amino acid mutation; in some embodiments, the PD1 ECD contains an amino acid mutation, where alanine at position 132 is mutated to leucine, increasing the affinity of the PD1 ECD for PDL1. The amino acid sequence of the mutated ECD is shown in SEQ ID NO:9. Co-stimulatory molecules of the present application include CD28, 4-1BB, ICOS, CD27, IL-12R, CD3, OX40, and combinations thereof. The ICDs of the aforementioned co-stimulatory molecules may contain at least one amino acid mutation. In some embodiments, the ICD of the present application is CD28 ICD; in some embodiments, the ICD of the present application is 4-1BB ICD; in some embodiments, the CD28 ICD sequence may have at least one amino acid mutation; in some embodiments, the 4-1BB ICD sequence may have at least one amino acid mutation; in some embodiments, the CD28 ICD amino acid sequence is as shown in SEQ ID NO: 12; in some embodiments, the 4-1BB ICD amino acid sequence is as shown in SEQ ID NO: 13.In some embodiments, the ECD and the ICD are connected by a transmembrane region (TM) sequence; the transmembrane region comprises a transmembrane domain of a protein selected from the group consisting of: α, R, or β chains of T cell receptors, CD28, CD3α, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154, and combinations thereof, wherein the transmembrane domain of the above proteins may have at least one amino acid mutation. In some embodiments, the transmembrane region sequence is a CD8 transmembrane region sequence or a CD28 transmembrane region sequence; in some embodiments, the CD8 transmembrane region sequence is as shown in SEQ ID NO: 10; in some embodiments, the CD28 transmembrane region sequence is as shown in SEQ ID NO: 11. In some embodiments, the chimeric switch receptor is PD1 (CD)-CD8 (TM)-4-1BB (ICD) (abbreviated as PD1-BB); in some embodiments, the PD1-BB amino acid sequence is as shown in SEQ ID NO: 15; in some embodiments, the chimeric switch receptor is PD1 (CD)-CD28 (TM)-CD28 (ICD) (abbreviated as PD1-28); in some embodiments, the PD1-28 amino acid sequence is as shown in SEQ ID NO: 16. Nucleic Acid Molecules The nucleic acid molecules of the present application comprise a nucleic acid sequence encoding a TCR molecule targeting hepatitis B surface antigen of the present application; and / or comprise a nucleic acid sequence encoding a chimeric switch receptor of the present application. The present application provides nucleic acid molecules encoding the TCR molecules described herein or fragments thereof, wherein the fragments may be one or more CDRs, variable regions of the alpha and / or alpha chains, and alpha and / or beta chains. In some embodiments, the nucleic acid encodes one or more structural features for increasing and / or stabilizing the association between the expressed TCR alpha and beta chains. In some embodiments, the features may be specific amino acids or amino acid sequences. In some embodiments, the nucleic acid may encode one or more non-natural cysteine ​​residues for forming one or more disulfide bonds between the TCR alpha and beta chains. In some embodiments, the nucleic acid may encode one or more non-native cysteine ​​residues in the constant domains of the TCR α and β. The nucleotide sequence of the nucleic acid molecule of the present application may be single-stranded or double-stranded, may be RNA or DNA, and may or may not contain introns. Preferably, the nucleotide sequence of the nucleic acid molecule of the present application does not contain introns but is capable of encoding the TCR of the present application and / or the chimeric switch receptor of the present application.The nucleotide sequence may be codon-optimized. Different cells vary in their specific codon usage, and codon usage in the sequence can be altered to increase expression based on the cell type. Codon usage tables for mammalian cells and various other organisms are well known to those skilled in the art. In some embodiments, the coding sequence of the present application is a single strand, with the TCRR chain coding sequence and the TCRa chain coding sequence linked via a P2A coding sequence, and then further linked to the chimeric switch receptor coding sequence via a T2A coding sequence; and the single-stranded encoding nucleotides are in the same reading frame. It should be understood that in gene cloning procedures, it is often necessary to design appropriate restriction sites, which inevitably introduces one or more irrelevant residues at the termini of the expressed amino acid sequence, but this does not affect the activity of the target sequence. To construct fusion proteins, promote recombinant protein expression, obtain recombinant proteins that are automatically secreted outside the host cell, or facilitate purification of recombinant proteins, it is often necessary to add amino acids to the N-terminus, C-terminus, or other appropriate regions within the recombinant protein. These include, but are not limited to, suitable linker peptides, signal peptides, leader peptides, and terminal extensions. Therefore, the amino or amino termini of the fusion proteins of the present invention may also contain one or more polypeptide fragments as protein tags. Any suitable tag may be used herein. For example, the tags may include FLAG, HA, HAL c-Myc, Poly-His, Poly-Arg, Strep-Tag, UL EE, T7, 4A6, g, B, gE, and Ty1. These tags can be used to purify proteins. This application also relates to vectors containing the nucleic acid sequences described herein and one or more regulatory sequences operably linked to these sequences. The nucleic acid molecules described herein can be manipulated in a variety of ways to ensure expression of the fusion proteins. Techniques for altering polynucleotide sequences using recombinant DNA methods are known in the art. The regulatory sequence can be a suitable promoter sequence. Promoter sequences are typically operably linked to the coding sequence for the protein to be expressed. The promoter can be any nucleotide sequence that exhibits transcriptional activity in the selected host cell, including mutant, truncated, and hybrid promoters, and can be obtained from genes encoding extracellular or intracellular polypeptides homologous or heterologous to the host cell. The regulatory sequence can also be a suitable transcription terminator sequence, a sequence recognized by the host cell to terminate transcription. The terminator sequence is operably linked to the 3' end of the nucleotide sequence encoding the polypeptide. Any terminator that is functional in the selected host cell can be used in the present invention. The regulatory sequence can also be a suitable leader sequence, an untranslated region of an mRNA that is important for translation by the host cell.The leader sequence is operably linked to the 5' end of the nucleotide sequence encoding the polypeptide. Any terminator that is functional in the selected host cell can be used in this application. The nucleic acid molecules of the present application can be cloned into many types of vectors. For example, they can be cloned into plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Furthermore, the vector is an expression vector. The expression vector can be provided to the cell in the form of a viral vector. Viral vector technology is well known in the art and is described in, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. Typically, suitable vectors contain an origin of replication functional in at least one organism, a promoter sequence, convenient restriction enzyme sites, and one or more selectable markers (e.g., WO 01 / 96584 and U.S. Pat. No. 6,326,193). For example, in certain embodiments, the present invention utilizes a lentiviral vector containing an origin of replication, a 3ITR, a 5ITR, a polynucleotide sequence described herein, and optionally a selectable marker. An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high-level expression of any polynucleotide sequence operably linked thereto. Another example of a suitable promoter is elongation growth factor-1a (EF-1a). However, other constitutive promoter sequences may also be used, including but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the heme promoter, and the creatine kinase promoter. Furthermore, the use of inducible promoters is also contemplated. The use of inducible promoters provides a molecular switch capable of turning on expression of a polynucleotide sequence operably linked to the inducible promoter when expression is desired, and turning off expression when expression is undesirable. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.To assess expression of the gene of interest, the expression vector introduced into the cell may also contain either or both a selectable marker gene and a reporter gene to facilitate identification and selection of expressing cells from a population of cells sought to be transfected or infected via a viral vector. Alternatively, the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both the selectable marker and the reporter gene may be flanked by appropriate regulatory sequences to enable expression in the host cell. Useful selectable markers include, for example, antibiotic resistance genes such as neo. Reporter genes are used to identify potentially transfected cells and to assess the functionality of regulatory sequences. After the DNA has been introduced into the recipient cells, reporter gene expression is measured at an appropriate time. Suitable reporter genes may include. Methods for introducing genes into cells and expressing genes in cells are known in the art. Vectors can be readily introduced into host cells, for example, mammalian, bacterial, yeast, or insect cells, by any method known in the art. For example, expression vectors can be transferred into host cells by physical, chemical, or biological means. Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Biological methods for introducing polynucleotides of interest into host cells include the use of DNA and RNA vectors. Chemical methods for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, and microspheres; and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. Biological methods for introducing polynucleotides into host cells include the use of viral vectors, particularly lentiviral vectors, which have become the most widely used method for inserting genes into mammalian, such as human, cells. Other viral vectors can be derived from poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, among others. Numerous viral-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. Selected genes can be inserted into vectors and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to the subject's cells in vivo or ex vivo. Many retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are known in the art. In one embodiment, lentiviral vectors are used. Immune cells The immune cells of the present application are selected from lymphocytes, dendritic cells, monocytes / macrophages, granulocytes, and mast cells. In some embodiments, the immune cells are lymphocytes; in some embodiments, the immune cells are NK cells; in some embodiments, the immune cells are B cells; and in some embodiments, the immune cells are TIL cells. In some embodiments, the immune cells are T cells, which may be derived from T cells isolated from a subject or may be a portion of a mixed cell population isolated from a subject, such as a peripheral blood lymphocyte (PBL) population. For example, the cells can be isolated from peripheral blood mononuclear cells (PBMCs) and can be CD4+ helper T cells or CD8+ cytotoxic T cells. The cells can be in a mixed population of CD4+ helper T cells / CD8+ cytotoxic T cells. Generally, the cells can be activated with antibodies (e.g., anti-CD3 antibodies) to make them more receptive to transfection.The modified immune cells described herein are immune cells comprising the aforementioned TCR receptor molecules and the aforementioned chimeric switch receptors. In some embodiments, the modified immune cells are constructed by introducing coding sequences encoding the aforementioned TCR receptor molecules and the aforementioned chimeric switch receptors, or vectors comprising such coding sequences, into isolated immune cells. In some embodiments, the modified immune cells are constructed by introducing coding sequences encoding the aforementioned TCR receptor molecules and the aforementioned chimeric switch receptors, or vectors comprising such coding sequences, into immune cells in vivo. In some embodiments, the coding sequences for the TCR receptor molecules and the chimeric switch receptors are expressed in tandem and in the same reading frame. Compositions: This application also provides compositions comprising the modified immune cells, nucleic acids, or vectors of the present invention. In some embodiments, the compositions are pharmaceutical compositions. In some embodiments, the compositions are compositions suitable for research, treatment, prevention, and / or diagnosis. In some embodiments, the modified immune cells, nucleic acids, or vectors of the present application are preferably formulated into a medicament or drug together with one or more other pharmaceutically acceptable ingredients known to those skilled in the art, including but not limited to pharmaceutically acceptable carriers, adjuvants, excipients, diluents, fillers, buffers, preservatives, antioxidants, lubricants, stabilizers, solubilizers, surfactants, masking agents, colorants, flavorings, and sweeteners. The term "pharmaceutically acceptable" as used herein refers to compounds, ingredients, materials, compositions, dosage forms, etc. that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of the subject in question (e.g., a human) without excessive toxicity, irritation, allergic reaction, or other problems or complications, commensurate with a reasonable benefit / risk ratio. Each carrier, adjuvant, excipient, etc. must also be "acceptable" in the sense of being compatible with the other ingredients of the formulation. Suitable carriers, adjuvants, excipients, etc. can be found in standard pharmaceutical textbooks, such as Remington's Pharmaceutical Sciences and the Handbook of Pharmaceutical Excipients. The pharmaceutical compositions of the present invention can be administered in a manner appropriate to the condition to be treated (or prevented). The amount and frequency of administration will be determined by factors such as the patient's condition, and the type and severity of the patient's condition. When an "immunologically effective amount," "anti-tumor effective amount," "tumor-suppressive effective amount," or "therapeutic amount" is specified, the precise amount of the composition of the present invention to be administered can be determined by a physician, taking into account individual differences in the patient's (subject's) age, weight, tumor size, degree of infection or metastasis, and condition.Generally speaking, pharmaceutical compositions comprising the T cells described herein can be administered at a dose of E4 to E9 cells / kg body weight, preferably E5 to E7 cells / kg body weight. T cell compositions can also be administered multiple times at these doses. Cells can be administered using infusion techniques well known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988). The optimal dosage and treatment regimen for a particular patient can be readily determined by one skilled in the medical field by monitoring the patient for signs of disease and adjusting treatment accordingly. Administration of the subject compositions can be performed by any convenient means, including by nebulization, injection, swallowing, infusion, implantation, or transplantation. The compositions described herein can be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intraspinal, intramuscularly, by intravenous injection, or intraperitoneally. Medical Uses In another aspect, the use of the modified immune cells, nucleic acids, vectors, or pharmaceutical compositions of the present application in the preparation of a medicament for treating or preventing a disease or condition is provided. In some embodiments, the modified immune cells, nucleic acids, vectors or pharmaceutical compositions of the present application can be used to prevent or treat diseases related to HBV infection. Diseases related to HBV infection include acute hepatitis (including fulminant hepatic failure), chronic hepatitis, liver fibrosis, liver cirrhosis, liver cancer such as hepatocellular carcinoma (HCC), or pancreatic cancer. Treatment and prevention methods can be performed by isolating T cells from patients or volunteers with HBV-related diseases, introducing the nucleic acid molecules or vectors of the present application into the above-mentioned T cells, and then returning these genetically modified cells to the patient for treatment. Therefore, the present application provides a method for treating HBV-related diseases, comprising: infusing isolated T cells expressing the TCR and chimeric switch receptor of the present application, preferably, the T cells are derived from the patient himself, into the patient. Generally, the method comprises (1) isolating T cells from the patient, (2) transducing the T cells in vitro with the nucleic acid molecules or vectors of the present application, and (3) infusing the genetically modified T cells into the patient. The number of cells to be isolated, transfected and returned can be determined by the physician. In some embodiments of the present invention, the modified immune cells, nucleic acids, vectors, or pharmaceutical compositions of the present invention may be combined with other therapies known in the art. Such therapies include, but are not limited to, chemotherapy, radiotherapy, immunosuppressants, and viral inhibitors. For example, treatment may be combined with nucleotide analogs or interferons known in the art for treating HBV-induced diseases. Nucleotide analogs include lamivudine, adefovir dipivoxil, telbivudine, entecavir, tenofovir disoproxil, and clavudine.

[0015] "Patient," "subject," "individual," and the like are used interchangeably herein to refer to living organisms, such as mammals, capable of inducing an immune response. Examples include, but are not limited to, humans, dogs, cats, mice, rats, and transgenic species thereof. The technical solutions of this application will be described clearly and completely below in conjunction with specific examples. Obviously, the examples described are only a portion of the embodiments of this application, and are not exhaustive. All other embodiments derived by those skilled in the art based on the examples herein without inventive effort are within the scope of protection of this application. It should be noted that the order in which the following examples are described does not constitute a limitation on the preferred order of the embodiments. Experimental methods in the following examples, where specific conditions are not specified, generally follow conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd edition, 2001), or the conditions recommended by the manufacturer. Unless otherwise noted, percentages and parts are calculated by weight. Unless otherwise noted, reagents and materials referred to herein are commercially available or can be prepared by those skilled in the art based on common knowledge. Any methods and materials similar or equivalent to those described herein may be used in this application. The preferred embodiments and materials herein are for exemplary purposes only and do not limit the scope of this application. Example 1: Construction of viral vectors expressing HBV TCR, HBV TCR-PD1-BB, and HBV TCR-PD1-28

[0016] The structural principles of HBV TCR-PD1-BB and HBV TCR-PD1-28 are shown in Figure 1 , and the sequence structures of HBV TCR, HBV TCR-PD1-28, and HBV TCR-PD1-BB are shown in Figure 2 . The coding sequence of the HBV TCR fragment (amino acid sequence shown in SEQ ID NO:14), the coding sequence of the HBV TCR-PD1-BB fragment (amino acid sequence shown in SEQ ID NO:17), and the coding sequence of the HBV TCR-PD1-28 fragment (amino acid sequence shown in SEQ ID NO:18) were synthesized in their entirety. The synthetic genes were double-enzyme digested and then inserted into a lentiviral vector to construct a plasmid. The resulting vectors were constructed. Structurally, the TCR consists of two chains, the α and R, each composed of a variable domain (responsible for binding to the MHC-peptide complex) and a constant domain (responsible for binding to both TCR chains). A PD-1 costimulatory domain fusion receptor (chimeric switch receptor) is added as an anti-exhaustion sequence. The TCR α and R chains are linked by a P2A self-cleaving peptide, which is then linked to the PD-1 costimulatory domain fusion receptor via a T2A self-cleaving peptide. The constant region undergoes sequence modification to reduce endogenous TCR mispairing.

[0017] HBV TCR-PD1-BB and HBV TCR-PD1-28 contain the T cell receptor β chain variable region (TCR R variable region), the T cell receptor R chain constant region (TCR R constant region), the T cell receptor α chain variable region (TCR α variable region), the T cell receptor α chain constant region (TCR α constant region), the PD1 extracellular domain, the CD8 or CD28 transmembrane domain, and the 41BB or CD28 intracellular signaling domain (Figure 2). Cloning the target gene into the backbone plasmid: To improve target gene expression efficiency, selecting an appropriate backbone plasmid is crucial. We used the publicly available pCDH-EF1-MCS-T2A-copGFP plasmid original sequence as a base (see Table 1 for restriction site information for the lentiviral backbone plasmid), replaced the ampicillin resistance gene with the kanamycin resistance gene, and then performed complete gene synthesis of the backbone plasmid. Primers were then designed to introduce EcoRI and Sall restriction sites at both ends of the target gene sequence. PCR amplification of the target gene fragment was performed, and the target gene band was selected based on the electrophoresis results of the PCR product for purification and recovery. Simultaneously, the recovered PCR product and the pCDH-EF1-MCS-T2A-copGFP backbone plasmid were digested with EcoRI and Sall. The two fragments were ligated using T4 DNA ligase, and the ligated product was transformed into Stbl3 competent cells. A single clone was selected for culture, and the plasmid was extracted and identified by double enzyme digestion, electrophoresis, and sequencing. The constructed target plasmid includes key functional elements such as the 5'LTR (long terminal repeat), HIV-1 (packaging and integration signal), RRE (Rev response element), cPPT / CTS (central polyadenylation tract / central termination sequence), EF-1a core promoter (human EF-1a core promoter), WPRE (woodchuck hepatitis virus post-transcriptional regulatory element), 3'LTR-SIN (self-inactivating long terminal repeat) (the key functional elements and locations of the lentiviral backbone plasmid are shown in Table 2), and the TCR-HBsAg gene. Table 1. Summary of restriction site information of lentiviral backbone plasmids. Table 2. Key functional elements and locations of lentiviral backbone plasmids Example 2: Lentivirus packaging

[0018] The lentiviral vectors used in the production of HBV TCR, HBV TCR-PD1-BB, and HBV TCR-PD1-28 cell products are third-generation, self-inactivating (SIN) lentiviral vectors derived from HIV-1 and featuring a VSV-G pseudoenvelope. They carry nucleic acid encoding the HBsAg-specific T cell receptor (HBsAg TCR). These lentiviral vectors are only infectious and replication-incompetent. Their particles are approximately 80-120 nm in diameter and have a roughly spherical or icosahedral shape. The viral outer membrane is a lipid envelope embedded with the VSV-G envelope protein. Inwardly, a spherical matrix is ​​formed by protein p17, and a semi-conical capsid is formed by protein p24. The capsid contains the RNA encoding the TCR. The functions of the various nucleic acid components of third-generation self-inactivating lentiviral vectors are described as follows: At either end of the TCR gene are truncated / chimeric long terminal repeats: the ASITR and the Δ3ITR. The Δ5ITR is a deletion of U3, replacing it with the enhancer and promoter of respiratory syncytial virus pneumonia (RSV), making the vector independent of Tat for replication. The Δ3ITR is a deletion of U3, eliminating its promoter / enhancer activity, making it self-inactivating.

[0019] RRE element function: A cis-acting element of Rev, promoting the transport of large, unspliced ​​mRNA molecules from the nucleus to the cytoplasm. cPPT element function: Improving vector transduction efficiency. EF-1a promoter function: Regulating the onset and extent of gene expression (transcription).

[0020] Function of the WPRE element: It upregulates polyadenylation of transcripts and promotes nuclear export, thereby enhancing target gene expression efficiency. Lentiviral vectors are obtained by transiently transfecting 293T cells with a third-generation four-plasmid system and then purified. The four-plasmid system consists of three packaging plasmids (also known as "helper plasmids") and a target plasmid. The packaging plasmid pGagPol-KanR encodes the viral structural protein Gag, which forms the viral core structure, and the reverse transcriptase Pol, which is required for RNA reverse transcription and integration. The packaging plasmid pRev-KanR encodes the Rev protein, which binds to RNA to promote mRNA transport and protein expression. The packaging plasmid pVSV-G-KanR encodes the vesicular stomatitis virus envelope protein VSV-G, which replaces the HIV envelope protein. This allows the lentiviral vector to infect cells from virtually all tissues and improves the stability of the lentiviral particles.

[0021] D10 complete cell culture medium was prepared as follows: DMEM (Gibco, 11965092-092), 10% FBS (Gibco, 10099141), 1% Sodium Pyruvate (Gibco, 11360070), and stored in a refrigerator at 4°C for later use.

[0022] Day 0: 293T cells were less than 20 passages and not overly confluent. 2×1.7 cells were plated on a 150 mm dish (Corning, 430599). 20 mL of D10 medium was added, the cells were thoroughly mixed, and cultured overnight at 37°C.

[0023] Day 1: Transfect 293T cells when they reach 60-80% confluency. The time from plating to transfection should be no longer than 24 hours. Prepare plasmid complexes for lentiviral packaging according to Table 3. Table 3. Materials Required for Plasmid Complexes While gently vortexing the plasmid, PEIpro was added dropwise, mixed thoroughly, and allowed to stand at room temperature for 15 minutes to form a plasmid-PEI complex. The complex was slowly added to a 150 mm dish of 293T cells, mixed thoroughly, and incubated at 37°C (2°C) in a CO2 incubator for 6 hours.

[0024] Day 1: Gently replace the medium of 293T cells transfected for 6 hours with 20 mL of fresh D10 medium.

[0025] Day 3: Collect the viral supernatant 48 hours after transfection and store it in a refrigerator at 4°C. Then add 20 mL of D10 medium.

[0026] Day 4: Collect the viral supernatant 72 hours after transfection and mix it with the viral supernatant collected 48 hours after transfection; centrifuge at 3000g for 10 minutes at 4 degrees Celsius, filter through a 0.45μm filter to remove debris and retain the supernatant, and concentrate the virus using a 100K ultrafiltration cup.

[0027] Centrifuge at 3000g at 4°C to the desired volume for virus concentration. After centrifugation, remove the centrifuge device, separate the filter cup from the filtrate collection cup below, and invert the filter cup onto the sample collection cup. Centrifuge at 1000g for 2 minutes at 4°C. The sample collection cup contains the virus concentrate. Collect the concentrate, aliquot, and store below -70°C. Virus titer determination: Prepare a 24-well plate. Add a certain amount of virus concentrate to a Jurkat plate at a dilution ratio of 1 x 10" well per 500 μl. Serial dilutions are performed. After incubation for 72 hours, the virus titer is determined by flow cytometry. Virus titer determination: Prepare flow cytometry buffer: DPBS (Gibco, 14190250) and 2% FBS (Gibco, 10099141). Refrigerate for 4 hours. Take Jurkat cells, centrifuge 1 x 10" cells per group at 400g for 5 minutes, discard the supernatant, and wash twice with flow cytometry buffer.

[0028] PE Dextramer HBV-S20 was diluted with flow cytometry buffer at a ratio of 1:100. 100 μl of antibody diluent was added to each sample, and the cells were incubated in the dark for 30 minutes. Flow cytometry buffer was added to wash the cells, and the cells were centrifuged at 400 g for 5 minutes, with the supernatant discarded. This was repeated twice. The cells were resuspended in 100 μl of flow cytometry buffer and loaded for flow cytometry analysis. Vector infection titer (TU / mL) = number of cells plated per well x positive rate (%) x dilution factor / titration volume (mL). As shown in Figure 3a, the titers of HBV TCR, HBV TCR-PD1-BB, and HBV TCR-PD1-28 lentiviral vectors were all above 1E7 TU / ml, indicating that all three HBV TCRs were successfully packaged into viruses at high titers. Example 3: Preparation of HBV TCR, HBV TCR-PD1-BB, and HBV TCR-PD1-28

[0029] T cell culture medium preparation: PRIME-XV- T cell CDM (Irvine, 91154), 400 IU / ml IL-2.

[0030] T cell cryopreservation solution preparation: 75% CS10 (ThermoFisher, A2596101) + 25% HS A (FLEXBUMIN, S20181007)

[0031] DayO: Isolate relatively pure CD3+ T cells from a single blood draw and adjust the cell concentration to 1x 10 using T cell culture medium. 6 / mL, according to Transact (CD3 / CD28 microspheres) (Macs, 6201000014): add activator to cell suspension at a ratio of 1:30, mix gently and thoroughly, then add interleukin-2 at a final concentration of 400 IU / mL, stimulate culture for 24 hours, and then infect with virus;

[0032] Day 1: Count cells and adjust the density of T cells to 5 x 10 5 / mL, virus solution was added;

[0033] Day 2-11: After cell infection, the cell status was observed every day, and T cell culture medium containing IL-2 400 IU / mL was added as appropriate to maintain the T cell density at 5 x 10 5 / mL, to expand cells;

[0034] Day 12: Cells were harvested by centrifugation at 300 g for 5 minutes, washed with saline solution containing 5% human albumin, and cryopreserved at an appropriate density using T cell cryopreservation buffer. Freeze using a programmed freezing device and store in liquid nitrogen. Example 4: Flow Cytometry Analysis of HBV TCR, HBV TCR-PD1-BB, and HBV TCR-PD1-28 MOI and TCR-Specific Expression

[0035] Preparation of flow cytometry buffer for HBV TCR, HBV TCR-PD1-BB, and HBV TCR-PD1-28 MOI detection: DPBS (Gibco, 14190250), 2% FBS (Gibco, 10099141), set at 4. Refrigerate for later use. HBV TCR, HBV TCR-PD1-BB, HBV TCR-PD1-28 cells, and UT cells (control group) were washed once with PBS, the supernatant discarded, and PE Dextramer HBV-S20 (Immudex, WB3290-PE) was added. Protected from light for 30 minutes, the cells were washed with PBS, resuspended, and finally analyzed by flow cytometry (Beckman CytoFLEX). The results are shown in Figure 3b. HBV TCR expression was higher than that of HBV TCR-PD1-BB and HBV TCR-PD1-28. At an MOI of 1.0 in each group, the TCR positivity rate was above 30%, and the HBV TCR positivity rate was as high as 60%, indicating that the three monoclonal numbers of HBV S20 TCR-T were stably expressed in the MOI gradient test.

[0036] HBV TCR-T specific expression and mismatch rate detection flow cytometry buffer preparation: DPBS, 2% FBS, placed in 4. (2) Refrigerate for later use. Prepared cells from each group of HBV TCR, HBV TCR-PD1-BB and HBV TCR-PD1-28 and UT cells (control group) were taken, washed once with PBS and the supernatant was discarded. Each group was divided into two parts, and PE Dextramer HBV-S20 / BV421 PD-1 and PE Dextramer HBV-S20 / APC TCR vp 5.1, 4 were added respectively. (2) After 30 minutes in the dark, the cells were washed three times with flow cytometry buffer, resuspended, and finally detected by flow cytometry. The results are shown in Figure 4. The mismatch rates of HBV TCR, HBV TCR-PD1-BB and HBV TCR-PD1-28 expression were all low, and the specific expression reached more than 30%, indicating that HBV TCR-T in each group can be specifically expressed with a low mismatch rate. Example 5: Expression of HBV TCR, HBV TCR-PD1-BB and HBV TCR-PD1-28 killing of liver cancer cells and cytokine detection

[0037] M10 cell complete culture preparation: DMEM, 10% FBS (Gibco, 10099141), 1% Sodium Pyruvate (Gibco, 11360070), 1% HEPES (Gibco, 15630080), 1% NEAA (Gibco, 11140-050) placed in a refrigerator at 4°C for later use.

[0038] T cell culture medium preparation: PRIME-XV- T cell CDM (Irvine, 91154), 400 IU / ml IL-2.

[0039] HBV TCR> HBV TCR-PD1-BB and HBV TCR-PD1-28 kill liver cancer cells (RTCA)

[0040] DO: HepG2-LMS-LG cells and HepG2 cells (negative control) with good growth status were taken and the cell density was adjusted to 4 x 10 5 / ml for standby use, take a 96-well plate for RTCA coated with collagen, add 50ul / well M10 culture medium for instrument baseline measurement, and then add 4x10 5 50ul of cells corresponding to / ml were placed in a static tube for about 5 minutes and then the growth curve was continuously monitored for about 16 hours.

[0041] D1: Take TCR-T and UT cells with measured TCR% and cell viability, prepare effector cells according to the positive rate, add 50ul / well of effector cells at effector to target ratios of 1:1 and 1:5, and continuously monitor the killing curve on the instrument.

[0042] D3: Copy the RTCA growth and killing curves, stop the culture, and collect the co-culture supernatant for cytokine analysis. Cytokine Assay: Take the CBA kit (Human Th1 / Th2 Cytokine Cytometric Bead Array Kit II, BD, 551809) and equilibrate to room temperature. Pipette 2ml of Assay Diluent to reconstitute the standard to a concentration of 5000pg / ml and equilibrate at room temperature for 30 minutes. Standard Preparation: Take the standard, labeled S1, and sequentially dilute standards S2-S9 by 2-fold. S10 serves as a blank. Prepare the Human Th1 / Th2 Cytokine Capture Beads mixture: Take microsphere solutions A1-A6, vortex to mix, and then mix to equal volumes. A 96-well L-bottom plate was prepared, and 50 μl of the Human Th1 / Th2 Cytokine Capture Beads mixture was added. 50 μl of Human Th1 / Th2 PE Detection Reagent (cytokine detection sample) was then added and centrifuged at 400 g for 5 minutes. 50 μl of each sample was added to the sample detection wells, and 50 μl of the S10-S10 preparation was added to the marker wells. The mixture was incubated at room temperature in the dark for 180 minutes. 100 μl of Wash buffer was added, and the mixture was centrifuged at 300 g for 5 minutes. The supernatant was discarded. The cells were resuspended in 100 μl of 2nt Wash buffer, loaded onto the plate, and analyzed by flow cytometry. The results showed that after 48 hours of co-incubation, HBV TCR, HBV TCR-PD1-BB, and HBV TCR-PD1-28, at an effector-target ratio of 1:5, all achieved a cytotoxicity rate exceeding 90% against HepG2-LMS-LG target cells. UT showed no significant cytotoxicity against target cells (Figure 5). At an effector-target ratio of 1:1, HBV TCR, HBV TCR-PD1-BB, HBV TCR-PD1-28, and UT secreted almost no cytokines when co-incubated with negative target cells (HepG2). When co-incubated with positive target cells, IFN-γ cytokine secretion by HBV TCR-PD1-BB was approximately 10 times that of HBV TCR-PD1-28 and 20 times that of the HBV TCR group, while UT had no significant cytokine secretion (Figure 5).Example 6: Multi-round Target Cell Stimulation Assay for Tumor Exhaustion Using HBV TCR, HBV TCR-PD1-BB, and HBV TCR-PD1-28. To address the practical difficulty of solid tumors escaping PD-L1, we conducted multi-round tumor cell stimulation experiments to simulate in vivo T cell exhaustion caused by PD-L1 signaling on the tumor cell surface. We compared the tumor cell killing abilities of HBV TCR-PD1-BB and HBV TCR-PD1-28 (TCR-resistant T cells) and HBV TCR (T cells expressing only the TCR) to validate the role of the PD-1 helper sequence. Multi-round Target Cell Stimulation:

[0043] T cell culture medium preparation: PRIME-XV- T cell CDM (Irvine, 91154), 400 IU / ml IL-2.

[0044] D1: Take the HBV TCR, HBV TCR-PD1-BB, HBV TCR-PD1-28 and UT cells whose cell viability has been measured, count them, and adjust the cell density to 5x10 5 / ml, 4ml was added to a 6-well plate, and an equal number of target cells HepAD38-PDL1 were added;

[0045] D5: Count and adjust the cell volume to 5x 10 5 / ml, 4ml, add equal amount of target cells for the second round of stimulation;

[0046] D10: Harvest cells, count, and conduct subsequent experiments; Cell killing function test

[0047] M10 cell culture complete preparation: DMEM, 10% FBS (Gibco, 10099141), 1% Sodium Pyruvate (Gibco, 11360070), 1% HEPES (Gibco, 15630080), 1% NEAA (Gibco, 11140-050) placed in refrigerator at 4°C for later use.

[0048] DO: Target cells HepAD38-PDL1 were taken and digested and the cell density was adjusted to 4 x 10 5 / m 1 for standby, take a collagen-coated RTCA 96-well plate, add 50ul / well of M10 culture medium for instrument baseline measurement, then add 50ul of cells corresponding to a density of 4x105 / ml to each well, let it stand for about 5 minutes, and then continuously monitor the growth curve for about 24 hours;

[0049] D1: Take HBV TCR, HBV TCR-PD1-BB, HBV TCR-PD1-28 and UT with measured TCR% and cell viability, and count 4x10 5 / ml to prepare effector cells, add 50ul / well of effector cells according to the effector-target ratio of 1:1, and continuously detect the killing curve on the instrument;

[0050] D3: Copy the RTCA growth and killing curves, stop the culture, and collect the co-culture supernatant for cytokine detection. Cytokine detection is performed according to the cytokine detection steps in Example 5. The results showed that after two rounds of tumor cell stimulation, only HBV TCR-PD1-BB and HBV TCR-PD1-28 still had a significant killing effect on the target cells HepAD38-PDL1, while HBV TCR had basically lost its function. This indicates that HBV TCR-PD1-BB and HBV TCR-PD1-28 can still maintain their anti-tumor effects after two rounds of tumor cell stimulation, and can still kill nearly 80% of tumor cells after 48 hours, indicating that their killing ability has not been affected or weakened (Figure 6). At the same time, no cytokine secretion was detected in all groups of negative target cells (HepG2). When co-incubated with positive target cells (HepAD38-PDL1), HBV TCR and UT almost did not secrete cytokines, while HBV TCR-PD1-BB and HBV TCR-PD1-28 could detect the cytokines IFN-γ and TNF-α, and the HBV TCR-PD1-BB group had significantly higher secretion of cytokines than HBV. TCR-PD1-28 group (Figure 7). Based on the above analysis, HBV TCR-PD1-BB and HBV TCR-PD1-28 were able to maintain their anti-tumor effects and cytotoxicity after multiple rounds of tumor stimulation. This is because their anti-exhaustion sequence (PD-1 auxiliary sequence) can convert the braking signal (PD-L1) on the surface of tumor cell lines into a stimulatory signal, preventing the cells from prematurely entering a state of exhaustion. This provides them with anti-exhaustion ability and a sustained cytotoxic effect. Example 7: Detection of HBV TCR and HBV TCR-PD1-BB's Killing Function and Cytokine Release on Various Target Cells. We next analyzed the anti-tumor effects of HBV TCR-PD1-BB. Because the activation signals carried by HBV TCR-PD1-BB include HBsAg and PD-L1, in order to verify whether HBV TCR-PD1-BB has different killing abilities for tumor cells expressing HBsAg and PD-L1, we constructed four liver cancer cell lines: HBsAg+ / PD-Ll+, HBsAg+ / PD-L1-, HBsAg- / PD-LI+, and HBsAg- / PD-L1- for functional evaluation.

[0051] T cell culture medium preparation: PRIME-XV- T cell CDM (Irvine, 91154), 400 IU / ml IL-2.

[0052] M10 cell complete culture preparation: DMEM, 10% FBS (Gibco, 10099141), 1% Sodium Pyruvate (Gibco, 11360070), 1% HEPES (Gibco, 15630080), 1% NEAA (Gibco, 11140-050) placed in a refrigerator at 4°C for later use.

[0053] DO: Take the above four target cells and digest them and adjust the cell density to 4x10 5 / ml for standby use, take a 96-well plate for RTCA coated with C ollagen, add 50ul / well M10 culture medium for instrument baseline measurement, and then add 4x10 5 50ul of cells corresponding to / ml were placed in a static tube for about 5 minutes and then the growth curve was continuously monitored for about 16 hours.

[0054] D1: Take the HBV TCR, HBV TCR-PD1-BB and UT with measured TCR% and cell viability, and count 4x10 positive live cells. 5 / mU 0.8xl0 5 / ml to prepare effector cells, add 50ul / well of effector cells according to the effector-target ratio of 1:1 and 1:5, and continuously detect the killing curve on the instrument;

[0055] D3: RTCA growth and killing curves were copied, the culture was stopped, and the co-culture supernatant was collected for cytokine analysis. Cytokine analysis was performed according to the cytokine detection procedures in Example 5. The results (Figure 8) showed that untransfected T cells did not significantly kill either target cell type, and HBV TCR-PD1-BB and HBV TCR had no significant killing effect on HBsAg- / PD-L1+ and HBsAg- / PD-L1- target cells. At an effector-target ratio of 1:5, both HBV TCR-PD1-BB and HBV TCR killed HBsAg+ target cells by more than 60% within 24 hours, and HBV TCR-PD1-BB was more potent than HBV TCR in killing HBsAg+ / PD-L1+ and HBsAg+ / PD-L1- cells. HBV TCR-PD1-BB was more potent in killing HBsAg+ / PD-L1+ target cells than HBsAg+ / PD-L1- cells, while the opposite was true for HBV TCR. In addition, cytokine detection results showed that HBV TCR-PD1-BB secreted almost no cytokines when co-cultured with HBsAg- / PD-Ll+ or HBsAg- / PD-L1- target cells. When co-cultured with HBsAg+ / PD-Ll- target cells, the IFN-γ secreted was comparable to that of HBV TCR, while the IFN-γ secreted when co-cultured with HBsAg+ / PD-Ll+ target cells was significantly higher than that of HBV TCR-. The above experiments indicate that HBV TCR-PD1-BB can target and kill HBsAg+ liver cancer cells. Because HBV TCR-PD1-BB can convert the inhibitory signal of PD-1 into an activation stimulation signal, the anti-tumor effect of HBV TCR-PD1-BB is more prominent for HBsAg+ liver cancer cells that highly express PD-L1, as manifested by a significant increase in cytokine secretion and stronger tumor killing ability. Furthermore, HBV TCR-PD1-BB only targets and kills liver cancer cells expressing HBsAg. It has no non-specific killing effect on tumor cells that only express PD-L1 but not HBsAg. Example 8: Detection of Proliferation Ability of HBV TCR-PD1-BB and HBV TCR Target Cells After Multiple Rounds of Stimulation. The ability of T cells to proliferate and expand again after multiple rounds of stimulation is also an important indicator for evaluating anti-tumor activity. CPDF450 (Cell Proliferation Dye eFluor™ 450) is a purple fluorescent dye that can be used to monitor single cell division.This fluorescent dye binds to any cellular protein containing primary amines. As cells divide, the dye is evenly distributed between daughter cells, and the number of cell divisions can be measured by the successive halvings of the dye's fluorescence intensity. Multiple rounds of stimulation of target cells:

[0056] T cell culture medium preparation: PRIME-XV- T cell CDM (Irvine, 91154), 400 IU / ml IL-2.

[0057] D1: Take the HBV TCR, HBV TCR-PD1-BB and UT cells whose cell viability has been measured, count them, and adjust the cell volume to 5x10 5 / ml, 4ml was added to a 6-well plate, and an equal number of target cells HepAD38-PDL1 were added;

[0058] D2: Count cells, observe cell status, and replenish fluids when appropriate;

[0059] D5: Count and adjust the cell volume to 5x10 5 / ml, 4ml, and an equal number of target cells were added for the second round of stimulation. This was repeated for a total of four rounds of stimulation. The proliferation fold was calculated based on the total cell count and culture volume. The results showed that after three and four rounds of stimulation, HBV TCR-PD1-BB cells maintained high proliferation activity, with a total proliferation fold exceeding 200-fold, significantly higher than those in the HBV TCR and UT groups (Figure 9). Cell proliferation assay: Flow cytometry buffer was prepared: DPBS and 2% FBS were placed in a refrigerator at 4°C (2°F). After two rounds of stimulation, HBV TCR, HBV TCR-PD1-BB, and UT cells were centrifuged at 400g for 5 minutes, and the supernatant was discarded. CPDF450 was added and the cells were stained in a dark incubator for 30 minutes. The cells were washed with flow cytometry buffer and centrifuged at 400g for 5 minutes, and the supernatant was discarded. This was repeated twice. The cells were washed twice with T cell culture medium.

[0060] Day 1: Count and adjust the cell volume to 5x10 5 / ml, 4ml was added to a 6-well plate, and an equal number of target cells HepAD38-PDL1 were added;

[0061] Day 5: Cells were collected and centrifuged at 400g for 5 minutes to discard the supernatant. Flow cytometry buffer was added to wash the cells. The cells were resuspended in 100ul of flow cytometry buffer and loaded for flow cytometry. The results showed that after two rounds of stimulation, only about 15% of the cells in the negative control cell group and the HBV TCR group with positive target cells had proliferation activity. At the same time, more than 70% of the cells in the HBV TCR-PD1-BB group with positive target cells still had proliferation activity (Figure 9). Example 9: In vivo efficacy and efficacy test of HBV TCR-PD1-BB in an immunodeficient mouse HBsAg+ human HCC transplant tumor model. In order to understand the killing effect of HBV TCR-PD1-BB on tumor cells in an in vivo environment, we used immunodeficient mice and HBsAg+ liver cancer cells for modeling and constructed a mouse CDX model (tumor cell line transplant model) to simulate the killing and clearance effect of HBV TCR-PD1-BB cells on liver cancer cells in the body after clinical liver cancer patients received different doses of HBV TCR-PD1-BB cells under ablation chemotherapy. We used NSG immunodeficient mice and inoculated 12 HBsAg+ tumor cells on the right side of the mouse body on day 12. On day 0, the tumor size reached approximately 120 mm. 3 We divided the mice into five groups, three of which received injections of different doses of HBVTCR-PD1-BB cells, namely the high-dose group (1x10 7 The HBV TCR-PD1-BB cells were administered at a dose of 10 cells / mouse, a medium-dose group (3x10 cells / mouse), and a low-dose group (1x10 cells / mouse). The remaining two groups served as control groups, receiving cryopreserved cells (Control) and untransfected T cells (UT), respectively. Tumor size was measured and data collected every 3 to 4 days. The results showed that different doses of HBV TCR-PD1-BB cells significantly inhibited tumor growth. Tumor volumes in mice receiving 1x10 cells were significantly smaller than those in the other two dose groups (Figure 10a). Furthermore, in mice receiving 1x10 cells, tumors almost completely disappeared within three weeks, and the HBV TCR-PD1-BB mice showed no significant weight loss within 6 days (Figure 10b). This indicates that HBV TCR-PD1-BB cells maintain their anti-cancer effects in mice, and that a certain concentration of HBV TCR-PD1-BB cells is significantly effective in clearing tumor cells.

[0062] In vivo persistence of HBV TCR-PD1-BB mice: To detect the persistence of HBV TCR-PD1-BB in mice, qPCR was performed on the blood samples of mice on the 20th day after cell injection, and WPRE was used as a characteristic fragment to determine the gene copy number of transfected HBV TCR-PD1-BB T cells in the peripheral blood of mice. 6 HBV TCR-PD1-BB group, 3 x 10 6 Significant cell persistence was detected in the blood of mice in the HBV TCR-PD1-BB and bio' HBV TCR-PD1-BB groups. Peripheral blood VCN copy numbers were 91.56 ± 37.34 copies / µg, 1256.35 ± 661.43 copies / µg, and 6330.33 ± 4662.88 copies / µg, respectively (P < 0.01), demonstrating the presence of active HBV TCR-PD1-BB cells 20 days after transfusion (Figure 10c). Furthermore, the degree of HBV TCR-PD1-BB expansion (VCN copy number) in mice showed a significant negative correlation with tumor size. Tumors in mice with a higher VCN copy number exhibited significantly smaller volumes than those in the other groups. These results indicate that greater HBV TCR-PD1-BB expansion in mice correlates with a stronger tumor-killing and inhibitory effect. Example 10: In vivo long-term efficacy experiment of HBV TCR-PD1-BB in the HBsAg+PDL1+ transplanted tumor model of immunodeficient mice Purpose: To evaluate the pharmacodynamics of the test TCR-T cells in the subcutaneous xenograft NCG female mouse model of human liver cancer HepAD38-PDL1-LG-G5 cell line. Experimental animals: NCG mice, female, 6-8 weeks old, weighing 18-22 grams. Tumor cell inoculation: The target cell line was subcutaneously inoculated into the right foreleg axilla of NCG mice, with an inoculation volume of 1x " cells (PBS with Matrigel, 1:1, 0.2 ml). The average tumor volume reached 100-150 mm 3 The dosing schedule for each group is as follows: The results showed that the HBVTCR-PD1-BB high-dose group (2*10 7 ) completely cleared the tumor in the mice and there was no recurrence for 42 days. The low-dose group (0.2*10 7 ) also has a certain anti-tumor effect; HBV TCR high-dose group (2*107 ) showed strong tumor suppression ability, but the tumor recurred significantly one month later, while no tumor suppression effect was observed in the HBV TCR low-dose group, negative control group (UT) and blank control group (Cryoprotectant) ( Figure 11 ).

[0063] HBVTCR-PD1-BB has better in vivo efficacy than HBVTCR-T in the HBsAg+PDL1+ xenograft tumor model, can overcome in vivo immunosuppression, and achieve long-term tumor suppression without recurrence. This application has been described in detail above. Specific examples are used herein to illustrate the principles and implementations of this application. The above examples are intended only to facilitate understanding of the methods and core concepts of this application. Furthermore, those skilled in the art will appreciate that variations in the specific implementations and scope of application are possible based on the principles of this application. Therefore, this specification should not be construed as limiting this application.

Claims

Claims 1. A modified immune cell targeting hepatitis B surface antigen, characterized in that: The modified immune cells contain a T cell receptor (TCR) targeting hepatitis B surface antigen and a chimeric converting receptor; the chimeric converting receptor contains the extracellular domain (ECD) of an immunosuppressive receptor and the intracellular domain (ICD) of a co-stimulatory molecule that mediates immune cell activation signals; the ECD binds to its ligand to produce an immune cell activation signal rather than an immune cell inactivation signal in the modified immune cells.

2. The modified immune cell according to claim 1, characterized in that The TCR comprises TCRα One or two amino acids in the TCRβ chain variable region are replaced by other amino acids; and the amino acid sequence of PCDR3 in the TCRβ chain variable region is as shown in SEQ ID NO: 6, or a variant thereof, wherein one or two amino acids in the TCRβ chain variable region are replaced by other amino acids.

3. The modified immune cell according to claim 2, characterized in that The TCRα chain variable region comprises complementarity determining regions aCDR1, aCDR2 and aCDR3, and the TCRβ chain variable region comprises complementarity determining regions pCDRK, PCDR2 and RCDR3, wherein: aCDR1, aCDR2 and aCDR3 are shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, and pCDRK, RCDR2 and pCDR3 are shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively; or the above-mentioned CDR variants, wherein one or two amino acids in one or more CDRs are replaced by other amino acids.

4. The modified immune cell according to claim 2, characterized in that The TCR α chain variable region comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 7; and / or the TCR β chain variable region comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:

8.

5. The modified immune cell according to any one of claims 1 to 4, characterized in that The immunosuppressive receptor includes any one of PD1, CTLA4, BTLA, TIM3, TIGIT, TGFp receptor and any other protein having immunosuppressive function or associated with immunosuppressive signaling pathways, and combinations thereof, and the immunosuppressive receptor ECD sequence may have at least one amino acid mutation; and / or the costimulatory molecule includes: CD28, 4-1BB, ICOS, CD27, IL-12R, CD3, OX40 and combinations thereof, and the costimulatory molecule ICD sequence may have at least one amino acid mutation.

6. The modified immune cell according to claim 5, characterized in that The ECD is PD1 ECD; preferably, the alanine at position 132 of the PD1 ECD is mutated to leucine, and the mutated PD1 ECD amino acid 27 The sequence is shown in SEQ ID NO:

9.

7. The modified immune cell according to claim 5, characterized in that The ICD is CD28 ICD or 4-1BB ICD; preferably, the amino acid sequence of the CD28 ICD is shown in SEQ ID NO: 12, and the amino acid sequence of the 4-1BB ICD is shown in SEQ ID NO:

13.

8. The modified immune cell according to claim 5, characterized in that The ECD and the ICD are connected by a transmembrane region sequence; the transmembrane region comprises a transmembrane domain of a protein selected from the group consisting of: α, B or β chains of T cell receptors, CD28, CD3α, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154, and combinations thereof, and the transmembrane region sequence may have at least one amino acid mutation; preferably, the transmembrane region sequence is a CD8 transmembrane region sequence or a CD28 transmembrane region sequence.

9. The modified immune cell according to claim 8, characterized in that The CD8 transmembrane region sequence is shown in SEQ ID NO: 10, and the CD28 transmembrane region sequence is shown in SEQ ID NO:

11.

10. The modified immune cell according to claim 1, characterized in that The immune cells are selected from lymphocytes, dendritic cells, macrophages, granulocytes, and mast cells; preferably, the immune cells are T cells.

11. A nucleic acid molecule, characterized in that The nucleic acid molecule comprises a nucleic acid sequence encoding the TCR molecule according to any one of claims 1 to 10; and / or comprises a nucleic acid sequence encoding the chimeric switch receptor according to any one of claims 1 to 10.

12. A carrier, characterized in that The vector comprises the nucleic acid molecule according to claim 11.

13. A pharmaceutical composition, characterized in that The composition contains a pharmaceutically acceptable carrier and the modified immune cell according to any one of claims 1 to 10, the nucleic acid molecule according to claim 11, and the vector according to claim 12.

14. Use of the modified immune cell according to any one of claims 1 to 10, the nucleic acid molecule according to claim 11, the vector according to claim 12, or the pharmaceutical composition according to claim 13 in the preparation of a medicament for preventing or treating diseases related to HBV infection.

15. The use according to claim 14, characterized in that The HBV infection-related diseases include one or more of hepatitis, liver fibrosis, cirrhosis, and liver cancer.

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