HBV surface antigen-specific T cell receptor and its use

Abstract

Provided are an HLA-A02 restricted T cell receptor (TCR) that specifically targets the HBV surface antigen, which is the S20 peptide consisting of FLLTRILTI, and a T cell expressing the TCR. Also provided are a nucleic acid molecule and a vector encoding the TCR, and their medical use in reducing HBV antigen-positive tumor cells and in treating or preventing HBV infection and related diseases.

Description

Technical Field

[0001] The present invention relates to the technical field of immunotherapy, and in particular, to T cell receptors targeting HBV surface antigen, fragments thereof, TCR polypeptides, pharmaceutical compositions and their use.

Background Art

[0002] 1.1 Global Prevalence of HBV HBV is a non-cytopathic double-stranded DNA virus that is mainly transmitted through blood, mother-to-child transmission or sexual transmission. Although highly effective vaccines and antiviral drugs are currently available, HBV still causes a significant disease burden worldwide. In 2015, the number of people living with HBV worldwide was 257 million, and the number of deaths caused by complications of chronic HBV infection exceeded 884,000. Epidemiological studies suggest that Asian and African countries have a higher proportion of HBV infections than the United States or Europe. Furthermore, because HBV lacks the proofreading function of reverse transcriptase, HBV is prone to mutation and is likely to produce different genotypes. Currently, there are 10 different HBV genotypes distributed in different geographical regions.

[0003] 1.2 The Most Common HBV Genotypes in Asians.

[0004] In Asian countries, the genotypes of HBV are mainly B and C subtypes. Since evidence has been reported that HBV genotypes affect clinical outcomes, researchers have focused on the prevalence of different genotypes in different regions. For example, the progression from cirrhosis to HCC is much more likely to occur in genotypes C and D than in other genotypes.

[0005] 1.3 HCC and Its Carcinogenic Pathways HCC (hepatocellular carcinoma) is the primary liver cancer and is the most common and fatal liver cancer among Asians. In 2017, HCC caused approximately 470,000 deaths due to long-term complications of chronic hepatitis infection. The molecular pathology of HCC is a complex process involving various molecular abnormalities and genetic mutations, ultimately leading to the development of malignant diseases and the onset of HCC.

[0006] The integration of HBV-DNA is found in 80 - 90% of HBV-related HCC. At the molecular level, HBV interferes with the gene expression networks of infected hepatocytes and directs them towards cancer progression pathways. The integration of HBV-DNA increases genomic instability by affecting gene expression at the insertion site, thereby activating specific oncogenic pathways such as phosphatidylinositol 3-kinase / Akt, myc, Wnt / β-catenin, c-Met, and hedgehog. Previous studies have suggested that the activation of Akt signaling can inhibit transforming growth factor (TGF)-β-induced apoptosis and promote tumor formation, and is also associated with β-catenin signaling, thereby inducing hepatocellular carcinoma. The molecular changes caused by the integration of HBV-DNA also affect DNA damage checkpoints, leading to tumor formation in cirrhosis. These molecular changes include the loss of function of the p53 tumor suppressor gene, the inactivation of the p27 cell cycle regulator, the loss of heterozygosity at the insulin-like growth inhibitor 2 receptor site, and the loss of expression of the p16 cell cycle arrest protein.

[0007] At the same time, the virus can also inhibit or block innate immunity, thereby affecting the development of the adaptive immune response. This chronic persistent immune response appears at the pathological level as hepatitis and long-term fibrosis, ultimately leading to cirrhosis and liver cancer.

[0008] 1.4 Current Status of HCC Treatment Currently, surgical resection and liver transplantation are the most effective treatment methods for HCC. However, less than 30% of HCC patients meet the criteria for these surgeries, and the postoperative recurrence rate of eligible patients reaches 80% within 5 years. The waiting time for liver donation from a donor is very long, and the waiting list continues to grow. As a result, nearly 25% of patients cannot receive transplantation due to tumor progression.

[0009] The most commonly used palliative therapy is transarterial chemoembolization (TACE), but this therapy is contraindicated in advanced cirrhosis and liver decompensation. This is because ischemic injury associated with embolism can lead to an increase in ascites and death.

[0010] Currently, the targeted drugs approved by the FDA for the treatment of HCC are sorafenib and regorafenib, both of which are tyrosine kinase inhibitors. However, clinical trial data in patients with advanced HCC suggest that these drugs can lead to an increased risk of bleeding and arteriovenous thrombosis. Furthermore, the survival advantage in the indicated population is only 2 to 3 months, and it is very common for the disease to progress over time.

[0011] Some combinations with chemotherapeutic agents, such as PIAF (cisplatin, interferon, doxorubicin, and 5-fluorouracil), GEMOX (gemcitabine and oxaliplatin), and FOLFOX4 (fluorouracil, calcium folinate, and oxaliplatin) are still under development, but chemotherapy generally does not provide a significant survival advantage to HCC patients. However, clinical trials have not been successful and the results are incomplete.

[0012] In recent years, significant breakthroughs have been made in the immunotherapy of HCC. According to the Phase III first-choice immunotherapy trial of Mbrave150, the results showed that the combination of atezolizumab and bevacizumab (T+A) significantly reduced the risk of death and disease progression compared to the standard treatment of sorafenib alone. However, the ORR (RECIST v1.1) was only 27.3%, and the mPFS was only 6.8 months. Among Chinese patients with advanced HCC who had a worse prognosis, the mPFS was only 5.7 months. The above data indicate that although the immunotherapy of HCC has made great progress compared to the past, the effectiveness is still insufficient, and further research on the immunotherapy of HCC is needed.

[0013] Patients with HBV-related HCC require antiviral therapy to inhibit virus replication, reduce serum virus levels, alleviate intrahepatic inflammatory responses, and improve the prognosis associated with cirrhosis. Antiviral therapy can effectively inhibit HBV replication and reduce hepatitis symptoms, but it is noteworthy that the virus cannot be removed and suppressed after treatment is stopped. Nucleosides and nucleoside analogs as well as interferon are often used in the clinical practice of antiviral therapy for the prevention and treatment of HBV-related HCC, but the actual therapeutic effectiveness of these products is uncertain, and the clinical outcomes regarding overall survival and disease recurrence still remain controversial. Furthermore, the long-term use of antiviral drugs is associated with the occurrence of drug resistance, and continuous use may render these drugs ineffective. For patients treated with chemotherapy, prophylactic antiviral therapy using entecavir or tenofovir is recommended due to the possibility of HBV reactivation.

[0014] In conclusion, due to the limitations and high risks of currently available treatment methods and drugs, new treatment strategies are needed to provide more options for patients with chronic hepatitis B and HBV-related HCC.

[0015] 1.5 The role of T cells in the immunotherapy of HBV-related HCC and the theoretical basis for using TCR T cells T cells are immune cells derived from bone marrow and lymph and mature in the thymus. T cells express T cell antigen receptors (TCRs) on their surface and play an important role in removing infections and cancer cells in cell-mediated immunity. TCRs recognize and specifically bind to target epitopes presented by major histocompatibility complex (MHC) molecules. When T cells recognize their target, they can kill the target cells by massive proliferation, cytokine release, and cytotoxicity.

[0016] Adoptive T cell immunotherapy has been attempted to treat human malignancies such as leukemia, as well as viral diseases such as cytomegalovirus (CMV) and Epstein-Barr virus (Epstein-Barr virus, EBV). However, the isolation and proliferation of virus- or tumor-specific T cells from patients' blood are difficult and time-consuming. Therefore, by adopting a new treatment strategy and introducing a T cell receptor (TCR) or TCRα / β heterodimer targeting a specific antigen, gene-edited T lymphocytes are enabled to act on specific viruses or tumor antigens, and T cells with high antigen specificity are obtained.

[0017] Hepatitis B virus synthesizes HBsAg protein in infected hepatocytes, aggregates in the endoplasmic reticulum to form virus (sub-virus) particles, which are either secreted or reach the cell surface via physiological exchange of the membrane. HBsAg expression is also commonly found in HCC cells integrated with HBV-DNA.

[0018] Clinical evidence suggests that adoptive immunotherapy with HBV-specific T cells can control HBV replication or tumor growth. Leukemia patients can even acquire immunity against HBV after bone marrow transplantation by receiving bone marrow from donors who have acquired immunity specifically against HBV (either from HBV vaccination or from self-immunity to achieve recovery from HBV infection). Similarly, transplantation of HBV-positive livers in subjects with immunity specific to HBV can also remove HBV infection in the transplanted liver.

[0019] Clinical evidence suggests that adoptive immunotherapy using HBV-specific T cells can control HBV replication and tumor growth. Leukemia patients can even acquire immunity against HBV after bone marrow transplantation by being vaccinated against HBV or receiving bone marrow from HBV-specific donors who rely on autoimmunity to achieve recovery from HBV infection. Similarly, transplantation of HBV-positive livers in subjects with immunity specific to HBV can also eliminate HBV infection in the transplanted liver.

[0020] However, HBV-specific T cell immunity is severely impaired in many patients with chronic hepatitis B and HBV-related HCC. In these patients, HBV-specific T cells are functionally defective, depleted, and prone to exhaustion. Due to these functional defects, HBV-specific T cells are rarely detected by in vitro analysis of the patient's blood.

[0021] Therefore, constructing high-affinity TCR-T cells for reinfusion therapy has become a new option. This involves using genetic manipulation and other biological methods to develop specific TCRs targeting the HBV surface antigen, isolating patient T cells, and transfecting them in vitro. However, currently, safe and effective TCR-Ts for the treatment of diseases caused by HBV infection, especially HBV-induced liver cancer, have not been proven.

Summary of the Invention

Problems to be Solved by the Invention

[0022] Through extensive and thorough research, the present application provides a T cell receptor (TCR) and modified TCR-T cells that specifically target the HBV surface antigen and result in the elimination of liver cancer cells caused by HBV infection.

Means for Solving the Problems

[0023] This application provides a novel TCR molecule or a fragment thereof that specifically binds to the hepatitis B surface antigen FLLTRILTI-HLA-A2 complex. The T cell receptor (TCR) or a fragment thereof is Amino acid sequence: αCDR3: ATDERDDMR (SEQ ID NO: 3), or a variant thereof in which one or two amino acids are replaced by another amino acid A TCR α-chain variable region containing an αCDR3 having And Amino acid sequence: βCDR3: ASSLNTEAF (SEQ ID NO: 6) or a variant thereof in which one or two amino acids are replaced by another amino acid A TCR β-chain variable region containing a βCDR3 having Including.

[0024] In other embodiments, the amino acid sequence: αCDR3: GADTSTDKLI (SEQ ID NO: 15), or a variant thereof in which one or two amino acids are replaced by another amino acid A TCR α-chain variable region containing an αCDR3 having And The TCR β-chain variable region has the amino acid sequence: βCDR3: ASSHGGAYEQY (SEQ ID NO: 18) or a variant thereof in which one or two amino acids are replaced by another amino acid Including a βCDR3 having.

[0025] In other embodiments, the amino acid sequence: αCDR3: ATDAYGQNFV (SEQ ID NO: 24), or a variant thereof in which one or two amino acids are replaced by another amino acid A TCR α-chain variable region containing an αCDR3 having And Amino acid sequence: βCDR3: ASGSNTEAF (SEQ ID NO: 25) or a variant thereof in which one or two amino acids are replaced by another amino acid It is a variable region of the TCRβ chain containing a βCDR3 having [conditions not specified].

[0026] In some embodiments, a variable region of the TCRα chain containing αCDR1, αCDR2, and αCDR3 shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and a variable region of the TCRβ chain containing βCDR1, βCDR2, and βCDR3 shown in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively, or a variant thereof in which one or two amino acids in one or more of the CDRs are replaced with another amino acid.

[0027] In some embodiments, a variable region of the TCRα chain containing αCDR1, αCDR2, and αCDR3 shown in SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, respectively, as well as a variable region of the TCRβ chain containing βCDR1, βCDR2, and βCDR3 shown in SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively, or a variant thereof in which one or two amino acids in one or more of the CDRs are replaced with another amino acid.

[0028] In some embodiments, a variable region of the TCRα chain containing αCDR1, αCDR2, and αCDR3 shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 24, respectively, as well as a variable region of the TCRβ chain containing βCDR1, βCDR2, and βCDR3 shown in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 25, respectively, or a variant thereof in which one or two amino acids in one or more of the CDRs are replaced with another amino acid.

[0029] In some embodiments, the variable region of the TCRα chain contains an amino acid sequence that is at least 90% identical to any one of SEQ ID NO: 7, SEQ ID NO: 19, and SEQ ID NO: 26.

[0030] In some embodiments, the variable region of the TCRβ chain comprises an amino acid sequence that is at least 90% identical to any one of SEQ ID NO: 8, SEQ ID NO: 20, and SEQ ID NO: 27.

[0031] In some embodiments, the variable region of the TCRα chain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 7. In some embodiments, the variable region of the TCRβ chain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 8.

[0032] In some embodiments, the variable region of the TCRα chain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 19. In some embodiments, the variable region of the TCRβ chain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 20.

[0033] In some embodiments, the variable region of the TCRα chain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 26. In some embodiments, the variable region of the TCRβ chain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 27.

[0034] In some embodiments, the variable region of the TCRα chain comprises the amino acid sequence set forth in SEQ ID NO: 7, and the variable region of the TCRβ chain comprises the amino acid sequence set forth in SEQ ID NO: 8. In some embodiments, the variable region of the TCRα chain comprises the amino acid sequence set forth in SEQ ID NO: 19, and the variable region of the TCRβ chain comprises the amino acid sequence set forth in SEQ ID NO: 20. In some embodiments, the variable region of the TCRα chain comprises the amino acid sequence set forth in SEQ ID NO: 26, and the variable region of the TCRβ chain comprises the amino acid sequence set forth in SEQ ID NO: 27.

[0035] In some embodiments, the TCR comprises the TCRα chain constant region TRAC * 01 and the TCRβ chain constant region TRBC1 * 01 or TRBC2 *It is an αβ heterodimer containing 01. The constant region can be selected from the human TCR constant region sequence or the mouse constant region sequence. In some embodiments, by introducing at least one additional artificial disulfide bond into the constant region, the stability of the heterodimer is increased and the proportion of mismatches with the endogenous chain is reduced.

[0036] In some embodiments, the amino acid sequence of the TCR α chain is set forth in SEQ ID NO: 10. In some embodiments, the TCR β chain amino acid sequence is set forth in SEQ ID NO: 11. In some embodiments, the amino acid sequence of the TCR α chain is set forth in SEQ ID NO: 21. In some embodiments, the TCR β chain amino acid sequence is set forth in SEQ ID NO: 22. In some embodiments, the amino acid sequence of the TCR α chain is set forth in SEQ ID NO: 28. In some embodiments, the TCR β chain amino acid sequence is set forth in SEQ ID NO: 29.

[0037] In some embodiments, the TCR is a single-chain fusion protein. In some embodiments, the single-chain TCR is formed by linking the TCR β chain and the TCR α chain via P2A.

[0038] In some embodiments, from the N-terminus to the C-terminus, the TCR comprises a T cell receptor β chain variable region (TRBV), a T cell receptor β chain constant region (TRBC), a P2A peptide, a T cell receptor α chain variable region (TRAV), and a T cell receptor α chain constant region (TRAC). In some embodiments, the amino acid sequence of the P2A peptide comprises the amino acid sequence set forth in SEQ ID NO: 9. In some embodiments, the amino acid sequence of the single-chain TCR is set forth in SEQ ID NO: 12. In some embodiments, the amino acid sequence of the single-chain TCR is set forth in SEQ ID NO: 23. In other embodiments, the amino acid sequence of the single-chain TCR is set forth in SEQ ID NO: 30.

[0039] In some embodiments, the cysteine residues form at least one artificial disulfide bond between the α-chain constant region and the β-chain constant region of the TCR.

[0040] In some embodiments, the TCR is soluble.

[0041] In some embodiments, the TCR comprises (a) all or a portion of the TCRα chain excluding the transmembrane region, and (b) all or a portion of the TCRβ chain excluding the transmembrane region, wherein both (a) and (b) either comprise a functional variable region or comprise a functional variable region and at least a portion of the TCR chain constant region.

[0042] In some embodiments, it is a TCR or fragment thereof that can bind to a polypeptide of the HBV surface antigen presented by HLA-A2. In some embodiments, the polypeptide comprises or consists of the amino acid sequence FLLTRILTI (SEQ ID NO: 31) or FLLTKILTI (SEQ ID NO: 32).

[0043] In some embodiments, at least one conjugate is bound to the C-terminus or N-terminus of the α-chain and / or β-chain of the TCR, and the conjugate bound to the T cell receptor may be a detectable label, a therapeutic agent, a PK modification moiety, or any combination thereof.

[0044] This application further provides a nucleic acid molecule encoding the above TCR or fragment.

[0045] In some embodiments, the expression of the TCR gene can be successfully improved by using codon optimization.

[0046] In some embodiments, the coding sequence of TRAV is set forth in SEQ ID NO: 33. In some embodiments, the coding sequence of TRBV is set forth in SEQ ID NO: 34. In some embodiments, the coding sequence of TRAV is set forth in SEQ ID NO: 38. In some embodiments, the coding sequence of TRBV is set forth in SEQ ID NO: 39. In some embodiments, the coding sequence of TRAV is set forth in SEQ ID NO: 43. In some embodiments, the coding sequence f of TRBV is set forth in SEQ ID NO: 44.

[0047] In some embodiments, the coding sequence of the TCRα chain is set forth in SEQ ID NO: 35. In some embodiments, the coding sequence of the TCRβ chain is set forth in SEQ ID NO: 36. In some embodiments, the coding sequence of the TCRα chain is set forth in SEQ ID NO: 40. In some embodiments, the coding sequence of the TCRβ chain is set forth in SEQ ID NO: 41. In some embodiments, the coding sequence of the TCRα chain is set forth in SEQ ID NO: 45. In some embodiments, the coding sequence of the TCRβ chain is set forth in SEQ ID NO: 46.

[0048] In some embodiments, the coding sequence of the TCR is single-stranded, and the coding sequences of the TCRβ chain and the TCRα chain are linked by a P2A coding sequence. In some embodiments, the single-stranded coding sequence of the TCR is set forth in SEQ ID NO: 37, SEQ ID NO: 42, or SEQ ID NO: 47.

[0049] This application further provides an expression vector comprising the above nucleic acid molecule.

[0050] In some embodiments, the vector is selected from the group consisting of plasmids, binary vectors, DNA vectors, mRNA vectors, retroviral vectors, lentiviral vectors, transposon-based vectors, and artificial chromosomes. In some embodiments, the vector is a viral vector, and in some embodiments, the vector is a lentiviral vector.

[0051] This application further provides a polypeptide encoded by the above nucleic acid molecule or vector.

[0052] This application further provides a host cell comprising the above TCR or fragment, nucleic acid molecule, vector, or polypeptide.

[0053] In some embodiments, the cell is a stem cell, in some embodiments, the cell is an NK cell, in some embodiments, the cell is a T cell, and in some embodiments, the cell is CD4 +T cells, and in some embodiments, the cells are CD8 + T cells.

[0054] This application further provides a pharmaceutical composition comprising a TCR or fragment, nucleic acid molecule, vector, polypeptide, or host cell, and a pharmaceutically acceptable excipient, diluent, or carrier.

[0055] This application further provides the use of a TCR or fragment thereof, nucleic acid molecule, vector, polypeptide, host cell or pharmaceutical composition in the manufacture of a medicament for preventing or treating HBV infection and other related diseases. In some embodiments, the diseases caused by HBV infection are hepatitis, liver fibrosis, cirrhosis, or liver cancer.

[0056] This application further provides a TCR or fragment, nucleic acid molecule, vector, polypeptide, cell, or pharmaceutical composition for use in the treatment of HBV infection or diseases caused by HBV infection. In some embodiments, the diseases caused by HBV infection include one or more of hepatitis, liver fibrosis, cirrhosis, and liver cancer.

[0057] It should be understood that within the scope of this application, the above-described technical features of this application and the technical features specified below (e.g., embodiments) can be combined with each other to form novel or preferred technical solutions. Due to space limitations, not all of them can be described here.

Advantages of the Invention

[0058] This application provides a novel specific TCR or fragment targeting the HBV surface antigen. It is surprising to find that the TCR of this application can target and recognize four genotypes of the HBV surface antigen S20-28, including genotypes A, B, C, and D. The TCR can recognize the most common HLA allele subtypes in the HBV epidemic population. Therefore, the population applicable to the TCR of this application has a wider range. The TCR-T cells of this application have very strong antiviral ability in vitro and in vivo, can specifically target and kill HBV-infected cells. Therefore, the TCR can be used for the treatment of HBV infection and related diseases including hepatitis, liver fibrosis, cirrhosis, and liver cancer without off-target side effects.

[0059] On the other hand, this application modifies the constant region by introducing an inter-chain disulfide bond via cysteine, improves the stability of the transduced TCR / heterodimer, and reduces the mispairing with the endogenous TCR chain. Furthermore, this application optimizes the expression codons of the TCR to improve the expression efficiency.

Brief Description of the Drawings

[0060]

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Mode for Carrying Out the Invention

[0061] Definitions To more easily understand the present invention, specific technical terms and scientific terms are specifically defined below. Unless specifically defined elsewhere in this specification, all other technical terms and scientific terms used in this specification have the meanings generally understood by those skilled in the technical field to which the present invention pertains.

[0062] As used herein, "hepatitis B surface antigen T cell receptor (TCR)" refers to a TCR that binds to a complex of a major histocompatibility complex (MHC) and a HBV surface antigen and induces a helper response or a cytotoxic response. Specifically, the HBV surface antigen may be HBs20-28, which can be used interchangeably with HBs20, HBs20-28, S20-28, and S20 in this application. Unless otherwise specified, the HBV surface antigen refers to the S20-28 antigens of genotypes A and D having the amino acid sequence FLLTRILTI. In some embodiments, the HBV surface antigen is HBV genotypes A and D, and in some embodiments, the HBV surface antigen is HBV genotypes B and C. In some embodiments, the HBV surface antigen contains the FLLTRILTI (SEQ ID NO: 31) amino acids, and in some embodiments, the HBV surface antigen contains the FLLTKILTI (SEQ ID NO: 32) amino acids. In some embodiments, the HBV surface antigen has the amino acids of FLLTRILTI (SEQ ID NO: 31), and in some embodiments, the HBV surface antigen has the amino acids of FLLTKILTI (SEQ ID NO: 32).

[0063] The term "MHC molecule" refers to a protein of the immunoglobulin superfamily that may be a class I or class II MHC molecule. MHC molecules are specific for antigen presentation, and different individuals have different MHCs and can present different short peptides in protein antigens on the surface of APC cells. Human MHC is often referred to as the HLA gene or HLA complex.

[0064] TCR is a glycoprotein found on the surface of the T cell membrane, existing as an α-chain / β-chain or γ-chain / δ-chain heterodimer. The δTCR heterodimer consists of an α-chain and a β-chain in 95% of T cells, while 5% of T cells have a TCR composed of a γ-chain and a δ-chain. Natural heterodimeric TCR has an α-chain and a β-chain, and the α-chain and β-chain are subunits of the heterodimeric TCR. In a broad sense, the α-chain and β-chain each contain a variable region, a linker, and a constant region. The β-chain also typically contains a short variable region between the variable region and the linker region, and the short variable region is often regarded as part of the linker region. The variable region contains CDR1, CDR2, and CDR3, which are three CDRs (complementary determining regions). The CDRs are chimerized in the framework region. The CDR regions of the α-chain and β-chain in this application are delimited using the IMGT numbering system. The CDR regions determine the binding of the TCR to the pMHC complex. The sequences of the TCR constant regions can be found in the public database of IMGT. For example, the constant region sequence of the α-chain is "TRAC*01", and the constant domain sequences of the β-chain are "TRBC1*01" or "TRBC2*01".

[0065] In this application, the terms "T cell receptor", "TCR", and "TCR molecule" are used interchangeably.

[0066] TCR molecule The TCR or fragment of this application recognizes the HBV surface antigen that is HLA-A2 restricted. Approximately 50% of the general population expresses the MHC class I molecule HLA-A2, HLA-A serotype. Therefore, HLA-A2 restricted TCRs may find extensive therapeutic use. In particular, the subtypes are HLA-A * 0201, * 0202, * 0203, * 0206, and * 0207 gene products, including many HLA-A *The gene products of allelic genes can be identified. There may be a clear difference in subtypes between the white population and the Asian population, but more than 95% of the HLA-A2 positive white population is HLA-A0201. The HLA-A2 positive Chinese population can be classified into 23% HLA-A0201, 45% HLA-A0207, 8% HLA-A0206, and 23% HLA-A0203.

[0067] In some embodiments, the TCR comprises a variable region of the TCRα chain and a variable region of the TCRβ chain, each having three complementarity determining regions (CDRs).

[0068] In some embodiments, an amino acid sequence: αCDR3: ATDERDDMR (SEQ ID NO: 3), or an αCDR3 having a variant thereof in which one or two amino acids are replaced by another amino acid, is included in the variable region of the TCRα chain, and / or and an amino acid sequence: βCDR3: ASSLNTEAF (SEQ ID NO: 6) or a βCDR3 having a variant thereof in which one or two amino acids are replaced by another amino acid, is included in the variable region of the TCRβ chain.

[0069] In other embodiments, an amino acid sequence: αCDR3: GADTSTDKLI (SEQ ID NO: 15), or an αCDR3 having a variant thereof in which one or two amino acids are replaced by another amino acid, is included in the variable region of the TCRα chain, and / or and an amino acid sequence: βCDR3: ASSHGGAYEQY (SEQ ID NO: 18) or a βCDR3 having a variant thereof in which one or two amino acids are replaced by another amino acid, is included in the variable region of the TCRβ chain.

[0070] In other embodiments, an amino acid sequence: αCDR3: ATDAYGQNFV (SEQ ID NO: 24), or an αCDR3 having a variant thereof in which one or two amino acids are replaced by another amino acid, is included in the variable region of the TCRα chain, and / or Amino acid sequence: βCDR3: ASGSNTEAF (SEQ ID NO: 25) or a TCRβ chain variable region comprising a βCDR3 having a variant thereof in which one or two amino acids are replaced with another amino acid.

[0071] In some embodiments, a variable region of the TCRα chain comprising αCDR1, αCDR2, and αCDR3 shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and / or a variable region of the TCRβ chain comprising βCDR1, βCDR2, and βCDR3 shown in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively, or a variant thereof in which one or two amino acids in one or more of the CDRs are replaced with another amino acid.

[0072] In some embodiments, a variable region of the TCRα chain comprising αCDR1, αCDR2, and αCDR3 shown in SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, respectively, and / or a variable region of the TCRβ chain comprising βCDR1, βCDR2, and βCDR3 shown in SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively, or a variant thereof in which one or two amino acids in one or more of the CDRs are replaced with another amino acid.

[0073] In some embodiments, a variable region of the TCRα chain comprising αCDR1, αCDR2, and αCDR3 shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 24, respectively, and / or a variable region of the TCRβ chain comprising βCDR1, βCDR2, and βCDR3 shown in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 25, respectively, or a variant thereof in which one or two amino acids in one or more of the CDRs are replaced with another amino acid.

[0074] As long as the framework structure is compatible with the CDR region, a chimeric TCR can be prepared by inserting the described amino acid sequence of the CDR region of the present application into any suitable framework structure. Those skilled in the art can design or synthesize a TCR molecule with corresponding functions based on the CDR regions disclosed in the present application. Therefore, the TCR in the present application refers to a TCR comprising the above-described α and / or β chain CDR region amino acid sequences and any suitable framework structure. The amino acid sequence of the variable region of the TCR α chain of the present application having at least 90%, preferably 95%, more preferably 98% sequence identity with SEQ ID NO: 7, SEQ ID NO: 19 or SEQ ID NO: 26 and / or the amino acid sequence of the variable region of the TCR β chain having at least 90%, preferably 95%, more preferably 98% sequence identity with SEQ ID NO: 8, SEQ ID NO: 20 or SEQ ID NO: 27.

[0075] In some embodiments, the variable region of the TCR α chain comprises the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, the variable region of the TCR β chain comprises the amino acid sequence set forth in SEQ ID NO: 8. In some embodiments, the variable region of the TCR α chain comprises the amino acid sequence set forth in SEQ ID NO: 19. In some embodiments, the variable region of the TCR β chain comprises the amino acid sequence set forth in SEQ ID NO: 20. In some embodiments, the variable region of the TCR α chain comprises the amino acid sequence set forth in SEQ ID NO: 26. In some embodiments, the variable region of the TCR β chain comprises the amino acid sequence set forth in SEQ ID NO: 27.

[0076] In some embodiments, the amino acid sequence of the variable region of the TCR α chain is as set forth in SEQ ID NO: 7, and the amino acid sequence of the variable region of the TCR β chain is as set forth in SEQ ID NO: 8. In some embodiments, the amino acid sequence of the variable region of the TCR α chain is as set forth in SEQ ID NO: 19, and the amino acid sequence of the variable region of the TCR β chain is as set forth in SEQ ID NO: 20. In some embodiments, the amino acid sequence of the variable region of the TCR α chain is as set forth in SEQ ID NO: 26, and the amino acid sequence of the variable region of the TCR β chain is as set forth in SEQ ID NO: 27.

[0077] In some embodiments, the TCR is an αβ heterodimer comprising a TCR α-chain constant region and a TCR β-chain constant region. In some embodiments, the constant region of the TCR molecule of the present application is a human constant region. One skilled in the art can know or obtain the human constant region amino acid sequence by referring to relevant books or the publicly available IMGT database. For example, the constant region sequence of the α-chain is "TRAC * 01", and the constant region sequence of the β-chain is "TRBC1 * 01" or "TRBC2 * 01". In some embodiments, additional disulfide bonds are introduced into the constant region to improve stability and reduce the mismatch between the exogenous TCR molecule and the endogenous TCR molecule. The constant region may also be a mouse constant region. By replacing TRAC and TRBC with constant domains derived from mice, the mismatch between the exogenous TCR molecule and the endogenous TCR molecule can be avoided. This effect is the same as the purpose of the exogenous introduction of artificial disulfide bonds.

[0078] In some embodiments, the amino acid sequence of the α-chain of the TCR is set forth in SEQ ID NO: 10, and the amino acid sequence of the β-chain of the TCR is set forth in SEQ ID NO: 11. In some embodiments, the amino acid sequence of the α-chain of the TCR is set forth in SEQ ID NO: 21, and the amino acid sequence of the β-chain of the TCR is set forth in SEQ ID NO: 22. In some embodiments, the amino acid sequence of the α-chain of the TCR is set forth in SEQ ID NO: 28, and the amino acid sequence of the β-chain of the TCR is set forth in SEQ ID NO: 29.

[0079] In some embodiments, the TCR molecule of the present application is a single chain consisting of part or all of the α chain and / or part or all of the β chain. In some embodiments, the single-chain TCR is formed by linking the amino acid sequence of the TCRβ chain and the amino acid sequence of the TCRα chain via P2A. In a preferred embodiment of the present application, the T cell antigen receptor polypeptide contains TRBV, TRBC, P2A, TRAV, and TRAC from the N-terminus to the C-terminus. In another preferred embodiment of the present application, the T cell antigen receptor polypeptide contains TRAV, TRAC, P2A, TRBV, and TRBC from the N-terminus to the C-terminus.

[0080] In some embodiments, the α-chain variable region of the single-chain TCR molecule contains CDR1 (SEQ ID NO: 1), CDR2 (SEQ ID NO: 2), and CDR3 (SEQ ID NO: 3), and preferably, is the α-chain variable region shown in SEQ ID NO: 7. The β-chain variable region of the single-chain TCR molecule contains CDR1 (SEQ ID NO: 4), CDR2 (SEQ ID NO: 5), and CDR3 (SEQ ID NO: 6), and preferably, is the β-chain variable region shown in SEQ ID NO: 8.

[0081] In some embodiments, the α-chain variable region of the single-chain TCR molecule contains CDR1 (SEQ ID NO: 13), CDR2 (SEQ ID NO: 14), and CDR3 (SEQ ID NO: 15), and preferably, is the α-chain variable region shown in SEQ ID NO: 19. The β-chain variable region of the single-chain TCR molecule contains CDR1 (SEQ ID NO: 16), CDR2 (SEQ ID NO: 17), and CDR3 (SEQ ID NO: 18), and preferably, is the β-chain variable region shown in SEQ ID NO: 20.

[0082] In some embodiments, the α-chain variable region of the single-chain TCR molecule contains CDR1 (SEQ ID NO: 1), CDR2 (SEQ ID NO: 2), and CDR3 (SEQ ID NO: 24), and preferably, is the α-chain variable region shown in SEQ ID NO: 26. The β-chain variable region of the single-chain TCR molecule contains CDR1 (SEQ ID NO: 4), CDR2 (SEQ ID NO: 5), and CDR3 (SEQ ID NO: 25), and preferably, is the β-chain variable region shown in SEQ ID NO: 27.

[0083] In some embodiments, the amino acid sequence of the single-chain TCR is set forth in SEQ ID NO: 12. In some embodiments, the amino acid sequence of the single-chain TCR is set forth in SEQ ID NO: 23. In some embodiments, the amino acid sequence of the single-chain TCR is set forth in SEQ ID NO: 30.

[0084] Natural TCRs are membrane proteins stabilized by their transmembrane regions. Similar to immunoglobulins (antibodies) as antigen recognition molecules, TCRs can also be developed for diagnostic and therapeutic applications where it is necessary to obtain soluble TCR molecules.

[0085] Soluble TCR molecules do not contain their transmembrane domains. Soluble TCR molecules have a wide range of applications not only for studying the interaction between TCR and pMHC, but also as diagnostic tools for detecting infections or as markers for autoimmune diseases. Soluble TCRs can be used to deliver therapeutic agents (e.g., cytotoxic or immunostimulatory compounds) to cells presenting specific antigens. Soluble TCRs can also be conjugated to other molecules (e.g., anti-CD3 antibodies) to redirect T cells to target cells presenting specific antigens.

[0086] In some embodiments, the TCR is soluble.

[0087] In some embodiments, the TCR comprises (a) all or part of the TCR α-chain excluding the transmembrane domain, and (b) all or part of the TCR β-chain excluding the transmembrane domain, wherein both (a) and (b) contain a functional variable domain or contain a functional variable domain and at least part of the TCR chain constant domain.

[0088] In some embodiments, an artificial disulfide bond exists between the α-chain constant region and the β-chain constant region of the soluble TCR.

[0089] Soluble TCRs can be prepared by any method known in the art. Examples of processes that can be used to prepare soluble TCRs can include constructing multimeric receptor chains where the immunoglobulin heavy chain variable region from at least one phosphocholine-specific antibody can be replaced with TCRα and β variable regions, introducing a translation termination codon upstream of the TCR transmembrane region, or replacing the transmembrane domains of TCRα and β chain cDNAs with a glycosylphosphatidylinositol (GPI)-linking signal from the carboxy terminus of the GPI-linked protein Thy-1, but are not limited thereto.

[0090] The soluble TCRs of the present application may be used alone or may be combined with a conjugate, preferably by covalent bonding such as covalent bonding. The conjugate can include a detectable label, a therapeutic agent, a PK-modifying moiety, or any combination or conjugation thereof.

[0091] Examples of detectable labels for diagnostic purposes can include fluorescent or chemiluminescent labels, radioactive labels, contrast agents for MRI (magnetic resonance imaging) or CT (computed tomography), or enzymes capable of producing a detectable product, but are not limited thereto.

[0092] Therapeutic agents that can be conjugated to the TCRs of the present application can include chemotherapeutic agents (e.g., cisplatin), prodrug-activating enzymes, cytokines, toxins (e.g., PE38, calicheamicin, or diphtheria toxin), immunomodulatory antibody fragments (e.g., anti-CD3 or anti-CD16, Fc fragments, scFv), radionuclides, virus particles, liposomes, gold nanoparticles, nanomagnetic particles, or nanoparticles in any form, but are not limited thereto.

[0093] The soluble TCR can be linked to at least one antiviral drug. The antiviral drug can target HBV. For example, the antiviral drug can include, but is not limited to, adefovir dipivoxil, interferon α-2b, pegylated interferon α-2a, lamivudine, entecavir, telbivudine, etc.

[0094] The TCRs of the present application can also be provided in the form of multivalent complexes. The multivalent TCR complexes of the present application include two, three, four, or more multimers formed by combining the TCRs of the present application. For example, using the tetramerization domain of p53, tetramers, or complexes formed by binding one or more TCRs of the present application to another molecule can be generated. Compared with the non-multimeric wild-type or TCR heterodimers of T cells of the present application, the binding ability of the multivalent TCR complexes of the present application to the FLLTRILTI-HLA-A * 02 complex can be enhanced. Therefore, the multivalent complexes of the TCRs of the present application also belong to the present application. The TCR complexes of the present application can be used to track or target cells presenting specific antigens in vitro or in vivo, and to generate intermediates for other multivalent TCR complexes having such uses.

[0095] Nucleic acid molecule The present application provides a nucleic acid molecule encoding the TCR molecule described above in the present specification, or a fragment thereof, which may be one or more CDRs, variable regions of the α and / or β chains, or the α and / or β chains.

[0096] In some embodiments, the nucleic acid encodes one or more structural features for increasing and / or stabilizing the association between the expressed TCR α-chain and β-chain. In some embodiments, the feature may be a specific amino acid or amino acid sequence. 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 α-chain and β-chain. In some embodiments, the nucleic acid may encode one or more non-natural cysteine residues in the constant domains of the TCR α-chain and β-chain.

[0097] The nucleic acid molecule of the present application may be single-stranded or double-stranded, the nucleic acid molecule may be RNA or DNA, and may or may not contain introns. Preferably, the nucleic acid molecule of the present application does not contain introns, but can encode the TCR of the present application or a fragment thereof.

[0098] The expression of the TCR gene could be successfully improved by codon optimization. Different species prefer codons with different biases. Depending on the cell type, the codons in the sequence can be changed to increase the expression level. Codon usage frequency tables for mammalian cells, as well as various other organisms, are known to those skilled in the art.

[0099] A nucleic acid sequence encoding TRAV of the present application as set forth in SEQ ID NO: 33, SEQ ID NO: 38, or SEQ ID NO: 43, and / or a nucleic acid sequence encoding TRBV as set forth in SEQ ID NO: 34, SEQ ID NO: 39, or SEQ ID NO: 44.

[0100] A nucleic acid sequence encoding the TCR α-chain of the present application as set forth in SEQ ID NO: 35, SEQ ID NO: 40, or SEQ ID NO: 45, and / or a nucleic acid sequence encoding the TCR β-chain as set forth in SEQ ID NO: 36, SEQ ID NO: 41, or SEQ ID NO: 46.

[0101] In some embodiments, the coding sequence of the TCR is single-stranded, the coding sequence of the TCRβ chain and the coding sequence of the TCRα chain are linked by a P2A coding sequence, and the single-stranded coding nucleic acid is within the same reading frame. In some embodiments, the single-stranded coding sequence of the TCR is set forth in SEQ ID NO: 37, SEQ ID NO: 42, or SEQ ID NO: 47.

[0102] Expression vector This application provides at least one "vector" (DNA or RNA) as a medium for transferring exogenous nucleic acid into cells. The vector may be an expression vector for expressing nucleic acid within the cell. Such a vector may include a promoter sequence operably linked to the nucleic acid encoding the sequence to be expressed. The vector may also include a stop codon and an expression enhancer.

[0103] In some embodiments, this application provides at least one construct comprising a polynucleotide of this application operably connected to at least one promoter. The coding sequences of the α and β chains of the TCR can be operably linked to at least one promoter that is functional in isolated cells. Suitable promoters may be constitutive and inducible promoters, and the selection of a suitable promoter may be well within the scope of those skilled in the art. For example, suitable promoters may include, but are not limited to, retroviral LTR, SV40 promoter, CMV promoter, and cellular promoters (e.g., β-actin promoter).

[0104] In some embodiments, the present application provides at least one vector comprising a construct according to the present application or a polynucleotide according to the present invention. In particular, the vector can include, but is not limited to, plasmids, binary vectors, mRNA vectors, lentiviral vectors, retroviral vectors, adenoviral vectors, adeno-associated viral vectors, and herpes simplex viral vectors. More specifically, as described in the examples, lentiviral vectors can be used for delivery of constructs either in vitro, ex vivo, or in vivo.

[0105] cell The present application also includes isolated cells that express a TCR and / or fragment, wherein the cells can be stem cells or immune cells. The immune cells can be T cells, natural killer cells, dendritic cells, or macrophages. In some embodiments, the immune cells are T cells. The T cells can be derived from T cells isolated from a subject or can be part of a mixed population of cells isolated from a subject, such as a peripheral blood lymphocyte (PBL) population. For example, the cells can be isolated from peripheral blood mononuclear cells (PBMC), CD4 + helper T cells, or CD8 + cytotoxic T cells. The cells can be present in a mixed population of CD4 + helper T cells / CD8 + cytotoxic T cells. Typically, the cells are activated with an antibody (e.g., anti-CD3 antibody) to be more receptive to transfection with a vector containing, for example, a nucleic acid sequence encoding a TCR molecule of the present application. In some embodiments, the cells of the present application can also be stem cells, such as hematopoietic stem cells (HSC). Since the CD3 molecule is not expressed on the surface of stem cells, transfer of the TCR gene into HSC does not result in expression of the TCR on the cell surface. However, when HSC differentiate into lymphoid precursors that migrate to the thymus, expression of the CD3 molecule initiates expression of the TCR molecule. Cells expressing a TCR or fragment of the present application can be suitable for use in adoptive transfer protocols for providing a particularly effective treatment modality. The cells of the present application can be HBV-specific CD8 +and CD4 + The problem that the cells are lacking or insufficiently functional can be overcome.

[0106] There are numerous methods suitable for transfection of T cells with DNA or RNA encoding the TCR or a fragment thereof of the present application (e.g., Robbins et al. (2008) J. Immunol. 180: 6116 - 6131). Methods for introducing polynucleotide molecules or vectors into cells are known in the art. Vectors can be readily introduced into host cells, such as 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.

[0107] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. Biological methods for introducing the polynucleotide of interest into host cells include the use of DNA and RNA vectors. Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems such as polymer complexes, nanocapsules, microspheres, as well as lipid - based systems including water - in - oil emulsions, micelles, mixed micelles, and liposomes.

[0108] The present application also provides a method for producing a TCR, fragment, or polypeptide according to the present application, which includes introducing a vector from the present invention into a cell and culturing the cell under conditions suitable for expression of the vector by the cell.

[0109] Any cell suitable for the expression of a polypeptide can be used to produce the TCRs, fragments, and polypeptides according to the present invention. The cell may be prokaryotic or eukaryotic. Suitable prokaryotic cells include Escherichia coli (E. coli). Examples of eukaryotic cells include yeast cells, plant cells, insect cells, or mammalian cells. In some cases, the cell is not a prokaryotic cell because some prokaryotic cells do not allow the same post-translational modifications as eukaryotes. Furthermore, very high expression levels can be achieved in eukaryotes, and the protein can be easily purified from eukaryotes using appropriate tags. Specific plasmids that enhance the secretion of the TCR, fragment, or polypeptide into the medium can also be utilized.

[0110] Composition This application also provides a composition comprising a TCR, fragment, nucleic acid, vector, polypeptide, or cell according to the present invention. In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the composition is a composition suitable for use in research, treatment, prevention, and / or diagnosis.

[0111] In some embodiments, the TCR, fragment, nucleic acid, vector, polypeptide, or cell according to the present invention is preferably formulated as a pharmaceutical or a medicament together with one or more other pharmaceutically acceptable components 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 (e.g., wetting agents), masking agents, colorants, flavoring agents, and sweetening agents. As used herein, the term "pharmaceutically acceptable" refers to a compound, component, material, composition, dosage form, etc. that, within the scope of sound medical judgment, has a reasonable benefit / risk ratio and is suitable for use in contact with the tissues of the subject (e.g., human) without excessive toxicity, irritation, allergic reaction, or other problems or complications. Each carrier, adjuvant, excipient, etc. must also be "acceptable" in the sense of being compatible with the other components of the formulation. Suitable carriers, adjuvants, excipients, etc. can be found in standard pharmaceutical textbooks, such as Remington’s Pharmaceutical Sciences, 18th Edition, Mack Publishing Company, Easton, Pa., 1990, and Handbook of Pharmaceutical Excipients, 2nd Edition, 1994.

[0112] Pharmaceutical use In another aspect, there is provided the use of the TCR or fragment, nucleic acid, vector, polypeptide, cell, or pharmaceutical composition of the present application in the preparation of a pharmaceutical for the treatment or prevention of a disease or disorder.

[0113] In some embodiments, the TCR or fragment, nucleic acid, vector, polypeptide, cell, or pharmaceutical composition of the present application can be used to prevent or treat diseases caused by HBV infection. Diseases caused by HBV infection include acute hepatitis (including fulminant hepatitis), chronic hepatitis, liver fibrosis, liver cirrhosis, liver cancers such as hepatocellular carcinoma (HCC), or pancreatic cancer.

[0114] Methods of Treatment and Prevention Treatment or prevention can be carried out by isolating T cells from patients or volunteers suffering from diseases caused by HBV infection. The TCR of the present application is introduced into the above T cells, and then these genetically engineered cells are injected back into the patient. Accordingly, the present application provides a method for treating diseases caused by HBV infection by injecting isolated T cells expressing the TCR of the present application into a patient. Preferably, the T cells are derived from the patient. Generally, the method includes (1) isolating T cells from a patient, (2) transducing the T cells in vitro using a nucleic acid molecule or vector capable of encoding the TCR molecule of the present application, and (3) injecting the genetically engineered T cells into the patient in vivo. The number of cells to be isolated, transfected, and reinjected can be determined by a physician.

[0115] Hereinafter, with reference to specific embodiments, the technical solution of the present application will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work are included in the protection scope of the present application. It should be noted that the following order of description of the embodiments is not intended to limit the preferred order of the embodiments.

[0116] In the following examples, experimental methods for which specific conditions are not described generally follow conventional conditions as described in Molecular cloning: Laboratory Manual by Sambrook et al. (Molecular cloning - A Laboratory manual), or follow the methods proposed by the manufacturer. Percentages and parts are by weight unless otherwise indicated. Unless otherwise specified, the reagents and materials included in the text can be obtained commercially or can be prepared by those skilled in the art according to general knowledge. Any methods and materials similar or equivalent to those described can be used in this application. The preferred embodiments and materials in this specification are for illustrative purposes only and do not limit the content of this application.

Example

[0117] Obtaining the target gene of HBV S20 TCR and constructing its vector Obtaining the target gene of HBV S20 TCR: Peripheral blood mononuclear cells (PBMCs) were isolated from fresh blood of volunteers (HLA - A2+) who had recovered from HBV infection. 1 nM of S20 polypeptide (FLLTRILTI) and T2 cells (American typical Species collection) were incubated at 37 °C for 2 hours. Then, 1×10 6 individual PBMCs were stimulated with 1×10 5 individual S20 - loaded T2 cells for 14 days, and the medium was supplemented with IL - 7 (Peprotech, Hamburg, Germany) and IL - 15 (Peprotech, Hamburg, Germany) at a final concentration of 10 ng / mL and Aldesleukin (Novartis Pharmaceuticals) at a final concentration of 50 U / mL.

[0118] T cells were stained with HLA - A * 02 - S20 multimer, and CD8 * bound to HLA - A +T cells were isolated and enriched by flow cytometry. The T cells were further screened for S20 epitope-specific clones. S20 epitope-specific clones were extracted for RNA sequencing to obtain the sequences of both chains of the TCR and construct an HBV S20-specific TCR library. Three clones, A01 / B01 / C01, were selected. These TCRs have high affinity and do not require modification of the variable region (e.g., affinity maturation). The amino acid sequences of CDR1, CDR2, CDR3, the TCRα variable region, and the TCRβ variable region of the α and β chains corresponding to A01 / B01 / C01 are shown in Table 1.

[0119]

Table 1

[0120] Furthermore, at least one additional disulfide bond was introduced into the TCR constant region to improve stability and reduce the mismatch between the internal and external TCR chains. On the other hand, the expression of the TCR gene could be successfully improved by codon optimization. The amino acid sequences of the modified TCRα and TCRβ chains of A01, B01, and C01, as well as the coding sequences of the modified TCRα variable region, TCRβ variable region, TCRα chain, and TCRβ chain, are shown in Table 2. The TCRα and β chains are linked by the P2A self-cleaving peptide element to ensure that each transduced cell expresses the α and β chains at the same level. The amino acid sequences of the single-chain TCR molecules corresponding to A01, B01, and C01 are shown in SEQ ID NO: 12, SEQ ID NO: 23, and SEQ ID NO: 30, and the coding sequences are shown in SEQ ID NO: 37, SEQ ID NO: 42, and SEQ ID NO: 47, respectively.

[0121]

Table 2

[0122] Cloning of the target gene into the pCDH plasmid: To improve the expression efficiency of the TCR gene, it is very important to select an appropriate vector plasmid. Based on the original sequence of the general pCDH-EF1-MCS-T2A-copGFP plasmid (the information on the restriction sites on the plasmid is shown in Table 3), the inventors replaced the ampicillin resistance gene with the kanamycin resistance gene and performed total plasmid gene synthesis. Then, in order to introduce EcoRI and SalI restriction sites at both ends of the target gene, primers at both ends of the target gene were designed. PCR amplification of the target gene (A01 / B01 / C01 single-stranded TCR coding sequence) was performed to obtain an amplification product with a length of about 1800 bp. The electrophoretic band of the target gene was purified and ligated to the linearized pCDH-EF1-MCS-T2A-copGFP plasmid by double digestion with EcoRI and SalI. The ligation product was transformed into Stbl3 competent cells, and single clones were picked and cultured. The target plasmid was extracted and then identified by double enzyme digestion, electrophoresis, and sequencing. The target construct contains 5’LTR, HIV-1 Ψ, RRE, cPPT / CTS, EF-1α core promoter, WPRE, 3’LTR-SIN, and other major functional elements (the important functional elements and positions of the pCDH plasmid are shown in Table 4).

[0123]

Table 3

[0124]

Table 4

Example

[0125] Lentivirus packaging The lentiviral vector used in A01 / B01 / C01 TCR-T cells was a third-generation "self-inactivating (SIN)" lentiviral vector derived from HIV-1 with a VSV-G pseudomembrane, loaded with nucleic acid encoding a targeted HBsAg-specific T cell receptor (HBsAg TCR). The lentiviral vector had only infectious activity, no replication ability, a particle diameter of about 80-120 nm, and a shape approximately spherical or icosahedral symmetric structure. The outer membrane of the virus was a lipid-like envelope embedded with the VSV-G envelope protein. Inside were a spherical matrix (matrix) formed by the protein p17 and a semi-conical capsid (capsid) formed by the protein p24. RNA nucleic acid information containing the TCR coding sequence was present in the capsid.

[0126] The functions of each element of the third-generation self-inactivating lentiviral vector are described as follows.

[0127] At both ends of the TCR gene, there are truncated / chimeric long terminal repeats, Δ5’LTR and Δ3’LTR, respectively. Among them, U3 is removed from the Δ5’LTR and replaced by the enhancer and promoter of the RS virus pneumonia (RSV). The replication of the vector no longer depends on "tat". After U3 is removed from the Δ3’LTR, there is no longer any priming / enhancing activity. And it becomes a self-inactivating vector.

[0128] Function of RRE: A cis-acting element of Rev that promotes the transport of large unspliced mRNA molecules from the nucleus to the cytoplasm.

[0129] Function of cPPT: Improves the transduction efficiency of the vector.

[0130] Function of EF-1α promoter: Regulates the start time and degree of gene expression (transcription).

[0131] Function of WPRE: It can up-regulate the polyadenylation of transcripts, promote the nuclear export of transcripts, and improve the expression efficiency of target genes.

[0132] The lentiviral vector is obtained by transient transfection of 293T cells using a third-generation four-plasmid system consisting of three packaging plasmids (also known as "helper plasmids") and a shuttle plasmid.

[0133] The packaging plasmid pGagPol-KanR encodes the viral structural protein Gag and the reverse transcriptase Pol. The former forms the viral core structure, and the latter is necessary for RNA reverse transcription and integration.

[0134] The plasmid pRev-KanR encodes the Rev protein that binds to RNA and promotes mRNA transport and protein expression.

[0135] The plasmid pVSV-G-KanR encodes the vesicular stomatitis virus envelope protein VSV-G that replaces the HIV viral envelope protein, enables the lentiviral vector to infect cells from almost all tissues, and improves the stability of lentiviral particles.

[0136] Preparation of D10 cell complete medium: DMEM, 10% FBS (v / v), 1% sodium pyruvate, and placed in a refrigerator at 4°C for later use.

[0137] Day 0: The 293T cells used were less than 20 passages old, the cells did not completely fill the dish, 2 × 10 7 cells were placed in a 150 mm dish, and 20 mL of D10 medium was added while mixing well, and cultured overnight at 37°C.

[0138] Day 1: When the confluence reached 60 - 80%, the 293T cells were transfected, and the time from when the cells were cultured in monolayer to transfection was 24 hours or less.

[0139] A plasmid complex for lentiviral packaging was prepared according to Table 5.

[0140]

Table 5

[0141] While gently vortexing the plasmid, PEIpro was added dropwise while mixing well, and then left standing 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 well, and then incubated at 37 °C for 6 hours in a carbon dioxide incubator.

[0142] Day 1: The transfection medium of 293T cells (6 hours later) was gently replaced with complete D10 medium.

[0143] Day 3: The viral supernatant of the 48-hour transfection was collected and temporarily stored in a refrigerator at 4 °C. Then, 20 mL of D10 medium was added.

[0144] Day 4: The viral supernatant of the 72-hour transfection was collected, mixed with the viral supernatant of the 48-hour transfection, and then centrifuged at 3000 g for 10 minutes at 4 °C. Debris was removed through a 0.45 μm filter, and the supernatant was retained. A 100K ultrafiltration cup was used for virus concentration.

[0145] After centrifuging at 3000 g to the desired volume of virus concentration at 4 °C, the centrifuge was removed, and the filter cup was separated from the filtrate collection cup. The filter cup was placed upside down on the sample collection cup. The virus concentrate in the sample collection cup was collected after centrifuging at 1000 g for 2 minutes at 4 °C and stored at less than -70 °C after packaging.

[0146] Detection of virus solution titer: 1×10 5Jacket cells of cells / holes were seeded in a 24-well plate. A specific amount of virus concentrate diluted in a gradient pattern was added to the jacket cells. After culturing for 72 hours, the virus titer was detected by flow cytometry.

[0147] Virus titer detection: Preparation of flow cytometry buffer: DPBS, 2% FBS, stored at 4 °C for later use.

[0148] After centrifugation at 400 g for 5 minutes, the supernatant of 1×10 6 jacket cells in each group was discarded and washed twice with flow buffer.

[0149] PE Dextramer HBV-S20 cells were diluted with flow buffer at a ratio of 1:100. 100 μL of antibody dilution was added to each sample, and flow buffer was added. After incubating in the dark at 4 °C for 30 minutes, the cells were washed, and then after centrifugation at 400 g for 5 minutes, the supernatant was discarded, and this process was repeated twice.

[0150] The cells were suspended in 100 μL of flow cytometry buffer and detected by flow cytometry.

[0151] Vector infectivity titer (TU / mL) = Number of cells per well × Positive rate (%) × Dilution rate / Titration volume (mL) The results are shown in Figure 3. The lentivirus titers of A01, B01, and C01 TCRs all exceeded 1×10 8 TU / mL, indicating that all three HBV S20 TCRs can successfully package the virus at high titers.

Example

[0152] Preparation of HBV S20 TCR-T Preparation of T cell culture medium: PRIME-XV-T cell CDM, 400 IU / mL of IL-2.

[0153] Preparation of T cell cryopreservation: 75% CS10 + 25% HSA.

[0154] Day 0: Pure CD3 from apheresis + Isolate T cells and adjust the cell concentration to 1×10 6 cells / mL in T cell medium. While mixing well, add the activator according to transact (CD3 / CD28 microsphere): cell suspension = 1:30, and then add interleukin 2 with a final concentration of 400 IU / mL to stimulate the cells 24 hours before virus infection.

[0155] Day 1: Adjust the density of T cells to 5×10 5 cells / mL and add the virus solution.

[0156] Days 2 - 11: After infection, observe the cell state daily and timely add T cell culture medium containing 400 IU / mL of IL-2 to maintain the density of T cells at 5×10 5 / mL to proliferate the cells.

[0157] Day 12: Collect the cells by centrifugation at 300 g for 5 minutes, wash the cells with physiological saline containing 5% human serum albumin, freeze them with a special freezing solution for T cells according to the appropriate density, freeze them with a programmed cooler, and then store them in liquid nitrogen.

Example

[0158] Detection of MOI of S20 TCR-T and TCR-specific expression Detection of MOI of HBV S20 TCR Preparation of flow buffer (FACS buffer): DPBS, 2% FBS (v / v), store at 4°C for later use.

[0159] Collect TCR-T cells and NT cells (control group) infected with lentiviruses A01, B01, and C01, wash them with FACS buffer, stain them with PE Dextramer HBV-S20, and incubate them in the dark at 4°C for 30 minutes. Wash the cells with FACS buffer and analyze them by flow cytometry.

[0160] As shown in Figure 4, the expression levels of A01, B01, and C01 TCR-Ts were consistent with each other. In each group, when MOI HBV S20 TCR = 1.2, the TCR-T positive rate exceeded 60%, indicating that all three S20 TCR clones were stably expressed in TCR-T cells infected with gradient MOI.

[0161] Detection of specific expression of HBV S20 TCR-T Cells prepared from each group of A01 TCR-T, B01 TCR-T, and C01 TCR-T and NT cells (control group) were collected, washed with FACS buffer, stained with PE Dextramer HBV-S20 and APC TCR vβ5.1, and incubated in the dark at 4°C for 30 minutes. The cells were washed with FACS buffer and applied to flow cytometry.

[0162] As shown in Figure 5, the mispairing rates of A01, B01, and C01 TCR-T were all low, and the specific expression reached over 50%, indicating that the three clones of HBV S20 TCR-T can be specifically expressed with a low mispairing rate.

Example

[0163] Cytolysis and cytokine analysis of S20 TCR-T against HepG2-LMS-LG cells M10 complete medium preparation: DMEM, 10% FBS (v / v), 1% sodium pyruvate, 1% HEPES, 1% NEAA, stored at 4°C for later use.

[0164] Preparation of HepG2-LMS-LG target cells Three wells of HLA-A02 subtype HepG2 cells were plated at 1×10 in a 24-well plate pre-coated with collagen 5Seeded with the amount of cells / well, and then added 25 μL, 5 μL, 1 μL of the LMS-LG lentiviral vector encoding the full-length HBsAg protein (including the FLLTRILTI sequence) fused with luciferase and GFP protein to prepare the target HepG2-LMS-LG cells. When the cell confluence reached 90%, they were transferred to a 6-well plate and grown. When the cell confluence reached about 90% again, flow detection was performed. Cells with a GFP positive rate exceeding 95% were selected for growth culture, and a seed cell bank of the target cells was established.

[0165] As shown in Figure 6, the positive rate of HepG2-LMS-LG in the target cells exceeded 99%, and the expression was uniform, indicating that the target cells were successfully constructed.

[0166] Killing of A01 TCR-T against HepG2-LMS-LG target cells (chemiluminescence method) Day 0: HepG2-LMS-LG cells were seeded in a 96-well plate pre-coated with collagen at a density of 1×10 4 / well, 100 μL / well. 100 μL of A01 TCR-T cells were added at effector-to-target (E:T) ratios of 8:1, 4:1, 2:1, 1:1, 1:2, 1:4, 1:8 respectively, and 100 μL of culture medium was added to the wells containing A01 TCR-T cells, and then cultured at 37 °C for 24 hours.

[0167] Day 1: 50 μL of the ONE-Glo™ Luciferase Assay System was added to each well while mixing well and applied to a microplate reader.

[0168] Calculation method: Killing efficiency = (1 - average value of effector cells and target cells / average value of target cells) × 100%.

[0169] As shown in Figure 7, the killing efficiency of A01 TCR-T against target cells reached over 80% at E:T ratios of 8:1 and 4:1. As the E:T ratio decreased, the killing efficiency decreased, and the killing effect could still be detected until the E:T ratio reached 1:4. As a negative control, no killing function was observed in HepG2 vs. A01 TCR-T and UT vs. HepG2-LMS-LG cells, indicating that A01 TCR-T could specifically kill HBsAg-positive target cells.

[0170] Killing function of B01 / C01 TCR-T against HepG2-LMS-LG cells (real-time cell analysis, RTCA).

[0171] Day 0: After digestion, the cell density of HepG2-LMS-LG cells was adjusted to 4×10 5 / mL. To prime the determination of the instrument baseline of RTCA, 50 μL / well of M10 medium was added to the wells of a 96-well plate pre-coated with collagen. Then, 50 μL of cells (4×10 5 / mL) was added to each well. After a rest of about 5 minutes, the growth curve was continuously detected for about 16 hours.

[0172] Day 1: The TCR positivity rate and cell viability of B01 / C01 TCR-T cells were detected.

[0173] Effector cells were prepared at concentrations of 8×10 5 positive cells / mL, 2.0×10 5 / mL, 0.5×10 5 / mL, and then 50 μL / well of effector cells was added at effector-target ratios of 2:1, 1:2, 1:8, and the killing curve was continuously monitored.

[0174] Day 3: After 72 hours, RTCA monitoring was terminated for data copying and killing curve fitting, and the co-culture supernatant was collected for cytokine analysis.

[0175] The detection of cytokines was performed according to the kit instructions.

[0176] The CBA kit (Human Th1 / Th2 Cytokine Cytometric Bead Array Kit II, BD, 551809) was taken out and equilibrated to room temperature.

[0177] Using 2 mL of assay diluent, the standard substance was dissolved to a concentration of 5000 pg / mL and equilibrated at room temperature for 30 minutes.

[0178] Standard preparation: The above standard was designated as S1, serially diluted 2-fold to obtain S2 - S9, and S10 was used as the blank.

[0179] To constitute the human Th1 / Th2 cytokine capture beads mixture, the microsphere solutions A1 - A6 were thoroughly vortexed and mixed in a ratio of 1:1:1:1:1:1.

[0180] 50 μL of the Human Th1 / Th2 Cytokine Capture Beads mixture was added to a 96-well U-bottom plate, and 50 μL of the human Th1 / Th2 PE detection reagent was added.

[0181] After centrifugation at 400 g for 5 minutes, 50 μL of the supernatant was added to each detection well, or 50 μL of S10 - S1 was added to the standard wells.

[0182] Incubated in the dark at room temperature for 180 minutes, 100 μL of wash buffer was added, centrifuged at 300 g for 5 minutes, and then the supernatant was discarded.

[0183] Resuspended with 100 μL of FACS buffer, the sample was loaded onto a flow cytometer for detection, and the results were analyzed.

[0184] As shown in FIGS. 9A and 9B, the killing rates after co-culturing B01 TCR-T with target cells at E:T ratios of 2:1, 1:2, and 1:8 for 48 hours all reached over 60%, and UT had no obvious function against target cells. The IFN-γ secretion in the B01 TCR-T treatment group was significant, while that in the UT group was slight (see FIG. 9C). The killing rates after co-culturing C01 TCR-T with target cells at E:T ratios of 2:1, 1:2, and 1:8 for 48 hours reached over 60%. UT did not show a cell lysis function against target cells, and C01 TCR-T showed little function against negative cells. Regarding FIG. 10A, the IFN-γ secretion of C01 TCR-T was obvious in target cells but low in negative cells. Furthermore, the UT treatment group had no obvious cytokine secretion (see FIG. 10B).

[0185] Killing function of A01 / B01 / C01 TCR-T against HepG2-LMS-LG cells Day 0: Seed HepG2-LMS-LG cells at a density of 1×10 4 / well, 100 μL / well into a 96-well plate pre-coated with collagen, then add 100 μL of A01, B01, and C01 TCR-T cells at E:T ratios of 1:1, 1:2, and 1:4, and supplement the control wells with 100 μL of T cell culture medium, and co-culture for 24 hours in an incubator at 37°C.

[0186] Day 1: Add 50 μL of ONE-Glo™ Luciferase Assay System to each well while mixing thoroughly, and apply it to a microplate reader for detection.

[0187] Calculation method: Killing efficiency = (1 - average value of effector cells and target cells / average value of target cells) × 100%.

[0188] As shown in FIG. 11, A01, B01, and C01 TCR-T all showed a significant specific killing effect dose-dependently against HepG2-LMS-LG, but had little killing effect against HBsAg-negative target cells.

Example

[0189] Functionality of Different T Cell Subsets of HBV S20 TCR-T against Target Cells CD4 + T cells are helper T lymphocytes whose main function is to enhance anti-infection mediated by phagocytes and to enhance the humoral immune response mediated by B cells. CD8 + T cells are suppressor / killer T lymphocytes whose main function is to directly and specifically kill target cells. The inventors isolated the CD4 + / CD8 + cell subsets of HBV S20 TCR-T to examine the anti-tumor activity of HBV S20 TCR-T.

[0190] Cell Sorting Add 25 μL of Dynabeads® CD4 and Dynabeads® CD8 positive magnetic beads to 1 × 10 6 cells of HBV S20 TCR-T, spray evenly while mixing well respectively, then transfer to a separation column and incubate at 2 - 8 °C for 20 minutes.

[0191] Put the separation column into a magnetic stand for 2 minutes. After adsorbing the cells attached to the magnetic beads to the tube wall, discard the supernatant in the tube. Remove the separation column, add 1 mL of washing solution (buffer 1) and rinse 2 - 3 times, then place it on the magnetic stand for 2 minutes again. Repeat the above process 5 times.

[0192] Resuspend the cells in 100 μL of buffer 2.

[0193] After adding 10 μL of DETACHaBEAD®, incubate at room temperature for 45 minutes while gently mixing.

[0194] Put the separation column into a magnetic stand for 1 minute and transfer the T cells in the supernatant to a new test tube.

[0195] The cells were thoroughly washed with 4 mL of buffer 2, and the supernatant was discarded by centrifugation at 400 g for 5 minutes.

[0196] The obtained high-purity CD4 without magnetic beads + and CD8 + viable cells were used for subpopulation phenotyping by flow cytometry and subsequent functional experiments.

[0197] Detection of cell subpopulations: FACS buffer preparation: DPBS, 2% FBS, placed in a refrigerator at 4 °C for later use.

[0198] The selected CD4 + and CD8 + cells were evenly pipetted, centrifuged at 400 g for 5 minutes, and washed twice with FACS buffer.

[0199] PE-Cy7-CD4 (BIOLEGEND, 300512) / PerCP-Cy5.5-CD8 (BIOLEGEND, 301032) / PE Dextramer antibodies were diluted 1:100 with FACS buffer, 100 μL of the detection antibody was added to each sample, and then incubated at 4 °C for 30 minutes in the dark. The samples were washed twice with FACS and centrifuged at 400 g for 5 minutes in buffer before analysis.

[0200] The cells were resuspended in 100 μL of FACS buffer and detected by flow cytometry.

[0201] As shown in Figure 11A, the purity of the selected CD4 + cells and CD8 + cells was over 95%, and the positive rate of CD4 + cells was slightly higher than that of CD8 + cells.

[0202] Cytotoxic function of S20 TCR-T against HepG2-LMS-LG cells (RTCA) Target cell complete medium (M10): DMEM, 10% FBS, 1% sodium pyruvate, 1% HEPES, 1% NEAA, and stored in a 4°C refrigerator for later use.

[0203] T cell complete medium (TCM): To an appropriate amount of PRIME-XV culture in a 50 mL centrifuge tube, IL-2 was added while mixing well at a final concentration of 400 IU / mL and stored at 2 - 8°C for later use.

[0204] Day 0: The cell density of HepG2-LMS-LG cells in good growth condition was adjusted to 4×10 5 / mL after digestion. 50 μL / well of M10 medium was added to the 96-well holes coated with collagen plate for RTCA baseline determination. Then, 50 μL of cells (4×10 5 / mL) was added to each well. After a rest for about 5 minutes, the growth curve was continuously detected for about 16 hours.

[0205] Day 1: The TCR positive rate and cell viability of TCR-T cells sorted by CD4 + and CD8 + were detected, with unsorted TCR-T cells as the control. 50 μL / well of effector cells was added at effector:target ratios of 4:1, 1:1, and 1:4, and the killing curve was continuously detected through the machine.

[0206] Day 3: After 72 hours, RTCA monitoring was terminated for data copying and killing curve fitting, and the co-culture supernatant was collected for cytokine analysis.

[0207] The detection of cytokines was performed according to the cytokine detection procedure in Example 5.

[0208] As shown in Figure 11B, CD8 + cells showed excellent killing ability against tumor cells at three effector:target ratios, and the half-killing time of CD8 + cells was only half of that of the control group. Under the condition of a high target ratio, CD4 +The cells also had a good killing effect on the target cells. When the effector:target ratio was 1:1, CD8 + The cytokine release level of the cells was the same as that of the control group, but higher than that of the CD4 + cell group (see Figures 11C and 11D).

[0209] As a conclusion, these results suggest that CD8 + TCR-T cells have a stronger killing ability and cytokine release ability against tumor cells than CD4 + TCR-T cells.

Example

[0210] Detection of cross-reactivity of A01 / B01 / C01 TCR-T against the human polypeptide database Due to the sequence diversity of TCRs, epitopes, and MHC / HLA molecules, TCRs can have cross-reactivity that leads to potential off-target toxicity. Alanine scanning peptide libraries can be used to identify specific amino acid sites that are closely related to the function, stability, and conformation of polypeptides. Each amino acid residue in the HBV S20 epitope peptide was individually mutated to alanine to test the cross-recognition ability of HBV S20 TCR-T on the mutated epitope.

[0211] Identification of important amino acids recognized by HBV S20 TCR-T Target cell complete medium (R10) was prepared by thoroughly mixing RPMI 1640:FBS at a ratio of 10:1 and stored at 2 - 8°C for later use.

[0212] T cell complete medium (TCM) was prepared by adding IL-2 at a final concentration of 400 IU / mL to an appropriate amount of PRIME-XV culture in a 50 mL centrifuge tube while thoroughly mixing and stored at 2 - 8°C for later use.

[0213] Peptide Preparation: Positive control S20-A / D (No. 1), the amino acid sequence of No. 1 is FLLTRILTI. Negative control C18-A / D (No. 2), the amino acid sequence of No. 2 is FLLTKILTI. S20 mutant polypeptides (No. 3 - 11) with mutations in F1A, L2A, L3A, T4A, R5A, I6A, L7A, T8A, and I9A respectively compared to the amino acid sequence of FLLTRILTI. 2 mg of each peptide was dissolved in 170 μL of DMSO to a final concentration of 10 mM. 3 μL of the 10 mM solution was diluted to 100 μM with 297 μL of TCM and then serially diluted 10-fold to 10 μM.

[0214] Preparation of Target Cells: T2 cells were harvested and resuspended in R10 medium. Cell viability and density were measured. 1.8×10 6 T2 cells were resuspended in 6 mL of TCM medium according to the counting results. 100 μL / well of T2 cells (3×10 4 / well) were added to a 96-well U-bottom plate. The corresponding peptide solution was added to the plate at a final concentration of 1 μM. The 96-well U-bottom plate was placed in an incubator at 37 °C for 2 hours, and then effector cells were added.

[0215] Preparation of Effector Cells: The viability and density of HBV S20 TCR-T cells were measured. 2.6×10 6 HBV S20 TCR-T cells were resuspended in 3 mL of TCM medium, and then the density of positive cells was adjusted to 6×10 5 / mL. 50 μL / well of effector cells were added to each well, and the plate was placed in an incubator at 37 °C. The cytokine levels in the supernatant were measured by flow cytometry after 24 hours of co-incubation.

[0216] Cytokine detection was performed according to the procedure of Example 5. As a result, the specific recognition function of HBV S20 TCR-T for the peptide having amino acid mutations at positions 3 / 4 / 5 / 6 was significantly reduced, and cytokine secretion was undetectable or much lower than that of the original S20 peptide group (in Figure 12, the histograms from left to right represent the results of peptide numbers 1-11 in order). An important amino acid motif (3 / 4 / 5 / 6, LTRI) of the HBV S20 TCR-T recognition epitope was identified. The four amino acid residues were considered to play important roles in direct binding to S20 TCR or in controlling the spatial structure in the binding. These results provided guidance for computer prediction analysis to evaluate the cross-reactivity of other similar human self-antigen peptides and further evaluate the specificity and safety of epitope recognition of HBV S20 TCR-T.

[0217] The inventors obtained 14 peptide sequences having 6 amino acids identical to the S20 epitope peptide based on computer prediction, sequence alignment in the database, and BLAST sequence alignment of the human peptide library. No human peptides with more than 6 amino acids identical to S20 were found. Using T2, different concentrations of peptides were loaded and then incubated with HBV S20 TCR-T to detect cytokine secretion.

[0218] Peptide preparation: Positive control S20-A / D (No. 1), negative control C18-A / D (No. 2), and 14 peptide sequences (No. 14-27) having 6 amino acids identical to the S20 epitope peptide. 2 mg of each peptide was dissolved in 200 μL of DMSO to a final concentration of 10 mg / mL. 10 μL of the 10 mg / mL solution was diluted to 1 mg / mL with 90 μL of TCM and then serially diluted 10-fold to 100 ng / mL.

[0219] Preparation of target cells: After subculturing several times at 500 g / 5 min, T2 cells in good growth condition were centrifuged and the supernatant was removed. The cells were resuspended in R10 medium. The cell viability and density were measured. 1.8×10 6 T2 cells were resuspended in 9 mL of TCM medium according to the counting result. 100 μL / well of T2 cells (2×10 4 cells / well) were added to a 96-well U-bottom plate. The corresponding peptide solution was added to the plate at final concentrations of 0.1 μg / mL and 1 μg / mL. After the 96-well U-bottom plate was placed in an incubator and incubated at 37°C for 2 hours, effector cells were added.

[0220] Preparation of effector cells: T2 cells were collected and resuspended in R10 medium. After uniformly spraying HBV S20 TCR-T cells, the cell viability and density were measured. 4.4×10 6 HBV S20 TCR-T cells were resuspended in 4.5 mL of TCM medium according to the counting result, and then the density of positive cells was adjusted to 1×10 6 / mL. 50 μL / well of effector cells were seeded into each well, and the plate was placed in an incubator at 37°C. The cytokine levels in the supernatant were measured by flow cytometry after 24 hours of co-incubation.

[0221] Cytokine detection was performed according to the procedure of Example 5.

[0222] As shown in Figure 13, A01 / B01 / C01 TCR-T did not have cross-reactivity with 14 polypeptides.

[0223] In conclusion, the inventors of the present invention demonstrated that the important amino acid motif recognized by HBV S20 TCR-T is located at positions 3 to 6 (LTRI). When the amino acid residues in this region changed, the recognition function of the TCR for the mutant polypeptide decreased significantly. By comparing human peptide libraries using bioinformatics prediction algorithms, 14 human peptide sequences identical to the S20 epitope peptide in 6 amino acids were obtained. The cross-reaction of HBV S20 TCR-T with these human self-antigenic peptides was not observed by the T2 cell loading method, indicating that the potential off-target toxicity risk of HBV S20 TCR-T against human endogenous antigens is very low.

Example

[0224] Detection of the binding ability of A01 / B01 / C01 TCR-T to S20 with different genotypes T cells recognize tumor antigens mainly through TCR recognition of tumor antigens and HLA-peptide complexes on the surface of target cells. Activation signals are transmitted through the specific binding of TCR-T to tumor antigens, thereby resulting in a targeted killing function against tumor cells. To study the function of HBV S20 TCR-T against HBV with different genotypes, T2 cells were loaded with different concentrations of S20-gt A / D (genotype A or D with the amino acid sequence FLLTRILTI) or S20-gt B / C (genotype B or C with the amino acid sequence FLLTKILTI).

[0225] Target cell complete medium (R10): Prepared by mixing RPMI 1640 and FBS at a ratio of 10:1 while thoroughly mixing, and stored at 2 - 8 °C for later use.

[0226] T cell complete medium (TCM): IL-2 was added to an appropriate amount of PRIME-XV culture in a 50 mL centrifuge tube at a final concentration of 400 IU / mL while thoroughly mixing, and stored at 2 - 8 °C for later use.

[0227] Peptide Preparation: 2 mg of S20-AD, S20-BC, and C18-AD peptides were each dissolved in 170 μL of DMSO to a final concentration of 10 mM. 3 μL of the 10 mM solution was diluted to 100 μM with 297 μL of TCM and then serially diluted 10-fold to 10 μM.

[0228] Target Cell Preparation: T2 cells were harvested and resuspended in R10 medium. Cell viability and density were measured. 2.5×10 6 T2 cells were resuspended in 5 mL of TCM medium according to the counting results. 100 μL / well of T2 cells (5×10 4 / well) were added to a 96-well U-bottom plate, and 100 μL of TCM was added to each well of the negative control group. The corresponding peptide solution was added to the plate at a final concentration of 10 -5 M to 10 -9 M. The 96-well U-bottom plate was placed in an incubator at 37 °C for 2 hours, and then effector cells were added.

[0229] Effector Cell Preparation: T2 cells were collected and resuspended in R10 medium. 3.6×10 6 HBV S20 TCR-T cells were resuspended in 2.5 mL of TCM medium according to the counting results, and then the density of positive cells was adjusted to 1×10 6 / mL. 50 μL / well of effector cells were seeded into each well, and the plate was placed in an incubator at 37 °C. Cytokine levels in the supernatant were measured by flow cytometry after 24 hours of co-incubation.

[0230] Cytokine detection was performed according to the procedure of Example 5.

[0231] As shown in Figure 14, A01 / B01 / C01 TCR-T had significant cytokine secretion in T2 cells loaded with HBV S20 gt A / D polypeptide or S20 gt B / C polypeptide. The secretion of IFN-γ was such that the concentration of the loaded polypeptide was 10 -5When reaching M, it exceeded 5 ng / mL. It had no function against C18 - 27 gt A / D. And the A01 / B01 / C01 TCR - cytokine was secreted in a dose - dependent manner of S20.

[0232] In conclusion, HBV S20 TCR - T has obvious functions against HBV with different genotypes. HBV S20 TCR - T can cover most HBV virus subtypes and has good functions against several common genotypes.

Example

[0233] A01 / B01 / C01 TCR - T recognized different subtypes of HLA - A02.

[0234] When the TCR specifically recognizes the antigenic peptide presented by the MHC molecule, it is restricted by the HLA subtype. HepG2 is a human hepatoma cell line of HLA - A * 02:01 / 24:02, and SW403 is a human hepatoma cell line of HLA - A * 02:05 / 03:01, and KATO III is a human gastric cancer cell line of HLA - A * 02:01 / 02:07, and SNU - 1 is a human gastric cancer cell line of HLA - A * 02:07 / 30. After loading the above cells with the S20 epitope peptide, the ability of HBV S20 TCR - T to stimulate and kill target cells and secrete cytokines was tested, and the binding ability of the S20 epitope peptide - MHC complex presented by different subtypes of target cells of HLA - A02 and HBV S20 TCR - T was evaluated in vitro.

[0235] Target cell complete medium (R10) was prepared by thoroughly mixing RPMI 1640 and FBS at a ratio of 10:1, and stored at 2 - 8 °C for later use.

[0236] T cell complete medium (TCM): An appropriate amount of PRIME-XV culture in a 50 mL centrifuge tube was added with IL-2 while mixing well at a final concentration of 400 IU / mL, and stored at 2 - 8 °C for later use.

[0237] Peptide preparation: 2 mg of S20 peptide was dissolved in 200 μL of DMSO to achieve a final concentration of 10 mg / mL. After diluting 10 μL of the solution to 1 mg / mL with 90 μL of TCM, it was serially diluted 10-fold to 1 μg / mL.

[0238] Target cell preparation: HepG2, SW403, KATO III, and SNU-1 cells were collected and resuspended in R10 medium. Cell viability and density were measured. 8×10 5 cells were resuspended in 4 mL of TCM medium according to the counting results. 2×10 4 / well of target cells (100 μL / well) were added to a 96-well U-bottom plate, and 100 μL of TCM was added to the group of T cells without peptide. The corresponding peptide solution was added to each well at a final concentration of 1 μg / mL. The 96-well U-bottom plate was placed in an incubator at 37 °C for 2 hours, and then effector cells were added.

[0239] Effector cell preparation: After uniformly spraying HBV S20 TCR-T cells, cell viability and density were measured. The density of the cells was adjusted with TCM according to the counting and positive rate results. 50 μL / well of effector cells (E:T = 2:1) were seeded, and the plate was placed in an incubator at 37 °C. Cytokine levels were measured by flow cytometry after 24 hours of co-incubation.

[0240] As shown in Figure 15, HLA-A * 02:01 (HepG2) was used as a reference, and HLA-A *Under the limitation of 02:07, significant cytokine secretion could be detected after incubating A01 TCR-T, B01 TCR-T, and C01 TCR-T with KATO III and SNU-1 loaded with the S20 polypeptide, but no related cytokine secretion could be detected after incubating A01 TCR-T, B01 TCR-T, and C01 TCR-T with target cells without S20 polypeptide loading. HLA-A * Cytokine secretion of SW403 with or without S20 polypeptide loading could not be detected under the limitation of 02:07.

[0241] HLA-A * 02:01 or HLA-A * Under the limitation of 02:07, HepG2, KATO III, and SNU-1 cells loaded with the S20 epitope peptide can effectively activate HBV S20 TCR-T to secrete related cytokines and show significant specific binding activity. However, due to the restriction of HLA subtype, SW403 (HLA-A * 02:05) cells loaded with the S20 epitope peptide cannot activate the killing function of HBV S20 TCR-T. Multiple HLA-A * 02:01 and HLA-A * The cross-recognition activity of HBV S20 TCR-T against the S20 epitope peptide presented by the 02:07 allele means that there is a widely applicable patient population covered by HBV S20 TCR-T.

Example

[0242] Pharmacodynamics and pharmacokinetics experiments of A01 TCR-T in the HepG2-LMS-LG xenograft model To understand the antitumor activity of A01 TCR-T against tumor cells in vivo, the inventors used HBsAg +A mouse CDX model was constructed using liver cancer cells (HepG2-LMS-LG) transplanted into immunodeficient mice (Shanghai Model Organisms Center, Inc.) to evaluate the tumor elimination effect of A01 TCR-T cells against liver cancer.

[0243] 1×10 7 tumor cells were subcutaneously inoculated into the right axilla of NPG mice on day -6. On day 0, the tumor size reached approximately 100 mm 3 , and the mice were divided into 6 groups of 10 each. Among them, 4 groups, namely, the high-dose group (2×10 7 cells / mouse), the medium-dose group (1×10 7 cells / mouse), the low-dose group (0.5×10 7 cells / mouse), and the ultra-low-dose group (0.2×10 7 cells / mouse) were injected with different doses of A01 TCR-T cells. The remaining 2 groups were used as control groups treated with cell cryopreservation (vehicle) and non-transfected T cells (UT) (1.5×10 7 cells / mouse), respectively. Tumor and pharmacokinetic data were collected, and the tumor size was measured twice a week.

[0244] Compared with the control group, different doses of A01 TCR-T cells had a significant inhibitory effect on tumor growth. The tumor volumes in the four treatment groups decreased significantly (see Figure 16a), and the mice showed good tolerance even after intravenous injection, with no weight loss (see Figure 16b). The copy number of A01 TCR-T could be detected in all tissues 1 day after administration, and then showed a tendency to gradually decrease, reaching the lowest level 7 days after administration. However, 14 and 21 days after administration, A01 TCR-T cells regrew in tissues rich in blood flow such as the liver, spleen, lung, and heart, and the number of A01 TCR T cells reached a peak 21 days after administration (see Figure 16c).

[0245] Based on the above results, A01 TCR-T has anti-tumor activity against the HepG2-LMS-LG xenograft model, and its efficacy is positively correlated with the dose.

[0246] TCR-T cells in tissues, specifically in tissues such as the liver, spleen, lung, and heart with rich blood flow, significantly proliferated, and no other systemic toxicities related to the test article were found. Since mononuclear cell infiltration can occur in multiple organs / tissues of the animal model caused by graft-versus-host disease (GvHD), the test period was controlled to 21 days. Since HBV-S20 TCR-T cells were prepared in clinical trials using patient-derived autologous T cells, GvHD was avoided.

Example

[0247] In vivo pharmacodynamic experiment of B01 TCR-T in HepG2-LMS-LG xenograft model The inventors of the present invention used HBsAg + An immunodeficient mouse (Shanghai Model Organisms Center, Inc.) transplanted with liver cancer cells (HepG2-LMS-LG) was used to construct a mouse CDX model developed to evaluate the tumor elimination effect of B01 TCR-T cells against liver cancer. 1×10 7 tumor cells were subcutaneously inoculated into the right axilla of NPG mice on day -6. On day 0, the mice were divided into 6 groups of 10 mice each. Among them, 4 groups, namely, the high-dose group (2×10 7 cells / mouse), the medium-dose group (1×10 7 cells / mouse), the low-dose group (0.5×10 7 cells / mouse), and the ultra-low-dose group (0.2×10 7 cells / mouse) were injected with different doses of B01 TCR-T cells. The remaining 2 groups were used as control groups treated with cell cryopreservation (vehicle) and non-transfected T cells (Mock-T) (4.3×10 7 cells / mouse), respectively. Thereafter, the tumor size was measured twice a week, and data were collected.

[0248] Compared with the control group, different doses of B01 TCR-T cells had a significant inhibitory effect on tumor growth, and the tumor volumes in the four treatment groups decreased significantly (see Figure 17a). The tumor inhibition rates all exceeded 90%, and the mice showed good tolerance without weight loss after intravenous injection (see Figure 17b).

[0249] In conclusion, there was an obvious killing effect of B01 TCR-T on tumor cells in vivo, and no adverse reactions were observed.

Example

[0250] In vivo pharmacodynamics experiment of C01 TCR-T HepG2-LMS-LG xenograft model The inventors used immunodeficient mice (Shanghai Model Organisms Center, Inc.) transplanted with liver cancer cells (HepG2-LMS-LG) to construct a mouse CDX model developed to evaluate the tumor elimination effect of C01 TCR-T cells on liver cancer. + 1×10

[0251] individual tumor cells were subcutaneously inoculated into the right axilla of NPG mice on day -6. On day 0, the mice were divided into 4 groups of 5 mice each. Among them, 2 groups, namely, the high-dose group (2×10 7 cells / mouse) and the low-dose group (5×10 7 cells / mouse), were injected with different doses of C01 TCR-T cells. The remaining 2 groups were used as control groups treated with cell cryopreservation (vehicle) and non-transfected T cells (Mock-T) (2.9×10 6 cells / mouse), respectively. The tumor size was continuously monitored, and then the data were collected. 7

[0252] ​Compared with the control group, high-dose C01 TCR-T cells had a more significant inhibitory effect on tumor growth than the low-dose group, and the tumor volume decreased significantly (see Figure 18a). Mice in different dose groups showed good tolerance without weight loss after intravenous injection (see Figure 18b). Higher copy numbers of C01 TCR-T (by qPCR method) could be detected in the animal tissues of the high-dose group compared with the control group, indicating the survival and proliferation of TCR-T cells in animals. The degree of cell proliferation showed a significant negative correlation with tumor size. More obvious proliferation of C01 TCR-T in mice indicated a stronger killing and inhibitory effect on tumors. (See Figure 18c).

[0253] In conclusion, higher copy numbers of C01 TCR-T existed in vivo. C01 TCR-T had an obvious killing effect on tumor cells in vivo, and no obvious adverse reactions were observed.

[0254] This application has been introduced in detail above. Regarding the principle and implementation mode of this application, specific examples have been used in this specification for explanation. The description of the above embodiments is only used to help understand the method and core concept of this application. Those skilled in the art may make changes to specific embodiments and application scopes according to the concept of this application. Therefore, the content of this specification should not be construed as a limitation of this application.

Claims

1. A T cell receptor (TCR) or a fragment thereof, Amino acid sequence: αCDR3: ATDERDDRMR (SEQ ID NO: 3), or a variant thereof in which one or two amino acids are replaced with another amino acid. a TCR alpha chain variable region comprising an alpha CDR3 having and A TCR β chain variable region comprising a βCDR3 having the amino acid sequence: βCDR3: ASSLNTEAF (SEQ ID NO: 6) or a variant thereof in which one or two amino acids are replaced with another amino acid; or Amino acid sequence: αCDR3: GADTSTDKLI (SEQ ID NO: 15), or a variant thereof in which one or two amino acids are replaced with another amino acid. a TCR alpha chain variable region comprising an alpha CDR3 having and Amino acid sequence: βCDR3: ASSHGGAYEQY (SEQ ID NO: 18) or a variant thereof in which one or two amino acids are replaced with another amino acid a TCR β chain variable region comprising a βCDR3 having or Amino acid sequence: αCDR3: ATDAYGQNFV (SEQ ID NO: 24), or a variant thereof in which one or two amino acids are replaced with another amino acid. a TCR alpha chain variable region comprising an alpha CDR3 having and Amino acid sequence: βCDR3: ASGSNTEAF (SEQ ID NO: 25) or a variant thereof in which one or two amino acids are replaced by another amino acid; a TCR β chain variable region comprising a βCDR3 having A T cell receptor (TCR) or a fragment thereof comprising:

2. a TCR alpha chain variable region comprising alpha CDR1, alpha CDR2 and alpha CDR3 as set forth in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively; and a TCR β chain variable region comprising βCDR1, βCDR2 and βCDR3 as set forth in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively; or a variant thereof in which one or two amino acids in one or more of the CDRs are replaced with another amino acid; or a TCR alpha chain variable region comprising alpha CDR1, alpha CDR2 and alpha CDR3 as set forth in SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15, respectively; and a TCR β chain variable region comprising βCDR1, βCDR2 and βCDR3 as set forth in SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, respectively; or a variant thereof in which one or two amino acids in one or more of the CDRs are replaced with another amino acid; or αCDR1, αCDR2, and αCDR3 are shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 24, respectively. a TCR α chain variable region comprising αCDR2 and αCDR3; and a TCR β chain variable region comprising βCDR1, βCDR2 and βCDR3 as set forth in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:25, respectively; or a variant thereof in which one or two amino acids in one or more of the CDRs are replaced with another amino acid; 2. The TCR or fragment of claim 1, comprising:

3. 2. The TCR or fragment of claim 1, wherein the TCR alpha chain variable region comprises an amino acid sequence that is at least 90% identical to any one of SEQ ID NO:7, SEQ ID NO:19 and SEQ ID NO:

26.

4. 2. The TCR or fragment of claim 1, wherein the TCR beta chain variable region comprises an amino acid sequence that is at least 90% identical to any one of SEQ ID NO:8, SEQ ID NO:20 and SEQ ID NO:

27.

5. 2. The TCR or fragment of claim 1, wherein the TCR alpha chain comprises the amino acid sequence set forth in SEQ ID NO: 10, SEQ ID NO: 21 or SEQ ID NO:

28.

6. 2. The TCR or fragment of claim 1, wherein the TCR beta chain comprises the amino acid sequence set forth in SEQ ID NO: 11, SEQ ID NO: 22 or SEQ ID NO:

29.

7. 7. A TCR or fragment according to any one of claims 1 to 6, capable of binding to a polypeptide of an HBV surface antigen presented by HLA-A2.

8. 8. The TCR or fragment of claim 7, wherein the polypeptide comprises or consists of the amino acid sequence FLLTRILTI (SEQ ID NO: 31) or FLLTKILTI (SEQ ID NO: 32).

9. 2. The TCR or fragment of claim 1, wherein the C-terminus or N-terminus of the alpha and / or beta chain of the TCR is combined with a conjugate, wherein the conjugate is a detectable marker, a therapeutic agent, a PK-modifying moiety, or a combination thereof.

10. A nucleic acid molecule encoding a TCR molecule or fragment according to claim 1 or 2.

11. the coding sequence of the variable region of the TCR alpha chain is set forth in SEQ ID NO: 33, SEQ ID NO: 38 or SEQ ID NO: 43; and / or The nucleic acid molecule of claim 10, wherein the coding sequence of the variable region of the TCR β chain is set forth in SEQ ID NO: 34, SEQ ID NO: 39 or SEQ ID NO:

44.

12. the coding sequence of the TCR alpha chain is set forth in SEQ ID NO: 35, SEQ ID NO: 40 or SEQ ID NO: 45; and / or The nucleic acid molecule of claim 10, wherein the coding sequence of the TCR β chain is set forth in SEQ ID NO: 36, SEQ ID NO: 41 or SEQ ID NO:

46.

13. An expression vector comprising the nucleic acid molecule of claim 10.

14. 11. An isolated polypeptide encoded by the nucleic acid molecule of claim 10.

15. A host cell comprising the TCR or fragment of claim 1 or 2.

16. A pharmaceutical composition comprising the TCR or fragment of claim 1 or 2 and a pharmaceutically acceptable excipient, diluent, or carrier.

17. 17. The pharmaceutical composition of claim 16 for use in the treatment of a disease caused by HBV infection.

18. 18. The pharmaceutical composition of claim 17, wherein the diseases caused by HBV infection include hepatitis, liver fibrosis, cirrhosis, and liver cancer.

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