A Practical Approach to Immunotherapy of Hepatocellular Carcinoma Using T Cells Redirected Against Hepatitis B Virus

Abstract

Hepatocellular carcinoma (HCC) cells often have hepatitis B virus (HBV)-DNA integration and can be targeted by HBV-specific T cells. The use of viral vectors to introduce exogenous HBV-specific T-cell receptors (TCR) on T cells to redirect their specificity is complex and expensive to implement in clinical trials. Moreover, it raises safety concerns related to insertional mutagenesis and potential toxicity of long-lived HBV-specific T cells in patients with persistent infection. To develop a more practical and safer approach to cell therapy of HCC, we used electroporation of mRNA encoding anti-HBV TCR. Approximately 80% of CD8+ T cells expressed functional HBV TCR 24 hours postelectroporation, an expression efficiency much higher than that obtained by retroviral transduction (~18%). Antigen-specific cytokine production of electroporated T cells was efficient within 72-hour period, after which the redirected T cells lost their HBV-specific function. Despite this transient functionality, the TCR-electroporated T cells efficiently prevented tumor seeding and suppressed the growth of established tumors in a xenograft model of HCC. Finally, we established a method for large-scale TCR mRNA electroporation that yielded large numbers of highly functional clinical-grade anti-HBV T cells. This method represents a practical approach to cell therapy of HCC and its inherently self-limiting toxicity suggests potential for application in other HBV-related pathologies. Hepatocellular carcinoma (HCC) cells often have hepatitis B virus (HBV)-DNA integration and can be targeted by HBV-specific T cells. The use of viral vectors to introduce exogenous HBV-specific T-cell receptors (TCR) on T cells to redirect their specificity is complex and expensive to implement in clinical trials. Moreover, it raises safety concerns related to insertional mutagenesis and potential toxicity of long-lived HBV-specific T cells in patients with persistent infection. To develop a more practical and safer approach to cell therapy of HCC, we used electroporation of mRNA encoding anti-HBV TCR. Approximately 80% of CD8+ T cells expressed functional HBV TCR 24 hours postelectroporation, an expression efficiency much higher than that obtained by retroviral transduction (~18%). Antigen-specific cytokine production of electroporated T cells was efficient within 72-hour period, after which the redirected T cells lost their HBV-specific function. Despite this transient functionality, the TCR-electroporated T cells efficiently prevented tumor seeding and suppressed the growth of established tumors in a xenograft model of HCC. Finally, we established a method for large-scale TCR mRNA electroporation that yielded large numbers of highly functional clinical-grade anti-HBV T cells. This method represents a practical approach to cell therapy of HCC and its inherently self-limiting toxicity suggests potential for application in other HBV-related pathologies. IntroductionHepatocellular carcinoma (HCC) is the third leading cause of cancer deaths worldwide.1Parkin DM Bray F Ferlay J Pisani P Estimating the world cancer burden: Globocan 2000.Int J Cancer. 2001; 94: 153-156Crossref PubMed Scopus (3283) Google Scholar In Asian countries, where chronic hepatitis B virus (HBV) infection is frequent, HBV is a major etiologic agent, accounting for at least 80% of HCC.2El-Serag HB Epidemiology of viral hepatitis and hepatocellular carcinoma.Gastroenterology. 2012; 142: 1264-1273.e1Abstract Full Text Full Text PDF PubMed Scopus (2344) Google Scholar HBV predisposes to the development of HCC by causing liver chronic inflammation but also by integrating into the genome of hepatocytes; a high frequency of HBV integrations has been observed in HBV-related HCC tumors3Sung WK Zheng H Li S Chen R Liu X Li Y et al.Genome-wide survey of recurrent HBV integration in hepatocellular carcinoma.Nat Genet. 2012; 44: 765-769Crossref PubMed Scopus (623) Google Scholar resulting in the expression of HBV antigens on tumor cells.4Brechot C Pourcel C Louise A Rain B Tiollais P Presence of integrated hepatitis B virus DNA sequences in cellular DNA of human hepatocellular carcinoma.Nature. 1980; 286: 533-535Crossref PubMed Scopus (467) Google Scholar,5Edman JC Gray P Valenzuela P Rall LB Rutter WJ Integration of hepatitis B virus sequences and their expression in a human hepatoma cell.Nature. 1980; 286: 535-538Crossref PubMed Scopus (204) Google ScholarThe therapeutic options for HCC are limited, particularly in patients with advanced disease, who almost invariably succumb to their disease.6Llovet JM Burroughs A Bruix J Hepatocellular carcinoma.Lancet. 2003; 362: 1907-1917Abstract Full Text Full Text PDF PubMed Scopus (3757) Google Scholar Tumors expressing viral peptides are attractive targets for cell therapies because their expression is limited to infected cells. In principle, it should be possible to isolate T lymphocytes with antiviral specificities from patients and expand them ex vivo before reinfusion,7Straathof KC Bollard CM Popat U Huls MH Lopez T Morriss MC et al.Treatment of nasopharyngeal carcinoma with Epstein-Barr virus–specific T lymphocytes.Blood. 2005; 105: 1898-1904Crossref PubMed Scopus (295) Google Scholar,8Rooney CM Smith CA Ng CY Loftin SK Sixbey JW Gan Y et al.Infusion of cytotoxic T cells for the prevention and treatment of Epstein-Barr virus-induced lymphoma in allogeneic transplant recipients.Blood. 1998; 92: 1549-1555Crossref PubMed Google Scholar but this process is slow, laborious, and often unsuccessful. Moreover, we found that patients with HBV-related HCC, like those with only chronic hepatitis B, have a profound defect of HBV-specific T cells.9Gehring AJ Ho ZZ Tan AT Aung MO Lee KH Tan KC et al.Profile of tumor antigen-specific CD8 T cells in patients with hepatitis B virus-related hepatocellular carcinoma.Gastroenterology. 2009; 137: 682-690Abstract Full Text Full Text PDF PubMed Scopus (127) Google ScholarGenetic modification of peripheral blood T cells with T-cell receptors (TCRs) can rapidly endow T cells with a defined antigen specificity10June CH Blazar BR Riley JL Engineering lymphocyte subsets: tools, trials and tribulations.Nat Rev Immunol. 2009; 9: 704-716Crossref PubMed Scopus (169) Google Scholar,11Park TS Rosenberg SA Morgan RA Treating cancer with genetically engineered T cells.Trends Biotechnol. 2011; 29: 550-557Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar and represents an attractive approach to cell therapy of tumors expressing viral peptides, like HBV-related HCC. Indeed, we recently demonstrated that T cells with redirected specificity toward HBV envelope antigens can recognize and lyse HCC lines with natural HBV-DNA integration.12Gehring AJ Xue SA Ho ZZ Teoh D Ruedl C Chia A et al.Engineering virus-specific T cells that target HBV infected hepatocytes and hepatocellular carcinoma cell lines.J Hepatol. 2011; 55: 103-110Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar However, a specific concern regarding this approach is that HBV antigen expression is not exclusive to transformed hepatocytes; nontumor hepatocytes might also express HBV antigens and adoptive T-cell therapy could trigger severe liver damage.13Ando K Moriyama T Guidotti LG Wirth S Schreiber RD Schlicht HJ et al.Mechanisms of class I restricted immunopathology. A transgenic mouse model of fulminant hepatitis.J Exp Med. 1993; 178: 1541-1554Crossref PubMed Scopus (414) Google Scholar,14Maini MK Boni C Lee CK Larrubia JR Reignat S Ogg GS et al.The role of virus-specific CD8(+) cells in liver damage and viral control during persistent hepatitis B virus infection.J Exp Med. 2000; 191: 1269-1280Crossref PubMed Scopus (668) Google Scholar,15Bertoletti A Maini MK Protection or damage: a dual role for the virus-specific cytotoxic T lymphocyte response in hepatitis B and C infection?.Curr Opin Microbiol. 2000; 3: 387-392Crossref PubMed Scopus (65) Google Scholar The traditional method to genetically engineer T cells, i.e., viral transduction, may compound this by T cells which express anti-HBV specificity and may be to into Moreover, viral vectors the of therapy a in PubMed Scopus Google S A J A et in patients after therapy of PubMed Scopus Google A C F D S et integration expression but has on the and of T PubMed Scopus Google Scholar Finally, the and of viral transduction in clinical trials to the of this of cell the of to the cell therapy of HBV-related HCC, we anti-HBV T cells, expressing anti-HBV could be by mRNA expression and in T cells engineered by this method and their in cells in and in an model in to cells. Finally, we the of a large-scale clinical-grade mRNA electroporation method to rapidly large numbers of anti-HBV redirected T cells for clinical of TCR by mRNA mRNA encoding the and of the HBV and used electroporation to introduce it into T cells from of TCR was by and hours after of CD8+ T cells expressed the The TCR expression was at 24 hours postelectroporation, where of CD8+ T cells expressed the TCR TCR expression and was not after hours The of expression at 24 hours was much higher than that by retroviral transduction electroporated T cells not expression of TCR and a control for functional an was also expressed on T cells by mRNA electroporation of and of TCR expressed by electroporation or retroviral electroporated T cells for their to in response to cells human cell at from to The of was at 24 hours postelectroporation, with the of expression TCR not CD8+ T cells in to T cells where of CD8+ T cells expression in electroporated T cells after of CD8+ T cells or mRNA electroporated T cells not in response to cells the mRNA electroporated CD8 and T cells a of to the T cells and are to efficiently and of electroporated or T cells with cells with not of electroporated T cells and in to only of T cells the of and and the of that could be in of electroporated T cells was or higher than that by T cells of TCR expression and of mRNA electroporated T of expression of at of or CD8 T cells from after with of or CD8 T cells from after with mRNA retroviral expression of at or CD8 T cells from after with cells. in a the of TCR expressed by mRNA electroporation was to that of TCR expressed by retroviral T cells from the electroporated or with and for to specific T-cell found that the functional of electroporated and T cells was to the HBV-specific CD8 T-cell from which the TCR was and could be observed at to functional HBV-specific can be with high efficiency into T cells by mRNA and of mRNA electroporated and T cells. of T-cell T cells by mRNA electroporation with retroviral are of response obtained from cytokine of targets and or targets and by electroporated T cells with T cells are of of and on a T cells electroporated or with after hours with cells. with cells control of and are mRNA electroporated T cells have of lymphocytes CD8+ T cells and T cells in mRNA electroporated T cells with T cells The of and cells within lymphocytes or the CD8+ or by into subsets: and expressed their cytotoxic T cells with the HCC cell which expressed HBV antigen and or HBV antigen expressing at for 24 hours and was by in target cells after with with cells of the transduction method In cells by of engineered T cells In the of electroporated T cells was higher in than that of T cells, with at the expression of and by the engineered T cells, and found in expression but a higher expression of in electroporated T cells also mRNA T-cell than 80% of the electroporated lymphocytes from T cells lymphocytes of and cells. of antigen-specific T cells, more than 80% of electroporated CD8 higher than that of T cells The of T cells was with transduction with the mRNA electroporated T cells have in the of T cells redirected we established a mouse xenograft tumor model cells expressing HBV antigen and in the of growth was by In we used cells to the of T cells for and and CD8 cells CM Smith CA Ng CY Loftin SK Sixbey JW Gan Y et al.Infusion of cytotoxic T cells for the prevention and treatment of Epstein-Barr virus-induced lymphoma in allogeneic transplant recipients.Blood. 1998; 92: 1549-1555Crossref PubMed Google P S of cytotoxic T cells during adoptive of virus can be prevented by B cells or T J Immunol. PubMed Scopus Google CA A et T cell a is by T cells and by T Immunol. 2005; PubMed Scopus Google C therapy with CD8(+) T it may by with a from its PubMed Scopus Google Scholar found that CD8 was and tumors in in of CD8 tumor cells in the by of cells to only a in tumor growth with control with T cells. Moreover, in that we also observed that T cells to the tumor that expressed the target antigen and tumor This was also by of and tumor CD8 cytotoxic and T cells. tumor cells by in after tumor with CD8 CD8 CD8 or T cells with T cells In tumor was by and are of mouse and the of is of the Tumors in with CD8 with CD8 or T cells. from of from with T cells at after adoptive cell tumor cells expressing and CD8 CD8 T cells in the tumor and in at used the model to the of T cells electroporated with anti-HBV TCR mRNA electroporated T cells, of CD8 of T cells of electroporated T cells tumor growth and therapy was after the tumor the of T-cell of mRNA electroporated T cells control tumor growth and tumor cells by in after tumor with of mRNA electroporated T cells with T cells was by and of the the in vivo we electroporated T cells could HCC in with and numbers of mRNA electroporated T cells hours In T cells, tumor seeding and growth was numbers of T cells tumor development in with numbers of T cells of HCC tumor cells seeding by mRNA electroporated T cells. tumor cells by in hours or with of mRNA electroporated T cells with T cells was by and of the of large-scale method to express anti-HBV TCR in T that mRNA electroporation could be used to express functional anti-HBV TCR in T cells, which in To this to large-scale that can be used in HCC we used a T cells in a hours after of CD8+ T cells In we obtained antigen-specific T cells cells and after HBV-specific was to that of T cells electroporated in the it is possible to large of functional antigen-specific T of TCR expression and of mRNA electroporated T cells in clinical-grade A cell and of clinical-grade electroporation of T cells. of CD8 and in mRNA electroporated T cells at 24 hours the frequency of and cells of CD8 or electroporated T of this the potential of T cells expressing anti-HBV TCR by mRNA The of cells expressing the receptors 24 hours after electroporation was much higher than that by retroviral TCR electroporation antigen-specific in of the in response to HBV-specific and tumor cells expressing HBV antigen in The anti-HBV TCR mRNA electroporated cells demonstrated their also in vivo by the growth of human cells in of the and antigen-specific of the T cells after electroporation was transient and after of electroporated T cells may be to clinical not this a major because the transduction method is and only a hours in a large-scale redirected T cells have that to production of T cells expressing the TCR of antigen-specific T J Li J et of lymphocyte with cancer in patients cell Immunol. PubMed Scopus Google J Rosenberg SA of T-cell is with tumor in a adoptive cell 2005; PubMed Scopus Google Scholar and of T C MC C of CD8+ T cells from cells persistent T cell in PubMed Scopus Google CA C K et of in the in vivo of CD8+ T 2005; PubMed Scopus Google CA P K et CD8+ T cells with T 2005; PubMed Scopus Google Scholar might have a higher therapeutic A is that T cells expressing TCR have the potential to to chronic to by of and TCR R R et T cell PubMed Scopus Google Scholar In the of HCC, hepatocytes may also be and or of the transient TCR expression resulting from mRNA should be Finally, with mRNA is of insertional mutagenesis with the use of integrating viral therapy a in PubMed Scopus Google S A J A et in patients after therapy of PubMed Scopus Google A C F D S et integration expression but has on the and of T PubMed Scopus Google to the of in where electroporated cells to be more than cells, the more efficient in could cells in HCC xenograft mouse model of electroporated cells to tumor cell growth but not to with with antigen T Y C CM Liu X et of electroporated T cells expressing a antigen of human PubMed Scopus Google Scholar and are to the transient of TCR expression after is possible that higher of T cells a more could have been more the potential of TCR-electroporated T cells was demonstrated by the that of them was to of cells in mouse that are particularly HCC therapy on liver JM Burroughs A Bruix J Hepatocellular carcinoma.Lancet. 2003; 362: 1907-1917Abstract Full Text Full Text PDF PubMed Scopus (3757) Google JM and liver for hepatocellular 2005; PubMed Scopus Google Scholar and HCC in transplant patients to the seeding of HCC cells often HBV B et carcinoma is with an of hepatitis B virus after liver Full Text Full Text PDF PubMed Scopus Google Scholar in the liver or in The of model are that cells and expand in the and not in the and cells are the only cells that express model not have In this the model might the after liver in chronic HBV patients with HCC. mRNA electroporated T cells expressing anti-HBV TCR might a potential to the of tumor cells. a TCR specificity be in clinical to HCC seeding is have also specific for HBV and AJ Xue SA Ho ZZ Teoh D Ruedl C Chia A et al.Engineering virus-specific T cells that target HBV infected hepatocytes and hepatocellular carcinoma cell lines.J Hepatol. 2011; 55: 103-110Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar that can be used to T cells of The of mRNA TCR electroporation might an production of T the TCR expression in a large of cells that this approach can be into clinical The with mRNA electroporation should be than those the production and clinical use of viral Moreover, the and for mRNA production are than those to clinical-grade viral C D et a for PubMed Scopus Google H D T P et method to the of natural cells 2012; Full Text Full Text PDF PubMed Scopus Google Scholar The electroporation can be within Liu S C R J et of antigen receptors in natural cells with a PubMed Scopus Google Scholar at an of than This is in to retroviral transduction, a of retroviral for a with expressing HCC at least more and in and and in to of the TCR mRNA electroporation method should the clinical application of anti-HBV cell therapy of HCC. practical and their also potential for other to antiviral T-cell therapy in patients with chronic hepatitis Finally, it should be possible to the method to TCR other viral peptides, like for treatment of other and or expressing in with and was to cells in cells in and blood cells from blood cells with and in human for and used for retroviral T cells used for mRNA electroporation the for and was to before transduction, and of CD8+ T cells. The HBV TCR and and into was and T cells AJ Xue SA Ho ZZ Teoh D Ruedl C Chia A et al.Engineering virus-specific T cells that target HBV infected hepatocytes and hepatocellular carcinoma cell lines.J Hepatol. 2011; 55: 103-110Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar hours after transduction, expression of TCR was by T cells and cells, cells with peripheral blood cells and in human T cells with and and used for adoptive cell T cells for CD8 and expression before adoptive cell In T cells to for of CD8 T cells a the The of the cell from to by for cell obtained from and for cytokine obtained from and cytokine was by and cells with was or and was with the of TCR mRNA and electroporation the TCR from a that we and it into the The was from the and the The DNA was used to the TCR mRNA the or mRNA was to was with was by and The resulting was by and in electroporation with the peripheral blood cells for with and in human in of and TCR mRNA was at The was in a and electroporated cells in human and at and electroporation was T cells in electroporation mRNA was at The was in a electroporated to the cells at for and in human and at and and cells with of for at and T cells or mRNA electroporated T cells with cells for hours in the of and for CD8 and of CD8+ the functional or electroporated T cells with cells in the of A and for and or expressing in to with and with T cells or electroporated T cells at target in in with human for 24 was by expression in target cells. was and of was to and for to cell was with a cells used a for expressed after and of vivo xenograft tumor model and adoptive cell and specific by the and of in with the of the and and the for of to with expressing mouse in the a C S C et of established liver tumor by Immunol. PubMed Scopus Google with infected a mouse model for Google Scholar was to for In T cells or electroporated T cells in and growth was by in vivo with of in with tumors on before tumor with and for in the tumor was and expressed and in in and at into and on for and with and with for at with mouse CD8 at by with with and was by the was in more than was the with for with P of a with in by electroporated T cells to T cells after with cells or cells with of of and IntroductionHepatocellular carcinoma (HCC) is the third leading cause of cancer deaths worldwide.1Parkin DM Bray F Ferlay J Pisani P Estimating the world cancer burden: Globocan 2000.Int J Cancer. 2001; 94: 153-156Crossref PubMed Scopus (3283) Google Scholar In Asian countries, where chronic hepatitis B virus (HBV) infection is frequent, HBV is a major etiologic agent, accounting for at least 80% of HCC.2El-Serag HB Epidemiology of viral hepatitis and hepatocellular carcinoma.Gastroenterology. 2012; 142: 1264-1273.e1Abstract Full Text Full Text PDF PubMed Scopus (2344) Google Scholar HBV predisposes to the development of HCC by causing liver chronic inflammation but also by integrating into the genome of hepatocytes; a high frequency of HBV integrations has been observed in HBV-related HCC tumors3Sung WK Zheng H Li S Chen R Liu X Li Y et al.Genome-wide survey of recurrent HBV integration in hepatocellular carcinoma.Nat Genet. 2012; 44: 765-769Crossref PubMed Scopus (623) Google Scholar resulting in the expression of HBV antigens on tumor cells.4Brechot C Pourcel C Louise A Rain B Tiollais P Presence of integrated hepatitis B virus DNA sequences in cellular DNA of human hepatocellular carcinoma.Nature. 1980; 286: 533-535Crossref PubMed Scopus (467) Google Scholar,5Edman JC Gray P Valenzuela P Rall LB Rutter WJ Integration of hepatitis B virus sequences and their expression in a human hepatoma cell.Nature. 1980; 286: 535-538Crossref PubMed Scopus (204) Google ScholarThe therapeutic options for HCC are limited, particularly in patients with advanced disease, who almost invariably succumb to their disease.6Llovet JM Burroughs A Bruix J Hepatocellular carcinoma.Lancet. 2003; 362: 1907-1917Abstract Full Text Full Text PDF PubMed Scopus (3757) Google Scholar Tumors expressing viral peptides are attractive targets for cell therapies because their expression is limited to infected cells. In principle, it should be possible to isolate T lymphocytes with antiviral specificities from patients and expand them ex vivo before reinfusion,7Straathof KC Bollard CM Popat U Huls MH Lopez T Morriss MC et al.Treatment of nasopharyngeal carcinoma with Epstein-Barr virus–specific T lymphocytes.Blood. 2005; 105: 1898-1904Crossref PubMed Scopus (295) Google Scholar,8Rooney CM Smith CA Ng CY Loftin SK Sixbey JW Gan Y et al.Infusion of cytotoxic T cells for the prevention and treatment of Epstein-Barr virus-induced lymphoma in allogeneic transplant recipients.Blood. 1998; 92: 1549-1555Crossref PubMed Google Scholar but this process is slow, laborious, and often unsuccessful. Moreover, we found that patients with HBV-related HCC, like those with only chronic hepatitis B, have a profound defect of HBV-specific T cells.9Gehring AJ Ho ZZ Tan AT Aung MO Lee KH Tan KC et al.Profile of tumor antigen-specific CD8 T cells in patients with hepatitis B virus-related hepatocellular carcinoma.Gastroenterology. 2009; 137: 682-690Abstract Full Text Full Text PDF PubMed Scopus (127) Google ScholarGenetic modification of peripheral blood T cells with T-cell receptors (TCRs) can rapidly endow T cells with a defined antigen specificity10June CH Blazar BR Riley JL Engineering lymphocyte subsets: tools, trials and tribulations.Nat Rev Immunol. 2009; 9: 704-716Crossref PubMed Scopus (169) Google Scholar,11Park TS Rosenberg SA Morgan RA Treating cancer with genetically engineered T cells.Trends Biotechnol. 2011; 29: 550-557Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar and represents an attractive approach to cell therapy of tumors expressing viral peptides, like HBV-related HCC. Indeed, we recently demonstrated that T cells with redirected specificity toward HBV envelope antigens can recognize and lyse HCC lines with natural HBV-DNA integration.12Gehring AJ Xue SA Ho ZZ Teoh D Ruedl C Chia A et al.Engineering virus-specific T cells that target HBV infected hepatocytes and hepatocellular carcinoma cell lines.J Hepatol. 2011; 55: 103-110Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar However, a specific concern regarding this approach is that HBV antigen expression is not exclusive to transformed hepatocytes; nontumor hepatocytes might also express HBV antigens and adoptive T-cell therapy could trigger severe liver damage.13Ando K Moriyama T Guidotti LG Wirth S Schreiber RD Schlicht HJ et al.Mechanisms of class I restricted immunopathology. A transgenic mouse model of fulminant hepatitis.J Exp Med. 1993; 178: 1541-1554Crossref PubMed Scopus (414) Google Scholar,14Maini MK Boni C Lee CK Larrubia JR Reignat S Ogg GS et al.The role of virus-specific CD8(+) cells in liver damage and viral control during persistent hepatitis B virus infection.J Exp Med. 2000; 191: 1269-1280Crossref PubMed Scopus (668) Google Scholar,15Bertoletti A Maini MK Protection or damage: a dual role for the virus-specific cytotoxic T lymphocyte response in hepatitis B and C infection?.Curr Opin Microbiol. 2000; 3: 387-392Crossref PubMed Scopus (65) Google Scholar The traditional method to genetically engineer T cells, i.e., viral transduction, may compound this by T cells which express anti-HBV specificity and may be to into Moreover, viral vectors the of therapy a in PubMed Scopus Google S A J A et in patients after therapy of PubMed Scopus Google A C F D S et integration expression but has on the and of T PubMed Scopus Google Scholar Finally, the and of viral transduction in clinical trials to the of this of cell the of to the cell therapy of HBV-related HCC, we anti-HBV T cells, expressing anti-HBV could be by mRNA expression and in T cells engineered by this method and their in cells in and in an model in to cells. Finally, we the of a large-scale clinical-grade mRNA electroporation method to rapidly large numbers of anti-HBV redirected T cells for clinical