RESUMEN
BACKGROUND AND AIMS: Liver transplantation (LT) is the primary curative option for cirrhotic patients with early-stage hepatocellular carcinoma (HCC). However, tumor recurrence occurs in 15-20% of cases with unfavorable prognosis. We have developed a library of T cell receptors (TCRs) specific for different hepatitis B virus (HBV) antigens, restricted by different molecules of human leucocyte antigen (HLA)-class I, to redirect T cells against HBV antigens (Banu in Sci Rep 4:4166, 2014). We further demonstrated that these transiently functional T cells specific for HBV obtained through messenger RNA (mRNA) electroporation can eliminate HCC cells expressing HBV antigens in vitro and in vivo (Kah in J Clin Invest 127:3177-3188, 2017). A phase I clinical trial for patients with HCC recurrence post-liver transplant was conducted to assess the safety, tolerability, and anti-tumor efficacy of transiently functional HBV-TCR T cells. Here, we report the clinical findings with regard to the safety and anti-tumor efficacy of mRNA electroporated HBV-specific TCR-T cells. (ClinicalTrials.gov identifier: NCT02719782). PATIENTS AND METHODS: A total of six patients with HBV-positive recurrent HCC post-liver transplant and HLA-matched to TCR targeting hepatitis B surface antigen (HBsAg) or hepatitis B core antigen (HBcAg) (HLA-A*02:01/HBsAg, HLA-A*11:01/HBcAg, HLA-B*58:01/HBsAg or HLA-C*08:01/HBsAg) were enrolled in this study. The primary objective was to assess the safety of short-lived mRNA electroporated HBV-TCR T cells based on the incidence and severity of the adverse event (AE) graded per National Cancer Institute Common Terminology Criteria for Adverse Events (NCI CTCAE), Version 4.0. The secondary objective was to determine the effectiveness of HBV-TCR T cells as per RECIST 1.1 criteria. Patients were followed up for survival for 2 years post-end of treatment. RESULTS: The median age of the six patients was 35.5 years (range: 28-47). The median number of HBV-TCR T cell infusions administered was 6.5 (range: 4-12). The treatment-related AE included grade 1 pyrexia. This study reported no cytokine release syndrome nor neurotoxicity. One patient remained alive and five were deceased at the time of the data cutoff (30 April 2020). CONCLUSION: This study has demonstrated that multiple infusions of mRNA electroporated HBV-specific TCR T cells were well-tolerated in patients with HBV-positive recurrent HCC post-liver transplant.
Asunto(s)
Carcinoma Hepatocelular , Hepatitis B , Neoplasias Hepáticas , Trasplante de Hígado , Humanos , Adulto , Persona de Mediana Edad , Virus de la Hepatitis B/genética , Antígenos de Superficie de la Hepatitis B , Neoplasias Hepáticas/patología , Antígenos del Núcleo de la Hepatitis B/uso terapéutico , ARN Mensajero , Recurrencia Local de Neoplasia/terapia , Recurrencia Local de Neoplasia/complicaciones , Receptores de Antígenos de Linfocitos T/genética , Hepatitis B/complicacionesRESUMEN
The application of hepatitis B virus (HBV)-T-cell receptor (TCR) T-cell immunotherapy in patients with HBV-related hepatocellular carcinoma (HBV-HCC) has been apathetic, as the expression of HBV antigens by both normal HBV-infected hepatocytes and HCC cells with HBV-DNA integration increases the risk of on-target off-tumor severe liver inflammatory events. To increase the safety of this immunotherapeutic approach, we developed messenger RNA (mRNA) HBV-TCR-redirected T cells that-due to the transient nature of mRNA-are functionally short lived and can be infused in escalating doses. The safety of this approach and its clinical potential against primary HBV-HCC have never been analyzed in human trials; thus, we studied the clinical and immunological parameters of 8 patients with chronic HBV infection and diffuse nonoperable HBV-HCC treated at weekly intervals with escalating doses (1 × 104 , 1 × 105 , 1 × 106 , and 5 × 106 TCR+ T cells/kg body weight) of T cells modified with HBV-TCR encoding mRNA. The treatment was well tolerated with no severe systemic inflammatory events, cytokine storm, or neurotoxicity observed in any of these patients throughout treatment. Instead, we observed a destruction of the tumor lesion or a prolonged stable disease in 3 of 8 patients. Importantly, the patients without clinically relevant reductions of HCC did not display any detectable peripheral blood immunological alterations. In contrast, signs of transient localized liver inflammation, activation of the T-cell compartment, and/or elevations of serum chemokine (C-X-C motif) ligand (CXCL) 9 and CXCL10 levels were detected in patients with long-term clinical benefit. Conclusion: We show that despite the reduced in vivo half-life (3-4 days), adoptive transfer of mRNA HBV-TCR T cells into patients with HBV-HCC show long-term clinical benefit that was associated with transient immunological alterations.
Asunto(s)
Carcinoma Hepatocelular , Neoplasias Hepáticas , Carcinoma Hepatocelular/terapia , Virus de la Hepatitis B/genética , Humanos , Inmunoterapia , Neoplasias Hepáticas/terapia , ARN Mensajero , Receptores de Antígenos de Linfocitos T/genética , Linfocitos TRESUMEN
BACKGROUND & AIMS: Immunotherapy with hepatitis B virus (HBV)-specific TCR redirected T (HBV-TCR-T) cells in HBV-related hepatocellular carcinoma (HBV-HCC) patients after liver transplantation was reported to be safe and had potential therapeutic efficacy. We aim to investigate the safety of HBV-TCR-T-cell immunotherapy in advanced HBV-HCC patients who had not met the criteria for liver transplantation. METHODS: We enrolled eight patients with advanced HBV-HCC and adoptively transferred short-lived autologous T cells expressing HBV-specific TCR to perform an open-label, phase 1 dose-escalation study (NCT03899415). The primary endpoint was to evaluate the safety of HBV-TCR-T-cell therapy according to National Cancer Institute Common Terminology Criteria for Adverse Events (version 4.03) during the dose-escalation process. The secondary endpoint was to assess the efficacy of HBV-TCR-T-cell therapy by evaluating the anti-tumor responses using RECIST criteria (version 1.1) and the overall survival. RESULTS: Adverse events were observed in two participants among the 8 patients enrolled. Only one patient experienced a Grade 3 liver-related adverse event after receiving a dose of 1 × 105 HBV-TCR-T cells/kg, then normalized without interventions with immunosuppressive agents. Among the patients, one achieved a partial response lasting for 27.7 months. Importantly, most of the patients exhibited a reduction or stabilization of circulating HBsAg and HBV DNA levels after HBV-TCR-T-cell infusion, indicating the on-target effects. CONCLUSIONS: The adoptive transfer of HBV-TCR-T cells into advanced HBV-HCC patients were generally safe and well-tolerated. Observations of clinical efficacy support the continued development and eventual application of this treatment strategy in patients with advanced HBV-related HCC. CLINICAL TRIALS REGISTRATION: This study was registered at ClinicalTrials.gov (NCT03899415).
Asunto(s)
Carcinoma Hepatocelular , Neoplasias Hepáticas , Carcinoma Hepatocelular/terapia , Virus de la Hepatitis B , Humanos , Inmunoterapia , Neoplasias Hepáticas/terapia , Receptores de Antígenos de Linfocitos T , Linfocitos TRESUMEN
Modification of specificity of T cells for the use in adoptive transfer (CAR- or TCR-redirected T cells) has revolutionized the therapy of liquid tumors and some infectious diseases. However, several obstacles are still hampering the efficacy of such potent therapy, hence concurrent modification of the function is also required to obtain successful results. Here we show the use of splice-switching antisense oligonucleotides (SSOs) as a tool to transiently modify T cell function. We demonstrate the possibility to transfect SSOs and an exogenous TCR into primary human T cells in the same electroporation reaction, without affecting viability and function of the transfected T lymphocytes. Moreover, we show that SSOs targeting T cell-specific mRNAs induce the skipping of the targeted exons, and the reduction of the protein and consequent modification of T cell function. This technical work paves the way to the use of SSOs in immune cells, not only for the knockdown of the functional isoform of the targeted proteins, but also for the protein manipulation by elimination of specific domains encoded by targeted exons.
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Supervivencia Celular/efectos de los fármacos , Oligonucleótidos Antisentido/farmacología , Linfocitos T/inmunología , Supervivencia Celular/inmunología , Exones/efectos de los fármacos , Exones/genética , Humanos , Mutación/genética , Oligonucleótidos Antisentido/genética , Oligonucleótidos Antisentido/inmunología , Empalme del ARN/genética , Empalme del ARN/inmunología , ARN Mensajero/genética , Linfocitos T/efectos de los fármacosRESUMEN
BACKGROUND AND AIMS: HBV-specific T-cell receptor (HBV-TCR) engineered T cells have the potential for treating HCC relapses after liver transplantation, but their efficacy can be hampered by the concomitant immunosuppressive treatment required to prevent graft rejection. Our aim is to molecularly engineer TCR-T cells that could retain their polyfunctionality in such patients while minimizing the associated risk of organ rejection. APPROACH AND RESULTS: We first analyzed how immunosuppressive drugs can interfere with the in vivo function of TCR-T cells in liver transplanted patients with HBV-HCC recurrence receiving HBV-TCR T cells and in vitro in the presence of clinically relevant concentrations of immunosuppressive tacrolimus (TAC) and mycophenolate mofetil (MMF). Immunosuppressive Drug Resistant Armored TCR-T cells of desired specificity (HBV or Epstein-Barr virus) were then engineered by concomitantly electroporating mRNA encoding specific TCRs and mutated variants of calcineurin B (CnB) and inosine-5'-monophosphate dehydrogenase (IMPDH), and their function was assessed through intracellular cytokine staining and cytotoxicity assays in the presence of TAC and MMF. Liver transplanted HBV-HCC patients receiving different immunosuppressant drugs exhibited varying levels of activated (CD39+ Ki67+ ) peripheral blood mononuclear cells after HBV-TCR T-cell infusions that positively correlate with clinical efficacy. In vitro experiments with TAC and MMF showed a potent inhibition of TCR-T cell polyfunctionality. This inhibition can be effectively negated by the transient overexpression of mutated variants of CnB and IMPDH. Importantly, the resistance only lasted for 3-5 days, after which sensitivity was restored. CONCLUSIONS: We engineered TCR-T cells of desired specificities that transiently escape the immunosuppressive effects of TAC and MMF. This finding has important clinical applications for the treatment of HBV-HCC relapses and other pathologies occurring in organ transplanted patients.
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Carcinoma Hepatocelular/cirugía , Rechazo de Injerto/prevención & control , Neoplasias Hepáticas/cirugía , Recurrencia Local de Neoplasia/terapia , Linfocitos T/trasplante , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/virología , Técnicas de Cocultivo , Resistencia a Medicamentos/genética , Rechazo de Injerto/inmunología , Rechazo de Injerto/patología , Células Hep G2 , Hepatitis B/patología , Hepatitis B/cirugía , Hepatitis B/virología , Virus de la Hepatitis B/inmunología , Virus de la Hepatitis B/metabolismo , Humanos , Inmunoterapia Adoptiva/métodos , Hígado/efectos de los fármacos , Hígado/inmunología , Hígado/patología , Hígado/virología , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/virología , Trasplante de Hígado/efectos adversos , Ácido Micofenólico/farmacología , Ácido Micofenólico/uso terapéutico , Recurrencia Local de Neoplasia/inmunología , Recurrencia Local de Neoplasia/patología , Ingeniería de Proteínas , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Tacrolimus/farmacología , Tacrolimus/uso terapéuticoRESUMEN
BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is often associated with hepatitis B virus (HBV) infection. Cells of most HBV-related HCCs contain HBV-DNA fragments that do not encode entire HBV antigens. We investigated whether these integrated HBV-DNA fragments encode epitopes that are recognized by T cells and whether their presence in HCCs can be used to select HBV-specific T-cell receptors (TCRs) for immunotherapy. METHODS: HCC cells negative for HBV antigens, based on immunohistochemistry, were analyzed for the presence of HBV messenger RNAs (mRNAs) by real-time polymerase chain reaction, sequencing, and Nanostring approaches. We tested the ability of HBV mRNA-positive HCC cells to generate epitopes that are recognized by T cells using HBV-specific T cells and TCR-like antibodies. We then analyzed HBV gene expression profiles of primary HCCs and metastases from 2 patients with HCC recurrence after liver transplantation. Using the HBV-transcript profiles, we selected, from a library of TCRs previously characterized from patients with self-limited HBV infection, the TCR specific for the HBV epitope encoded by the detected HBV mRNA. Autologous T cells were engineered to express the selected TCRs, through electroporation of mRNA into cells, and these TCR T cells were adoptively transferred to the patients in increasing numbers (1 × 104-10 × 106 TCR+ T cells/kg) weekly for 112 days or 1 year. We monitored patients' liver function, serum levels of cytokines, and standard blood parameters. Antitumor efficacy was assessed based on serum levels of alpha fetoprotein and computed tomography of metastases. RESULTS: HCC cells that did not express whole HBV antigens contained short HBV mRNAs, which encode epitopes that are recognized by and activate HBV-specific T cells. Autologous T cells engineered to express TCRs specific for epitopes expressed from HBV-DNA in patients' metastases were given to 2 patients without notable adverse events. The cells did not affect liver function over a 1-year period. In 1 patient, 5 of 6 pulmonary metastases decreased in volume during the 1-year period of T-cell administration. CONCLUSIONS: HCC cells contain short segments of integrated HBV-DNA that encodes epitopes that are recognized by and activate T cells. HBV transcriptomes of these cells could be used to engineer T cells for personalized immunotherapy. This approach might be used to treat a wider population of patients with HBV-associated HCC.
Asunto(s)
Carcinoma Hepatocelular/terapia , ADN Viral , Virus de la Hepatitis B/genética , Inmunoterapia Adoptiva/métodos , Neoplasias Hepáticas/terapia , Neoplasias Pulmonares/terapia , Recurrencia Local de Neoplasia/genética , Linfocitos T/inmunología , Transcriptoma/inmunología , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/secundario , Carcinoma Hepatocelular/virología , Línea Celular Tumoral , Electroporación , Epítopos de Linfocito T/biosíntesis , Epítopos de Linfocito T/genética , Epítopos de Linfocito T/inmunología , Antígenos de la Hepatitis B/genética , Antígenos de la Hepatitis B/inmunología , Humanos , Inmunoterapia Adoptiva/efectos adversos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/virología , Trasplante de Hígado , Neoplasias Pulmonares/diagnóstico por imagen , Neoplasias Pulmonares/secundario , Masculino , Persona de Mediana Edad , Biosíntesis de Proteínas , ARN Viral/genética , Receptores de Antígenos de Linfocitos T , Integración Viral , alfa-Fetoproteínas/metabolismoRESUMEN
BACKGROUND & AIMS: Strategies to develop virus-specific T cells against hepatic viral infections have been hindered by safety concerns. We engineered nonlytic human T cells to suppress replication of hepatitis B virus (HBV) and hepatitis C virus (HCV) without overt hepatotoxicity and investigated their antiviral activity. METHODS: We electroporated resting T cells or T cells activated by anti-CD3 with mRNAs encoding HBV or HCV-specific T-cell receptors (TCRs) to create 2 populations of TCR-reprogrammed T cells. We tested their ability to suppress HBV or HCV replication without lysis in 2-dimensional and 3-dimensional cultures of HepG2.2.15 cells and HBV-infected HepG2-hNTCP cells. We also injected TCR-reprogrammed resting and activated T cells into HBV-infected urokinase-type plasminogen activator/severe combined immunodeficiency disease/interleukin 2γ mice with humanized livers and measured levels of intrahepatic and serological viral parameters and serum alanine aminotransferase. Livers were collected for analysis of gene expression patterns to determine effects of the TCR-reprogrammed T cells. RESULTS: TCR-reprogrammed resting T cells produced comparable levels of interferon gamma but lower levels of perforin and granzyme than activated T cells and did not lyse HCV- or HBV-infected hepatoma cells. Although T-cell secretion of interferon gamma was required to inhibit HCV replication, the HBV-specific TCR-reprogrammed resting T cells reduced HBV replication also through intracellular activation of apolipoprotein B mRNA editing enzyme, catalytic polypeptide 3 (APOBEC3). The mechanism of APOBEC3 intracellular activation involved temporal expression of lymphotoxin-ß receptor ligands on resting T cells after TCR-mediated antigen recognition and activation of lymphotoxin-ß receptor in infected cells. CONCLUSIONS: We developed TCR-reprogrammed nonlytic T cells capable of activating APOBEC3 in hepatoma cells and in HBV-infected human hepatocytes in mice, limiting viral infection. These cells with limited hepatotoxicity might be developed for treatment of chronic HBV infection.
Asunto(s)
Citosina Desaminasa/inmunología , Hepacivirus/inmunología , Virus de la Hepatitis B/inmunología , Hepatitis B Crónica/terapia , Hígado/metabolismo , Activación de Linfocitos/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunología , Desaminasas APOBEC , Animales , Citidina Desaminasa , Electroporación , Células Hep G2 , Hepatocitos , Humanos , Interferón gamma/inmunología , Ratones , Ratones SCID , ARN Mensajero , ARN Viral , Receptores de Antígenos de Linfocitos T/genéticaRESUMEN
Although therapy for chronic hepatitis C virus infection has delivered remarkable cure rates, curative therapies for hepatitis B virus (HBV) may only be available in the distant future. The possibility to eliminate or at least stably maintain low levels of HBV replication under the control of a functional anti-host response has stimulated the development of specific immunotherapies for HBV infection. We reviewed the development of T-cell therapy for HBV, highlighting its potential antiviral efficiency but also its potential toxicities in different groups of chronic HBV patients. Hepatitis B virus (HBV) and hepatitis C virus (HCV) infections are the only two communicable diseases in which there have been increases in related morbidity and mortality over the past 20 years [1]. Both viruses are chronically infecting about 500 million people (HBV ~350 million, HCV ~150 million) and represent the seventh most frequent cause of death worldwide [1]. HBV and HCV are hepatotropic, non-cytopathic viruses able to establish persistent infections that cause different degrees of hepatic inflammation (chronic hepatitis), leading to the development of liver cirrhosis and hepatocellular carcinoma (HCC). The two viruses are unrelated and virologically different. HCV remains prevalent in North America and Europe, whereas chronic hepatitis B is prevalent in Asia and sub-Saharan Africa [1,2]. HCV is an RNA virus belonging to the Flaviviridae family, and HBV is a DNA virus of the Hepadnaviridae family and uses reverse transcriptase to synthesize its DNA from a pre-genomic RNA form [3]. HCV is able to activate in the infected host a classical type I interferon (IFN)-mediated innate response [3], whereas HBV generally escapes innate immune recognition and does not activate type I IFN-mediated immunity. Chronic HBV and HCV infections are both characterized by quantitative and functional defects of virus-specific T-cell response [4,5]. The frequency of virus-specific T cells is extremely low, and virus-specific T cells show features of exhaustion in both chronic HBV and HCV patients [6]. However, the quantitative and functional defects are more pronounced in HBV infections, with T cells virtually undetectable in the blood of many chronic HBV patients by ex vivo analysis [7-9]. In addition, while frequency and impact of viral mutations in T cell epitopes are frequently detectable in HCV infections [10], viral mutations affecting CD8 T-cell epitopes are scarcer in chronic HBV patients [6,11,12]. Of extreme practical importance in relation to the potential impact of T-cell therapy for HBV and HCV are the efficacies of currently available treatments. New therapies for HCV have delivered remarkable cure rates, with more than 90% of patients achieving viral clearance with all oral direct-acting antivirals [13]. In contrast, curative therapies for HBV will not be available until the distant future (14). Thus, although it is difficult to see a possible therapeutic advantage of a new T-cell-based therapy in chronic HCV patients, the fact that current therapies for HBV only partially suppress but do not eliminate HBV from the infected host has encouraged research for new and more radical therapies designed to eliminate or at least stably maintain low levels of HBV replication under the control of a functional anti-host response. For these reasons, in this review, we concentrate on the development of T-cell therapy for HBV. T-cell therapy for HCV chronic infection is certainly important for understanding the mechanisms of T-cell antiviral control [15,16], but their use for therapy appears unlikely.
Asunto(s)
Trasplante de Células/efectos adversos , Trasplante de Células/métodos , Hepatitis B Crónica/terapia , Linfocitos T/trasplante , Epítopos de Linfocito T/genética , Hepatitis C Crónica/terapia , Humanos , Linfocitos T/inmunologíaRESUMEN
Adoptive transfer of T cells engineered to express a hepatitis B virus-specific (HBV-specific) T cell receptor (TCR) may supplement HBV-specific immune responses in chronic HBV patients and facilitate HBV control. However, the risk of triggering unrestrained proliferation of permanently engineered T cells raises safety concerns that have hampered testing of this approach in patients. The aim of the present study was to generate T cells that transiently express HBV-specific TCRs using mRNA electroporation and to assess their antiviral and pathogenetic activity in vitro and in HBV-infected human liver chimeric mice. We assessed virological and gene-expression changes using quantitative reverse-transcriptase PCR (qRT-PCR), immunofluorescence, and Luminex technology. HBV-specific T cells lysed HBV-producing hepatoma cells in vitro. In vivo, 3 injections of HBV-specific T cells caused progressive viremia reduction within 12 days of treatment in animals reconstituted with haplotype-matched hepatocytes, whereas viremia remained stable in mice receiving irrelevant T cells redirected toward hepatitis C virus-specific TCRs. Notably, increases in alanine aminotransferase levels, apoptotic markers, and human inflammatory cytokines returned to pretreatment levels within 9 days after the last injection. T cell transfer did not trigger inflammation in uninfected mice. These data support the feasibility of using mRNA electroporation to engineer HBV TCR-redirected T cells in patients with chronic HBV infection.
Asunto(s)
Traslado Adoptivo , Hepatitis B/inmunología , Linfocitos/citología , Receptores de Antígenos de Linfocitos T/metabolismo , Alanina Transaminasa/metabolismo , Animales , Carcinoma Hepatocelular/inmunología , Carcinoma Hepatocelular/virología , Técnicas de Cocultivo , Electroporación , Femenino , Perfilación de la Expresión Génica , Granzimas/metabolismo , Haplotipos , Células Hep G2 , Hepatitis B/terapia , Antígenos de Superficie de la Hepatitis B/inmunología , Virus de la Hepatitis B , Hepatitis B Crónica/inmunología , Hepatitis B Crónica/terapia , Humanos , Inflamación , Interferón gamma/metabolismo , Hígado/metabolismo , Neoplasias Hepáticas/inmunología , Neoplasias Hepáticas/virología , Masculino , Ratones , ARN Mensajero/metabolismo , Linfocitos T/virologíaRESUMEN
The tumor microenvironment imposes physical and functional constraints on the antitumor efficacy of adoptive T cell immunotherapy. Preclinical testing of different T cell preparations can help in the selection of efficient immune therapies, but in vivo models are expensive and cumbersome to develop, while classical in vitro 2D models cannot recapitulate the spatiotemporal dynamics experienced by T cells targeting cancer. Here, we describe an easily customizable 3D model, in which the tumor microenvironment conditions are modulated and the functionality of different T cell preparations is tested. We incorporate human cancer hepatocytes as a single cell or as tumor cell aggregates in a 3D collagen gel region of a microfluidic device. Human T cells engineered to express tumor-specific T cell receptors (TCR-T cells) are then added in adjacent channels. The TCR-T cells' ability to migrate and kill the tumor target and the profile of soluble factors were investigated under conditions of varying oxygen levels and in the presence of inflammatory cytokines. We show that only the 3D model detects the effect that oxygen levels and the inflammatory environment impose on engineered TCR-T cell function, and we also used the 3D microdevice to analyze the TCR-T cell efficacy in an immunosuppressive scenario. Hence, we show that our microdevice platform enables us to decipher the factors that can alter T cell function in 3D and can serve as a preclinical assay to tailor the most efficient immunotherapy configuration for a specific therapeutic goal.
RESUMEN
Therapy with genetically modified autologous T cells has shown great promise in cancer therapy. For an efficient control of hepatitis C virus (HCV) infection, cytotoxic T cells (CTL) are pivotal, but persistence of activated T cells may lead to liver toxicity. Here, anti-HCV T cell receptors (TCRs) recognizing the HCV nonstructural (NS) NS3 or NS5 viral peptide target were examined by mRNA transfection of human peripheral blood lymphocytes (PBLs) derived from healthy donors as well as chronically infected HCV patients. Immunological analysis shows that while the CTLs expressing the NS5-specific TCR reduced HCV RNA replication by a noncytotoxic mechanism, the NS3-specific TCR-redirected CTLs were polyfunctional and inhibited HCV RNA replication through antigen-specific cytotoxicity. Transcriptome signatures from these two types of CTL responses revealed uniquely expressed gene clusters upon encountering hepatoma target cells presenting endogenously expressed HCV proteins. The NS3 TCR induced a rapid expression of apoptotic signaling pathways and formation of embryonic gene clusters, whereas the NS5A TCR activation induced extended proliferative and metabolic pathways as the HCV target cells survived. Our results provide detailed insights into basic HCV T cell immunology and have clinical relevance for redirecting T cells to target virally infected hepatoma cells.IMPORTANCE Due to the protective ability of HCV-specific T cells and the hepatotoxic potential that they possess, there is a great need for the understanding of the functional aspects of HCV-specific T cells. To circumvent the low level of precursor frequency in patients, we engineered primary CD8+ T cells by mRNA TCR vectors to confer HCV specificity to new T cells. HCV TCRs that differ in antigen specificity and polyfunctionality were examined. mRNA TCR engineering of peripheral blood lymphocytes from healthy donors or chronically infected HCV patients resulted in strikingly high levels of HCV TCR expression and HCV-specific responses. While a cytotoxicity response from a polyfunctional T cell activation caused hepatotoxicity and the rapid induction of apoptotic signaling pathways, the noncytotoxic T cell activation showed extended proliferative, metabolic pathways and persistence of HCV target cells. Our results provide detailed insights into basic HCV T cell immunology and have clinical relevance for immune protection of HCV-associated diseases.
Asunto(s)
Hepacivirus/inmunología , Activación de Linfocitos/inmunología , Receptores de Antígenos de Linfocitos T/genética , Linfocitos T Citotóxicos/inmunología , Proteínas no Estructurales Virales/inmunología , Apoptosis/genética , Apoptosis/inmunología , Línea Celular , Proliferación Celular , Técnicas de Cocultivo , Citotoxicidad Inmunológica/inmunología , Técnicas de Transferencia de Gen , Hepacivirus/genética , Hepatitis C Crónica/virología , Humanos , ARN Mensajero/genética , ARN Viral/biosíntesis , ARN Viral/genética , Receptores de Antígenos de Linfocitos T/biosíntesis , Proteínas no Estructurales Virales/genéticaRESUMEN
Autologous T lymphocytes genetically modified to express T cell receptors or chimeric antigen receptors have shown great promise in the treatment of several cancers, including melanoma and leukemia. In addition to tumor-associated antigens and tumor-specific neoantigens, tumors expressing viral peptides can also be recognized by specific T cells and are attractive targets for cell therapy. Hepatocellular carcinoma cells often have hepatitis B virus DNA integration and can be targeted by hepatitis B virus-specific T cells. Here, we describe a method to engineer hepatitis B virus-specific T cell receptors in primary human T lymphocytes based on electroporation of hepatitis B virus T cell receptor messenger RNA. This method can be extended to a large scale therapeutic T cell production following current good manufacturing practice compliance and is applicable to the redirection of T lymphocytes with T cell receptors of other virus specificities such as Epstein-Barr virus, cytomegalovirus, and chimeric receptors specific for other antigens expressed on cancer cells.
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ARN Mensajero/inmunología , Receptores de Antígenos de Linfocitos T/genética , Linfocitos T/inmunología , Antígenos Virales/inmunología , Células Cultivadas , Electroporación , Virus de la Hepatitis B/inmunología , Humanos , ARN Mensajero/genética , Receptores de Antígenos de Linfocitos T/inmunología , Especificidad del Receptor de Antígeno de Linfocitos T , Linfocitos T/citologíaRESUMEN
Cancer immunotherapy using a patient's own T cells redirected to recognize and kill tumor cells has achieved promising results in metastatic melanoma and leukemia. This technique involves harnessing a patient's T cells and then delivering a gene that encodes a new T cell receptor (TCR) or a chimeric antigen receptor (CAR) that allow the cells to recognize specific cancer antigens. The prospect of using engineered T cell therapy for persistent viral infections like hepatitis B virus (HBV) and their associated malignancies is promising. We recently tested in a first-in-man clinical trial, the ability of HBV-specific TCR-redirected T cells to target HBsAg-productive hepatocellular carcinoma (HCC) and demonstrated that these redirected T cells recognized HCC cells with HBV-DNA integration [1] We discuss here the possibility to use HBV-specific TCR-redirected T cells targeting hepatitis B viral antigens as a tumor specific antigen in patients with HBV-related HCC, and the potential challenges facing the development of this new immunotherapeutic strategy.
RESUMEN
Hepatitis B virus (HBV) can replicate within hepatocytes without causing direct cell damage. The host immune response is, therefore, not only essential to control the spread of virus infection, but it is also responsible for the inflammatory events causing liver pathologies. In this review, we discuss how HBV deals with host immunity and how we can harness it to achieve virus control and suppress liver damage.
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Virus de la Hepatitis B/inmunología , Hepatitis B/inmunología , Inmunidad Celular/inmunología , Inmunidad Innata/inmunología , Inmunidad Adaptativa/fisiología , Femenino , Hepatitis B/virología , Hepatitis B Crónica/inmunología , Hepatitis B Crónica/virología , Humanos , Masculino , Sensibilidad y Especificidad , Índice de Severidad de la EnfermedadRESUMEN
Therapeutic vaccination for the treatment of chronic hepatitis B has thus far been unsatisfactory. In this review, we discuss potential new therapeutic vaccination strategies and other immunotherapeutic approaches that aim to achieve efficient restoration of HBV immunity in chronically infected patients.
Asunto(s)
Inmunidad Adaptativa , Virus de la Hepatitis B/inmunología , Hepatitis B Crónica/inmunología , Hepatitis B Crónica/terapia , Inmunidad Innata , Inmunoterapia/métodos , Linfocitos T/inmunología , HumanosAsunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Traslado Adoptivo/métodos , Neoplasias de los Conductos Biliares/terapia , Conductos Biliares Intrahepáticos , Linfocitos T CD4-Positivos/inmunología , Colangiocarcinoma/terapia , Linfocitos Infiltrantes de Tumor/trasplante , Células TH1/trasplante , Femenino , HumanosRESUMEN
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.Molecular Therapy-Nucleic Acids (2013) 2, e114; doi:10.1038/mtna.2013.43; published online 13 August 2013.
RESUMEN
Hepatitis C virus (HCV) is a major cause of severe liver disease, and one major contributing factor is thought to involve a dysfunction of virus-specific T-cells. T-cell receptor (TCR) gene therapy with HCV-specific TCRs would increase the number of effector T-cells to promote virus clearance. We therefore took advantage of HLA-A2 transgenic mice to generate multiple TCR candidates against HCV using DNA vaccination followed by generation of stable T-cell-BW (T-BW) tumour hybrid cells. Using this approach, large numbers of non-structural protein 3 (NS3)-specific functional T-BW hybrids can be generated efficiently. These predominantly target the genetically stable HCV genotype 1 NS3(1073-1081) CTL epitope, frequently associated with clearance of HCV in humans. These T-BW hybrid clones recognized the NS3(1073) peptide with a high avidity. The hybridoma effectively recognized virus variants and targeted cells with low HLA-A2 expression, which has not been reported previously. Importantly, high-avidity murine TCRs effectively redirected human non-HCV-specific T-lymphocytes to recognize human hepatoma cells with HCV RNA replication driven by a subgenomic HCV replicon. Taken together, TCR candidates with a range of functional avidities, which can be used to study immune recognition of HCV-positive targets, have been generated. This has implications for TCR-related immunotherapy against HCV.
Asunto(s)
Hepacivirus/inmunología , Epítopos Inmunodominantes/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T Citotóxicos/inmunología , Proteínas no Estructurales Virales/inmunología , Animales , Donantes de Sangre , Células Cultivadas , Antígeno HLA-A2/genética , Antígeno HLA-A2/inmunología , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Receptores de Antígenos de Linfocitos T/genética , Proteínas no Estructurales Virales/genéticaRESUMEN
T cell functional plasticity helps tailor antiviral immunity during different phases of infections. We tested whether, during different phases of HBV infection, virus-specific T cells can acquire specific proinflammatory functions that could drive granulocyte/mononuclear cell liver infiltration. Multifunctional analysis of HBV-specific T cells during acute and chronic HBV infection revealed that HBV-specific T cells had the capacity to produce the neutrophil chemokine CXCL-8 but not IL-17. CXCL-8 producing T cells were detectable in the liver of chronic HBV patients with active hepatitis; while in acute HBV patients CXCL-8 production by T cells was temporally limited to the acute phase of disease, concomitant with the peak of liver inflammation. Characterization of the conditions necessary for the development of CXCL-8 producing T cells showed a requirement for IL-7 and IL-15 during T cell expansion. These data show that functional plasticity of virus-specific T cells spontaneously occurs during HBV infection and that an environment rich IL-7 and IL-15 can license T cells with the ability to produce CXCL-8 and potentially influence liver pathology.
Asunto(s)
Quimiocinas/metabolismo , Virus de la Hepatitis B/inmunología , Hepatitis B/inmunología , Interleucina-8/metabolismo , Neutrófilos/inmunología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Enfermedad Aguda , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Células Cultivadas , Hepatitis B/virología , Virus de la Hepatitis B/efectos de los fármacos , Humanos , Interleucina-15/farmacología , Interleucina-17/farmacología , Hígado/efectos de los fármacos , Hígado/inmunología , Hígado/patología , Hígado/virología , Neutrófilos/efectos de los fármacos , Péptidos/inmunología , Especificidad de la Especie , Linfocitos T/efectos de los fármacos , Proteínas Virales/inmunologíaRESUMEN
BACKGROUND & AIMS: Virus-specific T cells capable of controlling HBV and eliminating hepatocellular carcinoma (HCC) expressing HBV antigens are deleted or dysfunctional in patients with chronic HBV or HBV-related HCC. The goal of this study was to determine if T cell receptor (TCR) gene transfer can reconstitute HBV-specific T cell immunity in lymphocytes of chronic HBV patients and investigate whether HCC cells with natural HBV-DNA integration can be recognized by genetically modified T cells. METHODS: We used vector-mediated gene transfer to introduce HLA-A2-restricted, HBV-specific TCRs into T cells of chronic HBV as well as HBV-related HCC patients. RESULTS: The introduced TCRs were expressed on the cell surface, evidenced by Vß and pentamer staining. TCR transduced T cells produced IFN-γ, TNF-α, IL-2, and lysed HBV infected hepatocyte-like cell lines. Furthermore, HCC cell lines with natural HBV-DNA integration could be recognized by HBV-specific TCR-re-directed T cells. CONCLUSIONS: TCR re-directed HBV-specific T cells generated from PBMC of chronic HBV and HBV-related HCC patients were multifunctional and capable of recognizing HBV-infected cells and HCC tumor cells expressing viral antigens from naturally integrated HBV DNA. These genetically modified T cells could be used to reconstitute virus-specific T cell immunity in chronic HBV patients and target tumors in HBV-related HCC.