Immunosuppressive Drug-Resistant Armored T-Cell Receptor T Cells for Immune Therapy of HCC in Liver Transplant Patients.

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.

Use of Expression Profiles of HBV-DNA Integrated Into Genomes of Hepatocellular Carcinoma Cells to Select T Cells for Immunotherapy.

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.

Spatiotemporal Differences in Presentation of CD8 T Cell Epitopes during Hepatitis B Virus Infection.

Distinct populations of hepatocytes infected with hepatitis B virus (HBV) or only harboring HBV DNA integrations coexist within an HBV chronically infected liver. These hepatocytes express HBV antigens at different levels and with different intracellular localizations, but it is not known whether this heterogeneity of viral antigen expression could result in an uneven hepatic presentation of distinct HBV epitopes/HLA class I complexes triggering different levels of activation of HBV-specific CD8+ T cells. Using antibodies specific to two distinct HLA-A*02:01/HBV epitope complexes of HBV nucleocapsid and envelope proteins, we mapped their topological distributions in liver biopsy specimens of two anti-hepatitis B e antigen-positive (HBe+) chronic HBV (CHB) patients. We demonstrated that the core and envelope CD8+ T cell epitopes were not uniformly distributed in the liver parenchyma but preferentially located in distinct and sometimes mutually exclusive hepatic zones. The efficiency of HBV epitope presentation was then tested in vitro utilizing HLA-A*02:01/HBV epitope-specific antibodies and the corresponding CD8+ T cells in primary human hepatocyte and hepatoma cell lines either infected with HBV or harboring HBV DNA integration. We confirmed the existence of a marked variability in the efficiency of HLA class I/HBV epitope presentation among the different targets that was influenced by the presence of gamma interferon (IFN-γ) and availability of newly translated viral antigens. In conclusion, HBV antigen presentation can be heterogeneous within an HBV-infected liver. As a consequence, CD8+ T cells of different HBV specificities might have different antiviral efficacies.IMPORTANCE The inability of patients with chronic HBV infection to clear HBV is associated with defective HBV-specific CD8+ T cells. Hence, the majority of immunotherapy developments focus on HBV-specific T cell function restoration. However, knowledge of whether distinct HBV-specific T cells can equally target all the HBV-infected hepatocytes of a chronically infected liver is lacking. In this work, analysis of CHB patient liver parenchyma and in vitro HBV infection models shows a nonuniform distribution of HBV CD8+ T cell epitopes that is influenced by the presence of IFN-γ and availability of newly translated viral antigens. These results suggest that CD8+ T cells recognizing different HBV epitopes can be necessary for efficient immune therapeutic control of chronic HBV infection.

Immunoprevalence and immunodominance of HLA-Cw*0801-restricted T cell response targeting the hepatitis B virus envelope transmembrane region.

HLA-C-restricted T cells have been shown to play an important role in HIV control, but their impact on protection or pathogenesis in other viral infections remains elusive. Here, we characterized the hierarchy of HLA class I-restricted hepatitis B virus (HBV) epitopes targeted by CD8 T cells in HBV-infected subjects. The frequency of CD8 T cells specific for a panel of 18 HBV epitopes (restricted by HLA-A∗0201/03/07 [hereinafter HLA-A0201/03/07], -A1101, -A2402/07, -B5801, -B4001, -B1301, and -Cw0801) was quantified in a total of 59 subjects who resolved HBV infection. We found that the HLA-Cw0801-restricted epitope comprised of Env residues 171 to 180 (Env171-180) is immunoprevalent in the Southeast Asian subjects (10/17 HLA-Cw0801-positive subjects) and immunodominant in the majority of HLA-Cw0801-positive subjects able to control HBV infection. HLA-Cw0801-restricted Env171-180-specific CD8 T cells recognized endogenously produced HBV surface antigen (HBsAg) and tolerated amino acid variations within the epitope detected in HBV genotypes B and C. In conclusion, we demonstrate that the HLA-Cw0801-restricted Env171-180 T cell response is an important component of the HBV-specific adaptive T cell immunity in Asians infected with HBV. Thus, HLA-C restricted T cells might play an important role in various viral infections.

Targeted delivery of interferon-α to hepatitis B virus-infected cells using T-cell receptor-like antibodies.

UNLABELLED: During antiviral therapy, specific delivery of interferon-α (IFNα) to infected cells may increase its antiviral efficacy, trigger a localized immune reaction, and reduce the side effects caused by systemic administration. Two T-cell receptor-like antibodies (TCR-L) able to selectively bind hepatitis B virus (HBV)-infected hepatocytes of chronic hepatitis B patients and recognize core (HBc18-27) and surface (HBs183-91) HBV epitopes associated with different human leukocyte antigen (HLA)-A*02 alleles (A*02:01, A*02:02, A*02:07, A*02:11) were generated. Each antibody was genetically linked to two IFNα molecules to produce TCR-L/IFNα fusion proteins. We demonstrate that the fusion proteins triggered an IFNα response preferentially on the hepatocytes presenting the correct HBV-peptide HLA-complex and that the mechanism of the targeted IFNα response was dependent on the specific binding of the fusion proteins to the HLA/HBV peptide complexes through the TCR-like variable regions of the antibodies. CONCLUSION: TCR-L antibodies can be used to target cytokines to HBV-infected hepatocytes in vitro. Fusion of IFNα to TCR-L decreased the intrinsic biological activity of IFNα but preserved the overall specificity of the protein for the cognate HBV peptide/HLA complexes. This induction of an effective IFNα response selectively in HBV-infected cells might have a therapeutic advantage in comparison to the currently used native or pegylated IFNα.

Immunological alterations after immunotherapy with short lived HBV-TCR T cells associates with long-term treatment response in HBV-HCC.

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.

Engineering virus-specific T cells that target HBV infected hepatocytes and hepatocellular carcinoma cell lines.

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.

Profile of tumor antigen-specific CD8 T cells in patients with hepatitis B virus-related hepatocellular carcinoma.

BACKGROUND & AIMS: Tumor and viral antigens are expressed by hepatocellular carcinoma (HCC) in patients with chronic hepatitis B, but little is known about the immunodominance and function of tumor- and virus-specific CD8+ T cells in these patients. METHODS: HLA-A2-restricted T-cell responses to 16 tumor antigens and hepatitis B virus (HBV) proteins were tested using 49 previously described epitopes. Cells from 30 HLA-A2+, HBV-infected patients (10 with HCC, 10 with HBV cirrhosis, and 10 HBV but no cirrhosis) were analyzed, after expansion, by enzyme-linked immunosorbent spot (ELISPOT). Interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, and interleukin (IL)-2 production, as well as expression of the degranulation marker CD107a on tumor-specific CD8+ T cells, were evaluated. RESULTS: Cells from all groups had tumor-specific responses. The tumor antigens NY-ESO-1 and SSX-2 were most frequently targeted and were immunogenic in the HLA-A2 subtypes that are characteristic of Asian ethnicity. Tumor-specific T cells had low affinities; T cells from non-HCC patients were polyfunctional (IFN-gamma+, TNF-alpha+, CD107a+) and those from HCC patients displayed an exhausted phenotype (IFN-gamma+, CD107a+). Programmed Death 1 (PD-1) was expressed at higher levels on T cells from tumor and liver than peripheral blood from HCC patients and might contribute to T-cell exhaustion. Blocking PD-1/PD-L1 increased the frequency of tumor-specific T cells in HCC patients but did not restore T cell function. CONCLUSIONS: Patients with or without HCC have a quantitative and functional hierarchy of tumor-specific T cells. HLA-A2-restricted T cells from HCC patients target NY-ESO-1, but exist in an exhausted state that might require additional activation to restore function.

Use of Single Chain MHC Technology to Investigate Co-agonism in Human CD8+ T Cell Activation.

Non-stimulatory self peptide MHC (pMHC) complexes do not induce T cell activation and effector functions, but can enhance T cell responses to agonist pMHC, through a process termed co-agonism. This protocol describes an experimental system to investigate co-agonism during human CD8+ T cell activation by expressing human MHC class I molecules presenting pre-determined peptides as single polypeptides (single chain MHC) in a xenogeneic cell line. We expressed single chain MHCs under conditions where low levels of agonist single chain p-MHC complexes and high levels of non-stimulatory single chain p-MHC complexes were expressed. Use of this experimental system allowed us to compare CD8+ T cell responses to agonist pMHC in the presence or absence of non-stimulatory pMHC. The protocol describes cell line transfection with single chain MHC constructs, generation of stable cell lines, culture of hepatitis B virus-specific human CD8+ T cells and T cell activation experiments simultaneously quantifying cytokine production and degranulation. The presented methods can be used for research on different aspects of CD8+ T cell activation in human T cell systems with known peptide MHC specificity.

Nonstimulatory peptide-MHC enhances human T-cell antigen-specific responses by amplifying proximal TCR signaling.

Foreign antigens are presented by antigen-presenting cells in the presence of abundant endogenous peptides that are nonstimulatory to the T cell. In mouse T cells, endogenous, nonstimulatory peptides have been shown to enhance responses to specific peptide antigens, a phenomenon termed coagonism. However, whether coagonism also occurs in human T cells is unclear, and the molecular mechanism of coagonism is still under debate since CD4 and CD8 coagonism requires different interactions. Here we show that the nonstimulatory, HIV-derived peptide GAG enhances a specific human cytotoxic T lymphocyte response to HBV-derived epitopes presented by HLA-A*02:01. Coagonism in human T cells requires the CD8 coreceptor, but not T-cell receptor (TCR) binding to the nonstimulatory peptide-MHC. Coagonists enhance the phosphorylation and recruitment of several molecules involved in the TCR-proximal signaling pathway, suggesting that coagonists promote T-cell responses to antigenic pMHC by amplifying TCR-proximal signaling.

Optimization of 3-D hepatocyte culture by controlling the physical and chemical properties of the extra-cellular matrices.

Hepatocytes are anchorage-dependent cells sensitive to microenvironment; the control of the physicochemical properties of the extra-cellular matrices may be useful to the maintenance of hepatocyte functions in vitro for various applications. In a microcapsule-based 3-D hepatocyte culture microenvironment, we could control the physical properties of the collagen nano-fibres by fine-tuning the complex-coacervation reaction between methylated collagen and terpolymer of hydroxylethyl methacrylate-methyl methacrylate-methylacrylic acid. The physical properties of the nano-fibres were quantitatively characterized using back-scattering confocal microscopy to help optimize the physical support for hepatocyte functions. We further enhanced the chemical properties of the collagen nano-fibres by incorporating galactose onto collagen, which can specifically interact with the asialoglycoprotein receptor on hepatocytes. By correlating a range of collagen nano-fibres of different physicochemical properties with hepatocyte functions, we have identified a specific combination of methylated and galactosylated collagen nano-fibres optimal for maintaining hepatocyte functions in vitro. A model of how the physical and chemical supports interplay to maintain hepatocyte functions is discussed.

Building and optimizing a virus-specific T cell receptor library for targeted immunotherapy in viral infections.

Restoration of antigen-specific T cell immunity has the potential to clear persistent viral infection. T cell receptor (TCR) gene therapy can reconstitute CD8 T cell immunity in chronic patients. We cloned 10 virus-specific TCRs targeting 5 different viruses, causing chronic and acute infection. All 10 TCR genetic constructs were optimized for expression using a P2A sequence, codon optimization and the addition of a non-native disulfide bond. However, maximum TCR expression was only achieved after establishing the optimal orientation of the alpha and beta chains in the expression cassette; 9/10 TCRs favored the beta-P2A-alpha orientation over alpha-P2A-beta. Optimal TCR expression was associated with a significant increase in the frequency of IFN-gamma+ T cells. In addition, activating cells for transduction in the presence of Toll-like receptor ligands further enhanced IFN-gamma production. Thus, we have built a virus-specific TCR library that has potential for therapeutic intervention in chronic viral infection or virus-related cancers.

Mobilizing monocytes to cross-present circulating viral antigen in chronic infection.

Selection of antigens for therapeutic vaccination against chronic viral infections is complicated by pathogen genetic variations. We tested whether antigens present during persistent viral infections could provide a personalized antigenic reservoir for therapeutic T cell expansion in humans. We focused our study on the HBV surface antigen (HBsAg), which is present in microgram quantities in the serum of chronic HBV patients. We demonstrated by quantitative fluorescent microscopy that, out of 6 professional APC populations in the circulation, only CD14 monocytes (MNs) retained an HBsAg depot. Using TCR-redirected CD8+ T cells specific for MHC-I-restricted HBV epitopes, we showed that, despite being constantly exposed to antigen, ex vivo-isolated APCs did not constitutively activate HBV-specific CD8+ T cells. However, differentiation of HBsAg+ CD14 MNs from chronic patients to MN-derived DCs (moDCs) induced cross-presentation of the intracellular reservoir of viral antigen. We exploited this mechanism to cross-present circulating viral antigen and showed that moDCs from chronically infected patients stimulated expansion of autologous HBV-specific T cells. Thus, these data demonstrate that circulating viral antigen produced during chronic infection can serve as a personalized antigenic reservoir to activate virus-specific T cells.

A practical approach to immunotherapy of hepatocellular carcinoma using T cells redirected against hepatitis B virus.

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.

Binding of TCR multimers and a TCR-like antibody with distinct fine-specificities is dependent on the surface density of HLA complexes.

Class I Major Histocompatibility Complex (MHC) molecules evolved to sample degraded protein fragments from the interior of the cell, and to display them at the surface for immune surveillance by CD8(+) T cells. The ability of these lymphocytes to identify immunogenic peptide-MHC (pMHC) products on, for example, infected hepatocytes, and to subsequently eliminate those cells, is crucial for the control of hepatitis B virus (HBV). Various protein scaffolds have been designed to recapitulate the specific recognition of presented antigens with the aim to be exploited both diagnostically (e.g. to visualize cells exposed to infectious agents or cellular transformation) and therapeutically (e.g. for the delivery of drugs to compromised cells). In line with this, we report the construction of a soluble tetrameric form of an αß T cell receptor (TCR) specific for the HBV epitope Env(183-191) restricted by HLA-A*02:01, and compare its avidity and fine-specificity with a TCR-like monoclonal antibody generated against the same HLA target. A flow cytometry-based assay with streptavidin-coated beads loaded with Env(183-191)/HLA-A*02:01 complexes at high surface density, enabled us to probe the specific interaction of these molecules with their cognate pMHC. We demonstrate that the TCR tetramer has similar avidity for the pMHC as the antibody, but they differ in their fine-specificity, with only the TCR tetramer being capable of binding both natural variants of the Env(183-191) epitope found in HBV genotypes A/C/D (187Arg) and genotype B (187Lys). Collectively, the results highlight the promiscuity of our soluble TCR, which could be an advantageous feature when targeting cells infected with a mutation-prone virus, but that binding of the soluble oligomeric TCR relies considerably on the surface density of the presented antigen.

Licensing virus-specific T cells to secrete the neutrophil attracting chemokine CXCL-8 during hepatitis B virus infection.

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.