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.

Intrahepatic CD206+ macrophages contribute to inflammation in advanced viral-related liver disease.

BACKGROUND & AIMS: Liver inflammation is key in the progression of chronic viral hepatitis to cirrhosis and hepatocellular carcinoma. The magnitude of viral replication and the specific anti-viral immune responses should govern the degree of inflammation, but a direct correlation is not consistently found in chronic viral hepatitis patients. We aim to better define the mechanisms that contribute to chronic liver inflammation. METHODS: Intrahepatic CD14+ myeloid cells from healthy donors (n=19) and patients with viral-related liver cirrhosis (HBV, HBV/HDV or HCV; n=15) were subjected to detailed phenotypic, molecular and functional characterisation. RESULTS: Unsupervised analysis of multi-parametric data showed that liver disease was associated with the intrahepatic expansion of activated myeloid cells mainly composed of pro-inflammatory CD14+HLA-DRhiCD206+ cells, which spontaneously produced TNFα and GM-CSF. These cells only showed heightened pro-inflammatory responses to bacterial TLR agonists and were more refractory to endotoxin-induced tolerance. A liver-specific enrichment of CD14+HLA-DRhiCD206+ cells was also detected in a humanised mouse model of liver inflammation. This accumulation was abrogated following oral antibiotic treatment, suggesting a direct involvement of translocated gut-derived microbial products in liver injury. CONCLUSIONS: Viral-related chronic liver inflammation is driven by the interplay between non-endotoxin-tolerant pro-inflammatory CD14+HLA-DRhiCD206+ myeloid cells and translocated bacterial products. Deciphering this mechanism paves the way for the development of therapeutic strategies specifically targeting CD206+ myeloid cells in viral-related liver disease patients. Lay summary: Viral-related chronic liver disease is driven by intrahepatic pro-inflammatory myeloid cells accumulating in a gut-derived bacterial product-dependent manner. Our findings support the use of oral antibiotics to ameliorate liver inflammation in these patients.

A rapid method to assess the in vivo multi-functionality of adoptively transferred engineered TCR T cells.

Introduction: The clinical efficacy of chimeric antigen and T cell receptor (TCR) T cell immunotherapies is attributed to their ability to proliferate and persist in vivo. Since the interaction of the engineered T cells with the targeted tumour or its environment might suppress their function, their functionality should be characterized not only before but also after adoptive transfer. Materials and methods: We sought to achieve this by adapting a recently developed Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) rapid whole blood T cell assay to stimulate engineered TCR T cells in small volumes of whole blood (<1 ml) without in vitro cellular purification. As a proof-of-concept, we used this method to longitudinally study two patients with primary Hepatitis B Virus (HBV)-related hepatocellular carcinoma who received multiple dose-escalating infusions of transiently functional mRNA-engineered HBV-TCR T cells. Results: We demonstrated that a simple pulsing of whole blood with a peptide corresponding to the epitope recognized by the specific HBV-TCR elicited Th1 cytokine secretion in both patients only after HBV-TCR T cell treatment and not before. The amount of cytokines secreted also showed an infusion-dose-dependent association. Discussions: These findings support the utility of the whole blood cytokine release assay in monitoring the in vivo function and quantity of engineered T cell products following adoptive transfer.

Distinct roles for the NK cell-activating receptors in mediating interactions with dendritic cells and tumor cells.

NK cells are innate immune cells that are important in tumor immunity, but also have the ability to modulate the adaptive immune system through cytokine production or direct cell-cell interactions. This study investigates the interaction of NK cells with dendritic cells (DCs) and tumor cells, and the role of specific NK cell-activating receptors in this process. Primary rat NK cells and an NK cell line produced IFN-γ when cocultured with either DCs or the rat hepatoma cell line McA-RH7777 (McA). This NK cell activation by DCs and McA required cell-cell contact and was dependent on distinct NK-activating receptors. Silencing NK cell expression of NKp46 and NKp30 significantly diminished DC- and McA-mediated NK cell IFN-γ production, respectively. NK cells killed immature and mature DCs independently of NKp46, NKp30, and NKG2D; however, cytotoxicity against McA cells was dependent on NKp30 and NKG2D. Thus, we have shown in this study that NKp30 plays dual activating roles in NK-McA tumor interactions by mediating cytokine production and cytotoxicity. More importantly, NK cells are activated by both DCs and hepatoma cells to produce IFN-γ, but require distinct NK cell-activating receptors, NKp46 and NKp30, respectively. Our data suggest that therapeutics could be developed specifically to target NK-DC interactions without compromising NK tumor immunity.

Messenger RNA electroporated hepatitis B virus (HBV) antigen-specific T cell receptor (TCR) redirected T cell therapy is well-tolerated in patients with recurrent HBV-related hepatocellular carcinoma post-liver transplantation: results from a phase I trial.

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.

Lytic efficiency of immunosuppressive drug-resistant armoured T cells against circulating HBV-related HCC in whole blood.

Recurrence of hepatitis B virus-related hepatocellular carcinoma (HBV-HCC) after liver transplant (LT) is mediated by circulating tumour cells (CTCs) and exacerbated by the immunosuppressants required to prevent graft rejection. To circumvent the effects of immunosuppressants, we developed immunosuppressive drug-resistant armoured HBV-specific T-cell receptor-redirected T cells (IDRA HBV-TCR). However, their ability to eliminate HBV-HCC circulating in the whole blood has never been tested, and whether their lytic efficacy is compatible with the number of adoptively transferred T cells in vivo has never been measured. Hence, we developed a microscopy-based assay to quantify CTCs in whole blood. The assay was then used to quantify the efficacy of IDRA HBV-TCRs to lyse free-floating HBV-HCC cells in the presence of Tacrolimus and Mycophenolate Mofetil (MMF). We demonstrated that a panel of antibodies (AFP, GPC3, Vimentin, pan-Cytokeratin, and CD45) specific for HCC tumour antigens and immune cells can effectively differentiate HCC-CTCs in whole blood. Through dose-titration experiments, we observed that in the presence of immunosuppressive drugs, a minimum of 20 000 IDRA HBV-TCR T cells/ml of whole blood is necessary to lyse ~63.5% of free-floating HBV-HCC cells within 16 hours. In conclusion, IDRA HBV-TCR T cells can lyse free-floating HBV-HCC cells in whole blood in the presence of Tacrolimus and MMF. The quantity of IDRA-HBV TCR T cells required can be achieved by the adoptive transfer of 5 × 106 IDRA-HBV TCR-T cells/kg, supporting the utilisation of IDRA HBV-TCR T cells to eliminate CTCs as prophylaxis against recurrence after LT.

Engineering immunosuppressive drug-resistant armored (IDRA) SARS-CoV-2 T cells for cell therapy.

Solid organ transplant (SOT) recipients receive immunosuppressive drugs (ISDs) and are susceptible to developing severe COVID-19. Here, we analyze the Spike-specific T-cell response after 3 doses of mRNA vaccine in a group of SOT patients (n = 136) treated with different ISDs. We demonstrate that a combination of a calcineurin inhibitor (CNI), mycophenolate mofetil (MMF), and prednisone (Pred) treatment regimen strongly suppressed the mRNA vaccine-induced Spike-specific cellular response. Such defects have clinical consequences because the magnitude of vaccine-induced Spike-specific T cells was directly proportional to the ability of SOT patients to rapidly clear SARS-CoV-2 after breakthrough infection. To then compensate for the T-cell defects induced by immunosuppressive treatment and to develop an alternative therapeutic strategy for SOT patients, we describe production of 6 distinct SARS-CoV-2 epitope-specific ISD-resistant T-cell receptor (TCR)-T cells engineered using the mRNA electroporation method with reactivity minimally affected by mutations occurring in Beta, Delta, Gamma, and Omicron variants. This strategy with transient expression characteristics marks an improvement in the immunotherapeutic field and provides an attractive and novel therapeutic possibility for immunosuppressed COVID-19 patients.

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.

Identification of the rat NKG2D ligands, RAE1L and RRLT, and their role in allograft rejection.

NKG2D is a receptor expressed by NK cells and subsets of T lymphocytes. On NK cells, NKG2D functions as a stimulatory receptor that induces effector functions. We cloned and expressed two rat NKG2D ligands, both members of the RAE1 family, RAE1L and RRLT, and demonstrate that these ligands can induce IFN-gamma secretion and cytotoxicity by rat NK cells. To examine changes in expression of NKG2D and the NKG2D ligands RAE1L and RRLT after transplantation, we used a Dark Agouti (DA)-->Lewis rat model of liver transplantation. NKG2D expression was significantly increased in allogeneic liver grafts by day 7 post-transplant. Ligands of NKG2D, absent in normal liver, were readily detected in both syngeneic and allogeneic liver grafts by day 1 post-transplant. By day 7 post-transplant, hepatocyte RAE1L and RRLT expression was significantly and specifically increased in liver allografts. In contrast to acute rejection that develops in the DA-->Lewis model, transplantation of Lewis livers into DA recipients (Lewis-->DA) results in spontaneous tolerance. Interestingly, expression of RAE1L and RRLT is low in Lewis-->DA liver allografts, but significantly increased in DA-->Lewis liver allografts undergoing rejection. In conclusion, our results suggest that expression of NKG2D ligands may be important in allograft rejection.

Liver fibrosis and CD206+ macrophage accumulation are suppressed by anti-GM-CSF therapy.

BACKGROUND & AIMS: Chronic liver inflammation leads to fibrosis and cirrhosis and is associated with an accumulation of intrahepatic TNFα-secreting CD206+ macrophages, which may participate in maintaining chronic liver disease in a GM-CSF-dependent manner. We aimed to elucidate the exact role of GM-CSF in the development and progression of chronic liver disease. METHODS: Liver immunohistochemistry and serum quantification were performed in patients with viral and non-viral-related liver disease to compare CD206+ monocyte/macrophages, fibrosis and GM-CSF. This was followed by functional validations in vitro and in vivo in humanised mice. RESULTS: Using multiplex immunofluorescence and histo-cytometry, we show that highly fibrotic livers had a greater density of CD206+ macrophages that produced more TNFα and GM-CSF in the non-tumour liver regions of patients with hepatocellular carcinoma (n = 47), independent of aetiology. In addition, the absolute number of CD206+ macrophages strongly correlated with the absolute number of GM-CSF-producing macrophages. In non-HCC chronic HCV+ patients (n = 40), circulating GM-CSF levels were also increased in proportion to the degree of liver fibrosis and serum viral titres. We then demonstrated in vitro that monocytes converted to TNFα-producing CD206+ macrophage-like cells in response to bacterial products (lipopolysaccharide) in a GM-CSF-dependent manner, confirming the in vivo normalisation of serum GM-CSF concentration following oral antibiotic treatment observed in HBV-infected humanised mice. Finally, anti-GM-CSF neutralising antibody treatment reduced intrahepatic CD206+ macrophage accumulation and abolished liver fibrosis in HBV-infected humanised mice. CONCLUSIONS: While the direct involvement of CD206+ macrophages in liver fibrosis remains to be demonstrated, these findings show that GM-CSF may play a central role in liver fibrosis and could guide the development of anti-GM-CSF antibody-based therapy for the management of patients with chronic liver disease. LAY SUMMARY: Liver fibrosis is a major driver of liver disease progression. Herein, we have shown that granulocyte-macrophage colony-stimulating factor (GM-CSF) plays an important role in the development of liver fibrosis. Our findings support the use of anti-GM-CSF neutralising antibodies for the management of patients with chronic liver disease resulting from both viral and non-viral causes.

Rapamycin, but not cyclosporine or FK506, alters natural killer cell function.

Infiltration of natural killer (NK) cells into solid organ allografts is observed in clinical and experimental transplantation. Studies suggest a role for NK cells in acute and chronic rejection of solid organ allografts; however, the effects of immunosuppressive agents on NK cells are not clearly established. Rat NK cell lines were analyzed for proliferation and cytotoxicity in the presence of cyclosporine, FK506, or rapamycin. Lewis recipients of DA liver allografts received immunosuppressive agents after transplantation. NK cells demonstrated robust function both in the absence and presence of cyclosporine and FK506. In contrast, rapamycin significantly inhibited proliferation and cytotoxicity of NK cells. NK cell numbers remained stable in graft recipients treated with cyclosporine and FK506, whereas there was a significant decrease in NK cells in rapamycin-treated recipients. These data indicate that immunosuppressive drugs have differential effects on NK cell function that may impact the immune response of transplant recipients.

In silico modeling of cancer cell dissemination and metastasis.

Metastasis is the main cause of cancer-related death. It is surprising then that the exact nature of metastasis-the process by which cancer cells leave the primary tumor to reach distant organs, and resume proliferation-is not fully understood. Moreover, the different conditions under which the immune system can either promote or suppress metastasis are only now beginning to be uncovered. In recent years, our understanding of metastasis as a genocentric, cell-autonomous process has shifted toward a systemic model in which interactions between cancer cells and their surrounding microenvironments lead to dissemination and metastasis. In silico modeling of the various steps involved in metastasis can help provide an understanding of how tumor properties emerge from the complex interplays between tumor cells and their microenvironment. In silico models can also be useful in identifying the selective forces that favor the outcomes of cancer cells with metastatic potential.

Immune predictors of cancer progression.

The immune system has multiple, complex, and sometimes opposing roles during cancer progression. While immune-compromised individuals have a higher incidence of cancers, inflammation is also associated with increased risk of disease progression. It is becoming apparent that simple measures of immune responses in the blood are of limited use in cancer. Instead, the importance of the exact identity and functional characteristics of tumor-infiltrating immune cells is increasingly recognized. This realization has led to recent studies that have revealed a critical role for chemokine expression in the tumor microenvironment and suggested a therapeutic potential of manipulating intratumoral expression of chemokines to alter the local immune milieu.