RESUMO
Chimeric antigen receptor (CAR) T cell therapy is a promising novel therapeutic approach for cancer but also for chronic infection. We have developed a fully human, second-generation CAR directed against the envelope protein of hepatitis B virus on the surface of infected cells (S-CAR). The S-CAR contains a human B cell-derived single-chain antibody fragment and human immunoglobulin G (IgG) spacer, CD28- and CD3-signaling domains that may be immunogenic in mice. Because immunosuppression will worsen the clinical course of chronic hepatitis B, we aimed at developing a preclinical mouse model that is immunocompetent and mimics chronic hepatitis B but nevertheless allows evaluating efficacy and safety of a fully human CAR. The S-CAR grafted on T cells triggered antibody responses in immunocompetent animals, and a co-expressed human-derived safeguard, the truncated epidermal growth factor receptor (EGFRt), even induced B and T cell responses, both limiting the survival of S-CAR-grafted T cells. Total body irradiation and transfer of T cells expressing an analogous, signaling-deficient S-CAR decoy and the safeguard induced immune tolerance toward the human-derived structures. S-CAR T cells transferred after immune recovery persisted and showed long-lasting antiviral effector function. The approach we describe herein will enable preclinical studies of efficacy and safety of fully human CARs in the context of a functional immune system.
Assuntos
Hepatite B/terapia , Receptores de Antígenos Quiméricos/imunologia , Anticorpos de Cadeia Única/imunologia , Proteínas do Envelope Viral/imunologia , Animais , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Antígenos CD28/imunologia , Complexo CD3/imunologia , Modelos Animais de Doenças , Hepatite B/genética , Hepatite B/imunologia , Hepatite B/virologia , Vírus da Hepatite B/genética , Vírus da Hepatite B/patogenicidade , Humanos , Imunocompetência/efeitos dos fármacos , Imunoglobulina G/imunologia , Imunoglobulina G/farmacologia , Camundongos , Receptores de Antígenos Quiméricos/administração & dosagem , Receptores de Antígenos Quiméricos/genética , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/farmacologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Proteínas do Envelope Viral/antagonistas & inibidoresRESUMO
To design new CARs targeting hepatitis B virus (HBV), we isolated human monoclonal antibodies recognizing the HBV envelope proteins from single B cells of a patient with a resolved infection. HBV-specific memory B cells were isolated by incubating peripheral blood mononuclear cells with biotinylated hepatitis B surface antigen (HBsAg), followed by single-cell flow cytometry-based sorting of live, CD19+ IgG+ HBsAg+ cells. Amplification and sequencing of immunoglobulin genes from single memory B cells identified variable heavy and light chain sequences. Corresponding immunoglobulin chains were cloned into IgG1 expression vectors and expressed in mammalian cells. Two antibodies named 4D06 and 4D08 were found to be highly specific for HBsAg, recognized a conformational and a linear epitope, respectively, and showed broad reactivity and neutralization capacity against all major HBV genotypes. 4D06 and 4D08 variable chain fragments were cloned into a 2nd generation CAR format with CD28 and CD3zeta intracellular signaling domains. The new CAR constructs displayed a high functional avidity when expressed on primary human T cells. CAR-grafted T cells proved to be polyfunctional regarding cytokine secretion and killed HBV-positive target cells. Interestingly, background activation of the 4D08-CAR recognizing a linear instead of a conformational epitope was consistently low. In a preclinical model of chronic HBV infection, murine T cells grafted with the 4D06 and the 4D08 CAR showed on target activity indicated by a transient increase in serum transaminases, and a lower number of HBV-positive hepatocytes in the mice treated. This study demonstrates an efficient and fast approach to identifying pathogen-specific monoclonal human antibodies from small donor cell numbers for the subsequent generation of new CARs.
Assuntos
Antígenos de Superfície da Hepatite B , Vírus da Hepatite B , Humanos , Vírus da Hepatite B/imunologia , Vírus da Hepatite B/genética , Animais , Camundongos , Antígenos de Superfície da Hepatite B/imunologia , Receptores de Antígenos Quiméricos/imunologia , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/metabolismo , Anticorpos Monoclonais/imunologia , Imunoterapia Adotiva , Hepatite B/imunologia , Hepatite B/virologia , Anticorpos Amplamente Neutralizantes/imunologia , Linfócitos B/imunologia , Linfócitos T/imunologiaRESUMO
Despite the availability of an effective prophylactic vaccine, 820,000 people die annually of hepatitis B virus (HBV)-related liver disease according to WHO. Since current antiviral therapies do not provide a curative treatment for the 296 million HBV carriers around the globe, novel strategies to cure HBV are urgently needed. A promising approach is the redirection of T cells towards HBV-infected hepatocytes employing chimeric antigen receptors or T-cell engager antibodies. We recently described the effective redirection of T cells employing a second-generation chimeric antigen receptor directed against the envelope protein of hepatitis B virus on the surface of infected cells (S-CAR) as well as bispecific antibodies that engage CD3 or CD28 on T cells employing the identical HBV envelope protein (HBVenv) binder. In this study, we added a trispecific antibody comprising all three moieties to the tool-box. Cytotoxic and non-cytolytic antiviral activities of these bi- and trispecific T-cell engager antibodies were assessed in co-cultures of human PBMC with HBV-positive hepatoma cells, and compared to that of S-CAR-grafted T cells. Activation of T cells via the S-CAR or by either a combination of the CD3- and CD28-targeting bispecific antibodies or the trispecific antibody allowed for specific elimination of HBV-positive target cells. While S-CAR-grafted effector T cells displayed faster killing kinetics, combinatory treatment with the bispecific antibodies or single treatment with the trispecific antibody was associated with a more pronounced cytokine release. Clearance of viral antigens and elimination of the HBV persistence form, the covalently closed circular (ccc) DNA, through cytolytic as well as cytokine-mediated activity was observed in all three settings with the combination of bispecific antibodies showing the strongest non-cytolytic, cytokine-mediated antiviral effect. Taken together, we demonstrate that bi- and trispecific T-cell engager antibodies can serve as a potent, off-the-shelf alternative to S-CAR-grafted T cells to cure HBV.
Assuntos
Anticorpos Biespecíficos , Receptores de Antígenos Quiméricos , Humanos , Receptores de Antígenos Quiméricos/genética , Vírus da Hepatite B , Antivirais , Proteínas do Envelope Viral/genética , Linfócitos T , Antígenos CD28/genética , Leucócitos Mononucleares , DNA Circular , Citocinas/genéticaRESUMO
T-cell therapy with T cells that are re-directed to hepatitis B virus (HBV)-infected cells by virus-specific receptors is a promising therapeutic approach for treatment of chronic hepatitis B and HBV-associated cancer. Due to the high number of target cells, however, side effects such as cytokine release syndrome or hepatotoxicity may limit safety. A safeguard mechanism, which allows depletion of transferred T cells on demand, would thus be an interesting means to increase confidence in this approach. In this study, T cells were generated by retroviral transduction to express either an HBV-specific chimeric antigen receptor (S-CAR) or T-cell receptor (TCR), and in addition either inducible caspase 9 (iC9) or herpes simplex virus thymidine kinase (HSV-TK) as a safety switch. Real-time cytotoxicity assays using HBV-replicating hepatoma cells as targets revealed that activation of both safety switches stopped cytotoxicity of S-CAR- or TCR-transduced T cells within less than one hour. In vivo, induction of iC9 led to a strong and rapid reduction of transferred S-CAR T cells adoptively transferred into AAV-HBV-infected immune incompetent mice. One to six hours after injection of the iC9 dimerizer, over 90% reduction of S-CAR T cells in the blood and the spleen and of over 99% in the liver was observed, thereby limiting hepatotoxicity and stopping cytokine secretion. Simultaneously, however, the antiviral effect of S-CAR T cells was diminished because remaining S-CAR T cells were mostly non-functional and could not be restimulated with HBsAg. A second induction of iC9 was only able to deplete T cells in the liver. In conclusion, T cells co-expressing iC9 and HBV-specific receptors efficiently recognize and kill HBV-replicating cells. Induction of T-cell death via iC9 proved to be an efficient means to deplete transferred T cells in vitro and in vivo containing unwanted hepatotoxicity.
Assuntos
Transferência Adotiva , Caspase 9/biossíntese , Antígenos da Hepatite B/imunologia , Vírus da Hepatite B/imunologia , Hepatite B Crônica/terapia , Linfócitos T/transplante , Transferência Adotiva/efeitos adversos , Animais , Caspase 9/genética , Morte Celular , Linhagem Celular , Técnicas de Cocultura , Citocinas/metabolismo , Citotoxicidade Imunológica , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Indução Enzimática , Feminino , Vírus da Hepatite B/patogenicidade , Hepatite B Crônica/imunologia , Hepatite B Crônica/metabolismo , Hepatite B Crônica/virologia , Humanos , Subunidade gama Comum de Receptores de Interleucina/genética , Subunidade gama Comum de Receptores de Interleucina/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/metabolismo , Simplexvirus/enzimologia , Simplexvirus/genética , Linfócitos T/enzimologia , Linfócitos T/imunologia , Linfócitos T/patologia , Timidina Quinase/genética , Timidina Quinase/metabolismo , Transdução GenéticaRESUMO
CD4+ T cells play an important role in the immune response against cancer and infectious diseases. However, mechanistic details of their helper function in hepatitis B virus (HBV) infection in particular, or their advantage for adoptive T cell therapy remain poorly understood as experimental and therapeutic tools are missing. Therefore, we identified, cloned, and characterized a comprehensive library of 20 MHC class II-restricted HBV-specific T cell receptors (TCRs) from donors with acute or resolved HBV infection. The TCRs were restricted by nine different MHC II molecules and specific for eight different epitopes derived from intracellularly processed HBV envelope, core, and polymerase proteins. Retroviral transduction resulted in a robust expression of all TCRs on primary T cells. A high functional avidity was measured for all TCRs specific for epitopes S17, S21, S36, and P774 (half-maximal effective concentration [EC50] <10 nM), or C61 and preS9 (EC50 <100 nM). Eight TCRs recognized peptide variants of HBV genotypes A to D. Both CD4+ and CD8+ T cells transduced with the MHC II-restricted TCRs were polyfunctional, producing interferon (IFN)-γ, tumor necrosis factor (TNF)-α, interleukin (IL)-2, and granzyme B (GrzB), and killed peptide-loaded target cells. Our set of MHC class II-restricted TCRs represents an important tool for elucidating CD4+ T cell help in viral infection with potential benefit for T cell therapy.
RESUMO
Chronic hepatitis B virus (HBV) infection remains a major global concern due to its high prevalence and the increased probability of progressing toward cirrhosis and hepatocellular carcinoma (HCC). While currently available therapies are effective in controlling HBV replication, they rarely achieve functional cure. Similarly, effective treatment options for HBV-related HCC (HBV-HCC) are limited and primarily applicable only for early stages of the disease. With the general success of chimeric antigen receptor T-cell immunotherapy against B-cell leukemia, adoptively transferring engineered autologous T cells specific for HBV or HCC antigens might represent a promising therapeutic approach for both chronic HBV infection and HBV-HCC. This review will describe the novel T cell-related immunotherapies being developed for both indications and discuss the approach of each strategy, their considerations and limitations when applied for treatment of chronic HBV infection (CHB) and HBV-HCC.
Assuntos
Transferência Adotiva , Carcinoma Hepatocelular/terapia , Hepatite B Crônica/terapia , Neoplasias Hepáticas/terapia , Linfócitos T/imunologia , Animais , Carcinoma Hepatocelular/imunologia , Carcinoma Hepatocelular/virologia , Ensaios Clínicos como Assunto , Vírus da Hepatite B , Hepatite B Crônica/imunologia , Humanos , Cirrose Hepática/patologia , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/virologia , CamundongosRESUMO
T-cell therapy of chronic hepatitis B is a novel approach to restore antiviral T-cell immunity and cure the infection. We aimed at identifying T-cell receptors (TCR) with high functional avidity that have the potential to be used for adoptive T-cell therapy. To this end, we cloned HLA-A*02-restricted, hepatitis B virus (HBV)-specific T cells from patients with acute or resolved HBV infection. We isolated 11 envelope- or core-specific TCRs and evaluated them in comprehensive functional analyses. T cells were genetically modified by retroviral transduction to express HBV-specific TCRs. CD8+ as well as CD4+ T cells became effector T cells recognizing even picomolar concentrations of cognate peptide. TCR-transduced T cells were polyfunctional, secreting the cytokines interferon gamma, tumor necrosis factor alpha and interleukin-2, and effectively killed hepatoma cells replicating HBV. Notably, our collection of HBV-specific TCRs recognized peptides derived from HBV genotypes A, B, C and D presented on different HLA-A*02 subtypes common in areas with high HBV prevalence. When co-cultured with HBV-infected cells, TCR-transduced T cells rapidly reduced viral markers within two days. Our unique set of HBV-specific TCRs with different affinities represents an interesting tool for elucidating mechanisms of TCR-MHC interaction and dissecting specific anti-HBV mechanisms exerted by T cells. TCRs with high functional avidity might be suited to redirect T cells for adoptive T-cell therapy of chronic hepatitis B and HBV-induced hepatocellular carcinoma.