RESUMEN
Exposure to replication-competent lentivirus (RCL) is a theoretical safety concern for individuals treated with lentiviral gene therapy. For certain ex vivo gene therapy applications, including cancer immunotherapy trials, RCL detection assays are used to screen the vector product as well as the vector-transduced cells. In this study, we reviewed T cell products screened for RCL using methodology developed in the National Gene Vector Biorepository. All trials utilized third-generation lentiviral vectors produced by transient transfection. Samples from 26 clinical trials totaling 460 transduced cell products from 375 subjects were evaluated. All cell products were negative for RCL. A total of 296 of the clinical trial participants were screened for RCL at least 1 month after infusion of the cell product. No research subject has shown evidence of RCL infection. These findings provide further evidence attesting to the safety of third-generation lentiviral vectors and that testing T cell products for RCL does not provide added value to screening the lentiviral vector product.
Asunto(s)
Vectores Genéticos/genética , Lentivirus/genética , Linfocitos T/inmunología , Linfocitos T/metabolismo , Replicación Viral/genética , Traslado Adoptivo , Línea Celular , Estudios de Seguimiento , Terapia Genética , Humanos , Lentivirus/fisiología , Transducción GenéticaRESUMEN
The cancer-testis antigen NY-ESO-1 is expressed by many solid tumors and has limited expression by mature somatic tissues, making it a highly attractive target for tumor immunotherapy. Targeting NY-ESO-1 using engineered T cells has demonstrated clinical efficacy in the treatment of some adult tumors. Neuroblastoma is a significant cause of cancer mortality in children, and is a tumor type shown to be responsive to immunotherapies. We evaluated a large panel of primarily resected neuroblastoma samples and demonstrated that 23% express NY-ESO-1. After confirming antigen-specific activity of T cells genetically engineered to express an NY-ESO-1 directed high-affinity transgenic T cell receptor in vitro, we performed xenograft mouse studies assessing the efficacy of NY-ESO-1-targeted T cells in both localized and disseminated models of neuroblastoma. Disease responses were monitored by tumor volume measurement and in vivo bioluminescence. After delivery of NY-ESO-1 transgenic TCR T cells, we observed significant delay of tumor progression in mice bearing localized and disseminated neuroblastoma, as well as enhanced animal survival. These data demonstrate that NY-ESO-1 is an antigen target in neuroblastoma and that targeted T cells represent a potential therapeutic option for patients with neuroblastoma.
RESUMEN
Despite recent therapeutic advances, multiple myeloma (MM) remains largely incurable. Here we report results of a phase I/II trial to evaluate the safety and activity of autologous T cells engineered to express an affinity-enhanced T cell receptor (TCR) recognizing a naturally processed peptide shared by the cancer-testis antigens NY-ESO-1 and LAGE-1. Twenty patients with antigen-positive MM received an average 2.4 × 10(9) engineered T cells 2 d after autologous stem cell transplant. Infusions were well tolerated without clinically apparent cytokine-release syndrome, despite high IL-6 levels. Engineered T cells expanded, persisted, trafficked to marrow and exhibited a cytotoxic phenotype. Persistence of engineered T cells in blood was inversely associated with NY-ESO-1 levels in the marrow. Disease progression was associated with loss of T cell persistence or antigen escape, in accordance with the expected mechanism of action of the transferred T cells. Encouraging clinical responses were observed in 16 of 20 patients (80%) with advanced disease, with a median progression-free survival of 19.1 months. NY-ESO-1-LAGE-1 TCR-engineered T cells were safe, trafficked to marrow and showed extended persistence that correlated with clinical activity against antigen-positive myeloma.
Asunto(s)
Antígenos de Neoplasias/inmunología , Proteínas de la Membrana/inmunología , Mieloma Múltiple/terapia , Receptores de Antígenos de Linfocitos T/fisiología , Linfocitos T/inmunología , Anciano , Antígenos de Neoplasias/genética , Antígenos de Superficie/genética , Antígenos de Superficie/inmunología , Femenino , Ingeniería Genética , Humanos , Masculino , Proteínas de la Membrana/genética , Persona de Mediana Edad , Mieloma Múltiple/inmunología , Mieloma Múltiple/mortalidad , Sindecano-1/análisisRESUMEN
BACKGROUND: CCR5 is the major coreceptor for human immunodeficiency virus (HIV). We investigated whether site-specific modification of the gene ("gene editing")--in this case, the infusion of autologous CD4 T cells in which the CCR5 gene was rendered permanently dysfunctional by a zinc-finger nuclease (ZFN)--is safe. METHODS: We enrolled 12 patients in an open-label, nonrandomized, uncontrolled study of a single dose of ZFN-modified autologous CD4 T cells. The patients had chronic aviremic HIV infection while they were receiving highly active antiretroviral therapy. Six of them underwent an interruption in antiretroviral treatment 4 weeks after the infusion of 10 billion autologous CD4 T cells, 11 to 28% of which were genetically modified with the ZFN. The primary outcome was safety as assessed by treatment-related adverse events. Secondary outcomes included measures of immune reconstitution and HIV resistance. RESULTS: One serious adverse event was associated with infusion of the ZFN-modified autologous CD4 T cells and was attributed to a transfusion reaction. The median CD4 T-cell count was 1517 per cubic millimeter at week 1, a significant increase from the preinfusion count of 448 per cubic millimeter (P<0.001). The median concentration of CCR5-modified CD4 T cells at 1 week was 250 cells per cubic millimeter. This constituted 8.8% of circulating peripheral-blood mononuclear cells and 13.9% of circulating CD4 T cells. Modified cells had an estimated mean half-life of 48 weeks. During treatment interruption and the resultant viremia, the decline in circulating CCR5-modified cells (-1.81 cells per day) was significantly less than the decline in unmodified cells (-7.25 cells per day) (P=0.02). HIV RNA became undetectable in one of four patients who could be evaluated. The blood level of HIV DNA decreased in most patients. CONCLUSIONS: CCR5-modified autologous CD4 T-cell infusions are safe within the limits of this study. (Funded by the National Institute of Allergy and Infectious Diseases and others; ClinicalTrials.gov number, NCT00842634.).
Asunto(s)
Linfocitos T CD4-Positivos/trasplante , Terapia Genética , Infecciones por VIH/terapia , Transfusión de Linfocitos , Receptores CCR5/genética , Adulto , Terapia Antirretroviral Altamente Activa , Transfusión de Sangre Autóloga , Linfocitos T CD4-Positivos/química , Terapia Combinada , ADN Viral/sangre , Femenino , Terapia Genética/efectos adversos , Terapia Genética/métodos , VIH/genética , VIH/aislamiento & purificación , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/inmunología , Humanos , Recuento de Linfocitos , Masculino , Persona de Mediana Edad , ARN Viral/sangre , Recto/inmunología , Carga ViralRESUMEN
MAGE A3, which belongs to the family of cancer-testis antigens, is an attractive target for adoptive therapy given its reactivation in various tumors and limited expression in normal tissues. We developed an affinity-enhanced T cell receptor (TCR) directed to a human leukocyte antigen (HLA)-A*01-restricted MAGE A3 antigen (EVDPIGHLY) for use in adoptive therapy. Extensive preclinical investigations revealed no off-target antigen recognition concerns; nonetheless, administration to patients of T cells expressing the affinity-enhanced MAGE A3 TCR resulted in a serious adverse event (SAE) and fatal toxicity against cardiac tissue. We present a description of the preclinical in vitro functional analysis of the MAGE A3 TCR, which failed to reveal any evidence of off-target activity, and a full analysis of the post-SAE in vitro investigations, which reveal cross-recognition of an off-target peptide. Using an amino acid scanning approach, a peptide from the muscle protein Titin (ESDPIVAQY) was identified as an alternative target for the MAGE A3 TCR and the most likely cause of in vivo toxicity. These results demonstrate that affinity-enhanced TCRs have considerable effector functions in vivo and highlight the potential safety concerns for TCR-engineered T cells. Strategies such as peptide scanning and the use of more complex cell cultures are recommended in preclinical studies to mitigate the risk of off-target toxicity in future clinical investigations.
Asunto(s)
Presentación de Antígeno/inmunología , Antígenos de Neoplasias/inmunología , Conectina/química , Reacciones Cruzadas/inmunología , Antígeno HLA-A1/inmunología , Proteínas de Neoplasias/inmunología , Péptidos/inmunología , Linfocitos T/metabolismo , Secuencia de Aminoácidos , Antígenos de Neoplasias/química , Antineoplásicos/farmacología , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Conectina/inmunología , Reacciones Cruzadas/efectos de los fármacos , Células HEK293 , Humanos , Activación de Linfocitos/efectos de los fármacos , Datos de Secuencia Molecular , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Proteínas de Neoplasias/química , Péptidos/química , Ingeniería de Proteínas , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/efectos de los fármacosRESUMEN
An obstacle to cancer immunotherapy has been that the affinity of T-cell receptors (TCRs) for antigens expressed in tumors is generally low. We initiated clinical testing of engineered T cells expressing an affinity-enhanced TCR against HLA-A*01-restricted MAGE-A3. Open-label protocols to test the TCRs for patients with myeloma and melanoma were initiated. The first two treated patients developed cardiogenic shock and died within a few days of T-cell infusion, events not predicted by preclinical studies of the high-affinity TCRs. Gross findings at autopsy revealed severe myocardial damage, and histopathological analysis revealed T-cell infiltration. No MAGE-A3 expression was detected in heart autopsy tissues. Robust proliferation of the engineered T cells in vivo was documented in both patients. A beating cardiomyocyte culture generated from induced pluripotent stem cells triggered T-cell killing, which was due to recognition of an unrelated peptide derived from the striated muscle-specific protein titin. These patients demonstrate that TCR-engineered T cells can have serious and not readily predictable off-target and organ-specific toxicities and highlight the need for improved methods to define the specificity of engineered TCRs.
Asunto(s)
Enfermedades Cardiovasculares/complicaciones , Melanoma/sangre , Mieloma Múltiple/sangre , Proteínas Musculares/metabolismo , Miocardio/patología , Proteínas Quinasas/metabolismo , Linfocitos T/citología , Alelos , Secuencias de Aminoácidos , Antígenos de Neoplasias/metabolismo , Técnicas de Cultivo de Célula , Conectina , Citocinas/metabolismo , Epítopos/metabolismo , Antígenos HLA-A/metabolismo , Humanos , Inmunoterapia Adoptiva , Células Madre Pluripotentes Inducidas/citología , Masculino , Melanoma/terapia , Persona de Mediana Edad , Mieloma Múltiple/terapia , Miocardio/inmunología , Proteínas de Neoplasias/metabolismo , Péptidos/metabolismo , Ingeniería de Proteínas , Receptores de Antígenos de Linfocitos T/inmunologíaRESUMEN
Since HIV requires CD4 and a co-receptor, most commonly C-C chemokine receptor 5 (CCR5), for cellular entry, targeting CCR5 expression is an attractive approach for therapy of HIV infection. Treatment of CD4(+) T cells with zinc-finger protein nucleases (ZFNs) specifically disrupting chemokine receptor CCR5 coding sequences induces resistance to HIV infection in vitro and in vivo. A chimeric Ad5/F35 adenoviral vector encoding CCR5-ZFNs permitted efficient delivery and transient expression following anti-CD3/anti-CD28 costimulation of T lymphocytes. We present data showing CD3/CD28 costimulation substantially improved transduction efficiency over reported methods for Ad5/F35 transduction of T lymphocytes. Modifications to the laboratory scale process, incorporating clinically compatible reagents and methods, resulted in a robust ex vivo manufacturing process capable of generating >10(10) CCR5 gene-edited CD4+ T cells from healthy and HIV+ donors. CD4+ T-cell phenotype, cytokine production, and repertoire were comparable between ZFN-modified and control cells. Following consultation with regulatory authorities, we conducted in vivo toxicity studies that showed no detectable ZFN-specific toxicity or T-cell transformation. Based on these findings, we initiated a clinical trial testing the safety and feasibility of CCR5 gene-edited CD4+ T-cell transfer in study subjects with HIV-1 infection.
Asunto(s)
Enzimas de Restricción del ADN/genética , Vectores Genéticos/normas , Infecciones por VIH/genética , Infecciones por VIH/inmunología , Receptores CCR5/genética , Dedos de Zinc/genética , Adenovirus Humanos/genética , Traslado Adoptivo , Animales , Antígenos CD28/inmunología , Complejo CD3/inmunología , Enzimas de Restricción del ADN/metabolismo , Femenino , Vectores Genéticos/administración & dosificación , Vectores Genéticos/efectos adversos , Vectores Genéticos/genética , Infecciones por VIH/terapia , Humanos , Activación de Linfocitos/inmunología , Masculino , Ratones , Fenotipo , Receptores CCR5/inmunología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Transducción Genética/métodos , Transducción Genética/normas , Trasplante HeterólogoRESUMEN
We report the safety and tolerability of 87 infusions of lentiviral vectormodified autologous CD4 T cells (VRX496-T; trade name, Lexgenleucel-T) in 17 HIV patients with well-controlled viremia. Antiviral effects were studied during analytic treatment interruption in a subset of 13 patients. VRX496-T was associated with a decrease in viral load set points in 6 of 8 subjects (P = .08). In addition, A â G transitions were enriched in HIV sequences after infusion, which is consistent with a model in which transduced CD4 T cells exert antisense-mediated genetic pressure on HIV during infection. Engraftment of vector-modified CD4 T cells was measured in gut-associated lymphoid tissue and was correlated with engraftment in blood. The engraftment half-life in the blood was approximately 5 weeks, with stable persistence in some patients for up to 5 years. Conditional replication of VRX496 was detected periodically through 1 year after infusion. No evidence of clonal selection of lentiviral vectortransduced T cells or integration enrichment near oncogenes was detected. This is the first demonstration that gene-modified cells can exert genetic pressure on HIV. We conclude that gene-modified T cells have the potential to decrease the fitness of HIV-1 and conditionally replicative lentiviral vectors have a promising safety profile in T cells.
Asunto(s)
Linfocitos T CD4-Positivos/trasplante , Terapia Genética/métodos , Infecciones por VIH/terapia , VIH-1/genética , Lentivirus/genética , Oligonucleótidos Antisentido/farmacología , Traslado Adoptivo/métodos , Adulto , Antivirales/efectos adversos , Antivirales/metabolismo , Antivirales/farmacología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/fisiología , Femenino , Terapia Genética/efectos adversos , Vectores Genéticos/efectos adversos , Vectores Genéticos/metabolismo , Vectores Genéticos/farmacología , Infecciones por VIH/genética , Infecciones por VIH/inmunología , VIH-1/inmunología , Humanos , Lentivirus/metabolismo , Lentivirus/fisiología , Masculino , Persona de Mediana Edad , Oligonucleótidos/administración & dosificación , Oligonucleótidos/genética , Oligonucleótidos Antisentido/administración & dosificación , Oligonucleótidos Antisentido/efectos adversos , Oligonucleótidos Antisentido/genética , Transducción Genética/métodos , Carga Viral/efectos de los fármacos , Replicación Viral/genéticaRESUMEN
The success of adoptive T cell gene transfer for treatment of cancer and HIV is predicated on generating a response that is both durable and safe. We report long-term results from three clinical trials to evaluate gammaretroviral vector-engineered T cells for HIV. The vector encoded a chimeric antigen receptor (CAR) composed of CD4 linked to the CD3ζ signaling chain (CD4ζ). CAR T cells were detected in 98% of samples tested for at least 11 years after infusion at frequencies that exceeded average T cell levels after most vaccine approaches. The CD4ζ transgene retained expression and function. There was no evidence of vector-induced immortalization of cells; integration site distributions showed no evidence of persistent clonal expansion or enrichment for integration sites near genes implicated in growth control or transformation. The CD4ζ T cells had stable levels of engraftment, with decay half-lives that exceeded 16 years, in marked contrast to previous trials testing engineered T cells. These findings indicate that host immunosuppression before T cell transfer is not required to achieve long-term persistence of gene-modified T cells. Further, our results emphasize the safety of T cells modified by retroviral gene transfer in clinical application, as measured in >500 patient-years of follow-up. Thus, previous safety issues with integrating viral vectors are hematopoietic stem cell or transgene intrinsic, and not a general feature of retroviral vectors. Engineered T cells are a promising form of synthetic biology for long-term delivery of protein-based therapeutics. These results provide a framework to guide the therapy of a wide spectrum of human diseases.