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BACKGROUNDRecessive dystrophic epidermolysis bullosa (RDEB) is a severe form of skin fragility disorder due to mutations in COL7A1 encoding basement membrane type VII collagen (C7), the main constituent of anchoring fibrils (AFs) in skin. We developed a self-inactivating lentiviral platform encoding a codon-optimized COL7A1 cDNA under the control of a human phosphoglycerate kinase promoter for phase I evaluation.METHODSIn this single-center, open-label phase I trial, 4 adults with RDEB each received 3 intradermal injections (~1 × 106 cells/cm2 of intact skin) of COL7A1-modified autologous fibroblasts and were followed up for 12 months. The primary outcome was safety, including autoimmune reactions against recombinant C7. Secondary outcomes included C7 expression, AF morphology, and presence of transgene in the injected skin.RESULTSGene-modified fibroblasts were well tolerated, without serious adverse reactions or autoimmune reactions against recombinant C7. Regarding efficacy, there was a significant (P < 0.05) 1.26-fold to 26.10-fold increase in C7 mean fluorescence intensity in the injected skin compared with noninjected skin in 3 of 4 subjects, with a sustained increase up to 12 months in 2 of 4 subjects. The presence of transgene (codon-optimized COL7A1 cDNA) was demonstrated in the injected skin at month 12 in 1 subject, but no new mature AFs were detected.CONCLUSIONTo our knowledge, this is the first human study demonstrating safety and potential efficacy of lentiviral fibroblast gene therapy with the presence of COL7A1 transgene and subsequent C7 restoration in vivo in treated skin at 1 year after gene therapy. These data provide a rationale for phase II studies for further clinical evaluation.TRIAL REGISTRATIONClincalTrials.gov NCT02493816.FUNDINGCure EB, Dystrophic Epidermolysis Bullosa Research Association (UK), UK NIHR Biomedical Research Centre at Guy's and St Thomas' NHS Foundation Trust and King's College London, and Fondation René Touraine Short-Exchange Award.
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Epidermólisis Ampollosa Distrófica/terapia , Fibroblastos , Terapia Genética , Lentivirus/genética , Adulto , Colágeno Tipo VII/genética , Femenino , Fibroblastos/metabolismo , Fibroblastos/trasplante , Terapia Genética/efectos adversos , Terapia Genética/métodos , Humanos , Masculino , Persona de Mediana Edad , Resultado del TratamientoRESUMEN
Hematopoietic stem cell gene therapy is a promising therapeutic strategy for the treatment of neurological disorders, since transplanted gene-corrected cells can traffic to the brain, bypassing the blood-brain barrier, to deliver therapeutic protein to the CNS. We have developed this approach for the treatment of Mucopolysaccharidosis type IIIA (MPSIIIA), a devastating lysosomal storage disease that causes progressive cognitive decline, leading to death in early adulthood. In a previous pre-clinical proof-of-concept study, we demonstrated neurological correction of MPSIIIA utilizing hematopoietic stem cell gene therapy via a lentiviral vector encoding the SGSH gene. Prior to moving to clinical trial, we have undertaken further studies to evaluate the efficiency of gene transfer into human cells and also safety studies of biodistribution and genotoxicity. Here, we have optimized hCD34+ cell transduction with clinical grade SGSH vector to provide improved pharmacodynamics and cell viability and validated effective scale-up and cryopreservation to generate an investigational medicinal product. Utilizing a humanized NSG mouse model, we demonstrate effective engraftment and biodistribution, with no vector shedding or transmission to germline cells. SGSH vector genotoxicity assessment demonstrated low transformation potential, comparable to other lentiviral vectors in the clinic. This data establishes pre-clinical safety and efficacy of HSCGT for MPSIIIA.
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Lentiviral vectors (LVs) have recently witnessed an increasing demand in research and clinical applications. Their current purification processes represent the main bottleneck in their widespread use, as the methods used are cumbersome and yield low recoveries. We aimed to develop a one-step method to specifically purify LVs, with high yields and reduced levels of impurities, using the biotin-streptavidin system. Herein, packaging HEK293T cells were genetically engineered with a cyclical biotin-mimicking peptide displayed on a CD8α stalk, termed cTag8. LVs were modified with cTag8 by its passive incorporation onto viral surfaces during budding, without viral protein engineering or hindrance on infectivity. Expression of cTag8 on LVs allowed complete capture of infectious particles by streptavidin magnetic beads. As cTag8 binds streptavidin in the nanomolar range, the addition of micromolar concentrations of biotin resulted in the release of captured LVs by competitive elution, with overall yields of ≥60%. Analysis of eluted LVs revealed high purity with a >3-log and 2-log reduction in DNA contamination and host cell proteins, respectively. This one-step purification was also tested for scalable vector processing using monolith affinity chromatography, with an encouraging preliminary overall yield of 20%. This method will be of valuable use for both research and clinical applications of LVs.
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Autologous T cells engineered to express chimeric antigen receptor against the B cell antigen CD19 (CAR19) are achieving marked leukemic remissions in early-phase trials but can be difficult to manufacture, especially in infants or heavily treated patients. We generated universal CAR19 (UCART19) T cells by lentiviral transduction of non-human leukocyte antigen-matched donor cells and simultaneous transcription activator-like effector nuclease (TALEN)-mediated gene editing of T cell receptor α chain and CD52 gene loci. Two infants with relapsed refractory CD19+ B cell acute lymphoblastic leukemia received lymphodepleting chemotherapy and anti-CD52 serotherapy, followed by a single-dose infusion of UCART19 cells. Molecular remissions were achieved within 28 days in both infants, and UCART19 cells persisted until conditioning ahead of successful allogeneic stem cell transplantation. This bridge-to-transplantation strategy demonstrates the therapeutic potential of gene-editing technology.
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Leucemia-Linfoma Linfoblástico de Células Precursoras B/terapia , Linfocitos T/citología , Nucleasas de los Efectores Tipo Activadores de la Transcripción/genética , Alemtuzumab/uso terapéutico , Antígenos CD19/metabolismo , Antígeno CD52/metabolismo , Ensayos de Uso Compasivo , Femenino , Edición Génica , Trasplante de Células Madre Hematopoyéticas , Humanos , Lactante , Lentivirus/genética , Recurrencia Local de Neoplasia , Receptores de Antígenos de Linfocitos T/genética , Inducción de Remisión , Trasplante de Células Madre , Efectores Tipo Activadores de la Transcripción , Trasplante HomólogoRESUMEN
In 1993, we described an English family with beta-thalassaemia that was not linked to the beta-globin locus. Whole genome sequence analyses revealed potential causative mutations in 15 different genes, of which 4 were consistently and uniquely associated with the phenotype in all 7 affected family members, also confirmed by genetic linkage analysis. Of the 4 genes, which are present in a centromeric region of chromosome 1, ASH1L was proposed as causative through functional mRNA knock-down and chromatin-immunoprecipitation studies in human erythroid progenitor cells. Our data suggest a putative role for ASH1L (Trithorax protein) in the regulation of globin genes.
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Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Factores de Transcripción/metabolismo , Globinas beta/genética , Talasemia beta/genética , Talasemia beta/metabolismo , Línea Celular , Mapeo Cromosómico , Células Precursoras Eritroides/citología , Células Precursoras Eritroides/metabolismo , Silenciador del Gen , Ligamiento Genético , Variación Genética , Genoma Humano , Secuenciación de Nucleótidos de Alto Rendimiento , N-Metiltransferasa de Histona-Lisina , Histonas/metabolismo , Humanos , Fenotipo , Interferencia de ARN , Talasemia beta/diagnósticoRESUMEN
Non-integrating lentiviral vectors or also known as integrase-defective lentiviral (IDLV) hold a great promise for gene therapy application. They retain high transduction efficiency for efficient gene transfer in various cell types both in vitro and in vivo. IDLV is produced via a combined mutations introduced on the HIV-based lentiviral to disable their integration potency. Therefore, IDLV is considered safer than the wild-type integrase-proficient lentiviral vector as they could avoid the potential insertional mutagenesis associated with the nonspecific integration of transgene into target cell genome afforded by the wild-type vectors.Here we describe the system of IDLV which is produced through mutation in the integrase enzymes at the position of D64 located within the catalytic core domain. The efficiency of the IDLV in expressing the enhanced green fluorescent protein (GFP) reporter gene in transduced human monocyte (U937) cell lines was investigated. Expression of the transgene was driven by the spleen focus-forming virus (SFFV) LTRs. Transduction efficiency was studied using both the IDLV (ID-SFFV-GFP) and their wild-type counterparts (integrase-proficient SFFV-GFP). GFP expression was analyzed by fluorescence microscope and FACS analysis.Based on the results, the number of the GFP-positive cells in ID-SFFV-GFP-transduced U937 cells decreased rapidly over time. The percentage of GFP-positive cells decreased from ~50 % to almost 0, up to 10 days post-transduction. In wild-type SFFV-GFP-transduced cells, GFP expression is remained consistently at about 100 %. These data confirmed that the transgene expression in the ID-SFFV-GFP-transduced cells is transient in dividing cells. The lack of an origin of replication due to mutation of integrase enzymes in the ID-SFFV-GFP virus vector has caused the progressive loss of the GFP expression in dividing cells.Integrase-defective lentivirus will be a suitable choice for safer clinical applications. It preserves the advantages of the wild-type lentiviral vectors but with the benefit of transgene expression without stable integration into host genome, therefore reducing the potential risk of insertional mutagenesis.
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Proteínas Fluorescentes Verdes/genética , Lentivirus/genética , Transducción Genética/métodos , Vectores Genéticos , Células HEK293 , Humanos , Integrasas/genética , Transgenes/genéticaRESUMEN
Cells therapies, engineered to secrete replacement proteins, are being developed to ameliorate otherwise debilitating diseases. Recessive dystrophic epidermolysis bullosa (RDEB) is caused by defects of type VII collagen, a protein essential for anchoring fibril formation at the dermal-epidermal junction. Whereas allogeneic fibroblasts injected directly into the dermis can mediate transient disease modulation, autologous gene-modified fibroblasts should evade immunological rejection and support sustained delivery of type VII collagen at the dermal-epidermal junction. We demonstrate the feasibility of such an approach using a therapeutic grade, self-inactivating-lentiviral vector, encoding codon-optimized COL7A1, to transduce RDEB fibroblasts under conditions suitable for clinical application. Expression and secretion of type VII collagen was confirmed with transduced cells exhibiting supranormal levels of protein expression, and ex vivo migration of fibroblasts was restored in functional assays. Gene-modified RDEB fibroblasts also deposited type VII collagen at the dermal-epidermal junction of human RDEB skin xenografts placed on NOD-scid IL2Rgamma(null) recipients, with reconstruction of human epidermal structure and regeneration of anchoring fibrils at the dermal-epidermal junction. Fibroblast-mediated restoration of protein and structural defects in this RDEB model strongly supports proposed therapeutic applications in man.
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Colágeno Tipo VII/genética , Epidermólisis Ampollosa Distrófica/genética , Epidermólisis Ampollosa Distrófica/terapia , Fibroblastos/trasplante , Animales , Codón , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Vectores Genéticos , Xenoinjertos , Humanos , Lentivirus/genética , Masculino , Ratones , Ratones SCID , Distribución Aleatoria , Trasplante de Piel/métodos , Ingeniería de Tejidos , Cicatrización de Heridas/fisiologíaRESUMEN
The herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) encodes several microRNAs. One of these, miR-H2, overlaps and is antisense to the ICP0 gene and appears to decrease expression of the ICP0 protein. To determine if miR-H2 plays a role in the HSV-1 latency-reactivation cycle, we constructed a mutant, McK-ΔH2, in which this microRNA has been disrupted without altering the predicted amino acid sequence of ICP0. McK-ΔH2 produced increased amounts of ICP0. Although replication of McK-ΔH2 was similar to that of its wild-type (wt) McKrae parental virus in RS cells and mouse eyes, McK-ΔH2 was more neurovirulent in Swiss-Webster mice than McKrae based on the percent of mice that died from herpes encephalitis following ocular infection. In addition, using a mouse trigeminal ganglia (TG) explant model of induced reactivation, we show here for the first time that miR-H2 appears to play a role in modulating HSV-1 reactivation. Although the percent of TG from which virus reactivated by day 10 after explant was similar for McK-ΔH2, wt McKrae, and the marker-rescued virus McK-ΔH2Res, at earlier times, significantly more reactivation was seen with McK-ΔH2. Our results suggest that in the context of the virus, miR-H2 downregulates ICP0 and this moderates both HSV-1 neurovirulence and reactivation.
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Regulación Viral de la Expresión Génica/genética , Herpes Simple/genética , MicroARNs/genética , ARN Viral/genética , Activación Viral/genética , Animales , Modelos Animales de Enfermedad , Femenino , Herpesvirus Humano 1/patogenicidad , Herpesvirus Humano 1/fisiología , Proteínas Inmediatas-Precoces/biosíntesis , Proteínas Inmediatas-Precoces/genética , Immunoblotting , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ubiquitina-Proteína Ligasas/biosíntesis , Ubiquitina-Proteína Ligasas/genética , Virulencia , Latencia del Virus/genéticaRESUMEN
HBV-DNA integration frequently occurs in HBV-related hepatocellular carcinoma (HCC), but whether HBV antigens are expressed in HCC cells and can be targeted by immune therapeutic strategies remains controversial. Here, we first characterized HBV antigen expression in HCC metastases, occurring in a patient who had undergone liver transplantation for HBV-related HCC. We then deployed for the first time in HCC autologous T cells, genetically modified to express an HBsAg specific T cell receptor, as therapy against chemoresistant extrahepatic metastases. We confirmed that HBV antigens were expressed in HCC metastases (but not in the donor liver) and demonstrated that tumour cells were recognized in vivo by lymphocytes, engineered to express an HBV-specific T cell receptor (TCR). Gene-modified T cells survived, expanded and mediated a reduction in HBsAg levels without exacerbation of liver inflammation or other toxicity. Whilst clinical efficacy was not established in this subject with end-stage metastatic disease, we confirm the feasibility of providing autologous TCR-redirected therapy against HCC and advocate this strategy as a novel therapeutic opportunity in hepatitis B-associated malignancies.
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Carcinoma Hepatocelular/secundario , Antígenos de Superficie de la Hepatitis B/metabolismo , Inmunoterapia/métodos , Neoplasias Hepáticas/patología , Receptores de Antígenos de Linfocitos T/uso terapéutico , Tacrolimus/uso terapéutico , Anciano , Carcinoma Hepatocelular/inmunología , Carcinoma Hepatocelular/terapia , Resultado Fatal , Estudios de Seguimiento , Humanos , Inmunosupresores/uso terapéutico , Neoplasias Hepáticas/inmunología , Neoplasias Hepáticas/terapia , Masculino , Metástasis de la NeoplasiaRESUMEN
Netherton syndrome (NS) is a serious inherited skin disorder caused by mutations in the serine protease inhibitor Kazal type 5 gene (SPINK5), which encodes for a serine protease inhibitor lymphoepithelial Kazal type-related inhibitor (LEKTI). Patients with NS have defective keratinization, hair shaft defects, recurrent infections, atopy, and a predisposition to skin malignancies. Historically, 1 in 10 infants has died before their first birthday. Currently, there are no proven treatments to cure this condition. A SIN-lentiviral vector encoding the codon-optimized SPINK5 gene under the control of a 572 bp element derived from the human involucrin promoter can confer compartment-specific LEKTI expression in NS keratinocytes with restoration of normal skin architecture. Here we detail a study protocol for a phase I trial for feasibility and safety evaluations of autologous epidermal sheets generated from ex vivo gene-corrected keratinocyte stem cells, which will be grafted onto patients with mutation-proven NS.
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Terapia Genética , Lentivirus/genética , Síndrome de Netherton/terapia , Ensayos Clínicos Fase I como Asunto/métodos , Femenino , Humanos , Queratinocitos/metabolismo , Queratinocitos/trasplante , Masculino , Proteínas Inhibidoras de Proteinasas Secretoras/genética , Proteínas Inhibidoras de Proteinasas Secretoras/metabolismo , Inhibidor de Serinpeptidasas Tipo Kazal-5RESUMEN
UNLABELLED: Suicide gene modified donor T cells can improve immune reconstitution after allogeneic haematopoietic stem cell transplantation (SCT), but can be eliminated in the event of graft versus host disease (GVHD) through the administration of prodrug. Here we report the production and first-in-man use of mismatched donor T cells modified with a gamma-retroviral vector expressing a herpes simplex thymidine kinase (HSVTK):truncated CD34 (tCD34) suicide gene/magnetic selection marker protein. A stable packaging cell line was established to produce clinical grade vector pseudotyped with the Gibbon Ape Leukaemia Virus (GALV). T cells were transduced in a closed bag system following activation with anti-CD3/CD28 beads, and enriched on the basis of CD34 expression. Engineered cells were administered in two escalating doses to three children receiving T-depleted, CD34 stem cell selected, mismatched allogeneic grafts. All patients had pre-existing viral infections and received chemotherapy conditioning without serotherapy. In all three subjects cell therapy was tolerated without acute toxicity or the development of acute GVHD. Circulating gene modified T cells were detectable by flow cytometry and by molecular tracking in all three subjects. There was resolution of virus infections, concordant with detectable antigen-specific T cell responses and gene modified cells persisted for over 12 months. These findings highlight the suitability of tCD34 as a GMP compliant selection marker and demonstrate the feasibility, safety and immunological potential of HSVTK-tCD34 suicide gene modified donor T cells. TRIAL REGISTRATION: ClinicalTrials.gov NCT01204502
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Genes Transgénicos Suicidas/genética , Terapia Genética/métodos , Linfocitos T/metabolismo , Timidina Quinasa/genética , Antígenos CD34/genética , Antígenos CD34/metabolismo , Niño , Preescolar , Femenino , Citometría de Flujo , Ganciclovir/administración & dosificación , Vectores Genéticos/genética , Enfermedad Injerto contra Huésped/genética , Enfermedad Injerto contra Huésped/metabolismo , Enfermedad Injerto contra Huésped/terapia , Trasplante de Células Madre Hematopoyéticas/métodos , Humanos , Lactante , Recién Nacido , Virus de la Leucemia del Gibón/genética , Activación de Linfocitos/inmunología , Masculino , Simplexvirus/enzimología , Linfocitos T/inmunología , Linfocitos T/trasplante , Timidina Quinasa/metabolismo , Trasplante Homólogo , Resultado del TratamientoRESUMEN
ABC294640, a selective inhibitor of sphingosine kinase-2, inhibits the formation of sphingosine 1-phosphate (S1P), a signaling lipid implicated in promoting tumor survival. We investigated the anticancer activity of ABC294640 in two ovarian cancer cell lines, BG-1 and Caov-3. ABC294640 dose-dependently inhibited clonogenic survival and cell viability of both ovarian cancer lines in vitro. Using enzyme-linked immunosorbant assays and western blot detection in chemoresistant Caov-3 cells, treatment with ABC294640 alone also potentiated bcl-2-associated X-protein and caspase-9 transcription levels, although it did not significantly increase apoptotic cell death. Interestingly, ABC294640 administered to Caov-3 ovarian cancer cells in conjunction with paclitaxel induced apoptotic cell death through activation of caspase-9. Induction of apoptosis may mediate the anticancer effect of ABC294640 in ovarian cancer, although its precise antitumor mechanism is unclear. Ultimately, through its inhibition of S1P formation and subsequent effects on critical survival signaling cascades, ABC294640 may prove to be a useful adjunct to help re-sensitize tumors to standard therapy.
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Adamantano/análogos & derivados , Antineoplásicos/uso terapéutico , Resistencia a Antineoplásicos , Neoplasias Ováricas/tratamiento farmacológico , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Piridinas/uso terapéutico , Adamantano/farmacología , Adamantano/uso terapéutico , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Femenino , Humanos , Neoplasias Ováricas/enzimología , Neoplasias Ováricas/patología , Piridinas/farmacología , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Despite being of the myeloid lineage, acute myeloid leukaemia (AML) blasts are of low immunogenicity, probably because they lack the costimulatory molecule CD80 and secrete immunosuppressive factors. We have previously shown that in vitro stimulation of autologous peripheral blood mononuclear cells (PBMCs) with primary AML cells modified to express CD80 and IL-2 promotes proliferation, secretion of Th1 cytokines and expansion of activated CD8(+) T cells. In this study, we show that allogeneic effector cells (from a healthy donor or AML patients) when stimulated with IL-2/CD80 modified AML blasts were able to induce the lysis of unmodified AML blasts. Effector cells stimulated with IL-2/CD80AML blasts had higher lytic activity than cells stimulated with AML cells expressing CD80 or IL-2 alone. Similarly, AML patient PBMCs primed with autologous IL-2/CD80 AML cells had a higher frequency of IFN-gamma secreting cells and show cytotoxicity against autologous, unmodified blasts. Crucially, the response appears to be leukaemia specific, since stimulated patient PBMCs show higher frequencies of IFN-gamma secreting effector cells in response to AML blasts than to remission bone marrow cells from the same patients. Although studied in a small number of heterogeneous patient samples, the data are encouraging and support the continuing development of vaccination for poor prognosis AML patients with autologous cells genetically modified to express IL-2/CD80.
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Antígeno B7-1/genética , Interleucina-2/genética , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/inmunología , Adulto , Anciano , Antígeno B7-1/inmunología , Células Cultivadas , Femenino , Terapia Genética/métodos , Humanos , Interleucina-2/inmunología , Masculino , Persona de Mediana Edad , Neutrófilos/inmunología , Transducción GenéticaRESUMEN
Immunotherapeutic strategies may promote T and/or natural killer (NK) cell cytotoxicity. NK cells have the potential to exert a powerful anti-leukaemia effect, as demonstrated by studies of allogeneic transplantation. We have previously shown that CD80/interleukin 2 (IL2) lentivirus (LV)-transduced AML cells stimulate in-vitro T cell activation. The present study demonstrated that allogeneic and autologous culture of peripheral blood mononuclear cells with CD80/IL2-expressing AML cells also promoted NK cell cytotoxicity. Expression of the activation receptors NKp30, NKp44, CD244, CD25, CD69 and HLA-DR significantly increased following allogeneic culture and a consistent increased expression of NKp30, NKp44, NKp46, NKG2D, NKG2C and CD69, and up-regulation of the cytolytic marker CD107a was detected following autologous culture with LV-CD80/IL2 AML cells. Furthermore, increased NK cell lysis of K562 and primary AML blasts was detected. The lytic activity increased by twofold against K562 (from 46.6% to 90.4%) and allogeneic AML cells (from 11.8% to 20.1%) following in-vitro stimulation by CD80/IL2-expressing AML cells. More importantly for potential therapeutic applications, lysis of primary AML cells by autologous NK cells increased by more than 40-fold (from 0.4% to 22.5%). These studies demonstrated that vaccination of patients with CD80/IL2-transduced AML cells could provide a powerful strategy for T/NK cell-mediated stimulation of anti-leukaemic immunological responses.
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Antígeno B7-1/genética , Inmunoterapia Adoptiva/métodos , Interleucina-2/genética , Leucemia Mieloide Aguda/terapia , Células T Asesinas Naturales/inmunología , Transducción Genética/métodos , Adulto , Anciano , Estudios de Casos y Controles , Citotoxicidad Inmunológica , Femenino , Citometría de Flujo , Vectores Genéticos/administración & dosificación , Vectores Genéticos/genética , Humanos , Inmunofenotipificación , Células K562 , Lentivirus/genética , Leucemia Mieloide Aguda/inmunología , Activación de Linfocitos , Masculino , Persona de Mediana Edad , Estadísticas no Paramétricas , Células Tumorales Cultivadas , Adulto JovenRESUMEN
Immunotherapeutic strategies are increasingly being explored as a method of enhancing anti-tumour immune responses in patients with acute myeloid leukaemia (AML). Regulatory CD4(+) T cells (Tregs) suppress effector T and natural killer (NK) cells and therefore pose a potential challenge to the efficacy of immunotherapy. AML cells transduced with a lentivirus expressing CD80 (B7.1) and IL2 (LV-CD80/IL2) are capable of stimulating T and NK cell cytotoxicity in vitro. This study examines the effect of CD80/IL2 modified AML cells on Treg number and function. We report a significant increase in the number of CD8(+) T cells (P = 0.046) CD3(-)CD56(+) NK cells (P = 0.028) and CD3(+)CD4(+)CD25(high)Foxp3(+) Tregs (P = 0.043) following stimulation for 7 days with allogeneic LV-CD80/IL2 AMLs. In contrast, autologous LV-CD80/IL2 AML cell cultures provide a weaker stimulation with a lower number of CD8(+) T cells (P = 0.011) and no change in NK cell or Treg numbers. However, an increase in cytotoxic CD8(+) T cells and NK cells are detected following both allogeneic and autologous LV-CD80/IL2 stimulation as demonstrated by an increase in IFN-gamma and CD107a expression. Despite the presence of increased numbers of Tregs with suppressive activity in a subset of cultures, increased lysis of unmodified AMLs was still achieved following allogeneic (day 0, 2.2%; day 7, 20.4%) and more importantly, autologous LV-CD80/IL2 culture in which AML patients had recently received intensive chemotherapy (day 0, 0%; day 7, 16%). Vaccination with LV-CD80/IL2 therefore provides a potential strategy to enhance anti-leukaemia immune responses without a concomitant stimulation of Treg-mediated inhibition of cytotoxic immunological responses.
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Antígeno B7-1/genética , Interleucina-2/genética , Lentivirus/genética , Leucemia Mieloide Aguda/inmunología , Linfocitos T Reguladores/inmunología , Transducción Genética , Adulto , Anciano , Anticuerpos Monoclonales/inmunología , Linfocitos T CD8-positivos/inmunología , Citometría de Flujo , Humanos , Interferón gamma/metabolismo , Células Asesinas Naturales/inmunología , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Persona de Mediana Edad , Transgenes/fisiología , Células Tumorales CultivadasRESUMEN
Whole-cell vaccines allow the induction of anti-tumor immune responses without the need to define tumor antigens. We wished to directly compare, for the first time, the capacity of B7-1, B7-2 and 4-1BB ligand (4-1BBL) costimulatory molecules to convert murine and human acute myeloid leukemia (AML) cells into whole vaccines. 32Dc-kit is a murine myeloid cell line, which develops an AML-like disease over a protracted period, emulating human AML disease development. 32Dc-kit cells were modified to express elevated levels of B7-1, B7-2 or 4-1BBL, and each led to tumor rejection, although only mice injected with 32Dc-kit/B7-2 cells were able to reject subsequent parental tumor cell challenge. T-cell deficient nude mice were able to reject the 32Dc-kit variants, but they could not reject parental cell challenge; however, we found no evidence of cytotoxic T lymphocyte or natural killer (NK) activity ex vivo suggesting that tumor cell killing was mediated by an immune response that could not be recapitulated using purified NK or T cells as lone effectors. In human allogeneic mixed lymphocyte reactions (MLRs), we found no single costimulatory molecule was more effective, suggesting that the induction of a universal anti-tumor response will require a combination of costimulatory molecules.
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Vacunas contra el Cáncer , Inmunidad , Inmunoterapia Adoptiva/métodos , Leucemia Mieloide Aguda/terapia , Ligando 4-1BB/inmunología , Ligando 4-1BB/uso terapéutico , Animales , Antígeno B7-1/inmunología , Antígeno B7-1/uso terapéutico , Antígeno B7-2/inmunología , Antígeno B7-2/uso terapéutico , Línea Celular Tumoral , Humanos , Activación de Linfocitos , Prueba de Cultivo Mixto de Linfocitos , RatonesRESUMEN
Cell based therapies for acute myeloid leukaemia (AML) have made significant progress in the last decade benefiting the prognosis and survival of patients with this aggressive form of leukaemia. Due to advances in haematopoietic stem cell transplantation (HSCT) and particularly the advent of reduced intensity conditioning (RIC), the scope of transplantation has now extended to those patients previously ineligible due to age and health restrictions and has been associated with a decrease in transplant related mortality. The apparent graft versus leukaemia (GvL) effect observed following HSCT demonstrates the potential of the immune system to target and eradicate AML cells. Building on previously published pre-clinical studies by ourselves and others, we are now initiating a Phase I clinical study in which lentiviral vectors are used to genetically modify AML cells to express B7.1 (CD80) and IL-2. By combining allogeneic HSCT with immunisation, using the autologous AML cells expressing B7.1 and IL-2, we hope to stimulate immune eradication of residual AML cells in poor prognosis patients that have achieved donor chimerism. In this report we describe the background to cell therapy based approaches for AML, and discuss difficulties associated with the deployment of a chronically stimulated, hence exhausted/depleted immune system to eradicate tumour cells that have already escaped immune surveillance.
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Inmunoterapia/métodos , Leucemia Mieloide/inmunología , Leucemia Mieloide/terapia , Enfermedad Aguda , Ensayos Clínicos como Asunto , Femenino , Vectores Genéticos , Humanos , Lentivirus/genética , Escape del Tumor/inmunologíaRESUMEN
We describe the modification of tumour cells to enhance their capacity to act as antigen presenting cells with particular focus on the use of costimulatory molecules to do so. We have been involved in the genetic modification of tumour cells to prepare a whole cell vaccine for nearly a decade and we have a particular interest in acute myeloid leukaemia (AML). AML is an aggressive and difficult to treat disease, especially, for patients for whom haematopoietic stem cell (HSC) transplant is not an option. AML patients who have a suitable donor and meet HSC transplant fitness requirements, have a 5-year survival of 50%; however, for patients with no suitable donor or for who age is a factor, the prognosis is much worse. It is particularly poor prognosis patients, who are not eligible for HSC transplant, who are likely to benefit most from immunotherapy. It would be hoped that immunotherapy would be used to clear residual tumour cells in these patients in the first remission following standard chemotherapy treatments and this will extend the remission and reduce the risk of a second relapse associated with disease progression and poor mortality rates. In this symposia report, we will focus on whole cell vaccines as an immunotherapeutic option with particular reference to their use in the treatment of AML. We will aim to provide a brief overview of the latest data from our group and considerations for the use of this treatment modality in clinical trials for AML.
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Vacunas contra el Cáncer/inmunología , Inmunoterapia/métodos , Leucemia Mieloide/inmunología , Leucemia Mieloide/terapia , Ligando 4-1BB , Enfermedad Aguda , Animales , Células Presentadoras de Antígenos , Autoinmunidad , Modelos Animales de Enfermedad , Humanos , Ratones , Pronóstico , Factores de Necrosis Tumoral/inmunología , Regulación hacia ArribaRESUMEN
Nonviral, host-derived proteins on lentiviral vector surfaces can have a profound effect on the vector's biology as they can both promote infection and provide resistance to complement inactivation. We have exploited this to engineer a specific posttranslational modification of a "nonenvelope," virally associated protein. The bacterial biotin ligase (BirA) and a modified human DeltaLNGFR have been introduced into HEK293T cells and their protein products directed to the lumen of the endoplasmic reticulum. The BirA then couples biotin to an acceptor peptide that has been fused to the DeltaLNGFR. This results in the covalent linkage of biotin to the extracellular domain of the DeltaLNGFR expressed on the cell surface. Lentiviral vectors from these cells are metabolically labeled with biotin in the presence of free biotin. These biotinylated lentiviral vectors have a high affinity for streptavidin paramagnetic particles and, once captured, are easily manipulated in vitro. This is illustrated by the concentration of lentiviral vectors pseudotyped with either the VSV-G or an amphotropic envelope in excess of 4500-fold. This new cell line has the potential for widespread application to envelope pseudotypes compatible with lentiviral vector production.