RESUMEN
Patients with primary central nervous system lymphoma (PCNSL) are treated with high-dose methotrexate-based chemotherapy, which requires hospitalization and extensive expertise to manage related toxicity. The use of R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone) could overcome these difficulties, but blood-brain barrier (BBB) penetration of related drugs is poor. Tumor necrosis factor-α coupled with NGR (NGR-hTNF), a peptide targeting CD13+ vessels, induces endothelial permeabilization and improves tumor access of cytostatics. We tested the hypothesis that NGR-hTNF can break the BBB, thereby improving penetration and activity of R-CHOP in patients with relapsed/refractory PCNSL (NCT03536039). Patients received six R-CHOP21 courses, alone at the first course and preceded by NGR-hTNF (0.8 µg/m2) afterward. This trial included 2 phases: an "explorative phase" addressing the effect of NGR-hTNF on drug pharmacokinetic parameters and on vessel permeability, assessed by dynamic contrast-enhanced magnetic resonance imaging and 99mTc-diethylene-triamine-pentacetic acid-single-photon emission computed tomography, and the expression of CD13 on tumor tissue; and an "expansion phase" with overall response rate as the primary end point, in which the 2-stage Simon Minimax design was used. At the first stage, if ≥4 responses were observed among 12 patients, the study accrual would have continued (sample size, 28). Herein, we report results of the explorative phase and the first-stage analysis (n = 12). CD13 was expressed in tumor vessels of all cases. NGR-hTNF selectively increased vascular permeability in tumoral/peritumoral areas, without interfering with drug plasma/cerebrospinal fluid concentrations. The NGR-hTNF/R-CHOP combination was well tolerated: there were only 2 serious adverse events, and grade 4 toxicity was almost exclusively hematological, which were resolved without dose reductions or interruptions. NGR-hTNF/R-CHOP was active, with 9 confirmed responses (75%; 95% confidence interval, 51-99), 8 of which were complete. In conclusion, NGR-hTNF/R-CHOP was safe in these heavily pretreated patients. NGR-hTNF enhanced vascular permeability specifically in tumoral/peritumoral areas, which resulted in fast and sustained responses.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Barrera Hematoencefálica/efectos de los fármacos , Neoplasias del Sistema Nervioso Central/tratamiento farmacológico , Linfoma no Hodgkin/tratamiento farmacológico , Proteínas Recombinantes de Fusión/farmacocinética , Factor de Necrosis Tumoral alfa/farmacocinética , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Biomarcadores , Barrera Hematoencefálica/diagnóstico por imagen , Antígenos CD13/metabolismo , Permeabilidad de la Membrana Celular , Neoplasias del Sistema Nervioso Central/diagnóstico , Neoplasias del Sistema Nervioso Central/metabolismo , Neoplasias del Sistema Nervioso Central/mortalidad , Ciclofosfamida/efectos adversos , Ciclofosfamida/uso terapéutico , Doxorrubicina/efectos adversos , Doxorrubicina/uso terapéutico , Femenino , Humanos , Inmunohistoquímica , Linfoma no Hodgkin/diagnóstico , Linfoma no Hodgkin/metabolismo , Linfoma no Hodgkin/mortalidad , Masculino , Neuroimagen/métodos , Prednisona/efectos adversos , Prednisona/uso terapéutico , Proteínas Recombinantes de Fusión/administración & dosificación , Proyectos de Investigación , Rituximab/efectos adversos , Rituximab/uso terapéutico , Tomografía Computarizada de Emisión de Fotón Único , Resultado del Tratamiento , Factor de Necrosis Tumoral alfa/administración & dosificación , Vincristina/efectos adversos , Vincristina/uso terapéuticoRESUMEN
Cells in the tumor microenvironment may be reprogrammed by tumor-derived metabolites. Cholesterol-oxidized products, namely oxysterols, have been shown to favor tumor growth directly by promoting tumor cell growth and indirectly by dampening antitumor immune responses. However, the cellular and molecular mechanisms governing oxysterol generation within tumor microenvironments remain elusive. We recently showed that tumor-derived oxysterols recruit neutrophils endowed with protumoral activities, such as neoangiogenesis. Here, we show that hypoxia inducible factor-1a (HIF-1α) controls the overexpression of the enzyme Cyp46a1, which generates the oxysterol 24-hydroxycholesterol (24S-HC) in a pancreatic neuroendocrine tumor (pNET) model commonly used to study neoangiogenesis. The activation of the HIF-1α-24S-HC axis ultimately leads to the induction of the angiogenic switch through the positioning of proangiogenic neutrophils in proximity to Cyp46a1+ islets. Pharmacologic blockade or genetic inactivation of oxysterols controls pNET tumorigenesis by dampening the 24S-HC-neutrophil axis. Finally, we show that in some human pNET samples Cyp46a1 transcripts are overexpressed, which correlate with the HIF-1α target VEGF and with tumor diameter. This study reveals a layer in the angiogenic switch of pNETs and identifies a therapeutic target for pNET patients.
Asunto(s)
Transformación Celular Neoplásica/metabolismo , Hidroxicolesteroles/metabolismo , Tumores Neuroendocrinos/etiología , Tumores Neuroendocrinos/metabolismo , Neoplasias Pancreáticas/etiología , Neoplasias Pancreáticas/metabolismo , Animales , Transformación Celular Neoplásica/genética , Colestanotriol 26-Monooxigenasa/genética , Colestanotriol 26-Monooxigenasa/metabolismo , Colesterol 24-Hidroxilasa , Citocinas/metabolismo , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Activación Enzimática , Femenino , Técnica del Anticuerpo Fluorescente , Proteínas Activadoras de GTPasa/genética , Proteínas Activadoras de GTPasa/metabolismo , Expresión Génica , Regulación Neoplásica de la Expresión Génica , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Inmunohistoquímica , Masculino , Ratones , Ratones Transgénicos , Neovascularización Patológica/genética , Tumores Neuroendocrinos/patología , Neoplasias Pancreáticas/patología , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Memory stem T cells (TSCM) have been proposed as key determinants of immunologic memory. However, their exact contribution to a mounting immune response, as well as the mechanisms and timing of their in vivo generation, are poorly understood. We longitudinally tracked TSCM dynamics in patients undergoing haploidentical hematopoietic stem cell transplantation (HSCT), thereby providing novel hints on the contribution of this subset to posttransplant immune reconstitution in humans. We found that donor-derived TSCM are highly enriched early after HSCT. We showed at the antigen-specific and clonal level that TSCM lymphocytes can differentiate directly from naive precursors infused within the graft and that the extent of TSCM generation might correlate with interleukin 7 serum levels. In vivo fate mapping through T-cell receptor sequencing allowed defining the in vivo differentiation landscapes of human naive T cells, supporting the notion that progenies of single naive cells embrace disparate fates in vivo and highlighting TSCM as relevant novel players in the diversification of immunological memory after allogeneic HSCT.
Asunto(s)
Trasplante de Células Madre Hematopoyéticas , Memoria Inmunológica , Linfopoyesis , Linfocitos T/inmunología , Linfocitos T/fisiología , Adulto , Donantes de Sangre , Diferenciación Celular/inmunología , Proliferación Celular , Haplotipos , Humanos , Memoria Inmunológica/inmunología , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Especificidad del Receptor de Antígeno de Linfocitos T/inmunología , Trasplante HomólogoRESUMEN
Tumor-derived metabolites dampen tumor-infiltrating immune cells and antitumor immune responses. Among the various metabolites produced by tumors, we recently showed that cholesterol oxidized products, namely oxysterols, favor tumor growth through the inhibition of DC migration toward lymphoid organs and by promoting the recruitment of pro-tumor neutrophils within the tumor microenvironment. Here, we tested different drugs capable of blocking cholesterol/oxysterol formation. In particular, we tested efficacy and safety of different administration schedules, and of immunotherapy-based combination of a class of compounds, namely zaragozic acids, which inhibit cholesterol pathway downstream of mevalonate formation, thus leaving intact the formation of the isoprenoids, which are required for the maturation of proteins involved in the immune cell function. We show that zaragozic acids inhibit the in vivo growth of the RMA lymphoma and the Lewis lung carcinoma (LLC) without inducing side effects. Tumor growth inhibition requires an intact immune system, as immunodeficient tumor-bearing mice do not respond to zaragozic acid treatment. Of note, the effect of zaragozic acids is accompanied by a marked reduction in the LXR target genes Abcg1, Mertk, Scd1 and Srebp-1c in the tumor microenvironment. On the other hand, zoledronate, which blocks also isoprenoid formation, did not control the LLC tumor growth. Finally, we show that zaragozic acids potentiate the antitumor effects of active and adoptive immunotherapy, significantly prolonging the overall survival of tumor-bearing mice treated with the combo zaragozic acids and TAA-loaded DCs. This study identifies zaragozic acids as new antitumor compounds exploitable for the treatment of cancer patients.
Asunto(s)
Antineoplásicos/uso terapéutico , Compuestos Bicíclicos Heterocíclicos con Puentes/uso terapéutico , Carcinoma Pulmonar de Lewis/terapia , Células Dendríticas/inmunología , Inmunoterapia Adoptiva/métodos , Linfoma de Células T/terapia , Ácidos Tricarboxílicos/uso terapéutico , Animales , Carcinoma Pulmonar de Lewis/inmunología , Colesterol/metabolismo , Terapia Combinada , Células Dendríticas/trasplante , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Linfoma de Células T/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones SCID , Oxiesteroles/metabolismo , Escape del Tumor , Microambiente TumoralRESUMEN
Genetically targeted T cells promise to solve the feasibility and efficacy hurdles of adoptive T-cell therapy for cancer. Selecting a target expressed in multiple-tumor types and that is required for tumor growth would widen disease indications and prevent immune escape caused by the emergence of antigen-loss variants. The adhesive receptor CD44 is broadly expressed in hematologic and epithelial tumors, where it contributes to the cancer stem/initiating phenotype. In this study, silencing of its isoform variant 6 (CD44v6) prevented engraftment of human acute myeloid leukemia (AML) and multiple myeloma (MM) cells in immunocompromised mice. Accordingly, T cells targeted to CD44v6 by means of a chimeric antigen receptor containing a CD28 signaling domain mediated potent antitumor effects against primary AML and MM while sparing normal hematopoietic stem cells and CD44v6-expressing keratinocytes. Importantly, in vitro activation with CD3/CD28 beads and interleukin (IL)-7/IL-15 was required for antitumor efficacy in vivo. Finally, coexpressing a suicide gene enabled fast and efficient pharmacologic ablation of CD44v6-targeted T cells and complete rescue from hyperacute xenogeneic graft-versus-host disease modeling early and generalized toxicity. These results warrant the clinical investigation of suicidal CD44v6-targeted T cells in AML and MM.
Asunto(s)
Antígenos de Neoplasias/inmunología , Receptores de Hialuranos/inmunología , Inmunoterapia Adoptiva , Leucemia Mieloide Aguda/terapia , Terapia Molecular Dirigida , Mieloma Múltiple/terapia , Subgrupos de Linfocitos T/inmunología , Animales , Antígenos de Neoplasias/genética , Antígenos CD28/inmunología , Complejo CD3/inmunología , Línea Celular Tumoral/inmunología , Línea Celular Tumoral/trasplante , Citotoxicidad Inmunológica , Genes Transgénicos Suicidas , Enfermedad Injerto contra Huésped/terapia , Humanos , Receptores de Hialuranos/genética , Interleucina-15/inmunología , Interleucina-15/farmacología , Interleucina-7/inmunología , Interleucina-7/farmacología , Leucemia Mieloide Aguda/inmunología , Leucemia Mieloide Aguda/patología , Leucemia Mielomonocítica Aguda/inmunología , Leucemia Mielomonocítica Aguda/patología , Leucemia Mielomonocítica Aguda/terapia , Activación de Linfocitos , Ratones , Mieloma Múltiple/inmunología , Mieloma Múltiple/patología , Trasplante de Neoplasias , Estructura Terciaria de Proteína , ARN Interferente Pequeño/farmacología , Proteínas Recombinantes de Fusión/inmunología , Especificidad del Receptor de Antígeno de Linfocitos T , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Long-living memory stem T cells (T(SCM)) with the ability to self-renew and the plasticity to differentiate into potent effectors could be valuable weapons in adoptive T-cell therapy against cancer. Nonetheless, procedures to specifically target this T-cell population remain elusive. Here, we show that it is possible to differentiate in vitro, expand, and gene modify in clinically compliant conditions CD8(+) T(SCM) lymphocytes starting from naive precursors. Requirements for the generation of this T-cell subset, described as CD62L(+)CCR7(+)CD45RA(+)CD45R0(+)IL-7Rα(+)CD95(+), are CD3/CD28 engagement and culture with IL-7 and IL-15. Accordingly, T(SCM) accumulates early after hematopoietic stem cell transplantation. The gene expression signature and functional phenotype define this population as a distinct memory T-lymphocyte subset, intermediate between naive and central memory cells. When transplanted in immunodeficient mice, gene-modified naive-derived T(SCM) prove superior to other memory lymphocytes for the ability to expand and differentiate into effectors able to mediate a potent xenogeneic GVHD. Furthermore, gene-modified T(SCM) are the only T-cell subset able to expand and mediate GVHD on serial transplantation, suggesting self-renewal capacity in a clinically relevant setting. These findings provide novel insights into the origin and requirements for T(SCM) generation and pave the way for their clinical rapid exploitation in adoptive cell therapy.
Asunto(s)
Memoria Inmunológica , Interleucina-15/metabolismo , Interleucina-7/metabolismo , Células Precursoras de Linfocitos T/metabolismo , Subgrupos de Linfocitos T/metabolismo , Animales , Diferenciación Celular , Proliferación Celular , Supervivencia Celular , Análisis por Conglomerados , Femenino , Perfilación de la Expresión Génica , Humanos , Inmunofenotipificación , Interleucina-15/genética , Interleucina-7/genética , Selectina L/metabolismo , Antígenos Comunes de Leucocito/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones SCID , Células Precursoras de Linfocitos T/citología , Células Precursoras de Linfocitos T/trasplante , Subgrupos de Linfocitos T/citología , Subgrupos de Linfocitos T/trasplante , Receptor fas/metabolismoRESUMEN
The genetic modification of T cells with a suicide gene grants a mechanism of control of adverse reactions, allowing safe infusion after partially incompatible hematopoietic stem cell transplantation (HSCT). In the TK007 clinical trial, 22 adults with hematologic malignancies experienced a rapid and sustained immune recovery after T cell-depleted HSCT and serial infusions of purified donor T cells expressing the HSV thymidine kinase suicide gene (TK+ cells). After a first wave of circulating TK+ cells, the majority of T cells supporting long-term immune reconstitution did not carry the suicide gene and displayed high numbers of naive lymphocytes, suggesting the thymus-dependent development of T cells, occurring only upon TK+ -cell engraftment. Accordingly, after the infusions, we documented an increase in circulating TCR excision circles and CD31+ recent thymic emigrants and a substantial expansion of the active thymic tissue as shown by chest tomography scans. Interestingly, a peak in the serum level of IL-7 was observed after each infusion of TK+ cells, anticipating the appearance of newly generated T cells. The results of the present study show that the infusion of genetically modified donor T cells after HSCT can drive the recovery of thymic activity in adults, leading to immune reconstitution.
Asunto(s)
Terapia Genética/métodos , Neoplasias Hematológicas/terapia , Trasplante de Células Madre Hematopoyéticas/métodos , Linfocitos T/metabolismo , Adulto , Terapia Combinada , Expresión Génica , Genes Transgénicos Suicidas/genética , Neoplasias Hematológicas/sangre , Neoplasias Hematológicas/genética , Humanos , Interleucina-7/sangre , Recuento de Linfocitos , Estudios Prospectivos , Radiografía Torácica , Regeneración/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Linfocitos T/trasplante , Timidina Quinasa/genética , Timidina Quinasa/metabolismo , Timo/metabolismo , Timo/fisiopatología , Tomografía Computarizada por Rayos X , Resultado del TratamientoRESUMEN
Allogeneic hematopoietic stem cell transplantation (HSCT) from a human leukocyte antigen (HLA)-haploidentical family donor (haplo-HSCT) is a readily available and potentially curative option for high-risk leukemia. In haplo-HSCT, alloreactivity plays a major role in the graft-versus-leukemia (GVL) effect, which, however, is frequently followed by relapse due to emerging leukemic cell variants that have lost the unshared HLA haplotype as a mechanism of immune escape. We report that stimulation of HLA-haploidentical donor T lymphocytes with leukemic antigen-presenting cells (L-APCs) expands a population of leukemia-reactive T cells, which, besides alloreactivity to unshared HLAs, contain leukemia-associated specificities restricted by shared HLAs. According to a preferential central-memory (T(CM)) phenotype and to high interleukin (IL)-7Rα expression, these T cells persist in vivo and sustain a major GVL effect in a clinically relevant xenograft model. Moreover, we demonstrate that modifying L-APC-expanded T cells to express the herpes simplex virus thymidine kinase (HSV-tk) suicide gene enables their elimination with the prodrug ganciclovir (GCV), therefore providing a safety switch in case of graft-versus-host disease (GVHD). These results warrant the clinical investigation of L-APC-expanded T cells modified with a suicide gene in the setting of haplo-HSCT.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Genes Transgénicos Suicidas/genética , Efecto Injerto vs Leucemia/genética , Antígenos HLA/genética , Leucemia/genética , Linfocitos T/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Animales , Línea Celular Tumoral , Modelos Animales de Enfermedad , Citometría de Flujo , Ganciclovir/farmacología , Genes Transgénicos Suicidas/inmunología , Genes del Tumor de Wilms , Terapia Genética , Enfermedad Injerto contra Huésped/genética , Enfermedad Injerto contra Huésped/inmunología , Enfermedad Injerto contra Huésped/terapia , Antígenos HLA/inmunología , Trasplante de Células Madre Hematopoyéticas/métodos , Humanos , Leucemia/patología , Leucemia/terapia , Ratones , Ratones SCID , Persona de Mediana Edad , Linfocitos T/trasplante , Adulto JovenRESUMEN
Cancer vaccines have recently been shown to induce some clinical benefits. The relationship between clinical activity and anti-vaccine T cell responses is somewhat controversial. Indeed, in many trials it has been documented that the induction of vaccine-specific T cells exceeds the clinical responses observed. Here, we evaluate immunological and clinical responses in 23 MAGE-A3(+) melanoma patients treated with autologous lymphocytes genetically engineered to express the tumor antigen MAGE-A3 and the viral gene product thymidine kinase of the herpes simplex virus (HSV-TK). HSV-TK was used as safety system in case of adverse events and as tracer antigen to monitor the immune competence of treated patients. The increase of anti-TK and anti-MAGE-A3 T-cells after vaccination was observed in 90 and 27% of patients, respectively. Among 19 patients with measurable disease, we observed a disease control rate of 26.3%, with one objective clinical response, and four durable, stable diseases. Three patients out of five with no evidence of disease (NED) at the time of vaccination remained NED after 73+, 70+ and 50+ months. Notably, we report that only patients experiencing MAGE-A3-specific immune responses showed a clinical benefit. Additionally, we report that responder and non-responder patients activate and expand T cells against the tracer antigen TK in a similar way, suggesting that local rather than systemic immune suppression might be involved in limiting clinically relevant antitumor immune responses.
Asunto(s)
Antígenos de Neoplasias/inmunología , Vacunas contra el Cáncer/uso terapéutico , Terapia Genética , Melanoma/inmunología , Proteínas de Neoplasias/inmunología , Linfocitos T/inmunología , Adulto , Anciano , Neoplasias Óseas/inmunología , Neoplasias Óseas/mortalidad , Neoplasias Óseas/terapia , Ensayos Clínicos Fase II como Asunto , Femenino , Estudios de Seguimiento , Humanos , Hipersensibilidad Tardía , Neoplasias Hepáticas/inmunología , Neoplasias Hepáticas/mortalidad , Neoplasias Hepáticas/terapia , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/mortalidad , Neoplasias Pulmonares/terapia , Masculino , Melanoma/mortalidad , Melanoma/terapia , Persona de Mediana Edad , Estadificación de Neoplasias , Neoplasias Cutáneas/inmunología , Neoplasias Cutáneas/mortalidad , Neoplasias Cutáneas/terapia , Linfocitos T/metabolismo , Timidina Quinasa/inmunología , Timidina Quinasa/metabolismoRESUMEN
In allogeneic hematopoietic cell transplantation (HSCT), donor T lymphocytes mediate the graft-versus-leukemia (GVL) effect, but induce graft-versus-host disease (GVHD). Suicide gene therapy-that is, the genetic induction of a conditional suicide phenotype into donor T cells-allows dissociating the GVL effect from GVHD. Genetic modification with retroviral vectors after CD3 activation reduces T-cell alloreactivity. We recently found that alloreactivity is maintained when CD28 costimulation, IL-7, and IL-15 are added. Herein, we used the minor histocompatibility (mH) antigens HA-1 and H-Y as model alloantigens to directly explore the antileukemia efficacy of human T cells modified with the prototypic suicide gene herpes simplex virus thymidine kinase (tk) after activation with different stimuli. Only in the case of CD28 costimulation, IL-7, and IL-15, the repertoire of tk(+) T cells contained HA-1- and H-Y-specific CD8(+) cytotoxic T cells (CTL) precursors. Thymidine kinase-positive HA-1- and H-Y-specific CTLs were capable of self-renewal and differentiation into potent antileukemia effectors in vitro, and in vivo in a humanized mouse model. Self-renewal and differentiation coincided with IL-7 receptor expression. These results pave the way to the clinical investigation of T cells modified with a suicide gene after CD28 costimulation, IL-7, and IL-15 for a safe and effective GVL effect.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Diferenciación Celular , Proliferación Celular , Genes Transgénicos Suicidas/inmunología , Leucemia/diagnóstico , Receptores de Interleucina-7/fisiología , Animales , Biomarcadores/metabolismo , Linfocitos T CD8-positivos/metabolismo , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Células Cultivadas , Femenino , Expresión Génica/fisiología , Terapia Genética/métodos , Vectores Genéticos/inmunología , Humanos , Inmunoterapia Adoptiva/métodos , Leucemia/genética , Leucemia/inmunología , Leucemia/terapia , Ratones , Ratones Endogámicos NOD , Ratones SCID , Pronóstico , Receptores de Interleucina-7/genética , Receptores de Interleucina-7/metabolismo , Especificidad del Receptor de Antígeno de Linfocitos T/genética , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/metabolismo , Linfocitos T Citotóxicos/fisiología , Trasplante HomólogoRESUMEN
BACKGROUND: We investigated the long-term outcome of gene therapy for severe combined immunodeficiency (SCID) due to the lack of adenosine deaminase (ADA), a fatal disorder of purine metabolism and immunodeficiency. METHODS: We infused autologous CD34+ bone marrow cells transduced with a retroviral vector containing the ADA gene into 10 children with SCID due to ADA deficiency who lacked an HLA-identical sibling donor, after nonmyeloablative conditioning with busulfan. Enzyme-replacement therapy was not given after infusion of the cells. RESULTS: All patients are alive after a median follow-up of 4.0 years (range, 1.8 to 8.0). Transduced hematopoietic stem cells have stably engrafted and differentiated into myeloid cells containing ADA (mean range at 1 year in bone marrow lineages, 3.5 to 8.9%) and lymphoid cells (mean range in peripheral blood, 52.4 to 88.0%). Eight patients do not require enzyme-replacement therapy, their blood cells continue to express ADA, and they have no signs of defective detoxification of purine metabolites. Nine patients had immune reconstitution with increases in T-cell counts (median count at 3 years, 1.07x10(9) per liter) and normalization of T-cell function. In the five patients in whom intravenous immune globulin replacement was discontinued, antigen-specific antibody responses were elicited after exposure to vaccines or viral antigens. Effective protection against infections and improvement in physical development made a normal lifestyle possible. Serious adverse events included prolonged neutropenia (in two patients), hypertension (in one), central-venous-catheter-related infections (in two), Epstein-Barr virus reactivation (in one), and autoimmune hepatitis (in one). CONCLUSIONS: Gene therapy, combined with reduced-intensity conditioning, is a safe and effective treatment for SCID in patients with ADA deficiency. (ClinicalTrials.gov numbers, NCT00598481 and NCT00599781.)
Asunto(s)
Adenosina Desaminasa/genética , Antígenos CD34/genética , Terapia Genética , Trasplante de Células Madre Hematopoyéticas , Inmunodeficiencia Combinada Grave/terapia , Adenosina Desaminasa/deficiencia , Células de la Médula Ósea/inmunología , Preescolar , Terapia Combinada , Estudios de Seguimiento , Vectores Genéticos , Humanos , Lactante , Recuento de Linfocitos , Retroviridae , Inmunodeficiencia Combinada Grave/inmunología , Transducción Genética , Acondicionamiento PretrasplanteRESUMEN
BACKGROUND: Transplantation of hematopoietic stem cells from partially matched family donors is a promising therapy for patients who have a hematologic cancer and are at high risk for relapse. The donor T-cell infusions associated with such transplantation can promote post-transplantation immune reconstitution and control residual disease. METHODS: We identified 43 patients who underwent haploidentical transplantation and infusion of donor T cells for acute myeloid leukemia or myelodysplastic syndrome and conducted post-transplantation studies that included morphologic examination of bone marrow, assessment of hematopoietic chimerism with the use of short-tandem-repeat amplification, and HLA typing. The genomic rearrangements in mutant variants of leukemia were studied with the use of genomic HLA typing, microsatellite mapping, and single-nucleotide-polymorphism arrays. The post-transplantation immune responses against the original cells and the mutated leukemic cells were analyzed with the use of mixed lymphocyte cultures. RESULTS: In 5 of 17 patients with leukemia relapse after haploidentical transplantation and infusion of donor T cells, we identified mutant variants of the original leukemic cells. In the mutant leukemic cells, the HLA haplotype that differed from the donor's haplotype had been lost because of acquired uniparental disomy of chromosome 6p. T cells from the donor and the patient after transplantation did not recognize the mutant leukemic cells, whereas the original leukemic cells taken at the time of diagnosis were efficiently recognized and killed. CONCLUSIONS: After transplantation of haploidentical hematopoietic stem cells and infusion of donor T cells, leukemic cells can escape from the donor's antileukemic T cells through the loss of the mismatched HLA haplotype. This event leads to relapse.
Asunto(s)
Efecto Injerto vs Leucemia/genética , Antígenos HLA/genética , Trasplante de Células Madre Hematopoyéticas , Prueba de Histocompatibilidad , Leucemia Mieloide Aguda/terapia , Linfocitos T/inmunología , Adulto , Células Cultivadas , Cromosomas Humanos Par 6 , Efecto Injerto vs Leucemia/inmunología , Haplotipos , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/inmunología , Complejo Mayor de Histocompatibilidad , Mutación , Síndromes Mielodisplásicos , Recurrencia , Estudios Retrospectivos , Quimera por TrasplanteAsunto(s)
Enfermedades Genéticas Congénitas/terapia , Terapia Genética/tendencias , Células Madre Hematopoyéticas/fisiología , Leucemia/terapia , Animales , Enfermedades Genéticas Congénitas/genética , Ingeniería Genética , Regulación Gubernamental , Trasplante de Células Madre Hematopoyéticas , Humanos , Leucemia/genética , MercadotecníaRESUMEN
For decades, transplantation of haematopoietic stem cells--either unmodified, or genetically modified to correct genetic disorders--has been used to treat disorders of the blood and immune systems. The present challenge is to reduce the risk of such transplants and increase the number of patients who can safely access this treatment. In developing countries, such 'one-shot' treatments are highly desirable because chronic treatments are difficult to sustain. To make these therapies more accessible and effective it will be important to improve clinical protocols and gene-delivery vectors, and to gain a deeper understanding of stem cells.
Asunto(s)
Tratamiento Basado en Trasplante de Células y Tejidos , Enfermedades Hematológicas/patología , Enfermedades Hematológicas/terapia , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/citología , Terapia Genética , Salud , Células Madre Hematopoyéticas/fisiología , HumanosRESUMEN
Duchenne muscular dystrophy remains an untreatable genetic disease that severely limits motility and life expectancy in affected children. The only animal model specifically reproducing the alterations in the dystrophin gene and the full spectrum of human pathology is the golden retriever dog model. Affected animals present a single mutation in intron 6, resulting in complete absence of the dystrophin protein, and early and severe muscle degeneration with nearly complete loss of motility and walking ability. Death usually occurs at about 1 year of age as a result of failure of respiratory muscles. Here we report that intra-arterial delivery of wild-type canine mesoangioblasts (vessel-associated stem cells) results in an extensive recovery of dystrophin expression, normal muscle morphology and function (confirmed by measurement of contraction force on single fibres). The outcome is a remarkable clinical amelioration and preservation of active motility. These data qualify mesoangioblasts as candidates for future stem cell therapy for Duchenne patients.
Asunto(s)
Células Madre Adultas/trasplante , Distrofia Muscular Animal/terapia , Distrofia Muscular de Duchenne/terapia , Trasplante de Células Madre , Células Madre Adultas/inmunología , Animales , Terapia Combinada , Creatina Quinasa/sangre , Perros , Distrofina/biosíntesis , Distrofina/genética , Distrofina/inmunología , Terapia Genética , Humanos , Masculino , Células Musculares , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Trasplante Autólogo , Trasplante HeterólogoRESUMEN
Dendritic cell (DC) targeting in vivo has recently been shown to confer strong and protective cytotoxic T lymphocyte (CTL)-based immunity in tumor murine models. Our group has recently demonstrated in preclinical models that the infusion of genetically modified lymphocytes (GMLs) expressing the self/tumor antigen TRP-2 is able to elicit functional TRP-2-specific effectors with antitumor activity by targeting DCs in vivo. Here we have analyzed vaccine- and tumor-specific immune responses of 10 melanoma patients treated with autologous GMLs expressing the cancer germline gene MAGE-A3. Three of 10 patients treated with MAGE-A3-GML showed an increase of circulating anti-MAGE-A3 T cells, and developed skin delayed-type hypersensitivity to MAGE-A3. Interestingly, in 2 of these patients, with progressive and measurable tumors at study entry, anti-MAGE-A3 T cells were detected not only in the blood but also within tumors resected after vaccination. These results demonstrate that the infusion of MAGE-A3-GML elicits antitumor T cells, which are capable of trafficking to inflamed tissues and of infiltrating tumors. Clinical studies on a larger group of patients are needed to evaluate the clinical efficacy of such a strategy.
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Antígenos de Neoplasias/genética , Antígenos de Neoplasias/inmunología , Vacunas contra el Cáncer/administración & dosificación , Terapia Genética/métodos , Melanoma/terapia , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/inmunología , Neoplasias Cutáneas/terapia , Traslado Adoptivo , Animales , Células COS , Vacunas contra el Cáncer/efectos adversos , Línea Celular Tumoral , Chlorocebus aethiops , Humanos , Hipersensibilidad Tardía/inmunología , Melanoma/inmunología , Melanoma/patología , Estadificación de Neoplasias , Proyectos Piloto , Neoplasias Cutáneas/inmunología , Neoplasias Cutáneas/patología , Linfocitos T/citología , Linfocitos T/fisiología , Linfocitos T/trasplante , Timidina Quinasa/genética , Timidina Quinasa/inmunología , TransfecciónRESUMEN
Long-term clinical remissions of leukemia, after allogeneic hematopoietic stem cell transplantation, depend on alloreactive memory T cells able to self-renew and differentiate into antileukemia effectors. This is counterbalanced by detrimental graft-versus-host disease (GVHD). Induction of a selective suicide in donor T cells is a current gene therapy approach to abrogate GVHD. Unfortunately, genetic modification reduces alloreactivity of lymphocytes. This associates with an effector memory (T(EM)) phenotype of gene-modified lymphocytes and may limit antileukemia effect. We hypothesized that alloreactivity of gene-modified lymphocytes segregates with the central memory (T(CM)) phenotype. To this, we generated suicide gene-modified T(CM) lymphocytes with a retroviral vector after CD28 costimulation and culture with IL-2, IL-7, or a combination of IL-7 and IL-15. In vitro, suicide gene-modified T(CM) cells self-renewed upon alloantigen stimulation and resisted activation-induced cell death. In a humanized mouse model, only suicide gene-modified T cells cultured with IL-7 and IL-15 persisted, differentiated in T(EM) cells, and were as potent as unmanipulated lymphocytes in causing GVHD. GVHD was halted through the activation of the suicide gene machinery. These results warrant the use of suicide gene-modified T(CM) cells cultured with IL-7 and IL-15 for the safe exploitation of the alloreactive response against cancer.
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Genes Transgénicos Suicidas/inmunología , Enfermedad Injerto contra Huésped/inmunología , Memoria Inmunológica , Interleucina-15/farmacología , Interleucina-7/farmacología , Trasplante de Células Madre , Linfocitos T/inmunología , Animales , Muerte Celular/genética , Muerte Celular/inmunología , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Células Cultivadas , Genes Transgénicos Suicidas/genética , Enfermedad Injerto contra Huésped/genética , Enfermedad Injerto contra Huésped/terapia , Humanos , Memoria Inmunológica/genética , Interleucina-15/inmunología , Interleucina-2/genética , Interleucina-2/inmunología , Interleucina-7/inmunología , Isoantígenos/genética , Isoantígenos/inmunología , Activación de Linfocitos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Neoplasias/genética , Neoplasias/inmunología , Neoplasias/terapiaRESUMEN
The exploitation of the physiologic processing and presenting machinery of DCs by in vivo loading of tumor-associated antigens may improve the immunogenic potential and clinical efficacy of DC-based cancer vaccines. Here we show that lymphocytes genetically modified to express self/tumor antigens, acting as antigen carriers, efficiently target DCs in vivo in tumor-bearing mice. The infusion of tyrosinase-related protein 2-transduced (TRP-2-transduced) lymphocytes induced the establishment of protective immunity and long-term memory in tumor-bearing mice. Analysis of the mechanism responsible for the induction of such an immune response allowed us to demonstrate that cross-presentation of the antigen mediated by the CD11c(+)CD8alpha(+) DC subset had occurred. Furthermore, we demonstrated in vivo and in vitro that DCs had undergone activation upon phagocytosis of genetically modified lymphocytes, a process mediated by a cell-to-cell contact mechanism independent of CD40 triggering. Targeting and activation of secondary lymphoid organ-resident DCs endowed antigen-specific T cells with full effector functions, which ultimately increased tumor growth control and animal survival in a therapeutic tumor setting. We conclude that the use of transduced lymphocytes represents an efficient method for the in vivo loading of tumor-associated antigens on DCs.
Asunto(s)
Antígenos de Neoplasias/genética , Autoantígenos/genética , Células Dendríticas/inmunología , Neoplasias/inmunología , Linfocitos T/inmunología , Animales , Presentación de Antígeno , Células Presentadoras de Antígenos/inmunología , Antígenos CD40/inmunología , Comunicación Celular , Línea Celular Tumoral , Movimiento Celular , Oxidorreductasas Intramoleculares/genética , Ratones , Ratones Transgénicos , Fagocitosis , Linfocitos T/trasplanteRESUMEN
Gene transfer into HSCs is an effective treatment for SCID, although potentially limited by the risk of insertional mutagenesis. We performed a genome-wide analysis of retroviral vector integrations in genetically corrected HSCs and their multilineage progeny before and up to 47 months after transplantation into 5 patients with adenosine deaminase-deficient SCID. Gene-dense regions, promoters, and transcriptionally active genes were preferred retroviral integrations sites (RISs) both in preinfusion transduced CD34(+) cells and in vivo after gene therapy. The occurrence of insertion sites proximal to protooncogenes or genes controlling cell growth and self renewal, including LMO2, was not associated with clonal selection or expansion in vivo. Clonal analysis of long-term repopulating cell progeny in vivo revealed highly polyclonal T cell populations and shared RISs among multiple lineages, demonstrating the engraftment of multipotent HSCs. These data have important implications for the biology of retroviral vectors, the dynamics of genetically modified HSCs, and the safety of gene therapy.