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1.
Circulation ; 135(15): 1417-1428, 2017 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-28209728

RESUMO

BACKGROUND: Atherosclerotic peripheral artery disease affects 8% to 12% of Americans >65 years of age and is associated with a major decline in functional status, increased myocardial infarction and stroke rates, and increased risk of ischemic amputation. Current treatment strategies for claudication have limitations. PACE (Patients With Intermittent Claudication Injected With ALDH Bright Cells) is a National Heart, Lung, and Blood Institute-sponsored, randomized, double-blind, placebo-controlled, phase 2 exploratory clinical trial designed to assess the safety and efficacy of autologous bone marrow-derived aldehyde dehydrogenase bright (ALDHbr) cells in patients with peripheral artery disease and to explore associated claudication physiological mechanisms. METHODS: All participants, randomized 1:1 to receive ALDHbr cells or placebo, underwent bone marrow aspiration and isolation of ALDHbr cells, followed by 10 injections into the thigh and calf of the index leg. The coprimary end points were change from baseline to 6 months in peak walking time (PWT), collateral count, peak hyperemic popliteal flow, and capillary perfusion measured by magnetic resonance imaging, as well as safety. RESULTS: A total of 82 patients with claudication and infrainguinal peripheral artery disease were randomized at 9 sites, of whom 78 had analyzable data (57 male, 21 female patients; mean age, 66±9 years). The mean±SEM differences in the change over 6 months between study groups for PWT (0.9±0.8 minutes; 95% confidence interval [CI] -0.6 to 2.5; P=0.238), collateral count (0.9±0.6 arteries; 95% CI, -0.2 to 2.1; P=0.116), peak hyperemic popliteal flow (0.0±0.4 mL/s; 95% CI, -0.8 to 0.8; P=0.978), and capillary perfusion (-0.2±0.6%; 95% CI, -1.3 to 0.9; P=0.752) were not significant. In addition, there were no significant differences for the secondary end points, including quality-of-life measures. There were no adverse safety outcomes. Correlative relationships between magnetic resonance imaging measures and PWT were not significant. A post hoc exploratory analysis suggested that ALDHbr cell administration might be associated with an increase in the number of collateral arteries (1.5±0.7; 95% CI, 0.1-2.9; P=0.047) in participants with completely occluded femoral arteries. CONCLUSIONS: ALDHbr cell administration did not improve PWT or magnetic resonance outcomes, and the changes in PWT were not associated with the anatomic or physiological magnetic resonance imaging end points. Future peripheral artery disease cell therapy investigational trial design may be informed by new anatomic and perfusion insights. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT01774097.


Assuntos
Terapia Baseada em Transplante de Células e Tecidos , Doença Arterial Periférica/terapia , Idoso , Aldeído Desidrogenase/metabolismo , Células da Medula Óssea/metabolismo , Transplante de Medula Óssea , Terapia Baseada em Transplante de Células e Tecidos/efeitos adversos , Terapia Baseada em Transplante de Células e Tecidos/métodos , Comorbidade , Exercício Físico , Extremidades/irrigação sanguínea , Feminino , Seguimentos , Humanos , Claudicação Intermitente/terapia , Masculino , Pessoa de Meia-Idade , Perfusão , Doença Arterial Periférica/diagnóstico , Doença Arterial Periférica/metabolismo , Qualidade de Vida , Fatores de Risco , Resultado do Tratamento
2.
Blood ; 125(26): 4103-13, 2015 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-25977584

RESUMO

To test the feasibility of a single T-cell manipulation to eliminate alloreactivity while sparing antiviral and antitumor T cells, we infused 12 haploidentical hematopoietic stem cell transplant patients with increasing numbers of alloreplete haploidentical T cells expressing the inducible caspase 9 suicide gene (iC9-T cells). We determined whether the iC9-T cells produced immune reconstitution and if any resultant graft-versus-host disease (GVHD) could be controlled by administration of a chemical inducer of dimerization (CID; AP1903/Rimiducid). All patients receiving >10(4) alloreplete iC9-T lymphocytes per kilogram achieved rapid reconstitution of immune responses toward 5 major pathogenic viruses and concomitant control of active infections. Four patients received a single AP1903 dose. CID infusion eliminated 85% to 95% of circulating CD3(+)CD19(+) T cells within 30 minutes, with no recurrence of GVHD within 90 days. In one patient, symptoms and signs of GVHD-associated cytokine release syndrome (CRS-hyperpyrexia, high levels of proinflammatory cytokines, and rash) resolved within 2 hours of AP1903 infusion. One patient with varicella zoster virus meningitis and acute GVHD had iC9-T cells present in the cerebrospinal fluid, which were reduced by >90% after CID. Notably, virus-specific T cells recovered even after AP1903 administration and continued to protect against infection. Hence, alloreplete iC9-T cells can reconstitute immunity posttransplant and administration of CID can eliminate them from both peripheral blood and the central nervous system (CNS), leading to rapid resolution of GVHD and CRS. The approach may therefore be useful for the rapid and effective treatment of toxicities associated with infusion of engineered T lymphocytes. This trial was registered at www.clinicaltrials.gov as #NCT01494103.


Assuntos
Caspase 9/genética , Doença Enxerto-Hospedeiro/prevenção & controle , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Linfócitos T/transplante , Adolescente , Criança , Pré-Escolar , Feminino , Citometria de Fluxo , Genes Transgênicos Suicidas , Haplótipos , Transplante de Células-Tronco Hematopoéticas/métodos , Humanos , Transtornos Linfoproliferativos/cirurgia , Masculino , Pessoa de Meia-Idade , Compostos Orgânicos/uso terapêutico , Reação em Cadeia da Polimerase em Tempo Real , Adulto Jovem
3.
Blood ; 123(25): 3895-905, 2014 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-24753538

RESUMO

Adoptive transfer of donor-derived T lymphocytes expressing a safety switch may promote immune reconstitution in patients undergoing haploidentical hematopoietic stem cell transplant (haplo-HSCT) without the risk for uncontrolled graft versus host disease (GvHD). Thus, patients who develop GvHD after infusion of allodepleted donor-derived T cells expressing an inducible human caspase 9 (iC9) had their disease effectively controlled by a single administration of a small-molecule drug (AP1903) that dimerizes and activates the iC9 transgene. We now report the long-term follow-up of 10 patients infused with such safety switch-modified T cells. We find long-term persistence of iC9-modified (iC9-T) T cells in vivo in the absence of emerging oligoclonality and a robust immunologic benefit, mediated initially by the infused cells themselves and subsequently by an apparently accelerated reconstitution of endogenous naive T lymphocytes. As a consequence, these patients have immediate and sustained protection from major pathogens, including cytomegalovirus, adenovirus, BK virus, and Epstein-Barr virus in the absence of acute or chronic GvHD, supporting the beneficial effects of this approach to immune reconstitution after haplo-HSCT. This study was registered at www.clinicaltrials.gov as #NCT00710892.


Assuntos
Caspase 9/genética , Transplante de Células-Tronco Hematopoéticas/métodos , Linfócitos T/transplante , Transgenes/genética , Adolescente , Aspergilose/imunologia , Aspergilose/microbiologia , Aspergilose/prevenção & controle , Aspergillus fumigatus/imunologia , Caspase 9/biossíntese , Criança , Pré-Escolar , Indução Enzimática/efeitos dos fármacos , Feminino , Seguimentos , Doença Enxerto-Hospedeiro/imunologia , Doença Enxerto-Hospedeiro/prevenção & controle , Humanos , Imunoterapia Adotiva/métodos , Linfoma Anaplásico de Células Grandes/imunologia , Linfoma Anaplásico de Células Grandes/terapia , Masculino , Síndromes Mielodisplásicas/imunologia , Síndromes Mielodisplásicas/terapia , Compostos Orgânicos/administração & dosagem , Compostos Orgânicos/uso terapêutico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/imunologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/terapia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/imunologia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/terapia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Fatores de Tempo , Transplante Homólogo , Resultado do Tratamento , Viroses/imunologia , Viroses/prevenção & controle , Viroses/virologia
4.
N Engl J Med ; 365(18): 1673-83, 2011 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-22047558

RESUMO

BACKGROUND: Cellular therapies could play a role in cancer treatment and regenerative medicine if it were possible to quickly eliminate the infused cells in case of adverse events. We devised an inducible T-cell safety switch that is based on the fusion of human caspase 9 to a modified human FK-binding protein, allowing conditional dimerization. When exposed to a synthetic dimerizing drug, the inducible caspase 9 (iCasp9) becomes activated and leads to the rapid death of cells expressing this construct. METHODS: We tested the activity of our safety switch by introducing the gene into donor T cells given to enhance immune reconstitution in recipients of haploidentical stem-cell transplants. Patients received AP1903, an otherwise bioinert small-molecule dimerizing drug, if graft-versus-host disease (GVHD) developed. We measured the effects of AP1903 on GVHD and on the function and persistence of the cells containing the iCasp9 safety switch. RESULTS: Five patients between the ages of 3 and 17 years who had undergone stem-cell transplantation for relapsed acute leukemia were treated with the genetically modified T cells. The cells were detected in peripheral blood from all five patients and increased in number over time, despite their constitutive transgene expression. A single dose of dimerizing drug, given to four patients in whom GVHD developed, eliminated more than 90% of the modified T cells within 30 minutes after administration and ended the GVHD without recurrence. CONCLUSIONS: The iCasp9 cell-suicide system may increase the safety of cellular therapies and expand their clinical applications. (Funded by the National Heart, Lung, and Blood Institute and the National Cancer Institute; ClinicalTrials.gov number, NCT00710892.).


Assuntos
Caspase 9/genética , Genes Transgênicos Suicidas , Doença Enxerto-Hospedeiro/terapia , Imunoterapia Adotiva , Linfócitos T/transplante , Proteínas de Ligação a Tacrolimo/genética , Adolescente , Apoptose , Caspase 9/metabolismo , Criança , Pré-Escolar , Feminino , Técnicas de Transferência de Genes , Humanos , Leucemia/terapia , Masculino , Compostos Orgânicos/uso terapêutico , Recidiva , Transplante de Células-Tronco , Linfócitos T/imunologia
5.
Cytotherapy ; 16(8): 1048-58, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24726657

RESUMO

BACKGROUND: The use of bone marrow-derived mesenchymal stromal cells (MSCs) as a cellular therapy for various diseases, such as graft-versus-host disease, diabetes, ischemic cardiomyopathy and Crohn's disease, has produced promising results in early-phase clinical trials. However, for widespread application and use in later phase studies, manufacture of these cells must be cost-effective, safe and reproducible. Current methods of manufacturing in flasks or cell factories are labor-intensive, involve a large number of open procedures and require prolonged culture times. METHODS: We evaluated the Quantum Cell Expansion System for the expansion of large numbers of MSCs from unprocessed bone marrow in a functionally closed system and compared the results with a flask-based method currently in clinical trials. RESULTS: After only two passages, we were able to expand a mean of 6.6 × 10(8) MSCs from 25 mL of bone marrow reproducibly. The mean expansion time was 21 days, and cells obtained were able to differentiate into all three lineages: chondrocytes, osteoblasts and adipocytes. The Quantum was able to generate the target cell number of 2.0 × 10(8) cells in an average of 9 fewer days and in half the number of passages required during flask-based expansion. We estimated that the Quantum would involve 133 open procedures versus 54,400 in flasks when manufacturing for a clinical trial. Quantum-expanded MSCs infused into an ischemic stroke rat model were therapeutically active. CONCLUSIONS: The Quantum is a novel method of generating high numbers of MSCs in less time and at lower passages when compared with flasks. In the Quantum, the risk of contamination is substantially reduced because of the substantial decrease in open procedures.


Assuntos
Células da Medula Óssea/citologia , Técnicas de Cultura de Células , Terapia Baseada em Transplante de Células e Tecidos , Células-Tronco Mesenquimais/citologia , Animais , Reatores Biológicos , Diferenciação Celular/genética , Linhagem da Célula , Humanos , Transplante de Células-Tronco Mesenquimais , Ratos
6.
Cytotherapy ; 15(4): 416-22, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23480951

RESUMO

Mesenchymal stromal cells (MSCs) are multipotent progenitor cells capable of differentiating into adipocytes, osteoblasts and chondroblasts as well as secreting a vast array of soluble mediators. This potentially makes MSCs important mediators of a variety of therapeutic applications. They are actively under evaluation for immunomodulatory purposes such as graft-versus-host disease and Crohn's disease as well as regenerative applications such as stroke and congestive heart failure. We report our method of generating clinical-grade MSCs together with suggestions gathered from manufacturing experience in our Good Manufacturing Practices facility.


Assuntos
Células da Medula Óssea/citologia , Técnicas de Cultura de Células/métodos , Leucócitos Mononucleares/citologia , Células-Tronco Mesenquimais/citologia , Diferenciação Celular , Células Cultivadas , Ensaios Clínicos Fase I como Assunto , Criopreservação , Humanos , Transplante de Células-Tronco Mesenquimais/métodos
7.
Cytotherapy ; 14(9): 1131-43, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22900959

RESUMO

BACKGROUND AIMS: Interest in natural killer (NK) cell-based immunotherapy has resurged since new protocols for the purification and expansion of large numbers of clinical-grade cells have become available. METHODS: We have successfully adapted a previously described NK expansion method that uses K562 cells expressing interleukin (IL)-15 and 4-1 BB Ligand (BBL) (K562-mb15-41BBL) to grow NK cells in novel gas-permeable static cell culture flasks (G-Rex). RESULTS: Using this system we produced up to 19 × 10(9) functional NK cells from unseparated apheresis products, starting with 15 × 10(7) CD3(-) CD56 (+) NK cells, within 8-10 days of culture. The G-Rex yielded a higher fold expansion of NK cells than conventional gas-permeable bags and required no cell manipulation or feeding during the culture period. We also showed that K562-mb15-41BBL cells up-regulated surface HLA class I antigen expression upon stimulation with the supernatants from NK cultures and stimulated alloreactive CD8 (+) T cells within the NK cultures. However, these CD3 (+) T cells could be removed successfully using the CliniMACS system. We describe our optimized NK cell cryopreservation method and show that the NK cells are viable and functional even after 12 months of cryopreservation. CONCLUSIONS: We have successfully developed a static culture protocol for large-scale expansion of NK cells in the gas permeable G-Rex system under good manufacturing practice (GMP) conditions. This strategy is currently being used to produce NK cells for cancer immunotherapy.


Assuntos
Imunoterapia Adotiva , Células K562/citologia , Células Matadoras Naturais/citologia , Linfócitos T , Ligante 4-1BB/metabolismo , Remoção de Componentes Sanguíneos , Técnicas de Cultura de Células , Sobrevivência Celular , Criopreservação , Citometria de Fluxo , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Interleucina-15/metabolismo , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Ativação Linfocitária/imunologia , Linfócitos T/citologia , Linfócitos T/imunologia , Linfócitos T/metabolismo
8.
Biol Blood Marrow Transplant ; 16(2): 253-62, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19822219

RESUMO

To test the hypothesis that the outcome of hematopoietic stem cell (HSC) grafts is at least partially determined by the cellular composition of the graft, the National Marrow Donor Program (NMDP) analyzed the correlation of cellular phenotypes of unrelated grafts with graft outcome. Samples from 94 bone marrow (BM) and 181 peripheral blood progenitor cell (PBPC) grafts for transplantations at 40 U.S. transplant centers between 2003 and 2005 were analyzed at a single immunophenotyping reference laboratory. Samples were shipped from transplant centers upon receipt of graft. Graft cellular composition included analysis of leukocyte total cell numbers, and subsets of myeloid [CD34(+), CD34(+) CD38(-)], lymphoid [CD3(+), CD3(+) CD4(+), CD3(+) CD8(+)], and activated lymphoid cells [CD3(+) CD25(+), CD3(+) CD69(+), CD3(+) HLA-DR(+)] coexpressing CD3(+). There was substantial variability in the cellular composition of BM and PBPC grafts before and after graft processing by red blood cell (RBC) removal or plasma depletion in preparation for transplant. With BM grafts, cellular composition was not associated with hematopoietic recovery, graft-versus-host disease (GVHD), or survival. With PBPC grafts, survival rates were higher with CD34(+)>5 x 10(6)/kg, 59% compared to 34% with CD34(+)< or =5 x 10(6)/kg at 1 year. Platelet recovery was higher with PBPC containing CD3(+) CD8(+) >8 x 10(7)/kg. Neutrophil recovery or GVHD could not be predicted by any cellular subsets of PBPC grafts. Although survival was superior with PBPC grafts containing >5 x 10(6) CD34(+)/kg, an optimal graft mix of myeloid, lymphoid, and activated lymphoid subsets was not identified.


Assuntos
Transplante de Medula Óssea , Subpopulações de Linfócitos/transplante , Células Mieloides/transplante , Transplante de Células-Tronco de Sangue Periférico , Antígenos CD/metabolismo , Transplante de Medula Óssea/métodos , Transplante de Medula Óssea/mortalidade , Seleção do Doador , Feminino , Citometria de Fluxo , Sobrevivência de Enxerto , Doença Enxerto-Hospedeiro/epidemiologia , Transplante de Células-Tronco Hematopoéticas/métodos , Transplante de Células-Tronco Hematopoéticas/mortalidade , Transplante de Células-Tronco Hematopoéticas/estatística & dados numéricos , Humanos , Imunofenotipagem , Contagem de Leucócitos , Subpopulações de Linfócitos/metabolismo , Masculino , Células Mieloides/classificação , Células Mieloides/metabolismo , Transplante de Células-Tronco de Sangue Periférico/métodos , Transplante de Células-Tronco de Sangue Periférico/mortalidade , Fenótipo , Sistema de Registros , Reprodutibilidade dos Testes , Análise de Sobrevida , Resultado do Tratamento
10.
J Immunother Cancer ; 3: 5, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25734008

RESUMO

BACKGROUND: Virus-specific T-cells (VSTs) proliferate exponentially after adoptive transfer into hematopoietic stem cell transplant (HSCT) recipients, eliminate virus infections, then persist and provide long-term protection from viral disease. If VSTs behaved similarly when modified with tumor-specific chimeric antigen receptors (CARs), they should have potent anti-tumor activity. This theory was evaluated by Cruz et al. in a previous clinical trial with CD19.CAR-modified VSTs, but there was little apparent expansion of these cells in patients. In that study, VSTs were gene-modified on day 19 of culture and we hypothesized that by this time, sufficient T-cell differentiation may have occurred to limit the subsequent proliferative capacity of the transduced T-cells. To facilitate the clinical testing of this hypothesis in a project supported by the NHLBI-PACT mechanism, we developed and optimized a good manufacturing practices (GMP) compliant method for the early transduction of VSTs directed to Epstein-Barr virus (EBV), Adenovirus (AdV) and cytomegalovirus (CMV) using a CAR directed to the tumor-associated antigen disialoganglioside (GD2). RESULTS: Ad-CMVpp65-transduced EBV-LCLs effectively stimulated VSTs directed to all three viruses (triVSTs). Transduction efficiency on day three was increased in the presence of cytokines and high-speed centrifugation of retroviral supernatant onto retronectin-coated plates, so that under optimal conditions up to 88% of tetramer-positive VSTs expressed the GD2.CAR. The average transduction efficiency of early-and late transduced VSTs was 55 ± 4% and 22 ± 5% respectively, and early-transduced VSTs maintained higher frequencies of T cells with central memory or intermediate memory phenotypes. Early-transduced VSTs also had higher proliferative capacity and produced higher levels of TH1 cytokines IL-2, TNF-α, IFN-γ, MIP-1α, MIP-1ß and other cytokines in vitro. CONCLUSIONS: We developed a rapid and GMP compliant method for the early transduction of multivirus-specific T-cells that allowed stable expression of high levels of a tumor directed CAR. Since a proportion of early-transduced CAR-VSTs had a central memory phenotype, they should expand and persist in vivo, simultaneously protecting against infection and targeting residual malignancy. This manufacturing strategy is currently under clinical investigation in patients receiving allogeneic HSCT for relapsed neuroblastoma and B-cell malignancies (NCT01460901 using a GD2.CAR and NCT00840853 using a CD19.CAR).

11.
J Immunol ; 168(2): 909-18, 2002 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-11777989

RESUMO

Adoptive immunotherapy with EBV-specific CTL (EBV-CTL) effectively prevents and treats EBV-driven lymphoproliferation in immunocompromised hosts. EBV-seronegative solid organ transplant recipients are at high risk of EBV-driven lymphoproliferation because they lack EBV-specific memory T cells. For the same reason, standard techniques for generating EBV-CTL in vitro from EBV-naive individuals are unsuccessful. To overcome this problem, we compared several methods of expanding EBV-CTL from seronegative adults and children. First, the standard protocol, using EBV-transformed lymphoblastoid B cell lines (LCL) as the source of APC, was compared with protocols using EBV-Ag-loaded dendritic cells as APC. Surprisingly, the standard protocol effectively generated CTL from all seronegative adults. The additional finding of EBV-DNA in the peripheral blood of three of these four adults suggested that some individuals may develop cellular, but not humoral, immune responses to EBV. By contrast, LCL failed to reactivate EBV-CTL from any of the six EBV-seronegative children. EBV-Ag-loaded dendritic cells could expand EBV-CTL, but only in a minority of children. However, the selective expansion of CD25-expressing T cells, 9-11 days after activation with LCL alone, proved to be a simple and reliable method for generating EBV-CTL from all seronegative children. The majority of these CTL were CD4(+) (71 +/- 26%) and demonstrated HLA class II-restricted, EBV-specific killing. Our results suggest that a negative EBV serology does not accurately identify EBV-negative individuals. In addition, our method for selecting EBV-specific CTL from naive individuals by precursor cell enrichment may be applicable to the immunotherapy of cancer patients with a low frequency of tumor- or virus-specific CTL.


Assuntos
Antígenos CD4/biossíntese , Citotoxicidade Imunológica , Epitopos de Linfócito T/imunologia , Herpesvirus Humano 4/imunologia , Ativação Linfocitária , Subpopulações de Linfócitos T/imunologia , Linfócitos T Citotóxicos/imunologia , Adulto , Portador Sadio/imunologia , Portador Sadio/virologia , Linhagem Celular Transformada , Criança , Pré-Escolar , Meios de Cultivo Condicionados , Testes Imunológicos de Citotoxicidade/normas , Testes Imunológicos de Citotoxicidade/estatística & dados numéricos , Células Dendríticas/imunologia , Células Dendríticas/virologia , Infecções por Vírus Epstein-Barr/imunologia , Infecções por Vírus Epstein-Barr/virologia , Feminino , Sangue Fetal/imunologia , Humanos , Lactente , Masculino , Receptores de Interleucina-2/biossíntese , Reprodutibilidade dos Testes , Subpopulações de Linfócitos T/metabolismo , Subpopulações de Linfócitos T/virologia , Linfócitos T Citotóxicos/metabolismo , Linfócitos T Citotóxicos/virologia
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