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1.
Leukemia ; 33(9): 2195-2207, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30816327

RESUMO

Successful adoptive chimeric antigen receptor (CAR) T-cell therapies against hematological malignancies require CAR-T expansion and durable persistence following infusion. Balancing increased CAR-T potency with safety, including severe cytokine-release syndrome (sCRS) and neurotoxicity, warrants inclusion of safety mechanisms to control in vivo CAR-T activity. Here, we describe a novel CAR-T cell platform that utilizes expression of the toll-like receptor (TLR) adaptor molecule, MyD88, and tumor-necrosis factor family member, CD40 (MC), tethered to the CAR molecule through an intentionally inefficient 2A linker system, providing a constitutive signal that drives CAR-T survival, proliferation, and antitumor activity against CD19+ and CD123+ hematological cancers. Robust activity of MC-enhanced CAR-T cells was associated with cachexia in animal models that corresponded with high levels of human cytokine production. However, toxicity could be successfully resolved by using the inducible caspase-9 (iC9) safety switch to reduce serum cytokines, by administration of a neutralizing antibody against TNF-α, or by selecting "low" cytokine-producing CD8+ T cells, without loss of antitumor activity. Interestingly, high basal activity was essential for in vivo CAR-T expansion. This study shows that co-opting novel signaling elements (i.e., MyD88 and CD40) and development of a unique CAR-T architecture can drive T-cell proliferation in vivo to enhance CAR-T therapies.

2.
Clin Transl Gastroenterol ; 8(11): e126, 2017 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-29095427

RESUMO

OBJECTIVES: Flow cytometry (FC) aids in characterization of cellular and molecular factors involved in pathologic immune responses. Although FC has potential to facilitate early drug development in inflammatory bowel disease, interlaboratory variability limits its use in multicenter trials. Standardization of methods may address this limitation. We compared variability in FC-aided quantitation of T-cell responses across international laboratories using three analytical strategies. METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated from three healthy donors, stimulated with phorbol 12-myristate 13-acetate and ionomycin at a central laboratory, fixed, frozen, and shipped to seven international laboratories. Permeabilization and staining was performed in triplicate at each laboratory using a common protocol and centrally provided reagents. Gating was performed using local gating with a local strategy (LGLS), local gating with a central strategy (LGCS), and central gating (CG). Median cell percentages were calculated across triplicates and donors, and reported for each condition and strategy. The coefficient of variation (CV) was calculated across laboratories. Between-strategy comparisons were made using a two-way analysis of variance adjusting for donor. RESULTS: Mean interlaboratory CV ranged from 1.8 to 102.1% depending on cell population and gating strategy (LGLS, 4.4-102.1%; LGCS, 10.9-65.6%; CG, 1.8-20.9%). Mean interlaboratory CV differed significantly across strategies and was consistently lower with CG. CONCLUSIONS: Central gating was the only strategy with mean CVs consistently lower than 25%, which is a proposed standard for pharmacodynamic and exploratory biomarker assays.

3.
Cancer Discov ; 7(11): 1306-1319, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28801306

RESUMO

Adoptive immunotherapy with T cells expressing chimeric antigen receptors (CAR) has had limited success for solid tumors in early-phase clinical studies. We reasoned that introducing into CAR T cells an inducible costimulatory (iCO) molecule consisting of a chemical inducer of dimerization (CID)-binding domain and the MyD88 and CD40 signaling domains would improve and control CAR T-cell activation. In the presence of CID, T cells expressing HER2-CARζ and a MyD88/CD40-based iCO molecule (HER2ζ.iCO T cells) had superior T-cell proliferation, cytokine production, and ability to sequentially kill targets in vitro relative to HER2ζ.iCO T cells without CID and T cells expressing HER2-CAR.CD28ζ. HER2ζ.iCO T cells with CID also significantly improved survival in vivo in two xenograft models. Repeat injections of CID were able to further increase the antitumor activity of HER2ζ.iCO T cells in vivo Thus, expressing MyD88/CD40-based iCO molecules in CAR T cells has the potential to improve the efficacy of CAR T-cell therapy approaches for solid tumors.Significance: Inducible activation of MyD88 and CD40 in CAR T cells with a small-molecule drug not only enhances their effector function, resulting in potent antitumor activity in preclinical solid tumors, but also enables their remote control post infusion. Cancer Discov; 7(11); 1306-19. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 1201.


Assuntos
Antígenos CD40/genética , Imunoterapia Adotiva/métodos , Fator 88 de Diferenciação Mieloide/genética , Neoplasias/terapia , Receptores de Antígenos de Linfócitos T/uso terapêutico , Animais , Antígenos CD40/imunologia , Antígenos CD40/uso terapêutico , Linhagem Celular Tumoral , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica/imunologia , Humanos , Camundongos , Fator 88 de Diferenciação Mieloide/imunologia , Fator 88 de Diferenciação Mieloide/uso terapêutico , Neoplasias/genética , Neoplasias/imunologia , Receptor ErbB-2/genética , Receptor ErbB-2/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/imunologia , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/genética , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/imunologia , Ensaios Antitumorais Modelo de Xenoenxerto
4.
Mol Ther ; 25(9): 2176-2188, 2017 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-28697888

RESUMO

Anti-tumor efficacy of T cells engineered to express chimeric antigen receptors (CARs) is dependent on their specificity, survival, and in vivo expansion following adoptive transfer. Toll-like receptor (TLR) and CD40 signaling in T cells can improve persistence and drive proliferation of antigen-specific CD4+ and CD8+ T cells following pathogen challenge or in graft-versus-host disease (GvHD) settings, suggesting that these costimulatory pathways may be co-opted to improve CAR-T cell persistence and function. Here, we present a novel strategy to activate TLR and CD40 signaling in human T cells using inducible MyD88/CD40 (iMC), which can be triggered in vivo via the synthetic dimerizing ligand, rimiducid, to provide potent costimulation to CAR-modified T cells. Importantly, the concurrent activation of iMC (with rimiducid) and CAR (by antigen recognition) is required for interleukin (IL)-2 production and robust CAR-T cell expansion and may provide a user-controlled mechanism to amplify CAR-T cell levels in vivo and augment anti-tumor efficacy.


Assuntos
Antígenos CD28/metabolismo , Antígenos CD40/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Proteínas Recombinantes de Fusão , Linfócitos T/imunologia , Linfócitos T/metabolismo , Animais , Antígenos CD28/genética , Antígenos CD40/genética , Proliferação de Células , Sobrevivência Celular , Análise por Conglomerados , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Humanos , Imunoterapia Adotiva/métodos , Leucemia/genética , Leucemia/imunologia , Leucemia/metabolismo , Leucemia/terapia , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Camundongos , Receptores de Antígenos de Linfócitos T/genética , Transdução de Sinais , Linfócitos T/efeitos dos fármacos , Receptores Toll-Like/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
5.
Cancer Immunol Immunother ; 66(10): 1345-1357, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28608115

RESUMO

This phase I trial reports the safety and activity of BPX101, a second-generation antigen-targeted autologous antigen presenting cell (APC) vaccine in men with metastatic castration-resistant prostate cancer (mCRPC). To manufacture BPX101, APCs collected in a single leukapheresis were transduced with adenoviral vector Ad5f35 encoding inducible human (ih)-CD40, followed by incubation with protein PA001, which contains the extracellular domain of human prostate-specific membrane antigen. The ih-CD40 represents a modified chimeric version of the dendritic cell (DC) co-stimulatory molecule, CD40, which responds to a bioinert membrane-permeable activating dimerizer drug, rimiducid (AP1903), permitting temporally controlled, lymphoid-localized, DC-specific activation. Eighteen men with progressive mCRPC following ≤1 prior chemotherapy regimen were enrolled to evaluate three doses of BPX101 (4 × 106, 12.5 × 106 and 25 × 106 cells) administered intradermally every 2-4 weeks followed by rimiducid (0.4 mg/kg) intravenous (IV) infusion 24 h after each BPX101 dose. There were no dose-limiting toxicities. Immune upregulation as well as anti-tumor activity was observed with PSA declines, objective tumor regressions and robust efficacy of post-trial therapy. This novel antigen-targeted and in vivo activated immunotherapy platform may warrant further development as monotherapy and as a component of rational combinations.


Assuntos
Antígenos CD40/metabolismo , Vacinas Anticâncer/imunologia , Células Dendríticas/imunologia , Neoplasias da Próstata/imunologia , Idoso , Vacinas Anticâncer/uso terapêutico , Estudos de Coortes , Humanos , Masculino
6.
PLoS One ; 12(3): e0173176, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28257518

RESUMO

Although the role of T cells in autoimmunity has been explored for many years, the mechanisms leading to the initial priming of an autoimmune T cell response remain enigmatic. The 'hit and run' model suggests that self-antigens released upon cell death can provide the initial signal for a self-sustaining autoimmune response. Using a novel transgenic mouse model where we could induce the release of self-antigens via caspase-dependent apoptosis. We tracked the fate of CD8+ T cells specific for the self-antigen. Our studies demonstrated that antigens released from apoptotic cells were cross-presented by CD11c+ cells in the draining lymph node. This cross-presentation led to proliferation of self-antigen specific T cells, followed by a transient ability to produce IFN-γ, but did not lead to the development of autoimmune diabetes. Using this model we examined the consequences on T cell immunity when apoptosis was combined with dendritic cell maturation signals, an autoimmune susceptible genetic background, and the deletion of Tregs. The results of our study demonstrate that autoimmune diabetes cannot be initiated by the presentation of antigens released from apoptotic cells in vivo even in the presence of factors known to promote autoimmunity.


Assuntos
Autoantígenos/administração & dosagem , Linfócitos T CD8-Positivos/imunologia , Diabetes Mellitus Tipo 1/imunologia , Linfócitos T Reguladores/imunologia , Animais , Apresentação do Antígeno/genética , Apresentação do Antígeno/imunologia , Apoptose/genética , Autoantígenos/imunologia , Autoimunidade/genética , Células Dendríticas/imunologia , Diabetes Mellitus Tipo 1/etiologia , Diabetes Mellitus Tipo 1/patologia , Modelos Animais de Doenças , Humanos , Tolerância Imunológica , Interferon gama/genética , Camundongos , Camundongos Transgênicos/imunologia
7.
PLoS One ; 11(10): e0164547, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27741278

RESUMO

Therapeutic DNA-based vaccines aim to prime an adaptive host immune response against tumor-associated antigens, eliminating cancer cells primarily through CD8+ cytotoxic T cell-mediated destruction. To be optimally effective, immunological adjuvants are required for the activation of tumor-specific CD8+ T cells responses by DNA vaccination. Here, we describe enhanced anti-tumor efficacy of an in vivo electroporation-delivered DNA vaccine by inclusion of a genetically encoded chimeric MyD88/CD40 (MC) adjuvant, which integrates both innate and adaptive immune signaling pathways. When incorporated into a DNA vaccine, signaling by the MC adjuvant increased antigen-specific CD8+ T cells and promoted elimination of pre-established tumors. Interestingly, MC-enhanced vaccine efficacy did not require direct-expression of either antigen or adjuvant by local antigen-presenting cells, but rather our data supports a key role for MC function in "atypical" antigen-presenting cells of skin. In particular, MC adjuvant-modified keratinocytes increased inflammatory cytokine secretion, upregulated surface MHC class I, and were able to increase in vitro and in vivo priming of antigen-specific CD8+ T cells. Furthermore, in the absence of critical CD8α+/CD103+ cross-priming dendritic cells, MC was still able to promote immune priming in vivo, albeit at a reduced level. Altogether, our data support a mechanism by which MC signaling activates an inflammatory phenotype in atypical antigen-presenting cells within the cutaneous vaccination site, leading to an enhanced CD8+ T cell response against DNA vaccine-encoded antigens, through both CD8α+/CD103+ dendritic cell-dependent and independent pathways.


Assuntos
Células Apresentadoras de Antígenos/imunologia , Antígenos CD40/genética , Fator 88 de Diferenciação Mieloide/genética , Vacinas de DNA/imunologia , Animais , Células Apresentadoras de Antígenos/citologia , Células Apresentadoras de Antígenos/metabolismo , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Vacinas Anticâncer/imunologia , Vacinas Anticâncer/uso terapêutico , Linhagem Celular , Proliferação de Células , Citocinas/análise , Citocinas/metabolismo , Feminino , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Queratinócitos/citologia , Queratinócitos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , MicroRNAs/metabolismo , Células NIH 3T3 , Neoplasias/imunologia , Neoplasias/terapia , Vacinas de DNA/uso terapêutico
8.
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
9.
Stem Cells ; 33(1): 91-100, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25330775

RESUMO

The high risk of insertional oncogenesis reported in clinical trials using integrating retroviral vectors to genetically modify hematopoietic stem and progenitor cells (HSPCs) requires the development of safety strategies to minimize risks associated with novel cell and gene therapies. The ability to ablate genetically modified cells in vivo is desirable, should an abnormal clone emerge. Inclusion of "suicide genes" in vectors to facilitate targeted ablation of vector-containing abnormal clones in vivo is one potential safety approach. We tested whether the inclusion of the "inducible Caspase-9" (iCasp9) suicide gene in a gamma-retroviral vector facilitated efficient elimination of vector-containing HSPCs and their hematopoietic progeny in vivo long-term, in an autologous non-human primate transplantation model. Following stable engraftment of iCasp9 expressing hematopoietic cells in rhesus macaques, administration of AP1903, a chemical inducer of dimerization able to activate iCasp9, specifically eliminated vector-containing cells in all hematopoietic lineages long-term, suggesting activity at the HSPC level. Between 75% and 94% of vector-containing cells were eliminated by well-tolerated AP1903 dosing, but lack of complete ablation was linked to lower iCasp9 expression in residual cells. Further investigation of resistance mechanisms demonstrated upregulation of Bcl-2 in hematopoietic cell lines transduced with the vector and resistant to AP1903 ablation. These results demonstrate both the potential and the limitations of safety approaches using iCasp9 to HSPC-targeted gene therapy settings, in a model with great relevance to clinical development.


Assuntos
Caspase 9/genética , Genes Transgênicos Suicidas , Transplante de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/citologia , Animais , Apoptose/fisiologia , Caspase 9/biossíntese , Terapia Genética , Vetores Genéticos , Células-Tronco Hematopoéticas/enzimologia , Macaca mulatta
10.
Vaccine ; 32(33): 4228-33, 2014 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-24923637

RESUMO

Over the past 20 years, dendritic cells (DCs) have been utilized to activate immune responses capable of eliminating cancer cells. Currently, ex vivo DC priming has been the mainstay of DC cancer immunotherapies. However, cell-based treatment modalities are inherently flawed due to a lack of standardization, specialized facilities and personnel, and cost. Therefore, direct modes of DC manipulation, circumventing the need for ex vivo culture, must be investigated. To facilitate the development of next-generation, in vivo targeted DC vaccines, we characterized the DC interaction and activation potential of the Tobacco Mosaic virus (TMV), a plant virus that enjoys a relative ease of production and the ability to deliver protein payloads via surface conjugation. In this study we show that TMV is readily taken up by mouse bone marrow-derived DCs, in vitro. Footpad injection of fluorophore-labeled TMV reveals preferential uptake by draining lymph node resident DCs in vivo. Uptake leads to activation, as measured by the upregulation of key DC surface markers. When peptide antigen-conjugated TMV is injected into the footpad of mice, DC-mediated uptake and activation leads to robust antigen-specific CD8(+) T cell responses, as measured by antigen-specific tetramer analysis. Remarkably, TMV priming induced a greater magnitude T cell response than Adenovirus (Ad) priming. Finally, TMV is capable of boosting either Ad-induced or TMV-induced antigen-specific T cell responses, demonstrating that TMV, uniquely, does not induce neutralizing self-immunity. Overall, this study elucidates the in vivo DC delivery and activation properties of TMV and indicates its potential as a vaccine vector in stand alone or prime-boost strategies.


Assuntos
Apresentação do Antígeno , Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/imunologia , Vírus do Mosaico do Tabaco/imunologia , Adenoviridae/imunologia , Animais , Células Dendríticas/metabolismo , Feminino , Linfonodos/imunologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Baço/imunologia
11.
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
12.
Cancer Res ; 74(2): 609-20, 2014 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-24305876

RESUMO

The reactive stroma surrounding tumor lesions performs critical roles ranging from supporting tumor cell proliferation to inducing tumorigenesis and metastasis. Therefore, it is critical to understand the cellular components and signaling control mechanisms that underlie the etiology of reactive stroma. Previous studies have individually implicated fibroblast growth factor receptor 1 (FGFR1) and canonical WNT/ß-catenin signaling in prostate cancer progression and the initiation and maintenance of a reactive stroma; however, both pathways are frequently found to be coactivated in cancer tissue. Using autochthonous transgenic mouse models for inducible FGFR1 (JOCK1) and prostate-specific and ubiquitously expressed inducible ß-catenin (Pro-Cat and Ubi-Cat, respectively) and bigenic crosses between these lines (Pro-Cat × JOCK1 and Ubi-Cat × JOCK1), we describe WNT-induced synergistic acceleration of FGFR1-driven adenocarcinoma, associated with a pronounced fibroblastic reactive stroma activation surrounding prostatic intraepithelial neoplasia (mPIN) lesions found both in in situ and reconstitution assays. Both mouse and human reactive stroma exhibited increased transforming growth factor-ß (TGF-ß) signaling adjacent to pathologic lesions likely contributing to invasion. Furthermore, elevated stromal TGF-ß signaling was associated with higher Gleason scores in archived human biopsies, mirroring murine patterns. Our findings establish the importance of the FGFR1-WNT-TGF-ß signaling axes as driving forces behind reactive stroma in aggressive prostate adenocarcinomas, deepening their relevance as therapeutic targets.


Assuntos
Neoplasias da Próstata/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Proteínas Wnt/metabolismo , Adenocarcinoma/metabolismo , Animais , Biópsia , Linhagem Celular Tumoral , Proliferação de Células , Transformação Celular Neoplásica , Modelos Animais de Doenças , Fibroblastos/metabolismo , Humanos , Inflamação , Masculino , Camundongos , Camundongos Nus , Camundongos Transgênicos , Transdução de Sinais , Especificidade da Espécie , Células Estromais/metabolismo
13.
Mol Ther Methods Clin Dev ; 1: 14053, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-26052521

RESUMO

Induced pluripotent stem cell (iPSC) therapies offer a promising path for patient-specific regenerative medicine. However, tumor formation from residual undifferentiated iPSC or transformation of iPSC or their derivatives is a risk. Inclusion of a suicide gene is one approach to risk mitigation. We introduced a dimerizable-"inducible caspase-9" (iCasp9) suicide gene into mouse iPSC (miPSC) and rhesus iPSC (RhiPSC) via a lentivirus, driving expression from either a cytomegalovirus (CMV), elongation factor-1 α (EF1α) or pluripotency-specific EOS-C(3+) promoter. Exposure of the iPSC to the synthetic chemical dimerizer, AP1903, in vitro induced effective apoptosis in EF1α-iCasp9-expressing (EF1α)-iPSC, with less effective killing of EOS-C(3+)-iPSC and CMV-iPSC, proportional to transgene expression in these cells. AP1903 treatment of EF1α-iCasp9 miPSC in vitro delayed or prevented teratomas. AP1903 administration following subcutaneous or intravenous delivery of EF1α-iPSC resulted in delayed teratoma progression but did not ablate tumors. EF1α-iCasp9 expression was downregulated during in vitro and in vivo differentiation due to DNA methylation at CpG islands within the promoter, and methylation, and thus decreased expression, could be reversed by 5-azacytidine treatment. The level and stability of suicide gene expression will be important for the development of suicide gene strategies in iPSC regenerative medicine.

14.
PLoS One ; 8(12): e82742, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24358223

RESUMO

Modification of T cells with chimeric antigen receptors (CAR) has emerged as a promising treatment modality for human malignancies. Integration of co-stimulatory domains into CARs can augment the activation and function of genetically targeted T cells against tumors. However, the potential for insertional mutagenesis and toxicities due to the infused cells have made development of safe methods for removing transferred cells an important consideration. We have genetically modified human T cells with a lentiviral vector to express a CD20-CAR containing both CD28 and CD137 co-stimulatory domains, a "suicide gene" relying on inducible activation of caspase 9 (iC9), and a truncated CD19 selectable marker. Rapid expansion (2000 fold) of the transduced T cells was achieved in 28 days after stimulation with artificial antigen presenting cells. Transduced T cells exhibited effective CD20-specific cytotoxic activity in vitro and in a mouse xenograft tumor model. Activation of the iC9 suicide switch resulted in efficient removal of transduced T cells both in vitro and in vivo. Our work demonstrates the feasibility and promise of this approach for treating CD20(+) malignancies in a safe and more efficient manner. A phase I clinical trial using this approach in patients with relapsed indolent B-NHL is planned.


Assuntos
Antígenos CD20/genética , Caspase 9/genética , Genes Transgênicos Suicidas , Imunoterapia Adotiva/métodos , Linfoma/terapia , Receptores de Antígenos de Linfócitos T/genética , Linfócitos T/transplante , Animais , Células Cultivadas , Humanos , Células Jurkat , Linfoma/genética , Linfoma/imunologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Células NIH 3T3 , Proteínas Recombinantes de Fusão/genética , Linfócitos T/metabolismo , Resultado do Tratamento , Ensaios Antitumorais Modelo de Xenoenxerto
15.
Cell Stem Cell ; 11(5): 676-88, 2012 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-23122291

RESUMO

The role of Notch signaling in the maintenance of adult murine prostate epithelial homeostasis remains unclear. We found that Notch ligands are mainly expressed within the basal cell lineage, while active Notch signaling is detected in both the prostate basal and luminal cell lineages. Disrupting the canonical Notch effector Rbp-j impairs the differentiation of prostate basal stem cells and increases their proliferation in vitro and in vivo, but does not affect luminal cell biology. Conversely, ectopic Notch activation in adult prostates results in a decrease in basal cell number and luminal cell hyperproliferation. TGFß dominates over Notch signaling and overrides Notch ablation-induced proliferation of prostate basal cells. However, Notch confers sensitivity and positive feedback by upregulating a plethora of TGFß signaling components including TgfßR1. These findings reveal crucial roles of the self-enforced positive reciprocal regulatory loop between TGFß and Notch in maintaining prostate basal stem cell dormancy.


Assuntos
Próstata/citologia , Receptores Notch/metabolismo , Células-Tronco/citologia , Fator de Crescimento Transformador beta/metabolismo , Animais , Diferenciação Celular , Proliferação de Células , Masculino , Camundongos , Microscopia Eletrônica de Transmissão e Varredura , Próstata/metabolismo , Transdução de Sinais , Células-Tronco/metabolismo
16.
Oncoimmunology ; 1(3): 362-363, 2012 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-22737615

RESUMO

To better control the "licensing" of pro-Th1 dendritic cells (DCs), Spencer and colleagues have developed a synthetic ligand-inducible chimeric receptor, iMyD88/CD40 (iMC), incorporating synergistic Toll-like receptor (TLR) and costimulatory signaling elements, permitting DC regulation in vivo within the context of an immunological synapse. This novel technology results in potent anti-cancer activity.

17.
Mol Ther ; 20(7): 1462-71, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22434138

RESUMO

Recent modest successes in ex vivo dendritic cell (DC) immunotherapy have motivated continued innovation in the area of DC manipulation and activation. Although ex vivo vaccine approaches continue to be proving grounds for new DC manipulation techniques, the intrinsic limits of ex vivo therapy, including high cost, minimal standardization, cumbersome delivery, and poor accessibility, incentivizes the development of vaccines compatible with in vivo DC targeting. We describe here a method to co-deliver both tumor-specific antigen (TSA) and an iMyD88/CD40 adjuvant (iMC), to DCs that combines toll-like receptor (TLR) and CD40 signaling. In this study, we demonstrate that simple TSA delivery via adenoviral vectors results in strong antitumor immunity. Addition of iMC delivered in a separate vector is insufficient to enhance this effect. However, when delivered simultaneously with TSA in a single bicistronic vector (BV), iMC is able to significantly enhance antigen-specific cytotoxic T-cell (CTL) responses and inhibit established tumor growth. This study demonstrates the spatial-temporal importance of concurrent DC activation and TSA presentation. Further, it demonstrates the feasibility of in vivo molecular enhancement of DCs necessary for effective antitumor immune responses.


Assuntos
Antígenos de Neoplasias/imunologia , Antígenos CD40/imunologia , Citotoxicidade Imunológica , Células Dendríticas/imunologia , Melanoma Experimental/terapia , Fator 88 de Diferenciação Mieloide/imunologia , Linfócitos T Citotóxicos/imunologia , Adjuvantes Imunológicos , Animais , Antígenos de Neoplasias/biossíntese , Antígenos de Neoplasias/genética , Antígenos CD40/metabolismo , Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/metabolismo , Dependovirus , Feminino , Imunoterapia , Interleucina-12/metabolismo , Ativação Linfocitária , Melanoma Experimental/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Fator 88 de Diferenciação Mieloide/biossíntese
18.
PLoS One ; 7(1): e30814, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22303459

RESUMO

Wnt signaling is crucial for a variety of biological processes, including body axis formation, planar polarity, stem cell maintenance and cellular differentiation. Therefore, targeted manipulation of Wnt signaling in vivo would be extremely useful. By applying chemical inducer of dimerization (CID) technology, we were able to modify the Wnt co-receptor, low-density lipoprotein (LDL)-receptor-related protein 5 (LRP5), to generate the synthetic ligand inducible Wnt switch, iLRP5. We show that iLRP5 oligomerization results in its localization to disheveled-containing punctate structures and sequestration of scaffold protein Axin, leading to robust ß-catenin-mediated signaling. Moreover, we identify a novel LRP5 cytoplasmic domain critical for its intracellular localization and casein kinase 1-dependent ß-catenin signaling. Finally, by utilizing iLRP5 as a Wnt signaling switch, we generated the Ubiquitous Activator of ß-catenin (Ubi-Cat) transgenic mouse line. The Ubi-Cat line allows for nearly ubiquitous expression of iLRP5 under control of the H-2K(b) promoter. Activation of iLRP5 in isolated prostate basal epithelial stem cells resulted in expansion of p63(+) cells and development of hyperplasia in reconstituted murine prostate grafts. Independently, iLRP5 induction in adult prostate stroma enhanced prostate tissue regeneration. Moreover, induction of iLRP5 in male Ubi-Cat mice resulted in prostate tumor progression over several months from prostate hyperplasia to adenocarcinoma. We also investigated iLRP5 activation in Ubi-Cat-derived mammary cells, observing that prolonged activation results in mammary tumor formation. Thus, in two distinct experimental mouse models, activation of iLRP5 results in disruption of tissue homeostasis, demonstrating the utility of iLRP5 as a novel research tool for determining the outcome of Wnt activation in a precise spatially and temporally determined fashion.


Assuntos
Homeostase , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Multimerização Proteica , Via de Sinalização Wnt , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sequência de Aminoácidos , Animais , Proteína Axina/metabolismo , Caseína Quinase I/metabolismo , Proteínas Desgrenhadas , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Feminino , Humanos , Espaço Intracelular/metabolismo , Masculino , Neoplasias Mamárias Animais/patologia , Microdomínios da Membrana/metabolismo , Camundongos , Dados de Sequência Molecular , Fosfoproteínas/metabolismo , Fosforilação , Próstata/patologia , Próstata/transplante , Ligação Proteica , Transporte Proteico , Células Estromais/metabolismo , Células Estromais/patologia , Relação Estrutura-Atividade , Proteínas Supressoras de Tumor/metabolismo , beta Catenina/metabolismo
19.
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
20.
J Gene Med ; 13(12): 680-91, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22009763

RESUMO

BACKGROUND: Previous studies have shown that Herpes Simplex Virus thymidine kinase (HSV-tk)/ganciclovir (GCV) comprised the most commonly used suicide gene therapy for prostate cancer, with modest results being obtained. However, novel suicide genes, such as Escherichia coli purine nucleoside phosphorylase (PNP), have been utilized to demonstrate more potent tumor killing and an enhanced bystander effect on local, non-expressing cells compared to HSV-tk. METHODS: PNP/fludarabine (Fludara®; fludarabine phosphate; Berlex Labs, Richmond, CA, USA) was deliveried by prostate-specific, rat probasin-based promoter, ARR2PB. After infection of various cell lines with ADV.ARR(2) PB-PNP and administration of androgen analog, R1881, expression of PNP mRNA was detected; in vivo, the antitumor effect of the ARR(2) PB-PNP/Fludara system was monitored and analyzed, as well as animal survival. RESULTS: After in vitro infection with ADV.ARR(2) PB-PNP (multiplicity of infection = 10), LNCaP cells were more sensitive to a lower concentration Fludara (LD(50) , approximately 0.1 µg/ml) in the presence of R1881. Furthermore, robust bystander effects after R1881/Fludara treatment were observed in LNCaP cells after infection with bicistronic vector ADV.ARR2PB/PNP-IRES-EGFP in contrast to a much weaker effect in cells treated with ADV.CMV-HSV-tk/GCV. In vivo, tumor size in the ADV.ARR2PB-PNP/Fludara treatment group was dramatically smaller than in the control groups, and the mice treated with our system had a significantly prolonged survival, with three of eight mice surviving up to the 160-day termination point, as well as no systemic toxicity. CONCLUSIONS: The ARR(2) PB-PNP/Fludara system induced massive tumor cell death and a prolonged life span without systemic cytotoxicity; therefore, it might be a more attractive strategy for suicide gene therapy of prostate cancer.


Assuntos
Genes Transgênicos Suicidas , Terapia Genética , Neoplasias da Próstata , Purina-Núcleosídeo Fosforilase/genética , Fosfato de Vidarabina/análogos & derivados , Animais , Arrestinas/genética , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Linhagem Celular Tumoral , Escherichia coli , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Genes Transgênicos Suicidas/genética , Vetores Genéticos , Proteínas de Fluorescência Verde/metabolismo , Humanos , Masculino , Metribolona/administração & dosagem , Camundongos , Regiões Promotoras Genéticas , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Neoplasias da Próstata/terapia , Purina-Núcleosídeo Fosforilase/uso terapêutico , Ratos , Fosfato de Vidarabina/uso terapêutico , beta-Arrestinas
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