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1.
J Immunother Cancer ; 11(3)2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36918221

RESUMO

BACKGROUND: Chimeric antigen receptor (CAR) T-cell therapies have demonstrated transformational outcomes in the treatment of B-cell malignancies, but their widespread use is hindered by technical and logistical challenges associated with ex vivo cell manufacturing. To overcome these challenges, we developed VivoVec, a lentiviral vector-based platform for in vivo engineering of T cells. UB-VV100, a VivoVec clinical candidate for the treatment of B-cell malignancies, displays an anti-CD3 single-chain variable fragment (scFv) on the surface and delivers a genetic payload that encodes a second-generation CD19-targeted CAR along with a rapamycin-activated cytokine receptor (RACR) system designed to overcome the need for lymphodepleting chemotherapy in supporting successful CAR T-cell expansion and persistence. In the presence of exogenous rapamycin, non-transduced immune cells are suppressed, while the RACR system in transduced cells converts rapamycin binding to an interleukin (IL)-2/IL-15 signal to promote proliferation. METHODS: UB-VV100 was administered to peripheral blood mononuclear cells (PBMCs) from healthy donors and from patients with B-cell malignancy without additional stimulation. Cultures were assessed for CAR T-cell transduction and function. Biodistribution was evaluated in CD34-humanized mice and in canines. In vivo efficacy was evaluated against normal B cells in CD34-humanized mice and against systemic tumor xenografts in PBMC-humanized mice. RESULTS: In vitro, administration of UB-VV100 resulted in dose-dependent and anti-CD3 scFv-dependent T-cell activation and CAR T-cell transduction. The resulting CAR T cells exhibited selective expansion in rapamycin and antigen-dependent activity against malignant B-cell targets. In humanized mouse and canine studies, UB-VV100 demonstrated a favorable biodistribution profile, with transduction events limited to the immune compartment after intranodal or intraperitoneal administration. Administration of UB-VV100 to humanized mice engrafted with B-cell tumors resulted in CAR T-cell transduction, expansion, and elimination of systemic malignancy. CONCLUSIONS: These findings demonstrate that UB-VV100 generates functional CAR T cells in vivo, which could expand patient access to CAR T technology in both hematological and solid tumors without the need for ex vivo cell manufacturing.


Assuntos
Receptores de Antígenos Quiméricos , Linfócitos T , Humanos , Animais , Cães , Camundongos , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos de Linfócitos T , Leucócitos Mononucleares , Distribuição Tecidual , Engenharia Celular/métodos
2.
Cell Rep ; 34(12): 108888, 2021 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-33761349

RESUMO

During development, progenitors often differentiate many cell generations after receiving signals. These delays must be robust yet tunable for precise population size control. Polycomb repressive mechanisms, involving histone H3 lysine-27 trimethylation (H3K27me3), restrain the expression of lineage-specifying genes in progenitors and may delay their activation and ensuing differentiation. Here, we elucidate an epigenetic switch controlling the T cell commitment gene Bcl11b that holds its locus in a heritable inactive state for multiple cell generations before activation. Integrating experiments and modeling, we identify a mechanism where H3K27me3 levels at Bcl11b, regulated by methyltransferase and demethylase activities, set the time delay at which the locus switches from a compacted, silent state to an extended, active state. This activation delay robustly spans many cell generations, is tunable by chromatin modifiers and transcription factors, and is independent of cell division. With their regulatory flexibility, such timed epigenetic switches may broadly control timing in development.


Assuntos
Divisão Celular/genética , Proteínas do Grupo Polycomb/metabolismo , Ativação Transcricional/genética , Animais , Linhagem da Célula/genética , Epigênese Genética , Loci Gênicos , Histonas/metabolismo , Humanos , Lisina/metabolismo , Metilação , Camundongos Endogâmicos C57BL , Conformação Proteica , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Linfócitos T/citologia , Fatores de Tempo , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/metabolismo
3.
Elife ; 72018 11 20.
Artigo em Inglês | MEDLINE | ID: mdl-30457103

RESUMO

Cell fate decisions occur through the switch-like, irreversible activation of fate-specifying genes. These activation events are often assumed to be tightly coupled to changes in upstream transcription factors, but could also be constrained by cis-epigenetic mechanisms at individual gene loci. Here, we studied the activation of Bcl11b, which controls T-cell fate commitment. To disentangle cis and trans effects, we generated mice where two Bcl11b copies are tagged with distinguishable fluorescent proteins. Quantitative live microscopy of progenitors from these mice revealed that Bcl11b turned on after a stochastic delay averaging multiple days, which varied not only between cells but also between Bcl11b alleles within the same cell. Genetic perturbations, together with mathematical modeling, showed that a distal enhancer controls the rate of epigenetic activation, while a parallel Notch-dependent trans-acting step stimulates expression from activated loci. These results show that developmental fate transitions can be controlled by stochastic cis-acting events on individual loci.


Assuntos
Diferenciação Celular , Epigênese Genética , Proteínas Repressoras/biossíntese , Linfócitos T/fisiologia , Transcrição Gênica , Proteínas Supressoras de Tumor/biossíntese , Animais , Genes Reporter , Microscopia Intravital , Proteínas Luminescentes/análise , Proteínas Luminescentes/genética , Camundongos , Modelos Teóricos , Coloração e Rotulagem , Fatores de Tempo
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