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1.
Anal Chem ; 96(26): 10780-10790, 2024 Jul 02.
Artículo en Inglés | MEDLINE | ID: mdl-38889002

RESUMEN

This study introduces a T cell enrichment process, capitalizing on the size differences between activated and unactivated T cells to facilitate the isolation of activated, transducible T cells. By employing multidimensional double spiral (MDDS) inertial sorting, our approach aims to remove unactivated or not fully activated T cells post-activation, consequently enhancing the efficiency of chimeric antigen receptor (CAR) T cell manufacturing. Our findings reveal that incorporating a simple, label-free, and continuous MDDS sorting step yields a purer T cell population, exhibiting significantly enhanced viability and CAR-transducibility (with up to 85% removal of unactivated T cells and approximately 80% recovery of activated T cells); we found approximately 2-fold increase in CAR transduction efficiency for a specific sample, escalating from ∼10% to ∼20%, but this efficiency highly depends on the original T cell sample as MDDS sorting would be more effective for samples possessing a higher proportion of unactivated T cells. This new cell separation process could augment the efficiency, yield, and cost-effectiveness of CAR T cell manufacturing, potentially broadening the accessibility of this transformative therapy and contributing to improved patient outcomes.


Asunto(s)
Separación Celular , Activación de Linfocitos , Receptores Quiméricos de Antígenos , Linfocitos T , Linfocitos T/citología , Humanos , Receptores Quiméricos de Antígenos/metabolismo , Separación Celular/métodos , Técnicas Analíticas Microfluídicas/instrumentación , Inmunoterapia Adoptiva/métodos
2.
bioRxiv ; 2024 May 09.
Artículo en Inglés | MEDLINE | ID: mdl-38766252

RESUMEN

Chimeric antigen receptor therapies have demonstrated potent efficacy in treating B cell malignancies, but have yet to meaningfully translate to solid tumors. Here, we utilize our pooled screening platform, CARPOOL, to expedite the discovery of CARs with anti-tumor functions necessary for solid tumor efficacy. We performed selections in primary human T cells expressing a library of 1.3×10 6 3 rd generation CARs targeting IL13Rα2, a cancer testis antigen commonly expressed in glioblastoma. Selections were performed for cytotoxicity, proliferation, memory formation, and persistence upon repeated antigen challenge. Each enriched CAR robustly produced the phenotype for which it was selected, and one enriched CAR triggered potent cytotoxicity and long-term proliferation upon in vitro tumor rechallenge. It also showed significantly improved persistence and comparable antigen-specific tumor control in a microphysiological human in vitro model and a xenograft model of human glioblastoma. Taken together, this work demonstrates the utility of extending CARPOOL to diseases beyond hematological malignancies and represents the largest exploration of signaling combinations in human primary cells to date.

3.
bioRxiv ; 2024 May 02.
Artículo en Inglés | MEDLINE | ID: mdl-38746119

RESUMEN

The anti-tumor function of engineered T cells expressing chimeric antigen receptors (CARs) is dependent on signals transduced through intracellular signaling domains (ICDs). Different ICDs are known to drive distinct phenotypes, but systematic investigations into how ICD architectures direct T cell function-particularly at the molecular level-are lacking. Here, we use single-cell sequencing to map diverse signaling inputs to transcriptional outputs, focusing on a defined library of clinically relevant ICD architectures. Informed by these observations, we functionally characterize transcriptionally distinct ICD variants across various contexts to build comprehensive maps from ICD composition to phenotypic output. We identify a unique tonic signaling signature associated with a subset of ICD architectures that drives durable in vivo persistence and efficacy in liquid, but not solid, tumors. Our findings work toward decoding CAR signaling design principles, with implications for the rational design of next-generation ICD architectures optimized for in vivo function.

5.
Nat Biomed Eng ; 6(7): 855-866, 2022 07.
Artículo en Inglés | MEDLINE | ID: mdl-35710755

RESUMEN

The immunostimulatory intracellular domains (ICDs) of chimaeric antigen receptors (CARs) are essential for converting antigen recognition into antitumoural function. Although there are many possible combinations of ICDs, almost all current CARs rely on combinations of CD3𝛇, CD28 and 4-1BB. Here we show that a barcoded library of 700,000 unique CD19-specific CARs with diverse ICDs cloned into lentiviral vectors and transduced into Jurkat T cells can be screened at high throughput via cell sorting and next-generation sequencing to optimize CAR signalling for antitumoural functions. By using this screening approach, we identified CARs with new ICD combinations that, compared with clinically available CARs, endowed human primary T cells with comparable tumour control in mice and with improved proliferation, persistence, exhaustion and cytotoxicity after tumour rechallenge in vitro. The screening strategy can be adapted to other disease models, cell types and selection conditions, and could be used to improve adoptive cell therapies and to expand their utility to new disease indications.


Asunto(s)
Neoplasias , Receptores de Antígenos de Linfocitos T/análisis , Receptores Quiméricos de Antígenos , Animales , Antígenos CD28/metabolismo , Humanos , Inmunoterapia Adoptiva , Ratones , Neoplasias/metabolismo , Receptores Quiméricos de Antígenos/genética , Receptores Quiméricos de Antígenos/metabolismo , Linfocitos T
6.
Cell Rep ; 35(1): 108960, 2021 04 06.
Artículo en Inglés | MEDLINE | ID: mdl-33826890

RESUMEN

The tumor microenvironment encompasses an intertwined ensemble of both transformed cancer cells and non-transformed host cells, which together establish a signaling network that regulates tumor progression. By conveying both homo- and heterotypic cell-to-cell communication cues, tumor-derived extracellular vesicles (tEVs) modulate several cancer-associated processes, such as immunosuppression, angiogenesis, invasion, and metastasis. Herein we discuss how recent methodological advances in the isolation and characterization of tEVs may help to broaden our understanding of their functions in tumor biology and, potentially, establish their utility as cancer biomarkers.


Asunto(s)
Vesículas Extracelulares/metabolismo , Neoplasias/metabolismo , Animales , Apoptosis , Biomarcadores/metabolismo , Humanos , Microfluídica , Modelos Biológicos
7.
Nat Commun ; 10(1): 5408, 2019 11 27.
Artículo en Inglés | MEDLINE | ID: mdl-31776331

RESUMEN

At the interface between the innate and adaptive immune system, dendritic cells (DCs) play key roles in tumour immunity and hold a hitherto unrealized potential for cancer immunotherapy. Here we review the role of distinct DC subsets in the tumour microenvironment, with special emphasis on conventional type 1 DCs. Integrating new knowledge of DC biology and advancements in cell engineering, we provide a blueprint for the rational design of optimized DC vaccines for personalized cancer medicine.


Asunto(s)
Vacunas contra el Cáncer/inmunología , Vacunas contra el Cáncer/farmacología , Células Dendríticas/inmunología , Inmunoterapia/métodos , Animales , Vacunas contra el Cáncer/genética , Técnicas de Cultivo de Célula , Diferenciación Celular , Separación Celular/métodos , Células Dendríticas/fisiología , Humanos , Neoplasias/inmunología , Linfocitos T/inmunología
8.
Adv Healthc Mater ; 8(2): e1801188, 2019 01.
Artículo en Inglés | MEDLINE | ID: mdl-30549244

RESUMEN

Biomaterial properties that modulate T cell activation, growth, and differentiation are of significant interest in the field of cellular immunotherapy manufacturing. In this work, a new platform technology that allows for the modulation of various activation particle design parameters important for polyclonal T cell activation is presented. Artificial antigen presenting cells (aAPCs) are successfully created using supported lipid bilayers on various cell-templated silica microparticles with defined membrane fluidity and stimulating antibody density. This panel of aAPCs is used to probe the importance of activation particle shape, size, membrane fluidity, and stimulation antibody density on T cell outgrowth and differentiation. All aAPC formulations are able to stimulate T cell growth, and preferentially promote CD8+ T cell growth over CD4+ T cell growth when compared to commercially available pendant antibody-conjugated particles. T cells cultured with HeLa- and red blood cell-templated aAPCs have a less-differentiated and less-exhausted phenotype than those cultured with spherical aAPCs with matched membrane coatings when cultured for 14 days. These results support continued exploration of silica-supported lipid bilayers as an aAPC platform.


Asunto(s)
Células Presentadoras de Antígenos/citología , Técnicas de Cultivo de Célula/instrumentación , Técnicas de Cultivo de Célula/métodos , Membrana Dobles de Lípidos/química , Activación de Linfocitos , Anticuerpos , Células Presentadoras de Antígenos/fisiología , Linfocitos T CD4-Positivos/citología , Linfocitos T CD8-positivos/citología , Diferenciación Celular , Eritrocitos/citología , Células HeLa , Humanos , Tamaño de la Partícula , Prueba de Estudio Conceptual , Dióxido de Silicio
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