RESUMEN
Adoptive cell therapy using chimeric antigen receptor (CAR) T cells has proven to be lifesaving for many cancer patients. However, its therapeutic efficacy has been limited in solid tumors. One key factor for this is cancer-associated fibroblasts (CAFs) that modulate the tumor microenvironment (TME) to inhibit T cell infiltration and induce "T cell dysfunction." Additionally, the sparsity of tumor-specific antigens (TSA) and expression of CAR-directed tumor-associated antigens (TAA) on normal tissues often results in "on-target off-tumor" cytotoxicity, raising safety concerns. Using TALEN-mediated gene editing, we present here an innovative CAR T cell engineering strategy to overcome these challenges. Our allogeneic "Smart CAR T cells" are designed to express a constitutive CAR, targeting FAP+ CAFs in solid tumors. Additionally, a second CAR targeting a TAA such as mesothelin is specifically integrated at a TCR signaling-inducible locus like PDCD1. FAPCAR-mediated CAF targeting induces expression of the mesothelin CAR, establishing an IF/THEN-gated circuit sensitive to dual antigen sensing. Using this approach, we observe enhanced anti-tumor cytotoxicity, while limiting "on-target off-tumor" toxicity. Our study thus demonstrates TALEN-mediated gene editing capabilities for design of allogeneic IF/THEN-gated dual CAR T cells that efficiently target immunotherapy-recalcitrant solid tumors while mitigating potential safety risks, encouraging clinical development of this strategy.
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Chimeric antigen receptor T-cell (CAR T-cell) therapy has been shown to be clinically effective for managing a variety of hematological cancers. However, CAR T-cell therapy is associated with multiple adverse effects, including neurotoxicity and cytokine release syndrome (CRS). CRS arises from massive cytokine secretion and can be life-threatening, but it is typically managed with an anti-IL-6Ra mAb or glucocorticoid administration. However, these treatments add to a patient's medication burden and address only the CRS symptoms. Therefore, alternative strategies that can prevent CRS and neurotoxicity associated with CAR T-cell treatment are urgently needed. Here, we explored a therapeutic route aimed at preventing CRS rather than limiting its consequences. Using a cytokine-profiling assay, we show that granulocyte-macrophage colony-stimulating factor (GMCSF) is a key CRS-promoting protein. Through a combination of in vitro experiments and gene-editing technology, we further demonstrate that antibody-mediated neutralization or TALEN-mediated genetic inactivation of GMCSF in CAR T-cells drastically decreases available GMCSF and abolishes macrophage-dependent secretion of CRS biomarkers, including monocyte chemoattractant protein 1 (MCP-1), interleukin (IL) 6, and IL-8. Of note, we also found that the genetic inactivation of GMCSF does not impair the antitumor function or proliferative capacity of CAR T-cells in vitro We conclude that it is possible to prevent CRS by using "all-in-one" GMCSF-knockout CAR T-cells. This approach may eliminate the need for anti-CRS treatment and may improve the overall safety of CAR T-cell therapies for cancer patients.
Asunto(s)
Citocinas/inmunología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/inmunología , Neoplasias Hematológicas/inmunología , Neoplasias Hematológicas/terapia , Inmunoterapia Adoptiva , Monocitos , Proteínas de Neoplasias/inmunología , Antineoplásicos Inmunológicos/inmunología , Antineoplásicos Inmunológicos/farmacología , Citocinas/genética , Edición Génica , Técnicas de Silenciamiento del Gen , Glucocorticoides/farmacología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/patología , Humanos , Monocitos/inmunología , Monocitos/patología , Proteínas de Neoplasias/genética , Receptores Quiméricos de Antígenos/genética , Receptores Quiméricos de Antígenos/inmunologíaRESUMEN
BACKGROUND: Engineered therapeutic cells have attracted a great deal of interest due to their potential applications in treating a wide range of diseases, including cancer and autoimmunity. Chimeric antigen receptor (CAR) T-cells are designed to detect and kill tumor cells that present a specific, predefined antigen. The rapid expansion of targeted antigen beyond CD19, has highlighted new challenges, such as autoactivation and T-cell fratricide, that could impact the capacity to manufacture engineered CAR T-cells. Therefore, the development of strategies to control CAR expression at the surface of T-cells and their functions is under intense investigations. RESULTS: Here, we report the development and evaluation of an off-switch directly embedded within a CAR construct (SWIFF-CAR). The incorporation of a self-cleaving degradation moiety controlled by a protease/protease inhibitor pair allowed the ex vivo tight and reversible control of the CAR surface presentation and the subsequent CAR-induced signaling and cytolytic functions of the engineered T-cells using the cell permeable Asunaprevir (ASN) small molecule. CONCLUSIONS: The strategy described in this study could, in principle, be broadly adapted to CAR T-cells development to circumvent some of the possible hurdle of CAR T-cell manufacturing. This system essentially creates a CAR T-cell with an integrated functional rheostat.
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Antígenos CD19/inmunología , Expresión Génica/inmunología , Receptores Quiméricos de Antígenos/inmunología , Linfocitos T/inmunología , Antígenos CD19/genética , Antígenos CD19/metabolismo , Línea Celular Tumoral , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Expresión Génica/efectos de los fármacos , Expresión Génica/genética , Humanos , Isoquinolinas/farmacología , Inhibidores de Proteasas/farmacología , Receptores Quiméricos de Antígenos/genética , Receptores Quiméricos de Antígenos/metabolismo , Sulfonamidas/farmacología , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismoRESUMEN
The adoptive transfer of chimeric antigen receptor (CAR) T cell represents a highly promising strategy to fight against multiple cancers. The clinical outcome of such therapies is intimately linked to the ability of effector cells to engraft, proliferate, and specifically kill tumor cells within patients. When allogeneic CAR T-cell infusion is considered, host versus graft and graft versus host reactions must be avoided to prevent rejection of adoptively transferred cells, host tissue damages and to elicit significant antitumoral outcome. This work proposes to address these three requirements through the development of multidrug-resistant T cell receptor αß-deficient CAR T cells. We demonstrate that these engineered T cells displayed efficient antitumor activity and proliferated in the presence of purine and pyrimidine nucleoside analogues, currently used in clinic as preconditioning lymphodepleting regimens. The absence of TCRαß at their cell surface along with their purine nucleotide analogues-resistance properties could prevent their alloreactivity and enable them to resist to lymphodepleting regimens that may be required to avoid their ablation via HvG reaction. By providing a basic framework to develop a universal T cell compatible with allogeneic adoptive transfer, this work is laying the foundation stone of the large-scale utilization of CAR T-cell immunotherapies.
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Tratamiento Basado en Trasplante de Células y Tejidos , Resistencia a Múltiples Medicamentos/genética , Inmunoterapia Adoptiva , Receptores de Antígenos de Linfocitos T/genética , Proteínas Recombinantes de Fusión/genética , Linfocitos T/inmunología , Linfocitos T/metabolismo , Antígenos CD19/genética , Antineoplásicos/administración & dosificación , Antineoplásicos/farmacología , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Terapia Combinada , Citotoxicidad Inmunológica , Desoxicitidina Quinasa/deficiencia , Desoxicitidina Quinasa/genética , Expresión Génica , Silenciador del Gen , Humanos , Inmunoterapia Adoptiva/efectos adversos , Inmunoterapia Adoptiva/métodos , Concentración 50 Inhibidora , Activación de Linfocitos/genética , Activación de Linfocitos/inmunología , Prueba de Cultivo Mixto de Linfocitos , Neoplasias/genética , Neoplasias/inmunología , Neoplasias/terapia , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores de Antígenos de Linfocitos T alfa-beta/deficiencia , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Proteínas Recombinantes de Fusión/metabolismo , Linfocitos T/efectos de los fármacos , Trasplante HomólogoRESUMEN
The ability to specifically engineer the genome of living cells at precise locations using rare-cutting designer endonucleases has broad implications for biotechnology and medicine, particularly for functional genomics, transgenics and gene therapy. However, the potential impact of chromosomal context and epigenetics on designer endonuclease-mediated genome editing is poorly understood. To address this question, we conducted a comprehensive analysis on the efficacy of 37 endonucleases derived from the quintessential I-CreI meganuclease that were specifically designed to cleave 39 different genomic targets. The analysis revealed that the efficiency of targeted mutagenesis at a given chromosomal locus is predictive of that of homologous gene targeting. Consequently, a strong genome-wide correlation was apparent between the efficiency of targeted mutagenesis (≤ 0.1% to ≈ 6%) with that of homologous gene targeting (≤ 0.1% to ≈ 15%). In contrast, the efficiency of targeted mutagenesis or homologous gene targeting at a given chromosomal locus does not correlate with the activity of individual endonucleases on transiently transfected substrates. Finally, we demonstrate that chromatin accessibility modulates the efficacy of rare-cutting endonucleases, accounting for strong position effects. Thus, chromosomal context and epigenetic mechanisms may play a major role in the efficiency rare-cutting endonuclease-induced genome engineering.
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Efectos de la Posición Cromosómica , Enzimas de Restricción del ADN/metabolismo , Animales , Células CHO , Línea Celular , Cricetinae , Cricetulus , Enzimas de Restricción del ADN/química , Marcación de Gen , Ingeniería Genética , Genoma Humano , Humanos , MutagénesisRESUMEN
Solid tumors, such as triple-negative breast cancer (TNBC), are biologically complex due to cellular heterogeneity, lack of tumor-specific antigens, and an immunosuppressive tumor microenvironment (TME). These challenges restrain chimeric antigen receptor (CAR) T cell efficacy, underlining the importance of armoring. In solid cancers, a localized tumor mass allows alternative administration routes, such as intratumoral delivery with the potential to improve efficacy and safety but may compromise metastatic-site treatment. Using a multi-layered CAR T cell engineering strategy that allowed a synergy between attributes, we show enhanced cytotoxic activity of MUC1 CAR T cells armored with PD1KO, tumor-specific interleukin-12 release, and TGFBR2KO attributes catered towards the TNBC TME. Intratumoral treatment effectively reduced distant tumors, suggesting retention of antigen-recognition benefits at metastatic sites. Overall, we provide preclinical evidence of armored non-alloreactive MUC1 CAR T cells greatly reducing high TNBC tumor burden in a TGFB1- and PD-L1-rich TME both at local and distant sites while preserving safety.
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Inmunoterapia Adoptiva , Mucina-1 , Receptores Quiméricos de Antígenos , Neoplasias de la Mama Triple Negativas , Microambiente Tumoral , Mucina-1/metabolismo , Mucina-1/genética , Mucina-1/inmunología , Neoplasias de la Mama Triple Negativas/terapia , Neoplasias de la Mama Triple Negativas/patología , Neoplasias de la Mama Triple Negativas/inmunología , Neoplasias de la Mama Triple Negativas/metabolismo , Humanos , Femenino , Animales , Receptores Quiméricos de Antígenos/inmunología , Receptores Quiméricos de Antígenos/metabolismo , Receptores Quiméricos de Antígenos/genética , Ratones , Inmunoterapia Adoptiva/métodos , Línea Celular Tumoral , Linfocitos T/inmunología , Linfocitos T/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Integrative gene transfer is widely used for bioproduction, drug screening, and therapeutic applications but usual viral methods lead to random and multicopy insertions, contribute to unstable transgene expression and can disturb endogenous gene expression. Homologous targeting of an expression cassette using rare-cutting endonucleases is a potential solution; however the number of studied loci remains limited. Furthermore, the behavior and performance of various types of gene cassettes following gene targeting is poorly defined. Here we have evaluated three loci for gene targeting, including one locus compatible with the proposed Safe Harbor criteria for human translational applications. Using optimized conditions for homologous gene targeting, reporter genes under the control of different promoters were efficiently inserted at each locus in both sense and antisense orientations. Sustainable expression was achieved at all three loci without detectable disturbance of flanking gene expression. However, the promoter, the integration locus and the cassette orientation have a strong impact on transgene expression. Finally, single targeted integrations exhibited greatly improved transgene expression stability versus multicopy or random integration. Taken together, our data suggest a potential set of loci for site-specific transgene integration, suitable for a variety of biotechnological applications.
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Expresión Génica , Marcación de Gen , Mutagénesis Insercional/métodos , Transgenes , Biotecnología/métodos , Línea Celular , Genes Reporteros , Inestabilidad Genómica , Humanos , Regiones Promotoras GenéticasRESUMEN
Adoptive cell therapy based on chimeric antigen receptor (CAR)-engineered T-cells has proven to be lifesaving for many cancer patients. However, its therapeutic efficacy has so far been restricted to only a few malignancies, with solid tumors proving to be especially recalcitrant to efficient therapy. Poor intra-tumor infiltration by T cells and T cell dysfunction due to a desmoplastic, immunosuppressive microenvironment are key barriers for CAR T-cell success against solid tumors. Cancer-associated fibroblasts (CAFs) are critical components of the tumor stroma, evolving specifically within the tumor microenvironment (TME) in response to tumor cell cues. The CAF secretome is a significant contributor to the extracellular matrix and a plethora of cytokines and growth factors that induce immune suppression. Together they form a physical and chemical barrier which induces a T cell-excluding 'cold' TME. CAF depletion in stroma rich solid tumors can thus provide an opportunity to convert immune evasive tumors susceptible to tumor-antigen CAR T-cell cytotoxicity. Using our TALEN-based gene editing platform we engineered non-alloreactive, immune evasive CAR T-cells (termed UCAR T-cells) targeting the unique CAF marker Fibroblast Activation Protein, alpha (FAP). In an orthotopic mouse model of triple-negative breast cancer (TNBC) composed of patient derived-CAFs and tumor cells, we demonstrate the efficacy of our engineered FAP UCAR T-cells in CAF depletion, reduction of desmoplasia and successful tumor infiltration. Furthermore, while previously resistant, pre-treatment with FAP UCAR T-cells now sensitized these tumors to Mesothelin (Meso) UCAR T-cell infiltration and anti-tumor cytotoxicity. Combination therapy of FAP UCAR, Meso UCAR T cells and the checkpoint inhibitor anti-PD-1 significantly reduced tumor burden and prolonged mice survival. Our study thus proposes a novel treatment paradigm for successful CAR T-cell immunotherapy against stroma-rich solid tumors.
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Receptores Quiméricos de Antígenos , Animales , Ratones , Receptores Quiméricos de Antígenos/genética , Receptores Quiméricos de Antígenos/metabolismo , Nucleasas de los Efectores Tipo Activadores de la Transcripción/metabolismo , Inmunoterapia , Linfocitos T , Antígenos de NeoplasiasRESUMEN
Despite the remarkable success of autologous chimeric antigen receptor (CAR) T cells, some patients relapse due to tumor antigen escape and low or uneven antigen expression, among other mechanisms. Therapeutic options after relapse are limited, emphasizing the need to optimize current approaches. In addition, there is a need to develop allogeneic "off-the-shelf" therapies from healthy donors that are readily available at the time of treatment decision and can overcome limitations of current autologous approaches. To address both challenges simultaneously, we generated a CD20xCD22 dual allogeneic CAR T cell. Herein, we demonstrate that allogeneic CD20x22 CAR T cells display robust, sustained and dose-dependent activity in vitro and in vivo, while efficiently targeting primary B-cell non-Hodgkin lymphoma (B-NHL) samples with heterogeneous levels of CD22 and CD20. Altogether, we provide preclinical proof-of-concept data for an allogeneic dual CAR T cell to overcome current mechanisms of resistance to CAR T-cell therapies in B-NHL, while providing a potential alternative to CD19 targeting.
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Trasplante de Células Madre Hematopoyéticas , Linfoma de Células B , Humanos , Receptores de Antígenos de Linfocitos T , Recurrencia Local de Neoplasia , Linfocitos T , Linfocitos B , Inmunoterapia Adoptiva , Antígenos CD19RESUMEN
The development of gene editing technologies over the past years has allowed the precise and efficient insertion of transgenes into the genome of various cell types. Knock-in approaches using homology-directed repair and designer nucleases often rely on viral vectors, which can considerably impact the manufacturing cost and timeline of gene-edited therapeutic products. An attractive alternative would be to use naked DNA as a repair template. However, such a strategy faces challenges such as cytotoxicity from double-stranded DNA (dsDNA) to primary cells. Here, we sought to study the kinetics of transcription activator-like effector nuclease (TALEN)-mediated gene editing in primary T cells to improve nonviral gene knock-in. Harnessing this knowledge, we developed a rapid and efficient gene insertion strategy based on either short single-stranded oligonucleotides or large (2 Kb) linear naked dsDNA sequences. We demonstrated that a time-controlled two-step transfection protocol can substantially improve the efficiency of nonviral transgene integration in primary T cells. Using this approach, we achieved modification of up to Ë 30% of T cells when inserting a chimeric antigen receptor (CAR) at the T-cell receptor alpha constant region (TRAC) locus to generate 'off-the shelf' CAR-T cells.
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Edición Génica , Linfocitos T , Electroporación/métodos , Edición Génica/métodos , Mutagénesis Insercional , Linfocitos T/metabolismo , TransfecciónRESUMEN
Universal CAR T-cell therapies are poised to revolutionize cancer treatment and to improve patient outcomes. However, realizing these advantages in an allogeneic setting requires universal CAR T-cells that can kill target tumor cells, avoid depletion by the host immune system, and proliferate without attacking host tissues. Here, we describe the development of a novel immune-evasive universal CAR T-cells scaffold using precise TALEN-mediated gene editing and DNA matrices vectorized by recombinant adeno-associated virus 6. We simultaneously disrupt and repurpose the endogenous TRAC and B2M loci to generate TCRαß- and HLA-ABC-deficient T-cells expressing the CAR construct and the NK-inhibitor named HLA-E. This highly efficient gene editing process enables the engineered T-cells to evade NK cell and alloresponsive T-cell attacks and extend their persistence and antitumor activity in the presence of cytotoxic levels of NK cell in vivo and in vitro, respectively. This scaffold could enable the broad use of universal CAR T-cells in allogeneic settings and holds great promise for clinical applications.
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Edición Génica , Nucleasas de los Efectores Tipo Activadores de la Transcripción , Humanos , Inmunoterapia Adoptiva , Receptores de Antígenos de Linfocitos T/genética , Linfocitos TRESUMEN
The prelude to type-1 diabetes is leukocyte infiltration into the pancreatic islets, or insulitis. This process begins in pancreatic lymph nodes when T lymphocytes reactive to islet beta cells encounter antigen-presenting cells (APCs) displaying peptides derived from beta cell proteins. We show here that a ripple of physiological beta cell death, which occurs at 2 wk of age in all mouse strains, precipitates the arrival of such APCs, and that the relevant APC is a dendritic cell of CD11c+CD11b+CD8alpha- phenotype. These findings have significant implications concerning the nature of the diabetes-provoking deficits in NOD mice, the identity of the primordial diabetogenic antigens, and our understanding of the balance between immunity and tolerance in a pathological context.
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Células Dendríticas/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Islotes Pancreáticos/metabolismo , Linfocitos T/metabolismo , Animales , Linfocitos B , Muerte Celular/fisiología , Modelos Animales de Enfermedad , Linfocitos T/inmunologíaRESUMEN
Here, we developed a straightforward methodology to generate TCRαß negative (allogeneic) cells for CAR-T cell therapies. With an early and transient expression of an anti-CD3 CAR in the engineered donor T cells, we programmed these cells to self-eliminate the TCR+ cell population and obtained an ultrapure TCRαß- population (99-99.9%) at the end of the CAR-T production. This novel and easy-to-implement procedure preserves the production yield and cell fitness and has the potential to streamline the manufacturing of "off-the-shelf" CAR T-cell therapies.
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An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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Endowing chimeric antigen receptor (CAR) T cells with additional potent functionalities holds strong potential for improving their antitumor activity. However, because potency could be deleterious without control, these additional features need to be tightly regulated. Immune pathways offer a wide array of tightly regulated genes that can be repurposed to express potent functionalities in a highly controlled manner. Here, we explore this concept by repurposing TCR, CD25 and PD1, three major players of the T cell activation pathway. We insert the CAR into the TCRα gene (TRACCAR), and IL-12P70 into either IL2Rα or PDCD1 genes. This process results in transient, antigen concentration-dependent IL-12P70 secretion, increases TRACCAR T cell cytotoxicity and extends survival of tumor-bearing mice. This gene network repurposing strategy can be extended to other cellular pathways, thus paving the way for generating smart CAR T cells able to integrate biological inputs and to translate them into therapeutic outputs in a highly regulated manner.
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Sistema Inmunológico/metabolismo , Receptores Quiméricos de Antígenos/inmunología , Linfocitos T/inmunología , Animales , Línea Celular Tumoral , Edición Génica , Humanos , Interleucina-12/genética , Activación de Linfocitos/inmunología , Ratones , Neoplasias/inmunología , Neoplasias/patología , Receptores de Antígenos de Linfocitos T/metabolismo , Nucleasas de los Efectores Tipo Activadores de la Transcripción/metabolismoRESUMEN
Pyruvate Kinase Deficiency (PKD) is a rare erythroid metabolic disease caused by mutations in the PKLR gene, which encodes the erythroid specific Pyruvate Kinase enzyme. Erythrocytes from PKD patients show an energetic imbalance and are susceptible to hemolysis. Gene editing of hematopoietic stem cells (HSCs) would provide a therapeutic benefit and improve safety of gene therapy approaches to treat PKD patients. In previous studies, we established a gene editing protocol that corrected the PKD phenotype of PKD-iPSC lines through a TALEN mediated homologous recombination strategy. With the goal of moving toward more clinically relevant stem cells, we aim at editing the PKLR gene in primary human hematopoietic progenitors and hematopoietic stem cells (HPSCs). After nucleofection of the gene editing tools and selection with puromycin, up to 96% colony forming units showed precise integration. However, a low yield of gene edited HPSCs was associated to the procedure. To reduce toxicity while increasing efficacy, we worked on i) optimizing gene editing tools and ii) defining optimal expansion and selection times. Different versions of specific nucleases (TALEN and CRISPR-Cas9) were compared. TALEN mRNAs with 5' and 3' added motifs to increase RNA stability were the most efficient nucleases to obtain high gene editing frequency and low toxicity. Shortening ex vivo manipulation did not reduce the efficiency of homologous recombination and preserved the hematopoietic progenitor potential of the nucleofected HPSCs. Lastly, a very low level of gene edited HPSCs were detected after engraftment in immunodeficient (NSG) mice. Overall, we showed that gene editing of the PKLR gene in HPSCs is feasible, although further improvements must to be done before the clinical use of the gene editing to correct PKD.
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Edición Génica/métodos , Células Madre Hematopoyéticas/citología , Piruvato Quinasa/genética , Nucleasas de los Efectores Tipo Activadores de la Transcripción/genética , Regiones no Traducidas 3' , Regiones no Traducidas 5' , Animales , Células Cultivadas , Células HEK293 , Células Madre Hematopoyéticas/química , Humanos , RatonesRESUMEN
DEVELOPMENT OF CAR T-CELLS IN SOLID TUMORS: CHALLENGES AND PERSPECTIVES: While Chimeric Antigen Receptor (CAR) T-cells have shown outstanding results in some hematologic malignancies, studies in solid tumors are less encouraging with poor response rates. Several factors can account for this lack of efficiency in solid tumors: heterogeneous expression or absence of specific target antigen (and so higher risk of toxicity), immunosuppressive microenvironment, homing and tumoral trafficking issues or lack of CAR T-cell persistence. Different approaches can be considered to overcome these resistance mechanisms: bispecific CARs, use of logic gates, combination with immune checkpoint inhibitors, engineered CAR T-cells resistant to immunosuppressive molecules, addition of chemokines or enzymes, combination with oncolytic virus, intra-tumoral administration, selection of memory T cell subpopulations and development of armored CAR T-cells secreting cytokines such as IL-12, -15 or -18. Last generation optimized CAR T-cell design should thus improve therapeutic efficiency. CAR-T cells may represent in a near future a therapeutic breakthrough also in solid tumors, especially in cold tumors and/or tumors lacking MHC class I expression. Cet article fait partie du numéro supplément Les cellules CAR-T : une révolution thérapeutique ? réalisé avec le soutien institutionnel des partenaires Gilead : Kite et Celgene.
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Inmunoterapia Adoptiva/métodos , Neoplasias/terapia , Receptores Quiméricos de Antígenos , Especificidad de Anticuerpos/inmunología , Ensayos Clínicos como Asunto , Humanos , Interleucina-12/metabolismo , Interleucina-15/metabolismo , Interleucina-18/metabolismo , Neoplasias/inmunología , Receptores Quiméricos de Antígenos/inmunología , Receptores Mensajeros de Linfocitos/inmunología , Subgrupos de Linfocitos T/inmunología , Linfocitos T/inmunología , Resultado del Tratamiento , Microambiente Tumoral/inmunologíaRESUMEN
CAR T-cell therapies hold great promise for treating a range of malignancies but are however challenged by the complexity of their production and by the adverse events related to their activity. Here we report the development of the CubiCAR, a tri-functional CAR architecture that enables CAR T-cell detection, purification and on-demand depletion by the FDA-approved antibody Rituximab. This novel architecture has the potential to streamline the manufacturing of CAR T-cells, allow their tracking and improve their overall safety.
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Inmunoterapia Adoptiva , Neoplasias Experimentales/inmunología , Neoplasias Experimentales/cirugía , Receptores Quiméricos de Antígenos/inmunología , Rituximab/farmacología , Animales , Línea Celular Tumoral , Humanos , Ratones , Ratones Endogámicos BALB C , Neoplasias Experimentales/patologíaRESUMEN
To detect and characterize autoreactive T cells in diabetes-prone NOD mice, we have developed a multimeric MHC reagent with high affinity for the BDC-2.5 T cell receptor, which is reactive against a pancreatic autoantigen. A distinct population of T cells is detected in NOD mice that recognizes the same MHC/peptide target. These T cells are positively selected in the thymus at a surprisingly high frequency and exported to the periphery. They are activated specifically in the pancreatic LNs, demonstrating an autoimmune specificity that recapitulates that of the BDC-2.5 cell. These phenomena are also observed in mouse lines that share with NOD the H-2g7 MHC haplotype but carry diabetes-resistance background genes. Thus, a susceptible haplotype at the MHC seems to be the only element required for the selection and emergence of autoreactive T cells, without requiring other diabetogenic loci from the NOD genome.
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Alelos , Autoinmunidad , Linfocitos T CD4-Positivos/inmunología , Diabetes Mellitus Tipo 1/inmunología , Complejo Mayor de Histocompatibilidad/genética , Animales , Autoantígenos/inmunología , Linfocitos T CD4-Positivos/fisiología , Diabetes Mellitus Tipo 1/metabolismo , Femenino , Haplotipos , Hibridomas/citología , Hibridomas/inmunología , Hibridomas/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones Transgénicos , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Timo/citología , Timo/metabolismoRESUMEN
A key to the success of chimeric antigen receptor (CAR) T-cell based therapies greatly rely on the capacity to identify and target antigens with expression restrained to tumor cells. Here we present a strategy to generate CAR T-cells that are only effective locally (tumor tissue), potentially also increasing the choice of targetable antigens. By fusing an oxygen sensitive subdomain of HIF1α to a CAR scaffold, we generated CAR T-cells that are responsive to a hypoxic environment, a hallmark of certain tumors. Along with the development of oxygen-sensitive CAR T-cells, this work also provides a basic framework to use a multi-chain CAR as a platform to create the next generation of smarter self-decision making CAR T-cells.