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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 are 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 Tumor Associated Antigen (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 which efficiently target immunotherapy-recalcitrant solid tumors while mitigating potential safety risks, encouraging clinical development of this strategy.
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One of the most recent advances in the genome editing field has been the addition of "TALE Base Editors", an innovative platform for cell therapy that relies on the deamination of cytidines within double strand DNA, leading to the formation of an uracil (U) intermediate. These molecular tools are fusions of transcription activator-like effector domains (TALE) for specific DNA sequence binding, split-DddA deaminase halves that will, upon catalytic domain reconstitution, initiate the conversion of a cytosine (C) to a thymine (T), and an uracil glycosylase inhibitor (UGI). We developed a high throughput screening strategy capable to probe key editing parameters in a precisely defined genomic context in cellulo, excluding or minimizing biases arising from different microenvironmental and/or epigenetic contexts. Here we aimed to further explore how target composition and TALEB architecture will impact the editing outcomes. We demonstrated how the nature of the linker between TALE array and split DddAtox head allows us to fine tune the editing window, also controlling possible bystander activity. Furthermore, we showed that both the TALEB architecture and spacer length separating the two TALE DNA binding regions impact the target TC editing dependence by the surrounding bases, leading to more restrictive or permissive editing profiles.
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Citosina , Edición Génica , Timina , Edición Génica/métodos , Humanos , Citosina/metabolismo , Citosina/química , Timina/metabolismo , Timina/química , Efectores Tipo Activadores de la Transcripción/metabolismo , Efectores Tipo Activadores de la Transcripción/genética , ADN/metabolismo , ADN/genética , Células HEK293RESUMEN
Sickle cell disease is a devastating blood disorder that originates from a single point mutation in the HBB gene coding for hemoglobin. Here, we develop a GMP-compatible TALEN-mediated gene editing process enabling efficient HBB correction via a DNA repair template while minimizing risks associated with HBB inactivation. Comparing viral versus non-viral DNA repair template delivery in hematopoietic stem and progenitor cells in vitro, both strategies achieve comparable HBB correction and result in over 50% expression of normal adult hemoglobin in red blood cells without inducing ß-thalassemic phenotype. In an immunodeficient female mouse model, transplanted cells edited with the non-viral strategy exhibit higher engraftment and gene correction levels compared to those edited with the viral strategy. Transcriptomic analysis reveals that non-viral DNA repair template delivery mitigates P53-mediated toxicity and preserves high levels of long-term hematopoietic stem cells. This work paves the way for TALEN-based autologous gene therapy for sickle cell disease.
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Anemia de Células Falciformes , Edición Génica , Terapia Genética , Células Madre Hematopoyéticas , Nucleasas de los Efectores Tipo Activadores de la Transcripción , Anemia de Células Falciformes/terapia , Anemia de Células Falciformes/genética , Edición Génica/métodos , Animales , Células Madre Hematopoyéticas/metabolismo , Humanos , Femenino , Ratones , Terapia Genética/métodos , Nucleasas de los Efectores Tipo Activadores de la Transcripción/metabolismo , Nucleasas de los Efectores Tipo Activadores de la Transcripción/genética , Trasplante de Células Madre Hematopoyéticas , Globinas beta/genética , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Reparación del ADN , Mutación , Talasemia beta/terapia , Talasemia beta/genética , Modelos Animales de Enfermedad , Técnicas de Transferencia de GenRESUMEN
Gene therapy in hematopoietic stem and progenitor cells (HSPCs) shows great potential for the treatment of inborn metabolic diseases. Typical HSPC gene therapy approaches rely on constitutive promoters to express a therapeutic transgene, which is associated with multiple disadvantages. Here, we propose a novel promoterless intronic gene editing approach that triggers transgene expression only after cellular differentiation into the myeloid lineage. We integrated a splicing-competent eGFP cassette into the first intron of CD11b and observed expression of eGFP in the myeloid lineage but minimal to no expression in HSPCs or differentiated non-myeloid lineages. In vivo, edited HSPCs successfully engrafted in immunodeficient mice and displayed transgene expression in the myeloid compartment of multiple tissues. Using the same approach, we expressed alpha-L-iduronidase (IDUA), the defective enzyme in Mucopolysaccharidosis type I, and observed a 10-fold supraendogenous IDUA expression exclusively after myeloid differentiation. Edited cells efficiently populated bone marrow, blood, and spleen of immunodeficient mice, and retained the capacity to secrete IDUA ex vivo. Importantly, cells edited with the eGFP and IDUA transgenes were also found in the brain. This approach may unlock new therapeutic strategies for inborn metabolic and neurological diseases that require the delivery of therapeutics in brain.
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Edición Génica , Células Madre Hematopoyéticas , Intrones , Células Mieloides , Nucleasas de los Efectores Tipo Activadores de la Transcripción , Transgenes , Animales , Edición Génica/métodos , Ratones , Células Madre Hematopoyéticas/metabolismo , Humanos , Células Mieloides/metabolismo , Nucleasas de los Efectores Tipo Activadores de la Transcripción/genética , Nucleasas de los Efectores Tipo Activadores de la Transcripción/metabolismo , Diferenciación Celular/genética , Terapia Genética/métodos , Iduronidasa/genética , Iduronidasa/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Expresión Génica , Linaje de la Célula/genética , Antígeno CD11b/genética , Antígeno CD11b/metabolismo , Trasplante de Células Madre Hematopoyéticas/métodos , Mucopolisacaridosis I/terapia , Mucopolisacaridosis I/genéticaRESUMEN
Gain-of-function mutations in the PIK3CD gene result in activated phosphoinositide 3-kinase δ syndrome type 1 (APDS1). This syndrome is a life-threatening combined immunodeficiency and today there are neither optimal nor long-term therapeutic solutions for APDS1 patients. Thus, new alternative treatments are highly needed. The aim of the present study is to explore one therapeutic avenue that consists of the correction of the PIK3CD gene through gene editing. Our proof-of-concept shows that TALEN-mediated gene correction of the mutated PIK3CD gene in APDS1 T cells results in normalized phospho-AKT levels in basal and activated conditions. Normalization of PI3K signaling was correlated to restored cytotoxic functions of edited CD8+ T cells. At the transcriptomic level, single-cell RNA sequencing revealed corrected signatures of CD8+ effector memory and CD8+ proliferating T cells. This proof-of-concept study paves the way for the future development of a gene therapy candidate to cure activated phosphoinositide 3-kinase δ syndrome type 1.
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TALE base editors are a recent addition to the genome editing toolbox. These molecular tools are fusions of a transcription activator-like effector domain (TALE), split-DddA deaminase halves, and an uracil glycosylase inhibitor (UGI) that have the distinct ability to directly edit double strand DNA, converting a cytosine (C) to a thymine (T). To dissect the editing rules of TALE-BE, we combined the screening of dozens of TALE-BE targeting nuclear genomic loci with a medium/high throughput strategy based on precise knock-in of TALE-BE target site collections into the cell genome. This latter approach allowed us to gain in depth insight of the editing rules in cellulo, while excluding confounding factors such as epigenetic and microenvironmental differences among different genomic loci. Using the knowledge gained, we designed TALE-BE targeting CD52 and achieved very high frequency of gene knock-out (up to 80% of phenotypic CD52 knock out). We further demonstrated that TALE-BE generate only insignificant levels of Indels and byproducts. Finally, we combined two molecular tools, a TALE-BE and a TALEN, for multiplex genome engineering, generating high levels of double gene knock-out (â¼75%) without creation of translocations between the two targeted sites.
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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
Therapies to treat patients infected with human immunodeficiency virus (HIV) aim at preventing viral replication but fail to eliminate the virus. Although transplantation of allogeneic CCR5Δ32 homozygous stem cell grafts provided a cure for a few patients, this approach is not considered a general therapeutic strategy because of potential side effects. Conversely, gene editing to disrupt the C-C chemokine receptor type 5 (CCR5) locus, which encodes the major HIV coreceptor, has shown to confer resistance to CCR5-tropic HIV strains. Here, an engineered transcription activator-like effector nuclease (TALEN) that enables efficient CCR5 editing in hematopoietic cells is presented. After transferring TALEN-encoding mRNA into primary CD4+ T cells, up to 89% of CCR5 alleles are disrupted. Genotyping confirms the genetic stability of the CCR5-edited cells, and genome-wide off-target analyses established the absence of relevant mutagenic events. When challenging the edited T cells with CCR5-tropic HIV, protection in a dose-dependent manner is observed. Functional assessments reveal no significant differences between edited and control cells in terms of proliferation and their ability to secrete cytokines upon exogenous stimuli. In conclusion, a highly active and specific TALEN to disrupt CCR5 is successfully engineered, paving the way for its clinical application in hematopoietic stem cell grafts.
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Infecciones por VIH , VIH-1 , Receptores CCR5 , Nucleasas de los Efectores Tipo Activadores de la Transcripción , Resistencia a la Enfermedad , Infecciones por VIH/genética , Infecciones por VIH/prevención & control , VIH-1/genética , Humanos , Receptores CCR5/genética , Nucleasas de los Efectores Tipo Activadores de la Transcripción/genética , Nucleasas de los Efectores Tipo Activadores de la Transcripción/farmacología , Efectores Tipo Activadores de la TranscripciónRESUMEN
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
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
Clinical success of autologous CD19-directed chimeric antigen receptor T cells (CAR Ts) in acute lymphoblastic leukemia and non-Hodgkin lymphoma suggests that CAR Ts may be a promising therapy for hematological malignancies, including multiple myeloma. However, autologous CAR T therapies have limitations that may impact clinical use, including lengthy vein-to-vein time and manufacturing constraints. Allogeneic CAR T (AlloCAR T) therapies may overcome these innate limitations of autologous CAR T therapies. Unlike autologous cell therapies, AlloCAR T therapies employ healthy donor T cells that are isolated in a manufacturing facility, engineered to express CARs with specificity for a tumor-associated antigen, and modified using gene-editing technology to limit T cell receptor (TCR)-mediated immune responses. Here, transcription activator-like effector nuclease (TALEN) gene editing of B cell maturation antigen (BCMA) CAR Ts was used to confer lymphodepletion resistance and reduced graft-versus-host disease (GvHD) potential. The safety profile of allogeneic BCMA CAR Ts was further enhanced by incorporating a CD20 mimotope-based intra-CAR off switch enabling effective CAR T elimination in the presence of rituximab. Allogeneic BCMA CAR Ts induced sustained antitumor responses in mice supplemented with human cytokines, and, most importantly, maintained their phenotype and potency after scale-up manufacturing. This novel off-the-shelf allogeneic BCMA CAR T product is a promising candidate for clinical evaluation.
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Antígeno de Maduración de Linfocitos B/inmunología , Trasplante de Células/métodos , Inmunoterapia Adoptiva/métodos , Mieloma Múltiple/terapia , Receptores Quiméricos de Antígenos/inmunología , Linfocitos T/inmunología , Linfocitos T/trasplante , Animales , Antineoplásicos Inmunológicos/uso terapéutico , Antígeno de Maduración de Linfocitos B/genética , Donantes de Sangre , Línea Celular Tumoral , Trasplante de Células/efectos adversos , Citotoxicidad Inmunológica/genética , Edición Génica , Vectores Genéticos , Enfermedad Injerto contra Huésped/terapia , Humanos , Inmunoterapia Adoptiva/efectos adversos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Mieloma Múltiple/patología , Supervivencia sin Progresión , Receptores Quiméricos de Antígenos/genética , Receptores Quiméricos de Antígenos/metabolismo , Rituximab/uso terapéutico , Linfocitos T/metabolismo , Nucleasas de los Efectores Tipo Activadores de la Transcripción/genética , Transducción Genética , Trasplante Homólogo/métodosRESUMEN
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
Using a TALEN-mediated gene-editing approach, we have previously described a process for the large-scale manufacturing of "off-the-shelf" CAR T cells from third-party donor T cells by disrupting the gene encoding TCRα constant chain (TRAC). Taking advantage of a previously described strategy to control TALEN targeting based on the exclusion capacities of non-conventional RVDs, we have developed highly efficient and specific nucleases targeting a key T cell immune checkpoint, PD-1, to improve engineered CAR T cells' functionalities. Here, we demonstrate that this approach allows combined TRAC and PDCD1 TALEN processing at the desired locus while eliminating low-frequency off-site processing. Thus, by replacing few RVDs, we provide here an easy and rapid redesign of optimal TALEN combinations. We anticipate that this method can greatly benefit multiplex editing, which is of key importance especially for therapeutic applications where high editing efficiencies need to be associated with maximal specificity and safety.
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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.
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Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Inmunoterapia Adoptiva/métodos , Neoplasias/terapia , Oxígeno/metabolismo , Receptores de Antígenos de Linfocitos T/genética , Proteínas Recombinantes de Fusión/genética , Linfocitos T Citotóxicos/fisiología , Antígenos de Neoplasias/inmunología , Línea Celular Tumoral , Citotoxicidad Inmunológica , Ingeniería Genética , Humanos , Activación de Linfocitos , Neoplasias/inmunología , Especificidad del Receptor de Antígeno de Linfocitos T , Linfocitos T Citotóxicos/trasplante , Microambiente TumoralRESUMEN
BACKGROUND: Rosetting, namely the capacity of the Plasmodium falciparum-infected red blood cells to bind uninfected RBCs, is commonly observed in African children with severe malaria. Rosetting results from specific interactions between a subset of variant P. falciparum erythrocyte membrane protein 1 (PfEMP1) adhesins encoded by var genes, serum components and RBC receptors. Rosette formation is a redundant phenotype, as there exists more than one var gene encoding a rosette-mediating PfEMP1 in each genome and hence a diverse array of underlying interactions. Moreover, field diversity creates a large panel of rosetting-associated serotypes and studies with human immune sera indicate that surface-reacting antibodies are essentially variant-specific. To gain better insight into the interactions involved in rosetting and map surface epitopes, a panel of monoclonal antibodies (mAbs) was investigated. METHODS: Monoclonal antibodies were isolated from mice immunized with PfEMP1-VarO recombinant domains. They were characterized using ELISA and reactivity with the native PfEMP1-VarO adhesin on immunoblots of reduced and unreduced extracts, as well as SDS-extracts of Palo Alto 89F5 VarO schizonts. Functionality was assessed using inhibition of Palo Alto 89F5 VarO rosette formation and disruption of Palo Alto 89F5 VarO rosettes. Competition ELISAs were performed with biotinylated antibodies against DBL1 to identify reactivity groups. Specificity of mAbs reacting with the DBL1 adhesion domain was explored using recombinant proteins carrying mutations abolishing RBC binding or binding to heparin, a potent inhibitor of rosette formation. RESULTS: Domain-specific, surface-reacting mAbs were obtained for four individual domains (DBL1, CIDR1, DBL2, DBL4). Monoclonal antibodies reacting with DBL1 potently inhibited the formation of rosettes and disrupted Palo Alto 89F5 VarO rosettes. Most surface-reactive mAbs and all mAbs interfering with rosetting reacted on parasite immunoblots with disulfide bond-dependent PfEMP1 epitopes. Based on competition ELISA and binding to mutant DBL1 domains, two distinct binding sites for rosette-disrupting mAbs were identified in close proximity to the RBC-binding site. CONCLUSIONS: Rosette-inhibitory antibodies bind to conformation-dependent epitopes located close to the RBC-binding site and distant from the heparin-binding site. These results provide novel clues for a rational intervention strategy that targets rosetting.
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Anticuerpos Monoclonales/metabolismo , Moléculas de Adhesión Celular/metabolismo , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/metabolismo , Animales , Anticuerpos Monoclonales/farmacología , Ensayo de Inmunoadsorción Enzimática , Ratones , Plasmodium falciparum/efectos de los fármacos , Unión ProteicaRESUMEN
The ability to control T cells engineered to permanently express chimeric antigen receptors (CARs) is a key feature to improve safety. Here, we describe the development of a new CAR architecture with an integrated switch-on system that permits to control the CAR T-cell function. This system offers the advantage of a transient CAR T-cell for safety while letting open the possibility of multiple cytotoxicity cycles using a small molecule drug.
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Citotoxicidad Inmunológica/efectos de los fármacos , Ingeniería Genética/métodos , Proteínas Mutantes Quiméricas/genética , Receptores de Antígenos de Linfocitos T/genética , Linfocitos T/inmunología , Antígenos CD8/genética , Antígenos CD8/inmunología , Línea Celular Tumoral , Técnicas de Cocultivo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/inmunología , Humanos , Proteínas Mutantes Quiméricas/inmunología , Dominios Proteicos , Receptores de Antígenos de Linfocitos T/inmunología , Anticuerpos de Cadena Única/genética , Sirolimus/análogos & derivados , Sirolimus/farmacología , Linfocitos T/citología , Linfocitos T/efectos de los fármacos , Tacrolimus/farmacología , TransfecciónRESUMEN
Adhesion of Plasmodium falciparum-infected red blood cells (iRBC) to human erythrocytes (i.e. rosetting) is associated with severe malaria. Rosetting results from interactions between a subset of variant PfEMP1 (Plasmodium falciparum erythrocyte membrane protein 1) adhesins and specific erythrocyte receptors. Interfering with such interactions is considered a promising intervention against severe malaria. To evaluate the feasibility of a vaccine strategy targetting rosetting, we have used here the Palo Alto 89F5 VarO rosetting model. PfEMP1-VarO consists of five Duffy-Binding Like domains (DBL1-5) and one Cysteine-rich Interdomain Region (CIDR1). The binding domain has been mapped to DBL1 and the ABO blood group was identified as the erythrocyte receptor. Here, we study the immunogenicity of all six recombinant PfEMP1-VarO domains and the DBL1- CIDR1 Head domain in BALB/c and outbred OF1 mice. Five readouts of antibody responses are explored: ELISA titres on the recombinant antigen, VarO-iRBC immunoblot reactivity, VarO-iRBC surface-reactivity, capacity to disrupt VarO rosettes and the capacity to prevent VarO rosette formation. For three domains, we explore influence of the expression system on antigenicity and immunogenicity. We show that correctly folded PfEMP1 domains elicit high antibody titres and induce a homogeneous response in outbred and BALB/c mice after three injections. High levels of rosette-disrupting and rosette-preventing antibodies are induced by DBL1 and the Head domain. Reduced-alkylated or denatured proteins fail to induce surface-reacting and rosette-disrupting antibodies, indicating that surface epitopes are conformational. We also report limited cross-reactivity between some PfEMP1 VarO domains. These results highlight the high immunogenicity of the individual domains in outbred animals and provide a strong basis for a rational vaccination strategy targeting rosetting.
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Adhesinas Bacterianas/inmunología , Plasmodium falciparum/inmunología , Proteínas Protozoarias/inmunología , Adhesinas Bacterianas/química , Adhesinas Bacterianas/genética , Animales , Anticuerpos Antiprotozoarios/sangre , Antígenos de Protozoos/química , Antígenos de Protozoos/genética , Reacciones Cruzadas , Epítopos/química , Epítopos/genética , Eritrocitos/parasitología , Femenino , Humanos , Vacunas contra la Malaria/química , Vacunas contra la Malaria/genética , Vacunas contra la Malaria/inmunología , Malaria Falciparum/inmunología , Malaria Falciparum/parasitología , Malaria Falciparum/prevención & control , Ratones , Ratones Endogámicos BALB C , Plasmodium falciparum/genética , Plasmodium falciparum/patogenicidad , Estructura Terciaria de Proteína , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Formación de RosetaRESUMEN
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.