ABSTRACT
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.
ABSTRACT
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.
Subject(s)
Anemia, Sickle Cell , Gene Editing , Genetic Therapy , Hematopoietic Stem Cells , Transcription Activator-Like Effector Nucleases , Anemia, Sickle Cell/therapy , Anemia, Sickle Cell/genetics , Gene Editing/methods , Animals , Hematopoietic Stem Cells/metabolism , Humans , Female , Mice , Genetic Therapy/methods , Transcription Activator-Like Effector Nucleases/metabolism , Transcription Activator-Like Effector Nucleases/genetics , Hematopoietic Stem Cell Transplantation , beta-Globins/genetics , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , DNA Repair , Mutation , beta-Thalassemia/therapy , beta-Thalassemia/genetics , Disease Models, Animal , Gene Transfer TechniquesABSTRACT
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.
Subject(s)
Gene Editing , Hematopoietic Stem Cells , Introns , Myeloid Cells , Transcription Activator-Like Effector Nucleases , Transgenes , Animals , Gene Editing/methods , Mice , Hematopoietic Stem Cells/metabolism , Humans , Myeloid Cells/metabolism , Transcription Activator-Like Effector Nucleases/genetics , Transcription Activator-Like Effector Nucleases/metabolism , Cell Differentiation/genetics , Genetic Therapy/methods , Iduronidase/genetics , Iduronidase/metabolism , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Gene Expression , Cell Lineage/genetics , CD11b Antigen/genetics , CD11b Antigen/metabolism , Hematopoietic Stem Cell Transplantation/methods , Mucopolysaccharidosis I/therapy , Mucopolysaccharidosis I/geneticsABSTRACT
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.
ABSTRACT
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.
Subject(s)
Receptors, Chimeric Antigen , Animals , Mice , Receptors, Chimeric Antigen/genetics , Receptors, Chimeric Antigen/metabolism , Transcription Activator-Like Effector Nucleases/metabolism , Immunotherapy , T-Lymphocytes , Antigens, NeoplasmABSTRACT
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.
Subject(s)
Gene Editing , Transcription Activator-Like Effector Nucleases , Humans , Immunotherapy, Adoptive , Receptors, Antigen, T-Cell/genetics , T-LymphocytesABSTRACT
Recent advances in genome editing tools, especially novel developments in the clustered regularly interspaced short palindromic repeats associated to Cas9 nucleases (CRISPR/Cas9)-derived editing machinery, have revolutionized not only basic science but, importantly, also the gene therapy field. Their flexibility and ability to introduce precise modifications in the genome to disrupt or correct genes or insert expression cassettes in safe harbors in the genome underline their potential applications as a medicine of the future to cure many genetic diseases. In this review, we give an overview of the recent progress made by French researchers in the field of therapeutic genome editing, while putting their work in the general context of advances made in the field. We focus on recent hematopoietic stem cell gene editing strategies for blood diseases affecting the red blood cells or blood coagulation as well as lysosomal storage diseases. We report on a genome editing-based therapy for muscular dystrophy and the potency of T cell gene editing to increase anticancer activity of chimeric antigen receptor T cells to combat cancer. We will also discuss technical obstacles and side effects such as unwanted editing activity that need to be surmounted on the way toward a clinical implementation of genome editing. We propose here improvements developed today, including by French researchers to overcome the editing-related genotoxicity and improve editing precision by the use of novel recombinant nuclease-based systems such as nickases, base editors, and prime editors. Finally, a solution is proposed to resolve the cellular toxicity induced by the systems employed for gene editing machinery delivery.
Subject(s)
CRISPR-Cas Systems , Gene Editing , CRISPR-Cas Systems/genetics , Endonucleases/genetics , Gene Transfer Techniques , Genetic TherapyABSTRACT
Superoxide reductases (SORs) are mononuclear non-heme iron enzymes involved in superoxide radical detoxification in some microorganisms. Their atypical active site is made of an iron atom pentacoordinated by four equatorial nitrogen atoms from histidine residues and one axial sulfur atom from a cysteinate residue, which plays a central role in catalysis. In most SORs, the residue immediately following the cysteinate ligand is an asparagine, which belongs to the second coordination sphere and is expected to have a critical influence on the properties of the active site. In this work, in order to investigate the role of this asparagine residue in the Desulfoarculus baarsii enzyme (Asn117), we carried out, in comparison with the wild-type enzyme, absorption and resonance Raman (RR) studies on a SOR mutant in which Asn117 was changed into an alanine. RR analysis was developed in order to assign the different bands using excitation in the (Cys116)-S-â Fe3+ charge transfer band. By investigating the correlation between the (Cys116)-S-â Fe3+ charge transfer band maximum with the frequency of each RR band in different SOR forms, we assessed the contribution of the ν(Fe-S) vibration among the different RR bands. The data showed that Asn117, by making hydrogen bond interactions with Lys74 and Tyr76, allows a rigidification of the backbone of the Cys116 ligand, as well as that of the neighboring residues Ile118 and His119. Such a structural role of Asn117 has a deep impact on the S-Fe bond. It results in a tight control of the H-bond distance between the Ile118 and His119 NH peptidic moiety with the cysteine sulfur ligand, which in turn enables fine-tuning of the S-Fe bond strength, an essential property for the SOR active site. This study illustrates the intricate roles of second coordination sphere residues to adjust the ligand to metal bond properties in the active site of metalloenzymes.
Subject(s)
Bacterial Proteins/chemistry , Cysteine/chemistry , Iron/chemistry , Oxidoreductases/chemistry , Amino Acid Sequence , Catalysis , Catalytic Domain , Hydrogen Bonding , Ligands , Mutagenesis, Site-Directed , Protein Conformation , Spectrophotometry, Ultraviolet , Spectrum Analysis, Raman , Sulfur/chemistryABSTRACT
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.
ABSTRACT
Patients with relapsed or refractory acute myeloid leukemia (AML) have a dismal prognosis and limited treatment options. Chimeric antigen receptor (CAR) T cells have achieved unprecedented clinical responses in patients with B cell leukemias and lymphomas and could prove highly efficacious in AML. However, a significant number of patients with AML may not receive treatment with an autologous product due to manufacturing failures associated with low lymphocyte counts or rapid disease progression while the therapeutic is being produced. We report the preclinical evaluation of an off-the-shelf CAR T cell therapy targeting Fms-related tyrosine kinase 3 (FLT3) for the treatment of AML. Single-chain variable fragments (scFvs) targeting various epitopes in the extracellular region of FLT3 were inserted into CAR constructs and tested for their ability to redirect T cell specificity and effector function to FLT3+ AML cells. A lead CAR, exhibiting minimal tonic signaling and robust activity in vitro and in vivo, was selected and then modified to incorporate a rituximab-responsive off-switch in cis. We found that allogeneic FLT3 CAR T cells, generated from healthy-donor T cells, eliminate primary AML blasts but are also active against mouse and human hematopoietic stem and progenitor cells, indicating risk of myelotoxicity. By employing a surrogate CAR with affinity to murine FLT3, we show that rituximab-mediated depletion of FLT3 CAR T cells after AML eradication enables bone marrow recovery without compromising leukemia remission. These results support clinical investigation of allogeneic FLT3 CAR T cells in AML and other FLT3+ hematologic malignancies.
Subject(s)
Immunotherapy, Adoptive , Leukemia, Myeloid, Acute/immunology , Leukemia, Myeloid, Acute/therapy , Receptors, Chimeric Antigen/immunology , T-Lymphocytes/immunology , fms-Like Tyrosine Kinase 3/immunology , Animals , Bone Marrow/immunology , Bone Marrow/metabolism , Disease Models, Animal , Humans , Immunotherapy, Adoptive/adverse effects , Immunotherapy, Adoptive/methods , Leukemia, Myeloid, Acute/diagnosis , Mice , Receptors, Chimeric Antigen/genetics , T-Cell Antigen Receptor Specificity , T-Lymphocytes/metabolism , Treatment Outcome , Xenograft Model Antitumor Assays , fms-Like Tyrosine Kinase 3/antagonists & inhibitorsABSTRACT
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
ABSTRACT
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.
Subject(s)
Immune System/metabolism , Receptors, Chimeric Antigen/immunology , T-Lymphocytes/immunology , Animals , Cell Line, Tumor , Gene Editing , Humans , Interleukin-12/genetics , Lymphocyte Activation/immunology , Mice , Neoplasms/immunology , Neoplasms/pathology , Receptors, Antigen, T-Cell/metabolism , Transcription Activator-Like Effector Nucleases/metabolismABSTRACT
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.
Subject(s)
Antigens, CD19/immunology , Gene Expression/immunology , Receptors, Chimeric Antigen/immunology , T-Lymphocytes/immunology , Antigens, CD19/genetics , Antigens, CD19/metabolism , Cell Line, Tumor , Cell Membrane/drug effects , Cell Membrane/metabolism , Gene Expression/drug effects , Gene Expression/genetics , Humans , Isoquinolines/pharmacology , Protease Inhibitors/pharmacology , Receptors, Chimeric Antigen/genetics , Receptors, Chimeric Antigen/metabolism , Sulfonamides/pharmacology , T-Lymphocytes/drug effects , T-Lymphocytes/metabolismABSTRACT
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.
Subject(s)
B-Cell Maturation Antigen/immunology , Cell Transplantation/methods , Immunotherapy, Adoptive/methods , Multiple Myeloma/therapy , Receptors, Chimeric Antigen/immunology , T-Lymphocytes/immunology , T-Lymphocytes/transplantation , Animals , Antineoplastic Agents, Immunological/therapeutic use , B-Cell Maturation Antigen/genetics , Blood Donors , Cell Line, Tumor , Cell Transplantation/adverse effects , Cytotoxicity, Immunologic/genetics , Gene Editing , Genetic Vectors , Graft vs Host Disease/therapy , Humans , Immunotherapy, Adoptive/adverse effects , Mice , Mice, Inbred NOD , Mice, SCID , Multiple Myeloma/pathology , Progression-Free Survival , Receptors, Chimeric Antigen/genetics , Receptors, Chimeric Antigen/metabolism , Rituximab/therapeutic use , T-Lymphocytes/metabolism , Transcription Activator-Like Effector Nucleases/genetics , Transduction, Genetic , Transplantation, Homologous/methodsABSTRACT
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.
Subject(s)
Cytokines/immunology , Granulocyte-Macrophage Colony-Stimulating Factor/immunology , Hematologic Neoplasms/immunology , Hematologic Neoplasms/therapy , Immunotherapy, Adoptive , Monocytes , Neoplasm Proteins/immunology , Antineoplastic Agents, Immunological/immunology , Antineoplastic Agents, Immunological/pharmacology , Cytokines/genetics , Gene Editing , Gene Knockdown Techniques , Glucocorticoids/pharmacology , Granulocyte-Macrophage Colony-Stimulating Factor/genetics , Hematologic Neoplasms/genetics , Hematologic Neoplasms/pathology , Humans , Monocytes/immunology , Monocytes/pathology , Neoplasm Proteins/genetics , Receptors, Chimeric Antigen/genetics , Receptors, Chimeric Antigen/immunologyABSTRACT
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.
Subject(s)
Immunotherapy, Adoptive , Neoplasms, Experimental/immunology , Neoplasms, Experimental/surgery , Receptors, Chimeric Antigen/immunology , Rituximab/pharmacology , Animals , Cell Line, Tumor , Humans , Mice , Mice, Inbred BALB C , Neoplasms, Experimental/pathologyABSTRACT
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.
Subject(s)
Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Immunotherapy, Adoptive/methods , Neoplasms/therapy , Oxygen/metabolism , Receptors, Antigen, T-Cell/genetics , Recombinant Fusion Proteins/genetics , T-Lymphocytes, Cytotoxic/physiology , Antigens, Neoplasm/immunology , Cell Line, Tumor , Cytotoxicity, Immunologic , Genetic Engineering , Humans , Lymphocyte Activation , Neoplasms/immunology , T-Cell Antigen Receptor Specificity , T-Lymphocytes, Cytotoxic/transplantation , Tumor MicroenvironmentABSTRACT
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.
Subject(s)
Cytotoxicity, Immunologic/drug effects , Genetic Engineering/methods , Mutant Chimeric Proteins/genetics , Receptors, Antigen, T-Cell/genetics , T-Lymphocytes/immunology , CD8 Antigens/genetics , CD8 Antigens/immunology , Cell Line, Tumor , Coculture Techniques , Gene Expression , Genetic Vectors/chemistry , Genetic Vectors/immunology , Humans , Mutant Chimeric Proteins/immunology , Protein Domains , Receptors, Antigen, T-Cell/immunology , Single-Chain Antibodies/genetics , Sirolimus/analogs & derivatives , Sirolimus/pharmacology , T-Lymphocytes/cytology , T-Lymphocytes/drug effects , Tacrolimus/pharmacology , TransfectionABSTRACT
Homing endonucleases recognize and generate a DNA double-strand break, which has been used to promote gene targeting. These enzymes recognize long DNA stretches; they are highly sequence-specific enzymes and display a very low frequency of cleavage even in complete genomes. Although a large number of homing endonucleases have been identified, the landscape of possible target sequences is still very limited to cover the complexity of the whole eukaryotic genome. Therefore, the finding and molecular analysis of homing endonucleases identified but not yet characterized may widen the landscape of possible target sequences. The previous characterization of protein-DNA interaction before the engineering of new homing endonucleases is essential for further enzyme modification. Here we report the crystal structure of I-CvuI in complex with its target DNA and with the target DNA of I-CreI, a homologue enzyme widely used in genome engineering. To characterize the enzyme cleavage mechanism, we have solved the I-CvuI DNA structures in the presence of non-catalytic (Ca(2+)) and catalytic ions (Mg(2+)). We have also analyzed the metal dependence of DNA cleavage using Mg(2+) ions at different concentrations ranging from non-cleavable to cleavable concentrations obtained from in vitro cleavage experiments. The structure of I-CvuI homing endonuclease expands the current repertoire for engineering custom specificities, both by itself as a new scaffold alone and in hybrid constructs with other related homing endonucleases or other DNA-binding protein templates.
Subject(s)
Chlorella vulgaris/enzymology , Deoxyribonuclease I/chemistry , Plant Proteins/chemistry , Chlorella vulgaris/genetics , Crystallography, X-Ray , Deoxyribonuclease I/genetics , Plant Proteins/genetics , Protein Structure, Tertiary , Structure-Activity RelationshipABSTRACT
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.