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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.

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 CAR T-cells scaffold that evades NK cell and alloresponsive T-cell attacks and imparts efficient antitumor activityin vitroandin vivo. This scaffold could enable the broad use of universal CAR T-cells in allogeneic settings and holds great promise for future powerful clinical applications.

International audience; 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.

Publisher: The Royal Society of Chemistry; 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−→ Fe3+ charge transfer band. By investigating the correlation between the (Cys116)–S− → 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.

International audience; Host versus graft reaction is a major impediment to CAR-T cell immune therapy in allogeneic settings. Authors show here that CAR-T cells, engineered to be deficient in MHC I expression but to express the NK inhibitor HLA-E, are resistant to destruction by both T and NK cells of the host. 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 TCR alpha beta- 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.

Here, we developed a straightforward methodology to generate TCRαβ 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 TCRαβ- 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.

Sickle cell disease (SCD) is one of the most common inherited diseases affecting millions of people worldwide. SCD stems from a single point mutation (A>T) in exon 1 of the HBB gene which results in sickle hemoglobin. The only available curative treatment of SCD is allogeneic hematopoietic stem cell transplant, which is only viable for ~20% of SCD patients. Ex-vivo gene therapy approaches have shown to be a promising therapeutic option for patients. Most products currently in the clinic have focused on methods to enhance functional hemoglobin production (e.g., disruption of BCL11A to encourage β-globin gene switching or direct insertion of a functional β-globin gene into patient HSPCs). The Cellectis approach is to directly repair the mutated HBB gene in order to restore HbA production. TALGlobin01 is an autologous HSPC-based gene therapy product designed with a TALEN ® optimized to cleave the sickle HBB gene (TALEN-HBB01) and an AAV based engineering process leading to highly efficient HBB gene correction via endogenous homology directed repair (HDR), while mitigating potential risks of HBB gene knock-out (KO). Use of TALGlobin01 resulted in up to 70% of HDR-mediated HBB gene correction (56% mean frequency) in homozygous sickle (HbSS) patient HSPCs with only 20% of NHEJ-dependent insertion/deletion (indels) events detected. This gene correction process did not affect cell viability, hematopoietic stem/progenitor immunophenotype or differentiation potential of corrected HSPCs. Allelic editing at clonal resolution in single BFU-E colonies showed that up to 72% (with a mean of 50%) of progenitors contained at least one corrected allele, while only 23% were either not corrected or had indels on one allele. Notably, our optimized engineering process led to only 9% of colonies harboring bi-allelic indels events. To evaluate the ability of TALGlobin01 to prevent the sickling phenotype associated with SCD, we performed in vitro differentiation of HbSS patient-edited cells into late-stage erythroid cells and assessed HbA protein production by HPLC. We observed that HbA accounted for up to 60% (with a mean of 49%) of the total Hb with a concomitant decrease of HbS production from 90% to 19%. Interestingly, our gene correction process maintained a balanced α chain/non-α chain ratio, consistent with our genotyping results showing a low frequency of clones harboring bi-allelic indels. More importantly, efficient expression of HbA was translated into a sharp decrease of hypoxia-induced sickling rate of in vitro-generated erythrocytes when comparing unedited to TALGlobin01 edited cells (from 95% to 13%, respectively). When injected in vivo, engineered HSPCs from non-mobilized SCD patients retained the capacity to engraft into immunodeficient NSG mice with levels of allelic correction comparable to the input (~40%) and still detectable at 16-17 weeks post-transplantation. An unbiased genome wide approach (OCA), coupled to target enrichment high-throughput sequencing screening, confirmed the TALEN-HBB01 cleavage activity at only one off-target site located at the HBD locus. TALEN-HBB01 cleavage activity was assessed at this off-target site in patient HSPCs engineered with TALGlobin01 and found to be very low compared to the on-site cleavage activity (50.7% indels at the on-site versus 1.2% at the off-site). Furthermore, we developed a clinical-scale TALGlobin01 manufacturing process that achieved up to 60% of HDR-mediated HBB gene correction and less than 10% of NHEJ-mediated HBB gene inactivation in healthy donor derived HSPCs. These results are the first demonstration that a TALEN-based engineering process could be used to efficiently correct the SCD mutated HBB gene in HbSS patient-derived CD34+ HSPCs. Taken together, the high level of HbA expression and reversion of sickling phenotype, the efficient in vivo long-term engraftment potential of TALGlobin01 edited cells and the low levels of HBB KO or off-target cleavage generated by our gene correction process, warrant the clinical evaluation of TALGlobin01 to treat SCD patients. Disclosures Moiani: Cellectis, S.A.: Current Employment, Current equity holder in publicly-traded company. Hong: Cellectis, Inc.: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Letort: Cellectis, S.A.: Current Employment, Current equity holder in publicly-traded company. Lizot: Cellectis, S.A.: Current Employment, Current equity holder in publicly-traded company. Chirinos: Cellectis, S.A.: Current Employment. Temburni-Blake: Cellectis, Inc.: Current Employment, Current equity holder in publicly-traded company. Mayer: Cellectis, Inc.: Current Employment, Current equity holder in publicly-traded company. Leduc: Cellectis, S.A.: Current Employment, Current equity holder in publicly-traded company. Pinard: Cellectis, S.A.: Current Employment, Current equity holder in publicly-traded company. Foray: Cellectis, S.A.: Current Employment, Current equity holder in publicly-traded company. Boyne: Cellectis, Inc.: Current Employment, Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months, Patents & Royalties. Kazancioglu: Cellectis, Inc.: Current Employment, Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months. Dusseaux: Cellectis, S.A.: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Gouble: Cellectis SA: Current Employment, Current equity holder in publicly-traded company, Current holder of stock options in a privately-held company. Frattini: Celgene/BMS: Current equity holder in publicly-traded company, Ended employment in the past 24 months; Cellectis, Inc.: Current Employment, Current equity holder in publicly-traded company. Brownstein: BMS: Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months, Ended employment in the past 24 months; Cellectis, Inc.: Current Employment, Current equity holder in publicly-traded company. Duclert: Cellectis, S.A.: Current Employment, Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months. Shiffer-Mannoui: Cellectis, S.A.: Current Employment, Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months. Juillerat: Cellectis, Inc.: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Duchateau: Cellectis, S.A.: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Valton: Cellectis, S.A.: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties.

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.

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.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Emerging CAR T-cell therapies have shown high remission rates in patients with hematologic malignancies. Despite the success of these novel immunotherapies, multiple adverse effects are associated with CAR T-cell infusions. Cytokine release syndrome (CRS) is one of the most common CAR T cell-associated toxicities and results from the rapid elevation of inflammatory cytokines, which can be life threatening. CRS is commonly managed in the clinic with anti-IL-6R antibodies and glucocorticoids. While efficacious, these treatments address CRS only after it has initiated; therefore, alternative therapeutic strategies preventing CRS onset remain an urgent need. Using cytokine profiling arrays, we identified that secreted granulocyte macrophage colony stimulating factor (GM-CSF) contributes to CRS, since elevated GM-CSF levels promote macrophage-dependent secretion of key CRS biomarkers including MCP-1, IL-6, and IL-8. Modulation of GM-CSF levels either via genetic engineering of CAR T-cells using TALEN® technology or via neutralizing-antibodies resulted in a marked reduction of macrophage-dependent release of CRS-associated cytokines, while preserving antitumor activity. Our work proposes a novel therapeutic strategy to improve the overall safety of CAR T-cell therapies in cancer patients by using GM-CSF knockout CAR T cells. Citation Format: Mohit Sachdeva, Beatriz Aranda-Orgilles, Philippe Duchateau, Laurent Poirot, Julien Valton. GM-CSF modulation restricts the secretion of main cytokines associated with CAR T-cell induced cytokine release syndrome [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A60.

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.

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.

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 αβ-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 TCRαβ 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.

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.

TALEN is one of the most widely used tools in the field of genome editing. It enables gene integration and gene inactivation in a highly efficient and specific fashion. Although very attractive, the apparent simplicity and high success rate of TALEN could be misleading for novices in the field of gene editing. Depending on the application, specific TALEN designs, activity assessments and screening strategies need to be adopted. Here we report different methods to efficiently perform TALEN-mediated gene integration and inactivation in different mammalian cell systems including induced pluripotent stem cells and delineate experimental examples associated with these approaches.

Autologous chimeric antigen receptor (CAR) T cells have achieved unprecedented clinical responses in patients with B-cell leukemias, lymphomas and multiple myeloma, raising interest in using CAR T cell therapies in AML. These therapies are produced using a patient's own T cells, an approach that has inherent challenges, including requiring significant time for production, complex supply chain logistics, separate GMP manufacturing for each patient, and variability in performance of patient-derived cells. Given the rapid pace of disease progression combined with limitations associated with the autologous approach and treatment-induced lymphopenia, many patients with AML may not receive treatment. Allogeneic CAR T (AlloCAR T) cell therapies, which utilize cells from healthy donors, may provide greater convenience with readily available off-the-shelf CAR T cells on-demand, reliable product consistency, and accessibility at greater scale for more patients. To create an allogeneic product, the TRAC and CD52 genes are inactivated in CAR T cells using Transcription Activator-Like Effector Nuclease (TALEN®) technology. These genetic modifications are intended to minimize the risk of graft-versus-host disease and to confer resistance to ALLO-647, an anti-CD52 antibody that can be used as part of the conditioning regimen to deplete host alloreactive immune cells potentially leading to increased persistence and efficacy of the infused allogeneic cells. We have previously described the functional screening of a library of anti-FLT3 single-chain variable fragments (scFvs) and the identification of a lead FLT3 CAR with optimal activity against AML cells and featuring an off-switch activated by rituximab. Here we characterize ALLO-819, an allogeneic FLT3 CAR T cell product, for its antitumor efficacy and expansion in orthotopic models of human AML, cytotoxicity in the presence of soluble FLT3 (sFLT3), performance compared with previously described anti-FLT3 CARs and potential for off-target binding of the scFv to normal human tissues. To produce ALLO-819, T cells derived from healthy donors were activated and transduced with a lentiviral construct for expression of the lead anti-FLT3 CAR followed by efficient knockout of TRAC and CD52. ALLO-819 manufactured from multiple donors was insensitive to ALLO-647 (100 µg/mL) in in vitro assays, suggesting that it would avoid elimination by the lymphodepletion regimen. In orthotopic models of AML (MV4-11 and EOL-1), ALLO-819 exhibited dose-dependent expansion and cytotoxic activity, with peak CAR T cell levels corresponding to maximal antitumor efficacy. Intriguingly, ALLO-819 showed earlier and more robust peak expansion in mice engrafted with MV4-11 target cells, which express lower levels of the antigen relative to EOL-1 cells (n=2 donors). To further assess the potency of ALLO-819, multiple anti-FLT3 scFvs that had been described in previous reports were cloned into lentiviral constructs that were used to generate CAR T cells following the standard protocol. In these comparative studies, the ALLO-819 CAR displayed high transduction efficiency and superior performance across different donors. Furthermore, the effector function of ALLO-819 was equivalent to that observed in FLT3 CAR T cells with normal expression of TCR and CD52, indicating no effects of TALEN® treatment on CAR T cell activity. Plasma levels of sFLT3 are frequently increased in patients with AML and correlate with tumor burden, raising the possibility that sFLT3 may act as a decoy for FLT3 CAR T cells. To rule out an inhibitory effect of sFLT3 on ALLO-819, effector and target cells were cultured overnight in the presence of increasing concentrations of recombinant sFLT3. We found that ALLO-819 retained its killing properties even in the presence of supraphysiological concentrations of sFLT3 (1 µg/mL). To investigate the potential for off-target binding of the ALLO-819 CAR to human tissues, tissue cross-reactivity studies were conducted using a recombinant protein consisting of the extracellular domain of the CAR fused to human IgG Fc. Consistent with the limited expression pattern of FLT3 and indicative of the high specificity of the lead scFv, no appreciable membrane staining was detected in any of the 36 normal tissues tested (n=3 donors). Taken together, our results support clinical development of ALLO-819 as a novel and effective CAR T cell therapy for the treatment of AML. Disclosures Sommer: Allogene Therapeutics, Inc.: Employment, Equity Ownership. Cheng:Allogene Therapeutics, Inc.: Employment, Equity Ownership. Yeung:Pfizer Inc.: Employment, Equity Ownership. Nguyen:Allogene Therapeutics, Inc.: Employment, Equity Ownership. Sutton:Allogene Therapeutics, Inc.: Employment, Equity Ownership. Melton:Allogene Therapeutics, Inc.: Employment, Equity Ownership. Valton:Cellectis, Inc.: Employment, Equity Ownership. Poulsen:Allogene Therapeutics, Inc.: Employment, Equity Ownership. Djuretic:Pfizer, Inc.: Employment, Equity Ownership. Van Blarcom:Allogene Therapeutics, Inc.: Employment, Equity Ownership. Chaparro-Riggers:Pfizer, Inc.: Employment, Equity Ownership. Sasu:Allogene Therapeutics, Inc.: Employment, Equity Ownership.

In this study, we asked whether CpG methylation could influence the DNA binding affinity and activity of meganucleases used for genome engineering applications. A combination of biochemical and structural approaches enabled us to demonstrate that CpG methylation decreases I-CreI DNA binding affinity and inhibits its endonuclease activity in vitro. This inhibition depends on the position of the methylated cytosine within the DNA target and was almost total when it is located inside the central tetrabase. Crystal structures of I-CreI bound to methylated cognate target DNA suggested a molecular basis for such inhibition, although the precise mechanism still has to be specified. Finally, we demonstrated that the efficacy of engineered meganucleases can be diminished by CpG methylation of the targeted endogenous site, and we proposed a rational design of the meganuclease DNA binding domain to alleviate such an effect. We conclude that although activity and sequence specificity of engineered meganucleases are crucial parameters, target DNA epigenetic modifications need to be considered for successful gene editions.

The “flavin destructase” enzyme BluB catalyzes the unprecedented conversion of flavin mononucleotide (FMN) to 5,6-dimethylbenzimidazole (DMB), a component of vitamin B12. Because of its unusual chemistry, the mechanism of this transformation has remained elusive. This study reports the identification of 12 mutant forms of BluB that have severely reduced catalytic function, though most retain the ability to bind flavin. The “flavin destructase” BluB is an unusual enzyme that fragments the flavin cofactor FMNH2 in the presence of oxygen to produce 5,6-dimethylbenzimidazole (DMB), the lower axial ligand of vitamin B12 (cobalamin). Despite the similarities in sequence and structure between BluB and the nitroreductase and flavin oxidoreductase enzyme families, BluB is the only enzyme known to fragment a flavin isoalloxazine ring. To explore the catalytic residues involved in this unusual reaction, mutants of BluB impaired in DMB biosynthesis were identified in a genetic screen in the bacterium Sinorhizobium meliloti. Of the 16 unique point mutations identified in the screen, the majority were located in conserved residues in the active site or in the unique “lid” domain proposed to shield the active site from solvent. Steady-state enzyme assays of 12 purified mutant proteins showed a significant reduction in DMB synthesis in all of the mutants, with eight completely defective in DMB production. Ten of these mutants have weaker binding affinities for both oxidized and reduced FMN, though only two have a significant effect on complex stability. These results implicate several conserved residues in BluB's unique ability to fragment FMNH2 and demonstrate the sensitivity of BluB's active site to structural perturbations. This work lays the foundation for mechanistic studies of this enzyme and further advances our understanding of the structure-function relationship of BluB.

Autologous chimeric antigen receptor (CAR) T cells targeting B-Cell Maturation Antigen (BCMA) have demonstrated promising clinical activity, inducing durable responses in patients with relapsed/refractory multiple myeloma (MM). Development of autologous CAR T therapies is however limited by logistical challenges and the time required for manufacturing, which has to be done for each patient. In addition, manufacturing may not be feasible in some patients. An allogeneic approach that utilizes engineered cells from a healthy donor could potentially expand patient access to these therapies by providing a readily available off-the-shelf product. We have previously described the screening of a library of single chain variable fragments (scFvs) with high affinity to human BCMA and the identification of candidate BCMA CARs with potent antitumor activity. Here we sought to further characterize ALLO-715, our lead allogeneic BCMA CAR T cell product, for its specificity to human BCMA, antitumor efficacy in vitro using a long-term killing assay and in xenograft mouse models with physiologic levels of human IL-7 and IL-15, and suitability for scale-up manufacturing. Allogeneic ALLO-715 CAR T cells were generated by lentiviral transduction with a second generation CAR construct incorporating a novel scFv derived from a fully-human antibody with high affinity to BCMA (KD value ~ 5 nM, determined at 37°C) and featuring a rituximab-driven off-switch. Transduced T cells were then transfected with mRNAs encoding Transcription Activator-Like Effector Nucleases (TALEN®) designed to specifically disrupt the T cell receptor alpha chain and CD52 loci. These modifications result in a cell product with a lower risk of TCR-mediated graft-versus-host disease and resistance to the CD52 antibody alemtuzumab, a lymphodepleting agent. BCMA CAR T cells exhibited robust cell expansion, with low levels of tonic signaling that resulted in minimal differentiation (> 50% Tscm/Tcm phenotype). In in vitro assays, ALLO-715 CAR T cells displayed potent cytotoxic activity when co-cultured with the target cell lines MM.1S, Molp-8, and BCMA-REH but negligible cytotoxicity against BCMA-negative REH cells. The high proliferative potential indicated by the high frequency of memory T cells was validated in long-term killing assays, where ALLO-715 CAR T cells showed substantial expansion in the presence of MM.1S cells with no evidence of exhaustion or diminished cytolytic activity after seven days of continuous exposure to target. The potency of ALLO-715 CAR T cells was unaffected by high concentrations of soluble BCMA (>10 ug/mL), which has been shown previously to interfere with the activity of some BCMA-specific CARs. In MM xenograft mouse models, ALLO-715 CAR T cells were highly efficacious at single dose. High serum IL-15 levels have been associated with CAR T cell expansion in clinical trials. To evaluate the impact of homeostatic cytokines on CAR T cell survival and antitumor activity in our xenograft models, mice were administered adeno-associated viruses (AAV) for the expression of human IL-7 and IL-15. In the presence of physiological concentrations of these cytokines, enhanced BCMA CAR T cell expansion and anti-tumor activity were observed. To assess potential off-target interactions of ALLO-715 CAR, tissue cross-reactivity studies were carried out on standard human tissue panels using a scFv-human IgG fusion protein. Consistent with the limited expression pattern of BCMA, reactivity was seen on scattered cells in lymphoid tissues such as tonsil and abundantly on BCMA-expressing cell lines, but no appreciable staining was detected in other tissues. We examined BCMA CAR T cells manufactured following a proprietary GMP-like clinical scale process and found that cell expansion and viability, T cell phenotype and in vivo antitumor efficacy were preserved. These results demonstrate the potential of ALLO-715 as a novel allogeneic BCMA CAR T therapy for the treatment of relapsed/refractory MM and other BCMA-positive malignancies. Disclosures Sommer: Allogene Therapeutics: Employment, Equity Ownership, Patents & Royalties. Boldajipour:Pfizer Inc.: Employment, Patents & Royalties. Valton:Cellectis.Inc: Employment, Equity Ownership, Patents & Royalties. Galetto:Cellectis SA: Employment, Equity Ownership, Patents & Royalties. Bentley:Allogene Therapeutics: Employment, Equity Ownership. Sutton:Allogene Therapeutics: Employment, Equity Ownership. Ni:Allogene Therapeutics: Employment, Equity Ownership. Leonard:Allogene Therapeutics: Employment, Equity Ownership. Van Blarcom:Allogene Therapeutics: Employment, Equity Ownership. Smith:Cellectis. Inc: Employment, Patents & Royalties. Chaparro-Riggers:Pfizer Inc.: Employment, Patents & Royalties. Sasu:Allogene Therapeutics: Employment, Equity Ownership, Patents & Royalties.

CAR T-cell therapies hold great promise for treating a range of liquid malignancies but are however challenged to access and eradicate solid tumors. To overcome this hurdle, CAR T-cell were engineered to secrete different cytokines known to improve T-cell antitumor activity, prevent T-cell anergy and reduce activation induced cell death. While cytokine-expressing CAR T-cell were shown to be be highly active against solid tumor in in vivo models, they have also led to toxicity associated with the systemic release of cytokine. Therefore, new engineering strategies enabling the fine tuning of cytokine secretion by CAR T-cell are warranted. We sought to explore one of these engineering strategies by integrating an IL-12 chimeric heterodimer expression cassette under the control of the endogenous promoters regulating PD1 or CD25. Because both genes are known to be activated upon tumor engagement by CAR T-cells, they could be repurposed to secrete cytokine only in the vicinity of a given tumor. This approach would reduce the potential side effects induced by their systemic secretion while maintaining their capacity to improve antitumor activity. By combining TALEN® technology with AAV6 repair vectors delivering the CAR to the TRAC locus and the IL-12 to the CD25 or PD1 loci, we have engineered CAR and IL-12 expressions under the respective control of TCR and CD25 or PD1 regulatory elements. This double targeted insertion led to the disruption of PD1 and TRAC genes, to the expression of a tool CAR and to the conditional secretion of IL-12 in the media. Such secretion was found to be transient, dependent on tumor engagement and to follow the regulation patterns of CD25 or PD1 genes, commonly observed upon T-cell activation. In addition, it was also found to enhance the antitumor activity and the proliferative capacities of CAR T-cells. Similar results were obtained when IL-15 was substituted for IL-12. This proof of concept paves the way for seamless multi-repurposing of immune pathways to generate smarter CAR T-cells able to sense and react to their environment in a highly regulated and specific manner. Citation Format: Mohit Sachdeva, Brian Busser, Sonal Temburni, Alexandre Juillerat, Laurent Poirot, Philippe Duchateau, Julien Valton. Repurposing endogenous immune pathways to improve chimeric antigen receptor T-cells potency [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-107.

International audience; The ActVA-ActVB system from Streptomyces coelicolor is a two-component flavin-dependent monooxygenase involved in the antibiotic actinorhodin biosynthesis. ActVB is a NADH:flavin oxidoreductase that provides a reduced FMN to ActVA, the monooxygenase that catalyzes the hydroxylation of dihydrokalafungin, the precursor of actinorhodin. In this work, using stopped-flow spectrophotometry, we investigated the mechanism of hydroxylation of dihydrokalafungin catalyzed by ActVA and that of the reduced FMN transfer from ActVB to ActVA. Our results show that the hydroxylation mechanism proceeds with the participation of two different reaction intermediates in ActVA active site. First, a C(4a)-FMN-hydroperoxide species is formed after binding of reduced FMN to the monooxygenase and reaction with O(2). This intermediate hydroxylates the substrate and is transformed to a second reaction intermediate, a C(4a)-FMN-hydroxy species. In addition, we demonstrate that reduced FMN can be transferred efficiently from the reductase to the monooxygenase without involving any protein.protein complexes. The rate of transfer of reduced FMN from ActVB to ActVA was found to be controlled by the release of NAD(+) from ActVB and was strongly affected by NAD(+) concentration, with an IC(50) of 40 microm. This control of reduced FMN transfer by NAD(+) was associated with the formation of a strong charge.transfer complex between NAD(+) and reduced FMN in the active site of ActVB. These results suggest that, in Streptomyces coelicolor, the reductase component ActVB can act as a regulatory component of the monooxygenase activity by controlling the transfer of reduced FMN to the monooxygenase.

Chimeric antigen receptor (CAR)-redirected T-cells have given rise to long-term durable remissions and remarkable objective response rates in patients with refractory leukemia, raising hopes that a wider application of CAR technology may lead to a new paradigm in cancer treatment. Similarly, transcription activator-like effector nuclease (TALEN™)-mediated gene editing has emerged as powerful strategy to introduce targeted mutations and holds great promise in therapeutics and offers multiple opportunities to improve CAR T-cell therapies. The knockout of the TCR alpha gene eliminates TCR expression and abrogates the donor T-cell's potential for graft-versus-host disease (GvHD) while maintaining a potent anti-tumoral activity. Thus it is possible to manufacture T-cells from third-party healthy donors to generate allogeneic “off-the-shelf” engineered CAR T-cells. Disruption of CD52 or deoxycytidine kinase genes may be a useful approach to makes T-cells compatible with concurrent oncology treatments such as alemtuzumab or Fludarabine while the bypass of key immune checkpoint regulators such PD1/PDL1 by inactivation of T-cells receptors would potentiate the antitumor T-cell response by impairing the interaction of the inhibitory receptor PD-1 on T cells with PD-L1 expressed on tumor cells. Here, we will present in vitro and in vivo proof of concepts demonstrating the potential of TALEN™-mediated gene editing for adoptive T-cell therapy.

International audience; The flavin reductase ActVB is involved in the last step of actinorhodin biosynthesis in Streptomyces coelicolor. Although ActVB can be isolated with some FMN bound, this form was not involved in the flavin reductase activity. By studying the ferric reductase activity of ActVB, we show that its FMN-bound form exhibits a proper enzymatic activity of reduction of iron complexes by NADH. This shows that ActVB active site exhibits a dual property with regard to the FMN. It can use it as a substrate that goes in and off the active site or as a cofactor to provide an electron transferase activity to the polypeptide.

The ActVA-ActVB system from Streptomyces coelicolor isatwo-component flavin-dependent monooxygenase that belongs to an emerging class of enzymes involved in various oxidation reactions in microorganisms. The ActVB component is a NADH:flavin oxidoreductase that provides a reduced FMN to the second component, ActVA the proper monooxygenase. In this work, we demonstrate that the ActVA-ActVB system catalyzes the aromatic monohydroxylation of dihydrokalafungin by molecular oxygen. In the presence of reduced FMN and molecular oxygen, the ActVA active site accommodates and stabilizes an electrophilic flavin FMN-OOH hydroperoxide intermediate species as the oxidant. Surprisingly, we demonstrate that the quinone form of dihydrokalafungin is not oxidized by the ActVA-ActVB system, whereas the corresponding hydroquinone is an excellent substrate. The enantiomer of dihydrokalafungin, nanaomycin A, as well as the enantiomer of kalafungin, nanaomycin D, are also substrates in their hydroquinone forms. The previously postulated product of the ActVA-ActVB system, the antibiotic actinorhodin, was not found to be formed during the oxidation reaction.

International audience; The two-component flavin-dependent monooxygenases belong to an emerging class of enzymes involved in oxidation reactions in a number of metabolic and biosynthetic pathways in microorganisms. One component is a NAD(P)H:flavin oxidoreductase, which provides a reduced flavin to the second component, the proper monooxygenase. There, the reduced flavin activates molecular oxygen for substrate oxidation. Here, we study the flavin reductase ActVB and ActVA-ORF5 gene product, both reported to be involved in the last step of biosynthesis of the natural antibiotic actinorhodin in Streptomyces coelicolor. For the first time we show that ActVA-ORF5 is a FMN-dependent monooxygenase that together with the help of the flavin reductase ActVB catalyzes the oxidation reaction. The mechanism of the transfer of reduced FMN between ActVB and ActVA-ORF5 has been investigated. Dissociation constant values for oxidized and reduced flavin (FMNox and FMNred) with regard to ActVB and ActVA-ORF5 have been determined. The data clearly demonstrate a thermodynamic transfer of FMNred from ActVB to ActVA-ORF5 without involving a particular interaction between the two protein components. In full agreement with these data, we propose a reaction mechanism in which FMNox binds to ActVB, where it is reduced, and the resulting FMNred moves to ActVA-ORF5, where it reacts with O2 to generate a flavinperoxide intermediate. A direct spectroscopic evidence for the formation of such species within ActVA-ORF5 is reported.

A key issue when designing and using DNA-targeting nucleases is specificity. Ideally, an optimal DNA-targeting tool has only one recognition site within a genomic sequence. In practice, however, almost all designer nucleases available today can accommodate one to several mutations within their target site. The ability to predict the specificity of targeting is thus highly desirable. Here, we describe the first comprehensive experimental study focused on the specificity of the four commonly used repeat variable diresidues (RVDs; NI:A, HD:C, NN:G and NG:T) incorporated in transcription activator-like effector nucleases (TALEN). The analysis of > 15 500 unique TALEN/DNA cleavage profiles allowed us to monitor the specificity gradient of the RVDs along a TALEN/DNA binding array and to present a specificity scoring matrix for RVD/nucleotide association. Furthermore, we report that TALEN can only accommodate a relatively small number of position-dependent mismatches while maintaining a detectable activity at endogenous loci in vivo, demonstrating the high specificity of these molecular tools. We thus envision that the results we provide will allow for more deliberate choices of DNA binding arrays and/or DNA targets, extending our engineering capabilities.

The last few years have seen the increasing development of new DNA targeting molecular tools and strategies for precise genome editing. However, opportunities subsist to either improve or expand the current toolbox and further broaden the scope of possible biotechnological applications. Here we report the discovery and the characterization of BurrH, a new modular DNA binding protein from Burkholderia rhizoxinica that is composed of highly polymorphic DNA targeting modules. We also engineered this scaffold to create a new class of designer nucleases that can be used to efficiently induce in vivo targeted mutagenesis and targeted gene insertion at a desired locus.

Background: The past decade has seen the emergence of several molecular tools that render possible modification of cellular functions through accurate and easy addition, removal, or exchange of genomic DNA sequences. Among these technologies, transcription activator-like effectors (TALE) has turned out to be one of the most versatile and incredibly robust platform for generating targeted molecular tools as demonstrated by fusion to various domains such as transcription activator, repressor and nucleases. Results: In this study, we generated a novel nuclease architecture based on the transcription activator-like effector scaffold. In contrast to the existing Tail to Tail (TtT) and head to Head (HtH) nuclease architectures based on the symmetrical association of two TALE DNA binding domains fused to the C-terminal (TtT) or N-terminal (HtH) end of FokI, this novel architecture consists of the asymmetrical association of two different engineered TALE DNA binding domains fused to the N- and C-terminal ends of FokI (TALE:: FokI and FokI:: TALE scaffolds respectively). The characterization of this novel Tail to Head (TtH) architecture in yeast enabled us to demonstrate its nuclease activity and define its optimal target configuration. We further showed that this architecture was able to promote substantial level of targeted mutagenesis at three endogenous loci present in two different mammalian cell lines. Conclusion: Our results demonstrated that this novel functional TtH architecture which requires binding to only one DNA strand of a given endogenous locus has the potential to extend the targeting possibility of FokI-based TALE nucleases.

Methods and kits for detecting nucleic acid sequences of interest using dna-binding protein domain

Multiple myeloma (MM) is a hematological disease of plasma B cells that remains incurable despite the availability of numerous therapies. The plasma cell-specific expression of the TNF superfamily receptor BCMA may allow targeting of normal and malignant plasma cells. Genetically engineered chimeric antigen-receptor T cells (CAR T) have shown tremendous promise in the treatment of several hematological diseases, including MM. However, conventional autologous CAR T therapies use patient-derived T cells and the logistics of on-demand CAR T manufacture limits their availability to a broad patient pool. Here we describe the preclinical evaluation of an allogeneic CAR T therapy targeting BCMA that has the potential for a readily available, off-the-shelf therapy for MM and other malignancies expressing BCMA. Human T cells were transduced with recombinant lentiviral vectors encoding three BCMA CAR candidates designed with fully human anti-BCMA scFvs, CD8a transmembrane domains and the intracellular signaling domains of 4-1BB and CD3zeta. All CAR T efficiently killed BCMA-expressing multiple myeloma cell lines (KMS12BM, MM1.S, Molp-8 and OPM-2), but not BCMA-negative REH cells in vitro and in vivo. Whereas 2 of the 3 candidates exhibited target-independent cytokine production, accelerated T cell differentiation and reduced target cell-induced expansion in vitro, the third candidate did not exhibit this scFv-induced autoactivation and was chosen as the lead molecule. Due to the allogeneic nature of this T cell therapy, the possibility of graft-versus-host (GvH) reactions can be a safety concern. We applied Cellectis' know-how and TALEN® technology for the gene inactivation of the T cell receptor (TCR) alpha chain to significantly reduce the probability for TCR-mediated GvH reactions and found that TCR knockout did not affect CAR T activity in vitro or in vivo. Furthermore, we incorporated intra-CAR rituximab-recognition domains into the CAR molecule to enable depletion of CAR T cells from patients when necessary. We found that this modified CAR retained anti-BCMA CAR T activity and enabled CAR T depletion by rituximab. Another aspect of allogeneic CAR T therapies is the rejection of the CAR T by host-versus-graft (HvG) reactions. Lymphodepletion prior to CAR T infusion enhances CAR T efficacy in autologous CAR T trials and may also prevent anti-CAR HvG reactions in allogeneic therapy settings. Engineering lymphodepletion resistance into CAR T cells could therefore enable sustained lymphodepletion for enhanced allogeneic CAR T persistence and efficacy. CD52 is expressed on all lymphocytes and administration of the anti-CD52 antibody alemtuzumab for prolonged lymphodepletion is an approved treatment for multiple sclerosis. TALEN®-mediated knockout of CD52 protected BCMA CAR T from alemtuzumab-induced cytotoxicity and did not alter BCMA CAR T anti-tumor activity. Taken together these results support allogeneic BCMA CAR T as an off-the-shelf adoptive immunotherapy for the treatment of multiple myeloma and other BCMA-positive malignancies. Disclosures Boldajipour: Pfizer: Employment. Galetto:Cellectis SA: Employment. Sommer:Pfizer Inc.: Employment. Pertel:Pfizer Inc.: Employment. Valton:Cellectis Inc.: Employment. Park:Pfizer Inc.: Employment. Gariboldi:Cellectis SA: Employment. Chen:Alexo Therapeutics: Employment. Geng:Kodiak Sciences: Employment. Dong:Pfizer Inc.: Employment. Boucher:Pfizer Inc.: Employment. Van Blarcom:Pfizer Inc.: Employment. Chaparro-Riggers:Pfizer Inc.: Employment. Rajpal:Pfizer Inc.: Employment. Smith:Cellectis SA: Employment. Kuo:Pfizer Inc.: Employment. Sasu:Pfizer Inc.: Employment.

Adoptive CAR T cell therapies have been reported to be highly efficient at killing tumor cells and are on the verge of revolutionizing the field of cancer treatment. However, there are safety concerns with CAR T cell treatments which would require intervention, including on-target off-tumor effects, on-tumor toxicities such as cytokine release syndrome and tumor lysis syndrome. Avoiding the aformentionned acute and long term adverse events appears to be mandatory to improve the safety of adoptive cell immunotherapies. To address this issue, different teams developed molecular suicide switch systems to remove the transferred cell population. While these different systems are efficient at promoting eradication of adoptively transferred cells, they are expressed independent of the CAR. This could potentially lead to the generation of a CAR T population devoid of the suicide switch system raising concerns about their utility in the clinic. To address this issue, we developed a novel CAR architecture containing an integrated suicide switch component. We demonstrated that this CAR architecture was efficiently depleted by an FDA-approved molecule, retained its cytolytic potential and allowed for universal enrichment and detection of CAR T cells by GMP compatible kit. We will present its development and in-depth characterization. Note: This abstract was not presented at the conference. Citation Format: Julien Valton. An intrinsic safeguard for chimeric antigen receptor adoptive T-cell immunotherapies [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B100.

7039Background: Adoptive CAR T cell therapies have been reported to be highly efficient at killing tumor cells and are on the verge of revolutionizing the field of cancer treatment. However, there ...

Adoptive immunotherapy using engineered T-cells has emerged as a powerful approach to treat cancer. The potential of this approach relies on the ability to redirect the specificity of T cells through genetic engineering. Novel specificities in T cells have been typically implemented through the genetic transfer of the so-called chimeric antigen receptors (CARs). CARs are synthetic receptors composed of an extracellular targeting moiety and one or more intracytoplasmic signaling domain derived from lymphocyte activation receptors. Present CAR architectures are designed to combine all relevant domains within a single polypeptide, thereby; they combine advantages of MHC unrestricted target recognition to the potent native effector mechanisms of the T cell. Although adoptive transfer of CAR T cells is proven to be an effective cancer therapy, potential adverse effects such as cytokine release syndrome (CRS) and/or the risk of on-target off-tumor targeting are still a major concern.Synthetic biology applies many of the principles of engineering to the field of biology in order to create biological devices which can ultimately be integrated into increasingly complex systems. Our ability to engineer synthetic systems in primary T-cells that function as Boolean logic gates responding to multiple inputs would benefit adoptive immunotherapy using engineered T-cells. Exogenous or endogenous environmental signal integration by a modular AND gate may represent an important advancement in improving our control of the safety of the CAR T-cell technology.Here, we describe the development of novel CAR designs that integrate new components directly within the CAR architecture to improve our capacity to spatiotemporally control and switch the CAR T-cells functions between on and off states. In particular, we showed that such a system can be engineered to control the CAR through addition of an exogenous small molecule (Rapamycin or synthetic rapalogs) ultimately inducing the cytolytic properties of the engineered T-cell. Alternatively, properties of the tumor microenvironment can also be used as additional endogenous input to the target antigen recognition. We showed that low oxygen levels can be used to trigger the CAR surface presentation, creating a so called “self-decision making” CAR T-cell.

The adoptive transfer of 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 TCR-deficient CAR T cells. We demonstrate in vitro and in an in vivo xenograft mice model, that these engineered T cells displayed efficient antitumor activity and proliferated in the presence of single or muliple nucleotide analogues, currently used in clinic as preconditioning lymphodepleting regimens and antineoplastic agents. The absence of TCR at their cell surface along with their 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 frame work 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.

Chimeric antigen receptor (CAR)-redirected T-cells have given rise to long-term durable remissions and remarkable objective response rates in patients with refractory leukemia, raising hopes that a wider application of CAR technology may lead to a new paradigm in cancer treatment. Similarly, transcription activator-like effector nuclease (TALEN™)-mediated gene editing has emerged as powerful strategy to introduce targeted mutations and holds great promise in therapeutics and offers multiple opportunities to improve CAR T-cell therapies. The knockout of the TCR alpha gene eliminates TCR expression and abrogates the donor T-cell's potential for graft-versus-host disease (GvHD) while maintaining a potent anti-tumoral activity. Thus it is possible to manufacture T-cells from third-party healthy donors to generate allogeneic “off-the-shelf” engineered CAR T-cells. Disruption of CD52 or deoxycytidine kinase genes may be a useful approach to makes T-cells compatible with concurrent oncology treatments such as alemtuzumab or Fludarabine while the bypass of key immune checkpoint regulators such PD1/PDL1 by inactivation of T-cells receptors would potentiate the antitumor T-cell response by impairing the interaction of the inhibitory receptor PD-1 on T cells with PD-L1 expressed on tumor cells. Here, we will present in vitro and in vivo proof of concepts demonstrating the potential of TALEN™-mediated gene editing for adoptive T-cell therapy. Citation Format: Julien Valton, Anne-Sophie Gautron, Alexandre Juillerat, Philippe Duchateau, Laurent Poirot. Targeted genome modifications for improved adoptive immunotherapy. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A080.

The "flavin destructase" enzyme BluB catalyzes the unprecedented conversion of flavin mononucleotide (FMN) to 5,6-dimethylbenzimidazole (DMB), a component of vitamin B(12). Because of its unusual chemistry, the mechanism of this transformation has remained elusive. This study reports the identification of 12 mutant forms of BluB that have severely reduced catalytic function, though most retain the ability to bind flavin. The "flavin destructase" BluB is an unusual enzyme that fragments the flavin cofactor FMNH(2) in the presence of oxygen to produce 5,6-dimethylbenzimidazole (DMB), the lower axial ligand of vitamin B(12) (cobalamin). Despite the similarities in sequence and structure between BluB and the nitroreductase and flavin oxidoreductase enzyme families, BluB is the only enzyme known to fragment a flavin isoalloxazine ring. To explore the catalytic residues involved in this unusual reaction, mutants of BluB impaired in DMB biosynthesis were identified in a genetic screen in the bacterium Sinorhizobium meliloti. Of the 16 unique point mutations identified in the screen, the majority were located in conserved residues in the active site or in the unique "lid" domain proposed to shield the active site from solvent. Steady-state enzyme assays of 12 purified mutant proteins showed a significant reduction in DMB synthesis in all of the mutants, with eight completely defective in DMB production. Ten of these mutants have weaker binding affinities for both oxidized and reduced FMN, though only two have a significant effect on complex stability. These results implicate several conserved residues in BluB's unique ability to fragment FMNH(2) and demonstrate the sensitivity of BluB's active site to structural perturbations. This work lays the foundation for mechanistic studies of this enzyme and further advances our understanding of the structure-function relationship of BluB.

Homing endonucleases represent protein scaffolds that provide powerful tools for genome manipulation, as these enzymes possess a very low frequency of DNA cleavage in eukaryotic genomes due to their high specificity. The basis of protein-DNA recognition must be understood to generate tailored enzymes that target the DNA at sites of interest. Protein-DNA interaction engineering of homing endonucleases has demonstrated the potential of these approaches to create new specific instruments to target genes for inactivation or repair. Protein-DNA interface studies have been focused mostly on specific contacts between amino acid side chains and bases to redesign the binding interface. However, it has been shown that 4 bp in the central DNA sequence of the 22-bp substrate of a homing endonuclease (I-CreI), which do not show specific protein-DNA interactions, is not devoid of content information. Here, we analyze the mechanism of target discrimination in this substrate region by the I-CreI protein, determining how it can occur independently of the specific protein-DNA interactions. Our data suggest the important role of indirect readout in this substrate region, opening the possibility for a fully rational search of new target sequences, thus improving the development of redesigned enzymes for therapeutic and biotechnological applications.