Abstract: Chimeric Antigen Receptor (CAR) T cell therapy is emerging as a potential treatment for solid tumors, even if only limited activity has been observed for CAR T therapies to date. Cellular therapies face indeed many hurdles in solid tumors, such as the immunosuppressive microenvironment. TGFβ is an important growth factor of the tumor microenvironment and has been shown to suppress anti-tumor immunity. Gene editing represents a powerful way to enhance properties of CAR T cells and can be used to circumvent the effect of TGFβ signaling. The tumor associated antigen mesothelin (MSLN) is an attractive target for cellular therapy; being expressed at high levels in several tumor types (e.g., pleural mesothelioma and pancreatic cancer) while only modestly expressed in healthy tissues. Methods UCARTMeso, an allogeneic CAR T cell product targeting MSLN expressing cells is being developed by Cellectis. UCARTMeso bears an anti-MSLN CAR and a triple gene knock-out (KO) for TRAC, CD52 and TGFBR2 genes, all generated using TALEN® gene-editing technology. TRAC KO limits the risk of GvHD, while CD52 KO allows the use of alemtuzumab in the preconditioning regimen. The additional KO of TGFBR2 confers resistance to the immunomodulatory effects of TGFβ within the solid tumor microenvironment. Results Preclinical studies showed high specificity of the anti-MSLN CAR, as well as potent anti-tumor activity in vitro against different cell lines expressing MSLN. In addition, this activity was confirmed in mouse studies against pancreatic and pleural mesothelioma tumor models, with comparable activities observed in the latest model upon i.v. or intra-pleural administration of UCARTMeso. Also, we observed that TGFBR2 edited anti-MSLN CAR T cells displayed a blockade in the TGFβ signaling pathway, being able to respond to antigen stimulation in the presence of TGFβ (figure 1). Abstract 143 Figure 1 Left panel: TGFβ-induced SMAD2/3 phosphorylation in anti-MSLN CAR T cells. UCARTMeso cells were stained with mesothelin recombinant protein for CAR detection and anti-pSMAD2/3 one hour post exposure to TGFβ. The lack of SMAD2/3 phosphorylation in TGFBR2 KO cells indicates that they are unable to trigger TGFβ signaling. Right panel: Antigen-induced anti-MSLN CAR T cell activation in the presence (blue histogram) or absence (red histogram) of TGFβ. CAR T cells were stained with anti-CD25 antibody and analyzed by flow cytometry 5 days post exposure to antigen ± TGFβ. The data shows that cells not edited at the TGFBR2 locus are unable to be activated upon target exposure in the presence of TGFβ, while edited cells were activated in the presence of TGFβ, triggering CD25 expression at similar levels as those of cells activated in the absence of TGFβ. Conclusions Altogether, we have demonstrated potent antitumor activity in vitro and in vivo, and that addition of the third knock-out of TGFBR2 gene provide valuable additional properties to UCARTMeso cells, representing a very attractive strategy for their use in the treatment of solid tumors.

Abstract: Adoptive immunotherapy with autologous T-cells expressing chimeric antigen receptors (CARs) targeting CD19 has achieved long-term remissions in patients with B cell leukemia, pointing out that CAR technology may become a new alternative in cancer treatment. In this work we assessed the feasibility of targeting the CS1 antigen (SLAMF7) for the treatment of Multiple Myeloma (MM). MM is a B-cell neoplasia characterized by clonal expansion of malignant plasma cells in the bone marrow. Even if currently available therapies can improve overall survival, MM still remains incurable in most patients. Immunotherapy against MM is therefore an area in which extensive research is being made, with novel antigenic targets being considered. Among these is the CS1 glycoprotein, which is highly expressed on tumor cells from most patients with MM. However, CS1 is also expressed on normal CD8+ T-cells, which may be problematic for a CAR-based approach as antigen-expressing T cells will be targeted, impacting both the number and the phenotype of the final CAR T cell population. To circumvent this issue we have used our highly-efficient transcription activator-like effector nuclease (TALEN) gene-editing technology to inactivate CS1 in T-cells prior to transduction with a viral vector encoding an anti-CS1 CAR. Our results demonstrate that while non-gene-edited T-cells expressing an anti-CS1 CAR display limited cytolytic activity against MM cell lines, and resulted in a progressive loss of CD8+ T-cells. CS1-gene-edited CAR cells display significantly increased cytotoxic activity, with the percentage of CD8+ T-cells remaining unaffected. In addition, experiments in an orthotopic MM mouse model showed that CS1 disrupted T-cells were able to mediate an in vivo anti-tumoral activity. Subsequently, we have utilized this strategy for CS1 in the context of our allogeneic "off-the-shelf" engineered CAR+ T-cell platform. This allogenic platform utilizes TALEN gene editing technology to inactivate the TCRα constant (TRAC) gene, eliminating their potential to mediate Graft versus Host Disease (GvHD). We have previously shown that editing of the TRAC gene can be achieved at high frequencies, allowing efficient production of TCR-deficient T-cells that no longer mediate alloreactivity in a xeno-GvHD mouse model. Our results also show that multiplex genome editing is possible and can lead to the production of double KO (TRAC and CS1) T-cells, allowing large scale manufacturing of allogeneic, non alloreactive CS1 specific T-cells with enhanced antitumor activity. Moreover, these allogenic T-cells could be easily available for administration to a large number of MM patients. Disclosures Galetto: Cellectis SA: Employment. Chion-Sotinel:Cellectis SA: Employment. Gouble:Cellectis SA: Employment. Smith:Cellectis: Employment, Patents & Royalties.

Abstract: Autologous T-cells engineered to express chimeric antigen receptors (CARs) that target specific tumor antigens are known to be of high potential in treating different kinds of cancer. However, they must be generated on a “per patient” basis, thereby limiting the population of patients that could benefit from this approach. In particular, immune homeostasis may be affected in heavily pre-treated patients, such that autologous T-cells may be low in number, not fully functional, or unable to expand, thereby restricting the amount of cells that could be manufactured. The use of allogeneic T-cells isolated from healthy third party donors could constitute an easy-to-scale-up alternative, producible in advance, with potential for standardized quality controls, better batch consistency, and immediate availability for administration to a larger number of patients. In this context, we have established a highly efficient, 18-day, good manufacturing practice (GMP)–compatible process to produce CAR T-cells from healthy donor peripheral blood mononuclear cells (PBMCs). To circumvent the potential of allogeneic T-cells inducing graft-versus-host disease (GvHD) in recipient patients, the TCR alpha constant (TRAC) gene was inactivated using a proprietary transcription activator-like effector nuclease (TALEN™)-mediated gene editing technology. The CD52 gene was also disrupted using another specific TALEN™ to allow the administration of engineered T-cells following an alemtuzumab-based lymphodepleting therapy. The antitumor activity of these double-knockout CAR T-cells was shown to be as potent as non-nuclease-edited cells expressing the same CAR in vitro. The current manufacturing process is highly reproducible, making it suitable for use in a larger scale manufacturing platform for administration as “off-the-shelf” immunopharmaceuticals. We estimate that a single production run, starting from a healthy volunteer leukapheresis product containing 109 PBMCs, would allow the production of up to 500 doses of CAR double-knockout T-cells, at 2x107 cells per dose, allowing the extension of CAR therapies to a larger number of patients. Our results provide the proof of concept for the general applicability of this approach as a platform for large-scale GMP–compliant manufacturing of allogeneic, off-the-shelf, non-alloreactive, frozen CAR T-cells. From this manufacturing platform, we produced UCART19 cells, which are TCR/CD52-deficient, RQR8+ (as a safety attribute), and anti-CD19 CAR+, to investigate their potential in the treatment of CD19+ B cell leukemias. We believe this adaptable manufacturing platform offers multiple opportunities to improve CAR T-cell therapies through multiplex genome editing, such as rendering UCART cells resistant to standard chemotherapy or to tumor evasion mechanisms. Disclosures Derniame: Cellectis SA: Employment. Poirot:Cellectis SA: Employment. Schiffer-Mannioui:Cellectis SA: Employment. Galetto:Cellectis SA: Employment. Beurdeley:Cellectis SA: Employment. Reynier:Cellectis SA: Employment. Arnould:Cellectis SA: Employment. Smith:Cellectis SA: Employment.

Abstract: Background: CD123, the trans-membrane alpha chain of the interleukin-3 receptor (IL-3RA) is overexpressed in acute myeloid leukemia (AML) and distinguishes leukemia stem cells from their normal counterparts. There are several novel therapeutics under development to target CD123 in AML, including CD123 fused to Diphtheria toxin, a recombinant chimeric anti-CD123 MoAb, CD3/CD123 bi-specific T cell engagers, and engineered T cells that express chimeric antigen receptors (CARs). Thus, accurate detection and quantification of CD123 is critical for newly diagnosed and relapsed patients, and to follow minimal residual disease for patients in remission. Our data suggest that the evaluation of CD123 by flow cytometry varies significantly with different antibody clones. Objective: To identify the most accurate flow cytometry method for evaluation of CD123 expression in patients with AML to evaluate CD123 targeting therapies. Methods: 51 AML patient samples and 7 normal cord blood or bone marrow samples were stained with five different commercially available monoclonal antibodies to detect CD123 (7G3, 6H6, 9F5, AC145 and FAB301P), as well as CD45 and CD5, for evaluation by multiparameter flow cytometry. CD123 gene expression was also compared between these primary AML samples and bone marrow samples from healthy donors. Cell surface expression (by percentage and MFI) was evaluated relative to transcriptional expression and sensitivity to known therapeutics (cytarabine, parthenolide, and HSP90 inhibitors). Results: We observed CD123 surface expression patterns varied between the antibody clones tested. For the 9F5 and 6H6 clones, 93% and 82% of the samples, respectively, showed 〉 60% CD123+ cells whereas for the 7G3, FAB 301P and AC145 clones, 71 to 76% of the samples showed 〉 60% positivity. Also, surface expression of CD123 using 7G3, AC 145 and FAB 301P did not correlate with transcript levels for IL3RA assessed using qPCR, while surface expression of CD123 using 9F5 and 6H6 did correlate with transcript levels of IL3RA, using both mean fluorescence intensity (MFI) and percentage. For example, the correlation between CD123 surface expression as measured by percentage and IL3RA transcripts was most significant using the 9F5 and 6H6 clone (R2=0.1084, p=0.0183, R2=0.1588, p=0.0038 respectively) whereas the correlation for 7G3 (R2=0.0004, p=0.8945), FAB301P (R2=0.0027, p=0.7151) and AC145 (R2=0.0392, p=0.1638) were not significant. Surface expression of CD123 evaluated with 7G3 antibody did not correlate with overall sensitivity to in vitro treatment with cytarabine (R2=0.03767, p= 0.6451). However, using the 9F5 antibody, we found that higher levels of surface CD123 were associated with resistance to cytarabine in vitro (R2= 0.5502, p= 0.0351). Differences were noted for other experimental therapeutics including parthenolide and PU-H71. Most importantly, when we tested the ability of a novel allogeneic anti-CD123 CAR T-cell therapy (UCART123) to eliminate CD123+ AML cells, we found that CD123 positivity as measured by the 7G3 clone was not predictive of sensitivity to UCART123 in vitro or in vivo AML patient derived xenotransplants. Conclusions: Several novel therapeutic modalities targeting CD123 in AML are under development, including allogeneic anti-CD123 CAR T-cell therapy. Accurate, quantitative assessment of CD123 expression is thus of utmost importance for patient selection in clinical trials as well as disease monitoring. We found discrepancies between antibody clones, and such discrepancies may alter patient selection and data interpretation regarding patient response to CD123 based therapies. For therapies targeting CD123, protocol design and antibody selection should be done considering the results in this study. Based on our findings we recommend 9F5 or 6H6 antibody clones as well as the utilization of qPCR along side flow cytometry for adequate detection. Flow cytometry findings should be reported as percent positive cells. If utilizing the 9F5 clone, samples with 〉 60% CD123+ should be considered positive for CD123. A comparison in a large cohort may be warranted to determine the impact of multiple CD123 measurements on disease outcome. Disclosures Galetto: Cellectis SA: Employment. Gouble:Cellectis: Employment. Smith:Cellectis SA: Employment. Roboz:Agios, Amgen, Amphivena, Astex, AstraZeneca, Boehringer Ingelheim, Celator, Celgene, Genoptix, Janssen, Juno, MEI Pharma, MedImmune, Novartis, Onconova, Pfizer, Roche/Genentech, Sunesis, Teva: Consultancy; Cellectis: Research Funding. Guzman:Cellectis: Research Funding.

Abstract: Adoptive immunotherapy using autologous T-cells endowed with chimeric antigen receptors (CARs) has emerged as a promising new approach to treating cancer. However, a limitation of this approach is that CAR T-cells must be generated on a bespoke basis. To overcome this limitation, we have developed an allogeneic based platform for large scale production of “off-the-shelf” CAR T-cells from unrelated 3rd party donors. This platform utilizes Transcription Activator-Like Effector Nuclease (TALENTM) gene editing technology to inactivate the TCRα constant (TRAC) gene, eliminating the potential for T-cells bearing alloreactive TCR’s to mediate Graft versus Host Disease (GvHD). We have previously demonstrated that editing of the TRAC gene can be achieved at high frequencies, obtaining up to 80% of TCRαβ negative cells. This allows us to efficiently produce TCR-deficient T-cells that have been shown to no longer mediate alloreactivity in a xeno-GvHD mouse model. Furthermore, the capacity to perform efficient multiplex genome editing using TALENTM offers the possibility of simultaneously rendering cells resistant to standard chemotherapy or to tumor evasion mechanisms. In this work we present the adaptation of this allogeneic platform to the production of T cells targeting CD123, the transmembrane alpha chain of the interleukin-3 receptor, which is expressed in tumor cells from the majority of patients with Acute Myeloid Leukemia (AML). In a first step, we have screened human primary T-cells harboring CARs with different antigen recognition domains in the context of multiple CAR architectures in order to identify candidates displaying specific activity against cell lines expressing variable levels of the CD123 antigen. To provide proof of concept for the general applicability of the allogeneic approach we have manufactured a TCR-deficient CD123 CAR T-cell (UCART123) and demonstrated that this product maintains a potent anti-tumoral activity in vitro. Experiments in an orthotopic AML mouse model using UCART123 cells are currently ongoing, in order to establish the absence of alloreactivity and the anti-tumoral activity in vivo. The ability to carry out large scale manufacturing of allogeneic, non alloreactive CD123 specific T Cells from a single healthy donor could thus offer the possibility of an off-the-shelf treatment that would be immediately available for administration to a large number of AML patients. Disclosures Galetto: Cellectis SA: Employment. Lebuhotel:Cellectis SA: Employment. Gouble:Cellectis SA: Employment. Schiffer-Mannioui:Cellectis SA: Employment. Smith:Cellectis SA: Employment.

Abstract: Background: Acute myeloid leukemia (AML) is the most common form of acute leukemia in adults, with an incidence that increases with age, and a generally poor prognosis. The prognosis remains especially grim for those who are older, have secondary AML, or relapsed or refractory (R/R) disease, in which 5-year OS is 5-10%. Therefore, novel therapeutic approaches are needed. CD123(IL3Rα) is a cell surface target that is expressed on normal, committed hematopoietic progenitor cells, and a variety of hematological neoplasms, including AML, myelodysplastic syndrome (MDS), and blastic plasmacytoid dendritic cell neoplasm (BPDCN). UCART123 is genetically modified, allogeneic ("off-the-shelf"), anti-CD123 CAR T cell product candidate in which the TCR alpha constant gene is disrupted to reduce the risk of GvHD, and the CD52 gene is disrupted to permit the use of alemtuzumab for selective and prolonged host lymphodepletion. Also, the CAR is co-expressed with a suicide mechanism (RQR8), which can be activated by using rituximab. In vitro data have demonstrated that UCART123 efficiently targets primary AML cells, with minimal effect on normal progenitors. Also, in PDX mouse models of AML, UCART123 cells can eliminate tumor cells in vivo, prevent relapse, and improve survival; in a competitive BM/AML PDX model, UCART123 cells demonstrated preferential targeting of AML blasts (Guzman; Blood 2016). Methods: AMELI-01 is a phase 1, multi-center clinical trial of UCART123 that employs an mTPI design to evaluate the safety, tolerability and preliminary anti-leukemia activity of UCART123 in patients (pts) with R/R AML. Additional objectives include determination of the MTD; characterization of the expansion, trafficking and persistence of UCART123; assessment of cytokine, chemokine and CRP levels after UCART123 infusion; and assessment of immune cell depletion, reconstitution and immune response. Dose escalation will include up to 28 pts. The dose expansion portion follows a Simon 2-stage design and will enroll up to an additional 37 pts. Eligible pts must be ≤ 65 years of age with R/R AML, adequate organ function, a confirmed donor for potential back-up stem cell transplantation, and no ongoing & gt; G1 toxicity from prior treatment. Pts with APL, prior gene or cellular therapy, & gt; 1 allogeneic SCT, or those with a clinically relevant CNS disorder (including CNS leukemia) are not eligible. Pts receive a lymphodepletion (LD) regimen of either fludarabine and cyclophosphamide (FC) or fludarabine, cyclophosphamide plus alemtuzumab (FCA) starting on Day -5, followed by an infusion of UCART123 at one of 5 dose levels on Day 0. Pts are evaluated for the presence of dose-limiting toxicities (DLT) during a 28-day observation period, which extends to 42 days in the setting of marrow aplasia and/or persistent clinically significant cytopenias without residual AML. DL1 has cleared safety without DLT, and enrollment at the next dose levels are proceeding. ClinicalTrials.gov Identifier: NCT03190278 Monica L. Guzman, et al; Allogeneic TCRα/β Deficient CAR T-Cells Targeting CD123 Prolong Overall Survival of AML Patient-Derived Xenografts. Blood 2016; 128 (22): 765. doi: https://doi.org/10.1182/blood.V128.22.765.765 Disclosures Roboz: Orsenix: Consultancy; Otsuka: Consultancy; Takeda: Consultancy; Trovagene: Consultancy; Cellectis: Research Funding; Jasper Therapeutics: Consultancy; Epizyme: Consultancy; Helsinn: Consultancy; MEI Pharma: Consultancy; Novartis: Consultancy; Janssen: Consultancy; Pfizer: Consultancy; Agios: Consultancy; Celgene: Consultancy; Astex: Consultancy; Amphivena: Consultancy; Abbvie: Consultancy; Array BioPharma: Consultancy; Bayer: Consultancy; Celltrion: Consultancy; Eisai: Consultancy; Jazz: Consultancy; Roche/Genentech: Consultancy; Sandoz: Consultancy; Actinium: Consultancy; Argenx: Consultancy; Astellas: Consultancy; Daiichi Sankyo: Consultancy; AstraZeneca: Consultancy. DeAngelo:Blueprint Medicines Corporation: Consultancy, Research Funding; Forty-Seven: Consultancy; Amgen: Consultancy; Abbvie: Research Funding; Glycomimetics: Research Funding; Shire: Consultancy; Takeda: Consultancy; Pfizer: Consultancy; Novartis: Consultancy, Research Funding; Jazz: Consultancy; Autolos: Consultancy; Incyte Corporation: Consultancy; Agios: Consultancy. Sallman:Agios, Bristol Myers Squibb, Celyad Oncology, Incyte, Intellia Therapeutics, Kite Pharma, Novartis, Syndax: Consultancy; Celgene, Jazz Pharma: Research Funding. Guzman:SeqRx: Honoraria; Cellectis: Research Funding. Kantarjian:Delta Fly: Honoraria; Novartis: Honoraria, Research Funding; Adaptive biotechnologies: Honoraria; Ascentage: Research Funding; Amgen: Honoraria, Research Funding; BMS: Research Funding; Daiichi-Sankyo: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding; Jazz: Research Funding; Actinium: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sanofi: Research Funding; BioAscend: Honoraria; Janssen: Honoraria; Oxford Biomedical: Honoraria; Immunogen: Research Funding; Aptitute Health: Honoraria. Konopleva:Genentech: Consultancy, Research Funding; Rafael Pharmaceutical: Research Funding; Ascentage: Research Funding; AstraZeneca: Research Funding; Agios: Research Funding; Amgen: Consultancy; Sanofi: Research Funding; F. Hoffmann La-Roche: Consultancy, Research Funding; Stemline Therapeutics: Consultancy, Research Funding; Forty-Seven: Consultancy, Research Funding; Eli Lilly: Research Funding; Kisoji: Consultancy; Ablynx: Research Funding; AbbVie: Consultancy, Research Funding; Reata Pharmaceutical Inc.;: Patents & Royalties: patents and royalties with patent US 7,795,305 B2 on CDDO-compounds and combination therapies, licensed to Reata Pharmaceutical; Calithera: Research Funding; Cellectis: Research Funding. Bejanyan:Kiadis Pharma: Membership on an entity's Board of Directors or advisory committees. Esteva:Cellectis: Current Employment. Garton:Cellectis: Current Employment. Backhouse:Cellectis: Current Employment. Galetto:Cellectis: Current Employment. Brownstein:Cellectis: Current Employment. Pemmaraju:Blueprint Medicines: Honoraria; Roche Diagnostics: Honoraria; MustangBio: Honoraria; AbbVie: Honoraria, Research Funding; SagerStrong Foundation: Other: Grant Support; Plexxikon: Research Funding; Novartis: Honoraria, Research Funding; LFB Biotechnologies: Honoraria; Stemline Therapeutics: Honoraria, Research Funding; Celgene: Honoraria; Affymetrix: Other: Grant Support, Research Funding; Incyte Corporation: Honoraria; DAVA Oncology: Honoraria; Samus Therapeutics: Research Funding; Cellectis: Research Funding; Daiichi Sankyo: Research Funding; Pacylex Pharmaceuticals: Consultancy.

Abstract: 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. A limitation of the current autologous approach is that CAR T-cells must be manufactured on a "per patient basis". We have developed a standardized platform for manufacturing T-cells from third-party healthy donors to generate allogeneic "off-the-shelf" engineered CD19-CAR+ T-cell–based frozen products. Our platform involves the use of transcription activator-like effector nucleases (TALEN™), which mediate the simultaneous inactivation of two genes through genome editing. The knockout of the TCR alpha gene eliminates TCR expression and is intended to abrogate the donor T-cell’s potential for graft-versus-host disease (GvHD), while knocking out the CD52 gene makes donor T-cells resistant to the lymphodepleting agent alemtuzumab. In addition, our T-cells are engineered to coexpress the RQR8 gene as a safety feature, with the aim of rendering them sensitive to the monoclonal antibody rituximab. We previously provided proof-of-concept for the application of this approach by manufacturing TCR/CD52-deficient RQR8+ and CD19-CAR+ T-cells (UCART19) using a good manufacturing practice–compatible process, and we also demonstrated that the resulting UCART19 cells were functional using in vitro assays. Here we report the ability of UCART19 cells to engraft into an orthotopic human CD19+ lymphoma xenograft immunodeficient mouse model. UCART19 cells exhibited antitumor activity equivalent to that of standard CD19 CAR T-cells. We also demonstrated that UCART19 cells did not mediate alloreactivity in a xeno-GvHD mouse model. Furthermore, the effectiveness of the rituximab-induced depletion mechanism of RQR8+ cells was shown in an immunocompetent mouse model. In conclusion, our work significantly enlarges upon previous results by showing in vivo that (1) concomitant inactivation of a second gene has no deleterious effects on T-cells, (2) the antitumor potency of manufactured TCR/CD52-deficient CD19–CAR+ T-cells is similar to that of standard CD19-CAR+ T-cells, (3) TCR gene inactivation is efficient at preventing potential graft-versus-host reaction, and (4) allogeneic T-cells can be depleted by the use of rituximab. This valuable dataset supports the development of allogeneic CAR T-cells, and UCART19 will be investigated in an exploratory, first-in-human, clinical trial where refractory/relapsed CD19+ B-cell leukemia patients are to be enrolled. Disclosures Gouble: Cellectis SA: Employment. Poirot:Cellectis SA: Employment. Schiffer-Mannioui:Cellectis SA: Employment. Galetto:Cellectis SA: Employment. Derniame:Cellectis SA: Employment. Arnould:Cellectis SA: Employment. Desseaux:Cellectis SA: Employment. Smith:Cellectis SA: Employment.