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Off-the-shelf allogeneic CAR T cells derived from healthy donor cells have the potential to overcome many of the issues associated with the time-consuming manufacturing of autologous CAR T cells. However, adoptive transfer of allogeneic T cells carries the risk of graft-versus-host disease (GvHD). Most of the clinical experience with allogeneic CAR T cells is based on gene editing to eliminate T cell receptor (TCR) to mitigate the risk of GvHD. While clearly effective, the downsides of gene editing include multiple manufacturing steps requiring multiple clinical grade reagents, thus extending culture times, which can be associated with T cell exhaustion. As an alternative, we have explored short hairpin RNA (shRNA) as a means to knockdown TCR expression at the mRNA level. This shRNA is co-expressed along with the CAR in a single clinical grade vector, therefore requiring just one step of genetic modification. CYAD-211 is an allogeneic anti-BCMA CAR T that co-expresses a shRNA targeting CD3z which results in reduction of cell surface TCR expression. IMMUNICY-1 is an ongoing open-label Phase 1 trial (NCT04613557) designed to evaluate CYAD-211 in adult patients with refractory or relapsed multiple myeloma (MM) following at least two prior MM regimens. Patients receive non-myeloablative preconditioning (cyclophosphamide 300 mg/m²/day and fludarabine 30 mg/m²/day, for 3 days) followed by a single CYAD-211 infusion in a 3+3 dose escalation design evaluating three dose-levels (DL): 30x10 6, 100x10 6 and 300x10 6 cells/infusion. As of July 29, 2021, nine patients were enrolled across the 3 DLs. Patients had received a median of four prior lines of treatment. Seventy-eight percent of patients were previously exposed to all three major MM drug classes (proteasome inhibitors, immunomodulatory drugs, and anti-CD38 antibody therapy). Eight patients had prior autologous stem cell transplantation. CYAD-211 was well tolerated. One patient developed grade 1 cytokine release syndrome. Two patients had Grade ≥ 3 hematologic toxicities possibly related to the experimental treatment. Two patients experienced infectious adverse events (1 grade 1 rhinitis and 1 grade 2 upper respiratory infection). There was no neurologic toxicity and no GvHD. There was no dose-limiting toxicity. Eight patients were evaluated for activity per IMWG criteria. Two patients achieved partial response at dose-levels 1 and 2 while 5 patients had stable disease (SD). One patient with an ongoing SD (3 months +) showed evidence of reduction in size of plasmacytomas. Analysis of peripheral blood samples by molecular methods confirmed the engraftment of CYAD-211. All patients had detectable CAR T cells. However, the engraftment was short lasting (3-4 weeks). There was a correlation between the depth of lymphodepletion and engraftment. There was also a dose-response in terms of CYAD-211 kinetics with a level neighboring 8,000 copies of CAR T per microgram of input DNA in patients at DL3. These early data indicate that CYAD-211 is well tolerated with a good safety profile. While further study is required to fully understand the anti-BCMA potency of the CAR used in this trial, the lack of observed GvHD despite engraftment of CYAD-211 provides proof of concept of the safe administration of CAR T using a shRNA-allogeneic platform. The lack of sustained engraftment of CYAD-211 can be explained by rejection of the allogeneic cells by the recovering immune system of the recipient and calls for exploring the role of augmented lymphodepletion. Furthermore, given the ability to include multiple shRNA within the single CAR vector, future strategies will also examine knocking down other molecules that are important in driving immune rejection. Disclosures Al-Homsi: BMS: Other: Independent Medical Education Grant; Daichii Sankyo: Consultancy; Celyad Oncology: Other: Advisory Board. Deeren: Alexion: Consultancy; BMS: Consultancy; Incyte: Consultancy; Novartis: Consultancy; Sanofi: Consultancy, Research Funding; Sobi: Consultancy; Takeda: Consultancy. Nishihori: Karyopharm: Research Funding; Novartis: Research Funding. Meuleman: iTeos Therapeutics: Consultancy. Abdul-Hay: Amgen: Membership on an entity's Board of Directors or advisory committees; Takeda: Speakers Bureau; Abbvie: Consultancy; Jazz: Other: Advisory Board, Speakers Bureau; Servier: Other: Advisory Board, Speakers Bureau. Braun: Celyad Oncology: Current Employment. Lonez: Celyad Oncology: Current Employment. Dheur: Celyad Oncology: Current Employment. Alcantar-Orozco: Celyad Oncology: Current Employment. Gilham: Celyad Oncology: Current Employment. Flament: Celyad Oncology: Current Employment. Lehmann: Celyad Oncology: Current Employment.

While CAR T cell therapy has delivered impressive clinical efficacy in B cell malignancies, similar levels of clinical activity have not been demonstrated in acute myeloid leukemia (AML). One underlying reason for this lack of translation is the relative paucity of validated CAR T targets. Major Histocompatibility Complex Class 1 related proteins MICA and MICB along with the UL16 binding proteins 1-6 (ULBP1-6) are frequently over-expressed on AML and myelodysplastic syndrome (MDS) blasts. The Natural Killer Group 2D (NKG2D) is a single receptor able to bind MICA, MICB and ULBP1-6, thus providing a potentially powerful approach that could be exploited to target this family of targets thereby providing a novel CAR T approach for AML/MDS. Expressing the NKG2D receptor fused to the human CD3z cytoplasmic domain generates a CAR that, when expressed in primary T cells, generates a CAR T product (termed CYAD-01) that showed good anti-tumor activity in preclinical models. In clinical trials, CYAD-01 showed a good tolerability profile but with a disappointing level of clinical activity when combined with cyclophosphamide / fludarabine preconditioning (DEPLETHINK trial, NCT03466320). We hypothesised that the transient expression of MICA/MICB on the CAR T itself may inhibit the activity of the NKG2D CAR through self-targeting and consequent fratricide. We developed a single shRNA able to knockdown both MICA and MICB. Co-expression of this shRNA with the NKG2D CAR generated a second generation CAR T product termed CYAD-02 which was examined in a highly similar AML/MDS patient population and preconditioning regimen as employed in the DEPLETHINK trial. CYAD-02 was evaluated in the the first-in-human CYCLE-1 trial (NCT04167696) in patients with r/r AML/MDS. The dose-escalation phase evaluated three dose-levels (DL) (1x10 8, 3x10 8 and 1x10 9 total cells per infusion), administered as a single infusion after standard preconditioning chemotherapy (cyclophosphamide 300 mg/m²/day and fludarabine 30 mg/m²/day, daily for 3 days) according to a classic 3+3 design. As of July 2021, 11 patients have been treated with CYAD-02 at the different dose-levels (3 at each DL1 and 2 and 5 at DL3). In terms of safety and tolerability, there was no evidence of a differential safety profile of CYAD-02 within the CYCLE-1 trial as compared to that observed in the CYAD-01 clinical trial. Of note, five patients enrolled in all 3 DLs experienced Grade ≥ 3 adverse events (AE) at least possibly related to CYAD-02 (cytokine release syndrome, febrile neutropenia, white blood cell count decreased, infusion related reaction). With respect to clinical activity, seven patients in total achieved stable disease (2 at DL1, 3 at DL2 and 2 at DL3) with 4 demonstrating evidence of transient blast reduction or anti-leukemic activity (decrease of at least 50% of the bone marrow blasts). In addition, two MDS patients at DL3 achieved a marrow complete response. Overall, this suggests dose dependent response to CYAD-02. Furthermore, patients receiving CYAD-02 cells seem to display a greater duration of response and stronger blast reduction as compared to patients who received CYAD-01 in DEPLETHINK. Interestingly, peak engraftment levels were higher for CYAD-02 compared to CYAD-01 at the same dose (DEPLETHINK trial). At DL3, CYAD-02 cells could be readily detected in peripheral blood of 4/5 patients through month 2. In addition, and unlike what is reported in B cell CAR T-cells trials, there was very limited evidence of elevated homeostatic cytokines (IL-7/IL-15) following CyFlu administration with IL-15 not detected in any of the patients and IL-7 showing only a minor increase in 2 patients. The knockdown of MICA/MICB appears to have a positive contribution to the initial clinical activity of CYAD-02 as compared to that achieved with the first generation CYAD-01 CAR T, together with good safety and tolerability. The lack of homeostatic cytokines after preconditioning, likely limiting the engraftment and activity of CAR T cells, may be related to the biology of myeloid malignancies. One approach to further drive the potency of NKG2D-based CAR T cells would likely be armoring the CAR T through using the T cell as a vehicle to secrete cytokines alongside the CAR. Overall, shRNA knockdown technology provides a means to modify CAR T function and here shows that single shRNA can target two independent genes to enhance the phenotype of the CAR Ts. Disclosures Deeren: Alexion: Consultancy; BMS: Consultancy; Incyte: Consultancy; Novartis: Consultancy; Sanofi: Consultancy, Research Funding; Sobi: Consultancy; Takeda: Consultancy. Lin: AbbVie, Aptevo Therapeutics, Astellas Pharma, Bio-Path Holdings, Celgene, Celyad, Genentech-Roche, Gilead Sciences, Incyte, Jazz Pharmaceuticals, Novartis, Ono Pharmaceutical, Pfizer, Prescient Therapeutics, Seattle Genetics, Tolero, Trovagene: Research Funding. Alcantar-Orozco: Celyad Oncology: Current Employment. Dheur: Celyad Oncology: Current Employment. Breman: Celyad Oncology: Current Employment. Braun: Celyad Oncology: Current Employment. Lonez: Celyad Oncology: Current Employment. Gilham: Celyad Oncology: Current Employment. Flament: Celyad Oncology: Current Employment. Lehmann: Celyad Oncology: Current Employment.

Background CYAD-01 is a T-cell product engineered to express a chimeric antigen receptor (CAR) based on the NKG2D receptor (NKG2D CAR) which binds 8 ligands (MICA/B, ULBP1-6) over-expressed by a large variety of malignancies, including acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). The phase I THINK study (NCT03018405) evaluated the safety and clinical activity of multiple injections of CYAD-01 infused every 2 weeks, without preconditioning chemotherapy, in 13 relapsed/refractory (r/r) AML and MDS patients. While an encouraging objective response rate according to ELN2017 (AML) or revised IPSS (MDS) and reduction in bone marrow blasts were seen with good safety profile, the responses were short-lived (≤ 3 months - see ASH 2019, poster 3826). To enhance CAR T-cell persistence, we evaluated a weekly dose schedule without preconditioning (THINK study) or the addition of cyclophosphamide and fludarabine (CyFlu) as a preconditioning regimen prior to CAR T-cell infusion (phase I DEPLETHINK study, NCT03466320). Aim To further increase persistence and potency of the T-cell product, optimization of the previously used mAb manufacturing process was performed by shortening the duration of production along with modification of PI3K inhibitor. This optimized manufacturing process (termed "OptimAb") aimed to generate CYAD-01 cells with a higher frequency of early memory T-cells with high cytokine secretion upon activation, as compared to the original "mAb" process. Results As compared to the previous mAb manufacturing process, the OptimAb manufacturing process generates a product that secretes higher levels of IFN-γ upon co-culture with tumor cells and contains a higher frequency of CD62L+ T-cells in vitro, characteristic of an early memory phenotype. In an in vivo aggressive AML (THP-1) model, CYAD-01 OptimAb displayed a strong improvement in long-term anti-tumor activity as compared to the CYAD-01 mAb at the same dose chosen to have a minimal anti-tumor activity (stress-test dose, see figure). Based on these results, both THINK and DEPLETHINK clinical studies were amended to evaluate the OptimAb process. As of August 2020, 5 patients have been treated with multiple infusions of the OptimAb CYAD-01 as standalone treatment at the dose of 3x108 cells/infusion in the small expansion segment of the THINK study. 7 patients were treated with a single infusion of OptimAb CYAD-01 administered after a CyFlu preconditioning in the dose-escalation segment at the doses of 3x108 cells/infusion or 1x109 cells/infusion in the DEPLETHINK study. To date, the results demonstrate the safety and tolerability for CYAD-01 OptimAb with or without a prior lymphodepletion in patients with r/r AML and MDS. Preliminary data of the clinical and pharmacokinetics evaluation of CYAD-01 manufactured with the improved OptimAb process, as compared with the mAb process at the same dose, in two Phase I studies will be provided at the time of presentation. Conclusion/summary The autologous CYAD-01, a first generation NKG2D CAR T-cell product is currently investigated in r/r AML/MDS patients, a difficult to target disease due in part to the absence of truly AML-specific surface antigens, its rapid clinical progression and the absence of disease control by the CyFLu preconditioning. CYAD-01 manufactured using an optimized process, OptimAb, aims to improve CAR T-cell persistence and clinical responses. The data analysis of the same CAR-T product with different manufacturing processes, with or without preconditioning chemotherapy, will provide the medical community with clinical and scientific insights to guide the future of this therapeutic modality. Figure Disclosures Sallman: Agios, Bristol Myers Squibb, Celyad Oncology, Incyte, Intellia Therapeutics, Kite Pharma, Novartis, Syndax: Consultancy; Celgene, Jazz Pharma: Research Funding. Al-Homsi:Celyad: Membership on an entity's Board of Directors or advisory committees. Pollyea:Janssen: Consultancy; 47: Consultancy, Research Funding; Amgen: Consultancy; Genentech: Consultancy; Novartis: Consultancy; Karyopharm: Consultancy; Syndax: Consultancy; Syros: Consultancy; Abbvie: Consultancy, Research Funding; Daiichi Sankyo: Consultancy; Takeda: Consultancy; Pfizer: Consultancy; Celgene/BMS: Consultancy; Agios: Consultancy; Glycomimetics: Other. Wang:Abbvie: Consultancy; Pfizer: Speakers Bureau; Genentech: Consultancy; Stemline: Speakers Bureau; PTC Therapeutics: Consultancy; Macrogenics: Consultancy; Astellas: Consultancy; Bristol Meyers Squibb (Celgene): Consultancy; Jazz Pharmaceuticals: Consultancy. Demoulin:Celyad Oncology: Current Employment. Sotiropoulou:Celyad Oncology: Current Employment. Alcantar-Orozco:Celyad Oncology: Current Employment. Breman:Celyad Oncology: Current Employment. Dheur:Celyad Oncology: Current Employment. Braun:Celyad Oncology: Current Employment. Lonez:Celyad Oncology: Current Employment. Gilham:Celyad Oncology: Current Employment. Flament:Celyad Oncology: Current Employment. Lehmann:Celyad Oncology: Current Employment.

Background Autologous CAR T-cell therapy targeting the B-cell maturation antigen (BCMA) has shown impressive objective response rates in patients with advanced multiple myeloma (MM). Clinical grade manufacturing of autologous CAR T-cells has limitations including vein-to-vein delivery time delay and potentially sub-optimal immunological capability of T-cells isolated from patients with advanced disease. Allogeneic CAR T-cell products, whereby cells from healthy third-party donors are used to generate an "off-the-shelf" CAR T-cell product, have the potential to overcome some of these issues. To circumvent the primary potential risk of graft-versus-host disease (GvHD) associated with the use of allogeneic T-cells, abrogation of the T-cell receptor (TCR) expression in the CAR T-cells, via gene editing, is being actively pursued. To avoid the potential safety risks and manufacturing challenges associated with gene editing, the allogeneic CYAD-211 CAR T-cell product exploits short hairpin RNA (shRNA) interference technology to down-regulate TCR expression thus avoiding the risk of life-threatening GvHD. Aim The aim is to generate a BCMA-specific allogeneic CAR T-cell product using a non-gene editing approach and study its activity both in vitro and in vivo. CYAD-211 combines a BCMA-specific CAR with a single optimized shRNA targeting the TCR CD3ζ subunit. Downregulation of CD3ζ impairs the TCR expression on the surface of the donor T-cells, preventing their reactivity with the normal host tissue cells and potential GvHD induction. Maintaining all the elements required for the therapy within a single vector (all-in-one vector) provides some significant manufacturing advantages, as a solitary selection step will isolate cells expressing all the desired traits. Results CYAD-211 cells produce high amounts of interferon-gamma (IFN-γ) during in vitro co-cultures with various BCMA-expressing MM cell lines (i.e., RPMI-8226, OPM-2, U266, and KMS-11). Cytotoxicity experiments confirmed that CYAD-211 efficiently kills MM cell lines in a BCMA-specific manner. The anti-tumor efficacy of CYAD-211 was further confirmed in vivo, in xenograft MM models using the RPMI-8226 and KMS-11 cell lines. Preclinical data also showed no demonstrable evidence of GvHD when CYAD-211 was infused in NSG mice confirming efficient inhibition of TCR-induced activation. Following FDA acceptance of the IND application, IMMUNICY-1, a first-in-human, open-label dose-escalation phase I clinical study evaluating the safety and clinical activity of CYAD-211 for the treatment of relapsed or refractory MM patients to at least two prior MM treatment regimens, is scheduled to begin recruitment. IMMUNICY-1 will evaluate three dose-levels of CYAD-211 (3x107, 1x108 and 3x108 cells/infusion) administered as a single infusion after a non-myeloablative conditioning (cyclophosphamide 300 mg/m²/day and fludarabine 30 mg/m²/day, daily for 3 days) according to a classical Fibonacci 3+3 design. Description of the study design and preliminary safety and clinical data from the first cohort will be presented at ASH 2020. Conclusion CYAD-211 is the first generation of non-gene edited allogeneic CAR T-cell product based on shRNA technology. The IMMUNICY-1 clinical study seeks to provide proof of principle that single shRNA-mediated knockdown can generate fully functional allogeneic CAR T-cells in humans without GvHD-inducing potential. We anticipate that subsequent generations of this technology will incorporate multiple shRNA hairpins within a single vector system. This will enable the production of allogeneic CAR T-cells in which multiple genes of interest are modulated simultaneously thereby providing a platform approach that can underpin the future of this therapeutic modality. Figure 1 Disclosures Al-Homsi: Celyad: Membership on an entity's Board of Directors or advisory committees. Brayer:Janssen: Consultancy; Bristol-Myers Squibb, WindMIL Therapeutics: Research Funding; Bristol-Myers Squibb, Janssen, Amgen: Speakers Bureau. Nishihori:Novartis: Other: Research support to institution; Karyopharm: Other: Research support to institution. Sotiropoulou:Celyad Oncology: Current Employment. Twyffels:Celyad Oncology: Current Employment. Bolsee:Celyad Oncology: Current Employment. Braun:Celyad Oncology: Current Employment. Lonez:Celyad Oncology: Current Employment. Gilham:Celyad Oncology: Current Employment. Flament:Celyad Oncology: Current Employment. Lehmann:Celyad Oncology: Current Employment.

Background T-cells engineered to express a chimeric antigen receptor (CAR) based on the NKG2D receptor (NKG2D CAR) targeting the 8 NKG2D ligands (MICA/B, ULBP1-6) over-expressed by a large variety of malignancies have been developped to treat patients, including patients with acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Previously, CYAD-01, the first generation of NKG2D CAR T-cell products, was evaluated in several Phase I clinical trials and showed initial signals of objective clinical responses in patients with r/r AML and MDS, albeit with short durability. Preclinical data have shown that NKG2D ligands MICA and MICB are transiently upregulated on activated CAR T-cells, and target-dependent killing of CAR T-cells post-infusion can potentially occur, leading to short in vivo persistence. In an effort to increase the persistence and potency of the NKG2D CAR T-cells, CYAD-02 was developed as a next-generation product using a non-gene editing approach to silence the expression of MICA and MICB. Aim MICA and MICB were down-regulated by inserting a single optimized short hairpin RNA (shRNA) targeting both MICA and MICB within the NKG2D CAR construct. This next-generation NKG2D CAR T-cell product is manufactured with the OptimAb process, resulting in CAR T-cells with a higher frequency of early memory T-cells secreting high levels of cytokines upon activation, and is referred to as CYAD-02. Results As compared to CYAD-01, CYAD-02 cell expansion in vitro was 3-fold increased. In an in vivo AML model, CYAD-02 showed 10-fold higher engraftment 1 week after injection and improved anti-tumor activity as compared to CYAD-01 manufactured with the initial mAb process. This led to a 2.6-fold increase of mouse survival as compared to CYAD-01 in a stress-test aggressive AML model where the dose of CYAD-01 was titrated down for minimal activity (figure). The first-in-human study evaluating CYAD-02, the CYCLE-1 study (NCT04167696), has been initiated in early 2020 in patients with r/r AML/MDS. The study evaluates three dose-levels of CYAD-02 (1x108, 3x108 and 1x109 cells/infusion), administered as a single infusion after non-myeloablative preconditioning chemotherapy (cyclophosphamide 300 mg/m²/day and fludarabine 30 mg/m²/day, daily for 3 days, CyFlu) according to a classical Fibonacci 3+3 design. As of August 2020, 6 patients have been treated with CYAD-02 at the dose of 1x108 or 3x108 cells/infusion. To date, the results demonstrate the safety and tolerability for CYAD-02 in patients with r/r AML and MDS with no dose-limiting toxicity observed. The study is currently enrolling at 1x109 cells/infusion. The CYAD-02 safety profile and preliminary clinical activity data together with the pharmacokinetics evaluation from the complete dose escalation segment will be provided at the time of presentation. Conclusion/summary The CYAD-02 is the first autologous CAR T-cell product based on the non-gene edited shRNA technology used to treat patients. This next generation NKG2D CAR T-cell product is currently investigated in the CYCLE-1 Phase I study in r/r AML/MDS patient population, a difficult to target disease due in part to the absence of truly AML-specific surface antigens, its rapid clinical progression and the absence of disease control by the CyFlu preconditioning. Both the anti-MICA and MICB shRNA hairpin and the OptimAb manufacturing process for CYAD-02 aim to improve CAR T-cell persistence and clinical responses. Figure Disclosures Lin: Mateon Therapeutics: Research Funding; Aptevo: Research Funding; Abbvie: Research Funding; Ono Pharmaceutical: Research Funding; Incyte: Research Funding; Gilead Sciences: Research Funding; Jazz: Research Funding; Astellas Pharma: Research Funding; Bio-Path Holdings: Research Funding; Celgene: Research Funding; Celyad: Research Funding; Genetech-Roche: Research Funding; Seattle Genetics: Research Funding; Tolero Pharmaceuticals: Research Funding; Trovagene: Research Funding; Prescient Therapeutics: Research Funding; Pfizer: Research Funding. Demoulin:Celyad Oncology: Current Employment. Fontaine:Celyad Oncology: Current Employment. Sotiropoulou:Celyad Oncology: Current Employment. Alcantar-Orozco:Celyad Oncology: Current Employment. Breman:Celyad Oncology: Current Employment. Dheur:Celyad Oncology: Current Employment. Braun:Celyad Oncology: Current Employment. Lonez:Celyad Oncology: Current Employment. Gilham:Celyad Oncology: Current Employment. Flament:Celyad Oncology: Current Employment. Lehmann:Celyad Oncology: Current Employment.

Introduction: CYAD-01 is a chimeric antigen receptor T-cell (CAR-T) product based on the receptor NKG2D with specificity for a broad range of ligands (MICA, MICB and ULBP1-6) expressed on most tumors. In vivo preclinical studies showed long-term anti-tumor activity of CYAD-01, whilst not only targeting tumor cells but also cells from the tumor neo-vasculature and immunosuppressive environment in the absence of pre-conditioning therapy. Methods: Exploiting this unique mode of action of CYAD-01, the THINK trial (NCT03018405) is an open-label Phase I study assessing the safety and clinical activity of multiple CYAD-01 administrations without prior preconditioning in 2 parallel cohorts: one in patients (pt) with metastatic solid tumors and the other one in hematological malignancies, including relapsing/refractory (r/r) acute myeloid leukemia (AML), multiple myeloma (MM) and myelodysplastic syndrome (MDS). The dose escalation segment of the study evaluates 3 dose levels (DL; 3x108, 1x109 and 3x109 cells per injection) of one cycle of 3 CYAD-01 administration with 2-weeks intervals. The study has been amended at the stage of DL-2 to authorize a second cycle of 3 CYAD-01 administrations in case of no progressive disease after 2 months. Results: As of July 31, 2018, 12 pts in the hematological cohort (8 AML, 3 MM and 1 MDS) have been enrolled at the 3 DLs (6 pts in DL-1, 3 in DL-2 and 3 in DL-3) without prior preconditioning. Median age was 64 (range 29-83) and median number of prior therapies was 3. DL-3 (3 pts) has been fully accrued as of data cutoff. Over 34 injections, 5 pts experienced grade (G) 3/4 treatment-related AEs: in DL-1, one pt experienced G3 lymphopenia and a second pt experienced G4 lymphopenia and G4 pneumonitis in DL-2, one pt experienced G3 lymphopenia and G3 thrombocytopenia and two other pts experienced G3 cytokine release syndrome (CRS). Treatment related AEs occurring in ≥ 1pt include pyrexia, CRS, hypoxia, lymphopenia, fatigue and nausea. CRS occurred in 5 pts, three G1/2 and two G3 AEs, with rapid resolution to appropriate treatment including tocilizumab. No neurotoxicity AEs have been observed to date. Out of the 8 r/r AML pts enrolled, 7 were response evaluable (2 at DL-1, 3 at DL-2 and 2 at DL-3). The third DL-3 pt has just initiated the first cycle of CYAD-01 treatment. Overall response rate in r/r AML pts was 42% (3/7 patients) with 1 complete remission with partial hematologic recovery (CRh) in DL-1 and 2 CR with incomplete marrow recovery (CRi; 1 in DL-1 and 1 in DL-3). All responding pts achieved response by day 29 (i.e. after 2 CYAD-01 administrations). The AML pt with CRh in DL-1 was bridged to allogeneic hematopoietic stem cell transplantation (allo-HSCT) on day +97 post CYAD-01 and is in durable complete molecular remission (CRMRD-) for more than 1 year (ongoing). The two other responding pts had CRi for 1 month. Two other AML patients at DL-2 had clinical benefit/disease stabilization with hematologic improvement and bone marrow blasts decrease: one pt for 3 months and with a decrease from 24% to 10% and the second pt for at least 4 months (ongoing) and with a decrease from 9.8% to 5.5%. The first patient in CRh had a relative increase in the systemic levels of SDF-1, RANTES and MCP-1 which correlated with the timing of injections. CYAD-01 engraftment kinetics, NKG2D ligand expression (including blasts and soluble ligand expression), and the kinetics of cytokine induction will be correlated with patient's responses. The 3 MM and the MDS pt, all in DL-1, did not present any sign of clinical activity. Conclusions: We have demonstrated the feasibility and safety of multiple injections of CYAD-01 without preconditioning chemotherapy. We evidenced promising anti-leukemic activity with 42% ORR in r/r AML with 5/7 pts having clinical benefit. Rates of G3/4 CRS were low and manageable. Updated safety, activity and correlative science data of the complete dose-escalation segment will be presented. Disclosures Sallman: Celgene: Research Funding, Speakers Bureau. Kerre:Celyad: Consultancy; BMS: Consultancy; Celgene: Consultancy, Research Funding. Davila:Celyad: Consultancy, Membership on an entity's Board of Directors or advisory committees. Wang:Jazz: Speakers Bureau; Jazz: Speakers Bureau; Amgen: Consultancy; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Speakers Bureau; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Speakers Bureau; Amgen: Consultancy; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees. Dekker:Celyad: Employment. Snykers:Celyad: Employment. Sotiropoulou:Celyad: Employment. Breman:Celyad: Employment. Braun:Celyad: Employment. Lonez:Celyad: Employment. Verma:Celyad: Employment. Lehmann:GSK: Patents & Royalties; Celyad: Employment, Honoraria, Patents & Royalties.

Introduction: Conventional CAR-T cells express a single chain antibody variable fragment that restricts recognition to one tumor antigen and a limited set of cancers. This study employs a novel CAR fusing full-length human NKG2D with the CD3z signaling domain. In autologous transduced CM-CS1 T cells, NKG2D CAR receives endogenous costimulation via DAP10 to target multiple NKG2D-ligands that are upregulated in solid and hematologic malignancies but absent or poorly expressed on healthy tissues. Methods: A phase 1 dose-escalation study to establish safety and feasibility of a single infusion of CM-CS1 T cells without lymphodepleting conditioning enrolled subjects with AML/MDS-RAEB or relapsed/refractory progressive multiple myeloma (MM) without standard therapy options (NCT02203825). Eligibility criteria included suitable organ function, no CNS disease, no prior allogeneic SCT or adoptive T-cell therapy, no therapy within 3 weeks prior to infusion, no immune suppression, and no uncontrolled infection. Dose-escalation spanned 4 cohorts [half-log increments from 1x106 to 3x107 CM-CS1 T cells] according to a 3+3 design. DLTs included ≥ Grade 3 non-hematologic toxicity or ≥ Grade 2 autoimmune toxicity related to CAR T cells. Initial assessment was at 28 days. At least 1 AML/MDS and 1 MM subject were mandated in each dose level. Manufacturing included PBMC stimulation with OKT3 and IL-2 followed by 2 rounds of retroviral transduction at DFCI's Cell Manipulation Core Facility. Vector copy number (VCN) and replication-competent retrovirus (RCR) testing were performed on whole blood and PBMCs, respectively, using quantitative PCR. Results: From April 2015 to July 2016, 11 subjects were infused, and 10 completed the DLT period. Eight of 11 were male, 6 had AML/MDS, and median age was 70 (range 44 to 79) (Panel A). Median WBC was 2.3 (range 0.7 to 7.2 K/uL); median ALC was 0.74 (range 0.09-2.37 K/uL). Five had cells manufactured from peripheral blood; 6 underwent apheresis. Median percentage of blasts in bone marrow for AML/MDS patients was 50% (range 4-68%). All myeloma patients had undergone ≥ 5 therapies including ≥1 autologous SCT. Four of the 6 AML/MDS patients had secondary disease, 3 had complex cytogenetics, 3 had p53 mutations, and 1 had a FLT3-ITD mutation. Dose-escalation proceeded from 1x106 to 3x107 CM-CS1 T cells. All 11 products passed release criteria, and there were no infusion reactions. Products consisted of median 97.2% CD3+ cells and 31.0% CD8+ cells, with vector-specific NKG2D expression on median 74.6% of CD3+ and 66.3% of CD8+ cells (Panel B). The first 10 subjects completed their 28 day evaluation period without DLTs. There were no cases of cytokine release syndrome, cell-related neurotoxicity, auto-immunity, or CAR T-related death. SAEs included a Grade 4 intracochlear bleed and an episode each of grade 4 neutropenia and thrombocytopenia deemed related to disease progression. Forty percent of patients experienced some Grade 3 toxicity, all related to underlying disease or a complication thereof (Panel C). At these initial cell doses, no patient to date has had objective tumor response at the 28 day evaluation mark. Nine initiated subsequent therapies; there have been 4 deaths secondary to disease or complications of subsequent therapies. However, cases of unexpected survival without further therapy and responses to subsequent treatments were noted. For example a patient with p53-mutated AML survived 4 months despite 50% blasts at infusion, and another entered PR at 6+months after cells on an IDH-1 inhibitor with Conclusion: In the first 3+ dose-escalation cohorts of patients with AML/MDS and myeloma, a single dose of CM-CS1 T cells without lymphodepletion was feasible and well-tolerated, with no DLTs. CAR T cells generally have not persisted beyond 1 week, consistent with pre-clinical models. Correlative analyses including post-infusion immunophenotyping are in process. Future studies of multiple infusions of NKG2D CAR T cells in both hematologic malignancies and solid tumors at the higher cell doses associated with efficacy in pre-clinical models are in planning. Table Table. Disclosures Murad: Celdara Medical, LLC: Employment. Reder:Celdara Medical, LLC: Employment. Sentman:Celdara Medical, LLC: Membership on an entity's Board of Directors or advisory committees, Other: Holds patents on this technology. Wade:Celdara Medical, LLC: Employment. Schmucker:Celdara Medical, LLC: Employment. Lehmann:Celyad, SA: Employment. Snykers:Celyad, SA: Employment. Allen:Celyad, SA: Employment. Stone:Celator: Consultancy; Jansen: Consultancy; Novartis: Consultancy; Merck: Consultancy; ONO: Consultancy; Sunesis Pharmaceuticals: Consultancy; Roche: Consultancy; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Juno Therapeutics: Consultancy; Xenetic Biosciences: Consultancy; Agios: Consultancy; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Consultancy; Pfizer: Consultancy; Karyopharm: Consultancy; Amgen: Consultancy. Soiffer:Kiadis: Membership on an entity's Board of Directors or advisory committees; Juno: Consultancy. Dranoff:Novartis: Employment. Ritz:Kiadis: Membership on an entity's Board of Directors or advisory committees.