Background CYAD-01 is an autologous chimeric antigen receptor (CAR) T-cell product based on the natural killer (NK) group 2D (NKG2D) receptor, which binds eight ligands that are overexpressed in a wide range of haematological malignancies but are largely absent on non-neoplastic cells. Initial clinical evaluation of a single infusion of CYAD-01 at a low dose in patients with relapsed or refractory acute myeloid leukaemia, myelodysplastic syndromes, and multiple myeloma supported the feasibility of the approach and prompted further evaluation of CYAD-01. The aim of the present study was to determine the safety and recommended phase 2 dosing of CYAD-01 administered without preconditioning or bridging chemotherapy.Methods The multicentre THINK study was an open-label, dose-escalation, phase 1 study for patients with relapsed or refractory acute myeloid leukaemia, myelodysplastic syndromes, or multiple myeloma, after at least one previous line of therapy. Patients were recruited from five hospitals in the USA and Belgium. The dose-escalation segment evaluated three dose levels: 3 x 108 (dose level one), 1 x 109 (dose level two), and 3 x 109 (dose level three) cells per infusion with a 3 + 3 Fibonacci study design using a schedule of three infusions at 2-week intervals followed by potential consolidation treatment consisting of three additional infusions. The occurrence of dose-limiting toxicities post-CYAD-01 infusion was assessed as the primary endpoint in the total treated patient population. The trial was registered with ClinicalTrials.gov, NCT03018405, and EudraCT, 2016-003312-12, and has been completed.Findings Between Feb 6, 2017, and Oct 9, 2018, 25 patients were registered in the haematological dose-escalation segment. Seven patients had manufacturing failure for insufficient yield and two had screening failure. 16 patients were treated with CYAD-01 (three with multiple myeloma and three with acute myeloid leukaemia at dose level one; three with acute myeloid leukaemia at dose level two; and six with acute myeloid leukaemia and one with myelodysplastic syndromes at dose level three). Median follow-up was 118 days (IQR 46-180). Seven patients (44%) had grade 3 or 4 treatment-related adverse events. In total, five patients (31%) had grade 3 or 4 cytokine release syndrome across all dose levels. One dose-limiting toxicity of cytokine release syndrome was reported at dose level three. No treatment-related deaths occurred, and the maximum tolerated dose was not reached. Three (25%) of 12 evaluable patients with relapsed or refractory acute myeloid leukaemia or myelodysplastic syndromes had an objective response. Among responders, two patients with acute myeloid leukaemia proceeded to allogeneic haematopoietic stem-cell transplantation (HSCT) after CYAD-01 treatment, with durable ongoing remissions (5 and 61 months).Interpretation Treatment with a multiple CYAD-01 infusion schedule without preconditioning is well tolerated and shows anti-leukaemic activity, although without durability outside of patients bridged to allogeneic HSCT. These phase 1 data support the proof-of-concept of targeting NKG2D ligands by CAR T-cell therapy. Further clinical studies with NKG2D-based CAR T-cells are warranted, potentially via combinatorial antigen targeted approaches, to improve anti-tumour activity.Funding Celyad Oncology.Copyright (c) 2023 Elsevier Ltd. All rights reserved.

NKG2D-CAR T Cell Product Transduction Assessment

TPS227 Background: The allogeneic chimeric antigen receptor (CAR) T-cell treatment CYAD-101 utilizes the NKG2D receptor that targets eight ligands expressed on tumor cells and also on stromal and immunosuppressive immune cells of the tumor microenvironment. CYAD-101 co-expresses a T-cell receptor (TCR) inhibitory peptide with the aim to eliminate the potential of graft versus host disease (GvHD), the main safety risk associated with engineered cells of allogeneic origin. In the previous alloSHRINK trial (NCT03692429), CYAD-101 was administered post FOLFOX preconditioning chemotherapy to 15 patients with progressive metastatic colorectal cancer (mCRC) who were previously treated with FOLFOX. Overall, the treatment was well tolerated with no evidence of GvHD. The disease control rate was 73.3% with two patients presenting a confirmed partial response per RECIST’s criteria (4 months and 8 months of duration from first CYAD-101 infusion) and nine had stable disease with a median duration of 4.6 months. Evidence of increase in the TCR repertoire and modulation of the cytokine profile post-treatment with CYAD-101 were observed implying that CYAD-101 may also be modulating the immune suppressive environment in patients mirroring what was demonstrated in pre-clinical models. We considered that a sequential therapy with the anti-PD1 monoclonal antibody pembrolizumab to further release the anti-tumor potential of this expanded T-cell population may drive deeper, more durable and new clinical responses beyond that currently demonstrated with the CAR T-cells alone. Methods: The phase 1b KEYNOTE-B79 trial (NCT04991948) will evaluate, according to a Simon’s two stage study design, the safety and clinical activity of three consecutive infusions of CYAD-101 (1x10 9 cells per infusion) post FOLFOX preconditioning chemotherapy with two-week interval between cycles, followed by pembrolizumab treatment (200 mg administered every three weeks for up to two years) in microsatellite stable/mismatch-repair proficient mCRC patients with recurrent/progressing disease after at least one metastatic line of therapy, which must include FOLFOX chemotherapy. The pembrolizumab treatment will be initiated 3 weeks after the last CYAD-101 infusion to fall outside the classical time window of potential CAR T-cell toxicities (e.g., cytokine release syndrome). The co-primary endpoints of the trial are the occurrence of dose-limiting toxicities (DLTs) at any time from the first FOLFOX preconditioning treatment up to 3 weeks after the first pembrolizumab treatment and the objective response rate (ORR) at the tumor assessment planned 6 weeks after the first pembrolizumab treatment administration. The KEYNOTE-B79 study will be initiated in Q4-2021 in five sites in USA and Europe. Clinical trial information: NCT04991948.

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.

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.

74 Background: CYAD-101 is a first-in-class, non-gene edited allogeneic CAR T-cell product that combines the broad breadth of tumor targeting of the NKG2D-based chimeric antigen receptor (CAR) with a peptide-based approach that controls graft versus host disease (GvHD). NKG2D binds eight ligands commonly over-expressed across many tumors while the co-expressed T-cell receptor (TCR) inhibitory (TIM) peptide interferes with signaling by the endogenous TCR. A bank of CYAD-101 cells was produced from a single donor and evaluated in the AlloSHRINK phase 1 study (NCT03692429) in patients with unresectable metastatic colorectal cancer (mCRC). Methods: Three CYAD-101 infusions, each administered following a FOLFOX standard cycle as preconditioning chemotherapy, were tested in a 3+3 dose-escalation study (dose-levels (DL): 10 8 , 3x10 8 and 10 9 T-cells per infusion) in patients with relapsed/refractory mCRC who progressed after previous treatment with oxaliplatin-based chemotherapy, with or without irinotecan-based chemotherapy. Results: Fifteen patients (pts) were enrolled (3 pts at DL-1, 3 pts at DL-2, 9 pts at DL-3). No dose-limiting toxicity (DLT), Grade ≥ 3 related adverse events or GvHD were reported after any of the CYAD-101 infusions, thus confirming the overall good safety profile of CYAD-101 post FOLFOX. Encouraging anti-tumor activity was observed with 2 confirmed partial responses (PR), including one response in a KRAS mutated patient. In addition, 9 pts achieved stable disease (SD), with 7 SD lasting at least 3 months. The median progression-free survival in this heavily pre-treated population was 3.9 months (95% CI). Whilst engraftment of the CYAD-101 cells was observed after each infusion, the relative level of systemic cytokines appeared to be primarily modulated by cell dose with some suggestion that the magnitude of modulation might be associated with clinical response. Interestingly, preliminary analysis of the T-cell repertoire identified some evidence of TCR diversity after therapy in the patient showing the most durable partial response. Conclusions: These clinical results demonstrate the safety and tolerability of a fist-in-human non-gene edited allogeneic CAR T-cell treatment with early promising anti-tumor activity in advanced mCRC pts. Preliminary translational analysis present intriguing observations that the modulation of systemic cytokine levels may be associated with dose which is uncommon in CAR T-cell therapies reported to date while limited T-cell clonal diversification in the best responding patient underscores the likely central role of the adoptively transferred T-cell in driving therapeutic response in this particular patient. Extension cohort evaluating CYAD-101 following other preconditioning chemotherapy is expected to be initiated end 2020. Clinical trial information: NCT03692429.

Background The peptide-based allogeneic chimeric antigen receptor (CAR) T-cell treatment CYAD-101 utilizes an NKG2D receptor that targets eight ligands expressed on tumor cells and non-malignant stromal cells of many cancer types. CYAD-101 also co-expresses a peptide intended to eliminates the potential of graft versus host disease (GvHD). In the phase 1 alloSHRINK study (NCT03692429), CYAD-101 was administered with FOLFOX preconditioning chemotherapy to 15 patients with metastatic colorectal cancer (mCRC). The treatment was well tolerated with no evidence of GvHD, no treatment-related adverse events ≥ Grade 3 and only two patients who presented a cytokine release syndrome grade 1. By contrast, encouraging clinical activity was observed including two partial responses. Evidence of changes in the TCR repertoire and modulation of the cytokine profile four months post-treatment with CYAD-101 were also observed implying that the NKG2D CAR T may also be modulating the immune suppressive environment in patients reflecting that seen in pre-clinical models (ASCO GI 2021 abstract #74).Given the expansion of the T cell repertoire after CYAD-101 therapy, we considered that employing a checkpoint inhibitor to release this expanded T cell population may drive more durable clinical responses beyond that currently seen with the CAR T alone. Methods The KEYNOTE-B79 trial evaluates the safety and clinical activity of multiple infusions of CYAD-101, administered post FOLFOX preconditioning chemotherapy, then followed three weeks after CYAD-101 by a pembrolizumab consolidation treatment (200 mg every three weeks for a maximum two years total treatment duration) in microsatellite stable/mismatch-repair proficient mCRC patients with recurrent/progressing disease after at least one metastatic line of therapy which must include FOLFOX chemotherapy.The schedule of administration of three CYAD-101 infusions at the dose 1x109 cells/infusion Q2W post-FOLFOX preconditioning chemotherapy are based on the alloSHRINK study.This sequencing of checkpoint inhibitor at a timepoint after CYAD-101 therapy ensures that the modulated endogenous immune response is enabled by pembrolizumab. This study is not focused on impacting the CAR T cell itself largely since CYAD-101 cells at the time of manufacture show negligible expression of PD-1 and that this sequencing ensures no overlap of potential toxicities that could arise from the CAR T or checkpoint inhibitor therapies.The KEYNOTE-B79 study is planned to be initiated in Q4-2021. Ethics Approval The study was approved by all relevant authorities and submitted to Institution's Ethics Boards for their approval before study initiation.

3032 Background: In contrast to autologous CAR-T cell therapies, allogeneic donor-derived CAR-T cells can be banked and used in a timely fashion overcoming the critical time delay of just in time autologous cell manufacture. CYAD-101 is an allogeneic CAR-T that uses a non-gene edited peptide-based technology (TIM) to control graft versus host disease (GvHD) combined with a NKG2D-based CAR. Pre-clinical studies confirmed that CYAD-101 maintained CAR-directed anti-tumor activity in the absence of the induction of GvHD. Clinical grade CYAD-101 cells were produced for the phase 1 alloSHRINK trial (NCT03692429). Methods: A bank of clinical grade CYAD-101 cells was generated through two production runs using a single donor apheresis. Together, the bank generated > 53 billion CYAD-101 cells suitable for the entire dose escalation segment and short expansion phase of the trial (15 patients in total). Both runs showed high consistency with the CYAD-101 product generated composed mainly of CD4+ T cells (>85%) with a transduction level of > 92%, low relative expression of CD69/CD25 and largely absent expression levels of PD-1/LAG-3. The CYAD-101 cells were predominantly (>80%) CD45RA−/ CD62L−/ CD27− suggestive of an effector memory T cell population. Results: Upon co-culture with target K562 cells, CYAD-101 readily produced IFN-γ that was blocked by a NKG2D blocking antibody confirming specificity of the CAR. CYAD-101 cells showed in vitro cytotoxicity against tumor cells and produced an array of Th1 (IFN-γ, IL-2 and TGF-β) and Th2 (IL-4, IL-5) cytokines. Importantly, minimal IFN-γ was produced upon TCR stimulation while stimulation with a non-TCR mitogen (PMA + ionomycin) lead to high levels of IFN-γ. Together, these data show that clinical grade CYAD-101 cells were able to functionally respond through the CAR but showed minimal TCR-driven activation. Fifteen refractory metastatic CRC patients who had previously failed at least one line of oxaliplatin—containing therapy were treated with three doses of CYAD-101 cells given on Day 3 of three successive FOLFOX chemotherapy cycles. Updated clinical results continue to demonstrate an encouraging clinical activity (2 patients with partial response and 9 with stable disease) and the absence of GvHD in the context of CYAD-101 cell engraftment. Conclusions: These early clinical results demonstrate the safety and tolerability of a non-gene edited predominantly CD4+ CAR-T therapeutic approach. The initial observations of clinical activity in metastatic CRC patients warrants the continued development of this therapy. Clinical trial information: NCT03692429 .

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.

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.

Chimeric antigen receptor-T cells (CAR-Ts) are an exciting new cancer treatment modality exemplified by the recent regulatory approval of two CD19-targeted CAR-T therapies for certain B cell malignancies. However, this success in the hematological setting has yet to translate to a significant level of objective clinical responses in the solid tumor setting. The reason for this lack of translation undoubtedly lies in the substantial challenges raised by solid tumors to all therapies, including CAR-T, that differ from B cell malignancies. For instance, intravenously infused CAR-Ts are likely to make rapid contact with cancerous B cells since both tend to reside in the same vascular compartments within the body. By contrast, solid cancers tend to form discrete tumor masses with an immune-suppressive tumor microenvironment composed of tumor cells and non-tumor stromal cells served by abnormal vasculature that restricts lymphocyte infiltration and suppresses immune function, expansion, and persistence. Moreover, the paucity of uniquely and homogeneously expressed tumor antigens and inherent plasticity of cancer cells provide major challenges to the specificity, potency, and overall effectiveness of CAR-T therapies. This review focuses on the major preclinical and clinical strategies currently being pursued to tackle these challenges in order to drive the success of CAR-T therapy against solid tumors.

Abstract NKG2D ligands are widely expressed in solid and hematologic malignancies but absent or poorly expressed on healthy tissues. We conducted a phase I dose-escalation study to evaluate the safety and feasibility of a single infusion of NKG2D-chimeric antigen receptor (CAR) T cells, without lymphodepleting conditioning in subjects with acute myeloid leukemia/myelodysplastic syndrome or relapsed/refractory multiple myeloma. Autologous T cells were transfected with a γ-retroviral vector encoding a CAR fusing human NKG2D with the CD3ζ signaling domain. Four dose levels (1 × 106–3 × 107 total viable T cells) were evaluated. Twelve subjects were infused [7 acute myeloid leukemia (AML) and 5 multiple myeloma]. NKG2D-CAR products demonstrated a median 75% vector-driven NKG2D expression on CD3+ T cells. No dose-limiting toxicities, cytokine release syndrome, or CAR T cell–related neurotoxicity was observed. No significant autoimmune reactions were noted, and none of the ≥ grade 3 adverse events were attributable to NKG2D-CAR T cells. At the single injection of low cell doses used in this trial, no objective tumor responses were observed. However, hematologic parameters transiently improved in one subject with AML at the highest dose, and cases of disease stability without further therapy or on subsequent treatments were noted. At 24 hours, the cytokine RANTES increased a median of 1.9-fold among all subjects and 5.8-fold among six AML patients. Consistent with preclinical studies, NKG2D-CAR T cell–expansion and persistence were limited. Manufactured NKG2D-CAR T cells exhibited functional activity against autologous tumor cells in vitro, but modifications to enhance CAR T-cell expansion and target density may be needed to boost clinical activity.

OBJECTIVES:Tumor-associated antigens (TAAs) are frequently overexpressed in several cancer types. The aim of this study was to investigate the expression of TAAs in breast cancer. MATERIAL AND METHODS:A total of 250 selected invasive breast cancers including 50 estrogen receptor (ER)-positive (Luminal B like), 50 triple-negative (TN), 50 ER-positive lobular type, 50 ER- and progesterone receptor (PgR)-positive (Luminal A like) and 50 cerbB2-positive breast cancers, were assessed for New York esophageal squamous cell carcinoma-1 (NY-ESO-1), Wilms tumor antigen (WT-1) and PReferentially expressed Antigen of MElanoma (PRAME) antigen expression by immunohistochemistry (IHC). RESULTS:A significantly higher expression of cancer testis (CT)-antigens NY-ESO-1 and WT-1 antigen was detected in TN breast cancers compared with ER-positive tumors. NY-ESO-1 overexpression (score 2 + and 3+) assessed by monoclonal and polyclonal antibodies was detected in 9 (18%) TN cancers as compared to 2 (4%) ER-positive tumors (p = 0.002). WT1 over-expression (score 2 + and 3+) was confirmed in 27 (54%) TN tumor samples as compared to 6 (12%) ER-positive (p < 0.0001). PRAME over-expression (score 2 + and 3+) was detected in 8 (16%) HER2 positive tumor samples as compared to no TN and ER-positive cancers (p = 0.0021). CONCLUSIONS:NY-ESO-1 and WT1 antigens are overexpressed in TN breast cancers. Because of the limited therapeutic options for this patient subgroup, CT antigen-based vaccines might prove to be useful for patients with this phenotype of breast cancer.

Background: Chimeric antigen receptor T-cell (CAR-Ts) therapies have yet to demonstrate positive results in the context of solid tumors likely because of the inability of classical CAR-Ts to infiltrate into the tumor and overcome the hostile immune suppressive tumor microenvironment (TME). CYAD-01, a NKG2D receptor-based CAR-T, is currently evaluated in the ongoing THINK study (NCT03018405) without preconditioning therapy in both hematologic and solid indications, and demonstrated encouraging signs of clinical stabilization in refractory metastatic colorectal (mCRC) and ovarian cancer patients. These preliminary results prompted us to design a clinical development plan evaluating CYAD-01 in multiple settings. Trial design: To improve the likeliness to observe clinical activity of CYAD-01 in metastatic solid tumors, the SHRINK (NCT03310008) and the LINK (NCT03370198) trials have been designed to address, respectively, the challenge related to the immunosuppressive TME and difficulty of CAR-T cells to access the site of metastases. The SHRINK trial is evaluating the multiple infusion CYAD-01 treatment i.v. administered concurrently to a standard-of-care FOLFOX neoadjuvant treatment for the treatment of mCRC disease with potentially resectable liver metastases, with the aim to (i) favor infiltration into the immunosuppressive TME but also (ii) provide an opportunity for the CYAD-01 cells to better engraft due to the lymphodepletion induced by the FOLFOX, and likely (iii) increase the NKG2D ligand expression in tumor tissues targeted by CYAD-01. The LINK trial is evaluating multiple hepatic transarterial administrations of CYAD-01 in mCRC patients with unresectable liver metastases with the potential advantage of a lower systemic toxicity and higher and more persistent concentration of the infused cells on the TME compared to systemic administration and difference in blood supply between uninvolved liver parenchyma and metastases. Finally, the boosting of the adaptive immune response by CYAD-01 might control distant lesions due to a possible abscopal effect. Both studies were initiated in 2018 and the first patients were successfully enrolled. Clinical trial identification: NCT03310008, EudraCT: 2017-000616-41; NCT03370198, EudraCT 2017-000959-11. Legal entity responsible for the study: Celyad SA. Funding: Celyad SA. Disclosure: A. Flament, F.F. Lehmann, C. Lonez: Employee: Celyad SA. All other authors have declared no conflicts of interest.

BACKGROUND AIMS:Adoptive cell therapy employing natural killer group 2D (NKG2D) chimeric antigen receptor (CAR)-modified T cells has demonstrated preclinical efficacy in several model systems, including hematological and solid tumors. We present comprehensive data on manufacturing development and clinical production of autologous NKG2D CAR T cells for treatment of acute myeloid leukemia and multiple myeloma (ClinicalTrials.gov Identifier: NCT02203825). An NKG2D CAR was generated by fusing native full-length human NKG2D to the human CD3ζ cytoplasmic signaling domain. NKG2D naturally associates with native costimulatory molecule DAP10, effectively generating a second-generation CAR against multiple ligands upregulated during malignant transformation including MIC-A, MIC-B and the UL-16 binding proteins. METHODS:CAR T cells were infused fresh after a 9-day process wherein OKT3-activated T cells were genetically modified with replication-defective gamma-retroviral vector and expanded ex vivo for 5 days with recombinant human interleukin-2. RESULTS:Despite sizable interpatient variation in originally collected cells, release criteria, including T-cell expansion and purity (median 98%), T-cell transduction (median 66% CD8+ T cells), and functional activity against NKG2D ligand-positive cells, were met for 100% of healthy donors and patients enrolled and collected. There was minimal carryover of non-T cells, particularly malignant cells; both effector memory and central memory cells were generated, and inflammatory cytokines such as granulocyte colony-stimulating factor, RANTES, interferon-γ and tumor necrosis factor-α were selectively up-regulated. CONCLUSIONS:The process resulted in production of required cell doses for the first-in-human phase I NKG2D CAR T clinical trial and provides a robust, flexible base for further optimization of NKG2D CAR T-cell manufacturing.

Abstract Introduction: CYAD-01 is a chimeric antigen receptor T-cell (CAR-T) product based on the receptor NKG2D which demonstrated anti-tumor efficacy through different mechanisms of action in numerous preclinical models. A comprehensive clinical program was developed with the aim to define the optimal CYAD-01 treatment in acute myeloid leukemia (AML). Methods: The ongoing Phase I THINK trial (NCT03018405) evaluates multiple CYAD-01 injections in 2 parallel cohorts: one in patients (pt) with metastatic solid tumors and the other one in relapsing/refractory hematological malignancies. 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 at 2 weeks apart. As of July 30, 2018, 12 pts in the THINK hematological cohort (8 AML, 3 MM and 1 MDS) have been enrolled at the 3 DLs without prior preconditioning. No dose-limiting toxicity occurred. We evidenced promising anti-leukemic activity with 42% ORR in r/r AML with 5/7 pts having clinical benefit. Based on the initial data generated into the THINK study, two additional Phase I clinical trials have been developed to evaluate 3 DLs (1x108, 3x108 and 1x109 cells per injection) post preconditioning regimen or in association with standard of care (SoC). The DEPLETHINK study (NCT03466320) aims at evaluating the CYAD-01 treatment administered after a cyclophosphamide and fludarabine preconditioning regimen to r/r AML patients. The preconditioning chemotherapy should (i) favor the expansion and engraftment of CYAD-01, (ii) increase NKG2D ligand expression on tumor cells and (iii) help to overcome the strong immunosuppressive microenvironment. The EPITHINK study (EudraCT 2018-000745-39) aims at assessing the safety of the CYAD-01 treatment administered concurrently with SoC 5-azacytidine treatment in treatment-naïve AML pts ineligible for intensive treatment. Similar to the preconditioning treatment, the epigenetic treatment could contribute to favor engraftment of CYAD-01 cells and increase target antigen expression while better controlling the disease progression of patients early in the treatment. Results: Description of the study design and preliminary clinical data from the first cohorts of the EPITHINK and DEPLETHINK studies will be presented. The preliminary data in term of CYAD-01 engraftment kinetics, NKG2D ligand expression (including blasts and soluble ligands), and the kinetics of cytokine induction will be correlated with the concurrent treatment (preconditioning or epigenetic treatment) versus the stand alone therapeutic approach. Conclusions: We have demonstrated the feasibility and safety of multiple injections of CYAD-01 without preconditioning chemotherapy (THINK). Preliminary safety, activity and correlative science data will be presented according a prior preconditioning (DEPLETHINK) and concurrent administration with SoC epigenetic treatment (EPITHINK). Disclosures Sallman: Celgene: Research Funding, Speakers Bureau. Kerre:BMS: Consultancy; Celgene: Consultancy, Research Funding; Celyad: Consultancy. Wang:Novartis: Speakers Bureau; Amgen: Consultancy; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz: Speakers Bureau; Novartis: Speakers Bureau; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy; 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; Jazz: Speakers Bureau. Selleslag:Kiadis Pharma: Other: Financial support for study-related issues. Beguin:Kiadis Pharma: Consultancy. Al-Homsi:Celyad: Membership on an entity's Board of Directors or advisory committees. Dekker:Celyad: Employment. Breman:Celyad: Employment. Sotiropoulou:Celyad: Employment. Snykers:Celyad: Employment. Braun:Celyad: Employment. Lonez:Celyad: Employment. Verma:Celyad: Employment. Lehmann:Celyad: Employment, Honoraria, Patents & Royalties; GSK: Patents & Royalties. Davila:Celyad: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Abstract 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.

Celyad recently initiated several clinical trials with the CYAD-01 product, a natural killer group 2D (NKG2D)-based chimeric antigen receptor (CAR), in both solid and hematologic tumor types. This review discusses the unique properties of CYAD-01, expecting to provide a new paradigm to fight against solid tumors.