The persistence and reactivity of CAR T cells were enhanced by adding co-stimulatory domains, which is the basis of currently approved CAR-T cell therapies. However, this comes at the expense of increasing toxicities from the strong cytokine release effect. This is the first report from anti-CD19 CAR-T cell therapy with a single activation domain to show a favourable safety profile and clinical efficacy with two patients who achieved durable responses up to 28 months in a cohort with heavily pretreated B cell malignancies.

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

Allogeneic chimeric antigen receptor (CAR) T holds the promise of taking this therapeutic approach to broader patient populations while avoiding the intensive manufacturing demands of autologous cell products. One limitation to delivering an allogeneic CAR T is T-cell receptor (TCR) driven toxicity. In this work, the expression of a peptide to interfere with TCR signaling was assessed for the generation of allogeneic CAR T cells. The expression of a truncated CD3ζ peptide was shown to incorporate into the TCR complex and to result in blunted TCR responses. When coexpressed with a natural killer group 2D (NKG2D) CAR, the allogeneic T cells (called CYAD-101) failed to induce graft-versus-host disease in mouse models while maintaining antitumor activity driven by the CAR in vitro and in vivo. Two clinical grade discrete batches of CYAD-101 cells were produced of single donor apheresis resulting in 48 billion CAR T cells sufficient for the entire dose-escalation phase of the proposed clinical trial. The 2 batches showed high consistency producing a predominantly CD4+ T-cell population that displayed an effector/central memory phenotype with no evidence of exhaustion markers expression. These clinical grade CYAD-101 cells secreted cytokines and chemokines in response to ligands expressing target cells in vitro, demonstrating effector function through the CAR. Moreover, CYAD-101 cells failed to respond to TCR stimulation, indicating a lack of allogeneic potential. This bank of clinical grade, non-gene-edited, allogeneic CYAD-101 cells are used in the alloSHRINK clinical trial (NCT03692429).

BackgroundManipulating protein expression to generate cells with a specific desired phenotype is one of the central goals of engineered cell therapy. Short Hairpin RNA (shRNA) is a well-established approach to reduce protein expression through the targeted degradation of messenger RNA transcripts. However, the use of shRNA in the Chimeric Antigen Receptor (CAR) T cell therapy has been limited. We have recently shown that single shRNA incorporated into a CAR expression vector can knockdown expression of the target antigen when expressed on the CAR T itself to avoid fratricide or expression of some elements of the T cell receptor (TCR) to generate allogeneic CAR T cells. An attraction of the shRNA approach is to express multiple shRNA from the same vector that can regulate protein expression thereby optimzing CAR T cell phenotype.MethodsRetroviral vectors encoding a CAR targeting a well-studied antigen (generally BCMA) co-expressing a tag for cell enrichment and identification along with shRNA multiplexed were generated. The shRNA multiplexed were inserted within a microRNA (miR) framework to enable expression from a single PolII promoter (the retroviral LTR promoter). Functional assessment of target knockdown target in T cells along with retroviral titers was determinedResultsOur products in ongoing clinical development have employed a miR196a2 scaffold enabling the expression of the desired shRNA driven by the same promoter as that used for the CAR and other transgenes. Multiplexing the miR196a2 scaffold to express multiple shRNA (targeting CD247, beta 2 Microglobulin and CD95) was successful in terms of target knockdown but an obvious reduction in retroviral titer was observed. These titer reductions were variable between the duplex and triplex shRNA constructs examined but were uniformly low when considering clinical development. A proprietary scaffold was developed that coupled expression of duplexed and triplexed shRNA while also elevating vector titer by at least 2-3x.ConclusionsMultiplexing shRNA within a single vector format with scaffolds that ensure co-linked expression of the shRNA with therapeutic transgenes is a highly attractive approach to generate CAR T cells with bespoke, desired phenotypes. However, simply multiplexing shRNA using a currently clinical-used scaffold (miR196a2) resulted in reductions in vector titer. Engineering further proprietary scaffolds were produced that maintained shRNA expression but elevated retroviral titer to a level which does not preclude clinical development. These developments now provide the opportunity to develop second generation clinical candidates using shRNA multiplexed technology.

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.

BackgroundWhilst delivering impressive clinical efficacy in certain hematological malignancies, Chimeric Antigen Receptor (CAR) T cell therapy has yet to deliver significant clinical impact across a broader array of cancer indications. Armoring CAR T through the co-expression of immune modifying cytokines is an approach that may aid anti-cancer activity but is currently at an embryonic stage of development. In this study, the potential benefit of expressing IL-18 alongside a NKG2D CAR was assessed.MethodsA series of retroviral vectors encoding the NKG2D CAR (a fusion of NKG2D with CD3z), a cell surface tag to facilitate cell selection and tracking (truncated CD19) either with or without full length IL-18 were compared. In certain vectors, a single shRNA targeting CD3z was included to generate allogeneic CAR T versions. All transgenes were delivered as a single vector expressed under the control of the retroviral promoter with individual 2A elements ensuring equimolar levels of protein expression. T cells transduced with the individual vectors were challenged in vitro and in vivo to determine the impact of IL-18 upon NKG2D CAR directed function.ResultsArmored NKG2D CAR T cells that included the IL-18 transgene showed high levels of IL-18 secretion in culture and increased levels of interferon gamma secretion upon antigen challenge as compared to non-armored NKG2D CAR T cells. Armored NKG2D CAR T cells also showed prolonged sequential target cell killing as compared to non-armored CAR T versions. Importantly, in an in vivo stress test where the dose of non-armored NKG2D T cells was reduced to a level where minimal anti-tumor activity and survival above control was seen using an established THP-1 model, armored CAR T cells showed enhanced anti-tumor activity (as determined by bioluminescence) and overall survival. Interestingly, at high doses of armored CAR T cells, toxicity was seen in some tumor bearing models. This toxicity was abrogated by systemic infusion of human IL-18 binding protein (IL-18BP).ConclusionsArmoring NKG2D CAR T cells with IL-18 resulting in increased in vitro and in vivo target-dependent anti-tumor activity. The transient toxicity observed with high doses of the armored CAR T in tumor bearing models was eliminated by IL-18BP. Together, these observations imply that armoring NKG2D CAR T cells with IL-18 is likely to drive improved anti-tumor activity of the CAR T cell in line with previous publications1 2 while the presence of systemic IL-18BP3 should negate possible toxicities arising from high level constitutive expression of the cytokine.ReferencesChmielewski M, Abken H. Cell Reports 2017;21(11): 3205–32192.Hu B, Ren J, Luo Y, Keith B, Young R, Scholler J, Zhao Y, June C. Cell Reports 2017; 20(13): 3025–30333.Dinarello C, Novick D, Kim S, Kaplamski G. Frontiers in Immunology 2013;4;289

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.

3103 Background: Engineered T cells expressing chimeric antigen receptors (CAR) are now delivering clinically relevant results in patients with advanced hematological malignancies. One critical area for future development is to modulate gene expression thereby endowing the engineered T cell with specific desired features that enhance anti-tumor activity. Methods: Short-hairpin RNA (shRNA) were cloned individually or multiplexed within micro-RNA scaffolds that enabled the co-expression of the individual shRNA with a CAR and a selectable marker all driven by a PolII promoter within a single retroviral vector. Primary human T cells transduced with the CAR-shRNA vectors were selected, expanded in vitro, subjected to negative selection to eliminate any remaining TCR+ cells and examined for target gene expression and functional activity. Results: A 500bp DNA fragment incorporating a shRNA-specific for CD3ζ cloned into a retroviral vectoreffectively knocked down expression of CD3ζ in transduced BCMA-specific CAR T cells. The consequent reduction of cell surface TCR expression resulted in minimal cytokine production upon TCR stimulation in vitro providing a potential allogeneic CAR T approach. These CAR T cells showed no demonstrable evidence of GvHD induction when infused in NSG mice yet maintained BCMA-specific CAR activity in KMS-11 and RPMI-8226 established myeloma models. Initial studies further confirmed that two shRNA could be expressed from a single retroviral vector to modulate the expression of multiple genes. Further engineering of the microRNA framework reduced the size of the transgene load to 394bp while enabling the expression of up to 4 shRNA within a single vector. shRNA specific for CD3ζ, beta-2-microglobulin, CD52 and diacylglycerol kinase alpha were engineered into the framework downstream of a CD19-CAR. Transduced Jurkat cells showed concurrent knockdown of the respective gene products at the mRNA and protein levels. Conclusions: A first-in-human clinical trial evaluating the first-generation single shRNA-vector in the context of a BCMA-targeting CAR as a non-gene edited approach to allogeneic CAR T cell therapy will be initiated in 2020. The proof of principle study here shows that multiple shRNAs are active within a single viral vector thereby avoiding the need for bespoke individual clinical reagents to target multiple genes. The multiplexed shRNA vector system is now in further development to explore whether this strategy can enhance the therapeutic potential of CAR T cells.

Engineering T cells to express chimeric antigen receptors (CARs) specific for antigens on hematological cancers has yielded remarkable clinical responses, but with solid tumors, benefit has been more limited. This may reflect lack of suitable target antigens, immune evasion mechanisms in malignant cells, and/or lack of T cell infiltration into tumors. An alternative approach, to circumvent these problems, is targeting the tumor vasculature rather than the malignant cells directly. CLEC14A is a glycoprotein selectively overexpressed on the vasculature of many solid human cancers and is, therefore, of considerable interest as a target antigen. Here, we generated CARs from 2 CLEC14A-specific antibodies and expressed them in T cells. In vitro studies demonstrated that, when exposed to their target antigen, these engineered T cells proliferate, release IFN-γ, and mediate cytotoxicity. Infusing CAR engineered T cells into healthy mice showed no signs of toxicity, yet these T cells targeted tumor tissue and significantly inhibited tumor growth in 3 mouse models of cancer (Rip-Tag2, mPDAC, and Lewis lung carcinoma). Reduced tumor burden also correlated with significant loss of CLEC14A expression and reduced vascular density within malignant tissues. These data suggest the tumor vasculature can be safely and effectively targeted with CLEC14A-specific CAR T cells, offering a potent and widely applicable therapy for cancer.

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.