Chimeric Antigen Receptor Vector Research Articles

Potent and Target Specific Cytotoxicity of PRAME TCR Mimic CAR T Cells in KMT2A-Rearranged AML Patient-Derived Xenograft Model

Chimeric antigen receptor (CAR) T cell therapy was a resounding success in CD19+ ALL despite the fact that it is essential for B-cell maturation because it is not expressed during normal myeloid hematopoiesis. Normal and leukemic B-cells are targeted by CD19+ CAR T-cells without causing myeloid hematopoietic toxicity. Advancing such therapy to acute myeloid leukemia (AML) has been challenging in part due to a paucity of “dispensable” targets or targets whose expression is limited to the leukemic cells. Using a large AML and normal hematopoiesis transcriptome database, we pursued discovery of “AML-Restricted Targets”; genes that are silent in normal hematopoiesis but expressed in AML. This broad discovery effort yielded a library of AML-restricted targets that included PRAME (Preferentially Expressed Antigen in Melanoma), an intracellular protein expressed via MHC on the cell surface, to be a highly expressed in AML including the aggressive KMT2A-rearranged (KMT2A-r) AML. We used a TCR mimic (mTCR) antibody, which recognizes the PRAME peptide/HLA-A2 complex on the tumor cell surface, to develop a PRAME directed TCR mimic CAR T (PRAME mTCRCAR T) for pre-clinical studies. Studies in cell lines and CDX models have shown significant efficacy of this CAR T (Kirkey, Bld Adv. 2022). To conduct final IND-enabling studies, we generated a KMT2A-r HLA-A2+/PRAME+ patient-derived xenograft (PDX) model to treat with PRAME mTCRCAR T-cells. Here we demonstrate the in vivo activity of PRAME mTCRCAR T cells against this unique PDX AML model. VL and VH sequences from the PRAME specific TCR mimic antibody (Pr20) were used to construct the single-chain fragment variable domain into the 41-BB/CD3ζ CAR vector. The PDX model was derived from a PRAME +/HLA-A2 + pediatric patient with KMTA2-r AML. PDX cells were transduced with lentiviral ffluciferase for noninvasive bioluminescent IVIS imaging to monitor leukemic progression. Mice were transplanted with 1x10 6 PDX leukemia cells then 1 week later, PDX leukemia-bearing mice were treated with unmodified T cells or PRAME mTCRCAR T cells at 5x10 6 cells (1:1 CD4:CD8) per mouse. Leukemia burden was measured by IVIS imaging and regular peripheral blood analysis. Mice who received unmodified T cells had rapid disease progression by day 50 and all died by day 105. In contrast, PRAME mTCRCAR T-treated mice rapidly cleared disease and all remained alive and leukemia-free >180 days post-treatment (p=0.001). Average leukemia burden in the control cohort was 3.5% at week 6 and 41.75% at week 11, while no leukemia was detected following CAR T-cell treatment. In addition, the control arm had a marked expansion of leukemia with a 7.75- and 429.6-fold increase in radiance by IVIS at 6 and 11 weeks post-treatment, respectively. No significant increase in radiance was seen in the PRAME mTCRCAR T cell treated group. We further evaluated hematopoietic toxicity of PRAME mTCRCAR T cells in humanized mice. Sub-lethally irradiated NSG-SGM3 mice were reconstituted with HLA-A2+ CD34 selected human cord blood stem cells with simultaneous and allowed to engraft. Human hematopoietic cell engraftment was evaluated following treatment with unmodified T cells vs. PRAME mTCRCAR T cells. Evaluation of human CD45+ cells in the peripheral blood via flow cytometry showed no difference in the levels of hematopoietic engraftment in mice treated with PRAME mTCRCAR T cell (8%) vs. unmodified T cells (6%) vs. an untreated control cohort (5%), demonstrating lack of toxicity of PRAME mTCRCAR T cells against HLA-A2 hematopoiesis. We demonstrate that PRAME mTCRCAR T cells can irradicate AML in a target specific manner in aggressive KMT2A-r AML without toxicity. We show potent efficacy with eradication of leukemia in PDX-bearing mice following treatment with PRAME mTCRCAR T cells resulting in prolonged survival, without hematopoietic toxicity. These results provide strong rationale for advancing this therapeutic approach to clinical development.

Cellular Mechanisms Affecting Allogeneic CAR T Cell Expansion and Rejection in Large B-Cell Lymphoma

Allogeneic chimeric antigen receptor (CAR) T cell therapies hold promise to improve CAR T access. However, limited expansion and persistence due to rejection in some recipients may impact efficacy. Elucidation of cellular mechanisms governing allogeneic CAR T expansion and persistence may inform patient management and technology development. We hypothesized that cell-extrinsic and intrinsic mechanisms differentially regulate rejection and expansion of allogeneic CAR T cells in patients with large B-cell lymphoma. We identified 11 patients with relapsed/refractory large B-cell lymphoma treated on the ALPHA-2 phase 1 trial (NCT04416984) with ALLO-501A, a healthy donor-derived CD19 CAR T with T cell receptor (TCR) knockout. ALLO-501A uses Cellectis technologies. Despite treatment with a single ALLO-501A lot, we observed heterogeneity in CAR T expansion and clinical outcomes. Six patients had robust expansion (expanders), and five patients had relatively poor expansion (non-expanders) [median maximum CAR vector copy number 21,571 (range 10,296-104,784) versus 141 (range 55-502) copies/µg DNA, p < 0.01]. As with autologous therapies, expanders exhibited longer CAR T persistence and a higher frequency of clinical responses than non-expanders. To elucidate cell-extrinsic mechanisms involved in allogeneic CAR T rejection, we hypothesized that recipient-derived alloreactive T cells limit expansion in non-expanders. We adapted a mixed lymphocyte reaction assay to identify alloreactive CD4 + and CD8 + T cell clones from pre-lymphodepletion PBMC samples. Briefly, labeled recipient PBMCs were incubated with irradiated ALLO-501A cells from the treatment lot. Proliferating recipient CD4 + and CD8 + T cells were isolated and underwent TCR sequencing. Alloreactive clones were defined as those enriched in this sample compared to an unstimulated sample from the same patient (p < 0.05 by Fisher's test) and were tracked post-treatment. Two expanders and two non-expanders were selected for analysis. Non-expanders exhibited more robust in vitro T cell proliferation in response to ALLO-501A than expanders (85 vs. 40% proliferating; p = 0.03). Thus, more alloreactive clones were identified in non-expanders (median 762 CD4 + clones, 314 CD8 + clones) than expanders (median 313 CD4 + clones, 90 CD8 + clones). Non-expanders also had higher median alloreactive CD8 + T cell frequencies than expanders at days 7 and 14 following allogeneic CAR T infusion ( Figure A). A similar pattern was not observed for alloreactive CD4 + clones or non-alloreactive T cell clones, highlighting the potential role of recipient alloreactive CD8 + T cells in the rejection process and the need to control this process for the success of allogeneic CAR T. Next, to define the role of cell-intrinsic mechanisms in allogeneic CAR T expansion, we tracked clonal evolution of the CAR T repertoire through TCR sequencing of the infusion product (IP) and post-treatment PBMC samples in all patients. At day 14 (peak allogeneic CAR T expansion), all expanders had numerous IP-derived clones and substantial clonotype overlap with each other. In contrast, no clonotype overlap was observed among non-expanders ( Figure B). Furthermore, we identified the same high-frequency IP-derived clones in multiple expanders and their relative abundance reflected the original clonal hierarchy of the IP. This pattern differs from autologous therapies, where clonal hierarchy is not maintained after infusion (Sheih, et al. Nat. Comm. 2020), and suggests that cell-intrinsic mechanisms may influence allogeneic CAR T expansion potential. Single-cell RNA and proteomic studies to characterize these expanding clones are ongoing. In conclusion, we compared expansion kinetics across 11 allogeneic CAR T recipients to elucidate the cellular mechanisms governing allogeneic CAR T expansion and rejection. We revealed the impact of recipient alloreactive CD8 + T cells in allogeneic CAR T rejection and identified expansion of identical allogeneic CAR T clonotypes with retained clonal hierarchy in multiple patients. While patient factors such as tumor burden and target antigen expression likely play a role as well, our results suggest that strategies to eliminate alloreactive recipient cells and enhance allogeneic CAR T fitness through improved product design, manufacturing and lymphodepletion could improve allogeneic CAR T expansion, persistence and efficacy.

T lymphocytes expressing the switchable chimeric Fc receptor CD64 exhibit augmented persistence and antitumor activity

Chimeric antigen receptor (CAR) Tcell therapy lacks persistent efficacy with "on-target, off-tumor" toxicities for treating solid tumors. Thus, an antibody-guided switchable CAR vector, the chimeric Fc receptor CD64 (CFR64), composed of a CD64 extracellular domain, is designed. Tcells expressing CFR64 exert more robust cytotoxicity against cancer cells than CFR Tcells with high-affinity CD16 variant (CD16v) or CD32A as their extracellular domains. CFR64 Tcells also exhibit better long-term cytotoxicity and resistance to Tcell exhaustion compared with conventional CAR Tcells. With trastuzumab, the immunological synapse (IS) established by CFR64 is more stable with lower intensity induction of downstream signaling than anti-HER2 CAR Tcells. Moreover, CFR64 Tcells exhibit fused mitochondria in response to stimulation, while CARH2 Tcells contain predominantly punctate mitochondria. These results show that CFR64 Tcells may serve as a controllable engineered Tcell therapy with prolonged persistence and long-term antitumor activity.

Validation of a High-Sensitivity Assay for Detection of Chimeric Antigen Receptor T-Cell Vectors Using Low-Partition Digital PCR Technology

Although invivo engraftment, expansion, and persistence of chimeric antigen receptor (CAR) T cells are pivotal components of treatment efficacy, quantitative monitoring has not been implemented in routine clinical practice. We describe the development and analytical validation of a digital PCR assay for ultrasensitive detection of CAR constructs after treatment, circumventing known technical limitations of low-partitioning platforms. Primers and probes, designed for detection of axicabtagene, brexucabtagene, and Memorial Sloan Kettering CAR constructs, were employed to validate testing on the Bio-Rad digital PCR low-partitioning platform; results were compared with Raindrop, a high-partitioning system, as reference method. Bio-Rad protocols were modified to enable testing of DNA inputs as high as 500 ng. Using dual-input reactions (20 and 500 ng) and a combined analysis approach, the assay demonstrated consistent target detection around 1 × 10-5 (0.001%) with excellent specificity and reproducibility and 100% accuracy compared with the reference method. Dedicated analysis of 53 clinical samples received during validation/implementation phases showed the assay effectively enabled monitoring across multiple time points of early expansion (day 6 to 28) and long-term persistence (up to 479 days). CAR vectors were detected at levels ranging from 0.005% to 74% (vector versus reference gene copies). The highest levels observed in our cohort correlated strongly with the temporal diagnosis of grade 2 and 3 cytokine release syndrome diagnosis (P<0.005). Only three patients with undetectable constructs had disease progression at the time of sampling.

Preclinical evaluation of targeting PRAME with TCR mimic CAR T cells in AML.

2553 Background: Chimeric antigen receptor (CAR) T cell therapy has revolutionized cancer treatment, but has had limited success against AML in part due to overlap of cell surface antigens expressed in AML and normal hematopoietic cells. To identify AML-restricted targets, we interrogated the transcriptome from over 3000 AML patients and found PRAME (Preferentially Expressed Antigen in Melanoma), an intracellular protein, to be a highly expressed AML-restricted target. Using a novel approach to target intracellular antigens, we developed a PRAME CAR T cell using a TCR mimic (mTCR) antibody, which recognizes the PRAME peptide/HLA-A2 complex on the tumor cell surface. To conduct final IND-enabling studies, we generated an in vivo patient derived xenograft (PDX) model to treat with PRAME mTCRCAR T cells. Here we demonstrate the in vivo activity of PRAME mTCRCAR T cells against a PDX AML model. Methods: We used the VL and VH sequences from the PRAME TCR mimic antibody (Pr20) to construct the single chain fragment variable domain into the 41-BB/CD3ζ CAR vector. The PDX model was derived from a PRAME+/HLA-A2+ pediatric patient with AML. PDX cells were transduced with ffluciferase for noninvasive bioluminescent IVIS imaging to monitor leukemic progression. PDX leukemia-bearing mice were treated with unmodified T-cells or PRAME mTCRCAR T cells at 5x106 cells (1:1 CD4:CD8) per mouse 1 week after leukemia injection. Leukemia burden was measured by IVIS imaging and peripheral blood analysis. Results: Treatment with PRAME mTCRCAR T cells led to eradication of leukemia with all mice alive &gt;100 days post-treatment, while control mice (unmodified T cells) had disease progression at Day 50 (p=0.001). Average leukemia burden in the control cohort was 3.5% at week 6 and 41.75% at week 11, while no leukemia was detected following CAR T cell treatment. In addition, the control arm had a marked expansion of leukemia with a 7.75 and 429.6 fold increase in radiance by IVIS at 6 and 11 weeks post-treatment. No significant increase in radiance was seen in the PRAME mTCRCAR T cell treated group. Human T cells (CD45+/CD3+) were detectable in the peripheral blood at Day 7 and 14 post-treatment in both the unmodified and CAR T cell treated groups. Conclusions: We demonstrate the therapeutic potential of targeting PRAME with mTCRCAR T cells in AML. We show potent efficacy with eradication of leukemia in PDX-bearing mice following treatment with PRAME mTCRCAR T cells resulting in prolonged survival. These results provide a novel approach to target PRAME with CAR T cells and provide compelling data to evaluate PRAME mTCRCAR T cells for use in clinical trials against AML. [Table: see text]

Supraphysiological FOXP3 expression in human CAR-Tregs results in improved stability, efficacy, and safety of CAR-Treg products for clinical application

The forkhead family transcription factor (FOXP3) is an essential regulator for the development of regulatory T cells (Tregs) and orchestrates both suppressive function and Treg lineage identity. Stable expression of FOXP3 enables Tregs to maintain immune homeostasis and prevent autoimmunity. However, under pro-inflammatory conditions, FOXP3 expression in Tregs can become unstable, leading to loss of suppressive function and conversion into pathogenic T effector cells. Therefore, the success of adoptive cell therapy with chimeric antigen receptor (CAR) Tregs is highly dependent on the stability of FOXP3 expression to ensure the safety of the cell product. To warrant the stable expression of FOXP3 in CAR-Treg products, we have developed an HLA-A2-specific CAR vector that co-expresses FOXP3. The transduction of isolated human Tregs with the FOXP3-CAR led to an increase in the safety and efficacy of the CAR-Treg product. In a hostile microenvironment, under pro-inflammatory and IL-2-deficient conditions, FOXP3-CAR-Tregs showed a stable expression of FOXP3 compared to Control-CAR-Tregs. Furthermore, additional exogenous expression of FOXP3 did not induce phenotypic alterations and dysfunctions such as cell exhaustion, loss of functional Treg characteristics or abnormal cytokine secretion. In a humanized mouse model, FOXP3-CAR-Tregs displayed an excellent ability to prevent allograft rejection. Furthermore, FOXP3-CAR-Tregs revealed coherent Treg niche-filling capabilities. Overexpression of FOXP3 in CAR-Tregs has thereby the potential to increase the efficacy and reliability of cellular products, promoting their clinical use in organ transplantation and autoimmune diseases.

Preclinical optimization of a GPC2-targeting CAR T-cell therapy for neuroblastoma

BackgroundAlthough most patients with newly diagnosed high-risk neuroblastoma (NB) achieve remission after initial therapy, more than 50% experience late relapses caused by minimal residual disease (MRD) and succumb to their...

Genetic Engineering and Enrichment of Human NK Cells for CAR-Enhanced Immunotherapy of Hematological Malignancies.

The great clinical success of chimeric antigen receptor (CAR) T cells has unlocked new levels of immunotherapy for hematological malignancies. Genetically modifying natural killer (NK) cells as alternative CAR immune effector cells is also highly promising, as NK cells can be transplanted across HLA barriers without causing graft-versus-host disease. Therefore, off-the-shelf usage of CAR NK cell products might allow to widely expand the clinical indications and to limit the costs of treatment per patient. However, in contrast to T cells, manufacturing suitable CAR NK cell products is challenging, as standard techniques for genetically engineering NK cells are still being defined. In this study, we have established optimal lentiviral transduction of primary human NK cells by systematically testing different internal promoters for lentiviral CAR vectors and comparing lentiviral pseudotypes and viral entry enhancers. We have additionally modified CAR constructs recognizing standard target antigens for acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) therapy—CD19, CD33, and CD123—to harbor a CD34-derived hinge region that allows efficient detection of transduced NK cells in vitro and in vivo and also facilitates CD34 microbead-assisted selection of CAR NK cell products to >95% purity for potential clinical usage. Importantly, as most leukemic blasts are a priori immunogenic for activated primary human NK cells, we developed an in vitro system that blocks the activating receptors NKG2D, DNAM-1, NKp30, NKp44, NKp46, and NKp80 on these cells and therefore allows systematic testing of the specific killing of CAR NK cells against ALL and AML cell lines and primary AML blasts. Finally, we evaluated in an ALL xenotransplantation model in NOD/SCID-gamma (NSG) mice whether human CD19 CAR NK cells directed against the CD19+ blasts are relying on soluble or membrane-bound IL15 production for NK cell persistence and also in vivo leukemia control. Hence, our study provides important insights into the generation of pure and highly active allogeneic CAR NK cells, thereby advancing adoptive cellular immunotherapy with CAR NK cells for human malignancies further.

High efficiency closed-system gene transfer using automated spinoculation

BackgroundGene transfer is an important tool for cellular therapies. Lentiviral vectors are most effectively transferred into lymphocytes or hematopoietic progenitor cells using spinoculation. To enable cGMP (current Good Manufacturing Practice)-compliant cell therapy production, we developed and compared a closed-system spinoculation method that uses cell culture bags, and an automated closed system spinoculation method to decrease technician hands on time and reduce the likelihood for microbial contamination.MethodsSepax spinoculation, bag spinoculation, and static bag transduction without spinoculation were compared for lentiviral gene transfer in lymphocytes collected by apheresis. The lymphocytes were transduced once and cultured for 9 days. The lentiviral vectors tested encoded a CD19/CD22 Bispecific Chimeric Antigen Receptor (CAR), a FGFR4-CAR, or a CD22-CAR. Sepax spinoculation times were evaluated by testing against bag spinoculation and static transduction to optimize the Sepax spin time. The Sepax spinoculation was then used to test the transduction of different CAR vectors. The performance of the process using healthy donor and a patient sample was evaluated. Functional assessment was performed of the CD19/22 and CD22 CAR T-cells using killing assays against the NALM6 tumor cell line and cytokine secretion analysis. Finally, gene expression of the transduced T-cells was examined to determine if there were any major changes that may have occurred as a result of the spinoculation process.ResultsThe process of spinoculation lead to significant enhancement in gene transfer. Sepax spinoculation using a 1-h spin time showed comparable transduction efficiency to the bag spinoculation, and much greater than the static bag transduction method (83.4%, 72.8%, 35.7% n = 3). The performance of three different methods were consistent for all lentiviral vectors tested and no significant difference was observed when using starting cells from healthy donor versus a patient sample. Sepax spinoculation does not affect the function of the CAR T-cells against tumor cells, as these cells appeared to kill target cells equally well. Spinoculation also does not appear to affect gene expression patterns that are necessary for imparting function on the cell.ConclusionsClosed system-bag spinoculation resulted in more efficient lymphocyte gene transfer than standard bag transductions without spinoculation. This method is effective for both retroviral and lentiviral vector gene transfer in lymphocytes and may be a feasible approach for gene transfer into other cell types including hematopoietic and myeloid progenitors. Sepax spinoculation further improved upon the process by offering an automated, closed system approach that significantly decreased hands-on time while also decreasing the risk of culture bag tears and microbial contamination.

A Phase I/II Clinical Trial of Piggybac-Modified GMR CAR-T Cell Therapy for CD116 Positive Relapsed/Refractory Myeloid Malignancies

Background: The prognosis of relapsed/refractory (R/R) myeloid malignancies remains poor, and the development of novel treatment strategies is crucial. Although chimeric antigen receptor (CAR)-modified T cell therapy for B cell malignancies has shown excellent clinical efficacy, the use of CAR-T cells for myeloid malignancies has been more challenging, partly due to the heterogeneous expression of candidate target antigens in leukemia cells and shared expression of those antigens in normal myeloid cells or progenitor cells. We previously developed the piggyBac-modified chimeric antigen receptor (CAR)-T cells targeting CD116, also known as GM-CSF receptor alpha chain (GMR) (Nakazawa Y, et al. J Hematol Oncol. 2016 and Hasegawa A, et al. Clin Transl Immunology. 2021). GMR CAR-T cells showed substantial antitumor effects against both acute myeloid leukemia and juvenile myelomonocytic leukemia. Moreover, modulation of the spacer and antigen recognition site of the CAR vector further enhanced the anti-tumor effects of GMR CAR-T cells. GMR CAR-T cells showed an acceptable safety profile with limited cytotoxicity on normal hematopoietic cells except monocytes. Based on these results, we have initiated a first-in-human clinical trial of GMR CAR-T cell therapy in March 2021 in Japan.Study Design and Methods: The study is a phase I/II, single-center, dose-escalation study with a traditional 3+3 dose-escalation design (Table 1). Maximum of 18 patients will be recruited. Primary objectives of this study are to determine the safety and severe adverse events of piggyBac-modified GMR CAR-T cells for CD116 + relapsed/refractory myeloid malignancies by assessing the dose-limiting toxicity within 28 days from the single infusion of GMR CAR-T cells.The patients with CD116 + myeloid malignancies aged more than 1 year with myeloid malignancies who experienced an induction failure or a relapse after hematopoietic stem cell transplantation (HSCT) will be recruited. CD116 is defined as positive when a CD116 relative mean fluorescence (RFI) in leukemia cells ≥ 2. RFI was calculated by dividing the MFI of samples with that of the isotype control. Major exclusion criteria are as follows, acute promyelocytic leukemia, acute graft-versus-host disease (GVHD) (Grade ≥ 2), extensive chronic GVHD, and concurrent treatment with corticosteroid (≥ 6 mg/m2). Statistical analysis will be performed when the data will be fixed.Peripheral blood mononuclear cells will be harvested from the patient by leukapheresis and then will be transduced with GMR CAR vector by piggyBac transposon system. All manufacturing process of GMR CAR-T cells is performed in Cell Processing Center in Shinshu University Hospital under good manufacturing practice conditions. The patient will be treated with lymphodepleting chemotherapy consisting of fludarabine and cyclophosphamide, followed by CAR-T cell infusion. The dose of CAR-T cells will be 3 x 10 5 and 1 x 10 6 in cohorts 1 & 2 and cohort 3, respectively (Table 1). Kinetics of GMR CAR-T cells will be determined by quantifying the GMR CAR gene in the peripheral blood using real-time PCR after the CAR-T cell infusion. All the patients will be required to receive HSCT by day 56 following CAR-T cell infusion. The primary endpoint of the study is the safety, pharmacokinetics, and efficacy of GMR CAR-T therapy (Trial registration: jRCT2033210029).Conclusion: We herein described the protocol of first-in-human GMR CAR-T cells for relapsed/refractory myeloid malignancies. By employing the optimized CAR vector and production protocol, the safety and efficacy of GMR CAR-T cells will be evaluated in this study. [Display omitted] DisclosuresSaito: Toshiba corporation: Research Funding. Yagyu: AGC Inc.: Research Funding. Nakazawa: AGC Inc.: Research Funding; Toshiba Corporation: Research Funding.

Targeting PRAME with TCR-Mimic CAR T Cells in AML

Chimeric antigen receptor (CAR) Ts have been effective in pre-B ALL, but their efficacy in AML has yet to be established. A significant barrier to effective CAR T therapy for AML is the substantial overlap of cell surface antigens expressed on AML and normal hematopoietic cells. To overcome this barrier, we profiled the transcriptome of over 3000 AML cases in children and young adults and contrasted this to normal hematopoietic tissues in search for AML-restricted targets (high expression in AML, silence in normal hematopoiesis). This led to the discovery of over 200 AML-restricted genes. Of these, Preferentially Expressed Antigen in Melanoma (PRAME) is among one of the highest expressing AML-restricted genes (Figure 1A) and, given its previous track record as a target for a variety of cancers, we selected this target for further assessment and therapeutic development in AML. However, PRAME is intracellular and therefore is inaccessible for targeting with conventional CAR T. Recently, a novel approach to target intracellular antigens was developed using TCR mimic (mTCR) antibodies, which recognize peptide/human leukocyte antigen (HLA) complexes on the tumor cell surface in a similar mode of recognition as authentic T Cell Receptors (TCRs). The Pr20 antibody was developed to recognize the PRAME ALY peptide in the context of HLA-A*02. Utilizing this Pr20 antibody, we developed a mTCR CAR T targeting PRAME and evaluated its preclinical efficacy in AML.The VL and VH sequences from Pr20 were used to construct the single-chain fragment variable domain of the 41-BB/CD3ζ CAR vector. We evaluated PRAME mTCR CAR T cells against OCI-AML-2 and THP-1 AML cell lines (PRAME +/HLA-A*02 +), K562 CML cell line (PRAME +/HLA-A*02 -) and HEK293T (293T) (PRAME -/HLA-A*02 +). Using a PE-conjugated Pr20 antibody, we confirmed that OCI-AML2 and THP-1 express PRAME ALY: HLA-A*02 but not K562 and 293T by flow cytometry (Figure 1B). As further confirmation, AML blasts in primary patient samples also stained with the Pr20 antibody (Figure 1C). For in-vivo studies, leukemia-bearing mice were treated with unmodified T or PRAME mTCR CAR T cells at 5x10 6 cells (1:1 CD4:CD8) per mouse 1 week following leukemia injection. Leukemia burden was measured weekly by bioluminescence IVIS imaging. Cells were treated with 10ng/mL of IFN-γ prior to co-incubation with T cells for 16 hours.PRAME mTCR CAR T cells demonstrated potent cytolytic activity against OCI-AML2 and THP1 but not against K562 or 293T cells, following co-incubation with target cells for 24 hours (Figure 1D). Consistent with potent, target-specific reactivity against PRAME ALY: HLA-A*02 positive cells, increased levels of IFN-γ, IL-2 and TNF-α were detected in cocultures of CAR T cells with OCI-AML2 and THP1 but not with K562 and 293T cells (Figure 1D). The cytolytic activity of PRAME mTCR CAR T cells extended to primary AML specimens expressing the PRAME ALY: HLA-A*02 antigen (data not shown). In-vivo efficacy of PRAME mTCR CAR T was demonstrated in OCI-AML2 and THP-1 CDX models (Figure 1E). Treatment with CAR T cells induced leukemia clearance and significantly reduced leukemia burden in OCI-AML2 and THP-1 xenograft mice, respectively, while treatment with unmodified T cells exhibited leukemia progression (Figure 1E). The anti-leukemia activity of CAR T cells resulted in enhanced survival in OCI-AML2 (p=0.0035) and THP-1 (p=0.0047) xenografts (Figure 1F). The in-vivo activity of PRAME mTCR CAR T cells was target specific, as treatment with CAR T cells did not affect leukemia burden and survival in K562 xenograft mice (Figure 1F). Given that IFN-γ promotes PRAME presentation, we investigated whether treatment of IFN-γ would enhance cytolytic activity of PRAME mTCR CAR T cells. OCI-AML2 and THP-1 cells pretreated with IFN-γ were more sensitive to cytolysis compared to untreated controls (Figure 1G).In this study, we demonstrate the therapeutic potential of targeting PRAME with mTCR CAR T cells in AML. We show potent, target-specific reactivity of PRAME mTCR CAR T cells against PRAME ALY: HLA-A*02 positive AML cells, both in-vitro and in-vivo. We further demonstrate that the activity of PRAME mTCR CAR T cells can be enhanced with IFN-γ treatment, providing a useful strategy to increase efficacy. Thus, the results presented provide a novel approach to target PRAME with CAR T cells and compelling data to evaluate PRAME mTCR CAR T cells in AML clinical trials. [Display omitted] DisclosuresPardo: Hematologics, Inc.: Current Employment. Hylkema: Quest Diagnostics Inc: Current equity holder in publicly-traded company; Moderna: Current equity holder in publicly-traded company. Scheinberg: Eureka Therapeutics: Current equity holder in publicly-traded company.

Bispecific CAR-T Cells Targeting Both BCMA and CD24: A Potentially Treatment Approach for Multiple Myeloma

Introduction: Anti-myeloma BCMA-specific chimeric antigen receptor (CAR) T-cell therapies represent a promising new treatment strategy, with high response rates observed in the early stages of therapy. However, the responses are not durable. One known mechanism of relapse has been traced to the loss of BCMA expression following long-term CAR-T therapy. Another potential reason is that while BCMA CAR-T cells eliminate the bulk of BCMA-positive MM cells, a small subset of BCMA-negative, very drug-resistant MM cells, such as tumor-initiating cells (TICs) survive and seed relapses. There is a strong correlation between the presence of MM TICs with minimal residual disease, acquired drug resistance and relapse. This suggests that TIC-targeted therapies could improve outcomes. We have previously demonstrated that MM cells expressing CD24 also exhibit features of TICs, e.g. self-renewal, increased expression of embryonic stem cell genes and drug resistance. We have generated bispecific CAR-T cells which recognize both BCMA and CD24 antigens and have tested their therapeutic efficacy in MM cells in vitro and in vivo models.Methods: We constructed a bispecific BCMA-CD24 CAR vector, with 2 complete CAR units: BCMA CAR and CD24 CAR. P2A was inserted between these two CARs. The BCMA CAR contains a safety switch in the hinge region, and a CD28 co-activation domain with CD3ζ. The CD24 CAR contained a 4-1BB co-activation domain with CD3ζ. To decrease the risk of severe immunological side effects, we integrated RQR8, an immunological safety switch with epitopes for CD34 and CD20 as a suicide molecule into the hinge region. Lentivirus particles were used to transduce primary human T cells. CAR-T cells were detected on day 7 by flow cytometry using antibodies to CD34. We performed co-culture killing assays, detected the T cell activation marker CD69 and measured the cytokines in the supernatant. We determined whether BCMA-CD24 CAR-T cells targeted the TIC population by flow cytometry and microscopy. The NOD. Cg-Prkdc scidIl2rg tm1Wjl/SzJ (NSG) xenograft mouse model was used for in vivo studies. 8-week-old NSG mice were administered 2 × 10 6 MM cells by intravenous injection. On day 7 after MM cells injection, 1 × 10 6 CAR-T cells were administered. Mice were weighed and monitored for signs of distress every two days. Bioluminescence images were acquired 10 min after D-luciferin injection. Myeloma progression was monitored every 7 days until the mice develop hind limb paralysis or the bioluminescence signal (ROI) is more than 2 × 10 10.Results: CAR-T cells were detected by flow cytometry using the RQR8-specific CD34 antibody. The BCMA-CD24 CAR was found to be expressed on roughly 13% of T-cells. To determine the selective lysis by the CAR-T cells, we performed co-culture killing assays in which MM cell lines over-expressing CD24 (ARP-1 CD24OE or OCI CD24OE cells) were incubated with CAR-T cells. When the CAR-T: MM ratio was 5:1, the lysis percentage of target cells was 99% (ARP-1 CD24OE) and 89% (OCI CD24OE). CAR-T cell activation was determined by increased CD69 expression and IL-2 production. As expected, exposure to CD24 + MM cells resulted in strong activation of CAR-T cells, and CAR-T cells did target and kill the TIC population. Bioluminescence imaging showed CAR-T mediated antitumor activity, yielding near-complete tumor clearance. Additionally, mice treated with CAR-T cells exhibited increased survival compared with mice in the control groups.Conclusion and Significance: This study developed a BCMA-CD24 CAR-T, a novel MM immunotherapy. We have demonstrated strong cytotoxic activity and selectivity for MM cells in vitro and in vivo. Future studies will be aimed at determining if BCMA-CD24 CAR-T can target TIC-mediated relapses. DisclosuresNo relevant conflicts of interest to declare.

PL03.3.A Development and characterization of CD317-specific CAR T cells as an innovative immunotherapeutic strategy against glioblastoma

Abstract BACKGROUND Due to the limited success of existing therapies for gliomas, innovative therapeutic options are urgently needed. Chimeric antigen receptor (CAR) T cell therapy has been successful in patients with hematological malignancies. However, using this treatment against solid tumors such as glioblastomas is more challenging. Here, we generated CAR T cells targeting the transmembrane protein CD317 (BST-2, HM1.24) which is overexpressed in glioma cells and may therefore serve as a novel target antigen for CAR T cell-based immunotherapy. MATERIAL AND METHODS CAR T cells targeting CD317 were generated by lentiviral transduction of human T cells from healthy donors. The anti-glioma activity of CD317-CAR T cells was determined in lysis assays using different glioma target cell lines with varying CD317 expression levels. The efficiency of CD317-CAR T cells to control tumor growth in vivo was evaluated in clinically relevant orthotopic xenograft glioma mouse models. RESULTS We created a second-generation CAR construct targeting CD317 and observed strong anti-glioma activity of CD317-CAR T cells in vitro. Glioma cells with a CRISPR/Cas9-mediated CD317 knockout were resistant to CD317-specific CAR T cells, demonstrating their target antigen-specificity. Since CD317 is also expressed by T cells, transduction with a CD317-directed CAR resulted in fratricide of the transduced T cells. Silencing of CD317 in CAR T cells by integrating a specific shRNA into the CAR vector significantly increased the viability, proliferation and cytotoxic function of the CAR T cells. Importantly, intratumoral treatment with CD317-CAR T cells prolonged the survival and cured a significant fraction of glioma-bearing nude mice. CONCLUSION We demonstrate strong CD317-specific anti-tumor activity of CD317-CAR T cells against various glioma cell lines in vitro and in xenograft glioma models in vivo. These data lay a scientific basis for the subsequent evaluation of this therapeutic strategy in clinical neuro-oncology.

BATF3/MYC axis drives hyper-proliferation of TET2 deficient CAR T cells

Abstract A recent report described a patient presenting a dominant chimeric antigen receptor (CAR) T cell clone at the height of anti-tumor response wherein a TET2 allele was disrupted by the integration of the lentiviral 4-1BB CAR vector (Fraietta et. al. Nature 2018). The enhanced proliferative ability conferred by this chance TET2 disruption opened up the possibility of generating TET2 edited CAR T cells for an enhanced anti-tumor efficacy. We modeled and evaluated loss of TET2 in human T cells engineered to express different CAR receptors in a murine model of human acute lymphoblastic leukemia (ALL). Our results indicate that CAR design plays a critical role in determining anti-tumor efficacy and early stage T cell phenotype upon TET2 disruption. We further observe that TET2 edited CAR T cells, over time, enter a hyper-proliferative state with a near total loss of effector function. The frequency with which TET2 edited T cells achieve hyper-proliferation is dependent on the signaling properties of the CAR receptor. This state is in contrast to the canonical exhaustion state wherein loss of proliferative ability precedes loss of effector function. This hyper-proliferative state shares some transcriptional features with T cell leukemia/lymphoma with sustained upregulation of cell cycle associated factors. Exome analysis identified point mutations and chromosomal aberrations in hyper-proliferative cells, but they were not conserved across different populations. Chromatin accessibility analysis revealed that loss of TET2 sets an epigenetic state that allows for sustained elevated levels of BATF3 which in turn drives a MYC-dependent proliferative program in TET2 deficient CAR T cells.

APDL1-CART cells exhibit strong PD-L1-specific activity against leukemia cells.

Chimeric antigen receptor (CAR) T cells target specific tumor antigens and lyse tumor cells in an MHC-independent manner. However, the efficacy of CAR-T cell and other cancer immunotherapies is limited by the expression of immune-checkpoint molecules such as programmed death-ligand 1 (PD-L1) on tumor cells, which binds to PD-1 receptors on T cells leading to T cell inactivation and immune escape. Here, we incorporated a PD-L1-targeted single-chain variable fragment (scFv) fusion protein sequence into a CAR vector to generate human anti-PD-L1-CAR-T cells (aPDL1-CART cells) targeting the PD-L1 antigen. Unlike control T cells, aPDL1-CART cells significantly halted the expansion and reduced the viability of co-cultured leukemia cells (Raji, CD46, and K562) overexpressing PD-L1, and this effect was paralleled by increased secretion of IL-2 and IFN-γ. The antitumor efficacy of aPDL1-CART cells was also evaluated in vivo by co-injecting control T cells or aPDL1-CART cells along with PDL1-CA46 cells to generate subcutaneous xenografts in NCG mice. Whereas large tumors developed in mice inoculated with PDL1-CA46 cells alone or together with control T cells, no tumor formation was detected in xenografts containing aPDL1-CART cells. Our data suggest that immune checkpoint-targeted CAR-T cells may be useful for controlling and eradicating immune-refractory hematological malignancies.

Mutated GM-CSF-based CAR-T cells targeting CD116/CD131 complexes exhibit enhanced anti-tumor effects against acute myeloid leukaemia.

ObjectivesAs the prognosis of relapsed/refractory (R/R) acute myeloid leukaemia (AML) remains poor, novel treatment strategies are urgently needed. Clinical trials have shown that chimeric antigen receptor (CAR)‐T cells for AML are more challenging than those targeting CD19 in B‐cell malignancies. We recently developed piggyBac‐modified ligand‐based CAR‐T cells that target CD116/CD131 complexes, also known as the GM‐CSF receptor (GMR), for the treatment of juvenile myelomonocytic leukaemia. This study therefore aimed to develop a novel therapeutic method for R/R AML using GMR CAR‐T cells.MethodsTo further improve the efficacy of the original GMR CAR‐T cells, we have developed novel GMR CAR vectors incorporating a mutated GM‐CSF for the antigen‐binding domain and G4S spacer. All GMR CAR‐T cells were generated using a piggyBac‐based gene transfer system. The anti‐tumor effect of GMR CAR‐T cells was tested in mouse AML xenograft models.ResultsNearly 80% of the AML cells predominant in myelomonocytic leukaemia were found to express CD116. GMR CAR‐T cells exhibited potent cytotoxic activities against CD116+ AML cells in vitro. Furthermore, GMR CAR‐T cells incorporating a G4S spacer significantly improved long‐term in vitro and in vivo anti‐tumor effects. By employing a mutated GM‐CSF at residue 21 (E21K), the anti‐tumor effects of GMR CAR‐T cells were also improved especially in long‐term in vitro settings. Although GMR CAR‐T cells exerted cytotoxic effects on normal monocytes, their lethality on normal neutrophils, T cells, B cells and NK cells was minimal.ConclusionsGMR CAR‐T cell therapy represents a promising strategy for CD116+ R/R AML.

Interleukin-6-knockdown of chimeric antigen receptor-modified T cells significantly reduces IL-6 release from monocytes

BackgroundT cells expressing a chimeric antigen receptor (CAR) engineered to target CD19 can treat leukemia effectively but also increase the risk of complications such as cytokine release syndrome (CRS) and CAR T cell related encephalopathy (CRES) driven by interleukin-6 (IL-6). Here, we investigated whether IL-6 knockdown in CART-19 cells can reduce IL-6 secretion from monocytes, which may reduce the risk of adverse events.MethodsSupernatants from cocultures of regular CART-19 cells and B lymphoma cells were added to monocytes in vitro, and the IL-6 levels in monocyte supernatants were measured 24 h later. IL-6 expression was knocked down in regular CART-19 cells by adding a short hairpin RNA (shRNA) (termed ssCART-19) expression cassette specific for IL-6 to the conventional CAR vector. Transduction efficiency and cell proliferation were measured by flow cytometry, and cytotoxicity was measured by evaluating the release of lactate dehydrogenase into the medium. Gene expression was assessed by qRT-PCR and RNA sequencing. A xenograft leukemia mouse model was established by injecting NOD/SCID/γc-/- mice with luciferase-expressing B lymphoma cells, and then the animals were treated with regular CART-19 cells or ssCART-19. Tumor growth was assessed by bioluminescence imaging.ResultsBoth recombinant IL-6 and CART-19 derived IL-6 significantly triggered IL-6 release by monocytes. IL-6 knockdown in ssCART-19 cells dramatically reduced IL-6 release from monocytes in vitro stduy. In vivo study further demonstrated that the mice bearing Raji cells treated with ssCART-19 cells showed significant lower IL-6 levels in serum than those treated with regular CART-19 cells, but comparable anti-tumor efficacy between the animal groups.ConclusionCAR T-derived IL-6 is one of the most important initiators to amplify release of IL-6 from monocytes that further drive sCRS development. IL-6 knockdown in ssCART-19 cells by shRNA technology provide a promising strategy to improve the safety of CAR T cell therapy.

Application of droplet digital PCR for the detection of vector copy number in clinical CAR/TCR T cell products

BackgroundGenetically engineered T cells have become an important therapy for B-cell malignancies. Measuring the efficiency of vector integration into the T cell genome is important for assessing the potency and safety of these cancer immunotherapies.MethodsA digital droplet polymerase chain reaction (ddPCR) assay was developed and evaluated for assessing the average number of lenti- and retroviral vectors integrated into Chimeric Antigen Receptor (CAR) and T Cell Receptor (TCR)-engineered T cells.ResultsThe ddPCR assay consistently measured the concentration of an empty vector in solution and the average number of CAR and TCR vectors integrated into T cell populations. There was a linear relationship between the average vector copy number per cell measured by ddPCR and the proportion of cells transduced as measured by flow cytometry. Similar vector copy number measurements were obtained by different staff using the ddPCR assay, highlighting the assays reproducibility among technicians. Analysis of fresh and cryopreserved CAR T and TCR engineered T cells yielded similar results.ConclusionsddPCR is a robust tool for accurate quantitation of average vector copy number in CAR and TCR engineered T cells. The assay is also applicable to other types of genetically engineered cells including Natural Killer cells and hematopoietic stem cells.

The Effect of Apoptosis Inhibitor Blockade Agents on the Third Generation CD19 CAR T Cells

Background: Chimeric antigen receptor (CAR) engineered T cell therapy is currently revolutionizing the field of cancer immunotherapy. However, further enhancement of the function of CAR T cells either through modification of CAR structure or through combination with other therapies is intensively investigated. Resistance to apoptosis is one of the hallmarks of cancer biology. Overexpression of anti-apoptotic proteins of the Bcl-2 family proteins, such as Bcl-2, Mcl-1 and Bcl-xl is considered primarily responsible for the increased apoptosis resistance and prolonged survival of hematological tumor cells. Apoptosis inhibitor blockade agents (AIBAs) such as Bcl-2, Mcl-1 and COX inhibitors could sensitize malignant cells to apoptosis. Therefore, combining CAR T cell therapy with AIBAs might be a promising approach to increase the therapeutic response. However, the third generation of CAR T cells including a 4-1BB co-stimulatory domain which triggers a signaling cascade with the upregulation of anti-apoptotic molecules might be hampered by AIBAs. Therefore, in this study we analyzed the influence of AIBAs on third generation CD19 CAR T cells. Materials and methods: CD19 CAR T cells were manufactured using a 3rd generation CAR vector containing both CD28 and 4-1BB co-stimulatory domains. The expression of anti-apoptotic Bcl-2 family members in leukemia/lymphoma cell lines and in CD19 CAR T cells have been assessed by quantitative real time polymerase chain reaction (qRT-PCR), western blot (WB) and flow cytometery. The optimal concentrations of inhibitors have been determined by CellTiter Glo™ assay. The effect of inhibitors on CAR T cells and tumor cells has been evaluated by chromium-51 (51Cr) release assay, Calcein AM™ assay and FACS-based apoptosis assay. Intracellular cytokine staining and surface marker staining were performed to determine the function of CAR T cells. Results: qRT-PCR and WB showed that leukemia/lymphoma cell lines 380 and U698M had the highest Bcl-2 (qRT-PCR: 1.73 ± 0.04, p = 0.001; WB: 1.82 ± 0.54, p = 0.016) and Mcl-1 (qRT-PCR: 12.39 ± 1.37, p = 0.000; WB: 1.92 ± 1.08, p = 0.142) expression levels, respectively,. These findings were confirmed by anti-apoptotic BCL-2 family protein intracellular staining. Elevated protein expression of Mcl-1, Bcl-2 and Bcl-xl was observed in CAR T cells upon 380 cell stimulation. The manufactured CD19 CAR T cells exhibited specific killing on CD19+ tumor cells. A slightly enhanced killing efficiency of CAR T cells was observed when 380 cells were co-cultured with CAR T cells in the presence of ABT199 (0 µM vs. 1 µM: 54.21 ± 3.23 vs. 69.06 ± 2.17, p = 0.0041). This result could be explained by a higher sensitivity of 380 cells to ABT199 than CART cells and by an increased activation of CAR T cells. Even though the killing efficiency of CAR T cells was not improved by S63845, the CD107a+TNF-α+IFN-γ+ CAR T cells were increased as well as the proliferation and persistence of CAR T cells were strengthened. Of note, pretreatment of tumor cells with inhibitors could significantly enhance the killing efficiency of CAR T cells via upregulation of CD19 antigen on tumor cells (ABT199-pretreated 380 cells: 0 µM vs.1 µM, 42.59 ± 5.82 vs. 54.58 ± 4.87, p = 0.0493; S63845-pretreated-U698M cells: 0 µM vs. 0.3 µM, 31.88 ± 4.77 vs. 66.35 ± 8.09, p = 0.000). Conclusion: The quantity of CAR T cells can be negatively affected by Apoptosis inhibitor blockade agents. However, the quality of CAR T cells could be improved. The cytotoxic effect of CAR T cells can be further enhanced by pretreatment of tumor cells with inhibitors. Disclosures Müller-Tidow: MSD: Membership on an entity's Board of Directors or advisory committees. Dreger:Neovii, Riemser: Research Funding; MSD: Membership on an entity's Board of Directors or advisory committees, Other: Sponsoring of Symposia; AbbVie, AstraZeneca, Gilead, Janssen, Novartis, Riemser, Roche: Consultancy; AbbVie, Gilead, Novartis, Riemser, Roche: Speakers Bureau. Schmitt:MSD: Membership on an entity's Board of Directors or advisory committees, Other: Sponsoring of Symposia; Therakos Mallinckrodt: Other: Financial Support. Schmitt:Therakos Mallinckrodt: Other: Financial Support.

Next Generation NKG2D-based CAR T-cells (CYAD-02): Co-expression of a Single shRNA Targeting MICA and MICB Improves Cell Persistence and Anti-Tumor Efficacy in vivo

Background The Natural Killer Group 2D (NKG2D) receptor is a NK cell activating receptor that binds to eight different ligands (NKG2DL) commonly over-expressed in cancer, including MICA and MICB. The product candidate CYAD-01 are chimeric antigen receptor (CAR) T-cells encoding the full length human NKG2D fused to the intracellular domain of CD3ζ. Data from preclinical models have shown that CYAD-01 cells specifically target solid and hematological tumors. Encouraging preliminary results from the Phase I clinical trial THINK, assessing CYAD-01 safety, showed initial signals of objective clinical responses in patients with r/r AML and MDS. The clinical development of CAR T-cells has been limited by several challenges including achieving sufficient numbers of cells for clinical application. We have previously shown that NKG2D ligands are transiently expressed on activated T cells and that robust cell yields are generated through the addition of a blocking antibody and a PI3K inhibitor during cell manufacture. Here, we investigated the ability of an optimized short hairpin RNA (shRNA) technology to modulate NKG2DL expression on CYAD-01 cells and to determine if there is an increase in the anti-tumor activity of NKG2D-based CAR T-cells (termed CYAD-02). Methods Molecular and cellular analyses identified MICA and MICB as the key NKG2DL expressed on activated T-cells and highly likely to participate in driving fratricide. In silico analysis and in vitro screening allowed the identification of a single shRNA targeting the conserved regions of MICA and MICB, thus downregulating both MICA and MICB expression. The selected shRNA was incorporated in the NKG2D-based CAR vector, creating the next-generation NKG2D-based CAR T-cell candidate, CYAD-02. In addition, truncated versions of the NKG2D receptor were generated to explore the mechanisms of action of NKG2D receptor activity in vivo. The in vivo persistence and anti-tumor activity of CYAD-02 cells was evaluated in an aggressive preclinical model of AML. Results Injection of CAR T-cells bearing truncated forms of the NKG2D-CAR in immunosuppressed mice resulted in similar persistence to the control T-cells. In contrast, CYAD-01 cells had reduced persistence, suggesting that the recognition of the NKG2DL by the NKG2D receptor could contribute to this effect. Analysis of cell phenotype upon CAR T-cell activation showed that MICA and MICB were transiently expressed on T-cells during manufacturing. These results collectively suggested that downregulating MICA and MICB expression in CYAD-01 cells could be a mean to increase CAR T-cell persistence in vivo. Candidate shRNA were screened for efficient targeting of both MICA and MICB at the mRNA and protein level. T-cells transduced with a single vector encoding for the NKG2D-based CAR and the selected shRNA targeting MICA and MICB (CYAD-02) demonstrated 3-fold increased expansion during in vitro culture in the absence of the blocking antibody used to increase cell yield during manufacture. When injected into immunosuppressed mice, CYAD-02 cells generated with the Optimab process showed 10-fold higher engraftment one week after injection and potent anti-tumor activity resulting in 2.6-fold increase of mouse survival in an aggressive AML model. Conclusions By using a single vector encoding the NKG2D-based CAR next to a shRNA targeting MICA and MICB and combined with improved cell culture methods, CYAD-02, the next-generation of NKG2D-based CAR T-cells, demonstrated enhanced in vivo persistence and anti-tumor activity. Following FDA acceptance of the IND application, a Phase 1 dose-escalation trial evaluating the safety and clinical activity of CYAD-02 for the treatment of r/r AML and MDS is scheduled to start in early 2020. Disclosures Fontaine: Celyad: Employment. Demoulin:Celyad: Employment. Bornschein:Celyad: Employment. Raitano:Celyad: Employment. Machado:Horizon Discovery: Employment. Moore:Avvinity Therapeutics: Employment, Other: Relationship at the time the work was performed; Horizon Discovery: Employment, Equity Ownership, Other: Relationship at the time the work was performed; Centauri Therapeutics: Consultancy, Other: Current relationship. Sotiropoulou:Celyad: Employment. Gilham:Celyad: Employment.