cesnicabtagene autoleucel (ARI0002h)

We optimized different dual-targeting approaches, including co-transduction of two lentiviral vectors, bicistronic, tandem, loop and pool strategies, based on our academic anti-CD19 (ARI0001) and anti-BCMA (ARI0002h) CAR-T cells. ARI0003 CAR-T cells were effectively manufactured under Good Manufacturing Practice conditions, with reduced risk of genotoxicity compared to other dual-targeting approaches. A first-in-human phase I clinical trial (CARTD-BG-01, NCT06097455) has been initiated to evaluate the safety and efficacy of ARI0003 in NHL.

Lymphodepletion (LD) with cyclophosphamide and fludarabine was used...Tocilizumab and steroids were administered in 68% (mainly for persistent grade 1 CRS) and 30% of pts, respectively... Results from 30 additional pts and a longer follow-up of the first cohort confirm the deep and durable responses obtained with ARI0002h. The booster dose may be partially responsible for the improvement of responses over time and exhaustion may play a role in relapse.

To shed some light on specific transcriptomic programs activated after CAR-T cell administration, we interrogated longitudinal samples of CAR-T cells collected from patients enrolled in CARTBCMA-HCB-01 (NCT04309981) and academic clinical trial in patients with RRMM (Oliver-Caldes A, et al... Overall, our analysis combining scRNA-seq/scTCR-seq with novel machine learning models, allowed us to characterize key transcriptional differences observed between patients, infusion products and in vivo infused CAR-T, as well as between CAR-T according to their location (PB vs. BM). Importantly, our results identify IL10 as a regulatory mechanism promoting CAR-T cell dysfunction, representing a potential target to be modulated for the development of improved CAR-T therapies for MM.

To shed light on the potential mechanisms driving CAR-T cell persistence we performed longitudinal transcriptomic analysis, at single cell level, of CAR-T cells from a patient enrolled in CARTBCMA-HCB-01 clinical trial (NCT04309981) (Oliver-Caldes A, et al... Our analysis clearly showed that most CAR-T cells after administration presented expression of key markers related to T cell memory and survival without clonal expansion, suggesting that memory induction is crucial for long-term persistence. Interestingly, prior to tumor relapse, we observed reactivation of CAR-T cell with the enrichment of effector and activation signatures, and GRN analysis identified FOS and NR4A2 regulons as potential drivers involved in CAR-T cell reactivation. Overall, scRNA-seq/scTCR-seq coupled with novel machine learning models provides relevant mechanistic insights that could uncover useful targets to be modulated for the development of long-term persistent CAR-T cells.

ARI0002h administered in a fractioned manner with a booster dose after 3 months can provide deep and sustained responses in patients with relapsed or refractory multiple myeloma, with a low toxicity, especially in terms of neurological events, and with the possibility of a point-of-care approach.

At our institution, we have developed an academic BCMA-BBζ CART product (ARI2h; NCT04309981) for relapsed/refractory MM patients with encouraging results... This is the first bispecific BCMA/CD229 CART cell that adequately controls BCMA-expressing and non-expressing myeloma cells. This approach could mitigate disease escape due to biallelic loss of BCMA.

Our In-house developed CARs targeting CD19 (ARI0001) and BCMA (ARI0002h) have proven safe and efficacious in the treatment of patients with B-cell malignancies and multiple myeloma, respectively. Here we present co-transduction as a feasible strategy to obtain dual CD19/BCMA CAR-T cells. ARI0003 responds better to lower CD19 levels than ARI0001 and presents advantages compared to the bicistronic strategy in terms of efficacy and to the pool in terms of time and cost of manufacturing. Our studies provide strong pre-clinical data to test ARI0003 dual-CAR-T cells in clinical trials to treat NHL patients.

CAR-T cells were FACS-isolated from infusion products (IP) as well as from bone marrow (BM) and peripheral blood (PB), at different times after cell infusion (1 and 3 months), from three MM patients enrolled in the academic clinical trial assessing ARI0002h... Overall, our data show that multiomic analysis are useful tools to characterize CAR-T cell dynamics after infusion, that presented different characteristics regarding their location (PB vs BM) and a heterogeneous behavior within the patients. Moreover, GRN analysis at single cell level offers an important tool to understand mechanisms modulating CAR-T cell response and to identify possible mechanisms of relapse.

In this work we performed phenotypic and functional characterization of CAR-T generated from healthy donors and MM patients at different disease stage.Method ology: Second generation CAR-T cells targeting BCMA and co-expressing BFP as a reporter gene (modified from ARI0002h CAR-T sequence1), were generated by lentiviral transduction of aCD3/aCD28 activated T cells, from each group of donor/patients, and expanded in the presence of IL-7/IL-15...Conclusion s : Our results would indicate that CAR-T generated from patients at later stages of the disease present a more differentiated phenotype, with reduced functionality and increased exhaustion features, that could compromise antitumor efficacy. Transcriptomic analysis allows the identification of molecular mechanisms that could be modulated for the development of improved therapies.

A BCMA-BBζ CAR T cell product (ARI2h; NCT04309981) has been developed at our Institution for relapsed/refractory MM patients with encouraging results (Fernández de Larrea, EHA 2022)...We have developed the first bispecific CD229/BCMA CAR T cell against MM, demonstrating that a single bicistronic vector encoding BCMA- and CD229-targeted CAR T cells effectively controls BCMA-expressing and non-expressing MM cells. This could be translated into complete mitigation of escape mediated by BCMA loss.

All pts had previous triple-exposure to bortezomib, lenalidomide and daratumumab, and 37% were penta-exposed (addition of carfilzomib and pomalidomide). ARI0002h-related neutropenia is clinically relevant and has a slower recovery profile compared to CD19-CART therapy for lymphoma, but with three differentiated phenotypes with a similar clinical behavior. CRP level and CRS duration were the only variables associated with the duration of neutropenia.

Stimulated T cells transduced with ARI2h, an in-house anti-BCMA CAR that is currently being explored in a clinical trial for relapsed/refractory MM (NCT04309981), were electroporated with either Cas9 alone (WT) or Cas9 + Prdm1-targeting sgRNA (Blimp-1 KO)... Genetic disruption of Blimp-1 caused anti-BCMA CAR-T cells to maintain a memory-like phenotype that retained effector function, even following repeated challenges with tumour cells. As a result, Blimp-1 KO CAR-T cells were considerably more effective than WT CAR-T cells at treating advanced MM in a murine model. Overall, we conclude that using gene editing to restrict Blimp-1 expression represents a promising strategy to enhance the sustained efficacy of anti-BCMA CAR-T cells.

Methodology: CAR-T cells were FACS-isolated from infusion products (IP) as well as from bone marrow (BM) and peripheral blood (PB) from three MM patients (HCB08, IBS08 and HVA02), enrolled in the academic clinical trial CARTBCMA-HCB-01 (NCT04309981) assessing ARI0002h, an autologous CAR T-cell product targeting BCMA1, at different times after cell infusion (1 and 3 months)... Overall, our data show that multiomic analysis are useful tools to characterize CAR-T cells and their dynamics after infusion, that presented different characteristics regarding their location (PB vs BM) and a heterogeneous behavior within the patients. Moreover, GRN analysis at single cell level offers an important tool to understand mechanisms modulating CAR-T cell response and to identify possible mechanisms of relapse.

CAR constructs targeting CD19 (ARI0001) or BCMA (ARI0002h) were engineered in our institution and both have demonstrated safety and efficacy for treating patients with B-cell lymphoid neoplasms and multiple myeloma, respectively. Co-transduction is advantageous with respect to the pool since it only requires one manufacturing process, decreasing the cost of the therapy and increasing patients' access to these novel therapies. Our studies provide a strong rational to test CD19/BCMA dual-CAR-T cells in clinical trials for the treatment of NHL patients.

ARI0002h production is fast and feasible. The peak of CART expansion occurs after 14d of infusion and also in some pts after reinfusion, regardless of LR, which suggests that LR may not be mandatory to allow further expansion after booster dose. Responses obtained are deep and construct immunogenicity seems to be low.