Efficacy and safety of universal (TCRKO) ARI-0001 CAR-T cells for the treatment of B-cell lymphoma

Abstract

Autologous T cells expressing the Chimeric Antigen Receptor (CAR) have been approved as advanced therapy medicinal products (ATMPs) against several hematological malignancies. However, the generation of patient-specific CAR-T products delays treatment and precludes standardization. Allogeneic off-the-shelf CAR-T cells are an alternative to simplify this complex and time-consuming process. Here we investigated safety and efficacy of knocking out the TCR molecule in ARI-0001 CAR-T cells, a second generation αCD19 CAR approved by the Spanish Agency of Medicines and Medical Devices (AEMPS) under the Hospital Exemption for treatment of patients older than 25 years with Relapsed/Refractory acute B cell lymphoblastic leukemia (B-ALL). We first analyzed the efficacy and safety issues that arise during disruption of the TCR gene using CRISPR/Cas9. We have shown that edition of TRAC locus in T cells using CRISPR as ribonuleorproteins allows a highly efficient TCR disruption (over 80%) without significant alterations on T cells phenotype and with an increased percentage of energetic mitochondria. However, we also found that efficient TCRKO can lead to on-target large and medium size deletions, indicating a potential safety risk of this procedure that needs monitoring. Importantly, TCR edition of ARI-0001 efficiently prevented allogeneic responses and did not detectably alter their phenotype, while maintaining a similar anti-tumor activity ex vivo and in vivo compared to unedited ARI-0001 CAR-T cells. In summary, we showed here that, although there are still some risks of genotoxicity due to genome editing, disruption of the TCR is a feasible strategy for the generation of functional allogeneic ARI-0001 CAR-T cells. We propose to further validate this protocol for the treatment of patients that do not fit the requirements for standard autologous CAR-T cells administration.

Combining CRISPR-Cas9 and TCR exchange to generate a safe and efficient cord blood-derived T cell product for pediatric relapsed AML

“… 29 53 54 However, when using CRISPR/Cas9 for single or multiple targets, it is important to take the probability of off-target effects, such as translocations and off-target insertion/deletions, into consideration. The probability of off-target effects appears to be low 55 ; however, further investigation with improved methods for quantitative evaluation is required. 56 The complexity of CRISPR/Cas9 in combination with lentiviral transduction provides opportunities to optimize the application and to minimize the probability for off-target effects to occur, for example using more specific high-fidelity nuclease proteins, minimizing exposure to gRNAs, and controlling dosage.…”

Generating universal anti-CD19 CAR T cells with a defined memory phenotype by CRISPR/Cas9 editing and safety evaluation of the transcriptome

“…Creative Biolabs supplied a third-generation anti-CD19 CAR (clone FCM63, αCD19-28-BB-zz) which incorporates the intracellular domains of CD28, 4-1BB and CD3z, along with a truncated EGFR as a selection marker. Lentiviral vectors carrying this CAR were manufactured in our lab by co-transfection of HEK-293T cells with the plasmid of interest, the plasmid pCMVDR8.91 and the p-MD-G plasmid as previously described by our team ( 36 ).…”

CAR-T in the Treatment of Acute Myeloid Leukemia: Barriers and How to Overcome Them

“…Moreover, progress is being made in the development of allogeneic “universal” CAR T‐cell therapies. These off‐the‐shelf CAR‐T cells can be manufactured in batches instead of on‐demand, resulting in economies of scale, which may lower the cost for the healthcare payers 88 . Finally, the costs of associated care could also potentially be reduced through a reduction/shift in side effects with novel CAR T‐cell therapies.…”

“…These off-the-shelf CAR-T cells can be manufactured in batches instead of on-demand, resulting in economies of scale, which may lower the cost for the healthcare payers. 88 Finally, the costs of associated care could also potentially be reduced through a reduction/shift in side effects with novel CAR T-cell therapies. Ultimately, it may become possible in the future to reprogram T cells in vivo, thereby avoiding ex-vivo manufacturing costs.…”