In vitro evidence supporting the use of exogenous IL-2 support for allogeneic UCART22 cells to treat heavily pretreated R/R B-ALL patients Free

Introduction: Clinical and preclinical studies suggest that exogenous low dose Interleukin-2 (IL-2) administration can significantly boost CAR-T efficacy without exacerbating toxicity. IL-2 plays a critical role in enhancing the expansion and persistence of CAR-T cells by supporting T-cell proliferation, survival, and effector function. IL-2 can amplify the initial expansion phase by promoting the clonal growth of activated T cells, particularly CD8⁺ cytotoxic subsets. It also supports the formation and maintenance of memory-like CAR-T cells, contributing to prolonged persistence and sustained anti-tumor activity. UCART22 is an allogeneic non-alloreactive T-cell product gene edited with messenger ribonucleic acids (mRNAs) encoding Transcription Activator Like Effector Nucleases (TALEN®) to disrupt T-cell receptor alpha constant chain (TRAC) and CD52 genes and transduced with 3rd generation self-inactivated lentiviral vector to express anti-CD22 chimeric antigen receptor (CAR) and RQR8 (a depletion ligand which confers susceptibility to rituximab). It is currently at phase 2 clinical development in heavily pretreated R/R Acute Lymphoblastic Leukemia. An in vitro study investigated the impact of exogenous IL2 on proliferation and persistence and target cell elimination with repeated re-activation using a CD22-positive target cell line.

Methods: A CD22-positive Raji cell line engineered to express EGFP and NanoLuc^TM, was transduced with a R&D grade lentiviral vector co-expressing the NanoLuc^TM and the EGFP protein. At Day 0, 1x10^6 CD22CAR+ T cells were seeded with 250 000 CD22-postive target cells. The cells were passaged and rechallenged on Day 4, 7, 11, 14 and the experiment stopped on Day 18. At each timepoint of passaging, 1x10^6 T cells were reseeded in 1 mL of medium with 250 000 fresh CD22-expressing target cells; except on Day 14 where the number of added target cells was doubled to 500 000 cells. Live cell concentration was determined at each timepoint using a Nucleocounter-200. To quantify each cell population ( UCART22 drug product and the CD22-positive target cell line Raji_NanoLuc_GFP_Clone1), flow cytometry was performed to quantify the percentage of EGFP-positive cells, corresponding to the CD22-positive target cell line, and the percentage of EGFP-negative cells, corresponding to the UCART22 drug product cells . The cumulative fold expansion (CFE) of UCART22 cells and the absolute number of live Raji cells was calculated for each timepoint.

Results: During the first 4 challenges target Raji cells were efficiently killed regardless of presence and concentration of exogenous IL2. When the amount of Raji cells was doubled at day 18, CAR T cells were only able to show killing activity in the presence of exogenous IL2. Lower concentration of IL2 appeared to result in improved maintenance of killing when compared to the higher concentration at Day 18. Administration of IL2 was associated with increased expansion UCART22 cells throughout the repeated challenge resulting in increased persistence of cells at day 18. Of note, lower IL2 concentrations resulted in later peak expansion and higher UCART22 levels at the end of the study while allowing improved target killing.Conclusion: This in vitro study suggests that exogenous IL2 may improve expansion and persistence of UCART22 which may enhance tumour killing. Whether or not this observation will translate into improved clinical outcomes for patients warrants further evaluation in the clinical setting.