IKZF1 Deletion Confers Resistance to CAR19 in r/r B-ALL Via CD19 Intron Retention

In recent years, CD19-directed chimeric antigen receptor T-cell (CAR19) therapy has provided a promising outcome for hematological malignancies, but a considerable number of patients ultimately have progressive disease. Our previous study also found that tumor intrinsic IKZF1 deletion (IKZF1^del) conferred an inferior event-free survival (EFS) in refractory and/or refractory (r/r) B-ALL patients receiving CAR19 therapy, with 1-year EFS of 27.8 ± 12.4%, compared with 53.6 ± 6.1% of the IKZF1^wt group (P=0.011). [28th EHA oral presentation; 2023] However, the underlying impact of tumor intrinsic IKZF1^del on the resistance to CAR19 therapy remains elusive.

Previous studies have emphasized the importance of IKZF1 in the process of B cell development, as well as the abnormal alternative splicing of CD19 in CAR19 resistance. herein we presumed that IKZF1^del mediated CAR19 resistance via CD19 introns retention.

Firstly, we performed RNA-Seq analysis using bone marrow mononuclear cells from patients newly diagnosed with B-ALL. A total of 113 patients (IKZF1^del (37 of 113, 32.7%) vs. IKZF1^WT (76 of 113, 67.3%)) was collected to analyze the frequency of CD19 introns retention (IR). Expectedly, we found that the average frequency of IR was higher in the IKZF1^Del group, compared with the IKZF1^WT group.

Furthermore, we conducted a protein-structure prediction, which showed a premature termination codon in CD19-IR2, and caused a truncated CD19 protein with a dys-function. And the contact of CAR scFv structure (FMC63) and target antigen was predicted, suggesting an unstable binding region of FMC63 and CD19-IR2 structure, which yielded a lower affinity of -8.4 kcal/mol, compared to -11.4 kcal/mol in the structure of CAR and wildtype CD19.

Additionally, to verify whether tumor intrinsic IKZF1^Del mediated CAR19 resistance via an unstable contact structure, we generated Nalm6 leukemic cells with IKZF1 deletion subtype (Nalm6_IK6). Nalm6_IK6 cells showed a higher level of non-functional IK6 subtype and lower lever of functional IK1 subtype, and the expression of CD19-IR2 was higher in Nalm6_IK6 cells. Then we tested their avidity to CAR19 cells using a single-cell avidity measurement technology, z-MOVI, which determined the precise binding force between the effector and target cells. Inspiring, after a 5-minute co-culture period, Nalm6_IK6 cells showed a significantly lower binding capability followed by acoustic force exposure compared to Nalm6_WT cells. The cytotoxic capacity of CAR19 to these tumor cells was also investigated. Notably, after a 24-hour co-culture period, CAR19 cells showed weaker lytic activity of Nalm6_IK6 cells than Nalm6_WT cells.

In conclusion, herein we suggested that tumor intrinsic IKZF1^del conferred an affinity-dependent resistance to CAR19 via CD19 introns retention. These data not only enriched the field of how tumor suppressor gene-IKZF1^del mediated CAR19 immunotherapy resistance, but offered a theoretical basis for optimal strategy to these IKZF1^del r/r B-ALL patients.