Dual Split-Signaling TIM3+CLEC12a Targeting CAR T-Cells with Optimized Signaling As a Safe Potential Therapy for Acute Myeloid Leukemia

Background:

The success of chimeric antigen receptor (CAR) T-cell therapies targeting B cell maturation antigen (BCMA) in multiple myeloma and CD19 in B-cell malignancies has demonstrated the immense potential of this immunotherapeutic strategy. However, the translation of CAR T-cell therapy to acute myeloid leukemia (AML) patients has encountered challenges due to the absence of surface antigens specifically or preferentially expressed on AML cells. To overcome this hurdle, we explored the feasibility and safety of simultaneously targeting the AML-associated antigens CLEC12a and TIM3 using an AND-gate strategy in which the essential stimulatory and co-stimulatory T-cell activation signals are segregated into separate CARs. To improve the safety of our strategy we also attenuated signaling through the stimulatory anti-CLEC12a CAR via affinity-optimization or through targeted mutations in its immunoreceptor tyrosine-based activation motif (ITAM) regions.

Methods:

To identify the most optimal dual-split CAR T-cell configuration, we designed several chimeric antigen receptor constructs in which we targeted CLEC12a with stimulatory CARs (sCARs) with high (C^hi) or low (C^low) affinities. In addition, the high affinity C^hi stimulatory CAR was mutated in the second and third ITAM regions (denoted as C^hixx) to attenuate the CD3ζ stimulatory signal. TIM3 was targeted by a costimulatory CAR (cCAR) containing both CD28 and 41BB signaling domains. The dual-split CARs were generated using two separate retroviral constructs. After the assessment of the CAR expression and specific surface markers associated with memory phenotype and exhaustion, dual-split CAR T-cells were functionally tested using an isogenic cell line model for specific lysis against dual CLEC12a/TIM3 positive or single CLEC12a or TIM3 positive isogenic target cells as well as for dual antigen-specific cytokine secretion. The dual-split CAR T-cells were also tested for their efficiency to specifically eliminate leukemic stem/progenitor cells by the colony formatting unit (CFU) assay. Moreover, anti-AML activity and persistence of the dual-split CAR T-cells was assessed using an AML PDX mouse model in which human primary AML cells expressing TIM3 and CLEC12a were injected.

Results:

Using the isogenic cell line model, we revealed that the dual-split CAR T-cells with the combination of the high affinity CLEC12a sCAR and TIM3 cCAR (C^hi+T) show antigen-specific cytokine secretion and cytotoxicity, and that this specificity was enhanced when the CLEC12a sCAR was attenuated through ITAM mutations (C^hixx+T). In CFU assays, all tested CLEC12a/TIM3 dual-split CAR T-cell combinations exhibited potent inhibition (up to 95%) of leukemia blast viability and leukemia stem/progenitor self-renewal. In vivo, although AML did not engraft in all mice, the best-performing dual-split C^hixx+T1 CAR T-cells displayed potent anti-leukemic activity, as they prevented engraftment in two additional mice compared to control groups (n=6 each group). Furthermore, in contrast to control groups dual-split CAR T-cells exhibited prolonged persistence in the blood up to 20 weeks after injection.

Conclusion:

We successfully generated highly specific dual-targeting CAR T-cells by splitting the T-cell activation signals into two simultaneously expressed CARs, and using a novel approach of attenuating the sCAR through mutations in ITAM regions. Overall, we show that dual-split CAR T cells targeting CLEC12a and TIM3, particularly the combination C^hixx+T CAR T-cells, have specific and promising anti-AML activity. Our findings underscore the potential of innovative dual-split CAR T-cell therapies as a novel and effective immunotherapeutic approach for combating AML.