Paper Details

ABSTRACT

CD19/20 KIR-CAR is a CD19 and CD20 dual targeted multi-chain killer immunoglobulin-like receptor (KIR)-based chimeric antigen receptor (CAR) with 2 independent membrane-anchored scFv proteins and a separate DAP12 intracellular activator domain derived from NK cells. CD19/20 KIR-CAR binds either target to induce tumor lysis, broadening potential patient (pt) population while eliminating tumor antigen loss.

Up to half of B cell non-Hodgkin lymphoma (B-NHL) patients treated with approved CD19-targeted CAR T will experience tumor progression within 2 years, due in part to CD19 antigen loss. To combat CD19-negative relapse, CARs targeting 2 separate tumor antigens have been developed with early clinical success, though durability and toxicity are ongoing concerns. Commonly, dual-targeting CAR T cells comprise 2 different binders in tandem as a single polypeptide, retaining single-chain architecture. Dual-targeting CAR T cells can also be generated by expressing 2 single-chain CARs in the same T cell, either via a single bicistronic construct or multiple vectors. In either case, this added complexity increases the potential for on- and off-target T cell activity and exhaustion.

Compared to single-chain FM63-41BBζ or FMC63-CD28ζ CAR designs, CD19-targeted DS191-KIR-CAR cells comprise separate targeting and signaling KIR/DAP12 domains, which function only upon antigen binding. We have demonstrated that KIR-CAR T cells have reduced tonic signaling and off-target activity vs single chain CAR T cells. In vivo we have shown that DS191-KIR-CAR has superior tumor control in xenograft models vs benchmark FM63-41BBζ or FMC63-CD28ζ CAR T, while paradoxically secreting lower levels of cytokines. DS191-KIR-CAR is currently being evaluated in a FIH phase 1 clinical trial for pts with CAR-treated or naïve relapsed/refractory B cell non-Hodgkin lymphomas (NCT06544265).

CD19/20 dual KIR-CAR builds upon the DS191-KIR-CAR to demonstrate proof-of concept for the modular design of dual targeting KIR-CAR. Identical KIR2DS2 transmembrane (TM) domains were used for each binder to preserve equivalent signaling capacity via DAP12, and DNA sequences were optimized to eliminate lentiviral genetic deletions. Given the potential differences in distance between CD19 and CD20 scFv-binding epitopes to the tumor cell membrane, we optimized the immune synapse length to maximize on-target T cell activation.

We found that KIR-CAR functionality was highly dependent on the length of the stem region between the TM domain and scFv, particularly for the CD20-targeting Leu16 scFv, which yielded poor lysis with the 26-amino acid (aa) stem domain in DS191-KIR-CAR. Since Leu16 recognizes a membrane proximal epitope, we increased this region to 65aa  (Leu16_Long-KIR-CAR), which substantially increased CD20+ tumor lysis (up to 100-fold vs the short stem) in vitro. We also showed that CD19/20_Long dual KIR-CAR effectively lysed Raji tumors expressing both CD19 and CD20 in vitro, on par with single antigen-targeted KIR-CAR constructs without additive cytokine production. In contrast to a previously published CD19/20 bicistronic conventional CAR T benchmark design, CD19/20 dual KIR-CAR T lysed tumors effectively with 4-fold reduced levels of IFN? and TNFα, and a 7-fold reduced level of IL-2. In vivo, CD19/20_Long dual KIR-CAR effectively treated a CD19-antigen-loss lymphoma model in NOD-SCID IL2Rγc^–/– mice engrafted with mixed CD20+ CD19+/- Raji tumors. While both CD19/20 dual-targeted conventional CAR and KIR-CAR controlled tumors, CD19/20 KIR-CAR did so with a 3-fold reduction in levels of IFN? and TNFα, and over a 10-fold reduction of IL-2 in mouse plasma. We investigated the potential for CD19/20 dual KIR-CAR T cell generation through both ex vivo and in vivo lentiviral delivery, demonstrating the adaptability of the KIR-CAR construct for different delivery platforms.

In summary, we have demonstrated preclinical proof-of-concept for a novel dual-targeting multi-chain KIR-CAR in vitro and in vivo in mouse xenograft tumor models. Generated using a modular platform, CD19/20 dual KIR-CAR T cells conferred effective control of antigen-heterogenous B cell lymphomas, with substantially reduced cytokine secretion over single-chain CAR T cells, suggesting potential for less CRS or ICANS and an overall improved risk/benefit ratio. These results support further development of ex vivo and in vivo CD19/20 dual KIR-CAR generation for translation into clinical trials for patients with B-NHL.