Synkir-310 split-signaling based KIR-CAR T cell therapy achieves faster and deeper anti-B cell tumor efficacy with reduced cytokine levels

Abstract

Abstract Currently, five CD19-targeting chimeric antigen receptor (CAR) T cells are FDA-approved for treatment of B cell malignancies. These CAR T share a single-chain CD3-based signaling structure incorporating a co-stimulation motif and FMC63 single-chain variable fragment (scFv) to target CD19. While these CAR T demonstrate robust clinical responses in 60-80% of patients with B cell non-Hodgkin lymphomas (B-NHL), approximately half of patients relapse within one year (Cappell Nat Rev Clin Oncol. 2023), demonstrating a need for more durable therapies. We designed SynKIR-310 using our multi-chain natural killer immunoglobulin-like receptor (KIR)-CAR T cell platform (Wang Cancer Immunol Res. 2015) targeting CD19 with a novel canine-derived, more human-like scFv, DS191. SynKIR-310 uses more physiologically-relevant separated antigen binding and activation signals derived from natural killer (NK) cells. This CD19-targeting KIR-CAR was designed to reduce the constitutive T cell activation and exhaustion observed with conventional single-chain CAR, and to confer prolonged anti-tumor functionality and deeper anti-tumor responses. SynKIR-310 was compared in vitro and in vivo with a CD3-based single-chain CAR T analogous to tisagenlecleucel (FMC63-41BBζ CAR) for antigen specificity, sensitivity, and anti-tumor functions including cytokine production and target cell lysis. In vitro, SynKIR-310 and FMC63-41BBζ CAR T showed comparable results in all functional co-culture assays (Yucel ASGCT 2024; Blair ASGCT 2025).This data supported the specificity and sensitivity of SynKIR-310 for CD19. To evaluate SynKIR-310 in vivo, we engraftedimmunocompromised NOD-SCID-IL2Rγc–/– (NSG) mice with CD19+ NALM6 tumors by intravenous (IV) injection on Day 0 and performed a dose escalation study of SynKIR-310 versus FMC63-41BBζ CAR T cells. T cells were IV injected 5 days after tumor injection, and tumor progression was monitored by bioluminescent imaging (BLI). At higher doses of T cells, both SynKIR-310 and FMC63-41BBζ CAR T performed comparably overall against tumors, although SynKIR-310 showed a more rapid onset of anti-tumor effect. At the low dose, FMC63-41BBζ CAR T cells showed no anti-tumor impact, while SynKIR-310 significantly reduced tumor progression throughout the duration of the study. Intriguingly, both CAR T types remained detectable in the periphery at comparable levels, although only KIR-CAR showed anti-tumor efficacy. Importantly, there were no overt toxicities detected or significant differences between SynKIR-310 and FMC63-41BBζ CAR at low or high dose-treated mice in a toxicology panel performed on terminal serum collected at the end of the study. To determine whether the improved anti-tumor efficacy in vivo resulted from use of the novel DS191 scFv binder, or from different signaling in KIR-CAR versus 41BBζ CAR, we compared SynKIR-310, FMC63-KIR-CAR and FMC63-41BBζ CAR T cells at the same low dose in the NALM6-NSG in vivo model. SynKIR-310 and FMC63-KIR-CAR T were comparable in controlling the tumors earlier than FMC63-41BBζ CAR T and both demonstrated deeper tumor control than FMC63-41BBζ CAR T, highlighting that the multichain NK-based KIR-CAR signaling was responsible for the differential responses, and all 3 groups had similar T cell persistence. Evaluation of circulating cytokines at early (Day 8) and later (Day 13) time points also revealed notable differences. While all treatment groups produced similar levels of cytokines early on, at the later time point, FMC63-41BBζ CAR treatment, with a poorer anti-tumor response, paradoxically showed significantly more IFNγ (37-fold), TNFα (27-fold) and IL-2 (17-fold) compared to both SynKIR-310 and FMC63-KIR-CAR treatments. Overall, these results demonstrate SynKIR-310 achieves faster tumor regression and greater tumor control, with reduced systemic levels of cytokine production. These results may translate into improved anti-tumor activity along with a reduced risk of cytokine-mediated toxicities typically observed in conventional CD3-based CAR T cell therapies, supporting clinical translation of SynKIR-310 for patients with relapsed/refractory B-NHL, currently in an ongoing Phase 1 clinical trial (NCT06544265).