1369 CIS-demasking cytokine linker technology allows selective cytokine delivery to activated immune cells while sparing others and peripheral toxicity

BackgroundImmunocytokines are promising new therapeutic approaches to enhance T-cell response by delivering interleukins into the tumoral site. Fusion of wild-type or attenuated interleukins to anti-PD-1 antibody has shown some efficacy to preferentially cis-activate PD1-expressing T-cells but on-target/off-tumor activity on PD-1-negative cells is still observed due to the high affinity of the cytokine to its receptor leading a broad systemic effect, toxicity, high clearance and low tumor biodistribution limiting the potential of some immunocytokines (e.g. anti-PD-1/IL-2/IL-15/IL-21). Various strategies have emerged to achieve localized cytokine activation using conditional strategies (e.g. allosteric modulators, MMP-cleavable linkers or pH-dependent binding) that remain challenging due to high dependency to specific TME composition (MMP, acidosis...).We developed the Cytomask Platform, a universal and innovative linker technology allowing exclusive cytokine CIS-demasking upon binding of the fused antibody to its target without TRANS-activation associated with undesired effects(e.g. Toxicity).MethodsA series of different linkers in term of length, composition, charge... were screened for cis-activation of cytokine on PD1-expressing vs PD1-negative T-cells using different cytokines fused to a high-affinity anti-PD1. Iterative screening cycles wer performed to select optimal composition. In vivo, pharmacokinetic/pharmacodynamic studies have been performed using humanized PD1KI tumor-bearing mice.ResultsCytomask linker technology decreases IL-2 or IL-15 cytokine activity (pSTAT5 signaling) on PD1-negative cells while maintaining high activation of PD1-transduced T-cells even in co-culture. On primary naïve (PD1-) vs activated (PD1+) human T-cells, the difference was more striking, since no or very low activation has been observed in naïve T-cells while high potentiation of PD-1+activated T-cells was induced. Importantly, Cytomask linker technology does not induce TRANS-activation on PD1-negative T-cells illustrating an innovative and strict CIS-demasking mechanism of action. In vivo, Anti-PD-1/IL-15 and/or anti-PD1/IL-2 Cytomask molecules illustrated better pharmacokinetic compared to conventional linkers, strong reduction of peripheral T-cell proliferation and reduced toxicity after single or multiple injections(2mg/kg). High T-cell proliferation has been observed in tumor-micro-environment where PD1-expressing T cells are located. Low T-cell proliferation i