Background Immunocytokines 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).

Methods A 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.

Results Cytomask 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 in periphery was seen, illustrating the potential of CIS-demasking technology to target the right T-cells at the right place.

Conclusions The Cytomask linker technology illustrates specific intrinsic property to mask cytokine on naïve peripheral immune cells not expressing the target of the antibody while allowing selective CIS-demasking of the cytokine and CIS-activation of activated immune cells expressing (e.g. PD-1). This linker technology could be used with a broad range of cytokine to abrogate OFF-tumor cytokine activity associated with toxicity while selectively CIS-activating activated immune cells in the TME.