Abstract B131: NXC03, an AI-designed, affinity-attenuated IL-21 mutein with half-life extension enhances antitumor immunity
Authors: Yuan Puwei , Liu Junli , Zhang Fei , Lei Ying , Zhu Yanan , Gao Songsong , Liu Fan , Guo Taylor B 
Published: December 2023

Abstract
Background:IL-21 is a pleotropic cytokine with actions including functional programming of CD8+ T cells towards a central memory phenotype with enhanced activation, expansion and survival. Indeed, recombinant IL-21 exhibited antitumor activity in preclinical and clinical studies, but its clinical development has been hindered due to short serum half-life and high dose-limiting toxicities such as hepatotoxicity and neutropenia. We hypothesized that an IL-21 mutein with weaker binding to IL-21 receptor (IL-21R) and increased stability and half-life may achieve an optimal therapeutic index for development. Here we describe the discovery and preclinical profile of such an AI-designed IL-21-Fc fusion mutein, NXC03.
Methods:We computationally evaluated ~400,000 combinatorial mutations of IL-21 to look for those with desired affinity and stability profile via multiple techniques, including direct coupling analysis and physical energy functions. Following iterative in silico and in vitro screening, we selected NXC03-9804 out of the top-20 hits and fashioned it as an IgG4 Fc fusion protein (NXC03). We measured the affinity of NXC03 to human IL-21R by biolayer interferometry and its melting temperature (Tm) by label-free nano differential scanning fluorimetry. We determined the bioactivity of NXC03 by assessing human primary T-cell proliferation and STAT3 phosphorylation (pSTAT3) in huT78 cells. We evaluated the antitumor efficacy of NXC03 alone or in combination with a murine PD-1 mAb (aPD1) in MC38 and anti-PD-1 resistant B16-F10 syngeneic tumor models in hIL21R knock-in mice. We also studied the safety profiles and pharmacokinetics of NXC03 following subcutaneous (SC) administrations in monkeys.
Results:NXC03 differs from wildtype (WT) IL-21 by 4 amino acid residues and displays a unique fast-on fast-off binding kinetics to IL-21R with a 1000-fold larger KD than that of WT IL-21. It has a Tm of 62.0 °C, notably higher than that of WT IL-21 (48.5 °C). Despite a ~10-fold reduction in potency in inducing pSTAT3 in huT78 cells, NXC03 was comparable to WT IL-21 in enriching CCR7+CD45RO-CD8+ stem memory T cells (TSCM) in PBMC culture. In both MC38 and B16-F10 mouse models, NXC03 monotherapy demonstrated strong, dose-dependent antitumor activity and was more potent than WT IL-21. Combination treatment of NXC03 with aPD1 further increased efficacy compared to either alone. Fusion of NXC03 and aPD1 had similar effect. Moreover, NXC03 treatment produced lasting memory response that protected against tumor rechallenge following drug cessation. NXC03 exhibited a half-life in monkeys (14.1 h, IV; 55.5h, SC) much greater than that of WT IL-21 (0.8 h, IV). NXC03 was well tolerated with no neutropenia or hepatotoxicity observed in monkeys.
Conclusion:NXC03, an AI-designed attenuated IL-21 mutein, exhibits strong antitumor activity and holds promise as an immunostimulatory agent for combination with checkpoint inhibitors. Given its excellent drug developability profile, NXC03 is being fast-tracked for development towards clinical testing.
Citation Format: Puwei Yuan, Junli Liu, Fei Zhang, Ying Lei, Yanan Zhu, Songsong Gao, Fan Liu, Taylor B Guo. NXC03, an AI-designed, affinity-attenuated IL-21 mutein with half-life extension enhances antitumor immunity [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B131.