Abstract LB333: Cytokine affinity tuning using the AlphaSeq platform to generate targeted immuno-oncology therapeutics

Abstract

Abstract Here, we demonstrate a novel approach for generating affinity detuned cytokines that can be fused to antibodies for selective immune cell activation to improve anti-tumor responses and mitigate systemic toxicity. Although cytokine therapies have demonstrated curative effects in some cancer patients, clinical use remains limited with current toxicity profiles accompanying systemic administration. Next generation cytokine approaches include conditional signaling focused to sites of interest, including the tumor microenvironment or specific immune cell populations. In this presentation, we share a novel approach for generating detuned cytokine therapeutic candidates using the AlphaSeq platform, which involves the repurposing of yeast agglutination and mating to quantitatively measure protein-protein interactions at a library-on-library scale. We show, for the first time, how AlphaSeq can be used to measure interactions between cytokines and their receptors as well as generate engineered cytokines with a broad range of affinities. A saturated mutational library was created for a cytokine of interest and subsequently screened against a second library consisting of axis receptor chains, their species orthologs and off-target receptors. Cytokine variants with lower affinity than parental were recombinantly expressed as Fc fusion proteins to orthogonally measure affinity with biolayer interferometry and characterize potency with an in vitro human PBMC phosflow assay. Finally, detuned cytokine candidates were fused to antibodies recognizing surface antigens of interest to demonstrate cell population-specific signaling. Our results show the AlphaSeq platform can accurately quantitate thousands of cytokine variant affinities simultaneously against multiple relevant receptors which translates to signaling potency in primary human cells. When engineered as antibody fusion proteins, candidate immunocytokines are identified that only induce signaling in intended cell populations. AlphaSeq’s rapid, comprehensive affinity determination can be utilized to develop a portfolio of clinically relevant therapeutic immunocytokines. Citation Format: Ryan Swanson, Davis Goodnight, Jeff Adamo, Kyle Minch, Emily Engelhart, Randolph Lopez, David Younger. Cytokine affinity tuning using the AlphaSeq platform to generate targeted immuno-oncology therapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB333.