Abstract 5487: IndEx-2: An <i>in vitro</i> model to evaluate the impact of target antigen density on antibody and immune therapeutics

Abstract The density of cell-surface receptors targeted by therapeutic agents profoundlyinfluences their safety, efficacy, and pharmacokinetics. Low receptor expression on healthy cells can result in on-target, off-tumor toxicities, while tumor heterogeneity in receptor density may allow low-expressing clones to evade treatment. Traditional invitro evaluation relies on panels of cancer cell lines with varying receptor levels, anapproach that is both logistically challenging and experimentally inconsistent. This limitation is particularly pronounced for therapeutics targeting two receptors.To address this, we developed IndEx-2, an in vitro cell line platform that employs chemical proximity promoters to achieve a high dynamic range of tuneable and independent expression of up to two target antigens within a single, uniform cell line background. We have demonstrated precise control of antigen density for several receptors ranging from a few hundred up to a million receptors, including a cell line thatallows independent control of two target antigens (CD19 and CD22). We report herethree examples of the utility of the system. In the first, we have compared CLN978, a novel CD19- TCE developed for B-cell malignancies and autoimmune diseases, with Blinatumomab and demonstrated that the former retained its activity in terms of cytotoxicity and cytokine production at much lower levels of CD19 expression and estimate the threshold for each molecule in a cohort of 10 healthy donors. In the second, we assessed the ability of two HER2-targeting CAR-T cells, employing high and low antigen binders, and estimate the number of receptors that resulted in half maximal response (R50) for cytotoxicity and IFNγ production, demonstrating the utility of the system as an “on-target, off-tumor” hazard assessment screening tool. Lastly, using our CD19/CD22 dual-antigen cell line, we compared tandem (“OR”) CD19/CD22-CD3zCD28 and dual (“AND”) CD19-CD3z and CD22-CD28 CAR constructs targetingboth antigens. The tandem CAR exhibited enhanced cytotoxicity when either or both antigens were expressed, whereas the dual CAR required co-expression of both antigens for optimal T-cell activation. These results underscore IndEx-2's robustcapability to evaluate the influence of antigen density on therapeutic safety andefficacy, providing a powerful tool for the development of antibody-based and immune-targeting cancer therapies. Citation Format: Gavin Meehan, Agapitos Patakas, Hannah Findlay, Vivek Senthivel, Timothy London. IndEx-2: An in vitro model to evaluate the impact of target antigen density on antibody and immune therapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5487.