Abstract 3119: Next-generation multi-specific and conditionally activated CD3 Switch-DARPins with CD2 co-stimulation to tackle the current limitations of T cell engagers in solid tumors

Abstract Introduction: T-cell engagers (TCE) are powerful and clinically validated cancer therapeutics but are limited by high toxicity, lack of tumor-specific targets, and presence of exhausted/dysfunctional T cells particularly in solid tumors. Our CD3 Switch-DARPin platform provides a logic-gated “on/off” function (“Switch”) to increase tumor specificity and enable strong CD3 engagement and activation without compromising safety. Additionally, a DARPin with a co-stimulatory function can be added to enhance T-cell responses, especially in the context of an adverse tumor microenvironment. Using ovarian cancer (OC) as a proof-of-concept indication, we designed a multi-specific CD3 Switch-DARPin with CD2 co-engagement (CD2 x CD3 Switch-DARPin) which targets two tumor-associated antigens (TAAs); mesothelin and EpCAM. The logic-gated function of our Switch-DARPin ensures that the CD3 moiety is able to bind and activate T cells only when the TAAs are expressed on target cells. Herein we present a Switch-DARPin TCE that safely sustains anti-tumor efficacy, providing new TAA options and expanding TCE use in solid tumors. Methods: RNAseq phenotypic analyses of OC patient samples were validated on resected tumors and ascites using flow cytometry and immunohistochemistry. The specific activity and safety of our CD2 x CD3 Switch-DARPin were assessed in vitro and ex vivo using reporter cells, primary human T cells from healthy donors and OC patient ascites, whole blood cytokine release assays, and in vivo in PBMC-humanized NXG mice engrafted subcutaneously with mesothelin- and EpCAM-expressing cells. Results: Our analysis confirmed that mesothelin and EpCAM are highly co-expressed in OC tumors. Furthermore, reduced expression of CD28 compared to CD2 on tumor-infiltrating lymphocytes, combined with CD58 downregulation on primary tumors and ascites, support the use of CD2 as a co-stimulatory receptor for T cell engagement in OC. Functionally, the CD2 x CD3 Switch-DARPin abrogated TAA-independent T-cell activation and reduced potential on-target off-tumor toxicity, while CD2 co-engagement sustained T-cell response and prevented T-cell dysfunction. Notably, in vivo, the CD2 x CD3 Switch-DARPin induced significant tumor regression without causing systemic release of cytokines in the blood. In contrast, a control molecule without the Switch led to systemic cytokine release and showed no antitumor efficacy, likely due to fratricide-mediated depletion of T cells. Conclusions: Our CD3 Switch-DARPin platform provides a novel approach for sustained tumor-specific T-cell engagement, by combining a logic-gated on/off Switch mechanism with CD2 co-stimulation. This allows immune activation to be directed to tumors, increasing efficacy and safety and paving the way for new opportunities for cancer treatment. Citation Format: Matteo Bianchi, Marco Franchini, Tamara Lekishvili, Eleni Tselempi, Joanna Robinson, Yvonne Kaufmann, Guillaume Ems, Chloé Friang, Clara Tissier, Mischa R. Müller, Alexander Link, Christophe Borg, Amelie Croset, Valérie Calabro, Anne Goubier, Marcela Guzman Ayala. Next-generation multi-specific and conditionally activated CD3 Switch-DARPins with CD2 co-stimulation to tackle the current limitations of T cell engagers in solid tumors [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 3119.