Abstract 5231: NK and T cells armed with EVE16, a novel synthetic immune receptor, mediate robust anti-tumor responses through dual direct cytotoxicity and ADCC with minimal trogocytosis and exhaustion

Abstract Despite the major advances, cancer patients treated with targeted chimeric antigen receptor (CAR)- based T and NK cell therapies often face the challenge of frequent disease relapse, and their efficacy in solid tumors is low. This is attributed to the inherent potency of the starting cell material, unique characteristics of CAR engineering resulting in trogocytosis and tonic signaling, and the notable absence of homogenously expressed surface markers, particularly in solid tumors. To address these limitations, we engineered a novel synthetic immune receptor termed Enhanced-Valency Engineered CD16A (EVE16) and evaluated it in memory-like NK cells and T cells against liquid and solid tumors. We hypothesized that post-activation cleavage of CD16A by ADAM17 plays a crucial role in reducing trogocytosis and tonic signaling. EVE16 utilizes the inherent potential of CD16A to trigger an activation signaling cascade through the endogenous CD3ζ adaptor signaling protein. Unexpectedly, we discovered that, aside from the CD16A transmembrane domain, the Fc-binding domain is essential for proper surface expression. After multiple rounds of optimization, we developed a receptor that surpassed the activation potency of endogenous CD16A and allowed both direct binding to the tumor targets and retained antibody-dependent cellular cytotoxicity (ADCC), and this dual 'CAR-like' and ADCC function opens the potential for combination with clinically approved antibodies to prevent immune escape. Furthermore, in contrast to the conventional CAR(BBζ), the optimized EVE16 receptor exhibited minimal tonic signaling in both T and NK cells. This translated to enhanced sensitivity towards low antigen-density targets, as demonstrated by a cytotoxicity assay performed on polyclonal cancer cells engineered with target antigen knock-down. Additionally, EVE16, but not CAR transduced, protected T and NK cells from trogocytosis and allowed for the efficient disengagement of cytotoxic lymphocytes from cancer targets due to CD16A cleavage by ADAM17 upon immune cell activation. In in vivo mouse xenograft models, CD19-targeted EVE16 memory-like NK cells, rapidly manufactured using our approach, demonstrate outstanding performance in terms of robust expansion, prolonged persistence, and effective leukemic clearance, underscoring the effectiveness of our method. In summary, we here describe a novel synthetic immune receptor called EVE16 which enables both direct (CAR-like) and ADCC functionality while preventing trogocytosis and exhaustion. Clinical trials are being planned for the near future. Citation Format: Alaa Ali, James D. Ham, Faten Zairi, Vaishvi Patel, Mubin Tarannum, Khanhlinh Dinh, Yasmin Abdulhamid, Juliana Vergara, Robert Soiffer, Jerome Ritz, John Beadle, Nikola A. Ivica, Jianzhu Chen, Rizwan Romee. NK and T cells armed with EVE16, a novel synthetic immune receptor, mediate robust anti-tumor responses through dual direct cytotoxicity and ADCC with minimal trogocytosis and exhaustion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5231.