Abstract 2900: To develop a next generation NK therapy with deep and durable response by engineered CD16 enhancing NK cytotoxicity

Abstract Introduction: Allogeneic NK cell-based therapies have been demonstrated to elicit clinical efficacy against hematopoietic malignancies. Unlike T-cell therapies, allogeneic NK cells do not cause toxicities such as serious cytokine release syndrome, neurotoxicity, or graft-vs-host-disease, and are less expensive. We believe NK cell-based therapies provide an important platform for “off-shelf” allogeneic cell therapies, so we designed a panel of arming strategies to enhance NK cytotoxicity and elicit deep and durable response. CD16a is a transmembrane protein that co-localizes with CD3ζ and FcεRIγ on NK cells and binds to the Fc portion of IgG antibodies. Upon ligation, it induces a potent series of signals resulting in cytokine production and cytotoxic effector activity via ADCC. Here we use the term CD16 to refer to CD16a on NK cells. CD16 is a potent activating receptor and is necessary for NK cell cytotoxic effector, so engineered CD16 is the cornerstone of NK enhancing strategy. CD16 displays a functional allelic dimorphism generating allotypes with either a phenylalanine (F) or a valine (V) residue at amino acid position 158. CD16VV is associated with higher ADCC activity and clinical response than CD16VF and CD16FF. However, only 15% of the population is CD16VV that maximizes ADCC. Several studies reported that tumor microenvironment may cause the shedding of CD16 which suppresses ADCC though ADAM17-mediated proteolytic cleavage upon NK cell activation. Therefore, selective inhibition of ADAM17 can reduce the shedding of CD16 in NK cells. Method: We designed a library of CD16 point mutations in the cleavage region of ADAM17 to screen for ADAM17-resistant CD16 mutations to prevent CD16 shedding and improve the efficacy of NK therapy. NK92 cells lacking expression of endogenous CD16 were transduced to express wild type CD16 or CD16 mutations, then activated with PMA. The surface expression of CD16 on these cells was then examined using flow cytometry. Result: We found 3 mutations effectively blocked CD16 cleavage in cell-based assays, while wild-type CD16 and other CD16 mutations underwent a marked down-regulation of expression. We also demonstrated these 3 mutations were resistant to cleavage when expressed in the human primary NK cells and did not disrupt IgG binding. Conclusion: We developed a non-cleavable CD16 with high affinity to Fc receptor, which can be expressed in engineered NK cells to develop a next generation NK therapy to elicit more potent and durable cytotoxic response. Citation Format: Cuiqing Yang, Fuwei Jiang, Yifang Wang, Tingting Liu, Qingyang Wang, Qin Wang, Gang Ye, Renhong Tang, Zhuoxiao Cao. To develop a next generation NK therapy with deep and durable response by engineered CD16 enhancing NK cytotoxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2900.