Abstract 2761: FT573: Preclinical development of multiplexed-engineered iPSC-derived NK cells expressing a novel camelid nanobody chimeric antigen receptor (CAR) targeting pan-cancer antigen B7-H3
Authors: Zorko Nicholas , Cichocki Frank , Goulding John , Hancock Bryan , Blum Robert , Pribadi Mochtar , Gaertner Bjoern , Lee Tom , Felices Martin , Bjordahl Ryan , Valamehr Bahram , Miller Jeffrey S. 
Published: June 2022

Abstract
Introduction: B7-H3 (CD276) has gained significant clinical interest as a pan-tumor target antigen for development of various immuno-oncology agents. Due to its broad expression on a wide variety of solid tumors and minimal expression on normal tissues, B7-H3 is an ideal tumor antigen target. Additionally, high levels of B7-H3 are found on “immunologically cold” tumors such as glioblastoma multiforme, prostate cancer, head and neck cancer and soft tissue sarcomas, which typically have poor response to approved immune therapies. To effectively target B7-H3 with an off-the-shelf cellular therapy, we describe here the development of camB7-H3 CAR-NK cell utilizing our iPSC platform to engineer multiple modalities into a clonal iPSC line, which can serve as the starting cell source for mass production of off-the-shelf, iPSC-derived CAR-NK cells (CAR-iNK cells).
Methods: A camelid nanobody specific for human B7-H3 (camB7-H3) was discovered using a phage display library and validated in functional assays. camB7-H3 CAR-iNK cells were designed to 1) express membrane-bound IL-15/IL-15 receptor fusion for enhanced persistence, 2) have a CD38 knockout to improve metabolic fitness, 3) express a high-affinity non-cleavable CD16 to maximize ADCC when combined with a therapeutic antibody, and 4) express an anti-camB7-H3 CAR optimized for NK cell signaling. As the initial preclinical study, camB7-H3 CAR-iNK cells were assessed using flow cytometry-based functional assays evaluating CD107a and IFNγ or xCelligence target killing assays against B7-H3 transgenic or naturally expressing tumor cell lines.
Results: The camelid single domain B7-H3 initially tested in CAR-T exhibited B7-H3 specific binding and specific activity against several solid tumor cell lines (breast, ovarian, prostate, lung). We next produced camB7-H3 CAR-iNK cells and demonstrated superior B7-H3-specific target elimination compared to untransduced iNK cells. These results were seen across multiple tumor lines representing various solid tumor indications. Further enhancement of anti-tumor efficacy was seen when combined with therapeutic antibodies, including trastuzumab and cetuximab.
Conclusions: We have successfully produced and validated the specificity and function of an engineered camB7-H3 CAR-iNK cell exhibiting robust target killing and on-target specificity. To our knowledge, this is the first camelid nanobody antigen recognition domain reported in a CAR-NK cell to be used as an off-the-shelf immunotherapy. The combining camB7-H3 CAR-NK with monoclonal antibodies targeting HER2 and EGFR as a dual targeting approach will add additional tumor specificity, further increase the efficacy of tumor cell elimination and prevent antigen escape. Additional camB7-H3 CAR-iNK cell preclinical studies are in process and will be discussed.
Citation Format: Nicholas Zorko, Frank Cichocki, John Goulding, Bryan Hancock, Robert Blum, Mochtar Pribadi, Bjoern Gaertner, Tom Lee, Martin Felices, Ryan Bjordahl, Bahram Valamehr, Jeffrey S. Miller. FT573: Preclinical development of multiplexed-engineered iPSC-derived NK cells expressing a novel camelid nanobody chimeric antigen receptor (CAR) targeting pan-cancer antigen B7-H3 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2761.