Abstract 601: Chimeric antigen receptors incorporating novel binding domains targeting CD123 direct potent antitumor activity of T cells: Correlation between affinity and activity
Authors: LaFleur David W. , Qin Haiying , Edwards Justin P. , Zaritskaya Liubov , Gupta Ankit , Mu C. Jenny , Richman Laura K. , Fry Terry J. , Hilbert David M. 
Published: September 2020

Abstract
Unlike the tumor targets associated with initial CAR T cell successes, the vast majority of tumor associated antigens are not unique, but rather expressed at elevated levels compared to normal tissue. For example, while CD123 is overexpressed on many AML tumor cells, concerns regarding on-target, off-tumor toxicities - often associated low-level CD123 expression on normal endothelial cells - has hindered clinical success. Therefore, future generations of CAR T therapies will require novel strategies that have the ability to discriminate between normal and elevated antigen levels - potentially through mechanisms that involve 1) the simultaneous or sequential targeting of multiple tumor associated antigens and 2) agents that contain affinity-tuned targeting domains. Due to the large inventory of validated antibodies against a variety of therapeutic targets, scFv naturally emerged as the obvious and justifiable targeting domain for chimeric antigen receptors. However, many of the characteristics that have made antibodies versatile and effective recombinant therapeutics (e.g., high affinity, bivalency, antibody-dependent cytotoxicity, complement-dependent cytotoxicity, FcRn recycling, and low renal filtration rates) are not advantageous for membrane associated chimeric receptors. Furthermore, scFv are not native protein structures and their development, particularly as it pertains to solubility and aggregation, can be challenging. Therefore, we sought to develop a simple, highly selective targeting domain that could be engineered into complex, potentially multispecific therapeutics. We describe the design and development of non-scFv-derived binding domains. Using phage display and targeted mutagenesis, we identify a series of binding domains that target CD123 with high specificity - as characterized by functional and tissue binding studies. These domains exhibit affinities that range over 2 logs and demonstrate in vitro potencies that correlate with their affinities. CARs comprised of higher affinity binding domains mediate potent T-cell activation and cytolysis of CD123-expressing target cells and induce complete durable remission in two AML xenograft models. We also describe a strategy of engineering less immunogenic binding domains through the identification and removal of putative T cell epitopes and demonstrate that the resultant variants retain biological activity. Finally, we demonstrate further potential of our binding domains by generating functional, bi-specific CARs comprised of a CD123-specific binding domain and a CD19-specific scFv. The ability to incorporate our target-specific binding domains with a range of affinities into complex chimeric receptors, affords a viable alternative to scFv as targeting domains in CAR T cell therapeutics.
Citation Format: David W. LaFleur, Haiying Qin, Justin P. Edwards, Liubov Zaritskaya, Ankit Gupta, C. Jenny Mu, Laura K. Richman, Terry J. Fry, David M. Hilbert. Chimeric antigen receptors incorporating novel binding domains targeting CD123 direct potent antitumor activity of T cells: Correlation between affinity and activity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 601.