Abstract 1871: A differentiated CD73 blocking antibody with a unique intra-dimer binding mechanism for cancer immunotherapy

Abstract

Abstract Introduction: CD73 is an emerging target for cancer immunotherapy owing to its critical role for the generation of an adenosine-enriched tumor microenvironment. Unlike other CD73 antibodies which adopt bivalent binding to two separate CD73 dimers at the N-terminal domain (termed inter-dimer binding), typically resulting in a “hook effect”, we describe a differentiated CD73 antibody, TJD5, that achieves full inhibition through monovalent binding to the C-terminal domain of a single CD73 dimer (termed intra-dimer binding), leading to a more favorable pharmacologic profile. Methods: TJD5 was discovered through a series of screening assays, including CD73 binding and enzyme activity inhibition. The critical residues for antibody binding were identified by a shotgun mutagenesis library and structure modeling. We are further analyzing the structure of TJD5 Fab and CD73 dimer complex by cryogenic electron microscopy (cryoEM). The inhibitory potency of TJD5 was compared with other antibodies in both in vitro enzymatic assay and in situ histochemical assay. We also profiled the immuno-modulatory properties of TJD5 via in vitro T cell and B cell activation assays as well as anti-tumor activities in human CD73 transgenic mouse and tumor xenograft model. Results: Comparing with other CD73 antibodies with epitopes in the N-terminal region which reportedly adopt an inter-dimer binding mode to separate CD73 dimers, TJD5 binds to the outside edge of the CD73 dimer interaction surface at the C-terminal domain. While we are analyzing the TJD5:CD73 complex by cryoEM, modeling data indicate an intra-dimer binding of TJD5 to a single CD73 dimer which confers the antibody with two unique features. First, TJD5 showed a stronger potency with the ability to completely inhibit CD73 enzyme as opposed to other antibodies exhibited a “hook effect” as manifested by a U-shaped inhibition curve. Second, TJD5 Fab could also inhibit CD73 activity, indicating a monovalent interaction is sufficient for the inhibition whereas others require a bivalent interaction. Consistently, a complete inhibition of intra-tumor CD73 activity by TJD5 could be demonstrated in vivo. Immunologically, in addition to the reversal of adenosine mediated T cell suppression, TJD5 directly increased B cell activation and antigen presentation as evidenced by upregulation of CD69, CD83, CD86 and HLA-DR that was unaffected by the addition of adenosine. Combination of TJD5 with a PD-(L)1 antibody showed synergistic anti-tumor activities both in vitro and in vivo. Conclusion: TJD5 is a humanized anti-CD73 antibody with a unique C-terminal domain epitope that enables a novel intra-dimer binding mode, which strongly differentiates it from other CD73 antibodies both in terms of mechanism of action and inhibitory potency. TJD5 is now in phase I clinical development both in the US and China to validate these differentiated properties and anti-tumor activity. Citation Format: Zhengyi Wang, Wei Cao, Yanni Zhang, Cong Xu, Yuan Meng, Taylor B. Guo. A differentiated CD73 blocking antibody with a unique intra-dimer binding mechanism for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1871.