In vivo testing of an agonistic anti-GITR mAb that induces cell surface clustering to augment signal transduction and a 2nd Gen bispecific anti-GITR/PD1 Ab derivative for improved anti-tumor activity

Project Summary The durability of clinical responses to immune checkpoint inhibitors targeting PD1 has generated tremendous interest in immunotherapy of cancers. However, not all patients demonstrate a complete or durable response and thus, there is a need to develop additional therapeutic antibodies (mAbs) that target other immune pathways

and can be used in combination with anti-PD1 therapies. Though the majority of the focus to date has been on other inhibitory pathways such as CTLA4 and TIGIT, recent developments have suggested a role for mAbs targeting co-stimulatory receptors, allowing for the introduction of therapies that not only release the proverbial

brakes of the immune system, but also apply the gas to get the immune response moving. One such costimulatory receptor is GITR (TNFRSF18), a TNFR superfamily member surface receptor that is upregulated on activated T cells and endogenously expressed on both Tregs and NK, though basal expression on NK cells

is considerably lower. As with many stimulatory receptors, GITR requires receptor clustering to reach the activation threshold for signal transduction and cellular activation and thus, many studies involving agonistic anti- GITR antibodies failed to show a significant clinical benefit in humans. Though mutant antibody Fc domains have

been developed that enable spontaneous hexamerization upon cell surface binding, the hexamerization also enhances C1q binding and complement deposition, leading to an immunodepleting molecule with potent effector functions. To broaden the applications of this platform, our lab has used a structure guided engineering approach

to identify additional mutations that can abrogate C1q binding, leading to a hexamerizing antibody with negligible effector functions and one that is suitable for targeting and activating stimulatory receptors. In vitro luciferase based bioassays demonstrated that when combined with a hexamerizing Fc domain, our agonistic anti-GITR

antibody demonstrates significantly enhanced stimulation and a unique synergistic effect with GITR-Ligand. The first aim of this proposal looks to examine the in vivo efficacy of this agonistic, hexamerizing anti-GITR antibody with augmented stimulatory capabilities using an established and well characterized orthotopic renal cell

carcinoma model in a humanized mouse. Additionally, advanced analytical techniques including single cell RNA sequencing, immunohistochemistry, and multiparameter FACS will enable the in-depth evaluation of the mechanistic and cellular differences resulting from therapeutic use of hexamerizing antibodies targeting

stimulatory receptors. The second aim further advances the therapeutic potential of this antibody by engineering and testing in vivo, a hexamerizing bispecific antibody that targets both GITR and PD1. This molecule will both rescue exhausted immune cells and stimulate activated T cells, allowing for remodeling of the tumor

microenvironment and restoration of anti-tumor immunity. This proposal not only will evaluate the therapeutic potential of a novel Fc domain and bispecific antibody, but also provide valuable insight into the mechanistic differences resulting from enhanced receptor signaling and combination therapies.