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.
