Dimerization of the 4Ig isoform of B7-H3 in tumor cells mediates enhanced proliferation and tumorigenic signaling.

B7-H3 (CD276) has two isoforms (2Ig and 4Ig), no confirmed cognate receptor, and physiological functions that remain elusive. While differentially expressed on many solid tumors correlating with poor survival, mechanisms of how B7-H3 signals in cis (tumor cell) versus in trans (immune cell co-regulator) to elicit pro-tumorigenic phenotypes remain poorly defined. Herein, we characterized a tumorigenic and signaling role for tumor cell-expressed 4Ig-B7-H3, the dominant human isoform, in gynecological cancers that could be abrogated upon CRISPR/Cas9 knockout of B7-H3; tumorigenesis was rescued upon re-expression of 4Ig-B7-H3. Size exclusion chromatography revealed dimerization states for the extracellular domains of both human 4Ig- and murine 2Ig-B7-H3. mEGFP lifetimes of expressed 4Ig-B7-H3-mEGFP fusions determined by FRET-FLIM assays confirmed close-proximity interactions of 4Ig-B7-H3 and identified two distinct homo-FRET lifetime populations, consistent with monomeric and homo-dimer interactions. In live cells, bioluminescence imaging of 4Ig-B7-H3-mediated split luciferase complementation showed dimerization of 4Ig-B7-H3. To separate basal from dimer state activities in the absence of a known receptor, C-terminus (cytosolic) chemically-induced dimerization of 4Ig-B7-H3 increased tumor cell proliferation and cell activation signaling pathways (AKT, Jak/STAT, HIF1α, NF-κß) significantly above basal expression of 4Ig-B7-H3 alone. These results revealed a new, dimerization-dependent intrinsic tumorigenic signaling role for 4Ig-B7-H3, likely acting in cis, and provide a therapeutically-actionable target for intervention of B7-H3-dependent tumorigenesis.

Evaluation of 89Zr-Labeled Anti-PD-L1 Monoclonal Antibodies Using DFO and Novel HOPO Analogues as Chelating Agents for Immuno-PET.

Programmed death ligand 1 (PD-L1) is a type 1 transmembrane immunosuppressive protein that is expressed on a wide range of cell types, including cancer cells. Anti-PD-L1 antibodies have revolutionized cancer therapy and have led to improved outcomes for subsets of cancer patients, including triple-negative breast cancer (TNBC) patients. As a result, PET imaging of PD-L1 protein expression in cancer patients has been explored for noninvasive detection of PD-L1 expressing tumors as well as monitoring response to anti-PD-L1 immune checkpoint therapy. Previous studies have indicated that the in vivo stability and in vivo target detection of antibody-based radio-conjugates can be dramatically affected by the chelator used. These reports demonstrated that the chelator HOPO diminishes 89Zr de-chelation compared to DFO. Herein, we report an improved HOPO synthesis and evaluated a series of novel analogues for thermal stability, serum stability, PD-L1-specific binding using the BT-549 TNBC cell line, PET imaging in vivo, as well as biodistribution of 89Zr-labeled anti-PD-L1 antibodies in BT-549 xenograft murine models. A new chelator, C5HOPO, demonstrated high stability in vitro and afforded effective PD-L1 targeting in vivovia immuno-PET. These results demonstrated that an improved HOPO chelator is an effective chelating agent that can be utilized to image therapeutically relevant targets in vivo.