Fluorescence lifetime imaging for quantitative assessment of in vivo PD-L1 target engagement. - Abstract Immune checkpoint inhibitors (ICIs) have significantly improved outcomes in patients with head and neck squamous cell carcinoma (HNSCC), yet only a subset achieve durable clinical benefit. All FDA-approved PD-L1 therapeutics are monoclonal antibodies (mAbs), which, due to their large size, exhibit limited tumor penetration. Tumor concentrations of mAbs are heavily influenced by dynamic target expression, intrinsic (e.g., interstitial pressure) and extrinsic factors tied to clearance and nonspecific tissue uptake. Current companion diagnostics, such as programmed death ligand 1 (PD-L1) immunohistochemistry (IHC) does not determine whether a therapeutic mAb reaches and binds to its target (target engagement) within the tumor microenvironment (TME)— a prerequisite for biological activity. This limits our ability to distinguish whether a lack of ICI response reflects insufficient drug delivery, lack of target binding, or ineffective signaling despite target engagement. Therefore, there is an urgent need for a clinically actionable biomarker that quantifies receptor-bound drug in vivo and reflects downstream immune activation. This R21 application proposes a novel, translational imaging approach using fluorescence lifetime (FL) imaging of a near-infrared (NIR) fluorophore labeled PD-L1 therapeutic antibody (Atezolizumab-IRDye800CW, “Atezo-800”) to measure PD-L1 target engagement in vivo. Our preliminary data demonstrate that the FL of antibody-conjugated NIR dyes increases upon target binding in the TME, allowing discrimination of bound versus unbound mAb via in vivo FL imaging (FLI). Building on the distinct FLs of target- bound and unbound mAbs, we hypothesize that FLI can provide a quantitative in vivo measure of PD-L1 target engagement that correlates with downstream immune activation and predicts therapeutic response. Unlike the traditional dual-probe or peptide-based methods, which suffer from substantial translational barriers, our approach employs a single, FDA approved mAb conjugated to a NIR dye. Aim 1 will establish FLI-based metrics to quantify PD-L1 binding in vivo. Aim 2 will evaluate these target engagement metrics as predictive biomarkers of immune activation and ICI therapy response in preclinical HNSCC models. A key innovation of this proposal is the use of FLI of a single, FDA-approved therapeutic mAb to quantify target engagement, eliminating the need for a second reference agent. Importantly, the imaging probe, Atezo-800, is already advancing through the investigational new drug (IND) approval process in our collaborator’s laboratory, offering a clear route to human application. FLI of Atezo-800 may serve as a robust, noninvasive biomarker of PD-L1 binding, with immediate applications in patient stratification, dose optimization, and drug development. Dr. Pal leads this project in collaboration with Dr. Rosenthal, an expert in FDA regulatory pathways for antibody-dye conjugates, and Dr. Sade-Feldman, a leading expert in immune profiling of HNSCC. This R21 will generate key imaging and mechanistic data supporting two future R01s: one focused on translating FLI into early-phase clinical trials, and another investigating the mechanisms of ICI therapy resistance.
