(575b) Granzyme B Nanoreporter for Early Monitoring of Tumor Response to PD-L1 Checkpoint Blockade

Problem: Immune checkpoint blockade (ICB) therapies such as anti PD-L1, anti PD-1 and anti CTLA-4 have emerged as major breakthrough in treating cancer. Despite recent advancements in these therapies, accurate monitoring of treatment efficacy is challenging due to heterogenous immune response. In order to monitor the therapy efficacy, traditional imaging techniques such as MRI, CT and PET/CT scanning are routinely used, however, they lack the sensitivity and specificity to correctly validate early response of the tumor. This is due to the dependence on anatomic or metabolic readouts of tumor which can be misled by pseudo-progression phenomenon instead of the actual outcome. In more detail, the productive immune cells infiltration together with tumor growth post immunotherapy can be revealed as disease progression based on the Response Evaluation Criteria in Solid Tumors (RECIST) criteria. Therefore, existing clinical prognosis tools are still short of the ability to differentiate responders from non-responders early on, and ineffective treatment in the long term can have a negative impact on patients’ quality of life. Hence, it is important to develop an optimal method to directly report the outcome of therapeutic activity in the tumor in real time.

Statement of Purpose: In this study, we engineered Granzyme B nanoreporter (GNR) with dual function of delivering anti PD-L1 antibody to the tumor site and tracking time-sensitive Granzyme B (GrB) activity as a direct way to capture T cell response. Anti PD-L1 antibody conjugated GNRs efficiently blocked the PD-1/PD-L1 interactions and consequently promoted GrB release from cytotoxic T cells that could be imaged using an activatable imaging probe that was co-delivered by GNRs. GNRs enabled monitoring of the anti-PD-L1 therapy in preclinical models and differentiated highly responsive (highly immunogenic) from poorly responsive (poorly immunogenic) tumors. Taken together, these findings indicate that GNR has the potential to serve as a tool for sensitive and noninvasive evaluation of immunotherapy efficacy. Early assessment of GrB activity assists to identify responding vs. non-responding patients, avoid adverse effects of ineffective treatment, and allow patients to switch to a more effective regimen early on.

Methods: The GrB nanoreporter (GNR) comprises a polymeric backbone conjugated with anti PD-L1 ICBs and activatable GrB reporter probes. We synthesized the reporter probe using a short peptide containing a GrB-cleavable (IEPD) sequence that holds a FRET pair that provided fluorescence signal followed by cytotoxic T-lymphocyte activity in tumors. After synthesizing IEPD substrate, we conjugated to the sequence an NIR-FRET pair including Dylight 755 dye (donor) and Dylight 766Q quencher (acceptor) via thiol-maleimide and carbodiimide formation, respectively, at either side of the substrate. To synthesize GNRs, we first conjugated Carboxy-(PEG)8-amine and reporter element with polymer at optimized molar ratios, followed by ultrasonication of the construct in water for 10 minutes to form a nanoparticle. The anti-PD-L1 antibody was then tagged to PEG-carboxyl on the nanoparticle surface using EDC-NHS chemistry to obtain complete GNRs. Next, we performed physicochemical characterizations of the GNRs (e.g., size, morphology, stability, toxicity, etc.). We next tested if GNRs could accumulate to solid tumors for therapeutic and imaging agent delivery purposes. Finally, we investigated if GNRs enabled the monitoring of immunotherapy response in real-time in preclinical studies using B16/F10 melanoma and MC38 colon adenocarcinoma models. We further explored the ability to early differentiate responding tumor from non-responding tumor using these two cancer models.

Results: We have shown that we were able to synthesize GNRs by conjugating immunotherapy drug and enzyme-responsive imaging agent to the polymer backbone at an optimized molar ratio. High-resolution Transmission Electron Microscopy (HR-TEM) showed spherical nanostructures of average particle size ~200 nm and Dynamic Light Scattering (DLS) showed the structural stability of GNRs over time by measuring hydrodynamic size as well as zeta potential. To evaluate GNRs efficacy in reporting treatment response in a preclinical study, we intravenously injected αPDL1-tagged GNRs and control IgG-tagged GNRs into mice bearing B16/F10 and MC38 tumors. We next imaged the animals at different time points between the treatments using In Vivo Imaging System (IVIS). We observed that αPD-L1 GNRs exhibited a significantly higher fluorescence signal in the MC38 tumor as compared to control GNRs and the signal was detected as early as 24 h after the first treatment. More importantly, B16/F10 tumors exhibited minimal to no signal throughout the whole study (Fig. 1), correlating well to its poor immunogenicity when compared to MC38. Interestingly, we did not observe any significant difference in the tumor volume between the treatment groups even at the end of the trial.

Conclusions: We demonstrated that coping PD-L1 immune checkpoint inhibitor to nanocarrier induced a potent immune response that could be directly reported by the GrB imaging probe. Delivering an immunotherapeutic agent and imaging probe by a single nanoparticle allowed exceptional assistance in the early determination of responding and non-responding tumors by synchronizing the spatial and temporal distribution of both therapeutic and reporting elements. Therefore, this self-reporting platform technology is a potential translational application for early-on tracking of tumor response to different immunotherapeutic regimens and more accurate assessment of disease progression than measurements that depend on changes in anatomical configurations of tumor volume. Such a tool will help clinicians choose an appropriate treatment for patients, thus avoiding over-treatment and unnecessary toxicity; this can significantly impact patients’ quality of life.

References: Nguyen A.et. al. “Granzyme-B nanoreporter for early monitoring of tumor response to immunotherapy.” Science Advances, 2020; 4: eabc2777.