Fluorescent Imaging of Atezolizumab to Identify Intratumoral Drug Delivery

Introduction: Immunotherapy has shown promising results in head and neck squamous cell carcinoma (HNSCC), however only 30% of all patients respond with even fewer having complete remission of tumor. In patients with partial response, some tumor nests are observed which show no response to the therapeutic agent. The goal of this study is to identify whether no penetration of Atezolizumab (PD-L1 Inhibitor) within tumor nests co-relates with a diminished immune response.

Methods: A flank tumor xenograft model with the MOC-1 and MOC-2 cell lines was used in mice to evaluate the delivery of Atezolizumab labelled with IRDye 800CW. Tumors were allowed to grow till 100mm3 before being randomized into a control (injected with water) and treatment arm. 200ug of Atezolizumab was given intraperitoneally. Tumor volume was measured over 30 days. Macro distribution of antibody was analyzed via ex-vivo imaging on a Li-Cor imaging device and distribution of antibody within tumor was analyzed using an Olympus slide scanner. Immunohistochemistry (IHC) was done for PD-L1,CD-8 and Cytokeratin. PD-L1 expression was also evaluated in cell lines using flow cytometry.

Results: PD-L1 was expressed roughly on 45% of cells by flow cytometry in both cell lines. Treated MOC-1 tumors had a slower growth curve (p < 0.05) when compared to the control group however no difference was observed when comparing MOC-2 tumor growth with its control. In-vivo near infrared imaging revealed localization of antibody to tumor within 24 hours in both cell lines with no difference observed in fluorescence signal intensity. Fluorescence slide imaging revealed lower drug delivery within MOC-2 tumors. Ongoing preliminary analysis reveals decreased penetration of drug in fibrotic tumor nests. Intra-tumoral distribution of drug was not significantly associated with CD-8 IHC expression.

Conclusions: This study demonstrates the utility of using labelled Atezolizumab in tracking intra-tumoral distribution of drug and suggests that the mechanism of why some tumor nests in partial responders do not respond to therapy are partly influenced by penetration of drug in these tumor nests.