Abstract 5111: Bio-distribution and tumor targeting of a P-cadherin x CD3 bi-specific redirected T-cell molecule using fluorescence molecular tomography imaging
Authors: Giddabasappa Anand , Gupta Vijay , Fisher Timothy S. , David John , Rand Norberg , Rohner Allison , Cohen Justin , Clark Tracey , Haddish-Berhane Nahor , Root Adam , May Chad 
Published: August 2015

Introduction: Previously we have shown the utility of Fluorescent Molecular Tomography (FMT) imaging in evaluating bio-distribution of biologics. P-cadherin LP-DART is a bi-specific Dual Affinity Re-Targeting (DART®) molecule targeting CD3 expressed on T-cells and P-cadherin expressed on tumors. In this study we evaluated the bio-distribution and tumor targeting of P-cadherin LP-DART using FMT imaging in a colorectal xenograft model.

Methods: NSG or athymic nude mice with subcutaneous HCT-116 xenografts were used. Studies that included engraftment of T-cells received either PBMNCs or T-cells isolated from healthy human volunteers. Bio-distribution studies were initiated when the tumors reached 300-500 mm3. P-cadherin LP-DART or a negative control-DART (non-targeted domain x CD3 binding domain) was conjugated with a near-infrared fluorophore VivoTag680XL (VT680), and the labeling efficiency was determined by spectrophotometer. T-cells used in trafficking studies were labeled with CellVue815. Cell surface P-cadherin expression and P-cadherin LP-DART binding was determined by flow cytometry. T-cell activity was measured with cytotoxic T-lymphocyte (CTL) assays. FMT imaging was performed longitudinally post injection of labeled bi-specifics. Data was analyzed using TrueQuant software. Plasma and tissues were collected for PK analysis by ELISA or histology.

Results: VT680 conjugation to P-cadherin LP-DART did not significantly affect the binding to P-cadherin, whereas CD3 binding was decreased. In vivo FMT imaging revealed high levels of P-cadherin LP-DART accumulation in the tumors. The in vivo kinetics revealed that the peak accumulation in tumors was 96hrs post-injection. At 240hrs post-injection, there was still measurable P-cadherin LP-DART detected in tumors. Ex vivo imaging showed 20-25 fold increase in accumulation of P-cadherin LP-DART compared to negative control DART. Comparison of P-cadherin LP-DART accumulation between PBMNC engrafted and non-engrafted model showed no significant difference in quantity or kinetics. There was no significant difference in the kinetics of elimination in the whole-body, heart or liver between P-cadherin LP-DART or negative control. Ex vivo comparison of accumulation in various organs showed no difference between P-cadherin LP-DART or negative control. Cell trafficking studies with CellVue labeled T-cells showed the co-localization of T-cells and P-cadherin LP-DART in tumors.

Conclusion: FMT imaging showed that P-cadherin LP-DART specifically targeted HCT-116 tumors. Cell trafficking studies showed that engrafted T-cells accumulated in tumors. This study shows the utility of FMT in bio-distribution studies of biologics and in vivo cell trafficking.

Citation Format: Anand Giddabasappa, Vijay Gupta, Timothy S. Fisher, John David, Norberg Rand, Allison Rohner, Justin Cohen, Tracey Clark, Nahor Haddish-Berhane, Adam Root, Chad May. Bio-distribution and tumor targeting of a P-cadherin x CD3 bi-specific redirected T-cell molecule using fluorescence molecular tomography imaging. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5111. doi:10.1158/1538-7445.AM2015-5111