Abstract 2861: Dose-dependent tissue distribution and tumor targeting of Notch3-ADC using fluorescence molecular tomography imaging
Authors: Giddabasappa Anand , Gupta Parul , Leal Mauricio , Golas Jonathan , Li Fengping , Yang Bing , Esparza Antonio , Winkelmann Christopher , Geles Kenneth 
Published: July 2017

Background: NOTCH3, a cell surface receptor involved in cell-cell communications, is over-expressed or amplified in certain human tumors. NOTCH3 is known to regulate proliferation, differentiation and survival of cancer cells or cancer stem cells and thus an important therapeutic target. NOTCH3 antibody drug conjugate (ADC) is comprised of humanized anti-NOTCH3 antibody conjugated to an auristatin based cytotoxic payload. NOTCH3-ADC has shown promising results in pre-clinical tumor models. In this study we evaluated the kinetics, dose-dependent tissue distribution, tumor accumulation and targeting specificity of the NOTCH3-ADC in OVCAR3 xenograft model using fluorescence molecular tomography (FMT) imaging. The NOTCH3-ADC is mouse cross-reactive thus providing an accurate assessment of biodistribution.

Methods: NOTCH3-ADC was conjugated to the near-infrared dye, AlexaFluor680 (AF680). The in vitro cellular binding was evaluated by cell-based ELISA. In vivo biodistribution was evaluated using OVCAR3 subcutaneous xenograft model. NOTCH3-ADC-AF680 (1mg/kg; 3mg/kg and 10mg/kg) was injected when the tumors were ~300mm3 and imaged 5 min, 24, 48, 96, 168 and 240 h post-injection. Ex vivo FMT imaging, pharmacokinetic analysis and immunohistochemistry (IHC) was performed at 48 and 240 h after whole-body perfusion. An in vivo receptor competition FMT study was performed by injecting excess of unconjugated anti-NOTCH3 antibody (Ab) or a non-targeted control Ab.

Results: In vitro cell binding studies showed that conjugation of AF680 to NOTCH3-ADC did not change its binding ability. A dose-dependent tumor regression was also observed after a single injection of NOTCH3-ADC-AF680. In vivo FMT imaging showed dose-dependent whole-body clearance kinetics of NOTCH3-ADC. Dose-dependent accumulation in the tumors was observed with peak accumulation at 24-48 h post-injection and a slow decline at later time-points. A maximum accumulation of ~10 %ID/g was observed which was independent of the dose of NOTCH3-ADC-AF680. Ex vivo FMT quantitation of tumor was consistent with the IHC for antibody and LC/MS analysis of released payload. Pharmacological competition with excess unlabeled control Ab did not block tumor accumulation of NOTCH3-ADC-AF680, whereas excess unlabeled NOTCH3-Ab blocked ~47% of NOTCH3-ADC-AF680 accumulation. There was no significant specific accumulation of NOTCH3-ADC in other organs as observed by FMT imaging or IHC.

Conclusions: These imaging studies provided understanding of the kinetics, tumor accumulation and biodistribution of NOTCH3-ADC. Further this work showcase the utility of non-invasive FMT imaging in better understanding of pharmacology and behavior of biologic drugs.

Citation Format: Anand Giddabasappa, Parul Gupta, Mauricio Leal, Jonathan Golas, Fengping Li, Bing Yang, Antonio Esparza, Christopher Winkelmann, Kenneth Geles. Dose-dependent tissue distribution and tumor targeting of Notch3-ADC using fluorescence molecular tomography imaging [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2861. doi:10.1158/1538-7445.AM2017-2861