Evaluation of a Centyrin-Based Near-Infrared Probe for Fluorescence-Guided Surgery of Epidermal Growth Factor Receptor Positive Tumors

Abstract

Tumor-targeted near-infrared fluorescent dyes have the potential to improve cancer surgery by enabling surgeons to locate and resect more malignant lesions where good visualization tools are required to ensure complete removal of malignant tissue. Although the tumor-targeted fluorescent dyes used in humans to date have been either small organic molecules or high molecular weight antibodies, low molecular weight protein scaffolds have attracted significant attention because they penetrate solid tumors almost as efficiently as small molecules, but can be infinitely mutated to bind almost any antigen. Here we describe the use of a 10 kDa protein scaffold, a Centyrin, to target a near-infrared fluorescent dye to tumors that overexpress the epidermal growth factor receptor (EGFR) for fluorescence-guided surgery (FGS). We have developed and optimized the dose and time required for imaging small tumor burdens with minimal background fluorescence in real-time fluorescence-guided surgery of EGFR-expressing tumor xenografts in murine models. We demonstrate that the Centyrin-near-infrared dye conjugate (CNDC) binds selectively to human EGFR cancer cells with an EC of 2 nM, localizes to EGFR tumor xenografts in athymic nude mice and that uptake of the dye in xenografts is significantly reduced when EGFR are blocked by preinjection of excess unlabeled Centyrin. Taken together, these data suggest that CNDCs can be used for intraoperative identification and surgical removal of EGFR-expressing lesions and that Centyrins targeted to other tumor-specific antigens should prove similarly useful in fluorescence guided surgery of cancer. In addition, we demonstrate that the CNDC is detected in the NIR region of the spectrum and can be utilized for fluorescence-guided surgery (FGS). In addition, we propose that with its eventual complete clearance from EGFR-negative tissues and its quantitative retention in the tumor mass for >24 h, a Centyrin-targeted NIR dye should provide excellent tumor contrast when injected at least 6-8 h before initiation of cancer surgery in human patients.

Specific Targeting of Somatostatin Receptor Subtype-2 for Fluorescence-Guided Surgery

“…This has indeed been shown in a dose-escalation study in patients with IR800conjugated cetuximab where higher TBRs were obtained with the highest injection dose (30), as well as with a fluorescent diabody targeting an antiprostate stem cell antigen (31). However, numerous studies have reported better TBRs with lower doses because increasing the injection amount also leads to more nontarget uptake that reduces contrast, increases the number of falsepositives, and negatively affects specificity (32)(33)(34)(35). In order to examine this effect with Ga-MMC(IR800)-TOC, xenografts were injected with 0.5 or 2 nmol and showed a direct correlation between injected dose and absolute fluorescence in tumor and normal tissues.…”

Development and Differentiation in Monobodies Based on the Fibronectin Type 3 Domain

“…This was further confirmed after loading the Adnectin with 64 Cu where the monobody was able to provide same-day PET visualisation of tumour cells, resulting in an increase in the monobody uptake in tumours over 24 h post-injection [62,69]. This concept has been taken even further with a Centyrin domain evolved to bind EGFR and conjugated with a fluorescent dye, which was then applied to guide surgical removal of tumour cells [70].…”

Development and Differentiation in Monobodies Based on the Fibronectin Type 3 Domain

“…This was further confirmed after loading the Adnectin with 64 Cu where the monobody was able to provide same-day PET visualisation of tumour cells, resulting in an increase in the monobody uptake in tumours over 24 hours postinjection [61,68]. This concept has been taken even further with a Centyrin domain evolved to bind EGFR and conjugated with a fluorescent dye, which was then applied to guide surgical removal of tumour cells [69].…”