Activatable fluorescent cys-diabody conjugated with indocyanine green derivative: consideration of fluorescent catabolite kinetics on molecular imaging

Kohei Sano,Insook Kim,Towhid Ali,Anna M Wu,Peter L Choyke,Chang H Paik,Derek W Bartlett,Hisataka Kobayashi,Takahito Nakajima

doi:10.1117/1.jbo.18.10.101304

Abstract

Antibody fragments including diabodies have more desirable pharmacokinetic characteristics than whole antibodies. An activatable optical imaging probe based on a cys-diabody targeting prostate-specific membrane antigen conjugated with the near-infrared fluorophore, indocyanine green (ICG), was designed such that it can only be activated when bound to the tumor, leading to high signal-to-background ratios. We employed short polyethylene glycol (PEG) linkers between the ICG and the reactive functional group (Sulfo-OSu group), resulting in covalent conjugation of ICG to the cys-diabody, which led to lower dissociation of ICG from cys-diabody early after injection, reducing hepatic uptake. However, unexpectedly, high and long-term fluorescence was observed in the kidneys, liver, and blood pool more than 1 h after injection of the cys-diabody PEG-ICG conjugate. A biodistribution study using I125-labeled cys-diabody-ICG showed immediate uptake in the kidneys followed by a rapid decrease, while gastric activity increased due to released radioiodine during rapid cys-diabody-ICG catabolism in the kidneys. To avoid this catabolic pathway, it would be preferable to use antibody fragments large enough not to be filtered through glomerulus or to conjugate the fragments with fluorescent dyes that are readily excreted into urine when cleaved from the cys-diabody to achieve high tumor-specific detection.

Highlights

Monoclonal antibodies have been used for targeted therapy and imaging because of their high affinity for a diverse number of antigens overexpressed on cancer cells.[1,2] Molecular imaging based on mAbs labeled with radioisotopes can localize tumor within the whole body and characterize it for drug targeting.[3,4]
The radioactivity observed in the kidneys was sharply reduced within 6 h, while that in the stomach was dramatically increased. These results suggest that the cys-diabody is dehalogenated and possibly catabolized in the kidneys, and the fragment of cys-diabody including the iodine ion can be slowly released into the circulation followed by gradual uptake in the stomach and thyroid
We have shown that indocyanine green (ICG) with short polyethylene glycol (PEG) linkers can be covalently conjugated to immunoglobulin G (IgG) with superior efficacy compared with ICG-Sulfo-OSu without a PEG linker.[14]

Summary

Introduction

Monoclonal antibodies (mAbs) have been used for targeted therapy and imaging because of their high affinity for a diverse number of antigens overexpressed on cancer cells.[1,2] Molecular imaging based on mAbs labeled with radioisotopes can localize tumor within the whole body and characterize it for drug targeting.[3,4]. The prolonged clearance time of whole mAbs reduces the target-to-background ratio, lowering sensitivity and specificity of labeled mAbs.[5] To overcome this problem, mAbs have been subjected to enzymatic cleavage or have been genetically modified to create fragments of various sizes, including variable region fragments (Fvs), diabody, fragments, antigen binding (Fabs), minibody, F(ab)’[2] fragment, and CH2-deleted mAb.[6,7] If these fragments are too small, their renal clearance will be too rapid, compromising tumor uptake. Intermediate-size Fabs with mild prolongation of clearance but with bivalent binding to antigen, such as diabodies (50 to 60 kDa) or minibodies (75 to 80 kDa), would seem more likely to provide the highest tumor-to-background ratios.[7]

Methods

Results

Conclusion