Tri-functional platform for construction of modular antibody fragments for in vivo 18F-PET or NIRF molecular imaging.

Raymond F. Gamache,Wen-Ting K. Tsai,Jennifer M. Murphy,Kirstin A. Zettlitz,Jeffrey Collins,Anna M. Wu

doi:10.1039/c9sc05007h

Abstract

Positron emission tomography (PET) molecular imaging is a powerful tool for interrogating physiological and biochemical processes to understand the biology of disease and advance therapeutic developments. Near-infrared fluorescence (NIRF) optical imaging has become increasingly popular for intraoperative staging to enable cellular resolution imaging of tumor margins during surgical resection. In addition, engineered antibody fragments have emerged as promising molecular imaging agents given their exquisite target selectivity, rapid systemic clearance and site-selective chemical modification. We report a tri-functional platform for construction of a modular antibody fragment that can rapidly be labeled with radionuclides or fluorophores for PET or NIRF molecular imaging of prostate stem cell antigen (PSCA).

Highlights

Molecular imaging techniques such as positron emission tomography (PET) and near-infrared uorescence (NIRF) optical imaging are highly valuable tools for diagnosis, staging and therapy monitoring of disease
Recombinant antibody fragments called cys-diabodies contain a C-terminal cysteine that forms a disul de bridge to stabilize dimerization which can be preferentially reduced to unveil free thiols for wellde ned, site-speci c conjugation.5b,6 Importantly, the cysteine modi cation is located away from the antigen-binding site to preserve immunoreactivity of the protein conjugate. These constructs are powerful imaging agents due to their small size, high tumor uptake, thiol-speci c conjugation and fast blood clearance which results in high tumor to background ratios within 2 hours of probe injection.6b Labeled with positron-emitting radionuclides (18F, 64Cu, 124I and 89Zr), recombinant diabody/cys-diabody constructs have been employed as immunoPET probes for a variety of tumor types including breast, pancreatic, prostate and colorectal cancers as well as for tumor-in ltrating CD8+ T cells.5b,6a,7 In addition, uorescently labeled antibody fragments have been evaluated in vivo for their potential use as targeted optical imaging probes in xenogra models.[8]
The inherent nature of the inverse electron demand Diels–Alder (IEDDA) cycloaddition[12] between tetrazine and trans-cyclooctene – catalyst-free, extremely rapid, highly selective, modular, robust – make it an attractive reaction for protein labeling applications and provided the rationale to incorporate it into the tri-functional platform design and construction.[13]

Summary

Introduction

Molecular imaging techniques such as positron emission tomography (PET) and near-infrared uorescence (NIRF) optical imaging are highly valuable tools for diagnosis, staging and therapy monitoring of disease. For proof-of-concept, a cys-diabody against prostate stem cell antigen (PSCA) was conjugated to the tri-functional platform and immunoPET and optical imaging were performed to access whole-body biodistribution as well as tumor targeting potential in mice carrying prostate cancer xenogra s.

Results

Conclusion