Streamlined Development of Fully Human Antibody Fragments for ImmunoPET Imaging Using Phage Display Technology

Exploiting antibodies' highly specific binding affinities to various biomarkers, immuno-positron emission tomography (immunoPET) provides a non-invasive way to obtain whole body information on in vivo molecular events that may be crucial for diagnosis and prognosis, which is increasingly important for diagnostics and prognostics. Small, fully human or humanized antibody fragments are preferred for immunoPET imaging probe development due to their favorable pharmacokinetic properties and low risk of immunogenicity. Phage display technology provides a convenient approach to rapidly generate fully human antibody candidates, showing the potential to accelerate and streamline the development process of antibody fragment based immunoPET imaging probes, which is strongly needed as increasing numbers of targeted therapies are being developed every year. Chapter 2 and Chapter 3 describe the use of a traditional fully human scFv phage display library for developing anti-MET antibody fragments for both immunoPET imaging and potential therapeutic application. Multiple fully human anti-MET scFv clones were isolated and reformatted. Three cys-diabody clones showed high affinities (0.7 to 5.1 nM) and inhibitory effect on MET over-expressing cells. The H2 cys-diabody and minibody were able to distinguish a MET over-expressing gefitinib resistant tumor from the parental tumor at as early as 4 hours post injection, indicating the potential of same day imaging for patient stratification. To further exploit the benefit of the phage display technology and streamline the antibody fragment devlopment, as described in Chapter 4 and Chapter 5, two novel fully human scFv phage display libraries with customized linkers and restriction sites were constructed to simplify the antibody fragment reformatting process. The libraries were used for selections against several different targets and successfully generated multiple positive clones. ScFvs from these libraries were rapidly reformatted into diabodies using the restriction sites in the customized linkers. Size exclusion chromatography analysis of these antibody fragments proved the shortened linkers can successfully induce dimerization for the majority of the diabody clones. Together, the studies presented in this dissertation highlight a successful example of the use of phage display technology for developing fully human antibody fragments for immunoPET imaging and how phage display libraries can be improved to further streamline this process