Bicyclic Peptides as a New Modality for Imaging and Targeting of Proteins Overexpressed by Tumors.

Molecular imaging of cancers using probes specific for tumor-associated target proteins offers a powerful solution for providing information regarding selection of targeted therapy, patient stratification, and response to therapy. Here we demonstrate the power of bicyclic peptides as targeting probes, exemplified with the tumor-overexpressed matrix metalloproteinase MT1-MMP as a target. A bicyclic peptide with subnanomolar affinity towards MT1-MMP was identified, and its radioconjugate showed selective tumor uptake in an HT1080 xenograft mouse model. Proteolytic stabilization of the peptide by chemical modification significantly enhanced the in vivo tumor signal [from 2.5%ID/g to 12%ID/g at 1 hour post injection (p.i.)]. Studies using mouse xenograft models with different cell lines show a robust correlation between tumor signals and in vivo MT1-MMP expression levels. Fatty acid modification of the bicyclic peptide extended its circulating half-life, resulting in increased tumor signals (36%ID/g at 6 hours p.i.). Comparative work with an equipotent radiolabeled MT1-MMP targeting antibody demonstrated starkly differential biodistribution and tumor accumulation properties, with the tumor signal slowly increasing to 6.2%ID/g within 48 hours. The rapid tumor penetration characteristics of bicyclic peptides, coupled with high potency and chemical versatility, thus offer high-contrast imaging probes for clinical diagnostics with compelling additional potential in targeted therapy.Significance: This work demonstrates the potential of bicyclic peptides as a platform for the development of high-contrast imaging probes for potential use in clinical cancer diagnostics and molecularly targeted therapeutics.

A novel in vivo method for isolating antibodies from a phage display library by neuronal retrograde transport selectively yields antibodies against p75(NTR.).

The neurotrophin receptor p75(NTR) is utilized by a variety of pathogens to gain entry into the central nervous system (CNS). We tested if this entry portal might be exploited using a phage display library to isolate internalizing antibodies that target the CNS in vivo. By applying a phage library that expressed human single chain variable fragment (scFv) antibodies on their surface to a transected sciatic nerve, we showed that (1) phage conjugated to anti-p75(NTR) antibody or phage scFv library pre-panned against p75(NTR) are internalized by neurons expressing p75(NTR); (2) subsequent retrograde axonal transport separates internalized phage from the applied phage; and, (3) internalized phage can be recovered from a proximal ligature made on a nerve. This approach resulted in 13-fold increase in the number of phage isolated from the injured nerve compared with the starting population, and isolation of 18 unique internalizing p75(NTR) antibodies that were transported from the peripheral nerve into the spinal cord, through the blood-brain barrier. In addition, antibodies recognizing other potentially internalized antigens were identified through in vivo selection using a fully diverse library. Because p75(NTR) expression is upregulated in motor neurons in response to injury and in disease, the p75(NTR) antibodies may have substantial potential for cell-targeted drug/gene delivery. In addition, this novel selection method provides the potential to generate panels of antibodies that could be used to identify further internalization targets, which could aid drug delivery across the blood-brain barrier.