In vivocharacterization of activatable cell penetrating peptides for targeting protease activity in cancer

Abstract

Activatable cell penetrating peptides (ACPPs) are novel in vivo targeting agents comprised of a polycationic cell penetrating peptide (CPP) connected via a cleavable linker to a neutralizing polyanion (Fig. 1A). Adsorption and uptake into cells are inhibited until the linker is proteolyzed1–3. An ACPP cleavable by matrix metalloproteinase-2 (MMP-2) in vitro was the first one demonstrated to work in a tumor model in vivo, but only HT-1080 xenografts and resected human squamous cell carcinomas were tested. Generality to other cancer types, in vivo selectivity of ACPPs for MMPs, and spatial resolution require further characterization. We now show that ACPPs can target many xenograft tumor models from different cancer sites, as well as a well thoroughly studied transgenic model of spontaneous breast cancer (mouse mammary tumor virus promoter driving polyoma middle T antigen, MMTV-PyMT). Pharmacological inhibitors and genetic knockouts indicate that current ACPPs are selective for MMP-2 and MMP-9 in the above in vivo models. In accord with the known local distribution of MMP activity, accumulation is strongest at the tumor-stromal interface in primary tumors and associated metastases, indicating better spatial resolution (<50 µm) than other currently available MMP-cleavable probes4. We also find that background uptake of ACPPs into normal tissues such as liver and kidney can be decreased by appending inert macromolecules of 30–50 KDa to the polyanionic inhibitory domain. Our results validate an approach that should generally deliver imaging agents and chemotherapeutics to sites of invasion, tumor-promoting inflammation, and metastasis.

Surgery with molecular fluorescence imaging using activatable cell-penetrating peptides decreases residual cancer and improves survival

“…lacking protease activity (typically 6 h for free ACPPs and 48 h for ACPPDs). All tumors retained the Cy5-labeled free ACPP and ACPPD to a greater extent than normal tissues or tissue treated with protease-resistant controls (12,13). Furthermore, tumor-tonormal tissue ratios from all tumor types were greater with ACPPD compared to free ACPP (Table 1).…”

“…To test whether ACPPs are taken up differentially between tumor and normal tissue, we examined four tumor models: isografts derived from spontaneous mammary adenocarcinoma tumors in transgenic mice (line 8119, MMTV-PyMT) (11,12), the murine melanoma cell line B16F10 transplanted into immune-competent mice, and human cancer cell lines (MDA-MB 435 melanoma and HT1080 fibrosarcoma) xenografted into nude mice. Both unadorned ACPPs not conjugated to macromolecules [free ACPPs (12)] and ACPPs conjugated to a generation 5 poly(amidoamine) dendrimer [ACPPs conjugated to dendrimers (ACPPDs) (13)] were separately injected i.v. and imaged by the deep red fluorescence of their Cy5 labels after sufficient time for elimination from normal tissues To whom correspondence should be addressed.…”

Opening windows on new biology and disease mechanisms: development of real-time in vivo sensors

“…In these cases, high spatial resolution, specificity and selectivity become more important. There have been exciting advances in the application of cancer protease-activated biosensors based on fluorescent proteins for surgery and diagnostics [44]. In the future, we believe that biosensors of different classes will both complement and supplement one another to obtain the maximum useful amount of information for facilitating clinically relevant decisions.…”

Ratiometric Activatable Cell‐Penetrating Peptides Provide Rapid In Vivo Readout of Thrombin Activation

“…This method provides detection of spatially localized enzymatic activity in living tissues through the accumulation of cleaved probe. ACPPs have been previously reported that target MMPs [16,17] and elastases [20] in tumors. A thrombin-activated ACPP with cleavage sequence DPRSFL, from the PAR1 receptor was recently reported for monitoring thrombin activation in atherosclerotic plaques.…”

“…[15] Activatable cell-penetrating peptides (ACPPs) target various cargos, including fluorescent imaging agents, to sites of protease activity in vivo. [16][17][18][19] ACPPs consist of a polycationic cell-penetrating peptide attached to a cargo and a polyanionic inhibitory domain with a protease-cleavable linker. Probe activation and cargo uptake depend on localized proteolysis of the linker sequence that connects the polyanionic and polycationic domains, which converts the probe to an adherent form.…”