In vivo screening of phage libraries in tumor-bearing mice has been used to identify peptides that direct phage homing to a tumor. The power of in vivo phage screening is illustrated by the recent discovery of peptides with unique tumor-penetrating properties. These peptides activate an endocytic transport pathway related to but distinct from macropinocytosis. They do so through a complex process that involves binding to a primary, tumor-specific receptor, followed by a proteolytic cleavage, and binding to a second receptor. The second receptor, neuropilin-1 (or neuropilin-2) activates the transport pathway. This trans-tissue pathway, dubbed the C-end Rule (CendR) pathway, mediates the extravasation transport through extravascular tumor tissue of payloads ranging from small molecule drugs to nanoparticles. The CendR technology provides a solution to a major problem in tumor therapy, poor penetration of drugs into tumors. Targeted delivery with tumor-penetrating peptides has been shown to specifically increase the accumulation of drugs, antibodies and nanotherapeutics in experimental tumors in vivo, and in human tumors ex vivo. Remarkably the payload does not have to be coupled to the peptide; the peptide activates a bulk transport system that sweeps along a drug present in the blood. Treatment studies in mice have shown improved anti-tumor efficacy and less damage to normal tissues with drugs ranging from traditional chemotherapeutics to antibodies, and to nanoparticle drugs.
The iRGD peptide homes to tumors and accumulates in them through a 3-step process (Fig. 1): First, the integrin-binding RGD sequence motif binds to αvβ3 and αvβ5 integrins, which are specifically expressed in tumor endothelial cells. Other cells in tumors also express these integrins, which is likely to be important for the spreading of the peptide within tumor tissue, but the vascular endothelium is the gateway to the tumor for the peptide. Second, a protease cleavage event activates the CendR motif (R/KXXR/K). This protease(s) has not been identified, but is likely a furin or furin-like enzyme because the CendR motif is a preferred recognition motif for these proteases. In principle, any protease that cuts after a basic residue can activate iRGD. We have used trypsin and urokinase in vitro for this purpose. The protease cleavage requires the integrin binding; a peptide that has the CendR motif but does not bind to integrins (CRGEKGPDC) is not activated. The requirement for integrin binding limits the activation of iRGD to tumors. Third, the CendR motif binds to neuropilin-1 (NRP-1) or neuropilin-2 (NRP-2), and the interaction activates an endocytotic/exocytotic transport pathway named the CendR pathway. This pathway is responsible for the enhanced transport of drugs into tumors triggered by iRGD.
Using an in vivo screening procedure designed to probe tumor lymphatic vessels, we identified a peptide that specifically accumulated in tumor lymphatics and not in normal lymphatics. We now know that this peptide, LyP-1, primarily accumulates in a myeloid cell/macrophage in tumors, when intravenously injected into tumor-bearing mice. Some of these cells incorporate into tumor lymphatics, causing LyP-1 accumulation in the endothelium of these vessels. Endothelial cells of tumor blood vessels and tumor cells also bind LyP-1, but much less of the peptide accumulates in these cells than in tumor macrophages. The macrophages are particularly abundant in hypoxic areas of tumors, which are low on blood vessels but contain abundant, albeit dysfunctional lymphatic vasculature. Remarkably, the phage carrying the LyP-1 peptide reaches these areas within minutes of systemic injection. The ability of this peptide to reach poorly vascularized parts of tumors remained a mystery for several years, until we discovered another peptide with similar tumor-penetrating properties, and set out to uncover the underlying mechanism. (Adv Drug Deliv Rev. 2017 Feb; 110-111: 3–12.)