Vascular ligand-receptor mapping by direct combinatorial selection in cancer patients

Abstract

Molecules differentially expressed in blood vessels among organs or between damaged and normal tissues, are attractive therapy targets; however, their identification within the human vasculature is challenging. Here we screened a peptide library in cancer patients to uncover ligand-receptors common or specific to certain vascular beds. Surveying ∼2.35 × 10 6 motifs recovered from biopsies yielded a nonrandom distribution, indicating that systemic tissue targeting is feasible. High-throughput analysis by similarity search, protein arrays, and affinity chromatography revealed four native ligand-receptors, three of which were previously unrecognized. Two are shared among multiple tissues (integrin α4/annexin A4 and cathepsin B/apolipoprotein E3) and the other two have a restricted and specific distribution in normal tissue (prohibitin/ annexin A2 in white adipose tissue) or cancer (RAGE/leukocyte proteinase-3 in bone metastases). These findings provide vascular molecular markers for biotechnology and medical applications. To discover or analyze functional ligand-receptor interactions in blood vessels under disease conditions, we have used combinatorial screenings based on phage display, which has enabled the targeted delivery of agents to specific vascular beds (8,9). This approach allows the selection of homing peptides to specific organs in vivo after systemic administration of random peptide libraries (10, 11). We have isolated ligand peptides and identified their tissue-specific receptors in rodents and in a patient, and have developed a ligand-receptor in prostate cancer (12, 13) that serves as the basis for an ongoing first-in-man trial.Over the past few years we have developed a tripartite approach to enable serial combinatorial selections to humans. First, we established an ethical framework to ensure respectful research in patients who were brain-dead or whose families decided to terminate life support (14,15). Second, we adapted techniques that were validated in rodents (16) to enable synchronous selection of ligands to multiple organs. Third, we integrated genomic tools, in which recovery of 10 6 peptides is ∼1;000-fold faster and ∼250-fold cheaper (17). Here, these quantitative and qualitative methods enabled refined combinatorial selections in patients and identified unique ligand-receptors.

Vascular-targeted nanotherapy for obesity: Unexpected passive targeting mechanism to obese fat for the enhancement of active drug delivery

“…This result clearly indicates that vascular-targeted nanotherapy integrating antiangiogenesis with nanotechnology is highly promising strategy for effective control of obesity. Moreover, the ligand receptor prohibitin was also expressed in human WFV [19]. Therefore, this work may, in the near future, offer a paradigm shift in the discovery and development of anti-obesity drugs for the treatment of human obesity.…”

Proteasome activator complex PA28 identified as an accessible target in prostate cancer by in vivo selection of human antibodies

“…The bias toward the more abundant tumor cells is compensated in the ensuing in vivo selection that will target only the systemically accessible antigens. This procedure has already been carried out successfully using peptide phage display in tumor-bearing mice and in other disease models (9,33,34); however, there are very few examples of human antibodies selected with a similar procedure, and always against vascular targets: thymic endothelium (35) and atherosclerotic endothelial and subendothelial tissues (36).…”

Autonomous self-navigating drug-delivery vehicles: From Science Fiction to Reality

“…Development of phage screening technique in human patients is believed to lead to the molecular mapping of human blood vasculature toward a targeted medicines [64]. In another set of phage screening experiments in humans, Krag et al [62] used minor coat protein p3-fused peptide/scFv libraries constructed in the fd-tet/fUSE5 vector system [65].…”

“…However, distribution of clones in different organs with fused random peptides was strongly biased, as was proved by assessments of the peptide 3-mer motif occurrences in a certain organ and in the native library. Optimization of phage survey by replacement of phage tittering and TU-counting with DNA extraction and quantitative (q)PCR allowed obtaining precise phage homing results in a few hours after removal of tissue, for dozens of samples in one setup [63].Development of phage screening technique in human patients is believed to lead to the molecular mapping of human blood vasculature toward a targeted medicines [64]. In another set of phage screening experiments in humans, Krag et al [62] used minor coat protein p3-fused peptide/scFv libraries constructed in the fd-tet/fUSE5 vector system [65].…”