Targeting the tumor vasculature with engineered cystine-knot miniproteins

Abstract

The extra domain B splice variant (EDB) of human fibronectin selectively expressed in the tumor vasculature is an attractive target for cancer imaging and therapy. Here, we describe the generation and characterization of EDB-specific optical imaging probes. By screening combinatorial cystine-knot miniprotein libraries with phage display technology we discover exquisitely EDB-specific ligands that share a distinctive motif. Probes with a binding constant in the picomolar range are generated by chemical oligomerization of selected ligands and fluorophore conjugation. We show by fluorescence imaging that the probes stain EDB in tissue sections derived from human U-87 MG glioblastoma xenografts in mice. Moreover, we demonstrate selective accumulation and retention of intravenously administered probes in the tumor tissue of mice with U-87 MG glioblastoma xenografts by in vivo and ex vivo fluorescence imaging. These data warrants further pursuit of the selected cystine-knot miniproteins for in vivo imaging applications.

Milking the Cow: Cattle-Derived Chimeric Ultralong CDR-H3 Antibodies and Their Engineered CDR-H3-Only Knobbody Counterparts Targeting Epidermal Growth Factor Receptor Elicit Potent NK Cell-Mediated Cytotoxicity

“…In this respect, yeast surface display has proven to be versatile for engineering different antibody derivatives (36)(37)(38)(39)(40)(41) but also more complex molecules such as cytokines (42), ligands (43), or cysteine-rich miniproteins (44,45). More recently, Sahin and colleagues could demonstrate that disulfide-rich peptides can also be enriched by phage display (46), substantiating the versatility of display technologies for engineering of more complex molecules. After immunization, an immune response was detected in all immunized cattle.…”

Linking molecular evolution to molecular grafting

“…The structural and functional diversity of the ICK motif show that it has acquired different mutations and activity over evolutionary time, suggesting that peptides with this motif are highly innovable. Indeed, they have attracted wide interest as therapeutic modalities ( 48 , 49 , 50 , 51 , 52 ). For example, analogs of the scorpion peptide chlorotoxin ( 53 ) and the engineered R01-MG peptide ( 54 ) have undergone clinical trials as imaging tools to guide surgical oncology, and an analog of the cyclotide kalata B1 is entering trials as an immunomodulatory agent ( 55 ).…”

Screening of tumor-targeting peptides for diagnosis and therapy through phage display technology

“…Peptide CNWMINKEC is non-cytotoxic to cells and is stable in serum up to 24 hours, indicated as a useful probe for imaging and diagnosing tumors with high expression of KIM-1 in vivo [41] . In addition to targeting tumor cells, and considering the significance of tumor angiogenesis in tumorigenesis, some receptors in the tumor vasculature have been chosen as targets for design imaging probes to optimize tumor diagnosis and treatment [42] . The extra domain B splice variant (EDB), which is selectively expressed in the tumor vasculature, was also considered as a target for imaging and treatment of cancer tissues, and some selected specific ligands for EDB have been delivered through phage display technology [43] .…”