Screening of a Novel Peptide Targeting the Proteoglycan-Like Region of Human Carbonic Anhydrase IX

Abstract

The extracellular domain of human carbonic anhydrase IX (CA IX) is extended by a proteoglycan-like region (PGLR). The aim of the present study was the development of novel molecules with specificity for PGLR, which may be used for tumor targeting and imaging. PGLR was chemically synthesized, and phage display biopanning was performed. The identified ligand PGLR-P1 was labeled with 125I and characterized for target binding and metabolic stability. In vitro characterization included kinetic, competition, and internalization studies on CA IX-positive renal cell carcinoma SKRC 52 cells. The CA IX-negative cell lines HEK293 wt and BxPC3 were used as negative controls. In vitro binding experiments revealed an increasing affinity of 125I-PGLR-P1 to SKRC 52 cells but not to negative control HEK293 wt and BxPC3 cells. Internalization studies indicated an exclusive cell membrane binding. Biodistribution analysis demonstrated a higher accumulation in SKRC 52 tumors than in most normal tissues after perfusion. In vivo blocking led to a significant decrease in tumor uptake. Our findings indicate that PGLR-P1 is a promising lead structure for the development of new peptide-based ligands targeting the PGLR of CA IX and reveal challenges that need to be considered for peptide-related molecular imaging.

Past, Present, and Future: Development of Theranostic Agents Targeting Carbonic Anhydrase IX

“…Citing concerns of isoform selectivity, the same research group directed a screen against the PG-like domain of CA-IX and isolated the dodecapeptide NMPKDVTTRMSS (PGLR-P1) 72 . A C-terminal D-tyrosine was again introduced to facilitate radiolabeling with 125 I/ 131 I.…”

Microsecond Simulation of the Proteoglycan-like Region of Carbonic Anhydrase IX and Design of Chemical Inhibitors Targeting pH Homeostasis in Cancer Cells

“…61 There is an earlier study on peptides targeting the proteoglycan region based on phage display and biopanning. 62 Moreover, our group has also shown oligopeptides derived from M75 antibody to be efficient inhibitors of CAIX in HeLa cells. 63 As the proteoglycan-like region is unique to CAIX, it becomes essential to know about its conformational landscape for designing selective inhibitors targeting the tumor cells.…”

“…Recent studies have also shown a monoclonal antibody M75 to selectively target an epitope at the proteoglycan region . There is an earlier study on peptides targeting the proteoglycan region based on phage display and biopanning . Moreover, our group has also shown oligopeptides derived from M75 antibody to be efficient inhibitors of CAIX in HeLa cells .…”

New Developments in Carbonic Anhydrase IX-Targeted Fluorescence and Nuclear Imaging Agents

“…However, tumor uptake of 131 I-CaIX-P1-4-10 was not significantly improved (~2.5% ID/g at 1 h PI), and SKRC-52 tumors could not be distinguished from the background. To improve the isoform selectivity, Rena et al identified a new linear dodecapeptide PGLR-P1 (NMPKDVTTRMSS), which targets the region of the extracellular proteoglycan (PG)-like domain of CAIX with no homology to other Cas [ 30 ]. The [ 125 I]I-labeled PGLR-P1 showed a higher selectivity toward CAIX (1.8% applied dose) and the PGLR domain of CAIX (10% applied dose) than CAII and CAXII (both < 0.1% applied dose).…”