A New Peptide Ligand for Targeting Human Carbonic Anhydrase IX, Identified through the Phage Display Technology

Abstract

Carbonic anhydrase IX (CAIX) is a transmembrane enzyme found to be overexpressed in various tumors and associated with tumor hypoxia. Ligands binding this target may be used to visualize hypoxia, tumor manifestation or treat tumors by endoradiotherapy.MethodsPhage display was performed with a 12 amino acid phage display library by panning against a recombinant extracellular domain of human carbonic anhydrase IX. The identified peptide CaIX-P1 was chemically synthesized and tested in vitro on various cell lines and in vivo in Balb/c nu/nu mice carrying subcutaneously transplanted tumors. Binding, kinetic and competition studies were performed on the CAIX positive human renal cell carcinoma cell line SKRC 52, the CAIX negative human renal cell carcinoma cell line CaKi 2, the human colorectal carcinoma cell line HCT 116 and on human umbilical vein endothelial cells (HUVEC). Organ distribution studies were carried out in mice, carrying SKRC 52 tumors. RNA expression of CAIX in HCT 116 and HUVEC cells was investigated by quantitative real time PCR.Results In vitro binding experiments of 125I-labeled-CaIX-P1 revealed an increased uptake of the radioligand in the CAIX positive renal cell carcinoma cell line SKRC 52. Binding of the radioligand in the colorectal carcinoma cell line HCT 116 increased with increasing cell density and correlated with the mRNA expression of CAIX. Radioligand uptake was inhibited up to 90% by the unlabeled CaIX-P1 peptide, but not by the negative control peptide octreotide at the same concentration. No binding was demonstrated in CAIX negative CaKi 2 and HUVEC cells. Organ distribution studies revealed a higher accumulation in SKRC 52 tumors than in heart, spleen, liver, muscle, intestinum and brain, but a lower uptake compared to blood and kidney.ConclusionsThese data indicate that CaIX-P1 is a promising candidate for the development of new ligands targeting human carbonic anhydrase IX.

A novel small peptide as an epidermal growth factor receptor targeting ligand for nanodelivery in vitro

“…Screening of phage display libraries, 8,23 CAD, 10 hydropathic complementarity, 24 hydropathic analysis, and comparative sequence/structure analysis 7 have been used to design small peptide ligands. In this study, we demonstrated different methods of screening for small molecule peptide ligands.…”

Identification of Ligands and Translation to Clinical Applications

“…This approach has been used in various applications, such as in the mapping and mimicking of epitopes, the identification of new receptors and natural ligands, high-affinity antibodies and analogs, the isolation of specific antigens binding to bioactive compounds, the production of novel enzyme inhibitors and DNA-binding proteins, and the probing of cellular and tissue-specific processes. Phage display has been successfully applied for the identification of peptides with a high specificity for target tumor neovasculature or a variety of human tumor cells (16,(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33).…”

“…This degradation inevitably leads to a stability problem, with a plasma half-life in the range of several minutes (26)(27)(28)(29)(30). Stability may be increased by shortening the peptide after identification of the essential binding sequence using an alanine scan, exchange of single amino acids, introduction of D-amino acids, peptide cyclization, and coupling to chelators such as DOTA.…”

Mechanistic and high-throughput approaches for the design of molecular imaging probes and targeted therapeutics

“…Phage display has successfully been applied for the identification of novel peptides with high specificity for target proteins overexpressed in the tumor neovasculature, such as delta-like ligand 4 (Dll4), or for human lung carcinoma, mammary carcinoma, prostate carcinoma and neuroblastoma cells [1,2,[21][22][23][24][25][26].…”