CAIX is a tumor-associated member of the family of carbonic anhydrases that contributes to the acidification of extracellular pH and neutralization of intracellular pH protecting tumor cells from acidic pericellular microenvironment. In this study we present the results of the evaluation of CaIXP1, a
ar dodecapeptide with specificity for the extracellular domain of human carbonic anhydrase IX, identified through the technology of phage display. Binding experiments on the immobilized extracellular domain of CAIX revealed a higher accumulation on carbonic anhydrase IX, compared to the extracellular domain of the epidermal growth factor receptor, which was used as negative control target-protein. Tracer accumulation was found to be inhibited by the unlabeled CaIXP1 peptide, which is evidence for a specific binding to the target. The hypothesis of a specific binding is supported by the results of the in vitro experiments, while the binding of the peptide was reduced to the background level for the cell line CaKi 2 which has been described as CAIX negative. In addition, a dependence of binding of the radiolabeled peptide on the CAIX mRNA expression was shown for the colorectal carcinoma cell line HCT 116 and for human Torin 1 mTOR inhibitor umbilical vein endothelial cells. Both mRNA expression of carbonic anhydrase IX and binding capacity of the CaIX-P1 peptide were higher for HCT 116 compared to the HUVEC cells. For the colorectal carcinoma HCT 116 cells the qRT-PCR analysis revealed a cell density dependent expression of CAIX, which also correlated to the binding of 125I-labeled CaIX-P1. Further evidence for a specific accumulation of the CaIX-P1 peptide was found in competition experiments revealing that the uptake of the radiolabeled ligand in CAIX positive SKRC 52 cells was reduced with increasing concentration of the unlabeled peptide, whereas octreotide as unspecific competitor at the same concentration had no effect. The results of the internalization experiments demonstrated a quick internalization of the peptide at 37uC, which thereafter decreased with progression of time. Experiments at 4uC revealed a strongly reduced internalization to the background level. Those results are in concert with the results of previous studies, investigating CAIX specific antibodies. In particular, Chrastina et al. demonstrated a quick internalization of 125I-labeled monoclonal antibody M75, which is specific for human carbonic anhydrase IX, in human colorectal carcinoma cells. Furthermore, the authors of this study point to the fact that antibodies, iodinated through the tyrosine residues, are dehalogenated after internalization and that the radioactive metabolites are excreted from the cells. Such a process of intracellular dehalogenation or degradation, leading to radioactive products that are excreted by the cells, might also explain the time dependent reduction of both internalized activity and total cellular uptake of 125I-labeled CaIX-P1 at 37uC.