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A new peptide ligand for targeting human carbonic anhydrase IX, identified through the phage display technology
Vasileios Askoxylakis1, Regine Garcia-Boy, Shoaib Rana
1Department of Radiooncology and Radiation Therapy, University of Heidelberg, Heidelberg, Germany. vasileios.askoxylakis@med.uni-heidelberg.de
Unlabelled:
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.
Methods:
Phage 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 (125)I-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.
Conclusions:
These data indicate that CaIX-P1 is a promising candidate for the development of new ligands targeting human carbonic anhydrase IX.
Insights
A novel peptide, CaIX-P1, targets carbonic anhydrase IX (CAIX), an enzyme overexpressed in tumors. This peptide shows promise for visualizing tumors and developing targeted cancer therapies.
Area of Science:
- Biochemistry
- Oncology
- Molecular Biology
Background:
- Carbonic anhydrase IX (CAIX) is a transmembrane enzyme frequently overexpressed in various tumors.
- CAIX overexpression is strongly associated with tumor hypoxia, a condition that promotes tumor growth and resistance to therapy.
- Targeting CAIX offers potential for improved cancer diagnostics and therapeutics, including tumor imaging and endoradiotherapy.
Purpose of the Study:
- To identify and characterize novel peptide ligands that specifically bind to human carbonic anhydrase IX (CAIX).
- To evaluate the potential of the identified peptide, CaIX-P1, for applications in cancer diagnosis and therapy.
- To assess the in vitro and in vivo binding characteristics and biodistribution of CaIX-P1.
Main Methods:
- Phage display technology was employed to screen a peptide library against the extracellular domain of human CAIX.
- The selected peptide, CaIX-P1, was synthesized and tested for binding affinity and specificity using various cancer cell lines (CAIX-positive and -negative) and human umbilical vein endothelial cells (HUVEC).
- In vivo studies in mice bearing human renal cell carcinoma xenografts were conducted to assess tumor accumulation and organ distribution of radiolabeled CaIX-P1.
Main Results:
- The peptide CaIX-P1 demonstrated specific binding to CAIX-positive cells (SKRC 52) and CAIX-expressing colorectal cancer cells (HCT 116), with binding intensity correlating with CAIX mRNA levels.
- Radioligand uptake was significantly inhibited by unlabeled CaIX-P1 but not by a control peptide, confirming specificity.
- In vivo studies showed preferential accumulation of CaIX-P1 in tumors compared to several organs, although kidney and blood showed higher uptake.
Conclusions:
- The peptide CaIX-P1 exhibits specific binding to carbonic anhydrase IX.
- CaIX-P1 is a promising candidate for developing targeted diagnostic and therapeutic agents for CAIX-expressing tumors.
- Further development of CaIX-P1-based ligands could enhance cancer visualization and treatment strategies.
