novel VHH nanobody against the active site (the CA domain) of tumor-associated, carbonic anhydrase isoform IX and its usefulness for cancer diagnosis

• Published in: Biotechnology letters Vol. 36; no. 1; pp. 21 - 28
• Main Authors: Araste, Fatemeh, Ebrahimizadeh, Walead, Rasooli, Iraj, Rajabibazl, Masoumeh, Mousavi Gargari, Seyed Latif
• Format: Journal Article
• Language: English
• Published: Dordrecht Springer-Verlag 2014; Springer Netherlands; Springer Nature B.V
• Subjects: active sites; Affinity; anaerobic conditions; Animals; Antigens, Neoplasm - chemistry; Antigens, Neoplasm - immunology; Antigens, Neoplasm - metabolism; Applied Microbiology; bacteriophages; Binding; Biochemistry; Biomarkers, Tumor - chemistry; Biomarkers, Tumor - immunology; Biomarkers, Tumor - metabolism; Biomedical and Life Sciences; Biotechnology; camels; Camelus; Cancer therapies; carbonate dehydratase; Carbonic anhydrase; Carbonic Anhydrase IX; Carbonic Anhydrases - chemistry; Carbonic Anhydrases - immunology; Carbonic Anhydrases - metabolism; Catalytic Domain; Cell Surface Display Techniques; Chromatography, Affinity; complementary DNA; Diagnosis; Enzymatic activity; enzyme activity; enzyme-linked immunosorbent assay; HeLa Cells; Humans; Hypoxia; Immunization; Life Sciences; Lymphocytes; Male; Microbiology; Nanocomposites; Nanomaterials; Nanostructure; neoplasms; Neutralization; Original Research Paper; polymerase chain reaction; Single-Domain Antibodies - immunology; Single-Domain Antibodies - isolation & purification; Single-Domain Antibodies - metabolism; Studies; Tumors; Iran; Carbonic anhydrase IX; Phage display; VHH nanobody; Cancer diagnosis; Cancer treatment
• ISSN: 0141-5492; 1573-6776
• DOI: 10.1007/s10529-013-1340-1

Expression of carbonic anhydrase IX (CAIX) significantly increases under hypoxic conditions in tumor cells. CAIX activity is executed by the catalytic domain (CA) located on the extracellular part of the enzyme. Neutralization of CAIX enzymatic activity reduces malignancy and survival of tumor cells. To inhibit the enzymatic activity, a VHH nanobody was developed against the CA domain of CAIX using phage display technology. Following immunization of a camel with the recombinant CAIX, VHH fragments were isolated by nested PCR on lymphocyte cDNA. Binding affinity of isolated nanobodies was tested by ELISA. A clone (K24) with the highest binding affinity was expressed in a soluble form. Affinity of K24 nanobody was determined to be approx. 2.3 × 10⁻⁵. K24 nanobody recognized the expressed CAIX in the HeLa cell lines with high selectivity and specificity. These findings thus have usefulness for the diagnosis and treatment of cancers.