Two human genes, GenBank accession DI084180 and DI075739, were 94.7% and 92.2% identical to DB32-6 V H and V L , respectively. Humanized DB32-6 V H consisted of the modified FR1 to FR4 from the accession DI084180 gene, and the CDR1 to CDR3 of the DB32-6 V H , respectively, while humanized DB32-6 V L consisted of the modified FRs from the accession DI075739 gene and the CDRs of the DB32-6 V L . Both were synthesized (GENEART) and amplified by PCR using pfu Turbo DNA polymerase (EMD Bioscience). The resulting V H was cloned into modified expression vector pcDNA3.1 (Invitrogen) with a signal peptide and human IgG1 constant region, while the V L was cloned into modified expression vector pSecTag (Invitrogen). We generated a variant of humanized DB32-6 (hDB32-6 variant) in which leucine residues at positions 1.2 and 1.3 of C H 2 domain were substituted with alanine residues [38] . The V H and V L plasmids were cotransfected into CHO-K1 cells and selected by G 418 and puromycin for 2-3 weeks. Transformed cells were limit diluted in 96-well plates. After two weeks, stable clones produced humanized antibodies in the McCoy's 5A medium (Sigma-Aldrich), as identified by ELISA. Humanized antibodies were produced by CELLine AD 1000 (INTEGRA Biosciences) according to manufacturer's directions.