Humanized E16 VH consists of the framework segments from the human germline VH1-18 VH segment and JH6 segment 43, 44 , and the CDR regions of the E16 VH, respectively. The humanized E16 VL consists of the framework segments of the human germline VK-B3 VL segment and JK2, (refs. 45-47) segment and the CDR regions of E16 VL. The humanized VH segments were assembled de novo from oligonucleotides and amplified by PCR. The humanized VL segments were assembled by PCR and overlapping PCR. The resulting VH and VL segments were subsequently combined by overlapping PCR with a leader sequence and the appropriate constant region segment and cloned into the expression vector pCI-neo as NheI-EcoRI fragments. We confirmed the DNA sequence of the resulting plasmids by sequence analysis. Site-directed mutagenesis was then performed to substitute mouse for human residues at key framework positions VH-71 (T71A) and VL-49 (Y49S). The resulting plasmids were cotransfected into human 293 cells using lipofectamine-2000 and humanized antibody was recovered from the resulting conditioned medium and purified by protein A and size-exclusion chromatography.