Cloning and humanization of E16. E16 heavy-and light-chain RNA was isolated from hybridoma cells after guanidinium thiocyanate and phenol-chloroform extraction, and converted to cDNA by reverse transcription. The VH and VL segments were amplified by PCR using the 5′ rapid amplification of cDNA ends (RACE) system (Invitrogen). Gene-specific primers (GSP) for VH and VL were as follows: VH-GSP1: 5´-GGTCACTGTCACTGGCTCAGGG-3´; VH-GSP2: 5´-AGGCGGATCCAGGGGCCAGTGGATAGAC-3´; VL-GSP1: 5´-GCACACG ACTGAGGCACCTCCAGATG-3´; and VL-GSP2: 5´ CGGATCCGATGGATAC AGTTGGTGCAGCATC-3´. The PCR products were inserted into the plasmid pCR2.1-TOPO using the TopoTA kit (Invitrogen). We then subjected the resulting plasmids to DNA sequencing to determine the VH and VL sequences for E16. The cDNA sequences were translated and the predicted amino acid sequence determined. From these sequences the framework and CDR regions were identified as previously defined 42 . We joined the mouse VH to a human C-γ1 constant region and an Ig leader sequence, and inserted it into pCI-neo for mammalian expression. We joined the mouse VL to a human Cκ segment and an Ig leader sequence and also cloned it into pCI-neo for mammalian expression of chimeric E16 (Ch-E16). For Ch-E16, site-directed mutagenesis was also performed to change residue 297 from asparagine to glutamine of the heavy chain to eliminate the single glycosylation site on the γ1 Fc.