Humanization Of Mouse Anti-bladder Cancer McAb By CDR3 Directed Phage Antibody Library

On the basis of epitope guided selection, we explored a new mouse mAb humanization method-CDR3 directed phage antibody library.First, Fd andK genes of an anti-human bladder carcinoma mAb BDI were cloned by RT-PCR. Sequencing analysis showed that the Vk and VH were derived from MK IV and MH3D respectively. Fd and K genes were then cloned into the expressing vector p3MH and expressed in E. coli. Using PCR mediated mutagenesis, the N-terminal sequence of VH region was corrected and the biding activity was obviously improved after the correction. ELISA and immunohistochemistry analysis demonstrated that expressed Fab bind to bladder cancer cells specifically.Secend, According to the original sequences of mAb BDI, oligonucleotide primers containing the original CDR3 sequences of mAb BDIwere synthesized. Through overlap PCR, the mouse VH and V K, CDR3s were splice to human diverse Fd and K genes and cloned into a phage antibody expression vector pAL, in which the Fd are flanked by two non-homologous loxp sites, obtaining a primary phage antibody library of 2 x 107 in size. The library phagemid was transferred into a Cre+ bacteria BS1365 with high multiplicity of infection (MOI). Through cre-loxp mediated recombination, the Fd and Kchains at different vectors were shuffled and a large humanized library with 1 x 1010 in size was obtained. After 4 round of panning against bladder cancer cells EJ, 4 clones were found to be able to bind EJ cells specifically. Competition ELISA studies showed that 3 clones had the same specifity as the parental tnAb. The DNA sequencing of the variable regions demonstrated that their VH and VL derived from VHI, VHII, and Vk I, Vk VI families respectively. Immunohistochemistry study showed that the humanize Fab were able to bind to bladder cancer specimens.Our results demonstrated that through CDR3 directed phage antibody library with very large size, human counterpart of mouse mAb could be selected without the parental mouse V region as template. This will facilitate the humanization of mouse mAb and can also find its use in constructing new antibody-like molecules containing a short stretch of sequence involving in specific binding to a particelar target.