Humanization Of Mouse Anti-human Integrin αvβ3 McAb E10 By CDR3 Directed Phage Antibody Library

Molecular cloning of antibody repertoire in combinatorial phage display vector has turned out to be a powerfur technique for obtaining human monoclonal antibody genes and probing the antibody immunoresponse against a given antigen .In this studis, On the basis of epitope guided selection,we explored a new mouse mAb humanization method-CDR3 directed phage antibody library.1. the variable region of the heavy chain (VH) and light chain (VL ) genes of an anti-human integrinανβ3 mAb E10 were cloned by RT-PCR. Sequencing analysis showed that the Vk and VH were derived from VKIII and IgG1 respectively.2. VH and VL genes of mAb E10 were ligated with human heavy chain CH1 and human CK genes.The chimeric Fab expression vector was constructed by cloning the chimeric heavy chain Fd and light chain genes into pComb3.Fab antibody were expressed on phage by superinfection with the helper phage VCSM13, indirect ELISA and competitive inhibition ELISA analysis demonstrated that expressed Fab antibody had binding activity with human integrinανβ3 specifically. 3.According to the original sequences of mAb E10, oligonucleotide primers containing the original CDR3 sequences of mAb E10 were synthesized. Through overlap PCR, the mouse VH and VK CDR3 were splice to human diverse Fd and K genes. then E10 chimeric Fd genes and human K chain genes were cloned into phagemid pComb3 and transformed into the E.Coli to construct a hybrid phage antibody library ,which is consisted of 2.1×106 colony forming units.Under the super-infection of helper phage VCSM13,the hybrid phage antibody titer is about 8.96×1014,about 80% clones possess light chain genes. After 4 round of panning against human integrinανβ3 , the eluted phages were 10-fold enriched.10 clones were found to be able to human integrin ανβ3 specifically. competitive inhibition ELISA studies showed that 3 clones had the same specifity as the parental mAb.The strongest anti-human integrin ανβ3 reactive B12 clone was sequenced,Comparison of the B12 light chain gene with Kabat database shows that the light chain is a member of VKIII family,the germ-line gene is DPK21 and JK4.the light genes encode 213 aa.4.the B12 clone light gene was paired as a template with human heavy chain Fd genes.and display on the filamentous phage ,forming one human phage display antibody library which contained 2×107 separate clones, the human phage antibody titer is about 2.14×1014,about 50% clones possess heavy chain Fd genes. After 3 round of panning against human integrin ανβ3 , indirect ELISA and competitive inhibition ELISA studies showed that 3 clones had the same specifity as the parental mAb.The strongest anti-human integrinανβ3 reactive D5 clone was sequenced,the DNA sequence reveals that the heavy chain Fd gene belonges to IgG1 subclass,and its variable region is derived form rearranged germ-line gene of 7-2+ and JH4. Our results demonstrated that through CDR3 directed phage antibody library epitope guided selection is a promising strategy in murine mAb humanization.