Generation and selection of a post-alloHSCT human Fab library by phage display. (A) By using a total of 186 different and separate combinations, human Vκ, Vλ, and VH encoding sequences were amplified by reverse transcription PCR from alloHSCT (after 6m) PBMCs derived from patient A, assembled into Vκ-Cκ-VH and Vλ-Cλ-VH cassettes by 3-fragment overlap extension PCR, and cloned into phagemid pC3C by asymmetric SfiI ligation. The resulting post-alloHSCT human Fab library was subsequently converted from phagemid to phage by transformation of E coli and the addition of helper phage. (B) The post-alloHSCT human Fab library was selected by 3 rounds of panning on PBMCs derived from patient α with untreated B-CLL (round 2, round 3, and round 5). Two additional rounds of panning on immobilized rat anti-HA mAb were carried out (round 1 and round 4). The steps of 1 cell panning round are shown.
