Tailoring in vitro selection for a picomolar affinity human antibody directed against XXXX receptor 2 for enhanced neutralizing activity.

@entity1 @entity462 @entity7 @entity5 @entity319 @entity1778 @entity1334 @entity3
@entity7 ( @entity7 ) and its receptors have been implicated in promoting solid @entity5 growth and @entity3 via stimulating @entity5 -associated angiogenesis. We previously identified several fully human neutralizing anti- @entity7 receptor 2 (or @entity319 ( @entity319 )) antibodies from a large antibody phage display library. These antibodies bind specifically to @entity319 , block @entity7 / @entity319 interaction, and inhibit @entity7 -induced proliferation of @entity1 endothelial cells and migration of @entity319 + @entity1778 cells. Three of these antibodies, interestingly, share an identical heavy chain variable (VH) sequence. In this report, we constructed a new library comprising the single VH paired with the variable light chain (VL) repertoire obtained from the original na  ve @entity1 library. Initial in vitro selection revealed that the single VH could pair with a number of different VL while retaining its specificity for @entity319 . However, a consensus VH/VL pair, clone 1121, was identified after three or four rounds of selection by tailoring the stringency of the panning conditions. Clone 1121 showed a >30-fold higher binding affinity to @entity319 (Kd, 100 pm) because of improvement on both association and dissociation constants and blocked @entity7 / @entity319 interaction with an IC50 of approximately 1 nm, compared with that of 3-4 nm for the parent @entity1334 fragments. Further, clone 1121 was more potent in inhibiting @entity7 -stimulated @entity319 phosphorylation in endothelial cells. A binding epitope mapping study on clone 1121 and one of the parent clones, 2C6, demonstrated that both antibodies interacted with the third immunoglobulin domain within the extracellular region of @entity319 . Several peptide phage display libraries were utilized to further examine the fine binding specificities of the two antibodies. All of the 2C6-binding peptides are @entity462 -constrained, whereas clone 1121 binds to both @entity462 -constrained and linear peptides. It is noteworthy that most of the 2C6-binding peptides also cross-react with clone 1121, but none of the clone 1121-specific peptides binds to 2C6, indicating that clone 1121 retained part of the original binding epitope(s) of 2C6 while gaining new binding specificity. Taken together, our observation suggests that clone 1121 may have great clinical potential in anti-angiogenesis therapy. It further underscores the efforts to identify antibodies of high affinity for enhanced biological activities.
