Efficient in vitro affinity maturation of phage antibodies using BIAcore guided selections

Abstract

Selection of higher affinity mutant phage antibodies has proven less than straightforward due to sequence dependent differences in phage antibody expression, toxicity to Escerichia coli, and difficulty in eluting the highest affinity phage. These differences lead to selection for increaseid levels of expression or decreased toxicity rather than for higher affinity. In this work, we demonstrate how surface plasmon resonance as employed in the BIAcore can be used to increase the efficiency of phage antibody 1elections, yielding greater increments in affinity from a single library. A mutant phage antibody library WaSf’ rea ted by randomizing nine amino acids located in the VL CDR3 of C6.S, a human scFv which binds the tumor antigen c-erbB-2 with a Kd of 1.6 X 10-8 M. The library was subjected to five rounds of selection in sol tion using decreasing concentrations of biotinylated c-erbB-2. After each round of selection, polyclonal phag. were prepared and the rate of binding to c-erbB-2 determined in a BIAcore under mass transport limited condiltions. Determination of the rate of binding permitted calculation of the concentration, and hence percent, of binding phage present. Results were used to select the antigen concentration for the next round of selection. Tol determine the optimal eluent, polyclonal phage was injected in a BIAcore and eluted using one offive different solutions (10 mM HCI, SO mM HCI, 100 mM HCI, 100 mM triethylamine, 2.6 M MgCI2). Differences were observed in eluent efficacy, which was reflected in significant differences in the affinities of phage antibodies isolated from the library after a round of selection using the different eluents. Use of the BIAcore to determine the optimal eluent and guide the antigen concentration used for selection yielded a C6.S mutant with a 16 fold reduction in Kd (Kd = 1.0 x 10-9 M). This represents at least a twofold greater increment in affinity JhanpreviouFly obtainedfrom a single library of phage antibodies which bind antigens. [Hum Antibod Hybridomas 1996; 7: 97-105]. © 1996, IOS Press. All rights reserved.