NEUTRALIZING MURINE MONOCLONAL-ANTIBODIES TO HUMAN IL-5 ISOLATED FROMHYBRIDOMAS AND A FILAMENTOUS PHAGE FAB DISPLAY LIBRARY

Conventional hybridomas and combinatorial Ab libraries were used to de velop neutralizing murine mAbs to human IL-5. Mice were immunized with rlL-5. Spleens from two mice were used to generate hybridomas. Spleen s from an additional three mice were used to construct a combinatorial library. In both instances, Abs were identified and selected by ELISA using 96-well plates coated with rlL-5. These Abs were tested for the ability to block binding of iodinated rIL-5 to the ac-chain of the hu man IL-5 receptor (IL-5R alpha) and to inhibit proliferation of IL-5-d ependent cells. By hybridoma technology, 16 mAbs were obtained, 11 of which blocked binding to IL-5R alpha, including three that inhibited p roliferation. Quantitative binding assays and sequence analysis reveal ed that these latter three mAbs were closely related. Combinatorial cl oning and selection by phage display was used to isolate 24 bacterial colonies secreting Fabs that bound to I-125-rIL-5 and to rIL-5-coated plates. Sequencing of 10 of the Fabs indicated that four unique Abs we re obtained, comprising one predominant V-H paired with one of two dif ferent V-L. The sequence of the Fabs was distinct from the sequences o f the neutralizing mAbs. In contrast to the mAbs, none of the Fabs blo cked binding of I-125-IL-5 to IL-5R alpha or neutralized the biologic activity of IL-5. The inability to identify neutralizing Fabs was show n not to result from their monovalency, because a Fab derived from one of the neutralizing mAbs, by cloning and expression of its Fd and kap pa light chains, retained neutralizing activity. By chain shuffling, p airing of the Fd fragment of the heavy chain of one of the neutralizin g mAbs (2B6), with the light chain library derived from the IL-5-immun ized mice, neutralizing Fabs were obtained. These Fabs contained light chain sequences closely related to the original light chain of 2B6. H ence, chain shuffling allowed detection of a light chain sequence that was not evident upon two-chain combinatorial selection. The results r eveal differences in the Abs obtained from a combinatorial library vs hybridomas and demonstrate how these approaches can be used in concert to select mAbs with neutralizing activity.