Use of combinatorial mutagenesis to select for multiply substituted human interleukin-3 variants with improved pharmacologic properties

A combinatorial mutagenesis strategy was used to create a collection of nea rly 500 variants of human interleukin 3 (IL-3), each with four to nine amin o acid substitutions clustered within four linear, nonoverlapping regions o f the polypeptide. The variants were secreted into the periplasm of Escheri chia coli and supernatants were assayed for IL-3 receptor-dependent cell pr oliferation activity. Sixteen percent of the variants, containing "region-r estricted" substitutions, retained substantial proliferative activity throu gh two rounds of screening. A subset of these was combined to yield variant s with substitutions distributed through approximately half of the polypept ide. With one exception, "half-substituted" variants exhibited proliferativ e activity within 3.5-fold of native IL-3. A subset of the "half-substitute d" variants was combined to yield "fully substituted" IL-3 variants having 27 or more substitutions. The combination of the substitutions resulted in a set of polypeptides, some of which exhibit increased proliferative activi ty relative to native IL-3. The elevated hematopoietic potency was confirme d in a methylcellulose colony-forming unit assay using freshly isolated hum an bone marrow cells. A subset of the multiply substituted proteins exhibit ed only a modest increase in inflammatory mediator (leukotriene C-4) releas e. The molecules also exhibited 40- to 100-fold greater affinity for the al pha subunit of the IL-3 receptor and demonstrated a 10-fold faster associat ion rate with the alpha-receptor subunit. The multiply substituted IL-3 var iants described in this study provide a unique collection of molecules from which candidates for clinical evaluation may be defined and selected. (C) 1999 International Society for Experimental Hematology. Published by Elsevi er Science Inc.