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

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 nearly 500 variants of human interleukin 3 (IL-3), each with four to nine amino acid substitutions clustered within four linear, nonoverlapping regions of the polypeptide. The variants were secreted into the periplasm of Escheri...

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Bibliographic Details
Published in:Experimental hematology Vol. 27; no. 12; pp. 1746 - 1756
Main Authors: Klein, Barbara K., Olins, Peter O., Bauer, S.Christopher, Caparon, Maire H., Easton, Alan M., Braford, Sarah R., Abrams, Mark A., Klover, Jon A., Paik, Kumnan, Thomas, John W., Hood, William F., Shieh, Jeng-Jong, Polazzi, Joseph O., Donnelly, Ann M., Zeng, David L., Welply, Joseph K., McKearn, John P.
Format: Journal Article
Language:English
Published: Netherlands Elsevier Inc 01-12-1999
Subjects:
Amino Acid Substitution
Escherichia coli
Hematopoietic cytokine
Humans
Interleukin 3
Interleukin-3 - chemistry
Interleukin-3 - genetics
Interleukin-3 - pharmacology
Leukotriene C 4
Mutagenesis
Protein Engineering
Structure-Activity Relationship
Protein engineering
Hematopoietic cytokine
Interleukin 3
Leukotriene C 4
Escherichia coli
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Summary:A combinatorial mutagenesis strategy was used to create a collection of nearly 500 variants of human interleukin 3 (IL-3), each with four to nine amino acid substitutions clustered within four linear, nonoverlapping regions of the polypeptide. The variants were secreted into the periplasm of Escherichia coli and supernatants were assayed for IL-3 receptor-dependent cell proliferation activity. Sixteen percent of the variants, containing “region-restricted” substitutions, retained substantial proliferative activity through two rounds of screening. A subset of these was combined to yield variants with substitutions distributed through approximately half of the polypeptide. With one exception, “half-substituted” variants exhibited proliferative activity within 3.5-fold of native IL-3. A subset of the “half-substituted” 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 activity relative to native IL-3. The elevated hematopoietic potency was confirmed in a methylcellulose colony-forming unit assay using freshly isolated human bone marrow cells. A subset of the multiply substituted proteins exhibited only a modest increase in inflammatory mediator (leukotriene C 4) release. The molecules also exhibited 40- to 100-fold greater affinity for the α subunit of the IL-3 receptor and demonstrated a 10-fold faster association rate with the α-receptor subunit. The multiply substituted IL-3 variants described in this study provide a unique collection of molecules from which candidates for clinical evaluation may be defined and selected.
ISSN:0301-472X
1873-2399
DOI:10.1016/S0301-472X(99)00118-6