A deletion variant of human interleukin-3, hIL-3 15-125 , was produced in the periplasmic space of Escherichia coli and had full activity in an AML193.1.3 cell proliferation assay. Libraries of random single-amino acid substitutions were constructed at each of 105 positions in the gene for hIL-3 . Approximately eight single-site substitutions at each position were produced in osmotic shock fractions and screened for activity. 15 mutants were found with bioactivity of 5-26-fold greater than that of native hIL-3. The majority of amino acids in hIL-3 15-125 could be substituted without substantial loss of activity. Substitution of residues predicted to be in the hydrophobic core of the protein often resulted in reduced activity and/or low accumulation levels. Only five residues predicted to be on the surface of the protein were intolerant of substitution and hence are candidates for sites of interaction with the receptor. We therefore propose that the majority of residues in hIL-3 serve a structural role and permit the display of a few key residues in the correct configuration for recognition by the receptor.Human interleukin-3 (hIL-3) 1 is a multilineage hematopoietic cytokine acting in the bone marrow to promote the growth of most lineages of blood cell precursors (1). Recently, exogenously administered hIL-3 has shown promise for the clinical relief of neutropenia and thrombocytopenia induced by cancer chemotherapy (2, 3). Sequence homology comparisons of hIL-3 with other proteins indicate that it is a member of the hematopoietic cytokine family (4 -6) and that it adopts a four-␣-helix bundle topology (7-10). The protein binds to a receptor comprising at least two nonidentical subunits (11, 12). Although the precise nature of interaction between hIL-3 and its receptor is not known, studies using site-specific mutants have shed some light on which portions of the protein are important for function (8,(13)(14)(15)(16)(17). In particular, mutagenesis of the adjacent helices A and D indicate that these regions are important for interaction with the receptor. This is similar to the findings for human interleukin-5 and human granulocyte-macrophage colony stimulating factor, whose receptors share a common ␤ subunit with the hIL-3 receptor (11,18,19). Other members of the hematopoietic cytokine family also have important residues in helices A and D (19 -23) and in helix C (20,22,24,25).In this paper we have undertaken an extensive mutagenesis of hIL-3 in order to discover mutants with enhanced proliferative activity and to define residues necessary for activity. Although alanine scanning mutagenesis has been successfully used to derive structure-activity information (20, 26 -32), we chose to perform a more extensive mutagenesis, permitting the incorporation of any of the possible 19 substitutions (33). MATERIALS AND METHODS Production of hIL-3 and Variants in the Escherichia coli Cytoplasm-General techniques for manipulation of DNA are described elsewhere (34). The hIL-3 gene (35) was obtained from British Biotechnolo...

Myelopoietins comprise a class of chimeric cytokine receptor agonists consisting of an hIL-3 (human interleukin-3) receptor agonist and an hG-CSF (human granulocyte colony-stimulating factor) receptor agonist linked head-to-tail at their respective carboxy and amino termini. The combination of an early acting cytokine (hIL-3) with a late acting one (hG-CSF) allows efficient hematopoeitic reconstruction following myeloablative insult, and drives differentiation of non-myelocytic lineages (ie thrombocytic lineages) that are inaccessible using hG-CSF alone, in both preclinical models and clinical settings. A myelopoietin species was displayed and mutagenized on filamentous bacteriophage: both component agonists of myelopoietin were presented in biologically functional conformations as each recognized its corresponding receptor. Five amino acid positions in a short region of the hG-CSF receptor agonist module of myelopoietin that had been identified as important for proliferative activity were mutagenized. Display was used because it allows very 'deep' mutagenesis at selected residues: Ͼ10 5 substitution variants were affinity-screened using the hG-CSF receptor and 130 new, active variants of myelopoietin were identified and characterized. None of the selected variants were significantly more active than the parental myelopoietin species in a hG-CSF-dependent cell proliferation assay, though many were as active. Many of these relatively high-activity variants contained parental amino acids at several positions, suggesting the parental sequence may already be optimal at these positions for the assays used, and potentially accounting for the failure to identify enhanced bioactivity variants. Analysis of substitutions of high-activity variants complements and extends previous alanine scanning, and other genetic and biochemical data for hG-CSF variants. Leukemia (2001) 15, 1277-1285.

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A deletion derivative of the cytokine human interleukin-3 (hIL-3(15-125), comprising amino acids 15-125 of the native protein) was produced as a fusion to the filamentous phage surface protein pIII. The cytokine was detected in association with phage particles by protein immunoblotting. Compared to an equivalent quantity of soluble-cytokine, phage-presented hIL-3(15-125) exhibited reduced biological activity in a hIL-3-dependent cell proliferation assay. The reduction in activity was attributable to presence of phage particles in the assay, rather than directly owing to physical incorporation of the cytokine into the phage particle. Owing to the position of the amber codon in the phagemid vector, the phagemid-produced free hIL-3(15-125) species (designated hIL-3(15-125) epsilon) had 20 amino acids appended to its C-terminus; hIL-3(15-125) epsilon did not exhibit reduced bioactivity. hIL-3(15-125)-presenting phage were affinity-selected with either a hIL-3-reactive polyclonal antibody or with cells expressing the heterodimeric hIL-3 receptor. These data are consistent with the use of phage-display technology for the affinity selection of hIL-3 variants with modified biological properties.

Here we describe a post-translational modification of SC-63032, a variant of the species restricted, multi-lineage hematopoeitic factor human interleukin-3 (hIL-3). We have made two new dendritic polymer (polyamidoamine or PAMAM dendrimers, generation 5)-SC-63032 bioconjugates. Using two distinct chemistries (one of which is novel to this work), we achieved site-specific conjugation with respect to the amino acid in the proteins ligated to the dendrimers. In both bioconjugates, conjugated cytokine maintains its ability to bind the hIL-3 alpha receptor subunit, but is significantly (about 10-fold) less potent in inducing hIL-3 dependent in vitro cell proliferation than is the free cytokine. In vivo data indicates that conjugation decreases the immunogenicity of the conjugated cytokine modestly. In the absence of pharmacokinetic or biodistribution effects associated with the bioconjugates that increase their potency in vivo (which can only be tested in a higher primate, due to the species restriction of hIL-3 and its derivatives), these immune mitigation effects may be too small to be therapeutically significant. Though unmodified PAMAM dendrimers fail to elicit an antibody response in mice, protein conjugation to dendrimers haptenizes them, and a dendrimer-specific antibody response is produced. In toto, the principal limitation of the dendrimer-cytokine bioconjugates herein is in their reduced receptor affinity and potency in vitro. Were the in vivo potency of the bioconjugates to parallel the in vitro potency of the conjugates reported here, it is likely that particular dendrimer bioconjugates could not justify their higher costs of goods relative to the parent SC-63032 molecule, though retention of SC-63032 biological activities in conjugates suggests that other cytokine-dendrimer bioconjugates may be bioactive. This is good news to the nanotechnology community, in as much as PAMAM dendrimers are among the monodisperse polymeric nanomaterials available, and these results show that they can be used successfully in conjugates to bioactive proteins.