No abstract

SUMMARYThe type 2 helper T cell (T H 2) cytokine interleukin (IL)-4 is thought to play a central role in the early stages of asthma. In an effort to develop an antibody treatment for asthma that neutralizes the effects of IL-4, a murine monoclonal antibody, 3B9, was generated with specificity for human IL-4. In vitro studies demonstrated that 3B9 inhibited IL-4-dependent events including IL-5 synthesis, T H 2 cell activation and up-regulation of immunoglobulin E expression. 3B9 was then humanized (pascolizumab, SB 240683) to reduce immunogenicity in humans. SB 240683 demonstrated species specificity for both monkey and human IL-4 with no reactivity to mouse, rat, cow, goat or horse IL-4. Pascolizumab inhibited the response of human and monkey T cells to monkey IL-4 and effectively neutralized IL-4 bioactivity when tested against several IL-4-responsive human cell lines. Affinity studies demonstrated rapid IL-4 binding by pascolizumab with a slow dissociation rate. In vivo pharmacokinetic and chronic safety testing in cynomolgus monkeys demonstrated that pascolizumab was well tolerated, and no adverse clinical responses occurred after up to 9 months of treatment. Three monkeys developed an anti-idiotypic response that resulted in rapid pascolizumab clearance. However, in the chronic dosing study the antibody response was transient and not associated with clinical events. In conclusion, pascolizumab is a humanized anti-IL-4 monoclonal antibody that can inhibit upstream and downstream events associated with asthma, including T H 2 cell activation and immunoglobulin E production. Clinical trials are under way to test the clinical efficacy of pascolizumab for asthma.

Human interleukin 5 (hIL5) and soluble forms of its receptor alpha subunit were expressed in Drosophila cells and purified to homogeneity, allowing a detailed structural and functional analysis. B cell proliferation confirmed that the hIL5 was biologically active. Deglycosylated hIL5 remained active, while similarly deglycosylated receptor alpha subunit lost activity. The crystal structure of the deglycosylated hIL5 was determined to 2.6-A resolution and found to be similar to that of the protein produced in Escherichia coli. Human IL5 was shown by analytical ultracentrifugation to form a 1:1 complex with the soluble domain of the hIL5 receptor alpha subunit (shIL5R alpha). Additionally, the relative abundance of ligand and receptor in the hIL5.shIL5R alpha complex was determined to be 1:1 by both titration calorimetry and SDS-polyacrylamide gel electrophoresis analysis of dissolved cocrystals of the complex. Titration microcalorimetry yielded equilibrium dissociation constants of 3.1 and 2.0 nM, respectively, for the binding of hIL5 to shIL5R alpha and to a chimeric form of the receptor containing shIL5R alpha fused to the immunoglobulin Fc domain (shIL5R alpha-Fc). Analysis of the binding thermodynamics of IL5 and its soluble receptor indicates that conformational changes are coupled to the binding reaction. Kinetic analysis using surface plasmon resonance yielded data consistent with the Kd values from calorimetry and also with the possibility of conformational isomerization in the interaction of hIL5 with the receptor alpha subunit. Using a radioligand binding assay, the affinity of hIL5 with full-length hIL5R alpha in Drosophila membranes was found to be 6 nM, in accord with the affinities measured for the soluble receptor forms. Hence, most of the binding energy of the alpha receptor is supplied by the soluble domain. Taken with other aspects of hIL5 structure and biological activity, the data obtained allow a prediction for how 1:1 stoichiometry and conformational change can lead to the formation of hIL5.receptor alpha beta complex and signal transduction.

Cassette mutagenesis was used to identify side chains in human interleukin 5 (hIL-5) that mediate binding to hIL-5 receptor a chain . A series of single alanine substitutions was introduced into a stretch of residues in the C-terminal region, including helix D, which previously had been implicated in receptor ca chain recognition and which is aligned on the IL-5 surface so as to allow the topography of receptor binding residues to be examined. hIL-5 and single site mutants were expressed in COS cells, their interactions with hIL-5Ra were measured by a sandwich surface plasmon resonance biosensor method, and their biological activities were measured by an IL-5-dependent cell proliferation assay. A pattern of mutagenesis effects was observed, with greatest impact near the interface between the two four-helix bundles of IL-5, in particular at residues , and least at the distal ends of the D helices. This pattern suggests the possibility that residues near the interface of the two four-helix bundles in hIL-5 comprise a central patch or hot spot, which constitutes an energetically important a chain recognition site. This hypothesis suggests a structural explanation for the 1:1 stoichiometry observed for the complex of hIL-5 with hIL-5Ra.Interleukin 5 (IL-5) plays a central role in the control of eosinophilia and as such is a major contributor to the tissue damage seen in asthma and other eosinophil-related disorders (1-4). The high-resolution crystallographic structure of human IL-5 (hIL-5) has been determined (5, 6). It contains a core of two four-helix bundles in the IL-5 dimer, with each of the four-helix units similar to that in other cytokines (7-11). However, the arrangement of bundles in IL-5 is unusual in that helix D of one monomer combines with helices A, B, and C of the second monomer and vice versa.The hIL-5 receptor is composed of two types of subunits, denoted a and ,3 (hIL-SRa and -13) (12). observed (6, 14).The mechanism of IL-5 recognition of receptor a chain and the structural origin of 1:1 stoichiometry have formed a major focus for our mechanistic studies of hIL-5R recognition. In this study, we chose to investigate the effects of mutagenesis of residues in the C-terminal region of hIL-5 that includes helix D. This was based on a previous report (19) that used data from hybrid molecules of mouse-human IL-5 to suggest that the C-terminal region interacts directly with IL-5Rca and confers the species specificity of IL-5. We made sequence changes over a stretch of surface that extends from the distal ends of the two four-helix bundles of the IL-5 dimer inward to the interface between the bundles. This mutagenesis series allowed us to investigate both the importance of specific residues in the C-terminal region and the overall topography of the receptor recognition site on IL-5. The results show that the greatest impact of mutagenesis on receptor binding occurs for residues close together at the bundle interface, in particular Glu-110 and Trp-111. This observation suggests that a central reco...