Mapping and characterization of the epitope(s) of Sch 55700, a humanized mAb, that inhibits human IL-5.

mAb against human IL-5 inhibit pulmonary eosinophilia, tissue damage and airway hyper-reactivity in allergic animal models. Sch 55700 is a humanized, neutralizing anti-IL-5 antibody. To better understand the molecular mechanism by which Sch 55700 blocks IL-5 bioactivity, we have mapped its epitope by scanning IL-5 with synthetic peptides. Those peptides containing a region, ERRRV, corresponding to amino acids 89-93 of IL-5 specifically interact with both Sch 55700 and its parental rat IgG, 39D10. Among the five residues of this region, all three arginine residues were particularly critical for interaction of these peptides with Sch 55700. We further characterized this region by alanine scanning using site-directed mutagenesis. Examination of COS-expressed IL-5 mutants by Western blot showed that single mutations of E(89), R(90), R(91) or R(92) to alanine caused a loss of IL-5 binding to both Sch 55700 and 39D10. We further demonstrated in surface plasmon resonance studies using a BIAcore biosenosor that E(89), R(90) or R(91) are involved in the interaction between IL-5 and its receptor alpha subunit. Based upon the findings here and previously reported structures of the IL-5 and 39D10 variable region, we propose a model suggesting that the molecular interactions between the IL-5 and Sch 55700 mainly involve several ion pair interactions. We conclude that Sch 55700 occupies a region, ERRR, on IL-5 that is essential for its interaction with the receptor and thereby blocks IL-5 bioactivity.

Molecular views of viral polyprotein processing revealed by the crystal structure of the hepatitis C virus bifunctional protease-helicase.

BACKGROUND: Hepatitis C virus (HCV) currently infects approximately 3% of the world's population. HCV RNA is translated into a polyprotein that during maturation is cleaved into functional components. One component, nonstructural protein 3 (NS3), is a 631-residue bifunctional enzyme with protease and helicase activities. The NS3 serine protease processes the HCV polyprotein by both cis and trans mechanisms. The structural aspects of cis processing, the autoproteolysis step whereby the protease releases itself from the polyprotein, have not been characterized. The structural basis for inclusion of protease and helicase activities in a single polypeptide is also unknown. RESULTS: We report here the 2.5 A resolution structure of an engineered molecule containing the complete NS3 sequence and the protease activation domain of nonstructural protein 4A (NS4A) in a single polypeptide chain (single chain or scNS3-NS4A). In the molecule, the helicase and protease domains are segregated and connected by a single strand. The helicase necleoside triphosphate and RNA interaction sites are exposed to solvent. The protease active site of scNS3-NS4A is occupied by the NS3 C terminus, which is part of the helicase domain. Thus, the intramolecular complex shows one product of NS3-mediated cleavage at the NS3-NS4A junction of the HCV polyprotein bound at the protease active site. CONCLUSIONS: The scNS3-NS4A structure provides the first atomic view of polyprotein cis processing. Both local and global structural rearrangements follow the cis cleavage reaction, and large segments of the polyprotein can be folded prior to proteolytic processing. That the product complex of the cis cleavage reaction exists in a stable molecular conformation suggests autoinhibition and substrate-induced activation mechanisms for regulation of NS3 protease activity.

Effect of Sch 55700, a humanized monoclonal antibody to human interleukin-5, on eosinophilic responses and bronchial hyperreactivity.

This report describes the development and the biology of Sch 55700, a humanized monoclonal antibody to human IL-5 (hIL-5). Sch 55700 was synthesized using CDR (complementarity determining regions) grafting technology by incorporating the antigen recognition sites for hIL-5 onto consensus regions of a human IgG4 framework. In vitro, Sch 55700 displays high affinity (Kd = 20 pmol/l) binding to hIL-5, inhibits the binding of hIL-5 to Ba/F3 cells (IC50 = 0.5 nmol/l) and blocks IL-5 mediated proliferation of human erythroleukemic TF-1 cells. In allergic mice, Sch 55700 (0.1-10 mg/kg, i.p. or i.m.) inhibits the influx of eosinophils in the lungs, demonstrates long duration of activity and the anti-inflammatory activity of this compound is additive with oral prednisolone. In allergic guinea pigs, Sch 55700 (0.03-30 mg/kg i.p.) inhibits both the pulmonary eosinophilia and airway hyperresponsiveness and at 30 mg/kg, i.p. inhibited allergic, but not histamine-induced bronchoconstriction. In allergic rabbits, Sch 55700 blocks cutaneous eosinophilia. Sch 55700 (0.1-1 mg/kg i.p.) also blocks the pulmonary eosinophilia and neutrophilia caused by tracheal injection of hIL-5 in guinea pigs. In allergic cynomolgus monkeys, a single dose of Sch 55700 (0.3 mg/kg i.v.) blocks the pulmonary eosinophilia caused by antigen challenge for up to six months. Sch 55700 is, therefore, a potent antibody against IL-5 in vitro and in a variety of species in vivo that could be used to establish the role of IL-5 in human eosinophilic diseases such as asthma.

A novel recombinant single-chain hepatitis C virus NS3-NS4A protein with improved helicase activity.

Hepatitis C virus (HCV) nonstructural protein 3 (NS3) has been shown to possess protease and helicase activities and has also been demonstrated to spontaneously associate with nonstructural protein NS4A (NS4A) to form a stable complex. Previous attempts to produce the NS3/NS4A complex in recombinant baculovirus resulted in a protein complex that aggregated and precipitated in the absence of nonionic detergent and high salt. A single-chain form of the NS3/NS4A complex (His-NS4A21-32-GSGS-NS3-631) was constructed in which the NS4A core peptide is fused to the N-terminus of the NS3 protease domain as previously described (Taremi et al., 1998). This protein contains a histidine tagged NS4A peptide (a.a. 21-32) fused to the full-length NS3 (a.a. 3-631) through a flexible tetra amino acid linker. The recombinant protein was expressed to high levels in Escherichia coli, purified to homogeneity, and examined for NTPase, nucleic acid unwinding, and proteolytic activities. The single-chain recombinant NS3-NS4A protein possesses physiological properties equivalent to those of the NS3/NS4A complex except that this novel construct is stable, soluble and sixfold to sevenfold more active in unwinding duplex RNA. Comparison of the helicase activity of the single-chain recombinant NS3-NS4A with that of the full-length NS3 (without NS4A) and that of the helicase domain alone suggested that the presence of the protease domain and at least the NS4A core peptide are required for optimal unwinding activity.

Construction, expression, and characterization of a novel fully activated recombinant single-chain hepatitis C virus protease.

Efficient proteolytic processing of essential junctions of the hepatitis C virus (HCV) polyprotein requires a heterodimeric complex of the NS3 bifunctional protease/helicase and the NS4A accessory protein. A single-chain recombinant form of the protease has been constructed in which NS4A residues 21-32 (GSVVIVGRIILS) were fused in frame to the amino terminus of the NS3 protease domain (residues 3-181) through a tetrapeptide linker. The single-chain recombinant protease has been overexpressed as a soluble protein in E. coli and purified to homogeneity by a combination of metal chelate and size-exclusion chromatography. The single-chain recombinant protease domain shows full proteolytic activity cleaving the NS5A-5B synthetic peptide substrate, DTEDVVCCSMSYTWTGK with a Km and k(cat) of 20.0 +/- 2.0 microM and 9.6 +/- 2.0 min(-1), respectively; parameters identical to those of the authentic NS3(1-631)/NS4A(1-54) protein complex generated in eukaryotic cells (Sali DL et al., 1998, Biochemistry 37:3392-3401).

Human interleukin 4 receptor complex: neutralization effect of two monoclonal antibodies.

The interaction of human interleukin 4 with the extracellular domain of its receptor alpha-subunit (shuIL-4R alpha) was characterized in studies utilizing chemical cross-linking, size exclusion chromatography, and Western blot analysis. A 1:1 stoichiometric complex could be demonstrated over a wide range (0.04-2.7) of ligand-receptor concentration ratios. It could also be cross-linked with bifunctional reagents containing a minimum chain length of eight methylene residues or the equivalent (11.4 angstroms). Using surface plasmon resonance, (SPR) technology, we established the high-affinity of human interleukin 4 (huIL-4) to shuIL-4R alpha which was immobilized on a BIAcore sensor chip (K(d) = 46 pM). The mechanisms of action of neutralizing monoclonal antibodies (Mab) 25D2 and 35F2 [Abrams et al. (1991) U.S. Patent 5,041,381; Ramanathan et al. (1990) in Advances in Gene Technology: The Molecular Biology of Immune Diseases and the Immune Response (Streilein, J. W., et al., Eds.) p 163, IRL Press, Oxford; DeKruyff et al. (1989) J. Exp. Med. 170, 1477-1493] were subsequently evaluated on the basis of their interaction with huIL-4 in the presence of shuIL-4R alpha. SPR studies showed that Mab 25D2 binds to huIL-4 and reduces its affinity for shuIL-4R alpha by 54-fold. Formation of a ternary complex between Mab 25D2 and the huIL-4/shuIL-4R alpha complex was demonstrated in size exclusion chromatography experiments. In contrast, Mab 35F2 which also binds huIL-4 failed to form a stable ternary complex with huIL-4 and shuIL-4 alpha during size exclusion chromatography. SPR studies supported this finding and showed that the interactions of Mab 35F2 and shuIL-4R alpha to huIL-4 are mutually exclusive. These data are consistent with results of previous epitope mapping studies showing that Mabs 25D2 and 35F2 bind to huIL-4 at two different sites [Ramanathan et al. (1993) Biochemistry 32, 3549-3556]. Together, the results suggest that Mab 25D2 binds to a domain in huIL-4 including helix D and exerts its inhibitory effect through a dual action. It decreases the affinity of huIL-4 for huIL-4R alpha and potentially blocks interaction with a secondary receptor subunit such as the IL-2R gamma [Reusch et al. (1994) Eur. J. Biochem. 222, 491-499]. Mab 35F2 operates through a direct and simpler mechanism, binding to helix C and inhibiting huIL-4 activity by sterically excluding all interaction with huIL-4R alpha.

Characterization of recombinant human interleukin 4 receptor from CHO cells: role of N-linked oligosaccharides.

Interleukin 4 (IL-4) mediates its biological activities through interaction with its receptor on the cell surface. A recombinant extracellular domain of the alpha subunit of human interleukin 4 receptor was expressed in CHO cells and purified to homogeneity by a combination of ion exchange and immunoaffinity chromatography. Analysis of the purified protein by MALDI MS provided an average mass of 38,241 Da while microsequencing identified the site of the signal sequence processing to be Ser23-Gly24. The receptor was highly glycosylated, containing N-linked complex oligosaccharides with bi-, tri-, and tetraantennary structures. Five of the six potential glycosylation sites could be assigned to Asn residues 53, 98, 128, 134 and 176. N-deglycosylation increased aggregation and reduced solubility of the receptor but did not affect its IL-4 binding activity. These observations provide preliminary insights into the role of N-linked oligosaccharides in IL-4 receptor biosynthesis and function at the cell surface.

Synthetic mimics of juxtaposed amino- and carboxyl-terminal peptide domains of human gamma interferon block ligand binding to human gamma interferon receptor.

The epitopes of two neutralizing antibodies (47N3-6 and 47N30A35) raised against rhuIFN-gamma each mapped both to amino-terminal regions (22-29 and 12-19, respectively) and to a carboxyl-terminal region 131-139, suggesting the juxtaposition of these two domains in the native protein. Three novel peptides were designed to mimic a conformation of rhuIFN-gamma that places the two regions in close proximity (discontinuous peptides 1 (15-21-GGG-132-138), 2 (15-29...111-118...130-138), and 3 (15-21-CGPGC-130-138)), by bridging the amino- and carboxyl-terminal regions of gamma interferon. Each discontinuous peptide inhibits biological or receptor binding activities with an IC50 of 15-50 microM and produces a neutralizing antibody when used as an immunogen. Neutralizing rabbit polyclonal antibody (P616) raised against discontinuous peptide 1 was used as immunogen to generate an anti-idiotypic response. This anti-idiotypic antibody inhibits receptor binding and recognizes soluble gamma interferon receptor on direct enzyme-linked immunosorbent assay. The anti-idiotypic response suggests that juxtaposed regions at the amino and carboxyl termini serve as the receptor-ligand binding site of human gamma interferon.

Transient optical spectroscopy of single crystals of the reaction center from Rhodobacter sphaeroides wild-type 2.4.1.

The photoactivity of the crystallized reaction centers from Rhodobacter sphaeroides wild-type strain 2.4.1 has been examined by light-induced absorption spectral changes associated with charge separation and triplet state formation in the reaction center. Upon excitation of a crystal at ambient redox potential, the primary donor 865 nm band bleaches reversibly. The kinetics of its recovery were found to be biphasic with rate constants 11.5 +/- 1.3 s-1 and 0.9 +/- 0.4 s-1 which correspond to lifetimes of 87.0 +/- 9.0 ms and 1.0 +/- 0.7 s, respectively. The ratio of the fast-to-slow component preexponential terms was 3.5 +/- 1.1 suggesting that the majority (78.9 +/- 13.0%) of the reaction centers in the crystals lack the secondary quinone, QB. The addition of sodium ascorbate to the crystals attenuates the 865 nm absorption change, and gives rise to strong carotenoid triplet-triplet absorption changes at 547 nm. These data indicate that the reaction center-bound carotenoid in the crystals is capable of accepting triplet energy from the primary donor triplet.

Linear dichroism of single crystals of the reaction center from Rhodobacter sphaeroides wild type strain 2.4.1.

The linear dichroism of single crystals of the photochemical reaction center from Rhodobacter sphaeroides 2.4.1, expressed as the anisotropy (or polarization) ratio, p = (A ‖ - A ⊥)/A ‖ + A ⊥, relative to the long morphological axis of the crystals, has been measured to be -0.12±0.03 for the primary donor Q y and -0.15±0.8 for the carotenoid. These dichroic effects can be predicted using data obtained from magnetophotoselection (Frank et al. 1979, McGann and Frank 1985) and electron spin resonance (ESR)(Frank et al. 1988a, Budil et al. 1988) experiments. Magnetophotoselection data yield the projections of the transition moments onto the primary donor triplet state principal magnetic axis system. The single crystal triplet state ESR experiments provide the Euler matrix for the transformation from the principal magnetic axis system to the crystal unit cell axis system. Thus, the projections of the transition moments (site 1) onto the crystal units cell axes (a, b, c) are determined to be-0.39, 0.90 and 0.18, respectively. The projections of the carotenoid transition moment (site 1) onto the crystal unit cell axes (a, b, c) are determined to be -0.60, 0.02 and 0.80, respectively. This information used in conjunction with the crystalline space group symmetry (P212121) and the morphology of the crystals allows one to predict the observed anisotropy ratios.

Crystallization and preliminary X-ray and optical spectroscopic characterization of the photochemical reaction center from Rhodobacter sphaeroides strain 2.4.1.

The photochemical reaction center from Rhodobacter sphaeroides 2.4.1 has been crystallized. The crystals were obtained in a solution of beta-octylglucoside by the vapor diffusion technique using polyethylene glycol 4000 as the precipitant at 22 degrees C. The orthorhombic crystals (space group P2(1)2(1)2(1)) have cell constants a = 142.5 A, b = 136.1 A, c = 78.5 A, and diffract to 3.7 A. The crystals display pronounced linear dichroism in the carotenoid absorption spectral region.