Prevention of Human Rhinovirus Infection by Multivalent Fab Molecules Directed against ICAM-1

Abstract

We have developed a technology for improving avidity by making bivalent, trivalent, or tetravalent recombinant polypeptides. We designed tripartite proteins consisting of the Fab fragment of an antibody fused with a hinge derived from human immunoglobulin D that was further linked to polymerization domains derived from human coiled-coil proteins. We report here on the application of this method with a Fab domain directed against the major human rhinovirus receptor, intercellular adhesion molecule 1 (ICAM-1). Multivalent anti-ICAM-1 molecules were produced in bacteria and purified as soluble preassembled homogeneous proteins at high yield. These proteins successfully blocked rhinovirus infection in vitro, with the efficiency increasing from monomer to dimer, trimer, and tetramer. The diminished dissociation rate of these multivalent antibodies and their improved efficacy in preventing rhinovirus infection provide a foundation for producing prophylactic and therapeutic molecules against human rhinovirus, the causative agent of the majority of common colds.Human rhinoviruses (HRVs) are the major cause of the common cold (23,24) and are among the most frequently occurring human pathogens. These viruses infect cells of the nasal epithelium by binding to cell surface receptors. On the basis of their cellular receptor specificities, the more than 100 HRV serotypes can be divided into two groups. The major group contains about 90% of all serotypes and uses intercellular adhesion molecule 1 (ICAM-1) as its receptor (25). A receptor-blocking approach has shown that anti-ICAM-1 monoclonal antibody (MAb) 1A6 prevents HRV infection of cells in vitro (3). In human clinical trials, the antibody diminished cold symptoms but failed to prevent onset of the disease (8). The limited efficacy of MAb 1A6 is most likely due to its low functional affinity (or avidity) for ICAM-1 compared to that of the multivalent HRV particles. Consistent with this interpretation was a study in which several MAbs against ICAM-1 were shown to dissociate from ICAM-1 at the same rate as HRV itself (i.e., they had equal dissociation rate constants [k off s]) (2).For over a decade, science has tried to improve on nature by using multivalency to increase the avidities of antibodies. The majority of these proteins were engineered by fusing the antigen-binding scFv fragment of an antibody with a polymerization domain, such as streptavidin (10), the yeast protein GCN4 leucine zipper domain (7, 18), or the tetramerization domain of p53 (18). Fab fragments of antibodies, however, generally exhibit significantly higher binding affinities than their corresponding scFv fragments. Because of the greater complexity of assembling multichain proteins, little work has been done on making trimeric and tetrameric Fab molecules. Our method involves the generation of tripartite proteins consisting of a humanized Fab, a linker sequence derived from the human immunoglobulin D (IgD) hinge (13), and a multimerization domain derived from either of the human transcription factors...

The Role of p38 MAPK in Rhinovirus-Induced Monocyte Chemoattractant Protein-1 Production by Monocytic-Lineage Cells

“…Previous studies have shown that anti-ICAM-1 Ab can effectively block HRV16 from binding to the ICAM-1 receptor (47,48). Monocytes were pretreated with two different concentrations of anti-ICAM-1 Ab for 30 min (10 and 15 g/ml) followed by 30 min of exposure to HRV16 at an MOI of 10, (Fig.…”

Rhinovirus and the developing lung

“…In theory, preventing HRV binding to ICAM1, the most common HRV receptor, would be an effective way of arresting HRV infection at an early stage. Experimental evidence using anti-ICAM1 antibodies showed early promise [113,114]. Likewise, capsid binders, such as pleconaril, have been shown to be potent anti-HRV agents experimentally via the inhibition of host cell attachment [11].…”

Adaptation of an ICAM-1-Tropic Enterovirus to the Mouse Respiratory Tract

“…We recapitulated this effect with monoclonal anti-ICAM-1 antibody RR1/1, previously used to block HRV binding in vitro (5). Pretreatment of HeLa H1 cells with RR1/1 resulted in an ϳ80-fold or ϳ4-fold reduction of progeny at 8 h postinfection with 40 nM or 4 nM antibody, respectively (Fig.…”