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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...

Human rhinoviruses are the major causative agents of the common cold. Because there are greater than 100 viral serotypes, little immunological protection is afforded to humans by prior rhinovirus exposure, which accounts for the high incidence of infection. In most cases, rhinovirus leads to a short self-limiting illness. However, for asthmatics, the elderly and immunocompromised patients, rhinovirus infection can lead to life-threatening complications. This has spurred a consistent effort over recent decades to identify effective treatments and preventions for rhinovirus infection. While some work has focused on alleviating the symptoms induced as a result of inflammatory pathways stimulated by rhinoviruses, the majority of the research has been focused on limiting or preventing viral infection altogether. Various approaches have been taken to halt rhinovirus infection. Prevention of virus-cell interaction has been the aim of research on viral capsid binders and cell receptor blockers. Interference with correct viral protein processing is the goal of the design and testing of protease inhibitors. Current work is attempting to interfere with viral RNA replication by testing silencing RNA molecules. In this review, we will discuss recent advances in the development and testing of human rhinovirus therapeutics.

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