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.

FIG. 1.

Illustration of our conception of the tetrameric Fab anti-ICAM-1 molecule. A homology model of Fab19 was attached to a modeled representation of a hinge and multimerization domain to form a tetravalent Fab protein. Molecular models were constructed by using the programs Swiss Model and Swiss PDB Viewer (6). Each polypeptide chain is represented by a different color.

FIG. 2.

Competitive ELISA results. The graph shows the percent inhibition of tracer antibody binding as a function of the amount of each protein added to wells coated with soluble human ICAM-1. Triangles, CFY196; squares, CFY195; diamonds, Fab19.

FIG. 3.

Protection of HeLa cells from HRV infection by proteins that are multimers of anti-ICAM-1 Fab19. Percent protection was calculated as indicated in Materials and Methods, with 100% protection being equivalent to the staining of uninfected cells and 0% protection being equivalent to the staining of infected untreated cells. Circles, MAb RR1; triangles, CFY193B; squares, CFY202; diamonds, CFY196. The points represent mean values from one experiment in which the samples were tested in triplicate. The error bars represent standard deviations.

FIG. 4.

Reduction of viral titer in protected respiratory epithelial cells. Viral titers from HRV serotype 15-infected BEAS-2B cells before infection and 48 h after infection are shown. Each percentage is the mean from two independent experiments measured from cells pretreated with the indicated proteins. The error bars represent the standard deviations. A value of 100% corresponds to the titer of virus produced from infected cells with no protective pretreatment.