Biochemical and Biological Characterization of a Dodecameric CD4-Ig Fusion Protein

Abstract

Drug toxicities associated with HAART lend urgency to the development of new anti-HIV therapies. Inhibition of viral replication at the entry stage of the viral life cycle is an attractive strategy because it prevents de novo infection. Soluble CD4 (sCD4), the first drug in this class, failed to suppress viral replication in vivo. At least three factors contributed to this failure: sCD4 demonstrated poor neutralizing activity against most primary isolates of HIV in vitro; it demonstrated an intrinsic capacity to enhance viral replication at low concentrations; and it exhibited a relatively short half-life in vivo. Many anti-gp120 monoclonal antibodies, including neutralizing monoclonal antibodies also enhance viral replication at suboptimal concentrations. Advances in our understanding of the events leading up to viral entry suggest strategies by which this activity can be diminished. We hypothesized that by constructing a sCD4-based molecule that is large, binds multiple gp120s simultaneously, and is highly avid toward gp120, we could remove its capacity to enhance viral entry. Here we describe the construction of a polymeric CD4-IgG1 fusion protein. The hydrodynamic radius of this molecule is ϳ12 nM. It can bind at least 10 gp120 subunits with binding kinetics that suggest a highly avid interaction toward virion-associated envelope. This protein does not enhance viral replication at suboptimal concentrations. These observations may aid in the design of new therapeutics and vaccines.

Cryoelectron Tomographic Analysis of an HIV-neutralizing Protein and Its Complex with Native Viral gp120

“…We show here that D1D2-IgP, a multimeric CD4-immunoglobulin construct with extreme neutralization activity against primary isolates of HIV-1 (6,12), is heterogeneous in size and structure. Cryoelectron tomography studies reveal that the monomer units, or arms, of D1D2-IgP appear to have no preferred orientation, in contrast to those of IgM imaged under identical conditions.…”

“…D1D2-IgP is a multimeric CD4-immunoglobulin fusion construct with exemplary neutralization activity against primary isolates of HIV-1 (6,12). We imaged the three-dimensional structure of individual unfixed, unstained D1D2-IgP particles in vitreous ice using cryoelectron tomography.…”

“…The clustered CD4 may mediate an avidity effect, so that soluble CD4 competes poorly with target cell CD4 for viral gp120 (6). To mimic the clustering of cell surface CD4, Arthos et al (12) designed a multimeric CD4 construct, D1D2-Ig␣tp (which we abbreviate as D1D2-IgP, where "P" indicates the tendency of this construct to polymerize), achieving a similar avidity effect. D1D2-IgP is a multidomain protein that comprises the two extracellular N-terminal domains of human CD4, D1 and D2, fused to the hinge, C␥2, and C␥3 domains of human IgG1, fused in turn to the 18-amino acid human IgA ␣ secretory tailpiece.…”

“…Analytical ultracentrifugation and dynamic light scattering revealed that D1D2-IgP molecules are typically multimeric species that carry 6 -8 or more Ig 2 (CD4) 2 units (i.e. 12-14 CD4 units) and have sizes ranging from ϳ600 to ϳ1200 kDa, with a 12-nm average hydrodynamic radius (12). D1D2-IgP neutralizes minimally passaged primary clinical isolates of HIV-1 at Ͻ3 nM IC 90 values (6,12), lower than those reported of monoclonal antibodies, polyclonal sera, or other CD4 constructs (6,13,14).…”

“…It has been hypothesized that the steric bulk of D1D2-IgP, which is many times larger than a monoclonal antibody, prevents approach and binding of gp120 to target cell CD4 (12). However, the large mass of D1D2-IgP is also a disadvantage, as it would likely result in undesirable pharmacologic properties such as poor distribution and immunogenicity.…”