Functional expression of the CD4 protein after cross-linking to red blood cells with a bifunctional reagent

We used a bifunctional reagent for the design of a new therapeutic agent constructed by cross-linking a soluble form of the CD4 protein to red blood cell membranes. CD4 is a member of the immunoglobulin gene superfamily and is the receptor for the AIDS virus, HIV. We produced soluble CD4 in eucaryotic cells transfected with a soluble CD4 expression vector, and purified it by cation-exchange chromatography. Flow cytometry studies demonstrated that CD4-coated red blood cells were specifically stained with an anti-CD4 monoclonal antibody, whereas intact red blood cells and intermediates obtained during the coupling procedure were not stained. By comparison, with CD4+ lymphoid cells, the number of soluble CD4 molecules per CD4-expressing red blood cells was estimated to be approx. 100,000. We present evidence that, unlike the classical chromium chloride coupling method, large amounts of soluble CD4 were efficiently and uniformly coupled to RBCs. CD4 red blood cells bind specifically HIV particles, and inhibit the binding of HIV particles to target cells, the initial step of HIV life cycle. The anti-HIV activity of CD4-bearing red blood cells was found to be at least 20-times higher than that of free soluble CD4. Our results demonstrate that proteins can be efficiently coupled to red blood cells using bifunctional reagents. They also suggest that CD4-coated RBC are promising CD4-based anti-HIV agents.