Avidin/Biotin-Mediated Conjugation of Antibodies to Erythrocytes: An Approach for in vivo Immunoerythrocyte Exploration

Summary

Antibody-carrying red blood cells (immunoerythrocytes) may be useful as a vehicle for drug targeting and for selective elimination of antigens from the bloodstream. Such immunoerythrocytes have to circulate in the bloodstream for a prolonged time without elimination and possess high affinity to the target antigen. Streptavidin/biotin technology is useful for attachment of biotinylated antibodies (b-Ab) to biotinylated red blood cells (b-RBC). Modification of RBC with biotin hydroxysuccinimide ester provides covalent coupling of biotin residues to amino groups on the RBC membrane. b-RBC are stable in serum in vitro and in the bloodstream. Binding of streptavidin (SA) to b-RBC, however, leads to their lysis by complement in vitro and fast elimination from circulation. The mechanism of lysis is streptavidin-induced cross-linking of membrane regulators of complement. Reduction of the surface density of biotin residues on RBC membrane prevents lysis. Surface density of biotin residues on RBC may be regulated by varying biotin ester concentration during RBC biotinylation (b_n RBC). SA specifically binds to b_n-RBC (e.g., 10⁵ SA molecules per rat RBC biotinylated with 20 μM biotin, b₂₀-RBC). Modest and intermediate degrees of RBC biotinylation provide effective attachment of b-IgG to streptavidin-coated b-RBC (SA/b-RBC). SA/b₂₀-RBC bind 5 × 10⁴ b-IgG molecules per cell. In contrast, SA attached to extensively biotinylated RBC (b₇₀₀-RBC) does not bind biotinylated IgG. b-Ab/SA/b₂₀-RBC are stable in serum and bind to immobilized antigen in vitro. ⁵¹Cr-labeled serum-stable b-Ab/SA/b₂₀-RBC circulate without marked elimination and/or lysis in the bloodstream after iv. injection in rats. Biodistribution of b-Ab/SA/b₂₀-RBC in rats is similar to that of control RBC. Therefore, b-Ab/SA/b₂₀-RBC satisfy major requirements of a vehicle for drug targeting (i.e., high affinity to the target and biocompatibility) and can be explored in vivo.