ABSTRACT

We prepared a fusion protein containing a humanized monoclonal antibody (mAb), mAb H22, with specificity for the human high affinity Fc receptor for IgG (FcγRI, CD64), fused to HIV-1 gp120. This fusion protein construct was produced by joining the cDNA for the full length H22 heavy chain gene in frame to the cDNA for gp120. This construct, which also expressed a selectable marker, was stably transfected into a murine myeloma cell line that expressed the previously transfected H22 kappa light chain. The resulting fusion protein, (H22 x gp120), was secreted from the myeloma cell line and was purified by affinity chromatography. Flow cytometric analysis revealed that H22 x gp120 bound with high affinity via the Fab portion of H22 to CD64 expressed on monocytes and macrophages from both humans and human CD64- expressing transgenic mice. Western blot analysis revealed that the 390 kDa fusion protein reacted with both anti-human IgG and anti-gp120 mAbs. Incubation of a monocyte cell line with this fusion protein at 37°C resulted in internalization of the complex as determined by flow cytometric analysis. Immunization of human CD64 transgenic mice with the purified H22 x gp120 fusion protein induced higher titers of anti-gp120 serum antibodies compared to immunization of non-transgenic littermates. Targeting gp120 to CD64-expressing antigen presenting cells (APC) in vivo may augment immune responses and enhance protective immunity.