We have previously used bispecific antibodies (bsAb) to redirect cytotoxic cells against tumor and other target cells and to study triggering mechanisms. In a collaboration with GenVec, we are developing a new application for bsAbs, the selective targeting of cells for adenovirus infection. A bsAb with anti-CD3 and anti-FLAG activity induces the infection of human T cell with adenovirus that contains a genetically introduced FLAG peptide and expresses - galactosidase as a reporter gene. T cells are not infected without the bsAb, both CD4 and CD8 T cells are infected, and CD3- cells are not infected. Other bsAbs are being produced to see which types of molecules on cell surfaces can be used to induce bsAb dependent infections. BsAb directed infection could prove important for gene therapy applications. We have produced single chain (sc) bsAbs by concatenating two scFvs. These molecules are secreted as active protein by mammalian cells, or are produced by bacteria and refolded in vitro. We have found that inclusion of a glycosylation site on an anti-DNP scFv enhances its rate of secretion, without affecting its stability. More recently we have found that in the ER, the glycosylation site increases the rate of protein folding as measured by the appearance of anti-DNP binding activity. Preliminary studies, in collaboration with Kelly Kearse, showed that the glycosylated form of the scFv binds calnexin. We will use this system to see if calnexin aids the scFv in gaining anti-DNP activity. In our studies of in vitro protein folding we found that proteins that are normally secreted require special procedures for producing a native molecule. To see how a cytoplasmic protein would fold in vitro, we, in collaboration with Charles Zacharchuk, expressed the cytoplasmic portion of bcl-2 as inclusion bodies in bacteria. We found that after denaturation with guanidine and dialysis, the bcl-2 protein adopted a biologically active form that consisted of 2 non-covalently interacting regions separated by a long, exposed, protease-sensitive loop. The hydrodynamic properties of bcl-2 suggest that its structure is similar to the recently published X- ray structure of bcl-x. This first example suggests that cytoplasmic proteins adopt their native configuration more readily than secreted ones. Formerly Z01 CB 10028-01 EIB.
