Construction and characterization of a fusion protein which mediates cytolysis of her2-overexpressing tumor cells by type i fc gamma receptor (fcjri)-expressing effector cells

Heregulin (HRG) is a ligand for the HER3 and HER4 molecules. Both of these receptors may form heterodimers with HER2, a molecule which is overexpressed in some breast cancer cells. The affinity of HRG for HER3 and HER4 dramatically increases when these molecules form heterodimers with HER2. We have constructed by genetic means a bispeciflc reagent which binds to the HER3 and HER4 molecules and to the high affinity receptor for the Fc portion of IgG, FcrRI, which is a cytotoxic trigger molecule expressed by myeloid cells. For this construction, genomic DNA encoding the Fd fragment of humanized anti-FcrRI mAb, H₂2, which binds FcyRI at an epitope that is distinct from the Fc binding site, was fused to cDNA encoding the EGF domain of the β2 form of HRG. The resulting H₂2Fd-HRG expressing vector was transfected into a myeloma cell line which was previously transfected with a vector containing DNA encoding the H₂2 kappa light chain. The resultant fusion protein was expressed predominantly as H₂2Fab-HRG monomers, even though there exists a free Cys residue in the hinge region of the H₂2 Fab component. Using flow cytometry It was found that this molecule was able to bind to the HER2 overexpresslng tumor cell line, SKBR-3, as well as to FcrRI-expressIng cells. This H₂2-HRG fusion protein can inhibit the growth of SKBR-3 tumor cells and can mediate fusion protein dependent cytotoxicity of these cells in the presence of FoyRI-bearlng cytotoxic effector cells. These results suggest that this fusion protein could mediate anti-tumor cytotoxic activities under physiologic conditions, and that it may have therapeutic utility.