Monoclonal Antibody FC-5.01,Directed Against CD63 Antigen,is Internalized into Cytoplasmic Vesicles in the IIB-BR-G Human Breast Cancer Cell Line

Monoclonal antibody (MAb) FC-5.01, raised against the undifferentiated breast cancer cell line IIB-BR-G, has been recently shown to react with CD63. The antigen (Ag) recognized by MAb FC-5.01 is expressed in plasma membranes of IIB-BR-G and other neoplastic cells, as well as in activated platelets and endothelial cells, as detected by indirect immunofluorescence performed at 4°C on live cells. In permeabilized cells, MAb FC-5.01 colocalizes with acridine orange in acidic vesicles (lysosomal/endosomal compartment). Scatchard plot analysis performed on IB-BR-G cells demonstrated a 1.4 ± 0.4 × 10⁷ M^-1 affinity constant and 2.1 × 10⁶ antigenic sites per cell. MAb FC-5.01 is not able to mediate C fixation or ADCC toward CD63⁺ cells, but the FC-5.01-CD63 complex is efficiently internalized into cytoplasmic vesicles, as shown by an acid wash immunofluorescence assay. Cellular catabolism of the antibody bound by IIB-BR-G cells was studied using [¹²⁵I]-FC-5.01. At 18 h, >70% of the radioactivity was present in the supernatant as degraded fragments (TCA-soluble). After internalization, rapid Ag re-expression could be demonstrated in IIB-BR-G cells. MAb FC-5.01 diminished migration of CD63⁺ cells in a Boyden chamber assay. Some of the above-mentioned properties would enable the use of MAb FC-5.01 as a vehicle to target different compounds inside CD63⁺ cells.