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 brea st cancer cell line DB-BR-G, has been recently shown to react with CD63, Th e antigen (Ag) recognized by MAb FC-5.01 is expressed in plasma membranes o f IIB-BR-G and other neoplastic cells, as well as in activated platelets an d endothelial cells, as detected by indirect immunofluorescence performed a t 4 degrees C on live cells. In permeabilized cells, MAb FC-5.01 colocalize s with acridine orange in acidic vesicles (lysosomal/endosomal compartment) , Scatchard plot analysis performed on IB-BR-G cells demonstrated a 1.4 +/- 0.4 x 10(7) M-1 affinity constant and 2.1 x 10(6) antigenic sites per cell . MAb FC-5.01 is not able to mediate C fixation or ADCC toward CD63(+) cell s, but the FC-5.01-CD63 complex is efficiently internalized into cytoplasmi c vesicles, as shown by an acid wash immunofluorescence assay. Cellular cat abolism of the antibody bound by IIB-BR-G cells was studied using [I-125]-F C-5.01, At 18 h, >70% of the radioactivity was present in the supernatant a s degraded fragments (TCA-soluble). After internalization, rapid Ag re-expr ession could be demonstrated in IIB-BR-G cells. MAb FC-5.01 diminished migr ation of CD63(+) cells in a Boyden chamber assay. Some of the above-mention ed properties would enable the use of MAb FC-5.01 as a vehicle to target di fferent compounds inside CD63(+) cells.