Targeted Lysis of Human Breast Cancer Cells by Human Effector Cells Armed with Bispecific Antibody 2B1 (ANTI-c-erbB-2 / ANTI-Fcγ Receptor III)

Abstract

Hybridomas 520C9 (murine IgG1 recognizing the proto-oncogene product c-erbB-2) and 3G8 (murine IgG1 recognizing human FcγR III or CD16) were stained with vital fluorescent dyes and fused with PEG; hybrid hybridomas were isolated by dual fluorescence on a cell sorter. Cultures whose supernatants promoted lysis of SK-Br-3 breast cancer cells by human total mononuclear cells were chosen for subcloning. The immunoglobulin species produced by clone 2B1 could be resolved on anion exchange chromatography into parental antibody 520C9, a middle peak containing bispecific antibody, and parental antibody 3G8. The bispecific peak further resolved into major and minor peaks on cation exchange chromatography. The major peak appeared bispecific on the basis of SDS PAGE, isoelectric focusing, immunofluorescent binding to both tumor and effector cells, and ability to promote targeted cytolysis. The minor peak also appeared bispecific by gel criteria, but lacked tumor cell binding and cytolytic targeting ability. We tentatively suggest that the minor species of bispecific antibody is more heavily glycosylated at the hinge region in a manner that sterically interferes with activity of the 520C9 but not the 3G8 binding site. No evidence for imrruпoglobulin species containing heterologous light/heavy chain pairing was observed. Purified 2B1 bispecific antibody (active form) promoted lysis of erbB-2 positive breast, ovarian and lung cancer cells by human LGLs (large granular lymphocytes) and macrophages. Half maximal targeting activity was usually observed at antibody concentrations between 10 and 100 ng/ml. Target cell lysis was not blocked by the level of irrelevant immunoglobulin found in 100% autologous human serum. Lysis by human LGLs was somewhat reduced in the presence of high numbers of human PMNs, but substantial targeted killing due to 2B1 could be observed in unfractionated human blood.