Polymorphonuclear Granulocytes Induce Antibody-Dependent Apoptosis in Human Breast Cancer Cells

Abstract

Recent studies in HER-2/neu-targeted immunotherapy demonstrated that polymorphonuclear neutrophils (PMN) mediated Ab-dependent cellular cytotoxicity against HER-2/neu-positive breast cancer cell lines. However, the mechanism of cell death remained unclear. We used several assays to analyze the induction of apoptosis in the breast cancer cell line SK-BR-3 via PMN-dependent Ab-dependent cellular cytotoxicity. In the presence of the HER-2/neu Ab 520C9 and PMN from healthy donors, apoptosis occurred as detected by annexin V binding and disappearance of euploid SK-BR-3 nuclei, which can be differentiated from PMN nuclei by their increased DNA contents. Apoptosis induction was observed with E:T cell ratios as low as 10:1. Laser scanning fluorescence microscopy of TUNEL tumor cells or staining for cleaved cytokeratin-18 further confirmed apoptosis of the SK-BR-3 breast cancer cells. Killing via 520C9 was dependent on the interaction with FcR on PMN, because 1) F(ab′)2 fragments of 520C9 mediated no cytotoxicity, 2) target cell death was influenced by a biallelic polymorphism of FcγRIIa on the effector cells, and 3) a bispecific Ab against HER-2/neu and the IgA receptor (FcαRI) expressed on effector cells significantly induced apoptosis. Thus, PMN induce Ab-dependent apoptosis against human breast cancer cells targeted with HER-2/neu-directed mAbs or FcR directed bispecific Abs.

A Recombinant Bispecific Single-Chain Fragment Variable Specific for HLA Class II and FcαRI (CD89) Recruits Polymorphonuclear Neutrophils for Efficient Lysis of Malignant B Lymphoid Cells

“…Accordingly, this cell line is highly refractory to apoptosis induction by conventional chemotherapeutics and CD95 Abs (31), reflecting a different sensitivity of the cells to proapoptotic signals. ADCC induced by PMNs generally occurs through the induction of apoptosis in the target cells (32). Interestingly, our bispecific construct, targeting the identical epitope on HLA class II as the FcgR-directed Abs, was able to eliminate these highly resistant cell lines, although they expressed lower Ag densities.…”

Mac-1 Promotes FcγRIIA-Dependent Cell Spreading and Migration on Immune Complexes

“…However, FcγRIIA, which possesses an ITAM motif within its cytoplasmic domain and can signal in the absence of other ITAM-containing subunits, still depends on Mac-1 for its ADCC activity toward the IC-tagged targets (1,6,8). To understand the role of Mac-1 in FcγRIIA-mediated functions, we first evaluated whether Mac-1 is required for cell adhesion to ICs, using human 293 cells (which do not express endogenous Mac-1 or FcγRIIA) that express either Mac-1 alone or Mac-1 plus FcγRIIA.…”

“…A number of clinical studies have demonstrated that FcγRIIA (CD32), a major FcR that recognizes the IgG subclass, is essential to the efficacy of several therapeutic antibody-based drugs, including Herceptin for HER-2/neu-positive breast cancer cells, Rituxan for non-hodgkins lymphoma, and the antibodies for melanoma (1,2,9). Yet, inappropriate engagement of FcγRIIA also causes autoimmune diseases in patients undergoing treatment using these therapeutic antibodies (10), and in transgenic mice overexpressing FcγRIIA (11).The FcγRIIA-mediated antibody-dependent cytotoxicity (ADCC) is dependent on both FcγRIIA and the integrin Mac-1 (α M β 2 , CR3, CD11b/CD18) (1,8,12,13). Despite the importance of Mac-1 in the FcγRIIA-mediated leukocyte functions, and the extensive studies conducted on Mac-1 interaction with various FcRs, including FcγRIIIB (CD16), FcαRI (CD89) and FcεRII (CD23), in addition to [14][15][16], the molecular basis underlying Mac-1 recognition of these FcRs is still less understood.…”

“…A number of clinical studies have demonstrated that FcγRIIA (CD32), a major FcR that recognizes the IgG subclass, is essential to the efficacy of several therapeutic antibody-based drugs, including Herceptin for HER-2/neu-positive breast cancer cells, Rituxan for non-hodgkins lymphoma, and the antibodies for melanoma (1,2,9). Yet, inappropriate engagement of FcγRIIA also causes autoimmune diseases in patients undergoing treatment using these therapeutic antibodies (10), and in transgenic mice overexpressing FcγRIIA (11).…”

Immature Neutrophils Mediate Tumor Cell Killing via IgA but Not IgG Fc Receptors

“…In vitro, neutrophils have been shown to exert potent cytolytic capacity against a variety of tumor cells in the presence of antitumor mAb, and in vivo studies support a role for neutrophils in tumor rejection (7,9,10). It has been furthermore demonstrated that neutrophils can induce Ab-dependent apoptosis in human breast cancer cells (11). Additionally, neutrophils represent the most populous Fc␥R-expressing leukocyte subset within the blood, and their numbers can be increased by treatment with G-CSF (12).…”