The monocyte network is important for therapeutic efficacy of antibody therapies against cancer. One mechanism which monocytes/macrophages use to kill cancer cells is phagocytosis. Using trastuzumab and human breast cancer cell lines as a model, we used flow cytometry to evaluate the importance of avidity, antigen density, Fcγ receptor (FcγR) expression, and FcγR polymorphisms in human monocyte phagocytosis. By increasing avidity for the tumor through the addition of pertuzumab to trastuzumab, there was a two-to-threefold increase in phagocytosis potency against the HCC1419 cell line compared to antibodies alone, while NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC) failed to increase tumor cell death. Consistent with increasing the avidity through multiple antibodies, antigen density significantly enhanced phagocytosis with breast cancer cell lines that were HER2 gene-amplified compared to non-amplified tumor cells. Confirmation that high antigen density enhanced phagocytosis was obtained when HER2 was overexpressed in HER2 non-amplified cell lines. In contrast, NK cell ADCC failed to distinguish differences in tumor cell death when comparing gene-amplified and non-amplified breast cancer cell lines. The level of phagocytosis was influenced by FcγRIIa and FcγRIIIa expression. Most monocytes are FcγRIIIa, and the induction of the receptor significantly enhances antibody-dependent phagocytosis. Although both receptors are involved, when blocked FcγRIIIa had a greater influence on phagocytosis. Furthermore, the polymorphism FcγRIIIa 158V significantly enhanced phagocytosis; whereas FcγRIIa 131H polymorphism appeared to improve phagocytosis but was not statistically significant. Targeting of monocytes for enhanced phagocytosis may improve the effectiveness of therapeutic antibodies to improve clinical outcomes.
Tumor-targeting antibodies have been successful in the treatment of various types of cancers. Antibodies engage the immune system with their Fc, stimulating immune cell effector function. In the clinic, FcγRIIIa polymorphisms with higher affinity for the Fc of antibodies were shown to improve response rates and overall survival. Efforts have been made to modify the Fc to enhance affinity to Fc receptors and thereby improve effector function. An alternative for improving immune effector function may be to increase the level of tumor antigen expression. In this study, tamoxifen was used to increase HER2/neu protein level to determine whether increased tumor antigen expression could enhance NK cell-mediated antibody-dependent cytotoxicity (ADCC). Tamoxifen was found to increase HER2/neu 1.5-fold to threefold in breast cancer cell lines that were HER2/neu non-amplified. Using flow cytometry to simultaneously evaluate NK cell degranulation and tumor cell death, the increase in HER2/neu enhanced NK cell-mediated ADCC. However, in cells that had HER2/neu gene amplification and estrogen receptor expression, tamoxifen elevated HER2/neu but failed to improve NK cell function. The quantity of HER2/neu on the tumor cell surface was approximately double that of the number of Fc receptors found on NK cells. This appears to reflect a ceiling at which increasing antigen expression fails to improve NK cell effector function. This has clinical implications as trying to increase antigen expression to enhance NK cell function may be useful for patients with antigen-low tumors, but not in those whose tumors have gene amplification or high levels of antigen expression.
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