Using the Allergic Immune System to Target Cancer: Tumor Specific IGE Antibodies as Cancer Therapeutics.

The branch of the immune system that responds to immunoglobulin E (IgE) antibodies is involved in the body's defense against large extracellular parasites. IgE-activated effector cells such as mast cells, basophils and perhaps eosinophils are potent triggers of inflammation, and sources of toxic mediators. Yet, the potential of this system for tumor immunotherapy is under-explored. We hypothesized that IgE effector cells can elicit effective anti-tumor responses. To test this hypothesis, we constructed mouse-human chimeric IgE antibodies. Here, we describe the production of anti-human CD20 and antihuman mucin-1 (MUC1) IgEs, and investigate their anti-tumor efficacies. Methods. To create human IgE versions of our mouse-anti-hCD20 and hMUC1 antibodies, we cloned the variable regions of the parent mouse antibodies, and grafted them onto human kappa and epsilon constant regions. Heavy and light chain vectors were transfected into Chinese Hamster Ovary cells, and stable clones were selected. CHO clones were cultured in a 5L cellbag using the Wave Bioreactor(TM), and IgE purified from the supernatants via an affinity column. The final products were analyzed via SDS-PAGE, flow cytometry and ELISA. To investigate the anti-tumor effects of the anti-hCD20 IgE in vitro, human effector cells (monocytes, mast cells or eosinophils) were cultured with CFSE-labeled hCD20+ tumor cells, in the presence of control or anti-hCD20 IgE. After 24hr, propidium iodide was added to label dead cells, and the mixture analyzed by flow cytometry. To study the in vivo effects of anti-hMUC1 IgE, mice bearing 4T1.hMUC1 tumors (implanted either subcutaneously or intra-peritoneally) were treated with either control or anti-hMUC1 IgE. The mice were then monitored for tumor growth, morbidity and survival. Results. The Wave Bioreactor(TM) enabled rapid and efficient scale-up of protein production. The use of large disposable cell bags, a sterile feed-harvest system, and temperature modulation at high cell densities, resulted in a dramatic increase in cell densities, viability and IgE production. The final products are free of major protein contaminants, and retain the antigen-specificities of the parent mouse antibodies. Next, we observed that hCD20-specific IgE antibodies activated human monocytes, mast cells and eosinophils when co-cultured with hCD20+ tumor cells, and compared to control IgE, induced increased tumor cell death in vitro. Eosinophil-mediated cytotoxicity involves the death receptor TRAIL, and cationic proteins released from eosinophil granules. Mast cell mediated cytotoxicity involves the release of tumor necrosis factor. Activated mast cells also secrete many other inflammatory and chemotactic factors. Finally, administration of anti-hMUC1 IgE antibodies led to modest reductions in 4T1.hMUC1 tumor growth and mouse survival, compared to mice treated with control IgE. Further work is needed to optimize our in vivo protocols, with a focus on increasing recruitment of effector cells into tumor sites. Conclusion. From these data, we conclude that allergic effector cells can indeed modulate tumor growth. The use of tumor-specific antibodies of the epsilon isotype yielded encouraging results in vitro and in vivo, and represents a novel strategy in the treatment of cancer with antibodies.