Abstract 4736: Generation of tumor-specific antibodies from sentinel lymph nodes of breast cancer patients using hybridoma technology.
Authors: Shukla Girja S. , Pero Stephanie C. , Sun Yu-Jing , Carman Chelsea , Krag David N. 
Published: August 2014

Abstract
The field of targeted therapy is providing an entirely new generation of highly active anticancer drugs. For example, addition of a targeted drug to a chemotherapy regimen has shown improved survival in the treatment of breast cancer (Herceptin) and colorectal cancer (Avastin). Main problems with this kind of therapy are that many patients are not candidates because their tumors do not overexpress the drug target and that patient develop resistance to the targeted drug. A method to rapidly develop different sets of therapeutic antibodies (Abs) would greatly contribute to the field of targeted anticancer therapy. Even more novel would be methods that can develop Abs derived from individual patients. The rationale behind this concept is solidly supported by a large number of SEREX studies demonstrating that the sera of cancer patients do contain autoantibodies against their own tumors. These autoantibodies may show greater cancer specificity as tolerance mechanisms usually inhibit Ab production against normal cells. We generated hybridomas from the sentinel nodes of 29 breast cancer patients. Surgically removed fresh breast tumors and sentinel nodes were used for this study. Sentinel lymph node cells were harvested by either scraping the cut surface or by removing a 2 mm slice of the nodes. Lymph nodes were mechanically disaggregated and sieved before Ficoll gradient centrifugation. The yield of lymphocytes varied from 0.5 to 50x106 cells. Following stimulation, mononuclear cells were fused with plasmacytoma P3X63.Ag8.653 under hypo-osmolar condition, using BTX Electro Cell Manipulator and microslide electrodes. Each immunoglobulin secreting clone was evaluated with ELISA for binding to a panel of breast cancer cell lines representing the 4 subclasses of breast cancer, and 2 non-cancer breast epithelial cell lines. Tumor cell lysate-binding was observed in 28% of the hybridoma clones. The lysate-binding clones were further evaluated with whole cell imaging, flow cytometry, and Western blotting. Approximately 10% of the tumor-binding clones were found to bind whole tumor cells with unique binding patterns. These clones were also found to bind to the patient's own tumor. Thus, we have identified several tumor-specific autoantibodies from breast cancer patients. Successful outcome of these experiments demonstrate the feasibility of rapidly generating human monoclonal Abs that have a great potential for tumor target discovery and their use in cancer therapy and diagnosis. The innovative aspect of this strategy is the exploitation of the cancer patient's own immune repertoire for generating therapeutic Abs. This is in contrast to most of the currently available therapeutic Abs for clinical use, which are derived from chimerization and humanization of rodent Abs. No currently available antibody is developed through the selective and regulatory processes of the human immune system.
Citation Format: Girja S. Shukla, Stephanie C. Pero, Yu-Jing Sun, Chelsea Carman, David N. Krag. Generation of tumor-specific antibodies from sentinel lymph nodes of breast cancer patients using hybridoma technology. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4736. doi:10.1158/1538-7445.AM2013-4736