Abstract 1277: Tumor-specific human monoclonal antibodies isolated from cancer patients that bind antigens expressed on the outer plasma membrane of cancer cells
Authors: Heimbach Baron , Swider Cezary , Zhao Huiwu , Simon Paul , Alpaugh R. Katherine , Walker Michael , Krag David , Knight David , Chan Tung , Borghaei Hossein , Dessain Scott 
Published: August 2015

Abstract
Introduction: Cancer cells express novel antigens that reflect their transformed state and may engender an antibody response. We hypothesize that some of these auto-antibodies detect neo-antigens that are shared by diverse tumor types and that a subset of these are expressed on the outer plasma membrane (PM) of cancer cells, but not normal cells. Here, we describe two human mAbs, isolated from lymph node B-cells of breast cancer patients, that bind antigens expressed only on the surface of cancer cells.
Methods: We have optimized a hybridoma method for cloning human IgG monoclonal antibodies (mAbs) by screening for those that bind to live, heterologous cancer cell lines. We use whole cell immunofluorescence binding assays with human cancer cell lines, detecting cell-reactive human IgG with the Operetta. We use B-cells from lymph nodes obtained at surgery from stage I, ER+PR+ breast cancer patients and Stage I or II lung cancer patients, as well as peripheral blood B-cells from lung cancer patients responsive to PD-1 inhibitors. We used cDNA library expression methods to identify the tumor antigens and characterized the tumor-specific activities of two mAbs using immunohistochemistry (IHC), gene transfection, flow cytometry, immunoblotting, and invasion assays.
Results: Two of the mAbs isolated from lymph nodes of patients with breast cancer bind to antigens that are only expressed on the outer PM of cancer cells. The antigen recognized by mAb 9H2 is known to be expressed on the outer PM of metastatic cells, while the antigen recognized by mAb 2B9 is a cytoplasmic protein not previously recognized to exist on the outer PM. IHC demonstrates that the 9H2 antigen is expressed only rarely on normal cells, but is commonly on breast and colon cancers (3 of 4 samples tested for each tumor type). Binding was also seen on 2 of 4 small cell lung cancers samples and 1 of 3 pancreatic cancers. The existence of the 2B9 antigen on the outer PM was confirmed by study of 293T cells transiently transfected with MYC-tagged antigen expression constructs and analyzed by flow cytometry with the mAb 2B9 and an anti-myc mAb. Treatment of MDA-MB-231 cells with the mAb 2B9 substantially reduced invasion in the transwell migration assay. An additional panel of 12 mAbs exhibited internalizing activity and is being evaluated for efficacy as antibody-drug conjugates
Conclusion: Our panel of mAbs from cancer patients supports the hypothesis that patients with malignancies make antibodies that bind novel cancer-specific antigens. Two mAbs bind antigens expressed only on the surface of malignant cells; one has not previously been detected on the outer PM. The ability of mAb 2B9 to inhibit breast cancer cell invasion suggests that the anti-tumor immune response may be functional. Studying the anti-cancer antibody repertoire may identify new targets for mAb-based cancer diagnostics and therapeutics.
Citation Format: Baron Heimbach, Cezary Swider, Huiwu Zhao, Paul Simon, R. Katherine Alpaugh, Michael Walker, David Krag, David Knight, Tung Chan, Hossein Borghaei, Scott Dessain. Tumor-specific human monoclonal antibodies isolated from cancer patients that bind antigens expressed on the outer plasma membrane of cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1277. doi:10.1158/1538-7445.AM2015-1277