Introduction: New cancer-specific antigens are needed to fully realize the potential of antibody-based cancer therapies. Cancer cells express novel antigens that are characteristic of the transformed state and may engender an antibody response in patients. We hypothesize that some of these antibodies detect neo-antigens shared by diverse tumor types. We optimized a hybridoma method for cloning human IgG monoclonal antibodies (mAbs) by screening for binding to heterologous cell lines. Here, we describe four mAbs, isolated from cancer patients, that bind to cancer cells.

Methods: We developed immunofluorescence binding assays with the A431, MCF-7, and MDA-MB-231 cell lines, detecting cell-reactive human IgG with the Operetta (Perkin Elmer). The assays were adapted for extracellular binding as well as internalization using specific culture conditions. We tested sera from 59 lung cancer patients with diverse clinical features. 24 of the samples contained IgG reactivity with A431 cells at > 1:100 titer, and 17 had evidence of IgG internalization. We generated hybridomas using peripheral blood B-cells isolated from two of these lung cancer patients, as well as B-cells from a lymph node of a stage I, ER+PR+ breast cancer patient. We characterized the binding of four mAbs using immunohistochemistry (IHC), microscopy, flow cytometry and immunoblotting. One mAb was used to identify its cognate antigen by immunoprecipitation and mass spectrometry.

Results: In a panel of 59 lung cancer patient sera, 24 had IgG that bound the A431 cell line, and 17 of these had evidence of internalization. We isolated three human mAbs from two lung cancer patients and one mAb from the breast cancer lymph node. We performed IHC with three of the mAbs in formalin fixed, paraffin embedded normal and tumor tissues and found primarily tumor cell binding to a variety of tumor types. The 5A6 (lung) bound 3 of 4 non-small cell lung, 3 of 4 colon, 2 of 4 ovarian and 1 of 4 small cell lung cancers. 19F10 (lung) bound 1 of 4 colon and 2 of 4 small cell lung. 2G4 (breast) bound 4 of 4 non-small cell lung, 3 of 4 prostate, 3 of 4 ovarian, and 2 of 4 colon cancers. Some normal tissue binding was observed, such as small intestine and distal renal tubule (2G4 and 19F10), pancreatic islet (19F10), and prostate (2G4). The 6A10 mAb (lung) bound to the constitutive heat shock protein, HSC70, which was localized in the plasma membrane of cancer cell lines.

Conclusion: We have cloned 4 human mAbs from cancer patients. This supports the hypothesis that patients with active malignancies make antibodies capable of binding cancer-specific antigens. We identified HSC70, a protein that has an altered cell localization in cancer cells, as one such antigen. The three mAbs tested by IHC bound to diverse tumor types that often differed from the tumors of the patients where the B-cells originated. The human anti-cancer antibody repertoire holds promise as a tool to identify new targets for mAb-based cancer diagnostics and therapeutics.

Citation Format: Huiwu Zhao, Jiping Zhang, Ramdev Puligedda, Cezary Swider, Paul Simon, Baron Heimbach, Sharad Adekar, Maureen Murphy, Hossein Borghaei, Scott Dessain. Tumor-specific human monoclonal antibodies isolated from cancer patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3625. doi:10.1158/1538-7445.AM2014-3625