Abstract 2661: Patient-derived breast cancer targeting heavy chain antibodies

Abstract

Abstract The emergence of tumor-targeting antibodies such as Trastuzumab, Rituximab and anti-CD47 antibody has highlighted the significant role of antibodies for effective cancer therapy. The most common approach to generate tumor specific antibodies is to select an established tumor specific antigen and generate antibodies to that target. The drawback with this approach is that it does not lead to identification of novel antigens or antigenic epitopes. Therefore, we developed a strategy that will lead to the identification of both tumor specific antibodies and novel tumor antigens. The approach uses the patient's immune system to guide us to define tumor specific antigens. In order to achieve this, we identified reactive germinal centers in the tumor draining sentinel lymph nodes from breast cancer patients. Immunohistochemical analysis of reactive germinal centers defined the B-cell regions for mRNA isolation and generation of cDNA molecules for the variable heavy chain segment (VH) of antibodies. The VH cDNA molecules were cloned into a mammalian expression vector to generate bivalent heavy chain antibodies with a C-terminus mouse Fc tag. At present, we have generated about 250 antigen-driven variable heavy chain clones from 27 breast cancer patient lymph node samples. We screened 46 heavy chain antibodies against a panel of breast cancer cell lines using immunofluorescence and identified 8 antibodies that showed strong and specific staining. Of these 8 antibodies, one of them (VH6-93) showed membrane staining preferentially on MDA-MB-231 cells in comparison to MCF7 cells. VH6-93 antibody also showed strong staining on conditionally reprogrammed tumor cells derived from a breast cancer patient with only moderate staining on normal breast epithelial cells derived from the same patient. VH6-93 also revealed preferential binding to breast tumor tissues as compared to normal ductal tissues evidenced by immunofluorescence and immunohistochemistry. To identify the target antigens, a multi-tiered approach was used, which included immunoprecipitation of target antigen from cell lysates followed by peptide sequencing with mass spectrometry as well as utilizing Nucleic Acid Programmable Protein Array (NAPPA) technology. A comprehensive analysis of two patient-derived antibodies has produced a panel of potential antigen targets, which are undergoing specificity and validation analysis. In summary, this methodology of using activated B-cells from sentinel lymph nodes is a unique way to enrich for tumor-specific antibody clones. The findings from this research will not only lead to tumor specific antibodies but will also reveal novel tumor specific antigens. Identification of these novel antigens can give rise to additional therapeutic strategies such as peptide or protein vaccination. Citation Format: Charan Kumar V. Devarakonda, Daniel Kita, Kathryn N. Phoenix, Dewey M. Magee, Kevin P. Claffey. Patient-derived breast cancer targeting heavy chain antibodies. [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 2661. doi:10.1158/1538-7445.AM2014-2661