Date of Completion

7-27-2015

Embargo Period

1-12-2016

Keywords

Sentinel lymph node, single domain antibodies, heavy chain antibodies, heat shock protein 90, MDA-MB-231, breast cancer, calnexin

Major Advisor

Kevin P. Claffey

Associate Advisor

Linda H. Shapiro

Associate Advisor

David K. Han

Associate Advisor

Robert B. Clark

Associate Advisor

Daniel W. Rosenberg

Field of Study

Biomedical Science

Degree

Doctor of Philosophy

Open Access

Campus Access

Abstract

Emergence of tumor-targeting antibodies such as Trastuzumab, Rituximab, and Ipilimumab has highlighted the significant role of antibodies in effective cancer therapy. These examples validate the need for identifying additional tumor specific antibodies. To identify tumor specific antibodies, a unique strategy was previously established to capture B-cell responses against breast tumor antigens from patient-derived sentinel lymph nodes. Initial application of this approach led to identification of a tumor specific single domain antibody (sdAb). In this study, 46 different sdAbs were screened by ELISA and immunofluorescence leading to the identification of a potential tumor targeting antibody (VH6-93). But single domain antibodies lack Fc region and hence cannot mediate effector functions such as antibody dependent cell mediated cytotoxicity and complement mediated cytotoxicity. To overcome these deficiencies, VH6-93 along with 7 different sdAbs were converted to heavy chain antibodies (HCAbs). VH6-93 now termed as HCAb2 was screened against normal breast cells (MCF10A, HMEC) and tumor cell lines (MCF7, MDA-MB-231) to determine specific binding to the surface of tumor cells. Flow cytometry screen revealed that HCAb2 selectively bound to the surface of MDA-MB-231 cells in comparison to MCF10A and MCF7 cells. Immunofluorescence analysis with HCAb2 revealed punctate staining on cell membrane of MDA-MB-231 cells. Results observed with cell lines were validated by screening a cohort of primary human breast normal and tumor tissues using immunofluorescence. HCAb2 demonstrated preferential binding to human breast tumor tissues in comparison to normal breast tissues. In primary breast tumor tissues, HCAb2 showed positive binding to both E-cadherin positive and negative tumor cells. HCAb2 also specifically bound to bladder and ovarian tumor cells in comparison to normal cells. Mass spectrometry analysis identified the target antigen of HCAb2 to be Heat shock protein 90 (HSP90). Binding of HCAb2 to cell surface HSP90 reduced the migration of MDA-MB-231 cells in an in vitro scratch assay. HCAb2 also demonstrated in vivo tumor targeting capability by homing to MDA-MB-231 xenograft tumors in NOD scid gamma mice with little targeting to mouse normal tissues. In conclusion, this screening methodology identified HCAb2 as a tumor specific heavy chain antibody that targets cell surface HSP90.

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