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Patient-derived heavy chain antibody targets cell surface HSP90 on breast tumors

BACKGROUND: Monoclonal antibodies have been used to effectively treat various tumors. We previously established a unique strategy to identify tumor specific antibodies by capturing B-cell response against breast tumor antigens from patient-derived sentinel lymph nodes. Initial application of this ap...

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Autores principales: Devarakonda, Charan V., Kita, Daniel, Phoenix, Kathryn N., Claffey, Kevin P.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4559304/
https://www.ncbi.nlm.nih.gov/pubmed/26334999
http://dx.doi.org/10.1186/s12885-015-1608-z
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author Devarakonda, Charan V.
Kita, Daniel
Phoenix, Kathryn N.
Claffey, Kevin P.
author_facet Devarakonda, Charan V.
Kita, Daniel
Phoenix, Kathryn N.
Claffey, Kevin P.
author_sort Devarakonda, Charan V.
collection PubMed
description BACKGROUND: Monoclonal antibodies have been used to effectively treat various tumors. We previously established a unique strategy to identify tumor specific antibodies by capturing B-cell response 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. In this paper we optimized our previous strategy by generating heavy chain antibodies (HCAbs) to overcome the deficiencies of single domain antibodies. Here we identified and characterized a heavy chain antibody (HCAb2) that targets cell surface HSP90 antigen on breast tumor cells but not normal cells. METHODS: Eight HCAbs derived from 4 breast cancer patients were generated using an in vitro expression system. HCAbs were screened against normal breast cells (MCF10A, HMEC) and tumor cell lines (MCF7, MDA-MB-231) to identify cell surface targeting and tumor specific antibodies using flow cytometry and immunofluorescence. Results observed with cell lines were validated by screening a cohort of primary human breast normal and tumor tissues using immunofluorescence. Respective antigens for two HCAbs (HCAb1 and HCAb2) were identified using immunoprecipitation followed by mass spectrometry. Finally, we generated MDA-MB-231 xenograft tumors in NOD scid gamma mice and performed in vivo tumor targeting analysis of HCAb1 and HCAb2. RESULTS: Flow cytometry screen revealed that HCAb2 selectively bound to the surface of MDA-MB-231 cells in comparison to MCF10A and MCF7 cells. HCAb2 showed punctate membrane staining on MDA-MB-231 cells and 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. We identified and validated the target antigen of HCAb2 as Heat shock protein 90 (HSP90). HCAb2 also selectively targeted MDA-MB-231 xenograft tumor cells in vivo with little targeting to mouse normal tissues. Finally, HCAb2 specifically targeted calnexin negative xenograft tumor cells. CONCLUSIONS: From our screening methodology, we identified HCAb2 as a breast tumor specific heavy chain antibody targeting cell surface HSP90. HCAb2 also targeted MDA-MB-231 tumor cells in vivo suggesting that HCAb2 could be an ideal tumor targeting antibody. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12885-015-1608-z) contains supplementary material, which is available to authorized users.
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spelling pubmed-45593042015-09-04 Patient-derived heavy chain antibody targets cell surface HSP90 on breast tumors Devarakonda, Charan V. Kita, Daniel Phoenix, Kathryn N. Claffey, Kevin P. BMC Cancer Research Article BACKGROUND: Monoclonal antibodies have been used to effectively treat various tumors. We previously established a unique strategy to identify tumor specific antibodies by capturing B-cell response 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. In this paper we optimized our previous strategy by generating heavy chain antibodies (HCAbs) to overcome the deficiencies of single domain antibodies. Here we identified and characterized a heavy chain antibody (HCAb2) that targets cell surface HSP90 antigen on breast tumor cells but not normal cells. METHODS: Eight HCAbs derived from 4 breast cancer patients were generated using an in vitro expression system. HCAbs were screened against normal breast cells (MCF10A, HMEC) and tumor cell lines (MCF7, MDA-MB-231) to identify cell surface targeting and tumor specific antibodies using flow cytometry and immunofluorescence. Results observed with cell lines were validated by screening a cohort of primary human breast normal and tumor tissues using immunofluorescence. Respective antigens for two HCAbs (HCAb1 and HCAb2) were identified using immunoprecipitation followed by mass spectrometry. Finally, we generated MDA-MB-231 xenograft tumors in NOD scid gamma mice and performed in vivo tumor targeting analysis of HCAb1 and HCAb2. RESULTS: Flow cytometry screen revealed that HCAb2 selectively bound to the surface of MDA-MB-231 cells in comparison to MCF10A and MCF7 cells. HCAb2 showed punctate membrane staining on MDA-MB-231 cells and 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. We identified and validated the target antigen of HCAb2 as Heat shock protein 90 (HSP90). HCAb2 also selectively targeted MDA-MB-231 xenograft tumor cells in vivo with little targeting to mouse normal tissues. Finally, HCAb2 specifically targeted calnexin negative xenograft tumor cells. CONCLUSIONS: From our screening methodology, we identified HCAb2 as a breast tumor specific heavy chain antibody targeting cell surface HSP90. HCAb2 also targeted MDA-MB-231 tumor cells in vivo suggesting that HCAb2 could be an ideal tumor targeting antibody. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12885-015-1608-z) contains supplementary material, which is available to authorized users. BioMed Central 2015-09-03 /pmc/articles/PMC4559304/ /pubmed/26334999 http://dx.doi.org/10.1186/s12885-015-1608-z Text en © Devarakonda et al. 2015 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Research Article
Devarakonda, Charan V.
Kita, Daniel
Phoenix, Kathryn N.
Claffey, Kevin P.
Patient-derived heavy chain antibody targets cell surface HSP90 on breast tumors
title Patient-derived heavy chain antibody targets cell surface HSP90 on breast tumors
title_full Patient-derived heavy chain antibody targets cell surface HSP90 on breast tumors
title_fullStr Patient-derived heavy chain antibody targets cell surface HSP90 on breast tumors
title_full_unstemmed Patient-derived heavy chain antibody targets cell surface HSP90 on breast tumors
title_short Patient-derived heavy chain antibody targets cell surface HSP90 on breast tumors
title_sort patient-derived heavy chain antibody targets cell surface hsp90 on breast tumors
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4559304/
https://www.ncbi.nlm.nih.gov/pubmed/26334999
http://dx.doi.org/10.1186/s12885-015-1608-z
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