NDLI: High-content Analysis of Antibody Phage-display Library Selection Outputs Identifies Tumor Selective Macropinocytosis-dependent Rapidly Internalizing Antibodies*

Content Provider	PubMed Central
Author	Ha, Kevin D. Bidlingmaier, Scott M. Zhang, Yafeng Yang, Su Liu, Bin
Copyright Year	2014
Abstract	Many forms of antibody-based targeted therapeutics, including antibody drug conjugates, utilize the internalizing function of the targeting antibody to gain intracellular entry into tumor cells. Ideal antibodies for developing such therapeutics should be capable of both tumor-selective binding and efficient endocytosis. The macropinocytosis pathway is capable of both rapid and bulk endocytosis, and recent studies have demonstrated that it is selectively up-regulated by cancer cells. We hypothesize that receptor-dependent macropinocytosis can be achieved using tumor-targeting antibodies that internalize via the macropinocytosis pathway, improving potency and selectivity of the antibody-based targeted therapeutic. Although phage antibody display libraries have been utilized to find antibodies that bind and internalize to target cells, no methods have been described to screen for antibodies that internalize specifically via macropinocytosis. We hereby describe a novel screening strategy to identify phage antibodies that bind and rapidly enter tumor cells via macropinocytosis. We utilized an automated microscopic imaging-based, High Content Analysis platform to identify novel internalizing phage antibodies that colocalize with macropinocytic markers from antibody libraries that we have generated previously by laser capture microdissection-based selection, which are enriched for internalizing antibodies binding to tumor cells in situ residing in their tissue microenvironment (Ruan, W., Sassoon, A., An, F., Simko, J. P., and Liu, B. (2006) Identification of clinically significant tumor antigens by selecting phage antibody library on tumor cells in situ using laser capture microdissection. Mol. Cell. Proteomics. 5, 2364–2373). Full-length human IgG molecules derived from macropinocytosing phage antibodies retained the ability to internalize via macropinocytosis, validating our screening strategy. The target antigen for a cross-species binding antibody with a highly active macropinocytosis activity was identified as ephrin type-A receptor 2. Antibody-toxin conjugates created using this macropinocytosing IgG were capable of potent and receptor-dependent killing of a panel of EphA2-positive tumor cell lines in vitro. These studies identify novel methods to screen for and validate antibodies capable of receptor-dependent macropinocytosis, allowing further exploration of this highly efficient and tumor-selective internalization pathway for targeted therapy development.
Related Links	http://dx.doi.org/10.1074/mcp.m114.039768
Ending Page	3331
Page Count	12
Starting Page	3320
File Format	PDF
ISSN	15359476
e-ISSN	15359484
Journal	Molecular & Cellular Proteomics : MCP
Issue Number	12
Volume Number	13
Language	English
Publisher	The American Society for Biochemistry and Molecular Biology
Publisher Date	2014-12-01
Access Restriction	Open
Rights Holder	The American Society for Biochemistry and Molecular Biology
Subject Keyword	Analytical Chemistry Biochemistry Molecular Biology Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Medicine Analytical Chemistry Molecular Biology Biochemistry

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