Monoclonal antibodies represent the vanguard of targeted therapy due to their specificity and versatility. Aside from disrupting tumor cell survival and proliferation through cell surface receptor interactions, antibodies can induce antibody-dependent cellular cytotoxicity (ADCC) through immune effector cells, such as natural killer (NK) cells. Although clinical evidence suggests a role for ADCC in antibody therapy of cancer, molecular determinants of sensitivity and resistance to ADCC have not been well characterized. As resistance to antibody therapy is common, we aimed to uncover molecular determinants of ADCC response. We utilized two approaches to assess sensitivity and resistance, respectively, in an in vitro model system consisting of an NK-like cell line (NK92-CD16V), an anti-EGFR monoclonal antibody (cetuximab), and EGFR-expressing A431 cells.
We screened 60 genes from an EGFR gene network by RNA interference (RNAi) to assess for genes whose knockdown enhanced ADCC. First, we developed a high-throughput siRNA-based functional genomics screening platform for assessing ADCC. Primary and secondary screens identified three genes whose knockdown enhanced ADCC: GRB7, PRKCE, and ABL1. Characterization studies of these three genes revealed different putative mechanisms by which knockdown enhanced ADCC. We focused our efforts on ABL1, as c-Abl represents a highly translational target. ABL1 knockdown reduced the proliferation of A431 cells, in addition to its effects on enhanced ADCC. Inhibition of c-Abl kinase activity enhanced ADCC, phenocopying the effects of ABL1 knockdown and providing pre-clinical evidence for combining c-Abl inhibitors and cetuximab.
We also derived an ADCC-resistant cell population, A431/ADCCR, by successive ADCC treatment of A431 cells. Characterization of A431/ADCCR cells revealed reduced EGFR expression, diminished cell proliferation, and distinctive morphology compared to parental A431 cells. Whole genome gene expression analysis revealed over 330 differentially-expressed genes in A431/ADCCR cells. Generation and characterization of the A431/ADCCR cell line has yielded a valuable tool for probing resistance to ADCC, particularly acquired resistance through loss of EGFR.
Together, these two complementary approaches have revealed molecular determinants of sensitivity and resistance to ADCC. Enhancing tumor sensitivity to ADCC could translate into increased monoclonal antibody efficacy in the clinic, while unraveling resistance could inform therapeutic advances in the future.
|Advisor:||Weiner, Louis M.|
|Commitee:||Campbell, Kerry S., Hurley, Carolyn K., Pishvaian, Michael J., Waldman, Todd A., Wellstein, Anton|
|School Location:||United States -- District of Columbia|
|Source:||DAI-B 74/11(E), Dissertation Abstracts International|
|Subjects:||Molecular biology, Cellular biology, Immunology|
|Keywords:||Adcc, Egfr, Monoclonal antibody, Nk cell, Resistance, Sensitivity|
Copyright in each Dissertation and Thesis is retained by the author. All Rights Reserved
The supplemental file or files you are about to download were provided to ProQuest by the author as part of a
dissertation or thesis. The supplemental files are provided "AS IS" without warranty. ProQuest is not responsible for the
content, format or impact on the supplemental file(s) on our system. in some cases, the file type may be unknown or
may be a .exe file. We recommend caution as you open such files.
Copyright of the original materials contained in the supplemental file is retained by the author and your access to the
supplemental files is subject to the ProQuest Terms and Conditions of use.
Depending on the size of the file(s) you are downloading, the system may take some time to download them. Please be