Development of EMab-51, a Sensitive and Specific Anti-Epidermal Growth Factor Receptor Monoclonal Antibody in Flow Cytometry, Western Blot, and Immunohistochemistry

The epidermal growth factor receptor (EGFR) is a member of the human epidermal growth factor receptor (HER) family of receptor tyrosine kinases and is involved in cell growth and differentiation. EGFR homodimers or heterodimers with other HER members, such as HER2 and HER3, activate downstream signaling cascades in many cancers. In this study, we developed novel anti-EGFR monoclonal antibodies (mAbs) and characterized their efficacy in flow cytometry, Western blot, and immunohistochemical analyses. First, we expressed the full-length or ectodomain of EGFR in LN229 glioblastoma cells and then immunized mice with LN229/EGFR or ectodomain of EGFR, and performed the first screening using enzyme-linked immunosorbent assays. Subsequently, we selected mAbs according to their efficacy in flow cytometry (second screening), Western blot (third screening), and immunohistochemical (fourth screening) analyses. Among 100 mAbs, only one clone EMab-51 (IgG1, kappa) reacted with EGFR in Western blot analysis. Finally, immunohistochemical analyses with EMab-51 showed sensitive and specific reactions against oral cancer cells, warranting the use of EMab-51 to detect EGFR in pathological analyses of EGFR-expressing cancers.


Introduction
T he epidermal growth factor receptor (EGFR) is a transmembrane receptor that is involved in cell growth and differentiation. (1)(2)(3) EGFR is a member of the human epidermal growth factor receptor (HER) family of receptor tyrosine kinases. EGFR homodimers or heterodimers with other HER members, such as HER2 and HER3, activate downstream signaling cascades and control many biological processes. These pathways are frequently dysregulated via overexpression of EGFR in many cancers, including brain tumors, head and neck cancers, lung cancers, breast cancers, pancreatic cancers, kidney cancers, prostate cancers, ovary cancers, bladder cancers, and colorectal cancers. (4) EGFR is the first receptor target against which monoclonal antibodies (mAbs), including cetuximab (a mouse-human chimeric mAb; IgG 1 ) against colorectal cancers and head and neck cancers, panitumumab (a fully human mAb; IgG 2 ) against colorectal cancers, and necitumumab (a fully human mAb; IgG 1 ) against nonsmall cell lung cancers, have been developed for cancer treatment. (5)(6)(7) Anti-EGFR mAbs possess several functional mechanisms, including antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), blocking dimerization or ligand binding, and EGFR endocytosis.
We have produced antipodoplanin (PDPN) cancer-specific mAbs (CasMabs), clone LpMab-2 (8,9) and LpMab-23, (10,11) which specifically recognize cancer-type PDPN in tumor tissues. For this technology, it is critical that immunogens are produced using cancer cell lines, such as LN229 glioblastoma cells, which express cancer-specific glycan-attached membrane proteins. This method could be used to develop useful mAbs against multiple membrane proteins. In this study, we produced sensitive and specific mAbs against EGFR using this technology.

Animals
Female 4-week-old BALB/c mice were purchased from CLEA Japan (Tokyo, Japan). Animals were housed under specific pathogen-free conditions. The Animal Care and Use Committee of Tohoku University approved all the animal experiments described in this study.
The resulting hybridomas were grown in RPMI medium supplemented with hypoxanthine, aminopterin, and thymidine selection medium supplement (Thermo Fisher Scientific, Inc.). Culture supernatants were screened using enzyme-linked immunosorbent assay (ELISA) with EGFRec, and mAbs were selected using flow cytometry (second screening), Western blot (third screening), and immunohistochemical analyses (fourth screening). MAbs were purified from supernatants of hybridomas cultured in Hybridoma-SFM medium (Thermo Fisher Scientific, Inc.) using Protein G Sepharose 4 Fast Flow (GE Healthcare UK Ltd., Buckinghamshire, England).
Determination of the binding affinity using flow cytometry LN229 and A431 (2 · 10 5 cells) were suspended in 100 mL of serially diluted mAbs (6 ng/mL-50 mg/mL), and Alexa Fluor 488-conjugated anti-mouse IgG (1:200; Cell Signaling Technology, Inc.) was then added. Fluorescence data were collected using a cell analyzer (EC800; Sony Corp.). The dissociation constants (K D ) were calculated by fitting the binding isotherms using the built-in one-site binding models in GraphPad PRISM 6 (GraphPad software, Inc., La Jolla, CA).

Immunohistochemical analyses
Oral cancer tissues were purchased from US Biomax, Inc. (Rockville, MD). Histologic sections of 4-mm thickness were deparaffinized in xylene and were then rehydrated and autoclaved in EnVision FLEX Target Retrieval Solution High pH (Agilent Technologies, Inc.) for 20 minutes. Sections were then incubated with 5 mg/mL of EMab-51 for 1 hour at room temperature and were then treated using an EnVision+ Kit (Agilent Technologies, Inc.) for 30 minutes. Color was developed using 3,3-diaminobenzidine tetrahydrochloride (Agilent Technologies, Inc.) for 2 minutes, and sections were then counterstained with hematoxylin (Wako Pure Chemical Industries Ltd.). The intensity of staining was evaluated as -, 1+, 2+, 3+.

Production of anti-EGFR mAbs
In this study, we immunized mice with LN229/EGFR or purified recombinant EGFRec from culture supernatants of LN229/EGFRec cells. A booster i.p. injection of LN229/ EGFR or EGFRec was also administered, and culture supernatants were then screened for binding to purified EGFRec using ELISA. As a second screening, we used flow cytometry to assess reactions with LN229 and LN229/EGFR cells. LN229 cells express endogenous EGFR (15) ; therefore, a stronger reaction against LN229/EGFR was required.
A total of 38 clones were generated from immunizations with LN229/EGFR, and 62 were generated from immuniza-tions with purified EGFRec. Of 100 mAbs, 94 clones reacted with A431 cells, which express endogenous EGFR. (16) Subsequently, mAbs were further selected according to their efficacy on Western blot analysis. These analyses identified only one clone EMab-51 (IgG 1 , kappa) from EGFRec immunizations that were useful for Western blot analysis.  ITAI ET AL.

Immunohistochemical analysis against oral cancers
We investigated the immunohistochemical utility of EMab-51 in human oral cancers because high EGFR expression was observed in oral cancer cell lines, such as HSC-2 and HSC-3 (Fig. 1C). As depicted in Figure 3, EMab-51 stained the membranes of cancer cells of oral squamous cell carcinomas. EMab-51 stained 6 of 38 (15.8%) squamous cell carcinomas in oral cancer tissue array (Supplementary  Table S1) and did not stain any other tumor types of oral cancers (Supplementary Table S2).

Discussion
Establishment of mAbs, which are useful for Western blot, flow cytometry, and immunohistochemical analyses, is often difficult because the design of immunogens and screening methods to develop specific mAbs is different between Western blot and immunohistochemical applications. Furthermore,
Among 100 anti-EGFR mAbs, 90 clones (90%) were identified as belonging to the IgG 1 subclass, 5 to the IgG 2a subclass, 1 to the IgG 2b subclass, and 4 to the IgM class. EMab-51 was also determined to be of the IgG 1 subclass, precluding confirmation of ADCC or CDC using EMab-51. Thus, in future studies, we will convert the subclass of EMab-51 into mouse IgG 2a or IgG 2b subclasses or human IgG 1 and assess their applications for measuring ADCC/CDC activities. (9,24,32) In this study, we used EnVision FLEX Target Retrieval Solution (high pH) for antigen retrieval. (33)(34)(35) For immunohistochemical analysis, we typically used citrate buffer (pH 6.0) for antigen retrieval; however, citrate buffer did not improve the signal from EMab-51 use (data not shown).
In conclusion, of 100 clones of anti-EGFR mAbs, EMab-51 was highly efficacious in Western blot analyses and produced strong staining in oral cancers. Hence, EMab-51 could be useful in all present experiments and will likely be an advantageous tool for the pathological identification of EGFR in many cancers.