Antibodies specifically targeting a locally misfolded region of tumor associated EGFR

Abstract

Epidermal Growth Factor Receptor (EGFR) is involved in stimulating the growth of many human tumors, but the success of therapeutic agents has been limited in part by interference from the EGFR on normal tissues. Previously, we reported an antibody (mab806) against a truncated form of EGFR found commonly in gliomas. Remarkably, it also recognizes full-length EGFR on tumor cells but not on normal cells. However, the mechanism for this activity was unclear. Crystallographic structures for Fab:EGFR 287-302 complexes of mAb806 (and a second, related antibody, mAb175) show that this peptide epitope adopts conformations similar to those found in the wtEGFR. However, in both conformations observed for wtEGFR, tethered and untethered, antibody binding would be prohibited by significant steric clashes with the CR1 domain. Thus, these antibodies must recognize a cryptic epitope in EGFR. Structurally, it appeared that breaking the disulfide bond preceding the epitope might allow the CR1 domain to open up sufficiently for antibody binding. The EGFR C271A/C283A mutant not only binds mAb806, but binds with 1:1 stoichiometry, which is significantly greater than wtEGFR binding. Although mAb806 and mAb175 decrease tumor growth in xenografts displaying mutant, overexpressed, or autocrine stimulated EGFR, neither antibody inhibits the in vitro growth of cells expressing wtEGFR. In contrast, mAb806 completely inhibits the ligand-associated stimulation of cells expressing EGFR C271A/C283A. Clearly, the binding of mAb806 and mAb175 to the wtEGFR requires the epitope to be exposed either during receptor activation, mutation, or overexpression. This mechanism suggests the possibility of generating antibodies to target other wild-type receptors on tumor cells.cancer ͉ cryptic ͉ epitope ͉ therapeutic antibody ͉ structure

Characterization of ABT-806, a Humanized Tumor-Specific Anti-EGFR Monoclonal Antibody

“…The different epitopes that these antibodies recognize underlie their distinct mechanisms of action and consequent efficacies against human xenograft tumors overexpressing EGFRvIII. Previous studies suggest that binding to the ABT-806 epitope blocks receptor dimerization and subsequent activation (8). In contrast, cetuximab exerts its antitumor activity, at least in part, by binding to its epitope on domain III of the EGFR preventing ligand binding (27).…”

“…Details of EGFR protein forms (based on numbering from accession# NP_005219.2) are as follows: wild-type EGFR comprised the entire extracellular binding domain (amino acids 1-645); EGFRvIII contained an in-frame deletion generating a truncated protein with a novel glycine (residues 1-29 and G-298-645); EGFR 1-501 (the first 1-525 amino acids inclusive 24 amino acid leader); and a double mutant EGFR C271A,C283A (containing EGFR 1-645, C295A, C307A) replacing cysteine at 271 and 283 in the processed protein with alanine (8) …”

“…The targeted epitope is accessible in tumors with wild-type EGFR amplification or in tumors that express EGFRvIII, the most common deletion mutant of EGFR that lacks the ligand-binding domain of exons 2-7 and retains constitutive kinase activity (8)(9)(10). Thus mAb806 has tumor-specific binding properties and would not be expected to elicit the side effects, including skin rash, observed with other cetuximab-like EGFR targeting (mAbs) associated with targeting of normal tissues.…”

Targeting a Cancer-Specific Epitope of the Epidermal Growth Factor Receptor in Triple-Negative Breast Cancer

“…Additionally, this epitope is not known to be targeted by current anti-EGFR therapeutics such as cetuximab (21,22). The m806 antibody displayed no targeting of normal tissues and produced minimal toxicities in a phase I clinical trial (23), while demonstrating activity against cancers exhibiting resistance to cetuximab or with EGFR kinase domain mutations (19,24). These properties suggest that m806 might be uniquely suited for delivery of cytotoxins to cancers that overexpress EGFR while sparing healthy cells.…”

“…The m806 epitope is not accessible when the receptor is in an inactive monomer or activated dimer but is exposed in the locally misfolded (transitional), or "untethered," conformation, which preferentially occurs under oncogenic conditions (19,20). Additionally, this epitope is not known to be targeted by current anti-EGFR therapeutics such as cetuximab (21,22).…”