Epidermal growth factor receptor (EGFR) is a well known and validated target for monoclonal antibody (mAb) therapeutics. Three anti-EGFR antibodies are currently marketed, cetuximab, necitumumab, and panitumumab. Cetuximab, a recombinant chimeric (human-mouse) monoclonal antibody (mAb) was the first approved, in February 2004, for treatment of colorectal cancer in patients who failed to respond to irinotecan-based chemotherapy.  By binding to EGFR with high affinity, the anti-EGFR antibodies prevent EGF, the ligand to EGFR, binding, and therefore block receptor activation and subsequent pro-survival and proliferation-associated signaling pathways. Therefore, in tumors that depend on this receptor to grow, blocking EFGR can halt tumor progression. This is critical, as patients whose tumors had elevated levels of EGFR/EGF were more likely to have aggressive disease, and therefore a poorer prognosis. 
Patients commonly become resistant to anti-EGFR antibody therapies through mutational escape. Cetuximab, necitumumab, and panitumumab bind relatively close epitopes and even share epitope regions on EGFR domain III. [3-5] Whilst a mutation in EGFR can make tumors resistant to one antibody but still susceptible to the remaining two such as in the case of S492R that blocks cetuximab binding but panitumumab remains able , there are many mutations that can block a tumor’s susceptibility to all three antibodies simultaneously. 
Another anti-EGFR mAb, derived from mouse and known as 528, was first reported in the early 1980s. [7,8] Makabe and colleagues  recently reported that, while 528 also binds EGFR domain III, its epitope includes a loop formed by residues 353–362 that is not part of the binding sites of cetuximab, necitumumab, and panitumumab. Thus, tumors that are resistant to all three of the currently available antibodies could in theory be susceptible to 528. Although additional studies are required to accurately deduce the interaction of EGFR and 528, compare 528 to the existing therapies, and assess the effects of various EGFR mutations, these initial findings by Makabe and colleagues are intriguing and represent a worthwhile avenue to explore.
Scientists have also investigated 528’s anti-EGFR binding capabilities in bispecific formats that may have therapeutic potential. Humanized versions of 528’s variable region and the anti-CD3 variable region derived from OKT-3, an immunosuppressant drug, were used to construct a bispecific molecule, hEx3, with the aim of bridging T cells to EGFR on cancer cells, thereby targeting the cancer cells for destruction.  This bispecific construct was shown to form functional tetramers.  The cytotoxicity of hEx3 could be enhancement by affinity maturation , by rearranging the variable domain order [13, 14] and by generating Fc fusions. [14, 15 Taken together, the findings of these studies are intriguing. The simple rearrangement of the heavy and light domains from heavy-light to light-heavy substantially enhanced the cytotoxic anti-tumor activity of the hEx3 diabody, as did the introduction of a LH-HY52W mutation hypothesised to increasing affinity of the 528 variable region and its target, EGFR. Moreover, the engineered molecules had enhanced anti-tumour killing in vivo.  This result may be associated with increased valency or perhaps through the reduction of serum clearance, which is currently an obstacle to use of non-native, truncated antibody formats. 
Overall, anti-EGFR based antibody therapeutics utilizing 528’s epitope-binding region may present new avenues of attack due to its distanced binding site compared to existing therapies. Importantly, nuanced changes to antibody structures, including simple domain rearrangements and alteration of the amino acid sequence, could translate into substantial changes to efficacy.
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