Abstract
The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase whose proliferative effects can contribute to the development of many types of solid tumors when overexpressed. For this reason, EGFR inhibitors such as cetuximab can play an important role in treating cancers such as colorectal cancer and head and neck cancer. Cetuximab is a chimeric monoclonal antibody containing mouse variable regions that bind to EGFR and prevent it from signaling. Although cetuximab has been used clinically since 2004 to successfully control solid tumors, advances in protein engineering have created the opportunity to address some of its shortcomings. In particular, the presence of mouse sequences could contribute to immunogenicity in the form of anti-cetuximab antibodies, and an occupied glycosylation site in FR3 can contribute to hypersensitivity reactions and product heterogeneity. Using simple framework graft or sequence-/structure-guided approaches, cetuximab was humanized onto 11 new frameworks. In addition to increasing humanness and removing the VH glycosylation site, dynamic light scattering revealed increases in stability, and bio-layer interferometry confirmed minimal changes in binding affinity, with patterns emerging across the humanization method. This work demonstrates the potential to improve the biophysical and clinical properties of first-generation protein therapeutics and highlights the advantages of computationally guided engineering.
Highlights
Cetuximab is a chimeric IgG1 monoclonal antibody that was approved in 2004 for the treatment of colorectal cancer and in 2006 for the treatment of squamous cell carcinoma of the head and neck [1,2,3]
By binding to domain III of the extracellular domain of the epidermal growth factor receptor (EGFR), which is overexpressed on tumor cells, cetuximab competitively inhibits the binding of EGF and other ligands, preventing the dimerization of EGFR [1]
The immunogenicity of cetuximab appears low based on the low incidence of anti-cetuximab IgG responses (5%), hypersensitivity is a common occurrence due largely to pre-existing IgE antibodies against the galactose-α-1,3-galactose oligosaccharide that modifies the VH when expressed in Sp2/0 cells [10,11,12]
Summary
Cetuximab is a chimeric IgG1 monoclonal antibody that was approved in 2004 for the treatment of colorectal cancer and in 2006 for the treatment of squamous cell carcinoma of the head and neck [1,2,3]. In 2020, cetuximab saratolacan, an antibody– dye conjugate that photosensitizes EGFR-bearing tumors, was approved in Japan for the treatment of head and neck cancer, demonstrating the potential of cetuximab binding domains to be used in alternative formats, such as antibody–drug conjugates and multipolicy antibodies [5]. Humanization can increase the stability of antibodies by making the framework regions more compatible [7] Another concern is the occupied glycan site at VH N85 (Kabat), where Fab glycosylation could affect the biological properties of the antibody as well as introducing glycan heterogeneity, which must be well controlled during manufacturing [8,9]. The immunogenicity of cetuximab appears low based on the low incidence of anti-cetuximab IgG responses (5%), hypersensitivity is a common occurrence due largely to pre-existing IgE antibodies against the galactose-α-1,3-galactose oligosaccharide that modifies the VH when expressed in Sp2/0 cells [10,11,12]
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