Improving the efficacy and side effect profile of therapeutic antibodies
Abstract
Therapeutic antibodies exhibit side effects from binding non-target tissues that limit their effectiveness or cause harm, including acneiform folliculitis and gastrointestinal upset (cetuximab and matuzumab) and cardiotoxicity (trastuzumab). These antibodies are approved to treat tumors wherein proteases are aberrantly expressed, including matrix metalloprotease-9 (MMP-9). To limit the side effects of these otherwise potent therapeutics, "masks" that block antibody binding systemically and that are removed by protease exposure are designed, developed and explored to enhance tumor specificity. Herein, a design is demonstrated that leverages synergy in tumor cell death identified for the combination of anti-Epidermal Growth Factor Receptor (EGFR) antibodies cetuximab and matuzumab to create a cross-masked antibody design. Specifically, the antibodies' epitope (domain III of the EGFR) was fused N-terminally to a single chain variable fragment (scFv) construct of cetuximab and 425 (matuzumab's murine precursor) through an MMP-9 site containing linker. In each mask, mutations were introduced into EGFR domain III (EGFRdIII) to reduce the intramolecular affinity—S460P/G461N for 425 as determined herein and mutations from the literature for cetuximab. These mask-antibody fusions were mixed to form a heterodimer or cross-masked structure. MMP-9 exposure increased the affinity 8-fold in surface plasmon resonance (SPR) analysis using immobilized EGFRdIII. Flow cytometry confirmed higher binding after MMP-9 exposure. We also explored small peptides as masks to replace the EGFRdIII. Using diffraction methods, it was shown that previously described (cetuximab binding) peptides bind to a novel framework binding site, but not the expected complementarity determining regions (CDRs). Size exclusion chromatography and SPR confirmed simultaneous binding of cetuximab, EGFRdIII and the mimotope. Further, the mimotope bound the fragment antigen binding [F(ab)'], but not a scFv construct of cetuximab that places a linker across the peptide binding site. While the intention was to use these peptides to occlude the CDRs, these peptides could also be used as an anchor to sterically occlude tumor-derived antigen and thus create a non-covalent mask. Initial studies define critical parameters in implementing this concept. Taken together, modulation of antibody affinity through covalent/non-covalent masking represents a novel and potentially powerful method to improve the targeting of therapeutic antibodies to the tumor.
Subject Area
Biochemistry|Medicine|Biophysics
Recommended Citation
Donaldson, Joshua Michael, "Improving the efficacy and side effect profile of therapeutic antibodies" (2012). ProQuest ETD Collection - Thomas Jefferson University. AAI3498429.
https://jdc.jefferson.edu/dissertations/AAI3498429
