Antibody drug conjugates (ADCs) are a rapidly expanding class of therapeutics that combine the targeting capabilities of monoclonal antibodies with the cytotoxicity of highly potent chemotherapeutics. Six of the ten current FDA approved ADCs received approval in the past two years alone. However, all current formulations of ADCs rely on drug conjugation methods that result in heterogeneous ADC populations, both in terms of the number of drugs conjugated to the antibody (drug to antibody ratio, DAR) and the site of conjugation. Variability in DAR and heterogeneity in the sites of conjugation both affect the pharmacokinetics and efficacies of these molecules. There is a significant need for a site specific and stoichiometric method of conjugation that is stable in plasma and does not require the use of harsh reaction conditions which could lead to decreased antibody affinity or downstream toxicity.
Supramolecular assembly through the use of coiled-coil domains has been demonstrated through the formation of fibers, hydrogels, peptide “origami,” multimeric single chain variable fragments (scFvs), and split, universal, and programmable chimeric antigen receptor (SUPRA CAR) T cells. Here, we describe the use of a heterotetrameric coiled-coil system in which one set of coiled-coil peptides is expressed on the C termini of an antibody and the other set is chemically conjugated to the highly cytotoxic microtubule inhibitor monomethyl auristatin E (MMAE). The peptides are either positively charged or negatively charged to discourage homotetramerization, but when mixed under physiological conditions, will form a heterotetrameric coiled-coil. In this way, we control the loading of two drug molecules onto the antibody in a site-specific manner.
We explore the use of this coiled-coil system for the treatment of non-small cell lung cancer (NSCLC), which only has a 25% five-year survival rate. GPR87 is a receptor overexpressed in NSCLC in both adenocarcinoma and squamous cell carcinoma, as we demonstrate by the staining of primary NSCLC patient samples. We have prepared an αGPR87-MMAE ADC using coiled-coil based supramolecular assembly. Our ADC is non-cytotoxic to a control GPR87 negative cell line and exhibits in vitro IC50s of 6.3 nM and 9.0 nM in the NSCLC cell lines NCI-H358 and NCI-H520, respectively. Preliminary in vivo experiments using the corresponding antibody fluorophore conjugate (AFC) show higher accumulation of the AFC in a stage 4 NSCLC patient derived xenograft tumor vs. other major organs, such as the liver, kidney, heart, and lungs, 72 hours following injection. / 2023-09-24T00:00:00Z
| Identifer | oai:union.ndltd.org:bu.edu/oai:open.bu.edu:2144/43084 |
| Date | 25 September 2021 |
| Creators | Berry, Samantha Marie |
| Contributors | Grinstaff, Mark W. |
| Source Sets | Boston University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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