Abstract
Antibody-drug conjugates (ADC) have generated significant interest as targeted therapeutics for cancer treatment, demonstrating improved clinical efficacy and safety compared with systemic chemotherapy. To extend this concept to other tumor-targeting proteins, we conjugated the tubulin inhibitor monomethyl-auristatin-F (MMAF) to 2.5F-Fc, a fusion protein composed of a human Fc domain and a cystine knot (knottin) miniprotein engineered to bind with high affinity to tumor-associated integrin receptors. The broad expression of integrins (including αvβ3, αvβ5, and α5β1) on tumor cells and their vasculature makes 2.5F-Fc an attractive tumor-targeting protein for drug delivery. We show that 2.5F-Fc can be expressed by cell-free protein synthesis, during which a non-natural amino acid was introduced into the Fc domain and subsequently used for site-specific conjugation of MMAF through a noncleavable linker. The resulting knottin-Fc-drug conjugate (KFDC), termed 2.5F-Fc-MMAF, had approximately 2 drugs attached per KFDC. 2.5F-Fc-MMAF inhibited proliferation in human glioblastoma (U87MG), ovarian (A2780), and breast (MB-468) cancer cells to a greater extent than 2.5F-Fc or MMAF alone or added in combination. As a single agent, 2.5F-Fc-MMAF was effective at inducing regression and prolonged survival in U87MG tumor xenograft models when administered at 10 mg/kg two times per week. In comparison, tumors treated with 2.5F-Fc or MMAF were nonresponsive, and treatment with a nontargeted control, CTRL-Fc-MMAF, showed a modest but not significant therapeutic effect. These studies provide proof-of-concept for further development of KFDCs as alternatives to ADCs for tumor targeting and drug delivery applications. Mol Cancer Ther; 15(6); 1291-300. ©2016 AACR.
Highlights
Antibody–drug conjugates (ADC) have risen to the forefront of cancer therapy in recent years due to their ability to combine the tumor-targeting selectivity of mAbs with the delivery of potent cytotoxic agents [1]
EETI 2.5F was genetically fused to a human IgG1 Fc domain to create a construct termed 2.5F–Fc, which was produced by cell-free protein synthesis using an E. coli–based extract [14]
A nonnatural amino acid was selectively incorporated into the Fc domain by replacing position F404 with an amber stop codon that allowed incorporation of pAMF using a modified aminoacyl tRNA synthetase. 2.5F–Fc proteins were expressed at high levels using cell-free protein synthesis, and following purification were analyzed by SDS-PAGE and gel filtration chromatography (Fig. 1B and C)
Summary
Antibody–drug conjugates (ADC) have risen to the forefront of cancer therapy in recent years due to their ability to combine the tumor-targeting selectivity of mAbs with the delivery of potent cytotoxic agents [1]. This enthusiasm has been led by recent FDA approvals of ADCs including Adcetris (Seattle Genetics) and Kadcyla Adcetris (brentuximab vedotin), which targets the cell surface protein CD30, is approved for relapsed or post–stem cell transplant Hodgkin lymphoma and systemic anaplastic large cell lymphoma [3]. Numerous other ADCs, Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/). Ackerman are co-first authors and contributed to this article
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