title: Targeted Drug Delivery with an Integrin-Binding Knottin-Fc-MMAF Conjugate Produced by Cell-Free Protein Synthesis.
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.
authors.affiliation: Division of Pediatric Hematology/Oncology, Stanford Medical School, Stanford, California.
authors.forename: Nicolas V
authors.initials: NV
authors.lastname: Currier
authors.affiliation: Department of Bioengineering, Stanford University, Stanford, California.
authors.forename: Shelley E
authors.initials: SE
authors.lastname: Ackerman
authors.affiliation: Department of Bioengineering, Stanford University, Stanford, California.
authors.forename: James R
authors.initials: JR
authors.lastname: Kintzing
authors.affiliation: Sutro Biopharma, Inc., South San Francisco, California.
authors.forename: Rishard
authors.initials: R
authors.lastname: Chen
authors.affiliation: Department of Bioengineering, Stanford University, Stanford, California.
authors.forename: Maria
authors.initials: M
authors.lastname: Filsinger Interrante
authors.affiliation: Sutro Biopharma, Inc., South San Francisco, California.
authors.forename: Alexander
authors.initials: A
authors.lastname: Steiner
authors.affiliation: Sutro Biopharma, Inc., South San Francisco, California.
authors.forename: Aaron K
authors.initials: AK
authors.lastname: Sato
authors.affiliation: Department of Bioengineering, Stanford University, Stanford, California. Department of Chemical Engineering, Stanford University, Stanford, California. jennifer.cochran@stanford.edu.
authors.forename: Jennifer R
authors.initials: JR
authors.lastname: Cochran
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002474:Cell-Free System; D057771:Cystine-Knot Miniproteins / Q000737:chemistry*; D016503:Drug Delivery Systems; D006801:Humans; D018796:Immunoconjugates / Q000737:chemistry / Q000494:pharmacology*; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry; D016023:Integrins / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D009369:Neoplasms / Q000188:drug therapy*; D009842:Oligopeptides / Q000737:chemistry / Q000494:pharmacology*; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D023041:Xenograft Model Antitumor Assays
chemical_list: D057771:Cystine-Knot Miniproteins; D018796:Immunoconjugates; D007141:Immunoglobulin Fc Fragments; D016023:Integrins; D009842:Oligopeptides; D010455:Peptides; C513576:monomethylauristatin F