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 tumorassociated integrin receptors. The broad expression of integrins (including avb3, avb5, and a5b1) 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 cellfree 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 proofof-concept for further development of KFDCs as alternatives to ADCs for tumor targeting and drug delivery applications.

Integrins as Therapeutic Targets: Successes and Cancers

“…In this review we provide an update on the progress of integrin-specific drugs in late-stage clinical trial. Two targeting concepts form the basis for integrin therapies: inhibition of integrin function; and the use of integrin expression patterns to specify drug delivery, either as addressed microencapsulated drug [ 13 ], or in the form of affinity-binder-drug conjugates [ 14 ]. Direct inhibition of integrin function has so far dominated therapeutic strategies that have reached the clinic, and is the only form of anti-integrin therapy so far shown to work in patients, so we focus here on this therapeutic form.…”

Peptibodies: An elegant solution for a long‐standing problem

“…Finally, in the Fc domain (3), such as homodimerization of the two Fc molecules, increasing avidity by including a minimum of two peptides per peptibody . In addition to therapy, the peptide can act as an active targeting agent by decreasing off‐target effects, as it can recognizes specific receptors . In a LaFleur et al study four different peptides where added to a monoclonal antibody (mAb), named Zybody, to increase specificity …”

“…Currier et al have successfully designed an integrin‐binding knottin‐Fc‐MMAF conjugate. The peptibody efficiently managed to inhibit proliferation of human glioblastoma, ovarian, and breast cancer cells to a greater extent than the peptide‐Fc or drug alone or added in combination …”

“…[21] In addition to therapy, the peptide can act as an active targeting agent by decreasing off-target effects, as it can recognizes specific receptors. [34] In a LaFleur et al study four different peptides where added to a monoclonal antibody (mAb), named Zybody, to increase specificity. [6] The overall goal of Fc domain in the peptibody construct is to prolong the peptibodies half-life through the FcRn salvage pathway.…”

“…The peptibody efficiently managed to inhibit proliferation of human glioblastoma, ovarian, and breast cancer cells to a greater extent than the peptide-Fc or drug alone or added in combination. [34] The large scale production of peptibodies adopting different techniques distinct from recombinant DNA technology is yet another challenge. To date, the use of unmodified peptides simplified their production using either bacteria or mammalian cells expressing a recombinant protein, obtaining high expression levels and fast manufacturing process at reasonable costs.…”