Abstract
BackgroundCystine-knot miniproteins, also known as knottins, have shown great potential as molecular scaffolds for the development of targeted therapeutics and diagnostic agents. For this purpose, previous protein engineering efforts have focused on knottins based on the Ecballium elaterium trypsin inhibitor (EETI) from squash seeds, the Agouti-related protein (AgRP) neuropeptide from mammals, or the Kalata B1 uterotonic peptide from plants. Here, we demonstrate that Agatoxin (AgTx), an ion channel inhibitor found in spider venom, can be used as a molecular scaffold to engineer knottins that bind with high-affinity to a tumor-associated integrin receptor.Methodology/Principal FindingsWe used a rational loop-grafting approach to engineer AgTx variants that bound to αvβ3 integrin with affinities in the low nM range. We showed that a disulfide-constrained loop from AgRP, a structurally-related knottin, can be substituted into AgTx to confer its high affinity binding properties. In parallel, we identified amino acid mutations required for efficient in vitro folding of engineered integrin-binding AgTx variants.Molecular imaging was used to evaluate in vivo tumor targeting and biodistribution of an engineered AgTx knottin compared to integrin-binding knottins based on AgRP and EETI.Knottin peptides were chemically synthesized and conjugated to a near-infrared fluorescent dye. Integrin-binding AgTx, AgRP, and EETI knottins all generated high tumor imaging contrast in U87MG glioblastoma xenograft models. Interestingly, EETI-based knottins generated significantly lower non-specific kidney imaging signals compared to AgTx and AgRP-based knottins.Conclusions/SignificanceIn this study, we demonstrate that AgTx, a knottin from spider venom, can be engineered to bind with high affinity to a tumor-associated receptor target. This work validates AgTx as a viable molecular scaffold for protein engineering, and further demonstrates the promise of using tumor-targeting knottins as probes for in vivo molecular imaging.
Highlights
There is a critical need for in vivo molecular imaging agents that bind and with high affinity to clinical targets of interest, while displaying desirable pharmacokinetics and tissue biodistribution properties [1,2]
We showed that engineered integrin-binding knottins based on AgTx, Agouti-related protein (AgRP), and elaterium trypsin inhibitor (EETI) can be conjugated to a near-infrared fluorescent dye and used for in vivo optical imaging of tumors in mice, but that EETI-based knottins had lower non-specific accumulation in the kidneys
EETI 2.5F is an engineered knottin that binds to avb3, avb5, and a5b1 integrins [26], while AgRP 7C is an engineered knottin that binds only to avb3 integrin [27]
Summary
There is a critical need for in vivo molecular imaging agents that bind and with high affinity to clinical targets of interest, while displaying desirable pharmacokinetics and tissue biodistribution properties [1,2]. Ideal molecular imaging agents are ones that exhibit robust tumor localization and rapid clearance from non-target tissues and organs [3,4]. Knottins share a common disulfide-bonded framework, and contain loops of variable length and composition that are constrained to a core of anti-parallel beta-strands (Fig. 1) [8] This structure confers high thermal, chemical, and proteolytic stability [9,10], which is desirable for in vivo biomedical applications. Cystine-knot miniproteins, known as knottins, have shown great potential as molecular scaffolds for the development of targeted therapeutics and diagnostic agents. We demonstrate that Agatoxin (AgTx), an ion channel inhibitor found in spider venom, can be used as a molecular scaffold to engineer knottins that bind with high-affinity to a tumor-associated integrin receptor
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