Abstract | BACKGROUND: METHODOLOGY/PRINCIPAL FINDINGS: We 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/SIGNIFICANCE: In 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.
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Authors | Sarah J Moore, Cheuk Lun Leung, Heidi K Norton, Jennifer R Cochran |
Journal | PloS one
(PLoS One)
Vol. 8
Issue 4
Pg. e60498
( 2013)
ISSN: 1932-6203 [Electronic] United States |
PMID | 23573262
(Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, Non-P.H.S.)
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Chemical References |
- Agatoxins
- Carbocyanines
- Fluorescent Dyes
- Integrin alphaVbeta3
- Cysteine
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Topics |
- Agatoxins
(chemistry, genetics)
- Amino Acid Substitution
- Animals
- Carbocyanines
(chemistry)
- Cysteine
(genetics)
- Cystine Knot Motifs
- Female
- Fluorescent Dyes
(chemistry)
- Humans
- Integrin alphaVbeta3
(metabolism)
- K562 Cells
- Mice
- Mice, Nude
- Mutagenesis, Site-Directed
- Neoplasm Transplantation
- Neoplasms
(diagnosis)
- Protein Binding
- Protein Engineering
- Protein Folding
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