Abstract
BackgroundThe Ecballium elaterium trypsin inhibitor (EETI-II), a 28-amino acid member of the knottin family of peptides, contains three interwoven disulfide bonds that form multiple solvent-exposed loops. Previously, the trypsin binding loop of EETI-II has been engineered to confer binding to several alternative molecular targets. Here, EETI-II was further explored as a molecular scaffold for polypeptide engineering by evaluating the ability to mutate two of its structurally adjacent loops.Methodology/Principal FindingsYeast surface display was used to engineer an EETI-II mutant containing two separate integrin binding epitopes. The resulting knottin peptide was comprised of 38 amino acids, and contained 11- and 10-residue loops compared to wild-type EETI-II, which naturally contains 6- and 5-residue loops, respectively. This knottin peptide bound to αvβ3 and αvβ5 integrins with affinities in the low nanomolar range, but bound weakly to the related integrins α5β1 and αiibβ3. In addition, the engineered knottin peptide inhibited tumor cell adhesion to vitronectin, an extracellular matrix protein that binds to αvβ3 and αvβ5 integrins. A 64Cu radiolabeled version of this knottin peptide demonstrated moderate serum stability and excellent tumor-to-muscle and tumor-to-blood ratios by positron emission tomography imaging in human tumor xenograft models. Tumor uptake was ∼3–5% injected dose per gram (%ID/g) at one hour post injection, with rapid clearance of probe through the kidneys.Conclusions/SignificanceWe demonstrated that multiple loops of EETI-II can be mutated to bind with high affinity to tumor-associated integrin receptors. The resulting knottin peptide contained 21 (>50%) non-native amino acids within two mutated loops, indicating that extended loop lengths and sequence diversity were well tolerated within the EETI-II scaffold. A radiolabeled version of this knottin peptide showed promise for non-invasive imaging of integrin expression in living subjects. However, reduced serum and metabolic stability were observed compared to an engineered integrin-binding EETI-II knottin peptide containing only one mutated loop.
Highlights
Cystine-knot miniproteins, known as knottins, are small polypeptides (20–60 amino acids) that have an interwoven disulfide-bonded framework, triple-stranded b-sheet fold, and possess one or more solvent exposed loops that mediate binding to diverse targets [1,2]
We recently used yeast surface display, a combinatorial method, to identify elaterium trypsin inhibitor (EETI-II)-based knottin peptides that bound with high affinity to avb3/avb5 or avb3/avb5/a5b1 integrins [8], which are overexpressed on tumors or their neovasculature and mediate angiogenesis and metastasis [18,19,20,21]
Combinatorial libraries were created in which EETI-II Loop 3 was substituted with XXXRGDXXX, XXXRGDXXXX, and XXXRGDXXXXX, where X can be any amino acid (Figure 1B and Table S1)
Summary
Cystine-knot miniproteins, known as knottins, are small polypeptides (20–60 amino acids) that have an interwoven disulfide-bonded framework, triple-stranded b-sheet fold, and possess one or more solvent exposed loops that mediate binding to diverse targets [1,2]. The Ecballium elaterium trypsin inhibitor (EETI-II) knottin contains three disulfide bonds and binds to and inhibits trypsin through a single 6-amino acid loop [13,14]. In mouse tumor models, radiolabeled versions of these integrin-binding knottin peptides exhibited high tumor uptake with low background in non-target tissue (i.e. liver and kidney) [22,23]. The Ecballium elaterium trypsin inhibitor (EETI-II), a 28-amino acid member of the knottin family of peptides, contains three interwoven disulfide bonds that form multiple solvent-exposed loops. EETI-II was further explored as a molecular scaffold for polypeptide engineering by evaluating the ability to mutate two of its structurally adjacent loops
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