Engineering agatoxin, a cystine-knot peptide from spider venom, as a molecular probe for in vivo tumor imaging

Abstract

Background: Cystine-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 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.

Figure 1. AgTx, AgRP, and EETI knottins engineered to bind tumor-associated integrins.

(A) Native knottin structures. AgTx IVB (PDB 1OMB), truncated AgRP (PDB 1MR0), and EETI-II (PDB 2ETI), with disulfide bonds shown in gold, and native loops that were mutated to bind tumor-associated integrins shown in red. Structures were rendered in PyMOL. (B) Schematic of protein engineering strategy and sequences of native and engineered knottins used in this study. Conserved cysteine residues are shown in gold, and bars indicate disulfide bond connectivity. The N- and C-termini of AgTx were truncated and the sequences of isoforms IVA and IVB were combined to create a knottin scaffold with no lysine residues (cyan), allowing for site-specific conjugation of AF680 at the N-terminal amino group. The integrin-binding loop from AgRP 7C was grafted into the structurally analogous loop of this new scaffold to create AgTx 7C. Mutated loops are underlined and shown in red. * indicates knottins used for in vivo imaging. EETI RDG contains a scrambled sequence that does not bind integrins, and was used as a negative control.

Figure 2. Synthesis and folding of AgTx 7C indicated a deletion product.

(A–C) RP-HPLC chromatograms. (A) The crude peptide from solid-phase peptide synthesis had two major peaks, one with the expected mass and the other with a loss of 156 Da, indicating possible deletion of an arginine residue. (B) Folding of crude peptide yielded a sharp peak which was 156 Da less than the expected mass. (C) Purified, folded peptide exhibited a single, sharp peak. (D) Expected and observed masses of indicated HPLC peaks as analyzed by MALDI-TOF mass spectrometry. Note that there is an 8 Da difference between unfolded and folded AgTx 7C due to the formation of 4 disulfide bonds.

Figure 3. Engineered AgTx 7C variants bind to K562-α_vβ₃ cells with similar IC₅₀ values.

Varying concentrations of AgRP 7C and AgTx 7C variants were incubated with FLAG-AgRP 7A and allowed to compete for binding to integrin receptors expressed on the surface of K562-α_vβ₃ cells. Representative competition binding curves are shown, and are plotted as knottin concentration versus the fraction of FLAG-AgRP 7A bound. IC₅₀ values reported as mean of three experiments ± SD.

Figure 4. Unlabeled and AF680-labeled knottins bind U87MG cells with high affinity.

Unlabeled (open squares, dashed line) and AF680-labeled (closed circles, solid line) knottins similarly compete off FLAG-AgRP 7A knottin binding to α_vβ₃ integrins expressed on U87MG glioblastoma cells. Representative competition binding curves shown for (A) AgTx 7C ΔR21, (B) AgRP 7C, (C) EETI 2.5F, and (D) EETI RDG control. IC₅₀ values reported as mean of three experiments ± SD.

Figure 5. Non-invasive in vivo imaging of AF680-labeled knottins in U87MG tumor xenografts.

(A) Representative whole-body fluorescent images of murine U87MG tumor xenografts injected via tail vein with 1.5 nmol AF680-labeled knottins AgTx 7C ΔR21, AgRP 7C, EETI 2.5F, and EETI RDG control. Tumors (white arrow) and kidneys (K) are indicated. Radiant efficiency [ = ] (p/s/cm²/sr)/(μW/cm²). (B–C) Quantification of imaging signals, reported as the total radiant efficiency, in the (B) tumor and (C) kidney over 24 hr. Total radiant efficiency [ = ] (p/s)/(μW/cm²). (D) Imaging contrast, reported as the ratio of fluorescent signals for tumor versus normal tissue. There is no statistical difference in imaging contrast between AgTx 7C ΔR21 and AgRP 7C at all time points measured (p>0.05). Error bars represent ± SE, n = 4 for all knottins.

Figure 6. Ex vivo imaging of tissue and organs from U87MG tumor xenografts.

(A) Representative ex vivo images of tumor, kidney, liver, muscle, and blood at 4 hr post injection of 1.5 nmol AF680-labeled knottins. (B) Quantification of total imaging signal per organ, normalized to organ mass, corroborates high kidney signals for AgTx 7C ΔR21 and AgRP 7C compared to EETI-based knottins. No fluorescent signal was detectable in blood samples at 4 hr post probe injection. Error bars represent ± SE. For all knottins, n = 9, except EETI RDG (n = 4).