Pharmacokinetically Stabilized Cystine Knot Peptides That Bind Alpha-v-Beta-6 Integrin with Single-Digit Nanomolar Affinities for Detection of Pancreatic Cancer

Integrins are a family of heterodimeric cell surface receptors that mediate cellular adhesion to extracellular matrix proteins. Integrins also serve as bidirectional signal transducers to regulate differentiation, migration, proliferation, and cell death (1, 2). Integrin α_vβ₃ promotes tumor neovascularization (3, 4). However in certain cancers, other integrins, such as α_vβ₆, become highly overexpressed on cell surfaces (2, 5, 6). Therefore, this biomarker is being validated for detection of colon, liver, ovarian, pancreatic, and squamous cell cancers (7–9). Molecular imaging of integrin α_vβ₆ may be used to gauge receptor expression levels to determine prognosis and guide therapy (10–12). Here, we have developed potent integrin α_vβ₆ binders for clinical translation as radiotracers for early cancer detection.

Previously, several integrin α_vβ₆ binders were identified from natural and combinatorial sources. Linear α_vβ₆-binding peptides derived from the coat protein of foot-and-mouth disease viruses (FMDV) generally suffered poor in vivo stability, which raise concerns about their potential immunogenicity. ⁶⁴Cu-labeled versions of FMDV peptides showed extremely high renal retention, which suggests that these peptides may not be ideal translational candidates. An alternative to radiometals is the use of radiohalogens (7, 9, 13). Phage display systems have identified several linear and disulfide-cyclized peptides that bind α_vβ₆ (14, 15). In one study, a radio-iodinated linear peptide HBP-1 showed rapid degradation in serum (16). One-bead-one-compound libraries have identified many binders, of which 43 ¹⁸F-labeled linear peptides were tested in a high throughput live animal imaging survey (17). Although some of these peptides show promising small animal data, linear peptides or even simple disulfide-bonded peptides with stability problems may discourage translation (17). Peptide fragments can be highly immunogenic thus rendering the parent peptide untranslatable (18). For these reasons, we sought to generate high-affinity binders that are very stable in physiologic media, show low off-target accumulation and effectively detect cancer in living subjects.

Engineered cystine knot peptides (knots) have shown promise for cancer imaging with α_vβ₃ as a target (19–21). The cystine knot is a rigid molecular scaffold of 3 to 4 kDa that owes its exceptionally high stability to 3 interwoven disulfide bonds and a centrally located β-sheet. Potent receptor-binding activities have been engineered into the scaffold's solvent exposed loops (22). The knotted structure helps to resist degradation/denaturation in hostile biologic, chemical, and physical environments such as strong acids and boiling water (23). Cystine knots have shown exceptional structural stability during prolonged incubation in serum (19). Moreover, their use in humans as uterogenics has not led to reports of adverse side effects, albeit without formal published studies (24, 25). Binding potency remains high for engineered knots that we have subjected to long-term storage (>1 year) in water at 4°C or stored in lyophilized form at room temperature. The N-terminus provides a sole primary amine for site-specific conjugation of imaging labels, bioactive cargo, or pharmacokinetic stabilizers. Collectively, these characteristics bode well for clinical translation.

In this study, we developed new cystine knots that bind integrin α_vβ₆ expressed on pancreatic tumors grown in mice. The binders showed single-digit nanomolar dissociation constants similar to antibodies used clinically for imaging and therapy. These new knots rapidly accumulated in α_vβ₆-positive tumors, which led to excellent tumor-to-normal contrast. The new knots did not accumulate in tumors that do not express integrin α_vβ₆. The peptides were specific for the targeted integrin α_vβ₆ receptors expressed on orthotopic pancreatic tumors and various xenografts used in this study. In addition, pharmacokinetic stabilization strategies endowed knots with rapid renal clearance, which significantly reduced off-target dosing. This study indicates a broadly applicable way to engineer new binders and to stabilize their pharmacokinetics for enhanced clinical performance.

The Supplementary Material section provides additional details.

BxPC-3 pancreatic cancer cells were obtained from American Type Culture Collection (ATCC) and grown in RPMI-1640 media (ATCC). A431 epidermoid cancer cells, human embryonic kidney 293T cells (293), U87MG (malignant glioma), and MDA-MB-435 (breast cancer) cells were obtained from frozen lab stocks and grown in Dulbecco's Modified Eagle's Medium supplemented with 10% FBS and penicillin/streptomycin (Invitrogen). Recombinant human integrins α_vβ₆, α_vβ₃, α_vβ₅, and α₅β₁ were purchased from R&D Systems. All other chemicals were obtained from Fisher Scientific unless otherwise specified. Yeast media, growth and induction conditions, and integrin-binding buffer (IBB) are previously described (22).

The open reading frames encoding cystine knot peptides were generated by overlap-extension PCR using yeast-optimized codons. Positions for randomization, denoted by “X” were constructed with NNB degenerate codons (Supplementary materials). PCR products were amplified using primers with overlap to the pCT yeast display plasmid upstream or downstream of the NheI and BamHI restriction sites, respectively. For each library, approximately 40 μg of DNA insert and 4 μg of linearized pCT vector were electroporated into the Saccharomyces cerevisiae strain EBY100 by homologous recombination as previously described (26). For libraries L2 and L3, approximately 5 × 10⁶ to 7 × 10⁶ transformants per sub-library were combined for a total diversity of approximately 1 × 10⁷ clones as estimated by serial dilution plating and colony counting. For libraries L2.1 and L3.1, approximately 2 × 10⁶ transformants per sub-library were combined for a total diversity of approximately 4 × 10⁶ clones.

For library screening, various concentrations of recombinant α_vβ₆ integrin were added to yeast suspended in IBB for various times at room temperature. Next, a 1:250 dilution of chicken anti-cMyc IgY antibody (Invitrogen) was added for 1 hour at 4°C. The cells were washed with ice-cold IBB and incubated with a 1:25 dilution of fluorescein-conjugated anti-α_v integrin antibody (Biolegend) and a 1:100 dilution of Alexa 555–conjugated goat anti-chicken IgG secondary antibody (Invitrogen) for 0.5 hours at 4°C. Cells were washed in IBB and α_vβ₆ integrin binders were isolated using a Becton Dickinson FACS Aria III instrument. For the first round of sorting, approximately 2 × 10⁷ yeast clones were screened with 100 nmol/L α_vβ₆ integrin. To increase sort stringency, integrin concentrations were successively decreased to 1 nmol/L in later sort rounds, and a diagonal sort gate was used to isolate yeast cells with enhanced integrin binding (FITC fluorescence) for a given protein expression level (Alexa 555 fluorescence). For the second diversification round, approximately 5 × 10⁶ yeast clones were screened as described earlier with a final concentration of 300 pmol/L α_vβ₆ integrin coupled with 3 days of washing in IBB at 37°C. Plasmid DNA was recovered by Zymoprep (Zymo Research), amplified in Max Efficiency DH5α Escherichia coli cells (Invitrogen) and sequenced.

S₀2 and R₀1 were synthesized, folded, and purified as previously described with a modification to the folding buffer, which included the addition of an equal volume of isopropanol and 800 mmol/L guanidium hydrochloride (22). A20 was similarly prepared without folding. R₀2 and E₀2 were biosynthesized using Pichia pastoris (Supplementary Materials). Peptides were conjugated through their N-terminus amine to DOTA-NHS and radiolabeled with ⁶⁴Cu²⁺ as previously described (19). The radiochemical purity was determined by high-performance liquid chromatography (HPLC) to be more than 95%. The radiochemical yield was usually more than 80%. The specific activity of the probe was approximately 500 Ci/mmol. Molecular masses were confirmed by matrix-assisted laser desorption/ionization–mass spectrometry (MALDI-MS; ABI 5800, Supplementary Table S2).

Various concentrations of integrin α_vβ₆, α_vβ₅, α_vβ₃, and α₅β₁ were incubated with 10⁵ yeast cells expressing R₀1, R₀2, R₀3, S₀1, S₀2, S₀3, or 2.5F in the presence of 10⁶ uninduced yeast cells as previously described (22, 27). Prior to flow cytometric analysis, yeast cells were processed, stained, and washed as described earlier in Library Synthesis and Screening.

Neutravidin-coated wells (Pierce) were saturated with biotinylated peptides A20, R₀1, S₀2, or 2.5F. A concentration of 10 nmol/L of recombinant integrins or 10⁵ cells in IBB were added to wells at room temperature for 2 hours. Wells were washed 3 times with ice-cold IBB. Integrins were detected with mouse anti-α_v or anti-α₅ primary antibodies (Biolegend) and anti-mouse Alexa-488 (Cellsignal). Cells were detected with crystal violet (22). Signals were quantified with a plate reader (Tecan). For competition binding assays, integrin α_vβ₆ or BxPC-3 cells were preincubated with R₀1 or S₀2 for 2 hours prior to introduction into A20-coated wells.

Aliquots of ⁶⁴Cu-DOTA peptides were incubated in an equal volume of mouse or human serum for up to 24 hours. Samples were acidified with trifluoroacetic acid and centrifuged to remove precipitants. In addition, ⁶⁴Cu-DOTA peptides were extracted from full mouse bladders 1.5 to 2 hours after injection. Soluble fractions were filtered with a Spin-X 0.2 μm filter (Corning) when necessary. All samples were analyzed by radio-HPLC on a Dionex C₄ column.

Animal procedures were conducted per protocols 11580 and 21637 (Stanford University Administrative Panels on Laboratory Animal Care). Female athymic nude mice, 4 to 6 weeks old (Charles River), were subcutaneously shoulder-injected with 10⁷ cells suspended in 100 μL PBS. Orthotopic tumors were generated with 10⁶ cells/20 μL Matrigel (Supplementary materials). Mice were used for imaging/biodistribution studies when xenografts or orthotopic tumors reached approximately 10 or 5 mm, respectively, in diameter.

Tumor-bearing mice (n = 3 for each probe) were injected with approximately 50 to 100 μCi (∼0.15 nmol) of probe via the tail vein and imaged with a microPET R4 rodent model scanner (Siemens) using 5 minute static scans. Images were reconstructed by a 2-dimensional ordered expectation maximum subset algorithm and calibrated as previously described (28). Region of interests (ROI) were drawn over the tumor on decay-corrected whole body images using ASIPro VM software (Siemens). ROIs were converted to counts/g/min, and %ID/g values were determined assuming a tissue density of 1 g/mL. No attenuation correction was conducted.

Anesthetized nude mice bearing xenograft/orthotopic tumors were injected with approximately 50 to 100 μCi (∼0.15 nmol) of ⁶⁴Cu-DOTA peptides via tail vein and euthanized after 1 or 24 hours. Tissues were removed, weighed, and measured by scintillation counting (19, 29). Radiotracer uptake in tissues was reported as %ID/g and represents the mean ± SD of experiments carried out on 3 mice.

Nine phage display–derived lead-motif (RTDLXXL) containing sequences were engrafted into loop-1 of an acyclized cystine knot scaffold Momordica cochinchinensis Trypsin Inhibitor-II (MCoTI-II) from squash (14, 30, 31). These chimeric binders are referred to as the “R-knots” (R₀) as MCoTI-II has high arginine content (Fig. 1). Optimal loop-1 length and motif position were determined, which enabled design of libraries X₃RTDLXXLX₃ and X₄RTDLXXLX₃ (L2 and L3 Fig. 1B; Supplementary Fig. S1), which were pooled and sorted by fluorescence-activated cell sorting (FACS). The X₄RTDLXXLX₃ library dominated sort-round five (1 nmol/L target). Frequent occurrence of arginine or lysine residues at certain loop positions suggested favorable binding interactions. Arginine was fixed at these positions in the second libraries (L2.1 and L3.1) to ensure a sole N-terminus amine for chemical labeling. The remaining loop positions were randomized (Fig. 1, Schematic 1A). The final sort-round was conducted in 300 pmol/L target followed by 3 days of washing at 37°C. The top 3 most-represented binders (1, 2, and 3) were characterized.

A novel α_vβ₆-binding activity, “2,” was grafted into several arginine-rich scaffolds (R_0–3), glutamic acid–rich scaffolds (E_0-4) and serine-rich scaffolds (S_0–3). α_vβ₆ binding was determined by flow cytometry (Fig. 1B; Supplementary Fig. S2B and Schematic S1) to be comparable across scaffolds; maintenance of high-affinity binding to integrin α_vβ₆ indicates that engineered loops and knotted scaffolds can function independently and interchangeably. Novel integrin α_vβ₆-binding activities called “1,” “2,” and “3” were each tested in the context of both the R₀ and S₀ scaffolds for their ability to bind target. We measured the K_D of R₀1, R₀2, and R₀3 to be 3.6 ± 0.9, 3.2 ± 2.7, and 3.6 ± 1.6 nmol/L, respectively, by flow cytometry using soluble integrin α_vβ₆ (Fig. 2A). Interestingly, the K_D of the S₀1 and S₀2 was only slightly less at 6.5 ± 2.0 and 6.0 ± 0.1 nmol/L, respectively, whereas the K_D of S₀3 (3.1 ± 0.5 nmol/L) matched that of its parent (Fig. 2B). These results suggest that the knotted structure is tightly maintained in engineered R₀ and S₀ binders, so that novel activities can be trans-grafted into other wild-type or rationally designed knots without notable loss of potency (Supplementary Fig. S2B). We also tested cross-reactivity of peptides used throughout these studies to other integrins. Peptides show very low binding to integrins α_vβ₃, α_vβ₅, and α₅β₁ (Fig. 2C). Scrambled peptides did not bind integrin α_vβ₆ (Supplementary Fig. S2A).

Biotinylated peptides A20, R₀1, S₀2, and 2.5F were immobilized onto neutravidin-coated plates. All four peptides captured recombinant integrin α_vβ₆, but only knottin 2.5F captured integrins α_vβ₃, α_vβ₅, and α₅β₁. Engineered binders show different levels of specificity for their targets (Fig. 3A). Biotinylated A20, precoated onto microwell plates, engaged in competitive binding with soluble R₀1 or S₀2 for recombinant integrin α_vβ₆. Dose-dependent inhibition indicated competition between peptides for a specific target-binding site (Fig. 3B). A20, R₀1, and S₀2 also captured cells that express native integrin α_vβ₆ (Fig. 3C). Flow cytometry showed all tested cell lines (BxPC-3, A431, U87MG, MDA-MB-435) but the 293 cells express integrin α_vβ₆ (Supplementary Fig. S3A and S3B). Peptides R₀1 and S₀2 blocked adhesion of BxPC-3 cells onto A20-coated wells confirming specific binding between peptides and functionally active integrins expressed on cellular surfaces (Fig. 3D). ⁶⁴Cu-DOTA–labeled peptides also showed binding to target-expressing cells and not to 293 negative controls (Supplementary Fig. S4).

Most wild-type and engineered knots resist degradation/denaturation in physiologic media such as serum and urine (19, 23, 29). However, loop engineering can compromise stability. For example, R₀2 was approximately 80% degraded during 24 hours serum incubation (Fig. 4A). Comparatively, ⁶⁴Cu-DOTA–labeled R₀1 showed much greater stability and approximately 20% degradation occurred during 24 hours serum incubation. These 2 peptides share identical primary sequences in loops 2 through 5 of their structural frames (Supplementary Schematic S1). In contrast, ⁶⁴Cu-DOTA-S₀2 and -E₀2 showed exceptionally high (>95%) stability during 24 hour serum incubation (Fig. 4B and C). For completeness, the linear control ⁶⁴Cu-DOTA-A20 was more than 90% degraded after 24 hours of incubation in serum (Fig. 4D). Urine samples drawn from mice 1.5 to 2 hours after injection showed approximately 20% degradation of ⁶⁴Cu-DOTA-R₀1 and less than 5% degradation of ⁶⁴Cu-DOTA S₀2, our current lead translational candidates (Supplementary Fig. S4A and S4B). These results show that nonbinding portions of peptide scaffolds may be used to increase in vivo stability.

Radiolabeled versions of knots were evaluated by positron emission tomography (PET) in mice bearing integrin α_vβ₆-expressing BxPC-3 (pancreatic cancer) or A431 (epidermoid cancer) xenografts and α_vβ₆-negative 293 tumors. Mice were injected with approximately 75 μCi of ⁶⁴Cu-DOTA–labeled peptides. R₀2, E₀2, S₀2, R₀1, and the positive control A20 rapidly accumulated in α_vβ₆-positive A431 tumors and generated excellent tumor-to-muscle contrast ratios of 6 to 11 at 1 hour postinjection (Fig. 5A and B). Absolute uptake in A431 tumors was highest for the 2 R₀-based binders R₀1 (∼5%ID/g) and R₀2 (∼4%ID/g) than for E₀2 (∼1.5%ID/g), S₀2 (∼2%ID/g), and A20 (∼2%ID/g) at 1 hour postinjection (Fig. 5A and B).

PET studies of BxPC-3 pancreatic xenografts confirmed these results. Importantly, significantly less radiotracer accumulated in α_vβ₆-negative 293 xenografts (P < 0.05, n = 3 mice; Fig. 5D). Comparatively, tumor uptake of R₀2 measured 4.3%ID/g ± 0.7%ID/g in BxPC-3 xenografts compared with 1.3%ID/g ± 0.1%ID/g in 293 xenografts. S₀2 and R₀1 corroborated these results (Fig. 5D). Collectively, these results show that the α_vβ₆-specific probes selectively bind α_vβ₆-positive (T+) tumors (A431 and BxPC-3) and do not accumulate in the α_vβ₆-negative (T−) 293 tumors (Fig. 5A and D). Unfortunately, conclusive identification of orthotopic BxPC-3 tumors by PET remained elusive.

Biodistribution analysis of R₀1 and S₀2 in BxPC-3 xenograft tumors showed 4.13%ID/g ± 1.01%ID/g and 1.80%ID/g ± 0.50%ID/g 1 hour postinjection. These data closely matched corresponding PET data (Table 1 and Fig. 5D). Importantly, S₀2 effectively accumulated in BxPC-3 orthotopic tumors (1.85%ID/g ± 0.11%ID/g, 1 hour postinjection) compared with normal pancreas (∼0.2%ID/g–0.3%ID/g, 1 hour postinjection, Tables 1 and 2). However, substantial amounts of S₀2 accumulated in liver, stomach, intestines, and kidneys as measured by biodistribution analysis and seen by PET imaging (Tables 1 and 2 and Fig. 5).

R₀2 and R₀1 accumulated to greater levels in A431 tumors than E₀2 and S₀2. However, these arginine-rich binders cleared slowest from surrounding muscle tissue. Biodistribution analysis indicated approximately 0.6%ID/g muscle at 1 hour for the R₀2, whereas approximately half (∼0.3%ID/g) was observed for E₀2 and S₀2 (Supplementary Table S1). Therefore, tumor-to-muscle contrast was comparable for each of the engineered knots.

Renal retention varied by binding activity and scaffold (Fig. 5C). R₀1 ranged from approximately 45%ID/g (1 hour) to approximately 20%ID/g (24 hours), whereas R₀2 ranged from approximately 75%ID/g (1 hour) to approximately 28%ID/g (24 hours). This difference is attributed to the higher arginine content of activity “2.” However, transfer of activity “2” to the carboxyl-rich scaffold (E₀2) did not decrease kidney signal [∼72%ID/g (1 hour) and ∼16%ID/g (24 hours)]. These results suggest that in addition to arginine residues, the kidneys also actively retain carboxyl-containing residues. Importantly, when the “2” activity was tested in the S₀ scaffold (S₀2), significantly lower renal retention was measured [∼18%ID/g (1 hour) and ∼7%ID/g (24 hours)]. The large differences in renal uptake suggest that scaffold pharmacokinetics may be optimized to evade renal reuptake and allow a binder to pass freely into the bladder. Arginine is one of the most versatile amino acids in animal cells, serving as a precursor for the synthesis of proteins, nitric oxide, urea, polyamines, proline, glutamate, creatine, and agmatine (32). Therefore, arginine recycling by the kidneys is beneficial to the organism. By substituting pharmacokinetically favorable amino acids in the diversity-tolerant cystine knot framework, nonspecific uptake by off-target tissues may be reduced.

Integrin α_vβ₆ is being explored as a clinical target because of its elevated expression in several cancers (2). In this study, yeast surface display and high throughput screening by FACS produced many high-affinity binders that selectively accumulate in α_vβ₆-positive pancreatic and epidermoid cancer models but not in α_vβ₆-negative tumors actively growing in mice. By grafting the loop known as activity “2” (RSLARTDLDHLRGR) across different cystine knot scaffolds with biased amino acid content (R₀, E₀, and S₀), we achieved significantly lower renal signal without compromising α_vβ₆ binding affinity or specificity. This study suggests that primary sequence composition drives pharmacokinetics, so that it may be possible to optimize tumor uptake, tumor-to-muscle contrast, and off-target accumulation in tissues by fine-tuning the non-target–binding portions of the scaffold in addition to engineering target-binding loops.

Absolute ⁶⁴Cu-DOTA signal intensity at the tumor was greatest for the 2 R₀ binders, R₀1 and R₀2 (∼ 4.5%ID/g at 1 hour postinjection). ⁶⁴Cu-DOTA–labeled E₀2, S₀2, and A20 generated approximately half the tumor signal intensity compared with the R-rich scaffolds. Given the similar affinities of the engineered knots (Fig. 2A and B), we rationalize that higher tumor signal of R₀s benefited from the inherent “stickiness” of arginine. However, independent of a scaffold's compositional differences, each of the activity “2” knots (R₀2, E₀2, and S₀2) showed similarly useful tumor-to-normal contrast enabling precise delineation of integrin α_vβ₆-positive tumors (T+) from α_vβ₆-negative tumors (T−) shown in Fig. 5.

For clinical imaging of pancreatic cancer, S₀2 has emerged as our lead candidate. ⁶⁴Cu-DOTA-S₀2 showed an excellent tumor-to-background ratio (∼10 at 1 hour), rapid renal clearance (∼25%ID/g 1 hour postinjection to <10%ID/g after 24 hours), and high serum stability (>95% after 24 hours). In contrast, ⁶⁴Cu-DOTA-R₀2 rapidly degrades in serum (<20% intact 24 hours) and accumulates to a much higher degree in the kidneys (∼75%ID/g 1 hour). ⁶⁴Cu-DOTA-E₀2 also suffers from prolonged and high renal retention (∼75%ID/g 1 hour). Therefore, to take advantage of lower renal background of S₀s, we are conducting affinity maturation to achieve higher tumor uptake comparable with R₀ binders. We are also exploring ¹⁸F labeling strategies as the pharmacokinetics of these binders support a shorter lived isotope for clinical translation.

Biodistribution studies indicated that ⁶⁴Cu-DOTA-S₀2 also effectively accumulated in orthotopic pancreatic tumors (∼1.8%ID/g 1 hour postinjection) compared with normal pancreas (∼0.2%ID/g–0.3%ID/g). However, tumor-to-liver/kidney/gut ratios were suboptimal (Tables 1 and 2). The proximity of these PET-avid tissues is approximately 1 to 3 mm from the pancreas and is beyond the spatial resolution of the PET scanner. Therefore, definitive identification of orthotopic tumors by PET was limited by closely packed mouse organs. However, human scanning should not suffer from these limitations.

The engineered knots exhibit specific binding to integrin α_vβ₆. They do not bind related integrins α_vβ₃, α_vβ₅, or α₅β₁, and the scrambled (RDTLXXL) versions do not bind α_vβ₆ even at high concentrations (Figs. 2C and 3A; Supplementary Fig. S2A). Low uptake of α_vβ₆-specific radiotracers by 293 xenografts correlated to their limited expression of both the α_v and β₆ integrin subunits (Fig. 5, Supplementary Fig. S3A). In a very clear example, ⁶⁴Cu-DOTA-S₀2 specificity was shown in a mouse bearing both BxPC-3 and 293 xenografts (Fig. 5A, far right). Here, signal intensity for the α_vβ₆-positive tumor (T+, left shoulder) was approximately 2%ID/g, versus approximately 0.5%ID/g for the α_vβ₆-negative tumor (T−, right shoulder) at 1 hour postinjection. The “stickier” R₀ binders also showed selectivity for integrin α_vβ₆-expressing cells/tumors and recombinant protein (Figs. 2, 3, and 5; Supplementary Fig. S3). However, muscle washout was slower for the R₀s, so that contrast was comparable across all binders tested (Fig. 5B and D).

Renal clearance can potentially be controlled and optimized. This study provides insight about the significant differences in renal uptake reported for peptides previously engineered to bind integrin α_vβ₃, a vascular target (19). Knottins 2.5D and 2.5F, based on the cystine knot Ecballium elaterium Trypsin Inhibitor (EETI-II), contain a minimal number of arginines. The renal signals of ⁶⁴Cu-DOTA-2.5D and -2.5F measured less than 10%ID/g from 1 to 24 hours. In contrast, ⁶⁴Cu-DOTA-AgRP-7C, derived from the agouti-related peptide, which contains many complex nitrogen- and oxygen-containing residues, showed significantly higher renal retention of more than 65%ID/g at the same time points (29). Renal uptake of radiometal-labeled affibodies has also been reported to be very high (>100%ID/g) at multiple time points for binders 002, 1907, and 2377 (33–35). Affibody sequence inspection reveals the presence of many K, D/E, and Q/N residues, which can contribute to high renal retention (36–38). The kidneys appear to actively survey for the more complex, charge-rich amino acids (D/E/K/R) while allowing rapid clearance of the simpler noncharged amino acids (e.g., A, G, S). The development of stable, pharmacokinetically optimized scaffolds is highly desirable. Our results suggest one way to gain precise control of binder pharmacokinetics.

In summary, cystine knots are inherently stable scaffolds that are potentially well suited for clinical molecular imaging. Loop-grafting and directed evolution experiments generated single-digit nanomolar binders. PET imaging studies produced clinically useful tumor-to-normal contrast within 1 hour. We showed that simple scaffold swapping could enhance knot stability and stabilize pharmacokinetics to evade renal surveillance and approach renal stealth. Importantly, most knots are very well behaved during long-term storage. Collectively, these data suggest that cystine knots warrant further exploration for clinical translation.

No potential conflicts of interest were disclosed.

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