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

Canary Center for Cancer, Early Detection, Molecular Imaging Program, Department of Radiology, Stanford University School of Medicine, Stanford, California 94305, USA.
Clinical Cancer Research (Impact Factor: 8.72). 12/2011; 18(3):839-49. DOI: 10.1158/1078-0432.CCR-11-1116
Source: PubMed


Detection of pancreatic cancer remains a high priority and effective diagnostic tools are needed for clinical applications. Many cancer cells overexpress integrin α(v)β(6), a cell surface receptor being evaluated as a novel clinical biomarker.
To validate this molecular target, several highly stable cystine knot peptides were engineered by directed evolution to bind specifically and with high affinity (3-6 nmol/L) to integrin α(v)β(6). The binders do not cross-react with related integrin α(v)β(5), integrin α(5)β(1), or tumor-angiogenesis-associated integrin, α(v)β(3).
Positron emission tomography showed that these disulfide-stabilized peptides rapidly accumulate at tumors expressing integrin α(v)β(6). Clinically relevant tumor-to-muscle ratios of 7.7 ± 2.4 to 11.3 ± 3.0 were achieved within 1 hour after radiotracer injection. Minimization of off-target dosing was achieved by reformatting α(v)β(6)-binding activities across various natural and pharmacokinetically stabilized cystine knot scaffolds with different amino acid content. We show that the primary sequence of a peptide scaffold directs its pharmacokinetics. Scaffolds with high arginine or glutamic acid content suffered high renal retention of more than 75% injected dose per gram (%ID/g). Substitution of these amino acids with renally cleared amino acids, notably serine, led to significant decreases in renal accumulation of less than 20%ID/g 1 hour postinjection (P < 0.05, n = 3).
We have engineered highly stable cystine knot peptides with potent and specific integrin α(v)β(6)-binding activities for cancer detection. Pharmacokinetic engineering of scaffold primary sequence led to significant decreases in off-target radiotracer accumulation. Optimization of binding affinity, specificity, stability, and pharmacokinetics will facilitate translation of cystine knots for cancer molecular imaging.


Available from: Marybeth Pysz, Apr 28, 2014
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    • "To develop a more stable and effective agent for imaging integrin αvβ6 cysteine knot peptides were engineered which demonstrated nanomolar affinity for this integrin [44]. Four DOTA-derivatized compounds were labeled with 64Cu and metabolic stability was studied in mouse serum. "
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    ABSTRACT: Noninvasive determination of integrin expression has become an interesting approach in nuclear medicine. Since the discovery of the first (18)F-labeled cyclic RGD peptide as radiotracer for imaging integrin α v β 3 expression in vivo, there have been carried out enormous efforts to develop RGD peptides for PET imaging. Moreover, in recent years, additional integrins, including α 5 β 1 and α v β 6, came into the focus of pharmaceutical radiochemistry. This review will discuss the tracers already evaluated in clinical trials and summarize the preliminary outcome. It will also give an overview on recent developments to further optimize the first-generation compounds such as [(18)F]Galacto-RGD. This includes recently developed (18)F-labeling strategies and also new approaches in (68)Ga-complex chemistry. Furthermore, the approaches to develop radiopharmaceuticals targeting integrin α 5 β 1 and α v β 6 will be summarized and discussed.
    BioMed Research International 06/2014; 2014(5158):871609. DOI:10.1155/2014/871609 · 2.71 Impact Factor
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    • "In addition to having different scaffold sequences, the engineered loops of EETI 2.5F (PRPRGDNPPLT) and EETI RDG (VTGRDGSPASS) are markedly different from that of AgRP/AgTx 7C (YGRGDNDLR). In agreement with this hypothesis, our colleagues recently reported that highly charged residues, particularly arginine and glutamic acid, of Momordica cochinchinensis trypsin inhibitor-II knottins contribute greatly to non-specific kidney retention [67]. In addition, an 111In-labeled version of one of our alternative engineered integrin-binding AgRP knottins (AgRP 6E, engineered loop sequence: VERGDGNRR) had an approximately 50% reduction in kidney signal compared to 111In-labeled AgRP 7C [35], demonstrating the influence of the engineered loop on tissue biodistribution. "
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    PLoS ONE 04/2013; 8(4):e60498. DOI:10.1371/journal.pone.0060498 · 3.23 Impact Factor
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    • "Recently, a αvβ6-binding cysteine knot was engineered to improve in vivo stability 39, 40. This ligand displays similar affinity and specificity for αvβ6 as the A20FMDV peptide. "
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