Radiolabeling of substrates with 2-[18F]fluoroethylazide exploits the rapid kinetics, chemical selectivity, and mild conditions of the copper-catalyzed azide-alkyne cycloaddition reaction. While this methodology has proven to result in near-quantitative labeling of alkyne-tagged precursors, the relatively small size of the fluoroethylazide group makes separation of the 18F-labeled radiotracer and the unreacted precursor challenging, particularly with precursors >500 Da (e.g., peptides). We have developed an inexpensive azide-functionalized resin to rapidly remove unreacted alkyne precursor following the fluoroethylazide labeling reaction and integrated it into a fully automated radiosynthesis platform. We have carried out 2-[18F]fluoroethylazide labeling of four different alkynes ranging from <300 Da to >1700 Da and found that >98% of the unreacted alkyne was removed in less than 20 min at room temperature to afford the final radiotracers at >99% radiochemical purity with specific activities up to >200 GBq/μmol. We have applied this technique to label a novel cyclic peptide previously evolved to bind the Her2 receptor with high affinity, and demonstrated tumor-specific uptake and low nonspecific background by PET/CT. This resin-based methodology is automated, rapid, mild, and general allowing peptide-based fluorine-18 radiotracers to be obtained with clinically relevant specific activities without chromatographic separation and with only a minimal increase in total synthesis time.

Peptides typically have poor biostabilities, and natural sequences cannot easily be converted into drug-like molecules without extensive medicinal chemistry. We have adapted mRNA display to drive the evolution of highly stable cyclic peptides while preserving target affinity. To do this, we incorporated an unnatural amino acid in an mRNA display library that was subjected to proteolysis prior to selection for function. The resulting "SUPR (scanning unnatural protease resistant) peptide" showed ≈500-fold improvement in serum stability (t1/2 =160 h) and up to 3700-fold improvement in protease resistance versus the parent sequence. We extended this approach by carrying out SUPR peptide selections against Her2-positive cells in culture. The resulting SUPR4 peptide showed low-nanomolar affinity toward Her2, excellent specificity, and selective tumor uptake in vivo. These results argue that this is a general method to design potent and stable peptides for in vivo imaging and therapy.

Abstract In recent years, patients with Her2-positive breast cancer have benefitted greatly from targeted therapies such as Trastuzumab and Pertuzumab. However, there are currently no FDA-approved Her2-targeted imaging agents to diagnose and monitor Her2-positive breast cancer leaving physicians to rely on biopsies to determine Her2 status. Whole-body visualization of Her2 would allow the noninvasive identification of all Her2 primary and metastatic lesions as well as serve as a powerful tool to monitor the effectiveness of Her2-targeted therapeutics. We recently described the generation of Her2-targeted SUPR (Scanning Unnatural Protease Resistant) peptides that selectively bind the Her2 receptor in vitro with low nanomolar affinity. Cy5-labeled SUPR peptides showed rapid and Her2-specific tumor uptake and minimal retention in non-tumor tissues after 24 hours of washout. In this study, we describe 18F radiolabeling of Her2-targeted SUPR peptides and their evaluation as PET radiotracers to visualize Her2 expression in vivo. The lead compound, SUPR4, was labeled with 18F-fluoroethylazide in high radiochemical yield and specific activity on an automated radiochemical synthesis platform. The resulting radiotracer (SUPR-18F) showed rapid and Her2-selective tumor uptake between 30-60 minutes post-injection with minimal liver uptake. The majority of the tracer was cleared by the kidneys at 2 hours post-injection although some activity was observed in the GI tract suggesting hepatobiliary excretion. No significant uptake was observed in the brain. 90 minute dynamic PET scans were performed to estimate the rate of tumor uptake and clearance in major organ systems and the biodistribution quantified by autoradiography post-mortem. Pre-injection of unlabeled SUPR peptide, Trastuzumab, and Pertuzumab followed by PET imaging with SUPR-18F was used to confirm tumor uptake as a function of specific binding to the Her2 receptor. Having established the utility of SUPR-18F in pre-clinical mouse models, we anticipate that this class of PET tracers could be employed in same-day imaging procedures throughout the administration of Her2-targeted therapy. Citation Format: Lindsay E. Kelderhouse, Amanda Hardy, Federica Pisaneschi, Joshua P. Gray, Seth Gammon, Richard W. Roberts, Terry T. Takahashi, Steve Fiacco, Steven W. Millward. SUPR-PET: Nuclear imaging of Her2-positieve breast cancer with SUPR peptides. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4236.

Abstract Ideal molecular imaging agents would combine the affinity and selectivity of monoclonal antibodies with the rapid clearance and pharmacokinetics of small molecules Here we describe the use of directed evolution to design Scanning Unnatural Protease Resistant (SUPR) peptides as novel molecular scaffolds for in vivo optical and PET imaging of the Her2 receptor. SUPR peptides were obtained through selection against Her2-expressing breast cancer cells in culture using focused biological display peptide libraries pre-selected for protease resistance. SUPR peptide libraries were programmed with unnatural amino acids for enhanced stability and chemically cyclized after translation to enhance affinity. Peptides isolated from selection selectively bind the Her2 receptor in vitro with low nanomolar affinity and do not compete with Trastuzumab or Pertuzumab. Cy5-labeled SUPR peptides show rapid and Her2-specific tumor uptake in vivo and minimal retention in non-tumor tissues after 24 hours of washout. SUPR peptides can be efficiently labeled with 18F via the copper-catalyzed azide-alkyne cycloaddtion and the resulting radiotracers used for nuclear imaging of Her2-postive tumors by PET/CT. Unlike antibodies and their derivatives, SUPR peptides are synthesized chemically and show rapid clearance from systemic circulation. SUPR peptides also show dramatically enhanced stability relative to linear peptides obtained from phage display or combinatorial chemistry while still retaining affinities compatible with molecular imaging applications. While SUPR peptides are selected from biological display libraries, they share similar chemical composition and structure with peptides derived from non-ribosomal peptide synthesis. We believe that this combination of affinity, stability, evolvability, and synthetic accessibility make SUPR peptide technology a general approach for generating highly potent compounds for targeted molecular imaging of cancer biomarkers. Citation Format: Lindsay Kelderhouse, Amanda Hardy, Terry T. Takahashi, Argentina Ornelas, Seth Gammon, Shannon Howell, Peiying Yang, Pin Wang, Richard W. Roberts, Steve Fiacco, Steven W. Millward. SUPR peptides as novel targeted molecular imaging agents for Her2. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1499. doi:10.1158/1538-7445.AM2015-1499

Abstract The implementation of personalized medicine for Her2-positive breast cancer treatment (Trastuzumab and Pertuzumab) has made a dramatic effect on overall patient outcome. Unfortunately, there are currently no FDA-approved imaging agents to monitor Her2-positive breast cancer leaving physicians to rely on invasive biopsies and anatomical imaging to monitor treatment with Her2-targeted therapeutics. There is also increasing evidence that women initially diagnosed with Her2-negative breast can present with Her2-positive disease upon recurrence. Noninvasive, whole-body visualization of Her2 would identify Her2-recurrent disease and serve as a powerful tool to monitor the effectiveness of new and emerging Her2-targeted therapeutics (e.g. TDM-1). While many Her2-antibody based imaging agents have been used in preclinical applications, their long circulating half-lives, high liver uptake, and poor synthetic accessibility pose a challenges for clinical translation. Here we describe the use of highly stable cyclic peptides derived from biological display as imaging agents for Her2-positive breast cancer. These peptides have antibody-like high affinity (&amp;lt;10 nM) for Her2-positive tumors and their size (&amp;lt;2 kDa) allows for higher rates of clearance, lower background signal, and consequently higher image sensitivity. Anti-Her2 peptides were pre-optimized for affinity, protease resistance and bio-stability prior to labeling with the Cy7.5 near-infrared dye. Her2-specificity was determined by in vitro assays with Her2-postive (BT474 and SKOV3), HER2-normal (MCF7) and Her2-negative (MDA-MB-231) cell lines. Sequences that showed superior affinity and selectivity were used to acquire fluorescent images of ectopic and orthotopic murine models of Her2-positive breast cancer. Our preliminary imaging data suggests that this scaffold has excellent translational potential for targeted molecular imaging of Her2-postive breast cancer. Citation Format: Lindsay E Kelderhouse, Amanda Hardy, Yong Pan, Patrea Rhea, Argentina Ornelas, Seth Gammon, Peiying Yang, Stephen Fiacco, Steven W Millward. In vitro and in vivo imaging of Her2 with cyclic peptides derived from directed evolution [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-01-10.

This 533-MHz BiCMOS very large scale integration (VLSI) implementation of the PowerPC architecture contains three pipelines and a large on-chip secondary cache to achieve a peak performance of 1600 MIPS. The 15 mm/spl times/10 mm die contains 2.7 M transistors (2M CMOS and 0.7 M bipolar) and dissipates less than 85 W. The die is fabricated in a six-level metal, 0.5-/spl mu/m BiCMOS process and requires 3.6 and 2.1 V power supplies.