Cochran Lab
Cochran Lab
RESEARCH

Engineering Proteins as Molecular Imaging Agents

Introduction

In vivo molecular imaging enables non-invasive visualization of biological processes within living subjects, and holds great promise for diagnosis and monitoring of disease. The ability to create new agents that bind to molecular targets is critically important to further advance this field (1). Our research group has a strong interest in developing protein- and peptide-based molecular imaging probes that target and illuminate tumors for applications in cancer such as diagnosis, clinical staging and disease management, monitoring disease progression and response to therapy, and surgical guided resection.

Figure 1Fig. 1. EETI-II (grey), a knottin peptide found in the seeds of the squirting cucumber Ecballium elaterium, was engineered to bind with high affinity and unique specificities to tumor-associated integrin receptors (red loop). After chemical probe. conjugation (yellow star), these engineered knottins were used for a variety of molecular imaging applications

 

Engineered Knottins as a New Class of Molecular Imaging Agents

We recently established engineered knottin peptides as a new class of molecular imaging agents. We showed that the small size of knottins and their high stability translated into desirable pharmacokinetic and biodistribution properties for molecular imaging applications, namely high tumor uptake and rapid clearance from non-target tissues (2,3). Knottins, engineered to bind to integrin receptors expressed on tumors and the tumor vasculature, have shown promise as diagnostic agents for imaging integrin expression in living subjects. Using combinatorial methods, EETI-II and AgRP knottin scaffolds were engineered to bind with low nanomolar to sub-nanomolar affinity and unique specificities to alpha5 beta1, alphav beta5 and/or alphav beta3 integrins (4,5). Positron emission tomography (PET) imaging with 64Cu- or 18F-radiolabeled versions of these knottins showed high tumor uptake and fast circulation clearance in murine tumor xenograft or spontaneous models (6-11). Interestingly, EETI-II based knottins exhibited extremely low liver and renal uptake (6-9) which appears to be unique among protein and peptide-based PET imaging agents. Moreover, an engineered EETI-II knottin detected small ( ~3 mm) lung tumors with enhanced contrast compared to the current clinical standard 18F-fluorodeoxyglucose (FDG), which is taken up non-specifically in highly metabolic tissues such as the heart (10). Engineered EETI-II knottins were conjugated to both optical and PET probes for dual-modality imaging (12), and were also used to target echogenic microbubbles to the tumor vasculature for contrast-enhanced ultrasound-based imaging (13). In addition, preliminary evaluation of an engineered EETI-II knottin labeled with 177Lu showed potential for radiotherapy applications (14).

Collaborators in the School of Medicine

Zhen Cheng, Juergen Wilmann, Dean Felsher, Sanjiv Sam Gambhir

Selected Imaging Publications

  1. Cochran, F.V., and Cochran, J.R. (2010) Phage Display and Molecular Imaging: Expanding Fields of Vision in Living Subjects. Biotechnology and Genetic Engineering Reviews. 27, 57-94. Article
  2. Moore, S. J. and Cochran, J. R. (2012) Engineering Knottins as Novel Binding Agents. Methods in Enzymology. 503, 223-251. Article
  3. Moore, S. J. et al. (2011) Knottins: Disulfide-bonded Therapeutic and Diagnostic Peptides. In Press, Drug Discovery Today: Technologies. Article
  4. Kimura, R. H. et al. (2009) Engineered Cystine Knot Peptides that Bind alphav beta3, alphav beta5, and alpha5 beta1 Integrins with Low Nanomolar Affinity. Proteins: Structure, Function, and Bioinformatics. 77, 359-69. Article
  5. Silverman, A. P. et al. (2009) Engineered Cystine-Knot Peptides That Bind alphav beta3 Integrin with Antibody-Like Affinities. Journal of Molecular Biology, 385, 1064-75. Article
  6. Kimura, R. H. et al. (2009) Engineered Knottin Peptides: A New Class of Agents for Imaging Integrin Expression in Living Subjects. Cancer Research, 69, 2435-42. Article
  7. Miao, Z. et al. (2009) An Engineered Knottin Peptide Labeled with 18F for PET Imaging of Integrin Expression. Bioconjugate Chemistry, 20, 2342-7. Article
  8. Liu, S. et al. (2011) PET imaging of Integrin Positive Tumors using 18F-labeled knottin peptides. Theranostics. 1, 403-12. Article
  9. Kimura, R. H. et al. (2011) Functional Mutation of Multiple Solvent-Exposed Loops in the Ecballium elaterium Trypsin Inhibitor-II Cystine Knot Miniprotein. PLoS ONE, 6, e16112. Article
  10. Nielsen, C.H. et al. (2010) PET Imaging of Tumor Neovascularization in a Transgenic Mouse Model Using a Novel 64-Cu-DOTA-Knottin Peptide. Cancer Research, 70, 9022-30. Article
  11. Jiang, L. et al. (2010) Evaluation of a 64Cu-Labeled Cystine-Knot Peptide Based on Agouti Related Protein for PET Imaging of Tumors Expressing alphav beta3 Integrin. Journal of Nuclear Medicine, 51, 251-8. †co-corresponding authors. Article
  12. Kimura, R. H. et al. (2010) A Dual-Labeled Knottin Peptide for PET and Near-Infrared Fluorescence Imaging of Integrin Expression in Living Subjects. Bioconjugate Chemistry, 21, 436-44. Article
  13. Willmann, J. K. et al. (2010) Targeted Contrast-Enhanced Ultrasound Imaging of Tumor Angiogenesis with Contrast Microbubbles Conjugated to Integrin-Binding Knottin Peptides. Journal of Nuclear Medicine, 51, 433-40. Article | Press Release
  14. Jiang, L. et al. (2011) Preliminary evaluation of (177)Lu-labeled knottin peptides for integrin receptor-targeted radionuclide therapy. Eur J Nucl Med Mol Imaging. 38, 613-22. Article