Phage display and molecular imaging: expanding fields of vision in living subjects

Abstract

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 and deliver imaging probes to desired locations in the body is critically important to further advance this field. To address this need, phage display, an established technology for the discovery and development of novel binding agents, is increasingly becoming a key component of many molecular imaging research programs. This review discusses the expanding role played by phage display in the field of molecular imaging with a focus on in vivo applications. Furthermore, new methodological advances in phage display that can be directly applied to the discovery and development of molecular imaging agents are described. Various phage library selection strategies are summarized and compared, including selections against purified target, intact cells, and ex vivo tissue, plus in vivo homing strategies. An outline of the process for converting polypeptides obtained from phage display library selections into successful in vivo imaging agents is provided, including strategies to optimize in vivo performance. Additionally, the use To whom correspondence may be addressed (cochran1@stanford.edu) Abbreviations: scFv, single chain variable fragment; Fab, fragment antigen binding; ELISA, Enzyme-Linked Immunosorbent Assay; NEB, New England Biolabs; PET, positron emission tomography; SPECT, single photon emission computed tomography; NIR, near infrared; PEG, polyethylene glycol; SPARC, secreted protein acidic and rich in cysteine; VCAM-1, vascular cell adhesion molecule; MRI, magnetic resonance imaging; DOTA, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid; IC 50 , half maximal inhibitory concentration; CT, computed tomography; K D , equilibrium dissociation constant; EGFR, epidermal growth factor receptor; EGFR-ECD, EGFR extracellular domain; Aβ 42 , amyloid-beta; MMP, matrix metalloprotease; uPAR, urokinase-type plasminogen activator receptor; VEGF, vascular endothelial growth factor; IL-11, Interleukin-11; IL-11αR, Interleukin-11 α-receptor. 58F.V. CoChran and J.r. CoChran of phage particles as imaging agents is also described. In the latter part of the review, a survey of phage-derived in vivo imaging agents is presented, and important recent examples are highlighted. Other imaging applications are also discussed, such as the development of peptide tags for site-specific protein labeling and the use of phage as delivery agents for reporter genes. The review concludes with a discussion of how phage display technology will continue to impact both basic science and clinical applications in the field of molecular imaging.

Engineering Agatoxin, a Cystine-Knot Peptide from Spider Venom, as a Molecular Probe for In Vivo Tumor Imaging

“…Similar results were obtained with imaging probe conjugation to the N-terminus of AgRP 7C and EETI 2.5F. In comparison, small molecules and disulfide-constrained cyclic peptides present binding epitopes within a more limited framework that can sometimes be disrupted upon imaging probe conjugation [53], [55].…”

“…Based on structural similarities between AgRP and AgTx knottins, we next grafted a disulfide-bonded integrin-binding loop from an engineered AgRP variant into AgTx, and showed that high-affinity integrin binding was conferred with this new construct. A similar approach could potentially be used to incorporate disulfide-bonded cyclic peptides, such as those identified from phage display libraries [53], into knottin scaffolds.…”

Serum Stability and Affinity Optimization of an M2 Macrophage-Targeting Peptide (M2pep)

“…In vivo applications of peptides as therapeutic or imaging agents are usually limited by their short serum half-life, typically on the order of a few minutes, due to degradation by serum proteases as well as clearance via kidneys and liver 19 - 21 . In regard to serum stability, several strategies including N-terminal acetylation, C-terminal amidation, D-amino acid substitution, and peptide stapling/macrocyclization have been utilized to impart peptide resistance against peptidase degradation 19 , 22 . In this study, serum stability of M2pep was first evaluated to determine peptidase-susceptible sites.…”

Challenges in the development of nanoparticle‐based imaging agents: Characterization and biology

“…Among the most well‐known are Humira®, an antibody against TNF‐alpha used in the treatment of rheumatoid arthritis, Portrazza®, an antibody against EGFR used in the treatment of non‐small‐cell lung carcinoma, and Cyramza®, an antibody against VEGFR used in the treatment of advanced gastric or gastric‐esophageal adenocarcinoma and metastatic non‐small‐cell carcinoma (Frenzel, Schirrmann, & Hust, 2016; Nixon, Sexton, & Ladner, 2014). Phage display screens can be performed in vitro, on proteins previously identified as biomarkers, on intact cells, on ex vivo tissues, and, recently, in vivo as well (Brinton, Bauknight, Dasa, & Kelly, 2016; Cochran & Cochran, 2010; Siva Sai Krishna Dasa et al, 2015; Deramchia et al, 2012). Phage display provides a simple, easy and cost‐effective strategy that enables high throughput screening of billions of peptides and antibody fragments against a wide variety of target molecules, including cell surface receptors.…”