Alternative non-antibody protein scaffolds for molecular imaging of cancer

doi:10.1016/j.coche.2013.08.009

Current Opinion in Chemical Engineering

Volume 2, Issue 4, November 2013, Pages 425-432

https://doi.org/10.1016/j.coche.2013.08.009 Get rights and content

Highlights

•
Molecular imaging is a valuable clinical tool for cancer detection and treatment.
•
Non-antibody protein scaffolds are an effective source of molecular imaging agents.
•
Affibodies, knottins, fibronectins, and DARPins have imaged many cancer biomarkers.
•
Ligand biophysical properties can be modulated to optimize in vivo performance.

The development of improved methods for early detection and characterization of cancer presents a major clinical challenge. One approach that has shown excellent potential in preclinical and clinical evaluation is molecular imaging with small-scaffold, non-antibody based, engineered proteins. These novel diagnostic agents produce high contrast images due to their fast clearance from the bloodstream and healthy tissues, can be evolved to bind a multitude of cancer biomarkers, and are easily functionalized by site-specific bioconjugation methods. Several small protein scaffolds have been verified for in vivo molecular imaging including affibodies and their two-helix variants, knottins, fibronectins, designed ankyrin repeat proteins (DARPins), and several natural ligands. Further, the biodistribution of these engineered ligands can be optimized through rational mutation of the conserved regions, careful selection and placement of chelator, and modification of molecular size.

Graphical abstract

Introduction

Molecular imaging can provide critical clinical information regarding the presence, concentration, and localization of cancer biomarkers in vivo. The data, which can be obtained dynamically or longitudinally if desired, empowers early detection, patient stratification, and treatment monitoring. A major challenge in this field is the development of high affinity, specific ligands for the multitude of important biomarkers. Directed evolution and other strategies position engineered proteins to offer a robust, high throughput means for ligand generation (Figure 1). Many of these engineered proteins have shown promising results in preclinical evaluation as well as early clinical results. While antibodies and their fragments have been explored [1], their large size and slow clearance are not ideal due to the functional requirement of reduced background signal necessary for imaging contrast; moreover, for targeting of poorly vascularized tumors, such as those in early formation, large size slows extravasation and delivery [2^•]. For these reasons, as well as benefits in stability, production, and chemical conjugation, alternative protein topologies have been studied as scaffolds for molecular imaging. Short linear and cyclic peptides have exhibited significant success as molecular imaging agents [3], although their robustness in engineering high affinity toward novel targets is limited. Thus, this review will focus on advances in molecular imaging of cancer, particularly positron emission tomography (PET), single-photon emission computed tomography (SPECT), and gamma-camera imaging using engineered, folded, non-antibody proteins (Figure 2 and Table 1). The majority of the research has been performed using subcutaneous xenografted tumors in mice. There are exceptions to these experimental systems, including clinical data, which will be noted.

Section snippets

Affibody

The affibody is a 58 amino acid, three helical bundle [4]. Typically, randomization of 13 amino acids on the surface of helices 1 and 2 is used to generate novel binding ligands. The most extensively studied class of affibodies is those targeting human epidermal growth factor receptor 2 (HER2). The second generation HER2-binding affibody, Z_HER2:342, was engineered to bind with 22 pm affinity [5^•], and has successfully imaged HER2-expressing tumor xenografts in mice when labeled with ¹²⁵I [5^•],

Designing delivery

It is evident that disparate protein topologies can be engineered for in vivo molecular recognition and delivery of radioisotopes to tumors. As the field continues to mature, we should strive to identify the ideal molecule for the application of interest, to maximize tumor uptake, minimize off-target retention, and tune kinetics for efficacy, logistics, and patient safety. Quantitative design rules would be tremendously useful to optimize performance. Select studies have been performed that

References and recommended reading

Papers of particular interest, published within the period of review, have been highlighted as:

• of special interest

Acknowledgements

This work was funded by the National Institutes of General Medical Science Biotechnology Training Grant (L.A.S.) and the University of Minnesota.

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View full text

Alternative non-antibody protein scaffolds for molecular imaging of cancer

Highlights

Graphical abstract

Introduction

Section snippets

Affibody

Designing delivery

References and recommended reading

Acknowledgements

Methods Mol Biol

Eur J Nucl Med Mol Imaging

J Nucl Med

Q J Nucl Med Mol Imaging

Nucl Med Biol

Eur J Nucl Med Mol Imaging

Advances in immuno-positron emission tomography: antibodies for molecular imaging in oncology

J Clin Oncol

A modeling analysis of the effects of molecular size and binding affinity on tumor targeting

Mol Cancer Ther

Molecular imaging targeting peptide receptors

Methods

Tumor imaging using a picomolar affinity HER2 binding affibody molecule

Cancer Res

Pre-clinical evaluation of [111In]-benzyl-DOTA-Z(HER2:342), a potential agent for imaging of HER2 expression in malignant tumors

Int J Mol Med

Tc-99m-maEEE-Z(HER2:342), an affibody molecule-based tracer for the detection of HER2 expression in malignant tumors

Bioconjug Chem

[18F]FBEM-ZHER2:342 — affibody molecule — a new molecular tracer for in vivo monitoring of HER2 expression by positron emission tomography

Eur J Nucl Med Mol Imaging

Evaluation of [(111/114m)In]CHX-A″-DTPA-ZHER2:342, an affibody ligand coniugate for targeting of HER2-expressing malignant tumors

Q J Nucl Med Mol Imaging

On the selection of a tracer for PET imaging of HER2-expressing tumors: direct comparison of a 124-labeled affibody molecule and trastuzumab in a murine xenograft model

J Nucl Med

(68)Ga-DOTA-affibody molecule for in vivo assessment of HER2/neu expression with PET

Eur J Nucl Med Mol Imaging

HER2-positive tumors imaged within 1 hour using a site-specifically 11C-labeled Sel-tagged affibody molecule

J Nucl Med

Molecular imaging of HER2-expressing malignant tumors in breast cancer patients using synthetic 111In- or 68Ga-labeled affibody molecules

J Nucl Med

Affibody molecules for epidermal growth factor receptor targeting in vivo: aspects of dimerization and labeling chemistry

J Nucl Med

Imaging of EGFR expression in murine xenografts using site-specifically labelled anti-EGFR 111In-DOTA-Z EGFR:2377 Affibody molecule: aspect of the injected tracer amount

Eur J Nucl Med Mol Imaging

In vivo and in vitro uptake of 111In, delivered with the affibody molecule (ZEGFR:955)2, in EGFR expressing tumour cells

Oncol Rep

Small-animal PET imaging of human epidermal growth factor receptor positive tumor with a 64Cu labeled affibody protein

Bioconjug Chem

PET of EGFR expression with an 18F-labeled affibody molecule

J Nucl Med

Imaging of insulinlike growth factor type 1 receptor in prostate cancer xenografts using the affibody molecule 111In-DOTA-ZIGF1R:4551

J Nucl Med

[99mTc(CO)3]+-(HE)3-ZIGF1R:4551, a new Affibody conjugate for visualization of insulin-like growth factor-1 receptor expression in malignant tumours

Eur J Nucl Med Mol Imaging

Evaluation of backbone-cyclized HER2-binding 2-helix Affibody molecule for in vivo molecular imaging

Nucl Med Biol

Engineered two-helix small proteins for molecular recognition

ChemBioChem

A 2-helix small protein labeled with 68Ga for PET imaging of HER2 expression

J Nucl Med

A novel 18F-labeled two-helix scaffold protein for PET imaging of HER2-positive tumor

Eur J Nucl Med Mol Imaging

Engineering knottins as novel binding agents

Methods Enzymol

Engineered knottin peptides: a new class of agents for imaging integrin expression in living subjects

Cancer Res

Pre-clinical evaluation of [¹¹¹In]-benzyl-DOTA-Z(HER2:342), a potential agent for imaging of HER2 expression in malignant tumors

[¹⁸F]FBEM-ZHER2:342 — affibody molecule — a new molecular tracer for in vivo monitoring of HER2 expression by positron emission tomography

On the selection of a tracer for PET imaging of HER2-expressing tumors: direct comparison of a ¹²⁴-labeled affibody molecule and trastuzumab in a murine xenograft model

HER2-positive tumors imaged within 1 hour using a site-specifically ¹¹C-labeled Sel-tagged affibody molecule

Molecular imaging of HER2-expressing malignant tumors in breast cancer patients using synthetic ¹¹¹In- or ⁶⁸Ga-labeled affibody molecules

Imaging of EGFR expression in murine xenografts using site-specifically labelled anti-EGFR ¹¹¹In-DOTA-Z EGFR:2377 Affibody molecule: aspect of the injected tracer amount

In vivo and in vitro uptake of ¹¹¹In, delivered with the affibody molecule (ZEGFR:955)2, in EGFR expressing tumour cells

Small-animal PET imaging of human epidermal growth factor receptor positive tumor with a ⁶⁴Cu labeled affibody protein

PET of EGFR expression with an ¹⁸F-labeled affibody molecule

Imaging of insulinlike growth factor type 1 receptor in prostate cancer xenografts using the affibody molecule ¹¹¹In-DOTA-ZIGF1R:4551

[^99mTc(CO)3]+-(HE)3-ZIGF1R:4551, a new Affibody conjugate for visualization of insulin-like growth factor-1 receptor expression in malignant tumours

A 2-helix small protein labeled with ⁶⁸Ga for PET imaging of HER2 expression

A novel ¹⁸F-labeled two-helix scaffold protein for PET imaging of HER2-positive tumor