Novel Small Proteins for PET Imaging of Breast Cancer

Institution: Stanford University
Investigator(s): Zhen Cheng, Ph.D. -
Award Cycle: 2008 (Cycle 14) Grant #: 14IB-0091 Award: $261,806
Award Type: IDEA
Research Priorities
Detection, Prognosis and Treatment>Imaging, Biomarkers, and Molecular Pathology: improving detection and diagnosis



Initial Award Abstract (2008)

Human epidermal growth factor receptor type-2 (HER2) is a protein over-expressed in 25-30% of human breast cancers. HER2 targeted breast cancer therapy such as Trastuzumab (Herceptin) is commonly used in clinic. It also serves as a prognostic indicator of patient survival and a predictive marker of response to anti-tumor treatment. Thus a technology for accurate testing HER2 status is very important and has great impact on clinical management of breast cancer patients. We are particularly interested in developing positron emission tomography (PET) agents for HER2 imaging because of the high sensitivity, high spatial resolution, strong quantification ability, good safety (radiation no more than an ordinary X-ray test) and great potential for clinical translation of PET.

This project is initially focused on the design, synthesis and evaluation of small proteins with novel structure for their HER2 binding affinities. This will be measured by Surface Plasmon Resonance (SPR) Biacore analysis. This technology allows fully automated, real time measurement of binding kinetics between two or more molecules. Different strategies including sequence modifications and protein cyclization through disulfide bridges will be applied to achieve small proteins with high HER2 binding affinity. The most promising small proteins will then be labeled with a PET radioisotope, 18F. Finally, we will investigate the HER2 targeting ability of our probes using breast cancer xenografts (human tumor cell lines grown in mice) using small animal PET imaging. Finally, the PET probe with the best tumor imaging property will be studied in more detail for its ability to more accurately determine the HER2 status in tumors. The correlation between the tumor uptakes obtained from quantitation analysis of PET images and tumor HER2 levels measured by immunohistochemistry and Western blot analysis will be analyzed. In summary, this project represents the first trial to test the small protein scaffold as a platform for the imaging of HER2 expression. The HER2 targeted PET molecular probe developed in this research will hopefully provide a real-time, non-invasive assay of the overall tumor HER2 expression in patients.

If successful, a new PET imaging agent envisioned in this project will have broad implication in clinic, such as stratification of breast cancer patients for HER2 targeted drug therapy and surveillance of therapeutic efficacy. If the imaging agent demonstrates promising results in breast cancer animal models, we will further test their safety profile and move into clinical breast cancer imaging trial.




Final Report (2010)

Human epidermal growth factor receptor type 2 (HER2) is an important breast cancer biomarker. HER2 targeted therapy has shown significant benefits in breast cancer patients. More importantly, HER2 is an important prognostic indicator of cancer patients. The goal of this project is to develop a positron emission tomography (PET) probe based on the Affibody protein scaffold for non-invasively imaging HER2 status in breast cancer small animal models and ultimately translate this technique into clinical application.

Affibody molecules* (7 kDa, with 3-helix structure) are a new class of scaffold proteins being developed into a generalizable approach to target tumors. We have developed strategies to engineer smaller Affibody proteins with 2-helix structure and high HER2 binding affinity. One 2-hleix protein, called MUT-DS, was successfully discovered and then site-specifically labeled with a variety of radionuclides including 68Ga, 64Cu, 111In or 18F (an isotpe of Fluorine). In vitro and in vivo studies have demonstrated that the resulting probes all exhibited excellent tumor targeting ability and favorable pharmacokinetics. High mouse serum stability was found for the radiometals labeled MUT-DS, while some degree of decomposition was observed for the protein labeled with 4-18F-fluorobenzaldehyde (4-18F-FBA) at 1 h incubation. The 18F-FBA labeled 3-helix Affibody was also prepared and demonstrated ability to image HER2 expression in small animal models.

Our study shows that the radiometal labeled MUT-DS could be promising probes for imaging HER2, while new strategies and 18F radiosynthesis for MUT-DS labeling are needed for further optimization of the 18F-2-helix Affibody probe for imaging HER2 expression.

* Affibody affinity ligands are unique research reagents, produced using innovative protein-engineering technologies. They are small, simple proteins composed of a three-helix bundle based on the scaffold of one of the IgG-binding domains of Protein A. Protein A is a surface protein from the bacterium Staphylococcus aureus. This scaffold has excellent features as an affinity ligand and can be designed to bind with high affinity to any given target protein. The domain consists of 58 amino acids, 13 of which are randomized to generate Affibody libraries with a large number of ligand variants. Thus, the libraries consist of a multitude of protein ligands with an identical backbone and variable surface-binding properties. (Copied from http://www.abcam.com)




Symposium Abstract (2010)

G. Ren, 1 Z. Miao, 1 J.M. Webster,2 R. Zhang,2 S. S. Gambhir,1 F. A. Syud,2 Z. Cheng1
1Molecular Imaging Program at Stanford (MIPS), Department of Radiology, Bio-X Program, Stanford University, and 2Global Research, General Electric Company.

Affibody molecules are novel scaffold proteins and suitable for rapidly developing molecular probes for a variety of tumor targets. It has been shown that Affibody molecules against human epidermal growth factor type 2 (HER2) can be labeled with different radionuclides and can specifically image HER2 expressions and modulations both in cells and in preclinical animal models. In our research, we have further developed strategies for discovery even smaller Affibody analogs, 2-helix proteins, for in vivo HER2 molecular imaging .

Methods: A library of anti-HER2 2-helix Affibody analogs were chemically synthesized using solid phase peptide synthesizer. Helices 1 and 2 that contain the binding domain were preserved while a third helix was truncated. A number of both sequence mutations and synthetic strategies to optimize the affinity of 2-helix proteins were developed. The lead candidate cyclic peptide was then selected and was site specifically radiolabeled with several positron emission tomography (PET) radionuclides including 18F, 68Ga or 64Cu. The resulting molecular probes were then evaluated for microPET imaging of HER2 in SKOV3 tumor mice.

Results: Several constrained 2-helix peptides with high HER2 affinity were successfully identified (for example: MUT-DS, KD = 5 nM). Conjugation with a metal chelator or radiofluorination agent still preserved high binding affinity. Radiolabeled (68Ga, 64Cu or 18F) probes displayed high stability in mouse serum and specificity towards HER2 in cell culture. Biodistribution and microPET imaging studies further revealed that the probes had rapid, high and specific HER2 positive tumor uptakes and excellent tumor imaging ability.

Conclusions: 2-helix small proteins with high affinity to tumor biomarkers can be discovered and used for in vivo molecular imaging. PET radionuclides labeled MUT-DS showed promising results for imaging HER2 expression and modulation in vivo.



Engineered two-helix small proteins for molecular recognition.
Periodical:ChemBioChem
Index Medicus: Chembiochem
Authors: Webster JM, Zhang R, Gambhir SS, Cheng Z, Syud FA.
Yr: 2009 Vol: 10 Nbr: 9 Abs: Pg:1293-6

A 2-helix small protein labeled with 68Ga for PET imaging of HER2 expression.
Periodical:Journal of Nuclear Medicine
Index Medicus: J Nucl Med
Authors: Ren G, Zhang R, Liu Z, Webster JM, Miao Z, Gambhir SS, Syud FA, Cheng Z
Yr: 2009 Vol: 50 Nbr: 9 Abs: Pg:1492-9