Cheng Lab

Molecular Probe Development

Molecular probe is an agent used to visualize, characterize and quantify biological processes in living systems. Molecular probes are the major driving force of the molecular imaging research. Many different types of platforms have been explored in molecular probe discovery, including simple radioisotopes, small molecules, peptides, proteins, aptamers, nanoparticles, etc.

 

Radioisotopes

Copper (Cu) is a key nutrient to human cells. Human Cu transporter 1 (CTR1), a 190-amino acid protein of 28 kDa with three transmembrane domains, is the primary protein responsible for import of Cu in mammal. Interestingly, Cu metabolism is also essential for many cancers, and it has been found that CTR1 is over-expressed in a variety of cancer cells including non-small cell lung cancer (NSCLC) and liver cancer. Over-expression of CTR1 has also been found to be associated with better cisplatin based chemotherapy response and favorable treatment outcome in lung cancer patients. Traditionally, 64Cu2+ has been widely used in labeling of peptides, proteins, antibodies, nanoparticles and other biologically relevant small molecules through a variety of bifunctional chelators such as DOTA and TETA. Recently, with the better understanding the role of CTR1 in tumor biology, 64CuCl2 has been reported as a novel and promising PET probe for imaging of Wilson’s disease, prostate cancer, and hepatic carcinoma, though further studies are required to verify and validate the CTR1 targeting specificity and sensitivity. Considering the unique advantages of 64CuCl2 (easily available, clinical translatable, simple radiochemistry and no complex radiolabeling process involved, high stability and no issue of degradation, high bioactivity towards CTR1, possibility for both imaging and therapy, etc.), we have further tested the imaging quality and treatment efficacy of 64CuCl2 for melanoma. Our study highlights that 64CuCl2 can be used for imaging and treatment both melanotic and amelanotic melanoma.

 

Small Molecules

Cutaneous malignant melanoma is one of the most lethal cancers. The most important approach for improvement of survival of melanoma patients still remains early diagnosis, along with accurate staging of disease extent. Positron emission tomography (PET) is a very promising technology for non-invasively imaging tumor micrometastases. PET coupled with a proper imaging probe may provide oncologists a highly sensitive procedure for the accurate staging of high-risk melanomas.

Though 18F-fluorodeoxyglucose (18F-FDG) PET has been widely used clinically for melanoma imaging, other approaches to specifically identify, characterize, monitor and guide therapeutics for malignant melanoma are still needed. Consequently, many probes targeting general molecular events including metabolism, angiogenesis, hypoxia and apoptosis in melanoma have been successfully developed. Our research has focused on developing novel small molecules based PET probes targeting melanoma associated specific targets such as melanin, etc.  Small molecule benzamide analogs can bind with melanin overproduced in malignant melanoma. Through rational design of the probes and further in vivo evaluation of several PET radionuclide labeled compounds, we have successfully identified 18F-benzamide analogs 18F-FBZA, 18F-P3BZA, 18F-FPDA which display excellent properties for melanoma imaging. Especially, 18F-P3BZA was found to specifically target melanoma with high tumor uptakes and good tumor to normal tissue ratios. It represents a potential PET probe for imaging melanotic malignant melanoma and its metastases, and we would like to move this agent into clinical evaluation (See Figure 2).

 

Scaffold Proteins

Recently, significant advancement has been made in the in vitro display technologies and rational protein engineering. Many novel protein scaffolds that specifically bind to targeted biomarkers have been discovered. A common feature of these protein scaffolds is that they are highly structured, generally displayed fast in vivo clearance, rapid tumor accumulation, sufficient in vivo stability and good bioavailability, and low immunogenicity and toxicity. Protein mutants derived from these scaffolds that recognize different tumor biomarkers can be obtained in relatively short time. The in vivo behavior of these mutants is likely to have many similarities with the parent protein they’re derived from. The versatile production, consistent in vivo performance, and ability to recognize a variety of biomarkers highlight the importance of using protein scaffold mutants for developing probes for molecular imaging.

My research group is interested in studying: 1) whether the protein scaffolds could potentially be used to develop imaging agents that bind to a great variety of important molecular targets associated with cancers; 2) whether the protein scaffold based approach could become a generalizable strategy for facilitating the molecular probe development. Thus over the past several years, we have focused our work on studying two new emerging protein scaffolds for their diagnostic applications: Affibody and Cystine knot miniproteins (knottins).

Affibody molecules, derived from one of the IgG-binding domains of staphylococcal protein A, are composed of a relatively small, engineered protein scaffold with 58-amino acid residues and a three-helix bundle scaffold structure. Cystine knot proteins are small constrained polypeptides that share a common disulfide-bonded framework and a triple-stranded b-sheet fold. We have synthesized and evaluated a variety of radiolabeled (18F, 68Ga, 64Cu, 111In, etc) or optical dye labeled Affibody (3-hellix and 2-helix) and Cystine knot miniproteins. These imaging agents could be used to image several important tumor targets such as human epidermal growth factor receptor type 2 (HER2), epidermal growth factor receptor (EGFR), tumor angiogenesis target integrin receptor avb3. Overall, our studies have clearly demonstrated that Affibody and Cystine knot protein scaffolds can be used as excellent platforms for molecular probes development. Those Affibody and Cystine knot based probes are worthy of further evaluation and optimization for the development of positron emission tomography (PET) probes for clinical HER2, EGFR, avb3 and avb6 imaging. The Affibody based PET probe developed for EGFR imaging is now being translated into clinical cancer imaging applications.

    Please read our publications for more information about affibody imagings:

    Please read our publications for more information about knottin imagings:

 

Monoclonal Antibodies

The heparin sulfate proteoglycan glypican-3 (GPC3) is a potentially valuable molecular target for diagnostic imaging of hepatocellular carcinoma (HCC). We have studied the use of a positron emission tomography (PET) molecular probe based on a GPC3-specific monoclonal antibody (MAb) to accurately identify GPC3-positive HCC cells in vitro and in vivo. Our results demonstrate that PET imaging targeting GPC3 is a feasible and translatable approach for diagnostic imaging of HCC lesions.