Jingjuan Qiao

HER2 Targeted Molecular MR Imaging Using a De Novo Designed Protein Contrast Agent

The application of magnetic resonance imaging (MRI) to non-invasively assess disease biomarkers has been hampered by the lack of desired contrast agents with high relaxivity, targeting capability, and optimized pharmacokinetics. We have developed a novel MR imaging probe targeting to HER2, a biomarker for various cancer types and a drug target for anti-cancer therapies. This multimodal HER20targeted MR imaging probe integrates a de novo designed protein contrast agent with a high affinity HER2 affibody and a near IR fluorescent dye. Our probe can differentially monitor tumors with different expression levels of HER2 in both human cell lines and xenograft mice models. In addition to its 100-fold higher dose efficiency compared to clinically approved non-targeting contrast agent DTPA, our developed agent also exhibits advantages in crossing the endothelial boundary, tissue distribution, and tumor tissue retention over reported contrast agents as demonstrated by even distribution of the imaging probe across the entire tumor mass. This contrast agent will provide a powerful tool for quantitative assessment of molecular markers, and improved resolution for diagnosis, prognosis and drug discovery.

Design of a novel class of protein-based magnetic resonance imaging contrast agents for the molecular imaging of cancer biomarkers

Magnetic resonance imaging (MRI) of disease biomarkers, especially cancer biomarkers, could potentially improve our understanding of the disease and drug activity during preclinical and clinical drug treatment and patient stratification. MRI contrast agents with high relaxivity and targeting capability to tumor biomarkers are highly required. Extensive work has been done to develop MRI contrast agents. However, only a few limited literatures report that protein residues can function as ligands to bind Gd(3+) with high binding affinity, selectivity, and relaxivity. In this paper, we focus on reporting our current progress on designing a novel class of protein-based Gd(3+) MRI contrast agents (ProCAs) equipped with several desirable capabilities for in vivo application of MRI of tumor biomarkers. We will first discuss our strategy for improving the relaxivity by a novel protein-based design. We then discuss the effect of increased relaxivity of ProCAs on improving the detection limits for MRI contrast agent, especially for in vivo application. We will further report our efforts to improve in vivo imaging capability and our achievement in molecular imaging of cancer biomarkers with potential preclinical and clinical applications.

Protein MRI contrast agent with unprecedented metal selectivity and sensitivity for liver cancer imaging

Significance Primary and metastatic liver cancers that are associated with high mortality rates and poor treatment responses are only diagnosed at late stages, due to the lack of highly sensitive contrast agents and robust imaging methodologies. We have developed a protein MRI contrast agent (ProCA32) by engineering high-affinity Gd3+-binding pockets in rat and human α-parvalbumin. ProCA32 can function as both a T1- and T2-weighted contrast agent, which enables noninvasive detection of early-stage micrometastatic liver tumors with sizes as small as 0.24 mm using T1- and T2-weighted or T2/T1 ratio MRI. Our protein-based MRI contrast agents and imaging methodology are expected to provide robust results for the early detection of liver cancer as well as other liver diseases. With available MRI techniques, primary and metastatic liver cancers that are associated with high mortality rates and poor treatment responses are only diagnosed at late stages, due to the lack of highly sensitive contrast agents without Gd3+ toxicity. We have developed a protein contrast agent (ProCA32) that exhibits high stability for Gd3+ and a 1011-fold greater selectivity for Gd3+ over Zn2+ compared with existing contrast agents. ProCA32, modified from parvalbumin, possesses high relaxivities (r1/r2: 66.8 mmol−1⋅s−1/89.2 mmol−1⋅s−1 per particle). Using T1- and T2-weighted, as well as T2/T1 ratio imaging, we have achieved, for the first time (to our knowledge), robust MRI detection of early liver metastases as small as ∼0.24 mm in diameter, much smaller than the current detection limit of 10–20 mm. Furthermore, ProCA32 exhibits appropriate in vivo preference for liver sinusoidal spaces and pharmacokinetics for high-quality imaging. ProCA32 will be invaluable for noninvasive early detection of primary and metastatic liver cancers as well as for monitoring treatment and guiding therapeutic interventions, including drug delivery.

Pyruvate Kinase M2 in Blood Circulation Facilitates Tumor Growth by Promoting Angiogenesis

Background: It is known that PKM2 is present in cancer patient blood. It is not known if PKM2 functions in cancer progression. Results: PKM2 in blood facilitates tumor growth by promoting tumor angiogenesis via increasing angiogenic endothelial cell migration and ECM attachment. Conclusion: Extracellular PKM2 promotes tumor angiogenesis. Significance: We reveal a novel mechanism of cancer angiogenesis that could potentially be a new target for anti-angiogenesis. It is long known that pyruvate kinase isoform M2 (PKM2) is released into the circulation of cancer patients. The PKM2 levels in patients have been suggested as a diagnostic marker for many types of cancers. However, it is not known how PKM2 is released in the blood, and whether the circulating PKM2 has any physiological function(s) in tumor progression. In this report, we demonstrate that PKM2 in the blood facilitates tumor growth by promoting tumor angiogenesis. Our experiments show that PKM2 promotes tumor angiogenesis by increasing endothelial cell proliferation, migration, and cell-ECM adhesion. Only the dimeric PKM2 possess the activity in promoting tumor angiogenesis, which is consistent with the observations that PKM2 in circulation of cancer patients is a dimer form.

PEGylation of Protein-based MRI Contrast Agents Improves Relaxivities and Biocompatibilities

Magnetic resonance imaging (MRI) has emerged as a leading diagnostic technique in clinical and preclinical settings. However, the application of MRI to assess specific disease markers for diagnosis and monitoring drug effect has been severely hampered by the lack of desired contrast agents with high relaxivities, and optimized in vivo retention time. We have reported the development of protein-based MRI contrast agents (ProCA1) by rational design of Gd(3+) binding sites into a stable protein resulting in significantly increased longitudinal (r(1)) and transverse (r(2)) relaxivities compared to Gd-DTPA. Here, we report a further improvement of protein contrast agents ProCA1 for in vivo imaging by protein modification with various sizes of polyethylene glycol (PEG) chain. PEGylation results in significant increases of both r(1) and r(2) relaxivities (up to 200%), and these high relaxivities persist even at field strengths up to 9.4 T. In addition, our experimental results demonstrate that modified contrast agents have significant improvement of in vivo MR imaging and biocompatibilities including dose efficiency, protein solubility, blood retention time and decreased immunogenicity. Such improvement can be important to the animal imaging and pre-clinical research at high or ultra-high field where there is an urgent need for molecular imaging probes and optimized contrast agent.

Design of ProCAs (protein-based Gd(3+) MRI contrast agents) with high dose efficiency and capability for molecular imaging of cancer biomarkers.

Magnetic resonance imaging (MRI) is the leading imaging technique for disease diagnostics, providing high resolution, three‐dimensional images noninvasively. MRI contrast agents are designed to improve the contrast and sensitivity of MRI. However, current clinically used MRI contrast agents have relaxivities far below the theoretical upper limit, which largely prevent advancing molecular imaging of biomarkers with desired sensitivity and specificity. This review describes current progress in the development of a new class of protein‐based MRI contrast agents (ProCAs) with high relaxivity using protein design to optimize the parameters that govern relaxivity. Further, engineering with targeting moiety allows these contrast agents to be applicable for molecular imaging of prostate cancer biomarkers by MRI. The developed protein‐based contrast agents also exhibit additional in vitro and in vivo advantages for molecular imaging of disease biomarkers, such as high metal‐binding stability and selectivity, reduced toxicity, proper blood circulation time, and higher permeability in tumor tissue in addition to improved relaxivities.

Molecular imaging of EGFR/HER2 cancer biomarkers by protein MRI contrast agents

Epidermal growth factor receptor (EGFR) and HER2 are major prognosis biomarkers and drug targets overexpressed in various types of cancer cells. There is a pressing need to develop MRI contrast agents capable of enhancing the contrast between normal tissues and tumors with high relaxivity, capable of targeting tumors, and with high intratumoral distribution and minimal toxicity. In this review, we first discuss EGFR signaling and its role in tumor progression as a major drug target. We then report our progress in the development of protein contrast agents with significant improvement of both r1 and r2 relaxivities, pharmacokinetics, in vivo retention time, and in vivo dose efficiency. Finally, we report our effort in the development of EGFR-targeted protein contrast agents with the capability to cross the endothelial boundary and with good tissue distribution across the entire tumor mass. The noninvasive capability of MRI to visualize spatially and temporally the intratumoral distribution as well as quantify the levels of EGFR and HER2 would greatly improve our ability to track changes of the biomarkers during tumor progression, monitor treatment efficacy, aid in patient selection, and further develop novel targeted therapies for clinical application.

GRPR-targeted Protein Contrast Agents for Molecular Imaging of Receptor Expression in Cancers by MRI

Gastrin-releasing peptide receptor (GRPR) is differentially expressed on the surfaces of various diseased cells, including prostate and lung cancer. However, monitoring temporal and spatial expression of GRPR in vivo by clinical MRI is severely hampered by the lack of contrast agents with high relaxivity, targeting capability and tumor penetration. Here, we report the development of a GRPR-targeted MRI contrast agent by grafting the GRPR targeting moiety into a scaffold protein with a designed Gd3+ binding site (ProCA1.GRPR). In addition to its strong binding affinity for GRPR (Kd = 2.7 nM), ProCA1.GRPR has high relaxivity (r1 = 42.0 mM−1s−1 at 1.5 T and 25 °C) and strong Gd3+ selectivity over physiological metal ions. ProCA1.GRPR enables in vivo detection of GRPR expression and spatial distribution in both PC3 and H441 tumors in mice using MRI. ProCA1.GRPR is expected to have important preclinical and clinical implications for the early detection of cancer and for monitoring treatment effects.

Towards the Molecular Imaging of Prostate Cancer Biomarkers Using Protein-based MRI Contrast Agents

Prostate cancer is the most common cancer for man with a high mortality rate due to a lack of non-invasive accurate and sensitive molecular diagnostic methods. The molecular imaging of cancer biomarkers using MRI with its spatial and temporal resolution, however, is largely limited by the lack of contrast agents with high sensitivity, targeting specificity and deep tumor penetration. In this review, we will first overview the current stage of prostate cancer diagnosis and then review prostate cancer biomarkers and related imaging techniques. Since biomarker targeting moieties are essential for molecular imaging, we will use prostate-specific membrane antigen (PSMA) as an example to discuss different methods to characterize the interaction between biomarker and targeting moieties. At the end, we will review current progress of the development of targeted protein-based MRI contrast agents (ProCAs) for prostate cancer biomarkers with improved relaxivity and targeting capability.