The Precision Medicine Resource Center has four Technology Research and Development cores, commonly referred to as TR&Ds, listed below:

• Molecular imaging Reagants for Prostate Cancer Theranostics (TR&D 1)
• Optimizing and Humanizing a Reporter Gene for Imaging in Deep Tissue (TR&D 2)
• Imaging Agents for Inflammatory Components of Malignancy (TR&D 3)
• CEST MRI Agents for Receptor Imaging

TR&D 1: Molecular Imaging Reagants for Prostate Cancer Theranostics

Co-PI: Zaver M. Bhujwalla, M.Sc., Ph.D.
Co-PI: Dmitri Artemov, Ph.D.

This program is focused on the development of theranostic agents, which will uncover aspects of the tumor microenvironment (TME) while leveraging the TME to treat cancers using photodynamic therapy. Down-regulation of checkpoints will occur through targeted (PSMA) delivery of nucleic acids and prodrug enzymes.

Specific Aims:

• Develop a rapidly translatable molecular imaging probe to image focal adhesion kinase (FAK) expression in prostate cancer
• Develop theranostic reagents to perform phototherapy of CAFs
• Synthesize PSMA targeted nanoparticles that deliver siRNA and the prodrug enzyme bacterial cytosine deaminase (bCD)

Prostate cancer (PCa) is the second leading cause of death from cancer in men in the U.S. The vast majority of men dying of PCa continue to succumb to metastatic castration-resistant disease. There is a compelling need to find effective treatments for metastatic PCa.

Our purpose in TR&D1 of the Precision Medicine Resource Center is to successfully develop prototype theranostic molecular imaging platforms to pursue novel avenues of (i) detecting and targeting the focal adhesion kinase (FAK) mechanotransduction pathway that allow cells to migrate (Aims 1a and b), (ii) detecting and eliminating activated cancer associated fibroblasts (CAFs) that play an important role in the formation of a prometastatic extracellular matrix (ECM) in PCa (Aim 2), and (iii) developing prostate specific antigen (PSMA)-targeted nanoparticles (NPs) to deliver siRNA to downregulate programmed death ligand 1 (PD-L1) together with a prodrug enzyme, to exploit the activation of the immune system, together with localized cell killing, in locally advanced and metastatic PCa (Aim 3). Optical and PET imaging reporters will be integrated into the platforms to achieve spatial and temporal visualization of the NPs in vivo for precision medicine. PSMA, a type II integral membrane protein that is abundantly expressed on the surface of PCa in castration-resistant, advanced and metastatic disease, provides a unique advantage to deliver PSMA-specific NPs for effective control and treatment of locally advanced or metastatic PCa.

These studies will result in the accelerated development of FAK PET imaging probes with near term clinical translation that will have a direct impact on the selection of patients for ongoing FAK inhibitor treatment trials. Mechanical movement of cancer cells is a prerequisite for invasion and metastasis. NPs that achieve PCa-specific downregulation of FAK using PSMA-specific delivery will provide cancer-specific downregulation of cell migration, a key step in the metastatic cascade. Similarly, detection of CAFs in tumors with imaging will provide a distinct advantage over biopsy specimens in evaluating CAF numbers as a marker of aggressiveness. CAF elimination with phototherapy may provide a strategy to reduce or eliminate PCa metastasis. The development of NPs to improve immunotherapy in PCa through theranostics and their translation will represent a significant advance in this field since PCa has traditionally not responded well to immunotherapy. TR&D1 will also serve as the Pre-Clinical Validation Core that will, through close interactions with the CPs and other TR&Ds, develop and disseminate novel molecular imaging theranostic agents that will advance precision medicine of cancer worldwide.

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TR&D 2: Optimizing and Humanizing a Reporter Gene for Imaging in Deep Tissue

Co-PI: Jeff Bulte, M.D., Ph.D.
Co-PI: Assaf A. Gilad, Ph.D.

Specific Aims:

• Synthesis of next generation LRP reporters
• Optimize MRI pulse sequences and data processing for visualizing next generation LRP reporters
• Humanization of the next generation LRP

We will develop novel synthetic, non-metallic reporter genes that can be detected with chemical exchange saturation transfer (CEST) MRI for precise visualization and pinpointing of biological processes in living organisms. Using a lysine-rich protein (LRP) as such a prototype gene, we have previously demonstrated that 1) We can image rapidly dividing tumor cells without the limitation of a label dilution effect that currently exists with conventional MR contrast agents; 2) We can image promoter-driven specific gene expression; and 3) we can image oncolytic virotherapy. In this TR&D, we aim to dramatically improve the CEST contrast and biocompatibility of LRP for further dissemination to the scientific community, and to create a pathway towards eventual clinical translation.

Using advanced, rational design-based molecular-genetic engineering approaches, we will first develop a so- called “enhanced” LRP, or eLRP (Aim 1a). Enhancement is defined by transcription and translation efficiency, protein refolding, optimal proton exchange rate, and strongest CEST contrast. For the latter, a custom-designed high-throughput screening methodology will be used to determine optimal peptide sequence configurations. Next, we will “humanize” eLRP to create heLRP, using an array of immunological assays (Aim 1b). We will use established algorithms to identify epitopes that induce a T cell and/or humoral response in reverse. Re- engineered LRPs will undergo reiterated screening processes until all immunogenicity has been eliminated without compromising CEST contrast. Alternatively, we will use human protamine-1 (hPRM1) as a starting template to create chimeric LRP/hPRM1 constructs through DNA shuffling. The absence of serum polyclonal antibodies from heLRP-immunized rabbits will be used as a final key criteria for TR&D 2 dissemination.

During this immunological screening process, we will simultaneously identify the counterpart of heLRP, i.e., an “immunogenic” LRP or iLRP. Following in vivo transfection, this iLRP will be used as a new theranostic vector to simultaneously induce an anti-tumor immune response and visualize subsequent tumor cell regression (Aim 2). Finally, we aim to demonstrate how eLRP can be used to provide a unique dynamic insight into biological processes and as defined by cell-cell interactions. We have chosen dendritic cell (DC) immunotherapy as an example. Following a validation study to confirm that constitutively expressed eLRP DCs can be detected in vivo when migrating to lymph nodes following vaccination (Aim 3a), we will investigate when and where DC activation occurs upon presenting antigen to CD4+ cells. We aim to accomplish this using IL-12 promoter-driven specific expression (Aim 3b). Concurrently, we will assess the time course and whole body distribution of activated, Ova- specific CD4+ transgenic cells using BLI in an Ova-expressing melanoma mouse model.

We believe that our LRP reporters will have many applications in the study of basic cell biology and cell malfunctioning in a wide variety of disease models, as they can be designed de novo and in silico, and hence have unlimited potential for manipulation and fine-tuning as needed for the precise visualization of the biological process in question.

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TR&D 3: Imaging Agents for Inflammatory Components of Malignancy

Co-PI: Martin G. Pomper, M.D., Ph.D.
Co-PI: Andrew Horti, Ph.D.
Co-PI: Sridhar Nimmagadda, Ph.D.

Specific Aims:

• Synthesis and validation of agents targeting PD-L1
• In vivo validation of imaging agents for the complement system
• Translation of these promising agents to clinic

We will develop imaging agents to detect inflammation and immunity, with a focus on small molecule PET agents to monitor immunotherapy, an increasing medical need. Inflammation has long been recognized as a promoter of tumor growth. More recently, harnessing innate and adaptive immunity to treat cancer through immune checkpoint inhibition and vaccines has captivated the community of cancer researchers and clinicians alike. We will develop and disseminate agents that address two different aspects of cancer and its relationships to inflammation and immunity, namely, checkpoint inhibition and complement. The immune checkpoint protein programmed death-ligand 1 (PD-L1) and its receptor PD-1 are preferred targets for cancer immunotherapy. PD-L1 is expressed by a variety of tumors, and its over-expression is induced in tumor cells to adapt to tumor infiltrating cytotoxic T cells. PD-L1 immunohistochemistry (IHC) is the best predictive biomarker for therapeutic monitoring of PD-L1/PD-1 targeted therapies. However, PD-L1 IHC is fraught with use of discordant antibodies, intra- and inter-tumoral heterogeneity of expression as well as limited bio-specimen availability such that we believe non-invasive imaging can help. Furthermore, despite the promise of immune checkpoint therapy, the majority of patients do not respond for reasons unclear. The complement system is central to recruiting inflammatory cells and promoting release of factors that can promote tumor growth and progression, confounding immunotherapy.

We will synthesize, validate and disseminate agents targeting PD-L1 (Aim 1) and complement receptors C3aR and C5aR (Aim 2), which are bound by their cognate tumor-promoting anaphylatoxins. In Aim 3 we will validate – with correlation to post-imaging surgical tissue – a current BTRC lead PD-L1 imaging agent in patients undergoing immunotherapy for pancreas cancer through support from the Bloomberg-Kimmel Institute for Immunotherapy. Once validated in this ultimate fashion we will be confident to disseminate that agent for human studies elsewhere. TR&D 3 will also serve as the Clinical Validation Core, a hub that will disseminate not only valuable new human agents as noted above, but will also provide precursor and standard for other agents, allow cross-referencing of BTRC INDs and provide analysis to meet the evolving needs of the driving Collaborative Projects and service recipients.

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TR&D 4: CEST MRI Agents for Receptor Imaging

Co-PI: Michael McMahon, Ph.D.
Co-PI: Xing Yang, Ph.D,
Co-PI: Peter C.M. van Zijl, Ph.D.

Specific Aims:

• Targeted CEST PSMA Agents
• MR Methods for Acquisition and Analysis
• Identification of New Targets

The overarching goal of TR&D 4 is to introduce concepts of precision medicine to chemical exchange saturation transfer (CEST) magnetic resonance imaging (MRI). CEST MRI amplifies signal from macromolecular species that contain protons in exchange with water. Currently CEST leverages the suitable chemical shift frequencies and exchange rates of amide or hydroxyl protons. One may use CEST for detection of proteins that naturally contain many such functionalities or for exogenously administered, artificial agents, such as the lysine-rich reporter from TR&D 2. As such CEST is not an inherently targeted (to phenotypically expressed proteins) or precision method. TR&D 4 of the BTRC will generate and disseminate precision CEST agents. We will develop non-metallic, targeted MR agents for receptor imaging with a focus on polymeric species to ensure adequate signal. Currently, about one third of all MRI scans rely on administration of non-specific relaxation-based gadolinium contrast agents to aid in the clinical differentiation of healthy and diseased tissues, however in view of recent concerns about accumulation of gadolinium in brain and bone, there is an increasing demand for non- metallic agents. CEST MRI allows the imaging of low-concentration compounds with the molar sensitivity of MRI and also has the advantages that CEST agents can be designed to be biodegradable. We will synthesize translatable CEST MRI agents for receptor-based imaging together with acquisition and analysis approaches that are optimized for the properties of each individual agent.

To accomplish this, in Aim 1, we will conjugate a PSMA targeting ligand to highly sensitive CEST polymers, including dextran polymers as biocompatible agents and salicylic acid polymers as higher sensitivity polymers. We will then optimize pulse sequences for detecting the polymeric agents in terms of exchange rate and chemical shift (Aim 2). The pulse sequences developed in Aim 2 will also be applied to TR&D 2. In Aim 3 we will further proceed to investigate two other clinically relevant receptors, carbonic anhydrase IX (CA-IX), the expression of which has implications for the tumor microenvironment, and Axl tyrosine kinase. CA-IX is an antigen expressed on clear cell renal cell carcinoma (ccRCC), and could provide valuable information for radical nephrectomy and renal cancer surveillance. In Aim 4, we will generate cGMP-grade material of the most promising item in the Center for Translational Molecular Imaging on the pathway to human use. For this step, the agents generated will be tested in preliminary toxicity studies will be performed prior to GLP toxicity testing. 

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