The Thomas Reiner Lab: Research Overview

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The Reiner Lab research program revolves around the development, validation and translation of novel imaging agents. Our research is firmly rooted in the preclinical research space, but we also aggressively pursue translational clinical projects. One of our key achievements is the development of a quantitative small molecule imaging platform for PARP1, yielding several translated imaging agents that have originated in our lab. One of our imaging agents, PARPi-FL, a fluorescent intraoperative probe, entered clinical trials in March 2017 (IND#133,109, NCT03085147). A second translated drug, [18F]PARPi, a quantitative PET imaging agent, was approved for clinical trials in August 2018 and November 2019 (IND#139974; NCT03631017 and NCT04173104, respectively). Phase I data for both agents has been published in Nature Biomedical Engineering (Kossatz et al 2020) and Clinical Cancer Research (Schöder et al, 2020). A radiotherapeutic version of our PARP1 imaging agents is being tested in mouse models of cancer.

While our previous work was in large part focused on the detection, delineation and treatment of cancer, we also interrogate other physiologically relevant markers. These agents help us to understand the molecular mechanisms of drug action, shedding new light on tumor cell pathology and helping to predict why some patients respond well to a particular treatment regimen, while others do not.

PARPi-FL Transformation of a small molecule into a diagnostic screening tool.

While oral squamous cell carcinoma (OSCC) can frequently be cured in early stages (83% 5-year survival for local disease), diagnosis is often delayed, also because tumors remain asymptomatic despite their superficial location, and almost two thirds of all patients present with advanced stage disease at the time of diagnosis. There is therefore a significant diagnostic delay, and better and earlier detection of this disease are unmet clinical needs. However, a screening platform for detecting oral cancer in its early stages does not exist. Based on this unmet clinical need, we have developed a screening method for oral cancer which works like a mouthwash, allowing physicians to delineate oral cancer early and non-invasively (Kossatz et al., 2016, Kossatz et al., 2019). This mouthwash uses a fluorescent molecule that can be applied topically and adheres to cancer cells inside the oral cavity. Any cancer cells in the mouth will therefore appear fluorescent, and can be detected by us with a custom made fluorescent laparoscope.

Our lead imaging tracer is called PARPi-FL, is a fluorescent dye (excitation wavelength 488 nm, emission wavelength 525 nm) and has low IC50 values for inhibition of PARP1 activity in an enzymatic assay (12.2 nM vs. 6.0 nM; (Thurber et al., 2013; Reiner et al., 2012). The small molecule is metabolically stable in mice and accumulates within minutes in the nuclei of cancer cells, where it is retained for several hours. Quantification of the nuclear fluorescence demonstrated that almost all cancer cells (> 99.8%) were targeted and that the average concentration in the nucleus is high when compared to other imaging targets (1.9 ± 0.5 μM).

This formerly preclinical project has now been translated. Our investigator-initiated Phase I/II clinical protocol (IRB# 15-366, IND#133,109, NCT03085147) is the first of a series of clinical protocols, in which we aim to show that PARPi-FL imaging has better sensitivity and specificity than existing diagnostic methods in oral cancer, resulting in improved overall survival and quality of life for patients. We recently published encouraging initial results (Kossatz et al. 2020), confirming our central hypothesis, and are excited to drive this project through Phase II and beyond. PARPi-FL is the first molecularly targeted drug where this is being attempted.

parpi18

Fig 1: First-in-human imaging of PARPi-FL. (A) PARPi-FL first-in-human imaging (NCT03085147), showing a patient who presented with a malignant recurrent squamous cell carcinoma (yellow arrow) and an adjacent benign granuloma (blue arrow). (B) Using a Quest Spectrum imaging device with a laparoscopic camera and PARPi-FL optimized laser/filter system, the tumour area of the patient was imaged after gargling a 500 nM PARPi-FL solution. Granuloma (blue circles) and tumour (yellow circles) areas showed distinct patterns of PARPi-FL accumulation, and specific PARPi-FL uptake was only detectable in the tumour. The white circles highlight a non-malignant region which showed autofluorescence, but no PARPi-FL uptake. (C) Tumor-to-margin ratios of fluorescence imaging for patients in the Phase I dose escalation cohorts (100 nM, 250 nM, 500 nM, and 1000 nM).

[18F]PARPi – A small molecule for guiding treatment in primary and metastatic brain tumor patients

Positron emission tomography (PET) scans of the brain are often performed to identify and distinguish cancers from infection and from other non-cancer entities at diagnosis, most importantly from alterations related to prior treatment. Currently, [18F]fluorodeoxyglucose (FDG) is the most commonly used radiotracer in the United States; however, its sensitivity and specificity are known to be limited due to the high glucose uptake of the normal brain and also prominent uptake with postoperative and treatment related inflammatory changes. Because of the high physiologic uptake in normal gray matter, FDG-avid brain cancers are also often indistinct on PET brain scans.

Using a radiolabeled PARP1-selective small molecule, we demonstrated superior cancer visualization and superior lesion-to-contralateral uptake ratios. Unlike FDG, cancer detection with [18F]PARPi is not based on metabolic activity but rather on the presence of the DNA-repair enzyme poly (ADP-ribose) polymerase1 (PARP1) inside the cancer cell nuclei. The poly (ADP-ribose) polymerase (PARP) family of DNA repair enzymes is overexpressed in many solid cancers including brain metastases and high grade gliomas. This overexpression is thought to represent a cellular response to the genomic instability and the frequent cell division occurring in cancer cells. The difference in PARP1 expression found in between cancer and normal tissue generates the contrast that can be seen on a PET scan after injection of [18F]PARPi. We are not only focusing on glioblastoma, and our efforts are spanning other malignancies as well (including large B cell lymphoma, tumors oif the head&neck and small cell lung cancer). A [18F]PARPi Phase I safety and radiation dosimetry trial (IRB#18-241, IND#139974, NCT03631017) was published in early 2020 (Schöder et al., 2020).

First-in-human imaging of [18F]PARPi

Fig 2: First-in-human imaging of [18F]PARPi. (A) Specific nuclear PARPi-FL uptake was seen in all cancers (lesions 2, 6 and 7). Faint signal was seen in treatment related changes (e.g. necrosis, lesion #5). This differential PARP1 expression in cancer versus treatment related changes was also observed on immunohistochemistry between patients. (B) Differences in quantified PARP1 expression was seen in cancer (lesions #2, 6 and 7 – in blue) when compared to the tissue that only presented treatment related changes (lesion 5 – in pink). Lesion #5, with no viable cancer, had median PARP1 expression over total tissue area of 3%, which was significantly lower than the expression in all cancer specimens, lesions #2, 6 and 7 - 7%, 10% and 14%, respectively, p < 0.001, Kruskal-Wallis test. (C) Correlation of PARP1 expression and the SUVmax of [18F]PARPi at 60 minutes post injection. (D) Coronal PET/MR images of the parietal lobe taken with [18F]PARPi tracer. Difference in uptake seen on imaging (arrow points to high uptake) is believed to be due to difference in PARP1 expression seen at histology between areas of cancer and areas of treatment change. Scale bar in slides with high magnification correspond to 50 μm and overviews correspond to 0.5 cm.

[123I]MAPi – A precision therapeutic for treating brain tumors with limited off-target toxicity.

The Auger electron emitting iodine isotope 123I has distinct advantages over other therapeutic radionuclides because of its intense local energy deposition, which is principally confined to a 10 nanometer sphere around the decay site. As a result, Auger electron emitting radionuclides can exhibit high linear energy transfer (LET) and radiotoxicity when confined within the nucleus and associated with DNA, but not when in the extracellular space or cytosol. Consequently, Auger emitting radiopharmaceuticals that are excreted via the kidneys or the liver are less likely to damage these organs than α- or β-emitting radionuclides. So far, however, the development of a radiopharmaceutical, which could harness this compelling physical effect has had only limited success. In 2015, we began synthesizing libraries of iodinated small molecules that bind to the DNA repair enzyme poly [ADP-ribose] polymerase 1 (PARP1) and accumulate in the nuclei of cancer cells (Salinas et al., 2015). Their radiotherapeutic 123I-labeled versions capitalize on the radiotoxic potential of Auger emitters targeted to DNA. We showed in mouse models of glioblastoma that organ-specific toxicity associated with the metabolism and excretion of these PARP1-trageted emitters will be low, and that these novel drugs could provide a significant clinical benefit by widening the therapeutically relevant window between tumor and dose limiting normal tissues. Dose estimates and survival studies showed very encouraging RBE values (Pirovano et al. 2020). Clinically, PARP1 targeted small molecules could be an ideal vector to deliver therapeutic radioisotopes to tumor tissues without damaging healthy surrounding tissue.

PARP-targeted Auger therapeutics

Fig 3: PARP-targeted Auger therapeutics. (A) Clonogenic survival of U251 GBM cells following in vitro Auger 123I-MAPi therapy in comparison to external irradiation. (B) Representative image showing immunofluorescence of γ-H2AX (red), DAPI nuclear staining (blue), and merged images after treatment with 123I-MAPi. All images from the experiment were analyzed and quantified and showed significant increase of the number of γ-H2AX foci within the nucleus of cells after treatment with 123I-MAPi (n = 85), 127I-PARPi (n = 125), and vehicle control (n = 140). ***p-value < 0.001, Kruskal-Wallis test. (C) 3D dosimetry estimates calculated by Monte Carlo simulation for subcutaneous pump administration. Equivalent doses assume the measured deterministic RBE of 48.4 for 123I-MAPi tumor tissue and assume RBE of 1.0 elsewhere. (D) In vivo characterization of induced DSB after topical treatment with 123I-MAPi in mice bearing TS543 patient-derived glioblastoma cells. H&E was used to confirm tumor presence and γ-H2AX staining to investigate DSB formation at 1-hour post injection of 123I-MAPi or vehicle as a control. (E) Kaplan-Meier survival study of pump implanted mice shows an improvement of survival of 123I-MAPi treated mice (n = 8) when compared to control (n = 8). Treatment mice osmotic pumps were loaded with 481 ± 111 kBq. Log-Rank (Mantel-Cox) test, *p-value < 0.05.