Jacob L. Houghton

Underscoring the influence of inorganic chemistry on nuclear imaging with radiometals

Over the past several decades, radionuclides have matured from largely esoteric and experimental technologies to indispensible components of medical diagnostics. Driving this transition, in part, have been mutually necessary advances in biomedical engineering, nuclear medicine, and cancer biology. Somewhat unsung has been the seminal role of inorganic chemistry in fostering the development of new radiotracers. In this regard, the purpose of this Forum Article is to more visibly highlight the significant contributions of inorganic chemistry to nuclear imaging by detailing the development of five metal-based imaging agents: (64)Cu-ATSM, (68)Ga-DOTATOC, (89)Zr-transferrin, (99m)Tc-sestamibi, and (99m)Tc-colloids. In a concluding section, several unmet needs both in and out of the laboratory will be discussed to stimulate conversation between inorganic chemists and the imaging community.

(18)F-Based Pretargeted PET Imaging Based on Bioorthogonal Diels-Alder Click Chemistry.

A first-of-its-kind 18F pretargeted PET imaging approach based on the bioorthogonal inverse electron demand Diels–Alder (IEDDA) reaction between tetrazine (Tz) and trans-cyclooctene (TCO) is presented. As proof-of-principle, a TCO-bearing immunoconjugate of the anti-CA19.9 antibody 5B1 and an Al[18F]NOTA-labeled tetrazine radioligand were harnessed for the visualization of CA19.9-expressing BxPC3 pancreatic cancer xenografts. Biodistribution and 18F-PET imaging data clearly demonstrate that this methodology effectively delineates tumor mass with activity concentrations up to 6.4 %ID/g at 4 h after injection of the radioligand.

Pretargeted Immuno-PET of Pancreatic Cancer: Overcoming Circulating Antigen and Internalized Antibody to Reduce Radiation Doses

5B1 is a fully human, monoclonal antibody that has shown promise for the PET imaging of cancers expressing carbohydrate antigen 19.9 (CA19.9)—a carbohydrate prevalent in cells with aberrant glycosylation and an established effector of metastasis. The long physiologic half-life of the antibody and interference from circulating CA19.9 may increase the time required to generate quality images as well as the risk of radiation exposure to healthy tissues during repeated PET imaging. Pretargeting methodologies are an effective approach to expeditiously acquire PET images, but in this case, the pretargeting approach is complicated by the internalization of 5B1 by CA19.9-expressing cells. We sought to adapt and optimize a pretargeting strategy that exploits the bioorthogonal reaction between transcyclooctene (TCO) and tetrazine (Tz) to overcome these complications. Methods: 5B1 was modified with TCO, and a novel NOTA-PEG7-Tz radioligand was synthesized with the goal of improving on a previously reported analog. BxPC3 and Capan-2 cells were evaluated for their ability to internalize anti-CA19.9 antibodies using a fluorometric assay, and xenografts of the same lines were used for in vivo studies. The pretargeting approach was optimized, and the 2 radioligands were compared using biodistribution and PET imaging in murine models of pancreatic cancer. Results: BxPC3 and Capan-2 cells were shown to rapidly internalize anti-CA19.9 monoclonal antibodies, including 5B1. 64Cu-NOTA-PEG7-Tz showed improved in vivo pharmacokinetics relative to 64Cu-NOTA-Tz using 5B1-TCO as the targeting vector. PET imaging and biodistribution studies showed that injecting the radioligand 72 h after the administration of 5B1-TCO resulted in the best uptake (8.2 ± 1.7 percentage injected dose per gram at 20 h after injection) and tumor-to-background activity concentration ratios. Dosimetry calculations revealed that the pretargeting system produced a greater than 25-fold reduction in total body radiation exposure relative to 89Zr-desferrioxamine-5B1. PET/CT imaging in an orthotopic Capan-2 xenograft model—which secretes large amounts of CA19.9 and more rapidly internalizes anti-CA19.9 antibodies—showed that this approach is viable even in the difficult circumstances presented by a circulating antigen and internalized targeting vector. Conclusion: The 5B1-TCO and 64Cu-NOTA-PEG7-Tz system evaluated in these studies can delineate CA19.9-positive xenografts in murine models of pancreatic cancer despite the challenges posed by the combination of circulating antigen and internalization of the 5B1-TCO.

Site-specifically labeled CA19.9-targeted immunoconjugates for the PET, NIRF, and multimodal PET/NIRF imaging of pancreatic cancer

Significance Pancreatic cancer will soon be the second leading cause of cancer deaths annually, yet no adequate molecular imaging tools exist to aid in the staging, monitoring, and treatment of the disease. Here we describe the development and preclinical evaluation of three unique immunoconjugates for positron emission tomography, near-infrared fluorescent optical imaging, and multimodal imaging of pancreatic ductal adenocarcinoma (PDAC). The probes were developed using a site-specific, chemoenzymatic methodology that is robust, reproducible, and modular. By targeting CA19.9, the most abundant antigen in >90% of PDAC tumors, we were able to obtain high-quality images in multiple murine models of PDAC, suggesting these constructs could be the core of a molecular imaging toolkit aimed at improving outcomes for patients with PDAC. Molecular imaging agents for preoperative positron emission tomography (PET) and near-infrared fluorescent (NIRF)-guided delineation of surgical margins could greatly enhance the diagnosis, staging, and resection of pancreatic cancer. PET and NIRF optical imaging offer complementary clinical applications, enabling the noninvasive whole-body imaging to localize disease and identification of tumor margins during surgery, respectively. We report the development of PET, NIRF, and dual-modal (PET/NIRF) imaging agents, using 5B1, a fully human monoclonal antibody that targets CA19.9, a well-established pancreatic cancer biomarker. Desferrioxamine (DFO) and/or a NIRF dye (FL) were conjugated to the heavy-chain glycans of 5B1, using a robust and reproducible site-specific (ss) labeling methodology to generate three constructs (ssDFO-5B1, ssFL-5B1, and ssdual-5B1) in which the immunoreactivity was not affected by the conjugation of either label. Each construct was evaluated in a s.c. xenograft model, using CA19.9-positive (BxPC3) and -negative (MIAPaCa-2) human pancreatic cancer cell lines. Each construct showed exceptional uptake and contrast in antigen-positive tumors with negligible nonspecific uptake in antigen-negative tumors. Additionally, the dual-modal construct was evaluated in an orthotopic murine pancreatic cancer model, using the human pancreatic cancer cell line, Suit-2. The ssdual-5B1 demonstrated a remarkable capacity to delineate metastases and to map the sentinel lymph nodes via tandem PET-computed tomography (PET/CT) and NIRF imaging. Fluorescence microscopy, histopathology, and autoradiography were performed on representative sections of excised tumors to visualize the distribution of the constructs within the tumors. These imaging tools have tremendous potential for further preclinical research and for clinical translation.

Establishment of the In Vivo Efficacy of Pretargeted Radioimmunotherapy Utilizing Inverse Electron Demand Diels-Alder Click Chemistry

The pretargeting system based on the inverse electron demand Diels-Alder reaction (IEDDA) between trans-cyclooctene (TCO) and tetrazine (Tz) combines the favorable pharmacokinetic properties of radiolabeled small molecules with the affinity and specificity of antibodies. This strategy has proven to be an efficient method for the molecularly targeted delivery of pharmaceuticals, including isotopes for radiological imaging. Despite encouraging results from in vivo PET imaging studies, this promising system has yet to be thoroughly evaluated for pretargeted radioimmunotherapy (PRIT). Toward that end, we synthesized two novel 177Lu-labeled tetrazine-bearing radioligands. Next, we compared the usefulness of our ligands for PRIT when paired with TCO-modified 5B1—a human, anti-CA19.9 mAb—in preclinical murine models of pancreatic cancer. The exemplary ligand, 177Lu-DOTA-PEG7-Tz, showed rapid (4.6 ± 0.8% ID/g at 4 hours) and persistent (16.8 ± 3.9% ID/g at 120 hours) uptake in tumors while concurrently clearing from blood and nontarget tissues. Single-dose therapy studies using 5B1-TCO and varying amounts of 177Lu-DOTA-PEG7-Tz (400, 800, and 1,200 μCi) showed that our system elicits a dose-dependent therapeutic response in mice bearing human xenografts. Furthermore, dosimetry calculations suggest that our approach is amenable to clinical applications with its excellent dosimetric profile in organs of clearance (i.e., liver and kidneys) as well as in dose-limiting tissues, such as red marrow. This study established that a pretargeted methodology utilizing the IEDDA reaction can rapidly and specifically deliver a radiotherapeutic payload to tumor tissue, thus illustrating its excellent potential for clinical translation. Mol Cancer Ther; 16(1); 124–33. ©2016 AACR.

Challenges of Pancreatic Cancer

AbstractThe development of novel molecular cancer imaging agents has considerably advanced in recent years. Numerous cancer imaging agents have demonstrated remarkable potential for aiding the diagnosis, staging, and treatment planning at the preclinical stage, which in turn has led to a number of agents being approved for human trials. Pancreatic ductal adenocarcinoma is currently the most deadly common carcinoma with an overall 5-year survival rate of about 6%. As detection technologies progress, the need for molecular imaging tools that will allow the diagnosis at an early stage will be crucial to improving patient outcomes. In this review, we will highlight agents that illuminate various cell populations that comprise the tumor: epithelial, endothelial, and stromal tumor cells.

Dual-Modality Imaging of Prostate Cancer with a Fluorescent and Radiogallium-Labeled Gastrin-Releasing Peptide Receptor Antagonist

Gastrin-releasing peptide (GRP) receptors (GRPr) are frequently overexpressed in human prostate cancer, and radiolabeled GRPr affinity ligands have shown promise for in vivo imaging of prostate cancer with PET. The goal of this study was to develop a dual-modality imaging probe that can be used for noninvasive PET imaging and optical imaging of prostate cancer. Methods: We designed and synthesized an IRDye 650 and DOTA-conjugated GRPr antagonist, HZ220 (DOTA-Lys(IRDye 650)-PEG4-[D-Phe6, Sta13]-BN(6-14)NH2), by reacting DOTA-Lys-PEG4-[D-Phe6, Sta13]-BN(6-14)NH2 (HZ219) with IRDye 650 N-hydroxysuccinimide (NHS) ester. Receptor-specific binding of gallium-labeled HZ220 was characterized in PC-3 prostate cancer cells (PC-3), and tumor uptake in mice was imaged with PET/CT and fluorescence imaging. Receptor binding affinity, in vivo tumor uptake, and biodistribution were compared with the GRPr antagonists HZ219, DOTA-PEG4-[D-Phe6, Sta13]-BN(6-14)NH2 (DOTA-AR), and DOTA-(4-amino-1-carboxymethyl-piperidine)-[D-Phe6, Sta13]-BN(6-14)NH2 (DOTA-RM2). Results: After hydrophilic–lipophilic balance cartridge purification, 68Ga-HZ220 was obtained with a radiochemical yield of 56% ± 8% (non–decay-corrected), and the radiochemical purity was greater than 95%. Ga-HZ220 had a lower affinity for GRPr (inhibitory concentration of 50% [IC50], 21.4 ± 7.4 nM) than Ga-DOTA-AR (IC50, 0.48 ± 0.18 nM) or Ga-HZ219 (IC50, 0.69 ± 0.18 nM). Nevertheless, 68Ga-HZ220 had an in vivo tumor accumulation similar to 68Ga-DOTA-AR (4.63 ± 0.31 vs. 4.07 ± 0.29 percentage injected activity per mL [%IA/mL] at 1 h after injection) but lower than that of 68Ga-DOTA-RM2 (10.4 ± 0.4 %IA/mL). The tumor uptake of 68Ga-HZ220 was blocked significantly with an excessive amount of GRP antagonists. IVIS spectrum imaging also visualized PC-3 xenografts in vivo and ex vivo with a high-contrast ratio. Autoradiography and fluorescent-based microscopic imaging with 68Ga-HZ220 consistently colocated the expression of GRPr. 68Ga-HZ220 displayed a higher kidney uptake than both 68Ga-DOTA-AR and 68Ga-DOTA-RM2 (16.9 ± 6.5 vs. 4.48 ± 1.63 vs. 5.01 ± 2.29 %IA/mL). Conclusion: 68Ga-HZ220 is a promising bimodal ligand for noninvasive PET imaging and intraoperative optical imaging of GRPr-expressing malignancies. Bimodal nuclear/fluorescence imaging may not only improve cancer detection and guide surgical resections, but also improve our understanding of the uptake of GRPr ligands on the cellular level.

Preloading with Unlabeled CA19.9 Targeted Human Monoclonal Antibody Leads to Improved PET Imaging with 89Zr-5B1

CA19.9 is one of the most commonly occurring and highest density antigens in >90% of pancreatic cancers, making it an excellent target for monoclonal antibody (mAb)-based imaging and therapy applications. Preloading of unlabeled antibodies to enhance targeting of a radiolabeled mAb has been previously described both for imaging and radioimmunotherapy studies for other targets. We investigated the effect of preloading with the unmodified anti-CA19.9 antibody 5B1 on the uptake and contrast of the PET tracer 89Zr-5B1 in subcutaneous and orthotopic murine models of pancreatic cancer utilizing Capan-2 xenografts, known to both express CA19.9 and shed antigen into circulation. Biodistribution and PET imaging studies with 89Zr-5B1 alone showed high levels in the liver, spleen, and lymph nodes of mice with subcutaneous Capan-2 tumor xenografts when administered without preinjection of 5B1. When unlabeled 5B1 was administered prior to 89Zr-5B1, the tracer significantly enhanced image contrast and tumor to tissue ratios in the same model, and the improvement was related to the time interval between the injections. Moreover, tumors were clearly delineated in an orthotopic pancreatic cancer model using our optimized approach. Taken together, these data suggest that preloading with 5B1 can improve 89Zr-5B1 imaging of disease in a Capan-2 mouse model and that exploration of preloading may have clinical utility for ongoing clinical investigations.

Leveraging PET to image folate receptor α therapy of an antibody-drug conjugate

BackgroundThe folate receptor α (FRα)-targeting antibody-drug conjugate (ADC), IMGN853, shows great antitumor activity against FRα-expressing tumors in vivo, but patient selection and consequently therapy outcome are based on immunohistochemistry. The aim of this study is to develop an antibody-derived immuno-PET imaging agent strategy for targeting FRα in ovarian cancer as a predictor of treatment success.MethodsWe developed [89Zr]Zr-DFO-M9346A, a humanized antibody-based radiotracer targeting tumor-associated FRα in the preclinical setting. [89Zr]Zr-DFO-M9346A’s binding ability was tested in an in vitro uptake assay using cell lines with varying FRα expression levels. The diagnostic potential of [89Zr]Zr-M9346A was evaluated in KB and OV90 subcutaneous xenografts. Following intravenous injection of [89Zr]Zr-DFO-M9346A (~90 μCi, 50 μg), PET imaging and biodistribution studies were performed. We determined the blood half-life of [89Zr]Zr-DFO-M9346A and compared it to the therapeutic, radioiodinated ADC [131I]-IMGN853. Finally, in vivo studies using IMG853 as a therapeutic, paired with [89Zr]Zr-DFO-M9346A as a companion diagnostic were performed using OV90 xenografts.ResultsDFO-M9346A was labeled with Zr-89 at 37 °C within 60 min and isolated in labeling yields of 85.7 ± 5.7%, radiochemical purities of 98.0 ± 0.7%, and specific activities of 3.08 ± 0.43 mCi/mg. We observed high specificity for binding FRα positive cells in vitro. For PET and biodistribution studies, [89Zr]Zr-M9346A displayed remarkable in vivo performance in terms of excellent tumor uptake for KB and OV xenografts (45.8 ± 29.0 %IA/g and 26.1 ± 7.2 %IA/g), with low non-target tissue uptake in other organs such as kidneys (4.5 ± 1.2 %IA/g and 4.3 ± 0.7 %IA/g). A direct comparison of the blood half life of [89Zr]Zr-M9346A and [131I]-IMGN853 corroborated the equivalency of the radiopharmaceutical and the ADC, paving the way for a companion PET imaging study.ConclusionsWe developed a new folate receptor-targeted 89Zr-labeled PET imaging agent with excellent pharmacokinetics in vivo. Good tumor uptake in subcutaneous KB and OV90 xenografts were obtained, and ADC therapy studies were performed with the precision predictor.

Abstract 5204: Preclinical development of MVT-1075 as radioimmunotherapy for pancreatic cancer and other CA19-9 positive malignancies

Rationale: The CA19-9 antigen is frequently overexpressed in pancreatic and other GI tumors. MVT-5873 (HuMab-5B1), a fully human monoclonal antibody currently in phase I study, targets the sialyl Lewis A (sLea) epitope on CA19-9, and is a promising platform for development of a targeted radioimmunotherapy (RIT). MVT-5873 was conjugated with the chelator CHX-A″-DTPA and radiolabeled with the beta-emitting isotopes Lutetium -177 (177Lu) or Yttrium-90 (90Y) to form the RIT agents MVT-1075 (177Lu- CHX-A″-DTPA-HuMAb-5B1) and MVT-1916 (90Y- CHX-A″-DTPA-HuMAb-5B1), respectively. The antitumor efficacy of each of the constructs was studied in nude mice bearing BxPC3 human pancreatic tumor xenografts, known to express CA19-9. Methods: The initial dose-finding studies utilized doses of MVT-1075 of 75-450 μCi and MVT-1916 of 25-250 μCi, administered to groups of mice (n = 8) bearing subcutaneous (subQ) BxPC3 tumors (~ 150 mm3). Further studies focused on MVT-1075 and assessed antitumor effect in an orthotopic xenograft model, the effect of dose fractionation, and biodistribution in nontumor bearing (normal) and BxPC3 tumor-bearing mice. Results: A single dose of MVT-1075 at 75, 150, 300, or 450 μCi significantly inhibited subQ BxPC3 tumor growth at all dose levels, with sustained suppression with higher doses. MVT-1916 produced similar results. MVT-1075 was selected based on the favorable half-life of 177Lu (6.7 d) and its utility for clinical biodistribution assessments. In an orthotopic BxPC3 tumor model, treatment with a single dose of MVT-1075 at 300 μCi significantly inhibited tumor growth, with Day 20 tumor volume approximately 50% that of the initial starting volume. A third BxPC3 xenograft study evaluated fractionated dosing schedules, (150 μCi x 1, 75 μCi x 2, 50 μCi x3), with both single-dose and fractionated schedules effectively inhibiting subQ BxPC3 tumor growth. Biodistribution studies in normal mice showed an expected gradually decreasing activity in blood, heart, and lungs, with low uptake in normal pancreas. In subQ BxPC3 tumor-bearing mice, tumor uptake was rapid, reaching 69% ID/g by 24 h and 86% ID/g by 120 h. Otherwise, the biodistribution pattern paralleled that of normal mice, with relative %ID/g values within about ± 25% of normal mice across all time points comparing blood, heart, lungs, kidneys, and pancreas, with slightly higher uptake in liver and slightly lower uptake in spleen. Conclusions: MVT-1075 demonstrates promising antitumor activity in a human pancreatic cancer xenograft model, with efficacy shown in both single dose and fractionated schedules. Biodistribution shows rapid and substantial tumor uptake, with much lower uptake in normal organs. These findings support the phase I clinical trial of MVT-1075 in patients with CA19-9 positive pancreatic cancers planned to begin in early 2017. Citation Format: Jacob L. Houghton, Ryan Lanning, Dayla Abdel-atti, Toni Jun, Christine M. Kearns, Michael Schlosser, Wolfgang Scholz, Jason S. Lewis, Paul W. Maffuid. Preclinical development of MVT-1075 as radioimmunotherapy for pancreatic cancer and other CA19-9 positive malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5204. doi:10.1158/1538-7445.AM2017-5204