Investigation of Copper-64-Based Host-Guest Chemistry Pretargeted Positron Emission Tomography.

Pretargeting is a technique that uses macromolecules as targeting agents for nuclear imaging and therapy with the goal of reducing the radiation toxicity to healthy tissues often associated with directly radiolabeled macromolecules. In pretargeting, a macromolecule is radiolabeled in vivo at the target site using a radiolabeled small molecule (radioligand) that interacts with the macromolecule with high specificity. We report an investigation of host-guest chemistry-driven pretargeting using copper-64 radiolabeled ferrocene (Fc; guest) compounds and a cucurbit[7]uril (CB7; host) molecule functionalized carcinoembryonic antigen targeting hT84.66-M5A monoclonal antibody (CB7-M5A). Two novel ferrocene-based radioligands ([64Cu]Cu-NOTA-PEG3-Fc and [64Cu]Cu-NOTA-PEG7-Fc) were prepared, and their in vitro stability, pharmacokinetic in vivo profile in healthy mice, and pretargeting performance in a subcutaneous BxPC3 human pancreatic cancer cell xenograft mouse model were compared. The antibody dosing was optimized using a zirconium-89 radiolabeled M5A antibody ([89Zr]Zr-DFO-M5A) in a BxPC3 xenograft model, and the dosimetry of [89Zr]Zr-DFO-M5A and the pretargeting approach were compared. Finally, the effects of varying lag times up to 9 days between CB7-M5A and radioligand injection were investigated. In vivo pretargeting studies with both ferrocene radioligands resulted in specific tumor uptake (p = 0.0006 and p = 0.003) and also showed that the host-guest-based pretargeting approach excels with extended lag times up to 9 days with good tumor localization, suggesting that host-guest pretargeting may be suitable for use without clearing agents which have complicated clinical application of this technique. To our knowledge, the reported lag time of 9 days is the longest investigated lag time in any reported pretargeting studies.

Cucurbituril-Ferrocene: Host-Guest Based Pretargeted Positron Emission Tomography in a Xenograft Model.

Pretargeted positron emission tomography is a macromolecule-driven nuclear medicine technique that involves targeting a preadministered antigen target-bound macromolecule with a radioligand in vivo, aiming to minimize the overall radiation dose. This study investigates the use of antibody based host-guest chemistry methodology for pretargeted positron emission tomography. We hypothesize that the novel pretargeting approach reported here overcomes the challenges the current pretargeting platforms have with the in vivo stability and modularity of the pretargeting components. A cucurbit[7]uril host molecule modified, anti-carcinoembryonic antigen antibody (M5A; CB7-M5A) and a 68Ga-radiolabeled ferrocene guest radioligand ([68Ga]Ga-NOTA-PEG3-NMe2-Fc) were studied as potential host-guest chemistry pretargeting agents for positron emission tomography in BxPC3 xenografted nude mice. The viability of the platform was studied via in vivo biodistribution and positron emission tomography. Tumor uptake of [68Ga]Ga-NOTA-PEG3-NMe2-Fc was significantly higher in mice which received CB7-M5A prior to the radioligand injection (pretargeted) (3.3 ± 0.7%ID/g) compared to mice which only received the radioligand (nonpretargeted) (0.2 ± 0.1%ID/g).

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

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 ((ss)DFO-5B1, (ss)FL-5B1, and (ss)dual-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 (ss)dual-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.

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.

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

A first-of-its-kind (18)F 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[(18)F]NOTA-labeled tetrazine radioligand were harnessed for the visualization of CA19.9-expressing BxPC3 pancreatic cancer xenografts. Biodistribution and (18)F-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.

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.

MIB Guides: Measuring the Immunoreactivity of Radioimmunoconjugates.

Immunoglobulins, both full-length antibodies and smaller antibody fragments, have long been regarded as effective platforms for diagnostic and therapeutic radiopharmaceuticals. The construction of radiolabeled immunoglobulins (i.e., radioimmunoconjugates) requires the manipulation of the biomolecule through the attachment of a radiohalogen or the bioconjugation of a chelator that is subsequently used to coordinate a radiometal. Both synthetic approaches have historically relied upon the stochastic modification of amino acids within the immunoglobulin, a process which poses a risk to the structural and functional integrity of the biomolecule itself. Not surprisingly, radioimmunoconjugates with impaired antigen binding capacity will inevitably exhibit suboptimal in vivo performance. As a result, the biological characterization of any newly synthesized radioimmunoconjugate must include an assessment of whether it has retained its ability to bind its antigen. Herein, we provide straightforward and concise protocols for three assays that can be used to determine the immunoreactivity of a radioimmunoconjugate: (1) a cell-based linear extrapolation assay; (2) a cell-based antigen saturation assay; and (3) a resin- or bead-based assay. In addition, we will provide a critical analysis of the relative merits of each assay, an examination of the inherent limitations of immunoreactivity assays in general, and a discussion of other approaches that may be used to interrogate the biological behavior of radioimmunoconjugates.

AMP-activated protein kinase and ATP-citrate lyase are two distinct molecular targets for ETC-1002, a novel small molecule regulator of lipid and carbohydrate metabolism.

ETC-1002 (8-hydroxy-2,2,14,14-tetramethylpentadecanedioic acid) is a novel investigational drug being developed for the treatment of dyslipidemia and other cardio-metabolic risk factors. The hypolipidemic, anti-atherosclerotic, anti-obesity, and glucose-lowering properties of ETC-1002, characterized in preclinical disease models, are believed to be due to dual inhibition of sterol and fatty acid synthesis and enhanced mitochondrial long-chain fatty acid ß-oxidation. However, the molecular mechanism(s) mediating these activities remained undefined. Studies described here show that ETC-1002 free acid activates AMP-activated protein kinase in a Ca(2+)/calmodulin-dependent kinase ß-independent and liver kinase ß 1-dependent manner, without detectable changes in adenylate energy charge. Furthermore, ETC-1002 is shown to rapidly form a CoA thioester in liver, which directly inhibits ATP-citrate lyase. These distinct molecular mechanisms are complementary in their beneficial effects on lipid and carbohydrate metabolism in vitro and in vivo. Consistent with these mechanisms, ETC-1002 treatment reduced circulating proatherogenic lipoproteins, hepatic lipids, and body weight in a hamster model of hyperlipidemia, and it reduced body weight and improved glycemic control in a mouse model of diet-induced obesity. ETC-1002 offers promise as a novel therapeutic approach to improve multiple risk factors associated with metabolic syndrome and benefit patients with cardiovascular disease.

Chemical and structural insights into the regioversatility of the aminoglycoside acetyltransferase Eis.

A recently discovered cause of tuberculosis resistance to a drug of last resort, the aminoglycoside kanamycin, results from modification of this drug by the enhanced intracellular survival (Eis) protein. Eis is a structurally and functionally unique acetyltransferase with an unusual capability of acetylating aminoglycosides at multiple positions. The extent of this regioversatility and its defining protein features are unclear. Herein, we determined the positions and order of acetylation of five aminoglycosides by NMR spectroscopy. This analysis revealed unprecedented acetylation of the 3''-amine of kanamycin, amikacin, and tobramycin, and the γ-amine of the 4-amino-2-hydroxybutyryl group of amikacin. A crystal structure of Eis in complex with coenzyme A and tobramycin revealed how tobramycin can be accommodated in the Eis active site in two binding modes, consistent with its diacetylation. These studies, describing chemical and structural details of acetylation, will guide future efforts towards designing aminoglycosides and Eis inhibitors to overcome resistance in tuberculosis.

Unexpected N-acetylation of capreomycin by mycobacterial Eis enzymes.

OBJECTIVES: The enhanced intracellular survival (Eis) protein from Mycobacterium tuberculosis (Eis_Mtb), a regio-versatile N-acetyltransferase active towards many aminoglycosides (AGs), confers resistance to kanamycin A in some cases of extensively drug-resistant tuberculosis (XDR-TB). We assessed the activity of Eis_Mtb and of its homologue from Mycobacterium smegmatis (Eis_Msm) against a panel of anti-tuberculosis (TB) drugs and lysine-containing compounds. METHODS AND RESULTS: Both enzymes acetylated capreomycin and some lysine-containing compounds, but not other non-AG non-lysine-containing drugs tested. Modelling studies predicted the site of modification on capreomycin to be one of the two primary amines in its ß-lysine side chain. Using Eis_Mtb, we established via nuclear magnetic resonance (NMR) spectroscopy that acetylation of capreomycin occurs on the ε-amine of the ß-lysine side chain. Using Msm, we also demonstrated for the first time to our knowledge that acetylation of capreomycin results in deactivation of the drug. CONCLUSIONS: Eis is a unique acetyltransferase capable of inactivating the anti-TB drug capreomycin, AGs and other lysine-containing compounds.

Pretargeting with Cucurbituril-Adamantane Host-Guest Pair in Xenograft Models.

The goal of reducing the total-body radiation dose of macromolecule-based nuclear medicine with a 2-step pretargeting strategy has been achieved with several pretargeting methodologies in preclinical and clinical settings. However, the lack of modularity, biocompatibility, and in vivo stability in existing pretargeting agents obstructs their respective platforms' wide clinical use. We hypothesized that host-guest chemistry would provide an optimal pretargeting methodology. A cucurbit[7]uril host and an adamantane guest molecule form a high-affinity host-guest complex (association constant, ∼1014 M-1), and in this work, we explored the use of this noncovalent interaction as the basis for antibody-based pretargeted PET. Along with the straightforward modularity of these agents, cucurbit[7]uril and adamantane are recognized to have high in vivo stability and suitability for human use, which is why we proposed this methodology as the ideal approach for pretargeted nuclear medicine. Methods: Three 64Cu-labeled adamantane guest radioligands were developed, and their in vitro stability, lipophilicity, and in vivo blood half-lives were compared. The adamantane radioligands were analyzed for pretargeting using a cucurbit[7]uril-modified carcinoembryonic antigen-targeting full-length antibody, hT84.66-M5A, as the macromolecule pretargeting agent with 2 different dosing schedules. These molecules were evaluated for pretargeting in human pancreatic cancer BxPC3 and MIAPaCa-2 mouse xenografts using PET and in vivo biodistribution studies. The dosimetry of the cucurbit[7]uril-adamantane (CB7-Adma) pretargeting approach in men was calculated and compared with that of the directly 89Zr-labeled hT84.66-M5A. Results: The adamantane radioligands possessed high in vitro stability up to 24 h (>90%). Pretargeted PET with CB7-Adma methodology resulted in specific tumor uptake (P < 0.05) with low background signal. The in vivo formed CB7-Adma complex was demonstrated to be stable, with high tumor uptake up to 24 h after radioligand injection (12.0 ± 0.9 percentage injected dose/g). The total-body radiation dose of the pretargeting strategy was only 3.3% that of the directly 89Zr-labeled hT84.66-M5A. Conclusion: The CB7-Adma strategy is highly suitable for pretargeted PET. The exceptional stability of the pretargeting agents and the specific and high tumor uptake of the pretargeted adamantane radioligands provide great potential for the platform.

Current Landscape in Clinical Pretargeted Radioimmunoimaging and Therapy.

The principle of pretargeted radioimmunoimaging and therapy has been investigated over the past 30 y in preclinical and clinical settings with the aim of reducing the radiation burden of healthy tissue for antibody-based nuclear medicine techniques. In the past few decades, 4 pretargeting methodologies have been proposed, and 2 of them-the bispecific antibody-hapten and the streptavidin-biotin platforms-have been evaluated in humans in phase 1 and 2 studies. With this review article, we aim to survey clinical pretargeting studies in order to understand the challenges that these platforms have faced in human studies and to provide an overview of how the clinical approval of the pretargeting system has proceeded in the past several decades. Additionally, we will discuss the successes of the pretargeting human studies and compare and highlight the pretargeting approaches and conditions that will advance clinical translation of the pretargeting platform in the future.

Dissecting the cosubstrate structure requirements of the Staphylococcus aureus aminoglycoside resistance enzyme ANT(4').

Aminoglycosides are important antibiotics used against a wide range of pathogens. As a mechanism of defense, bacteria have evolved enzymes able to inactivate these drugs by regio-selectively adding a variety of functionalities (acetyl, phospho, and nucelotidyl groups) to their scaffolds. The aminoglycoside nucleotidyltransferase ANT(4') is one of the most prevalent and unique modifying-enzymes. Here, by TLC, HRMS, and colorimetric assays, we demonstrate that the resistance enzyme ANT(4') from Staphylococcus aureus is highly substrate and cosubstrate promiscuous. We show that deoxy-ribonucleotide triphosphates (dNTPs) are better cosubstrates than NTPs. We demonstrate that the position of the triphosphate group (5' and not 3') on the ribose/deoxyribose ring is important for recognition by ANT(4'), and that NTPs with larger substituents at the 3'-position of the ribose ring are not cosubstrates for ANT(4'). We confirm that for all aminoglycosides tested, the respective nucleotidylated products are completely inactive. These results provide valuable insights into the development of strategies to combat the ever-growing bacterial resistance problem.

The future of aminoglycosides: the end or renaissance?

Although aminoglycosides have been used as antibacterials for decades, their use has been hindered by their inherent toxicity and the resistance that has emerged to these compounds. It seems that such issues have relegated a formerly front-line class of antimicrobials to the proverbial back shelf. However, recent advances have demonstrated that novel aminoglycosides have a potential to overcome resistance as well as to be used to treat HIV-1 and even human genetic disorders, with abrogated toxicity. It is not the end for aminoglycosides, but rather, the challenges faced by researchers have led to ingenuity and a change in how we view this class of compounds, a renaissance.

Exploring the substrate promiscuity of drug-modifying enzymes for the chemoenzymatic generation of N-acylated aminoglycosides.

Aminoglycosides are broad-spectrum antibiotics commonly used for the treatment of serious bacterial infections. Decades of clinical use have led to the widespread emergence of bacterial resistance to this family of drugs limiting their efficacy in the clinic. Here, we report the development of a methodology that utilizes aminoglycoside acetyltransferases (AACs) and unnatural acyl coenzyme A analogues for the chemoenzymatic generation of N-acylated aminoglycoside analogues. Generation of N-acylated aminoglycosides is followed by a simple qualitative test to assess their potency as potential antibacterials. The studied AACs (AAC(6')-APH(2'') and AAC(3)-IV) show diverse substrate promiscuity towards a variety of aminoglycosides as well as acyl coenzyme A derivatives. The enzymes were also used for the sequential generation of homo- and hetero-di-N-acylated aminoglycosides. Following the clinical success of the N-acylated amikacin and arbekacin, our chemoenzymatic approach offers access to regioselectively N-acylated aminoglycosides in quantities that allow testing of the antibacterial potential of the synthetic analogues making it possible to decide which molecules will be worth synthesizing on a larger scale.

ImmunoPET Predicts Response to Met-targeted Radioligand Therapy in Models of Pancreatic Cancer Resistant to Met Kinase Inhibitors.

Background: Pancreatic ductal adenocarcinoma (PDAC) has limited standard of care therapeutic options. While initially received with enthusiasm, results from targeted therapy with small molecule tyrosine kinases inhibitors (TKIs) have been mixed, in part due to poor patient selection and compensatory changes in signaling networks upon blockade of one or more kinase of tumors. Here, we demonstrate that in PDACs otherwise resistant to rational kinase inhibition, Met-directed immuno-positron emission tomography (immunoPET) can identify targets for cell-signaling independent targeted radioligand therapy (RLT). In this study, we use Met-directed immunoPET and RLT in models of human pancreatic cancer that are resistant to Met- and MEK-selective TKIs, despite over-expression of Met and KRAS-pathway activation. Methods: We assessed cell membrane Met levels in human patient samples and pancreatic ductal adenocarcinoma (PDAC) cell lines (BxPC3, Capan2, Suit2, and MIA PaCa-2) using immunofluorescence, flow cytometry and cell-surface biotinylation assays. To determine whether Met expression levels correlate with sensitivity to Met inhibition by tyrosine kinase inhibitors (TKIs), we performed cell viability studies. A Met-directed imaging agent was engineered by labeling Met-specific onartuzumab with zirconium-89 (Zr-89) and its in vivo performance was evaluated in subcutaneous and orthotopic PDAC xenograft models. To assess whether the immunoPET agent would predict for targeted RLT response, onartuzumab was then labeled with lutetium (Lu-177) as the therapeutic radionuclide to generate our [177Lu]Lu-DTPA-onartuzumab RLT agent. [177Lu]Lu-DTPA-onartuzumab was administered at 9.25MBq (250µCi)/20µg in three fractions separated by three days in mice subcutaneously engrafted with BxPC3 (high cell-membrane Met) or MIA PaCa-2 (low cell-membrane Met). Primary endpoints were tumor response and overall survival. Results: Flow cytometry and cell-surface biotinylation studies showed that cell-membrane Met was significantly more abundant in BxPC3, Capan2, and Suit2 when compared with MIA PaCa-2 pancreatic tumor cells. Crizotinib and cabozantinib, TKIs with known activity against Met and other kinases, decreased PDAC cell line viability in vitro. The TKI with the lowest IC50 for Met, capmatinib, had no activity in PDAC lines. No additive effect was detected on cell viability when Met-inhibition was combined with MEK1/2 inhibition. We observed selective tumor uptake of [89Zr]Zr-DFO-onartuzumab in mice subcutaneously and orthotopically engrafted with PDAC lines containing high cell-surface levels of Met (BxPC3, Capan2, Suit2), but not in mice engrafted with low cell-surface levels of Met (MIA PaCa-2). Significant tumor growth delay and overall survival benefit were observed in both BxPC3 and MIA PaCa-2 engrafted animals treated with RLT when compared to controls, however, the benefit was more pronounced and more durable in the BxPC3 engrafted animals treated with [177Lu]Lu-DTPA-onartuzumab RLT. Conclusions: Our findings demonstrate that while over-expression of Met is not predictive of Met-directed TKI response, immunoPET can detect Met over-expression in vivo and predicts for therapeutic response to Met-selective RLT. This phenomenon can be exploited for other Met-overexpressing tumor types specifically, and to any differentially overexpressed surface molecule more broadly.

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

BACKGROUND: The 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. METHODS: We 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. RESULTS: DFO-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. CONCLUSIONS: We 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.

Tumor-Specific Zr-89 Immuno-PET Imaging in a Human Bladder Cancer Model.

PURPOSE: Tumor-specific molecular imaging is an important tool for assessing disease burden and treatment response. CA19.9 is an important tumor-specific marker in several malignancies, including urothelial carcinoma. [89Zr]DFO-HuMab-5B1 (MVT-2163) is a CA19.9-specific antibody-based construct that has been validated in preclinical animal models of lung, colorectal, and pancreatic malignancies for positron emission tomography (PET) imaging and is currently in a phase I trial for pancreatic cancer (NCT02687230). Here, we examine whether [89Zr]DFO-HuMab-5B1 may be useful in defining urothelial malignancies. PROCEDURES: Surface expression of CA19.9 was confirmed in the human bladder cancer line HT 1197. The radioimmunoconjugate [89Zr]DFO-HuMab-5B1 was injected into mice bearing HT 1197 xenografts, and followed by PET imaging, ex vivo experiments including biodistribution, histology and autoradiography, and analysis of blood samples for shed antigen levels were performed. RESULTS: [89Zr]DFO-HuMab-5B1 specifically accumulates in HT 1197 engrafted tumors when imaged with PET. Ex vivo biodistribution of organs and autoradiography of engrafted tumors confirm our construct's specific tumor binding. The target antigen CA19.9 was not found to be shed in vitro or in vivo. CONCLUSIONS: [89Zr]DFO-HuMab-5B1 can be used to delineate urothelial carcinomas by PET imaging and may provide tumor-specific information prior to, during, and after systemic therapies.

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.