immuno-PET Agents Research Articles

UPAR Immuno-PET in Pancreatic Cancer, Aging, and Chemotherapy-Induced Senescence.

Identifying cancer therapy resistance is a key time-saving tool for physicians. Part of chemotherapy resistance includes senescence, a persistent state without cell division or cell death. Chemically inducing senescence with the combination of trametinib and palbociclib (TP) yields several tumorigenic and prometastatic factors in pancreatic cancer models with many potential antibody-based targets. In particular, urokinase plasminogen activator receptor (uPAR) has been shown to be a membrane-bound marker of senescence in addition to an oncology target. Methods: Here, 2 antibodies against murine uPAR and human uPAR were developed as immuno-PET agents to noninvasively track uPAR antigen abundance. Results: TP treatment increased cell uptake both in murine KPC cells and in human MiaPaCa2 cells. In vivo, subcutaneously implanted murine KPC tumors had high tumor uptake with the antimurine uPAR antibody independently of TP in young mice, yet uPAR uptake was maintained in aged mice on TP. Mice xenografted with human MiaPaCa2 tumors showed a significant increase in tumor uptake on TP therapy when imaged with the antihuman uPAR antibody. Imaging with either uPAR antibody was found to be more tumor-selective than imaging with [18F]FDG or [18F]F-DPA-714. Conclusion: The use of radiolabeled uPAR-targeting antibodies provides a new antibody-based PET imaging candidate for pancreatic cancer imaging as well as chemotherapy-induced senescence.

CEACAM5-Targeted Immuno-PET in Androgen Receptor-Negative Prostate Cancer.

The incidence of androgen receptor (AR)-negative (AR-) prostate cancer, including aggressive neuroendocrine prostate cancer (NEPC), has more than doubled in the last decade, but its timely diagnosis is difficult as it lacks typical prostate cancer hallmarks. The carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) has recently been identified as an upregulated surface antigen in NEPC. We developed an immuno-PET agent targeting CEACAM5 and evaluated its ability to delineate AR- prostate cancer invivo. Methods: CEACAM5 expression was evaluated in a panel of prostate cancer cell lines by immunohistochemistry and Western blotting. The CEACAM5-targeting antibody labetuzumab was conjugated with the chelator desferrioxamine (DFO) and radiolabeled with 89Zr. The invivo distribution of the radiolabeled antibody was evaluated in xenograft prostate cancer models by PET imaging and ex vivo organ distribution. Results: The NEPC cell line H660 exhibited strong CEACAM5 expression, whereas expression was limited in the AR- cell lines PC3 and DU145 and absent in the AR-positive cell line LNCaP. [89Zr]Zr-DFO-labetuzumab imaging was able to clearly delineate both neuroendocrine H660 xenografts and AR- DU145 invivo but could not detect the AR-positive xenograft LNCaP. Conclusion: Immuno-PET imaging with [89Zr]Zr-DFO-labetuzumab is a promising diagnostic tool for AR- prostate cancer.

Nanobody-Based ImmunoPET for Hepatocellular Carcinoma

HCC accounts for 75-90% of all primary liver cancers, the majority of which are treated with liver-directed therapy. Treatment response and recurrence are difficult to discern using conventional imaging with MR/CT. Tumor-selective PET imaging could help with clinical management in this setting. Here, we engineer HN3, a single-domain antibody (nanobody) specific to GPC3, a histopathologically-defining HCC marker, as an immunoPET agent. We compared both conventional and sortase-based site-specific modification methods for synthesizing HN3 immunoPET tracers. Stochastic lysine conjugation with deferoxamine (DFO-NCS) was done to synthesize nHN3-DFO. ssHN3-DFO was engineered utilizing sortase-mediated conjugation of HN3 containing an LPETG C-terminal tag and a triglycine-DFO chelator. Biolayer interferometry (BLI) and radioligand saturation assays were done to determine binding affinity pre- and post-Zirconium-89 labeling. Following, PET/CT with a terminal 3-hour biodistribution was done in mice inoculated with isogenic A431 and A431-GPC3+ xenografts to determine conjugate specificity for GPC3. Finally, conjugates were evaluated in a HepG2 liver cancer model via ex vivo biodistribution studies and a comparative PET/CT study in mice bearing HepG2 tumors that were imaged with both [18F]FDG and 89Zr-ssHN3. Both conjugates exhibited nanomolar binding affinity for GPC3 in vitro (11-30 nM for nHN3 and 10-15 nM for ssHN3). A431 and A431-GPC3+ PET/CT and biodistribution studies showed specificity to GPC3 by both probes, with more favorable tumor uptake by 89Zr-ssHN3 at 3 hours post-injection (14% IA/g vs. 7% IA/g for nHN3). Both tracers also displayed uptake in HepG2 (GPC3+) liver tumors, again with the site specifically conjugated probe having higher tumor accumulation and lower liver signal than the conventionally modified HN3 (7% IA/g vs. 5 % IA/g for tumor and 2% IA/g vs. 4% IA/g for liver at 1-hour post-injection). PET/CT studies in mice imaged with [18F]FDG and 89Zr-ssHN3 demonstrated more consistent tumor accumulation for the nanobody conjugate (4/4 mice had uptake by the tumor vs. 1/4 for FDG). We successfully designed, synthesized, and characterized novel GPC3-selective nanobody PET probes that can image liver tumors in vivo. The site-specifically conjugated tracer showed more favorable biodistribution and pharmacokinetic properties, resulting in a much higher tumor: liver signal compared to 89Zr-nHN3. We also show the superiority of the 89Zr-ssHN3 imaging over conventional [18F]FDG, highlighting a clear advantage in using targeted tumor imaging for this cancer type. Successful translation of the site-specifically conjugated nanobody may ultimately aid in characterizing lesions following liver-directed therapy and allow for more comprehensive screening, early diagnosis, and post-treatment surveillance of HCC.

Detection of IL12/23p40 via PET Visualizes Inflammatory Bowel Disease.

Inflammatory bowel disease (IBD), which includes both Crohn disease and ulcerative colitis, is a relapsing inflammatory disease of the gastrointestinal tract. Long-term chronic inflammatory conditions elevate the patient's risk of colorectal cancer (CRC). Currently, diagnosis requires endoscopy with biopsy. This procedure is invasive and requires a bowel-preparatory regimen, adding to patient burden. Interleukin 12 (IL12) and interleukin 23 (IL23) play key roles in inflammation, especially in the pathogenesis of IBD, and are established therapeutic targets. We propose that imaging of IL12/23 and its p40 subunit in IBD via immuno-PET potentially provides a new noninvasive diagnostic approach. Methods: Our aim was to investigate the potential of immuno-PET to image inflammation in a chemically induced mouse model of colitis using dextran sodium sulfate by targeting IL12/23p40 with a 89Zr-radiolabeled anti-IL12/23p40 antibody. Results: High uptake of the IL12/23p40 immuno-PET agent was exhibited by dextran sodium sulfate-administered mice, and this uptake correlated with increased IL12/23p40 present in the sera. Competitive binding studies confirmed the specificity of the radiotracer for IL12/23p40 in the gastrointestinal tract. Conclusion: These promising results demonstrate the utility of this radiotracer as an imaging biomarker of IBD. Moreover, IL12/23p40 immuno-PET can potentially guide treatment decisions for IBD management.

Abstract 5035: Preclinical development of a 89Zr-labeled human antibody as a novel immuno-PET agent for noninvasive cancer detection and monitoring response to treatment

Abstract Lung cancer is the leading cause of cancer death worldwide. There is an unmet need for therapies to improve lung cancer outcomes without causing additional toxicities. Tax-interacting protein 1 (TIP1) is a radiation-inducible antigen that plays a role in lung cancer progression and resistance to therapy. TIP1 is a targetable cell surface protein. We developed human antibodies (Ab) that bind specifically to lung cancer. This study aimed to evaluate our lead anti-TIP1 human antibody targeting lung cancer in vitro and in vivo. Anti-TIP1 IgG1 Ab was developed by biopanning phage display human antibody library. The purified Ab was characterized by size-exclusion chromatography-HPLC (SEC-HPLC), mass spectrometry, ELISA, BIAcore and flow cytometry. Ab was labeled with 89Zr using desferrioxamine chelator. The surface binding of 89Zr-Ab was evaluated in the A549 and H460 lung cancer cells. Stability of the 89Zr-Ab was assessed in human serum. Mice bearing 3 patient-derived xenografts (PDX) lung tumors or A549 tumors were injected with 50 µCi of 89Zr-Ab. Small animal PET imaging and biodistribution were conducted over 7 days. Specific tumor uptake was evaluated in biodistribution studies on day 5 by injecting cold Ab before 89Zr-Ab. We obtained 95% pure anti-TIP1 Ab by SEC-HPLC. Mass spectrometry confirmed the intact mass and glycosylation pattern of the anti-TIP1 IgG1. The Ab is bound to recombinant TIP1 protein and cancer cell surface with high affinity. The radiochemical purity of the 89Zr-Ab was 99.9%, and the molar activity was 11.54 MBq/nmol with a radiolabeling yield of 91.4%. Both A549 and H460 cell lines demonstrated specific binding as >70% inhibition was observed when the cold Ab was added. The ratio of SUV tumor to SUV muscle increased from 5.8 at 2 days to 6.3 at 7 days in PDX. In biodistribution studies, we found significantly higher (p<0.05) tumor uptake of the anti-TIP1 Ab (9.15±3.13 %ID/gm) vs. isotype control (5.72±1.02 %ID/gm). Cold anti-TIP1 Ab significantly blocked (p<0.0001) binding of the 89Zr-Ab in A549 tumors. The anti-TIP1 Ab targets lung cancer cells in vitro and in vivo. The Ab retained antigen-binding potential following labeling with 89Zr. This Ab can detect TIP1 positive tumors for therapy when conjugated with therapeutic radionuclides. IND enabling studies with the lead anti-TIP1 Ab are underway. Citation Format: Abhay Kumar Singh, Calvin D. Lewis, Cristian AWV Boas, Philipp Diebolder, Prashant N. Jethva, Aaron Rhee, Jong Hee Song, Young Ah Goo, Shunqiang Li, Yongjian Liu, Buck Rogers, Vaishali Kapoor, Dennis E. Hallahan. Preclinical development of a 89Zr-labeled human antibody as a novel immuno-PET agent for noninvasive cancer detection and monitoring response to treatment. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5035.

Treatment of Prostate Cancer with CD46-targeted 225Ac Alpha Particle Radioimmunotherapy.

Radiopharmaceutical therapy is changing the standard of care in prostate cancer and other malignancies. We previously reported high CD46 expression in prostate cancer and developed an antibody-drug conjugate and immunoPET agent based on the YS5 antibody, which targets a tumor-selective CD46 epitope. Here, we present the preparation, preclinical efficacy, and toxicity evaluation of [225Ac]DOTA-YS5, a radioimmunotherapy agent based on the YS5 antibody. [225Ac]DOTA-YS5 was developed, and its therapeutic efficiency was tested on cell-derived (22Rv1, DU145), and patient-derived (LTL-545, LTL484) prostate cancer xenograft models. Biodistribution studies were carried out on 22Rv1 tumor xenograft models to confirm the targeting efficacy. Toxicity analysis of the [225Ac]DOTA-YS5 was carried out on nu/nu mice to study short-term (acute) and long-term (chronic) toxicity. Biodistribution study shows that [225Ac]DOTA-YS5 agent delivers high levels of radiation to the tumor tissue (11.64% ± 1.37%ID/g, 28.58% ± 10.88%ID/g, 29.35% ± 7.76%ID/g, and 31.78% ± 5.89%ID/g at 24, 96, 168, and 408 hours, respectively), compared with the healthy organs. [225Ac]DOTA-YS5 suppressed tumor size and prolonged survival in cell line-derived and patient-derived xenograft models. Toxicity analysis revealed that the 0.5 μCi activity levels showed toxicity to the kidneys, likely due to redistribution of daughter isotope 213Bi. [225Ac]DOTA-YS5 suppressed the growth of cell-derived and patient-derived xenografts, including prostate-specific membrane antigen-positive and prostate-specific membrane antigen-deficient models. Overall, this preclinical study confirms that [225Ac]DOTA-YS5 is a highly effective treatment and suggests feasibility for clinical translation of CD46-targeted radioligand therapy in prostate cancer.

Pharmacokinetics and Biodistribution of 89Zr-Miltuximab and Its Antibody Fragments as Glypican-1 Targeting Immuno-PET Agents in Glioblastoma.

Glioblastoma (GBM) is the most aggressive form of primary brain cancer, accounting for about 85% of all primary central nervous system (CNS) tumors. With standard treatment strategies like surgery, radiation, and chemotherapy, the median survival time of patients with GBM is only 12-15 months from diagnosis. The poor prognosis of GBM is due to a very high tumor recurrence rate following initial treatment, indicating a dire need for improved diagnostic and therapeutic alternatives for this disease. Antibody-based immunotheranostics holds great promise in treating GBM, combining the theranostic applications of radioisotopes and target-specificity of antibodies. In this study, we developed and validated antibody-based positron emission tomography (PET) tracers targeting the heparan sulfate proteoglycan, glypican-1 (GPC-1), for noninvasive detection of disease using diagnostic molecular imaging. GPC-1 is overexpressed in multiple solid tumor types, including GBM, and is a promising biomarker for novel immunotheranostics. Here, we investigate zirconium-89 (89Zr)-conjugated Miltuximab (a clinical stage anti-GPC-1 monoclonal antibody developed by GlyTherix, Ltd.) and engineered fragments for their potential as immuno-PET tracers to detect GPC-1positive GBM tumors in preclinical models. We explore the effects of molecular size, avidity, and Fc-domain on the pharmacokinetics and biodistribution in vivo, by comparing in parallel the full-length antibody (Miltuximab), Fab'2, Fab, and single-chain variable fragment (scFv) formats. High radiolabeling efficiency (>95%) was demonstrated by all the formats and the stability post-radiolabeling was higher for larger constructs of Miltuximab and the Fab. Receptor-mediated internalization of all 89Zr-labeled formats was observed in a human GBM cell line in vitro, while full-length Miltuximab demonstrated the highest tumor retention (5.7 ± 0.94% ID/g, day-9 postinjection (p.i.)) and overall better tumor-to-background ratios than the smaller Fc-less formats. Results from in vivo PET image quantification and ex vivo scintillation counting were highly correlated. Altogether, 89Zr-DFO-Miltuximab appears to be an effective immuno-PET imaging agent for detecting GPC-1positive tumors such as GBM and the current results support utility of the Fc containing whole mAb format over smaller antibody fragments for this target.

68Ga-Labeled Trastuzumab Fragments for ImmunoPET Imaging of Human Epidermal Growth Factor Receptor 2 Expression in Solid Cancers.

Background: Trastuzumab, the first humanized antibody approved for therapeutic use has shown promising results for the treatment of patients with human epidermal growth factor receptor 2 (HER2) positive cancers. The aim of this study was to formulate immunoPET agents based on trastuzumab fragments and demonstrate their potential for early diagnosis of HER2-positive tumors. Materials and Methods: F(ab')2 and F(ab') fragments of trastuzumab were prepared by enzymatic digestion and conjugated with chelator NOTA for labeling with 68Ga. For comparison, intact trastuzumab was also radiolabeled. In vitro stability, immunoreactivity, and binding affinity of radio formulations toward HER2 receptors were evaluated by performing in vitro studies in cancer cell lines. Biodistribution and PET imaging studies were performed in animal model bearing tumors. Results: 68Ga-NOTA-F(ab')-trastuzumab, 68Ga-NOTA-F(ab')2-trastuzumab, and 68Ga-NOTA-trastuzumab could be prepared with >98% radiochemical purity (% RCP) and were found to be stable when studied up to 4 h. In vitro binding studies revealed high affinity and specificity of formulations toward HER2 receptors. Specific tumor uptake of 68Ga-NOTA-F(ab')-trastuzumab and 68Ga-NOTA-F(ab')2-trastuzumab in HER2-positive tumors was observed in biodistribution and PET imaging studies. Conclusions: This study describes optimization of protocol for the formulation of 68Ga-NOTA-F(ab')-trastuzumab and 68Ga-NOTA-F(ab')2-trastuzumab for targeting HER2-overexpressing tumors. Further studies with these radioformulations are warranted to confirm their potential as immunoPET agents for management of HER2-positive breast and other solid tumors.

Abstract A032: Targeting pancreatic cancer senescence with ImmunoPET

Abstract Pancreatic cancer is the fourth leading cause of cancer-related deaths for men and women, with five-year overall survival rates around 10 percent. Despite years of cancer research and new immunotherapy solutions, pancreatic cancer is still mainly treated by chemotherapies, starting with gemcitabine. Many cytostatic drug combinations have been shown, in a wide range of cancers, to induce senescence, a persistent, anti-apoptotic process characterized almost exclusively by beta galactosidase activity. Studies inducing senescence with the combination of trametinib and palbociclib (TP) have linked several tumorigenic and pro-metastatic factors in murine pancreatic cancer models to the senescence-associated secretory profile (SASP), giving rise to new antigen targets during the induction of senescence. Accordingly, senescence has recently been elevated as a new hallmark of cancer. There is thus a critical need for antigen-based tools to noninvasively identify markers of senescence in vivo. Senescence targets comprise of shed antigens (VEGF and IL-6) in the context of senescence-inducing therapy with trametinib and palbociclib (TP), when membrane bounds targets such as urokinase plasminogen activated receptor (uPAR) are potentially enriched. We show that VEGF and IL-6 targeting antibodies labeled with zirconium-89 deferoxamine (89Zr-DFO) bind cells treated with TP though in vivo, marked reduction in tumor targeting is seen. Preloading and targeting antigen shed are being actively considered as alternative imaging approaches. For membrane bound antigens associated with senescence, here we report the latest work developing uPAR immunoPET agents. First, we have shown that attachment of 89Zr-DFO preserves the binding function of both murine (muPAR) and human uPAR (huPAR) antibodies with high cross-reactivity of the murine uPAR (muPAR) antibody with huPAR. Cell uptake was increased two-fold in murine KPC cells with senescence-inducing TP treatment. In vivo, accumulation in subcutaneously implanted murine KPC pancreatic tumors was observed with TP-treated mice; however lower liver and blood activity was imaged by PET/CT and confirmed by terminal biodistribution at 144h. Human uPAR-targeting antibody in nude mice bearing flank MiaPaCa2 tumors showed strong antibody uptake by 144 h, with increased tumor uptake for mice undergoing TP therapy. Preloading studies at 10 µg, 40 µg, and 100µg at 4h prior to injection of 40µg 89Zr-DFO-huPAR showed improved tumor uptake kinetics with the highest preloading level, though overall tumor uptake was decreased at the higher radiolabeled antibody dose used, thus more optimization is needed. The development and optimization of 89Zr-DFO-uPAR antibodies for human and murine targets provides a superior means of identifying pancreatic cancer and pancreatic cancer senescence than that afforded by shed antigens. Future directions include further dose optimization and testing uptake in other pancreatic models, senescence-inducing drug combinations, and possible antibody-conjugated senolytic or endoradiotherapy strategies. Citation Format: Edwin C. Pratt, Riccardo Mezzadra, Tara Viray, Spencer Kaminsky, Scott W. Lowe, Jason S. Lewis. Targeting pancreatic cancer senescence with ImmunoPET [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr A032.

Polyazamacrocycle Ligands Facilitate 89Zr Radiochemistry and Yield 89Zr Complexes with Remarkable Stability.

Over the last three decades, the chemistry of zirconium has facilitated antibody development and the clinical management of disease in the precision medicine era. Scientists have harnessed its reactivity, coordination chemistry, and nuclear chemistry to develop antibody-based radiopharmaceuticals incorporating zirconium-89 (89Zr: t1/2 = 78.4 h, β+: 22.8%, Eβ+max = 901 keV; EC: 77%, Eγ = 909 keV) to improve disease detection, identify patients for individualized therapeutic interventions. and monitor their response to those interventions. However, release of the 89Zr4+ ion from the radiopharmaceutical remains a concern, since it may confound the interpretation of clinical imaging data, negatively affect dosimetric calculations, and hinder treatment planning. In this report, we relate our novel observations involving the use of polyazamacrocycles as zirconium-89 chelators. We describe the synthesis and complete characterization of zirconium 2,2',2″,2‴-(1,4,7,10-tetraazacyclotridecane-1,4,7,10-tetrayl)tetraacetic acid (Zr-TRITA), zirconium 3,6,9,15-Tetraazabicyclo[9.3.1] pentadeca-1(15),11,13-triene-3,6,9-triacetic acid (Zr-PCTA), and zirconium 2,2',2″-(1,4,7-triazacyclononane-1,4,7-triyl)triacetic acid (Zr-NOTA). In addition, we elucidate the solid-state structure of each complex using single-crystal X-ray diffraction analysis. Finally, we found that [89Zr]Zr-PCTA and [89Zr]Zr-NOTA demonstrate excellent stability in vitro and in vivo and provide a rationale for these observations. These innovative findings have the potential to guide the development of safer and more robust immuno-PET agents to improve precision medicine applications.

ImmunoPET as Stoichiometric Sensor for Glypican-3 in Models of Hepatocellular Carcinoma

Background Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide. While conventional imaging approaches like ultrasound, CT, and MRI play critical roles in the diagnosis and surveillance of HCC, improved methods for detection and assessment of treatment response are needed. One promising approach is the use of radiolabeled antibodies for positron emission tomography (immunoPET) imaging. Glypican-3 (GPC3) is a proteoglycan that is highly expressed in the majority of HCC tumors. GPC3-specific antibodies are used to diagnose HCC histopathologically, and have been proposed as a treatment of HCC. Here, we design, synthesize and demonstrate that our humanized immunoPET agent, [89Zr]Zr-DFO-TAB-H14, can stoichiometrically bind to models of human liver cancer with varied GPC3 expression. Methods: The GPC3-specific monoclonal humanized IgG1, TAB-H14, was used as a scaffold for engineering our immunoPET agent. Fluorescent and deferroxamine (DFO) chelate conjugates of TAB-H14 were characterized using mass spectrometry. Binding affinity of TAB-H14 and conjugates for GPC3 was determined in cell-free biolayer interferometry, and cell-based radioimmunoassays. GPC3-expression was assessed by flow cytometry and immunofluorescence using commercially available anti-GPC3 antibodies and TAB-H14 in GPC3−(A431) and GPC3+ cell lines including an engineered line (A431-GPC3+, G1) and liver cancer lines (HepG2, Hep3B, and Huh7). DFO-TAB-H14, was radiolabeled with Zr-89. Mice were subcutaneously engrafted with the aforementioned cell lines and in vivo target engagement of the immunoPET agent [89Zr]Zr-DFO-TAB-H14 was determined using PET/CT, quantitative biodistribution, and autoradiography. Results: TAB-H14 demonstrated subnanomolar to nanomolar affinity for human GPC3. Fluorescently tagged TAB-H14 was able to bind to GPC3 on cell membranes of GPC3-expressing lines by flow cytometry. These results were confirmed by immunofluorescence staining of A431, G1 HepG2, Hep3B, and Huh7 tumor sections. ImmunoPET imaging with [89Zr]Zr-DFO-TAB-H14 showed stoichiometric tumor uptake corresponding to the cell surface expression levels. Autoradiography and immunostaining confirmed in vivo findings. Conclusion: We systematically demonstrate that the humanized immnoPET agent [89Zr]Zr-DFO-TAB-H14 specifically and stoichiometrically binds to GPC3 in several models of human liver cancer, serving as a promising in vivo GPC3 sensor. This agent may provide utility in HCC diagnosis and surveillance, and the selection of candidates for GPC3-directed therapies.

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.

89Zr-Chloride Can Be Used for Immuno-PET Radiochemistry Without Loss of Antigen Reactivity In Vivo.

89Zr immuno-PET continues to be assessed in numerous clinical trials. This report evaluates the use of 89Zr-chloride in the radiolabeling of monoclonal antibodies conjugated with desferrioxamine B (DFO), describes its effects on radiopharmaceutical reactivity toward antigen, and offers guidance on how to ensure long-term stability and purity. Methods:89Zr-DFO-trastuzumab and 89Zr-DFO-cetuximab were prepared using 89ZrCl4 The stability of each was evaluated for 7 d in 20 mM histidine/240 mM sucrose buffer, 0.25 M sodium acetate (NaOAc) buffer containing 5 mg·mL-1n-acetyl-l-cysteine (NAC), or 0.25 M NaOAc containing 5 mg·mL-1 l-methionine (L-MET). To assess antigen reactivity, 89Zr-DFO-trastuzumab was evaluated using the Lindmo method and tested in PET/CT imaging of mouse models of human epidermal growth factor receptor 2-positive or -negative lung cancer. Results: Using 89ZrCl4, 89Zr-DFO-trastuzumab and 89Zr-DFO-cetuximab were prepared with increased specific activity and retained purities of 95% after 3 d when formulated in NaOAc buffer containing L-MET. Based on Lindmo analysis and small-animal PET/CT imaging, 89Zr-DFO-trastuzumab remained reactive toward antigen after being prepared with 89ZrCl4Conclusion:89ZrCl4 facilitated the radiosynthesis of 89Zr immuno-PET agents with increased specific activity. L-MET enhanced long-term solution stability better than all other formulations examined, and 89Zr-DFO-trastuzumab remained reactive toward antigen. Although further evaluation is necessary, these initial results suggest that 89ZrCl4 may be useful in immuno-PET radiochemistry as radiolabeled monoclonal antibodies are increasingly integrated into precision medicine strategies.

Labeling Single Domain Antibody Fragments with Fluorine-18 Using 2,3,5,6-Tetrafluorophenyl 6-[18F]Fluoronicotinate Resulting in High Tumor-to-Kidney Ratios.

ImmunoPET agents are being investigated to assess the status of epidermal growth factor receptor 2 (HER2) in breast cancer patients with the goal of selecting those likely to benefit from HER2-targeted therapies and monitoring their progress after these treatments. We have been exploring the use of single domain antibody fragments (sdAbs) labeled with 18F using residualizing prosthetic agents for this purpose. In this study, we have labeled two sdAbs that bind to different domains on the HER2 receptor, 2Rs15d and 5F7, using 2,3,5,6-tetrafluorophenyl 6-[18F]fluoronicotinate ([18F]TFPFN) and evaluated their HER2 targeting properties in vitro and in vivo. The overall decay-corrected radiochemical yield for the synthesis of [18F]TFPFN-2Rs15d and [18F]TFPFN-5F7 was 5.7 ± 3.6 and 4.0 ± 2.0%, respectively. The radiochemical purity of labeled sdAbs was >95%, immunoreactive fractions were about 60%, and affinity was in the low nanomolar range. Intracellularly trapped activity from [18F]TFPFN-2Rs15d and [18F]TFPFN-5F7 in HER2-expressing SKOV-3 ovarian and BT474M1 breast carcinoma cells were similar to the sdAbs labeled using the previously validated radioiodination residualizing prosthetic agents N-succinimidyl 4-guanidinomethyl-3-[125I]iodobenzoate ([125I]SGMIB) and N-succinimidyl 3-guanidinomethyl-5-[125I]iodobenzoate ( iso-[125I]SGMIB). Intracellular activity was about 2-fold higher for radiolabeled 5F7 compared with 2Rs15d for both 18F and 125I. While tumor uptake of both [18F]TFPFN-2Rs15d and [18F]TFPFN-5F7 was comparable to those for the coadministered 125I-labeled sdAb, renal uptake of the 18F-labeled sdAbs was substantially lower. In microPET images, the tumor was clearly delineated in SKOV-3 and BT474 xenograft-bearing athymic mice with low levels of background activity in normal tissues, except the bladder. These results indicate that the [18F]TFPFN prosthetic group could be a valuable reagent for developing sdAb-based immunoPET imaging agents.

A comprehensively revised strategy that improves the specific activity and long-term stability of clinically relevant 89Zr-immuno-PET agents.

Zirconium-89 is currently being used in numerous clinical trials involving monoclonal antibodies and positron emission tomography. This report describes a revised strategy that reduces preparation time while increasing the specific activity of clinically relevant immuno-PET agents. Additionally, it demonstrates that n-acetyl-l-cysteine acts as a superior radioprotective agent that improves long-term stability without compromising antigen affinity in vivo.

Production, applications and status of zirconium-89 immunoPET agents

Zirconium-89 has attracted huge interests and is used in tracing and quantification of slow biological processes and labeling of long half-live biomolecules such as monoclonal antibodies for pharmacokinetic studies and clinical trials. In this review, a concise introduction to targetry, irradiation data, separation and coordination chemistry of zirconium-89 has been presented. A detailed overreviwew on bi-functional ligands conjugation and 89Zr radiolabeling been addressed. The latest status of preclinical as well as clinical trials using 89Zr radioimmunomolecules in various human diseases has been presented since 2012.

Production and radiochemical separation of a potential immuno-PET imaging agent 89Zr from proton irradiated natY target

Zirconium-89 (89Zr) is an interesting immuno-PET imaging agent due to its favorable physical characteristics of 78.4 h half-life and a relatively low positron energy of 0.9 MeV. Radiochemical separation of 89Zr from irradiated yttrium target matrix has been achieved by different separation methodologies like anion-exchange chromatography, cation-exchange chromatography and size exclusion chromatography (Sephadex G-25 resin). A novel and simple method based on size exclusion chromatography (Sephadex G-25) for radiochemical separation of 89Zr with no harmful substances has been explored. The experimental yield of 89Zr from natural yttrium target was found to be 27 MBq/μAh using 12 MeV proton. The radiochemical separation yield, radionuclidic purity and chemical purity of 89Zr using an anion exchanger, Dowex-1 resin/2 M HCl column system and Sephadex G-25 resin column was found to be high enough.

Zirconium tetraazamacrocycle complexes display extraordinary stability and provide a new strategy for zirconium-89-based radiopharmaceutical development.

We report our initial investigations into the use of tetraazamacrocycles as zirconium-89 chelators. We describe the synthesis and complete characterization of several Zr tetraazamacrocycle complexes, and definitively describe the first crystal structure of zirconium 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (Zr-DOTA) using single crystal X-ray diffraction analysis. After evaluating several radioactive analogs, we found that 89Zr-DOTA is superior to 89Zr-DFO, the only 89Zr-complex to be used clinically in 89Zr-radiopharmaceutical applications. Finally, we provide a rationale for the unanticipated and extraordinary stability of these complexes in vitro and in vivo. These results may inform the development of safer and more robust immuno-PET agents for precision medicine applications.

Abstract IA16: New methods of PET imaging pancreas cancer

Abstract The medical need to properly diagnose, stage, and treat PDAC is staggering despite research efforts in recent years. PDAC is one of the most aggressive cancers, ranking fourth as a cause of cancer mortality. Most patients do not present with symptoms until the late stages of the disease, and for that reason, 80% of patients have metastatic disease at the time of diagnosis. The overall five-year survival rate is approximately 5%, and for those who undergo surgical resection the 5-year survival rate is only 25% due to the high incidence of undiscovered metastases that are not detected using imaging modalities such as MRI and CT. The clinical options available to PDAC patients are dependent on disease progression at the time of diagnosis. Resection offers the best chance of survival, but the presence of metastases, which often escape detection using MRI and CT due to their lack of specificity, precludes patients from resection. Patients are regularly misdiagnosed or understaged, complicating treatment strategies and preventing enrollment in clinical trials. Many of these problems could be avoided if adequate diagnostic and staging procedures were available. Positron emission tomography (PET) is a promising technological platform for the detection and staging of PDAC. Currently, PET imaging of PDAC generally utilizes the imaging agent 2-deoxy-2-[18F]-fluoro-D-glucose (18F -FDG), relying on increased tumor metabolism relative to nonmalignant cells (Warburg effect). However, despite the success of FDG as a diagnostic tool in some cancers, it lacks the sensitivity and specificity required for the staging of small primary lesions (<7 mm) and liver metastases (<1 cm). Additionally, in the case of PDAC, it is generally incapable of differentiating benign disease (i.e. pancreatitis, jaundice) from malignancy. The use of FDG as a diagnostic tool for PDAC may be supplemented by assaying biomarkers from biological fluids. The diagnosis of PDAC is often based partially on the detection of elevated levels of circulating CA19.9 (also known as sialyl Lewisa) antigen in sera. CA19.9 is a ligand for epithelial leukocyte adhesion molecules, and its overexpression is a key event in invasion and metastasis of many tumors, including PDAC tumors. However, the specificity of the assays for PDAC are predominantly attenuated by antigen secretion from benign pathologies among distant organs (e.g., jaundice in the liver) or disorders of the pancreas (e.g., pancreatitis) that FDG is also not able to delineate. Thus, CA19.9 assays are useful screening tools but are not an accepted diagnostic tool due to the high occurrence of false-positive tests. Thus, the development of a radiotracer that is capable of targeting the antigen at its tissue of origin and distinguishing benign disease from malignancy is necessary to supplement antibody-based serum biomarker measurements. Radiolabeled antibodies or antibody fragments especially those specific to surface-bound antigens expressed by tumor cells can provide the necessary specificity and sensitivity. The development of high affinity imaging agents could enhance our ability to monitor and detect micro-metastases and slow growing epithelial cancers. In the case of PDAC, the CA19.9 antigen is attractive because it is the most highly expressed tumor antigen in pancreatic cancer and is minimally expressed in healthy pancreas tissue. Moreover, multiple copies of CA19.9 are expressed on many membrane proteins, providing an advantage in terms of specificity and sensitivity compared to other tumor antigens. For those reasons, several antibody development programs have been recently initiated, leading to the development of the fully human monoclonal antibody (mAb) 5B1. The recombinant mAb 5B1 potently binds an extracellular epitope on the CA19.9 protein, providing an ideal platform for developing an immunoPET agent for imaging and staging of PDAC. One concern with 5B1 was the potential for circulating antigen to diminish its usefulness. However, initial experiments with 5B1 indicated that it was not a major concern in the case of 5B1, which provided exceptional PET images. By targeting tumor-associated CA19.9 via non-invasive PET, we will directly address these needs effectively and efficiently. Citation Format: Jason S. Lewis. New methods of PET imaging pancreas cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr IA16.

Quantitative ImmunoPET of Prostate Cancer Xenografts with 89Zr- and 124I-Labeled Anti-PSCA A11 Minibody

Prostate stem cell antigen (PSCA) is expressed on the cell surface in 83%-100% of local prostate cancers and 87%-100% of prostate cancer bone metastases. In this study, we sought to develop immunoPET agents using (124)I- and (89)Zr-labeled anti-PSCA A11 minibodies (scFv-CH3 dimer, 80 kDa) and evaluate their use for quantitative immunoPET imaging of prostate cancer. A11 anti-PSCA minibody was alternatively labeled with (124)I- or (89)Zr-desferrioxamine and injected into mice bearing either matched 22Rv1 and 22Rv1×PSCA or LAPC-9 xenografts. Small-animal PET data were obtained and quantitated with and without recovery coefficient-based partial-volume correction, and the results were compared with ex vivo biodistribution. Rapid and specific localization to PSCA-positive tumors and high-contrast imaging were observed with both (124)I- and (89)Zr-labeled A11 anti-PSCA minibody. However, the differences in tumor uptake and background uptake of the radiotracers resulted in different levels of imaging contrast. The nonresidualizing (124)I-labeled minibody had lower tumor uptake (3.62 ± 1.18 percentage injected dose per gram [%ID/g] 22Rv1×PSCA, 3.63 ± 0.59 %ID/g LAPC-9) than the residualizing (89)Zr-labeled minibody (7.87 ± 0.52 %ID/g 22Rv1×PSCA, 9.33 ± 0.87 %ID/g LAPC-9, P < 0.0001 for each), but the (124)I-labeled minibody achieved higher imaging contrast because of lower nonspecific uptake and better tumor-to-soft-tissue ratios (22Rv1×PSCA:22Rv1 positive-to-negative tumor, 13.31 ± 5.59 (124)I-A11 and 4.87 ± 0.52 (89)Zr-A11, P = 0.02). Partial-volume correction was found to greatly improve the correspondence between small-animal PET and ex vivo quantification of tumor uptake for immunoPET imaging with both radionuclides. Both (124)I- and (89)Zr-labeled A11 anti-PSCA minibody showed high-contrast imaging of PSCA expression in vivo. However, the (124)I-labeled A11 minibody was found to be the superior imaging agent because of lower nonspecific uptake and higher tumor-to-soft-tissue contrast. Partial-volume correction was found to be essential for robust quantification of immunoPET imaging with both (124)I- and (89)Zr-labeled A11 minibody.