Development and characterization of CD54-targeted immunoPET imaging in solid tumors.
Wei Weijun,Jiang Dawei,Lee Hye Jin,Li Miao,Kutyreff Christopher J,Engle Jonathan W,Liu Jianjun,Cai Weibo
European journal of nuclear medicine and molecular imaging
PURPOSE:Intercellular adhesion molecule-1 (ICAM-1, CD54) is an emerging therapeutic target for a variety of solid tumors including melanoma and anaplastic thyroid cancer (ATC). This study aims to develop an ICAM-1-targeted immuno-positron emission tomography (immunoPET) imaging strategy and assess its diagnostic value in melanoma and ATC models. METHODS:Flow cytometry was used to screen ICAM-1-positive melanoma and ATC cell lines. Melanoma and ATC models were established using A375 cell line and THJ-16T cell line, respectively. An ICAM-1-specific monoclonal antibody (R6-5-D6) and a nonspecific human IgG were radiolabeled with Cu and the diagnostic efficacies were interrogated in tumor-bearing mouse models. Biodistribution and fluorescent imaging studies were performed to confirm the specificity of the ICAM-1-targeted imaging probes. RESULTS:ICAM-1 was strongly expressed on melanoma and advanced thyroid cancer cell lines. Cu-NOTA-ICAM-1 immunoPET imaging efficiently delineated A375 melanomas with a peak tumor uptake of 21.28 ± 6.56 %ID/g (n = 5), significantly higher than that of Cu-NOTA-IgG (10.63 ± 2.58 %ID/g, n = 3). Moreover, immunoPET imaging with Cu-NOTA-ICAM-1 efficiently visualized subcutaneous and orthotopic ATCs with high clarity and contrast. Fluorescent imaging with IRDye 800CW-ICAM-1 also visualized orthotopic ATCs and the tumor uptake could be blocked by the ICAM-1 parental antibody R6-5-D6, indicating the high specificity of the developed probe. Finally, blocking with the human IgG prolonged the circulation of the Cu-NOTA-ICAM-1 in R2G2 mice without compromising the tumor uptake. CONCLUSION:ICAM-1-targeted immunoPET imaging could characterize ICAM-1 expression in melanoma and ATC, which holds promise for optimizing ICAM-1-targeted therapies in the future.
10.1007/s00259-020-04784-0
Light-induced synthesis of protein conjugates and its application in photoradiosynthesis of Zr-radiolabeled monoclonal antibodies.
Guillou Amaury,Earley Daniel F,Patra Malay,Holland Jason P
Nature protocols
Efficient methods to functionalize proteins are essential for the development of many diagnostic and therapeutic compounds, such as fluorescent probes for immunohistochemistry, zirconium-89 radiolabeled mAbs (Zr-mAbs) for positron emission tomography and antibody-drug conjugates (ADCs). This protocol describes a step-by-step procedure for the light-induced functionalization of proteins with compounds bearing the photochemically active aryl azide group. As an illustration of the potential utility of our approach, this protocol focuses on the synthesis of Zr-mAbs using photoactivatable derivatives of the metal ion binding chelate desferrioxamine B (DFO). The light-induced synthesis of Zr-mAbs is a unique, one-pot process involving simultaneous radiolabeling and protein conjugation. The photoradiochemical synthesis of purified Zr-mAbs, starting from unmodified proteins, [Zr][Zr(CO)] (Zr-oxalate), and a photoactivatable DFO derivative, can be performed in <90 min. The method can be easily adapted to prepare other radiolabeled proteins, ADCs or fluorescently tagged proteins by using drug molecules or fluorophores functionalized with photoactive moieties.
10.1038/s41596-020-0386-5
Anti-human PD-L1 Nanobody for Immuno-PET Imaging: Validation of a Conjugation Strategy for Clinical Translation.
Bridoux Jessica,Broos Katrijn,Lecocq Quentin,Debie Pieterjan,Martin Charlotte,Ballet Steven,Raes Geert,Neyt Sara,Vanhove Christian,Breckpot Karine,Devoogdt Nick,Caveliers Vicky,Keyaerts Marleen,Xavier Catarina
Biomolecules
Immune checkpoints, such as programmed death-ligand 1 (PD-L1), limit T-cell function and tumor cells use this ligand to escape the anti-tumor immune response. Treatments with monoclonal antibodies blocking these checkpoints have shown long-lasting responses, but only in a subset of patients. This study aims to develop a Nanobody (Nb)-based probe in order to assess human PD-L1 (hPD-L1) expression using positron emission tomography imaging, and to compare the influence of two different radiolabeling strategies, since the Nb has a lysine in its complementarity determining region (CDR), which may impact its affinity upon functionalization. The Nb has been conjugated with the NOTA chelator site-specifically via the Sortase-A enzyme or randomly on its lysines. [Ga]Ga-NOTA-(hPD-L1) Nbs were obtained in >95% radiochemical purity. In vivo tumor targeting studies at 1 h 20 post-injection revealed specific tumor uptake of 1.89 ± 0.40%IA/g for the site-specific conjugate, 1.77 ± 0.29%IA/g for the random conjugate, no nonspecific organ targeting, and excretion via the kidneys and bladder. Both strategies allowed for easily obtaining Ga-labeled hPD-L1 Nbs in high yields. The two conjugates were stable and showed excellent in vivo targeting. Moreover, we proved that the random lysine-conjugation is a valid strategy for clinical translation of the hPD-L1 Nb, despite the lysine present in the CDR.
10.3390/biom10101388
Photochemical Conjugation and One-Pot Radiolabelling of Antibodies for Immuno-PET.
Patra Malay,Eichenberger Larissa S,Fischer Gregor,Holland Jason P
Angewandte Chemie (International ed. in English)
Monoclonal antibodies (mAbs), immunoglobulin fragments, and other proteins are important scaffolds in the development of radiopharmaceuticals for diagnostic immuno-positron emission tomography (immuno-PET) and targeted radioimmunotherapy (RIT). Conventional methods for radiolabelling proteins with metal ions such as Ga, Cu, Zr, and Y require multi-step procedures involving pre-purification, functionalisation with a chelate, and subsequent radiolabelling. Standard coupling chemistries are time-consuming, difficult to automate, and involve synthesis, isolation, and storage of an intermediate, new molecular entity (the conjugated mAb) whose biochemical properties can differ from those of the parent protein. To circumvent these issues, we developed a photoradiochemical approach that uses fast, chemoselective, light-induced protein modification under mild conditions with novel metal-ion-binding chelates derivatised with aryl azide (ArN ) groups. Experiments show that one-pot photochemical conjugation and radiolabelling of formulated mAbs can be achieved in <20 min.
10.1002/anie.201813287
PD-L1 detection using Zr-atezolizumab immuno-PET in renal cell carcinoma tumorgrafts from a patient with favorable nivolumab response.
Vento Joseph,Mulgaonkar Aditi,Woolford Layton,Nham Kien,Christie Alana,Bagrodia Aditya,de Leon Alberto Diaz,Hannan Raquibul,Bowman Isaac,McKay Renee M,Kapur Payal,Hao Guiyang,Sun Xiankai,Brugarolas James
Journal for immunotherapy of cancer
BACKGROUND:Programmed death-ligand 1 (PD-L1) expression in metastatic renal cell carcinoma (RCC) correlates with a worse prognosis, but whether it also predicts responsiveness to anti-PD-1/PD-L1 therapy remains unclear. Most studies of PD-L1 are limited by evaluation in primary rather than metastatic sites, and in biopsy samples, which may not be representative. These limitations may be overcome with immuno-positron emission tomography (iPET), an emerging tool allowing the detection of cell surface proteins with radiolabeled antibodies. Here, we report iPET studies of PD-L1 in a preclinical tumorgraft model of clear cell RCC (ccRCC) from a patient who had a favorable response to anti-PD-1 therapy. CASE PRESENTATION:A 49-year-old man underwent a cytoreductive nephrectomy in 2017 of a right kidney tumor invading into the adrenal gland that was metastatic to the lungs and a rib. Histological analyses revealed a ccRCC of ISUP grade 4 with extensive sarcomatoid features. IMDC risk group was poor. Within two hours of surgery, a tumor sample was implanted orthotopically into NOD/SCID mice. Consistent with an aggressive tumor, a renal mass was detected 18 days post-implantation. Histologically, the tumorgraft showed sarcomatoid differentiation and high levels of PD-L1, similar to the patient's tumor. PD-L1 was evaluated in subsequently transplanted mice using iPET and the results were compared to control mice implanted with a PD-L1-negative tumor. We labeled atezolizumab, an anti-PD-L1 antibody with a mutant Fc, with zirconium-89. iPET revealed significantly higher Zr-atezolizumab uptake in index than control tumorgrafts. The patient was treated with high-dose IL2 initially, and subsequently with pazopanib, with rapidly progressive disease, but had a durable response with nivolumab. CONCLUSIONS:To our knowledge, this is the first report of non-invasive detection of PD-L1 in renal cancer using molecular imaging. This study supports clinical evaluation of iPET to identify RCC patients with tumors deploying the PD-L1 checkpoint pathway who may be most likely to benefit from PD-1/PD-L1 disrupting drugs.
10.1186/s40425-019-0607-z
Retooling a Blood-Based Biomarker: Phase I Assessment of the High-Affinity CA19-9 Antibody HuMab-5B1 for Immuno-PET Imaging of Pancreatic Cancer.
Lohrmann Christian,O'Reilly Eileen M,O'Donoghue Joseph A,Pandit-Taskar Neeta,Carrasquillo Jorge A,Lyashchenko Serge K,Ruan Shutian,Teng Rebecca,Scholz Wolfgang,Maffuid Paul W,Lewis Jason S,Weber Wolfgang A
Clinical cancer research : an official journal of the American Association for Cancer Research
PURPOSE:In patients with cancer who have an abnormal biomarker finding, the source of the biomarker in the bloodstream must be located for confirmation of diagnosis, staging, and therapy planning. We evaluated if immuno-PET with the radiolabeled high-affinity antibody HuMab-5B1 (MVT-2163), binding to the cancer antigen CA19-9, can identify the source of elevated biomarkers in patients with pancreatic cancer. PATIENTS AND METHODS:In this phase I dose-escalating study, 12 patients with CA19-9-positive metastatic malignancies were injected with MVT-2163. Within 7 days, all patients underwent a total of four whole-body PET/CT scans. A diagnostic CT scan was performed prior to injection of MVT-2163 to correlate findings on MVT-2163 PET/CT. RESULTS:Immuno-PET with MVT-2163 was safe and visualized known primary tumors and metastases with high contrast. In addition, radiotracer uptake was not only observed in metastases known from conventional CT, but also seen in subcentimeter lymph nodes located in typical metastatic sites of pancreatic cancer, which were not abnormal on routine clinical imaging studies. A significant fraction of the patients demonstrated very high and, over time, increased uptake of MVT-2163 in tumor tissue, suggesting that HuMab-5B1 labeled with beta-emitting radioisotopes may have the potential to deliver therapeutic doses of radiation to cancer cells. CONCLUSIONS:Our study shows that the tumor antigen CA19-9 secreted to the circulation can be used for sensitive detection of primary tumors and metastatic disease by immuno-PET. This significantly broadens the number of molecular targets that can be used for PET imaging and offers new opportunities for noninvasive characterization of tumors in patients.
10.1158/1078-0432.CCR-18-3667
Development of a Theranostic Convergence Bioradiopharmaceutical for Immuno-PET Based Radioimmunotherapy of L1CAM in Cholangiocarcinoma Model.
Song In Ho,Jeong Mun Sik,Hong Hyo Jeong,Shin Jong Il,Park Yong Serk,Woo Sang-Keun,Moon Byung Seok,Kim Kwang Il,Lee Yong Jin,Kang Joo Hyun,Lee Tae Sup
Clinical cancer research : an official journal of the American Association for Cancer Research
PURPOSE:Cholangiocarcinoma is a malignancy of bile duct with a poor prognosis. Conventional chemotherapy and radiotherapy are generally ineffective, and surgical resection is the only curative treatment for cholangiocarcinoma. L1-cell adhesion molecule (L1CAM) has been known as a novel prognostic marker and therapeutic target for cholangiocarcinoma. This study aimed to evaluate the feasibility of immuno-PET imaging-based radioimmunotherapy using radiolabeled anti-L1CAM antibody in cholangiocarcinoma xenograft model. EXPERIMENTAL DESIGN:We prepared a theranostic convergence bioradiopharmaceutical using chimeric anti-L1CAM antibody (cA10-A3) conjugated with 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) chelator and labeled with Cu or Lu and evaluated the immuno-PET or SPECT/CT imaging and biodistribution with Cu-/Lu-cA10-A3 in various cholangiocarcinoma xenograft models. Therapeutic efficacy and response monitoring were performed by Lu-cA10-A3 and F-FDG-PET, respectively, and immunohistochemistry was done by TUNEL and Ki-67. RESULTS:Radiolabeled cA10-A3 antibodies specifically recognized L1CAM , clearly visualized cholangiocarcinoma tumors in immuno-PET and SPECT/CT imaging, and differentiated the L1CAM expression level in cholangiocarcinoma xenograft models. Lu-cA10-A3 (12.95 MBq/100 μg) showed statistically significant reduction in tumor volumes ( < 0.05) and decreased glucose metabolism ( < 0.01). IHC analysis revealed Lu-cA10-A3 treatment increased TUNEL-positive and decreased Ki-67-positive cells, compared with saline, cA10-A3, or Lu-isotype. CONCLUSIONS:Anti-L1CAM immuno-PET imaging using Cu-cA10-A3 could be translated into the clinic for characterizing the pharmacokinetics and selecting appropriate patients for radioimmunotherapy. Radioimmunotherapy using Lu-cA10-A3 may provide survival benefit in L1CAM-expressing cholangiocarcinoma tumor. Theranostic convergence bioradiopharmaceutical strategy would be applied as imaging biomarker-based personalized medicine in L1CAM-expressing patients with cholangiocarcinoma.
10.1158/1078-0432.CCR-19-1157
Macrophage cell tracking PET imaging using mesoporous silica nanoparticles via in vivo bioorthogonal F-18 labeling.
Jeong Hyeon Jin,Yoo Ran Ji,Kim Jin Kwan,Kim Min Hwan,Park Su Hong,Kim Haebin,Lim Jae Wook,Do Sun Hee,Lee Kyo Chul,Lee Yong Jin,Kim Dong Wook
Biomaterials
We introduce an efficient cell tracking imaging protocol using positron emission tomography (PET). Since macrophages are known to home and accumulate in tumor tissues and atherosclerotic plaque, we design a PET imaging protocol for macrophage cell tracking using aza-dibenzocyclooctyne-tethered PEGylated mesoporous silica nanoparticles (DBCO-MSNs) with the short half-life F-18-labeled azide-radiotracer via an in vivo strain-promoted alkyne azide cycloaddition (SPAAC) covalent labeling reaction inside macrophage cells in vivo. This PET imaging protocol for in vivo cell tracking successfully visualizes the migration of macrophage cells into the tumor site by the bioorthogonal SPAAC reaction of DBCO-MSNs with [F]fluoropentaethylene glycolic azide ([F]2) to form F-labeled aza-dibenzocycloocta-triazolic MSNs (F-DBCOT-MSNs) inside RAW 264.7 cells. The tissue radioactivity distribution results were consistent with PET imaging findings. In addition, PET images of atherosclerosis in ApoE mice fed a western diet for 30 weeks were obtained using the devised macrophage cell-tracking protocol.
10.1016/j.biomaterials.2019.01.043
Granzyme B PET imaging of immune-mediated tumor killing as a tool for understanding immunotherapy response.
LaSalle Thomas,Austin Emily E,Rigney Grant,Wehrenberg-Klee Eric,Nesti Sarah,Larimer Benjamin,Mahmood Umar
Journal for immunotherapy of cancer
BACKGROUND:Cancer immunotherapy research is expanding to include a more robust understanding of the mechanisms of treatment response and resistance. Identification of drivers of pro-tumor and anti-tumor immunity during treatment offers new strategies for effective alternative or combination immunotherapies. Currently, tissue or blood samples are collected and analyzed, then dichotomized based on clinical end points that may occur months or years after tissue is collected. While overall survival is ultimately the desired clinical outcome, this dichotomization fails to incorporate the nuances that may occur during an anti-tumor response. By failing to directly measure immune activation at the time of sampling, tumors may be misclassified and potentially obscure important biological information. Non-invasive techniques, such as positron emission tomography (PET), allow for global and quantitative measurements of cancer specific processes and are widely used clinically to help manage disease. METHODS:We have previously developed a novel PET agent that can non-invasively quantify granzyme B release in tumors and have demonstrated its ability to predict response to checkpoint inhibitor therapy in multiple murine models of cancer. Here, we used the quantitative measurement of granzyme B release as a direct and time-matched marker of immune cell activation in order to determine immune cell types and cytokines that correlate with effective checkpoint inhibitor therapy in both tumors and tumor-draining lymph nodes. RESULTS:Through PET imaging, we were able to successfully distinguish distinct microenvironments, based on tumor type, which influenced immune cell subpopulations and cytokine release. Although each tumor was marked by functionally distinct pathways of immune cell activation and inflammation, they also shared commonalities that ultimately resulted in granzyme B release and tumor killing. CONCLUSIONS:These results suggest that discrete tumor immune microenvironments can be identified in both responsive and non-responsive tumors and offers strategic targets for intervention to overcome checkpoint inhibitor resistance.
10.1136/jitc-2019-000291
Harnessing Cu/Cu for a theranostic approach to pretargeted radioimmunotherapy.
Proceedings of the National Academy of Sciences of the United States of America
Over the past decade, theranostic imaging has emerged as a powerful clinical tool in oncology for identifying patients likely to respond to targeted therapies and for monitoring the response of patients to treatment. Herein, we report a theranostic approach to pretargeted radioimmunotherapy (PRIT) based on a pair of radioisotopes of copper: positron-emitting copper-64 (Cu, = 12.7 h) and beta particle-emitting copper-67 (Cu, = 61.8 h). This strategy is predicated on the in vivo ligation between a trans-cyclooctene (TCO)-bearing antibody and a tetrazine (Tz)-based radioligand via the rapid and bioorthogonal inverse electron-demand Diels-Alder reaction. Longitudinal therapy studies were conducted in a murine model of human colorectal carcinoma using an immunoconjugate of the huA33 antibody modified with TCO (huA33-TCO) and a Cu-labeled Tz radioligand ([Cu]Cu-MeCOSar-Tz). The injection of huA33-TCO followed 72 h later by the administration of 18.5, 37.0, or 55.5 MBq of [Cu]Cu-MeCOSar-Tz produced a dose-dependent therapeutic response, with the median survival time increasing from 68 d for the lowest dose to >200 d for the highest. Furthermore, we observed that mice that received the highest dose of [Cu]Cu-MeCOSar-Tz in a fractionated manner exhibited improved hematological values without sacrificing therapeutic efficacy. Dual radionuclide experiments in which a single administration of huA33-TCO was followed by separate injections of [Cu]Cu-MeCOSar-Tz and [Cu]Cu-MeCOSar-Tz revealed that the positron emission tomography images produced by the former accurately predicted the efficacy of the latter. In these experiments, a correlation was observed between the tumoral uptake of [Cu]Cu-MeCOSar-Tz and the subsequent therapeutic response to [Cu]Cu-MeCOSar-Tz.
10.1073/pnas.2009960117
Nanoparticle PET-CT imaging of macrophages in inflammatory atherosclerosis.
Nahrendorf Matthias,Zhang Hanwen,Hembrador Sheena,Panizzi Peter,Sosnovik David E,Aikawa Elena,Libby Peter,Swirski Filip K,Weissleder Ralph
Circulation
BACKGROUND:Macrophages participate centrally in atherosclerosis, and macrophage markers (eg, CD68, MAC-3) correlate well with lesion severity and therapeutic modulation. On the basis of the avidity of lesional macrophages for polysaccharide-containing supramolecular structures such as nanoparticles, we have developed a new positron emission tomography (PET) agent with optimized pharmacokinetics to allow in vivo imaging at tracer concentrations. METHODS AND RESULTS:A dextranated and DTPA-modified magnetofluorescent 20-nm nanoparticle was labeled with the PET tracer 64Cu (1 mCi/0.1 mg nanoparticles) to yield a PET, magnetic resonance, and optically detectable imaging agent. Peak PET activity 24 hours after intravenous injection into mice deficient in apolipoprotein E with experimental atherosclerosis mapped to areas of high plaque load identified by computed tomography such as the aortic root and arch and correlated with magnetic resonance and optical imaging. Accumulated dose in apolipoprotein E-deficient aortas determined by gamma counting was 260% and in carotids 392% of respective wild-type organs (P<0.05 both). Autoradiography of aortas demonstrated uptake of the agent into macrophage-rich atheromata identified by Oil Red O staining of lipid deposits. The novel nanoagent accumulated predominantly in macrophages as determined by fluorescence microscopy and flow cytometry of cells dissociated from aortas. CONCLUSIONS:This report establishes the capability of a novel trimodality nanoparticle to directly detect macrophages in atherosclerotic plaques. Advantages include improved sensitivity; direct correlation of PET signal with an established biomarker (CD68); ability to readily quantify the PET signal, perform whole-body vascular surveys, and spatially localize and follow the trireporter by microscopy; and clinical translatability of the agent given similarities to magnetic resonance imaging probes in clinical trials.
10.1161/CIRCULATIONAHA.107.741181
64Cu-DOTA-trastuzumab PET imaging for HER2-specific primary lesions of breast cancer.
Sasada S,Kurihara H,Kinoshita T,Yoshida M,Honda N,Shimoi T,Shimomura A,Yunokawa M,Yonemori K,Shimizu C,Hamada A,Kanayama Y,Watanabe Y,Fujiwara Y,Tamura K
Annals of oncology : official journal of the European Society for Medical Oncology
10.1093/annonc/mdx227
In Vivo Imaging of the Tumor-Associated Enzyme NCEH1 with a Covalent PET Probe.
Chang Jae Won,Bhuiyan Mohammed,Tsai Hsiu-Ming,Zhang Hannah J,Li Gang,Fathi Shaghayegh,McCutcheon David C,Leoni Lara,Freifelder Richard,Chen Chin-Tu,Moellering Raymond E
Angewandte Chemie (International ed. in English)
Herein, we report the development of an F-labeled, activity-based small-molecule probe targeting the cancer-associated serine hydrolase NCEH1. We undertook a focused medicinal chemistry campaign to simultaneously preserve potent and specific NCEH1 labeling in live cells and animals, while permitting facile F radionuclide incorporation required for PET imaging. The resulting molecule, [ F]JW199, labels active NCEH1 in live cells at nanomolar concentrations and greater than 1000-fold selectivity relative to other serine hydrolases. [ F]JW199 displays rapid, NCEH1-dependent accumulation in mouse tissues. Finally, we demonstrate that [ F]JW199 labels aggressive cancer tumor cells in vivo, which uncovered localized NCEH1 activity at the leading edge of triple-negative breast cancer tumors, suggesting roles for NCEH1 in tumor aggressiveness and metastasis.
10.1002/anie.202004762
PET imaging of occult tumours by temporal integration of tumour-acidosis signals from pH-sensitive Cu-labelled polymers.
Huang Gang,Zhao Tian,Wang Chensu,Nham Kien,Xiong Yahong,Gao Xiaofei,Wang Yihui,Hao Guiyang,Ge Woo-Ping,Sun Xiankai,Sumer Baran D,Gao Jinming
Nature biomedical engineering
Owing to the diversity of cancer types and the spatiotemporal heterogeneity of tumour signals, high-resolution imaging of occult malignancy is challenging. F-fluorodeoxyglucose positron emission tomography allows for near-universal cancer detection, yet in many clinical scenarios it is hampered by false positives. Here, we report a method for the amplification of imaging contrast in tumours via the temporal integration of the imaging signals triggered by tumour acidosis. This method exploits the catastrophic disassembly, at the acidic pH of the tumour milieu, of pH-sensitive positron-emitting neutral copolymer micelles into polycationic polymers, which are then internalized and retained by the cancer cells. Positron emission tomography imaging of the Cu-labelled polymers detected small occult tumours (10-20 mm) in the brain, head, neck and breast of mice at much higher contrast than F-fluorodeoxyglucose, C-methionine and pH-insensitive Cu-labelled nanoparticles. We also show that the pH-sensitive probes reduce false positive detection rates in a mouse model of non-cancerous lipopolysaccharide-induced inflammation. This macromolecular strategy for integrating tumour acidosis should enable improved cancer detection, surveillance and staging.
10.1038/s41551-019-0416-1