Development of 89Zr-Ontuxizumab for in vivo TEM-1/endosialin PET applications.
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ABSTRACT: Purpose
The complexity of sarcoma has led to the need for patient selection via in vivo biomarkers. Tumor endothelial marker-1 (TEM-1) is a cell surface marker expressed by the tumor microenvironment. Currently MORAb-004 (Ontuxizumab), an anti-TEM-1 humanized monoclonal antibody, is in sarcoma clinical trials. Development of positron emission tomography (PET) for in vivo TEM-1 expression may allow for stratification of patients, potentially enhancing clinical outcomes seen with Ontuxizumab.Results
Characterization of cell lines revealed clear differences in TEM-1 expression. One high expressing (RD-ES) and one low expressing (LUPI) cell line were xenografted, and mice were injected with 89Zr-Ontuxizumab. PET imaging post-injection revealed that TEM-1 was highly expressed and readily detectable in vivo only in RD-ES. In vivo biodistribution studies confirmed high radiopharmaceutical uptake in tumor relative to normal organs.Experimental design
Sarcoma cell lines were characterized for TEM-1 expression. Ontuxizumab was labeled with 89Zr and evaluated for immunoreactivity preservation. 89Zr-Ontuxizumab was injected into mice with high or null expressing TEM-1 xenografts. In vivo PET imaging experiments were performed.Conclusion
89Zr-Ontuxizumab can be used in vivo to determine high versus low TEM-1 expression. Reliable PET imaging of TEM-1 in sarcoma patients may allow for identification of patients that will attain the greatest benefit from anti-TEM-1 therapy.
SUBMITTER: Lange SE
PROVIDER: S-EPMC4914343 | biostudies-literature | 2016 Mar
REPOSITORIES: biostudies-literature
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Project description:UNLABELLED:Tumor endothelial marker 1 (TEM1/endosialin) is a tumor vascular marker highly overexpressed in multiple human cancers with minimal expression in normal adult tissue. In this study, we report the preparation and evaluation of (124)I-MORAb-004, a humanized monoclonal antibody targeting an extracellular epitope of human TEM1 (hTEM1), for its ability to specifically and sensitively detect vascular cells expressing hTEM1 in vivo. METHODS:MORAb-004 was directly iodinated with (125)I and (124)I, and in vitro binding and internalization parameters were characterized. The in vivo behavior of radioiodinated MORAb-004 was characterized in mice bearing subcutaneous ID8 tumors enriched with mouse endothelial cells expressing hTEM1 and by biodistribution and small-animal immuno-PET studies. RESULTS:MORAb-004 was radiolabeled with high efficiency and isolated in high purity. In vitro studies demonstrated specific and sensitive binding of MORAb-004 to MS1 mouse endothelial cells expressing hTEM1, with no binding to control MS1 cells. (125)I-MORAb-004 and (124)I-MORAb-004 both had an immunoreactivity of approximately 90%. In vivo biodistribution experiments revealed rapid, highly specific and sensitive uptake of MORAb-004 in MS1-TEM1 tumors at 4 h (153.2 ± 22.2 percentage injected dose per gram [%ID/g]), 24 h (127.1 ± 42.9 %ID/g), 48 h (130.3 ± 32.4 %ID/g), 72 h (160.9 ± 32.1 %ID/g), and 6 d (10.7 ± 1.8 %ID/g). Excellent image contrast was observed with (124)I-immuno-PET. MORAb-004 uptake was statistically higher in TEM1-positive tumors than in control tumors. Binding specificity was confirmed by blocking studies using excess nonlabeled MORAb-004. CONCLUSION:In our preclinical model, with hTEM1 exclusively expressed on engineered murine endothelial cells that integrate into the tumor vasculature, (124)I-MORAb-004 displays high tumor-to-background tissue contrast for detection of hTEM1 in easily accessible tumor vascular compartments. These studies strongly suggest the clinical utility of (124)I-MORAb-004 immuno-PET in assessing TEM1 tumor-status.
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Project description:Endosialin (Tumor Endothelial Marker-1 (TEM-1), CD248) is primarily expressed on pericytes of tumor-associated microvasculature, tumor-associated stromal cells and directly on tumors of mesenchymal origin, including sarcoma and melanoma. While the function of endosialin/TEM-1 is incompletely understood, studies have suggested a role in supporting tumor growth and invasion thus making it an attractive therapeutic target. In an effort to further understand its role in cancer, we previously developed a humanized anti-endosialin/TEM-1 monoclonal antibody (mAb), called ontuxizumab (MORAb-004) for testing in preclinical and clinical studies. We herein report on the generation of an extensive panel of recombinant endosialin/TEM-1 protein extracellular domain (ECD) fragments and novel mAbs against ECD motifs. The domain-specific epitopes were mapped against ECD sub-domains to identify those that can detect distinct structural motifs and can be potentially formatted as probes suitable for diagnostic and functional studies. A number of mAbS were shown to cross-react with the murine and human protein, potentially allowing their use in human animal models and corresponding clinical trials. In addition, pairing of several mAbs supported their use in immunoassays that can detect soluble endosialin/TEM-1 (sEND) in the serum of healthy subjects and cancer patients.
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Project description:UnlabelledHepatocellular carcinoma (HCC) is a devastating malignancy in which imperfect imaging plays a primary role in diagnosis. Glypican-3 (GPC3) is an HCC-specific cell surface proteoglycan overexpressed in most HCCs. This paper presents the use of (89)Zr-conjugated monoclonal antibody against GPC3 ((89)Zr-αGPC3) for intrahepatic tumor localization using PET.MethodsPolymerase chain reaction confirmed relative GPC3 expression in cell lines. In vitro binding, in vivo biodistribution, and small-animal PET studies were performed on GPC3-expressing HepG2 and non-GPC3-expressing HLF and RH7777 cells and orthotopic xenografts.Results(89)Zr-αGPC3 demonstrated antibody-dependent, antigen-specific tumor binding. HepG2 liver tumors exhibited high peak uptake (836.6 ± 86.6 percentage injected dose [%ID]/g) compared with background liver (27.5 ± 1.6 %ID/g). Tumor-to-liver contrast ratio was high and peaked at 32.5. The smallest HepG2 tumor (<1 mm) showed lower peak uptake (42.5 ± 6.4 %ID/g) and tumor-to-liver contrast (1.57) but was still clearly visible on PET. Day 7 tissue activity was still substantial in HepG2 tumors (466.4 ± 87.6 %ID/g) compared with control RH7777 tumors (3.9 ± 1.3 %ID/g, P < 0.01), indicating antigen specificity by (89)Zr-αGPC3. HepG2 tumor treated with unlabeled αGPC3 or heat-denatured (89)Zr-αGPC3 demonstrated tumor activity (2.1 %ID/g) comparable to that of control xenografts, confirming antibody dependency.ConclusionThis study demonstrated the feasibility of using (89)Zr-αGPC3 to image HCC in the liver, as well as the qualitative determination of GPC3 expression via small-animal PET. The ability to clarify the identity of small liver lesions with an HCC-specific PET probe would provide clinicians with vital information that could significantly alter patient management, warranting further investigation for clinical translation.
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Project description:Tumor survival is influenced by interactions between tumor cells and the stromal microenvironment. One example is Endosialin (Tumor Endothelial Marker-1 (TEM-1) or CD248), which is expressed primarily by cells of mesenchymal origin and some tumor cells. The expression, as a function of architectural masking, of TEM-1 and its pathway-associated proteins was quantified and examined for association with five-year disease-specific survival on a colorectal cancer (CRC) cohort divided into training (n=330) and validation (n=164) sets. Although stromal expression of TEM-1 had prognostic value, a more significant prognostic signature was obtained through linear combination of five compartment-specific expression scores (TEM-1 Stroma, TEM-1 Tumor Vessel, HIF2? Stromal Vessel, Collagen IV Tumor, and Fibronectin Stroma). This resulted in a single continuous risk score (TAPPS: TEM-1 Associated Pathway Prognostic Signature) which was significantly associated with decreased survival on both the training set [HR=1.76 (95%CI: 1.44-2.15); p<0.001] and validation set [HR=1.38 (95%CI: 1.02-1.88); p=0.04]. Importantly, since prognosis is a critical clinical question in Stage II patients, the TAPPS score also significantly predicted survival in the Stage II patient (n=126) cohort [HR=1.75 (95%CI: 1.22-2.52); p=0.002] suggesting the potential of using the TAPPS score to assess overall risk in CRC patients, and specifically in Stage II patients.
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Project description:Chimeric antigen receptor (CAR) T cell therapy is a promising clinical approach for reducing tumor progression and prolonging patient survival. However, improvements in both the safety and the potency of CAR T cell therapy demand quantitative imaging techniques to determine the distribution of cells after adoptive transfer. The purpose of this study was to optimize 89Zr-oxine labeling of CAR T cells and evaluate PET as a platform for imaging adoptively transferred CAR T cells. Methods: CAR T cells were labeled with 0-1.4 MBq of 89Zr-oxine per 106 cells and assessed for radioactivity retention, viability, and functionality. In vivo trafficking of 89Zr-oxine-labeled CAR T cells was evaluated in 2 murine xenograft tumor models: glioblastoma brain tumors with intracranially delivered IL13R?2-targeted CAR T cells, and subcutaneous prostate tumors with intravenously delivered prostate stem cell antigen (PSCA)-targeted CAR T cells. Results: CAR T cells were efficiently labeled (75%) and retained more than 60% of the 89Zr over 6 d. In vitro cytokine production, migration, and tumor cytotoxicity, as well as in vivo antitumor activity, were not significantly reduced when labeled with 70 kBq/106 cells. IL13R?2-CAR T cells delivered intraventricularly were detectable by PET for at least 6 d throughout the central nervous system and within intracranial tumors. When intravenously administered, PSCA-CAR T cells also showed tumor tropism, with a 9-fold greater tumor-to-muscle ratio than for CAR-negative T cells. Conclusion: 89Zr-oxine can be used for labeling and imaging CAR T cells while maintaining cell viability and function. On the basis of these studies, we conclude that 89Zr-oxine is a clinically translatable platform for real-time assessment of cell therapies.
| S-EPMC6167529 | biostudies-literature
Project description:UnlabelledHepatocellular carcinoma (HCC) is an increasingly lethal malignancy for which management is critically dependent on accurate imaging. Glypican-3 (GPC3) is a cell surface receptor overexpressed in most HCCs and provides a unique target for molecular diagnostics. The use of monoclonal antibodies (mAbs) that target GPC3 (αGPC3) in PET imaging has shown promise but comes with inherent limitations associated with mAbs such as long circulation times. This study used (89)Zr-conjugated F(ab')2 fragments directed against GPC3 ((89)Zr-αGPC3-F(ab')2) to evaluate the feasibility of the fragments as a diagnostic immuno-PET imaging probe.MethodsImmobilized ficin was used to digest αGPC3, creating αGPC3-F(ab')2 fragments subsequently conjugated to (89)Zr. In vivo biodistribution and PET studies were performed on GPC3-expressing HepG2 and GPC3-nonexpressing RH7777 orthotopic xenografts.ResultsReliable αGPC3-F(ab')2 production via immobilized ficin digestion was verified by high-performance liquid chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis. (89)Zr-αGPC3-F(ab')2 demonstrated F(ab')2-dependent, antigen-specific cell binding. HepG2 tumor uptake was higher than any other tissue, peaking at 100 ± 21 percentage injected dose per gram (%ID/g) 24 h after injection, a value 33- to 38-fold higher than GPC3-nonexpressing RH7777 tumors. The blood half-life of the (89)Zr-αGPC3-F(ab')2 conjugate was approximately 11 h, compared with approximately 115 h for historic mAb controls. This shorter half-life enabled clear tumor visualization on PET 4 h after administration, with a resultant peak tumor-to-liver contrast ratio of 23.3. Blocking antigen-expressing tumors with an excess of nonradiolabeled αGPC3 resulted in decreased tumor uptake similar to native liver. The kidneys exhibited high tissue uptake, peaking at 24 h with 83 ± 12 %ID/g. HepG2 tumors ranging from 1.5 to 7 mm were clearly visible on PET, whereas larger RH7777 tumors displayed signal lower than background liver tissue.ConclusionThis study demonstrates the feasibility of using (89)Zr-αGPC3-F(ab')2 for intrahepatic tumor localization with small-animal PET. Faster blood clearance and lower background liver uptake enable excellent signal-to-noise ratios at early time points. Increased renal uptake is similar to that as has been seen with clinical radioactive peptide imaging. (89)Zr-αGPC3-F(ab')2 addresses some of the shortcomings of whole-antibody immuno-PET probes. Further optimization is warranted to maximize probe sensitivity and specificity in the process of clinical translation.
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