AMG 110, a bispecific T cell engager (BiTE) antibody construct, induces T cell-mediated cancer cell death by cross-linking epithelial cell adhesion molecule (EpCAM) on tumor cells with a cluster of differentiation 3 ε (CD3ε) on T cells. We labeled AMG 110 with 89 Zr or nearinfrared fluorescent dye (IRDye) 800CW to study its tumor targeting and tissue distribution. Methods: Biodistribution and tumor uptake of 89 Zr-AMG 110 was studied up to 6 d after intravenous administration to nude BALB/c mice bearing high EpCAM-expressing HT-29 colorectal cancer xenografts. Tumor uptake of 89 Zr-AMG 110 was compared with uptake in head and neck squamous cell cancer FaDu (intermediate EpCAM) and promyelocytic leukemia HL60 (EpCAM-negative) xenografts. Intratumoral distribution in HT-29 tumors was studied using 800CW-AMG 110. Results: Tumor uptake of 89 Zr-AMG 110 can be clearly visualized using small-animal PET imaging up to 72 h after injection. The highest tumor uptake of 89 Zr-AMG 110 at the 40-μg dose level was observed at 6 and 24 h (respectively, 5.35 ± 0.22 and 5.30 ± 0.20 percentage injected dose per gram; n 5 3 and 4). Tumor uptake of 89 Zr-AMG 110 was EpCAM-specific and correlated with EpCAM expression. 800CW-AMG 110 accumulated at the tumor cell surface in viable EpCAM-expressing tumor tissue. Conclusion: PET and fluorescent imaging provided real-time information about AMG 110 distribution and tumor uptake in vivo. Our data support using 89 Zr and IRDye 800CW to evaluate tumor and tissue uptake kinetics of bispecific T cell engager antibody constructs in preclinical and clinical settings.
The human epidermal growth factor receptor 3 (HER3) is an interesting target for antitumor therapy. For optimal HER3 signaling inhibition, a biparatopic Nanobody construct (MSB0010853) was developed that binds 2 different HER3 epitopes. In addition, MSB0010853 contains a third HER3 epitope that binds albumin to extend its circulation time. MSB0010853 is cross-reactive with HER3 and albumin of mouse origin. We aimed to gain insight into MSB0010853 biodistribution and tumor uptake by radiolabeling the Nanobody construct with 89 Zr. Methods: MSB0010853 was radiolabeled with 89 Zr. Dose-and time-dependent tumor uptake was studied in nude BALB/c mice bearing a subcutaneous HER3 overexpressing H441 non-small cell lung cancer xenograft. Dose-dependent biodistribution of 89 Zr-MSB0010853 was assessed ex vivo at 24 h after intravenous injection. Protein doses of 5, 10, 25, 100, and 1,000 mg were used. Time-dependent biodistribution of MSB0010853 was analyzed ex vivo at 3, 6, 24, and 96 h after intravenous administration of 25 mg of 89 Zr-MSB0010853. PET imaging and biodistribution were performed 24 h after administration of 25 mg of 89 Zr-MSB0010853 to mice bearing human H441, FaDu (high HER3 expression), or Calu-1 (no HER3 expression) tumor xenografts. Results: Radiolabeling of MSB0010853 with 89 Zr was performed with a radiochemical purity of greater than 95%. Ex vivo biodistribution showed protein dose-and time-dependent distribution of 89 Zr-MSB0010853 in all organs. Uptake of 89 Zr-MSB0010853 in H441 tumors was only time-dependent. Tumor could be visualized up to at least 96 h after injection. The highest mean SUV of 0.6 6 0.2 was observed at 24 h after injection of 25 mg of 89 Zr-MSB0010853. 89 Zr-MSB0010853 tumor uptake correlated with HER3 expression and was highest in H441 (6.2 6 1.1 percentage injected dose per gram [%ID/g]) and lowest in Calu-1 (2.3 6 0.3 %ID/g) xenografts. Conclusion: 89 Zr-MSB0010853 organ distribution and tumor uptake in mice are time-dependent, and tumor uptake correlates with HER3 expression. In contrast to tumor uptake except for kidney uptake, organ distribution of 89 Zr-MSB0010853 is protein dose-dependent for the tested doses. 89 Zr-MSB0010853 PET imaging gives insight into the in vivo behavior of MSB0010853.
Background: AMG 211 is a novel, bispecific single-chain antibody of the bispecific T-cell engager (BiTE® antibody construct) class. AMG 211 targets carcinoembryonic antigen (CEA) on tumor cells and the CD3 epsilon subunit of the human T-cell receptor complex on T-cells. CEA is a glycosylated human oncofetal antigen and is abundantly expressed by a variety of tumors; especially those in the gastrointestinal tract. We radiolabeled AMG 211 with zirconium-89 (89Zr) to study the tumor targeting and tissue distribution of AMG 211 in preclinical mouse tumor xenograft models. Material and Methods: AMG 211 was conjugated with desferal for 89Zr labeling and quality control was performed. A tracer amount of 10 μg (5 MBq) 89Zr-AMG 211 was administered to mice bearing subcutaneously-implanted CEA-expressing LS174T human colorectal adenocarcinoma. MicroPET imaging was performed at 0.5, 3, 6, 24, 48 and 72 h after tracer injection. A dose-escalation biodistribution study was performed 6 h after injection of 2, 10, 50, 100 or 500 μg 89Zr-AMG 211. 89Zr-AMG 211 was also evaluated in a CEA-negative HL60 promyelocytic leukemia xenograft. Furthermore, the non-CEA-binding BiTE® antibody construct, MEC 14, served as a negative control in LS174T tumors. Ex vivo gel electrophoresis and autoradiography was used to determine 89Zr-AMG 211 integrity in plasma and tumor lysate. Results: In vitro analysis of desferal-conjugated AMG 211 showed similar CEA binding as unlabeled AMG 211. Radiochemical purity of 89Zr-AMG 211 exceeded 95%, with a maximum specific activity of 500 MBq/mg. Size-exclusion high-performance liquid chromatography showed no degradation products and less than 5% of aggregates. MicroPET imaging revealed time-dependent tumor uptake of 89Zr-AMG 211 in LS174T bearing mice with the highest uptake 3 h after injection and a prolonged imageable tumor retention up to 48 h. 89Zr-AMG 211 rapidly decreased in blood (T1/2 = 1 h). Dose-escalation showed a dose-dependent tumor uptake 6 hours after injection with the highest tumor uptake observed with 2 μg (7.5 ± 1.5%ID/g) and the lowest tumor uptake with 500 μg (3.9 ± 0.1%ID/g). Mice bearing CEA-positive LS174T versus CEA-negative HL60 tumors showed higher tumor uptake (6.0 ± 1.3 vs 0.5 ± 0.1%ID/g) and tumor-to-blood ratios (21.0 ± 4.0 vs 1.6 ± 0.2) after injection of 10 μg 89Zr-AMG 211. In addition, the non-specific 89Zr-MEC 14 showed low tumor (0.5 ± 0.2%ID/g) uptake and tumor-to-blood ratio (5.5 ± 2.1) that is similar to tissue background in the LS174T model. Ex vivo autoradiography showed intact 89Zr-AMG 211 in all plasma samples. Moreover, in line with biodistribution data, intact 89Zr-AMG 211 was present only in the LS174T xenograft lysates and not detected in HL60 lysate. 89Zr-MEC14 did not bind CEA and was not detected in LS174T lysates. Conclusions: This study showed dose-dependent CEA-specific targeting of 89Zr-AMG 211. In addition, intact labeled AMG 211 was present in plasma and LS174T tumor lysates indicating in vivo tracer integrity. Our data support the use of 89Zr-ImmunoPET in the clinical setting to assess the distribution and tumor-targeting properties of AMG 211 and BiTE® antibody constructs. Citation Format: S.J.H. Waaijer, F.J. Warnders, M.N. Lub-de Hooge, S. Stienen, M. Friedrich, A. Sternjak, P.C. Pieslor, K. Cheung, A.G.T. Terwisscha van Scheltinga, C.P. Schröder, E.G.E. de Vries. Preclinical evaluation of the radiolabeled bispecific T-cell engager 89Zr-AMG 211 targeting CEA-positive tumors. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A85.
AMG 211, a bispecific T-cell engager (BiTE) antibody construct, targets carcinoembryonic antigen (CEA) and the CD3 epsilon subunit of the human T-cell receptor. AMG 211 was labeled with zirconium-89 (Zr) or fluorescent dye to evaluate the tumor-targeting properties. Zr-AMG211 was administered to mice bearing CEA-positive xenograft tumors of LS174T colorectal adenocarcinoma or BT474 breast cancer cells, as well as CEA-negative HL-60 promyelocytic leukemia xenografts. Biodistribution studies with 2- to 10-μgZr-AMG211 supplemented with unlabeled AMG 211 up to 500-μg protein dose were performed. A BiTE that does not bind CEA, Zr-Mec14, served as a negative control.Zr-AMG211 integrity was determined in tumor lysates Intratumoral distribution was studied with IRDye800CW-AMG211. Moreover,Zr-AMG211 was manufactured according to Good Manufacturing Practice (GMP) guidelines for clinical trial NCT02760199 Zr-AMG211 demonstrated dose-dependent tumor uptake at 6 hours. The highest tumor uptake was observed with a 2-μg dose, and the lowest tumor uptake was observed with a 500-μg dose. After 24 hours, higher uptake of 10-μgZr-AMG211 occurred in CEA-positive xenografts, compared with CEA-negative xenografts. Although the blood half-life of Zr-AMG211 was approximately 1 hour, tumor retention persisted for at least 24 hours.Zr-Mec14 showed no tumor accumulation beyond background level. autoradiography revealed time-dependent disintegration ofZr-AMG211. 800CW-AMG211 was specifically localized in CEA-expressing viable tumor tissue. GMP-manufactured Zr-AMG211 fulfilled release specifications. Zr-AMG211 showed dose-dependent CEA-specific tumor targeting and localization in viable tumor tissue. Our data enabled its use to clinically evaluate AMG 211 behavior. .
<p>Supplementary Figures S1-S4 and Supplementary Table S1 including Supplementary Figure legend and Supplementary Table caption Figure S1. Flow chart of the drug substance (N-sucDf-AMG211) manufacturing process and drug product (89Zr-AMG211) formulation and filling process. Figure S2. Quality control of 89Zr AMG211. Representative size exclusion high performance liquid chromatography chromatogram of 89Zr AMG211 with 280 nm signal (top panel), radiochemical signal (middle panel) and the overlay (lower panel). Figure S3. Immunoreactivity of 89Zr AMG211. A) Representative competition assay using an effective N sucDf:AMG 211 ratio of 2:1. Curve fit with 95% confidence interval is visualized. B) Immunoreactivity towards CEA of different ratios N sucDf:AMG 211. Data are mean {plus minus} SD. Figure S4. Membrane binding and internalization of 89Zr AMG211 after CEA binding on LS174T cells (n = 3). Membrane bound and internalized 89Zr-AMG211 are expressed as percentage of initial cell associated activity. Data are mean {plus minus} SD. Figure S5. Expression of CEA on LS174T, BT474 and HL 60 cell lines (n = 3). Membrane expression is expressed as percentage of LS174T signal. Data are mean {plus minus} SD Table S1. GMP manufacturing of N sucDf AMG211 and 89Zr AMG211. Release criteria are fulfilled for batch 1, 2, and 3. In addition stability are shown for N sucDf AMG211 stored at 80{degree sign}C for 6 months, all quality criteria are still met.</p>
<div>Abstract<p><b>Purpose:</b> AMG 211, a bispecific T-cell engager (BiTE) antibody construct, targets carcinoembryonic antigen (CEA) and the CD3 epsilon subunit of the human T-cell receptor. AMG 211 was labeled with zirconium-89 (<sup>89</sup>Zr) or fluorescent dye to evaluate the tumor-targeting properties.</p><p><b>Experimental Design:</b> <sup>89</sup>Zr-AMG211 was administered to mice bearing CEA-positive xenograft tumors of LS174T colorectal adenocarcinoma or BT474 breast cancer cells, as well as CEA-negative HL-60 promyelocytic leukemia xenografts. Biodistribution studies with 2- to 10-μg <sup>89</sup>Zr-AMG211 supplemented with unlabeled AMG 211 up to 500-μg protein dose were performed. A BiTE that does not bind CEA, <sup>89</sup>Zr-Mec14, served as a negative control. <sup>89</sup>Zr-AMG211 integrity was determined in tumor lysates <i>ex vivo</i>. Intratumoral distribution was studied with IRDye800CW-AMG211. Moreover, <sup>89</sup>Zr-AMG211 was manufactured according to Good Manufacturing Practice (GMP) guidelines for clinical trial NCT02760199.</p><p><b>Results:</b> <sup>89</sup>Zr-AMG211 demonstrated dose-dependent tumor uptake at 6 hours. The highest tumor uptake was observed with a 2-μg dose, and the lowest tumor uptake was observed with a 500-μg dose. After 24 hours, higher uptake of 10-μg <sup>89</sup>Zr-AMG211 occurred in CEA-positive xenografts, compared with CEA-negative xenografts. Although the blood half-life of <sup>89</sup>Zr-AMG211 was approximately 1 hour, tumor retention persisted for at least 24 hours. <sup>89</sup>Zr-Mec14 showed no tumor accumulation beyond background level. <i>Ex vivo</i> autoradiography revealed time-dependent disintegration of <sup>89</sup>Zr-AMG211. 800CW-AMG211 was specifically localized in CEA-expressing viable tumor tissue. GMP-manufactured <sup>89</sup>Zr-AMG211 fulfilled release specifications.</p><p><b>Conclusions:</b> <sup>89</sup>Zr-AMG211 showed dose-dependent CEA-specific tumor targeting and localization in viable tumor tissue. Our data enabled its use to clinically evaluate AMG 211 <i>in vivo</i> behavior. <i>Clin Cancer Res; 24(20); 4988–96. ©2018 AACR</i>.</p></div>
<p>Supplementary Figures S1-S4 and Supplementary Table S1 including Supplementary Figure legend and Supplementary Table caption Figure S1. Flow chart of the drug substance (N-sucDf-AMG211) manufacturing process and drug product (89Zr-AMG211) formulation and filling process. Figure S2. Quality control of 89Zr AMG211. Representative size exclusion high performance liquid chromatography chromatogram of 89Zr AMG211 with 280 nm signal (top panel), radiochemical signal (middle panel) and the overlay (lower panel). Figure S3. Immunoreactivity of 89Zr AMG211. A) Representative competition assay using an effective N sucDf:AMG 211 ratio of 2:1. Curve fit with 95% confidence interval is visualized. B) Immunoreactivity towards CEA of different ratios N sucDf:AMG 211. Data are mean {plus minus} SD. Figure S4. Membrane binding and internalization of 89Zr AMG211 after CEA binding on LS174T cells (n = 3). Membrane bound and internalized 89Zr-AMG211 are expressed as percentage of initial cell associated activity. Data are mean {plus minus} SD. Figure S5. Expression of CEA on LS174T, BT474 and HL 60 cell lines (n = 3). Membrane expression is expressed as percentage of LS174T signal. Data are mean {plus minus} SD Table S1. GMP manufacturing of N sucDf AMG211 and 89Zr AMG211. Release criteria are fulfilled for batch 1, 2, and 3. In addition stability are shown for N sucDf AMG211 stored at 80{degree sign}C for 6 months, all quality criteria are still met.</p>
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