作者: Wuensche, Thomas E ; Jensen, Allan ; Mes, Iris ; Beaino, Wissam ; Windhorst, Albert D ; Verlaan, Mariska ; Vugts, Danielle J ; Bang-Andersen, Benny ; Stergiou, Natascha ; Janssen, Bart ; Kooijman, Esther J M ; Dongen, Guus A M S ; Asuni, Ayodeji A ; Schreurs, Maxime

The accelerated approval of the monoclonal antibody (mAb) aducanumab as a treatment option for Alzheimer's Disease and the continued discussions about its efficacy have shown that a better understanding of immunotherapy for the treatment of neurodegenerative diseases is needed. ⁸⁹Zr-immuno-PET could be a suitable tool to open new avenues for the diagnosis of CNS disorders, monitoring disease progression, and assessment of novel therapeutics. Herein, three different ⁸⁹Zr-labeling strategies and direct radioiodination with ¹²⁵I of a bispecific anti-amyloid-beta aducanumab derivate, consisting of aducanumab with a C-terminal fused anti-transferrin receptor binding single chain Fab fragment derived from 8D3 (Adu-8D3), were compared ex vivo and in vivo with regard to brain uptake and target engagement in an APP/PS1 Alzheimer's disease mouse model and wild type animals. Methods: Adu-8D3 and a negative control antibody, based on the HIV specific B12 antibody also carrying C-terminal fused 8D3 scFab (B12-8D3), were each conjugated with NCS-DFO, NCS-DFO*, or TFP-N-suc-DFO-Fe-ester, followed by radiolabeling with ⁸⁹Zr. ¹²⁵I was used as a substitute for ¹²⁴I for labeling of both antibodies. 30 µg of radiolabeled mAb, corresponding to approximately 6 MBq ⁸⁹Zr or 2.5 MBq ¹²⁵I, were injected per mouse. PET imaging was performed 1, 3 and 7 days post injection (p.i.). All mice were sacrificed on day 7 p.i. and subjected to ex vivo biodistribution and brain autoradiography. Immunostaining on brain tissue was performed after autoradiography for further validation. Results: Ex vivo biodistribution revealed that the brain uptake of [⁸⁹Zr]Zr-DFO*-NCS-Adu-8D3 (2.19 ±0.12 %ID/g) was as high as for its ¹²⁵I-analog (2.21 ±0.15 %ID/g). [⁸⁹Zr]Zr-DFO-NCS-Adu-8D3 and [⁸⁹Zr]Zr-DFO-N-suc-Adu-8D3 showed significantly lower uptake (< 0.65 %ID/g), being in the same range as for the ⁸⁹Zr-labeled controls (B12-8D3). Autoradiography of [⁸⁹Zr]Zr-DFO*-NCS-Adu-8D3 and [¹²⁵I]I-Adu-8D3 showed an amyloid-beta related granular uptake pattern of radioactivity. In contrast, the [⁸⁹Zr]Zr-DFO-conjugates and the control antibody groups did not show any amyloid-beta related uptake pattern, indicating that DFO is inferior for ⁸⁹Zr-immuno-PET imaging of the brain in comparison to DFO* for Adu-8D3. This was confirmed by day 7 PET images showing only amyloid-beta related brain uptake for [⁸⁹Zr]Zr-DFO*-NCS-Adu-8D3. In wild type animals, such an uptake was not observed. Immunostaining showed a co-localization of all administered Adu-8D3 conjugates with amyloid-beta plaques. Conclusion: We successfully demonstrated that ⁸⁹Zr-immuno-PET is suitable for imaging and quantifying amyloid-beta specific brain uptake using a bispecific aducanumab brain shuttling antibody, Adu-8D3, but only when using the novel chelator DFO*, and not DFO, for labeling with ⁸⁹Zr.

2018-06-04·Molecular Pharmaceutics2区 · 医学

Evaluation of Anti-LGR5 Antibodies by ImmunoPET for Imaging Colorectal Tumors and Development of Antibody–Drug Conjugates

2区 · 医学

Article

作者: Hernandez Vargas, Servando ; Azhdarinia, Ali ; Simien, Jo A. ; Ghosh, Sukhen C. ; Liu, Qingyun ; Yu, Wangsheng A. ; Carmon, Kendra S. ; Voss, Julie ; Cui, Jie

Leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) is highly expressed in colorectal tumors and marks colon cancer stem cells that drive tumor growth and metastasis. Recently, we showed that LGR5 is a promising target for antibody-drug conjugate (ADC) therapy. However, it is important to identify LGR5-positive tumors that would respond to ADC treatment. Prior to drug conjugation, we evaluated two different anti-LGR5 monoclonal antibodies (mAbs), 8F2 and 9G5, using ⁸⁹Zr-immunoPET to select the optimal mAb for ADC development and tumor imaging. Binding, specificity, and internalization were compared, and mAbs were prescreened as ADC candidates against colon cancer cells using secondary ADCs. Both mAbs demonstrated strong, specific binding in 293T-LGR5 cells but not 293T-vector cells. In DLD-1 colorectal cancer cells, which express high levels of LGR5, the mAbs rapidly internalized into lysosomes and promoted ADC-induced cytotoxicity, with 8F2 exhibiting slightly higher potency. No binding was detected in DLD-1-shLGR5 (LGR5 knockdown) cells. ⁸⁹Zr-DFO-LGR5 mAbs were generated and shown to retain high affinity and LGR5-dependent uptake in vitro. PET/CT imaging of DLD-1 tumors was performed 5 days postinjection of ⁸⁹Zr-DFO-LGR5 mAbs, and findings were consistent with biodistribution data, which showed significantly higher tumor uptake (%ID/g) for ⁸⁹Zr-DFO-8F2 (17.9 ± 2.2) compared to ⁸⁹Zr-DFO-9G5 (5.5 ± 1.2) and ⁸⁹Zr-DFO-IgG (3.8 ± 1.0). No significant uptake was observed in DLD-1-shLGR5 tumors. This study identifies 8F2 as the optimal candidate for ADC development and provides initial evidence that ⁸⁹Zr-DFO-LGR5 mAbs may be utilized to stratify tumors which would respond best to LGR5-targeted ADC therapy.

International Journal of Molecular Sciences2区 · 生物学

Biodistribution PET/CT Study of Hemoglobin-DFO-89Zr Complex in Healthy and Lung Tumor-Bearing Mice

2区 · 生物学

ArticleOA

作者: Savino, Carmelinda ; Sas, Zuzanna ; Król, Magdalena ; Braniewska, Agata ; Kucharzewska, Paulina ; Rygiel, Tomasz P. ; Taciak, Bartłomiej ; Clarkson, Richard ; Strzemecki, Damian ; Boffi, Alberto ; Bonamore, Alessandra ; Kiraga, Łukasz ; Montemiglio, Linda Celeste ; Paisey, Stephen J. ; Seaton, Gillian ; Turnham, Daniel ; Cerutti, Gabriele ; Dabkowski, Adam M.

Proteins, as a major component of organisms, are considered the preferred biomaterials for drug delivery vehicles. Hemoglobin (Hb) has been recently rediscovered as a potential drug carrier, but its use for biomedical applications still lacks extensive investigation. To further explore the possibility of utilizing Hb as a potential tumor targeting drug carrier, we examined and compared the biodistribution of Hb in healthy and lung tumor-bearing mice, using for the first time 89Zr labelled Hb in a positron emission tomography (PET) measurement. Hb displays a very high conjugation yield in its fast and selective reaction with the maleimide-deferoxamine (DFO) bifunctional chelator. The high-resolution X-ray structure of the Hb-DFO complex demonstrated that cysteine β93 is the sole attachment moiety to the αβ-protomer of Hb. The Hb-DFO complex shows quantitative uptake of 89Zr in solution as determined by radiochromatography. Injection of 0.03 mg of Hb-DFO-89Zr complex in healthy mice indicates very high radioactivity in liver, followed by spleen and lungs, whereas a threefold increased dosage results in intensification of PET signal in kidneys and decreased signal in liver and spleen. No difference in biodistribution pattern is observed between naïve and tumor-bearing mice. Interestingly, the liver Hb uptake did not decrease upon clodronate-mediated macrophage depletion, indicating that other immune cells contribute to Hb clearance. This finding is of particular interest for rapidly developing clinical immunology and projects aiming to target, label or specifically deliver agents to immune cells.

100 项与 89Zr-DFO*-Adu-8D3 相关的药物交易

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