Advancing ⁸⁹Zr-immuno-PET in neuroscience with a bispecific anti-amyloid-beta monoclonal antibody - The choice of chelator is essential

Abstract

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. 89 Zr-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 89 Zr-labeling strategies and direct radioiodination with 125 I 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 89 Zr. 125 I was used as a substitute for 124 I for labeling of both antibodies. 30 µg of radiolabeled mAb, corresponding to approximately 6 MBq 89 Zr or 2.5 MBq 125 I, 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 [ 89 Zr]Zr-DFO*-NCS-Adu-8D3 (2.19 ±0.12 %ID/g) was as high as for its 125 I-analog (2.21 ±0.15 %ID/g). [ 89 Zr]Zr-DFO-NCS-Adu-8D3 and [ 89 Zr]Zr-DFO- N -suc-Adu-8D3 showed significantly lower uptake (< 0.65 %ID/g), being in the same range as for the 89 Zr-labeled controls (B12-8D3). Autoradiography of [ 89 Zr]Zr-DFO*-NCS-Adu-8D3 and [ 125 I]I-Adu-8D3 showed an amyloid-beta related granular uptake pattern of radioactivity. In contrast, the [ 89 Zr]Zr-DFO-conjugates and the control antibody groups did not show any amyloid-beta related uptake pattern, indicating that DFO is inferior for 89 Zr-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 [ 89 Zr]Zr-DFO*-NCS-Adu-8D3. In wild type animals, such an uptake wa...

Indium-111 radiolabelling of a brain-penetrant Aβ antibody for SPECT imaging

“…Previous research has also demonstrated high brain uptake of 111 In-labelled TfR targeting antibodies in wild-type mice ( 29 ). In addition, Aβ/TfR bispecific antibodies radiolabelled with zirconium-89 have also demonstrated a relatively high background signal in wild-type mice and a smaller difference in brain retention between Aβ-expressing and wild-type mice compared to radioiodinated versions ( 30 , 31 ). Multiple mechanisms could explain the prolonged retention of 111 In (and other radiometals) in the wild-type mouse brain.…”

Development of brain-penetrable antibody radioligands for in vivo PET imaging of amyloid-β and tau

“…Our ex vivo imaging study using the radiolabeled bispecific antibody ligands in two different mouse models with Aβ pathology visualized Aβ in the brain. The PET signal increases with age and closely correlates with brain Aβ levels, demonstrating that bispecific antibody PET ligands can be successfully used for the brain imaging of Aβ pathology ( 8 – 10 ). We have also previously reported on the diagnostic imaging potential of the tau ligands 6B2G12 and scFv235 in tauopathy mice, e.g., in vivo imaging system (IVIS) imaging, in which they selectively detect pathological tau lesions in vivo ( 11 ).…”

Good practices for 89Zr radiopharmaceutical production and quality control

“…Sample preparation time, however, can be extensive since deglycosylation, buffer exchange, and salt removal may be necessary in the case of MALDI-TOF. In addition, when higher p -NCS-Bz-DFO/DFO*-to-mAb ratios are reached, some MS methods can become inaccurate or impaired (Chomet et al 2021 ; Wuensche et al 2022 ). An alternative to MS methods is a titration with carrier-added [ 89 Zr]Zr-oxalate (known amounts of non-radioactive Zr-oxalate with trace amounts of [ 89 Zr]Zr-oxalate) (Vugts et al 2017 ).…”