Summary

This study developed copper-64-labeled PET tracers to assess TREM2 expression for monitoring microglia activation in neurological diseases. Using an antibody transport vehicle (ATV) that binds to the human transferrin receptor (hTfR), the researchers improved brain uptake of the radiotracers, especially in microglia-rich regions. The enhanced brain penetration makes [64Cu]Cu-NODAGA-ATV:4D9 a promising tool for non-invasive imaging of TREM2, with potential applications in Alzheimer’s disease, glioma, and chronic inflammation.

Neurodegenerative diseases (NDDs) involve the gradual loss of neurons in the CNS, leading to cognitive and motor dysfunction, with conditions like Alzheimer's and Parkinson's linked to amyloid protein accumulation and neuroinflammation. Microglia, the brain's resident immune cells, transition from a homeostatic to disease-associated state (DAM) in response to amyloid pathology, with TREM2 playing a key role in their activation. PET imaging of TREM2, a more specific microglia marker than TSPO, could enable non-invasive monitoring of microglial activation, especially in Alzheimer's disease, but challenges exist in delivering tracers across the blood-brain barrier (BBB). This study developed copper-64-labeled TREM2-targeting PET tracers, enhanced with an antibody transport vehicle (ATV) to cross the BBB, enabling the imaging of TREM2 in an AD mouse model.

Materials and Methods

Experimental details, including patient tissues, assays, and imaging data, are available in the supplemental materials. Animal studies followed German regulations and used mouse cohorts from Jackson Laboratory and Charles River, housed in ventilated cages. Cohorts included 5xFAD, wild-type (WT), and human transferrin receptor (TfR) expressing mice. Antibodies were modified with p-NCS-benzyl-NODAGA, purified, and validated using HPLC and a spectrometric arsenazo assay.

For radiolabeling, NODAGA-conjugated antibodies were incubated with [64Cu]CuCl2, purified, and analyzed by radio-TLC and HPLC. Autoradiography involved incubating mouse brain sections with [64Cu]Cu-NODAGA-ATV:4D9, with excess unlabeled antibody for specificity. Sections were processed and analyzed using imaging software with reference regions for normalization. Human brain sections followed a similar process, using white matter as the reference.

Small animal PET

Mice from cohorts 1 and 2 were injected intravenously with either [64Cu]Cu-NODAGA-4D9 (13.1 ± 3.2 MBq) or [64Cu]Cu-NODAGA-ATV:4D9 (10.2 ± 1.3 MBq), and PET scans (Mediso Nanoscan PET/CT, 70 kvp/650 µA, exposure time 300 ms, Helical 1.0 pitch, with coincidence mode 1-5 in 1 scan position) were conducted at 2, 20, and 40 hours post-injection with. Images were analyzed using PMOD software, aligned to an MRI brain atlas, and tracer uptake was calculated as %ID/g in the hippocampus and cortex. PET-to-biodistribution correlation was assessed via voxel-wise SPM analysis, with p < 0.05 considered significant. Mice from cohort 3 received 38.1 ± 1.2 MBq [64Cu]Cu-NODAGA-ATV:4D9, and microglial uptake was evaluated using scRadiotracing.

Results with nanoScan PET/CT

NODAGA-modified 4D9 and ATV:4D9 antibodies were successfully labeled with [64Cu], yielding highly stable radiotracers with high radiochemical purity and specific activity. In vitro autoradiography showed that [64Cu]Cu-NODAGA-ATV:4D9 specifically binds to its target in 5xFAD;TfRmu/hu mouse brain sections, with higher uptake in the cortex and hippocampus compared to wild-type mice. Biodistribution studies revealed that brain uptake of [64Cu]Cu-NODAGA-ATV:4D9 is significantly enhanced due to hTfR-mediated transcytosis, with 80% of the brain signal attributed to hTfR binding.

PET imaging using [64Cu]Cu-labeled 4D9 and ATV:4D9 antibodies showed elevated TREM2 signals in 5xFAD;TfRmu/hu mice compared to WT;TfRmu/hu controls, with higher tracer uptake in the frontal cortex and hippocampus at 20 hours post-injection. This TREM2-dependent signal enhancement correlated with higher TREM2 protein levels in the 5xFAD brain. Although signal-to-noise ratios decreased at 40 hours due to decay, TREM2 PET demonstrated superior %ID/g and SUVR ratios compared to TSPO PET tracers.

Figure 3: TREM2 PET imaging in 5xFAD and wild-type mice. (A) Schematic representation of the PET/CT workflow. (B) Group average PET images of 5xFAD;TfRmu/hu, 5xFAD, WT;TfRmu/hu and WT mouse brains at 20 h p.i. overlaid on an MRI template. Red arrows indicate highest uptake in the frontal cortex and the hippocampus of 5xFAD;TfRmu/hu animals. (C, D) Quantitative tracer uptake (%ID/g) in predefined VOIs of the frontal cortex (CTX) and the hippocampus (HIP). Striped bars illustrate the fraction of hTfR-related binding determined by biodistribution using 64Cu-labeled ATV:ISO (Figure S4). One-way ANOVA/Tukey’s multiple comparison test, p ≤ 0.05 (*), p ≤ 0.01 (**), p ≤ 0.001 (***), and p ≤ 0.0001 (****), mean ± SD.

To minimize skull spill-in affecting PET imaging, we conducted voxel-wise correlation analysis between brain biodistribution and TREM2 PET uptake. The results identified specific cortical regions, particularly the frontal cortex, where tracer uptake in 5xFAD;TfRmu/hu mice was significantly higher than in WT;TfRmu/hu controls at 20 hours post-injection. This time point demonstrated the strongest linear correlation with biodistribution data, while correlations were lower at 2 and 40 hours, confirming 20 hours as the optimal time for TREM2 PET imaging with antibody-labeled tracers.

Figure 4: PET-to-biodistribution associations. (A) Images from voxel-wise regression analysis of biodistribution brain uptake with PET images using statistical parametric mapping (SPM). Color scale shows TREM2 PET voxels with highest correlation to biodistribution in red using all four genotypes. (B) TREM2 PET signals in data-driven cortical cluster VOIs derived from the regression analysis in correlation with brain uptake from biodistribution at 2 h, 20 h and 40 h p.i. (linear regression, α = 0.05, 95% CI), (C) Group comparison of TREM2 PET results in data-driven cortical cluster VOIs across genotypes. One-way ANOVA/Tukey’s multiple comparison test, p ≤ 0.01 (**), p ≤ 0.001 (***), and p ≤ 0.0001 (****), mean ± SD.

Figure 5: Ex vivo autoradiography confirmation of regional TREM2 PET signals. (A) Ex vivo autoradiography of 5xFAD;TfRmu/hu, WT;TfRmu/hu, 5xFAD and WT brain sections at 2 h and 20 h p.i. (B,C) Higher cortex-to-cerebellum ratios were observed in 5xFAD;TfRmu/hu mice compared to WT;TfRmu/hu mice at the 2 h p.i. time point (5xFAD;TfRmu/hu: 2 mice, 22 sections; WT;TfRmu/hu: 2 mice, 15 sections; unpaired t-test, p < 0.0001 (****), boxplot min to max) and the 20 h p.i. time point (5xFAD;TfRmu/hu: 2 mice, 19 sections; WT;TfRmu/hu: 2 mice, 19 sections; unpaired t-test, p < 0.0001 (****), boxplot min to max).

Ex vivo autoradiography was conducted on brain sections from perfused 5xFAD;TfRmu/hu and WT;TfRmu/hu mice at 2 and 20 hours post-injection to validate TREM2 PET imaging patterns. The cortex-to-cerebellum ratios were significantly higher in 5xFAD;TfRmu/hu mice (2.04 ± 0.18 at 2 h and 1.48 ± 0.15 at 20 h) compared to WT controls (1.05 ± 0.24 at 2 h and 1.16 ± 0.12 at 20 h, both p < 0.0001), mirroring PET data trends. Additionally, cell sorting after PET tracer injection in AppSAA;TfRmu/hu mice demonstrated that [64Cu]Cu-NODAGA-ATV:4D9 selectively binds to microglia, showing a significant signal in the microglia-enriched fraction while negligible in the depleted fraction, confirming the specificity of TREM2 PET.

Figure 6: scRadiotracing demonstrates specificity of [64Cu]Cu-NODAGA-ATV:4D9 to microglia. (A) Experimental workflow, including TREM2 PET at 20 h p.i., brain dissociation and cell sorting as well as flow cytometry and gamma emission recording to calculate radioactivity per cell (microglia = turquoise, astrocyte = pink, neuron = yellow, oligodendrocyte = gray). (B) TREM2 PET results are shown as axial (frontal cortex, hippocampus) and sagittal (forebrain, hindbrain) regional difference maps (n = 4 AppSAA;TfRmu/hu mice compared to n = 5 WT;TfRmu/hu of Fig.3) projected upon an MRI template. (C, D) Flow cytometry indicates high purity of CD11b-positive cells in microglia-enriched fractions and absence of CD11b-positive cells in the microglia-depleted fractions of n = 4 individual AppSAA;TfRmu/hu mice. Data are shown as mean fluorescence intensity. (E) Relative cellular abundance of microglial cells in AppSAA;TfRmu/hu (n = 4) mice compared to WT (n = 12) mice. Unpaired t-test, p = 0.0002 (***), mean ± SD. (F) TREM2 radiotracer uptake of microglia-enriched vs microglia-depleted (i.e. mixed fraction of neurons, astrocytes, oligodendrocytes) fractions in AppSAA;TfRmu/hu mice, confirming high specificity to microglia. Unpaired t-test, p < 0.0001 (****), mean ± SD. (G) Estimation of the PET signal percentage which is explained by microglial uptake as a product of microglial abundance and microglial tracer uptake per cell. Based on (E) and 7.4 × 106 microglia in WT brains taken from literature, we calculated 24.7 × 106 microglia in AppSAA;TfRmu/hu brains.

Figure 7: [64Cu]Cu-NODAGA-14D3 ARG signal represents TREM2 IHC signal in Alzheimer’s disease patients. (A) Representative TREM2 immunohistochemistry (IHC) and in vitro autoradiography (ARG) of frontal brain sections derived from a patient with Alzheimer’s disease (AD) revealed cortical binding of [64Cu]Cu-NODAGA-14D3. TREM2 IHC and tracer binding in ARG co-localized. Autoradiography of a blocked brain slice demonstrated a negligible signal. (B) Cortex-to-white matter ratios were consistent in IHC and ARG and significantly higher than in blocked ARG (One-way ANOVA/Tukey’s multiple comparison test, p ≤ 0.001 (***), boxplot min to max). (C) ARG-blocking resulted in a cortical signal reduction (unpaired t-test, p < 0.0001 (****), boxplot min to max.

They explored the potential translation of our preclinical findings into clinical applications by conducting immunohistochemistry and in vitro autoradiography on human Alzheimer’s disease (AD) brain sections using the anti-human TREM2 antibody 14D3. This antibody was modified with the NODAGA chelator and labeled with copper-64, achieving high radiochemical purity (RCP > 99%). The results showed co-localization of TREM2 immunohistochemistry and [64Cu]Cu-NODAGA-14D3 autoradiography, with enriched binding in the cortex, indicating high regional TREM2 expression linked to AD pathology. The consistency of cortex-to-white matter ratios in both methods and a significant reduction in signal upon blocking further supports the potential of the ATV-enabled 14D3 antibody for future TREM2 PET imaging in clinical settings.

Conclusion

In conclusion, we introduce the first microglia-specific PET radiotracer designed for imaging TREM2-related microglial activation in the central nervous system using a mouse model of Alzheimer's disease (AD). By utilizing ATV technology, we enhanced the tracer's delivery efficiency across the blood-brain barrier. Preliminary experiments with a radiotracer targeting human TREM2 demonstrated promising results through immunohistochemistry and in vitro autoradiography on brain sections from AD patients, providing a solid foundation for translating this research into clinical applications. TREM2 PET imaging holds significant promise for verifying target engagement and monitoring therapy responses in antibody-based TREM2 agonistic treatments.

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