Introduction

Pathological transactive response DNA-binding protein 43 kDa (TDP-43) is the main component of inclusions found in several neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD)1,2, limbic predominant age-related TDP-43 encephalopathy (LATE)3, and found as co-pathology in Alzheimer’s disease (AD)4, dementia with Lewy bodies (DLB)5 and chronic traumatic encephalopathy (CTE)6. The mislocalization from nucleus to cytoplasm and aggregation of TDP-43 in neurons and glial cells drives neurodegeneration, resulting in progressive loss of motor and cognitive functions7,8.

TDP-43 proteinopathy is found postmortem in about 45% of FTD cases, being reported as FTLD-TDP to differentiate from FTLD characterized by accumulation of aggregated Tau (FTLD-Tau) or fused in sarcoma (FUS)–Ewing sarcoma–TAF15 family (FTLD-FUS or FTLD-FET) proteins2,9,10,11. Clinically, FTD represents a heterogeneous spectrum characterized by changes in behavior, personality, and language12. The three major FTD phenotypes, behavior variant (bvFTD), semantic variant primary progressive aphasia (svPPA), and the non-fluent agrammatic primary progressive aphasia (nfPPA), are each associated with the involvement of distinct parts of the frontal and temporal lobes. The availability of reliable biomarkers to support early differential diagnosis of FTD would fill a large knowledge gap.

ALS is a progressive, fatal motor neuron disease (MND) characterized by neuronal death leading to the loss of motor function, with an average life expectancy of 2–5 years after diagnosis13. In ALS, TDP-43 proteinopathy is found in approximately 97% of patients, originating in the motor cortex, brainstem, and spinal cord14. ALS and FTD represent a disease spectrum sharing clinical features with up to 50% ALS patients showing some executive function deficits15 and up to 15% of bvFTD patients showing motor dysfunction16, further highlighting the need for reliable biomarkers for accurate diagnosis.

LATE describes an aging-associated disease entity with TDP-43 proteinopathy (LATE-neuropathological changes (NC)) with or without the presence of amyloid plaques and Tau neurofibrillary tangles (AD neuropathological change, ADNC) in the aging brain, which mimics the clinical features of AD3,17. Large autopsy series indicate that LATE-NC affects ~1/3rd of individuals past 85 years of age and is strongly associated with cognitive impairment18,19. While the clinical diagnostic guidelines for LATE are being developed20,21, a definitive diagnosis is currently only possible post-mortem, and development of sensitive molecular-specific TDP-43 biomarkers has been specifically noted as a high priority in the field3.

The histopathology underlying the above-mentioned TDP-43 proteinopathies is described by four main subtypes: FTLD-TDP types A, B, C, and D9,22,23 based on morphology, localization, and association with clinical and genetic variants. Clinically, type A is associated with bvFTD, naPPA, AD23, and LATE24 and found in patients with mutations in progranulin (GRN), C9orf72 and TBK1 genes. Type B is common in FTD-MND and associated with mutations in the C9orf72 and TBK1 genes. Type C is found in svPPA, with no associated genes identified so far. Type D is rare and associated with valosin-containing protein (VCP) mutations. In addition to intracellular morphology and location, distinct folds, seeding capacity, and toxicity for FTLD-TDP type A25, type B26, and type C[27](https://www.nature.com/articles/s41467-025-64540-6#ref-CR27 “Arseni, D. et al. Heteromeric amyloid filaments of ANXA11 and TDP-43 in FTLD-TDP Type C. Nature, https://doi.org/10.1038/s41586-024-08024-5

(2024).“) fibrils have been established28,29, confirming structural and biochemical heterogeneity. Despite a growing understanding of the pathomechanism, sensitive biomarkers for visualization, spatial distribution, and spreading patterns of pathological TDP-43 in the living brain are not available.

Positron emission tomography (PET) is a powerful imaging modality that enables visualization and quantification of molecular pathology in the brain. Significant progress has been made in the development of PET tracers targeting specific misfolded proteins such as amyloid beta (Aβ)30,31, Tau32,33, and a-synuclein34, significantly improving our understanding of neurodegenerative disorders. Aβ and Tau PET tracers provided data unraveling the temporal evolution of AD pathology accumulation in the brain. Indeed, the Aβ PET imaging allowed for the approval of Aβ immunotherapies35,36: Aβ PET positivity was applied for the inclusion of AD subjects in the studies, and a decrease in Aβ PET signal was used as a surrogate biomarker for measuring clinical effect. In addition, employing a Tau PET tracer facilitated patient classification and enrollment in anti-Aβ clinical trials and ultimately enabled the interpretation of their outcomes32,37,38. Beyond use as a diagnostic, prognostic, and staging biomarker, a TDP-43 PET tracer would be utilized for patient selection, proof of target engagement, as well as pharmacodynamic readout, enabling the assessment of efficacy in therapeutic trials in the ALS-FTD spectrum, LATE-NC, or other TDP-43 proteinopathy-related diseases. In addition, it will allow patient stratification in AD, where TDP-43 often appears as an aggravating co-pathology. Also, TDP-43 PET imaging would complement and validate the development of fluid biomarkers, while furthermore providing critical insight into the spatial distribution of pathological TDP-43 in the brain.

In this study, we report the characterization of first-in-class TDP-43 PET tracers, [18F]ACI-19278 and [18F]ACI-19626. Both compounds demonstrate high affinity to aggregated TDP-43, excellent selectivity over other amyloids found as co-pathologies, and a suitable pharmacokinetic profile for PET imaging in the human brain. ACI-19626 was prioritized for first-in-human (FIH) evaluation in healthy volunteers and patients with TDP-43 proteinopathies. Based on the data presented here, both compounds have the potential to detect TDP-43 by PET in the brains of living patients.

Results

[³H]ACI-19278 and [³H]ACI-19626 bind to patient brain-derived pathological TDP-43 aggregates

ACI-19278 and ACI-19626 (Supplementary Fig. 1a) are small molecular weight compounds identified by rational design leveraging the AC Immune Morphomer® platform. This is a large library of CNS-penetrant small molecular weight compounds designed to bind specifically to conformationally altered and beta-sheet-rich structures in protein aggregates commonly found in proteinopathies. Taking advantage of the autofluorescence properties of some compounds available in the library, initial hits were identified using simple target engagement screening assays. Identified hits were then improved through a multi-parameter medicinal chemistry optimization approach, radiolabeled with tritium, and their binding properties were characterized in orthogonal assays using patient-derived material. Using classical autoradiography (ARG), binding of [3H]ACI-19278 and [3H]ACI-19626 was assessed in brain sections obtained from the frontal cortex of FTLD-TDP type A, frontal and temporal cortex of FTLD-TDP type B, frontal and temporal cortex of FTLD-TDP type C, hippocampus of LATE-NC with ADNC (LATE-NC + ADNC), motor cortex of ALS and frontal and temporal cortex of healthy control cases (Fig. 1). Strong binding, substantially above the control tissues, was observed for the samples from FTLD-TDP type A and B, as well as LATE-NC (Fig. 1a, Total). When evaluating the signal in the presence of excess non-labeled compound, displacement was observed for the FTLD-TDP type A and B as well as LATE-NC tissues (Fig. 1a, NSB), indicating specific binding. [3H]ACI-19278 showed significant differentiation of FTLD-TDP type A tissue samples from control tissue, while the specific binding for [3H]ACI-19626 was significantly greater for both FTLD-TDP types A and B compared to that of the control tissues (Fig. 1b).

Fig. 1: Characterization of binding of [³H]ACI-19278 and [³H]ACI-19626 to brain samples from TDP-43 proteinopathies.

a Autoradiographic images of [3H]ACI-19278 and [3H]ACI-19626 binding in brain tissue sections from control (temporal cortex), FTLD-TDP (type A, frontal cortex; type B, temporal cortex), and LATE-NC + ADNC (hippocampus) cases. Total: total binding; Non-specific binding (NSB): residual binding in the presence of 2 μM unlabeled ACI-19278 or ACI-19626. Scale bar, 5 mm. Seven cases were tested for FTLD-TDP type A, five ([3H]ACI-19278) or seven ([3H]ACI-19626) cases for type B, and one case from LATE-NC + ADNC in independent experiments. b Specific binding (for ACI-19278: n = 9 control; n = 7 type A; n = 5 type B; n = 5 type C; n = 1 LATE-NC; n = 5 ALS. For ACI-19626: n = 7 control; n = 7 type A; n = 6 type B; n = 4 type C; n = 1 LATE-NC; n = 3 ALS). Data is shown as mean ± SD. One-way ANOVA with Dunnett’s multiple comparisons test; for ACI-19278 **p = 0.006 for control vs type A; for ACI-19626 *p = 0.01 for control vs type A, and **p = 0.009 for control vs type B. c Representative autoradiograms of [3H]ACI-19626 with higher magnification images of selected areas in adjacent sections following labeling with anti-phospho-TDP-43 antibody. Top row (images 1, 3, and 5), high-density TDP-43 pathology; bottom row (images 2, 4, 6), mid-low density TDP-43 pathology. Scale bar, 20 μm. d Saturation binding studies with [3H]ACI-19278 and [3H]ACI-19626 on human brain tissue sections from an FTLD-TDP type A donor. Data from one experiment. e Saturation binding studies with [3H]ACI-19278 and [3H]ACI-19626 in brain homogenates from control (n = 1), FTLD-TDP type A, type B, and type C cases. Data is shown as mean ± SD for [3H]ACI-19626 on type A (six independent experiments) and for both compounds on type C (three independent experiments). In other cases, representative data from two independent experiments is shown. f Kd values from saturation binding studies in e and Supplementary Fig. 1e. Mean ± SD is reported for FTLD-TDP type A and B across two cases. n.a., no fit or R2 < 0.85. Kd values in panels d and f were assessed by different techniques and on different tissue preparations. Source data are provided as a Source Data file.

Low to no displaceable binding for both compounds was detected in brain sections from FTLD-TDP type C despite abundant phospho-TDP-43 (pTDP-43)-positive dystrophic neurites characteristic of this subtype of FTLD-TDP39 (Supplementary Fig. 1b). When tissue sections from ALS cases were incubated with [3H]ACI-19278 or [3H]ACI-19626, no displaceable signal was detected, possibly due to the very low density of pTDP-43 pathology in ALS tissue (Supplementary Fig. 1c), at least in the motor cortex region tested. Both compounds displayed an ARG signal comparable to that of the control on tissue from FTLD-TDP type C and ALS cases (Fig. 1b). Brain samples used for classical ARG included both grey and white matter (Supplementary Fig. 1e). Some non-specific white matter binding was observed for ACI-19278 whereas ACI-19626 showed no non-specific retention to white matter regions that could affect the interpretation of the tracer binding to TDP-43 inclusions present in the white matter areas.

The binding specificity for TDP-43 aggregates in FTLD-TDP type A and B as well as LATE-NC + ADNC tissue was confirmed by the association of the ARG signal intensity with the distribution and density of TDP-43 pathology across the same tissue, as visualized using anti-pS409-410 labeling (ACI-19626 ARG data and immunofluorescence in adjacent tissue sections in Fig. 1c). A stronger ARG signal was observed in regions dense in pTDP-43 aggregates (Fig. 1c, inserts 1, 3, and 5) while signal was less intense in regions with low levels of TDP-43 pathology (Fig.1c, inserts 2, 4, 6).

To measure affinity to pathological aggregated TDP-43, saturation binding studies were performed with [3H]ACI-19278 and [3H]ACI-19626 using ARG on FTLD-TDP type A frontal cortex brain sections (Fig. 1d). The equilibrium dissociation constant (Kd) and in vitro binding potential or target occupancy (ratio of target density, Bmax, over Kd) were evaluated. In brain sections from two FTLD-TDP type A donors (one sporadic case and one genetic case with mutation in the progranulin gene), [3H]ACI-19278 showed a mean Kd value of 25 ± 25 nM with Bmax/Kd of 16 ± 4 and [3H]ACI-19626 a mean Kd value of 18 ± 1 nM with Bmax/Kd of 13 ± 1 (Fig. 1d and Supplementary Fig. 1f). A Kd of 27 nM was calculated for LATE-NC + ADNC tissue for ACI-19626 (Supplementary Fig. 1g). The high binding affinity to FTLD-TDP aggregates was further confirmed in an orthogonal radiobinding assay using sarkosyl-insoluble brain extracts from samples with FTLD-TDP pathology (Fig. 1e–f and Supplementary Fig. 1h). Across two FTLD-TDP extracts tested (one from a sporadic case and one from a genetic case with mutation in the progranulin gene), [3H]ACI-19278 and [3H]ACI-19626 showed a mean Kd value of 37 ± 4 nM and 24 ± 1 nM, respectively, for FTLD-TDP type A aggregates. Furthermore, [3H]ACI-19278 and [3H]ACI-19626 displayed a mean Kd value of 38 ± 25 nM and 25 ± 18 nM, respectively, in two C9orf72 mutation cases with FTLD-TDP type B pathology. Low levels of non-saturable binding were detected for both ligands in FTLD-TDP type C tissue (Fig. 1e, f), in agreement with the autoradiography studies. Limited, non-saturable levels of binding were detected in ALS brain homogenates (Supplementary Fig. 1i) in which the levels of pathological aggregates were notably lower compared with those from FTLD-TDP type A and B tissue (Supplementary Fig. 1j). Finally, no relevant, saturable binding was observed in control brain-derived homogenates, further highlighting the tracer specificity for pathological TDP-43.

Taken together, these data demonstrate that ACI-19278 and ACI-19626 bind to pathological TDP-43 in FTLD-TDP type A and B with affinity values in the nanomolar range. In addition, both compounds display specific binding to LATE-NC + ADNC tissue.

Binding specificity of ACI-19278 and ACI-19626 for aggregated versus physiological TDP-43

To assess target engagement of [3H]ACI-19278 and [3H]ACI-19626 to individual TDP-43 inclusions, high-resolution ARG studies were performed, allowing for a resolution of ~1 µm. No signal was observed in the absence of TDP-43 pathology in temporal cortex from control (Fig. 2a and Supplementary Fig. 2a). Immunofluorescent labeling of pTDP-43 aggregates (Fig. 2a and Supplementary Fig. 2a, top panel) and high-resolution ARG with [3H]ACI-19626 (Fig. 2b, bottom panel) or [3H]ACI-19278 (Supplementary Fig. 2a, bottom panel) performed on the same sections showed an extensive co-localization in FTLD-TDP type A and type B frontal and temporal cortex tissue, respectively, demonstrating target engagement of both compounds to TDP-43 inclusions. No ARG signal was observed on TDP-43 aggregates in temporal cortex sections from FTLD-TDP type C cases for both compounds, despite the abundance of pTDP-43 pathology. Notably, [3H]ACI-19278 (Supplementary Fig. 2a) and [3H]ACI-19626 (Fig. 2a, Supplementary Fig. 2b) displayed target engagement to pTDP-43 inclusions present in LATE-NC + ADNC tissue from multiple cases, even in case of low abundance of TDP-43 pathology. Finally, high-resolution autoradiography studies in motor cortex sections from ALS cases showed target engagement to TDP-43 inclusions in this tissue, regardless of their low density (Fig. 2a, Supplementary Fig. 2c). Altogether, these high-resolution ARG data are consistent with the classical ARG findings and radiobinding studies and further highlight binding specificity to pathological TDP-43 for ACI-19278 and ACI-19626.

Fig. 2: Binding specificity of ACI-19278 and ACI-19626 to aggregated TDP-43.

a High-resolution autoradiography with [³H]ACI-19626 (20 nM) in tissue from FTLD-TDP type A, type B and type C, LATE-NC + ADNC, ALS cases and a healthy control. Immunofluorescence with phospho-TDP-43 pS409/410 (pTDP-43) antibody (top panels, white arrowheads). Accumulation of silver grains on pTDP-43 inclusions on the same section (bottom panels, red arrowheads), showing co-labeling of pTDP-43 aggregates. Scale bar, 10 μm. Representative images of two independent experiments and donors for each tissue type (except for LATE-ND + ADNC, 3 cases tested). b Surface plasmon resonance studies for the determination of binding affinity of ACI-19278 and ACI-19626 to aggregated and soluble TDP-43. Single-cycle kinetics analysis was performed on immobilized brain-derived TDP-43 aggregates from an FTLD-TDP type A donor and on full-length recombinant TDP-43. Response signals (RU) are plotted against ACI-19278 or ACI-19626 concentration and steady-state affinity fits to calculate Kd. Data from one out of four independent experiments are shown. c ACI-19278 efficiently binds to TDP-43 fibrils and not to nuclear physiological TDP-43. Top, representative immunofluorescence image showing SH-SY5Y cells treated with TDP-43 fibrils (fLCD, yellow) and endogenous TDP-43 (magenta) and with ACI-19278 (cyan). Scale bar, 5 μm. Bottom, quantification depicting the percentage of overlapping area of exogenous TDP-43 aggregates (fLCD) and ACI-19278. Data is shown as mean ± SD from two independent experiments (n = 11). d ACI-19278 does not interfere with the physiological function of TDP-43. RT-PCR amplification and capillary electrophoresis analysis of CFTR exon 9 inclusion in CTFR minigene transfected SH-SY5Y cells in the presence of ACI-19728 (0.1–1 µM). Results from four independent experiments) (bottom panel), data shown as mean ± SD. One-way ANOVA with Dunnett’s multiple comparisons test showed no statistically significant differences. Source data are provided as a Source Data file.

Additionally, we evaluated the binding specificity to pathological versus physiological soluble TDP-43 in an orthogonal, label-free assay using surface plasmon resonance (SPR) spectroscopy. More specifically, insoluble fractions from the frontal cortex of FTLD-TDP type A brain and recombinant soluble TDP-43 were immobilized on the biosensor surface to assess binding of ACI-19278 and ACI-19626 (Fig. 2b and Supplementary Fig. 3). Both ligands showed binding specificity to aggregated TDP-43 with mean Kd values of 60 ± 9 nM for ACI-19278 and 80 ± 26 nM for ACI-19626 and no binding to soluble TDP-43.

To further explore the specificity of binding to aggregated versus physiological TDP-43 in a cellular environment[40](https://www.nature.com/articles/s41467-025-64540-6#ref-CR40 “Scialo, C. et al. Seeded aggregation of TDP-43 induces its loss of function and reveals early pathological signatures. Neuron, https://doi.org/10.1016/j.neuron.2025.03.008

(2025).“), the inherent fluorescent properties of ACI-19278 were exploited to evaluate target engagement. In agreement with the high-resolution ARG and SPR studies, the fluorescence signal from ACI-19278 colocalized with the fluorescence signal of internalized, cytoplasmic TDP-43 fibrils (fLCD) but not with endogenous physiological nuclear TDP-43 (Fig. 2c and Supplementary Fig. 4a). Notably, ACI-19278 showed an average colocalization of approximately 50% with the fLCD signal, indicating significant overlap with TDP-43 aggregates and therefore, extensive target engagement (Fig. 2c).

Finally, to rule out potential interference with physiological function of TDP-43, we evaluated ACI-19278 in a cystic fibrosis transmembrane conductance regulator (CFTR) exon 9 splicing assay in SH-SY5Y cells, surrogate for the function of TDP-43 in vitro41 (Supplementary Fig. 4b). Incubation of CFTR minigene-transfected cells with a range of concentrations of ACI-19278 (0.1–1 µM) showed no interference with the natural TDP-43 controlled RNA splicing of exon 9 (Fig. 2d). In contrast, a positive control comprising siRNA-mediated knockdown of TDP-43 resulted in increased inclusion of exon 9, as expected (Supplementary Fig. 4c).

The findings for ACI-19278 in cellular models are expected to also apply for ACI-19626 since the two ligands have a close chemical structure (Supplementary Fig. 1a), display a similar binding profile and ACI-19278 can compete with ACI-19626 (Supplementary Fig. 4d). Collectively, these data generated using patient-derived brain material, cellular models, and multiple orthogonal experimental techniques, show that ACI-19278 and ACI-19626 bind specifically to pathological TDP-43 inclusions and neither bind to soluble TDP-43 nor alter the physiological function of TDP-43.

[³H]ACI-19278 and [³H]ACI-19626 are selective for aggregated TDP-43 over common brain amyloid co-pathologies

Having shown desirable on-target binding to TDP-43 of ACI-19278 and ACI-19626, we next evaluated selectivity for TDP-43 versus other amyloidogenic proteins by assessing binding to Aβ and Tau aggregates on frontal cortex sections from an AD brain devoid of TDP-43 pathology (Fig. 3a). Radiolabeled reference molecules for Aβ (Aβ ref cmpd which is structurally related to Pittsburg compound B and can be displaced by it (Supplementary Fig. 5a) and Tau (PI-262042) displayed strong, displaceable binding (Fig. 3a, middle and bottom panel) that correlated with the distribution of Aβ plaques and Tau aggregates, as indicated by immunofluorescence labeling on the same tissue sections (Fig. 3a, top panel). In contrast, [3H]ACI-19278 and [3H]ACI-19626 displayed minimal to no retention on this tissue (Fig. 3a), indicating their preferential binding to TDP-43 over Aβ and Tau aggregates.

Fig. 3: ACI-19278 and ACI-19626 selectivity for TDP-43 over common co-pathologies.

a Autoradiography in Alzheimer’s disease (AD) tissue containing amyloid and Tau aggregates with an Aβ reference compound ([³H]Aβ ref cmpd) and Tau reference ligand ([³H]PI-2620). NSB: non-specific binding. Immunolabeling of adjacent sections with phospho-TDP-43 (orange), phospho-Tau (green) or Aβ (red) antibody. Scale bar, 2 mm. Representative autoradiograms from one donor from 3 independent experiments. b High-resolution autoradiography in the entorhinal cortex sections of an AD patient. [3H]PI-2620 included for reference. Thioflavin S staining in an adjacent section (left, green). Scale bar, 50 µm. Representative data of two independent experiments. c High-resolution autoradiography in FTLD-Tau tissue sections. Immunolabeling with phospho-Tau (top panels, green). Scale bar, 50 µm. Representative data of two donors tested in one experiment. d High-resolution autoradiography in tissue sections from a LATE-NC + ADNC case. Immunolabeling of the same sections with phospho-TDP-43 (orange), phospho-Tau (green), or Aβ (red) antibodies. Autoradiography images from adjacent sections with [3H]PI-2620 and [3H]Aβ reference compounds are shown on the bottom. Scale bar, 20 µm. Representative data of two donors tested in one experiment. White arrowheads: pTDP-43 pathology; red arrowheads: compound binding. e Saturation binding studies with [³H]ACI-19278 and [³H]ACI-19626 in brain homogenates from: From left to right, AD brain tissue containing Aβ and Tau aggregates (AD, insoluble fraction), AD brain tissue enriched for insoluble Tau paired helical filaments (AD, Tau PHF), and Parkinson’s disease (PD) brain tissue enriched for α-synuclein aggregates (PD, α -syn enriched). Reference ligands specific for Aβ (Aβ ref cmpd), Tau (PI-2620, Tau ref cmpd), and α-synuclein (ACI-12589, α-syn ref cmpd), respectively, were assessed in each homogenate. Representative data of at least two independent experiments for [³H]ACI-19278 and [³H]ACI-19626, and mean ± SD of three independent experiments for the [³H]Aβ ref cmpd in AD brain homogenates. Data from one out of two donors tested in independent experiments are shown for PHF and PD brain homogenates. Dotted line shows the limit under which binding is considered not relevant. Source data are provided as a Source Data file.

To confirm these findings, binding to pathological Tau present in different Tauopathies was assessed by high-resolution ARG combined with immunolabeling of phosphorylated Tau (pTau, Fig. 2b–c). While the Tau tracer, [3H]PI-2620, provided the positive control for a specific signal of the Tau tangles in AD entorhinal cortex tissue, [3H]ACI-19278 and [3H]ACI-19626 displayed no binding (Fig. 3b). Furthermore, no signal was detected for [3H]ACI-19278 or [3H]ACI-19626 on the pTau inclusions in FTLD-Tau frontal cortex tissue (Fig. 3c and Supplementary Fig. 5b), further confirming their selectivity over aggregated Tau. Evaluation of binding to non-AD 3R and 4R tauopathies has also showed no binding for ACI-19626 (Supplementary Fig. 5b).

Next, selectivity was assessed for TDP-43 over Aβ and Tau aggregates in the context of mixed protein pathologies. For this, high-resolution ARG experiments were conducted in LATE-NC + ADNC hippocampal tissue. The ARG signal from [3H]ACI-19278 and [3H]ACI-19626 (Fig. 3d, middle panels) colocalized with immunolabeling of pTDP-43 (Fig. 3d, top panels, white arrows), demonstrating binding to distinct aggregates of a smaller size as compared to the larger Aβ- or Tau-immunopositive aggregates in the same section (Fig. 3d, top panels, Aβ and pTau). Minimal co-localization with Aβ-positive structures was observed for [3H]ACI-19278, while no co-localization with Aβ or Tau-positive aggregates was observed for [3H]ACI-19626. In contrast, as expected, the Aβ binding reference compound ([3H]Aβ ref cmpd) and Tau binding reference compound, [3H]PI-2620, respectively revealed the characteristic forms of Aβ plaques and Tau tangles in adjacent sections (Fig. 3d, bottom panels).

Further assessment of the selectivity towards TDP-43 versus other aggregation-prone proteins was made by evaluating the binding affinity of [3H]ACI-19278 and [3H]ACI-19626 in brain homogenates derived from: a) AD brain tissue with confirmed burden of Aβ pathology (AD insoluble fraction, Fig. 3e, b) AD brain tissue enriched for insoluble Tau paired helical filaments (AD Tau PHF, Fig. 3e, and c) Parkinson’s disease (PD) brain tissue enriched for α-synuclein aggregates (PD α-syn enriched, Fig. 3e). Neither compound showed any relevant, saturable binding in any of these conditions, suggesting that both [3H]ACI-19278 and [3H]ACI-19626 are indeed selective over co-pathologies such as Aβ, Tau and α-syn aggregates. In contrast, the reference molecules for Aβ (Aβ ref cmpd), Tau (PI-2620), and α-syn (ACI-1258934, α-syn ref cmpd) displayed binding with high affinity to their respective targets with Kd values of 26 nM, 21 nM, and 66 nM, respectively.

Finally, we evaluated the potential off-target binding of ACI-19278 and ACI-19626 against a panel of more than 100 receptors, enzymes, ion channels, and transporters (Supplementary Table 1) at a concentration of 1 µM. No relevant binding to any of the tested proteins was observed, indicating excellent selectivity. Since binding to monoamine oxidase (MAO) A and B has been reported as a frequent off-target liability for Tau PET tracers43, displacement assays with the radiolabeled MAO-A inhibitor, [3H]Harmine, and the MAO-B inhibitor, [3H]deprenyl, were performed using microsomes expressing recombinant human MAO-A or MAO-B enzyme. No significant competition was measured for ACI-19278 or ACI-19626 (Supplementary Table 2), indicating no binding to MAO-A or B. Taken together, ACI-19278 and ACI-19626 display excellent selectivity for TDP-43 versus other aggregation-prone proteins, including pathological Aβ, Tau, and α-syn aggregates, and have clean off-target profiles.

Evaluation of the pharmacokinetic profile of [18F]ACI-19278 and [18F]ACI-19626 in rhesus macaques

Prior to the evaluation of the radiotracer pharmacokinetic (PK) profile in rhesus macaques, the PK of ACI-19278 and ACI-19626 was assessed in mice (Supplementary Fig. 6a). Both molecules showed brain uptake and fast washout. Next, to evaluate the suitability of the pharmacokinetic (PK) profile for use as a PET tracer, ACI-19278 and ACI-19626 were radiolabeled with fluorine-18 ([18F], Supplementary Fig. 6b) and administered intravenously in separate sessions to the same rhesus macaque. Dynamic PET scans (0–180 min) were conducted to assess brain uptake, distribution, and washout parameters in vivo. Both ligands entered the brain quickly, showed good brain permeability and exhibited a fast washout (Fig. 4). Specifically, [18F]ACI-19278 demonstrated a peak whole brain uptake at 5.5 min with standardized uptake value (SUV) of 2.9 (2.7% of injected dose, ID) while [18F]ACI-19626 reached the peak uptake at 4.5 min with SUV of 1.1 (1% ID) (Fig. 4b, c and Supplementary Fig. 6c, d). [18F]ACI-19626 showed lower brain uptake compared to [18F]ACI-19278 (Fig. 4b and Supplementary Fig. 6c), which is consistent with its lower lipophilicity as compared to ACI-19278. Conversely, [18F]ACI-19278 demonstrated slightly higher retention across various brain regions, including the dentate nucleus, cortical white matter and thalamus. [18F]ACI-19626 displayed a more homogeneous distribution with no obvious regional retention (Fig. 4a, b). Nonetheless, the washout of both ligands occurred rapidly, with a brain concentration ratio between peak and 60 min at 5.4 for [18F]ACI-19278 and 15.5 for [18F]ACI-19626 (Fig. 4b, c). Finally, the two ligands were quickly metabolized in a similar manner, with ~30% parent remaining at 30 min post tracer administration (Supplementary Fig. 6d).

Fig. 4: Assessment of the pharmacokinetic profile of [18F]ACI-19278 and [18F]ACI-19626 as brain PET tracers in rhesus macaques.

The same rhesus macaque, receiving either [18F]ACI-19278 or [18F]ACI-19626 intravenously at different points in time, was imaged by PET. A magnetic resonance imaging (MRI) of the brain was acquired on a separate day for anatomical reference and image analysis. a Transverse, sagittal and coronal views for MRI and PET images are provided to illustrate the determination of standardized uptake values (SUV) averaged over 0–30 min (middle row) and 30–90 min post-injection (bottom row). b Time activity curves (TACs) generated from the data obtained by quantification of the SUV for either [18F]ACI-19278 or [18F]ACI-19626 from different brain regions. Data from one experiment. c Comparison of the TACs for [18F]ACI-19278 and [18F]ACI-19626 averaged across the whole brain and reported as a percentage of the highest uptake (SUVmax) for each molecule. Source data are provided as a Source Data file.

Finally, metabolite identification for ACI-19626 was performed in vitro in rat, monkey, and human hepatocytes (Supplementary Table 3). Sixteen metabolites were detected in human hepatocytes, with oxidation and dehydrogenation to acid in methylpiperidine (M7) being the most abundant. The carboxylic acid moiety in M7 makes it highly unlikely to be brain penetrant and thus mitigates the risk of having a radioactive metabolite interfering with the parent’s signal.

Discussion

In vivo imaging of aggregated TDP-43 would open a new era for the development of disease-modifying therapies for TDP-43 proteinopathies. Here, we report for the first time the characterization of two compounds, [18F]ACI-19278 and [18F]ACI-19626, which exhibit all the desired characteristics for the successful visualization of TDP-43 pathology in the human brain by PET.

In the absence of available TDP-43 binding reference compounds, the development of TDP-43 PET tracers has proven challenging. This is at least in part due to the low density of TDP-43 inclusions as compared to the relatively higher Aβ and Tau pathology burden[44](#ref-CR44 “Cordts, I. et al. TDP-43 Proteinopathy specific biomarker development. Cells 12, https://doi.org/10.3390/cells12040597

(2023).“),45,46. The data provided in this paper support the potential successful translation of the in vitro*/*ex vivo binding signal to in vivo PET, as the in vitro techniques/assays that were used were optimally selected based on their translational value. First, ACI-19278 and ACI-19626 show strong displaceable binding to brain sections with FTLD-TDP pathology by ARG, a technique with a resolution comparable to PET. Second, the intensity of the ARG signal correlates with the abundance and distribution of TDP-43 aggregates, reflecting a faithful quantification of pathology. Third, ACI-19278 and ACI-19626 display Kd values below 20 nM, and the in vitro binding potentials (Bmax/Kd ratios) estimated using ARG on FTLD-TDP brain sections are above 10, which is higher than the threshold considered optimal for the successful translation of PET tracers to in vivo studies47 (>5). These parameters compare favorably to those of the first a-syn PET tracer ACI-12589 (with an estimated Bmax/Kd ratio of 12.5 in MSA), able to visualize a-syn pathology in MSA patient brain[34](https://www.nature.com/articles/s41467-025-6454