Targeting amyotrophic lateral sclerosis by neutralizing seeding-competent TDP-43 in CSF

Abstract

In amyotrophic lateral sclerosis, a disease driven by abnormal transactive response DNA-binding protein of 43 kDa aggregation, CSF may contain pathological species of transactive response DNA-binding protein of 43 kDa contributing to the propagation of pathology and neuronal toxicity. These species, released in part by degenerating neurons, would act as a template for the aggregation of physiological protein contributing to the spread of pathology in the brain and spinal cord. In this study, a robust seed amplification assay was established to assess the presence of seeding-competent transactive response DNA-binding protein of 43 kDa species in CSF of apparently sporadic amyotrophic lateral sclerosis patients. These samples resulted in a significant acceleration of substrate aggregation differentiating the kinetics from healthy controls. In parallel, a second assay was developed to determine the level of target engagement that would be necessary to neutralize such species in human CSF by a therapeutic monoclonal antibody targeting transactive response DNA-binding protein of 43 kDa. For this, evaluation of the pharmacokinetic/pharmacodynamic effect for the monoclonal antibody, ACI-5891.9, in vivo and in vitro confirmed that a CSF concentration of ≍1100 ng/mL would be sufficient for sustained target saturation. Using this concentration in the seed amplification assay, ACI-5891.9 was able to neutralize the transactive response DNA-binding protein of 43 kDa pathogenic seeds derived from amyotrophic lateral sclerosis patient CSF. This translational work adds to the evidence of transmission of transactive response DNA-binding protein of 43 kDa pathology via CSF that could contribute to the non-contiguous pattern of clinical manifestations observed in amyotrophic lateral sclerosis and demonstrates the ability of a therapeutic monoclonal antibody to neutralize the toxic, extracellular seeding-competent transactive response DNA-binding protein of 43 kDa species in the CSF of apparently sporadic amyotrophic lateral sclerosis patients.

Keywords: ALS; RT-QuIC; TDP-43; biomarker; immunotherapy.

Graphical Abstract

Figure 1

Detection of seeding-competent extracellular TDP-43 in CSF of ALS patients. (A) Aggregation kinetics of a seed-free TDP-43 peptide (352–414) used as the substrate for the SAA. Formation/accumulation of β-sheet-rich structures for a range of concentrations of substrate was evaluated over time by measuring the ThT fluorescence every 30 min. (B) Kinetics of a seed-free (grey) and seeded (red) aggregation where the final product of the 15 µM condition described in A was used as synthetic seeds. (C) Electron micrographs of assay substrate fibrils obtained after amplification reaction. Scale bar = 200 nm. (D) CSF from apparently sporadic ALS patients (n = 20, black) and age-matched HCs (n = 12, blue) were used as seeds in the SAA with 10 µM of substrate. An arbitrary amplification threshold was defined in the exponential phase at 3000 rfu (dotted line). (E) Quantification of the time to reach 3000 rfu. Data shown as mean ± standard error. Unpaired Student’s t-test (two-tailed), ****P < 0.0001.

Figure 2

Pharmacologically relevant concentrations to saturate TDP-43 in human CSF by ACI-5891.9 achieved in vivo. (A) A SIMOA®-based assay to assess target engagement in CSF of NHPs (used for B). ACI-5891 was used as a capture antibody after coating to magnetic beads. A biotinylated version of ACI-5965 was used as a detection antibody. Arrows on the left indicate the ability of the assay to measure free TDP-43 as opposed to unmeasured immune complexes (shown by arrows on the right). (B) ACI-5891.9 IC₅₀ estimation in human CSF using the assay described in A. Free TDP-43 signal shown on y-axis by measuring AEB in human CSF (n = 3) with increasing concentration of ACI-5891.9 (x-axis). (C) ACI-5891.9 PK/PD relationship in NHPs (n = 4) administered with ACI-5891.9 (single intravenous dose at 40 mg/kg). Left and right y-axes represent ACI-5891.9 exposure (circles) and free TDP-43 fold change (triangles), respectively. NHP#1 (open circles and triangles) presented ADA response. The dotted line represents ACI-5891.9 K_D (126 pM or 18.9 ng/mL). (D) ACI-5891.9 exposure in CSF of the same four NHPs (described in C) following single-dose intravenous administration at 40 mg/kg. Open circle in 1344 h time point indicates the animal with ADA.

Figure 3

ACI-5891.9 neutralizes TDP-43 seeding species in CSF of ALS patients. (A) Apparently sporadic ALS patients or HCs. (B) CSF were pre-incubated for 30 min at RT with 1000 ng/mL of ACI-5891.9 (filled symbols) or 1000 ng/mL of the isotype control antibody (open symbols) prior to be used as seeds in the SAA. (C) Quantification of the fluorescence at 12 h. Data shown as mean ± standard error, for ALS (n = 13) and HC (n = 9). An ordinary one-way ANOVA followed by a Tukey’s test for post hoc analysis, *P < 0.05.