A brain-shuttled antibody targeting alpha synuclein aggregates for the treatment of synucleinopathies

Abstract

Parkinson's disease and multiple system atrophy are members of a class of devastating neurodegenerative diseases called synucleinopathies, which are characterized by the presence of alpha-synuclein (α-Syn) rich aggregates in the brains of patients. Passive immunotherapy targeting these aggregates is an attractive disease-modifying strategy, which must not only demonstrate target selectivity towards α-Syn aggregates, but also achieve appropriate brain exposure to have the desired therapeutic effect. Here we present preclinical data for SAR446159, a next-generation antibody for the treatment of synucleinopathies. SAR446159 is a bispecific antibody composed of an α-Syn-binding immunoglobulin and an engineered insulin-like growth factor receptor 1 binding single-chain variable fragment, acting as a shuttle to transport an antibody across the blood-brain barrier. SAR446159 binds tightly and preferentially to α-Syn aggregates and prevents their seeding capacity in vitro and in vivo. The binding properties of SAR446159 combined with its brain-shuttle technology make it a potent immunotherapeutic for treating synucleinopathies.

Fig. 1. Characteristics of SAR446159.

A Schematic diagram of SAR446159 and its mechanism of action. Images were generated with the assistance of Biorender. B The structure of α-Syn, showing the N-terminal (green) amphipathic region, the hydrophobic NAC (blue) region, and the acidic C-terminal (red) region (PDB: 1XQ8). C Sensograms of SAR446159 and 1E4 binding to monomeric and aggregated α-Syn.

Fig. 2. Staining of human brain tissue.

Staining of α-Syn pathology in postmortem Parkinson’s disease (PD) and multiple system atrophy (MSA) patient brain tissues. In the PD patient staining, the brain regions are A cerebral cortex, B subcortical white matter, C substantia nigra (SN), D substantia nigra pars compacta (SNpc), E midbrain. In the MSA patient staining, the brain regions are A cerebral cortex, B hippocampus, C midbrain, D SNpc, E olivary nucleus in brainstem. Black arrows are α-Syn-expressing neurons (PD) or cytoplasmic glial inclusion (MSA); red arrows, Lewy bodies (LBs); blue arrows, Lewy neurites (LNs).

Fig. 3. PFF blocking with SAR446159.

A Human iPSC-derived dopaminergic neurons or human microglia treated with α-Syn PFFs conjugated to pHrodo (5 μg/mL), with (+ SAR 10 μg/mL) or without SAR446159 (No SAR). The data were quantified at 24 h and normalized for relative confluence. B Mouse cortical neuron survival following high-dose PFF treatment. One-way ANOVA, followed by Dunnett’s multiple comparison’s test. C Cytokine secretion from human iPSC-derived dopaminergic neuron-astrocyte-microglia tricultures following treatment with α-Syn PFFs with or without antibodies. PFF = 10 μg/ml, antibodies = 10 μg/ml. One-way ANOVA, Sidak correction for multiple comparisons test. Error bars represent the mean ± S.D.

Fig. 4. Rat and NHP PK.

Antibody exposure of SAR446159 (red circles) and 1E4 (white circles) in the serum, brain, and CSF as a function of time following a single IV dose in A rats (n = 5–7 animals for serum and CSF, n = 3–4 animals for brain) and B non-human primates (NHPs, n = 3 animals). Error bars represent the mean ± S.D.

Fig. 5. Short-term efficacy study of SAR446159.

mThy-1 human α-Syn transgenic mice (male, 9-month-old) were randomized into three groups and treated intraperitoneally every three days with test articles for a total of 4 doses. A Human IgG staining of brain tissue from 9-month-old mThy-1 human α-Syn transgenic mice treated with hIgG, 1E4, or SAR446159, with representative images from the SNpc are shown. N = 7 for hIgG treatment and n = 8 for 1E4 and SAR446159 treatment. B Immunostaining for pSer129 α-Syn positivity in SNpc is shown. For each animal, average values measured from 2 fields were used. C Co-localization of SAR446159 (hIgG stain, green) with neurons (NeuN, purple), astrocytes (GFAP, purple), and microglia (Iba1, white). DAPI was used to stain nuclei (blue). D α-Syn-PFF enhanced localization of anti-α-Syn antibodies to neurons. DIV10 tricultures were treated with 10 μg/ml 1E4 or SAR446159 with or without 2 μg/ml α-Syn-PFF for 48 h. Cells were fixed and IF stained against human IgG (green), neuronal marker Tuj1 (red), and DAPI (blue). E Live cell imaging of iPSC derived dopaminergic neurons treated with Fabfluor (red when internalized) or SAR446159 labeled with Fabfluor (red when internalized, 3 μg/ml) and α-Syn PFFs (2.5 μg/ml). Statistics: all statistical analyses involved a one-way ANOVA followed by Dunnett’s multiple comparisons test. Error bars represent the mean ± S.D.

Fig. 6. Long-term efficacy study of SAR446159.

Animals were injected with PFFs at 3 months of age. One week following the PFF administration, animals were randomized and treated with weekly intraperitoneal doses of antibodies for 6 months, a total of 24 doses. The mIgG group was treated with at dose of 15 mg/kg and the non-PFF group was treated with saline. A LB/LN-like inclusions in the ipsilateral SNpc were counted as the average number in 4 fields (2 field per section, 2 sections per animal). B LB/LN-like inclusions in the ipsilateral amygdala (Amy) were counted as the average number in 2 fields (1 field per section, 2 sections per animal). C TH expression in the ipsilateral striatum (STR) was measured as optical density in 2 sections per animal. D Cells expressing p-α-Syn in the ipsilateral cortex were counted as the average number in 6 fields (3 fields per section, 2 sections per animal). E TH-positive cells in both the contralateral and the ipsilateral SNpc were counted as the sum of total TH-positive cells in 9 consecutive sections approximately 120 μm apart per animal. F Latency to fall in rotarod and wire hang tests were averaged over two consecutive testing days. All experiments were performed by investigators blinded to the treatment status. Statistical significance was determined using a one-way ANOVA followed by Dunnett’s multiple comparisons test. Error bars represent the mean ± S.D.