Targeting the glycine-rich domain of TDP-43 with antibodies prevents its aggregation in vitro and reduces neurofilament levels in vivo

Abstract

Cytoplasmic aggregation and concomitant nuclear clearance of the RNA-binding protein TDP-43 are found in ~ 90% of cases of amyotrophic lateral sclerosis and ~ 45% of patients living with frontotemporal lobar degeneration, but no disease-modifying therapy is available. Antibody therapy targeting other aggregating proteins associated with neurodegenerative disorders has shown beneficial effects in animal models and clinical trials. The most effective epitopes for safe antibody therapy targeting TDP-43 are unknown. Here, we identified safe and effective epitopes in TDP-43 for active and potential future passive immunotherapy. We prescreened 15 peptide antigens covering all regions of TDP-43 to identify the most immunogenic epitopes and to raise novel monoclonal antibodies in wild-type mice. Most peptides induced a considerable antibody response and no antigen triggered obvious side effects. Thus, we immunized mice with rapidly progressing TDP-43 proteinopathy (“rNLS8” model) with the nine most immunogenic peptides in five pools prior to TDP-43ΔNLS transgene induction. Strikingly, combined administration of two N-terminal peptides induced genetic background-specific sudden lethality in several mice and was therefore discontinued. Despite a strong antibody response, no TDP-43 peptide prevented the rapid body weight loss or reduced phospho-TDP-43 levels as well as the profound astrogliosis and microgliosis in rNLS8 mice. However, immunization with a C-terminal peptide containing the disease-associated phospho-serines 409/410 significantly lowered serum neurofilament light chain levels, indicative of reduced neuroaxonal damage. Transcriptomic profiling showed a pronounced neuroinflammatory signature (IL-1β, TNF-α, NfκB) in rNLS8 mice and suggested modest benefits of immunization targeting the glycine-rich region. Several novel monoclonal antibodies targeting the glycine-rich domain potently reduced phase separation and aggregation of TDP-43 in vitro and prevented cellular uptake of preformed aggregates. Our unbiased screen suggests that targeting the RRM2 domain and the C-terminal region of TDP-43 by active or passive immunization may be beneficial in TDP-43 proteinopathies by inhibiting cardinal processes of disease progression. Graphical Abstract

A patient-derived amyotrophic lateral sclerosis blood-brain barrier cell model reveals focused ultrasound-mediated anti-TDP-43 antibody delivery

“…Together these results provide important proof-of-concept evidence for non-invasive, FUS +MBfacilitated antibody transport across the in vitro ALS BBB, which could aid in improving the delivery of large molecule drugs and TDP-43-directed immunotherapies (96)(97)(98)(99) in ALS. With no such platform reported to date, our study provides the first patient-cell-derived model for investigation of FUS +MB bioeffects and screening of FUS +MB -deliverable drug formats most compatible with disease subtypes, supporting personalised medicine approaches to ALS drug delivery.…”

“…With BBB limiting an estimated 99.8 % of peripherally administered large molecule drugs such as therapeutics antibodies (95), we next tested the possibility of enhancing the permeabilisation of ALS BBB to large molecules with FUS +MB . As a proof-of-concept, we trialled the delivery of anti-TDP-43 antibody, considering promising preclinical development of TDP-43 targeted immunotherapies (96)(97)(98)(99) as well as the recent clinical success of therapeutic antibodies in the treatment of other neurodegenerative disorders (100)(101)(102)(103)(104). Although the utilised anti-TDP-43 antibody has not yet proved therapeutically beneficial, we were previously successful in delivering Aducanumab (Aduhelm TM ) and anti-tau therapeutic antibodies with FUS +MB in human Alzheimer's disease in vitro models (37,41); and a recent clinical study demonstrated beneficial effects of Aducanumab combined with FUS +MB in Alzheimer's disease patients, as compared to Aducanumab alone (104).…”

Generation and characterization of monoclonal antibodies against pathologically phosphorylated TDP-43

“…Importantly, immunotherapy using mAbs to target misfolded proteins has been widely investigated to treat neurodegenerative diseases. In fact, it has recently been reported that immunization with pS409/410-TDP-43 peptides, as a passive immunotherapy approach, reduces neuroaxonal damage in a TDP-43 mouse model [ 27 ]. We therefore believe our antibodies have therapeutic potential for the treatment of FTD/ALS and other TDP-43 proteinopathies.…”

Reliable detection of RNA in hippocampus sections of mice by FISH up to a post-mortem delay of 24 h

“…For the other approach, we used a 1:100 dilution of the recombinant monoclonal guinea pig Iba1 antibody from Synaptic Systems (catalog no. 234 308, Göttingen, Germany) against the allograft inflammatory factor 1 in the cytoplasm of the microglia (Glotfelty et al 2023 ; Riemenschneider et al 2023 ). To detect the primary antibody binding, we used an Alexa 488-coupled secondary antibody from Invitrogen (A11034 and A11073, Darmstadt, Germany) matching the species of the primary antibodies.…”