Rational Generation of Monoclonal Antibodies and Intrabodies Selective for Pathogenic TDP-43

TAR DNA-binding protein 43 (TDP-43), encoded by the TARDBP gene, is a ribonucleoprotein associated with the pathogenesis of amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and Alzheimer’s disease (AD). Under physiological conditions, TDP-43 is predominantly localized in the nucleus, where it participates in a variety of cellular functions related to RNA splicing, transport, and stability, as well as miRNA biogenesis. In disease, it is disproportionately mislocalized to the cytoplasm where it forms aggregates, which contribute to neurotoxicity and prion-like cell-to-cell propagation of pathogenic TDP-43. Targeting of misfolded aggregates of TDP-43 represents an attractive therapeutic strategy. However, development of effective immunotherapeutic agents remains a challenge, as they require stringent selectivity for misfolded TDP-43 in order to maintain the essential functions of physiologically native TDP-43. To address this issue, monoclonal antibodies (mAbs) and intrabodies were generated against an epitope in the N-terminal domain of TDP-43 that is only exposed when the protein is misfolded, but not in its properly folded form. We show that mouse and rabbit mAbs against this epitope displayed high binding affinities by surface plasmon resonance analysis and selectively reacted with pathological TDP-43 in post-mortem tissues from ALS, FTD, and AD patients. In a cell line system, human embryonic kidney (HEK) 293T cells, mAbs and corresponding intrabodies specifically reacted with cytoplasmic aggregates of transfected misfolded TDP-43 lacking the nuclear localization signal, TDP-43 ^ΔNLS . Functionally, mAbs inhibited cell-to-cell transmission of misfolded TDP-43 and the seeding activity of misfolded TDP-43 from FTLD brain homogenates by a novel RT-QuIC assay. Intrabodies promoted the degradation of intracellular aggregates of TDP-43 in HEK293T cells and in induced pluripotent stem cell-derived motor neurons (iPSC-MNs) from ALS patients. The results provide proof-of-concept evidence that supports selective targeting of misfolded toxic aggregates of TDP-43 as a potentially safe and effective avenue to treat neurodegenerative diseases associated with TDP-43 proteinopathy.