Targeting of misfolded, pathogenic TDP‐43 with rationally designed antibodies

Background Misfolded, aggregated TDP‐43 has been implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS), frontotemporal lobar dementia (FTLD) and limbic‐predominant age‐related TDP‐43 encephalopathy (LATE) through direct toxicity, loss of function of normal TDP‐43, induction of misfolding of other neuronal proteins, and prion‐like, cell‐to‐cell propagation of disease. We sought to generate antibodies selectively targeting the misfolded, pathogenic form of TDP‐43 while sparing physiological forms of TDP‐43 important for normal cell function. Method Mice and rabbits were immunized with an unfolded N‐terminal domain (NTD) linear epitope predicted to become exposed in cytosolically mislocalized, aggregated TDP‐43 but otherwise buried in natively folded TDP‐43. Selectivity of polyclonal antibody (pAb) for misfolded NTD was confirmed by studies with denaturing and native gel electrophoresis followed by immunoblotting. Monoclonal antibody (mAb) affinity for the immunizing peptide was measured by surface plasmon resonance (SPR). mAb selectivity for pathogenic, aggregated TDP43 was assessed by immunocytochemistry (ICC) of HEK293FT cells transfected with mutant TDP‐43 lacking a functional nuclear localization signal (DNLS) or upon arsenite stress, and by immunohistochemistry (IHC) on patient samples. The ability of mAbs to inhibit cell‐to‐cell transmission of DNLS TDP‐43 was evaluated in cell culture. Result Affinity‐purified pAb from immunized animals displayed reactivity with recombinant NTD under denaturing but not native conditions, indicating selectivity for unfolded NTD. mAb clones displayed pM affinity for the NTD epitope by SPR. ICC showed mAb reactivity with cytoplasmic aggregates of DNLS‐TDP‐43 but not wild‐type nuclear TDP‐43. Antibodies also did not recognize TDP‐43 in physiological stress granules in HEK‐293FT cells. IHC of ALS and FTLD CNS sections, but not normal control, confirmed selective immunoreactivity of mAbs with pathogenic TDP‐43. In cell culture, mAbs inhibited transmission of misfolding TDP‐43 from the conditioned medium of donor HEK293FT cells transfected with DNLS‐TDP‐43 to naïve recipient cells. Conclusion Immunization with an NTD epitope of misfolded TDP‐43 gave rise to mAbs selective for pathogenic vs physiologically important forms of TDP‐43. The mAbs were capable of inhibiting cell‐to‐cell propagation of misfolding TDP43 in vitro. Such antibodies may have value against extracellular transmission of misfolding TDP‐43 or as pathogenic TDP43‐specific intrabodies expressed intracellularly.