Generation of Antibodies Selective for Misfolded Disease-associated TDP-43

Objective: To develop a rational and robust antibody family specific for disease-misfolded TDP-43. Background: Misfolded molecular species of TDP-43 have been implicated in the neurotoxicity and prion-like cell-to-cell propagation in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar dementia (FTLD). We have found that a tryptophan (Trp68) in the TDP-43 N-terminal domain (NTD) participates in the cross-seeding of SOD1 misfolding propagation (Pokrishevsky et al, submitted), despite being inaccessible in the natively folded NTD (e.g., Afroz et al, Nature Comm 2017). We hypothesized that NTD Trp68 becomes exposed when TDP-43 is cytosolically mislocalized/aggregated. Design/Methods: We immunized rabbits with an unfolded NTD linear peptide epitope including Trp68 to generate polyclonal and monoclonal antibodies (pAb, mAb). mAb antibody affinity to the immunizing peptide was determined by surface plasmon resonance (SPR). NTD was expressed in E. coli, and properly folded monomer status was confirmed by size exclusion chromatography, followed by studies with denaturing and native gel electrophoresis and immunoblotting. Antibody specificity was confirmed by immunohistochemistry (IHC) on patient samples, and immunocytochemistry (ICC) of HEK293 cells transfected with TDP-43 triple-tandem mutation of the nuclear localization sequence (ΔNLS). Results: SPR of mAbs revealed picomolar affinity to the epitope. Recombinant NTD displayed pAb immunoreactivity only under denaturing conditions. IHC of ALS/FTLD CNS sections, but not normal CNS, was reactive to antibodies. ICC revealed immunoreactivity for mislocalized/aggregated ΔNLS-TDP-43, but not nuclear wild-type TDP-43. Antibodies also failed to recognize TDP-43 in physiologic stress granules in HEK293 cells. A ΔNLS-TDP-43 construct in which Trp68 was mutated to serine did not display Immunoreactivity in transfected cells, indicating that Trp68 is immunodominant in the immunizing peptide. Conclusions: We have developed a family of antibodies sensitive to solvent exposure of NTD Trp68 that are selective for misfolded/aggregated, disease-associated TDP-43 while sparing physiologically important molecular species, which may find utility in biomarker and immunotherapy applications for TDP-43 associated diseases. Disclosure: Dr. Cashman has received personal compensation for consulting, serving on a scientific advisory board, speaking, or other activities with ProMIS Neurosciences. Dr. Cashman has received compensation for serving on the Board of Directors of ProMIS Neurosciences. Dr. Cashman has received royalty, license fees, or contractual rights payments from ProMIS Neurosciences. Dr. Cashman holds stock and/or stock options in ProMIS Neurosciences which sponsored research in which Dr. Cashman was involved as an investigator. Dr. Cashman holds stock and/or stock options in ProMIS Neurosciences. Dr. Cashman has received research support from ProMIS Neurosciences. Dr. Louadi has nothing to disclose. Dr. Roman has nothing to disclose. Dr. Gibbs has received personal compensation for consulting, serving on a scientific advisory board, speaking, or other activities with ProMIS Neurosciences. Dr. Dijkstra has nothing to disclose. Dr. Kaplan has received personal compensation for consulting, serving on a scientific advisory board, speaking, or other activities with ProMIS Neurosciences. Dr. Kaplan holds stock and/or stock options in ProMIS Neurosciences which sponsored research in which Dr. Kaplan was involved as an investigator. Dr. Kaplan has received research support from ProMIS Neurosciences.