Abstract Hexanucleotide G 4 C 2 repeat expansions in the C9ORF72 gene are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Dipeptide repeat proteins (DPRs) generated by translation of repeat-containing RNAs show toxic effects in vivo as well as in vitro and are key targets for therapeutic intervention. We generated human antibodies that bind DPRs with high affinity and specificity. Anti-GA antibodies engaged extra- and intracellular poly-GA and reduced aggregate formation in a poly-GA over-expressing human cell line. However, antibody treatment in human neuronal cultures synthesizing exogenous poly-GA resulted in the formation of large extracellular immune complexes and did not affect accumulation of intracellular poly-GA aggregates. Treatment with antibodies was also shown to directly alter the morphological and biochemical properties of poly-GA and to shift poly-GA/antibody complexes to more rapidly sedimenting ones. These alterations were not observed with poly-GP and have important implications for accurate measurement of poly-GA levels including the need to evaluate all centrifugation fractions and disrupt the interaction between treatment antibodies and poly-GA by denaturation. Targeting poly-GA and poly-GP in two mouse models expressing G 4 C 2 repeats by systemic antibody delivery for up to 16 months was well-tolerated and led to measurable brain penetration of antibodies. Long term treatment with anti-GA antibodies produced improvement in an open field movement test in aged C9ORF72 450 mice. However, chronic administration of anti-GA antibodies in AAV-(G 4 C 2 ) 149 mice was associated with increased levels of poly-GA detected by immunoassay and did not significantly reduce poly-GA aggregates or alleviate disease progression in this model. Significance Immunotherapy has been proposed for neurodegenerative disorders including Alzheimer’s or Parkinson’s diseases. Recent reports using antibodies against poly-GA or active immunization suggested similar immunotherapy in ALS/FTD caused by repeat expansion in the C9ORF72 gene (1, 2). Here, we systematically characterized human antibodies against multiple DPR species and tested the biological effects of antibodies targeting poly-GA in different cellular and mouse models. Target engagement was shown in three independent cellular models. Anti-GA antibodies reduced the number of intracellular poly-GA aggregates in human T98G cells but not in cultured human neurons. Whereas chronic anti-GA treatment in BAC C9ORF72 450 mice did not impact poly-GA levels and modestly improved one behavioral phenotype, poly-GA levels detected by immunoassays were increased and disease progression was unaltered in AAV-(G 4 C 2 ) 149 mice.

Comprehensive preclinical evaluation of human-derived anti-poly-GA antibodies in cellular and animal models of C9ORF72 disease