Characterization of a humanized amyloid beta oligomer monoclonal antibody (5E3) under preclinical development for passive immunotherapy of Alzheimer’s disease

Soluble Amyloid βeta oligomers (AβOs) trigger a cascade of toxic signaling in neurons that disrupt neuronal communication, behavior dysfunction and memory loss in Alzheimer's disease (AD). A murine monoclonal antibody (m5E3) has been developed, with demonstrated specificity and selectivity that targets soluble AβOs in vivo and treatment reduced levels in CSF and brain homogenates in Tg2576 and APP/PS1 treated mice. Affinity has been retained across humanized isotype variants and reduced effector functionality has been incorporated by design. Preclinical evaluation is described including a number of physicochemical attributes and pharmacokinetic properties of purified humanized 5E3 in mice. Expression vectors encoding for the IgG1, IgG2 and IgG4 variants were used to transfect CHO K1SV by electroporation or Lipofectiamine®. Immunoreactivity of purified antibodies was characterized by ELISA and immunoblotting, and affinity confirmed against cyclic-SNK (cSNK) and synthetic AβO epitope (Octet analysis and Biacore™ 300). Biodistribution and pharmacokinetic properties were determined in ApoE4 mice using unlabelled and labeled mAbs. Purified isotype variants retained similar affinity and selectivity towards the cSNK epitope and synthetic Aβ42 oligomers. The naturally occurring target recognized by m5E3 in human AD biospecimens was also recognized by all humanized variants. The biodistribution of fluorescent and radio-labelled m5E3 and humanized mAbs was assessed following i.p. injection in mice. The distribution of antibodies was assessed to determine penetration in the CNS and clearance profile in the absence of target (i.e. soluble AβOs). In order to meet the manufacture requirements and advance selection, stable cell lines have been established for each isotype to support manufacturing at commercially relevant levels. The humanization of m5E3 to various isotypic frameworks was completed and affinity to the conformationally constrained cSNK epitope and the selective target of AβO was retained. Systemic administration (i.p.) of humanized variants was assessed to determine penetration across the BBB and clearance. Together, these data provide reassurance towards preclinical evaluation for lead candidate selection and IND enabling studies.