Introduction: After stroke, penumbral cells express neoepitopes that are recognized by natural IgM antibodies (NIgM) leading to complement (C) activation and propagation of inflammation. We previously identified NIgMs that specifically recognize ischemic cells, and showed that whereas antibody-deficient Rag1-/- mice are protected from ischemic stroke, administration of B4IgM, a NIgM that recognizes modified annexin-4, restored C activation and injury. We developed a site-targeted C inhibitor by fusing a single chain antibody derived from B4mAb to the C inhibitor Crry. We hypothesize that the targeted fusion construct B4Crry will inhibit the neurodegenerative effects of C and improves outcome after stroke.
Methods: Stroke was induced in adult and aged mice by 60 min right middle cerebral artery occlusion. Animals were examined for infarct volume and performance on motor and cognitive tasks. Brains were extracted for histological analyses.
Results: A single dose of B4Crry 6hrs post-ischemia specifically targeted to the ischemic hemisphere and inhibited C activation and IgM deposition in the penumbra, yielding significant reduction in infarct volumes, neuronal loss and neurological deficits (p<0.01) at 24hrs in adult male, female and aged mice. B4Crry-mediated neuroprotection persisted through 15 days of recovery yielding improved performance on corner task, and improved spatial learning and memory retention (p<0.05). Vehicle treated animals showed a sustained chronic inflammatory response with continuous IgM and C3d deposition and robust proinflammatory microglial activation. Acute administration of B4Crry interrupted the chronic inflammatory neurodegenerative cycle by significantly inhibiting C and IgM deposition, and shifting microglial polarity to an anti-inflammatory phenotype at 15 days. Simultaneously, B4Crry resulted in pronounced increase in neurogenesis and neuronal migration. We also showed that B4Crry bound specifically to the ischemic penumbra of postmortem brain samples obtained from acute stroke patients, but not to normal brain tissue from the same patient.
Conclusions: B4Crry is a novel therapeutic agent that provides local inhibition of C and interrupts neurodegenerative inflammatory cascades after stroke.