Inhibition of microfibrillar-associated protein 4 as a potential therapy targeting choroidal neovascularization in age-related macular degeneration Bartosz Pilecki Institute of Molecular Medicine University of Southern Denmark 20.01.2016

Age-Related Macular Degeneration (AMD) Leading cause of blindness in people over 50 Affects > 30 million people worldwide Characterized by the loss of central vision due to the gradual deterioration of the macula (angiogenesis, inflammation, fibrosis) Dry AMD atropy of Retinal epithelial cells – blurred central vision Wet AMD rapid worsening af vision, blind spots

RPE – retinal pigment epithelium AMD pathogenesis Photo receptors RPE BM Choroid RPE – retinal pigment epithelium BM – Bruchs membrane

Current treatment options Intravitreal injection of vascular endothelial growth factor (VEGF) inhibitors has become the standard treatment for wet AMD Anti-VEGF therapy does not affect inflammation and fibrosis and gives rise to side effects Laser-based therapies are the only solution for the subpopulations of non-responders to anti-VEGF monotherapy (15-25%) New treatment strategies are warranted Luc: 21,4 letter more with 0.5 mg luc in trial III Eylea: ph III: samme efficacy as luc Ref. Datamonitor

Microfibrillar-associated protein 4 Extracellular matrix elastin-binding protein (Pilecki, Holm et al 2016) RGD-dependent integrin ligand Promotes integrin-dependent smooth muscle cell proliferation and macrophage chemotaxis (Schlosser, Pilecki et al 2016; Pilecki et al, 2015) Mediates endothelial cell adhesion and integrin signaling activation (Ormhøj et al, unpublished) Suggests a potential involvement of MFAP4 in AMD pathogenesis

Neovascularization and inflammation of the eye αMFAP4 actions in AMD Endothelial cells Inflammatory cells Extracellular matrix Neovascularization and inflammation of the eye Integrin receptor Activation MFAP4 MFAP4 MFAP4 blocking antibody

Choroidal neovascularization (CNV) rodent model CNV is the hallmark lesion of advanced AMD Similar to the CNV that occurs in human ocular disease Laser-induced injury Administration of αVEGF, αMFAP4 and IgG control at days 0 and 7 Endpoints analyzed at days 7 and 14 after surgery Brown Norway rats, 5 microliters, app. 60 micrograms – (12 mg/ml, 2 EU/ml)   1 injection; hyperemia (rødfarvning), chemosis (swelling), discharge (liquid flows from the eye) (Draize examination) uptill 23 days Electroretinogram Weight Behaviour HE histology Figure adapted from Singh-SR, Gene Therapy, 2009

αMFAP4 reduces lesion size at day 7, in contrast to αVEGF Trend for superiority of high dose αMFAP4 fundus flourescein angiogr

αMFAP4 andαVEGF have similar effects in reduction of lesion size at day 14 Trend for superiority of high doseαMFAP4

αMFAP4 andαVEGF have similar effects in reduction of inflammation at day 14 Trend for superiority of high doseαMFAP4

Summary: No adverse tolerability, assessed by electroretinography, physical defects, gross observations of ocular irritation and histological observation of retinal tissue Treatment with αMFAP4, particularly at higher dose, was able to consistently reduce lesion size and density and limit leukocyte infiltration into the burn area Effects of αMFAP4 were already detectable 7 days after lesion formation αMFAP4-based therapy can potentially be used to treat the pathological angiogenesis and inflammation in AMD

Perspectives: Efficacy of prophylactic αMFAP4 treatment Combinational αMFAP4/αVEGF treatment In vitro MFAP4/anti-MFAP4 effects on ocular angiogenesis, vessel permeability, cell proliferation and cytokine secretion

School of Medicine, University of Nottingham, UK: Acknowledgements: School of Medicine, University of Nottingham, UK: Zoe Blackley Nikita Ved David Bates UFFE GRITH ANDERS KARIN CHARLOTTE KIRSTEN VICKI BARTEK SEBASTIAN LINE CHRISTINE KIMMIE SOFIE ANDERS ANITA