ARVO 2014 Annual Meeting Abstracts 161 AMD and CNV: Preclinical and Clinical Studies Sunday, May 04, 2014 3:15 PM–5:00 PM Exhibit/Poster Hall SA Poster Session Program #/Board # Range: 1173–1205/C0201–C0233 Organizing Section: Retinal Cell Biology Contributing Section(s): Retina Program Number: 1173 Poster Board Number: C0201 Presentation Time: 3:15 PM–5:00 PM The Apolipoprotein E4 Targeted Replacement (APOE4 TR) Mouse Model of Age-Related Macular Degeneration (AMD) Exhibits Anti-Retinal Autoreactivity Albert H. Alhatem1, Nataliya Lenchik1, 2, Sarka Beranova-Giorgianni4, Mikael Klingeborn5, David D. New1, Francesco Giorgianni4, Ivan Gerling2, Marko Radic3, Catherine Bowes Rickman5, 6, Alessandro Iannaccone1. 1Ophthalmology, Hamilton Eye Institute, Univ. Tennessee HSC, Memphis, TN; 2Medicine/ Endocrinology, Univ. Tennessee HSC, Memphis, TN; 3Microbiology, Immunology and Biochemistry, Univ. Tennessee HSC, Memphis, TN; 4Pharmaceutical Sciences, University of TN Health Science Center, Memphis, TN; 5 Dept. Ophthalmology/Duke Eye Center, Duke University Medical Center, Durham, NC; 6Cell Biology, Duke University, Durham, NC. Purpose: To test the hypothesis that auto-antibodies (AAbs) recognizing ocular tissue antigens develop in the APOE4 TR mouse model of AMD. Methods: APOE4 TR mice aged at least 65 weeks (~15 mo) develop an AMD-like phenotype after being fed high fat cholesterol-enriched diet (HFCD) for 8-10 wks (Malek et al. PNAS 2005; 102: 11900-05). Western blots (WBs) were performed by reacting sera collected at 24 mos from APOE4 TR-HFCD mice and control APOE4 TR mice on normal diet (ND) against retina/RPE/BM/choroid tissue lysates of adult C57BL/6 mice. Lysates (10 mg) were loaded on gels, incubated with 5mL of serum, and developed. The intensity of the bands seen on WB was quantified with the Odyssey system and compared by student’s T-test. Immunohistochemistry (IHC) was performed against anti-mouse IgG antibody by fluorescence microscopy following incubation of adult C57BL/6 mouse retina sections with mouse sera. To identify the autoantigens, APOE4 TR-HFCD sera were immunoprecipitated, followed by 2-dimension electrophoresis (2DE) and liquid chromatography-tandem mass spectrometry (LC-MS/ MS), performed on spots seen on 2DEs with APOE4 TR-HFCD sera following previously reported methods (Lenchik et al. ARVO 2013, Abs. 4103). Results: Significantly more intensely reactive bands were observed in sera of APOE4 TR-HFCD compared to APOE4 TR-ND mice. The most robust reactivities were seen at 17kDa (p= 0.005 after 1-hr incubation, p= 0.000007 after 3 hrs) and 13kDa (p= 0.005, 1 hr; p=0.001, 3 hrs). Additional reactivities were seen after 1-hr incubation at 79kDa (p= 0.013), 47kDa (p= 0.028), and 20kDa (p=0.046). IHC studies showed moderate and diffuse staining of C57BL/6 mouse retinal sections at the outer nuclear layer level. No staining was seen with APOE4 TR-ND sera. Differentially reactive spots were also observed on 2DE between APOE4 TR-HFCD and APOE4 TR-ND sera, and IDs are being investigated by LC-MS/MS. Conclusions: As we have previously shown in the serum of human AMD (Iannaccone et al. Adv. Exp. Med. Biol. 2012; 723:11-6), AAbs recognizing retinal targets develop in this animal model of AMD, suggesting a stereotyped response to AMD-like retinal degenerative events that incites a secondary autoimmune component and that has the potential to further retinal damage. Commercial Relationships: Albert H. Alhatem, None; Nataliya Lenchik, None; Sarka Beranova-Giorgianni, None; Mikael Klingeborn, None; David D. New, None; Francesco Giorgianni,

None; Ivan Gerling, None; Marko Radic, None; Catherine Bowes Rickman, None; Alessandro Iannaccone, None Support: Grants from NEI/NIH R01 EY022706 (AI), R01 EY019038 (CBR) and P30 EY005722 (Duke Eye Center); Research to Prevent Blindness, Inc. New York, NY (Physician Scientist Award to AI, unrestricted grants to UTHSC Ophthalmology/Hamilton Eye Institute and Duke Eye Center); Edward N. & Della L. Thome Memorial Foundation Award (CBR). Program Number: 1174 Poster Board Number: C0202 Presentation Time: 3:15 PM–5:00 PM Dual inhibition of angiopoietin-2 and vascular endothelial growth factor-A with Crossmab RG7716 suppressed laser-induced choroidal neovascularization in a non-human primate model Gemmy C. Cheung1, Veluchamy A. Barathi1, Bo Bo Tun1, Say Wei Yeo1, Pei Pei Gan1, Chan lwin Nyein1, Jorg Regula2, Guido Hartman2. 1 Singapore Eye Research Institute, Singapore, Singapore; 2Pharma Research & Early Development, Hoffmann La Roche, Basel, Switzerland. Purpose: RG7716 is a bispecific antibody developed with CrossMab technology to tightly bind VEGF-A on one arm and angiopoietin (Ang)-2 on the other arm. We evaluated in vivo efficacy of RG7716 in a nonhuman primate model of laser-induced choroidal neovascularization (CNV). Methods: CNV was induced by laser photocoagulation on Day 0 in both eyes of 30 cynomolgus monkeys. Laser-induced lesions were confirmed; their severity was scored using fluorescein angiography from grade 1 (no hyperfluorescence) to grade 4 (bright hyperfluorescence and leakage) by a masked investigator on Day 14. Intravitreal injection was administered in both eyes on Day 15. Three active-treatment groups (high-dose RG7716, 90ug/0.05mL; low-dose RG7716, 30ug/0.05mL; and anti-Ang-2, 90ug/0.05mL), one active control group (anti-VEGF-A [ranibizumab, the best characterized anti-VEGF inhibitor], 30ug/0.05mL, equimolar to high-dose RG7716), and one inactive control group (isotype at 90ug/0.05mL) were included. Change in CNV grade was determined by comparing fluorescein angiography on Days 28 vs 14. Aqueous humor was collected from all eyes at baseline and on Days 15 and 32. Cytokine analysis of aqueous was performed by Multiplex ELISA system measuring IL-6, IL-8, MCP1, VEGF-A, PDGF-B, Ang-2 and bFGF. Results: A single intravitreal injection of high- and low-dose RG7716 reduced mean CNV lesion grade by 0.99-fold and -0.68 fold respectively. Treatment with anti-VEGF-A (Ranibizumab) resulted in reduction of mean CNV lesion grade by 0.63-fold. anti ANG2 treatment results in a reduction of mean CNV lesion grade by -0.46 –fold. At an equimolar number of binding sites, RG7716 reduced lesion severity significantly more than anti-VEGF alone (analysis of variance followed by Tukey’s multiple comparison, P100 ohms/cm2, suggesting tight electrical contacts between neighboring cells. RPE cells on PLGA show improved responses to changes in intracellular calcium and for their ability to transport fluid from apical to basal side on the monolayer. Conclusions: Successful validation of artificial RPE tissue indicates its suitability for implantation into the subretinal space. We are currently testing these scaffolds in animal models. This work will provide a GMP-ready protocol for generating RPE tissues on a scaffold for transplantation as treatment for diseases like AMD. Commercial Relationships: Vladimir Khristov, None; Juliet Hartford, None; Qin Wan, None; Mostafa R. Lotfi, None; Kiyoharu J. Miyagishima, None; Arvydas Maminishkis, None; Juan Amaral, None; Sheldon S. Miller, None; Janine Davis, None; Kapil Bharti, None Program Number: 1176 Poster Board Number: C0204 Presentation Time: 3:15 PM–5:00 PM Anti-C5 mAb: In Vivo Effects Following Intravitreal Administration to Cynomolgus Monkeys Mark Milton, Laura Dill Morton, Birgit Jaitner, David A. Shaw, Timothy MacLachlan. Novartis Institutes For BioMedical Research, Cambridge, MA.

Purpose: To characterize the in vivo effects of LFG316, a recombinant anti-C5 IgG1 monoclonal antibody after intravitreal (IVT) administration to cynomolgus monkeys Methods: Male and female cynomolgus monkeys were administered repeated (0, 3, or 5 mg/eye every two weeks) 50 mL intravitreal injections of LFG316 for either 3 or 6 months Results: After intravitreal administration, LFG316 distributed slowly out of the eye and into the systemic circulation with the maximum serum concentrations of total LFG316 occurring at ~ 5 days post dose. The shape of the total LFG316 serum concentration versus time curve was typical for the extravascular administration of a monoclonal antibody. At all dose levels, LFG316 could be detected throughout the dose interval and the duration of the study. Appearance of LFG316 in serum is accompanied by a similar molar increase in total C5 serum concentration; indicating that LFG316 in serum is mostly complexed to C5. Consequently, there was no evidence of pharmacodynamic activity (as measured by the hemolysis of rabbit red blood cells by serum samples) from systemic exposure to LFG316, since apparent free C5 concentration in serum (approximated by total C5 minus total LFG316) is not affected. AntiLFG316 antibodies were detected in 1/24 and 3/20 monkeys in the 3-month and 6-month studies, respectively. There were no treatmentrelated mortalities or findings regarding clinical observations, body weights, estimated food consumption, ophthalmoscopy, intra-ocular pressure, electroretinography, electrocardiography, blood pressure, immunophenotyping, hematology, clinical chemistry, urine analysis, organ weights or histopathology that could be attributed to treatment with LFG316 Conclusions: LFG316 was well tolerated in cynomolgus monkeys after multiple intravitreal injections up to a dose of 5 mg/eye every other week for up to 6 months. LFG316 distributed from the eye into the serum, bound to C5 in the serum but the amount of target (C5) capture was low, leading to no observable impact on the activity of the alternative complement pathway in the serum Commercial Relationships: Mark Milton, Novartis (E); Laura Dill Morton, Novartis (E); Birgit Jaitner, Novartis (E); David A. Shaw, Novartis (E); Timothy MacLachlan, Novartis (E) Program Number: 1177 Poster Board Number: C0205 Presentation Time: 3:15 PM–5:00 PM AAV2.Flt23k Intraceptor Inhibits VEGF and CNV in Mice Xiaohui Zhang, Hironori Uehara, Subrata K. Das, Austin Bohner, Balamurali K. Ambati. Moran Eye Center, University of Utah, Salt Lake City, UT. Purpose: To determine the efficacy and safety of long-term expression of a virus mediated intraceptor vascular endothelial growth factor (VEGF) inhibitor, AAV2.Flt23k, on murine choroidal neovascularization (CNV) induced by laser photocoagulation. Methods: To evaluate the long-term expression of AAV2 mediated gene therapy, AAV2.AcGFP was subretinal injected 1 ml (5x108 vg) and screened by Heidelberg Spectralis at 2 weeks, 1 month, 3 months and 6 months. In an experimental model of laser induced CNV, AAV2.Flt23k, and control AAV2.AcGFP and PBS were subretinal injected one month prior to laser induction. After two weeks, CNV volume was measured. VEGF level was measured with ELISA. To evaluate safety, electroretinography (ERG) and OCT retinal thickness were assessed 6 months after subretinal injection. Results: The AAV2.AcGFP expression was at 2 weeks and sustained expression for at least 6 months. The mean CNV volume was significantly smaller in the AAV2.Flt23k (7.60 ± 1.15 × 104 mm3) group compared with control mice treated with AAV2.AcGFP (19.81 ± 4.10 × 104 mm3, p