Targeting key angiogenic pathways with a bispecific Cross MA b optimized for neovascular eye diseases
2016; Springer Nature; Volume: 8; Issue: 11 Linguagem: Inglês
10.15252/emmm.201505889
ISSN1757-4684
AutoresJörg T. Regula, Peter Lundh von Leithner, Richard Foxton, Veluchamy A. Barathi, Chui Ming Gemmy Cheung, Sai Bo Bo Tun, Yeo Sia Wey, Daiju Iwata, Miroslav Dostálek, Jörg Moelleken, Kay Stubenrauch, Everson Nogoceke, Gabriella Widmer, Pamela Strassburger, Michael Koss, Christian Klein, David T. Shima, Guido Hartmann,
Tópico(s)Corneal Surgery and Treatments
ResumoResearch Article14 October 2016Open Access Transparent process Targeting key angiogenic pathways with a bispecific CrossMAb optimized for neovascular eye diseases Jörg T Regula Jörg T Regula Roche Pharma Research and Early Development, Roche Innovation Center München, Penzberg, Germany Search for more papers by this author Peter Lundh von Leithner Peter Lundh von Leithner Department of Ocular Biology and Therapeutics, UCL London, Institute of Ophthalmology, London, UK Search for more papers by this author Richard Foxton Richard Foxton Department of Ocular Biology and Therapeutics, UCL London, Institute of Ophthalmology, London, UK Roche Pharma Research and Early Development, Roche Innovation Center Basel, F. Hoffmann-La Roche Ltd, Basel, Switzerland Search for more papers by this author Veluchamy A Barathi Veluchamy A Barathi Translational Pre-Clinical Model Platform, Singapore Eye Research Institute, The Academia, Singapore, Singapore The Ophthalmology & Visual Sciences Academic Clinical Program, DUKE-NUS Graduate Medical School, Singapore, Singapore Search for more papers by this author Chui Ming Gemmy Cheung Chui Ming Gemmy Cheung Translational Pre-Clinical Model Platform, Singapore Eye Research Institute, The Academia, Singapore, Singapore Search for more papers by this author Sai Bo Bo Tun Sai Bo Bo Tun Translational Pre-Clinical Model Platform, Singapore Eye Research Institute, The Academia, Singapore, Singapore Search for more papers by this author Yeo Sia Wey Yeo Sia Wey Translational Pre-Clinical Model Platform, Singapore Eye Research Institute, The Academia, Singapore, Singapore Search for more papers by this author Daiju Iwata Daiju Iwata Department of Ocular Biology and Therapeutics, UCL London, Institute of Ophthalmology, London, UK Search for more papers by this author Miroslav Dostalek Miroslav Dostalek Roche Pharma Research and Early Development, Roche Innovation Center Basel, F. Hoffmann-La Roche Ltd, Basel, Switzerland Search for more papers by this author Jörg Moelleken Jörg Moelleken Roche Pharma Research and Early Development, Roche Innovation Center München, Penzberg, Germany Search for more papers by this author Kay G Stubenrauch Kay G Stubenrauch Roche Pharma Research and Early Development, Roche Innovation Center München, Penzberg, Germany Search for more papers by this author Everson Nogoceke Everson Nogoceke Roche Pharma Research and Early Development, Roche Innovation Center Basel, F. Hoffmann-La Roche Ltd, Basel, Switzerland Search for more papers by this author Gabriella Widmer Gabriella Widmer Roche Pharma Research and Early Development, Roche Innovation Center Basel, F. Hoffmann-La Roche Ltd, Basel, Switzerland Search for more papers by this author Pamela Strassburger Pamela Strassburger Roche Pharma Research and Early Development, Roche Innovation Center Basel, F. Hoffmann-La Roche Ltd, Basel, Switzerland Search for more papers by this author Michael J Koss Michael J Koss Department of Ophthalmology, Goethe University, Frankfurt am Main, Germany Department of Ophthalmology, Ruprecht Karls University, Heidelberg, Germany Search for more papers by this author Christian Klein Christian Klein Roche Pharma Research and Early Development, Roche Innovation Center Zürich, F. Hoffmann-La Roche Ltd, Zürich, Switzerland Search for more papers by this author David T Shima David T Shima Department of Ocular Biology and Therapeutics, UCL London, Institute of Ophthalmology, London, UK Search for more papers by this author Guido Hartmann Corresponding Author Guido Hartmann [email protected] orcid.org/0000-0002-5967-9711 Roche Pharma Research and Early Development, Roche Innovation Center Basel, F. Hoffmann-La Roche Ltd, Basel, Switzerland Search for more papers by this author Jörg T Regula Jörg T Regula Roche Pharma Research and Early Development, Roche Innovation Center München, Penzberg, Germany Search for more papers by this author Peter Lundh von Leithner Peter Lundh von Leithner Department of Ocular Biology and Therapeutics, UCL London, Institute of Ophthalmology, London, UK Search for more papers by this author Richard Foxton Richard Foxton Department of Ocular Biology and Therapeutics, UCL London, Institute of Ophthalmology, London, UK Roche Pharma Research and Early Development, Roche Innovation Center Basel, F. Hoffmann-La Roche Ltd, Basel, Switzerland Search for more papers by this author Veluchamy A Barathi Veluchamy A Barathi Translational Pre-Clinical Model Platform, Singapore Eye Research Institute, The Academia, Singapore, Singapore The Ophthalmology & Visual Sciences Academic Clinical Program, DUKE-NUS Graduate Medical School, Singapore, Singapore Search for more papers by this author Chui Ming Gemmy Cheung Chui Ming Gemmy Cheung Translational Pre-Clinical Model Platform, Singapore Eye Research Institute, The Academia, Singapore, Singapore Search for more papers by this author Sai Bo Bo Tun Sai Bo Bo Tun Translational Pre-Clinical Model Platform, Singapore Eye Research Institute, The Academia, Singapore, Singapore Search for more papers by this author Yeo Sia Wey Yeo Sia Wey Translational Pre-Clinical Model Platform, Singapore Eye Research Institute, The Academia, Singapore, Singapore Search for more papers by this author Daiju Iwata Daiju Iwata Department of Ocular Biology and Therapeutics, UCL London, Institute of Ophthalmology, London, UK Search for more papers by this author Miroslav Dostalek Miroslav Dostalek Roche Pharma Research and Early Development, Roche Innovation Center Basel, F. Hoffmann-La Roche Ltd, Basel, Switzerland Search for more papers by this author Jörg Moelleken Jörg Moelleken Roche Pharma Research and Early Development, Roche Innovation Center München, Penzberg, Germany Search for more papers by this author Kay G Stubenrauch Kay G Stubenrauch Roche Pharma Research and Early Development, Roche Innovation Center München, Penzberg, Germany Search for more papers by this author Everson Nogoceke Everson Nogoceke Roche Pharma Research and Early Development, Roche Innovation Center Basel, F. Hoffmann-La Roche Ltd, Basel, Switzerland Search for more papers by this author Gabriella Widmer Gabriella Widmer Roche Pharma Research and Early Development, Roche Innovation Center Basel, F. Hoffmann-La Roche Ltd, Basel, Switzerland Search for more papers by this author Pamela Strassburger Pamela Strassburger Roche Pharma Research and Early Development, Roche Innovation Center Basel, F. Hoffmann-La Roche Ltd, Basel, Switzerland Search for more papers by this author Michael J Koss Michael J Koss Department of Ophthalmology, Goethe University, Frankfurt am Main, Germany Department of Ophthalmology, Ruprecht Karls University, Heidelberg, Germany Search for more papers by this author Christian Klein Christian Klein Roche Pharma Research and Early Development, Roche Innovation Center Zürich, F. Hoffmann-La Roche Ltd, Zürich, Switzerland Search for more papers by this author David T Shima David T Shima Department of Ocular Biology and Therapeutics, UCL London, Institute of Ophthalmology, London, UK Search for more papers by this author Guido Hartmann Corresponding Author Guido Hartmann [email protected] orcid.org/0000-0002-5967-9711 Roche Pharma Research and Early Development, Roche Innovation Center Basel, F. Hoffmann-La Roche Ltd, Basel, Switzerland Search for more papers by this author Author Information Jörg T Regula1,‡, Peter Lundh von Leithner2,‡, Richard Foxton2,3, Veluchamy A Barathi4,5, Chui Ming Gemmy Cheung4, Sai Bo Bo Tun4, Yeo Sia Wey4, Daiju Iwata2, Miroslav Dostalek3, Jörg Moelleken1, Kay G Stubenrauch1, Everson Nogoceke3, Gabriella Widmer3, Pamela Strassburger3, Michael J Koss6,7, Christian Klein8, David T Shima2 and Guido Hartmann *,3 1Roche Pharma Research and Early Development, Roche Innovation Center München, Penzberg, Germany 2Department of Ocular Biology and Therapeutics, UCL London, Institute of Ophthalmology, London, UK 3Roche Pharma Research and Early Development, Roche Innovation Center Basel, F. Hoffmann-La Roche Ltd, Basel, Switzerland 4Translational Pre-Clinical Model Platform, Singapore Eye Research Institute, The Academia, Singapore, Singapore 5The Ophthalmology & Visual Sciences Academic Clinical Program, DUKE-NUS Graduate Medical School, Singapore, Singapore 6Department of Ophthalmology, Goethe University, Frankfurt am Main, Germany 7Department of Ophthalmology, Ruprecht Karls University, Heidelberg, Germany 8Roche Pharma Research and Early Development, Roche Innovation Center Zürich, F. Hoffmann-La Roche Ltd, Zürich, Switzerland ‡These authors contributed equally to this work *Corresponding author. Tel: +41 616874588; Fax: +41 616880382; E-mail: [email protected] EMBO Mol Med (2016)8:1265-1288https://doi.org/10.15252/emmm.201505889 Correction(s) for this article Targeting key angiogenic pathways with a bispecific CrossMAb optimized for neovascular eye diseases22 May 2017 Targeting key angiogenic pathways with a bispecific CrossMAb optimized for neovascular eye diseases30 April 2019 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Anti-angiogenic therapies using biological molecules that neutralize vascular endothelial growth factor-A (VEGF-A) have revolutionized treatment of retinal vascular diseases including age-related macular degeneration (AMD). This study reports preclinical assessment of a strategy to enhance anti-VEGF-A monotherapy efficacy by targeting both VEGF-A and angiopoietin-2 (ANG-2), a factor strongly upregulated in vitreous fluids of patients with retinal vascular disease and exerting some of its activities in concert with VEGF-A. Simultaneous VEGF-A and ANG-2 inhibition was found to reduce vessel lesion number, permeability, retinal edema, and neuron loss more effectively than either agent alone in a spontaneous choroidal neovascularization (CNV) model. We describe the generation of a bispecific domain-exchanged (crossed) monoclonal antibody (CrossMAb; RG7716) capable of binding, neutralizing, and depleting VEGF-A and ANG-2. RG7716 showed greater efficacy than anti-VEGF-A alone in a non-human primate laser-induced CNV model after intravitreal delivery. Modification of RG7716's FcRn and FcγR binding sites disabled the antibodies' Fc-mediated effector functions. This resulted in increased systemic, but not ocular, clearance. These properties make RG7716 a potential next-generation therapy for neovascular indications of the eye. Synopsis The ratio of angiopoietins ANG-1 and ANG-2 regulates vessel quiescence and neoangiogenesis as well as barrier function. It also acts as a switch from health to neovascular eye diseases. ANG-2, but not ANG-1 levels are strongly upregulated in neovascular eye diseases. ANG-2 works in concert with VEGF-A to mediate pathological angiogenesis and endothelial dysfunction. Combined inhibition of ANG-2 and VEGF-A is more efficacious than anti-VEGF-A monotherapy in rodent models of choroidal neovascularization. The combination prevents vessel leakiness and neovascularization and its consequences such as increased retinal apoptosis or loss of retinal functionality. A bispecific antibody (CrossMAb RG7716) was generated with a modified Fc region optimized for use in ophthalmology. It reduced lesion severity better than anti-VEGF-A monotherapy in a laser-induced model of CNV in non-human primates. Introduction The retina is supplied with oxygen, nutrients, and waste exchanges by two distinct vascular beds: the retinal and choroidal capillary networks. Abnormal leakage and/or neovascularization from retinal vessels are the hallmarks of diseases such as diabetic macular edema (DME), diabetic retinopathy (DR), and retinal vein occlusion (RVO) (Penn et al, 2008; Wang & Hartnett, 2016). Wet age-related macular degeneration (wAMD) develops when new vessels grow into the subretinal space from the underlying choroidal capillary network underneath the outer retina (Campochiaro, 2013). Microangiopathy, neovascularization, and vascular leakage from choroidal and retinal capillaries are the most common causes of moderate and severe vision loss in developed countries (WHO, 2014). The importance of vascular endothelial growth factor (VEGF)-A in pathological angiogenesis, particularly in oncology and eye diseases, led to the development of several agents for clinical intervention. Anti-VEGF-A-based therapies are now the standard of care for a variety of solid tumor indications and ocular neovascular diseases, such as wAMD, DME, and RVO. One way of enhancing the efficacy of anti-VEGF-A-based therapies is adding the inhibition of another soluble factor to the therapeutic reagent that is also essential for angiogenesis (Jo et al, 2006). The angiopoietins ANG-1 and ANG-2 are growth factors that play a key role in vessel homeostasis, angiogenesis, and vascular permeability. Both ligands interact with the Tie-2 transmembrane receptor tyrosine kinase. Tie-2 is preferentially expressed on endothelial cells and a subset of myeloid cells. Loss- and gain-of-function experiments have demonstrated the critical contributions of the angiopoietin/Tie-2 system in vascular development and vascular diseases (Puri et al, 1995; Asahara et al, 1998; Augustin et al, 2009). Tie-2 receptor-deficient mice die by embryonic day 10.5 as a result of vessel immaturity and incorrect vessel organization (Dumont et al, 1994; Sato et al, 1995). ANG-1-deficient mice show a phenotype that is reminiscent of the Tie-2-deficient phenotype (Suri et al, 1996), while transgenic overexpression of ANG-2 mimics the phenotype of Tie-2- and ANG-1-deficient mice, suggesting that ANG-1 and ANG-2 have opposing activities. Both Tie-2 ligands bind to the receptor with similar affinity, whereas only ANG-1 induces strong phosphorylation of the kinase domain and subsequent signaling events. ANG-2 has been characterized as a partial Tie-2 agonist that competitively inhibits ANG-1 signaling (Maisonpierre et al, 1997; Saharinen et al, 2008). Structural studies have identified an agonistic loop (P-domain) in ANG-1 that is capable of converting ANG-2 into a full agonist on chimerization (Yu et al, 2013). ANG-2 also amplifies proapoptotic signals in pericytes under stress from elevated glucose conditions or proinflammatory cytokines like TNF-α (Cai et al, 2008; Park et al, 2014). Overall data suggest that ANG-1 protects from pathological angiogenesis and drives toward a quiescent, mature vessel phenotype (Nambu et al, 2004; Hammes et al, 2011; Lee et al, 2014), whereas ANG-2 promotes vascular leakage and hypotensive, abnormal vessel structure (Ziegler et al, 2013). Interaction of the angiopoietins with the VEGF-A pathway has been studied in ocular setting using double transgenic mice co-expressing ANG-1 and VEGF-A. ANG-1 expression, when initiated simultaneously with VEGF-A, suppressed VEGF-A-induced neovascularization and prevented retinal detachment (Nambu et al, 2005). Although ANG-1 effectively blocked the initiation and progression of VEGF-A-induced neovascularization, no impact on previously established lesions was observed (Nambu et al, 2005). Conversely, co-expression of ANG-2 with VEGF-A in the developing retina and in ischemic retina models showed accelerated neovascularization compared to VEGF-A expression alone (Oshima et al, 2004, 2005). Further pathophysiological significance arises from reports of elevated levels of VEGF-A and ANG-2 in vitreous samples from diabetic patients undergoing vitrectomy, which correlate with each other and with disease severity (Watanabe et al, 2005; Loukovaara et al, 2013). Therefore, VEGF-A and ANG-2 appear strongly linked to normal vascular development, but also to pathological neovascularization and vascular permeability, which are the hallmarks of ocular neovascular disease. Several bispecific antibodies are currently in clinical development, including an anti-VEGF-A/ANG-2 bispecific antibody targeting neovascularization in tumors (Kienast et al, 2013). The aim of this study was to explore the potential of dual VEGF-A and ANG-2 inhibition in the management of retinal vascular diseases. We tested monotherapy and dual inhibition of VEGF-A/ANG-2 using a species crossreactive CrossMAb in the spontaneous model of CNV in JR5558 mice. We then developed a human bispecific CrossMAb (RG7716), which binds and neutralizes human VEGF-A and ANG-2 with high potency and tested it in the laser-induced model of CNV in non-human primates. Our data suggest that dual inhibition of these angiogenic signaling circuits improves outcomes in preclinical models of ocular neovascular disease. Results ANG-2 levels are elevated in human retinal vascular diseases To investigate whether ANG-2 is a suitable pharmacological target for human retinal vascular diseases, we measured the levels of ANG-1 and ANG-2 in the vitreous of patients newly diagnosed with wet AMD, DR, proliferative DR, or RVO in comparison with macular hole as controls, as described in Koss et al (2011). In human vitreous samples, ANG-1 levels were weakly elevated in RVO (71.1 up to 107 pg/ml) and decreased in proliferative DR (down to 36.3 pg/ml) compared to controls (Fig 1A). However, levels of ANG-2 were significantly elevated in all four retinal vascular diseases investigated compared to controls (Fig 1B). From control levels of 68.4 pg/ml, ANG-2 increased to 139 pg/ml in wet AMD, to 302 pg/ml in DR, to 1,140 pg/ml in RVO, and to 1,625 pg/ml in proliferative DR. Figure 1. Vitreous concentrations of angiopoietins in patients newly diagnosed with retinal diseases and cell model of barrier breakdown testing the interaction of VEGF-A and angiopoietins A, B. Box plots of vitreal ANG-1 (A) and ANG-2 (B) levels from newly diagnosed patients with wAMD, DR, proliferative DR and RVO compared to controls (macular hole). The interquartile range of the data is indicated by the box. A nonparametric Kruskal–Wallis analysis followed by Dunn's method for multiple comparisons was used to show significant differences of the groups to control which are indicated by asterisks. ANG-1 levels did not differ significantly, but ANG-2 levels were significantly different: control vs. AMD (*, P = 0.0451), vs. DR (****, P < 0.0001), pDR (****, P < 0.0001), and RVO (****, P < 0.0001). C. Schematic of cellular experiments to measure transendothelial resistance in human endothelial cells using transwell filters and CellZscope technology. D. 24-h concentration of ANG-2 in supernatants of the basal side of the culture stimulated with 5 ng/ml of VEGF-A, 200 ng/ml of ANG-1, or the combination. Error bars show SEM with one-sided ANOVA (P < 0.0001) and Tukey's multiple t-test for five independent experiments indicating significance for control vs. VEGF-A (**, P = 0.0028); VEGF-A vs. ANG-1 (****, P < 0.0001); ANG-1 vs. VEGF-A and ANG1 (**, P = 0.0061); VEGF-A vs. VEGF-A and ANG-1 (ns, P = 0.0625). E. Human endothelial cells plated on filters were assessed for endothelial barrier function over time after the addition of 5 ng/ml VEGF-A alone or in combination with 200 ng/ml ANG-1 or 10 μg/ml anti-ANG-2 or all three test items. The final time point was used for statistical analysis. Error bars show SEM with one-sided ANOVA (P < 0.0001) and Tukey's multiple t-test for three independent experiments indicating significance of VEGF-A vs. untreated (****, P < 0.0001); vs. VEGF-A and ANG-1 and anti-ANG-2 (***, P = 0.0004); vs. VEGF-A and anti-ANG-2 (*, P = 0.038); vs. VEGF-A and ANG-1 (*, P = 0.040). Furthermore, VEGF-A and anti-ANG-2 and ANG-1 are significantly different vs. VEGF-A and ANG-1 (*, P = 0.0451); vs. VEGF-A and anti-ANG-2 (*, P = 0.0474); vs. untreated (*, P = 0.0492). Finally untreated is significantly different vs. VEGF-A and ANG-1 (***, P = 0.0009) and VEGF-A and anti-ANG-2 (***, P = 0.0008). Data information: ANOVA, analysis of variance; SEM, standard error of the mean; ANG-2, angiopoietin-2; DR, diabetic retinopathy; RVO, retinal vein occlusion; wAMD, wet age-related macular degeneration; pDR, proliferative diabetic retinopathy; vs., versus. Download figure Download PowerPoint Interplay of VEGF-A and angiopoietins in a model of endothelial barrier breakdown We then investigated the interplay between VEGF-A and the angiopoietins in a barrier breakdown model in human primary endothelial cells, in order to better understand some of the cellular aspects leading to hyperpermeability in patients (Fig 1C–E). Human endothelial cells in culture secrete large amounts of ANG-2 into the supernatant. VEGF-A increases the amount of ANG-2 even further, whereas ANG-1 reduces the secretion of ANG-2. Combined exposure of endothelial cells to VEGF-A and ANG-1 showed a trend to reduce ANG-2 production induced by VEGF-A (Fig 1D). Endothelial barrier breakdown is a key event in retinal eye disease, with edema being a major driver of pathology in the retina. VEGF-A is a known inducer of endothelial barrier breakdown and, indeed, we measured progressive loss of endothelial barrier function over time in our model. We then tested whether ANG-2 present in the culture contributes to VEGF-A-induced barrier breakdown. When adding anti-ANG-2 together with VEGF-A, barrier breakdown was reduced. This suggests that VEGF-A, at least partly, signals via ANG-2 to trigger endothelial barrier breakdown. The addition of ANG-1 also reduced VEGF-A-induced barrier breakdown, demonstrating an improved endothelial monolayer integrity function of ANG-1. Combined addition of anti-ANG-2 and ANG-1 reduced the VEGF-A-induced barrier breakdown even further (Fig 1E). The dynamic nature of VEGF-A-induced barrier breakdown was demonstrated when a bispecific anti-VEGF-A/ANG-2 was added 18 h after the addition of VEGF-A. TEER values reverted back to values before the addition of VEGF-A to the culture (Appendix Fig S1). The results confirm the concept of VEGF-A and ANG-2 being drivers of endothelial barrier breakdown, while ANG-1 counteracts these activities and promotes normal vessel integrity. Monotherapy of anti-VEGF-A vs. combination therapy of anti-VEGF-A and anti-ANG-2 in rodent models of ocular neoangiogenesis We then turned to a model of spontaneous CNV (sCNV) in the JR5558 mouse strain to further probe the potential for anti-VEGF-A/ANG-2 combination therapy. The sCNV model develops leaky, neovascular tufts arising from the choriocapillaries, which resemble human choroidal lesions. The pathological consequences of these leaky neovessels distorting the retinal architecture can be seen by increased neuroretinal cell death and loss of retinal functionality observed using electroretinography. The model also shows increased expression of VEGF mRNA in the RPE/choroid and retina when compared to wild-type mice. Inhibition of VEGF receptor-2 using a blocking antibody reduced the severity of neovascularization, making this a suitable model to test for combination therapies that enhance the efficacy of anti-VEGFs (Nagai et al, 2014). We first probed for ANG-2 expression in the model and found that ANG-2 mRNA was elevated in the choroid/RPE, but not retina, of JR5558 mice compared to wild-type mice starting at day 27 and increasing further as measured at days 50 and 62 (Fig 2A and B). Figure 2. Inhibition of vessel leakiness and lesion number by combined inhibition of VEGF-A and ANG-2 in the model of sCNV in JR5558 mice using fluorescence angiography A, B. Real-time qPCR analysis of ANG-2 levels in retina and RPE/choroid complexes of JR5558 and C57BL/6J (C57) mice. (A) Relative expression levels of ANG-2 were significantly increased in the RPE/choroid complexes of JR5558 mice in comparison with C57, at 50 and 62 days old (*, P = 0.022 at D50 and *, P = 0.042 at D62). (B) By contrast, retina levels of ANG-2 were not significantly different between C57 and JR5558 mice, indicating neovascularization is driven locally by ANG-2. Asterisk (*) denotes statistical significance of JR5558 mice compared to wild-type C57BL/6 using unpaired t-test for each time point analyzed separately. C. Schematic presentation of experimental design. Mice received IP injections of CrossMAb anti-VEGF-A/ANG-2 (species crossreactive, B20-4.1 and LC10), anti-ANG-2 (LC10), an anti-VEGF-A IgG1 (B20-4.1), or IgG control at postnatal D14 and D19 followed by fluorescence angiography at D26 (early intervention), results demonstrated in (D–K). Alternatively, mice received antibody at postnatal D47 and D55 followed by fluorescence angiography at D60 (late intervention), results demonstrated in (L) and (M). D–I. Representative examples of fluorescence angiograms of IgG control (D), anti-VEGF-A (E), anti-ANG-2 (F) (all at 5 mg/kg), and three doses of anti-VEGF-A/ANG-2 (G, H, and I) (at 3, 5, and 10 mg/kg). J, K. Bar graph of numbers of spontaneously occurring lesions (J) and area by fluorescence angiography (K) after two weekly doses of antibody (antibodies at 5 mg/kg IP and anti-VEGF-A/ANG-2 at 10 [high], 5 [mid], and 3 [low] mg/kg IP) followed by analysis a week later in the early intervention model. L, M. Bar graph of numbers of spontaneously occurring lesions (L) and area by fluorescence angiography (M) after two weekly doses of antibody (3 mg/kg IP) followed by analysis a week later in the late intervention model. Data information: SEM is shown as error bars with n = 4 (A, B) or n = 8 (J–M) animals per group and significance indicated by * using ANOVA (J, K: P < 0.0001; L: P < 0.0018; M: P < 0.031 followed by Tukey's multiple t-test in J–M). In (J), IgG control is significant against anti-VEGF-A (****, P < 0.0001), anti-ANG-2 (**, P = 0.0069), anti-VEGF-A/ANG-2 low (*, P = 0.0194), mid and high (****, P < 0.0001). Furthermore, anti-VEGF-A/ANG-2 high is significant against anti-VEGF-A (*, P = 0.0428), anti-ANG-2 (****, P < 0.0001), and anti-VEGF-A/ANG-2 low (****, P < 0.0001). Finally, anti-ANG-2 is significantly different from anti-VEGF-A/ANG-2 mid (**, P < 0.0041). In (K), IgG control is significant against anti-VEGF-A (***, P = 0.0003) and anti-VEGF-A/ANG-2 mid and high (both ****, P < 0.0001). Furthermore, anti-VEGF-A/ANG-2 high is significantly different from anti-VEGF-A (**, P = 0.0037), anti-ANG-2 (****, P < 0.0001), and anti-VEGF-A/ANG-2 low (***, P = 0.0001). Finally, anti-ANG-2 is significantly different from anti-VEGF-A/ANG-2 mid (**, P < 0.0022) and anti-VEGF-A/ANG-2 low against mid (*, P = 0.022). In (L), IgG control is significantly different from anti-ANG-2 (*, P = 0.023) and anti-VEGF-A/ANG-2 (*, P = 0.014). Vehicle is different from anti-ANG-2 (*, P = 0.024) and anti-VEGF-A/ANG-2 (*, P = 0.016). In (M), IgG control is significantly different from anti-ANG-2 (*, P = 0.044) and anti-VEGF-A/ANG-2 (*, P = 0.049). D, day; IP, intraperitoneal; sCNV, spontaneous choroidal neovascularization. Download figure Download PowerPoint Next, we directly tested the concept of combined inhibition of VEGF-A and ANG-2 being more efficacious than VEGF-A inhibition alone in preventing neovascularization and its associated endothelial dysfunction. In all rodent experiments, we used the rodent crossreactive B20-4.1 Fab arm (Liang et al, 2006) and the anti-ANG2-LC10 antibody generated as CrossMAbs or IgGs, as indicated (Schaefer et al, 2011). Since neovascular tufts arise from the choriocapillaris between postnatal day (D)10 and D15 and lesion numbers peak at approximately D30 in this model (Nagai et al, 2014), administration of antibodies was initiated at D14 and D19 by intraperitoneal (IP) injection (termed early intervention mode; Fig 2C). At D26, fluorescein angiography (FA) was performed, and lesion number and size were evaluated. Anti-VEGF-A and anti-ANG-2 were tested at 5 mg/kg while the anti-VEGF-A/ANG-2 antibody was tested in a dose–response with 10, 5, and 3 mg/kg. A normal IgG1 can bind two molecules of VEGF-A or ANG-2 (one per Fab arm), but an anti-VEGF-A/ANG-2 CrossMAb only one of each ligand. Therefore, the 10 mg/kg high dose of the anti-VEGF-A/ANG-2 CrossMAb has an equal molar concentration of binding sites as 5 mg/kg of standard IgG1 (anti-VEGF-A, anti-ANG-2, or IgG control). Anti-VEGF-A reduced the number and lesion area significantly compared to IgG control, while anti-ANG-2 alone showed a trend toward reduction. The highest doses of anti-VEGF-A/ANG-2 CrossMAb significantly reduced the number and size of lesions compared to IgG control and anti-VEGF-A or anti-ANG-2 monotherapy alone (Fig 2D–K). Choroidal neovascularization progressively damages the retina, so later time points were investigated in the mouse model to more closely reflect the clinical situation in which a patient presents with established disease. In the late intervention model, antibody is given when a considerable number of lesions with parainflammation and neuronal cell death are already established (Nagai et al, 2014). Antibody was given once a week for 2 weeks from D47 onwards, with examination at D60. In these experiments, we compared both anti-VEGF-A and anti-ANG-2 alone to bispecific VEGF-A/ANG-2 CrossMAb antibody (CrossMAb and IgG control at 3 mg/kg dose, VEGF-A and ANG-2 antibodies at 1.5 mg/kg). The reduction in the number of lesions with this late intervention regimen was less compared to early intervention when antibodies were administered while lesions were developing (Fig 2L). However, despite a small reduction in lesion number by anti-ANG-2 and anti-VEGF-A/ANG-2, the reduction did reach statistical significance compared to IgG control and vehicle (Fig 2L). In contrast to lesion number, pronounced reductions in the area of lesions were apparent for anti-VEGF-A/ANG-2
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