A Fully Human Monoclonal Antibody Targeting Ckit Is a Potent Inhibitor of Pathological Choroidal Neovascularization in Mice

pharmaceutics

Article A Fully Human Monoclonal Antibody Targeting cKIT Is a Potent Inhibitor of Pathological Choroidal Neovascularization in Mice

Songyi Seo 1,2, Koung Li Kim 2, Yeongju Yeo 1,2, Ryul-I Kim 1,2, Hayoung Jeong 1,2, Jin-Ock Kim 3, Sun-Hwa Song 3, Mi-Jin An 4 , Jung-Woong Kim 4 , Hye Kyoung Hong 5, Min Hee Ham 5, Se Joon Woo 5 , Jong-Hyuk Sung 6, Sang Gyu Park 3,* and Wonhee Suh 1,2,*

1 Department of Global Innovative Drug, Graduate School of Chung-Ang University, Seoul 06974, Korea; [email protected] (S.S.); [email protected] (Y.Y.); [email protected] (R.-I.K.); [email protected] (H.J.) 2 College of Pharmacy, Chung-Ang University, Seoul 06974, Korea; [email protected] 3 College of Pharmacy, Ajou University, Suwon 16499, Korea; [email protected] (J.-O.K.); [email protected] (S.-H.S.) 4 Department of Life Science, Chung-Ang University, Seoul 06974, Korea; [email protected] (M.-J.A.); [email protected] (J.-W.K.) 5 Department of Ophthalmology, Seoul National University College of Medicine, Seoul National University Bundang Hospital, Seongnam 13620, Korea; [email protected] (H.K.H.); [email protected] (M.H.H.); [email protected] (S.J.W.) 6 College of Pharmacy, Yonsei University, Incheon 21983, Korea; [email protected] * Correspondence: [email protected] (S.G.P.); [email protected] (W.S.); Tel.: +82-31-219-3491 (S.G.P.); +82-2-820-5960 (W.S.) Citation: Seo, S.; Kim, K.L.; Yeo, Y.; Kim, R.-I.; Jeong, H.; Kim, J.-O.; Song, Abstract: Stem cell factor (SCF) and its receptor, cKIT, are novel regulators of pathological neo- S.-H.; An, M.-J.; Kim, J.-W.; Hong, H.K.; et al. A Fully Human vascularization in the eye, which suggests that inhibition of SCF/cKIT signaling may be a novel Monoclonal Antibody Targeting cKIT pharmacological strategy for treating neovascular age-related macular degeneration (AMD). This Is a Potent Inhibitor of Pathological study evaluated the therapeutic potential of a newly developed fully human monoclonal antibody Choroidal Neovascularization in targeting cKIT, NN2101, in a murine model of neovascular AMD. In hypoxic human endothelial Mice. Pharmaceutics 2021, 13, 1308. cells, NN2101 substantially inhibited the SCF-induced increase in angiogenesis and activation of the https://doi.org/10.3390/ cKIT signaling pathway. In a murine model of neovascular AMD, intravitreal injection of NN2101 pharmaceutics13081308 substantially inhibited the SCF/cKIT-mediated choroidal neovascularization (CNV), with efficacy comparable to aflibercept, a vascular endothelial growth factor inhibitor. A combined intravitreal Academic Editors: Monica M. injection of NN2101 and aflibercept resulted in an additive therapeutic effect on CNV. NN2101 neither Jablonski and Uday B. Kompella caused ocular toxicity nor interfered with the early retinal vascular development in mice. Ocular pharmacokinetic analysis in rabbits indicated that NN2101 demonstrated a pharmacokinetic profile Received: 9 July 2021 Accepted: 17 August 2021 suitable for intravitreal injection. These findings provide the first evidence of the potential use of the Published: 20 August 2021 anti-cKIT blocking antibody, NN2101, as an alternative or additive therapeutic for the treatment of neovascular AMD.

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in Keywords: age-related macular degeneration; choroidal neovascularization; cKIT; fully human published maps and institutional afﬁl- monoclonal antibody; stem cell factor iations.

1. Introduction

Copyright: © 2021 by the authors. Neovascular age-related macular degeneration (AMD) is a common cause of irre- Licensee MDPI, Basel, Switzerland. versible vision loss predominantly affecting the elderly population [1]. The pathology of This article is an open access article neovascular AMD is characterized by choroidal neovascularization (CNV), where new distributed under the terms and immature blood vessels exhibit growth from the underlying choriocapillary into the sub- conditions of the Creative Commons retinal pigment epithelium or subretinal space [2]. These immature blood vessels present Attribution (CC BY) license (https:// with leakage of ﬂuids and blood into and under the retina, leading to vision loss or distor- creativecommons.org/licenses/by/ tion. Vascular endothelial growth factor (VEGF) plays a pivotal role in pathological NV 4.0/).

Pharmaceutics 2021, 13, 1308. https://doi.org/10.3390/pharmaceutics13081308 https://www.mdpi.com/journal/pharmaceutics Pharmaceutics 2021, 13, 1308 2 of 14

during the development of neovascular AMD, and anti-VEGF therapy (such as aflibercept, ranibizumab, and bevacizumab) is considered a standard treatment for neovascular AMD in clinical practice [2–4]. However, there have been concerns regarding the potential long-term side effects of anti-VEGF therapy because of the inhibition of physiological VEGF function [5–11]. Therefore, there is an immense need for identifying previously unrecognized disease targets and for developing novel therapeutics. We previously identified stem cell factor (SCF) and its receptor, cKIT, as novel targets for neovascular eye diseases [12]. At hypoxia, cKIT was highly expressed in vascular endothelial cells, and it enhanced SCF-mediated angiogenesis through the glycogen synthase kinase (GSK)-3β/β-catenin pathway. The hypoxia-induced increase in SCF/cKIT signaling mediated pathological ocular NV in murine models of neovascular AMD and proliferative diabetic retinopathy (PDR). In addition, there is substantial evidence implicating SCF and cKIT in human neovascular eye diseases [13,14]. The expression of SCF and cKIT is highly upregulated in patients with active PDR compared to that in patients with quiescent PDR or nondiabetic patients [13,14]. These findings suggest that anti-SCF/cKIT therapy is a promising therapeutic strategy for treating neovascular eye diseases. Recently, NN2101, a fully human monoclonal antibody (IgG) targeting cKIT, was generated and characterized [15]. NN2101 binds to the SCF-binding regions of human cKIT with a dissociation constant (KD) in the picomolar range. Western blotting and competitive enzyme-linked immunosorbent assay (ELISA) indicated that NN2101 completely inhibited the binding of cKIT to SCF and blocked the activation of cKIT downstream signaling molecules in human leukemic cells. In addition, NN2101 exhibits high specificity to cKIT, reduced effector function, and low immunogenicity. In this study, we investigated the therapeutic efficacy of NN2101 in a murine model of neovascular AMD and determined the ocular toxicity and pharmacokinetics (PK) of NN2101.

2. Materials and Methods 2.1. Cell Culture Human umbilical vein endothelial cells (HUVECs; Lonza, Walkersville, MD, USA), murine endothelial cells (American Type Culture Collection, Manassas, VA, USA), human retinal microvascular endothelial cells (HRMEC; Cell Systems, Kirkland, WA, USA), human ocular choroid ﬁbroblast (HOCF; ScienCell, Carlsbad, CA, USA), human corneal ﬁbroblast (HK; ScienCell, Carlsbad, CA, USA), and human retinal pigmented epithelial cells (ARPE- 19; American Type Culture Collection, Manassas, VA, USA) were cultured with appropriate culture media, according to the manufacturer’s protocol. For hypoxic treatment, cells were placed in a modular incubator chamber (MIC-101; Billups-Rothenberg Inc., Del Mar, CA, USA) in an atmosphere consisting of 93% N2, 5% CO2, and 2% O2.

2.2. Western Blotting Cells and tissues were lysed with lysis buffer (Thermo Scientiﬁc, Rockford, IL, USA). The proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophore- sis and then transferred to a nitrocellulose membrane. Nuclear and cytosolic fractions of β-catenin were separated using nuclear and cytoplasmic extraction reagent (Thermo Scientiﬁc, Rockford, IL, USA), according to the manufacturer’s instructions. Blots were hybridized with the appropriate primary IgG, followed by incubation with horseradish peroxidase-conjugated secondary IgG. Immunoreactive bands were visualized using a chemiluminescent reagent (Amersham Biosciences, Piscataway, NJ, USA). The IgGs used in the experiment are listed in Supplementary Table S1. Densitometry was performed using ImageJ software (National Institute of Health, Bethesda, MD, USA).

2.3. In Vitro Angiogenesis Assay To analyze the inhibitory effect of NN2101 on SCF-induced in vitro angiogenesis at hypoxia, tube formation, scratch wound migration, and cell proliferation assays were performed as previously described [16]. For the tube formation assay, HUVECs were Pharmaceutics 2021, 13, 1308 3 of 14

seeded in 24-well plates coated with Matrigel (Corning, Tewksbury, MA, USA) and were then subjected to treatment with endothelial basal medium (EBM, Lonza, Walkersville, MD, USA) containing 1% fetal bovine serum (FBS, Lonza, Walkersville, MD, USA) or supplemented with recombinant human SCF (rh SCF; R&D Systems, Minneapolis, MN, USA; 50 ng/mL) and/or NN2101 (1 µg/mL) for 6 h. Tube networks were quantified by measuring the tube lengths. For the scratch wound migration assay, confluent monolayers of HUVECs were wounded using pipette tips and incubated for 24 h in EBM containing 1% FBS or supplemented with rh SCF (50 ng/mL) and/or NN2101 (1 µg/mL). Cell migration was assessed by measuring the area of cells that had migrated from the wound edges. For the cell proliferation assay, HUVECs were seeded into 96-well plates and incubated for 2 days in EBM containing 1% FBS or supplemented with rh SCF (50 ng/mL) and/or NN2101 (1 µg/mL). Cell proliferation was quantified using a cell counting kit-8 assay (Dojindo Molecular Technology, Inc., Rockville, MD, USA). In addition, in vitro angiogenesis assays were performed similarly in murine endothelial cells that were subjected to treatment with or without recombinant murine SCF (rm SCF; R&D Systems, Minneapolis, MN, USA; 50 ng/mL) and NN2101 (1 µg/mL).

2.4. Animals Six- to eight-week-old male mice and New Zealand white male rabbits weighing 1.5–2 kg were purchased from Orient Co. (Seoul, Korea). The animals were cared for in accordance with the Guide for the Care and Use of Laboratory Animals published by the United States National Institutes of Health. All animal procedures used in this study were approved by the Institutional Animal Care and Use Committees (IACUC) of Chung-Ang University (IACUC number: 201800044, start date: 4 May 2018) and Seoul National Univer- sity Bundang Hospital (IACUC number: 2018BA1801-239/003/02, start date: 9 January 2018). For the surgical procedures, mice were anesthetized with an intraperitoneal injection of a mixture of ketamine (100 mg/kg; Yuhan Co., Seoul, Korea) and xylazine (6 mg/kg; Bayer Korea, Ltd., Seoul, Korea). Pupils were dilated using topical drops of phenylephrine and tropicamide (Santen, Osaka, Japan). Rabbits were anesthetized with an intramuscular injection of Zoletil (a mixture of tiletamine and zolazepam; 15 mg/kg; Virbac Laboratories, Carros, France) and xylazine (5 mg/kg) and the pupils were dilated with ophthalmic drops of 1% proparacaine (Alcon Laboratories, Inc., Fort Worth, TX, USA). The level of anesthesia was monitored by toe-pinch reﬂex. Euthanasia was performed by cervical dislocation following in anesthesia.

2.5. Animal Model of Laser-Induced CNV Laser photocoagulation (wavelength: 532 nm; fixed diameter: 50 µm; intensity: 300 mW; duration: 0.70 s) was induced to create experimental CNV lesions in mice using an image-guided laser system (Micron IV, Phoenix Research Laboratories, Pleasanton, CA, USA), as previously described [17]. Immediately after laser injury, mice received an intravitreal injection of NN2101, aflibercept (Regeneron Pharmaceuticals, Tarrytown, NY, USA), a combination of NN2101 and aflibercept, or phosphate buffered saline (PBS) vehicle. For the quantification of laser-induced CNV lesions, eyes were enucleated 7 days after laser photocoagulation. The posterior eye-cups consisting of the retinal pigment epithelium (RPE), choroid, and sclera were dissected. The dissected RPE-choroid-sclera were subjected to treatment with blocking solution (5% bovine serum albumin, 5% normal goat serum, and 0.5% Triton X-100 in PBS) for 1 h at room temperature and stained with Alexa Fluor® 594-conjugated isolectin Griffonia simplicifolia IB4 (1:50 dilution; Invitrogen, Carlsbad, CA, USA) overnight at 4 ◦C and flat-mounted onto slides. Images of CNV lesions were obtained using a confocal microscope (Carl Zeiss, Jena, Germany), and the fluorescence intensity of each image was analyzed using ImageJ software (version 1.52, National Institutes of Health, Bethesda, MD, USA) [17]. CNV areas of samples were expressed relative to that of controls. Pharmaceutics 2021, 13, 1308 4 of 14

2.6. Combination Index (CI) CI analysis on dose-response data was performed to classify drug interactions into the categories of synergy, additivity, or antagonism. Dose-response data for NN2101 and aﬂibercept were plotted and analyzed using GraphPad Prism software (GraphPad Software Inc., San Diego, CA, USA), and the CI value was calculated using the formula proposed by Chou and Talalay [18]. A CI of less than, equal to, or more than 1 indicates antagonism, additivity, or synergy, respectively.

2.7. Immunohistochemistry and Terminal Deoxynucleotidyl Transferase dUTP Nick End-Labeling (TUNEL) Assay Eyes were enucleated, fixed, and embedded in paraffin. After quenching endogenous peroxidase activity and blocking with 10% normal goat serum, tissue sections were stained with anti-glial fibrillary acidic protein (GFAP) IgG and fluorescent secondary IgG. Nuclei were stained with 40,6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA, USA). Images were obtained using a confocal microscope. All images shown are representative of >3 independent experiments. TUNEL assays were performed on paraffin sections using the in situ cell death detection kit (Roche Applied Science, Mannheim, Germany). Tissue sections subjected to treatment with DNase I (1500 U/mL, Roche Ap- plied Science) were used as a positive control. Images were obtained using confocal or fluorescence microscope (Olympus, Tokyo, Japan). Six to ten sections were examined per group.

2.8. Live and Dead Cell Double Staining Assay Cells were seeded in 96-well plates and incubated in complete growth medium supplemented with or without NN2101. Three days later, cells were stained with calcein-AM (Sigma-Aldrich, St. Louis, MO, USA; 2 µg/mL), propidium iodide (PI; Sigma-Aldrich; 2 µg/mL), and Hoechst (Sigma-Aldrich; 20 µM) to determine the percentage of live or dead cells using Celigo (Nexelom Bioscience, Lawrence, MA, USA).

2.9. Ocular Pharmacokinetics (PK) Ocular PK analysis was performed as previously described [19]. In brief, NN2101 (600 µg in 30 µL) was intravitreally injected into the right eyes of anesthetized and pupil- dilated rabbits, 1 mm behind the surgical limbus in the superotemporal quadrant using a 30-gauge needle and a Hamilton syringe. Left eyes received no intravitreal injection. Three rabbits were sacriﬁced at each time point (1 h, 1, 3, 6, 9, 14, and 30 days after injection). The serum was harvested, and both the right and left eyes were enucleated and immediately frozen at −80 ◦C. The frozen eyes were separated into three parts: vitreous humor, aqueous humor, and retina/choroid. The concentration of NN2101 was measured using an indirect ELISA. The concentration-time data of NN2101 were ﬁt using a standard non-compartmental analysis to determine the PK parameters using WinNonlin software version 6.4 (Certara, Ronceton, NJ, USA).

2.10. Retinal Vascular Development NN2101 was injected intravitreally into mice at postnatal day (P) 2, and retinal blood vessels were analyzed at P6. Radial length of retinal blood vessels was measured as the shortest distance from the optic disc to the peripheral vascular front in each quadrant of the retina. Number of sprouting endothelial cells was measured in 0.02 mm2 areas of vascular front in each retina. Vascular density in whole-mounted retina was measured as an IB4-positive area divided by measured total area of the retina; it was presented as a percentage. For each quantiﬁcation, at least four images per whole-mount retina were obtained and analyzed using ImageJ software or LSM Image software (Carl Zeiss, Jena, Germany). Pharmaceutics 2021, 13, x FOR PEER REVIEW 5 of 14

areas of vascular front in each retina. Vascular density in whole-mounted retina was measured as an IB4-positive area divided by measured total area of the retina; it was presented as a percentage. For each quantification, at least four images per whole-mount Pharmaceutics 2021, 13, 1308 5 of 14 retina were obtained and analyzed using ImageJ software or LSM Image software (Carl Zeiss, Jena, Germany).

2.11.2.11. StatisticalStatistical Analysis GraphPadGraphPad PrismPrism softwaresoftware was usedused to analyze the data. data. All All data data in in the the article article passed passed thethe normalitynormality andand equalequal variancevariance tests.tests. Statistical Statistical significance signiﬁcance was was evaluated evaluated using using an an unpairedunpaired Student’sStudent’s tt-test-test or one-wayone-way ANOVA with Bonferroni’s Bonferroni’s post post hoc hoc multiple multiple com- com- parisonparison test.test. TheThe datadata areare presentedpresented asas thethe meanmean ± SEMSEM with with pp-values-values < < 0.05 0.05 considered considered signiﬁcant.significant. TheThe number of samples is indicated using using nn..

3.3. ResultsResults 3.1.3.1. TheThe Anti-cKITAnti-cKIT Blocking IgG, NN2101, Inhibits SCF/cKIT-Mediated SCF/cKIT-Mediated Angiogenic Angiogenic Signaling Signaling andand AngiogenesisAngiogenesis inin HumanHuman Endothelial Cells at Hypoxia OurOur previousprevious studystudy showedshowed that,that, inin hypoxichypoxic endothelialendothelial cells, SCF/cKIT signaling signaling β β enhancesenhances angiogenesisangiogenesis byby activatingactivating thethe GSK-3GSK-3β/β-catenin-catenin pathway pathway [12]. [12]. To To investigate investigate whetherwhether thethe anti-cKIT anti-cKIT IgG, IgG, NN2101, NN2101, blocked blocked the the activation activation of the of SCF/cKITthe SCF/cKIT signaling signaling pathway,pathway, we performed we performed Western Western blotting blotting and found and found that treatment that treatment with NN2101 with NN2101 substantially sub- β inhibitedstantially the inhibited SCF-mediated the SCF-mediated phosphorylation phosphorylation of cKIT, AKT, of and cKIT, GSK-3 AKT,in and hypoxic GSK-3 humanβ in β endothelialhypoxic human cells endothelial (Figure1A). cells The phosphorylation(Figure 1A). The phosphorylation of GSK-3 induces of GSK-3 the phosphorylationβ induces the β β andphosphorylation nuclear translocation and nuclear of translocation-catenin, leading of β-catenin, to the transcription leading to the of transcription-catenin target of genesβ-catenin (Figure target1B,C). genes Therefore, (Figure the1B,C). treatment Therefore, with the NN2101 treatment abrogated with NN2101 the SCF-induced abrogated β β increasethe SCF-induced in the nuclear increase fraction in the of nuclear-catenin fraction and the of β protein-catenin expression and the protein of -catenin expression target genesof β-catenin related target to angiogenesis genes related (VEGFA, to angiogenesis IL-8, c-Myc, (VEGFA, and IL-8, CyclinD1) c-Myc, in and hypoxic CyclinD1) human in endothelialhypoxic human cells (Figureendothelial1B,C). cells Moreover, (Figure the1B,C treatment). Moreover, ofhuman the treatment endothelial of human cells with en- NN2101dothelial completely cells with NN2101 inhibited completely the SCF-induced inhibited increase the SCF-induced in tube formation, increase scratch in tube wound for- migration,mation, scratch and cellwound proliferation migration, at and hypoxia cell proliferation (Figure1D–F). at hypoxia (Figure 1D–F).

Figure 1. Cont.

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Figure 1. TheFigure anti-cKIT 1. The blocking anti-cKIT IgG, blocking NN2101, IgG, inhibits NN2101, SCF/cKIT-mediated inhibits SCF/cKIT angiogenic-mediated signalingangiogenic and signaling angiogenesis and angiogenesis in in human endothelialhuman cells endothelial at hypoxia. cells (A at) NN2101hypoxia. inhibits(A) NN2101 the SCF-induced inhibits the SCF-induced phosphorylation phosph of cKIT,orylation AKT, of andcKIT, GSK-3 AKT,β andin GSK-3β in hypoxic endothelialhypoxic cells. endothelial HUVECs cells. cultured HUVECs at hypoxia cultured were at hypoxia subjected were to treatmentsubjected to with treatment or without with recombinant or without recombinant human human SCF (rh SCF) (50SCF ng/mL) (rh SCF) and (50 NN2101 ng/mL) (5 µandg/mL). NN2101 Phosphorylation (5 μg/mL). Phosphorylation of cKIT (p-cKIT; arrowhead),of cKIT (p-cKIT; AKT (p-AKT),arrowhead), and GSK-3AKT (p-AKT),β and (p-GSK-3β; Ser9)GSK-3 wasβ assessed(p-GSK-3 usingβ; Ser9) Western was assessed blotting. using Band Western intensities blotting. of phosphorylated Band intensities proteins of phosphorylated were normalized proteins to were normalized to those of total proteins and expressed as the fold increase ± SEM, compared to those in the untreated control those of total proteins and expressed as the fold increase ± SEM, compared to those in the untreated control group. (set as 1; group. (set as 1; one-way ANOVA with Bonferroni post hoc multiple comparison test, * p < 0.05, ** p < 0.001, n = 3). (B) one-way ANOVA with Bonferroni post hoc multiple comparison test, * p < 0.05, ** p < 0.001, n = 3). (B) NN2101 blocks the NN2101 blocks the SCF-induced increase in the nuclear translocation of β-catenin in hypoxic endothelial cells. Nuclear SCF-induced increaseand cytosolic in the fractions nuclear translocationof β-catenin in of cellsβ-catenin subjected in hypoxic to treatment endothelial as described cells. Nuclear in (A) were and cytosolic determined fractions by Western blot- of β-catenin inting cells analysis. subjected PARP to treatment and β-Actin as described were used in as (A internal) were determined loading controls by Western for nuclear blotting and analysis. cytosol fractions, PARP and respectively. β-Actin were usedBand as intensities internal loading of the nuclear controls fraction for nuclear of β-catenin and cytosol were fractions, normalized respectively. to the PARP Band band intensities intensities of the and nuclear expressed as the fraction of β-cateninfold increase were normalized ± SEM, compared to the PARPto those band in the intensities untreated and control expressed group as (set the as fold 1; one-way increase ±ANOVASEM, compared with Bonferroni post to those in thehoc untreated multiple control comparison group test, (set as*** 1; p one-way< 0.001, n ANOVA = 3). (C with) NN2101 Bonferroni blocks postthe SCF-induced hoc multiple in comparisoncrease in the test, expression of *** p < 0.001, nβ=-catenin 3). (C) target NN2101 genes blocks in hypoxic the SCF-induced endothelial increase cells. The in protein the expression levels of VEGFA, of β-catenin IL-8, targetc-Myc, genes and CyclinD1 in hypoxic were assessed as described in (A) (one-way ANOVA with Bonferroni post hoc multiple comparison test, * p < 0.05, ** p < 0.01, n = 3). endothelial cells. The protein levels of VEGFA, IL-8, c-Myc, and CyclinD1 were assessed as described in (A) (one-way (D–F) NN2101 suppresses the SCF-induced angiogenesis in hypoxic endothelial cells. Tube formation (D), scratch wound ANOVA withmigration Bonferroni (E post), and hoc cellmultiple proliferation comparison assays (F test,) were * p performed< 0.05, ** withp < 0.01, HUVECsn = 3). subjecte (D–F)d NN2101to treatment suppresses with or without rh the SCF-inducedSCF angiogenesis (50 ng/mL) and in hypoxicNN2101 endothelial (1 μg/mL) at cells. hypoxia. Tube formationTube formation (D), scratchand scrat woundch wound migration migration (E), andwere cell quantified by proliferation assaysmeasuring (F) were the tube performed length and with relative HUVECs area subjected covered by to cells treatment that had with migrated or without from rh the SCF wound (50 ng/mL)edges, respectively. and Cell NN2101 (1 µg/mL)proliferation at hypoxia. was Tubedetermined formation using and a scratchcell counting wound kit-8 migration assay. wereThe data quantiﬁed are expressed by measuring as the the fold tube increase length ± SEM com- and relative areapared covered to that by in cells the thatuntreated had migrated control group from the(Cont) wound (one-way edges, ANOVA respectively. with CellBonferroni proliferation post hoc was multiple determined comparison test, using a cell counting* p < 0.05, kit-8 ** p assay.< 0.01, Then > 4). data Scale are bars expressed = 200 μm. as the fold increase ± SEM compared to that in the untreated control group (Cont) (one-way ANOVA with Bonferroni post hoc multiple comparison test, * p < 0.05, ** p < 0.01, n > 4). 3.2. Intravitreal Administration of NN2101 Suppresses SCF/cKIT Signaling and the Pathological Scale bars = 200 µm. CNV in a Murine Model of Neovascular AMD

3.2. Intravitreal AdministrationNN2101 not only of NN2101 binds strongly Suppresses to SCF/cKIThuman cKIT Signaling (KD = 2.83 and the× 10 Pathological−12 M), but also binds CNV in a Murineto mouse Model cKIT of Neovascular with a high AMD affinity (KD = 11.5 × 10−9 M) [15]. Indeed, NN2101 completely −12 NN2101blocked not only the binds SCF-induced strongly toincrease human in cKIT migration (KD = 2.83and ×tube10 formationM), but of also murine binds endothelial −9 to mouse cKITcells with (Supplementary a high affinity Figure (KD = 11.5S1). ×Therefore,10 M) [15the]. cross-species Indeed, NN2101 reactivity completely of NN2101 al- blocked thelowed SCF-induced the assessment increase of in the migration efficacy and of NN2 tube101 formation in vivo. ofWe murine investigated endothelial the therapeutic cells (Supplementaryeffect of NN2101 Figure S1). on Therefore,pathological the NV cross-species in the choroid reactivity using of a NN2101common allowed murine model of the assessmentneovascular of the efficacy AMD of [17]. NN2101 Immediatelyin vivo. Weafter investigated laser photocoagulation, the therapeutic mice effect were of intravitre- NN2101 on pathologicalally injected with NV in NN2101 the choroid or an using equivalent a common volume murine of PBS model vehicle of neovascular(Figure 2A). Quantita- AMD [17]. Immediatelytive analysis of after CNV laser lesions photocoagulation, revealed that intravitreal mice were administration intravitreally injectedof NN2101 signifi- with NN2101cantly or an reduced equivalent the area volume of CNV of PBS lesions vehicle compared (Figure 2withA). Quantitativethe vehicle (Figure analysis 2B). The cho- of CNV lesionsroidal revealed tissues that of intravitrealNN2101-injected administration CNV mice of NN2101exhibited significantly a substantial reduced decrease in the the area of CNVphosphorylation lesions compared of cKIT, with GSK-3 theβ vehicle, and β-catenin (Figure2 toB). levels Thechoroidal as low as tissuesthat in the of choroidal NN2101-injectedtissues CNV of normal mice exhibitedmice (Figure a substantial 2C). Consistently, decrease protein in the levels phosphorylation of β-catenin oftarget genes cKIT, GSK-3(VEGFA,β, and β -cateninIL-8, and to levelsc-Myc) as lowwere as markedly that in the reduced choroidal in tissues the choroidal of normal tissues of mice (FigureNN2101-injected2C). Consistently, CNV protein mice levels (Figure of β 2D).-catenin These target results genes demonstrate (VEGFA, IL-8, that and NN2101 effi- c-Myc) wereciently markedly blocked reduced SCF/cKIT in the signaling choroidal and tissues reduced of the NN2101-injected pathological CNV CNV in a mice murine model (Figure2D ).of These neovascular results demonstrate AMD. that NN2101 efficiently blocked SCF/cKIT signaling and reduced the pathological CNV in a murine model of neovascular AMD.

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FigureFigure 2. Intravitreal2. Intravitreal administration administration of NN2101 of suppressesNN2101 suppre SCF/cKITsses signaling SCF/cKIT and signaling the pathological and the patholog- CNVical inCNV murine in modelmurine of neovascularmodel of neovascular AMD. (A) schematic AMD. diagram(A) schematic of laser-induced diagram CNV of experi-laser-induced CNV ments.experiments. Immediately Immediately after laser-induced after la ruptureser-induced of Bruch’s rupture membrane, of Bruch’s mice received membrane, an intravitreal mice received an in- injectiontravitreal of NN2101injection (1 ofµg NN2101 in 1 µL) or(1 anμg equivalentin 1 μL) or volume an equivalent of PBS vehicle. volume On of day PBS 7 aftervehicle. laser On day 7 after photocoagulation,laser photocoagulation, choroidal tissues choroidal were harvestedtissues were for further harvested analysis; for (B )further representative analysis; images (B of) representative flat-mountedimages of choroidsflat-mounted with CNV choroids lesions andwith quantification CNV lesions of CNV and lesion quantification area. Choroidal of flatCNV mounts lesion area. Cho- were stained with IB4 (red) and the relative CNV area was quantified by measuring the fluorescence roidal flat mounts were stained with IB4 (red) and the relative CNV area was quantified by meas- intensity of images with IB4-positive areas (unpaired Student’s t-test, * p < 0.05, n > 34 laser spots, uring the fluorescence intensity of images with IB4-positive areas (unpaired Student’s t-test, * p < n > 12 mice per group). Scale bar = 100 µm. (C) The phosphorylation of cKIT (p-cKIT; arrowhead), 0.05, n > 34 laser spots, n > 12 mice per group). Scale bar = 100 μm. (C) The phosphorylation of cKIT GSK-3β (p-GSK-3β; Ser9), and β-catenin (p-β-catenin; Ser33/37/Thr41) and (D) the protein levels (p-cKIT; arrowhead), GSK-3β (p-GSK-3β; Ser9), and β-catenin (p-β-catenin; Ser33/37/Thr41) and of β-catenin target genes (VEGFA, IL-8, and c-Myc) were assessed using Western blotting and are (D) the protein levels of β-catenin target genes (VEGFA, IL-8, and c-Myc) were assessed using expressed as the fold increase ± SEM compared to those in the normal (Nor) control group (one-way ANOVAWestern with blotting Bonferroni and post are hoc expressed multiple comparison as the fold test, in *creasep < 0.05, ± **SEMp < 0.01,compared *** p < 0.001, to thosen = 3). in the normal β(Nor)-Actin wascontrol used group as a loading (one-way control. ANOVA All data arewith presented Bonferroni as the meanpost hoc± SEM. multiple comparison test, * p < 0.05, ** p < 0.01, *** p < 0.001, n = 3). β-Actin was used as a loading control. All data are presented as 3.3.the A mean Combination ± SEM. Therapy of NN2101 and Aflibercept Additively Inhibits Pathological CNV in Mice 3.3.We A Combination next compared Therapy the therapeutic of NN2101 efficacies and Afliberc of NN2101ept Additively and aflibercept, Inhibits anPathological anti- CNV in VEGF agent used in clinical practice. At a dose of 1 µg, NN2101 significantly reduced Mice the pathological CNV with an efficacy comparable to that of the human equivalent dose (2.5 µg)We of afliberceptnext compared in mice the (Figure therapeutic3A). When effica relativecies CNV of areasNN2101 were plottedand aflibercept, as a an anti-VEGF agent used in clinical practice. At a dose of 1 μg, NN2101 significantly reduced the pathological CNV with an efficacy comparable to that of the human equivalent dose (2.5 μg) of aflibercept in mice (Figure 3A). When relative CNV areas were plotted as a function of logarithm of doses, we noted that both NN2101 and aflibercept exhibited a linear correlation between the dose and therapeutic effect (Figure 3B). We then investigated whether a combination of NN2101 and aflibercept would induce a synergistic, additive, or antagonistic effect. We conducted the constant ratio drug combination assay based on the Chou–Talalay CI method [18]. A combination of suboptimal doses of

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function of logarithm of doses, we noted that both NN2101 and aﬂibercept exhibited a Pharmaceutics 2021, 13, x FOR PEER REVIEW linear correlation between the dose and therapeutic effect (Figure3B). We8 thenof 14 investigated

whether a combination of NN2101 and aflibercept would induce a synergistic, additive, or antagonistic effect. We conducted the constant ratio drug combination assay based aflibercept (0.5on μ theg) and Chou–Talalay NN2101 (0.28 CI method μg) significantly [18]. A combination blocked the of pathological suboptimal dosesCNV of aflibercept µ µ with an effect(0.5 comparableg) and NN2101to that of (0.28 2.5 μgg) of significantly aflibercept (Figure blocked 3C). the Furthermore, pathological the CNV CI with an effect comparable to that of 2.5 µg of aflibercept (Figure3C). Furthermore, the CI value was value was calculated to be close to 1, which indicated that a combination therapy using calculated to be close to 1, which indicated that a combination therapy using NN2101 and NN2101 and aflibercept additively improved the therapeutic efficacy against the patho- aflibercept additively improved the therapeutic efficacy against the pathological CNV. logical CNV.

Figure 3. A combinationFigure 3. A therapycombination of NN2101 therapy and of aflibercept NN2101 and additively afliberc inhibitsept additively pathological inhibits CNV pathological in mice. (A )CNV Quantification of CNV lesioninarea mice. of ( miceA) Quantification subjected to treatment of CNV withlesion an area intravitreal of mice injectionsubjected of to PBS treatment or the indicated with an doseintravitreal of either NN2101 or afliberceptinjection (one-way of ANOVA PBS or withthe Bonferroniindicated dose post hocof eith multipleer NN2101 comparison or aflibercept test, * p < (one-way 0.05, ** p < ANOVA 0.01 vs. PBS, withn > 17 laser spots, n > 6 miceBonferroni per group); post hoc (B) multiple dose–response comparison data oftest, NN2101 * p < 0.05, and ** aflibercept p < 0.01 vs. in PBS, (A) weren > 17 plotted laser spots, with superimposedn > 6 mice per group); (B) dose–response data of NN2101 and aflibercept in (A) were plotted with su- linear regression lines; (C) representative images of flat-mounted choroids with CNV lesions and quantification of CNV perimposed linear regression lines; (C) representative images of flat-mounted choroids with CNV lesion area of mice subjected to treatment with an intravitreal injection of NN2101, aflibercept, combination of NN2101 and lesions and quantification of CNV lesion area of mice subjected to treatment with an intravitreal aflibercept, orinjection PBS (one-way of NN2101, ANOVA aflibercept, with Bonferroni combination post of hoc NN2101 multiple and comparison aflibercept, test, or PBS ns = (one-way not significant, ANOVA * p < 0.05 vs. PBS, n > 21 laserwith spots,Bonferronin > 7 micepost perhoc group).multiple Scale comparison bar = 50 µtest,m. Allns data= not are significant, presented * asp < the 0.05 mean vs. ±PBS,SEM. n > 21 laser spots, n > 7 mice per group). Scale bar = 50 μm. All data are presented as the mean ± SEM. 3.4. NN2101 Does Not Induce Ocular Toxicity 3.4. NN2101 Does NotWe Induce investigated Ocular whetherToxicity an intravitreal injection of NN2101 induced retinal toxicity We investigatedin mice. whether Normal an adult intravitreal mice were injection intravitreally of NN2101 injected induced with retinal NN2101 toxicity at a dose of 20 µg, in mice. Normalwhich adult was mice 20-fold were higher intravitreally than the injected effective with dose NN2101 (1 µg) at in a CNV dose mice.of 20 μ Twog, weeks after which was 20-foldtreatment, higher TUNEL than the assay effective and GFAP dose immunohistochemistry (1 μg) in CNV mice. Two were weeks performed after to assess cell treatment, TUNELapoptosis assay and and retinal GFAP tissue immunohi stress, respectively.stochemistry Examination were performed of retinal to assess tissues revealed no cell apoptosisdifference and retinal in celltissue apoptosis stress, andresp GFAPectively. expression Examination between of theretinal NN2101-injected tissues re- and control vealed no differenceeyes (Figure in cell4 A,B).apoptosis These and results GFAP indicate expression the absence between of the intraocular NN2101-injected toxicity of NN2101 in and control eyesnormal (Figure adult 4A,B). mice. These In addition, results weindicate tested the whether absence NN2101 of intraocular exerted toxicity any antiproliferative of NN2101 in normal adult mice. In addition, we tested whether NN2101 exerted any antiproliferative or cytotoxic effect in human ocular cells, including retinal endothelial cells, choroidal and corneal fibroblasts, and RPE cells. The live and dead cell double staining assay showed that NN2101 did not impair the cell viability and induced no cy-

Pharmaceutics 2021, 13, 1308 9 of 14

Pharmaceutics 2021, 13, x FOR PEER REVIEW 9 of 14 or cytotoxic effect in human ocular cells, including retinal endothelial cells, choroidal and corneal ﬁbroblasts, and RPE cells. The live and dead cell double staining assay showed that NN2101 did not impair the cell viability and induced no cytotoxicity in any of the cells totoxicity in anysubjected of the cells to subjected treatment to with treatment NN2101 with at aNN concentration2101 at a concentration of 400 µg/mL, of 400 which was 2-fold μg/mL, which wasgreater 2-fold than greater the vitreousthan the concentrationvitreous concentration of NN2101 of NN2101 in CNV in mice CNV (1 miceµg NN2101 in 5 µL (1 μg NN2101 inmurine 5 μL murine vitreous vitreous ﬂuid corresponds fluid corresponds to 0.2 mg/mL) to 0.2 mg/mL) (Figure (Figure4C,D). 4C,D).

Figure 4. NN2101Figure 4. does NN2101 not induce does not ocular induce toxicity. ocular ( Atoxicity.,B) an intravitreal (A,B) an intravitreal injection ofinjection NN2101 of inducesNN2101 no induces ocular toxicity in normal adultno mice.ocular Mice toxicity received in normal a single adult intravitreal mice. Mice injection received of NN2101a single intravitreal (20 µg in 1 µinjectionL) or an of equivalent NN2101 volume(20 of PBS. Eyes wereμ harvestedg in 1 μL) two or an weeks equivalent after injection. volume TUNELof PBS. Eyes assay were (A) and harvested immunofluorescence two weeks after staining injection. with TUNEL anti-GFAP IgG (B) were performedassay ( onA) paraffinand immunofluorescence sections of the whole staining eyes. with In the anti-GFAP TUNEL assay,IgG (B DNase) were I-treatedperformed sections on paraffin were included as sections of the whole eyes. In the TUNEL assay, DNase I-treated sections were included as positive positive controls. In the immunofluorescence staining with anti-GFAP IgG, sections stained with irrelevant nonspecific IgGs controls. In the immunofluorescence staining with anti-GFAP IgG, sections stained with irrelevant were included as negative controls (NC). The nuclei are shown in blue (DAPI). Representative images in (A,B) were selected nonspecific IgGs were included as negative controls (NC). The nuclei are shown in blue (DAPI). from threeRepresentative independent experiments images in with(A,B) similar were selected results. Allfrom scale three bars independent = 50 µm. (C ,Dexperiments) NN2101 induces with similar no cytotoxicity in human ocularresults. cells. All HRMECs, scale bars HOCF, = 50 HK, μm. and (C APRE-19,D) NN2101 cells induces were incubated no cytotoxicity with NN2101 in human (400 µ g/mL)ocular orcells. PBS for 3 days, and the percentagesHRMECs, ofHOCF, calcein-AM-positive HK, and APRE-19 live cellscells (wereC) and incubated PI-positive with dead NN2101 cells (D (400) were μg/mL) assessed. or PBS The for data 3 are shown as the meandays,± SEM and (unpairedthe percentages Student’s of calcein-AM-positivet-test, ns = not significant, live cellsn = ( 6).C) and PI-positive dead cells (D) were assessed. The data are shown as the mean ± SEM (unpaired Student’s t-test, ns = not significant, n = 6). 3.5. NN2101 Does Not Affect Early Retinal Vascular Development in Mice We evaluated the effect of NN2101 on normal retinal vascular development in mice. 3.5. NN2101 DoesPreviously, Not Affect weEarly showed Retinal that Vascular depletion Development of SCF using in Mice an anti-SCF neutralizing IgG does not We evaluateddelay the the effect superficial of NN2101 retinal on norm vascularal retinal development vascular indevelopment mice, and cKit in mice.mutant (KitW-sh/W-sh) Previously, we showedmice displayed that depletion normal of retinalSCF using vascular an anti-SCF development neutralizing [12]. IgG Accordingly, does not we found no delay the superficialsignificant retinal difference vascular between development mice treated in mice, intravitreally and cKit mutant with NN2101 (KitW-sh/W-sh and) PBS vehicle, in mice displayedterms normal of vascularretinal vascular density, radial development length, and [12]. number Accordingly, of sprouting we cellsfound (Figure no 5). These data significant differenceindicate between that NN2101 mice treated did not intravitreally impair the early with physiological NN2101 and retinal PBS vehicle, vascular development in terms of vascularin mice. density, radial length, and number of sprouting cells (Figure 5). These data indicate that NN2101 did not impair the early physiological retinal vascular development in mice.

PharmaceuticsPharmaceutics2021 2021, ,13 13,, 1308 x FOR PEER REVIEW 1010 of of 14 14

FigureFigure 5.5. NN2101NN2101 doesdoes notnot affectaffect earlyearly retinalretinal vascularvascular developmentdevelopment inin mice.mice. ((AA)) representativerepresentative imagesimages ofof retinaretina vasculaturevasculature ofof micemice thatthat receivedreceived anan intravitrealintravitreal injectioninjection of of NN2101NN2101 (20(20µ μg)g) oror PBSPBS atat postnatalpostnatal dayday (P)(P) 2.2. MiceMice werewere maintainedmaintained underunder normoxianormoxia untiluntil P6,P6, andand thethe retinalretinal vasculaturevasculature was visualized by staining with IB4 (black). High-magnification views of the growth fronts of was visualized by staining with IB4 (black). High-magnification views of the growth fronts of vessels vessels are shown on the right and vascular sprouts are marked with white dots. Scale bars = 100 are shown on the right and vascular sprouts are marked with white dots. Scale bars = 100 µm; μm; (B) quantification of the radial length of blood vessels, number of sprouting endothelial cells, (andB) quantification vascular density of the in the radial whole-mounted length of blood retinas vessels, of mice number in (A of). sproutingThe data are endothelial shown as cells, the mean and vascular± SEM (unpaired density in Student’s the whole-mounted t-test, ns = retinasnot significant, of mice in n (=A 4). mice The dataper group). are shown as the mean ± SEM (unpaired Student’s t-test, ns = not significant, n = 4 mice per group). 3.6. PK of Intravitreal NN2101 in the Rabbit Eyes and Serum 3.6. PK of Intravitreal NN2101 in the Rabbit Eyes and Serum As drugs administered intravitreally should have a long half-life (T1/2) to avoid fre- As drugs administered intravitreally should have a long half-life (T ) to avoid quent administration, we investigated the PK of NN2101 after a single intravitreal1/2 injec- frequent administration, we investigated the PK of NN2101 after a single intravitreal in- jectiontion into into rabbits. rabbits. No Noadverse adverse events events or signs or signs of ocular of ocular inflammation inflammation were were observed observed after afterintravitreal intravitreal administration. administration. The PK The paramete PK parametersrs of NN2101 of NN2101 in the invitreous the vitreous humor, humor, aque- aqueousous humor, humor, retina/choroid, retina/choroid, and blood and bloodserum serumare summarized are summarized in Table in 1. Table In the1. vitreous In the vitreoushumor, the humor, concentration the concentration of NN2101 of NN2101peaked 1 peaked h after 1injection h after injection(maximum (maximum concentration con- (Cmax) = 378.3 μg/mL) and declined with a T1/2 of 103.3 h, which is similar to that of centration (Cmax) = 378.3 µg/mL) and declined with a T1/2 of 103.3 h, which is similar aflibercept (T1/2 = 94.1 h) (Figure 6A) [19]. Likewise, the mean residence time (MRT) of to that of aflibercept (T1/2 = 94.1 h) (Figure6A) [ 19]. Likewise, the mean residence time (MRT)NN2101 of (131.3 NN2101 h) was (131.3 comparable h) was comparable to that of aflibercept to that of aflibercept (135.8 h). Shortly (135.8 h).after Shortly intravitreal after intravitrealinjection, substantial injection, substantialamounts of amounts NN2101 of were NN2101 detected were in detected the aqueous in the aqueoushumor and humor reti- andna/choroid, retina/choroid, which suggests which suggests that NN2101 that NN2101 in the vitreous in the vitreous humor humorwas rapidly was rapidly distributed dis- either to the aqueous chamber or to the retina/choroid (Figure 6B,C). The T1/2 of NN2101 tributed either to the aqueous chamber or to the retina/choroid (Figure6B,C). The T1/2 of NN2101in the aqueous in the aqueoushumor and humor retina/choroid and retina/choroid were 203.0 were h and 203.0 128.9 h and h, respectively. 128.9 h, respectively. One day Oneafter day intravitreal after intravitreal injection, injection, NN2101 NN2101reached reachedthe maximum the maximum serum concentration serum concentration (Cmax = (C0.3max μg/mL),= 0.3 µ whichg/mL), was which approximately was approximately 1200-fold 1200-fold lower than lower that than in thatthe vitreous in the vitreous humor humor(Figure (Figure 6D). 6D).

Pharmaceutics 2021, 13, 1308 11 of 14

Table 1. PK parameters of NN2101 in the vitreous humor, aqueous humor, retina/choroid, and serum of rabbits.

PK Parameters Vitreous Aqueous Retina/Choroid Serum

T1/2, h 103.3 203.0 128.9 MRT, h 131.3 208.5 243.7 311.6 V/F, mL 1.3 12.0 98.1 CL/F, mL/h 0.009 0.041 0.527 Cmax, µg/mL 378.3 100.8 3.2 0.3 AUC, h × µg/mL 63,921.0 12,771.9 1101.6 132.7 Pharmaceutics 2021, 13, x FOR PEER REVIEW 11 of 14 T1/2, half-life; MRT, mean residence time; V/F, apparent volume of distribution; CL/F, apparent clearance; Cmax, maximum concentration; AUC, area under the concentration time curve.

Figure 6. Time-concentrationFigure 6. Time-concentration plots for NN2101 plots in for the NN2101 eyes and in serum the ey ofes rabbits and serum after intravitreal of rabbits injection.after intravitreal Concentration of NN2101 in vitreousinjection. humor Concentration (A), aqueous of humorNN2101 (B in), retinavitreous and humor choroid (A tissues), aqueous (C), andhumor serum (B), (D retina) of rabbits and choroid after intravitreal injection of NN2101tissues ( (600C), andµg inserum 30 µ L)(D is) of shown. rabbits Samples after intravitreal were taken injection from the of injected NN2101 right (600 (solid μg in line) 30 μ andL) is uninjected shown. Samples were taken from the injected right (solid line) and uninjected left (dashed line) left (dashed line) eyes over 30 days, and the concentration of NN2101 was assessed using indirect ELISA. The data are eyes over 30 days, and the concentration of NN2101 was assessed using indirect ELISA. The data expressed as the mean ± SEM (n = 3 rabbits per time point). are expressed as the mean ± SEM (n = 3 rabbits per time point). 4. Discussion Table 1. PK parameters of NN2101 in the vitreous humor, aqueous humor, retina/choroid, and serum of rabbits. Recently, we reported the novel roles of SCF and cKIT in the regulation of pathological NV in the eyes [12]. Our previous study showed that cKIT and SCF were highly expressed PK Parametersin the angiogenic Vitreous choroidal vasculature Aqueous and nearby Retina/Choroid RPE layers inSerum a murine model of T1/2, h neovascular AMD, 103.3 and they promoted 203.0 pathological NV 128.9 by activating the cKIT/GSK-3β/β- MRT, hcatenin signaling 131.3 pathway and by 208.5 enhancing the β-catenin-mediated 243.7 311.6 transcription of the V/F, mL 1.3 12.0 98.1 genes related to angiogenesis. These ﬁndings prompted us to investigate the potential CL/F, mL/h 0.009 0.041 0.527 of a fully human monoclonal IgG targeting cKIT, NN2101, as an alternative or additive Cmax, μg/mL 378.3 100.8 3.2 0.3 therapeutic agent for treating neovascular AMD. AUC, h × μg/mL 63921.0 12771.9 1101.6 132.7 T1/2, half-life; MRT, mean residence time; V/F, apparent volume of distribution; CL/F, apparent clearance; Cmax, maximum concentration; AUC, area under the concentration time curve.

4. Discussion Recently, we reported the novel roles of SCF and cKIT in the regulation of pathological NV in the eyes [12]. Our previous study showed that cKIT and SCF were highly expressed in the angiogenic choroidal vasculature and nearby RPE layers in a murine model of neovascular AMD, and they promoted pathological NV by activating the cKIT/GSK-3β/β-catenin signaling pathway and by enhancing the β-catenin-mediated transcription of the genes related to angiogenesis. These findings prompted us to investigate the potential of a fully human monoclonal IgG targeting cKIT, NN2101, as an alternative or additive therapeutic agent for treating neovascular AMD.

Pharmaceutics 2021, 13, 1308 12 of 14

Both in vitro and in vivo experiments confirmed that NN2101 considerably inhibited the activation of SCF/cKIT signaling and reduced the expression of VEGFA, IL-8, and c-Myc, to levels as low as that in normal mice. Intravitreal administration of 1 µg NN2101 significantly decreased the area of CNV lesions with an efficacy comparable to that of the human equivalent dose of aflibercept (2.5 µg). Moreover, the present study revealed that a combination of suboptimal doses of aflibercept and NN2101 inhibited CNV formation with an effect comparable to that of 2.5 µg of aflibercept, which suggested that a combination therapy using aflibercept and NN2101 could help reduce the therapeutic dosages and possible side effects of anti-VEGF agents. NN2101 exhibited no deleterious effect on various human ocular cells. When a high dose of NN2101 was intravitreally administered into normal adult mice, there were no evident changes in the levels of cell apoptosis and retinal tissue stress. Moreover, an intravitreal injection of NN2101 did not affect the early retinal vascular development in mice. These results suggest that anti-SCF/cKIT therapy may efficiently alleviate the pathological CNV without interrupting the normal vascular and neuronal cell homeostasis in the eye. Considering the important role of physiological VEGF in the eye, NN2101 targeting SCF/cKIT signaling would have an advantage over anti-VEGF agents in terms of safety [5–11]. In the clinical practice of neovascular AMD, an intravitreal injection is the most appropriate administration strategy that can be considered to maximize the therapeutic efficacy of drugs in the posterior segment of the eye and minimize the systemic exposure of drugs [20,21]. Therefore, drugs should present with a long T1/2 to achieve a sustained therapeutic concentration in the vitreous humor and to reduce the administration fre- quency. The present data revealed that NN2101 had an adequate PK profile for intravitreal administration. When compared with aflibercept (m.w. = 145 kDa) whose PK data were previously obtained under experimental settings same as that of the present study, NN2101 (m.w. = 180 kDa) exhibited similar PK, including T1/2, MRT, and apparent clearance in the vitreous humor of a rabbit model. As the vitreous T1/2 values of aflibercept in humans are estimated to be approximately 2.4–2.8-fold greater than those in rabbits, the T1/2 of NN2101 in the human vitreous humor might range from 10.3 to 12.1 days, which allows for monthly or bimonthly intravitreal injections during neovascular AMD treatment [19,22,23]. Shortly after an intravitreal injection, a substantial amount of NN2101 was found in the aqueous humor and retina/choroid tissues, which indicated that NN2101 in the vitreous humor was distributed and removed through the anterior and posterior routes. Moreover, NN2101 was detected in the serum one day after intravitreal administration. However, Cmax and area under the concentration time curve of NN2101 in the serum were approximately 1000-fold and 500-fold lower than those in the vitreous humor, respectively, which suggested that systemic exposure after an intravitreal injection of NN2101 might be too low to interfere with the normal extraocular functions of SCF/cKIT signaling. Nevertheless, further studies are necessary to investigate whether the repeated and long-term administration of NN2101 dysregulates SCF/cKIT signaling and leads to the occurrence of any systemic adverse event in hematopoiesis, fertility, and melanogenesis [24–26]. To our knowledge, this is the first study to demonstrate the therapeutic potential of anti-cKIT blocking IgG, NN2101, in the treatment of neovascular AMD. The present study not only revealed that NN2101 suppressed the pathological CNV with a comparable to that of aflibercept, but also suggested that a combination of NN2101 and aflibercept could help confer an additive therapeutic effect on CNV. Moreover, NN2101 showed desirable PK and no ocular toxicity, which is essential for the clinical application of therapeutic IgGs. These findings suggest that NN2101 may be considered a novel promising therapeutic agent for the treatment of neovascular AMD.

Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/pharmaceutics13081308/s1, Figure S1: NN2101 effectively inhibits SCF-mediated in vitro angiogenesis in murine endothelial cells at hypoxia; Table S1: IgGs used for Western blotting and immunohistochemistry. Pharmaceutics 2021, 13, 1308 13 of 14

Author Contributions: Conceptualization, S.G.P. and W.S.; Validation, S.S., K.L.K. and S.-H.S.; Formal Analysis, S.S., K.L.K., M.-J.A., J.-W.K., H.K.H., M.H.H. and S.J.W.; Investigation, S.S., K.L.K., Y.Y., R.-I.K., J.-O.K., S.-H.S., M.-J.A., J.-W.K., H.K.H., M.H.H., S.J.W., J.-H.S. and H.J.; Resources, J.-O.K., J.-H.S. and S.G.P.; Writing—Original Draft Preparation, S.S. and W.S.; Supervision, W.S.; Project Administration and Funding Acquisition, S.G.P. and W.S. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by the National Research Foundation funded by the Korea (NRF) grant funded by the Korea government (2020R1A4A4079817, 2018M3A9H2019045, 2020R1A2C1012930). Institutional Review Board Statement: The study was conducted according to the guideline to the Declaration of Helsinki, and approved by the Institutional Animal Care and Use Committees of Chung-Ang University and Seoul National University Bundang Hospital. Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are available from the corresponding author on reasonable request. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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