Establishment of monoclonal antibodies against the extracellular domain that block binding of NMO-IgG to AQP4

Journal of Neuroimmunology 260 (2013) 107–116

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Establishment of monoclonal antibodies against the extracellular domain that block binding of NMO-IgG to AQP4 Kaori Miyazaki a, b, Yoichiro Abe a,⁎, Hiroko Iwanari e, Yota Suzuki a, Takahiro Kikuchi a, Takashi Ito a, 1, Jungo Kato a, c, Osamu Kusano-Arai e, f, Toshiyuki Takahashi g, Shuhei Nishiyama g, Hiroko Ikeshima-Kataoka a, Shoji Tsuji a, 2, Takeshi Arimitsu a, d, Yasuhiro Kato a, Toshiko Sakihama e, Yoshiaki Toyama b, Kazuo Fujihara g, Takao Hamakubo e, Masato Yasui a a

Department of Pharmacology, School of Medicine, Keio University, 35 Shinanomachi, Shinjyuku-ku, Tokyo, 160-8582, Japan Department of Orthopaedic Surgery, School of Medicine, Keio University, Japan c Department of Anesthesiology, School of Medicine, Keio University, Japan d Department of Pediatrics, School of Medicine, Keio University, Japan e Department of Molecular Biology and Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo, 153-8904, Japan f Institute of Immunology Co. Ltd., 1-1-10 Koraku, Bunkyo, Tokyo, 112-0004, Japan g Department of Neurology, Tohoku University Graduate School of Medicine, 1-1 Seiryomachi, Aoba-ku, Sendai, 980-8574, Japan b

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Article history: Received 19 April 2012 Received in revised form 2 March 2013 Accepted 15 March 2013 Keywords: Aquaporin-4 NMO-IgG Nueromyelitis optica Monoclonal antibody

a b s t r a c t Neuromyelitis optica is a demyelinating disease characterized by a disease-specific autoantibody designated as NMO-IgG that specifically recognizes aquaporin-4, and the binding of NMO-IgG to AQP4 causes the progress of the disease. Prevention of the binding of NMO-IgG, therefore, may alleviate the disease. Here we have developed monoclonal antibodies against AQP4 with a baculovirus display system in order to obtain high affinity monoclonal antibodies against the extracellular domains of AQP4. Our monoclonal antibodies can block the binding of NMO-IgG in spite of their heterogeneity. Taken together, we propose that our monoclonal antibodies can be applied in clinical therapy for NMO patients. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Neuromyelitis optica (NMO) is a demyelinating disease characterized by optic neuritis and acute myelitis (Wingerchuk et al., 2006). It has long been categorized as a subtype of multiple sclerosis (MS). However, a disease-specific autoantibody designated as NMO-IgG (Lennon et al., 2004) has been detected in the patients' plasma, and was found to bind to the extracellular domains of aquaporin-4 (AQP4), a water channel protein expressed in astrocyte foot processes at the blood–brain barrier (Lennon et al., 2005). Consistent with this finding, extensive loss of AQP4 and glial fibrillary acidic protein (GFAP) immunoreactivities, as well as perivascular deposition of immunoglobulins and activated complements, were observed in NMO lesions (Misu et al., 2007; Roemer et al., 2007). The complement-dependent cytotoxicity of NMO-IgG

⁎ Corresponding author. Tel.: +81 3 5363 3750; fax: +81 3 3359 8889. E-mail address: [email protected] (Y. Abe). 1 Current address: Department of Community-based Perinatal and Emergency medicine, Kitasato University school of medicine, 1-15-1 Kitasato Minamiku Sagamihara Kanagawa, 252-0374 Japan. 2 Current address: Department of Pediatrics, Kansai Medical University, 2-5-1 Shin-machi, Hirakata-shi Osaka, 573-1010, Japan. 0165-5728/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jneuroim.2013.03.003

was also demonstrated in vitro using AQP4-expressing cells, including primary cultured astrocytes (Hinson et al., 2007; Kinoshita et al., 2009; Sabater et al., 2009), and ex vivo spinal cord slices (Zhang et al., 2011). More recently, intra-cerebral injection of NMO-IgG in combination with human complement induces the loss of AQP4 immunoreactivity followed by demyelination (Saadoun et al., 2010), which is T-cell independent (Saadoun et al., 2011). These observations strongly suggest that, distinct from MS, the binding of NMO-IgG to astrocytes through AQP4 causes disruption of astrocytes followed by demyelination (Fujihara, 2011). Therefore, prevention of the binding of NMO-IgG to AQP4 is one of the therapeutic options for NMO. AQP4, the target antigen of NMO-IgG, has two dominant isoforms designated as M1 and M23. Whereas M23 is transcribed from the transcriptional start site upstream of exon 1, M1 is transcribed from that upstream of exon 0, which is spliced to exon 1 in frame resulting in the addition of an extra 22 amino acids to the N terminus of the M23 isoform (Yang et al., 1995; Lu et al., 1996; Ma et al., 1996). These two isoforms constitute heterotetramers and have a role in the formation of the orthogonal array of particles (OAPs), a unique feature of AQP4, with opposing effects. Whereas M23 tends to form OAPs, M1 does not form OAPs and tends to disrupt them (Furman et al., 2003).

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Recent reports have demonstrated that the epitopes of NMO-IgG are varied (Nicchia et al., 2009; Tani et al., 2009; Mader et al., 2010; Crane et al., 2011; Pisani et al., 2011; Iorio et al., 2013). In most cases, NMO-IgG preferentially recognizes 3D conformation of membrane-integrated M23 isoform, suggesting that formation of OAP is required for antibody recognition (Nicchia et al., 2009; Mader et al., 2010; Crane et al., 2011; Pisani et al., 2011). Some NMO-IgGs recognize 3D conformation of both M1 and M23 equally (Crane et al., 2011) or both M1 and M23 even in the denaturing condition (Iorio et al., 2013). Therefore, it is more feasible to develop molecules that block the binding site of NMO-IgG, such as monoclonal antibody against AQP4, than to develop neutralizing molecules that block the antigen-binding site of NMO-IgG. However, it is difficult to develop high affinity antibodies against the extracellular domains of membrane-integrated AQP4 since AQP4 is a six-transmembrane protein, whose N- and C-terminal domains are oriented cytoplasm, and whose three extracellular loops are suggested to constitute a tightly packed conformation. Recently, Saitoh et al. have demonstrated a new method efficiently generating high affinity monoclonal antibodies against the extracellular domains of multipass membrane proteins using a baculovirus display system, in which the viruses display the foreign multipass membrane proteins as immunogens (Saitoh et al., 2007). We therefore used a baculovirus display system to develop monoclonal antibodies that recognize the extracellular domains of human AQP4 and successfully demonstrated that they blocked the binding of a variety of NMO-IgGs to AQP4, which potentially provides a therapeutic option for NMO and NMO spectrum disorders. 2. Materials and methods 2.1. Isolating IgG from NMO patient serum Ig fractions were obtained from the plasma exchange material of five patients with AQP4-antibody-positive NMO. The use of the patients' plasma for this study was approved by the Ethics Committee of Tohoku University School of Medicine (No. 2007-327). 2.2. Establishment of monoclonal antibodies against the extracellular domains of hAQP4 Monoclonal anti-AQP4 extracellular domain antibodies were established according to the previous report (Saitoh et al., 2007). In brief, the cDNA encoding the human AQP4 M23 isoform was inserted into a pBlueBac4.5 plasmid transfer vector (Invitrogen, Carlsbad, CA, USA). gp64 transgenic mice were immunized intraperitoneally with budded baculovirus (BV) expressing either both hAQP4 M23 and mouse C3d (for the C94 and D15 series) or hAQP4 alone (for the D12 series) in PBS in the presence of pertussis toxin. After the primary screening by flow cytometry (for the C94 series) or with an ELISA (for the D15 and D12 series) using CHO cells stably expressing hAQP4 M23, one out of 576, 173 out of 756, and 271 out of 1152 clones were obtained for the C94, D12 and D15 series, respectively. After the second screening with flow cytometry using hAQP4 M23-expressing the CHO-cell clone, 1, 2, and 61 clones for the C94, D12, and D15 series, respectively, were obtained and clones C9401, D12092, D15107, and D15129 were chosen. 2.3. Plasmids construction Wild-type human AQP4-M23 cDNA was purchased from Toyobo (Osaka, Japan). Mouse AQP4 (mAQP4) M1 and M23 cDNAs were amplified from total RNA extracted from a primary culture of mouse astrocytes using either a sense primer for M1 5′-GAAGGCATGAGTGACAGAGCTGCG GCAAGG-3′; or for M23 5′-ACTATGGTGGCTTTCAAAGGAGTCTGGACT-3′; and an antisense primer 5′-TAGTCATACGGAAGACAATACCTCTCCCGA-3′ followed by subcloning into a pGEM-T Vector (Promega Corporation, Madison, WI, USA) for sequencing. T62S/K64N, M149T, and E228A

mutations of hAQP4 M23 isoform were introduced by a QuickChange site-directed mutagenesis kit (Agilent Technologies, Santa Clara, CA, USA) using primers 5′-ACCATCAACTGGGGTGGATCAGAAAATCCTTTAC CGGTCG-3′ and 5′-CGACCGGTAAAGGATTTTCTGATCCACCCCAGTTGATG GT-3′; 5′-AGGCCTGGGAGTCACCACGGTTCATGGAAATC-3′ and 5′-GATT TCCATGAACCGTGGTGACTCCCAGGCCT-3′; and 5′-CAGTTATCATGGGAA ATTGGGCAAACCATTGGATATATTGGGT-3′ and 5′-ACCCAATATATCCAATG GTTTGCCCAATTTCCCATGATAACTG-3′, respectively. Obtained cDNA were cloned between the NheI and the SacII sites of pIRES2-EGFP (Clontech Laboratories, Mountain View, CA, USA). 2.4. Cell culture and transfection CHO cells were grown in Ham's F12 nutrient mixture supplemented with 10% fetal bovine serum and 50 units/ml penicillin and 50 μg/ml streptomycin. Cells were seeded onto 60-mm dishes at a density of 1 × 105 cells/dish and transfected with AQP4 and its derivatives inserted into the pIRES2-EGFP expression vector using Lipofectamine and plus reagents (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. Forty-eight hours after transfection, cells were subjected to flow cytometry. For Western blotting, cells were lysed with a buffer containing 20 mM Tris–Cl (pH7.4), 1 mM EDTA, 1% Triton X-100 and Complete™ protease inhibitor cocktail tablets (Roche Diagnostics, Indianapolis, IN, USA). 2.5. Establishment of stable CHO cell clones The pIRES2-EGFP vector in the presence or absence of hAQP4-M23 cDNA was linearized with AflII and transfected into CHO cells seeded onto a 35-mm dish. Forty-eight hours after transfection, cells were trypsinized and reseeded onto ten 100-mm dishes and further cultured for 7–10 days in the medium containing G418 (500 μg/ml). Several colonies were picked up based on the intensity of the EGFP fluorescence and amplified. Expression of AQP4 and EGFP was confirmed with Western blotting. 2.6. Immunofluorescence microscopy Mouse cortical acute slices (P14) were fixed with 4% paraformaldehyde in PBS for 1 h, washed four times with PBS, permeabilized with 0.1% TritonX-100 in PBS, and finally blocked with 0.1% BSA in PBS. Slices were incubated with an anti-AQP4 C-terminal domain antibody (Sigma, St. Louis, MO, USA) or the monoclonal anti-AQP4 extracellular domain antibody for 1 h, washed four times with PBS, followed by incubation with Alexa Fluor 488-conjugated goat antirabbit IgG and Alexa-Fluor 555-conjugated goat anti-mouse IgG (Invitrogen, Carlsbad, CA, USA) for 1 h. CHO cells stably expressing hAQP4 M23 were incubated with 20 μg of purified patients' IgG in growth medium at 37 °C for 2 h. Then 10 μg of monoclonal anti-hAQP4 extracellular antibodies were added to the medium, and the cells were further incubated for 24 h. The cells were fixed with 4% paraformaldehyde in PBS for 1 h, washed two times with PBS, permeabilized with 0.1% TritonX-100 in PBS, and finally blocked with 0.1% BSA in PBS. NMO-IgG and monoclonal antibodies bound to the cells were stained with Alexa Fluor 568conjugated goat anti-human IgG and Alexa Fluor 555-conjugated goat anti-mouse IgG (Invitrogen, Carlsbad, CA, USA), respectively. Immunostained slices and cells were observed with a confocal microscope (Olympus, Tokyo, Japan). 2.7. Immunoprecipitation A confluent monolayer of CHO cells stably expressing hAQP4 was scraped and lysed with Blue Native (BN)-buffer (1% Triton X-100, 12 mM NaCl, 500 mM 6-aminohexanoic acid, 20 mM BisTris, pH7.0, 2 mM EDTA, 10% glycerol and protease inhibitor mixture), vortexed

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on ice, frozen once at −80 °C, and centrifuged at 20,000 ×g for 5 min at 4 °C. Supernatants were collected, and the total protein content was measured using the BCA protein assay kit (Pierce, Rockford, IL, USA). Two hundred micrograms of protein was incubated in the presence of 10 μg of either a rabbit anti-AQP4 C-terminal antibody or each monoclonal anti-AQP4 extracellular domain antibody on a mechanical rotator overnight at 4 °C. The next day, 40 μl of pre-washed nProtein A Sepharose 4 Fast Flow beads (GE healthcare, Waukesha, WI, USA) were added to the samples. To isolate the immunocomplexes, the samples were centrifuged at 11,000 ×g for 2 min at 4 °C, and the beads were washed four times with Washing Buffer containing 0.2% Triton X-100, 10 mM Tris-Cl (pH 7.4), 150 mM NaCl, 1 mM EDTA, and 1 mM EGTA. AQP4 was eluted by adding 20 μl of 3 × Laemmli Buffer at 37 °C for 30 min and subjected to Western blotting. 2.8. Western blotting Samples were subjected to SDS-PAGE followed by transfer to a polyvinylidene difluoride membrane. Transferred membranes were incubated with the anti-AQP4 antibody or the monoclonal antibodies. Signals were visualized with a Chemi-Lumi One L system (Nacalai Tesque, Kyoto, Japan) and detected with an Image Quant LAS system (GE healthcare, Waukesha, WI, USA). The antibodies used were a polyclonal rabbit anti-AQP4 C-terminal domain antibody (Sigma, St. Louis, MO, USA); and HRP-conjugated goat anti-mouse and goat anti-rabbit antibodies (Sigma, St. Louis, MO, USA). 2.9. Reconstitution of recombinant human AQP4 into liposomes and measurement of the water permeability of AQP4 proteoliposomes The Sf9 insect cell/baculovirus system was used to express the hAQP4 for purification and reconstitution into liposomes as described previously (Yang et al., 1997; Yukutake et al., 2009). The water permeability of hAQP4 proteoliposomes was measured with the carboxyfluorescein quenching method (Zeidel et al., 1992) using a stopped-flow apparatus with a dead time of b3 ms (Unisoku, Hirakata, Japan). The fitting parameters were then used to determine the Pf by first applying the linear conversion from relative fluorescence into relative volume and then iteratively solving the Pf equation: P f ¼ ðdVðtÞ=dtÞ=fðSAVÞðMVWÞðCin−CoutÞg; where Pf is the osmotic water permeability, V(t) is the relative intravesicular volume as a function of time, SAV is the vesicular surface area to volume ratio, MVW is the molar volume of water (18 cm3/mol), and Cin and Cout are the initial concentrations of total solute inside and outside the vesicle, respectively. 2.10. Confirmation of specific binding of anti-hAQP4 mouse monoclonal antibodies to AQP4 in liposomes The specific binding of monoclonal antibodies to AQP4 in liposomes was confirmed with Western blotting as previously described (Yukutake et al., 2008). In brief, AQP4 proteoliposomes were incubated with 20 μg/ml of monoclonal anti-AQP4 extracellular domain antibody at room temperature for 1 h. The reactant was harvested by centrifugation (liposomes: 45 min at 50,000 rev./min) and resuspended in the buffer solution for three cycles to remove residual IgG. The liposomes were solubilized with an SDS-PAGE sample buffer containing 16 mg/ml SDS. 2.11. Flow cytometry CHO cells either stably or transiently expressing AQP4 and its derivatives were trypsinized and suspended in 100 μl of 0.1% BSA in PBS. Then cells were incubated with either 2 μg of monoclonal anti-hAQP4

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extracellular domain antibodies or 10 μg of purified patients' IgG on ice for 1 h. Cells were washed with 0.1% BSA in PBS and stained with PE-conjugated mouse anti-human IgG (1:5, Biolegend, San Diego, CA, USA) or PE-conjugated goat anti-mouse IgG (1:100, Biolegend, San Diego, CA, USA) for 1 h. Bound antibodies were estimated with FACSCalibur (BD, Franklin Lakes, NJ, USA). 3. Results 3.1. Characterization of NMO-IgG To investigate the epitope of NMO-IgG, we first prepared expression constructs, in which the M1 or M23 isoform of human or mouse AQP4 (hAQP4 or mAQP4) cDNA was connected to enhanced green fluorescent protein (EGFP) cDNA with an internal ribosomal entry site (IRES). In these constructs, the intensity of the fluorescence of EGFP is used to reflect the level of expression of AQP4. The binding property of purified NMO-IgGs derived from five patients was examined using CHO cells transiently expressing AQP4. As shown in Fig. 1, all five NMO-IgGs bound to the cell surface expressing the M23 isoform of hAQP4, and their binding increased as the intensity of the EGFP fluorescence did (Fig. 1). Two out of the five NMO-IgGs derived from patients 1 and 2 also bound to the cell surface expressing the M23 isoform of mAQP4 (Fig. 1). All NMO-IgGs failed to recognize the M1 isoform regardless of the difference in species except for the one derived from patient 1 (Fig. 1). These results indicate that NMO-IgGs are heterogeneous and their epitopes are varied, which is consistent with the previous reports (Nicchia et al., 2009; Tani et al., 2009; Mader et al., 2010; Crane et al., 2011; Pisani et al., 2011; Iorio et al., 2013). 3.2. Development of the monoclonal antibodies against the extracellular domain of AQP4 Recent reports have demonstrated that the binding of NMO-IgG to astrocytes via AQP4 causes the disease (Hinson et al., 2007; Misu et al., 2007; Roemer et al., 2007; Kinoshita et al., 2009; Sabater et al., 2009; Saadoun et al., 2010; Zhang et al., 2011). Therefore, prevention of the binding of NMO-IgG to AQP4 may improve the disease prognosis. We therefore tried to develop monoclonal antibodies that interfered with the binding of NMO-IgGs to AQP4 without affecting AQP4 function. The baculoviral display method was used in order to obtain monoclonal antibodies against the extracellular domain of AQP4 (Saitoh et al., 2007). Four independent clones were obtained, and their binding affinity and specificity to AQP4 were examined with flow cytometry using CHO cells transiently transfected with AQP4 cDNA constructs. All the monoclonal antibodies recognized both M1 and M23 isoforms of human AQP4 (hAQP4) as expected (Fig. 2a, b, e, f, i, j, m, n). While two of them (C9401 and D12092) only recognized hAQP4 (Fig. 2c, d, g, h), the other two (D15107 and D15129) also recognized the M23 but not the M1 isoform of mouse AQP4 (mAQP4) (Fig. 2k, l, o, p). The affinities of D15107 and D15129 for the M23 isoform of mAQP4 was 8–16-fold lower than those of hAQP4, and serial dilution of D15107 and D15129 showed that these antibodies recognized the M1 and M23 isoforms of hAQP4 with almost the same affinity (data not shown). Since D15107 and D15129 recognize the M23 isoform of mAQP4, we evaluated whether these antibodies could be used for immunohistological analysis with mouse tissues. The antibodies clearly stained perivascular vessels in mouse cortical acute slice tissues with immunofluorescence which was identical to the staining pattern with polyclonal anti-AQP4 C-terminal antibodies (Fig. 3A). Although all four monoclonal antibodies are applicable for immuno precipitation (Fig. 3B), none of them recognized denatured AQP4 as determined by Western blotting (Fig. 3C). Taken together, we concluded that all the monoclonal antibodies could recognize the 3D structure of the extracellular domains of

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Fig. 1. Binding properties of purified NMO-IgGs derived from five patients. NMO-IgG obtained from five patients (Pt1–Pt5) by plasma exchange were added to CHO cells transiently transfected with either hAQP4 M23 (a, e, i, m, and q), mAQP4 M23 (b, f, j, n, and r), hAQP4 M1 (c, g, k, o, and s), or mAQP4 M1 (d, h, l, p, and t) cDNA connected to EGFP cDNA with IRES, and bound antibodies were estimated with flow cytometry.

AQP4 and that the 3D structure is slightly different between mAQP4 and hAQP4 as well as between M1 and M23. Having identified that C9401 and D12092 recognized hAQP4 but not mAQP4, mutant hAQP4 M23 isoforms, in which one of the three extracellular loops was changed to the corresponding region of mouse AQP4, were constructed and transiently transfected into CHO cells in order to identify the epitopes of the antibody recognition. While replacement of

neither loop C nor loop E of hAQP4 with that of mAQP4 affected the binding of C9401 and D12092 (Fig. 4, c, d, g, h), replacement of loop A drastically reduced the binding of these antibodies to hAQP4 (Fig. 4, b and f), indicating that the epitope of the monoclonal antibodies includes loop A. Unexpectedly, the binding of D15107 and D15129, which recognized mAQP4 M23 isoform, was also affected by swapping of the loop A (Fig. 4, j and n).

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Fig. 2. Binding properties of four established monoclonal antibodies using flow cytometry. Established anti-AQP4 extracellular domain antibodies, including C9401 (a–d), D12092 (e–h), D15107 (i–l), and D15129 (m–p), were added to CHO cells transiently transfected with either hAQP4 M1 (a, e, i, and m), hAQP4 M23 (b, f, j and n), mAQP4 M1 (c, g, k, and o), or mAQP4 M23 (d, h, l, and p) cDNA connected to EGFP cDNA with IRES, and bound antibodies were estimated with flow cytometry.

Next we examined whether binding of the monoclonal antibodies to AQP4 would inhibit the water permeability of AQP4, for which we performed a functional assay using liposomes incorporated with hAQP4. As shown in Fig. 5, the osmotic water permeability of AQP4 was not inhibited by the monoclonal antibodies. The binding of the antibodies to AQP4 in this assay was confirmed by Western blotting with a secondary antibody that recognized mouse IgG (Fig. 5). The swelling assays of Xenopus oocytes also indicated that the monoclonal antibodies did not inhibit AQP4 function (data not shown). 3.3. The monoclonal antibodies blocked the binding of NMO-IgGs Having identified that the monoclonal antibodies did not affect AQP4 function as a water channel, we examined whether these antibodies would interfere with the binding of NMO-IgG to AQP4. For this purpose, we established CHO cell clones stably expressing the M23

isoform of hAQP4 and EGFP. All five NMO-IgGs bound to the cells expressing hAQP4 M23 but not to cells expressing EGFP alone. The presence of either D12092 or D15107 significantly reduced the binding of each NMO-IgG, indicating that the monoclonal Abs blocked the bindings of all five NMO-IgGs in spite of their heterogeneity (Fig. 6). These results prompted us to examine whether addition of monoclonal antibodies can replace the binding of NMO-IgG to AQP4 using the CHO cells stably expressing hAQP4 M23. The AQP4 expressing cells were initially incubated with 20 μg of NMO-IgG from patient 1 or 2 for 2 h. At this time point, NMO-IgGs surely bound to the surface of the cells (Fig. 7a and not shown). Then 10 μg of each monoclonal antibody was added to the medium and the cells were further incubated in the presence of NMO-IgG for 24 h. Cells incubated with NMO-IgGs in the absence of monoclonal antibodies still showed binding of NMO-IgG to the surface (Fig. 7b and not shown). In contrast, addition of either D12092 or D15107 to the medium drastically reduced

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Fig. 3. Monoclonal anti-AQP4 extracellular domain antibodies do not recognize denatured AQP4. (A) Immunofluorescence of mouse brain slice culture using a monoclonal anti-AQP4 extracellular domain antibody. P14 mouse brain slice culture was double-stained with D15129 (red) and anti-AQP4 C-terminal antibodies (green). Both AQP4 extracellular and C-terminal antibodies stained the perivascular structure. (B) Immunoprecipitation of hAQP4-M23 with monoclonal anti-AQP4 extracellular domain antibodies. Cell lysates from CHO cells stably expressing hAQP4-M23 were immunoprecipitated with either a rabbit anti-AQP4 C-terminal antibody (lane 2), C9401 (lane 3), D12092 (lane 4), D15107 (lane 5), or D15129 (lane 6) followed by Western blotting using a goat anti-AQP C-terminal antibody. One hundred milligrams of lysate was also electrophoresed as an input (lane 1). hAQP4-M23 is indicated with an arrow. (C) Western blotting of hAQP4-M23 using monoclonal anti-AQP4 extracellular domain antibodies. Cell lysate from CHO cells (lanes 2, 4, and 6) or those stably expressing hAQP4-M23 (lanes 1, 3, and 5) was subjected to SDS-PAGE followed by immunoblotting using rabbit anti-AQP4 C-terminal antibody (lanes 1, 2), C9401 (lanes 3, 4), D12092 (lanes 5, 6), D15107 (lanes 7, 8), or D15129 (lanes 9, 10). hAQP4-M23 is indicated with an arrow.

the binding of NMO-IgGs to the cells (Fig. 7c and d and not shown). The added monoclonal antibodies were detected on the surface of AQP4-expressing cells after 24 h of incubation (Fig. 7f and g), indicating that the monoclonal antibodies replaced the binding of NMO-IgGs even in the presence of NMO-IgG in the medium. During these experiments, we did not see obvious morphological change by incubation with monoclonal antibodies, unless complement was added to the medium. We also confirmed that the addition of monoclonal antibodies in the absence of complement did not affect the morphology of primary cultured mouse astrocytes as well as HEK293 cells transiently expressing human AQP4 M23 isoform up to 2 h (Supplemental Fig. 1).

4. Discussion Many groups demonstrated that NMO-IgGs recognize the 3D structure of the extracellular domains of AQP4, although the epitopes are varied (Nicchia et al., 2009; Tani et al., 2009; Mader et al., 2010; Crane et al., 2011; Pisani et al., 2011; Iorio et al., 2013). Consistent with the recent reports, our present studies showed that NMO-IgGs preferentially recognized the M23 isoform of hAQP4 with higher affinity than the M1 isoform of hAQP4 or either isoform of mAQP4 (Fig. 1). Two different mechanisms that could explain difference in the binding affinity of most NMO-IgGs to AQP4 between two isoforms have been proposed. One is

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Fig. 4. Effect of amino acid substitutions in the extracellular loops of hAQP4 on binding of monoclonal antibodies. C9401 (a–d), D12092 (e–h), D15107 (i–l), and D15129 (m–p) were added to CHO cells transiently transfected with either wild-type hAQP4 M23 (a, e, i, and m), T62S/K64N-hAQP4 M23 (b, f, j and n), M149T-hAQP4 M23 (c, g, k, and o), or E228A-hAQP4 M23 (d, h, l, and p) cDNA connected to EGFP cDNA with IRES, and bound antibodies were estimated with flow cytometry.

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Fig. 5. The monoclonal antibodies have no effect on water permeability of AQP4. (A) The functional assay of hAQP4 incorporated into liposomes. Water permeability was estimated by the functional assay using liposomes incorporating His-tagged hAQP4 M23 in the presence or absence of the monoclonal antibodies D12092 and D15107. (B) Western blotting of monoclonal antibodies bound to liposomes. Lysate from liposomes with (lanes 2, 4, and 6) or without (lanes 1, 3, and 5) hAQP4 treated with vehicle (lanes 1, 2), D12092 (lanes 3, 4), or D15107 (lanes 5, 6) was subjected to Western blotting using an HRP-labeled anti-mouse IgG or anti-AQP4 C-terminal antibody followed by HRP-labeled anti-rabbit IgG. His-tagged hAQP4-M23 is indicated with an arrow.

that a tetramer/tetramer interface forms the epitope upon OAP assembly (Crane et al., 2011; Pisani et al., 2011). In addition, Pisani et al. also suggested that interaction of AQP4 tetramers in OAPs results in conformational change of AQP4, affecting the interaction of the three extracellular loops within AQP4 monomers. NMO-IgGs used in this study were purified IgG fraction of patient plasma, in which several different types of NMO-IgGs may be mixed. Actually, Crane et al. cloned different types of NMO-IgG from a patient: one preferentially recognizes M23 isoform (rAb-53), and one equally recognizes both M1 and M23 isoforms (rAb-58) (2011). Therefore, to address which mechanism explains binding pattern of our NMO-IgG, it is necessary to clone each NMO-IgG. In the present study, the monoclonal antibodies were established by the baculovirus display method (Saitoh et al., 2007), in which gp64 transgenic mice expressing wild-type AQP4 were immunized with hAQP4 expressed on the budding baculovirus, in order to produce the antibodies that would recognize the extracellular domains of hAQP4. We succeeded in developing four monoclonal antibodies

against the extracellular domains of AQP4 that were divided into two types: one which recognized only hAQP4 (C9401 and D12092) and the other one which recognized not only hAQP4 but also the M23 isoform of mAQP4 (D15107 and D15129) (Fig. 2). All the monoclonal antibodies equally recognized both the M1 and M23 isoforms of hAQP4, indicating that the binding of monoclonal antibodies do not require OAP formation. This is a characteristic similar to rAb-58 cloned by Crane et al. (2011) but different from NMO-IgGs we examined so far with the exception of the one from patient 1 (Fig. 1). This result excluded the possibility that the epitope of these monoclonal antibodies is the tetramer/tetramer interface. Since C9401 and D12092 distinguished human and mouse AQP4, we examined which extracellular domains were responsible for antibody recognition by changing the amino acid(s) on each extracellular loop of hAQP4 M23 to those of mAQP4. Sequences for human and mouse AQP4 demonstrated that only 23 amino acids were different from each other. The differences in the extracellular loops between hAQP4 and mAQP4 were Thr62 to Ser and Lys64 to Asn on loop A; Met149 to Thr on loop C; and Glu 228 to Ala on loop E. Both C9401 and D12092 failed to bind to the cells expressing hAQP4 M23 with mouse loop A but not loop C or loop E, indicating that at least loop A is included in the epitope of both C9401 and D12092 (Fig. 4). We excluded the possibility that hAQP4 M23 with mouse loop A did not fold properly since the localization of hAQP4 M23 with mouse loop A and wild type is indistinguishable as observed with confocal microscopy (data not shown). Thus we conclude that Thr62 and/or Lys 64 in loop A were involved in the epitope of the monoclonal antibodies. Although D15107 and D15129 did not distinguish between the M1 and M23 isoforms of hAQP4, they recognized the M23 isoform, but not the M1 isoform, of mAQP4. Interestingly, although they recognized mAQP4 M23, they failed to interact with hAQP4 M23 with mouse loop A (Fig. 4). These results strongly support the notion that OAP formation results in a conformational change of AQP4 affecting the position of each loop in the 3D structure. In addition, three extracellular loops of AQP4 may interact with each other to form a particular 3D structure, and changing the amino acid(s) in only one of the loops influenced the position of each loop. Thus, similar to NMO-IgGs, the monoclonal antibodies may recognize a particular conformation formed by the three loops, but in contrast to NMO-IgGs, a region conserved between M1 and M23 of human AQP4. This interpretation is also supported by the fact that Western blotting was inapplicable in all the monoclonal antibodies (Fig. 3). If this is the case, although we did not observe any effect of changing the amino acids in loop C or loop E on binding of all monoclonal antibodies, we cannot exclude the possibility that loop C and/or loop E also contain the epitopes of monoclonal antibodies. None of the monoclonal antibodies we obtained blocked the water permeability of AQP4 as determined by the carboxyfluorescein quenching method using a stopped-flow apparatus (Fig. 5). Recent papers suggest that the epitopes of some NMO-IgGs include loop E, which is involved in water pore formation (Tani et al., 2009; Pisani et al., 2011). Our preliminary data also indicated that the epitopes of NMO-IgGs from patients 4 and 5 included loop E. Nevertheless, these antibodies did not affect the water permeability of AQP4. One possible reason is that all mutations that affected the binding of NMO-IgG were concentrated in the C-terminal half of loop E, which may contribute less to the water permeability of AQP4 than the N-terminal half of loop E. As described above, AQP4 is highly conserved between man and mouse. Therefore, if we immunized mice with an AQP4 knockout background instead of the wild type, we would be able to establish a greater variety of monoclonal antibodies, hopefully including an antibody that could block water permeability, which would be applicable in the treatment of brain edema under certain conditions. Recently, Ratelade and Verkman (2012) pointed out that, since NMO-IgG is large compared to AQP4, it is sterically impossible to accommodate more than one NMO-IgG per tetramer, which contains four monomers and thus four separate water pores. If this is the case,

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Fig. 6. Effect of treatment with monoclonal antibodies on the binding of NMO-IgG to cells expressing hAQP4. NMO-IgGs (Pt1-5) were added to CHO cells stably transfected with hAQP4 M23-IRES-EGFP cDNA in the presence (black line histogram) or absence (gray zone histogram) of pretreatment with the monoclonal antibody D12092 and D15107, and bound NMO-IgGs were estimated with flow cytometry. The horizontal axis shows the antibodies binding and the vertical axis shows the cell counts.

it is reasonable that our monoclonal antibodies did almost completely block the binding of all NMO-IgGs examined so far (Fig. 6), although the epitopes of the NMO-IgGs are varied. Interestingly, the monoclonal antibodies added to the AQP4-expressing cells that bound NMO-IgG drastically reduced the binding of NMO-IgGs to the surface of cells, even in the presence of NMO-IgG in the medium (Fig. 7). There is at least two possibilities: one is the affinity of monoclonal antibodies to the AQP4 is high enough to displace NMO-IgG from AQP4, and the other is NMO-IgG bound to AQP4 was endocytosed and monoclonal antibodies bound to the AQP4 newly transported to the cell surface. In any case, monoclonal antibodies can remove NMO-IgG from the surface of AQP4-expressing cells even in the presence of NMO-IgG. Therefore, we propose that this might be one of the new therapeutic options for NMO. It should be noted that the subclass of the monoclonal antibodies is either IgG2b or IgG2a, which can activate complement. So it is highly

likely that administration of these antibodies induces similar symptoms induced by NMO-IgG. As expected we observed complement-dependent cytotoxicity by the monoclonal antibodies (data not shown). Thus to apply these monoclonal antibodies in the treatment of NMO, it would be necessary to remove the Fc region by making F(ab′)2 or to humanize the monoclonal antibodies. During preparation of this manuscript we noticed a report demonstrating that a non-pathogenic monoclonal NMO-IgG, which was cloned from clonally expanded plasma blast populations in the CSF of NMO patients and was introduced point mutations into the Fc portion, prevented the development of NMO lesions in ex vivo and in vivo models (Tradtrantip et al., 2012). These observations supported our idea that blocking the binding of NMO-IgG to AQP4 by monoclonal antibodies would be applicable in the treatment of NMO as well as NMO spectrum disorders. Our strategy, in which monoclonal antibodies against the extracellular domain of AQP4 with higher affinity

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Fig. 7. Monoclonal antibodies replace the binding of NMO-IgG on the cell expressing AQP4. CHO cells stably transfected with AQP4 M23-IRES-EGFP were incubated with NMO-IgG from patient 2 for 2 h (a), then they were treated with D12092 (c and f) or D15107 (d and g) in the presence of NMO-IgG and further incubated for 24 h. Cells incubated with NMO-IgG for 26 h were also examined (b and e). Bound NMO-IgG and the monoclonal antibodies were detected by Alexa Fluor 568-cinjugated anti-human IgG (a–d) and Alexa Fluor 555-conjugated anti-mouse IgG (e–g), respectively.

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