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Antibody Inhibition of Properdin Prevents Complement-Mediated Intravascular and Extravascular Hemolysis

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Antibody Inhibition of Properdin Prevents Complement-Mediated Intravascular and Extravascular Hemolysis Antibody Inhibition of Properdin Prevents Complement-Mediated Intravascular and Extravascular Hemolysis This information is current as Damodar Gullipalli, Fengkui Zhang, Sayaka Sato, of September 29, 2021. Yoshiyasu Ueda, Yuko Kimura, Madhu Golla, Takashi Miwa, Jianxiang Wang and Wen-Chao Song J Immunol 2018; 201:1021-1029; Prepublished online 13 June 2018; doi: 10.4049/jimmunol.1800384 Downloaded from http://www.jimmunol.org/content/201/3/1021 References This article cites 37 articles, 18 of which you can access for free at: http://www.jimmunol.org/content/201/3/1021.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists by guest on September 29, 2021 • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2018 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Antibody Inhibition of Properdin Prevents Complement-Mediated Intravascular and Extravascular Hemolysis Damodar Gullipalli,* Fengkui Zhang,† Sayaka Sato,* Yoshiyasu Ueda,* Yuko Kimura,* Madhu Golla,* Takashi Miwa,* Jianxiang Wang,† and Wen-Chao Song* Paroxysmal nocturnal hemoglobinuria (PNH) is a serious blood disorder characterized by dysregulated complement activation on blood cells. Eculizumab, the current standard therapy and a humanized anti-C5 mAb, relieves anemia and thrombosis symptoms of PNH patients by preventing complement-dependent intravascular hemolysis (IVH). However, up to 20% of PNH patients on long-term eculizumab treatment still suffer from significant anemia and are transfusion dependent because of extravascular hemolysis (EVH) of C3-opsonized PNH erythrocytes. In this study, we show that function-blocking anti-properdin (P) mAbs dose-dependently inhibited autologous, complement-mediated hemolysis induced by factor H dysfunction. Furthermore, anti– human P (hP) mAbs potently and dose-dependently inhibited acidified serum–induced hemolysis of PNH erythrocytes (Ham test). Downloaded from In contrast to erythrocytes rescued by anti-C5 mAb, nonlysed PNH erythrocytes rescued by anti-P mAb incurred no activated C3 fragment deposition on their surface. These results suggested that anti-P mAbs may prevent EVH as well as IVH of PNH erythrocytes. To test the in vivo efficacy of anti-hP mAbs in preventing EVH, we generated a P humanized mouse by transgenic expression of hP in P knockout mice (hP-Tg/P2/2). In a murine EVH model, complement-susceptible erythrocytes were completely eliminated within 3 d in control mAb-treated hP-Tg/P2/2 mice, whereas such cells were protected and persisted in 2 2 hP-Tg/P / mice treated with an anti-hP mAb. Collectively, these data suggest that anti-P mAbs can inhibit both IVH and EVH http://www.jimmunol.org/ mediated by complement and may offer improved efficacy over eculizumab, the current standard therapy for PNH. The Journal of Immunology, 2018, 201: 1021–1029. aroxysmal nocturnal hemoglobinuria (PNH) is an ac- remarkable success of eculizumab in the clinic, however, up to quired blood disorder caused by somatic mutations in 20% of PNH patients on the drug remain transfusion dependent P hematopoietic stem cells of the gene encoding phos- (5), suggesting that eculizumab is not completely effective in all phatidylinositol glycan class A, a key enzyme involved in the patients (6). biosynthesis of GPI anchor of membrane proteins (1, 2). Among The major reason for the lack of complete efficacy of eculi- by guest on September 29, 2021 the proteins affected in PNH are two membrane complement zumab in some PNH patients relates to its mode of action and the regulators: decay-accelerating factor (DAF, CD55) and CD59 mechanism of PNH pathogenesis. The absence of DAF, a C3 (1). Because of the lack of these proteins on them, affected PNH convertase inhibitor, and CD59, a membrane attack complex blood cells are highly susceptible to complement-mediated at- (MAC) inhibitor, renders PNH erythrocytes susceptible to both tack and injury, leading to debilitating symptoms in the patient C3 activation and injury by MAC (1). Although eculizumab can that include hemolytic anemia, platelet activation, thrombotic effectively block MAC-mediated intravascular hemolysis (IVH), attack, and stroke (1, 3). Until about a decade ago when a hu- it does not inhibit C3 activation on PNH erythrocytes (1). Indeed, manized anti-C5 mAb (eculizumab) was approved and became C3 fragment (C3b/iC3b/C3d)–opsonized PNH erythrocytes were the standard therapy, PNH was largely untreatable and carried detected abundantly in the circulation of patients after eculizu- a significant risk of mortality and morbidity (4). Despite the mab treatment (7–9). It is believed that C3 fragment–opsonized erythrocytes have a much shorter t1/2 because of complement *Department of Systems Pharmacology and Translational Therapeutics, Perelman receptor–mediated phagocytosis, a process commonly referred to School of Medicine, University of Pennsylvania, Philadelphia, PA 19010; and as extravascular hemolysis (EVH) (10). Thus, although eculi- † Institute of Hematology, Chinese Academy of Medical Sciences, Tianjin 300020, zumab is effective at inhibiting IVH, it does not address the China problem of EVH, which becomes unmasked after eculizumab ORCIDs: 0000-0001-5355-5307 (Y.U.); 0000-0001-9437-9151 (J.W.). treatment, potentially explaining its lack of complete efficacy in Received for publication March 13, 2018. Accepted for publication May 18, 2018. some patients. This work is supported in part by National Institutes of Health Grants AI-103965, In the current study, we have tested the concept of blocking pro- AI-44970, and AI-085596. perdin (P) as an alternative and more effective therapeutic strategy for Address correspondence and reprint requests to Dr. Wen-Chao Song, Department of Systems Pharmacology and Translational Therapeutics, Perelman School PNH. P is a plasma glycoprotein and a component of the alternative of Medicine, University of Pennsylvania, Room 1254 BRBII/III, 421 Curie Boulevard, pathway (AP) of complement activation (11). It is the only known Philadelphia, PA 19104. E-mail address: [email protected] positive regulator of AP and works by stabilizing the AP C3 con- Abbreviations used in this article: aNHS, acidified NHS; AP, alternative pathway; vertase C3bBb (12, 13). Using in vitro hemolytic assays of normal DAF, decay-accelerating factor; EVH, extravascular hemolysis; FH, factor H; fP, factor properdin; GVB, gelatin veronal buffer; hP, human P; hP-Tg, hP transgenic; and PNH erythrocytes as a surrogate of IVH and a murine model of IVH, intravascular hemolysis; KO, knockout; MAC, membrane attack complex; EVH, we demonstrated that anti-P mAbs can potently inhibit both NHS, normal human serum; P, properdin; PBST, PBS and 0.05% Tween 20; PNH, IVH and EVH mediated by complement. Our findings suggest that paroxysmal nocturnal hemoglobinuria; RT, room temperature; WT, wild-type. anti-P mAbs may offer improved efficacy over eculizumab, the cur- Copyright Ó 2018 by The American Association of Immunologists, Inc. 0022-1767/18/$35.00 rent standard therapy for PNH. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1800384 1022 PROPERDIN INHIBITION AND PNH HEMOLYSIS Materials and Methods LPS AP assay Generation and screening of anti-human P mAbs Microtiter plates were coated with LPS (Salmonella typhosa LPS; 2 2 m To generate anti-human factor properdin (fP) mAbs, P knockout (KO) (P / ) Sigma-Aldrich) (2 g/well) in PBS overnight at 4˚C. After washing the mice (14) were each i.p. immunized with 50 mg (in 100 ml PBS) purified plated wells with PBS and 0.05% Tween 20 (PBST) three times, wells m were treated with a blocking buffer (1% BSA in PBS) for 1 h at RT. NHS human fP (CompTech) emulsified with 100 l TiterMax adjuvant (Sigma- ++ Aldrich). At day 14 and day 21, mice were again immunized with 50 mg diluted to 50% with GVB buffer (Sigma-Aldrich) supplemented with Mg2+ (5 mM)2EGTA (20 mM), with or without preincubation with purified human fP emulsified with TiterMax adjuvant. One week later, the m mice were examined for serum anti-fP titer. Mice with an Ab titer of anti-P mAbs, was then added to the plated wells (50 l/well). NHS 1:10,000 or higher were used for hybridoma fusion experiments. Two days samples diluted in the same way but containing EDTA (20 mM) were prior to fusion experiment, mice were injected (i.p.) again with 50 mg used as positive controls of complete inhibition of AP complement purified human fP without any adjuvant (in 100 ml PBS). Spleen was activation. Preincubation of NHS with anti-hP mAbs was performed at harvested, and isolated single cells were fused with P3-X63-Ag8.653 my- 4˚C for 1 h. AP complement activation in plated wells was allowed to proceed for 1 h at 37˚C, and reaction was stopped by addition of cold eloma cells (American Type Culture Collection). Positive clones were se- m lected by human P (hP) binding and inhibitory activity in AP complement 10 mM EDTA in PBS (100 l/well). After washing three times with activation assay. Single clones were grown in culture flasks in PBST, plated wells were incubated with an HRP-conjugated goat anti- hypoxanthine–thymidine medium (Sigma-Aldrich) containing Hybridoma- human C3 polyclonal Ab (catalog no. 0855237; MP Biomedicals) SFM (Life Technologies) and supplemented with FBS (HyClone Labora- (1:4000 diluted in blocking buffer) for 1 h at RT.
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