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

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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. Wells were washed tories), gentamicin (Life Technologies), and penicillin/streptomycin (Life three times with PBST and developed with HRP substrate (100 ml tet- Technologies). mAbs were purified from serum-free culture supernatant by ramethylbenzidine peroxidase substrate [BD Pharmingen]). After 5 min, protein G affinity chromatography using the A¨ KTAFPLC system (GE reaction was stopped with 2 N H2SO4 and plated wells were read at 450 Healthcare Life Sciences). To screen for hP-binding mAbs, MaxiSorp nm in a microplate reader. plates (Nunc, Roskilde, Denmark) were coated overnight at 4˚C with 50 ml Downloaded from hP (2 mg/ml). Wells were washed three times in washing buffer (PBS/ hP ELISA m 0.05% Tween 20) and then treated with 200 l 1% BSA/PBS for 1 h at Microtiter plates were coated with 50 ml of anti-hP mAb clone 8.1 (2 mg/ml, room temperature (RT). The plates were then incubated with 1:5 diluted generated in house) in PBS overnight at 4˚C. After washing the plates with m hybridoma supernatant (50 l/well) in 1% BSA/PBS for 1 h at RT. Wells PBST three times, 200 ml blocking buffer (1% BSA in PBS) was added to were washed three times and incubated for 1 h at RT with 50 ml HRP–anti- each well, and the plate was incubated for 1 h at RT. Afterwards, hP mouse IgG (1:4000; Sigma-Aldrich) in 1% BSA/PBS. After washing three transgenic (hP-Tg) mouse serum (diluted to 10% with PBS containing 1% times, wells were incubated at RT for 5 min with 100 ml tetrame- BSA and 20 mM EDTA) was added to the plate wells (50 ml/well) and http://www.jimmunol.org/ thylbenzidine peroxidase substrate (BD Pharmingen, San Jose, CA). Re- incubated for 1 h at RT. Plates were then washed three times with PBST, m action was stopped with 50 l2NH2SO4, and OD of the samples was and captured hP was detected using a biotinylated goat anti-hP Ab (catalog measured at 450 nm. no. A239; CompTech) and standard HRP-based ELISA detection system. NHS and wild-type (WT) mouse serum served as a positive and negative Production of anti-human C5 mAb control, respectively, in this assay.

An anti-human C5 mAb (QDC5) bearing the same V region H chain/V Generation of hP-Tg mice region L chain sequences as described in Thomas et al. (15) and human IgG4 Fc region with S229P mutation (16) were expressed in Chinese Previous work in our laboratory has shown that hP can function effectively hamster ovary cells using the pFuse hIgG4/pFuse CLIg hK vectors (Invi- in the mouse AP complement system (14). To generate a P humanized voGen, San Diego, CA) and purified by protein G affinity chromatography mouse, we subcloned the hP cDNA [nucleotide no. NM_002621 (20)] into by guest on September 29, 2021 using the AKTA FPLC system. the eukaryotic expression vector pCAGGS, which has been used by others for transgenic studies (21–23). The pCAGGS-hP plasmid was digested Hemolytic assays with restriction enzymes Sal I and Hind III to release the hP cDNA along with the CMV immediate early enhancer, chicken b-actin promoter, and Hemolytic assays of normal human erythrocytes were performed using rabbit globin poly(A) sequence. The DNA was then injected into C57BL/ previously published methods (17, 18). Normal human erythrocytes (Valley 6J mouse zygotes and transferred into pseudopregnant female recipient Biomedical, Winchester, VA) at 5 3 106 cells per reaction were added to a C57BL/6J mice. Pups were screened using hP-specific primers solution (100 ml final volume) composed of gelatin veronal buffer (GVB++) (59-ATCAGAG-GCCTGTGACACC-39 and 59- CTGCCCTTGTAGCT- (Sigma-Aldrich) supplemented with Mg2+ (5 mM)–EGTA (10 mM), 50% CCTCA-39) and tail DNA as well as by hP ELISA of plasma samples. normal human serum (NHS) in the presence or absence of 30 mM hP-positive mice were then bred with P2/2 mice to generate hP-Tg mice recombinant human fH19-20 (17, 19), and anti-human CD55 (clone on P2/2 background (P humanized mice). All animal studies were BRIC216) and CD59 (MEM-43) Abs (final concentration: 7.5 or 10 mg/ml approved by the Institutional Animal Care and Use Committee of the as specified in figures; AbD Serotec). In assays with anti-P Ab (final University of Pennsylvania. concentration: 5–20 mg /ml as specified in figures), undiluted NHS was incubated with anti-P Ab for 30 min at 4˚C before use. Reaction mixtures In vivo functional assays of anti-hP and anti-mouse C5 mAbs in were centrifuged (1500 rpm for 5 min) to pellet nonlysed erythrocytes. P humanized mice Degree of erythrocyte lysis was measured by OD at 405 nm of an aliquot of the recovered supernatant. Percentage lysis was calculated by normal- To test the efficacy of the anti-hP mAb 19.1 in inhibiting AP complement izing the OD value to that of a completely lysed erythrocyte sample (hy- activity and the efficacy of an anti-mouse C5 mAb (BB5.1) (24) in potonic lysis in water, considered as 100%). inhibiting terminal complement activity in vivo, P humanized mice were For hemolytic assays of PNH erythrocytes, blood was collected from two i.p. injected with either 500 mg of 19.1 per mouse or an isotype control PNH patients who had never received eculizumab therapy and from healthy mAb (MOPC) or 1 mg/mouse of the anti-mouse C5 mAb BB5.1. Serum volunteers after informed consent and with approval from the appropriate was collected at different time points, and LPS-based AP complement institutional committee of the Institute of Hematology, Chinese Academy of activation assay was performed as described above to check the blocking Medical Sciences, in compliance with the Declaration of Helsinki. NHS ability of mAb 19.1 in vivo. P KO mouse serum was used as control for (from ABO blood type–compatible healthy donors) were diluted to 50% this assay. The effect of inhibition was expressed as the percentage of AP ++ with GVB buffer (0.15 mM CaCl2 and 0.5 mM MgCl2) (Sigma-Aldrich), complement activity prior to mAb 19.1 injection in the same mice. To acidified to pH 6.4 (acidified NHS [aNHS]), and used for assays with fresh assess the efficacy of anti-mouse C5 mAb in inhibiting terminal comple- PNH RBCs (5 3 106 cells per assay in 100 ml final volume). Samples were ment activity, mouse serum was diluted to 10% with GVB++ buffer, mixed incubated at 37˚C for 30 min, and reaction was stopped by adding 200 ml 1:1 with similarly diluted 10% C5-deficient human serum, and incubated of cold 20 mM EDTA in PBS. In Ab blocking experiments, anti-P or anti- with Ab-sensitized chicken RBC (catalog no. R401-0050; Rockland Im- C5 Abs were incubated with aNHS for 30 min in 4˚C before adding to the munochemicals) (2.5 3 106 cells per reaction, final volume 50 ml) at 37˚C PNH RBC reaction mixture. Lysis reaction processing and percentage lysis for 30 min. Ab-sensitized chicken RBCs were prepared by incubating the calculation were the same as described above. Nonlysed PNH erythro- cells with a rabbit anti-chicken RBC Ab (150 mg/ml, catalog no. 103-41390; cytes from these assays were stained with FITC-labeled goat anti-human Rockland Immunochemicals) on ice for 1 h (24). Percentage of lysis was C3 Ab (catalog no. 0855167; MP Biomedicals) (1:100) and analyzed by calculated by normalizing the cell-free hemoglobin OD value to that of a FACSCalibur. completely lysed RBC sample through hypotonic lysis in water. The Journal of Immunology 1023

EVH assay Anti-P mAb prevents acidified serum–induced lysis and EVH test was performed as previously described (25). Briefly, DAF2/2/Crry2/2/ activated C3 fragment opsonization of PNH erythrocytes 2/2 C3 mouse erythrocytes (from 150 ml blood for each recipient mouse) were We next tested the efficacy of mAb 19.1 in preventing acidified m labeled ex vivo with CFSE (Molecular Probes) in 1 ml PBS containing 5 M serum–induced lysis of PNH RBCs (Ham test). RBCs from two CFSE. The labeling reaction was carried out at RT for 5 min, and then cells were washed several times in PBS. CFSE-labeled DAF2/2/Crry2/2/C32/2 PNH patients were used for this experiment. FACS analysis erythrocytes were transfused intravenously (via tail vein or retro-orbital showed that the patients’ RBCs contained more than 50% PNH route)intohP-Tgmicepretreated6hbeforeerythrocytetransfusionwith clones (DAF negative) (Fig. 2A). As shown in Fig. 2B and 2C, 2 mg/mouse of either anti-hP mAb clone 19.1 or an isotype control mAb significant lysis of PNH RBCs occurred in 50% aNHS, and this (MOPC) or 1 mg/mouse of the anti-mouse C5 mAb clone BB5.1 (24, 26). Blood was collected from the tail vein at 5 min and various sub- lysis was inhibited by QDC5, a recombinant anti-human C5 mAb. sequent time points as indicated, and the percentage of labeled eryth- Notably, the inhibitory effect of the anti-C5 mAb reached a pla- rocytes present was calculated after FACS and normalized to values at 5 teau at 30–40 mg/ml (0.2–0.26 mM), and further increase in Ab minineachmouse. concentration up to 50 mg/ml (0.33 mM) did not completely eliminate residual hemolysis (Fig. 2B, 2C). In contrast, the anti-P Detection of activated C3 fragment deposited on mAb 19.1 caused near complete inhibition of PNH RBC lysis at transfused erythrocytes 5 mg/ml (0.03 mM) (Fig. 2B, 2C). As expected, when lysis-resistant For detection of activated C3 fragment deposition in vivo on transfused 2/2 2/2 2/2 PNH RBCs rescued by anti-C5 or anti-P mAb treatment were ana- DAF /Crry /C3 mouse erythrocytes, cells were labeled with lyzed by FACS for activated C3 fragment deposition, we found CellTrace Far Red (catalog no. C34564; Thermo Fisher Scientific) according m that those from anti-C5 mAb treatment incurred activated C3 to manufacturer’s instructions and injected i.v. (from 150 l blood for each Downloaded from recipient mouse, retro-orbital route) into hP-Tg mice pretreated with either 2 fragment opsonization, whereas those subjected to anti-P treat- mg of anti-hP mAb 19.1 or an isotype control mAb (MOPC). Blood was ment showed no such deposition on their surface (Fig. 2D). To- collected from the tail vein at 5 and 30 min, and erythrocytes were stained gether, these results suggested that blocking P at the C3 activation with a FITC-conjugated mouse anti-mouse C3/C3b/iC3b mAb (clone 10C7/ catalog no. CL7631F, used at 5 mg/ml; Cedarlane). Transfused cells were step was more effective at inhibiting PNH RBC lysis than gated (CellTrace Far Red) and analyzed for activated C3 deposition (FITC) blocking C5 at the terminal step, and anti-P mAb had a further by FACSCalibur. advantage over anti-C5 mAb in that it could also prevent activated C3 fragment opsonization of PNH RBCs. http://www.jimmunol.org/ Western blot Anti-P mAb prevents complement-mediated EVH in a Proteins were resolved on 4–12% gradient gel under nonreducing condi- humanized mouse model tions and transferred to polyvinylidene difluoride membranes. P was detected with mouse anti-hP 19.1 (2 mg/ml) for 1 h, followed by HRP- To test the effectiveness of anti-hP mAb in preventing EVH in vivo, conjugated rabbit anti-mouse IgG (1:4000 dilution; Sigma-Aldrich) and we used a murine model whereby RBCs from DAF, Crry, and C3 the ECL chemiluminescent detection system (Amersham Pharmacia Bio- tech, Uppsala, Sweden) triple KO mice, when adoptively transferred into WT mouse re- cipients, were rapidly eliminated from the circulation by an AP complement-dependent EVH mechanism (28). With the use of Results C3- and C5-deficient mice as recipients, this mouse model was by guest on September 29, 2021 Generation and characterization of function-blocking mouse previously established to involve an EVH rather than an IVH anti-hP mAbs mechanism (28). Because none of our anti-hP mAbs cross-reacted We generated mouse anti-hP mAbs by immunizing P KO mice with mouse P, we generated a P humanized mouse by first creating (P2/2) with hP protein, followed by standard hybridoma fusion a hP-Tg mouse, followed by crossing with the P KO (P2/2) mouse and screening procedures (27). By ELISA-based binding assays, (14). We constructed a hP transgene plasmid consisting of the hP we identified a number of hybridoma clones that secreted mAbs cDNA under the regulation of chicken b-actin promoter/CMV displaying high P-binding property (Fig. 1A). Four of these mAbs immediate early enhancer and the rabbit b-globin polyadenylation were shown to have strong function-blocking activities in a P- signal sequence (Fig. 3A). Injection of the transgenic construct dependent AP complement activation assay using 10% NHS into the pronuclei of fertilized eggs of C57BL6 mice resulted in (Fig. 1B). From these clones, we chose mAb 19.1 for subsequent five hP-Tg mouse founders (Fig. 3B). ELISA analysis confirmed hemolytic experiments. In a Western blot of proteins separated by the presence of hP in the sera of all five founder lines (Fig. 3C). nonreducing SDS-PAGE gel, mAb 19.1 recognized purified P as We then crossed hP-Tg founder line no. 15 with a P2/2 mouse and well as plasma P in their native form (Fig. 1C) and showed dose- generated hP-Tg/P2/2 mice. Fig. 3D shows that, as previously dependent inhibition of AP complement activity in 50% NHS, described (14), serum from a P2/2 mouse had no LPS-dependent achieving complete AP inhibition at 5 mg/ml (0.03 mM) AP complement activity, whereas sera from hP-Tg/P2/2 mice (Fig. 1D). We then tested the inhibitory activity of mAb 19.1 in an displayed AP complement activity similar to that of a WT mouse, induced autologous hemolysis assay. Normal human RBCs are suggesting that transgenically expressed hP was fully functional resistant to autologous complement lysis because of the presence in mice. of regulators on the cell surface and in the plasma. However, We next used the hP-Tg/P2/2 mice to test AP complement- when the activity of such complement regulators is blocked ex- blocking function of the anti-hP mAb 19.1 in vivo. We treated perimentally, autologous, complement-mediated hemolysis could 3 hP-Tg/P2/2 mice with mAb 19.1 (0.5 mg/mouse) and assessed occur (17, 19). Fig. 1E shows that combined inhibition of DAF on AP complement activity in their sera at various time intervals. human RBC by a neutralizing mAb and factor H (FH) by a Fig. 4A shows that injection of mAb 19.1 completely inhibited hP dominant-negative FH fragment (SCR19-20) rendered normal hu- function in hP-Tg/P2/2 mice for up to 48 h. Finally, when man RBCs susceptible to AP complement lysis in 50% NHS. In this complement-susceptible RBCs from DAF2/2Crry2/2C32/2 KO induced hemolytic assay, we found mAb 19.1 to be completely mice were transfused into hP-Tg/P2/2 mice treated with an effective at inhibiting human RBC lysis (Fig. 1E). Further tests isotype control mAb (MOPC), they were efficiently removed within showed that mAb 19.1 also caused concentration-dependent 5 d (Fig. 4C). However, such RBCs persisted in hP-Tg/P2/2 mice inhibition of normal human RBC lysis when DAF, CD59, and treated with anti-P mAb 19.1 (Fig. 4C). In contrast to the effec- FH were blocked (Fig. 1F). tiveness of anti-P mAb, an anti-mouse C5 mAb did not prevent the 1024 PROPERDIN INHIBITION AND PNH HEMOLYSIS Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 1. Generation and characterization of anti-hP Abs. (A) Screening of anti-hP mAbs by ELISA. Culture supernatant from four hybridoma clones (mAb 19.1, 22.1, 25, and 30) showed strong binding to plate-immobilized hP, in which supernatant from a negative mAb clone 27.1 showed no binding. (B) Inhibition of LPS-induced AP complement activation by affinity-purified mAb 19.1, 22.1, 25, and 30. All four clones of mAbs effectively inhibited AP complement activation when added to 10% NHS at a final concentration of 1 mg/ml. A sample with EDTA added (EDTA) served as a positive control for inhibition. A sample with no mAb added (NHS) served as the baseline AP complement activation. OD 450 value represents ELISA reading of relative C3 fragment deposition (i.e., activated and plate-bound C3). (C) Western blot confirming that mAb clone 19.1 specifically recognizes purified P and P in NHS. Lane 1: 20 ng of purified hP; Lane 2: 1 ml of NHS separated on nonreducing SDS-PAGE. mAb 19.1 was used at 2 mg/ml in Western blotting. (D) Further functional characterization of mAb clone 19.1 showing it concentration-dependently inhibiting LPS-induced AP complement activity in 50% NHS. NHS was diluted 1:1 (i.e., to 50%) in EGTA–Mg2+-supplemented GVB++ buffer and then pretreated with or without mAb (1, 2.5, and 5 mg/ml) for 1 h at 4˚C before adding to LPS-coated plates. It completely blocked AP complement activity at 5 mg/ml in this assay. EDTA serum (NHS plus EDTA) was used as a positive control for complement inhibition. (E and F). Human erythrocytes were lysed by 50% NHS in the presence of human FH short consensus repeats 19–20 and anti-DAF Ab [7.5 mg/ml, (E)] or anti-DAF and anti-CD59 Abs [10 mg/ml for both, (F)]. Lysis was prevented by EDTA or mAb 19.1 [5 mg/ml for (E)] and as indicated in (F). Assays were performed in triplicates and percentage lysis was normalized to hypotonic lysis (100%) with double-distilled water (DDW). All data are representative of at least three independent experiments, and values are expressed as mean (SD) of at least three replicate assays per data point. *p , 0.001 compared with sample without mAb 19.1 treatment (NHS), one-way ANOVA. elimination of transfused DAF2/2Crry2/2C32/2 RBCs from Collectively, these data demonstrated that anti-P mAb, but not anti- hP-Tg/P2/2 mice (Fig. 4C) despite its demonstrated activity to C5 mAb, was capable of preventing complement-mediated EVH. completely inhibit lytic complement activity in hP-Tg/P2/2 mice (Fig. 4B). FACS analysis showed significantly higher amounts of Discussion activated C3 fragment deposition on RBCs transfused into control In this study, we have generated function-blocking anti-hP mAbs mAb-treated mice than those into anti-P mAb-treated mice (Fig. 4D). and used them to demonstrate the critical role of P in AP The Journal of Immunology 1025 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 2. Anti-hP mAb 19.1 effectively blocks lysis of PNH RBCs in aNHS. (A) FACS analysis of DAF expression on RBCs of a healthy volunteer (HV) and two PNH patients (Pt 1, Pt 2) showing both patients having .50% of PNH cells. (B and C) Inhibition of aNHS-induced lysis of PNH patient’s RBCs by anti-P (19.1) and anti-C5 (QDC5) mAbs. With RBCs from both patients, anti-P mAb 19.1 effectively inhibited lysis at 5 mg/ml (0.03 mM) concentration, whereas anti-C5 mAb (QDC5) concentration-dependently inhibited lysis, but it reached a plateau effect around 30–40 mg/ml (0.2–0.26 mM) and failed to completely prevent lysis. Two independent experiments of duplicate assays were performed with similar results. Data from the two exper- iments were combined and presented as mean (SD). aNHS was used at 50%. (D) FACS analysis of activated C3 fragment deposition on RBCs from assays in (B) and (C). In each patient, a subpopulation of cells with very high C3 fragment staining was present in anti-C5 mAb–treated but not mAb 19.1–treated sample. A sample of nontreated and nonstained cells from each patient was used as a negative control for FACS analysis. Results shown are representative of two independent analyses. *p , 0.001, two-way ANOVA. complement-mediated IVH and EVH. P promotes AP comple- complement activation was completely abrogated in P2/2 ment activation and is the only known positive regulator of the mouse serum (14), and in several murine models of AP complement system. Although it has not been considered to be an complement-driven diseases, P deficiency significantly attenu- indispensable component of the AP, recent studies of P2/2 mice ated disease severity (27, 29–31). These findings raised have revealed a surprisingly critical role of P in several settings the possibility that P may represent an attractive therapeutic of AP complement activation. For example, LPS-induced AP target. 1026 PROPERDIN INHIBITION AND PNH HEMOLYSIS Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 3. Generation and characterization of hP-Tg mice. (A) hP cDNA construct used for hP-Tg mouse generation. The cDNA cassette is composed of the chicken b-actin promoter with CVM-IE enhancer, hP cDNA, and the rabbit b-globin poly(A) tail for stable expression of the cDNA in eukaryotic cells. (B) Genotyping of hP-Tg mice by PCR using hP-specific primers and tail DNA. Of 40 mice screened, five transgene-positive founders were identified by the presence of a PCR fragment of approximately 800 bp (#15, #20, #24, #27, and #32). (C) hP protein was detected by ELISA in the sera of the five transgene-positive mice but not in a transgene-negative mouse (#29). Normal mouse serum (NMS; i.e., serum from WT mouse) and serum from P2/2 mouse were also negative for hP, whereas NHS was positive for hP. One-way ANOVA comparing with NMS or P2/2 mouse serum, *p , 0.0001. (D) Transgenically expressed hP restored AP complement activity to P2/2 mice. In contrast to P2/2 mouse serum, which showed no LPS-dependent AP complement activity, sera from two P2/2 mice with hP transgene expression (HuPTg1 and HuPTg2) showed similar AP complement activity to that of a WT mouse. Data in (C) and (D) are representative of two to three independent experiments. Mean (SD) of triplicate assays (C) or average of duplicate assays (D) is shown.

Our data in this study has confirmed that P plays a critical role in finding that is consistent with previous data on eculizumab activity human AP complement activation (Fig. 1) and suggested that (32, 33). In striking contrast, an anti-P mAb achieved complete anti-P mAbs may be effective in treating human PNH disease. hemolysis inhibition at 5–15 mg/ml (0.03–0.1 mM) (Fig. 2). A Eculizumab, the current standard therapy for PNH, has clinical similar level of efficacy of anti-P mAbs was observed in pre- limitations due to its inability to prevent EVH mediated by venting autologous lysis of normal human RBCs when DAF or phagocytosis of activated C3 fragment–opsonized PNH erythro- DAF/CD59 and FH functions were blocked (Fig. 1). cytes. Additionally, in the setting of strong complement activation The differential potency of anti-P and anti-C5 mAbs in pre- leading to deposition of high-density C3b on the activating sur- venting autologous, complement-mediated hemolysis is likely face, an anti-C5 inhibitor such as eculizumab may be outcompeted related to quantitative as well as mechanistic differences between by the C5 convertase assembled on the cell surface for C5 binding, the two target proteins. The plasma concentration of P has been making residual C5 cleavage and hemolysis unavoidable (32, 33). reported to be in the range of 5–15 mg/ml (0.09–0.27 mM based on Indeed, in an acidified serum–induced hemolytic assay of PNH monomeric form) whereas that of C5 is ∼80 mg/ml (0.42 mM) erythrocytes, we observed that an eculizumab-like recombinant (34). In addition, P works by stabilizing the AP C3 convertase, anti-C5 mAb failed to completely inhibit hemolysis when used at and thus plays a facilitating and, in some cases, critical role in a saturation concentration of 50 mg/ml (0.33 mM) (Fig. 2), a enabling AP complement activation to occur (12, 13). Accordingly, The Journal of Immunology 1027 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 4. Therapeutic efficacy of anti-hP mAb 19.1 in a murine model of EVH. (A) Pharmacodynamics of mAb 19.1 in hP-Tg mice. Each mouse was treated with 0.5 mg mAb 19.1 (n = 3 mice). Serum samples were collected before and at various time points after mAb treatment and assessed for LPS- dependent AP complement activity. At this dosage of mAb 19.1, AP complement activity was suppressed to background (P2/2) level for 2 d. EDTA: time 0 sample with EDTA (20 mM) added. NS comparing 8-, 24-, and 48-h samples with fP2/2 or EDTA-treated serum or comparing 72-h sample with 0-h sample. One-way ANOVA. (B) Pharmacodynamics of anti-mouse C5 mAb (BB5.1) in hP-Tg mice. Each mouse was treated with 1 mg of anti-mouse C5 mAb (n = 3). Serum samples were collected before and at various time points after mAb treatment and assessed for lytic activity using Ab-sensitized chicken RBCs. C5 KO mouse serum was used as a control for C5 inhibition. Percentage of chicken RBC (cRBC) lysis was normalized to a sample completely lysed by hypotonic shock in double-distilled water. NS compared with C5KO serum. One-way ANOVA. (C) Effect of anti-hP mAb 19.1 on the survival of transfused CFSE-labeled DAF2/2/Crry2/2/C32/2 mouse erythrocytes in hP-Tg mice. Recipient mice were treated with anti-hP mAb 19.1 (n = 4), an isotype control mAb (n = 4), or anti-mouse C5 mAb (n = 3) 6 h prior to RBC transfusion, and blood samples were taken at 5 min, 6 h, and then daily for 5 d. The percentage of CFSE-labeled RBCs was measured by FACS and normalized to that determined at 5 min (100%). Transfused DAF2/2/Crry2/2/C32/2 mouse RBCs were rapidly eliminated in control mAb– or anti-C5 mAb–treated hP-Tg mice, but such an outcome was prevented by mAb 19.1 treatment. Two-way ANOVA, *p , 0.001. (D) FACS analysis of activated C3 fragment deposition on DAF2/2/Crry2/2/C32/2 RBCs at 5 and 30 min after their transfusion into control mAb– or mAb 19.1–treated hP-Tg/P2/2 mice (representative data from two recipient mice are shown). At both time points, C3 fragment deposition was significantly higher on RBCs transfused into control mAb–treated than mAb 19.1–treated hP-Tg/P2/2 mice. The reason for the marked reduction in C3 fragment deposition on RBCs in control mAb–treated hP-Tg/P2/2 mice between 5 and 30 min is unknown, but it could be caused by C3 fragment degradation to C3d, shedding from the cell surface, or rapid removal of the C3-opsonized cells. Data in (A)–(C) are presented as mean (SD) with indicated n numbers. blocking P function would be expected to be more effective; by complement amplification and terminal complement activation. 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