CN103796664B - Anti-properdin antibody and its application - Google Patents

Abstract

The present invention relates to the use of the Selective depression of the complement system alternative pathway (AP) of anti-properdin antibody.In particular it relates to treat the pathology of AP mediation in individuality or the method for the situation of AP mediation by making individual and anti-properdin antibody contact.

Description

Anti-properdin antibody and its application

Background of the invention

The complement system provides the first line of host defense against invading pathogens. Complement also plays a pathogenic role in human inflammatory diseases. Activation of the complement system occurs through three different pathways: the classical pathway (CP), the lectin pathway (LP) and the alternative pathway (AP). CP is initiated by antigen-antibody binding. LP is triggered when mannose-binding lectin (MBL) interacts with surface sugar molecules on microorganisms. Activation of both pathways leads to assembly of the CCP C3 convertase C4b2a, although direct cleavage of C3 by MBL-related serine proteases can also occur. AP is a self-amplifying loop driven by the AP C3 convertase, C3bBb. AP activation can occur secondary to CP or LP activation, or be initiated independently. In the latter case, low levels of spontaneous C3 'tick-over' generate an initial C3bBb that rapidly propagates AP in the absence of sufficient regulation. Therefore, it is often assumed that AP activation with no or insufficient negative regulation on the surface of non-self is considered a default process, whereas autologous cells usually avoid this outcome with the help of a variety of membrane-bound and fluid-phase complement inhibitory proteins. In certain instances, altered, damaged or stressed autologous cells and tissues can also activate APs and cause inflammatory damage.

Unlike the presence of numerous arrestin proteins, the plasma protein properdin is the only known positive regulator of the complement activation cascade. Properdin is a plasma glycoprotein of approximately 53 kDa with an estimated blood concentration of 5-10 μg/ml. It exists mostly in a head-to-tail conformation as fixed ratio dimers, trimers and tetramers. The currently held view on the function of properdin is that it promotes AP activation by prolonging the half-life of the nascent C3bBb convertase. According to this view, properdin plays a facilitative but not essential role in AP activation. Since activation of CP and LP will invariably trigger the AP amplification loop, it is expected that properdin will also indirectly promote CP- and LP-mediated complement activation. Therefore, based on the general knowledge prior to the present invention, it was not possible to consider properdin as an attractive target for anti-complement therapy since it lacks specificity and is not essential for complement activation.

Although all three complement activation pathways contribute to host defense against microbial infections, recent studies have shown that complement-mediated pathologies in humans, such as age-related macular degeneration, atypical hemolytic uremic syndrome, paroxysmal Nocturnal hemoglobinuria (PNH), rheumatoid arthritis, allergic asthma and ischemia-reperfusion injury are mainly mediated by AP. Accordingly, there remains a need in the art for anti-complement compositions and methods for treating human inflammatory diseases by selectively inhibiting AP while leaving CP and LP unaffected to combat pathogens and protect the host from infection. The present invention fulfills this need.

overview

The present invention relates to anti-properdin antibodies and methods of inhibiting the alternative pathway of complement (AP) using anti-properdin antibodies.

In one embodiment, the invention is a composition comprising an antibody that specifically binds properdin. In a preferred embodiment, properdin is human properdin. In some embodiments, antibodies of the invention are monoclonal antibodies. In some embodiments, antibodies of the invention are humanized antibodies. In some embodiments, antibodies of the invention are chimeric antibodies.

In one embodiment, an antibody of the invention comprises at least one CDR selected from the group consisting of: VH-CDR1: SEQ ID NO:3; VH-CDR2: SEQ ID NO:4; VH-CDR3: SEQ ID NO:5; VL - CDR1: SEQ ID NO:8; VL-CDR2: SEQ ID NO:9; and VL-CDR3: SEQ ID NO:10. In another embodiment, an antibody of the invention comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:2. In one embodiment, an antibody of the invention comprises a light chain comprising the amino acid sequence of SEQ ID NO:7. In another embodiment, an antibody of the invention comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:2 and a light chain comprising the amino acid sequence of SEQ ID NO:7. In one embodiment, an antibody of the invention specifically binds an epitope comprising at least one amino acid of SEQ ID NO:52.

In one embodiment, an antibody of the invention comprises at least one CDR selected from the group consisting of: VH-CDR1: SEQ ID NO: 13; VH-CDR2: SEQ ID NO: 14; VH-CDR3: SEQ ID NO: 15; VL - CDR1: SEQ ID NO: 18; VL-CDR2: SEQ ID NO: 19; and VL-CDR3: SEQ ID NO: 20. In another embodiment, an antibody of the invention comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:12. In one embodiment, an antibody of the invention comprises a light chain comprising the amino acid sequence of SEQ ID NO:17. In another embodiment, an antibody of the invention comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:12 and a light chain comprising the amino acid sequence of SEQ ID NO:17. In one embodiment, an antibody of the invention specifically binds an epitope comprising at least one amino acid of SEQ ID NO:53.

In one embodiment, an antibody of the invention comprises at least one CDR selected from the group consisting of: VH-CDR1: SEQ ID NO:23; VH-CDR2: SEQ ID NO:24; VH-CDR3: SEQ ID NO:25; VL - CDR1: SEQ ID NO:28; VL-CDR2: SEQ ID NO:29; and VL-CDR3: SEQ ID NO:30. In another embodiment, an antibody of the invention comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:22. In one embodiment, an antibody of the invention comprises a light chain comprising the amino acid sequence of SEQ ID NO:27. In another embodiment, an antibody of the invention comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:22 and a light chain comprising the amino acid sequence of SEQ ID NO:27.

In one embodiment, an antibody of the invention comprises at least one CDR selected from the group consisting of: VH-CDR1: SEQ ID NO:33; VH-CDR2: SEQ ID NO:34; VH-CDR3: SEQ ID NO:35; VL - CDR1: SEQ ID NO:38; VL-CDR2: SEQ ID NO:39; and VL-CDR3: SEQ ID NO:40. In another embodiment, an antibody of the invention comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:32. In one embodiment, an antibody of the invention comprises a light chain comprising the amino acid sequence of SEQ ID NO:37. In another embodiment, an antibody of the invention comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:32 and a light chain comprising the amino acid sequence of SEQ ID NO:37.

In one embodiment, an antibody of the invention comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:42. In another embodiment, an antibody of the invention comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:44. In one embodiment, an antibody of the invention comprises a light chain comprising the amino acid sequence of SEQ ID NO:47. In one embodiment, an antibody of the invention comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:42 and a light chain comprising the amino acid sequence of SEQ ID NO:47. In another embodiment, an antibody of the invention comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:44 and a light chain comprising the amino acid sequence of SEQ ID NO:47.

In one embodiment, an antibody of the invention comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:49. In one embodiment, an antibody of the invention comprises a light chain comprising the amino acid sequence of SEQ ID NO:51. In another embodiment, an antibody of the invention comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:49 and a light chain comprising the amino acid sequence of SEQ ID NO:51.

In one embodiment, an antibody of the invention comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:2 and SEQ ID NO:63. In one embodiment, an antibody of the invention comprises a light chain comprising the amino acid sequence of SEQ ID NO:7 and SEQ ID NO:64. In another embodiment, an antibody of the invention comprises a heavy chain - comprising the amino acid sequences of SEQ ID NO: 2 and SEQ ID NO: 63 - and a light chain - comprising SEQ ID NO: 7 and SEQ ID NO: 64 amino acid sequence.

In one embodiment, an antibody of the invention comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:12 and SEQ ID NO:63. In one embodiment, an antibody of the invention comprises a light chain comprising the amino acid sequence of SEQ ID NO:17 and SEQ ID NO:64. In another embodiment, an antibody of the invention comprises a heavy chain - comprising the amino acid sequences of SEQ ID NO: 12 and SEQ ID NO: 63 - and a light chain - comprising SEQ ID NO: 17 and SEQ ID NO: 64 amino acid sequence.

In one embodiment, an antibody of the invention comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:22 and SEQ ID NO:63. In one embodiment, an antibody of the invention comprises a light chain comprising the amino acid sequence of SEQ ID NO:27 and SEQ ID NO:64. In another embodiment, an antibody of the invention comprises a heavy chain - comprising the amino acid sequences of SEQ ID NO: 22 and SEQ ID NO: 63 - and a light chain - comprising SEQ ID NO: 27 and SEQ ID NO: 64 amino acid sequence.

In one embodiment, an antibody of the invention comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:32 and SEQ ID NO:63. In one embodiment, an antibody of the invention comprises a light chain comprising the amino acid sequence of SEQ ID NO:37 and SEQ ID NO:64. In another embodiment, an antibody of the invention comprises a heavy chain - comprising the amino acid sequences of SEQ ID NO: 32 and SEQ ID NO: 63 - and a light chain - comprising SEQ ID NO: 37 and SEQ ID NO: 64 amino acid sequence.

In one embodiment, an antibody of the invention is an antibody that binds properdin and competes with binding of at least one anti-properdin antibody described herein. In another embodiment, the antibody of the invention is an antibody that binds properdin and competes with the binding of the antibody designated mAbl9.1 to properdin. In another embodiment, the antibody of the invention is an antibody that binds properdin and competes with the binding of the antibody designated mAb25 to properdin. In another embodiment, the antibody of the invention is an antibody that binds properdin and competes with the binding of the antibody designated mAb22.1 to properdin. In another embodiment, the antibody of the invention is an antibody that binds properdin and competes with the binding of the antibody designated mAb30 to properdin.

In another embodiment, the invention is a method of treating an alternative pathway (AP)-mediated pathology in an individual comprising administering to said individual at least one anti-properdin antibody described herein. In various embodiments, the alternative pathway (AP)-mediated pathology is selected from at least the following: macular degeneration, ischemia-reperfusion injury, arthritis, rheumatoid arthritis, paroxysmal nocturnal hemoglobinuria (PNH) syndrome syndrome, atypical hemolytic uremic syndrome (aHUS) syndrome, asthma, organ transplant sepsis, inflammation, glomerulonephritis, lupus, and combinations thereof. In some embodiments, an anti-properdin antibody selectively inhibits the alternative pathway, but not the classical and lectin pathways. In some embodiments, the anti-properdin antibody does not affect the AP amplification loop of the classical and lectin pathways. In some embodiments, the anti-properdin antibody inhibits the production of C3bBb protein.

In one embodiment, the invention is a transgenic mouse expressing human properdin (eg, SEQ ID NO:67; SEQ ID NO:54) but not murine properdin.

Brief description of the drawings

The foregoing summary, together with the following detailed description of the preferred embodiment of the invention, will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the invention, a preferred presented embodiment is shown in the drawings. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings. In the attached picture:

Figure 1 is a schematic diagram of the complement pathway. Complement can be activated through three pathways: the classical pathway, the lectin pathway and the alternative pathway. The classical pathway is activated when C1q binds an antibody linked to an antigen, activating C1r and C1s that split C4 and C2. When mannose-binding lectin (MBL) encounters a conserved pathogenic carbohydrate motif, the lectin pathway is activated, activating MBL-associated serine proteases (MASPs) and cleaving C4 and C2 again. C4 and C2 cleavage products form the classical and lectin pathway C3 convertase, C4bC2a, which cleaves C3 into C3b and C3a. A second molecule of C3b can associate with C4bC2a to form the C5 convertase of the classical and lectin pathways, C4bC2aC3b. The alternative pathway (AP) is activated when C3 undergoes spontaneous hydrolysis and forms the initial AP C3 convertase, C3(H2O)Bb, leading to additional C3 cleavage and AP C3 conversion in the presence of factors B and D Final formation of enzyme (C3bBb) and AP C5 convertase (C3bBbC3b). Properdin promotes AP activation by stabilizing AP convertase. All three pathways culminate in the formation of convertases, which in turn generate the major effectors of the complement system: anaphylatoxins (C4a/C3a/C5a), membrane attack complexes (MAC) and opsonins (eg C3b). Anaphylatoxins are potent pro-inflammatory molecules derived from the C4, C3 and C5 splits. MAC is a late assembly of complement components C5b to C9 that can directly cleave targeted surfaces. C3b causes phagocytosis of opsonized targets and also serves to amplify complement activation by AP.

Figure 2 depicts the results of experiments showing dose-dependent inhibition of LPS-induced AP complement activation by mAb 19.1, 22.1, 25 and 30. All four mAb clones potently inhibited AP complement activation when added to 50% normal human serum (NHS) at a final concentration of 5 μg/ml. Samples spiked with EDTA (NHSEDTA) served as negative controls (EDTA blocks complement activation). Samples without added mAb (OAb) served as baseline AP complement activation. Experiments were performed in GVB-EGTA-Mg++ buffer. ELISA plates were coated overnight with LPS. NHS was pre-incubated with mAbs prior to addition to the plate. AP complement activation was detected by measuring the amount of C3 deposited on the plate (OD450).

Figure 3 depicts the results of experiments showing that anti-human properdin mAbs inhibit human red blood cell (RBC) lysis caused by dysfunctional fH and DAF. Human RBCs are not lysed in the absence of human serum (Eh only). They were also resistant to lysis by human serum (50%) in the absence of fH19-20, a recombinant fH fragment that prevents fH from interacting with autologous cells (Ofh1920). However, about 70 % of RBCs lysed. The lysis was completely inhibited by 5 μg/ml of each of the 4 anti-properdin mAbs (19.1, 22.1, 25, 30). RBC samples treated with distilled water (Eh+DDW) caused complete lysis and served as a positive control. RBC samples in normal human serum treated with EDTA (NHSEDTA) served as negative controls (no lysis because EDTA blocks complement activation). Lysis assays were performed in Mg++-EGTA GVB++ buffer to allow only AP complement activation.

Figure 4 depicts the results of an experiment evaluating antibody-sensitized sheep RBCs incubated with 50% normal human serum (NHS) in the absence or presence of 5 μg/ml anti-properdin mAbs. RBCs samples without addition of human serum (ShEs only) showed no lysis; RBCs incubated with 50% NHS without addition of anti-properdin mAb showed complete lysis (50% NHS); with 50% NHS and 5 μg/ml mAbs19 RBCs incubated at .1, 22.1, 25 or 30 were also completely lysed, showing no inhibitory effect of mAbs on classical pathway-mediated complement lysis of sensitized ovine RBCs. RBCs incubated with 50% NHS in the presence of EDTA (NHSEDTA) did not lyse, showing that lysis was complement mediated; sheep RBCs treated with distilled water (Es+DDW) served as 100% lysis control.

Figure 5 - comprising Figures 5A-5C - depicts the results of epitope mapping for mAb 19.1 and 25 and generation of human properdin (fP) deletion mutants. Figure 5a depicts the deduced amino acid sequence of human properdin (SEQ ID NO:54). The signal peptide is underlined. The mature protein begins at residue 28. Figure 5b depicts the amino acid sequence of the seven thrombospondin repeat (TSR) domains of human properdin. They are as follows: TSR0 (SEQ ID NO:55), TSR1 (SEQ ID NO:56), TSR2 (SEQ ID NO:57), TSR3 (SEQ ID NO:58), TSR4 (SEQ ID NO:59), TSR5 (SEQ ID NO:59) ID NO:60), TSR6 (SEQ ID NO:61). Figure 5c depicts the results of epitope mapping of mAb 19.1 and 25 and generation of human properdin (fP) deletion mutants. Human properdin (fP) consists of seven thrombospondin repeat (TSR) domains numbered 0 to 6. A single TSR domain (and in some instances both TSR domains) has been deleted and the mutant proteins expressed in Chinese Hamster Ovary (CHO) cells. All TSR deletion mutants were expressed at the expected size except for the TSR5 deletion mutant which was substantially smaller than the expected size. It is possible that the TSR5 deletion mutant underwent proteolysis. The size of the TSR5 deletion was similar to that of the TSR5+6 deletion mutant, suggesting that TSR6 may have been proteolytically removed in the TSR5 deletion mutant. CHO cell lysates were analyzed by Western blot using a polyclonal goat anti-human fP antibody. M: molecular weight (MW) marker.

Figure 6 depicts epitope mapping for mAb 19.1 and 25 and the results of an ELISA assay for mAb 19.1 and 25 binding to human properdin deletion mutants. CHO cell lysates were coated onto ELISA plates and detected with mAbl 9.1 or 25. A third mAb 29.3 binding to a different epitope of 19.1 and 25 was used as a control to confirm protein expression. The results showed that both mAb 19.1 and 25 reacted with the following deletion mutants: dTSR0, dTSR1, dTSR2, dTSR3, dTSR4. Therefore, it can be concluded that the epitopes for mAb 19.1 and 25 are not located in TSR0-4. In addition, mAbl9.1 lost binding to dTSR5 and dTSR5+6, but retained binding to dTSR6, suggesting that its epitope is located in TSR5. mAb25 lost binding to dTSR5, dTSR5+6 and dTSR6, suggesting that its epitope is located in TSR5-6. However, since dTSR5 has undergone proteolytic degradation, which leads to the possible removal of TSR6 (Figure 5), the epitope for mAb25 may be located in TSR6. HuP refers to full-length human properdin transfectant, which was used as a positive control. Con refers to untransfected CHO cell lysate, which was used as a negative control for lack of binding. All mutant proteins contained a 6xHis tag at the C-terminus.

Figure 7 depicts the results of epitope mapping showing that the epitope of mAbl9.1 was mapped to the C-terminal half of TSR5 with the following amino acid sequence: RGRTCRGRKFDGHRCAGQQQDIRHCYSIQHCP (SEQ ID NO:52). Because TSR0-4 (i.e., dTSR5+6) did not react with mAb19.1, while TSR0-5 (i.e., dTSR6) reacted with 19.1 (Figure 6), further deletion mutants were generated: TSR0-4+1/4TSR5, TSR0 -4+1/2TSR5 and TSR0-4+3/4TSR5. Mutant, but not complete properdin proteins contain a 6xHis tag at their C-terminus. Western blot analysis using anti-human fP and anti-His tag antibodies showed insufficient expression of TSR0-4+3/4TSR5. Two other mutants, TSR0-4+1/4TSR5 and TSR0-4+1/2TSR5 were confirmed to be expressed, but none of them were recognized by mAb19.1, suggesting that they had lost their epitopes against mAb19.1. Therefore, it can be concluded that the key epitope residues for mAb 19.1 are located within the C-terminal half of TSR5 (SEQ ID NO:52).

Figure 8 - comprising Figures 8A and 8B - depicts the results of epitope mapping showing the epitope of mAb25. In Figure 8A, the data indicate an epitope localized to the C-terminal quarter of TSR6, having the amino acid sequence: LVVEEKRPCLHVPACKDPEEEEL (SEQ ID NO:53). Because TSR0-5 (ie, dTSR6) lost binding to mAb25, it was concluded that TSR6 constitutes at least a partial epitope of mAb25. Additional mutants that produced TSR6 were as follows: TSR0-5+1/4TSR6, TSR0-5+1/2TSR6 and TSR0-5+3/4TSR6. Mutant, but not complete properdin proteins contain a 6xHis tag at their C-terminus. All three mutants were successfully expressed as shown by Western blot with anti-human fP and anti-His tag antibodies. ELISA binding experiments showed that all three mutants lost binding to mAb25. As a positive control, all mutant proteins reacted with mAb19.1. This result suggested that the last quarter of TSR6 (having the sequence specified by SEQ ID NO: 53) constitutes a critical part of the mAb25 epitope. HuP refers to CHO cells transfected with full-length (intact) human fP as a positive control; ConLysate refers to untransfected CHO cells as a negative control for binding. In Figure 8B, the data indicate that the epitope of mAb25 is dependent on two cysteine residues in TSR6 (SEQ ID NO: 61, as shown in Figure 5B). These are cysteine 62 (C62) and cysteine 78 (C78) of TSR6. Single mutations of alanine (A) at C62 or C78 in full-length human properdin did not abolish mAb25 binding, but double mutations of C62A and C78A abolished mAb25 binding. As a positive control for mutant protein expression, mAb 19.1 showed reactivity for all samples. This result suggests that C78 within the last quarter of TSR6 (with the sequence specified by SEQ ID NO: 53) and C62 outside of SEQ ID NO: 53 but within TSR6 (SEQ ID: 61) constitute the epitope of mAb25. Two key residues. Binding assays for Abs 19.1 and 25 were performed on ELISA plates using homogenates of transfected CHO cells. HuP refers to CHO cells transfected with full-length (intact) human fP as a positive control; Con refers to untransfected CHO cells as a negative control for binding. Other samples were CHO cells transfected with mutant human fP cDNA containing single or double C62A and C78A mutations.

Figure 9 depicts the nucleotide and amino acid sequences of the heavy chain (SEQ ID NO:1; SEQ ID NO:2) and light chain (SEQ ID NO:6; SEQ ID NO:7) variable region sequences of mAb19.1, Including CDRs (VH-CDR1: SEQ ID NO:3; VH-CDR2: SEQ ID NO:4; VH-CDR3: SEQ ID NO:5; VL-CDR1: SEQ ID NO:8; VL-CDR2: SEQ ID NO:9 ; VL-CDR3: SEQ ID NO: 10).

Figure 10 depicts the nucleotide and amino acid sequences of the heavy chain (SEQ ID NO:11; SEQ ID NO:12) and light chain (SEQ ID NO:16; SEQ ID NO:17) variable region sequences of mAb25, including the CDRs ( VH-CDR1: SEQ ID NO: 13; VH-CDR2: SEQ ID NO: 14; VH-CDR3: SEQ ID NO: 15; VL-CDR1: SEQ ID NO: 18; VL-CDR2: SEQ ID NO: 19; VL-CDR3: SEQ ID NO: 20).

Figure 11 depicts the nucleotide and amino acid sequences of the heavy chain (SEQ ID NO:21; SEQ ID NO:22) and light chain (SEQ ID NO:26; SEQ ID NO:27) variable region sequences of mAb22.1, including CDRs (VH-CDR1: SEQ ID NO:23; VH-CDR2: SEQ ID NO:24; VH-CDR3: SEQ ID NO:25; VL-CDR1: SEQ ID NO:28; VL-CDR2: SEQ ID NO: 29; VL-CDR3: SEQ ID NO: 30).

Figure 12 depicts the nucleotide and amino acid sequences, including CDRs, of the variable region sequences of the heavy chain (SEQ ID NO:31; SEQ ID NO:32) and light chain (SEQ ID NO:36; SEQ ID NO:37) of mAb30 (VH-CDR1: SEQ ID NO:33; VH-CDR2: SEQ ID NO:34; VH-CDR3: SEQ ID NO:35; VL-CDR1: SEQ ID NO:38; VL-CDR2: SEQ ID NO:39 ; VL-CDR3: SEQ ID NO: 40).

Figure 13 depicts mAb19.1 heavy chain variable after CDR grafting using two human germline VH sequences (human VH4-59-01 (SEQ ID NO:41); human VH3-66-04 (SEQ ID NO:43)). Humanized amino acid sequence of the region (humanized 19.1VH-4-59-01 (SEQ ID NO:42); humanized 19.1VH-3-66-04 (SEQ ID NO:44)).

Figure 14 depicts the humanized amino acids of the mAbl 9.1 light chain variable region following CDR grafting using human germline VL sequences (human VL4-1-01 (SEQ ID NO:45); human JK2 (SEQ ID NO:46)) Sequence (Humanized 19.1VL-4-1-01 (SEQ ID NO:47)).

Figure 15 depicts the humanized amino acid sequence of the mAb25 heavy chain variable region (humanized 25-VH-1- 69-06 (SEQ ID NO: 49)).

Figure 16 depicts the humanized amino acid sequence of the mAb25 light chain variable region after CDR grafting using the human germline VL sequence (human VL1-69-06 (SEQ ID NO:50)); human Jk3 (SEQ ID NO:62) ( Humanized 25-VL-1-69-06 (SEQ ID NO:51)).

Figure 17 - comprising Figures 17A and 17B - depicts the results of experiments evaluating recombinant chimeric and humanized 19.1 mAbs. Figure 17A depicts the amino acid sequence of the human IgG4 heavy chain constant region with serine 229 mutated to proline (SEQ ID NO:63), and the human light chain kappa constant region (SEQ ID NO:64). These sequences were used to construct chimeric (mouse variable regions + human constant regions) and humanized (humanized mouse variable regions + human constant regions) anti-properdin antibodies. Figure 17B depicts the results of experiments evaluating the expression of recombinant chimeric and humanized 19.1 mAbs. SDS PAGE analysis of recombinant chimeric 19.1 mAb and two humanized 19.1 mAbs. The chimeric 19.1 heavy chain construction was obtained by linking the VH region of 19.1 with the human IgG4 heavy chain constant region. The chimeric 19.1 light chain was constructed by linking the VL region of 19.1 with the human kappa chain constant region. The humanized 19.1 heavy chain and light chain were constructed in the same way, that is, the humanized VH region was connected to the human IgG4 heavy chain constant region, and the humanized light chain was connected to the human κ chain constant region. CHO cells were co-transfected with heavy and light chain cDNAs and established stable lineages by drug moiety. For both humanized mAbs, each of the two humanized heavy chains was paired with the same humanized light chain for transfection. Expressed mAbs were purified from CHO cell culture media by protein G affinity columns.

Figure 18 depicts the results of experiments using Biacore to measure the antigen binding affinities of 19.1, chimeric 19.1 and humanized 19.1 mAbs. Purified human fP was coupled to a CM4 chip using an amine coupling method. Biacore analysis was performed on a Biacore-2000 instrument. Chips were regenerated between each binding using 50 mM NaOH.

Figure 19 depicts the results of experiments measuring the antigen binding affinities of mAbs 25, 22.1 and 30, as determined by Biacore analysis. Purified human fP was coupled to a CM4 chip using an amine coupling method. Biacore analysis was performed on a Biacore-2000 instrument. Chips were regenerated between each binding using 50 mM NaOH.

Figure 20 depicts the results of experiments evaluating the relative activity of 19.1, chimeric 19.1 and humanized 19.1 mAbs in blocking LPS-induced human AP complement activation. ELISA plates were coated with LPS overnight, 50% normal human serum (NHS) diluted in GVB-Mg++-EGTA was added and incubated at 37°C for 1 hr, then C3 deposition was detected using anti-C3 antibody. NHS without added antibody served as a positive control (NHS), and NHS with EDTA added served as a negative control (NHSEDTA). For 19.1 mAb, concentrations of 5 μg/ml and 10 μg/ml were sufficient to inhibit complement activation. For the chimeric and two humanized 19.1 mAbs, a concentration of 5 μg/ml was not sufficient to inhibit complement activation. However, concentrations of 10 μg/ml and 20 μg/ml were effective in blocking AP complement activation.

Figure 21 depicts the results of experiments evaluating the relative activity of 19.1, chimeric 19.1 and humanized 19.1 mAbs in blocking human RBC lysis by human AP complement in the setting of dysfunctional fH and DAF. Human RBCs were incubated with 50% normal human serum in the presence of fH19-20 (30 μM) and anti-DAF antibody (7.5 μg/ml). Human sera were diluted in GVB-Mg++-EGTA and incubated at 37°C for 1 hr. Human serum was pre-incubated with increasing concentrations of 19.1, chimeric 19.1 and humanized 19.1 mAbs (1-15 μg/ml) for 1 hr at 4°C before addition to RBCs. All 4 mAbs had dose-dependent inhibition of RBC lysis. However, the EC50 of chimeric and humanized 19.1 mAbs was higher than 19.1 mAb. This result is consistent with the data shown in Figure 20.

Figure 22 depicts the results of an experiment evaluating the relative activity of 19.1, chimeric 19.1 and humanized 19.1 mAbs in blocking LPS-induced rhesus AP complement activation. The ELISA plate was coated with LPS overnight, 50% normal Rhesus monkey serum (NRS) diluted in GVB-Mg++-EGTA was added and incubated at 37°C for 1 hr, then C3 deposition was detected by anti-human C3 antibody. NRS without antibody served as a positive control (NRS) and NRS with EDTA added as a negative control (NRSEDTA). For 19.1 and chimeric 19.1 mAbs, concentrations of 10-40 μg/ml were sufficient to inhibit rhesus complement activation. Concentrations of 30 or 40 μg/ml effectively inhibited complement activation for both humanized 19.1 mAbs. Concentrations of 10 or 20 μg/ml also substantially inhibited rhesus AP complement activation.

Figure 23 depicts the results of experiments evaluating the relative activity of 19.1, chimeric 19.1 and humanized 19.1 mAbs in blocking LPS-induced AP complement activation in cynomolgus monkeys. ELISA plates were coated with LPS overnight, 50% normal cynomolgus monkey serum (NCS) diluted in GVB-Mg++-EGTA was added and incubated at 37°C for 1 hr, then C3 deposition was detected using anti-human C3 antibody. NCS without antibody served as a positive control (NCS), and NCS with EDTA added as a negative control (NCSEDTA). For 19.1 mAb, a concentration of 10-40 μg/ml was sufficient to inhibit cynomolgus AP complement activation. For the chimeric 19.1 mAb, a concentration of 20-40 μg/ml was sufficient to inhibit cynomolgus AP complement activation, but a concentration of 10 μg/ml also significantly inhibited complement activation. Concentrations of 30 or 40 μg/ml effectively inhibited complement activation in cynomolgus monkeys for both humanized 19.1 mAbs. However, a concentration of 20 μg/ml also substantially inhibited cynomolgus AP complement activation. The concentration of 10 μg/ml also partially inhibited the complement activity of AP in cynomolgus monkeys.

Figure 24 depicts the results of experiments evaluating the inhibition (Ham's test) of mAb 19.1, 25 and humanized 19.1 on lysis of erythrocyte acidified serum from PNH patients. RBCs from patients with paroxysmal nocturnal hemoglobinuria (PNH) were subjected to Ham's acidified serum test in the presence or absence of mAbs. RBCs were incubated with autologous serum (final concentration 83%) for 2 hr at 37C and percent lysis was calculated by measuring the OD405 of the supernatant, normalized to a sample of RBCs completely lysed by distilled water (Eh DDW). The incubation mixture consisted of: 240 μl serum, 25 μl 1/6N HCL (or 25 μl saline for negative control), 12.5 μl 50% (v/v) RBC suspension, 10 μl mAb in saline. A sample of RBCs incubated with non-acidified autologous serum (NHS) was used as a negative control (background lysis). In the absence of mAbs, about 50% of RBCs were lysed by acidified serum. The cleavage was completely inhibited by mAb 19.1 at concentrations of 8 μg/ml and greater, humanized 19.1 mAb (#459) at concentrations of 20 μg/ml and mAb25 at concentrations of 8 μg/ml and greater.

Figure 25 -comprising Figures 25A-25E-describes generation of properdin humanized mice. The human fP expression vector was constructed as shown in the schematic diagram in Figure 25A, using chicken β-actin promoter with CVM-IE enhancer and rabbit β-globin polyA tail for cDNA expression in eukaryotic cells stable expression. This plasmid was linearized and microinjected into fertilized eggs of C57BL/6 mice to generate human fP transgenic founder mice. Positive founder mice ( A human fP cDNA fragment of approximately 800 bp is shown). Of the 40 mice analyzed, five (#15, 20, 24, 27 and 32) were positive (Figure 25B, red arrows). An ELISA assay was performed to detect human fP in transgene positive mice (Fig. 25C). Plates were coated with a non-blocking mAb against human fP (clone 8.1). After incubation with diluted serum (10%), human fP was detected by ELISA using an HRP-conjugated goat anti-human fP antibody. Normal human serum (NHS) was used as a positive control. As can be seen, human fP was detected in the NHS and in the sera of five transgenic mice, but not in normal (i.e., non-transgenic) mouse sera (NMS), transgene negative (#29) or in fP ^-/- Not detected in mouse serum. Founder mice #32 were bred with WT mice and pups were screened by PCR as described above. Three representative F1 mice—one PCR-negative (F1-429) and two PCR-positive (F1-430 and F1-431)—were tested for the presence of human fP in their sera by ELISA ( Figure 25D). As shown, human fP was detected in two PCR-positive mice, but not in PCR-negative mice. Sera from the NHS and founder parent (#32) were used as positive controls. This result suggests that the transgene is stable and transmittable through the germline. Founder mice #32 were then bred with fP ^−/− mice to generate fP ^−/− − human fP transgenic+ mice. LPS-induced AP complement activation assays showed that fP ^-/- -human fP transgenic+ mice but not fP ^-/- mice had indistinguishable serum AP complement activity from WT mice (Fig. 25E), suggesting that transgenicly expressed human fP Can rescue the phenotype of fP ^-/- mice. WT sera treated with EDTA were used as negative controls. This result confirms the generation of a properdin humanized mouse strain.

Figure 26 depicts experiments examining the in vivo activity and kinetics of mAb25 in "properdin humanized" mice. Properdin humanized mice (fP ^−/− -human fP transgene+) were injected with 0.5 mg (ip) mAb25. Serum samples were collected prior to injection (0 hr) and then at various time points after injection and assayed for LPS-induced AP complement activation. As shown, there was no AP complement activity in fP ^−/− mouse sera or in EDTA-treated WT sera. In contrast, AP complement activity was detected in WT sera and in fP humanized mouse sera at time Oh (before mAb treatment). AP complement activity in humanized mice remained undetectable at 8, 24 and 48 hr after mAb treatment, but was detectable at 72, 96 and 120 hr. These results suggest that at a dose of 0.5 mg/mouse, mAb25 can inhibit AP complement activity in vivo for 48 hr.

Figure 27 depicts the results of experiments showing that anti-human properdin mAb 19.1 prevents extravascular hemolysis (EVH). In this EVH model, properdin humanized mice (n=4 per experimental group) were infused with red blood cells (RBC) from Crry/DAF/C3 triple knockout (TKO) mice. Recipient mice (properdin humanized mice) were treated with mAb19.1 (2 mg/mouse, i.p.) or control mouse IgG1 mAb (MOPC, purified from MoPC31C hybridoma from ACTT) for 6 hr prior to RBC transfer. RBCs were harvested from donor TKO mice, washed in PBS and labeled with CFSE before injection (via tail vein) into recipient mice according to a previously published procedure (Miwa et al., 2002, Blood99:3707-3716). . Each recipient mouse received RBCs equal to 100 μl of blood. At 5 minutes and 6, 24, 48, 72, 96, 120 hours after RBC infusion, recipient mice were bled and RBCs were analyzed to determine the number of CFSE-labeled (ie, infused) RBCs remaining in circulation. The number of CFSE-labeled RBCs in each recipient was normalized (as %) to the number of RBCs detected at the 5 min time point. In control IgG (MOPC)-treated recipient mice, TKO RBCs were rapidly eliminated by EVH, consistent with previous findings (Miwa et al., 2002, Blood 99:3707-3716). However, in recipient mice treated with anti-human properdin 19.1 mAb, EVH did not develop and the infused RBCs persisted, showing that anti-properdin mAb was effective in preventing EVH.

Figure 28 depicts the nucleic acid sequence of human properdin cDNA (SEQ ID NO: 67) used to generate human properdin transgenic mice.

Detailed description of the invention

The present invention relates to inhibition of the alternative complement pathway (AP) using anti-properdin antibodies. In various embodiments, the invention relates to compositions and methods for treating AP-mediated pathologies or AP-mediated conditions in an individual by contacting the individual with an anti-properdin antibody. AP-mediated pathologies and conditions that can be treated with the compositions and methods of the present invention include, but are not limited to, macular degeneration, ischemia-reperfusion injury, arthritis, rheumatoid arthritis, asthma, paroxysmal nocturnal hemoglobinuria ( PNH) syndrome, atypical hemolytic uremic syndrome (aHUS) syndrome, sepsis, organ transplantation, inflammation (including, but not limited to, inflammation associated with cardiopulmonary bypass surgery and renal dialysis), glomerulonephritis (Including, but not limited to, anti-neutrophil cytoplasmic antibody (ANCA)-mediated glomerulonephritis, lupus, and combinations thereof.

definition

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described.

As used herein, each of the following terms has the meaning associated with it in this section.

The articles "a" and "an" are used herein to refer to one or more than one (ie, at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

As used herein, the terms "inhibit" and "inhibition" mean to reduce, inhibit, decrease or block an activity or function by at least about 10% relative to a control value. Preferably, the activity is inhibited or blocked by 50%, more preferably 75% and even more preferably 95% compared to the control value.

The terms "effective amount" and "pharmaceutically effective amount" refer to a sufficient amount of an agent to provide a desired biological result. The result may be reduction and/or alleviation of the signs, symptoms or causes of a disease or disorder, or any other desired alteration of a biological system. An appropriate effective amount in any individual case can be determined by one of ordinary skill in the art using routine experimentation.

The terms "patient", "subject", "individual" and the like are used interchangeably herein and refer to any animal, preferably a mammal, most preferably a human, having a complement system, including conditions requiring treatment, or susceptible conditions or sequelae thereof affect people. Individuals can include, for example, dogs, cats, pigs, cows, sheep, goats, horses, rats, monkeys, and mice, as well as humans.

When used in the context of an organism, tissue, cell or component thereof, the term "abnormal" refers to a difference in at least one observable or detectable characteristic (e.g., age, treatment, time of day, etc.) Normal" (desired/steady-state) those organisms, tissues, cells or components thereof of those organisms, tissues, cells or components thereof, respectively. A property that is normal or expected for one cell, tissue type, or subject may be abnormal for a different cell or tissue type.

A "disease" is a state of animal health in which the animal cannot be maintained at homeostasis, and in which the animal's health continues to deteriorate if the disease does not improve.

In contrast, a "condition" in an animal is a state of health in which the animal is able to maintain homeostasis, but wherein the animal's state of health is less favorable than it would be in the absence of the condition. Without treatment, the condition does not necessarily lead to a further reduction in the animal's state of health.

A disease or condition is "alleviated" if the severity of the signs or symptoms of the disease or condition, the frequency with which such signs or symptoms are experienced by the patient, or both are reduced.

An "effective amount" or "therapeutically effective amount" of a compound is an amount of the compound sufficient to provide a beneficial effect to the subject to whom the compound is administered.

As used herein, "instructional material" includes publications, records, diagrams or publications that can be used to demonstrate the effectiveness of the compounds, compositions, vectors or delivery systems of the invention in kits for achieving relief from the various diseases or conditions described herein. any other expression medium. Optionally or alternatively, the instructional material may describe one or more methods of alleviating a disease or condition in a mammalian cell or tissue. Instructional material for a kit of the invention may, for example, be attached to or shipped with a container containing an identified compound, composition, vector or delivery system of the invention. Alternatively, the instructional material may be shipped separately from the container, with the intent that the instructional material and compound be used cooperatively by the recipient.

A "therapeutic" treatment is a treatment applied to a subject exhibiting signs of pathology for the purpose of reducing or eliminating those signs.

As used herein, "treating a disease or disorder" means reducing the frequency and/or severity experienced by a patient of signs and/or symptoms of a disease, disorder or pathology. Disease, disorder and pathology are used interchangeably herein.

As used herein, the phrase "biological sample" is intended to include any sample - including cells, tissues or bodily fluids - in which expression of a nucleic acid or polypeptide can be detected. Examples of such biological samples include, but are not limited to, blood, lymph, bone marrow, biopsies and smears. Samples that are liquid by nature are referred to herein as "body fluids." Biological samples can be obtained from a patient by a number of techniques including, for example, by scraping or swabbing an area or by using a needle to obtain bodily fluids. Methods of taking various bodily samples are well known in the art.

The term "antibody", as used herein, refers to an immunoglobulin molecule that is capable of specifically binding a particular epitope of an antigen. Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins. Antibodies of the invention can exist in a variety of forms, including, for example, polyclonal antibodies, monoclonal antibodies, intrabodies ("intrabodies"), Fv, Fab, Fab', F(ab)2, and F( ab')2, and single-chain antibodies (scFv), heavy-chain antibodies such as camelid antibodies and humanized antibodies (Harlow et al., 1999, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA85: 5879-5883; Bird et al., 1988, Science242: 423-426).

The term "synthetic antibody", as used herein, means an antibody produced using recombinant DNA techniques, such as, for example, an antibody expressed by phage, as described herein. The term should also be construed as referring to an antibody which has been produced by the synthesis of an antibody-encoding DNA molecule, and whose DNA molecule expresses the antibody protein, or a defined amino acid sequence of the antibody, wherein the DNA or amino acid sequence has been used Obtained by synthetic DNA or amino acid sequence techniques available and well known in the art.

As used herein, the term "heavy chain antibody" or "heavy chain antibodies" includes immunoglobulin molecules derived from camelid species, either by immunization with peptides followed by isolation of serum, or by cloning and Nucleic acid sequences encoding such antibodies are expressed. The term "heavy chain antibody" further includes immunoglobulin molecules isolated from animals with heavy chain disease, or prepared by cloning and expressing VH (variable heavy immunoglobulin) genes from animals.

"Chimeric antibody" refers to a type of engineered antibody that contains naturally occurring variable regions (light and heavy chains) derived from a donor antibody in combination with light and heavy chain constant regions derived from a recipient antibody.

"Humanized antibody" refers to a type of engineered antibody that has CDRs derived from a non-human donor immunoglobulin, the remainder of the molecule derived from an immunoglobulin source portion derived from one (or more) human immunoglobulins (or multiple). In addition, framework support residues can be altered to maintain binding affinity (see, e.g., 1989, Queen et al., Proc. Natl. Acad Sci USA, 86:10029-10032; 1991, Hodgson et al., Bio/Technology, 9 :421). A suitable human recipient antibody can be one selected from conventional databases, eg, KABAT database, Los Alamos database, and Swiss protein database, using nucleotide and amino acid sequence homology to the donor antibody. Human antibodies characterized by framework region homology (on an amino acid basis) to the donor antibody may be suitable for providing heavy chain constant regions and/or heavy chain variable framework regions for insertion of the donor CDRs. Suitable acceptor antibodies capable of donating light chain constant or variable framework regions can be selected in a similar manner. It should be noted that the recipient antibody heavy and light chains need not be derived from the same recipient antibody. The prior art describes several methods of producing such humanized antibodies (see eg EP-A-0239400 and EP-A-054951).

The term "donor antibody" refers to an antibody (monoclonal and/or recombinant) that donates the amino acid sequence of its variable regions, CDRs or other functional fragments, or analogs thereof, to a primary immunoglobulin partner for An altered immunoglobulin coding region is provided and the resulting expressed altered antibody has the antigen specificity and neutralizing activity properties of the donor antibody.

The term "recipient antibody" refers to an antibody (monoclonal and/or recombinant) heterologous to the donor antibody that encodes all (or any portion, but in some embodiments all) of its heavy chain and/or The amino acid sequence of the light chain framework regions and/or its heavy and/or light chain constant regions is contributed to the first immunoglobulin partner. In certain embodiments, the human antibody is a recipient antibody.

"CDRs" are defined as the amino acid sequences of the complementarity determining regions of antibodies, which are the hypervariable regions of the heavy and light chains of immunoglobulins. See, eg, Kabat et al., Sequences of Proteins of Immunological Interest, 4th Ed., U.S. Department of Health and Human Services, National Institutes of Health (1987). There are three heavy chain and three light chain CDRs (or CDR regions) in the variable portion of an immunoglobulin. Thus, "CDRs" as used herein refers to all three heavy chain CDRs or all three light chain CDRs (or both all heavy chain and all light chain CDRs, as appropriate). The structure and protein folding of antibodies may refer to other residues that are considered part of the antigen binding region and will be understood by the skilled artisan to be so. See eg Chothia et al., (1989) Conformations of immunoglobulin hypervariable regions; Nature 342, p877-883.

As used herein, "immunoassay" refers to any binding assay that utilizes antibodies capable of specifically binding a target molecule to detect and quantify the target molecule.

The term "specifically binds", as used herein with respect to an antibody, refers to an antibody that recognizes a particular antigen, but does not substantially recognize or bind other molecules in a sample. For example, an antibody that specifically binds an antigen from one species may also bind antigens from one or more species. However, such cross-species reactivity does not by itself change the classification of an antibody to be specific. In another example, an antibody that specifically binds an antigen may also bind a different allelic form of the antigen. However, such cross-reactivity does not by itself change the class of an antibody to be specific.

In some instances, the term "specific binding (or specifically binding)" may be used with reference to the interaction of an antibody, protein or peptide with a second chemical species to mean that the interaction is dependent on a specific structure on the chemical species (e.g., an antigenic determination). clusters or epitopes); for example, antibodies typically recognize and bind specific protein structures rather than proteins. If the antibody is specific for epitope "A", then the presence of a molecule containing epitope A (or free, unlabeled A) in a reaction containing labeled "A" and the antibody will reduce the label bound to the antibody The amount of A.

The "coding region" of a gene consists of nucleotide residues of the coding strand of the gene, and the nucleotides of the non-coding strand of the gene are homologous or complementary to the coding region of the mRNA molecule produced by gene transcription.

The "coding region" of an mRNA molecule also consists of nucleotide residues of the mRNA molecule that match the anti-codon region of the transfer RNA molecule during translation of the mRNA molecule, or that encode a stop codon. The coding region may thus include nucleotide residues that include codons for amino acid residues that are not present in the mature protein encoded by the mRNA molecule (eg, amino acid residues in a protein export signal sequence).

As used herein, reference to "complementarity" of nucleic acids refers to the broad concept of sequence complementarity between regions of two nucleic acid strands or between two regions of the same nucleic acid strand. It is known that an adenine residue in a first nucleic acid region can form specific hydrogen bonds ("base pairing" ). Similarly, it is known that a cytosine residue of a first nucleic acid strand is capable of base pairing with a residue of a second nucleic acid strand that is antiparallel to the first strand if the residue is guanine. If a first region of a nucleic acid is complementary to a second region of the same or different nucleic acid, then when the two regions are arranged in an antiparallel fashion, at least one nucleotide residue of the first region is compatible with a residue of the second region base pairing. Preferably, the first region comprises a first portion and the second region comprises a second portion whereby, when the first and second portions are arranged in an antiparallel fashion, the first portion is at least about 50% and preferably at least about 75%, at least about 90%, or at least about 95%, of the nucleotide residues are capable of base pairing with nucleotide residues in the second portion. More preferably, all nucleotide residues in the first portion are capable of base pairing with nucleotide residues in the second portion.

The term "DNA" as used herein is defined as deoxyribonucleic acid.

"Encoding" refers to the inherent property of a specific sequence of nucleotides in a polynucleotide, such as a gene, cDNA or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes - having a defined sequence of nucleotides (i.e., rRNA , tRNA and mRNA) or defined amino acid sequences and biological properties derived therefrom. Thus, a gene encodes a protein if transcription and translation of the corresponding gene's mRNA produces the protein in a cell or other biological system. Both the coding strand—whose nucleotide sequence is identical to the mRNA sequence and is usually given in the Sequence Listing—and the noncoding strand, which serves as a template for transcription of a gene or cDNA, can both be referred to as encoding a protein or other product of that gene or cDNA.

Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and encode the same amino acid sequence. To the extent that the phrase a nucleotide sequence encoding a protein or RNA may also include introns, a nucleotide sequence encoding a protein may, in some forms, contain intron(s).

"Isolated" means altered or removed from the natural state. For example, a nucleic acid or peptide naturally occurring in its normal environment in a living animal is not "isolated", but the same nucleic acid or peptide partially or completely separated from coexisting materials of its natural environment is "isolated". An isolated nucleic acid or protein can exist in substantially purified form, or it can exist in a non-native environment such as, for example, a host cell.

"Isolated nucleic acid" refers to a nucleic acid segment or fragment that has been separated from the sequences flanking it in its naturally occurring state, i.e., has been separated from the sequences that normally adjoin the segment—i.e., the sequences adjacent to the segment in its naturally occurring genome - Removed DNA fragments. The term also applies to a nucleic acid that has been substantially purified from other components that naturally accompany it, ie, RNA or DNA or proteins, that naturally accompany it in cells. The term thus includes, for example, recombinant DNA incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genome of a prokaryote or eukaryote, or as a separate molecule (i.e., as cDNA or genome or by PCR or cDNA fragments produced by restriction enzyme digestion) exist independently of other sequences. It also includes recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequences.

In the context of the present invention, the following abbreviations for commonly occurring nucleic acid bases are used. "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine and "U" refers to uridine.

The term "polynucleotide" as used herein is defined as a chain of nucleotides. Additionally, nucleic acids are polymers of nucleotides. Accordingly, nucleic acid and polynucleotide as used herein are interchangeable. It is common knowledge of those skilled in the art that nucleic acids are polynucleotides, which can be hydrolyzed into monomeric "nucleotides". Monomeric nucleotides can be hydrolyzed into nucleosides. As used herein, polynucleotides include, but are not limited to, methods available in the art - including without limitation, recombinant methods, i.e., cloning of nucleic acid sequences from recombinant libraries or the genome of cells using common cloning techniques and PCR, and the like. Methods - and all nucleic acid sequences obtained by synthetic methods.

As used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to a compound consisting of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can make up the sequence of a protein or peptide. Polypeptides include any peptide or protein - comprising two or more amino acids linked to each other by peptide bonds. As used herein, the term refers to short chains, which are also commonly referred to in the art as eg peptides, oligopeptides and oligomers, and longer chains, commonly referred to in the art as proteins, of which there are many varieties. "Polypeptide" includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, Fusion proteins, etc. Polypeptides include natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

The term "progeny" as used herein refers to descendent or offspring, and includes mammalian offspring, and also includes differentiated or undifferentiated descendent cells from a parent cell. In one usage, the term progeny refers to a descendent cell that is genetically identical to a parent. In another application, the term progeny refers to progeny cells that are genetically and phenotypically identical to a parent. In another usage, the term progeny refers to a progeny cell that has differentiated from a parent cell.

The term "RNA" as used herein is defined as ribonucleic acid.

The term "recombinant DNA" as used herein is defined as DNA produced by joining pieces of DNA from different sources.

The term "recombinant polypeptide" as used herein is defined as a polypeptide produced by the use of recombinant DNA methods.

As used herein, "linked" refers to the covalent attachment of one molecule to a second molecule.

As the term is used herein, a "variant" is a nucleic acid or peptide sequence that differs in sequence from a reference nucleic acid or peptide sequence, respectively, but retains the essential biological properties of the reference molecule. Alterations in the sequence of a variant nucleic acid may not alter the amino acid sequence of the peptide encoded by the reference nucleic acid, or may result in amino acid substitutions, additions, deletions, fusions and truncations. Changes in the sequence of a peptide variant are usually restricted or conserved so that the sequences of the reference peptide and the variant are very similar overall and identical in many regions. Variant and reference peptides may differ in amino acid sequence by one or more substitutions, additions, deletions in any combination. Variants of a nucleic acid or peptide may be naturally occurring, such as allelic variants, or may be variants that are not known to occur naturally. Variants of non-naturally occurring nucleic acids and peptides can be prepared by mutagenesis techniques or by direct synthesis.

The term "reproduction" as used herein refers to the reproduction of a species, resulting in at least one offspring.

The term "natural reproduction" as used herein refers to the reproduction of a species through sexual union.

The term "inbred animal" is used herein to refer to an animal that has been interbreeded with other similar animals of the same species in order to maintain and fix certain traits or prevent other traits from being introduced into the breeding population.

The term "outbred animal" as used herein refers to an animal that has been bred with any other animal of the same species without regard to maintaining certain characteristics.

Ranges: Throughout this disclosure, various aspects of this invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all possible subranges as well as individual values within that range. For example, the description of a range such as 1 to 6 should be understood to have specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc. as well as subranges within that range. Single numbers, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the width of the range.

describe

The present invention relates to inhibition of the alternative complement pathway (AP) using anti-properdin antibodies. In one embodiment, the invention relates to a method of treating an AP-mediated pathology or AP-mediated condition in an individual by contacting the individual with an anti-properdin antibody.

In one embodiment, the invention is a method of treating AP-mediated pathology in an individual comprising the step of administering to said individual an anti-properdin antibody, thereby inhibiting the production of the C3bBb protein complex. Examples of complement-mediated pathologies that may be treated using the methods of the invention include, but are not limited to, macular degeneration, ischemia-reperfusion injury, arthritis, rheumatoid arthritis, paroxysmal nocturnal hemoglobinuria (PNH) syndrome, Atypical hemolytic uremic syndrome (aHUS) syndrome, asthma, inflammation, glomerulonephritis, lupus, organ transplant sepsis, or a combination thereof.

The ability of the immune system to distinguish "self" from "non-self" antigens is central to the functioning of the immune system as a specific defense against invading microorganisms. "Non-self" antigens are those antigens on substances that enter or are presented in the body that are detectably different or heterogeneous from the animal's own components, while "self" antigens are those in a healthy animal that are not identical to its own components Those antigens were not detectably different or heterogeneous. In various embodiments of the methods described herein, the inhibited AP activation is activation triggered by at least one of: microbial antigens, non-biological foreign surfaces, altered autologous tissue, or combinations thereof. An example of a non-biological heterogeneous surface is a blood transfusion vessel such as those used in cardiopulmonary bypass surgery or kidney dialysis. Examples of altered native tissues include apoptotic, necrotic and ischemic stressed tissues and cells, or combinations thereof.

In some embodiments, an anti-properdin antibody of the invention selectively inhibits AP but does not inhibit the classical pathway (CP) or the lectin pathway (LP). Generally, CP is initiated by antigen-antibody complexes, LP is activated by the binding of lectins to sugar molecules on the surface of microorganisms, and AP is constitutively activated at low levels, but due to the lack of regulatory proteins, it can be activated in bacteria, viruses and parasitoids. Biological cells are rapidly amplified on the surface. Host cells are normally protected from AP complement activation by regulatory proteins. But in some cases, such as when regulatory proteins are deficient or lost, AP can also become uncontrollably activated on the host cell, leading to complement-mediated pathology. CP is composed of components C1, C2, C4 and joins AP at the C3 activation step. LP is composed of mannose-binding lectins (MBLs) and MBL-associated serine proteases (Masps) and shares components C4 and C2 with CP. AP consists of component C3 and several factors such as factor B, factor D, properdin and the fluid phase regulator factor H. Complement activation consists of three phases: (a) recognition; (b) enzyme activation and (c) membrane attack leading to cell death. The first phase of CP complement activation begins at C1. C1 is composed of three distinct proteins: the recognition subunit, C1q, and the serine protease subcomponents C1r and C1s - which are bound together in a calcium-dependent tetrameric complex, C1r2s2. An intact C1 complex is required for physiological activation of C1. Activation occurs when the intact C1 complex binds immunoglobulin complexed with antigen. This binding activates C1s, which then cleaves the C4 and C2 proteins, producing C4a and C4b, and C2a and C2b. The C4b and C2a fragments combine to form the C3 convertase, C4b2a, which in turn cleaves C3 to form C3a and C3b. Activation of LP is initiated by MBL binding certain sugars on the target surface, and this triggers the activation of Masps, which then cleave C4 and C2 in a manner similar to the C1s activity of CP, resulting in the production of the C3 convertase, C4b2a . Thus, CP and LP are activated by different mechanisms, but they share the same components C4 and C2, and both pathways lead to the production of the same C3 convertase, C4b2a. C4b2a-induced splitting of C3 into C3b and C3a is a central event in the complement pathway for two causes. It initiates the AP amplification loop because surface-deposited C3b is the main intermediate of AP. Both C3a and C3b are biologically important. C3a is pro-inflammatory and together with C5a is known as an anaphylatoxin. C3b and its further cleavage products also bind complement receptors presented on neutrophils, eosinophils, monocytes and macrophages, thereby promoting phagocytosis and clearance of C3b-opsonized particles. Finally, C3b can associate with C4b2a to form the C5 convertase of CP and LP, activating the terminal complement sequence, leading to the production of C5a - a potent pro-inflammatory mediator - and the cleavage of the membrane attack complex (MAC) - C5-C9 - the assembly.

Because CP and AP have critical roles in host defense, and many complement-dependent human pathologies are mediated by AP, it is desirable to selectively inhibit AP in the treatment of such human pathologies. Accordingly, in preferred embodiments of the methods described herein, the immunity provided by CP and LP is maintained while AP is selectively inhibited. Accordingly, in various embodiments, the anti-properdin antibodies used in the methods described herein do not inhibit CP and LP. In certain embodiments, the anti-properdin antibodies described herein are distinct from previously developed anti-properdin antibodies that inhibit AP and CP.

AP is thought to be constitutively active at low levels because C3 is naturally hydrolyzed to form C3(H2O). C3(H2O) behaves like C3b in that it can associate with fB, which makes fB susceptible to fD cleavage and activation. The resulting C3(H2O)Bb then cleaves C3 to generate C3b and C3a, thereby initiating the AP cascade via the AP-forming C3 convertase—C3bBb. An amplification loop is established as the initial C3 convertase produces increasing amounts of C3b. It should be noted that since CP and LP also generate C3b, which can bind factor B and engage AP, once these pathways are activated, the AP amplification loop also engages CP and LP. AP thus consists of two functional entities: an independent complement activation pathway independent of CP or LP and an amplification process that does participate and promotes the full manifestation of CP and LP. In one embodiment, the anti-properdin antibodies used in the methods described herein selectively inhibit the AP activation process and do not interfere with the AP amplification loop of CP and LP.

Properdin is structurally composed of an N-terminal domain and six thrombospondin type I repeat (TSR) domains. Under physiological conditions, it exists in plasma as cyclic polymers (dimers, trimers, tetramers), formed by head-to-tail association of monomers. Human properdin is encoded on the short arm of the X chromosome, and its deficiency, especially when combined with C2, MBL or IgG2 deficiency, constitutes a risk factor for fatal Neisserial infection.

It appears that the requirement for properdin in AP priming is variable and dependent on the nature of the activation surface. As a non-limiting example, properdin appears to be essential for LPS- and LOS-induced AP activation as well as for AP-mediated autologous tissue damage such as extravascular hemolysis of red blood cells in Crry-deficient mice (see U.S. Patent Application No. 2010/0263061). The invention described herein discloses that properdin is essential for human AP complement-mediated erythrocyte lysis in the context of fH and DAF dysfunction/blockade (Figure 3). The invention described herein also discloses that properdin is essential for autologous serum lysis of PNH erythrocytes (Figure 24). On the other hand, zymosan-induced AP activation is modestly impaired by properdin deficiency, and properdin does not appear to play an important role in CVF- and CP-triggered AP amplification (see US Patent Application No. 2010/0263061). AP activation on a given surface may represent a balance between properdin-dependent promotion of C3bBb stabilization and Factor H (fH)-dependent inhibition of C3 'spinning'. AP activators - for which properdin is not essential - may have limited interaction with fH as a result of lack of sufficient fH-dependent repression, and spontaneous C3 activation and amplification may occur as a default process without Auxiliary of properdin. Thus, in various embodiments of the invention described herein, inhibition of properdin function by the anti-properdin antibodies of the invention offers several advantages, including: it does not compromise the AP amplification loops of CP and LP, enabling These pathways are fully active for host defense; it does not completely eliminate AP complement activation because not all AP activators (i.e., pathogens) require properdin to trigger the pathway when combined with AP inhibitors such as anti-fB and anti-fD antibodies This reduces the extent of damage in the host defense when compared to other methods.

In one embodiment, the AP activity inhibited by the method of the invention is AP activation caused by at least one of the following: lipopolysaccharide (LPS), lipooligosaccharide (LOS), pathogen-associated molecular patterns (PAMPs) and risk - Associated Molecular Patterns (DAMPs). In another embodiment, the AP activity inhibited using the methods of the invention is the production of the C3bBb protein complex. In another embodiment, the AP activity inhibited by the methods of the invention is properdin dependent.

In some embodiments, the methods of the invention preserve the individual's ability to fight infection through CP and LP. In one embodiment, the invention is a method of inhibiting AP activation by bacterial lipooligosaccharides (LOS) in an individual comprising the steps of: administering to said individual an anti-properdin antibody; and thereby inhibiting bacterial LOS in the individual The resulting AP activation. In another embodiment, provided herein are methods of inhibiting AP activation by bacterial LPS. In certain embodiments, AP activation is caused by Salmonella typhi (S.typhosa) LPS, and the inhibitors used in the methods provided herein do not inhibit activation of APs by Salmonella minnesota (S.minnesota) LPS or E.coli LPS. induced AP activity. In various embodiments, the anti-properdin antibodies of the invention selectively inhibit AP, but do not inhibit CP-triggered complement activation, LP-triggered complement activation, zymosan-induced activation, or cobra venom factor- caused activation.

In one embodiment, provided herein is a method of inhibiting pathogen-associated molecular pattern-mediated AP activation in an individual comprising the step of: administering to said individual an anti-properdin antibody, thereby inhibiting PAMP-mediated activation in the individual AP is activated. Examples of PAMPs whose AP activation can be inhibited by the methods of the present invention include, but are not limited to, muramyl dipeptide (MDP), CpG motifs from bacterial DNA, double-stranded viral RNAs, peptidoglycan, lipophospho Muric acid, outer surface protein A from Borrelia burgdorferi, synthetic mycoplasma macrophage-activated lipoprotein-2, tripalmitoyl-cysteinyl-seryl-(lysyl)3- Lysine (P3CSK4), dipalmitoyl-CSK4 (P2-CSK4), monopalmitoyl-CSK4 (PCSK4), amphotericin B, triacylated or diacylated bacterial polypeptides, and combinations thereof.

In one embodiment, the invention is a method of inhibiting the initiation of AP activation in an individual comprising the step of: administering to said individual an anti-properdin antibody, thereby inhibiting the initiation of AP activation in the individual. In another embodiment, provided herein is a method of inhibiting amplification of AP activation in an individual comprising the step of administering to said individual an inhibitor of AP, thereby inhibiting amplification of AP activation in the individual. Examples of these embodiments are PNH patients suffering from AP complement-mediated hemolysis and individuals suffering from AP complement-mediated aHUS, asthma, ischemia/reperfusion injury, rheumatoid arthritis, and ANCA-mediated renal disease . In various embodiments of the invention, diseases and conditions that may be treated using the compositions and methods of the invention include, but are not limited to, AP complement-mediated hemolysis, AP complement-mediated aHUS, asthma, ischemia/ Reperfusion injury, rheumatoid arthritis and ANCA-mediated renal disease.

In various embodiments, provided herein are methods of identifying potential antibodies that have an inhibitory effect on AP comprising the steps of: a) administering an anti-properdin antibody to an individual; b) measuring the resulting phenotype in the individual; and c) incorporating The resulting individual phenotypes were compared to those of properdin ^-/- knockout animals (see US Patent Application No. 2010/0263061). In another embodiment, anti-properdin antibodies for use in the methods provided herein are identified by a method of selecting potential therapeutic compounds using properdin ^-/- knockout animals (see US Patent Application No. 2010/0263061) .

In various other embodiments, provided herein are methods of identifying potential anti-properdin antibodies that have an inhibitory effect on AP. One such method involves the steps of: a) coating the plate with lipopolysaccharide (LPS); b) washing the plate to remove unbound LPS; c) adding bovine serum albumin (BSA) in phosphate buffered saline (PBS) ; d) washing the plate to remove unbound BSA; e) adding a mixture of candidate anti-properdin antibody compounds mixed into human serum; f) washing the plate; g) adding anti-human C3 antibody; h) washing the plate to remove unbound BSA Bound antibody; i) addition of TMB substrate reagent; j) addition of sulfuric acid to stop reaction; k) measurement of optical density at 450 nm; l) comparison of optical density of plates containing candidate anti-properdin antibody compounds to positive controls and The optical density of the negative comparison control was compared; wherein the anti-properdin antibody was recognized when the optical density was reduced compared to the positive comparison control.

anti-properdin antibody

In some embodiments, the invention includes compositions comprising antibodies that specifically bind properdin. In one embodiment, the anti-properdin antibody is a polyclonal antibody. In another embodiment, the anti-properdin antibody is a monoclonal antibody. In some embodiments, the anti-properdin antibody is a chimeric antibody. In a further embodiment, the anti-properdin antibody is a humanized antibody. In a preferred embodiment, properdin is human properdin.

In one embodiment, the anti-properdin antibody comprises at least one CDRs selected from the group consisting of: VH-CDR1: SEQ ID NO:3; VH-CDR2: SEQ ID NO:4; VH-CDR3: SEQ ID NO:5; VL-CDR1: SEQ ID NO:8; VL-CDR2: SEQ ID NO:9; and VL-CDR3: SEQ ID NO:10. In another embodiment, the anti-properdin antibody comprises all of the following CDRs: VH-CDR1: SEQ ID NO:3; VH-CDR2: SEQ ID NO:4; VH-CDR3: SEQ ID NO:5; VL- CDR1: SEQ ID NO:8; VL-CDR2: SEQ ID NO:9; and VL-CDR3: SEQ ID NO:10.

In some embodiments, an anti-properdin antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:2. In other embodiments, the anti-properdin antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO:7. In another embodiment, the anti-properdin antibody is a monoclonal antibody designated mAbl9.1. The monoclonal anti-properdin antibody designated mAbl9.1 comprised a heavy chain - comprising the amino acid sequence of SEQ ID NO:2 - and a light chain - comprising the amino acid sequence of SEQ ID NO:7. In some embodiments, the monoclonal anti-properdin antibody designated mAbl9.1 is humanized.

In some embodiments, an anti-properdin antibody of the invention binds an epitope that includes at least one amino acid of TSR5 (SEQ ID NO:60). In some embodiments, an anti-properdin antibody of the invention specifically binds an epitope comprising at least one amino acid of the amino acid sequence RGRTCRGRKFDGHRCAGQQQDIRHCYSIQHCP (SEQ ID NO:52). In some embodiments, the anti-properdin antibody that specifically binds an epitope comprising at least one amino acid of SEQ ID NO:52 is the mAb designated mAbl9.1. In some embodiments, the anti-properdin antibody is an antibody that competes for binding with the antibody designated mAbl9.1. In various embodiments, the epitope to which an antibody of the invention binds is a linear epitope or a conformational epitope.

In one embodiment, the anti-properdin antibody comprises at least one CDRs selected from the group consisting of: VH-CDR1: SEQ ID NO: 13; VH-CDR2: SEQ ID NO: 14; VH-CDR3: SEQ ID NO: 15; VL-CDR1: SEQ ID NO:18; VL-CDR2: SEQ ID NO:19; and VL-CDR3: SEQ ID NO:20. In another embodiment, the anti-properdin antibody comprises all of the following CDRs: VH-CDR1: SEQ ID NO: 13; VH-CDR2: SEQ ID NO: 14; VH-CDR3: SEQ ID NO: 15; VL- CDR1: SEQ ID NO: 18; VL-CDR2: SEQ ID NO: 19; and VL-CDR3: SEQ ID NO: 20.

In some embodiments, an anti-properdin antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:12. In other embodiments, the anti-properdin antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO:17. In another embodiment, the anti-properdin antibody is a monoclonal antibody designated mAb25. The monoclonal anti-properdin antibody designated mAb25 comprised a heavy chain - comprising the amino acid sequence of SEQ ID NO: 12 - and a light chain - comprising the amino acid sequence of SEQ ID NO: 17. In some embodiments, the monoclonal anti-properdin antibody designated mAb25 is humanized.

In some embodiments, an anti-properdin antibody of the invention binds an epitope that includes at least one amino acid of TSR5 (SEQ ID NO:60) and/or TSR6 (SEQ ID NO:61). In some embodiments, an anti-properdin antibody of the invention specifically binds an epitope comprising at least one amino acid of the amino acid sequence LVVEEKRPCLHVPACKDPEEEEL (SEQ ID NO:53). In some embodiments, an anti-properdin antibody of the invention specifically binds an epitope comprising cysteine 62 (C62) present in SEQ ID NO:61. In some embodiments, an anti-properdin antibody of the invention specifically binds an epitope comprising cysteine 78 (C78) present in SEQ ID NO:53. In some embodiments, the anti-properdin antibody that specifically binds an epitope comprising the amino acids of SEQ ID NO:53 is a mAb designated mAb25. In some embodiments, the anti-properdin antibody is an antibody that competes for binding with the antibody designated mAb25. In various embodiments, the epitope to which an antibody of the invention binds is a linear epitope or a conformational epitope.

In one embodiment, the anti-properdin antibody comprises at least one CDRs selected from: VH-CDR1: SEQ ID NO:23; VH-CDR2: SEQ ID NO:24; VH-CDR3: SEQ ID NO:25; VL-CDR1: SEQ ID NO:28; VL-CDR2: SEQ ID NO:29; and VL-CDR3: SEQ ID NO:30. In another embodiment, the anti-properdin antibody comprises all of the following CDRs: VH-CDR1: SEQ ID NO:23; VH-CDR2: SEQ ID NO:24; VH-CDR3: SEQ ID NO:25; VL- CDR1: SEQ ID NO:28; VL-CDR2: SEQ ID NO:29; and VL-CDR3: SEQ ID NO:30.

In some embodiments, an anti-properdin antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:22. In other embodiments, the anti-properdin antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO:27. In another embodiment, the anti-properdin antibody is a monoclonal antibody designated mAb22.1. The monoclonal anti-properdin antibody designated mAb22.1 comprised a heavy chain - comprising the amino acid sequence of SEQ ID NO:22 - and a light chain - comprising the amino acid sequence of SEQ ID NO:27. In other embodiments, the anti-properdin antibody is an antibody that competes for binding with the antibody designated mAb22.1. In some embodiments, the monoclonal anti-properdin antibody designated mAb22.1 is humanized.

In one embodiment, the anti-properdin antibody comprises at least one CDRs selected from the group consisting of: VH-CDR1: SEQ ID NO:33; VH-CDR2: SEQ ID NO:34; VH-CDR3: SEQ ID NO:35; VL-CDR1: SEQ ID NO:38; VL-CDR2: SEQ ID NO:39; and VL-CDR3: SEQ ID NO:40. In another embodiment, the anti-properdin antibody comprises all of the following CDRs: VH-CDR1: SEQ ID NO:33; VH-CDR2: SEQ ID NO:34; VH-CDR3: SEQ ID NO:35; VL- CDR1: SEQ ID NO:38; VL-CDR2: SEQ ID NO:39; and VL-CDR3: SEQ ID NO:40.

In some embodiments, an anti-properdin antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:32. In other embodiments, the anti-properdin antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO:37. In another embodiment, the anti-properdin antibody is a monoclonal antibody referred to as mAb30. The monoclonal anti-properdin antibody designated mAb30 comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:32 and a light chain comprising the amino acid sequence of SEQ ID NO:37. In other embodiments, the anti-properdin antibody is an antibody that competes for binding with the antibody designated mAb30. In some embodiments, the monoclonal anti-properdin antibody designated mAb30 is humanized.

In other embodiments, the anti-properdin antibody comprises a humanized heavy chain comprising the amino acid sequence of SEQ ID NO:42. In some embodiments, an anti-properdin antibody comprises a humanized heavy chain comprising the amino acid sequence of SEQ ID NO:44. In still other embodiments, the anti-properdin antibody comprises a humanized light chain comprising the amino acid sequence of SEQ ID NO:47. In some embodiments, an anti-properdin antibody comprises a humanized antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO:42 and a light chain comprising the amino acid sequence of SEQ ID NO:47. In other embodiments, an anti-properdin antibody comprises a humanized antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO:44 and a light chain comprising the amino acid sequence of SEQ ID NO:47. In a further embodiment, the anti-properdin antibody is an antibody that competes for binding with the humanized antibodies described herein.

In some embodiments, an anti-properdin antibody comprises a humanized heavy chain comprising the amino acid sequence of SEQ ID NO:49. In other embodiments, the anti-properdin antibody comprises a humanized light chain comprising the amino acid sequence of SEQ ID NO:51. In some embodiments, an anti-properdin antibody comprises a humanized antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO:49 and a light chain comprising the amino acid sequence of SEQ ID NO:51. In a further embodiment, the anti-properdin antibody is an antibody that competes for binding with the humanized antibodies described herein.

In some embodiments, an anti-properdin antibody comprises a chimeric heavy chain comprising the amino acid sequence of SEQ ID NO: 2 and a human heavy chain constant region, such as, by way of non-limiting example, a human IgG4 constant region comprising SEQ ID NO: 2 NO: Amino acid sequence of 63. In other embodiments, the anti-properdin antibody comprises a chimeric light chain - comprising the amino acid sequence of SEQ ID NO: 7 and a human light chain constant region, such as, by way of non-limiting example, a human kappa light chain constant region - The amino acid sequence of SEQ ID NO:64 is included. In a certain embodiment, an anti-properdin antibody comprises a chimeric heavy chain - comprising the amino acid sequences of SEQ ID NO: 2 and SEQ ID NO: 63 - and a chimeric light chain - comprising SEQ ID NO: 7 and Amino acid sequence of SEQ ID NO:64.

In some embodiments, an anti-properdin antibody comprises a chimeric heavy chain comprising the amino acid sequence of SEQ ID NO: 12 and a human heavy chain constant region, such as, by way of non-limiting example, a human IgG4 constant region comprising SEQ ID NO: 12 NO: Amino acid sequence of 63. In other embodiments, the anti-properdin antibody comprises a chimeric light chain - comprising the amino acid sequence of SEQ ID NO: 17 and a human light chain constant region, such as, by way of non-limiting example, a human kappa light chain constant region - The amino acid sequence of SEQ ID NO:64 is included. In a certain embodiment, an anti-properdin antibody comprises a chimeric heavy chain - comprising the amino acid sequences of SEQ ID NO: 12 and SEQ ID NO: 63 - and a chimeric light chain - comprising SEQ ID NO: 17 and Amino acid sequence of SEQ ID NO:64.

In some embodiments, an anti-properdin antibody comprises a chimeric heavy chain comprising the amino acid sequence of SEQ ID NO: 22 and a human heavy chain constant region, such as, by way of non-limiting example, a human IgG4 constant region comprising SEQ ID NO: 22 NO: Amino acid sequence of 63. In other embodiments, the anti-properdin antibody comprises a chimeric light chain - comprising the amino acid sequence of SEQ ID NO: 27 and a human light chain constant region, such as, by way of non-limiting example, a human kappa light chain constant region - The amino acid sequence of SEQ ID NO:64 is included. In a certain embodiment, an anti-properdin antibody comprises a chimeric heavy chain - comprising the amino acid sequences of SEQ ID NO: 22 and SEQ ID NO: 63 - and a chimeric light chain - comprising SEQ ID NO: 27 and Amino acid sequence of SEQ ID NO:64.

In some embodiments, an anti-properdin antibody comprises a chimeric heavy chain comprising the amino acid sequence of SEQ ID NO: 32 and a human heavy chain constant region, such as, by way of non-limiting example, a human IgG4 constant region comprising SEQ ID NO: 32 NO: Amino acid sequence of 63. In other embodiments, the anti-properdin antibody comprises a chimeric light chain - comprising the amino acid sequence of SEQ ID NO: 37 and a human light chain constant region, such as, by way of non-limiting example, a human kappa light chain constant region - The amino acid sequence of SEQ ID NO:64 is included. In a certain embodiment, an anti-properdin antibody comprises a chimeric heavy chain - comprising the amino acid sequences of SEQ ID NO: 32 and SEQ ID NO: 63 - and a chimeric light chain - comprising SEQ ID NO: 37 and Amino acid sequence of SEQ ID NO:64.

screening test

The invention has application in a variety of screening assays, including determining whether candidate anti-properdin antibodies inhibit AP.

In some embodiments, the level of AP activity in the presence of a candidate anti-properdin antibody is compared to the AP activity detected in a positive comparison control. Positive comparison controls included AP activation in the absence of added test compound. In some embodiments, a candidate anti-properdin antibody is identified as an AP inhibitor when the AP activity in the presence of the candidate anti-properdin antibody is less than about 70% of the AP activity detected in the positive comparison control; this is consistent with Greater than about 30% inhibition of AP activity corresponds to the presence of the test compound. In other embodiments, a candidate anti-properdin antibody is identified as an AP inhibitor when the AP activity in the presence of the candidate anti-properdin antibody is less than about 80% of the AP activity detected in the positive comparison control; this is consistent with This corresponds to greater than about 20% inhibition of AP activity in the presence of the test compound. In still other embodiments, the candidate anti-properdin antibody is identified as an AP inhibitor when the AP activity in the presence of the candidate anti-properdin antibody is less than about 90% of the AP activity detected in the positive comparison control; this Corresponds to greater than about 10% inhibition of AP activity in the presence of the test compound. In some embodiments, the level of inhibition of AP by a candidate anti-properdin antibody is compared to the level of inhibition detected in a negative comparison control.

A variety of immunoassay formats, including competitive and noncompetitive immunoassay formats, antigen capture assays, double antibody sandwich assays, and triple antibody sandwich assays, are useful methods of the present invention (Self et al., 1996, Curr. Opin. Biotechnol. 7:60-65). The present invention should not be construed as limited to any one type of known or hitherto unknown assay, so long as the assay detects AP inhibition.

Hemolysis assays are included in the methods of the invention. In various embodiments, red blood cells (RBCs) are obtained from normal (healthy) individuals or from individuals showing signs or symptoms of a disease or disorder such as, for example, PNH. In various embodiments, RBCs are cultured in the presence of recombinant fH fragments - including complement control protein (CCP) repeats 19 and 20 of fH (fH19-20, 5-50 μM) - and anti-DAF neutralizing antibody (3-20 μg /ml) was lysed with 5% to 75% normal human serum (NHS). In some embodiments, RBCs from an individual exhibiting signs or symptoms of a disease or disorder, such as PNH, are lysed with acidified serum. In some embodiments, the hemolysis assay is performed in GVB++-Mg++-EGTA buffer to allow only AP complement activation, but the skilled artisan will understand that other suitable buffers can be used as long as the buffer only allows AP complement activation. In one embodiment, the extent of lysis is calculated by measuring the OD410 of the supernatant of the incubation mixture as a measure of the release of hemoglobin into solution. In various embodiments, at least one anti-properdin antibody is added at a concentration of about 1 μg/ml to about 50 μg/ml and pre-incubated with serum to measure the degree of inhibition of hemolysis of RBCs.

Enzyme-linked immunosorbent assays (ELISAs) are used in the methods of the invention. Enzymes such as, but not limited to, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase or urease can be linked to, for example, an anti-C3 antibody or a secondary antibody for use in the present invention Methods. The horseradish peroxidase detection system can, for example, be used with the chromogenic substrate tetramethylbenzidine (TMB), which in the presence of hydrogen peroxide produces a soluble product detectable at 450 nm. Other convenient enzyme-linked systems include, for example, the alkaline phosphatase detection system, which can be used with the chromogenic substrate p-nitrophenylphosphate to yield a soluble product that is readily detectable at 405 nm. Similarly, the β-galactosidase detection system can be used with the chromogenic substrate o-nitrophenyl-β-D-galactopyranoside (ONPG) to generate a soluble product. Alternatively, a urease detection system can be used with a substrate such as urea-bromocresol purple (Sigma Immunochemicals, St. Louis, MO). Useful enzyme-linked primary and secondary antibodies are available from any number of commercial sources.

Chemiluminescent detection was also used to detect AP inhibition. Chemiluminescent secondary antibodies are available from any number of commercial sources.

Fluorescence detection was also used to detect AP inhibition. Useful fluorescent dyes include, but are not limited to, DAPI, fluorescein, Hoechst 33258, R-phycocyanin, B-phycocyanin, R-phycoerythrin, rhodamine, Texas Red, and Lissamine-fluorescein - or rhodamine-labeled antibody.

Radioimmunoassays (RIAs) are also useful in the methods of the invention. Such assays are well known in the art and described, for example, by Brophy et al. (1990, Biochem. Biophys. Res. Comm. 167:898-903) and Guechot et al. (1996, Clin. Chem. 42:558-563 )describe. Radioimmunoassays, for example, are performed using iodine-125-labeled primary or secondary antibodies (Harlow et al., supra, 1999).

Analyze the signal from a detectable antibody, for example, using a spectrophotometer to detect color from a chromogenic substrate; using a radiation counter to detect radiation, such as a gamma counter for iodine-125; or using a fluorometer in the presence of a certain Fluorescence is detected in the absence of wavelengths of light. When using an enzyme-linked assay, quantitative analysis is performed using a spectrophotometer. It is understood that the assays of the present invention can be performed manually or, if desired, can be automated and signals from multiple samples can be detected simultaneously in a number of commercially available systems.

The methods of the invention also include the use of capillary electrophoresis-based immunoassays (CEIAs), which can be automated if desired. Immunoassays can also be used with laser-induced fluorescence, as described, for example, by Schmalzing et al. describe. Liposome immunoassays, such as flow injection liposome immunoassays and liposome immunosensors (Rongen et al., 1997, J. Immunol. Methods 204:105-133), can also be used according to the methods of the present invention. White

Quantitative Western blots can also be used to determine the level of AP inhibition in the methods of the invention. Western blots are quantified using well known methods such as scanning densitometry (Parra et al., 1998, J. Vasc. Surg. 28:669-675).

Application method

The methods of the invention comprise administering a therapeutically effective amount of at least one anti-properdin antibody to an individual determined to have an AP-mediated pathology. In preferred embodiments, the individual is a mammal with an AP system. In a more preferred embodiment, the individual is a human.

The methods of the invention may comprise administering at least one of any of the anti-properdin antibodies described herein, but the invention should in no way be construed as being limited to the anti-properdin antibodies described herein, but should be construed as Including any anti-properdin antibodies that reduce or reduce AP activation - known and unknown.

The methods of the invention comprise administering to a subject a therapeutically effective amount of at least one anti-properdin antibody, wherein compositions of the invention comprising anti-properdin antibodies or combinations thereof are used alone or in combination with other therapeutic agents. The invention may be used in combination with other treatment modalities, such as, for example, anti-inflammatory treatments and the like. Examples of anti-inflammatory treatments that may be used in combination with the methods of the invention include, for example, treatment with steroidal drugs, and treatment with non-steroidal drugs.

Pharmaceutical Compositions and Treatments

Administration of anti-properdin antibodies in the treatment methods of the invention can be accomplished in a number of different ways using methods known in the art. The therapeutic and prophylactic methods of the present invention thus include the use of pharmaceutical compositions, including anti-properdin antibodies. Pharmaceutical compositions useful in practicing the invention may be administered to deliver doses of between 1 ng/kg/day and 100 mg/kg/day. In one embodiment, the invention envisions administering a dose that results in an anti-properdin antibody concentration of the invention in the individual of between 1 μM and 10 μM. In another embodiment, the invention contemplates administering a dose that results in a concentration of an anti-properdin antibody of the invention in the individual's plasma of between 1 μM and 10 μM.

Typically, dosages that can be administered to animals, preferably humans, in the methods of the invention are 0.5 [mu]g to about 50 mg per kilogram of animal body weight. The precise dose administered will vary depending on any number of factors including, but not limited to, the species of animal and the type of disease state being treated, the age of the animal, and the route of administration. Preferably, the dosage of the compound will vary from about 1 [mu]g to about 10 mg per kilogram of animal body weight. More preferably, dosage will vary from about 3 [mu]g to about 1 mg per kilogram of animal body weight.

The compound may be administered to the animal frequently several times a day, or it may be administered less frequently, such as once a day, once a week, once every two weeks, once a month, or even less frequently, such as once every few months or even a year Once or less frequently. Dosage frequency will be apparent to the skilled artisan and depends on any number of factors such as, but not limited to, the type and severity of the disease being treated, the species and age of the animal, and the like. The formulations of the pharmaceutical compositions described herein can be prepared by any method known or later developed in the art of pharmacology. In general, such methods of preparation comprise the steps of bringing into association the active ingredient with the carrier or one or more other adjuvants, and then, if necessary or desired, shaping or packaging the product into desired single- or multi-dose units. .

Although the description of pharmaceutical compositions provided herein primarily refers to pharmaceutical compositions suitable for ethical administration to humans, the skilled artisan will appreciate that such compositions are generally suitable for administration to all species of animals. It is well understood that pharmaceutical compositions suitable for administration to humans are modified so that the compositions are suitable for administration to various animals, and that a skilled veterinary pharmacologist can design and conduct such a procedure using only ordinary experimentation, if any. grooming. Individuals contemplated for administration of the pharmaceutical compositions of the invention include, but are not limited to, humans and other primates, mammals - including commercially relevant mammals such as non-human primates, cattle, pigs, horses, sheep, cats and dogs.

Pharmaceutical compositions for use in the methods of the invention may be prepared, packaged, or sold in formulations suitable for the following routes of administration: ophthalmic, oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal , buccal route of administration. Other contemplated formulations include engineered nanoparticles, liposomal formulations, re-encapsulated erythrocytes containing active ingredients, and immune-based formulations.

The pharmaceutical compositions of the invention may be prepared, packaged or sold in large quantities, as a single unit dose, or as a plurality of single unit doses. A unit dose is an individual quantity of a pharmaceutical composition that includes a predetermined quantity of an active ingredient. The amount of active ingredient is usually equal to the dose of active ingredient to be administered to the individual, or a convenient fraction of such a dose such as, for example, one half or one third of such a dose.

The relative amounts of active ingredients, pharmaceutically acceptable carrier and any additional ingredients of the pharmaceutical compositions of this invention will vary depending on the characteristics, size and condition of the individual to be treated, and further depending on the route by which the composition will be administered. As an example, the composition may comprise between 0.1% and 100% (w/w) active ingredient.

In addition to the active ingredient, the pharmaceutical composition of the present invention may further include one or more additional pharmaceutically active agents.

Controlled or sustained release formulations of the pharmaceutical compositions of the invention can be prepared using conventional techniques.

Parenteral administration of a pharmaceutical composition includes any route of administration characterized by physical disruption of individual tissues and administration of the pharmaceutical composition through a breach in the tissue. Parenteral administration thus includes, but is not limited to, administration of the pharmaceutical composition by injection of the composition, administration of the composition through a surgical incision, administration of the composition by tissue penetration through non-surgical trauma, and the like. In particular, parenteral administration is contemplated including, but not limited to, intravenous, intraocular, intravitreal, subcutaneous, intraperitoneal, intramuscular, intrasternal injection, and intratumoral.

Formulations of pharmaceutical compositions suitable for parenteral administration include the active ingredient in combination with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or continuous administration. Injectable formulations can be prepared, packaged, or sold in unit dosage form, eg, in ampoules or in multi-dose containers with a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents. In one embodiment of the formulation for parenteral administration, the active ingredient is provided in dry (i.e. powder or granular) form for use with a suitable vehicle (e.g. sterile pyrogen-free water) prior to parenteral administration in a reconstituted composition. Do the refactoring.

Pharmaceutical compositions can be prepared, packaged or sold in the form of sterile injectable aqueous or oily suspensions or solutions. Such suspensions or solutions may be formulated according to known techniques, and may contain, besides the active ingredient, additional ingredients such as dispersing, wetting or suspending agents as herein described. Such sterile injectable preparations can be prepared using nontoxic parenterally acceptable diluents or solvents, such as, for example, water or 1,3-butanediol. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or diglycerides. Other useful parenterally administrable formulations include those comprising the active ingredient in microcrystalline form in liposomal formulations, or as a component of biodegradable polymer systems. Compositions for sustained release or implantation may include pharmaceutically acceptable polymeric or hydrophobic materials such as emulsions, ion exchange resins, sparingly soluble polymers or sparingly soluble salts.

The pharmaceutical compositions of the present invention may be prepared, packaged or sold in a formulation suitable for buccal and pulmonary administration. Such formulations may include dry particles comprising the active ingredient and having a diameter in the range of about 0.5 to about 7 nanometers, and preferably about 1 to about 6 nanometers. Such compositions are readily available using devices—including dry powder reservoirs to which the propellant stream can be directed to disperse the powder—or using self-propelling solvent/powder dispersion vessels such as containing dissolved or suspended low-boiling propellants in sealed containers. The means of ingredients are applied in dry powder form. Preferably, such a powder comprises particles wherein at least 98% by weight of the particles have a diameter greater than 0.5 nm and at least 95% by number have a diameter of less than 7 nm. More preferably, at least 95% by weight of the particles have a diameter greater than 1 nanometer and at least 90% by number of the particles have a diameter of less than 6 nanometers. Dry powder compositions preferably include a solid finely divided diluent such as sugar and are conveniently presented in unit dosage form.

Low boiling propellants generally include liquid propellants - having a boiling point below 65°F at atmospheric pressure. Typically, the propellant may constitute 50 to 99.9% (w/w) of the composition and the active ingredient may constitute 0.1 to 20% (w/w) of the composition. The propellant may further comprise additional ingredients such as liquid nonionic or solid anionic surfactants or solid diluents (preferably of the same order of particle size as the particles comprising the active ingredient).

Pharmaceutical compositions of the invention formulated for pulmonary delivery may also provide the active ingredient in the form of droplets of a solution or suspension. Such formulations may be prepared, packaged or sold as aqueous or dilute alcoholic solutions or suspensions, optionally sterile, including the active ingredient, and may be conveniently administered using any spraying or atomizing device. Such formulations may further comprise one or more additional ingredients including, but not limited to, flavoring agents such as sodium saccharin, volatile oils, buffers, surfactants, or preservatives such as methylparaben. The droplets provided by this route of administration preferably have an average diameter in the range of about 0.1 to about 200 nanometers.

The formulations described herein for pulmonary delivery are also useful for intranasal delivery of the pharmaceutical compositions of the invention.

Another formulation suitable for intranasal administration is a coarse powder comprising the active ingredient and having an average particle size of about 0.2 to 500 microns. Such formulations are administered in the manner employed by snuff, ie, rapid inhalation through the nasal passages from a powder container held close to the nostrils.

Formulations suitable for nasal administration may, for example, comprise as little as about 0.1% (w/w) and as much as 100% (w/w) active ingredient, and may further comprise one or more additional ingredients as described herein.

The pharmaceutical compositions of the present invention may be prepared, packaged or sold in formulations suitable for oral administration. Such formulations may, for example, be in the form of tablets or lozenges prepared by conventional methods, and may contain, for example, 0.1 to 20% (w/w) active ingredient, the balance comprising orally soluble or degradable and, optionally, one or more additional ingredients described herein. Alternatively, formulations suitable for oral administration may include powdered or nebulized solutions or suspensions containing the active ingredient. When dispersed, such powdered, aerosolized or aerosolized formulations preferably have an average particle or droplet size in the range of about 0.1 to about 200 nanometers, and may further comprise one or more of the herein described additional ingredients.

As used herein, "additional ingredients" include, but are not limited to, one or more of the following: excipients; surfactants; dispersants; inert diluents; granulating and disintegrating agents; binders; lubricants ; sweeteners; flavoring agents; coloring agents; preservatives; physiologically degradable compositions such as gelatin; aqueous carriers and solvents; oily carriers and solvents; suspending agents; dispersing or wetting agents; emulsifiers, emollients; buffers salts; thickeners; fillers; emulsifiers; antioxidants; antibiotics; antifungal agents; stabilizers; and pharmaceutically acceptable polymeric or hydrophobic materials. Other "additional ingredients" that may be included in the pharmaceutical compositions of the present invention are known in the art, for example as described in Remington's Pharmaceutical Sciences (1985, Genaro, ed., Mack Publishing Co., Easton, PA), which Incorporated herein by reference.

human properdin mouse

The invention also includes transgenic mice that express human properdin but not mouse properdin. In order to establish transgenic mice, the nucleic acid encoding human properdin protein can be incorporated into a recombinant expression vector in a form suitable for expression of human properdin protein in host cells. The term "in a form suitable for expression of the fusion protein in a host cell" is intended to refer to a recombinant expression vector that includes one or more regulatory sequences in a manner that allows transcription of the nucleic acid into mRNA and translation of the mRNA into the human properdin protein Operably linked to a nucleic acid encoding human properdin protein. The term "regulatory sequence" is art recognized and is intended to include promoters, enhancers and other expression control elements (eg, polyadenylation signals). Such regulatory sequences are known to those skilled in the art and described in 1990, Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. It will be appreciated that the design of the expression vector may depend on such factors as the choice of host cell to be transfected and/or the amount of human properdin protein to be expressed.

Transgenic mice can be produced, for example, by introducing nucleic acid encoding human properdin protein (usually linked to appropriate regulatory elements, such as constitutive or tissue-specific enhancers) into oocytes, for example, by microinjection, and allowed the oocytes to develop in female foster mice. Intronic sequences and polyadenylation signals may also be included in the transgene to increase the efficiency of transgene expression. Methods of producing transgenic animals, particularly animals such as mice, have become routine in the art and are described, for example, in U.S. Patent Nos. 4,736,866 and 4,870,009 and 1986, Hogan et al., A Laboratory Manual, Cold Spring Harbor, N.Y., Cold Spring Harbor Laboratory is described. Transgenic founder mice can be used to breed additional animals that carry the transgene. Transgenic mice carrying a transgene encoding a properdin protein of the invention can be further bred into other transgenic mice carrying other transgenes, or into other knockout mice, e.g., knockouts that do not express the mouse properdin gene Mice, such as those described in US Patent Application No. 2010/0263061. It should be understood that in addition to transgenic mice, the system described herein can be used to generate other human properdin expressing animals.

In one embodiment, the transgenic mice of the present invention express human properdin from the chicken β-actin promoter with a CVM-IE enhancer, but the skilled artisan will understand that the transgenic mice of the present invention include those from other promoters. The promoter and enhancer express human properdin. Examples of promoters useful in the present invention include, but are not limited to, DNA pol II promoter, PGK promoter, ubiquitin promoter, albumin promoter, globulin promoter, ovalbumin promoter, SV40 early promoter , Ruth sarcoma virus (RSV) promoter, reverse transcription LTR and lentiviral LTR. Promoter and enhancer expression systems useful in the present invention also include inducible and/or tissue-specific expression systems.

In some embodiments, the human properdin inserted into the mouse genome includes the nucleic acid sequence of SEQ ID NO:67 and the amino acid sequence of SEQ ID NO:54.

Reagent test kit

The invention also includes kits comprising an anti-properdin antibody of the invention, or a combination thereof, and instructional material which describes, for example, administering an anti-properdin antibody or combination thereof to an individual, as described elsewhere herein Therapeutic treatment or non-therapeutic use described. In one embodiment, the kit further comprises a (preferably sterile) suitable for dissolving or suspending a therapeutic composition, including, for example, an anti-properdin antibody of the invention or a combination thereof, prior to administering the antibody to the individual. pharmaceutically acceptable carrier. Optionally, the kit includes an applicator for administering the antibody.

Experimental Example

The invention will now be described with reference to the following examples. These examples are provided for the purpose of illustration only, and the invention should in no way be construed as being limited to these examples, but rather should be construed to encompass any and all modifications which become apparent as a result of the teaching provided herein.

Without further description, it is believed that one skilled in the art, using the preceding description and the following illustrative examples, can make and utilize the compounds of the invention and perform the claimed methods. The following working examples thus specify preferred embodiments of the invention and should not be construed as limiting the remainder of the disclosure in any way.

Example 1

Anti-human properdin monoclonal antibodies were generated using the hybridoma method first described by Kohler et al. (1975, Nature, 256:495), with some modifications. Properdin knockout mice (fP ^−/− ) (8 weeks old) were immunized intraperitoneally with 50 μg (in 100 μl PBS) of purified human properdin (CompTech Inc) emulsified in 100 μl Titermax adjuvant (from Sigma) . On days 14 and 21, mice were immunized again with 50 μg of purified human properdin emulsified in Titermax adjuvant. One week later, the serum anti-properdin titer of the mice was detected. Mice with antibody titers of 1:10,000 or higher were used for hybridoma fusion experiments. Two days before the fusion experiment, mice were injected again (ip) with 50 μg of purified human properdin (in 100 μl PBS). Mice were sacrificed by cervical dislocation and spleens were isolated for preparation of single cell suspensions by mechanical disruption. The spleen cell suspension was washed once with HYB-SFM (Invitrogen) + 10% FBS medium, and the cells were counted and mixed with X63-Ag8.653 myeloma cells (ATCC) at a ratio of 2:1. The cell mixture was washed again with HYB-SFM medium, and a cell pellet was prepared by centrifugation (1000 rpm x 5 min). The cell pellet was gently agitated and loosened, then the cells were brought to confluency by the slow addition of polyethylene glycol (PEG1500) ( ³ x 108 cells, 1.5 ml PEG). The cells were placed at 37° C. for 1 min, and then 20 ml of HYB-SFM medium was added to the cells within 3 min (1 ml for the first minute, 3 ml for the second minute, and 16 ml for the third minute). Centrifuge the mixture at 1000 rpm for 5 min, and plate the cells in 24-well plate HAT medium (10 ml HAT [Sigma H0262], 5 ml penicillin/streptomycin, 500 μl gentamicin and 10% FBS in 500 ml HYB-SFM medium) middle. After 2 weeks, supernatants from wells with visible clones were taken and screened by ELISA for reactivity with purified human properdin. Positive clones were picked and plated in a 96-well plate by limiting dilution, and single clones were obtained after the second round of screening by ELISA. Positive clones were expanded in HT-medium (10 ml HT, 5 ml penicillin/streptomycin 500 μl gentamicin and 10% FBS in 500 ml HYB-SFM medium). Hybridoma cells were transferred to serum-free medium (HYB-SFM) for 2-3 days prior to antibody collection. Cell cultures were harvested for mAb purification by protein G affinity chromatography.

For cloning of cDNAs of anti-properdin mAbs, total RNA was isolated from hybridoma cells by TRizol reagent (Sigma). First-strand cDNA was synthesized by reverse transcription using oligonucleotide (dT) primers. To amplify the heavy chain cDNA (for IgG1, IgG2a/b), the following primers were used in the PCR reaction: 5'-GAGGTGAAGCTGGTGGAG(T/A)C(T/A)GG-3' (SEQ ID NO:68) and 5'-GGGGCCAGTGGATAGAC-3' (SEQ ID NO: 69). To amplify the kappa light chain, the following primers were used: a mixture of 4 upstream primers: 5'-CCAGTTCCGAGCTCCAGATGACCCAGACTCCA-3' (SEQ ID NO: 70); 5'-CCAGTTCCGAGCTCGTGCTCACCCAGTCTCCA-3' (SEQ ID NO: 71); 5 '-CCAGTTCCGAGCTCCAGATGACCCAGTCTCCA-3' (SEQ ID NO:72); 5'-CCAGTTCCGAGCTCGTGATGACACAGTCTCCA-3' (SEQ ID NO:73); Downstream primer: 5'-GTTGGTGCAGCATCAGC-3' (SEQ ID NO:74). PCR amplicons were cloned into pCRTOPO TA2.1 vector (Invitrogen) and sequenced. To obtain the signal peptide (leader) sequence of mAbs, the 5'-RACE method was used using a kit from Invitrogen (GeneRacer). The complete variable region cDNA was amplified using specific primers determined from 5'-RACE and initial sequencing data.

Example 2

The dose-dependent inhibition of LPS-induced AP complement activation by mAb 19.1, 22.1, 25 and 30 was examined. All four mAb clones potently inhibited AP complement activation when added to 50% normal human serum (NHS) at a final concentration of 5 μg/ml (see Figure 2). ELISA plates (96-well, Nunc) were coated with 50 μl of LPS solution (40 μg/ml in phosphate buffered saline [PBS]) overnight at 4°C. The next day, the plate was washed 3 times with PBS containing 0.05% Tween-20 (PBS-T), and 50 μl of 50% normal human that had been incubated with 1-5 μg/ml anti-properdin mAb for 1 hour at 4 °C was added. Serum (NHS). NHS was diluted with GVB-EGTA-Mg++ (containing 10 mM GTA and 2.5 mM Mg++ final concentration). The plate was incubated at 37° C. for 1 hour, washed 3 times with PBS-T, then 50 μl of HRP-conjugated goat anti-human C3 antibody (1:4000, Cappel) was added and the plate was left at room temperature for 1 hour. Plates were washed 3 times with PBS-T and then developed with BD Pharmingen A+B reagents. The reaction was terminated after 45 min with 2N H2SO4. AP complement activation was detected by measuring the amount of C3 deposited on the plate (OD450). Samples spiked with EDTA (NHSEDTA) served as negative controls (EDTA blocks complement activation). Samples without mAb added (OAb) served as baseline AP complement activation.

Example 3

Experiments were performed showing that anti-human properdin mAbs inhibit human red blood cell (RBC) lysis caused by dysfunctional fH and DAF (see Figure 3). Normal human RBCs (5× ¹⁰ cells) were treated with 100 μl 50% NHS (diluted with GVB-EGTA-Mg++ containing 10 mM EGTA and 2.5 mM Mg++ final concentration) in the presence of 30 μM recombinant fH19-20 and 7.5 μg mouse anti-human DAF (from AbD Serotec) at 37C for 20 min (2006, Ferreira et al., J Immunol. 177:6308-6316). The lysis reaction was stopped by adding 200 μl of 20 mM EDTA in ice-cold PBS. The incubation mixture was centrifuged at 1500 g for 5 min, and the supernatant was collected for measurement of OD420nm. NHS was pre-incubated with 0 or 5 μg/ml anti-properdin antibody for 1 hour at 4C before adding RBCs. Samples without NHS or fH19-20, or with EDTA were used as negative lysis controls, and RBCs samples that were completely lysed with 100 μl distilled water were used as positive controls (100% lysis), against which the % lysis in other samples was performed standardization.

Example 4

Experiments evaluating antibody-sensitized sheep RBCs incubated with 50% normal human serum (NHS) in the absence or presence of 5 μg/ml anti-properdin mAbs (see Figure 4). Antibody-sensitized sheep RBCs (5×10 ⁶ cells, from CompTech Inc) were incubated with 100 μl of 50% NHS (diluted with GVB++ buffer) at 37° C. for 20 min. NHS was pre-incubated with 0 or 5 μg/ml anti-properdin antibody for 1 hour at 4C before adding sheep RBCs. The lysis reaction was stopped by adding 200 μl of 20 mM EDTA in ice-cold PBS. The incubation mixture was centrifuged at 1500 g for 5 min, the supernatant was collected and the OD420nm was measured. Samples without NHS or with EDTA were used as negative lysis controls, and samples of sheep RBCs completely lysed with 100 μl of distilled water were used as positive controls (100% lysis), against which the % lysis in the other samples was normalized.

Example 5

Generation of human properdin deletion mutants and verification of their expression in CHO cells by Western blot (see Figure 5). Human properdin (fP) consists of seven thrombospondin repeat (TSR) domains, which are numbered 0 to 6. Individual TSR domains 0 to 5 (see SEQ ID NOs: 55, 56, 57, 58, 59 and 60) are missing (1989, Hemsley et al., Nucleic Acid. 5 Res. 17:6545). To delete TSR6 (SEQ ID NO: 61 ) or TSR5-6, normal PCR methods were used, followed by cloning into expression vectors (using the pCAGGS vector). Deletion mutants were transfected into CHO cells in Optimem medium in 6-well plates using Lipofectamine reagent (Invitrogen). After 48 hours, the cells were treated with 50 mM Tris-HCL containing 150 mM NaCl, 10% glycerol, 1 mM EDTA and protease inhibitor cocktail (Roche) and 1% Triton (Triton) X-100, pH7.4 (250 μl per well ) cleavage. The lysate was centrifuged at 10,000 rpm for 10 min, and the protein concentration was determined by BCA protein assay. Approximately 100 μg of total protein from each sample was used for SDS-PAGE analysis.

Example 6

A sandwich ELISA assay of mAb 19.1 and 25 binding to a human properdin deletion mutant was performed for epitope mapping of mAb 19.1 and 25. (See Figure 6). ELISA plates were coated with 50 μl of the relevant mAb at 2 μg/ml overnight at 4°C. Plates were washed 3 times with PBS-T, then 25 μg (in 50 μl PBS containing 1% BSA) of CHO cell lysate protein was added to the wells and the plate was incubated for 1 hour at room temperature. Plates were washed 3 times with PBS-T, then captured proteins were detected by biotinylated goat anti-human properdin antibody and HRP-avidin system. A third mAb 29.3 binding to a different epitope of 19.1 and 25 was used as a control to verify mutein expression.

Example 7

Epitope mapping showed that the epitope of mAbl 9.1 mapped to the C-terminal half of TSR5, had the following amino acid sequence: RGRTCRGRKFDGHRCAGQQQDIRHCYSIQHCP (SEQ ID NO: 52) (see Figure 7). Three mutants of human properdin including TSR0-4+1/4TSR5, TSR0-4+1/2TSR5 or TSR0-4+3/4TSR5 were generated by conventional PCR. They were cloned into pCAGGS and expressed in CHO cells as described in Example 5. Protein expression was verified by Western analysis using goat anti-human properdin antibody. Blots were stripped and reprobed with a mouse anti-His-tag antibody (Qiagen) to confirm the presence of a C-terminal His-tag (no C-terminal proteolysis). As described in Example 6, a sandwich ELISA assay was performed to determine the reactivity with mAb 19.1. mAb29.3, which binds to a different epitope than 19.1, was used as a control to confirm mutein expression.

Example 8

Epitope mapping showed that the epitope of mAb25 mapped to the C-terminal quarter of TSR6 had the following amino acid sequence: LVVEEKRPCLHVPACKDPEEEEL (SEQ ID NO: 53) (see Figure 8). Three human properdin mutants including TSR0-5+1/4TSR6, TSR0-5+1/2TSR6 or TSR0-5+3/4TSR6 were generated by conventional PCR. They were cloned into pCAGGS and expressed in CHO cells as described in Example 5. Protein expression was verified by Western analysis using goat anti-human properdin antibody. The blot was stripped and reprobed with a mouse anti-His-tag antibody (Qiagen) to confirm the presence of the C-terminal His-tag (no C-terminal proteolysis). As described in Example 6, a sandwich ELISA assay was performed to determine reactivity with mAb25. mAb 19.1 binding to 25 different epitopes was used as a control to confirm mutein expression. Epitope mapping also showed that the epitope of mAb25 is dependent on two cysteine residues in TSR6 (SEQ ID NO: 61, shown in Figure 5B). These are cysteine 62 (C62) and cysteine 78 (C78) of TSR6. Single mutations at C62 or C78 to alanine (A) in full-length human properdin did not abolish mAb25 binding, but double mutations at C62A and C78A abolished mAb25 binding. As a positive control for mutein expression, mAb 19.1 showed reactivity to all samples. This result suggests that C78 within the last quarter of TSR6 (with the sequence specified by SEQ ID NO: 53), and C62 located outside of SEQ ID NO: 53 but within TSR6 (SEQ ID: 61) constitute the expression of mAb25. Two key residues of the position. Binding assays of mAbs 19.1 and 25 were performed on ELISA plates using homogenates of transfected CHO cells. HuP refers to full-length (intact) human fP transfected CHO cells as a positive control; Con refers to untransfected CHO cells as a negative control for binding. Other samples were CHO cells transfected with mutant human fP cDNA containing single or double C62A and C78A mutations.

Example 9

Experiments were performed to evaluate the expression of recombinant chimeric and humanized 19.1 mAbs in CHO cells (see Figure 17). The mAb19.1 variable region (SEQ ID NO: 1) was cloned into the pFUSE-CHIg-hG4 vector (from InvivoGen, containing the human IgG4 heavy chain constant region in which serine 229 was mutated to proline) using EcoRI/NheI sites. ) to construct chimeric 19.1 heavy chain cDNA. The chimeric 19.1 light chain cDNA was constructed by cloning the mAb19.1 variable region (SEQ ID NO:6) into the pFUSE2-CLIg-hk vector (from InvivoGen, containing the human kappa light chain constant region) using AgeI/BsiWI sites . Using EcoRI/NheI sites, by cloning the humanized heavy chain variable region of 19.1 (cDNAs encoding SEQ ID NO:42 and SEQ ID NO:44, synthesized by Genescript) into the pFUSE-CHIg-hG4 vector (from InvivoGen , containing the human IgG4 heavy chain constant region in which serine 229 was mutated to proline) to construct humanized 19.1 heavy chain cDNAs. Using the AgeI/BsiWI site, the pFUSE2-CLIg-hk vector (from InvivoGen, containing the human kappa light chain constant region) was cloned by cloning the 19.1 humanized light chain variable region (cDNA encoding SEQ ID NO: 47, synthesized by Genescript). ) to construct the humanized 19.1 light chain cDNA. Using Lipofectamine reagent, use 19.1 chimeric heavy chain and light chain or 19.1 humanized heavy chain and light chain (two humanized heavy chains paired with the same humanized light chain) to co- Transfect CHO cells. After transfection, CHO cells were selected with Geocine (1 mg/ml) and Blastcidine (10 μg/ml) for about 7 days. Drug-resistant cell colonies were picked, trypsinized and cultured in limiting dilution in 96-well plates in the presence of the same selection drug. After the cells became confluent in the 96-well plate, the medium was tested for reactivity with human properdin by ELISA, and positive clones were expanded. For antibody production, stably transfected CHO cell lines were grown in 150 cm flasks in DMEM:F12 medium with 10% FBS and after reaching confluency they were transferred to serum-free CD-CHO medium (Invitrogen). After 3 days, the medium was collected and the mAbs were purified by protein G chromatography. Aliquots of purified mAbs were analyzed by SDS-PAGE.

Example 10

Experiments were performed to measure the antigen binding affinities of mAb19.1, chimeric mAb19.1, humanized mAb19.1, mAb25, mAb22.1 and mAb30 (see Figures 18 and 19). The association and dissociation rate constants of anti-human properdin mAb binding to immobilized human properdin were measured using surface plasmon resonance analysis using a BIAcore2000 instrument (Biacore AB, Uppsala, Sweden). Biacore experiments were performed at 25°C. The carboxylated dextran matrix of the CM4 sensor chip was used to attach purified human properdin via amine coupling chemistry to achieve a surface density of 200RU. mAbs were diluted to 150, 75, 35.5, 17.75, 8.87 and OnM in HBSET (HEPES buffered saline with EDTA and Tween20) buffer and samples were injected onto the properdin surface at 30 μl/min (60 μl injection) for continuous 120s, allowing the dissociation of bound analyte to proceed for 900s. Data were analyzed by BIA Evaluation software 3.2 assuming a bivalent binding model. Regeneration of the surface was achieved by injecting 50 μl of 50 mM NaOH (50 μl/min).

Example 11

Experiments were performed to evaluate the relative activity of 19.1, chimeric 19.1 and two humanized 19.1 mAbs in blocking LPS-induced human AP complement activation (see Figure 20). ELISA plates (96 wells, Nunc) were coated with 50 μl of LPS solution (40 μg/ml in PBS, overnight at 4° C.). The next day, plates were washed 3 times with PBS containing 0.05% PBS-T and 50 μl of 50% NHS incubated with 0, 5, 10 or 20 μg/ml anti-properdin mAb for 1 hour at 4°C was added. NHS was diluted with GVB-EGTA-Mg++ (containing 10 mM EGTA and 2.5 mM Mg++, final concentration). The plate was placed at 37°C for 1 hour, washed 3 times with PBS-T, then 50 μl of HRP-conjugated goat anti-human C3 antibody (1:4000, Cappel) was added and the plate was left at room temperature for 1 hour. Plates were washed 3 times with PBS-T and then developed with BD Pharmingen A+B reagents. The reaction was stopped after 5 min with 2N H2SO4. AP complement activation was detected by measuring the amount of C3 deposited on the plate (OD450). Samples spiked with EDTA (NHSEDTA) served as negative controls (EDTA blocks complement activation). Samples without added mAb (NHS) served as baseline AP complement activation.

Example 12

Experiments were performed to evaluate the relative activity of 19.1, chimeric 19.1 and two humanized 19.1 mAbs in blocking human RBC lysis by human AP complement in the context of dysfunctional fH and DAF (see Figure 21). Normal human RBCs (5 × ¹⁰ cells) were treated with 100 μl 50% NHS (diluted with GVB-EGTA-Mg++ containing 10 mM EGTA and 2.5 mM Mg++ final concentration) in the presence of 30 μM recombinant fH19-20 and 7.5 μg mouse anti-human DAF ( Incubate at 37°C for 20 min in the case of ADB Serotec (2006, Ferreira et al., J Immunol. 177:6308-6316). NHS was pre-incubated with 1-15 μg/ml of various anti-properdin mAbs at 4°C for 1 hour before adding RBCs. The lysis reaction was stopped by adding 200 μl of 20 mM EDTA in ice-cold PBS. The incubation mixture was centrifuged at 1500 g for 5 min, the supernatant was collected and the OD420nm was measured. Samples without addition of NHS or fH19-20, or with addition of EDTA were used as negative lysis controls, and samples of RBCs completely lysed with 100 μl of distilled water were used as positive controls (100% lysis), against which the % lysis in other samples was normalized.

Example 13

Experiments were performed to evaluate the relative activities of 19.1, chimeric 19.1 and two humanized 19.1 mAbs in blocking LPS-induced AP complement activation in rhesus and cynomolgus monkeys (see Figures 22 and 23). ELISA plates (96 wells, Nunc) were coated with 50 μl of LPS solution (40 μg/ml in PBS, overnight at 4° C.). The next day, the plate was washed 3 times with PBS containing 0.05% PBS-T, and 50 μl of 50% 50% anti-properdin mAb that had been preincubated with 0, 10, 20, 30 or 40 μg/ml anti-properdin mAb at 4 °C for 1 hour was added. Normal rhesus serum (NRS) or normal cynomolgus serum (NCS). NRS or NCS was diluted with GVB-EGTA-Mg++ (containing 10 mM EGTA and 2.5 mM Mg++, final concentration). Incubate the plate at 37°C for 1 hour, wash 3 times with PBS-T, then add 50 μl of HRP-conjugated goat anti-human C3 antibody (1:4000, Cappel, cross-reactive with monkey C3), and leave the plate at room temperature 1 hour. Plates were washed 3 times with PBS-T and then developed with BD Pharmingen A+B reagents. The reaction was stopped after 5 min with 2NH2SO4. AP complement activation was detected by measuring the amount of C3 deposited on the plate (OD450). EDTA-added samples (NRSEDTA or NCSEDTA) served as negative controls (EDTA blocks complement activation). Samples without added mAb (NRS or NCS) served as baseline AP complement activation.

Example 14

Experiments were performed to evaluate the inhibition of acidified serum lysis of PNH erythrocytes (Ham's test) by mAb 19.1,25 and humanized 19.1-459 (see Figure 24). RBCs from patients with paroxysmal nocturnal hemoglobinuria (PNH) were subjected to Ham's acidified serum test in the presence or absence of mAbs. RBCs were incubated with autologous serum (83% final concentration) at 37°C for 2 hrs, and the lysis percentage was calculated by measuring the OD405 of the supernatant, normalized to the RBCs sample completely lysed in distilled water (Eh DDW). The incubation mixture consisted of: 240 μl serum, 25 μl 1/6N HCL (or 25 μl saline for negative control), 12.5 μl 50% (v/v) RBC suspension, 10 μl mAb in saline. A sample of RBCs incubated with non-acidified autologous serum (NHS) was used as a negative control (background lysis). In the absence of mAbs, about 50% of RBCs were lysed by acidified serum. The cleavage was completely inhibited by mAb 19.1 at concentrations of 8 μg/ml and above, humanized 19.1 mAb (#459) at concentrations of 20 μg/ml and mAb25 at concentrations of 8 μg/ml and above.

Example 15

Properdin humanized mice were generated as follows (see Figure 25). The human fP expression vector was constructed with the pACGGS plasmid, as shown in the schematic diagram in Figure 25A, using the chicken β-actin promoter with the CVM-IE enhancer and the rabbit β-globin polyA tail for expression in eukaryotic cells stable expression of cDNA. The human properdin cDNA sequence and its encoded protein sequence used to construct the expression vector are shown in SEQ ID NO:67 and SEQ ID NO:68. The plasmid was linearized by restriction enzyme digestion and microinjected into fertilized eggs of C57BL/6 mice to generate human fP transgenic founder mice. Screening by PCR (using primers 5'-ATCAGAGGCCTGTGACACC-3' (SEQ ID NO:65) and 5'-CTGCCCTTGTAGCTCCTCA-3' (SEQ ID NO:66) specific to human fP and genomic DNAs isolated from mouse tail), Positive founder mice (displaying a human fP cDNA fragment of about 800 bp) could be identified. Of the 40 mice analyzed, five (#15, 20, 24, 27 and 32) were positive (Figure 25B, red arrows). A sandwich ELISA assay was performed to detect human fP in transgene positive mice (Fig. 25C). Plates were coated with a non-blocking mAb against human fP (clone 8.1). After incubation with diluted mouse serum (10%), human fP was detected by using an HRP-conjugated goat anti-human fP antibody. Normal human serum (NHS) was used as a positive control. By this method, human fP should be detectable in the NHS and in the serum of transgene-positive mice (e.g. 15, 20, 24, 27, 32), but not in normal (i.e. non-transgenic, e.g. 29) mice or fP ^-/- - detected in mouse serum. Transgene-positive founder mice are then bred to WT mice to establish germline transmission. Selection from such mated F1 mice was accomplished by detection of the transgene by PCR of tail DNA, and detection of human properdin in serum by sandwich ELISA, as described above. Following confirmation of germline transmission, founder mice were bred to fP ^−/− mice to generate fP ^−/− − human fP transgenic+ mice. Restoration of AP complement activity in fP ^−/− −human fP transgenic+ mice was assessed by LPS-induced AP activation assay, as described in Example 2. In this assay, AP complement activity should be detected in serum of WT mice and in serum of fP ^−/− mice positive for the human fP transgene, but not in fP ^−/− mice. In this assay, serum from WT mice treated with EDTA will serve as a negative control for AP complement activation.

Example 16

Experiments were performed to examine the in vivo activity and kinetics of mAb25 in "properdin-humanized" mice (Figure 26). Properdin humanized mice (fP ^−/− -human fP transgene+) were injected with 0.5 mg (ip) mAb25. Blood samples (50-75 μl) and serum preparations were collected by retro-orbital bleeding before injection (Ohr) and then at various time points after injection. AP complement activation induced by LPS in serum samples was detected. For the assay, ELISA plates (96 wells, Nunc) were coated with 50 μl of LPS solution (40 μg/ml in PBS overnight at 4° C.). The next day, the plates were washed 3 times with PBS containing 0.05% Tween-20 (PBS-T), and 50 μl of serially diluted (starting at 1:10) mouse serum was added to each well. Mouse serum was diluted with GVB-EGTA-Mg++ (containing 10 mM EGTA and 2.5 mM Mg++ final concentration). The plate was incubated at 37° C. for 1 hour, washed 3 times with PBS-T, then 50 μl of HRP-conjugated rabbit anti-mouse C3 antibody (1:2000, Cappel) was added and the plate was left at room temperature for 1 hour. Plates were washed 3 times with PBS-T and then developed with BD Pharmingen A+B reagents. The reaction was stopped after 5 min with 2N H2SO4. AP complement activation was detected by measuring the amount of C3 deposited on the plate (OD450). In this illustrative example, AP complement activity was absent in fP ^−/− mouse sera or WT sera treated with EDTA. In contrast, AP complement activity was detected in WT serum and in fP humanized mouse serum at 0 hr (before mAb25 treatment). AP complement activity in humanized mice was still inhibited 8, 24 and 48 hr after mAb25 treatment, but became detectable at 72, 96 and 120 hr. These results show that at a dose of 0.5 mg/mouse, mAb25 can inhibit AP complement activity in vivo for at least 48 hr.

Example 17

Experiments were performed to evaluate the effect of anti-human properdin mAb 19.1 on extravascular hemolysis (EVH). In this EVH model, properdin humanized mice (n=4 per experimental group) were infused with red blood cells (RBC) from Crry/DAF/C3 triple knockout (TKO) mice. Recipient mice (properdin humanized mice) were treated with mAb19.1 (2 mg/mouse, i.p.) or control mouse IgG1 mAb (MOPC, purified from MoPC31C hybridoma, from ACTT) for 6 hr prior to RBC transfer . RBCs were obtained from donor TKO mice, washed in PBS and labeled with CFSE before injection (via tail vein) into recipient mice, according to a previously published procedure (Miwa et al., 2002, Blood99:3707-3716) . Each recipient mouse received RBCs equal to 100 μl of blood. At 5 minutes and 6, 24, 48, 72, 96, 120 hours after RBC infusion, recipient mice were bled and RBCs analyzed to determine the number of CFSE-labeled (ie, infused) RBCs remaining in circulation. The number of CFSE-labeled RBCs in each recipient was normalized (as %) to that detected at the 5 min time point. In control IgG (MOPC)-treated recipient mice, TKO RBCs were rapidly depleted by EVH, consistent with previous findings (Miwa et al., 2002, Blood 99:3707-3716). However, in recipient mice treated with anti-human properdin 19.1 mAb, EVH did not develop and the infused RBCs persisted, showing that anti-properdin mAb was effective in preventing EVH (Figure 27).

The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated by reference in their entirety.

While this invention has been disclosed with reference to specific embodiments, it is evident that other embodiments and modifications of this invention can be devised by those skilled in the art without departing from the true spirit and scope of this invention. It is intended that the appended claims be construed to cover all such embodiments and equivalents.

Claims (9)

1. A composition comprising an antibody that specifically binds to human properdin, wherein the antibody is at least one selected from the group consisting of: a. Antibody comprising: VH-CDR1: SEQ ID NO:3; VH-CDR2: SEQ ID NO:4; VH-CDR3: SEQ ID NO:5; VL-CDR1: SEQ ID NO:8; VL-CDR2: SEQ ID NO:9; and VL-CDR3: SEQ ID NO:10, b. an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO:2 and a light chain comprising the amino acid sequence of SEQ ID NO:7, c. an antibody that specifically binds to an epitope in the amino acid sequence of SEQ ID NO:52, d. an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 42 and a light chain comprising the amino acid sequence of SEQ ID NO: 47, e. an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO:44 and a light chain comprising the amino acid sequence of SEQ ID NO:47, and f. an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO:2 and SEQ ID NO:63 and a light chain comprising the amino acid sequence of SEQ ID NO:7 and SEQ ID NO:64 . 2. A composition comprising an antibody that binds to human properdin and competes with the binding of the antibody of claim 1 to human properdin. 3. Use of the antibody of any one of claims 1 and 2 for the manufacture of a medicament for inhibiting the alternative complement pathway (AP) in a subject with a complement alternative pathway (AP)-mediated pathology. 4. The application of claim 3, wherein the pathology is at least one selected from the group consisting of macular degeneration, ischemia-reperfusion injury, paroxysmal nocturnal hemoglobinuria (PNH) syndrome, atypical hemolytic uremia (aHUS) syndrome, asthma, organ transplant sepsis, inflammation and lupus. 5. The use of claim 4, wherein the inflammation is arthritis or glomerulonephritis. 6. The use of claim 5, wherein the arthritis is rheumatoid arthritis. 7. The use of claim 3, wherein the antibody selectively inhibits the alternative pathway (AP), but not the classical and lectin pathways. 8. The use of claim 3, wherein the antibody does not affect the alternative pathway (AP) amplification loop of the classical pathway and the lectin pathway. 9. The use of claim 3, wherein the administration of the antibody inhibits the production of C3bBb protein.