HK1196954A - Anti-properdin antibodies and uses thereof - Google Patents

Description

Anti-properdin antibodies and uses thereof

Background

The complement system provides the first 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 results in the assembly of the CP C3 convertase C4b2a, although direct cleavage of C3 by MBL-related serine proteases may also occur. AP is a self-amplifying loop driven by AP C3 convertase, C3 bBb. AP activation may occur secondary to CP or LP activation, or initiated independently. In the latter case, low-level spontaneous C3 'freewheeling (tick-over)' produces the original C3bBb, which rapidly propagates the AP in the absence of sufficient regulation. Thus, it is generally assumed that no or insufficient negatively regulated AP activation on non-self surfaces is considered a deletion (default) process, whereas autologous cells usually avoid this result with the help of various membrane-bound and fluid-phase complement inhibitory proteins. In certain instances, altered, damaged or stressed autologous cells and tissues may also activate AP and cause inflammatory damage.

Unlike the presence of numerous inhibitory proteins, the plasma protein properdin is the only known positive regulator of the complement activation cascade. Properdin is an approximately 53kDa plasma glycoprotein with estimated blood concentrations of 5-10. mu.g/ml. It exists mostly in head-to-tail conformation as a fixed ratio of dimers, trimers and tetramers. The current view held on properdin function is that it promotes AP activation by prolonging the half-life of the nascent C3bBb convertase. From this perspective, properdin plays a promoting 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, properdin cannot be considered an attractive anti-complement therapeutic target because it lacks specificity and is not essential for complement activation.

While all three complement activation pathways contribute to host resistance to microbial infection, recent studies have shown 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 primarily mediated by AP. Thus, there remains a need in the art for anticomplementary compositions and methods of treating inflammatory diseases in humans by selectively inhibiting AP while not affecting CP and LP to combat pathogens and protect the host from infection. The present invention fulfills this need.

SUMMARY

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

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, the antibodies of the invention are monoclonal antibodies. In some embodiments, the antibodies of the invention are humanized antibodies. In some embodiments, the antibodies of the invention are chimeric antibodies.

In one embodiment, the antibody of the invention comprises at least one CDR selected from: VH-CDR 1: 3, SEQ ID NO; VH-CDR 2:4, SEQ ID NO; VH-CDR 3: 5, SEQ ID NO; VL-CDR 1: 8 in SEQ ID NO; VL-CDR 2: 9, SEQ ID NO; and VL-CDR 3: 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, the antibody of the invention specifically binds to an epitope comprising at least one amino acid of SEQ ID NO 52.

In one embodiment, the antibody of the invention comprises at least one CDR selected from: VH-CDR 1: 13 in SEQ ID NO; VH-CDR 2: 14, SEQ ID NO; VH-CDR 3: 15, SEQ ID NO; VL-CDR 1: 18 in SEQ ID NO; VL-CDR 2: 19 in SEQ ID NO; and VL-CDR 3: 20 in SEQ ID NO. 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, the 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, the antibody of the invention specifically binds to an epitope comprising at least one amino acid of SEQ ID NO 53.

In one embodiment, the antibody of the invention comprises at least one CDR selected from: VH-CDR 1: 23, SEQ ID NO; VH-CDR 2: 24 is SEQ ID NO; VH-CDR 3: 25 in SEQ ID NO; VL-CDR 1: 28 in SEQ ID NO; VL-CDR 2: 29 in SEQ ID NO; and VL-CDR 3: 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, the 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, the antibody of the invention comprises at least one CDR selected from: VH-CDR 1: 33, SEQ ID NO; VH-CDR 2: 34 in SEQ ID NO; VH-CDR 3: 35 in SEQ ID NO; VL-CDR 1: 38, SEQ ID NO; VL-CDR 2: 39 in SEQ ID NO; and VL-CDR 3: 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, the 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, the 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, the antibody of the invention comprises a heavy chain comprising the amino acid sequences of SEQ ID NO 2 and SEQ ID NO 63. In one embodiment, the antibody of the invention comprises a light chain comprising the amino acid sequences 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 the amino acid sequences of SEQ ID NO 7 and SEQ ID NO 64.

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

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

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

In one embodiment, an antibody of the invention is an antibody that binds to properdin and competes with the binding of at least one anti-properdin antibody described herein. In another embodiment, the antibody of the invention is an antibody that binds to properdin and competes with the binding of an antibody called mab19.1 to properdin. In another embodiment, the antibody of the invention is an antibody that binds to properdin and competes with the binding of an antibody called mAb25 to properdin. In another embodiment, the antibody of the invention is an antibody that binds to properdin and competes with the binding of an antibody called mab22.1 to properdin. In another embodiment, the antibody of the invention is an antibody that binds to properdin and competes with the binding of an antibody called 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 the 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, atypical hemolytic uremic (aHUS) syndrome, asthma, organ transplant septicemia, inflammation, glomerulonephritis, lupus, and combinations thereof. In some embodiments, the anti-properdin antibody selectively inhibits the alternative pathway, but does not inhibit the classical pathway and the lectin pathway. In some embodiments, the anti-properdin antibody does not affect the AP amplification loop of the classical pathway and the lectin pathway. In some embodiments, the anti-properdin antibody inhibits production of C3bBb protein.

In one embodiment, the invention is a transgenic mouse that expresses human properdin (e.g., SEQ ID NO: 67; SEQ ID NO:54) but does not express murine properdin.

Brief Description of Drawings

The foregoing summary, as well as the following detailed description of preferred embodiments of the invention, will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there are shown in the drawings embodiments which are presently preferred. 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 drawings:

FIG. 1 is a schematic of the complement pathway. Complement can be activated by three pathways: the classical pathway, the lectin pathway and the alternative pathway. When C1q binds to an antigen-linked antibody, the classical pathway is activated, activating C1r and C1s, which 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. The C4 and C2 cleavage products form the classical and lectin pathway C3 convertase, C4bC2a, which cleaves C3 into C3b and C3 a. A second molecule of C3b may associate with C4bC2a, forming the C5 convertase, C4bC2aC3b, of the classical and lectin pathways. When C3 undergoes spontaneous hydrolysis and forms the initial AP C3 convertase, C3(H2O) Bb, the Alternative Pathway (AP) is activated, leading to additional C3 cleavage and eventual formation of the AP C3 convertase (C3bBb) and the AP C5 convertase (C3bBbC3B) in the presence of factors B and D. Properdin promotes AP activation by stabilizing AP convertases. All three pathways culminate in the formation of convertases, which in turn produce the major effectors of the complement system: anaphylatoxin (C4a/C3a/C5a), Membrane Attack Complex (MAC), and opsonin (e.g., C3 b). Anaphylatoxins are potent pro-inflammatory molecules derived from C4, C3, and C5 divisions. MACs are late assemblies of complement components C5b to C9 that can directly lyse the targeted surface. C3b elicits phagocytosis of opsonin-acting targets and also serves to amplify complement activation by AP.

Figure 2 depicts the results of experiments showing the dose-dependent inhibition of AP complement activation by LPS caused by mabs19.1, 22.1, 25 and 30. When added to 50% Normal Human Serum (NHS) at a final concentration of 5. mu.g/ml, all 4 mAb clones effectively inhibited AP complement activation. The EDTA-added sample (NHSEDTA) served as a negative control (EDTA blocked complement activation). Samples without mAb (0Ab) added served as baseline AP complement activation. The experiments were performed in GVB-EGTA-Mg + + buffer. ELISA plates were coated with LPS overnight. NHS was pre-incubated with mAb prior to plate addition. AP complement activation was detected by measuring the amount of C3 deposited on the plate (OD 450).

FIG. 3 depicts the results of experiments showing that anti-human properdin mAbs inhibit human Red Blood Cell (RBC) lysis caused by fH and DAF dysfunction. Human RBCs are not lysed in the absence of human serum (Eh only). They are also resistant to lysis by human serum (50%) in the absence of fH19-20, a recombinant fH fragment that prevents fH interaction with autologous cells (0fH 1920). However, when human RBCs were incubated with 50% human serum in the presence of 30 μ M fH19-20 and 7.5 μ g/ml function-blocking anti-aging accelerator factor (DAF, membrane complement regulator) mAb (fH1920+ anti cd55), about 70% of the RBCs were lysed. This lysis was completely inhibited by each of 4 properdin mAbs (19.1, 22.1, 25, 30) at 5. mu.g/ml. RBC samples treated with distilled water (Eh + DDW) caused complete lysis and served as positive controls. RBC samples in normal human serum treated with EDTA (nhsedta) served as a negative control (no lysis, as EDTA blocks complement activation). The lysis assay was performed in Mg + + -EGTA GVB + + buffer to allow only AP complement activation.

FIG. 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. mu.g/ml anti-properdin mAbs. RBCs samples without human serum (only ShEs) added showed no lysis; RBCs incubated with 50% NHS without addition of properdin mAb showed complete lysis (50% NHS); RBCs incubated with 50% NHS and 5 μ g/ml mAbs19.1, 22.1, 25 or 30 were also completely lysed, showing that mAbs have no inhibitory effect on classical pathway-mediated complement lysis of sensitized sheep RBCs. RBCs incubated with 50% NHS in the presence of EDTA (NHSEDTA) did not lyse, indicating that lysis is mediated by complement; sheep RBCs treated with distilled water (Es + DDW) served as 100% lysis controls.

FIG. 5, which includes FIGS. 5A-5C, depicts the results of epitope mapping against mAbs19.1 and 25 and the generation of human properdin (fP) deletion mutants. FIG. 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. FIG. 5b depicts the amino acid sequence of the 7 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:60), TSR6(SEQ ID NO: 61). Figure 5c depicts the results of epitope mapping of mab19.1 and 25 and the generation of human properdin (fP) deletion mutants. Human properdin (fP) consists of 7 thrombospondin repeat (TSR) domains numbered 0 to 6. A single TSR domain (and in some examples two TSR domains) has been deleted and the mutant protein 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 was proteolytically processed. The TSR5 deletion was similar in size to 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) markers.

Figure 6 depicts the results of epitope mapping against mab19.1 and 25 and ELISA assays of mab19.1 and 25 binding to human properdin deletion mutants. CHO cell lysates were coated onto ELISA plates and detected with mabs19.1 or 25. A third mab29.3, which binds to a different epitope of 19.1 and 25, was used as a control to confirm protein expression. The results show that both mabs19.1 and 25 reacted with the following deletion mutants: dTSR0, dTSR1, dTSR2, dTSR3 and dTSR 4. Thus, it can be concluded that the epitopes for mabs19.1 and 25 are not located in TSR 0-4. In addition, mab19.1 lost binding to dTSR5 and dTSR5+6, but retained binding to dTSR6, suggesting that its epitope is located in TSR 5. mAb25 lost binding to dTSR5, dTSR5+6 and dTSR6, suggesting that its epitope is located in TSR 5-6. However, because dTSR5 has undergone proteolytic degradation leading to possible TSR6 removal (fig. 5), the epitope for mAb25 is likely located in TSR 6. HuP refers to full-length human properdin transfection, which served 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 6 × His tag at the C-terminus.

Fig. 7 depicts the results of epitope mapping, showing that the epitope of mab19.1 is positioned 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 (fig. 6), further deletion mutants were generated: TSR0-4+1/4TSR5, TSR0-4+1/2TSR5, and TSR0-4+3/4TSR 5. Mutant, but not intact properdin proteins contain a 6 × His tag at their C-terminus. Western blot analysis with anti-human fP and anti-His tag antibodies showed that TSR0-4+3/4TSR5 was not fully expressed. The other two mutants, TSR0-4+1/4TSR5 and TSR0-4+1/2TSR5, were shown to be expressed, but none were recognized by mAb19.1, suggesting that they had lost the epitope for mAb 19.1. Thus, it can be concluded that the critical epitope residue for mAb19.1 is located within the C-terminal half of TSR5(SEQ ID NO: 52).

Figure 8, including figures 8A and 8B, depicts epitope mapping results showing the epitope of mAb 25. In fig. 8A, the data indicate an epitope located to the C-terminal quarter of TSR6, having the amino acid sequence: LVVEEKRPCLHVPACKDPEEEEL (SEQ ID NO: 53). Because TSR0-5 (i.e., dTSR6) lost binding to mAb25, it was concluded that TSR6 constitutes at least a partial epitope of mAb 25. Additional mutants that produced TSR6 were as follows: TSR0-5+1/4TSR6, TSR0-5+1/2TSR6, and TSR0-5+3/4TSR 6. Mutant, but not intact properdin proteins contain a 6 × His tag at their C-terminus. All three mutants were successfully expressed as shown by western blotting with anti-human fP and anti-His tag antibodies. ELISA binding experiments showed that all three mutants lost binding to mAb 25. As a positive control, all mutant proteins reacted with mab 19.1. This result suggests that the last quarter of TSR6 (having the sequence designated by SEQ ID NO:53) constitutes a critical part of the epitope of mAb 25. HuP refers to full-length (intact) human fP transfected CHO cells as a positive control; ConLysate refers to untransfected CHO cells, as a negative control for binding. In FIG. 8B, the data indicate that the epitope of mAb25 is dependent on two cysteine residues in TSR6(SEQ ID NO:61, as shown in FIG. 5B). These are cysteine 62(C62) and cysteine 78(C78) of TSR 6. Single mutations of alanine (a) at C62 or C78 in full-length human properdin did not abrogate mAb25 binding, but double mutations at C62A and C78A abrogated mAb25 binding. As a positive control for mutant protein expression, mab19.1 showed reactivity to all samples. This result suggests that C78 within the last quarter of TSR6 (having 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 two key residues of the mAb25 epitope. Binding assays of abs19.1 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 two C62A and C78A mutations.

FIG. 9 depicts the nucleotide and amino acid sequences of the variable region sequences of the heavy (SEQ ID NO: 1; SEQ ID NO:2) and light (SEQ ID NO: 6; SEQ ID NO:7) chains of mAb19.1, including the CDRs (VH-CDR 1: SEQ ID NO: 3; VH-CDR 2: SEQ ID NO: 4; VH-CDR 3: SEQ ID NO: 5; VL-CDR 1: SEQ ID NO: 8; VL-CDR 2: SEQ ID NO: 9; VL-CDR 3: SEQ ID NO: 10).

FIG. 10 depicts the nucleotide and amino acid sequences of the variable region sequences of the heavy (SEQ ID NO: 11; SEQ ID NO:12) and light chains (SEQ ID NO: 16; SEQ ID NO:17) of mAb25, including the CDRs (VH-CDR 1: SEQ ID NO: 13; VH-CDR 2: SEQ ID NO: 14; VH-CDR 3: SEQ ID NO: 15; VL-CDR 1: SEQ ID NO: 18; VL-CDR 2: SEQ ID NO: 19; VL-CDR 3: SEQ ID NO: 20).

FIG. 11 depicts the nucleotide and amino acid sequences of the variable region sequences of the heavy (SEQ ID NO: 21; SEQ ID NO:22) and light (SEQ ID NO: 26; SEQ ID NO:27) chains of mAb22.1, including the CDRs (VH-CDR 1: SEQ ID NO: 23; VH-CDR 2: SEQ ID NO: 24; VH-CDR 3: SEQ ID NO: 25; VL-CDR 1: SEQ ID NO: 28; VL-CDR 2: SEQ ID NO: 29; VL-CDR 3: SEQ ID NO: 30).

FIG. 12 depicts the nucleotide and amino acid sequences of the variable region sequences of the heavy (SEQ ID NO: 31; SEQ ID NO:32) and light (SEQ ID NO: 36; SEQ ID NO:37) chains of mAb30, including the CDRs (VH-CDR 1: SEQ ID NO: 33; VH-CDR 2: SEQ ID NO: 34; VH-CDR 3: SEQ ID NO: 35; VL-CDR 1: SEQ ID NO: 38; VL-CDR 2: SEQ ID NO: 39; VL-CDR 3: SEQ ID NO: 40).

FIG. 13 depicts the humanized amino acid sequence of the heavy chain variable region of mAb19.1 following 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 19.1VH-4-59-01(SEQ ID NO: 42); humanized 19.1VH-3-66-04(SEQ ID NO: 44)).

FIG. 14 depicts the humanized amino acid sequence of the light chain variable region of mAb19.1 (humanized 19.1VL-4-1-01(SEQ ID NO:47)) following CDR grafting using the human germline VL sequence (human VL4-1-01(SEQ ID NO: 45); human JK2(SEQ ID NO: 46)).

FIG. 15 depicts the humanized amino acid sequence of the heavy chain variable region of mAb25 (humanized 25-VH-1-69-06(SEQ ID NO:49)) following CDR grafting using the human germline VH sequence (human VH1-69-06(SEQ ID NO: 48)).

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

FIG. 17, comprising FIGS. 17A and 17B, depicts the results of an experiment evaluating recombinant chimeric and humanized 19.1 mAbs. FIG. 17A depicts the amino acid sequence of the human IgG4 heavy chain constant region in which serine 229 is 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 region + human constant region) and humanized (humanized mouse variable region + human constant region) anti-properdin antibodies. FIG. 17B depicts the results of an experiment evaluating the expression of recombinant chimeric and humanized 19.1 mAbs. SDS PAGE analysis of recombinant chimeric 19.1mAb and two humanized 19.1 mAbs. Construction of the chimeric 19.1 heavy chain was achieved by linking the VH domain of 19.1 to the human IgG4 heavy chain constant region. Construction of the chimeric 19.1 light chain was achieved by linking the VL region of 19.1 to the human kappa chain constant region. The humanized 19.1 heavy and light chains were constructed in the same manner, i.e., the humanized VH domain was linked to the human IgG4 heavy chain constant region and the humanized light chain was linked to the human kappa chain constant region. CHO cells were co-transfected with heavy and light chain cDNAs and stable lineages were established by the drug moiety. For both humanized mAbs, each of the two humanized heavy chains was paired with the same humanized light chain for transfection. The expressed mAbs were purified from CHO cell culture medium by protein G affinity column.

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

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

FIG. 20 depicts the results of experiments evaluating the relative activity of 19.1, chimeric 19.1 and humanized 19.1mAbs in blocking LPS-induced complement activation of human AP. ELISA plates were coated overnight with LPS, 50% Normal Human Serum (NHS) diluted in GVB-Mg + + -EGTA was added and incubated for 1hr at 37 ℃ before detecting C3 deposition using anti-C3 antibody. NHS without antibody was used as positive control (NHS) and EDTA was used as negative control (NHSEDTA). For the 19.1mAb, concentrations of 5. mu.g/ml and 10. mu.g/ml were sufficient to inhibit complement activation. For chimeric and two humanized 19.1mAbs, the concentration of 5. mu.g/ml was insufficient to inhibit complement activation. However, concentrations of 10. mu.g/ml and 20. mu.g/ml were effective in blocking AP complement activation.

FIG. 21 depicts the results of experiments evaluating the relative activity of 19.1, chimeric 19.1 and humanized 19.1mAbs in blocking human RBC lysis by human AP complement in cases of fH and DAF dysfunction. Human RBCs were incubated with 50% normal human serum in the presence of fH19-20 (30. mu.M) and anti-DAF antibody (7.5. mu.g/ml). Human serum was diluted in GVB-Mg + + -EGTA and incubated at 37 ℃ for 1 hr. Prior to addition to RBCs, human serum was preincubated with increasing concentrations of 19.1, chimeric 19.1 and humanized 19.1mAbs (1-15. mu.g/ml) for 1hr at 4 ℃. All 4 mAbs had dose-dependent inhibition of RBC lysis. However, the EC50 of the chimeric and humanized 19.1mAbs was higher than that of the 19.1 mAb. The results are consistent with the data shown in fig. 20.

FIG. 22 depicts the results of experiments evaluating the relative activity of 19.1, chimeric 19.1 and humanized 19.1mAbs in blocking LPS-induced complement activation of Rousian AP. ELISA plates were coated overnight with LPS, 50% normal cynomolgus monkey serum (NRS) diluted in GVB-Mg + + -EGTA was added and incubated at 37 ℃ for 1hr, followed by detection of C3 deposition using anti-human C3 antibody. NRS without antibody served as positive control (NRS) and EDTA-added NRS served as negative control (nrsetta). For 19.1 and chimeric 19.1mAbs, a concentration of 10-40. mu.g/ml was sufficient to inhibit cynomolgus complement activation. For both humanized 19.1mAbs, concentrations of 30 or 40 μ g/ml were effective in inhibiting complement activation. Concentrations of 10 or 20 μ g/ml also substantially inhibited macaque AP complement activation.

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

FIG. 24 depicts the results of experiments evaluating the inhibition of acidification of erythrocytes by mAb19.1,25 and humanised 19.1 (Ham 'stest, Hamm's test) in PNH patients. RBCs from Paroxysmal Nocturnal Hemoglobinuria (PNH) patients were subjected to hams' acidified serum trials in the presence or absence of mAbs. RBCs were incubated with autologous serum (final concentration 83%) at 37 ℃ for 2hr and percent lysis was calculated by measuring OD405 of the supernatant, normalized to a sample of RBCs completely lysed by distilled water (Eh DDW). The incubation mixture consisted of: mu.l serum, 25. mu.l 1/6N HCL (or 25. mu.l saline for negative control), 12.5. mu.l 50% (v/v) RBC suspension, 10. mu.l mAb in saline. RBCs samples incubated with non-acidified autologous serum (NHS) were used as negative controls (background lysis). In the absence of mAbs, approximately 50% of RBCs were lysed by acidification of the serum. This lysis was completely inhibited by mAb19.1 at concentrations of 8. mu.g/ml and greater, humanized 19.1mAb (#459) at a concentration of 20. mu.g/ml, and mAb25 at concentrations of 8. mu.g/ml and greater.

FIG. 25, comprising FIGS. 25A-25E, depicts the generation of properdin humanized mice. The human fP expression vector was constructed as schematically shown in fig. 25A, using the chicken β -actin promoter with CVM-IE enhancer and rabbit β -globin polya tail for stable expression of cDNA in eukaryotic cells. This plasmid was linearized and microinjected into fertilized eggs of C57BL/6 mice to generate human fP transgenic founder mice (foundermice). Positive founder mice (showing a human fP cDNA fragment of approximately 800 bp) were identified by PCR screening (using primers 5'-ATCAGAGGCCTGTGACACC-3' (SEQ ID NO:65) and 5'-CTG CCCTTGTAGCTCCTCA-3' (SEQ ID NO:66) specific for human fP). Of the 40 mice analyzed, five (#15, 20, 24, 27, and 32) were positive (fig. 25B, red arrows). ELISA assays were performed to detect human fP in transgenic positive mice (fig. 25C). Plates were coated with non-blocking mAb against human fP (clone 8.1). After incubation with diluted serum (10%), human fP was detected by ELISA using HRP-conjugated goat anti-human fP antibody. Normal Human Serum (NHS) was used as a positive control. It can be seen that human fP was detected in NHS and in the sera of 5 transgenic mice, but in normal (i.e. non-transgenic) mouse sera (NMS), transgene negative (#29) or in fP^-/-No detection was observed in mouse serum. Founder mouse #32 was 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 (fig. 25D). As shown, human fP was detected in two PCR-positive mice, but not in PCR-negative mice. Sera from NHS and the founder parent (#32) were used as positive controls. This result suggests that the transgene is stable and transmissible through the germline. Founder mouse #32 was then placed with fP^-/-Mice bred together to produce fP^-/-Human fP transgene + mouse. LPS-induced AP complement activation assay showed fP^-/--human fP transgene+ mice instead of fP^-/-Mice had serum AP complement activity indistinguishable from WT mice (FIG. 25E), suggesting that human fP expressed from the transgene could rescue fP^-/-Phenotype of mice. WT serum treated with EDTA was used as a negative control. This result confirmed the generation of properdin humanized mouse strains.

Figure 26 describes the examination of "properdin humanized" mice in vivo activity and kinetics of mAb25 experiment. Humanised mice (fP) were given properdin^-/-Human fP transgene +) with 0.5mg (i.p.) mAb 25. Serum samples were taken prior to injection (0hr) and then at various time points after injection and tested for LPS-induced AP complement activation. As shown, at fP^-/-There was no AP complement activity in mouse serum or in EDTA-treated WT serum. In contrast, AP complement activity was detected in WT serum and in fP humanized mouse serum at time 0hr (prior to mAb treatment). AP complement activity in humanized mice remained undetectable at 8, 24 and 48hr after mAb treatment, but was detectable at 72, 96 and 120 hr. These results suggest that mAb25 was able to inhibit AP complement activity in vivo for 48hr at a dose of 0.5 mg/mouse.

Figure 27 depicts the results of an experiment showing that anti-human properdin mab19.1 prevents extravascular hemolysis (EVH). In this EVH model, properdin humanized mice (n =4 per experimental group) were infused with Red Blood Cells (RBCs) from Crry/DAF/C3 Triple Knockout (TKO) mice. Prior to RBC transfer, recipient mice (properdin humanized mice) were treated with mab19.1(2 mg/mouse, i.p.) or control mouse IgG1mAb (MOPC, purified from MOPC31C hybridoma, from ACTT) for 6 hr. Prior to injection (via the tail vein) into recipient mice, RBCs were harvested from donor TKO mice, washed in PBS and labeled with CFSE according to the previously disclosed procedure (Miwa et al, 2002, Blood 99: 3707-. Each recipient mouse received RBCs in an amount equal to 100. mu.l of blood. At 5 minutes and 6, 24, 48, 72, 96, 120 hours after RBC infusion, recipient mice are bled and RBCs are analyzed to determine the number of CFSE-labeled (i.e., infused) RBCs remaining in circulation. The number of CFSE-labeled RBCs in each recipient was normalized (% to%) to the number of RBCs detected at the 5min 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-. However, in recipient mice treated with anti-human properdin 19.1mAb, no EVH appeared and infused RBCs remained, indicating that anti-properdin mAb was effective in preventing EVH.

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

Detailed Description

The present invention relates to inhibition of the alternative complement pathway (AP) using anti-properdin antibodies. In various embodiments, the present invention relates to compositions and methods for treating an AP-mediated pathology or AP-mediated condition in a subject by contacting the subject with an anti-properdin antibody. AP-mediated pathologies and conditions that may 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 (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 antibodies (ANCA) -mediated glomerulonephritis, lupus, and combinations thereof.

Definition of

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 to more than one (i.e., to 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 a reduction, inhibition, reduction, or blocking of 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 a 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 can be a 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 the complement system, including a human in need of treatment, or susceptible to a condition or sequela thereof. Individuals may include, for example, dogs, cats, pigs, cows, sheep, goats, horses, rats, monkeys, and mice, as well as humans.

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

"disease" is a state of health of an animal in which the animal is unable to maintain homeostasis, and in which the animal's health continues to deteriorate if the disease does not improve.

In contrast, a "disorder" in an animal is a health state in which the animal is able to maintain homeostasis, but in which the health state of the animal is less favorable than in the absence of the disorder. Left untreated, the condition does not necessarily cause a further reduction in the health status of the animal.

A disease or disorder is "alleviated" if the severity of the signs or symptoms of the disease or disorder, the frequency of such signs or symptoms experienced by the patient, or both, is 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 which the compound is administered.

As used herein, "instructional material" includes a publication, a record, a diagram, or any other medium of expression that can be used to show the effectiveness of the compounds, compositions, vectors, or delivery systems of the invention in a kit for effecting the alleviation of the various diseases or conditions described herein. Optionally or alternatively, the instructional material may describe one or more methods of alleviating a disease or disorder in a mammalian cell or tissue. The instructional material of the kit of the invention may, for example, be attached to or shipped with the container containing the identified compound, composition, vector or delivery system of the invention. Alternatively, the instructional material may be shipped separately from the container for the purpose of instructing the cooperative use of the material and the compound by the recipient.

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

As used herein, "treating a disease or disorder" means experiencing a reduction in the frequency and/or severity of signs and/or symptoms of a disease, disorder or pathology by a patient. Diseases, disorders, and pathologies 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 the 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. A sample that is naturally a liquid is referred to herein as a "body fluid". Biological samples can be obtained from a patient by a variety of techniques, including, for example, by scraping or swabbing an area or by using a needle to obtain bodily fluids. Methods of collecting a variety of body samples are well known in the art.

The term "antibody", as used herein, refers to an immunoglobulin molecule that specifically binds to a particular epitope of an antigen. The antibody may be an intact immunoglobulin derived from a natural source or derived from a recombinant source and may be an immunoreactive part of an intact immunoglobulin. The Antibodies of the invention may exist in a variety of forms including, for example, polyclonal Antibodies, monoclonal Antibodies, intracellular Antibodies ("intrabodies"), Fv, Fab ', F (ab)2 and F (ab') 2, as well as single chain Antibodies (scFv), heavy chain Antibodies such as camelid Antibodies (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, Science 426: 423).

The term "synthetic antibody," as used herein, means an antibody produced using recombinant DNA techniques, such as, for example, an antibody expressed by a bacteriophage, as described herein. The term should also be construed to refer to antibodies that have been produced by synthesis of a DNA molecule encoding the antibody and which DNA molecule expresses an antibody protein, or the amino acid sequence of a determined antibody, wherein the DNA or amino acid sequence has been obtained using 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 and subsequent isolation of serum, or by cloning and expression of nucleic acid sequences encoding such antibodies. 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 chain 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 an acceptor antibody.

"humanized antibody" refers to a type of engineered antibody having CDRs derived from a non-human donor immunoglobulin, the remainder of the molecule from immunoglobulin origin being derived from one (or more) human immunoglobulin(s). 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-. Suitable human acceptor antibodies may be antibodies selected from conventional databases, e.g., the KABAT database, the Los Alamos database, and the Swiss protein database, using nucleotide and amino acid sequence homology to the donor antibody. A human antibody characterized by homology (on an amino acid basis) to the framework regions of the donor antibody may be suitable to provide heavy chain constant regions and/or heavy chain variable framework regions for insertion of the donor CDRs. Suitable acceptor antibodies capable of contributing light chain constant or variable framework regions may be selected in a similar manner. It should be noted that the acceptor antibody heavy and light chains need not be derived from the same acceptor antibody. The prior art describes several methods for producing such humanized antibodies (see, e.g., EP-A-0239400 and EP-A-054951).

The term "donor antibody" refers to an antibody (monoclonal and/or recombinant) that donates the amino acid sequences of its variable regions, CDRs or other functional fragments or analogs thereof to a first immunoglobulin partner to provide altered immunoglobulin coding regions and resulting expressed altered antibodies-with the antigen-specific and neutralizing activity characteristics of the donor antibody.

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

"CDRs" are defined as the complementarity determining region amino acid sequences of an antibody that are hypervariable regions of immunoglobulin heavy and light chains. See, for example, 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 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, if appropriate). The structure and protein folding of an antibody may refer to other residues considered part of the antigen binding region and will be understood by the skilled person to be such. See, e.g., Chothia et al, (1989) formulations of immunogenic hypervariable regions, Nature342, p 877-883.

As used herein, "immunoassay" refers to any binding assay that utilizes an antibody 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 to an antigen of one species may also bind to an antigen from one or more species. However, such cross species reactivity does not itself change the classification of antibodies to be specific. In another example, an antibody that specifically binds to an antigen can also bind to different allelic forms of the antigen. However, such cross-reactivity does not itself change the classification of the antibody to be specific.

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

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

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 (e.g., amino acid residues in a protein export signal sequence).

As used herein, "complementary" with reference to 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 if the residue is thymine or uracil, an adenine residue of a first nucleic acid region can form a specific hydrogen bond ("base pairing") with a residue of a second nucleic acid region that is antiparallel to the first region. Similarly, it is known that a cytosine residue of a first nucleic acid strand is capable of forming base pairing with a residue of a second nucleic acid strand which 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 a different nucleic acid, then at least one nucleotide residue of the first region is capable of base pairing with a residue of the second region when the two regions are arranged in an antiparallel manner. 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 anti-parallel manner, at least about 50% and preferably at least about 75%, at least about 90% or at least about 95% of the nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion. More preferably, all nucleotide residues of the first part are capable of base pairing with nucleotide residues in the second part.

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

"encoding" refers to the inherent property of a particular 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 a defined sequence of amino acids and biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of the mRNA corresponding to the gene produces the protein in a cell or other biological system. The coding strand, whose nucleotide sequence is identical to the mRNA sequence and is usually provided in the sequence listing, and the non-coding strand, which serves as a transcription template for a gene or cDNA, can both be referred to as encoding a protein or other product of the gene or cDNA.

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

"isolated" refers to altered or removed from the natural state. For example, a nucleic acid or peptide that naturally occurs in a living animal in its normal environment is not "isolated," but the same nucleic acid or peptide that is partially or completely isolated from the coexisting materials of its natural environment is "isolated. An isolated nucleic acid or protein may exist in a substantially purified form, or may exist in a non-natural environment such as, for example, a host cell.

An "isolated nucleic acid" refers to a nucleic acid segment or fragment that has been separated from sequences that flank it in a naturally occurring state, i.e., a DNA fragment that has been removed from sequences that are normally adjacent to the fragment, i.e., sequences that are adjacent to the fragment in its naturally occurring genome. The term also applies to nucleic acids that have been substantially purified from other components that naturally accompany the nucleic acid, i.e., RNA or DNA or proteins, that naturally accompany it in a cell. The term thus includes, for example, recombinant DNA, which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genome of a prokaryote or eukaryote, or which exists as a separate molecule (i.e., as a cDNA or genome or a cDNA fragment produced by PCR or restriction enzyme digestion), independent of other sequences. It also includes recombinant DNA, which 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 strand of nucleotides. In addition, the nucleic acid is a nucleotide polymer. Thus, nucleic acids and polynucleotides as used herein are interchangeable. The person skilled in the art has the general knowledge that nucleic acids are polynucleotides, which can be hydrolyzed into monomeric "nucleotides". Monomeric nucleotides can be hydrolyzed to nucleosides. As used herein, polynucleotides include, but are not limited to, all nucleic acid sequences obtained by methods available in the art, including, without limitation, recombinant methods, i.e., cloning of nucleic acid sequences from recombinant libraries or cell genomes using common cloning techniques and PCR, and the like, and 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. The 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 the protein or peptide. Polypeptides include any peptide or protein, including 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, for example, peptides, oligopeptides and oligomers, and longer chains, which are commonly referred to in the art as proteins, of which there are many kinds. "polypeptide" includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, and the like. The polypeptide includes a natural peptide, a recombinant peptide, a synthetic peptide, or a combination thereof.

The term "progeny" as used herein refers to progeny (desintent or offset) and includes mammalian progeny, and also includes differentiated or undifferentiated progeny cells from the parent cell. In one usage, the term progeny (progeny) refers to a progeny cell (descementcell) that is genetically identical to the parent. In another application, the term progeny (progeny) refers to a progeny cell that is genetically and phenotypically identical to the parent. In another usage, the term progeny (progeny) refers to a progeny cell that has been 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 ligating DNA fragments of different origin.

The term "recombinant polypeptide" as used herein is defined as a polypeptide produced by using 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 sequence or peptide sequence that differs in sequence from a reference nucleic acid sequence or peptide sequence, respectively, but retains the necessary biological properties of the reference molecule. Changes in the sequence of a nucleic acid variant may not alter the amino acid sequence of a peptide encoded by the reference nucleic acid, or may result in amino acid substitutions, additions, deletions, fusions and truncations. Changes in the sequence of peptide variants are often limited or conserved, so that the sequences of the reference peptide and the variant are very similar overall and identical in many regions. The 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, the result being at least one offspring.

The term "natural reproduction" as used herein refers to the reproduction of species by sexual association.

The term "close relative reproductive animal" as used herein refers to an animal that has been cross bred with other similar animals of the same species to maintain and fix certain traits or prevent the introduction of other traits into the reproductive population.

The term "distant breeding animal" as used herein refers to an animal that breeds with any other animal of the same species without regard to maintaining certain characteristics.

The range is as follows: throughout this disclosure, various aspects of the invention may be presented in a range format. It is to 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 the possible sub-ranges as well as individual numerical values within that range. For example, a description of a range such as 1 to 6 should be understood to have specifically disclosed sub-ranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numbers within that range, e.g., 1,2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

Description of the invention

The present invention relates to the 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 an AP-mediated pathology in an individual comprising the step of administering to the individual an anti-properdin antibody, thereby inhibiting production of a C3bBb protein complex. Examples of complement-mediated pathologies that can be treated using the methods of the present 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), asthma, inflammation, glomerulonephritis, lupus, organ transplant sepsis, or combinations thereof.

The ability of the immune system to distinguish between "self" and "non-self" antigens is extremely important to the immune system as a specific defense against invading microorganisms. "non-self" antigens are those antigens that enter or are present on a substance in the body that is detectably different or heterogeneous to the animal's own components, whereas "self" antigens are those antigens that are not detectably different or heterogeneous to its own components in a healthy animal. In various embodiments of the methods described herein, the inhibited AP activation is an activation triggered by at least one of: a microbial antigen, a non-biological heterogeneous surface, an altered self-tissue, or a combination thereof. An example of a non-biological heterogeneous surface is a blood delivery vessel such as those used for cardiopulmonary bypass surgery or renal dialysis. Examples of altered self tissues include apoptotic, necrotic and ischemia stressed tissues and cells, or combinations thereof.

In some embodiments, the anti-properdin antibodies of the invention selectively inhibit AP, but do not inhibit the Classical Pathway (CP) or the Lectin Pathway (LP). In general, CP is initiated by antigen-antibody complexes, LP is activated by the binding of lectins to sugar molecules on the surface of microorganisms, while AP is constitutively activated at low levels, but can be rapidly amplified on the surface of bacterial, viral and parasitic cells due to the lack of regulatory proteins. The host cell is typically protected from AP complement activation by regulatory proteins. However, in some cases, such as when regulatory proteins are defective or lost, AP may also be uncontrollably activated on the host cell, leading to complement-mediated pathologies. CP consists of components C1, C2, C4 and associates with AP at the C3 activation step. LP consists of mannose-binding lectins (MBLs) and MBL-associated serine proteases (masss) and shares components C4 and C2 with CP. AP consists of component C3 and several factors such as factor B, factor D, properdin, and fluid phase regulator factor H. Complement activation consists of three phases: (a) identifying; (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 different proteins: the recognition subunit, C1q, and the serine protease subcomponents C1r and C1s, which are bound together in a calcium-dependent tetrameric complex, C1r2s 2. The intact C1 complex is essential for the physiological activation of C1. Activation occurs when the intact C1 complex binds to an immunoglobulin complexed with an antigen. This binding activates C1s, which then cleaves C4 and C2 proteins, producing C4a and C4b, and C2a and C2 b. The C4b and C2a fragments combine to form a C3 convertase, C4b2a, which in turn cleaves C3 to form C3a and C3 b. Activation of LP is initiated by MBL binding to certain sugars on the target surface, and this triggers activation of masss, which then cleaves C4 and C2 in a manner similar to the C1s activity of CP, resulting in the production of the C3 convertase, C4b2 a. 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, C4b2 a. The cleavage of C3 into C3b and C3a by C4b2a is a central event of the complement pathway for two reasons. It starts the AP amplification loop because the 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 called an anaphylatoxin. C3b and its further division products also bind complement receptors present on neutrophils, eosinophils, monocytes, and macrophages, thereby promoting phagocytosis and clearance of C3 b-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 assembly of the cleaved Membrane Attack Complex (MAC), C5-C9.

Because CP and AP have key 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 the CP and LP is maintained while selectively inhibiting AP. Thus, in various embodiments, an anti-properdin antibody for use in the methods described herein does not inhibit CP and LP. In certain embodiments, the anti-properdin antibodies described herein are different from previously developed anti-properdin antibodies that inhibit AP and CP.

Since C3 naturally hydrolyzes to form C3(H2O), AP is believed to be constitutively active at low levels. C3(H2O) behaves like C3b, and it can associate with fB, which makes fB susceptible to fD cleavage and activation. The resulting C3(H2O) Bb then cleaves C3, producing C3b and C3a, initiating the AP cascade via the AP-forming C3 convertase, C3 bBb. As the initial C3 convertase produced increasing amounts of C3b, an amplification loop was established. It should be noted that since CP and LP also produce C3B, where C3B may bind factor B and participate in AP, once these pathways are activated, the AP amplification loop also participates in CP and LP. Thus, an AP consists of two functional entities: independent complement activation pathways independent of CP or LP and amplification processes that do participate in and promote full expression of CP and LP. In one embodiment, the anti-properdin antibody used in the methods described herein selectively inhibits the AP activation process and does not interfere with the AP amplification loop of CP and LP.

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

It appears that the need for properdin in AP priming is variable and dependent on the nature of the activating surface. As non-limiting examples, properdin appears to be essential for LPS-and LOS-induced AP activation and for AP-mediated autologous tissue damage such as Crry-deficient extravascular haemolysis of mouse erythrocytes (see U.S. patent application No. 2010/0263061). The invention described herein discloses that properdin is essential for human AP complement-mediated red blood cell lysis in the context of fH and DAF dysfunction/blockade (fig. 3). The invention described herein also discloses that properdin is essential for autologous serolysis of PNH red blood cells (fig. 24). On the other hand, zymosan-induced AP activation is moderately impaired by properdin deficiency, and properdin does not appear to play a significant role in CVF-and CP-triggered AP amplification (see U.S. patent application No. 2010/0263061). AP activation on a given surface may represent a balance between properdin-dependent promotion of stabilization by C3bBb and factor h (fh) -dependent inhibition of C3 'freewheeling'. AP activators, to which properdin is not essential, may have limited interaction with fH, and as a result of the lack of sufficient fH-dependent inhibition, spontaneous C3 activation and amplification may occur as a deletion process without the aid of properdin. Thus, in various embodiments of the invention described herein, inhibition of properdin function by an anti-properdin antibody of the invention provides several advantages, including: it does not harm the AP amplification loops of CP and LP, making these pathways fully active against host defense; it does not completely eliminate AP complement activation because not all AP activators (i.e., pathogens) require properdin to trigger this pathway, which reduces the extent of damage in host defense when compared to other approaches in which AP inhibits such as anti-fB and anti-fD antibodies.

In one embodiment, the AP activity inhibited using the methods of the invention is AP activation by at least one selected from the group consisting of: lipopolysaccharide (LPS), Lipooligosaccharide (LOS), pathogen-associated molecular patterns (PAMPs), and hazard-associated molecular patterns (DAMPs). In another embodiment, the AP activity inhibited using the methods of the invention is production of a C3bBb protein complex. In another embodiment, the AP activity inhibited using the methods of the invention is properdin-dependent.

In some embodiments, the methods of the invention maintain the ability of an individual to combat infection by both CP and LP. In one embodiment, the invention is a method of inhibiting AP activation by bacterial Lipooligosaccharide (LOS) in an individual comprising the steps of: administering to the individual an anti-properdin antibody; and thereby inhibiting AP activation by bacterial LOS in the individual. In another embodiment, provided herein is a method of inhibiting AP activation by bacterial LPS. In certain embodiments, the AP activation is caused by salmonella typhi (s.typhosa) LPS, and the inhibitor for use in the methods provided herein does not inhibit AP activity caused by salmonella minnesota (s.minnesota) LPS or escherichia coli (e.coli) LPS. In various embodiments, an anti-properdin antibody of the invention selectively inhibits AP, but does not inhibit CP-triggered complement activation, LP-triggered complement activation, zymosan-induced activation, or cobra venom factor-induced activation.

In one embodiment, provided herein is a method of inhibiting pathogen-associated molecular pattern-mediated AP activation in an individual, comprising the steps of: administering an anti-properdin antibody to the individual, thereby inhibiting PAMP-mediated AP activation in the individual. Examples of PAMPs, the activation of which AP can be inhibited by the methods of the invention, include, but are not limited to, Muramyl Dipeptide (MDP), CpG motifs from bacterial DNA, double-stranded viral RNAs, peptidoglycans, lipoteichoic acid, exoprotein a from Borrelia burgdorferi (Borrelia burgdorferi), synthetic mycoplasma macrophage-activating lipoprotein-2, tripalmitoyl-cysteinyl-seryl- (lysyl) 3-lysine (P3CSK4), dipalmitoyl-CSK 4(P2-CSK4), monopalmitoyl-CSK 4(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 steps of: administering an anti-properdin antibody to the individual, thereby inhibiting the initiation of AP activation in the individual. In another embodiment, provided herein is a method of inhibiting AP activation amplification in an individual, comprising the steps of: administering to the individual an inhibitor of AP, thereby inhibiting amplification of AP activation in the individual. Examples of such 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 kidney disease. In various embodiments of the invention, diseases and disorders 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 having inhibitory effects on AP, comprising the steps of: a) administering to the individual an anti-properdin antibody; b) measuring the resulting phenotype of the individual; and c) combining the individual phenotype produced with properdin^-/-The phenotype of the knockout animals (see U.S. patent application No. 2010/0263061) was compared. In another embodiment, an anti-properdin antibody for use in a method provided herein utilizes properdin^-/-Knockout animals are identified by methods that select for potential therapeutic compounds (see U.S. 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 comprises the steps of: a) coating the plate with Lipopolysaccharide (LPS); b) washing the plate to remove unbound LPS; c) bovine Serum Albumin (BSA) in Phosphate Buffered Saline (PBS) was added; d) washing the plate to remove unbound BSA; e) adding a mixture of candidate anti-properdin antibody compounds mixed with human serum; f) washing the plate; g) adding anti-human C3 antibody; h) washing the plate to remove unbound antibody; i) adding a TMB substrate reagent; j) adding sulfuric acid to terminate the reaction; k) measuring the optical density at 450 nm; l) comparing the optical density of the plate containing the candidate anti-properdin antibody compound to the optical density of a positive comparison control and a negative comparison control; wherein the anti-properdin antibody is recognized when the optical density is reduced compared to a positive control.

Anti-properdin antibodies

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-CDR 1: 3, SEQ ID NO; VH-CDR 2:4, SEQ ID NO; VH-CDR 3: 5, SEQ ID NO; VL-CDR 1: 8 in SEQ ID NO; VL-CDR 2: 9, SEQ ID NO; and VL-CDR 3: SEQ ID NO 10. In another embodiment, the anti-properdin antibody comprises all of the following CDRs: VH-CDR 1: 3, SEQ ID NO; VH-CDR 2:4, SEQ ID NO; VH-CDR 3: 5, SEQ ID NO; VL-CDR 1: 8 in SEQ ID NO; VL-CDR 2: 9, SEQ ID NO; and VL-CDR 3: SEQ ID NO 10.

In some embodiments, the 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 mab 19.1. The monoclonal properdin antibody, designated mAb19.1, includes 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, a monoclonal anti-properdin antibody, designated mab19.1, is humanized.

In some embodiments, an anti-properdin antibody of the invention binds to an epitope comprising at least one amino acid of TSR5(SEQ ID NO: 60). In some embodiments, an anti-properdin antibody of the invention specifically binds to an epitope comprising at least one amino acid of amino acid sequence RGRTCRGRKFDGHRCAGQQQDIRHCYSIQHCP (SEQ ID NO: 52). In some embodiments, the anti-properdin antibody that specifically binds to an epitope comprising at least one amino acid of SEQ ID No. 52 is a mAb referred to as mAb 19.1. In some embodiments, the anti-properdin antibody is an antibody that competes for binding with an antibody called mab 19.1. In various embodiments, the epitope to which an antibody of the invention can bind 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-CDR 1: 13 in SEQ ID NO; VH-CDR 2: 14, SEQ ID NO; VH-CDR 3: 15, SEQ ID NO; VL-CDR 1: 18 in SEQ ID NO; VL-CDR 2: 19 in SEQ ID NO; and VL-CDR 3: 20 in SEQ ID NO. In another embodiment, the anti-properdin antibody comprises all of the following CDRs: VH-CDR 1: 13 in SEQ ID NO; VH-CDR 2: 14, SEQ ID NO; VH-CDR 3: 15, SEQ ID NO; VL-CDR 1: 18 in SEQ ID NO; VL-CDR 2: 19 in SEQ ID NO; and VL-CDR 3: 20 in SEQ ID NO.

In some embodiments, the 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 mAb 25. The monoclonal properdin antibody, designated mAb25, includes 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, a monoclonal anti-properdin antibody, designated mAb25, is humanized.

In some embodiments, an anti-properdin antibody of the invention binds to an epitope comprising 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 to an epitope comprising at least one amino acid of amino acid sequence LVVEEKRPCLHVPACKDPEEEEL (SEQ ID NO: 53). In some embodiments, the anti-properdin antibodies of the invention specifically bind to an epitope comprising cysteine 62(C62) present in SEQ ID NO: 61. In some embodiments, the anti-properdin antibodies of the invention specifically bind to an epitope comprising cysteine 78(C78) present in SEQ ID NO: 53. In some embodiments, the anti-properdin antibody that specifically binds to an epitope comprising amino acids of SEQ ID No. 53 is a mAb referred to as mAb 25. In some embodiments, the anti-properdin antibody is an antibody that competes for binding with an antibody designated mAb 25. In various embodiments, the epitope to which an antibody of the invention can bind 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-CDR 1: 23, SEQ ID NO; VH-CDR 2: 24 is SEQ ID NO; VH-CDR 3: 25 in SEQ ID NO; VL-CDR 1: 28 in SEQ ID NO; VL-CDR 2: 29 in SEQ ID NO; and VL-CDR 3: SEQ ID NO 30. In another embodiment, the anti-properdin antibody comprises all of the following CDRs: VH-CDR 1: 23, SEQ ID NO; VH-CDR 2: 24 is SEQ ID NO; VH-CDR 3: 25 in SEQ ID NO; VL-CDR 1: 28 in SEQ ID NO; VL-CDR 2: 29 in SEQ ID NO; and VL-CDR 3: SEQ ID NO 30.

In some embodiments, the 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 mab 22.1. The monoclonal properdin antibody, designated mAb22.1, includes 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 an antibody called mab 22.1. In some embodiments, a 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-CDR 1: 33, SEQ ID NO; VH-CDR 2: 34 in SEQ ID NO; VH-CDR 3: 35 in SEQ ID NO; VL-CDR 1: 38, SEQ ID NO; VL-CDR 2: 39 in SEQ ID NO; and VL-CDR 3: SEQ ID NO 40. In another embodiment, the anti-properdin antibody comprises all of the following CDRs: VH-CDR 1: 33, SEQ ID NO; VH-CDR 2: 34 in SEQ ID NO; VH-CDR 3: 35 in SEQ ID NO; VL-CDR 1: 38, SEQ ID NO; VL-CDR 2: 39 in SEQ ID NO; and VL-CDR 3: SEQ ID NO 40.

In some embodiments, the 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 designated mAb 30. The monoclonal properdin antibody, designated mAb30, includes 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 an antibody called mAb 30. In some embodiments, a 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, the 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, the 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, the 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 antibody described herein.

In some embodiments, the 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, the 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 antibody described herein.

In some embodiments, the 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 the amino acid sequence of SEQ ID NO. 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 comprising the amino acid sequence of SEQ ID NO. 64. In a certain embodiment, the 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 the amino acid sequences of SEQ ID NO. 7 and SEQ ID NO. 64.

In some embodiments, the 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 the amino acid sequence of SEQ ID NO. 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 comprising the amino acid sequence of SEQ ID NO 64. In a certain embodiment, the 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 the amino acid sequences of SEQ ID NO. 17 and SEQ ID NO. 64.

In some embodiments, the 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 the amino acid sequence of SEQ ID NO. 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 comprising the amino acid sequence of SEQ ID NO 64. In a certain embodiment, the 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 the amino acid sequences of SEQ ID NO:27 and SEQ ID NO: 64.

In some embodiments, the 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 the amino acid sequence of SEQ ID NO. 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 comprising the amino acid sequence of SEQ ID NO 64. In a certain embodiment, the 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 the amino acid sequences of SEQ ID NO:37 and SEQ ID NO: 64.

Screening assays

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

In some embodiments, the level of AP activity in the presence of the candidate anti-properdin antibody is compared to the AP activity detected in a positive comparison control. A positive comparison control included AP activation without added test compound. In some embodiments, a candidate anti-properdin antibody is identified as an AP inhibitor when AP activity detected in a positive comparison control has less than about 70% AP activity in the presence of the candidate anti-properdin antibody; this corresponds to greater than about 30% inhibition of AP activity in the presence of the test compound. In other embodiments, a candidate anti-properdin antibody is identified as an AP inhibitor when AP activity detected in a positive comparison control has less than about 80% AP activity in the presence of the candidate anti-properdin antibody; this corresponds to greater than about 20% inhibition of AP activity in the presence of the test compound. In still other embodiments, a candidate anti-properdin antibody is identified as an AP inhibitor when AP activity detected in a positive comparison control has less than about 90% AP activity in the presence of the candidate anti-properdin antibody; this corresponds to greater than about 10% inhibition of AP activity in the presence of the test compound. In some embodiments, the level of AP inhibition of the candidate anti-properdin antibody is compared to the level of inhibition detected in a negative comparison control.

A wide variety of immunoassay formats, including competitive and non-competitive immunoassay formats, antigen capture assays, double antibody sandwich assays, and triple antibody sandwich assays, are useful methods of the 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 heretofore unknown assay, so long as the assay is capable of detecting AP inhibition.

Hemolytic assays are included in the methods of the present invention. In various embodiments, Red Blood Cells (RBCs) are obtained from a normal (healthy) individual or from an individual exhibiting signs or symptoms of a disease or disorder such as, for example, PNH. In various embodiments, RBCs are lysed with 5% to 75% Normal Human Serum (NHS) in the presence of recombinant fH fragments, including Complement Control Protein (CCP) repeats of fH19 and 20(fH19-20, 5-50 μ M), and an anti-DAF neutralizing antibody (3-20 μ g/ml). 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 hemolytic assay is performed in GVB + + -Mg + + -EGTA buffer to allow only AP complement activation, but the skilled person will appreciate that other suitable buffers may be used as long as the buffer allows only 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 hemoglobin release into the solution. In various embodiments, at least one anti-properdin antibody is added at a concentration of about 1 μ g/ml to about 50 μ g/ml and pre-incubated with serum to measure the extent of hemolysis inhibition 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, beta-galactosidase, or urease can be linked to, for example, an anti-C3 antibody or a secondary antibody for use in the methods of the invention. The horseradish peroxidase detection system can, for example, be used with a chromogenic substrate, Tetramethylbenzidine (TMB), which produces a soluble product detectable at 450nm in the presence of hydrogen peroxide. Other convenient enzyme systems include, for example, the alkaline phosphatase detection system, which can be used with the chromogenic substrate p-nitrophenylphosphate, resulting in 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), resulting in a soluble product detectable at 410 nm. Alternatively, the 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 may be obtained from any number of commercial sources.

Chemiluminescence detection was also used to detect AP inhibition. Chemiluminescent secondary antibodies may be obtained from any number of commercial sources.

Fluorescence detection is also used to detect AP inhibition. Useful fluorescent dyes include, but are not limited to, DAPI, fluorescein, Hoechst33258, R-phycocyanin, B-phycocyanin, R-phycoerythrin, rhodamine, Texas Red, and Lissamine-fluorescein-or rhodamine-labeled antibodies.

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

Analyzing the signal emitted from the detectable antibody, e.g., detecting color from the chromogenic substrate using a spectrophotometer; detecting radiation using a radiation counter, such as a gamma counter for iodine-125 detection; or detecting fluorescence in the presence of light of a certain wavelength using a fluorometer. When the enzyme-linked assay is used, quantitative analysis is performed by using a spectrophotometer. It will be appreciated that the assays of the invention can be performed manually or, if desired, can be automated and that signals from multiple samples can be detected simultaneously in many commercially available systems.

The methods of the invention also include the use of capillary electrophoresis based immunoassays (CEIA), which can be automated if desired. Immunoassays can also be used with laser induced fluorescence, as described, for example, by Schmalzing et al (1997, electrophoresinis 18:2184-2193) and Bao (1997, J.Chromatogr.B.biomed.Sci.699: 463-480). Liposome immunoassays, such as flow injection liposome immunoassays and liposome immunosensors (Rongen et al, 1997, J.Immunol.Methods204:105-133), can also be used according to the methods of the invention. White colour (Bai)

Quantitative western blotting may also be used to determine the level of AP inhibition in the methods of the invention. The western blot is quantified using well known methods such as scanning densitometry (Parra et al, 1998, J.Vasc.Surg.28: 669-.

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 a preferred embodiment, the individual is a mammal having an AP system. In a more preferred embodiment, the individual is a human.

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

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

Pharmaceutical compositions and treatments

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

Generally, a dose of 0.5 μ g to about 50mg per kilogram of animal body weight can be administered to an animal, preferably a human, in the methods of the invention. 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 μ g to about 10mg per kilogram of animal body weight. More preferably, the dosage will vary from about 3 μ g to about 1mg 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 several months or even once a year or less frequently. The dosage frequency will be apparent to the skilled artisan and will depend on any number of factors, such as, but not limited to, the type and severity of the disease being treated, the type and age of the animal, and the like. The pharmaceutical composition formulations described herein may be prepared by any method known in the pharmacological arts or developed in the future. In general, such a preparation method comprises the following steps: the active ingredient is combined with a carrier or one or more other adjuvants and the product is then, if necessary or desired, shaped or packaged into the desired single or multiple dosage units.

Although the description of the pharmaceutical compositions provided herein primarily relates to pharmaceutical compositions suitable for ethical administration to humans, the skilled artisan will appreciate that such compositions are generally suitable for administration to all kinds of animals. It is well understood that pharmaceutical compositions suitable for administration to humans are modified to render the compositions suitable for administration to various animals, and that only ordinary experimentation, if any, may be utilized by the skilled veterinary pharmacologist to design and perform such modifications. Administration of the pharmaceutical compositions of the present invention contemplates individuals including, but not limited to, humans and other primates, mammals, including commercially relevant mammals such as non-human primates, cows, pigs, horses, sheep, cats, and dogs.

The pharmaceutical compositions used in the methods of the present invention may be prepared, packaged or sold in formulations suitable for the following routes of administration: ocular, oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal, buccal routes of administration. Other contemplated formulations include engineered nanoparticles, liposomal formulations, re-encapsulated red blood cells containing active ingredients, and immune-based formulations.

The pharmaceutical compositions of the present invention may be prepared, packaged or sold in bulk, as a single unit dose, or as multiple single unit doses. A unit dose is an individual amount of a pharmaceutical composition comprising a predetermined amount of an active ingredient. The amount of active ingredient is generally equal to the dose of active ingredient to be administered to an 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 the active ingredients, pharmaceutically acceptable carriers and any additional ingredients of the pharmaceutical compositions of the invention will vary according to the characteristics, size and condition of the individual being treated, and further according to the route by which the composition is to 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 comprise 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 the tissue of the individual and administration of the pharmaceutical composition through a breach in the tissue. Parenteral administration thus includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, administration of the composition through a surgical incision, administration of the composition through tissue penetration of a non-surgical wound, and the like. In particular, parenteral administration is contemplated to include, but is not limited to, intravenous, intraocular, intravitreal, subcutaneous, intraperitoneal, intramuscular, intrasternal injection, and intratumoral.

Pharmaceutical composition formulations 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 preparations may be prepared, packaged or sold in unit dose form, e.g. in ampoules or in multi-dose containers containing 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 include one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in dry (i.e., powder or granular) form for reconstitution with a suitable carrier (e.g., sterile, pyrogen-free water) prior to parenteral administration of the reconstituted composition.

The pharmaceutical compositions may be prepared, packaged or sold in the form of sterile injectable aqueous or oily suspensions or solutions. The suspension or solution may be formulated according to known techniques and may include, in addition to the active ingredient, additional ingredients such as dispersing, wetting or suspending agents as described herein. Such sterile injectable formulations may be prepared using non-toxic 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 non-volatile oils such as synthetic mono-or diglycerides. Other useful parenterally administrable formulations include those which comprise the active ingredient in microcrystalline form in a liposomal formulation, or as a component of a biodegradable polymer system. 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 pulmonary administration via the oral cavity. Such formulations may include dry particles that include an active ingredient and which have a diameter in the range of about 0.5 to about 7 nanometers and preferably about 1 to about 6 nanometers. Such compositions are conveniently administered in dry powder form using a device comprising a dry powder reservoir to which a propellant stream can be directed to disperse the powder, or using a self-propelled solvent/powder dispersion container such as a device containing the active ingredient dissolved or suspended in a low boiling propellant in a sealed container. Preferably, such a powder comprises particles, wherein at least 98% by weight of the particles have a diameter of more than 0.5 nm and at least 95% by number of the particles 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 less than 6 nanometers. The dry powder composition preferably includes a solid finely divided diluent such as sugar and is conveniently provided in unit dosage form.

Low boiling propellants typically include liquid propellants, having a boiling point below 65 ° F at atmospheric pressure. Typically, the propellant may constitute from 50 to 99.9% (w/w) of the composition and the active ingredient may constitute from 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 having the same particle size as the particles comprising the active ingredient).

The pharmaceutical compositions of the present 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 diluted alcoholic solutions or suspensions, optionally sterile, including the active ingredient, and may be conveniently applied using any spraying or atomising device. Such formulations may further include one or more additional ingredients including, but not limited to, flavoring agents such as sodium saccharin, volatile oils, buffering agents, 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.

Formulations described herein for pulmonary delivery are also useful for intranasal delivery of the pharmaceutical compositions of the present 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 form of a powder inhaler (snuff), i.e. a rapid inhalation through the nasal passage from a powder container held close to the nostril.

Formulations suitable for nasal administration may, for example, comprise as little as about 0.1% (w/w) and as much as 100% (w/w) of the active ingredient, and may further comprise one or more of the additional ingredients 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 using conventional methods and may have, for example, 0.1 to 20% (w/w) active ingredient(s), the balance comprising an orally-dissolvable or degradable composition and, optionally, one or more additional ingredients described herein. Alternatively, formulations suitable for oral administration may comprise powders or atomized solutions or suspensions containing the active ingredient. When dispersed, such powdered, atomized, or aerosolized formulations preferably have an average particle or droplet size in the range of from about 0.1 to about 200 nanometers, and may further include one or more additional ingredients described herein.

As used herein, "additional ingredients" include, but are not limited to, one or more of the following: an excipient; a surfactant; a dispersant; an inert diluent; granulating and disintegrating agents; a binder; a lubricant; a sweetener; a flavoring agent; a colorant; a preservative; physiologically degradable compositions such as gelatin; an aqueous carrier and a solvent; an oily vehicle and a solvent; a suspending agent; dispersing or wetting agents; emulsifiers, demulcents; a buffering agent; salt; a thickener; a filler; an emulsifier; an antioxidant; (ii) an antibiotic; an antifungal agent; a stabilizer; and a pharmaceutically acceptable polymeric or hydrophobic material. Other "additional ingredients" that may be included in the Pharmaceutical compositions of the present invention are known in the art and are described, for example, in Remington's Pharmaceutical Sciences (1985, Genaro, ed., mack publishing co., Easton, PA), which is incorporated herein by reference.

Human properdin mouse

The invention also includes transgenic mice that express human properdin but not mouse properdin. To create transgenic mice, nucleic acids encoding human properdin protein may be incorporated into recombinant expression vectors 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 comprising one or more regulatory sequences operably linked to a nucleic acid encoding human properdin protein in a manner that allows transcription of the nucleic acid into mRNA and translation of the mRNA into human properdin protein. The term "regulatory sequence" is art-recognized and is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are known to those skilled in the art and are described in 1990, Goeddel, Gene Expression Technology, Methods in Enzymology185, Academic Press, San Diego, Calif. It will be appreciated that the design of the expression vector may depend on factors such as the choice of host cell to be transfected and/or the amount of human properdin protein to be expressed.

Transgenic mice can be generated, for example, by introducing a nucleic acid encoding a human properdin protein (usually linked to appropriate regulatory elements, such as constitutive or tissue-specific enhancers) into an oocyte, for example, by microinjection, and allowing the oocyte to develop in female breeding mice. Intron sequences and polyadenylation signals may also be included in the transgene to increase the efficiency of transgene expression. Methods for generating transgenic animals, particularly animals such as mice, have become routine in the art and are described, for example, in U.S. Pat. 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. Transgenic founder mice can be used to breed additional animals carrying the transgene. Transgenic mice carrying a transgene encoding a properdin protein of the invention may be further bred into other transgenic mice carrying other transgenes, or into other knockout mice, e.g., knockout mice that do not express the murine properdin gene, such as those described in U.S. patent application No. 2010/0263061. It will be appreciated that the system described herein can be used to produce other human properdin expressing animals in addition to transgenic mice.

In one embodiment, the transgenic mice of the invention express human properdin from a chicken β -actin promoter with a CVM-IE enhancer, but the skilled artisan will appreciate that transgenic mice of the invention include expression of human properdin from other promoters and enhancers. Examples of promoters useful in the present invention include, but are not limited to, the DNA pol II promoter, the PGK promoter, the ubiquitin promoter, the albumin promoter, the globin promoter, the ovalbumin promoter, the SV40 early promoter, the Rous Sarcoma Virus (RSV) promoter, the reverse transcription LTR, and the lentivirus 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 comprises the nucleic acid sequence of SEQ ID NO 67 and the amino acid sequence of SEQ ID NO 54.

Reagent kit

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

Experimental examples

The invention will now be described with reference to the following examples. These examples are provided for illustrative purposes only and the present invention should in no way be construed as limited to these examples but rather construed to include any and all variations which become apparent in light of the teachings provided herein.

Without further elaboration, it is believed that one skilled in the art can, using the preceding description and the following illustrative examples, make and use the compounds of the present invention and practice the claimed methods. The following working examples thus particularly point out 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. Knockout mice of properdin(fP^-/-) (8 weeks old) were immunized intraperitoneally with 50 μ g (in 100 μ l PBS) of purified human properdin (ComTech Inc) emulsified with 100 μ l Titermax adjuvant (from Sigma). On days 14 and 21, mice were again immunized with 50 μ g of purified human properdin emulsified with Titermax adjuvant. After one week, the mouse serum anti-properdin titers were measured. Mice with an antibody titer of 1:10,000 or higher were used for hybridoma fusion experiments. Two days before the fusion experiment, mice were again injected with 50 μ g of purified human properdin (in 100 μ l PBS) (i.p). Mice were sacrificed by cervical dislocation and spleens were isolated for preparation of single cell suspensions by mechanical disruption. Spleen cell suspensions were washed once with HYB-sfm (invitrogen) +10% FBS medium and cells were counted and mixed with X63-ag8.653 myeloma cells (ATCC) at a 2:1 ratio. The cell mixture was washed again with HYB-SFM medium and cell pellet was prepared by centrifugation (1000rpm × 5 min). The cell pellet was gently agitated and loosened, then treated by slow addition of polyethylene glycol (PEG1500) (3X 10)⁸Cells, 1.5ml PEG) caused cell fusion. Cells were placed at 37 ℃ for 1min, then 20ml of HYB-SFM medium was added to the cells over 3min (1 ml for the first minute, 3ml for the second minute, 16ml for the third minute). The mixture was centrifuged at 1000rpm for 5min, and cells were plated in HAT medium (10 ml HAT [ Sigma H0262 ] in 500ml HYB-SFM medium) in 24-well plates]5ml penicillin/streptomycin, 500. mu.l gentamicin and 10% FBS). After 2 weeks, supernatants were taken from wells with visible clones for screening for reactivity with purified human properdin by ELISA. Positive clones were picked and plated in 96-well plates by limiting dilution, and single clones were obtained after a second round of screening by ELISA. Positive clones were expanded in HT-medium (10 ml HT in 500ml HYB-SFM medium, 5ml penicillin/streptomycin 500. mu.l gentamicin and 10% FBS). Prior to antibody collection, hybridoma cells were transferred to serum-free medium (HYB-SFM) for 2-3 days. Cell cultures were collected for mAb purification by protein G affinity chromatography.

To clone cDNAs against properdin mAbs, total RNA was isolated from hybridoma cells by TRizol reagent (Sigma). First strand cDNA was synthesized by reverse transcription using an oligonucleotide (dT) primer. For amplification of 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: mixture of 4 forward 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); a downstream primer: 5'-GTTGGTGCAGCATCAGC-3' (SEQ ID NO: 74). The PCR amplicons were cloned into the pCRTOPO TA2.1 vector (Invitrogen) and sequenced. To obtain the signal peptide (leader) sequences of mAbs, 5' -RACE method was used with 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

Dose-dependent inhibition of LPS-induced AP complement activation by mabs19.1, 22.1, 25 and 30 was examined. When added to 50% Normal Human Serum (NHS) at a final concentration of 5. mu.g/ml, all 4 mAbs clones effectively inhibited AP complement activation (see FIG. 2). ELISA plates (96-well, Nunc) were coated with 50. mu.l LPS solution (40. mu.g/ml in phosphate buffered saline [ PBS ]) overnight at 4 ℃. The next day, plates were washed 3 times with PBS containing 0.05% Tween-20 (PBS-T) and 50 μ l50% Normal Human Serum (NHS) that had been incubated with 1-5 μ g/ml anti-properdin mAb for 1 hour at 4 ℃ was added. NHS was diluted with GVB-EGTA-Mg + + (containing 10mM EGTA and 2.5mM Mg + + final concentration). The plates were incubated at 37 ℃ for 1 hour, washed 3 times with PBS-T, then 50. mu.l of HRP-conjugated goat anti-human C3 antibody (1:4000, Cappel) was added and the plates were left at room temperature for 1 hour. Plates were washed 3 times with PBS-T and then developed using BD Pharmingen A + B reagent. The reaction was terminated after 45min with 2N H2 SO. AP complement activation was detected (OD450) by measuring the amount of C3 deposition on the plate. The EDTA-added sample (NHSEDTA) served as a negative control (EDTA blocked complement activation). Samples without mAb added (0Ab) 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 fH and DAF dysfunction (see figure 3). Normal human RBCs (5X 10)⁶Cells) were incubated with 100 μ l50% NHS (diluted with GVB-EGTA-Mg + + containing 10mM EGTA and 2.5mM Mg + + final concentrations) for 20min at 37C in the presence of 30 μ M recombinant fH19-20 and 7.5 μ g mouse anti-human DAF (from AbD Serotec) (2006, Ferreira et al, J Immunol.177: 6308-6316). The lysis reaction was stopped by adding 200. mu.l of 20mM EDTA in ice-cold PBS. The incubation mixture was centrifuged at 1500g for 5min and the supernatant was collected and subjected to beading for OD420nm measurement. Prior to addition of RBC, NHS was preincubated with 0 or 5. mu.g/ml anti-properdin antibody at 4C for 1 hour. Samples without NHS or fH19-20 added, or EDTA added, were used as negative lysis controls, RBCs samples that were completely lysed with 100 μ l of distilled water were used as positive controls (100% lysis), and% lysis in other samples was normalized to this positive control.

Example 4

Experiments to evaluate antibody-sensitized sheep RBCs incubated with 50% Normal Human Serum (NHS) in the absence or presence of 5 μ g/ml anti-properdin mAbs (see fig. 4). Antibody-sensitized sheep RBCs (5X 10)⁶Cells, from ComTech Inc) were incubated with 100. mu.l 50% NHS (diluted in GVB + + buffer) for 20min at 37 ℃. NHS was preincubated with 0 or 5. mu.g/ml anti-properdin antibody at 4C for 1 hour prior to addition of sheep RBCs. The lysis reaction was stopped by adding 200. mu.l of 20mM EDTA in ice-cold PBS. The incubation mixture was centrifuged at 1500g for 5min, the supernatant was collected and OD420nm was measured. Samples without NHS or EDTA added were used as negative lysis controls and sheep RBCs samples that were completely lysed with 100 μ l of distilled water were used as positive controls (100% lysis) against which% lysis in other samples was normalized.

Example 5

Generation of human properdin deletion mutants and their expression in CHO cells was verified by western blot (see figure 5). Human properdin (fP) consists of 7 thrombospondin repeat (TSR) domains, which are numbered 0 to 6. Single TSR domains 0 to 5 (see SEQ ID NOS: 55, 56, 57, 58, 59 and 60) were deleted by inverse PCR using the full-length human properdin cDNA (SEQ ID NO:67) in the pCMV vector (from Origene) as a template (1989, Hemsley et al, Nucleic acid.5Res.17: 6545). For deletion of TSR6(SEQ ID NO:61) or TSR5-6, normal PCR methods were used followed by cloning into an expression vector (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, cells were lysed with 50mM Tris-HCl, pH7.4 (250. mu.l per well) containing 150mM NaCl, 10% glycerol, 1mM EDTA and protease inhibitor cocktail (Roche) and 1% Triton X-100. The lysate was centrifuged at 10,000rpm for 10min and the protein concentration was determined by the BCA protein assay method. Approximately 100. mu.g total protein from each sample was analyzed by SDS-PAGE.

Example 6

Sandwich ELISA assays of mab19.1 and 25 binding to human properdin deletion mutants were performed for epitope mapping of mab19.1 and 25. (see fig. 6). ELISA plates were coated with 50. mu.l of 2. mu.g/ml of the relevant mAb overnight at 4 ℃. The plate was washed 3 times with PBS-T, then 25. mu.g (in 50. mu.l PBS containing 1% BSA) CHO cell lysate protein was added to the wells and the plate was incubated for 1 hour at room temperature. The plates were washed 3 times with PBS-T and then the captured protein was detected by a biotinylated goat anti-human properdin antibody and HRP-avidin system. A third mab29.3, which binds to a different epitope of 19.1 and 25, was used as a control to verify mutein expression.

Example 7

Epitope mapping showed the epitope of mab19.1, located to the C-terminal half of TSR5, with the following amino acid sequence: RGRTCRGRKFDGHRCAGQQQDIRHCYSIQHCP (SEQ ID NO:52) (see FIG. 7). Three properdin mutants, 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. The blot was stripped and re-probed with mouse anti-His tag antibody (Qiagen) to confirm the presence of the C-terminal His tag (no C-terminal proteolysis). A sandwich ELISA assay was performed to determine reactivity with mab19.1 as described in example 6. Mab29.3, which binds 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, which is located to the C-terminal quarter of TSR6, has the following amino acid sequence: LVVEEKRPCLHVPACKDPEEEEL (SEQ ID NO:53) (see FIG. 8). Three 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 re-probed with mouse anti-His tag antibody (Qiagen) to confirm the presence of the C-terminal His tag (no C-terminal proteolysis). A sandwich ELISA assay was performed to determine reactivity with mAb25 as described in example 6. Mab19.1, which binds a different epitope of 25, was used as a control to confirm mutein expression. Epitope mapping also showed that the epitope of mAb25 was dependent on two cysteine residues in TSR6(SEQ ID NO:61, shown in FIG. 5B). These are cysteine 62(C62) and cysteine 78(C78) of TSR 6. Single mutations at C62 or C78 to alanine (a) in full-length human properdin did not abrogate mAb25 binding, but double mutations at C62A and C78A abrogated mAb25 binding. As a positive control for mutein expression, mab19.1 showed reactivity to all samples. This result suggests that C78 within the last quarter of TSR6 (having the sequence specified by SEQ ID NO:53) and C62 located outside of SEQ ID NO:53 but within TSR6(SEQ ID: 61) constitute two key residues of the epitope of mAb 25. Binding assays for mabs19.1 and 25 were performed on ELISA plates using transfected CHO cell homogenates. 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 19.1mAbs in CHO cells (see FIG. 17). Chimeric 19.1 heavy chain cDNA was constructed using the EcoRI/NheI site by cloning the mAb19.1 variable region (SEQ ID NO:1) into the pFUSE-CHIg-hG4 vector (from InvivoGen containing the human IgG4 heavy chain constant region in which serine 229 was mutated to proline). A 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 the AgeI/BsiWI site. Humanized 19.1 heavy chain cDNAs were constructed 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) using the EcoRI/NheI sites. A humanized 19.1 light chain cDNA was constructed by cloning the pFUSE2-CLIg-hk vector (from InvivoGen, containing the human kappa light chain constant region) with the humanized light chain variable region of 19.1 (cDNA encoding SEQ ID NO:47, synthesized by Genescript) using the AgeI/BsiWI site. CHO cells were co-transfected with either the chimeric heavy and light chains of 19.1 or the humanized heavy and light chains of 19.1 (both humanized heavy chains paired with the same humanized light chain) using Lipofectamine reagent (Lipofectamine reagent). After transfection, CHO cells were selected with Geocine (1mg/ml) and blasticidin (Blastcidine) (10. mu.g/ml) for approximately 7 days. Drug-resistant cell colonies were picked, trypsinized and cultured in a 96-well plate for limiting dilution in the presence of the same selection drug. After cells became confluent in 96-well plates, the media was tested for reactivity with human properdin by ELISA, and positive clones were expanded. For antibody production, stably transfected CHO cell lines were cultured in 150cm culture flasks in DMEM with 10% FBS: f12 medium, and after reaching confluence, 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 affinity of mAb19.1, chimeric mAb19.1, humanized mAb19.1, mAb25, mAb22.1, and mAb30 (see fig. 18 and 19). The binding 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). The Biacore experiment was performed at 25 ℃. The carboxylated dextran matrix of the CM4 sensor chip was used to link purified properdin by amine coupling chemistry to obtain a 200RU surface density. mAbs were diluted to 150, 75, 35.5, 17.75, 8.87 and 0nM in HBSET (HEPES buffered saline with EDTA and Tween 20) buffer and samples were injected at 30. mu.l/min (60. mu.l injection) onto properdin surface for 120s, allowing dissociation of bound analyte to proceed for 900 s. Data were analyzed by BIA evaluation software 3.2 postulated bivalent binding model. Regeneration of the surface was obtained by injection of 50. mu.l of 50mM NaOH (50. mu.l/min).

Example 11

Experiments were performed to evaluate the relative activity of 19.1, chimeric 19.1 and two humanized 19.1mAbs in blocking LPS-induced human AP complement activation (see figure 20). ELISA plates (96 wells, Nunc) were coated with 50. mu.l LPS solution (40. mu.g/ml in PBS, overnight at 4 ℃). The following day, plates were washed 3 times with PBS containing 0.05% PBS-T and 50 μ l50% NHS that had been incubated with 0, 5, 10 or 20 μ g/ml anti-properdin mAb for 1 hour at 4 ℃ was added. NHS was diluted with GVB-EGTA-Mg + + (containing 10mM EGTA and 2.5mM Mg + +, final concentration). The plate was left at 37 ℃ for 1 hour, washed 3 times with PBS-T, then 50. mu.l 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 using BD Pharmingen A + B reagent. The reaction was stopped 5min after 2N H2SO 4. AP complement activation was detected by measuring the amount of C3 deposition on the plate (OD 450). The EDTA-added sample (NHSEDTA) served as a negative control (EDTA blocked complement activation). Samples without mAb added (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 19.1mAbs in blocking human RBC lysis by human AP complement in the context of fH and DAF dysfunction (see figure 21). Normal human RBCs (5X 10)⁶Cells) were incubated with 100 μ l50% NHS (diluted with GVB-EGTA-Mg + + containing 10mM EGTA and 2.5mM Mg + + final concentrations) for 20min at 37 ℃ in the presence of 30 μ M recombinant fH19-20 and 7.5 μ g mouse anti-human DAF (from ADB Serotec) (2006, Ferreira et al, JImmunol.177: 6308-6316). Prior to addition of RBCs, NHS was preincubated with 1-15. mu.g/ml of various mAbs against properdin for 1 hour at 4 ℃. The lysis reaction was stopped by adding 200. mu.l of 20mM EDTA in ice-cold PBS. The incubation mixture was centrifuged at 1500g for 5min, the supernatant was collected and OD420nm was measured. Samples without NHS or fH19-20, or EDTA addition were used as negative lysis controls, RBCs samples that were completely lysed with 100. mu.l of distilled water were used as positive controls (100% lysis), and% lysis in other samples was normalized to this positive control.

Example 13

Experiments were performed to evaluate the relative activity of 19.1, chimeric 19.1 and two humanized 19.1mAbs in blocking LPS-induced complement activation in cynomolgus and cynomolgus monkey AP (see fig. 22 and 23). ELISA plates (96 wells, Nunc) were coated with 50. mu.l LPS solution (40. mu.g/ml in PBS, overnight at 4 ℃). The following day, plates were washed 3 times with PBS containing 0.05% PBS-T and 50 μ l50% normal cynomolgus monkey serum (NRS) or normal cynomolgus monkey serum (NCS) preincubated with 0, 10, 20, 30 or 40 μ g/ml of anti-properdin mAb for 1 hour at 4 ℃ were added. NRS or NCS was diluted with GVB-EGTA-Mg + + (containing 10mM EGTA and 2.5mM Mg + +, final concentration). The plates were incubated at 37 ℃ for 1 hour, washed 3 times with PBS-T, then 50. mu.l of HRP-conjugated goat anti-human C3 antibody (1:4000, Cappel, monkey C3 cross-react) was added and the plates were left at room temperature for 1 hour. Plates were washed 3 times with PBS-T and then developed using BD Pharmingen A + B reagent. The reaction was stopped 5min after 2NH2SO 4. AP complement activation was detected by measuring the amount of C3 deposition on the plate (OD 450). Samples with EDTA added (nrsetta or ncsetta) served as negative controls (EDTA blocked complement activation). Samples without mAb added (NRS or NCS) served as baseline AP complement activation.

Example 14

Experiments were performed to evaluate the inhibition of acidified serolysis of PNH erythrocytes by mab19.1,25 and humanized 19.1-459 (hamm's test) (see figure 24). RBCs from Paroxysmal Nocturnal Hemoglobinuria (PNH) patients were subjected to hams' acidified serum trials in the presence or absence of mAbs. RBCs were incubated with autologous serum (final concentration 83%) at 37 ℃ for 2hr, and percent lysis was calculated by measuring OD405 of the supernatant, normalized to a sample of RBCs fully lysed in distilled water (Eh DDW). The incubation mixture consisted of: mu.l serum, 25. mu.l 1/6N HCL (or 25. mu.l saline for negative control), 12.5. mu.l 50% (v/v) RBC suspension, 10. mu.l mAb in saline. RBCs samples incubated with non-acidified autologous serum (NHS) were used as negative controls (background lysis). In the absence of mAbs, approximately 50% of RBCs were lysed by acidified serum. This lysis was completely inhibited by mAb19.1 at concentrations of 8. mu.g/ml and above, humanized 19.1mAb (#459) at concentrations of 20. mu.g/ml and mAb25 at concentrations of 8. mu.g/ml and above.

Example 15

Properdin humanized mice were generated as follows (see fig. 25). The human fP expression vector was constructed with pACGGS plasmid, as shown schematically in fig. 25A, using the chicken β -actin promoter with CVM-IE enhancer and rabbit β -globin polya tail for stable expression of cDNA in eukaryotic cells. 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. Plasmids were linearized by restriction enzyme digestion and microinjected into fertilized eggs of C57BL/6 mice to generate human fP transgenic founder mice. Positive founder mice (showing a human fP cDNA fragment of about 800 bp) can be identified by PCR screening (using primers 5'-ATCAGAGGCCTGTGACACC-3' (SEQ ID NO:65) and 5'-CTGCCCTTGTAGCTCCTCA-3' (SEQ ID NO:66) specific for human fP and genomic DNAs isolated from mouse tails). Of the 40 mice analyzed, five (#15, 20, 24, 27, and 32) were positive (fig. 25B, red arrow). Sandwich ELISA assays were performed to detect human fP in transgenic positive mice (fig. 25C). Plates were coated with non-blocking mAb against human fP (clone 8.1). After incubation with diluted mouse serum (10%), human fP was detected by using HRP-conjugated goat anti-human fP antibody. Normal Human Serum (NHS) was used as a positive control. By this method, human fP should be detected in NHS and in the serum of transgenic 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. The transgenic positive founder mice and WT mice were then propagated to establish germline transmission. Screening from such mated F1 mice was accomplished by PCR detection of the transgene through the tail DNA, and human properdin in its serum was detected by sandwich ELISA, as described above. After confirmation of germline transmission, founder mice were compared with fP^-/-Mouse reproduction to produce fP^-/-Human fP transgene + mouse. fP^-/-Restoration of AP complement activity in 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 positive for fP transgenically positive in WT mouse serum and in human fP^-/-It was detected in mouse serum, but not in fP^-/-Detected in mouse serum. In this assay, serum from WT mice treated with EDTA will be used 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 (fig. 26). Humanizing properdin mouse (fP)^-/-Human fP transgene +) injection with 0.5mg (i.p.) mAb 25. Blood samples (50-75 μ l) were collected by retro-orbital bleeding and serum was prepared before injection (0hr) and then at various time points after injection. Serum samples were tested for LPS-induced AP complement activation. For this assay, ELISA plates (96-well, Nunc) were coated with 50. mu.l LPS solution (40. mu.g/ml in PBS overnight at 4 ℃). The following day, plates were washed 3 times with PBS containing 0.05% Tween-20 (PBS-T), and 50. mu.l of serial dilutions (from 1:10) of mouse serum were added to each well. Mouse sera were diluted with GVB-EGTA-Mg + + (containing 10mM EGTA and 2.5mM Mg + + final concentration). The plate was incubated at 37 ℃ for 1 hour, washed 3 times with PBS-T, then 50. mu.l 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 using BD Pharmingen A + B reagent. The reaction was stopped 5min after 2N H2SO 4. AP complement activation was detected by measuring the amount of C3 deposition on the plate (OD 450). In this exemplary embodiment, fP^-/-AP complement activity was absent in mouse serum or in WT serum treated with EDTA. In contrast, AP complement activity was detected in WT serum and in fP humanized mouse serum at 0hr (prior to mAb25 treatment). AP complement activity in humanized mice was still inhibited at 8, 24 and 48hr after mAb25 treatment, but was detected at 72, 96 and 120hr onset. These results show that at a dose of 0.5 mg/mouse,mAb25 inhibited AP complement activity in vivo for at least 48 hr.

Example 17

Experiments were performed to evaluate the effect of anti-human properdin mab19.1 on extravascular haemolysis (EVH). In this EVH model, properdin humanized mice (n =4 per experimental group) were infused with Red Blood Cells (RBCs) from Crry/DAF/C3 Triple Knockout (TKO) mice. Prior to RBC transfer, recipient mice (properdin humanized mice) were treated with mab19.1(2 mg/mouse, i.p.) or control mouse IgG1mAb (MOPC, purified from MOPC31C hybridoma, from ACTT) for 6 hr. RBCs were harvested from donor TKO mice, washed in PBS and labeled with CFSE prior to injection (via tail vein) into recipient mice, according to previously published procedures (Miwa et al, 2002, Blood 99: 3707-. Each recipient mouse received RBCs in an amount equal to 100. mu.l of blood. Recipient mice were bled and RBCs analyzed to determine the number of CFSE-labeled (i.e., infused) RBCs remaining in circulation 5 minutes and 6, 24, 48, 72, 96, 120 hours after RBC infusion. The number of CFSE-labeled RBCs in each recipient was normalized (as%) to the 5min 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-one 3716). However, in recipient mice treated with anti-human properdin 19.1mAb, no EVH occurred and infused RBCs remained, showing that anti-properdin mAb was effective in preventing EVH (fig. 27).

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

Although the invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of the invention can be devised by those skilled in the art without departing from the true spirit and scope of the invention. It is intended that the following claims be interpreted to embrace all such embodiments and equivalent variations.

Claims (58)

1. A composition comprising an antibody that specifically binds properdin.

2. The composition of claim 1, wherein said properdin is human properdin.

3. The composition of claim 1, wherein the antibody is a monoclonal antibody.

4. The composition of claim 1, wherein the antibody is a humanized antibody.

5. The composition of claim 1, wherein the antibody is a chimeric antibody.

6. The composition of claim 1, wherein the antibody comprises at least one CDRs selected from the group consisting of: VH-CDR 1: 3, SEQ ID NO; VH-CDR 2:4, SEQ ID NO; VH-CDR 3: 5, SEQ ID NO; VL-CDR 1: 8 in SEQ ID NO; VL-CDR 2: 9, SEQ ID NO; and VL-CDR 3: SEQ ID NO 10.

7. The composition of claim 1, wherein the antibody comprises CDRs: VH-CDR 1: 3, SEQ ID NO; VH-CDR 2:4, SEQ ID NO; VH-CDR 3: 5, SEQ ID NO; VL-CDR 1: 8 is shown in SEQ ID NO; VL-CDR 2: 9, SEQ ID NO; and VL-CDR 3: SEQ ID NO 10.

8. The composition of claim 1, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID No. 2.

9. The composition of claim 1, wherein the antibody comprises a light chain comprising the amino acid sequence of SEQ ID No. 7.

10. The composition of claim 1, wherein the antibody 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.

11. The composition of claim 1, wherein the antibody specifically binds to an epitope comprising at least one amino acid of SEQ ID NO 52.

12. The composition of claim 1, wherein the antibody comprises at least one CDRs selected from the group consisting of: VH-CDR 1: 13 in SEQ ID NO; VH-CDR 2: 14, SEQ ID NO; VH-CDR 3: 15, SEQ ID NO; VL-CDR 1: 18 in SEQ ID NO; VL-CDR 2: 19 in SEQ ID NO; and VL-CDR 3: 20 in SEQ ID NO.

13. The composition of claim 1, wherein the antibody comprises CDRs: VH-CDR 1: 13 in SEQ ID NO; VH-CDR 2: 14, SEQ ID NO; VH-CDR 3: 15, SEQ ID NO; VL-CDR 1: 18 is SEQ ID NO; VL-CDR 2: 19 in SEQ ID NO; and VL-CDR 3: 20 in SEQ ID NO.

14. The composition of claim 1, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID No. 12.

15. The composition of claim 1, wherein the antibody comprises a light chain comprising the amino acid sequence of SEQ ID No. 17.

16. The composition of claim 1, wherein the antibody 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.

17. The composition of claim 1, wherein the antibody specifically binds to an epitope comprising at least one amino acid of SEQ ID NO 53.

18. The composition of claim 1, wherein the antibody comprises at least one CDRs selected from the group consisting of: VH-CDR 1: 23, SEQ ID NO; VH-CDR 2: 24 is SEQ ID NO; VH-CDR 3: 25 in SEQ ID NO; VL-CDR 1: 28 in SEQ ID NO; VL-CDR 2: 29 in SEQ ID NO; and VL-CDR 3: SEQ ID NO 30.

19. The composition of claim 1, wherein the antibody comprises CDRs: VH-CDR 1: 23, SEQ ID NO; VH-CDR 2: 24 is SEQ ID NO; VH-CDR 3: 25 in SEQ ID NO; VL-CDR 1: 28 is SEQ ID NO; VL-CDR 2: 29 in SEQ ID NO; and VL-CDR 3: SEQ ID NO 30.

20. The composition of claim 1, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID No. 22.

21. The composition of claim 1, wherein the antibody comprises a light chain comprising the amino acid sequence of SEQ ID No. 27.

22. The composition of claim 1, wherein the antibody 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.

23. The composition of claim 1, wherein the antibody comprises at least one CDRs selected from the group consisting of: VH-CDR 1: 33, SEQ ID NO; VH-CDR 2: 34 in SEQ ID NO; VH-CDR 3: 35 in SEQ ID NO; VL-CDR 1: 38, SEQ ID NO; VL-CDR 2: 39 in SEQ ID NO; and VL-CDR 3: SEQ ID NO 40.

24. The composition of claim 1, wherein the antibody comprises CDRs: VH-CDR 1: 33, SEQ ID NO; VH-CDR 2: 34 in SEQ ID NO; VH-CDR 3: 35 in SEQ ID NO; VL-CDR 1: SEQ ID NO: 38; VL-CDR 2: 39 in SEQ ID NO; and VL-CDR 3: SEQ ID NO 40.

25. The composition of claim 1, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO 32.

26. The composition of claim 1, wherein the antibody comprises a light chain comprising the amino acid sequence of SEQ ID No. 37.

27. The composition of claim 1, wherein the antibody 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.

28. The composition of claim 1, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID No. 42.

29. The composition of claim 1, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO 44.

30. The composition of claim 1, wherein the antibody comprises a light chain comprising the amino acid sequence of SEQ ID No. 47.

31. The composition of claim 1, wherein the antibody 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.

32. The composition of claim 1, wherein the antibody 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.

33. The composition of claim 1, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO. 49.

34. The composition of claim 1, wherein the antibody comprises a light chain comprising the amino acid sequence of SEQ ID No. 51.

35. The composition of claim 1, wherein the antibody 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.

36. The composition of claim 1, wherein the antibody comprises a heavy chain comprising the amino acid sequences of SEQ ID NO 2 and SEQ ID NO 63.

37. The composition of claim 1, wherein the antibody comprises a light chain comprising the amino acid sequences of SEQ ID NO 7 and SEQ ID NO 64.

38. The composition of claim 1, wherein the antibody comprises a heavy chain comprising the amino acid sequences of SEQ ID NO 2 and SEQ ID NO 63 and a light chain comprising the amino acid sequences of SEQ ID NO 7 and SEQ ID NO 64.

39. The composition of claim 1, wherein the antibody comprises a heavy chain comprising the amino acid sequences of SEQ ID NO 12 and SEQ ID NO 63.

40. The composition of claim 1, wherein the antibody comprises a light chain comprising the amino acid sequences of SEQ ID NO 17 and SEQ ID NO 64.

41. The composition of claim 1, wherein the antibody comprises a heavy chain comprising the amino acid sequences of SEQ ID NO 12 and SEQ ID NO 63 and a light chain comprising the amino acid sequences of SEQ ID NO 17 and SEQ ID NO 64.

42. The composition of claim 1, wherein the antibody comprises a heavy chain comprising the amino acid sequences of SEQ ID NO 22 and SEQ ID NO 63.

43. The composition of claim 1, wherein the antibody comprises a light chain comprising the amino acid sequences of SEQ ID NO 27 and SEQ ID NO 64.

44. The composition of claim 1, wherein the antibody comprises a heavy chain comprising the amino acid sequences of SEQ ID NO 22 and SEQ ID NO 63 and a light chain comprising the amino acid sequences of SEQ ID NO 27 and SEQ ID NO 64.

45. The composition of claim 1, wherein the antibody comprises a heavy chain comprising the amino acid sequences of SEQ ID NO 32 and SEQ ID NO 63.

46. The composition of claim 1, wherein the antibody comprises a light chain comprising the amino acid sequences of SEQ ID NO 37 and SEQ ID NO 64.

47. The composition of claim 1, wherein the antibody comprises a heavy chain comprising the amino acid sequences of SEQ ID NO 32 and SEQ ID NO 63 and a light chain comprising the amino acid sequences of SEQ ID NO 37 and SEQ ID NO 64.

48. A composition comprising an antibody that binds to properdin and competes with the binding of the antibody of claim 1 to properdin.

49. A composition comprising an antibody that binds properdin and competes with the binding of an antibody designated mab19.1 to properdin.

50. A composition comprising an antibody that binds to properdin and competes with the binding of an antibody designated mAb25 to properdin.

51. A composition comprising an antibody that binds properdin and competes with the binding of an antibody designated mab22.1 to properdin.

52. A composition comprising an antibody that binds to properdin and competes with the binding of an antibody designated mAb30 to properdin.

53. A method of treating an Alternative Pathway (AP) -mediated pathology in an individual comprising the step of administering to said individual an anti-properdin antibody according to claim 1.

54. The method of claim 53, wherein said pathology is selected from at least the following: macular degeneration, ischemia reperfusion injury, arthritis, rheumatoid arthritis, Paroxysmal Nocturnal Hemoglobinuria (PNH) syndrome, atypical hemolytic uremic (aHUS) syndrome, asthma, organ transplant septicemia, inflammation, glomerulonephritis, lupus, and combinations thereof.

55. The method of claim 53, wherein the anti-properdin antibody selectively inhibits the alternative pathway but not the classical pathway and the lectin pathway.

56. The method of claim 53, wherein the anti-properdin antibody does not affect the AP amplification loop of the classical pathway and the lectin pathway.

57. The method of claim 53, wherein administration of the anti-properdin antibody inhibits production of C3bBb protein.

58. A transgenic mouse, wherein the transgenic mouse expresses human properdin, wherein the nucleic acid sequence of the human properdin is SEQ ID No. 67 and the amino acid sequence of the human properdin is SEQ ID No. 54, and wherein the transgenic mouse does not express mouse properdin.