INHIBITION OF THE COMPLEMENT SYSTEM

Abstract

Agents and compounds which can be used to modulate the activity of the complement system, novel biological targets associated with such modulation, and pharmaceutical compositions, medicaments and methods of treatment for use in preventing, ameliorating or treating diseases that are characterised by inappropriate complement activity. These diseases include age-related macular degeneration (AMD), meningitis, renal disease, autoimmune disease and inflammation. Therapeutic antibodies and screening assays for identifying agents useful in treating these diseases are also provided.

Claims

1. A method of treating, preventing or ameliorating a disease characterized by excessive complement activation in a subject, the method comprising administering, to a subject in need of such treatment, a therapeutically effective amount of an antibody or antigen binding fragment thereof, which: (i) reduces the concentration or activity of at least one complement factor H-related (CFHR) protein selected from the group consisting of: CFHR1, CFHR2, CFHR3, CFHR4 and CFHR5; or (ii) reduces or inhibits dimerization or higher order assembly of at least one CFHR protein selected from the group consisting of: CFHR1, CFHR2, CFHR3, CFHR4 and CFHR5, to treat, prevent or ameliorate a disease characterized by excessive complement activation in the subject.

2. The method according to claim 1, wherein the antibody or antigen binding fragment thereof is used to treat, prevent or ameliorate meningitis, renal disease, C3 glomerulopathy, autoimmune disease conditions, inflammation including conditions, rheumatoid arthritis, asthma, lupus nephritis, ischemia-reperfusion injury, atypical hemolytic uremic syndrome, thrombotic thrombocytopenic purpura, paroxysmal nocturnal hemoglobinuria, Membranoproliferative glomerulonephritis, hemolytic uremic syndrome, Hypocomplementemic glomerulonephritis, dense deposit disease, macular degeneration, age-related macular degeneration (AMD), spontaneous fetal loss, Pauci-immune vasculitis, epidermolysis bullosa, recurrent fetal loss, multiple sclerosis, traumatic brain injury, Degos' disease, myasthenia gravis, cold agglutinin disease, dermatomyositis, Graves' disease, Hashimoto's thyroiditis, type I diabetes, psoriasis, pemphigus, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, Goodpasture syndrome, antiphospholipid syndrome, Infective endocarditis, or injury resulting from myocardial infarction, cardiopulmonary bypass or hemodialysis.

3. The method according to claim 1, wherein the antibody or antigen binding fragment thereof reduces the concentration or activity of, or reduces or inhibits dimerization or higher order assembly of, at least one CFHR protein comprising an amino acid sequence substantially as set out in SEQ ID NO:2, 4, 6, 8, 9 or 11, or a functional variant or fragment thereof.

4. The method according to claim 1, wherein the antibody or antigen binding fragment thereof binds to domain 1 and 2 of any of SEQ ID NO:2, 4, 6, 8, 9 or 11, or a fragment of variant thereof, and thereby reduces the concentration or activity of, or reduces or inhibits dimerization or higher order assembly of, the at least one CFHR protein.

5. The method according to claim 1, wherein the antibody or antigen binding fragment thereof binds to a CFHR protein to reduce the concentration of the CFHR dimers from the subject, but does not prevent dimerization.

6. The method according to claim 5, wherein the antibody or antigen binding fragment thereof binds to SEQ ID No.12, SEQ ID No: 13 or SEQ ID No.27, or a fragment or variant thereof, to reduce the concentration of the CFHR dimers from the subject, but does not prevent dimerization.

7. The method according to claim 1, wherein the antibody or antigen binding fragment thereof binds to SEQ ID No.12, SEQ ID No: 13 or SEQ ID No.27, or a fragment or variant thereof, and thereby reduces the concentration or activity of, or reduces or inhibits dimerization or higher order assembly of, the at least one CFHR protein.

8. The method according to claim 1, wherein the antibody or antigen binding fragment thereof binds to a region of SEQ ID No.12, or a fragment or variant thereof, other than that which is represented by SEQ ID No.13, and thereby reduces the concentration or activity of, or reduces or inhibits dimerization or higher order assembly of, the at least one CFHR protein.

9. The method according to claim 1, wherein the antibody or antigen binding fragment thereof: (a) reduces binding between a CFHR and a C3 fragment; (b) increases binding between CFH and a C3 fragment; (c) binds to a CFHR to reduce its biological activity; or (d) decreases expression of a CFHR.

10. The method according to claim 1, wherein the antibody or antigen binding fragment thereof is raised against any of SEQ ID NO:2, 4, 6, 8, 9 or 11, or a fragment of variant thereof, acting as an antigen.

11. The method according to claim 10, wherein the antibody or antigen binding fragment thereof is raised against domains 1 and 2 of any of SEQ ID NO:2, 4, 6, 8, 9 or 11, or a fragment of variant thereof, acting as antigen.

12. The method according to claim 10, wherein the antibody or antigen binding fragment thereof is raised against SEQ ID No.12, SEQ ID No.13 or SEQ ID No.27, acting as antigen.

13. The method according to claim 1, wherein the antibody is recombinant.

14. The method according to claim 13, wherein the recombinant antibody is chimeric, humanized or fully human.

15. The method according to claim 1, wherein the antigen-binding fragment is selected from the group consisting of VH, VL, Fd, Fab, Fab, scFv, F(ab).sub.2 and Fc fragments.

16. A method for identifying an agent that modulates dimerization or higher order assembly of at least one complement factor H-related (CFHR) protein selected from a group consisting of: CFHR1, CFHR2, CFHR3, CFHR4 and CFHR5, the method comprising: (i) contacting, in the presence of a test agent, a first protein selected from a group consisting of: CFHR1, CFHR2, CFHR3, CFHR4 and CFHR5, with a second protein selected from a group consisting of: CFHR1, CFHR2, CFHR3, CFHR4 and CFHR5; and (ii) detecting binding between the first and second proteins, wherein an alteration in binding as compared to a control is an indicator that the agent modulates dimerization or higher order assembly of at least one complement factor H-related (CFHR) protein selected from a group consisting of: CFHR1, CFHR2, CFHR3, CFHR4 and CFHR5.

17. An assay for identifying an agent that modulates dimerisation or higher order assembly of at least one complement factor H-related (CFHR) protein selected from a group consisting of: CFHR1, CFHR2, CFHR3, CFHR4 and CFHR5, the method comprising: (i) a first protein selected from a group consisting of: CFHR1, CFHR2, CFHR3, CFHR4 and CFHR5; (ii) a second protein selected from a group consisting of: CFHR1, CFHR2, CFHR3, CFHR4 and CFHR5; and (iii) a vessel configured to permit contacting of at least one test agent with the first and/or second agent.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0145] FIG. 1 shows that CFHR1, CFHR2 and CFHR5 contain an identical novel dimerization motif. FIG. 1(a) Alignment of the SCR domains of CFHR1, CFHR2 and CFHR5 with CFH. These proteins are comprised of subunits termed short consensus repeat (SCR) domains and domains have been aligned according to the CFH domain with which they share the highest amino acid similarity, percentage identity indicated. Red boxing denotes domains for which novel X-ray structures are presented in this manuscript. The complement regulatory domains of CFH reside within the first four amino-terminal domains (cyan). None of the CFHR proteins contain domains similar to these. CFH surface recognition domains which contain C3b/C3d and glycosaminoglycan (GAG) binding sites reside within the carboxyl-terminal two domains (CFH.sub.19-20) and all three CFHR proteins contain highly similar domains. Mapping of the conserved residues onto the existing structure of CFH.sub.19-20 suggests that GAG but not C3b/C3d binding is altered or lost within CFHR2.sub.3-4 (see FIGS. 6 & 7). The first two amino-terminal domains of CFHR1, CFHR2 and CFHR5 are highly conserved and have previously been described as CFH.sub.67-like, although the level of identity is less than 40%. FIG. 1(b) X-ray crystal structure of CFHR1.sub.1-2. The two copies of CFHR1.sub.12 that form the head-to-tail dimer are shown as grey cartoons with a semi-transparent surface. Residues Tyr34, Ser36 and Tyr39 that are critical in stabilising the dimer are shown in a ball-and-stick representation (Figure drawn using program PyMol, www.pymol.org). FIG. 1(c) Sequence alignment of CFHR1.sub.1-2, CFHR2.sub.1-2 and CFHR5.sub.1-2 with CFH.sub.6-7. The dimerization interface is conserved between these CFHR proteins but not in CFH. (interface residues determined using PISA; residues Tyr34, Ser36 and Tyr39 indicated by *; other interface residues by ). Red boxed residues=non-conservative, green boxed residues=conservative variation and yellow boxed residues=residues unique to CFH.sub.67. FIG. 1(d) Mapping sequence variation onto the molecular surface of one copy of CFHR1.sub.12. This analysis confirmed that the dimerization interface is conserved amongst CFHR1.sub.12, CFHR2.sub.12 and CFHR5.sub.12 but not in CFH.sub.67 (positions of Tyr34, Ser36 and Tyr 39 indicated with *);

[0146] FIG. 2 shows that CFHR1, CFHR2 and CFHR5 are dimeric in serum. FIG. 2(a) Multi-angle light scattering analyses (MALS) of a (i) serum fraction containing CFHR1, CFHR2 and CFHR5 and (ii) recombinant CFHR1.sub.1-2. MALS analysis of this fraction (red) demonstrates that this mixture contains a mass range between 65 and 80 kDa. MALS using recombinant CFHR1.sub.1-2 (blue trace and mass profile) demonstrates that it forms a homogenous dimer in both solution and crystal. FIG. 2 (b) Immunoprecipitation of CFHR2 in serum reveals the presence of CFHR1-CFHR2 heterodimers in vivo. Serum was immunoprecipitated using a specific anti-CFHR2 antibody (MBC22) and western blot analysis of the immunoprecipitated material with anti-CFHR1/2/5 antibody (MBC125) performed. This revealed the presence of CFHR1 (lane 1) which was absent in serum from an individual homozygous for the CFHR3-1 deletion polymorphism (lane 3). Lane 2 and 4 represent control sera in which no anti-CFHR2 antibody was used. The detection of CFHR2-CFHR5 heterodimers was not possible due to non-specific bands in the CFHR5 region. FIG. 2(c) Immunoprecipitation of CFHR5 in serum reveals the presence of CFHR1-CFHR5 heterodimers in vivo. Serum was immunoprecipitated using an anti-CFHR5 antibody and western blot analysis of the immunoprecipitated material with anti-CFHR1/2/5 antibody performed. This revealed the presence of CFHR1 (lane 1) which was absent in serum from an individual homozygous for the CFHR3-1 deletion polymorphism (lane 3). Lane 2 and 4 represent control sera in which no anti-CFHR5 antibody was used. The detection of CFHR2-CFHR5 heterodimers was not possible due to non-specific bands in the CFHR2 region. FIG. 2(d) ELISA assay to detect CFHR2-CFHR5 heterodimers in vivo. Using an anti-CFHR5 capture antibody and an anti-CFHR2 detection antibody, positive signal was demonstrable in two individuals homozygous for the CFHR3-1 deletion polymorphism. A much weaker signal was detectable in individuals without this deletion. No signal was seen when recombinant CFHR5 was tested indicating that the anti-CFHR2 detection antibody does not cross-react with CFHR5;

[0147] FIG. 3 shows that dimerisation enhances the interaction of CFHR5 with complement c3 in vivo. FIG. 3(a) Generation of a CFHR5 protein lacking critical amino acids within the dimerisation motif. Monomeric CFHR5 (CFHR5.sup.dimer mutant) was generated by mutating the three stabilizing amino acids (Tyr34Ser, Ser36Tyr, Tyr39Glu) within the dimerisation motif to the corresponding amino acids within CFH. FIG. 3(b) Analysis of recombinant CFHR5 and CFHR5.sup.dimer mutant using SDS PAGE gel electrophoresis. Both the wild type and dimer mutants were purified to single homogenous species as visualized by denaturing electrophoresis. FIG. 3(c) Analysis of recombinant CFHR5 and CFHR5.sup.dimer mutant using size exclusion chromatography. Size exclusion chromatography was performed on a Superdex200 10/30 column (GE Healthcare) equilibrated in 50 mM Tris.HCl, pH 7.5, 150 mM NaCl at 0.4 ml/min. The column was followed in-line by an Optilab-Rex refractive index monitor (Wyatt Technologies). The CFHR5 dimer elutes early from the column (blue trace) whilst the monomeric CFHR5.sup.dimer mutant protein elutes at a larger column volume (red trace). FIG. 3(d) Interaction of CFHR5 and CFHR5.sup.dimer mutant with renal-bound mouse C3 in vivo. When recombinant CFHR5.sup.dimer mutant was injected at identical concentration to that of CFHR5, CFHR5.sup.dimer mutant binding to glomerular C3 was significantly reduced compared to that of wild-type CFHR5;

[0148] FIG. 4 shows that CFHR1 and CHFR5 de-regulate complement activation by competitively inhibiting the interaction of cfh with c3b. FIG. 4(a) CFH binding to C3b is inhibited by either recombinant CFHR5 or serum-derived CFHR1. ELISA wells were coated with C3b and 0.07 M CFH was incubated with increasing amounts of either CFHR1 (0.014 to 1.8 M) or CFHR5 (0.005 to 0.6 M). Both proteins reduced the CFH-C3b interaction in a dose-dependent manner. Similar results were obtained when recombinant CFHR1.sub.345 (0.14 to 18 M) and CFHR2.sub.34 (0.13 to 16 M) were used. FIG. 4(b) CFH-dependent alternative pathway haemolytic assay. Using a CFH dose that reduced lysis of Guinea-Pig erythrocytes to 50%, the addition of increasing concentrations of CFHR1.sub.35, CFHR2.sub.34, serum-derived CFHR1 and recombinant CFHR5 resulted in a dose-dependent increase in lysis. Full length, dimeric, CFHR1 and CFHR5 were orders of magnitude more potent with respect to the monomeric CFHR1 and CFH2 fragments lacking the dimerisation motif. FIG. 4(c) Enhanced de-regulation by plasma-derived preparations containing CFHR1, CFHR2 and CFHR5 from individuals with familial C3 glomerulopathy due to a CFHR5 mutation. Using the haemolytic assay described in (b) serum-derived preparations from patients with CFHR5 mutation associated with C3 glomerulopathy showed significantly greater haemolysis than controls;

[0149] FIG. 5 shows that modulation of complement in vivo by CFHR1, CFHR2 and CFHR5. These proteins compete with CFH for interaction with C3b. Unlike CFH, they are devoid of intrinsic complement regulatory activity under physiological conditions. However, their interaction with C3b prevents the binding of C3b to CFH and thereby prevents inactivation of C3b by CFH. This process is termed de-regulation. Whether or not C3b interacts with CFH or components of the CFHR family will be influenced by factors such as C3b density, surface polyanions and the local concentrations of CFH and CFHR proteins. In this way, CFHR proteins provide a sophisticated means through which complement activation can be modulated in vivo. Inset: A general schematic for the functionally important portions of CFHR1, CFHR2 and CFHR5 is shown;

[0150] FIG. 6 shows that the C3b interface is conserved in the C-terminal domains but not the GAG binding surface. FIG. 6(a) Crystal structure of CFHR234 suggests GAG binding is altered or lost. The electrostatic potential (contoured at +3 kT/eblue, 3 kT/ered; calculated using the APBS plugin within Pymol: www.pymol.org) is mapped onto the surface and that of CFH19-20, PDB 3OXU, shown for comparison. CFH charged GAG-binding surface is ablated (right image, GAG binding surface=yellow dashed outline). FIG. 6(b) Crystal structure of CFHR234 suggests C3b binding maintained despite sequence variation. Sequence variation (identical residuesgrey; variationyellow) between CFHR2 and CFH (PDB 3OXU) mapped onto the CFHR234 structure shows high conservation of the C3b binding site despite the relatively low level of amino acid conservation in these domains between these proteins;

[0151] FIG. 7 shows that CFHR1 interacts with heparin via its C-terminal domains (domains 3-5) but CFHR2 does not. Approximately 0.5 mg CFHR1345 and CFHR234 in 50 mM Tris, 10 mM NaCl, pH 7.5 was loaded onto a 1 ml HiTrap Heparin column (GE Healthcare) using an AKTAfplc (GE healthcare). Non-bound material was washed out with 5 CV 50 mM Tris, 10 mM NaCl, pH 7.5 prior to a gradient elution of 50% 50 mM Tris, 1M NaCl, pH 7.5 over 15 CV. CFHR234 did not bind and was washed out during wash step. FHR1345 eluted at 29.6 mS/cm;

[0152] FIG. 8 shows that binding of CFHR5 to C3 in vivo is dose-dependent and targets CFHR5 to the kidney. FIG. 8(a) Binding of CFHR5 to C3 in vivo is dose-dependent. Glomerular CFHR5 staining was reduced when decreased doses of CFHR5 (30, 15, 7.5 and 3.8 g) were injected into CFH/ mice. No staining was observed in mice injected with PBS (negative control). FIG. 8(b) Targeting of CFHR5 to the kidney is dependent on C3. Ex vivo binding of CFHR5 to kidney sections of CFH/ mice and animals with combined deficiency of either Cfh and C3 (CFH.C3/), or CFH and C5 (CfH.C5/). Glomerular CFHR5 staining was evident only in the presence of C3;

[0153] FIG. 9 shows that Surface Plasmon Resonance (SPR) analysis of CFHR5 and CFH binding to C3b. FIG. 9(a) Binding of CFHR5 and CFH to low levels of C3b coupled through amine groups (no clustering of C3b). CFH (from 4 M) or CFHR5 (from 6.6 M) were flowed across immobilized C3b (400 RU) at different concentrations. Affinity was calculated by steady state analysis. Analysis assumes a 1:1 binding interaction and for CFHR5 calculations used molarity of binding sites. FIG. 9(b) Binding to C3b coupled through the thiolester. C3b (150 RU) was amine-coupled to a CM5 Biacore chip and used as a nidus for convertase formation. Further C3b was deposited on the chip surface by flowing fB and fD to form C3bBb followed by C3 as convertase substrate. Cleavage of C3 to C3b followed by nucleophilic attack on the C3 thioester by CM groups on the chip surface resulted in covalent binding of C3b (625 RU). CFH (from 4 M) and CFHR5 (from 1.35 M) were flowed across the surface and binding was analysed at steady state. Binding of CFH was very heterogeneous, likely due to crosslinking between multiple C3b-binding sites on fH and clusters of deposited C3b molecules. Affinity could not be calculated under these conditions. CFHR5 bound to this surface 10-fold more tightly than to the amine-coupled C3b, although binding heterogeneity was increased (see 2nd value). Comparison of (a) and (b) reveals differences in the binding caused by avidity; when C3b is clustered on the chip surface (b), multiple C3b-binding domains within one molecule of CFH or within the CFHR5 dimer can bind and cross-link C3b. Data were calculated using the following values: CFH, mass 155 kDa, extinction coefficient 1.95 cm-1(mg/mL)-1; CFHR5, mass 65 kDa, extinction coefficient 1.55 cm-1(mg/mL)-1;

[0154] FIG. 10 shows that surface plasmon resonance analysis of CFHR5 and CFH binding to the inactivation fragments, iC3b and C3dg. FIG. 10(a) C3b deposited in FIG. 7(b) was converted to iC3b by on-chip incubation with fH (30 mg/ml) and fI (10 mg/ml). Binding of CFH (from 5.3 M) and CFHR5 (from 2.7 M) to iC3b was assessed by flowing across the surface and evaluating at steady state. Binding of CFHR5 to iC3b was comparable to C3b (although more heterogeneous); binding of fH was vastly reduced compared to C3b and was 10-fold weaker than CFHR5. FIG. 10(b) Binding of CFHR5 and CFH to C3dg coupled through the thiolester was assessed by treating the iC3b surface with CR1 and fI to convert to C3dg, C3c was released from the chip surface. CFH (from 5.3 M) and CFHR5 (from 2.7 M) were flowed across the surface and binding evaluated at steady state. Binding of CFHR5 to C3dg was comparable to iC3b and C3b; binding affinity of fH was very weak and could not be calculated under these concentrations. Data were calculated using the following values: CFH, mass 155 kDa, extinction coefficient 1.95 cm-1(mg/mL)-1; CFHR5, mass 65 kDa, extinction coefficient 1.55 cm-1(mg/mL)-1;

[0155] FIG. 11 shows that CFHR5 does not have fluid-phase factor I (fI) cofactor activity for the proteolytic inactivation of either C3b (a) or iC3b (b). (a) C3b was deposited on the chip surface and convertase formation monitored by flowing CFB and FD (left panel, solid line). The surface was then treated twice with CFHR5 (first cycle 0.18 M and second cycle 0.44 M) and FI (10 g/ml constant) for 120 seconds each cycle. Convertase was formed by flowing CFB and FD exactly as before (left panel, dashed line). The amount of convertase formed was identical before and after treatment. In contrast, treatment of the surface with CFH (0.1 M) and fI ablated convertase formation (right panel). Moreover, no cleavage of the 65 chain of iC3b was observed after the incubation of iC3b (2 g) with CFHR5 (0.42 g) and fI (0.12 g) at 37 C. (b);

[0156] FIG. 12 shows that CFHR1 interacts with C3b and not C5 via its C-terminal domains (domains 3-5). (a) CFHR1 purified from serum or (b) recombinant CFHR1 domains 1 and 2 are immobilised on the sensor chip surface via primary amine coupling (CFHR1-2300 RU; CFHR112-750 RU) and C5 at concentrations between 50 and 400 nM is flowed across. No significant interaction is seen at any concentration. (c) 400 nM C3b is flown over surfaces with either serum-purified CFHR1, recombinant N-terminal CFHR112 or recombinant C-terminal CFHR1345 (CFHR1-2300 RU; CFHR112-750 RU; CFHR1345-1800 RU). C3b interacts only with the full-length or C-terminal fragments. (Flow rate 20 l/min all panels);

[0157] FIG. 13 shows that CFHR1 does not act as a complement regulator. Alternative pathway haemolysis assays were performed in a total volume of 200 l containing 20% serum and approximately 106 guinea pig erythrocytes in 100 mM HEPES, 150 mM NaCl, 8 mM EGTA, 5 mM MgCl2, 0.1% gelatin, pH 7.5. Haemolysis was measured by the absorbance at 405 nm after 60 minutes at 370 C and appropriate control subtraction. Haemolysis using NHS and fH deficient serum was measured in the presence and absence of 700 nM CFHR1. All measurements were taken in triplicate and the control (no CFHR1 added) is taken as 100%. A significant increase in haemolysis was observed in factor H sufficient serum upon addition of CFHR1 (p=0.037);

[0158] FIG. 14 shows the analysis of recombinant CFHR51212-9. (a) Multi-angle light scattering analysis of CFHR51212-9. Purified recombinant CFHR51212-9 elutes as multiple species from an analytical gel filtration column with masses that range from approximately 130-950 kDa indicative of the formation of higher-order assemblies than dimers. (b) Comparison of CFHR51212-9 versus CFHR5 wild-type binding to C3b coupled through the thioester. Dilutions of the proteins (from 0.8 M) were flowed across immobilised C3b (408 RU amine-coupled C3b and 500 RU coupled through the thioester) and binding monitored. CFHR51212-9 demonstrated a different binding kinetics compared to wild-type CFHR5;

[0159] FIG. 15 shows a summary of the identities and activities of homodimeric species formed between CFHR1, CFHR2 and CFHR5. (a) A summary of the activities of each homo-dimeric species formed by CFHR1, CFHR2 and CFHR5 in serum. (b) Summary of the heterodimeric species formed between CFHR1, CFHR2 and CFHR5 which will have the properties of both components; and

[0160] FIG. 16 shows deregulation of complement by CFHR1, CFHR2, CFHR3, CFHR4 and CFHR5 (all monomeric forms).

MATERIALS AND METHODS

[0161] Protein Expression and Purification

[0162] The gene encoding CFHR112 was amplified and inserted into the pKLAC2 vector using primers CFHR1.sub.1.sub._For [SEQ ID NO:14] and CFHR1.sub.2.sub._Rev [SEQ ID NO:15] prior to transformation into Kluyveromyces lactis and selection of successful integrants as per the manufacturers instructions (New England Biosciences).

[0163] Primers

TABLE-US-00017 CFHR1.sub.1_For [SEQIDNO:14] 5-gctgacaaggatgatctcgagaaaagagaagcaacattttgtgattt tcc-3 CFHR1.sub.2_Rev [SEQIDNO:15] 5-gccgcccatggacatctaagtggacctgcatttgg-3 CFHR1.sub.3_For [SEQIDNO:16] 5-gagatataccatgggcacttcctgtgtgaatccgcccacagtac-3 CFHR1.sub.5_Rev [SEQIDNO:17] 5-gccggatcctctatctttttgcacaagttggatactccagtttccc- 3 CFHR2.sub.3_For [SEQIDNO:18] 5-tataccatgggcgaaaaatgtgggccccctccacctattgacaatg g-3 CFHR2.sub.4_Rev [SEQIDNO:19] 5-cgtgccggatcctatttttcttcacaactgggatataccagtttcc c-3 CFHR5.sub.1_For [SEQIDNO:20] 5-caagttcctacaggggaagttttctcttactactgtgaagagaattt tgtgtctccttcaaaatcct-3 CFHR5.sub.2_Rev [SEQIDNO:21] 5-aggattttgaaggagacacaaaattctcttcacagtagtaagagaaa acttcccctgtaggaacttg-3

[0164] K. lactis expressing CFHR112 was grown in a minimal media and the secreted target protein purified from the culture supernatant using size exclusion chromatography (Column; S75 16/60 (GE Healthcare) followed by ion exchange chromatography (Column; Mono Q 5/50 (GE Healthcare). Buffer A; 25 mM Tris, 10 mM NaCl, pH 7.5. Buffer B; 25 mM Tris, 1M NaCl, pH 7.5).

[0165] CFHR1345 and CFHR234 were amplified and inserted into the pET-15b vector (Novagen) using primers CFHR1.sub.3.sub._For [SEQ ID NO:16], CFHR1.sub.5.sub._Rev [SEQ ID NO:17], CFHR2.sub.3.sub._For [SEQ ID NO:18] and CFHR2.sub.4.sub._Rev [SEQ ID NO:19]. Both proteins were expressed in Escherichia coli strain BL21(DE3) and refolded from inclusion bodies based on the protocol by White et al with the substitution of the published refold buffer for 1 mM Cysteine, 2 mM Cystine, 20 mM Ethanolamine, 1 mM EDTA, pH 11.0. Refolded proteins were further purified using size exclusion chromatography (Column; S75 16/60 (GE Healthcare). Buffer: 50 mM Tris, 150 mM NaCl, pH 7.5). Full-length CFHR5 cDNA was cloned into a modified version pCAGGS plasmid. CFHR5dimer mutant was generated by multi site-directed mutagenesis (Stratagene) according to manufacturer's instructions using primers CFHR5.sub.1.sub._For [SEQ ID NO:20] and CFHR52_Rev [SEQ ID NO:21]. Recombinant CFHR5 and CFHR5dimer mutant proteins were expressed in HEK293 cells. Recombinant proteins were purified by a single affinity chromatography step. Wild-type CFHR5 supernatant was applied onto a Hitrap NHSactivated HP (GE Healthcare) column coated with MBC125 mouse monoclonal anti-CFHR1/2/5 antibody. CFHR5dimer mutant supernatant was applied onto a Hitrap NHS-activated HP column coated with rabbit anti-human CFHR5 antibody (a gift from Dr. J. McRae). After extensive washes with PBS and 0.5M NaCl-containing buffers, bound protein was eluted with 50 mM diethylamine and fractions were neutralized with 1/10 volume of 1M Tris pH7.

[0166] EDTA-plasma derived CFHR1, CFHR2 and CFHR5 used for haemolytic assays were co-purified using the Hitrap NHS-activated HP column coated with MBC125 mouse monoclonal anti-CF HR1/2/5 2 antibody following the same method as described above for recombinant CFHR5. Identical EDTA plasma volume was used for the purification for each sample. Native CFHR1, CFHR2 and CFHR5 used for MALS were co-purified using the Hitrap NHS-activated HP column coated with MBC125 mouse monoclonal anti-CFHR1/2/5 antibody as above but omitting the NaCl wash step. Following elution from MBC125 affinity column, protein was dialysed against to mM sodium phosphate pH7.8 and loaded onto a Mono Q column (GE Healthcare) in the same buffer. Protein was eluted using a gradient to 300 mM NaCl over 25 column volumes (CVs) and the major peak (eluting at approximately 120 mM NaCl) was used for subsequent analysis using MALS.

[0167] Crystallisation and X-Ray Data Collection

[0168] Crystals were grown using the sitting drop vapour diffusion method from 0.2 L protein+0.2 L mother liquor drops at 210 C using protein stocks at A280=3.3 and 7.8 for CFHR112 and CFHR234 respectively. CFHR112 crystals grew from a mother liquor containing 36% PEG 2000 MME, 0.1M MES pH 6.5. CFHR234 crystals grew in 30% PEG 8000, 0.2M ammonium sulphate. Crystals were plunge cooled in liquid nitrogen following cryoprotection in 20% and 15% ethylene glycol for CFHR112 and CFHR234, respectively. Data were collected at both the ESRF and DIAMOND using the rotation method with oscillation ranges of 0.150 or 0.20 at 120 K. CFHR112 data were collected at beamline ID29 (ESRF, Grenoble) with =1.7105 . CFHR234 data were collected at beamline 104-1 (DIAMOND, UK) with =0.9173 . Data were integrated and scaled using XIA2 19 with the 3dii option to enforce usage of XDS 20 for integration and SCALA for scaling 21.

[0169] Structure Solution and Refinement

[0170] The structures of CFHR112 and CFHR234 were solved by molecular replacement using PHASER 22 with models derived from fH67 (PDB id: 2UWN) and fH19-20 (PDB id: 2G7I) respectively. Models were refined iteratively with manual rebuilding in COOT 23 and refinement using autoBUSTER 24. Data collection and refinement statistics are shown in Table 1. Ramachandran plots show that for CFHR112 93.4% of residues are in the favoured and 0.4% in the disallowed and for CFHR234 98.4% favoured, 0% disallowed.

[0171] C3b Binding Competition Assay

[0172] C3b at 25 g/ml in 0.1M NaHCO3 pH 9.5 buffer was immobilised in microtiter well plate (NUNC) overnight at 4 C. After blocking for 1 hour at room temperature with PBS containing 2% BSA, 0.073 M of CFH alone or in combination with serial dilutions of CFHR5, CFHR1, CFHR1345 and CFHR234 (starting at 0.584 M, 1.8 M, 18 M and 16 M, respectively) were incubated for 2 hours at room temperature. A monoclonal anti-CFH (OX24) antibody was used as a detection antibody. Optical density (OD) values at 450 nm were corrected and expressed as a percentage of CFH binding considering 100% those OD values where CFH was incubated in the absence of CFHR proteins.

[0173] Fluid-Phase CFI Cofactor Activity Assays

[0174] CFH or soluble complement receptor 1 (sCR1) CFI cofactor activity for the cleavage of either C3b or iC3b was done as previously described 25. CFHR5 cofactor activity was tested under the same conditions.

[0175] Detection of Heterodimers by Immunoprecipitation and ELISA Assays

[0176] Detection of heterodimers CFHR1-CFHR2 and CFHR1-CFHR5 were identified by immunoprecipitation. 50 l of serum from an individual with 2 copies of the CFHR3-1 genes or from an individual lacking these genes (CFHR3-1 homozygote) were diluted 1/10 in PBS and incubated with either a monoclonal anti-CFHR2 antibody (MBI-18) or with a monoclonal anti-CFHR5 (R&D Systems) antibody for 1 h at 4 C. In parallel, as a negative control for the immunoprecipitation, samples were not incubated with any antibody. Protein A/G sepharose beads previously washed with PBS were added and incubated overnight at 4 C. After extensive washes of the beads with PBS, bound proteins were eluted in protein loading buffer, separated using SDS-PAGE and analysed by western blotting using the anti-CFHR1/2/5 antibody (MBC125) followed by a HRP-conjugated rabbit anti-mouse IgG antibody (DAKO). Detection of heterodimer CFHR2-CFHR5 from serum was identified by enzyme-linked immunosorbent assay using rabbit anti-human CFHR5 (Abcam) and mouse anti-human CFHR2 (MBI-18) antibodies as capture and detection antibodies, respectively.

[0177] Administration of CFHR5 to Cfh/ Mice and Immunohistochemistry Studies

[0178] Cfh/ mice were injected intravenously with 30 g of either recombinant CFHR5 or CFHR5dimer mutant protein. Mice were sacrificed 2 hours post-injection and immunostaining performed on snap-frozen renal tissue performed as previously described 11. Mouse C3 was detected using a FITC-conjugated goat anti-mouse C3 antibody (MP Biomedicals, CA, USA). CFHR5 staining was performed using a polyclonal rabbit anti-human CFHR5 antibody (Abcam). Glomerular fluorescence intensity was calculated using image analysis software (Image-Pro Plus 7.0) and an Olympus U-TV1X-2 camera. We assessed 20 glomeruli from mice injected with identical concentration of either recombinant CFHR5 (n=2) or CFHR5dimer mutant protein (n=2). The median arbitrary fluorescence was significantly different between the two groups when calculated using either the total glomeruli counted in each group (n=40, p<0.05, unpaired t test) or when comparing per animal (n=2 per group, 20 glomeruli per animal, p<0.05, unpaired t test). The experiment was repeated with a separate batch of recombinant CFHR5 or CFHR5dimer mutant protein and glomerular binding of the CFHR5dimer mutant protein was again reduced.

[0179] Haemolytic Assays

[0180] Alternative pathway haemolysis assays were performed in a total volume of 200 l containing 20% serum and approximately 106 guinea pig erythrocytes in 100 mM HEPES, 150 mM NaCl, 8 mM EGTA, 5 mM MgCl2, 0.1% gelatin, pH 7.5. Haemolysis was measured by the absorbance at 405 nm after 60 minutes at 370 C and appropriate control subtraction. Dilution series of CFHR1, CFHR1345, CFHR234 and CFHR5, ranging from 1 nM to 9 M, were added to reactions that had been supplemented with 140 nM CFH. All measurements were recorded in triplicate and are presented as haemolysis relative to the level of lysis in the absence of any CFHR proteins (0%) and 100% lysis by H2O. The effect of the CFHR51212-9 mutation upon deregulation was assessed by comparison to the level of haemolysis by the wild type protein. CFHR1, CFHR2 and CFHR5 were co-purified from individuals with wild-type or mutant CFHR5 and haemolysis was measured using the same protocol described above with the addition of the CFHRs to reconstitute the serum levels in each individual. All measurements were performed in triplicate and are reported as percentages of maximum lysis by H2O. Haemolysis using normal human sera and CFH-deficient serum was measured in the presence and absence of 700 nM CFHR1 using the same protocol without the addition of CFH. All measurements were performed in triplicate and are reported as percentages of maximum lysis by H.sub.2O.

[0181] Heparin Binding

[0182] Approximately 0.5 mg CFHR1345 and CFHR234 in 50 mM Tris, 10 mM NaCl, pH 7.5 was loaded onto a 1 ml HiTrap Heparin column (GE Healthcare) using an AKTAfplc (GE Healthcare). Non-bound material was washed out with 5 CVs 50 mM Tris, 10 mM NaCl, pH 7.5 prior to a gradient elution of 50% 50 mM Tris, 1M NaCl, pH 7.5 over 15 CVs. The conductivity at which the peak elutes was recorded for each sample.

[0183] Multi Angle Laser Light Scattering

[0184] 100 g of sample was injected onto an S200 16/60 column (GE Healthcare. Buffer: 50 mM tris, 150 mM NaCl, pH 7.5) and the elution monitored using a Dawn Helios II (Wyatt Technology) and an Optilab TrEX (Wyatt Technology). All data and were analysed using ASTRA (Wyatt Technology).

[0185] Surface Plasmon Resonance

[0186] All data in FIGS. 9-11 were gathered using a Biacore T100 (GE Healthcare). A reference channel that was mock activated-deactivated was included on each chip. For kinetic studies, samples were injected using the KINJECT command, in HBS/P (10 mM HEPES pH7.4, 150 mM NaCl, 0.05% surfactant-P20) flowed at 30 l/min and analysed at 25 C. All kinetic data were double-referenced (data from reference cell and blank injection subtracted). The chip surface was regenerated between cycles using to mM sodium acetate pH 4.0, 1 M NaCl. C3b (Comptech, Tyler, USA) was primary amine-coupled (deposition levels=150-400 RU) to a CM5 chip following manufacturer's instructions (GE Healthcare). Where binding to clustered C3b was under investigation, further C3b was deposited by forming C3 convertase on amine-coupled C3b by flowing 100 g/ml FB and 1 g/ml factor D using the same buffer supplemented with 1 mM MgCl2, followed by C3 as substrate 26 resulting in 625 RU of nascent C3b covalently bound to the chip surface. To generate iC3b, the surface was treated with 3 successive cycles of CFH (15.5 g/ml) and factor I (10 g/ml) until C3 convertase could no longer be formed. To generate C3dg, the iC3b surface was treated with soluble CR1 (gift from T Cell Sciences, 3 cycles at 5 g/ml, 3 cycles at 50 g/ml) and factor I (10 g/ml). For kinetic analyses, CFH or CFHR5 were dialysed into HBS/P and each was flowed across the surface at a range of concentrations as indicated (1:2 serial dilution), with a regeneration step between each cycle. Data were analysed by steady state equilibrium analysis. Cofactor activity was assessed by flowing CFHR5 (0.18 M and 0.44 M over two 120 s cycles) with factor I (10 g/ml) across the surface for 2 mins at 10 l/min. As a positive control, CFH (0.1 M) was flowed with factor I for 120 s. The capacity of C3b on the surface to form a convertase was assessed before and after CFH/CFHR5/factor I injection by flowing CFB and factor D, decrease in convertase formation indicated cleavage of C3b to iC3b. All data in FIG. 12 were collected on a Biacore 3000 instrument (GE Healthcare) using CM5 chips to which proteins were immobilised via standard primary amine coupling protocols. A reference channel that was mock activated-deactivated was included on each chip. HBS-EP buffer was used throughout. 2300 RU CFHR1, 750 RU CFHR112 and 1800 RU CFHR1345 were immobilized on a chip. 50 l of 400 nM C3b (Calbiochem) was flowed over the surface at 20 l/min using the KINJECT command with a dissociation time of 400 seconds. A dilution series of C5 (Calbiochem) between 50 nM and 400 nM was injected in an identical manner. All curves were reference subtracted and analysed using BIAEVALUATION (GE Healthcare).

EXAMPLES

[0187] The complement system is a key component of the early, innate, immune system. Genetic variation in complement regulation influences susceptibility to age-related macular degeneration (AMD), meningitis and kidney disease. Variation includes genomic rearrangements within the complement factor H-related (CFHR) locus. Unfortunately, up until now, elucidating the mechanism underlying these associations has been hindered by the lack of understanding of the biological role of CFHR proteins. In the following examples, however, the inventors present unique structural data demonstrating that at least three of the CFHR proteins (CFHR1, 2 and 5) contain a shared dimerisation motif and that this hitherto unrecognised structural property enables formation of both homodimers and heterodimers. The examples also show that dimerisation confers avidity for tissue-bound complement fragments and enables these proteins to efficiently compete with the physiological complement inhibitor, complement factor H (CFH), for ligand binding. The data go on to demonstrate that these CFHR proteins function as competitive antagonists of CFH to modulate complement activation in vivo and explain why variation in the CFHRs predisposes to disease.

Example 1CFHR1, CFHR2 and CFHR5, Contain a Novel Dimerization Motif

[0188] Comparing the amino acid conservation between CFHR1, CFHR2 and CFHR5 and CFH demonstrated that the CFHR proteins do not possess the residues implicated in the complement regulatory activity of CFH (cyan, FIG. 1a) but that these CFHRs shared a unique pair of highly conserved N-terminal domains (>85% sequence identity, FIG. 1a). The inventors therefore determined the crystal structure of the first two SCR domains of CFHR1 (CFHR1.sub.12), which revealed that these domains assemble as a tight head-to-tail dimer with residues Tyr34, Ser36 and Tyr39 playing key roles in stabilising the assembly (FIG. 1b-d, Table 1).

TABLE-US-00018 TABLE 1 Data collection and refinement statistics CFHR1.sub.32 CFHR2.sub.34 Data collection Space group P2.sub.12.sub.12.sub.1 P2 Cell dimensions a, b, c () 45.3, 46.9, 111.7 53.0, 25.2, 95.7 , , () 90.0, 90.0, 90.0 90.0, 93.8, 90.0 Resolution () 55.8-2.0 (2.1-2.0) 95.5-2.0 (2.1-2.0) R.sub.merge 0.09 (0.54) 0.05 (0.26) I/I 11.2 (2.9) 15.2 (4.0) Completeness (%) 96.6 (90.6) 96.7 (85.8) Redundancy 6.2 (6.4) 3.2 (2.6) Refinement Resolution () 1.99-55.83 (1.99-2.13) 2.00-19.09 (2.00-2.12) No. Reflection 16261 (2724) 16963 (2567) R.sub.workf/R.sub.free 0.22/0.25 (0.22/0.26) 0.21/0.24 (0.21/0.27) No. atoms Protein 1973 1952 Ligand/ion 166 117 Water 102 77 B-factors (.sup.2) Protein 52 27 Ligand/ion 53 40 Water 50 25 R.m.s deviations Bond lengths () 0.008 0.010 Bond angle () 0.98 1.10 *Highest resolution shall is shown in parenthesis.

[0189] The recombinant CFHR1.sub.12 fragment was also homogenously dimeric in solution (FIG. 2a) and the only conditions under which the chains can be separated is by reducing SDS-PAGE (FIG. 2a). Surprisingly, the dimer interface is highly conserved amongst CFHR1, CFHR2 and CFHR5 (FIGS. 1c and d). This conservation, together with the structural data, shows that CFHR1, CFHR2 and CFHR5 can assemble as hetero- as well as homo-dimers. The inventors next looked for the presence of these species in vivo.

Example 2Plasma CFHR1, CFHR2 and CFHR5 Exist as Dimeric Species In Vivo

[0190] The inventors purified CFHR1, CFHR2 and CFHR5 from serum using a monoclonal antibody (MBC125; anti-CFHR1/2/5) that recognizes a shared epitope within the first two SCR domains of these proteins. When this purified preparation was analysed in solution by multi-angle laser light scattering (FIG. 2a) the observed mass range was 65-80 kDa. The lowest observed mass exceeded the predicted molecular mass of the smallest protein (CFHR2, predicted Mr=30 kDa), whilst the largest observed mass exceeded that of the largest protein (CFHR5, predicted Mr=64 kDa). This demonstrated that CFHR2 is not monomeric in vivo and was consistent with CFHR1, CFHR2 and CFHR5 dimerisation.

[0191] To look for heterodimers in vivo the inventors performed serum immunoprecipitation using either a specific anti-CFHR2 (MBC22; FIG. 2b) or anti-CFHR5 (FIG. 2c) antibody. In both assays, sera from individuals with and without the CFHR3-1 deletion polymorphism were used and probed with the anti-CFHR1/2/5 antibody. This revealed the presence of CFHR1-CFHR2 (FIG. 2b) and CFHR1-CFHR5 (FIG. 2c) heterodimers in serum. The specificity of these assays was supported by the lack of these heterodimers in sera from individuals with the CFHR3-1 deletion polymorphism. Detection of CFHR2-5 heterodimers using these assays was not possible because of the presence of non-specific bands in the region of CFHR5 (FIG. 2b) and CFHR2 (FIG. 2c). The inventors therefore designed an ELISA assay using anti-CFHR5 as a capture antibody and anti-CFHR2 as a detection antibody (FIG. 2d). This showed a strong signal using sera from two individuals homozygous for the CFHR3-1 deletion whilst a weak or absent signal resulted when sera from individuals without this polymorphism was used. This demonstrated that the relative abundance of CFHR1, CFHR2 and CFHR5 influences the pattern of dimers present in vivo.

Example 3Dimerisation Enhances the Interaction of CFHR5 with Renal-Bound Mouse Complement C: In Vivo

[0192] The inventors next explored the functional consequences of dimerisation. They predicted that dimerisation would enhance ligand interaction through avidity. To test this they generated monomeric and dimeric CFHR5 proteins. Monomeric CFHR5 (CFHR5.sup.dimer mutant) was generated in vitro by mutating the three key amino acids within the dimerisation motif to the corresponding amino acids within CFH (Tyr34Ser, Ser36Tyr, Tyr39Glu, FIGS. 3a and b). CFHR5.sup.dimer mutant was demonstrated to be monomeric using MALS (FIG. 3c). Next they examined the interaction of monomeric and dimeric CFHR5 with tissue-bound complement in a mouse model. Gene-targeted CFH-deficient mice have florid deposition of activated mouse C3 along the glomerular basement membrane (GBM) within the kidney. Human CFHR5 was able to interact with the GBM-bound C3 in a specific and dose-dependent manner (FIG. 8). Using this model the interaction of intravenously administered monomeric CFHR5 with GBM-bound mouse C3 was significantly reduced compared to that of the dimeric protein (median glomerular staining=227 and 95 arbitrary fluorescence units, for wild-type and dimer mutant respectively, P<0.05, unpaired t test, FIG. 3d). This indicated that dimerisation of CFHR5 enhanced its ability to interact with mouse C3 in vivo.

Example 4Dimerisation Enhances the Ability of CFHR1 and CFHR5 to Compete with CFH for C2b Binding In Vitro

[0193] The inventors next speculated that dimerisation of CFHR1, CFHR2 and CFHR5 would enable these proteins to efficiently compete with CFH for interaction with C3 in vivo. Since CFH, CFHR1 and CFHR5 contain the same carboxyl-terminal C3b/C3d binding site (FIG. 1a, FIG. 6), the inventors developed an ELISA assay to determine if CFHR1 and CFHR5 influence the interaction of CFH with C3b. This demonstrated that the CFH-C3b interaction was inhibited in a dose dependent manner at physiologically relevant concentrations by native dimers of CFHR5 (dose range 0.005 to 0.6 M) and CFHR1 (dose range 0.014 to 1.8 M) (FIG. 4a). Monomeric constructs of CFHR1 and CFHR2 that lack the dimerization domains (denoted CFHR1.sub.345 and CFHR2.sub.34, respectively) could also inhibit CFH binding but at higher concentrations (FIG. 4a).

Example 5CFHR1 and CFHR De-Regulate Complement Activation by Acting as Competitive Antagonists of CFH

[0194] To determine the physiological relevance of the competition between CFHR1/CFHR5 and CFH for C3b binding the inventors have studied the ability of CFHR1 and CFHR5 to regulate C3. Using surface plasmon resonance (SPR), in which the sensor surface was coated with either amine or thioester coupled C3b (monomeric or clustered C3b respectively; FIG. 9), or thioester-coupled iC3b and C3dg (FIG. 10), CFHR5 bound to C3b, iC3b and C3dg but there was no evidence of fluid-phase factor I cofactor activity (FIG. 11). CFHR1 has previously been reported to inhibit the C5 not C3 convertase by binding to C5/C5b6 but the inventors were unable to detect any significant interaction with C5 (FIG. 12). Moreover, they were unable to detect any evidence of complement regulatory activity when CFHR1 was investigated in alternative pathway haemolysis assays (FIG. 13). These data indicated that CFHR1 and CFHR5 have no intrinsic C3 or C5 regulatory activity at physiological concentrations. They therefore hypothesized that these proteins, through their ability to compete with CFH for binding to C3b, actually prevent CFH-mediated complement regulation.

[0195] To test this, the inventors utilized a complement-dependent haemolytic assay comprising unopsonised guinea-pig erythrocytes (a complement activating surface) incubated with 20% normal human sera. The addition of 100 nM CFH resulted in 50% inhibition of cell lysis and therefore enabled us to determine if exogenous CFHR proteins increased or decreased haemolysis. Using these conditions, in which the total CFH concentration in the assay was approximately 0.5 M (100 nM added to assay in addition to 20% normal human sera), they added increasing concentrations of concentrations of CFHR1.sub.345, CFHR2.sub.34, serum-derived CFHR1 and recombinant CFHR5 (FIG. 4b). Surprisingly, these preparations increased rather than decreased haemolysis in a dose-dependent fashion. Importantly, the IC50 was significantly lower for the dimeric CFHR1 (0.7 M) and CFHR5 (0.15 M) compared to the monomeric CFHR1 (3.6 M) and CFHR2 (4.7 M) fragments. These data demonstrated that CFHR1 and CFHR5 can interfere with the C3b inhibitory actions of CFH by acting as competitive antagonists and that this interference is enhanced by dimerisation. The inventors refer to this process as complement de-regulation because it emphasizes the point that these proteins have no ability to influence complement regulation in the absence of CFH.

Example 6De-Regulation by CFHR Mutation Associated with Familial C3 Glomerulopathy

[0196] In patients with familial complement-mediated kidney disease, termed C3 glomerulopathy, there is a heterozygous CFHR5 mutation in which the initial two N-terminal domains are duplicated. The data presented here reveal that this results in duplication of the dimerisation motif (denoted CFHR5.sub.1212-9). When they generated recombinant CFHR5.sub.1212-9 it was clear that the purified preparation readily aggregated and was associated with atypical C3 binding kinetics using SPR (FIG. 14). When they elucidated the dimerisation domain, they re-interpreted this aggregation as a direct consequence of duplicated dimerisation domains (enabling multimeric interaction) rather than an in vitro artefact. A further consequence of the structural data was that examination of the isolated recombinant CFHR5.sub.1212-9 was irrelevant pathophysiologically since it was likely that CFHR5.sub.1212-9 interacted with CFHR1, CFHR2 and the wild-type CFHR5 (derived from the unaffected allele) in vivo. Consequently, they tested whether de-regulation is influenced in these patients by comparing plasma preparations containing all CFHR1, CFHR2 and CFHR5 species from affected individuals and healthy controls without the CFHR3-1 deletion polymorphism (FIG. 4c). This showed that patient preparations resulted in significantly greater haemolysis than that of controls.

DISCUSSION

[0197] The data presented herein provide compelling evidence that CFHR1, CFHR2 and CFHR5 at physiologically relevant concentrations interfere with the complement inhibitory activities of CFH. This process, which the inventors term de-regulation, is influenced by the ability of these proteins to form dimers (FIG. 5). This structural property confers avidity enabling these dimeric molecules to compete with CFH for ligand due to the fact that the C-terminal C3b/C3d recognition sites are essentially conserved between the CFHR proteins and CFH. The shared dimerisation domain between CFHR1, CFHR2 and CFHR5 enabled the formation of both homo- and heterodimers. The dimerisation motif that has been characterized is not present within CFHR3 and CFHR4 but it has been suggested that CFHR4, at least (and possibly also CFHR3), may also exist as a dimer. Accordingly, CFHR3 and CFHR4 are also believed to form dimers and behave as competitive antagonists of CFH.

[0198] The inventors were able to demonstrate heterodimers within CFHR1, CFHR2 and CFHR5 and the specificity of these interactions was evident when comparing sera from individuals with and without CFHR1. A priori the inventors predicted that homo and heterodimers containing CFHR1 would predominate in sera from individuals without the CFHR3-1 deletion polymorphism since this protein is most abundant with a mean serum concentration equimolar to that of CFH (CFH=116-562 g/ml, 0.7-3.6 M, mean 2.1 M (13), CFHR1=70-100 g/ml, 1.7-2.5 M, mean 2.1 M (11)). In contrast the median concentration of CFHR5 (3-6 g/ml, 0.05-0.09 M, mean 0.07 M (14)) is much lower. The inventors are not aware of published estimates for the circulating concentration of CFHR2 but the data suggest its concentration is intermediate between CFHR1 and CFHR5 (Coomassie gel inset, FIG. 2a). Consistent with the predominance of CFHR1-containing dimers, CFHR2-CFHR5 heterodimers were only readily detectable in sera from patients deficient in CFHR1 (those with the CFHR3-1 deletion polymorphism).

[0199] The inventors were unable to demonstrate C3 regulatory activity for CFHR5 and were unable to demonstrate an interaction between CFHR1 and C5. Interestingly, although CFHR3 has previously been reported as a regulator of complement (in non-physiological conditions), other experiments reported in the same paper demonstrate that, as shown here for CFHR1, CFHR2 and CFHR5, CFHR3 can also de-regulate CFH. Recently, CFHR4 was shown to be devoid of intrinsic complement activity but able to act as a platform on which complement activation could proceed unhindered. Therefore, if CFHR4 was able to compete for CFH ligands then it too has the potential to de-regulate CFH activity. Taken together, the data suggest that the CFHR1, CFHR2 and CFHR5 modulate complement activation by competing with CFH for C3b binding. In contrast to CFH-C3b interaction which prevents further C3b generation (negative regulation), the interaction of these CFHR proteins with C3b enables C3b amplification to proceed unhindered. The ability of CFHR proteins to de-regulate CFH would be predicted to be influenced by many factors including (1) the concentration and composition of the CFHR proteins relative to CFH in the vicinity of complement activation, (2) the spatial density of deposited C3 (for example, they speculate that the action of large dimers such as CFHR5-CFHR5 may be important when spatial density is low), (3) the polyanion composition of the surface upon which complement is activated since the polyanion affinities of the different CFHR proteins may vary and (4) the flow rate across the site of complement activation in surfaces in contact with blood (the enhanced avidity of dimeric species would favour their interaction with ligand relative to CFH under high flow) such as within the kidney.

[0200] The data had obvious implications for how one considers the impact of the C3 glomerulopathy-associated CFHR5 mutation in which there is duplication of the dimerisation domain (duplication of SCR1 and SCR2, CFHR5.sub.1212-9)(8). Theoretically, this duplication would result in trimeric or higher order complexes. However, since CFHR1 is abundant in vivo, the inventors speculate that the most common species would be trimeric and composed of two molecules of CFHR1 complexed with CFHR5.sub.1212-9. When they purified CFHR1, CFHR2, CFHR5 and CFHR5.sub.1212-9 from an affected individual, this serum fraction was more potent in de-regulation than serum fractions from healthy controls. If it is assumed that CFH plays a physiological role in protecting the GBM from C3 activation, the data would suggest that C3 glomerulopathy develops in individuals since the presence of CFHR5.sub.1212-9 results in a greater degree of CFHR-mediated de-regulation.

[0201] CFH serum levels are not actively regulated in an individual, varying only under extreme conditions such as meningococcal sepsis where tight interactions with the bacterium deplete CFH. The inventors believe that fine-tuning of complement activation (complement modulation) can be achieved by altering CFHR levels. It is notable that in otitis media with effusion, where complement is strongly activated in the middle ear effusion fluid, CFHR5 levels were noted to be high and it was proposed that competition between CFHR5 and CFH might be relevant in this circumstance. This requires further study but the data presented here would predict that a local increase in CFHR protein concentration would, through enhanced CFH de-regulation, enable rapid enhancement of complement activation. The opposite might be achieved by down-regulating CFHR concentrations thereby reducing de-regulation.

[0202] In summary, the inventors clearly show that these proteins can bind bivalently to adjacent molecules of C3b (or iC3b/C3dg/C3d) deposited on the membrane, and that these dimers are not artifacts of expression in P. pastoris, but occur in the plasma. In addition, the inventor have demonstrated, using surface Plasmon resonance (SPR), that CFHR5 (that has several modules between its dimerisation site and its C3b-binding site) binds surprisingly well to clustered C3b molecules, but not so well to spaced-apart C3b molecules, and this may suggest that CFHR1-5 are sensitive to the distribution of C3b molecules, and can therefore modulate the regulatory activity of CFH accordingly. These observations have revealed an exciting and novel function of the CFHR proteins. The inventors propose that these molecules have evolved to enable complement to be modulated at a sophisticated level under diverse circumstances. Understanding how these proteins modulate activation during infection, tissue injury and inflammation will enable us not only to gain further understanding of the role of complement in disease but also to devise novel strategies to increase or decrease complement activation therapeutically.

Example 7CFHR1, CFHR2, CFHR3, CFHR4 and CFHR De-Regulate Complement Activation by Acting as Competitive Antagonists of CFH

[0203] In Example 5, the inventors have already shown that CFHR1 and CFHR5, through their ability to compete with CFH for binding to C3b, prevent CFH-mediated complement regulation. The inventors then set out to test CFHR3 and CFHR4, using a complement-dependent haemolytic assay comprising unopsonised guinea-pig erythrocytes (a complement activating surface) incubated with 20% normal human sera (Goicoechea de Jorge et al., Dimerization of complement factor H-related proteins modulates complement activation in vivo. Proc Natl Acad Sci USA. 2013 Mar. 19; 110 (12):4685-90). The addition of 100 nM CFH resulted in 50% inhibition of cell lysis and therefore enabled them to determine if exogenous CFHR proteins increased or decreased haemolysis. Using these conditions, in which the total CFH concentration in the assay was approximately 0.5 M (100 nM added to assay in addition to 20% normal human sera), they added increasing concentrations of concentrations of CFHR1, CFHR2, CFHR3, CFHR4 and CFHR5. The results are shown in FIG. 16.

[0204] Surprisingly, these preparations increased rather than decreased haemolysis in a dose-dependent fashion. Importantly, the IC50 are within the physiological range of these proteins. Accordingly, these data show that CFHR3 and CFHR4 de-regulate, and so validates the hypothesis that deregulation applies to all five of the CFHR proteins.

REFERENCES

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