POLYPEPTIDES FOR THE DIAGNOSIS AND THE TREATMENT OF C3 NEF ASSOCIATED C3 GLOMERULOPATHY
20180162911 · 2018-06-14
Inventors
Cpc classification
C12Y304/21047
CHEMISTRY; METALLURGY
G01N33/564
PHYSICS
C07K14/00
CHEMISTRY; METALLURGY
International classification
C07K14/00
CHEMISTRY; METALLURGY
Abstract
The present invention relates to polypeptides for the diagnosis and treatment of C3 NeF associated C3 Glomerulopathy. In particular, the present invention is defined by the claims. In particular, the present invention relates to a polypeptide that is capable of inhibiting the binding of C3 NeF to C3 convertase and which comprises a first segment which consists of n consecutive amino acids selected in a first amino acid sequence set forth in SEQ ID NO:1 fused to a second segment which consists of n consecutive amino acids selected in a second amino acid sequence set forth in SEQ ID NO:2, wherein n and n represent integer number, n and n3 and n+n10.
Claims
1. A polypeptide that is capable of inhibiting the binding of C3 NeF to C3 convertase and which comprises a first segment which consists of n consecutive amino acids selected in a first amino acid sequence set forth in SEQ ID NO:1 (GQDEENQKQCQDLGAFTESMVVF) fused to a second segment which consists of n consecutive amino acids selected in a second amino acid sequence set forth in SEQ ID NO:2 (LKHDEYNIENLQKTVWD), wherein n and n represent an integer number, n and n3 and n+n10.
2. The polypeptide of claim 1 which comprises at least 10, 11, 12, 13, 14, 15, 16, 17, 18; 19; 20; 21; 22; 23; 24; 25; 26; 27; 28; 29; 30; 31; 32; 33; 34; 35; 36; 37; 38; 39; or 40 amino acids.
3. The polypeptide of claim 1 which comprises less than 15 amino acids.
4. The polypeptide of claim 1 wherein the first segment consists of 3; 4; 5; 6; 7; 8; 9; 10; 11; 12; 13; 14; 15; 16; 17; 18; 19; 20; 21; 22; or 23 consecutive amino acids selected in the first amino acid sequence set forth in SEQ ID NO:1.
5. The polypeptide of claim 1 wherein the second segment consists of 3; 4; 5; 6; 7; 8; 9; 10; 11; 12; 13; 14; 15; 16; or 17 consecutive amino acids selected in the first amino acid sequence set forth in SEQ ID NO:2.
6. The polypeptide of claim 1 wherein the first and second segments are fused directly or via a spacer.
7. The polypeptide of claim 1 which consists of an amino acid sequence set forth in SEQ ID NO:3, 4, 5, 6, 7, 10 or 11.
8. A nucleic acid molecule encoding for the polypeptide of claim 1.
9. A vector which comprises the nucleic acid molecule of claim 8.
10. A host cell genetically transformed with the nucleic acid molecule claim 8.
11. A method of treating C3 NeF associated C3 Glomerulopathy or Barraquer-Simons syndrome in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the polypeptide of claim 1 or a nucleic acid molecule encoding the polypeptide.
12. A pharmaceutical composition comprising the polypeptide of claim 1 or a nucleic acid molecule encoding the polypeptide and a pharmaceutically acceptable carrier.
13. A method for detecting the presence of C3 Nef in a sample comprising contacting the sample with the polypeptide of claim 1 under conditions that allow an immunocomplex of the polypeptide and an antibody to form, and detecting the presence of the immunocomplex.
14. The method of claim 13 wherein the sample is a blood sample.
15. The method of claim 13 wherein an assay for the detection of immunocomplexes utilizes a solid phase or substrate to which the polypeptide that is capable of inhibiting the binding of C3 NeF to C3 convertase and which comprises a first segment which consists of n consecutive amino acids selected in a first amino acid sequence set forth in SEQ ID NO:1 (GQDEENQKQCQDLGAFTESMVVF) fused to a second segment which consists of n consecutive amino acids selected in a second amino acid sequence set forth in SEQ ID NO:2 (LKHDEYNIENLQKTVWD), wherein n and n represent an integer number, n and n3 and n+n10, is directly or indirectly attached.
16. A kit for detecting C3Nef in a sample which comprises the polypeptide of claim 1 and means for determining binding of the polypeptide to C3Nef in a sample.
Description
FIGURES
[0036]
[0037]
[0038]
[0039]
EXAMPLE 1
[0040] Methods
[0041] Patients
[0042] Between 2001 and 2013, 149 patients were included in the French C3 glomerulopathy registry and EDTA blood samples were sent to the laboratory of Immunology (Hpital Europen Georges Pompidou, Paris, France) for complement assessment. The inclusion criteria were adapted to the recent classification of C3 glomerulopathy. All kidneys biopsy reports were reviewed and patients were selected according to the identification of the immune reactant by immunofluorescence study. Patients were included if they demonstrated the presence of glomerular staining of C3 with at least two order of magnitude of intensity for others immune reactants by immunofluorescence. In France, the electron microscopy is not performed routinely for GN characterisation and thus EM analysis of biopsy was missing in 90% of cases. Therefore the electron-dense appearance of deposits in glomeruli, which correspond to the C3 detected by IF was unavailable. Using light microscopy morphological features and Immunofluorescence of a biopsy sample pathologists recognized 44 cases of Dense Deposits diseases and 77 cases of C3 glomerulonephritis with predominant C3 deposits.
[0043] We excluded for this study patients with monoclonal gammopathy detected by immunofixation (n=28 patients) and patients with identified mutations in Factor H (n=12), Factor I (n=3), MCP (n=1), C3 (n=5) and CFHR 5 (n=1). Two out the 20 patients carried combined mutations in C3 and Factor I. None of the patients carry a mutation in Factor B gene. We also excluded 12 patients in whom complement assessment had been performed during the time of transplantation to avoid the influence of immunosuppressive drug on the antibody titres. Plasma samples were available for 89 patients for C3NeF screening and for assessment of complement biomarkers. For 36/89 samples insufficient volume of plasma or IgG available prevented us from performing of haemolytic assay with Properdin (data not shown). None of the patients presented anti-Properdin and anti-FB antibodies.
[0044] Genetic Analysis and Complement Plasma Proteins Levels
[0045] The DNA was extracted from whole blood using proteinase K/phenol method (Dragon-Durey et al. JASN 2003) for direct sequencing of all CFH, CFI, MCP, C3, FB and CFHR5 exons. EDTA plasma samples were used for measurement of C3, FH and FI concentrations as previously described (Roumenina et al. J Immunol Methods 2011). Soluble C5b9 level determination was done using the MicroVue sC5b-9 Plus EIA Assay (Quidel, San Diego, Calif.), according to manufacturer instructions. Normal values were evaluated testing plasma from 50 healthy donors.
[0046] Assays for C3NeF Detection.
[0047] The ability of patient' IgG to stabilize a pre-formed membrane bound C3bBb was measured with EAC3bBb generated by the incubation of EC3b with appropriate concentration of FB and FD. Briefly, IgG was partially purified from plasma by chromatography on DEAE-cellulose and subsequently quantified by nephelometry. DEAE eluate contains IgG, and also low levels of contaminants such as Properdin and Factor H. Sheep erythrocytes EAC34b were incubated in presence of FB (Complement Technology) and FD (Sigma) to form the alternative convertase C3bBb. The convertase was allowed to decay in EDTA-containing buffer in presence of 400 g of patient' IgG. Lysis was developed by the addition of rat serum. C3NeF stabilization was measured as the percentage of the residual convertase compared to the reference control without decay. The cut-off for positive C3NeF was determined as the mean+2 sd of the C3NeF stabilization of IgG from 30 healthy individuals.
[0048] The ability of patient' IgG to stabilize a pre-formed membrane bound C3bBbP was measured with EAC3bBb generated by the incubation of EAC3b with appropriate concentrations of FB FD and Properdin (Calbiochem). This assay is similar to the previous one, but the C3bBbP convertase was let decay for 30 minutes at 30 C. The cut-off for positive C3NeF was determined as the mean+2 sd of the C3NeF stabilization of IgG from 30 healthy individuals.
[0049] Assessment of C3NeF Binding to the Convertase by Surface Plasmon Resonance (SPR)
[0050] C3NeF binding to the convertase was studied by surface plasmon resonance (SPR) with ProteOn XPR36 equipment (BioRad). C3b was coupled to the GLC biosensor chip and C3 convertases C3bBb and C3bBbP were formed by flowing on chip respectively FB (3 g/ml) and FD (0.3 g/ml) or FB (1 g/ml), FD (0.1 /ml) and FP (0.5 g/ml) in a Mg.sup.2+ containing buffer (10 mM Hepes pH 7.4, 40 mM NaCl, 10 mM MgCl.sub.2, 0.005% surfactant P20). After convertase formation, 100 ng of purified C3NeF positive IgGs or control IgG from healthy donors were injected over the convertase containing flow cell and one flow cell in which buffer was flowed instead of FB, FD and/or FP, used as control for background bindings to coated C3b. The association of IgG to the convertase and the dissociation of the complex were followed in real-time for 300 seconds and 600 seconds respectively. Data were analyzed using ProteOn Manager Software and the data from the control flow cell were subtracted.
[0051] Competition Assay
[0052] Membrane-bound convertase was formed as in the C3bBb assay. During the convertase decay step, cells were incubated with FP alone at a fixed dose (800 ng); 4 different doses of IgG alone and a mix of both IgG and FP at the same concentration. The maximal concentration of IgG applied in the test was chosen depending on their C3NeF activity. Similarly, the competition was made with a fixed amount of IgG and different doses of FP.
[0053] Peptides Design
[0054] 3 peptides, reproducing conformational neo-epitopes on the C3 convertase C3bBb and containing sequences from the C345C domain of C3 and the vWF type A domain of FB were designed by mapping the surface areas, spanning the putative Properdin binding site. The Properdin binding area was deduced from the previously published electron microscopy images (Alcorlo et al., PNAS, 2013). Different peptides were selected manually by visualization of the surface area of the C3bBb complex with SCIN (PDB ID 2WIN), after removal of the sequence corresponding to SCIN. Peptide 1 covered 22 residues of C3b (1616-1637) and 11 residues of Bb (308-318) (G-QDEENQKQCQDLGAFTESMVVFNIENLQKTVWD) (SEQ ID NO:5). Peptide 2 consisted of 3 parts of C3b (1609-1614; 1544-1547; 1635-1636) and of one part of the vWFA domain of Bb (292-297) (G-EDEEPWEQITVV YPKIWV) (SEQ ID NO: 8). Peptide 3 included 9 adjacent residues (1508-1516) and 1 detached residue (1641) of C3b and 10 adjacent residues (254-263) and 1 detached residue (368) of Bb (G-EERLDKACENNDSIGASNFTG) (SEQ ID NO: 9). A glycine was added at the beginning of each peptide to avoid the presence of problematic aminoacids at the N-terminus. Peptides were synthesized by ThermoFisher Scientific with a purity of >90% and re-suspended in PBS/DMSO 5% in order to obtain a final concentration of 1 mg/ml.
[0055] Peptides Inhibition Assay
[0056] Different mixes of 50 ng of Properdin and 10 g of peptides were incubated at room temperature for 2 hours with gentle shaking in EDTA-containing buffer. As a control, Properdin was incubated with the peptide's dilution buffer. Membrane-bound convertase was formed as in the C3bBb assay. During the convertase decay step, cells were incubated with the mix of FP and peptides. FP amount was chosen in order to obtain 50% of residual convertase after the decay. Residual convertase was measured and data were expressed as percentage of inhibition (percentage of residual convertase compared to the control without peptide).
[0057] The test was performed as described earlier, but 80 g of peptides was utilized and the incubation of IgG and peptides was made at 4 C. overnight. IgG amount was determined depending on their stabilization capacity, in order to obtain 50% of residual convertase after the decay.
[0058] Results
[0059] Identification of Patients with C3NeF Using the C3b-Coated Erythrocytes, Based on Alternative Pathway C3 Convertase Stabilization Assays
[0060] To evaluate the ability of C3Nef to stabilize the C3 convertase, we used a fixed concentration of 400 g patient or control IgG and determined the residual number of lytic sites per cell (Z) after 20 minutes of incubation with EAC3bBb cells with the IgG preparations. The results were calculated and expressed as percent of lytic sites per cell (Z) corresponding to the % of C3bBb stabilization. In presence of the same amount of IgG prepared from healthy donors the residual convertase at 20 minutes was less than 20% of convertase generated at the initial convertase. Of the 89 patients with C3G, 69 (78%) were positive for C3NeF in this assay. The percentage of residual lytic sites varies from 20 to 100% of the initial convertase. The % of stabilization was between 20% to 40%, 40% to 80% and 80% to 100% for 44% (n=30), 36% (n=25) and 20% (n=14) respectively (data not shown). The stabilization of the C3 convertase is IgG dose dependant and 50% of stabilization is observed with concentration of 1 g to 400 g of total IgG in the assay depending on the patient tested (data not shown).
[0061] Identification of Patients with C3NeF Using C3b-Coated Erythrocytes Based Assay of Properdin-Stabilized Alternative Pathway C3 Convertase
[0062] To evaluate the ability of C3NeF to bind to and stabilize the C3 convertase in presence of Properdin we developed a new haemolytic test. We used a fixed amount of patient or control IgG (400 g) to determine the residual lytic sites after 30 minutes of incubation of the IgG preparations with EAC3bBbP cells. Less than 20% of lytic sites per cell was observed at 30 minutes with the same amount of IgG prepared from healthy donors (n=30).
[0063] Of the 55 tested patients, 28 (51%) were positive for C3NeF in this assay. The percentage of residual lytic sites here varies from 20 to 100% of the initial convertase. The % of stabilization was between 20% and 40%, 40% to 80% and 80% to 100% for 22% (n=6), 39% (n=11) and 39% (n=11) respectively (data not shown). The capacity of C3NeF to stabilize the C3bBbP is IgG dose dependant.
[0064] Of the 55 tested samples, 44 (80%) were positive in C3bBb and/or C3bBbP stabilization assay (data not shown). Patients' IgG stabilized both the C3bBb and the C3bBbP convertases in 50% of positive C3NeF samples (n=22). C3bBb stabilization convertase assays detected C3NeF which were negative in C3bBbP stabilization convertase assays (36% of positive C3NeF, n=16). C3bBbP stabilization convertase assays detected C3NeF which were negative in C3bBb stabilization convertase assays (14% of positive C3NeF, n=6).
[0065] Analysis of C3NeF Binding to the Convertase by Surface Plasmon Resonance
[0066] C3NeF positive IgG were tested for their capacity to bind to the convertases C3bBb and C3bBbP in real time by SPR. Tested IgG were representative of the range of characteristics observed in C3NeF functional assay (2 patients' IgG positive in the C3bBb based C3NeF haemolytic assay (P1, P2) and 3 patients' IgG positive in both of the assays (P3, P4, P5). IgG from three healthy donors were used as controls.
[0067] C3b was immobilized on chip and convertase was formed by flowing FD and FB in one case and FD, FB and Properdin in another case. After convertases formation, IgG were flowed on the chip and their binding to the complex was followed. Binding signal was increased when P1 and P2 IgG were flowed in the presence of C3bBb but no binding signal was observed in the presence of C3bBbP convertase (data not shown). Binding signal was increased when P3, P4 and P5 IgG were flowed on both the C3bBb and on the C3bBbP convertases (data not shown).
[0068] Biomarkers of Complement Activation in C3G Patients According to the C3Nef Status
[0069] To evaluate the influence of the presence of different types of C3Nef on the level of complement activation in patients, we measured C3 and sC5b9 plasmatic levels. C3 levels were significantly lower in patients with C3NeF compared to patients without. The C3 levels were below the normal range (<660 mg/ml) for 52% of patients with C3Nef against the C3bBb (including 11 (16%) patients with C3 level below 200 mg/ml), and for 25% of patients with no detectable C3NeF (data not shown). The concentration of sC5b9 was not significantly different between the groups of patients with and without C3NeF against the C3bBb (data not shown).
[0070] In the case of C3NeF stabilizing C3bBbP, 41% of patients with C3NeF had significantly lower C3 levels compared to patients without (data not shown). Contrary to the previous one, in presence of C3NeF against C3bBbP, levels of sC5b9 were strongly increased (data not shown).
[0071] C3 levels were significantly lower in patients with C3NeF against both the C3bBb and the C3bBbP convertases or only C3bBbP convertase when compared to C3NeF negative patients (data not shown). The sC5b9 was significantly higher in the groups of C3NeF directed against the C3bBbP or C3bBb and C3bBbP than in the group of C3Nef against the C3bBb alone (data not shown).
[0072] Characterisation of the Binding Area of C3NeF
[0073] We raised the possibility that the area of the C3Nef binding site drive the functional consequences on the complement activation. Since both C3Nef and Properdin stabilize the C3 convertase and because we found C3Nef samples which stabilize the C3bBb convertase and have no effect on the C3bBbP convertase stabilization, we hypothesized that the area of C3NeFs binding site is located within the Properdin binding site in the C3bBb convertase (Properdin-like C3NeF). However C3NeF which stabilize the C3bBb and C3bBbP convertases should bind the surface of the C3 convertase at a different site compared to Properdin. In addition, this binding area should not be affected by the conformational change induce by the binding of Properdin to the C3 convertase (Properdin Independent C3NeF). Lastly we proposed that C3NeF which stabilize the C3bBbP convertase and has no effect on the C3bBb convertase stabilization is dependent on the presence of Properdin on the C3 convertase (Properdin Dependent C3NeF).
[0074] To test the hypothesis that C3NeF would share the same epitope as Properdin, we performed a competitive assay between patient IgG and C3NeF. In the C3b Erythrocyte based convertase stabilization assays, 60% of residual C3bBb lytic site is obtained with 800 ng of Properdin. We used four representative C3NeF samples which stabilize only the C3bBb (n=2, P1 and P2) and both the C3bBb and C3bBbP (n=2, P3 and P4).
[0075] Patients' IgG (P1, P2, P3 and P4) added at the time of dissociation step increase the percentage of stabilization in a dose dependent manner. 50, 300, 150 and 300 microG of IgG induce 50% of convertase stabilisation for P1, P2, P3 and P4 IgG respectively. Properdin added at the same time of P3 and P4 IgG increases the percentage of stabilization in a IgG dose dependant manner and rich a maximum of stabilization with 20 microG of IgG (data not shown). Our data show that Properdin and C3NeF cooperate in stabilizing the C3 convertase.
[0076] At opposite, increasing amount of P1 and P2 IgG induce a decrease % of stabilisation of fixed amount of Properdin (data not shown). Moreover, in presence of P1 IgG the % of stabilisation doesn't change whatever the concentration of Properdin added in the assay (data not shown). Our data show that Properdin and C3NeF compete in stabilizing the C3 convertase.
[0077] Identification of C3NeF and Properdin Binding Site
[0078] To better define the binding site of Properdin on the C3 convertase, we designed 3 different peptides: one reproducing the putative Properdin binding site (peptide 1) as deduced by the pseudo atomic structure proposed by Alcorlo et al.; two reproducing regions on the C3 convertase on the right (peptide 2) and on the left (peptide 3) side (data not shown). We tested the ability of these peptides to inhibit the stabilization of the C3 convertase mediated by Properdin (peptide inhibition assay). Properdin was pre-incubated with the peptides and this mixture was added at the time of convertase dissociation step. Properdin incubated with peptides' excipient reached 40% of stabilization. In presence of peptide 1 this effect was almost completely abrogated, reaching 80% inhibition of the Properdin stabilization capacity (n=2 experiments). The inhibition was dose-dependent. Peptide 2 and 3 had no effect (
[0079] Having mapped the putative Properdin binding site on C3bBb to the sequence corresponding to Peptide 1, we tested the capacity of the peptides to reduce also C3NeF stabilization. We firstly screened C3NeF sample from P1, able to stabilize only C3bBb. IgG from P1 and peptides were pre-incubated overnight and then added during the convertase decay step. As for Properdin, we compared stabilization of IgG alone and of the IgG-peptide complexes. The % stabilization of 100 g of IgG (taken as 100% in absence of peptide) was decreased with 40% in the case of peptide 1 (n=3 experiments), but remained in the same range for peptides 2 and 3. Peptide 1 inhibited IgG stabilization in a dose dependent manner. (
[0080] We tested peptide 1 inhibition also for other C3NeF samples: 3 which were positive for the C3bBb and negative for the C3bBbP test and 3, which are positive in both tests. In all cases C3NeF stabilization was diminished, with a range of 20-40% for the former group and of 20-70% for the latter. (
TABLE-US-00002 TABLE 1 IgG binding residual and K.sub.d of convertase after IgG injection. IgG binding on the convertases is calculated subtracting the median value of RU of NHIgG to the RU of C3NeF positive taken at the end of the IgG injection (at 900 seconds). K.sub.d of the convertase were calculated using ProteOn Manager Software by fitting sensorgrams into two state interaction model. IgG binding, residual (RU) K.sub.d C3bBb C3BbBP C3bBb P1 324 42 1.96E04 P2 107 67 7.62E04 P3 123 266 2.50E03 P4 217 122 1.83E03 P5 448 223 6.38E04 NHIgGs.sub.m 1 13
EXAMPLE 2
[0081] Therapeutic inhibition of C3 NeF using peptides may serve as promising treatment option in C3G. The inventors designed and extensively characterized three peptides that interfere with the Properdin and/or C3NeF in the stabilization of the C3 convertase. Since 1) both C3NeF and properdin stabilize the AP C3 convertase and 2) some C3NeF samples have no effect on the C3bBbP convertase stabilization, the inventors raised the hypothesis that the area of C3NeF binding site is located within the properdin binding site in the C3bBb convertase. Their first task is to identify the properdin binding site on C3b and on the C3bBb convertase using peptides inhibition assays. The binding area of Properdin on the convertase is unknown.
A/ Peptides Design
Strategy
[0082] The Properdin binding area was deduced from the previously published electron microscopy images. Different peptides were selected manually by visualization of the surface area of the C3bBb complex with SCIN (PDB ID 2WIN), after removal of the sequence corresponding to SCIN. To better define the binding site of Properdin on the C3 convertase, the inventors already designed 3 different peptides: one reproducing the putative Properdin binding site (peptide 1) as deduced by the pseudo atomic structure proposed by Alcorlo et al.; two reproducing regions on the C3 convertase on the right (peptide 2) and on the left (peptide 3) side. They tested the ability of these peptides to inhibit the stabilization of the C3 convertase mediated by Properdin (peptide inhibition assay). Properdin was pre-incubated with the peptides and this mixture was added at the time of convertase dissociation step. Properdin incubated with peptides' excipient reached 40% of stabilization. Peptide 1 covered 22 residues of C3b (1616-1637) and 11 residues of Bb (308-318) (G-QDEENQKQCQDLGAFTESMVVFNIENLQKTVWD). In presence of peptide 1 this effect was almost completely abrogated, reaching 80% inhibition of the Properdin stabilization capacity (n=2 experiments). The inhibition was dose-dependent. Peptide 2 and 3 had no effect.
New Design
[0083] Four peptides, reproducing conformational neo-epitopes on the C3 convertase and containing sequences from the C345C domain of C3 and the vWF type A domain of FB were designed by mapping the surface areas, spanning the putative Properdin binding site. The peptides are depicted in
B/ Identification of Properdin Binding Site
Methods:
Epitope Competition Assay:
[0084] To investigate whether Properdin binds the peptides biotinylated peptides was coated onto ELISA plates and increased among of Properdin were incubated before adding anti human Properdin.
Peptides Inhibition Assay
[0085] Different mixes of 50 ng of Properdin and 10 g of peptides were incubated at room temperature for 2 hours with gentle shaking in EDTA-containing buffer. As a control, Properdin was incubated with the peptide's dilution buffer. Membrane-bound convertase was formed as in the C3bBb assay. During the convertase decay step, cells were incubated with the mix of FP and peptides. FP amount was chosen in order to obtain 50% of residual convertase after the decay. Residual convertase was measured and data were expressed as percentage of inhibition (percentage of residual convertase compared to the control without peptide).
Results: Characterisation of the Binding Area of Proderdin on the C3bBb Convertase (FIG. 3):
[0086] To better define the binding site of Properdin on the C3 convertase, the inventors tested the ability of these peptides to binds Properdin and to inhibit the stabilization of the C3 convertase mediated by Properdin. Properdin was pre-incubated with the peptides and this mixture was added at the time of convertase dissociation step. Properdin incubated with peptides' excipient reached 40% of stabilization. In presence of peptide 1 and BbExt this effect was almost completely abrogated, reaching 80% inhibition of the Properdin stabilization capacity (n=2 experiments). The inhibition was dose-dependent. Bb C3bExt and controls peptides had no effect. The ELISA measures the capacity of peptides to bind Properdin. The inventors found that BbExt and Pep1 binds the Properdin confirming the presence of a major epitope within the peptide derived from the C3bBb that interacts with Properdin. Importantly they identified two essential sequences for the binding of Properdin to the C3bBb convertases: NLQKTVWD on Bb and SMVVF on C3b.
C/ Identification of C3NeF Epitopes Binding Site
[0087] The inventors use the term of C3NeF for all IgG capable to stabilize the C3 convertases C3bBb and C3bBbP. They identified two types of C3Nefs with different binding sites on the C3 convertase. C3NeF of one of the groups was found to bind at the Properdin binding site and to compete with it, sharing the same effect (Properdin independent C3NeF). The majority of C3NeF bind in an area, adjacent to the Properdin binding site and have an additive effect on the C3 convertase stabilization (Properdin enhancer C3 NeFs). Having mapped the putative Properdin binding site on C3bBb to the sequence corresponding to Peptide 1 and BbExt, but not Short and C3b Ext they tested the capacity of the peptides to reduce also C3NeF stabilization.
Methods:
[0088] Membrane-bound convertase was formed as in the C3bBb assay. During the convertase decay step, cells were incubated with a fixed dose of Peptides (80 g) and patient' IgG (amount determined by the concentration of IgG leading to 50% of residual convertase). The concentration of IgG applied in the test was chosen depending on their C3NeF activity.
Results (FIG. 4)
[0089] The inventors tested 3 patients' Ig which were positive for the C3bBb and negative for the C3bBbP test and 4, which are positive in both tests. In presence of 3 out 4 peptides the stabilization was diminished, with a range of 20-40% for the Properdin like C3NeF. At the opposite sole the peptides Short diminished the stabilization of the Convertase, with a range of 40-80% for the Properdin enhancer C3NeF. In presence of Properdin_like, the percentage of inhibition was significantly higher in presence of peptide1 vs control (p=0.005), of BbExt vs control (p=0.005), of C3bExt vs control (p=0.005). No significant difference was observed between control and short (short vs control (p=0.32). In presence of Properdin_enhancer, the percentage of inhibition was significantly higher in presence of short vs control (p=0.001). No significant difference was observed between control and Pep1, Bbext and C3bExt (p=0.25; 0.46; 0.61). Peptide inhibition experiments indicate this subtype of C3 NeF did not recognize the same area on the C3 convertase. These results confirm that one sub type of C3NeF and properdin, which both increase the AP activity, share the same binding site on the C3bBb convertase. By functional assays, the inventors demonstrated that three peptides inhibits the binding of patients' IgG on the C3 convertase to induce their functional effect. In cases of Properdin-enhancer C3 NeF the Peptides neutralizing Properdin binding will stop the amplification of the C3 convertase stabilization and limits the deleterious effect of the C5 activation. Lastly Peptides neutralizing Properdin binding will inhibit the Properdin-dependant C3 NeF binding in this context.
CONCLUSION
[0090] C3NeF were found in approximately 65% of the patients of C3G which include the Dense Deposit Disease (DDD) and the C3 Glomerulonephritis (C3GN) and are associated with an overactivation of the alternative pathway (AP) of the complement. The antigen-driven expansion of self reactive B cell clones in response to a presence of C3 convertase in the kidney may explain the permanent production of C3 NeF and the echec of the Conventional Immunosuppresive therapy. The inventors propose to inhibit first the C3bBb target which will induce secondary the decrease of the polyclonal B cell response. For this, they proposed to target the C3bBb Convertase. Their results suggest that properdin participates to the uncontrolled activation of C3 and C5 convertases in patients with C3 NeF stabilizing the convertases with and without Properdin. Therefore therapeutic properdin inhibition (using antibodies or peptides) will have a benefit effect. At opposite, they demonstrated that C3 Nef autoantibodies which bind sole the C3bBb convertase have the same binding site with Properdin on the enzyme. Therefore specific therapeutic inhibition of properdin would be detrimental in this setting. The identification of different types of C3NeF with distinct functional specificities in vivo may have consequences for patients' management. Using the peptides therapeutic strategy, both Properdin and C3 NeF will not bind the Convertase. This inhibition of binding could induce secondary the disappearance of the C3bBb enzymes (decrease of the half life of the enzymes) and in the same time of the antigen-driven B cell clone producing the C3 NEf. These results show that inhibiting the binding of C3Nef and/Or Properdin to the enzyme will be a good strategy to treat Dense Deposit Disease (DDD), the C3 Glomerulonephritis (C3GN) and Barraquer Simons (partial lipodystrophy). More, the designed peptides could be used to design a new in vitro assay for the screening of C3 NeF.
REFERENCES
[0091] Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.