Polypeptides for inhibiting complement activation

Abstract

The invention relates to a polypeptide comprising a C3 convertase effector domain, a C5 convertase effector domain and optionally a terminal complex inhibitory effector domain which is resistant to deregulation by physiologic FHR-Proteins and has a dimerization motif, and to its therapeutic use.

Claims

1. A method of treating or preventing a disorder related to or associated with the complement system, which comprises administering to patient in need thereof, a polypeptide comprising an inhibitory C3 convertase effector domain and an inhibitory C5 convertase effector domain.

2. The method according to claim 1, wherein said disorder related to or associated with the complement system is selected from the group consisting of atypical hemolytic uremic syndrome (aHUS), thrombotic microangiopathy (TMA), C3 glomerulopathy (C3G), IgA nephropathy, systemic lupus erythematosus nephritis, transplant rejection, paroxysmal nocturnal hemoglobinuria (PNH), age-related macular degeneration (AMD), infectious diseases, sepsis, SIRS, trauma injury, ischemia/reperfusion damage and myocardial infarction.

3. The method of claim 1, wherein said inhibitory C3 convertase effector domain confers C3 convertase inhibition by decay-accelerating and cofactor activity.

4. The method of claim 1, wherein said polypeptide has decay-accelerating and cofactor activity.

5. The method of claim 1, wherein said inhibitory C3 convertase effector domain is a fragment of Factor H (FH).

6. The method of claim 1, wherein said inhibitory C3 convertase effector domain comprises Short Consensus Repeats (SCRs) 1 to 4 of FH.

7. The method of claim 1, wherein said inhibitory C3 convertase effector domain comprises SCRs 1-4 of FHR2.

8. The method of claim 1, wherein said inhibitory C5 convertase effector domain is a fragment of Factor H-related protein 1 (FHR1).

9. The method of claim 1, wherein said inhibitory C5 convertase effector domain comprises SCR1 and SCR2 of FHR1.

10. The method of claim 1, wherein C5 activation and cleavage into C5a and C5b is inhibited by binding of the polypeptide to C5.

11. The method of claim 1, wherein said polypeptide further comprises a domain that is capable of binding to cellular surfaces.

12. The method of claim 11, wherein said domain that is capable of binding to cellular surfaces comprises SCR19 and SCR20 of FH.

13. The method of claim 1, wherein said polypeptide is a multimer.

14. The method of claim 12, wherein said polypeptide comprises at least one dimerization motif from SCR1 of FHR1.

15. The method of claim 1, wherein said polypeptide is capable of inhibiting TCC (C5b-9) formation.

16. The method of claim 15, wherein said TCC formation is inhibited by binding of the polypeptide to C5b-6.

17. The method of claim 1, wherein said polypeptide comprises the structure A-B-C, wherein A is an inhibitory C5 convertase effector domain, B is an inhibitory C3 convertase effector domain, and C is absent or a domain that is capable of binding to cellular surfaces.

18. The method of claim 1, wherein A and B are fused directly or via a linker.

19. The method of claim 1, wherein B and C are fused directly or via a linker.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1: Structure and characterization of MFHR1. A) Structural assembly of MFHR1. MFHR1 is a fusion protein composed of SCR1-2 of FHR1 (FHR1 1-2) N-terminally linked to SCR1-4 and SCR19-20 of hFH termed as FH1-4 and FH19-20, respectively. A penta-histidine tag (N-terminal) was used as means for purification. FHR1-2 contains a dimerization motif, inhibits C5 convertase activity and MAC assembly. FH has decay acceleration and cofactor activity (FH1-4) and binding sites for C3b and cell surfaces (FH19-20). B) SDS-PAGE and Coomassie (left) or silver (right) staining of MFHR1 purified from supernatants of baculovirus infected SF9 cells via Ni-affinity and size exclusion chromatography. MFHR1 appears with the calculated molecular weight of 58.65 kDa under reducing conditions. Faster mobility of MFHR1 under non-reducing conditions (Coomassie stain, right lane) indicates the presence of disulfide bounds. C) Immunodetection using polyclonal anti-human FH, anti-FH1-4 or monoclonal anti-C18 and anti-FHR1-2 antibodies indicates the correct integrity of FHR1-2, FH1-4 and FH2O in recombinant MFHR1 (I.). Recombinant FH1-4; 19-20 (II.), full-length FHR1 (III.), FH1-4 (IV.) or human plasma derived hFH (V.) served as controls.

(2) FIG. 2. MFHR1 binds to C3b, displays cofactor activity and dissociates C3 convertases. A) Interaction of MFHR1 to C3b was analyzed by ELISA. C3b was immobilized on microtiter plates and serial dilutions of MFHR1 or hFH were added and binding was detected using anti-FH primary and HRP-labelled anti-goat secondary antibodies. BSA coated wells were used as controls. Data are mean±SD from n=3 experiments. Maximum binding of hFH to C3b was set to 100% relative binding. B) MFHR1 efficiently dissociates C3bBb (C3 convertase) complexes. Convertases were assembled on microtiter plates in presence of C3b, Factor B (FB) and Factor D (FD) and hFH or MFHR1 were added and incubated at 37° C. Intact C3bBb and dissociation of these complexes was measured by the relative amount of FB. OD450 values of control wells (C3b+FB+FD without regulators) were set to 100%. Negative control was performed without adding FD. Data are mean±SD from n=3 experiments. C) MFHR1 displays cofactor activity. Cofactor testing was performed by incubation of C3b and Factor I (FI) with increasing concentrations of MFHR1 or hFH (5-25 0 nM) for 30 min at 37° C. SDS-PAGE and Coomassie blue staining was used to visualise a-chain cleavage and α′68, α′43 and α′46 fragments. D) For quantification of cofactor activity, C3b α-chain band intensity was determined by densitometry and intact C3b α-chain was normalized to β-chain and set to 100%. Data represent mean values±SD from n=5 experiments.

(3) FIG. 3. MFHR1 binds C5 and regulates terminal pathway activation by inhibition of the C5 convertase/C5 cleavage and MAC formation. A) MFHR1 binds to C5 as determined by ELISA. Equimolar amounts of MFHR1, full length FHR1, hFH or BSA (133 nM) were immobilized to Nunc plates and incubated with increasing concentrations (10, 20, 40 μg/ml) of C5. Binding was detected using monoclonal C5 antibodies and HRP-labelled secondary antibodies. Data are mean±SD from n=3 experiments B) MFHR1 inhibits AP activation by blockade of C5 cleavage. Cobra venom factor (CVF) C5 convertase was generated on sheep erythrocytes (sE) after adding Factor B (FB) and Factor D (FD). Hemolysis was induced after adding C5, C6, C7, C8 and C9 components and detected at 414 nm. Preincubation of C5 with FHR1, MFHR1 or Eculizumab significantly inhibited hemolysis while hFH or BSA showed now inhibition. Controls without CVF convertase (-FB/FD) or C5 did not induce hemolysis. C) MFHR1 inhibits formation of the membrane attack complex (MAC) on sE. MAC formation on sE was induced by incubation with C5b6, C7, C8 and C9 components and detected by hemolysis of cells. Preincubation of C5b6 with MFHR1, FHR1 or Eculizumab inhibited hemolysis at significant levels compared to hFH or BSA. Samples without C9 did not induce hemolysis. In B and C control without regulator (w/o) was set as 100%. All data represent mean values±SD of 3 separate experiments. ***P<0.001 vs. w/o control, one-way ANOVA with Bonferroni multiple comparison test.

(4) FIG. 4. MFHR1 inhibits complement alternative pathway (AP) and classical pathway (CP) activation and reduces anaphylatoxin release in human serum at clinically relevant levels. MFHR1, hFH, FHR1 or Eculizumab were added into human serum and A) C3b depositions or B) C5b-9 complex formation was measured following alternative pathway (AP) activation was proceeded by LPS using specific buffer conditions. A) MFHR1 reduces surface C3b depositions with higher efficiency than hFH as measured by ELISA using anti-human C3 and HRP-labelled secondary antibodies. As expected, eculizumab did not inhibit C3b depositions. B) MFHR1 inhibits C5b-9 with higher efficiency than hFH or eculizumab as determined using the complement AP-ELISA (WIESLAB®). Untreated control serum was set to 100% for each individual experiment. Data represent single values from n=4 assays±SEM using sera of 4 individual healthy donors. In addition, MFHR1 efficiently blocks generation of C) C3a and D) C5a as assayed in the supernatant of AP activated sera using specific C3a, C5a ELISA (Quidel) (n=3 assays±SEM). E) Inhibition of classical pathway (CP) by MFHR1 was assessed using the complement CP-ELISA (WIESLAB®). AP activity of untreated control serum was set to 100% for each individual experiment. Data represent single values from n=3 assays±SEM using sera of 3 individual healthy 3 donors. See Table 1 for calculated IC50 values.

(5) FIG. 5. Multimeric complexes increase AP regulatory activity of MFHR1 in human serum. A) Size exclusion chromatography (Superdex 200 10/300 GL) of bovine serum albumin (BSA), serum plasma derived FH (hFH) and MFHR1. The three compositions of BSA mixture presented different retention volumes based on molecular mass, which was I. BSA trimer (198 kDa, 10.35 ml), II. BSA dimer (132 kDa, 11.45 ml) and III. BSA monomer (66 kDa, 13.4 ml). Under the same condition, hFH (9.3 ml) showed that the peak of protein species migrates as dimeric proteins at approximately 300 kDa. MFHR1 showed a peak (I.) at retention volume of 10 ml indicating that MFHR1 migrates predominantly in a multimeric state in the fluid phase. Theoretical trimer (II. 10.7), dimer (III. 11.4) intermediates (IV.-V.) or monomeric (IV. 13.5) MFHR1 are indicated in the elution profile. B) Analysis of MFHR1 after elution from SEC column as performed in A. 20 μl protein from fractions was loaded on a 10% SDS-PAGE and silver stained C) Afterwards, single or pooled MFHR1 SEC fractions were added into human serum at 10 nM and AP activation was proceeded by incubation in LPS coated wells using AP specific buffer conditions. Regulatory efficiency of MFHR1 fractions was analyzed by measuring C5b-9 complex formation. AP activity of untreated control serum was set to 100%.

(6) FIG. 6. MFHR1 is resistant to deregulation by FHR1 and FHR5. A) MFHR1 binding to C3b is not competitively inhibited by FHR1 or FHR5. MFHR1 (triangles) or hFH (square) (termed as test protein) was added to C3b coated microtiter plates alone or with increasing concentrations of FHR1 (black line) or FHR5 (grey line) ranging from equimolar amounts to 100-fold excess. MFHR1 or hFH bound to C3b was detected using specific antibodies. Average of 3 independent assays with SD is shown. B) MFHR1 mediated protection of sheep erythrocytes (sE) from serum induced AP-activation is not attenuated by FHR5, while AP regulatory function of hFH was dose dependent deregulated by FHR5. MFHR1 or hFH was used at concentrations that reduce FH-depleted serum induced lysis of sE to 50% and increasing concentrations FHR5 ranging from equimolar amounts to 100-fold excess were added. Hemolysis was determined at 414 nm and data are expressed as relative increase over samples where no FHR5 has been added. Data represent single values from n=3 assays±SEM.

(7) FIG. 7. MFHR1 reduces uncontrolled complement activation in modeling treatment of atypical haemolytic uremic syndrome (aHUS) and C3 Glomerulopathy (C3G) A) Mutations in FH may be causative for familial aHUS and loss of complement regulation in sera of these patients mediate hemolysis if added to sheep erythrocytes (sE). MFHR1 added to serum of an aHUS patient (pat. #1 FH R1215Q (Gerber et al. 2003)) protects sE from complement mediated MAC formation and lysis with higher efficiency than eculizumab. Data represent mean values±SD from n=3 experiments. B) Serum C3 is ubiquitously increased in C3G duo to an excessive complement activation and inhibition uncontrolled complement activity may offer an alternative treatment option in C3G. MFHR1 added to serum of a C3Neph positive patient efficiently inhibits AP activation and C5b-9 formation after LPS stimulation.

(8) FIG. 8. MFHR1 shows significant therapeutic benefit against uncontrolled complement activation in C3 Glomerulopathy (C3G) in vivo. A-C) FH-deficient mice (FH-/-) develop abnormal glomerular C3 accumulation and low serum C3 levels due AP overactivation thus providing a useful C3G model for testing therapeutic efficiency of complement targeted drugs. A) MFHR1 significantly increases C3 serum levels at indicated time points after intraperitoneal injection of 0.5 mg MFHR1 at comparable levels to hFH. Mean values are shown as bars with plotted individual data points. B) MFHR1 significantly reduces abnormal glomerular C3 accumulation. Glomerular C3 fluorescence intensity was determined 24 hours after administration of MFHR1, hFH or PBS treated FH-/- mice. Sections of untreated wild-type mice were used as negative control. Means are shown as bars±SD with plotted individual data points expressed as arbitrary fluorescence units (AFU). C) Representative images of glomerular C3 depositions. Scale bars; 50 μm. ***P<0.001 vs. PBS group, one-way ANOVA with Bonferroni test.

DETAILED DESCRIPTION

(9) The invention pertains to a polypeptide comprising an inhibitory C3 convertase effector domain, an inhibitory C5 convertase effector domain (C5 binding domain), and optionally an inhibitory TCC formation domain and/or a dimerization motif.

(10) An “inhibitory C3 convertase effector domain” in connection with the present invention refers to an amino acid sequence capable of inhibiting C3 convertase, i.e. of inhibiting C3 convertase formation. Inhibition of C3 convertase is typically present if deposition of C3b is inhibited or if there is a reduced formation of C3a after complement alternative pathway activation relative to a control. Formation of C3b and C3a can be determined in assays as depicted in FIG. 4A or FIG. 4C, respectively. In another embodiment, the inhibitory C3 convertase effector domain has C3 convertase decay-accelerating activity and cofactor activity. C3 convertase decay-accelerating activity can be determined in an assay as depicted in FIG. 2B. Cofactor activity can be determined in an assay as depicted in FIG. 2C-2D.

(11) In certain embodiments the inhibitory C3 convertase effector domain is fragment of FH. In accordance with the present invention, FH denotes a protein having at least 70% sequence identity with the amino acid sequence as shown in SEQ ID NO:1. Preferably, the FH has an amino acid sequence identity with the amino acid sequence as shown in SEQ ID NO:1 of at least 80%, more preferably of at least 90%, more preferably of at least 95%. Most preferably, FH comprises or consists of an amino acid sequence as shown in SEQ ID NO:1.

(12) The comparison of sequences and determination of percent identity (and percent similarity) between two amino acid sequences can be accomplished using any suitable program, e.g.

(13) the program “BLAST 2 SEQUENCES (blastp)” (Tatusova et al. (1999) FEMS Microbiol. Lett. 174, 247-250) with the following parameters: Matrix BLOSUM62; Open gap 11 and extension gap 1 penalties; gap x_dropoff50; expect 10.0 word size 3; Filter: none.

(14) The C3 convertase effector domain preferably comprises or consists of SCR1-4 of FH. In one embodiment, the C3 convertase effector domain comprises or consists of amino acids 19-264 of SEQ ID NO:1.

(15) In another embodiment, the inhibitory C3 convertase effector domain is fragment of FHR2. In accordance with the present invention, FHR2 denotes a protein having at least 70% sequence identity with the amino acid sequence as shown in SEQ ID NO:3. Preferably, the amino acid sequence of the FHR2 has a sequence identity with the amino acid sequence as shown in SEQ ID NO:3 of at least 80%, more preferably of at least 90%, more preferably of at least 95%. Most preferably, FHR2 comprises or consists of an amino acid sequence as shown in SEQ ID NO:3.

(16) The C3 convertase effector domain may comprise or consist of SCR1-4 of FHR2. In one embodiment, the C3 convertase effector domain comprises or consists of amino acids 22-268 of SEQ ID NO:3.

(17) An “inhibitory C5 convertase effector domain” in connection with the present invention refers to an amino acid sequence capable of inhibiting C5 convertase. Inhibition of C5 convertase (prevention of C5 cleavage) is typically present if there is a reduced formation of C5a. Formation of C5a can be determined in an assay as depicted in FIG. 4D. In an alternative experimental approach binding of an inhibitory effector domain to C5 is shown (FIG. 3 A). This prevents C5 cleavage to C5a and C5b by experimental C5 convertases and inhibits MAC formation and lysis of cells as depicted in (FIG. 3B).

(18) In certain embodiments the inhibitory C5 convertase effector domain is fragment of FHR1. In accordance with the present invention, FHR1 denotes a protein having at least 70% sequence identity with the amino acid sequence as shown in SEQ ID NO:2. Preferably, the amino acid sequence of the FHR1 has a sequence identity with the amino acid sequence as shown in SEQ ID NO:2 of at least 80%, more preferably of at least 90%, more preferably of at least 95%. Most preferably, FHR1 comprises or consists of an amino acid sequence as shown in SEQ ID NO:2. Besides binding to C5 and preventing C5 cleavage by C5 convertases, SCR 1-2 from FHR1 comprise a dimerization motif, which leads to multimerisation of MFHR1 amplifying function of the polypeptide as shown in FIG. 5. Besides C5 convertase inhibition, SCR 1-2 from FHR1 inhibit MAC formation by binding C5b-6 (FIG. 3C).

(19) The inhibitory C5 convertase effector domain preferably comprises or consists of SCR1 and SCR2 of FHR1. In one embodiment, the inhibitory C5 convertase effector domain comprises or consists of amino acids 22-142 of SEQ ID NO:2.

(20) The inhibitory C3 convertase effector domain, the inhibitory C5 convertase and the inhibitory MAC formation effector domain may be fused directly or via a linker. The linker may be a peptidic linker or a non-peptidic linker. Preferably, the linker consists of 1 to 100 amino acids, more preferably of 1 to 50 amino acids, more preferably of 1 to 20 amino acids, more preferably of 1 to 10 amino acids, most preferably of 1 to 5 amino acids. The linker sequence is typically heterologous to amino acid sequence of the C3 convertase effector domain and to the amino acid sequence of the inhibitory C5 convertase effector domain.

(21) The polypeptide of the invention may have the structure A-B, wherein A is the C3 convertase effector domain as defined herein, and B is a C5 convertase effector domain as defined herein. In another embodiment, the polypeptide of the invention may have the structure B-A, wherein A is the C3 convertase effector domain as defined herein, and B is a C5 convertase effector domain as defined herein.

(22) In a particular embodiment, the polypeptide of the invention comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7.

(23) In certain embodiments the polypeptide of the invention comprises a third domain, said third domain having cell surface binding properties. Preferably, said third domain comprises or consists of a fragment of FH. More preferably, the third domain comprises or consists of SCR19 and SCR20 of FH. In one embodiment, the third domain comprises or consists of amino acids 1107-1230 of SEQ ID NO:1.

(24) The polypeptide of the invention may have the structure A-B-C, wherein A is the C5 convertase effector domain as defined herein, B is a C3 convertase effector domain as defined herein, and C is the third domain as defined herein. In another embodiment, the polypeptide of the invention may have the structure A-C-B, B-A-C, B-C-A, C-A-B, or C-B-A, wherein the meanings of A, B and C are as defined herein. The order of the letters A, B and C indicates the sequence from N-terminus to C-terminus of the polypeptide, e.g. in A-B-C, domain A is at the N-terminus, and domain C is at the C-terminus.

(25) The third domain can be fused directly to the C3 convertase effector domain and/or to the C5 effector domain and MAC effector domain, or via a linker as defined hereinabove.

(26) In a particular embodiment, the polypeptide of the invention comprises or consists of the amino acid sequence as shown in SEQ ID NO:8. In another embodiment, the polypeptide of the invention comprises or consists of the amino acid sequence as shown in SEQ ID NO:9.

(27) The polypeptide of the invention can be used to treat or prevent disorders related to and/or associated with the complement system. These disorders include, but are not limited to, atypical hemolytic uremic syndrome (aHUS), thrombotic microangiopathy (TMA), C3 glomerulopathy (C3G), IgA nephropathy, systemic lupus erythematosus nephritis, humoral rejections in kidney transplanted patients, tissue damage after ischemia-reperfusion events, e.g. after renal transplantation, excessive complement activation, tissue damage, e.g. under hemodialysis, Paroxysmal nocturnal hemoglobinuria (PNH), age-related macular degeneration (AMD), infectious diseases, sepsis, SIRS, trauma injury, myocardial infarction are diseases and conditions where complement activation is made responsible for additional local or systemic damage.

(28) Preferably, the disorder to be treated or prevented is selected from the group consisting of aHUS, TMA, C3G, IgA nephropathy, systemic lupus erythematosus nephritis, PNH and AMD.

(29) The invention further provides nucleic acids encoding the polypeptide of the invention, which can be inserted into suitable plasmids and vectors for expression in host cells.

(30) The nucleic acid encoding the polypeptide to be expressed can be prepared according to methods known in the art. Based on the cDNA sequences of FH (NCBI Reference Sequence: NM_000186.3) FHR1 (NCBI Reference Sequence: NM_002113.2) and FHR2 (NCBI Reference Sequence: NP_005657.1) recombinant DNA encoding the above-mentioned polypeptides can be designed and generated.

(31) Constructs in which the cDNA contains the entire open reading frame inserted in the correct orientation into an expression plasmid may be used for protein expression. Typical expression vectors contain promoters that direct the synthesis of large amounts of mRNA corresponding to the inserted nucleic acid in the plasmid-bearing cells. They may also include an origin of replication sequence allowing for their autonomous replication within the host organism, and sequences that increase the efficiency with which the synthesized mRNA is translated. Stable long-term vectors may be maintained as freely replicating entities by using regulatory elements of, for example, viruses (e.g., the OriP sequences from the Epstein Barr Virus genome). Cell lines may also be produced that have integrated the vector into the genomic DNA, and in this manner the gene product is produced on a continuous basis. Typically, the cells to be provided are obtained by introducing the nucleic acid encoding the polypeptide into suitable host cells.

(32) Any host cell susceptible to cell culture and to expression of polypeptides may be utilized in accordance with the present invention. In certain embodiments, the host cell is mammalian.

(33) In other embodiments, the host cell is an insect cell, e.g. an Sf9 cell), or a physcomitrella cell (e.g. physcomitrella Patens). Alternatively, the host cell may be a bacterial cell.

(34) In general, it will typically be desirable to isolate and/or purify glycoproteins expressed according to the present invention. In certain embodiments, the expressed glycoprotein is secreted into the medium and thus cells and other solids may be removed, as by centrifugation or filtering for example, as a first step in the purification process.

(35) The expressed polypeptide may be isolated and purified by standard methods including, but not limited to, chromatography (e.g., ion exchange, affinity, size exclusion, and hydroxyapatite chromatography), gel filtration, centrifugation, or differential solubility, ethanol precipitation and/or by any other available technique for the purification of proteins (See, e.g., Scopes, Protein Purification Principles and Practice 2nd Edition, Springer-Verlag, New York, 1987; Higgins, S. J. and Hames, B. D. (eds.), Protein Expression: A Practical Approach, Oxford Univ Press, 1999; and Deutscher, M. P., Simon, M. I., Abelson, J. N. (eds.), Guide to Protein Purification: Methods in Enzymology (Methods in Enzymology Series, Vol. 182), Academic Press, 1997, each of which is incorporated herein by reference).

(36) One of ordinary skill in the art will appreciate that the exact purification technique will vary depending on the character of the polypeptide to be purified, the character of the cells from which the polypeptide is expressed, and/or the composition of the medium in which the cells were grown.

(37) Another aspect of the invention is a pharmaceutical composition comprising the polypeptide of the invention, and a pharmaceutically acceptable excipient or carrier. The pharmaceutical composition may comprise the polypeptide in an effective amount for treating or preventing a complement-related disorder in a subject.

(38) Therapeutic formulations of the polypeptide of the invention suitable in the methods described herein can be prepared for storage as lyophilized formulations or aqueous solutions by mixing the glycoprotein having the desired degree of purity with optional pharmaceutically-acceptable carriers, excipients or stabilizers typically employed in the art (all of which are referred to herein as “carriers”), i.e., buffering agents, stabilizing agents, preservatives, isotonifiers, non- ionic detergents, antioxidants, and other miscellaneous additives. See, Remington's Pharmaceutical Sciences, 16th edition (Osol, ed. 1980). Such additives must be nontoxic to the recipients at the dosages and concentrations employed.

(39) The pharmaceutical compositions of the present invention may be formulated for oral, sublingual, intranasal, intraocular, rectal, transdermal, mucosal, topical or parenteral administration. Parenteral administration may include intradermal, subcutaneous, intramuscular (i.m.), intravenous (i.v.), intraperitoneal (i.p.), intra-arterial, intramedullary, intracardiac, intraarticular (joint), intrasynovial, intracranial, intraspinal, and intrathecal (spinal fluids) injection or infusion, preferably intraperitoneal (i.p.) injection in mouse and intravenous (i.v.) in human. Any device suitable for parenteral injection or infusion of drug formulations may be used for such administration. For example, the pharmaceutical composition may be contained in a sterile pre-filled syringe.

(40) Determination of the effective dosage, total number of doses, and length of treatment with a soluble polypeptide of the invention is well within the capabilities of those skilled in the art, and can be determined using a standard dose escalation study. The dosage of a soluble polypeptide of the invention to be administered will vary according to the particular soluble polypeptide, the subject, and the nature and severity of the disease, the physical condition of the subject, the therapeutic regimen (e.g., whether a second therapeutic agent is used), and the selected route of administration; the appropriate dosage can be readily determined by a person skilled in the art.

(41) Summary of the amino acid sequences:

(42) TABLE-US-00001 SEQ ID NO: Description 1 Amino acid sequence of FH 2 Amino acid sequence of FHR1 3 Amino acid sequence of FHR2 4 Amino acid sequence of construct MMFHR1 with His tag His-tag - FHR1(SCR1-2) - FH(SCR1-4) 5 Amino acid sequence of construct MMFHR1 without His tag FHR1(SCR1-2) - FH(SCR1-4) 6 Amino acid sequence of construct FHR1-FHR2 with His tag His-tag - FHR1(SCR1-2) - FHR2(SCR1-4) 7 Amino acid sequence of construct FHR1-FHR2 without His tag FHR1(SCR1-2) - FHR2(SCR1-4) 8 Amino acid sequence of construct MFHR1 with His tag His-tag - FHR1(SCR1-2) - FH(SCR1-4) - FH(SCR19-20) 9 Amino acid sequence of construct MFHR1 without His tag FHR1(SCR1-2) - FH(SCR1-4) - FH(SCR19-20)

Examples

(43) MFHR1 consists of the N-terminal FH regulatory active domains SCR1-4 (C3/C5 convertase decay accelerating- and cofactor activity) with C-terminal surface recognition domains (SCR19-20) of FH in combination with the N-terminal domains of FHR1 (SCR1-2) [FIG. 1]. FHR1 is the only known endogenous complement regulator of the C5 convertase. FHR1 SCR1-2 binds to C5 and thereby inhibits C5 cleavage into C5a and C5b. Additionally, FHR1 SCR1-2 inhibits the terminal complement pathway by binding to C5b6. Through the combination of these domains the complement activation is inhibited at multiple effector sites of the cascade (C3b degradation, inhibition of C3/C5-convertases, inhibition of C5 cleavage and C5b-9 formation). This also leads to the suppression and formation of the anaphylatoxins C3a and C5a, which are thought to contribute in disease progression in AP related diseases.

(44) To produce MFHR1, cDNA fragments containing the requested sequence of the FH and FHR1 domains mentioned above were amplified by PCR and subsequently assembled by self-priming overlap PCR. The DNA was cloned into the pFastbac gp67-10xHis baculo expression vector and MFHR1 was expressed in SF9 insect cells. The amino acid sequence of MFHR1 is shown in SEQ ID NO:8. The purification of the protein was performed by affinity chromatography and size exclusion chromatographie. SDS-PAGE and Commassie or silver stained gel showed a single 58.65 kDa band corresponding to the calculated molecular weight of MFHR1 [FIG. 1B]. The successful fusion of FHR1 SCR1-2 and FH SCR 1-4 and SCR 19-20 was confirmed by immunodetection using specific antibodies for the detection of FHR1 derived domains SCR1-2, anti-FH1-4 for detection of FH derived domains 1-4, monoclonal anti-C18 for detection of FH derived domain 20 and polyclonal FH antibodies [FIG. 1C].

(45) MFHR1 binds to C3b [FIG. 2A], dissociates C3 convertases [FIG. 2B] and displays cofactor activity [FIG. 2C-2D]. In contrast to plasma purified FH (FHplasma), FHR1 and MFHR1 show the ability to bind C5, mediated by SCR1-2 from FHR1 [FIG. 3A]. FHR1 and MFHR1 regulate terminal pathway activation by inhibition of the C5 convertase/ C5 cleavage [FIG. 3B] and MAC formation [FIG. 3C]. This demonstrates that our novel fusion protein MFHR1 not only retains the complement regulatory activity of FH [FIG. 2] and FHR1 [FIG. 3], but moreover, combines the properties of both. MFHR1 inhibits aHUS serum induced hemolysis of sheep erythrocytes more efficient than eculizumab, demonstrating that, in addition to its regulatory activity, surface recognition derived from FH domains 19-20 is retained in MFHR1 [FIG. 7A]. The high potency of MFHR1 was further emphasized in ELISA based complement activity assays. Following activation of the complement cascade in human serum spiked with MFHR1, its AP regulatory activity was determined by measuring C3b depositions [FIG. 4A] or C5b6-9 [FIG. 4B] and its CP regulatory efficiency was determined by measuring C5b6-9 using the WIESLAB® CP-ELISA [FIG. 4E]. The addition of MFHR1 to normal human serum (NHS) or serum from a C3G patient (patient under relapse, C3Nef autoantibody positive) efficiently inhibits complement complement activation in a dose dependent manner [FIG. 4A-B, 4E, 7B]. As in the hemolytic assay, MFHR1 showed higher inhibitory efficiency on a molar basis than hFH (Table 1), eculizumab and other relevant inhibitors that are under investigation in a preclinical stadium (IC50 values summarized in Table 1). Furthermore, we demonstrate that MFHR1 inhibits the generation of C3a [FIGS. 4C] and C5a [FIG. 4D] in human serum more efficiently than eculizumab.

(46) The use of a dimerization motif (mediated by SCR1 of FHR1) facilitates the generation of multimeric complexes of MFHR1. We showed that multimeric complexes have higher regulatory activity, presumably by increasing local concentration of the regulators [FIG. 5]. Furthermore, MFHR1 is resistant to FHR mediated deregulation [FIG. 6A-B].

(47) Therapeutic value of MFHR1 was proven in two modeling treatment approaches in vitro and a murine model of C3G in vivo. The addition of MFHR1 efficiently reduces uncontrolled complement activation in sera of aHUS [FIGS. 7A] and C3G [FIG. 7B] patients. In FH-/- deficient mice, showing a C3G-like phenotype, the administration of MFHR1 resulted in the rapid normalization of plasma C3 levels [FIG. 8A] and resolution of the glomerular C3 deposition [FIG. 8B-C].

(48) TABLE-US-00002 TABLE1 Summary of estimated IC50 values for inhibitory activity in human serum CP ELISA AP ELISA (IC50) (IC50) Regulator C3b depositions C5b-9 C5b-9 MFHR1 0.015 ± 0.009 0.0033 ± 0.001  0.021 ± 0.01 hFH 0.537 ± 0.238 (0.53*) 0.177 ± 0.08 (3.0 ± 1.6**) n.t. Eculizumab n.e. 0.015 ± 0.0025 n.t. mini-FH  0.05 ± 0.027 (0.04*) 0.016 ± 0.013  n.t. TT30 0.014***  0.04 ± 0.005**   2.1 ± 0.7** Compstatin 1.42.sup.# 1.07.sup.# n.t. Values are in μM. IC50 values were calculated using log(inhibitor) vs. response --variable slope equation after transformation of the X data to Log X using GraphPad Prism software, n.t. = not tested, n.e. = no effect. Bold letters, AG Häffner, *Schmidt et al. 2013, **Hareli et al. 2011, ***Schmidt et al. 2015, .sup.#Gorham et al. 2013.

(49) Taken together, our results demonstrate that combinatorial approaches to intercept complement activation on multiple effector sites in the complement cascade (C3b degradation, inhibition of C3/C5-convertases, inhibition of C5 cleavage and TCC formation) simultaneously and by its ability to multimerize (or respectively both) has several beneficial advantages compared to “conventional” approaches in order to improve regulators of complement activation. MFHR1, comprising different functional properties from FH and FHR1 regulates complement activity on the level of C3 and C5 convertases and by blocking the terminal complement pathway and is likely more active compared to FH or eculizumab or other published clinical relevant complement inhibitors. Notably, MFHR1 is particularly resistant to deregulation by FHR1 and FHR5 and multimeric complexes may increase its regulatory efficiency.

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