Fragment of complement factor h related protein 5 which restores complement regulation

Abstract

The present invention relates to a peptide therapy to modulate the complement system. The invention provides a novel fragment of Complement Factor H Related protein 5 which restores complement regulation. Pharmaceutical compositions, medicaments and methods of treatment for use in preventing, ameliorating or treating diseases that are characterised by inappropriate complement activity are also described.

Description

[0001] FRAGMENT OF COMPLEMENT FACTOR H RELATED PROTEIN 5 WHICH RESTORES COMPLEMENT REGULATION

[0002] Field of Invention

[0003] The present invention relates to an isolated polypeptide for use as a peptide therapy to modulate the complement system. More particularly, the invention provides a novel fragment of Complement Factor H Related protein 5 which restores complement regulation. Also provided are pharmaceutical compositions, medicaments and methods of treatment for use in preventing, ameliorating or treating diseases that are characterised by inappropriate complement activity.

[0004] Background

[0005] Complement is a key component of innate immunity and host defence. Dysregulation of complement activation, which is designed to be destructive to a pathogen, can damage host cells and has been increasingly implicated in disease pathology. In vertebrates, over 60 proteins comprise 3 activation pathways - classical, alternative and lectin1 4. Although each is individually triggered, all pathways merge at the step of Complement 3 (C3) activation. C3 has various roles in the complement cascade and effector functions56. To prevent host tissue damage, the activation of C3 is strictly regulated by membrane-bound and plasma regulatory molecules7.

[0006] Complement Factor H (FH) is a key circulating regulator of the complement system that can bind to and inhibit C3 deposited on host surfaces, protecting the organism from damage8. Locally released FH in tissues may help to limit complement activation and maintain an antiinflammatory environment. Factor H regulates complement activation by inhibiting the assembly of the alternative pathway complement components. The regulatory activities of FH are modulated by a family of 5 Complement Factor H Related proteins9.

[0007] Complement Factor H Related protein 5 (FHR5) is composed of nine Short Consensus Repeat (SCR) domains, which can form a homodimer via the two N-terminal SCR domains (domains 1 and 2)10 13. The C-terminal domains (domains 8 and 9 in each dimer) can bind to C3 on cell surfaces. As such the dimers are bivalent. FHR5 dimers confer increased avidity for complement at surfaces compared with the predominantly monovalent FH. FHR5 is a potent competitive antagonist to FH, but lacks FH’s N-terminal C3 regulating domain and will therefore de-regulate complement. This phenomenon is predicted to occurwhen C3 is densely deposited on a surface, as bivalent binding is only possible when neighbouring C3 molecules are within ~20nm on a surface (the length of the FHR5 dimers end-to-end).

[0008] The inventors have previously shown that dimerization of FHR5 significantly enhances the avidity of this protein for ligand which enable it to out-compete FH. This dimerization-driven avidity enables FHR5 to function as a deregulator of complement by acting as a competitive antagonist of FH.

[0009] It will be appreciated that there are many disease conditions that are associated with inappropriate complement activation, and particularly excessive complement activity14. The kidney appears particularly sensitive to the effects of complement activation15. Indeed, activation of complement in the kidney by antibodies is an important disease mechanism in IgA nephropathy and other common, severe and untreatable kidney diseases such as lupus nephritis and antibody mediated transplant rejection16 18. It has previously been shown that genetic variation across the CFHR gene family locus influences susceptibility to disease. Several rare variants within the CFHR locus are associated with familial C3 glomerulopathy19 23and a common variant at this locus affects risk of IgA nephropathy24. Glomerulonephritis is commonly associated with deposition of electron dense material in the glomerulus which contains complement components, including C3. Among individuals with Cypriot ancestry, CFHR5 nephropathy segregates with a heterozygous internal duplication of the CFHR5 gene which results in an elongated FHR5 protein25. This mutant FHR5 can form oligomers that possess additional binding sites for C3 on surfaces, conferring increased avidity for C3-coated surfaces and therefore increased potency as a FH antagonist compared with WT FHR5. The disease is endemic in people of Cypriot ancestry and is characterised by haematuria and can be associated with acute deterioration in renal function that can be triggered by otherwise trivial infections elsewhere in the body26 30. There is no proven therapy for the CFHR5 nephropathy. Other complement-mediated diseases (such as IgA nephropathy, immune complex glomerulonephritides or antibody mediated kidney transplant rejection) are treated either by suppressing the overall immune response. However, clinical trials of systemic Factor B inhibition or systemic C3 inhibition are currently underway. Analysis of the Finngen database reveals that rare loss of function variants in CFHR5 are associated with reduced risk of age-related macular degeneration, suggesting that FHR5 also plays an important role in mediating damage in this disorder31

[0010] Thus, the aim of the present invention is to provide a novel peptide therapeutic to modulate complement activation in order to treat the above-mentioned diseases. The inventors set out to achieve this by focusing their studies on the function of the SCR 1 and 2 domains of human FHR5 and the mechanism of binding and subsequent inhibition of FHR5 by disruption of bivalent FHR5 homodimer formation. The resulting novel mechanism of complement inhibition by FHR5 de-dimerisation represents an attractive target to modulate complement activation in diseased states by restoring complement regulation at sites where, due to inflammation and consequent complement activation, C3d has accumulated above a critical density on a surface. Summary of the Invention

[0011] In one aspect, the invention relates to an isolated polypeptide comprising domains 1 and 2 of human complement factor H-related 5 (FHR5) protein.

[0012] In one embodiment, the isolated polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 1 or a sequence with at least 80% sequence identity thereto.

[0013] In one embodiment, the isolated polypeptide comprises an amino acid sequence with at least 85%, 90%, 95%, 98%, 99% or 100% sequence identity with the sequence set forth in SEQ ID NO: 1.

[0014] In one embodiment, the isolated polypeptide forms a homodimer.

[0015] In one embodiment, the isolated polypeptide forms a heterodimer with endogenous FHR5.

[0016] In one embodiment, the isolated polypeptide reduces the concentration or activity of endogenous FHR5.

[0017] In one embodiment, the isolated polypeptide prevents endogenous FHR5 homodimer formation.

[0018] In one embodiment, the isolated polypeptide inhibits cell surface deposition of Complement 3 (C3).

[0019] In one embodiment, the isolated polypeptide inhibits FHR5-mediated competitive antagonism of Factor H.

[0020] In one embodiment, the isolated polypeptide comprises a purification tag, wherein the purification tag is suitable for purifying the isolated polypeptide from mammalian, other eukaryotic, and / or bacterial cells.

[0021] In one embodiment, the isolated polypeptide comprises a linker region.

[0022] In one embodiment, the isolated polypeptide comprises at least one post-translational modification, preferably wherein at least one of the post-translational modifications is selected from PEGylation, phosphorylation, acetylation, methylation, glycosylation, succinylation, ubiquitination, hydroxylation, sumoylation, amidation, or glutathionylation, In another aspect, the invention relates to a nucleic acid sequence that encodes the amino acid sequence of the isolated polypeptide as described herein.

[0023] In another aspect, the invention relates to a vector comprising a nucleic acid sequence as described herein.

[0024] In another aspect, the invention relates to a host cell comprising the nucleic acid sequence or the vector as described herein.

[0025] In one embodiment, the invention relates to the isolated polypeptide described herein or the nucleic acid sequence described herein or the vector described herein for use in the treatment of a disease characterised by excessive complement activation.

[0026] In one embodiment, the invention relates to a method of treating of a disease characterised by excessive complement activation in a subject, comprising administering to the subject the isolated polypeptide described herein or the nucleic acid sequence described herein or the vector described herein.

[0027] In one embodiment, the invention relates to the use of the isolated polypeptide described herein or the nucleic acid described herein or the vector described herein for the manufacture of a medicament for the treatment of a disease characterised by excessive complement activation in a subject.

[0028] In one embodiment, the disease is selected from meningitis, renal disease, autoimmune disease or inflammation including conditions, such as rheumatoid arthritis, asthma, lupus nephritis, membranous nephropathy, infection-associated glomerulonephritis, post-infectious glomerulonephritis, paraprotein-associated glomerulopathy, Alport syndrome-associated nephritis, ischemia-reperfusion injury, atypical haemolytic uremic syndrome, thrombotic thrombocytopenic purpura, paroxysmal nocturnal haemoglobinuria, Membranoproliferative glomerulonephritis, hemolytic uremic syndrome, hypocomplementemic glomerulonephritis, dense deposit disease, macular degeneration (e.g. age-related macular degeneration, AMD), spontaneous foetal loss, Pauci-immune vasculitis, epidermolysis bullosa, recurrent foetal 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 anaemia, idiopathic thrombocytopenic purpura, Goodpasture syndrome, antiphospholipid syndrome, Infective endocarditis, or injury resulting from myocardial infarction, cardiopulmonary bypass and hemodialysis. In one embodiment, the renal disease is selected from glomerulonephritis, C3 glomerulopathy, mesangiocapillary glomerulonephritis type 1 , 2 or 3, membranoproliferative glomerulonephritis type 1 , 2 or 3, dense deposit disease, C3 glomerulonephritis, immune complex membranoproliferative glomerulonephritis, immune complex glomerulonephritis, nephritis, lupus nephritis, CFHR5 nephropathy, IgA nephropathy or antibody mediated kidney transplant rejection. The invention may therefore be for use in the treatment of a renal disease as listed above or a method for the treatment of a renal diseases as listed above.

[0029] In another aspect, the invention relates to a pharmaceutical composition comprising the isolated polypeptide as described herein or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.

[0030] In one embodiment, the pharmaceutical further comprises one or more additional active agents, a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.

[0031] In another aspect, the invention relates to the isolated polypeptide or pharmaceutical composition as described herein for use as a medicament.

[0032] In one embodiment, the isolated polypeptide or the pharmaceutical composition is administered intravenously, subcutaneously, intramuscularly, or intradermally.

[0033] In another aspect, the invention relates to a kit comprising an isolated polypeptide or a pharmaceutical composition as described herein and instructions for use.

[0034] In another aspect, the invention relates to an intro, in vivo or ex vivo method to reduce the concentration or activity of FHR5 protein comprising contacting a biological sample with the isolated polypeptide as described herein.

[0035] In another aspect, the invention relates to an in vitro, in vivo or ex vivo method of inhibiting FHR5 dimerisation comprising contacting a biological sample with the isolated polypeptide as described herein.

[0036] In another aspect, the invention relates to an in vitro, in vivo or ex vivo method of inhibiting C3 deposition comprising contacting a biological sample with the isolated polypeptide as described herein.

[0037] In another aspect, the invention relates to a method of monitoring the therapeutic efficacy of a treatment for a disease characterised by excessive complement activation, comprising determining the level of FHR5 dimerisation prior to administration of the isolated polypeptide or the pharmaceutical composition descried herein, and determining the level of FHR5 dimerisation after administration.

[0038] In another aspect, the invention relates to a method of monitoring the therapeutic efficacy of a treatment for a disease characterised by excessive complement activation, comprising; determining the cell surface level of C3 prior to administration of the isolated polypeptide or the pharmaceutical composition described herein and determining the cell surface level of C3 after administration.

[0039] Figures

[0040] The invention is further described in the following non-limiting figures.

[0041] Figure 1. Silver stain of electrophoresis gel containing denatured recombinantly produced FHR5 protein and selected of its constituent Short Complement Regulator domains (SCRs): Full length human FHR5 WT (65 kDa); human FHR5 SCR1 / 2 domains (15 kDa); human FHR5 SCR1 domain (9 kDa)

[0042] Figure 2. Sedimentation rates of recombinant proteins: A. full length WT FHR5; B. FHR5 SCR1 / 2 domains; C. FHR5 SCR1 only showing dimeric sedimentation (A, 131 kDa) and B, 28.3 kDa) and monomeric sedimentation (C, 8.6 kDa).

[0043] Figure 3. A. A streptavidin-coated Surface Plasmon Resonance (SPR) chip was incrementally loaded with biotinylated C3d (panel i) resulting in various density of C3d available for analysis of FHR5 binding kinetics. Sensorgrams for 79RU, 91 RU, 618RU and 836RU C3d at various concentrations of FHR5 are shown (raw data in panels ii, iv, vi, viii; bivalent fitted data Hi, v, vii, ix). B. Steady state responses at various concentrations of FHR5 demonstrated progressive reduction in Kd as the C3d density on the chip increased.

[0044] Figure 4. A. Biolayer interferometry using 8 different densities of immobilized C3d. B. Binding avidity of FHR5 varied with C3d density. C. Data were fitted to a sigmoid curve.

[0045] Figure 5. A mouse model of induced nephrotoxic nephritis (NTN) was injected with either 45 pg human FHR5-His protein or 45 pg albumin-His in 0.9 % saline 1 hour before sacrificing and harvesting kidneys. Immunofluorescence staining was performed on 10 pm cryosections of the kidneys to assess the co-localisation of FHR5 to complement C3 in the glomeruli. Frozen sections were blocked with 10% donkey serum (Sigma-Aldrich) followed by rabbit anti-6xhis (Abeam, ab9108) and then donkey anti-rabbit Alexa Fluor 647 (green) to detect either FHR5-His or Albumin-His. FITC-conjugated (violet) goat anti-C3 antibody (MP Biomedicals) was used to stain for complement C3 deposition. Images were taken using a 65x oil objective on a Leica Confocal Microscope and show co-localisation of (murine) C3 (violet) with human FHR5 (green, top right) but not human albumin (green, bottom right).

[0046] Figure 6. Analytical ultracentrifugation of A. full length FHR5 alone (top panel), SCR1 / 2 alone (middle panel) and 2:1 (SCR1 / 2:FHR5) mixture (lower panel) demonstrating heterodimerization of SCR1 / 2 with full length FHR5 following mixing. B. When FHR5 (top panel) was mixed with SCR1 alone (middle panel) heterodimerization was not seen (lower panel).

[0047] Figure 7. Intensity of fluorescence for C3 on mammalian cells was increased when cells were incubated with serum supplemented with recombinant FHR5 at two different doses compared with cells incubated with serum alone.

[0048] Figure 8. A. Increase in fluorescence intensity of C3 staining on mammalian cells incubated with FHR5 was inhibited by co-incubation with SCR12 (R5I / 2) in molar ratios of 3:1 and 10:1 , showing a dose-response relationship. B. No consistent change in fluorescence intensity of C3 staining on mammalian cells was observed when FHR5 was co-incubated with SCR1 only, even at higher molar ratios (6.5 and 19.5)

[0049] Figure 9. Reduction of glomerular deposition of human FHR5 in the mouse nephrotoxic nephritis model is seen when the FHR5 is mixed with FHR5 SCR1 / 2 using a 1 :20 molar ratio.

[0050] Figure 10. FHR5 and SCR1 / 2 administered separately to mice with NTN (n=3 for each group).

[0051] Detailed description

[0052] The embodiments of the invention will now be described. In the following passages, different embodiments are described. In the following passages, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

[0053] Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, pathology, oncology, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. The methods and techniques of the present disclosure are generally performed according to conventional methods well-known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, e.g., Green and Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012) Therapeutic Monoclonal Antibodies: From Bench to Clinic, Zhiqiang An (Editor), Wiley, (2009); and Antibody Engineering, 2nd Ed., Vols 1 and 2, Kontermann and Dubel, eds., Springer-Verlag, Heidelberg (2010).

[0054] Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly accomplished in the art or as described herein. The nomenclatures used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

[0055] In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the cell culture, molecular genetics, nucleic acid chemistry, and immunology laboratory operation steps used herein are all routine steps widely used in the corresponding fields. Meanwhile, for a better understanding of the present invention, definitions and explanations of related terms are provided below.

[0056] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. While the foregoing disclosure provides a general description of the subject matter encompassed within the scope of the present disclosure, including methods, as well as the best mode thereof, of making and using this disclosure, the following examples are provided to further enable those skilled in the art to practice this disclosure. However, those skilled in the art will appreciate that the specifics of these examples should not be read as limiting on the invention, the scope of which should be apprehended from the claims and equivalents thereof appended to this disclosure. Various further aspects and embodiments of the present disclosure will be apparent to those skilled in the art in view of the present disclosure.

[0057] Amino acid sequences are presented herein in the 5' to 3' direction, from left to right, unless specifically indicated otherwise. Amino acids are represented herein in the manner recommended by the IUPAC-IUB Biochemical Nomenclature Commission, by either the one- letter code, or the three-letter code. As used in the description of the invention and the appended claims, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0058] Also as used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[0059] The terms “polypeptide(s)” and “protein(s)” are used interchangeably throughout the application and denote at least two covalently attached amino acids, thus may signify proteins, polypeptides, oligopeptides, peptides, and fragments thereof. The protein may be made up of naturally occurring amino acids and peptide bonds, or synthetic peptidomimetic structures. Hence, “amino acid(s)” or “peptide residue(s)”, as used herein, denote both naturally occurring and synthetic amino acids. In some cases, the immunoglobulin proteins of the present invention may be synthesized using any in vivo or in vitro protein synthesis technique known in the art.

[0060] Isolated polypeptide

[0061] According to one aspect of the invention, there is provided an isolated polypeptide, comprising domains 1 and 2 of human complement factor H-related 5 (FHR5) protein.

[0062] Complement plays a key role in the pathophysiology of numerous common and rare kidney diseases, including glomerulonephritis and transplant rejection for which current standard of care is (unlicensed) immunosuppression that has major adverse effects. Complement inhibition is potentially much safer and is a logical way to prevent or ameliorate kidney damage in immune disorders. Further, the use of an isolated polypeptide comprising domains 1 and 2 of humans FHR5 targets a layer of complement regulation that appears to be highly specific to the inflamed kidney. Unlike complement terminal blockade with eculizumab, which is currently the only licensed complement therapeutic for kidney disease, the inventor’s novel approach directly inhibits the central alternative pathway uniquely in the kidneys, while leaving the key effector mechanisms of complement, which are important in fighting serious bacterial infections, intact. Therefore on-target adverse effects are predicted to be relatively mild.

[0063] Full length human FHR5 is a 62 kDa, 9 domain protein. The isolated polypeptide comprising domains 1 and 2 of human FHR5 of the present invention is a soluble, 15 kDa fragment of a native human protein and is therefore not expected to be immunogenic since it contains no novel epitopes. For the same reason, off target adverse effects are unlikely. The term "isolated polypeptide" is one that has been identified and separated and / or recovered from a component of its natural environment.

[0064] In one embodiment, the isolated polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 1 or a sequence with at least 80% sequence identity thereto.

[0065] Domains 1 and 2 of FHR5 (SEQ ID NO: 1): EGTLCDFPKIHHGFLYDEEDYNPFSQVPTGEVFYYSCEYNFVSPSKSFWTRITCTEEGWSPT PKCLRMCSFPFVKNGHSESSGLIHLEGDTVQIICNTGYSLQNNEKNISCVERGWSTPPICSFTK G

[0066] As used herein, the term "homology" or “identity” generally refers to the percentage of amino acid residues in a sequence that are identical with the residues of the reference polypeptide with which it is compared, after aligning the sequences and in some embodiments after introducing gaps, if necessary, to achieve the maximum percent homology, and not considering any conservative substitutions as part of the sequence identity. Thus, the percent homology between two amino acid sequences is equivalent to the percent identity between the two sequences. Neither N- or C-terminal extensions, tags or insertions shall be construed as reducing identity or homology. Methods and computer programs for the alignment are well known. The percent identity between two amino acid sequences can be determined using well known mathematical algorithms.

[0067] Sequence homology / identity as used above can be at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88% 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% for example at least 95%, 96%, 97%, 98% or 99% sequence homology / identity.

[0068] The isolated polypeptide may be a variant of SEQ ID NO.1 having one or more amino acid substitutions, deletions, insertions or other modifications, and which retains a biological function of the polypeptide, that is regulating endogenous FHR5. Thus, variant isolated polypeptide can be sequence engineered. Modifications may include one or more substitution, deletion or insertion of one or more codons encoding the polypeptide that results in a change in the amino acid sequence as compared with the native polypeptide. Amino acid substitutions can be the result of replacing one amino acid with another amino acid having similar structural and / or chemical properties, such as the replacement of a leucine with a serine, i.e., conservative amino acid replacements.

[0069] In one embodiment, the isolated polypeptide forms a homodimer. In one embodiment, the isolated polypeptide forms a heterodimer with endogenous FHR5. In one embodiment, the isolated polypeptide comprising domains 1 and 2 of human FHR5 may reversibly form homodimers as well as heterodimers with endogenous FHR5.

[0070] In one embodiment, the isolated polypeptide reduces the concentration or activity of endogenous FHR5. The inventors have previously shown that reducing the concentration of FHR5 homodimers, or heterodimers with other FHR family members will reduce or inhibit the activity of said dimers. This results in a decrease of complement activation, which is required for the effective treatment of certain diseases. The present invention has interestingly shown that this effect can be achieved with a fragment as small as domains 1 and 2 of FHR5.

[0071] Reduction of polypeptide concentration can be referred to as protein depletion, and reduction of protein activity can be referred to as protein neutralisation or inhibition.

[0072] In one embodiment, the isolated polypeptide prevents endogenous FHR5 homodimer formation. The inventors have found that the isolated polypeptide of the present invention occupies the dimerisation domains of endogenous FHR5, which prevents self-dimerisation.

[0073] In one embodiment, the isolated polypeptide inhibits cell surface deposition of Complement 3 (C3). The isolated polypeptide of the present invention lacks domains 8 and 9 of the endogenous FHR5, the domains responsible for binding to C3. Therefore, the isolated polypeptide is unable to bind to C3. Further, mutant FHR5 which has a duplication of domains 1 and 2 is no longer able to form multimers and oligomers when it forms a dimer with the isolated polypeptide. The phenotype of multimer and oligomer formation is responsible for the gain-of-function effect of increased antagonism of FH in FHR5 nephropathy.

[0074] Endogenous FHR5 is bivalent; the N-terminal domains 1 and 2 can form homodimers, whilst the C-terminal domains 8 and 9 can bind to C3. Endogenous FHR5 which has formed a dimer with the isolated polypeptide of the present invention is monovalent, reducing the ability of endogenous FHR5 to bind to C3. Therefore, in one embodiment, the isolated polypeptide inhibits FHR5-mediated competitive antagonism of Factor H which in turn prevents cell surface deposition of Complement 3 (C3). Thus, the isolated polypeptide comprising domains 1 and 2 of human complement factor H-related 5 (FHR5) protein inhibits cell surface deposition of Complement 3 (C3).

[0075] In one embodiment, the isolated polypeptide comprises a purification tag, wherein the purification tag is suitable for purifying the isolated polypeptide from mammalian, insect, yeast other eukaryotic and / or bacterial cells. Such tags are well known in the art and non-limiting examples are described herein. Purification tags may be selected from, for example, a His-tag, T7 tag, GST tag, Halo tag, HiBiT peptide tag, Maltose binding protein tag or Strep-tag. In one embodiment, the isolated polypeptide comprises a linker region. The term "linker" refers to a peptide comprising one or more amino acids. It will be understood by those skilled in the art that a linker region allows for the purification tag to be biochemically cleaved after polypeptide isolation. A peptide linker comprises 1 to 50, for example 1 to 20 amino acids. Peptide linkers are known in the art and non-limiting examples are described herein. Suitable, non-immunogenic linker peptides are, for example, linkers that include G and / or S residues, such as GSG, (G4S)n, (SG4)n or G4(SG4)n peptide linkers, wherein "n" is generally a number between 1 and 10, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0076] Recombinant protein production to produce isolated polypeptides are standard techniques in the art. The production tags and linker regions may be utilised for recombinant protein product in mammalian cells, as set forth in SEQ ID NO: 2. This contains a GSG linker and a His-tag. The production tags may be utilised for recombinant protein production in bacterial cells, as set forth in SEQ ID NO: 3. This contains a T7-tag.

[0077] Domains 1 and 2 of FHR5 for expression in mammalian cells (SEQ ID NO: 2) MLLLFSVILISWVSTVGGEGTLCDFPKIHHGFLYDEEDYNPFSQVPTGEVFYYSCEYNFVSPSK SFWTRITCTEEGWSPTPKCLRMCSFPFVKNGHSESSGLIHLEGDTVQIICNTGYSLQNNEKNI SCVERGWSTPPICSFTKGHHHHHH

[0078] Domains 1 and 2 of FHR5 for expression in bacteria (SEQ ID NO: 3): MASMTGGQQMGRGSEGTLCDFPKIHHGFLYDEEDYNPFSQVPTGEVFYYSCEYNFVSPSKS FWTRITCTEEGWSPTPKCLRMCSFPFVKNGHSESSGLIHLEGDTVQIICNTGYSLQNNEKNIS CVERGWSTPPICSFTKGE

[0079] In one embodiment the isolated polypeptide comprises at least one post-translational modification, preferably wherein at least one of the post-translational modifications is selected from PEGylation, phosphorylation, acetylation, methylation, glycosylation, succinylation, ubiquitination, hydroxylation, sumoylation, amidation, or glutathionylation,

[0080] The invention also relates to a nucleic acid sequence that encodes an amino acid sequence that encodes domains 1 and 2 of human FHR5. The invention also relates to a vector comprising a nucleic acid sequence as described herein. The invention also relates to a host cell comprising the nucleic acid sequence as described herein or a vector as described herein. The host cell may be a mammalian, insect, yeast, other eukaryotic or bacterial cell.

[0081] Nucleotide sequence of domains 1 and 2 of FHR5 for expression in mammalian cells (SEQ ID

[0082] NO: 5) atgttgctcttattcagtgtaatcctaatctcatgggtatccactgttgggggagaaggaacactttgtgattttccaaaaatacaccatgg atttctgtatgatgaagaagattataaccctttttcccaagttcctacaggggaagttttctattactcctgtgaatataattttgtgtctccttc aaaatccttttggactcgcataacatgcacagaagaaggatggtcaccaacaccgaagtgtctcagaatgtgttcctttccttttgtga aaaatggtcattctgaatcttcaggactaatacatctggaaggtgatactgtacaaattatttgcaacacaggatacagccttcaaaac aatgagaaaaacatttcgtgtgtagaacggggctggtccactcctcccatatgcagcttcactaaaggacatcatcatcatcatcattg a

[0083] Nucleotide sequence of domains 1 and 2 of FHR5 for expression in bacteria (SEQ ID NO: 6): atggctagcatgactggtggacagcaaatgggtcgcggatccgaaggaacactttgtgattttccaaaaatacaccatggatttctgt atgatgaagaagattataaccctttttcccaagttcctacaggggaagttttctattactcctgtgaatataattttgtgtctccttcaaaatc cttttggactcgcataacatgcacagaagaaggatggtcaccaacaccgaagtgtctcagaatgtgttcctttccttttgtgaaaaatg gtcattctgaatcttcaggactaatacatctggaaggtgatactgtacaaattatttgcaacacaggatacagccttcaaaacaatgag aaaaacatttcgtgtgtagaacggggctggtccactcctcccatatgcagcttcactaaaggagaatga

[0084] Therapy

[0085] In one embodiment, the invention discloses the isolated polypeptide described herein or the nucleic acid sequence described herein or the vector described herein for use in the treatment of a disease characterised by excessive complement activation. In one embodiment, the invention also discloses a method of treating of a disease characterised by excessive complement activation in a subject, comprising administering to the subject the isolated polypeptide described herein or the nucleic acid sequence described herein or the vector described herein. In one embodiment, the invention also discloses the use of the isolated polypeptide described herein or the nucleic acid sequence described herein or the vector described herein for the manufacture of a medicament for the treatment of a disease characterised by excessive complement activation in a subject.

[0086] The disease characterised by excessive complement activation is used to mean any disease, disorder or syndrome in which complement is incorrectly regulated. Complement may be inappropriately activated or may not be activated when required. Alternatively, the magnitude of any complement response may be inappropriate. Complement related diseases include some renal diseases, and certain autoimmune disorders. In particular, complement related diseases may include meningitis, renal disease, autoimmune disease or inflammation including conditions, such as rheumatoid arthritis, asthma, lupus nephritis, membranous nephropathy, infection-associated glomerulonephritis, post-infectious glomerulonephritis, paraprotein- associated glomerulopathy, Alport syndrome-associated nephritis, ischemia-reperfusion injury, atypical haemolytic uremic syndrome, thrombotic thrombocytopenic purpura, paroxysmal nocturnal haemoglobinuria, Membranoproliferative glomerulonephritis, hemolytic uremic syndrome, hypocomplementemic glomerulonephritis, dense deposit disease, macular degeneration (e.g. age-related macular degeneration, AMD), spontaneous foetal loss, Pauci- immune vasculitis, epidermolysis bullosa, recurrent foetal 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 anaemia, idiopathic thrombocytopenic purpura, Goodpasture syndrome, antiphospholipid syndrome, Infective endocarditis, or injury resulting from myocardial infarction, cardiopulmonary bypass and hemodialysis.

[0087] Many renal disorders are known to be due to complement dysregulation. Therefore, in one embodiment, the renal disease is selected from glomerulonephritis, C3 glomerulopathy, mesangiocapillary glomerulonephritis type 1 , 2 or 3, membranoproliferative glomerulonephritis type 1 , 2 or 3, dense deposit disease, C3 glomerulonephritis, immune complex membranoproliferative glomerulonephritis, immune complex glomerulonephritis, nephritis, lupus nephritis, CFHR5 nephropathy, IgA nephropathy or antibody mediated kidney transplant rejection.

[0088] In one embodiment, as part of the above-mentioned treatment of a disease, the nucleic acid and / or vector may transduce the subject’s own cells for producing domains 1 and 2 of human FHR5. This transduction may occur systemically in the liver or locally in the kidney.

[0089] In one embodiment, as part of the above-mentioned treatment of disease, the vector may comprise a viral mRNA therapy.

[0090] In one embodiment, the isolated polypeptide forms a homodimer. In one embodiment, the isolated polypeptide forms a heterodimer with endogenous FHR5. As part of the above- mentioned treatment of a disease the isolated polypeptide comprising domains 1 and 2 of human FHR5 may reversibly form homodimers as well as heterodimers with endogenous FHR5.

[0091] In one embodiment, as part of the above-mentioned treatment of a disease, the isolated polypeptide reduces the concentration or activity of endogenous FHR5.

[0092] In one embodiment, as part of the above-mentioned treatment of a disease, the isolated polypeptide prevents endogenous FHR5 homodimer formation.

[0093] In one embodiment, as part of the above-mentioned treatment of a disease, the isolated polypeptide inhibits cell surface deposition of Complement 3 (C3).

[0094] In one embodiment, as part of the above-mentioned treatment of a disease, the isolated polypeptide inhibits FHR5-mediated competitive antagonism of Factor H. The proposed mechanism of providing an isolated polypeptide comprising domains 1 and 2 FHR5 neither depletes or inhibits an abundant activating protein (e.g., C5 or C3 which circulate in gram scale quantities) nor is it supplementation of an already moderately abundant regulator (e.g., FH), where a high dose would be needed to provide clinical efficacy (i.e., to manipulate complement pharmacologically). The inventors have therefore provided an elegant solution to treat a disease characterised by excessive complement activation by inhibiting a very low abundant yet highly potent de-regulator.

[0095] Pharmaceutical Composition

[0096] Generally, unless indicated otherwise herein, the isolated polypeptide referred to herein will be intended for use in prophylaxis or treatment of diseases or disorders in man (and / or optionally also in warm-blooded animals and in particular mammals). Thus, they can be used as, and / or can suitably be a part of, a (biological) drug or other pharmaceutically or therapeutically active compound and / or of a pharmaceutical product or composition.

[0097] Thus, the invention also relates to a pharmaceutical composition comprising an isolated polypeptide of domains 1 and 2 of human FHR5, or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient. In another aspect, the invention relates to the isolated polypeptide or pharmaceutical composition as described herein for use as a medicament.

[0098] The pharmaceutical composition may optionally comprise one or more additional active agents, a pharmaceutically acceptable carrier, diluent, excipient or adjuvant. The isolated polypeptide can be administered by any convenient route, including but not limited to oral, topical, parenteral, sublingual, rectal, vaginal, ocular, intranasal, pulmonary, intradermal, intravitreal, intramuscular, intraperitoneal, intravenous, subcutaneous, intracerebral, transdermal, transmucosal, by inhalation, or topical, particularly to the ears, nose, eyes, or skin or by inhalation.

[0099] Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intraperitoneal, intranasal, rectal, intravesical, intradermal, topical or subcutaneous administration. Preferably, the compositions are administered parenterally. In one embodiment, the isolated polypeptide or the pharmaceutical composition is administered intravenously, subcutaneously, intramuscularly, or intradermally.

[0100] The pharmaceutically acceptable carrier or vehicle can be particulate, so that the compositions are, for example, in tablet or powder form. The term "carrier" refers to a diluent, adjuvant or excipient, with which a drug antibody conjugate of the present invention is administered. Such pharmaceutical carriers can be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. The carriers can be saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. In addition, auxiliary, stabilizing, thickening, lubricating and coloring agents can be used. Water is a preferred carrier when the drug antibody conjugates of the present invention are administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical carriers also include excipients such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The present compositions, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.

[0101] The pharmaceutical composition of the invention can be in the form of a liquid, e.g., a solution, emulsion or suspension. The liquid can be useful for delivery by injection, infusion (e.g., IV infusion) or sub-cutaneously. When intended for oral administration, the composition is preferably in solid or liquid form, where semi-solid, semi-liquid, suspension and gel forms are included within the forms considered herein as either solid or liquid.

[0102] As a solid composition for oral administration, the composition can be formulated into a powder, granule, compressed tablet, pill, capsule, chewing gum, wafer or the like form. Such a solid composition typically contains one or more inert diluents. In addition, one or more of the following can be present: binders such as carboxymethylcellulose, ethyl cellulose, microcrystalline cellulose, or gelatin; excipients such as starch, lactose or dextrins, disintegrating agents such as alginic acid, sodium alginate, corn starch and the like; lubricants such as magnesium stearate; glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin; a flavoring agent such as peppermint, methyl salicylate or orange flavoring; and a coloring agent. When the composition is in the form of a capsule (e. g. a gelatin capsule), it can contain, in addition to materials of the above type, a liquid carrier such as polyethylene glycol, cyclodextrin or a fatty oil.

[0103] The composition can be in the form of a liquid, e. g. an elixir, syrup, solution, emulsion or suspension. The liquid can be useful for oral administration or for delivery by injection. When intended for oral administration, a composition can comprise one or more of a sweetening agent, preservatives, dye / colorant and flavor enhancer. In a composition for administration by injection, one or more of a surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer and isotonic agent can also be included. Compositions can take the form of one or more dosage units. It may be desirable to administer the composition by intravenous injection or infusion.

[0104] The amount of the therapeutic that is effective / active in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. In addition, in vitro or in vivo assays can optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the compositions will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Factors like age, body weight, sex, diet, time of administration, rate of excretion, condition of the host, drug combinations, reaction sensitivities and severity of the disease shall be taken into account.

[0105] Typically, the amount is at least about 0.01 % of an isolated polypeptide of the present invention by weight of the composition. When intended for oral administration, this amount can be varied to range from about 0.1 % to about 80% by weight of the composition. Preferred oral compositions can comprise from about 4% to about 50% of the isolated polypeptide of the present invention by weight of the composition.

[0106] Preferred compositions of the present invention are prepared so that a parenteral dosage unit contains from about 0.01 % to about 2% by weight of the isolated polypeptide of the present invention.

[0107] For administration by injection, the composition can comprise from about typically about 0.1 mg / kg to about 250 mg / kg of the subject’s body weight, preferably, between about 0.1 mg / kg and about 20 mg / kg of the animal's body weight, and more preferably about 1 mg / kg to about 10 mg / kg of the animal's body weight. The composition may be administered at a dose of about 1 to 30 mg / kg, e.g., about 5 to 25 mg / kg, about 10 to 20 mg / kg, about 1 to 5 mg / kg, or about 3 mg / kg. The dosing schedule can vary from e.g., once a day to once every 1 , 2, 3, or 4 weeks.

[0108] As used herein, "treat", "treating" or "treatment" means inhibiting or relieving a disease or disorder. For example, treatment can include a postponement of development of the symptoms associated with a disease or disorder, and / or a reduction in the severity of such symptoms that will, or are expected, to develop with said disease. The terms include ameliorating existing symptoms, preventing additional symptoms, and ameliorating or preventing the underlying causes of such symptoms. Thus, the terms denote that a beneficial result is being conferred on at least some of the mammals, e.g., human patients, being treated. Many medical treatments are effective for some, but not all, patients that undergo the treatment. The term "subject" or "patient" refers to an animal which is the object of treatment, observation, or experiment. By way of example only, a subject includes, but is not limited to, a mammal, including, but not limited to, a human or a non-human mammal, such as a non-human primate, murine, bovine, equine, canine, ovine, or feline.

[0109] The molecules or pharmaceutical composition of the invention may be administered as the sole active ingredient or in combination with one or more other therapeutic agent. A therapeutic agent is a compound or molecule which is useful in the treatment of a disease. Examples of therapeutic agents include antibodies, antibody fragments, drugs, toxins, nucleases, hormones, immunomodulators, pro-apoptotic agents, anti-angiogenic agents, boron compounds, photoactive agents or dyes and radioisotopes.

[0110] According to an aspect of the invention, there is provided a kit comprising the isolated polypeptide according to one aspect of the invention or a pharmaceutical composition according to another aspect of the invention. The kit is not limited to these components and further components of the it may be apparent to the skilled person.

[0111] Methods

[0112] The invention also relates to an intro, in vivo or ex vivo method to reduce the concentration or activity of FHR5 protein comprising contacting a biological sample with the isolated polypeptide of claims 1 to 3.

[0113] The biological sample which is contacted may be selected from tissue, saliva, urine, blood including whole blood and plasma. The biological sample may be obtained from a healthy subject. The biological sample may be obtained from a subject having or suspected of a disease characterised by excessive complement activation.

[0114] The invention also relates to an in vitro, in vivo or ex vivo method of inhibiting FHR5 dimerisation comprising contacting a biological sample with the isolated polypeptide of the present invention.

[0115] The invention also relates to an in vitro, in vivo or ex vivo method of inhibiting C3 deposition comprising contacting a biological sample with the isolated polypeptide of the present invention.

[0116] The invention also relates to a method of monitoring the therapeutic efficacy of a treatment for a disease characterised by excessive complement activation, comprising determining the level of FHR5 dimerisation prior to administration of the therapy and determining the level of FHR5 dimerisation after administration of the therapy. The results from the sample obtained prior to therapy may provide a reference value to compare the results from the biological sample obtained after the subject has received therapy.

[0117] The invention also relates to a method of monitoring the therapeutic efficacy of a treatment for a disease characterised by excessive complement activation, comprising; determining the cell surface level of C3 prior to administration of the therapy and determining the cell surface level of C3 after administration of the therapy.

[0118] All documents mentioned in this specification are incorporated herein by reference in their entirety, including references to gene accession numbers, scientific publications and references to patent publications.

[0119] EXAMPLES

[0120] The invention is now further described in the non-limiting examples.

[0121] Example 1. Materials and Methods

[0122] His-tagged FHR5 was cloned into pCI-neo mammalian expression vector (Promega) and transiently transfected into Expi293F™ cells using Expifectamine™ 293 Transfection Kit (ThermoFisher) as per manufacturer’s instructions. 5 days post-transfection, cell media was separated from the cells by centrifugation at 3000 x g, 20 mins with the resultant supernatant subject to further filtration through a 0.45 pm filter and dilution 1 :1 with equilibration buffer (20 mM Sodium Phosphate, 300 mM NaCI, 10 mM Imidazole, pH 7.4) containing 1 complete EDTA- free protease inhibitor tablet (MERCK Life Sciences) per 100 mL.

[0123] 1 mL Ni-NTA resin (Qiagen) was added to the cell media preparation per 100 mL media preparation and incubated mixing gently at room temperature for 30 minutes. The mixture was poured into an empty spin column (Thermo Scientific) and filtered under vacuum to leave a packed Ni-NTA resin. The resin was washed with wash buffer (20 mM Sodium Phosphate, 300 mM NaCI, 25 mM Imidazole, pH 7.4) and protein eluted with the addition of 2 column volumes elution buffer (20 mM Sodium Phosphate, 300 mM NaCI, 250 mM Imidazole, pH 7.4) and centrifugation at 700 x g, 2 mins, 4° C. This elution step was repeated 5x.

[0124] The elution fractions were concentrated to a volume <500 pl and loaded on to a Superdex 200 10 / 300 size exclusion column (Cytiva) and washed through with 50 mM HEPES, 137 mM NaCI, pH 7.4. SCR1 / 2-His was cloned into pCI-neo mammalian expression vector (Promega) and transiently transfected into Expi293F™ cells using Expifectamine™ 293 Transfection Kit (ThermoFisher) as per manufacturer’s instructions. 5 days post-transfection, cell media was separated from the cells by centrifugation at 3000 x g, 20 mins with the resultant supernatant subject to further filtration through a 0.45 pm filter and dilution 1 :1 with equilibration buffer (20 mM sodium phosphate, 300 mM NaCI, 10 mM Imidazole, pH 7.4) containing 1 complete EDTA-free protease inhibitor tablet (MERCK Life Sciences) per 100 mL.

[0125] 1 mL Ni-NTA resin (Qiagen) was added to the cell media preparation per 100 mL media preparation and incubated mixing gently at room temperature for 30 minutes. The mixture was poured into an empty spin column (Thermo Scientific) and filtered under vacuum to leave a packed Ni-NTA resin. The resin was washed with wash buffer (20 mM Sodium Phosphate, 300 mM NaCI, 25 mM Imidazole, pH 7.4) and protein eluted with the addition of 2 column volumes elution buffer (20 mM Sodium Phosphate, 300 mM NaCI, 250 mM Imidazole, pH 7.4) and centrifugation at 700 x g, 2 mins, 4° C. This elution step was repeated 5x.

[0126] The elution fractions were concentrated to a volume <500 pl and loaded on to a Superdex 75 10 / 300 size exclusion column (Cytiva) and washed through with 50 mM HEPES, 137 mM NaCI, pH 7.4.

[0127] SCR1 / 2 was cloned into pET21 a ampicillin resistant bacterial expression vector (Novagen) and transformed into BL21 (DE3) E.coli (New England Biolabs). Overnight cultures were prepared by inoculating 2 x 10mL LB, 100 pg / mL ampicillin, 1 % glucose with single colonies of SCR1 / 2 pET21 a BL21 (DE3). These were incubated overnight, 37° C, 225 rpm. The following day, the overnight cultures were used to inoculate 2 x 1 L LB, 100 pg / mL ampicillin, 1 % glucose. The cultures were grown for ~3hrs at 37° C, 225 rpm until ODeoo = 0.6-0.8, at which point protein expression was induced by the addition of a final concentration of 1 mM IPTG and the cultures incubated for a further 4 hours at 37° C, 225 rpm. Bacterial cells were harvested by centrifugation at 6000 x g, 20 minutes, 4° C.

[0128] Bacterial pellets were resuspended in lysis buffer (20 mM Tris, 150 mM NaCI, 1 mM EDTA, pH 8.0 with 1 complete EDTA-free protease inhibitor cocktail tablet (MERCK Life Sciences) and 0.5 mg / mL PefablocOSC (Sigma-Aldrich). Following complete resuspension of the cells, deoxycholate was added to a final concentration of 0.4 % (W / V) and the mixture incubated at room temperature for 10 minutes. The next step in the lysis process was the addition of MgCI2 to a final concentration of 40 mM, 15 units of DNAse I (New England Biolabs) and 10 mg lysozyme and further incubation of 30 minutes at room temperature. Sonication of the sample to ensure maximum cell lysis was performed on ice using a Hielscher UP200S Ultrasonicator (200 W, 24 kHz); 7 cycles, 30 s on, 30 s off at 60 % amplitude. Homogenate was centrifuged at 10,000 x g, 30 min, 4° C.

[0129] Resulting inclusion body pellet was washed 2 x by resuspension in 20 mL PBS, Tween-20 0.05% and centrifugation 10,000 x g, 30 min, 4° C. Inclusion body pellet was solubilised in 8M Urea, 1 mM EDTA, 100 mM Tris-HCI, 25 mM DTT, pH 8.0 and incubated at room temperature for 2 hrs. Cell debris was removed from the preparation by centrifugation at 10,000 x g, 30 min, 4° C.

[0130] Protein was refolded using a rapid dilution method whereby the protein preparation was added dropwise at a rate of ~1 mL / hour into refolding buffer (0.5 M L-arginine, 1 mM L-cysteine, 2 mM L-cystine, 20 mM ethanolamine, pH 11.0) over ~20 hrs.

[0131] The refolded protein solution was concentrated and dialysed against 2 x 2 L 50 mM Tris, 137 mM NaCI, 1 mM EDTA, pH 8.5 at 4° C over a 24 hour period using 3.5 kDa MWCO dialysis membrane (Thermo Scientific). Anion Exchange Chromatography (AEC) was used as the first step of protein purification. The protein sample was passed through a Hi-Trap QFF column (Cytiva) and the bound protein eluted on a gradient of 137 - 500 mM NaCI. The corresponding protein peak elution fractions for SCR1 / 2 were concentrated and passed through a Superdex 75 10 / 300 Size Exclusion Chromatography column (Cytiva) in 50 mM Tris, 150 mM NaCI, 1 mM EDTA, pH 7.5 buffer.

[0132] The corresponding SCR1 / 2 peak was subjected to sequential ammonium sulphate precipitations; 1 .5 M and 2 M. The resultant precipitate at 2 M was re-solubilised in 50 mM Tris, 150 mM NaCI, 1 mM EDTA, pH 7.5 buffer and passed through a Superose 6 10 / 300 Increase size exclusion column.

[0133] Identities of all recombinant proteins were confirmed using liquid chromatography electrospray ionisation mass spectrometry. Data were obtained using an Acquity UPLC coupled to an Acquity SQD2 mass spectrometer. Proteins were dialysed into 100mM ammonium acetate at approximately 0.2mg / ml. Raw data was converted to zero charge mass spectra using the maximum entropy deconvolution algorithm in MassLynx or Masshunter

[0134] Incremental immobilisation of C3d-biotin on SA-surface plasmon resonance (SPR) chips: First immobilisation was run using the in-built wizard for an SA chip immobilisation, using C3d at 50nM. The chip was conditioned with three injections of 1 M NaCI, immobilised C3d to a level of 400 RU, and the system was then washed with 1 M NaCI, 50% isopropanol. For subsequent immobilisations, the system was switched to manual mode. Flow was switched to Fc2 at 30ul / min and C3d injected for 2 minutes, which gave a reliable addition to the response of the chip of approximately 400RU. Standard SPR runs for measuring avidity used a multicycle wizard with a two-solution regeneration strategy. Ligand was immobilised in Fc2 whilst Fc1 was kept as a reference. Analyte was injected for 70s to observe association and allowed to disassociate for 15 minutes. System was run at a flow rate of 30ul / min and a temperature of 20C. Running buffer for all experiments, unless stated otherwise, consisted of 10mM HEPES, 137mM NaCI, 0.05% Tween20 at pH 7.4. Sample was dialysed into running buffer overnight to ensure proper buffer matching and to alleviate “buffer jump” effects. For the regeneration conditions, 1 M NaCI and 10mM sodium acetate at pH 4.5 were used. Each run began with three start up cycles of buffer and regeneration conditions. Samples were run over 7 or 8 different concentrations, from the lowest to the highest concentrations with blank samples interspersed. The concentration closest to the estimated Kd was ran a second time at the end of the experiment to check that the responsiveness of the chip was the same. Chips were docked the day before use and equilibrated in running buffer overnight. Samples were capped to prevent evaporation. Kinetic data were analysed using a Langmuir kinetic fit models in the Biacore X100 evaluation software. Quality of fit was assessed by the Chi-squared value. Affinity data were analysed by fitting steady state responses to a Michaelis-Menten curve in Origin.

[0135] Biolayer interferometry was performed using an Octet RED96e system at 25C. Sample dilutions were made directly into a 96 well plate, to a volume of 200ul per well. Running buffer was 10mM HEPES, 137mM NaCI, 0.05% Tween20. Ligand was immobilised onto disposable streptavidin octet biosensors, each one used for one surface density of C3d, at one concentration. For each surface density of C3d tested, protein was loaded simultaneously onto 8 individual probes by dipping into wells containing biotinylated ligand until the desired nanometre shift had been obtained (300 seconds). Probes were then returned to running buffer for 180 seconds. Probes were dipped into analyte at 8 different concentrations (including blank) for 100 seconds to observe association, then placed into buffer for 600s to observe dissociation. Up to three different ligand densities per plate could be measured in this way. Binding was monitored in realtime by observing the nanometre shift of the biosensors. Affinity data were analysed by fitting steady state responses to a Michaelis-Menten curve in Origin.

[0136] Analytical ultracentrifugation was performed using the Beckman Optima or Beckman XL-I analytical ultracentrifuges at 20C and at 50,000rpm. Standard 12mm height double sector cells were used with sapphire windows. 400ul of sample or bufferwas pipetted into each sector, which were then sealed with a gasket and plug. Cells were then gently inserted into the ANTi50 rotor and radially aligned. The Optima was calibrated during an initial 3000rpm stage and baseline measurements were made. Once the samples had equilibrated to 20C, they were spun at 50,000rpm for approximately 17 hours. Absorbance at 280nm and interference optics were measured throughout. All experiments were performed in 10mM HEPES, 137mM NaCI pH7.4 measured by an Anton Paar DMA 5000 density meter at 20C to have a buffer density of 1 ,0053g / ml. The viscosity of this buffer was measured by an Anton Paar AMVn automated microviscometer to be 0.01002 poise. The partial specific volumes used in the data were determined by SLUV (PERKINS, 1986). Sedimentation boundary scans were fitted to the Lamm equation with SEDFIT, after visual inspection to mark the approximate location of the meniscus and to check for leaks. Absorbance data were fitted to the size distribution model (Schuck, 2000) with frictional ratio, meniscus position, and bottom position floated as parameters. Goodness of fit was assessed by Root-Mean-Squared-Deviation (RMSD) and visual inspection of the residuals bitmap. A good fit was judged to be an RMSD of below 0.009 and a monochromatic bitmap.

[0137] C3 deposition assay was performed using 70-80% confluent HK2 cells gently detached with EDTA and incubated for 2 hours with MHC class I (W6 / 32) monoclonal antibody 1 :15 in DMEM- / - which were then washed and incubated for 1 hour at 37C with normal human serum diluted 15:1 in VBS supplemented with recombinant proteins that had been incubated at 37C for 2 hours. Cells were then washed with cold PBS and incubated on ice for 1 hour with anti-C3 fluorescently labelled polyclonal antibody, washed twice with PBS and then subjected to FACS analysis to measure fluorescence intensity.

[0138] Day 0: 10-12 week old female C57BI / 6 mice were subcutaneously injected with 200 pl of a 1 :1 emulsion of 0.2 mg / mL Sheep IgG in 0.9 % sterile saline: Complete Freund’s Adjuvant. Day 5: The same mice were injected intravenously with 200 pl of a 1 :1 mixture of sheep nephrotoxic serum (a kind gift from Alan Salama):10 pg / mL Lipopolysaccharide (E.Coli R515), (Hycult Biotech) in 0.9% sterile saline. Day 9: Mice were transferred to metabolic cages for 16hrs overnight to collect urine samples. Day 10: Mice were injected intravenously with either 200 pL 0.9% sterile saline, 45 pg FHR5-His in 0.9 % sterile saline or 45 pg Albumin-His (Abeam) in 0.9 % sterile saline. Animals were sacrificed using schedule 1 method 1 hour after injection and kidneys harvested.

[0139] Example 2. FHR5 exists as a dimer in solution

[0140] Analytical ultracentrifugation in a solution with physiological salt concentration demonstrates that full length wild-type FHR5 protein sediments at 6.1 S, consistent with a molecular mass of 131 kDa, approximately double the molecular mass computed from the amino acid sequence and confirmed by electrophoresis of denatured recombinant protein (Figure 1). This is evidence that FHR5 exists as a dimer, with no monomer detected experimentally (Figure 2A). Analysis of recombinant fragments of FHR5 comprising SCR1 / 2 and SCR1 alone demonstrate that SCR1 / 2 is dimeric but SCR1 is a monomer (Figure 2B and 2C). Example 3. Avidity changes with C3d density

[0141] A streptavidin-coated Surface Plasmon Resonance (SPR) chip was incrementally loaded with C3d biotinylated (at a molar ratio of 2-3 biotin per C3d molecule) by injection of 50 nM C3d over 2 minutes. This achieved reliable addition to the response of around 400 RU allowing various densities of C3d (Figure 3A panel i) to be used to test FHR5 binding kinetics. Sensorgrams for 79RU, 91 RU, 618RU and 836RU C3d at various concentrations of FHR5 are shown (Figure 4A panels ii, iv, vi, viii, with the bivalent fitted data shown in Figure 3A panels Hi, v, vii, ix). Steady state responses at various concentrations of FHR5 (Figure 3B) demonstrated progressive reduction in Kd as the C3d density on the chip increased.

[0142] To compare avidity across a greater range of C3d densities, similar experiments were performed using Bio-Layer Interferometry using 8 different densities of C3d (Figure 4A). This demonstrated 2 orders of magnitude difference in Kd between the lowest (Kd = 2952 nM) and highest (Kd = 42 nM) densities (Figure 4B) that fitted a sigmoid curve (Figure 4C). The circulating concentration of FHR5 is around 70 nM so these biophysical data provide direct evidence that avidity of FHR5 for a surface coated in C3d is dependent on the density of C3d on the surface and that a threshold C3d density is needed for the Kd to reach a physiologically relevant range.

[0143] Example 4. FHR5 co-localises with mouse C3 in vivo

[0144] Immunoglobulins purified from the serum of sheep that had been immunised against mouse glomerular basement membrane protein were injected into mice that had been pre-immunised against sheep immunoglobulin resulting in binding of the sheep immunoglobulin to the mouse glomerular capillary walls and inflammation resulting from their recognition by the mouse immune system. This results in glomerulonephritis (‘nephrotoxic nephritis”) in which complement C3 is deposited at the site of inflammation. Mice were injected with His-tagged human FHR5 protein or His-tagged human albumin and killed 1 hour later. Immunofluorescence studies on the mouse glomeruli revealed co-localisation of the human FHR5 (but not albumin) with murine C3 (Figure 5).

[0145] Example 5. R512 inhibits CFHR5 dimerization in vitro

[0146] Buffer containing full length wild type FHR5 protein was mixed with that containing R512 (i.e., FHR5 SCR1 / 2) at a molar ratio of 1 :2 (FHR5:R512) for one hour at 37 degrees Celsius. The mixture was then subjected to analytical ultracentrifugation which resulted in detection of a species sedimenting at a rate intermediate between each pure protein (Figure 6A) consistent with reduction of full-length homodimers due to generation of heterodimers comprising FHR5 and R512. This indicates that circulating FHR5 homodimers are likely amenable to disruption pharmacologically in solution. When full length FHR5 was similarly incubated with (monomeric) SCR1 only domain protein at similar or even greater molar ratios, evidence of heterodimerization was not observed (Figure 6B).

[0147] Example 6. R512 fragment reduces C3 deposition in the presence of FHR5

[0148] Mammalian cells were decorated with monoclonal lgG1 antibody recognising MHO molecules on their surface. After incubation with human serum (with or without supplemental purified human C3, recombinant FHR5 and / or FHR5 SCR1 / 2 proteins) cells were washed and stained with FITC-labelled goat-anti human C3 immunoglobulin and fluorescence was measured with a flow cytometer to quantify C3 deposited on each cell. Mean fluorescence intensity was increased by supplementation with FHR5 at two different doses, and the effect was potentiated by coincubation with purified human C3 protein (Figure 7).

[0149] When serum was supplemented with SCR1 / 2 in addition to FHR5 (at molar ratios of 3:1 and 10:1) fluorescence intensity of C3 staining decreased in a dose-dependent fashion to below levels seen when serum with no FHR5 supplementation was used, raising the possibility of inhibition by SCR1 / 2 of endogenous FHR5 protein contained in the serum (Figure 8A). When cells were supplemented with SCR1 alone in addition to FHR5, even at higher doses, no inhibition of C3 deposition was observed (Figure 8B).

[0150] Example 7. Modulation of FHR5 deposition in mouse nephrotoxic nephritis model

[0151] Materials and Methods

[0152] DNA preparation:

[0153] Human CFHR5 DNA with a 6 x His 3’ addition was synthesised and cloned into the pCI-Neo mammalian expression vector (GenScript).

[0154] SCR1 / 2 - A length of DNA representing the first two short consensus repeat (SCR) sequences of CFHR5 with a 6 x His 3’ addition was synthesised and cloned into the pCI-neo mammalian expression vector as described above.

[0155] 10-50 ng of CFHR5-His pCI-neo or SCR1 / 2-His pCI-neo were transformed into XL10 Gold ultracompetent cells, as per manufacturers protocol (Stratagene). The transformed cells were plated onto LB agar containing 100 pg / mL ampicillin and incubated at 37°C overnight to allow selection of successful transformants. A single colony was selected from the transformation selection plates and used to inoculate LB broth containing 100 pg / mL ampicillin to be used for DNA amplification and purification using Promega PureYieldTM Plasmid Midiprep Kit, as per manufacturers protocol (Promega).

[0156] The correct gene sequence was confirmed by DNA sequencing (Eurofins Genomics) and the remaining DNA was used for transfection into Expi293FTM cells for protein expression.

[0157] Protein expression:

[0158] Plasmid DNA was transfected into Expi293FTM cells (Thermo Fisher Scientific) using ExpiFectamineTM 293 transfection kit as per manufacturers protocol (Thermo Fisher Scientific).

[0159] Protein purification:

[0160] Five days post-transfection expressed protein was purified from the cell culture media in a two- step process; affinity purification using Ni-NTA agarose (Qiagen) prior to size exclusion chromatography on a Superdex 200 Increase 10 / 300 column (Cytiva).

[0161] Ni-NTA purification:

[0162] Expression cell media was cleared by centrifugation at 700 g, 10 minutes, the resultant supernatant was passed through a 0.45 pm vacuum filter to remove cell debris. The cell supernatant was mixed 1 :1 with Ni-NTA equilibration buffer (50 mM Sodium Phosphate, 300 mM NaCI, 10 mM Imidazole, pH 7.4) with complete EDTA free protease inhibitor tablets added (Merck Life Science UK), 1 tablet per 100 mL.

[0163] Ni-NTA agarose was equilibrated with equilibration buffer and added to the cleared supernatant, 1 mL Ni-NTA agarose / 100 mL cleared supernatant, and mixed at RT for 30 minutes. Ni-NTA agarose was poured into a spin column and washed with Ni-NTA Wash buffer (50 mM Sodium Phosphate, 300 mM NaCI, 25 mM Imidazole, pH 7.5). Bound protein was eluted using Ni-NTA elution buffer (50 mM Sodium Phosphate, 300 mM NaCI, 250 mM Imidazole, pH 7.4). The presence of recombinant protein in the elution fractions was confirmed by Western Blot using an anti-6xHis antibody.

[0164] Following Ni-NTA purification, the eluted protein was concentrated prior to loading onto a Superdex 200 Increase 10 / 300 size exclusion column attached to an AKTA chromatography system (Cytiva). The protein sample was washed through the column with 50 mM Hepes, 137 mM NaCI, 1 mM EDTA, pH7.4.

[0165] Mouse model of nephrotoxic nephritis:

[0166] Carried out under Home Office Project Licence PP2388079. Day 1 - Pre-immunisation: 8-12 week old female C57BI / 6 mice were injected subcutaneously with up to 200 pL (maximum 10 mL / Kg) of a 1 :1 emulsion of Complete Freund’s Adjuvant (Sigma-Aldrich) : Sheep IgG (Sigma- Aldrich) 0.2 mg / mL in 0.9% saline. 3 mice were untreated. Day 6: Mice that had been preimmunised were given iv injections of up to 100 pL (maximum 5 mL / Kg) of a 1 :1 solution of sheep nephrotoxic serum (a kind gift from Alan Salama) : 10 pg / mL Lipopolysaccharide (E.coli R515) in 0.9 % Saline (Cambridge Bioscience). Day 9: Mice were transferred to metabolic cages for 16 hours overnight for urine collection. Day 10 - For glomerular complement C3 and FHR5 co-localisation studies: Mice were injected iv with either 1 pg, 5 pg, 20 pg or 45 pg FHR5-His in 0.9 % Saline or 45 pg Albumin-His (Abeam) 1 hr prior to killing by cervical dislocation.

[0167] For inhibition studies of FHR5 co-localisation to glomerular C3 deposits by SCR1 / 2:

[0168] Mice were injected iv with either 20 pg FHR5-His, 94 pg SCR1 / 2-His or a mix of 20 pg FHR5- His & 94 pg SCR1 / 2-His (1 :20 molar ratio) that had been pre-incubated at 37C for 3 hrs.

[0169] OR

[0170] Mice were injected ivwith a single dose of either 2 pg FHR5-His in 0.9 % saline, 9.4 pg SCR1 / 2- His in 0.9 % saline or sequential injections of 9.4 pg SCR1 / 2-His in 0.9 % saline followed immediately by a second injection of 2 pg FHR5-His in 0.9 % saline.

[0171] Controls used were mice untreated for NTN but with iv injections of FHR5-His or SCR1 / 2-His or both and NTN mice that had 0.9 % saline iv instead of protein.

[0172] Mice were killed by cervical dislocation 1 hour after iv injection of either FHR5, SCR1 / 2 or a combination of protein. Kidneys were harvested immediately and prepared for histological examination.

[0173] Preparation of kidneys for histological examination:

[0174] Following harvesting, the mouse kidneys were fixed in Paraformaldehyde Lysine Periodate (PLP) fixative (0.1 M lysine monohydrochloride, 0.05 M disodium hydrogen orthophosphate, 1 % paraformaldehyde, 10 mM sodium metaperiodate, pH 7.4) for 4 hours at 4°C. Following fixation, the mouse kidneys were incubated in 10 % sucrose overnight at 4°C before embedding in OCT and snap freezing in isopentane cooled with liquid nitrogen.

[0175] Immunofluorescence:

[0176] 10 pm sections of OCT embedded mouse kidney were cut on a Bright OTF5000 cryostat and collected on Polysine coated slides (VWR).

[0177] Slides were thawed at room temperature for 10 minutes then rehydrated in Phosphate Buffered Saline for 10 minutes. Tissue sections were blocked with 10 % Donkey Serum (Sigma-Aldrich) in Phosphate Buffered Saline + 0.05 % Triton X-100 for 30 minutes, room temperature. Tissue sections were incubated with 100 pl of 0.01 pg / pl rabbit polyclonal to 6 x His tag primary antibody (Abeam) at room temperature for 1 hour.

[0178] Slides were washed with PBS + 0.05 % Triton X-100 and, where necessary, incubated with 100 pl of 0.01 pg / pl Donkey F(ab’)2 anti-rabbit IgG H&L (AlexaFluor647) pre-adsorbed secondary antibody (Abeam) at room temperature for 1 hour.

[0179] Slides were washed with PBS +0.05 % Triton X-100 and, where necessary, incubated with 100 pl of a 1 :50 dilution of anti-human complement C3 goat IgG fraction (MP Biomedicals), fluorescein-conjugated primary antibody at room temperature for 1 hour.

[0180] Slides were washed with PBS + 0.05 % Triton X-100 followed by PBS alone.

[0181] Coverslips were mounted with Prolong Gold antifade mountant (Thermo Fisher Scientific) and samples allowed to cure for a minimum of 24 hours before imaging.

[0182] Slides were imaged on a Zeiss LSM 700 confocal microscope.

[0183] Immunofluorescence signals were quantified using Imaged by isolating glomerular tufts as the region of interest and measuring the mean fluorescence intensity density. Threshold values were set using images of kidney sections from NTS-only treated mice as a control and values were corrected for signal from mice receiving SCR1 / 2 but no full length FHR5.

[0184] Results

[0185] Pre-mixing 20 pg human FHR5 (full length) with 94 pg SCR1 / 2 (a molar ratio of 1 :20) resulted in reduction in localisation of FHR5 to inflamed mouse glomeruli, consistent with FHR5 inhibition by de-dimerization (Figure 9, N=3 for each treatment group. Immunofluorescence images shown correspond to the medians in each group shown in the bar chart). Quantification of the immunofluorescence signal across 8 representative glomeruli in 2 sections showed a 61 % reduction in intensity of FHR5 staining the presence of SCR1 / 2 (p<0.001).

[0186] When 9.4 pg SCR1 / 2 was injected intravenously immediately prior to 2 pg full length tagged recombinant human FHR5 (i.e. a molar ratio of 20:1) a substantial reduction in accumulation of glomerular FHR5 was observed compared with FHR5 injection in the absence of SCR1 / 2 (Figure 10, N=3 for each treatment group. Immunofluorescence images shown correspond to the medians in each group shown in the bar chart). Quantification of the immunofluorescence signal across 8 representative glomeruli in 2 sections showed a 49% reduction in the intensity of FHR5 staining in the presence of SCR1 / 2 (p=0.044). References

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[0219] Sequences

[0220] Domains 1 and 2 of FHR5 (SEQ ID NO: 1):

[0221] EGTLCDFPKIHHGFLYDEEDYNPFSQVPTGEVFYYSCEYNFVSPSKSFWTRITCTEEGWSPT PKCLRMCSFPFVKNGHSESSGLIHLEGDTVQIICNTGYSLQNNEKNISCVERGWSTPPICSFTK G

[0222] Domains 1 and 2 of FHR5 for expression in mammalian cells (SEQ ID NO: 2) MLLLFSVILISWVSTVGGEGTLCDFPKIHHGFLYDEEDYNPFSQVPTGEVFYYSCEYNFVSPSK SFWTRITCTEEGWSPTPKCLRMCSFPFVKNGHSESSGLIHLEGDTVQIICNTGYSLQNNEKNI SCVERGWSTPPICSFTKGHHHHHH

[0223] Domains 1 and 2 of FHR5 for expression in bacteria (SEQ ID NO: 3):

[0224] MASMTGGQQMGRGSEGTLCDFPKIHHGFLYDEEDYNPFSQVPTGEVFYYSCEYNFVSPSKS FWTRITCTEEGWSPTPKCLRMCSFPFVKNGHSESSGLIHLEGDTVQIICNTGYSLQNNEKNIS CVERGWSTPPICSFTKGE Full length human FHR5 (SEQ ID NO: 4):

[0225] MLLLFSVILISWVSTVGGEGTLCDFPKIHHGFLYDEEDYNPFSQVPTGEVFYYSCEYNFV SPSKSFWTRITCTEEGWSPTPKCLRMCSFPFVKNGHSESSGLIHLEGDTVQIICNTGYSL QNNEKNISCVERGWSTPPICSFTKGECHVPILEANVDAQPKKESYKVGDVLKFSCRKNLI RVGSDSVQCYQFGWSPNFPTCKGQVRSCGPPPQLSNGEVKEIRKEEYGHNEWEYDCNPN FIINGPKKIQCVDGEWTTLPTCVEQVKTCGYIPELEYGYVQPSVPPYQHGVSVEVNCRNE YAMIGNNMITCINGIWTELPMCVATHQLKRCKIAGVNIKTLLKLSGKEFNHNSRIRYRCS DIFRYRHSVCINGKWNPEVDCTEKREQFCPPPPQIPNAQNMTTTVNYQDGEKVAVLCKEN YLLPEAKEIVCKDGRWQSLPRCVESTAYCGPPPSINNGDTTSFPLSVYPPGSTVTYRCQS FYKLQGSVTVTCRNKQWSEPPRCLDPCVVSEENMNKNNIQLKWRNDGKLYAKTGDAVEFQ CKFPHKAMISSPPFRAICQEGKFEYPICE

[0226] Nucleotide sequence of domains 1 and 2 of FHR5 for expression in mammalian cells (SEQ ID NO: 5) atgttgctcttattcagtgtaatcctaatctcatgggtatccactgttgggggagaaggaacactttgtgattttccaaaaatacaccatgg atttctgtatgatgaagaagattataaccctttttcccaagttcctacaggggaagttttctattactcctgtgaatataattttgtgtctccttc aaaatccttttggactcgcataacatgcacagaagaaggatggtcaccaacaccgaagtgtctcagaatgtgttcctttccttttgtga aaaatggtcattctgaatcttcaggactaatacatctggaaggtgatactgtacaaattatttgcaacacaggatacagccttcaaaac aatgagaaaaacatttcgtgtgtagaacggggctggtccactcctcccatatgcagcttcactaaaggacatcatcatcatcatcattg a

[0227] Nucleotide sequence of domains 1 and 2 of FHR5 for expression in bacteria (SEQ ID NO: 6): Atggctagcatgactggtggacagcaaatgggtcgcggatccgaaggaacactttgtgattttccaaaaatacaccatggatttctgt atgatgaagaagattataaccctttttcccaagttcctacaggggaagttttctattactcctgtgaatataattttgtgtctccttcaaaatc cttttggactcgcataacatgcacagaagaaggatggtcaccaacaccgaagtgtctcagaatgtgttcctttccttttgtgaaaaatg gtcattctgaatcttcaggactaatacatctggaaggtgatactgtacaaattatttgcaacacaggatacagccttcaaaacaatgag aaaaacatttcgtgtgtagaacggggctggtccactcctcccatatgcagcttcactaaaggagaatga

[0228] CFHR5-His DNA sequence (Includes 5’ Sal I restriction site and 3’ GSG linker, 6xHis tag & Not I restriction site) (SEQ ID NO: 7):

[0229] GTCGACATGTTGCTCTTATTCAGTGTAATCCTAATCTCATGGGTATCCACTGTTGGGGGAG AAGGA

[0230] ACACTTTGTGATTTTCCAAAAATACACCATGGATTTCTGTATGATGAAGAAGATTATAAC CCTTTTTCCCAAGTTCCTACAGGGGAAGTTTTCTATTACTCCTGTGAATATAATTTTGTG TCTCCTTCAAAATCCTTTTGGACTCGCATAACATGCACAGAAGAAGGATGGTCACCAACA CCGAAGTGTCTCAGAATGTGTTCCTTTCCTTTTGTGAAAAATGGTCATTCTGAATCTTCA GGACTAATACATCTGGAAGGTGATACTGTACAAATTATTTGCAACACAGGATACAGCCTT CAAAACAATGAGAAAAACATTTCGTGTGTAGAACGGGGCTGGTCCACTCCTCCCATATGC

[0231] AGCTTCACTAAAGGAGAATGTCATGTTCCAATTTTAGAAGCCAATGTAGATGCTCAGCCA AAAAAAGAAAGCTACAAAGTTGGAGACGTGTTGAAATTCTCCTGCAGAAAAAATCTTATA

[0232] AGAGTTGGATCAGACTCAGTTCAATGTTACCAATTTGGGTGGTCACCTAACTTTCCAACA

[0233] TGCAAAGGACAAGTACGATCATGTGGTCCACCTCCTCAACTCTCCAATGGTGAAGTTAAG

[0234] GAGATAAGAAAAGAGGAATATGGACACAATGAAGTAGTGGAATATGATTGCAATCCTAAT

[0235] TTTATAATAAACGGGCCTAAGAAAATACAATGTGTGGATGGAGAATGGACAACTTTACCC

[0236] ACTTGTGTTGAACAAGTGAAAACATGTGGATACATACCTGAACTCGAGTACGGTTATGTT

[0237] CAGCCGTCTGTCCCTCCCTATCAACATGGAGTTTCAGTCGAGGTGAATTGCAGAAATGAA

[0238] TATGCAATGATTGGAAATAACATGATTACCTGTATTAATGGAATATGGACAGAGCTTCCT

[0239] ATGTGTGTTGCAACACACCAACTTAAGAGGTGCAAAATAGCAGGAGTTAATATAAAAACA

[0240] TTACTCAAGCTATCTGGGAAAGAATTTAATCATAATTCTAGAATACGTTACAGATGTTCA

[0241] GACATCTTCAGATACAGGCACTCAGTCTGTATAAACGGGAAATGGAATCCTGAAGTAGAC

[0242] TGCACAGAAAAAAGGGAACAATTCTGCCCACCGCCACCTCAGATACCTAATGCTCAGAAT

[0243] ATGACAACCACAGTGAATTATCAGGATGGAGAAAAAGTAGCTGTTCTCTGTAAAGAAAAC

[0244] TATCTACTTCCAGAAGCAAAAGAAATTGTATGTAAAGATGGACGATGGCAATCATTACCA

[0245] CGCTGTGTTGAGTCTACTGCATATTGTGGGCCCCCTCCATCTATTAACAATGGAGATACC

[0246] ACCTCATTCCCATTATCAGTATATCCTCCAGGGTCAACAGTGACGTACCGTTGCCAGTCC

[0247] TTCTATAAACTCCAGGGCTCTGTAACTGTAACATGCAGAAATAAACAGTGGTCAGAACCA

[0248] CCAAGATGCCTAGATCCATGTGTGGTATCTGAAGAAAACATGAACAAAAATAACATACAG

[0249] TTAAAATGGAGAAACGATGGAAAACTCTATGCAAAAACAGGGGATGCTGTTGAATTCCAG

[0250] TGTAAATTCCCACATAAAGCGATGATATCATCACCACCATTTCGAGCAATCTGTCAGGAA

[0251] GGGAAATTTGAATATCCTATATGTGAAGGTAGTGGTCATCATCATCATCATCATTGAGCGG CCGC

[0252] SCR1 / 2-His DNA sequence (Includes 5’ Sal I restriction site and 3’ GSG linker, 6xHis tag & Not

[0253] I restriction site) (SEQ ID NO: 8):

[0254] GTCGACATGTTGCTCTTATTCAGTGTAATCCTAATCTCATGGGTATCCACTGTTGGGGGAG

[0255] AAGGAACACTTTGTGATTTTCCAAAAATACACCATGGATTTCTGTATGATGAAGAAGATTAT

[0256] AACCCTTTTTCCCAAGTTCCTACAGGGGAAGTTTTCTATTACTCCTGTGAATATAATTTTGT

[0257] GTCTCCTTCAAAATCCTTTTGGACTCGCATAACATGCACAGAAGAAGGATGGTCACCAAC

[0258] ACCGAAGTGTCTCAGAATGTGTTCCTTTCCTTTTGTGAAAAATGGTCATTCTGAATCTTCA

[0259] GGACTAATACATCTGGAAGGTGATACTGTACAAATTATTTGCAACACAGGATACAGCCTTC

[0260] AAAACAATGAGAAAAACATTTCGTGTGTAGAACGGGGCTGGTCCACTCCTCCCATATGCA GCTTCACTAAAGGAGGTAGTGGTCATCATCATCATCATCATTGAGCGGCCGC

Claims

Claims1 . An isolated polypeptide comprising domains 1 and 2 of human complement factor H-related 5 (FHR5) protein.

2. The isolated polypeptide of claim 1 , wherein the isolated polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 1 or a sequence with at least 80% sequence identity thereto.

3. The isolated polypeptide of claim 1 or 2, wherein the isolated polypeptide comprises an amino acid sequence with at least 85%, 90%, 95%, 98%, 99% or 100% sequence identity with the sequence set forth in SEQ ID NO: 1 .

4. The isolated polypeptide according to any preceding claim, wherein the isolated polypeptide forms a homodimer.

5. The isolated polypeptide according to any preceding claim, wherein the isolated polypeptide forms a heterodimer with endogenous FHR5.

6. The isolated polypeptide according to any preceding claim, wherein the isolated polypeptide reduces the concentration or activity of endogenous FHR5.

7. The isolated polypeptide according to any preceding claim, wherein the isolated polypeptide prevents endogenous FHR5 homodimer formation.

8. The isolated polypeptide according to any preceding claim, wherein the isolated polypeptide inhibits cell surface deposition of Complement 3 (C3).

9. The isolated polypeptide according to any preceding claim, wherein the isolated polypeptide inhibits FHR5-mediated competitive antagonism of Factor H.

10. The isolated polypeptide according to any preceding claim, wherein the isolated polypeptide comprises a purification tag, wherein the purification tag is suitable for purifying the isolated polypeptide from mammalian and / or bacterial cells .11 . The isolated polypeptide according to any preceding claim, wherein the isolated polypeptide comprises a linker region.

12. The isolated polypeptide according to any preceding claim, wherein the isolated polypeptide comprises at least one post-translational modification, preferably wherein at least one of thepost-translational modifications is selected from PEGylation, phosphorylation, acetylation, methylation, glycosylation, succinylation, ubiquitination, hydroxylation, sumoylation, amidation, or glutathionylation,13. A nucleic acid sequence that encodes the amino acid sequence of the isolated polypeptide according to any of claims 1 to 12.

14. A vector comprising a nucleic acid sequence according to claim 13.

15. A host cell comprising the nucleic acid sequence according to claim 13 or the vector of claim 14.

16. The isolated polypeptide comprising domains 1 and 2 of human FHR5 according to any of claims 1 to 12 or the nucleic acid sequence according to claim 13 or the vector according to claim 14 for use in the treatment of a disease characterised by excessive complement activation.

17. A method of treating of a disease characterised by excessive complement activation in a subject, comprising administering to the subject the isolated polypeptide comprising domains 1 and 2 of human FHR5 according to any of claims 1 to 12 or the nucleic acid sequence according to claim 13 or the vector according to claim 14.

18. Use of the isolated polypeptide according to any of claims 1 to 12 or the nucleic acid sequence according to claim 13 or the vector according to claim 14 comprising domains 1 and 2 of human FHR5 for the manufacture of a medicament for the treatment of a disease characterised by excessive complement activation in a subject.

19. The isolated polypeptide or the nucleic acid or the vector for use according to claim 16, the method according to claim 17, or the use according to claim 18, wherein the disease is selected from meningitis, renal disease, autoimmune disease or inflammation including conditions, such as rheumatoid arthritis, asthma, lupus nephritis, membranous nephropathy, infection-associated glomerulonephritis, post-infectious glomerulonephritis, paraprotein-associated glomerulopathy, Alport syndrome-associated nephritis, ischemia-reperfusion injury, atypical haemolytic uremic syndrome, thrombotic thrombocytopenic purpura, paroxysmal nocturnal haemoglobinuria, Membranoproliferative glomerulonephritis, hemolytic uremic syndrome, hypocomplementemic glomerulonephritis, dense deposit disease, macular degeneration (e.g. age-related macular degeneration, AMD), spontaneous foetal loss, Pauci-immune vasculitis, epidermolysis bullosa, recurrent foetal 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 anaemia, idiopathic thrombocytopenicpurpura, Goodpasture syndrome, antiphospholipid syndrome, Infective endocarditis, or injury resulting from myocardial infarction, cardiopulmonary bypass and hemodialysis20. The isolated polypeptide or the nucleic acid or the vector for use or the method or the use according to claim 19, wherein the renal disease is selected from glomerulonephritis, C3 glomerulopathy, mesangiocapillary glomerulonephritis type 1 , 2 or 3, membranoproliferative glomerulonephritis type 1 , 2 or 3, dense deposit disease, C3 glomerulonephritis, immune complex membranoproliferative glomerulonephritis, immune complex glomerulonephritis, nephritis, lupus nephritis, CFHR5 nephropathy, IgA nephropathy or antibody mediated kidney transplant rejection.21 . A pharmaceutical composition comprising the isolated polypeptide according to any claims 1 to 12 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.

22. A pharmaceutical composition according to claim 21 , further comprising one or more additional active agents, a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.

23. The isolated polypeptide according to any of claims 1 to 12, or the pharmaceutical composition according to claim 21 for use as a medicament.

24. The isolated polypeptide for use according to any of claims 1 to 12, or the pharmaceutical composition according to claim 21 wherein the isolated polypeptide or the pharmaceutical composition is administered intravenously, subcutaneously, intramuscularly, or intradermally.

25. A kit comprising an isolated polypeptide according to any of claims 1 to 12 or a pharmaceutical composition according to claim 21 or 22 and instructions for use.

26. An intro, in vivo or ex vivo method to reduce the concentration or activity of FHR5 protein comprising contacting a biological sample with the isolated polypeptide according to any of claims 1 to 12.

27. An in vitro, in vivo or ex vivo method of inhibiting FHR5 dimerisation comprising contacting a biological sample with the isolated polypeptide according to any of claims 1 to 12.

28. An in vitro, in vivo or ex vivo method of inhibiting C3 deposition comprising contacting a biological sample with the isolated polypeptide according to any of claims 1 to 12.

29. A method of monitoring the therapeutic efficacy of a treatment for a disease characterised by excessive complement activation, comprising determining the level of FHR5 dimerisation prior to administration of the isolated polypeptide according to any one of claims 1 to 12 or the pharmaceutical composition according to claims 21 or 22 and determining the level of FHR5 dimerisation after administration.

30. A method of monitoring the therapeutic efficacy of a treatment for a disease characterised by excessive complement activation, comprising; determining the cell surface level of C3 prior to administration of the isolated polypeptide according to any one of claims 1 to 12 or the pharmaceutical composition according to claims 21 or 22 and determining the cell surface level of C3 after administration.

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