WO2023175099A1 - Agents pour le traitement de troubles liés au complément - Google Patents

Agents pour le traitement de troubles liés au complément Download PDF

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WO2023175099A1
WO2023175099A1 PCT/EP2023/056794 EP2023056794W WO2023175099A1 WO 2023175099 A1 WO2023175099 A1 WO 2023175099A1 EP 2023056794 W EP2023056794 W EP 2023056794W WO 2023175099 A1 WO2023175099 A1 WO 2023175099A1
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complement
fhr1
fhr5
fhr3
fhr2
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Paul Bishop
Richard Unwin
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Complement Therapeutics Limited
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/472—Complement proteins, e.g. anaphylatoxin, C3a, C5a
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70596—Molecules with a "CD"-designation not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to the fields of molecular biology and medicine. More specifically, the present invention relates to agents for treating complement-related disorders and methods for detecting complement proteins to inform treatment strategies.
  • the complement system contributes to innate host immune defence by assisting in the rapid recognition and elimination of microbial intruders.
  • dysregulation of complement can contribute to inflammatory, immune-related, and age-related conditions.
  • inappropriate regulation of the complement system has been implicated in a wide variety of diseases in humans e.g. diseases of the eye and kidney, as well as neurological diseases and cancer (Morgan, B.P., Semin Immunopathol, 2018. 40(1): p. 113-124; Halbgebauer, R., et al., Semin Immunol, 2018. 37: p. 12-20; Ma, Y., et al., Aging Dis, 2019. 10(2): p. 429-462; and Kleczko, E.K., et al., Front Immunol, 2019. 10: p. 954.
  • Complement pathway activation and control is regulated by a complex interplay between pathway activators and inhibitors.
  • These activators and inhibitors are commonly enzymes which cleave and inactivate complement molecules on biological surfaces and/or in solution to maintain steady regulation of complement activating species.
  • the complement pathways are in a constant state of flux and balance, and disturbances to this balance can lead to inappropriate activation and the consequences above.
  • C3 complement component 3
  • C3 comprises a p chain and an a’ chain which associate through interchain disulphide bonds.
  • C3 is cleaved to generate two functional fragments, C3a and C3b.
  • C3a is a potent anaphylatoxin.
  • Deposition of C3b on biological surfaces, e.g. extracellular matrix and cell surfaces, is the central activating mechanism of the alternative pathway.
  • C3b is a potent opsonin, targeting pathogens, antibody-antigen immune complexes and apoptotic cells for phagocytosis by phagocytes and NK cells.
  • C3b also reacts with other complement proteins to form active convertase enzymes that are able to produce further (surface-attachable) C3b molecules, serving to activate and amplify complement responses (Clark, S.J., et al., J Immunol, 2014. 193(10): p. 4962-70).
  • C3b associates with Factor B to form the C3bBb-type C3 convertase and with C3bBb to form the C3bBb3b-type C5 convertase.
  • Proteolytic cleavage of C3 also produces C3a and C3b through the classical complement pathway and the lectin pathway.
  • C3b activation of complement is regulated by complement protein factor I (Fl).
  • Fl prevents complement activation by cleaving C3b to a proteolytically-inactive form, designated iC3b, which is unable to participate in convertase assembly, and further to downstream products iC3dg and C3d.
  • Fl requires the presence of a cofactor, examples of which include the blood-borne Factor H (FH) protein and the membrane-bound surface co-factor ‘complement factor 1 ’ (CR1 ; CD35).
  • FH and CR1 also help to exert decay-accelerating activity, which can assist in the deconstruction of already formed C3 convertases.
  • FH is encoded by the CFH gene on human chromosome 1q32 within the RCA (regulators of complement) gene cluster.
  • FH-like protein 1 FHL-1
  • FHL-1 FH-like protein 1
  • the CFHR1-5 genes encode a group of five secreted plasma proteins (FHR1 to FHR5) synthesised primarily by hepatocytes and found in the circulation.
  • the FHR proteins retain some sequence homology with C3b binding domains of FH and are thought to enhance complement activation (Skerka et al., Mol Immunol. 2013, 56:170-180).
  • the five FHR proteins are thought to counter the inhibitory effects of FH and FHL-1 , and thus contribute to the pathology of complement-related disorders (Clark, S.J. and P.N. Bishop, J Clin Med, 2015. 4(1): p. 18-31).
  • Elevated levels of one or more of the five FHR proteins have been implicated in a variety of complement- related disorders affecting different tissues, such as those in the kidney (see e.g. Medjeral-Thomas et al., Kidney Int Rep. 2019, 4(10):1387-1400;Wong & Kavanagh, Semin Immunopathol. 2018; 40(1): 49-64; Sethi et al., Kidney Int. 2009, 75(9):952-60; Abrera-Abeleda et al., J Med Genet. 2006, 43(7): 582-589), autoimmune diseases (see e.g.
  • AMD age-related macular degeneration
  • Macular degeneration is believed to be driven in part by complement-mediated attack on ocular tissues.
  • a major driver of AMD risk is genetic variation at the RCA locus resulting in dysregulation of the complement cascade.
  • AMD is the leading cause of blindness in the developed world: currently responsible for 8.7% of all global blind registrations. It is estimated that 196 million people will be affected by 2020, increasing to 288 million by 2040 (Wong et al. Lancet Glob Heal (2014) 2:e106-16). AMD manifests as the progressive destruction of the macula, the central part of the retina at the back of the eye, leading to loss of central visual acuity.
  • choriocapillaris a layer of capillaries found in the choroid (a highly vascularized layer that supplies oxygen and nutrition to the outer retina).
  • the choriocapillaris is separated from the metabolically active retinal pigment epithelium (RPE) by Bruch’s membrane (BrM); a thin (2-4 pm), acellular, five-layered sheet of extracellular matrix.
  • the BrM serves two major functions: the substratum of the RPE and a blood vessel wall. The structure and function of BrM is reviewed e.g.
  • Drusen are formed from the accumulation of lipids, proteins and cellular debris, and include a swathe of complement activation products (Anderson et al., Prog Retin Eye Res 2009, 29:95-112; Whitcup et al., Int J Inflam 2013, 1-10).
  • the presence of drusen within BrM disrupts the flow of nutrients from the choroid across this extracellular matrix to the RPE cells, which leads to cell dysfunction and eventual death, leading to the loss of visual acuity.
  • FHL-1 predominates at BrM, suggesting an important role for this variant in protection of retinal tissue from complement-mediated attack (Clark, S.J., et al., supra).
  • FH is found in the blood at a higher concentration than FHL-1 .
  • Both FH and FHL-1 protect against complement over-activation in the ECM of the choroid (the capillary network underlying BrM).
  • WO2019/215330 describes that FHR4 is a positive regulator of complement activation and prevents FH-mediated C3b breakdown, leading to the formation of C3 convertase and the progression of the complement activation loop.
  • the present invention seeks to address these issues.
  • the present invention relates to treating diseases, disorders and conditions associated with pathological levels of Complement Factor H-related proteins (FHR proteins).
  • FHR proteins Complement Factor H-related proteins
  • Methods provided herein may involve detecting complement proteins, e.g. one or more FHR proteins, and using the results of said detection to identify the risk of onset of complement-related disorders, inform treatment and treat said disorders.
  • the present application provides nucleic acids and polypeptides that are useful in treating the complement- related disorders.
  • the present application describes a method which is capable of distinguishing between Complement Factor H, FHL-1 and the five Complement Factor H related (FHR) proteins, despite their sequence similarity.
  • the method can also distinguish between breakdown products derived from C3, and other complement-related proteins with high sequence similarity.
  • nucleic acid for use in a method of treating or preventing a complement-related disorder in a subject, wherein the nucleic acid comprises a nucleotide sequence, and wherein the nucleotide sequence comprises or consists of a sequence having at least 80% sequence identity to SEQ ID NO:176 or SEQ ID NO:177.
  • nucleic acid comprising a nucleotide sequence, wherein the nucleotide sequence comprises or consists of a sequence having at least 80% sequence identity to SEQ ID NO:176 or SEQ ID NO:177, in the manufacture of a medicament for treating or preventing a complement-related disorder.
  • the complement-related disorder is characterised by elevated levels of one or more of FHR1 , FHR2, FHR3, FHR4 and/or FHR5.
  • the level of one of more FHR proteins may be detected or determined as described herein.
  • the complement-related disorder is selected from macular degeneration, Age- related Macular Degeneration (AMD), Geographic Atrophy (‘dry’ or non-exudative AMD), early AMD, early onset macular degeneration (EOMD), intermediate AMD, late/advanced AMD, ‘wet’ (neovascular or exudative) AMD, choroidal neovascularisation (CNV), retinal dystrophy, autoimmune uveitis, Haemolytic Uremic Syndrome (HUS), atypical Haemolytic Uremic Syndrome (aHUS), DEAP HUS (Deficiency of FHR plasma proteins and Autoantibody Positive form of Hemolytic Uremic Syndrome), kidney injury/damage/dysfunction, glomerular diseases, Membranoproliferative Glomerulonephritis Type II (MPGN II), sepsis, Henoch-Schbnlein purpura (HSP), IgA nephropathy, chronic kidney disease, paroxysmal nocturnal hemoglobin
  • the subject has elevated levels of one or more of FHR1 , FHR2, FHR3, FHR4 and/or FHR5. In some embodiments the subject has been determined to have elevated levels of one or more of FHR1 , FHR2, FHR3, FHR4 and/or FHR5, for example using the methods provided herein, optionally in a blood or plasma sample obtained from the subject.
  • the method prior to the administration of the nucleic acid, the method comprises:
  • step (a) comprises determining the level of two, three, four or five of FHR1 , FHR2, FHR3, FHR4 and/or FHR5.
  • the method comprises determining the level of: i. FHR1 and FHR2; ii. FHR1 and FHR3;
  • the level of FHR1 , FHR2, FHR3, FHR4 and/or FHR5, and optionally FH and/or FHL-1 is determined by mass spectrometry.
  • step (a) comprises:
  • the treatment and/or prevention comprises modifying at least one cell of a subject to express or comprise the nucleic acid.
  • the at least one cell may be a cell of the eye, kidney, vascular system, blood, muscle, skin, oesophagus, small or large intestine, intestinal tract, pharynx, trachea, lungs, bronchi, bronchioles, or central nervous system.
  • the at least one cell may be a cancer cell or a tumor cell.
  • the treatment and/or prevention comprises administering a vector comprising the nucleic acid.
  • the vector may be an AAV vector as described herein.
  • the treatment and/or prevention comprises administering a recombinant AAV as described herein, comprising the nucleic acid.
  • the nucleotide sequence encodes a polypeptide that is capable of binding to C3b.
  • nucleotide sequence encodes a polypeptide that is capable of acting as a cofactor for Fl-mediated breakdown of C3b. In some embodiments the nucleotide sequence encodes a polypeptide comprising a portion of CR1. In some embodiments the nucleotide sequence encodes a polypeptide, the polypeptide comprising an amino acid sequence, and wherein said amino acid sequence consists of a sequence having at least 80% sequence identity to SEQ ID NO:146.
  • the nucleic acid comprises a nucleotide sequence encoding a signal peptide.
  • the nucleotide sequence encoding a signal peptide has at least 80% sequence identity to SEQ ID NO:181.
  • the signal peptide encoded by said nucleotide sequence may have at least 80% sequence identity to SEQ ID NQ:180.
  • nucleotide sequence consists of SEQ ID NO:176 or SEQ ID NO:177.
  • nucleotide sequence comprises or consists of a sequence having at least 80% sequence identity to SEQ ID NO:176 or SEQ ID NO:177, the method comprising:
  • the invention relates to treating and preventing diseases and conditions associated with dysregulated complement using nucleic acids, nucleotide sequences and vectors. Also disclosed herein is the treatment and/or prevention of particular patients characterised by FHR status, based on the measured observation that circulating levels of all five Factor H-related (FHR) proteins in human blood are elevated in a population of individuals suffering from a number of complement-related disorders, such as those in the eye, kidney, CNS and in cancers. Circulating levels of these FHR proteins derive exclusively from the liver as their only known source of expression in the human body. Expression of said proteins is to a large extent genetically driven.
  • FHR Factor H-related
  • FHR proteins to out-compete the negative regulators of complement activation (i.e. FH and FHL-1) means their increased concentration can pre-dispose a patient to be more complement-active.
  • the detection of overexpression of one or more FHR proteins, e.g. as described herein, is therefore predictive of an individual’s likelihood of developing a complement-related disorder. Increased levels of such proteins can also reveal a worse clinical outcome.
  • FHR proteins prevent Factor l-mediated breakdown of C3b, leading to pathological complement over-activation.
  • polypeptides and nucleic acids encoding said polypeptides that comprise C3b binding domains that are able to compete for C3b binding with the FHR proteins, thus helping to promote C3b breakdown to iC3b and further to e.g., C3dg.
  • the polypeptides outcompete FHR proteins and can reduce the pathological effects of C3b accumulation in affected tissues.
  • Methods disclosed herein also relate to the detection and quantification of complement related proteins, particularly one or more FHR proteins and optionally FHL-1 and/or FH. Such methods are useful to identity and stratify patients with disorders related to over-activity of the complement system due to increased levels of FHR proteins. Such methods may be used to stratify patients based on their risk of developing or having complement-related disorders.
  • MS mass spectrometry
  • proteins e.g. in a sample are routinely digested into peptides using a specific protease.
  • the industry standard protease for this purpose is trypsin.
  • Other enzymes that are commonly used to digest proteins for MS analysis include elastase, chymotrypsin or LysN.
  • Trypsin cleaves C-terminal to all K and R residues, provided they are not followed by a proline residue, and yields peptides which retain a basic group at their C-terminus which subsequently helps ionisation and transmission of peptides into the gas phase in a mass spectrometer.
  • Peptides digested by trypsin tend to be ionised more efficiently during MS and thus produce a larger signal than peptides digested by non-trypsin enzymes.
  • MS individual peptides in the sample digest can be detected with a signal proportional to its abundance.
  • the concentration of the parent protein can be derived from the relative abundance (signal) of endogenous peptide compared to an exogenous ‘standard’ peptide e.g. containing a stable isotope.
  • FH and FHL-1 Trypsin digestion of complement proteins FH and FHL-1 does not produce peptides that can be detected individually using MS alone.
  • the only FHL-1 specific tryptic peptide is a 4-amino acid C-terminal sequence which is too small to be detected reliably by MS techniques.
  • the FHR proteins also share substantial sequence identity, meaning that it is hard to distinguish between them and measure them specifically using e.g. antibody-based assays.
  • Also described herein is a targeted mass spectrometry assay using a non-standard proteolytic enzyme, GluC (V8 protease), to produce distinct proteotypic peptides for all the FHR proteins, as well as proteotypic peptides that can be used to distinguish between FHL-1 and FH, which can be used for the simultaneous detection and accurate measurement in plasma of all seven key regulatory proteins encoded from the CFH gene cluster using a single MS assay: FH, FHL-1 , and FHR1 , FHR2, FHR3, FHR4 and FHR5.
  • GluC V8 protease
  • FHL-1 is a distinct biological entity from FH.
  • the proteins have a similar action but the size of FHL-1 means that its distribution in the body is likely to be distinct from FH. This is apparent in the eye where FHL-1 can cross to the retinal side of Bruch’s membrane, e.g. where drusen form, but the larger FH protein cannot, see e.g. Clark et al., J Immunol 2014, 193(10) 4962-4970 and Clark et al., Frontiers in Immunology 2017 8:1778, which are hereby incorporated by reference in their entirety. In this respect, there is evidence that FHL-1 is the prime driver of complement C3b turnover in the eye, meaning that levels of FHL-1 are likely to better inform disease risk than levels of FH.
  • GluC is also able to produce proteotypic peptides for C3b and Fl, enabling direct measurement of C3b itself as well as levels of its proteolytic enzyme and required fluid-phase cofactors.
  • the methods described herein mean that all these complement proteins can be measured using a single assay.
  • C3 turnover can be measured using the MS approach of the present invention because GluC digestion also produces proteotypic neopeptides from many C3 inactivation and breakdown products generated during inactivating cleavages.
  • the inventors demonstrate herein that a series of products produced as a result of C3/C3b cleavage can be detected and quantified using the same single GluC/MS assay. This allows the concentrations of all known C3 fragments e.g.
  • iC3b, C3c, C3dg and C3d can not only measure absolute levels of regulatory complement proteins, but can also track protein products resulting from C3 inactivation and thus assess complement activation and the progression of the amplification loop.
  • complementome a single methodology for concurrent determination of the presence, absolute levels and relative molar ratios of up to seven individual complement-related proteins from the CFH family plus C3b-inactivating enzyme Fl, central complement component C3, and seven proteins derived from C3 breakdown, which may be referred to herein as the “complementome”.
  • the ability to detect absolute levels of so many complement-related proteins in one assay is critical for the successful detection, diagnosis and treatment of complement-related diseases using the nucleic acid and polypeptide agents provided herein.
  • Complement is a central part of the innate immunity that serves as a first line of defence against foreign and altered host cells. Complement is activated upon infection with microorganisms to induce inflammation and promote elimination of the pathogens.
  • the complement system is composed of plasma proteins produced mainly by the liver or membrane proteins expressed on cell surface. Complement operates in plasma, in tissues, or within cells.
  • the complement system can be activated via three distinct pathways: the classical pathway (CP), alternative pathway (AP) and lectin binding pathway (LP).
  • CP classical pathway
  • AP alternative pathway
  • LP lectin binding pathway
  • C3b molecules bound to host cells are inactivated rapidly by a group of membrane-bound or plasma complement regulators.
  • complement proteins become sequentially activated in an enzyme cascade: the activation of one protein enzymatically cleaves and activates the next protein in the cascade.
  • C3 convertase which cleaves the central complement component C3 into activation products C3b, a large fragment that acts as an opsonin (binds to foreign microorganisms to increase their susceptibility to phagocytosis), and C3a, an anaphylatoxin that promotes inflammation.
  • C3b forms the C3 convertase (C3bBb) which cleaves further C3 molecules, generates more C3b and C3a, and amplifies C3b deposition on cell surfaces. This is the complement amplification loop.
  • C3b deposition and activation of complement may occur on acellular structures (i.e. on extracellular matrix), such as Bruch’s membrane (BrM) and the intercapillary septa of the choriocapillaris in the eye.
  • Activated C3 can trigger the lytic pathway, which can damage the plasma membranes of cells and some bacteria.
  • C5a another anaphylatoxin produced by this process, attracts macrophages and neutrophils and also activates mast cells.
  • complement activation products e.g. C3b
  • C3b complement activation products
  • a number of soluble as well as membrane bound complement regulators ensure regulation of complement activation at the surface of host cells and control different activation phases and sites of action (Skerka et al., Mol Immunol 2013, 56:170-180). Complement regulators are described further herein.
  • “Complement protein” may be used interchangeably herein with “complement regulator”, “a regulator of complement”, or “protein of the complement system” and refers to a protein component of the complement system or complement cascade, e.g. as described in Merle et al., Front. Immunol., 2015, 6:262 and Merle et al., Front. Immunol., 2015, 6:257, which are hereby incorporated by reference in their entirety.
  • a “complement protein” referred to herein may be involved in any of the three complement pathways and/or in the amplification loop. In some embodiments a “complement protein” referred to herein is involved in the alternative pathway and/or the complement activation loop. In some embodiments, a “complement protein” referred to herein is involved in the breakdown, turnover and/or inactivation of C3 or C3b, or is a product of said breakdown, turnover and/or inactivation.
  • a “complement protein” as used herein may refer to one or more of FH, FHL-1 , FHR1 , FHR2, FHR3, FHR4, FHR5, Fl, C3, C3b, C3a, iC3b, C3f, C3c, C3dg, and/or C3d.
  • Factor H regulates the alternative complement pathway and the amplification loop. It inhibits C3 convertase formation by competing with FB binding to C3b and also acts as a cofactor for C3b inactivation to iC3b by Factor I (Fl), thus preventing inappropriate complement activation and inflammation. FH also exerts decay-accelerating activity, which can assist in the deconstruction of already formed C3 convertases, see e.g. Clark et al., J Immunol 2014, 193(10) 4962-4970, which is hereby incorporated by reference in its entirety.
  • Human FH comprises 20 CCP domains.
  • the CFH gene also produces a truncated form of FH, called FHL-1 , comprising only the first seven CCP domains before terminating with a unique 4-amino acid C terminus (Clark et al, 2014 supra).
  • the sequence of human FHL-1 (Uniprot: P08603-2) is provided herein as SEQ ID NO:2.
  • FH protein is found on the choroidal side of Bruch’s membrane (BrM), with particular accumulation in the choriocapillaris (capillary layer in the choroid). Small amounts have also been found in patches on the RPE side of the BrM, but no FH was observed in the BrM itself.
  • FHL-1 on the other hand has been observed throughout BrM and other ECM structures e.g. drusen (Clark et al, 2014 supra). It is likely that FHL-1 confers greater complement protection to BrM than does FH, whereas FH provides the main protection for the ECM of the choroid. It is thought that FHL-1 is therefore a major regulator of complement in the BrM (a key site in AMD pathogenesis). The methods described herein allow for the individual detection and quantitation of FH and FHL-1.
  • FH, FHL-1 and FHR1-FHR5 are described in e.g. Clark et al., J Clin Med, 2015. 4(1): 18-31 , which is hereby incorporated by reference in its entirety.
  • FHR1 , FHR2, FHR3, FHR4 and FHR5, encoded by the CFHR genes are also described in e.g. Skerka et al., Mol Immunol 2013, 56:170-180, which is hereby incorporated by reference in its entirety. These proteins are highly related and share a high degree of sequence identity. The N termini share 36-94% sequence identity, whilst the C-terminal domains are very similar to the FH C-terminus (36-100%). The high amino acid identity among family members is demonstrated by the fact that antibodies raised against FH can detect multiple FHR proteins in plasma and that antibodies generated against FHR proteins cross-react with the other FHRs. This cross-reactivity presents a challenge for purification of FHR proteins from plasma, as well as determining their concentration.
  • FHR proteins are divided into two groups depending on their conserved domains.
  • FHR1 SEQ ID NO:3
  • FHR2 SEQ ID NO:3, 4
  • FHR5 SEQ ID NQ:10
  • Group I is characterised by their conserved N-termini. They exist in plasma as homo- and heterodimers, mediated by the conserved N- terminal domains.
  • Group II contains FHR3 (SEQ ID NO:6, 7) and FHR4 (SEQ ID NO:8, 9) which lack the N-terminal 12imerization domains, but which show a high degree of sequence similarity to portions of FH.
  • All five FHR proteins comprise C-termini sequences that act to recognise and bind C3b, and which are very similar to the C-terminus of FH.
  • FHR1 is known to compete with FH and FHL-1 for binding to C3b. It is also reported to bind to C3b components of the C5 convertase and interfere with the assembly of the MAC (see e.g. Heinen S et al., Blood (2009) 114 (12): 2439-2447 and Hannan JP et al., PloS One. 2016; 11 (11):e0166200, which are hereby incorporated by reference in their entirety).
  • FHR1 includes at least one of FHR1 (SEQ ID NO:3; FHRA) and a second FHR1 isoform (FHRB) with 3 point mutations, and preferably includes both FHR1 isoforms.
  • FHR1 refers to FHR1 from any species and includes isoforms, fragments, variants or homologues of FHR1 from any species. In preferred embodiments, “FHR1” refers to human FHR1 .
  • FHR2 may inhibit C3 convertase activity, acting to inhibit the amplification loop, but may also activate the amplification loop.
  • the protein has two glycosylated forms, a single glycosylated form (24 kDa) and a double glycosylated form (28 kDa).
  • the term “FHR2” includes at least one of the two isoforms or at least one of the glycosylated forms, and preferably includes both isoforms and any glycosylated forms.
  • FHR2 refers to FHR2 from any species and includes isoforms, fragments, variants or homologues of FHR2 from any species. In preferred embodiments, “FHR2” refers to human FHR2.
  • FHR3 binds to C3b and C3d and may have low cofactor activity for Fl-mediated cleavage of C3b. FHR3 may also upregulate complement. There are two FHR3 isoforms (SEQ ID NO:6 and 7). FHR3 is detected in plasma in multiple variants (ranging from 35 to 56 kDa), reflecting the existence of four different glycosylated variants of FHR3. As used herein, the term “FHR3” includes at least one of the two isoforms or at least one of the glycosylated variants of FHR3, and preferably includes both isoforms and any glycosylated forms. “FHR3” refers to FHR3 from any species and includes isoforms, fragments, variants or homologues of FHR3 from any species. In preferred embodiments, “FHR3” refers to human FHR3.
  • the human CFHR4 gene encodes two proteins: FHR4A (SEQ ID NO:8) and FHR4B (SEQ ID NO:9), an alternative splice variant.
  • FHR4A SEQ ID NO:8
  • FHR4B SEQ ID NO:9
  • WO 2019/215330 A1 describes that FHR4 is a positive regulator of complement activation and prevents FH-mediated C3b breakdown.
  • High levels of FHR4 in tissues are likely to promote local inflammatory responses and cell lysis, leading to disorders associated with complement activation, and circulating FHR4 levels can be used as an indicator of risk of developing complement-related disorders, see e.g. Cipriani et al., Nat Commun 11 , 778 (2020), hereby incorporated by reference in its entirety.
  • FHR4 includes at least one of FHR4A isoform 1 , FHR4A isoform 2 (G20 point deletion from isoform 1) or FHR4B, and preferably includes FHR4A isoforms 1 and 2 as well as FHR4B.
  • FHR4 refers to FHR4 from any species and includes isoforms, fragments, variants or homologues of FHR4 from any species. In preferred embodiments, “FHR4” refers to human FHR4.
  • FHR5 also recognises and binds to C3b on self surfaces. FHR5 appears as a glycosylated protein of 62 kDa. As used herein, the term “FHR5” includes any glycosylated variants of FHR5, and preferably includes all isoforms and any glycosylated forms. As used herein, “FHR5” refers to FHR5 from any species and includes isoforms, fragments, variants or homologues of FHR5 from any species. In preferred embodiments, “FHR5” refers to human FHR5.
  • CFH family members particularly FHR1-5, can also be used as biomarkers for diagnosing or predicting disorders in which dysregulation of complement is pathologically implicated.
  • C3 is the central complement component.
  • the pathways by which C3 is processed into various downstream products can lead to activation of complement, e.g. including inflammation and immune responses, or to the inactivation and regulation of complement. It is therefore important in terms of complement pathogenesis and treatment of complement-related disorders to be able to detect and measure the levels, including relative levels, of C3, C3b and their downstream components/processing products.
  • Human C3 (UniProt: P01024; SEQ ID NO:12) comprises a 1 ,663 amino acid sequence (including an N-terminal, 22 amino acid signal peptide). Amino acids 23 to 667 encode C3 p chain (SEQ ID NO:13), and amino acids 749 to 1 ,663 encode C3b a’ chain (SEQ ID NO:14).
  • C3 p chain and C3 a’ chain associate through interchain disulphide bonds (formed between cysteine 559 of C3 p chain, and cysteine 816 of the C3 a’ chain) to form C3b.
  • C3a is a 77 amino acid fragment corresponding to amino acid positions 672 to 748 of C3 (SEQ ID NO:15), generated by proteolytic cleavage of C3 to form C3b.
  • iC3b comprises the C3 p chain, C3 a’ chain fragment 1 and C3 a’ chain fragment 2 (associated via disulphide bonds).
  • Cleavage of the a’ chain also liberates C3f, which corresponds to amino acid positions 1304 to 1320 of C3 (SEQ ID NO:18).
  • iC3b is processed further to C3c comprising the C3 p chain, C3 a’ chain fragment 2 and C3c a’ chain fragment 1 (corresponding to amino acid positions 749-954 of C3; SEQ ID NO:19).
  • This cleavage event produces fragment C3dg (corresponding to amino acid positions 955-1303 of C3; SEQ ID NO:142), which is itself broken down into fragments C3g (corresponding to amino acid positions 955-1001 of C3; SEQ ID NO:143) and C3d (corresponding to amino acid positions 1002-1303 of C3; SEQ ID NO:144).
  • Complement Factor I (Fl; encoded in humans by the gene CFI).
  • Human Complement Factor I (UniProt: P05156; SEQ ID NO:11) has a 583 amino acid sequence (including an N-terminal, 18 amino acid signal peptide).
  • Amino acids 340 to 574 of the light chain encode the proteolytic domain of Fl, which is a serine protease containing the catalytic triad responsible for cleaving C3b to produce iC3b (Ekdahl et al., J Immunol (1990) 144 (11): 4269-74).
  • Proteolytic cleavage of C3b by Fl to yield iC3b is facilitated by co-factors, including FH, CR1 and possibly some of the FHR proteins.
  • Co-factors for Fl typically bind to C3b and/or Fl, and potentiate processing of C3b to iC3b by Fl.
  • any reference to a complement protein refers to said protein from any species and include isoforms, fragments, variants or homologues of said protein from any species.
  • the protein is a mammalian protein (e.g. cynomolgus, human and/or rodent (e.g. rat and/or murine) protein).
  • Isoforms, fragments, variants or homologues of the complement proteins described herein may optionally be characterised as having at least 70%, preferably one of 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of the immature or mature protein from a given species, e.g. human protein sequences provided herein.
  • Isoforms, fragments, variants or homologues of complement proteins described herein may optionally be functional isoforms, fragments, variants or homologues, e.g. having a functional property/activity of the reference protein, as determined by analysis by a suitable assay for the functional property/activity.
  • the invention relates to molecules, such as nucleic acids and polypeptides, for example that are useful for treating complement-related disorders, as described herein.
  • the complement-related disorders may be disorders associated with elevated levels or expression of FHR proteins.
  • the nucleic acids and polypeptides described herein may be used with any method of detection, determination or treatment provided herein.
  • Vectors, cells and compositions comprising the nucleic acids and polypeptides are also envisaged for use in the methods described herein.
  • nucleic acid sequences that encode polypeptides which comprise or consist of portions of human Complement Receptor 1 (CR1). Any polypeptide, or portion of polypeptide, described herein may be encoded by, or partly encoded by, a nucleic acid as described herein.
  • polypeptide or ‘polypeptides’ herein may be taken to apply to a polypeptide encoded by a nucleic acid or nucleotide sequence, such as those disclosed herein.
  • nucleic acid or ‘nucleotide sequence’ herein may be taken to apply to a nucleic acid or nucleotide sequence that encodes a polypeptide(s) such as those disclosed herein.
  • Complement Receptor 1 refers to CR1 from any species and includes isoforms, fragments, variants or homologues of CR1 from any species.
  • the CR1 is mammalian CR1 (e.g. cynomolgus, human and/or rodent (e.g. rat and/or murine) CR1).
  • CR1 transcript variant S mRNA is provided at NCBI NM_000651 .6 (Gl 1731160520, version 6).
  • CR1 transcript variant F mRNA is provided at NCBI NM_000573.4 (Gl 1677499597, version 4).
  • Human CR1 (UniProt: P17927 (Entry version 205 (23 Feb 2022), Sequence version 3 (02 Mar 2010)); SEQ ID NO:1) has a 2,039 amino acid sequence (including an N-terminal, 41 amino acid signal peptide), and comprises 30 complement control protein (CCP) domains (also known as sushi domains or short consensus repeats (SCRs)), with the N-terminal 28 CCPs organised into four long homologous repeat (LHR) domains each comprising 7 CCPs: LHR-A, LHR-B, LHR-C and LHR-D.
  • CCP complement control protein
  • LHR long homologous repeat
  • CCPs 8-10 in LHR-B (UniProt: P17927 positions 491 to 684; SEQ ID NO:146), and CCPs 15-17 in LHR-C (UniProt: P17927 positions 941 to 1134; SEQ ID NO:147).
  • CCPs 8-10 and 15-17 differ in sequence by three amino acid residues.
  • nucleic acid sequences that encode polypeptides that are capable of binding to C3b.
  • the polypeptides can act as cofactors for Factor I, e.g. during Factor l-mediated breakdown/inactivation of C3b. Further functional properties of the nucleic acids and polypeptides are described hereinbelow.
  • the polypeptides may comprise or consist of C3b-binding portions of CR1 (indicated in SEQ ID NO: 184).
  • the polypeptides may comprise or consist of CR1 CCP domains that bind to C3b.
  • the polypeptides may comprise or consist of a sequence corresponding to CR1 LHR-B (e.g. positions 491-939 of SEQ ID NO:184), or a portion thereof, and/or CR1 LHR-C (positions 941-1389), or a portion thereof.
  • nucleic acid encoding a polypeptide that comprises or consists of an amino acid sequence corresponding to CCPs 8-10 of CR1 (i.e. SEQ ID NO:146).
  • nucleic acid encoding a polypeptide that comprises or consists of an amino acid sequence corresponding to CCPs 15- 17 of CR1 i.e. SEQ ID NO:147.
  • a nucleic acid of the present disclosure may comprise or consist of a nucleotide sequence corresponding to SEQ ID NO: 178 or 179.
  • a nucleic acid of the present disclosure may comprise or consist of a nucleotide sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:178 or 179.
  • the nucleic acid sequence may be codon-optimised to improve the codon composition without altering the encoded amino acid sequence.
  • a nucleic acid may comprise or consist of a nucleotide sequence corresponding to or based on SEQ ID NO:178, but wherein the codons have been optimised for expression in particular cells.
  • the cells may be human cells.
  • the cells may be any cells that are affected by a complement related disorder, e.g. as described herein.
  • the cells may be in a tissue or organ affected by a complement related disorder.
  • the nucleic acid sequence may be codon-optimised for expression in human cells of the eye, kidney, vascular system, blood, muscle, skin, oesophagus, small or large intestine, intestinal tract, pharynx, trachea, lungs, bronchi, bronchioles, central nervous system, or brain.
  • the cells may be RPE cells.
  • the cells may be epithelial cells.
  • the cells may be endothelial cells.
  • the cells may be kidney cells, such as glomerular endothelial cells, macula densa, mesangial cells, parietal epithelial cells, podocytes, or tubule epithelial cells.
  • a nucleic acid of the present disclosure may comprise or consist of a nucleotide sequence corresponding to SEQ ID NO: 176.
  • a nucleic acid of the present disclosure may comprise or consist of a nucleotide sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:176.
  • nucleic acid described herein may comprise additional nucleotide sequence(s), in addition to a nucleotide sequence described hereinabove. Sequence identity of a nucleotide sequence to a SEQ ID NO provided herein may be assessed over the whole nucleic acid. Alternatively, sequence identity may be assessed over the specified nucleotide sequence only (e.g. over the sequence of SEQ ID NO:176, 177, 178 or 179 only). In some cases, the nucleic acid may comprise additional nucleotide sequence(s) that are not taken into account when assessing sequence identity, e.g. to SEQ ID NO:176, 177, 178 and/or 179.
  • a nucleic acid sequence, or nucleotide sequence, described herein may be defined by its length.
  • a nucleic acid may be defined by its ‘total length’, e.g. including any nucleotide sequence comprised therein and any additional sequence(s), from 5’ to 3’.
  • a nucleic acid disclosed herein may be defined by the length of the nucleotide sequence comprised therein (i.e. the nucleotide sequence comprises or consisting of a sequence having at least 70% sequence identity to SEQ ID NO:176, 177, 178 and/or 179).
  • the nucleic acid may comprise additional nucleotide sequence(s) that renders the sequence of the entire nucleic acid longer than the specified length of said nucleotide sequence having at least 70% sequence identity to SEQ ID NO:176, 177, 178 and/or 179.
  • the nucleic acid, or the nucleotide sequence contained within the nucleic acid may comprise or consist of an nucleotide sequence that is 12 kb or fewer, 11 .5 kb or fewer, 11 kb or fewer, 10.5 kb or fewer, 10 kb or fewer, 9.5 kb or fewer, 9 kb or fewer, 8.5 kb or fewer, 8 kb or fewer, 7.5 kb or fewer, 7 kb or fewer, 6.5 kb or fewer, 6 kb or fewer, 5.5 kb or fewer, 5 kb or fewer, 4.5 kb or fewer, 4 kb or fewer, 3.3 kb or fewer, 3 kb or fewer, 2.5 kb or fewer, 2 kb or fewer, 1 .5 kb or fewer ,or 1 kb or fewer in length.
  • the nucleic acid, or the nucleotide sequence contained within the nucleic acid may comprise or consist of a nucleotide sequence of 1000 bp or fewer, 990 bp or fewer, 980 bp or fewer, 970 bp or fewer, 960 bp or fewer, 950 bp or fewer, 940 bp or fewer, 930 bp or fewer, 920 bp or fewer, 910 bp or fewer, 900 bp or fewer, 890 bp or fewer, 880 bp or fewer, 870 bp or fewer, 860 bp or fewer, 850 bp or fewer, 840 bp or fewer, 830 bp or fewer, 820 bp or fewer, 810 bp or fewer, 800 bp or fewer, 790 bp or fewer, 780 bp or fewer, 770 bp or fewer, 760 bp
  • nucleic acid or nucleotide sequence as above, that comprises or consists of a nucleotide sequence having ‘up to’ any length of bp described herein, e.g. up to 600 bp, up to 610 bp etc. Any two end points described herein may be used to make a range of lengths for the nucleic acid or nucleotide sequence, for example 600-800 bp in length or any combination of end points above.
  • the nucleic acid, or the nucleotide sequence contained within the nucleic acid may comprise or consist of a nucleotide sequence that is 500-1000 bp, 500-950 bp, 500-900 bp, 500-850 bp, 500-800 bp, 500-750 bp, 500-700 bp, 500-650 bp, 525-800 bp, 550-700 bp, of 550-650 bp in length.
  • the nucleic acid, or nucleotide sequence contained within the nucleic acid has a length of 550-600 bp or 620-660 bp.
  • a nucleic acid described herein may be longer than the nucleotide sequence comprised therein (said nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 176, 177, 178 and/or 179).
  • a nucleic acid provided herein may have one length above, whilst said nucleotide sequence may be of a shorter length. Any of the lengths and/or ranges above can be combined.
  • a nucleic acid may be 10 kb or fewer in length, whilst the nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 176, 177, 178 and/or 179 may be 700 bp or fewer in length.
  • a nucleic acid may be 1000 bp or fewer in length, whilst the nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 176, 177, 178 and/or 179 may be 550- 700 bp in length.
  • Polypeptides provided herein may comprise or consist of an amino acid sequence corresponding to CCPs 8-10 of CR1 (SEQ ID NO:146). Polypeptides provided herein may comprise or consist of an amino acid sequence corresponding to CCPs 15-17 of CR1 (SEQ ID NO:147). Polypeptides provided herein may comprise or consist of an amino acid sequence corresponding to SEQ ID NO:182 or 183.
  • a polypeptide described herein may comprise or consist of an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:146, 147, 182 and/or 183. The sequence identity may be assessed over the whole polypeptide sequence.
  • a polypeptide described herein may comprise an amino acid sequence, wherein said amino acid sequence comprises or consists of a sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:146, 147, 182 and/or 183. That is, the polypeptide may comprise an amino acid sequence over which sequence identity is assessed with respect to SEQ ID NO:146, 147, 182 or 183.
  • the polypeptide may also comprise additional sequence that is not taken into account when assessing sequence identity with SEQ ID NO:146, 147, 182 or 183.
  • the polypeptide may comprise additional sequence at the N terminus and/or C terminus of the amino acid sequence above.
  • the additional sequence(s) may be sequence from CR1 or may be unrelated to CR1 .
  • a polypeptide provided herein e.g. that is/can be encoded by a nucleic acid described herein, may be defined by its length.
  • the size of the polypeptide is important so that it can access sites of pathological complement activation. For example, the polypeptide may need to traverse Bruch’s membrane to reach sites of unwanted complement activation in the eye, or may need to traverse the glomerular basement membrane to reach sites of unwanted complement activation in the kidney.
  • the polypeptide may be defined by its ‘total length’, e.g. including any amino acid sequence comprised therein and any additional sequence(s), from the N-terminus to the C-terminus.
  • the ‘total length’ may include or exclude any moieties attached or conjugated to said polypeptide, e.g. that are used to target the polypeptide to particular cells, tissues or sites of complement activation.
  • the ‘total length’ may include or exclude other amino acid sequences or protein domains that are fused to said polypeptide.
  • the polypeptide may be defined by the length of the amino acid sequence comprised therein (i.e. the amino acid sequence comprises or consisting of a sequence having at least 70% sequence identity to SEQ ID NO:146, 147, 182 or 183).
  • the polypeptide may comprise additional sequence(s) that renders the sequence of the entire polypeptide longer than the specified length of said amino acid sequence.
  • the polypeptide, or the amino acid sequence contained within the polypeptide may be 1000 amino acids or fewer in length.
  • the polypeptide, or said amino acid sequence may comprise or consist of an amino acid sequence of 990 or fewer, 980 or fewer, 970 or fewer, 960 or fewer, 950 or fewer, 940 or fewer, 930 or fewer, 920 or fewer, 910 or fewer, 900 or fewer, 890 or fewer, 880 or fewer, 870 or fewer, 860 or fewer, 850 or fewer, 840 or fewer, 830 or fewer, 820 or fewer, 810 or fewer, 800 or fewer, 790 or fewer, 780 or fewer, 770 or fewer, 760 or fewer, 750 or fewer, 740 or fewer, 730 or fewer, 720 or fewer, or 710 or fewer amino acids.
  • the polypeptide, or the amino acid sequence contained within the polypeptide may be 700 amino acids or fewer in length.
  • the polypeptide, or said amino acid sequence may comprise or consist of an amino acid sequence of 690 or fewer, 680 or fewer, 670 or fewer, 660 or fewer, 650 or fewer, 640 or fewer, 630 or fewer, 620 or fewer, 610 or fewer, 600 or fewer, 590 or fewer, 580 or fewer, 570 or fewer, 560 or fewer, 550 or fewer, 540 or fewer, 530 or fewer, 520 or fewer, 510 or fewer, 500 or fewer, 490 or fewer, 480 or fewer, 470 or fewer, 460 or fewer, 450 or fewer, 440 or fewer, 430 or fewer, 420 or fewer, 410 or fewer, 400 or fewer, 390 or fewer, 380 or fewer, 370 or fewer, 360 or fewer, 350 or fewer,
  • polypeptide, or amino acid sequence as above that comprises or consists of an amino acid sequence having ‘up to’ any length of amino acids described herein, e.g. up to 1000 amino acids or up to 700 amino acids. Any two end points described herein may be used to make a range of lengths for the polypeptide or amino acid sequence, for example 200-700 amino acids or any combination of end points above.
  • the polypeptide, or the amino acid sequence contained within the polypeptide may be 100-700 amino acids in length.
  • the polypeptide, or the amino acid sequence contained within the polypeptide may be 100-690, 100-680, 100-670, 100-660, 100-650, 100-640, 100-630, 100-620, 100-610, 100-600, 100-590,
  • a polypeptide described herein may be longer than the CR1 amino acid sequence comprised therein (said amino acid sequence having at least 70% sequence identity to SEQ ID NO: 146, 147, 182 and/or 183).
  • a polypeptide provided herein may have one length above, whilst said amino acid sequence may be of a shorter length. Any of the lengths and/or ranges above can be combined.
  • a polypeptide may be 700 or fewer amino acids in length, whilst the amino acid sequence having at least 70% sequence identity to SEQ ID NO: 146, 147, 182 and/or 183 may be 400 or fewer amino acids in length.
  • a polypeptide may be 620 or fewer amino acids in length, whilst the amino acid sequence having at least 70% sequence identity to SEQ ID NO: 146, 147, 182 and/or 183 may be 100- 220 amino acids in length.
  • polypeptide or the amino acid sequence contained within the polypeptide, may have a length of 194 or 212 amino acids.
  • Nucleic acids and polypeptides provided herein may comprise modifications and/or additional nucleotide or amino acid sequences.
  • the additional sequences may or may not contribute to the length/total length of the nucleic acid/polypeptide as described herein.
  • Nucleotide sequences described herein may comprise a stop codon, e.g. TAA, at the 3’ end.
  • a nucleic acid herein may comprise a nucleotide sequence encoding a secretory pathway sequence (also known as a signal peptide or signal sequence).
  • a secretory pathway sequence is an amino acid sequence which directs secretion of a polypeptide.
  • the secretory pathway sequence may be cleaved from the mature protein once export of the polypeptide chain across the rough endoplasmic reticulum is initiated.
  • Polypeptides secreted by mammalian cells generally have a signal peptide fused to the N-terminus of the polypeptide, which is cleaved from the translated polypeptide to produce a "mature" form of the polypeptide.
  • nucleotide sequence encoding a secretory pathway sequence may be part of or joined to another nucleotide sequence described herein (e.g. with sequence identity to SEQ ID NO:176 or 177), or may be arranged separately to said nucleotide sequence within the nucleic acid.
  • Secretory pathway/signal peptide sequences normally consist of a sequence of 5-30 hydrophobic amino acids, which form a single alpha helix. The sequence may be present in the newly-translated polypeptide (e.g. prior to processing to remove the sequence).
  • Secretory pathway/signal peptide sequences are known for many proteins, and are recorded in databases such as GenBank, UniProt, Swiss-Prot, TrEMBL, Protein Information Resource, Protein Data Bank, Ensembl, and InterPro, and/or can be identified/predicted e.g. using amino acid sequence analysis tools such as SignalP (Petersen et al., 2011 Nature Methods 8: 785-786) or Signal-BLAST (Frank and Sippl, 2008 Bioinformatics 24: 2172-2176).
  • SignalP Petersen et al., 2011 Nature Methods 8: 785-786
  • Signal-BLAST Frank and Sippl, 2008 Bioinformatics 24: 2172-2176.
  • the secretory pathway sequence is derived from Complement Factor H (FH).
  • the nucleic acid may comprise a nucleotide sequence having at least 80% sequence identity (e.g. at least 85%, 90%, 95%, 97%, 99% or 100%) to SEQ ID NO: 185.
  • the secretory pathway sequence may be codon-optimised.
  • the nucleic acid may comprise a nucleotide sequence having at least 80% sequence identity (e.g. at least 85%, 90%, 95%, 97%, 99% or 100%) to SEQ ID NO: 181 .
  • the polypeptide may comprise a secretory pathway sequence comprising or consisting of SEQ ID NO: 180.
  • a nucleic acid or polypeptide according to the present invention may additionally comprise a cleavage site for removing the secretory pathway sequence from the polypeptide.
  • the cleavage site for removing the secretory pathway sequence from the polypeptide is a cleavage site for an endoprotease.
  • the cleavage site is for an endoprotease expressed by the cell in which the nucleic acid or polypeptide is expressed.
  • the cleavage site is a signal peptidase cleavage site.
  • the cleavage site is a protease cleavage site, e.g.
  • a polypeptide according to the present disclosure is preferably soluble. Preferably, it can pass through extracellular membranes, such as Bruch’s membrane in the eye or the glomerular basement membrane (GBM) in the kidney, i.e. so it can reach sites of complement activation.
  • extracellular membranes such as Bruch’s membrane in the eye or the glomerular basement membrane (GBM) in the kidney, i.e. so it can reach sites of complement activation.
  • a polypeptide according to the present invention is not glycosylated. In some embodiments, a polypeptide according to the present invention lacks one or more sites for glycosylation. In some embodiments, the polypeptide of the present invention lacks one or more sites for N-linked glycosylation. In some embodiments, a polypeptide according to the present invention lacks N-linked glycans. In some embodiments, a polypeptide according to the present invention is expressed and/or secreted by cells that are unable to glycosylate or fully glycosylate polypeptides. For example, cells may lack functional glycosyl transferase enzymes. In some embodiments, the polypeptide is aglycosyl (i.e.
  • the polypeptide is not glycosylated).
  • the polypeptide has been deglycosylated, e.g. by treatment with a glycosidase (e.g. Peptide N-Glycosidase).
  • Deglycosylation is preferably non-denaturing.
  • a polypeptide according to the present invention is partially glycosylated, non-glycosylated or de-glycosylated.
  • a polypeptide described herein may comprise amino acid sequence(s) to facilitate expression, folding, trafficking, processing, purification or detection of the polypeptide.
  • the polypeptide may comprise a sequence encoding a protein tag, e.g. a His, (e.g. 6XHis), FLAG, Myc, GST, MBP, HA, E, or Biotin tag, optionally: at the N- or C- terminus of the polypeptide; in a linker; or at the N- or C- terminus of a linker.
  • the polypeptide comprises a detectable moiety, e.g.
  • the detectable moiety facilitates detection of the polypeptide in a sample obtained from a subject, e.g. following administration to the subject of the polypeptide, nucleic acid, vector, cell or pharmaceutical composition according to the present invention.
  • the sample may be any biological sample obtained from a subject.
  • the sample is a liquid biopsy, such as ocular fluid (tear fluid, aqueous humour, or vitreous), blood, plasma, etc.
  • the sample is a cytological sample or a tissue sample such as a surgical sample, e.g. of ocular cells/tissue.
  • a polypeptide described herein may be conjugated to other moieties, such as other therapeutic agents.
  • a polypeptide described herein may be conjugated to targeting molecules for administration to particular cells or tissues.
  • Targeting molecules may include, for example, nanoparticles, liposomes, micelles, beads, polymers, metal particles, dendrimers, antibodies, aptamers, nanotubes or micro-sized silica rods.
  • Such targeting molecules may be attached to the polypeptide after expression from a nucleic acid provided herein.
  • Targeting molecules may also be expressed as part of the polypeptide, e.g. from one nucleic acid. In such cases, the targeting molecule may not be considered part of the polypeptide for the assessment of its length, as described herein.
  • any additional amino acid sequence described herein (i.e. outside the region of amino acid sequence having sequence identity with SEQ ID NO: 146 or 147) lacks substantial sequence identity to one or more of amino acid sequences 1-490, 685-940 and/or 1135-2039 of human CR1 (SEQ ID NO:184, numbered according to Uniprot P17927; Entry version 205 (23 Feb 2022), Sequence version 3 (02 Mar 2010)).
  • the polypeptide/additional amino acid sequence lacks substantial sequence identity to CR1 CCP domains 1-7, 11-14 and/or 18-30.
  • the additional amino acid sequence has less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, or less than 5% sequence identity to one or more of amino acid sequences at positions 1-490, 685-940 and/or 1135-2039 of human CR1 (shown in SEQ ID NO:184).
  • polypeptide/additional amino acid sequence lacks substantial sequence identity to CR1 long homologous repeat (LHR) domains LHR-A (amino acid positions 1-489 of SEQ ID NO:184) and/or LHR-D (amino acid positions 1394-1842 of SEQ ID NO:184).
  • polypeptide/additional amino acid sequence does not comprise a sequence corresponding to LHR-A (amino acid positions 1-489 of SEQ ID NO:184) and/or LHR-D (amino acid positions 1394-1842 of SEQ ID NO:184).
  • polypeptide does not comprise or consist of the whole extracellular domain of CR1 (e.g. sCR1), e.g. as described in US 8664176 B2.
  • polypeptide/additional amino acid sequence does not comprise a sequence corresponding to CCPs 11-15 of CR1 , or amino acid positions 685 to 940 of SEQ ID NO: 184 herein.
  • a nucleic acid or nucleotide sequence described herein lacks substantial sequence identity to one or more nucleotide sequences of CR1 mRNA or cDNA outside the sequences provided in SEQ ID NO:176, 177, 178 or 179.
  • the polypeptide is a detached/discrete/separate/individual/isolated molecule. In some embodiments, the polypeptide is not a multi-domain polypeptide. In some embodiments, the polypeptide is a single contiguous amino acid sequence that is unconnected, i.e. not joined, fused or attached, to another amino acid sequence. In some embodiments the polypeptide is not attached by an amino acid linker or a non-amino acid linker to another polypeptide or amino acid sequence. In some embodiments the polypeptide is not a section, part or region of a longer amino acid sequence, i.e. it is not part of an amino acid sequence that exceeds the maximum, specified, polypeptide length.
  • polypeptide is not part of, or does not form a section of, a fusion protein.
  • polypeptide may comprise a sequence provided herein and one or more additional amino acids, as long as the maximum length of the polypeptide is not exceeded. The short length of the polypeptides described herein enables the polypeptides to pass through the BrM and reach sites of complement activation.
  • the polypeptide/additional amino acid sequence does not comprise a domain or amino acid sequence that binds to VEGF.
  • the polypeptide may not comprise a domain or amino acid sequence that inhibits VEGF.
  • the polypeptide may not comprise a half-life prolonging domain, e.g. an Fc domain as described in WO 2013/082563 A1.
  • the polypeptide does not comprise, or is not conjugated to, an antibody or antigen-binding molecule.
  • the polypeptide does not comprise, or is not conjugated to, an antibody or antigen-binding molecule that binds to C3d, e.g. as described in US11053305.
  • the polypeptide does not comprise all or part of a convertase decay accelerating domain (e.g. from DAF or CD55) and/or all or part of a host cell recognition domain, e.g. as described in WO 2018/002131 A1 .
  • a convertase decay accelerating domain e.g. from DAF or CD55
  • a host cell recognition domain e.g. as described in WO 2018/002131 A1 .
  • the polypeptide/additional amino acid sequence does not comprise an amino acid sequence from Factor H (except for, in some embodiments, the FH signal peptide sequence). That is, in some embodiments the polypeptide/additional amino acid sequence does not comprise an amino acid sequence from the mature form of Factor H from which the signal peptide has already been removed. In some embodiments, the polypeptide/additional amino acid sequence lacks substantial sequence identity to SEQ ID NO:1 herein.
  • the polypeptide/additional amino acid sequence has less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, or less than 5% sequence identity to SEQ ID NO:1 herein. In some embodiments, the polypeptide/additional amino acid sequence lacks substantial sequence identity to amino acids 19 to 1213 of SEQ ID NO:1 herein.
  • the polypeptide/additional amino acid sequence has less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, or less than 5% sequence identity to amino acids 19 to 1213, amino acids 19 to 264, amino acids 324 to 507, amino acids 987 to 1230 and/or amino acids 1107 to 1230 of SEQ ID NO:1 herein.
  • polypeptide/additional amino acid sequence has less than 25% sequence identity to CCPs 1-4 of Factor H (positions 19 to 264 of SEQ ID NO:1 herein). In some embodiments the polypeptide/additional amino acid sequence has less than 20% sequence identity to CCPs 6-8 of Factor H (positions 324 to 507 of SEQ ID NO:1 herein). In some embodiments the polypeptide/additional amino acid sequence has less than 10% sequence identity to CCPs 19-20 of Factor H (positions 1107 to 1230 of SEQ ID NO:1 herein).
  • nucleic acids and polypeptides provided herein may be isolated and/or substantially purified, e.g. from other nucleic acid or naturally-occurring biological material.
  • Nucleic acids described herein are preferably provided for introduction into a cell, e.g. a human cell.
  • a nucleic acid described herein may be contained in a vector.
  • a nucleic acid described herein may be a vector.
  • reference to a nucleic acid should also be taken to refer to a vector comprising or consisting of said nucleic acid.
  • a vector comprising a nucleic acid as described herein is provided herein.
  • a “vector” as used herein is a nucleic acid molecule used as a vehicle to transfer exogenous nucleic acid into a cell.
  • the vector may be a vector for expression of the nucleic acid in the cell, i.e. an expression vector.
  • Such vectors may include a promoter sequence operably linked to the nucleotide sequence encoding the sequence to be expressed.
  • a vector may also include a termination codon and expression enhancers.
  • a vector may include regulatory elements, such as a polyadenylation site. Any suitable vectors, promoters, enhancers and termination codons known in the art may be used to express a peptide or polypeptide from a vector according to the invention.
  • Suitable vectors include plasmids, binary vectors, DNA vectors, mRNA vectors, viral vectors (e.g. gammaretroviral vectors (e.g. murine Leukemia virus (MLV)-derived vectors), lentiviral vectors, retroviral vectors, adenovirus vectors, adeno-associated virus (AAV) vectors, vaccinia virus vectors and herpesvirus vectors, e.g. Herpes Simplex Virus vectors), transposon-based vectors, and artificial chromosomes (e.g. yeast artificial chromosomes), e.g.
  • viral vectors e.g. gammaretroviral vectors (e.g. murine Leukemia virus (MLV)-derived vectors), lentiviral vectors, retroviral vectors, adenovirus vectors, adeno-associated virus (AAV) vectors, vaccinia virus vectors and herpesvirus vectors, e.g. Herpe
  • the lentiviral vector may be pELNS, or may be derived from pELNS.
  • the vector may be a vector encoding CRISPR/Cas9.
  • a nucleic acid or nucleotide sequence provided herein comprises inverted terminal repeat (ITR) sequences for use in an AAV or adenovirus vector.
  • ITRs are 145 nucleotide, palindromic sequences located at the termini of an adenovirus or AAV genome, see e.g. Earley et al., Hum Gene Ther. February 2020; 31 (3-4): 151-162, which is hereby incorporated by reference in its entirety.
  • ITRs are important for the regulation and priming of viral DNA replication and contain secondary structures including the Rep binding element (RBE) and a terminal resolution site (TRS), which together constitute the AAV origin of replication.
  • RBE Rep binding element
  • TRS terminal resolution site
  • a nucleic acid or nucleotide sequence described herein comprises at least one ITR sequence.
  • the nucleic acid or nucleotide sequence comprises a 5’ ITR and/or a 3’ ITR.
  • the 5’ and/or 3’ ITR is selected from an AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAB7, AAV8, AAV9, AAV10, AAV11 , AAV12, AAV-DJ, AAV-DJ/8, AAV-rh10, AAVrh.39, AAV-retro, AAV-PHP.B, AAV8-PHP.eB, AAV-PHP.S, AAV-Anc80, AAV2.5, AAV SparkWO, R100, AAV2.7m8, AAV-LK05 and AAVtYF ITR.
  • the 5’ and/or 3’ ITR is selected from an AAV1 , AAV2, AAV4, AAV5, or AAV8 ITR.
  • the 5’ and 3’ ITRs may be from the same AAV serotype, or from different AAV serotypes.
  • the adeno-associated virus (AAV) vector is selected from AAV serotype 1 , 2, 3, 4,
  • the AAV vector is an AAV serotype 2 (AAV- 2) vector, or a hybrid and/or mutant thereof.
  • the AAV vector is an AAV serotype 8 (AAV-8) vector, or a hybrid and/or mutant thereof.
  • the AAV vector exhibits tropism to eye tissue/cells. In some embodiments, the AAV vector is selected from serotype 1 , 2, 4, 5 or 8. In some embodiments, the AAV vector exhibits tropism to kidney tissue/cells. In some embodiments, the AAV vector is selected from serotype AAV2, AAV-DJ, AAV-LK05, and AAV-Anc80.
  • the expression of a nucleic acid or a nucleic acid contained in a vector, according to the present invention is driven by a promoter that drives expression in a specific retinal cell type, e.g. rods, cones, RPE, or ganglion cells, as described for example in Beltran WA, et al. Gene Ther. 2010; 17:1162-74 and Boye SE, et al. Hum Gene Ther. 2012; 23:1101-15, which are hereby incorporated by reference in their entirety.
  • a promoter that drives expression in a specific retinal cell type, e.g. rods, cones, RPE, or ganglion cells, as described for example in Beltran WA, et al. Gene Ther. 2010; 17:1162-74 and Boye SE, et al. Hum Gene Ther. 2012; 23:1101-15, which are hereby incorporated by reference in their entirety.
  • the expression of a nucleic acid or a nucleic acid contained in a vector, according to the present invention is driven by a promoter that drives expression of that nucleic acid in retinal pigment epithelial (RPE) cells, or kidney cells such as those described herein.
  • the promoter is an RPE65 or VMD2 promoter, or modified version thereof.
  • the promoter is a chicken p actin promoter.
  • the promoter is a podocyte-specific promoter.
  • the promoter is a podocin, nephrin, NPT2a, NPHS1 , NKCC2, AQP2, SGLT2, or Ksp-cadherin promoter.
  • the vector may be a eukaryotic vector, e.g. a vector comprising the elements necessary for expression of protein from the vector in a eukaryotic cell.
  • the vector may be a mammalian vector, e.g. comprising a cytomegalovirus (CMV) or SV40 promoter to drive protein expression.
  • CMV cytomegalovirus
  • the vector comprises an inducible promoter, i.e. gene expression is activated by the promoter only in the presence or absence of a particular molecule.
  • inducible promoters will be known to the skilled person. Examples of inducible promoters are described in e.g. Le at al. Invest Ophthalmol Vis Sci. 2008, 49(3): 1248-1253 and McGee Sanftner et al. Mol Ther. 2001 . 3(5): 688-696; which are hereby incorporated by reference in their entirety.
  • AAV comprising:
  • nucleic acid or nucleotide sequence as described herein e.g. comprising at least one ITR;
  • At least one capsid protein e.g. of a serotype described herein.
  • a polypeptide described herein may possess or demonstrate one or more of the following properties (e.g. as determined in an appropriate assay for said property).
  • a nucleic acid described herein may encode a polypeptide that possesses one or more of said properties.
  • a polypeptide or nucleic acid with one or more such properties may be described as being ‘capable of demonstrating said property/properties.
  • a polypeptide e.g. encoded by a nucleic acid
  • Binds to C3b binds to C3b in the region of C3b bound by a cofactor for Fl; binds to C3b in the region of C3b bound by Complement Receptor 1 (or a fragment thereof); binds to C3b in the region of C3b bound by Complement Receptor 1 CCP domains 8-10 and/or
  • the alternative complement pathway diffuses through extracellular membranes, such as Bruch’s membrane (BrM) and/or the glomerular basement membrane (GBM); displays superior ability to diffuse through EC membranes compared to a co-factor for Fl (or a fragment thereof); displays similar ability to diffuse through EC membranes compared to a co-factor for Complement Factor I (or a fragment thereof); displays superior ability to diffuse through EC membranes compared to FH; displays similar ability to diffuse through EC membranes compared to FHL-1 ; displays superior ability to diffuse through EC membranes compared to FHL-1 ; displays similar ability to diffuse through EC membranes compared to soluble CR1 ; displays superior ability to diffuse through EC membranes compared to soluble CR1 ; displays superior ability to diffuse through EC membranes compared to a multi-domain fusion protein; acts as a co-factorto enable F-mediated reduction/prevention of the formation of a functional C3bBb-type C3 convertase; acts as a co-factorto
  • binding to a given target can be determined and quantified.
  • the binding may be the response detected in a given assay.
  • a polypeptide according to the present invention displays binding to C3b in such an assay which is greater than 1 times, e.g. one of >1 .01 , >1 .02, >1 .03, >1 .04, >1 .05, >1 .06, >1 .07, >1.08, >1.09, >1.1 , >1.2, >1.3, >1.4, >1.5, >1.6, >1.7, >1.8, >1.9, >2, >3, >4, >5, >6, >7, >8, >9, >10, >15, >20, >25, >30, >35, >40, >45, >50, >60, >70, >80, >90, or >100 times the level of binding signal detected in such an assay to a negative control molecule to which the polypeptide does not bind.
  • a polypeptide according to the present invention is capable of binding to C3b with an affinity of binding which is higher than the affinity of binding to C3b displayed by a co-factor for Complement Factor I (or a fragment thereof) in a given assay. In some embodiments a polypeptide according to the present invention is capable of binding to C3b with an affinity of binding which is at least
  • a polypeptide according to the present invention is capable of binding to C3b with an affinity of binding which is 2, 3, 4, 5, 6, 7, 8, 9 or 10 order(s) of magnitude greater than the affinity of binding to C3b displayed by a co-factor for Complement Factor I (or a fragment thereof) in a given assay.
  • the co-factor for Complement Factor I is Complement Factor H or truncated FH isoform FHL-1 .
  • the co-factor for Complement Factor I may be CR1 .
  • a polypeptide according to the present invention is capable of binding to C3b with an affinity of binding which is higher than the affinity of binding to C3b displayed by one or more FHR proteins, e.g. one or more of FHR1 , FHR2, FHR3, FHR4 and/or FHR5.
  • FHR proteins e.g. one or more of FHR1 , FHR2, FHR3, FHR4 and/or FHR5.
  • a polypeptide according to the present invention is capable of binding to C3b with an affinity of binding which is at least 1 .5 times, at least 2 times, at least 2.5 times, at least 3 times, at least 3.5 times, at least 4 times, at least 4.5 times, at least 5 times, at least 5.5 times, at least 6 times, at least 6.5 times, at least 7 times, at least 7.5 times, at least 8 times, at least 8.5 times, at least 9 times, at least 9.5 times, at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 30 times, at least 35 times, at least 40 times, at least 45 times, at least 50 times, at least 75 times, at least 100 times, at least 150 times, at least 200 times, at least 250 times, at least 300 times, at least 350 times, at least 400 times, at least 450 times, at least 500 times, at least 550 times, at least 600 times, at least 650 times, at least 700 times, at least 750 times, at least 800 times, at least
  • the level of complement activation/over-activation may be determined by the assays described herein, e.g. abnormal levels of complement components, or by tests/assays that are known by one skilled in the art, e.g. as described in Shih and Murali Am. J. Hematol. 2015, 90: 1180-1186; Kirschfink and Mollnes, Clin Diagn Lab Immunol. 2003, 10(6): 982-989; Nilsson and Ekdahl, Clinical and Developmental Immunology, 2012, Article ID 962702; which are hereby incorporated by reference in their entirety.
  • the ability of a given polypeptide to diffuse through extracellular membranes can be analysed e.g. in vitro, e.g. as described in Clark et al J. Immunol (2014) 193, 4962-4970, hereby incorporated by reference in its entirety.
  • the diffusion through such membranes may be detected by measuring the rate of diffusion through to the diffusate chamber and/or detecting the proportion of polypeptide present in the diffusate chamber at the end of the experiment.
  • a similar ability to diffuse through EC membranes may be indicated by detecting a rate of diffusion through to the diffusate chamber which is within 30%, e.g.
  • a superior ability to diffuse through EC membranes may be indicated by detecting a rate of diffusion through to the diffusate chamber which is higher (e.g.
  • the invention provides methods for treating and/or preventing a complement related disorder, i.e. a disorder associated with complement dysregulation, such as complement overactivation.
  • a complement related disorder i.e. a disorder associated with complement dysregulation, such as complement overactivation.
  • the invention relates to methods for selecting treatment for and/or treating subjects/patients that have a complement-related disorder or have been identified as having a complement-related disorder, e.g. by determining the level of a complement protein as described herein.
  • the methods described herein may be diagnostic, prognostic and/or predictive of the risk of onset or progression of a complement-related disorder. Diagnostic methods can be used to determine the diagnosis or severity of a disease, prognostic methods help to predict the likely course of disease in a defined clinical population under standard treatment conditions, and predictive methods predict the likely response to a treatment in terms of efficacy and/or safety, thus supporting clinical decision-making. Such methods may be used prior to administration of a therapeutic agent, such as a nucleic acid or polypeptide provided herein.
  • complement-related disorder refers to disorders, diseases or conditions that comprise or arise from deficiencies or abnormalities in the complement system.
  • the complement-related disorder is a disorder driven by complement activation or complement over-activation.
  • developer e.g. of a disorder, as used herein refer both to the onset of a disease as well as the progression, exacerbation or worsening of a disease state.
  • biomarker(s) refers to one or more measurable indicators of a biological state or condition.
  • the disorder is one in which the complement system, or activation/over-activation/dysregulation thereof, is pathologically implicated.
  • the complement related disorder may be any disorder described herein. “Pathologically implicated” as used herein may refer to a protein level which is raised or lowered in the disorder compared with a reference value, and/or where the protein contributes towards the pathology of the disorder. The selection or combination of complement protein(s) detected/determined may depend on the complement-related disorder of interest and the complement protein(s) that are useful biomarkers for said disorder.
  • Subjects with elevated levels of FHR1 , FHR2, FHR3, FHR4 and/or FHR5 may derive therapeutic or prophylactic benefit from the activity levels of any one or more of said proteins being reduced, e.g. using any nucleic acid or polypeptide described herein.
  • Subjects with elevated levels of FHR1 , FHR2, FHR3, FHR4 and/or FHR5, and optionally FHL-1 and/or FH, and/or increased expression of a gene(s) encoding one or more of said proteins, may derive therapeutic or prophylactic benefit from said expression levels being reduced. This may be achieved with a complement-targeted therapy or complement-targeted therapeutic agent.
  • a method for selecting a subject for treatment with a therapeutic agent comprising:
  • a therapeutic agent e.g. a complement-targeted therapy or therapeutic agent as described herein, for a subject, the method comprising:
  • the subject in the methods above may then be treated with a nucleic acid, nucleotide sequence, vector, rAAV, or polypeptide as described herein.
  • the subject may be suspected of having, or may have been determined to have, a complement-related disorder, e.g. using the methods described herein.
  • the level(s) of FHR1 , FHR2, FHR3, FHR4 and/or FHR5, may be elevated compared to the level of FH and/or FHL-1 .
  • a complement protein for selecting a subject for treatment with a nucleic acid or polypeptide or complement-targeted therapy, or for selecting a therapeutic agent for a subject, the use comprising the steps disclosed hereinabove.
  • Also provided is a method of treatment comprising:
  • nucleic acid or polypeptide as provided herein, or a complement-targeted therapy or therapeutic agent.
  • nucleic acid or polypeptide as provided herein, or a complement-targeted therapy or therapeutic agent, for use in a method of treatment, wherein the method comprises:
  • nucleic acid or polypeptide as provided herein, or a complement-targeted therapy or therapeutic agent, in the manufacture of a medicament for the treatment of a complement- related disorder, e.g. Macular Degeneration, wherein the method of treatment comprises:
  • Any method described herein may comprise one or more of the steps of:
  • the level of the complement protein e.g. one or more of FHR1 , FHR2, FHR3, FHR4 and/or FHR5, and optionally FHL-1
  • the level of the complement protein e.g. one or more of FHR1 , FHR2, FHR3, FHR4 and/or FHR5, and optionally FHL-1
  • step (c) treating the subject by targeting the complement protein(s) that were found to be elevated in step (b).
  • the methods may comprise determining whether the level of a complement protein is altered, e.g. increased or decreased, as compared to the level of that complement protein in blood in a control subject that does not have a complement-related disorder, or a subject that has a complement- related disorder that is not associated with an altered level of said protein.
  • the methods may comprise determining the relative concentrations of complement proteins compared to each other, e.g. the level of a complement protein may be elevated as compared to the level of a different complement protein, which may be unaltered or decreased, in the same subject or as compared to a control subject.
  • Targeting the complement protein(s) refers to using any suitable agent, such as those described herein, to reduce the level(s) of the complement protein(s) that were found to be elevated in the subject, for example by reducing gene expression, preventing mRNA transcription, sequestering the protein(s), or preventing the normal activity of the protein(s).
  • Treatment may refer to treating, preventing, or reducing the likelihood of a complement-related disorder, such as those described herein.
  • Treatment of a complement-related disorder as described herein may involve modifying at least one cell of a subject to express or comprise a nucleic acid, nucleotide sequence, vector or rAAV provided herein.
  • Treatment of a complement-related disorder as described herein may involve modifying at least one cell of a subject to express or comprise a polypeptide provided herein, e.g. via a nucleic acid, nucleotide sequence or vector provided herein.
  • the at least one cell may be a cell, e.g. human cell, of the eye, kidney, vascular system, blood, muscle, skin, oesophagus, small or large intestine, intestinal tract, pharynx, trachea, lungs, bronchi, bronchioles, or central nervous system.
  • a cell e.g. human cell, of the eye, kidney, vascular system, blood, muscle, skin, oesophagus, small or large intestine, intestinal tract, pharynx, trachea, lungs, bronchi, bronchioles, or central nervous system.
  • Treatment of a complement-related disorder as described herein may involve administering to a subject a vector comprising or consisting of a nucleic acid as described herein.
  • Treatment of a complement-related disorder as described herein may involve administering to a subject a vector comprising or consisting of a nucleic acid encoding a polypeptide as described herein.
  • the level of a complement protein is determined using any suitable technique known in the art and available to a skilled person. In some embodiments the level of a complement protein is determined by mass spectrometry and/or digesting the protein with endoproteinase GluC, e.g. as described herein.
  • the methods described herein are performed in vitro or ex vivo.
  • a sample may be obtained from a subject of interest, or a control subject, and the steps that involve determining the level of a complement protein, determining whether a subject has or is at risk of developing a complement-related disorder, and digesting at least one complement protein are performed in vitro or ex vivo. Steps of the methods that involve treating a subject may be performed in vivo.
  • the methods described herein may be useful in monitoring the success of treatment, including past or ongoing treatment, for complement-related disorders.
  • the methods described herein may comprise treating a subject who has/has been determined to have a complement-related disorder, and then re-determining the level of one or more of FHR1 , FHR2, FHR3, FHR4 and/or FHR5, and optionally FH and/or FHL-1 , after treatment.
  • Such methods are useful for determining the efficacy of treatment and the progression of the disorder.
  • a complement-related disorder according to the present disclosure is one in which the complement system, or activation/over-activation/dysregulation thereof, is pathologically implicated.
  • the complement-related disorder may comprise disruption of the classical, alternative and/or lectin complement pathways.
  • the disorder may be associated with deficiencies in, abnormalities in, or absence of regulatory components of the complement system.
  • the disorder may be a disorder associated with the alternative complement pathway, disruption of the alternative complement pathway and/or associated with deficiencies in, abnormalities in, or absence of regulatory components of the alternative complement pathway.
  • the disorder is associated with the complement amplification loop.
  • the disorder is associated with inappropriate activation, over-activation, or dysregulation of the complement system, in whole or in part, e.g. C3 convertase assembly, C3b production, C3b deposition, and/or the amplification loop.
  • the present invention provides methods for determining whether a complement-related disorder is associated with over-activation of the complement system. In some cases, the methods are capable of determining whether elevated levels of FHR1 , FHR2, FHR3, FHR4 and/or FHR5 are contributing to complement over-activation and/or complement-related disorders, e.g. as compared to a control subject that does not have a complement-related disorder.
  • the disorder is characterised by elevated levels of any one or more FH family proteins, e.g. any one or more of FHR1 , FHR2, FHR3, FHR4 and/or FHR5.
  • the elevated levels may be in a subject. That is, the subject to be assessed or treated may have (or be/have been determined to have) elevated levels of one or more of FHR1 , FHR2, FHR3, FHR4 and/or FHR5, e.g. assessed by a method provided herein.
  • the disorder may be characterised by elevated circulating levels of one or more of FHR1 , FHR2, FHR3, FHR4 and/or FHR5, i.e. in a blood- or plasma-derived sample as described herein.
  • the disorder may be characterised by elevated expression of one or more of FHR1 , FHR2, FHR3, FHR4 and/or FHR5 by hepatocytes.
  • the disorder may be characterised by elevated levels of one or more of FHR1 , FHR2, FHR3, FHR4 and/or FHR5 detected in a tissue of interest, e.g. eye, kidney, brain, lung, tumor, vascular tissue. Elevated levels can be determined by comparison to a control value(s)/subject(s) as described herein.
  • a complement-related disorder may have elevated levels of one or more FHR proteins.
  • some subjects with a complement-related disorder may have elevated levels of one or more FHR proteins, and some subjects with the same complement-related disorder may not.
  • the presence of elevated levels of one or more FHR proteins can indicate a worse prognosis. Determining the levels of one or more FHR proteins therefore may provide a distinct population of patients who will benefit in particular from treatment with the nucleic acids and proteins described herein, e.g. as compared to patients with normal levels of FHR proteins.
  • the complement-related disorder may be characterised by altered levels of FH and/or FHL-1 , either up or down, e.g. in addition to the elevated levels of one or more FHR proteins.
  • the disorder is associated with any one or more of C3, C3b, iC3b, Fl, FH, FHL-1 , or FHR1-FHR5. In some cases, the disorder is associated with deficiencies or abnormalities in the activity of any one or more of C3, C3b, iC3b, Fl, FH, FHL-1 , or FHR1-FHR5. In some cases one or more of these proteins are pathologically implicated in the disorder, e.g. have raised or lower levels compared with a reference value.
  • the disorder is associated with one or more of CR1 , CD46, CD55, C4BP, Factor B (FB), Factor D (FD), SPICE, VCP (or VICE) and/or MOPICE.
  • the disorder is associated with deficiencies or abnormalities in the activity of one or more of CR1 , CD46, CD55, C4BP, Factor B, Factor D, SPICE, VCP (or VICE) and/or MOPICE, or where one or more of these proteins are pathologically implicated.
  • the disorder may be a disorder associated with C3 or a C3-containing complex, an activity/response associated with C3 or a C3-containing complex, or a product of an activity/response associated with C3 or a C3-containing complex. That is, in some embodiments, the disorder is a disorder in which C3, a C3-containing complex, an activity/response associated with C3 or a C3-containing complex, or the product of said activity/response is pathologically implicated.
  • the disorder may be associated with an increased level of C3 or a C3-containing complex, an increased level of an activity/response associated with C3 or a C3-containing complex, or an increased level of a product of an activity/response associated with C3 or a C3-containing complex as compared to the control state.
  • the disorder may be associated with a decreased level of C3 or a C3-containing complex, a decreased level of an activity/response associated with C3 or a C3-containing complex, or a decreased level of a product of an activity/response associated with C3 or a C3-containing complex as compared to the control state.
  • the disorder may be a disorder associated with C3b or a C3b-containing complex, an activity/response associated with C3b or a C3b-containing complex, or a product of an activity/response associated with C3b or a C3b-containing complex. That is, in some embodiments, the disorder is a disorder in which C3b, a C3b-containing complex, an activity/response associated with C3b or a C3b-containing complex, or the product of said activity/response is pathologically implicated.
  • the disorder may be associated with an increased level of C3b or a C3b-containing complex, an increased level of an activity/response associated with C3b or a C3b-containing complex, or increased level of a product of an activity/response associated with C3b or a C3b-containing complex as compared to the control state.
  • the disorder may be associated with a decreased level of C3b or a C3b-containing complex, a decreased level of an activity/response associated with C3b or a C3b-containing complex, or a decreased level of a product of an activity/response associated with C3b or a C3b-containing complex as compared to the control state.
  • the disorder may be a disorder associated with any one or more of FH, FHL-1 , Fl, FHR1-FHR5, FB, FD, CR1 and/or CD46, an activity/response associated with any one or more of FH, FHL-1 , Fl, FHR1-FHR5, FB, FD, CR1 and/or CD46 or a product of an activity/response associated with any one or more of FH, FHL-1 , Fl, FHR1-FHR5, FB, FD, CR1 and/or CD46.
  • the disorder is a disorder in which any one or more of FH, FHL-1 , Fl, FHR1-FHR5, FB, FD, CR1 and/or CD46, an activity/response associated with any one or more of FH, FHL-1 , Fl, FHR1-FHR5, FB, FD, CR1 and/or CD46, or the product of said activity/response is pathologically implicated.
  • the disorder may be associated with a decreased level of any one or more of FH, FHL-1 , Fl, FHR1-FHR5, FB, FD, CR1 and/or CD46, a decreased level of an activity/response associated with any one or more of FH, FHL-1 , Fl, FHR1-FHR5, FB, FD, CR1 and/or CD46, or a decreased level of a product of an activity/response associated with any one or more of FH, FHL-1 , Fl, FHR1-FHR5, FB, FD, CR1 and/or CD46 as compared to a control state.
  • the disorder may be associated with an increased level of any one or more of FHR1 , FHR2, FHR3, FHR4 and/or FHR5, an increased level of an activity/response associated with any one or more of FHR1 , FHR2, FHR3, FHR4 and/or FHR5, or an increased level of a product of an activity/response associated with any one or more of FHR1 , FHR2, FHR3, FHR4 and/or FHR5 as compared to a control state, see e.g. Zhu et al., Kidney Int. 2018 Jul;94(1):150-158; Pouw et al., Front Immunol. 2018 Apr 24;9:848; both hereby incorporated by reference in their entirety.
  • the methods may comprise determining the systemic level of any combination of FHR1 to FHR5.
  • the disorder may be associated with an increased level of any one or more of FHR1 , FHR2 and/or FHR3, an increased level of an activity/response associated with any one or more of FHR1 , FHR2 and/or FHR3, or an increased level of a product of an activity/response associated with any one or more of FHR1 , FHR2 and/or FHR3.
  • the disorder may be associated with an increased level of FHR4, an increased level of an activity/response associated with FHR4, or an increased level of a product of an activity/response associated with FHR4 as compared to a control state, see e.g. WO 2019/215330 and Cipriani et al., Nat Commun 11 , 778 (2020), both hereby incorporated by reference in their entirety.
  • the disorder may be associated with an increased level of FHL-1 .
  • the disorder is associated with increased levels of any one or more of C3, C3b, C3 convertase and/or C3bBb as compared to a control state. In some embodiments the disorder is associated with decreased levels of any one or more of C3, C3b, C3 convertase and/or C3bBb as compared to a control state. In some embodiments, the disorder is associated with increased levels of iC3b as compared to a control state. In some embodiments, the disorder is associated with decreased levels of iC3b as compared to a control state.
  • the disorder is associated with increased levels of any one or more of C3a, C3f, C3c, C3dg, C3d, and/or C3g as compared to a control state. In some embodiments the disorder is associated with decreased levels of any one or more of C3a, C3f, C3c, C3dg, C3d, and/or C3g as compared to a control state.
  • the methods described herein find use in diagnosing, treating or preventing, or selecting a subject for treatment or prevention of, a disorder which would benefit from one or more of: a reduction in the level or activity of one or more of C3bBb-type C3 convertase, C3bBb3b-type C5 convertase and/or C4b2a3b-type C5 convertase; a reduction in the level of one or more of C3, C3b, C3a, iC3b, FHR1 , FHR2, FHR3, FHR4, FHR5, C5b and/or C5a; or an increase in the level of one or more of iC3b, C3f, C3c, C3dg, C3d, C3g, FH, FHL-1 , Fl, FH, FHL-1 , FHR1 , FHR2, FHR3, FHR4 and/or FHR5 as compared to reference value(s).
  • the methods described herein find use in treating or preventing, or selecting a subject for treatment or prevention of, a disorder which would benefit from a reduction in the level or activity of one or more of FHR1 , FHR2, FHR3, FHR4 and/or FHR5, and optionally FHL-1 .
  • the disorder may be an ocular disorder.
  • a disease or condition to be assessed, diagnosed, treated or prevented as described herein is a complement-related ocular disease.
  • the disorder is macular degeneration.
  • the disorder may be selected from, i.e. is one or more of, age-related macular degeneration (AMD), choroidal neovascularisation (CNV), macular dystrophy, and diabetic maculopathy.
  • AMD age-related macular degeneration
  • CNV choroidal neovascularisation
  • AMD diabetic maculopathy
  • AMD includes early AMD, intermediate AMD, late/advanced AMD, geographic atrophy (‘dry’ (i.e. non-exudative) AMD), and ‘wet’ (i.e.
  • exudative or neovascular AMD each of which may be a disorder in its own right that is detected, treated and/or prevented as described herein.
  • the disease or condition to be treated or prevented is a combination of the diseases/conditions above, e.g. ‘dry’ and ‘wet’ AMD.
  • the disease or condition to be treated or prevented is not ‘wet’ AMD or choroidal neovascularisation.
  • AMD is commonly-defined as causing vision loss in subjects age 50 and older.
  • a subject to be treated is age 50 or older, i.e. is at least 50 years old.
  • early AMD refers to a stage of AMD characterised by the presence of medium-sized drusen, commonly having a diameter of up to ⁇ 200 pm, within Bruch’s membrane adjacent to the RPE layer. Subjects with early AMD typically do not present with significant vision loss.
  • intermediate AMD refers to a stage of AMD characterised by large drusen and/or pigment changes in the retina. Intermediate AMD may be accompanied by some vision loss.
  • late AMD refers to a stage of AMD characterised by the presence of drusen and vision loss, e.g. severe central vision loss, due to damage to the macula.
  • ‘reticular pseudodrusen’ (RPD) or ‘reticular drusen’ may be present, referring to the accumulation of extracellular material in the subretinal space between the neurosensory retina and RPE.
  • “Late AMD” encompasses ‘dry’ and ‘wet’ AMD.
  • ‘dry’ AMD also known as geographic atrophy
  • ‘wet’ AMD also known as choroidal neovascularization, neovascular and exudative AMD
  • abnormal blood vessels grow underneath and into the retina. These vessels can leak fluid and blood which can lead to swelling and damage of the macula and subsequent scar formation. The damage may be rapid and severe.
  • ‘Dry’ AMD also known as geographic atrophy, represents around 50% of late-stage AMD cases.
  • CNV choroidal neovascularisation
  • VEGF vascular endothelial growth factor
  • the disorder is early-onset macular degeneration (EOMD).
  • EOMD refers to a phenotypically severe sub-type of macular degeneration that demonstrates a much earlier age of onset than classical AMD and results in many more years of substantial visual loss.
  • Sufferers may show an early-onset drusen phenotype comprising uniform small, slightly raised, yellow subretinal nodules randomly scattered in the macular, also known as ‘basal laminar drusen’ or ‘cuticular drusen’.
  • EOMD may also be referred to as “middle-onset macular degeneration”.
  • the EOMD subset is described in e.g. Boon CJ et al.
  • EOMD is related to complement dysregulation and disrupted Factor H activity.
  • a subject to be treated is age 49 or younger.
  • a subject to be treated is between ages 15 and 49, i.e. is between 15 and 49 years old.
  • the disease or condition to be treated is a macular dystrophy.
  • a macular dystrophy can be a genetic condition, usually caused by a mutation in a single gene, that results in degeneration of the macula.
  • the methods described herein may be used for determining whether a subject is at risk of onset of macular degeneration, e.g. EOMD and/or AMD, and/or is at risk of EOMD and/or AMD progression.
  • the disorder is selected from EOMD, AMD, geographic atrophy (‘dry’ (i.e. non-exudative) AMD), early AMD, intermediate AMD, late/advanced AMD, ‘wet’ (neovascular or exudative) AMD, choroidal neovascularisation (CNV) and retinal dystrophy.
  • the subject has or is suspected to have a complement-related disorder.
  • the disorder is AMD.
  • the disorder is EOMD.
  • the present invention provides a method for determining whether a subject is at risk of developing macular degeneration, e.g. EOMD and/or AMD, the method comprising:
  • Also provided herein is a method for assessing the propensity or predisposition of a subject to develop a complement-related disorder, comprising steps (a) to (d) above.
  • the disorder is one associated with the kidney, e.g. nephropathy/a nephropathic disorder.
  • the disorder is a neurological and/or neurodegenerative disorder.
  • the disorder is associated with autoimmunity, e.g. an autoimmune disease.
  • the disorder is associated with inflammation, e.g. an inflammatory disease.
  • the disorder is characterised by the deposition of C3, e.g. the glomerular pathologies (see e.g. Skerka et al 2013, supra).
  • FHR proteins have been implicated in complement-related kidney disorders.
  • FH, FHL-1 , FHR1 , FHR2, FHR3 and FHR5 have been implicated in IgA nephropathy (see e.g. Poppelaars et al., J Clin Med. 2021 , 10(20):4715; Zhu et al., Kidney Int. 2018 Jul, 94(1):150-158).
  • Poppelaars et al suggest that FHR1 and FHR5 compete with the regulatory function of Factor H, such that the FHR proteins amplify alternative pathway activation and thereby stimulate development and progression of IgA nephropathy.
  • FHR5 has been implicated in C3 glomerulopathy and renal impairment (see e.g. Medjeral-Thomas et al., Kidney Int Rep. 2019, 4(10):1387-1400), as well as glomerular damage and kidney injury (e.g. Malik et al., PNAS, 2021 , 118(13) e2022722118).
  • Abnormal FHR hybrid proteins have also been reported in C3 glomerulopathy, and are thought to compete with FH for C3b binding and regulation (see e.g. Wong & Kavanagh, Semin Immunopathol. 2018; 40(1): 49-64).
  • FH, FHR1 and FHR3 were detected in the glomeruli of patients with Dense Deposit Disease (DDD)/membranoproliferative glomerulonephritis type II, see e.g. Sethi et al., Kidney Int. 2009, 75(9):952-60, and Abrera-Abeleda et al., J Med Genet. 2006, 43(7): 582-589. Elevated FHR1 levels have been implicated in ANCA vasculitis (see e.g. Skerka et al., Br J Pharmacol. 2021 Jul;178(14):2823-2831).
  • the disorder may be selected from Haemolytic Uremic Syndrome (HUS), atypical Haemolytic Uremic Syndrome (aHUS), DEAP HUS (Deficiency of FHR plasma proteins and Autoantibody Positive form of Hemolytic Uremic Syndrome), autoimmune uveitis, Membranoproliferative Glomerulonephritis Type II (MPGN II), sepsis, Henoch-Schbnlein purpura (HSP), IgA nephropathy, chronic kidney disease, paroxysmal nocturnal hemoglobinuria (PNH), autoimmune hemolytic anemia (AIHA), systemic lupus erythematosus (SLE), Sjogren’s syndrome (SS), rheumatoid arthritis (RA), C3 glomerulopathy (C3G), dense deposit disease (DDD), C3 nephritic factor glomerulonephritis (C3 NF GN), FHR5 nephropathy, hereditary angio
  • the disorder is cancer. Any cancer may be treated as described herein.
  • the cancer may be a liquid or blood cancer, such as leukemia, lymphoma or myeloma.
  • the cancer is a solid cancer, such as breast cancer, lung cancer, liver cancer, colorectal cancer, nasopharyngeal cancer, kidney cancer or glioma.
  • the cancer is located in the liver, bone marrow, lung, spleen, brain, pancreas, stomach or intestine.
  • the cancer is lung cancer.
  • the complement-related disorder is an indoleamine 2,3-dioxygenase 1 (IDO)-expressing cancer.
  • Complement activation plays a role in the development and progression of cancer.
  • DeCordova et al., Immunobiology. 2019, 224(5):625-631 reports that FHR5 is secreted by primary tumor cells derived from Glioblastoma multiforme (GBM) patients and may be used by the cells to resist complement mediated lysis.
  • GBM Glioblastoma multiforme
  • Afshar-Kharghan, J Clin Invest. 2017, 127(3):780-789 reports that expression of complement factors is increased in malignant tumors, including the FHR proteins which would outcompete FH and lead to complement dysregulation.
  • FH has been reported as a biomarker for lung cancer, squamous lung cancer, bladder cancer, ovarian cancer, liver cancer and SCC (e.g. Revel et al., Antibodies (Basel). 2020, 9(4): 57).
  • the cancer is glioblastoma e.g. glioblastoma multiforme (GBM).
  • GBM glioblastoma
  • CNS central nervous system
  • GBM is among the malignancies that are uniquely unresponsive to cancer immunotherapy.
  • indoleamine 2,3-dioxygenase 1 (IDO) activity in tumour cells increased expression of FH and FHL-1 , which were found to be associated with expression of immunosuppressive genes, suppression of anti-tumour immune responses, poorer survival outcomes for glioma patients and a faster rate to GBM recurrence.
  • the complement-related disorder is I DO-expressing GBM.
  • the complement-related disorder is isocitrate dehydrogenase (IDH)-expressing GBM.
  • the GBM expresses both IDO and IDH.
  • the cancer e.g. GBM, comprises tumour cells with increased expression of FH and/or FHL-1 .
  • the present disclosure provides a method for determining whether a subject has, is at risk of developing, or is at risk of progression of, glioblastoma e.g. glioblastoma multiforme (GBM), the method comprising:
  • the disorder is neurodegeneration or neurodegenerative disease.
  • the disorder may comprise progressive atrophy and loss of function of neurons.
  • the disorder may be selected from Parkinson’s disease, Alzheimer’s disease, dementia, stroke, Lewy body disease, Amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), Huntington’s disease and prion diseases.
  • FHR proteins as FH competitors and reviews the adverse effect of complement activation in the central nervous system, such as in the context of Alzheimer’s disease, Parkinson’s disease, schizophrenia, myasthenia gravis (MG), amyotrophic lateral sclerosis (ALS), and Guillain-Barre syndrome (GBS).
  • MG myasthenia gravis
  • ALS amyotrophic lateral sclerosis
  • GBS Guillain-Barre syndrome
  • the complement-related disorder that may be treated and/or prevented i.e. that is characterised by elevated levels of one or more of FHR1 , FHR2, FHR3, FHR4 and/or FHR5 is selected from macular degeneration, age related macular degeneration (AMD), geographic atrophy (‘dry’ (i.e.
  • AMD non-exudative non-exudative AMD
  • AMD early AMD
  • EOMD early onset macular degeneration
  • intermediate AMD late/advanced AMD
  • wet neovascular or exudative AMD
  • CNV choroidal neovascularisation
  • retinal dystrophy Haemolytic Uremic Syndrome (HUS), atypical Haemolytic Uremic Syndrome (aHUS), DEAP HUS (Deficiency of FHR plasma proteins and Autoantibody Positive form of Hemolytic Uremic Syndrome)
  • autoimmune uveitis kidney injury/damage/dysfunction, glomerular diseases
  • MPGN II Membranoproliferative Glomerulonephritis Type II
  • HSP Henoch-Schbnlein purpura
  • IgA nephropathy chronic kidney disease
  • ANCA vasculitis autoimmune hemolytic anemia (AIHA)
  • the complement-related disorder is an infectious disease.
  • Complement is a major component of the innate immune system involved in defending against foreign pathogens, including bacteria, viruses, fungi and parasites. Activation of complement leads to robust and efficient proteolytic cascades, which result in opsonization and lysis of the pathogen as well as in the generation of the classical inflammatory response through the production of potent proinflammatory molecules.
  • the role of complement in innate and adaptive immune responses is reviewed in e.g. Bisberger, J., Song, WC. Cell Res 2010; 20, 34-50, and Rus H et al., Immunol Res. 2005; 33(2):103-12, which are hereby incorporated by reference in their entirety.
  • the complement-related disorder is infection by severe acute respiratory syndrome-related coronavirus (SARSr-CoV). In some embodiments the complement-related disorder is infection with SARS-CoV-2. In some embodiments the complement-related disorder is a disease/condition caused or exacerbated by SARS-CoV-2 infection, e.g. COVID-19 or another disease/condition for which infection with SARS-CoV-2 is a contributing factor.
  • SARSr-CoV severe acute respiratory syndrome-related coronavirus
  • SARSr-CoV is a species of coronavirus of the genus Betacoronavirus and subgenus Sarbecoronavirus that infects humans, bats and certain other mammals. It is an enveloped positive-sense single-stranded RNA virus.
  • SARS-CoV severe acute respiratory syndrome
  • SARS-CoV-2 which has caused the coronavirus disease 2019 (COVID-19) pandemic.
  • SARS-CoV-2 refers to the SARSr-CoV having the nucleotide sequence of GenBank: MN996527.1 (“Severe acute respiratory syndrome coronavirus 2 isolate WIV02, complete genome”), reported in Zhou et al., Nature (2020) 579: 270-273, and encompasses variants thereof having a nucleotide sequence with at least 85% sequence identity (e.g.
  • Variants of SARS-CoV-2 of particular interest include: (i) the variant designated VUI-202012/01 , which belongs to the B.1 .1 .7 lineage, having the canonical nucleotide sequence of GISAID accession EPI_ISL_601443; (ii) the variant designated 501 Y.V2/B.1 .351 , having the canonical nucleotide sequence of GISAID accession EPI_ISL_768642; (iii) the variant known as B.1 .1 .248/P.1 , having the canonical nucleotide sequence of GISAID accession EPI_ISL_792680; (iv) the variant known as B.1 .617.1 ; and (v) the variant known as B.1 .617.2.
  • the complement-related disorder is a disease/condition caused by infection of SARS-CoV-2 variants B.1 .1.7, B.1 .1 .248/P.1 , B.1.617.1 and/or B.1.617.2.
  • COVID-19 Report 19 May 2020: ISARIC; 2020 and Certy et al., BMJ (2020) 369:m1985, which are hereby incorporated by reference in their entirety.
  • Common symptoms include cough, fever, headache, dyspnoea, anosmia, pharyngitis, nasal obstruction, rhinorrhoea, asthenia, myalgia, joint pain, gustatory dysfunction, abdominal pain, vomiting, and diarrhoea.
  • ARDS acute respiratory distress syndrome
  • the complement- related disorder is ARDS or acute respiratory failure.
  • Complement activation has been implicated in the pathogenesis of severe SARS-CoV-2 infection. Circulating markers of complement activation are elevated in patients with COVID-19 compared to those with influenza and to patients with non-COVID-19 respiratory failure. Patients hospitalized with COVID-19 reportedly have significantly higher median plasma sC5b-9, C5a, and Factor B levels compared to those with influenza, pneumonia or sepsis, and certain markers of complement activation have been associated with worse outcomes in COVID-19 patients, see e.g. Ma L et al., Sci Immunol. 2021 May 13; 6(59): eabh2259. Patients requiring ICU treatment, or who died from COVID-19 infection, were found to have significantly higher Factor D levels.
  • SARS-CoV-2 antigens and SARS-CoV-2 RNA in the blood reportedly correlates with the level of IL-6, inflammation, respiratory failure and death, see e.g. Brasen CL et al., Clin Chem Lab Med. 2021 Aug 27. Doi: 10.1515/cclm-2021 -0694.
  • FHL1 , FHR1 , FHR2, FHR3, FHR4 and FHR5 were observed to correlate with increasing COVID-19 severity, with the highest levels being observed in subjects with clinically severe COVID-19 requiring assisted ventilation.
  • detection of one or more of FHL1 , FHR1 , FHR2, FHR3, FHR4 and/or FHR5 may predict the likelihood of a subject developing severe COVID-19. Appropriate treatment and monitoring can thus be deployed.
  • the methods described herein are useful for predicting the risk of development of conditions associated with SARS-CoV-2 infection, as well as predicting the severity of such infection, e.g. the likelihood of developing severe or critical COVID-19 and associated complications such as ARDS.
  • the present disclosure provides a method for determining/identifying whether a subject has, or is at risk of developing, a complement-related disorder associated with SARS-CoV-2 infection, e.g. COVID-19 or ARDS, the method comprising:
  • Methods provided herein may be useful for determining the risk of a subject developing a serious complement-related disorder, e.g. the methods are useful for distinguishing between subjects who may develop a mild complement-related disorder and subjects who are at risk of serious disease, and/or identifying subjects who are likely to develop serious disease.
  • the methods described herein can be used to identify subjects that are at risk of developing a severe disorder associated with SARS-CoV-2 infection, e.g. severe COVID-19 or critical COVID-19.
  • a severe disorder associated with SARS-CoV-2 infection e.g. severe COVID-19 or critical COVID-19.
  • Cases of COVID-19 can generally be categorised into five groups: asymptomatic, mild, moderate, severe and critical. Severe COVID-19 includes pneumonia and patients may require supplemental oxygen.
  • Critical COVID-19 includes severe pneumonia and ARDS, and in some cases sepsis. Patients with critical COVID-19 require assisted ventilation.
  • the present invention provides methods of predicting, based on the analysis described herein of a sample from a subject, whether a subject is at risk of developing a complement-related disorder, has a complement-related disorder, is in need of treatment for a complement-related disorder, will respond to treatment for a complement-related disorder, and/or is responding/has responded to treatment for a complement-related disorder.
  • the methods may be used for determining whether a subject is at risk of onset of the disorder, and/or is at risk of progression, exacerbation or worsening of the disorder.
  • Methods described herein may also be useful for assessing whether treatment for a complement-related disorder is/has been effective or successful.
  • the methods described herein may be useful for determining whether a subject is likely to respond or not respond to a therapeutic treatment, or whether a subject is responding to a therapeutic treatment.
  • the methods should enable patients to receive the most effective therapy fortheir particular pathological requirements.
  • the subject has or is suspected to have a complement-related disorder.
  • the disorder is AMD.
  • the disorder is EOMD.
  • the disorder is glioblastoma e.g. glioblastoma multiforme (GBM).
  • the disorder is a complement-related disorder associated with SARS-CoV-2 infection, e.g. COVID-19 or ARDS.
  • the present invention provides a method for treating or preventing a complement-related disorder in a subject, the method comprising administering an effective amount of a complement-targeted therapy/therapeutic agent, wherein the subject to be treated has been determined to have atypical presence or levels of one or more complement proteins, e.g. detected/d etermined as described herein, as compared to a control subject and/or reference value(s).
  • the subject has been determined to be at risk of developing a complement-related disorder, and/or identified as having a complement-related disorder.
  • the present invention provides a complement-targeted therapy/therapeutic agent for use in a method of treating or preventing a complement-related disorder in a subject, the method comprising administering an effective amount of the therapy/therapeutic agent, wherein the subject has/has been determined to have atypical presence or levels of one or more complement proteins, e.g. determined as described herein, as compared to a reference value(s).
  • the subject has been determined to be at risk of developing a complement-related disorder, and/or identified as having a complement- related disorder.
  • a complement-targeted therapy/therapeutic agent in the manufacture of a medicament for treating or preventing a complement-related disorder in a subject, wherein the subject has/has been determined to have atypical presence or levels of one or more complement proteins, e.g. determined as described herein, as compared to a reference value(s).
  • the subject has been determined to be at risk of developing a complement-related disorder, and/or identified as having a complement-related disorder.
  • the subject has been determined to be at risk of developing a complement-related disorder, and/or identified as having a complement-related disorder.
  • the subject to be treated has atypical presence or levels of at least one complement protein, preferably one or more of FH, FHL-1 , FHR1 , FHR2, FHR3, FHR4, FHR5, Fl, C3, C3b, C3a, iC3b, C3f, C3c, C3dg, and/or C3d.
  • the subject to be treated has atypical presence of levels of one or more of FHR1 , FHR2, and/or FHR3, and optionally FHR4 and/or FHR5, and/or FHL-1 .
  • the subject may benefit from treatment to reduce the level of any complement proteins that are increased as compared to a reference value(s) and/or from treatment to increase the level of any complement proteins that are decreased as compared to a reference value(s).
  • treatment may, for example, be reduction in the development or progression of a disease/condition, alleviation of the symptoms of a disease/condition or reduction in the pathology of a disease/condition.
  • Treatment or alleviation of a disease/condition may be effective to prevent progression of the disease/condition, e.g. to prevent worsening of the condition or to slow the rate of development.
  • treatment or alleviation may lead to an improvement in the disease/condition, e.g. a reduction in the symptoms of the disease/condition or reduction in some other correlate of the severity/activity of the disease/condition.
  • Prevention/prophylaxis of a disease/condition may refer to prevention of a worsening of the condition or prevention of the development of the disease/condition, e.g.
  • Methods provided herein for assessing the risk of development i.e. the onset or risk of progression of, or for identifying subjects having/at risk of, a complement-related disorder may be performed in conjunction with additional diagnostic methods and/or tests for such disorders that will be known to one skilled in the art.
  • methods for assessing the risk of development of a complement-related disorder comprise further techniques selected from: CH50 or AH50 measurement via haemolytic assay, measurement of neoantigen formation during MAC complex (C5b, C6, C7, C8, C9) generation, C3 deficiency screening, mannose-binding lectin assays, immunochemical assays to quantify individual complement components, flow cytometry to assess cell-bound regulatory proteins e.g. CD55, CD59 and CD35, and/or renal function tests, see e.g. Shih AR and Murali MR, Am. J. Hematol.
  • methods provided herein for assessing the risk of development of AMD and/or EOMD comprise further assessment techniques selected from: dark adaptation testing, contrast sensitivity testing e.g. Pelli Robson, visual acuity testing using e.g. a Snellen chart and/or Amsler grid, Farnsworth- Munsell 100 hue test and Maximum Color Contrast Sensitivity test (MCCS) for assessing colour acuity and colour contrast sensitivity, preferential hyperacuity perimetry (PHP), fundus photography of the back of the eye, fundus examination, fundus autofluorescence, optical coherence tomography, angiography e.g.
  • fluorescence angiography fluorescence angiography
  • fundus fluorescein angiography fundus fluorescein angiography
  • indocyanine green angiography optical coherence tomography angiography
  • adaptive optics retinal imaging deep learning analysis of fundus images
  • electroretinogram methods and/or methods to measure histological changes such as atrophy, retinal pigment changes, exudative changes e.g. hemorrhages in the eye, hard exudates, subretinal/sub- RPE/intraretinal fluid, and/or the presence of drusen.
  • Methods described herein may take into account lifestyle factors known to contribute to risk of developing complement-related disorders.
  • lifestyle factors that may cause or contribute to AMD include smoking, being overweight, high blood pressure and having a family history of AMD.
  • the methods provided herein may comprise determining in a subject the presence or absence of a genetic profile characterised by polymorphisms in the subject’s genome associated with complement dysregulation.
  • the polymorphisms may be found within or near genes such as CCL28, FBN2, ADAM12, PTPRC, IGLC1 , HS3ST4, PRELP, PPID, SPOCK, APOB, SLC2A2, COL4A1 , MYOC, ADAM19, FGFR2, C8A, FCN1 , IFNAR2, C1 NH, C7 and ITGA4.
  • a genetic profile associated with complement dysregulation may comprise one or more, often multiple, single nucleotide polymorphisms, e.g. as set out in Tables I and II of US 2010/0303832, which is hereby incorporated by reference in its entirety.
  • a complement-related disorder described herein may comprise a genetic element and/or a genetic risk factor.
  • a method of identifying a subject having a complement-related disorder or at risk of developing a complement-related disorder comprising determining in a subject the presence or absence of one or more genetic factors associated with AMD and/or EOMD, e.g. one or more AMD- or EOMD-associated genetic variants.
  • any method provided herein may comprise determining in a subject the presence or absence of one or more genetic factors associated with AMD and/or EOMD, e.g. one or more AMD- or EOMD-associated genetic variants.
  • the methods comprise screening (directly or indirectly) for the presence or absence of the one or more genetic factors.
  • the genetic factor(s) are genetic risk factor(s).
  • the subject has been determined to have one or more such risk factors.
  • the methods of the present invention involve determining whether a subject possesses one or more such risk factors, e.g. by obtaining a sample from the subject, or in a sample obtained from the subject.
  • the one or more genetic factors may be located on chromosome 1 at or near the RCA locus, e.g. in the CFH/CFHR genes/the CFH locus.
  • the presence of one or more CFH locus AMD-risk variants increase disease risk via increase of FHR protein levels.
  • the one or more genetic factors may be located in one or more of: CFH e.g. selected from Y402H (i.e. rs1061170 c ), rs1410996 c , I62V (rs800292), A473A (rs2274700), R53C, D90G, D936E (rs1065489), R1210C, IVS1 (rs529825), IVS2 insTT, IVS6 (rs3766404), A307A (rs1061147), IVS10 (rs203674), rs3753396, R1210C, rs148553336, rs191281603, rs35292876, and rs800292; CFHR4 e.g.
  • a genetic factor is Y402H (i.e. rs1061170 c ). In some embodiments, a genetic factor is rs3753396. In some embodiments, a genetic factor is rs6685931 and/or rs1409153. In some embodiments, a genetic factor is at intronic KCNT2 rs61820755. In some embodiments, a genetic factor is not rs6685931.
  • a genetic factor is rs61820755, and may be associated with FHL-1 .
  • the genetic risk factors may be present in combination with elevated levels of one or more FHR proteins.
  • the one or more genetic factors at the CFH locus may be selected from intergenic CFHR1/CFHR4 rs149369377 and/or rs61820755 for FHR-1 , CFHR2 rs4085749 for FHR-2, intronic CFH rs70620 for FHR-3, rs12047098 for FHR-4, intronic KCNT2 rs72732232 for FHR-5.
  • the presence of any one or more of these genetic factors indicates that the subject has or is likely to develop a complement-related disorder.
  • the one or more genetic risk factors may be selected from rs10922109, rs570618, rs121913059 (R1210C), rs148553336, rs187328863, rs61818925, rs35292876, and rs191281603.
  • the one or more genetic factors may be selected from one or more of rs113721756 on chromosome 10, rs111260777 on chromosome 11 , rs117468955 on chromosome 12, rs200404865 on chromosome 13, rs4790395 on chromosome 17 and rs117115124 on chromosome 19. These factors may be present separately, or in addition to, genetic factors at the CFH locus. These factors may be present in combination with elevated FHR-3 levels.
  • the methods described herein may involve detecting combinations of risk factors to assess the risk of a subject developing a complement related disorder, e.g. if one or both of the risk factors are present in a subject.
  • a complement related disorder e.g. if one or both of the risk factors are present in a subject.
  • Assessment of the presence of any genetic risk factor provided herein may be combined with the detection of any one or more of FHR1 , FHR2, FHR3, FHR4 and/or FHR5, and optionally FHL-1 as described herein.
  • the presence of genetic factor rs10922109 may be assessed in combination with the detection of any one or more of FHR-1 , FHR-2, FHR-3, and/or FHR-4; rs570618 may be assessed in combination with the detection of FHR-1 and/or FHR-2; rs61818925 may be assessed in combination with the detection of FHR-2 and/or FHR-4; and rs148553336 may be assessed in combination with the detection of FHR-5.
  • the subject may comprise high risk CFH polymorphism T1277C.
  • a method according to the present disclosure does not comprise detecting the T1277C polymorphism.
  • the subject does not comprise high risk CFH polymorphism T1277C.
  • the method may comprise a step of determining that the subject has or is likely to develop a complement-related disorder if one or more genetic factors, e.g. those described herein, are present.
  • a method of identifying a subject having a complement-related disorder or at risk of developing a complement-related disorder comprising assessing the subject for one or more genetic risk factors, e.g. any of those described herein or others, and determining that the subject has or is likely to develop a complement-related disorder if the one or more genetic risk factors are present in the subject.
  • a method of determining whether a subject has, or is at risk of developing, a complement-related disorder the method comprising assessing the subject for one or more genetic risk factors, e.g. any of those described herein or others, and determining that the subject has or is likely to develop a complement-related disorder if the one or more genetic risk factors are present in the subject.
  • Also provided is a method for selecting a subject for treatment with a therapeutic agent e.g. a complement-targeted therapy or therapeutic agent
  • a therapeutic agent e.g. a complement-targeted therapy or therapeutic agent
  • a method for selecting a therapeutic agent e.g. a complement-targeted therapy or therapeutic agent, for a subject
  • methods of treatment e.g. a complement-targeted therapy or therapeutic agent
  • a complement- targeted therapy or therapeutic agent for use in a method of treatment
  • the use of a complement- targeted therapy or therapeutic agent in the manufacture of a medicament for the treatment of a complement-related disorder
  • the method uses the steps above to assess genetic risk factors (either alone or in combination with determining the level of a complement protein e.g.
  • Any such method comprising detecting and assessing genetic risk factors may comprise a treatment step as described herein, e.g. treating a subject that has been determined to have or be likely to develop a complement-related disorder.
  • the methods provided herein further comprise determining in a subject the presence or absence of one or more genetic factors associated with EOMD, e.g. one or more EOMD-associated genetic variants. In some cases, the methods comprise screening (directly or indirectly) for the presence or absence of the one or more genetic factors.
  • the genetic factor(s) are genetic risk factor(s).
  • the subject has been determined to have one or more such risk factors. In some embodiments, the methods of the present invention involve determining whether a subject possesses one or more such risk factors. In some embodiments the subject may possess one or more risk factors for early-onset macular degeneration (EOMD).
  • EOMD early-onset macular degeneration
  • EOMD is thought to be caused by monogenic inheritance of rare variants of the CFH gene (see e.g. Boon CJ et al. Am J Hum Genet 2008; 82(2):516-23; van de Ven JP, et al. Arch Ophthalmol 2012;130(8):1038- 47; Yu Y et al. Hum Mol Genet 2014; 23(19):5283-93; Duwari MR, et al. Mol Vis 2015; 21 :285-92;
  • the subject may possess one or more of EOMD-associated genetic variants.
  • EOMD-associated genetic variants are described in e.g. Servais A et al. Kidney Int, 2012; 82(4):454-64 and Dragon-Durey MA, et al. J Am Soc Nephrol 2004; 15(3):787-95; which are hereby incorporated by reference in their entirety.
  • the subject may possess one or more of the following EOMD-associated genetic variants: CFH c.1243del, p.(Ala415Profs*39) het; CFH c.350+1G>T het; CFH c.619+1G>A het; CFH c.380G>A, p.(Arg127His); CFH C.694OT, p.(Arg232Ter); or CFH c.1291T>A, p.(Cys431 Ser).
  • the methods provided herein comprise screening for deletions within the RCA locus (a region of DNA sequence located on chromosome one that extends from the CFH gene through to the CD46 (MCP) gene) that are associated with AMD and/or EOMD risk or protection.
  • RCA locus a region of DNA sequence located on chromosome one that extends from the CFH gene through to the CD46 (MCP) gene
  • Methods for determining the presence or absence of genetic factors include restriction fragment length polymorphism identification (RFLPI) of genomic DNA, random amplified polymorphic detection (RAPD) of genomic DNA, amplified fragment length polymorphism detection (AFLPD), multiple locus variable number tandem repeat (VNTR) analysis (MLVA), SNP genotyping, multilocus sequence typing, PCR, DNA sequencing e.g. Sanger sequencing or Next-Generation sequencing, allele specific oligonucleotide (ASO) probes, and oligonucleotide microarrays or beads.
  • RFLPI restriction fragment length polymorphism identification
  • RAPD random amplified polymorphic detection
  • AFLPD amplified fragment length polymorphism detection
  • VNTR multiple locus variable number tandem repeat
  • SNP genotyping multilocus sequence typing
  • PCR DNA sequencing e.g. Sanger sequencing or Next-Generation sequencing
  • ASO allele specific oligonucleotide
  • microarrays or beads
  • the subject is selected for therapeutic or prophylactic treatment with a complement-targeted therapy/therapeutic agent based on their being determined to possess one or more genetic factors for AMD and/or EOMD, e.g. one or more AMD-associated and/or EOMD-associated genetic variants, or for a macular dystrophy.
  • the subject has been determined to have one or more such genetic factors.
  • the methods provided herein comprise determining whether a subject possesses one or more such genetic factors. Such methods and genetic factors are described herein.
  • a method of diagnosing, treating or preventing a complement-related disorder in a subject wherein the subject has/has been determined/is determined to possess one or more genetic factors for AMD and/or EOMD, and wherein the subject has/has been determined/is determined to have atypical presence or levels of one or more complement proteins, e.g. detected/determined as described herein, as compared to a reference value(s); optionally wherein the method comprises administering a complement-targeted therapy/therapeutic agent.
  • subject refers to a subject, patient or individual and may be any animal or human.
  • the subject is preferably mammalian, more preferably human.
  • the subject may be a non-human mammal, but is more preferably human.
  • the subject may be male or female.
  • the subject may be a patient.
  • Therapeutic uses may be in human or animals (veterinary use).
  • the subject to be treated with a therapeutic substance described herein may be a subject in need thereof.
  • the subject may be identified, or may have been identified, as having a complement-related disorder or being at risk of developing a complement-related disorder, e.g. by a method described herein.
  • a subject described herein may belong to a patient subpopulation i.e. the subject may be part of an identifiable, specific portion or subdivision of a population.
  • the population and/or subpopulation may have or be suspected to have a complement-related disorder.
  • the subpopulation may display atypical presence or levels of one or more complement proteins, e.g. detected/d etermined as described herein, as compared to the population as a whole.
  • the population and/or subpopulation may have or be suspected to have AMD, EOMD or a macular dystrophy.
  • the subject is characterised as having an atypical presence or level of one or more complement proteins, e.g. detected/determined/measured as described herein.
  • a subject may have, have been determined to have, or be characterised as having elevated levels of a complement protein selected from one or more of FHR1 , FHR2, FHR3, FHR4 and/or FHR5, and optionally FHL-1 .
  • a method of treating or preventing a complement-related disorder in a subject wherein the subject is characterised as having an atypical presence or levels of one or more complement proteins, e.g. detected/determined as described herein.
  • complement-targeted therapeutic agent for use in a method of treating or preventing a complement-related disease in a subject, wherein the subject is characterised as having an atypical presence or levels of one or more complement proteins, e.g. detected/determined as described herein.
  • Methods according to the present invention may be performed outside the human or animal body.
  • Methods according to the present invention may be performed, or products may be present, in vitro, ex vivo, or in vivo.
  • in vitro is intended to encompass experiments with materials, biological substances, cells and/or tissues in laboratory conditions or in culture whereas the term “in vivo” is intended to encompass experiments and procedures with intact multi-cellular organisms.
  • Ex vivo refers to something present or taking place outside an organism, e.g. outside the human or animal body, which may be on tissue (e.g. whole organs) or cells taken from the organism.
  • the determining, detecting, measuring, quantifying, predicting and/or diagnosing steps of the methods provided herein are performed in vitro.
  • the complement related disorder may be any disorder in which the complement system, or activation/over-activation/dysregulation thereof, is pathologically implicated.
  • the complement related disorder may be any disorder described herein.
  • the methods described herein may be useful in monitoring the success of treatment, including past or ongoing treatment, for complement- related disorders. Such treatment may involve the nucleic acids and/or polypeptides as described herein.
  • a method of identifying a subject having a complement-related disorder or at risk of developing a complement-related disorder comprising:
  • a method of determining whether a subject has, or is at risk of developing, a complement-related disorder comprising:
  • step (a) comprises determining the level of two of the complement proteins selected from FHR1 , FHR2 and/or FHR3. In some embodiments step (a) comprises determining the level of three of the complement proteins selected from FHR1 , FHR2 and FHR3.
  • step (a) comprises, or further comprises, determining the level of FHR4 and/or FHR5.
  • the methods described herein may comprise determining that the subject has or is likely to develop a complement-related disorder if the level of FHR4 and/or FHR5 is elevated as compared to the level of that complement protein in blood in a control subject that does not have a complement-related disorder.
  • step (a) comprises, or further comprises, determining the level of FH and/or FHL-1.
  • the method may comprise determining the level of FHL-1 , alone or in combination with other complement protein(s), and determining that the subject has or is likely to develop a complement-related disorder if the level of FHL-1 is altered, e.g. elevated, as compared to the level of FHL-1 in blood in a control subject that does not have a complement-related disorder.
  • the level of FH and/or FHL-1 may be increased or decreased compared to a control subject.
  • Determining the level of two or more complement proteins may be performed simultaneously, concurrently or sequentially.
  • the complement proteins may be detected in the same assay, or in one or more separate assays.
  • Determining the level of a second or subsequent complement protein may be performed concurrently with, prior to or after determining the level of a first complement protein.
  • steps (a) and (b) may be repeated one or more times on the same subject at appropriate time intervals in order to assess the progression of a complement-related disorder.
  • Any aspect or embodiment described herein may comprise determining the level of any one of the following proteins, e.g. in a subject: a) FHR1 ; b) FHR2; c) FHR3; d) FHR4; e) FHR5; f) FHR1 and FHR2; g) FHR1 and FHR3; h) FHR1 and FHR4; i) FHR1 and FHR5; j) FHR2 and FHR3; k) FHR2 and FHR4; l) FHR2 and FHR5; m) FHR3 and FHR4; n) FHR3 and FHR5; o) FHR4 and FHR5; p) FHR1 , FHR2 and FHR3; q) FHR1 , FHR2 and FHR4; r) FHR1 , FHR2 and FHR5; s) FHR1 , FHR3 and FHR4; t) FHR1 , FHR3 and F
  • reference to ‘FHR1 ’ herein may refer to the detection of either one or both of FHR1a and/or FHR1 b.
  • the complement protein(s) to be detected/determined is not FHR3.
  • the complement protein(s) to be detected/determined is not FHR4.
  • the complement protein(s) to be detected/determined is not FH.
  • the complement protein(s) to be detected/determined is not FHL-1.
  • complement protein(s) detected may depend on the complement-related disorder of interest and the complement protein(s) that are useful biomarkers for an individual disorder. For example, detecting one or more of FHR1 , FHR2, FHR3, FHR4, FHR5 and/or FHL-1 is predictive of AMD risk, whereas other particular complement proteins and combinations thereof are predictive for other complement-related disorders, see e.g. the disorders and references described herein.
  • the present disclosure allows the precise detection and distinction of any one or more of the complement proteins described herein, thus allowing the absolute levels of said proteins to inform the likelihood of disorder onset and/or progression according to the variations in protein levels in each disorder.
  • the complement protein(s) may be detected in a sample obtained from a subject.
  • the sample may be obtained to inform appropriate treatment and/or progression of the disorder.
  • any aspect described herein may comprise determining the level of any one or more complement proteins selected from FHR1 , FHR2, FHR3, FHR4, FHR5, FH and/or FHL-1 , e.g. in a blood sample obtained from a subject, and then determining that the subject has or is likely to develop a complement-related disorder if the level of the complement protein(s) is altered as compared to the level of that complement protein(s) in blood in a control subject that does not have a complement-related disorder.
  • the term “altered” as used herein refers to the level of the complement protein(s) increasing or decreasing, e.g.
  • the level of one or more complement proteins may be higher or lower as compared to the level of those complement proteins in blood in a control subject that does not have a complement-related disorder. In some cases, the level of the complement protein may be decreased as compared to the level of that complement protein in blood in a control subject that does not have a complement-related disorder. In some cases, where the level of two or more complement proteins is determined, the level of one or more complement proteins may be elevated whilst the level of one or more different complement proteins may be decreased as compared to the levels of those complement proteins in blood in a control subject that does not have a complement-related disorder.
  • the level of a complement protein is determined using any suitable technique known in the art and available to a skilled person. In some embodiments the level of a complement protein is determined by mass spectrometry and/or digesting the protein with endoproteinase GluC, e.g. as described herein. Determining the level of a complement protein(s) may involve detecting any combination of peptides produced by digestion with GluC, as described herein.
  • the level of a complement protein may be determined using, for example, an enzyme-linked immunosorbent assay (ELISA/EIA) e.g. as described in van Beek et al., Front Immunol. 2017; 8: 1328; van Beek et al. Front Immunol.
  • ELISA/EIA enzyme-linked immunosorbent assay
  • the level of a complement protein may be determined using, for example, Western blotting or dot blotting with appropriate antibodies, HPLC, protein immunoprecipitation or immunoelectrophoresis.
  • Any method described herein may comprise an initial step of obtaining a sample and/or at least one protein, e.g. complement protein, from the subject. Suitable sources of samples are described herein.
  • the methods described herein may comprise determining the level of circulating FHR1 , FHR2, and/or FHR3, circulating FHR4 and/or FHR5, and optionally circulating FH and/or FHL-1. Circulating proteins may be present in e.g. blood or lymph.
  • Any method described herein may comprise determining the level of one or more of C3, C3b, C3a, iC3b, C3f, C3c, C3dg, and/or C3d, e.g. as described herein.
  • a method of determining whether a subject has, or is at risk of developing, a complement-related disorder comprising:
  • the method further comprises digesting one or both of FH and/or FHL-1 with endoproteinase GluC to obtain one or more peptides, determining the level of the one or more peptides by mass spectrometry and/or using the results of the mass spectrometry to determine the level of FH and/or FHL-1 .
  • Exemplary combinations of complement proteins for use in the methods of the present invention are described above.
  • a complement protein selected from one or more of FHR1 , FHR2, FHR3, FHR4 and/or FHR5, and optionally FH and/or FHL-1 , for identifying a subject having a complement-related disorder or at risk of developing a complement-related disorder, or for determining whether a subject has or is at risk of developing a complement-related disorder, the use comprising:
  • a complement protein selected from one or more of FHR1 , FHR2, FHR3, FHR4 and/or FHR5, and optionally FH and/or FHL-1 , as a biomarker e.g. for identifying a subject having a complement-related disorder or at risk of developing a complement-related disorder, or for determining whether a subject has or is at risk of developing a complement-related disorder, the use comprising the steps described hereinabove.
  • endoproteinase GluC in a method for determining the presence and/or level of a complement protein, e.g. in a sample or a subject, e.g. according to the methods described herein.
  • endoproteinase GluC in a method of identifying a subject having a complement-related disorder or at risk of developing a complement-related disorder, the method comprising: a) digesting at least one complement protein in a sample obtained from the subject with endoproteinase GluC to obtain one or more peptides; b) determining the presence and/or level of the one or more peptides by mass spectrometry; and c) using the results of (b) to determine whether the subject has or is likely to develop a complement-related disorder.
  • GluC in a method of selecting a subject for treatment of a complement-related disorder with a complement-targeted therapeutic, the method comprising: a) digesting at least one complement protein in a sample obtained from the subject with endoproteinase GluC to obtain one or more peptides; b) determining the presence and/or level of the one or more peptides by mass spectrometry; and c) using the results of (b) to determine whether the subject is in need of a complement-targeted therapeutic.
  • GluC in the diagnosis of a complement-related disorder, comprising: a) digesting at least one complement protein in a sample obtained from the subject with endoproteinase GluC to obtain one or more peptides; b) determining the presence and/or level of the one or more peptides by mass spectrometry; and c) using the results of (b) to determine whether the subject is in need of a complement-targeted therapeutic.
  • the methods described herein are performed in vitro or ex vivo.
  • a sample may be obtained from a subject of interest, and/or a control subject, and the determining steps are performed in vitro or ex vivo.
  • the level of the complement protein(s) is compared to the level of a reference value or level, sometimes called a control.
  • the level of the complement protein(s) is compared to the level of the same complement protein in a control subject that does not have a complement-related disorder.
  • a reference value may be obtained from a control sample, which itself may be obtained from a control subject. Data or values obtained from the individual to be tested, e.g. from a sample, can be compared to data or values obtained from the control sample. In some cases, the control is a spouse, partner, or friend of the subject.
  • the level of the complement protein(s) that are determined may be elevated (i.e. higher, increased, greater) compared to the reference value or level. That is, there may be more of the complement protein(s) in the sample tested compared to the reference value. There may be a higher amount or concentration of the complement protein(s) in the tested sample compared to the reference value or in a control sample.
  • the level of the complement protein(s) that are determined may be reduced (i.e. lower, decreased) compared to the reference value or level. That is, there may be less of the complement protein(s) in a tested sample tested compared to the reference value. There may be a lower amount or concentration of the complement protein(s) in a tested sample compared to the reference value or in a control sample.
  • the term “reference value” refers to a known measurement value used for comparison during analysis.
  • the reference value is one or a set of test values obtained from an individual or group in a defined state of health.
  • the reference value may be one or a set of test values obtained from a control.
  • the reference value is/has been obtained from determining the level of complement proteins in subjects known not to have a complement-related disorder.
  • the reference value is/has been obtained from determining the level of complement proteins in subjects which have a complement-related disorder that is not associated with elevated levels of FHR protein(s), e.g. a subset of subjects in which FHR proteins are not considered to be a pathological factor.
  • the reference value is set by determining the level or amount of a complement protein previously from the individual to be tested e.g. at an earlier stage of disease progression, or prior to onset of the disease.
  • the reference value may be taken from a sample obtained from the same subject, or a different subject or subject(s).
  • the sample may be derived from the same tissue/cells/bodily fluid as the sample used by the present invention.
  • the reference value may be a standard value, standard curve or standard data set. Values/levels which deviate significantly from reference values may be described as atypical values/levels.
  • control may be a reference sample or reference dataset, or one or more values from said sample or dataset.
  • the reference value may be derived from a reference sample or reference dataset.
  • the reference value may be derived from one or more samples that have previously been obtained from one or more subjects that are known not to have a complement-related disorder and/or known or expected not to be at risk of developing a complement-related disorder.
  • the reference value may be derived from one or more samples that have previously been obtained from one or more subjects that are known to have a complement-related disorder.
  • the reference value may be derived from one or more samples that have previously been obtained from one or more subjects that are known to be at risk of developing a complement-related disorder.
  • the reference value may be consensus level or an average, or mean, value calculated from a reference dataset, e.g. a mean protein level.
  • the reference dataset/value may be obtained from a large-scale study of subjects known to have a complement-related disorder, such as AMD, e.g. as described herein.
  • the reference value may be derived from one or more samples that have previously been obtained from one or more subjects that are in the same family as the subject of interest, or from one or more subjects that are not in the same family as the subject of interest.
  • the reference value may be derived from one or more samples that have previously been obtained and/or analysed from the individual/subject/patient to be tested, e.g. a sample was obtained from the individual when they were at an earlier stage of a complement-related disorder, or a sample was obtained from the individual before the onset of a complement-related disorder.
  • the reference value may be obtained by performing analysis of the sample taken from a control subject in parallel with a sample from the individual to be tested.
  • the control value may be obtained from a database or other previously obtained value.
  • the reference value may be determined concurrently with the methods disclosed herein, or may have been determined previously.
  • Control subjects from which samples are/have been obtained may have undergone treatment for a complement-related disorder and/or received a complement-related therapy/therapeutic agent.
  • Controls may be positive controls in which the target molecule is known to be present, or expressed at high level, or negative controls in which the target molecule is known to be absent or expressed at low level.
  • Samples from one or more control subjects may comprise any one, two, three, four, five, six of seven of FHR1 , FHR2, FHR3, FHR4, FHR5, FH and/or FHL-1 .
  • each complement protein is in a separate control sample.
  • a control sample contains multiple complement proteins.
  • the methods described herein comprise comparing the level of one of more complement proteins determined as described herein to different, e.g. one or more, samples, each sample containing one or more complement proteins.
  • the methods described herein comprise comparing the level of one or more complement proteins determined as described herein to a single sample, wherein the sample contains one or more complement proteins.
  • control samples are obtained from the same tissue(s) as the sample obtained from the individual to be tested. In some cases control samples are obtained from different tissue(s) as the sample obtained from the individual to be tested. Control samples may be obtained from control subjects at certain time(s) of day, or on certain days. Sample(s) obtained from the individual to be tested are preferably obtained at the same time(s) of day and/or day(s) as the control samples.
  • an increase/decrease of a complement protein, e.g. as described herein, as compared to a reference value indicates an increased risk of developing a complement-related disorder.
  • an increase/decrease of a complement protein, e.g. as described herein indicates an increased risk of developing the disorder when compared to a reference value taken from the same subject at an earlier stage of the disorder, e.g. in a sample from the same subject.
  • a method described herein may comprise determining the level of two or more complement proteins and comparing their values e.g. concentrations. The values may be compared to each other, as well as to reference values, e.g.
  • increased levels of C3 and C3b compared to stationary or decreased levels of iC3b and further C3b breakdown products may be indicative of a higher risk of development of a complement-related disorder and/or the need to treat a subject for a complement- related disorder.
  • Decreased levels of C3 and C3b compared to stationary or increased levels of iC3b and further C3b breakdown products may be indicative of a lower risk of development of a complement- related disorder and/or that treatment for a complement-related disorder is effective.
  • a method described herein may comprise comparing the levels of any one or more of FHR1 , FHR2, FHR3, FHR4 and/or FHR5, to the level of FH and/or FHL-1 in the subject tested. For example, elevated levels of FHR1 , FHR2, FHR3, FHR4 and/or FHR5, and optionally FHL-1 , compared to stationary levels of FH (i.e. a statistically non-significant change) in a subject may be indicative of a higher risk of the subject developing a complement-related disorder and/or the need to treat the subject for a complement-related disorder.
  • a method provided herein comprises a step of correlating the presence of an atypical or altered amount/level of a complement protein with an increased risk of the subject developing or having a complement-related disorder.
  • Examples of reference values for complement proteins in human subjects known not to have a complement-related disorder include: a) FH: ⁇ 150 to 500 pg/ml in human blood (Clark et al., J Immunol 2014. 193(10):4962-70 and unpublished data);
  • mean reference values for circulating FH, FHL-1 and FHR1-5 in human subjects known not to have a complement-related disorder, e.g. AMD include the following (95% Cl in parentheses): a) FH, nM: 737.3 (718.2 - 756.5) b) FHL-1 , nM: 10.4 (10.1 - 10.8) c) FHR-1 , nM: 31 .2 (29.4 - 32.9) d) FHR-2, nM: 45.3 (43.1 - 47.6) e) FHR-3, nM: 24.1 (21 .7 - 26.5) f) FHR-4, nM: 46.1 (42.7 - 49.6) g) FHR-5, nM: 25.5 (24.5 - 26.5).
  • An ‘elevated’ level of a complement protein may be elevated/increased/higher when compared to a reference value forthat protein, e.g. as above.
  • a ‘reduced’ level of a complement protein e.g. in a sample, may be reduced/decreased/lower when compared to a reference value forthat protein, e.g. as above.
  • the relative concentrations of one complement protein to another can be determined using their reference values. For example, the ratio of the level of one complement protein to the level of another, or others, can be inferred from the concentrations provided above, e.g. FH:FHL-1 , C3:iC3b, C3:C3b etc.
  • the relative concentrations and/or ratios of the level of one complement protein to another, or others, may be altered in complement-related disorders.
  • the methods provided herein involve detecting two or more complement proteins and determining how the levels of the complement proteins change with respect to one another as compared to a reference value(s). For example, the level of a first complement protein may increase as compared to the level of a second complement protein, or vice versa, e.g. FHL-1 vs FH, FHR1 to FHR5 vs FH and/or FHL-1 , C3 vs iC3b, C3 vs C3b.
  • the methods of the present invention include treating a subject who is at risk of developing, predicted to be at risk of developing, who has been determined or identified to be at risk of developing, or who has, has been identified as having, has been determined to have, or has been diagnosed as having a complement-related disorder, e.g. as described herein.
  • Such treatment may comprise administering a therapy or agent, e.g. a nucleic acid, nucleotide sequence, vector, rAAV or polypeptide described herein, that targets the complement system and/or specific complement components.
  • a therapy or agent e.g. a nucleic acid, nucleotide sequence, vector, rAAV or polypeptide described herein, that targets the complement system and/or specific complement components.
  • an agent that “targets” a complement component acts to inhibit, degrade, silence, knock down, reduce or otherwise decrease expression and/or activity of said component.
  • any method described herein for determining/identifying whether a subject has, or is at risk of developing, a complement-related disorder may additionally comprise a treatment step to treat said disorder.
  • a method provided herein for identifying whether a subject has or is at risk of developing a complement-related disorder may comprise a treatment step to treat or prevent said disorder, wherein the subject has been determined to have atypical presence or levels of one or more complement proteins, e.g. detected/determined as described herein, as compared to a reference value(s).
  • the treatment is only administered if the subject has been determined to have, or be likely to have/at risk of having/at risk of developing, a complement-related disorder by a method described herein.
  • a treatment step may comprise administering to a subject a therapeutically or prophylactically effective amount of one or more nucleic acids, nucleotide sequences, vectors, rAAV or polypeptides as described herein.
  • a nucleic acid or polypeptide described herein may be used in conjunction with another complement- targeted therapy’ or ‘complement-targeted therapeutic agent’.
  • the terms ‘complement-targeted therapy’ and ‘complement-targeted therapeutic agent’ may be used interchangeably herein.
  • a complement-targeted therapeutic agent may also be a complement-targeted therapy, and vice versa.
  • Complement-targeted therapeutic agents may be referred to herein as ‘therapeutic agents’ or ‘agents’.
  • a nucleic acid or polypeptide described herein may be administered (e.g. simultaneously or sequentially) with a complement-targeted therapeutic agent.
  • a complement-targeted therapeutic for use in the methods provided herein comprises a peptide or a polypeptide, e.g. that targets one or more complement proteins.
  • a complement-targeted therapeutic agent for use in the methods provided herein comprises a polypeptide comprising a C3b binding region and a C3b inactivating region, e.g. as described in WO 2018/224663 (hereby incorporated by reference in its entirety), e.g. wherein the C3b inactivating region comprises, or consists of, an amino acid sequence having at least 65% sequence identity to the amino acid sequence of SEQ ID NO:149 and/or wherein the C3b binding region comprises, or consists of, an amino acid sequence having at least 65% sequence identity to the amino acid sequence of SEQ ID NO:150, 151 or 152.
  • SEQ ID NO:149 to 152 described herein correspond to SEQ ID NO:9, 11 , 13 and 14, respectively, described in WO 2018/224663.
  • the polypeptide may comprise a linker between the C3b binding region and the C3b inactivating region.
  • the polypeptide comprises a sequence comprising or consisting of an amino acid sequence having at least 65% sequence identity to the amino acid sequence of SEQ ID NO:32, 33 or 34 disclosed in WO 2018/224663.
  • the complement-targeted therapeutic may have one or more of the following properties: binds to C3b, binds to C3b in the region of C3b bound by a cofactor for Fl, acts as a cofactor for Fl, enables Fl-mediated inactivation of C3b, reduces the amount of C3b via Fl, increases the amount of C3b breakdown products e.g. iC3b, C3dg, C3d, C3f, e.g. via Fl, and/or diffuses through BrM.
  • a complement-targeted therapeutic agent may be, for example, one or more C1 inhibitors, C5 inhibitors, C5a inhibitors, C5aR antagonists, C3 inhibitors, C3a inhibitors, C3b inhibitors, C3aR antagonists, classical pathway inhibitors, alternative pathway inhibitors, FH- supplementation therapy and/or MBL pathway inhibitors.
  • Specific complement-targeted therapeutics include without limitation one or more of human C1 esterase inhibitor (C1-INH), eculizumab (Soliris®, Alexion; a humanized monoclonal lgG2/4-antibody targeting C5), APL-2 (Apellis), mubodina (Adienne Pharma and Biotech), ergidina (Adienne Pharma and Biotech), POT-4 (a cyclic peptide inhibitor of C3; Alcon), rituximab (Biogen personal, Genentech/Roche), ofatumumab (Genmab, GSK), compstatin analogues, soluble and targeted forms of CD59, PMX53 and PMX205, (Cephalon/Teva), JPE-1375 (Jerini), CCX168 (ChemoCentryx), NGD-2000-1 (former Neurogen), Cinryze (Shire), Berinert (CSL Behring), Cetor (Sanquin), Ruconest/Conestat
  • a nucleic acid or polypeptide described herein may be administered (e.g. simultaneously or sequentially) with a complement-targeted therapeutic agent that is capable of increasing the level of FH and/or FHL-1 in the subject, e.g. a nucleic acid encoding FH or FHL-1 , or a polypeptide corresponding to or derived from FH or FHL-1 .
  • a complement-targeted therapeutic agent that is capable of increasing the level of FH and/or FHL-1 in the subject, e.g. a nucleic acid encoding FH or FHL-1 , or a polypeptide corresponding to or derived from FH or FHL-1 .
  • a nucleic acid or polypeptide described herein may be administered (e.g. simultaneously or sequentially) with any suitable agent that decreases the level of FH and/or FHL-1 and/or decreases the level of expression of the CFH gene.
  • Subjects with elevated levels of FHR1 , FHR2, FHR3, FHR4 and/or FHR5, (and optionally FHL-1) and/or increased expression of a gene(s) encoding one or more FHR proteins/FHL-1 may also derive therapeutic or prophylactic benefit from said levels being reduced (e.g. in conjunction with treatment using a nucleic acid or polypeptide described herein). This may be achieved by administering any suitable agent that decreases the level of one or more of the FHR proteins/FHL-1 and/or decreases the level of expression of one or more CFHR/CFH genes.
  • RNA-seq RNA-seq
  • RPA ribonuclease protection assay
  • Methods described herein may also comprise administering one or more agents that decrease the level of and/or decrease the expression of one or more complement proteins that are/have been determined to be elevated.
  • Agents may be referred to a “complement-targeted therapeutic agents”.
  • the agent is capable of decreasing the level of FHR1 , FHR2, FHR3, FHR4 and/or FHR5, and/or capable of decreasing the level of expression of a gene encoding FHR1 , FHR2, FHR3, FHR4 and/or FHR5.
  • the agent inhibits, degrades, silences, knocks down, reduces or otherwise decreases expression and/or activity of FHR mRNA or protein.
  • the agent is capable of decreasing the level of FHL-1 , and/or capable of decreasing the level of expression of a gene encoding FHL-1. In some cases, the agent inhibits, degrades, silences, knocks down, reduces or otherwise decreases expression and/or activity of FHL-1 mRNA or protein.
  • the complement-targeting therapeutic agent targets the same complement protein that is detected using a method described herein.
  • the complement protein detected is FHR1 and the agent decreases the level of FHR1 and/or decreases the level of expression of a gene encoding FHR1 .
  • the complement protein detected is FHR2 and the agent decreases the level of FHR2 and/or decreases the level of expression of a gene encoding FHR2.
  • the complement protein detected is FHR3 and the agent decreases the level of FHR3 and/or decreases the level of expression of a gene encoding FHR3.
  • the complement protein detected is FHR4 and the agent decreases the level of FHR4 and/or decreases the level of expression of a gene encoding FHR4.
  • the complement protein detected is FHR5 and the agent decreases the level of FHR5 and/or decreases the level of expression of a gene encoding FHR5.
  • the complement protein detected is FHL-1 and the agent decreases the level of FHL-1 and/or decreases the level of expression of a gene encoding FHL-1 .
  • the present disclosure is intended to encompass targeting any one or combination of FHR1 , FHR2, FHR3, FHR4 and/or FHR5, and optionally FHL-1 , or genes encoding said proteins, using any suitable targeting agent(s).
  • the complement-targeting therapeutic agent targets a different complement protein to the protein detected using a method described herein.
  • a method may identify a subject as having a complement-related disorder by determining the level of one or more of FHR1 to FHR5, and then said subject may be treated with an agent that targets a different complement protein, such as FH, FHL-1 , C3, C3b etc, such as the complement-targeted therapeutics described in WO 2018/224663 and/or WO 2019/138137.
  • a method may identify a subject as having a complement-related disorder by determining an elevated level of FHR1 , and said subject may be treated with an agent that decreases the level of one or more of FHR2, FHR3, FHR4 and/or FHR5 (or an equivalent situation with other combinations of FHR proteins).
  • a complement-targeting therapeutic agent e.g. for use in tandem with a nucleic acid or polypeptide described herein, may possess one or more of the following properties: acts to inhibit expression of one or more of the CFHR genes; interferes with transcription of the CFHR genes; interferes with translation of mRNA encoding FHR1 , FHR2, FHR3, FHR4 and/or FHR5 protein; degrades mRNA encoding FHR1 , FHR2, FHR3, FHR4 and/or FHR5; binds to FHR1 , FHR2, FHR3, FHR4 and/or FHR5 protein; sequesters FHR1 , FHR2, FHR3, FHR4 and/or FHR5 protein; sequesters FHR1 , FHR2, FHR3, FHR4 and/or FHR5 protein in the blood; competes for binding of FHR1 , FHR2, FHR3, FHR4 and/or FHR5 protein; blocks activity of FHR1 , FHR2, FHR3, FHR4 and
  • FHR1 , FHR2, FHR3, FHR4 and/or FHR5 protein reduces their ability to enter tissue; reduces the ability of FHR1 , FHR2, FHR3, FHR4 and/or FHR5 protein to reach the eye; reduces the amount of FHR1 , FHR2, FHR3, FHR4 and/or FHR5 protein that enters the eye; reduces the amount of FHR1 , FHR2, FHR3, FHR4 and/or FHR5 protein in the eye; reduces the ability of FHR1 , FHR2, FHR3, FHR4 and/or FHR5 protein to enter or cross BrM; inhibits FHR1-, FHR2-, FHR3-, FHR4- and/or FHR5-mediated signalling; modulates a reaction involving C3b; modulates a reaction involving FHR1 , FHR2, FHR3, FHR4 and/or FHR5 and C3b; reduces the ability of FHR1 , FHR2, FHR3, FHR4 and/or FHR5
  • C3b in the region of C3b bound by a cofactor for Factor I; acts as a co-factor to enable Complement Factor l-mediated inactivation of C3b; reduces the amount of C3b and/or increases the amount C3b breakdown products, reduces C3 convertase activation; reduces production of C3bBb; increases C3 deactivation; reduces C3 activation; cleaves or helps to cleave C3b e.g. via Fl; increases production of iC3b; decreases complement activation; and/or inactivates a complement pathway e.g. alternative complement pathway.
  • a cofactor for Factor I acts as a co-factor to enable Complement Factor l-mediated inactivation of C3b; reduces the amount of C3b and/or increases the amount C3b breakdown products, reduces C3 convertase activation; reduces production of C3bBb; increases C3 deactivation; reduces C3 activation; cleaves or helps to cle
  • “inhibits”, “inhibition”, “reduces” or “reduction” refers to a reduction, decrease or lessening relative to a control condition.
  • “decreases the level of FHR1 , FHR2, FHR3, FHR4 and/or FHR5” refers to reduction or lessening relative to a control condition.
  • the level of FHR1 , FHR2, FHR3, FHR4 and/or FHR5 may be measured by determining the level, amount or concentration of FHR1 , FHR2, FHR3, FHR4 and/or FHR5 in the blood of a subject relative to a reference level.
  • a decrease in the level of FHR1 , FHR2, FHR3, FHR4 and/or FHR5 and/or a decrease in the level of expression of a gene encoding FHR1 , FHR2, FHR3, FHR4 and/or FHR5 may be measured by determining the level of the protein/the level of expression of a gene encoding the protein in the subject after treatment with the agent and comparing it to the level in the subject before treatment or comparing it with the level of the protein/the level of expression of a gene encoding the protein in a control subject that does not have a complement-related disorder, e.g. the level may be less elevated after treatment as compared to the control subject when compared to the elevation before treatment.
  • the decrease in the level of FHR1 , FHR2, FHR3, FHR4 and/or FHR5 may also refer to the sequestration or binding of FHR1 , FHR2, FHR3, FHR4 and/or FHR5 by agents in such a way that circulating levels/amount/concentration of FHR1 , FHR2, FHR3, FHR4 and/or FHR5 are decreased.
  • an agent may be described as an agent that decreases the level of circulating FHR1 , FHR2, FHR3, FHR4 and/or FHR5.
  • the complement-targeted therapeutic agent e.g. for use in tandem with a nucleic acid or polypeptide described hereinabove, may be nucleic acid-based or comprise nucleic acid elements.
  • the agent may promote silencing of gene expression via RNA-mediated interference (RNAi) or antisense degradation mechanisms, e.g. via Rnase H.
  • RNAi RNA-mediated interference
  • Rnase H antisense degradation mechanisms
  • the agent is, or comprises, an antisense nucleic acid.
  • An “antisense nucleic acid” as referred to herein is a nucleic acid (e.g. DNA or RNA molecule) that is complementary to at least a portion of a specific target nucleic acid (e.g. an mRNA translatable into a protein, such as an FHR protein) and is capable of reducing transcription of the target nucleic acid (e.g. mRNA from DNA), reducing the translation of the target nucleic acid (e.g. mRNA) or altering transcript splicing (e.g. by a single stranded morpholino oligo).
  • Antisense nucleic acids may be single stranded, e.g.
  • Antisense nucleic acids are capable of hybridizing to (e.g. selectively hybridizing to) a target nucleic acid (e.g. target mRNA) via Watson-Crick base pairing. In some cases the antisense nucleic acids specifically bind to the target nucleic acid. In some cases, the antisense nucleic acid hybridizes to the target nucleic acid sequence (e.g. mRNA) under stringent hybridization conditions. In some cases, the antisense nucleic acid hybridizes to the target nucleic acid (e.g. mRNA) under moderately stringent hybridization conditions.
  • nucleotide sequence of an antisense nucleic acid is sufficiently complementary to the target nucleic acid of interest such that it binds or hybridises to the target nucleic acid.
  • sequence of the target nucleic acid it is easy and routine to design a suitable antisense nucleic acid that will hybridize to the target to achieve the desired effect.
  • the target RNA may be an mRNA that encodes for all or part of a complement protein, such as FHR1 , FHR2, FHR3, FHR4 or FHR5, or FHL-1 , mRNA.
  • a nucleic acid that acts as a complement-targeted therapeutic agent may target one of the five FHR mRNAs/proteins, i.e. specifically target FHR1 , FHR2, FHR3, FHR4 or FHR5, or it may target more than one of FHR1 , FHR2, FHR3, FHR4 or FHR5 as a result of the sequence similarity between the FHR proteins.
  • RNAi uses small doublestranded RNA molecules to cause degradation of target mRNA.
  • antisense nucleic acids for use as agents according to the present invention include siRNAs (including their derivatives or pre-cursors, such as nucleotide analogs), short hairpin RNAs (shRNA), micro RNAs (miRNA, including their long primary transcripts (pri-miRNAs) and partially processed 60-70 base pair hairpin transcripts (pre-miRNAs)), saRNAs (small activating RNAs) and small nucleolar RNAs (snoRNA) or certain of their derivatives or pre-cursors.
  • siRNAs including their derivatives or pre-cursors, such as nucleotide analogs
  • shRNA short hairpin RNAs
  • miRNA micro RNAs
  • pri-miRNAs long primary transcripts
  • pre-miRNAs partially processed 60-70 base pair hairpin transcripts
  • saRNAs small activating RNAs
  • Antisense nucleic acid molecules may stimulate RNA interference (RNAi).
  • siRNA nucleic acids are ⁇ 21-25 nucleotides in length and comprise a guide strand which hybridizes with the target mRNA, plus a complementary passenger strand (e.g., each complementary sequence of the double stranded siRNA is 21-25 nucleotides in length, and the double stranded siRNA is about 21-25 base pairs in length). They promote degradation of the target mRNA via RISC. Structure and function of siRNAs are well known in the art and are described in e.g. Kim and Rossi, Biotechniques. 2008 Apr; 44(5): 613-616.
  • Suitable siRNA molecules for use in the methods of the present invention may be designed by schemes known in the art, see for example Elbashire et al., Nature, 2001 411 :494-8; Amarzguioui et al., Biochem. Biophys. Res. Commun. 2004 316(4):1050-8; and Reynolds et al., Nat. Biotech. 2004, 22(3):326-30. Details for making siRNA molecules can be found in the websites of several commercial vendors such as Ambion, Dharmacon, GenScript, Invitrogen and OligoEngine.
  • siRNAs can be expressed from a vector and/or produced chemically or synthetically. Synthetic RNAi can be obtained from commercial sources, for example, Invitrogen (Carlsbad, Calif.). RNAi vectors can also be obtained from commercial sources, for example, Invitrogen. microRNAs (miRNAs) also regulate gene expression via RISC.
  • miRNAs are initially expressed as long primary transcripts (pri-miRNAs), which are processed within the nucleus into 60-70 nucleotide hairpins (pre-miRNAs), which are further processed in the cytoplasm into small double stranded nucleic acids that interact with RISC and target mRNA.
  • miRNAs comprise “seed sequences” that are essential for binding to target mRNA.
  • seed sequences usually comprise six nucleotides and are situated at positions 2-7 at the miRNA 5’ end.
  • the agent comprises a double stranded nucleic acid molecule in which one strand is wholly or partially complementary to, or hybridizes with, an mRNA sequence encoding all or part of FHR1 , FHR2, FHR3, FHR4 or FHR5, or FHL-1 .
  • the agent comprises a siRNA molecule comprising a guide strand complementary to, or that hybridizes with, a portion of an mRNA sequence that encodes all or part of FHR1 , FHR2, FHR3, FHR4 or FHR5, or FHL-1 .
  • the agent comprises a miRNA molecule (pri-, pre- or mature miRNA) comprising a seed sequence capable of hybridizing to a portion of an mRNA sequence that encodes all or part of FHR1 , FHR2, FHR3, FHR4 and/or FHR5, or FHL-1 .
  • a miRNA molecule (pri-, pre- or mature miRNA) comprising a seed sequence capable of hybridizing to a portion of an mRNA sequence that encodes all or part of FHR1 , FHR2, FHR3, FHR4 and/or FHR5, or FHL-1 .
  • the agent is a single stranded antisense oligonucleotide (ASO).
  • ASOs modify expression of a target RNA, either by altering splicing or by recruiting Rnase H to degrade the target RNA.
  • Rnase H recognises DNA:RNA hybrids formed when the ASO binds to the target RNA.
  • ASOs tend to be 18-30 base pairs in length.
  • Many ASOs are designed as chimeras, comprising a mix of bases with different chemistries, or as gapmers, comprising a central DNA portion surrounded by ‘wings’ of modified bases.
  • ASOs are described in e.g. Scoles et al., Neurol Genet. 2019 Apr; 5(2): e323.
  • Antisense nucleic acids may comprise naturally occurring nucleotides or modifications such as e.g. phosphorothioate linkages, phosphorodiamidate linkages, methoxyethyl nucleotide modifications e.g. 2- MOE, ‘locked’ nucleic acids e.g. LNAs, peptide nucleic acids (PNAs), and/or 5’-methylcytosine modifications.
  • naturally occurring nucleotides or modifications such as e.g. phosphorothioate linkages, phosphorodiamidate linkages, methoxyethyl nucleotide modifications e.g. 2- MOE, ‘locked’ nucleic acids e.g. LNAs, peptide nucleic acids (PNAs), and/or 5’-methylcytosine modifications.
  • the agent comprises an antisense oligonucleotide that is capable of hybridizing to a portion of an mRNA sequence that encodes all or part of FHR1 , FHR2, FHR3, FHR4 and/or FHR5, or FHL-1.
  • Antisense nucleic acids described herein may comprise or consist of nucleotide sequences having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% complementarity to their target nucleic acid. Complementarity may be calculated over the whole length of the antisense nucleic acids and/or over all or part of the target nucleic acid to which the antisense nucleic acid binds.
  • a nucleic acid for use as a complement-targeted therapeutic agent may be an siRNA as described in WO 2019/215330 A1 , which is hereby incorporated by reference in its entirety.
  • a nucleic acid for use as a complement-targeted therapeutic agent may be an antisense molecule, e.g. siRNA, comprising SEQ ID NO: 174 and/or SEQ ID NO: 175.
  • the nucleic acid molecule may be an aptamer.
  • aptamer refers to oligonucleotides (e.g. short oligonucleotides or deoxyribonucleotides), that bind (e.g. with high affinity and specificity) to proteins, peptides, and small molecules. Aptamers typically have defined secondary or tertiary structure owing to their propensity to form complementary base pairs and, thus, are often able to fold into diverse and intricate molecular structures. The three-dimensional structures are essential for aptamer binding affinity and specificity, and specific three-dimensional interactions drives the formation of aptamer-target complexes.
  • Aptamers can be selected in vitro from very large libraries of randomized sequences by the process of systemic evolution of ligands by exponential enrichment (SELEX as described in Ellington AD, Szostak JW, Nature 1990, 346:818-822; Tuerk C, Gold L. Science 1990, 249:505-510) or by developing SOMAmers (slow off-rate modified aptamers) (Gold L et al. (2010) Aptamer-based multiplexed proteomic technology for biomarker discovery. PloS ONE 5(12):e15004).
  • SOMAmers are short, single stranded deoxyoligonucleotides with protein-like properties thanks to functional groups that mimic amino acid side chains. Applying the SELEX and the SOMAmer technology includes for instance adding functional groups that mimic amino acid side chains to expand the aptamer’s chemical diversity. As a result high affinity aptamers for a target may be enriched and identified.
  • Aptamers may be DNA or RNA molecules and may be single stranded or double stranded.
  • the aptamer may comprise chemically modified nucleic acids, for example in which the sugar and/or phosphate and/or base is chemically modified. Such modifications may improve the stability of the aptamer or make the aptamer more resistant to degradation and may include modification at the 2' position of ribose.
  • Aptamers may be synthesised by methods which are well known to the skilled person.
  • aptamers may be chemically synthesised, e.g. on a solid support.
  • Solid phase synthesis may use phosphoramidite chemistry. Briefly, a solid supported nucleotide is detritylated, then coupled with a suitably activated nucleoside phosphoramidite to form a phosphite triester linkage. Capping may then occur, followed by oxidation of the phosphite triester with an oxidant, typically iodine. The cycle may then be repeated to assemble the aptamer (e.g., see Sinha, N.
  • Aptamers may be peptides selected or engineered to bind specific target molecules. Peptide aptamers and methods fortheir generation and identification are reviewed in Reverdatto et al., Curr Top Med Chem. (2015) 15(12):1082-101 , which is hereby incorporated by reference in its entirety. Peptide aptamers may optionally have a minimum length of one of 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids. Peptide aptamers may optionally have a maximum length of one of 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 amino acids. Suitable peptide aptamers may optionally have a length of one of 2-30, 2-25, 2-20, 5-30, 5-25 or 5-20 amino acids.
  • Aptamers may have KD’S in the nM or pM range, e.g. less than one of 500nM, 10OnM, 50nM, 10nM, 1 nM, 500pM, 100pM.
  • An aptamer or SOMAmer suitable for use as described herein may bind to FHR1 , FHR2, FHR3, FHR4 and/or FHR5.
  • An aptamer or SOMAmer suitable for use as described herein may display specific binding for FHR1 , FHR2, FHR3, FHR4 or FHR5.
  • the aptamer may inhibit the function of FHR1 , FHR2, FHR3, FHR4 or FHR5, for example blocking their binding to other complement proteins such as C3b.
  • the agent is an antibody or antigen-binding molecule (both referred to herein as “antigen-binding molecule”) e.g. an anti-FHR1 antibody.
  • the antigen-binding molecule is specific for FHR1 , FHR2, FHR3, FHR4 and/or FHR5.
  • the antigen-binding molecule displays specific binding to FHR1 , FHR2, FHR3, FHR4 and/or FHR5.
  • the antigen-binding molecule displays specific binding to FHR1 , FHR2, FHR3, FHR4 and/or FHR5, and optionally to FH and FHL-1 .
  • An antibody that acts as a complement-targeted therapeutic agent may target one of the five FHR mRNAs/proteins, i.e. specifically target FHR1 , FHR2, FHR3, FHR4 or FHR5, or it may target more than one of FHR1 , FHR2, FHR3, FHR4 or FHR5 e.g. as a result of the sequence similarity between the FHR proteins.
  • the antigen-binding molecule is specific for C3b. In some cases, the antigen-binding molecule displays specific binding to C3b. In some cases, the antigen-binding molecule displays specific binding to FH and/or FHL-1 .
  • specific binding refers to binding which is selective for the antigen, and which can be discriminated from non-specific binding to non-target antigen.
  • An antigen- binding molecule that specifically binds to a target molecule preferably binds the target with greater affinity, and/or with greater duration than it binds to other, non-target molecules.
  • the antigen-binding molecule displays specific binding for FHR1 , FHR2, FHR3, FHR4 or FHR5, and optionally FH and/or FHL-1 , over other complement proteins.
  • An antigen-binding molecule may bind to human FHR1 , FHR2, FHR3, FHR4 and/or FHR5, and optionally to FH and FHL-1 , with a KD of 1 pM or less, preferably one of ⁇ 1 pM, ⁇ 10OnM, ⁇ 1 OnM, ⁇ 1 nM or ⁇ 1 OOpM.
  • Anti-FHR antigen-binding molecules may be antagonist antigen-binding molecules that inhibit or reduce a biological activity of FHR1 , FHR2, FHR3, FHR4 and/or FHR5.
  • Anti-FHR antigen-binding molecules may be neutralising antigen-binding molecules that neutralise the biological effect of FHR1 , FHR2, FHR3, FHR4 and/or FHR5, e.g. an ability to stimulate production of C3 convertase via C3b.
  • the antigen-binding molecule may bind to a particular region of interest of FHR1 , FHR2, FHR3, FHR4 and/or FHR5, FH/FHL-1 or C3b.
  • the antigen-binding region of an antigen-binding molecule may bind to a linear epitope of FHR1 , FHR2, FHR3, FHR4 and/or FHR5, FH/FHL-1 or C3b, consisting of a contiguous sequence of amino acids (i.e. an amino acid primary sequence).
  • the antigenbinding region molecule may bind to a conformational epitope of FHR1 , FHR2, FHR3, FHR4 and/or FHR5, FH/FHL-1 or C3b, consisting of a discontinuous sequence of amino acids of the amino acid sequence.
  • the antigen-binding molecule may be a multispecific antigen-binding molecule.
  • multispecific it is meant that the antigen-binding molecule displays specific binding to more than one target.
  • the antigen-binding molecule is a bispecific antigen-binding molecule.
  • the antigen-binding molecule comprises at least two different antigen-binding domains (i.e. at least two antigen-binding domains, e.g. comprising non-identical VHs and VLs).
  • Multispecific antigenbinding molecules may be provided in any suitable format, such as those formats described in described in Brinkmann and Kontermann Mabs (2017) 9(2): 182-212, which is hereby incorporated by reference in its entirety.
  • the antigen-binding molecule binds to FHR1 , FHR2, FHR3, FHR4 and/or FHR5, and optionally FH/FHL-1 , and another target (e.g. an antigen other than the FHR/FH proteins), and so is at least bispecific.
  • a target e.g. an antigen other than the FHR/FH proteins
  • bispecific means that the antigen-binding molecule is able to bind specifically to at least two distinct antigenic determinants.
  • the ability of a given polypeptide to bind specifically to a given molecule or another given peptide/polypeptide can be determined by analysis according to methods known in the art, such as by ELISA, Surface Plasmon Resonance (SPR; see e.g. Hearty et al., Methods Mol Biol 2012, 907:411-442), Bio-Layer Interferometry (see e.g. Lad et al., J Biomol Screen. 2015, 20(4): 498-507), flow cytometry, or by a radiolabeled antigen-binding assay (RIA) enzyme-linked immunosorbent assay.
  • ELISA Surface Plasmon Resonance
  • SPR Surface Plasmon Resonance
  • Bio-Layer Interferometry see e.g. Lad et al., J Biomol Screen. 2015, 20(4): 498-507
  • flow cytometry or by a radiolabeled antigen-binding assay (RIA) enzyme-linked immunosorbent assay.
  • the binding may be the response detected in a given assay.
  • Binding affinity may be expressed in terms of dissociation constant (KD).
  • KD dissociation constant
  • the region of a peptide/polypeptide to which an antibody binds can be determined by the skilled person using various methods well known in the art, including X-ray co-crystallography analysis of antibodyantigen complexes, peptide scanning, mutagenesis mapping, hydrogen-deuterium exchange analysis by mass spectrometry, phage display, competition ELISA and proteolysis-based ‘protection’ methods. Such methods are described, for example, in Gershoni et al., BioDrugs, 2007, 21 (3):145-156, which is hereby incorporated by reference in its entirety.
  • the antigen-binding molecule decreases the concentration of FHR1 , FHR2, FHR3, FHR4 and/or FHR5 in the blood. In some embodiments, the antigen-binding molecule decreases the amount of circulating FHR1 , FHR2, FHR3, FHR4 and/or FHR5 e.g. in the blood. In some embodiments, the antigen-binding molecule may sequester FHR1 , FHR2, FHR3, FHR4 and/or FHR5 protein.
  • the antigen-binding molecule binds to FHR1 , FHR2, FHR3, FHR4 and/or FHR5 and reduces the ability of FHR1 , FHR2, FHR3, FHR4 and/or FHR5 to reach the eye, enter the BrM, and/or enter the intercapillary septa of the choriocapillaris. In some embodiments, the antigen-binding molecule reduces binding of FHR1 , FHR2, FHR3, FHR4 and/or FHR5 to C3b.
  • the ability of an antigen-binding molecule to inhibit interaction between two binding partners can also be determined by analysis of the downstream functional consequences of such interaction.
  • the ability of an antigen-binding molecule to inhibit interaction of FHR1 , FHR2, FHR3, FHR4 and/or FHR5 and C3b may be determined by analysis of production of C3bBb and/or iC3b in an appropriate assay e.g. by detecting the production of protein from a reaction using ELISA, Western blotting or electrophoresis methods.
  • a person skilled in the art will be able to produce suitable antigen binding molecules using e.g. techniques as described herein or those known in the art, see e.g. Chiu and Gilliland, Curr Opin Struct Biol. 2016, 38:163-173, Jakobovits A, Curr Opin Biotechnol. 1995 Oct;6(5):561-6, and Bruggemann M et al., Arch Immunol TherExp (Warsz). 2015; 63(2): 101-108.
  • One suitable technique is phage display technology, see e.g. Hammers and Stanley, J Invest Dermatol. 2014, 134(2): e17 and Bazan J et al., Hum Vaccin Immunother.
  • Antigen-binding polypeptide chains may also be produced by techniques such as chemical synthesis (see e.g. Chandrudu et al., Molecules (2013), 18: 4373-4388), recombinant expression such as the techniques set out in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4 th Edition), Cold Spring Harbor Press, 2012, and in Nat Methods. (2008); 5(2): 135- 146, or cell-free-protein synthesis (CFPS; see e.g., Zemella et al. Chembiochem (2015) 16(17): 2420- 2431), all of which are hereby incorporated by reference in their entirety.
  • chemical synthesis see e.g. Chandrudu et al., Molecules (2013), 18: 4373-4388
  • recombinant expression such as the techniques set out in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4 th Edition), Cold Spring Harbor Press, 2012, and in Nat Methods. (2008); 5(2): 135-
  • the antigen-binding molecule may be monoclonal, i.e. a homogenous population of antibodies specifically targeting a single epitope on an antigen.
  • Monoclonal antibodies to selected antigens may be prepared by known techniques, for example those disclosed in “Monoclonal Antibodies: A manual of techniques “, H Zola (CRC Press, 1988) and in “Monoclonal Hybridoma Antibodies: Techniques and Applications “, J G R Hurrell (CRC Press, 1982). Chimaeric antibodies are discussed by Neuberger et al (1988, 8 th International Biotechnology Symposium Part 2, 792-799). Suitable polyclonal antibodies can also be prepared using methods well known in the art.
  • the antigen-binding portion may be a part of an antibody (for example a Fab fragment) or a synthetic antibody fragment (for example a single chain Fv fragment [ScFv]).
  • Antigen-binding fragments of antibodies, such as Fab and Fab2 fragments may also be used/provided as can genetically engineered antibodies and antibody fragments.
  • the variable heavy (VH) and variable light (VL) domains of the antibody are involved in antigen recognition, a fact first recognised by early protease digestion experiments. Further confirmation was found by “humanisation” of rodent antibodies.
  • Variable domains of rodent origin may be fused to constant domains of human origin such that the resultant antibody retains the antigenic specificity of the rodent parented antibody (Morrison et al (1984) Proc. Natl. Acad. Sd. USA 81 , 6851-6855).
  • Antibodies and antigen-binding fragments according to the present disclosure comprise the complementarity-determining regions (CDRs) of an antibody which is capable of binding to the relevant target molecule, i.e. a complement protein such as those described herein.
  • CDRs complementarity-determining regions
  • Antibodies to a given target protein e.g. FHR1 , FHR2 etc
  • model species e.g. rodents, lagomorphs
  • one or more amino acids of monoclonal antibodies raised by immunisation of model species can be substituted to arrive at an antibody sequence which is more similar to human germline immunoglobulin sequences (thereby reducing the potential for anti-xenogenic antibody immune responses in the human subject treated with the antibody).
  • Modifications in the antibody variable domains may focus on the framework regions in order to preserve the antibody paratope.
  • Antibody humanisation is a matter of routine practice in the art of antibody technology, and is reviewed e.g.
  • Phage display techniques may also be employed to the identification of antibodies to a given target protein (e.g. IL-11 or IL-11 Ra), and are well known to the skilled person.
  • a given target protein e.g. IL-11 or IL-11 Ra
  • the use of phage display for the identification of fully human antibodies to human target proteins is reviewed e.g. in Hoogenboom, Nat. Biotechnol. (2005) 23, 1105-1116 and Chan et al., International Immunology (2014) 26(12): 649-657, which are hereby incorporated by reference in their entirety.
  • Suitable antibodies for use as complement-targeted therapies may include those described in e.g.
  • An agent may be a sequestering agent, e.g. of FHR1 , FHR2, FHR3, FHR4 and/or FHR5.
  • the agent may be a protein molecule.
  • An example is an antigen-binding molecule e.g. as described herein, which sequesters FHR1 , FHR2, FHR3, FHR4 and/or FHR5 in the blood.
  • the agent may be a small molecule.
  • the small molecule may bind to FHR1 , FHR2, FHR3, FHR4 and/or FHR5 protein and prevent/reduce the ability of FHR1 , FHR2, FHR3, FHR4 and/or FHR5 to reach sites of complement activation and/or prevent/reduce an interaction between FHR1 , FHR2, FHR3, FHR4 and/or FHR5 and a normal binding partner e.g. C3b.
  • the small molecule may prevent/reduce correct folding of the FHR1 , FHR2, FHR3, FHR4 and/or FHR5 protein.
  • the small molecule prevents/reduces binding between FHR1 , FHR2, FHR3, FHR4 and/or FHR5 and a binding partner. In some cases, the small molecule binds to FHR1 , FHR2, FHR3, FHR4 and/or FHR5.
  • An agent may be a decoy receptor.
  • a decoy receptor refers to a peptide or polypeptide capable of binding FHR1 , FHR2, FHR3, FHR4 and/or FHR5.
  • the receptor may be a receptor, including fragments and derivatives thereof, for FHR1 , FHR2, FHR3, FHR4 and/or FHR5.
  • a decoy receptor may be able to recognise and bind a specific ligand but may not be able to signal or activate a subsequent response.
  • a decoy receptor may bind FHR1 , FHR2, FHR3, FHR4 and/or FHR5 to form a complex.
  • a decoy receptor may act as an inhibitor of FHR1 , FHR2, FHR3, FHR4 and/or FHR5 by binding FHR1 , FHR2, FHR3, FHR4 and/or FHR5 and preventing/reducing the ability or availability of the proteins to bind to their receptor(s).
  • the agent may be a molecule which binds FHR1 , FHR2, FHR3, FHR4 and/or FHR5 to prevent activation of C3/C3b.
  • a decoy receptor may act as an inhibitor of FHR1 , FHR2, FHR3, FHR4 and/or FHR5 by binding to a binding partner of one or more of the proteins, e.g.
  • the agent may be based on C3b, for example the receptor may be an inactive form of C3b.
  • the agent may be based on C3c and/or C3d.
  • the agent may be administered to/present in the blood, or attached to a tissue e.g. in or near the eye.
  • the receptor may be capable of inhibiting complement activation.
  • the receptor may be capable of inhibiting interaction between FHR1 , FHR2, FHR3, FHR4 and/or FHR5 and C3b.
  • the receptor may be capable of inhibiting the activation of C3b, and/or inhibiting the formation of C3 convertase.
  • a decoy receptor may be soluble (not membrane bound), or may be membrane bound e.g. expressed on a cell surface.
  • Decoy receptors may be presented and/or administered on a surface of a nanocarrier, for example, a nanoparticle, liposome, bead, polymer, metal particle, dendrimer, nanotube or micro-sized silica rods, see e.g. Wilczewska AZ et al., Pharmacol Rep. 2012, 64(5):1020-1037.
  • a nanocarrier for example, a nanoparticle, liposome, bead, polymer, metal particle, dendrimer, nanotube or micro-sized silica rods, see e.g. Wilczewska AZ et al., Pharmacol Rep. 2012, 64(5):1020-1037.
  • Agents that decrease the amount of FHR1 , FHR2, FHR3, FHR4 and/or FHR5 and/or decrease expression of a gene encoding FHR1 , FHR2, FHR3, FHR4 and/or FHR5 may fall into more than one of the categories above.
  • an antigen binding molecule or decoy receptor may also be a sequestering agent.
  • nucleic acids, nucleotide sequences, vectors, rAAV, polypeptides or agents described herein may be optionally isolated and/or substantially purified.
  • a nucleic acid, nucleotide sequence, vector, rAAV, polypeptide and/or other complement-targeted therapeutic agent described herein may be formulated as pharmaceutical compositions or medicaments for clinical use and may comprise a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.
  • methods are also provided for the production of pharmaceutically useful compositions, such methods of production may comprise one or more steps selected from: isolating a nucleic acid, nucleotide sequence, vector, rAAV, polypeptide and/or agent; and/or mixing a nucleic acid, polypeptide and/or agent with a pharmaceutically acceptable carrier, adjuvant, excipient or diluent.
  • the nucleic acid, nucleotide sequence, vector, rAAV, polypeptide, agent or composition may be formulated for topical, parenteral, systemic, intracavitary, intravenous, intra-arterial, intramuscular, intrathecal, intraocular, intravitreal, intraconjunctival, subretinal, suprachoroidal, subcutaneous, intradermal, intrathecal, oral, nasal or transdermal routes of administration which may include injection or infusion, or administration as an eye drop (i.e. ophthalmic administration).
  • Suitable formulations may comprise the agent in a sterile or isotonic medium.
  • Medicaments and pharmaceutical compositions may be formulated in fluid, including gel, form.
  • Fluid formulations may be formulated for administration by injection or infusion (e.g. via catheter) to a selected organ or region of the human or animal body.
  • a further aspect of the present invention relates to a method of formulating or producing a medicament or pharmaceutical composition for use in a method of medical treatment, the method comprising formulating a pharmaceutical composition or medicament by mixing a nucleic acid, polypeptide and/or other agent with a pharmaceutically acceptable carrier, adjuvant, excipient or diluent.
  • the nucleic acid, nucleotide sequence, vector, rAAV, polypeptide and/or agent is administered to the liver, e.g. to one or more hepatocytes. In some cases, the nucleic acid, nucleotide sequence, vector, rAAV, polypeptide and/or agent is administered to the blood (i.e. intravenous/intra- arterial administration). In some cases, the nucleic acid, polypeptide and/or agent is administered subcutaneously. In some cases, the nucleic acid, nucleotide sequence, vector, rAAV, polypeptide and/or agent is administered to the eye, e.g. to one or more RPE cells.
  • the nucleic acid, nucleotide sequence, vector, rAAV, polypeptide and/or agent is administered to the kidney, e.g. to one or more glomerular endothelial cells, macula densa, mesangial cells, parietal epithelial cells, podocytes, or tubule epithelial cells.
  • methods provided herein comprise targeted delivery of the nucleic acid, nucleotide sequence, vector, rAAV, polypeptide, and/or other agent i.e. wherein the concentration of the nucleic acid, nucleotide sequence, vector, rAAV, polypeptide, or agent in the subject is increased in some parts of the body relative to other parts and/or wherein the nucleic acid, nucleotide sequence, vector, rAAV, polypeptide, or agent is delivered via a controlled-release technique.
  • the methods comprise intravenous, intra-arterial, intramuscular or subcutaneous administration and wherein the nucleic acid, nucleotide sequence, vector, rAAV, polypeptide, or agent is formulated in a targeted agent delivery system.
  • Suitable targeted agent delivery systems include, for example, nanoparticles, liposomes, micelles, beads, polymers, metal particles, dendrimers, antibodies, aptamers, nanotubes or micro-sized silica rods.
  • Such systems may comprise a magnetic element to direct the nucleic acid, polypeptide or agent to the desired organ or tissue.
  • Suitable nanocarriers and delivery systems will be apparent to one skilled in the art.
  • the nucleic acid, nucleotide sequence, vector, rAAV, polypeptide or agent is formulated for targeted delivery to a specific organ(s) or tissue(s).
  • the nucleic acid, nucleotide sequence, vector, rAAV, polypeptide or agent is delivered to the liver, eye or kidney.
  • the methods comprise intravenous, intra-arterial, intramuscular or subcutaneous administration and wherein the nucleic acid, nucleotide sequence, vector, rAAV, polypeptide or agent is formulated for targeted delivery to the liver, eye or kidney.
  • RNA e.g. nanoparticle based formulations
  • RNA may be formulated for pulmonary administration for subsequent delivery to non-lung tissues, see e.g. US 2015/0157565 A1 , which is herein incorporated in its entirety.
  • the particular mode and/or site of administration may be selected in accordance with the location where reduction of FHR protein levels/activity and/or reduction of the level of CFHR expression is required.
  • the methods comprise intravenous and/or intra-arterial administration.
  • the methods comprise administration to the eye or kidney. Should reduction of CFHR expression be required, then an agent that decreases expression of one or more CFHR genes may be administered to the liver. In some cases, the agent is delivered to one or more hepatocytes.
  • RNA may be delivered naked, or by using nanoparticles, polymers, peptides e.g. cell-penetrating peptides, or by ex vivo transfection.
  • Nanoparticles may be organic, e.g.
  • Nanoparticles may be inorganic such as nanotubes or metal particles, optionally with organic molecules added. Viruses present another nanoparticle delivery option. Nanoparticles may be optimised to improve rate of endocytosis, avoid renal clearance and filtration, improve thermal stability, improve pH stability, prevent toxic effects, and improve RNA loading efficiency. Further encapsulation methods are described in e.g. US 2015/0157675 A1 .
  • Administration is preferably in a “therapeutically effective amount”, this being sufficient to show benefit to the individual.
  • the actual amount administered, and rate and time-course of administration, will depend on the nature and severity of the disease being treated. Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington’s Pharmaceutical Sciences, 20 th Edition, 2000, pub. Lippincott, Williams & Wilkins.
  • Administration may be alone or in combination with other treatments (e.g. other therapeutic or prophylactic intervention), either simultaneously or sequentially depending on the condition to be treated.
  • a nucleic acid or polypeptide described herein and a complement-targeted therapeutic agent may be administered simultaneously or sequentially.
  • two or more complement-targeted therapeutic agents, e.g. as described herein, are administered simultaneously or sequentially.
  • Simultaneous administration refers to administration of the nucleic acid, nucleotide sequence, vector, rAAV, or polypeptide and the complement-targeted therapeutic agent together, for example as a pharmaceutical composition containing both agents (combined preparation), or immediately after each other and optionally via the same route of administration, e.g. to the same tissue, artery, vein or other blood vessel.
  • Sequential administration refers to administration of one of the nucleic acid, nucleotide sequence, vector, rAAV, or polypeptide followed after a given time interval by separate administration of the other agent. It is not required that the two agents are administered by the same route, although this is the case in some embodiments.
  • the time interval may be any time interval.
  • therapeutic agents or techniques suitable for use with the present invention may comprise nutritional therapy, photodynamic therapy (PDT), laser photocoagulation, anti-VEGF (vascular endothelial growth factor) therapy, and/or additional therapies known in the art, see e.g. Al-Zamil WM and Yassin SA, Clin Interv Aging. 2017 Aug 22;12:1313-1330).
  • Anti-VEGF therapy may comprise agents such as ranibizumab (Lucentis, made by Genentech/Novartis), Avastin (Genentech), bevacizumab (off label A vastin), and aflibercept (Eylea®/VEGF Trap-Eye from Regeneron/Bayer).
  • agents ortechniques suitable for use with the present invention include APL-2 (Apellis), AdPEDF (GenVec), encapsulated cell technology (ECT; Neurotech), squalamine lactate (EVIZONTM, Genaera), OT-551 (antioxidant eye drops, Othera), anecortave actate (Retaane®, Alcon), bevasiranib (siRNA, Acuity Pharmaceuticals), pegaptanib sodium (Macugen®), and AAVCAGsCD59 (Clinical trial identifier: NCT03144999).
  • Multiple doses of a complement-targeted therapeutic agent may be provided.
  • One or more, or each, of the doses may be accompanied by simultaneous or sequential administration of another therapeutic agent.
  • Multiple doses may be separated by a predetermined time interval, which may be selected to be one of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days, or 1 , 2, 3, 4, 5, or 6 months.
  • doses may be given once every 7, 14, 21 or 28 days (plus or minus 3, 2, or 1 days).
  • An agent described herein may be formulated in a sustained release delivery system, in order to release the polypeptide, nucleic acid, vector or composition at a predetermined rate. Sustained release delivery systems may maintain a constant drug/therapeutic concentration for a specified period of time.
  • an agent described herein is formulated in a liposome, gel, implant, device, or drug- polymer conjugate e.g. hydrogel.
  • the present invention may involve detecting the presence of, and/or determining the level of, one or more complement proteins using suitable analytical techniques, e.g. as described herein.
  • a method described herein comprises contacting the complement protein with endoproteinase GluC to obtain one or more peptides, and detecting the one or more peptides by mass spectrometry.
  • a method described herein comprises contacting, e.g. digesting, the protein with GluC to obtain one or more peptides, and determining the level of the one or more peptides by mass spectrometry.
  • the methods involves both detecting a complement protein and determining the level of a complement protein.
  • the protein may be the same protein, or the methods may involve detection of a first complement protein and determining the level of a second complement protein.
  • the step of detecting/determining the level of the one or more peptides consists of detecting/determining the level of/measuring the peptide(s) by mass spectrometry. That is, the step of detecting/determining the level of/measuring the peptide(s) is performed by mass spectrometry only. Measuring the peptide(s) may include detecting the presence or absence of the one or more peptides, and/or determining the level, amount and/or concentration of each peptide in the sample.
  • the step of determining in any method described herein comprises:
  • the step of determining in any method described herein comprises:
  • obtaining a blood-derived sample from a subject comprising at least one complement protein that has been digested with endoproteinase GluC to obtain one or more peptides;
  • a blood-derived sample from a subject e.g. a subject that has or is suspected to have a complement-related disorder, that comprises at least one complement protein that has been digested with endoproteinase GluC. That is, the sample comprises peptides from complement proteins that have been digested with GluC.
  • the complement protein(s) may be selected from one or more of FHR1 , FHR2, FHR3, FHR4 and/or FHR5, in any combination as described herein.
  • the presence and/or level of FH and/or FHL-1 may also be determined.
  • digesting refers to placing the protein in contact with GluC under suitable conditions, e.g. temperature, pH etc, and for a suitable time such that the protein is digested, i.e. cleaved, into two or more fragments.
  • the digesting involves incubating the protein with GluC under suitable conditions, e.g. as described herein.
  • the protein e.g. a complement-related protein according to the present disclosure, may be contacted with GluC. That is, the methods provided herein may comprise a step of contacting the protein to be digested with GluC, e.g. at a concentration suitable for digesting the protein into peptides detectable by mass spectrometry.
  • a method for preparing a complement protein for analysis comprising contacting/digesting the protein with endoproteinase GluC to obtain one or more peptides.
  • the method comprises preparing a complement protein for subsequent analysis.
  • the one or more peptides may then be subjected to an analytical technique, e.g. mass spectrometry or any other suitable analytical technique.
  • the method comprises preparing a complement protein for analysis by mass spectrometry.
  • the analytical technique may be used to detect the presence and/or level of the one or more peptides.
  • complement protein is referred to herein in the singular (i.e. “a/the complement protein”)
  • pluralities/groups/populations of different complement proteins are also contemplated.
  • any disclosure herein comprising a complement protein also comprises more than one complement protein, i.e. at least one protein, or one or more proteins.
  • a/the complement protein may refer to “at least one complement protein”.
  • Detecting a protein as used herein refers to identifying/observing the presence, existence or level of the protein, e.g. in a sample, cell, tissue or subject.
  • the “level” of a complement protein used herein refers to the level, amount or concentration of said protein, e.g. in a sample, cell, tissue or subject.
  • the term “determining the level”, e.g. of a protein, used herein refers to the measurement and/or quantification of the level, amount or concentration of a protein. In some cases, “determining the level” includes calculating the level, amount or concentration of a protein in a sample. The sample may be from a subject. In some cases, “determining the level” includes calculating the level, amount or concentration of a protein in a subject, e.g. using a sample taken from the subject.
  • Determining the level” of a protein may include digesting the protein with GluC to obtain one or more peptides, detecting the one or more peptides as described herein and then calculating the level, amount or concentration of the protein/peptide, e.g. in a sample.
  • determining the level comprises quantifying, i.e. measuring the quantity of, the level, amount or concentration of a protein e.g. in a sample or in a subject. “Determining the level” may include determining the concentration of a protein. Quantification/measuring may include comparing the level, amount or concentration of a protein with a reference value, and/or comparing the level, amount or concentration of a protein with that in a control sample e.g. taken from the subject at a different time point, or taken from a healthy subject, e.g. one known not to have a complement-related disorder.
  • the methods comprise detecting/determining the level of a complement protein in a sample.
  • the sample may be in vitro or ex vivo.
  • a sample may have been taken from a subject, e.g. from a subject of interest or from a control subject.
  • a sample may be taken from any tissue or bodily fluid.
  • the sample is taken from a bodily fluid, more preferably one that circulates through the body.
  • the sample may be referred to as a circulating sample.
  • the sample may be a blood sample or lymph sample.
  • the sample is a blood sample or blood-derived sample.
  • the blood-derived sample may be a selected fraction of a subject’s blood, e.g. a selected cell-containing fraction or a plasma or serum fraction.
  • a selected serum fraction may comprise the fluid portion of the blood obtained after removal of the fibrin clot and blood cells.
  • the sample may comprise or may be derived from a tissue sample, biopsy or isolated cells from said individual.
  • the sample may be taken from the eye, kidney, brain or liver, e.g. comprising cells from the eye, kidney, brain or liver.
  • the sample may comprise retinal tissue.
  • the sample may comprise RPE cells or tissue from Bruch’s membrane or the choroid.
  • the sample may comprise drusen or other deposits of complement-related components.
  • the methods described herein comprise taking or obtaining a sample from a subject, e.g. blood, tissue etc. In some embodiments the methods described herein are performed on a sample that has been obtained/was obtained from a subject, e.g. that has been obtained previously and stored prior to use. Storage of samples, e.g. tissue and/or blood samples, are well known to a skilled person.
  • the sample is a blood sample. The blood sample may undergo/have undergone processing to obtain a plasma sample or a serum sample.
  • the methods comprise obtaining a blood-derived sample from a subject.
  • the methods comprise obtaining a plasma or serum sample from a subject.
  • the methods comprise isolating protein, e.g. total protein, from the sample. Suitable techniques to isolate protein from biological samples are well known in the field. In some embodiments the methods do not comprise isolating protein from the sample, e.g. the methods are performed on the unprocessed sample.
  • the methods are performed in vitro.
  • the presence, level, amount and/or concentration of the complement protein(s) may be detected/determined in vitro.
  • the methods involve determining the presence, level, amount and/or concentration of the complement protein(s) in a subject. This may involve performing the methods described herein in vitro, and using the results to calculate the presence, level, amount and/or concentration of the protein(s) in the subject.
  • a method for detecting at least one complement protein in a sample comprising digesting the protein(s) in the sample with endoproteinase GluC to obtain one or more peptides; and using mass spectrometry to detect the one or more peptides in the sample.
  • Any method described herein may comprise a step of detecting at least one complement protein, e.g. detecting the presence of the complement protein.
  • Also provided is a method for determining the level of at least one complement protein in a sample comprising digesting the protein(s) in the sample with endoproteinase GluC to obtain one or more peptides and using mass spectrometry to determine the level of the one or more peptides in the sample.
  • mass spectrometry to detect one or more peptides in a sample, or detecting and/or determining the level of one or more peptides by mass spectrometry, e.g. by the methods described herein, may include applying a mass spectrometry technique to the sample, e.g. by putting the sample in a mass spectrometer, and instructing the mass spectrometer to analyse the sample.
  • a mass spectrometry technique e.g. by putting the sample in a mass spectrometer, and instructing the mass spectrometer to analyse the sample.
  • the methods described herein may comprise both detecting at least one complement protein and determining the level of at least one complement protein.
  • the complement protein may be the same protein, and/or the methods may comprise detecting a least a first complement protein and determining the level of at least a second complement protein.
  • the methods described herein comprise detecting/determining the level of one complement protein. In some embodiments, the methods described herein comprise detecting/determining the level of at least one complement protein, one or more complement proteins, and/or groups or complement proteins e.g. as provided herein.
  • the complement protein is encoded from the RCA (regulators of complement) gene cluster, or RCA locus, on human chromosome 1 .
  • the RCA cluster is located on chromosome 1q32 and includes the CFH and CFHR1-5 genes.
  • the gene cluster also includes the membrane bound proteins CR1 (CD35), CR2 (CD21), decay-accelerating factor (DAF; CD55), and membrane cofactor protein (MCP; CD46), as well as soluble C4b-binding protein (C4bp).
  • the methods described herein are suitable for detecting/determining the level of multiple complement proteins via a single assay: i.e. using a single enzyme, GluC, to obtain analysable peptides and then using a single analytical technique, mass spectrometry, to detect and/or determine the levels of said peptides.
  • a single assay i.e. using a single enzyme, GluC, to obtain analysable peptides and then using a single analytical technique, mass spectrometry, to detect and/or determine the levels of said peptides.
  • GluC a single enzyme
  • mass spectrometry mass spectrometry
  • the methods described herein comprise detecting/determining the level of any one or more, e.g. any or all combinations, of FH, FHL-1 , FHR1 , FHR2, FHR3, FHR4, and/or FHR5.
  • the complement protein(s) is/are selected from the group consisting of FH, FHL-1 , FHR1 , FHR2, FHR3, FHR4, and/or FHR5.
  • the methods comprise detecting/determining the level of any one, two, three, four, five, six and/or seven of FH, FHL-1 , FHR1 , FHR2, FHR3, FHR4, and FHR5, alone or in combination.
  • the methods described herein are able to differentiate (i.e. distinguish, discriminate, separate) between the presence of (or levels of) each of FH, FHL-1 , FHR1 , FHR2, FHR3, FHR4 and/or FHR5.
  • the complement protein is any one or more, e.g. any or all combinations, of FHR1 , FHR2, FHR3, FHR4 and/or FHR5.
  • the complement protein is FHR1 .
  • the complement protein is FHR2.
  • the complement protein is FHR3.
  • the complement protein is FHR4.
  • the complement protein is FHR5.
  • the methods described herein are able to differentiate (i.e. distinguish, discriminate, separate) between the presence of (or levels of) each of FHR1 , FHR2, FHR3, FHR4 and/or FHR5. Exemplary combinations of FHR proteins that may be detected in the present invention are described herein.
  • the methods described herein permit or allow the detection of FHR1 alone, i.e. without detecting FHR2-FHR5. In some cases, the methods described herein permit or allow the detection of FHR2 alone, i.e. without detecting FHR1 or FHR3-FHR5. In some cases, the methods described herein permit or allow the detection of FHR3 alone, i.e. without detecting FHR1 , FHR2, FHR4, or FHR5. In some cases, the methods described herein permit or allow the detection of FHR4 alone, i.e. without detecting FHR1-FHR3 or FHR5. In some cases, the methods described herein permit or allow the detection of FHR5 alone, i.e. without detecting FHR1-FHR4.
  • the complement protein is to be detected/determine the level of FH and/or FHL-1 .
  • the methods described herein comprise detecting/determining the level of both FH and FHL- 1 .
  • the methods described herein differentiate (i.e. distinguish, discriminate, separate) between the presence of FH and the presence of FHL-1 and/or between the level/concentration of FH and the level/concentration of FHL-1 .
  • the methods described herein permit or allow the detection of FH alone, i.e. without detecting FHL-1 .
  • the methods described herein permit or allow the detection of FHL-1 alone, i.e. without detecting FH.
  • the complement protein to be detected/the level of which is determined is involved with breakdown, turnover and/or inactivation of C3/C3b.
  • the complement protein is produced by the breakdown and/or inactivation of C3/C3b, i.e. is a product of C3b inactivation/breakdown.
  • the methods described herein include determining the presence, rate and/or progression of C3b turnover.
  • the methods described herein involve detecting/determining the level of a protein involved in, or produced as a result of, the complement amplification loop.
  • the methods described herein involve detecting/determining the level of a protein involved in the generation or breakdown of C3 convertase.
  • the protein is a cofactor for Fl, e.g. FH, CR1 , or the FHR proteins.
  • Any method disclosed herein e.g. a method for detecting at least one complement protein in a sample comprising digesting proteins with GluC and detecting the resulting peptides by mass spectrometry, may be described in the alternative as a method for detecting C3 turnover, a method for detecting C3 breakdown, a method for measuring C3b turnover or C3b breakdown, or a method for measuring the progress of C3b turnover or C3b breakdown.
  • the present invention provides a method for detecting turnover or breakdown of C3b, comprising the steps described herein, e.g. digesting at least one complement protein with endoproteinase GluC to obtain one or more peptides and detecting the peptide(s) by mass spectrometry.
  • the method comprises digesting and then detecting at least two, three, four or more, up to 16, of the 16 complement proteins described herein.
  • the methods described herein comprise/further comprise detecting/determining the level of Fl, either alone or in combination with other complement proteins such as those described herein.
  • the methods described herein comprise detecting/determining the level of any one or more, e.g. any or all combinations, of C3, C3b, C3a, iC3b, C3f, C3c, C3dg, and/or C3d.
  • the complement protein(s) is/are selected from the group consisting of C3, C3b, C3a, iC3b, C3f, C3c, C3dg, and/or C3d.
  • the methods comprise detecting/determining the level of any one, two, three, four, five, six, seven and/or eight of C3, C3b, C3a, iC3b, C3f, C3c, C3dg, and/or C3d, in any combination.
  • the methods described herein comprise detecting/determining the level of one or more of C3, C3a, C3f, C3c, and/or C3d.
  • the methods described herein comprise determining the presence and/or level of C3b, iC3b, and/or C3dg, e.g. via the methodology in Table 3.
  • the methods described herein comprise detecting/determining the level of C3, C3b and/or iC3b.
  • the methods described herein are able to differentiate (i.e. distinguish, discriminate, separate) between the presence of (or levels of) two or more, or all, of C3, C3b, C3a, iC3b, C3f, C3c, C3dg, and/or C3d.
  • the methods described herein can detect multiple complement proteins, and distinguish between said complement proteins, using one enzyme e.g. GluC and one analytical method e.g. mass spectrometry.
  • the methods described herein may be used to detect/determine the level of any one of the individual proteins described herein, as well as any and all combinations of FH, FHL-1 , FHR1 , FHR2, FHR3, FHR4, FHR5, Fl, C3, C3b, C3a, iC3b, C3f, C3c, C3dg, and/or C3d, i.e. any one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen and/or sixteen of these proteins in any combination.
  • the complement protein(s) is/are selected from the group consisting of FH, FHL-1 , FHR1 , FHR2, FHR3, FHR4, FHR5, Fl, C3, C3b, C3a, iC3b, C3f, C3c, C3dg, and/or C3d.
  • the methods described herein may be used to detect/determine the level of FHL-1 and to detect/determine the level of any one or more of FH, FHR1 , FHR2, FHR3, FHR4, FHR5, Fl, C3, C3b, C3a, iC3b, C3f, C3c, C3dg, and/or C3d.
  • the method comprises distinguishing (i.e.
  • the methods provided herein allow for simultaneous detection of one or more of FH, FHL- 1 , FHR1 , FHR2, FHR3, FHR4, FHR5, Fl, C3, C3b, C3a, iC3b, C3f, C3c, C3dg, and/or C3d, including any combination thereof.
  • the methods provided herein allow for detection/determination of the level of one or more of FH, FHL-1 , FHR1 , FHR2, FHR3, FHR4, FHR5, Fl, C3, C3b, C3a, iC3b, C3f, C3c, C3dg, and/or C3d, including any combination thereof, in a single assay.
  • the methods provided herein allow for distinct, separable and detectable peptides to be produced from every protein listed above such that the presence and/or level of each protein can be distinguished from the others.
  • the methods provided herein allow for simultaneous detection of one or more of FHR1 , FHR2, FHR3, FHR4, FHR5, FH, and/or FHL-1 , including any combination thereof. In some cases the methods provided herein allow for detection/determination of the level of one or more of FHR1 , FHR2, FHR3, FHR4, FHR5, FH, and/or FHL-1 , including any combination thereof, in a single assay.
  • the present invention provides a method for detecting and/or determining the level of at least two complement proteins in a sample simultaneously and/or in one assay, the method comprising: digesting the proteins with endoproteinase GluC to obtain one or more peptides; and detecting and/or determining the level of the one or more peptides by mass spectrometry.
  • the complement protein may be any protein involved in one or more of the complement system pathways.
  • the complement protein may be one or more of C1 , C2, C4b2a C4, C4a, C5, C5a, FB, FD, C3Bb, MASP1 , MASP2, C1q, C1 r, C1s, C6, C7, C8, C9, CD59, Clusterin, Properdin, and/or Compstatin.
  • the complement protein to be detected is not one or more of C1 , C2, C4b2a C4, C4a, C5, C5a, FB, FD, C3Bb, MASP1 , MASP2, C1q, C1 r, C1s, C6, C7, C8, C9, CD59, Clusterin, Properdin, and/or Compstatin.
  • the present invention provides endoproteinase GluC for preparing at least one complement protein for detection by mass spectrometry. In some cases the invention provides endoproteinase GluC for preparing at least two, i.e. multiple or a plurality of, complement proteins for detection by mass spectrometry.
  • the at least two complement proteins may be any two, three, four or more, up to 16, of FH, FHL-1 , FHR1 , FHR2, FHR3, FHR4, FHR5, Fl, C3, C3b, C3a, iC3b, C3f, C3c, C3dg, and/or C3d, in any combination, as described herein.
  • Endoproteinase GluC also known as glutamyl endopeptidase, is a serine proteinase which preferentially cleaves peptide bonds C-terminal to glutamic acid residues. It also cleaves at aspartic acid residues at a rate 100-300 times slower than at glutamic acid residues.
  • the specificity of GluC depends on the pH and the buffer composition. At pH 4, the enzyme preferentially cleaves at the C terminus of E, whereas at pH 8 it additionally cleaves at D residues.
  • the sequence of GluC is provided in SEQ ID NO:153 and 154.
  • the methods described herein use GluC alone (i.e. only GluC) to digest the one or more complement proteins.
  • a step of digesting the protein(s) in the described methods consists of digesting the protein(s) with GluC.
  • the methods may comprise digesting the protein(s) with GluC and trypsin, e.g. to distinguish between FHR1a and FHR1 b.
  • any method described herein does not employ/use any other protease alone or in combination with GluC.
  • the digestion step of any method described herein does not use, or is not performed by, any one or more of the following enzymes or agents: trypsin, chymotrypsin (high specificity or low specificity), Lys-C, Lys-N, Arg-C, Asp-N, elastase, LysargiNase, pepsin, Sap9, OmpT, BNPS-skatole, any caspase, clostripain (clostridiopeptidase B), CNBr, enterokinase, factor Xa, granzymeB, neutrophil elastase, proteinase K, thermolysin, non-GluC glutamyl endopeptidase e.g. GluBI or GluSGB, proline endopeptidase, TEV protease, thrombin
  • GluC is obtainable from standard reagent providers e.g. Sigma Aldrich, NEB etc, and may be used according to the accompanying instructions or according to protocols well known in the field. An example protocol is described herein. Obtaining proteins from biological samples and suitable buffers to prepare samples/proteins for GluC digestion will also be known to the skilled person.
  • An example cell lysis buffer comprises: 8 M urea (4.8 g per 10 ml) in 50 mM NH4HCO3 and 20mM methylamine, diluted to a urea concentration of ⁇ 2 M, pH 8 (40 mg per 10 ml), containing 1 tablet of completeTM Mini EDTA-free protease inhibitor cocktail per 10 ml of lysis buffer.
  • a complement protein is contacted/incubated/digested with GluC enzyme for at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21 , at least 22, at least 23, or at least 24 hours.
  • a complement protein is contacted/incubated/digested with GluC enzyme for at least 12 hours.
  • a complement protein is contacted/incubated/digested with GluC enzyme for about 12 hours, e.g. 12 hours.
  • a complement protein is contacted/incubated/digested with GluC enzyme for about 16 hours, e.g. 16 hours.
  • the terms contacted, incubated and digested are used interchangeably herein.
  • a complement protein is contacted/incubated/digested with GluC enzyme at a temperature of at least 20°C, at least 21 °C, at least 22°C, at least 23°C, at least 24°C, at least 25°C, at least 26°C, at least 27°C, at least 28°C, at least 29°C, or at least 30°C.
  • a complement protein is contacted/incubated/digested with GluC enzyme at a temperature of at least 25°C.
  • a complement protein is contacted/incubated/digested with GluC enzyme at a temperature of about 25°C, e.g. 25°C.
  • a complement protein is contacted/incubated/digested with GluC enzyme at a pH of at least 7.0, at least 7.1 , at least 7.2, at least 7.3, at least 7.4, at least 7.5, at least 7.6, at least 7.7, at least 7.8, at least 7.9, at least 8.0, at least 8.1 , at least 8.2, at least 8.3, at least 8.4, at least 8.5, at least 8.6, at least 8.7, at least 8.8, at least 8.9, or at least 9.0.
  • a complement protein is contacted/incubated/digested with GluC enzyme at a pH of at least 8.0.
  • a complement protein is contacted/incubated/digested with GluC enzyme at a pH of about 8.0, e.g. a pH of 8.0.
  • the GluC enzyme and complement protein are contacted/incubated at a wt/wt ratio of 1/75.
  • the incubation step may comprise gentle shaking, e.g. at 400 rpm.
  • the methods described herein may comprise a contacting/incubation/digestion step comprising any combination of temperature, pH, and/or time as described above. In some cases, contacting/incubating/digesting is performed at 25°C at pH8 for 12 hours.
  • the invention provides a method for detecting and/or determining the level of at least one complement protein e.g. in a sample, the method comprising: digesting the protein(s) with endoproteinase GluC to obtain one or more peptides, the digesting comprising incubating the protein(s) with GluC at 25°C at pH8 for up to 12 hours; and detecting the one or more peptides by mass spectrometry.
  • the invention provides a method for detecting and/or determining the level of at least one complement protein e.g. in a sample, the method comprising: digesting the protein(s) with endoproteinase GluC to obtain one or more peptides, the digesting comprising incubating the protein(s) with GluC at 25°C at pH8 for up to 16 hours; and detecting the one or more peptides by mass spectrometry.
  • the following peptides may be produced by GluC digestion of complement proteins, e.g. as described herein.
  • the methods described herein comprise detecting/determining the level of any one or more of these peptides, i.e. any one or more of SEQ ID NO:20 to 141 , or 155, 156 or 157, in any combination. All combinations of peptides are envisaged.
  • the mass of peptides represented by SEQ ID NOs 20-27 can be found in Table 1 .
  • the FH peptide is VTYKCFE (SEQ ID NO:20). In some embodiments the FH peptide is any one or more of SNTGSTTGSIVCGYNGWSDLPICYE (SEQ ID NO:112; mass 2623.1206), NGWSPTPRCIRVKTCSKSSIDIE (SEQ ID NO:113; mass 2576.2839), LPKIDVHLVPDRKKDQYKVGE (SEQ ID NO:114; mass 2476.3801), YYCNPRFLMKGPNKIQCVDGE (SEQ ID NO:115; mass 2474.1545), NYNIALRWTAKQKLYSRTGE (SEQ ID NO:116; mass 2411.2709), KWSHPPSCIKTDCLSLPSFE (SEQ ID NO:117; mass 2274.0813), HGWAQLSSPPYYYGDSVE (SEQ ID NO:118; mass 2054.9010), ISHGVVAHMSDSYQYGEE (SEQ ID NO:119;
  • the FHL-1 peptide is NGWSPTPRCIRVSFTL (SEQ ID NO:21).
  • the FHR1 peptide is ATFCDFPKINHGILYGEE (SEQ ID NO:22).
  • the FHR1 peptide is NYNIALRWTAKQKLYLRTGE (SEQ ID NO:91 ; mass 2437.3230).
  • the FHR2 peptide is RGWSTPPKCRSTISAE (SEQ ID NO:23).
  • the FHR2 peptide is AMFCDFPKINHGILYDEE (SEQ ID NO:24). In some embodiments the FHR2 peptide is YNFVSPSKSFWTRITCAEE (SEQ ID NO:92; mass 2264.0572).
  • the FHR3 peptide is VACHPGYGLPKAQTTVTCTE (SEQ ID NO:25).
  • the FHR3 peptide is any one or more of KGWSPTPRCIRVRTCSKSDIE (SEQ ID NO:93; mass 2418.2260), NGYNQNYGRKFVQGNSTE (SEQ ID NO:94; mass 2074.9457), QVKPCDFPDIKHGGLFHE (SEQ ID NO:95; mass 2066.0043), FMCKLGYNANTSILSFQAVCRE (SEQ ID NO:96; mass 2494.1807), or YQCQPYYE (SEQ ID NO:97; mass 1092.4222).
  • the FHR4 peptide is YQCQSYYE (SEQ ID NO:26).
  • the FHR4 peptide is any one or more of NSRAKSNGMRFKLHDTLDYE (SEQ ID NO: 1]
  • the FHR5 peptide is RGWSTPPICSFTKGE (SEQ ID NO:27).
  • the FHR5 peptide is any one or more of GTLCDFPKIHHGFLYDEE (SEQ ID NQ:102; mass 2119.9673), YAMIGNNMITCINGIWTE (SEQ ID NQ:103; mass 2042.9264), YGYVQPSVPPYQHGVSVE (SEQ ID NQ:104; mass 2004.9581), GDTVQIICNTGYSLQNNE (SEQ ID NQ:105; mass 1967.8895), IVCKDGRWQSLPRCVE (SEQ ID NQ:106; mass 1887.9447), DYNPFSQVPTGE (SEQ ID NQ:107; mass 1352.5884), QVKTCGYIPE (SEQ ID NQ:108; mass 1136.5536), ANVDAQPKKE (SEQ ID NQ:109; mass 1098.5669), WTTLPTCVE (SEQ ID NQ:110; mass 1048.4899), or KVAVLCKE (SEQ ID NO:111
  • the methods described herein comprise detecting/determining the presence and/or level of one or more of SEQ ID NOs 21-27, in any combination.
  • any method described herein may comprise detecting/determining the level of one or more of SEQ ID NOs 28-37, 156 or 157, in any combination.
  • the methods provided herein are used to detect C3, C3b and breakdown products using one or more or all of the peptides in Table 2 in any combination, plus optionally SEQ ID NO:156 and/or 157, for example according to the methodology in Table 3.
  • the Fl peptide is any one or more of VKLVDQDKTMFICKSSWSMRE (SEQ ID NO:45; mass 2531.2455), VKLISNCSKFYGNRFYE (SEQ ID NO:46; mass 2068.0320), CLHPGTKFLNNGTCTAE (SEQ ID NO:47; mass 1805.8309), NYNAGTYQNDIALIE (SEQ ID NO:48; mass 1698.7969), GKFSVSLKHGNTDSE (SEQ ID NO:49; mass 1605.7867), VGCAGFASVTQEE (SEQ ID NQ:50; mass 1297.5729), VGCAGFASVTQE (SEQ ID NO:155; mass 1168.272), MKKDGNKKDCE (SEQ ID NO:51 ; mass 1295.6082), YVDRIIFHE (SEQ ID NO:52; mass 1191 .6156), CLHVHCRGLE (SEQ ID NO:53; mass 1166.5557), RVFSL
  • the Fl peptide is any one or more of CAGTYDGSIDACKGDSGGPLVCMDANNVTYVWGWSWGE (SEQ ID NO:38; mass 3996.7183), GTCVCKLPYQCPKNGTAVCATNRRSFPTYCQQKSLE (SEQ ID NO:39; mass 3994.8853), FPGVYTKVANYFDWISYHVGRPFISQYNV (SEQ ID NQ:40; mass 3467.7211), ANVACLDLGFQQGADTQRRFKLSDLSINSTE (SEQ ID NO:41 , mass 3397.6804), LPRSIPACVPWSPYLFQPNDTCIVSGWGRE (SEQ ID NO:42, mass 3388.6605), KKCLAKKYTHLSCDKVFCQPWQRCIE (SEQ ID NO:43; mass 3155.5773), LCCKACQGKGFHCKSGVCIPSQYQCNGE (SEQ ID NO:44; mass 2991.2861), VK
  • Peptides detected by the methods described herein may optionally have at least 70%, preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequences of the peptides described herein, e.g. any one of SEQ ID NOs 21- 141 .
  • Other suitable peptides may be readily determined by a skilled person and may be employed in the methods described herein.
  • the peptides used according to the methods herein permit mass spectrometry techniques to distinguish or differentiate between two or more complement proteins in a sample.
  • Methods provided herein comprising detecting and/or determining the levels of proteins, e.g. complement proteins, may involve using mass spectrometry to detect and/or determine the levels of proteins in a sample.
  • any method provided herein may comprise performing mass spectrometry to determine the presence and/or level of one or more peptides as described herein. That is, any step described herein that comprises determining the level of one or more proteins/peptides may comprise performing mass spectrometry to determine the presence and/or level of the one or more proteins/peptides.
  • any method described herein involves using only mass spectrometry (i.e. mass spectrometry alone) to detect/determine the level of the one or more peptides. That is, in some embodiments, the methods provided herein do not employ multiple analytical techniques and the peptide(s) are detected/determined/measured using a single assay. In preferred embodiments, the methods described herein do not detect/determine the level of/measure the peptide(s) using mass spectrometry in combination with another analytical technique suitable for detecting proteins/peptides. In preferred embodiments, detection/determination of the level of the one or more peptides is not performed at any stage using a non-mass spectrometry technique, e.g.
  • HPLC high performance liquid chromatography
  • immunological-based methods such as quantitative enzyme-linked immunosorbent assays (ELISA), Western blotting, protein immunoprecipitation, dot blotting or immunoelectrophoresis, electrophoresis or autoradiography.
  • LC/MS liquid chromatography-mass spectrometry
  • detecting and/or determining the level of’ e.g. a complement protein or peptide is the same as “using mass spectrometry to detect and/or determine the level of’ e.g. a complement protein or peptide.
  • Mass spectrometry is a well-known analytical technique for analysing a sample that typically comprises generating ions from the sample, optionally fragmenting the ions, separating the ions according to their mass/charge ratio (in time and/or space), and detecting the ions to provide information regarding the content of the sample.
  • At least one fragmentation step may be included.
  • Mass spectrometry techniques are well-known in the field and any suitable mass spectrometry technique may be employed for detecting and/or determining the levels of proteins in a sample, e.g. LC/MS, GC/MS, tandem mass spectrometry (MS/MS), quadrupole MS e.g. triple quadrupole MS (TQMS), time-of- flight MS e.g. MALDI-TOF, targeted MS e.g. selected reaction monitoring MS (SRM-MS)Zmultiple reaction monitoring (MRM-MS), parallel-reaction monitoring (PRM-MS), trapped-ion based methods e.g.
  • quadrupole trap MS three- dimensional quadrupole ion traps (“dynamic” traps) and ion cyclotron resonance mass spectrometers (“static” traps), quadrupole trap MS, hybrid linear trap orbitrap MS, quadrupole-Orbitrap MS, electrospray Ionization mass spectrometry (ESI-MS), or electron transfer dissociation MS (ETD).
  • dynamic traps three- dimensional quadrupole ion traps
  • static traps quadrupole trap MS
  • hybrid linear trap orbitrap MS hybrid linear trap orbitrap MS
  • quadrupole-Orbitrap MS quadrupole-Orbitrap MS
  • ETD electron transfer dissociation MS
  • the mass spectrometry technique may be a liquid chromatography-selected reaction monitoring mass spectrometry (LC-SRM-MS)-based assay.
  • LC-SRM-MS liquid chromatography-selected reaction monitoring mass spectrometry
  • Fragmenting the ions may be achieved using any suitable fragmentation technique, e.g. collision-induced dissociation (CID)Zcollisionally activated dissociation (CAD), electron-capture dissociation (ECD), electron transfer dissociation (ETD), in-source decay (ISD), infrared multiple photon dissociation (IRMPD) etc.
  • CID collision-induced dissociation
  • ECD electron-capture dissociation
  • ETD electron transfer dissociation
  • ISD in-source decay
  • IRMPD infrared multiple photon dissociation
  • the mass spectrometry techniques useful in the present invention may comprise quantitative analysis.
  • Mass spectrometry methods comprising quantitative analysis may comprise a targeted approach to detect and measure peptides of interest and their corresponding fragments. This may allow for greater specificity and sensitivity for quantification.
  • Quantitative mass spectrometry in proteomics is reviewed in e.g. Bantscheff, M., et al. Anal Bioanal Chem 2007, 389, 1017-1031 , which is hereby incorporated by reference in its entirety.
  • input peptides may undergo fragmentation in a collision cell, thus generating product ions exclusive to the peptides.
  • product ions exclusive to the peptides.
  • Both the intact peptide mass and one or more specific fragment ions of that peptides can be monitored over the course of an MS experiment e.g. using SRM/MRM, PRM etc.
  • transition i.e. a mass pair representing the m/z of an analyte (the parent ion) and the m/z of one of its product ions which is formed upon fragmentation of the parent ion.
  • Tables 7 and 8 provide examples of transitions for the complement proteins described herein, based on fragmentation of synthetic versions of each peptide of interest. Suitable alternative transitions may also be used, the identification of which is well within the routine remit of a skilled person.
  • Quantitation can be achieved by ‘spiking’ the sample with known quantities of labelled synthetic peptides.
  • the combination of retention time, peptide mass, and fragment mass practically eliminates ambiguities in peptide assignments and extends the quantification range to 4-5 orders of magnitude.
  • the methods provided herein comprise a step of determining optimised MS settings and/or quantitation reference values using stable isotopic standards.
  • Mass spectrometry techniques that may be used in the present invention may comprise targeted or semitargeted MS workflows and/or data-dependent acquisition (DDA) or data-independent acquisition (DIA) techniques.
  • DDA data-dependent acquisition
  • DIA data-independent acquisition
  • DDA uses knowledge obtained during the acquisition to decide which MS1 peptide precursors to subject for fragmentation (MS/MS) in the collision cell.
  • DIA performs predefined MS/MS fragmentation and data collection regardless of sample content, which allows for more sensitive and accurate protein quantification compared to DDA.
  • DIA strategies can be further segregated into targeted or untargeted acquisitions.
  • Targeted DIA methods fragment predefined precursor ions that correspond to the peptide analytes, usually at known (measured or predicted) retention times.
  • Targeted DIA has become widely used in academic, pharmaceutical, and biotechnology research for quantification of small molecules (metabolites), peptides, and post-translational modifications (PTMs).
  • SRM selected- reaction monitoring
  • Suitable DIA methods include e.g. Sequential Window Acquisition of All Theoretical mass spectrometry (SWATH MS; see e.g. Ludwig et al., Mol Syst Biol (2016)14:e8126), SONAR (Waters.com), or Online Parallel Accumulation-Serial Fragmentation (PASEF; see e.g. Meier et al., J Proteome Res. 2015 Dec 4;14(12):5378-87 and Meier et al., Mol Cell Proteomics. 2018 Dec; 17(12): 2534-2545).
  • SWATH MS Sequential Window Acquisition of All Theoretical mass spectrometry
  • SONAR Waters.com
  • PASEF Online Parallel Accumulation-Serial Fragmentation
  • the methods according to the present disclosure do not employ SWATH MS, i.e. the mass spectrometry technique is not SWATH MS.
  • kits for performing such methods.
  • the present invention provides a kit comprising endoproteinase GluC for use in a method of detecting and/or determining the level of one or more complement protein(s) e.g. in a sample.
  • the kit may be used for any of the methods described herein and/or for detecting/determining the level of any one or combination of proteins described herein.
  • the kit may be suitable for, used for, or intended/sold/distributed for detecting at least one complement protein in a sample, determining the level of at least one complement protein in a sample, preparing at least one complement protein for analysis and/or detection, determining the presence and/or level of a complement protein in a subject, determining whether a subject is at risk of developing a complement-related disorder, identifying a subject having a complement-related disorder, selecting a subject for treatment of a complement-related disorder, and/or treating a subject who is suspected to have a complement-related disorder.
  • the kit and components thereof may be suitable for use with MS techniques.
  • kits provided herein comprises one, two, or more components suitable for performing the methods described herein, in whole or in part.
  • the kit may comprise standards or controls, e.g. labelled peptide standard(s) for each protein to be detected using the kit.
  • the kit may comprise a predetermined quantity of labelled peptide standards.
  • the kit may comprise a predetermined quantity of GluC enzyme, optionally with the necessary buffers and reagents for enzyme digestion.
  • the components of the kit may be provided in a single composition, or may be provided as plural compositions.
  • the kit may be suitable for a point-of-care in vitro diagnostic test. It may be a kit for laboratory-based testing.
  • the kit may include instructions for use, such as an instruction booklet or leaflet.
  • the instructions may include a protocol for performing any one or more of the methods described herein e.g. for enzyme digestion, recommended MS settings, and/or data analysis templates.
  • the kit may comprise components for separating proteins in a sample and/or performing MS techniques e.g. liquid chromatography columns.
  • the kit may be adapted for use with dry samples, wet samples, frozen samples, fixed samples, urine samples, saliva samples, tissue samples, blood samples, or any other type of sample, including any of the sample types disclosed herein.
  • the kit may comprise a device for obtaining or processing a blood, serum, plasma, cell or tissue sample.
  • nucleic acid, nucleotide or amino acid sequence which corresponds to a reference nucleic acid, nucleotide or amino acid sequence may comprise at least 60%, e.g. one of at least 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the reference sequence.
  • Pairwise and multiple sequence alignment for the purposes of determining percent identity between two or more amino acid or nucleic acid sequences can be achieved in various ways known to a person of skill in the art, for instance, using publicly available computer software such as ClustalOmega (Sbding, J. 2005, Bioinformatics 21 , 951-960), T-coffee (Notredame et al. 2000, J. Mol. Biol. (2000) 302, 205-217), Kalign (Lassmann and Sonnhammer 2005, BMC Bioinformatics, 6(298)) and MAFFT (Katoh and Standley 2013, Molecular Biology and Evolution, 30(4) 772-780 software.
  • the default parameters e.g. for gap penalty and extension penalty, are preferably used.
  • phase “and/or” as used herein encompasses each member of the list individually, as well as any combination of one or members of the list up to and including every member of the list.
  • a method for detecting at least one complement protein in a sample comprising: digesting the protein(s) with endoproteinase GluC to obtain one or more peptides; and detecting the one or more peptides by mass spectrometry.
  • a method for determining the level of at least one complement protein in a sample comprising: digesting the protein(s) with endoproteinase GluC to obtain one or more peptides; and determining the level of the one or more peptides by mass spectrometry.
  • a method for preparing at least one complement protein for analysis comprising digesting the protein(s) with endoproteinase GluC to obtain one or more peptides.
  • complement protein(s) is one or more of C3, C3b, C3a, iC3b, C3f, C3c, C3dg, and/or C3d.
  • a method according to any one of paras 1 to 11 wherein the method comprises a step of obtaining the sample from a subject.
  • VTYKCFE SEQ ID NO:20
  • NGWSPTPRCIRVSFTL SEQ ID NO:21
  • RGWSTPPKCRSTISAE SEQ ID NO:23
  • AMFCDFPKINHGILYDEE SEQ ID NO:24
  • VACHPGYGLPKAQTTVTCTE (SEQ ID NO:25);
  • a method of determining the presence and/or level of a complement protein in a subject comprising performing a method for detecting at least one complement protein in a sample according to any one of paras 1 to 15.
  • a method of identifying a subject having a complement-related disorder or at risk of developing a complement-related disorder comprising: d) digesting at least one complement protein in a sample obtained from the subject with endoproteinase GluC to obtain one or more peptides; e) determining the presence and/or level of the one or more peptides by mass spectrometry; and f) using the results of (b) to determine whether the subject has or is likely to develop a complement-related disorder.
  • a method according to para 18, comprising (d) treating a subject who has been determined to be at risk of developing, or to have, a complement-related disorder, optionally wherein treating a subject comprises administering a therapeutically effective amount of a complement-targeted therapeutic to the subject.
  • a method of selecting a subject for treatment of a complement-related disorder with a complement-targeted therapeutic comprising: a) digesting at least one complement protein in a sample obtained from the subject with endoproteinase GluC to obtain one or more peptides; b) determining the presence and/or level of the one or more peptides by mass spectrometry; and c) using the results of (b) to determine whether the subject is in need of a complement-targeted therapeutic.
  • a complement-targeted therapeutic for use in a method of treating a complement-related disorder in a subject, the method comprising: a) digesting at least one complement protein in a sample obtained from the subject with endoproteinase GluC to obtain one or more peptides; b) determining the presence and/or level of the one or more peptides by mass spectrometry; and c) based on the results of (b), administering an effective amount of the complement-targeted therapeutic.
  • a method according to any one of paras 17 to 21 wherein the method comprises obtaining a sample from a subject comprising at least one complement protein, optionally wherein the sample comprises or is derived from blood, lymph, plasma, serum, tissue or cells.
  • a method according to any one of paras 18 to 22, wherein the complement-related disorder is selected from macular degeneration, age related macular degeneration (AMD), geographic atrophy (‘dry’ (i.e. non-exudative) AMD), early AMD, early onset macular degeneration (EOMD), intermediate AMD, late/advanced AMD, ‘wet’ (neovascular or exudative) AMD, choroidal neovascularisation (CNV), retinal dystrophy, Haemolytic Uremic Syndrome (HUS), atypical Haemolytic Uremic Syndrome (aHUS), DEAP HUS (Deficiency of FHR plasma proteins and Autoantibody Positive form of Hemolytic Uremic Syndrome), autoimmune uveitis, Membranoproliferative Glomerulonephritis Type II (MPGN II), sepsis, Henoch-Schbnlein purpura (HSP), IgA nephropathy, chronic kidney disease, paroxysmal nocturnal hemoglobin
  • a kit for use in a method of detecting and/or determining the level of one or more complement protein(s) in a sample the kit comprising endoproteinase GluC.
  • a method of identifying a subject having a complement-related disorder or at risk of developing a complement-related disorder comprising:
  • step (a) comprises determining the level of two or three of the complement proteins selected from FHR1 , FHR2 and/or FHR3.
  • step (a) further comprises determining the level of FHR4 and/or FHR5.
  • D The method according to any one of paras A-C, wherein the method further comprises determining the level of FHL-1 and/or FH, optionally determining that the subject has or is likely to develop a complement-related disorder if the level of FHL-1 is elevated as compared to the level of FHL-1 in blood in a control subject that does not have a complement-related disorder.
  • ALD Macular Degeneration
  • AMD Geographic Atrophy
  • EOMD early onset macular degeneration
  • intermediate AMD late/advanced AMD
  • wet neovascular or exudative AMD
  • CNV choroidal neovascularisation
  • retinal dystrophy and/or autoimmune uveitis.
  • HUS Haemolytic Uremic Syndrome
  • aHUS atypical Haemolytic Uremic Syndrome
  • DEAP HUS DeAP HUS
  • FHR Plasma proteins and Autoantibody Positive form of Hemolytic Uremic Syndrome
  • glomerular diseases Membranoproliferative Glomerulonephritis Type II (MPGN II)
  • HSP Henoch-Schbnlein purpura
  • IgA nephropathy chronic kidney disease
  • PNH paroxysmal nocturnal hemoglobinuria
  • AIHA autoimmune hemolytic anemia
  • SLE systemic lupus erythematosis
  • Sjogren’s syndrome SS
  • rheumatoid arthritis RA
  • C3G C3 glomerulopathy
  • DDD dense deposit disease
  • C3 nephritic factor glomerulonephritis C3 NF GN
  • FHR Haemolytic Uremic Syndrome
  • aHUS atypical Haemolytic
  • a method for selecting a subject for treatment with a complement-targeted therapy comprising:
  • a method for selecting a therapeutic agent for a subject comprising:
  • a method of treatment comprising: (a) determining the level of a complement protein selected from one or more of FHR1 , FHR2, FHR3, FHR4 and/or FHR5, and optionally FHL-1 , in a blood sample obtained from the subject;
  • a complement-targeted therapeutic agent for use in a method of treatment comprising:
  • the therapy or therapeutic agent is an siRNA, miRNA, shRNA, antisense oligonucleotide, gapmer, antibody, aptamer, SOMAmer, or small molecule.
  • a method of determining whether a subject has, or is at risk of developing, a complement-related disorder comprising:
  • T A method according to any one of paras K to S, wherein the level of a complement protein is determined by mass spectrometry.
  • FIG. 1 Schematic showing the C3 proteolytic cascade and the proteolytic events leading to the generation, breakdown and inactivation of C3b (modified from Maillard et al, J Am Soc Nephrol. 2015 Jul;26(7):1503-12). Proteoform-specific peptides for mass spectrometry are underlined.
  • FIG. 1 LC-SRM Trace showing detection of the heavy-labelled synthetic standard peptides of each individual RCA locus protein from a plasma sample.
  • FIG. 4 Data confirming that C3 and C3 breakdown products in human plasma can be detected by MS with sufficient specificity and sensitivity.
  • 4A Total ion chromatograph from SRM-MS analysis showing specific and simultaneous detection of C3b fragment-specific peptides.
  • 4B Linearity data for seven of the ten peptides spiked into a plasma background.
  • 4C Coomassie-stained electrophoresis gel of C3 breakdown products obtained in vitro.
  • 4D MS quantification of key C3 fragments from the in vitro assay products shown in 4C.
  • Figure 6 Scatterplot showing differences in protein levels between subjects with AMD and control individuals (mean and p values shown (p ⁇ 0.05 considered statistically significant)).
  • FIG. 7 Area under the Receiver Operating Characteristic curve for various models.
  • Figure 8. Receiver Operating Characteristic curve for a model that uses FHR1 , FHR2, FHR3, FHR4, FHR5, FHL-1 & CFH levels (with 2- & 3-way interactions) to predict whether an individual is an AMD case or a control subject.
  • FIGS 9A to 9F GWASs of circulating FHR-1 to FHR-5 protein levels reveal a strong genome-wide significant signal spanning the CFH locus.
  • Regional plots show the genome-wide significant (P-value ⁇ 5 x 10’ 8 ) association signals from the GWASs of FHR-1 to FHR-5 protein levels (panels A-E) at the CFH locus on chromosome 1 q31 .3.
  • Panel F shows the equivalent CFH region for the GWAS of FHL-1 protein levels (no genome-wide significant association regions observed). The most associated variant is denoted by a purple diamond and is labelled by its rs-number.
  • FIG. 10 Established AMD risk variants at the CFH locus are associated at genome-wide significance level with circulating FHR-1 , FHR-2, FHR-3 and FHR-4 protein levels in 252 Cambridge controls. Box plots of FHR protein levels by variant genotype for those established AMD risk variants at the CFH locus from the IAMDGC study that showed genome-wide significant (P-value ⁇ 5 x 10’ 8 ) associations in 252 controls from the Cambridge AMD cohort (Table 2). P-values and Beta values from Wald tests using linear regression models adjusted for sex, age and the first two genetic principal components (as estimated within the IAMDGC study) are indicated in the note at the bottom of each plot.
  • Mendelian randomization analysis shows highly significant elevation of circulating FHR-1 , FHR-2, FHR-4 and FHR-5 protein levels in advanced AMD.
  • Mendelian randomization estimates of the association of FHR-1 (panel A), FHR-2 (panel B), FHR-3 (panel C), FHR-4 (panel D) and FHR-5 (panel E) are presented together with the corresponding traditional epidemiologic odds ratio (OR) estimates obtained from logistic regression models (352 advanced AMD cases and 252 controls from the Cambridge AMD study).
  • the Mendelian randomization estimates were obtained using the Wald ratio (if a single instrument was available; FHR1 , FHR2, FHR4, FHR5) or the inverse-variance weighted (IVW) method under a fixed-effect model (if multiple instruments were available; FHR3).
  • Raw data used to calculate the Mendelian randomization estimates are provided in Table S7. The variance of each protein explained by its genetic instrument(s) is indicated in the note at the bottom of each plot.
  • Figure 12 Graphs showing the levels of the indicated complement proteins in the blood samples obtained from test 1 of COVID-19 patients having eventual asymptomatic disease (A), mild disease (B), disease requiring hospitalization but not supplemental oxygen (C), disease requiring hospitalization and low flow supplemental oxygen (D), disease requiring assisted ventilation €, or blood samples obtained from COVID-19-negative subjects (control).
  • Figure 13 ROC curves showing ability of the indicated complement proteins to predict severity of COVID disease, using first-test blood samples obtained from COVID-19 patients that eventually required assisted ventilation (Group E above) compared with healthy controls.
  • Figures 15A and 15B Breakdown of C3b in the presence of Factor I, FHR-4 and the CR1 polypeptide.
  • 15A Increasing concentrations of FHR-4 were added to monitor the effect on C3b breakdown.
  • 15B Increasing concentrations of CR1 polypeptide were added to pre-incubated C3b, Fl and FHR-4 to monitor the effect on C3b breakdown.
  • Figures 16A and 16B Graphs showing nucleic acid (A) and protein expression (B) of CR1 CCPs 8-10 in ARPE19 cells from plasmids containing codon-optimized nucleotide sequence (R3049 to R3052) compared to plasmid containing wild-type nucleotide sequence (R3053).
  • Figure 17 Graph showing nucleic acid expression of CR1 CCPs 8-10 in HEK293 cells from plasmids containing codon-optimized nucleotide sequence (R1 to R4) compared to plasmid containing wild-type nucleotide sequence (WT).
  • Example 1 Generation of peptides from complement proteins for mass spectrometry
  • GluC digestion was performed on FH, FHL-1 , FHR1-5, Fl, C3, C3b and C3b breakdown products to achieve distinct peptides for mass spectrometry. GluC digestion is described in Example 2.2.
  • Peptides that can be used to detect each protein or protein fragment are set out in Tables 1-4 below.
  • proteolytic events leading to the generation, breakdown and inactivation of C3 are shown in Figure 1 .
  • Proteoform-specific peptides produced by GluC digestion are underlined in Figure 1 and are shown in Table 2.
  • Table 3 shows how each protein can be detected individually using the peptides in Table 2.
  • Table 2. Peptide sequences for MS resulting from GluC digestion of C3, C3b and breakdown products.
  • GluC digestion of Factor I produced the candidate peptides in Table 5 for MS analysis.
  • SEQ ID NO:45 to 56 and 155 contain 8-21 amino acids and are a good length for MS analysis. Table 5.
  • a mixed SIS solution was prepared by firstly diluting stock solution of FHL-1 , FHR1 , FHR2, FHR3, FHR4 and FHR5 by tenfold (no dilution of CFH stock was required), then adding the appropriate amounts of each individual diluted solution to a final volume of 200 pL in 0.1 % TFA. This was then stored at -80 °C in 5 pL aliquots for further dilution immediately prior to spiking.
  • Spiking solution was prepared immediately prior to sample addition by adding 195 pL 50:50 acetonitrile:water to a 5 pL aliquot of the mixed SIS solution. 2 pL of this was carefully added to each digested sample prior to drying down.
  • Frozen plasma samples were allowed to thaw to room temperature before being vortexed hard for 5 minutes to dissolve any soluble material, then centrifuged at 13,300g for 30 min to settle any insoluble material.
  • the following gradient elution profile was used to separate the peptides (time: %B): 0 min: 5 % B; 2 min: 5 % B; 3 min: 12 % B; 12 min: 15 % B; 15 min: 20 % B; 30 min: 25 % B; 31 min: 90 % B; 39 min:90 % B; 40 min: 5 % B; 49 min: 5 % B.
  • a switching valve located between the column and source was diverted to the waste position at points in the chromatogram when the analyte peptides were not eluting. This allowed for six windows (two of the peptides, FHR-2 and FHL-1 , eluted within the same window) of acquisition, of approximately one minute each, to be acquired with the column on-line to the mass spectrometer. SRM data was processed using a dedicated project in Skyline (v19.1 .0.193; www.skyline.ms).
  • Figure 2 shows a LC-SRM Trace showing detection of the heavy-labelled synthetic standards of each individual RCA locus protein from a plasma sample. This demonstrates that the method is feasible, specific and has the required sensitivity to distinguish between peptides from these seven proteins, in particular between splice variants FH and FHL-1 .
  • Figure 3 shows linearity data for FH, FHL-1 , and FHR1-5. This demonstrates that the GluC digestion produces peptides that can be detected individually and specifically in native serum at endogenous levels. It also shows that the assay is capable of quantifying the level of each protein in the sample. Increasing amounts of protein increase the signal in a predictable manner, allowing determination of the levels, as well as the presence, of each of the proteins. Also demonstrated is that the assay is free from interference.
  • FH 25nM
  • FHL-1 0.25nM
  • FHR- 1 2nM
  • FHR-2 1 nM
  • FHR-3 1 nM
  • FHR-4 4nM
  • FHR-5 3nM.
  • Figure 4A shows that all peptides in Table 2 can be detected individually in a plasma sample by SRM-MS using at least three transitions.
  • the specificity of the assay for the peptides of interest is confirmed by the relative intensities of the transitions matching the relative intensity of the relevant product ions in an MS/MS scan.
  • Figure 4B confirms the specificity of the peptides, showing experiments in which the plasma sample was spiked with crude synthetic peptide which demonstrated the appropriate increase in signal.
  • C3b breakdown was further analysed in an in vitro assay.
  • C3b was incubated along with Fl and a fragment of cofactor CR1 , selected over FH as CR1 drives the reaction to cleavage of iC3b to C3c + C3dg, whereas FH will only support cleavage of C3b to iC3b.
  • Sequential samples were taken from the reaction and stopped by boiling.
  • Figure 4C shows the time course of the C3b breakdown via gel electrophoresis. Analysis using MS and the peptides of Table 2 demonstrates that the formation of C3b fragments iC3b, C3f and C3c, and loss of intact C3b can be clearly detected over time ( Figure 4D). Not all peptides are shown since some (e.g. C3a) will not be present in the in vitro set-up, and others represent multiple products.
  • a single assay which can measure all FH family, C3 fragments and Fl proteins allows for the simultaneous analysis of all key proteins in the complement amplification loop from just one sample and with efficient throughput.
  • Genome-wide association analyses were performed of the protein levels that were found to be elevated in advanced AMD cases (i.e ., FHL-1 and FHR-1 to FHR-5).
  • GWASs were performed for FH, FHL-1 and the five FHR levels (transformed to ensure normality) in controls only, using linear regression models adjusted for sex, age and the first two genetic principal components, and variants with Minor Allele Frequency, MAF > 1%.
  • the GWASs were carried out using the EPACTS software (http://genome.sph.umich.edu/wiki/EPACTS, version 3.3.2) and Wald tests were performed on the variant genotypes coded as 0, 1 and 2 according to the number of minor alleles for the directly typed variants or allele dosages for the imputed variants.
  • Manhattan and Q-Q plots were generated using the qqman R package (version 0.1.4). Regional plots of association were generated using LocusZoom.org.
  • LD linkage disequilibrium
  • the genetic associations with the outcome were obtained from the GWAS based on a logistic regression model with AMD status as outcome conducted on the Cambridge samples (252 controls and 353 cases). If multiple instruments were available for a protein, the inverse-variance weighted (IVW) method was used under a fixed-effect model. Instrument strength was evaluated using R2 as the proportion of the variance of the protein explained by the genetic variant(s).
  • the Mendelian randomization analysis was performed using the MendelianRandomization (version 0.4.2) and TwoSampleMR (version 0.5.5) R packages.
  • Figure 11 shows the Mendelian randomization estimates of the FHR protein levels obtained using the (one-sample) Wald ratio (if a single instrument was available; FHR1 , FHR2, FHR4, FHR5) or the IVW method (if multiple instruments were available; FHR3) together with the traditional epidemiologic estimates of the association of the levels with AMD obtained from logistic regression models and ORs (Table 1).
  • the variance of the FHR protein levels explained by the corresponding genetic instruments varied from 15% for FHR5 to 73% for FHR3.
  • the Mendelian randomization estimates were statistically significant and of concordant direction with the observational OR estimates for FHR-1 , FHR-2, FHR-4 and FHR-5, providing evidence in support of a causal effect ( Figure 11).
  • the Mendelian randomization estimate did not support an association of the protein levels with the disease (0.98, 95% Cl 0.87 - 1.10).
  • the GWAS of FHL-1 levels did not show any genome-wide significant signals to use as genetic instruments in the Mendelian randomization analysis.
  • CFH locus as a cis protein quantitative trait locus (c/s-pQTL) for the five FHR levels prompted the use of the available genetic data in a Mendelian randomization fashion to triangulate this evidence.
  • FHR-1 , FHR-2, FHR-4 and FHR-5 the support provided by Mendelian randomization analyses for a potential casual role in susceptibility to AMD is striking, with Mendelian randomization estimates corroborating the preliminary evidence shown by the observational OR estimates (see Table 1).
  • Example 4 Tumor cell IDO enhances immune suppression and decreases survival independent of tryptophan metabolism in glioblastoma
  • Example 4 of WO 2022/058447 A1 demonstrates that non-metabolic indoleamine 2,3-dioxygenase 1 (IDO) increases the expression of immunosuppressive complement factor H (CFH) expression and in-turn, suppressed the anti-tumor immune response and decreased the survival of experimental animals with brain tumors. Increased intratumoral CFH and FHL-1 levels were associated with poorer survival among glioma patients.
  • IDO non-metabolic indoleamine 2,3-dioxygenase 1
  • Figure 12 of WO 2022/058447 A1 shows that tumor cell IDO mediates immune suppression in-part through an IDO-dependent tryptophan metabolism-independent mechanism.
  • FIG 13 of WO 2022/058447 A1 shows that indoleamine 2,3 dioxygenase 1 (IDO) non-metabolically increases complement factor H (CFH) levels in human glioblastoma (GBM).
  • IDO indoleamine 2,3 dioxygenase 1
  • FIG 14 of WO 2022/058447 A1 shows that IDO and complement factor H (CFH) mRNA levels positively correlate with T cell infiltration in patient-resected glioblastoma (GBM).
  • CBM complement factor H
  • FIG 15 of WO 2022/058447 A1 shows that IDO enhances the expression of both complement factor H (CFH) isoforms in human glioblastoma (GBM).
  • CFG complement factor H
  • Figure 16 of WO 2022/058447 A1 shows that CFH increases immunosuppressive factor expression and decreases overall survival in a syngeneic mouse brain tumor model.
  • Figure 17 of WO 2022/058447 A1 shows systemic and local complement factor H levels and the relationship with other immunosuppressive factors in patient-resected glioblastoma.
  • Figure 18 of WO 2022/058447 A1 shows the overall survival of syngeneic mice with intracranial IDO-/- tGBM tumors and depleted for CD4+ T, CD8+ T, and NK1 .1 + immune cells.
  • Figure 19 of WO 2022/058447 A1 shows the histological evaluation of tumor progression and confirmation of IDO-mGFP expression in brain tumors.
  • Figure 22 of WO 2022/058447 A1 shows differentially expressed genes that possess the strongest correlation with IDO in human GBM cells.
  • Figure 23 of WO 2022/058447 A1 shows mass spectrometry parameters for detecting CFH and FHL-1 in plasma.
  • the inventors investigated the levels of complement proteins CFH, FHL1 , FHR1 , FHR2, FHR3, FHR4 and FHR5 in samples of blood obtained from 200 COVID-19 patients having varying severity of disease, and in samples of blood obtained from healthy control subjects (not having COVID- 19). Blood samples were obtained from subjects in April-July 2020 at the time of the first COVID test.
  • asymptomatic disease A
  • mild disease B
  • disease requiring hospitalization but not supplemental oxygen C
  • disease requiring hospitalization and low flow supplemental oxygen D
  • disease requiring assisted ventilation E
  • Detection of FH, FHL1 , FHR1 , FHR2, FHR3, FHR4 and FHR5 in samples from each group was performed using mass spectrometry as described in Examples 1 and 2.
  • Example 6 Small fragments of CR1 comprising CCPs 8-10 have therapeutic utility
  • WO 2019/138137 A1 hereby incorporated in its entirety, describes small polypeptides that comprise CCPs (SCRs) 8-10 of human CR1 (see SEQ ID NO:146 herein). These polypeptides can access sites of unwanted pathological complement activation, for example via diffusion between tissue compartments, and can facilitate Factor l-mediated breakdown of C3b into iC3b and further breakdown products, see e.g. Figure 1 herein. This helps to reduce undesirable accumulation of both C3b and iC3b.
  • iC3b is a pro- inflammatory molecule which acts to recruit immune cells to the site of complement activation, which in turn cause negative inflammatory effects.
  • Example 1 of WO 2019/138137 A1 characterises a polypeptide comprising CCPs 8-10 of CR1 attached to an FH signal sequence (designated CR1 a/nCR1 a in WO2019/138137 and referred to herein as SEQ ID NO:182).
  • FIG. 2 of WO 2019/138137 A1 shows that CR1 a/nCR1 a can act as cofactors to assist Factor I (Fl) in the cleavage of C3b to iC3b and then further to C3dg.
  • Factor I Factor I
  • the C3b breakdown using CR1 a/nCR1 a continues further than the normal native breakdown of C3b observed using Fl + FHL-1 , which can only produce inflammatory iC3b.
  • Figure 3 of WO 2019/138137 A1 shows that CR1 a and nCR1 a were able to cross Bruch’s membrane from the sample chamber while remaining functionally active cofactors for Fl-mediated breakdown of C3b to iC3b and beyond.
  • Example 6 of WO2019/138137 also demonstrates this property.
  • Example 3 of WO 2019/138137 A1 demonstrates that the binding affinity of CR1 a for C3b was 21 nM, see Figure 5 therein.
  • the binding affinity for C3b of FH was 580 nM and of FHL-1 was 1 .2 mM.
  • CR1 a therefore binds significantly more strongly to C3b than either of these native soluble complement regulators.
  • the strong binding affinity of CR1 a for C3b means that CR1a is a more effective agent to promote C3b break down than agents based on FH or FHL-1 .
  • a strong binding affinity enables CR1a to promote degradation of iC3b into desirable further downstream products e.g. C3dg.
  • FH and FHL-1 cannot cause degradation of C3b beyond iC3b. Further breakdown of iC3b to C3dg by CR1 a is thus advantageous and avoids further damage caused by the subject’s immune system.
  • Example 5 and Figure 7A of WO 2019/138137 A1 show that functional CR1a polypeptide secreted from human APRE-19 cells was able to act as a cofactor for Factor I leading to breakdown of C3b into iC3b and C3dg.
  • Figure 7B demonstrates that RPE cells expressing AAV-delivered CR1a were found to have increased C3b breakdown capacity to iC3b (product b; lane 3) compared to the native C3b turnover rate in RPE cells.
  • CR1a is secreted successfully from cells, e.g. retinal pigment epithelium cells, is functionally active as a Fl cofactor, enhances C3b break down to downstream products, is capable of complement regulation, and would provide therapeutic benefit for conditions involving over-activation of complement e.g. an excess of C3b.
  • Example 7 Further characterisation of polypeptides comprising CCPs 8-10 of CR1
  • the fluid-phase activity of a small polypeptide comprising CCPs 8-10 of CR1 was tested in a C3b breakdown assay.
  • 2pg of C3b, 0.04pg Fl and 1 pg of polypeptide were incubated together in a total volume of 20pl at 37°C for 30 min. Reactions were stopped by the addition of 5pl 5x SDS reducing sample buffer and boiling at 100°C for 10 minutes. Samples were run on a 4-12% NuPAGE Bis Tris gel at 200V for 60 minutes in order to maximise the separation of the C3b breakdown product bands.
  • Figure 14 shows C3b breakdown in the presence of Factor I and the CR1 polypeptide.
  • the C3b alphachain is cleaved by Fl to form iC3b, then C3dg and finally C3d.
  • the polypeptide acts as a ‘super Fl cofactor’, allowing Fl to cleave C3b down to the C3dg and C3d level, unlike native Fl cofactors which can only mediate cleavage down to the iC3b level.
  • Functional complement activity is assessed by ELISA experiments performed using the Wieslab® Complement system Screenkit (Svar Life Science).
  • Patient serum e.g. from patients known to have functional complement activity and/or elevated levels of FHR proteins, is diluted in the appropriate diluent to ensure that the appropriate complement pathway is activated.
  • the generation of the terminal MAC/TCC complex is detected with specific antibodies and is quantified by measuring absorbance (optical density) using the 405nm wavelength.
  • the CR1 polypeptide binds to C3b with a greater affinity than the FHR proteins, such that even in the presence of a large molar excess of FHR protein, it can still bind to C3b and facilitate the Fl-mediated cleavage of C3b.
  • This means that the CR1 polypeptide can drive complement inhibition even in the presence of elevated FHR protein concentrations, unlike other native soluble Fl co-factors (e.g. FH, FHL- 1).
  • the CR1 polypeptide finds utility in treating disorders involving undesirable complement activation associated with elevated levels of FHR proteins, which would otherwise out-compete FH and FHL-1 as cofactors for Fl-mediated C3b breakdown.
  • Plasmids were constructed comprising codon-optimised nucleic acid encoding human CR1 CCPs 8-10 (SEQ ID NO:176) attached to a codon-optimised sequence encoding a secretory pathway sequence from Factor H (SEQ ID NO:181). The entire codon-optimised nucleic acid sequence is shown in SEQ ID NO:177.
  • the plasmids express the amino acid sequence of CR1 CCPs 8-10 (shown in SEQ ID NO:182 with the secretory pathway sequence, and in SEQ ID NO:146 without said sequence).
  • wild type nucleic acid encoding human CR1 CCPs 8-10 (nucleotide sequence as in SEQ ID NO:178) was attached to wild type nucleic acid encoding the FH signal sequence (entire nucleotide sequence shown in SEQ ID NO:179).
  • RPE cells ARPE19 cells were cultured for 24 hours to 90% confluency before performing transient transfection of plasmids by lipofection. After 24 hours, media was removed and replaced with 500ul of Optimem +2% FBS. After a further 24 hours, the supernatant was aliquoted and stored at -80°C.
  • qPCR analysis cell pellets were harvested and total RNA extracted using a RNeasy mini kit (Qiagen). Samples were treated with TUrBOTM DNase and cDNA was produced using SuperscriptTM IV Reverse Transcriptase. qRT-PCR was performed using Fast SYBRTM Green in triplicate. Primer sequences were: 5'-CCTACCATCCACTCGACACA-3’ and 5’-TCAGTCAGTCAAGCTAGCAGT-3’ for CR1 CCPs 8-10; and 5’-TCCTCACCAACCTGCCAGA-3’ and 5’-TGAAGCCAGGGAACTGATTGA-3’ for snRNP (Housekeeping gene). ACt was calculated relative to snRNP, and AACt was calculated relative to the R3049 plasmid result.
  • Figure 16A shows that CR1 CCPs 8-10 was expressed 48 hours after transient transfection.
  • Plasmids R1 , R2, R3 and R4 contained codon-optimised nucleic acid sequence, which was expressed at higher levels in each case compared to the wild-type nucleotide sequence (WT). Error bars represent standard deviation from triplicate repeats.
  • Figure 16B shows higher expression of CR1 CCPs 8-10 protein from the plasmids containing codon- optimised nucleic acid (R1-R4), compared to plasmid containing wild type nucleic acid (WT).
  • HEK293 cells were cultured for 24 hours to 80% confluency before performing transient transfection of plasmids by lipofection. After 24 hours, media was removed and replaced with 1 ml of Optimem +2% FBS. After a further 24 hours, the supernatant was aliquoted and stored at -80°C. The cells were harvested using trypsin and pellets were stored at -80°C for RNA extraction and expression analysis by qPCR, as above.
  • Figure 17 shows higher expression of CR1 CCPs 8-10 nucleic acid from plasmids containing codon- optimised nucleic acid (R1-R4), compared to plasmid WT containing wild type nucleotide sequence.

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Abstract

Acides nucléiques et autres agents destinés à être utilisés dans le traitement de patients présentant des troubles liés au complément ou à risque de tels troubles. Des méthodes de sélection de patients pour un traitement avec de tels agents, ainsi que des méthodes de traitement de patients avec de tels agents sont également divulguées.
PCT/EP2023/056794 2022-03-16 2023-03-16 Agents pour le traitement de troubles liés au complément WO2023175099A1 (fr)

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