Complement Inhibitory Hybrid Protein

Abstract

Provided are hybrid proteins comprising a CFH functional unit and a DAF functional unit, nucleic acids encoding same, and methods and uses thereof for treating diseases or disorders associated with the complement system.

Description

[Technical field]

[0001] The present invention relates to hybrid proteins comprising a CFH functional unit and a DAF functional unit, nucleic acids encoding same, and methods and uses thereof for treating diseases or disorders associated with the complement system. [Background technology]

[0002] As part of the innate immune system, the complement system includes more than 30 soluble protein molecules and more than 30 other molecules, including endogenous complement components, various regulatory factors, and complement receptors. It is well known that the complement system acts directly on pathogens and promotes pathogen-specific adaptive immune responses. The complement system is also involved in cell differentiation and polarization, tissue regeneration, lipid metabolism, clearance of immune complexes, apoptosis, and so on. Since the absence or inappropriate regulation of complement activation will cause damage to host tissues, the complement system is tightly regulated by a series of proteins, and the complement activation regulator (RCA) family proteins are mainly involved in complement regulation; RCA proteins include membrane proteins such as decay accelerating factor (DAF; CD55), membrane cofactor protein (MCP; CD46), and complement receptor 1 (CR1; CD35), as well as fluid-phase proteins such as factor H (FH or CFH) and C4b-binding protein (C4BP). The structure of RCA proteins consists of complement control protein repeat (CCP) modules, of which 2–4 consecutive modules contribute regulatory functions known as decay accelerating activity (DAA) and cofactor activity (CFA). RCA proteins target the C3 / C5 convertases, which are central enzymes in the complement pathway. RCA proteins bind to these convertases or their noncatalytic subunits, inactivating them. DAA is found by irreversible dissociation of invertase into subunits after binding to RCA proteins, whereas CFA is found by cleavage and inactivation of invertase via recruitment of serine protease factor I (FI) after binding to noncatalytic subunits (C3b / C4b), thereby preventing the formation of C3 convertase. Factor H is an opsonin and ligand of complement receptors 2 and 3, and acts as a cofactor for factor I in catalytically cleaving C3b to iC3b, thereby inhibiting the amplification of C3b. Factor H promotes the irreversible dissociation of the C3 convertase C3bBb in the alternative pathway and may compete with factor B for C3b binding during the formation of the proconvertase. Factor H is a soluble complement regulator that is essential for protecting surfaces such as the extracellular matrix ECM.FH can bind to the peptide hormone adrenomedullin and prevent its degradation. FH plays a role in managing cellular senescence, stress, or injury through interactions with C-reactive protein, pentameric proteins, DNA, histones, annexin II, malondialdehyde-acetaldehyde adducts of proteins, and oxidized lipids. FHL1 also has factor I cofactor activity and C3bBb decay-accelerating activity (The Complement Facts Book. Scott Barnum and Theresa Schein, eds., Copyright 2018 Elsevier Ltd. All rights reserved. https: / / doi.org / 10.1016 / C2015-0-06595-9, Chapter 30).

[0003] DAF inhibits the formation and promotes the decay of classical and alternative C3 and C5 convertases, thereby inhibiting the cleavage of C3 and C5, essentially protecting host cells from self-complement attack. When purified DAF is added to cells, it can be incorporated into the cell membrane and exhibit functional activity. DAF has also been found to regulate T cell tolerance and negatively regulate several animal models of autoimmune disease (The Complement Facts Book. Scott Barnum and Theresa Schein, eds., Copyright 2018 Elsevier Ltd. All rights reserved. https: / / doi.org / 10.1016 / C2015-0-06595-9, Chapter 25).

[0004] The present invention relates to a hybrid protein having a superior complement regulating activity, which is expected to have advantages such as a superior therapeutic effect. Summary of the Invention

[0005] The present invention relates to a hybrid protein of CFH and DAF, in which CCP1 / SCR1 of CFH is fused to a CCP of DAF (eg, CCP3-4 / SCR3-4 of DAF) to form the hybrid protein.

[0006] The hybrid proteins have superior complement inhibitory activity, including activity that inhibits the alternative pathway (AP), the classical pathway (CP), and / or C3b deposition.

[0007] In some embodiments, the hybrid protein is effective to inhibit human complement hemolytic activity.

[0008] In some embodiments, the hybrid proteins of the invention are effective in inhibiting human classical complement pathway hemolysis and / or human alternative complement pathway hemolysis.

[0009] In some embodiments, the hybrid proteins of the invention are effective in inhibiting C3b deposition.

[0010] For example, the hybrid protein of the present invention contains fewer CCPs of DAF based on the CPP1 / SCR1 of CFH, so that a hybrid protein with a smaller molecular weight can be obtained. When used for therapeutic purposes such as ophthalmology, brain drugs, etc., this hybrid protein can be administered at the same mass concentration at a higher molar dose, and therefore has the potential for better therapeutic effects or longer administration intervals compared to other commonly used complement regulatory proteins with larger molecular weights (especially compared to hybrid proteins that also contain other CCPs of CFH). In addition, a smaller molecular weight may be advantageous for penetration into tissues such as tumor tissues and the blood-brain barrier.

[0011] In some embodiments, the present invention relates to the following specific aspects: 1. A hybrid protein comprising: (i) CCP1 of human complement factor H (CFH); (ii) CCP3 and CCP4 of human decay accelerating factor (DAF); Optionally, a signal peptide and / or tag, A hybrid protein comprising or consisting of:

[0012] 2. The hybrid protein of embodiment 1, wherein the CCP3 and CCP4 of said DAF are directly linked together to form CCP3-4.

[0013] 3. The hybrid protein according to embodiment 1 or 2, wherein CCP1 of said human CFH comprises or consists of the amino acid sequence of positions 19 to 82 of said human CFH protein, optionally wherein said CCP1 comprises a V62I mutation, and wherein said amino acid positions are numbered corresponding to the amino acid positions set out in SEQ ID NO:3.

[0014] 4. A hybrid protein according to embodiment 1 or 2, wherein CCP1 of said human CFH comprises or consists of the amino acid sequence of positions 19 to 84 of said human CFH protein, optionally wherein said CCP1 comprises a V62I mutation, and the numbering of said amino acid positions corresponds to the amino acid positions set out in SEQ ID NO:3.

[0015] 5. A hybrid protein described in any one of embodiments 1 to 4, wherein CCP3 of the human DAF comprises or consists of the amino acid sequence of positions 161 to 222 of the human DAF protein, and / or CCP4 of the human DAF comprises or consists of the amino acid sequence of positions 223 to 285 of the human DAF protein, and the numbers of the amino acid positions correspond to the amino acid positions described in SEQ ID NO:1.

[0016] 6. A hybrid protein according to any one of embodiments 1 to 4, wherein CCP3 of the human DAF comprises or consists of the amino acid sequence 163 to 222 of the human DAF protein, and / or CCP4 of the human DAF comprises or consists of the amino acid sequence 223 to 285 of the human DAF protein, and the numbers of the amino acid positions correspond to the amino acid positions set forth in SEQ ID NO:1.

[0017] 7. The hybrid protein of embodiment 2, wherein the CCP3-4 of the human DAF comprises or consists of the amino acid sequence of positions 161 to 285 of the human DAF protein, and the numbers of the amino acid positions correspond to the amino acid positions set forth in SEQ ID NO:1.

[0018] 8. A hybrid protein according to any one of embodiments 2 to 4, wherein the CCP3-4 of the human DAF comprises or consists of the amino acid sequence of positions 163 to 285 of the human DAF protein, and the numbers of the amino acid positions correspond to the amino acid positions set forth in SEQ ID NO:1.

[0019] 9.(1) the CCP1 of the human CFH comprises or consists of the amino acid sequence of positions 19 to 82 of the human CFH protein, the CCP3 of the human DAF comprises or consists of the amino acid sequence of positions 161 to 222 of the human DAF protein, and the CCP4 of the human DAF comprises or consists of the amino acid sequence of positions 223 to 285 of the human DAF protein; (2) the CCP1 of the human CFH comprises or consists of the amino acid sequence of positions 19 to 84 of the human CFH protein, the CCP3 of the human DAF comprises or consists of the amino acid sequence of positions 163 to 222 of the human DAF protein, and the CCP4 of the human DAF comprises or consists of the amino acid sequence of positions 223 to 285 of the human DAF protein; (3) the CCP1 of the human CFH comprises or consists of the amino acid sequence of positions 19 to 82 of the human CFH protein, and the CCP3-4 of the human DAF comprises or consists of the amino acid sequence of positions 161 to 285 of the human DAF protein; or (4) the CCP1 of the human CFH comprises or consists of the amino acid sequence of positions 19 to 84 of the human CFH protein, and the CCP3-4 of the human DAF comprises or consists of the amino acid sequence of positions 163 to 285 of the human DAF protein; 3. The hybrid protein of embodiment 2, wherein the amino acid positions of the human CFH protein are numbered corresponding to the amino acid positions set forth in SEQ ID NO:3, and the amino acid positions of the human DAF protein are numbered corresponding to the amino acid positions set forth in SEQ ID NO:1.

[0020] 10. The hybrid protein of embodiment 9, wherein CCP1 of said human CFH has a V62I mutation.

[0021] 11. The human CFH protein is a native human CFH protein, or (i) an amino acid sequence set forth in SEQ ID NO: 3 or 5; (ii) an amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 4 or 6; or (iii) an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of (i) or (ii); Contains, or It consists of an amino acid sequence according to any one of (i) to (iii). 11. A hybrid protein according to any one of embodiments 1 to 10.

[0022] 12. The human DAF protein is a native human DAF protein, or (i) the amino acid sequence set forth in SEQ ID NO:1; (ii) the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO:2; or (iii) an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of (i) or (ii); Contains, or It consists of an amino acid sequence according to any one of (i) to (iii). 12. A hybrid protein according to any one of embodiments 1 to 11.

[0023] 13. The CCP1 of human CFH comprises or consists of an amino acid sequence set forth in SEQ ID NO: 12, 13, 14, or 15, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 12, 13, 14, or 15; the CCP3 of human DAF comprises, or consists of, the amino acid sequence set forth in SEQ ID NO: 7 or 8, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 7 or SEQ ID NO: 8; the CCP4 of said human DAF comprises or consists of the amino acid sequence set forth in SEQ ID NO:9 or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO:9; and / or The CCP3-4 of the human DAF comprises an amino acid sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 11, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 11, or consists of any of these amino acid sequences; 13. A hybrid protein according to any one of embodiments 1 to 12.

[0024] 14. The hybrid protein according to any one of the preceding embodiments, wherein said tag is a purification tag, such as a hexahistidine tag or a biotin tag, for example comprising the amino acid sequence set forth in SEQ ID NO:12.

[0025] 15. The hybrid protein according to any one of the preceding embodiments, wherein the signal peptide is secretory and comprises, for example, the amino acid sequence set forth in SEQ ID NO: 17.

[0026] 16. A hybrid protein according to any one of the preceding embodiments, wherein the hybrid protein comprises or consists of an amino acid sequence of any one of SEQ ID NOs: 18 to 33, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity to this amino acid sequence.

[0027] 17. A nucleic acid molecule encoding a hybrid protein according to any one of embodiments 1 to 16.

[0028] 18. An expression vector comprising the nucleic acid molecule of embodiment 17, preferably, said expression vector is pcDNA3.1.

[0029] 19. A host cell comprising the nucleic acid molecule of embodiment 17 or the expression vector of embodiment 18, preferably said host cell being a prokaryotic or eukaryotic cell, such as, for example, a 293 cell, such as an Expi293 cell.

[0030] 20. A method for preparing a hybrid protein according to any one of embodiments 1 to 16, comprising culturing a host cell according to embodiment 19 under conditions suitable for expression of said hybrid protein, and optionally further comprising isolating said protein from said host cell or host cell culture and / or purifying said protein.

[0031] 21. A pharmaceutical composition or a medicine or a formulation comprising a hybrid protein according to any one of embodiments 1 to 16 or a nucleic acid molecule encoding the same, or a nucleic acid molecule according to embodiment 17; and optionally a pharma- ceutically acceptable excipient.

[0032] 22. A combination pharmaceutical product comprising a hybrid protein according to any one of embodiments 1 to 16 or a nucleic acid molecule encoding same, or a nucleic acid molecule according to embodiment 17; and an additional therapeutic agent.

[0033] 23. A method for preventing or treating a complement system-related disease or disorder in a subject, comprising administering to the subject an effective amount of a hybrid protein of any one of embodiments 1-16 or a nucleic acid molecule encoding same; or a nucleic acid molecule of embodiment 17; or a pharmaceutical composition or formulation of embodiment 21; or a combination pharmaceutical product of embodiment 22.

[0034] 24. The method of embodiment 23, wherein the disease or disorder is due to abnormal activation of the complement system or dysregulation of the complement system.

[0035] 25. The method of embodiment 24, wherein the abnormal activation of the complement system or dysregulation of the complement system is due to, for example, a microbial infection or an increase in autoimmune antibodies, or a decrease, deletion, incapacitation, or interference or blockade of the function of a complement regulatory protein.

[0036] 26. The method of embodiment 23, wherein the complement system-related disease or disorder is selected from diseases requiring inhibition of hemolysis, such as diseases requiring inhibition of hemolysis of the classical pathway of complement immunity and / or the alternative pathway of complement immunity; or diseases requiring inhibition of C3b deposition activity, such as dense deposit disease (DDD). [Brief description of the drawings]

[0037]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

[0038] Detailed Description of the Invention I. Definition It is to be understood that the present invention is not limited to the specific methodology, protocols, and reagents described herein. It is to be understood that the terms used herein are for the purpose of describing specific embodiments only, and are limited only by the scope of the appended claims, and are not intended to limit the scope of the present invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0039] Decay Accelerating Factor (DAF) Decay-accelerating factor (DAF, CD55) is a membrane-bound regulatory protein that protects autologous cells from the activation of autologous complement on their surface. DAF acts by rapidly dissociating C3 and C5 convertases (the central enzymes of the cascade). DAF has the most potent decay-accelerating activity among proteins involved in complement regulation, acting on classical pathway enzymes (C4b2a and C4b2a3b) and alternative pathway enzymes (C3bBb and C3BbC3b). However, DAF does not have a cofactor function.

[0040] Structural analysis of DAF reveals that starting from the N-terminus, it consists of units ranging from 4 to 60 amino acids in length, a highly O-glycosylated serine (S)- and threonine (T)-rich fragment (STP), followed by a post-translationally added glycosylphosphatidylinositol (GPI) anchor.

[0041] In some embodiments, the DAF is a human DAF having the following accession numbers: Genbank accession numbers M31516, M15799, M64653, S72858, or M643567.

[0042] In some embodiments, the DAF is human DAF. In some embodiments, the human DAF is native human DAF. In some embodiments, the amino acid sequence of native human DAF is set forth in SEQ ID NO:1, and the various modules are shown in Table 1 below. Four 60 amino acid long repeating units called complement control protein repeats (CCPs) or short consensus repeats (SCRs). CCP1 comprises amino acids 35-96, CCP2 comprises amino acids 96-160, CCP3 comprises amino acids 161-222, and CCP4 comprises amino acids 223-285. These provide all the regulatory activities of DAF. The highly O-glycosylated region acts as a cushion that suspends the CCP on the surface membrane at an appropriate distance. The GPI anchor allows DAF to move freely on the cell membrane and inactivate wherever the invertase complex is assembled. In this context, references to the amino acid positions of the modules of DAF are made to correspond to the amino acid position numbers set forth in SEQ ID NO:1.

[0043] [Table 1]

[0044] In some embodiments, the nucleotide sequence of a cDNA encoding DAF is set forth in SEQ ID NO:2.

[0045] Factor H (CFH or FH) "Complement factor H", "Factor H", "FH", "CFH protein" or "CFH" are used interchangeably and refer to an approximately 150 kDa protein that is a member of the regulators of complement activation family and a complement regulatory protein. CFH is a large soluble glycoprotein that circulates in human plasma and plays a role in regulating the alternative pathway of the complement system, ensuring that the complement system is directed against pathogens and other dangerous agents and does not cause damage to host tissues.

[0046] Factor H is primarily monomeric, but can associate weakly (KD=28 μM) and may oligomerize in the presence of glycosaminoglycans and high concentrations of metal ions. CFH is composed of 20 homologous units called complement regulatory protein repeats (CCPs) (SCRs or sushi domains), some of which function in cell adhesion and others in the exclusion of C3b from the cell surface. The 20 SCRs are each about 60 amino acids long, arranged head-to-tail, contain four cysteine ​​residues, and form two disulfide bonds per module. SCRs 19 and 20 are involved in binding C3b.

[0047] The splice variant FHL-1 consists of the first seven CCPs followed by the C-terminal sequence Ser-Pro-Leu-Thr. Each CCP contains approximately 60 residues, including four invariant cysteines, Cys I -Cys III , Cys II -Cys IV Adjacent modules are connected by sequences of 3 to 8 residues.

[0048] Negative stain electron microscopy images show that the FH molecule adopts a variety of conformations, but is primarily folded in half. Analytical ultracentrifugation, small angle X-ray scattering (depositions of modules in the PDB, e.g., 3 GAV), and chemical cross-linking also show that the modular chains themselves are bent. High-resolution structures of several FH fragments have been determined, alone or in complex with other molecules (PDB identifiers are listed in parentheses below): CCP1-2 (2RLP), 2-3 (2RLQ), 1-4 (2WII), 5, 6-7 (e.g., 2W80, 2YBY), 7 (2JGW, 2JGX), 6-8 (2UWN, 2V8E), 9 (4K12), 10-11 (4B2R), 11-12 (4B2S), 12-13 (2KMS), 15 (1HFI), 16 (1HCC), 15-16 (1HFH), 18-20 (3SWO), 19-20 (e.g., 2BZM, 2G7I, 4ONT). Each CCP resembles a prolate spheroid with a long axis of ∼4 nm and a short axis of ∼2 nm, containing antiparallel patches of β-strands roughly along the long axis, with the N- and C-termini positioned at opposite ends of the long axis, facilitating end-to-end arrangement of tandem CCPs with varying inter-modular contacts, tilts, and twists.

[0049] In some embodiments, the CFH is human CFH, e.g., native human CFH. In some embodiments, the amino acid sequence of CFH has the following accession numbers: HGNC:HGNC:4883, Ensembl.ENSG00000000971, HPRD:00601, MIM:134370, or Vega.OTTHUMG00000035607.

[0050] In some embodiments, the amino acid sequence of CFH is set forth in SEQ ID NO: 3. In some embodiments, the amino acid positions corresponding to each module of CFH are set forth in Table 2 below.

[0051] [Table 2]

[0052] In some embodiments, the nucleotide sequence of a cDNA encoding CFH is set forth in SEQ ID NO:4.

[0053] In some embodiments, the protein sequence encoding the CFH splice variant FHL-1 is set forth in SEQ ID NO: 5. In some embodiments, the nucleotide sequence of the cDNA encoding the CFH splice variant FHL-1 is set forth in SEQ ID NO: 6.

[0054] In this context, references to amino acid positions of modules of CFH are made to correspond to the amino acid position numbers set out in SEQ ID NO:3.

[0055] Other definitions For purposes of interpreting the specification, the following definitions will be used, and where appropriate, terms used in the singular will also include the plural and vice versa. It will be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0056] The term "about" used in conjunction with a numerical value is intended to encompass a numerical range having a lower limit of 5% less than the specified numerical value and an upper limit of 5% greater than the specified numerical value.

[0057] As used herein, the term "and / or" means any one of the alternatives, or two or more or all of the alternatives.

[0058] As used herein, the term "comprises" or "includes" means the inclusion of the recited elements, integers, or steps, but not the exclusion of other elements, integers, or steps. When the term "comprises" or "comprises" is used herein, it also encompasses combinations of the recited elements, integers, or steps, unless otherwise indicated. For example, reference to a protein that "comprises" a particular sequence is intended to encompass a protein consisting of that particular sequence.

[0059] As used herein, the terms "hybrid protein" and "chimeric polypeptide" are used interchangeably and refer to a larger polypeptide formed by the fusion of at least two heterologous polypeptide sequences, optionally via a linker. Hybrid proteins can be produced by recombinant expression.

[0060] "Percent identity" of an amino acid sequence refers to the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues of a specific amino acid sequence set forth herein, after aligning the candidate sequence with the specific amino acid sequence set forth herein and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering conservative substitutions that are included as part of the sequence identity. In some embodiments, the present invention contemplates variants of the proteins or polypeptides of the present invention having a substantial degree of identity, e.g., at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% or more identity, to the polypeptides or proteins specifically disclosed herein. Variants may include conservative changes.

[0061] In the context of polypeptide sequences, "conservative modifications" include substitutions, deletions, or additions that do not substantially alter the desired functional activity of the polypeptide sequence. For example, conservative substitutions often result in the replacement of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Eight groups of amino acids that are conservatively substituted for one another are listed below: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine ​​(C), methionine (M). In some embodiments, the term "conservative sequence modifications" is used to refer to amino acid modifications that do not significantly affect or alter the activity of the parent hybrid protein. For example, conservatively modified variants retain at least 80%, 85%, 90%, 95%, 98%, or 99% or more, e.g., 100-110% or more, of the activity of the parent polypeptide or parent hybrid protein.

[0062] The term "host cell" refers to a cell into which an exogenous polynucleotide has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells", including the primary transformed cell and its derived progeny. Host cells are any type of cell line that can be used to produce the hybrid proteins of the invention, including eukaryotic cells such as mammalian cells, insect cells, yeast cells; and prokaryotic cells such as E. coli cells. Host cells include cultured cells, as well as cells within transgenic animals, transgenic plants, cultured plant tissues or animal tissues.

[0063] The term "expression vector" refers to a vector that contains a recombinant polynucleotide that includes an expression control sequence operably linked to a nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression; other elements for expression can be provided by the host cell or an in vitro expression system. Expression vectors include all those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, viruses, and adeno-associated viruses) into which the recombinant polynucleotide is incorporated.

[0064] The terms "individual" or "subject" are used interchangeably and refer to mammals. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, horses), primates (e.g., humans, non-human primates such as monkeys), rabbits, and rodents (e.g., mice, rats). In particular, individuals are people.

[0065] The term "treating" includes administering a composition or hybrid polypeptide to prevent or delay the onset of symptoms, complications or biochemical manifestations of a disease, alleviate symptoms, or prevent or inhibit further progression of a disease, condition or disorder. The term "prevention" includes inhibiting the onset or progression of a disease or disorder, or the symptoms of a particular disease or disorder.

[0066] The term "pharmaceutical excipient" refers to a diluent, adjuvant (such as Freund's adjuvant (complete and incomplete)), excipient, carrier, stabilizer, or the like, with which an active agent is administered.

[0067] The term "pharmaceutical composition" refers to a composition that is in a form effective to permit expression of the biological activity of the active ingredients contained therein and that does not contain additional ingredients that are unacceptably toxic to the subject to which the composition is administered.

[0068] The term "effective amount" refers to the amount or dosage of the hybrid protein of the present invention or the nucleic acid encoding it, or a composition or combination thereof, which, when administered to a patient in a single or multiple doses, produces the desired effect in a patient in need of treatment or prevention.

[0069] A "therapeutically effective amount" refers to an amount effective to achieve a desired therapeutic result, at dosages and for periods of time necessary. A "therapeutically effective amount" is also an amount in which the therapeutically beneficial effects of a hybrid protein or composition or combination outweigh its toxic or detrimental effects. A "therapeutically effective amount" preferably inhibits a measurable parameter or improves a measurable parameter by at least about 40%, more preferably at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 100% relative to untreated subjects.

[0070] A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Generally, a prophylactically effective amount will be less than a therapeutically effective amount, since a prophylactic dose is administered to a subject prior to or at an earlier stage of disease.

[0071] These and other aspects and embodiments of the present invention are described in the accompanying drawings (a brief description of the drawings follows immediately below) and the following detailed description of the invention, and are illustrated in the following examples. Any or all of the features described above and throughout this application may be combined in various embodiments of the present invention. The following examples further illustrate the present invention, although it should be understood that the examples are set forth by way of illustration and not by way of limitation, and that various modifications may be made by those skilled in the art.

[0072] II. Hybrid Proteins The present invention relates to a hybrid protein of CFH and DAF, which comprises at least one functional unit derived from CFH and at least one functional unit derived from DAF.

[0073] A.DAF Functional Unit In certain embodiments, the hybrid protein of the invention preferably comprises a functional unit derived from DAF. Such a functional unit can dissociate C3 and C5 convertases and / or promote decay-accelerating activity against C3 convertase in the classical and / or alternative pathways. In some embodiments, the DAF functional unit comprises CCP3 and 4 of DAF.

[0074] The amino acid sequence of such a CCP may be identical to the native or naturally occurring amino acid sequence of DAF. Alternatively, the amino acid sequence of such a CCP may be slightly altered, particularly at the amino or carboxy terminus. This alteration occurs when a restriction enzyme site is incorporated into the polynucleotide encoding the CCP. This alteration also occurs when amino acids are deleted from the N-terminus or C-terminus of the functional unit. For example, in some embodiments, one to two amino acids may be deleted at the N-terminus of CCP3.

[0075] Some amino acid substitutions in the sequence, preferably conservative substitutions, can also be introduced without affecting the functional activity, e.g., by substituting charged amino acids for one another, hydrophilic amino acids for one another, hydrophobic amino acids for one another, or amino acids of similar mass for another.

[0076] In one embodiment, the DAF functional unit comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to CCP3 and / or CCP4 (or CCP3-4 with the C-terminus of CCP3 and the N-terminus of CCP4 directly linked together) of a human DAF protein. In some embodiments, the DAF functional unit comprises CCP3 and / or CCP4 (CCP3-4 with the C-terminus of CCP3 and the N-terminus of CCP4 directly linked together) of a human DAF protein. In some embodiments, the DAF functional unit comprises CCP3 and CCP4 of a human DAF protein. In some embodiments, the DAF functional unit is CCP3-4 with the C-terminus of CCP3 and the N-terminus of CCP4 directly linked together.

[0077] In some embodiments, the human DAF protein is a native human DAF protein. In some embodiments, the native human DAF protein comprises or consists of the amino acid sequence set forth as SEQ ID NO: 1. DAF comprises or consists of the amino acid sequence encoded by the DNA sequence set forth in SEQ ID NO:2.

[0078] In some embodiments, CCP3 comprises or consists of amino acids 161 to 222 of DAF. In some embodiments, 1 to 2 amino acids at the N-terminus of CCP3 may be deleted to add another functional unit, for example, CCP3 comprises or consists of amino acids 163 to 222 of DAF.

[0079] In some embodiments, CCP3 comprises an amino acid sequence set forth in SEQ ID NO:7 or 8, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO:7 or SEQ ID NO:8, or consists of any of these amino acid sequences.

[0080] SEQ ID NO:7: KSCPNPGEIRNGQIDVPGGILFGATISFSCNTGYKLFGSTSSFCLISGSSVQWSDPLPECRE

[0081] SEQ ID NO:8: CPNPGEIRNGQIDVPGGILFGATISFSCNTGYKLFGSTSSFCLISGSSVQWSDPLPECRE

[0082] In some embodiments, CCP4 comprises amino acids 223-285 of DAF or consists of amino acids 223-285 of DAF.

[0083] In some embodiments, CCP4 comprises or consists of the amino acid sequence set forth in SEQ ID NO:9, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO:9.

[0084] SEQ ID NO:9: IYCPAPPQIDNGIIQGERDHYGYRQSVTYACNKGFTMIGEHSIYCTVNNDEGEWSGPPECRG

[0085] In some embodiments, CCP3-4 comprises, or consists of, amino acids 161 to 285 or amino acids 163 to 285 of DAF. In some embodiments, the DAF functional unit CCP3-4 comprises, or consists of, an amino acid sequence set forth in SEQ ID NO:10 or SEQ ID NO:11, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO:10 or SEQ ID NO:11.

[0086] SEQ ID NO: 10: Human DAF CCP3-4 KSCPNPGEIRNGQIDVPGGILFGATISFSCNTGYKLFGSTSSFCLISGSSVQWSDPLPECRE IYCPAPPQIDNGIIQGERDHYGYRQSVTYACNKGFTMIGEHSIYCTVNNDEGEWSGPPECRG

[0087] SEQ ID NO: 11: Human DAF CCP3-4 (2 amino acids deleted from the N-terminus of CCP3) CPNPGEIRNGQIDVPGGILFGATISFSCNTGYKLFGSTSSFCLISGSSVQWSDPLPECRE IYCPAPPQIDNGIIQGERDHYGYRQSVTYACNKGFTMIGEHSIYCTVNNDEGEWSGPPECRG

[0088] When referring to an amino acid position of DAF, the amino acid sequence position corresponds to the amino acid position in SEQ ID NO:1.

[0089] B.CFH Functional Unit In a particular embodiment, the hybrid protein of the invention comprises a functional unit derived from CFH, which has the ability to dissociate the alternative pathway C3 convertase C3bBb and / or to bind to C3b. In a preferred embodiment, the CFH functional unit comprises CCP1 of CFH.

[0090] The amino acid sequence of such a CCP of CFH may be identical to the native or naturally occurring amino acid sequence of CFH. Alternatively, the amino acid sequence of such a CCP may be slightly altered, particularly at the amino and carboxy termini. This occurs when a restriction enzyme site is incorporated into the polynucleotide encoding the CCP. This also occurs when amino acids are deleted or added from the N-terminus or C-terminus of the functional unit.

[0091] For example, in some embodiments, one to two amino acids may be deleted at the C-terminus of CCP1.

[0092] In some embodiments, one or two amino acids of CCP2 may also be added to the C-terminus of CCP1 to attach a functional unit of DAF, and thus "CCP1" as described herein encompasses CCP1 with amino acids added to the C-terminus (e.g., one or two amino acids of CCP2). For example, when one or two amino acids are deleted from the N-terminus of the functional unit of DAF, one or two amino acids of CCP2 can be added to the C-terminus of CCP1 to attach the functional unit of DAF. In some embodiments, KS is deleted from CCP3 of DAF, and RP is added to the C-terminus of CCP1 of CFH, and the two are ligated to obtain a hybrid protein.

[0093] Some amino acid substitutions, preferably conservative substitutions, in the sequence can also be introduced without affecting the functional activity.Conservative substitutions can be made, for example, by substituting charged amino acids for each other, substituting hydrophilic amino acids for each other, substituting hydrophobic amino acids for each other, or substituting amino acids with similar masses for each other.For example, the CCP1 of the CFH of the present invention can contain the V62I mutation.

[0094] In one embodiment, the CFH functional unit comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity to CCP1 of the human CFH protein. In certain embodiments, the CFH functional unit comprises CCP1 of the human CFH protein.

[0095] In some embodiments, the human CFH protein is a native human CFH protein. In some embodiments, the native human CFH protein comprises, or consists of, the amino acid sequence set forth in SEQ ID NO: 3 or 5. In some embodiments, the CFH comprises, or consists of the amino acid sequence encoded by the DNA sequence set forth in SEQ ID NO: 4 or 6.

[0096] In some embodiments, CCP1 comprises, or consists of, amino acids 19-82 of CFH. In some embodiments, CCP1 comprises, or consists of amino acids 19-84 of CFH.

[0097] In some embodiments, the CFH functional unit comprises or consists of an amino acid sequence set forth in SEQ ID NO: 12, 13, 14, or 15, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 12, 13, 14, or 15.

[0098] SEQ ID NO: 12: Human CFH CCP1 (the two N-terminal amino acids RP of CCP2 are added to the C-terminus of CCP1) EDCNELPPRRNTEILTGSWSDQTYPEGTQAIYKCRPGYRSLGNVIMVCRKGEWVALNPLRKCQKRP

[0099] SEQ ID NO: 13: Human CFH CCP1 (CCP1) EDCNELPPRRNTEILTGSWSDQTYPEGTQAIYKCRPGYRSLGNVIMVCRKGEWVALNPLRKCQK

[0100] SEQ ID NO: 14: Human CFH CCP1 (the C-terminus of CCP1 is appended with the two N-terminal amino acids RP of CCP2 and contains V62I) EDCNELPPRRNTEILTGSWSDQTYPEGTQAIYKCRPGYRSLGNIIMVCRKGEWVALNPLRKCQKRP

[0101] SEQ ID NO: 15: Human CFH CCP1 (CCP1 contains V62I) EDCNELPPRRNTEILTGSWSDQTYPEGTQAIYKCRPGYRSLGNIIMVCRKGEWVALNPLRKCQK

[0102] When referring to an amino acid position of CFH, the amino acid sequence position corresponds to the amino acid position in SEQ ID NO:3.

[0103] C. Other units Optionally, the hybrid proteins of the invention may further comprise a tag, preferably of about 2-10 amino acids, added to the amino or carboxy terminus, e.g., the carboxy terminus, of the hybrid protein. Typically, such addition is made to stabilize the protein or to facilitate secretory expression or purification of the hybrid protein. Such tags are known in the art. Representative examples of such tags include sequences encoding a string of histidine residues (e.g., 2-10 histidines, e.g., 2, 3, 4, 5, 6, or 7 histidines), the epitope tag FLAG, herpes simplex glycoprotein D, β-galactosidase, maltose binding protein, or glutathione S-transferase.

[0104] In some embodiments, the tag is a histidine residue and can be added to the amino or carboxy terminus of the hybrid protein, for example the carboxy terminus.

[0105] In some embodiments, the tag is a 6xHis tag of GHHHHHH (SEQ ID NO: 16).

[0106] Optionally, the hybrid protein of the invention may also include a signal peptide, such as MGWSCIILFLVATATGVHS (SEQ ID NO: 17).

[0107] The present invention also encompasses hybrid proteins in which one or more amino acids have been modified by post-translational processes or synthetic methods. Examples of such modifications include, but are not limited to, glycosylation, iodination, myristoylation, and PEGylation.

[0108] D. Examples of Hybrid Proteins In some embodiments, the hybrid proteins of the invention comprise at least one functional unit derived from CFH and at least one functional unit derived from DAF.

[0109] In some embodiments, a hybrid protein of the invention comprises CCP1 of CFH and CCP3 and CCP4 of DAF. In some embodiments, a hybrid protein of the invention consists of CCP1 of CFH and CCP3 and CCP4 of DAF. In some embodiments, a hybrid protein of the invention comprises or consists of CCP1 of CFH and CCP3-4 of DAF.

[0110] In some embodiments, 1 to 2 amino acids are added to the C-terminus of CCP1 of CFH (e.g., 1 to 2 amino acids at the N-terminus of CCP2). In some embodiments, 1 to 2 amino acids are deleted from the N-terminus of CCP3 or CCP3-4 of DAF.

[0111] In some embodiments, a hybrid protein of the invention comprises CCP1 of CFH and CCP3-4 of DAF, with 1-2 N-terminal amino acids of CCP2 added to the C-terminus of CCP1 and correspondingly 1-2 amino acids deleted from the N-terminus of CCP3 of DAF. In some specific embodiments, a hybrid protein of the invention comprises CCP1 of CFH and CCP3-4 of DAF, with N-terminal amino acids RP of CCP2 added to the C-terminus of CCP1 and correspondingly amino acids KS deleted from the N-terminus of CCP3 of DAF.

[0112] In some embodiments, the CCP1 of the CFH of the present invention may comprise a V62I mutation.

[0113] In some embodiments, the hybrid protein of the invention further comprises a signal peptide at the N-terminus, such as the amino acid sequence set forth in SEQ ID NO: 13, and / or a tag at the C-terminus, such as a histidine tag, such as a 6xHis-tag, such as GHHHHHH.

[0114] In some embodiments, CCP3 of DAF comprises or consists of amino acids 161-222 of the DAF protein, and / or CCP4 comprises or consists of amino acids 223-285 of the DAF protein, wherein the numbering of the amino acid positions corresponds to the amino acid positions set forth in SEQ ID NO:1. In some embodiments, CCP3 of DAF is deleted by two amino acids from its N-terminus, e.g., comprises or consists of amino acids 163-222 of the DAF protein, wherein the numbering of the amino acid positions corresponds to the amino acid positions set forth in SEQ ID NO:1. In some embodiments, CCP3-4 of DAF comprises or consists of amino acids 161-285 of the DAF protein, wherein the numbering of the amino acid positions corresponds to the amino acid positions set forth in SEQ ID NO:1. In some embodiments, CCP3-4 of DAF is deleted by two amino acids at its N-terminus, e.g., comprises or consists of amino acids 163-285 of the DAF protein, wherein the numbering of the amino acid positions corresponds to the amino acid positions set forth in SEQ ID NO:1.

[0115] In some embodiments, CCP1 of CFH comprises or consists of amino acids 19-82 of the CFH protein, where the amino acid position numbers correspond to the amino acid positions set forth in SEQ ID NO: 3. In some embodiments, CCP1 of CFH comprises or consists of amino acids 19-84 of the CFH protein, where the amino acid position numbers correspond to the amino acid positions set forth in SEQ ID NO: 3.

[0116] In some embodiments, CCP3-4 of DAF comprises or consists of amino acids 161-285 of the DAF protein, where the numbering of the amino acid positions corresponds to the amino acid positions set forth in SEQ ID NO: 1. CCP1 of CFH comprises or consists of amino acids 19-82 of the CFH protein, where the numbering of the amino acid positions corresponds to the amino acid positions set forth in SEQ ID NO: 3.

[0117] In some embodiments, CCP3-4 of DAF comprises or consists of amino acids 163-285 of the DAF protein, where the amino acid position numbers correspond to the amino acid positions set forth in SEQ ID NO: 1. CCP1 of CFH comprises or consists of amino acids 19-84 of the CFH protein, where the amino acid position numbers correspond to the amino acid positions set forth in SEQ ID NO: 3.

[0118] In some specific embodiments, the DAF is a human DAF protein, such as a native human DAF protein. In some specific embodiments, the DAF protein comprises or consists of: (i) the amino acid sequence set forth in SEQ ID NO:1; (ii) an amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO:2; (iii) an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of (i) or (ii); or It consists of an amino acid sequence set forth in any one of (i) to (iii).

[0119] In some specific embodiments, the CFH is a human CFH protein, such as a native human CFH protein. (i) an amino acid sequence set forth in SEQ ID NO: 3 or 5; (ii) an amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 4 or 6; (iii) an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of (i) or (ii); or It consists of an amino acid sequence set forth in any one of (i) to (iii).

[0120] In some embodiments, the hybrid protein of the present invention comprises or consists of an amino acid sequence of any one of SEQ ID NOs: 18-21, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity to these amino acid sequences.

[0121] > Human CFH CCP1 or SCR1-Human DAF / CD55 CCP3-4 or SCR3-4 (bold): EDCNELPPRRNTEILTGSWSDQTYPEGTQAIYKCRPGYRSLGNVIMVCRKGEWVALNPLRKCQKRP CPNPGEIRNGQIDVPGGILFGATISFSCNTGYKLFGSTSSFCLISGSSVQWSDPLPECREIYCPAPPQIDNGIIQGERDHYGYRQSVTYACNKGFTMIGEHSIYCTVNNDEGEWSGPPPECRG (SEQ ID NO: 18)

[0122] EDCNELPPRRNTEILTGSWSDQTYPEGTQAIYKCRPGYRSLGNVIMVCRKGEWVALNPLRKCQK KSCPNPGEIRNGQIDVPGGILFGATISFSCNTGYKLFGSTSSFCLISGSSVQWSDPLPECREIYCPAPPQIDNGIIQGERDHYGYRQSVTYACNKGFTMIGEHSIYCTVNNDEGEWSGPPPECRG (SEQ ID NO: 19)

[0123] >Human CFH CCP1 or SCR1-Human DAF / CD55 CCP3-4 or SCR3-4 (bold) contains the V62I mutation: EDCNELPPRRNTEILTGSWSDQTYPEGTQAIYKCRPGYRSLGNIIMVCRKGEWVALNPLRKCQK KSCPNPGEIRNGQIDVPGGILFGATISFSCNTGYKLFGSTSSFCLISGSSVQWSDPLPECREIYCPAPPQIDNGIIQGERDHYGYRQSVTYACNKGFTMIGEHSIYCTVNNDEGEWSGPPPECRG (SEQ ID NO: 20)

[0124] EDCNELPPRRNTEILTGSWSDQTYPEGTQAIYKCRPGYRSLGNIIMVCRKGEWVALNPLRKCQKRPCPNPGEIRNGQIDVPGGILFGATISFSCNTGYKLFGSTSSFCLISGSSVQWSDPLPECREIYCPAPPQIDNGIIQGERDHYGYRQSVTYACNKGFTMIGEHSIYCTVNNDEGEWSGPPPECRG (SEQ ID NO: 21)

[0125] In some embodiments, the hybrid protein of the present invention comprises, or consists of, an amino acid sequence according to any one of SEQ ID NOs: 22-25, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity thereto.

[0126] >Human CFH CCP1 or SCR1-Human DAF / CD55 CCP3-4 or SCR3-4 (bold)-6×His (italics): EDCNELPPRRNTEILTGSWSDQTYPEGTQAIYKCRPGYRSLGNVIMVCRKGEWVALNPLRKCQKRP CPNPGEIRNGQIDVPGGILFGATISFSCNTGYKLFGSTSSFCLISGSSVQWSDPLPECREIYCPAPPQIDNGIIQGERDHYGYRQSVTYACNKGFTMIGEHSIYCTVNNDEGEWSGPPPECRGGHHHHHH (SEQ ID NO: 22)

[0127] EDCNELPPRRNTEILTGSWSDQTYPEGTQAIYKCRPGYRSLGNVIMVCRKGEWVALNPLRKCQK KSCPNPGEIRNGQIDVPGGILFGATISFSCNTGYKLFGSTSSFCLISGSSVQWSDPLPECREIYCPAPPQIDNGIIQGERDHYGYRQSVTYACNKGFTMIGEHSIYCTVNNDEGEWSGPPPECRGGHHHHHH (SEQ ID NO: 23)

[0128] > Human CFH CCP1 or SCR1-Human DAF / CD55 CCP3-4 or SCR3-4 (bold)-6×His (italics): containing the V62I mutation: EDCNELPPRRNTEILTGSWSDQTYPEGTQAIYKCRPGYRSLGNIIMVCRKGEWVALNPLRKCQKKSCPNPGEIRNGQIDVPGGILFGATISFSCNTGYKLFGSTSSFCLISGSSVQWSDPLPECREIYCPAPPQIDNGIIQGERDHYGYRQSVTYACNKGFTMIGEHSIYCTVNNDEGEWSGPPPECRGGHHHHHH (SEQ ID NO: 24)

[0129] EDCNELPPRRNTEILTGSWSDQTYPEGTQAIYKCRPGYRSLGNIIMVCRKGEWVALNPLRKCQKRP CPNPGEIRNGQIDVPGGILFGATISFSCNTGYKLFGSTSSFCLISGSSVQWSDPLPECREIYCPAPPQIDNGIIQGERDHYGYRQSVTYACNKGFTMIGEHSIYCTVNNDEGEWSGPPPECRGGHHHHHH (SEQ ID NO: 25)

[0130] In some embodiments, the hybrid protein of the present invention comprises or consists of an amino acid sequence according to any one of SEQ ID NOs: 26-29, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity thereto.

[0131] >Signal peptide-human CFH CCP1 or SCR1-human DAF / CD55 CCP3-4 or SCR3-4 (bold)-6×His (italics): MGWSCIILFLVATATGVHS EDCNELPPRRNTEILTGSWSDQTYPEGTQAIYKCRPGYRSLGNVIMVCRKGEWVALNPLRKCQKRP CPNPGEIRNGQIDVPGGILFGATISFSCNTGYKLFGSTSSFCLISGSSVQWSDPLPECREIYCPAPPQIDNGIIQGERDHYGYRQSVTYACNKGFTMIGEHSIYCTVNNDEGEWSGPPPECRGGHHHHHH (SEQ ID NO: 26)

[0132] MGWSCIILFLVATATGVHS EDCNELPPRRNTEILTGSWSDQTYPEGTQAIYKCRPGYRSLGNVIMVCRKGEWVALNPLRKCQKKSCPNPGEIRNGQIDVPGGILFGATISFSCNTGYKLFGSTSSFCLISGSSVQWSDPLPECREIYCPAPPQIDNGIIQGERDHYGYRQSVTYACNKGFTMIGEHSIYCTVNNDEGEWSGPPPECRGGHHHHHH (SEQ ID NO: 27)

[0133] MG > Signal peptide-human CFH CCP1 or SCR1-human DAF / CD55 CCP3-4 or SCR3-4 (bold)-6×His (italics): containing the V62I mutation: MGWSCIILFLVATATGVHS EDCNELPPRRNTEILTGSWSDQTYPEGTQAIYKCRPGYRSLGNIIMVCRKGEWVALNPLRKCQK KSCPNPGEIRNGQIDVPGGILFGATISFSCNTGYKLFGSTSSFCLISGSSVQWSDPLPECREIYCPAPPQIDNGIIQGERDHYGYRQSVTYACNKGFTMIGEHSIYCTVNNDEGEWSGPPPECRGGHHHHHH (SEQ ID NO: 28)

[0134] MGWSCIILFLVATATGVHS EDCNELPPRRNTEILTGSWSDQTYPEGTQAIYKCRPGYRSLGNIIMVCRKGEWVALNPLRKCQKRP CPNPGEIRNGQIDVPGGILFGATISFSCNTGYKLFGSTSSFCLISGSSVQWSDPLPECREIYCPAPPQIDNGIIQGERDHYGYRQSVTYACNKGFTMIGEHSIYCTVNNDEGEWSGPPPECRGGHHHHHH (SEQ ID NO: 29)

[0135] In some embodiments, the hybrid protein of the present invention comprises, or consists of, an amino acid sequence according to any one of SEQ ID NOs: 30-33, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity thereto.

[0136] >Signal peptide-human CFH CCP1 or SCR1-human DAF / CD55 CCP3-4 or SCR3-4 (bold): MGWSCIILFLVATATGVHS EDCNELPPRRNTEILTGSWSDQTYPEGTQAIYKCRPGYRSLGNVIMVCRKGEWVALNPLRKCQKRP CPNPGEIRNGQIDVPGGILFGATISFSCNTGYKLFGSTSSFCLISGSSVQWSDPLPECREIYCPAPPQIDNGIIQGERDHYGYRQSVTYACNKGFTMIGEHSIYCTVNNDEGEWSGPPPECRG (SEQ ID NO: 30)

[0137] MGWSCIILFLVATATGVHS EDCNELPPRRNTEILTGSWSDQTYPEGTQAIYKCRPGYRSLGNVIMVCRKGEWVALNPLRKCQK KSCPNPGEIRNGQIDVPGGILFGATISFSCNTGYKLFGSTSSFCLISGSSVQWSDPLPECREIYCPAPPQIDNGIIQGERDHYGYRQSVTYACNKGFTMIGEHSIYCTVNNDEGEWSGPPPECRG (SEQ ID NO: 31)

[0138] >Signal peptide-human CFH CCP1 or SCR1-human DAF / CD55 CCP3-4 or SCR3-4 (bold): Including the V62I mutation: MGWSCIILFLVATATGVHS EDCNELPPRRNTEILTGSWSDQTYPEGTQAIYKCRPGYRSLGNIIMVCRKGEWVALNPLRKCQK KSCPNPGEIRNGQIDVPGGILFGATISFSCNTGYKLFGSTSSFCLISGSSVQWSDPLPECREIYCPAPPQIDNGIIQGERDHYGYRQSVTYACNKGFTMIGEHSIYCTVNNDEGEWSGPPPECRG (SEQ ID NO: 32)

[0139] MGWSCIILFLVATATGVHS EDCNELPPRRNTEILTGSWSDQTYPEGTQAIYKCRPGYRSLGNIIMVCRKGEWVALNPLRKCQKRP CPNPGEIRNGQIDVPGGILFGATISFSCNTGYKLFGSTSSFCLISGSSVQWSDPLPECREIYCPAPPQIDNGIIQGERDHYGYRQSVTYACNKGFTMIGEHSIYCTVNNDEGEWSGPPPECRG (SEQ ID NO: 33)

[0140] III. Polynucleotides, Vectors, and Hosts The present invention provides a nucleic acid encoding any of the above hybrid proteins of the invention. Also provided is a vector comprising the nucleic acid. In one embodiment, the vector is an expression vector (e.g., a pcDNA vector such as pcDNA3.1). Also provided is a host cell comprising the nucleic acid or vector. In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from a yeast cell or a mammalian cell (e.g., a CHO cell or a 293 cell such as an Expi293 cell). In another embodiment, the host cell is a prokaryotic cell.

[0141] In one embodiment, the invention provides a nucleic acid encoding any of the above hybrid proteins, the polypeptide encoded by which, when expressed from a suitable expression vector, is capable of having DAF and / or CFH function, e.g., dissociation of C3 convertase and / or C5 convertase, decay accelerating activity for classical pathway C3 convertase and / or alternative pathway C3 convertase, dissociation of alternative pathway C3bBb or binding to C3b, and complement inhibitory activity (including activity to inhibit the alternative pathway (AP), classical pathway (CP), and / or C3b deposition).

[0142] To facilitate production and purification, the hybrid protein can be fused at its N-terminus to a secretion signal peptide and / or a tag peptide, such as a hexahistidine tag or a biotin tag.

[0143] As will be appreciated by those skilled in the art, each hybrid protein can be encoded by a variety of nucleic acid sequences due to codon degeneracy.

[0144] In some embodiments, the nucleic acid of the present invention comprises a nucleic acid encoding an amino acid sequence selected from any one of SEQ ID NOs: 18-33, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to an amino acid sequence selected from any one of SEQ ID NOs: 18-33.

[0145] Nucleic acid sequences encoding the molecules of the invention can be produced by methods well known in the art, such as de novo solid phase DNA synthesis or PCR amplification.

[0146] In one embodiment, one or more vectors are provided that comprise the nucleic acid of the present invention.In one embodiment, the vector is an expression vector, such as a prokaryotic expression vector or a eukaryotic expression vector.Vector includes, but is not limited to, virus, plasmid, cosmid, lambda phage, and yeast artificial chromosome (YAC).In a preferred embodiment, the expression vector is pcDNA, for example pcDNA3.1.

[0147] In one embodiment, a host cell is provided that comprises one or more polynucleotides of the present invention. In an embodiment, a host cell is provided that comprises an expression vector of the present invention. As used herein, the term "host cell" refers to any type of cell system that can be engineered to produce a protein of the present invention. Host cells suitable for replicating and supporting the expression of a protein of the present invention are well known in the art. Such cells can be transfected or transduced with a particular expression vector as desired, and the cells containing the vector can be cultured in large quantities to inoculate large-scale fermenters to obtain sufficient amounts of the protein of the present invention for clinical use.

[0148] Suitable host cells include prokaryotic microorganisms such as E. coli, eukaryotic microorganisms such as filamentous fungi and yeast, or various eukaryotic cells such as Chinese hamster ovary cells (CHO) and insect cells. Mammalian cell lines suitable for suspension culture can also be used. Examples of useful mammalian host cell lines include SV40 transformed monkey kidney CV1 line (COS-7). Human embryonic kidney lines (HEK293 or 293F or 293FT cells), baby hamster kidney cells (BHK), monkey kidney cells (CV1), Vero cells (VERO-76), human cervical carcinoma cells (HELA), canine kidney cells (MDCK), buffalo rat hepatocytes (BRL 3A), human lung cells (W138), human hepatocytes (Hep G2), myeloma cell lines such as CHO cells, NSO cells, YO, NS0, P3X63, Sp2 / 0, etc. Mammalian host cell lines suitable for protein production are known in the art. In a preferred embodiment, the host cell is a CHO cell, a HEK293 cell or an Expi293 cell.

[0149] IV. Production and Purification of the Molecules of the Invention In yet another aspect, the present invention provides a method for producing a hybrid protein of the present invention, the method comprising culturing a host cell comprising a nucleic acid molecule encoding said protein under conditions suitable for expression of said hybrid protein, and optionally isolating said protein from said host cell or host cell culture and / or purifying said protein.

[0150] For recombinant production, the polynucleotide encoding the protein of the present invention can be inserted into a vector for further cloning and / or expression in a host cell. Methods well known to those skilled in the art can be used to construct an expression vector. Once an expression vector containing a polynucleotide of the present invention is prepared for expression, the expression vector can be transfected or introduced into a suitable host cell. Various techniques can be used to achieve this goal, including, for example, protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, biolistics, liposome-based transfection, or other conventional techniques.

[0151] Proteins prepared as described herein may be purified by known prior art techniques such as nickel columns, high performance liquid chromatography, affinity chromatography, ion exchange chromatography, gel electrophoresis, etc. The actual conditions used to purify a particular protein will also depend on factors such as net charge, hydrophobicity, hydrophilicity, etc., and will be apparent to one of skill in the art.

[0152] The purity and quantity of the proteins of the present invention can be determined by any of a variety of well-known analytical methods.

[0153] V. Assay Methods The hybrid proteins provided herein may be identified, screened, or characterized for their physical / chemical properties and / or biological activity by a variety of assays known in the art.

[0154] The hemolytic inhibitory effect of the hybrid proteins of the invention can be measured by methods known in the art, such as in vitro assays and / or in vivo animal experiments. For example, hemolytic assays such as those described in Examples 4.1 and 4.2 can be used to test the hemolytic inhibitory effect of molecules on the classical and / or alternative pathways of complement immunity.

[0155] The inhibitory activity of the hybrid proteins of the invention against complement C3b deposition can be measured by methods known in the art, such as in vitro assays and / or in vivo animal assays. For example, a hemolytic assay, such as the method described in Example 4.3, can be used to test the inhibitory effect of molecules against C3b deposition on the surface of red blood cells.

[0156] VI. Pharmaceutical Compositions, Pharmaceutical Combinations, and Kits In one aspect, the invention provides a composition, such as a pharmaceutical composition, medicament, or formulation, comprising the hybrid protein of the invention or a nucleic acid molecule encoding same.

[0157] In one embodiment, the composition further comprises a pharmaceutical excipient, such as a pharmaceutical carrier, a pharmaceutical excipient including a buffering agent, as known in the art.

[0158] As used herein, the term "pharmaceutical carrier" includes any physiologically compatible solvent, dispersion medium, isotonicity agent, absorption delaying agent, etc. For the use of pharmaceutical excipients and their applications, see also "Handbook of Pharmaceutical Excipients", 8th Edition, RC Rowe, PJ Eskey and SCOwen, Pharmaceutical Press, London, Chicago.

[0159] The compositions or medicaments or formulations of the present invention can be in various forms. These forms include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injectable solutions), powders or suspensions, liposomal formulations, and suppositories. The preferred form depends on the intended mode of administration and therapeutic application.

[0160] In addition to the hybrid proteins of the invention or nucleic acids encoding same, the compositions or medicaments or formulations of the invention can also include additional therapeutic agents necessary for the particular indication being treated, preferably therapeutic agents that do not adversely affect the activity of each other. Thus, in one embodiment, a composition or formulation or medicament, e.g., a pharmaceutical composition, comprises a combination of one or more molecules of the invention and one or more additional therapeutic agents.

[0161] The composition or medicament or formulation of the present invention can be in various forms. These forms include, for example, liquids (e.g., injections), powders or suspensions, liposome preparations, and suppositories, as well as liquids, semisolids, and solids. The composition or medicament or formulation of the present invention is suitable for administration by intravenous bolus injection, intravenous infusion, intraperitoneal, intradermal, intramuscular, subcutaneous, and nasal routes (e.g., injection or infusion). The preferred form depends on the intended mode of administration and therapeutic application.

[0162] The invention also provides pharmaceutical combinations or pharmaceutical combination products comprising a hybrid protein of the invention or a nucleic acid encoding same and one or more other therapeutic agents.

[0163] The present invention also provides a kit of parts comprising a combination pharmaceutical agent, for example the kit of parts comprising in the same package: a first container containing a pharmaceutical composition comprising a hybrid protein of the invention or a nucleic acid encoding it; and -Optionally, a second container containing a pharmaceutical composition comprising one or more additional therapeutic agents (in some embodiments, two or more additional therapeutic agents are in the same container or in separate containers).

[0164] VII. Uses and Methods In one aspect of the present invention, there is provided a method for preventing or treating a complement system-related disease or disorder in a subject, the method comprising administering to the subject an effective amount of a hybrid protein of the present invention or a nucleic acid encoding the same, or a composition, medicament or formulation comprising the protein.

[0165] In some embodiments, the complement system-related disorder results from aberrant activation of the complement system or dysregulation of the complement system, which may result from, for example, microbial infection or an increase in autoimmune antibodies, or from reduced, deleted, disabled, impeded, or blocked function of a complement regulatory protein.

[0166] In some embodiments, the treatment of a disease would benefit from inhibiting the activity of the complement system.

[0167] In another aspect, the invention provides the use of a molecule of the invention, or a composition comprising same, in the manufacture or preparation of a medicament for a use as described herein, such as for the prevention or treatment of a complement system related disease or disorder as described herein.

[0168] In some embodiments, a complement system-related disease or disorder can be a disease requiring inhibition of hemolysis, for example a disease requiring inhibition of hemolysis of the classical pathway of complement immunity and / or the alternative pathway of complement immunity.

[0169] In some embodiments, the complement system-associated disease or disorder is a disease in which inhibition of C3b deposition activity is desired, such as dense deposit disease (DDD).

[0170] In some embodiments, the hybrid proteins of the invention or nucleic acids encoding same, or compositions or medicaments or formulations comprising such proteins or nucleic acids, delay the onset of a disorder and / or symptoms associated with the disorder.

[0171] In some embodiments, the hybrid proteins of the invention or nucleic acids encoding same, or compositions or medicaments or formulations comprising the proteins or nucleic acids, may also be administered in combination with one or more other therapies, e.g., therapeutic modalities and / or other therapeutic agents, for uses as described herein, e.g., for the prevention and / or treatment of associated diseases or disorders as described herein.

[0172] The route of administration of the hybrid proteins of the invention or the nucleic acids encoding them, or compositions, pharmaceuticals or formulations containing said proteins or nucleic acids, depends on known methods, such as injection or infusion.

[0173] The invention further encompasses the use of a hybrid protein of the invention or a nucleic acid encoding same, or a composition or formulation comprising the protein or nucleic acid, in the manufacture of a medicament for uses as described herein, such as for the prevention and / or treatment of an associated disease or disorder as described herein. EXAMPLES

[0174] Example 1. Molecular structure To construct protein expression vectors for the hybrid molecules of the present invention and the complement regulatory molecules of the prior art, DNA encoding each protein was subcloned into pcDNA3.1 expression vector (purchased from Biofeng) using synthesized gene DNA (GENEWIZ Inc., Suzhou / Sangon Biotech (Shanghai) Co., Ltd.) as a template by using conventional molecular cloning technology methods. The plasmid was confirmed by sequencing and then used for transient protein expression. Each encoded protein was added with a signal peptide MGWSCIILFLVATATGVHS at the N-terminus and a GHHHHHH6×His tag at the C-terminus to facilitate subsequent secretory expression in mammalian cells and nickel column purification.

[0175] The amino acid sequences of each protein are as follows: >CR13m (human CR1 CCP1-3 or SCR1-3, containing N29K, S37Y, G79D and D109N mutations, sequence from patent US9988611_B2): QCNAPEWLPFARPTNLTDEFEFPIGTYLKYECRPGYYGRPFSIICLKNSVWTGAKDRCRRKSCRNPPDPVNGMVHVIKDIQFGSQIKYSCTKGYRLIGSSSATCIISGNTVIWDNETPICDRIPCGLPPTITNGDFISTNRENFHYGSVVTYRCNPGSGGRKVFELVGEPSIYCTSNDDQVGIWSGPAPQCIGHHHHHH (SEQ ID NO: 34)

[0176] >D24 (human DAF / CD55 CCP2-4 or SCR2-4, the sequence can be referenced from Hui-fen Zhang et al., THE JOURNAL OF BIOLOGICAL CHEMISTRY, Vol. 276, No. 29, July 20, pp. 27290-27295, 2001): SCEVPTRLNSASLKQPYITQNYFPVGTVVEYECRPGYRREPSLSPKLTCLQNLKWSTAVEFCKKKSCPNPGEIRNGQIDVPGGILFGATISFSCNTGYKLFGSTSSFCLISGSSVQWSDPLPECREIYCPAPPQIDNGIIQGERDHYGYRQSVTYACNKGFTMIGEHSIYCTVNNDEGEWSGPPPECRGGHHHHHH (SEQ ID NO: 35)

[0177] >F15 (human CFH CCP1-5 or SCR1-5 containing V62I, sequences can be referenced from Masha Fridkis-Hareli et al., Blood. 2011 Oct 27; 118(17):4705-4713 and Agustin Tortajada et al., Hum Mol Genet. 2009 September 15; 18(18):3452-3461): EDCNELPPRRNTEILTGSWSDQTYPEGTQAIYKCRPGYRSLGNIIMVCRKGEWVALNPLRKCQK RPCGHPGDTPFGTFTLTGGNVFEYGVKAVYTCNEGYQLLGEINYRECDTDGWTNDIPICEVVKCLPVTAPENGKIVSSAMEPDREYHFGQAVRFVCNSGYKIEGDEEMHCSDGGFWSKEKPKCVEISCKSPDVINGSPISQKIIYKENERFQYKCNMGYEYSERGDAVCTESGWRPLPSCEEKSCDNPYIPNGDYSPLRIKHRTGDEITYQCRNGFYPATRGNTAKCTSTGWIPAPRCTLKPGHHHHHH (SEQ ID NO: 36)

[0178] >F1D34-1 (hybrid molecule 1 of the invention, human CFH CCP1 or SCR1-human DAF / CD55 CCP3-4 or SCR3-4): SEQ ID NO: 22, The sequence with the signal peptide linked to the N-terminus is SEQ ID NO:26.

[0179] >F1D34-2i (hybrid molecule 2 of the present invention, human CFH CCP1 or SCR1-human DAF / CD55 CCP3-4 or SCR3-4, containing V62I mutation): SEQ ID NO: 24, the amino acid sequence with a signal peptide sequence linked to the N-terminus is SEQ ID NO: 28.

[0180] >CR15D34 (hybrid molecule 3 of the invention, human CR1 CCP15 or SCR15-human DAF / CD55 CCP3-4 or SCR3-4): GHCQAPDHFLFAKLKTQTNASDFPIGTSLKYECRPEYYGRPFSITCLDNLVWSSPKDVCKR KSCPNPGEIRNGQIDVPGGILFGATISFSCNTGYKLFGSTSSFCLISGSSVQWSDPLPECREIYCPAPPQIDNGIIQGERDHYGYRQSVTYACNKGFTMIGEHSIYCTVNNDEGEWSGPPPECRGGHHHHHH (SEQ ID NO: 37)

[0181] >FH13 (human CFH CCP1-3 or SCR1-3, containing the V62I mutation and two additional amino acids at the C-terminus to attach a His purification tag): EDCNELPPRRNTEILTGSWSDQTYPEGTQAIYKCRPGYRSLGNIIMVCRKGEWVALNPLRKCQKRPCGHPGDTPFGTFTLTGGNVFEYGVKAVYTCNEGYQLLGEINYRECDTDGWTNDIPICEVVKCLPVTAPENGKIVSSAMEPDREYHFGQAVRFVCNSGYKIEGDEEMHCSDDGFWSKEKPKCVEISGHHHHHH (SEQ ID NO: 38)

[0182] >D14 (human DAF / CD55 CCP1-4 or SCR1-4, no leader sequence, with two extra amino acids at the C-terminus to attach a His purification tag): DCGLPPDVPNAQPALEGRTSFPEDTVITYKCEESFVKIPGEKDSVICLKGSQWSDIEEFCNRSCEVPTRLNSASLKQPYITQNYFPVGTVVEYECRPGYRREPSLSPKLTCLQNLKWSTAVEFCKKKSCPNPGEIRNGQIDVPGGILFGATISFSCNTGYKLFGSTSSFCLISGSSVQWSDPLPECREIYCPAPPQIDNGIIQGERDHYGYRQSVTYACNKGFTMIGEHSIYCTVNNDEGEWSGPPPECRGKSGHHHHHH (SEQ ID NO: 39)

[0183] Example 2. Protein Expression After amplifying the plasmid in DH5 alpha strain (Yeasen Biotechnology), the plasmid was prepared using NucleoBond Xtra Midi Plus (MACHEREY-NAGEL, see product manual for details). For transient expression of the protein, Expi293 cells (purchased from Thermo Fisher) were transfected with the plasmid prepared using PEI (Polyscience) (for transfection method, see Jager, V., et al., BMC Biotechnol 13, 52, 2013). The collected supernatant was purified by affinity chromatography using a nickel ion prepacked column (GE Life Sciences, HisTrap HP, see product manual for details), dialyzed against PBS for liquid exchange, and the protein sample for liquid exchange was sterilized by filtering through a 0.2 μm filter, and the protein was quantified according to the theoretical extinction coefficient using the NanoDrop (Thermofisher) A280 method.

[0184] Example 3. Detection of protein purity (SDS-PAGE) 2.5 μg of protein was taken and added proportionally to loading buffer containing reducing agent (Sangon Biotech) and loading buffer without reducing agent (Sangon Biotech). Samples using loading buffer containing reducing agent were boiled at 95°C for 5 minutes to completely denature the protein. Samples using loading buffer without reducing agent were not heat-treated. Electrophoresis was performed on 10% Precast-GLgel Tris-Glycine precast gels (Sangon Biotech) using a Mini-PROTEAN® Tetra electrophoresis system (BioRad). After electrophoresis, the gels were stained with 0.1% Coomassie brilliant blue staining solution and destained with ethanol-glacial acetic acid solution.

[0185] As a result of SDS-PAGE electrophoresis, except for the band of D14 protein, which was slightly diffuse, the bands of other proteins were relatively clear, indicating that the purity of the protein was good under both reducing and non-reducing conditions (Figure 2). Furthermore, a certain deviation was observed between the electrophoretic molecular weight and the theoretical molecular weight due to the reduction, denaturation, and modification state of the protein and the differences in electrophoretic conditions.

[0186] Example 4. Detection of complement inhibitory activity 4.1 Detection of inhibitory activity against the classical complement pathway (CP) In this experiment, the degree of inhibition of hemolysin-mediated hemolysis of sheep red blood cells by normal human serum complement activity was measured by testing different concentrations of the proteins to be tested, and the inhibitory activity of each protein against complement CP was analyzed and compared.

[0187] The experimental process can be briefly described as follows: (1) Gradient dilution of proteins: GVB++ buffer (Ca 2+ and Mg 2+ After preparing a 12,000 nM solution in gelatin flora buffer (containing TIANDZ, Cat. No.: 25-02080), a 4-fold gradient dilution was performed, and a total of 7 gradients were performed. (2) Serum dilution Normal human serum NHS (Shanghai Schbio Co., Ltd.) was removed from a -70°C refrigerator, thawed naturally at 4°C, and then diluted to 4% with GVB++ buffer. (3) Activation of sheep red blood cells: Sheep red blood cells (Nanjing SenBeiJia Biological Technology Co., Ltd.) were washed with GVB++ buffer until the supernatant was clear, then resuspended in GVB++ buffer, and hemolysin (BM351Y, Beijing Bersee Technology Co. Ltd.) was added to the red blood cell suspension at a ratio of 1:400, followed by incubation at 4°C for 15 minutes to activate the sheep red blood cells. The activated sheep red blood cells were washed twice with GVB++ and resuspended in GVB++ buffer. (4) Incubation: 25 μL each of diluted protein and serum were mixed in a 96-well plate, and then 50 μL of the red blood cell suspension obtained in step (3) was added to make the initial final protein concentration 3000 nM and the final serum concentration 1%. The mixture was mixed thoroughly and placed in a 37°C thermostatic incubator for 1 h, and a negative control (containing only serum) and a positive control (containing only serum and red blood cells) were added. (5) Termination: After incubation, 100 μL of 20 mM EDTA-GVB buffer (GVBE) was added to each well to terminate the reaction. (6) Reading: The 96-well plate was centrifuged at 3000 rpm for 5 minutes. 100 μL of the supernatant was added to a new 96-well flat-bottom plate. The absorbance at OD405 nm was measured with a multi-function plate reader and the data was stored. (7) Data processing: The obtained OD405nm readings were entered into the following formula to calculate the inhibition rate of red blood cell hemolysis. Hemolysis inhibition rate (%) = (positive control measurement value - experimental group measurement value) / (positive control measurement value - negative control measurement value) x 100

[0188] Next, the IC50 value of the protein was calculated by plotting a four-parameter fitting curve with the final protein concentration on the horizontal axis and the hemolysis inhibition rate on the vertical axis as a model. The results are shown in Figure 3.

[0189] The experimental test of CP hemolysis inhibitory activity showed that the CFH and DAF hybrid proteins F1D34-1 and F1D34-2i both had strong CP inhibitory activity, which was equivalent to the CR1 activity enhancing fragment CR13m and the DAF fragment D24, significantly superior to the CFH fragment FH15 and the DAF fragment D14, and far superior to the CR1 and DAF hybrid protein CR15D34. Only weak CP inhibitory activity was detected in CR15D34, while the CFH fragment FH13 had almost no activity (as shown in Figure 3), indicating that the hybrid protein obtained by domain hybridization has excellent CP inhibitory activity.

[0190] 4.2 Detection of inhibitory activity against the alternative complement pathway (AP) In this experiment, the degree of inhibition of rabbit erythrocyte hemolysis by normal human serum complement activity was measured by testing test proteins at different concentrations, and the inhibitory activity of each protein against complement AP was analyzed and compared.

[0191] The experimental process can be briefly described as follows: (1) Gradient dilution of protein: Protein was diluted in GVBMG buffer (Mg 2+ The solution was diluted 4-fold in a gelatin flora buffer containing EGTA (Cat. No. 25-02090, TIANDZ) to a concentration of 28,000 nM, and then diluted 4-fold in a gradient to perform a total of seven gradients. (2) Serum dilution Normal human serum NHS (Shanghai Schbio Co., Ltd.) was removed from a -70°C refrigerator, thawed naturally at 4°C, and then diluted to 60% with GVBMG. (3) Preparation of rabbit red blood cells: Rabbit red blood cells (Nanjing SenBeiJia Biological Technology Co., Ltd.) were washed with GVBMG until the supernatant was clear, and then resuspended in GVBMG. (4) Incubation: 25 μL each of the diluted protein and serum were mixed in a 96-well plate, and then 50 μL of the red blood cell suspension obtained in step (3) was added. The initial final protein concentration was 7000 nM and the final serum concentration was 15%. The mixture was thoroughly mixed and placed in a 37°C constant temperature incubator for 1 hour, and a negative control (containing only serum) and a positive control (containing only serum and red blood cells) were added. (5) Termination of reaction: After incubation, 100 μL of 10 mM EDTA-GVB buffer (GVBE) was added to each well to terminate the reaction. (6) Reading: The 96-well plate was centrifuged at 3000 rpm for 5 minutes. 100 μL of the supernatant was added to a new 96-well flat-bottom plate. The absorbance at OD405 nm was measured with a multi-function plate reader, and the data was stored. (7) Data processing: The obtained OD405nm measurement value was substituted into the following formula to calculate the inhibition rate of red blood cell hemolysis. Hemolysis inhibition rate (%) = (positive control measurement value - experimental group measurement value) / (positive control measurement value - negative control measurement value) x 100

[0192] Next, the IC50 value of the antibody was calculated by plotting a fitted curve consisting of four parameters with the final protein concentration on the horizontal axis and the hemolysis inhibition rate on the vertical axis. The results are shown in Figure 4.

[0193] Experimental tests of AP hemolysis inhibitory activity showed that the CFH and DAF hybrid proteins F1D34-1 and F1D34-2i both showed strong AP inhibitory activity, which was superior to that of the CFH fragments FH15 and FH13 and the DAF fragments D24 and D14, as well as to the CR1 and DAF hybrid protein CR15D34 and the CR1 activity-enhancing fragment CR13m (Figure 4), indicating that the hybrid proteins obtained by domain hybridization have superior AP inhibitory activity.

[0194] 4.3 Detection of inhibitory activity on complement C3b deposition In this experiment, we detected the inhibition of C3b deposition on the surface of rabbit erythrocytes after human complement activation by different complement regulatory proteins, and analyzed and compared the inhibitory activity of each protein against C3b deposition.

[0195] The experimental process can be briefly described as follows: (1) Gradient dilution of protein: GVBMG buffer (Mg 2+ The protein was diluted to 1400 using gelatin flora buffer (containing EGTA, Cat. No.: 25-02090, TIANDZ) and then diluted three-fold, for a total of seven gradient dilutions. (2) Serum dilution: To prevent complement activation from causing hemolysis and rupture of rabbit red blood cells, which would make it impossible to accurately detect C3b deposition on the cell surface, normal human serum from which C5 had been removed was used in the experiment. C5-Dpl NHS (Complement Technology) was removed from the -70°C refrigerator, and then naturally thawed at 4°C. The serum was diluted to 40% with GVBMG. (3) Preparation of rabbit red blood cells: 4% rabbit red blood cells (Nanjing SenBeiJia Biological Technology Co., Ltd.) were washed with GVBMG until the supernatant was clear, and then the cell density was 1x10 7 The cells were resuspended in GVBMG at 100 cells / mL. (4) Incubation: 25 μL each of the diluted protein and serum were mixed in a 96-well plate, and then 50 μL of the red blood cell suspension obtained in step (3) was added to make the initial final protein concentration 350 nM and the final serum concentration 10%, and the mixture was thoroughly mixed and placed in a constant temperature incubator at 37°C for 1 hour. A negative control (containing only complement-inactivated serum and red blood cells) and a positive control (containing only normal serum and red blood cells) were added. (5) Termination: After incubation, 100 μL of 10 mM EDTA-GVB buffer (GVBE) was added to each well to terminate the reaction. (6) Detection: The incubated cells were washed three times with PBS and stained with fluorescein-labeled anti-human C3b / iC3b antibody (APC anti-complement C3b / iC3b Antibody, Biolegend). They were then incubated at 4°C for 30 minutes and washed three times again. The APC fluorescence MFI (mean fluorescence intensity) of each sample was measured using a flow cytometer (Cytoflex, Beckman) and the data were stored. (7) Data processing: The rate of C3b deposition on the red blood cell surface was calculated using the following formula. Deposition rate (%) = MFI value of experimental group / MFI value of positive control × 100

[0196] The IC50 value of the antibody was then calculated by plotting a four-parameter fit curve with the final protein concentration on the horizontal axis and the deposition rate on the vertical axis. The results are shown in Figure 5.

[0197] Experimental tests on C3b deposition showed that the CFH and DAF hybrid proteins F1D34-1 and F1D34-2i both had strong C3b deposition inhibitory activity, superior to the CFH fragments FH15 and FH13 and the DAF fragments D24 and D14, as well as to the CR1 and DAF hybrid protein CR15D34 and the CR1 activity-enhancing fragment CR13m (shown in Figure 5), indicating that the hybrid proteins obtained by domain hybridization have superior C3b deposition inhibitory activity.

[0198] [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6] [Table 3-7] [Table 3-8] [Table 3-9] [Table 3-10]

Claims

1. It is a hybrid protein, (i) Human complement factor H (CFH) CCP1, (ii) Human DAFs CCP3 and CCP4, Optionally, signal peptide and / or tag, A hybrid protein containing or consisting of these.

2. The hybrid protein according to claim 1, wherein CCP3 and CCP4 of the DAF are both directly linked to form CCP3-4.

3. The hybrid protein according to claim 1 or 2, wherein the CCP1 of the human CFH includes or consists of the amino acid sequence from positions 19 to 82 of the human CFH protein, and optionally the CCP1 includes the V62I mutation, and the amino acid positions are numbered in correspondence with the amino acid positions described in Sequence ID No.

3.

4. The hybrid protein according to claim 1 or 2, wherein the CCP1 of the human CFH includes or consists of the amino acid sequence from positions 19 to 84 of the human CFH protein, and optionally the CCP1 includes the V62I mutation, and the amino acid positions are numbered in correspondence with the amino acid positions described in Sequence ID No.

3.

5. The hybrid protein according to claim 1 or 2, wherein the CCP3 of the human DAF includes or consists of the amino acid sequence from positions 161 to 222 of the human DAF protein, and / or the CCP4 of the human DAF includes or consists of the amino acid sequence from positions 223 to 285 of the human DAF protein, and the amino acid positions are numbered in correspondence with the amino acid positions described in Sequence ID No.

1.

6. The hybrid protein according to claim 1 or 2, wherein the CCP3 of the human DAF comprises or consists of amino acid sequences 163 to 222 of the human DAF protein, and / or the CCP4 of the human DAF comprises or consists of amino acid sequences 223 to 285 of the human DAF protein, and the amino acid positions are numbered in correspondence with the amino acid positions described in Sequence ID No.

1.

7. The hybrid protein according to claim 2, wherein the CCP3-4 of the human DAF includes or consists of the amino acid sequence from positions 161 to 285 of the human DAF protein, and the amino acid positions are numbered in correspondence with the amino acid positions described in Sequence ID No.

1.

8. The hybrid protein according to claim 2, wherein the CCP3-4 of the human DAF includes or consists of the amino acid sequence from positions 163 to 285 of the human DAF protein, and the amino acid positions are numbered in correspondence with the amino acid positions described in Sequence ID No.

1.

9. (1) The CCP1 of the human CFH contains or consists of the amino acid sequence from positions 19 to 82 of the human CFH protein, the CCP3 of the human DAF contains or consists of the amino acid sequence from positions 161 to 222 of the human DAF protein, and the CCP4 of the human DAF contains or consists of the amino acid sequence from positions 223 to 285 of the human DAF protein; (2) The CCP1 of the human CFH includes or consists of the amino acid sequence from positions 19 to 84 of the human CFH protein, the CCP3 of the human DAF includes or consists of the amino acid sequence from 163 to 222 of the human DAF protein, and the CCP4 of the human DAF includes or consists of the amino acid sequence from 223 to 285 of the human DAF protein; (3) The CCP1 of the human CFH includes or consists of the amino acid sequence from positions 19 to 82 of the human CFH protein, and the CCP3-4 of the human DAF includes or consists of the amino acid sequence from positions 161 to 285 of the human DAF protein; or (4) The CCP1 of the human CFH includes or consists of the amino acid sequence from positions 19 to 84 of the human CFH protein, and the CCP3-4 of the human DAF includes or consists of the amino acid sequence from positions 163 to 285 of the human DAF protein; The amino acid positions of the human CFH protein are numbered according to the amino acid positions described in Sequence ID No. 3, and the amino acid positions of the human DAF protein are numbered according to the amino acid positions described in Sequence ID No.

1. The hybrid protein according to claim 2.

10. The hybrid protein according to claim 9, wherein the CCP1 of the human CFH has the V62I mutation.

11. Whether the human CFH protein is a native human CFH protein, (i) The amino acid sequence described in Sequence ID No. 3 or 5; (ii) an amino acid sequence encoded by the nucleotide sequence described in SEQ ID NO: 4 or 6; or (iii) An amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in (i) or (ii); Includes or The amino acid sequence consists of any one of the following (i) to (iii): The hybrid protein according to claim 1 or 2.

12. Whether the human DAF protein is a native human DAF protein, (i) The amino acid sequence described in Sequence ID No. 1; (ii) The amino acid sequence encoded by the nucleotide sequence described in Sequence ID No. 2; or (iii) An amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in (i) or (ii); Includes or (iv) Consists of an amino acid sequence described in any one of (i) to (iii), The hybrid protein according to claim 1 or 2.

13. The CCP1 of the human CFH includes, or consists of, an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NOs: 12, 13, 14, or 15; The CCP3 of the human DAF contains, or consists of, an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 7 or SEQ ID NO: 8; The CCP4 of the human DAF contains, or consists of, an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 9; and / or The CCP3-4 of the human DAF contains, or consists of, an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 10 or SEQ ID NO:

11. The hybrid protein according to claim 1 or 2.

14. The hybrid protein according to claim 1 or 2, wherein the tag is a purified tag such as a hexahistidine tag or a biotin tag containing the amino acid sequence described in SEQ ID NO: 12, for example.

15. The hybrid protein according to claim 1 or 2, wherein the signal peptide is secreted and comprises, for example, the amino acid sequence described in SEQ ID NO:

17.

16. The hybrid protein according to claim 1 or 2, wherein the hybrid protein includes any one of the amino acid sequences of Sequence ID No. 18 to 33, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity with the aforementioned amino acid sequence, or consists of any of these amino acid sequences.

17. A nucleic acid molecule encoding the hybrid protein described in claim 1.

18. An expression vector comprising the nucleic acid molecule described in claim 17, wherein the expression vector is preferably pcDNA3.

1.

19. A host cell comprising the nucleic acid molecule described in claim 17 or the expression vector described in claim 18, preferably the host cell being a prokaryotic cell or a eukaryotic cell, such as a 293 cell including Expi293 cells.

20. A method for preparing the hybrid protein according to claim 1 or 2, wherein the method comprises culturing a host cell containing the nucleic acid molecule encoding the hybrid protein according to claim 17 or the expression vector according to claim 18 under conditions suitable for the expression of the hybrid protein, and optionally further comprising isolating the protein from the host cell or host cell culture medium and / or purifying the protein.

21. A pharmaceutical composition comprising the hybrid protein described in claim 1, or a nucleic acid molecule encoding the hybrid protein described in claim 1, and a pharmaceutically acceptable excipient.

22. A pharmaceutical composition comprising a hybrid protein according to claim 1, or a nucleic acid molecule encoding the hybrid protein according to claim 1, wherein the pharmaceutical composition is used in combination with an additional therapeutic agent.

23. A pharmaceutical composition according to claim 21 or 22 for preventing or treating complement system-related diseases or disorders in a subject.

24. The pharmaceutical composition according to claim 23, wherein the disease or disorder is due to abnormal activation of the complement system or dysregulation of the complement system.

25. The pharmaceutical composition according to claim 24, wherein the abnormal activation or dysregulation of the complement system is, for example, due to microbial infection or an increase in autoimmune antibodies, or due to a decrease, deletion, inactivation, interference, or blockage of the function of complement regulatory proteins.

26. The pharmaceutical composition according to claim 23, wherein the complement system-related disease or disorder is selected from diseases requiring hemolytic inhibition, such as diseases requiring inhibition of hemolysis in the classical pathway of complement immunity and / or alternative pathways of complement immunity; or diseases requiring inhibition of C3b deposition activity, such as dense deposit disease (DDD).

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