C3b-targeting bifunctional fusion protein, preparation method therefor and use thereof

Abstract

A C3b-targeting bifunctional fusion protein, a preparation method therefor and the use thereof. The C3b-targeting bifunctional fusion protein comprises a CRIg domain variant, an FH domain and a linker. The fusion protein has high affinity for human complement C3b, can effectively inhibit the activation of a plurality of complement pathways, can effectively inhibit the hemolytic reaction of complement pathway activation, and exhibits high affinity to FcRn.

Description

A bifunctional fusion protein targeting C3b and its preparation method and use Technical Field

[0001] The present invention relates to the field of fusion proteins, and in particular to a bifunctional fusion protein targeting C3b, and a preparation method and use thereof. Background Art

[0002] The complement system is composed of over 30 proteins found widely in serum, tissue fluid, and on cell membranes, including complement activators, inhibitors, and complement receptors. It participates in both specific and nonspecific immunity, manifesting as proinflammatory, opsonophagocytic, and cytolytic responses. The complement system is generally activated through three pathways: the classical pathway (CP), which is activated by IgG and IgM antibodies; the lectin pathway (LP), which is activated by mannose on the bacterial surface; and the alternative pathway (AP), which is activated by various components, including pathogen cell wall / membrane components or complement C3b analogs such as snake toxins. C3 is the central molecule of the complement system and the intersection of its three activation pathways. C3b, produced by the hydrolysis of C3 by C3 convertase, is a core component of C3 convertase (C3bBb) and C5 convertase (C3bBbC3b, C4bC2bC3b), and is a key protein influencing the activation of intermediate cascades and the terminal pathway of the complement pathway. During complement activation, multiple regulatory proteins participate in preventing damage to the body, including Factor H (FH), Factor I (FI), and Factor B (FB) in the blood circulation. FH not only promotes the inactivation of C3b by FI, but also competitively inhibits the binding of FB to C3b and dissociates C3b from C3bBb, thereby accelerating the inactivation of C3bBb and inhibiting complement pathway activation. The C3b receptor CRIg expressed on the cell membrane surface can specifically bind to the β chain of C3b and occupy its ability to bind to the substrate, thereby inhibiting the activity of C3 convertase and C5 convertase, thereby exerting the function of inhibiting complement pathway activation.

[0003] The complement system is a crucial component of the innate immune system. Imbalances in complement play a crucial role in the pathogenesis of a wide range of diseases, including both rare and common ones. These disorders have a wide range of impacts, from acute inflammatory conditions like eye and periodontal diseases to chronic conditions like cancer, autoimmune diseases, neurodegenerative diseases, kidney disease, and chronic hemolytic disorders. Currently approved complement therapies are primarily used to treat rare diseases such as paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), generalized myasthenia gravis (gMG), and neuromyelitis optica spectrum disorders (NMO). These therapies are gradually expanding into more common conditions such as age-related macular degeneration (AMD) and geographic atrophy (GA).

[0004] Given that currently marketed inhibitors targeting C3 and / or C5 proteins have certain side effects and unsatisfactory therapeutic effects, the present invention proposes to develop a bifunctional fusion protein targeting C3b based on the natural inhibitory proteins FH and CRIg, for the treatment of diseases associated with abnormal complement system activation. The resulting CRIg-FH fusion protein is expected to have fewer side effects and better therapeutic effects than currently marketed inhibitors targeting C3 and / or C5 proteins.

[0005] Summary of the Invention

[0006] The object of the present invention is to provide a sequence-engineered CRIg-FH fusion protein or CRIg-FH-Fc fusion protein.

[0007] The fusion protein of the present invention has significantly improved affinity and complement inhibitory activity. The present invention also provides a nucleic acid molecule encoding the fusion protein; an expression vector comprising the nucleic acid molecule; a host cell comprising the expression vector; a method for preparing the fusion protein; a pharmaceutical composition comprising the fusion protein; the use of the fusion protein or the pharmaceutical composition in preparing a drug for preventing or treating diseases associated with complement activation; and a method for preventing or treating diseases associated with complement activation using the fusion protein or the pharmaceutical composition.

[0008] In a first aspect of the present invention, a bifunctional fusion protein targeting C3b is provided, comprising:

[0009] a) a CRIg domain variant comprising a mutation relative to SEQ ID NO: 2 selected from the group consisting of: M86Y; or a combination of Q64R and M86Y; and

[0010] b) FH domain.

[0011] In another preferred embodiment, the C-terminus of the CRIg extracellular domain is directly or indirectly connected to the N-terminus of the FH domain, or the N-terminus of the CRIg extracellular domain is directly or indirectly connected to the C-terminus of the FH domain.

[0012] In another preferred embodiment, the FH domain comprises the amino acid sequence shown in SEQ ID NO: 4.

[0013] In another preferred embodiment, the bifunctional fusion protein targeting C3b further comprises:

[0014] c) A linker connecting a) and b).

[0015] In another preferred embodiment, the bifunctional fusion protein targeting C3b has the following connection sequence from N-terminus to C-terminus: CRIg domain variant-linker-FH domain.

[0016] In another preferred example, the C-terminus of the FH domain is directly or indirectly connected to the N-terminus of the Fc domain variant, or the C-terminus of the CRIg extracellular domain is directly or indirectly connected to the N-terminus of the Fc domain variant.

[0017] In another preferred embodiment, the linker is a flexible linker, preferably (GS4)n or (SG4)m, wherein n or m is a positive integer selected from 1, 2, 3, 4, and 5.

[0018] In another preferred embodiment, the bifunctional fusion protein targeting C3b further comprises:

[0019] d) Fc domain variants.

[0020] In another preferred embodiment, the Fc domain variant comprises an Fc domain variant selected from the group consisting of IgG1-Fc, IgG2-Fc, IgG3-Fc, and IgG4-Fc.

[0021] In another preferred embodiment, the Fc domain variant is IgG4-Fc; preferably, it includes an amino acid mutation S228P at position 228 of the human IgG4 constant region Fc sequence, and / or includes a mutation selected from the group consisting of: M252Y / S254T / T256E.

[0022] In another preferred embodiment, the bifunctional fusion protein retains the biological activity of the above elements a), b) and / or d).

[0023] In another preferred embodiment, the bifunctional fusion protein further has one or more of the following functions:

[0024] (a) Activity of binding to human complement C3b;

[0025] (b) inhibiting the alternative complement pathway;

[0026] (c) inhibition of the classical complement pathway;

[0027] (d) inhibition of the complement lectin pathway;

[0028] (e) inhibiting activation of the alternative and classical pathways;

[0029] (e) Inhibits erythrocyte lysis.

[0030] In a preferred embodiment, the bifunctional fusion protein is a single-chain structure.

[0031] In another preferred embodiment, the bifunctional fusion protein is a dimer; preferably a homologous or heterologous dimer.

[0032] In another preferred embodiment, the bifunctional fusion protein comprises a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises the CRIg domain variant, the first FH domain and the first Fc domain variant; the second polypeptide chain comprises the CRIg domain variant, the second FH domain and the second Fc domain variant.

[0033] The first Fc domain variant and the second Fc domain variant can interact with each other to form a dimer.

[0034] In another preferred embodiment, the first polypeptide chain further comprises a first linker connecting the CRIg domain variant and the first FH domain;

[0035] The second polypeptide chain further comprises a second linker connecting the CRIg domain variant and the second FH domain.

[0036] In another preferred embodiment, the first FH domain and the second FH domain are the same or different.

[0037] In another preferred embodiment, the first Fc domain variant and the second Fc domain variant are the same or different.

[0038] In another preferred embodiment, the first polypeptide chain and the second polypeptide chain are identical.

[0039] In another preferred embodiment, the first polypeptide chain and / or the second polypeptide chain of the bifunctional fusion protein has a structure shown in the following formula I or II: XLYZ (Formula I) YLXZ (Formula II)

[0040] Where,

[0041] X is a CRIg domain variant comprising a mutation selected from the group consisting of M86Y or a combination of Q64R and M86Y relative to SEQ ID NO: 2;

[0042] Y is the FH domain;

[0043] Z is none or an Fc domain variant;

[0044] L is none or a linker;

[0045] - represents a peptide bond or a peptide linker.

[0046] In another preferred embodiment, any two of X, Y, and Z are connected in a head-to-head, head-to-tail, or tail-to-tail manner.

[0047] In another preferred embodiment, the "head" refers to the N-terminus of a polypeptide or a fragment thereof, especially the N-terminus of a wild-type polypeptide or a fragment thereof.

[0048] In another preferred embodiment, the "tail" refers to the C-terminus of a polypeptide or a fragment thereof, especially the C-terminus of a wild-type polypeptide or a fragment thereof.

[0049] In another preferred embodiment, the sequence of X is as shown in SEQ ID NO.9, or SEQ ID NO.10, or SEQ ID NO.11.

[0050] In another preferred embodiment, the sequence of Y is shown in SEQ ID NO: 4.

[0051] In another preferred embodiment, the nucleotide sequence encoding Y is shown in SEQ ID NO: 3.

[0052] In another preferred embodiment, the sequence of L is (SG4)3 or (G4S)3.

[0053] In another preferred embodiment, the sequence of Z is as shown in SEQ ID NO: 6;

[0054] As shown in positions 440-663 of SEQ ID NO.7, or

[0055] As shown in positions 440-663 of SEQ ID NO.15.

[0056] In another preferred embodiment, the first polypeptide chain and the second polypeptide chain comprise the amino acid sequence shown in any one of SEQ ID NOs. 7-15.

[0057] In another preferred embodiment, the first polypeptide chain and the second polypeptide chain comprise the amino acid sequence shown in any one of SEQ ID NOs. 9, 10, 11, and 15.

[0058] In another preferred embodiment, the fusion protein is a homodimer and comprises amino acids selected from the following group: SEQ ID NO. 9, 10, 11, 15.

[0059] In another preferred embodiment, the fusion protein can inhibit the C3 signaling pathway.

[0060] In another preferred embodiment, the fusion protein exhibited an inhibitory effect on the complement activation pathway in a rat passive Heymann nephritis (PHN) model, with the urine protein level in the administration group being significantly lower than that in the model group, and exhibiting a dose-dependent effect.

[0061] In another preferred embodiment, the fusion protein exhibits an inhibitory effect on the complement pathway in cynomolgus monkeys with spontaneous periodontitis, significantly inhibiting the disease progression of cynomolgus monkeys with periodontitis.

[0062] In the second aspect of the present invention, a nucleic acid molecule is provided, encoding the fusion protein described in the first aspect of the present invention.

[0063] In the third aspect of the present invention, an expression vector is provided, comprising the nucleic acid molecule described in the second aspect of the present invention.

[0064] In a preferred embodiment, the expression vector includes: bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus, or other vectors.

[0065] In the fourth aspect of the present invention, a host cell is provided, comprising the expression vector described in the third aspect of the present invention or expressing the fusion protein described in the first aspect of the present invention.

[0066] In the fifth aspect of the present invention, a method for preparing the fusion protein described in the first aspect of the present invention is provided, comprising the following steps: synthesizing the fusion protein described in the first aspect of the present invention, and / or culturing the host cell described in the fourth aspect of the present invention under conditions for expressing the fusion protein described in the first aspect of the present invention.

[0067] In the sixth aspect of the present invention, a pharmaceutical composition is provided, comprising the fusion protein according to the first aspect of the present invention and optionally a pharmaceutically acceptable carrier.

[0068] In a preferred embodiment, the pharmaceutical composition is a liquid preparation.

[0069] In a preferred embodiment, the pharmaceutical composition is an injection or injection.

[0070] In the seventh aspect of the present invention, provided is a use of the fusion protein according to the first aspect of the present invention or the pharmaceutical composition according to the sixth aspect of the present invention in preparing a drug for preventing or treating diseases associated with complement activation.

[0071] In a preferred embodiment, the disease associated with complement activation is a disease of excessive complement activation.

[0072] In a preferred embodiment, the disease associated with complement activation is a disease associated with the C3 signaling pathway.

[0073] In a preferred embodiment, the fusion protein or pharmaceutical composition can inhibit, alleviate, improve or reduce pathological symptoms caused by excessive complement activation, such as 1) lysis or hemolysis of red blood cells; 2) increased proteinuria.

[0074] In another preferred embodiment, the diseases include paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), systemic myasthenia gravis (gMG), neuromyelitis optica spectrum disorders, age-related macular degeneration (AMD), geographic atrophy (GA), autolytic hemolytic anemia, autoimmune thrombocytopenia, aplastic anemia, systemic lupus erythematosus, rheumatoid arthritis, ankylosing spondylitis, atherosclerosis, Parkinson's disease, Alzheimer's disease (senile dementia), asthma, allergies, psoriasis, IgA nephropathy, glomerulonephritis, lupus nephritis, multiple sclerosis, acute respiratory distress syndrome, and inflammatory diseases of periodontal tissues (including gingivitis and periodontitis).

[0075] In another aspect, the present invention also provides a method for preventing or treating diseases related to complement activation using the above fusion protein or pharmaceutical composition.

[0076] In a preferred embodiment, the disease associated with complement activation is a disease of excessive complement activation.

[0077] In a preferred embodiment, the disease includes paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), generalized myasthenia gravis (gMG), neuromyelitis optica spectrum disorders, age-related macular degeneration (AMD), geographic atrophy (GA), autolytic hemolytic anemia, autoimmune thrombocytopenia, aplastic anemia, systemic lupus erythematosus, rheumatoid arthritis, ankylosing spondylitis, atherosclerosis, Parkinson's disease, Alzheimer's disease (senile dementia), asthma, allergies, psoriasis, IgA nephropathy, glomerulonephritis, lupus nephritis, multiple sclerosis, acute respiratory distress syndrome, and inflammatory diseases of periodontal tissues (including gingivitis and periodontitis).

[0078] In the eighth aspect of the present invention, a method for preventing or treating diseases related to complement activation is provided, comprising administering a pharmaceutically effective amount of the fusion protein according to the first aspect of the present invention or a pharmaceutical composition comprising the fusion protein to a subject in need.

[0079] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0080] FIG1 shows the binding affinity of the fusion proteins CRIg-FH-FCG4, CRIg-L(GS)-FH-FCG4, and CRIg-L-FH-FCG4 to human complement C3 as determined by ELISA.

[0081] FIG2 shows the binding affinity of the fusion proteins CRIg-FH-FCG4, CRIg-FH-FCG4-M86Y, CRIg-FH-FCG4-Q64R, and CRIg-FH-FCG4-Q64R-M86Y to human complement C3 as determined by ELISA.

[0082] Figure 3 shows the ELISA determination of the binding affinity of the fusion proteins CRIg-FH-FCG4, CRIg-L(GS)-FH-FCG4, CRIg-L(GS)-FH-FCG4-Q64R, CRIg-L(GS)-FH-FCG4-M86Y, and CRIg-L(GS)-FH-FCG4-Q64R-M86Y to human complement C3.

[0083] Figure 4 shows the inhibitory effects of the fusion proteins CRIg-FH-FCG4, CRIg-L(GS)-FH-FCG4, CRIg-L(GS)-FH-FCG4-M86Y, and CRIg-L(GS)-FH-FCG4-Q64R-M86Y on the lysis of erythrocytes activated by the complement AP pathway.

[0084] Figure 5 shows the inhibitory effects of the fusion proteins CRIg-FH-FCG4, CRIg-L(GS)-FH-FCG4, CRIg-L(GS)-FH-FCG4-M86Y, and CRIg-L(GS)-FH-FCG4-Q64R-M86Y on lysed erythrocytes activated by the complement CP pathway.

[0085] FIG6 shows the inhibitory effects of the fusion proteins CRIg-L(GS)-FH-FCG4, CRIg-L(GS)-FH-FCG4-M86Y, and CRIg-L(GS)-FH-FCG4-Q64R-M86Y on activation of the complement AP pathway.

[0086] FIG7 shows the inhibitory effects of the fusion proteins CRIg-L(GS)-FH-FCG4, CRIg-L(GS)-FH-FCG4-M86Y, and CRIg-L(GS)-FH-FCG4-Q64R-M86Y on activation of the complement CP pathway.

[0087] FIG8 shows the inhibitory effects of the fusion proteins CRIg-FH-FCG4, CRIg-L(GS)-FH-FCG4, and CRIg-L(GS)-FH-FCG4-M86Y on activation of the complement LP pathway.

[0088] FIG9 shows the inhibitory effects of the fusion proteins CRIg-FH-FCG4, CRIg-L(GS)-FH-FCG4, and CRIg-L(GS)-FH-FCG4-M86Y on activation of the complement AP pathway.

[0089] FIG10 shows the inhibitory effects of the fusion proteins CRIg-FH-FCG4, CRIg-L(GS)-FH-FCG4, and CRIg-L(GS)-FH-FCG4-M86Y on activation of the complement CP pathway.

[0090] FIG11 shows the ELISA method for determining the binding affinity of the fusion proteins biotin-CRIg-L(GS)-FH-FCG4-M86Y and biotin-CRIg-L(GS)-FH-FCG4-YTE-M86Y to human complement C3.

[0091] FIG12 shows the inhibitory effect of the fusion proteins CRIg-L(GS)-FH-FCG4-M86Y and CRIg-L(GS)-FH-FCG4-YTE-M86Y on the lysis of erythrocytes activated by the complement AP pathway.

[0092] FIG13 shows the inhibitory effect of the fusion proteins CRIg-L(GS)-FH-FCG4-M86Y and CRIg-L(GS)-FH-FCG4-YTE-M86Y on the lysis of erythrocytes activated by the complement CP pathway.

[0093] FIG14 shows the binding affinity of the fusion proteins CRIg-L(GS)-FH-FCG4-M86Y and CRIg-L(GS)-FH-FCG4-YTE-M86Y to FcRn at pH 7.0 (A) and pH 6.0 (B) as determined by ELISA.

[0094] FIG15 shows that CRIg-L(GS)-FH-FCG4-YTE-M86Y inhibits the 24-hour urine protein level in a rat PHN model in a dose-dependent manner. DETAILED DESCRIPTION

[0095] After extensive and intensive research and extensive screening, the present inventors have developed a series of fusion proteins containing functional portions of CRIg and FH with high affinity and complement inhibitory activity. The fusion proteins of the present invention may further comprise functional enhancing domains, such as Fc domains, as needed. Preferred fusion proteins exhibit an affinity for human complement C3b that is increased by at least 5-fold; more preferred fusion proteins exhibit an affinity of at least 20-fold, 30-fold, 40-fold, or 50-fold. The fusion proteins of the present invention can effectively inhibit activation of the complement AP, CP, and LP pathways, with preferred fusion proteins exhibiting approximately 3-5-fold increased inhibitory activity against the complement CP pathway. The fusion proteins of the present invention can effectively inhibit the erythrocyte lysis caused by activation of the complement AP and CP pathways, thereby protecting erythrocytes. The fusion proteins of the present invention also exhibit an approximately 6-20-fold increase in affinity for FcRn and are expected to have a longer half-life and improved pharmacokinetic properties. This work has led to the completion of the present invention.

[0096] the term

[0097] Unless defined otherwise, 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.

[0098] The term "about" can refer to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined.

[0099] As used herein, the terms "comprising" or "including" may be open, semi-closed, or closed. In other words, the terms also include "consisting essentially of" or "consisting of."

[0100] 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. As used herein, when used in reference to a specific recited value, the term "about" means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0101] As used herein, the term "optionally" or "optionally" means that the event or situation described subsequently may occur but need not occur. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that the antibody heavy chain variable regions of a specific sequence may have but need not have, and may have 1, 2, or 3.

[0102] As used herein, the terms “contain,” “have,” or “include” include “comprise,” “mainly consist of,” “substantially consist of,” and “consist of”; “mainly consist of,” “substantially consist of,” and “consist of” are subordinate concepts of “contain,” “have,” or “include.”

[0103] CRIg

[0104] CRIg was first discovered as a C3b / iC3b receptor expressed on the surface of macrophage membranes. It is a complement membrane regulatory protein that can produce an inhibitory effect early in the complement cascade by specifically recognizing C3b and inhibiting the activation of the C3 convertase. This inhibitory effect targets the alternative pathway of complement. The functional site of CRIg is located in the extracellular region. The CRIg domain described in the present application comprises the extracellular region of CRIg. An exemplary extracellular region of CRIg comprises the amino acid sequence shown in SEQ ID NO:2, or comprises an amino acid sequence that is at least 90% homologous to the amino acid sequence shown in SEQ ID NO.2, for example, any one of at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% homologous.

[0105] FH (factor H)

[0106] FH is a complement regulatory protein whose primary function is to regulate the alternative pathway of the complement system. FH contains several highly conserved motifs in its sequence, known as short consensus repeats (SCRs). Each SCR consists of approximately 60 amino acids, and the functional properties of FH are localized to the SCRs. As used herein, "FH domain" includes FH or variants thereof. Typically, FH comprises the SCR1-5 domains, and in some cases, FH may comprise additional SCR fragments. The FH domains described herein may include the full-length FH protein or fragments thereof (e.g., one or more SCRs), as well as various variants thereof (e.g., mutants, isoforms). Exemplary FH comprises the amino acid sequence set forth in SEQ ID NO:4, or an amino acid sequence that is at least 90% homologous to the amino acid sequence set forth in SEQ ID NO.4, for example, any amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homologous.

[0107] Fusion protein

[0108] The term "fusion protein" refers to a new polypeptide sequence derived from the fusion of two or more identical or different polypeptide sequences. The term "fusion" refers to the connection of two polypeptide sequences directly by peptide bonds or with the aid of one or more linkers (peptide linkers). The term "linker (peptide linker)" refers to a short peptide that can connect two polypeptide sequences, generally 1-30 amino acids in length.

[0109] Preferably, the peptide linker is a flexible peptide linker. Suitable examples of linkers include single glycine (Gly) or serine (Ser) residues, and the identity and sequence of the amino acid residues in the linker may vary depending on the type of secondary structural element to be achieved in the linker.

[0110] As used herein, the term "bifunctional fusion protein" refers to a fusion protein that can bind to two target proteins of different specificities. The bifunctional fusion protein of the present invention binds to CRIg and FH, wherein the CRIg domain variant of the bifunctional fusion protein is derived from the CRIg protein.

[0111] Variants

[0112] The term "variant" refers to a peptide that contains at least one amino acid substitution, deletion, or insertion relative to a wild-type or naturally occurring peptide. The fusion protein of the present invention also includes conservative variants thereof. The conservative amino acid mutation patterns can be found in Table A.

[0113] Table A

[0114] As used herein, a "CRIg domain variant" is derived from a CRIg domain, preferably comprising one or more amino acid mutations, substitutions, deletions, for example, a mutation selected from the group consisting of Q64R, M86Y, or a combination of Q64R and M86Y relative to the CRIg domain as shown in SEQ ID NO: 2.

[0115] As used herein, "Fc domain variant" is derived from an IgG Fc domain, which can be mutated to obtain a desired spatial structure, such as forming a knob-hole structure for better dimer formation; or comprising an S228P mutation that improves the stability of the IgG4 molecule; or a mutation that improves binding affinity to FcRn, such as the Fc domain variant comprising M252Y / S254T / T256E.

[0116] Complement activation-related diseases

[0117] The term "complement activation-related diseases" mainly refers to related diseases caused by abnormal or excessive complement activation, which may trigger an inflammatory response, as well as abnormal phagocytic and / or cell lysis effects. Diseases include all diseases and pathological conditions whose pathogenesis involves abnormal or excessive activation of the complement system, including diseases and pathological conditions that benefit from C3 convertase inhibition, including diseases and pathological conditions that benefit from complement alternative pathway inhibition. Complement-related diseases include, but are not limited to, inflammatory diseases and autoimmune diseases, covering acute inflammations such as eye diseases and periodontal diseases, to chronic diseases such as cancer, autoimmune diseases, neurodegenerative diseases, kidney diseases and chronic hemolytic diseases. Such diseases are such as C3-related nephropathy and TMA-related diseases. Such diseases include paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), generalized myasthenia gravis (gMG), neuromyelitis optica spectrum disorder, age-related macular degeneration (AMD), geographic atrophy (GA), autolytic hemolytic anemia, autoimmune thrombocytopenia, aplastic anemia, systemic lupus erythematosus, rheumatoid arthritis, ankylosing spondylitis, atherosclerosis, Parkinson's disease, Alzheimer's disease (senile dementia), asthma, allergies, psoriasis, IgA nephropathy, glomerulonephritis, lupus nephritis, multiple sclerosis, acute respiratory distress syndrome, and inflammatory diseases of periodontal tissues (including gingivitis and periodontitis).

[0118] The pathogenesis of aHUS primarily involves uncontrollable and sustained activation of the alternative complement pathway via a triggering event (such as infection or pregnancy), leading to the formation of the membrane attack complex, which in turn causes renal endothelial damage, activation of the coagulation cascade, and renal arteriolar microthrombosis, subsequently leading to clinical manifestations such as microangiopathic hemolytic anemia, thrombocytopenia, and acute renal failure. Some patients with aHUS harbor genetic mutations in complement-related factors (such as C3). Currently marketed drugs for the treatment of aHUS include eculizumab and crovalimab, which work by selectively inhibiting activation of terminal complement C5.

[0119] PNH is a rare hemolytic anemia caused by an autoimmune disorder in the patient's body. It is caused by an acquired gene mutation in hematopoietic stem cells, resulting in the loss of the cell surface protective proteins CD55 and CD59. Normally, CD55 and CD59 effectively defend against attacks by the complement system. However, when these proteins are missing, red blood cells lose their ability to protect themselves, ultimately leading to red blood cell lysis due to overactivation of the complement system, which in turn causes PNH. Currently marketed drugs for the treatment of PNH include the C5 inhibitors eculizumab and crovalimab, and the C3 inhibitor short peptide pegcetacoplan.

[0120] Gingivitis and periodontitis are common inflammatory diseases of the periodontium. Uncontrolled complement activation and the resulting chronic gingival inflammation are hallmarks of periodontal disease. Existing literature has reported that C3 inhibitors, such as AMY-101, can help reduce inflammatory bone loss in periodontitis. C3-targeted complement therapy may be considered a valuable adjunct to non-surgical periodontal treatment (C3 Targeted Complement Therapy for Chronic Periodontitis–A Scoping Review; 10.4103 / jispcd.jispcd_161_22). Results from a Phase II clinical trial (NCT0394444) of AMY-101 (cyclic peptide) for the treatment of periodontitis and gingivitis showed that the study met its primary and key secondary endpoints. Compared with placebo, AMY-101 demonstrated statistically significant and clinically meaningful efficacy in eliminating periodontal inflammation with good safety and tolerability (Phase IIa clinical trial of complement C3 inhibitor AMY-101 in adults with periodontal inflammation; 10.1172 / JCI152973).

[0121] Preventing and / or treating a disease associated with complement activation includes reducing the risk of developing the disease, reducing the likelihood of clinical symptoms associated with the disease, reducing the severity of the disease, and inhibiting the progression of the disease.

[0122] Sequence homology

[0123] Methods for determining sequence homology known to those of ordinary skill in the art include, but are not limited to, Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987, and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., Stockton Press, New York, 1991, and Carillo, H. and Lipman, D., SIAM J. Applied Biology. Math., 48:1073 (1988). The preferred method for determining identity is to obtain the largest match between the sequences tested. Methods for determining identity are compiled in publicly available computer programs. Preferred computer program methods for determining identity between two sequences include, but are not limited to, the GCG program package (Devereux, J. et al., 1984), BLASTP, BLASTN, and FASTA (Altschul, S, F. et al., 1990). The BLASTX program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S. et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S. et al., 1990). The well-known Smith-Waterman algorithm can also be used to determine identity.

[0124] Encoding nucleic acid and expression vector

[0125] The present invention also provides nucleic acid molecules encoding the above-mentioned fusion proteins. The nucleic acids of the present invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. DNA may be single-stranded or double-stranded. DNA may be a coding strand or a non-coding strand. The sequence of the DNA molecule encoding the fusion protein of the present invention may be obtained using conventional techniques, such as PCR amplification. In addition, the relevant sequence may also be synthesized using synthetic methods.

[0126] The present invention also relates to vectors comprising the above-mentioned appropriate DNA sequences and appropriate promoters or control sequences. These vectors can be used to transform appropriate host cells to enable protein expression. The vectors are conventional expression vectors in the art, meaning expression vectors comprising appropriate regulatory sequences, such as promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and / or sequences, and other appropriate sequences. The expression vectors can be viral or plasmids, such as appropriate phages or phagemids. For more technical details, see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, 1989. For many known techniques and protocols for nucleic acid manipulation, see Current Protocols in Molecular Biology, 2nd ed., Ausubel et al. The expression vectors of the present invention are preferably pcDNA3.4, pDR1, pcDNA3.1(+), pcDNA3.1 / ZEO(+), pDHFR, pcDNA4, pDHFF, pGM-CSF, or pCHO 1.0.

[0127] In the present invention, the term "host cell" refers to any host cell commonly used in the art, as long as the vector can stably replicate and the polynucleotide molecules carried can be effectively expressed. The host cells include prokaryotic expression cells and eukaryotic expression cells, and preferably include COS, CHO, NS0, sf9, sf21, DH5α, BL21 (DE3), TG1, BL21 (DE3), 293F or 293E cells.

[0128] Pharmaceutical compositions and applications

[0129] The present invention also provides a pharmaceutical composition. Preferably, the composition is a pharmaceutical composition comprising the above-mentioned fusion protein and a pharmaceutically acceptable carrier. Generally, these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 5-8, preferably about 6-8, although the pH value may vary depending on the properties of the formulated substance and the condition to be treated. The formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intravenous injection, intravenous drip, subcutaneous injection, local injection, intramuscular injection, intratumor injection, intraperitoneal injection (such as intraperitoneal injection), intracranial injection, or intracavitary injection. Pharmaceutically acceptable carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be compatible with the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, using physiological saline or an aqueous solution containing glucose and other adjuvants by conventional methods.

[0130] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples, for which detailed conditions are not specified, were generally performed under conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0131] The main advantages of the present invention include

[0132] (1) The fusion protein of the present invention has a higher affinity for C3b, a key protein in the human complement pathway; it can effectively inhibit the activation of multiple complement pathways and effectively inhibit the hemolytic reaction caused by complement pathway activation; and it also exhibits a high affinity for FcRn.

[0133] (2) The fusion protein of the present invention has an affinity for FcRn increased by about 6-20 times and is expected to have a longer half-life and better pharmacokinetic properties.

[0134] (3) The CRIg-FH fusion protein of the present invention is also sequence-engineered for the treatment of diseases associated with abnormal complement system activation. CRIg-FH primarily targets C3b, a key protein in the mid- and terminal activation of the complement pathway. C3b is produced transiently in the body and has a short half-life. The CRIg-FH fusion protein obtained by the present invention may have fewer side effects and a better therapeutic effect than currently marketed inhibitors targeting C3 and / or C5 proteins.

[0135] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples, for which detailed conditions are not specified, were generally performed under conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0136] Example 1 Activity determination of humanized fusion protein

[0137] 1.1 Preparation of humanized fusion protein

[0138] CRIg-L-FH-IgG4 is a fusion protein composed of the CRIg extracellular domain (G19-K137, NCBI Gene Accession No.: NM_007268.3), the linker and Factor H short consensus repeat domains (SCRs1-5, E19-K323, NCBI Gene Accession No.: NM_000186.4), and the human IgG4 constant region Fc. The CRIg-L-FH-IgG4 pcDNA3.4 plasmid was synthesized and cloned. The CRIg extracellular domain gene is 357 bp in length, encoding 119 amino acids, with a nucleotide sequence as shown in SEQ ID NO: 1 and an amino acid sequence as shown in SEQ ID NO: 2; the linker sequence is L(Ser1Gly4)3:(SG4)3; the Factor H short consensus repeat domain gene is 915 bp in length, encoding 305 amino acids, with a nucleotide sequence as shown in SEQ ID NO: 3 and an amino acid sequence as shown in SEQ ID NO: 4; and the human IgG4 constant region Fc gene is 675 bp in length, encoding 224 amino acids, with a nucleotide sequence as shown in SEQ ID NO: 5 and an amino acid sequence as shown in SEQ ID NO: 6.

[0139] Using the CRIg-L-FH-IgG4 plasmid as a template, primers were designed and PCR was used to replace serine 228 in the Fc region of human IgG4 constant region with proline (S228P). The resulting construct, named FCG4, was named. The extracellular domain of CRIg and the Factor H short consensus repeat domain were linked to FCG4 using molecular cloning techniques to construct the CRIg-FCG4-pcDNA3.4, FH-FCG4-pcDNA3.4, and FH-L(GS)-CRIg-FCG4-pcDNA3.4 plasmids.

[0140] The amino acid sequence of CRIg-L-FH-FCG4 is shown in SEQ ID NO: 7.

[0141] Using CRIg-L-FH-FCG4 plasmid as a template, primers were designed and the linker in the CRIg-L-FH-FCG4 sequence was deleted by PCR to obtain CRIg-FH-FCG4 plasmid.

[0142] Using CRIg-L-FH-FCG4 plasmid as a template, primers were designed and the linker sequence L(Ser1Gly4)3 was replaced with L(Gly4Ser1)3:(G4S)3 by PCR to obtain CRIg-L(GS)-FH-FCG4 plasmid, the amino acid sequence of which is shown in SEQ ID NO: 8.

[0143] Using the CRIg-L(GS)-FH-FCG4 plasmid as a template, primers were designed and PCR was performed to mutate the glutamine at position 64 of the CRIg sequence to arginine (Q64R), resulting in the CRIg-L(GS)-FH-FCG4-Q64R plasmid. The amino acid sequence of CRIg-L(GS)-FH-FCG4-Q64R is shown in SEQ ID NO: 9.

[0144] Using the CRIg-L(GS)-FH-FCG4 plasmid as a template, primers were designed and PCR was performed to mutate the methionine at position 86 of the CRIg sequence to tyrosine (M86Y), resulting in CRIg-L(GS)-FH-FCG4-M86Y. The amino acid sequence of CRIg-L(GS)-FH-FCG4-M86Y is shown in SEQ ID NO: 10.

[0145] Using the CRIg-L(GS)-FH-FCG4 plasmid as a template, PCR was performed to simultaneously mutate the glutamine at position 64 of the CRIg sequence to arginine (Q64R) and the methionine at position 86 to tyrosine (M86Y), resulting in CRIg-L(GS)-FH-FCG4-Q64R-M86Y. The amino acid sequence of CRIg-L(GS)-FH-FCG4-Q64R-M86Y is shown in SEQ ID NO: 11.

[0146] Using the CRIg-FH-FCG4 plasmid as a template, the glutamine at position 64 of the CRIg sequence was mutated to arginine (Q64R), and the methionine at position 86 was mutated to tyrosine (M86Y) by PCR. These two amino acid sites were mutated simultaneously to obtain three plasmids, CRIg-FH-FCG4-Q64R, CRIg-FH-FCG4-M86Y, and CRIg-FH-FCG4-Q64R-M86Y, whose amino acid sequences are shown in SEQ ID NOs: 12, 13, and 14, respectively.

[0147] The above-mentioned fusion protein pcDNA3.4 expression vectors were transfected into HEK-293F cells and expressed for 6 days. The cell supernatants were collected and purified by Protein A to obtain the fusion proteins. The molecular weights of the expressed proteins were confirmed by SDS-PAGE electrophoresis and SEC-HPLC to be consistent with the expectations, and the antibody purity was >95%. The cells were quantified, aliquoted, and stored at -80°C until use.

[0148] 1.2 ELISA determination of the binding affinity of each fusion protein to human complement C3

[0149] C3-his protein (purchased from Beijing Sino-Bio Technologies Co., Ltd., catalog number 13182-H08H) was diluted to 2 μg / mL and coated on ELISA plates at 100 μL / well for overnight at 4°C. The plates were washed three times with PBST (PBS containing 0.05% Tween 20), and blocked at room temperature for 2 h at 200 μL / well with 2% BSA prepared in PBST. After washing twice with PBST, 50 mM NiCl2 was diluted to 5 mM with 1% BSA prepared in PBST. The test protein was diluted in a 5-fold gradient using 1% BSA containing 5 mM NiCl2, with the highest concentration being 200 nM. The dilution gradient was 8 times, and the plates were added to the ELISA wells at 100 μL / well. The plates were incubated at room temperature for 1 h, and two replicates were prepared for each sample. The plates were washed three times with PBST, and the secondary antibody (Anti-Human IgG (Fc specific)-Peroxidase Antibody (purchased from Sigma-Aldrich LLC, catalog number: A0170) was added to ELISA wells at 100 μL / well and incubated at room temperature for 1 h. After washing three times with PBST, TMB color development solution was added at 100 μL / well and color was developed to the expected color. The color reaction was terminated with 2 M H2SO4 at 50 μL / well. Each reaction solution was shaken evenly and the OD 450 nm was measured on a microplate reader. The data were analyzed and the EC was calculated. 50 .

[0150] The experimental results are shown in Figure 1 and Table 1. The binding affinity of CRIg-L(GS)-FH-FCG4 to human complement C3 is significantly better than that of the single fusion proteins CRIg-FCG4 and FH-FCG4, and is also significantly better than that of the dual fusion proteins CRIg-FH-FCG4 and FH-L(GS)-CRIg-FCG4. The binding affinities of CRIg-L(GS)-FH-FCG4 and CRIg-L-FH-FCG4 to human complement C3 are similar.

[0151] Table 1: EC of fusion protein affinity for C3-His protein 50 value

[0152] As shown in Figure 2 and Table 2, the Q64R and M86Y mutations in CRIg significantly increased the binding affinity of the fusion protein to complement C3. 50 The value was about 2.5 times higher than that of CRIg-FH-FCG4. The EC value of CRIg-FH-FCG4-Q64R-M86Y for C3-His protein binding was 50 The value was about 3.5 times higher than that of CRIg-FH-FCG4. The EC value of CRIg-FH-FCG4-M86Y for C3-His protein binding was 50 The value was about 5.1 times higher than that of CRIg-FH-FCG4.

[0153] Table 2: EC of the fusion protein CRIg-FH-FCG4 and its mutant proteins for C3-His affinity 50 value

[0154] As shown in Figure 3 and Table 3, the introduction of Q64R and M86Y mutations into CRIg-L(GS)-FH-FCG4 significantly improved its binding affinity to complement C3. When the C3-his protein coating concentration was reduced to 1 μg / mL, the EC of CRIg-L(GS)-FH-FCG4-Q64R on C3-His protein binding was significantly improved. 50 The value was about 5.8 times higher than that of CRIg-L(GS)-FH-FCG4. The EC value of CRIg-L(GS)-FH-FCG4-Q64R-M86Y for C3-His protein binding was 50 The value was 52.8 times higher than that of CRIg-L(GS)-FH-FCG4. The EC value of CRIg-L(GS)-FH-FCG4-M86Y for C3-His protein binding was 50 The value was 39.8 times higher than that of CRIg-L(GS)-FH-FCG4.

[0155] Table 3: EC of the fusion protein CRIg-L(GS)-FH-FCG4 and its mutant proteins for C3-His affinity 50 value

[0156] Example 2 Fusion protein protects human erythrocytes from hemolysis induced by the alternative and classical complement pathways

[0157] 2.1 Fusion protein protects human erythrocytes from hemolysis induced by activation of the alternative complement pathway (AP)

[0158] Fresh human red blood cells (purchased from Shanghai Aoneng Biotechnology Co., Ltd., product number FTS-RBC-3) were added to Alsever's solution (Sigma, A3551) at a volume ratio of 1:1, mixed and stored in a refrigerator at 4°C. When used, the cells were counted; 3×10 9 Add 10 mL of 0.9% saline to the cells, mix well, centrifuge at 2000 rpm for 10 min, remove the supernatant, and repeat the above steps for three washes; prepare AP buffer (DPB + 5.5 mM MgCl2 + 0.15 mM CaCl2 + 8 mM EGTA) to resuspend the cells and count the total number of cells using trypan blue. The cell density was adjusted to 4E8 / mL, and 50 μL was added to each well of a 96-well cell culture plate. Normal human serum (Nomal Human Serum, purchased from Shanghai Aoneng Biotechnology Co., Ltd., catalog number FTS-SMA-50) was used to dilute each fusion protein in a 5-fold gradient, with the highest concentration being 10,000 nM (final concentration 5,000 nM). The fusion protein was diluted 11 times and added to red blood cells at 100 μL / well. 100 μL inactivated human serum was added to the NC wells, and 100 μL ddH2O was added to the PC wells. Two replicates were made for each sample, and the cells were incubated at 37°C for 15 min. CVF (C3- and C5-activating cobra venom factor, purchased from Quidel) was diluted with AP buffer. Corporation, Catalog No. A600) to 40 μg / mL (final concentration 10 μg / mL), 50 μL per well, mixed, and incubated at 37°C for 60 minutes. Centrifuged at 3000 rpm for 3 minutes, 100 μL of the supernatant was added to a new 96-well cell culture plate, and the absorbance was measured at 415 nm in a multi-function microplate reader for analysis. Erythrocyte lysis rate (%) = (Sample - NC) / (Sample (0 nM) - NC) * 100.

[0159] The experimental results are shown in Figure 4 and Table 4. The inhibitory effect of CRIg-L(GS)-FH-FCG4-M86Y on AP pathway activation and erythrocyte lysis is comparable to that of CRIg-L(GS)-FH-FCG4, and is superior to that of CRIg-FH-FCG4 and CRIg-L(GS)-FH-FCG4-Q64R-M86Y.

[0160] Table 4: IC values ​​of each fusion protein for inhibiting activation of the alternative complement pathway and lysis of erythrocytes 50 value

[0161] 2.2 Fusion protein protects human erythrocytes from hemolysis induced by activation of the classical complement pathway (CP)

[0162] Fresh human red blood cells (purchased from Shanghai Aoneng Biotechnology Co., Ltd., product number FTS-RBC-3) were added to Alsever's solution (Sigma, A3551) at a volume ratio of 1:1, mixed and stored in a refrigerator at 4°C. When used, the cells were counted; 3×10 9 The cells were added with 10 ml of 0.9% saline and centrifuged at 2000 rpm for 10 min. The supernatant was removed and the above steps were repeated for three washes. The cells were resuspended in CP buffer (DPBS + 0.5 mM MgCl2 + 0.15 mM CaCl2) and the total number of cells was counted using trypan blue. The cell density was adjusted to 4E8 / ml, and 50 μl was added to each well of a 96-well cell culture plate; normal human serum (Nomal Human Serum, purchased from Shanghai Aoneng Biotechnology Co., Ltd., product number FTS-SMA-50) was diluted with CP buffer to a concentration of 20% (final concentration 10%), and the fusion protein was diluted 5-fold using CP buffer containing 20% ​​human serum, with the highest concentration being 40,000 nM (final concentration 20,000 nM), and 11 dilution gradients were added to red blood cells, 100 μL / well, 100 μl 20% inactivated Human Serum was added to the NC well, and 100 μl ddH2O was added to the PC well. Two replicate wells were made for each sample, mixed, and incubated at 37°C for 15 min; Human Red Blood Cell Antibody (anti-human erythrocyte antibody, purchased from Rockland Immunochemicals, cat. no. 209-4139) to 600 μg / ml (final concentration 150 μg / ml), 50 μl per well, mixed, and incubated at 37°C for 30 min. Centrifuged at 3000 rpm for 3 min, 100 μl of supernatant was added to a new 96-well cell culture plate, and the absorbance was measured at 415 nm in a multi-function microplate reader for analysis. Erythrocyte lysis rate (%) = (Sample - NC) / (Sample (0 nM) - NC) * 100.

[0163] The experimental results are shown in Figure 5 and Table 5. Judging from the dose-effect curves for inhibiting the classical complement pathway activation and lysis of erythrocytes, CRIg-L(GS)-FH-FCG4-M86Y, CRIg-L(GS)-FH-FCG4-Q64R-M86Y, and CRIg-L(GS)-FH-FCG4 had similar inhibitory effects on the classical pathway activation and lysis of erythrocytes, and were superior to CRIg-FH-FCG4.

[0164] Table 5: IC values ​​of each fusion protein for inhibition of erythrocyte lysis by classical complement pathway activation 50value

[0165] Example 3 Inhibitory effect of fusion protein on activation of the alternative complement pathway and classical complement pathway

[0166] A commercial kit purchased from Svar was used Complement System Classical Pathway (COMPL CP310) and Complement Alternative Pathway (COMPL AP330) was used to detect the inhibitory effects of the fusion protein on the activation of the classical complement pathway and the alternative complement pathway.

[0167] 3.1 ELISA assay to determine the inhibitory effect of the fusion protein on the alternative complement pathway (AP)

[0168] Commercially available kits Complement Alternative Pathway (COMPL AP330) was used for ELISA testing. The kit was equilibrated to room temperature before use. Use ddH2O to dilute 30× wash buffer, dissolve PC on ice in 200μL ddH2O, store in aliquots at -80℃, do not freeze and thaw repeatedly, dilute PC and NC 19-fold with Diluent AP (15μL + 270μL); dilute normal human serum (Nomal Human Serum, purchased from Shanghai Aoneng Biotechnology Co., Ltd., product number FTS-SMA-50) to 5% with Diluent AP, and dilute the test protein 3-fold with Diluent AP containing 5% human serum, with the highest concentration being 10nM. After 8 dilutions, 100μL / well was added to the LPS-coated ELISA plate and incubated at 37℃ for 70min. Two replicates were made for each sample; wash three times with 300μL / well wash buffer, and pat the ELISA plate dry on absorbent paper to remove excess liquid; add conjugate containing alkaline phosphatase-labeled antibodies to C5b-9, 100 μL / well, incubate at room temperature for 30 min; wash three times with 300 μL / well wash buffer, and pat the ELISA plate dry on absorbent paper to remove excess liquid; add substrate solution, 100 μL / well, and incubate at room temperature for 30 min; add 5 mM EDTA, 100 μL / well, to terminate the reaction, and measure the absorbance at 405 nm in a multi-function microplate reader for analysis.

[0169] Data processing method: Inhibition rate (% BL) = (Sample-BK) / (Sample (0nM) -BK)*100

[0170] The experimental results are shown in Figure 6 and Table 6. The inhibitory activity of CRIg-L(GS)-FH-FCG4-M86Y on AP pathway activation is comparable to that of CRIg-L(GS)-FH-FCG4 and superior to that of CRIg-L(GS)-FH-FCG4-Q64R-M86Y.

[0171] Table 6: IC values ​​of each fusion protein for inhibition of activation of the alternative pathway 50 value

[0172] 3.2 ELISA assay for the inhibitory effect of fusion protein on the classical complement pathway (CP)

[0173] Commercially available kits ELISA was performed using the Complement System Classical Pathway (COMPL CP310). The kit was equilibrated to room temperature before use. 30× wash buffer was diluted with ddH2O. PC was dissolved on ice with 200 μL ddH2O and stored in a -80℃ refrigerator. No repeated freezing and thawing was allowed. PC and NC were diluted 102-fold with Diluent CP (3 μL + 303 μL). Normal human serum (purchased from Shanghai Aoneng Biotechnology Co., Ltd., catalog number FTS-SMA-50) was diluted to 1% with Diluent CP. The test protein was diluted 10-fold with Diluent CP containing 1% human serum. The highest concentration was 2000 nM. Eight dilutions were performed. 100 μL / well was added to the IgM-coated ELISA plate and incubated at 37℃ for 70 min. Two replicates were made for each sample. Wash three times with 300 μL / well wash buffer and pat the ELISA plate dry on absorbent paper to remove excess liquid. Conjμgate containing alkaline phosphatase-labeled antibodies was added to C5b-9, 100 μL / well, incubate at room temperature for 30 min; wash three times with wash buffer, and pat the ELISA plate dry on absorbent paper to remove excess liquid; add substrate solution, 100 μL / well, and incubate at room temperature for 30 min; add 5 mM EDTA, 100 μL / well, to terminate the reaction, and measure the absorbance at 405 nm in a multi-function microplate reader for analysis.

[0174] Data processing method: Inhibition rate (% BL) = (Sample-BK) / (Sample (0nM) -BK)*100

[0175] The experimental results are shown in Figure 7 and Table 7. Judging from the dose-effect curve, CRIg-L(GS)-FH-FCG4-M86Y and CRIg-L(GS)-FH-FCG4-Q64R-M86Y have comparable inhibitory activities on activation of the classical complement pathway and are superior to CRIg-L(GS)-FH-FCG4.

[0176] Table 7: IC of each fusion protein against classical pathway activation inhibition 50 value

[0177] Example 4 Inhibitory Effects of Fusion Protein on Activation of Complement Lectin Pathway, Alternative Pathway, and Classical Pathway

[0178] Based on the results of hemolysis experiments and complement activity inhibition experiments, CRIg-FH-FCG4-M86Y, which has a stronger binding affinity to complement C3 after mutation, was selected for further verification.

[0179] 4.1 ELISA assay for the inhibitory effect of the fusion protein on activation of the complement lectin pathway (LP)

[0180] Commercially available kits Complement system MBL pathway (COMPL MP320) was tested by ELISA. The kit was equilibrated to room temperature before use. 30× wash buffer was diluted with ddH2O. PC was dissolved on ice in 200 μL ddH2O and stored in a -80℃ refrigerator. No repeated freezing and thawing was allowed. PC and NC were diluted 102-fold with Diluent MP (3 μL + 303 μL). Normal human serum (purchased from Shanghai Aoneng Biotechnology Co., Ltd., catalog number FTS-SMA-50) was diluted to 1% with Diluent MP. The test protein was diluted 10-fold with Diluent MP containing 1% human serum. The highest concentration was 2000 nM. Eight dilution gradients were performed. 100 μL / well was added to the mannan-coated ELISA plate and incubated at 37℃ for 70 min. Two replicates were made for each sample. Wash three times with 300 μL / well wash buffer and pat the ELISA plate dry on absorbent paper to remove excess liquid. Conjugate containing alkaline phosphatase-labeled antibodies was added to C5b-9, 100 μL / well, incubate at room temperature for 30 min; wash three times with wash buffer and pat the ELISA plate dry on absorbent paper to remove excess liquid; add substrate solution, 100 μL / well, and incubate at room temperature for 30 min; add 5 mM EDTA, 100 μL / well, to terminate the reaction, and measure the absorbance at 405 nm in a multi-function microplate reader for analysis.

[0181] Data processing method: Inhibition rate (% BL) = (Sample-BK) / (Sample (0nM) -BK)*100

[0182] The experimental results are shown in FIG8 and Table 8. The inhibitory effect of CRIg-L(GS)-FH-FCG4-M86Y on the activation of the complement lectin pathway is slightly better than that of CRIg-L(GS)-FH-FCG4, and significantly better than that of CRIg-FH-FCG4, about 4 times.

[0183] Table 8: IC values ​​of each fusion protein for inhibition of lectin pathway activation 50 value

[0184] 4.2 ELISA assay for the inhibitory effect of the fusion protein on the alternative pathway (AP) and classical pathway (CP) of complement

[0185] The experimental method is similar to that of Examples 3.1 and 3.2.

[0186] The experimental results are shown in FIG9 and Table 9. The inhibitory activity of CRIg-L(GS)-FH-FCG4-M86Y on activation of the alternative complement pathway is equivalent to that of CRIg-L(GS)-FH-FCG4 and is superior to that of CRIg-FH-FCG4.

[0187] Table 9: IC values ​​of each fusion protein for inhibition of activation of the alternative complement pathway 50 value

[0188] The experimental results are shown in FIG10 and Table 10 . The inhibitory activity of CRIg-L(GS)-FH-FCG4-M86Y on activation of the classical complement pathway is significantly better than that of CRIg-L(GS)-FH-FCG4 and CRIg-FH-FCG4.

[0189] Table 10: IC values ​​of each fusion protein for inhibition of activation of the classical complement pathway 50 value

[0190] Example 5 Preparation of fusion protein CRIg-L(GS)-FH-FCG4-YTE-M86Y

[0191] Studies have shown that the M252Y / S254T / T256E amino acid mutations in the Fc region of IgG antibodies can reduce the elimination rate of antibodies in the circulation, enhance the binding affinity of IgG antibodies to FcRn at pH 6.0, and thereby prolong their half-life in serum.

[0192] Using CRIg-L(GS)-FH-FCG4-M86Y pcDNA3.4 plasmid as a template, the M252Y / S254T / T256E amino acid mutations in the Fc sequence were completed by PCR. The resulting product was named CRIg-L(GS)-FH-FCG4-YTE-M86Y, and the amino acid sequence is shown in SEQ ID NO: 15.

[0193] The fusion protein CRIg-L(GS)-FH-FCG4-YTE-M86Y pcDNA3.4 expression vector was transfected into HEK-293F cells for expression for 6 days. The supernatant was collected and purified by Protein A to obtain the humanized proteins. The molecular weight of the expressed fusion protein was determined to be approximately 148 kD by SDS-PAGE electrophoresis and SEC-HPLC, with a purity of >95%. The protein was quantified, aliquoted, and stored at -80°C until use.

[0194] Example 6 ELISA determination of the binding affinity of CRIg-L(GS)-FH-FCG4-YTE-M86Y to human complement C3

[0195] Protein biotinylation: According to EZ-Link TM NHS-LC-LC-biotin (purchased from Thermo Scientific TM Calculate the amount of biotinylation reagent required for CRIg-L(GS)-FH-FCG4-M86Y and CRIg-L(GS)-FH-FCG4-YTE-M86Y according to the instructions (Cat. No. 21343). Mix the protein and biotinylation reagent, incubate at room temperature for 1 hour, and then use protein ultrafiltration to wash away excess biotinylation reagent with PBS and quantify the amount.

[0196] C3-his protein (Complement component 3 Protein, Human, Recombinant (His Tag), purchased from Beijing Sino Biological Technology Co., Ltd., Cat. No. 13182-H08H) was diluted to 2 μg / mL and coated on an ELISA plate at 100 μL / well at 4°C overnight. The ELISA plate was washed three times with PBST (PBS containing 0.05% Tween 20) and blocked at room temperature for 2 h with 2% BSA prepared in PBST at 200 μL / well. After washing twice with PBST, 50 mM NiCl2 was diluted to a concentration of 5 mM with 1% BSA prepared in PBST and the plate was blocked with 5 mM NiCl2. Proteins were diluted 5-fold with 1% BSA in NiCl2 to a maximum concentration of 200 nM. Eight dilutions were added to the ELISA wells at 100 μL / well and incubated at room temperature for 1 h. Two replicates were prepared for each sample. The cells were washed three times with PBST. Secondary antibody (HRP-Streptavidin, purchased from BD Biosciences, catalog number: 554066) was diluted 1:8000 with 1% BSA in PBST and added to the ELISA wells at 100 μL / well and incubated at room temperature for 1 h. After washing three times with PBST, TMB color development solution was added at 100 μL / well and color was developed to the expected color. The color reaction was terminated with 2 M H2SO4 at 50 μL / well. Each reaction solution was vortexed evenly and the OD450 nm was measured on a microplate reader. The data were analyzed and the EC was calculated. 50 .

[0197] The experimental results are shown in FIG11 and Table 11 . The affinity of CRIg-L(GS)-FH-FCG4-M86 for C3 did not change after adding the YTE mutation to the Fc terminus.

[0198] Table 11: EC of fusion proteins binding to C3-His protein 50 value

[0199] Example 7 CRIg-L(GS)-FH-FCG4-YTE-M86Y protects human erythrocytes from hemolysis induced by activation of the alternative and classical complement pathways

[0200] The inhibitory effect of CRIg-L(GS)-FH-FCG4-YTE-M86Y on erythrocyte lysis was detected by hemolysis experiment. The experimental method was referred to Example 2.

[0201] The experimental results are shown in Figure 12 and Table 12. The addition of YTE mutation to the Fc terminus of CRIg-L(GS)-FH-FCG4-M86 did not change the inhibitory effect on hemolysis induced by activation of the alternative complement pathway.

[0202] Table 12: IC of the fusion proteins against the activation of the alternative complement pathway and the lysis of erythrocytes 50 value

[0203] Table 13: IC of the fusion proteins against the inhibition of erythrocyte lysis by the classical complement pathway 50 value

[0204] Example 8 Binding affinity of the fusion protein CRIg-L(GS)-FH-FCG4-YTE-M86Y to FcRn

[0205] 8.1 Binding experiments at pH 7.0

[0206] FcRn-his (purchased from Beijing Sino Biological Technology Co., Ltd., catalog number CT009-H08H) was biotinylated according to the method described in Example 6.

[0207] The proteins CRIg-L(GS)-FH-FCG4-M86Y and CRIg-L(GS)-FH-FCG4-YTE-M86Y were diluted to 5 μg / mL with ELISA coating solution and coated on ELISA plates at 100 μL / well. The plates were placed in a humidified chamber at 4°C for 16 h. The ELISA plates were washed three times with pH 7.0 PBST to remove unbound antigens, and the plates were patted dry on absorbent paper to remove excess liquid. The plates were then blocked with 2% BSA prepared in pH 7.0 PBS at 200 μL / well at room temperature for 2 h. The plates were washed once with pH 7.0 PBST to remove excess blocking solution, and the plates were patted dry to remove excess liquid. Biotin-FcRn-his was diluted 3-fold with 1% BSA prepared in pH 7.0 PBST. Protein, with a maximum concentration of 10,000 nM, was diluted 12 times and added to the ELISA wells at 100 μL / well. The plates were incubated at room temperature for 1 hour, and two replicates were prepared for each sample. Unbound or nonspecifically bound primary antibody was washed away with pH 7.0 PBST, and HRP-labeled Anti-Human SA secondary antibody was diluted 1:8000 with 1% BSA prepared in pH 7.0 PBST and added to the ELISA plate at 100 μL / well. The plates were incubated at room temperature for 0.5 hour. The plates were washed five times with pH 7.0 PBST, and the ELISA plates were patted dry on absorbent paper to remove excess liquid. TMB color development solution was added at 100 μL / well and color was developed to the appropriate depth. 2 M H2SO4 was added at 50 μL / well to stop color development. The absorbance was measured at a wavelength of 450 nm in a multifunctional microplate reader for data analysis.

[0208] 8.2 Binding experiments at pH 6.0

[0209] The binding experiment at pH 6.0 was performed in the same manner as that at pH 7.0, except that pH 7.0 PBST was replaced with pH 6.0 PBST.

[0210] The experimental results are shown in Figure 14 and Table 14. After the YTE mutation was added to the Fc terminus of CRIg-L(GS)-FH-FCG4-M86Y, the affinity for FcRn was significantly improved, and the affinity was stronger at pH 6.0. Therefore, in theory, the half-life of CRIg-L(GS)-FH-FCG4-YTE-M86Y in vivo should be longer.

[0211] Table 14: EC of fusion proteins binding to FcRn-His protein 50 value

[0212] Example 9 Therapeutic Effect of Fusion Protein CRIg-L(GS)-FH-FCG4-YTE-M86Y on Passive Heymann Nephritis (PHN) Model in Rats

[0213] Antibodies bind to specific antigens on the foot processes of rat glomerular epithelial cells to form immune complexes, which further activate the complement system through the bypass pathway and the lectin pathway, ultimately leading to large amounts of proteinuria in rats. In the present embodiment, 1 ml of sheep anti-rat Fx1A (rat kidney proximal tubule brush border antigen, purchased from PROBETEX, article number PTX-002S) antiserum was injected into SD male rats via the tail vein to establish a rat passive Heymann nephritis (PHN) model. 25 SD rats were randomly divided into 5 groups, including a blank control group, a PHN model group, and a CRIg-L (GS) -FH-FCG4-YTE-M86Y administration group at a dosage of 5 mg / kg, 10 mg / kg, and 20 mg / kg. Administered twice on the day of modeling and on the 4th day, by tail vein injection, the administration volume was 5 mg / kg. On the 7th day of modeling, 24-hour urine of rats was collected using a metabolic cage, the urine volume was recorded, the urine protein concentration was measured (urine protein quantitative test kit (CBB method) was purchased from Nanjing Jiancheng, product number C035-2-1), and the total 24-hour urine protein was calculated.

[0214] The results are shown in Figure 15. On the seventh day of modeling, the 24-hour urine protein level of the PHN model group rats was significantly increased compared with the blank control group. The urine protein level of the CRIg-L(GS)-FH-FCG4-YTE-M86Y-treated group was significantly lower than that of the model group, and showed a certain dose-dependency. This shows that CRIg-L(GS)-FH-FCG4-YTE-M86Y can effectively bind to C3b in rats, inhibit the activation of the complement system, and alleviate the proteinuria caused by nephritis.

[0215] discuss:

[0216] The present invention unexpectedly discovered that the M86Y mutation in a CRIg variant significantly enhances the binding affinity of the fusion protein for human complement C3 compared to the wild-type protein. Furthermore, the fusion protein containing the M86Y mutation significantly enhances its ability to inhibit erythrocyte lysis by the classical pathway, activation of the alternative pathway, and activation of the classical complement pathway, demonstrating unexpected technical benefits.

[0217] Sequences of the present invention

[0218] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. A bifunctional fusion protein targeting C3b, characterized in that: Include: a) a CRIg domain variant, said variant comprising a mutation selected from the group consisting of M86Y; or a combination of Q64R and M86Y relative to SEQ ID NO: 2; and b) FH domain.

2. The bifunctional fusion protein according to claim 1, characterized in that The bifunctional fusion protein targeting C3b further comprises: c) a linker connecting a) and b); and / or d) Fc domain variants.

3. The bifunctional fusion protein according to claim 1, characterized in that The bifunctional fusion protein comprises a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises the CRIg domain variant, the first FH domain and the first Fc domain variant; the second polypeptide chain comprises the CRIg domain variant, the second FH domain and the second Fc domain variant, The first Fc domain variant and the second Fc domain variant can interact with each other to form a dimer.

4. The bifunctional fusion protein according to claim 2, characterized in that: The Fc domain variant is IgG4-Fc; preferably, it includes an amino acid mutation S228P at position 228 of the Fc sequence of the human IgG4 constant region, and / or comprises a mutation selected from the group consisting of: M252Y / S254T / T256E.

5. The bifunctional fusion protein according to claim 3, characterized in that: The first polypeptide chain and the second polypeptide chain comprise the amino acid sequence shown in any one of SEQ ID NOs. 7-15.

6. A nucleic acid molecule, characterized in that Encoding the bifunctional fusion protein according to any one of claims 1 to 5.

7. An expression vector, characterized in that: Comprising the nucleic acid molecule of claim 6.

8. A host cell, characterized in that The invention comprises the expression vector of claim 7 or expresses the bifunctional fusion protein of any one of claims 1 to 5.

9. The method for preparing the bifunctional fusion protein according to claim 1, characterized in that: The method comprises the following steps: synthesizing the bifunctional fusion protein according to any one of claims 1 to 5, and / or culturing the host cell according to claim 9 under the condition of expressing the bifunctional fusion protein according to any one of claims 1 to 5.

10. A pharmaceutical composition, characterized in that Comprising the bifunctional fusion protein of claim 1 and optionally a pharmaceutically acceptable carrier.

11. Use of the bifunctional fusion protein according to any one of claims 1 to 5 or the pharmaceutical composition according to claim 10 in preparing a drug, characterized in that: The medicament is used for preventing or treating diseases associated with complement activation.

12. Use of the bifunctional fusion protein according to any one of claims 1 to 5 or the pharmaceutical composition according to claim 10 in preparing a drug, characterized in that: The drug is used to prevent or treat paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), generalized myasthenia gravis (gMG), neuromyelitis optica spectrum disease, age-related macular degeneration (AMD), geographic atrophy (GA), autohemolytic anemia, autoimmune thrombocytopenia, aplastic anemia, systemic lupus erythematosus, rheumatoid arthritis, ankylosing spondylitis, atherosclerosis, Parkinson's disease, Alzheimer's disease (senile dementia), asthma, allergy, psoriasis, IgA nephropathy, glomerulonephritis, lupus nephritis, multiple sclerosis, acute respiratory distress syndrome, Inflammatory diseases of the periodontium (including gingivitis and periodontitis).

13. A method for preventing or treating a disease associated with complement activation, characterized in that: The method comprises administering a pharmaceutically effective amount of the fusion protein according to any one of claims 1 to 5 or the pharmaceutical composition according to claim 10 to a subject in need thereof.

14. The method according to claim 13, characterized in that The disease associated with complement activation is selected from paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), generalized myasthenia gravis (gMG), neuromyelitis optica spectrum disease, age-related macular degeneration (AMD), geographic atrophy (GA), autohemolytic anemia, autoimmune thrombocytopenia, aplastic anemia, systemic lupus erythematosus, rheumatoid arthritis, ankylosing spondylitis, atherosclerosis, Parkinson's disease, Alzheimer's disease (senile dementia), asthma, allergies, psoriasis, IgA nephropathy, glomerulonephritis, lupus nephritis, multiple sclerosis, acute respiratory distress syndrome, and inflammatory diseases of periodontal tissues (including gingivitis and periodontitis).

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