CN109929026B - Human target complement inhibitor protein mCR2-DAF and application thereof - Google Patents

Abstract

The invention discloses a complement receptor 2 variant, a fusion protein of the complement receptor 2 variant and a complement inhibitor and application of the fusion protein in preparation of a medicament for treating autoimmune diseases. The complement receptor 2 variant is a molecular modified body obtained by computer modeling and amino acid replacement, has higher ligand binding and dissociation rate than a wild sequence thereof, and has better ligand binding force. Biological distribution experiments prove that the fusion protein provided by the invention can be rapidly highly aggregated at the arthritis part after entering a mouse model with rheumatoid arthritis, and has obvious anti-adhesion/anti-inflammatory targeted inhibition effect. In the treatment of MRL/lpr lupus erythematosus mice, the fusion protein can obviously improve the survival rate of the mice, and the symptoms of proteinuria, glomerular score, interstitial inflammation, vasculitis, crescent/necrosis and the like of the mice in a treatment group are obviously improved.

Description

Human target complement inhibitor protein mCR2-DAF and application thereof

Technical Field

The invention discloses a fusion protein, and belongs to the technical field of polypeptides.

Background

The complement system is composed of more than 30 soluble protein molecules, is a part of the natural immune system, and comprises more than 30 molecules such as complement inherent components, various regulatory factors, complement receptors and the like. The complement system can be activated through 3 relatively independent and interconnected pathways, thereby exerting various biological effects such as opsonophagocytosis, cell lysis, mediated inflammation, immunoregulation and immune complex removal, including phagocytosis enhancement and phagocyte chemotaxis enhancement; increase the permeability of blood vessels; neutralizing the virus; cell lysis; regulation of immune response, etc. Complement activation and its deposition on target structures can also indirectly cause cell or tissue destruction. Complement activation products that mediate tissue damage are produced at various points in the complement pathway. Inappropriate complement activation on host tissues plays an important role in the pathology of many autoimmune and inflammatory diseases, and is also responsible for many conditions associated with, for example, cardiopulmonary inflammation and bioincompatibility following transplant rejection. Complement inhibition is a potential therapeutic modality for the treatment of these immune-mediated diseases and conditions.

There are 3 pathways for complement activation, namely the classical pathway, the alternative pathway and the mannan-binding agglutination pathway. Components involved in the classical complement activation pathway include C1-C9. According to their role in the activation process, they are artificially divided into three groups, namely recognition units (Clq, Clr, Cls), activation units (C4, C2, C3) and membrane attack units (C5-C9), which play roles in different stages of activation, namely, recognition stage, activation stage and membrane attack stage, respectively. These 3 stages are generally performed at 3 different sites of the target cell membrane. During the activation process, C2, C3, C4 and C5 are all split into 2 or more fragments marked with symbols such as a and b, such as C3a, C3b and C3C. Wherein C2b, C3b, C4b and C5b are directly or indirectly bonded on target cells and participate in cytolytic process in the form of solid phase, and C3a and C5a are free in liquid phase. During the activation process, C5, C6 and C7 can be polymerized into C567 after being activated, and the C567 and C3a and C5a can play special biological functions together. The alternative activation pathway differs from the classical activation pathway in that activation crosses three components C1, C4, and C2, directly activates C3 and then completes the chain reaction of the components C5 to C9, and in that the activating substance is not an antigen-antibody complex but a cell wall component of bacteria, i.e., lipopolysaccharide, and polysaccharides, peptidoglycan, teichoic acid, and aggregated IgA and IgG 4. The alternative pathway can play an important role in resisting infection in the early stage of bacterial infection when no specific antibody is produced. Mannan-binding agglutination pathway mannan-binding lectin (MBL) in plasma directly recognizes N-galactosamine or mannose on the surface of various pathogenic microorganisms, and then sequentially activates MASP-1, MASP-2, C4, C2 and C3 to form C3 and C5 convertases which are the same as those of the classical pathway, and activates the activation pathway of complement cascade enzymatic reaction. The major activators of the MBL activation pathway are pathogenic microorganisms that contain mannose, fucose and N-galactosamine on their surface. The three pathways can produce C3 convertase, C3 molecule is cleaved into anaphylatoxin C3a by C3 convertase, and C3b with opsonization, C3b molecule can be linked with amine and hydroxyl on glycoprotein surface covalently, and the covalent is mediated by thioester group in C3b molecule. Thus, the molecule C3b can be adsorbed on the surface of a microorganism invaded in vivo, then combined with complement receptor 1(CR1/CD35), hydrolyzed under the action of serum H factor and I factor to form iC3b, and iC3b is subsequently cleaved to C3 d. The C3d fragment is the smallest fragment of complement C3 that is no longer enzymatically cleaved. Microorganisms that bind to the C3d molecule bind to complement receptor type II (CR2/CD 21).

Various activated fragments of C3 produced by complement activation act as ligands for various C3 receptors as complement opsonins. Complement receptor 2(CR2) is one of the complement receptors, and as a transmembrane protein, CR2 plays an important role in the survival of such cells, especially mature B cells, and the selection of high-affinity B cells, through expression on Follicular Dendritic Cells (FDC), B cells, and some T cells, and binding to immune complexes. CR2 is a member of the C3 family of binding proteins and consists of 15-16 Short Consensus Repeat (SCR) domains (a characteristic building block of the C3 family of binding proteins), with the C3 binding site contained in 2N-terminal SCRs. Unlike complement activation inhibitors (DAF, MCP, CR1 and Crry), CR2 is not a complement inhibitor and it does not bind C3 b. Natural ligands for CR2 are iC3b, C3dg and C3d, which are cell-binding lytic fragments of C3b that bind to the two N-terminal SCR domains of CR 2. Lysis of C3 initially caused the production of C3b and its deposition on the surface of activated cells. Fragment C3b is involved in the generation of an enzyme complex that expands the complement cascade. C3b is rapidly converted to inactive iC3b when C3b is deposited on the cell surface, particularly on the surface of a host containing complement activation modulators (i.e., most host tissues). Even in the absence of membrane bound complement regulators, fairly high levels of iC3b were formed. Subsequently, iC3b was digested by serum proteases into membrane-bound fragments C3dg and C3d, but this process was relatively slow. Thus, the C3 activating fragment ligand of CR2 is relatively long lived once it is produced and is present at high concentrations at the site of complement activation.

Down-regulation of complement activation has been shown in animal models and in vitro studies to be effective for the treatment of several disease indications, such as systemic lupus erythematosus and glomerulonephritis, rheumatoid arthritis, cardiopulmonary bypass and hemodialysis, hyperacute rejection in organ transplantation, myocardial infarction, reperfusion injury, and adult respiratory distress syndrome. In addition, other inflammatory conditions and autoimmune/immune complex diseases are also closely associated with complement activation, including thermal injury, severe asthma, anaphylactic shock, inflammatory bowel disease, rubella, angioedema, vasculitis, multiple sclerosis, myasthenia gravis, mesangial proliferative glomerulonephritis, and sjogren syndrome.

Two broad classes of membrane complement inhibitors have been demonstrated; inhibitors of the complement activation pathway (inhibiting C3 convertase formation), and inhibitors of the terminal complement pathway (inhibiting MAC formation). Membrane inhibitors of complement activation include complement receptor 1(CR1), Decay Accelerating Factor (DAF) and Membrane Cofactor Protein (MCP). They all have a protein structure consisting of a variable number of repeat units of about 60-70 amino acids called Short Consensus Repeats (SCR), a common feature of C3/C4 binding proteins.

Targeting complement inhibitors to complement activation and disease sites may improve their efficacy. Because complement plays an important role in host defense and immune complex catabolism, targeted complement inhibitors may also reduce potentially serious side effects, particularly with long-term complement inhibition. Recently, modified forms of sCR1 decorated with sialyl-Lewis x (sLex) were prepared and shown to bind to endothelial cells expressing P and E selectins. In rodent models of inflammatory disease, it was demonstrated that sCR1sLex is a more effective therapeutic than sCR 1. In vitro feasibility studies, antibody-DAF and antibody-CD 59 fusion proteins were shown to protect targeted cells more effectively from complement destruction than untargeted cells. Non-specific membrane targeting of recombinant complement inhibitors has also been achieved by coupling the inhibitor to a membrane insertion peptide.

The fusion of complement receptor 2, which has a specific targeting effect but no complement inhibitory effect, with a complement inhibitor, thereby exerting a specific inhibitory effect on complement activation is a new therapeutic approach. CN101563363B and CN100594037C report the beneficial effects of fusion of complement receptor 2 with complement inhibitors factor F, DAF and CD59, respectively, on inhibiting different activities. The invention aims to further carry out molecular modification on the complement receptor 2 through computer modeling and amino acid replacement so as to improve the specificity of the complement receptor 2 combined with the ligand thereof and improve the inhibitory effect of the complement inhibitor on complement activation.

Disclosure of Invention

Based on the above objects, the present invention provides a complement receptor 2 variant (mCR2), wherein the amino acid sequence of the complement receptor 2 variant is shown in SEQ ID No. 1.

Secondly, the invention also provides a nucleotide molecule for coding the complement receptor 2 variant, and the sequence of the nucleotide molecule is shown in SEQ ID NO. 2.

Thirdly, the invention also provides an expression vector containing the nucleotide molecule of claim 2, wherein the vector is pEE14.1.

In a fourth aspect, the present invention provides a fusion protein comprising the complement receptor 2 variant described above, wherein the fusion protein further comprises a complement activity modulator.

In a preferred embodiment, the complement activity modulator is a DAF molecule and the fusion protein is defined as mCR 2-DAF.

In a more preferred embodiment, the complement receptor 2 variant is linked to the DAF molecule with a short flexible peptide GGGSGGGGS.

More preferably, the amino acid sequence of the fusion protein is as set forth in SEQ ID NO. 3.

Fifth, the invention also provides a nucleotide molecule for coding the fusion protein, and the sequence of the nucleotide molecule is shown in SEQ ID NO. 4.

Finally, the invention provides the application of the fusion protein in preparing the medicine for treating the autoimmune disease.

In a preferred embodiment, the disease comprises rheumatoid arthritis and systemic lupus erythematosus.

The complement receptor 2 variant disclosed by the invention is a molecular modified body obtained by computer modeling and amino acid replacement, has higher ligand binding and dissociation rate than a wild sequence (CR2) thereof, and has better ligand binding force. The results of complement-mediated CHO cell and erythrocyte lysis experiments show that the mCR2-DAF has more obvious inhibition effect than CR 2-DAF. The inhibition efficiency is improved by 6 times when the concentration of mCR2-DAF is 15nmol/L and the concentration of CR2-DAF is 91 nmol/L. Biological distribution experiments prove that the fusion protein provided by the invention can be rapidly highly aggregated at the arthritis part after entering a mouse model with rheumatoid arthritis, and prove that the complement receptor 2 variant of the invention has specific targeting property to C3d, the mCR2-DAF has better improvement degree than CR2-DAF, and has obvious dose dependence relationship, and the mCR2-DAF is proved to have excellent anti-adhesion/anti-inflammatory targeting inhibition effect and good treatment effect on the inflammatory reaction of an organism. The mCR2-DAF disclosed by the invention can obviously improve the survival rate of mice in the treatment of MRL/lpr lupus erythematosus mice, the MRL/lpr lupus erythematosus mice can be completely protected in the whole treatment process, and the survival rate of an mCR2-DAF treatment group is 100%. And the symptoms of proteinuria, glomerular score, interstitial inflammation, vasculitis, crescent/necrosis and the like of the mCR2-DAF treatment group are obviously improved, which shows that the mCR2-DAF provided by the invention has excellent application prospect in preparing the autoimmune disease treatment medicine.

Drawings

FIG. 1 shows the sequence variation of mCR 2-DAF;

FIG. 2 is a schematic representation of the CR2-DAF sequence;

FIG. 3. 12% SDS-PAGE identification of mCR 2-DAF;

FIG. 4 shows the Western Blot identification of mCR 2-DAF;

FIG. 5 shows the results of the biological distribution experiment of mCR2-DAF in RA mice;

FIG. 6 results of experiments with mCR2-DAF treatment of RA mice;

FIG. 7 is a graph of the survival rate of MRL/lpr mice treated with mCR 2-DAF;

FIG. 8 is a graph comparing changes in proteinuria in MRL/lpr mice treated with mCR 2-DAF.

Detailed Description

The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. These examples are only illustrative and do not limit the scope of the present invention.

Computer modeling

The Builder program in the Insight II package was used to display short peptides in a de novo setting method. A molecular docking (docking) method is utilized to simulate the structure of a compound formed after the CR2 is combined with the C3d, and the stable conformation of the CR2/C3d compound is obtained through optimization of molecular mechanics and molecular dynamics. And analyzing the binding sites of the two, determining the core sequence of CR2 and C3d, and obtaining the interaction mode and energy of amino acids when the two are combined. According to the analysis results, replacing some amino acids of CR2, modeling again, obtained CR2 mutant peptide-C3 d complex stable conformation. The interaction mode and energy between the CR2 mutant peptide and C3d are analyzed, and the affinity size of the CR2 before and after mutation and the binding capacity of the CR 3d are compared.

The simulation of the CR2 and C3d complex was performed initially, and the following mutants were designed to improve their binding to C3 d:

1. thr86- -Ser; 2. phe130- - -Tyr; 3. glu133- - -Gln; 4. asp92- - -Glu; 5. thr 25-Ser (the amino acid sequence after CR2 mutation is shown as SEQ ID NO.1, the nucleotide sequence is shown as SEQ ID NO.2, the mutation site is shown as figure 1, and the CR2 wild sequence before mutation is shown as figure 2).

Example 1 preparation of CR2 mutant peptide-DAF fusion protein

1, selecting pEE14.1(Lonza biologics) as a material expression vector; CHO cells were used for protein expression, and the culture medium was DMEM containing 10% fetal bovine serum, purchased from Invitrogen. Murine anti-DAF mAbs 1H4 and 1A10, murine anti-human CR2mAb 171, anti-sheep red blood cell IgM and all secondary antibodies were purchased from Sigma.

2 method

2.1 preparation of antisera against CHO cell membranes and human DAF from rabbits the antisera were obtained according to the method described in Harlow, E., and Lane, D.antibodies: a laboratory manual, Cold Spring Harbor, New York, USA, 1988:726.

2.2 construction of expression recombinants and protein expression cDNA structural genes were made by linking 4N-terminal SCR units encoding CR2 with the sequence encoding the extracellular domain of DAF. The complement inhibitor sequence is 1-250 bases (Swissprot accession number P08174) encoding the mature DAF protein sequence. Computer modeling of CR2 and C3d complexes was performed to design mCR2 mutants with improved binding to C3d as follows: 1. thr86- -Ser; 2. phe130- - -Tyr; 3. glu133- - -Gln; 4. asp92- - -Glu; 5. thr25- -Ser.

Then, mCR2 and CR2 were linked to the complement inhibitor DAF, respectively, and the sequence was GGGSGGGGS at the N-terminus of mCR2 and CR2 (mCR2-DAF and CR 2-DAF). The gene frame is synthesized by PCR technology (the amino acid sequence of the fusion protein is shown as SEQ ID NO.3, and the nucleotide sequence is shown as SEQ ID NO. 4). All cloning steps were performed on the pee14.1 vector (see CN 104940953A). The pEE14.1 high-efficiency expression vector contains a special Glutamine (GS) gene expression system. The GS enzyme is responsible for the synthesis of glutamine using glutamyl and amine as substrates. Some mammalian cell lines do not contain the GS gene and therefore cannot survive in the absence of glutamine. For these cell lines, the transfected GS gene can be used as a selection marker to select for cells grown in glutamine-free medium. Methionine imine (MSX) inhibits the activity of GS enzyme, certain cell lines (CHO-K1) produce endogenous glutamine and can grow in glutamine-free medium containing MSX, and the medium added with sufficient MSX to inhibit GS enzyme activity provides screening pressure to screen out the desired cell lines. The mCR2-DAF and CR2-DAF fusion genes are synthesized by a PCR method, then are connected with a pEE14.1 vector after EcoRI and SmalI double enzyme digestion, the recombinant vectors pEE14.1-mCR2-DAF and pEE14.1-CR2-DAF are transfected into CHO-K1 cells by FuGENE 6, after 24 hours of transfection, a selective DMEM medium (without glutamine) is replaced, MSX is added until the final concentration is 50 mu M, positive clones are grown in about 2 weeks, and normal CHO cells without transfected plasmids gradually fall off from the bottle wall, and the cells are cracked and die. Selecting 3 cells, culturing with serum-containing medium and serum-free medium for 3 days, collecting cell supernatant, partially concentrating with PEG20000 for 5 times, and storing at-80 deg.C. The expression of mCR2-DAF and CR2-DAF was detected by ELISA (mCR2-DAF amino acid sequence is shown in SEQ ID NO.3 and FIG. 1, and CR2-DAF amino acid sequence is shown in FIG. 2). The recombinant protein was expressed in a secreted form in CHO cells. The expression level of the CHO cell stable expression strain cultured in the serum-free medium is higher than that of the CHO cell stable expression strain cultured in the serum-containing medium.

2.3 purification of recombinant proteins from cell culture supernatants were purified by affinity chromatography. The column was prepared by coupling anti-DAF mAb (1H4) to HiTrap NHS activated affinity column according to the manual. The pH of the recombinant protein-containing culture supernatant was adjusted to 8.0, and the supernatant was passed through the column at a rate of 0.5 mL/min. The column was then washed with 6-8 times the volume of PBS and the recombinant protein was eluted with 2-3 times the column volume of 0.1mol/L glycine (pH 2.4). The fusion protein was collected in 1mol/L Tris buffer (pH8.0), and dialyzed against PBS.

2.4 SDS-PAGE and Western blot identification

The purified protein was carried out on SDS-PAGE gels containing 100 g/L. The gel was stained with Coomassie Brilliant blue. Recombinant proteins were detected in a Westernblot experiment using murine anti-DAF mAb 1H 4.

3. Results

Recombinant proteins (mCR2-DAF, CR2-DAF) were isolated at 100-200. mu.g/L from culture supernatants of stably transfected CHO cells, and the results of SDS-PAGE (FIG. 3) and Western blot (FIG. 4) showed the appearance of target fragments of the expected size.

Example 2 analysis of kinetics of interaction of CR2 fusion protein with C3 ligand

Kinetic analysis of the interaction of the CR2 fusion protein with biotin-labeled C3dg (C3 dg-biotin) was detected using a Surface Plasmon Resonance (SPR) detection system (BIAcore3000 instrument). Human C3 dg-biotin (Guthridge, J.M., et al. structural students analysis of the recombinant N-terminal pair of short consensus/complementary domains of complementary receptors type 2(CR2/CD21) and interactions with ligands ligand 3 d. biochemistry.2001,40(20): 5931. 5941.) was injected at a rate of 2. mu.L/min onto a BIAcore streptavidin sensor chip for 20min, the buffer being 0.5 XPBS (pH7.4) (containing 0.5g/L Tween20) at an average amount of 50mg/L per flow cell. The BIAcore reaction units (ranging from 250 to 500) were generated from SPR signals acquired on captured C3dg. The group without the fusion protein was used as a control. Affinity was assessed by measuring the concentration of CR2 fusion protein (15.6-500 nmol/L) after washing at 25. mu.L/min with 0.5 XPBST (0.5g/LTween20) at 25. mu.L/min.

Kinetic analysis data showed that 1: 1 was best suited to fit the spherical detection parameters for the reaction model. SPR measurements showed that mCR2-DAF had higher binding and dissociation rates than it did at CR2-DAF (Table 1). Furthermore, mCR2-DAF has a higher affinity than CR 2-DAF.

TABLE 1 kinetic parameters for the binding of recombinant fusion proteins to C3 dg-biotin

Example 3 complement lysis assay

To determine complement inhibitory activity, 60% -80% of the fused CHO cells were separated with EDTA, washed 2 times with DMEM, and then resuspended in DMEM to a final concentration of 10⁶Individual cells/mL. Adding 100mL/L rabbit anti-CHO cell membrane antiserum into the cell suspension, and acting at 4 deg.C for 30min to sensitize the cells. The antiserum was then discarded and the cells resuspended in NHS diluted in DMEM to a final volume of 50. mu.L or 100. mu.L. The cells were incubated at 37 ℃ for 60min and finally cell viability was measured by the placental blue staining exclusion method (both live and dead cells were counted). The recombinant fusion protein was diluted with DEME, added to NHS, and then added to the CHO cell suspension. The final concentration was based on the control CHO cell lysis at which 100g/L NHS resulted in approximately 90% antibody sensitization. Complement-mediated inhibition of erythrolysis experiments sheep erythrocytes sensitized with antibody (EAs) were tested. Hemolysis assay was performed in gelatin phorona buffer (GVB)⁺⁺) In a final volume of 300. mu.L, containing 2.5 × 10⁷EAs, NHS were diluted 1: 300. The reaction mixture was incubated at 37 ℃ for 60min and finally stopped by adding 300. mu.L of a solution containing 10mmol/L EDTA-PBS. Centrifuging, collecting supernatant at 413nmAnd (3) quantitatively detecting the heme in the supernatant by using a spectral imager at the wavelength.

Detection of the activity of the fusion protein complement inhibitor: the results of complement-mediated CHO cell and erythrocyte lysis experiments show that CR2-DAF has a remarkable inhibiting effect compared with non-targeting sDAF, and mCR2-DAF has a more remarkable inhibiting effect compared with CR 2-DAF. The inhibition effect is 6 times by inhibiting 50% of CHO cell lysis, the concentration of mCR2-DAF is 15nmol/L, the concentration of CR2-DAF is 91nmol/L, the inhibition efficiency is increased by 415nmol/L for non-target sDAF, and the inhibition efficiency is increased by nearly 30 times (Table 2). In addition, sDAF has a stronger protective effect on erythrocytes than CHO cells in cytolytic inhibition experiments.

TABLE 2 concentration of complement inhibitors that inhibit lysis of 50% of cells

Example 4 in vivo experiment of CR2 mutant-DAF fusion protein RA:

1. biological profiling experiment

mCR2-DAF, CR2-DAF and DAF were labeled separately by the Iodogen method¹²⁵I, 150. mu.l of 50mmLo/L PBS (pH7.4), 100. mu.l (100. mu.g) of ScFv containing 1mg of BSA dissolved therein, and Na were sequentially added to an EP tube coated with 200. mu.g of Iodogen¹²⁵Washing the labeled mixture with methanol, double distilled water and 0.1% trifluoroacetic acid (TFA) for 5mL respectively at room temperature by slightly shaking the labeled tube at intervals for 15min, eluting the labeled mixture with SEP-PAK C18 column, eluting the labeled mixture with 0.1% TFA, collecting the first 1.5mL of eluent, freezing and drying, diluting with 1mg/mL of BSA-containing PBS solution, packaging, storing in a refrigerator at-80 deg.C, injecting 0.1mL of collagen II and complete Freund's adjuvant (purchased from Sigma USA) subcutaneously into the tail root of male DBA/1J mice, and building a rheumatoid arthritis (CIA) mouse model once on day 21 (model building refers to building of C57BL/6 mouse CIA model and preliminary screening of monitoring system, release of IV 29 th volume of Jun No. 6 2004), ① test and grouping method are as follows, the control group is¹²⁵I- -DAF (2ug), end-breaking and sacrifice after 24h, 48h, respectivelyBlood was collected, tissue organs were removed, weighed and radioactivity (μ Ci) determined, and the results were converted to ID%/g tissue ② arthritis group tail vein injection¹²⁵I- -DAF fusion protein (2ug), after 24h, 48h, 72h and 96h, respectively, the same treatment and detection were adopted, ③ mCR2-DAF arthritis treatment group, tail vein injection was performed once¹²⁵I-mCR2-DAF fusion protein (0.25ug), the same treatment and detection are adopted after 24h, ④ CR2-DAF arthritis treatment group is injected into tail vein once¹²⁵I-CR2-DAF fusion protein (0.25ug), and after 24h, the same treatment and detection are carried out. The results are shown in fig. 5, which indicates that the mCR2-DAF fusion protein of the present invention can be highly aggregated at the arthritis site (the top-down sequence of the legend in fig. 5 corresponds to the left-to-right sequence of the sublibraries in each of the grouped bar charts in fig. 5).

2. Treatment of rheumatoid arthritis (CIA) mouse model

The test was started from day 21 using the rheumatoid arthritis (CIA) mouse model (supra) and grouped as follows: injection of 50 μ l PBS control (N15). (ii) CR2-DAF low dose treatment group (N ═ 13) with CR2-DAF fusion protein (0.25) mg injected once a day. CR2-DAF high dose treatment group (N ═ 14) with two daily injections of CR2-DAF fusion protein (0.25) mg. mCR2-DAF low dose treatment group (N12) with one daily injection of CR2-DAF fusion protein (0.25) mg. mCR2-DAF high dose treatment group (N14) injected twice daily with CR2-DAF fusion protein (0.25) mg. Clinical scoring started on day 23 and animals were scored for arthritis according to the following criteria: 0 point, no arthritis; score 1, mild inflammation and redness of the paw; 2 points, severe erythema and swelling, affecting the function of the paw; in 3 minutes, the claws or joints become deformed, stiff and lose their functions. The maximum total limb score of each mouse is 12. The results are shown in figure 6, where the severity of arthritis was significantly lower in both mCR2-DAF fusion protein treatment groups than in the PBS group from day 23. Wherein the score of the fifth group (2 injections) is 3/5 or less of the fourth group (1 injection), 1/2 or less of the third group (CR2-DAF two injections), 1/3 or less of the fourth group (CR2-DAF one injection), and 1/4 or less of the fourth group (PBS control).

The experimental results prove that the mCR2-DAF fusion protein has specific targeting property on C3d, has better anti-adhesion/anti-inflammatory targeting inhibition effect, has good treatment effect on inflammatory reaction of organisms, and has a treatment effect obviously higher than that of CR 2-DAF.

Example 5 therapeutic Effect of mCR2-DAF and CR2-DAF in MRL/lpr lupus erythematosus mice

1. Improvement of survival rate

Comparison of survival rates of MRL/lpr mice from 16 to 24 weeks mCR2-DAF treated (n-24) versus CR2-DAF treated (n-26) and PBS control (n-24) mice

The MRL/lpr lupus erythematosus mouse model, which was first established in 1979 by Murphy and Roths, was made from multiple strains of mice through a complex hybridization process over 12 generations, and has 75% of its genes from LG/J, 12.6% from AKR/J, 12.1% from C3H/Di, and 0.3% from C57BL/6 strain mice. MRL/lpr mice contain recessive mutations in the Fas gene associated with spontaneous apoptosis of cells, the appearance of lymphoproliferative genes, resulting in T cell proliferation, generalized lymphadenectasis, and erosive arthritis, anti-DNA, anti-Sm, anti-Su, anti-nucleoside P antibodies, high titer ANA, hypergammaglobulinemia, and rheumatoid factor. The mouse was first developed at 8 weeks when autoantibodies were detectable in the serum. Lymphadenitis was observed at 12 weeks. At 12-16 weeks, MRL/lpr mice began to develop large amounts of autoantibodies, including anti-double stranded DNA antibodies. Multiple organs were involved at the age of approximately 16 weeks and stable deterioration of renal function characterized by severe proteinuria occurred. 16-24 weeks old mice develop proliferative immune complex mediated glomerulonephritis, vasculitis, and eventually death due to renal failure, with a mortality rate of 50%.

This example randomly groups 16-week-old MRL/lpr mice that had developed renal failure symptoms into three groups, the first group (n-24) received 0.2mg mCR2-DAF weekly from week 16-24, the second group (n-26) received 0.2mg cr2-DAF weekly from week 16-24, the third group (n-24) was a control group, and an equal amount of PBS weekly from week 16-24. The administration routes of the three groups are tail vein injection. The protection rate of mCR2-DAF and CR2-DAF on MRL/lpr lupus erythematosus mice was evaluated according to the survival rate of the administration group and the control group.

As shown in FIG. 7, the survival rate of MRL/lpr lupus erythematosus mice is significantly improved in the mice treated with CR2-DAF, since C3d in the complement activation pathway is effectively inhibited by CR2-DAF targeting C3d, the survival rate of MRL/lpr lupus erythematosus mice is completely protected by the whole treatment process of the mCR2-DAF treatment group, the survival rate is 100%, the survival rate of CR2-DAF group is maintained at 70% or more even at week 24, and the survival rate of mice in the treatment group from week 19 is significantly improved compared with the control group.

2. Improvement of renal function

The mice were placed in metabolic cages to study the effect of mCR2-DAF, CR2-DAF on urinary albumin secretion in MRL/lpr lupus erythematosus mice. 24 hour urine from mice was collected every two weeks starting at 16 weeks. To prevent bacterial growth ampicillin, gentamicin and chloramphenicol were added to the collection tubes. A standard curve is drawn by an ELISA method by using mouse albumin samples with known concentrations, urine albumin secretion of experimental mice is determined, and creatinine content in mouse urine is determined by using a biochemical analyzer. The final evaluation results are expressed as urinary albumin (mg) to creatinine (mg) ratio for 24 hours per experimental mouse. A higher urinary albumin creatinine ratio indicates impaired kidney function. As shown in fig. 8, the mCR2-DAF treated group (n ═ 18) in MRL/lpr mice showed significantly lower protein urine levels (P <0.01) than those in CR2-DAF treated group (n ═ 20) and PBS control group (n ═ 20) at week 22 and 24, and the mCR2-DAF treated group (n ═ 18) showed much lower protein urine levels (P <0.01) than those in CR2-DAF treated group (n ═ 20) at week 22 and week 24. Proved that the mCR2-DAF provided by the invention can obviously improve the symptoms of renal function injury.

3. Reduction of inflammatory response in kidney

After the experiment is finished, the kidney of the excised mouse is longitudinally dissected into two halves, wherein one half is subjected to immunofluorescence analysis, the other half is fixed by 10% neutral formaldehyde, the other half is subjected to solid paraffin embedding and sectioning, the sectioning of the kidney tissue processed by paraffin is dyed by a hematoxylin-eosin dyeing method and a periodic acid snowflake dyeing method, glomerulonephritis, hyperplasia, crescent moon formation and necrosis symptoms observed by the sectioning are respectively graded by a blind method, and meanwhile, the change of renal interstitium is also graded. The scores were divided into five grades of 0, 1, 2, 3 and 4, with 0 being no damage and 4 being severe damage. Perivascular inflammatory exudation was evaluated in a semi-quantitative manner by blinding two independent observers on more than 10 vessels per section. Inflammation was scored as 0-3, 0 as no inflammation, 1 as less than 50% of the vessels surrounded by 3 layers of cells, 2 as more than 50% of the vessels surrounded by 3-6 layers, 3 as most severely represented, more than 6 layers surrounded by cells. The evaluation results are shown in table 3.

TABLE 3 comparison of renal Damage in MRL/lpr mice between week 24 and week 23 after treatment and PBS control

Grouping	Glomerular score	Inflammation of the interstitium	Vasculitis	Crescentic/necrosis
					PBS control group (n ═ 14)	12.8±4.2	3.6±0.6	100％	65％
CR2-DAF treatment group (n ═ 16)	8.7±3.1	3.1±0.5	75％	50％
					mCR2-DAF treatment group (n ═ 14)	5.4±2.6	2.0±0.4	60％	20％

Compared with the control group, mCR2-DAF is more obviously reduced than CR2-DAF in glomerular integral, interstitial inflammation, vasculitis, crescent/necrosis and the like (P < 0.05).

Sequence listing

<110> Beijing Congpumet Innovation medicine science and technology, Limited liability company

<120> human source targeting complement inhibitor protein mCR2-DAF and application

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<170>SIPOSequenceListing 1.0

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<213>Homo sapiens

<400>1

Ile Ser Cys Gly Ser Pro Pro Pro Ile Leu Asn Gly Arg Ile Ser Tyr

1 5 10 15

Tyr Ser Thr Pro Ile Ala Val Gly Ser Val Ile Arg Tyr Ser Cys Ser

20 25 30

Gly Thr Phe Arg Leu Ile Gly Glu Lys Ser Leu Leu Cys Ile Thr Lys

35 40 45

Asp Lys Val Asp Gly Thr Trp Asp Lys Pro Ala Pro Lys Cys Glu Tyr

50 55 60

Phe Asn Lys Tyr Ser Ser Cys Pro Glu Pro Ile Val Pro Gly Gly Tyr

65 70 75 80

Lys Ile Arg Gly Ser Ser Pro Tyr Arg His Gly Glu Ser Val Thr Phe

85 90 95

Ala Cys Lys Thr Asn Phe Ser Met Asn Gly Asn Lys Ser Val Trp Cys

100 105 110

Gln Ala Asn Asn Met Trp Gly Pro Thr Arg Leu Pro Thr Cys Val Ser

115 120 125

Val Tyr Pro Leu Gln Cys Pro Ala Leu Pro Met Ile His Asn Gly His

130 135 140

His Thr Ser Glu Asn Val Gly Ser Ile Ala Pro Gly Leu Ser Val Thr

145 150 155 160

Tyr Ser Cys Glu Ser Gly Tyr Leu Leu Val Gly Glu Lys Ile Ile Asn

165 170 175

Cys Leu Ser Ser Gly Lys Trp Ser Ala Val Pro Pro Thr Cys Glu Glu

180 185 190

Ala Arg Cys Lys Ser Leu Gly Arg Phe Pro Asn Gly Lys Val Lys Glu

195 200 205

Pro Pro Ile Leu Arg Val Gly Val Thr Ala Asn Phe Phe Cys Asp Glu

210 215 220

Gly Tyr Arg Leu Gln Gly Pro Pro Ser Ser Arg Cys Val Ile Ala Gly

225 230 235 240

Gln Gly Val Ala Trp Thr Lys Met Pro Val Cys

245 250

<210>2

<211>753

<212>DNA

<213>Homo sapiens

<400>2

atttcttgtg gctctcctcc gcctatccta aatggccgga ttagttatta ttctaccccc 60

attgctgttg gttccgtgat aaggtacagt tgttcaggta ccttccgcct cattggagaa 120

aaaagtctat tatgcataac taaagacaaa gtggatggaa cctgggataa acctgctcct 180

aaatgtgaat atttcaataa atattcttct tgccctgagc ccatagtacc aggaggatac 240

aaaattagag gctcttcacc ctacagacat ggtgaatctg tgacatttgc ctgtaaaacc 300

aacttctcca tgaacggaaa caagtctgtt tggtgtcaag caaataatat gtgggggccg 360

acacgactac caacctgtgt aagtgtttac cctctccagt gtccagcact tcctatgatc 420

cacaatggac atcacacaag tgagaatgtt ggctccattg ctccaggatt gtctgtgact 480

tacagctgtg aatctggtta cttgcttgtt ggagaaaaga tcattaactg tttgtcttcg 540

ggaaaatgga gtgctgtccc ccccacatgt gaagaggcac gctgtaaatc tctaggacga 600

tttcccaatg ggaaggtaaa ggagcctcca attctccggg ttggtgtaac tgcaaacttt 660

ttctgtgatg aagggtatcg actgcaaggc ccaccttcta gtcggtgtgt aattgctgga 720

cagggagttg cttggaccaa aatgccagta tgt 753

<210>3

<211>510

<212>PRT

<213>Homo sapiens

<400>3

Ile Ser Cys Gly Ser Pro Pro Pro Ile Leu Asn Gly Arg Ile Ser Tyr

1 5 10 15

Tyr Ser Thr Pro Ile Ala Val Gly Ser Val Ile Arg Tyr Ser Cys Ser

20 25 30

Gly Thr Phe Arg Leu Ile Gly Glu Lys Ser Leu Leu Cys Ile Thr Lys

35 40 45

Asp Lys Val Asp Gly Thr Trp Asp Lys Pro Ala Pro Lys Cys Glu Tyr

50 55 60

Phe Asn Lys Tyr Ser Ser Cys Pro Glu Pro Ile Val Pro Gly Gly Tyr

65 70 75 80

Lys Ile Arg Gly Ser Ser Pro Tyr Arg His Gly Glu Ser Val Thr Phe

85 9095

Ala Cys Lys Thr Asn Phe Ser Met Asn Gly Asn Lys Ser Val Trp Cys

100 105 110

Gln Ala Asn Asn Met Trp Gly Pro Thr Arg Leu Pro Thr Cys Val Ser

115 120 125

Val Tyr Pro Leu Gln Cys Pro Ala Leu Pro Met Ile His Asn Gly His

130 135 140

His Thr Ser Glu Asn Val Gly Ser Ile Ala Pro Gly Leu Ser Val Thr

145 150 155 160

Tyr Ser Cys Glu Ser Gly Tyr Leu Leu Val Gly Glu Lys Ile Ile Asn

165 170 175

Cys Leu Ser Ser Gly Lys Trp Ser Ala Val Pro Pro Thr Cys Glu Glu

180 185 190

Ala Arg Cys Lys Ser Leu Gly Arg Phe Pro Asn Gly Lys Val Lys Glu

195 200 205

Pro Pro Ile Leu Arg Val Gly Val Thr Ala Asn Phe Phe Cys Asp Glu

210 215 220

Gly Tyr Arg Leu Gln Gly Pro Pro Ser Ser Arg Cys Val Ile Ala Gly

225 230 235 240

Gln Gly Val Ala Trp Thr Lys Met Pro Val Cys Gly Gly Gly Ser Gly

245 250255

Gly Gly Gly Ser Asp Cys Gly Leu Pro Pro Asp Val Pro Asn Ala Gln

260 265 270

Pro Ala Leu Glu Gly Arg Thr Ser Phe Pro Glu Asp Thr Val Ile Thr

275 280 285

Tyr Lys Cys Glu Glu Ser Phe Val Lys Ile Pro Gly Glu Lys Asp Ser

290 295 300

Val Ile Cys Leu Lys Gly Ser Gln Trp Ser Asp Ile Glu Glu Phe Cys

305 310 315 320

Asn Arg Ser Cys Glu Val Pro Thr Arg Leu Asn Ser Ala Ser Leu Lys

325 330 335

Gln Pro Tyr Ile Thr Gln Asn Tyr Phe Pro Val Gly Thr Val Val Glu

340 345 350

Tyr Glu Cys Arg Pro Gly Tyr Arg Arg Glu Pro Ser Leu Ser Pro Lys

355 360 365

Leu Thr Cys Leu Gln Asn Leu Lys Trp Ser Thr Ala Val Glu Phe Cys

370 375 380

Lys Lys Lys Ser Cys Pro Asn Pro Gly Glu Ile Arg Asn Gly Gln Ile

385 390 395 400

Asp Val Pro Gly Gly Ile Leu Phe Gly Ala Thr Ile Ser Phe Ser Cys

405 410 415

Asn Thr Gly Tyr Lys Leu Phe Gly Ser Thr Ser Ser Phe Cys Leu Ile

420 425 430

Ser Gly Ser Ser Val Gln Trp Ser Asp Pro Leu Pro Glu Cys Arg Glu

435 440 445

Ile Tyr Cys Pro Ala Pro Pro Gln Ile Asp Asn Gly Ile Ile Gln Gly

450 455 460

Glu Arg Asp His Tyr Gly Tyr Arg Gln Ser Val Thr Tyr Ala Cys Asn

465 470 475 480

Lys Gly Phe Thr Met Ile Gly Glu His Ser Ile Tyr Cys Thr Val Asn

485 490 495

Asn Asp Glu Gly Glu Trp Ser Gly Pro Pro Pro Glu Cys Arg

500 505 510

<210>4

<211>1530

<212>DNA

<213>Homo sapiens

<400>4

atttcttgtg gctctcctcc gcctatccta aatggccgga ttagttatta ttctaccccc 60

attgctgttg gttccgtgat aaggtacagt tgttcaggta ccttccgcct cattggagaa 120

aaaagtctat tatgcataac taaagacaaa gtggatggaa cctgggataa acctgctcct 180

aaatgtgaat atttcaataa atattcttct tgccctgagc ccatagtacc aggaggatac 240

aaaattagag gctcttcacc ctacagacat ggtgaatctg tgacatttgc ctgtaaaacc 300

aacttctcca tgaacggaaa caagtctgtt tggtgtcaag caaataatat gtgggggccg 360

acacgactac caacctgtgt aagtgtttac cctctccagt gtccagcact tcctatgatc 420

cacaatggac atcacacaag tgagaatgtt ggctccattg ctccaggatt gtctgtgact 480

tacagctgtg aatctggtta cttgcttgtt ggagaaaaga tcattaactg tttgtcttcg 540

ggaaaatgga gtgctgtccc ccccacatgt gaagaggcac gctgtaaatc tctaggacga 600

tttcccaatg ggaaggtaaa ggagcctcca attctccggg ttggtgtaac tgcaaacttt 660

ttctgtgatg aagggtatcg actgcaaggc ccaccttcta gtcggtgtgt aattgctgga 720

cagggagttg cttggaccaa aatgccagta tgtggaggtg ggtcgggtgg cggcggatcc 780

gactgtggcc ttcccccaga tgtacctaat gcccagccag ctttggaagg ccgtacaagt 840

tttcccgagg atactgtaat aacgtacaaa tgtgaagaaa gctttgtgaa aattcctggc 900

gagaaggact cagtgatctg ccttaagggc agtcaatggt cagatattga agagttctgc 960

aatcgtagct gcgaggtgcc aacaaggcta aattctgcat ccctcaaaca gccttatatc 1020

actcagaatt attttccagt cggtactgtt gtggaatatg agtgccgtcc aggttacaga 1080

agagaacctt ctctatcacc aaaactaact tgccttcaga atttaaaatg gtccacagca 1140

gtcgaatttt gtaaaaagaa atcatgccct aatccgggag aaatacgaaa tggtcagatt 1200

gatgtaccag gtggcatatt atttggtgca accatctcct tctcatgtaa cacagggtac 1260

aaattatttg gctcgacttc tagtttttgt cttatttcag gcagctctgt ccagtggagt 1320

gacccgttgc cagagtgcag agaaatttat tgtccagcac caccacaaat tgacaatgga 1380

ataattcaag gggaacgtga ccattatgga tatagacagt ctgtaacgta tgcatgtaat 1440

aaaggattca ccatgattgg agagcactct atttattgta ctgtgaataa tgatgaagga 1500

gagtggagtg gcccaccacc tgaatgcaga 1530

Claims (10)

1. A complement receptor 2 variant, wherein the amino acid sequence of said complement receptor 2 variant is as set forth in SEQ id No. 1.

2. A nucleotide molecule encoding the complement receptor 2 variant of claim 1, wherein the nucleotide molecule has the sequence set forth in SEQ ID No. 2.

3. An expression vector comprising the nucleotide molecule of claim 2, wherein said vector is pee 14.1.

4. A fusion protein comprising the complement receptor 2 variant of claim 1, wherein the fusion protein further comprises a complement activity modulator.

5. The fusion protein of claim 4, wherein the complement activity modulator is a DAF molecule.

6. The fusion protein of claim 5, wherein the complement receptor 2 variant is linked to the DAF molecule with a flexible short peptide GGGSGGGGS.

7. The fusion protein of claim 6, wherein the amino acid sequence of the fusion protein is as set forth in SEQ ID No. 3.

8. A nucleotide molecule encoding the fusion protein of claim 7, wherein the sequence of the nucleotide molecule is as set forth in SEQ ID No. 4.

9. Use of a fusion protein according to any one of claims 5 to 7 for the manufacture of a medicament for the treatment of an autoimmune disease.

10. The use of claim 9, wherein the disease comprises rheumatoid arthritis and systemic lupus erythematosus.

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