CN110128547B

CN110128547B - Human target complement inhibitor protein mCR2-fH and application thereof

Info

Publication number: CN110128547B
Application number: CN201910377299.5A
Authority: CN
Inventors: 唐晓敏; 杜兰英
Original assignee: Beijing Kangpu Meite Innovation Pharmaceutical Technology Co ltd
Current assignee: Beijing Kangpu Meite Innovation Pharmaceutical Technology Co ltd
Priority date: 2019-05-07
Filing date: 2019-05-07
Publication date: 2022-07-22
Anticipated expiration: 2039-05-07
Also published as: CN110128547A

Abstract

The invention discloses a fusion protein of a complement receptor 2 variant and a complement inhibitor fH and application of the fusion protein in preparation of a medicament for treating autoimmune diseases. The complement receptor 2 variant is a molecular modifier obtained by computer modeling and amino acid replacement, has higher ligand binding and dissociation rates 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 arthritis parts after entering a rheumatoid arthritis mouse model, and has a remarkable 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 source target complement inhibitor protein mCR2-fH and application

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 playing a plurality of biological effects of opsonophagocytosis, cell lysis, mediated inflammation, immunoregulation, immune complex removal and the like, 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 mannan-binding agglutination pathway and the alternative pathway. Components involved in the classical complement activation pathway include C1-C9. According to the role of the protein in the activation process, the protein is artificially divided into three groups, namely recognition units (Clq, Clr and Cls), activation units (C4, C2 and C3) and membrane attack units (C5-C9), which respectively play roles in different stages of activation, namely, a recognition stage, an activation stage and a membrane attack stage. 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 cracked into 2 or more fragments, which are 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 activated to polymerize into C567, and the C567 and C3a and C5a can play special biological functions. 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 MASP-1, MASP-2, C4, C2 and C3 are sequentially activated to form C3 and C5 convertase which are the same as those in the classical pathway, and the activation pathway of complement cascade enzymatic reaction is activated. The major activators of the MBL activation pathway are pathogenic microorganisms that contain mannose, fucose and N-galactosamine on their surface. The alternative pathway differs from the classical pathway in that activation crosses three components, C1, C4 and C2, C3 is directly activated and then the chain reaction of C5 to C9 is completed, and factor B is an important component in the alternative pathway of complement activation, also called C3 activator precursor. When circulating factor B binds to activated C3, activation of the alternative pathway is triggered. This complex is then cleaved by circulating factor D to produce the enzymatically active fragment C3 bBb. C3bBb cleaves C3 to produce C3b, which causes inflammation and also further amplifies the activation process, creating a positive feedback loop. The alternative activation pathway is also characterized by substances that activate not antigen-antibody complexes but bacterial cell wall components-lipopolysaccharides, as well as polysaccharides, peptidoglycans, 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.

Factor H is a factor that plays an important inhibitory role in the alternative pathway of complement activation. Factor H was discovered by Nilson et al (1965) and named as β 1H based on electrophoretic position, while Whaley and Ruddy named C3b inactivator accelerator factor. It has been determined that the entire length is a single-chain glycoprotein consisting of 1231 amino acids, with a molecular weight of 155kDa, having both long-rod and spherical regions. The function of the H factor includes the following aspects: (1) is a cofactor of the factor I, and can increase the sensitivity of C4b to the factor I. Factor I binds to C3b with at least 15-fold higher affinity in the presence of factor H than in the absence of factor H. The mechanism of factor I enhancement by factor H may be that after factor H binds to C3b, some conformational change occurs in C3b, increasing the affinity of binding to factor I. The active site for factor H binding to C3b is present in its N-terminal 35kDa portion. (2) Accelerate decay of the C3 convertase: factor H can remove factor B or Bb bound to C3B from C3 enzyme, and make it lose enzyme activity. (3) Preventing the formation of the initial and amplified C3 convertase in the alternative pathway. Factor H and factor B have been shown to have the same binding site on C3B, so factor H can compete with factor B or Bb for binding to C3B. In the presence of factor H, factor B is less likely to bind to C3(H2C) and C3B, and thus C3(H2C) Bb or C3bBb is less likely to form. However, there is a difference in the effect of factor H on C3b on the solid and liquid phases. Can be lysed or bound to C3b on the non-activator solid phase. For C3B immobilized on the surface of activator (such as zymosan, etc.), factor H has the same affinity to C3B as factor B, and as a result of competition, part of C3 convertase can be formed to ensure the activation of alternative pathway. Studies have reported that the chemical components on cell membranes that enhance the affinity of C3b for factor H are sialid and heparin aminopolysaccharides. Since most bacteria lack sialic acid on their surface, these bacteria, when invaded into the body, activate alternative pathways that help to control infection at an early stage. (4) Has certain effect on C3bBbP or C3bBbNeF which is combined with P factor or nephritis factor (NeF) to form stable C3bBbP or C3 bBbNeF. Factor H also inhibits the activity of the C5 convertase (C3bnBb or C3bBb 3b) and competes with C5 for binding to C3b, rendering C5 non-cleavable. In addition, in recent years, factor H has been found to induce IL-1 secretion from monocytes to participate in the regulation of immune responses.

The activated fragment of C3 generated by complement activation acts as a complement opsonin as a ligand for various C3 receptors. 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 (characteristic building blocks 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 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 rapidly converts to inactive iC3b on the cell surface, particularly when C3b is deposited 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. CR2 can therefore serve as an effective targeting vehicle for bringing molecules to the site of complement activation.

Diseases or conditions mediated by excessive or uncontrolled activation of the complement system, particularly by the alternative complement pathway, are causative agents of a variety of diseases or conditions. Downregulation of complement activation has been demonstrated 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, membranoproliferative glomerulonephritis, and sjogren's syndrome.

It has been demonstrated that the delivery of complement inhibitors to sites of complement activation and disease via a targeting vector is an effective therapeutic approach to fully exploit the effects of complement inhibitors. For example, in vitro feasibility studies, targeted CR2-DAF and CR2-CD59 fusion proteins have been shown to be more effective in protecting targeted cells from complement destruction than untargeted inhibitors. CN100594037C reports the beneficial effect of fusion of complement targeting factor CR2 with complement inhibitors DAF and CD59, respectively, on inhibiting different activities. CN101563363B reports the inhibition of alternative complement activation pathway and the therapeutic effect of CR2 and fH fusion protein on related diseases and symptoms. Among them, CR2-fH EC₅₀Between 20 and 30nM, which is 15-20 fold lower than the amount of fH present in this assay, demonstrating a clear benefit of targeting fH over endogenous fH. Also, in the absence of involvement of the classical complement pathway, the alternative complement pathway was inhibited using CR2-fHThere are significant benefits. However, the fusion protein of CR2 and fH still has a certain technical problem in the development of inhibitory activity, for example, the in vivo inhibitory effect is not as good as that of the in vitro inhibitory test, and the protective rate of the animal protection test needs to be further improved. Problems arising from the prior art suggest that the modified spatial conformation obtained by sequence variation of the fusion protein may be a means of promoting full complement inhibitor activity. Therefore, the modification of the fusion protein, especially the modification of the targeting factor CR2, is a new improvement idea.

The invention aims to further carry out molecular reconstruction on CR2 through computer modeling and amino acid replacement so as to improve the specificity of the combination of CR2 and a ligand, improve the molecular structure of CR2 and fH fusion protein as a whole and further improve the inhibition effect of CR2 and fH fusion protein as a targeted complement inhibitor on complement activation.

Disclosure of Invention

Based on the above purpose, the present invention firstly provides a fusion protein of complement receptor 2 variant (mCR2) and complement inhibitor fH, wherein the amino acid sequence of the complement receptor 2 variant is shown in SEQ ID No. 1.

In a preferred embodiment, the complement receptor 2 variant is linked to fH in the flexible short peptide GGGGSGGGGS.

More preferably, the amino acid sequence of the fusion protein is shown as SEQ ID NO.3, and the fusion protein is defined as mCR 2-fH.

Secondly, 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.

Thirdly, the invention also provides a recombinant expression vector containing the nucleotide molecule.

In a preferred embodiment, the vector is pEE14.1-mCR 2-fH.

Fourthly, the invention also provides a host engineering cell containing the expression vector.

In a preferred embodiment, the engineered cell is a CHO-K1-pEE14.1-mCR2-fH cell.

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 modifier obtained by computer modeling and amino acid replacement, has higher ligand binding and dissociation rates than a wild sequence (CR2) and has better ligand binding force. The results of complement-mediated CHO cell and erythrocyte lysis experiments show that mCR2-fH has more obvious inhibition effect than CR 2-fH. The inhibition of 50% CHO cell lysis, mCR2-fH concentration of 34nmol/L, and CR2-fH concentration of 88nmol/L, inhibition efficiency improved by 2 times. Biological distribution experiments prove that the fusion protein provided by the invention can be rapidly highly aggregated at arthritis parts 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, mCR2-fH has better improvement degree than CR2-fH, and has obvious dose dependence, and prove that mCR2-fH has excellent anti-adhesion/anti-inflammatory targeted inhibition effect and has good treatment effect on inflammatory reaction of organisms. The mCR2-fH 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 a mCR2-fH treatment group is 100%. And the symptoms of proteinuria, glomerular integral, interstitial inflammation, vasculitis, crescentic/necrosis and the like of the mCR2-fH treatment group are obviously improved, which shows that the mCR2-fH provided by the invention has excellent application prospect in preparation of autoimmune disease treatment medicines.

Drawings

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

FIG. 2.CR2-fH sequence diagram;

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

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

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

FIG. 6 results of an experiment of mCR2-fH treatment of RA mice;

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

FIG. 8 is a graph comparing the change in proteinuria in MRL/lpr mice treated with mCR 2-fH.

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 illustrative only and do not limit the scope of the present invention in any way.

Computer modeling

The short peptides were displayed in a de novo set-up using the Builder program in the Insight II package. The structure of the compound formed after the combination of CR2 and C3d is simulated by a molecular docking (docking) method, and the stable conformation of the CR2/C3d compound is obtained through the optimization of molecular mechanics and molecular dynamics. The binding sites of the two are analyzed, the core sequences of CR2 and C3d are determined, and the interaction mode and energy of amino acids are obtained when the two are combined. According to the results of the analysis, certain amino acids of CR2 were replaced, and modeled again, to obtain a stable conformation of the CR2 mutant peptide-C3 d complex. The mode and energy of interaction between the CR2 mutant peptide and C3d were analyzed, and the magnitude of affinity when binding to C3d before and after CR2 mutation was compared.

The simulation of the CR2 complex with C3d 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 wild sequence of CR2 before mutation is shown as figure 2).

Example 1 preparation of CR2 mutant (mCR2) -fH 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-fH 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 rabbit antiserum against CHO cell membranes and human fH was obtained according to the method described in Harlow, E., and Lane, D.antibodies: a laboratory Manual.Cold Spring Harbor laboratory.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 fH. The complement inhibitor sequence is 322 amino acids encoding the mature fH protein extracellular domain sequence (Swissprot accession No. P08603.4). Computer simulations of the CR2 and C3d complex were performed to design CR2 mutants that improved their binding to C3d as follows: 1. thr86- -Ser; 2. phe130- - -Tyr; 3. glu133- - -Gln; 4. asp92- - -Glu; 5. thr25- -Ser.

Then mCR2 and CR2 (wild sequences) were linked to complement inhibitor fH, respectively, and mCR2 and CR2 were linked at the N-terminus (mCR2-fH, CR2-fH) with the sequence GGGGSGGGGS. 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 an environment without 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) can inhibit the activity of GS enzyme, and some cell lines (CHO-K1) can produce endogenous glutamine, and can grow in a glutamine-free culture medium containing MSX, and the culture medium added with sufficient MSX to inhibit the activity of GS enzyme provides screening pressure, so as to screen the required cell strains. The mCR2-fH and CR2-fH fusion genes are synthesized by a PCR method and then are connected with pEE14.1 vectors after EcoRI and SmalI double enzyme digestion, the recombinant vectors pEE14.1-mCR2-fH and pEE14.1-CR2-fH are transfected into CHO-K1 host cells by FuGENE 6, and the host cells after transfection of the mCR2-fH are defined as CHO-K1-pEE14.1-mCR 2-fH. After 24 hours of transfection, selective DMEM medium (without glutamine) was changed and MSX was added to a final concentration of 50. mu.M, positive clones were grown for about 2 weeks, while normal CHO cells without the transfected plasmid gradually fell off the flask wall and were lysed and dead. 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-fH and CR2-fH was detected by ELISA (mCR2-fH amino acid sequence is shown in SEQ ID NO.3 and FIG. 1, and CR2-fH amino acid sequence is shown in FIG. 2). The recombinant protein was expressed in a secreted form in CHO cells. The expression amount of the stable CHO cell expression strain cultured in a serum-free medium is higher than that of the stable CHO cell expression strain cultured in a serum-containing medium.

2.3 purification of recombinant proteins in cell culture supernatants were purified by affinity chromatography. The column was prepared by coupling anti-fH 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.

2.4SDS-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 Western blot experiment using murine anti-fH mAb 1H 4.

3. As a result, the

Recombinant proteins (mCR2-fH, CR2-fH) were separated from the culture supernatant of stably transfected CHO cells at 100-200. mu.g/L, and identified by SDS-PAGE (FIG. 3) and Western blot (FIG. 4). In FIG. 3, lane 1 shows a protein molecular weight marker, lane 2 shows mCR2-CD59, and lane 3 shows CR2-CD 59. In FIG. 4, lane 4 shows mCR2-CD59, and lane 5 shows CR2-CD 59. The results show that the target fragment of the expected size appears.

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

Kinetic analysis of the interaction of 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 in the solution of the recombinant N-terminal pair of short consensus/complete domains of complex receptor type 2(CR2/CD21) and interactions with its ligand 3dg. biochemistry.2001,40(20): 5931. sup. 5941) was injected at a rate of 2. mu.L/min onto a BIAcore streptavidin sensor chip for 20min at an average of 50mg/L per flow cell, the buffer being 0.5 XPBS (pH7.4) (containing 0.5g/L Tween 20). 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 ℃ with 0.5 XPBST (0.5g/L Tween20) at a flow rate of 25. mu.L/min.

Kinetic analysis data showed that 1: 1 was most suitable for binding the spherical detection parameters for the reaction model. SPR measurements showed that mCR2-fH had higher binding and dissociation rates than CR2-fH (Table 1). Furthermore, mCR2-fH has a higher affinity than CR 2-fH.

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). Dilution of recombinant fusion proteins with DEMEThen added to NHS before adding 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 2.5X 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, and quantitatively detecting heme in the supernatant with a spectral imager at 413nm 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-fH has obvious inhibition effect compared with non-target fH (sfH), and mCR2-fH has more obvious inhibition effect compared with CR 2-fH. The inhibition of 50% CHO cell lysis, mCR2-fH concentration of 19nmol/L, CR2-fH concentration of 124nmol/L, inhibition efficiency improved by more than 6 times, non-target fH needs 435nmol/L, inhibition efficiency improved by nearly 23 times (Table 2). In addition, fH protects erythrocytes more strongly than CHO cells in cytolytic inhibition experiments.

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

Example 4 in vivo experiments with mCR2-fH fusion protein RA:

1. biological profiling experiment

mCR2-fH, CR2-fH and fH 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 Iodogen¹²⁵Solution I15. mu.l (185MBq) and the labeling tube gently shaken intermittently at room temperature for 15 min. The SEP-PAK C18 column was activated by washing with 5mL each of methanol, distilled water and 0.1% trifluoroacetic acid (TFA); the labeling mixture was loaded onto a column, rinsed with 0.1% TFA; eluting with 60% acetonitrile solution, and collecting the first 1.5mL eluate. Cooling by heatingAfter freeze drying, diluting with PBS solution containing 1mg/mLBSA, subpackaging, and storing in refrigerator at-80 deg.C for use. Male DBA/1J mice were injected subcutaneously into the caudal root of the tail with 0.1mL of collagen II and complete Freund's adjuvant (both purchased from Sigma, USA), and the establishment of a rheumatoid arthritis (CIA) mouse model was strengthened once on day 21 (refer to the establishment of a C57BL/6 mouse CIA model and the preliminary screening of a monitoring system, release military science journal 2004, 6.6.29, 472-. The test and grouping methods are as follows: control group tail vein injection¹²⁵I-fH (2ug), killed by decapitation after 24h, 48h, respectively, blood was collected, tissue organs were removed, weighed and measured for radioactivity (μ Ci), and the results were converted to ID%/g tissue. ② arthritis group tail vein injection¹²⁵And (2ug) treating and detecting the I-fH fusion protein after 24h, 48h, 72h and 96h respectively. ③ mCR2-fH arthritis treatment group, the tail vein injection is once¹²⁵I-mCR2-fH fusion protein (0.25ug), and the same treatment and detection are carried out after 24 h. (CR2-fH arthritis treatment group, tail vein injection once¹²⁵I-CR2-fH 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-fH fusion protein of the present invention is highly aggregated at the arthritic 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 (N ═ 18). (ii) CR2-fH low dose treatment group (N ═ 18) with daily injections of CR2-fH fusion protein (0.25) mg. (iii) CR2-fH high dose treatment group (N-16) injected twice daily with CR2-fH fusion protein (0.25) mg. mCR2-fH low dose treatment group (N18) with mCR2-fH fusion protein (0.25) mg injected once a day. Fifthly, mCR2-fH high dose treatment group (N-14) is injected with mCR2-fH fusion protein (0.25) mg twice a day. 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 paw redness; 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, and from day 23 onwards, the severity of arthritis was significantly lower in both treatment groups of mCR2-fH fusion protein than in the PBS group. Wherein the score of the fifth group (2 injections) is below 3/4 of the fourth group (1 injection), below 3/5 of the third group (CR2-fH two-injection group), below 1/2 of the fourth group (CR2-fH one-injection group), and below 1/3 of the fourth group (PBS control group).

The experimental results prove that the mCR2-fH 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 is obviously higher than the treatment effect of CR 2-fH.

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

1. Improvement of survival rate

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 produce 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%.

Comparison of mouse survival rates of MRL/lpr mice from 16 weeks to 24 weeks of mCR2-fH treated group (n-20), CR2-fH treated group (n-20), and PBS control group (n-22)

This example randomly groups 16-week MRL/lpr mice (shanghai srek) that had developed symptoms of renal failure into three groups, a first group (n-20) that received 0.2mg of mCR2-fH weekly from weeks 16-24, a second group (n-20) that received 0.2mg of CR2-fH weekly from weeks 16-24, a third group (n-22) that was a control group, and an equal amount of PBS weekly from weeks 16-24. The administration routes of the three groups are tail vein injection. The effect of mCR2-fH and CR2-fH targeting on inhibition of complement activation, as well as the protection rate against MRL/lpr lupus erythematosus mice, was evaluated by the administration group (0.2mg mCR 2-fH/week), 0.2mg CR 2-fH/week) and the control group (0.2mg PBS/week).

As shown in the experimental result of FIG. 7, in the mice treated with CR2-fH, C3d in the pathway of complement activation is effectively inhibited by CR2-fH targeting C3d, so the survival rate of MRL/lpr lupus erythematosus mice is obviously improved, the whole treatment process of the mCR2-fH treatment group can completely protect the MRL/lpr lupus erythematosus mice, the survival rate is 100%, the CR2-fH group can also maintain the survival rate of more than 60% even at the 24 th week, and compared with the control group, the survival rate of the mice in the treatment group from 19 weeks is obviously improved.

2. Improvement of renal function

The mice are placed in a metabolism cage to study the influence of mCR2-fH and CR2-fH on the urinary albumin secretion of 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 (Invitrogen Life Technologies) and chloramphenicol (Sigma-Aldrich) were added to the collection tubes. A standard curve is drawn by ELISA method using mouse albumin samples of known concentration, and urine albumin secretion of experimental mice is determined, and creatinine content in mouse urine is determined using a biochemical analyzer (Beckman Coulter). 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, MRL/lpr mice mCR2-fH treatment group (n-24), CR2-fH treatment group (n-22), and PBS control group (n-20) were compared for proteinuria: at weeks 22 and 24, protein urine levels were significantly reduced in the treatment group compared to the control group (P <0.01), and protein urine levels were more reduced in the mCR2-fH treatment group (n ═ 24) than in the CR2-fH treatment group (n ═ 24) (P < 0.01). Proved that mCR2-fH provided by the invention can improve the symptoms of renal function injury more obviously.

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 lesion and 4 being severe lesion. 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 the most severe manifestation, more than 6 layers of cells surrounding. The evaluation results are shown in table 3.

TABLE 3 comparison of Kidney Damage in MRL/lpr mice between week 24 after 16 to 23 weeks of treatment and PBS control

mCR2-fH reduced the renal inflammatory response better than CR2-fH in MRL/lpr lupus erythematosus mice. Compared with the control group, mCR2-fH showed more significant reductions in glomerular integral, interstitial inflammation, vasculitis, and crescentic/necrosis than CR2-fH (P < 0.05).

Sequence listing

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

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

<160> 4

<170> PatentIn version 3.3

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

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

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

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

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

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

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

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

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

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

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

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

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

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Gln Gly Val Ala Trp Thr Lys Met Pro Val Cys

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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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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Gly Gly Gly Gly Ser Lys Met Arg Leu Leu Ala Lys Ile Ile Cys Leu

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Met Leu Trp Ala Ile Cys Val Ala Glu Asp Cys Asn Glu Leu Pro Pro

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Arg Arg Asn Thr Glu Ile Leu Thr Gly Ser Trp Ser Asp Gln Thr Tyr

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Pro Glu Gly Thr Gln Ala Ile Tyr Lys Cys Arg Pro Gly Tyr Arg Ser

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Leu Gly Asn Val Ile Met Val Cys Arg Lys Gly Glu Trp Val Ala Leu

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Asn Pro Leu Arg Lys Cys Gln Lys Arg Pro Cys Gly His Pro Gly Asp

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Thr Pro Phe Gly Thr Phe Thr Leu Thr Gly Gly Asn Val Phe Glu Tyr

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Gly Val Lys Ala Val Tyr Thr Cys Asn Glu Gly Tyr Gln Leu Leu Gly

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Glu Ile Asn Tyr Arg Glu Cys Asp Thr Asp Gly Trp Thr Asn Asp Ile

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Pro Ile Cys Glu Val Val Lys Cys Leu Pro Val Thr Ala Pro Glu Asn

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Gly Gln Ala Val Arg Phe Val Cys Asn Ser Gly Tyr Lys Ile Glu Gly

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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 tgtggcggag gtgggtcggg tggcggcgga 780

tctaaaatga gacttctagc aaagattatt tgccttatgt tatgggctat ttgtgtagca 840

gaagattgca atgaacttcc tccaagaaga aatacagaaa ttctgacagg ttcctggtct 900

gaccaaacat atccagaagg cacccaggct atctataaat gccgccctgg atatagatct 960

cttggaaatg taataatggt atgcaggaag ggagaatggg ttgctcttaa tccattaagg 1020

aaatgtcaga aaaggccctg tggacatcct ggagatactc cttttggtac ttttaccctt 1080

acaggaggaa atgtgtttga atatggtgta aaagctgtgt atacatgtaa tgaggggtat 1140

caattgctag gtgagattaa ttaccgtgaa tgtgacacag atggatggac caatgatatt 1200

cctatatgtg aagttgtgaa gtgtttacca gtgacagcac cagagaatgg aaaaattgtc 1260

agtagtgcaa tggaaccaga tcgggaatac cattttggac aagcagtacg gtttgtatgt 1320

aactcaggct acaagattga aggagatgaa gaaatgcatt gttcagacga tggtttttgg 1380

agtaaagaga aaccaaagtg tgtggaaatt tcatgcaaat ccccagatgt tataaatgga 1440

tctcctatat ctcagaagat tatttataag gagaatgaac gatttcaata taaatgtaac 1500

atgggttatg aatacagtga aagaggagat gctgtatgca ctgaatctgg atggcgtccg 1560

ttgccttcat gtgaagaaaa atcatgtgat aatccttata ttccaaatgg tgactactca 1620

cctttaagga ttaaacacag aactggagat gaaatcacgt accagtgtag aaatggtttt 1680

tatcctgcaa cccggggaaa tacagccaaa tgcacaagta ctggctggat acctgctccg 1740

agatgtacc 1749

Claims

1. A fusion protein of a complement receptor 2 variant and a complement inhibitor fH, wherein the amino acid sequence of the complement receptor 2 variant is as set forth in SEQ ID No. 1.

2. The fusion protein of claim 1, wherein the complement receptor 2 variant is linked to fH in a flexible short peptide GGGGSGGGGS.

3. The fusion protein of claim 2, wherein the amino acid sequence of the fusion protein is set forth in SEQ ID No. 3.

4. A nucleotide molecule encoding the fusion protein of claim 3, wherein the sequence of the nucleotide molecule is as set forth in SEQ ID No. 4.

5. A recombinant expression vector comprising the nucleotide molecule of claim 4.

6. The expression vector of claim 5, wherein the vector is pEE14.1-mCR 2-fH.

7. A host engineered cell comprising the expression vector of claim 4.

8. The host cell of claim 7, wherein the engineered cell is a CHO-K1-pee14.1-mCR2-fH cell.

9. Use of the fusion protein of any one of claims 1-3 for the preparation of a medicament for the treatment of an autoimmune disease.

10. The use according to claim 9, wherein the diseases include rheumatoid arthritis and systemic lupus erythematosus.