CN110128547A - Source of people targeted complement inhibitor albumen mCR2-fH and application - Google Patents

Source of people targeted complement inhibitor albumen mCR2-fH and application Download PDF

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CN110128547A
CN110128547A CN201910377299.5A CN201910377299A CN110128547A CN 110128547 A CN110128547 A CN 110128547A CN 201910377299 A CN201910377299 A CN 201910377299A CN 110128547 A CN110128547 A CN 110128547A
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fusion protein
mcr2
gly
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ser
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唐晓敏
杜兰英
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Beijing Compmet Innovative Medicine Technology Co Ltd
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Abstract

The invention discloses the fusion proteins and the fusion protein of a kind of complement receptor 2 variant and complement inhibitor fH to prepare the application in Parmaceutical for treating disease of autoimmunization system object.The complement receptor 2 variant is the molecule modifier obtained after computer modeling, amino acid replacement, has higher ligand binding and dissociation rate than its wild type sequence, has better ligand binding affinity.Biodistribution is it is demonstrated experimentally that fusion protein provided by the invention enters after rheumatoid arthritis mouse model to have quickly in arthritis position high aggregation and significant anti-stick/anti-inflammatory targeted inhibition effect.In the treatment to MRL/lpr lupus erythematosus mouse, the fusion protein can be obviously improved the survival rate of mouse, the symptoms such as albuminuria, Glomerular score, interstitial inflammation, vasculitis and the crescent/necrosis for the treatment of group mouse be improved significantly.

Description

Source of people targeted complement inhibitor albumen mCR2-fH and application
Technical field
The invention discloses a kind of fusion proteins, belong to technical field of polypeptide.
Background technique
Complement system is a part of natural immune system, constituent by more than 30 soluble protein molecular compositions Including 30 different kinds of molecules such as complement proper constituent, a variety of regulatory factors and complement receptors.Complement system can be both relatively only by 3 The vertical approach connected each other again is activated, and exempts to play opsonophagocytosis, lytic cell, transmitting inflammation, immunological regulation and removing The various biologicals effect such as epidemic disease compound, including enhancing phagocytosis, enhance the chemotaxis of phagocyte；Increase the penetrating of blood vessel Property；Neutralize virus；Cytolysis；The adjustment effect etc. of immune response.Complement activation and its deposition on target structure Cell or tissue can be caused to destroy indirectly.The complement activation that each point in complement pathway generates mediating tissue damage produces Object.Unsuitable complement activation plays an important role in the pathology of many autoimmunity diseases and inflammatory disease on host tissue, It and is also the reason of causing many symptom related with the bio-incompatibility after such as cardiopulmonary inflammation and graft rejection.Complement Inhibition is to treat the potential treatment mode of these immune-mediated diseases and symptom.
The approach of complement activation has 3, i.e. classical pathway, mannan binding lectin approach and alternative pathway.It participates in mending The ingredient of body Classical pathway includes C1-C9.By its effect in activation, artificially it is divided into three groups, i.e. identification is single Position (Clq, Clr, Cls), activation unit (C4, C2, C3) and film challenging unit (C5-C9) are in the different phase of activation respectively It plays a role in cognitive phase, activation stage and film phase of the attack.This 3 stages are generally in 3 different parts of target cell membrane It carries out.Complement C2, C3, C4, C5 in activation are cracked into 2 or 2 or more segments respectively, are marked with a, b etc. respectively Symbol, such as C3a, C3b, C3c.Wherein C2b, C3b, C4b, C5b are directly or indirectly combined on target cell, in the form of solid phase Molten cell processes are participated in, C3a, C5a are free in liquid phase.Complement may also aggregate into after C5, C6, C7 are activated in activation C567, and special biological function is played together with C3a, C5a.Mannan binding lectin approach is by mannosan in blood plasma The N- amine-galactose on the Direct Recognition multiple pathogenic microorganisms surface binding lectin (mannan-binding lectin, MBL) Or mannose, and then MASP-1, MASP-2, C4, C2, C3 are successively activated, are formed and the identical C3 of classical pathway and C5 convertase, The activated channel of activating complement cascade enzymatic reaction.The primary activation object of MBL activated pathway is that mannose group, rock algae are contained in surface The pathogenic microorganism of sugar and N- amine-galactose.Alternative activation pathway is with Classical pathway the difference is that activation is to cross C1, C4, C2 tri- kinds of ingredients, directly activate C3 then to complete the chain reaction of each ingredient of C5 to C9, and Factor B is that complement bypass is living An important component in change approach, also known as complement 3 proactivator.When the Factor B of circulation combines the C3 of activation, just cause other Approach activation.Then D factor cutting that the complex is recycled generates enzymatic activity segment C3bBb.C3bBb cuts C3 and generates C3b causes inflammation and also further expansion activation, generates positive feedback loop.The characteristics of alternative activation pathway, also exists In activated material be not antigen antibody complex but the cell wall constituent-lipopolysaccharides and polysaccharide of bacterium, peptide glycan, phosphorus wall The substances such as acid and the IgA and IgG4 of cohesion.Alternative activation pathway is in bacterial infection early stage, when not yet generating specific antibody, Important anti-infectious function can be played.
In the alternative pathway of complement activation, the H factor is the factor for playing important inhibiting effect.The H factor by Nilson etc. (1965) discovery is named as β 1H according to electrophoresis position, and Whaley and Ruddy are then named as C3b inactivation Agent accelerated factor.It has been determined that its overall length is the single chain glycoprotein being made of 1231 amino acid, molecular weight 155kDa, existing length Stem portion also has spheric region.The function of the H factor includes the following aspects: (1) being the confactor of I factor, can increase Sensibility of the C4b to I factor.It is at least high by 15 when the binding affinity of i factor and C3b are compared with no H factor in the presence of having the H factor Times.The mechanism of H factor reinforcing i factor, it may be possible to after the H factor is with C3b ining conjunction with, make the certain conformation changes of C3b appearance, increase and The affinity that i factor combines.Active site of the H factor in conjunction with C3b is present in the part its N-terminal 35kDa.(2) accelerate C3 conversion The decay of enzyme: the H factor can will evict from from C3 enzyme with the C3b Factor B combined or Bb, and be allowed to lose enzymatic activity.(3) it prevents Initial and amplification C3 convertase formation in alternative route.Have proven to the H factor and Factor B has same binding site on C3b, therefore The H factor can be the same as the combination of Factor B or Bb competition and C3b.In the presence of having the H factor, Factor B is not easy to tie with C3 (H2C) and C3b It closes, therefore is not easy to form C3 (H2C) Bb or C3bBb.But there are difference for the C3b effect in H factor pair solid phase and in liquid phase.It can split Solve the C3b in liquid phase or being incorporated into inactive dose of solid phase.And for being fixed to activator (such as zymosan) surface C3b, the H factor are then suitable with Factor B to the affinity of C3b, that the two competes as a result, part C3 convertase can be formed, to guarantee The activation of alternative route.Studies have reported that can enhance on cell membrane C3b to the chemical component of H factor affinity be sialic acid and Heparin glycosaminoglycan.Since most of bacterium surfaces lack sialic acid, thus after these bacteriums intrusion body, substitution way can be activated Diameter facilitates the control in early stage to infection.(4) stable C3bBbP is formed to combining with the P factor or nephritis factor (NeF) Or C3bBbNeF, H factor pair they also have certain effect.The activity of H factor pair C5 convertase (C3bnBb or C 3bBb3b) Also there is inhibiting effect, and C5 can be prevented from cracking with C5 competitive binding C3b.In addition, the also inducible monokaryon of the discovery H factor is thin in recent years The adjusting of intracrine IL-1 participation immune response.
It is opsonic that the C3 activation segment that complement activation generates as the ligand of various C3 receptors plays the role of complement.Complement Receptor 2 (CR2) is one such complement receptors, as a kind of transmembrane protein, CR2 by dendritic cells,follicular (FDC), B cell and expression in some T cells and and immune complex survival of the combination for above-mentioned cell especially mature B cell And the selection of high-affinity B cell plays an important role.CR2 is the member of C3 binding protein family, and short total by 15-16 It is made of repetition (SCR) structural domain (the characteristic structural unit of C3 binding protein family), C3 binding site is included in 2 ends N It holds in SCR.Different from complement activation inhibitor (DAF, MCP, CR1 and Crry), CR2 is not complement inhibitor, and it is not tied Close C3b.The native ligand of CR2 is iC3b, C3dg and C3d, is the cell of the C3b in conjunction with the two N-terminal SCR structure domains CR2 In conjunction with crack fragment.The generation and its deposition on active cell surface that the cracking of C3 initially causes C3b.C3b segment participates in The generation of multienzyme complex, the multienzyme complex expand complement cascade.On cell surface, especially adjusted when containing complement activation When depositing C3b on the host surface (that is, most of host tissues) of agent, C3b is rapidly converted into the iC3b of inactivation.Even if not depositing In the case where the complement regulator that film combines, quite high-caliber iC3b is also formed.Then, iC3b is by haemocyanin enzymic digestion For film binding fragment C3dg and C3d, but this process is relatively slow.Therefore, the C3 activation segment ligand of CR2 is once after generation Relative longevity, and complement activation site is present in high concentration.Therefore CR2 can be served as and be taken molecule to complement activation site Efficient targeting carrier.
The disease or situation that complement system is excessive or uncontrolled activation mediates, especially by complement bypass it is excessive or not by The activation of control is the inducement of a variety of diseases or situation.The downward of complement activation is demonstrate,proved in animal model and in vitro study Disease indications several for treatment are effective in fact, such as systemic loupus erythematosus and glomerulonephritis, rheumatoid joint Scorching, cardiovascular shunt and hyperacute rejection, myocardial infarction, reperfusion injury and adult breathing in haemodialysis, organ transplant Distress Syndrome.In addition, other inflammatory conditions and autoimmunity disease/immune complex disease are also closely related with complement activation, packet Include thermal damage, severe asthma, anaphylactic shock, enteritis, rubeola, angioedema, vasculitis, multiple sclerosis, severe flesh Powerless, membranoprolifer ative glomerulonephritis and dry syndrome.
Existing research proves that complement inhibitor, which is transported to complement activation and disease site, by targeting vector becomes abundant Play a kind of effective treatment means of complement inhibitor effect.For example, in vitro in feasibility study, it has therefore proved that with the non-target of protection To inhibitor compare, CR2-DAF the and CR2-CD59 fusion protein of targeting more effectively protects the cell of targeting from complement Destruction.CN100594037C reports complement targeting factor CR2 respectively and is merged with complement inhibitor DAF and CD59 for inhibiting Inhibit the beneficial effect of different activities.CN101563363B then reports the fusion protein of CR2 and fH to complement bypass-activation way The inhibition of diameter and therapeutic effect to related disease and symptom.Wherein, the EC of CR2-fH50For 20-30nM, than in the test Present in fH amount it is 15-20 times low, this proves to compare endogenous fH, and the fH of targeting has apparent benefit.Also, do not having When being related to classic complement approach, inhibit alternative pathway of complement that there is apparent benefit using CR2-fH.But CR2 is merged with fH There is also certain technical problems in the performance of inhibitory activity for albumen, for example, inhibitory effect inhibits in fact not as good as external in vivo It tests, the protective rate of animal protection experiment also needs further to improve.The prompt of the prior art problem passes through fusion protein Sequence variations with the space conformation for changing structure obtained may be promote complement inhibitor give full play to an active means.Cause This is especially related to the structure that changes of fusion protein and is known as a kind of new improved though to the structure that changes of targeting factor CR2.
The present invention is quasi- by computer modeling, amino acid replacement, carries out further molecule to CR2 and changes structure, to improve CR2 With the specificity of ligand binding, and improvement CR2 and the molecular structure of fH fusion protein as a whole, further increase CR2 is with fH fusion protein as targeting complement inhibitor to the depression effect of complement activation.
Summary of the invention
Based on foregoing invention purpose, present invention firstly provides a kind of complement receptor 2 variant (mCR2) and Complement inhibitions The fusion protein of agent fH, the amino acid sequence of the complement receptor 2 variant is as described in SEQ ID NO.1.
In a preferred technical solution, the complement receptor 2 variant and fH are connected with flexible small peptide GGGGSGGGGS It connects.
It is further preferable that the amino acid sequence of the fusion protein, as shown in SEQ ID NO.3, the fusion protein defines For mCR2-fH.
Secondly, the present invention also provides the nucleic acid molecule for encoding above-mentioned fusion protein, the sequence of the nucleic acid molecule As described in SEQ ID NO.4.
Third, the present invention also provides a kind of recombinant expression carriers containing above-mentioned nucleic acid molecule.
In a preferred technical solution, the carrier is pEE14.1-mCR2-fH.
4th, the present invention also provides a kind of host's engineering cell containing above-mentioned expression vector.
In a preferred technical solution, the engineering cell is CHO-K1-pEE14.1-mCR2-fH cell.
Finally, the present invention provides fusion proteins above-mentioned to prepare the application in Parmaceutical for treating disease of autoimmunization system object.
In a preferred technical solution, the disease includes rheumatoid arthritis and systemic loupus erythematosus.
Complement receptor 2 variant disclosed by the invention is that the molecule obtained after computer modeling, amino acid replacement changes Structure body has higher ligand binding and dissociation rate than its wild type sequence (CR2), has better ligand binding affinity.Complement is situated between The inhibitory effect of the Chinese hamster ovary celI and red blood cell dissolution experiment led mCR2-fH ratio CR2-fH as the result is shown become apparent.Inhibit 50% Chinese hamster ovary celI dissolution, the concentration of mCR2-fH is 34nmol/L, and the concentration of CR2-fH is 88nmol/L, and efficiency is inhibited to improve 2 Times.Biodistribution is it is demonstrated experimentally that fusion protein provided by the invention can quickly exist after entering rheumatoid arthritis mouse model Arthritis position high aggregation, it was demonstrated that complement receptor 2 variant of the invention has special targeting, mCR2-fH ratio to C3d CR2-fH has better improvement degree, and has apparent dose-dependence, it was demonstrated that mCR2-fH have it is excellent it is anti-stick/ Anti-inflammatory targeted inhibition effect has good therapeutic effect to the inflammatory reaction of body.MCR2-fH disclosed by the invention is in MRL/ In the treatment of lpr lupus erythematosus mouse, it can be obviously improved the survival rate of mouse, entire therapeutic process can protect MRL/ completely Lpr lupus erythematosus mouse, mCR2-fH treatment group survival rate are 100%.And albuminuria, the glomerulus product of mCR2-fH treatment group Point, the symptoms such as interstitial inflammation, vasculitis and crescent/necrosis be improved significantly, show that mCR2-fH provided by the invention is making There is excellent application prospect in standby Parmaceutical for treating disease of autoimmunization system object.
Detailed description of the invention
Fig. 1 .mCR2-fH sequence variations schematic diagram；
Fig. 2 .CR2-fH sequence diagram；
The 12%SDS-PAGE qualification result of Fig. 3 .mCR2-fH；
The Western Blot qualification result of Fig. 4 .mCR2-fH；
Biodistribution experimental result of Fig. 5 .mCR2-fH in RA mouse；
Fig. 6 .mCR2-fH treats RA mouse test results；
The survival rate comparative diagram of Fig. 7 .mCR2-fH treatment MRL/lpr mouse；
The albuminuria that Fig. 8 .mCR2-fH treats MRL/lpr mouse changes comparative diagram.
Specific embodiment
The invention will now be further described with reference to specific embodiments, the advantages and features of the present invention will be with description and It is apparent.But examples are merely exemplary for these, does not constitute any restrictions to protection scope of the present invention.
Computer modeling
Using the Builder program in II software package of Insight, small peptide is shown in the method from the beginning built.Utilize molecule The method of docking (docking) is formed by the structure of compound after simulating CR2 in conjunction with C3d, through molecular mechanics and Molecule Motion Mechanics optimization obtains the Stable conformation of CR2/C3d compound.The binding site of the two is analyzed, determines that CR2 and C3d is tied The core sequence of conjunction, and obtain the mode to interact between amino acid when the two combines and energy.Based on the analysis results, it replaces Certain amino acid of CR2, mould is built again, obtains the Stable conformation of CR2 mutant peptide-C3d compound.Analyze CR2 mutant peptide and C3d Between the mode that interacts and energy, affinity size when comparing CR2 mutation front and back in conjunction with C3d.
The simulation of CR2 Yu C3d compound tentatively have been carried out, and has devised following mutant, has improved its knot with C3d It closes:
1,Thr86-----Ser；2,Phe130-----Tyr；3,Glu133-----Gln；4,Asp92-----Glu；5, Thr25-----Ser (CR2 mutation after amino acid sequence as shown in SEQ ID NO.1, nucleotide sequence such as SEQ ID NO.2 Shown, mutational site is as shown in Figure 1, the CR2 wild type sequence before mutation is as shown in Figure 2).
The preparation of embodiment 1.CR2 mutant (mCR2)-fH fusion protein
1 material expression carrier selects pEE14.1 (Lonza biologics)；Chinese hamster ovary celI is used for protein expression, culture Liquid is the DMEM containing 10% fetal calf serum, is purchased from Invitrogen company.Mouse anti-fH mAb 1H4 and 1A10, the anti-human CR2mAb of mouse 171, anti-sheep red blood cell IgM and all secondary antibodies are purchased from Sigma company.
2 methods
The sero-fast preparation of 2.1 rabbit-anti Chinese hamster ovary celI films and people fH according to document Harlow, E., and Lane, D.Antibodies:a laboratory manual.Cold Spring Harbor Laboratory.Cold Spring Harbor, New York, the method that USA.1988:726. is introduced obtain.
The buildings of 2.2 expression recombinants and protein expression cDNA structural gene by encode 4 end N- SCR units of CR2 with The sequence of coding fH extracellular region is connected.Complement inhibitor sequence is 322 of the protein extracellular encoding mature fH sequence Amino acid (Swissprot accession number is P08603.4).The computer simulation for carrying out CR2 and C3d compound, devises following CR2 mutant improves the combination of itself and C3d: 1, Thr86-----Ser；2,Phe130-----Tyr；3,Glu133----- Gln；4,Asp92-----Glu；5,Thr25-----Ser.
Then mCR2, CR2 (wild type sequence) are connect with complement inhibitor fH respectively, mCR2, CR2 (mCR2- at the end N- FH, CR2-fH) catenation sequence be GGGGSGGGGS.(amino acid sequence of fusion protein is such as with round pcr synthesis for gene frame Shown in SEQ ID NO.3, nucleotide sequence is as shown in SEQ ID NO.4).All cloning process are enterprising in pEE14.1 carrier Row (referring to CN104940953A).PEE14.1 efficient expression vector contains special glutamine (GS) gene expression system.GS Enzyme is responsible for that glutamy and amine is used to synthesize glutamine as substrate.Some mammal cell lines are free of GS gene, so not Adding cannot survive in the environment of glutamine.For these cell lines, the GS gene of transfection can be used as a kind of screening mark Note, can filter out the cell grown in no Glutamin medium.Methionine imines (Methioninesulphoximine, MSX the activity of GS enzyme) can be inhibited, certain cell lines (CHO-K1) can generate endogenous glutamin, can be in the nothing containing MSX It is grown in Glutamin medium, it is this culture medium of GS enzymatic activity to be inhibited to provide screening pressure added with enough MSX, to sieve Select required cell strain.MCR2-fH, CR2-fH fusion are after PCR method synthesizes with pEE14.1 carrier by passing through It is connected after EcoRI, SmalI double digestion, recombinant vector pEE14.1-mCR2-fH and pEE14.1-CR2-fH is transfected with FuGENE 6 CHO-K1 host cell, the host cell after transfecting mCR2-fH are defined as CHO-K1-pEE14.1-mCR2-fH.It is small to transfect 24 Shi Hou changes selective d MEM culture medium (without glutamine), and is added MSX to final concentration of 50 μM, grows within about 2 weeks positive gram Grand, the normal Chinese hamster ovary celI without transfected plasmids gradually falls off from bottle wall, and cell cracking is dead.3 plants of cells are selected, are used respectively There are blood serum medium and serum free medium culture 3 days, collects cell conditioned medium, it is partially spare through 5 times of PEG20000 concentration, Remaining -80 DEG C of preservations.With expression (the mCR2-fH amino acid sequence such as SEQ ID of ELISA method detection mCR2-fH, CR2-fH Shown in NO.3 and shown in Fig. 1, CR2-fH amino acid sequence is as shown in Figure 2).Recombinant protein table in Chinese hamster ovary celI with secreted form It reaches.It is higher than using the expression quantity of the Chinese hamster ovary celI stably expressing cell line of serum free medium culture and uses the culture medium culture containing serum Cell.
The purifying of 2.3 recombinant proteins purifies the albumen in cell culture supernatant using affinity chromatography.According to Operation manual, chromatographic column is by forming the affine column coupling for resisting fH mAb (1H4) and HiTrap NHS to activate.Recombination egg will be contained The pH value of white culture supernatant is adjusted to 8.0, crosses column with the rate of 0.5mL/min.Then pillar is washed with 6~8 times of volume PBS, recombinated Albumen is with 0.1mol/L glycine (pH2.4) elution of 2~3 times of column volumes.Fusion protein is collected into 1mol/L Tris buffering In liquid (pH8.0), and dialyse in PBS.
2.4SDS-PAGE and Western blot identification
The albumen of purifying carries out in the SDS-PAGE glue containing 100g/L.Gel coomassie brilliant blue staining.Western Recombinant protein is detected with mouse anti-fH mAb 1H4 in blot experiment.
3. result
100~200 μ of recombinant protein (mCR2-fH, CR2-fH) is separated to from the Chinese hamster ovary celI culture supernatant of stable transfection G/L is identified using SDS-PAGE (Fig. 3) and Western blot (Fig. 4).Swimming lane 1 is protein molecular weight mark in Fig. 3 Note, swimming lane 2 are mCR2-CD59, and swimming lane 3 is CR2-CD59.In Fig. 4, swimming lane 4 is mCR2-CD59, and swimming lane 5 is CR2-CD59.Knot There is the target fragment of expected size in fruit display.
Embodiment 2.CR2 fusion protein and C3 aglucon interaction dynamics are analyzed
The dynamic analysis surface of the C3dg (C3dg- biotin) of CR2 fusion protein and biotin labeling interaction Plasmon resonance (SPR) detection system is detected (BIAcore3000 instrument).It is average according to each flow cell The amount of 50mg/L, by people C3dg- biotin (Guthridge, J.M., et al.Structural studies in solution of the recombinant N-terminal pair of short consensus/complement repeat domains of complement receptor type 2(CR2/CD21)and interactions with Its ligand C3dg.Biochemistry.2001,40 (20): it 5931-5941.) is arrived with the speed injection of 2 μ L/min BIAcore streptavidin sensor core on piece acts on 20min, and buffer is that 0.5 × PBS (pH7.4) (contains 0.5g/L Tween20).The spr signal that is obtained from the C3dg of capture generate BIAcore reacton (range be 250 to 500).Fusion protein group is not added as control.25 DEG C, with the flow velocity of 25 μ L/min, with 0.5 × PBST (0.5g/L Tween20 after) washing, its affinity is assessed by detection CR2 fusion protein concentration (15.6~500nmol/L).
Dynamic analysis data show that be most suitable for engagement reaction model with spherical detection parameters at 1: 1.SPR testing result is aobvious Show, mCR2-fH ratio CR2-fH has higher combination and dissociation rate (table 1).And the affinity ratio CR2-fH high of mCR2-fH.
Kinetic parameter of 1 recombination fusion protein of table in conjunction with C3dg- biotin
The experiment of 3. complement lysis of embodiment
To measure the inhibitory activity to complement, the Chinese hamster ovary celI of 60%~80% fusion is separated with ethylenediamine tetra-acetic acid, is used DMEM is washed 2 times, is then resuspended in DMEM, make its final concentration of 106A cell/mL.100mL/L rabbit is added in cell suspension Anti- Chinese hamster ovary celI film antiserum, 4 DEG C of effect 30min, makes cell sensitization.Then antiserum is abandoned, cell is resuspended in diluted with DMEM In NHS, final volume is 50 μ L or 100 μ L.37 DEG C of effect 60min finally measure Cells viability with placenta orchid dyeing exclusive method (living cells and dead cell count).Recombination fusion protein is charged first in NHS after being diluted with DEME, is added to Chinese hamster ovary celI Suspension.Final concentration be subject to 100g/L NHS can lead to about 90% antibody sensitized control Chinese hamster ovary celI dissolution.Complement-mediated The sheep red blood cell (EAs) of red blood cell dissolution inhibition experiment antibody sensitized is measured.Hemolytic test is buffered in gelatin veronal Liquid (GVB++) in carry out, final volume be 300 μ L, include 2.5 × 107EAs, NHS are according to 1: 300 dilution.Reaction mixture exists 37 DEG C of incubation 60min are eventually adding the 300 μ L solution of EDTA-PBS containing 10mmol/L and terminate reaction.Centrifugation, takes supernatant, Quantitative detection is carried out to the ferroheme in supernatant with optical spectrum imagers under 413nm wavelength.
Fusion protein complement inhibitor Activity determination: the Chinese hamster ovary celI and red blood cell dissolution experiment of complement-mediated the results show that Inhibitory effect is obvious than non-targeted fH (s fH) by CR2-fH, and the inhibitory effect of mCR2-fH ratio CR2-fH becomes apparent.Inhibit 50%CHO cell dissolution, the concentration of mCR2-fH are 19nmol/L, and the concentration of CR2-fH is 124nmol/L, and efficiency is inhibited to improve More than 6 times, non-targeted fH needs 435nmol/L, and efficiency is inhibited to improve nearly 23 times (tables 2).In addition, inhibiting real in cell dissolution In testing, fH is stronger than Chinese hamster ovary celI to the protective effect of red blood cell.
The complement inhibitor concentration that table 2 can inhibit 50% cell to dissolve
Embodiment 4.mCR2-fH fusion protein RA experiment in vivo:
1. biodistribution is tested
MCR2-fH, CR2-fH and fH are marked respectively using Iodogen method125I is being coated with 200 μ g Iodogen's In EP pipe, the 100 μ l (100 of ScFv of 150 μ l of 50mmLo/L PBS (pH7.4), BSA containing the 1mg dissolution of existing preparation is sequentially added μg)、Na12515 μ l (185MBq) of I solution is interrupted gentle agitation at room temperature and marks pipe 15min.SEP-PAK C18 column uses first respectively Alcohol, distilled water and 0.1% trifluoroacetic acid (TFA) each 5mL, which are successively washed, makes its activation；Mark mixture upper prop, 0.1%TFA leaching It washes；60% acetonitrile solution elution, 1.5mL eluent before collecting.It is dilute with the PBS solution containing 1mg/mLBSA after freeze-dried It releases, dispense, -80 DEG C of freezer storages are spare.0.1mL collagen II and complete Freund assistant is subcutaneously injected in male DBA/1J mouse tail root Agent (is purchased from Sigma Co., USA), reinforces within the 21st day once establishing rheumatoid arthritis (CIA) mouse model (model foundation Referring to the foundation of C57BL/6 mouse CIA model and its preliminary screening of monitoring system, liberation army medical journal June in 2004 the The phase 472-474 of volume 29 the 6th).Test and group technology are as follows: 1. control group tail vein injection125I--fH (2ug), exists respectively For 24 hours, sacrificed by decapitation after 48h collects blood, takes out histoorgan, weighs and measure its radioactivity (μ Ci), be as a result scaled ID%/g tissue.2. arthritis group tail vein injection125I--fH fusion protein (2ug), respectively for 24 hours, after 48h, 72h, 96h, It takes and ibid handles, detects.3. mCR2-fH arthritis treatment group, tail vein injection are primary125I-mCR2-fH fusion protein (0.25ug) takes afterwards for 24 hours and ibid handles, detects.4. CR2-fH arthritis treatment group, tail vein injection are primary125I-CR2-fH Fusion protein (0.25ug) is taken afterwards for 24 hours and ibid handles, detects.As a result as shown in figure 5, showing mCR2-fH fusion of the invention Albumen can be in arthritis position high aggregation (each grouping column in the legend of the sequence from top to bottom of marginal data and Fig. 5 in Fig. 5 The sub- column figure of sequence from left to right in figure corresponds).
The treatment of rheumatoid arthritis 2. (CIA) mouse model
Using rheumatoid arthritis (CIA) mouse model (ibid), is tested since the 21st day, be grouped as follows: 1. injecting 50 μ l PBS control groups (N=18).2. CR2-fH low dose therapy group (N=18) injects a CR2-fH fusion protein daily (0.25)mg.3. CR2-fH high-dose therapy group (N=16) injects CR2-fH fusion protein (0.25) mg twice daily.④ MCR2-fH low dose therapy group (N=18) injects mCR2-fH fusion protein (0.25) mg daily.5. mCR2-fH high agent It measures treatment group (N=14), injects mCR2-fH fusion protein (0.25) mg twice daily.23rd day beginning clinical score, by following Standard scores to joint of animal inflammation degree: 0 point, no arthritis；1 point, light inflammation and claw are rubescent；2 points, Severe erythema and swollen It is swollen, influence claw function；It is 3 points, claw or dysarthrasis, stiff, lose function.Every mouse four limbs highest point total is 12 points. As a result as shown in fig. 6, since the 23rd day, the arthritis severity of mCR2-fH fusion protein Liang Ge treatment group is significantly lower than PBS group.Wherein the scoring of the 5. group (2 injection groups) be 4. group (1 injection group) 3/4 hereinafter, being the 3. group (CR2-fH Double injection group) 3/5 hereinafter, be 4. group (CR2-fH one injection group) 1/2 hereinafter, being the 1. group (PBS control group) 1/3 or less.
Above the results show mCR2-fH fusion protein of the invention has special targeting to C3d, has more preferable Ground, which resists, sticks/anti-inflammatory targeted inhibition effect, has good therapeutic effect to the inflammatory reaction of body, is significantly higher than CR2-fH's Therapeutic effect.
Therapeutic effect of the embodiment 5.mCR2-fH and CR2-fH in MRL/lpr lupus erythematosus mouse
1. the raising of survival rate
MRL/lpr lupus erythematosus mouse model is that Murphy and Roths were established in 1979 earliest, by multiple Strains of Mouse It is formed by the complicated hybrid process after 12 generations, the 75% of the murine genes of the model comes from from LG/J, 12.6% AKR/J, 12.1% come from C57BL/6 Strains of Mouse from C3H/Di and 0.3%.MRL/lpr mouse contains and cell spontaneity , there is lymphocytosis gene, leads to T cell hyperplasia, lymph nodes of body as a whole in the related Fas gene recessive mutation of programmed death Enlargement and erosive arthritis, anti-DNA, anti-Sm, anti-Su, anti-nucleosides P antibody, high titre ANA, hypergammaglobulinemia And rheumatoid factor.The mouse was fallen ill earliest in 8 weeks, and autoantibody can be detected in serum at this time.It is observed that at 12 weeks Lymphnoditis.12-16 weeks, MRL/lpr mouse started to generate a large amount of autoantibodies including anti-dsDNA antibody.Nearly 16 weeks Multiple organ is involved when age, and the stabilization renal function occurred characterized by serious albuminuria is degenerated.16-24 week old mouse is proliferated Property the glomerulonephritis that mediates of immune complex, vasculitis, and eventually lead to renal failure and dead, the death rate can achieve 50%.
MRL/lpr mouse week mCR2-fH treatment group (n=20), CR2-fH treatment group (n=20) and PBS from 16 weeks to 24 The comparison of the mouse survival rate of control group (n=22)
The 16 weeks MRL/lpr mouse (this Leco Corp. of Shanghai) for having already appeared renal failure symptom are randomly divided into three by the present embodiment Group, first group (n=20) received weekly 0.2mg mCR2-fH from the 16-24 weeks, and second group (n=20) from the 16-24 weeks Receive 0.2mg CR2-fH weekly, third group (n=22) is control group, receives the PBS of equivalent weekly from the 16-24 weeks.Three The administration route of group is tail vein injection.By according to administration group (0.2mg mCR2-fH/ week), 0.2mg CR2-fH/ week) The effect of mCR2-fH and CR2-fH targeted inhibition complement activation is evaluated with control group (0.2mg PBS/ week), and to MRL/lpr The protective rate of lupus erythematosus mouse.
Experimental result is as shown in fig. 7, receive the mouse of CR2-fH treatment, since the C3d in complement activation pathway is targeted The CR2-fH of C3d effectively inhibits, and therefore, the survival rate of MRL/lpr lupus erythematosus mouse significantly improves, and mCR2-fH treatment group is whole A therapeutic process can protect MRL/lpr lupus erythematosus mouse, survival rate 100% completely, even if CR2-fH group was at the 24th week Also the survival rate being able to maintain 60% or more is compared with control group, is significantly improved from the mouse survival rate of 19 Zhou Qi treatment groups.
2. renal function improvement
Mouse is placed in metabolic cage and studies mCR2-fH, CR2-fH to MRL/lpr lupus erythematosus mouse urinary albumin excretion Influence.Collect the twenty-four-hour urine liquid of a mouse every two weeks since 16 weeks.To prevent bacterial growth, it is added in collecting pipe Ampicillin, gentamicin (Invitrogen Life Technologies) and chloramphenicol (Sigma-Aldrich).It uses The Mouse albumin sample of known concentration draws standard curve by ELISA method, and determines the urinary albumin point of experiment mice Situation is secreted, and uses the creatinine content in biochemical instruments (Beckman Coulter) measurement mouse urine.Last evaluation result with 24 hours urinary albumins of every experiment mice (mg) and creatinine (mg) ratio indicate.The higher expression kidney of urinary albumin creatinine ratio Dirty function is damaged.As shown in figure 8, MRL/lpr mouse mCR2-fH treatment group (n=24), CR2-fH treatment group (n=22) It is compared with PBS control group (n=20) albuminuria situation: at the 22nd week and 24 weeks, compared with the control group, the albuminuria for the treatment of group Level is substantially reduced (P < 0.01), and mCR2-fH treatment group (n=24) is more than CR2-fH treatment group (n=24) Proteinuria level It reduces (P < 0.01).It proves mCR2-fH provided by the invention more and can significantly improve renal impairment symptom.
3. the inflammatory reaction of kidney mitigates
After experiment, longitudinally dissection is two halves to excision mouse kidney, and wherein half carries out immunofluorescence analysis, the other half 10% neutral formalin is fixed, solid paraffin embedded section, with hematoxylin eosin staining method and periodic acid Schiff stain method to paraffin The renal tissue slice of processing is dyed, using blind respectively to being sliced the glomerulus inflammation observed, hyperplasia, crescent certainly It is formed, downright bad symptom scores, while also scoring the variation of renal interstitial.It scores and is divided into 0,1,2,3,4 Pyatyis, 0 To be not damaged, 4 be serious damage.Perivascular inflammatory exudation evaluation uses semiquantitative way, by two independent observers' blind The blood vessel for being sliced 10 or more to every is evaluated.Inflammation is scored at 0-3, and 0 is no inflammation, and 1 is the blood vessel lower than 50% It is surrounded by 3 confluent monolayer cells, 2, which are 3-6 layers for the blood vessel greater than 50%, surrounds, and 3 show for most serious, and the cell more than 6 layers is surround.It comments The results are shown in Table 3 for valence.
Table 3.MRL/lpr mouse week treatment Hou24Zhou treatment group and PBS control group kidney damage situation from 16 weeks to 23 Comparison
MCR2-fH ratio CR2-fH is anti-to the inflammation for preferably alleviating kidney in the treatment of MRL/lpr lupus erythematosus mouse It answers.Compared with control group, mCR2-fH ratio CR2-fH Glomerular score, interstitial inflammation, vasculitis and crescent/necrosis etc. more Add and is substantially reduced (P < 0.05).
Sequence table
<110>Beijing Kang Pumeite innovates medical sci-tech Co., Ltd
<120>source of people targeted complement inhibitor albumen mCR2-fH and application
<160> 4
<170> PatentIn version 3.3
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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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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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Cys Leu Ser Ser Gly Lys Trp Ser Ala Val Pro Pro Thr Cys Glu Glu
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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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Tyr Ser Cys Glu Ser Gly Tyr Leu Leu Val Gly Glu Lys Ile Ile Asn
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Ala Arg Cys Lys Ser Leu Gly Arg Phe Pro Asn Gly Lys Val Lys Glu
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Gly Gly Gly Gly Ser Lys Met Arg Leu Leu Ala Lys Ile Ile Cys Leu
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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
305 310 315 320
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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atttcttgtg gctctcctcc gcctatccta aatggccgga ttagttatta ttctaccccc 60
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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 (10)

1. a kind of fusion protein of complement receptor 2 variant and complement inhibitor fH, which is characterized in that the complement receptor 2 becomes Allogeneic amino acid sequence is as described in SEQ ID NO.1.
2. fusion protein according to claim 1, which is characterized in that the complement receptor 2 variant and fH are with flexible short Peptide GGGGSGGGGS connection.
3. fusion protein according to claim 2, which is characterized in that the amino acid sequence of the fusion protein such as SEQ ID Described in NO.3.
4. a kind of nucleic acid molecule of fusion protein described in coding claim 3, which is characterized in that the sequence of the nucleic acid molecule Column are as described in SEQ ID NO.4.
5. a kind of recombinant expression carrier containing nucleic acid molecule described in claim 4.
6. expression vector according to claim 5, which is characterized in that the carrier is pEE14.1-mCR2-fH.
7. a kind of host's engineering cell containing expression vector described in claim 4.
8. host cell according to claim 7, which is characterized in that the engineering cell is CHO-K1-pEE14.1- MCR2-fH cell.
9. any fusion protein of claim 1-3 is preparing the application in Parmaceutical for treating disease of autoimmunization system object.
10. application according to claim 9, the disease includes rheumatoid arthritis and systemic loupus erythematosus.
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