WO2015032167A1 - 靶向特异性补体***抑制剂、其制备方法及应用 - Google Patents

靶向特异性补体***抑制剂、其制备方法及应用 Download PDF

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WO2015032167A1
WO2015032167A1 PCT/CN2014/000638 CN2014000638W WO2015032167A1 WO 2015032167 A1 WO2015032167 A1 WO 2015032167A1 CN 2014000638 W CN2014000638 W CN 2014000638W WO 2015032167 A1 WO2015032167 A1 WO 2015032167A1
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protein
complement
crig
acid sequence
nucleic acid
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French (fr)
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胡维国
乔倩
张鑫
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复旦大学附属肿瘤医院
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Priority to JP2016539395A priority Critical patent/JP2016535062A/ja
Priority to US14/916,800 priority patent/US9862757B2/en
Priority to AU2014317705A priority patent/AU2014317705B2/en
Publication of WO2015032167A1 publication Critical patent/WO2015032167A1/zh

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Definitions

  • the present invention relates to a complement system inhibitor in the field of biotechnology and pharmaceuticals, a preparation method and use thereof, and in particular to a targeted specific complement inhibitor and a preparation method and application thereof.
  • the complement system is a major component of innate immunity and an important regulator of acquired immunity. It plays an important role in immune surveillance, not only to eliminate invading pathogenic microorganisms and host cell debris, but also to coordinate the entire immune and inflammatory process. Complement can be activated by the classical pathway, the alternative pathway and the lectin pathway, and its physiological function is mainly carried out through the products formed after activation, including
  • C3b/iC3b is deposited on the surface of the attacked cell membrane, and immune effector cells such as monocytes are recruited to clear target cells by phagocytosis; localized inflammation is caused by anaphylatoxins such as C3a/C4a/C5a; and Membrane Attack Complex (MAC) , ⁇ C5b-9n) forms pores on the surface of the target cell membrane to eventually lyse the target cells.
  • MAC Membrane Attack Complex
  • C4BPCC4 binding protein factor I (factor I), factor H (factor H), S-protein (S protein) and Cl US teri n (clustered protein); and expression of complement membranes on the surface of cell membrane
  • CRIg is the first C3b/iC3b receptor found on the surface of macrophage membranes that binds to pathogens or other particles.
  • CRIg can produce an inhibitory effect in the early stages of the complement cascade by specifically recognizing C3b and inhibiting the activation of C3 converting enzyme, which is a substitute activation pathway for complement, but The inhibitory effect is lower than the classical complement replacement pathway factor H; meanwhile, the functional site of CRIg is located in the extracellular region. It has been found that there have been no reports on the development of targeted complement inhibitors using its properties in combination with C3b/iC3b.
  • complement regulatory proteins inhibit complement activation at different stages of the complement enzyme cascade
  • the complement system maintains a delicate balance between activation and suppression, and the body is in a healthy state.
  • this balance is broken, the occurrence, development and treatment of many diseases of the human body are closely related to this.
  • Numerous studies have shown that over-activation of the complement system participates in the development and progression of other diseases at different stages, such as auto-hemolytic anemia, autoimmune thrombocytopenia, aplastic anemia, systemic lupus erythematosus, rheumatoid arthritis, and rigidity.
  • complement system inhibitors Spondylitis, atherosclerosis, Parkinson's disease, Alzheimer's disease (senile dementia), asthma, allergies, psoriasis, myasthenia gravis, multiple sclerosis, Crohn's disease, etc. Therefore, drug development with the complement system as a therapeutic target, including the complement system inhibitors, has re-emphasized, and the development of complement system inhibitors, especially targeted inhibitors, has enormous social and economic value.
  • Compastatin has entered preclinical trials, but there is a potential risk of infection due to its ability to inhibit C3 at all sites.
  • Ekule has been successfully used to treat Paroxysmal Nocturnal Hemoglobinuria (PNH)
  • PNH Paroxysmal Nocturnal Hemoglobinuria
  • its intellectual property is foreign-owned and the price is extremely expensive. The price for one year is US$409,500.
  • Soliris had sales of $295 million and 2010 sales of $541 million.
  • PNH is a acquired hemolytic disease mainly caused by mutation of P/G- gene, which leads to the complement membrane of CD55 and CD59 normally anchored to erythrocyte membrane by glycosylphosphatidylinositol (GPI).
  • GPI glycosylphosphatidylinositol
  • the regulatory protein is no longer anchored to the blood cell membrane of PNH patients, so the blood cells of PNH patients are extremely sensitive to complement attack, which is prone to infection caused by leukopenia, hemolysis caused by rupture of red blood cells, and activation of platelets, eventually leading to repeated infections. , hemolysis, thrombosis, renal failure, bone marrow failure and elevated pulmonary arterial pressure, the disease is progressively aggravated, and there is basically no cure before the emergence of eculizumab, thus seriously threatening the lives of patients.
  • Eculizol The mechanism of action of Eculizol is to inhibit the activation of complement at the end of the complement cascade, thereby preventing the deposition of MAC on the blood cell membrane and the lysis of blood cells, especially red blood cells, and the clinical treatment of PNH patients. Received better results, can significantly reduce thrombosis, 66% of patients do not need blood transfusion within one year of treatment.
  • the condition of PNH patients after the application of eculizumab still cannot be completely relieved, because the C3 level of the complement cascade is still activated, producing C3b/iC3b and depositing with the surface of blood cells, so that these blood cells are mononuclear cells.
  • the object of the present invention is to provide a complement system inhibitor, and a preparation method thereof, and the use thereof in the preparation of a medicament for inhibiting complement activation, which can achieve the effect of targeting inhibition of complement activation, and can be used for various complements and complements.
  • Abnormal activation related to the treatment and prevention of human diseases.
  • the present invention provides a targeting specific complement inhibitor, wherein the targeting specific complement inhibitor is a protein having a function of inhibiting complement activation and the protein
  • the amino acid sequence comprises a CRIg extracellular domain and a complement inhibitory domain; the amino acid sequence of the protein is directly linked by the CRIg extracellular domain and the complement inhibitory domain, or is indirectly linked by a linker capable of linking the two;
  • the complement inhibitory domain is factor H, CI inhibitor (C1-INH), C4 binding protein (C4BP), factor 1 (factor 1), S protein (S- Protein), clusterin, complement membrane regulatory protein CD35/CR1, CD46/MCP, CD55 DAF, CD59 or a combination of functional fragments or full length of CRIg itself;
  • the linker is preferably a flexible linker peptide, but may be other linkers.
  • amino acid sequence of the protein is shown in SEQ ID NO 2 or SEQ ID NO 4.
  • the invention also provides a nucleic acid, wherein the nucleic acid sequence encodes the protein.
  • the nucleotide sequence of the nucleic acid is shown as SEQ ID NO 1 or SEQ ID NO 3.
  • the present invention also provides a vector, wherein the vector contains the nucleic acid.
  • the present invention also provides a method for preparing the targeted specific complement inhibitor, wherein the method comprises, by genetic engineering, linking a CRIg extracellular domain to a protein polypeptide of a complement inhibitory domain;
  • the amino acid sequence of the CRIg extracellular domain protein is as shown in SEQ ID NO: 10; the complement inhibitory domain is H factor, C1 inhibitory protein, C4 binding protein, factor I, S protein, cluster protein, complement Membrane regulatory protein CD35/CR CD46/MCP. CD55/DAF, a functional fragment of CD59 or CRIg itself or a combination of any one or more of full length;
  • the genetic engineering techniques include:
  • the nucleic acid sequence encoding the extracellular domain of CRIg and the nucleic acid sequence encoding the complement inhibitory domain are directly ligated by gene splicing by overlap extension PCR (SOE PCR), or indirectly through a nucleic acid sequence encoding a flexible linker peptide After ligation, the resulting fusion sequence is inserted into a eukaryotic expression vector, and the protein expression as a targeted specific complement inhibitor is induced by a eukaryotic protein expression system, and finally the resulting protein is purified.
  • SOE PCR overlap extension PCR
  • a protein expression system refers to a system consisting of a host, a foreign gene, a vector, etc., by which the expression of a foreign gene in a host can be achieved.
  • the host is an organism that expresses proteins, and may be bacteria, yeast, plant cells, animal cells, and the like. Due to the different characteristics of various organisms, the types of proteins suitable for expression are also different.
  • the type of the vector matches the host, and is classified into a prokaryotic (bacterial) expression vector, a yeast expression vector, a plant expression vector, a mammalian expression vector, an insect expression vector, and the like, depending on the host.
  • the vector contains a foreign gene fragment mediated by the vector, and the foreign gene can be expressed in the host.
  • the invention also provides the use of a targeted specific complement inhibitor for the preparation of a medicament for targeting inhibition of complement activation.
  • Interstitial hemoglobinuria in blood cells or other diseases such as mesangial proliferative glomerulonephritis, atypical hemolytic uremic syndrome, and age-related macular degeneration, in cells that suffer from complement attack in cells that are protected from complement attack. use.
  • the present invention also provides a medicament, wherein the active ingredient of the medicament is the protein which is a targeted specific complement inhibitor.
  • the drug is selected from the secretory expression and purification of the protein in the eukaryotic system, and it has been confirmed to have a targeted complement inhibitory function and good drug binding properties by in vitro and in vivo experiments. Among them, in vitro experiments confirmed that half of the effective drug concentration in patients with PNH was less than 100 nM, and in vivo experiments confirmed that it can effectively treat mesangial proliferative glomerulonephritis in rats.
  • the dosage form of the drug is preferably an injection.
  • Other dosage forms such as topical paints and the like can also be used.
  • the mode of administration of the drug includes:
  • the present invention produces a targeting CRIg by binding to a fragment C3b and/or iC3b formed after activation of complement component C3, and another component having a complement inhibitory effect such as factor H, either directly or using a flexible peptide.
  • (Gly4Ser) 3 and other methods are connected to prepare a fusion protein by genetic engineering, and finally achieve the effect of targeting inhibition of complement activation.
  • the present invention acts as a novel targeted complement inhibitor that specifically targets the complement activation site in vivo and potently inhibits complement activation and cell and tissue damage mediated by complement activation. Therefore, the targeted complement inhibitor provided by the present invention has great potential application value and development prospects, and the drug can be applied to the treatment and prevention of various human diseases related to abnormal activation of complement.
  • Figure 1 is a schematic diagram showing the expression and purification results of CRIg-fH and CRIg-L-fH.
  • Figure 2 is a schematic diagram showing the kinetic analysis and binding force determination results of the interaction between CRIg-L-fH and C3 activation and degradation products C3b, iC3b, C3c and C3d.
  • Figure 3 is a schematic diagram showing the kinetic analysis and binding force determination results of the interaction between CRIg-fH and C3 activation and degradation products C3b, iC3b, C3c, C3d.
  • Figure 4 is a graphical representation of the protective effect of CRIg-L-fH on hemolysis of red blood cells in seven PNH patients induced by the complement replacement pathway.
  • Figure 5 is a graphical representation of the protective effect of CRIg-L-fH on hemolysis of red blood cells in seven PNH patients induced by the classical complement pathway.
  • Figure 6 is a graph showing the results of the alleviation of pathological symptoms of elevated serum urea nitrogen induced by CRIg-L-fH in Thy-1N rat nephritis.
  • Figure 7 is a graph showing the results of the alleviation of pathological symptoms of serum creatinine induced by Thy- ⁇ rat nephritis by CRIg-L-fH.
  • Figure 8 is a graph showing the results of the alleviation of pathological symptoms of total protein leakage induced by Thy- ⁇ rat nephritis by CRIg-L-fH.
  • Figure 9 is a graph showing the effect of CRIg-L-fH on the alleviation of hematuria pathological symptoms caused by Thy- ⁇ rat nephritis.
  • Figure 10 is a schematic diagram showing the deposition results of IgG, C3 fragments, MAC and CRIg-L-fH in frozen sections of rats in each group by immunofluorescence. The best way to implement the invention
  • the present invention provides a targeted specific complement inhibitor which is a protein.
  • the protein has a function of inhibiting complement activation, and the amino acid sequence of the protein comprises a CRIg extracellular domain and a complement inhibitory domain; the amino acid sequence of the protein is directly linked by the CRIg extracellular domain and the complement inhibitory domain, or a connector capable of connecting the two indirectly connected;
  • the complement inhibitory domain is factor H, CI inhibitor (C1-INH), C4 binding protein (C4BP), factor 1 (factor 1), S-protein (S-protein), Clusterin, a functional fragment or full length of the complement membrane regulatory protein CD35/CR1, CD46/MCP, CD55/DAF, CD59 or CRIg itself;
  • the linker is preferably a flexible linker peptide, but may be other linkers.
  • the present invention provides a targeting specific complement inhibitor that utilizes two unique properties of CRIg (CRIg can specifically inhibit C3b and inhibit the activation of C3 converting enzyme, thereby producing an inhibitory effect in the early stage of the complement cascade;
  • CRIg can specifically inhibit C3b and inhibit the activation of C3 converting enzyme, thereby producing an inhibitory effect in the early stage of the complement cascade;
  • the functional site of CRIg is located in the extracellular domain, and its extracellular domain gene is cloned and expressed in fusion with other complement regulatory proteins to prepare a specific binding effect by CRIg and C3b/iC3b, and the recombinantly linked complement is regulated.
  • the protein is transported to the local complement activation site to achieve the purpose of inhibiting complement activation, preventing or treating the disease.
  • the gene sequence of the extracellular functional site of CRIg is shown in SEQ ID NO 9 of the Sequence Listing.
  • CRIg The amino acid sequence is shown in SEQ ID NO: 10 of the Sequence Listing.
  • other components linked to CRIg include all components that inhibit complement activation, such as the complement regulatory proteins C1-INH, C4BP, factor I, factor H, S-protein Clustering CD35/CR CD46/MCP, CD55/DAF, and CD59, Even full-length or partial sequences of CRIg itself, etc., in which the means of association include direct attachment, and indirect linkage through other molecules by other physical, chemical, and biological means.
  • CRIg and different complement inhibitors including Cl-INH (C1 inhibitor, C1 inhibitory protein), C4BP (C4 binding protein, C4 binding protein), factor I (factor I), factor H (present in the blood circulation) H factor), S-protein (S protein) and Clusterin (clustered protein); and expression of the complement membrane regulatory proteins CD35/CR1, CD46/MCP,
  • CRIg is directly linked to other complement inhibitors without any other link between the two;
  • the protein consists of CRIg and factor H (fH).
  • the present invention is indirectly directed to the CRIg functional extracellular domain and the factor H (fH) functional region (the recombinant protein is designated as CRIg-fH) or through the flexible linker peptide (SerlGly4) 3 (the recombinant protein is designated as CRIg-L-fH).
  • the targeted complement inhibitory effects of the recombinant proteins CRIg-fH and CRIg-L-fH are described.
  • the protein consists of a CRIg extracellular domain, factor H, and a linker between the two.
  • amino acid sequence of the protein is as set forth in SEQ ID NO: 2 or SEQ ID N04.
  • the invention provides nucleic acids encoding the above proteins.
  • the nucleotide coding sequence thereof is shown as SEQ ID NO 1 or SEQ ID NO 3.
  • the nucleic acid encodes the protein as a targeted specific complement inhibitor that inhibits complement activation and comprises a sequence encoding CRIg and a complement inhibitor.
  • the present invention also provides a vector comprising the above nucleic acid.
  • a vector comprising the above nucleic acid.
  • CRIg-fH and CRIg-(Gly4Ser) 3-fH as an example, the base sequence of the vector containing the nucleic acid is shown as SEQ ID NO 1 or SEQ ID NO 3.
  • the invention also provides a cell comprising the above nucleic acid.
  • the present invention also provides a method for preparing the targeted specific complement inhibitor, comprising directly or indirectly linking a protein polypeptide of a CRIg extracellular domain and a complement inhibitory domain by a genetic engineering technique;
  • the amino acid sequence of the CRIg extracellular domain protein is as shown in SEQ ID NO: 10;
  • the complement inhibitory domain is a functional fragment of factor H, C1 inhibitory protein, C4 binding protein, factor I, S protein, cluster protein, complement membrane regulatory protein CD35/CR1, CD46/MCP, CD55/DAF, CD59 or CRIg itself or a combination of any one or more of full length;
  • the genetic engineering technology is:
  • the nucleic acid sequence encoding the extracellular domain of CRIg and the nucleic acid sequence encoding the complement inhibitory domain are directly ligated by gene splicing by overlap extension PCR (SOE PCR), or indirectly linked by a nucleic acid sequence encoding a flexible linker peptide Then, the resulting fusion sequence is inserted into a eukaryotic expression vector, and the protein expression as a targeted specific complement inhibitor is induced by a eukaryotic protein expression system, and finally the resulting protein is purified.
  • SOE PCR overlap extension PCR
  • a protein expression system refers to a system consisting of a host, a foreign gene, a vector, etc., by which the expression of a foreign gene in a host can be achieved.
  • the host is an organism that expresses proteins, and may be bacteria, yeast, plant cells, animal cells, and the like. Due to the different characteristics of various organisms, the types of proteins suitable for expression are also different.
  • the type of the vector matches the host, and is classified into a prokaryotic (bacterial) expression vector, a yeast expression vector, a plant expression vector, a mammalian expression vector, an insect expression vector, and the like, depending on the host.
  • the vector contains a foreign gene fragment mediated by the vector, and the foreign gene can be expressed in the host.
  • the proteins of the invention may utilize genetic engineering techniques, preferably linking the CRIg protein to the factor H protein.
  • the amino acid sequence of the CRIg protein is shown in SEQ ID NO: 6; the amino acid sequence of the factor H protein is shown in SEQ ID NO: 8. Amino acid residues can also be sequentially linked in accordance with the sequence of the protein.
  • the nucleic acid of the present invention may also utilize genetic engineering techniques, preferably linking the coding sequences of the CRIg protein and the factor H protein; or sequentially linking the bases according to the sequence of the nucleic acid.
  • the present invention provides a targeted specific complement system inhibitor, i.e., the use of the above protein for the preparation of a medicament for inhibiting complement activation. It can also be used to prepare drugs that protect cells from complement attack and increase red blood cell pressure.
  • the invention provides the application of the above protein in preparing medicine for protecting blood cells of patients with paroxysmal nocturnal hemoglobinuria, and can also be used for preparing other diseases, such as mesangial proliferative glomerulonephritis, atypical hemolytic uremic syndrome and age.
  • the present invention also provides a medicament, wherein the active ingredient of the medicament is the protein which is a specific specific complement inhibitor, preferably a functional domain comprising CRIg and factor H.
  • This drug is a drug that inhibits complement activation.
  • the medicament produces a protein comprising an amino acid sequence such as SEQ ID NO 2 or SEQ ID NO 4.
  • the drug is selected as a target specific complement inhibitor obtained by secretory expression and purification in a eukaryotic system, and its in vivo and in vitro experiments have confirmed that it has a targeted complement inhibitory function and good drug binding properties. Among them, in vitro experiments confirmed that half of the effective drug concentration in patients with PNH was less than 100 nM, and in vivo experiments confirmed that it can effectively treat mesangial proliferative glomerulonephritis in rats.
  • the pharmaceutical dosage form is preferably an injection.
  • Other dosage forms such as topical paints and the like can also be used.
  • the mode of administration of the drug includes:
  • the present invention provides the complement system targeting a specific inhibitor, with another complement inhibitor CR2-CD59 targeting contrast, CRIg-fH binding constant Ka about three times smaller (CRIg-fH is 1.093xl0 4, CR2- CD59 is 3.45xl0 4 1/Ms), and the dissociation constant Kd is about 46 times smaller (CRIg-L-fH is 3.656xl0' 3 , CR2-CD59 is 0.169 1/s), and finally CRIg-L-fH
  • the equilibrium dissociation constant K D value is more than 15 times lower than CR2-CD59, and has better pharmacological activity, which means: CRIg-L-fH is slower than CR2-CD59 when combined with the drug target C3b/iC3b.
  • CRIg-Fc Another targeted complement inhibitor
  • CRIg does not inhibit the degradation of C3 converting enzymes, nor does the factor I-like factor I degrade C3b activity, so CRIg is only weak.
  • Complement inhibitory activity In contrast, CRIg-fH is linked to fH rather than the Fc portion of the antibody, so CRIg-fH will have a better complement inhibitory effect.
  • CRIg-fH Compared with the non-targeted complement inhibitor minifH, the advantage of CRIg-fH is more obvious. It not only has targeting, but also has a better binding constant, and its K D value is 3 times smaller than minifH.
  • Example 1 Construction, eukaryotic expression and purification of CRIg-fH and CRIg-L-fH protein expression vectors
  • CRIg is a complement receptor highly expressed on the surface of macrophages
  • total RNA was extracted from human histiocytic lymphoma cell line U937 by Trizol method and reverse transcribed into the first strand cDNA.
  • the corresponding protein sequences of human CRIg extracellular domain [102] (NP_001171759.1, residues 19-137, PDB: 2ICC_A) were found from NCBI's Protein database and found in the NCBI Gene database.
  • the nucleic acid sequence corresponding to the human CRIg gene (NM-001184830.1).
  • the upstream and downstream primer sequences of the extracellular domain of CRIg were designed and cloned.
  • the nucleic acid sequence of CRIg extracellular domain was amplified by PCR from U937 cDNA, ligated into pMD18 T vector, transformed, blue-white spot screening positive clone, positive selection The colonies were expanded and sent for sequencing.
  • the total RNA of human hepatoma cell line HepG2 is extracted by Trizol method and reverse transcribed into eDNA.
  • the factor H SCR1-5 domain NP- 000177.2, residues 19-323 [96]
  • the protein sequence was found from the NCBI Protein database
  • the human factor H gene was found from the NCBI Gene database.
  • the upstream and downstream primer sequences of the FH SCR1-5 domain were designed and cloned, and the nucleic acid sequence of the FH SCR1-5 domain was amplified by PCR from the cDNA of HepG2, and ligated into the PMD18 T vector, transformation, blue-white spot. Positive clones were screened, positive colonies were picked and expanded and sent for sequencing. The clones with the correct sequence in the sequencing results were selected, and the plasmid was extracted as a template for the next overlap extension PCR.
  • CRIg and the SCR1-5 domain of factor H are directly (CRIg-fH) or by a flexible linker sequence (SerGly4) 3 by using gene splicing by overlap extension PCR (SOE PCR). Indirect (CRIg-L-fH) is connected.
  • the principle of the overlap extension PCR method is to use primers with complementary ends to form a double strand containing overlapping regions in the PCR product, so that in the subsequent second round of PCR amplification reaction, the extension of the overlapping strand will not
  • the amplified fragments of the same source were spliced together, and a fusion sequence comprising the CRIg extracellular domain and the factor H SCR1-5 domain and the linker sequence (flexible linker sequence) (L, (SerlGly4) 3) (TCTGGTGGCGGTGGCTCCGGCGGAGGTGGGTCCGGTGGCGGCGGA) was constructed.
  • L, (SerlGly4) 3 TCTGGTGGCGGTGGCTCCGGCGGAGGTGGGTCCGGTGGCGGCGGA
  • VOV iDO ⁇ OLLWOO ⁇ Completion VOVOO ⁇ V ⁇ :) VULV ⁇ ID ⁇ 0 ⁇ OVDC OOO ⁇ ) OOJYD O ⁇ 3 O ⁇ VIVIYOVDO ⁇ O ⁇
  • the 293FT cells in logarithmic growth phase were uniformly plated in a cell culture dish of 15 cm in diameter at a cell density of 1.2 ⁇ 10 7 per dish, and transfected with pHLsec-CRIg-fH and pHLsec-CRIg-L-fH plasmids by PEI. At 37 ° C, 5 ° /. Replace 293 expression medium after 6 hours of culture in C0 2 incubator From invitrogen), the cells were collected for three days and centrifuged to remove cells and cell debris.
  • CRIg-fH and CRIg-L-fH fusion proteins were obtained by the above method using eukaryotic system to induce expression and purification.
  • PAGE electrophoresis polyacrylamide gel electrophoresis
  • concentration was concentrated after concentration. It reaches 1 ⁇ 2 mg/ml, as shown in Figure 1.
  • Biacore T200 protein interaction analyzer, purchased from GE Healthcare
  • Series S Sensor Chip NTA chip CRIg-L-fH protein solution
  • C3 activated and degraded protein components C3b, iC3b, C3c, C3d, purchased from Complement Technology
  • HBS-N solution available from GE Healthcare;
  • SPR Surface plasmon resonance
  • Chip pretreatment Embed the NTA sensor chip module into BIAcore instrument, prepare HBS-N running buffer (buffer), open BIAcore T200 protein interaction analyzer, set the program, first inject two needles HBS-N running buffer pair The chip was pre-cleaned at 25 ° C with a flow rate of 120 ⁇ 1 / min for 5 min per needle until the baseline was flattened;
  • Ni coupling a needle of Nickel solution (0.5 mM NiCl 2 /running buffer), 25 ° C, flow rate of 30 ⁇ 1 / min, for 60 sec, stable for 30 sec;
  • the two-shot Regeneration solution is injected to clean the chip until the detection line returns to the baseline level;
  • Example 3 CRIg-L-fH protects PNH erythrocytes from the complement replacement pathway and the classical pathway-induced hemolytic reaction
  • CVF cobra venom factor cobra venom Factor: lmg/ml, purchased from refinech), anti-human erythrocyte polyclonal antibody (purchased from Rockland), Bio-Tek synergy HT multi-function microplate reader (purchased from Finnish Labsystems), Minispin desktop high-speed centrifuge (purchased from Germany Eppendorf company), DK-8D type electric thermostatic water tank (purchased from Shanghai Jinghong Experimental Equipment Co., Ltd.)
  • Bio-Tek synergy HT multi-function microplate reader purchasedd from Finnish Labsystems
  • laser confocal microscope FV500 purchasedd from Olympus, Japan
  • BCA protein quantification kit purchased from Thermo Fisher:
  • Cobas 6000 analyzer fully automatic Biochemical analyzer purchased from Roche Company, SD rat, anti-C3b/iC3b-FITC antibody, anti-SC5b-9 antibody, anti-His antibody, rabbit anti-mouse (rabbit anti-mouse) IgG-FITC antibody
  • mice Male Sprague-Dawley rats were housed in SPF (Specific Pathogen Free) environment to a body weight of 150-200 g, and were randomly divided into three groups for modeling:
  • ATS group Rat anti-thymocyte serum (ATS) was injected into the tail vein of rats, and the injection dose was 1 ml/100 g body weight;
  • ATS+CRIg-L-fH group Rat anti-thymocyte serum (ATS) was injected into the tail vein of rats, and the injection dose was 1 ml/100 g body weight. Co-injection of CRIg-L-fH into the tail vein at a dose of 1 mg/100 g body weight;
  • the rats were housed in a rat metabolic cage. Blood samples were collected from the tail vein at 24 hr and 72 hr after injection and collected in standard coagulation tubes. The cells were placed at room temperature for 30 min, centrifuged at 3000 rpm for 10 min, and the supernatant (serum) was taken. The Cobas 6000 analyzer automatic biochemical analyzer was used to detect and collect renal function indicators (blood urea nitrogen and serum creatinine).
  • one rat was randomly selected from each group. After anesthesia with ether, the left ventricle was inserted into the heart for perfusion. The saline was perfused first. After the liver turned from red to white, reperfusion was more than 4%. POM, until the rat died. The left kidney was removed, immersed in PBS for several times, placed in a cell cryotube, placed in liquid nitrogen for 30 min, and then stored at -80 °C for later use.
  • the kidney tissue was embedded in the OCT, and the peripheral tissue was cut using a cryo-sectioner. The tissue area at the junction of the renal cortex and medulla was selected and a 6 um slice was attached to the slide. 4% paraformaldehyde was fixed for 15 min, rinsed with PBS. 10% normal goat serum was blocked and incubated overnight with anti-C3b/iC3b and anti-His anti-SC5b-9 antibodies at 4 degrees. The next day, PBS was rinsed three times and the secondary antibody was incubated. Rinse PBS, cover, microscopic examination.
  • CRIg-L-fH-treated Thy- ⁇ rats the levels of urea nitrogen and creatinine in the serum of the untreated group decreased, the total amount of protein in the urine and the amount of hemoglobin decreased, and a large amount of CRIg-L-fH was deposited.
  • the present invention provides a targeted specific complement inhibitor as a novel targeted complement inhibitor capable of specifically targeting a complement activation site in vivo, and long-term inhibition of complement activation and activation by complement.
  • Mediated cell and tissue damage which not only protects human-deficient PNH erythrocytes from hemolytic damage caused by complement attack, but also significantly attenuates complement-induced mesangial proliferative lesions in a rat model of Thy-1 nephritis .
  • the targeted specific complement inhibitor can effectively treat other diseases of hyperactivation of the complement system, such as age-related macular degeneration, atypical hemolytic uremic syndrome, rheumatoid arthritis, ankylosing spine Inflammation, lupus, etc. Therefore, the targeted specific complement inhibitors provided by the present invention have great potential application value and development prospects.

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Abstract

提供了一种靶向特异性补体抑制剂，该靶向特异性补体抑制剂为一种蛋白质，该蛋白质具有靶向抑制补体激活的功能，该蛋白质的氨基酸序列含有CRIg胞外功能区和补体抑制功能区；所述CRIg胞外功能区和补体抑制功能区直接相连，或通过连接物间接相连。还提供了所述靶向特异性补体抑制剂的制备方法和在制备靶向抑制补体激活的药物中的应用。

Description

靶向特异性补体***抑制剂、 其制备方法及应用 技术领域
本发明涉及生物技术和制药领域的一种补体***抑制剂及其制备方法和 用途， 具体地， 涉及一种靶向特异性补体抑制剂及其制备方法和应用。 背景技术
补体***是固有免疫的主要组成部分， 也是获得性免疫的重要调节者， 发挥着重要的免疫监视作用， 不仅可以清除入侵的病原菌微生物和宿主细胞 碎片， 还可以协调整个免疫和炎症过程。 补体可通过经典途径、 替补途径及 凝集素途径激活， 其发挥生理功能主要通过激活后形成的产物进行， 包括
C3b/iC3b沉积于被攻击细胞膜表面， 招募单核细胞等免疫效应细胞通过吞噬 作用而清除靶细胞； C3a/C4a/C5a等过敏毒素引起局部炎症； 以及补体膜攻 击复合物（Membrane Attack Complex, MAC, §卩 C5b-9n)在靶细胞膜表面形 成孔洞而最终裂解靶细胞。 在此过程中， 为了防止补体激活后对正常宿主细 胞的 "误伤"效应，机体在进化过程中形成了 10余种调控蛋白， 包括存在于 血液循环中的 C1-INH(C1 inhibitor; CI抑制蛋白 C4BPCC4 binding protein, C4结合蛋白）、 factor I (I因子）、 factor H (H因子）、 S-protein (S蛋白）和 ClUSterin (簇集蛋白)； 以及表达与细胞膜表面的补体膜调控蛋白 CD35/CR1、 CD46/MCP、 CD55/DAF和 CD59， 以及近期发现的另一种补体膜调控蛋白 CRIg。 CRIg是最早发现表达于巨噬细胞膜表面的一种 C3b/iC3b受体， 结合 后可吞噬病原体或其他颗粒。 进一步结构生物学研究还发现 CRIg可通过特 异性识别 C3b并抑制 C3转换酶的激活， 从而在补体级联反应的早期即产生 抑制效应——这种抑制作用为针对补体的替补激活途径， 但其抑制效果低于 经典的补体替补途径抑制剂 factor H; 同时， CRIg的功能位点位于胞外区。 査阅发现至今未有利用其与 C3b/iC3b结合的特性研发靶向性补体抑制剂的 报道。
由于补体调节蛋白在补体酶级联反应的不同阶段抑制补体的激活， 使得 补体***在激活和抑制之间维持一种精妙的平衡， 机体处于健康状况。但是， 这种平衡一旦被打破， 人体的很多疾病的发生、发展和治疗均与此密切相关。 大量研究表明， 补体***的过度激活不同程度不同阶段参与其他疾病的发生 与发展， 例如自身溶血性贫血、 自身免疫性血小板减少、 再生障碍性贫血、 ***性红斑狼疮、 类风湿性关节炎、 强直性脊柱炎、 动脉粥样硬化、 帕金森 氏病、 阿尔茨海默病（老年性痴呆)、 哮喘、 过敏、 银屑病、 重症肌无力、 多 发性硬化、 克隆氏肠病等。 因此， 以补体***为治疗靶点的药物研发， 包括 补体***抑制剂重新受到重视， 研发补体***抑制剂， 尤其是靶向性抑制剂 具有巨大的社会和经济价值。
研发补体***抑制剂经过了数十年的探索， 包括与 C3结合的蛋白质 Compastatin以及抗 C5的重组单抗 Eculizumab (依库珠， Soliris, 由亚力兄 制药公司 Alexion Pharmaceuticals生产）， 但仍然存在一些明显的不足。
Compastatin已经进入临床前期试验， 但是由于其抑制所有部位 C3的功能而 存在潜在的导致感染的风险。 另外， 尽管依库珠已经成功用于治疗阵发性睡 眠性（夜间）血红蛋白尿 (Paroxysmal Nocturnal Hemoglobinuria, PNH), 但 其知识产权为国外拥有， 价格异常昂贵， 使用一年的价格是 409,500美元， 2009年， Soliris销售额为 2.95亿美元， 2010年销售额 5.41亿美元。 PNH是 一种后天获得性溶血性疾病，主要由于 P/G- 基因的突变导致 CD55和 CD59 这两种正常通过糖基磷肌酰肌醇 （glycosylphosphatidylinositol，GPI)锚定于 红细胞膜上的补体膜调控蛋白不再锚定于 PNH患者的血细胞膜上，所以 PNH患者的血细胞对补体的攻击异常敏感， 极易产生白细胞下降导致的感 染、 红细胞破裂造成的溶血， 以及血小板被激活， 最终导致反复感染、溶血、 血栓形成、 肾功能衰竭、 骨髓衰竭和肺动脉压升高等， 疾病进行性加重， 在 依库珠单抗出现之前基本无药可治， 从而严重威胁患者的生命。 依库珠的作 用机理是与 C5结合后， 在补体级联反应的末期抑制补体的激活， 从而阻止 MAC在血细胞膜上的沉积和对血细胞尤其是对红细胞的裂解， 临床上对 PNH患者的治疗收到了较好的疗效， 能够显著减少血栓形成， 66%的患者在 治疗一年内不需再输血。然而， 理论上应用依库珠单抗后 PNH患者的病情仍 然无法得到完全缓解， 因为补体级联反应的 C3水平仍然存在激活， 产生 C3b/iC3b并沉积与血细胞表面， 使这些血细胞被单核细胞吞噬， 最终造成血 管外溶血， 这也是依库珠单抗治疗并不十分满意的原因。 因此， 在补体激活 更早期的 C3水平， 而不是像依库珠单抗的末期 C5水平， 阻止补体的激活， 将有效同时阻止 MAC介导的血管内和 C3b/iC3b介导的血管外溶血， 从而产 生更好的疗效。 发明的公开
本发明的目的是提供一种补体***抑制剂， 及其制备方法， 与其在制备 抑制补体激活药剂中的应用，该类型药剂可达到靶向性抑制补体激活的效果， 能够用于多种与补体异常激活有关的人类疾病的治疗和预防。
为了达到上述目的， 本发明提供了一种靶向特异性补体抑制剂， 其中， 该靶向特异性补体抑制剂为一种蛋白质， 该蛋白质具有靶向抑制补体激活的 功會 ^ 并且该蛋白质的氨基酸序列含有 CRIg胞外功能区和补体抑制功能区； 所述蛋白质的氨基酸序列由 CRIg胞外功能区和补体抑制功能区直接相连， 或者通过能够连接两者的连接物间接相连组成；
所述的补体抑制功能区为 H因子（factor H)、 CI抑制蛋白（CI inhibitor, C1-INH)、 C4结合蛋白 （C4 binding protein, C4BP)、 I因子 （factor 1)、 S 蛋白 （S-protein)、 簇集蛋白 （Clusterin ) , 补体膜调控蛋白 CD35/CR1、 CD46/MCP, CD55 DAF, CD59或者 CRIg本身的功能片段或全长的任意一 种或多种的组合；
所述的连接物优选为柔性连接肽段， 但也可为其他连接物。
该蛋白质的氨基酸序列如 SEQ ID NO 2或者 SEQ ID NO 4所示。
本发明还提供了一种核酸， 其中， 该核酸序列编码所述的蛋白质。
该核酸的碱基序列如 SEQ ID NO 1或者 SEQ ID NO 3所示。
本发明还提供了一种载体， 其中， 该载体含有所述的核酸。
本发明还提供了一种所述的靶向特异性补体抑制剂的制备方法， 其中， 该方法包含通过基因工程技术， 将 CRIg胞外功能区和补体抑制功能区的蛋 白质多肽相连；
所述的 CRIg胞外功能区蛋白质的氨基酸序列如 SEQ ID NO 10所示； 所述的补体抑制功能区为 H因子、 C1抑制蛋白、 C4结合蛋白、 I因子、 S蛋白、 簇集蛋白， 补体膜调控蛋白 CD35/CR CD46/MCP. CD55/DAF, CD59或者 CRIg本身的功能片段或全长的任意一种或多种的组合；
所述的基因工程技术包含：
通过重叠延伸 PCR法 (gene splicing by overlap extension PCR, SOE PCR), 将编码 CRIg胞外功能区的核酸序列和编码补体抑制功能区的核酸序 列直接连接， 或者通过编码柔性连接肽段的核酸序列间接连接， 然后将连接 所得的融合序列***到真核表达载体中， 通过真核蛋白表达***诱导该作为 靶向特异性补体抑制剂的蛋白质表达， 最后将所得的蛋白质纯化。
蛋白表达***是指由宿主、 外源基因、 载体等组成的体系， 通过这个体 系可以实现外源基因在宿主中表达的目的。 宿主是表达蛋白的生物体， 可以 为细菌、 酵母、 植物细胞、 动物细胞等。 由于各种生物的特性不同， 适合表 达蛋白的种类也不相同。 载体的种类与宿主相匹配， 根据宿主不同， 分为原 核 （细菌）表达载体、 酵母表达载体、 植物表达载体、 哺乳动物表达载体、 昆虫表达载体等。 载体中含有外源基因片段， 通过载体介导， 外源基因可以 在宿主中表达。
本发明还提供了一种所述的靶向特异性补体抑制剂在制备靶向抑制补体 激活的药物中的用途。 间血红蛋白尿 者血细胞或其他疾 ，、例如系膜增生性肾小球肾炎、 非典型 溶血性***综合征和年龄相关的黄斑变性等疾病中遭受补体攻击的细胞免 受补体攻击的药物中的用途。
本发明还提供了一种药物， 其中， 所述药物的活性成分为该作为靶向特 异性补体抑制剂的蛋白质。 该药物选用在真核***分泌表达并纯化获得的所 述蛋白质， 通过体内外实验均证实其具有靶向性补体抑制功能及良好的药物 结合性质。其中， 离体实验证实治疗 PNH患者的半数有效药物浓度低于 100 nM， 体内实验证实能有效治疗大鼠系膜增生性肾小球肾炎。
该药物的剂型优选为针剂。 也可采用其他剂型， 如外用涂剂等。
该药物的给药方式包括：
( 1 ) 直接给药。
(2) 通过能携带或表达该药物的载体***给药。
本发明与现有技术相比， 具有以下优点和技术效果- 本发明通过能与补体成分 C3激活后形成的片段 C3b和 /或 iC3b结合而产 生靶向作用的 CRIg， 与具有补体抑制效果的另一组分例如 H因子 （factor H) 直接或利用柔性肽段 （Gly4Ser) 3等方式相连， 通过基因工程的方法制备一 种融合蛋白， 最终达到靶向性抑制补体激活的效果。
本发明作为一种新型的靶向性补体抑制剂， 能够特异地靶向到体内补体 激活位点， 并长效地抑制补体激活和由补体激活所介导的细胞和组织损伤。 因此本发明提供的靶向性补体抑制剂， 具有巨大的潜在药物应用价值和开发 前景， 该类型药物可应用于多种与补体异常激活有关的人类疾病的治疗和预 防。 附图的简要说明
图 1为 CRIg-fH和 CRIg-L-fH的表达与纯化结果示意图。
图 2 为 CRIg-L-fH与 C3活化和降解产物 C3b、 iC3b、 C3c、 C3d相互作 用的动力学分析和结合力测定结果示意图。
图 3 为 CRIg-fH与 C3活化和降解产物 C3b、 iC3b、 C3c、 C3d相互作用 的动力学分析和结合力测定结果示意图。
图 4为 CRIg-L-fH对补体替补途径诱导的七例 PNH患者红细胞的溶血 起保护作用结果示意图。
图 5 为 CRIg-L-fH对补体经典途径诱导的七例 PNH患者红细胞的溶血 起保护作用结果示意图。
图 6为 CRIg-L-fH对 Thy-1N大鼠肾炎引发的血清尿素氮升高病理症状 的缓解作用结果示意图。
图 7为 CRIg-L-fH对 Thy-ΙΝ大鼠肾炎引发的血清肌酐升高病理症状的 缓解作用结果示意图。
图 8 为 CRIg-L-fH对 Thy-ΙΝ大鼠肾炎引发的尿液总蛋白渗漏病理症状 的缓解作用结果示意图。
图 9为 CRIg-L-fH对 Thy-ΙΝ大鼠肾炎引发的血尿病理症状的缓解作用 结果示意图。
图 10为免疫荧光检测各组大鼠肾脏冰冻切片的 IgG、 C3片段、 MAC和 CRIg-L-fH的沉积结果示意图。 实现本发明的最佳方式
以下结合附图对本发明的具体实施方式作进一步地说明。
本发明提供的靶向特异性补体抑制剂， 其为一种蛋白质。 该蛋白质具有 靶向抑制补体激活的功能， 并且该蛋白质的氨基酸序列含有 CRIg胞外功能 区和补体抑制功能区； 该蛋白质的氨基酸序列由 CRIg胞外功能区和补体抑 制功能区直接相连， 或通过能够连接两者的连接物间接相连组成；
该补体抑制功能区为 H因子 （factor H)、 CI抑制蛋白 （CI inhibitor, C1-INH) C4结合蛋白 （C4 binding protein, C4BP)、 I因子 （factor 1)、 S 蛋白 （S-protein)、 簇集蛋白 （Clusterin ) , 补体膜调控蛋白 CD35/CR1、 CD46/MCP, CD55/DAF, CD59或者 CRIg本身的功能片段或全长的任意一 种；
该连接物优选为柔性连接肽段， 但也可为其他连接物。
本发明提供的靶向特异性补体抑制剂，利用 CRIg两种独特的特性 (CRIg 可通过特异性识别 C3b并抑制 C3转换酶的激活， 从而在补体级联反应的早 期即产生抑制效应； 同时， CRIg的功能位点位于胞外区）， 克隆其胞外功能 区基因并与其他补体调控蛋白融合表达， 制备成一种通过 CRIg与 C3b/iC3b 特异性结合的靶向作用， 把重组联接的补体调控蛋白运送至局部补体激活部 位， 从而达到抑制补体激活， 预防或治疗疾病的目的。 CRIg的胞外功能部位 的基因序列见序列表的 SEQ ID NO 9, 氨基酸序列见序列表 SEQ ID NO 10。 另外， 与 CRIg相连的其他部分包括所有能抑制补体激活的成分， 例如补体 调控蛋白 C1-INH、 C4BP、 factor I、 factor H、 S-protein Clustering CD35/CR CD46/MCP、 CD55/DAF和 CD59、 甚至 CRIg本身等的全长或部分序列， 其 中相连的方式包括直接相连， 以及通过其他物理、 化学和生物等手段通过其 他分子间接相连。
即， CRIg与不同的补体抑制剂， 包括存在于血液循环中的 Cl-INH (C1 inhibitor, C1抑制蛋白）、 C4BP (C4 binding protein, C4结合蛋白）、 factor I (I因子）、 factor H (H因子）、 S-protein (S蛋白）和 Clusterin (簇集蛋白）； 以及表达与细胞膜表面的补体膜调控蛋白 CD35/CR1、 CD46/MCP、
CD55 DAF和 CD59甚至 CRIg本身，以及其他具有补体抑制功能的物质进行 连接。
CRIg与其他补体抑制剂的连接方式， 包括：
( 1 ) CRIg与其他补体抑制剂直接相连， 两者之间没有任何其他连接 物；
(2) CRIg与其他补体抑制剂通过生物方法连接；
(3 ) CRIg与其他补体抑制剂通过化学方法连接。
优选地， 该蛋白质由 CRIg和 factor H (fH) 组成。
本发明以 CRIg功能性胞外区与 factor H (fH)功能区直接（该重组蛋白 记为 CRIg-fH) 或通过柔性连接肽段 (SerlGly4)3间接 （该重组蛋白记为 CRIg-L-fH)相连为例， 描述该重组蛋白 CRIg-fH及 CRIg-L-fH的靶向补体 抑制效果。
在本发明的一个实施例中， 该蛋白质由 CRIg胞外功能区、 factor H以及 两者间的连接物组成。
在本发明的另一个实施例中，该蛋白质的氨基酸序列如 SEQ ID N02或 者 SEQ ID N04所示。
本发明提供了编码上述蛋白质的核酸。 优选地， 其核苷酸编码序列如 SEQ ID NO 1或者 SEQ ID NO 3所示。
该核酸编码具有抑制补体激活功能的该作为靶向特异性补体抑制剂的蛋 白质， 并且包含编码 CRIg和补体抑制剂的序列。
本发明还提供了一种含上述核酸的载体。以 CRIg-fH和 CRIg- (Gly4Ser) 3-fH为例， 该载体含有核酸的碱基序列如 SEQ ID NO 1或者 SEQ ID NO 3 所示。
本发明还提供了一种含上述核酸的细胞。
本发明还提供了一种该靶向特异性补体抑制剂的制备方法， 包含通过基 因工程技术， 将 CRIg胞外功能区和补体抑制功能区的蛋白质多肽直接或间 接相连；
CRIg胞外功能区蛋白质的氨基酸序列如 SEQ ID NO 10所示；
补体抑制功能区为 H因子、 C1抑制蛋白、 C4结合蛋白、 I因子、 S蛋白、 簇集蛋白， 补体膜调控蛋白 CD35/CR1、 CD46/MCP、 CD55/DAF、 CD59或 者 CRIg本身的功能片段或全长的任意一种或多种的组合； 该基因工程技术为：
通过重叠延伸 PCR法 (gene splicing by overlap extension PCR, SOE PCR), 将编码 CRIg胞外功能区的核酸序列和编码补体抑制功能区的核酸序 列直接连接， 或者通过编码柔性连接肽的核酸序列间接连接， 然后将连接所 得的融合序列***到真核表达载体中， 通过真核蛋白表达***诱导该作为靶 向特异性补体抑制剂的蛋白质表达， 最后将所得的蛋白质纯化。
蛋白表达***是指由宿主、 外源基因、 载体等组成的体系， 通过这个体 系可以实现外源基因在宿主中表达的目的。 宿主是表达蛋白的生物体， 可以 为细菌、 酵母、 植物细胞、 动物细胞等。 由于各种生物的特性不同， 适合表 达蛋白的种类也不相同。 载体的种类与宿主相匹配， 根据宿主不同， 分为原 核 （细菌）表达载体、 酵母表达载体、 植物表达载体、 哺乳动物表达载体、 昆虫表达载体等。 载体中含有外源基因片段， 通过载体介导， 外源基因可以 在宿主中表达。
本发明的蛋白质可以利用基因工程技术， 优选地连接 CRIg蛋白质和 factor H蛋白质。该 CRIg蛋白质的氨基酸序列如 SEQ ID NO 6所示；该 factor H蛋白质的氨基酸序列如 SEQ ID N0 8所示。也可以按照蛋白质的序列依次 连接氨基酸残基。
本发明的核酸也可以利用基因工程技术， 优选地连接 CRIg蛋白质和 factor H蛋白质的编码序列； 或者按照核酸的序列依次连接碱基。
本发明提供了靶向特异性补体***抑制剂， 即上述蛋白质在制备抑制补 体激活的药物中的应用。 也可以用于制备保护细胞免受补体攻击并提高红细 胞压积的药物。
本发明提供了上述蛋白质在制备保护阵发性夜间血红蛋白尿患者血细胞 的药物中的应用， 也可用于制备其他疾病， 例如系膜增生性肾小球肾炎、 非 典型溶血性***综合征和年龄相关的黄斑变性等疾病中使遭受补体攻击的 细胞免受补体攻击的药物或者药剂。
本发明还提供了一种药物， 药物的活性成分为该作为靶向特异性补体抑 制剂的蛋白质，优选地含有 CRIg和 factor H的功能域。该药物是抑制补体激 活的药物。该药物产生含有氨基酸序列如 SEQ ID NO 2或者 SEQ ID NO 4所 示的蛋白质。 该药物选用在真核***分泌表达并纯化获得的该作为靶向特异性补体抑 制剂的蛋白质， 体内外实验均证实其具有靶向性补体抑制功能及良好的药物 结合性质。其中， 离体实验证实治疗 PNH患者的半数有效药物浓度低于 100 nM， 体内实验证实能有效治疗大鼠系膜增生性肾小球肾炎。
该药物剂型优选为针剂。 也可采用其他剂型， 如外用涂剂等。
该药物的给药方式包括：
( 1 ) 直接给药；
(2) 通过能携带或表达该药物的载体***给药。
本发明提供的靶向特异性补体***抑制剂， 与另一种靶向性补体抑制剂 CR2-CD59对比， CRIg-fH的结合常数 Ka小 3倍左右（CRIg-fH为 1.093xl04 ， CR2-CD59为 3.45xl04 1/Ms), 而解离常数 Kd则要小 46倍左右（CRIg-L-fH 为 3.656xl0'3， CR2-CD59为 0.169 1/s)， 最终 CRIg-L-fH的平衡解离常数 KD 值比 CR2-CD59低 15倍多， 具有更好的药理学活性， 即意味着： 与药物靶 点 C3b/iC3b结合时， CRIg-L-fH虽然比 CR2-CD59慢 3倍， 但是 CRIg-L-fH 一旦结合后，则很难从药靶上解离下来，从而可减少给药次数，这对治疗 PNH 这类需长期给药的患者具有很大的优势。 由于缺少 CR2-ffl的数据， 同时两 种的靶向功能部位是 CRIg和 CR2, 而不是 fH和 CD59, 因此， 两者具有一 定程度的可比性。
目前缺少另一种靶向性补体抑制剂 CRIg-Fc的具体数据，但考虑到 CRIg 并不抑制 C3转换酶的降解，也没有类似 H因子的促 I因子降解 C3b的活性， 因此 CRIg只有很弱的补体抑制活性。比较而言 CRIg-fH连接的是 fH而不是 抗体的 Fc段， 因此 CRIg-fH将具有更好的补体抑制效果。
相对于非靶向性补体抑制剂 minifH, CRIg-fH的优势更为明显， 它不但 具有靶向性， 同时还具有更优的结合常数， 其 KD值比 minifH小 3倍。
更重要的是， 以上所有其他的补体抑制剂均为证实可以抑制补体的经典 途径， 而我们实验证实了 CRIg-L-fH不但可以抑制补体替补途径， 还可抑制 经典途径。
实施例 1 : CRIg-fH及 CRIg-L-fH蛋白表达载体的构建、 真核表达和纯 化
1.仪器与材料： Mastercycler pro-Eppendorf PCR仪 （购自德国 Eppendorf 公司）， DK-8D型电热恒温水槽 （购自上海精宏实验设备有限公司）， IQ350凝胶成 像***（购自美国 GE Healthcare公司）， C02细胞培养箱（购自美国 Thermo Scientific公司）， FR-980A生物电泳图像分析*** （购自复日科技公司）、 BioRAD Mini protein Tera system (购自美国 BioRAD 公司）、 NanoVue R A/DNA浓度 /纯度检测仪（购自德国 IKA公司）
2.实验方法：
2.1 基因克隆和载体构建
由于 CRIg是在巨噬细胞表面高丰度表达的补体受体， 因而应用 Trizol 法从人组织细胞淋巴瘤细胞系 U937中抽提总 RNA,反转录为第一链 cDNA。 根据已有的文献报道， 从 NCBI的 Protein数据库中找到人 CRIg胞外结构域 [102] (NP_001171759.1, residues 19-137, PDB: 2ICC_A)相应的蛋白序列， 并 从 NCBI的 Gene数据库中找到人 CRIg基因（NM— 001184830.1 )与之对应的 核酸序列。设计克隆 CRIg胞外结构域的上下游引物序列， 从 U937的 cDNA 中通过 PCR扩增得到 CRIg胞外结构域的核酸序列， 连接入 pMD18 T载体 中， 转化、 蓝白斑筛选阳性克隆、 挑取阳性菌落扩大培养并送测序。
同样地， 由于补体调节蛋白 factor H在肝脏细胞中表达量较高， 因而应 用 Trizol法抽提人肝癌细胞系 HepG2总 RNA, 反转录为 eDNA。 根据已有 的文献对 factor H SCR1-5结构域（NP— 000177.2， residues 19-323 )的报道 [96]， 从 NCBI的 Protein数据库中找到蛋白序列， 并从 NCBI的 Gene数据库找到 人 factor H基因中对应的核酸序列， 设计克隆 FH SCR1-5结构域的上下游引 物序列，从 HepG2的 cDNA中 PCR扩增得到 FH SCR1-5结构域的核酸序列， 连接入 PMD18 T载体中，转化、蓝白斑筛选阳性克隆、挑取阳性菌落扩大培 养并送测序。 选取测序结果中序列正确的克隆， 提取质粒作为下一步重叠延 伸 PCR的模板。
进一步， 应用重叠延伸 PCR法 ( gene splicing by overlap extension PCR, SOE PCR)将 CRIg的胞外结构域和 factor H的 SCR1-5结构域直接（CRIg-fH) 或者通过柔性连接肽序列 （SerGly4) 3间接（CRIg-L-fH)连接起来。 重叠 延伸 PCR法的原理是采用具有互补末端的引物， 使 PCR产物形成包含重叠 区域的双链，从而在随后的第二轮 PCR扩增反应中通过重叠链的延伸，将不 同来源的扩增片段重叠拼接起来， 构建包含 CRIg胞外结构域和 factor H SCR1-5结构域以及 linker序列 （柔性连接肽序列） （L, (SerlGly4)3) ( TCTGGTGGCGGTGGCTCCGGCGGAGGTGGGTCCGGTGGCGGCGGA ) 的融合序列。 ***到 pHLsec真核表达载体中， 双向测序鉴定***序列， 载 体命名为 pHLsec-CRIg-fH和 pHLsec-CRIg-L-fH。
CRIg核酸序列 (SEQ ID NO 5)：
ATGGGGATCTTACTGGGCCTGCTACTCCTGGGGCACCTAACAGTGGACA CTTATGGCCGTCCCATCCTGGAAGTGCCAGAGAGTGTAACAGGACCTT GGAAAGGGGATGTGAATCTTCCCTGCACCTATGACCCCCTGCAAGGCT ACACCCAAGTCTTGGTGAAGTGGCTGGTACAACGTGGCTCAGACCCTG TCACCATCTTTCTACGTGACTCTTCTGGAGACCATATCCAGCAGGCAAA GTACCAGGGCCGCCTGCATGTGAGCCACAAGGTTCCAGGAGATGTATC CCTCCAATTGAGCACCCTGGAGATGGATGACCGGAGCCACTACACGTG TGAAGTCACCTGGCAGACTCCTGATGGCAACCAAGTCGTGAGAGATAA GATTACTGAGCTCCGTGTCCAGAAACTCTCTGTCTCCAAGCCCACAGTG ACAACTGGCAGCGGTTATGGCTTCACGGTGCCCCAGGGAATGAGGATT AGCCTTCAATGCCAGGCTCGGGGTTCTCCTCCCATCAGTTATATTTGGTA TAAGCAACAGACTAATAACCAGGAACCCATCAAAGTAGCAACCCTAAG TACCTTACTCTTCAAGCCTGCGGTGATAGCCGACTCAGGCTCCTATTTCT GCACTGCCAAGGGCCAGGTTGGCTCTGAGCAGCACAGCGACATTGTGA AGTTTGTGGTCAAAGACTCCTCAAAGCTACTCAAGACCAAGACTGAGG CACCTACAACCATGACATACCCCTTGAAAGCAACATCTACAGTGAAGC AGTCCTGGGACTGGACCACTGACATGGATGGCTACCTTGGAGAGACCA GTGCTGGGCCAGGAAAGAGCCTGCCTGTCTTTGCCATCATCCTCATCAT CTCCTTGTGCTGTATGGTGGTTTTTACCATGGCCTATATCATGCTCTGTCG GAAGACATCCCAACAAGAGCATGTCTACGAAGCAGCCAGGGCACATGC CAGAGAGGCCAACGACTCTGGAGAAACCATGAGGGTGGCCATCTTCGC AAGTGGCTGCTCCAGTGATGAGCCAACTTCCCAGAATCTGGGCAACAA CTACTCTGATGAGCCCTGCATAGGACAGGAGTACCAGATCATCGCCCAG ATCAATGGCAACTACGCCCGCCTGCTGGACACAGTTCCTCTGGATTATG AGTTTCTGGCCACTGAGGGCAAAAGTGTCTGTTAA
CRI (SEQ ID NO 6)：
Figure imgf000013_0001
Factor H核酸序列 (SEQ ID NO 7)：
ATGAGACTTCTAGCAAAGATTATTTGCCTTATGTTATGGGCTATTTGTGT
AGCAGAAGATTGCAATGAACTTCCTCCAAGAAGAAATACAGAAATTCT
GACAGGTTCCTGGTCTGACCAAACATATCCAGAAGGCACCCAGGCTAT
CTATAAATGCCGCCCTGGATATAGATCTCTTGGAAATGTAATAATGGTAT
GCAGGAAGGGAGAATGGGTTGCTCTTAATCCATTAAGGAAATGTCAGA
AAAGGCCCTGTGGACATCCTGGAGATACTCCTTTTGGTACTTTTACCCTT
ACAGGAGGAAATGTGTTTGAATATGGTGTAAAAGCTGTGTATACATGTA
ATGAGGGGTATCAATTGCTAGGTGAGATTAATTACCGTGAATGTGACAC
AGATGGATGGACCAATGATATTCCTATATGTGAAGTTGTGAAGTGTTTAC
CAGTGACAGCACCAGAGAATGGAAAAATTGTCAGTAGTGCAATGGAAC
CAGATCGGGAATACCATTTTGGACAAGCAGTACGGTTTGTATGTAACTC
AGGCTACAAGATTGAAGGAGATGAAGAAATGCATTGTTCAGACGATGG
TTTTTGGAGTAAAGAGAAACCAAAGTGTGTGGAAATTTCATGCAAATC
CCCAGATGTTATAAATGGATCTCCTATATCTCAGAAGATTATTTATAAGGA
GAATGAACGATTTCAATATAAATGTAACATGGGTTATGAATACAGTGAA
AGAGGAGATGCTGTATGCACTGAATCTGGATGGCGTCCGTTGCCTTCAT
GTGAAGAAAAATCATGTGATAATCCTTATATTCCAAATGGTGACTACTCA
CCTTTAAGGATTAAACACAGAACTGGAGATGAAATCACGTACCAGTGT
AGAAATGGTTTTTATCCTGCAACCCGGGGAAATACAGCAAAATGCACA 3WOIOWOV03IVOOOIV9WO9IVWXV3OI3XOV0V0V3VXV99X VO9WOWWOOVOVIOIVOV3VIOOW1V3WIDIIWXV IVO0IIV VOO O 3WVVWIIIV O9VOII3VIVIIVVXIIVVV IVDIVV V39I9WOW1X3VWIVOV1W0O91OIOI9V0333II3W000VOO 丄 viovooxv丄丄 VIOKOYLL 丄 TOv v voo丄工 vo丄 wov丄丄丄 v iv
VOV iDO丄丄 OLLWOO丄 丄工 VOVOO丄 V丄：) VULV工工 ID 丄丄 0工 工工 OVDC OOO丄 ) OOJYD O工工 3 O工 VIVIYOVDO丄 O丄
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Figure imgf000016_0001
v^AAcr aoNdivMHJSciisioai^iDSidHS ^ao D^vivdoaiojoai
Figure imgf000016_0002
S0NA3d<m¾9M0AXHIA03¾0¾0^¾INS Hada¾ ¾THa31IITNSS
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Figure imgf000016_0004
Figure imgf000016_0005
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8£9000/M0iN3/X3d Z.91Z£0/S10J OAV （119aa) (SEQ ID NO 10)
Figure imgf000017_0001
QTPDGNQWRDKITELRVQK
FactorH SCR1-5结构域
核酸序列 （915bp) (SEQ ID NO 11):
Gaagattgcaatgaacttcctccaagaagaaatacagaaattctgacaggttcctggtctgaccaaacatatccagaa ggcacccaggctatctataaatgccgccctggatatagatctcttggaaatgtaataatggtatgcaggaagggagaa tgggttgctcttaatccattaaggaaatgtcagaaaaggccctgtggacatcctggagatactccttttggtacttttacc cttacaggaggaaatgtgtttgaatatggtgtaaaagctgtgtatacatgtaatgaggggtatcaattgctaggtgagatt aattaccgtgaatgtgacacagatggatggaccaatgatattcctatatgtgaagttgtgaagtgtttaccagtgacagc accagagaatggaaaaattgtcagtagtgcaatggaaccagatcgggaataccattttggacaagcagtacggtttgt atgtaactcaggctacaagattgaaggagatgaagaaatgcattgttcagacgatggtttttggagtaaagagaaacc aaagtgtgtggaaatttcatgcaaatccccagatgttataaatggatctcctatatctcagaagattatttataaggagaat gaacgatttcaatataaatgtaacatgggttatgaatacagtgaaagaggagatgctgtatgcactgaatctggatggc gtccgttgccttcatgtgaagaaaaatcatgtgataatccttatattccaaatggtgactactcacctttaaggattaaaca cagaactggagatgaaatcacgtaccagtgtagaaatggtttttatcctgcaacccggggaaatacagcaaaatgcac aagtactggctggatacctgctccgagatgtaccttgaaa
（305aa) (SEQ ID NO 12):
Figure imgf000017_0002
LK
2.2真核蛋白表达***的建立
取对数生长期的 293FT细胞， 以每皿 1.2X107的细胞密度均匀铺在直径 15cm的细胞培养皿中， PEI法分别转染 pHLsec-CRIg-fH和 pHLsec-CRIg-L-fH 大抽质粒，在 37°C， 5°/。C02培养箱中培养 6小时后更换 293表达培养基（购 自 invitrogen公司）， 继续培养三天后收取细胞上清， 离心去除细胞及细胞碎 片。
2.3蛋白亲和纯化、 透析和蔗糖浓縮
应用 Ni2+柱亲和纯化的方法，将细胞上清中 CRIg-fH和 CRIg-L-fH纯化 洗脱至洗脱缓冲液中， 转移至透析管中 （购自 Novagen公司）， 透析法将缓 冲液更换为 PBS (磷酸缓冲液）， 蔗糖法吸水浓缩后再用 PBS透析。
3.实验结果：
通过上述方法应用真核***诱导表达和纯化得到 CRIg-fH和 CRIg-L-fH 两种融合蛋白， PAGE 电泳 （聚丙烯酰胺凝胶电泳， polyacrylamide gelelectrophoresis)检测为单一条带， 浓縮后浓度可达到 1~2 mg/ml, 参见图 1所示。
所涉及相关序列如下：
CRIg-fH核酸序列 (SEQ ID NO 1)：
ATGGGCCGTCCCATCCTGGAAGTGCCAGAGAGTGTAACAGGACCTTGG AAAGGGGATGTGAATCTTCCCTGCACCTATGACCCCCTGCAAGGCTACA CCCAAGTCTTGGTGAAGTGGCTGGTACAACGTGGCTCAGACCCTGTCA CCATCTTTCTACGTGACTCTTCTGGAGACCATATCCAGCAGGCAAAGTA CCAGGGCCGCCTGCATGTGAGCCACAAGGTTCCAGGAGATGTATCCCT CCAATTGAGCACCCTGGAGATGGATGACCGGAGCCACTACACGTGTGA AGTCACCTGGCAGACTCCTGATGGCAACCAAGTCGTGAGAGATAAGAT TACTGAGCTCCGTGTCCAGAAAGAAGATTGCAATGAACTTCCTCCAAG AAGAAATACAGAAATTCTGACAGGTTCCTGGTCTGACCAAACATATCCA GAAGGCACCCAGGCTATCTATAAATGCCGCCCTGGATATAGATCTCTTGG AAATGTAATAATGGTATGCAGGAAGGGAGAATGGGTTGCTCTTAATCCA TTAAGGAAATGTCAGAAAAGGCCCTGTGGACATCCTGGAGATACTCCT TTTGGTACTTTTACCCTTACAGGAGGAAATGTGTTTGAATATGGTGTAAA AGCTGTGTATACATGTAATGAGGGGTATCAATTGCTAGGTGAGATTAATT ACCGTGAATGTGACACAGATGGATGGACCAATGATATTCCTATATGTGA AGTTGTGAAGTGTTTACCAGTGACAGCACCAGAGAATGGAAAAATTGT CAGTAGTGCAATGGAACCAGATCGGGAATACCATTTTGGACAAGCAGT ACGGTTTGTATGTAACTCAGGCTACAAGATTGAAGGAGATGAAGAAAT
GCATTGTTCAGACGATGGTTTTTGGAGTAAAGAGAAACCAAAGTGTGT
GGAAATTTCATGCAAATCCCCAGATGTTATAAATGGATCTCCTATATCTC
AGAAGATTATTTATAAGGAGAATGAACGATTTCAATATAAATGTAACATG
GGTTATGAATACAGTGAAAGAGGAGATGCTGTATGCACTGAATCTGGAT
GGCGTCCGTTGCCTTCATGTGAAGAAAAATCATGTGATAATCCTTATATT
CCAAATGGTGACTACTCACCTTTAAGGATTAAACACAGAACTGGAGAT
GAAATCACGTACCAGTGTAGAAATGGTTTTTATCCTGCAACCCGGGGAA
ATACAGCAAAATGCACAAGTACTGGCTGGATACCTGCTCCGAGATGTAC
CTTGAAATAA
CRI -fH蛋白序列 (SEQ ID NO 2)：
Figure imgf000019_0001
PATRGNTAKCTSTGWIPAPRCTLKStop
CRIg-L-fH核酸序列 (SEQ ID NO 3)：
ATGGGCCGTCCCATCCTGGAAGTGCCAGAGAGTGTAACAGGACCTTGG AAAGGGGATGTGAATCTTCCCTGCACCTATGACCCCCTGCAAGGCTACA CCCAAGTCTTGGTGAAGTGGCTGGTACAACGTGGCTCAGACCCTGTCA CCATCTTTCTACGTGACTCTTCTGGAGACCATATCCAGCAGGCAAAGTA CCAGGGCCGCCTGCATGTGAGCCACAAGGTTCCAGGAGATGTATCCCT CCAATTGAGCACCCTGGAGATGGATGACCGGAGCCACTACACGTGTGA AGTCACCTGGCAGACTCCTGATGGCAACCAAGTCGTGAGAGATAAGAT TACTGAGCTCCGTGTCCAGAAATCTGGTGGCGGTGGCTCCGGCGGAGG TGGGTCCGGTGGCGGCGGAGAAGATTGCAATGAACTTCCTCCAAGAAG AAATACAGAAATTCTGACAGGTTCCTGGTCTGACCAAACATATCCAGAA
GGCACCCAGGCTATCTATAAATGCCGCCCTGGATATAGATCTCTTGGAAA
TGTAATAATGGTATGCAGGAAGGGAGAATGGGTTGCTCTTAATCCATTA
AGGAAATGTCAGAAAAGGCCCTGTGGACATCCTGGAGATACTCCTTTT
GGTACTTTTACCCTTACAGGAGGAAATGTGTTTGAATATGGTGTAAAAG
CTGTGTATACATGTAATGAGGGGTATCAATTGCTAGGTGAGATTAATTAC
CGTGAATGTGACACAGATGGATGGACCAATGATATTCCTATATGTGAAG
TTGTGAAGTGTTTACCAGTGACAGCACCAGAGAATGGAAAAATTGTCA
GTAGTGCAATGGAACCAGATCGGGAATACCATTTTGGACAAGCAGTAC
GGTTTGTATGTAACTCAGGCTACAAGATTGAAGGAGATGAAGAAATGCA
TTGTTCAGACGATGGTTTTTGGAGTAAAGAGAAACCAAAGTGTGTGGA
AATTTCATGCAAATCCCCAGATGTTATAAATGGATCTCCTATATCTCAGA
AGATTATTTATAAGGAGAATGAACGATTTCAATATAAATGTAACATGGGT
TATGAATACAGTGAAAGAGGAGATGCTGTATGCACTGAATCTGGATGGC
GTCCGTTGCCTTCATGTGAAGAAAAATCATGTGATAATCCTTATATTCCA
AATGGTGACTACTCACCTTTAAGGATTAAACACAGAACTGGAGATGAA
ATCACGTACCAGTGTAGAAATGGTTTTTATCCTGCAACCCGGGGAAATA
CAGCAAAATGCACAAGTACTGGCTGGATACCTGCTCCGAGATGTACCTT
GAAATAA
CRI -L-fH蛋白序列 (SEQ ID NO 4)：
Figure imgf000020_0001
HRTGDEITYQCRNGFYPATRGNTAKCTSTGWIPAPRCTLKStop 实施例 2: CRIg-fH及 CRIg-L-fH与 C3活化和降解片段相互作用的动 力学分析和亲和力测定
1.仪器与材料：
Biacore T200 (蛋白互作分析仪， 购自 GE Healthcare公司）、 Series S Sensor Chip NTA芯片、 CRIg-L-fH蛋白溶液、 C3 活化和降解的蛋白组分 (C3b、 iC3b、 C3c、 C3d,购自 Complement Technology公司）、 HBS-N溶液（购 自 GE Healthcare公司；）
2.实验方法
表面等离子共振技术（Surface plasmon resonance, SPR)是一项用于分 析生物大分子之间的相互作用的技术， 既可以定性地判断两分子之间是否有 相互作用或者比较一种分子与多种分子之间相互作用的强弱， 也可以实时定 量地测定分子间相互作用的亲和力参数 （平衡常数）和动力学参数（速率常 数)， 甚至热力学参数 （反应的焓）。 该技术是利用了物理光学的原理， 在研 究两种分子相互作用时， 将一种分子固定在传感芯片表面， 而另一种分子的 溶液流过其表面， 两种分子的结合会使传感芯片表面的折射率发生改变， 因 此检测两分子间的相互作用。应用基于 SPR原理的蛋白互作*** BIAcore对 补体抑制剂 CRIg-fH与 C3 酶解产物相互作用动力学和亲和力分析。 由于 CRIg-fHHis标签， 因此选用 Series S Sensor NTA芯片。 实验方法如下：
( 1 )芯片预处理:将 NTA传感芯片模块嵌入 BIAcore仪器，配制 HBS-N running buffer (缓冲液）， 开启 BIAcore T200蛋白互作分析仪， 设置程序， 先注射两针 HBS-N running buffer对芯片进行预清洗， 25°C， 流速为 120 μ 1/min, 每针持续 5 min， 直至检测基线拉平；
(2) Ni偶联： 注射一针 Nickel溶液 （0.5 mM NiCl2/running buffer), 25°C， 流速 30 μ 1/min, 持续 60 sec, 稳定 30 sec;
(3 )配体偶联： 将 CRIg-fH融合蛋白用 HBS-N running buffer稀释为 0.2 u g/ml的配体溶液， 注射一针配体溶液， 25°C， 流速 30 1/min, 持续 60 sec, 稳定 30 sec?
(4)分析物进样： 用 HBS-N running buffer配制梯度稀释的 C3酶解产 物的分析溶液， 注射一针特定浓度的 C3酶解产物的分析溶液， 25°C， 流速 30 μ 1/min, 持续 2 min, 稳定 120 sec; (5 ) 芯片再生： 注射一针 Regeneration solution (再生溶液： 350 mM EDTA (乙二胺四乙酸）， pH8.3)， 25°C , 流速 30 1/min, 持续 60 sec;
(6) 重复步骤 （2) 到 （5) 进行 C3酶解产物第二个浓度的动力学曲 线测定；
(7)待 C3所有酶解产物的各个浓度都检测完毕，注射两针 Regeneration solution清洗芯片， 直到检测线回到基线水平；
(8)应用 BIAcore T200 v2.02软件进行动力学曲线的拟合和动力学参 数 （亲和常数 Ka、 解离常数 Kd和平衡解离常数 KD) 的测定。 亲和常数、 解离常数和平衡解离常数如表 1所示。 表 1
CRIQ-L-W k, ( 1 Mt) Kg ( M)
C3D 1 S48E4 3J50E-3 2.336E-7
iC3b 1.Q23E3 3.615E-3 3.513E-6
C3C 2479E3 6.15QE-3 2.492E-6
C3d - - -
k. ( 1/lis) Kg (M)
C3b 1.093E4 3.656E-3 3.346E-7
tC3 3.327E3 4 055E-3 1 219E-6
C3c 1.051 E3 7 155E-3 6.806E-8
C3d - - -
3. 实验结果显示，参见图 2(CRIg-L-fH与 C3活化和降解产物 C3b、iC3b、 C3c、 C3d相互作用）和图 3 (CRIg-fH与 C3b、 iC3b、 C3c、 C3d相互作用的 动力学分析和结合力测定结果示意图）， CRIg-L-fH和 CRIg-fH分别与 C3b、 iC3b和 C3c都有不同程度的结合，但是 CRIg-L-fH的结合力更强， 因此后续 的功能实验和动物模型选用 CRIg-L-fH。
实施例 3: CRIg-L-fH保护 PNH红细胞免受补体替补途径和经典途径诱 导的溶血反应
1. 仪器与材料：
七例 PNH患者红细胞、 正常人血清、 CVF (眼镜蛇毒因子 cobra venom factor: lmg/ml, 购自 comptech公司）、 抗人红细胞多抗（购自 Rockland公 司）、 Bio-Tek synergy HT 多功能酶标仪 （购自芬兰 Labsystems 公司）、 Minispin台式高速离心机 （购自德国 Eppendorf 公司）、 DK-8D型电热恒温 水槽 （购自上海精宏实验设备有限公司）
2. 实验方法：
2.1 CRIg-L-fH对补体替补途径诱导的 PNH红细胞溶血的抑制作用 七例 PNH患者末端采血收集红细胞 （RBC)， PBS洗三遍（5000rpm， 离心 3min)， 配成 4%RBC溶液； 200μ1反应体系， 加入 4%RBC 25μ1 (终浓 度 1%), CRIg-L-fH蛋白溶液倍比稀释后加入体系构建终浓度为 0-3μΜ的系 列浓度梯度， CVF ( 100 μ^/πιΏ 4 μ1， 正常人血清 20 μΐ ( 10%终浓度）， 同 时设置 blank (空白） 和 total lysis (全部裂解） 的对照， 37°C水浴 30min， 10,000rpm离心 lmin， 取 100 μΐ 上清， 酶标仪检测 OD414讓 吸光值。 2.2CRIg-L-fH对补体经典途径诱导的 PNH红细胞溶血的抑制作用
七例 PNH患者末端采血收集红细胞, PBS洗三遍（5,000 rpm,离心 3min)， 配成 4%RBC溶液； 200 μΐ反应体系， 加入 4% RBC 25 μΐ (终浓度 1%)， CRIg-L-fH蛋白溶液倍比稀释后加入体系构建终浓度为 0-3 μΜ的系列浓度梯 度， 抗人 RBC抗体（5mg/ml) 2μ1, 正常人血清 20 μΐ ( 10%终浓度）， 同时 设置 blank和 total lysis的对照， 37。C水浴 30min， 10,000 rpm离心 1 min， 取 100 μΐ上清， 酶标仪检测 OD414 nm吸光值。
2.3统计学处理方法
数据用均数 ±标准差 （mean ± SD)表示。 图表绘制应用 Excel软件。
3. 实验结果：
体外溶血实验结果表明， CRIg-L-fH蛋白对补体的替补途径和经典途径 诱导的 PNH红细胞溶血都有一定的抑制作用，其中对于补体替补途径的抑制 作用更为显著， 参见图 4和图 5所示。
实施例 4: CRIg-L-fH对 Thy-ΙΝ大鼠肾炎症状的缓解作用
1. 仪器与材料：
Bio-Tek synergy HT多功能酶标仪（购自芬兰 Labsystems公司）、 激光 共聚焦显微镜 FV500 (购自日本 Olympus 公司）、 BCA蛋白定量试剂盒（购 自 Thermo Fisher公司：)、 Cobas 6000 analyzer全自动生化分析仪（购自 Roche 公司）、 SD大鼠、 anti-C3b/iC3b-FITC抗体、 anti-SC5b-9抗体、 anti-His抗体、 rabbit anti-mouse (兔抗小鼠） IgG-FITC抗体
2. 实验方法：
2.1 Thy-l大鼠肾炎模型的建立
雄性 SD大鼠在 SPF (无特定病原体， Specefic pathogen Free)级环境中 饲养至体重达到 150-200 g， 随机分为三组进行建模：
( 1 ) NRS组（n=4): 大鼠尾静脉注射正常兔血清（normal rabbit serum, NRS), 注射剂量为 1 ml/100 g体重；
( 2 ) ATS 组 （n=4 ) : 大鼠尾静脉注射兔抗大鼠胸腺的抗血清 (anti-thymocyte serum, ATS), 注射剂量为 1 ml/100 g体重；
(3) ATS+CRIg-L-fH组（n=4): 大鼠尾静脉注射兔抗大鼠胸腺的抗血 清 （anti-thymocyte serum, ATS), 注射剂量为 1 ml/100 g体重， 随后尾静脉 联合注射 CRIg-L-fH， 注射剂量为 1 mg/100 g体重；。
2.2各组大鼠肾炎指标的检测
注射后， 将大鼠饲养于大鼠代谢笼中。 注射后第 24 hr和 72 hr， 通过尾 静脉采集血液样品， 收集于标准促凝管中。 室温放置 30 min， 3000 rpm离心 lO min, 取血液上清（血清）， 应用 Cobas 6000 analyzer全自动生化分析仪进 行肾功能指标 （血尿素氮和血清肌酸酐） 的检测和数据收集。
注射后第 24 hr、 48 hr和 72 hr，使用大鼠代谢笼收集 24小时尿液。尿液 原液经 50倍稀释后， 应用 BCA Protein Assay试剂盒测定尿液中的总蛋白浓 度， 总蛋白含量（mg/24 hr) =BCA法测定的蛋白浓度 x50倍（稀释倍数） x 尿液总体积。 取 100 μ ΐ稀释尿液加入到％孔微孔板中使用 Bio-Tek多功能 酶标仪直接读取 OD414nm吸光值， 由此测定尿液中的血红蛋白含量。
2.3免疫荧光检测各组大鼠肾脏冰冻切片的 IgG、 C3片段、 CRIg-L-fH的 沉积
注射后第三天和第七天， 每组分别随机选取一只大鼠, ***麻醉后， 左 心室插针进行心脏灌注， 先灌注生理盐水， 待肝脏由红变白后， 再灌注 4% 多聚甲醛， 直至大鼠死亡。 摘取左侧肾脏， 浸泡在 PBS中漂洗数次后， 放入 细胞冻存管中， 置于液氮固定 30 min， 随后放入 -80°C保存备用。
开启冷冻切片机， 待工作室内温度达到 -20°C以下时， 从 -80°C取出冻存 的肾脏组织，包埋在 OCT内， 使用冷冻切片机裁***组织，选取位于肾脏皮 质和髓质交界处的组织区域， 切为 6 u m的薄片贴附于载玻片上。 4%多聚 甲醛固定 15min， PBS漂洗。 10%正常羊血清封闭， 分别用 anti-C3b/iC3b、 anti-His anti-SC5b-9抗体 4度孵育过夜。 次日， PBS漂洗三遍后二抗孵育。 PBS漂洗， 封片， 镜检。
3. 实验结果：
CRIg-L-fH治疗的 Thy-ΙΝ大鼠相比未治疗组血清中的尿素氮和肌酐含 量下降， 尿液中的蛋白总量和血红蛋白量减少， 同时有大量的 CRIg-L-fH沉 积在肾小球系膜区， 并且几乎没有 C3b/iC3b的沉积， 可见 CRIg-L-fH能一定 程度的缓解大鼠的 Th-1N肾炎症状，参见图 6〜图 10所示（图 10中 *P<0.05, * * <0.01， ** * <0.001; n=4)0
综上所述 本发明提供的靶向特异性补体抑制剂作为一种新型的靶向性 补体抑制剂， 能够特异地靶向到体内补体激活位点， 并长效地抑制补体激活 和由补体激活所介导的细胞和组织损伤， 从而不仅能够保护人缺陷型 PNH 红细胞免受补体进攻引起的溶血性损伤， 而且还能在 Thy-1肾炎大鼠模型中 明显缓解补体诱导的系膜增生性病变。 同时， 也可预示该靶向特异性补体抑 制剂能有效治疗其他补体***过度激活的疾病， 例如年龄相关性视黄斑变 性、 非典型溶血性***综合征、 类风湿性关节炎、 强直性脊柱炎、 红斑狼 疮等。 因此， 本发明提供的靶向特异性补体抑制剂具有巨大的潜在药物应用 价值和开发前景。
尽管本发明的内容已经通过上述优选实施例作了详细介绍， 但应当认识 到上述的描述不应被认为是对本发明的限制。 在本领域技术人员阅读了上述 内容后， 对于本发明的多种修改和替代都将是显而易见的。 因此， 本发明的 保护范围应由所附的权利要求来限定。

Claims

权利要求
1.一种靶向特异性补体抑制剂， 其特征在于， 该靶向特异性补体抑制剂为一 种蛋白质， 该蛋白质具有靶向抑制补体激活的功能， 并且该蛋白质的氨基酸 序列含有 CRIg胞外功能区和补体抑制功能区； 所述蛋白质的氨基酸序列由 CRIg胞外功能区和补体抑制功能区直接相连，或通过能够连接两者的连接物 间接相连组成；
所述的补体抑制功能区为 H因子、 C1抑制蛋白、 C4结合蛋白、 I因子、 S 蛋白、簇集蛋白，补体膜调控蛋白 CD35/CR1、 CD46/MCP、 CD55/DAR CD59 或者 CRIg本身的功能片段或全长中的任意一种或多种的组合。
2.如权利要求 1所述的靶向特异性补体抑制剂， 其特征在于， 所述蛋白质的 氨基酸序列如 SEQ ID NO 2或者 SEQ ID NO 4所示。
3.—种核酸， 其特征在于， 该核酸序列编码权利要求 1所述的蛋白质。
4.如权利要求 3所述的核酸其特征在于，所述核酸的碱基序列如 SEQ ID NO 1或者 SEQ ID N0 3所示。
5.—种载体， 其特征在于， 该载体含有权利要求 3所述的核酸。
6.—种如权利要求 1所述的靶向特异性补体抑制剂的制备方法其特征在于， 该方法是通过基因工程技术， 将 CRIg胞外功能区和补体抑制功能区的蛋白 质多肽直接或间接连接；
所述的 CRIg胞外功能区蛋白质的氨基酸序列如 SEQ ID NO 10所示； 所述的补体抑制功能区为 H因子、 C1抑制蛋白、 C4结合蛋白、 I因子、 S 蛋白、簇集蛋白，补体膜调控蛋白 CD35/CR1、 CD46/MCP、 CD55/DAF, CD59 或者 CRIg本身的功能片段或全长中的任意一种或多种的组合。
7. 如权利要求 6所述的靶向特异性补体抑制剂的制备方法， 其特征在于， 所 述的基因工程技术为：
通过重叠延伸 PCR法， 将编码 CRIg胞外功能区的核酸序列和编码补体抑制 功能区的核酸序列直接连接， 或者通过编码柔性连接肽段的核酸序列间接连 接， 然后将连接所得的融合序列***到真核表达载体中， 通过真核蛋白表达 ***诱导该作为靶向特异性补体抑制剂的蛋白质表达， 最后将所得的蛋白质 纯化。
8.—种如权利要求 1所述的靶向特异性补体抑制剂在制备靶向抑制补体激活 的药物中的用途。
9.一种如权利要求 1所述的靶向特异性补体抑制剂在制备保护阵发性夜间血 红蛋白尿患者血细胞或其他疾病中遭受补体攻击的细胞免受补体攻击的药物 中的用途。
10.—种药物， 其特征在于， 该药物的活性成分为权利要求 1所述的蛋白质。
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