WO2021164527A1 - Humanized single-chain antibody of anti-complement c5 molecule and application thereof - Google Patents

Humanized single-chain antibody of anti-complement c5 molecule and application thereof Download PDF

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WO2021164527A1
WO2021164527A1 PCT/CN2021/074563 CN2021074563W WO2021164527A1 WO 2021164527 A1 WO2021164527 A1 WO 2021164527A1 CN 2021074563 W CN2021074563 W CN 2021074563W WO 2021164527 A1 WO2021164527 A1 WO 2021164527A1
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antibody
chain
variable region
seq
chain antibody
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唐晓敏
杜兰英
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北京康普美特创新医药科技有限责任公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/567—Framework region [FR]

Definitions

  • the present invention discloses a polypeptide, more specifically, the present invention discloses an antibody.
  • the complement system is composed of more than 30 soluble protein molecules and is a part of the natural immune system. Its components include more than 30 molecules such as the inherent components of complement, a variety of regulatory factors, and complement receptors.
  • the complement system can be activated through three relatively independent and interconnected pathways, so as to regulate phagocytosis, lyse cells, mediate inflammation, immune regulation and clear immune complexes and other biological effects, including enhancing phagocytosis and enhancing phagocytosis. Chemotaxis of cells, increased permeability of blood vessels, neutralization of viruses, cell lysis, immune response regulation, etc.
  • complement activation provides a valuable first-line defense against potential pathogens
  • complement activation that promotes a protective inflammatory response may also be a potential threat to the host.
  • Complement activation and its deposition on target structures can also indirectly cause cell or tissue destruction.
  • Complement activation products that mediate tissue damage are produced at various points in the complement pathway. Inappropriate complement activation on host tissues plays an important role in the pathology of many autoimmune and inflammatory diseases.
  • the classical complement activation pathway is activated by antigen-antibody complexes.
  • the components involved in this pathway include C1-C9. According to their role in the activation process, they are artificially divided into three groups, namely recognition units (Clq, Clr, Cls), The activation unit (C4, C2, C3) and the membrane attack unit (C5-C9) play a role in the different stages of activation, namely the recognition phase, the activation phase and the membrane attack phase.
  • Mannan-binding agglutination pathway is a change of the classical pathway.
  • Mannan-Binding Lectin (MBL) in plasma directly recognizes N-galactosamine or mannose on the surface of a variety of pathogenic microorganisms, and then activates them in turn MASP-1, MASP-2, C4, C2, C3, forming the same C3 and C5 convertases as the classical pathway, activate the activation pathway of the complement cascade enzymatic reaction.
  • the alternative activation pathway is activated by foreign substances, dead tissues, cells, bacteria, etc.
  • the alternative activation pathway is different from the classical activation pathway in that activation bypasses the three components of C1, C4, and C2, directly activates C3 and then completes C5. The chain reaction of each component to C9.
  • C3 convertase is composed of C3bC4b2a.
  • C3b produced by the complement system can combine with properdin and factor B to form a complex "PC3bB".
  • factor D cleaves factor B into Bb and Ba. This cleavage allows Ba to be released from the complex and forms the alternative pathway C3 convertase PC3bBb.
  • PC3bBb cuts C3 into C3a and C3b, thereby establishing an amplification loop of the bypass pathway.
  • the classical pathway and the mannan combined agglutination pathway can also provide an amplification loop for triggering the alternative pathway of complement activation.
  • the activated C3 convertase C3bC4b2a cleaves C3 into two active fragments: anaphylatoxin C3a and opsonizing C3b.
  • C3a is a powerful allergic toxin that can cause various clinical diseases.
  • C3a can activate neutrophils, monocytes, platelets, mast cells and T cells.
  • C3a has been shown to be essential for inducing paw edema.
  • the newly formed C3b can be added to the already produced C3 convertase to form C5 convertase, which can cleave C5 to produce C5b and C5a.
  • C5 is a glycosylated ⁇ -globulin with a molecular weight of 190kDa, and its concentration in serum is 75 ⁇ g/ml (0.4.mu.M), and 1.5-3% of the molecular weight is carbohydrates.
  • Mature C5 is a heterodimer, consisting of an ⁇ chain of 999 amino acids in length and about 115 kDa connected by disulfide bonds to 656 amino acids in length and 75 kDa ⁇ .
  • the C5 coding gene is a single copy, and its protein translation product is a C5 precursor protein with a length of 1659 amino acids and a leader peptide with a length of 18 amino acids.
  • the C5 precursor protein is cut at 655 to 659 amino acids to produce a ⁇ chain of 1-655 amino acids and an ⁇ chain of 660-1658 amino acid residues.
  • the four amino acid residues between the two are removed (Haviland et al. .J.Immunol.1991,146:362-368).
  • C5a is produced by the first 74 amino acid residues at the amino terminus of the ⁇ chain under the cleavage of C5 convertase. If there is a specific molecule binding at the cleavage site, the cleavage effect of C5 convertase is blocked.
  • the molecule can act as a complement inhibitor.
  • C5a is also a potent anaphylactic toxin, which can cause changes in smooth muscle, vascular tone and vascular permeability. It is also a potent chemokine and activator of neutrophils, monocytes, platelets, endothelial cells and T cells. C5a-mediated cell activation can significantly amplify the inflammatory response by inducing the release of other inflammatory mediators, including cytokines, hydrolases, arachidonic acid metabolites and reactive oxygen species.
  • Another cleavage product, C5b is inserted into the lipid bilayer on the surface of the target cell and becomes the core of C6, C7, C8 and C9 deposition, forming a C5b-9 complex.
  • C5b-9 is also called membrane attack complex (MAC).
  • MAC membrane attack complex
  • Single-chain antibodies are small-molecule antibodies prepared by genetic engineering methods. They are composed of flexible polypeptides (usually 12-15 amino acids) that combine the heavy chain variable region (VH) and light chain variable region (VL) of the antibody.
  • VH heavy chain variable region
  • VL light chain variable region
  • the molecular weight of the linked recombinant antibody is only one-sixth of the original natural antibody, but the single-chain antibody contains all the antigen-binding sites, so the single-chain antibody retains the antigen-binding activity of the antibody to the greatest extent.
  • a small fragment of the parent antibody's antigen binding activity which can make it reach the focus tissue that is difficult to reach by conventional antibodies.
  • the antibody library technology that emerged in the 1990s bypassed the hybridoma approach necessary in the previous monoclonal antibody development process, and even did not require immunization, bringing the preparation of humanized antibodies to a whole new level. More importantly, it makes people's long-awaited dream of obtaining therapeutic human antibodies a reality.
  • the phage antibody library is the earliest and most widely used antibody library. Phage display was first established by Smith (Science, 1985, 228(4705): 1315-1317), a method of fusing the foreign protein gene with the capsid protein gene of the phage to make the expression of the foreign protein appear on the surface of the phage. technology.
  • the phage antibody library uses the above principles to express antibodies with different specificities on the surface of different phages and screen them with antigens (Science, 1989, 246(4935): 1275-1281; PNAS, 1991, 88(18): 7978-7982, Human Antibodies, 1997, 8(4):155-168).
  • the target cells used to construct the phage antibody library can be hybridoma cells, immunized human B cells or unimmunized human B cells. Unimmunized human B lymphocytes are currently the most widely used target cells. They have a large reservoir capacity and theoretically contain all human antibody genes.
  • the screening of phage antibody library is to simulate the process of in vivo antibody affinity maturation.
  • the phage library expressing specific antibodies is adsorbed on the surface with solid-phased antigen, then free phages are eluted, and the antigen-adsorbed phages are used to infect the host bacteria after proliferation and amplification. Multiple rounds of "adsorption-elution-amplification" until the specific human antibody is screened.
  • the establishment of a large-capacity antibody library is the key to obtaining high-affinity human antibodies. If the capacity of the constructed phage antibody library is greater than 10 10 , it is possible to screen for specific antibodies with high affinity ( ⁇ 10 9 M -1 ).
  • complement activation provides a valuable first-line defense against potential pathogens
  • complement activation that promotes a protective inflammatory response may also be a potential threat to the host.
  • C3a and C5a anaphylactoxins can be recruited to the diseased site and activate neutrophils, monocytes, and platelets. These activated cells indiscriminately release destructive enzymes, which may cause organ damage. Therefore, the treatment of some inflammatory diseases caused by complement activation based on down-regulation or inhibition of complement activation has become a new attempt in this technical field. Current studies have shown that down-regulation or inhibition of complement activation is useful for the treatment of some disease indications in animal models and in vitro studies.
  • soluble complement inhibitors C1-inhibitor, soluble complement receptor 1 or sCR1
  • the anti-C5 recombinant human-derived monoclonal antibody can specifically bind to human terminal complement protein C5, and block the release of inflammatory factors C5a and the formation of C5b-9 by inhibiting the cleavage of human complement C5 to C5a and C5b.
  • the purpose of the present invention is to provide a human single-chain antibody that can specifically bind to C5 molecules through the phage antibody library technology, and further provide its application in the preparation of autoimmune disease therapeutic drugs.
  • the present invention first provides a humanized single-chain antibody against complement C5 molecule.
  • the amino acid sequences of CDR1, CDR2, and CDR3 of the variable region of the light chain of the antibody are as shown in SEQ ID NO.1, respectively.
  • -35, 51-57 and 90-99 amino acid sequences the amino acid sequences of CDR1, CDR2 and CDR3 of the variable region of the antibody heavy chain are shown in SEQ ID NO. 3 30-35, 50-66 and respectively
  • the amino acid sequence at positions 99-108 is shown.
  • amino acid sequence of the antibody light chain variable region is shown in SEQ ID NO. 1
  • amino acid sequence of the antibody heavy chain variable region is shown in SEQ ID NO. 3.
  • variable region of the light chain and the variable region of the heavy chain of the antibody are connected by a flexible polypeptide with an amino acid sequence as shown in SEQ ID NO.5.
  • the present invention also provides a polynucleotide encoding the above-mentioned single-chain antibody.
  • the sequence of the polynucleotide encoding the antibody light chain variable region is shown in SEQ ID NO. 2, and the antibody heavy chain can be encoded.
  • the sequence of the polynucleotide of the variable region is shown in SEQ ID NO.4.
  • the polynucleotide encoding the variable region of the antibody light chain and the polynucleotide encoding the variable region of the antibody heavy chain are composed of a flexible polypeptide whose sequence is shown in SEQ ID NO.6. Polynucleotide linkage.
  • the present invention also provides a vector for expressing the polynucleotide encoding the single-chain antibody.
  • the carrier is pEE14.1 / V L -Linker-V H .
  • the present invention provides a host cell containing the above-mentioned vector, and the cell is a CHO cell.
  • the present invention provides the application of the above-mentioned single-chain antibody in the preparation of drugs for the treatment of autoimmune diseases.
  • the disease is rheumatoid arthritis or systemic lupus erythematosus.
  • the light chain and heavy chain of the anti-C5 single-chain antibody disclosed in the present invention have unique CDR regions, show excellent antigen-binding activity in terms of antigen-binding ability, and the equilibrium dissociation constant K D (m) reaches 2.74 ⁇ 10 -11 .
  • the anti-C5 single-chain antibody disclosed in the present invention can significantly improve the survival rate of mice.
  • the entire treatment process of the high-dose treatment group can completely protect MRL/lpr lupus erythematosus mice. It is 100%, and the low-dose group can maintain a survival rate of more than 80% even in the 24th week.
  • the symptoms of proteinuria, glomerular integral, interstitial inflammation, vasculitis, and crescent/necrosis in the treatment group were significantly improved, indicating that the anti-C5 single-chain antibody provided by the present invention is useful in the preparation of autoimmune disease treatment drugs. Excellent application prospects.
  • the following methods are used to screen anti-C5 single-chain antibodies.
  • the specific method includes the following steps:
  • recombinant phage antibodies A polyethylene petri dish was coated with C5 antigen (Complement Technology, Inc; Item No.: A120Lot#, Accession#P06684), and the supernatant containing the recombinant phage was incubated in the petri dish at 37°C for 2 hours. Wash the plate 20 times with PBS, then wash the plate 20 times with PBST (PBS containing 0.05% Tween 20), and discard PBST. Add 10 mL of TG1 cells in the logarithmic growth phase and incubate at 37°C for 1 hour. Centrifuge, collect the supernatant, and proceed to the next round of screening. Repeat the "adsorption-elution-propagation" screening process twice. After superinfection with M13K07 helper phage, a phage surface display library of enriched clones can be generated.
  • C5 antigen Complement Technology, Inc; Item No.: A120Lot#, Accession#P
  • TG1 bacterial solution was diluted multiple times (stock solution, 1:10, 1:100, 1:1000) with 2 ⁇ YT, and then coated on SOBAG solid culture Incubate at 30°C overnight.
  • 94 single colonies were randomly picked from the plate and inoculated into 100 ⁇ l 2 ⁇ YTAG (containing 100 ⁇ g/mL ampicillin and 2% glucose) culture medium, and cultured at 30° C. overnight.
  • 2 ⁇ YTAG containing 100 ⁇ g/mL ampicillin and 2% glucose
  • the pelleted cells were resuspended in 200 ⁇ l 2 ⁇ YTAK (2 ⁇ YT containing 100 ⁇ g/mL ampicillin and 50 ⁇ g/mL kanamycin), and cultured overnight at 30°C. Centrifuge and collect the supernatant to obtain a monoclonal recombinant phage.
  • the enzyme-linked plate was coated with C5 antigen, 0.5% BSA was used as a negative control, and goat anti-M13 phage antibody was used as a positive control.
  • Block with 1% BSA at 37°C for 1 hour. Add 100 ⁇ l of an equal volume mixture of recombinant phage antibody supernatant and blocking solution to the enzyme-linked plate, and add M13 phage to the control wells. Incubate at 37°C for 1 hour, wash the plate 3 times with PBST (PBS containing 0.05% Tween 20), and wash the plate 3 times with PBS. Add 100 ⁇ l goat anti-M13 phage antibody IgG-HRP (1:2000) to each well, and incubate at 37°C for 1 hour.
  • PBST PBS containing 0.05% Tween 20
  • PBST and PBS wash the plate 3 times each time, add freshly prepared substrate H 2 O 2 -OPD, react at room temperature for 20 minutes, add 50 ⁇ l 2M H 2 SO 4 to stop the reaction, and detect the light absorption value of each well at A 490.
  • the light absorption value is 2.1 times or more of the negative control is the positive clone, from which the positive clone with the strongest C5 binding activity is selected.
  • the T7 DNA sequence TAATACGACTCACTATAGGG was used to determine the DNA sequence of the anti-C5 single-chain antibody on the positive recombinant plasmid.
  • the result showed that the gene has the nucleotide sequence of SEQ ID NO: 7 in the sequence list, consisting of 756 Base composition, the light chain variable region coding gene has the DNA sequence of SEQ ID NO: 2 in the sequence list, the flexible polypeptide linkage coding gene has the DNA sequence of SEQ ID NO: 6 in the sequence list, and the heavy chain variable region coding gene It has the DNA sequence of SEQ ID NO: 4 in the sequence listing.
  • V L -Linker-V H single-chain anti-C5 antibody encoded so as to have SEQ ID NO: 1-SEQ ID NO: 5-SEQ ID NO: 3 amino acid residue sequence in series.
  • the amino acid sequences of CDR1, CDR2, and CDR3 of the light chain variable region are as shown in the amino acid sequences 25-35, 51-57, and 90-99 of SEQ ID NO. 1, respectively.
  • the heavy chain is variable.
  • the amino acid sequences of CDR1, CDR2 and CDR3 of the region are shown in the amino acid sequences of positions 30-35, 50-66 and 99-108 of SEQ ID NO. 3, respectively.
  • step 1.4 to obtain the anti-C5 single chain antibody V L -Linker-V H fusion gene was cloned into eukaryotic expression vector pEE14 .1 (Lonza) to give an anti-C5 scFv carrying V L -Linker-V H gene fusion recombinant expression vector designated pEE14.1 / V L -Linker-V H . And then the recombinant plasmid by liposome pEE14.1 / V L -Linker-V H transfected into Chinese hamster ovary cells are CHO.
  • the medium was aspirated, and 10 mL of fresh selective medium DMEM+10% FCS+25 ⁇ m MSX was added. Incubate in a 37°C incubator with 5% CO 2 mixed gas and 98% humidity. Two weeks later, clones of approximately 1-2 mm appeared. Use the cloning ring to transfer the clones to a 24-well plate. Add 1 mL of selective medium DMEM+10% FCS+25 ⁇ m MSX to each well to continue the culture. After the transformant has grown to 5 days, the supernatant is aspirated. 100 ⁇ l of the supernatant was added to the C5 antigen-coated enzyme-linked plate, incubated at 37° C.
  • the highly expressed CHO cell line was enlarged and cultured, and the supernatant was harvested. The supernatant was slowly added to HiTrap N-hydroxysuccinimide column (Amersham Biosciences) to purify the single-chain antibody. Elute with 0.01mol/L pH 7.4 PBS at a flow rate of 1 mL/min until the eluate OD 280 ⁇ 0.02. Add 0.1 mol/L pH 2.4 glycine-HCl buffer at a flow rate of 1 mL/min, collect the adsorbed components, and immediately neutralize with 1 mol/L sodium carbonate to avoid protein denaturation. The results were identified by SDS-PAGE and Western Blot, and the results are shown in Figure 1 and Figure 2. After expression, the target protein of about 26KD was obtained, and the protein could specifically bind to C5, which was consistent with the expected results, indicating that a highly pure product was obtained. Anti-C5 single chain antibody.
  • the kinetic analysis of the interaction between the anti-C5 single-chain antibody and the C5 ligand was detected by the surface cytoplasmic genomic resonance (SPR) detection system.
  • SPR surface cytoplasmic genomic resonance
  • Reagents 1xPBST 500ml (filtered, 0.22uM membrane filter), EDC (now used now), NHS (now used now), 1M pH8.5 ethanolamine (5-10ml), 10mM pH2.0HCl (5-10ml) , 10mM pH2.0 Glycine (5-10ml).
  • 2.2.1.1 Dilute the protein with different pH sodium acetate to 10 ⁇ g/mL, 200 ⁇ L.
  • 2.2.2.2 Dilute the antigen with a suitable pH value of sodium acetate to 50 ⁇ g/mL, 200 ⁇ L, and fix it in a channel separately. 10 ⁇ L/min, 7min.
  • 2.2.3.1 Dilute the antibody to 100nM with PBST, 25 ⁇ L/min, bind for 3min, and dissociate for 5min.
  • 2.2.4.1 Dilute the antibody to 100nM with PBST, and then dilute 2 times, 7 gradients, 25 ⁇ L/min, bind for 3min, dissociate for 5min.
  • the SPR test results are shown in Table 2.
  • the test shows that the anti-C5 single chain antibody provided by the present invention shows excellent antigen binding efficiency.
  • the single chain antibody was diluted with DEME and added to the NHS first, and then added to the CHO cell suspension.
  • the final concentration of 100g/L NHS can cause about 90% of the antibody sensitized control CHO cells to be lysed as the standard.
  • the complement-mediated inhibition of red blood cell lysis was tested with antibody-sensitized goat red blood cells (EAs).
  • EAs antibody-sensitized goat red blood cells
  • the hemolysis test was performed in Gelatin Verona buffer (GVB ++ ), the final volume was 300 ⁇ L, containing 2.5 ⁇ 10 7 EAs, and NHS was diluted 1:300.
  • the reaction mixture was incubated at 37°C for 60 min, and finally 300 ⁇ L of 10 mmol/L EDTA-PBS solution was added to terminate the reaction. Centrifuge, take the supernatant, and quantitatively detect the hemoglobin in the supernatant with a spectral imager at a wavelength of 413nm.
  • Single-chain antibody complement inhibitor activity test The results of complement-mediated CHO cell and red blood cell lysis experiments show that in the cell lysis inhibition experiment, the anti-C5 single-chain antibody inhibiting antibody-sensitized CHO cells and red blood cells have a significant lysis effect (see table for details) 3), showing that the anti-C5 single-chain antibody can effectively inhibit the classical activation pathway of complement.
  • Example 4 Therapeutic effect of single-chain antibody targeting C5 on MRL/lpr lupus erythematosus mouse model
  • the MRL/lpr lupus erythematosus mouse model was first established by Murphy and Roths in 1979. It was formed by a complex hybridization process of multiple strains of mice after 12 generations. 75% of the mouse genes of this model came from LG/J, 12.6 % Is from AKR/J, 12.1% is from C3H/Di and 0.3% is from C57BL/6 strain mice.
  • MRL/lpr mice contain recessive mutations in the Fas gene related to spontaneous programmed cell death, and the presence of lymphocyte proliferation genes, leading to T cell proliferation, systemic lymph node enlargement, and erosive arthritis, anti-DNA, anti-Sm, anti- Su, anti-nucleoside P antibody, high titer ANA, hypergammaglobulinemia and rheumatoid factor.
  • the mouse first became ill at 8 weeks, at which time autoantibodies could be detected in the serum. Lymphadenitis can be observed at 12 weeks. At 12-16 weeks, MRL/lpr mice began to produce a large number of autoantibodies including anti-double-stranded DNA antibodies.
  • mice Nearly 16 weeks of age, multiple organ involvement and stable renal function degradation characterized by severe proteinuria appeared. 16-24 weeks old mice develop glomerulonephritis and vasculitis mediated by proliferative immune complexes, and eventually lead to renal failure and death, and the mortality rate can reach 50%.
  • 16-week-old MRL/lpr mice that had symptoms of renal failure were randomly divided into two groups.
  • the route of administration for both groups was tail vein injection. According to the survival rate of the administration group and the control group, the protection rate of the single-chain antibody targeting C5 on MRL/lpr lupus erythematosus mice was evaluated. The experimental results are shown in Figure 3.
  • MRL/lpr lupus erythematosus mice In mice treated with a single-chain antibody targeting C5, C5 in the complement activation pathway is effectively inhibited by a single-chain antibody targeting C5. Therefore, the survival of MRL/lpr lupus erythematosus mice
  • the high-dose treatment group can completely protect MRL/lpr lupus erythematosus mice during the whole treatment process, and the survival rate is 100%, while the low-dose group can maintain a survival rate of more than 80% even in the 24th week.
  • mice were placed in a metabolic cage to study the effect of single-chain antibody targeting C5 on urinary albumin secretion in MRL/lpr lupus erythematosus mice. From the 16th week, the mice’s 24-hour urine was collected every two weeks. To prevent bacterial growth, add ampicillin, gentamicin, and chloramphenicol to the collection tube. Using a mouse albumin sample of known concentration, a standard curve was drawn by ELISA method, and the urine albumin secretion of experimental mice was determined, and the creatinine content in the mouse urine was measured using a Beckman Coulter. The final evaluation result is expressed as the ratio of urine albumin (mg) to creatinine (mg) of each experimental mouse for 24 hours.
  • a high ratio of urine albumin to creatinine indicates that kidney function is impaired.
  • the mouse kidney was excised and longitudinally dissected into two halves, half of which was subjected to immunofluorescence analysis, and the other half was fixed with 10% neutral formaldehyde, and the sections were embedded in solid paraffin and stained with hematoxylin-eosin and periodic acid Schiff
  • the staining method was used to stain the paraffin-treated kidney tissue sections.
  • the glomerular inflammation, hyperplasia, crescent formation, and necrosis symptoms observed from the self-section were scored by blind method, and the changes in the renal interstitial were also scored. The score is divided into five levels: 0, 1, 2, 3, and 4, with 0 being no damage and 4 being severe damage.
  • the evaluation of perivascular inflammatory exudation adopts a semi-quantitative method.
  • the present invention provides a human-derived anti-complement C5 molecule single-chain antibody and its application in the preparation of autoimmune disease therapeutic drugs.
  • the single-chain antibody provided by the present invention is easy to industrially produce and has industrial applicability.

Abstract

Disclosed in the present invention is a humanized single-chain antibody of anti-complement C5 molecule. An equilibrium dissociation constant of the antibody reaches 2.74×10-11; during the treatment of MRL/lpr lupus erythematosus mice, the anti-C5 single-chain antibody provided by the present invention can significantly improve the viability of mice, symptoms such as the proteinuria, glomerular score, interstitial inflammation, vasculitis, and crescent/necrosis of a treatment group are significantly improved. The anti-C5 single-chain antibody provided by the present invention can be used for preparing drugs for treatment of autoimmune diseases.

Description

一种抗补体C5分子的人源化单链抗体及其应用Humanized single-chain antibody against complement C5 molecule and application thereof 技术领域Technical field
本发明公开了一种多肽，更具体地，本发明公开了一种抗体。The present invention discloses a polypeptide, more specifically, the present invention discloses an antibody.
背景技术Background technique
补体***由30余种可溶性蛋白分子组成，是天然免疫***的一部分，其组成成分包括补体固有成分、多种调节因子和补体受体等30多种分子。补体***可通过3条既相对独立又相互联系的途径被激活，从而发挥调理吞噬、裂解细胞、介导炎症、免疫调节和清除免疫复合物等多种生物学效应，包括增强吞噬作用、增强吞噬细胞的趋化性、增加血管的通透性、中和病毒、细胞溶解作用、免疫反应的调节作用等。虽然补体活化为抵抗潜在病原体提供了有价值的第一线防御力，但促进保护性炎症应答反应的补体激活也可能表现为对宿主的潜在威胁。补体激活和其在靶结构上的沉积也可以间接地引起细胞或组织破坏。在补体途径中的各个点产生介导组织损害的补体激活产物。宿主组织上不适当的补体激活在许多自身免疫病和炎性疾病的病理学中起重要作用。The complement system is composed of more than 30 soluble protein molecules and is a part of the natural immune system. Its components include more than 30 molecules such as the inherent components of complement, a variety of regulatory factors, and complement receptors. The complement system can be activated through three relatively independent and interconnected pathways, so as to regulate phagocytosis, lyse cells, mediate inflammation, immune regulation and clear immune complexes and other biological effects, including enhancing phagocytosis and enhancing phagocytosis. Chemotaxis of cells, increased permeability of blood vessels, neutralization of viruses, cell lysis, immune response regulation, etc. Although complement activation provides a valuable first-line defense against potential pathogens, complement activation that promotes a protective inflammatory response may also be a potential threat to the host. Complement activation and its deposition on target structures can also indirectly cause cell or tissue destruction. Complement activation products that mediate tissue damage are produced at various points in the complement pathway. Inappropriate complement activation on host tissues plays an important role in the pathology of many autoimmune and inflammatory diseases.
补体激活的途径有3个，即经典途径、甘露聚糖结合凝集途径和旁路途径。补体经典激活途径是通过抗原-抗体复合物激活的，参与该途径的成分包括C1-C9，按其在激活过程中的作用，人为地分成三组，即识别单位(Clq、Clr、Cls)、活化单位(C4、C2、C3)和膜攻击单位(C5-C9)，分别在激活的不同阶段即识别阶段、活化阶段和膜攻击阶段中发挥作用。甘露聚糖结合凝集途径是经典途径的一个变化，由血浆中甘露聚糖结合凝集素(Mannan-Binding Lectin，MBL)直接识别多种病原微生物表面的N-氨基半乳糖或甘露糖，进而依次活化MASP-1、MASP-2、C4、C2、C3，形成和经典途径相同的C3与C5转化酶，激活补体级联酶促反应的活化途径。旁路激活途径是通过外来物质、死亡组织、细胞、细菌等激活的，旁路激活途径与经典激活途径不同之处在于激活是越过了C1、C4、 C2三种成分，直接激活C3继而完成C5至C9各成分的连锁反应。C3活化后，在经典途径中涉及多种蛋白质，例如C1Q、C1r/C1s、C4和C2。经典途径C3转化酶由C3bC4b2a组成。在旁路途径活化中，补体***产生的C3b可以与备解素和因子B结合，从而形成复合物“PC3bB”。然后，在该复合物内，因子D将因子B切割成Bb和Ba。该切割使得Ba由复合物中释放，并形成旁路途径C3转化酶PC3bBb。PC3bBb将C3切割成C3a和C3b，从而建立旁路途径的放大环路。此外，旁路途径除了在补体激活中作为独立途径而被广泛认可的作用以外，经典途径和甘露聚糖结合凝集途径还可以为引发旁路途径的补体激活提供放大环。在这种旁路途径介导的放大机制中，激活产生的C3转化酶C3bC4b2a将C3切割为两个活性片段：过敏毒素C3a以及具有调理作用的C3b。C3a是一种强效过敏毒素，可导致各种临床疾病。C3a能激活中性粒细胞、单核细胞、血小板、肥大细胞和T细胞。在佐剂诱导的关节炎模型中已证明C3a对诱导脚爪浮肿至关重要。新形成的C3b加入已产生的C3转化酶可形成C5转化酶，后者可切割C5产生C5b和C5a。There are three pathways for complement activation, namely, the classical pathway, the mannan binding agglutination pathway and the alternative pathway. The classical complement activation pathway is activated by antigen-antibody complexes. The components involved in this pathway include C1-C9. According to their role in the activation process, they are artificially divided into three groups, namely recognition units (Clq, Clr, Cls), The activation unit (C4, C2, C3) and the membrane attack unit (C5-C9) play a role in the different stages of activation, namely the recognition phase, the activation phase and the membrane attack phase. Mannan-binding agglutination pathway is a change of the classical pathway. Mannan-Binding Lectin (MBL) in plasma directly recognizes N-galactosamine or mannose on the surface of a variety of pathogenic microorganisms, and then activates them in turn MASP-1, MASP-2, C4, C2, C3, forming the same C3 and C5 convertases as the classical pathway, activate the activation pathway of the complement cascade enzymatic reaction. The alternative activation pathway is activated by foreign substances, dead tissues, cells, bacteria, etc. The alternative activation pathway is different from the classical activation pathway in that activation bypasses the three components of C1, C4, and C2, directly activates C3 and then completes C5. The chain reaction of each component to C9. After C3 is activated, a variety of proteins are involved in the classical pathway, such as C1Q, C1r/C1s, C4 and C2. The classical pathway C3 convertase is composed of C3bC4b2a. In the activation of the alternative pathway, C3b produced by the complement system can combine with properdin and factor B to form a complex "PC3bB". Then, within the complex, factor D cleaves factor B into Bb and Ba. This cleavage allows Ba to be released from the complex and forms the alternative pathway C3 convertase PC3bBb. PC3bBb cuts C3 into C3a and C3b, thereby establishing an amplification loop of the bypass pathway. In addition, in addition to the widely recognized role of the alternative pathway as an independent pathway in complement activation, the classical pathway and the mannan combined agglutination pathway can also provide an amplification loop for triggering the alternative pathway of complement activation. In this alternative pathway-mediated amplification mechanism, the activated C3 convertase C3bC4b2a cleaves C3 into two active fragments: anaphylatoxin C3a and opsonizing C3b. C3a is a powerful allergic toxin that can cause various clinical diseases. C3a can activate neutrophils, monocytes, platelets, mast cells and T cells. In the adjuvant-induced arthritis model, C3a has been shown to be essential for inducing paw edema. The newly formed C3b can be added to the already produced C3 convertase to form C5 convertase, which can cleave C5 to produce C5b and C5a.
C5为分子量为190kDa的糖基化的β球蛋白，其在血清中的浓度为75μg/ml(0.4.mu.M)，分子量中的1.5-3％为碳水化合物。成熟的C5为异二聚体，由999氨基酸长度、约115kDa的α链以二硫键与656氨基酸长度75kDaβ连接。C5的编码基因为单拷贝，其蛋白翻译产物为一个1659氨基酸长度的C5前体蛋白以及18个氨基酸长度的前导肽。C5前体蛋白在655至659氨基酸处被切断产生出1-655氨基酸长度的β链，以及660-1658氨基酸残基的α链，两者中间的四个氨基酸残基则被切除(Haviland et al.J.Immunol.1991,146:362-368)。C5 is a glycosylated β-globulin with a molecular weight of 190kDa, and its concentration in serum is 75μg/ml (0.4.mu.M), and 1.5-3% of the molecular weight is carbohydrates. Mature C5 is a heterodimer, consisting of an α chain of 999 amino acids in length and about 115 kDa connected by disulfide bonds to 656 amino acids in length and 75 kDa β. The C5 coding gene is a single copy, and its protein translation product is a C5 precursor protein with a length of 1659 amino acids and a leader peptide with a length of 18 amino acids. The C5 precursor protein is cut at 655 to 659 amino acids to produce a β chain of 1-655 amino acids and an α chain of 660-1658 amino acid residues. The four amino acid residues between the two are removed (Haviland et al. .J.Immunol.1991,146:362-368).
C5a是在C5转化酶的切割作用下由α链的氨基端前74个氨基酸残基产生的，如果在该切割位点有特异性分子的结合，C5转化酶的切割效应被阻断，则该分子就可以作为一种补体抑制剂。C5a is produced by the first 74 amino acid residues at the amino terminus of the α chain under the cleavage of C5 convertase. If there is a specific molecule binding at the cleavage site, the cleavage effect of C5 convertase is blocked. The molecule can act as a complement inhibitor.
C5a也是一种强效过敏毒素，能引起平滑肌、血管紧张度和血管渗透性的改变。其还是中性粒细胞、单核细胞、血小板、内皮细胞和T细胞的强效趋化因子和激活剂。C5a介导的细胞激活通过诱导释放其它炎症介质，包括细胞因子、水解酶、花生四烯酸代谢产物和活性氧可显著放 大炎症应答反应。另一个切割产物C5b则***靶细胞表面的脂质双层中成为C6、C7、C8和C9沉积的核心，形成C5b-9复合物。C5b-9也称为膜攻击复合物(MAC)。现有证据显示在炎症中MAC起着重要作用，此外它还起着裂解细胞的孔形成复合物的作用。C5a is also a potent anaphylactic toxin, which can cause changes in smooth muscle, vascular tone and vascular permeability. It is also a potent chemokine and activator of neutrophils, monocytes, platelets, endothelial cells and T cells. C5a-mediated cell activation can significantly amplify the inflammatory response by inducing the release of other inflammatory mediators, including cytokines, hydrolases, arachidonic acid metabolites and reactive oxygen species. Another cleavage product, C5b, is inserted into the lipid bilayer on the surface of the target cell and becomes the core of C6, C7, C8 and C9 deposition, forming a C5b-9 complex. C5b-9 is also called membrane attack complex (MAC). Existing evidence shows that MAC plays an important role in inflammation. In addition, it also plays a role in lysing cell pores to form complexes.
单链抗体(ScFv)是用基因工程方法制备的小分子抗体，是由柔性多肽(一般为12-15个氨基酸)将抗体的重链可变区(VH)与轻链可变区(VL)连接而成的重组抗体，其分子量只相当于原天然抗体的六分之一，但单链抗体含有全部的抗原结合位点，所以单链抗体最大程度地保留了抗体的抗原结合活性，是具有亲本抗体抗原结合活性的小片段，从而可以使其达到常规抗体难以达到的病灶组织。Single-chain antibodies (ScFv) are small-molecule antibodies prepared by genetic engineering methods. They are composed of flexible polypeptides (usually 12-15 amino acids) that combine the heavy chain variable region (VH) and light chain variable region (VL) of the antibody. The molecular weight of the linked recombinant antibody is only one-sixth of the original natural antibody, but the single-chain antibody contains all the antigen-binding sites, so the single-chain antibody retains the antigen-binding activity of the antibody to the greatest extent. A small fragment of the parent antibody's antigen binding activity, which can make it reach the focus tissue that is difficult to reach by conventional antibodies.
在上世纪90年代出现的抗体库技术绕过了以往单抗研制过程中必需的杂交瘤途径，甚至不需要经过免疫，使人源化抗体的制备达到了一个全新的水平。更为重要的是，它使人们长期以来期望获得治疗性人源抗体的梦想成为了现实。噬菌体抗体库是最早出现也是目前应用最为广泛的抗体库。噬菌体展示是由Smith(Science,1985,228(4705):1315-1317)首先建立起来的一种将外源蛋白基因与噬菌体的衣壳蛋白基因相融合后使外源蛋白表达呈现于噬菌体表面的技术。噬菌体抗体库就是利用了以上原理使不同特异性的抗体表达在不同的噬菌体表面而用抗原对它们进行筛选(Science,1989，246(4935)：1275-1281；PNAS,1991，88(18)：7978-7982，Human Antibodies，1997，8(4)：155-168)。用于构建噬菌体抗体库的靶细胞可以是杂交瘤细胞、免疫的人B细胞或未经免疫的人B细胞。未经免疫的人B淋巴细胞是目前应用最多的靶细胞，它库容量大，理论上含有所有的人源抗体基因。噬菌体抗体库的筛选就是模拟体内抗体亲和力成熟的过程，用固相化抗原吸附表面表达特异性抗体的噬菌体库，然后洗脱游离的噬菌体，用抗原吸附的噬菌体感染宿主菌增殖扩增后再进行多轮的“吸附－洗脱－扩增”直至筛选到特异的人源抗体。大容量抗体库的建立是获得高亲和力人源抗体的关键，如果构建的噬菌体抗体库容量大于10 10，那就可能从中筛选得到高亲和力(≥10 9M －1)的特异性抗体。 The antibody library technology that emerged in the 1990s bypassed the hybridoma approach necessary in the previous monoclonal antibody development process, and even did not require immunization, bringing the preparation of humanized antibodies to a whole new level. More importantly, it makes people's long-awaited dream of obtaining therapeutic human antibodies a reality. The phage antibody library is the earliest and most widely used antibody library. Phage display was first established by Smith (Science, 1985, 228(4705): 1315-1317), a method of fusing the foreign protein gene with the capsid protein gene of the phage to make the expression of the foreign protein appear on the surface of the phage. technology. The phage antibody library uses the above principles to express antibodies with different specificities on the surface of different phages and screen them with antigens (Science, 1989, 246(4935): 1275-1281; PNAS, 1991, 88(18): 7978-7982, Human Antibodies, 1997, 8(4):155-168). The target cells used to construct the phage antibody library can be hybridoma cells, immunized human B cells or unimmunized human B cells. Unimmunized human B lymphocytes are currently the most widely used target cells. They have a large reservoir capacity and theoretically contain all human antibody genes. The screening of phage antibody library is to simulate the process of in vivo antibody affinity maturation. The phage library expressing specific antibodies is adsorbed on the surface with solid-phased antigen, then free phages are eluted, and the antigen-adsorbed phages are used to infect the host bacteria after proliferation and amplification. Multiple rounds of "adsorption-elution-amplification" until the specific human antibody is screened. The establishment of a large-capacity antibody library is the key to obtaining high-affinity human antibodies. If the capacity of the constructed phage antibody library is greater than 10 10 , it is possible to screen for specific antibodies with high affinity (≥ 10 9 M -1 ).
技术问题technical problem
虽然补体活化为抵抗潜在病原体提供了有价值的第一线防御力，但促进保护性炎症应答反应的补体激活也可能表现为对宿主的潜在威胁。例如，可将C3a和C5a过敏毒素招募到患病部位并激活中性粒细胞、单核细胞和血小板。这些激活的细胞不加选择地释放破坏性酶，可能引起器官损伤。因此，基于下调或者抑制补体激活治疗一些因补体激活所导致炎症性疾病成为本技术领域的一个新的尝试，目前研究显示在动物模型和体外研究中证实下调或者抑制补体激活对于治疗一些疾病适应症是有效的，例如类风湿性关节炎、***性红斑狼疮、肾小球肾炎等。已有几种作为重组蛋白的内源性可溶的补体抑制因子(C1-抑制因子、可溶的补体受体1或sCR1)在临床研究中得到了评价。抗C5重组人源型单克隆抗体能特异性地键合到人末端补体蛋白C5，通过抑制人补体C5向C5a和C5b的裂解以阻断炎症因子C5a的释放及C5b-9的形成。临床前研究表明该抗体对C5有高度亲和力,能阻断C5a和C5b-9的形成,并保护哺乳动物细胞不受C5b-9介导的损伤(Thomas et al.,Mol Immunol 1996,33:1389)，该文献公开的抗C5抗体仅仅显示了在亲和力和中和抑制方面的性能，对于在抑制补体介导的病理损伤方面还没有得到证实。该技术在2002年获得美国专利授权US6355245，2011年9月，美国食品和药物管理局(FDA)批准其作为治疗典型溶血性***(aHUS)患者的孤儿药，即抗C5抗体上市药物依库丽单抗(Eculizumab)。但是依库丽单抗在临床应用治疗溶血症尿毒综合征时的疗效还存在着争议。目前抗C5抗体抑制补体激活仍然存在着一些技术上的难题，比如难以获得高度特异性的抗C5抗体、现有技术制备的鼠源单克隆抗体或者嵌合抗体对人体所带来的免疫原性，以及抗体分子由于分子量的超限以及其空间构象的阻碍难以抵达一些病灶组织。这些技术问题不同程度限制了通过抗C5抗体抑制补体激活的实际应用。本领域仍然需要针对特定适应症开发出具有针对性抑制效果的抗体药物。Although complement activation provides a valuable first-line defense against potential pathogens, complement activation that promotes a protective inflammatory response may also be a potential threat to the host. For example, C3a and C5a anaphylactoxins can be recruited to the diseased site and activate neutrophils, monocytes, and platelets. These activated cells indiscriminately release destructive enzymes, which may cause organ damage. Therefore, the treatment of some inflammatory diseases caused by complement activation based on down-regulation or inhibition of complement activation has become a new attempt in this technical field. Current studies have shown that down-regulation or inhibition of complement activation is useful for the treatment of some disease indications in animal models and in vitro studies. It is effective, such as rheumatoid arthritis, systemic lupus erythematosus, glomerulonephritis, etc. Several endogenous soluble complement inhibitors (C1-inhibitor, soluble complement receptor 1 or sCR1) as recombinant proteins have been evaluated in clinical studies. The anti-C5 recombinant human-derived monoclonal antibody can specifically bind to human terminal complement protein C5, and block the release of inflammatory factors C5a and the formation of C5b-9 by inhibiting the cleavage of human complement C5 to C5a and C5b. Preclinical studies have shown that the antibody has a high affinity for C5, can block the formation of C5a and C5b-9, and protect mammalian cells from C5b-9-mediated damage (Thomas et al., Mol Immunol 1996, 33:1389 ), the anti-C5 antibody disclosed in this document only shows performance in terms of affinity and neutralization inhibition, and has not been confirmed in terms of inhibiting complement-mediated pathological damage. This technology was granted US patent US6355245 in 2002. In September 2011, the US Food and Drug Administration (FDA) approved it as an orphan drug for the treatment of patients with typical hemolytic uremic (aHUS), that is, the anti-C5 antibody marketed drug Eku Lizumab (Eculizumab). However, the efficacy of eculizumab in the treatment of hemolytic uremic syndrome is still controversial. At present, there are still some technical difficulties in inhibiting complement activation by anti-C5 antibodies, such as the difficulty of obtaining highly specific anti-C5 antibodies, the immunogenicity of mouse monoclonal antibodies or chimeric antibodies prepared by the prior art to the human body. , And because of the over-limit of molecular weight and the hindrance of its spatial conformation, it is difficult for antibody molecules to reach some focal tissues. These technical problems limit the practical application of inhibiting complement activation by anti-C5 antibodies to varying degrees. There is still a need in the art to develop antibody drugs with targeted inhibitory effects for specific indications.
本发明的目的就是通过噬菌体抗体库技术提供一种能够与C5分子特异性结合的人源单链抗体，进而提供其在制备自身免疫性疾病治疗药物的应用。The purpose of the present invention is to provide a human single-chain antibody that can specifically bind to C5 molecules through the phage antibody library technology, and further provide its application in the preparation of autoimmune disease therapeutic drugs.
技术解决方案Technical solutions
基于上述目的，本发明首先提供了一种抗补体C5分子的人源化单链抗体，所述抗体轻链可变区的CDR1、CDR2和CDR3的氨基酸序列分别如SEQ ID NO.1的第25-35、51-57和90-99位氨基酸序列所示，所述抗体重链可变区的CDR1、CDR2和CDR3的氨基酸序列分别如SEQ ID NO.3的第30-35、50-66和99-108位氨基酸序列所示。Based on the above objective, the present invention first provides a humanized single-chain antibody against complement C5 molecule. The amino acid sequences of CDR1, CDR2, and CDR3 of the variable region of the light chain of the antibody are as shown in SEQ ID NO.1, respectively. -35, 51-57 and 90-99 amino acid sequences, the amino acid sequences of CDR1, CDR2 and CDR3 of the variable region of the antibody heavy chain are shown in SEQ ID NO. 3 30-35, 50-66 and respectively The amino acid sequence at positions 99-108 is shown.
在一个优选的实施方案中，所述抗体轻链可变区的氨基酸序列如SEQ ID NO.1所示，所述抗体重链可变区的氨基酸序列如SEQ ID NO.3所示。In a preferred embodiment, the amino acid sequence of the antibody light chain variable region is shown in SEQ ID NO. 1, and the amino acid sequence of the antibody heavy chain variable region is shown in SEQ ID NO. 3.
在一个更为优选的实施方案中，所述抗体轻链可变区与重链可变区以氨基酸序列如SEQ ID NO.5的所示柔性多肽连接。In a more preferred embodiment, the variable region of the light chain and the variable region of the heavy chain of the antibody are connected by a flexible polypeptide with an amino acid sequence as shown in SEQ ID NO.5.
其次，本发明还提供了一种编码上述单链抗体的多核苷酸，编码所述抗体轻链可变区的多核苷酸的序列由SEQ ID NO.2所示，编码所述抗体重链可变区的多核苷酸的序列由SEQ ID NO.4所示。Secondly, the present invention also provides a polynucleotide encoding the above-mentioned single-chain antibody. The sequence of the polynucleotide encoding the antibody light chain variable region is shown in SEQ ID NO. 2, and the antibody heavy chain can be encoded. The sequence of the polynucleotide of the variable region is shown in SEQ ID NO.4.
在一个优选的实施方案中，编码所述抗体轻链可变区的多核苷酸与编码所述抗体重链可变区的多核苷酸由序列如SEQ ID NO.6所示的编码柔性多肽的多核苷酸连接。In a preferred embodiment, the polynucleotide encoding the variable region of the antibody light chain and the polynucleotide encoding the variable region of the antibody heavy chain are composed of a flexible polypeptide whose sequence is shown in SEQ ID NO.6. Polynucleotide linkage.
再次，本发明还提供了一种表达上述编码单链抗体的多核苷酸的载体。Third, the present invention also provides a vector for expressing the polynucleotide encoding the single-chain antibody.
在一个优选的实施方案中，所述载体为pEE14.1/V L-Linker-V H。 In a preferred embodiment, the carrier is pEE14.1 / V L -Linker-V H .
又次，本发明提供了一种含有上述载体的宿主细胞，所述细胞为CHO细胞。Again, the present invention provides a host cell containing the above-mentioned vector, and the cell is a CHO cell.
最后，本发明提供了上述单链抗体在制备自身免疫性疾病治疗药物中的应用。Finally, the present invention provides the application of the above-mentioned single-chain antibody in the preparation of drugs for the treatment of autoimmune diseases.
在一个优选的实施方案中，所述疾病为类风湿性关节炎或***性红斑狼疮。In a preferred embodiment, the disease is rheumatoid arthritis or systemic lupus erythematosus.
技术效果Technical effect
本发明公开的抗C5的单链抗体的轻链和重链具有独特的CDR区，在抗原结合能力上显示了优异的抗原结合活性，平衡解离常数K D(m)达 到2.74×10 -11。本发明公开的抗C5单链抗体在MRL/lpr红斑狼疮小鼠的治疗中，能够明显提升小鼠的存活率，高剂量治疗组整个治疗过程能够完全保护MRL/lpr红斑狼疮小鼠，存活率为100％，而低剂量组即使在第24周也能保持在80％以上的存活率。而且治疗组的蛋白尿、肾小球积分、间质炎症、血管炎和新月体/坏死等症状得到明显改善，显示本发明提供的抗C5单链抗体在制备自身免疫性疾病治疗药物中具有优异的应用前景。 The light chain and heavy chain of the anti-C5 single-chain antibody disclosed in the present invention have unique CDR regions, show excellent antigen-binding activity in terms of antigen-binding ability, and the equilibrium dissociation constant K D (m) reaches 2.74×10 -11 . In the treatment of MRL/lpr lupus erythematosus mice, the anti-C5 single-chain antibody disclosed in the present invention can significantly improve the survival rate of mice. The entire treatment process of the high-dose treatment group can completely protect MRL/lpr lupus erythematosus mice. It is 100%, and the low-dose group can maintain a survival rate of more than 80% even in the 24th week. In addition, the symptoms of proteinuria, glomerular integral, interstitial inflammation, vasculitis, and crescent/necrosis in the treatment group were significantly improved, indicating that the anti-C5 single-chain antibody provided by the present invention is useful in the preparation of autoimmune disease treatment drugs. Excellent application prospects.
附图说明Description of the drawings
图1.抗C5抗体的12％SDS-PAGE鉴定图谱；Figure 1. 12% SDS-PAGE identification map of anti-C5 antibody;
图2.抗C5抗体的Western Blot鉴定图谱；Figure 2. Western Blot identification map of anti-C5 antibody;
图3.靶向C5的单链抗体治疗MRL/lpr小鼠的存活率对照图；Figure 3. Survival rate control chart of MRL/lpr mice treated with single-chain antibody targeting C5;
图4.靶向C5的单链抗体治疗MRL/lpr小鼠的蛋白尿变化对照图。Figure 4. Control chart of proteinuria changes in MRL/lpr mice treated with C5 single-chain antibody.
本发明的实施方式Embodiments of the present invention
下面结合具体实施例来进一步描述本发明，本发明的优点和特点将会随着描述而更为清楚。但这些实施例仅是范例性的，并不对本发明权利要求限定的保护范围构成任何限制。The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer with the description. However, these embodiments are only exemplary, and do not constitute any limitation to the protection scope defined by the claims of the present invention.
实施例1、抗C5单链抗体的制备Example 1. Preparation of anti-C5 single chain antibody
用下述方法筛选抗C5单链抗体，具体方法包括以下步骤：The following methods are used to screen anti-C5 single-chain antibodies. The specific method includes the following steps:
1.1单链抗体噬菌体表达文库的构建及表达参见中国发明专利申请CN109575132A的实施例1，本申请通过引用，将CN109575132A的公开内容作为本申请的一部分并入到本申请说明书中。1.1 Construction and expression of single-chain antibody phage expression library refer to Example 1 of Chinese invention patent application CN109575132A. The disclosure of CN109575132A is incorporated into the specification of this application as a part of this application by reference.
1.2重组噬菌体抗体的筛选：用C5抗原(Complement Technology，Inc；货号：A120Lot#，Accession#P06684)包被聚乙烯培养皿，将含有重组噬菌体的上清与该培养皿中37℃孵育2小时。用PBS洗平皿20次，再用PBST(含0.05％Tween 20的PBS)洗平皿20次，弃PBST。加入10mL处于对数生长期TG1细胞，37℃培养1小时。离心，收集上清，进行下一轮筛选。重复“吸附-洗脱-繁殖”的筛选过程2次。再以M13K07辅助噬菌体超感染，就可以生成富集克隆的噬菌体表面呈现文库。1.2 Screening of recombinant phage antibodies: A polyethylene petri dish was coated with C5 antigen (Complement Technology, Inc; Item No.: A120Lot#, Accession#P06684), and the supernatant containing the recombinant phage was incubated in the petri dish at 37°C for 2 hours. Wash the plate 20 times with PBS, then wash the plate 20 times with PBST (PBS containing 0.05% Tween 20), and discard PBST. Add 10 mL of TG1 cells in the logarithmic growth phase and incubate at 37°C for 1 hour. Centrifuge, collect the supernatant, and proceed to the next round of screening. Repeat the "adsorption-elution-propagation" screening process twice. After superinfection with M13K07 helper phage, a phage surface display library of enriched clones can be generated.
1.3单克隆重组噬菌体的筛选与鉴定：第三轮筛选后，用2×YT将TG1菌液做倍数稀释(原液、1:10、1:100、1:1000)，然后涂布在SOBAG固体培养基(分子克隆，第三版，黄培堂等译)上，30℃培养过夜。从平板上随机挑取94个单菌落，分别接种于100μl 2×YTAG(含100μg/mL氨苄青霉素和2％葡萄糖)培养液中，30℃培养过夜。取20μl培养液，转接于200μl含5×10 8pfu/mL M13K07的2×YTAG培养液中，37℃培养2小时。离心，用200μl 2×YTAK(含100μg/mL氨苄青霉素和50μg/mL卡那霉素的2×YT)重悬沉淀细胞，30℃培养过夜。离心、收集上清，即为单克隆重组噬菌体。 1.3 Screening and identification of monoclonal recombinant phage: After the third round of screening, TG1 bacterial solution was diluted multiple times (stock solution, 1:10, 1:100, 1:1000) with 2×YT, and then coated on SOBAG solid culture Incubate at 30°C overnight. 94 single colonies were randomly picked from the plate and inoculated into 100 μl 2×YTAG (containing 100 μg/mL ampicillin and 2% glucose) culture medium, and cultured at 30° C. overnight. Take 20μl of culture medium, transfer it to 200μl of 2×YTAG culture medium containing 5×10 8 pfu/mL M13K07, and incubate at 37°C for 2 hours. After centrifugation, the pelleted cells were resuspended in 200 μl 2×YTAK (2×YT containing 100 μg/mL ampicillin and 50 μg/mL kanamycin), and cultured overnight at 30°C. Centrifuge and collect the supernatant to obtain a monoclonal recombinant phage.
用C5抗原包被酶联板，用0.5％BSA作为阴性对照，用羊抗M13噬菌体抗体作为阳性对照。1％BSA的37℃封闭1小时。把100μl重组噬菌体抗体上清和封闭液的等体积混合物加入到酶联板中，对照孔中加入M13噬菌体。37℃孵育1小时，PBST(含0.05％Tween 20的PBS)洗板3次，PBS洗板3次。每孔加入100μl羊抗M13噬菌体抗体IgG-HRP(1:2000)，37℃孵育1小时。PBST和PBS一次各洗板3次，加入新鲜配制的底物H 2O 2-OPD，室温反应20min，加入50μl 2M H 2SO 4终止反应，在A 490处检测每孔的光吸收值。光吸收值为阴性对照的2.1倍以上的为阳性克隆，从中挑选出与C5结合活性最强的阳性克隆。 The enzyme-linked plate was coated with C5 antigen, 0.5% BSA was used as a negative control, and goat anti-M13 phage antibody was used as a positive control. Block with 1% BSA at 37°C for 1 hour. Add 100 μl of an equal volume mixture of recombinant phage antibody supernatant and blocking solution to the enzyme-linked plate, and add M13 phage to the control wells. Incubate at 37°C for 1 hour, wash the plate 3 times with PBST (PBS containing 0.05% Tween 20), and wash the plate 3 times with PBS. Add 100 μl goat anti-M13 phage antibody IgG-HRP (1:2000) to each well, and incubate at 37°C for 1 hour. PBST and PBS wash the plate 3 times each time, add freshly prepared substrate H 2 O 2 -OPD, react at room temperature for 20 minutes, add 50 μl 2M H 2 SO 4 to stop the reaction, and detect the light absorption value of each well at A 490. The light absorption value is 2.1 times or more of the negative control is the positive clone, from which the positive clone with the strongest C5 binding activity is selected.
1.4阳性克隆重组质粒的DNA序列分析：用T7DNA序列TAATACGACTCACTATAGGG测定该阳性重组质粒上抗C5单链抗体的DNA序列，结果该基因具有序列表中SEQ ID NO：7的核苷酸序列，由756个碱基组成，其轻链可变区编码基因具有序列表中SEQ ID NO：2的DNA序列，柔性多肽连接编码基因具有序列表中SEQ ID NO：6的DNA序列，重链可变区编码基因具有序列表中SEQ ID NO：4的DNA序列。所述抗C5单链抗体编码基因的串联形式为SEQ ID NO：2-SEQ ID NO：6-SEQ ID NO：4，被命名为V L-Linker-V H(亦称ScFv)。V L-Linker-V H编码抗C5单链抗体使其具有SEQ ID NO：1-SEQ ID NO：5-SEQ ID NO：3串联氨基酸残基序列。通过进一步的序列分析，轻链可变区的CDR1、CDR2和CDR3的氨基酸序列分别如SEQ ID NO.1的第25-35、51-57和90-99位氨基酸序列所示，重链可变区的CDR1、CDR2和CDR3的氨基酸序列分别如SEQ ID NO.3的第30-35、50-66和99-108位氨基酸序列所 示。 1.4 DNA sequence analysis of the positive cloned recombinant plasmid: The T7 DNA sequence TAATACGACTCACTATAGGG was used to determine the DNA sequence of the anti-C5 single-chain antibody on the positive recombinant plasmid. The result showed that the gene has the nucleotide sequence of SEQ ID NO: 7 in the sequence list, consisting of 756 Base composition, the light chain variable region coding gene has the DNA sequence of SEQ ID NO: 2 in the sequence list, the flexible polypeptide linkage coding gene has the DNA sequence of SEQ ID NO: 6 in the sequence list, and the heavy chain variable region coding gene It has the DNA sequence of SEQ ID NO: 4 in the sequence listing. The tandem form of the gene encoding the anti-C5 single-chain antibody is SEQ ID NO: 2-SEQ ID NO: 6-SEQ ID NO: 4, which is named VL -Linker-V H (also known as ScFv). V L -Linker-V H single-chain anti-C5 antibody encoded so as to have SEQ ID NO: 1-SEQ ID NO: 5-SEQ ID NO: 3 amino acid residue sequence in series. Through further sequence analysis, the amino acid sequences of CDR1, CDR2, and CDR3 of the light chain variable region are as shown in the amino acid sequences 25-35, 51-57, and 90-99 of SEQ ID NO. 1, respectively. The heavy chain is variable. The amino acid sequences of CDR1, CDR2 and CDR3 of the region are shown in the amino acid sequences of positions 30-35, 50-66 and 99-108 of SEQ ID NO. 3, respectively.
1.5抗C5单链抗体ScFv真核表达载体的构建及高效表达细胞株的筛选1.5 Construction of eukaryotic expression vector of anti-C5 single-chain antibody ScFv and screening of high-efficiency expression cell lines
为了得到在分子结构、理化特性和生物学功能方面更接近于天然的高等生物蛋白质分子，把步骤1.4获得的抗C5单链抗体V L-Linker-V H融合基因克隆至高效真核表达载体pEE14.1(Lonza)中，得到携带抗C5单链抗体V L-Linker-V H融合基因的重组表达载体，命名为pEE14.1/V L-Linker-V H。再利用脂质体将重组质粒pEE14.1/V L-Linker-V H转染至中国仓鼠卵巢细胞CHO中。转染24小时后，吸去培养基，加入10mL新鲜的选择性培养基DMEM+10％FCS+25μm MSX。在含5％CO 2的混合气体，湿度为98％的37℃培养箱中培养。2周后，出现大约1-2mm的克隆，用克隆环将出现的克隆转接至24孔板中，每孔加入1mL选择性培养基DMEM+10％FCS+25μm MSX继续培养。转化子生长至5天后，吸取上清。将该上清100μl加入到用C5抗原包被的酶联板中，37℃孵育1小时，PBS洗板3次。每孔加入100μl HRP标记的二抗抗体IgG(1:2000)，37℃孵育1小时。PBS洗板3次，加入新鲜配制的底物H 2O 2-OPD，室温反应20min，加入50μl 2M H 2SO 4终止反应，在A 490处检测每孔的光吸收值。光吸收值为阴性对照的2.1倍以上的为阳性克隆，从中挑选出与C5结合活性最强的阳性克隆，即为高效表达抗C5单链抗体的CHO细胞株。 In order to get closer to the natural protein molecules in higher organisms molecular structure, physicochemical properties and biological functions, step 1.4 to obtain the anti-C5 single chain antibody V L -Linker-V H fusion gene was cloned into eukaryotic expression vector pEE14 .1 (Lonza) to give an anti-C5 scFv carrying V L -Linker-V H gene fusion recombinant expression vector designated pEE14.1 / V L -Linker-V H . And then the recombinant plasmid by liposome pEE14.1 / V L -Linker-V H transfected into Chinese hamster ovary cells are CHO. After 24 hours of transfection, the medium was aspirated, and 10 mL of fresh selective medium DMEM+10% FCS+25μm MSX was added. Incubate in a 37°C incubator with 5% CO 2 mixed gas and 98% humidity. Two weeks later, clones of approximately 1-2 mm appeared. Use the cloning ring to transfer the clones to a 24-well plate. Add 1 mL of selective medium DMEM+10% FCS+25μm MSX to each well to continue the culture. After the transformant has grown to 5 days, the supernatant is aspirated. 100 μl of the supernatant was added to the C5 antigen-coated enzyme-linked plate, incubated at 37° C. for 1 hour, and the plate was washed 3 times with PBS. Add 100μl of HRP-labeled secondary antibody IgG (1:2000) to each well, and incubate at 37°C for 1 hour. Wash the plate 3 times with PBS, add freshly prepared substrate H 2 O 2 -OPD, react at room temperature for 20 min, add 50 μl 2M H 2 SO 4 to stop the reaction, and detect the light absorption value of each well at A 490. The light absorption value is 2.1 times or more of the negative control is the positive clone, and the positive clone with the strongest C5 binding activity is selected from the positive clone, which is the CHO cell line that highly expresses the anti-C5 single-chain antibody.
1.6抗C5单链抗体的纯化1.6 Purification of anti-C5 single chain antibody
将高效表达的CHO细胞株放大培养，收获上清。将上清缓慢加入HiTrap N-hydroxysuccinimide column(Amersham Biosciences)中，纯化单链抗体。用0.01mol/L pH 7.4的PBS洗脱，流速为1mL/min，至洗出液OD 280<0.02为止。加入0.1mol/L pH 2.4的甘氨酸-HCl缓冲液，流速为1mL/min，收集吸附下来的成分，立即以1mol/L碳酸钠中和，以免蛋白变性。经SDS-PAGE和Western Blot鉴定，结果如图1和图2所示，经表达获得了约26KD的目的蛋白，且该蛋白可与C5特异结合，与预期结果相符，表明获得了纯度很高的抗C5单链抗体。 The highly expressed CHO cell line was enlarged and cultured, and the supernatant was harvested. The supernatant was slowly added to HiTrap N-hydroxysuccinimide column (Amersham Biosciences) to purify the single-chain antibody. Elute with 0.01mol/L pH 7.4 PBS at a flow rate of 1 mL/min until the eluate OD 280 <0.02. Add 0.1 mol/L pH 2.4 glycine-HCl buffer at a flow rate of 1 mL/min, collect the adsorbed components, and immediately neutralize with 1 mol/L sodium carbonate to avoid protein denaturation. The results were identified by SDS-PAGE and Western Blot, and the results are shown in Figure 1 and Figure 2. After expression, the target protein of about 26KD was obtained, and the protein could specifically bind to C5, which was consistent with the expected results, indicating that a highly pure product was obtained. Anti-C5 single chain antibody.
实施例2、抗C5单链抗体与C5配基相互作用动力学分析Example 2. Kinetic analysis of the interaction between anti-C5 single-chain antibody and C5 ligand
抗C5单链抗体与C5配基相互作用动力学分析用表面细胞质基因组共振(SPR)检测***进行检测。The kinetic analysis of the interaction between the anti-C5 single-chain antibody and the C5 ligand was detected by the surface cytoplasmic genomic resonance (SPR) detection system.
2.1实验仪器与试剂2.1 Experimental equipment and reagents
仪器：Reichert2SPR(Reichert公司)，芯片：SAM芯片(大分子检测)，(Reichert Inc公司，PART NO:13206061)。Instrument: Reichert2SPR (Reichert company), chip: SAM chip (macromolecule detection), (Reichert Inc. PART NO: 1326061).
试剂：1xPBST 500ml(过滤，0.22uM滤膜过滤)，EDC(现用现配)，NHS(现用现配)，1M pH8.5乙醇胺(5-10ml)，10mM pH2.0HCl(5-10ml)，10mM pH2.0甘氨酸(5-10ml)。Reagents: 1xPBST 500ml (filtered, 0.22uM membrane filter), EDC (now used now), NHS (now used now), 1M pH8.5 ethanolamine (5-10ml), 10mM pH2.0HCl (5-10ml) , 10mM pH2.0 Glycine (5-10ml).
2.2.实验步骤2.2. Experimental steps
2.2.1预富集2.2.1 Pre-enrichment
2.2.1.1将蛋白用不同的pH醋酸钠稀释到10μg/mL，200μL。2.2.1.1 Dilute the protein with different pH sodium acetate to 10μg/mL, 200μL.
表1.醋酸钠pH值选择表Table 1. Sodium acetate pH value selection table
蛋白名称Protein name 醋酸钠pH值Sodium acetate pH 固定通道Fixed channel
抗原antigen 6.0/5.5/5.0/4.5/4.06.0/5.5/5.0/4.5/4.0 某一通道A certain channel
2.2.1.2在某一通道中注射蛋白，25μL/min，2min。2.2.1.2 Inject protein in a certain channel, 25μL/min, 2min.
2.2.1.3选择合适的pH条件(pH5.0)。2.2.1.3 Choose appropriate pH conditions (pH5.0).
2.2.2蛋白固定2.2.2 Protein fixation
2.2.2.1 76.66mg EDC和11.52mg NHS溶解在1mL超纯水中，取200μL，活化左右两通道，10μL/min,7min。2.2.2.1 76.66mg EDC and 11.52mg NHS were dissolved in 1mL ultrapure water, 200μL was taken, and the left and right channels were activated at 10μL/min for 7min.
2.2.2.2将抗原用合适的pH值醋酸钠稀释到50μg/mL，200μL，单独固定在某个通道。10μL/min,7min。2.2.2.2 Dilute the antigen with a suitable pH value of sodium acetate to 50μg/mL, 200μL, and fix it in a channel separately. 10μL/min, 7min.
2.2.2.3如果一次固定量不够，重复注射抗体。2.2.2.3 If a fixed amount is not enough, repeat the injection of antibody.
2.2.2.4取200μL 1M乙醇胺(pH8.5)，封闭左右两个通道，10μL/min,7min。2.2.2.4 Take 200μL of 1M ethanolamine (pH8.5), seal the left and right channels, 10μL/min, 7min.
2.2.3抗体-抗原结合预实验2.2.3 Antibody-antigen binding preliminary experiment
2.2.3.1将抗体用PBST稀释至100nM，25μL/min,结合3min，解离5min。2.2.3.1 Dilute the antibody to 100nM with PBST, 25μL/min, bind for 3min, and dissociate for 5min.
2.2.3.2 10mM pH2.0HCl(或者10mM pH2.0甘氨酸)再生2min，解离2min。2.2.3.2 10mM pH2.0HCl (or 10mM pH2.0 glycine) regeneration for 2min, dissociation for 2min.
2.2.4正式实验2.2.4 Formal experiment
2.2.4.1将抗体用PBST稀释至100nM，再2倍稀释，7个梯度，25μL/min,结合3min，解离5min。2.2.4.1 Dilute the antibody to 100nM with PBST, and then dilute 2 times, 7 gradients, 25μL/min, bind for 3min, dissociate for 5min.
2.2.4.2 10mM pH2.0HCl(或者10mM pH2.0甘氨酸)再生2min，解离2min。2.2.4.2 10mM pH2.0HCl (or 10mM pH2.0 glycine) regeneration for 2min, dissociation for 2min.
SPR检测结果如表2显示，检测显示，本发明提供的抗C5单链抗体显示了优异的抗原结合效能。The SPR test results are shown in Table 2. The test shows that the anti-C5 single chain antibody provided by the present invention shows excellent antigen binding efficiency.
表2.抗C5单链抗体与C5结合的动力学参数Table 2. Kinetic parameters of anti-C5 single-chain antibody binding to C5
Figure PCTCN2021074563-appb-000001
Figure PCTCN2021074563-appb-000001
实施例3.抗C5单链抗体的体外抑制补体激活的实验Example 3. In vitro inhibition of complement activation by anti-C5 single-chain antibody
为测定对补体的抑制活性，60％～80％融合的CHO细胞用乙二胺四乙酸分开，用DMEM洗2次，然后重悬于DMEM中，使其终浓度为10 6个细胞/mL。在细胞悬液中加入100mL/L兔抗CHO细胞膜抗血清，4℃作用30min，使细胞致敏。然后弃抗血清，细胞重悬于用DMEM稀释的NHS中，终体积为50μL或100μL。37℃作用60min，最后用胎盘兰染色排除法测量细胞成活力(活细胞与死细胞均计数)。单链抗体用DEME稀释后先加入到NHS中，再加入至CHO细胞悬液。终浓度以100g/L NHS可导致大约90％抗体致敏的对照CHO细胞溶解为准。补体介导的红细胞溶解抑制实验用抗体致敏的羊红细胞(EAs)进行测定。溶血试验在明胶佛罗那缓冲液(GVB ++)中进行，终体积为300μL，包含有2.5×10 7EAs，NHS按照1:300稀释。反应混合物在37℃孵育60min，最后加入300μL含10mmol/L EDTA-PBS溶液终止反应。离心，取上清，在413nm波长下用光谱成像仪对上清中的血红素进行定量检测。 To determine the inhibitory activity of complement, 60% to 80% confluent CHO cells are separated with EDTA, washed twice with DMEM and resuspended in DMEM at a final concentration of 10 6 cells / mL. Add 100mL/L rabbit anti-CHO cell membrane antiserum to the cell suspension and react for 30min at 4°C to sensitize the cells. Then the antiserum was discarded, and the cells were resuspended in NHS diluted with DMEM to a final volume of 50 μL or 100 μL. Incubate at 37°C for 60 minutes, and finally measure the viability of cells with placental blue staining exclusion method (both live and dead cells are counted). The single chain antibody was diluted with DEME and added to the NHS first, and then added to the CHO cell suspension. The final concentration of 100g/L NHS can cause about 90% of the antibody sensitized control CHO cells to be lysed as the standard. The complement-mediated inhibition of red blood cell lysis was tested with antibody-sensitized goat red blood cells (EAs). The hemolysis test was performed in Gelatin Verona buffer (GVB ++ ), the final volume was 300 μL, containing 2.5×10 7 EAs, and NHS was diluted 1:300. The reaction mixture was incubated at 37°C for 60 min, and finally 300 μL of 10 mmol/L EDTA-PBS solution was added to terminate the reaction. Centrifuge, take the supernatant, and quantitatively detect the hemoglobin in the supernatant with a spectral imager at a wavelength of 413nm.
单链抗体补体抑制物活性检测：补体介导的CHO细胞和红细胞溶解实验结果显示，在细胞裂解抑制实验中，抗C5单链抗体抑制抗体致敏的CHO细胞和红细胞溶解效果明显(详见表3)，显示抗C5单链抗体能够有效抑制补体经典激活途径。Single-chain antibody complement inhibitor activity test: The results of complement-mediated CHO cell and red blood cell lysis experiments show that in the cell lysis inhibition experiment, the anti-C5 single-chain antibody inhibiting antibody-sensitized CHO cells and red blood cells have a significant lysis effect (see table for details) 3), showing that the anti-C5 single-chain antibody can effectively inhibit the classical activation pathway of complement.
表3.能抑制50％细胞发生溶解的补体抑制物浓度Table 3. Concentrations of complement inhibitors that can inhibit 50% cell lysis
Figure PCTCN2021074563-appb-000002
Figure PCTCN2021074563-appb-000002
实施例4.靶向C5的单链抗体对MRL/lpr红斑狼疮小鼠模型的治疗作用Example 4. Therapeutic effect of single-chain antibody targeting C5 on MRL/lpr lupus erythematosus mouse model
MRL/lpr红斑狼疮小鼠模型最早是Murphy和Roths于1979年建立，由多个品系小鼠经过历经12代的复杂杂交过程而成，该模型的小鼠基因的75％来自LG/J、12.6％来自AKR/J、12.1％来自C3H/Di及0.3％来自C57BL/6品系小鼠。MRL/lpr小鼠含有与细胞自发性程序性死亡有关的Fas基因隐性突变，出现淋巴细胞增生基因，导致T细胞增生，全身***肿大，以及侵蚀性关节炎，抗DNA、抗Sm、抗Su、抗核苷P抗体，高滴度ANA，高丙种球蛋白血症以及类风湿因子。该鼠最早发病于8周，此时血清中可检测到自身抗体。12周时可以观察到***炎。12－16周，MRL/lpr鼠开始产生包括抗双链DNA抗体在内的大量自身抗体。近16周龄时多器官受累，以及出现以严重蛋白尿为特征的稳定肾功能退化。16-24周龄鼠出现增殖性免疫复合物介导的肾小球肾炎，血管炎，并最终导致肾衰而死亡，死亡率可以达到50％。The MRL/lpr lupus erythematosus mouse model was first established by Murphy and Roths in 1979. It was formed by a complex hybridization process of multiple strains of mice after 12 generations. 75% of the mouse genes of this model came from LG/J, 12.6 % Is from AKR/J, 12.1% is from C3H/Di and 0.3% is from C57BL/6 strain mice. MRL/lpr mice contain recessive mutations in the Fas gene related to spontaneous programmed cell death, and the presence of lymphocyte proliferation genes, leading to T cell proliferation, systemic lymph node enlargement, and erosive arthritis, anti-DNA, anti-Sm, anti- Su, anti-nucleoside P antibody, high titer ANA, hypergammaglobulinemia and rheumatoid factor. The mouse first became ill at 8 weeks, at which time autoantibodies could be detected in the serum. Lymphadenitis can be observed at 12 weeks. At 12-16 weeks, MRL/lpr mice began to produce a large number of autoantibodies including anti-double-stranded DNA antibodies. Nearly 16 weeks of age, multiple organ involvement and stable renal function degradation characterized by severe proteinuria appeared. 16-24 weeks old mice develop glomerulonephritis and vasculitis mediated by proliferative immune complexes, and eventually lead to renal failure and death, and the mortality rate can reach 50%.
4.1靶向C5的单链抗体明显提高MRL/lpr红斑狼疮小鼠的存活率4.1 Single-chain antibodies targeting C5 significantly improve the survival rate of MRL/lpr lupus erythematosus mice
本实施例将已经出现肾衰症状的16周的MRL/lpr鼠随机分为两组，第一组为治疗组，从第16-24周起每周接受高剂量0.4mg/W(n＝30)和低剂量0.1mg/W(n＝30)的ScFv，第二组(n＝32)为对照组，每周接受等量的PBS。两组的给药途径均为尾静脉注射。根据给药组和对照组的存活率评价靶向C5的单链抗体对MRL/lpr红斑狼疮小鼠的保护率。实验结果如图3所示，接受靶向C5的单链抗体治疗的小鼠，由于补体激活途径中的C5被靶向C5的单链抗体有效抑制，因此，MRL/lpr红斑狼疮小鼠的存活率明显提高，高剂量治疗组整个治疗过程能够完全保护MRL/lpr红斑狼疮小鼠，存活率为100％，而低剂量组即使在第24周也能保持在80％以上的存活率。In this example, 16-week-old MRL/lpr mice that had symptoms of renal failure were randomly divided into two groups. The first group was the treatment group. From the 16th to the 24th week, they received a high dose of 0.4 mg/W (n=30). ) And a low dose of 0.1 mg/W (n=30) of ScFv. The second group (n=32) was the control group and received the same amount of PBS every week. The route of administration for both groups was tail vein injection. According to the survival rate of the administration group and the control group, the protection rate of the single-chain antibody targeting C5 on MRL/lpr lupus erythematosus mice was evaluated. The experimental results are shown in Figure 3. In mice treated with a single-chain antibody targeting C5, C5 in the complement activation pathway is effectively inhibited by a single-chain antibody targeting C5. Therefore, the survival of MRL/lpr lupus erythematosus mice The high-dose treatment group can completely protect MRL/lpr lupus erythematosus mice during the whole treatment process, and the survival rate is 100%, while the low-dose group can maintain a survival rate of more than 80% even in the 24th week.
4.2靶向C5的单链抗体明显改善MRL/lpr红斑狼疮小鼠蛋白尿症状4.2 Single-chain antibodies targeting C5 significantly improve the symptoms of proteinuria in MRL/lpr lupus erythematosus mice
将小鼠置于代谢笼里研究靶向C5的单链抗体对MRL/lpr红斑狼疮小 鼠尿白蛋白分泌的影响。从16周开始每两周收集一次小鼠的24小时尿液。为防止细菌生长，在收集管中加入氨苄青霉素、庆大霉素和氯霉素。使用已知浓度的小鼠白蛋白样品通过ELISA方法绘制标准曲线，并确定实验小鼠的尿白蛋白分泌情况，并使用Beckman生化仪(Beckman Coulter)测定小鼠尿液中的肌酐含量。最后的评价结果以每只实验小鼠24小时的尿白蛋白(mg)与肌酐(mg)比值表示。尿白蛋白肌酐比值偏高表示肾脏功能受到损害。如图4所示，接受靶向C5的单链抗体治疗的治疗组(0.1mg/W，n＝26)在第22周和24周时，其平均的尿白蛋白肌酐比值在2.8-3.8左右，而对照组(n＝26)在3.8-5.5左右，治疗组的蛋白尿水平明显降低(P<0.01)，证明本发明提供的靶向C5的单链抗体能够显著改善肾功能损伤症状。Mice were placed in a metabolic cage to study the effect of single-chain antibody targeting C5 on urinary albumin secretion in MRL/lpr lupus erythematosus mice. From the 16th week, the mice’s 24-hour urine was collected every two weeks. To prevent bacterial growth, add ampicillin, gentamicin, and chloramphenicol to the collection tube. Using a mouse albumin sample of known concentration, a standard curve was drawn by ELISA method, and the urine albumin secretion of experimental mice was determined, and the creatinine content in the mouse urine was measured using a Beckman Coulter. The final evaluation result is expressed as the ratio of urine albumin (mg) to creatinine (mg) of each experimental mouse for 24 hours. A high ratio of urine albumin to creatinine indicates that kidney function is impaired. As shown in Figure 4, the treatment group (0.1mg/W, n=26) receiving single-chain antibody therapy targeting C5 had an average urine albumin-creatinine ratio of about 2.8-3.8 at weeks 22 and 24. , While the control group (n=26) was around 3.8-5.5, the proteinuria level of the treatment group was significantly reduced (P<0.01), which proved that the single-chain antibody targeting C5 provided by the present invention can significantly improve the symptoms of renal damage.
4.3靶向C5的单链抗体明显改善MRL/lpr红斑狼疮小鼠肾脏炎症4.3 Single-chain antibodies targeting C5 significantly improve kidney inflammation in MRL/lpr lupus erythematosus mice
实验结束后，切除小鼠肾脏纵向解剖为两半，其中一半进行免疫荧光分析，另一半10％中性甲醛固定，固体石蜡包埋切片，以苏木精-伊红染色法和过碘酸雪夫染色法对石蜡处理的肾脏组织切片进行染色，采用盲法分别对自切片观察到的肾小球炎症、增生、新月体形成、坏死症状进行评分，同时对肾间质的变化也进行评分。评分共分为0、1、2、3、4五级，0为无损伤，4为严重损伤。血管周围炎性渗出评价采用半定量方式，由两个独立观察者盲法对每张切片10个以上的血管进行评价。炎症的得分为0-3，0为无炎症，1为低于50％的血管由3层细胞围绕，2为大于50％的血管为3-6层围绕，3为最严重表现，多于6层的细胞环绕。评价结果如表4所示。After the experiment, the mouse kidney was excised and longitudinally dissected into two halves, half of which was subjected to immunofluorescence analysis, and the other half was fixed with 10% neutral formaldehyde, and the sections were embedded in solid paraffin and stained with hematoxylin-eosin and periodic acid Schiff The staining method was used to stain the paraffin-treated kidney tissue sections. The glomerular inflammation, hyperplasia, crescent formation, and necrosis symptoms observed from the self-section were scored by blind method, and the changes in the renal interstitial were also scored. The score is divided into five levels: 0, 1, 2, 3, and 4, with 0 being no damage and 4 being severe damage. The evaluation of perivascular inflammatory exudation adopts a semi-quantitative method. Two independent observers blindly evaluate more than 10 blood vessels in each slice. The score for inflammation is 0-3, 0 means no inflammation, 1 means less than 50% of the blood vessels are surrounded by 3 layers of cells, 2 means more than 50% of the blood vessels are surrounded by 3-6 layers, 3 is the most serious manifestation, more than 6 Surrounded by layers of cells. The evaluation results are shown in Table 4.
表4.MRL/lpr小鼠从16周到23周治疗后第24周治疗组和PBS对照组肾脏损害情况对比Table 4. Comparison of renal damage between the treatment group and the PBS control group in the 24th week after treatment in MRL/lpr mice from 16 to 23 weeks
Figure PCTCN2021074563-appb-000003
Figure PCTCN2021074563-appb-000003
从表中可以看出，靶向C5的ScFv在MRL/lpr红斑狼疮小鼠中的治疗中减轻了肾脏的炎症反应。和对照组相比，治疗组(0.1mg/W)的肾小球积分、间质炎症、血管炎和新月体/坏死等明显降低(P<0.05)。It can be seen from the table that ScFv targeting C5 reduced the inflammation of the kidneys in the treatment of MRL/lpr lupus erythematosus mice. Compared with the control group, the glomerular score, interstitial inflammation, vasculitis and crescent/necrosis of the treatment group (0.1mg/W) were significantly reduced (P<0.05).
工业实用性Industrial applicability
本发明提供了一种人源抗补体C5分子的单链抗体，以及在制备自身免疫性疾病治疗药物中的应用，本发明提供的单链抗体易于工业化生产，具有工业实用性。The present invention provides a human-derived anti-complement C5 molecule single-chain antibody and its application in the preparation of autoimmune disease therapeutic drugs. The single-chain antibody provided by the present invention is easy to industrially produce and has industrial applicability.
序列表自由内容Sequence Listing Free Content
Figure PCTCN2021074563-appb-000004
Figure PCTCN2021074563-appb-000004
Figure PCTCN2021074563-appb-000005
Figure PCTCN2021074563-appb-000005
Figure PCTCN2021074563-appb-000006
Figure PCTCN2021074563-appb-000006
Figure PCTCN2021074563-appb-000007
Figure PCTCN2021074563-appb-000007
Figure PCTCN2021074563-appb-000008
Figure PCTCN2021074563-appb-000008

Claims (10)

  1. 一种抗补体C5分子的人源化单链抗体，其特征在于，所述抗体轻链可变区的CDR1、CDR2和CDR3的氨基酸序列分别如SEQ ID NO.1的第25-35、51-57和90-99位氨基酸序列所示，所述抗体重链可变区的CDR1、CDR2和CDR3的氨基酸序列分别如SEQ ID NO.3的第30-35、50-66和99-108位氨基酸序列所示。A humanized single-chain antibody against complement C5 molecule, characterized in that the amino acid sequences of CDR1, CDR2, and CDR3 of the light chain variable region of the antibody are as shown in SEQ ID NO.1 No. 25-35, 51- As shown in the amino acid sequences at positions 57 and 90-99, the amino acid sequences of CDR1, CDR2 and CDR3 of the variable region of the antibody heavy chain are shown as amino acids 30-35, 50-66 and 99-108 of SEQ ID NO.3, respectively The sequence is shown.
  2. 根据权利要求1所述的单链抗体，其特征在于，所述抗体轻链可变区的氨基酸序列如SEQ ID NO.1所示，所述抗体重链可变区的氨基酸序列如SEQ ID NO.3所示。The single-chain antibody of claim 1, wherein the amino acid sequence of the antibody light chain variable region is shown in SEQ ID NO. 1, and the amino acid sequence of the antibody heavy chain variable region is shown in SEQ ID NO. .3 shown.
  3. 根据权利要求2所述的单链抗体，其特征在于，所述抗体轻链可变区与重链可变区以氨基酸序列如SEQ ID NO.5所示的柔性多肽连接。The single-chain antibody according to claim 2, wherein the variable region of the light chain and the variable region of the heavy chain of the antibody are connected by a flexible polypeptide with an amino acid sequence as shown in SEQ ID NO.5.
  4. 一种编码权利要求1-3任一所述单链抗体的多核苷酸，其特征在于，编码所述抗体轻链可变区的多核苷酸的序列由SEQ ID NO.2所示，编码所述抗体重链可变区的多核苷酸的序列由SEQ ID NO.4所示。A polynucleotide encoding the single-chain antibody of any one of claims 1-3, wherein the sequence of the polynucleotide encoding the light chain variable region of the antibody is shown in SEQ ID NO. The sequence of the polynucleotide of the variable region of the antibody heavy chain is shown in SEQ ID NO.4.
  5. 根据权利要求4所述的多核苷酸，其特征在于，编码所述抗体轻链可变区的多核苷酸与编码所述抗体重链可变区的多核苷酸由序列如SEQ ID NO.6所示的编码柔性多肽的多核苷酸连接。The polynucleotide of claim 4, wherein the polynucleotide encoding the variable region of the antibody light chain and the polynucleotide encoding the variable region of the antibody heavy chain are composed of a sequence such as SEQ ID NO.6 The shown polynucleotides encoding flexible polypeptides are linked.
  6. 一种表达权利要求5所述的编码单链抗体的多核苷酸的载体。A vector for expressing the polynucleotide encoding the single-chain antibody of claim 5.
  7. 根据权利要求6所述的载体，其特征在于，所述载体为pEE14.1/V L-Linker-V H。 The vector of claim 6, wherein the vector is pEE14.1/V L -Linker-V H.
  8. 一种含有权利要求7所述载体的宿主细胞，其特征在于，所述细胞为CHO细胞。A host cell containing the vector of claim 7, wherein the cell is a CHO cell.
  9. 权利要求1-3任一所述的单链抗体在制备自身免疫性疾病治疗药物中的应用。Use of the single-chain antibody according to any one of claims 1 to 3 in the preparation of drugs for the treatment of autoimmune diseases.
  10. 根据权利要求9所述的应用，其特征在于，所述疾病为类风湿性关节炎或***性红斑狼疮。The use according to claim 9, wherein the disease is rheumatoid arthritis or systemic lupus erythematosus.
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