WO2021159939A1 - 一种抗补体c3分子的全人源单克隆抗体及应用 - Google Patents

一种抗补体c3分子的全人源单克隆抗体及应用 Download PDF

Info

Publication number
WO2021159939A1
WO2021159939A1 PCT/CN2021/073002 CN2021073002W WO2021159939A1 WO 2021159939 A1 WO2021159939 A1 WO 2021159939A1 CN 2021073002 W CN2021073002 W CN 2021073002W WO 2021159939 A1 WO2021159939 A1 WO 2021159939A1
Authority
WO
WIPO (PCT)
Prior art keywords
antibody
monoclonal antibody
seq
amino acid
light chain
Prior art date
Application number
PCT/CN2021/073002
Other languages
English (en)
French (fr)
Inventor
唐晓敏
杜兰英
Original Assignee
北京康普美特创新医药科技有限责任公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 北京康普美特创新医药科技有限责任公司 filed Critical 北京康普美特创新医药科技有限责任公司
Publication of WO2021159939A1 publication Critical patent/WO2021159939A1/zh

Links

Images

Classifications

    • 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 invention discloses an antibody, which belongs to the technical field of polypeptides.
  • 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; increase the permeability of blood vessels; neutralize viruses; cytolysis; regulation of immune response, 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 C3a and C3b, and thereby provides the C3b required in the C3 convertase C3bBb that forms the alternative pathway for antigen.
  • the C3 molecule is the core junction that connects the three complement activation pathways. It is a key molecule that determines the transition from upstream to downstream in complement activation, whether to form an amplification circuit, and the ultimate pathological damage path.
  • the intracellular form of the C3 precursor protein is a single-chain structure composed of 1663 amino acid residues, with a signal peptide composed of 22 amino acid residues at its amino terminus.
  • the secreted form of the C3 precursor protein has 1641 amino acid residues, which is cleaved by proteolytic enzymes and decomposed into two subunits, the ⁇ chain and the ⁇ chain.
  • the mature C3 molecule is the ⁇ chain and the ⁇ chain connected by disulfide bonds. constitute.
  • C3 convertase The above three pathways can produce C3 convertase, and the C3 molecule is cleaved at the alpha chain into two active fragments: anaphylatoxin C3a and opsonizing C3b.
  • C3a is a powerful allergic toxin, which means that it may 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.
  • C5a is also an 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.
  • C5b produced by cutting C5 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
  • complement activation provides a valuable first-line defense against potential pathogens
  • complement activation that promotes a protective inflammatory response can 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.
  • Eculizumab is a recombinant human monoclonal antibody that inhibits the activation of terminal complement components.
  • Chinese invention patent application CN104220453A discloses a humanized chimeric anti-C3b antibody, which can inhibit the rabbit red blood cell lysis experiment dependent on the alternative pathway, but does not inhibit the sheep erythrocyte lysis experiment sensitized by the classical pathway dependent antibody At the same time, the antibody does not inhibit the binding of properdin P and C3b to form C3/C5 convertase.
  • the experimental data in this application show that the binding site of the anti-C3 antibody to the antigen plays a key role in its specific role in inhibiting the complement activation pathway and its inhibitory efficacy.
  • the selective inhibition of the complement activation pathway can certainly eliminate the pathological damage but also incompletely eliminate the body's immune defenses.
  • the three activation pathways of complement that are related to each other as described above cannot achieve the desired inhibitory effect. There is still a need in the art to develop antibody drugs with targeted inhibitory effects for specific indications.
  • the purpose of the present invention is to provide a monoclonal antibody that specifically binds to C3 molecules, which can effectively inhibit the cleavage of C3 molecules and inhibit the production of anaphylactoxin C3a as its cleavage product to inhibit or reduce the degree of inflammation, and at the same time inhibit
  • the production of another cleavage product C3b, which is the core component of C3 convertase can effectively prevent the path of complement activation and amplification circuit at the same time, and provide the antibody for the treatment of autoimmune diseases such as lupus erythematosus, which is mainly damaged by immune complexes.
  • autoimmune diseases such as lupus erythematosus
  • the present invention first provides a fully human monoclonal antibody against complement C3 molecules.
  • the amino acid sequences of the CDR1, CDR2 and CDR3 regions of the light chain variable region of the antibody are as shown in SEQ ID NO:1, 25th-
  • the amino acid sequences of positions 37, 53-59 and 92-101 are shown in the amino acid sequences of the CDR1, CDR2 and CDR3 regions of the variable region of the antibody heavy chain as shown in SEQ ID NO: 5, 31-35, 50-66 and 99-108, respectively.
  • the amino acid sequence is shown in position.
  • amino acid sequence of the variable region of the antibody light chain is shown in SEQ ID NO: 1
  • amino acid sequence of the variable region of the antibody heavy chain is shown in SEQ ID NO: 5.
  • amino acid sequence of the constant region of the antibody light chain is shown in SEQ ID NO: 3
  • amino acid sequence of the constant region of the antibody heavy chain is shown in SEQ ID NO: 7.
  • the present invention also provides a polynucleotide encoding the heavy chain and/or light chain of the aforementioned monoclonal antibody.
  • polynucleotide sequence encoding the light chain variable region of the antibody is shown in SEQ ID NO: 2
  • polynucleotide sequence encoding the heavy chain variable region of the antibody is shown in SEQ ID. NO: shown in 6.
  • sequence of the polynucleotide encoding the antibody light chain constant region is shown in SEQ ID NO: 4, and the sequence of the polynucleotide encoding the antibody heavy chain constant region is shown in SEQ ID NO: 8 shows.
  • the present invention also provides a vector for expressing the above-mentioned polynucleotide encoding the heavy chain and/or light chain of the monoclonal antibody.
  • the present invention is not particularly limited to the vector expressing the polynucleotide encoding the heavy chain and/or light chain of the monoclonal antibody, as long as it is a eukaryotic expression vector capable of expressing the encoding polynucleotide, it is applicable.
  • the vector expressing the polynucleotide encoding the light chain of the monoclonal antibody is a pAB ⁇ vector
  • the vector expressing the polynucleotide encoding the heavy chain of the monoclonal antibody is the pABH vector.
  • the present invention also provides a host cell containing the above-mentioned vector, and the cell is an Expi 293F cell.
  • the present invention provides the application of the above-mentioned monoclonal antibodies in the preparation of drugs for the treatment of autoreactive diseases.
  • the autoimmune disease is lupus erythematosus.
  • the light chain and heavy chain of the anti-C3 antibody disclosed in the present invention have unique CDR regions and show excellent antigen-binding activity in terms of antigen-binding ability.
  • Ka(1/(M*S) is 8.72 ⁇ 10 4
  • Kd(1 /s) is 4.64 ⁇ 10 -5
  • K D (m) is 5.3 ⁇ 10 -10 .
  • the anti-C3 monoclonal antibody sensitized CHO cells and erythrocytes have a significant lysis effect, inhibiting 50%
  • the concentration of complement inhibitor for lysis of %CHO cells is 11 nmol/L
  • the concentration of complement inhibitor for lysis of 50% of red blood cells is 9 nmol/L, showing that anti-C3 monoclonal antibodies can effectively inhibit the classical activation pathway of complement.
  • Anti-C3 disclosed in the present invention In the treatment of MRL/lpr lupus erythematosus mice, the monoclonal antibody can significantly improve the survival rate of the mice.
  • the high-dose treatment group can completely protect the MRL/lpr lupus erythematosus mice during the whole treatment process, and the survival rate is 100%, while the survival rate is low.
  • the dose group can maintain a survival rate of more than 90% even in the 24th week.
  • the symptoms of proteinuria, glomerular score, interstitial inflammation, vasculitis, and crescent/necrosis in the treatment group have been significantly improved, showing that the The single-chain antibody of the anti-C3 monoclonal antibody provided by the invention has excellent application prospects in the preparation of therapeutic drugs for autoimmune diseases.
  • Figure 4 The kinetic analysis diagram of the interaction between anti-C3 antibody and target antigen
  • Figure 6 A control chart of proteinuria changes in MRL/lpr mice treated with a single-chain antibody targeting C3.
  • 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.
  • Take 20 ⁇ l culture medium transfer it to 200 ⁇ l 2 ⁇ YTAG culture medium containing 5 ⁇ 10 8 pfu/mL M13K07, and incubate at 37°C for 2 hours.
  • 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 C3 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 min, 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, and the positive clone with the strongest C3 binding activity is selected from it.
  • Figure 1 is an agarose gel identification diagram of amplified fragments, in which lane 1 is a light chain amplified product with a length of about 300 kb, and lane 2 is a heavy chain amplified product with a length of about 400 kb.
  • top10 Beijing Kangwei Century Biotechnology Co., Ltd., CW0807S
  • DH5 ⁇ can also be selected competent cells, resuscitate, and cultivate overnight in an ampicillin resistant culture plate to cultivate a single clone.
  • amino acid sequences of the CDR1, CDR2, and CDR3 regions of the light chain variable region are shown in the amino acid sequences 25-37, 53-59 and 92-101 of SEQ ID NO:1, respectively, and the polynucleotide sequence encoding the light chain variable region is shown in SEQ ID NO: 2; the amino acid sequence of the light chain constant region is shown in SEQ ID NO: 3, and the sequence of the polynucleotide encoding the light chain constant region is shown in SEQ ID NO: 4.
  • the amino acid sequence of the variable region of the antibody heavy chain is shown in SEQ ID NO: 5, and the amino acid sequence of the CDR1, CDR2, and CDR3 regions of the variable region of the antibody heavy chain are shown in SEQ ID NO: 5, 31-35, 50- respectively.
  • the amino acid sequence of positions 66 and 99-108 is shown, the polynucleotide sequence encoding the heavy chain variable region of the antibody is shown in SEQ ID NO: 6; the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO: 7
  • the sequence of the polynucleotide encoding the antibody heavy chain constant region is shown in SEQ ID NO: 8.
  • this expression system combines the high-expressing Expi 293 cell line, a serum-free medium with a clear chemical composition, a high-efficiency transfection reagent and a special enhancer. Take 15ug of heavy chain vector and 15ug of light chain vector and transfect Expi 293F cells, follow the instructions (ThermoFisher Scientific, A14635), harvest the culture solution 3 days later, centrifuge the supernatant about 30ml, use a volume of 5ml pre-packed Protein A affinity chromatography column, equilibrate with 20mM PBS before loading. After the conductivity shows to the baseline, inject the sample.
  • Lane 1 is the non-reducing SDS-PAGE electrophoresis result of the monoclonal antibody.
  • the theoretical value of the full molecular weight is about 150kDa. Because it is a glycosylated protein, the actual electropherogram is about 250kDa, and lane 2 is the molecular weight marker.
  • Lane 3 is the reduction SDS-PAGE electrophoresis result of the monoclonal antibody, the heavy chain (H) is expected to be 50kDa, and the light chain (Light chain, L) is expected to be 25kDa).
  • Goat anti-human antibody is used as the secondary antibody at a dilution ratio of 1:5000, and incubated at room temperature for 1 hour;
  • lane 1 is the molecular weight marker
  • lane 2 is the western-blotting identification band of the monoclonal antibody.
  • the theoretical value of the molecular weight of the monoclonal antibody is about 150kDa. Because it is a glycosylated protein, the actual electrophoresis is The figure is around 250kDa .
  • the kinetic analysis of the interaction between the anti-complement C3 monoclonal antibody and the C3 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 detection results are shown in Table 2, and the kinetic curve is shown in Figure 4.
  • the test shows that the anti-C3 monoclonal antibody provided by the present invention shows excellent antigen binding efficiency.
  • the monoclonal antibody was diluted with DEME and then 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 heme in the supernatant with a spectral imager at a wavelength of 413 nm.
  • Monoclonal antibody complement inhibitor activity test The results of complement-mediated CHO cell and red blood cell lysis experiments show that in the CHO cell lysis inhibition experiment, the anti-C3 monoclonal antibody inhibitory antibody-sensitized CHO cells and red blood cells have a significant lysis effect (see table for details) 3), showing that anti-C3 monoclonal antibodies can effectively inhibit the classical activation pathway of complement.
  • 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 for 12 generations. 75% of the mouse genes of this model are derived 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 three groups.
  • the route of administration for the three groups was tail vein injection.
  • the protection rate of ScFv-DA on MRL/lpr lupus erythematosus mice was evaluated according to the survival rate of the administration group and the control group.
  • mice treated with anti-C3 monoclonal antibodies because C3 in the complement activation pathway is effectively inhibited by anti-C3 monoclonal antibodies, it is difficult for the complement activation cascade to enter the amplification loop, and autoimmune inflammation is damaged. Effective inhibition, therefore, the survival rate of MRL/lpr lupus erythematosus mice is significantly improved.
  • the whole treatment process of the anti-C3 monoclonal antibody high-dose treatment group can completely protect MRL/lpr lupus erythematosus mice with a survival rate of 100%.
  • Anti-C3 monoclonal antibody The low-dose treatment group was able to maintain a survival rate of more than 90% even in the 24th week. Compared with the control group, the survival rate of the mice in the treatment group increased significantly from 18 weeks.
  • mice were placed in a metabolic cage to study the effect of anti-C3 monoclonal antibody 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, ampicillin, gentamicin (ampicillin, gentamicin; Invitrogen Life Technologies) and chloramphenicol (Sigma-Aldrich) were added to the collection tube.
  • the mouse albumin sample (Bethyl Laboratories) of known concentration to draw a standard curve by ELISA method, and determine the urine albumin secretion of experimental mice, and use the biochemical analyzer (Beckman Coulter) to determine the creatinine content in the mouse urine .
  • 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 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 anti-C3 monoclonal antibody treatment group (0.1mg/W) had significantly lower glomerular scores, interstitial inflammation, vasculitis and crescent/necrosis than the control group (P ⁇ 0.05).
  • the present invention provides a fully human monoclonal antibody against complement C3 molecule and its application in the preparation of autoimmune disease therapeutic drugs.
  • the monoclonal antibody provided by the present invention is easy to industrially produce and has industrial applicability.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Peptides Or Proteins (AREA)

Abstract

本发明公开了一种抗补体C3分子的全人源单克隆抗体，该抗体在CHO细胞裂解抑制实验中抑制抗体致敏的CHO细胞和红细胞溶解效果明显，在MRL/lPr红斑狠疮小鼠的治疗中能够明显提升小鼠的存活率。还公开了编码该单克隆抗体的多核苷酸、表达该多核苷酸的载体、含有该载体的宿主细胞、以及该单克隆抗体在制备自身免疫疾病治疗药物中的应用。

Description

一种抗补体C3分子的全人源单克隆抗体及应用 技术领域
本发明公开了一种抗体，属于多肽技术领域。
背景技术
补体系统由30余种可溶性蛋白分子组成，是天然免疫系统的一部分，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。补体系统可通过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。
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，补体系统产生的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切割成C3a和C3b，并由此提供抗原形成旁路途径的的C3转化酶C3bBb中所需的C3b。
C3分子是连接3种补体激活途径的核心交汇点，是决定补体激活从上游向下游过渡，是否形成放大回路，以及产生终极的病理损害路径的关键分子。细胞内形式的C3前体蛋白是由1663个氨基酸残基组成的单链结构，在其氨基端具有22个氨基酸残基组成的信号肽。分泌形式的C3前体蛋白具有1641个氨基酸残基，经蛋白水解酶切割后分解为两个亚单位，α链和β链，成熟的C3分子为以二硫键连接的α链和β链共同构成。以上三种途径均能产生C3转化酶，C3分子在α链处被C3转化酶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，意味可能导致各种临床疾病。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。C5a也是一种过敏毒素，能引起平滑肌、血管紧张度和血管渗透性的改变。它还是中性粒细胞、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。切断C5产生的C5b则插入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可能起着重要作用，此外它还起着裂解细胞的孔形成复合物的作用。
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以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，例如类风湿性关节炎、系统性红斑狼疮、肾小球肾炎等是有效的。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了评价。另外，已经上市了抑制补体级联反应中C5裂解的抗体药物(Thomas et al.,MolImmunol1996,33:1389)，即依库丽单抗(Eculizumab)。依库丽单抗是抑制末端补体成分活化的重组人源型单克隆抗体,其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。
技术问题
鉴于C3分子在补体激活的3个途径中的核心作用，对于C3分子在补体活化进程中所扮演的病理角色，以及对C3的调控能否作为抑制补体过度活化导致疾病的调控手段也得到了相应的研究。既往的C3抗体仅仅作为评价C3在炎症性疾病中的手段，对于C3抗体在抑制补体激活途径中的效果还知之甚少。中国发明专利申请CN104220453A公开了一种人源化的嵌合抗C3b的抗体，该抗体可以抑制旁路途径依赖的兔红细胞溶解实验，但对经典途径依赖的抗体致敏的绵羊红细胞溶解实验没有抑制作用，同时，该抗体并不抑制备解素P与C3b的结合，以形成C3/C5转化酶。该申请的实验数据显示，抗C3抗体与抗原的结合位点对于其在抑 制补体激活途径中的具体作用靶分子和抑制效能起着关键作用。对于补体激活途径的选择性抑制固然可以在消除病理学损伤的同时也能够不完全消除机体免疫防御力。但是对于处于如上所述相互关联的补体三种激活途径并不能取得理想的抑制效果。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。
本发明的目的就是提供一种特异性结合C3分子的单克隆抗体，以能够有效抑制C3分子的裂解，抑制作为其裂解产物的过敏毒素C3a的产生以达到抑制或者降低炎症反应的程度，同时抑制作为C3转化酶核心组分的另一裂解产物C3b的产生，进而同时有效阻止补体激活放大回路的路径，并进而提供所述抗体在免疫复合物损害为主的红斑狼疮等自身免疫性疾病治疗药物中的应用。
技术解决方案
基于上述发明目的，本发明首先提供了一种抗补体C3分子的全人源单克隆抗体，所述抗体轻链可变区CDR1、CDR2和CDR3区氨基酸序列分别如SEQ ID NO:1第25-37、53-59和92-101位氨基酸序列所示，所述抗体重链可变区CDR1、CDR2和CDR3区氨基酸序列分别如SEQ ID NO:5第31-35、50-66和99-108位氨基酸序列所示。
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:5所示。
在一个更为优选的实施方案中，所述抗体轻链恒定区的氨基酸序列如SEQ ID NO:3所示，所述抗体重链恒定区的氨基酸序列如SEQ ID NO:7所示。
其次，本发明还提供了一种编码上述单克隆抗体重链和/或轻链的多核苷酸。
在一个优选的实施方案中，编码所述抗体的轻链可变区的多核苷酸序列如SEQ ID NO:2所示，编码所述抗体的重链可变区的多核苷酸序列如SEQ ID NO:6所示。
在一个更为优选的实施方案中，编码所述抗体轻链恒定区的多核苷 酸的序列如SEQ ID NO:4所示，编码所述抗体重链恒定区的多核苷酸的序列如SEQ ID NO:8所示。
第三，本发明还提供了表达上述编码单克隆抗体重链和/或轻链的多核苷酸的载体。
本发明对于表达上述编码单克隆抗体重链和/或轻链的多核苷酸的载体并无特别限定，只要是能够表达出所述编码多核苷酸的真核表达载体均在可应用之列，在本发明的一个优选的实施方案中，表达编码单克隆抗体轻链的多核苷酸的载体为pABλ载体，表达编码单克隆抗体重链的多核苷酸的载体为pABH载体。
第四，本发明还提供了一种含有上述载体的宿主细胞，所述细胞为Expi 293F细胞。
最后，本发明提供了上述的单克隆抗体在制备自身反应性疾病治疗药物中的应用。
在一个优选的实施方案中，所述自身免疫性疾病为红斑狼疮。
技术效果
本发明公开的抗C3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，Ka(1/(M*S)为8.72×10 4，Kd(1/s)为4.64×10 -5，，K D(m)为5.3×10 -10。在CHO细胞裂解抑制实验中，抗C3单抗抑制抗体致敏的CHO细胞和红细胞溶解效果明显，抑制50％CHO细胞发生溶解的补体抑制物浓度是11nmol/L，抑制50％红细胞发生溶解的补体抑制物浓度是9nmol/L，显示抗C3单抗能够有效抑制补体经典激活途径。本发明公开的抗C3单抗在MRL/lpr红斑狼疮小鼠的治疗中，能够明显提升小鼠的存活率，高剂量治疗组在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90％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C3单抗的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。
附图说明
图1.轻链和重链基因扩增琼脂糖凝胶电泳鉴定图；
图2.抗C3抗体的12％SDS-PAGE鉴定结果；
图3.抗C3抗体的Western Blot鉴定结果；
图4.抗C3抗体与靶抗原的相互作用动力学分析图；
图5.靶向C3的单链抗体治疗MRL/lpr小鼠的存活率对照图；
图6.靶向C3的单链抗体治疗MRL/lpr小鼠的蛋白尿变化对照图。
本发明的实施方式
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，并不对本发明的权利要求所限定的保护范围构成任何限制。
实施例1.抗补体C3分子的全人源单克隆抗体的制备
1.1噬菌体抗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。
1.2重组噬菌体抗体的筛选：用C3抗原(Complement Technology，Inc；货号：A113，序列登记号K02765)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。在以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。
1.3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。
用C3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C3结合活性最强的阳性克隆。
1.4利用PCR技术分别扩增出结合活性最强的阳性克隆的轻链和重链片段，轻链和重链片段的上游带有BsrGI的酶切位点，下游带有BamHI的酶切位点；
轻链上游引物：
5’>CGCGTGTACAGGAAGCTGGGCCGATATCGTTCTGACTCAACCTC<3’
轻链下游引物：5’>CGCGAAGCTTGGTGCCACCGCCAAACAC<3’
重链上游引物：
5’>GCGCCCCTTAAGGGCGTGCAGTGCGAAGTGCAATTGGTGGAAAGC<3’
重链下游引物：
5’>CGGTGCTAGCGCTCGACACGGTCACCAGAGT<3’
PCR反应条件为95℃预变性5分钟，然后进入扩增循环，95℃变性50秒，68℃退火40秒，72℃延伸50秒，经过25个循环后72℃再延伸10分钟。图1为扩增片段的琼脂糖凝胶鉴定图，其中泳道1为轻链扩增产物，长度约300kb，泳道2为重链扩增产物，长度约400kb。
1.5利用试剂盒纯化片段；
1.6利用BsrGI和BamHI酶进行酶切片段；
将酶切过后的轻链片段和重链片段分别与pABλ载体(本实验室保存)以及pABH载体(本实验室保存)进行连接；
1.7将连接的片段，转到top10(北京康为世纪生物科技有限公司，CW0807S)(也可以选择DH5α)感受态细胞中，复苏，氨苄抗性培养板中培养过夜，培养出单克隆。
1.8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C3抗体的DNA序列，所述抗体轻链可变区的氨基酸序列如SEQ ID NO:1所示，所述抗体轻链可变区CDR1、CDR2和CDR3区氨基酸序列分别如SEQ ID NO:1第25-37、53-59和92-101位氨基酸序列所示，编码轻链可变区的多核苷酸序列如SEQ ID NO:2所示；轻链恒定区的氨基酸序列如SEQ ID NO:3所示，编码轻链恒定区的多核苷酸的序列如SEQ ID NO:4所示。所述抗体重链可变区的氨基酸序列如SEQ ID NO:5所示，所述抗体重链可变区CDR1、CDR2和CDR3区氨基酸序列分别如SEQ ID NO:5第31-35、50-66和99-108位氨基酸序列所示，编码所述抗体的重链可变区的多核苷酸序列如SEQ ID NO:6所示；重链恒定区的氨基酸序列如SEQ ID NO:7所示，编码所述抗体重链恒定区的多核苷酸的序列如SEQ ID NO:8所示。
1.9比对序列，将片段与载体连接正确的菌液，扩大培养，提取质粒；
1.10单抗的瞬时表达和亲和层析纯化
使用Expi293表达系统，此表达系统将高表达Expi 293细胞系、化学成分明确的无血清培养基和一种高效转染试剂与专用增强剂结合在一起。取15ug重链载体和15ug轻链载体混合后转染Expi 293F细胞，按照说明书进行操作(ThermoFisher Scientific，A14635)，3天后收获培养液，离心后上清约30ml，使用体积为5ml的预装Protein A亲和层析柱，上样前使用20mM PBS平衡，待电导显示到基线后进样，上样结束后，使用20mMPBS洗涤色谱柱至基线平稳，使用0.1M pH3.0的甘氨酸缓冲液洗脱目的蛋白，待OD 280近基线后，停止收集，使用至少3个柱体积的20mM的PBS洗涤色谱柱，至基线平稳后，用20％的乙醇洗涤色谱柱。单抗蛋白含量的测定使用紫外法，公式：蛋白含量＝1.54A 280-1.38A 260。亲和层析纯化后，取20μl纯化脱盐后的蛋白溶液，分别加入4μl的6×还原buffer和非还原的buffer，沸水浴5分钟，进行SDS-PAGE，然后脱色。SDS-PAGE检测结果见图2(泳道1为单抗的非还原SDS-PAGE电泳结 果，全分子量理论值在150kDa左右，由于是糖基化蛋白，实际电泳图在250kDa左右，泳道2为分子量标记，泳道3为单抗的还原SDS-PAGE电泳结果,重链(heavy chain,H)预期50kDa,轻链(Light chain,L)预期25kDa)。
1.11单克隆抗体的Western-Blotting鉴定
1.11.1 5％的浓缩胶，6％的分离胶；
1.11.2 1μl的1mg/ml抗原进行SDS-PAGE，70V浓缩胶，130V分离胶；
1.11.3转印到PVDF膜上，转印时间24min；
1.11.4 5％脱脂奶粉，0.1％Tween20的TBS封闭2h；
1.11.5 10ml浓度为5μg/ml的一抗，室温孵育2h；
1.11.6 TBST洗涤5次，5min/次；
1.11.7山羊抗人的抗体作为二抗，稀释比例为1:5000，室温孵育1h；
1.11.8 TBST洗涤5次，5min/次；
1.11.9显色。
Western-Blotting鉴定结果见图3，图3中，泳道1为分子量标记，泳道2为单抗的western-blotting鉴定条带，单抗分子量理论值在150kDa左右，由于是糖基化蛋白，实际电泳图在250kDa左右 。
实施例2.抗补体C3单克隆抗体与C3配基相互作用动力学分析
抗补体C3单克隆抗体与C3配基相互作用动力学分析用表面细胞质基因组共振(SPR)检测系统进行检测。
2.1实验仪器与试剂
仪器：Reichert2SPR(Reichert公司)，芯片：SAM芯片(大分子检测)，(Reichert Inc公司，PART NO:13206061)。
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)。
2.2.实验步骤
2.2.1预富集
2.2.1.1将蛋白用不同的pH醋酸钠稀释到10μg/mL，200μL。
表1.醋酸钠pH值选择表
Figure PCTCN2021073002-appb-000001
2.2.1.1在某一通道中注射蛋白，25μL/min，2min。
2.2.1.2选择合适的pH条件(pH5.0)。
2.2.2蛋白固定
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。
2.2.2.2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。
2.2.2.3如果一次固定量不够，重复注射抗体。
2.2.24取200μL 1M乙醇胺(pH8.5)，封闭左右两个通道，10μL/min,7min。
2.2.3抗体-抗原结合预实验
2.2.3.1将抗体用PBST稀释至100nM，25μL/min,结合3min，解离5min。
2.2.3.2 10mM pH2.0HCl(或者10mM pH2.0甘氨酸)再生2min，解离2min。
2.2.4正式实验
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。
2.2.4.2 10mM pH2.0HCl(或者10mM pH2.0甘氨酸)再生2min，解离2min。
SPR检测结果如表2显示，动力学曲线如图4所示。检测显示，本发明提供的抗C3单克隆抗体显示了优异的抗原结合效能。
表2.抗补体C3单克隆抗体与C3结合的动力学参数
Figure PCTCN2021073002-appb-000002
实施例3.抗C3全抗的体外抑制补体激活的实验
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)。单克隆抗体用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。
单克隆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CHO细胞裂解抑制实验中，抗C3单抗抑制抗体致敏的CHO细胞和红细胞溶解效果明显(详见表3)，显示抗C3单抗能够有效抑制补体经典激活途径。
表3.能抑制50％细胞发生溶解的补体抑制物浓度
Figure PCTCN2021073002-appb-000003
实施例4.抗C3单抗在MRL/lpr红斑狼疮小鼠中的治疗作用
1.生存率的提高
MRL/lpr小鼠从16周到24周抗C3单抗高剂量治疗组(n＝24)、ScFv-DAF低剂量治疗组(n＝24)和PBS对照组(n＝26)小鼠生存率的 对比。
MRL/lpr红斑狼疮小鼠模型最早是Murphy和Roths于1979年建立，由多个品系小鼠历经12代的复杂的杂交过程而成，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，全身淋巴结肿大，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周时可以观察到淋巴结炎。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％。
本实施例将已经出现肾衰症状的16周的MRL/lpr鼠随机分为三组，第一组(n＝24)为高剂量治疗组，从第16-24周起每周接受0.4mg/W的抗C3单抗，第二组(n＝24)为低剂量治疗组，从第16-24周起每周接受0.1mg/W的抗C3单抗，第三组(n＝26)为对照组，从第16-24周起每周接受等量的PBS。三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ScFv-DA对MRL/lpr红斑狼疮小鼠的保护率。
实验结果如图5所示，接受抗C3单抗治疗的小鼠，由于补体激活途径中的C3被抗C3单抗有效抑制，补体激活级联反应难以进入放大环路，自身免疫性炎症损害得到有效抑制，因此，MRL/lpr红斑狼疮小鼠的存活率明显提高，抗C3单抗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％，抗C3单抗低剂量治疗组即使在第24周也能保持在90％以上的存活率，和对照组相比，从18周起治疗组的小鼠生存率明显提高。
2.肾功能改善
将小鼠置于代谢笼里研究抗C3单抗对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(ampicillin, gentamicin；Invitrogen Life Technologies)和氯霉素(Sigma-Aldrich)。使用已知浓度的小鼠白蛋白样品(Bethyl Laboratories)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6所示，MRL/lpr小鼠抗C3单抗治疗组(0.1mg/W，n＝26)和PBS对照组(n＝26)蛋白尿情况对比在第22-24周时，与对照组相比，治疗组的蛋白尿水平明显降低(P<0.01)。证明本发明提供的抗C3单抗能够显著改善肾功能损伤症状。
3.肾脏的炎症反应减轻
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。
表4.MRL/lpr小鼠从16周到23周治疗后第24周抗C3单抗治疗组和PBS对照组肾脏损害情况对比
Figure PCTCN2021073002-appb-000004
和对照组相比，抗C3单抗治疗组(0.1mg/W)比对照组在肾小球积分、间质炎症、血管炎和新月体/坏死等更加明显降低(P<0.05)。
工业实用性
本发明提供了一种抗补体C3分子的全人源单克隆抗体，以及在制备自身免疫性疾病治疗药物中的应用，本发明提供的单克隆抗体易于工业化生产，具有工业实用性。
序列表自由内容
Figure PCTCN2021073002-appb-000005
Figure PCTCN2021073002-appb-000006
Figure PCTCN2021073002-appb-000007
Figure PCTCN2021073002-appb-000008
Figure PCTCN2021073002-appb-000009
Figure PCTCN2021073002-appb-000010
Figure PCTCN2021073002-appb-000011

Claims (10)

  1. 一种抗补体C3分子的全人源单克隆抗体，其特征在于，所述抗体轻链可变区CDR1、CDR2和CDR3区氨基酸序列分别如SEQ ID NO:1第25-37、53-59和92-101位氨基酸序列所示，所述抗体重链可变区CDR1、CDR2和CDR3区氨基酸序列分别如SEQ ID NO:5第31-35、50-66和99-108位氨基酸序列所示。
  2. 根据权利要求1所述的单克隆抗体，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:5所示。
  3. 根据权利要求2所述的单克隆抗体，其特征在于，所述抗体轻链恒定区的氨基酸序列如SEQ ID NO:3所示，所述抗体重链恒定区的氨基酸序列如SEQ ID NO:7所示。
  4. 一种编码权利要求1-3任一所述单克隆抗体重链和/或轻链的多核苷酸。
  5. 根据权利要求4所述的多核苷酸，其特征在于，编码所述抗体的轻链可变区的多核苷酸序列如SEQ ID NO:2所示，编码所述抗体的重链可变区的多核苷酸序列如SEQ ID NO:6所示。
  6. 根据权利要求5所述的多核苷酸，其特征在于，编码所述抗体轻链恒定区的多核苷酸的序列如SEQ ID NO:4所示，编码所述抗体重链恒定区的多核苷酸的序列如SEQ ID NO:8所示。
  7. 一种表达权利要求6所述编码单克隆抗体重链和/或轻链的多核苷酸的载体。
  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Expi 293F细胞。
  9. 权利要求1-3任一所述的单克隆抗体在制备自身免疫性疾病治疗药物中的应用。
  10. 根据权利要求9所述的应用，其特征在于，所述自身免疫性疾病为红斑狼疮。
PCT/CN2021/073002 2020-02-11 2021-01-21 一种抗补体c3分子的全人源单克隆抗体及应用 WO2021159939A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202010085564.5A CN111171147B (zh) 2020-02-11 2020-02-11 一种抗补体c3分子的全人源单克隆抗体及应用
CN202010085564.5 2020-02-11

Publications (1)

Publication Number Publication Date
WO2021159939A1 true WO2021159939A1 (zh) 2021-08-19

Family

ID=70647739

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/073002 WO2021159939A1 (zh) 2020-02-11 2021-01-21 一种抗补体c3分子的全人源单克隆抗体及应用

Country Status (2)

Country Link
CN (1) CN111171147B (zh)
WO (1) WO2021159939A1 (zh)

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111171148B (zh) * 2020-02-11 2021-07-20 北京康普美特创新医药科技有限责任公司 一种抗补体c3分子的人源化单链抗体及其应用
CN111171147B (zh) * 2020-02-11 2021-07-20 北京康普美特创新医药科技有限责任公司 一种抗补体c3分子的全人源单克隆抗体及应用
CN115215937B (zh) * 2021-04-15 2023-05-26 上海麦济生物技术有限公司 抗人masp-2抗体及其制备方法和应用
TW202317642A (zh) * 2021-06-30 2023-05-01 中國大陸商蘇州創勝醫藥集團有限公司 新穎之抗ｍａｓｐ－２抗體

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101848937A (zh) * 2007-11-02 2010-09-29 诺瓦提斯公司 调节补体组分的分子和方法
WO2011144640A1 (en) * 2010-05-18 2011-11-24 Syddansk Universitet Novel c3c epitope, antibodies binding thereto, and use thereof
CN103249432A (zh) * 2010-06-22 2013-08-14 科罗拉多大学董事会,法人团体 补体成分3的C3d片段的抗体
CN104220453A (zh) * 2012-04-03 2014-12-17 诺沃姆德治疗公司 人源化的嵌合抗因子c3抗体及其用途
CN111171147A (zh) * 2020-02-11 2020-05-19 北京康普美特创新医药科技有限责任公司 一种抗补体c3分子的全人源单克隆抗体及应用

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101580546B (zh) * 2009-06-04 2011-09-14 中国疾病预防控制中心病毒病预防控制所 人源抗人干扰素α抗体及其应用
EP3511342B1 (en) * 2010-03-10 2024-01-17 Genmab A/S Monoclonal antibodies against c-met
UY35620A (es) * 2013-06-21 2015-01-30 Novartis Ag Anticuerpos del receptor 1 de ldl oxidado similar a lectina y métodos de uso
PT3331910T (pt) * 2015-08-03 2020-03-24 Engmab Sarl Anticorpos monoclonais contra o antigénio de maturação de células b (bcma) humano
CN110240652B (zh) * 2019-06-05 2022-09-06 百奥泰生物制药股份有限公司 抗补体d因子抗体及其应用

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101848937A (zh) * 2007-11-02 2010-09-29 诺瓦提斯公司 调节补体组分的分子和方法
WO2011144640A1 (en) * 2010-05-18 2011-11-24 Syddansk Universitet Novel c3c epitope, antibodies binding thereto, and use thereof
CN103249432A (zh) * 2010-06-22 2013-08-14 科罗拉多大学董事会,法人团体 补体成分3的C3d片段的抗体
CN104220453A (zh) * 2012-04-03 2014-12-17 诺沃姆德治疗公司 人源化的嵌合抗因子c3抗体及其用途
CN111171147A (zh) * 2020-02-11 2020-05-19 北京康普美特创新医药科技有限责任公司 一种抗补体c3分子的全人源单克隆抗体及应用

Also Published As

Publication number Publication date
CN111171147A (zh) 2020-05-19
CN111171147B (zh) 2021-07-20

Similar Documents

Publication Publication Date Title
WO2021159939A1 (zh) 一种抗补体c3分子的全人源单克隆抗体及应用
WO2021164521A1 (zh) 一种抗补体c5分子的全人源单克隆抗体及应用
WO2021159946A1 (zh) 一种抗补体c3分子的人源化单链抗体及其应用
US20220372113A1 (en) Nano antibody for neutralizing toxicity of sars-cov-2 and preparation method and application thereof
WO2021109968A1 (zh) 一种抗C3d的靶向单链抗体和CD59的融合蛋白及应用
WO2021109953A1 (zh) 一种抗C3d的靶向单链抗体和DAF的融合蛋白及应用
CN109575132B (zh) 人源抗补体C3d分子的单链抗体及其应用
JPH10500289A (ja) 糸球体腎炎および他の炎症性疾患の治療のための組成物および方法
RU2742248C2 (ru) Однодоменные антитела, направленные против внутриклеточных антигенов
JP2014530360A (ja) 診断マーカーとしてのｏｘＭＩＦ
EP3992205A1 (en) Sars coronavirus-2 spike protein binding compounds
WO2021164527A1 (zh) 一种抗补体c5分子的人源化单链抗体及其应用
Abou Faycal et al. An adapted passive model of anti-MPO dependent crescentic glomerulonephritis reveals matrix dysregulation and is amenable to modulation by CXCR4 inhibition
WO2021004494A1 (zh) 低免疫原性低adcc/cdc功能的抗c5人源化单抗及其应用
TWI605059B (zh) Inflammatory disease treatment agent
CN110256562B (zh) Pd-1纳米抗体、制备方法及其应用
WO2023143425A1 (zh) 改善认知障碍的方法
WO2021233433A1 (zh) 抗SARS-CoV-2刺突蛋白单克隆抗体
CN106084042B (zh) 一种全人源抗MAGEA1的全分子IgG抗体及其应用
WO2007097461A1 (ja) Ｃ型肝炎治療用抗体
BR102012011493A2 (pt) Método de produção e obtenção de fragmento fab do anticorpo anti-digoxina monoclonal a partir da técnica de clonagem em biologia molecular
CN117247451B (zh) 一种针对人白介素6蛋白的单域抗体及其应用
CN113354736B (zh) 靶向人pd-l1单克隆抗体或其抗原结合片段及其应用
CN115850452A (zh) 一种抗非洲猪瘟病毒的单克隆抗体及其衍生产品和应用
AU2022379455A1 (en) Methods of treating the effects of cytokine storms

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21753668

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21753668

Country of ref document: EP

Kind code of ref document: A1