WO2021109953A1 - 一种抗C3d的靶向单链抗体和DAF的融合蛋白及应用 - Google Patents

一种抗C3d的靶向单链抗体和DAF的融合蛋白及应用 Download PDF

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WO2021109953A1
WO2021109953A1 PCT/CN2020/132588 CN2020132588W WO2021109953A1 WO 2021109953 A1 WO2021109953 A1 WO 2021109953A1 CN 2020132588 W CN2020132588 W CN 2020132588W WO 2021109953 A1 WO2021109953 A1 WO 2021109953A1
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fusion protein
antibody
daf
complement
scfv
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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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    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70596—Molecules with a "CD"-designation not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • 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
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    • 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]
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
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    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the invention relates to an antibody and a fusion protein, belonging to the technical field of polypeptides.
  • the complement system is composed of more than 30 soluble protein molecules and is part of the natural immune system. Its components include more than 30 molecules such as the inherent components of complement, various 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 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.
  • the components involved in the classical activation pathway of complement include C1-C9. According to their role in the activation process, they are artificially divided into three groups, namely recognition units (Clq, Clr, Cls), activation units (C4, C2, C3) and membrane attack units (C5-C9). The phases play a role in the recognition phase, the activation phase and the membrane attack phase.
  • the difference between the alternative activation pathway and the classical activation pathway is that activation bypasses the three components of C1, C4, and C2, directly activates C3 and then completes the chain reaction of each component from C5 to C9.
  • Mannan-Binding Lectin (MBL) in the plasma directly recognizes N-galactosamine or mannose on the surface of a variety of pathogenic microorganisms, and then activates MASP-1 and MASP-2 in turn.
  • the above three pathways can produce C3 invertase.
  • C3 molecules are cleaved by C3 invertase into anaphylactoxin C3a and C3b with opsonization.
  • C3b molecules can be covalently linked to amine groups and hydroxyl groups on the surface of glycoproteins.
  • C3b molecules can be adsorbed on the surface of microorganisms that invade the body, and then bind to type I complement receptors (CR1/CD35), and hydrolyze under the action of serum factors H and I to form iC3b, which is then cleaved into C3d.
  • the C3d fragment is the smallest fragment of complement C3 that can no longer be digested.
  • CD21, CD19, CD81 and CD225 (Leu-13) on the surface of B cells are linked by non-covalent bonds to form the activation co-receptors of B cells.
  • CD21 The extracellular region of CD21 binds to the antigen-bound complement component C3d, and the CD19 transmits stimulating signals to the cytoplasm, which can significantly reduce the threshold for antigen activation of B cells.
  • CD21 is also called type II complement receptor (CR2), which is mainly distributed on the surface of follicular dendritic cells (FDC), B cells and some T cells.
  • the decay accelerating factor (DAF, CD55) is composed of 4 SCRs plus a Ser/Thr rich region, so O-linked glycosylation can be extensively performed. DAF can be anchored to the cell membrane through a glycosyl phosphatidyl alcohol (GPI), and through the combination of its SCR and C4b/C3b, it accelerates the decay of the classical and alternative pathways C3/C5 convertase.
  • GPI glycosyl phosphatidyl alcohol
  • This treatment idea is based on the specific recognition of exogenous protein molecules as receptors and C3d molecules, thereby preventing the binding and activation of C3d molecules and CR2 during pathological processes.
  • this method still has certain limitations, for example, due to The molecular weight of the fusion protein of the targeting molecule and the inhibitor is too large, which increases the difficulty of the fusion protein to reach the lesion, especially in some hidden parts. It also has the disadvantages of reduced affinity and prolonged half-life, and even leads to unnecessary immunogens. Sex.
  • Single-chain antibodies are small-molecule antibodies prepared by genetic engineering methods. They are formed by connecting the variable region of the heavy chain (VH) of the antibody with the variable region of the light chain (VL) by a flexible connecting peptide (usually 12-15 amino acids). The resulting recombinant antibody has a molecular weight equivalent to 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 and is a parent antibody Small fragments of antigen binding activity, which can make it reach the focus tissue that is difficult to reach by conventional antibodies.
  • single-chain antibodies do not contain the Fc segment, they will not bind to Fc receptors on unrelated cells, avoiding immune complex reactions caused by the Fc segment. Therefore, inhibiting the binding of C3d and CR2 by anti-C3d antibodies, thereby exerting a specific inhibitory effect on complement activation, is a new treatment idea.
  • Chinese invention patent application CN109575132A discloses an anti-C3d antibody single-chain antibody, showing that the anti-C3d antibody has a certain technical effect in targeting inflammatory lesions and reducing the inflammatory response caused by complement activation.
  • anti-C3d antibodies can not only block the binding of C3d and CR2 Blockers, but also can target more potent complement inhibitors to inflammatory lesions to produce a synergistic effect that inhibits complement activation.
  • the prior art anti-C3d antibody single-chain antibody is still difficult to meet this technical requirement.
  • the fusion of anti-C3d antibody and different inhibitors will increase the molecular weight of the protein, which will change the spatial structure of the remodeled protein, thereby affecting Targeting effect of anti-C3d antibody.
  • the purpose of the present invention is to provide a new anti-C3d single-chain antibody with excellent targeting performance and a fusion protein of anti-C3d single-chain antibody and complement inhibitor DAF, so that the anti-C3d single-chain antibody is not only a blocking agent It blocks the binding of C3d and CR2, and can also target the complement inhibitor DAF to inflammatory lesions to produce excellent therapeutic effects.
  • the present invention first provides a human-derived anti-complement C3d molecule single-chain antibody.
  • 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.2.
  • the amino acid sequences at positions -35, 51-57, and 89-98 are shown in the amino acid sequences of CDR1, CDR2, and CDR3 of the variable region of the antibody heavy chain, respectively, as shown in SEQ ID NO. 4, 30-35, 50-66 and The amino acid sequence at positions 99-107 is shown.
  • amino acid sequence of the variable region of the antibody light chain is shown in SEQ ID NO.2
  • amino acid sequence of the variable region of the antibody heavy chain is shown in SEQ ID NO.4.
  • variable region of the light chain of the antibody and the variable region of the heavy chain are connected by a flexible polypeptide with an amino acid sequence as shown in SEQ ID NO.6.
  • the present invention also provides a fusion protein containing the aforementioned single-chain antibody, and the fusion protein also contains a complement activity regulator.
  • the complement activity regulator is a DAF molecule.
  • the single-chain antibody and the DAF molecule are linked with the flexible polypeptide shown in SEQ ID NO.8.
  • amino acid sequence of the fusion protein is as described in SEQ ID NO.10.
  • the present invention also provides a polynucleotide encoding the above-mentioned fusion protein, and the sequence of the polynucleotide is as described in SEQ ID NO.9.
  • the present invention provides the application of the above-mentioned fusion protein in the preparation of drugs for the treatment of autoimmune diseases.
  • the disease includes rheumatoid arthritis and systemic lupus erythematosus.
  • the fusion protein of the anti-C3d targeting single-chain antibody and DAF provided by the present invention has excellent antigen binding activity, and has a very high inhibitory efficiency for complement-mediated inhibition of CHO cell and red blood cell lysis. Biological distribution experiments have proved that the fusion protein provided by the present invention can quickly accumulate in the arthritis part after entering the rheumatoid arthritis mouse model, and has excellent anti-adhesion/anti-inflammatory targeting inhibition effect.
  • the fusion protein provided by the present invention can significantly improve the survival rate of mice, and the proteinuria, glomerular integral, interstitial inflammation, vasculitis and crescent of the treatment group Symptoms such as necrosis and necrosis are significantly improved, which shows that the fusion protein provided by the present invention has excellent application prospects in the preparation of autoimmune disease therapeutic drugs.
  • the following methods are used to screen anti-C3d single-chain antibodies.
  • the specific method includes the following steps:
  • Trizol reagent (Invitrogen) was used to extract the total RNA of the cells from the isolated human peripheral blood lymphocytes, and then mixed in the same ratio.
  • CDNA was reverse transcribed using cDNA Reverse Transcription Kit (Takara Company). The above steps are carried out in accordance with the instructions provided by the manufacturer.
  • the amplification system consists of 2 ⁇ l each of the upstream primer and downstream primer, 5 ⁇ l of 10 ⁇ PCR buffer, and a final concentration of dNTP of 2.5mM. After mixing, denature at 100°C for 5min, and add 2 units of Taq DNA polymerase. The total reaction volume is 50 ⁇ l. After mixing, add mineral oil. Carry out 30 cycles of reaction, each cycle conditions are: 94 °C denaturation 30s. Anneal at 55°C for 90s, and extend at 72°C for 90s. After the reaction proceeds to the last cycle, it is kept at 72°C for 10 minutes. After the reaction, 3 ⁇ l of the light chain and heavy chain variable region gene amplification products were taken out and subjected to 1.2% agarose electrophoresis detection.
  • variable region gene and the 357bp heavy chain variable region gene were obtained by PCR amplification.
  • the variable region genes were consistent with the expected results, and the rest were recovered and purified with Sephagels TM Bandprep Kit (Pharmacia).
  • the recovered heavy chain and light chain variable region genes are connected by a Linker sequence to form the ScFv gene.
  • the nucleotide sequence and amino acid sequence of the Linker sequence are shown in SEQ ID NO. 5 and 6, respectively.
  • the PCR amplification system consists of 2 ⁇ l of upstream primer and 2 ⁇ l of downstream primer, 2.5mM dNTP and 3 units of Taq DNA polymerase, 5 ⁇ l of 10 ⁇ PCR buffer, and a reaction volume of 50 ⁇ l. Carry out 30 PCR cycles, each cycle conditions are 94 °C denaturation 1 min, 55 °C annealing 2 min, 72 °C extension 2 min, the reaction proceeds to the last cycle and then kept at 72 °C 10 min. After the reaction, 3 ⁇ L 1.2% agarose electrophoresis test was taken from the PCR amplified product. The 750bp single-chain antibody gene was obtained by PCR amplification, which was consistent with the expected result. The rest was recovered and purified with SephagelsTM Bandprep Kit.
  • the enzyme-linked plate was coated with C3d 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 positive clones whose light absorption value was 2.1 times higher than that of the negative control were selected, and the positive clones with the strongest C3d binding activity were selected.
  • DNA sequence analysis of the positive cloned recombinant plasmid The DNA sequence of the anti-C3d single-chain antibody on the positive recombinant plasmid was determined with the T7 DNA sequence TAATACGACTCACTATAGGG, and the result was that the sequence consisted of 750 bases.
  • the light chain variable region coding gene has the DNA sequence of SEQ ID NO: 1 in the sequence list
  • the flexible polypeptide coding gene as a linker has the DNA sequence of SEQ ID NO: 5 in the sequence list
  • the heavy chain can be
  • the gene encoding the variable region has the DNA sequence of SEQ ID NO: 3 in the sequence list
  • the DNA sequence of the anti-C3d single-chain antibody encodes the amino acid residues of SEQ ID NO: 2-SEQ ID NO: 6-SEQ ID NO: 4 in the sequence list.
  • the base sequence of the protein, the antibody is named VL- Linker- VH (also known as ScFv).
  • step 1.7 to obtain the single-chain antibody against C3d V L -Linker-V H fusion gene was cloned into eukaryotic expression vector pEE14 .1 (Lonza) to give an anti-C3d carrying single chain antibody V L -Linker-V H gene fusion recombinant expression vector designated pEE14.1 / V L -Linker-V H .
  • pEE14.1 / V L -Linker-V H 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, and the clones were transferred to a 24-well plate with a cloning ring, and 1 mL of selective medium DMEM+10% FCS+25 ⁇ m MSX was added 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 enzyme-linked plate coated with C3d antigen, 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 target protein of about 26KD was obtained after expression, and the protein could specifically bind to C3d, which was consistent with the expected results, indicating that a highly pure anti-C3d single-chain antibody was obtained.
  • the expression vector is pEE14.1 (Lonza biologics); CHO cells are used for protein expression, and the culture medium is DMEM containing 10% fetal bovine serum, purchased from Invitrogen.
  • Mouse anti-DAF mAb 1H4 and 1A10, mouse anti-human CR2mAb 171, anti-goat red blood cell IgM and all secondary antibodies were purchased from Sigma.
  • the cDNA structural gene is formed by linking the sequence encoding the anti-C3d single-chain antibody with the sequence encoding the extracellular region of DAF.
  • the complement inhibitor sequence is a nucleotide sequence encoding 1-250 amino acid residues of the mature DAF protein sequence (Swissprot accession number P08174).
  • connection sequence is GGGSGGGGS (SEQ ID NO: 8).
  • the gene frame is synthesized by PCR technology (the amino acid sequence of the fusion protein is shown in SEQ ID NO. 10, and the nucleotide sequence is shown in SEQ ID NO. 9). All cloning steps were performed on the pEE14.1 vector (see CN104940953A).
  • the pEE14.1 high-efficiency expression vector contains a special glutamine (GS) gene expression system. GS enzyme is responsible for the synthesis of glutamine using glutamyl and amine as substrates.
  • the transfected GS gene can be used as a selection marker to select cells grown in glutamine-free medium.
  • Methionine sulphoximine (MSX) can inhibit the activity of GS enzyme, and some cell lines (CHO-K1) can produce endogenous glutamine, which can be used in glutamine-free medium containing MSX For growth, this medium with a sufficient amount of MSX to inhibit GS enzyme activity provides screening pressure to screen out the required cell lines.
  • the recombinant vector pEE14.1-ScFv-DAF was transfected into CHO-K1 cells with FuGENE 6 and 24 hours after transfection Change the selective DMEM medium (without glutamine), and add MSX to a final concentration of 50 ⁇ M, and positive clones will grow in about 2 weeks, but normal CHO cells without transfection plasmid gradually fall off the bottle wall, and the cells are lysed and died .
  • Three cell lines were selected and cultured in serum-containing medium and serum-free medium for 3 days respectively. The cell supernatant was collected, and part of it was concentrated 5 times with PEG20000 for use, and the rest were stored at -80°C.
  • the expression of ScFv-DAF was detected by ELISA method.
  • the recombinant protein is expressed in CHO cells in a secreted form.
  • the expression level of the stable expression strain of CHO cells cultured in serum-free medium was higher than that of cells cultured in serum-containing medium.
  • the chromatography column is formed by coupling the anti-DAF mAb (1H4) with the affinity column activated by HiTrap NHS.
  • the pH value of the culture supernatant containing the recombinant protein was adjusted to 8.0, and the column was passed through the column at a rate of 0.5 mL/min. Then the column was washed with 6 to 8 times the volume of PBS, and the recombinant protein was eluted with 2 to 3 times the column volume of 0.1 mol/L glycine (pH 2.4).
  • the fusion protein was collected in 1 mol/L Tris buffer (pH 8.0) and dialyzed in PBS.
  • the purified protein was carried out in SDS-PAGE gel containing 100g/L. The gel was stained with Coomassie Brilliant Blue.
  • mouse anti-DAF mAb 1H4 was used to detect recombinant proteins.
  • C3d-ScFV-CD59 with a concentration of 10 ⁇ g/ml and C3d-ScFV-DAF with a concentration of 20 ⁇ g/ml are used as the primary antibody and added to the 1 and 2 antigen tanks respectively, and the 3 and 4 antigen tanks are added without the primary antibody TBST skimmed milk powder, incubate at room temperature for 2h;
  • Anti-CD59 (rabbit monoclonal antibody) antibody, diluted according to the ratio of 1:5000, added to the 1 and 3 antigen tanks respectively, anti-DAF (murine monoclonal antibody) antibody, diluted according to the ratio of 1:5000, the decibel was added to 2 and Incubate in No. 4 antigen tank for 1 hour at room temperature;
  • the recombinant protein ScFv-DAF was isolated from the culture supernatant of stably transfected CHO cells, 100 ⁇ 200 ⁇ g/L.
  • the results of SDS-PAGE ( Figure 1) and Western blot ( Figure 2) showed that the target fragment (ScFv- The relative molecular weight of the DAF fusion protein is approximately 55KDa).
  • C3dg-biotin The kinetic analysis of the interaction between ScFv-DAF and biotin-labeled C3dg (C3dg-biotin) was detected with a surface cytoplasmic genome resonance (SPR) detection system (BIAcore 3000 instrument). According to the average amount of 50mg/L per flow cell, human C3dg-biotin (Guthridge, JM, et al.
  • the kinetic analysis data shows that 1:1 is the most suitable sphere detection parameter for the bonding reaction model.
  • SPR test results show that ScFv-DAF has a higher binding and dissociation rate than ScFv (Table 3). Moreover, the affinity of ScFv-DAF is higher than that of ScFv.
  • the kinetic parameters of the single-chain antibody of the anti-C3d antibody disclosed in the Chinese invention patent application CN109575132A were also measured.
  • the results showed that the Ka(1/Ms) of the antibody was 4.58 ⁇ 10 2 , Kd(1/s ) Is 0.023, and K D (nm) is 764 ⁇ 68.
  • the ScFv provided by the present invention greatly improves the kinetic parameters of C3dg-biotin binding.
  • the recombinant fusion protein 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.
  • mice After freeze-drying, it is diluted with PBS solution containing 1mg/mL BSA, aliquoted, and stored in a refrigerator at -80°C for later use.
  • Male DBA/1J mice were injected subcutaneously with 0.1 mL of collagen II and complete Freund’s adjuvant (both purchased from Sigma in the United States) at the base of the tail.
  • a mouse model of rheumatoid arthritis (CIA) was established (for model establishment, refer to C57BL). /6 The establishment of mouse CIA model and the preliminary screening of its monitoring system, Chinese Medical Journal of Chinese People's Liberation Army, June 2004, Vol. 29, No. 6, 472-474).
  • the test and grouping methods are as follows: 1 The control group was injected with 125 I-DAF (2ug) in the tail vein, and was killed by decapitation after 24h and 48h respectively. Blood was collected, tissues and organs were taken out, weighed and measured for radioactivity ( ⁇ Ci), and the results were converted into ID%/g organization. 2In the arthritis group, 125 I-DAF fusion protein (2ug) was injected into the tail vein. After 24h, 48h, 72h, and 96h, the same treatment and detection were taken. 3In the ScFv-DAF arthritis treatment group, 125 I-ScFv-DAF fusion protein (0.25ug) was injected into the tail vein once, and the same treatment and detection were taken 24 hours later.
  • Clinical scoring was started on the 23rd day, and the animal’s arthritis degree was scored according to the following criteria: 0 points, no arthritis; 1 point, slight inflammation and paw redness; 2 points, severe erythema and swelling, which affect paw function; 3 Points, the claws or joints are deformed, stiff, and lose function. The maximum total score of each mouse's limbs is 12 points.
  • the results are shown in Figure 4, starting from the 23rd day, the severity of arthritis in the four ScFv-DAF fusion protein treatment groups was significantly lower than that of the PBS group.
  • the score of group 5 (4 injections group) is less than 1/2 of group 4 (3 injections), and it is less than 1/4 of group 3 (ScFv-DAF 2 injections group), which is group 2 (ScFv-DAF 1 injection group) less than 1/5, and less than 1/7 of group 1 (PBS control group).
  • the ScFv-DAF fusion protein of the present invention has specific targeting to C3d, has a better anti-adhesion/anti-inflammatory targeting inhibitory effect, and has a good therapeutic effect on the body's inflammatory response, which is significantly higher than The therapeutic effect of ScFv-DAF.
  • 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 developed renal failure symptoms 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.
  • the experimental results are shown in Figure 5.
  • the ScFv-DAF high-dose treatment group can completely protect MRL/lpr lupus erythematosus mice during the whole treatment process, and the survival rate is 100%.
  • the ScFv-DAF low-dose treatment group can maintain a survival rate of more than 70% even in the 24th week. Compared with the control group, the survival rate of mice in the treatment group increased significantly from 18 weeks.
  • mice were placed in a metabolic cage to study the effect of ScFv-DAF on the urine albumin secretion of 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. Use mouse albumin samples of known concentrations to draw a standard curve by ELISA method, and determine the urine albumin secretion of experimental mice, and use a biochemical analyzer to determine the creatinine content in 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 ScFv-DAF treatment group had significantly lower glomerular scores, interstitial inflammation, vasculitis, and crescent/necrosis than the control group (P ⁇ 0.05).
  • the present invention provides a human-derived anti-complement C3d molecule single-chain antibody, a fusion protein of the single-chain antibody and the complement inhibitor DAF, and its application in the preparation of autoimmune disease therapeutic drugs.
  • the single-chain antibody provided by the present invention Antibodies and fusion proteins are easy to industrially produce and have industrial applicability.

Abstract

本发明公开了一种人源抗补体C3d分子的单链抗体以及所述单链抗体与补体抑制剂DAF的融合蛋白，所述抗体具有优异的抗原结合活性，生物学分布实验证明，本发明提供的融合蛋白进入类风湿关节炎小鼠模型后可快速在关节炎部位高度聚集，具有优异的抗黏附/抗炎靶向抑制效应。在对MRL/lpr红斑狼疮小鼠的治疗中，本发明提供的融合蛋白能够明显提升小鼠的存活率，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/坏死等症状明显得到改善，显示本发明提供的融合蛋白在制备自身免疫性疾病治疗药物中具有优异的应用前景。

Description

一种抗C3d的靶向单链抗体和DAF的融合蛋白及应用 技术领域
本发明涉及一种抗体及融合蛋白，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。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。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自身免疫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、系统性红斑狼疮、肾小球肾炎等。
补体激活的途径有3个，即经典途径、旁路途径和甘露聚糖结合凝集途径。参与补体经典激活途径的成分包括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。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。甘露聚糖结合凝集途径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。以上三种途径均能产生C3转化酶，C3分子被C3转化酶裂解为过敏毒素C3a以及具有调理作用的C3b，C3b分子能与糖蛋白表面的胺基与羟基共价连接，这种共价作用由C3b分子内部的硫酯基所介导。因而，C3b分子可吸附于侵入体内的微生物表面，再与I型补体受体(CR1/CD35)结合，在血清H因子和I因子的作用下水解形成iC3b，iC3b随后被裂解为C3d。C3d片段是补体C3的不能再酶解的最小片段。B细胞表面的CD21、CD19、CD81及CD225(Leu-13)以非共价键相联，形成B细胞的活化辅助受体。CD21的胞外区与结合于抗原的补体成分C3d结合，由CD19向胞浆传递刺激信号，可明显降低抗原激活B细胞的阈值。CD21也称为Ⅱ型补体受体(CR2)，其主要分布于滤泡树突状细胞(FDC)、B细胞和一些T细胞表面。
鉴于C3d分子和作为其受体的CR2在补体激活途径及体液免疫和细胞免疫激活进程中所扮演的重要角色，研究者开始关注阻断或者竞争性抑制C3d分子与CR2的结合进而下调及抑制补体激活以阻断自身免疫性疾病病理进程。将具有特异性靶向作用但不具有补体抑制作用的补体受体2与补体抑制剂融合，从而对补体激活发挥特异性抑制作用是自身免疫性疾病治疗的一个技术手段。中国发明专利CN101563363B和CN100594037C分别报道了补体受体2与补体抑制剂F因子、衰变加速因子或CD59的融合蛋白所表现出的补体抑制活性。衰变加速因子(DAF，CD55)是由4个SCRs加上一个Ser/Thr富含区组成，所以能够广泛进行O-连接的糖基化。DAF可通过一个糖基磷脂酰基醇(GPI)锚定在细胞膜上，并通过其SCR与C4b/C3b的结合，加速经典和替代途径C3/C5转化酶的衰变。该治疗思路是基于外源性蛋白质分子作为受体和C3d分子的特异性识别，从而阻止病理过程中C3d分子与CR2的结合和激活，在实际应用中该手段仍然具有一定的局限性，例如由于靶向分子和抑制剂的融合蛋白分子量过大而增加了融合蛋白到达病灶的难度，尤其是一些比较隐蔽的部位，而且还会产生亲和力降低，半衰期延长的弊端，甚至导致产生不必要的免疫原性。
单链抗体是用基因工程方法制备的小分子抗体，是由弹性连接肽(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。而且由于单链抗体不含有Fc段，因此不会跟无关细胞上的Fc受体结合，避免了Fc段所致的免疫复合物反应。因此，通过抗C3d抗体抑制C3d与CR2结合，从而对补体激活发挥特异性抑制作用是一种新的治疗思路。
技术问题
中国发明专利申请CN109575132A公开了一种抗C3d抗体的单链抗体，显示抗C3d抗体在靶向炎性病灶，降低补体激活造成的炎性反应具有一定的技术效果。但是，基于靶向治疗的要求，本领域仍然需要充分发挥抗C3d抗体的靶向功能，加强对补体激活途径的抑制效应的技术方案，即，抗C3d抗体不仅能够作为阻断C3d和CR2结合的阻断剂，而且还能够将更为有力的补体抑制剂靶向到炎症病灶，以产生抑制补体激活的协同效应。然而现有技术的抗C3d抗体的单链抗体还难以达到这种技术需要，抗C3d抗体与不同抑制剂的融合将会增加蛋白质的分子量，使得重构后的蛋白空间结构发生改变，从而影响了抗C3d抗体的靶向效应。
因此，本发明的目的就是提供一种新的具有优异靶向性能的抗C3d单链抗体以及抗C3d单链抗体与补体抑制剂DAF的融合蛋白，使得抗C3d单链抗体不仅是作为阻断剂阻断C3d和CR2结合，而且还能够将补体抑制剂DAF靶向至炎性病灶从而产生优异的治疗效果。
技术解决方案
基于上述发明目的，本发明首先提供了一种人源抗补体C3d分子的单链抗体，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.2的第25-35、51-57和89-98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.4的第30-35、50-66和99-107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.2所示，所述抗体重链可变区的氨基酸序列如SEQ ID NO.4所示。
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.6所示的柔性多肽连接。
其次，本发明还提供了一种含有上述单链抗体的融合蛋白，所述融合蛋白还含有补体活性调节剂。
在一个优选的实施方案中，所述补体活性调节剂为DAF分子。
在一个更为优选的实施方案中，所述单链抗体与DAF分子以SEQ ID NO.8所示的柔性多肽连接。
更为优选地，所述融合蛋白的氨基酸序列如SEQ ID NO.10所述。
第三，本发明还提供了一种编码上述融合蛋白的多核苷酸，所述多核苷酸的序列如SEQ ID NO.9所述。
最后，本发明提供了上述融合蛋白在制备自身免疫性疾病治疗药物中的应用。
在一个优选的实施方案中，所述疾病包括类风湿性关节炎和系统性红斑狼疮。
技术效果
本发明提供的抗C3d的靶向单链抗体和DAF的融合蛋白具有优异的抗原结合活性，对补体介导的CHO细胞和红细胞裂解抑制具有非常高的抑制效率。生物学分布实验证明，本发明提供的融合蛋白进入类风湿关节炎小鼠模型后可快速在关节炎部位高度聚集，具有优异的抗黏附/抗炎靶向抑制效应。在对MRL/lpr红斑狼疮小鼠的治疗中，本发明提供的融合蛋白能够明显提升小鼠的存活率，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/坏死等症状明显得到改善，显示本发明提供的融合蛋白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.ScFv-DAF的12％SDS-PAGE鉴定图；
图2.ScFv-DAF的Western Blot鉴定图；
图3.ScFv-DAF在RA小鼠的生物学分布柱状分析图；
图4.ScFv-DAF治疗RA小鼠的临床评分对照图；
图5.ScFv-DAF治疗MRL/lpr小鼠的存活率对照图；
图6.ScFv-DAF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，并不对本发明的保护范围构成任何限制。
实施例1、抗C3d单链抗体的制备
用下述方法筛选抗C3d单链抗体，具体方法包括以下步骤：
1.1 cDNA的制备
收集50份健康人的外周血各20ml，分别用淋巴细胞分离液(天津血研所)分离单个核细胞。用Trizol试剂(Invitrogen公司)从分离的人外周血淋巴细胞中提取细胞的总RNA，后按照相同的比例混合。用cDNA反转录试剂盒(Takara公司)反转录出cDNA。以上步骤按照厂家提供的说明书进行。
1.2抗体轻链和重链可变区基因的扩增：用PCR方法，以反转录合成的cDNA为模板，在PCR反应体系中分别加入扩增轻链和重链可变区基因的引物。根据GenBank中人类抗体基因序列，分别设计PCR扩增抗体重链、轻链可变区所需的引物(见表1)。
表1.抗体轻链和重链可变区基因的扩增引物
引物名称 序列
VHup1 CAGGTGCAGCTGCAGGAGTCSG
VHup2 CAGGTACAGCTGCAGCAGTCA
VHup3 CAGGTGCAGCTACAGCAGTGGG
VHup4 GAGGTGCAGCTGKTGGAGWCY
VHup5 CAGGTCCAGCTKGTRCAGTCTGG
VHup6 CAGRTCACCTTGAAGGAGTCTG
VHup7 CAGGTGCAGC TGGTGSARTCTGG
VHdown1 TGAGGAGACRGTGACCAGGGTG
VHdown2 TGAAGAGACG GTGACCATTGT
VH down3 TGAGGAGACG GTGACCAGGGTT
VH down4 TGAGGAGACG GTGACCGTGGTCC
VLup1 CAGTCTGTSBTGACGCAGCCGCC
VLup2 TCCTATGWGCTGACWCAGCCAC
VLup3 TCCTATGAGCTGAYRCAGCYACC
VLup4 CAGCCTGTGCTGACTCARYC
VLup5 CAGDCTGTGGTGACYCAGGAGCC
VLup6 CAGCCWGKGCTGACTCAGCCMCC
VLup7 TCCTCTGAGCTGASTCAGGASCC
VLup8 CAGTCTGYYCTGAYTCAGCCT
VLup9 AATTTTATGCTGACTCAGCCCC
VL down 1 TAGGACGGTSASCTTGGTCC
VL down 1 GAGGACGGTC AGCTGGGTGC
VKup1 GACATCCRGDTGACCCAGTCTCC
VKup2 GAAATTGTRWTGACRCAGTCTCC
VKup3 GATATTGTGMTGACBCAGWCTCC
VKup4 GAAACGACACTCACGCAGTCTC
VKdown1 TTTGATTTCCACCTTGGTCC
VKdown2 TTTGATCTCCASCTTGGTCC
VKdown3 TTTGATATCCACTTTGGTCC
VKdown4 TTTAA TCTCCAGTCGTGTCC
表1中的简并碱基B＝C或G，D＝G或A,K＝G或T，M＝A或C，R＝A或G，S＝G或C，W＝A或T，Y＝C或T。
扩增体系为上游引物和下游引物各2μl，10×PCR缓冲液5μl，dNTP终浓度为2.5mM，混匀后经100℃变性5min，加入2单位Taq DNA聚合酶。总反应体积为50μl。混匀后加矿物油。进行30个循环反应，每个循环条件是： 94℃变性30s。55℃退火90s，72℃延伸90s，反应进行到最后一个循环后在72℃保温10min。反应结束后，分别从轻链和重链可变区基因扩增产物中取出3μl进行1.2％的琼脂糖电泳检测，经PCR扩增获得了327bp的轻链可变区基因和357bp的重链可变区基因，与预期结果相符，其余的用Sephagels TM Bandprep Kit(Pharmacia)回收纯化。
1.3单链抗体基因的连接：将回收的重链和轻链可变区基因中间通过一段Linker序列连接起来，形成ScFv基因。Linker序列的核苷酸序列和氨基酸序列分别如SEQ ID NO.5和6所示。在抗体轻链和重链可变区基因的扩增引物5’端分别加上SfiI和NotI的酶切位点和数个保护碱基，3’端分别加上Linker序列(引物序列见表2)。
表2.用于抗体轻链和重链可变区基因连接的扩增引物
Figure PCTCN2020132588-appb-000001
Figure PCTCN2020132588-appb-000002
Figure PCTCN2020132588-appb-000003
PCR扩增体系为上游引物和下游引物各2μl，加入2.5mM的dNTP和3个单位的Taq DNA聚合酶，10×PCR缓冲液5μl，反应体积为50μl。进行30次PCR循环，每个循环条件为94℃变性1min，55℃退火2min，72℃延伸2min，反应进行到最后一个循环后在72℃保温10min。反应结束后从PCR扩增产物中取出3μL 1.2％的琼脂糖电泳检测，经PCR扩增获得了750bp的单链抗体基因，与预期结果相符，其余的用SephagelsTM Bandprep Kit回收纯化。
1.4单链抗体文库的构建及表达：将表达载体pCANTAB5E(Pharmacia)和回收后的单链抗体基因分别经Sfi 1和Not 1双酶切，在T4连接酶的作用下，将pCANTAB5E表达载体和单链抗体基因16℃连接过夜。将重组质粒转化大肠杆菌TG1感受态细胞，并把转化的细胞涂布在含100μg/mL氨苄青霉素LB培养板上，于37℃培养过夜。把生长在平板上的转化菌全部收集下来，用2×YTAG(含100μg/mL氨苄青霉素和2％葡萄糖)稀释细胞悬液至OD 600＝0.2，37℃培养培养至对数生长期(约OD 600＝0.4)，加入2×10 9pfu M13K07辅助噬菌体，37℃培养1小时，离心。用10mL2×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，37℃振荡培养过夜，离心后收集含有重组噬菌体的上清，即为 单链抗体噬菌体表达文库。
1.5重组噬菌体抗体的筛选：用C3d抗原(上海史瑞克生物科技有限公司)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。
1.6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100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C3d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C3d结合活性最强的阳性克隆。
1.7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C3d单链抗体的DNA序列，结果该序列由750个碱基组成。自5’端起，轻链可变区编码基因具有序列表中SEQ ID NO：1的DNA序列，作为连接子的柔性多肽 编码基因具有序列表中SEQ ID NO：5的DNA序列，重链可变区编码基因具有序列表中SEQ ID NO：3的DNA序列，抗C3d单链抗体的DNA序列编码具有序列表中SEQ ID NO：2-SEQ ID NO：6-SEQ ID NO：4的氨基酸残基序列的蛋白质，将该抗体命名为V L-Linker-V H(亦称ScFv)。
1.8抗C3d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
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1.7获得的抗C3d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C3d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C3d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C3d结合活性最强的阳性克隆，即为高效表达抗C3d单链抗体的CHO细胞株。
1.9抗C3d单链抗体的纯化
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，经表达获得了约26KD的目的蛋白，且该蛋白可与C3d特异结合，与预期结果相符，表明获得了纯度 很高的抗C3d单链抗体。
实施例2抗C3d单链抗体-DAF(ScFv-DAF)融合蛋白制备
1材料 表达载体选用pEE14.1(Lonza biologics)；CHO细胞用于蛋白表达，其培养液为含10％胎牛血清的DMEM，购自Invitrogen公司。鼠抗DAF mAb 1H4和1A10，鼠抗人CR2mAb 171，抗羊红细胞IgM及所有二抗均购自Sigma公司。
2方法
2.1兔抗CHO细胞膜和人DAF的抗血清的制备按照文献Harlow,E.,and Lane,D.Antibodies:a laboratory manual.Cold Spring Harbor Laboratory.Cold Spring Harbor,New York,USA.1988:726.介绍的方法获得。
2.2表达重组体的构建及蛋白表达cDNA结构基因由编码抗C3d单链抗体的序列与编码DAF编码胞外区的序列相连接而成。补体抑制物序列是编码成熟DAF蛋白序列1-250个氨基酸残基的核苷酸序列(Swissprot登录号为P08174)。
然后将抗C3d单链抗体与补体抑制物DAF连接，抗C3d单链抗体在N-端时(ScFv-DAF)连接序列为GGGSGGGGS(SEQ ID NO：8)。基因框架用PCR技术合成(融合蛋白的氨基酸序列如SEQ ID NO.10所示，核苷酸序列如SEQ ID NO.9所示)。所有克隆步骤均在pEE14.1载体上进行(参见CN104940953A)。pEE14.1高效表达载体含有特殊的谷氨酰胺(GS)基因表达系统。GS酶负责用谷氨酰和胺作为底物合成谷氨酰胺。一些哺乳动物细胞系不含GS基因，所以在没加谷氨酰胺的环境下不能存活。对于这些细胞系而言，转染的GS基因可以作为一种筛选标记，能筛选出无谷氨酰胺培养基中生长的细胞。甲硫氨酸亚胺(Methionine sulphoximine,MSX)能抑制GS酶的活性，某些细胞系(CHO-K1)能产生内源性谷氨酰胺，就能在含有MSX的无谷氨酰胺培养基中生长，这种加有足量MSX能抑制GS酶活性的培养基提供筛选压力，从而筛选出所需要的细胞株。ScFv-DAF融合基因经PCR方法合成后与pEE14.1载体均经过EcoRI、SmalI双酶切后连接，重组载体pEE14.1-ScFv-DAF用FuGENE 6转染CHO-K1细胞，转染24小时后，换选择性DMEM培养 基(不含谷氨酰胺)，并加入MSX至终浓度为50μM，约2周长出阳性克隆，而没有转染质粒的正常CHO细胞逐渐从瓶壁脱落，细胞裂解死亡。选择3株细胞，分别用有血清培养基及无血清培养基培养3天，收集细胞上清，部分经PEG20000浓缩5倍备用，其余-80℃保存。以ELISA方法检测ScFv-DAF的表达。重组蛋白以分泌形式在CHO细胞中表达。采用无血清培养基培养的CHO细胞稳定表达株的表达量高于采用含血清的培养基培养的细胞。
2.3重组蛋白的纯化利用亲和色谱法对细胞培养物上清中的蛋白进行纯化。按照操作手册，层析柱通过将抗DAF mAb(1H4)与HiTrap NHS活化的亲和柱偶联而成。将含重组蛋白培养上清的pH值调至8.0，以0.5mL/min的速率过柱。然后以6～8倍体积PBS洗柱子，重组蛋白以2～3倍柱体积的0.1mol/L甘氨酸(pH2.4)洗脱。将融合蛋白收集到1mol/L Tris缓冲液(pH8.0)中，并在PBS中透析。
2.4 SDS-PAGE和Western blot鉴定
纯化的蛋白在含100g/L的SDS-PAGE胶中进行。凝胶用考马斯亮蓝染色。
Western blot实验中用鼠抗DAF mAb 1H4对重组蛋白进行检测。
步骤：
①5％的浓缩胶，10％的分离胶；
②1μl的1mg/ml抗原进行SDS-PAGE，70V浓缩胶，130V分离胶；
③转印到PVDF膜上，转印时间24min；
④5％脱脂奶粉，0.1％Tween20的TBS封闭2h；
⑤10ml浓度为10μg/ml的C3d-ScFV-CD59,20μg/ml的C3d-ScFV-DAF的抗体作为一抗，分别加入到1和2抗原槽中，3和4抗原槽中加入不含有一抗的TBST脱脂奶粉，室温孵育2h；
⑥TBST洗涤5次，5min/次；
⑦抗CD59(兔单抗)的抗体，按照1:5000比例稀释，分别加入1和3号抗原槽中，抗DAF(鼠单抗)的抗体，按照1:5000比例稀释，分贝加入到2和4号抗原槽中，室温孵育1h；
⑧TBST洗涤5次，5min/次；
⑨山羊抗兔的抗体以及山羊抗鼠的抗体，按照1:5000稀释，分别加入到1和3号抗原槽以及2和4号抗原槽中，室温孵育1h；
⑩TBST洗涤5次，5min/次；
Figure PCTCN2020132588-appb-000004
显色。
3.结果
从稳定转染的CHO细胞培养上清中分离到重组蛋白ScFv–DAF，100～200μg/L，SDS-PAGE(图1)和Western blot(图2)结果显示出现预期大小的目标片段(ScFv-DAF融合蛋白的相对分子量大约为55KDa)。
实施例3.ScFv-DAF与C3配基相互作用动力学分析
ScFv-DAF与生物素标记的C3dg(C3dg-生物素)相互作用的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(BIAcore 3000仪器)。按照每个流动细胞平均50mg/L的量，将人C3dg-生物素(Guthridge,J.M.,et al.Structural studies in solution of the recombinant N-terminal pair of short consensus/complement repeat domains of complement receptor type 2(CR2/CD21)and interactions with its ligand C3dg.Biochemistry.2001,40(20):5931–5941.)以2μL/min的速度注射到BIAcore抗生物素蛋白链菌素传感器芯片上，作用20min，缓冲液是0.5×PBS(pH7.4)(含有0.5g/L Tween20)。从捕获的C3dg上获取的SPR信号产生BIAcore反应单位(范围是250到500)。以不加融合蛋白组作为对照。25℃，以25μL/min的流速，用0.5×PBST(0.5g/L Tween20)洗涤后，通过检测ScFv-DAF浓度(15.6～500nmol/L)来评估其亲和力。
动力学分析数据显示，1∶1最适合接合反应模型用球形检测参数。SPR检测结果显示，ScFv-DAF比ScFv有较高的结合与解离速率(表3)。而且ScFv-DAF的亲和力比ScFv高。
表3.重组融合蛋白与C3dg-生物素结合的动力参数
Figure PCTCN2020132588-appb-000005
在本研究中，对中国发明专利申请CN109575132A公开的抗C3d抗体的单链抗体也进行了动力参数的测定，结果显示该抗体的Ka(1/Ms)为4.58×10 2，Kd(1/s)为0.023，K D(nm)为764±68，相较于该抗体，本发明提供的ScFv极大改善了C3dg-生物素结合的动力参数。
实施例4.补体溶解实验
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)。重组融合蛋白用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。
融合蛋白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，ScFv-DAF比ScFv抑制效果明显，抑制效率提高了6倍多，在细胞裂解抑制实验中，ScFv-DAF对红细胞的保护作用比ScFv提高了3倍多(详见表4)。
表4.能抑制50％细胞发生溶解的补体抑制物浓度
Figure PCTCN2020132588-appb-000006
实施例5.ScFv-DAF融合蛋白RA体内实验：
1.生物学分布实验
采用Iodogen法将ScFv-DAF标记 125I，在涂布有200μg Iodogen的EP管中，依次加入现配制的50mmLo/L PBS(pH7.4)150μl、含1mg BSA溶解的ScFv 100μl(100μg)、Na 125I溶液15μl(185MBq)，室温下间断轻微摇动标记管15min。SEP-PAK C18柱分别用甲醇、双蒸水和0.1％三氟乙酸(TFA)各5mL依次洗涤使其活化；标记混合物上柱，0.1％TFA淋洗；60％的乙腈溶液洗脱，收集前1.5mL洗脱液。经冷冻干燥后，用含1mg/mL BSA的PBS溶液稀释、分装，-80℃冰箱贮存备用。雄性DBA/1J小鼠尾根部皮下注射0.1mL胶原Ⅱ和完全弗氏佐剂(均购于美国Sigma公司)，第21天加强一次建立类风湿关节炎(CIA)小鼠模型(模型建立参照C57BL/6小鼠CIA模型的建立及其监测体系的初步筛选，解放军医学杂志2004年6月第29卷第6期472-474)。试验和分组方法如下：①对照组尾静脉注射 125I-DAF(2ug)，分别在24h、48h后断头处死，收集血液，取出组织器官，称重并测定其放射性(μCi)，结果换算为ID％/g组织。②关节炎组尾静脉注射 125I-DAF融合蛋白(2ug)，分别在24h、48h、72h、96h后，采取同上处理、检测。③ScFv-DAF关节炎治疗组，尾静脉注射一次 125I-ScFv-DAF融合蛋白(0.25ug)，24h后采取同上处理、检测。结果如图3所示，表明本发明的ScFv-DAF融合蛋白可在关节炎部位高度聚集(图3中图例说明的自上而下顺序的图例与图3中每一分组柱图中的自左向右顺序的亚柱图一一对应)。
2.类风湿关节炎(CIA)小鼠模型的治疗
利用类风湿关节炎(CIA)小鼠模型(同上)，从第21天开始试验，分组如下：①注射50μl PBS对照组(N＝18)。②ScFv-DAF微剂量治疗组(N＝16)，每天注射一次ScFv-DAF融合蛋白(0.25)mg。③ScFv-DAF低剂量治疗组(N＝18)，每天注射两次ScFv-DAF融合蛋白(0.25)mg。④ScFv-DAF中剂量治疗组(N＝16)，每天注射三次ScFv-DAF融合蛋白(0.25)mg。⑤ScFv-DAF高剂量治疗组(N＝16)，每天注射四次ScFv-DAF融合蛋白(0.25)mg。第23天开始临床评分，按以下标准对动物关节炎程度计分：0分，无关节炎；1分，轻微炎症和爪部发红；2分，严重红斑和肿胀，影响爪部功能；3分，爪部或关节变形、僵硬，失去功能。每只 小鼠四肢最高总分为12分。结果如图4所示，从第23天开始，ScFv-DAF融合蛋白四个治疗组的关节炎严重程度明显低于PBS组。其中第⑤组(4次注射组)的评分是第④组(3次注射组)的1/2以下，是第③组(ScFv-DAF2次注射组)的1/4以下，是第②组(ScFv-DAF 1次注射组)的1/5以下，是第①组(PBS对照组)的1/7以下。
以上实验结果证明本发明的ScFv-DAF融合蛋白对C3d具有特异的靶向性，具有更好地抗黏附/抗炎靶向抑制效应，对机体的炎症反应具有很好的治疗作用，显著高于ScFv-DAF的治疗效果。
实施例6.ScFv-DAF融合蛋白在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ScFv-DAF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0.3mg/W的ScFv-DAF，第二组(n＝24)为低剂量治疗组，从第16-24周起每周接受0.15mg/W的ScFv-DAF，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ScFv-DAF治疗的小鼠，由于补体激活途径中的C3d被靶向C3d的ScFv-DAF有效抑制，因此，MRL/lpr红斑狼疮小鼠的存活率明显提高，ScFv-DAF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％，ScFv-DAF低剂量治疗组组即使在第24周也能保持在70％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ScFv-DAF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，并使用生化仪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ScFv-DAF治疗组(n＝22)和PBS对照组(n＝24)蛋白尿情况对比在第18-24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ScFv-DAF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5所示。
表5.MRL/lpr小鼠从16周到23周治疗后第24周ScFv-DAF治疗组和PBS对照组肾脏损害情况对比
Figure PCTCN2020132588-appb-000007
和对照组相比，ScFv-DAF治疗组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
本发明提供了一种人源抗补体C3d分子的单链抗体以及所述单链抗体与补体抑制剂DAF的融合蛋白，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和融合蛋白易于工业化生产，具有工业实用性。
序列表自由内容
Figure PCTCN2020132588-appb-000008
Figure PCTCN2020132588-appb-000009
Figure PCTCN2020132588-appb-000010
Figure PCTCN2020132588-appb-000011
Figure PCTCN2020132588-appb-000012
Figure PCTCN2020132588-appb-000013
Figure PCTCN2020132588-appb-000014
Figure PCTCN2020132588-appb-000015

Claims (10)

  1. 一种人源抗补体C3d分子的单链抗体，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.2的第25-35、51-57和89-98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.4的第30-35、50-66和99-107位氨基酸序列所示。
  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.2所示，所述抗体重链可变区的氨基酸序列如SEQ ID NO.4所示。
  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.6所示的柔性多肽连接。
  4. 一种含有权利要求3所述单链抗体的融合蛋白，其特征在于，所述融合蛋白还含有补体活性调节剂。
  5. 根据权利要求4所述的融合蛋白，其特征在于，所述补体活性调节剂为DAF分子。
  6. 根据权利要求5所述的融合蛋白，其特征在于，所述单链抗体与DAF分子以SEQ ID NO.8所示的柔性多肽连接
  7. 根据权利要求6所述的融合蛋白，其特征在于，所述融合蛋白的氨基酸序列如SEQ ID NO.10所述。
  8. 一种编码权利要求7所述融合蛋白的多核苷酸，其特征在于，所述多核苷酸的序列如SEQ ID NO.9所述。
  9. 权利要求4-7任一所述的融合蛋白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，所述疾病包括类风湿性关节炎和系统性红斑狼疮。
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