WO2010139196A1 - 人源抗人干扰素α抗体及其应用 - Google Patents

人源抗人干扰素α抗体及其应用 Download PDF

Info

Publication number
WO2010139196A1
WO2010139196A1 PCT/CN2010/000798 CN2010000798W WO2010139196A1 WO 2010139196 A1 WO2010139196 A1 WO 2010139196A1 CN 2010000798 W CN2010000798 W CN 2010000798W WO 2010139196 A1 WO2010139196 A1 WO 2010139196A1
Authority
WO
WIPO (PCT)
Prior art keywords
antibody
seq
amino acid
huifn
human
Prior art date
Application number
PCT/CN2010/000798
Other languages
English (en)
French (fr)
Inventor
梁米芳
孙志伟
王颖
龚斌
王双
李金枝
李川
孙丽娜
李德新
Original Assignee
中国疾病预防控制中心病毒病预防控制所
中国人民解放军军事医学科学院生物工程研究院
上海市免疫学研究院
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 中国疾病预防控制中心病毒病预防控制所, 中国人民解放军军事医学科学院生物工程研究院, 上海市免疫学研究院 filed Critical 中国疾病预防控制中心病毒病预防控制所
Publication of WO2010139196A1 publication Critical patent/WO2010139196A1/zh

Links

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
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/249—Interferons
    • 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
    • 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
    • C07K2317/60—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)

Definitions

  • the invention relates to the preparation and application of a human genetically engineered antibody for treatment, in particular to a therapeutic target human interferon a (huIFN-a) for systemic lupus erythematosus (SLE), which has blood for neutralizing lupus patients.
  • huIFN-a human interferon a
  • SLE systemic lupus erythematosus
  • the antibody drug developed by the blocking effect of antibody molecules is a new force developed in the field of biomedicine in recent years. According to statistics, since the birth of the first genetically engineered human-mouse chimeric antibody OKT3 in 1984, there are about 20 genetically engineered antibody drugs approved by the FDA, and another 150 are undergoing clinical research. It is estimated that by 2008, the revenue of antibody drugs will account for 30% of the total income of biotechnology products, so they have extremely broad application prospects in the treatment of tumors, cardiovascular diseases, autoimmune diseases and the like.
  • human antibody preparation technology is mainly human-derived antibody library technology (natural, semi-synthetic and total synthesis), transgenic mice, human-human hybridoma technology, human B cell immortalization technology.
  • human antibody libraries using phage antibody display library technology is one of the main methods. Screening for antibodies using phage antibody library technology does not require animal immunization, is easy to prepare antibodies to rare antigens, and screens for high-affinity antibodies that are fully human.
  • the human antibody library can be further divided into an immunological library and a non-immune library.
  • an immunological library In the presence of high titers of neutralizing antibodies in the periphery of some patients with acute infectious disease, it is of course desirable to obtain the desired antibodies by obtaining a pool of human antibodies that are effectively immunized.
  • key target molecules for cancer, autoimmune diseases and cardiovascular diseases are often composed of autoantigens, or it is difficult to induce an effective immune response in vivo, or the mechanism of action is related to immune blockade, so the non-immune antibody library It has broader applicability in screening therapeutic antibodies against target molecules associated with these diseases.
  • the non-immune antibody library includes a natural bulk antibody library and a synthetic antibody library, which is a technique for obtaining a genetically engineered antibody against an antigen without immunization.
  • Achim Knappik reported in detail the method of constructing a fully synthetic antibody library of human origin, and constructed a fully synthetic antibody library with a library capacity of 2 x lO 9 .
  • Non-immune antibody libraries, particularly synthetic antibody library technology have shown good promise in screening therapeutic antibody drugs.
  • SLE Systemic lupus erythematosus
  • SLE Systemic lupus erythematosus
  • the onset of SLE patients is characterized by an alternating recurrence and remission. Its pathogenesis is still not very clear. It has long been known that the level of type I interferon is significantly elevated in the serum of patients with SLE, and the degree of elevation is strongly correlated with the course of the disease; while patients with stable SLE or SLE are at Virus may cause recurrence or SLE after infection, suggesting possible viral induction A large amount of IFN- ⁇ is produced.
  • Medarex in the United States uses transgenic mouse technology to transfer human antibody genes into mice. This technology, combined with monoclonal antibody technology, has produced a large number of fully human-derived monoclonal antibodies, including all of huIFN- ⁇ .
  • a human-derived monoclonal antibody European Patent No. EP1781705, US Patent No.: 20070014724, which is a drug that can treat autoimmune diseases such as SLE and immune rejection.
  • One of the all-human monoclonals developed by Medarex has now entered Phase II clinical trials.
  • transgenic mouse technology as a platform for screening human monoclonal antibodies can effectively obtain therapeutic human monoclonal antibodies, bypassing the technical bottleneck of humanization of mouse antibodies, and speeding up the research and development efficiency of therapeutic monoclonal antibodies.
  • the human anti-huIFN-alb antibody obtained from the fully synthetic antibody library has relatively low technical difficulty and short antibody acquisition time.
  • the obtained human anti-huIFN-alb monoclonal antibody can also significantly reduce allergic reactions, increase antibody stability and biological activity, and will also become an effective drug for treating systemic lupus erythematosus. Summary of the invention
  • a first object of the present invention is to provide a human anti-human interferon a antibody and an active fragment thereof.
  • a second object of the present invention is to provide a gene encoding the above antibody or an active fragment thereof.
  • a third object of the present invention is to provide an application of the above antibody and an active fragment thereof for the preparation of a medicament for treating a disease caused by an excessive amount of interferon.
  • the invention utilizes phage surface presentation technology to obtain specific anti-human interferon alpha (huIFN- ⁇ ) by multiple rounds of bio-panning from a constructed fully synthetic human single-chain antibody library.
  • the scFv antibody obtained by screening includes 9 scFv antibodies, named separately For AIFNalscFvl, AIFNalscFv2, AIFNalscFv3, AIFNalscFv4, AIFNalscFv5, AIFNalscFv6, AIFNalscFv7, AIFNalscFv8, AIFNalscFv9.
  • These 9 recombinant antibodies are determined by the specific gene sequences of the hypervariable regions (CDRs) present in the variable regions of the antibody light and heavy chain genes, and are specifically expressed in prokaryotic cells to bind specifically to human interferon.
  • CDRs hypervariable regions
  • a (huIFN-a) functional antibody They specifically recognize human interferon alb (h U IFN-otlb) antigens, of which 3 are directed against human interferon huIFN-alb, and have significant immunological hybridization (West Blot, WB) and enzyme-linked immunosorbent assay with huIFN-alb ( The ELISA) reaction has a neutralizing activity function of blocking the binding of huIFN-alb to the receptor.
  • AIFNalscFvl, AIFNalscFv2, AIFNalscFv3, AIFNalscFv4, AIFNalscFv5, AIFNalscFv6, AIFNalscFv7, AIFNalscFv8> AIFNalscFv9-specific light and heavy chain variable region genes are derived from a specific enrichment screen for the human fully synthetic antibody gene pool.
  • the corresponding three CDR region sequence combinations of the light and heavy chain variable regions and the framework region sequences between the CDR regions constitute the sequence characteristics of each antibody variable region, AIFNalscFvl, AIFNalscFv2, AIFNalscFv3, AIFNalscFv4, AIFNalscFv5> AIFNalscFv6> AIFNalscFv7 affiliated to the antibody heavy chain family VH3, the antibody light chain family VL1, AIFNalscFv8, AIFNalscFv9 belong to the antibody heavy chain family VH3, the antibody light chain family VL3.
  • the function of the antibody protein is determined by the specific nucleotide sequence and its complement in the CDR1, CDR2 and CDR3 of the determinant complementary region of the light chain and heavy chain variable regions of the antibody gene, and the corresponding amino acid sequences of the CDR regions constitute the antibody.
  • the specific antigen binding region determines the antigen binding characteristics and anti-huIFN-cdb functional characteristics of each antibody in the present invention.
  • the amino acid detailed sequence of the antibody light chain and heavy chain variable region which determines the function of each neutralizing antibody and its comparison result are shown in Fig. 2.
  • the "-" symbol in the figure indicates the same amino acid as the first antibody sequence, and the shaded portion is CDR region.
  • amino acid sequences of the light chain variable region and the heavy chain variable region of the antibody are respectively shown in the following groups: the amino acid sequences shown in SEQ ID No. 1 and 2, and SEQ ID Nos. 3 and 4 are shown.
  • Group 1 leucine, isoleucine, norleucine, valine, norvaline, alanine, a 2- Aminobutanoic acid, 2-methionine, O-methyl serine, t-butyl glycine, t-butylalanine, tert-butylalanine Cyclohexylalanine (cyclohexylalanine);
  • group 2 aspartic acid (day Aspartate), glutamic acid, isoaspartic acid, isoglutamic acid, 2-aminoadipic acid, 2-aminosuberic acid (2-Aminosuberic acid);
  • Group 3 asparagine (asparagine), glutamine (glutamine);
  • Group 4 lysine, arginine, ornithine, 2,4-diaminobutanoic acid, 2,3-diaminopropionic acid ( 2, 3-diaminopropionic acid);
  • proline proline
  • 3-hydroxyproline 3-hydroxyproline
  • 4-hydroxyproiine 4-hydroxyproline
  • Group 6 serine (serine), threonine (threonine), homoserine (homoserine);
  • Group 7 phenylalanine (phenylalanine), tyrosine (tyrosine)
  • substitutions between individual amino acids within the set do not alter the activity of the antibody protein, and derived antibodies are derived from these changes.
  • the gene sequence encoding the above antibody can be modified in its coding region without changing the amino acid sequence to obtain a gene encoding the same antibody.
  • one skilled in the art can artificially engineer a gene based on the codon bias of the expression antibody host to increase the expression efficiency of the antibody.
  • the above-described gene encoding a single-chain antibody can be cloned into an expression vector, thereby transforming into a host, and a single-chain antibody can be obtained by inducing expression.
  • the light chain coding gene and the heavy chain coding gene of the above antibody can be cloned into a total anti-expression vector, respectively, and introduced into a host cell to obtain a total anti-interferon ⁇ -expressing anti-interferon ⁇ .
  • the light chain and heavy chain genes of the above three scFv antibodies (AIFN-scFvl. AIFN-scFv2, AIFN-scFv3) were cloned into the whole antibody expression vector pAC-K-CH3 and transfected into insect Sf cells, respectively.
  • Secreted expression of whole antibodies was achieved using a baculovirus/insect cell system, and whole antibodies AIFN1 IgG1, AIFN IgG2, and AIFN IgG3 were obtained.
  • the immunological properties of the obtained human monoclonal antibodies were identified by ELISA and Western Blot. The results indicated that all three human monoclonal antibodies were specific for huIFN-alb, but not for other structurally similar interferon family antigens such as huIFN-a2b and huIFN- ⁇ . Western Blotting results also showed that the three antibodies specifically recognized the denatured huIFN-alb protein.
  • the present invention obtains 9 anti-huIFN-ctlb scFv phage antibodies for the first time in the world, and expresses three scFv segment antibodies and whole antibodies in prokaryotic and eukaryotic systems, and completes the detection of in vitro immunoregulatory function of whole antibodies.
  • the achievement of a result brings hope to the treatment of systemic lupus erythematosus.
  • the human-neutralizing anti-huIFN-alb genetically engineered antibody variable region gene obtained above and the whole antibody gene under the characteristics of each of the above antibody genes can be used in prokaryotic cells, Expression and production of this antibody in yeast cells, eukaryotic cells, and any recombinant system, or any other gene containing the antibody gene based thereon, to obtain an antibody product having neutralizing huIFN-alb immunological activity, made It is clinically used to treat specific systemic drugs for systemic diseases caused by excessive huIFN-cdb in peripheral blood of patients with systemic lupus erythematosus, thus providing a new means for treating systemic lupus erythematosus.
  • an injectable antibody preparation can be prepared for the treatment of various autoimmune diseases caused by an excess of interferon in the human body.
  • Figure 1 shows the phage-ELISA to verify the binding specificity of 9 phage single-chain antibodies to huIFN-alb
  • Figure 2 shows a comparison of the amino acid sequences of the variable regions of the anti-human IFN-alb antibody of the present invention
  • Figure 3 shows the binding characteristics of five single-chain antibodies detected by ELISA
  • Figure 4 shows the binding characteristics of three single-chain antibodies expressed by prokaryotic expression by Western Blot
  • Figure 5 is a graph showing the specific binding of humans expressed by Pull Down
  • Figure 6 shows the SDS-PAGE electropherogram of purified IgG
  • Figure 7 shows the binding characteristics of 5 IgG whole antibodies detected by ELISA
  • Figure 8 is a graph showing the specific binding of three humans expressed by Western Blot
  • Figure 9 shows the changes of IFN-a-induced gene ISG15 after treatment of normal human PBMCs with IFN-a and anti-huIFN-alb antibodies;
  • Figure 11 shows the changes in IFN-cc-induced gene ISG15 in normal human PBMCs after addition to SLE patients (increased IFN-a) and normal human serum and anti-huIFN-alb antibody.
  • Example 1 The technical means used in the examples are conventional means well known to those skilled in the art unless otherwise specified.
  • Example 1 The technical means used in the examples are conventional means well known to those skilled in the art unless otherwise specified.
  • a human-derived fully synthetic genetically engineered antibody library was constructed using phage surface rendering technology.
  • the fully synthetic phage antibody library was enriched for screening with purified huIFN-alb and secreted for expression in E. coli.
  • the functional activity of scFv antibody specific binding to huIFN-alb was identified by ELISA, Western Blot and ⁇ ⁇ 11 Down, and sequenced.
  • the light chain and heavy chain variable region genes of the positive clones were then cloned into the whole antibody expression vector.
  • pAC-K-CH3 is transfected into insect Sf cells, and the secreted expression of whole antibodies is achieved by the baculovirus octopus cell system.
  • the immunological properties and immunomodulatory functions of the obtained human monoclonal antibodies were identified by ELISA and Western Blot. Materials and Methods
  • the human-derived fully synthetic antibody library was constructed by the Academy of Military Medical Sciences (Du Weishi, Wang Shuang, Sun Zhiwei et al. Construction of a fully synthetic human phage antibody library. Proceedings of the Academy of Military Medical Sciences, 2006, 30:319-322), screening antibody libraries
  • the antigen was constructed by prokaryotic expression of purified huIFN-alb (concentration of 1 mg/ml), and the strain was XLI-Blue (Stratagene, USA); the phage used was M13K07 (Invitrogene, USA).
  • the baculovirus expression vector is pAC-K-CH3 (PROGEN PR3003, Germany) (Liang, MF, Stefan, D., Li, DX, Queitsch, I" Li, W., and Bautz, EF Bac lovirus expression cassette vectors for rapid production Of complete human IgG from phage displayselected antibody fragments. Journal of Immunological Methods. 247: 119-130. ) , insect cells Sf and 293T cells from the American Cell Culture Center (ATCC). Construction and expression of huIFN-alb referenced the literature (Li Wu Ping, Lu Hongliang, Hou Yunde et al. High-efficiency expression, purification and antiviral activity of interferon-Plb.
  • PCR amplification of human interferon alb (huIFN -ALB See the) gene sequence, after sequencing, was cloned into prokaryotic expression vector pET30a (U.S. invitrogen), huIFN-alb by Ni metal by the recombinant plasmid was transformed into E. coli Rosseta TM (DE3) for protein expression. expression chelate affinity And chromatographic purification.
  • the screening antigen was huIFN-alb.
  • l x PBS NaCl 8g, C1 0.2g, Na 2 HP0 4 1.44g, KH 2 PO 4 0.24g dissolved in 800ml ddH 2 O, adjusted to pH 7.4 with HC1, constant volume Dilute to the working concentration to 1L
  • Enrichment screening methods are basically carried out according to the literature (Du Wei Shi, Wang Shuang, Sun Zhiwei et al. Construction of a fully synthetic human phage antibody library. Proceedings of the Academy of Military Medical Sciences, 2006, 30: 319-322), as follows:
  • M13K07 (titer 10 12 / ⁇ 1) 200 ⁇ 1, 37 ⁇ static set for 15-20min, shake culture at 37 °C l50rpm for 1 hour, add kanamycin (final concentration 5 ( ⁇ g / ml), 37 ° C culture overnight The cultured overnight bacterial suspension was centrifuged at 6500 rpm for 15 min, and the bacterial culture supernatant was collected.
  • the supernatant was transferred to a clean Erlenmeyer flask, 4% PEG 8000, 3% NaCl was added, and the solution was fully dissolved and lyophilized for 30 min or more. Centrifugation at 9000 rpm 20min, discard the supernatant. The pellet was dissolved in PBS, ice bathed for 15 min, centrifuged at 12000 rpm for 5 min, and the supernatant obtained was the first round of antibody library obtained. The supernatant was transferred to an Eppendorf tube and 3% BSA was frozen. After the titer of the phage was titrated, the next round of screening was performed. The screening was repeated three times. The clones obtained by the third round of enrichment screening were picked, and the binding activity of the phage antibody was identified by phage-ELISA. Positive clone, obtained by sequencing Gene sequences.
  • the PCR primers with Ncol and Xhol at the 5' end amplify the scFv antibody gene (upstream primer scFv-pET22-L: CATGCCATGGCCGATATCGTTCTGAC, downstream primer scFv-pET22-R: CCGCTCGAGGCTCGACACGGTCACCAGAG), and the PCR fragments were recovered and digested with Ncol and Xhol.
  • the recombinant double-stranded antibody expression plasmid was ligated with the same double-digested pET22b vector fragment, and transformed into BL-21 (DE3) competent cells. After overnight culture, the positive single colonies resistant to ampicillin were picked and inoculated with 2 ⁇ bacterial culture solution.
  • the huIFN-alb2 g/mL was diluted with a coating solution of 0.1 M NaHC0 3 (pH 9.6), and the ⁇ was added to a 96-well flat bottom plate of polyvinyl chloride, and coated at 4 ° C overnight; 4% skim milk was blocked, 37 ° C After incubation for 1 h, discard the supernatant and add the expressed scFv Bacterial antibody, after incubation at 37 ° C for 1 h, PBST was washed three times, added with enzyme-labeled anti-M13 secondary antibody (US Sigma, 1:4000 dilution), incubated at 37 ° C for 1 h; washed five times with PBST, added 100 L color The liquid (A+B) was developed, and finally the reaction was terminated by adding 2M H 2 S0 4 , and the absorbance A (450) value was measured by a microplate reader.
  • the secreted expression supernatant of the single-chain antibody was subjected to ultrafiltration-concentration and subjected to 10% SDS-PAGE electrophoresis, and the corresponding negative control was the carrier pET22b transformed bacteria.
  • the bacterial ultrafiltration-concentrated bacterial expression supernatant prepared in the same manner was used.
  • the protein after electrophoresis of the polyacrylamide gel was transferred to the NC membrane by a semi-dry method. After the transfer, the gel was subjected to Coomassie blue staining to check whether the protein was completely transferred. Lanes containing standard molecular weight proteins were cut and labeled with standard molecular weight reference protein positions.
  • the NC membrane was blocked in 1 x PBS containing 5% skim milk powder for 2 hours at room temperature, washed 3 times with 1 x PBST, and reacted with HPR-labeled murine anti-His antibody (Sigma, 1:500) for 1 hour at room temperature. After lxPBST was washed 3 times, it was placed in the DAB substrate to develop color, and the brown positive band appeared. The NC membrane was placed in double distilled water to terminate the reaction, and dried and protected from light.
  • the purified huIFN-ctlb, huIF-a2b and huIFN- ⁇ were used as antigens to identify the binding specificity of the single-chain antibody by Western Blotting.
  • the details are as follows: After the above purified three antigens (5 g) were subjected to SDS-PAGE electrophoresis, the gel was subjected to Coomassie blue staining after being transferred to the NC membrane by semi-dry method to check whether the protein was completely transferred. Standard molecular weight protein lanes were cut and the reference protein position of the molecular weight standard was indicated. The NC membrane was blocked with lxPBS containing 5 % skim milk chamber for 2 hours at room temperature.
  • the primary antibody working solution containing the AIFNalscFvl, AIFNalscFv2, and AIFNalscFv3 expression product stocks was incubated with the blocked NC membrane for 2 hours at room temperature.
  • lxPBST was washed 3 times and reacted with HPR-labeled anti-His antibody (Sigma, 1:1000) for 1 hour at room temperature, washed 3 times with lxTBST, placed in DAB substrate liquid color, and brown positive band appeared.
  • the NC membrane was placed in double distilled water to terminate the reaction in a timely manner, and stored in the dark after drying.
  • the pellet was resuspended in 20 ⁇ l of lxPBS, and boiled by protein loading buffer for 5 minutes, and subjected to 15 %SDS-PAGE electrophoresis, followed by conventional Coomassie blue staining and post-decoloration detection.
  • Plasmid DNA was prepared using Qiagen Miniprep Kit (QIAGEN, Germany) for nucleic acid sequence analysis.
  • the sequencing primer was 5'-AGCCCACCTCAACGCAATT-3.
  • the sequencing results were sequence aligned with the antibody gene sequences in the Internet V-Base gene pool.
  • the heavy chain of the antibody (3£0 10 0.20, 350 10 >40.22, 8 £ (3 10 ⁇ 0.24, SEQ ID N0.34> SEQ ID N0.36) PCR amplification with primer (VH3L: gtaactcgagAGCGGTGGCGGTCTGGTG, VHR: gaagctagcGCTCGACACGGTCACCAGAGTG) followed by double digestion with Xhol/Nhel, ligation into PAC-K-CH3 vector (PROGEN PR3003, Germany), and light chain (SEQ ID) ⁇ 19, SEQ ID NO.23. SEQ ID NO, 23, SEQ ID N0.33, SEQ ID N0.35) with primers
  • Transfection was performed using the Bac loGold co-transfection kit from Pharmogen, USA.
  • the method of operation is as follows: 5 g of recombinant plasmid DNA was mixed with 0.5 g of Bac loGold linear DNA, and transfected with transfection reagent to transfect Sf9 cells with a growth density of 50%, and cultured at 27 ° C for 4 days, collected and reconstituted. The viral cell culture supernatant of the virus is subjected to virus titration and amplification. See the Bac lovirus expression vector system manual for details.
  • the recombinant virus was infected with Sf9 cells with a growth density of about 70%, and 27 ⁇ was adsorbed for lh.
  • the SF-900 II serum-free culture solution was used, and the supernatant was collected after culturing for 27 to 5 days.
  • Direct expression of the induced expression supernatant was performed using Protein-A affinity chromatography (Amersham, USA) (Harlow E, Lane D. "Antibodies: A Laboratory Manual”. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988. ).
  • FITC-labeled anti-human IgG-Fc antibody solution (Sigma, USA 1:80) was added dropwise, incubated at 37 °C for 30 min, rinsed with lxPBS, air-dried, and the fluorescence expression rate and expression intensity of Sf cells were observed under the microscope.
  • the control cells were sf cells transfected with empty vector.
  • the purified huIFN-alb antigen was subjected to SDS-PAGE electrophoresis and transferred to the NC membrane by semi-dry method.
  • the NC membrane was blocked in 1 x PBS containing 5% skim milk powder for 2 hours at room temperature, and then placed in primary antibody containing AIFNallgGl, AIFNAL IgG2 and AIFNlg IgG3 expression products for 2 hours at room temperature, washed 3 times with l xPBST, and labeled with HPR.
  • the anti-IgG-Fc secondary antibody (Sigma, . 1: 1000) was reacted at room temperature for 1 hour. l After washing with xPBST for 3 times, it was placed in the liquid color of the DAB substrate, and the brown positive band appeared to stop the reaction in time, and stored in the dark.
  • peripheral blood mononuclear cells PBMCs
  • PBMCs peripheral blood mononuclear cells
  • synthetic human IFN was added.
  • -a l OOng/mL
  • different concentrations of anti-huIFN-alb antibody (( ⁇ g/mL, 3 ⁇ g/mL.
  • IFN- ⁇ -inducible genes ISG15 and IFIT-1
  • the collected cells were extracted with TRIzolTM reagent (Invitrogen).
  • the cDNA was obtained by reverse transcription (ReverAidTM ⁇ - ⁇ reverse transcriptase, Fermentas) with random primers, and the real-time quantitative PCR (SYBR premix Ex TaqTM, Takara) technique was used to detect the ISG15 and ITIF-1 genes in the sample.
  • the primer sequences of ISG15 and IFIT-1 are: ISG15 forward: 5,-GAG AGG CAG CGA ACT CAT CT-3'; Reverse: 5,-AGC TCT GAC ACC GAC ATG G-3'; IFIT-1 forward : 5 '-GCA GAA CGG CTG CCT AAT TT-3 '; Reverse: 5'-TCA GGC ATT
  • the primer sequence of the internal reference gene of TCA TCG TCA TC-3 ⁇ PCR reaction is as follows: GAPDH (200nM forward) 5 '-CTGGAACGGTGAAGGTGACA -3, , ( 200nM reverse ) 5'-
  • the experimental group also used SLE patient serum containing IFN-a, and normal human control serum (1 /10 volume ratio was added to the medium to treat normal human PBMCs, and anti-huIFN-alb antibody (24 g/mL) was added to neutralize the effect of IFN-a in serum, and incubated at 37 ° C and 5% CO 2 . After an hour, the collected cells were examined for differences in expression of the IFN-a-inducible gene (ISG15) to evaluate the immunological neutralizing activity of the anti-huIFN-alb antibody against IFN-a in serum.
  • ISG15 IFN-a-inducible gene
  • the collected cells were extracted with TRIzolTM reagent (Invitrogen), reverse transcribed by random primers (ReverAidTM ⁇ - ⁇ reverse transcriptase, Fermentas), and real-time quantitative PCR (SYBR) was used.
  • Premix Ex TaqTM, Takara) Technically tested the expression of the ISG15 gene in the sample.
  • the sequence of the ISG15 and the internal reference gene primer used was the same as above.
  • a statistical analysis of the average of the data obtained from the samples was performed.
  • the (alanine, A) at position (44) of the amino acid sequence shown in SEQ ID No. 1 is replaced with (valine, V), and the heavy chain thereof is variable.
  • the (40) position of the region (alanine, A) is replaced by (valine, V).
  • the light chain gene and the heavy chain gene were cloned into PAC-K-CH3 according to the above methods 6 to 10, and transfected into insect Sf9 cells, and the secreted expression of the whole antibody was achieved by the baculovirus/insect cell system, and This mutant was subjected to immunological detection. Result
  • the fully synthetic antibody library was screened with purified hnIFN-alb (1 (Vg/ml) as the antigen, and 1500 clones were randomly picked and collected to collect the supernatant after 3 rounds of screening.
  • Purified huIFN-alb antigen (2 g/ml) coated 96-well plate added to the supernatant of the sample to be tested, and detected the positive rate of antibody expression in the sample to be tested by HRP-labeled anti-human M13 secondary antibody (sigma, diluted 1:4000). After three rounds of screening, a total of 300 human-derived scFv-positive clones were obtained.
  • huIFN-alb purified huIFN-alb, huIFN-a2b and huIFN- ⁇ antigens were used for antibody by phage-ELISA.
  • the recombination assay was performed for binding specificity, and 100 clones specifically binding to huIFN-alb were obtained.
  • the DNASTAR and Mega3.0 sequence analysis software were used for analysis and processing, and the IgG sequences in the Internet V-Base gene pool were compared.
  • the sequence of 86 antibody sequences was sequenced in the above 100 human anti-huIFN-alb genetically engineered antibodies.
  • the clones belonged to 9 different antibody sequences, and 9 antibodies with different antibody light and heavy chain variable region sequences and their combinations were found.
  • the heavy chain variable region was mainly classified into the IgG VH3 family, and its light chain was variable.
  • the regions are mainly classified in the IgG VL1 and VL3 families.
  • Figure 2 is a comparison of the amino acid sequences of the variable region genes of 9 human anti-huIFN-alb genetically engineered antibodies.
  • the first line of antibody sequences is used as a standard sequence for comparison.
  • the "-" symbol indicates the first line.
  • the amino acid sequence of the antibody is the same, and the shaded part is the CDR region.
  • the protein sequence of the human single-chain antibody AIFNs scFv 1 is SEQ ID NO. 1 and 2
  • the protein sequence of the single-chain antibody AIFNalscFv2 is SEQ ID N 0.3 and 4
  • the protein sequence of the single-chain antibody AIFN-scFv3 is SEQ ID. NO. 5 and 6
  • the protein sequence of the single-chain antibody AIFNalscFv4 is SEQ ID N 0.7 and 8
  • the protein sequence of the single-chain antibody AIFN-scFv5 is SEQ ID NO. 9 and 10
  • the protein sequence of the single-chain antibody AIFN-scFv6 is SEQ ID.
  • the protein sequence of the single-chain antibody AIFN-scFv7 is SEQ ID NOs. 17 and 18.
  • phage-ELISA In order to identify whether these 9 different phage antibodies can stably bind huIFN-alb, we used phage-ELISA to verify the binding specificity and stability of 9 antibodies, and used phage-ELISA to detect 3 antigens. Hu IFNIFN-alb, huIFN-a2b and huIFN- ⁇ , ELISA results showed that 5 of them had higher ELISA values than huIFN-a2b and huIFN- ⁇ , and there were more than twice the difference.
  • the present invention further identified the functional activity of the prokaryotically expressed scFv antibody by ELISA, Western Blot and Pull Down.
  • the ELISA results indicated that the single-chain antibodies AIFNalscFvl, AIFNalscFv2, and AIFNalscFv3 secreted in bacteria reacted with huIFN-alb, but did not react with the recombinant proteins huIFN-a2b and huIFN- ⁇ , as shown in Fig. 3.
  • the light and heavy chain genes of three scFv antibodies (AIFNalscFvl, AIFN-scFv2, AIFN-alfFv3) that have been specifically verified by binding were cloned into the whole antibody expression vector PAC-K-CH3 transfected insect Sf cells, using baculovirus/insect
  • the cellular system achieves secretory expression of whole antibodies.
  • the expression supernatant was directly purified by Amersham's Protein-A affinity chromatography column.
  • the expression and purification of the whole antibody IgG were confirmed by SDS-PAGE. The results confirmed that the pure protein was obtained, and the antibody after melting was clearly observed.
  • the heavy chain located at about 28KD and 55KD, was analyzed by non-denaturing SDS-PAGE to obtain an unmelted antibody protein at about 150 KD, as shown in Fig. 6.
  • the present invention further identifies the functional activity of whole antibody IgG by ELISA and Western Blot.
  • the ELISA results were consistent with the verification of the functional activity of the scFv antibody.
  • the purified whole antibodies AIFNallgGl, AIFNal IgG2, and AIFNal IgG3 reacted with huIFN-alb but did not react with the recombinant proteins huIFN-a2b, huIFN- ⁇ and BSA. 7 is shown.
  • the antibody (AIFNal IgG1, AIFNAL IgG2, AIFNAL IgG3) obtained by the present invention acts on the normalized IFN- ⁇ treatment.
  • Human PBMCs showed that the expression of IFN- ⁇ -inducible genes ( ISG15 and IFIT-1 ) was significantly increased in normal human PBMCs under the action of IFN- ⁇ , but the expression of these two genes was significantly increased after the addition of anti-huIFN-alb antibody.
  • the light chain gene and heavy chain gene modified based on AIFNallgGl were cloned into PAC-K-CH3, and transfected into insect Sf cells, and full antibody secretion was achieved by baculovirus/insect cell system. Expression, the mutant AIFNallgGl' was obtained. The mutant was subjected to immunological detection. The ELISA results showed that the purified whole antibody AIFNallgGl' reacted with huIFN-alb but did not react with the recombinant proteins huIFN-a2b and huIFN- ⁇ , and the affinity and specificity were basically the same as AIFNallgGl'. Western Blotting results indicate that AIFNallgGl' can react with denatured IFN-alb. Industrial applicability
  • the human anti-huIFN-a antibody disclosed in the invention can be used as a specific antibody drug for treating systemic diseases caused by peripheral huIFN-alb excess in patients with systemic lupus erythematosus, thereby treating systemic erythema Lupus provides new means.
  • an injectable antibody preparation can be prepared for the treatment of various autoimmune diseases caused by an excess of interferon in the human body.

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)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Description

人源抗人干扰素 α 抗体及其应用
技术领域
本发明涉及治疗用人源基因工程抗体的制备及应用，尤其是特异性针对系统性红斑狼 疮 （Systemic lupus erythematosus , SLE ) 的治疗靶蛋白人干扰素 a(huIFN-a), 具有中和狼 疮病人血液中和 huIFN-a升高引起的全身性疾病的治疗用基因工程抗体。 背景技术
利用抗体分子的阻断作用研发的抗体药物是近年来生物医药领域中发展起来的新生 力量。据统计， 自 1984年第一个基因工程抗体人 -鼠嵌合抗体 OKT3诞生以来， 目前经 FDA 批准上巿的有约 20种基因工程改造的抗体药物， 另有 150种正在进行临床研究阶段， 预 计到 2008年， 抗体药物收入将占到整个生物技术类产品收入的 30%, 所以它们在针对肿 瘤、 心血管、 自身免疫性疾病等的治疗上有着极其广泛的应用前景。
回顾治疗性抗体的研究历程，早期的鼠源性抗体由于其相对于人类的异物性已经不适 用于临床， 所以制备人源性抗体（全人抗体）是目前治疗性抗体研发的主要策略。 在人源 性抗体研究领域， 人源抗体制备技术主要有人源抗体库技术 （天然、 半合成和全合成） ， 转基因小鼠、 人-人杂交瘤技术、 人 B 细胞永生化技术等。 利用噬菌体抗体展示库技术制 备人源抗体库是其中主要的方法之一。 釆用噬菌体抗体库技术筛选抗体不必进行动物免 疫， 易于制备稀有抗原的抗体及筛选全人源性的高亲和力抗体。 人源抗体库可进一步分为 免疫库和非免疫库。 在一些急性感染性疾病痊愈患者外周中如果存在高滴度的中和抗体， 那么可以通过获得有效免疫的人抗体库， 筛选获得所需要的抗体， 这当然是最理想的。 但 对于癌症、 自身免疫疾病和心血管疾病等的关键靶分子往往是由自身抗原组成， 或者很难 在体内诱导有效的免疫应答， 或者其作用机理是和免疫阻断有关， 所以非免疫抗体库在筛 选针对与这些疾病相关的靶分子的治疗性抗体中具有更广泛的应用性。 非免疫抗体库包括 天然大容量抗体库和合成抗体库， 它是一种不经过免疫就能获得针对某种抗原的基因工程 抗体的技术。 2000年 Achim Knappik详细报道了构建人源全合成抗体库的方法， 并构建了 库容量为 2 x lO9的全合成抗体库。非免疫抗体库特别是合成抗体库技术在筛选治疗性抗体 药物方面显示了良好的前景。
系统性红斑狼疮 (SLE)是一种以产生核抗体为特征， 并形成免疫复合物沉积于组织， 激活补体， 造成组织损伤的一种多系统受累的疾病。 SLE病人的发病特点是'复发与缓解交 替出现。 其发病机制目前仍不是很清楚。 很久以来人们就发现 SLE患者的血清中， I型干 扰素的水平明显升高， 而且升高的程度与病情的进程具有很强的关联性； 而稳定期的 SLE 病人或 SLE易感者， 在病毒感染后可能会复发或发生 SLE， 这一现象提示可能与病毒诱导 大量 IFN- α的产生有关。 在动物实验中， 当未发病的 NZB/W子一代小鼠中被注射 IFN- a 后， 则可以迅速导致这些小鼠产生严重的类似于 SLE 的病理特征， 由此直接证实了， IFN- oc在实验性 SLE发病中的作用。 由于 IFN- a本身具有多种免疫调节作用， 所以 SLE患 -者' (特别是活动期患者） 外周中高水平的 IFN- oc可能通过作用于多种免疫细胞， 如 T细胞、 B细胞参与 SLE发病中的多种临床表现. 虽然目前对于 SLE患者外周中髙水平 IFN- a产 生的确切机制尚不明确， 但是 IFN- a和 SLE临床的密切关系使其成为 SLE临床治疗中重 要的靶点分子， 通过设计分子阻断 IFN- c 和受体结合， 有望起到缓解和治疗 SLE的目的。
利用抗体库技术研究开发抗 huIFN-a 的基因工程治疗性抗体是获得治疗系统性红斑 狼疮的有效的抗体药物有效途径。 目前， 国际上有一例通过对鼠源抗 huIFN-a抗体进行人 源化改造，目的是得到治疗胰岛素依赖的糖尿病（ ίηβμΐίη dependent diabetes mellitus， IDDM) 和 SLE 的抗体药物的报道 ( Anan Chuntharapai, Jadine Lai, Xiaojian Huang et al. Characterization and humanization of a monoclonal antibody that neutralizes human leukocyte interferon: a candidate therapeutic for IDDM and SLE. CYTOKINE. 2001. 15: 250-260 )。 但鼠 源抗体的人源化改造尚不能完全消除宿主产生抗抗体引起的过敏反应。 美国的 Medarex公 司采用转基因鼠技术， 将人的抗体基因转入小鼠中， 这种技术与单克隆抗体技术结合， 得 到了大量全人源的单克隆抗体， 其中也包括针对 huIFN-α的全人源的单克隆抗体（欧洲专 利号 EP1781705, 美国专利号： 20070014724 ) ， 得到可以治疗自身免疫疾病 (如 SLE)和免 疫排斥反应的药物。 目前 Medarex公司开发的其中一株全人源的单克隆已经进入了二期临 床试验。 以转基因鼠技术为平台筛选人源单克隆抗体， 可以有效得到治疗用的人源单抗， 绕过了对鼠源抗体进行人源化的技术瓶颈， 加快了治疗用单抗的研发效率。 但是获得整合 了人抗体基因的转基因鼠从技术上讲难度较大， 相比较而言， 从全合成抗体库中得到的人 源抗 huIFN-alb抗体，技术难度相对较低，获得抗体的时间短，而且获得的人源抗 huIFN-alb 的单抗， 也能显著减少过敏反应， 增加抗体稳定性和生物学活性， 也将成为有效治疗系统 性红斑狼疮的抗体药物。 发明内容
本发明的第一个目的在于提供一种人源抗人干扰素 a抗体及其活性片段。
本发明的第二个目的在于提供编码上述抗体或其活性片段的基因。
本发明的第三个目的在于提供上述抗体及其活性片段在制备治疗干扰素过量引起的 疾病的药物中的应用。
本发明运用噬菌体表面呈现技术， 从已构建的全合成人源单链抗体库中， 通过多轮的 生物淘洗 （bio-panning ) , 筛选获得特异性抗人干扰素 α (huIFN-α) 的单链基因工程抗体 (single chain variable fragment， scFv)。 筛选获得的 scFv抗体包括 9株 scFv抗体， 分别命名 为 AIFNalscFvl、 AIFNalscFv2、 AIFNalscFv3、 AIFNalscFv4、 AIFNalscFv5、 AIFNalscFv6、 AIFNalscFv7、 AIFNalscFv8、 AIFNalscFv9。
这 9 株重组抗体是由存在于抗体轻链和重链基因可变区中的高变区 (CDRs)的特异性 基因序列决定的， 并在原核细胞中获得有效表达的特异性结合人干扰素 a (huIFN-a)的功能 性抗体。 它们特异性识别人干扰素 alb(hUIFN-otlb)抗原， 其中有 3 株针对人干扰素 huIFN-alb,与 huIFN-alb具有明显的免疫杂交反应（ Western Blot, WB )和酶联免疫 ( ELISA ) 反应， 具有阻断 huIFN-alb与受体结合的中和活性功能。
AIFNalscFvl 、 AIFNalscFv2 、 AIFNalscFv3 、 AIFNalscFv4 、 AIFNalscFv5 、 AIFNalscFv6、 AIFNalscFv7、 AIFNalscFv8> AIFNalscFv9特异性的轻链和重链可变区基 因来源于人源全合成抗体基因库的特异性富集筛选。其轻链和重链可变区相应的三个 CDR 区序列组合及其 CDR区之间框架区序列组成了每个抗体可变区序列特征， AIFNalscFvl、 AIFNalscFv2、 AIFNalscFv3、 AIFNalscFv4、 AIFNalscFv5> AIFNalscFv6> AIFNalscFv7 隶属于抗体重链家族 VH3, 抗体轻链家族 VL1 , AIFNalscFv8、 AIFNalscFv9隶属于抗体 重链家族 VH3， 抗体轻链家族 VL3 。 抗体蛋白功能由存在于抗体基因轻链和重链可变区 的决定族互补区域 CDR1、 CDR2和 CDR3中特异性核苷酸序列及其互补所决定， 6个相应 的 CDR 区氨基酸序列构成了抗体的特异性抗原结合区域， 决定本发明中每个抗体的抗原 结合特征和抗 huIFN-cdb功能特征。 决定每株中和抗体功能的抗体轻链和重链可变区氨基 酸详细序列及其比较结果如图 2所示， 图中" - "符号表示与第一行抗体序列相同的氨基酸， 阴影部分为 CDR区。
在本发明中， 抗体的轻链可变区和重链可变区氨基酸序列分别如以下各组所示： SEQ ID No.l和 2所示的氨基酸序列、 SEQ ID No.3和 4所示的氨基酸序列、 SEQ ID No.5和 6 所示的氨基酸序列、 SEQ ID No.7和 8所示的氨基酸序列、 SEQ ID No.9和 10所示的氨基 酸序列、 SEQ ID No.ll和 12所示的氨基酸序列、 SEQ ID No.13和 14所示的氨基酸序列、 SEQ ID No.15和 16所示的氨基酸序列以及 SEQ ID No.17和 18所示的氨基酸序列。
应当理解， 在不影响抗体活性的前提下， 本领域技术人员可根据序列表 SEQ ID Ν 1~18所示的氨基酸序列进行各种取代、 添加和 /或缺失一个或几个氨基酸获得具有同等 功能的氨基酸序列， 例如在非高变区将具有类似性质的氨基酸进行替换。 本发明包括所述 的抗体经过改造得到的各种衍生抗体。 氨基酸的替换按如下各组进行：
组 1: leucine (亮氨酸） , isoleucine (异亮氨酸） , norleucine (正亮氨酸） , valine (缬 氨酸） ， norvaline (正缬氨酸）， alanine (丙氨酸）， 2-aminobutanoic acid ( 2-氨基丁酸）， methionine (蛋氨酸） ， O-methyl serine ( 2-甲基丝氨酸） ， t-butyl glycine (叔丁基甘氨酸） ， t-butylalanine (叔丁基丙氨酸） ， cyclohexylalanine (环己基丙氨酸） ；组 2: aspartic acid (天 门冬氨酸）， glutamic acid (谷氨酸）， isoaspartic acid (异天门冬氨酸）， isoglutamic acid (异 谷氨酸）， 2- aminoadipic acid ( 2-氨基己二酸）， 2-aminosuberic acid ( 2-氨基辛二酸）； 组 3: asparagine (天门冬酰胺）， glutamine (谷氨酰胺）；
组 4： lysine (赖氛酸)， arginine (精氣酸)， ornithine (乌氣酸) , 2,4-diaminobutanoic acid ( 2，4-二氨基丁酸）， 2,3-diaminopropionic acid ( 2,3-二氨基丙酸）；
组 5: proline (脯氨酸）， 3-hydroxyproline ( 3-羟基脯氨酸）， 4-hydroxyproiine ( 4-羟基 脯氨酸）；
组 6: serine (丝氨酸）， threonine (苏氨酸）， homoserine (高丝氨酸）；
组 7： phenylalanine (苯丙氨酸）， tyrosine (酪氨酸）
组内各氨基酸之间的替换并不改变抗体蛋白的活性， 由这些改变得到衍生抗体。 此外， 考虑到密码子的简并性， 例如可在其编码区， 在不改变氨基酸序列的条件下， 对编码上述抗体的基因序列进行修改， 获得编码相同抗体的基因。 例如本领域技术人员可 以根据表达抗体宿主的密码子偏爱性， 人工合成改造基因， 以提高抗体的表达效率。
可将上述编码单链抗体的基因克隆到表达载体中， 进而转化宿主， 通过诱导表达获得 单链抗体。
此外， 可将上述抗体的轻链编码基因和重链编码基因分别克隆到全抗表达载体中， 并 导入宿主细胞中， 获得表达抗干扰素 α的全抗免疫球蛋白。
在本发明实施例中，将上述 3株 scFv抗体 ( AIFNal scFvl . AIFNal scFv2 , AIFNal scFv3 ) 的轻链和重链基因， 分别克隆入全抗体表达载体 pAC-K-CH3并转染昆虫 Sf 细胞， 利用 杆状病毒 /昆虫细胞系统实现了全抗体的分泌型表达， 得到全抗体 AIFNal IgGl、 AIFNal IgG2和 AIFNal IgG3。
用 ELISA和 Western Blot对所获人源单抗的免疫学特性进行鉴定。结果表明三株人源 单克隆抗体均特异性针对 huIFN-alb， 而对其他结构相近的干扰素家族抗原如 huIFN-a2b 和 huIFN-γ则没有反应性。 Western Blotting实验结果也表明， 3株抗体只特异性识别变性 的 huIFN-alb蛋白。
釆用体外细胞学实验测定了所获得的三株全抗体的免疫中和活性，结果显示其中一株 具有拮抗基因工程来源的 huIFN-odb和系统性红斑狼疮患者外周血清介导的干扰素刺激基 因的表达上调。
本发明在国际上首次获得 9株抗 huIFN-ctlb的 scFv噬菌体抗体， 并在原核与真核系 统中表达了 3株 scFv段抗体及其全抗体，完成了全抗体体外免疫调节功能的检测，这一结 果的获得为系统性红斑狼疮的治疗带来了希望。 利用上述获得的人源中和性抗 huIFN-alb 基因工程抗体可变区基因以及上述每个抗体基因特征下的全抗体基因， 可以在原核细胞、 酵母细胞、 真核细胞及任何重组系统中表达和生产此抗体或以此为基础的改建后的含有此 抗体基因的任何其他基因， 获得具有中和 huIFN-alb免疫学活性的抗体产物， 制成临床上 用于治疗由系统性红斑狼疮病人体内外周 huIFN-cdb过量引起的全身性疾病的特异性抗体 药物， 从而为治疗系统性红斑狼疮提供新的手段。 基于上述抗体基因及其直接或间接的基 因产物， 可制成注射型抗体制剂用于人体内由干扰素过量引起的多种自身免疫疾病的治 疗。 附图说明
图 1显示的是 phage-ELISA验证 9株噬菌体单链抗体对 huIFN-alb的结合特异性； 图 2显示的是本发明抗人 IFN-alb抗体可变区氨基酸序列的比较图；
图 3显示的是 ELISA检测 5株单链抗体的结合特性；
图 4显示的是用 Western Blot检测原核表达的 3株单链抗体的结合特性；
图 5显示的是用 Pull Down检测表达的株人的特异性结合情况图；
图 6显示的是纯化后 IgG的 SDS-PAGE电泳图；
图 7显示的是 ELISA检测 5株 IgG全抗体的结合特性；
图 8显示的是用 Western Blot检测表达的 3株人的特异性结合情况图；
图 9 显示的是 IFN- a及抗 huIFN-alb 抗体处理正常人 PBMCs后 IFN- a诱导基因 ISG15的变化情况；
图 10 IFN- a及抗 huIFN-alb抗体处理正常人 PBMCs后 IFN- a诱导基因 ITIF-1的变 化情况；
图 11显示的是加入 SLE病人（IFN-a升高）及正常人血清并用抗 huIFN-alb抗体中 和后正常人 PBMCs中 IFN- cc诱导基因 ISG15的变化情况。 具体实施方式
以下实施例进一步说明本发明的内容， 但不应理解为对本发明的限制。在不背离本发 明精神和实质的情况下， 对本发明方法、 步骤或条件所作的修改或替换， 均属于本发明的 范围。
若未特别指明， 实施例中所用的技术手段为本领域技术人员所熟知的常规手段。 实施例 1
本例运用噬菌体表面呈现技术， 构建了人源全合成基因工程抗体文库。 用纯化的 huIFN-alb 对全合成噬菌体抗体库进行富集筛选， 并在 E. coli 中进行分泌表达。 通过 ELISA 、 Western Blot及 Ρ μ 11 Down鉴定 scFv抗体对 huIFN-alb特异性结合的功能活性， 并进行序列测定。 然后将阳性克隆的轻链和重链可变区基因， 分别克隆入全抗体表达载体 pAC-K-CH3 转染昆虫 Sf 细胞， 利用杆状病毒八¾虫细胞系统实现全抗体的分泌型表达。 用 ELISA和 Western Blot对所获人源单抗的免疫学特性和免疫调节功能进行鉴定。 材料与方法
1. 细胞、 载体和抗原制备
人源全合成抗体库由军事医学科学院构建 （杜威世， 王双， 孙志伟等.全合成人源性噬 菌体抗体库的构建. 军事医学科学院院刊， 2006， 30:319-322 ) , 筛选抗体库的抗原为原核 构建表达纯化的 huIFN-alb (浓度为 lmg/ml ) , 菌株为 XLI-Blue (美国 Stratagene ) ； 所 用噬菌体为 M13K07 (美国 Invitrogene 公司）。 杆状病毒表达载体为 pAC-K-CH3 (德国 PROGEN PR3003 ) ( Liang, M. F., Stefan, D.， Li, D. X., Queitsch, I" Li, W., and Bautz, E. F. Bac lovirusexpression cassette vectors for rapid production of complete human IgG from phage displayselected antibody fragments. Journal of Immunological Methods.247: 119-130. ) , 昆虫 细胞 Sf 及 293T细胞来自美国细胞培养中心 （ATCC ) 。 huIFN-alb的构建与表达参考了 文献（李武平，吕宏亮，侯云德等. 干扰素 -Plb的高效表达、 纯化及抗病毒活性研究. 病毒学 报， 2002年 第 03期） ， 并作了修改， 具体方法如下： PCR 扩增人干扰素 alb ( huIFN-alb ) 基因序列， 测序鉴定后， 将其克隆入原核表达载体 pET30a (美国 invitrogen), 通过将重组质 粒转化大肠杆菌 RossetaTM(DE3)进行蛋白表达。 表达的 huIFN-alb经 Ni金属螯合亲和层析纯 化。
2. 噬菌体抗体库的富集筛选
筛选抗原为 huIFN-alb, 使用时用 l xPBS (NaCl 8g , C1 0.2g ， Na2HP04 1.44g ， KH2PO40.24g溶于 800ml ddH2O中， 用 HC1调 pH至 7.4, 定容到 1L)稀释至工作浓度， 包被免 疫管。 富集筛选方法基本按文献进行 （杜威世， 王双， 孙志伟等.全合成人源性噬菌体抗体 库的构建. 军事医学科学院院刊，2006， 30:319-322 ), 具体如下：
用 l xPBS稀释的纯化 huIFN-alb抗原 （ lO g/ml )溶液包被免疫管， 每孔 lml, 4'C过夜； 次日用 l xPBST ( l xPBS, 0.05%Tween20, pH7.2 )洗去未吸附的抗原， 用含 4 % BSA蛋白的 lxPBS 溶液 37°C封闭 1小时， 弃封闭液； 每孔加入 90μ1噬菌体抗体库（MOI为 1012, 各子库 按库容量以 1 : 1混合投入筛选）， 37°C孵育 2小时， 弃去孔中未结合的噬菌体， 并用 1.5ml 的 l xPBST水平震荡洗涤 （450rpm ), 共 10遍， 以充分去除未吸附的噬菌体； 最后用 l xPBS 洗三遍， 吸净孔中的液体； 后每孔加入 lml Glycine-HCl(100mM, pH2.2)的洗脱液， 室温震 荡洗涤（600rpm ) 10分钟。 加入适量的 2M Tris-HCl(100mM, pH=2.2), 以中和含噬菌体的 洗脱液； 将洗脱的噬菌体立即加入 5 ml新鲜制备的 XLI-Blue菌液中 （ OD6()() = 0.7 ), 室温孵 育 15 - 20分钟； 然后 150ipm, 37°C震荡培养 1小时， 立即取 10 μΐ涂布含 CTG(10(^g ml氯霉 素， 4(Vg/ml安苄青霉素， 40mM葡萄糖)的 LB培养平皿用以滴定噬菌体； 其余细菌根据噬 菌体的产出量涂 CTG培养平板， 37'C细菌培养箱培养过夜。 次曰用涂布棒将平板上长出的 细菌全部刮下，取部分刮下的细菌加入到 100ml的 CTG液体培养基中至 OD600-0.3 , 继续培 养细菌至浓度达到 OD600=0.5， 加入辅助噬菌体 M13K07(滴度为 1012/ιη1)200μ1 , 37Ό静置 15-20min后， 37°C l50rpm振荡培养 1小时， 加入卡那霉素（终浓度 5(^g/ml ), 37'C培养过夜。 将培养过夜的细菌悬液 6500rpm离心 15min, 收集细菌培养上清， 将上清转入干净的三角烧 瓶中， 加入 4%的 PEG8000,3%的 NaCl , 充分溶解后冰洛 30min以上。 9000rpm离心 20min, 弃上清。 沉淀用 PBS溶解， 冰浴 15min, 12000rpm离心 5min， 得到的上清即为第一轮富积 得到的抗体库， 将上清转入 Eppendorf管中， 加 3%的 BSA冻存备用。 滴定噬菌体滴度后， 进行下一轮筛选。 如此反复筛选 3次。 挑取第三轮富集筛选得到的克隆， 采用 phage-ELISA 对噬菌体抗体的结合活性进行鉴定， 对鉴定得到的阳性克隆， 测序获得抗体基因序列。
3. scFv段抗体的诱导表达与纯化
人源中和性抗 huIFN-alb基因工程 scFv抗体可溶性表达产物的制备基本按文献进行 ( Timothy J. LaRocca, Laura I. Katona, David G. Thanassi et al. Bactericidal Action of a Complement-Independent Antibody against Relapsing Fever Borrelia Resides in Its Variable Region. The Journal of Immunology, 2008, 180: 6222 -6228 ) , 具体为： 将带有阳性抗体轻、 重链基因插入的阳性克隆扩增后按常规方法提取质粒 DNA, 并用 5'端分别带有 Ncol和 Xhol 的 PCR 引 物 扩 增 scFv 抗 体 基 因 （ 上 游 引 物 scFv-pET22-L ： CATGCCATGGCCGATATCGTTCTGAC , 下 游 引 物 scFv-pET22-R ： CCGCTCGAGGCTCGACACGGTCACCAGAG ) , PCR片段回收后经 Ncol和 Xhol酶切， 与 相同双酶切的 pET22b载体片段连接， 获得重组单链抗体表达质粒， 转化 BL-21 ( DE3 )感 受态细胞， 过夜培养后， 挑取氨苄抗性的阳性单个菌落， 接种 2χΥΤ细菌培养液扩大培养， PCR鉴定片段插入完整后， 以 1/100比例将阳性工程菌在新的 2χΥΤ细菌培养液中培养至 OD6Q() = 0.7时， 加入 20μΜ IPTG, 20°C诱导表达过夜。 收获细菌， 离心后弃上清， 在沉淀 中加入原培养液 1/10体积的 PBS ( 0.02 M pH7.4 )重悬， 反复冻融三次, 4Ό 12000 rpm离心 30分钟， 上清中含有诱导表达的 scFv抗体， 可用于进一步的免疫学特性鉴定。 阴性对照为 载体 pET22b转化菌按同样方法制备的细菌裂解液。单链抗体的纯化采用 Ni金属螯合亲和层 析法， 按试剂盒中的常规步骤进行。
4. 人源抗 huIFN-alb单链抗体的免疫学特性检测
4.1 phage-ELISA检测 scFv噬菌体抗体的表达
用 0.1M NaHC03 ( pH9.6 ) 的包被液稀释 huIFN-alb2 g/mL, 取 ΙΟΟμΙ加入聚氯乙烯 96 孔平底板中， 4°C包被过夜； 4%脱脂奶封闭， 37°C孵育 lh后， 弃上清， 加入表达的 scFv噬 菌体抗体， 37°C孵育 lh后， PBST洗涤三次， 加入酶标抗 M13二抗（美国 Sigma, 1:4000稀 释使用）， 37°C孵育 lh; PBST洗涤五次后， 加入 100 L显色液（A+B )显色， 最后加入 2M H2S04终止反应， 酶标仪检测吸光度 A ( 450 ) 值。
4.2 Western Blot检测 scFv单链抗体的分泌表达
单链抗体分泌表达上清经超滤浓缩后进行 10%的 SDS-PAGE电泳， 对应的阴性对照为 载体 pET22b转化菌按同样方法制备的细菌超滤浓缩的细菌表达上清。采用半干法将聚丙烯 酰胺凝胶电泳后的蛋白质转移到 NC膜。转膜后对凝胶进行考马斯亮兰染色以检査蛋白质是 否转移完全。 将含标准分子量蛋白的泳道剪下， 标注标准分子量参照蛋白位置。 将 NC膜在 含 5 %脱脂奶粉的 1 xPBS中室温封闭 2小时， 1 xPBST洗涤 3次后与 HPR标记的鼠抗 His抗体 ( Sigma公司， 1 :500使用）室温反应 1小时。 lxPBST洗涤 3次后放入 DAB底物显液色中显色， 至棕色阳性条带出现， 将 NC膜置于双蒸水中终止反应， 干燥后避光保存。
4.3 Western Blot检测 scFv单链抗体的结合特异性
釆用纯化的 huIFN-ctlb、 huIF -a2b和 huIFN-γ为抗原用以 Western Blotting的方法鉴定单 链抗体的结合特异性。 具体如下： 将上述纯化的三种抗原 （5 g )经 SDS-PAGE电泳后， 釆 用半干法转移到 NC膜 转膜后对凝胶进行考马斯亮兰染色以检查蛋白质是否转移完全。 将 标准分子量蛋白泳道剪下， 标出分子量标准的参照蛋白位置。 对 NC膜采用含 5 %脱脂奶室 的 lxPBS温温封闭 2小时。 将含有 AIFNalscFvl、 AIFNalscFv2, AIFNalscFv3表达产物原液 的一抗工作液与封闭的 NC膜在室温孵育 2小时。 lxPBST洗涤 3次后与 HPR标记的抗 His抗体 ( Sigma公司， 1:1000使用）室温反应 1小时， lxTBST洗涤 3次后， 放入 DAB底物显液色中， 至棕色阳性条带出现， 将 NC膜置于双蒸水中适时终止反应， 干燥后避光保存。
4.4 P^l Down检测 scFv单链抗体的结合特异性
将 5(^g/ml单链抗体与 10(^g/ml huIFN-otlb于 4°C孵育 1小时， 后加入 4 g的 Ni-resin (结 合蛋白的能力为 8μ /μ1 ) 继续孵育 1小时， 3000rpm离心 5min, 弃上清， 沉淀以 lxPBS洗涤， 3000rpm离心 5min收集沉淀， 如此洗涤 3-5遍后， 用 20μ1 lxPBS重悬沉淀， 并加入蛋白上样 缓冲液煮沸处理 5分钟， 并进行 15%SDS-PAGE电泳， 然后常规的考马氏蓝染色和脱色后检 测。
5. 人源 scFv单链抗体可变区基因的核酸序列分析
用 Qiagen Miniprep Kit (德国 QIAGEN) 制备质粒 DNA进行核酸序列分析。测序引物为 5'-AGCCCACCTCAACGCAATT-3，。 测序结果和 Internet V-Base基因库中抗体基因序列进 行序列比对。
6. 全抗体重组表达质粒的构建
将获得的 3。？ 抗体的重链（3£0 10 0.20、350 10 >40.22、8£(3 10 ^0.24、 SEQ ID N0.34> SEQ ID N0.36 ) 以引物 （ VH3L:gtaactcgagAGCGGTGGCGGTCTGGTG， VHR: gaagctagcGCTCGACACGGTCACCAGAGTG ) PCR扩增后用 Xhol/Nhel双酶切， 连接入 PAC-K-CH3 载体（德国 PROGEN PR3003 ) , 再将轻链（ SEQ ID ΝΟ·19、 SEQ ID N0.21. SEQ ID NO,23、 SEQ ID N0.33 , SEQ ID N0.35 ) 以引物
( vy31:gtaagagctcACCCAGCCGCCGAGCGTG, VY1L: gtaagagctcACTCAACCTCCGTCTGT TTCTGG, VY3R: CTTGAAGCTTGGTGCCACCGCCAAAC )PCR扩增后经 Sacl/Hindlll 位点连接进去， 构建成全抗体表达载体。
7. 转染和扩毒
采用美国 Pharmogen 公司的 Bac loGold 共转染试剂盒进行转染。 操作方法略述如 下： 将 5 g的重组质粒 DNA与 0.5 g的 Bac loGold 线性 DNA 混合后， 并加入转染试剂转 染生长密度为 50 %的 Sf9细胞， 27°C培养 4天后， 收集含有重组病毒的毒种细胞培养上清进 行病毒滴定和扩增。 具体操作见 Bac lovirus expression vector system 手册。
8. 全抗体 IgG分泌表达和纯化
将重组病毒感染生长密度 70 %左右的 Sf9 细胞， 27Ό吸附 lh. 改用 SF-900 II无血清培 养液， 27Ό培养 3 ~ 5天后收集上清。 并采用 Protein-A亲和层析法（Amersham, 美国）直接 纯化诱导表达上清 ( Harlow E ， Lane D."Antibodies ： A Laboratory Manual". Cold Spring Harbor Laboratory Press , Cold Spring Harbor ,NY,1988. ) 。
9 . 抗 huIFN-alb人源 IgG抗体的免疫学特性检测
9.1 昆虫细胞表达 IgG抗体的免疫荧光 （IFA )检测
利用已获得的抗人 huIFN-alb的 IgG全抗体基因的重组质粒， 转染 Sf 细胞， 经过 3次扩 增获得重组杆状病毒， 感染 Sf 细胞， 27Ό培养约 4-5天， 收获细胞， l xPBS洗涤一次， 滴 加至载玻片上， 吹干， 在丙酮中固定 15min并干燥， 制成病毒抗原片。 滴加 FITC 标记的抗 人 IgG-Fc抗体溶液（美国 Sigma, 1 :80 ) , 37°C温育 30 min后， lxPBS冲洗， 晾干， 显微镜 下观察 Sf 细胞的荧光表达率和表达强度， 阴性对照细胞为转染空载体的 sf 细胞。
9.2 IgG全抗体特异性结合 huIFN-alb的 ELISA检测
用 0.1M NaHCO3 ( pH9.6 ) 的包被液稀释 huIFN-alb、 IFN-a2b、 IFN-γ. BSA至 2 g/ml, 取 ΙΟΟμΙ加入聚氯乙烯 96孔平底板中， 4°C包被过夜； 4%脱脂奶封闭， 37Ό孵育 lh后， 弃上 、 清， 加入 sf 细胞表达的全抗体 IgG上清， 37°C孵育 lh后， PBST洗涤三次， 加入 HRP标记 的抗 IgG-Fc二抗 （美国 Sigma, 1 ： 1000稀释使用） ， 37°C孵育 lh; PBST洗涤五次后, 加 入 100 L底物显色液（A+B )显色，最后加入 2M H2S04终止反应，酶标仪检测吸光度 (A=450) 值。
9.3 IgG全抗体特异性结合 huIFN-alb的 Western Blotting检测
将纯化的 huIFN-alb抗原经 SDS-PAGE电泳后， 半干法转移到 NC膜。 对 NC膜在含 5 % 脱脂奶粉的 1 xPBS室温封闭 2小时后， 分别置于含 AIFNallgGl、 AIFNalIgG2和 AIFNalIgG3 表达产物的一抗工作液中室温孵育 2小时， l xPBST洗涤 3次， 并于 HPR标记的抗 IgG-Fc二抗 ( Sigma公司， . 1 : 1000使用）室温反应 1小时。 l xPBST洗涤 3次后， 置于 DAB底物显液色中， 至棕色阳性条带出现适时终止反应， 避光保存。
10 . 抗 huIFN-alb人源 IgG抗体免疫中和活性的检测
10.1 抗 huIFN-alb人源 IgG抗体中和活性检测
收集正常人新鲜外周血标本， 密度梯度离心法分离获得外周血单个核细胞（PBMCs ), 将获得的 PBMCs 以 Ι χ ΙΟ5/孔的细胞量种入 ％ 孔培养板， 分别加入合成的人 IFN-a ( l OOng/mL )和不同浓度的抗 huIFN-alb抗体（(^g/mL、 3μg/mL. 12 g/mL ) ， 37°C和 5%C02条件下孵育 4小时，后收集细胞，检测上述不同培养条件下 IFN-a诱导基因（ISG15 和 IFIT-1 )的表达差异来评估抗 huIFN-alb抗体的免疫中和活性。收集的细胞采用 TRIzol™ 试剂 （Invitrogen公司）抽提 RNA, 以随机引物进行逆转录（ ReverAid™ Μ-Μμΐν逆转 录酶， Fermentas公司）获得 cDNA,并釆用实时定量 PCR ( SYBR premix Ex Taq™ , Takara 公司）技术检测样本中 ISG15和 ITIF-1基因的表达。 ISG15和 IFIT-1的引物序列分别为： ISG15 forward: 5，-GAG AGG CAG CGA ACT CAT CT-3'; Reverse: 5，-AGC TCT GAC ACC GAC ATG G-3'; IFIT-1 forward: 5 '-GCA GAA CGG CTG CCT AAT TT-3 '； Reverse: 5'-TCA GGC ATT TCA TCG TCA TC-3\ PCR反应的内参基因的引物序列如下： GAPDH ( 200nM forward ) 5 '-CTGGAACGGTGAAGGTGACA -3， ， ( 200nM reverse ) 5'-
AAGGGACTTCCTGTAACAATGCA -3，。 经 7500 Real-time PCR 系统 (Applied Biosystem) 进行 40个循环后计算得到 Ct值，以 2'Δα值代表目的基因的相对表达量（ -ACt=-(Ct n -Ct 对样品获得的数据进行平均值和 SD值的统计分析。
10.2 抗 huIFN-alb人源 IgG抗体中和 SLE病人血清 IFN-α的效应检测
为了评估抗 huIFN.-al b抗体是否具有临床应用的价值，在上述中和活性试验的基础上， 本实验组又利用含升髙的 IFN-a的 SLE病人血清及正常人对照血清（以 1/10体积比加入 培养基）处理正常人 PBMCs, 并加入抗 huIFN-alb抗体（ 24 g/mL )对血清中 IFN-a的作 用进行中和， 37°C和 5%C02条件下孵育 4小时后， 收集细胞检测 IFN-a诱导基因（ISG15 ) 的表达差异来评估抗 huIFN-alb抗体对血清中 IFN-a的免疫中和活性。 方法同上， 即收集 的细胞采用 TRIzol™ 试剂 （Invitrogen 公司） 抽提 RNA， 以随机引物进行逆转录 ( ReverAid™ Μ-Μμΐν逆转录酶， Fermentas公司）获得 cDNA,并采用实时定量 PCR( SYBR premix Ex Taq™ , Takara公司）技术检测样本中 ISG15基因的表达。 所用 ISG15及内参基 因引物序列同上。经 7500 Real-time PCR系统 (Applied Biosystem)进行 40个循环后得到 Ct 值， 以 2— AC i代表目的基因的相对表达量（-ACt=-(Ct s -Ct ^¾H) )。 对样品获得的数据 进行平均值的统计分析。
11. 非高变区突变后的抗体对 huIFN-odb抗性的研究
基于 AIFNallgGl轻链可变区氨基酸序列， 将 SEQ ID No.l所示氨基酸序列的第（44 ) 位的（丙氨酸， A )替换为 （缬氨酸， V ) , 将其重链可变区的第（40 )位的（丙氨酸， A ) 替换为 （纈氨酸， V )。 分别合成 AIFNallgGl的轻链编码核酸序列 （在相应位置将密码子 GCA替换为 GUU或 GUG或 GUA或 GUC )以及重链编码核酸序歹 ' 在相应位置将密码子 GCA 替换为 GUU或 GUG或 GUA或 GUC ) 。 按照上述 6~10的方法， 将轻链基因和重链基因克隆 到 PAC-K-CH3中， 并转染昆虫 Sf9 细胞， 利用杆状病毒 /昆虫细胞系统实现全抗体的分泌 型表达， 并对该突变体进行免疫学检测。 结果
1. 人源抗人 huIFN-alb抗体库的筛选
以纯化的 hnIFN-alb ( l(Vg/ml )为抗原对全合成抗体库进行筛选， 经 3轮筛选后随机挑 取 1500个克隆扩增培养收集上清。 以纯化的 huIFN-alb抗原（2 g/ml )包被 96孔板， 加入待 测样品上清， 通过 HRP标记的抗人 M13二抗（sigma公司， 1 :4000稀释使用）检测待测样品 中抗体表达阳性率。 结果显示， 经三轮筛选后， 共获得 300株人源 scFv表达阳性克隆。 300 株人源 scFv表达阳性克隆中， 通过 phage-ELISA, 釆用纯化的 huIFN-alb、 huIFN-a2b和 huIFN-γ抗原对抗体的结合特异性进行复筛检测， 获得 100个与 huIFN-alb特异性结合的克 隆。
2. 人源抗人 IFN抗^ scFv抗体的序列分析
用 DNASTAR、 Mega3.0序列分析软件进行分析处理， 比较 Internet V-Base基因库中的 IgG序列， 上述 100株人源抗 huIFN-alb基因工程抗体中， 对 86株抗体的序列进行了测序， 86个克隆分别属于 9个不同的抗体序列， 共发现 9株带有不同的抗体轻重链可变区序列及其 组合的抗体， 其重链可变区主要分类在 IgG VH3家族， 其轻链可变区主要分类在 IgG VL1 和 VL3家族。 图 2为 9株人源抗 huIFN-alb基因工程抗体的可变区基因的氨基酸序列及其相 互比较， 图中以第一行抗体序列为比较的标准序列， "-"符号表示与第一行抗体序列相同的 氨基酸，阴影部分为 CDR区。具体的，人源单链抗体 AIFNal scFv 1的蛋白序列如 SEQ ID NO.1 和 2， 单链抗体 AIFNalscFv2的蛋白序列如 SEQ ID N0.3和 4, 单链抗体 AIFNal scFv3的蛋白 序列如 SEQ ID NO.5和 6, 单链抗体AIFNalscFv4的蛋白序列如SEQ ID N0.7和8, 单链抗体 AIFNal scFv5的蛋白序列如 SEQ ID NO.9和 10 , 单链抗体 AIFNal scFv6的蛋白序列如 SEQ ID NO.l l和 12 , 单链抗体 AIFNalscFv7的蛋白序列如 SEQ ID N0.13和 14 , 单链抗体 AIFNalscFv8的蛋白序列如 SEQ ID N0.15和 16, 单链抗体 AIFNalscFv9的蛋白序列如 SEQ ID N0.17和 18。
3.人源抗 huIFN-alb噬菌体单链抗体的结合特性
为了鉴定这 9株不同的噬菌体抗体能否稳定结合 huIFN-alb, 我们采用 phage-ELISA对 9 株抗体的结合特异性和稳定性进行多次验证， phage-ELISA实验中釆用了 3种抗原， 分别为 huIFN-alb、 huIFN-a2b和 huIFN-γ, ELISA测定结果显 示， 其中有 5株抗体与 huIFN-alb结 合的 ELISA值明显高于 huIFN-a2b和 huIFN-γ, 有两倍以上差异， 结果表明， 最后获得 5株抗 体（AIFNalscFvl、 AIFNalscFv2> AIFNalscFv3、 AIFNalscFv8、 AIFNalscFv9 )能够稳定 而且特异性结合 huIFN-alb, 而与无关抗原 huIFN-a2b和 huIFN-γ无交叉反应， 结果见图 1。
4. 人源抗 huIFN-alb单链抗体的结合特异性
为了进一步证实所获得的 5株重组 scFv抗体是特异性的针对 huIFN-alb, 本发明进一步 通过 ELISA、 Western Blot和 Pull Down鉴定原核分泌表达的 scFv抗体的功能活性。 ELISA结 果表明细菌中分泌表达的单链抗体 AIFNalscFvl、 AIFNalscFv2、 AIFNalscFv3与 huIFN-alb 反应， 但与重组蛋白 huIFN-a2b、 huIFN-γ不反应， 如图 3所示。 Western Blotting结果表明 AIFNalscFvl、 AIFNalseFv2、 AIFNalscFv3可分别与变性的 huIFN-alb反应，但与重组蛋白 huIFN-a2b、 huIFN-γ不反应，如图 4所示。这提示 AIFNalscFvl、 AIFNalscFv2、 AIFNalscFv3 所针对的 huIFN-alb的抗原决定簇为线性抗原决定簇。 Pull Down结果表明表明 AIFNalscFvl . AIFNalscFv2、 AIFNalscFv3可与 huIFN-alb反应形成复合物， 如图 5所示。
5. 全抗体 IgG表达和纯化
将 3株经过结合特异性验证的 scFv抗体（AIFNalscFvl , AIFNalscFv2、 AIFNalscFv3 ) 的轻链和重链基因， 分别克隆入全抗体表达载体 PAC-K-CH3 转染昆虫 Sf 细胞， 利用 杆状病毒 /昆虫细胞系统实现全抗体的分泌型表达。 釆用 Amersham公司的 Protein-A 亲和 层析柱直接纯化表达上清， 通过 SDS-PAGE检验全抗体 IgG的表达及纯化情况， 结果证实 得到较纯蛋白， 可清晰观察到解链后的抗体轻、 重链， 分别位于约 28KD、 55KD处， 通过 非变性的 SDS-PAGE分析， 得到未解链的抗体蛋白， 位于 150 KD处左右， 如图 6所示。
6. 人源抗 huIFN-alb全抗体的结合特异性
为了证实所获得的重组 IgG抗体（AIFNalIgGl、 AIFNalIgG2 AIFNalIgG3 )是特异 性的针对 huIFN-alb,本发明进一步通过 ELISA和 Western Blot鉴定全抗体 IgG的功能活 性。 ELISA结果与验证 scFv抗体功能活性相一致，纯化的全抗体 AIFNallgGl、 AIFNalIgG2、 AIFNalIgG3与 huIFN-alb反应， 但与重组蛋白 huIFN-a2b、 huIFN-γ和 BSA不反应， 如图 7所示。 Western Blotting结果表明 AIFNalIgGl、 AIFNalIgG2、 AIFNalIgG3可分别与变 性的 IFN-alb反应。 这提示 AIFNalIgGl、 AIFNalIgG2、 AIFNalIgG3所针对的 IFN-alb 的抗原决定簇为线性抗原决定簇。 如图 8所示。
7. 人源抗 huIFN-alb全抗体的免疫中和活性
为了证实合成的人源抗 huIFN-alb抗体是否具有免疫中和活性及对其中和活性进行初 步的评估，本发明将获得的的抗体（ AIFNalIgGl、 AIFNalIgG2、 AIFNalIgG3 )作用于 IFN-α 处理过的正常人 PBMCs, 发现正常人 PBMCs在 IFN-α的作用下， IFN-a诱导基因（ ISG15 和 IFIT-1 ) 的表达明显升高， 但是加入抗 huIFN-alb抗体后这两个基因的表达都被明显的 抑制，并且呈现比较好的剂量依赖性， 即随着加入的抗 huIFN-alb抗体的增加，抑制 IFN-a. 诱导基因表达的作用就越明显。 由此说明本次制备的人源抗 huIFN-alb抗体具有良好的中 和活性。 其对 ISG15和 IFIT-1的作用分别如图 9和图 10所示。 抗 huIFN-alb抗体对血清 中 IFN-a的中和作用也很明显。 在病人血清 IFN-a的作用下， IFN-a诱导基因 ISG15的表 达明显升高， 在抗 huIFN-alb抗体的作用下则可降低到正常血清处理或无刺激培养条件下 的基因表达水平， 如图 11所示。 提示本发明具有良好的治疗性应用前景。
8、 非高变区突变后的抗体对 huIFN-alb抗性影响
按照上述 6~10的方法， 将基于 AIFNallgGl修改后的轻链基因和重链基因克隆到 PAC-K-CH3中， 并转染昆虫 Sf 细胞， 利用杆状病毒 /昆虫细胞系统实现全抗体的分泌型 表达， 得到突变体 AIFNallgGl'。 对该突变体进行免疫学检测， ELISA结果表明纯化的全 抗体 AIFNallgGl '与 huIFN-alb反应， 但与重组蛋白 huIFN-a2b和 huIFN-γ不反应， 且亲和力 和特异性与 AIFNallgGl'基本相同。 Western Blotting 结果表明 AIFNallgGl'可与变性的 IFN-alb反应。 工业实用性
本发明公开的人源抗 huIFN-a抗体,其可制成临床上用于治疗由系统性红斑狼疮病人 体内外周 huIFN-alb过量引起的全身性疾病的特异性抗体药物， 从而为治疗系统性红斑狼 疮提供新的手段。 基于上述抗体基因及其直接或间接的基因产物， 可制成注射型抗体制剂 用于人体内由干扰素过量引起的多种自身免疫疾病的治疗。

Claims

权 利 要 求 书
1、 人源抗 huIFN-α抗体， 其轻链 CDR1、 CDR2和 CDR3以及重链 CDR1、 CDR2和 CDR3的氨基酸序列选自如下各组中的一组：
L-CDR1 L-CDR2 L-CDR3 H-CDR1 H-CDR2 H-CDR3
1 ). SGSSSNIGSNYVS DNNQRPS QVRDNNENEW SYAMS AISGSGGSTYYADSVKG YIDFG匪 DF
2) SGSSSNIGSNYVS DNNQRPS QSRDDSDSLL SYAMS AISGSGGSTYYADSVKG GSDFGDSFAF
3 ) SGSSSNIGSNYVS DNNQRPS QARDSGVGAF SYAMS AISGSGGSTYYADSVKG HWWAAMDY
4) SGSSSNIGSNYVS DNNQRPS QVRD EEF SYAMS AISGSGGSTYYADSVKG VYNIFDA
5 ) SGSSSNIGSNYVS DNNQRPS QA DASLLF SYAMS AISGSGGSTYYADSVKG QTYGRMDY
6 ) SGSSSNIGSNYVS DNNQRPS QVRDNDDFSF SYAMS AISGSGGSTYYADSVKG QHYSVFAA
7 ) SGSSSNIGSNYVS DNNQRPS SS EGGAKA SYAMS AISGSGGSTYYADSVKG T WMIFDD
8) SGDALGDKYAS EDSKRPS QVRDGSLFAL SYAMS AISGSGGSTYYADSVKG WRRFIRGVDP
9) SGDALGD YAS EDSKRPS QSKSAALLR SYAMS AISGSGGSTYYADSVKG LWRSKRWVRS
2、 如权利要求 1所述的抗体， 其特征在于， 其轻链可变区氨基酸序列和重链可变区 氨基酸序列选自以下各组中的一组： SEQ ID No.l和 2所示的氨基酸序列、 SEQ ID No.3和 4所示的氨基酸序列、 SEQ ID N 5和 6所示的氨基酸序列、 SEQ ID No.7和 8所示的氨基 酸序列、 SEQ ID No.9和 10所示的氨基酸序列、 SEQ ID No.11和 12所示的氨基酸序列、 SEQ ID No.13和 14所示的氨基酸序列、 SEQ ID No.15和 16所示的氨基酸序列以及 SEQ ID No.17和 18所示的氨基酸序列。
3、 如权利要求 2所述的抗体， 其为单链抗体或全抗体免疫球蛋白 IgG。
4、 权利要求 1~3任一项所述的抗体经过改造得到的衍生抗体， 所述改造包括氨基酸 的缺失、 替换和 /或插入， 并且不改变抗体的活性， 所述氨基酸的替换按如下各组进行： 组 1: 亮氨酸，异亮氨酸,正亮氨酸,缬氨酸，正缬氨酸,丙氨酸 ,2-氨基丁酸,蛋氨酸 ,2-甲基 丝氨酸，叔丁基甘氨酸,叔丁基丙氨酸，环己基丙氨酸；
组 2: 天门冬氨酸,谷氨酸,异天门冬氨酸，异谷氨酸, 2-氨基己二酸, 2-氨基辛二酸； 组 3: 天门冬酰胺,谷氨酰胺；
组 4: 赖氨酸，精氨酸,乌氨酸 ,2，4-二氨基丁酸, 2,3-二氨基丙酸；
组 5: 脯氨酸 ,3-羟基脯氨酸， 4-羟基脯氨酸； 组 6: 丝氨酸,苏氨酸，高丝氨酸；
组 7 :苯丙氨酸,酪氨酸，
组内各氨基酸之间的替换并不改变抗体蛋白的活性， 由这些改变得到衍生抗体。
5、 编码权利要求 1~4任一项所述抗体的基因。
6、 如权利要求 5所述的基因， 其特征在于， 编码轻链可变区的核苷酸序列和编码重 链可变区的核苷酸序列选自以下各组中的一组： SEQ ID No.19和 SEQ ID No.20所示的核 苷酸序列、 SEQ ID No.21和 SEQ ID No.22所示的核苷酸序列、 SEQ ID No.23和 SEQ ID No.24所示的核苷酸序列、 SEQ ID No.25和 SEQ ID No.26所示的核苷酸序列、 SEQ ID No.27 和 SEQ ID No.28所示的核苷酸序列、 SEQ ID No.29和 SEQ ID No.30所示的核苷酸序列、 SEQ ID No.31和 SEQ ID No.32所示的核苷酸序列、 SEQ ID No.33和 SEQ ID No.34所示的 核苷酸序列、 SEQ ID No.35和 SEQ ID No.36所示的核苷酸序列。
7、 含有权利要求 5或 6所述基因的表达载体。
8、 含有权利要求 7所述表达载体的宿主。
9、 权利要求 1~4任一项所述抗体在制备治疗 huIFN-α过量引起的疾病的药物中的应 用， 尤其是系统性红斑狼疮。
10、 含有权利要求 1~4任一项所述抗体的药物或检测试剂。
PCT/CN2010/000798 2009-06-04 2010-06-04 人源抗人干扰素α抗体及其应用 WO2010139196A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN200910085232.0 2009-06-04
CN2009100852320A CN101580546B (zh) 2009-06-04 2009-06-04 人源抗人干扰素α抗体及其应用

Publications (1)

Publication Number Publication Date
WO2010139196A1 true WO2010139196A1 (zh) 2010-12-09

Family

ID=41362878

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2010/000798 WO2010139196A1 (zh) 2009-06-04 2010-06-04 人源抗人干扰素α抗体及其应用

Country Status (2)

Country Link
CN (1) CN101580546B (zh)
WO (1) WO2010139196A1 (zh)

Families Citing this family (8)

* 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
US10112995B2 (en) 2013-07-03 2018-10-30 Immunoqure Ag Human anti-IFN-α antibodies
CN104292331B (zh) * 2013-09-26 2017-11-17 中国人民解放军军事医学科学院生物工程研究所 人源抗人α干扰素抗体及其应用
CN106589122B (zh) * 2015-10-20 2020-10-27 中国人民解放军军事医学科学院生物工程研究所 人源抗人多亚型干扰素α抗体及其应用
CN110922480B (zh) * 2019-12-01 2021-06-11 北京康普美特创新医药科技有限责任公司 一种抗C3d的靶向单链抗体和DAF的融合蛋白及应用
CN110922489B (zh) * 2019-12-01 2021-05-04 北京康普美特创新医药科技有限责任公司 一种抗C3d的靶向单链抗体和CD59的融合蛋白及应用
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
WO2023183288A2 (en) * 2022-03-22 2023-09-28 The Board Of Trustees Of The Leland Stanford Junior University Monoclonal antibodies for targeting the cardiac conduction system

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006002177A2 (en) * 2004-06-21 2006-01-05 Medarex, Inc. Interferon alpha receptor 1 antibodies and their uses
WO2006086586A2 (en) * 2005-02-10 2006-08-17 Baylor Research Institute Anti-interferon alpha monoclonal antibodies and methods for use
CN101580546A (zh) * 2009-06-04 2009-11-18 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

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006002177A2 (en) * 2004-06-21 2006-01-05 Medarex, Inc. Interferon alpha receptor 1 antibodies and their uses
WO2006086586A2 (en) * 2005-02-10 2006-08-17 Baylor Research Institute Anti-interferon alpha monoclonal antibodies and methods for use
CN101580546A (zh) * 2009-06-04 2009-11-18 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

Also Published As

Publication number Publication date
CN101580546B (zh) 2011-09-14
CN101580546A (zh) 2009-11-18

Similar Documents

Publication Publication Date Title
CN111620945B (zh) 一种抗新型冠状病毒的单克隆抗体或其衍生体
WO2010139196A1 (zh) 人源抗人干扰素α抗体及其应用
WO2015085847A1 (zh) Pd-1抗体、其抗原结合片段及其医药用途
CN109071637B (zh) 结合严重发热伴血小板减少综合征病毒的包膜糖蛋白的抗体及其用途
CN113354729B (zh) 一种抗新型冠状病毒的单克隆抗体及其应用
CA2768204A1 (en) Gram-positive bacteria specific binding compounds
KR20120088551A (ko) 보체 단백질 ｃ３ｂ를 표적화하는 항체를 위한 조성물 및 방법
CN110317267B (zh) 针对狂犬病病毒的双特异性抗体及其用途
CA2711029A1 (en) Anti mif antibodies
WO2021104053A1 (zh) 一种tslp相关病症治疗剂的开发和应用
KR102504884B1 (ko) Sftsv에 결합 가능한 나노 항체 및 이의 응용
CN112969714B (zh) 抗cd40抗体、其抗原结合片段及其医药用途
AU2006281981A1 (en) Chimeric antibodies with new world primate regions
CN113354730B (zh) 一种抗新型冠状病毒的单克隆抗体及其应用
US9017668B2 (en) High affinity human antibodies to human cytomegalovirus (CMV) gB protein
CN115515976A (zh) 冠状病毒抗体
CN118184774A (zh) 中和SARS-CoV-2及变异株的全人源抗体及应用
CN112513088B (zh) 抗ox40抗体、其抗原结合片段及其医药用途
CN113101364A (zh) 一种自免疫抑制剂的开发和应用
JP2018506296A (ja) Ｉｌ−１７ｃに対する抗体
JP2014526886A (ja) マクロファージ遊走阻止因子（ｍｉｆ）とｄ−ドーパクロームトートメラーゼ（ｄ−ｄｔ）に交差反応性がある抗体
CN116554323B (zh) 人源化抗il21抗体的开发和应用
US20230094083A1 (en) Human anti-grp94 antibodies and uses
WO2011137569A1 (zh) 人源抗狂犬病毒糖蛋白中和性抗体rvfab3
KR20220087457A (ko) Lif에 특이적인 결합 분자 및 이의 용도

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: 10782900

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: 10782900

Country of ref document: EP

Kind code of ref document: A1