WO2017128880A1 - 一种特异性抑制连接蛋白26的全人抗体 - Google Patents

一种特异性抑制连接蛋白26的全人抗体 Download PDF

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WO2017128880A1
WO2017128880A1 PCT/CN2016/109847 CN2016109847W WO2017128880A1 WO 2017128880 A1 WO2017128880 A1 WO 2017128880A1 CN 2016109847 W CN2016109847 W CN 2016109847W WO 2017128880 A1 WO2017128880 A1 WO 2017128880A1
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antibody
connexin
fully human
chain variable
variable region
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French (fr)
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曲志虎
杨光
马马诺·法比奥
范思科
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ShanghaiTech University
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ShanghaiTech University
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Priority to US15/781,463 priority patent/US11345746B2/en
Priority to EP16887751.2A priority patent/EP3342784B1/en
Priority to EP20196319.6A priority patent/EP3778633A1/en
Priority to ES16887751T priority patent/ES2824176T3/es
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/51—Complete heavy chain or Fd fragment, i.e. VH + CH1
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/524—CH2 domain
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/526—CH3 domain
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—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)
    • C—CHEMISTRY; METALLURGY
    • 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
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • the present invention belongs to the field of biomedical technology, in particular, the present invention provides a fibronectin 26 (Connexin26) antigen which specifically recognizes human cochlea and skin tissue, and can repress the hemichannel single-chain antibody formed by the connexin 26 and express the antibody.
  • the plasmid vector, and the use of the single-chain antibody in the preparation of a medicament and a diagnostic kit for treating deafness, skin and tumor caused by mutation of connexin 26, the present invention also provides a diagnostic kit for preparing the antibody, which comprises The monoclonal antibody serves as an active ingredient.
  • Ion channel gap junction is a cell-to-cell connection. It binds to the cytoplasm of the cell, allowing smaller molecules such as ions, metabolites, and second messengers less than 1.8 kD to pass freely [1, 2] .
  • gap junction channels composed of connexin (Connexin) mediate ion, small molecule nutrient exchange and signal molecule propagation between adjacent cells.
  • Connexin connexin
  • the gap junction channels composed of different connexins have different permeability characteristics. Adjacent cells use gap junction-mediated intercellular communication or pathways that do not rely on gap junction channels to transmit developmental signals. Regulate cell proliferation, migration and differentiation during development.
  • Connexin is a family of proteins that are widely expressed in vertebrate cells.
  • the hexamers composed of members of this family are located on the cell membrane to form gap junction channels or hemichannels, mediating the exchange of substances between cells, between cells and extracellular matrices.
  • the gene encoding the connexin family of genes in the genome constitutes a connexin gene family [3] .
  • connexin genes Twenty-one connexin genes have been found in the human genome so far, and 20 connexin genes have been found in the mouse genome [3] . Intercellular communication mediated by gap junction channels and connexin hemichannels is important for many cellular functions. Roles, including regulation of cell growth, differentiation and development [4-6] . Mutations in the connexin gene are associated with many human diseases, including cardiovascular abnormalities, peripheral neuropathy, cataracts, and deafness. Connexin 26 (Cx26), connexin 30 (Cx30) is involved in the inner ear potassium ion cycle, and deletion of Cx26 and Cx30 causes very severe deafness in the developing cochlea with concomitant cell death [4,7,8] .
  • Cx26 and Cx30 are expressed by two adjacent genes (GJB2 and GJB6), and Cx26 and Cx30 are connected to non-sensory cells in the inner ear to form a multicellular body, and the two proteins are co-expressed in the inner ear [9] .
  • DNFB1 is located on chromosome 13q11–q12 [10] . 50% of idioms before deafness are related to DFNB1, and the Mediterranean are as high as 79% [10,11] . Deletion mutations in Cx26 and Cx30 have profound effects on organ formation in cochlear spirals [12-14] .
  • Point mutations in connexins cause the hemichannels that form the connexin to remain in a normally open or normally closed state, leading to deafness and skin disease [15] .
  • Antibodies that specifically recognize Cx26 and restore its function are expected to restore deafness and skin diseases caused by connexin mutations.
  • monoclonal antibodies play an important role in the diagnosis and treatment of diseases, especially for diseases expressing specific antigens.
  • the detection of monoclonal antibodies and antigens can be used as a "golden” standard for disease diagnosis, and monoclonal antibodies are also used as drugs. It plays an important role in autoimmune diseases and anti-tumor diseases.
  • the object of the present invention is to provide a single-chain antibody which can specifically bind to human connectin 26 antigen, thereby being useful for preparing a disease drug or a connexin 26-related diagnostic reagent for treating a connexin 26 mutation. box.
  • the present invention provides a fully human antibody which specifically inhibits connexin 26, which is characterized in that it is a recombinant immunoglobulin, the structure is scFv-Fc, and the scFv refers to a single-chain antibody, including a heavy chain.
  • the variable region and the light chain variable region, the amino acid sequence of the heavy chain variable region thereof is SEQ ID NO: 1
  • the amino acid sequence of the light chain variable region is SEQ ID NO: 2
  • Fc refers to the constant region.
  • said constant region comprises a CH2 constant region and a CH3 constant region.
  • the fully human antibody that specifically binds to connexin 26 specifically recognizes the 41st to 75th amino acid portions of the extracellular region of connexin 26.
  • the present invention also provides a genetically engineered antibody which has a homologous sequence of 30% or more with the above-described single-chain antibody which specifically inhibits the human antibody of connexin 26.
  • the genetically engineered antibody comprises one of a Fab fragment, an F(ab)' fragment, an Fd fragment, an Fv fragment and an Fc fragment, a combination of two or more of the above respective fragments, or at least A derivative formed with other proteins or peptide chains.
  • the heavy chain CDR 3 region sequence of the genetically engineered antibody is DFSWRGYYMDV, light chain CDR3 region sequence Listed as QQYGSSPRT.
  • the invention also provides a nucleic acid sequence encoding a fully human antibody that specifically binds to connexin 26, the sequence of which is SEQ ID NO: 3 or SEQ ID NO: 4.
  • the nucleotide sequence encodes a fully human antibody that specifically binds to connexin 26 as described above.
  • the present invention also provides the use of the above-described fully human antibody or genetically engineered antibody that specifically inhibits connexin 26 in the preparation of a medicament or kit for treating deafness, skin disease or tumor caused by connexin 26 mutation.
  • the present invention also provides a medicament for treating deafness, a skin disease or a tumor comprising the above-described fully human antibody or genetically engineered antibody which specifically inhibits connexin 26 as an active ingredient.
  • Anti-Cx26-scFvII-Fc The fully human antibody specific for the inhibition of connexin 26 of the present invention is named Anti-Cx26-scFvII-Fc, and the single-chain antibody is selectively bound to the connexin and can specifically inhibit the half-ion channel formed by the connexin. Obtained after multiple rounds of phage library screening.
  • Anti-Cx26-scFvII-Fc is a recombinant single-chain antibody and therefore has an antibody structure, biochemical properties and biological functions different from IgG.
  • Anti-Cx26-scFvII-Fc single-chain antibody has a molecular weight of approximately 100Kd, recognizes and binds to the target epitope, which is a junctional extracellular domain structure, specifically recognizes a cell line overexpressing connexin 26, and antibodies and connexins. 26 colocalizes on the cell surface.
  • Functional experiments showed that Anti-Cx26-scFvII-Fc was able to inhibit the activity of the connexin 26 half-ion channel. Immunohistochemical staining of the antibody alone can more accurately diagnose the expression of human connexin 26 in various tissues.
  • Anti-Cx26-scFvII-Fc single-chain antibody has potential application value in the treatment of deafness, skin diseases and tumors and connexin 26 diagnostic reagents.
  • the present invention uses the 41st-56th amino acid sequence of the extracellular region of human connexin 26 as an antigen, and the sequence is KEVWGDEQADFVCNTL. Obtained by single-chain antibody phage display library and screening technology, the antibody provided by the present invention can specifically recognize the connexin 26 and inhibit the formation of hemichannel activity by biochemical analysis and immunofluorescence identification of the antibody. Animal experiments showed that the antibody significantly inhibited the hemichannel activity of the cochlear tissue sections of mice. Therefore, the antibody can be used for the treatment of a disease associated with a connexin mutation.
  • Biotinylated antigen polypeptide (sequence: KEVWGDEQADFVCNTL, SEQ ID NO: 5, hereinafter referred to as antigen), synthesized from Kingsray.
  • Full-body single-chain antibody phage display library laboratory-owned.
  • Streptavidinized magnetic beads were purchased from Thermo Fisher.
  • High-adsorption enzyme-linked immunosorbent microplate was purchased from Corning Incorporated.
  • Anti-M13 HRP antibody was purchased from Thermo Fisher.
  • Helper phage was purchased from Life Technologies, Cat. No. 18311019.
  • XL1-Blue bacteria were purchased from Agilent Technologies, Cat. No. 200228.
  • Developer solution ABTS solution was purchased from Thermo Fisher Company, article number 002024.
  • phage display library expressing human single-chain antibody (containing 1 ⁇ 10 11 phage particles) was mixed with 5 ⁇ g of antigen and incubated for 30 minutes at room temperature, followed by addition of 50 ⁇ l of streptavidinized magnetic beads.
  • the antigen-bound phage was captured by streptavidin-treated magnetic beads, the unbound phage was removed by rinsing with PBST, and the phage stably bound to the magnetic beads was eluted with a solution of glycine hydrochloride (pH 2.2).
  • 200 ml of XL1-Blue bacteria (Agilent Technologies, Cat. No. 200228) was inoculated.
  • helper phage containing 1 ⁇ 10 11 helper phage particles
  • each plate has 100-500 clones, pick a single clone, and pass the phage enzyme-linked immunosorbent assay Each round of phage libraries after panning was verified.
  • the phage enzyme-linked immunological reaction step is to inoculate the bacteriophage-infected XL1-Blue monoclonal bacteria into a 200-microliter 96-well plate, shake at 200 rpm and 37 ° C for 4-6 hours, and after detecting the OD close to 0.6, add 1 ⁇ l.
  • the helper phage was shaken overnight at 30 °C. The next day, 3000 g was centrifuged to take the supernatant for use.
  • the polypeptide antigen CB was screened in a fully human antibody phage display library. After 4 rounds of panning, the signal of the enzyme-linked immunoreactivity was increased compared with the control group, and the pool was selected from the pool after panning. Take 700 monoclonal antibodies for enzyme-linked immunoreactivity verification, and finally determine that the enzyme-linked immunoreactivity reading is more than twice the clone as a positive clone, as shown in Figure 1B, 150 positive clones were sequenced and analyzed, and the comparison was repeated. The greater the number of times, the stronger the affinity of the antibody sequence, and the effective enrichment sequence is determined accordingly. The positive clone selected in the present invention was repeated 84 times in 150 clone sequences, indicating that the affinity was strong.
  • the picked clones were sequenced and the nucleic acid sequence of the selected positive clone was SEQ ID NO:3.
  • Sypro Orange dye was purchased from Thermo Fisher.
  • the pFUSE expression vector pFUSE-hIGg1-FC2 was purchased from Invivogen, Inc., item number pfuse-hg1fc2.
  • HiTrapProtein A HP columns were purchased from GE.
  • the protein purification instrument was purchased from GE.
  • Real-time quantitative PCR instrument was purchased from BioRad.
  • the plasmid extraction kit was purchased from Qiagen.
  • BCA Protein Assay Kit (Pierce TM BCA Protein Assay kit, Pierce # 23253).
  • BamH1 and BglII restriction enzymes were purchased from NEB Corporation.
  • Example 3 The positive sequence obtained in Example 1 (ie, SEQ ID NO: 3) was synthesized by whole gene (Nanjing Kingsray Biotech Co., Ltd.) and BamH1 and BglII restriction sites were added at both ends of the sequence, and the obtained nucleic acid sequence was subjected to reference NEB.
  • the instructions were inserted into the pFUSE-hIGg1-FC2 expression vector (Invivogen Co., Ltd., the procedure of which is described in the specification) after digestion with BamH1 and BglII, and a eukaryotic antibody expression plasmid of the Fc-segment single-chain antibody Anti-Cx26-scFvII-Fc was obtained.
  • the 293Fectin transfection reagent (Invitrogen, Cat. No. 12347500) was mixed with the eukaryotic antibody expression vector plasmid obtained above in a volume ratio of 30 ⁇ l: 15 ⁇ g, and then 30 ml of 293 Freestyle suspension cells (purchased from Thermo Fisher Scientific) was added. After shaking at 1200 rpm at 37 ° C overnight, the supernatant was collected after centrifugation using HiTrap Protein A HP columns at Antibody protein purification (Anti-Cx26-scFvII-Fc) was performed on a protein purifier, and finally the antibody concentration was measured using a BCA protein quantification kit (Pierce, Cat. No. 23252) with reference to the instructions.
  • the obtained purified antibody was subjected to Western blot analysis, specifically: 5 ⁇ g of the purified antibody sample was subjected to SDS-PAGE electrophoresis, electrophoresis voltage was 110 V, 60 minutes, and then transferred to a cellulose acetate membrane, and peroxidized with goat anti-human horseradish.
  • the enzyme secondary antibody purchased from Thermo Scientific
  • the obtained purified antibody was subjected to thermal stability test, specifically: the purified antibody was diluted to 0.1 mg/ml, and the Sypro Orange dye was added at a volume ratio of 1:2000, and then sealed.
  • the real-time quantitative PCR instrument BioRad was set at a temperature of 25 ° C. Rise to 90 ° C, 0.5 ° C per minute. The fluorescence value is also detected.
  • the obtained purified antibody was subjected to LC-MS mass spectrometry, specifically: 5 ⁇ g of the purified antibody was mixed with 1 ⁇ l of protein deglycosidase (purchased from NEB Corporation, Cat. No. P0709S) at 37 ° C for one hour, and then loaded to high resolution. Mass spectrometry was detected with an instrument Agilent 6230 TOF LC/MS.
  • the nucleic acid sequence (SEQ ID NO: 3) of the antibody obtained in Example 1 was digested into the pFUSE-hIGg1-FC2 expression vector to obtain an Fc-segment-containing scFv II DNA sequence (SEQ ID NO: 4), which was able to encode a specific A fully human single-chain antibody (Anti-Cx26-scFvII-Fc, sequence Seq ID NO6) which inhibits connexin 26, that is, the fully human antibody which specifically inhibits connexin 26 is a recombinant immunoglobulin, and the structure is scFv -Fc, as shown in Figure 2A, scFv refers to a single-chain antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region having the amino acid sequence of SEQ ID NO: 1 and a light chain thereof The amino acid sequence of the variable region is SEQ ID NO: 2, the heavy chain CDR 3 region sequence is DFSWRGYYMDV, the light
  • CBS antigen fixation solution was purchased from Thermo Fisher Scientific.
  • Anti-Human Fc HRP secondary antibody was purchased from Thermo Fisher.
  • Developing substrate ABST solution was purchased from Thermo Fisher Scientific.
  • Detection 96-well bottom plate was purchased from Corning.
  • the OD values of the negative control well enzyme-linked immunoreactive readings were all below 0.2, while the positive wells had an OD value of 0.4 or more. According to the results, 150 positive clone wells were obtained.
  • Octet RED instrument and its detection chip CM5 chip were purchased from Pall Company.
  • Anti-human IgG antibody was purchased from Thermo Fisher.
  • the affinity and kinetics of the antibody to the antigen were detected by a multi-cycle kinetic method.
  • the antibody is immobilized by the capture method.
  • the Anti-human IgG antibody is first coupled to the CM5 chip, and then the Anti-Cx26-scFv II-Fc antibody sample to be tested is diluted and passed through the surface of the chip, and the antibody to be tested is Captured by an anti-human Fc antibody that has been coupled.
  • an antigenic polypeptide (synthesized by Nanjing Kingsray, sequence Seq ID NO: 5) was injected, and the signal was detected and recorded after the antigen was bound to the antibody.
  • the antibody and antigen samples on the surface of the CM5 chip were completely eluted with a regenerating reagent (pH 1.7 glycine solution), and a new round of detection was performed.
  • the SRP assay detects interaction of the antibody with the Cx26 protein, showing that the anti-Cx26-scFv II-Fc antibody and Cx26 have a KD of 7.3E10-8M.
  • HeLa DH cells were purchased from Sigma.
  • the Cx26-Venus-YFP reporter plasmid was purchased from Addgene Corporation, Cat. No. 69016.
  • Fluorescent secondary antibody Alexa Fluor 594goat anti-human was purchased from Thermo Fisher Scientific.
  • Fluorescence confocal microscopy was purchased from Leica Corporation.
  • HeLa DH cells were transfected with plasmid 293Fectin (Sermerfly) according to the instructions of the plasmid Cx26-Venus-YFP, which expressed human Cx26 protein on the cell membrane with green fluorescence, fixed cells with 2% formaldehyde, and placed at room temperature for 10 minute. After rinsing, the cells were blocked by adding 2% BSA in PBS for 30 minutes, and the 1 mg/ml Anti-Cx26-scFvII-Fc antibody solution was diluted with 1:5% BSA/PBS at a dilution ratio of 1:500, and the cells were incubated for 4 to 5 hours.
  • plasmid 293Fectin Senderfly
  • the Anti-Cx26-scFv II-Fc antibody co-localized with the Cx26-GFP two proteins.
  • Example 6 Patch clamp technique detects antibody inhibition of Cx26 hemichannel activity
  • HeLa DH cells were purchased from Sigma.
  • the Cx26-Venus-YFP reporter plasmid was purchased from Addgene Corporation, Cat. No. 69016.
  • the automatic patch clamp system was purchased from MDC Corporation of the United States.
  • HeLa DH cells were transfected with plasmid 293Fectin (Sermerfly) according to the instructions of the protocol Cx26-Venus-YFP, patch clamps were used to record single cell membrane ion currents in whole cells, and 940 nM Anti-Cx26-scFv II was added to the cell supernatant.
  • Fc antibody control group Zn 2+ concentration 100uM, no antibody as a blank control group, increase the voltage to 40 mV to depolarize the cells leading to Cx26 hemichannel opening, record the antibody-added experimental group, positive control group and blank control The group's half-channel current, reducing the voltage to minus 40 mV, left the cells hyperpolarized and simultaneously recorded the half-channel currents of each group.

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Abstract

一种特异性抑制连接蛋白26的全人抗体，其为重组免疫球蛋白，结构为scFv-Fc，scFv指的是单链抗体，包括重链可变区和轻链可变区，其重链可变区的氨基酸序列为SEQ ID NO：1，其轻链可变区的氨基酸序列为SEQ ID NO：2，Fc指的是恒定区。用人连接蛋白26胞外区第41-56氨基酸序列为抗原，序列为KEVWGDEQADFVCNTL。通过单链抗体噬菌体展示库及筛选技术获得，通过对该抗体的生化分析和免疫荧光鉴定，确定其能特异性识别连接蛋白26，并抑制其形成的半通道活性。动物试验表明，该抗体对鼠耳蜗组织切片的半通道活性有显著抑制作用。因此，该抗体可用于连接蛋白突变相关疾病治疗。

Description

一种特异性抑制连接蛋白26的全人抗体 技术领域
本发明属于生物医药技术领域，具体而言本发明提供特异识别人耳蜗、皮肤组织表达的连接蛋白26(Connexin26)抗原，并能阻遏该连接蛋白26形成的半通道的单链抗体及表达该抗体的质粒载体，以及所述单链抗体在制备用于治疗连接蛋白26突变引起的耳聋、皮肤和肿瘤的药物和诊断试剂盒中的应用，本发明还提供利用该抗体制备诊断试剂盒，其包含所述单克隆抗体作为活性成分。
背景技术
离子通道间隙连接是一种细胞与细胞之间的连接。它连接细胞的胞质，允许小于1.8KD的较小分子如离子、代谢产物和第二信使等自由通过[1,2]。在脊椎动物，由连接蛋白(Connexin)组成的间隙连接通道介导相邻细胞之间离子、小分子营养物质交换及信号分子传播。哺乳动物发育早期已有多种连接蛋白表达，不同连接蛋白组成的间隙连接通道具有不同通透特征，相邻细胞利用间隙连接介导的细胞间通讯或者不依赖间隙连接通道的途径传递发育信号，调节发育过程中的细胞增殖、迁移和分化。连接蛋白是一个广泛表达于脊椎动物细胞的蛋白质家族。该家族成员组成的六聚体定位于细胞膜上，形成间隙连接通道或半通道，介导细胞之间、细胞与细胞外基质之间的物质交换。基因组中编码连接蛋白蛋白家族的基因组成连接蛋白基因家族[3]。
目前为止在人类基因组中已发现21种连接蛋白基因，小鼠基因组中发现20种连接蛋白基因[3]，由间隙连接通道和连接蛋白半通道介导的细胞间通讯对许多细胞功能具有重要的作用，包括调节细胞生长，分化和发育[4-6]。连接蛋白基因的突变与许多人类疾病相关联，包括心血管异常，周围神经病变，白内障和耳聋。连接蛋白26(Cx26)、连接蛋白30(Cx30)参与内耳钾离子循环，并且Cx26和Cx30的缺失引起正在发育中的耳蜗中柯蒂氏器极重度耳聋并伴随细胞死亡[4,7,8]。Cx26和Cx30由两个相邻基因(GJB2和GJB6)表达，Cx26、Cx30在内耳与非感应细胞连接形成多胞体，两种蛋白在内耳共表达[9]。DNFB1位于染色体13q11–q12[10]。50％的习语前失聪都与DFNB1有关，地中海人甚至高达79％[10,11]。Cx26和Cx30的缺失突变对耳蜗螺旋器的器官形成产生深远影响[12-14]。连接蛋白的点突变会导致连接蛋白形成的半通道一直处于常开或者常闭状态， 导致失聪和皮肤疾病的发生[15]。特异识别Cx26并恢复其功能的抗体有望恢复连接蛋白突变导致的失聪和皮肤疾病。
近年来单克隆抗体在疾病的诊断和治疗中发挥重要作用，特别是针对表达特异性抗原的疾病，单克隆抗体与抗原的反应检测可以作为疾病诊断的"金"标准，单克隆抗体作为药物也在自身免疫性疾病和抗肿瘤疾病中发挥重要的作用。
与其他常见疾病不同的是，连接蛋白突变导致的疾病由于发病率相对较低，治疗药物相对较少，另外在耳聋疾病中，由于抗体药物的给药方式存在困难，一直没有针对该靶点的抗体药物上市。用于临床诊断的试剂盒也没有，所以能够识别、结合连接蛋白的单链抗体将成为诊治结直肠癌的重要工具。
参考文献：
1.D.A.Goodenough,D.L.Paul,Gap junctions,Cold Spring HarbPerspectBiol 1(2009)a002576.
2.A.L.Harris,Connexin channel permeability to cytoplasmic molecules,ProgBiophysMolBiol 94(2007)120-143.
3.G.Sohl,K.Willecke,Gap junctions and the connexin protein family,Cardiovasc Res 62(2004)228-232.
4.C.J.Wei,X.Xu,C.W.Lo,Connexins and cell signaling in development and disease,Annu Rev Cell Dev Biol 20(2004)811-838.
5.A.B.Belousov,J.D.Fontes,Neuronal gap junctions:making and breaking connections during development and injury,Trends in Neurosciences 36(2013)227-236.
6.I.Fasciani,A.Temperan,L.F.Perez-Atencio,A.Escudero,P.Martinez-Montero,J.Molano,J.M.Gomez-Hernandez,C.L.Paino,D.Gonzalez-Nieto,L.C.Barrio,Regulation of connexinhemichannel activity by membrane potential and the extracellular calcium in health and disease,Neuropharmacology(2013).
7.D.W.Laird,Life cycle of connexins in health and disease,Biochem J 394(2006)527-543.
8.R.Dobrowolski,K.Willecke,Connexin-caused genetic diseases and corresponding mouse models,Antioxid Redox Signal 11(2009)283-295.
9.F.Ceriani,F.Mammano,A rapid and sensitive assay of intercellular coupling by voltage imaging of gap junction networks,Cell Commun Signal 11(2013)78.
10.F.J.del Castillo,I.del Castillo,The DFNB1subtype of autosomal recessive non-syndromic hearing impairment,Front Biosci 16(2011)3252-3274.
11.L.Zelante,P.Gasparini,X.Estivill,S.Melchionda,L.D'Agruma,N.Govea,M.Mila,M.D.Monica,J.Lutfi,M.Shohat,E.Mansfield,K.Delgrosso,E.Rappaport,S.Surrey,P.Fortina,Connexin26mutations associated with the most common form of non-syndromic neurosensory autosomal recessive deafness(DFNB1)in Mediterraneans,Hum Mol Genet 6(1997)1605-1609.
12.M.Cohen-Salmon,F.J.del Castillo,C.Petit,ConnexinsResponsiblbe for Hereditary Deafness-The Tale Unfolds,in:E.Winterhager(Ed.),Gap Junctions in Development and Disease,Springer-Verlag,Berlin,2005,pp.111-134.
13.M.Leibovici,S.Safieddine,C.Petit,Mouse models for human hereditary deafness,Curr Top Dev Biol 84(2008)385-429.
14.R.Dobrowolski,K.Willecke,Connexin-Caused Genetic Diseases and Corresponding Mouse Models,Antioxidants & Redox Signaling 11(2009)283-295.
15.F.J.del Castillo,I.del Castillo,The DFNB1subtype of autosomal recessive non-syndromic hearing impairment,Frontiers in Bioscience-Landmark 16(2011)3252-3274.
发明内容
本发明的目的是提供一种单链抗体，所述单链抗体可与人源连接蛋白26抗原特异性结合，从而可以用于制备治疗连接蛋白26突变导致的疾病药物或连接蛋白26相关诊断试剂盒。
为了达到上述目的，本发明提供了一种特异性抑制连接蛋白26的全人抗体，其特征在于，其为重组免疫球蛋白，结构为scFv-Fc，scFv指的是单链抗体，包括重链可变区和轻链可变区，其重链可变区的氨基酸序列为SEQ ID NO：1，其轻链可变区的氨基酸序列为SEQ ID NO：2，Fc指的是恒定区。
优选地，所述的恒定区包括CH2恒定区和CH3恒定区。
优选地，所述的特异性抑制连接蛋白26的全人抗体特异识别连接蛋白26的胞外区第41-75氨基酸部分。
本发明还提供了一种基因工程抗体，其特征在于，其与上述的特异性抑制连接蛋白26的全人抗体的单链抗体具有30％以上的同源序列。
优选地，所述的基因工程抗体包含Fab片段，F(ab)‘片段，Fd片段，Fv片段和Fc片段中的一种，上述各片段中两种以上的组合，或上述各片段中的至少一种与其它蛋白或肽链形成的衍生物。
优选地，所述的基因工程抗体的重链CDR 3区序列为DFSWRGYYMDV，轻链CDR3区序 列为QQYGSSPRT。
本发明还提供了一种编码特异性抑制连接蛋白26的全人抗体的核酸序列，其序列为SEQ IDNO：3或SEQ ID NO：4。
优选地，所述的核苷酸序列编码上述的特异性抑制连接蛋白26的全人抗体。
本发明还提供了上述的特异性抑制连接蛋白26的全人抗体或基因工程抗体在制备用于治疗与连接蛋白26突变导致的耳聋、皮肤病或者肿瘤的药物或试剂盒中的应用。
本发明还提供了一种用于治疗耳聋、皮肤病或者肿瘤的药物，其包含上述的特异性抑制连接蛋白26的全人抗体或基因工程抗体作为活性成份。
本发明的特异性抑制连接蛋白26的全人抗体命名为Anti-Cx26-scFvII-Fc，经过检测单链抗体对连接蛋白选择性结合，并可特异性抑制该连接蛋白形成的半离子通道，是经过多轮噬菌体库筛选获得。与目前临床应用常见IgG类抗体不同，Anti-Cx26-scFvII-Fc为重组单链抗体，因此具有不同于IgG的抗体结构、生化特性和生物学功能。我们的实验证明，Anti-Cx26-scFvII-Fc单链抗体分子量大约100Kd，识别、结合靶抗原表位为连接蛋白胞外区结构，特异识别过表达连接蛋白26的细胞系，且抗体与连接蛋白26在细胞表面共定位。功能实验显示Anti-Cx26-scFvII-Fc能够抑制连接蛋白26半离子通道的活性。单独使用该抗体的免疫组化染色即可较为准确地诊断各种组织中人源连接蛋白26的表达情况。综上Anti-Cx26-scFvII-Fc单链抗体在耳聋、皮肤病和肿瘤的治疗方面和连接蛋白26诊断试剂方面有潜在的应用价值。
本发明用人连接蛋白26胞外区第41-56氨基酸序列为抗原，序列为KEVWGDEQADFVCNTL。通过单链抗体噬菌体展示库及筛选技术获得，通过对该抗体的生化分析和免疫荧光鉴定，确定本发明提供的抗体能特异性识别连接蛋白26，并抑制其形成的半通道活性。动物试验表明，该抗体对鼠耳蜗组织切片的半通道活性有显著抑制作用。因此，该抗体可用于连接蛋白突变相关疾病治疗。
附图说明
图1.单链抗体的筛选和序列验证。
A.抗体富集后抗体库酶联免疫反应检测抗原结合活性
B.挑取抗体单克隆与抗原结合酶联免疫反应检测
图2.单链抗体的结构及生化活性鉴定。
A.scFv-Fc型免疫球蛋白结构示意图。
B.Western blot检测单链抗体分子量大小约为58KDa
C.scFv II-Fc抗体热稳定性检测
D.质谱分析双链结构Anti-Cx26-scFv II-Fc分子量大小为105140.3
图3.Anti-Cx26-scFv II-Fc抗体与抗原亲和力检测。
A.酶联免疫反应鉴定抗体scFvⅡ-Fc与抗原亲和力
B.SRP法检测抗体与Cx26蛋白相互作用，结果显示Anti-Cx26-scFv II-Fc抗体与Cx26的结果活性KD为7.3E10-8M
图4.免疫荧光检测Anti-Cx26-scFv II-Fc抗体与Cx26-GFP蛋白，结果显示两个蛋白共定位。
图5.去极化激活Cx26离子通道后，利用膜片钳技术检测Anti-Cx26-scFv II-Fc抗体阻碍Cx26离子半通道的活性。
具体实施方式
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。此外应理解，在阅读了本发明讲授的内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。
实施例1、单链抗体的筛选和单链抗体的表达纯化
1、所用材料：
(1)生物素化抗原多肽，(序列为：KEVWGDEQADFVCNTL，序列表SEQ ID NO：5，下称抗原)，合成自金斯瑞公司。(2)全人单链抗体噬菌体展示库，实验室自有。(3)链霉亲和素化的磁珠购自赛默飞公司。(4)高吸附酶联免疫反应微孔板购自康宁公司。(5)Anti-M13 HRP抗体购自赛默飞公司。(6)辅助噬菌体购自Life Technologies，货号18311019。(7)XL1-Blue细菌购自Agilent Technologies,货号200228。(8)显影液ABTS溶液购自赛默飞公司，货号002024。
2、实验方法：
表达全人单链抗体的噬菌体展示库200微升(含有噬菌体粒1×1011个)与5微克抗原混合后室温孵育30分钟，再加入50微升的链霉亲和素化的磁珠。与抗原结合的噬菌体被链霉亲和素化的磁珠捕获，未结合的噬菌体经PBST漂洗后去除，用盐酸甘氨酸(pH 2.2)溶液将与磁珠稳定结合的噬菌体洗脱待用。接种XL1-Blue细菌(Agilent Technologies,货号200228)200毫升，待OD达到0.6后，加入上述洗脱的噬菌体与XL1-Blue细菌37℃静置30分钟，后在30摄氏度条件下过夜扩增，离心收集细菌后，加入30微升辅助噬菌体(含有辅助噬菌体粒1×1011个)扩增后 进行下一轮淘选。重复上述筛选步骤3-4轮，将侵染噬菌体的XL1-Blue菌液经过充分稀释后涂10cm平板，每个板上有100-500个克隆，挑取单克隆，通过噬菌体酶联免疫反应对淘选后的各轮噬菌体库进行验证。
噬菌体酶联免疫反应步骤为，将侵染噬菌体的XL1-Blue单克隆菌接种进200微升的96孔板中，200rpm 37℃摇4-6小时，检测OD接近0.6后，加入1微升的辅助噬菌体，继续30℃摇过夜。第二天3000g离心取上清待用。取96低透平底微孔板，包被抗原过夜，第二天加入上一步制备好的噬菌体上清，室温孵育2小时，再加入Anti-M13HRP抗体孵育30分钟，后用PBST洗3次，加入50微升显影液ABTS。
由抗原的酶联免疫反应结果可以看出随着淘选的进行，酶联免疫反应的信号不断升高，而对照抗原(AcroBiosystems公司，货号CD0-H82F2)的信号仍然比较低，在第四轮淘选后，抗原的信号强度是对照抗原的多倍，说明经过4轮淘选，能够表达与抗原特异性结合抗体的噬菌体不断的被富集。从淘选后的第三、四轮库中挑取单克隆，最后确定酶联免疫反应读值是对照两倍以上的克隆作为阳性克隆，阳性克隆测序分析，经过比对分析，确定不同的抗体序列和富集情况。
3、实验结果：
如图1A所示，多肽抗原CB在全人抗体噬菌体展示库中进行筛选，经过4轮淘选后，酶联免疫反应的信号与对照组相比不断升高，从淘选后的库中挑取700个单克隆进行酶联免疫反应验证，最后确定酶联免疫反应读值是对照两倍以上的克隆作为阳性克隆，如图1B所示，150个阳性克隆测序分析，经过比对分析，重复次数越多，说明该抗体序列的亲和力越强，依此确定有效富集序列。本发明中所选的阳性克隆在150个克隆序列中重复84次，说明亲和力较强。
挑取的克隆经测序，所选的阳性克隆的核酸序列为SEQ ID NO：3。
实施例2、单链抗体纯化和生化特性检测
1、所用材料：
(1)Sypro Orange染料购自赛默飞公司。(2)pFUSE表达载体pFUSE-hIGg1-FC2购自Invivogen公司，货号pfuse-hg1fc2。(3)HiTrapProtein A HP columns购自GE公司。(4)
Figure PCTCN2016109847-appb-000001
蛋白纯化仪购自GE公司。(5)实时定量PCR仪购自BioRad公司。(6)质粒抽提试剂盒购自Qiagen公司。(7)BCA蛋白定量试剂盒(PierceTMBCA Protein Assay kit，Pierce#23253)。(8)BamH1和BglII限制性内切酶购自NEB公司。
2、实验方法：
将实施例1得到的阳性序列(即SEQ ID NO：3)由全基因合成(南京金斯瑞生物公司)并在序列两端加入BamH1和BglII酶切位点，将得到的核酸序列经过参照NEB说明书在BamH1和BglII酶切后插入pFUSE-hIGg1-FC2表达载体(Invivogen公司，操作步骤请参照说明书)，将获得具有Fc段的单链抗体Anti-Cx26-scFvⅡ-Fc的真核抗体表达质粒。利用293Fectin转染试剂(Invitrogen公司，货号12347500)与上述获得的真核抗体表达载体质粒按照体积质量比30微升：15微克的比例混合后加入30毫升的293Freestyle悬浮细胞(购自赛默飞公司)后，37℃1200rpm摇过夜，离心后收集上清，采用HiTrap Protein A HP columns在
Figure PCTCN2016109847-appb-000002
蛋白纯化仪上进行抗体蛋白纯化(Anti-Cx26-scFvⅡ-Fc)，最后用BCA法蛋白定量试剂盒(Pierce，货号23252)参照说明书检测抗体浓度。
将所得的纯化抗体进行Western blot检测，具体为：将5微克纯化抗体样品进行SDS-PAGE电泳，电泳电压110V，60分钟，后转印到醋酸纤维素膜上，用羊抗人辣根过氧化物酶二抗(购自赛默飞)杂交，洗膜加入显影液(Pierce公司，货号35055)成像。
将所得的纯化抗体进行热稳定性检测，具体为：将纯化抗体稀释至0.1毫克/毫升，按照体积比1:2000加入Sypro Orange染料后密封，实时定量PCR仪(BioRad)设置程序温度由25℃升至90℃，0.5℃每分钟。同时检测荧光值。
将所得的纯化抗体进行LC-MS质谱分析，具体为：将纯化的抗体5微克与1微升蛋白质脱糖酶(购自NEB公司，货号P0709S)混合37℃一小时后，上样到高分辨质谱用仪器Agilent 6230 TOF LC/MS检测。
3、实验结果：
将实施例1得到的抗体的核酸序列(SEQ ID NO：3)经过酶切插入pFUSE-hIGg1-FC2表达载体，得到含有Fc段的scFv II DNA序列(SEQ ID NO：4)，其能够编码特异性抑制连接蛋白26的全人单链抗体(Anti-Cx26-scFvⅡ-Fc，序列为Seq ID NO6)，即所述的特异性抑制连接蛋白26的全人抗体为重组免疫球蛋白，结构为scFv-Fc，如图2A所示，scFv指的是单链抗体，包括重链可变区和轻链可变区，其重链可变区的氨基酸序列为SEQ ID NO：1，其轻链可变区的氨基酸序列为SEQ ID NO：2，其重链CDR 3区序列为DFSWRGYYMDV，轻链CDR3区序列为QQYGSSPRT，Fc指的是恒定区，所述的恒定区包括CH2恒定区和CH3恒定区。所述的特异性抑制连接蛋白26的全人抗体特异识别连接蛋白26的胞外区第41-75氨基酸部分。
Western blot实验结果如图2B所示，胶图在58Kd处出现明显条带，说明抗体能特异识别Cx26 蛋白。热稳定性结果计算出该抗体的Tm值为65℃，如图2C所示，说明Anti-Cx26-scFvⅡ-Fc抗体具有高稳定性。如图2D所示，LC-MS质谱分析结果显示该抗体蛋白的分子量为105140道尔顿。
实施例3、酶联免疫反应检测单链抗体与连接蛋白26的结合
1、所用材料：
1.CBS抗原固定溶液购自赛默飞公司。2.Anti-Human Fc HRP二抗购自赛默飞公司。3.显影底物ABST溶液购自赛默飞公司。4.检测用96孔底透板购自Corning公司。
2、实验方法：
96孔底透板每孔加入50微升CBS抗原固定溶液稀释的Cx26抗原多肽(金斯瑞公司合成，序列请见Seq ID NO5)，抗原每孔0.05微克，4℃过夜孵育，室温震荡孵育30分钟，PBS漂洗三次，含5％牛奶的PBST溶液，37℃条件下封闭60分钟。PBS漂洗三次，加入实施例2所得的Anti-Cx26-scFvⅡ-Fc抗体，37℃条件下孵育60分钟。漂洗干燥后，加入Anti-Human Fc二抗，室温震荡孵育30分钟，PBS漂洗三次，最后加底物显色，酶标仪读值。
3、实验结果：
如图3A所示，阴性对照孔酶联免疫反应读值OD值均在0.2以下，而阳性孔OD值在0.4以上。根据结果获得150个阳性克隆孔。
实施例4、SRP法检测单链抗体与连接蛋白26的结合
1、所用材料：
1.Octet RED仪器及其检测芯片CM5芯片购自Pall公司。2.Anti-human IgG抗体购自赛默飞公司。
2、实验方法：
通过多循环动力学的方法检测抗体与抗原亲和力与动力学。抗体固定采用捕获法进行，先将Anti-human IgG抗体偶联在CM5芯片上，然后将待测的Anti-Cx26-scFv II-Fc抗体样品稀释梯度后流过芯片表面，待测抗体就会被已经偶联的抗人Fc抗体所捕获。随后再注入抗原多肽(由南京金斯瑞公司合成，序列为Seq ID NO：5)，抗原与抗体结合后信号被检测并记录。最后用再生试剂(pH1.7的甘氨酸溶液)将CM5芯片表面的抗体和抗原样品全部洗脱，并进行新一轮检测。
3、实验结果：
如图3B所示，SRP法检测抗体与Cx26蛋白相互作用，显示Anti-Cx26-scFv II-Fc抗体与Cx26的结果活性KD为7.3E10-8M。
实施例5、细胞免疫荧光检测抗体特异识别Cx26蛋白
1、所用材料：
(1)HeLa DH细胞购自西格玛公司。(2)Cx26-Venus-YFP报告基因质粒购买自Addgene公司，货号69016。(3)荧光二抗Alexa Fluor 594goat anti-human购自赛默飞公司。(4)荧光共聚焦显微镜购自徕卡公司。
2、实验方法：
HeLa DH细胞用293Fectin(赛默飞)参照说明书步骤转染质粒Cx26-Venus-YFP，该质粒可在细胞膜上表达人源Cx26蛋白且伴随绿色荧光，用2％甲醛固定细胞，室温条件下放置10分钟。漂洗后，加入含2％BSA的PBS溶液封闭细胞30分钟，1毫克/毫升Anti-Cx26-scFvII-Fc抗体溶液用1％BSA/PBS以稀释比1:500稀释后孵育细胞4～5小时，漂洗后，加入荧光Alexa Fluor 594 goat anti-human二抗在溶液1％BSA/PBS中以1:1000浓度稀释，室温条件下孵育60分钟后，加入DAPI(1微克/毫升)孵育5分钟，漂洗后，封片，激光共聚焦显微镜观察及拍照。
3、实验结果：
如图4所示，Anti-Cx26-scFv II-Fc抗体与Cx26-GFP两个蛋白共定位。
实施例6、膜片钳技术检测抗体抑制Cx26半通道活性
1、所用材料：
(1)HeLa DH细胞购自西格玛公司。(2)Cx26-Venus-YFP报告基因质粒购买自Addgene公司，货号69016。(3)全自动膜片钳系统购自美国MDC公司。
2、实验方法：
HeLa DH细胞用293Fectin(赛默飞)参照说明书步骤转染质粒Cx26-Venus-YFP，膜片钳采用全细胞式记录单个细胞膜离子电流，细胞处理上清中加入940nM的Anti-Cx26-scFv II-Fc抗体，对照组Zn2+浓度100uM，不加抗体作为空白对照组，加大电压至40毫伏使细胞去极化导致Cx26半通道开放，记录加入抗体的实验组，阳性对照组和空白对照组的半通道电流，降低电压至负40 毫伏使细胞处于超极化状态，并同时记录各组的半通道电流。
3、实验结果：如图5所示，抗体Anti-Cx26-scFv II-Fc可有效抑制Cx26半通道活性，是非特异抑制剂Zn2+的一百倍。
Figure PCTCN2016109847-appb-000003
Figure PCTCN2016109847-appb-000004
Figure PCTCN2016109847-appb-000005
Figure PCTCN2016109847-appb-000006

Claims (9)

  1. 一种特异性抑制连接蛋白26的全人抗体，其特征在于，其为重组免疫球蛋白，结构为scFv-Fc，scFv指的是单链抗体，包括重链可变区和轻链可变区，其重链可变区的氨基酸序列为SEQ ID NO：1，其轻链可变区的氨基酸序列为SEQ ID NO：2，Fc指的是恒定区。
  2. 如权利要求1所述的特异性抑制连接蛋白26的全人抗体，其特征在于，所述的恒定区包括CH2恒定区和CH3恒定区。
  3. 如权利要求1所述的特异性抑制连接蛋白26的全人抗体，其特征在于，所述的特异性抑制连接蛋白26的全人抗体特异识别连接蛋白26的胞外区第41-75氨基酸部分。
  4. 一种基因工程抗体，其特征在于，其与权利要求1-3中任一项所述的特异性抑制连接蛋白26的全人抗体的单链抗体具有30％以上的同源序列。
  5. 如权利要求4所述的基因工程抗体，其特征在于，所述的基因工程抗体包含Fab片段，F(ab)‘片段，Fd片段，Fv片段和Fc片段中的一种，上述各片段中两种以上的组合，或上述各片段中的至少一种与其它蛋白或肽链形成的衍生物。
  6. 如权利要求4所述的基因工程抗体，其特征在于，所述的基因工程抗体的重链CDR 3区序列为DFSWRGYYMDV，轻链CDR3区序列为QQYGSSPRT。
  7. 一种编码特异性抑制连接蛋白26的全人抗体的核苷酸序列，其序列为SEQ ID NO：3或SEQ ID NO：4。
  8. 权利要求1-3中任一项所述的特异性抑制连接蛋白26的全人抗体或权利要求4-6中任一项所述的基因工程抗体在制备用于治疗与连接蛋白26突变导致的耳聋、皮肤病或者肿瘤的药物或试剂盒中的应用。
  9. 一种用于治疗耳聋、皮肤病或者肿瘤的药物，其包含权利要求1-3中任一项所述的特异性抑制连接蛋白26的全人抗体或权利要求4-6中任一项所述的基因工程抗体作为活性成份。
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