Patent Application
20200299366
An ASCII text file of Sequence Listing is submitted separately, naming “GBSH006-POA-Sequence Listing-20200607”, created on Jun. 6, 2020, and sized 10 KB, and the ASCII text file is incorporated herein by reference.
The present invention belongs to the field of biotechnology medicine, in particular the present invention provides the single-chain antibody or plasmid expressing the antibody, which is capable of specifically recognizing Connexin26 expressed in human cochlea and skin tissue and inhibiting hemichannels of Connexin26, and the use of antibody in the preparation of a medicament and a diagnostic kit for treating deafness, skin disease and tumor caused by Connexin26 mutation. The present invention also provides the diagnostic kit prepared by using the antibody. The kit comprises the monoclonal antibody as an active ingredient.
Gap junction is a cell-cell junction formed by ion channels. Gap junction channels connect the cell-cell cytoplasm, allowing smaller molecules smaller than 1.8 KD to pass freely, such as ions, metabolites and second messengers[1,2]. In vertebrates, a gap junctional pathway composed of Connexin mediates the exchange of ions, small molecules, and signaling molecules between adjacent cells and are of high importance in the early stage of mammalian development. Gap junction channels can be composed by many different connexin proteins and display different permeability characteristics, which depend on the connexin composition. Adjacent cells use gap junction-mediated intercellular communication or gap-independent pathway to transmit developmental signals and regulate cell proliferation, migration and differentiation during development. Proteins of Connexin family are widely expressed in vertebrate cells. The hexamers of this family member are located on the cell membrane to form gap junctions or hemichannels and mediate the exchange of material between cells, and the extracellular matrix. The genes encoding connexin proteins in the genome make up the connexin gene family[3].
So far, 21 kinds of connexin genes have been found in human genome, and 20 kinds of the connexin gene[3] have been found in the mouse genome. Many important cell functions including cell growth regulation, differentiation and development is mediated by gap junction channels and connexin hemichannel communication[4-6]. Mutations in connexin genes are associated with many human diseases, including cardiovascular abnormalities, peripheral neuropathy, cataracts and deafness. Connexin 26 (Cx26) and Connexin 30 (Cx30) channels and hemichannels are involved in the potassium ions cycling of inner ear, and deletion of Cx26 and Cx30 causes profound hearing loss and concomitant cell death in developing organ of Corti in the cochlea[4,7,8]. Cx26 and Cx30 are expressed by two adjacent genes (GJB2 and GJB6), while Cx26 and Cx30 are connected with non-responsive cells in the inner ear to form polytopes. Both proteins are co-expressed in the inner ear[9]. DNFB1 is located on chromosome 13q11-q12[10]. Fifty percent of hereditary deafness is related to DFNB1, with up to 79% in Mediterranean[10,11] Deletion mutations at Cx26 and Cx30 have a profound effect on the organ formation of the cochlear spirals[12-14]. In some cases, point mutations in connexins can cause aberrant hemichannel opening or closing, which will result in deafness and skin diseases[15]. Antibodies that specifically recognize Cx26 and restore its function are expected to treat deafness and skin diseases caused by connexin mutations.
In recent years, monoclonal antibodies have been playing an important role in the diagnosis and treatment of diseases, especially for diseases that express specific antigens. The detection of the reaction of monoclonal antibodies and antigens can be used as the golden standard for disease diagnosis. Monoclonal antibodies also play an important role as drugs in autoimmune diseases and tumor diseases.
Diseases caused by connexin mutations have relatively low incidences and relatively few treatment drugs. Moreover, no antibody drug targeting connexin channels is available to treat deafness or skin diseases due to difficulties in the administration of antibody drugs. There are no kits for clinical diagnosis, so the single chain antibodies capable of recognizing and binding connexin will be an important tool in the diagnosis and treatment of colorectal cancer.
An object of the present invention is to provide a single chain antibody, which specially binds to human Connexin 26 antigen and thus can be used in the preparation of medicaments treating diseases caused by mutations in Connexin 26 or diagnostic kits relevant to Connexin 26.
To achieve the above object, the present invention provides a fully human antibody specifically inhibiting Connexin 26, characterized in that it is a recombinant immunoglobulin having the structure of scFv-Fc, wherein scFv refers to a single-chain antibody comprising a heavy chain variable region and a light chain variable region, the amino acid sequence of the heavy chain variable region is SEQ ID NO: 1, the amino acid sequence of the light chain variable region is SEQ ID NO: 2, and Fc refers to a constant region.
Preferably, the constant region comprises a CH2 constant region and a CH3 constant region.
Preferably, the fully human antibody specifically inhibiting Connexin 26 specifically recognizes the 41-75 amino acid portion of the extracellular region of Connexin 26.
The present invention also provides a genetically engineered antibody, characterized in that it has 30% or more homologous sequences to a single chain antibody of the fully human antibody specifically inhibiting Connexin 26.
Preferably, the genetically engineered antibody comprises one of a Fab fragment, a F(ab)′ fragment, an Fd fragment, an Fv fragment and an Fc fragment, a combination of two or more of the foregoing fragments, or a derivative of at least one of the foregoing fragments with other proteins or peptide chains.
Preferably, the heavy chain CDR 3 region sequence of the genetically engineered antibody is DFSWRGYYMDV, the light chain CDR3 region sequence is QQYGSSPRT.
The present invention also provides a nucleotide sequence encoding a fully human antibody specifically inhibiting Connexin 26, wherein the sequence thereof is SEQ ID NO: 3 or SEQ ID NO: 4.
Preferably, the nucleotide sequence encodes the fully human antibody specifically inhibiting Connexin 26 as described above.
The present invention also provides use of the fully human antibody specifically inhibiting Connexin 26 or the genetically engineered antibody in the manufacture of a medicament or kit for the treatment of deafness, skin disease or tumor caused by Connexin 26 mutation.
The present invention also provides a medicament for treating deafness, skin disease or tumor, wherein the medicament comprises the fully human antibody specifically inhibiting Connexin 26 or the genetically engineered antibody as an active ingredient.
The fully human antibody specifically inhibiting Connexin 26 of the present invention is named as Anti-Cx26-scFvII-Fc. Results show that the single chain antibody obtained by rounds of phage library screening can selectively bind to connexins and specifically inhibit hemichannel formed by the connexins. Anti-Cx26-scFvII-Fc is a recombinant single chain human antibody, which is different from the common IgG antibodies in clinical application. Anti-Cx26-scFvII-Fc has different antibody structure, biochemical characteristics and biological functions from IgG. Our experiments demonstrated that the Anti-Cx26-scFvII-Fc single chain antibody has a molecular weight of about 100 kd, recognizes and binds to the target antigen epitope of the connexin extracellular domain, specifically recognizes cells over-expressing connexin 26, and the antibody and connexin 26 are co-localizedon the cell surface. Functional experiments show that Anti-Cx26-scFvII-Fc can inhibit the activity of the Connexin 26 hemichannel. Immunohistochemically staining of this antibody alone can more accurately diagnose the expression of human Connexin 26 in various tissues. In summary, the Anti-Cx26-scFvII-Fc single chain antibody has potential applications in the treatment of deafness, skin diseases and tumors, and Connexin 26 diagnostic reagents.
The present invention uses the 41-56 amino acid sequence of human Connexin extracellular region as antigen, the amino acid sequence thereof is KEVWGDEQADFVCNTL. The antibody is obtained by single-chain antibody phage display library and screening technology. Through the biochemical analysis and immunofluorescence identification, it was confirmed that the antibody provided by the present invention specifically recognize connexin 26 and inhibit its formation of hemichannel activity. Animal experiments show that the antibody has a significant inhibitory effect on the hemichannel activity of mouse cochlear tissue sections. Therefore, the antibody can be used in the treatment of connexin-associated diseases.
A. Phage ELISA was used to detect antigen-binding activity after enriching the antibody.
B. ELISA assay of the binding between selected monoclonal antibody and antigen
B. The molecular weight of the single chain antibody is about 58KDa tested by western blot
C. scFv II-Fc antibody thermal stability test
D. Mass Spectrometry show that the Anti-Cx26-scFv II-Fc dimer has a molecular weight of 105140.3
A. ELISA to test the antibody/Cx26 protein binding affinity
B. SRP assay to test the antibody/Cx26 protein binding affinity, results showed that the anti-Cx26-scFv II-Fc antibody binding to Cx26 protein with a KD of 7.3E10-8M
The present invention is further described below with reference to specific embodiments. It should be understood that these examples are only for illustrating the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading of the present invention, those skilled in the art can make various modifications or changes to the present invention, and these equivalent forms also fall within the scope of the appended claims of the present application.
(1) biotinylated antigen polypeptide (sequence: KEVWGDEQADFVCNTL, SEQ ID NO: 5, hereinafter referred to as antigen) synthesized from Genscript. (2) laboratory's phage display human antibody library. (3) streptavidin magnetic beads purchased from Thermo Scientific. (4) high adsorption ELISA microplate purchased from Corning Corporation. (5) Anti-M13 HRP antibody purchased from Thermo Scientific. (6) Helper phage purchased from Life Technologies (Cat. No. 18311019). (7) XL1-Blue bacteria available from Agilent Technologies (Cat. NO. 200228). (8) developing solution ABTS solution purchased from Thermo Scientific, Cat. NO. 002024.
200 microliters of the phage display human antibody library expressing human single chain antibodies (containing 1×1011 phage particles) was mixed with 5 micrograms of antigen, incubated at room temperature for 30 minutes and then added to 50 microliters of the streptavidin magnetic beads. Phages bound to the antigen was captured by the streptavidin magnetic beads, the unbound phage was removed by PBST rinsing, and then the stably bound phages was eluted with glycine-HCl (pH 2.2) buffer. XL1-Blue bacteria (Agilent Technologies Cat. No. 200228) was inoculated into 200 mL of SB medium and when the OD reached 0.6, the eluted phages were added to the XL1-Blue bacteria, and incubated at 37° C. for 30 minutes, and then incubated overnight at 30° C. The bacteria were collected by centrifugation and added with 30 microliters of helper phage (containing 1×1011 helper phage particles). The next round of screening was performed after amplification. The above panning steps were repeated for 3-4 rounds. Then the phage-containing XL1-Blue broth was thoroughly diluted and plated onto 10 cm plates with 100-500 clones on each plate, and monoclones were picked. The phage ELISA test was carried out after each round of phage library panning.
The phage ELISA test comprises the following steps: inoculating the phage-containing XL1-Blue monoclonal bacteria into 200 microliters of SB medium in a 96 well plate, shaking at 37° C. with a speed of 200 rpm for 4-6 hours, adding 1 microliter helper phage when the OD is close to 0.6, and shaking at 30° C. overnight, centrifuging the next day to collect the supernatant at 3000 g for use, coating a 96 low-permeability microplate with the antigen overnight, adding the prepared phage in the supernatant to the microplate, incubating for 2 hours at room temperature, then adding the Anti-M13 HRP antibody and incubating for 30 minutes, washing for three times with PBST, and adding 50 microliter of ABTS as developing solution.
The result of the enzyme-linked immunosorbent assay (ELISA) shows that, as panning proceeds, the signal of the enzyme-linked immunosorbent rises continuously while the signal of the control antigen (Acro Biosystems, catalog number CDO-H82F2) is still low. After the fourth round panning, the signal intensity of the antigen was multiple times of that of the control antigen, indicating that after 4 rounds of panning, the phages capable of expressing the antigen-specific binding antibody were enriched. Monoclony was picked from the library of the third and fourth round, and the clone with ELISA results more than double of that of the control clone was selected as a positive clone. The sequence of the positive clone was analyzed, and different antibody sequences and enrichment situations were determined by comparison analysis.
As shown in
The selected positive clone was sequenced and the nucleic acid sequence of the selected positive clone was SEQ ID NO: 3.
(1) Sypro Orange dyes were purchased from Thermo Scientific. (2) pFUSE expression vector pFUSE-hIGg1-FC2 was purchased from Invivogen Company, catalog No. pfuse-hg1fc2. (3) HiTrap Protein A HP columns were purchased from GE. (4) ÄKTApurifier 100 protein purifier was purchased from GE. (5) Real-time quantitativePCR instrument was purchased from BioRad company. (6) Plasmid extraction kit was purchased from Qiagen company. (7) BCA Protein Assay Kit (Pierce™ BCA Protein Assay kit, Pierce #23253). (8) BamHI and BglII restriction enzymes were purchased from NEB.
The positive sequence obtained in Example 1 (i.e., SEQ ID NO: 3) was synthesized by gene synthesis method (Nanjing GenScript Ltd.) and added with the restriction sites BamHI and BglII at both ends of the sequence. The obtained nucleic acid sequence was inserted into the pFUSE-hIGg1-FC2 expression vector (Invivogen Corporation, with reference to the procedures described in the instruction manual) after enzyme digestion at the restriction sites BamHI and BglII with reference to the procedures described in the instruction manual of NEB to obtain an eukaryotic antibody expression plasmid of Anti-Cx26-scFvII-Fc, which is a single chain antibody having an Fc segment. 293Fectin reagent (Invitrogen Corporation, Cat. No. 12347500) was mixed with the eukaryotic antibody expression plasmid with a volume mass ratio of 30 microliter:15 micrograms. The obtained mixture was added to 30 mL of 293Freestyle suspension cells (available from Thermo Corporation) and incubated with shaking at 1200 rpm and 37° C. overnight. The supernatant was collected after centrifugation, and the antibody protein (Anti-Cx26-scFvII-Fc) was purifiedby using HiTrap Protein A HP columns on ÄKTApurifier 100 protein purifier. At last, the antibody concentration was tested by using the BCA Protein Assay Kit (Pierce, Cat #23252) with reference to the procedures described in the instruction manual.
The purified antibody was tested by Western blot: 5 micrograms of the purified antibody sample was loaded for SDS-PAGE electrophoresis, with electrophoresis voltage of 110V, 60 minutes, transferred to a cellulose acetate membrane, and then hybridized with horseradish peroxidase goat-anti-human secondary antibody (Thermo Scientific). The membrane was rinsed and then added with developing reagent (Pierce, Cat #35055) for imaging.
The purified antibody was subjected to heat stability test with the following steps: diluting the purified antibody to 0.1 mg/ml, adding Sypro Orange dye with a volume ratio of 1:2000 and sealing, loading onto the real-time quantitative PCR instrument (BioRad) with a heating program from 25° C. to 90° C., 0.5° C. per minute, and detecting fluorescence values.
The purified antibody was test on LC-MS mass spectrometry with the following steps: mixing 5 micrograms of the purified antibody with 1 microliter of PNGase enzyme (available from NEB Inc., Catalog No. P0709S) at 37° C. for one hour, and loading onto an Agilent 6230 TOF LC/MS instrument for high resolution mass spectrometry.
The nucleic acid sequence (SEQ ID NO:3) obtained in Example 1 was inserted into the pFUSE-hIGg1-FC2 expression vector after enzyme digestion to obtain scFv II DNA sequence (SEQ ID NO: 4) containing the Fc region. The scFv II DNA sequence is capable of encoding a fully human single chain antibody (Anti-Cx26-scFvII-Fc, SEQ ID NO:6) specifically inhibiting of Connexin 26, i.e., the fully human antibody specifically inhibiting Connexin 26, which is a recombinant immunoglobulin having the structure of scFv-Fc, shown in
The Western blot result is shown in
1. CBS antigen fixation solution was purchased from Thermo Scientific. 2. Anti-Human Fc HRP secondary antibody was purchased from Thermo Scientific. 3. Developing substrate ABST solution was purchased from ThermoScientific. 4.96-well flat bottom plate was purchased from Corning Corporation.
50 μl per well of the Cx26 polypeptide antigen (synthesized by GenScript, diluted with the CBS antigen fixation solution, SEQ ID NO:5) and 0.05 ug per well of the antigen were added to the 96-wellplate, incubated overnight at 4° C., and then incubated with shaking at room temperature for 30 min. The 96-well plate was rinsed for three times with PBS, blocked with PBST solution containing 5% milk at 37° C. for 60 minutes and rinsed with PBS for three times. The Anti-Cx26-scFv II-Fc antibody obtained in Example 2 was added and incubated at 37° C. for 60 minutes. After the 96-wellplate was rinsed and dried, the anti-Human Fc secondary antibody was added thereto and incubated for 30 minutes with shaking at room temperature. The 96-well plate was rinsed for three times with PBS, added with the developing substrate and eventually read with a microplate reader.
As shown in
1. Octet RED instrument and its detection chip (CM5 chip) was purchased from Pall Corporation. 2. Anti-human IgG antibody was purchased from Thermo Scientific.
The affinity and kinetics of the antibody and the antigen were detected by the method of multi cycle kinetics. Immobilization of the antibody was performed by capture method. First, the anti-human IgG antibody was coupled to the CM5 chip, and then the Anti-Cx26-scFv II-Fc antibody sample flowed through the chip surface after being serial diluted. The antibody to be detected was then captured by the coupled anti-human Fc antibody. Then the polypeptide antigen (synthesized by Nanjing Genscript Co. Ltd, sequence: Seq ID NO: 5) was added, the antigen was bound with the antibody, and the signal was detected and recorded. Finally, the antibody and the antigen samples on the CM5 chip surface were eluted by regeneration reagent (glycine solution, pH1.7) for a new round of testing.
As shown in
Recognized by the Antibody
(1) HeLa DH cells were purchased from Sigma company. (2) Cx26-Venus-YFP reporter plasmid was purchased from Addgene, Cat. No. 69016. (3) Fluorescent secondary antibody Alexa Fluor 594 goat anti-human was purchased from Thermo Scientific. (4) Fluorescence confocal microscope was purchased from Leica company.
Cx26-Venus-YFP plasmid was transfected into HeLa DH cells with 293Fectin (Thermo) with reference to the procedures described in the instruction manual. The plasmid Cx26-Venus-YFP can express human Cx26 protein on the cell membrane with green fluorescence. After fixed with 2% formalin, the cells kept at room temperature for 10 minutes. After rinsed, the cells were blocked with 2% BSA in PBS for 30 min, and then incubated for 4 to 5 hours in the Anti-Cx26-scFvII-Fc antibody solution, which was obtained by diluting 1 mg/mL Anti-Cx26-scFvII-Fc antibody solution with 1% BSA/PBS with a dilution ratio of 1:500. After rinsed, the cells were added with the fluorescent Alexa Fluor 594 goat anti-human secondary antibody, which was diluted in 1% BSA/PBS solution with a dilution ratio of 1:1000. The cells were incubated at room temperature for 60 min, added with DAPI (1 μg/mL) and incubated for 5 minutes, rinsed, mounted on a confocal laser scanning microscope, and detected.
As shown in
(1) HeLa DH cells were purchased from Sigma company. (2) Cx26-Venus-YFP reporter plasmid was purchased from Addgene, Cat. No. 69016. (3) Automatic patch clamp system was purchased from the United States MDC company.
Cx26-Venus-YFP plasmid was transfected into HeLa DH cells with 293Fectin (Thermo) with reference to the procedures described in the instruction manual. Whole cell patch clamp was used to record the single membrane ion currents. 940 nM Anti-Cx26-scFv II-Fc antibody was added into the supernatant of the cells, while 100 uM Zn′ was added in a positive control group and no antibody was added in a blank control group. The voltage was increased to 40 millivolts so that the cells were depolarized, which led to the opening of Cx26 hemichannel. The hemichannel currents of the experimental group added with the antibody, the positive control group and the blank control group were recorded. The voltage was decreased to minus 40 millivolts to hyperpolarize the cells and simultaneously recorded the hemichannel current in each group.
As shown in
| Number | Date | Country | Kind |
|---|---|---|---|
| 201610056295.3 | Jan 2016 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2016/109847 | 12/14/2016 | WO | 00 |
