CN107216388B - Preparation method and application of medicine for treating hepatitis C virus - Google Patents
Preparation method and application of medicine for treating hepatitis C virus Download PDFInfo
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- CN107216388B CN107216388B CN201710668064.2A CN201710668064A CN107216388B CN 107216388 B CN107216388 B CN 107216388B CN 201710668064 A CN201710668064 A CN 201710668064A CN 107216388 B CN107216388 B CN 107216388B
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Abstract
The invention discloses a fully human anti-HCV monoclonal neutralizing antibody. The verification shows that the antibody can be specifically combined with HCV, so that the antibody can be used for preparing products for diagnosing HCV. In addition, the antibody of the invention has better HCV neutralization activity, and can prevent related diseases caused by HCV infection, and the antibody of the invention is a fully human antibody which has lower immunogenicity than murine, chimeric and humanized antibodies, so the antibody of the invention can be used for preparing medicaments for treating or preventing the HCV related diseases.
Description
Technical Field
The invention belongs to the fields of cellular immunology and molecular biology, relates to a fully human monoclonal antibody medicament, and particularly relates to a fully human monoclonal neutralizing antibody medicament for resisting hepatitis C virus. In addition, the invention also relates to a preparation method and application of the neutralizing antibody medicament.
Background
Hepatitis c is caused by infection with Hepatitis C Virus (HCV), primarily transmitted from blood/body fluids. According to the world health organization, 1.7 million people are infected with HCV worldwide. The positive rate of anti-HCV of healthy people in China is 0.7-3.1%, and about 3800 ten thousand people. Due to various factors such as the biological characteristics of viruses and the immune function of a host, the immunity of the organism is difficult to effectively eliminate the viruses, so that about 50 to 80 percent of HCV infected persons develop chronic hepatitis, and 20 to 30 percent of the HCV infected persons develop liver cirrhosis. Hepatocellular carcinoma develops in 1-4% of cirrhosis patients each year.
At present, the therapeutic drugs for hepatitis C include interferon, nucleoside analogues, polypeptide drugs, Chinese herbal medicines, small molecule drugs and the like, and each drug has different degrees of curative effect, but cannot completely eradicate HCV. With the development of research, therapeutic antibodies have become a new direction for the treatment of hepatitis c.
Therapeutic antibodies are crude drug products which have been growing vigorously in recent years, and up to now, about 30 kinds of monoclonal antibodies have been approved for diagnosis and treatment of various diseases. In the treatment and research of various acute and chronic viral hepatitis, the monoclonal antibody has wide application and irreplaceable effect. In terms of development, monoclonal antibodies can be classified into three types of monoclonal antibodies, murine, humanized and fully human. Although the murine monoclonal antibody has the advantages of mature acquisition method, high affinity of the antibody and the like, the effective application of the antibody is limited along with the appearance of the defects that the internal antibody has short half-life in vivo, strong human anti-mouse rejection reaction (HAMA), inappropriate antibody-dependent cell cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) action excitation strength. Humanization of murine antibodies, i.e., chimeric antibodies, is a replacement of a portion of the sequence of a murine antibody with that of a human, and this approach reduces HAMA to some extent, but with a concomitant decrease in affinity. In order to obtain safer and more efficient monoclonal antibodies, researchers have tried various methods for obtaining fully human monoclonal antibodies, which are more common, such as the preparation of monoclonal antibodies using genetically engineered mice, phage antibody library technology, and the preparation of single B lymphocyte antibodies.
The invention relates to a single B lymphocyte antibody preparation technology, which is an in-vitro expression system of a monoclonal antibody formed by combining a single cell separation and identification technology with a plurality of PCR technologies, namely a method for directly obtaining a fully human monoclonal antibody from human B lymphocytes. The generated antibody has the advantages of full humanity, high antigen specificity, affinity and the like, and has unique advantages and good application prospects in the aspects of treating tumors, infectious diseases, autoimmune diseases, organ transplantation and the like. Therefore, it is very necessary to select the advanced and efficient monoclonal antibody preparation technology to prepare antibodies for treating and preventing HCV infection.
Disclosure of Invention
The purpose of the present invention is to provide a binding molecule for Hepatitis C Virus (HCV), which has an effective neutralizing effect on the hepatitis C virus.
In order to achieve the purpose, the invention adopts the following technical scheme:
the present invention provides an isolated binding molecule comprising:
(1) heavy chain CDR1 shown in SEQ ID NO. 2, heavy chain CDR2 shown in SEQ ID NO. 3, and heavy chain CDR3 shown in SEQ ID NO. 4; and/or
(2) Light chain CDR1 shown in SEQ ID NO. 6, light chain CDR2 shown in SEQ ID NO. 7, and light chain CDR3 shown in SEQ ID NO. 8.
As one aspect of the present invention, the binding molecule of the present invention comprises:
(1) a heavy chain variable region having an amino acid sequence set forth in SEQ ID NO 1; and/or
(2) And a light chain variable region having an amino acid sequence set forth in SEQ ID NO. 5.
"binding molecules" as used herein refers to antibody molecules and immunologically active fragments, i.e., molecules that contain an antigen binding site that immunospecifically binds to an antigen. The antibody molecules of the invention may be of any type (e.g. IgG, IgE, IgM, IgD, IgA), class (e.g. IgG1, IgG2, IgG3, IgG4, IgA1, hIgA2) or subclass. Immunologically active portions of antibody molecules include, but are not limited to, Fab 'and F (ab') 2, Fd, single chain fv (scfv), single chain antibodies, disulfide linked fv (sdfv), and single domain antibodies comprising a VL or VH domain. Antigen-binding antibody fragments, including single chain antibodies, may include variable regions alone or in combination with all or a portion of: hinge region, CH1, CH2 and CH3 domain. The invention also includes antigen binding fragments comprising any combination of the variable region and the hinge, CH1, CH2, and CH3 domains.
Binding molecules of the invention also include binding molecules recombinantly fused or chemically conjugated (including both covalent and non-covalent conjugations) to the polypeptide.
Examples of recombinant fusion of polypeptides include fusion of a binding molecule of the invention to a tag sequence, including but not limited to an HA tag, a 6XHis tag, a c-Myc tag, a flag tag.
Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β -galactosidase, or acetylcholinesterase, examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin, examples of suitable fluorescent materials include umbelliferone, fluorescein isothiocyanate, rhodamine, diazainide, examples of suitable luminescent materials include luminol, luciferase, and luciferase125I、131I、111In or99Tc。
The binding molecules of the invention also include functional variants of the binding molecules described above. A variant molecule is considered to be a functional variant of a binding molecule of the invention if it competes with the parent binding molecule for specific binding to hepatitis C virus or a protein fragment thereof. In other words, the functional variant is still capable of binding to hepatitis c virus or a protein fragment thereof. The functional variants may have conservative sequence modifications, including amino acid substitutions, additions, and deletions. These modifications can be introduced by standard techniques known in the art, such as site-directed mutagenesis and random PCR-mediated mutagenesis, and can comprise natural as well as unnatural amino acids. Furthermore, functional variants may comprise a truncation of the amino acid sequence at the amino terminus or the carboxy terminus or both. The functional variants of the invention may have the same or different, higher or lower binding affinity than the parent binding molecule, but still bind to hepatitis c virus or a fragment thereof. The functional variants of the invention have neutralizing activity against hepatitis c virus. The neutralizing activity may be the same or higher or lower than the parent binding molecule. Hereinafter, when the term "binding molecule" is used, it also covers functional variants of said binding molecule.
As a further aspect of the invention there is also provided a polynucleotide which binds to a molecule as hereinbefore described. The invention also includes polynucleotides that hybridize under stringent or less stringent conditions to a polynucleotide encoding a binding molecule of the invention.
Those skilled in the art will appreciate that functional variants of these polynucleotides are also part of the present invention. A functional variant is a nucleic acid sequence that can be directly translated using standard genetic code to provide the same amino acid sequence as translated from a parent nucleic acid molecule.
The polynucleotide sequence can be obtained and the nucleotide sequence of the polynucleotide determined using any method known in the art. For example, if the nucleotide sequence of an antibody is known, a polynucleotide encoding the antibody can be assembled from chemically synthesized oligonucleotides.
Alternatively, polynucleotides encoding the antibody may be produced from nucleic acids from a suitable source. If no clone containing a nucleic acid encoding a particular antibody is available, but the sequence of the antibody molecule is known, then the nucleic acid encoding the immunoglobulin can be obtained by chemical synthesis, or by PCR amplification from a suitable source (e.g., an antibody cDNA library, or a cDNA library generated from any tissue or cell expressing the antibody, e.g., hybridoma cells selected for expression of the antibody of the invention, or a nucleic acid isolated therefrom, preferably poly A + RNA) using synthetic primers hybridizable to the 3 'and 5' ends of the sequence, or by cloning using an oligonucleotide probe specific for the particular gene sequence to identify a cDNA clone encoding the antibody, e.g., from a cDNA library. The amplified nucleic acid produced by PCR can then be cloned into a replicable cloning vector using any method well known in the art.
Once the nucleotide sequence of the antibody and the corresponding amino acid sequence are determined, the nucleotide sequence of the antibody can be processed using methods well known in the art for processing nucleotide sequences, such as recombinant DNA techniques, site-directed mutagenesis, PCR, and the like, to generate antibodies having different amino acid sequences, such as amino acid substitutions, deletions, and/or insertions.
The amino acid sequences of the heavy and/or light chain variable domains can be examined to identify the sequence of the CDRs by well-known methods, for example, by comparison with known amino acid sequences of other heavy and light chain variable regions to determine regions of high variability of the sequences. Using conventional recombinant DNA techniques, one or more CDRs can be inserted into a framework region, for example into a human framework region to humanize a non-human antibody, as described above. The framework regions may be naturally occurring or common framework regions, and preferably human framework regions (see, e.g., the list of human framework regions in 293 Tzhiaeta l., J.mol.biol.278: 457-. Preferably, the polynucleotides produced by the combination of framework regions and CDRs encode antibodies that specifically bind to the polypeptides of the invention. Preferably, as discussed above, one or more amino acid substitutions may be made within the framework regions, and preferably the amino acid substitutions improve binding of the antibody to its antigen. In addition, one or more variable region cysteine residues involved in an intrachain disulfide bond may be substituted or deleted by these methods to produce an antibody molecule lacking one or more intrachain disulfide bonds. Other variations on the polynucleotides are encompassed by the present invention and are within the skill of those in the art.
The present invention also provides a recombinant expression vector comprising the polynucleotide as described above.
Recombinant expression vectors containing nucleotide sequences encoding binding molecules of the invention can be prepared using well-known techniques. The expression vector includes a nucleotide sequence operably linked to a suitable transcription or translation regulating nucleotide sequence, such as those derived from a mammalian, microbial, viral, or insect gene. Examples of regulatory sequences include transcriptional promoters, operators, enhancers, mRNA ribosome binding sites, and/or other suitable sequences that control transcription and translation initiation and termination. Nucleotide sequences are "operably linked" when the regulatory sequence is functionally related to the nucleotide sequence of a suitable polypeptide. Thus, a promoter nucleotide sequence is operably linked to, for example, an antibody heavy chain sequence if it controls the transcription of the appropriate nucleotide sequence.
The present invention also provides a host cell comprising a polynucleotide as described above or a recombinant expression vector as described above.
Host cells useful in the present invention include, but are not limited to, microorganisms such as bacteria (e.g., escherichia coli, bacillus subtilis) transformed with recombinant phage DNA, plasmid DNA, or cosmid DNA expression vectors containing antibody coding sequences; yeast such as Saccharomyces (Saccharomyces), Pichia (Pichia)) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant viral expression vectors (e.g., baculovirus) containing antibody coding sequences; plant cell systems infected with recombinant viral expression vectors (e.g., cauliflower mosaic virus (CaMV); Tobacco Mosaic Virus (TMV); or transformed with recombinant plasmid expression vectors containing antibody coding sequences (e.g., Ti plasmid), or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3 cells) carrying recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., the metallothionein promoter) or promoters derived from mammalian viruses (e.g., the adenovirus late promoter, the vaccinia virus 7.5K promoter).
In a particular embodiment of the invention, the host cell is a mammalian cell, more preferably a CHO cell.
Transformation and transfection of host cells with recombinant DNA may be carried out by conventional techniques well known to those skilled in the art. Some transformation, transfection methods that may be employed include, but are not limited to: conventional chemical methods such as calcium phosphate co-precipitation, PEI transfection, and conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, and the like.
The resulting transformants can be cultured by conventional methods to express the binding molecules of the invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culturing is performed under conditions suitable for growth of the host cell. After the host cells have been grown to an appropriate cell density, the selected promoter is induced by suitable means (e.g., temperature shift or chemical induction) and the cells are cultured for an additional period of time.
The binding molecules of the invention are preferably produced using mammalian cells, which typically require culture in serum-containing media. After the serum-free adaptation process of the cells is required, the cells can be normally grown in a serum-free medium.
The invention provides a pharmaceutical composition for inhibiting hepatitis C virus, or treating or preventing hepatitis C virus infection diseases, which comprises an effective amount of the binding molecule of the invention.
The invention also provides a pharmaceutical package or kit comprising one or more containers containing one or more components of the pharmaceutical composition of the invention. Optionally accompanying these containers may be instructions in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which instructions reflect certification by the agency regulating the manufacture, use or sale of pharmaceuticals for human administration.
The invention also provides an HCV detection product comprising a binding molecule as defined above.
The detection product includes, but is not limited to, a detection reagent, a kit, a chip or a test paper. Any assay product capable of detecting HCV comprising the aforementioned binding molecules is included within the scope of the present invention.
The present invention also provides a method for detecting HCV levels for non-diagnostic purposes, said method comprising the steps of:
(1) obtaining a sample containing HCV;
(2) contacting the sample obtained in step (1) with a binding molecule as described previously;
(3) detecting the binding reaction of the sample with the binding molecule.
The invention also provides a method for preparing said binding molecule comprising culturing a host cell comprising a vector of the invention under conditions suitable to cause expression of the protein from DNA encoding an antibody molecule of the invention, and isolating the binding molecule.
In particular, the invention provides a method of preparing a binding molecule as hereinbefore described using single B cell antibody preparation techniques. The single B cell antibody preparation technology is an in vitro expression system of a monoclonal antibody formed by combining a single cell separation identification technology with a plurality of PCR technologies, namely a method for directly obtaining a fully human monoclonal antibody from human B cells.
Further, the preparation method comprises the following steps:
(1) collecting a blood sample of a HCV infected person;
(2) isolating Peripheral Blood Mononuclear Cells (PBMCs);
(3) PBMC cells are sorted by using a flow cytometer, cell debris, adherent cells and dead cells are firstly removed, CD3-/CD14-/CD 16-/IgM-cells are obtained by fluorescent antibody staining, B cells expressing CD235a-/IgD-/CD20+ are selected, CD27ALL memory B cells are circled, and E2 dual-fluorescence labeled target cells are obtained by using an antigen specifically labeled with fluorescein.
(4) Amplifying the nucleotide fragments of the variable regions of the light chain and the heavy chain of the antibody in the single B cell obtained in the step (2) by using single-cell RT-PCR;
(5) fusing the nucleotide fragments of the variable regions of the light chain and the heavy chain of the antibody obtained in the step (3) into an expression vector containing a human antibody constant region to form a recombinant expression vector, and then introducing the recombinant expression vector into a host cell for expression;
(6) the binding molecules of the invention are screened for binding and neutralizing activity.
The invention also provides the application of the binding molecule in preparing HCV detection products.
The detection product comprises a binding molecule as described above; the detection product includes, but is not limited to, a detection reagent, a kit, a chip or a test paper. Any assay product that is capable of detecting HCV comprising the binding molecules described above is included within the scope of the present invention.
The invention also provides the use of a binding molecule as hereinbefore described in the preparation of a pharmaceutical composition as hereinbefore described.
The invention also provides the application of the binding molecule in preparing a medicinal preparation for inhibiting the hepatitis C virus or preventing or treating the hepatitis C virus infection diseases.
Further, the diseases include liver diseases such as hepatitis, liver cirrhosis or liver cancer; extrahepatic diseases such as skin disorders, e.g., cryoglobulinemia, delayed porphyria cutanea dermalis, leucocytoclastic vasculitis, reticularis, etc.
Further, the hepatitis is acute or chronic hepatitis c.
The binding molecule can also be combined with other medicines with the same or complementary functions, the combined application effect can be the sum of the functions of the binding molecule and other medicines, and can also be far greater than the sum of the functions of the binding molecule and other medicines, and the situation shows that the binding molecule and other medicines generate synergistic effect.
The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous population in which the individual antibodies comprised are identical except for a few naturally occurring mutations that may be present. The modifier "monoclonal" indicates only the identity of the antibody and is obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring any particular method for producing the antibody.
The term "variable" as used herein means that certain portions of the variable regions in an antibody differ in sequence, which results in the binding and specificity of each particular antibody for its particular antigen.variability is concentrated in three segments of the light and heavy chain variable regions, termed Complementarity Determining Regions (CDRs) or hypervariable regions.A native heavy and light chain variable regions each comprise four FR regions (the more conserved portions of the variable regions) which are approximately in an β -fold configuration, linked by three CDRs which form a connecting loop, which can form part of the β fold structure.
As used herein, an "effective amount" is a dose sufficient to alleviate one or more symptoms normally associated with HCV infection or any disease or condition caused by or associated with HBV infection. When considering prophylactic use, the term means a dose sufficient to prevent or delay establishment of HCV infection.
As used herein, "sample" encompasses a variety of sample types, including blood and other bodily fluid samples of biological origin, solid tissue samples such as biopsy tissue samples or tissue cultures, or cells derived therefrom or progeny thereof. The term also includes samples that have been treated by any means after they have been obtained, for example by treating with reagents, solubilizing, or enriching certain components such as proteins or polynucleotides. The term encompasses various clinical samples obtained from any species, also including cultured cells, cell supernatants and cell lysates.
The term "isolated" as used herein refers to a protein that is isolated from its natural environment (i.e., from at least one other component with which it is naturally associated).
All patents and publications mentioned herein are incorporated herein by reference to the extent allowed by law for the purpose of describing and disclosing the proteins, enzymes, vectors, host cells and methodologies reported in the patents and publications that might be used in connection with the invention. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.
Drawings
FIG. 1 is a SDS-PAGE and Western-blot detection chart of a TRN1008 antibody according to an embodiment of the present invention, wherein A: SDS-PAGE; b: western-blot;
FIG. 2 is a graph showing the detection of the binding activity of the TRN1008 antibody to 8 HCV antigens of different genotypes in the present example;
FIG. 3 is a graph showing the effect of neutralizing activity of the TRN1008 antibody against 7 HCV viruses of different subtypes according to the present invention;
FIG. 4 is a graph showing the measurement of the affinity activity of the TRN1008 antibody for the HCV antigen E2-core protein of 4 different genotypes in the present example, wherein a: e2-core-2 a; b: e2-core-4 a; c: e2-core-6 a; d: e2-core-7 a.
Detailed Description
The invention is not limited to the particular methodology, protocols, cell lines, vectors or reagents described herein, as these may vary. Furthermore, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention.
Unless otherwise defined, all technical and scientific terms and any abbreviations used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, the use of equipment and materials is described herein.
EXAMPLE 1 preparation of fully human anti-HCV monoclonal neutralizing antibody TRN1008
1. Sample acquisition: anticoagulated peripheral blood of HCV-infected patients was collected from kunming city, Yunnan, and the infection time was confirmed to be 2010.
2. Memory B cell sorting
Collecting blood samples of HCV-infected persons, separating Peripheral Blood Mononuclear Cells (PBMC), performing cell sorting by a BD FACCria flow cytometer (BD Biosciences, San Jose, CA), firstly removing cell debris, adhesive cells and dead cells, obtaining CD3-/CD14-/CD 16-/IgM-cells by fluorescent antibody staining, selecting B cells expressing CD235a-/IgD-/CD20+, trapping CD27ALL memory B cells, and obtaining E2 dual-fluorescence labeled target cells by using an antigen specifically labeled with fluorescein.
3. Single cell RT-PCR
0.5. mu.M of constant region primers for heavy and light chains of each subtype and Superscript IV reverse transcriptase (Invitrogen, Carlsbad, Calif.) were added to the PCR prediction mix in 96-well plates containing single B lymphocytes, with positive and negative controls set; reverse transcription PCR conditions: 60min at 55 ℃ and cooling to 4 ℃. The product cDNA is preserved for a long time at the temperature of 20 ℃.
4. Amplification of target genes in antibody variable regions
Using the reverse transcription product (cDNA) as a template, AmpliTaq Gold 360Master Mix (Invitrogen, Carlsbad, Calif.) and primers specific for each subtype of heavy and light chain antibody at 0.5. mu.M were added. Reaction conditions are as follows: pre-denaturation 95 ℃ for 5min, followed by 35 PCR cycles, each cycle: 94 ℃ X30 s, 58/60/64 (H/K/L). times.30 s, 72 ℃ X1.5 min, extension at 72 ℃ for 7min and then cooling to 10 ℃.
5. DNA gel electrophoresis identification
The identification was performed by electrophoresis experiments using 2% agarose gel. And mixing 2 mu L of PCR reaction product with 18 mu L of 0.1% Loading Buffer, Loading, and performing electrophoresis for 12min in an EG mode of an E-base electrophoresis system. The gel imaging system detects the size of the PCR product.
6. Construction of expression vector for recombinant antibody
Obtaining antibody heavy chain and light chain gene fragments (comprising a variable region and a constant region) which are detected to be positive by ELISA (enzyme-linked immunosorbent assay), respectively linking the heavy chain and light chain genes to pcDNA3.3 vectors by using a TA (polymerase chain reaction) cloning method, transforming the connection product into DH5 α competent bacteria, culturing overnight at 37 ℃ on a plate containing ampicillin, then picking 12 single colonies, carrying out PCR identification by using specific primers (PCR reaction conditions are: pre-denaturation at 94 ℃ for 3min, denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 1min, 40s, 28 cycles, and re-extension at 72 ℃ for 5min), taking 2 mu LPCR products for electrophoresis detection on 1% agarose gel, selecting colonies identified to be positive by PCR, carrying out gene sequencing, and obtaining the correct comparison result, namely the recombinant expression of the antibody heavy chain and light chain.
7. ELISA detection of expression of target Gene
Large-scale amplification expression of positive antibody heavy chain in Escherichia coli DH5 αAnd extracting the plasmid of the light chain gene by an endotoxin-free extraction kit. Transfecting CHO cell with transfection reagent Polyetherimide, adding fresh serum-free culture medium 4-6 hr after transfection, placing at 37 deg.C and 8% CO2Culturing in a constant temperature incubator for 96 hours, and collecting cell supernatant for detection.
After 72h of culture, cell supernatant was subjected to direct competition ELISA to detect the antibody expressed by the target gene. And (3) ELISA screening: different HCV envelope glycoproteins were used as antigens, and the antigens were diluted with 100ng/ml using a coating solution and coated on ELISA 96-well plates at 100. mu.l/well at 4 ℃ overnight. Blocking the mixture at 37 ℃ for 2 hours. After blocking, primary antibody is added, the initial concentration of the antibody is 25 mu g/ml, the primary antibody is diluted by 3 times of gradient, the volume of each pore is 100 mu l, the primary antibody is incubated for 1 hour at 37 ℃, meanwhile, the serum of HCV positive patients is used as a positive control, and the rabies antibody is used as a negative control. The cells were incubated with HRP-labeled goat anti-human IgG (1:2000 dilution) as a secondary antibody at 37 ℃ for 1 hour. Adding substrate color development solution (TMB), standing at 37 deg.C in dark for 5min, stopping reaction with 2M sulfuric acid, and performing color comparison with 450nm wavelength.
8. Antibody neutralization assay
The antibodies with binding activity screened by ELISA were subjected to neutralization assay. The coding sequences for the different HCV envelope glycoproteins were packaged into plasmids, the luciferin Luciferase gene was added to transfect 293T cells for packaging of HCV virus (HCVpp), and the supernatant was harvested for infection. Huh7 cells were plated in 96-well plates at approximately 1 x 10 cells per well4Each well was 100. mu.l in volume, and cultured overnight, with approximately 30% of cells confluent upon infection. HCVpp was mixed with the antibody and left at room temperature for 30 min. The Huh7 cells were infected by adding HCVpp and antibody mixtures to 96-well plates. The control group was added HCVpp only, and the medium was changed 24h after infection and incubation was continued for 1-2 days. The Luciferase activity was measured 2-3 days after infection by removing cell supernatant, using 30. mu.l of lysate per well, lysing sufficiently, adding 30. mu.l of substrate to 20. mu.l of cell lysate, and reading the fluorescence value. The antibody was compared to the control group and the neutralization efficiency was calculated.
9. Antibody mass expression and purification
An expression vector for neutralizing the antibody heavy chain and light chain (wherein, the heavy chain has neutralization activity) of the TRN1008 identified by the neutralization experimentThe amino acid sequence of the variable region is shown as SEQ ID NO. 1; the amino acid sequence of the antibody light chain variable region is shown in SEQID NO: 5) to cotransfect CHO cell (the cell density reaches 1.6 multiplied by 10)6One/ml), supplement medium 6-8 hours after transfection, 37 ℃, 8% CO2The cells were incubated for 120 hours on a shaker at 125 rpm. The transfection supernatant was then collected, centrifuged at 4000rp at 4 ℃ for 1 hour, the precipitate discarded and purified by Protein A affinity chromatography. The expression and purification results of the antibody TRN1008 are checked by SDS-PAGE and Western-blot, and the results are shown in figure 1, so that relatively pure protein is obtained, and light and heavy chains of the antibody after melting can be clearly observed.
Example 2 detection of binding Activity of TRN1008 antibody
1. Specific binding assay for TRN1008 antibody and HCV envelope glycoprotein
The binding specificity of the expressed purified antibodies was determined by ELISA as mentioned in example 1: the different HCV envelope glycoprotein coding sequences were packaged into plasmids, the luciferin Luciferase gene was added to transfect 293T cells for packaging of HCV virus (HCVpp), and the supernatant was harvested for infection. Huh7 cells were plated in 96-well plates at approximately 1 x 10 cells per well4Each well was 100. mu.l in volume, and cultured overnight, with approximately 30% of cells confluent upon infection. HCVpp was mixed with antibody HCV at different concentrations, starting at 75. mu.g/ml, diluted 3-fold and allowed to stand at room temperature for 30 min. The Huh7 cells were infected by adding HCVpp and antibody HCV mixtures to 96-well plates. The control group was added HCVpp only, and the medium was changed 24h after infection and incubation was continued for 1-2 days. The Luciferase activity was measured 2-3 days after infection by removing cell supernatant, using 30. mu.l of lysate per well, lysing sufficiently, adding 30. mu.l of substrate to 20. mu.l of cell lysate, and reading the fluorescence value.
As a result: the TRN1008 antibody can be specifically combined with HCV envelope glycoprotein by 100 percent, and the fluorescence value is reduced.
2. Detection of binding Activity of TRN1008 antibody to HCV antigens of different genotypes
The antigen was diluted to 100ng/ml with coating solution and coated on ELISA 96-well plate at 4 ℃ overnight at 100. mu.l/well using 8 different genotypes of HCV envelope glycoprotein. Blocking the mixture at 37 ℃ for 2 hours. After blocking, primary antibody was added in a volume of 100. mu.l per well and incubated at 37 ℃ for 1 hour. The cells were incubated with HRP-labeled goat anti-human IgG (1:2000 dilution) as a secondary antibody at 37 ℃ for 1 hour. Adding substrate color development solution (TMB)100 μ L/well, standing at 37 deg.C in dark for 5min, stopping reaction with 2M sulfuric acid, and performing color comparison with 450nm wavelength.
As a result, as shown in fig. 2, the TRN1008 antibody can bind to HCV antigens of different genotypes, and the TRN1008 has a very strong binding activity, wherein 1b, 1a, 2, 3, 4, 5, 6, and 7 in fig. 2 represent HCV1b, HCV1a, HCV2, HCV3, HCV4, HCV5, HCV6, and HCV7, respectively.
Example 3 detection of neutralizing Activity of the TRN1008 antibody with different strains of HCV Euviruses
Huh7 cells were plated in 96-well plates and cultured overnight with approximately 30% confluency of cells upon infection. HCV true virus strains (h77, Con1, JFH1, S52, ED43, SA13, HK6a) were mixed with antibodies of different concentrations, diluted 3-fold at an initial concentration of 25. mu.g/ml, and left at room temperature for 30 min. The mixed solution of HCV strains (h77, Con1, JFH1, S52, ED43, SA13 and HK6a) and antibody was added to a 96-well plate to infect Huh7 cells. Only the HCV strain 2G9 was added to the control group, and the culture was continued for 1 to 2 days after 24 hours of infection. Activity was measured 2-3 days after infection and fluorescence was read. The antibody was compared to the control group and the neutralization efficiency was calculated.
As shown in fig. 3, the neutralizing efficiency of the TRN1008 antibody against different HCV strains is shown in the following table, and the neutralizing efficiency of the TRN1008 antibody against HCV strains H77(1a), Con1(1b), JFH1(2a), S52(3a), ED43(4a), SA13(5a) and HK6a is very good, which shows a promising application prospect in immunotherapy and prophylaxis.
TABLE 1 neutralization efficiency of TRN1008 antibody with different HCV strains
Example 4 detection of affinity Activity of TRN1008 antibody with envelope glycoprotein of HCV
Analysis of the affinity and kinetics of TRN1008 binding to the HCV envelope glycoprotein (HCV-E2-core, antigen) was performed using BiaCore X-100: HCV-E2-core protein was diluted with HBS-EP buffer as the analyte, and the analyte was sequentially passed through the chip at increasing concentrations to obtain signal curves, respectively. And analyzing by BiaCore X-100System software to obtain the result of the affinity and kinetic parameters of the TRN 1008.
The results are shown in FIG. 4 and Table 2. The antibody against TRN1008 has KD values of 1.32E-11M, 1.12E-9M, 2E-11M and 2.98E-12M for the antigens E2-core-2a, E2-core-4a, E2-core-6a and E2-core-7a, respectively, and shows that the TRN1008 has high affinity activity.
TABLE 2 table of the kinetics results of the affinity of the TRN1008 antibody to HCV viral antigens
| Antigens | ka(1/Ms) | kd(1/s) | KD(M) |
| E2-core-2a | 13010 | 1.72E-07 | 1.32E-11 |
| E2-core-4a | 3.48E+04 | 3.89E-05 | 1.12E-09 |
| E2-core-6a | 24410 | 4.87E-07 | 2E-11 |
| E2-core-7a | 4.06E+04 | 1.21E-07 | 2.98E-12 |
Although only specific embodiments of the present invention have been described above, it will be understood by those skilled in the art that these are by way of illustration only, and that the scope of the invention is defined by the appended claims. Various changes or modifications to these embodiments may be made by those skilled in the art without departing from the principle and spirit of the invention, and these changes or modifications are within the scope of the invention.
SEQUENCE LISTING
<110> Tenuodi Biotechnology Ltd, Guangzhou
Zhuhai Tainuo Mibo Biotech Co., Ltd
River-south university
<120> preparation method and application of medicine for treating hepatitis C virus
<160>8
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Claims (13)
1. An isolated binding molecule, comprising:
(1) heavy chain CDR1 shown in SEQ ID NO. 2, heavy chain CDR2 shown in SEQ ID NO. 3, and heavy chain CDR3 shown in SEQ ID NO. 4; and
(2) light chain CDR1 shown in SEQ ID NO. 6, light chain CDR2 shown in SEQ ID NO. 7, and light chain CDR3 shown in SEQ ID NO. 8.
2. The binding molecule of claim 1, wherein the binding molecule comprises:
(1) a heavy chain variable region having an amino acid sequence set forth in SEQ ID NO 1; and
(2) and a light chain variable region having an amino acid sequence set forth in SEQ ID NO. 5.
3. The binding molecule of claim 1, wherein said binding molecule is an antibody molecule or an immunologically active fragment thereof.
4. A polynucleotide encoding the binding molecule of any one of claims 1-3.
5. A recombinant expression vector comprising the polynucleotide of claim 4.
6. A host cell comprising the polynucleotide of claim 4 or the recombinant expression vector of claim 5.
7. A pharmaceutical composition or HCV detection product comprising a therapeutically effective amount of a binding molecule according to any of claims 1 to 3.
8. A method for detecting HCV levels for non-diagnostic purposes, said method comprising the steps of:
(1) obtaining a sample containing HCV;
(2) contacting the sample obtained in step (1) with a binding molecule according to any one of claims 1 to 3;
(3) detecting binding of a sample to the binding molecule of any one of claims 1 to 3.
9. Use of a binding molecule according to any one of claims 1 to 3 for the preparation of a HCV detection product, or a medicament for inhibiting HCV, or a medicament for the prevention or treatment of a disease caused by HCV infection.
10. The use according to claim 9, wherein the disease caused by HCV infection comprises liver disease, extrahepatic disease.
11. The use according to claim 10, wherein the liver disease comprises hepatitis, cirrhosis or liver cancer.
12. The use according to claim 10, wherein the extrahepatic disorder comprises an abnormality of the skin.
13. The use of claim 12, wherein the skin disorder comprises cryoglobulinemia, porphyria cutanea tarda, thromboangiitis obliterans, reticularis.
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| US7605237B2 (en) * | 2006-10-02 | 2009-10-20 | Regeneron Pharmaceuticals, Inc. | High affinity human antibodies to human IL-4 receptor |
| CN106749644A (en) * | 2016-11-14 | 2017-05-31 | 广州泰诺迪生物科技有限公司 | A kind of neutralizing antibody TRN1001 of full people source HCV-Ab IgG |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US7605237B2 (en) * | 2006-10-02 | 2009-10-20 | Regeneron Pharmaceuticals, Inc. | High affinity human antibodies to human IL-4 receptor |
| CN106749644A (en) * | 2016-11-14 | 2017-05-31 | 广州泰诺迪生物科技有限公司 | A kind of neutralizing antibody TRN1001 of full people source HCV-Ab IgG |
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