WO2011085574A1 - 全人TNFα单克隆抗体及其制备方法与应用 - Google Patents

全人TNFα单克隆抗体及其制备方法与应用 Download PDF

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WO2011085574A1
WO2011085574A1 PCT/CN2010/073199 CN2010073199W WO2011085574A1 WO 2011085574 A1 WO2011085574 A1 WO 2011085574A1 CN 2010073199 W CN2010073199 W CN 2010073199W WO 2011085574 A1 WO2011085574 A1 WO 2011085574A1
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seq
monoclonal antibody
human tnfa
human
antibody
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倪健
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苏州工业园区晨健抗体组药物开发有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/241—Tumor Necrosis Factors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/04—Drugs for skeletal disorders for non-specific disorders of the connective tissue
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • 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
    • C07K2317/90—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

Definitions

  • the present invention relates to the field of biotechnology, and in particular to ⁇ 3 ⁇ 4 binding proteins and uses thereof. Background technique
  • RA Rheumatoid arthritis
  • RA rheumatoid arthritis
  • the prevalence of rheumatoid arthritis in China is about 0.32-0.36%, and the incidence rate in people over 60 years old is as high as 30% ⁇ 40%.
  • RA has a large number of patients and a large amount of drugs, making its market for therapeutic drugs huge. In 2005, the global market reached US$6 billion. At present, there are many clinical treatments for RA. It is preferred to use high-dose non-steroidal anti-inflammatory drugs to control symptoms. If it is not effective, clinically preferred immunosuppressive drugs such as methotrexate. About one-fourth of the patients will not relapse after the first onset of the disease, and about one-half of the patients will be relieved after the treatment, but they will recur, and some will eventually develop into joint dysfunction. One in four patients is ineffective for all current routine treatments.
  • Tumor necrosis factor-a (TNF- ⁇ ) is a cytokine that plays a major role in the pathogenesis of rheumatoid arthritis. Experimental and clinical results have confirmed that TNF is a suitable target for the treatment of this disease. Foreign studies on the pathogenesis of sputum have been carried out, and it is found that Tumor Necrosis Factor alpha (TNF-) plays a key role in the pathological process of the disease. Abnormal TNF levels in the joint cavity of patients or animal models Raise. TNFa and TNFp are a class of homologous secretory proteins in mammalian cells that share high homology with each other, both of which can have multiple effects on many cell types.
  • Tumor necrosis factor TNFo can cause hemorrhage and necrosis of tumor tissue and has many functions.
  • the mature molecule consists of 157 amino acids with two disulfide bonds and no glycosylation sites.
  • the precursor is a polypeptide consisting of 233 amino acids, and the polypeptide consisting of N-terminal 76 amino acids is not a signal peptide. It can immobilize the precursor on the cell membrane and retain the mature secretory form on the cell membrane.
  • the length of the mature mRNA of the precursor is 1. 7 kb. It is mainly produced by mononuclear macrophages in the body.
  • Inhibitors against TNF-a are by far the most successful treatments for RA.
  • Representative drugs such as Wyeth/Amgen Enbrel (etanercept), Johnson & Johnson, Schering Plough/Centocor company emicade (infliximab), Cambridge Antibody Technology/Abbott's Humira (adalimumab).
  • Enbrel The preparation developed for Amgen is a fusion protein of TNF- ⁇ receptor and Fc. It not only has a very good effect on rheumatoid arthritis, but also has obvious therapeutic effects on psoriasis and psoriatic arthritis, and promotes spondylitis. In the study of healing, Enbrel also entered the second phase of clinical.
  • Remicade is a chimeric anti-TNF- ⁇ monoclonal antibody that specifically binds to soluble and membrane-bound TNF- ⁇ . Indications include Crohn' disease, rheumatoid arthritis (RA), and severe tonicity. Spondylitis (AS). In 2003 and 2004, global sales were 2.27 billion and 2.89 billion US dollars respectively, which is a "blockbuster” in the biomedical field. In December 2002, Abbott's Humira became the first fully human monoclonal antibody approved for the relief of signs and symptoms of moderate to severe active RA and the progression of joint structural damage. Because it is a fully human antibody, it avoids the disadvantage that Remicade will produce an immune response.
  • Humira compared to Remicade and Enbrel is the convenience of the method of administration. Humira only needs to be injected twice a month, while Remicade and Enbrel must be injected twice a week. Humira's market share is increasing year by year, with sales in 2003 and 2004 of $280 million and $850 million, respectively. In 2006, the world's three major anti-TNF- ⁇ antagonists, Humira and Remicade and Enbrel, were nearly $8 billion. In 2008, Enbrel (64.9), Remicade (53.3), Humira (45.2), and Cimzia totaled $16.3 billion.
  • the new drugs currently in the final stage of development include UCB/Celltech CDP-870 (PEGylated humanized anti-TNF antibody Fab fragment), Wo B CDP571 (humanized anti-TNF monoclonal antibody), Bristol-Myers Squibb CTLA41g ( Abatacept), Roche's Rituxan and MRA.
  • CDP-870 The fastest-growing one is CDP-870, which has entered clinical phase III.
  • CDP-870 entered the market at the end of 2006.
  • CDP-870 is produced using E. coli, reducing its cost to four-quarters of other TNF inhibitor drugs.
  • TNF antibody drugs are considered to perform better than any other drug, they also have major drawbacks.
  • they are all derived from mammalian cells for expression and purification.
  • Remicade is a human-mouse chimeric monoclonal antibody containing 1/3 of the mouse protein component. Continuous injection can stimulate human resistance in more than 10% of patients. A murine protein antibody response or a human anti-chimeric protein antibody response affects the therapeutic effect and may result in serious adverse reactions.
  • the forthcoming CDP870 is a PEGylated humanized anti-TNF antibody Fab fragment [17] with a murine protein component down to 10% but still immunogenic.
  • the amino acid sequence of the variable region of Humir and the corresponding DNA sequence are derived from a human antibody library developed by CAT, and the coding region of the Fc fragment is derived from human IgG1. Thus, the variable region sequence of the product is not directly from humans, but from an antibody library derived from human B cells.
  • the human immune system is capable of producing extremely large types of antibodies
  • human or whole-body antibody technology has encountered bottlenecks: the abundance of precursor cells that produce specific antibodies in humans is low, even in antibody-positive individuals.
  • the precursor cells that produce specific antibodies are also much lower than the number of specific antibody cells required to produce human hybridoma technology. Therefore, isolation, purification, and enrichment of B cells that specifically secrete antibodies have become one of the key technologies for establishing a fully human antibody platform.
  • the invention adopts the whole human antibody technology to obtain the anti-human TNFa monoclonal antibody from the B cells of the rheumatoid arthritis patient body, and after fermentation and purification, it is modified and modified in vitro by the PEGylation (PEGylation) method, and the anti-inhibition is maintained. At the same time, TNF activity makes it more suitable for in vivo applications.
  • a first aspect of the invention discloses an anti-human TNFa monoclonal antibody comprising a heavy chain and a light chain, the light chain of which has the amino acid sequence of SEQ ID NO: 3, the heavy chain of which has SEQ ID NO: 4 or SEQ ID NO: The amino acid sequence of 6.
  • a second aspect of the invention discloses a polynucleotide comprising a polynucleotide sequence encoding a heavy chain and/or a light chain of the aforementioned anti-human TNFa monoclonal antibody.
  • sequence of the polynucleotide encoding the light chain of the aforementioned anti-human TNFa monoclonal antibody is SEQ ID NO: 1
  • sequence of the polynucleotide encoding the heavy chain of the aforementioned anti-human TNFa monoclonal antibody is SEQ ID NO: 2 Or SEQ ID NO: 5
  • sequence encoding the heavy and light chain polynucleotides of the anti-human TNFa monoclonal full-length antibody comprising both SEQ ID NO: 1 and SEQ ID NO: 2 or both SEQ ID NO: 1 SEQ ID NO: 5.
  • a third aspect of the invention discloses a genetically engineered host cell which is encoded with the aforementioned anti-human TNFa single gram
  • the vector of the heavy and light chain polynucleotides of the chimeric antibody is transformed or transduced.
  • Any cell suitable for expression of the expression cassette can be used as a host cell.
  • the host cell is a eukaryotic cell, and the mammalian host cell line which does not produce an antibody can be used, specifically: ovary cell CH0 of Chinese hamster; kidney cell of baby hamster (BHK, ATCC CCL 10); Sertol i cells of young rats (TM4, Mather, Biol. Reprod.
  • kidney cells of monkeys (COS cells); by SV40 (COS-7, ATCC CRL) 165 1) Kidney CVI cells of transformed monkeys; human embryonic kidney cells (HEK-293, Graham et al. J. Gen Virol. 36: 59 (1977)); monkey kidney cells (CVI ATCC CCL 70); African green Kidney cells of monkeys (VER0-76, ATCC CRL-1587); human cervical cancer cells (HELA, ATCC CCL 2), etc.
  • Other cell lines are also common in the literature of this subject. A variety of cell lines are available from the American Type Culture Collection Center (ATCC). There are many ways to introduce nucleic acids into cells.
  • the fourth aspect of the invention discloses a method for preparing an anti-human TNFa monoclonal antibody, comprising the following steps: 1) cultivating the host cell under conditions suitable for expressing an anti-human TNFa monoclonal antibody; 2) separating and purifying to obtain the monoclonal antibody antibody.
  • the antibody of interest can be produced by the following method.
  • a vector containing a nucleic acid encoding an antibody of interest is directly introduced into a host cell, and the cells are cultured under appropriate conditions to induce expression of the encoded antibody.
  • the anti-body molecules can be purified by conventional methods known to those skilled in the art. Specific purification methods include chromatography, such as ion exchange, affinity chromatography, especially affinity chromatography with specific antigens of protein A, as well as size column chromatography, centrifugal filtration, differential solubility, and the like. In many cases, antibodies secreted by the cells enter the culture and are harvested in the culture.
  • the anti-human TNFa monoclonal antibody is tested for its therapeutic effectiveness in animals by in vitro activity assay and animal experiment, and the pharmaceutical use of the above anti-human TNFa monoclonal antibody is further disclosed.
  • Anti-human TNFa monoclonal antibodies are useful in the preparation of a medicament for the treatment of TNFa-associated inflammation, such as the preparation of a medicament for the treatment of rheumatoid arthritis.
  • the conventional usage amount of the existing TNFa antibody can be referred to.
  • a pharmaceutical composition comprising a therapeutically effective amount of the monoclonal antibody of the present invention And a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carriers and components thereof include saccharides; cellulose and derivatives thereof; malt; gelatin; talc; solid lubricants; polyols; alginic acid; emulsifiers; wetting agents; Flavoring agent; tablet; antioxidant; preservative; pyrogen-free water; isotonic saline solution; phosphate buffer.
  • the pharmaceutical preparation should be matched to the mode of administration.
  • the pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants.
  • Pharmaceutical compositions such as tablets and capsules can be prepared by conventional methods.
  • Pharmaceutical compositions such as injections, solutions, tablets and capsules are preferably manufactured under sterile conditions.
  • the amount of active ingredient administered is a therapeutically effective amount.
  • the invention adopts the whole human antibody technology and obtains the anti-human TNFa fully human antibody.
  • the preliminary analysis of the physicochemical and biological activity of the antibody shows that the antibody has good affinity with TNF, and can effectively neutralize TNF to L929 cells in vitro. Killing.
  • the antibody since all of its amino acid sequences are completely identical to human antibodies, its immunogenicity to the human body is minimized.
  • PEGylation surface modification technology is adopted to reduce the probability of phagocytosis by the endothelial system, prolong the time in the blood circulation system, and achieve the purpose of sustained release in vivo, thereby avoiding the shortcomings of short half-life of small molecule antibodies.
  • While maintaining its anti-TNFa activity it is more suitable for in vivo applications.
  • the frequency of administration can be greatly reduced. This not only further reduces the patient's financial burden, but also greatly reduces the pain of patient injection and reduces labor loss. detailed description
  • 96-well plates were routinely coated with human TNFa (purchased from Shenzhen Jingmei Co., Ltd.), and 250 ng of the protein was used per well, and coated overnight. Then, it was blocked with 5% skim milk powder for 2 hours at room temperature, and the milk powder was prepared with pH 7.2 PBS. After washing, 100 ⁇ l of serum from different patients was added and incubated for 1 hour at room temperature. Peroxidase-labeled goat anti-human IgG was then added and allowed to stand at room temperature for 1 hour. After washing at least 5 times, TMD or other developer is added and treated at room temperature or 37 degrees for 20 minutes. Finally, a stop solution is added. After adding the peroxide chromogenic substrate for 10 minutes, use 50 ⁇ 1 The sulfuric acid of IN was terminated, and the 450 nm optical density value was read. Serum with positive and high 0D values was selected as the selected blood sample.
  • the secretion of anti-TNFa IgG was detected by ELISA.
  • the samples of the human B cell culture supernatant were screened. Among them, two positive clones having the best binding ability were selected and were IgG1.
  • the positive cloned cells were each 5000-10000, and total RNA was isolated by Trizol (GIBC0), and cDNA was obtained by MMLV reverse transcription (Promega). The above operations are carried out in accordance with the manufacturer's instructions. PCR was carried out using the following primers and conditions, and the amplification enzyme used was ClonTech's Pfu to ensure that possible mutations were reduced during amplification.
  • Light chain upstream primer GAAATTGTGCTCACACAGTC (SEQ ID NO: 7)
  • Heavy chain upstream primer GAAGTCCAGCTGGTCGAGAG (SEQ ID NO: 9)
  • Heavy chain downstream primer GTGAGTTTTGTCACMGATTTGGGCTC (SEQ ID NO: 10)
  • Main cycle 94 degrees 1 minute, 55 degrees 1 minute, 72 degrees 3 minutes.
  • PCR was carried out in accordance with the above conditions. After completion, the products were identified on 1% agarose gel electrophoresis. The two PCR products were about 650 bp and 670 bp, respectively, which was consistent with the theoretical size. The fragment was recovered using Promega's gel recovery kit and operated according to the manufacturer's instructions, each giving approximately 2 micrograms of DNA.
  • Example 2 Preparation of anti-human TNFa antibody 1. Acquisition of heavy chain and light chain coding sequences In order to perform gene expression of full-length antibodies, the full-length heavy and light chains of human IgG1 were synthesized according to the sequencing results of Example 1. The sequence is such that a complete anti-human TNFa antibody-01 gene is constructed.
  • the following fragment is the light chain coding region and has a length of 645b. (SEQ ID NO: 1)
  • the following fragment is the full-length heavy chain coding region with a length of 1353 bp. (SEQ ID NO: 2) GAAGTCCAGCTGGTCGAGAGCGGTGGCGGGCTGGTGCAACCCGGTGGATCACTGCGGCTCAGCTGCGCTGCTAGTGGC AGTAAGTCTATGAACTCCGCCACACACTATGCTGAAAGCGTGAAAGGGCGCTTCACAATCTCTAGAGACGATTCAAAG
  • the full-length amino acid sequence of the encoded heavy chain is (SEQ ID NO: 4):
  • TNFa antibody -02 There is also an antibody TNFa antibody -02, a light chain and heavy chain variable region and a TNFa antibody -01, which has three amino acid differences compared to TNFa antibody -02.
  • the full-length heavy chain coding region of TNFa antibody -02 is: (SEQ ID NO: 5)
  • the full-length amino acid sequence of the encoded heavy chain is (SEQ ID NO: 6):
  • the heavy chain full-length coding region of two TNF antibodies (SEQ ID NO: 2, SEQ ID NO: 5), the light chain coding region (SEQ ID NO: 1), polyA and PCMV were spliced according to conventional Overlapping PCR methods, respectively.
  • the spliced fragment was l ight chain+po 1 yA+PCMV+heavy chain.
  • the vectors constructed on the pCI vector cloned into Promega were named pCI-TNF-01 and pCI-TNF-02, respectively. 4. Transfection and Screening of CHO Cells In this study, CHO cells were transfected by liposome method (LipoFemine 2000, Invitrogen).
  • the transfection kit was purchased from Ir itrogen, and the above purified ones were separately taken during transfection. 100 ⁇ g of plasmids of heavy and light chains were transfected as CH0 cells as DNA samples, and the transfection procedure was performed according to the manufacturer's instructions.
  • Transfected CH0 cells were screened for 3 consecutive months of methotrexate (MTX) and screened with MTX 2nM, 5nM, 20nM, 40nM, 80nM, 120nM, 160nM, 200nM, 250nM, 300nM, every two weeks. Increasing the concentration once, each time the amount of MTX is about twice that of the previous one, depending on the cell growth.
  • the cell culture was carried out as usual, and the medium was: 15% fetal bovine serum (Gibco) + RPM1640/DEME.
  • the cells were cultured in a 37 ° 5% (3 ⁇ 4 incubator). In this process, the cell culture supernatant was taken for ELISA, and after three ELISA tests, positive mixed clones were obtained and some cells were frozen.
  • the monoclonalization was carried out according to the conventional extreme dilution method, and the mixed clone was subjected to the first monoclonal culture by the 96-well plate limiting dilution method, and the diluted cell density was 2.5 cells/ml, and 200 ul of the cell suspension was added to each well to select 200.
  • 89 positive clones were selected by ELISA.
  • the first 35 monoclonal clones in the ELISA results were amplified and partially frozen.
  • the monoclonal antibody was subjected to a second limiting dilution method for subcloning culture.
  • the cell culture supernatant was identified by ELISA twice to obtain 826 monoclonal clones, and the first 35 monoclonal clones were subjected to ELISA for the third subcloning. After clearing, 1026 monoclonal clones were obtained by ELISA. The first 30 of the ELISA results were amplified and frozen, and some cells were frozen. RNA was extracted from the monoclonal cells, and the target gene was detected, and the number of copies of the target gene of the obtained single cloned cells was confirmed, and it was confirmed to be an anti-human TNF antibody. The N-terminal sequencing of the antibody protein was performed, and the results were consistent with the designed amino acids.
  • the anti-human TNFa antibody obtained in Example 2 was structurally engineered into a free thiol group, after amino acid C at position 229. Add two protecting agents, amino acid AA, to form a free sulfhydryl group, and further PEGylate.
  • PEGylation and purification methods refer to AP Champman et al. [Reference: Therapeutic antibody fragments with prolonged in vivo half l ife. Nature Biotech. V17 1999 August, Page 780- The method of 783] was carried out to obtain a PEGylated anti-human TNFa monoclonal antibody. After ELISA identification, it was confirmed to be positive with TNFa.
  • the prepared PEGylated antibody was subjected to a protective test with L929 cells (ECACC 85011425) (refer to Example 5), and it was found that the PEGylated antibody has a good protective effect.
  • Example 4 Detection of biological activity of anti-human TNFa monoclonal antibody
  • TNFa was coupled to the chip according to the immobilize method in the kinetics/affinity assay of Biacore X100 control software. Set the coupling level to 3000RU.
  • the coupling level is reported based on the results of its automated operation and the coupling results are automatically reported.
  • the final coupling was 3847.5RU.
  • Example 5 Anti-human TNFa monoclonal antibody effector function -L929 cell protection test Determination method description:
  • the antibody was diluted by gradient filtration, and 50 ⁇ l was added to each well, supplemented with Actinomycin D to the final concentration of lug/ml;
  • Example 6 The acute toxicity and death of an anti-human TNFa monoclonal antibody after one-time administration to an animal were taken at a 100-fold clinical dose (50 mg/ml) against human TNFa-01 and anti-human TNFa-02, respectively. side effect.
  • ISRs Injection site reactions
  • mice 17-22g of healthy mice (the difference in body weight of the same test does not exceed 4g), the body weight of the rats is generally 120_150g (the difference between the same test body weight does not exceed 10g)
  • Test substance liquid or powder (insoluble in water, 0.5% sodium carboxymethylcellulose, a suspension made of 10% gum arabic).
  • Dose selection 4-5 dose groups are generally selected, and the spacing between each dose group should be determined according to the toxicity of the test substance and the pre-test results, generally preferably from 0.65 to 0.85.
  • Route and volume to the test substance two routes, one of which must be the route of administration for which the test article recommends clinical studies. 5, 0. 5, 0. 5, 0. 5, 0. 5, 0. 5, 0. 5, 0. 5, 0. 5, 0. 5, 0. 5, 0. 5ml, 0. 5ml, 0. 5ml, 0. 5ml It is appropriate.
  • the above route to the test subject should be less than or equal to 3, 1, 3 and 1 ml.
  • mice were randomly divided into 4-5 groups according to body weight, and each group had at least 10 animals (half male and female). Immediately after the test, the animal reaction was observed, and the observation was continued for 7-14 days. Record the toxic reaction of animals and the distribution of dead animals. The dead animals should be autopsied in time to record the lesions. Pathological examination is required if there is a visible change in the naked eye.
  • - primary antibody is anti-human TNFa monoclonal antibody and blank control
  • the secondary antibodies are rabbit anti-human Ig-FITC and goat anti-mouse Ig-FITC, respectively. Results: There was no binding to normal heart, brain, liver, lung, kidney, eye, esophagus, pancreas, breast, testis, ovarian tissue.
  • Example 8 Anti-human TNFa monoclonal antibody competitive inhibition assay
  • the experiment was carried out by ELISA, and the amount of human TNF a was 0. lug/well.
  • the working concentration of anti-human T Fa-01 and anti-human TNFa-02 is constant at 500 ng/ml.
  • the competitive concentration gradient of TNF a from various sources is 2000-15. 6ng/ml construct
  • Human TNF a has a good competitive inhibition effect against human TNFa-01 and anti-human TNFa-02 binding to human TNF ⁇ .
  • Monkey TNF ⁇ has a weak competitive inhibitory effect against human TNFa-01 and anti-human TNFa-02 binding to human TNFa.
  • Mouse TNF ⁇ has weak competitive inhibition
  • Anti-human TNFa-01 and anti-human TNFa-02 have different ability to bind TNF a from different sources.
  • mice were randomly selected and divided into one cage, a total of 4 cages, of which 4 was the control.
  • Group, No. 123 is the experimental group.
  • TNF-a total solution Dissolve 1 mg of TNF- ⁇ powder in 1 ml of sterile PBS to prepare a solution of 1 mg ml.
  • b. Prepare different concentrations of TNF-a solution according to the following methods, and intraperitoneally inject mice, each 0.5 ml.
  • Experimental group 1 100 g/0.5 ml/mouse: 300 ⁇ l ⁇ - ⁇ +1200 ⁇ 1 sterile PBS
  • Control group 4 1.5 ml sterile PBS (0.5 ml/mouse, 3 in total)
  • mice The death of the mice was observed 6 hours after the injection.
  • TKFa lethal dose ranged from 5mg/kg to 1.5mg/Kg.
  • TNFa to explore a small range of lethal doses in mice 6ml 20mg/ml D-galactosamine sensitizer preparation:
  • the experiment was set as the control group and the experimental group. Three mice were randomly selected and divided into one cage, a total of 4 cages, of which 4 was the control group and 123 was the experimental group.
  • mice of 1234 were intraperitoneally injected with D-galactosamine sensitizer, 500 mg/kg, that is, 0.5 ml per mouse.
  • TKF-a total solution 1 mg of TNF-a powder was dissolved in 1 ml of sterile PBS to prepare a solution of 1 mg ml.
  • b. Prepare different concentrations of TNF-a solution according to the following methods, and intraperitoneally inject mice, each weighing 20 g ⁇ lg, 0.5 ml each.
  • Experimental group 1 8 ( ⁇ g/0.5ml/mouse: 240 ⁇ 1 ⁇ - ⁇ +1260 ⁇ 1 sterile PBS
  • Control group 4 1.5 ml sterile PBS (0.5 ml/mouse, 3 in total)
  • mice The death of the mice was observed 12 hours after the injection.
  • TNFa The lethal dose of TNFa is optimal: 4mg/Kg Experiment 3.
  • Anti-tumor necrosis factor antibody protects mice, reagents and consumables
  • mice were randomly selected and divided into one cage, a total of 10 cages, of which 10 was the control group.
  • Nos. 1 to 9 are experimental groups.
  • mice of the No. 10 mice were intraperitoneally injected with D-galactosamine sensitizer, 10 mg/mouse, that is, 0.5 ml per mouse.
  • TKF-a total solution 1 mg of TNF-a powder was dissolved in 1 ml of sterile PBS to prepare a solution of 1 mg ml.
  • the concentration of TNF- ⁇ is set to 80 ⁇ ⁇ /0.5 ⁇ 1/mice
  • the antibody was injected 2 hours later. Each antibody dose was divided into three groups according to high, medium and low doses, and humiria antibody was set as positive control group. Set PBS as a negative control group. 6 mice in each group, each mouse weighing 20 ⁇ lg.

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Description

全人 TNFa单克隆抗体及其制备方法与应用 技术领域
本发明涉及生物技术领域， 具体涉及 ΤΝΪ¾结合蛋白及其应用。 背景技术
自身免疫性疾病是继癌症和心血管疾病的第三大类疾病。 类风湿性关节炎 （RA) 是最 常见的自身免疫病之一， 我国类风湿关节炎患病率约为 0.32-0.36%， 在 60岁以上人群发病 率高达 30 %〜40 %。 面对每人每年几十万元的进口药物治疗费用， 患者往往只能长期忍受 痛苦的化学治疗， 却又收效甚微。
RA的患病人群多， 用药量大， 使其治疗性药物的市场巨大， 2005年全球市场达到 60 亿美元。 目前临床治疗 RA的方案很多， 首选用大剂量非甾体抗炎药可控制症状， 如无效， 则临床首选氨甲蝶呤等免疫抑制药治疗。 约四分之一的患者首次发病后经及时治疗可终生 不再复发， 约二分之一的患者经治疔后症状暂时得以缓解， 但仍会反复发作， 部分最终发 展为关节功能障碍； 约四分之一的患者对目前所有的常规治疗均无效。 肿瘤坏死因子 - a ( TNF- α ) 是类风湿性关节炎病变过程中起主要作用的细胞因子， 实 验和临床结果均证实 TNF是治疗该病的适合靶点。国外对 Μ的发病机理进行了充分的研究， 发现肿瘤坏死因子 （Tumor Necrosis Factor alpha, TNF- ) 在该疾病的病理进程中起十 分关键的作用， 患者或动物模型病变关节腔内的 TNF水平异常升高。 TNFa和 TNFp是哺乳动 物细胞中的一类同源分泌型蛋白质， 它们相互之间具有很高的同源性， 二者都可以对许多 细胞类型造成多方面的影响。 这两种蛋白都可以通过与细胞膜上的受体作用而影响细胞的 生物学功能。 肿瘤坏死因子 TNFo能够引起肿瘤组织出血坏死并具有多方面功能。 成熟分子 由 157个氨基酸组成， 含有两个二硫键， 没有糖基化位点。 其前体为 233个氨基酸组成的 多肽， N-端 76个氨基酸组成的多肽不是信号肽， 它可以把前体固定在细胞膜上， 使成熟的 分泌型保留在细胞膜上。前体的成熟 mRNA长度为 1. 7kb。在体内主要由单核巨噬细胞产生。 针对 TNF- a 的抑制剂成为迄今为止最为成功的 RA 治疗药物。 代表药物如 Wyeth/Amgen公司 Enbrel(etanercept) , Johnson & Johnson, Schering Plough/Centocor公司 emicade(infliximab)， Cambridge Antibody Technology/ Abbott的 Humira(adalimumab)。 Enbrel 为 Amgen公司开发的制剂， 是 TNF- α受体和 Fc的融合蛋白， 不仅对风湿性关节炎有非常 不错的疗效， 且对牛皮癣及牛皮癣型关节炎也有明显的治疗作用， 同时在促进脊椎炎愈合 的研究上， Enbrel也进入了二期临床。 2004年销售额是 25亿美元， 预计 2008年可达 34亿 美元。 Remicade是一种人鼠嵌合性的抗 TNF- α单克隆抗体，可特异性结合可溶性及膜结合 型 TNF- α , 其适应症包括 Crohn' 病， 风湿性关节炎 (RA)， 重症强直性脊柱炎 （AS)。 在 2003年和 2004年全球销售额分别是 22.7亿和 28.9亿美元，是生物医药领域的"重磅炸弹"。 2002年 12月 Abbott公司的 Humira成为第一个获准用于减轻成人中到重度活动期 RA的病 征和症状及抑制关节结构损伤进展的全人单克隆抗体。 由于是全人抗体， 避免了 Remicade 会产生免疫反应的缺点。 Humira与 Remicade及 Enbrel相比的另一个优势是给药方法的便 利上。 Humira每月只需注射两次， 而 Remicade和 Enbrel必须每周注射两次。 Humira市场 占有率正逐年提高， 2003年和 2004年的销售额分别是 2.8亿和 8.5亿美元。 2006年， 全球 三大抗 TNF- α拮抗剂 Humira与 Remicade及 Enbrel 3个药物己近 80亿美元， 2008年 Enbrel ( 64.9)、 Remicade ( 53.3 )、 Humira (45.2)、 Cimzia合计为 163亿美元。 这三个代表药物的疗效均比较确定， 并且随着大规模临床试验的不断开展， 除 RA 以 外的克隆氏病、 强直性脊柱炎、 银屑病等其他自身免疫病也相继成为新的适应症， 使其市 场更加庞大。 这些生物产品的特异性高， 靶向性好使其副作用较其他同类药物明显降低， 逐渐地与甲氨蝶呤合用作为 A 的一线用药， 并增强它们对目前中至重度的风湿性疾病等 其他自身免疫病市场的渗透。 目前处于开发末期阶段的新药主要有 UCB/Celltech 公司的 CDP-870 (PEG化人源化抗 TNF 抗体 Fab片段） 禾 B CDP571 (人源化抗 TNF单抗)， Bristol -Myers Squibb公司的 CTLA41g (Abatacept), Roche公司的 Rituxan和 MRA等。 其中进展 最迅速的是 CDP-870, 目前己经进入临床 III期， CDP-870于 2006年底进入市场， CDP-870 应用大肠杆菌进行生产， 使其成本降低至其他 TNF 抑制剂药物的四分之一。 虽然现有的 TNF抗体类药物被认为较其它任何药物的效果都好， 但它们也存在很大的 缺点。 第一， 它们均来自哺乳动物细胞表达和纯化， 由于表达水平较低和生产能力有限远 远不能满足市场的需求，即便是 Amgen斥资 5亿美金创建了全球最大的 20,000升细胞培养 生物反应器， 这一问题依然存在（据 2004年美国 biopharma报告）； 第二， 这些抗体的高昂 成本使很大一部分患者被拒之门外， 据估计， 常规应用 Enbrel、 Remicade和 Humira治疗 RA一年的费用分别是 11400、 14200和 16776美金，且需反复使用（据 2004年美国 biopharma 报告）； 第三， 这些抗体所携带的 Fc段具有结合补体和 ADCC等活性， 体内应用会导致表 达 T F 的细胞的凋亡， 会产生一系列副作用； 第四， Remicade是人鼠嵌合性单抗， 含有 1/3的鼠源蛋白成分，连续注射可以在 10%以上的病人中激发人抗鼠源蛋白抗体反应或人抗 嵌合蛋白抗体反应，影响治疗效果并可能导致严重的不良反应。即将上市的 CDP870是 PEG 化人源化抗 TNF 抗体 Fab段 [17]， 其鼠源蛋白成分下降到 10 %， 但仍然有免疫原性。 而 Humir的可变区的氨基酸序列和相应的 DNA序列来自于 CAT公司开发的人源抗体库， 其 Fc 片段的编码区来自人的 I gGl。 由此可见， 该产品的可变区序列并非直接来自人， 而是来自 起源于人 B细胞的抗体库。 此外， 虽然人类免疫系统能够产生种类极其庞大的抗体， 但是人或全人抗体技术却遇 到了瓶颈： 人体内产生特异性抗体的前体细胞丰度很低， 即使在抗体阳性的个体中， 能够 产生特异性抗体的前体细胞也远远低于人杂交瘤技术所需要的能够产生特异性抗体细胞数 量。 因此， 分离、 纯化和富集特异性分泌抗体的 B细胞成为建立全人抗体平台的关键技术 之一。
发明内容
本发明用全人抗体技术从类风湿关节炎病人体的 B细胞中获得抗人 TNFa单克隆抗体， 发酵、 纯化后， 采用聚乙二醇化 （PEG化） 方法体外改造和修饰， 在保持其抗 TNF活性的 同时， 使之更适合体内应用。 本发明第一方面公开了一种抗人 TNFa单克隆抗体，包括重链与轻链，其轻链具有 SEQ ID N0 : 3的氨基酸序列， 其重链具有 SEQ ID N0 : 4或 SEQ ID N0 : 6的氨基酸序列。 进一步的， 所述抗人 TNFa单克隆抗体经 PEG化修饰。 本发明第二方面公开了一种多核苷酸， 该多核苷酸含有编码前述抗人 TNFa单克隆抗体 的重链和 /或轻链的多核苷酸序列。 进一步的，编码前述抗人 TNFa单克隆抗体的轻链的多核苷酸的序列为 SEQ ID NO : 1 , 编 码前述抗人 TNFa单克隆抗体的重链的多核苷酸的序列为 SEQ ID N0 : 2或 SEQ ID N0 : 5 , 编 码抗人 TNFa单克隆全长抗体的重链和轻链的多核苷酸的序列同时含有 SEQ ID NO : 1与 SEQ ID NO : 2或者同时含有 SEQ ID NO : 1与 SEQ ID N0 : 5。 本发明第三方面公开了一种遗传工程化的宿主细胞， 它被含有编码前述抗人 TNFa单克 隆抗体的重链和轻链的多核苷酸的载体所转化或转导。 任何适用于对表达盒进行表达的细 胞都可以作为宿主细胞。 例如， 酵母、 昆虫、 植物等的细胞。 优选的， 所述宿主细胞为真 核细胞， 可釆用不会产生抗体的哺乳动物宿主细胞系， 具体可以是： 中国仓鼠的卵巢细胞 CH0;幼仓鼠的肾脏细胞 (BHK, ATCC CCL 10)；幼鼠的塞尔托利细胞（ Sertol i cells ) (TM4, Mather, Biol. Reprod. 23 : 243-251 (1980) )； 猴的肾脏细胞（COS细胞）； 通过 SV40 (COS- 7， ATCC CRL 165 1)转化的猴的肾脏 CVI细胞; 人的胚肾细胞 （HEK- 293， Graham et al. J. Gen Virol. 36 : 59 (1977) )；猴肾脏细胞（CVI ATCC CCL 70)；非洲绿猴的肾脏细胞 （VER0- 76, ATCC CRL- 1587)； 人的子宫颈癌细胞 （HELA, ATCC CCL 2)等。 其他细胞系在本学科领域的文献 中也比较常见。 从美国模式菌种收集中心 (ATCC)可以获得多种细胞系。 把核酸导入细胞的方法有许多种。 比较常用的方法包括电穿孔技术、 粒子枪技术、 磷 酸钙沉淀、 直接显微注射等等。 具体方法的选择一般取决于被转化的细胞类型， 以及进行 转化所处的具体环境。 在某些情况下， 可以采用多阴离子转染脂质体和钙离子调节的细胞 质基因转化技术。 本发明第四方面公开了抗人 TNFa单克隆抗体的制备方法， 包括下列步骤： 1 )在适合 表达抗人 TNFa单克隆抗体的条件下培养所述宿主细胞； 2 )分离纯化获得所述单克隆抗体。
在获得编码本发明的抗体的核酸序列后， 可按照以下方法制备生产目的抗体。 例如将 含有编码目标抗体的核酸的载体直接导入宿主细胞， 细胞在适当的条件下进行培养， 从而 诱导出被编码抗体的表达。 一旦获得本发明所说的单克隆抗体， 就可以釆用本领域技术人员所知的常规方法对抗 体分子进行纯化。 具体纯化方法包括色谱法， 比如， 离子交换， 亲和层析， 尤其与蛋白质 A 的特定抗原进行亲和层析， 以及尺寸柱层析、 离心过滤法、 差异溶解度等。 在很多情况下， 细胞分泌的抗体进入培养液， 并在培养液中收获。
本发明第四方面对抗人 TNFa单克隆抗体通过体外活性测定和动物实验研究了该抗体在 动物体内的治疗有效性， 进一步公开了上述抗人 TNFa单克隆抗体的制药用途。 抗人 TNFa 单克隆抗体可用于制备治疗与 TNFa相关的炎症的药物，如制备治疗类风湿性关节炎的药物。 将本发明的抗人 TNFa抗体用于治疗类风湿性关节炎等与 TNFa相关的炎症时的用法用量， 可参照现有的 TNFa抗体的常规用法用量。 本发明第五方面， 还提供了一种医药组合物， 其包含治疗有效量的本发明的单克隆抗 体以及医药学上可接受的载体。 可作为医药学上可接受的载体及其组分的物质包括糖类； 纤维素及其衍生物； 麦芽； 明胶； 滑石； 固体润滑剂； 多元醇； 海藻酸； 乳化剂； 润湿剂； 着色剂； 调味剂； 压片剂； 抗氧化剂； 防腐剂； 无热原水； 等渗盐溶液； 磷酸盐缓冲液等。 药物制剂应与给药方式相匹配。 本发明的药物组合物可以被制成针剂形式， 例如用生理盐 水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。 诸如片剂和胶囊之类的药物 组合物， 可通过常规方法进行制备。 药物组合物如针剂、 溶液、 片剂和胶囊宜在无菌条件 下制造。 活性成分的给药量是治疗有效量。 本发明有益效果：
本发明采用了全人抗体技术， 获得了抗人 TNFa全人抗体， 对该抗体的理化及生物活性 的初步分析表明，该抗体与 TNF有较好的亲和力，体外能有效中和 TNF对 L929细胞的杀伤。 一方面， 由于其全部氨基酸序列均与人抗体完全一致， 使其对人体的免疫原性降低到最低。 其次， 采用 PEG化表面改性技术， 以减小其被内皮系统吞噬的概率， 延长在血液循环系统 中的时间， 同时又实现在体内缓释的目的， 避免了小分子抗体半衰期短的缺点， 在保持其 抗 TNFa活性的同时， 使之更适合体内应用。 与现有的单抗药物相比， 可以大大降低用药频 率。 这不但进一步降低了病人的经济负担， 而且大大减少了病人注射的痛苦， 减少了劳动 力损失。 具体实施方式
以下列举具体实施例进一歩阐述本发明， 应理解， 实施例并非用于限制本发明的保护范围。 实施例 1抗人 TNFa抗体基因的获得
取活动性类风湿关节炎病人外周血 5毫升， 用淋巴细胞分离液分离白细胞， 进行培养， 根据 ELISA结果鉴定阳性克隆。
1. 选择血样
为了获得分泌抗人 ΤΝΪ¾的人 Β细胞， 首先用人 TNFa (购自深圳晶美公司 )常规包被 96 孔板，每孔用该蛋白 250ng，包被过夜。然后，用 5%脱脂奶粉室温封闭 2小时，奶粉用 pH7. 2 PBS配制。 洗涤后， 加入不同病人血清 100微升， 室温温育 1小时。 然后加入过氧化物酶标 记的羊抗人 IgG， 室温放置 1小时。 洗涤至少 5遍后， 加入含 TMD或其他显色剂， 室温或 37度处理 20分钟。 最后， 加入终止液。 加入的过氧化物显色底物加入 10分钟后， 用 50μ1 IN的硫酸终止反应， 读取 450nm光密度值。 选取阳性且 0D值高的血清作为获选血样。
2. 分离并富集分泌抗 TNF抗体的人 B-细胞
3. 在适当条件下对分泌抗 TNF抗体的人 B-细胞进行培养
4. 抗体一反应 ELISA筛选
用 ELISA检测抗 TNFa IgG的分泌。 对人 B细胞培养液上清的样品进行筛选。 其中， 选 择出具有最好的结合能力的两个阳性克隆， 且为 IgGl。
取阳性克隆细胞各 5000-10000个， 分别用 Trizol (GIBC0) 分离总 RNA， 用 MMLV反转 录酶 （Promega公司产品） 以获取 cDNA。 上述操作均按照厂家说明书进行。 用下述引物和 条件进行 PCR, 所用扩增酶为 ClonTech的 Pfu以确保扩增过程中减少可能的突变。
轻链上游引物： GAAATTGTGCTCACACAGTC (SEQ ID NO : 7)
轻链下游引物： CTAACACTCTCCCCTGTTGAAGC (SEQ ID NO : 8)
重链上游引物： GAAGTCCAGCTGGTCGAGAG (SEQ ID NO : 9)
重链下游引物： GTGAGTTTTGTCACMGATTTGGGCTC (SEQ ID NO : 10)
PCR条件：
a) 反应体系的组成-
~Έ ^ 数量 （μΐ )
ΈΓ 百 ——— ——
25 mM Mg2S04 5
Pfu DNA聚合酶（ 5单位 /微升） 1
上游引物 （2微克 /微升） 2
下游引物 （2微克 /微升） 2
cDNA 1. 5μ1
灭菌三蒸水 至 50微升 b) 反应条件：
预变性： 94度 2分钟
主循环： 94度 1分钟， 55 度 1分钟， 72度 3分钟。
循环数： 20
后延伸： 72度 5分钟 PCR过程在 PROGENE Genium热循环仪上进行。
c) 电泳鉴定与胶回收
按照上述条件进行 PCR。完毕后将产物在 1%琼脂糖凝胶电泳上进行鉴定， 两个 PCR的产 物分别为 650bp和 670bp左右， 与理论大小相符。 用 Promega公司的胶回收试剂盒回收该 片段， 操作按照厂家的说明书进行， 各得 DNA约 2微克。
克隆与測序：
上述产物常规克隆到 PUC57, 鉴定出正确克隆后进行测序。 克隆方法釆用 MBI公司试剂 盒， 操作按照说明书进行。 对阳性克隆进行测序， 结果显示， 该两个克隆分别为人 IgGl抗 体的重链和轻链的编码基因。 实施例 2抗人 TNFa抗体的制备 1. 重链和轻链编码序列的获得 为了进行全长抗体的基因表达， 根据实施例 1的测序结果， 分别合成了人 IgGl的全长重链 及轻链序列， 以便构成完整的抗人 TNFa抗体 -01基因。
以下片段为轻链编码区， 长度为： 645b 。 ( SEQ ID NO : 1 )
Figure imgf000008_0001
GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN GEC
以下片段为全长的重链编码区， 长度为： 1353bp。 ( SEQ ID NO: 2 ) GAAGTCCAGCTGGTCGAGAGCGGTGGCGGGCTGGTGCAACCCGGTGGATCACTGCGGCTCAGCTGCGCTGCTAGTGGC AGTAAGTCTATGAACTCCGCCACACACTATGCTGAAAGCGTGAAAGGGCGCTTCACAATCTCTAGAGACGATTCAAAG
Figure imgf000009_0001
CTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCG
Figure imgf000009_0002
GAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAG GTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTG
Figure imgf000009_0003
CAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCG GAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGAC AAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCCCTGCACAACCACTACACGCAGAAG AGCCTCTCCCTGTCTCCGGGTAAATGA
其编码的重链全长氨基酸序列为 （SEQ ID NO : 4):
Figure imgf000009_0004
LNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
还有一个抗体 TNFa抗体 -02， 轻链及重链可变区和 TNFa抗体 -01—致， 重链恒定区与 TNFa抗体 -02相比有三个氨基酸不同。
TNFa抗体 -02的全长重链编码区为： （SEQ ID N0 : 5 )
GAAGTCCAGCTGGTCGAGAGCGGTGGCGGGCTGGTGCAACCCGGTGGATCACTGCGGCTCAGCTGCGCTGCTAGTGGCT TTCCCTTCTCTAACCACTGGATGAATTGGGTCCGGCAGGCTCCAGGAAAGGGTCTGGAGTGGGTGGGTGAGATCAGGAG TAAGTC
TCTCTG
CTTACGACCACTGGGGCCAAGGTACACTGGTCACCGTCTCGAGTGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGC GTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCC
CACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTC GCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGC
Figure imgf000010_0001
ACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGC AGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCC GGGTAAATGA
其编码的重链全长氨基酸序列为 （SEQ ID N0 : 6 ):
Figure imgf000010_0002
LNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK
TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
3. 抗人 TNFa单克隆抗体全长真核表达载体的构建
将两个 TNF抗体的重链全长编码区（SEQ ID NO : 2 , SEQ ID NO : 5)， 轻链编码区（SEQ ID NO: 1)，polyA和 PCMV按照常规 Overlapping PCR方法分别进行拼接，拼接后的片段为 l ight chain+po 1 yA+PCMV+heavy chain„ 克隆到 Promega 公司的 pCI载体上构建的载体分别命名 为 pCI- TNF- 01和 pCI- TNF- 02。 4. CHO细胞的转染与筛选 本研究采用脂质体法 （LipoFemine 2000, Invitrogen) 对 CHO细胞进行转染， 转染试 剂盒购自 Ir itrogen公司， 转染时分别取上述纯化的分别带有重链和轻链的质粒 100微克 作为 DNA样品对 CH0细胞进行转染， 转染操作程序按照厂家的说明书进行。
转染后的 CH0细胞经连续 3个月的氨甲喋吟（MTX)筛选， 并逐步添加 MTX 2nM、 5nM、 20nM、 40nM、 80nM、 120nM、 160nM、 200nM、 250nM、 300nM进行筛选， 每两周增加一 次浓度， 每次 MTX的用量约为前一次的 2倍， 具体视细胞生长情况而定。 细胞培养按照常规 进行， 培养基为： 15%胎牛血清 （Gibco) +RPM1640/DEME。 于 37度 5%(¾培养箱中培养。 在 此过程中取细胞培养上清进行 ELISA检测， 经过三次 ELISA检测， 得到阳性的混合克隆并 冻存部分细胞。
然后按照常规极度稀释法进行单克隆化， 对混合克隆用 96孔板极限稀释法进行第一次 单克隆培养， 稀释细胞密度为 2.5个细胞 /ml， 每孔加入 200ul细胞悬液， 挑选得到 200个单 克隆， 经过 ELISA检测挑选出 89个阳性克隆。 取 ELISA结果中前 35个单克隆进行放大培 养， 并分别冻存部分细胞。 对单克隆进行第二次极限稀释法进行亚克隆培养， 细胞培养上清 经过两次的 ELISA鉴定得到 826个单克隆，取 ELISA检测的前 35个单克隆进行第三次亚克 隆， 细胞培养上清经过 ELISA鉴定得到 1026个单克隆， 取 ELISA结果中前 30个进行放大 培养， 并冻存部分细胞。 取单克隆细胞提取 RNA， 进行目的基因的检测， 确认所得到的单 克隆细胞的目的基因拷贝数， 验证为抗人 TNF抗体。 进行抗体蛋白 N端测序， 结果与设计 氨基酸一致。
5. 基因表达水平的研究 经 ELISA上述部分克隆的表达强度进行了研究， 数据显示， 通过 MTX筛选， 获得了明显 提高的抗体表达量。
根据表达量， 确定具有较好的生长特性和表达量的克隆作为继续研究的对象。
将上述的表达细胞株培养后用 Protein A-Sepharose进行亲和层析， 获得纯度达 95%以 上的抗体蛋白。 实施例 3抗人 TNFa单克隆抗体的 PEG化
将实施例 2获得的抗人 TNFa抗体进行结构改造引入游离巯基， 在第 229位氨基酸 C后 加入两个保护剂氨基酸 AA形成游离巯基， 并进一步 PEG化， PEG化和纯化方法参照 AP Champman等 [参考文献： Therapeutic antibody fragments with prolonged in vivo half l ife. Nature Biotech. V17 1999 August, Page 780-783 ]的方法进行， 获得 PEG化的抗 人 TNFa单克隆抗体。 ELISA鉴定后证实与 TNFa反应呈阳性。 制备的 PEG化抗体以 L929细 胞 （ECACC 85011425) 进行保护性试验 （参考实施例 5)， 发现 PEG化抗体具有很好的保护 作用。 实施例 4 抗人 TNFa单克隆抗体生物学活性检测
1. ELISA测试方法：
1 ) 将抗体用 PBS稀释到同一个浓度 lmg/mL
2) 按照倍比稀释法将 lmg/mL的倍比稀释
3 ) 设立阴性对照， 空白对照以及阳性对照 （humiria)
4) 数据处理 0D450-稀释倍数
结论： 在以 0. lug/孔包被的酶标板中， 与一系列浓度梯度的抗体共温育， ELISA检测 OD值， 结果如下： ELISA检测到的 humira最低浓度为 0.25ug/mL; 抗人 TNFa抗体 -01和 抗人 TNFa抗体 -02检测的最低浓度为 0.125ug/mL， 是对照 humira的 1/2倍。 结果显示实施 例 3所获得的抗体活性明显高于对照。
3.用 Biacore T100分析所获得的抗人 TNFa单克隆抗体与抗原结合的亲和力
( 1 ) 芯片偶联
a、 用 pH5的 Acetate buffer稀释样品 （抗原 TNPa) 溶液， 使样品浓度分别为 2 g/ml。 b、 按照 Biacore X100 control software的 kinetics/affinity assay 禾呈序中 immobilize方法将 TNFa偶联至芯片上。 设置偶联水平为 3000RU。
根据其自动运行的结果报告偶联水平， 自动报告偶联结果。 最终偶联为 3847.5RU.
( 2) 抗体检测方法以及步骤
a、 将抗体分别用 HBS-EP缓冲液稀释抗体， 使抗体浓度分别为 0、 1， 2， 4, 8， 16， 32, 64， 400nmol/L
b、 进样： 结合时间 120s， 解离 900s.再生缓冲液为 50nmNaOH。 按照 Biacore X100 control software的 kinetics/affinity禾呈序运行 kinetics/affinity assay□
c、 数据分析
按照 Biacore X100 Evaluation软件分析结果。选择曲线比较平稳的浓度进行亲和力数据分 析， 至少选择 5个不同浓度的曲线。 分析目标是使 U值尽可能接近 1.如果 U值大于 15此结 果则不可信。
Figure imgf000013_0001
结果显示实施例 3所获得的抗体结合常数比较、 解离常数比较和亲和力比较明显高于 对照。 实施例 5 抗人 TNFa单克隆抗体效应功能 -L929细胞保护试验 测定方法说明：
1. 96孔板， 每孔 20000细胞， 培养过夜；
2.抗体用梯度稀释后过滤除菌， 每孔加入 50微升， 并辅以 Actinomycin D至终浓 度 lug/ml ;
3.加入 hTNFa至终浓度 80pg/ml；
4. 5%C02培养箱中 37度培养过夜；
5.用 Cel lTiter Aqueous One Solution Reagent显色后， 492讓测定光密度；
6.光密度对抗体浓度作图。
Figure imgf000013_0002
结果表明： 抗人 TNFa-01的 IC50大约是 humira的 1/5， 中和活性相当于对照 humira的 5倍， 抗人 TNFa-02的 IC50大约是 humira的 1/10， 中和活性相当于对照 humira的 10倍。 实施例 6 抗人 TNFa单克隆抗体一次给予动物后所产生的急性毒性反应和死亡情况 分别取抗人 TNFa-01和抗人 TNFa-02以 100倍临床剂量（50mg/ml)注射， 未发现明显 副作用。
--注射部位反应 (ISRs) : 瘙痒、 红肿等均轻微。
- -非注射部位反应： 萎靡不振等， 均轻微。
试验材料：
动物： 17-22g的健康小鼠（同次试验体重相差不超过 4g)， 大鼠体重一般 120_150g (同 次试验体重相差不超过 10g)
受试物： 液体或粉末剂 （不溶于水的可用 0. 5%羧甲基纤素钠， 10%阿拉伯胶制成的混悬 剂）。
试验方法：
1.剂量选择： 一般选用 4-5个剂量组，各剂量组间间距应根据受试物毒性大小和预试结 果而定， 一般以 0. 65- 0. 85为宜。
2.给受试物途径及体积：二种途径，其中一种必须是该受试物推荐临床研究的给药途径。 小鼠口服灌胃（po)、静脉注射（iv)、腹腔注射（ip )和皮下注射（sc)的体积，应以 0. 5-0. 8、 0. 5、 0. 5和 0. 5ml为宜。 大鼠上述途径给受试物体积应小于或等于 3、 1、 3和 1ml为好。
3.实验分组和观察： 试验时， 将动物按体重随机分成 4-5组， 每组至少 10只动物 （雌 雄各半）， 给受试物后即刻观察动物反应情况， 继续观察 7-14天， 记录动物毒性反应情况 和死亡动物分布， 死亡动物应及时进行尸检， 记录病变情况。 若有肉眼可见变化时则需进 行病理检查。
结果处理：
用 Bliss统计法计算 LD50值大于 900mg/kg。 实施例 7 抗人 TNFa单克隆抗体交叉性反应性实验
分别取抗人 T Fa-01和抗人 T Fa-02用人组织或细胞进行交叉反应性或非靶组织结合 的实验室检测， 西安陕西超英科技有限公司提供的正常多器官组织芯片 (NC00-01-003)。
*用间接免疫荧光法检测抗体在正常多器官组织芯片中的表达.
-一抗分别为抗人 TNFa单克隆抗体和空白对照，
-二抗为分别为兔抗人 Ig-FITC和山羊抗小鼠 Ig-FITC。 结果： 与正常的心、 脑、 肝、 肺、 肾、 眼、 食道、 胰腺、 乳腺、 睾丸、 卵巢组织没有结合。 实施例 8 抗人 TNFa单克隆抗体竞争抑制实验
方法概要：
1.实验采用 ELISA方法进行，包被人 TNF a量为 0. lug/孔。
2. 抗人 T Fa-01和抗人 TNFa-02的工作浓度恒定为 500ng/ml。
3.各种来源的 TNF a的竞争浓度梯度为 2000-15. 6ng/ml„
结果-
1.人 TNF a对抗人 TNFa-01和抗人 TNFa-02结合人 TNF α具有很好的竟争抑制 作用。
2.猴 TNF α对抗人 TNFa-01和抗人 TNFa-02结合人 TNF a具有弱的竟争抑制作 用。 小鼠 TNF α具有较弱的竟争抑制作用
3.大鼠 TNF α未体现出竟争抑制作用。
4. 抗人 TNFa-01和抗人 TNFa-02对不同来源的 TNF a的结合能力是人〉猴〉小鼠
>大鼠。
5.提示将来的药理 /毒理模型以猴为好。 实施例 9 抗人 TNFa单克隆抗体保护人 TNFa攻击小鼠试验 实验一、 T P对小鼠致死剂量大范围的探索
1、 试剂与耗材
D-半乳糖胺 批号： 079K1502 EC 217-198- 1
EDGE lml灭菌注射器 批号： YZB/国 0265-2007 有效期： 三年
Balb/c 小鼠 12只
2、 实验步骤
(1) 7ml 20mg/ml D-半乳糖胺致敏剂的制备：
准确称取 D-半乳糖胺粉末 140mg， 打开超净工作台， 紫外照射 30min， 在超净工作台中 量取 7ml 灭菌后 PBS， 将 140mg粉末溶于 PBS中， 用 0.22μηι过滤器精密过滤。
(2)、 实验准备
a、 实验设为对照组与实验组， 随机抓取 3只小鼠分为一笼， 共 4笼， 其中④号为对照 组， ①②③号为实验组。
b、 分别对①②③④号的小鼠腹腔注射 D-半乳糖胺致敏剂， 10mg/只， 即每只小鼠注射 0.5ml o
(3) 24h后腹腔注射 TNF-a
a、 TNF-a总溶液： 将 lmg的 TNF-α粉末用 1ml无菌 PBS溶解， 配制成 lmg ml的溶液。 b、 按照下列方法配制不同浓度的 TNF-a溶液， 并腹腔注射小鼠， 每只 0.5ml。
实验组①： 100 g/0.5ml/只小鼠： 300μ1 ΤΝΓ-α+1200μ1无菌 PBS
实验组②： 3C^g/0.5ml/只小鼠： 90μ1 ΤΜ?-α+1410μ1无菌 PBS
实验组③： lC^g/0.5ml/只小鼠： 30μ1 ΤΜ?-α+1470μ1无菌 PBS
对照组④： 1.5ml 无菌 PBS ( 0.5ml/只 小鼠， 共 3只）
注射 6小时后观察小鼠死亡情况。
、 实验结果
实验组①： 死亡 3只，
实验组②： 2只萎靡不振， 1只正常
实验组③： 3只正常
对照组无死亡
、 结论：
TKFa致死剂量范围在 5mg/kg〜1.5mg/Kg 实验二、 TNFa对小鼠致死剂量小范围的探索 、 6ml 20mg/ml D-半乳糖胺致敏剂的制备：
准确称取 D-半乳糖胺粉末 120mg， 打开超净工作台， 紫外照射 30min， 在超净工作台中 量取 6ml 灭菌后 PBS， 将 120mg粉末溶于 PBS中， 用 0.22μιη过滤器精密过滤。
、 实验准备：
(1)、 实验设为对照组与实验组， 随机抓取 3只小鼠分为一笼， 共 4笼， 其中④号为对照 组， ①②③号为实验组。
(2) . 分别对①②③④号的小鼠腹腔注射 D-半乳糖胺致敏剂， 500mg/kg只， 即每只小鼠 注射 0.5ml。
、 24h后腹腔注射 TNF-a:
a、 TKF-a总溶液： 将 lmg的 TNF-a粉末用 1ml无菌 PBS溶解， 配制成 lmg ml的溶液。 b、按照下列方法配制不同浓度的 TNF-a溶液， 并腹腔注射小鼠，每只小鼠体重 20g±lg， 每只 0.5ml。
实验组①： 8(^g/0.5ml/只小鼠： 240μ1 ΤΝΡ-α+1260μ1无菌 PBS
实验组②： 60 g/0.5ml/只小鼠： 180μ1 Τ Ρ-α+1320μ1无菌 PBS
实验组③： 4C^g/0.5ml/只小鼠： 120μ1 Τ Ρ-α+1380μ1无菌 PBS
对照组④： 1.5ml无菌 PBS (0.5ml/只 小鼠， 共 3只）
注射 12小时后观察小鼠死亡情况。
、 实验结果
实验组①： 48h后 3只全死亡
实验组②： 72h后 3只全死亡
实验组③： 无死亡
对照组④： 无死亡
、 结论：
TNFa的致死剂量最佳为： 4mg/Kg 实验三、 抗肿瘤坏死因子抗体对小鼠保护作用 、 试剂与耗材
D-半乳糖胺 批号： 079K1502 EC 217-198-1
EDGE lml灭菌注射器 批号： YZB/国 0265-2007 有效期： 三年
Balb/c 小鼠 60只
、 实验步骤
(1) 27ml 20mg/ml D-半乳糖胺致敏剂的制备：
准确称取 D-半乳糖胺粉末 540mg， 打开超净工作台， 紫外照射 30min， 在超净工作台中 量取 27ml 灭菌后 PBS, 将 540mg 粉末溶于无菌 PBS中， 用 0.22μπι过滤器精密过滤。
(2)、 实验准备：
实验设为对照组与实验组， 随机抓取 6只小鼠分为一笼， 共 10笼， 其中⑩号为对照组，
①〜⑨号为实验组。
(3)、分别对① ⑩号的小鼠腹腔注射 D-半乳糖胺致敏剂， lOmg/只，即每只小鼠注射 0.5ml。
(4)、 24h后腹腔注射 TNF-a
a、 TKF-a总溶液： 将 lmg的 TNF-a粉末用 lml无菌 PBS溶解， 配制成 lmg ml的溶液。 b、 根据肿瘤坏死因子致死剂量的探索后所得结果， 设定 TNF-α浓度为 80μ§/0.5ιη1/只小 鼠
(5)、 注射抗体
2小时后注射抗体。每种抗体剂量按照高中低剂量分为三组，设置 humiria抗体为阳性对 照组。 设置 PBS为阴性对照组。 每组小鼠 6只， 每只小鼠体重 20±lg.
、 结论
初步实验结论得出抗人 TNFa-01和抗人 TNFa-02保护剂量小于 11 ugZ小鼠 /lugTNFa。

Claims

权 利 要 求 书
1. 一种抗人 TNFa单克隆抗体， 包括重链与轻链， 其轻链具有 SEQ ID NO : 3的氨基酸序列， 其重链具有 SEQ ID N0: 4或 SEQ ID NO : 6的氨基酸序列。
2. 如权利要求 1所述抗人 TNFa单克隆抗体， 其特征在于， 所述抗人 TNFa单克隆抗体经 PEG化修饰。
3. 一种多核苷酸， 该多核苷酸含有编码权利要求 1所述抗人 TNFa单克隆抗体的重链和 / 或轻链的多核苷酸序列。
4. 如权利要求 3所述多核苷酸， 其特征在于， 所述编码抗人 TNFa单克隆抗体的轻链的多 核苷酸序列为 SEQ ID NO : 1， 编码抗人 TNFa单克隆抗体的重链的多核苷酸序列为 SEQ ID N0 : 2或 SEQ ID NO: 5, 编码抗人 TNFa单克隆抗体的重链和轻链的多核苷酸序列同时含 有 SEQ ID NO : 1与 SEQ ID NO : 2或者同时含有 SEQ ID NO : 1与 SEQ ID N0 : 5。
5. 一种遗传工程化的宿主细胞， 它被含有编码权利要求 1所述抗人 TNFa单克隆抗体的重 链和轻链的多核苷酸的载体所转化或转导。
6. 如权利要求 5所述遗传工程化的宿主细胞， 其特征在于， 所述编码抗人 TNFa单克隆抗 体的重链和轻链的多核苷酸的序列同时含有 SEQ ID N0 : 1与 SEQ ID NO : 2或者同时含有 SEQ ID N0 : 1与 SEQ ID NO : 5。
7. 如权利要求 5或 6所述遗传工程化的宿主细胞，其特征在于，所述宿主细胞为真核细胞。
8. 如权利要求 7所遗传工程化的宿主细胞， 其特征在于， 所述真核细胞为 CH0细胞。
9. 一种抗人 TNFa单克隆抗体的制备方法， 包括下列步骤： 1 ) 在适合表达抗人 TNFa单克 隆抗体的条件下培养权利要求 5-8任一权利要求所述宿主细胞； 2 ) 分离纯化获得所述 单克隆抗体。
10. 如权利要求 1或 2所述抗人 TNFa单克隆抗体的用途，为用于制备治疗与 TNFa相关的炎 症的药物。
11. 如权利要求 10所述抗人 TNFa单克隆抗体的用途， 其特征在于， 所述治疗与 TNFa相关 的炎症的药物为治疗类风湿性关节炎的药物。
12. 一种医药组合物， 其包含权利要求 1或 2所述抗人 TNFa单克隆抗体以及医药学上可接 受的载体。
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Publication number Priority date Publication date Assignee Title
WO2004063335A2 (en) * 2003-01-08 2004-07-29 Applied Molecular Evolution TNF-α BINDING MOLECULES
CN1613874A (zh) * 2003-11-06 2005-05-11 上海中信国健药业有限公司 全人源肿瘤坏死因子抗体，其制备方法以及药物组合物
CN101684156A (zh) * 2008-09-27 2010-03-31 苏州工业园区晨健抗体组药物开发有限公司 人TNFα单克隆抗体、其PEG化纳米颗粒及其应用

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004063335A2 (en) * 2003-01-08 2004-07-29 Applied Molecular Evolution TNF-α BINDING MOLECULES
CN1613874A (zh) * 2003-11-06 2005-05-11 上海中信国健药业有限公司 全人源肿瘤坏死因子抗体，其制备方法以及药物组合物
CN101684156A (zh) * 2008-09-27 2010-03-31 苏州工业园区晨健抗体组药物开发有限公司 人TNFα单克隆抗体、其PEG化纳米颗粒及其应用

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