WO2010105446A1 - 抗人肿瘤坏死因子受体α单抗及其应用 - Google Patents
抗人肿瘤坏死因子受体α单抗及其应用 Download PDFInfo
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Classifications
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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
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/52—Constant or Fc region; Isotype
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/55—Fab or Fab'
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
Definitions
- the present invention relates to the field of biotechnology, and in particular to anti-human TNFa monoclonal antibodies, alterations using molecular evolution techniques, and clinical applications of engineered structures.
- TNFa is an important inflammatory factor. Tumor necrosis factor TNFa can cause hemorrhage and necrosis of tumor tissue and has many functions.
- the precursor is a polypeptide consisting of 233 amino acids.
- the N-terminal 76 amino acid polypeptide is not a signal peptide. It can immobilize the precursor on the cell membrane.
- the mature secretory form is retained on the cell membrane.
- the mature mRNA of the precursor is 1. 7 kb in length. It is mainly produced by mononuclear macrophages in the body.
- the mature molecule consists of 157 amino acids with two disulfide bonds and no glycosylation sites. It has a different effect on different cells: killing cells, inhibiting, promoting or not affecting cell growth.
- this factor can inhibit hemorrhagic necrosis of tumors by affecting the blood vessels in the tumor area, and enhance the anti-tumor ability of the body by enhancing the immune function.
- the factor has important physiological effects on liver, bone, muscle, blood vessels and other systems.
- TNFa is associated with diseases such as rheumatoid arthritis. Excessive TNFa expression in rheumatoid arthritis patients is one of the main factors causing swelling and pain in the affected area, and TNFa is the most closely related to the disease in the TNF family. Protein.
- TNFa is indeed an ideal target for the treatment of a variety of autoimmune diseases.
- Rheumatoid arthritis referred to as Fengfengguan
- Fengfengguan Rheumatoid arthritis
- the main clinical manifestations are joint swelling, etc. About 20% of serious patients will suffer from joint bone damage, or even complete loss of labor ability, which will not only bring great inconvenience to personal life, but also bring huge burden to family and society.
- Psoriasis is another high-risk autoimmune disease.
- the new skin cells of the psoriasis patients grow too fast and the old skin cells do not fall off, causing inflammation, swelling, and scaling on the skin.
- Psoriasis varies in severity and can be very small or small, or it can be a large area of skin ulceration, mainly in the scalp, knees, elbows and torso. There is no difference in age, gender, ethnicity, etc. Its serious ulceration often makes the public think it is an infectious disease. Patients not only have troubles in daily treatment and care, but also often have serious psychological obstacles due to the rejection of the surrounding people, causing great obstacles to life and body and mind. If not treated or treated improperly, psoriasis can also cause or be associated with a wind, causing inconvenience or even complete loss of labor capacity.
- Autoimmune diseases with the same pathogenesis include tonic-type vertebral inflammation. This type of patient's spinal joints is not only limited by personal actions, but also loses the ability to work, or even completely loses the ability to take care of themselves, and it also causes severe physical pain.
- Therapeutic monoclonal antibodies have developed tremendously over the past decade, and several products have been successfully developed in anti-TNFa-associated autoimmune diseases, including TNFa-targeted Enbrel, Humira Remicade, and CD20.
- Target Rituxat These therapeutic monoclonal antibodies have achieved great clinical success because of their outstanding efficacy, and they have quickly become first-line treatments.
- Therapeutic monoclonal antibodies have also achieved unprecedented success in the fields of malignant tumors and infectious diseases. Due to their outstanding efficacy and the huge market demand created by them, these drugs are still being developed in high intensity.
- the methods for obtaining monoclonal antibodies are mainly hybridoma technology, mainly involving rodents (such as mice, rabbits), birds (such as chickens), primates (such as macaques, baboons, etc.) and human species.
- rodents such as mice, rabbits
- birds such as chickens
- primates such as macaques, baboons, etc.
- monoclonal antibodies have also been developed using mouse humanized antibodies.
- Another important requirement for being a candidate drug is whether it is easy to manufacture. This requires the production of antibodies to be produced on a large scale and at low cost. The most important indicator of this is the amount of antibody that can be produced per liter of culture medium, the so-called expression level.
- TNFa is one of the main factors causing swelling and pain in the affected area of rheumatoid arthritis, and a large number of studies have shown that it also causes septic shock syndrome, ankylosing spondylitis, psoriasis and rheumatoid arthritis.
- the main medium Elevated levels of TNFa in the serum of patients with septic shock syndrome and rheumatoid arthritis indicate an increase in mortality or disability.
- Passive input of TNFa antagonists can prevent shock and tissue damage caused by LPS and bacteria.
- Clinical application of TNFa antibodies or their receptors has obvious effects on septic shock syndrome, ankylosing spondylitis, psoriasis and rheumatoid arthritis.
- Humira is a monoclonal antibody drug developed by Abbott. The product was approved for marketing in 2002. The first indication is rheumatoid arthritis (RA). The target is TNFa. Due to its outstanding efficacy, global sales reached 20 in 2006. One hundred million U.S. dollars.
- the amino acid sequence of the variable region of the product and the corresponding DNA sequence are derived from the human antibody library developed by CAT. The coding region of its Fc fragment is derived from human IgGl. Thus, the variable region sequence of the product is not directly from humans, but from an antibody library derived from human B cells.
- the invention is based on the lymphocytes isolated from the peripheral blood of patients with rheumatoid arthritis, and adopts the antibody library technology as the core, and obtains a fully human antibody capable of neutralizing human TNFa through a series of panning processes.
- Fab form The use of molecular evolution technology to improve or change its affinity and specificity has met the requirements of important molecular markers such as affinity and specificity of therapeutic monoclonal antibodies, and successfully expressed biologically active full-length forms in CH0 cells. Therefore, the amino acid sequence of the anti-human TNFa antibody of the present invention and the corresponding DNA sequence are directly derived from humans. Moreover, compared with Humira, the anti-human TNFa antibody of the present invention has a significantly higher affinity than Humira, and its clinically effective dose can be significantly reduced. Summary of the invention
- the object of the present invention is to disclose an anti-human TNFa antibody and a gene encoding the same.
- Another object of the invention is to disclose the pharmaceutical use of the above anti-human TNFa antibodies.
- One aspect of the invention discloses an anti-human TNFa antibody, the light chain amino acid sequence comprising SEQ ID NO: 8, the heavy chain amino acid sequence comprising SEQ ID NO: 9 or SEQ ID NO: 10, preferably, the anti-human TNFa
- the antibody full length heavy chain amino acid is a polypeptide fragment of SEQ ID NO: 16 or SEQ ID NO: 18.
- the above SEQ ID NO: 8 is a light chain variable region amino acid sequence.
- Another aspect of the invention discloses a polynucleotide encoding the light chain of the above anti-human TNFa antibody, and preferably, the sequence of the polynucleotide comprises SEQ ID NO: 5.
- a third aspect of the invention discloses a polynucleotide encoding the full-length heavy chain of the above anti-human TNFa antibody, preferably, the sequence of the polynucleotide comprises SEQ ID NO: 15 or SEQ ID NO: 17 .
- the Fab form of the above anti-human TNFa antibody is disclosed.
- the above anti-human TNFa antibody or Fab antibody thereof for use in the preparation of a medicament for treating TNFa-related inflammation such as a medicament for treating rheumatoid arthritis
- a medicament for treating rheumatoid arthritis is disclosed.
- the anti-human TNFa antibody of the present invention or a Fab antibody thereof is used for the treatment of TNFa-related inflammation such as rheumatoid arthritis, it can be referred to the conventional usage amount of the existing TNFa antibody.
- TNF antibody drugs are considered to perform better than any other drug, they also have major drawbacks. First, they are all expressed and purified by mammalian cells. Due to their low expression levels and limited production capacity, they are far from meeting the market demand. Even Amgen has invested 500 million US dollars to create the world's largest 20,000 liter cell culture. Bioreactors, this problem still exists (according to the 2004 US biopharma report); second, the high cost of these antibodies has prevented a large proportion of patients from being excluded.
- the invention adopts the human hybridoma technology, and obtains the anti-human TNFa whole human antibody.
- the preliminary analysis of the physical and chemical activity and biological activity of the antibody indicates that the antibody has good affinity with TNF, and can effectively neutralize TNF to L929 in vitro. Cell killing.
- all of its amino acid sequences are identical to human antibodies, its immunogenicity to the human body is minimized.
- the expression of Fab small molecule antibodies by the E. coli expression system is greatly reduced, and there is no side effect caused by effects such as complement binding and ADCC.
- Figure 1 P 60Mb3H plasmid structure map.
- Figure 2 Expression vector pGPl plasmid map.
- Figure 3 Results of 6 Fab clone affinity tests for 7B4, 2H7, 4D3, 6F5, 3C9, 3H6, etc.
- the invention adopts the following experimental ideas:
- Peripheral blood was drawn from patients with active rheumatoid arthritis and leukocytes were separated using lymphocyte separation fluid.
- the isolated B cells were cultured, and positive clones were identified based on the ELISA results.
- the total RNA of the positive clone was isolated and reverse transcribed to obtain cDNA.
- Dohmen et al Journal of Immunological Methods 298 (2005) 9-20, Production of recombinant Ig molecules from antigen-selected single B cells and restricted usage of Ig-gene segments by anti-D antibodies), PCR amplification, cloning and sequencing to obtain anti-human TNFa antibodies
- the coding sequence for the heavy and light chain variable regions From this, the corresponding amino acid sequence can be inferred.
- the full-length heavy chain coding region and the light chain coding region were constructed and cloned into the expression vector pcDNA3.1 (+) (Invitrogen), and the vector was co-transfected into CH0 cells to express anti-human TNFa antibody. Increase the amount of antibody expression by MTX screening.
- Example 1 Obtainment of leukocytes secreting anti-human TNFa antibody 5 ml of peripheral blood of patients with active rheumatoid arthritis was taken, and leukocytes were separated by lymphocyte separation solution, cultured, and positive clones were identified based on ELISA results.
- 96-well plates were routinely coated with human TNFa (purchased from Shanghai Xinbino Bioengineering Co., Ltd.), and 250 ng of the protein was used per well, and coated overnight. Then, it was sealed 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, add TMD or other developer, and treat at room temperature or 37 degrees for 20 minutes. Finally, a stop solution is added. After the addition of the peroxide-developing substrate for 10 minutes, the reaction was terminated with 50 ⁇ l of IN sulfuric acid, and the 450 nm optical density value was read. Serum with positive and high 0D values was selected as the selected blood sample.
- red blood cell lysis is found to be incomplete, repeat steps 2 and 3 above. Usually very small amounts of red blood cells do not affect subsequent tests.
- wash 1-2 times Add appropriate amount of PBS, HBSS, normal saline or serum-free medium, resuspend the pellet, centrifuge at 400-500g for 2-3 minutes, discard the supernatant. It can be repeated one more time and washed a total of 1-2 times.
- the amount of the washing solution should generally be at least 5 times the volume of the cell pellet, and the precipitate obtained after centrifugation at 4 ° C is white blood cells.
- Trizol was added in a ratio of 1 ml of 100 mg or 10 7 leukocyte pellet, and immediately subjected to total RNA extraction purification or storage at _80 ° C for use.
- ⁇ LV reverse transcriptase (Invitrogen product, Cat. No. 28025-013) was used to obtain cDNA first. chain. 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 the amplification process.
- CTMCACTCTCCCCTGTTGAAGC (SEQ ID NO: 2)
- Heavy chain upstream primer GAGGTGCAGCTGGTGGAGTC (SEQ ID NO: 3)
- downstream primer (2 ⁇ g / 1 ⁇ L each, total 25 ⁇ L)
- Main cycle 94 degrees 1 minute, 55 degrees 1 minute, 72 degrees 3 minutes.
- the PCR process was performed on a Thermocycler thermal cycler.
- 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 20 micrograms of DNA.
- the carrier is a P C0Mb3H.
- the vector is a derivative derived from pCOMM3, and is suitable for expressing an antibody fragment, and the full sequence thereof is shown in GenBank, Accession No. AF268280, and Fig. 1 is a plasmid structure diagram made accordingly.
- Nissim, A Hoogenboom, H. R., Tomlinson, I. M., Flynn, G., Midgley, C., Lane, D. & Winter, G.
- the panning method was used to screen the Fab with high affinity from the above antibody library.
- the specific steps are as follows:
- Resuscitated antibody library strain 1 ml was added to fresh LB medium 14 ml, and cultured at 37 °C for 16 hours in a 50 ml flask.
- Its titer should be above 2X10 11 .
- the purified recombinant human TNFa protein (purchased from Shanghai Xinbino Bioengineering Co., Ltd.) was used as an antigen, and a 25 ml cell culture flask was coated in a conventional manner.
- the cells obtained above were diluted to 100,000 cells/ml and cultured on a 1.5% agar plate containing 0.1% ampicillin to obtain a monoclonal antibody.
- the clones on the above plates were cultured on a 96-well deep well plate, one clone per well, and a total of 960 clones were made.
- Humira Fab was used as a positive control, and 876 positive clones were screened by 4 rounds of panning, and 6 high-affinity Fab clones with affinity >10- w were obtained: 7B4, 2H7 4D3, 6F5, 3C9, 3H6, wherein the affinity of 7B4, 2H7 reaches the order of 10-12 , reaching or exceeding the level of Humira Fab. These 6 clones will be used to further identify the neutralizing ability.
- the neutralizing ability of the Fab antibody purified from the above clone to human TNFa was examined by the following method.
- the signal peptide coding sequence of the human IgG1 heavy chain is: (5' ⁇ 3') (SEQ ID NO: 11) DNA sequence of the Fc fragment of human IgG1 heavy chain (5 ' ⁇ 3') (SEQ ID NO: 12)
- the full-length DNA molecule of the light chain can be constructed.
- the encoded amino acid sequence is:
- the Nhel and EcoRI cut-points and protective bases are added to the 5'-end and the 3'-end to form the full-length DNA molecule of the light chain (SEQ ID NO: 19).
- CH0 cells were transfected by the liposome method (LipoFamine 2000, Invitrogen), and the transfection kit was purchased from Invitrogene, and 100 ⁇ g of the above-mentioned purified plasmids carrying heavy and light chains were respectively taken as transfection.
- the DNA samples were transfected into CH0 cells, and the transfection procedure was performed according to the manufacturer's instructions.
- the transfected CH0 cells were screened by G418 for 3 consecutive weeks, and the concentration was from 0. 05 ⁇ to 10 ⁇ , and the concentration was increased every two weeks, and the amount was 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. Incubate in a 37 degree 5% CO 2 incubator. Monoclonalization is then carried out according to the conventional extreme dilution method. The expression of the antibody was detected by ELISA, and the clone with higher expression was selected for preparation of the recombinant antibody expression product.
- Example 5 The ability of the full-length anti-human TNFa antibody to neutralize human TNFa The neutralization ability of the above purified full-length antibody against human TNFa was identified by the method of Example 3, and the positive control was Humira. The test results are shown in Figure 4.
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PCT/CN2009/070938 WO2010105446A1 (zh) | 2009-03-20 | 2009-03-20 | 抗人肿瘤坏死因子受体α单抗及其应用 |
EP09841729.8A EP2409992B1 (en) | 2009-03-20 | 2009-03-20 | A human anti-tumor necrosis factor alpha monoclonal antibody and use thereof |
CN2009800005464A CN101896502B (zh) | 2009-03-20 | 2009-03-20 | 抗人TNFα单抗、分子进化及其应用 |
US13/008,015 US8354108B2 (en) | 2009-03-20 | 2011-01-18 | Fully human antibody to human TNFα, molecular evolution and use thereof |
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EP2623515A1 (en) * | 2010-09-30 | 2013-08-07 | Chengdu Kanghong Biotechnologies Co., Ltd. | ANTI TNFalpha HUMANIZED ANTIBODY AND FRAGMENT ANTIGEN BINDING (FAB) AND USE THEREOF |
CN103773770A (zh) * | 2012-10-18 | 2014-05-07 | 高峰 | 一种可分泌的绿色荧光蛋白及其在重组蛋白高产表达细胞株筛选方面的应用 |
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WO2020018732A1 (en) * | 2018-07-19 | 2020-01-23 | Ab Studio Inc. | Novel systems for screening internalizing antibody and uses thereof |
CN114591432B (zh) * | 2022-03-25 | 2023-10-31 | 南京融捷康生物科技有限公司 | 抗TNFα的单域抗体及其用途 |
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- 2009-03-20 WO PCT/CN2009/070938 patent/WO2010105446A1/zh active Application Filing
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Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
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EP2623515A1 (en) * | 2010-09-30 | 2013-08-07 | Chengdu Kanghong Biotechnologies Co., Ltd. | ANTI TNFalpha HUMANIZED ANTIBODY AND FRAGMENT ANTIGEN BINDING (FAB) AND USE THEREOF |
JP2014503177A (ja) * | 2010-09-30 | 2014-02-13 | ツェンドゥー カンホン バイオテクノロジーズ カンパニー リミテッド | ヒト化抗ヒト腫瘍壊死因子α(TNF−α)抗体およびその抗原結合性フラグメント(Fab)およびその使用方法 |
EP2623515A4 (en) * | 2010-09-30 | 2014-03-26 | Chengdu Kanghong Biotechnologies Co Ltd | HUMANIZED ANTI-TNF ANTIBODIES AND FRAGMENT ANTIGEN BINDING AND THEIR USE |
US9096669B2 (en) | 2010-09-30 | 2015-08-04 | Chengdu Kanghong Biotechnologies Co., Ltd. | Humanized anti-TNF-α antibody and antigen-binding fragment (Fab) thereof and use of the same |
CN103773770A (zh) * | 2012-10-18 | 2014-05-07 | 高峰 | 一种可分泌的绿色荧光蛋白及其在重组蛋白高产表达细胞株筛选方面的应用 |
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US8354108B2 (en) | 2013-01-15 |
EP2409992B1 (en) | 2017-10-25 |
EP2409992A1 (en) | 2012-01-25 |
CN101896502A (zh) | 2010-11-24 |
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