WO2012009977A1 - Anticorps tnfα-fab entièrement humain et anticorps pégylé obtenu à partir dudit anticorps - Google Patents

Anticorps tnfα-fab entièrement humain et anticorps pégylé obtenu à partir dudit anticorps Download PDF

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WO2012009977A1
WO2012009977A1 PCT/CN2011/071717 CN2011071717W WO2012009977A1 WO 2012009977 A1 WO2012009977 A1 WO 2012009977A1 CN 2011071717 W CN2011071717 W CN 2011071717W WO 2012009977 A1 WO2012009977 A1 WO 2012009977A1
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antibody
fab
human
fab antibody
seq
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PCT/CN2011/071717
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倪健
朱向玲
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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
    • C07K2317/50—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
    • C07K2317/90—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 a Fab fragment of a TNFa antibody and a PEGylated antibody thereof. Background technique
  • autoimmune diseases The incidence of autoimmune diseases is high. About 355 million people worldwide suffer from various autoimmune diseases. In 1999, the World Health Organization listed rheumatism and cancer and cardiovascular diseases as the three major threats to human health. The incidence of rheumatism is high and the disability rate is high. In China, more than 10 million people are disabled due to rheumatism. Among them, rheumatoid arthritis (RA) and ankylosing spondylitis (AS) are the most harmful, and the disability rates of untreated RA and AS are as high as 70% and 30%, respectively.
  • RA rheumatoid arthritis
  • AS ankylosing spondylitis
  • the current method of combating autoimmune diseases is not to use steroids to slow down the inflammation caused by the immune system attacking tissues, but to suppress the immune system by using immuno-suppressant drugs. .
  • both of these methods can cause serious side effects, and they can only slow down the progression of the disease and not cure the disease.
  • anti-TNF antibody in biological agents, there is no specific drug treatment for rheumatism.
  • Non-steroidal anti-inflammatory analgesics and immunosuppressive agents that improve symptoms can only improve the symptoms of patients, improve the quality of life, and not completely alleviate the condition.
  • the use of such drugs alone exceeds 20 billion in China each year. Therefore, rheumatism seriously affects the patient's physical and mental health, and brings a heavy economic burden to the patient, which also affects the development of society.
  • TNF- ⁇ TNF- ⁇
  • IL-12 TNF- ⁇
  • antagonists of TNF-a include Enbrel (etanercept) from Amgeri, Remicade (infl iximab) from Centocor, and Humira (adal imumab) from Abbott.
  • Enbre is a fusion protein of human TNF- ⁇ receptor and Fc.
  • Remicade is a human-mouse chimeric anti-human TNF- ⁇ monoclonal antibody that specifically binds to soluble and membrane-bound TNF- ⁇ .
  • the intact monoclonal antibody has a large molecular weight, and the Fc segment complements the cytotoxic effector molecule function and prolongs the half-life in serum.
  • the complete monoclonal antibody has limited its application: 1. Large dosage and complicated preparation process. At present, only a few biological enterprises in the world have mastered the technology of large-scale expression of monoclonal antibodies; The Fc fragment has a large molecular weight and is not easy to penetrate cells; 3. It is expensive; 4. Sometimes Fc-induced cytokine inactivation, receptor blocking or virus neutralization are undesirable. In recent years, domestic and foreign studies have shown that under certain conditions, Fab fragments can replace intact monoclonal antibodies. Fab fragments have the following advantages: 1.
  • PEG polyethylene glycol
  • BSA bovine serum albumin
  • PEGylation has become a mature "gold standard” technology in modern medicine, and is widely used in the modification of proteins (peptides), enzymes, antibodies and small molecule drugs.
  • PEG can encapsulate protein drug molecules such as interferon to form a gel. Because of its large molecular weight, it is not easily excreted by the kidneys, so it can greatly prolong the action time of protein drugs in the body.
  • the PEG modification of protein drugs has been effective. In 1991, the first PEG-modified protein drug PEG-ADA was approved by the FDA.
  • Protein peptides have the following advantages after modification with polyethylene glycol: 1. Increased solubility and stability; 2. Immunogenicity and antigen Reduced sexual activity, reduced toxic side effects; 3, increased plasma half-life and increased systemic exposure; 4, increased biological activity and efficacy in vivo.
  • the object of the present invention is to disclose a Fab antibody of a human TNF ⁇ antibody and a gene encoding the same.
  • Another object of the present invention is to disclose a PEGylated product of a Fab antibody of the above human TNF ⁇ antibody.
  • a third object of the present invention is to disclose the pharmaceutical use of the Fab antibody of the above human TNF ⁇ antibody.
  • the invention discloses a Fab antibody of a human TNF ⁇ antibody, wherein the amino acid sequence of the light chain variable region is SEQ ID NO: 7, and the amino acid sequence of the heavy chain variable region is SEQ ID NO: 8. Further, the Fab antibody of the whole human TNF a antibody has the amino acid sequence of the light chain of SEQ ID NO: 12 and the amino acid sequence of the heavy chain of SEQ ID NO: 13.
  • the Fab antibody of the above human TNF ⁇ antibody is modified by PEGylation.
  • a polynucleotide encoding a Fab antibody heavy chain variable region, a light chain variable region, a heavy chain Fd segment or a full length light chain of the above human TNF a antibody is disclosed.
  • the nucleotide sequence of the polynucleotide of the Fab antibody light chain variable region encoding the human TNF a antibody is SEQ ID NO: 5, the multinuclear of the heavy chain variable region of the Fab antibody encoding the human TNF ⁇ antibody
  • the nucleotide sequence of the nucleotide sequence of SEQ ID NO: 6 the full-length light chain of the Fab antibody encoding the human TNF a antibody has the nucleotide sequence of SEQ ID NO: 11
  • the nucleotide sequence of the polynucleotide of the heavy chain Fd segment of the Fab antibody of the TNF ⁇ antibody is SEQ ID NO: 14.
  • the above polynucleotides can be used to construct engineered bacteria, expressing a fully human TNF ⁇ antibody or a Fab antibody thereof.
  • a genetically engineered host cell which is a host cell which is transformed or transduced with a vector comprising the aforementioned polynucleotide.
  • the host cell is a eukaryotic cell or a prokaryotic cell, which is a Chinese hamster egg cell and Escherichia coli, respectively.
  • the above host cells can be used to express a fully human TNF ⁇ antibody or a Fab antibody thereof.
  • the pharmaceutical use of the Fab antibody of the above human TNF a antibody and its PEGylated antibody is disclosed.
  • the Fab antibody of the human TNF ⁇ antibody can be used for the preparation of a medicament for treating inflammation associated with TNF ⁇ , such as a medicament for the treatment of rheumatoid arthritis.
  • the Fab antibody of the whole human TNF a antibody of the present invention or a PEGylated antibody thereof may be formulated by mixing with a conventional pharmaceutical carrier.
  • the conventional usage amount of the conventional TNF a antibody can be referred to.
  • the whole human TNF a antibody has good affinity with TNF, and can effectively neutralize the killing of L929 cells by TNF in vitro;
  • the Fab antibody of the invention can be expressed in large scale in E. coli, and the production cost is greatly reduced, and There are no side effects caused by effects such as complement binding and ADCC.
  • PEGylated surface modification technology is used to reduce the probability of engulfment by the endothelial system, prolong the time in the blood circulation system, and achieve sustained release in vivo. Objective, to avoid the shortcomings of short-term half-life of small molecule antibodies, while maintaining its anti-TNF a activity, making it more suitable for in vivo applications. detailed description
  • the invention adopts the following experimental ideas: Positive clones were obtained from peripheral blood leukocytes from patients with active rheumatoid arthritis.
  • RNA of the positive clone was isolated and reverse transcribed to obtain cDNA.
  • PCR amplification, cloning and sequencing yielded the coding sequences for the heavy and light chain variable regions of the anti-human TNFa antibody.
  • the full-length heavy chain coding region and the light chain coding region were constructed, cloned into the expression vector pci-neo (product of Invitrogen), and the vector was transfected into CH0 cells to express the human TNFa-Fab antibody. Increase the expression of antibodies by MTX screening.
  • the heavy chain coding region and the light chain coding region were cloned into the PC0M3H vector according to the conventional method, transformed into E. coli cells, and induced to express TNFa-Fab antibody, and the expression of the antibody was increased by adding glucose, sucrose and other optimization measures.
  • TNFa-Fab antibody was purified and PEGylated.
  • the invention is further illustrated by the following examples.
  • Example 1 Acquisition of B cells 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 conventionally 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. Then, peroxidase-labeled goat anti-human IgG was 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 ° for 20 minutes. Finally, a stop solution is added.
  • 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.
  • the secretion of anti-TNFa IgG was detected by ELISA.
  • a sample of the human B cell culture supernatant transformed by the EBV immortalization method was screened. Among them, a positive clone having the best binding ability was selected and was IgG1.
  • Example 2 Acquisition of the variable region of the whole human TNFa-Fab antibody gene 5000-10000 positive clone cells, total RNA was isolated using Trizol (GIBC0), using Superscript III First-strand reverse transcriptase (product of invitrogen) to obtain cDNA. 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 amplified enzyme used was KYO-plus of TOYOBO to ensure that possible mutations were reduced during the amplification.
  • Light chain upstream primer GCTAGCGCCGCCACCATGGACATGCGGG (SEQ ID N0: 1)
  • Heavy chain upstream primer CCGGAATTCGCCGCCACCATGGAGTT (SEQ ID NO: 3)
  • Heavy chain downstream primer ACTCGAGACGGTGACCAGTGTA (SEQ ID NO: 4)
  • downstream primer (2 ⁇ g / 1 ⁇ L each, total 25 ⁇ L)
  • Main cycle 94 degrees 20 seconds, 55 degrees 20 seconds, 72 degrees 1 minute.
  • the PCR process was performed on an Appl ied Biosystems thermal cycler.
  • PCR was carried out in accordance with the above conditions. After completion, the products were identified on 1% agarose gel electrophoresis. The products of the two PCRs were about 650 bp and 690 bp, respectively, which were consistent with the theoretical size. 5 ⁇ The DNA was collected in accordance with the manufacturer's instructions, the DNA was about 1. 5 micrograms.
  • the above product was routinely cloned into the PMD19-T vector according to the T-cloning method, and the correct clone was identified and sequenced.
  • the cloning method was carried out using the Tkara kit, and the operation was carried out according to the instructions.
  • the positive clones were sequenced and the results showed that the two clones were the variable region coding genes of the light and heavy chains of the human TNF-Fab antibody, respectively.
  • the sequencing results are as follows:
  • amino acid sequences of the encoded polypeptides are:
  • Example 3 Preparation of a fully human TNFa-Fab antibody 1. Acquisition of human IgG1 light chain constant region and heavy chain Fd fragment coding sequence
  • the TTAG human IgG1 heavy chain CHI gene coding sequence is as follows:
  • TNFa-Fab sequence was as follows (2923 bp): light chain Full length (SEQ ID NO: 11)
  • SEQ ID NO: 11 In the case where the whole human TNFa-Fab antibody gene heavy chain and light chain variable region are disclosed in Example 2, those skilled in the art can also obtain the above-mentioned heavy weight by means of total synthesis. Polynucleotide fragments of the variable regions of the strands and light chains, which in turn perform the above operations
  • Example 4 Transfection and Screening of CHO Cells CH0 cells were transfected by lipofectamine 2000 (Invitrogen).
  • the transfection kit was purchased from Invitrogen, and the purified heavy chain was taken during transfection. 100 ⁇ g of the plasmid of the light chain and the light chain were transfected into CH0 cells as a DNA sample, and the transfection procedure was carried out according to the manufacturer's instructions.
  • Transfected CH0 cells were screened for 3 consecutive months of methotrexate (MTX) and gradually screened with MTX 2nM, 5nM, 20nM, 40nM, 80nM, 120nM, 160nM, 200nM, 250nM, 300nM for every two weeks.
  • MTX methotrexate
  • the amount of MTX is about twice that of the previous one, depending on the growth of the cells.
  • 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. In the 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. 200 monoclonal clones were obtained, and 89 positive clones were selected by ELISA. The first 35 monoclonal clones in the ELISA results were amplified and frozen, and some cells were frozen. The monoclonal 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 clones detected by ELISA were subjected to a third subcloning, cell culture. 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 target gene copy number of the obtained monoclonal cells was confirmed, and it was confirmed to be an anti-human TNFa-Fab antibody. The N-terminal sequencing of the antibody protein was performed, and the results were consistent with the designed amino acids.
  • Example 5 Cloning and expression of whole human TNFa-Fab
  • the full-length and heavy-chain Fd fragments obtained in Example 3 were cloned into the PC0M3H vector according to a conventional method.
  • some bases were subjected to synonymous mutation without changing the amino acid sequence, and the amino acid sequence expressed after the mutation was SEQ ID NO: 13).
  • the 354th base of the heavy chain was mutated from the original C to A, and the 650th base was mutated from G to A.
  • the competent cell ToplOF' from New England Biolabs or Takara
  • the product was on the ampicillin plate. Initial screening and identification were carried out. 20 plaques were inoculated on liquid LB medium containing ampicillin for amplification.
  • the anti-human TNFa-Fab antibody obtained in Example 5 was introduced into the free sulfhydryl group at position 229, and the free amino acid C was added with two protective amino acids AA for PEGylation.
  • the PEGa-Fab PEGylation and purification method was referred to AP Champman et al. The method of therapeutic antibody fragments with prolonged in vivo half life. Nature Biotech. V17 1999 August, Page 780-783] was carried out to obtain a PEGylated anti-human TNFa-Fab antibody.
  • the purified PEGylated antibody was confirmed to be an anti-TNFa-Fab antibody by immunological ELISA.
  • Example 7 Detection of biological activity of TNFa-Fab antibody and PEGylated antibody
  • ELISA test method To determine the biological activity of TNFa-Fab by indirect ELISA, firstly dilute the detection TNF antigen with a coating buffer to a suitable concentration, and add a 100 ⁇ l package per well in a 96-well plate. Covered with liquid, 4 ° C refrigerator overnight. After the coating was completed, the washing liquid was continuously washed 5 times. 5%BSA ⁇ Accurately weighed 0. 5g BSA, dissolved in 100ml PBS, and mixed, which is 0. 5% BSA. 300 ⁇ l of blocking solution was added to each well and blocked again at 4 ° C overnight. The 96-well plate was taken out and washed five times on the plate washer.
  • TNFa was coupled to the chip according to the immobi l ize method in Biacore X100 control software ⁇ kinetics/affinity assay. 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 is 3847. 5RU.
  • the TNFa-Fab antibody and the TNFa-Fab antibody were diluted with HBS-EP buffer, respectively, so that the antibody concentrations were 0, 1, 2, 4, 8, 16, 32, 64, 400 nmol/L, respectively.
  • the binding time is 120s, the dissociation is 900s.
  • the regeneration buffer is 50nmNa0H.
  • the kinetics/aff inity assay was run according to the kinetics/affinity of Biacore X100 control software.
  • TNF- ⁇ has a killing effect on L929 cells, while TNFa-Fab specifically binds to TNF- ⁇ , thus protecting cells from killing. Therefore, TNF- ⁇ can be antagonized by TNFa-Fab against target cell L929 cell line. Killing to detect the biological activity of TNFa-Fab. L929 cells were used as target cells for TNF- ⁇ killing, and MTS/PMS staining was used to detect the protective effect of TNFa-Fab on killing.
  • the succinate dehydrogenase of living cell mitochondria can reduce MTS to a blue-black alpha compound, and this compound can be dissolved in tissue culture medium.
  • the formazan compound can directly measure its absorbance value 0D 49 at a wavelength of 490 nm. , indirectly reflects the number of living cells.
  • the absorbance value is 0D 49 .
  • the size of the TNFa-Fab is indirectly reflected by the introduction of a certain concentration gradient.
  • the TNFa-Fab standard is calibrated to calculate the biological activity of the assay sample.
  • the experimental procedure was as follows: L929 cells were cultured to logarithmic growth phase, cells were washed with PBS, trypsinized cell layers were added, cells were blown to disperse the cells, and cell counts were performed. The cell suspension was added to a 96-well plate, and the test sample was added when the cell density reached 90%. In order to determine the optimal cell plate density, the cells were seeded in a 96-well cell plate at 0. 25, 0.5, 1.0, 1. 5, 2. 0, 3. 0, 4. 0, 5. 0, 6 0, 7. OX 105cel ls/ml, 100 ⁇ /well, repeat 2 wells per concentration, terminate culture 24 hours after seeding, stain with MTS/PMS, and measure 0D at 490 nm.
  • the TNFa-Fab antibody and the PEGylated TNFa-Fab antibody were intravenously injected into rabbits, and blood samples were taken at 0, 0.5, 1, 2, 4, 8, 12, 24, 48 11 respectively, and the blood samples were centrifuged. After 30 min, the serum was separated, _2 (TC was stored for testing.
  • the collected serum was determined by ELISA: the serum to be tested was diluted with pbs to 1: 125, 1: 250, 1: 500, 1: 1 000, 1: 2 000 1, 4 000 was added to a 96-well culture plate, and 0.1 ml was added to the reaction well of each polystyrene plate.
  • the TNFa antigen was coated with a coating buffer overnight at 4 ° C (while making a blank well, Negative control wells and positive control wells. On the next day, discard the solution in the well and wash it 3 times with washing buffer for 3 min each time. Add blocking solution 0.1 ml to the coated wells above, set 37° C. Incubate for 1 h, then wash. Add the diluted test serum to each well, incubate for 1 h at 37 ° C. Then wash. Add the enzyme standard secondary antibody to each well, 0.1 ml. C incubated for 1 h, washed. Add the temporarily prepared opd substrate solution to each reaction well 0. 1 ml, 37 ° C protected from light 10 ⁇ 3 0 min, 2 mol / 1 sulfuric acid 0. 05 ml was added to terminate the reaction, and the absorbance at 495 nm was measured on a microplate reader.
  • Example 10 Acute toxicity and mortality of PEGylated anti-human TNFa-Fab monoclonal antibody after primary administration to animals
  • the PEGylated TNFa-Fab antibody was injected at a 100-fold clinical dose (50 mg/ml) and no significant side effects were observed.
  • ISRs Injection site reactions
  • a non-injection site reaction wilting, etc., all mild.
  • 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 (water-soluble suspension of 0.5% sodium carboxymethylcellulose, 10% gum arabic). experiment method:
  • Dose selection 4-5 dose groups are generally selected, the spacing between each dose group should be determined according to the toxicity of the test substance and the pre-test results, generally 0. 65-0. 85 is appropriate.
  • 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. 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 at least 10 animals in each group (male and female) Each half), observe the animal reaction immediately after the test object, continue to observe 7-14 days, record the animal toxicity 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.
  • Reagents, materials Human TNF a, Balb/c mice 8 test methods: 8 alb/c mice were randomly divided into 4 groups (2/group), and TNF a lOOug/mouse was administered via tail vein/abdominal cavity. 30ug/mouse, 10ug/mouse, Oug/mouse, observe the condition of the mouse within one week: death, hair luster, note, etc., and record the time of death.
  • Test method Balb/c mice were randomly divided into 3 groups. According to the dose of all deaths of the last experimental mice, 2 dose groups were designed 100/50, 30/15, 10/5ug/mouse, transvenous/peritoneal cavity. A specific dose of human TNF ⁇ was administered, and the condition of the mice was observed within one week: death, hair gloss, note, etc., and the time of death was recorded.
  • PEGylated anti-human TNFa-Fab monoclonal antibody protects mouse lethal dose human TNF alpha challenge
  • 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.
  • TNF- ⁇ total solution Dissolve 1 mg of TNF- ⁇ powder in 1 ml of sterile PBS to prepare a solution of lmg/ml.
  • concentration of TNF- ⁇ is set to 80 ⁇ g / 0. 5ml / mouse

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Abstract

La présente invention concerne un anticorps TNFα-Fab entièrement humain et l'anticorps pégylé obtenu à partir dudit anticorps. La séquence d'acides aminés de la région variable de la chaîne légère de l'anticorps TNFα-Fab entièrement humain selon la présente invention est SEQ ID NO: 7, et celle de la région variable de la chaîne lourde est SEQ ID NO: 8. La présente invention concerne en outre le gène codant ledit anticorps TNFα-Fab entièrement humain, ainsi que des cellules hôtes dans lesquelles le gène codant est exprimé. L'anticorps TNFα-Fab entièrement humain selon la présente invention peut être employé dans le traitement des inflammations liées à TNFα, comme la polyarthrite rhumatoïde.
PCT/CN2011/071717 2010-07-22 2011-03-11 Anticorps tnfα-fab entièrement humain et anticorps pégylé obtenu à partir dudit anticorps WO2012009977A1 (fr)

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CN201010234261.1A CN102336834B (zh) 2010-07-22 2010-07-22 全人TNFα-Fab抗体及其PEG化抗体

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