WO2009143689A1 - 一种可溶性tnf受体突变体 - Google Patents
一种可溶性tnf受体突变体 Download PDFInfo
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- WO2009143689A1 WO2009143689A1 PCT/CN2009/000037 CN2009000037W WO2009143689A1 WO 2009143689 A1 WO2009143689 A1 WO 2009143689A1 CN 2009000037 W CN2009000037 W CN 2009000037W WO 2009143689 A1 WO2009143689 A1 WO 2009143689A1
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- tnfrp75
- amino acid
- mutant
- tnf
- fusion protein
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- 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
- C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
- C07K14/7151—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for tumor necrosis factor [TNF], for lymphotoxin [LT]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/06—Antipsoriatics
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- 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
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- 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]
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- 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
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- 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
- A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- the invention belongs to the field of biomedical technology, and particularly relates to a tumor necrosis factor (TNF) receptor derivative and its application in medicine.
- TNF tumor necrosis factor
- Tumor necrosis factor a is a member of the tumor necrosis factor superfamily and has biological activities such as regulation of immune response, apoptosis and cell differentiation. TNF a has two intracellular receptors, TNF receptor 1 (TNFRp55) and TNF receptor 2 (TNFRp75). Overexpression of TNF a is an important factor in causing autoimmune diseases.
- TNF receptor 1 TNF receptor 1
- TNFRp75 TNF receptor 2
- Overexpression of TNF a is an important factor in causing autoimmune diseases.
- TNFRp55 TNF receptor 1
- TNFRp75 TNF receptor 2
- Receptor TNFRp75 Fc fusion protein (Etanercept). Both drugs showed significant therapeutic effects.
- Etanercept can simultaneously bind to TNF a and lymphotoxin (LT), but the clinical application dose is larger, about 25-50mg, subcutaneous injection is prone to erythema. Therefore, the development of the TNFRp75:Fc fusion protein, which is a high-affinity binding to TNF and lymphotoxin, has been extremely valuable. Summary of the invention
- One of the objects of the present invention is to provide a soluble receptor which is highly neutralized to TNFa and/or lymphotoxin, to reduce the dose of soluble receptor required for neutralizing TNFa, and to improve the therapeutic effect against autoimmune diseases. At the same time, reduce the cost of drug production.
- Another object of the present invention is to provide a fusion protein formed by a soluble receptor and other amino acid fragments having a stronger neutralizing effect on TNF a and/or lymphotoxin.
- Another object of the present invention is to provide a DNA sequence encoding the above soluble receptor or fusion protein.
- a further object of the present invention is to provide a pharmaceutical use of soluble receptors and fusion proteins thereof which have a stronger neutralizing effect on TNF a and/or lymphotoxin.
- the concept of the present invention is such that the structure of TNF receptor 2 and TNF a and LT is deeply studied by molecular simulation technology, and it is found that the 92nd amino acid of the natural sequence of TNF receptor 2 is very conjugated to TNF a and LT. Important, by rationalizing the design, point mutation of the 92nd amino acid to obtain high-intensity neutralization
- Soluble TNF receptor 2 mutants that act on TNF a and lymphotoxin Soluble TNF receptor 2 mutants that act on TNF a and lymphotoxin. .
- a soluble TNFRp75 mutant is disclosed in the wild type sequence (SEQ ID NO. l)
- the amino acid substitution at position 92 allows the cytotoxicity of the mutant to neutralize TNF a and lymphotoxin by more than 30% compared to the wild type.
- the glutamic acid (E) at position 92 is replaced by one of Asn, His, Ser, Ala, Lys or Gin.
- the 92nd amino acid E in the amino acid sequence described in SEQ ID NO. 1 is replaced by N, H, S, A, K or Q, respectively.
- the amino acid sequence of the TNFRp75 (E92H) mutant is as described in SEQ ID N0.2, wherein the 92nd position is histidine (His) and the N-terminal 1-22 amino acids are signal peptides.
- the amino acid sequence of the TNFRp75 (E92A) mutant is as described in SEQ ID N0.3, wherein the 92nd position is alanine (Ala) and the N-terminal 1-22 amino acids are signal peptides.
- the amino acid sequence of the TNFRp75 (E92N) mutant is as described in SEQ ID N0.4, wherein the 92nd position is asparagine (Asn) and the N-terminal 1-22 amino acids are signal peptides.
- the amino acid sequence of the TNFRp75 (E92S) mutant is as described in SEQ ID N0.5, wherein the 92nd position is serine (ser) and the N-terminal 1-22 amino acids are signal peptides.
- the amino acid tryptophan (Trp) at position 89 is also replaced by Tyr, Phe, His, Lys, Met or Leu, except that the glutamic acid (E) at position 92 is replaced, such as:
- the amino acid sequence of the TNFRp75 (E92N, W89Y) mutant is as described in SEQ ID N0.6, wherein amino acid 89 is tyrosine (Tyr), position 92 is asparagine (Asn), and N-terminal is 1-22 amino acids.
- amino acid 89 is tyrosine (Tyr)
- position 92 is asparagine (Asn)
- N-terminal is 1-22 amino acids.
- the TNFRp75 (E92S, W89Y) mutant amino acid sequence is as described in SEQ ID N0.7, wherein the 89th amino acid is tyrosine (Tyr), the 92nd is serine (Ser), and the N-terminal 1-22 amino acids are signals. Peptide.
- the TNF p75 (E92N, W89F) mutant amino acid sequence is as described in SEQ ID N0.8, wherein the 89th amino acid is phenylalanine (Phe), the 92nd position is asparagine (Asn), N-terminal 1-22
- the amino acids are signal peptides.
- a fusion protein of a soluble TNFRp75 mutant and other amino acid fragments is disclosed.
- the purpose of other amino acid fragments is to increase the stability of the mutant of TNFRp75 and prolong its biological half life.
- the other amino acid fragment is selected from the constant region (Fc) of human immunoglobulin (IgG) or one of the five functional regions of albumin.
- the additional amino acid fragment is 232 amino acids of the constant region (Fc) of human immunoglobulin (IgG).
- the soluble TNFRp75 mutant forms a fusion protein with 232 amino acid fragments of the C-terminal Fc fragment of human IgG, with or without other linker fragments, preferably without a linker fragment.
- TNFRp75 Fc mutant sequence amino acid sequence as set forth in SEQ ID NO.
- TNFRp75 Fc mutant amino acid sequence as set forth in SEQ ID NO.
- TNFRp75 (E92N): Fc mutant amino acid sequence as described in SEQ ID NO.
- TNF p75 (E92S): Fc mutant amino acid sequence as described in SEQ ID N0.12;
- TNFRp75 (E92N, W89Y): Fc mutant amino acid sequence as described in SEQ ID N0.13;
- TNFRp75 (E92S, W89Y): The Fc mutant amino acid sequence is as described in SEQ ID NO.
- TNF p75 (E92N, W89F): The Fc mutant amino acid sequence is as described in SEQ ID NO: 5.
- a DNA sequence encoding the above soluble receptor or fusion protein is disclosed.
- the DNA sequence encoding the soluble receptor and its fusion protein of the present invention can be changed according to the degeneracy of the codon and the codon preferences of different host cells, as long as the amino acid sequence translated by the DNA sequence does not change.
- the scope of the invention This is well known to those skilled in the art.
- the use of the above soluble TNFRp75 mutant and fusion protein thereof in the field of medicine is disclosed for the treatment of diseases related to overexpression of TNFa and/or lymphotoxin, including but not limited to: rheumatoid arthritis , psoriasis, scleroderma, Sjogren's syndrome, ankylosing spondylitis, lupus erythematosus, dermatomyositis, systemic lupus erythematoid-like syndrome, etc.
- a pharmaceutical composition comprising the above soluble TNFRp75 mutant and fusion protein thereof is disclosed.
- soluble TNFRp75 mutant of the present invention and its fusion protein have a certain degree of binding ability in combination with TNF and lymphotoxin.
- soluble TNFRp75 E92N: The ability of Fc to neutralize TNFa is 1.33 times that of wild-type soluble TNFRp75: Fc (ENBREL of AMGEN); while the ability to neutralize lymphotoxin is wild-type soluble TNFRp75: Fc (ENBREL of AMGEN) 2.77 times.
- the soluble TNFRp75 mutant of the present invention and the fusion protein thereof are used for treating TNFa and lymphotoxin-related diseases, and the activity can be reduced to reduce the dose of the drug, thereby reducing the incidence of erythema injection; and the dissociation time of TNFa is combined. Growth is conducive to prolonging the effective action of the drug.
- Figure 1-6 TNFRp75 mutant: Neutralization effect of Fc fusion protein on TNFa, LT 2 ⁇ 171 cell killing, wild type TNFRp75 : Fc fusion protein was used as a control in each group.
- FIG 1A TNFRp75 (E92 ⁇ ): Fc fusion protein and effect of TNFa;
- FIG. IB TNFRp75 (E92A): Fc fusion protein and the role of the LT 28- 171;
- TNFRp75 (E92H): Neutralization of TNFa by Fc fusion protein
- TNF p75 (E92H): Neutralization of Fc 28 . m by Fc fusion protein;
- FIG. 3A TNFRp75 (E92N): Neutralization of TNFa by Fc fusion protein
- Figure 3B TNFRp75 (E92N): Neutralization of LT 28-171 by Fc fusion protein;
- Figure 4A TNFRp75 (E92N, W89Y): Neutralization of TNFa by Fc fusion protein
- Figure 4B TNFRp75 (E92N, W89Y): Neutralization of LT 28-171 by Fc fusion protein
- Figure 5A TNFRp75 (E92S, W89Y): Neutralization of TNFa by Fc fusion protein
- Figure 5B TNFRp75 (E92S, W89Y): Neutralization of Fc 28 . m by Fc fusion protein;
- Figure 6A TNFRp75 (E92N, W89F): Neutralization of TNFa by Fc fusion protein;
- FIG 6B TNFRp75 (E92N, W89F) : Fc fusion protein and the role of the LT 28 171. detailed description
- TNF receptor 2 As used herein, the terms "TNF receptor 2", “TNFRp75”, “TNF 75 receptor” are used interchangeably and include human-derived TNF p75 receptors as well as homologs of murine, porcine, equine or bovine. Preferably, it is the human TNF p75 receptor.
- the native wild type human TNF p75 receptor amino acid sequence is set forth in SEQ ID NO.
- a soluble TNF p75 receptor refers to the extracellular portion of the TNF p75 receptor, a ligand-binding domain consisting of amino acids 1-257 of the N-terminal amino acid sequence of the wild-type human TNF p75 receptor, N-terminal 1
- the amino acid at position -22 is a signal peptide.
- soluble TNF p75 receptor or "soluble TNF p75 receptor mutant of the invention” refers to a TNF p75 receptor mutant which has improved binding ability to TNF, and more preferably 2 More than double; the ability to combine lymphotoxin is improved, and the ratio is improved by more than 10 times.
- Such a mutant can be obtained by amino acid insertion, deletion or substitution, preferably by amino acid substitution.
- E92H refers to the amino acid at position 92 being replaced by wild type Glu (glutamate) to His (histidine) of the mutant, and the amino acid sequence number is based on the wild type sequence.
- amino acid substitution refers to the replacement of one or more amino acids in a polypeptide or protein or protein fragment with another or several other amino acids by genetic engineering techniques or synthetic techniques.
- the soluble TNF p75 receptor mutein of the present invention can be prepared by: synthesizing primers according to the sequence of the published human soluble TNF p75 receptor, and amplifying the soluble TNF p75 receptor coding sequence by PCR, or artificially synthesizing the soluble The coding sequence for the TNF p75 receptor.
- the site-directed mutagenesis of the DNA sequence encoding the novel mutant protein of the present invention is ligated into a suitable expression vector and transferred to a suitable host cell. Finally, the transformed host cells are cultured, and the fusion protein of the present invention is obtained by isolation and purification.
- various carriers known in the art such as commercially available carriers can be used.
- a commercially available vector is selected, and then a nucleotide sequence encoding a novel mutein of the present invention is operably linked to an expression control sequence to form a protein expression vector.
- operably linked refers to a condition in which portions of a linear DNA sequence are capable of affecting the activity of other portions of the same linear DNA sequence. For example, if a signal peptide DNA is expressed as a precursor and is involved in the secretion of a polypeptide, then the signal peptide (secretion leader sequence) DNA is operably linked to the polypeptide DNA; if the promoter controls the transcription of the sequence, then it is operably linked to A coding sequence; if the ribosome binding site is placed at a position that enables translation, then it is operably linked to the coding sequence.
- “operably linked” means adjacent, and for secretory leader sequences means adjacent in the reading frame.
- the term "host cell” includes prokaryotic cells and eukaryotic cells.
- prokaryotic host cells include Escherichia coli, Bacillus subtilis and the like.
- eukaryotic host cells include yeast cells, insect cells, and mammalian cells.
- a method of making a soluble TNF p75 receptor: Fc fusion protein of the invention comprises the steps of:
- the soluble TNF p75 receptor of the present invention is useful for the treatment of diseases associated with TNF overexpression, including: rheumatoid arthritis, psoriasis, scleroderma, Sjogren's syndrome, ankylosing spondylitis, erythema Lupus, dermatomyositis, systemic lupus-like syndrome, etc. .
- the soluble TNF p75 receptor of the present invention The Fc fusion protein may be used alone or in combination with other drugs such as chemotherapeutic drugs.
- the invention also provides a pharmaceutical composition
- a pharmaceutical composition comprising an effective amount of one or more soluble TNF p75 of the invention Receptor: An Fc fusion protein, and at least one pharmaceutically acceptable carrier, diluent or excipient.
- the active ingredient is usually mixed with excipients, or diluted with excipients, or enclosed in a carrier in the form of a capsule or sachet.
- the excipient acts as a diluent, it can be a solid, semi-solid or liquid material as a vehicle for the excipient, carrier or active ingredient.
- the composition may be in the form of a tablet, a pill, a powder, a solution, a syrup, a sterile injectable solution or the like.
- excipients examples include: lactose, glucose, sucrose, sorbitol, mannitol, starch, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, and the like.
- the preparation may also include: a wetting agent, an emulsifier, a preservative (such as methyl and propyl hydroxybenzoate), a sweetener, and the like.
- the soluble TNF p75 receptor of the present invention The mode of administration of the Fc fusion protein and the pharmaceutical composition is not particularly shown, and can be administered orally, topically, parenterally, such as muscle, vein, or subcutaneous injection, or inhalation spray. Dosing. A preferred mode is oral administration.
- the dose is in the range of about 1 mg to 1000 mg for an adult having an average body weight of 60-70 kg, or parenterally in an injection manner.
- the dose is about 0.1 mg to 500 mg, which can be administered once or several times a day.
- the unit dose of the pharmaceutical composition usually comprises the active ingredient in the range of from 1 mg to 500 mg, typically 1 mg, 5 mg, 10 mg, 25 mg, 50 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg.
- the amount and dosage regimen of the therapeutically active ingredients employed in treating a particular condition with a composition of the invention will depend on a variety of factors including weight, age, sex, inevitable medical condition, severity of the disease, route of administration and frequency. This can be determined by the medical staff.
- the wild type human soluble TNF p75 receptor encoded by the present invention is used: Fc is commercially available from AMGEN (trade name ENBREL).
- the TNF a of the present invention was purchased from R&D Company.
- the LT 28-171 adopted by the present invention is in Chinese patent
- TNFR75 (E92H): Preparation of Fc fusion protein
- Wild-type human soluble TNFRp75 Fc fusion protein DNA fragment was used as a template
- the mutated soluble TNF p75 receptor DNA coding sequence was obtained by SOE-PCR (Splicing by Overlapping Extension PCR) technique. First, the DNA encoding the wild-type soluble TNFRp75: Fc fusion protein was used as a template, using primers.
- TNFRp75p aagcttatggctcccgtcgccgtctggg (SEQ ID NO. 16)
- E92HpFl TGCTTGAGCTGTGGCTCCCG (SEQ ID NO. 17)
- Fcp gaattcctatttacccggagacaggg (SEQ ID NO.18)
- E92HpRl CGGGAGCCACAGCTCAAGC AgtgGGGAA (SEQ ID NO. 19)
- Amplification of the soluble TNF p75 receptor comprises a mutation site and a DNA fragment of the second half of the Fc fragment.
- the above two PCR products were used as templates, and PCR amplification was carried out using the primers TNFRp75p and the primer Fcp to obtain the coding.
- TNFRp75 A DNA fragment of an Fc fusion protein.
- the gene fragment was ligated with Hindlll+EcoRI into the same restriction enzyme site as the expression vector pcDNA3 (purchased from Invitrogen). Enzyme digestion and ligation are performed according to the commercially available kit instructions.
- the above constructed mutant soluble TNF p75 receptor Fc fusion protein expression vector was transformed into Escherichia coli DH5a, and positive clones were picked and inoculated in 500 ml of LB medium for amplification.
- the purified DNA was extracted using Qiagen's Ultrapure Plasmid DNA Purification Kit according to the manufacturer's instructions.
- the above plasmid DNA was transfected into CHO-K1 (Chinese hamster ovary cells, purchased from ATCC) using Invitrogen's liposome kit, and the method was performed according to the manufacturer's instructions.
- the cell culture medium was changed to a screening medium containing G418 drug at 24-48 hours after transfection, and the screening medium was changed once every 3-4 days until cell clone formation.
- the cloned loop is used to pick up the monoclonal from the plate and transfer it into a 24-well plate.
- the culture supernatants of each clone were subjected to ELISA, and cell clones with high expression levels were selected for drug pressure amplification screening.
- concentration of the drug to be screened was raised to the highest, the expression level of each cloned single cell was detected, and two monoclonal cells with high expression level and good cell growth state were selected as seeds for preservation.
- the ELISA assay for cell expression was performed as follows:
- the coated antibody (anti-human TNFRp75-specific monoclonal antibody was purchased from R&D) was diluted to lug/ml with a coating solution (pH 9.6 CBS) and coated on a 96-well plate, 100 ul / L, at 5 ° Place C overnight. The liquid in the well was discarded, washed 3 times with PBST, dried, and then added with 400 ⁇ l/well blocking solution (1% BSA PBST), 2 hr at room temperature, washed 3 times with PBST, and dried.
- a coating solution pH 9.6 CBS
- the cells were expanded in a 500 ml culture flask at a cell concentration of l X 10 5 /ml, and cultured at 37 degrees for 3-4 days.
- Passage Take 2. The cell concentration of X 10 5 /ml was expanded and cultured in a 720 cm 2 roller bottle, and cultured for 3-4 days. Passage: The cells were expanded to a 1445 cm 2 roller bottle with a total number of cells of 4 X 10 7 and cultured for 6 days.
- Exchange The cells reached the plateau phase and the culture medium was serum-free medium (SFM, purchased from Gibco).
- SFM serum-free medium
- TNFRp75 Fc fusion protein of 8.7 mg.
- TNFRp75F aagcttatggctcccgtcgccgtctggg (SEQ ID NO. 16)
- Fcp gaattcctatttacccggagacaggg (SEQ ID NO.18)
- TNFRp75 Fc
- TNFRp75 E92N, W89Y
- Fc Fc
- TNFRp75 E92S, W89Y
- TNFRp75 (E92N, W89F): Specific primers for the Fc fusion protein encoding gene are shown in the table below:
- TNFRp75 E92S, W89Y
- Fc E92SW89YpFl SEQ ID N0.28
- TNFRp75 E92N, W89F
- Fc E92NW89FpFl SEQ ID NO. 30
- TNFRp75 mutant Neutralization of LT28-171 by Fc fusion protein
- E92A actinomycin D and lng/ml of LT 2S-171 and gradient TNFRp75
- control group added actinomycin D and lng/ml of LT 28-I 7I and gradient wild type rhTNFRp75 Fc.
- the above 96-well plate was placed in a 37 ° C, 5% carbon dioxide incubator for 24 h.
- the 96-well plate should be patted as much as possible so that no moisture remains in the plate; a decolorizing solution is added to the 96-well plate, 100 ul per well, and mixed, and the colorimetric instrument is 570 nm.
- TNFRp75 mutants The Fc fusion protein was tested in the same manner for the neutralization of LT ⁇ I 71 (hereinafter abbreviated as LT). The test results are shown in the table below. The "S" shape curve is plotted according to the test results as shown in Fig. 2-6.
- TNFRp75 Fc neutralizes LT
- TNFRp75 (E92A) : Fc 23.1 1/7.93 291 %
- TNFRp75 (E92H): Fc 44.05/3.74 1 177%
- TNFRp75 (E92N): Fc 21.99/7.94 277%
- TNFRp75 (E92N, W89Y) : Fc 54.49/3.74 1457%
- TNFRp75 (E92S, W89Y): Fc 23.1 1 /6.28. 368%
- TNFRp75 (E92N, W89F): Fc 23.28/7.56 308%
- TNFRp75 mutant Neutralization of TNF ⁇ by Fc fusion protein
- L929 cells 1 X 10 6 cells (1 96-well plate), actinomycin D and 10 ng/ml of TNF ⁇ and gradient TNFRp75 : Fc or its mutant were added.
- the above 96-well plate was placed in a 37-inch, 5% carbon dioxide incubator for 24 hours.
- the test results are shown in the table below.
- the abscissa of the figure is the logarithm of the fusion protein concentration (ng/ml), and the ordinate is the light absorption value of 570 nm.
- TNFRp75 (E92N, W89Y): Determination of binding constant of Fc-binding TNF ⁇ and lymphotoxin
- Ligand TNF ⁇ and LT, receptor rhTNFRp75 Fc
- TNFRp75 (E92N, W89Y) Fc 3.8mg/mL
- HBS-P buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% (v/v) surfactant P20, pH 7.4).
- the rhTNFRp75: Fc was coupled to the FC2 channel of the CM5 chip using the amino coupling method in the Biacore 3000 Control Software Wizard.
- HBS-P was used as working buffer, and Img/mL of rhTNFp75:Fc was diluted to a final concentration of 10 ( ⁇ g/mL with pH 4.0, 10 mM NaAC.
- the surface of the chip was mixed with 0.2 M EDC and 50 mM NHS 1:1 to 10 ⁇ .
- the flow rate of L/min was injected for 7 minutes, then the receptor solution was injected and injected for 7 minutes at pH 8.5, 1 M ethanolamine to block the surface of the activated chip.
- the final coupling amount of TNFRp75 was 7043.6 RU.
- TNFRp75 (E92N, W89Y): Fc was coupled to the FC4 channel of the CM5 chip using the amino coupling method in the Biacore 3000 Control Software Wizard.
- HBS-P as working buffer, 3.8 mg/mL TNFRp75 (E92N, W89Y): Fc was diluted with pH 4.0, 10 mM NaAC to a final concentration of 10 ( ⁇ g/mL.
- the surface of the chip was 0.2 M EDC and 50 mM NHS 1: 1 Mixing was injected at a flow rate of 10 ⁇ L/min for 7 minutes, then the receptor solution was injected and injected at pH 8.5, 1 M ethanolamine for 7 minutes to block the surface of the activated chip.
- TNFRp75 (E92N, W89Y): Final coupling of Fc The amount is 6275.0 RU.
- the binding activity of TNF, LT and receptor TNFRp75:Fc was determined by SPR (surface plasmon resonance) method.
- the ligands were diluted with HBS-P buffer to InM and 10 nM, respectively, and centrifuged and injected automatically to detect the binding activity of the ligands at different concentrations to the receptor TNFRp75. Detailed kinetic experiments were performed on ligands with binding activity.
- the LT mother liquor concentration was 2.25 mg/mL, and the molar concentration was 137 ⁇ . It was diluted with HBS-P buffer at a certain ratio to a concentration of 0, 0.3125, 0.625, 1.25, 2.5, 5.0, 10.0, 20.0 nM. ;
- Ligand pair TNFRp75 (E92N, W89Y): Fc primary screening and kinetics assay
- the ligands were diluted with HBS-P buffer to InM and 10 nM, respectively, and centrifuged and injected automatically to detect the binding activity of different concentrations of ligand to the receptor TNFRp75 (E92N, W89Y): Fc.
- Detailed kinetic experiments were performed on ligands with binding activity.
- the concentration of TNF a mother liquor was 10 g/inL, the molar concentration was 575 nM, and the concentration was 0, 0.3125, 0.625, 1.25, 2.5, 5.0, 10.0, 20.0 nM by using HBS-P buffer at a certain ratio;
- the concentration of LT mother liquor was 2.25 mg/mL, and the molar concentration was 137 ⁇ . It was diluted with HBS-P buffer at a certain ratio to make the concentrations 0, 0.3 125, 0.625, 1.25, 2.5, 5.0, 10.0, 20.0. nM;
- a rhTNFRp75: Fc and TNF a (abbreviated as TNF) and LT binding constants are shown in the following table.
- B TNFRp75 (E92N, W89Y): The binding constants of Fc to TNF a and LT are shown in the table below.
- TNFRp75 (E92N, W89Y): Fc binding to TNF ⁇ and LT binding dissociation constant (K D ) compared
- TNFRp75 Fc is 2-3 fold smaller, indicating that TNFRp75 (E92N, W89Y): Fc has a stronger ability to bind TNF a and LT.
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Priority Applications (9)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US12/745,724 US8454969B2 (en) | 2008-05-30 | 2009-01-12 | Soluble tumor necrosis factor receptor mutant |
JP2010544562A JP5445975B2 (ja) | 2008-05-30 | 2009-01-12 | Tnf受容体の可溶性変異体 |
BRPI0906044-8A BRPI0906044A2 (pt) | 2008-05-30 | 2009-01-12 | "mutante solúvel tnfrp75, proteína de fusão, sequências de dna, usos do mutante solúvel tnfrp75 e da proteína de fusão e composições farmacêuticas" |
CN200980101260.5A CN101883787B (zh) | 2008-05-30 | 2009-01-12 | 一种可溶性tnf受体突变体 |
AU2009253623A AU2009253623B2 (en) | 2008-05-30 | 2009-01-12 | A soluble tumor necrosis factor receptor mutant |
EP09753407.7A EP2221314B1 (en) | 2008-05-30 | 2009-01-12 | A soluble tumor necrosis factor receptor mutant |
RU2010141916/10A RU2478645C2 (ru) | 2008-05-30 | 2009-01-12 | Растворимый мутант рецептора фактора некроза опухоли |
CA2710040A CA2710040C (en) | 2008-05-30 | 2009-01-12 | A soluble tumor necrosis factor receptor mutant |
KR1020107015346A KR101243951B1 (ko) | 2008-05-30 | 2009-01-12 | 가용성 종양 괴사 인자 수용체 돌연변이 |
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CN200810038410.X | 2008-05-30 | ||
CNA200810038410XA CN101591388A (zh) | 2008-05-30 | 2008-05-30 | 一种可溶性tnf受体突变体 |
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US (1) | US8454969B2 (zh) |
EP (1) | EP2221314B1 (zh) |
JP (1) | JP5445975B2 (zh) |
KR (1) | KR101243951B1 (zh) |
CN (2) | CN101591388A (zh) |
AU (1) | AU2009253623B2 (zh) |
BR (1) | BRPI0906044A2 (zh) |
CA (1) | CA2710040C (zh) |
RU (1) | RU2478645C2 (zh) |
WO (1) | WO2009143689A1 (zh) |
Families Citing this family (6)
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KR101273893B1 (ko) * | 2010-09-13 | 2013-06-14 | 한올바이오파마주식회사 | 변형된 인간 종양 괴사 인자 수용체-1 폴리펩티드 또는 그의 절편 및 그의 제조방법 |
ES2626418T3 (es) | 2010-12-23 | 2017-07-25 | Hanall Biopharma Co., Ltd. | Polipéptido modificado del receptor-1 del factor de necrosis tumoral humano o fragmento del mismo y procedimiento de preparación del mismo |
BR112014031923B1 (pt) * | 2012-06-21 | 2022-02-08 | Daewoong Pharmaceutical Co., Ltd. | Uso de uma composição compreendendo pelo menos um tnfri modificado ou fragmento tnfri modificado, uso de uma formulação tópica e uso de uma composição |
CN104561022B (zh) * | 2014-12-22 | 2017-05-17 | 南京师范大学 | 家猪肿瘤坏死因子突变体的构建及蛋白表达纯化方法 |
CN108853482A (zh) * | 2017-05-12 | 2018-11-23 | 上海复旦张江生物医药股份有限公司 | 一种重组人TNFR-Fc融合蛋白突变体的用途 |
WO2022122005A1 (en) * | 2020-12-11 | 2022-06-16 | Adlai Nortye Biopharma Co., Ltd. | Anti-tnfr2 antibody and application thereof |
Citations (4)
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CN1502632A (zh) * | 2002-11-26 | 2004-06-09 | 广州绿阳生物工程有限公司 | 新型TNFR-Fc融合蛋白 |
US20040170975A1 (en) * | 1999-06-23 | 2004-09-02 | Compugen Ltd. | Variant of TNF-receptor |
CN101003575A (zh) * | 2007-01-12 | 2007-07-25 | 中国医学科学院血液学研究所泰达生命科学技术研究中心 | 人肿瘤坏死因子可溶性受体Ⅱ-抗体Fc段融合蛋白 |
CN101085813A (zh) * | 2006-06-05 | 2007-12-12 | 上海复旦张江生物医药股份有限公司 | 一种可溶性tnf受体突变体 |
Family Cites Families (2)
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JP2002514079A (ja) * | 1997-05-01 | 2002-05-14 | アムジエン・インコーポレーテツド | キメラopgポリペプチド |
WO2005012353A1 (en) * | 2003-08-01 | 2005-02-10 | Amgen Inc. | Crystalline tumor necrosis factor receptor 2 polypeptides |
-
2008
- 2008-05-30 CN CNA200810038410XA patent/CN101591388A/zh active Pending
-
2009
- 2009-01-12 EP EP09753407.7A patent/EP2221314B1/en not_active Not-in-force
- 2009-01-12 US US12/745,724 patent/US8454969B2/en not_active Expired - Fee Related
- 2009-01-12 AU AU2009253623A patent/AU2009253623B2/en not_active Ceased
- 2009-01-12 BR BRPI0906044-8A patent/BRPI0906044A2/pt not_active Application Discontinuation
- 2009-01-12 KR KR1020107015346A patent/KR101243951B1/ko active IP Right Grant
- 2009-01-12 CN CN200980101260.5A patent/CN101883787B/zh active Active
- 2009-01-12 JP JP2010544562A patent/JP5445975B2/ja not_active Expired - Fee Related
- 2009-01-12 CA CA2710040A patent/CA2710040C/en not_active Expired - Fee Related
- 2009-01-12 WO PCT/CN2009/000037 patent/WO2009143689A1/zh active Application Filing
- 2009-01-12 RU RU2010141916/10A patent/RU2478645C2/ru not_active IP Right Cessation
Patent Citations (4)
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US20040170975A1 (en) * | 1999-06-23 | 2004-09-02 | Compugen Ltd. | Variant of TNF-receptor |
CN1502632A (zh) * | 2002-11-26 | 2004-06-09 | 广州绿阳生物工程有限公司 | 新型TNFR-Fc融合蛋白 |
CN101085813A (zh) * | 2006-06-05 | 2007-12-12 | 上海复旦张江生物医药股份有限公司 | 一种可溶性tnf受体突变体 |
CN101003575A (zh) * | 2007-01-12 | 2007-07-25 | 中国医学科学院血液学研究所泰达生命科学技术研究中心 | 人肿瘤坏死因子可溶性受体Ⅱ-抗体Fc段融合蛋白 |
Also Published As
Publication number | Publication date |
---|---|
CN101591388A (zh) | 2009-12-02 |
US8454969B2 (en) | 2013-06-04 |
RU2478645C2 (ru) | 2013-04-10 |
EP2221314A4 (en) | 2010-12-29 |
EP2221314B1 (en) | 2013-07-03 |
EP2221314A1 (en) | 2010-08-25 |
CN101883787A (zh) | 2010-11-10 |
CA2710040A1 (en) | 2009-12-03 |
BRPI0906044A2 (pt) | 2015-07-07 |
KR20100135706A (ko) | 2010-12-27 |
US20110117082A1 (en) | 2011-05-19 |
AU2009253623A1 (en) | 2009-12-03 |
JP2011511627A (ja) | 2011-04-14 |
JP5445975B2 (ja) | 2014-03-19 |
RU2010141916A (ru) | 2012-04-20 |
AU2009253623B2 (en) | 2013-08-22 |
CA2710040C (en) | 2013-05-28 |
KR101243951B1 (ko) | 2013-03-13 |
CN101883787B (zh) | 2014-09-10 |
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