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Soluble TNF acceptor mutant

A soluble and mutant technology, applied to cytokine/lymphokine/interferon receptors, receptors/cell surface antigens/cell surface determinants, allergic diseases, etc., can solve the problem of decreased drug efficacy and increased serious infections Incidence rate, faster drug clearance rate, etc., to reduce inhibition and affinity reduction

Active Publication Date: 2012-01-25
上海复旦张江生物医药股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, Infliximab is a chimeric antibody, and about 10% of clinical patients will produce anti-Infliximab antibodies, which will speed up the drug clearance rate and reduce the drug effect, which limits the application of Infliximab to a certain extent
Etanercept can simultaneously bind with high affinity to TNF and lymphotoxin, although lymphotoxin plays an important role in immune regulation, it is not related to the pathogenesis of rheumatoid arthritis; binding to lymphotoxin not only partially neutralizes Etanercept (trade name ENBREL), and may increase the incidence of serious infections

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Embodiment 1, TNFR75 (W89L) mutant: the preparation of Fc fusion protein

[0068] (1) Preparation of TNFR75(W89L) mutant: Fc fusion protein encoding gene

[0069] Using the wild-type human soluble TNF p75 receptor: Fc fusion protein DNA fragment as a template, the mutant soluble TNF p75 receptor DNA coding sequence was obtained by SOE-PCR (Splicing by Overlapping Extension PCR) technology. First, the wild-type soluble TNFp75 receptor: Fc fusion protein coding DNA was used as a template, with primers

[0070] TNFR75p: aagcttatggctcccgtcgccgtctggg

[0071] W89LpFl: CTCAAGCACTCGGGAACcagGTTCC

[0072] Amplifies the DNA fragment of the first half of the selective soluble TNF p75 receptor containing the mutation site; with primers

[0073] Fcp: gaattcctatttacccggagacagggg

[0074] W89LpRl: GGAACctgGTTCCCGAGTGCTTGAG

[0075] Amplify selective soluble TNF p75 receptor DNA fragment including the mutation site and the second half of the Fc fragment. Finally, using the above...

Embodiment 2-8

[0086] Embodiment 2-8, other TNFR75 mutants: the preparation of Fc fusion protein

[0087] The shared primers used to prepare mutant TNFR75(W89H), TNFR75(W89Y), TNFR75(W89F), TNFR75(W89R), TNFR75(W89K), TNFR75(W89M), TNFR75(W89I) TNFR75: Fc fusion protein coding gene are as follows:

[0088] TNFR75F: aagcttatggctcccgtcgccgtctggg

[0089] Fcp: gaattcctatttacccggagacagggg

[0090] The specific primers used to prepare mutant TNFR75(W89H), TNFR75(W89Y), TNFR75(W89F), TNFR75(W89R), TNFR75(W89K), TNFR75(W89M), TNFR75(W89I) TNFR75:Fc fusion protein coding gene are as follows surface:

[0091] mutant name specific primer TNFR75(W89H)

[0092] TNFR75(W89I)

[0093] Other operations are the same as in Example 1.

Embodiment 9

[0094] Embodiment 9, TNFR75 mutant: determination of Fc fusion protein and ligand binding ability

[0095] 100ul of TNF and LT28-171 were coated onto the microtiter plate at a concentration of 10ug / ml, and 100ul of gradient TNFR75(W89H) mutant: Fc fusion protein was added to each well, and repeated wells were kept at 37°C for 1 hour. Add enzyme-linked antibody goat anti-humanIgG Fc-HRP (PIERCE company): 1:20000, 100ul / well, 37°C for 1 hour. OPD color development.

[0096] TNFR75(W89L), TNFR75(W89Y), TNFR75(W89F), TNFR75(W89R), TNFR75(W89K), TNFR75(W89M), TNFR75(W89I) were operated in the same way, and the results are shown in Table 1.

[0097] Table 1. Experimental results of ligand-selective receptor binding ability

[0098] Sample serial number

[0099] TNFR2-Fc W89M 180.45 17.12

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Abstract

The invention discloses a soluble TNF acceptor mutant, relating to tumor necrosis factor TNF acceptor derivant and its medical application. The amnio acid at 89th position of mutant is replaced and the ability of neutralizing cell toxicity of TNF alpha is retained, while ability of neutralizing LT is reduced by more than 10 times. The mutant can be used to treat over expressing disease of TNF alpha, because the affinity to lymphotoxin is reduced, the side effect caused by lymphotoxin neutralisation can be reduced, and the inhibitive effect to normal immunity function from medicine is aslo reduced.

Description

technical field [0001] The invention relates to tumor necrosis factor (TNF) receptor derivatives and their application in medicine. Background technique [0002] Both tumor necrosis factor α (TNF) and lymphotoxin α (LTα) are members of the tumor necrosis factor superfamily, which have biological activities such as regulating immune response, cell apoptosis and cell differentiation. TNF and lymphotoxin share two intracellular receptors, TNF receptor 1 (TNFR55) and TNF receptor 2 (TNFR75). Overexpression of TNF is an important factor causing rheumatoid arthritis. Currently, the most effective mainstream protein drugs for treating rheumatoid arthritis in the international market are chimeric antibodies (Infliximab) and soluble TNF that directly block the biological function of TNF? Receptor TNF R2:Fc fusion protein (Etanercept). Both drugs showed significant therapeutic effects. [0003] However, Infliximab is a chimeric antibody, and about 10% of clinical patients will prod...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/525C07K19/00A61K38/19A61K38/17A61P19/02A61P37/02A61P17/00C12N15/11C07K14/715C12N15/12
Inventor 杨彤刘彦君
Owner 上海复旦张江生物医药股份有限公司
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