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Fusion protein comprising an interleukin 4 and interleukin

a technology of interleukin and fusion protein, which is applied in the field of immunology and pharmacology, can solve the problems of limited therapeutic intervention, modification of the contour of adjacent articulating surfaces, and not all patients respond well to such type of therapeutic intervention

Inactive Publication Date: 2014-10-23
UMC UTRECHT HLDG BV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The patent describes a new therapy using a fusion protein that combines two cytokines, IL4 and IL10, to treat inflammation and pain. The fusion protein has superior biological activity compared to separate cytokines and can inhibit the expression of activating Fcγ receptors while inducing the expression of inhibitory Fcγ receptors. The therapy can be administered as an intravenous injection or a slow continuous infusion over a long period to reduce toxic side effects. The patent also describes the use of recombinant expression vectors and pharmaceutical compositions for the therapy.

Problems solved by technology

Although blocking TNFα and / or IL1β is success in many patients suffering from rheumatoid arthritis and inflammatory bowel diseases, not all patients respond well to such type of therapeutic interventions.
These structural alterations in the articular cartilage and peri-articular bone may lead to modification of the contours of the adjacent articulating surfaces.
Only very limited therapeutical interventions are proposed for the treatment of OA, and the few therapeutical interventions available are aimed at pain management rather than treatment of the functional impairments.

Method used

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  • Fusion protein comprising an interleukin 4 and interleukin
  • Fusion protein comprising an interleukin 4 and interleukin
  • Fusion protein comprising an interleukin 4 and interleukin

Examples

Experimental program
Comparison scheme
Effect test

example 1

Transfection of HEK293 Cells

[0124]Method:

[0125]HEK293 cells were transiently transfected according to standard procedures with a vector containing a transgene (Y Derocher et al., Nucleic Acids Research 2002, vol 30, no 2, e9). Briefly, the IL4-IL10 fusion protein insert was cloned in a pUPE expression vector, containing a cystatin signal sequence. HEK293E cells were then transfected with the pUPE expression vector containing the IL4-IL10 fusion protein of the present invention. At the same time cells were co-transfected with a vector carrying the transgene for beta-galactoside alpha-2,3-sialyltransferase 5 (SIAT 9) homo sapiens to optimize capping of the glycans with sialic acid. Cells were cultured in FreeStyle medium (Invitrogen) with 0.9% primatone and ˜0.04%, v / v, fetal calf serum. Five days after transfection, the conditioned medium was collected by low-speed centrifugation, after which it was concentrated over a 10 kDa QuixStand hollow fibre cartridge (GE Healthcare) and diafi...

example 2

[0126]ELISA Assays for Immunochemical Detection of IL4-IL10 Fusion Protein, IL4 and IL10

[0127]Method:

[0128]The IL4 and IL10 content in culture supernatant or chromatography fractions was measured by ELISA (IL4 PeliPair ELISA Kit; Sanquin, Amsterdam, the Netherlands; Cat# M9314 or IL10 PeliPair ELISA Kit; Sanquin; Cat# M9310) according to manufacturer's instructions. Briefly, catching antibodies against IL4 or IL10 were diluted 1:200 in phosphate buffered saline, pH 7.4 (PBS) and coated overnight onto an ELISA plate. All subsequent steps were performed in PBS supplemented with 0.1%, w / v, Tween-20 (PBS-T). A dose response curve consisting of serial dilutions to yield a range of 100 to 2 pg / ml of recombinant IL4 or IL10 was tested. Bound antibodies were detected with streptavidine-poly-HRP (Sanquin) followed by incubation with TMB (3,3′,5,5″-tetramethylbenzidine; Invitrogen, Carlsbad, Calif., USA; Cat# SB02). Reaction was stopped with 1M Sulphuric Acid (Chem Lab; Cat# CL05-2658-1000). ...

example 3

SDS-Page and Western Blotting

[0132]Method:

[0133]Samples were diluted 1:1 in sample buffer (Tris-HCl pH 6.8, 25%, w / v, Glycerol, 2%, w / v, SDS, 0.01%, w / v, bromophenol blue; BioRad, Richmond, Va., USA, Cat#161-0737), containing 710 mM 2-mercaptoethanol and incubated for 10 minutes at 100° C. Subsequently, samples were loaded on a 7.5%, w / v, polyacrylamide Tris / Glycine Gel (Mini-PROTEAN TGX Precast Gels without SDS; BioRad, Cat#456-1023). The molecular weight markers (WesternC Standard, 250-10 kD; BioRad; Cat#161-0376) were run on a separate lane. Electroporesis was performed under reducing conditions, using a Tris / glycine / SDS buffer (BioRad; Cat#161-0732). To identify the IL4-IL10 fusion protein, immunoblotting with anti-IL4 or anti-IL10 antibodies was performed. Proteins were separated on SDS-PAGE as described above, and then transferred to a PVDF-membrane (BioRad; Cat#161-0277) by Western blotting, using a Tris / glycine buffer (BioRad; Cat#161-0734) at 100V for 1 hour.

[0134]After blo...

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PUM

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Abstract

The invention is concerned with a fusion protein comprising interleukin 10 and interleukin 4, a nucleic acid molecule encoding such fusion protein, a vector comprising such nucleic acid molecule, and a host cell comprising such nucleic acid molecule or such vector. The invention further pertains to a method for producing such fusion protein. The fusion protein or a gene therapy vector encoding the fusion protein may be used in the prevention or treatment of osteoarthritis, chronic pain, a condition characterized by local or systemic inflammation, immune activation, and / or lymphoproliferation.

Description

FIELD OF THE INVENTION[0001]The present invention is in the field of immunology and pharmacology, particularly for treatment of osteoarthritis, chronic pain, inflammatory diseases or disorders, and related diseases and disorders. The invention particularly relates to a novel fusion protein comprising interleukin 4 (IL-4) and interleukin 10 (IL-10), optionally physically fused together by a linker. Particularly, the present invention provides an IL4-IL10 fusion protein endowed with a superior biological activity over a combination of the individual cytokines. The present invention also provides nucleic acid sequences encoding a IL4-IL10 fusion protein, expression vectors comprising such nucleic acid sequences, host cells or host organisms altered to harbour the nucleic acid sequence encoding the IL4-IL10 fusion protein and the IL4-IL10 fusion protein itself. The invention further provides methods for producing an IL4-IL10 fusion protein using a cell or organism harbouring such nuclei...

Claims

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Application Information

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IPC IPC(8): C07K14/54
CPCC07K14/5406C07K14/5428A61K38/00C07K2319/00A61P1/00A61P1/04A61P11/00A61P13/12A61P17/00A61P17/02A61P17/06A61P19/02A61P19/06A61P19/08A61P25/00A61P25/04A61P29/00A61P31/04A61P35/00A61P35/02A61P37/00A61P37/02A61P37/04A61P37/06A61P37/08A61P43/00A61P7/00A61P7/08A61P9/00A61P9/10A61P9/14A61P3/10A61K9/0029A61K9/0095A61K9/08A61K38/2026A61K38/2066A61K45/06
Inventor VAN ROON, JOEL ADRIANUS GIJSBERTHARTGRING, SARITA AIMEE YVONNEHACK, CORNELIS ERIKLOUWS, CHRISTINALAFEBER, FLORIS PAULUS JACOBUS GERARDUS
Owner UMC UTRECHT HLDG BV
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