Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Immunoglobulin fc variants

An immunoglobulin and globulin technology, applied in the field of immunoglobulin Fc variants, can solve the problems of inability to increase half-life and low biological activity, and achieve the effect of low administration frequency and increase in vivo half-life

Active Publication Date: 2014-09-10
HANMI PHARMA
View PDF8 Cites 53 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, even though it is linked to PEG or its fusion albumin, this peptide drug still shows relatively low biological activity or its in vivo half-life cannot be increased to a sufficient level

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Immunoglobulin fc variants
  • Immunoglobulin fc variants
  • Immunoglobulin fc variants

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Example 1: Construction of vectors expressing immunoglobulin Fc fragments

[0065] A position involved in the binding affinity of human FcRn was selected from the amino acid sequence (SEQ ID NO: 73) of HMC001 produced by Escherichia coli transformant HM11201 (KCCM-10660P, Korean Patent No. 10-0824505), and mutations were induced thereon to An amino acid at a corresponding position is replaced with another amino acid in order to increase FcRn binding affinity. To achieve this, use nucleotide-substituted primers and the QuikChange TM Site-directed mutagenesis kit (Stratagene). In this regard, primers used for site-directed mutagenesis are shown in Tables 1 to 3 below.

[0066] For site-directed mutagenesis by polymerase chain reaction (PCR), 50 ng of HMC001 expression vector, each primer pair, dNTP and PfuTurbo TM Polymerase (Stratagene) was added to the PCR tube and the reaction was performed as follows: 16 cycles of denaturation at 95°C for 30 seconds, 30 seconds a...

Embodiment 2

[0081] Example 2: Production of immunoglobulin Fc variants in E. coli

[0082]The expression vectors of immunoglobulin Fc variants prepared in Example 1 were respectively transformed into Escherichia coli BL21(DE3) competent cells (Invitrogen) by heat shock at 42°C for 1 minute, followed by solid culture in LB supplemented with ampicillin Colonies were cultured to select ampicillin-resistant colonies.

[0083] The colonies thus selected were inoculated on 500 ml of 2×LB medium (containing ampicillin) and cultured in a shaking incubator at 37° C. and 20 rpm for 15 hours. After transferring 100 ml of the culture broth to 500 ml of fresh 2×LB medium (containing ampicillin), the resulting solution was incubated until an optical density at 600 nm (OD600) of 4 to 5 was reached. Transfer 200ml of culture solution to a 5L-fermenter at a ratio of 10% (v / v), inject 2×LB medium (containing ampicillin) into it, and incubate at 37°C, 500-700rpm and 1vvm aeration rate A semi-automatic p...

Embodiment 3

[0084] Example 3: Isolation and purification of immunoglobulin Fc variants

[0085] The culture broth fermented in Example 2 was centrifuged at 12,000 g for 30 minutes to recover cell pellets. The cell pellet thus recovered was suspended in 10× volume of lysis buffer (20 mM Tris (pH 9.0), 1 mM EDTA (pH 8.0), 0.2 M NaCl, 0.5% triton X-100) and then heated at 15,000 psi Using a microfluidic machine (microfluidic chuck) under pressure three times. The cell lysates thus obtained were centrifuged at 6,000 g for 30 minutes to obtain only the inclusion bodies of the proteins expressed by the E. coli transformants.

[0086] Inclusion bodies were rinsed with 0.5% Triton X-100 and distilled water, and suspended in 8M urea solution containing 10× volume of 20 mM Tris (pH 9.0) for 2 hours to dissolve them. To separate undissolved solid impurities, the solution in which the inclusion bodies were dissolved was centrifuged at 12,000 g for 30 minutes to collect the supernatant. Then, L-c...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

PropertyMeasurementUnit
optical densityaaaaaaaaaa
Login to View More

Abstract

The present invention relates to immunoglobulin Fc variants having an increased binding affinity for FcRn, which is characterized by including one or more amino acid modifications selected from the group consisting of 307S, 308F, 380S, 380A, 428L, 429K, 430S, 433K and 434S (this numbering is according to the EU index) in the constant region of a native immunoglobulin Fc fragment. Owing to the high binding affinity for FcRn, the immunoglobulin Fc variants according to the present invention show more prolonged in vivo half-life, and thus can be used for the preparation of a long-acting formulation of protein drugs.

Description

technical field [0001] The present invention relates to immunoglobulin Fc variants with increased FcRn (neonatal Fc receptor) binding affinity and methods of using them to increase the in vivo half-life of physiologically active polypeptides. The immunoglobulin Fc variants of the present invention are characterized in comprising 307S, 308F, 380S, 380A, 428L, 429K, 430S, 433K and 434S selected from the constant region of the native immunoglobulin Fc fragment (the numbering is according to the EU index) One or more amino acid modifications. Background technique [0002] Antibodies are immune proteins that bind a specific antigen. Antibodies are composed of two polypeptide light chains and two polypeptide heavy chains. Each chain is composed of immunoglobulin domains and both have variable and constant regions. The variable regions between antibodies display significant sequence diversity and are responsible for binding the target antigen. Constant regions with relatively l...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C07K19/00A61K39/395
CPCA61K38/26C07K16/283C07K2319/30C07K2317/52C07K2319/00C07K16/00A61K47/48561C07K2317/94C07K2317/92A61K47/6849C07K16/28C07K19/00A61K39/395
Inventor 吴宜林许容豪黄祥渊崔仁荣郑圣烨权世昌
Owner HANMI PHARMA
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com