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Stabilized Fc polypeptides with reduced effector function and methods of use

A stable, number-specified technology, applied to medical preparations containing active ingredients, specific peptides, chemical instruments and methods, etc.

Inactive Publication Date: 2012-03-07
BIOGEN MA INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] The present invention solves the problem of prior art "effector-less" antibodies, indeed any Fc-containing "effector-less" protein, by providing an improved method for increasing the stability of the Fc region

Method used

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  • Stabilized Fc polypeptides with reduced effector function and methods of use
  • Stabilized Fc polypeptides with reduced effector function and methods of use
  • Stabilized Fc polypeptides with reduced effector function and methods of use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0624] Example 1. Rational design of stability-enhancing IgG Fc mutations

[0625] Aglycosylated antibodies represent an important class of therapeutic agents that do not require immune effector functions. However, it is clear that removal of CH2-associated oligosaccharides in IgG1 and IgG4 affects antibody conformation and stability. Loss of antibody stability can present process development challenges that adversely affect program timelines and resources. Here, we detail several methods for designing repertoires of amino acid positions in CH2 and CH3 to enhance IgG Fc stability.

[0626] A. Effector-less IgG: Associated Variation and Residue Frequency Design for the IgG4CH2 Domain

[0627] Correlative variation analysis was performed on different Cl class Ig fold sequence databases as previously described (Glaser et al., 2007; Wang et al., 2008). The compilation and structure / HMM-based alignment of the Cl class Ig fold sequences were also performed as previously describ...

Embodiment 2

[0657] Example 2. Thermostability screening of IgG Fc antibody domains produced in E. coli

[0658]A modified heat challenge assay described in US Patent Application Serial No. 11 / 725,970 was employed as a stability screen to determine the amount of soluble IgG Fc protein retained at 40°C after heat challenge at pH 4.5.

[0659] Escherichia coli strain W3110 (ATCC, Manassas, Va. Cat. #27325). Transformants were incubated at 30°C in SB (Teknova, Half Moon Bay, Ca.Cat.#S0140) supplemented with 0.6% glycine, 0.6% Triton X100, 0.02% arabinose and 50 μg / ml carbenicillin. Grow overnight in expression medium. Bacteria are pelleted by centrifugation and the supernatant collected for further processing.

[0660] After heat challenge, aggregated material was removed by centrifugation, and binding of soluble Fc samples retained in the treated clarified supernatants to protein A (delta P7837) was determined by DELFIA assay. Coat two 96-well plates (MaxiSorp, Nalge Nunc, Rochester, N...

Embodiment 3

[0665] Example 3. Production of stable IgG Fc antibodies

[0666] A. Mutagenesis, transient expression and purification of stable IgG Fc part in E. coli

[0667] Stability mutations were incorporated into the BRM13 construct previously detailed in Example 2 by site-directed mutagenesis using the Stratagene Quik-Change Lightning Mutagenesis Kit. Primers were designed to be 36-40 bases in length, with mutations in the appropriate sequence 10-15 bases flanking the middle, with a GC content of at least 40%, starting and ending at one or more C / G bases . All mutant constructs are listed in Table 3.1 below.

[0668] Table 3.1. IgG-Fc constructs expressed and purified from E. coli

[0669]

[0670]

[0671] After PCR using primers to introduce mutations, mutagenesis was digested with DpnI restriction endonuclease for 5 minutes at 37°C to completely digest the parental plasmid. Then the mutagenesis reaction was transferred to XL1 blue E. coli supercompetent cells. Ampicil...

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Abstract

The invention provides a method of producing Fc-containing polypeptides, such as antibodies, having stabilized Fc regions. The invention also provides stabilized Fc polypeptides produced according to these methods as well as methods of using such antibodies as therapeutics.

Description

[0001] related application [0002] This patent application claims the benefit of US Provisional Patent Application Serial No. 61 / 146,950, filed January 23, 2009, entitled "STABILIZED Fc POLYPEPTIDES WITH REDUCED EFFECTOR FUNCTION AND METHODS OF USE." The entire contents of the provisional patent applications mentioned above are incorporated herein by reference. Background of the invention [0003] The adaptive immune response is the mechanism by which the body protects itself from foreign organisms that cause the body to cause infection or disease. One mechanism is based on the ability of antibodies produced or administered to a host to bind antigen through their variable regions. Once the antibody binds the antigen, the antigen becomes a target for destruction, usually mediated at least in part through the antibody's constant or Fc region. [0004] There are several effector functions or activities that are mediated through the Fc region of an antibody. One effector funct...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/08C07K16/00
CPCA61K38/00C07K16/00C07K16/2875C07K2317/41C07K2317/52C07K2317/524C07K2317/526C07K2317/71C07K2317/92C07K2317/94
Inventor C.L.雷耶斯E.钱F.R.泰勒E.加伯B.R.米勒S.德马雷斯特S.格拉泽
Owner BIOGEN MA INC
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