Stable igg4 antibodies

a technology of igg4 antibodies and stabilized antibodies, which is applied in the field of new stabilized igg4 antibodies to achieve the effect of reducing the risk of immunogenicity

Inactive Publication Date: 2010-10-21
GENMAB AS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0011]The present invention relates to stabilized forms of IgG4 antibodies that have a reduced ability to undergo Fab-arm exchange. It has surprisingly been found that substitution of the Arg residue at position 409 or the Phe residue at position 405 in human IgG4 can prevent Fab arm exchange, and thus stabilize IgG4, even in the absence of a mutation of the core hinge region sequence to Cys-Pro-Pro-Cys. This was unexpected, because it was believed that elimination of the flexibility of the hinge region via a change of the core hinge sequence to Cys-Pro-Pro-Cys was a requirement for prevention of half-molecule exchange.
[0015]In several embodiments, the antibodies used in the invention have the advantage that they contain a minimal number of sequence changes in the constant region as compared to naturally occurring IgG4. This reduces the risk of immunogenicity when the antibody is used for human therapy.

Problems solved by technology

IgG4 antibodies therefore have unusual properties which are undesirable in vivo: IgG4 antibodies are unstable, dynamic, molecules which engage in Fab arm exchange.
The random nature of this process introduces unpredictability which is highly undesirable for human immunotherapy.

Method used

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Examples

Experimental program
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Effect test

example 1

Oligonucleotide Primers and PCR Amplification

[0176]Oligonucleotide primers were synthesized and quantified by Isogen Bioscience (Maarssen, The Netherlands). Primers were dissolved in H2O to 100 pmol / μl and stored at −20° C. A summary of all PCR and sequencing primers is given below. For PCR, PfuTurbo® Hotstart DNA polymerase (Stratagene, Amsterdam, The Netherlands) was used according to the manufacturer's instructions. Each reaction mix contained 200 μM mixed dNTPs (Roche Diagnostics, Almere, The Netherlands), 6.7 pmol of both the forward and reverse primer, 100 ng of genomic DNA or 1 ng of plasmid DNA and 1 unit of PfuTurbo® Hotstart DNA polymerase in PCR reaction buffer (supplied with polymerase) in a total volume of 20 μl. PCR reactions were carried out with a TGradient Thermocycler 96 (Whatman Biometra, Goettingen, Germany) using a 32-cycle program: denaturing at 95° C. for 2 min; 30 cycles of 95° C. for 30 sec, a 60-70° C. gradient (or another specific annealing temperature) fo...

example 2

Agarose Gel Electrophoresis

[0177]Agarose gel electrophoresis was performed according to Sambrook (Sambrook, Russell et al. 2000 Molecular cloning. A laboratory manual (third edition), Cold Spring Harbor Laboratory Press) using gels of 50 ml, in 1× Tris Acetate EDTA buffer. DNA was visualized by the inclusion of ethidium bromide in the gel and observation under UV light. Gel images were recorded by a CCD camera and an image analysis system (GeneGnome; Syngene, via Westburg B.V., Leusden, The Netherlands).

example 3

Analysis and Purification of PCR Products and Enzymatic Digestion

[0178]Purification of desired PCR fragments was carried out using a MinElute PCR Purification Kit (Qiagen, via Westburg, Leusden, The Netherlands; product #28006), according to the manufacturer's instructions. Isolated DNA was quantified by UV spectroscopy and the quality was assessed by agarose gel electrophoresis.

[0179]Alternatively, PCR or digestion products were separated by agarose gel electrophoresis (e.g. when multiple fragments were present) using a 1% Tris Acetate EDTA agarose gel. The desired fragment was excised from the gel and recovered using the QIAEX II Gel Extraction Kit (Qiagen; product #20051), according to the manufacturer's instructions.

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Abstract

The present invention relates to stabilized IgG4 antibodies, to methods of producing such antibodies and to uses of such antibodies as a medicament. In a main aspect, the invention relates to a stabilized IgG4 antibody, comprising a heavy chain and a light chain, wherein said heavy chain comprises a human IgG4 constant region having a substitution of the Arg residue at position (409), the Phe residue at position (405) or the Lys residue at position (370).

Description

[0001]All patents, patent applications and other publications cited herein are hereby incorporated by reference in their entirety.FIELD OF THE INVENTION[0002]The present invention relates to novel stabilized IgG4 antibodies, to methods of producing such antibodies and to uses of such antibodies as a medicament.BACKGROUND OF THE INVENTION[0003]Antibodies are being used as therapeutic agents for a number of diseases and disorders, including cancer and autoimmune diseases. Antibodies are immunoglobulins that recognize specific antigens and mediate their effects via several mechanisms, including inhibition of ligand-receptor interactions, inhibition of receptor activation, mediation of receptor internalization and activation of effector functions, such as complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC). There are five classes of immunoglobulins: IgG, IgA, IgM, IgD and IgE. The IgG class is further divided into subclasses IgG1, IgG2, IgG3 and I...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C07K16/24C07K16/18C12P21/08
CPCA61K47/48384C07K16/18A61K47/48507A61K47/48523A61K47/48546A61K47/48561A61K51/10A61K51/1006A61K51/1021A61K51/1027A61K51/1039C07K16/00C07K16/28C07K2317/526C07K2317/55C07K2317/94C07K16/16A61K47/48423A61K47/6803A61K47/6813A61K47/6835A61K47/6839A61K47/6845A61K47/6849A61P1/02A61P1/04A61P1/16A61P1/18A61P11/00A61P11/02A61P11/06A61P13/02A61P13/08A61P13/10A61P13/12A61P15/00A61P15/10A61P17/00A61P17/02A61P17/06A61P17/08A61P19/00A61P19/02A61P19/06A61P19/08A61P21/00A61P25/00A61P25/16A61P25/28A61P27/02A61P27/16A61P29/00A61P3/00A61P3/10A61P31/00A61P31/04A61P31/12A61P31/18A61P35/00A61P35/02A61P35/04A61P37/02A61P37/06A61P37/08A61P43/00A61P5/00A61P7/00A61P7/06A61P9/00A61P9/10A61K51/103A61K2039/505C07K16/2863C07K16/2887C07K2317/21C07K2317/24C07K2317/31C07K2317/53
Inventor VAN DE WINKEL, JANVINK, TOMSCHUURMAN, JANINEPARREN, PAULAALBERSE, ROBVAN DER NEUT KOLFSCHOTE, MARIJN
Owner GENMAB AS
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