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Antibody variants having modifications in the constant region

a constant region and antibody technology, applied in the field of modified antibodies, can solve the problems of inferior pharmacokinetics, dimerization may form undesirable immune complexes, and full-length antibodies may exhibit agonistic effects, and achieve the effect of preventing undeired fab arm exchang

Inactive Publication Date: 2015-12-24
GENMAB AS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The patent text explains how mutations can be made to prevent the formation of unwanted interactions between antibodies. By modifying certain parts of the antibody molecule, researchers have created monovalent antibodies that are less likely to dimerize through non-covalent interactions. These mutations also make the antibodies more stable and homogeneous, which makes them better suited for therapeutic applications. Additionally, the mutations at one specific position in the antibody molecule prevent the exchange of arms between two molecules. Overall, the patent describes how to modify antibodies to improve their properties and effectiveness in treating disease.

Problems solved by technology

Some full-length antibodies may exhibit agonistic effects (which may be considered to be undesirable) upon binding to the target antigen, even though the antibody works as an antagonist when used as a Fab fragment.
In the case of soluble antigens, dimerization may form undesirable immune complexes.
The presently available Fab fragments show inferior pharmacokinetics due to their small size resulting to filtration in the kidneys as well as their inability to interact with the Brambell receptor FcRn (Junghans R P et al., Proc Natl Acad Sci USA 93(11), 5512-6 (1996)), therefore being unstable in vivo and having very rapid clearance after administration.
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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  • Antibody variants having modifications in the constant region
  • Antibody variants having modifications in the constant region
  • Antibody variants having modifications in the constant region

Examples

Experimental program
Comparison scheme
Effect test

example 1

Structural Analysis of CH3-CH3 Interface

[0276]In human IgG1, the non-covalent interaction between the CH3 domains involves 16 residues located on four anti-parallel β-strands that make intermolecular contacts and burry 1090 Å2 from each surface (Deisenhofer, J.; Biochemistry, 1981. 20(9): p. 2361-70). Alanine scanning mutagenesis showed that stabilization of the IgG1 CH3-CH3 interaction was largely mediated by 6 of these residues, including K409 (Dall'Acqua, W., et al.; Biochemistry, 1998. 37(26): p. 9266-73). To get a better understanding of the role of K409 in the IgG1 CH3-CH3 interaction, the 1.65 Å 1 L6X crystal structure (Idusogie, E. E., et al.; J Immunol, 2000. 164(8): p. 4178-84) was studied in more detail using the Brugel modelling package (Delhaise, P., et al., J. Mol. Graph., 1984. 2(4): p. 103-106).

[0277]In order to propose mutations that should lead to a desired stabilization (or destabilization) of IgG4, a quantitative structure-based scoring methodology was employed (...

example 2

Water Hypothesis

[0278]In the IgG1 structure, K409 forms a hydrogen bond with D399′ on the opposite CH3 domain. Furthermore, K409 is part of a water-binding pocket together with S364 and T411 in the same CH3 domain and K370′ on the opposite CH3 domain. The presence of the water molecule prevents an electrostatic clash between K409 and K370′.

[0279]The K409R substitution (as in IgG4) was modelled in the 1 L6X structure by optimizing the side chain conformations of the arginine residue and its surrounding residues, using the FASTER algorithm (Desmet, J., et al.; Proteins, 2002. 48(1): p. 31-43). In this model, the guanidinium group of R409 takes up the position of the water molecule and causes an electrostatic clash with K370′. The side-chains of T411 and K370′ loose their interactions compared to the case with water present (as in IgG1), but D399 keeps its interaction with the side chain at position R409.

example 3

Destabilization of IgG4

[0280]The mutations in the Table below were made in order to destabilize the CH3-CH3 interaction of an IgG4.

[0281]KABAT indicates amino acid numbering according to Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). EU index indicates amino acid numbering according to EU index as outlined in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

Numbering of CH3 mutationsKABATEU index G4SEQ ID NO: 4370Y349R*Y217R*372L351N*L219N*372L351Q*L219Q*378E357AE225A378E357T*E225T*378E357V*E225V*378E357I*E225I*387S364R*S232R*387S364K*S232K*389T366AT234A389T366R*T234R*389T366K*T234K*389T366N*T234N*391L368AL236A391L368VL236V391L368E*L236E*391L368G*L236G*391L368S*L236S*391L368T*L236T*393K370AK238A393K370R*K238R*393K370TK238T427D399AD267A427D399T*D267T*427D399S*D267S*436F405AF273A436F...

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Abstract

The present invention relates to positions in the constant region of antibodies, in particular the CH3 region of lgG4, which affect the strength of CH3-CH3 interactions. Mutations that either stabilize or destabilize this interaction are disclosed.

Description

FIELD OF INVENTION[0001]The present invention relates to modified antibodies that may be used in therapeutic applications. The invention also relates to methods for producing the antibodies, pharmaceutical compositions comprising the antibodies and use thereof for different therapeutic applications.BACKGROUND OF THE INVENTION[0002]Native antibodies and immunoglobulins are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies between the heavy chains of different immunoglobulin isotypes. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region (abbreviated herein as CL). Each heavy chain is comprised of a heavy chain variable region (VH) and a heavy chain constant region (CH) consisting of three domain, CH1, C...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C07K16/28
CPCC07K16/2863C07K2317/21C07K2317/76C07K2317/526C07K2317/52A61K47/6849A61P29/00A61P35/00A61P37/02A61P43/00C07K16/00C07K2317/53A61K39/3955A61K51/103A61K2039/505C07K2317/524
Inventor LABRIJN, ARAN FRANKLOVERIX, STEFANPARREN, PAULVAN DE WINKEL, JANSCHUURMAN, JANINELASTER, IGNACE
Owner GENMAB AS
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