Dual Variable Region Antibody-Like Binding Proteins Having Cross-Over Binding Region Orientation

a technology variable regions, which is applied in the field of antibody-like binding proteins, can solve the problems of reduced avidity, short half-life, and difficult and expensive purification

Inactive Publication Date: 2012-10-04
SANOFI SA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Benefits of technology

[0058]Specific embodiments of the invention will become evident from the

Problems solved by technology

A disadvantage associated with the use of such fragments is that they have only one antigen binding site, leading to reduced avidity.
In addition, due to their small size, they are cleared very fast from the serum, and hence display a short half-life.
Bispecific antibodies were originally made by fusing two hybridomas, each capable of producing a different immunoglobulin (Milstein et al., 1983, Nature 305(5934): 537-40), but the complexity of species (up to ten different species) produced in cell culture made purification difficult and expensive (George et al., 1997, THE ANTIBODIES 4: 99-141 (Capra et al., ed., Harwood Academic Publishers)).
The resulting bispecific heterodimer is fully non-human, hence highly immunogenic, which could have deleterious side effects (e.g., “HAMA” or “HARA” reactions), and/or neutralize the therapeutic.
Despite the promising results obtained using heteroconjugates or bispecific antibodies produced from cell fusions as cited above, several factors made them impractic

Method used

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  • Dual Variable Region Antibody-Like Binding Proteins Having Cross-Over Binding Region Orientation
  • Dual Variable Region Antibody-Like Binding Proteins Having Cross-Over Binding Region Orientation
  • Dual Variable Region Antibody-Like Binding Proteins Having Cross-Over Binding Region Orientation

Examples

Experimental program
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Effect test

example 1

Design and Engineering of Bispecific Cross-Over Dual Variable Region Antibody-Like Binding Proteins

[0228]The cross-over dual variable region in an Fv format was described in U.S. Pat. No. 5,989,830 and was referred to as a cross-over double head (CODH) configuration. Molecular modeling predicted that the cross-over double-head (CODH) design results in a complex with both binding sites facing in opposite directions, without the restraints suggested for the Dual-Fv configuration. The CODH Fv format was examined to determine whether it could be converted into complete antibody-like molecules by adding a CL domain to the light chain and an Fc region to the heavy chain. A similar conversion was successful for the corresponding dual variable domains (DVD-Ig) and TBTI as described in U.S. Pat. No. 7,612,181 and International Publication No. WO 2009 / 052081. The arrangement of the variable regions in the CODH format is shown in the structures below, which indicate the amino to carboxyl orien...

example 2

Design of CODV-Ig Proteins by Molecular Modeling

[0241]To obtain fully functional antibody-like proteins utilizing the cross-over double head configuration that are amendable to incorporation of the Fc and CL1 domains, a molecular modeling protocol was developed for the inclusion and evaluation of different linkers between the constant and variable domains and between the dual variable domains on both the heavy and light chains. The question was whether the addition of unique linkers between each constant / variable domain interface and between the two variable / variable domain interfaces on both the heavy and light chains would allow proper protein folding to occur and produce functional antibody-like molecules in the cross-over dual variable region configuration (see FIG. 2). In other words, a total of four independent and unique linkers were evaluated (see FIG. 2). This molecular modeling protocol was based on protein-protein docking of homology models and experimental models of the ...

example 3

Generation of CODV-Ig Expression Plasmids

[0246]Nucleic acid molecules encoding the variable heavy and light chains of the six heavy chains and four light chains described in Table 4 were generated by gene synthesis at Geneart (Regensburg, Germany). The variable light chain domains were fused to the constant light chain (IGKC, GenBank Accession No. Q502W4) by digestion with the restriction endonucleases ApaLI and BsiWI and subsequently ligated into the ApaLI / BsiWI sites of the episomal expression vector pFF, an analogon of the pTT vector described by Durocher et al., (2002, Nucl. Acids Res. 30(2): E9), creating the mammalian expression plasmid for expression of the light chains.

[0247]The variable heavy chain domains were fused to the “Ted” variant of the human constant heavy chain (IGHG1, GenBank Accession No. 569F4), or alternatively, to a 6×His tagged CH1 domain from the human constant IGHG1 in order to create a bispecific Fab. Next, the VH domain was digested with the restriction ...

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Abstract

The invention provides antibody-like binding proteins comprising four polypeptide chains that form four antigen binding sites, wherein each pair of polypeptides forming an antibody-like binding protein possesses dual variable domains having a cross-over orientation. The invention also provides methods for making such antigen-like binding proteins.

Description

CROSS REFERENCE TO RELATED APPLICATIONS[0001]This application claims the benefit of priority from U.S. Provisional Application No. 61 / 468,276, filed Mar. 28, 2011, and French Patent Application No. 1160311, filed Nov. 14, 2011, the disclosures of which are incorporated by reference herein in their entirety.FIELD OF THE INVENTION[0002]The invention relates to antibody-like binding proteins comprising four polypeptide chains that form four antigen binding sites, wherein each pair of polypeptides forming the antibody-like binding protein possesses dual variable domains having a cross-over orientation. The invention also relates to methods for making such antigen-like binding proteins.BACKGROUND OF THE INVENTION[0003]Naturally occurring IgG antibodies are bivalent and monospecific. Bispecific antibodies having binding specificities for two different antigens can be produced using recombinant technologies and are projected to have broad clinical applications. It is well known that comple...

Claims

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Application Information

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IPC IPC(8): A61K39/395C12N15/13C07K19/00C12N5/10C12P21/02C12N15/62C07K16/18C12N15/63
CPCC07K16/00C07K16/46C07K16/244C07K16/247C07K16/2863C07K16/2866C07K16/32C07K16/461C07K16/468C07K2317/21C07K2317/24C07K2317/624C07K2317/64C07K2317/76C07K2317/92C07K2317/94C07K16/241C07K16/245C07K2317/626C07K2317/66C07K16/2803C07K16/2809C07K2317/55C07K2317/73C07K2317/31A61P37/02C07K2317/522C07K2317/524C07K2317/526C07K2317/53C07K2318/00
Inventor BAURIN, NICOLASBEIL, CHRISTIANCORVEY, CARSTENLANGE, CHRISTIANLI, DANXIMIKOL, VINCENTSTEINMETZ, ANKERAO, ERCOLE
Owner SANOFI SA
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