Methods For Making Fully Human Bispecific Antibodies Using A Common Light Chain

a technology of bispecific antibodies and light chains, which is applied in the direction of fluorescence/phosphorescence, instruments, peptides, etc., can solve the problems of bispecific antibodies having a suitable light chain component that can satisfactorily associate with each of the heavy chains of bispecific antibodies, and achieves enhanced methods for making bispecific antibodies, reduces the number of inappropriate species, and promotes stable interaction

Inactive Publication Date: 2013-02-21
REGENERON PHARM INC
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  • Abstract
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Benefits of technology

[0126]In various embodiments, methods for making bispecific antibodies are enhanced by employing a common light chain to pair with each heavy chain variable regions of the bispecific antibodies. In various embodiments, employing a common light chain as described herein reduces the number of inappropriate species of immunoglobulins lacking bispecificity as compared to employing original cognate light chains. In various embodiments, the heavy chain variable regions of the bispecific antibodies are identified from monospecific antibodies comprising a common light chain. In various embodiments, the heavy chain variable regions of the bispecific antibodies comprise human heavy chain variable gene segments that are rearranged in vivo within mouse B cells that have been previously engineered to express a limited human light chain repertoire, or a single human light chain, cognate with human heavy chains and, in response to exposure with an antigen of interest, generate a chimeric antibody repertoire containing a plurality of human heavy chain variable regions that are cognate with one or one of two possible human light chain variable regions, wherein the chimeric antibodies are specific for the antigen of interest.
[0127]In various aspects, a method of preparing a bispecific antibody is provided, the bispecific antibody compri...

Problems solved by technology

But making bispecific antibodies having a suitable light chain component that can satisfa...

Method used

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  • Methods For Making Fully Human Bispecific Antibodies Using A Common Light Chain
  • Methods For Making Fully Human Bispecific Antibodies Using A Common Light Chain
  • Methods For Making Fully Human Bispecific Antibodies Using A Common Light Chain

Examples

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example 1

Identification of Human VH Regions that Associate with Selected Human VL Regions

[0237]An in vitro expression system was constructed to determine if a single rearranged human germline light chain could be co-expressed with human heavy chains from antigen specific human antibodies.

[0238]Methods for generating human antibodies in genetically modified mice are known (see e.g., U.S. Pat. No. 6,596,541, Regeneron Pharmaceuticals, VELOCIMMUNE®). The VELOCIMMUNE® technology involves generation of a genetically modified mouse having a genome comprising human heavy and light chain variable regions operably linked to endogenous mouse constant region loci such that the mouse produces an antibody comprising a human variable region and a mouse constant region in response to antigenic stimulation. The DNA encoding the variable regions of the heavy and light chains of the antibodies produced from a VELOCIMMUNE® mouse are fully human. Initially, high affinity chimeric antibodies are isolated having ...

example 2

Generation of a Rearranged Human Germline Light Chain Locus

[0242]Various rearranged human germline light chain targeting vectors were made using VELOCIGENE® technology (see, e.g., U.S. Pat. No. 6,586,251 and Valenzuela et al. (2003) High-throughput engineering of the mouse genome coupled with high-resolution expression analysis, Nature Biotech. 21(6): 652-659) to modify mouse genomic Bacterial Artificial Chromosome (BAC) clones 302g12 and 254m04 (Invitrogen). Using these two BAC clones, genomic constructs were engineered to contain a single rearranged human germline light chain region and inserted into an endogenous κ light chain locus that was previously modified to delete the endogenous κ variable and joining gene segments.

[0243]Construction of Rearranged Human Germline Light Chain Targeting Vectors.

[0244]Three different rearranged human germline light chain regions were made using standard molecular biology techniques recognized in the art. The human variable gene segments used f...

example 3

Generation of Mice Expressing a Single Rearranged Human Light Chain

[0262]Targeted ES cells described above were used as donor ES cells and introduced into an 8-cell stage mouse embryo by the VELOCIMOUSE® method (see, e.g., U.S. Pat. No. 7,294,754 and Poueymirou et al. (2007) F0 generation mice that are essentially fully derived from the donor gene-targeted ES cells allowing immediate phenotypic analyses Nature Biotech. 25(1): 91-99. VELOCIMICE® independently bearing an engineered human germline Vκ1-39Jκ5 light chain region, a Vκ3-20Jκ1 light chain region or a VpreBJλ5 light chain region are identified by genotyping using a modification of allele assay (Valenzuela et al., supra) that detects the presence of the unique rearranged human germline light chain region.

[0263]Pups are genotyped and a pup heterozygous or homozygous for the unique rearranged human germline light chain region are selected for characterizing expression of the rearranged human germline light chain region.

[0264]Fl...

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Abstract

A genetically modified mouse is provided, wherein the mouse expresses an immunoglobulin light chain repertoire characterized by a limited number of light chain variable domains. Mice are provided that express just one or a few immunoglobulin light chain variable domains from a limited repertoire in their germline. Methods for making bispecific antibodies having universal light chains using mice as described herein, including human light chain variable regions, are provided. Methods for making human variable regions suitable for use in multispecific binding proteins, e.g., bispecific antibodies, and host cells are provided. Bispecific antibodies capable of binding first and second antigens are provided, wherein the first and second antigens are separate epitopes of a single protein or separate epitopes on two different proteins are provided.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application is a continuation-in-part of U.S. Ser. No. 13 / 412,936 filed Mar. 6, 2012, which is a continuation-in-part of U.S. Ser. No. 13 / 093,156 filed Apr. 25, 2011, which is a continuation-in-part of U.S. Ser. No. 13 / 022,759 filed Feb. 8, 2011, which is a nonprovisional application of U.S. Provisional Application Ser. No. 61 / 302,282, filed Feb. 8, 2010; which applications are hereby incorporated by reference in their entirety.FIELD[0002]A genetically modified mouse is provided that expresses antibodies having a common human variable / mouse constant light chain associated with diverse human variable / mouse constant heavy chains. A method for making a human bispecific antibody from human variable region gene sequences of B cells of the mouse is provided.BACKGROUND[0003]Antibodies typically comprise a homodimeric heavy chain component, wherein each heavy chain monomer is associated with an identical light chain. Antibodies having a hete...

Claims

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Application Information

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IPC IPC(8): C12P21/00G01N21/64G01N33/566
CPCA01K67/0278C07K2317/56C12N15/8509A01K2207/15A01K2217/072A01K2217/15A01K2227/105A01K2267/01C07K2317/21C07K2317/24C07K2317/515C07K2317/565C07K2317/567C07K2317/76C07K2317/92C07K2319/30C12N2800/204C07K16/28C07K16/468C07K2317/31C07K16/00
Inventor BABB, ROBERTMCWHIRTER, JOHNMACDONALD, LYNNSTEVENS, SEANDAVIS, SAMUELBUCKLER, DAVID R.HOSIAWA, KAROLINA A.MURPHY, ANDREW J.
Owner REGENERON PHARM INC
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