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Methods for making fully human bispecific antibodies using a common light chain

A bispecific, light chain technology, applied in the field of preparing fully human bispecific antibodies using common light chains

Active Publication Date: 2015-04-29
REGENERON PHARM INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But making bispecifics with appropriate light chain components that can satisfactorily bind each heavy chain of a bispecific has proven problematic

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

Experimental program
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Effect test

Embodiment 1

[0258] with the selected person V L human V H District identification

[0259] An in vitro expression system was constructed to determine whether a single rearranged human germline light chain could be co-expressed with a human heavy chain from an antigen-specific human antibody.

[0260] Methods for producing human antibodies in genetically modified mice are well known (see e.g. US 6,596,541, Regeneron Pharmaceuticals, ). The technique involves producing 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 responds to antigenic stimulation to produce human variable regions and Antibodies against mouse constant regions. Encoded from The DNA of the variable regions of the heavy and light chains of the mouse antibody is fully human. First, high-affinity chimeric antibodies with human variable regions and mouse constant regions are isolated. Antib...

Embodiment 2

[0272] Generation of rearranged human germline light chain loci

[0273] use technology (see, for example, US Patent 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 prepare multiple recombinant A row of human germline light chain targeting vectors was used to modify the mouse genome 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 endogenous κ that had been previously modified to delete endogenous κ variable and linking gene segments Light chain locus.

[0274] Construction of a rearranged human germline light chain targeting vector. Three different rearranged human germline light chain regions were prepared using standard art-recognized molecular biology techniques. The human v...

Embodiment 3

[0290] Generation of mice expressing a single rearranged human light chain

[0291] The targeted ES cells described above were used as donor ES cells and passed Methods (See, e.g., US Patent 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 analyzes Nature Biotech. 25(1):91-99) Introduce mouse embryos at the 8-cell stage. Independently engineered human germline VK1-39JK5 light chains were identified by genotyping using a modified allelic assay (Valenzuela et al., supra) that detects the presence of a unique rearranged human germline light chain region Region, Vκ3-20J1 light chain region or VpreBJλ5 light chain region

[0292] Pups were genotyped and mice that were heterozygous or homozygous for the only rearranged human germline light chain region were selected for characterization of expression of the rearranged human germline light chain region.

[0293] ...

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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

[0001] This application was filed as a PCT International Patent Application on June 5, 2013, and claims priority to U.S. Patent Application No. 13 / 488,628, filed June 5, 2012, the disclosure of which is incorporated by reference Incorporated into this article as a whole. technical field [0002] Genetically modified mice expressing antibodies having a common human variable region / mouse constant region light chain combined with a diversified human variable region / mouse constant region heavy chain are provided. Provided are methods for preparing human bispecific antibodies derived from human variable region gene sequences of mouse B cells. Background technique [0003] Antibodies typically comprise homodimeric heavy chain components, wherein each heavy chain monomer is associated with the same light chain. Antibodies with heterodimeric heavy chain components (eg, bispecific antibodies) are desired as therapeutic antibodies. But producing bispecific antibodies with appropriat...

Claims

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Application Information

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IPC IPC(8): A01K67/027C07K16/00C07K16/40C12N15/85
CPCC07K16/00C07K16/40C07K2317/51C07K2317/56C07K2317/565C07K2317/567C07K2317/33C07K2317/21C07K2317/31C07K2317/24C07K2317/92C07K2317/76A01K67/0278C12N15/8509A01K2207/15A01K2217/072A01K2217/15A01K2227/105A01K2267/01C07K16/468
Inventor R·巴布J·麦克维尔特L·麦克唐纳S·史蒂文斯S·戴维斯D·R·巴克勒K·A·霍西阿瓦A·J·墨菲
Owner REGENERON PHARM INC
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