Histidine Engineered Light Chain Antibodies and Genetically Modified Non-Human Animals for Generating the Same

a technology of light chain antibody and histidine, which is applied in the field of histidine engineered light chain antibody and genetically modified non-human animals for generating the same, can solve the problems of bispecific antibody having a suitable light chain component that can satisfactorily associate with each of the heavy chains of the bispecific antibody, requiring high doses to achieve the desired effect, and reducing binding

Inactive Publication Date: 2014-01-09
REGENERON PHARM INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Benefits of technology

[0010]In one aspect, a biological system is provided for generating an antibody or an antibody variable domain that binds a target antigen at a neutral pH but exhibits reduced binding of the same antigen at an acidic pH (e.g., pH 5.0-6.0). The biological system comprises a non-human animal, e.g., a rodent (e.g., a mouse or rat) that has a rearranged light chain sequence (e.g., a rearranged V-J) that comprises one or more histidine modifications. In various aspects, the one or more histidine modifications are in the light chain CDR3 codon. In various aspects, the non-human animal comprises a human or humanized heavy chain immunoglobulin locus. In vario

Problems solved by technology

But making bispecific antibodies having a suitable light chain component that can satisfactorily associate with each of the heavy chains of a bispecific antibody has proved pr

Method used

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  • Histidine Engineered Light Chain Antibodies and Genetically Modified Non-Human Animals for Generating the Same
  • Histidine Engineered Light Chain Antibodies and Genetically Modified Non-Human Animals for Generating the Same
  • Histidine Engineered Light Chain Antibodies and Genetically Modified Non-Human Animals for Generating the Same

Examples

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example 1

Identification of Histidine Residues in Antigen-Specific Human Light Chains

[0258]Generation of a common light chain mouse (e.g., Vκ1-39 or Vκ3-20 common light chain mouse) and antigen-specific antibodies in those mice is described in, e.g., U.S. patent application Ser. Nos. 13 / 022,759, 13 / 093,156, and 13 / 412,936 (Publication Nos. 2011 / 0195454, 2012 / 0021409, and 2012 / 0192300, respectively), incorporated by reference herein in their entireties. Briefly, rearranged human germline light chain targeting vector was 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, and 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 modifie...

example 2

Engineering and Characterization of Histidine-Substituted Human Universal Light Chain Antibodies

example 2.1

Engineering of Histidine Residues into a Germline Human Rearranged Light Chain

[0261]Histidine residues were engineered into a rearranged human Vκ1-39Jκ5 light chain using site directed mutagenesis primers specifically designed to introduce engineered histidine residues at Q105, Q106, Y108, and P111 positions of the human Vκ1-39Jκ5 light chain. Site directed mutagenesis was performed using molecular techniques known in the art (e.g., QuikChange II XL Site Directed Mutagenesis Kit, Agilent Technologies). Locations of the engineered residues in the CDR3 are shown in FIG. 2, the nucleic acid sequences of histidine-substituted CDR3's depicted in FIG. 2 are set forth in SEQ ID NOs: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, and 32 (corresponding amino acid sequences are set forth in SEQ ID NOs: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33). The nucleic acid and amino acid sequences of germline rearranged Vκ1-39Jκ5 CDR3 are set forth in SEQ ID NOs: 2 and 3, respect...

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Abstract

A genetically modified non-human animal is provided, wherein the non-human animal expresses an antibody repertoire capable of pH dependent binding to antigens upon immunization. A genetically modified non-human animal is provided that expresses human immunoglobulin light chain variable domains derived from a limited repertoire of human immunoglobulin light chain variable gene segments that comprise histidine modifications in their germline sequence. Methods of making non-human animals that express antibodies comprising histidine residues encoded by histidine codons introduced into immunoglobulin light chain nucleotide sequences are provided.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application is a continuation-in-part of U.S. patent application Ser. No. 13 / 832,247, filed Mar. 15, 2013, which claims benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 61 / 611,950, filed Mar. 16, 2012, and U.S. Provisional Application No. 61 / 736,930, filed Dec. 13, 2012, all incorporated by reference herein in their entireties.FIELD OF INVENTION[0002]A genetically modified non-human animal (e.g., rodent, e.g., mouse or rat) is provided that expresses antibodies capable of binding to an antigen in a pH dependent manner. A method for making modifications to immunoglobulin light chain variable region sequence of a non-human animal is provided, wherein the modifications include the mutagenesis of residues within the light chain variable region gene, e.g., nucleotides that encode one or more amino acids within a complementary determining region (CDR), to facilitate in vivo expression of antibodies comprising light chain...

Claims

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Application Information

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IPC IPC(8): C07K16/00C12N15/85A01K67/027
CPCC07K16/00A01K67/0275C12N15/8509A01K67/0278A01K2217/072A01K2217/075A01K2217/15A01K2227/105A01K2267/01C07K2317/515C07K2317/92C12N2800/204C07K2317/21C07K2317/94C07K2317/51C07K2317/24C07K2317/56
Inventor MCWHIRTER, JOHNMACDONALD, LYNNMURPHY, ANDREW J.
Owner REGENERON PHARM INC
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