Hybrid antibodies comprising human variable regions and mouse constant regions produced in a genetically modified mouse

• US 8,791,323 B2
• Filed: 11/09/2006
• Issued: 07/29/2014
• Est. Priority Date: 10/31/2000
• Status: Expired due to Fees

• Current Assignee: Regeneron Pharmaceuticals Incorporated
• Original Assignee: Regeneron Pharmaceuticals Incorporated
• Inventors: Murphy, Andrew J., Yancopoulos, George D., Karow, Margaret, Macdonald, Lynn, Stevens, Sean, Economides, Aris N., Valenzuela, David M.
• Primary Examiner(s): Noble, Marcia S
• Assistant Examiner(s): SGAGIAS, MAGDALENE K
• Application Number:
  US11/595,427
• Publication Number:
  US 20070061900A1
• Time in Patent Office: 2,819 Days
• Field of Search: 800/18, 436/548
• US Class Current: 800/18
• CPC Class Codes: A01K 2207/15 Humanized animals
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: A01K 2227/105 Murine
• CPC Class Codes: A01K 2267/01 Animal expressing industrially exogenous proteins
• CPC Class Codes: A01K 67/0275 Genetically modified vertebrates, e.g. transgenic
• CPC Class Codes: A01K 67/0278 Knock-in vertebrates, e.g. humanised vertebrates
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C07K 16/28 against receptors, cell surface antigens or cell surface determinants
• CPC Class Codes: C07K 16/462 Igs containing a variable region (Fv) from one specie and a constant region (Fc) from another
• CPC Class Codes: C07K 2317/10 characterized by their source of isolation or production
• CPC Class Codes: C07K 2317/21 from primates, e.g. man
• CPC Class Codes: C07K 2317/24 containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• CPC Class Codes: C07K 2317/51 Complete heavy chain or Fd fragment, i.e. VH + CH1
• CPC Class Codes: C07K 2317/515 Complete light chain, i.e. VL + CL
• CPC Class Codes: C07K 2317/56 variable (Fv) region, i.e. VH and/or VL
• CPC Class Codes: C12N 15/67 General methods for enhancing the expression
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 15/8509 for producing genetically modified animals, e.g. transgenic
• CPC Class Codes: C12N 15/902 using homologous recombination
• CPC Class Codes: C12N 15/907 in mammalian cells
• CPC Class Codes: C12N 2800/204 : of bacterial origin, e.g. BAC
• CPC Class Codes: C12P 21/00 : Preparation of peptides or proteins single cell protein C12N1/00

Abstract

A method for engineering and utilizing large DNA vectors to target, via homologous recombination, and modify, in any desirable fashion, endogenous genes and chromosomal loci in eukaryotic cells. These large DNA targeting vectors for eukaryotic cells, termed LTVECs, are derived from fragments of cloned genomic DNA larger than those typically used by other approaches intended to perform homologous targeting in eukaryotic cells. Also provided is a rapid and convenient method of detecting eukaryotic cells in which the LTVEC has correctly targeted and modified the desired endogenous gene(s) or chromosomal locus (loci) as well as the use of these cells to generate organisms bearing the genetic modification.

Claims

1. A method of producing an antibody, comprising:

(a) providing a genetically modified mouse having a genome comprising human heavy chain immunoglobulin V, D, and J gene segments and human light chain immunoglobulin V and J gene segments, wherein the human heavy chain V, D, and J gene segments and human light chain V and J gene segments replace mouse endogenous heavy and light chain immunoglobulin variable region gene segments at the endogenous mouse heavy and light chain immunoglobulin variable region gene segment loci, and the human heavy chain V, D, and J gene segments and human light chain V and J gene segments are linked to the endogenous mouse heavy and light chain immunoglobulin constant region gene loci to form a hybrid heavy chain locus and hybrid light chain locus, wherein the human heavy chain V, D, and J gene segments and the human light chain V and J gene segments are present in the germline of the mouse, whereby the hybrid heavy chain locus and hybrid light chain locus rearrange during B-cell development such that the mouse produces a serum containing an antibody comprising human heavy and light chain immunoglobulin variable regions and heavy and light chain immunoglobulin constant regions in response to antigenic stimulation, wherein the heavy and light chain immunoglobulin constant regions are endogenous mouse heavy and light chain constant regions;

(b) stimulating an immune response in the genetically modified mouse by administering antigen to the genetically modified mouse; and

,

(c) obtaining the hybrid antibody from the serum of the genetically modified mouse.

2. A method of producing a human antibody, comprising:

(a) providing a genetically modified mouse having a genome comprising human heavy chain immunoglobulin V, D, and J gene segments and human light chain immunoglobulin V and J gene segments, wherein the human heavy chain V, D, and J gene segments and human light chain V and J gene segments replace mouse endogenous heavy and light chain immunoglobulin variable region gene segments at the endogenous mouse heavy and light chain immunoglobulin variable region gene segment loci, and the human heavy chain V, D, and J gene segments and human light chain V and J gene segments are linked to the endogenous mouse heavy and light chain immunoglobulin constant region gene loci to form hybrid heavy chain locus and a hybrid light heavy chain locus, wherein the human heavy chain V, D, and J gene segments and the human light chain V and J gene segments are present in the germline of the mouse, whereby the hybrid heavy chain locus and hybrid light chain locus rearrange during B-cell development such that the mouse produces a serum containing a hybrid antibody comprising human heavy and light chain immunoglobulin variable regions and heavy and light chain immunoglobulin constant regions in response to antigenic stimulation, wherein the heavy and light chain immunoglobulin constant regions are endogenous mouse heavy and light chain constant regions;

(b) stimulating an immune response the genetically modified mouse by administering an antigen to the genetically modified mouse;

(c) preparing a hybridoma expressing the hybrid antibody from the genetically modified mouse stimulated with the antigen in step (b);

(d) isolating DNA encoding the human heavy and light chain immunoglobulin variable regions of the hybrid antibody from the hybridoma of step (c);

(e) operably linking in a cell the DNA encoding the human heavy chain immunoglobulin variable region of step (d) with the DNA encoding a human heavy chain immunoglobulin constant region and the DNA encoding the human light chain immunoglobulin variable region of step (d) with DNA encoding a human light chain immunoglobulin constant region;

(f) growing the cell under conditions such that the cell expresses a human antibody having the human heavy and light chain variable regions of the hybrid antibody of step (d) and human heavy and light chain constant regions of step (e); and

(g) recovering the antibody.

3. The method of claim 2, wherein the cell is a CHO cell.