Methods of modifying eukaryotic cells

• US 9,528,136 B2
• Filed: 09/25/2013
• Issued: 12/27/2016
• Est. Priority Date: 02/16/2001
• Status: Expired due to Term

• 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): BRISTOL, LYNN ANNE
• Application Number:
  US14/036,892
• Publication Number:
  US 20140018522A1
• Time in Patent Office: 1,189 Days
• Field of Search: 530/387.3
• US Class Current: 1/1
• 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 hybrid antibody produced by a method comprising exposing to an antigen a mouse comprising in its germline:

i. human unrearranged immunoglobulin heavy chain V, D and J gene segments, wherein the human unrearranged immunoglobulin heavy chain V, D and J gene segments in situ replace endogenous mouse immunoglobulin heavy chain V, D and J gene segments, and the human unrearranged immunoglobulin heavy chain V, D and J gene segments are operably linked to an endogenous mouse heavy chain constant region gene, wherein rearrangement of the human unrearranged immunoglobulin heavy chain V, D and J gene segments in the mouse results in a human rearranged heavy chain variable region gene linked to the mouse heavy chain constant region gene; and

ii. human unrearranged immunoglobulin kappa light chain V and J gene segments, wherein the human unrearranged immunoglobulin kappa light chain V and J gene segments in situ replace endogenous mouse immunoglobulin kappa light chain V and J gene segments, and the human unrearranged immunoglobulin kappa light chain V and J gene segments are operably linked to an endogenous mouse kappa chain constant region gene, wherein rearrangement of the human unrearranged immunoglobulin kappa light chain V and J gene segments in the mouse results in a human rearranged kappa light chain variable region gene linked to the endogenous mouse kappa light chain constant region gene;

such that the mouse produces an antibody to the antigen, wherein the antibody comprises a heavy chain comprising a human heavy chain variable region encoded by the human rearranged heavy chain variable region gene and a mouse heavy chain constant region encoded by the mouse heavy chain constant region gene, and a light chain comprising a human kappa light chain variable region encoded by the human rearranged kappa light chain variable region gene and a mouse kappa light chain constant region encoded by the mouse kappa light chain constant region gene.

2. The hybrid antibody of claim 1, wherein the human unrearranged immunoglobulin heavy chain V, D and J gene segments are contained on a human genomic DNA fragment that is larger than 20 kb and the human unrearranged immunoglobulin kappa light chain V and J gene segments are contained on a human genomic DNA fragment that is larger than 20 kb.

3. The hybrid antibody of claim 1, wherein the human unrearranged immunoglobulin heavy chain V, D and J gene segments are contained on a human genomic DNA fragment that is larger than 100 kb and the human unrearranged immunoglobulin kappa light chain V and J gene segments are contained on a human genomic DNA fragment that is larger than 100 kb.

4. The hybrid antibody of claim 1, wherein the endogenous murine immunoglobulin enhancer Eμ

remains intact upstream of the mouse heavy chain immunoglobulin constant region gene.

5. A hybrid antibody produced by a method comprising:

a) exposing to an antigen a mouse comprising in its germline;

i. human unrearranged immunoglobulin heavy chain V, D and J gene segments, wherein the human unrearranged immunoglobulin heavy chain V, D and J gene segments in situ replace endogenous mouse immunoglobulin heavy chain V, D and J gene segments, and the human unrearranged immunoglobulin heavy chain V, D and J gene segments are operably linked to an endogenous mouse heavy chain constant region gene, wherein rearrangement of the human unrearranged immunoglobulin heavy chain V, D and J gene segments in the mouse results in a human rearranged heavy chain variable region gene linked to the endogenous mouse immunoglobulin heavy chain constant region gene; and

ii. human unrearranged immunoglobulin kappa light chain V and J gene segments, wherein the human unrearranged immunoglobulin kappa light chain V and J gene segments in situ replace endogenous mouse immunoglobulin kappa light chain V and J gene segments, and the human unrearranged immunoglobulin kappa light chain V and J gene segments are operably linked to an endogenous mouse kappa light chain constant region gene, wherein rearrangement of the human unrearranged immunoglobulin kappa light chain V and J gene segments in the mouse results in a mouse rearranged kappa light chain variable region gene linked to the endogenous mouse immunoglobulin kappa light chain constant region gene;

b) making a hybridoma from the mouse, wherein the hybridoma produces an antibody to the antigen, wherein the antibody comprises a heavy chain comprising a human heavy chain variable region encoded by the human rearranged heavy chain variable region gene and a mouse heavy chain constant region encoded by the mouse heavy chain constant region gene, and a light chain comprising a human kappa light chain variable region encoded by the human rearranged kappa light chain variable region gene and a mouse kappa light chain constant region encoded by the mouse kappa light chain constant region gene.

6. The hybrid antibody of claim 5, wherein the human unrearranged immunoglobulin heavy chain V, D and J gene segments are contained on a human genomic DNA fragment that is larger than 20 kb and the human unrearranged immunoglobulin kappa light chain V and J gene segments are contained on a human genomic DNA fragment that is larger than 20 kb.

7. The hybrid antibody of claim 5, wherein the human unrearranged immunoglobulin heavy chain V, D and J gene segments are contained on a human genomic DNA fragment that is larger than 100 kb and the human unrearranged immunoglobulin kappa light chain V and J gene segments are contained on a human genomic DNA fragment that is larger than 100 kb.

8. The hybrid antibody of claim 5, wherein the hybridoma is made from the spleen of the mouse.

9. The hybrid antibody of claim 5, wherein the endogenous murine immunoglobulin enhancer Eμ

remains intact upstream of the mouse heavy chain immunoglobulin constant region gene.