Mice that make VL binding proteins

• US 9,516,868 B2
• Filed: 08/02/2011
• Issued: 12/13/2016
• Est. Priority Date: 08/02/2010
• Status: Active Grant

• Current Assignee: Regeneron Pharmaceuticals Incorporated
• Original Assignee: Regeneron Pharmaceuticals Incorporated
• Inventors: Hosiawa, Karolina A., Macdonald, Lynn, Stevens, Sean, Gurer, Cagan, Murphy, Andrew J.
• Primary Examiner(s): LEAVITT, MARIA GOMEZ
• Application Number:
  US13/195,951
• Publication Number:
  US 20120096572A1
• Time in Patent Office: 1,960 Days
• Field of Search: 800/8, 800/13, 800/4, 800/61
• US Class Current: 1/1
• CPC Class Codes: A01K 2217/072 maintaining or altering function, i.e. knock in
• 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 16/082 Hepadnaviridae, e.g. hepatitis B virus
• CPC Class Codes: C07K 16/461 Igs containing Ig-regions, -domains or -residues form different species
• CPC Class Codes: C07K 16/468 Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
• CPC Class Codes: C07K 2317/14 Specific host cells or culture conditions, e.g. components, pH or temperature
• CPC Class Codes: C07K 2317/21 from primates, e.g. man
• CPC Class Codes: C07K 2317/31 multispecific
• CPC Class Codes: C07K 2317/56 variable (Fv) region, i.e. VH and/or VL
• CPC Class Codes: C07K 2317/92 Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
• CPC Class Codes: C12N 15/8509 for producing genetically modified animals, e.g. transgenic
• CPC Class Codes: C12N 2015/8518 expressing industrially exogenous proteins, e.g. for pharmaceutical use, human insulin, blood factors, immunoglobulins, pseudoparticles

Abstract

Genetically modified mice and methods for making an using them are provided, wherein the mice comprise a replacement of all or substantially all immunoglobulin heavy chain V gene segments, D gene segments, and J gene segments with at least one light chain V gene segment and at least one light chain J gene segment. Mice that make binding proteins that comprise a light chain variable domain operably linked to a heavy chain constant region are provided. Binding proteins that contain an immunoglobulin light chain variable domain, including a somatically hypermutated light chain variable domain, fused with a heavy chain constant region, are provided. Modified cells, embryos, and mice that encode sequences for making the binding proteins are provided.

Claims

1. A mouse whose germline genome comprises a modified endogenous mouse immunoglobulin heavy chain locus comprising a replacement of all functional endogenous mouse immunoglobulin heavy chain variable (V_H) gene segments, all functional endogenous mouse immunoglobulin heavy chain diversity (D_H) gene segments and all functional endogenous mouse immunoglobulin heavy chain joining (J_H) gene segments at the endogenous mouse immunoglobulin heavy chain locus with a nucleotide sequence that comprises a plurality of contiguous unrearranged functional human immunoglobulin light chain variable V_κ

(hV_κ

) gene segments and all five unrearranged functional human immunoglobulin light chain joining gene segments (hJ_κ

1-hJ_κ

5),

wherein the plurality of unrearranged functional hV_κ

gene segments and the five unrearranged functional human immunoglobulin light chain joining gene segments (hJ_κ

1-hJ_κ

5) are operably linked to an intact endogenous mouse immunoglobulin heavy chain constant region at the endogenous mouse immunoglobulin heavy chain locus,

wherein the plurality of unrearranged functional hV_κ

gene segments and the five unrearranged functional human immunoglobulin light chain joining gene segments (hJ_κ

1-hJ_κ

5) rearrange in a B cell during B cell development to form a rearranged human immunoglobulin light chain variable region V_κ

/J_κ

nucleotide sequence operably linked to the endogenous mouse immunoglobulin heavy chain constant region at the endogenous mouse immunoglobulin heavy chain locus, and

wherein the mouse comprises a CD19⁺ B cell comprising the rearranged human immunoglobulin light chain variable region V_κ

/J_κ

nucleotide sequence operably linked to the endogenous mouse immunoglobulin heavy chain constant region at the endogenous mouse immunoglobulin heavy chain locus.

2. The mouse of claim 1, wherein all the functional endogenous mouse V_H, D_H, and J_H gene segments are replaced with at least 6 human V_κ

gene segments and the five unrearranged functional human immunoglobulin light chain joining gene segments (hJ_κ

1-hJ_κ

5).

3. The mouse of claim 1, wherein all the functional endogenous mouse V_H, D_H, and J_H gene segments are replaced with at least 16 human V_κ

gene segments and the five contiguous unrearranged human immunoglobulin light chain joining gene segments (hJ_κ

1-hJ_κ

5).

4. The mouse of claim 1, wherein all the functional endogenous mouse V_H, D_H, and J_H gene segments are replaced with at least 30 human V_κ

gene segments and the five contiguous unrearranged human immunoglobulin light chain joining gene segments (hJ_κ

1-hJ_κ

5).

5. The mouse of claim 1, wherein all the functional endogenous mouse V_H, D_H, and J_H gene segments are replaced with at least 40 human V_κ

gene segments and the five contiguous unrearranged human immunoglobulin light chain joining gene segments (hJ_κ

1-hJ_κ

5).

6. An isolated cell from the mouse of claim 1, wherein the cell comprises a modified endogenous mouse immunoglobulin heavy chain locus comprising a replacement of all functional endogenous mouse immunoglobulin heavy chain variable (V_H) gene segments, all functional endogenous mouse immunoglobulin heavy chain diversity (D_H) gene segments and all functional endogenous mouse immunoglobulin heavy chain joining (J_H) gene segments at the endogenous mouse immunoglobulin heavy chain locus with a nucleotide sequence that comprises a plurality of unrearranged functional human immunoglobulin light chain variable Vκ

(hV_κ

) gene segments and all five unrearranged functional human immunoglobulin light chain joining gene segments (hJ_κ

1-hJ_κ

5),

wherein the plurality of unrearranged functional hV_κ

gene segments and all five unrearranged functional human immunoglobulin light chain joining gene segments (hJ_κ

1-hJ_κ

5) are operably linked to an intact endogenous mouse immunoglobulin heavy chain constant region at the endogenous mouse immunoglobulin heavy chain locus,

wherein the plurality of unrearranged functional hV_κ

gene segments and the five unrearranged functional human immunoglobulin light chain joining gene segments (hJ_κ

1-hJ_κ

5) are capable of rearranging in a B cell during B cell development to form a rearranged human immunoglobulin light chain variable region V_κ

/J_κ

nucleotide sequence operably linked to the endogenous mouse immunoglobulin heavy chain constant region at the endogenous mouse immunoglobulin heavy chain locus.

7. The isolated cell of claim 6, wherein the cell is an embryonic stem cell.

8. A cell isolated from the mouse of claim 1, wherein the cell is the CD19⁺ B cell.

9. A hybridoma comprising a myeloma cell line fused with the CD19⁺ B cell of claim 8, wherein the hybridoma produces a polypeptide encoded by the rearranged human immunoglobulin light chain variable region V_κ

/J_κ

nucleotide sequence operably linked to the endogenous mouse immunoglobulin heavy chain constant region.

10. The mouse of claim 1, wherein the rearranged human immunoglobulin light chain variable region V_κ

/J_κ

gene sequence comprises at least one N addition.

11. The mouse of claim 1, wherein the mouse is homozygous or heterozygous for the modified endogenous immunoglobulin heavy chain locus.