Mice that make VL binding proteins

• US 9,686,970 B2
• Filed: 12/19/2013
• Issued: 06/27/2017
• Est. Priority Date: 08/02/2010
• Status: Active Grant

• Current Assignee: Regeneron Pharmaceuticals Incorporated
• Original Assignee: Regeneron Pharmaceuticals Incorporated
• Inventors: Murphy, Andrew J., Macdonald, Lynn, Stevens, Sean, Gurer, Cagan, Meagher, Karolina A.
• Primary Examiner(s): LEAVITT, MARIA GOMEZ
• Application Number:
  US14/135,510
• Publication Number:
  US 20140130193A1
• Time in Patent Office: 1,286 Days
• Field of Search: 800 4, 800 6, 800 13, 800 18, 4241311
• 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) 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 first nucleotide sequence that comprises (i) a first plurality of unrearranged functional human immunoglobulin light chain variable κ

(hVκ

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

gene segments (hJκ

1-hJκ

5),

wherein the first nucleotide sequence is operably linked to an intact mouse immunoglobulin heavy chain constant (C_H) region nucleic acid sequence at the endogenous mouse immunoglobulin heavy chain locus, and

(b) a modified endogenous immunoglobulin light chain κ

locus comprising a replacement of all functional endogenous mouse immunoglobulin light chain Vκ

gene segments and all functional endogenous mouse light chain Jκ

gene segments at the endogenous mouse immunoglobulin light chain κ

locus with a second nucleotide sequence that comprises (i) a second plurality of unrearranged functional human immunoglobulin light chain variable κ

(hVκ

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

1-hJκ

5),

wherein the second nucleotide sequence is operably linked to an intact mouse immunoglobulin light chain κ

constant (Cκ

) region nucleic acid sequence at the endogenous mouse light chain κ

immunoglobulin locus;

wherein in a B cell during B cell development the first nucleotide sequence rearranges to form a first rearranged human immunoglobulin light chain variable Vκ

/Jκ

nucleotide sequence operably linked to the intact mouse immunoglobulin heavy chain constant region nucleic acid sequence at the endogenous mouse immunoglobulin heavy chain locus, and the second nucleotide sequence rearranges to form a second rearranged human immunoglobulin light chain variable region Vκ

/Jκ

nucleotide sequence operably linked to the intact mouse immunoglobulin light chain κ

constant region nucleic acid sequence at the endogenous mouse κ

light chain immunoglobulin locus, and

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

/Jκ

nucleotide sequence operably linked to the intact mouse immunoglobulin heavy chain constant region nucleic acid sequence at the endogenous mouse heavy chain immunoglobulin locus and the second rearranged human immunoglobulin light chain variable region Vκ

/Jκ

nucleotide sequence operably linked to the intact mouse immunoglobulin light chain κ

constant region nucleic acid sequence at the endogenous mouse κ

light chain immunoglobulin locus.

2. The mouse of claim 1, wherein the first nucleotide sequence comprises

(a) six hVκ

gene segments and the five hJκ

1-hκ

5 gene segments;

(b) 16 hVκ

gene segments and the five hJκ

1-hκ

5 gene segments;

(c) 30 hVκ

gene segments and the five hJκ

1-hκ

5 gene segments;

or

(d) 40 hVκ

gene segments and the five hJκ

1-hκ

5 gene segments.

3. The mouse of claim 1, wherein the first rearranged hVκ

/hJκ

gene sequence operably linked to the intact mouse C_H region nucleic acid sequence comprises at least one N addition.

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

5. A cell isolated from the mouse of claim 1, wherein the cell is a germ cell.

6. A mouse embryonic stem cell comprising

(a) 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 first nucleotide sequence that comprises (i) a first plurality of unrearranged functional human immunoglobulin light chain variable κ

(hVκ

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

gene segments (hJκ

1-hJκ

5),

wherein the first nucleotide sequence is operably linked to an intact mouse immunoglobulin heavy chain constant (C_H) region nucleic acid sequence at the endogenous mouse immunoglobulin heavy chain locus, and

(b) a modified endogenous immunoglobulin light chain κ

locus comprising a replacement of all functional endogenous mouse immunoglobulin light chain Vκ

gene segments and all functional endogenous mouse light chain Jκ

gene segments at the endogenous mouse immunoglobulin light chain κ

locus with a second nucleotide sequence that comprises (i) a second plurality of unrearranged functional human immunoglobulin light chain variable κ

(hVκ

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

1-hJκ

5),

wherein the second nucleotide sequence is operably linked to an intact mouse immunoglobulin light chain κ

constant (Cκ

) region nucleic acid sequence at the endogenous mouse light chain κ

immunoglobulin locus;

wherein in a B cell during B cell development the first nucleotide sequence rearranges to form a first rearranged human immunoglobulin light chain variable Vκ

/Jκ

nucleotide sequence operably linked to the intact mouse immunoglobulin heavy chain constant region nucleic acid sequence at the endogenous mouse immunoglobulin heavy chain locus, and the second nucleotide sequence rearranges to form a second rearranged human immunoglobulin light chain variable region Vκ

/Jκ

nucleotide sequence operably linked to the intact mouse immunoglobulin light chain constant region nucleic acid sequence at the endogenous mouse immunoglobulin light chain κ

locus.