# US 10,246,509 B2 · Restricted immunoglobulin heavy chain mice

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- Number: US 10,246,509 B2
- Document ID: US10246509B2
- Status: Granted
- Kind: B2
- Application number: 13/653,456
- Filed: 2012-10-17
- Published: 2013-04-18
- Granted: 2019-04-02
- Earliest claimed: 2011-10-17
- Assignees: Regeneron Pharmaceuticals, Inc.
- Applicants: Regeneron Pharmaceuticals, Inc.
- Inventors: Lynn Macdonald; John McWhirter; Cagan Gurer; Karolina A. Hosiawa; Andrew J. Murphy
- Web page: https://patentgrep.com/patent/US10246509B2

## CPC

- C07K 16/28: against receptors, cell surface antigens or cell surface determinants
- A01K 67/0278: Knock-in vertebrates, e.g. humanised vertebrates
- A61P 31/04: Antibacterial agents
- A61P 31/10: Antimycotics
- A61P 31/12: Antivirals
- C07K 16/00: Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
- C07K 16/461: {Igs containing Ig-regions, -domains or -residues form different species}
- C12N 15/8509: {for producing genetically modified animals, e.g. transgenic}
- A01K 2217/072: maintaining or altering function, i.e. knock in
- A01K 2217/15: Animals comprising multiple alterations of the genome, by transgenesis or homologous recombination, e.g. obtained by cross-breeding
- A01K 2227/105: Murine
- A01K 2267/01: Animal expressing industrially exogenous proteins
- C07K 2317/10: characterized by their source of isolation or production
- C07K 2317/20: characterized by taxonomic origin
- C07K 2317/21: from primates, e.g. man
- C07K 2317/24: containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
- C07K 2317/515: Complete light chain, i.e. VL + CL
- C07K 2317/52: Constant or Fc region; Isotype
- C07K 2317/56: variable (Fv) region, i.e. VH and/or VL
- C07K 2317/565: Complementarity determining region [CDR]
- C07K 2317/567: Framework region [FR]
- C12N 2800/204: of bacterial origin, e.g. BAC
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- A01K: ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Mice having a restricted immunoglobulin heavy chain locus are provided, wherein the locus is characterized by a single polymorphic human VH gene segment, a plurality of human DH gene segments and a plurality of JH gene segments. Methods for making antibody sequences that bind an antigen (e.g., a viral antigen) are provided, comprising immunizing a mouse with an antigen of interest, wherein the mouse comprises a single human VH gene segment, a plurality of human DH gene segments and a plurality of JH gene segments, at the endogenous immunoglobulin heavy chain locus.

## Claims

### Claim 1

A rat or a mouse whose germline genome comprises:
a restricted immunoglobulin heavy chain locus characterized by the presence of a single human unrearranged VH1-69 gene segment, one or more human unrearranged DH gene segments, and one or more human unrearranged JH gene segments operably linked to a non-human immunoglobulin heavy chain constant region nucleic acid sequence comprising at least a non-human IgM gene at an endogenous heavy chain locus of the rat or mouse,
wherein the restricted immunoglobulin heavy chain locus is capable of rearranging and forming a plurality of distinct rearrangements, wherein each rearrangement is derived from the single human VH1-69 gene segment, one of the human DH segments, and one of the human JH segments, and wherein each rearrangement encodes a different heavy chain variable domain, and
wherein the rat or mouse further comprises a population of mature IgMintIgDhi B cells in its spleen, each B cell comprising a distinct rearranged human heavy chain variable region gene sequence derived from the restricted immunoglobulin heavy chain locus.

### Claim 2 (depends on 1)

The rat or mouse of claim 1, wherein the rat or mouse comprises a deletion of all or substantially all endogenous VH, DH, and JH gene segments.

### Claim 3 (depends on 1)

The rat or mouse of claim 1, wherein the non-human immunoglobulin constant region nucleic acid sequence is a mouse or rat immunoglobulin heavy chain constant region nucleic acid sequence.

### Claim 4 (depends on 1)

The rat or mouse of claim 1, wherein the genome of the rat or mouse further comprises one or more human immunoglobulin VL gene segments operably linked to one or more human JL gene segments.

### Claim 5 (depends on 4)

The rat or mouse of claim 4, wherein the one or more human VL gene segments and the one or more human JL gene segments are human Vκ and human Jκ gene segments.

### Claim 6 (depends on 4)

The rat or mouse of claim 4, wherein the one or more human immunoglobulin VL gene segments and one or more human JL gene segments are operably linked to a non-human light chain constant gene.

### Claim 7 (depends on 6)

The rat or mouse of claim 6, wherein the non-human light chain constant gene is selected from a mouse κ or λ constant region gene.

### Claim 8 (depends on 1)

A cell or tissue derived from the rat or mouse of claim 1.

### Claim 9 (depends on 4)

The rat or mouse of claim 4, wherein the one or more human VL gene segments and the one or more human JL gene segments are human Vλ and human Jλ, gene segments.

### Claim 10 (depends on 1)

The rat or mouse of claim 1, wherein the single human unrearranged VH gene segment comprises a plurality of polymorphs of the VH1-69 segment.

### Claim 11 (depends on 1)

The rat or mouse of claim 1, wherein the single human unrearranged VH gene segment consists of a single allele of the VH1-69 segment.

### Claim 12 (depends on 1)

The rat or mouse of claim 1, wherein the rat or mouse comprises a higher ratio of mature IgMintIgDhi B cells to immature IgDintIgMhi B cells in the spleen as compared to a control ratio of mature IgMintIgDhi B cells to immature IgDintIgMhi B cells in the spleen of a control rat or mouse comprising a plurality of human VH gene segments.

### Claim 13 (depends on 1)

The rat or mouse of claim 1, wherein the rat or mouse comprises at least 1×107 IgMintIgDhi B cells in its spleen.

### Claim 14 (depends on 1)

The rat or mouse of claim 1, wherein the number of CD19+cells in the population of mature IgMintIgDhi B cells in the spleen is within an order of magnitude of the number of CD19+cells in a population of mature IgMintIgDhi B cells in a spleen of a control rat or mouse comprising a plurality of human VH gene segments.

### Claim 15

A mouse whose germline genome comprises:
a replacement at an endogenous immunoglobulin heavy chain locus of all or substantially all endogenous VH, DH, and JH gene segments with a single human unrearranged VH1-69 gene segment, one or more human unrearranged DH gene segments, and one or more human unrearranged JH gene segments operably linked to an endogenous immunoglobulin heavy chain constant region nucleic acid sequence comprising at least an endogenous IgM gene at an endogenous heavy chain locus of the mouse,
wherein the restricted immunoglobulin heavy chain locus is capable of rearranging and forming a plurality of distinct rearrangements, wherein each rearrangement is derived from the single human VH1-69 gene segment, one of the human DH segments, and one of the human JH segments, and wherein each rearrangement encodes a different heavy chain variable domain, and
wherein the mouse further comprises a population of mature IgMintIgDhi B cells in the spleen, each B cell comprising a distinct rearranged human heavy chain variable region gene sequence derived from the single human unrearranged VH gene segment, the one or more human unrearranged DH, and the one or more human unrearranged JH gene segments.

### Claim 16 (depends on 15)

The mouse of claim 15, wherein the genome of the mouse further comprises:
a replacement at an endogenous immunoglobulin light chain locus of all or substantially all endogenous VL and JL gene segments with one or more human VL and one or more human JL gene segments, wherein the human VL and JL gene segments are operably linked with a non-human light chain constant region nucleic acid sequence.

### Claim 17 (depends on 16)

The mouse of claim 16, wherein the human VL and JL gene segments are human Vκand Jκgene segments which replace endogenous Vκand Jκgene segments, and wherein the human Vκand Jκgene segments are operably linked to a mouse Cκregion nucleic acid sequence.

### Claim 18 (depends on 16)

The mouse of claim 16, wherein the human VL and JL gene segments are human Vλand Jλgene segments which replace endogenous Vλand Jλgene segments, and wherein the human Vλand Jλgene segments are operably linked to a mouse Cλregion nucleic acid sequence.

### Claim 19 (depends on 15)

The mouse of claim 15, wherein the rat or mouse comprises a higher ratio of mature IgMintIgDhi B cells to immature IgDintIgMhi B cells in the spleen as compared to a control ratio of mature IgMintIgDhi B cells to immature IgDintIgMhi B cells in the spleen of a control mouse comprising a plurality of human VH gene segments.

### Claim 20 (depends on 15)

The mouse of claim 15, wherein the single human unrearranged VH gene segment comprises a plurality of polymorphs of the VH1-69 segment.

### Claim 21 (depends on 15)

The mouse of claim 15, wherein the single human unrearranged VH gene segment consists of a single allele of the VH1-69 segment.

### Claim 22 (depends on 15)

The mouse of claim 15, wherein the number of CD19+cells in the population of mature IgMintIgDhi B cells in the spleen is within an order of magnitude of the number of CD19+cells in a population of mature IgMintIgDhi B cells in a spleen of a control mouse comprising a plurality of human VH gene segments.

### Claim 23 (depends on 15)

The mouse of claim 15, wherein the rat or mouse comprises a higher ratio of mature IgMintIgDhi B cells to immature IgDintIgMhi B cells in the spleen as compared to a control ratio of mature IgMintIgDhi B cells to immature IgDintIgMhi B cells in the spleen of a control mouse comprising a plurality of human VH gene segments.

### Claim 24 (depends on 15)

The mouse of claim 15, wherein the mouse comprises at least 1×107 IgMintIgDhi B cells in its spleen.