# US 2013/0160153 A1 · Humanized Light Chain Mice

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- Number: US 2013/0160153 A1
- Document ID: US20130160153A1
- Status: Published application
- Kind: A1
- Application number: 13/716,238
- Filed: 2012-12-17
- Published: 2013-06-20
- Earliest claimed: 2011-12-20
- Assignees: Regeneron Pharmaceuticals, Inc.
- Inventors: Lynn Macdonald; Cagan Gurer; Karolina A. Hosiawa; Sean Stevens; Andrew J. Murphy
- Web page: https://patentgrep.com/patent/US20130160153A1

## CPC

- A01K 67/0278: Knock-in vertebrates, e.g. humanised vertebrates
- C07K 16/2866: {against receptors for cytokines, lymphokines, interferons}
- C07K 16/462: {Igs containing a variable region (Fv) from one specie and a constant region (Fc) from another}
- C12N 9/6489: {Metalloendopeptidases (3.4.24)}
- 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
- C12N 2800/204: of bacterial origin, e.g. BAC
- C12N 2800/30: Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT
- A01K: ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Non-human animals, tissues, cells, and genetic material are provided that comprise a modification of an endogenous non-human heavy chain immunoglobulin sequence and that comprise an ADAM6 activity functional in a mouse, wherein the non-human animals express a human immunoglobulin heavy chain variable domain and a cognate human immunoglobulin λ light chain variable domain.

## Claims

### Claim 1

A non-human animal comprising
(a) an insertion of one or more human Vλ and Jλ gene segments upstream of an non-human immunoglobulin light chain constant region,
(b) an insertion of one or more human VH, one or more human DH and one or more human JH gene segments upstream of an non-human immunoglobulin heavy chain constant region, and
(c) a nucleotide sequence that encodes an ADAM6 protein or a functional fragment thereof, wherein the ADAM6 protein is expressed from an ectopic ADAM6 nucleic acid sequence.

### Claim 2 (depends on 1)

The non-human animal of claim 1, wherein the non-human heavy and/or light chain constant regions are rodent constant regions.

### Claim 3 (depends on 2)

The non-human animal of claim 2, wherein the light chain constant region is a mouse Cκ or mouse Cλ region.

### Claim 4 (depends on 1)

The non-human animal of claim 1, wherein the non-human animal comprises at least 12 to at least 40 human Vλ gene segments and at least one human Jλ gene segment.

### Claim 5 (depends on 1)

The non-human animal of claim 1, wherein the at least one human Jλ gene segment is selected from Jλ1, Jλ2, Jλ3, Jλ7 and a combination thereof.

### Claim 6 (depends on 1)

The non-human animal of claim 1, wherein the non-human comprises at least four human Jλ gene segments.

### Claim 7 (depends on 6)

The non-human animal of claim 6, wherein the at least four human Jλ gene segments comprise at least Jλ1, Jλ2, Jλ3 and Jλ7.

### Claim 8 (depends on 1)

The non-human animal of claim 1, wherein the nucleotide sequence that encodes an ADAM6 protein or functional fragment thereof is present the same location as compared to a wild-type type non-human ADAM6 locus.

### Claim 9 (depends on 1)

The non-human animal of claim 1, wherein the nucleotide sequence that encodes an ADAM6 protein or functional fragment thereof is present within immunoglobulin gene segments.

### Claim 10 (depends on 9)

The non-human animal of claim 9, wherein the immunoglobulin gene segments are heavy chain gene segments.

### Claim 11 (depends on 10)

The non-human animal of claim 10, wherein the heavy chain gene segments are human.

### Claim 12 (depends on 10)

The non-human animal of claim 10, wherein the heavy chain gene segments are endogenous heavy chain gene segments of the non-human animal.

### Claim 13 (depends on 1)

The non-human animal of claim 1, wherein the non-human animal lacks an endogenous immunoglobulin VL and/or a JL gene segment at an endogenous immunoglobulin light chain locus.

### Claim 14 (depends on 1)

The non-human animal of claim 1, wherein the non-human animal comprises endogenous immunoglobulin VL and/or JL gene segments that are incapable of rearranging to form an immunoglobulin VL domain in the non-human animal.

### Claim 15 (depends on 14)

The non-human animal of claim 14, wherein all or substantially all endogenous immunoglobulin Vκ and Jκ gene segments are replaced with one or more human Vλ and Jλ gene segments.

### Claim 16 (depends on 14)

The non-human animal of claim 14, all or substantially all endogenous immunoglobulin Vλ and Jλ gene segments are replaced with one or more human Vλ and Jλ gene segments.

### Claim 17 (depends on 1)

The non-human animal of claim 1, wherein the non-human animal further comprises a human Vκ-Jκ intergenic region from a human κ light chain locus, wherein the human Vκ-Jκ intergenic region is contiguous with the one or more human Vλ and Jλ gene segments.

### Claim 18 (depends on 17)

The non-human animal of claim 17, wherein the human Vκ-Jκ intergenic region is placed between a human Vλ gene segment and a human Jλ gene segment.

### Claim 19

A genetically modified non-human animal that comprises
(a) one or more unrearranged human Vλ gene segments and one or more unrearranged human Jλ gene segments at an endogenous immunoglobulin light chain locus of the non-human animal,
(b) on or more unrearranged human VH gene segments, one or more unrearranged human DH gene segments, and one or more unrearranged human JH gene segments at an endogenous immunoglobulin heavy chain locus of the non-human animal, wherein the non-human animal is capable of expressing an ADAM6 protein or functional fragment thereof.

### Claim 20 (depends on 1)

A cell derived from the non-human animal of claim 1.

### Claim 21 (depends on 20)

The cell of claim 20, wherein the cell is a B cell.

### Claim 22 (depends on 21)

A hybridoma made from the B cell of claim 21.

### Claim 23 (depends on 19)

A cell derived from the non-human animal of claim 19.

### Claim 24 (depends on 23)

The cell of claim 23, wherein the cell is a B cell.

### Claim 25 (depends on 24)

A hybridoma made from the B cell of claim 24.

### Claim 26 (depends on 1)

A method for making an antibody that binds to an antigen of interest is provided, wherein the method comprises
(a) exposing a non-human animal of claim 1 to an antigen of interest,
(b) isolating one or more B lymphocytes of the non-human animal, wherein the one or more B lymphocytes express an antibody that binds the antigen of interest, and
(c) identifying a nucleic acid sequence that encodes an immunoglobulin light chain of the antibody that binds that antigen of interest, wherein the immunoglobulin light chain comprises a human Vλ domain and a non-human light chain constant domain, and
(d) employing the nucleic acid sequence of (c) with a human immunoglobulin light chain constant region nucleic acid sequence to make a human antibody that binds the antigen of interest.

### Claim 27 (depends on 26)

The method of claim 26, wherein the non-human light chain constant region is a mouse Cκ or

### Claim 28 (depends on 26)

The method of claim 26, wherein the non-human light chain constant region is a mouse Cλ.

### Claim 29 (depends on 26)

The method of claim 26, wherein the antibody comprises an immunoglobulin heavy chain comprising a human VH domain and a non-human CH domain.

### Claim 30 (depends on 29)

The method of claim 29, wherein the non-human CH domain is a mouse CH domain.

### Claim 31 (depends on 26)

The method of claim 26, wherein the non-human animal further comprises a human Vκ-Jκ intergenic region from a human κ light chain locus, wherein the human Vκ-Jκ intergenic region is contiguous with the one or more human Vλ and Jλ gene segments.

### Claim 32 (depends on 31)

The method of claim 31, wherein the human Vκ-Jκ intergenic region is placed between a human Vλ gene segment and a human Jλ gene segment

### Claim 33 (depends on 26)

The method of claim 26, wherein the non-human animal is a mouse.