# US 11,111,314 B2 · Non-human animals that select for light chain variable regions that bind antigen

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- Number: US 11,111,314 B2
- Document ID: US11111314B2
- Status: Granted
- Kind: B2
- Application number: 15/559,358
- Filed: 2016-03-18
- Published: 2018-08-30
- Granted: 2021-09-07
- Earliest claimed: 2015-03-19
- Assignees: Regeneron Pharmaceuticals, Inc.
- Applicants: Regeneron Pharmaceuticals, Inc.
- Inventors: Lynn Macdonald; Andrew J. Murphy; Cagan Gurer; Robert Babb
- Web page: https://patentgrep.com/patent/US11111314B2

## CPC

- C07K 16/468: {Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies}
- A01K 67/0278: Knock-in vertebrates, e.g. humanised vertebrates
- C07K 16/00: Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
- C07K 16/28: against receptors, cell surface antigens or cell surface determinants
- C07K 16/462: {Igs containing a variable region (Fv) from one specie and a constant region (Fc) from another}
- 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 2317/21: from primates, e.g. man
- C07K 2317/24: containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
- C07K 2317/31: multispecific
- C07K 2317/51: Complete heavy chain or Fd fragment, i.e. VH + CH1
- C07K 2317/515: Complete light chain, i.e. VL + CL
- C07K 2317/52: Constant or Fc region; Isotype
- C07K 2317/522: CH1 domain
- C07K 2317/524: CH2 domain
- C07K 2317/526: CH3 domain
- C07K 2317/528: CH4 domain
- C07K 2317/53: Hinge
- C07K 2317/55: Fab or Fab'
- 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]
- C07K 2317/569: Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
- C07K 2317/622: Single chain antibody (scFv)
- C07K 2317/92: Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
- 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

## Abstract

Non-human animals, cells, methods and compositions for making and using the same are provided, wherein the non-human animals and cells comprise an immunoglobulin heavy chain locus that includes unrearranged human immunoglobulin light chain gene segments and an immunoglobulin light chain locus that includes a single rearranged human light chain variable region nucleotide sequence. The unrearranged human light chain gene segments may be operably linked to a heavy chain constant region nucleotide sequence and the rearranged human immunoglobulin light chain variable region nucleotide sequence may be operably linked to a light chain constant region nucleotide sequence. Also provided are methods for obtaining nucleic acid sequences that encode immunoglobulin light chain variable domains capable of binding an antigen in the absence of a cognate variable domain, and expressing such nucleic acid sequences in a host cell, e.g., to generate a multispecific antigen-binding protein.

## Claims

### Claim 1

A mouse comprising in its germline genome:
(i) at an endogenous immunoglobulin (Ig) heavy chain locus, an unrearranged human Ig light chain variable kappa (Vκ) gene segment and an unrearranged human Ig light chain joining kappa (Jκ) gene segment operably linked to an endogenous Ig heavy chain constant region nucleic acid sequence comprising at least one intact Ig heavy chain constant region gene encoding a functional CH1 domain, wherein the at least one intact Ig heavy chain constant region gene is an Igμ gene, Igδ gene, Igγ gene, Igα gene or an Igε gene, wherein the unrearranged human Ig Vκ gene segment and the unrearranged human Ig Jκ gene segment rearrange in a B cell to form a hybrid sequence comprising a rearranged human Ig Vκ/Jκ gene sequence operably linked to the endogenous Ig heavy κ chain constant region nucleic acid sequence;
(ii) at an endogenous Ig light chain κ locus, a human universal Ig light chain variable region nucleotide sequence comprising a single human Ig Vκ gene segment rearranged with a single human Ig Jκ gene segment operably linked to an endogenous Ig light chain κ constant region nucleic acid sequence;
wherein the mouse expresses an antigen-binding protein that comprises a human Ig hybrid chain derived from the rearranged human Ig Vκ/Jκ gene sequence operably linked to the endogenous Ig heavy chain constant region nucleic acid sequence and a cognate light chain derived from the human universal Ig light chain variable region nucleotide sequence at the endogenous Ig light chain locus, wherein the human Ig hybrid chain comprises a human Ig light chain variable κ (hVκ/CHxULC) domain fused to an endogenous heavy chain constant IgM, IgD, IgG, IgE or IgA region comprising a functional CH1 domain, and wherein the cognate light chain comprises a human Ig light variable κ domain chain fused to an endogenous light chain κ constant domain.

### Claim 2 (depends on 1)

A B cell expressing the antigen-binding protein obtained from the mouse of claim 1.

### Claim 3 (depends on 1)

A method of making the mouse of claim 1, comprising modifying a genome of a mouse embryonic stem (ES) cell to comprise
(i) at an endogenous Ig heavy chain locus, an unrearranged human Ig Vκ gene segment and an unrearranged human Ig Jκ gene segment operably linked to an endogenous Ig heavy chain constant region nucleic acid sequence comprising at least one intact Ig heavy chain constant region gene encoding a functional CH1 domain, wherein the intact Ig heavy chain constant region gene is an Igμ gene, an Igδ gene, an Igγ gene, an Igα gene, or an Igε gene,
wherein the unrearranged human Ig Vκ gene segment and the unrearranged human Ig Jκ gene segment rearrange in a B cell to form a hybrid sequence comprising a rearranged human Ig Vκ/Jκ gene sequence operably linked to the endogenous Ig heavy chain constant region nucleic acid sequence; and
(ii) at an endogenous Ig light chain κ locus, a human universal Ig light chain variable region nucleotide sequence comprising a single human Ig Vκ gene segment rearranged with a single human Ig Jκ gene segment operably linked to an endogenous Ig light chain κ constant region nucleic acid sequence.

### Claim 4 (depends on 3)

The method of claim 3, wherein modifying the genome of the mouse ES cell comprises
(i) replacing endogenous Ig VH gene segments, endogenous Ig DH gene segments and endogenous Ig JH gene segments at the endogenous Ig heavy chain locus with the unrearranged human Ig Vκ gene segment and Jκ gene segment, and
(ii) replacing endogenous Ig Vκ gene segments and Jκ gene segments at the endogenous Ig light chain κ locus with the human universal Ig light chain variable region nucleotide sequence.

### Claim 5 (depends on 1)

A method of obtaining an hVκ/CHxULC domain or a nucleic acid encoding the hVκ/CHxULC domain, the method comprising
isolating from the mouse of claim 1 a cell expressing the nucleic acid that encodes the hVκ/CHxULC domain,
and obtaining from the cell the hVκ/CHxULC domain or the nucleic acid encoding the hVκ/CHxULC domain.

### Claim 6 (depends on 1)

The mouse of claim 1, wherein the unrearranged human Ig Vκ gene segment and the unrearranged human Ig Jκ gene segment comprises a plurality of unrearranged human Ig Vκ gene segments and a plurality of unrearranged human Ig Jκ gene segments, respectively, and wherein the pluralities of unrearranged human Ig Vκ ene segments and unrearranged human Ig Jκ gene segments replace endogenous Ig VH gene segments, endogenous Ig DH gene segments, and endogenous Ig JH gene segments at the endogenous Ig heavy chain locus.

### Claim 7 (depends on 1)

The mouse of claim 1, wherein the unrearranged human Ig Vκ gene segment and the unrearranged human Ig Jκ gene segment comprises a plurality of unrearranged human Ig Vκ gene segments and all unrearranged human Ig Jκ gene segments, respectively, and wherein the plurality of unrearranged human Ig Vκ gene segments and all unrearranged human Ig Jκ gene segments replace endogenous Ig VH gene segments, endogenous Ig DH gene segments, and endogenous Ig JH gene segments at the endogenous Ig heavy chain locus.

### Claim 8 (depends on 1)

The mouse of claim 1, wherein the unrearranged human Ig Vκ gene segment and the unrearranged human Ig Jκ gene segment comprises at least 40 unrearranged human Ig Vκ gene segments and all unrearranged human Ig Jκ gene segments, respectively, and wherein the at least 40 unrearranged human Ig Vκ gene segments and all unrearranged human Ig Jκ gene segments replace endogenous Ig VH gene segments, endogenous Ig DH gene segments, and endogenous Ig JH gene segments at the endogenous Ig heavy chain locus.

### Claim 9 (depends on 1)

The mouse of claim 1, wherein the human universal Ig light chain variable region nucleotide sequence is a rearranged Vκ1-39/Jκ gene sequence or a rearranged Vκ3-20/Jκ gene sequence.

### Claim 10 (depends on 1)

The mouse of claim 1,
wherein endogenous Ig Vκ gene segments and/or endogenous Ig Jκ gene segments at the endogenous Ig light chain κ locus are replaced with the human universal Ig light chain variable region nucleotide sequence.

### Claim 11 (depends on 10)

The mouse of claim 10, wherein the human universal Ig light chain variable region nucleotide sequence is a rearranged Vκ1-39/Jκ gene sequence or a rearranged Vκ3-20/Jκ gene sequence.

### Claim 12 (depends on 8)

The mouse of claim 8, wherein endogenous Ig Vκ gene segments and/or endogenous Ig Jκ gene segments at the endogenous Ig light chain κ locus are replaced with the human universal Ig light chain variable region nucleotide sequence.

### Claim 13 (depends on 12)

The mouse of claim 12, wherein the human universal Ig light chain variable region nucleotide sequence is a rearranged Vκ1-39/Jκ gene sequence or a rearranged Vκ3-20/Jκ gene sequence.

### Claim 14 (depends on 4)

The method of claim 4, comprising replacing endogenous Ig VH gene segments, endogenous Ig DH gene segments, and endogenous Ig JH gene segments at the endogenous Ig heavy chain locus with a plurality of unrearranged human Ig Vκ gene segments and a plurality of unrearranged human Ig Jκ gene segments.

### Claim 15 (depends on 4)

The method of claim 4, comprising replacing endogenous Ig VH gene segments, endogenous Ig DH gene segments, and endogenous Ig JH gene segments at the endogenous Ig heavy chain locus with a plurality of unrearranged human Ig Vκ gene segments and all unrearranged human Ig Jκ gene segments.

### Claim 16 (depends on 4)

The method of claim 4, comprising replacing endogenous Ig VH gene segments, endogenous Ig DH gene segments, and endogenous Ig JH gene segments at the endogenous Ig heavy chain locus with at least 40 unrearranged human Ig Vκ gene segments and all unrearranged human Ig Jκ gene segments.

### Claim 17 (depends on 4)

The method of claim 4, wherein the human universal Ig light chain variable region nucleotide sequence is a rearranged Vκ1-39/Jκ gene sequence or a rearranged Vκ3-20/Jκ gene sequence.

### Claim 18 (depends on 5)

The method of claim 5, wherein the unrearranged human Ig Vκ gene segment and the unrearranged human Jκ gene segment of the mouse comprises a plurality of unrearranged human Ig Vκ gene segments and a plurality of unrearranged human Ig Jκ gene segments, respectively, and wherein the pluralities of unrearranged human Ig Vκ gene segments and unrearranged human Ig Jκ gene segments replace endogenous Ig VH gene segments, endogenous Ig DH gene segments, and endogenous Ig JH gene segments at the endogenous Ig heavy chain locus.

### Claim 19 (depends on 5)

The method of claim 5, wherein the unrearranged human Ig Vκ gene segment and the unrearranged human Jκ gene segment of the mouse comprises a plurality of unrearranged human Ig Vκ gene segments and all unrearranged human Ig Jκ gene segments, respectively, and wherein the plurality of unrearranged human Ig Vκ gene segments and all unrearranged human Ig Jκ gene segments replace endogenous Ig VH gene segments, endogenous Ig DH gene segments, and endogenous Ig JH gene segments at the endogenous Ig heavy chain locus.

### Claim 20 (depends on 5)

The method of claim 5, wherein the unrearranged human Ig Vκ gene segment and the unrearranged human Jκ gene segment of the mouse comprises at least 40 unrearranged human Ig Vκ gene segments and all unrearranged human Ig Jκ gene segments, respectively, and wherein the at least 40 unrearranged human Ig Vκ gene segments and all unrearranged human Ig Jκ gene segments replace endogenous Ig VH gene segments, endogenous Ig DH gene segments, and endogenous Ig JH gene segments at the endogenous Ig heavy chain locus.

### Claim 21 (depends on 5)

The method of claim 5, wherein the human universal Ig light chain variable region nucleotide sequence of the mouse is a rearranged Vκ1-39/Jκ gene sequence or a rearranged Vκ3-20/Jκ gene sequence.

### Claim 22 (depends on 5)

The method of claim 5,
wherein endogenous Ig Vκ gene segments and/or endogenous Ig Jκ gene segments at the endogenous Ig light chain κ locus are replaced with the human universal Ig light chain variable region nucleotide sequence.

### Claim 23 (depends on 22)

The method of claim 22, wherein the human universal Ig light chain variable region nucleotide sequence of the mouse is a rearranged Vκ1-39/Jκ gene sequence or a rearranged Vκ3-20/Jκ gene sequence.

### Claim 24 (depends on 20)

The method of claim 20, wherein endogenous Ig Vκ gene segments and/or endogenous Ig Jκ gene segments at the endogenous Ig light chain κ locus are replaced with the human universal Ig light chain variable region nucleotide sequence.

### Claim 25 (depends on 24)

The mouse of claim 24, wherein the human universal Ig light chain variable region nucleotide sequence of the mouse is a rearranged Vκ1-39/Jκ gene sequence or a rearranged Vκ3-20/Jκ gene sequence.