# US 2016/0249592 A1 · Animal Models and Therapeutic Molecules

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- Number: US 2016/0249592 A1
- Document ID: US20160249592A1
- Status: Published application
- Kind: A1
- Application number: 15/095,315
- Filed: 2016-04-11
- Published: 2016-09-01
- Earliest claimed: 2012-03-28
- Assignees: Kymab Limited
- Applicants: Kymab Limited
- Inventors: Allan BRADLEY; E-Chiang LEE; Qi LIANG; Wei WANG; Dominik SPENSBERGER; Hui LIU; Jasper CLUBE
- Web page: https://patentgrep.com/patent/US20160249592A1

## CPC

- C07K 16/462: {Igs containing a variable region (Fv) from one specie and a constant region (Fc) from another}
- A01K 67/0278: Knock-in vertebrates, e.g. humanised vertebrates
- C07K 16/00: Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
- C07K 16/1239: {from Vibrionaceae (G)}
- C07K 16/1282: Clostridium (G)
- C07K 16/18: against material from animals or humans
- C07K 16/46: Hybrid immunoglobulins (hybrids of an immunoglobulin with a peptide not being an immunoglobulin C07K19/00)
- C12N 15/85: for animal cells
- C12N 15/8509: {for producing genetically modified animals, e.g. transgenic}
- A01K 2217/052: inducing gain of function
- A01K 2217/072: maintaining or altering function, i.e. knock in
- A01K 2217/075: inducing loss of function, i.e. knock out
- 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 2317/14: Specific host cells or culture conditions, e.g. components, pH or temperature
- 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 2317/51: Complete heavy chain or Fd fragment, i.e. VH + CH1
- C07K 2317/56: variable (Fv) region, i.e. VH and/or VL
- C07K 2317/92: Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
- C12N 2015/8518: {expressing industrially exogenous proteins, e.g. for pharmaceutical use, human insulin, blood factors, immunoglobulins, pseudoparticles}
- 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
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The invention discloses methods for the generation of chimaeric human—non-human antibodies and chimaeric antibody chains, antibodies and antibody chains so produced, and derivatives thereof including fully humanised antibodies; compositions comprising said antibodies, antibody chains and derivatives, as well as cells, non-human mammals and vectors, suitable for use in said methods.

## Claims

### Claim 1

A mouse having a genome comprising a homozygous recombinant immunoglobulin light chain locus,
said homozygous recombinant immunoglobulin light chain locus comprising unrearranged human V light chain gene segments positioned (i) at an endogenous mouse immunoglobulin light chain locus comprising an endogenous light chain enhancer and (ii) upstream of a constant region,
said homozygous recombinant light chain locus being functional to rearrange to express an Ig light chain comprising a human V region,
wherein said homozygous recombinant immunoglobulin light chain locus comprises human Vλ gene segments and Jλ gene segments operatively linked to an endogenous kappa light chain enhancer at an endogenous kappa locus,
wherein at least 80% of the immunoglobulin light chains that comprise λ variable regions expressed in said mouse comprise human λ variable regions,
wherein said mouse comprises IgG comprising human Vλ,
wherein said human Vλ and Jλ gene segments comprise at least the functional V and J gene segments from Vλ2-18 to Cλ7 of a human λ light chain locus,
wherein said mouse expresses endogenous λ light chain; and
wherein said mouse comprises B cells expressing an immunoglobulin chain comprising a human Vλ and B cells comprising an immunoglobulin chain comprising an endogenous Vλ and among said human and endogenous Vλ said human Vλ predominates.

### Claim 2 (depends on 1)

The mouse of claim 1, wherein said unrearranged human V light chain gene segments comprise a 3′ human V segment which is positioned within 100 kb of said endogenous light chain enhancer.

### Claim 3 (depends on 1)

The mouse of claim 1, wherein said endogenous light chain enhancer is in germline order relative to an endogenous constant region.

### Claim 4 (depends on 1)

The mouse of claim 1, wherein said endogenous kappa light chain enhancer comprises an iEκ or 3′ Ex enhancer or both.

### Claim 5 (depends on 1)

The mouse of claim 1, wherein said genome of said mouse further comprises a recombinant heavy chain locus, said heavy chain locus comprising one or more unrearranged human VH segments, one or more human DH segments and one or more human JH segments positioned upstream of a rodent IgH constant region comprising at least rodent Cμ and Cγ gene segments, wherein said recombinant heavy chain locus is functional to undergo class switching to produce a gamma heavy chain.

### Claim 6 (depends on 5)

The mouse of claim 5, wherein said mouse comprises antigen-specific antibodies comprising said human Vλ regions, wherein said a said antigen-specific antibody comprises an affinity of less than 50 nanomolar as determined by surface plasmon resonance.

### Claim 7 (depends on 1)

The mouse of claim 1, wherein the recombinant immunoglobulin light chain locus comprising human Vλ gene segments and Jλ gene segments is positioned upstream of an endogenous constant kappa region.

### Claim 8

A mouse having a genome comprising a homozygous recombinant immunoglobulin light chain locus,
said homozygous recombinant immunoglobulin light chain locus comprising unrearranged human V light chain gene segments positioned (i) at an endogenous mouse immunoglobulin light chain locus comprising an endogenous light chain enhancer and (ii) upstream of a constant region,
said homozygous recombinant light chain locus being functional to rearrange to express an Ig light chain comprising a human V region,
wherein said homozygous recombinant immunoglobulin light chain locus comprises human Vλ gene segments and Jλ gene segments operatively linked to an endogenous kappa light chain enhancer at an endogenous kappa locus,
wherein 80% of total splenic B cells comprise immunoglobulin light chains that comprise λ variable regions expressed in said mouse comprise human λ variable regions, and wherein said mouse comprises IgG comprising human Vλ,
wherein said human Vλ and Jλ gene segments comprise at least the functional V and J gene segments from Vλ2-18 to Cλ7 of a human λ light chain locus,
wherein said mouse expresses endogenous λ light chain;
wherein said mouse comprises B cells expressing an immunoglobulin chain comprising a human Vλ and B cells comprising an immunoglobulin chain comprising an endogenous Vλ, and among said human and endogenous Vλ said human Vλ predominates,
wherein the recombinant immunoglobulin light chain locus comprising human Vλ gene segments and Jλ gene segments is positioned upstream of an endogenous constant kappa region.

### Claim 9 (depends on 1)

The mouse of claim 1, wherein said IgG comprising human Vλ regions comprises antigen specific human Vλ.

### Claim 10 (depends on 1)

The mouse of claim 1, wherein said IgG comprising human Vλ regions comprises a repertoire of human λ variable regions.

### Claim 11 (depends on 5)

The mouse of claim 5, said recombinant locus being positioned at an endogenous heavy chain locus.

### Claim 12 (depends on 6)

The mouse of claim 6, wherein said antigen specific antibody comprises a γ constant region.

### Claim 13 (depends on 1)

The mouse of claim 1, wherein said mouse produces subsequent generation mice comprising unrearranged human Vλ and Jλ light chain gene segments positioned (i) at an endogenous mouse immunoglobulin light chain locus comprising an endogenous light chain enhancer and (ii) upstream of a constant region, said recombinant light chain locus being functional to rearrange to express an Ig light chain comprising a human Vλ region.

### Claim 14 (depends on 1)

The mouse of claim 1, wherein endogenous VK expression is inactive.

### Claim 15 (depends on 1)

The mouse of claim 1, wherein said mouse expresses λ immunoglobulin light chains comprising a plurality of a human λ variable regions encoded by a human Vλ and Jλ gene segments, and wherein said human Vλ gene segments comprise VAλ3 1, Vλ2-18, Vλ3-16, V2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, and Vλ4-3; and wherein said human Vλ and Jλ gene segments are included in said human λ locus DNA.