Animal models and therapeutic molecules

• US 9,445,581 B2
• Filed: 03/28/2012
• Issued: 09/20/2016
• Est. Priority Date: 03/28/2012
• Status: Active Grant

• Current Assignee: Kymab Limited
• Original Assignee: Kymab Limited
• Inventors: Bradley, Allan, Lee, E-Chiang, Liang, Qi, Wang, Wei, Spensberger, Dominik, Liu, Hui, Clube, Jasper
• Primary Examiner(s): Chen, Shin Lin
• Application Number:
  US13/433,084
• Publication Number:
  US 20130263293A1
• Time in Patent Office: 1,637 Days
• Field of Search: 800/6, 800/18, 435/328, 530/387.3
• US Class Current: 1/1
• CPC Class Codes: A01K 2217/052 inducing gain of function
• CPC Class Codes: A01K 2217/072 maintaining or altering function, i.e. knock in
• CPC Class Codes: A01K 2217/075 inducing loss of function, i.e. knock out
• CPC Class Codes: A01K 2217/15 Animals comprising multiple alterations of the genome, by transgenesis or homologous recombination, e.g. obtained by cross-breeding
• CPC Class Codes: A01K 2227/105 Murine
• CPC Class Codes: A01K 2267/01 Animal expressing industrially exogenous proteins
• 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 16/1239 from Vibrionaceae (G)
• CPC Class Codes: C07K 16/1282 from Clostridium (G)
• CPC Class Codes: C07K 16/18 against material from animals or humans
• CPC Class Codes: C07K 16/46 Hybrid immunoglobulins hybrids of an immunoglobulin with a peptide not being an immunoglobulin C07K19/00
• CPC Class Codes: C07K 16/462 Igs containing a variable region (Fv) from one specie and a constant region (Fc) from another
• 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 2317/24 containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• CPC Class Codes: C07K 2317/51 Complete heavy chain or Fd fragment, i.e. VH + CH1
• 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 15/85 for animal cells
• 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

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 humanized antibodies; compositions comprising the disclosed antibodies, antibody chains and derivatives, as well as cells, non-human mammals and vectors, suitable for use in the disclosed methods.

Claims

1. A mouse whose 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,

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 Vλ

2-18 to Cλ

7 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, wherein B cells of said mouse comprise immunoglobulin comprising human λ

variable region relative to immunoglobulin comprising mouse λ

variable region at a ratio of 80;

15.

2. A mouse whose 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,

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 Vλ

2-18 to Cλ

7 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, and wherein 80% of total splenic B cells of said mouse comprise human λ

variable regions.

3. A mouse whose 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,

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 Vλ

2-18 to Cλ

7 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, and wherein 84% of light chains of total splenic B cells of said mouse comprise human λ

variable regions.

4. A mouse whose 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,

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 Vλ

2-18 to Cλ

7 of a human v light chain locus,

wherein said mouse exhibits relative usage of Vλ

gene segments in splenic B cells typical of the relative usage of Vλ

gene segments in splenic B cells of humans.

5. The mouse of claim 4, 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.

6. The mouse of claim 4, wherein said endogenous light chain enhancer is in germline order relative to an endogenous constant region.

7. The mouse of claim 4, wherein said endogenous kappa enhancer comprises an iEK or 3′

EK enhancer or both.

8. The mouse of claim 4, 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, wherein the endogenous lambda loci are intact, wherein said mouse does not comprise endogenous kappa chain.

9. The mouse of claim 8, wherein B cells of said mouse comprise immunoglobulin comprising human λ

constant region relative to immunoglobulin comprising mouse λ

constant region at a ratio of 80;

15.

10. The mouse of claim 8, wherein 80% of total splenic B cells of said mouse comprise human λ

variable regions.

11. The mouse of claim 8, wherein 84% of light chains of total splenic B cells of said mouse comprise human λ

variable regions.

12. The mouse of claim 4, wherein said IgG comprising human Vλ

regions comprises antigen specific human Vλ

.

13. The mouse of claim 4, wherein said IgG comprising human Vλ

regions comprises a repertoire of human λ

variable regions.

14. The mouse of claim 4, 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 V region.

15. The mouse of claim 4, wherein endogenous VK expression is inactive,

wherein said mouse expresses λ

immunoglobulin light chains comprising a plurality of human λ

variable regions encoded by human Vλ and

Jλ

gene segments,

wherein said human Vλ

comprises VA3 1, Vλ

2-18, Vλ

3-16, V2-14, Vλ

3-12, Vλ

2-11, Vλ

3-10, Vλ

3-9, Vλ

2-8, and Vλ

4-3, and wherein said human Vλ and

Jλ

gene segments are included in said human λ

locus DNA.

16. A mouse whose 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 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,

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 Vλ

2-18 to Cλ

7 of a human λ

light chain locus,

and wherein said mouse expresses light chains comprising an endogenous Vλ

,

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 said mouse exhibits relative usage of Jλ

gene segments in splenic B cells typical of the relative usage of Jλ

gene segments in splenic B cells of humans.

17. The mouse of claim 16, 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.

18. The mouse of claim 16, wherein said endogenous light chain enhancer is in germline order relative to an endogenous constant region.

19. The mouse of claim 16, wherein said endogenous kappa enhancer comprises an iEK or 3′

EK enhancer or both.

20. The mouse of claim 16, 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, wherein the endogenous lambda loci are intact, wherein said mouse does not comprise endogenous kappa chain.

21. The mouse of claim 20 wherein B cells of said mouse comprise immunoglobulin comprising human λ

constant region relative to immunoglobulin comprising mouse λ

constant region at a ratio of 80;

15.

22. The mouse of claim 20, wherein 80% of total splenic B cells of said mouse comprise human λ

variable regions.

23. The mouse of claim 20, wherein 84% of light chains of total splenic B cells of said mouse comprise human λ

variable regions.

24. The mouse of claim 16, wherein said IgG comprising human Vλ

regions comprises antigen specific human Vλ

.

25. The mouse of claim 16, wherein said IgG comprising human Vλ

regions comprises a repertoire of human λ

variable regions.

26. The mouse of claim 16, 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 V region.

27. The mouse of claim 16, wherein endogenous VK expression is inactive, wherein said mouse expresses λ

immunoglobulin light chains comprising a plurality of human λ

variable regions encoded by human Vλ and

Jλ

gene segments, wherein said human Vλ

comprises Vλ

3 1, Vλ

2-18, Vλ

3-16, V2-14, Vλ

3-12, Vλ

2-11, Vλ

3-10, Vλ

3-9, Vv2-8, and Vλ

4-3, and wherein said human Vλ and

Jλ

gene segments are included in said human λ

locus DNA.