Animal models and therapeutic molecules

Patent metadata

Field	Value
Publication number	US-9788534-B2
Application number	US-201313846672-A
Country	US
Kind code	B2
Filing date	Mar 18, 2013
Priority date	Mar 18, 2013
Publication date	Oct 17, 2017
Grant date	Oct 17, 2017

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

First claim

The invention claimed is:

1. A transgenic mouse having a germline comprising a homozygous immunoglobulin heavy chain (IgH) locus,
wherein said homozygous IgH locus comprises unrearranged human IgH V gene segments, D gene segments and J gene segments at an endogenous IgH locus upstream of an enhancer and a constant (C) region comprising a mouse CH gene segment, wherein said human IgH gene segments join to mouse J/C intron DNA at a chimeric junction upstream of said enhancer,
said human IgH V gene segments, D gene segments and J gene segments are operably linked to said C region to permit expression of an Ig heavy chain polypeptide comprising a human variable region and a mouse constant region,
said unrearranged human IgH V gene segments, D gene segments and J gene segments comprise all the functional human D and JH gene segments and human V gene segments Vh2-5, Vh7-41, Vh-4-4, Vhf-3, Vhf-2 and Vh6-1,
wherein said human JH gene segments comprise a human 3′JH, said 3′ JH being contiguous with human J/C intronic DNA, wherein said 3′JH is less than 2 kb upstream of said chimeric DNA junction,
wherein said mouse J/C intron DNA between said chimeric junction and said enhancer comprises said mouse 129 strain or C57BL/6 strain DNA,
wherein bone marrow B cells of said mouse comprise more pro-B cells than pre-B cells, wherein said pro B cells express CD43 Med B220 Med and said pre B cells express CD43 low B220 high ,
wherein said transgenic mouse expresses immunoglobulin heavy chains comprising human variable regions.

2. The mouse of claim 1 , wherein the mouse expresses a normal relative proportion of serum IgG1, IgG2a, IgG2b and IgM antibodies.

3. The mouse of claim 1 , wherein the mouse expresses (i) serum IgG1 at a concentration of about 25-350 μg/ml; (ii) serum IgG2a at a concentration of about 0-200 μg/ml; (iii) serum IgG2b at a concentration of about 30-800 μg/ml; and (iv) serum IgM at a concentration of about 50-300 μg/ml; or (i) serum IgG1 at a concentration of about 10-600 μg/ml; (ii) serum IgG2a at a concentration of about 0-500 μg/ml; (iii) serum IgG2b at a concentration of about 20-700 μg/ml; and (iv) serum IgM at a concentration of about 50-700 μg/ml; as determined by Ig capture on a plate followed by incubation with anti-mouse isotype-specific labeled antibodies and quantification of Ig using the label.

4. The mouse of claim 1 , wherein the mouse produces a normal proportion or percentage of mature splenic B-cells and/or a normal proportion or percentage of bone marrow B-cell progenitor cells.

5. The mouse of claim 1 , wherein said human 3′ JH gene segments comprise human JH6, and said JH6 is less than 2 kb upstream of said chimeric junction.

6. The mouse of claim 1 , wherein DNA between said chimeric DNA junction and said enhancer comprises said mouse DNA of 129 strain.

7. The mouse of claim 1 , wherein said germline comprises all or part of said mouse heavy chain variable region inverted with respect to said heavy chain constant region.

8. The mouse of claim 1 , wherein said germline comprises all or part of said mouse heavy chain variable region away from said heavy chain constant region.

9. The mouse of claim 1 , wherein said germline comprises all, part or none of said mouse heavy chain variable region.

10. The mouse of claim 1 , wherein said transgenic mouse expresses serum antibodies comprising immunoglobulin heavy chains IgG1, IgG2b and IgM.

11. The mouse of claim 10 , wherein said transgenic mouse expresses serum antibodies further comprising immunoglobulin heavy chain IgG2a.

12. The mouse of claim 1 , wherein less than 10% of IgH chains comprise a mouse variable region.

13. The mouse of claim 12 , wherein said mouse does not express endogenous IgH heavy chain.

14. The mouse of claim 1 , further comprising the human V gene segments Vh2-26, Vhf-24, Vh3-23, Vh3-21, Vh3-20, Vh1-18, Vh3-15, Vh3-13, Vh3-11, Vh3-9, Vhf-8, and Vh3-7.

15. The mouse of claim 1 , further comprising the human V gene segments Vh3-13, Vh3-11, Vh3-9, Vhf-8, and Vh3-7, wherein said transgenic mouse expresses immunoglobulin heavy chains comprising human variable regions Vh3-13, Vh3-11, Vh3-9, Vhf-8, and Vh3-7.

16. The mouse of claim 1 , wherein 75% of IgH chains comprise a mouse variable region.

17. The mouse of claim 1 , wherein both endogenous heavy chain loci are inactive to express endogenous heavy chain polypeptide; said mouse comprises intact endogenous lambda loci to express endogenous lambda light chain polypeptide; and said mouse comprises endogenous kappa loci which are inactive to express endogenous kappa light chain polypeptide.

18. The mouse of claim 1 , wherein said mouse comprises human lambda variable region gene segments at each endogenous lambda locus.

19. The mouse of claim 1 , further comprising the human V gene segments Vh3-13, Vh3-11, Vh3-9, Vhf-8, and Vh3-7, wherein said transgenic mouse expresses immunoglobulin heavy chains comprising human variable regions Vh3-13, Vh3-11, Vh3-9, Vhf-8, and Vh3-7.

Assignees

• Kymab Ltd

Inventors

• Bradley Allan
• Lee E-Chiang
• Liang Qi
• Wang Wei
• Spensberger Dominik
• Liu Hui
• Clube Jasper

Classifications

• A01K2217/15

  Animals comprising multiple alterations of the genome, by transgenesis or homologous recombination, e.g. obtained by cross-breeding · CPC title

• C07K2317/92

  Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value · CPC title

• C07K16/1239

  from Vibrionaceae (G) · CPC title

• C12N15/8509

  for producing genetically modified animals, e.g. transgenic · CPC title

• C07K16/06

  from serum · CPC title