Animal Models and Therapeutic Molecules

• US 20150040250A1
• Filed: 10/16/2014
• Published: 02/05/2015
• Est. Priority Date: 07/08/2009
• Status: Active Grant

• Current Assignee: Kymab Limited
• Original Assignee: Kymab Limited
• Inventors: Bradley, Allan, Lee, E-Chiang, Liang, Qi, Wang, Wei, Friedrich, Glenn
• Granted Patent: US 10,064,398 B2
• Time in Patent Office: Days
• US Class Current: 800/6
• CPC Class Codes: A01K 2207/15 Humanized animals
• 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 67/0271 Chimeric vertebrates, e.g. comprising exogenous cells
• CPC Class Codes: A01K 67/0275 Genetically modified vertebrates, e.g. transgenic
• CPC Class Codes: A01K 67/0276 Knock-out vertebrates
• CPC Class Codes: A01K 67/0278 Knock-in vertebrates, e.g. humanised vertebrates
• CPC Class Codes: A61K 2039/505 comprising antibodies
• CPC Class Codes: A61K 39/107 Vibrio
• CPC Class Codes: A61K 39/35 Allergens
• 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 16/1203 from Gram-negative bacteria
• CPC Class Codes: C07K 16/1239 from Vibrionaceae (G)
• CPC Class Codes: C07K 16/18 against material from animals or humans
• 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 2317/515 : Complete light chain, i.e. VL + CL
• CPC Class Codes: C07K 2317/52 : Constant or Fc region; Isotype
• CPC Class Codes: C07K 2317/56 : variable (Fv) region, i.e. VH and/or VL
• CPC Class Codes: C07K 2317/565 : Complementarity determining region [CDR]
• CPC Class Codes: C07K 2317/567 : Framework region [FR]
• CPC Class Codes: C07K 2317/76 : Antagonist effect on antigen, e.g. neutralization or inhibition of binding
• CPC Class Codes: C07K 2317/92 : Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
• 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 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

1. A transgenic mouse having a germline with a homozygous immunoglobulin heavy chain (IgH) locus, a said homozygous IgH locus comprising unrearranged human IgH variable region gene segments positioned upstream of a constant (C) region comprising an endogenous C gene segment of an IgH locus;

wherein said homozygous IgH locus comprises in 5′

to 3′

transcriptional orientation a human JH gene segment, a human/mouse chimeric DNA junction, an enhancer, and said C region, wherein said homozygous IgH locus comprises mouse 129 strain DNA positioned downstream of said human/mouse chimeric DNA junction and upstream of said enhancer;

wherein a said homozygous IgH locus of said mouse is capable of undergoing V, D, J joining;

wherein said transgenic mouse is capable, upon stimulation with antigen, of producing an antibody comprising a chimeric Ig heavy chain comprising a human heavy chain V region; and

wherein said transgenic mouse is capable of breeding with a second transgenic mouse, said second transgenic mouse having a germline with a homozygous IgH locus comprising unrearranged human IgH variable region gene segments positioned upstream of a constant (C) region comprising an endogenous C segment of an IgH locus to provide subsequent generation mice comprising mice comprising males and females comprising in their germline an homozygous IgH locus comprising unrearranged human IgH variable region gene segments positioned upstream of a constant (C) region comprising an endogenous C segment of an IgH locus.

2. The transgenic mouse of claim 1 wherein said C gene segment comprises a mouse Cp segment.

3. The transgenic mouse of claim 1, wherein said unrearranged human IgH variable gene segments comprise one or more unrearranged human IgH variable (VH) gene segments, one or more unrearranged human IgH D gene segments, and one or more unrearranged human IgH joining (JH) gene segments, said C region comprising a plurality of C gene segments comprising a mouse C gene segment.

4. The transgenic mouse of claim 3, wherein said one or more unrearranged human IgH D gene segments comprises a plurality of unrearranged human IgH D gene segments and said one or more unrearranged human JH gene segments comprises a plurality of unrearranged human JH gene segments.

5. The transgenic mouse of claim 4, wherein said transgenic IgH locus comprises a plurality of unrearranged human IgH VH gene segments.

6. The transgenic mouse of claim 3, wherein said one or more unrearranged human JH gene segments comprises a 3′

human JH segment, wherein DNA between said 3′

human JH segment and said human/mouse chimeric DNA junction is less than 1 kb.

7. The transgenic mouse of claim 3, wherein said one or more unrearranged human JH gene segments comprises a 3′

human JH segment wherein DNA between said 3′

human JH segment and said human/mouse chimeric DNA junction is less than 2 kb.

8. The transgenic mouse of claim 1, wherein said germline comprises all or part of mouse IgH variable region DNA.

9. The transgenic mouse of claim 3, wherein said one or more unrearranged human VH gene segments are positioned in place of endogenous mouse Ig VH region segment(s) upstream of said endogenous C segment of an IgH locus.

10. The transgenic mouse of claim 3, wherein said one or more unrearranged human DH segments are positioned in place of endogenous mouse Ig DH region segment(s) upstream of said endogenous C segment of an IgH locus.

11. The transgenic mouse of claim 3, wherein said one or more unrearranged human JH segments are positioned in place of endogenous mouse Ig JH region segment(s) upstream of said endogenous C segment of an IgH locus.

12. The transgenic mouse of claim 1, wherein said subsequent generation mice comprise males and females having in their germline a homozygous IgH locus comprising unrearranged human IgH variable region gene segments positioned upstream of a constant (C) region comprising an endogenous C segment of an IgH locus.

13. The transgenic mouse of claim 12, wherein said males comprised in said subsequent generation mice are capable of breeding with females having in their germline a homozygous IgH locus comprising unrearranged human IgH variable region gene segments positioned upstream of a constant (C) region comprising an endogenous C segment of an IgH locus to provide further subsequent generation mice having in their germline a homozygous IgH locus comprising unrearranged human IgH variable region gene segments positioned upstream of an IgH constant (C) region comprising an endogenous C segment of an IgH locus.

14. The transgenic mouse of claim 12, wherein said male and female mice comprised in said subsequent generation mice are capable of breeding with a mouse having in their germline a homozygous IgH locus, comprising unrearranged human IgH variable region gene segments positioned upstream of a constant (C) region comprising an endogenous C segment of an IgH locus to provide further subsequent generation mice comprising mice having in their germline a homozygous IgH locus, comprising unrearranged human IgH variable region gene segments positioned upstream of a constant (C) region comprising an endogenous C segment of an IgH locus.

15. The transgenic mouse of claim 3, wherein said all or part of said mouse IgH variable region DNA is inverted with respect to its native chromosomal orientation.

16. The transgenic mouse of claim 3, wherein said all or part of said mouse IgH variable region DNA is positioned at a telomeric locus.

17. The transgenic mouse of claim 15, wherein said inverted all or part is positioned upstream of said unrearranged human IgH variable region gene segments.

18. A method for producing any one of an antibody specific to an antigen, a cell producing an antibody specific to an antigen, or serum comprising antigen-specific antibody, wherein said antibody comprises a chimeric Ig heavy chain comprising a human heavy chain variable region, the method comprising immunizing the transgenic mouse of claim 1 with said antigen, and recovering said antibody, said cell, or said serum.

19. The method of claim 18, further comprising isolating nucleic acid encoding said human heavy chain variable region, and operatively linking to said nucleic acid encoding said human heavy chain variable region nucleic acid encoding a human constant region.