ANIMAL MODELS AND THERAPEUTIC MOLECULES

• US 20140134156A1
• Filed: 12/20/2013
• Published: 05/15/2014
• 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, Legent, Anais, Kirby, Ian
• Granted Patent: US 9,434,782 B2
• Time in Patent Office: Days
• US Class Current: 424/132.1
• 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 method for producing an antibody specific for a selected antigen, the method comprising

(a) immunizing a transgenic mouse with said selected antigen for a time and under conditions which permit the immunized mouse to produce antibody specific for said antigen, wherein said transgenic mouse comprises in its germline a transgenic immunoglobulin heavy chain (IgH) locus functional to undergo VDJ joining and to produce a plurality of antigen-specific antibodies comprising human V regions, said transgenic IgH locus comprising, in 5′

to 3′

transcriptional orientation,

i) unrearranged human immunoglobulin heavy chain (IgH) variable region (VH) DNA comprising a human IgH V segment, one or more human D segments and one or more human JH segments comprising a human JH6 segment;

ii) a chimeric J/C intron comprising human intronic DNA contiguous with mouse intronic DNA,

iii) a mouse enhancer, and

iv) a switch element comprising (SEQ ID;

18 (gagtcgagtc))n, where n=3 or more repeats;

wherein said human intronic DNA of (ii) comprises human intronic DNA naturally contiguous with and downstream of said human JH6 segment and said mouse intronic DNA of (ii) comprises mouse intronic DNA naturally contiguous with and upstream of a mouse IgH Cμ

enhancer; and

v) constant (C) region DNA of a mouse comprising a C segment,

wherein said unrearranged human heavy chain DNA of (i), said chimeric J/C intron of (ii), said mouse enhancer of (iii), and said switch element of (iv) are operatively linked to said C region; and

(b) isolating from said immunized mouse one or more of;

said antibody, a cell producing said antibody, a nucleic acid encoding said antibody, and/or a biological sample comprising said antibody.

2. The method of claim 1, wherein said isolated cell comprises nucleic acid encoding a human immunoglobulin variable region, and said method further comprises the step of:

isolating nucleic acid encoding said human immunoglobulin variable region.

3. The method of claim 2, further comprising the step of:

joining said human immunoglobulin variable region encoding nucleic acid to a human constant region encoding nucleic acid, to provide nucleic acid encoding an antibody comprising said human variable region and a human constant region.

4. The method of claim 1, wherein said antibody comprises a human variable region and a mouse constant region and said method further comprises the step of:

isolating said antibody from said isolated biological sample so as to provide said antibody.

5. The method of claim 1, wherein said transgenic IgH locus is functional to undergo class switching to produce a plurality of antigen-specific immunoglobulin (Ig) isotypes.

6. The method of claim 5, wherein said antigen-specific antibody isotypes present in said transgenic mouse comprises IgG1, IgG2a, IgG2b and IgM.

7. The method of claim 1, wherein said transgenic mouse is homozygous for said transgenic IgH locus.

8. The method of claim 1, wherein said mouse intronic DNA comprises DNA of an IgH locus of a mouse 129 BAC library.

9. The method of claim 8, wherein said mouse 129 BAC library DNA is a DNA between an endogenous mouse IgH J region and a mouse IgH enhancer of a mouse 129 strain IgH locus.

10. The method of claim 8, wherein said mouse 129 BAC DNA is a DNA between the mouse J region and the mouse constant region of a mouse 129 IgH locus.

11. A mouse cell produced by the method of claim 1, said cell comprising a genomic DNA comprising said transgenic IgH locus comprising a chimeric J/C intron comprising mouse intronic DNA of a mouse 129 BAC library.

13. The method of claim 1, wherein said unrearranged human heavy chain V region DNA comprises gene segments V6-1 to JH6 in human germline DNA order.

14. The method of claim 1, wherein said mouse is functional to express said IgG antibody comprising a binding affinity ranging from 0.38 to 40.60 nM.

15. The method of claim 1, wherein said IgG antibody of claim (C) comprises a variable region corresponding to a variable region encoded by human VH2-5, human VDJH1, or human VDJH6.

16. The method of claim 1, wherein said C region is endogenous to said transgenic mouse.

17. The method of claim 1, wherein said chimeric J/C intron comprises 400 bp of human intronic DNA naturally contiguous with and downstream of said human JH6 segment.

18. The method of claim 1, wherein one or more of said mouse intronic DNA of said chimeric J/C intron of (ii), said enhancer of (iii) and/or said switch element of (iv) comprises DNA of an IgH locus of a 129 strain mouse, said DNA being naturally contiguous with and downstream of a mouse 129 strain chromosome 12 coordinate equivalent to a NCBIM37(C57Bl/6J strain) mouse chromosome 12 coordinate selected from the group consisting of:

114,666,183;

114,666,190;

114,666,283;

114,666,335;

114,666,385;

114,666,486;

114,666,536;

114,666,625; and

114,666,725.

19. The method of claim 1, wherein said one or more of said mouse intronic DNA of said chimeric J/C intron of (ii), said enhancer of (iii) and/or said switch element of (iv) comprises DNA of an IgH locus of a 129 strain mouse, said DNA being naturally contiguous with and downstream of mouse chromosome 12 coordinate 114,666,435 of NCBIM37 (C57Bl/6J strain) or mouse chromosome 12 coordinate 1,207,826 of NT114985.2 (129SvJ strain).

20. The method of claim 1, wherein one or more of said mouse intronic DNA of said chimeric J/C intron of (ii), said enhancer of (iii) and/or said switch element of (iv) comprises DNA of an IgH locus of a 129 strain mouse, said DNA being naturally contiguous with and downstream of a mouse 129 strain chromosome 12 coordinate equivalent to a C57Bl/6J strain (NCBIM37) mouse chromosome 12 coordinate located within a coordinates interval selected from the group consisting of:

interval (a) 114,666,183 and 114,666,725;

interval (b) 114,666,283 and 114,666,625;

interval (c) 114,666,335 and 114,666,536; and

interval (d) 114,666,385 and 114,666,486.

21. The method of claim 1, wherein said chimeric J/C intron comprises mouse intronic DNA of an IgH locus of an AB2.1 cell.

22. The method of claim 1, wherein said chimeric J/C intron comprises mouse intronic DNA of an IgH locus of a 129 strain mouse.

23. The method of claim 1, wherein one or both of said mouse enhancer and/or said switch element comprises mouse enhancer and/or said switch element DNA of an IgH locus of a 129 strain mouse.

24. The method of claim 1, said mouse being functional to breed to produce a subsequent generation mouse comprising said germline recombinant immunoglobulin heavy chain locus.

25. The method of claim 3, further comprising the step of:

expressing the antibody from said nucleic acid, wherein said nucleic acid encodes an antibody comprising said human variable region and a human constant region.

26. The method of claim 1, wherein said immunized mouse expresses antibody heavy chains having a CDR-H3 length of at least 17 amino acids.

27. The method of claim 26, wherein 5% or more of said antibody heavy chains have a CDR-H3 length of at least 17 amino acids.