Animal models and therapeutic molecules

• US 10,165,763 B2
• Filed: 11/14/2013
• Issued: 01/01/2019
• 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
• Primary Examiner(s): SINGH, ANOOP KUMAR
• Application Number:
  US14/080,630
• Publication Number:
  US 20140120582A1
• Time in Patent Office: 1,874 Days
• 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 humanized 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 whose genome comprises a homozygous immunoglobulin heavy chain (IgH) locus comprising a human heavy chain variable region at an endogenous IgH locus, upstream of a constant (C) region, said constant region comprising an endogenous C gene segment,

said human heavy chain variable region comprising a plurality of unrearranged human immunoglobulin heavy chain variable (VH) gene segments, a plurality of unrearranged human immunoglobulin heavy chain diversity (DH) gene segments and a plurality of unrearranged human immunoglobulin heavy chain joining (JH) gene segments comprising a 3′

JH gene segment, wherein the human variable region gene segments in said IgH locus are operably linked to said endogenous IgH C gene segment,

said IgH locus in a 5′

to 3′

transcriptional orientation comprising said 3′

JH gene segment, human DNA, a human/mouse DNA junction, mouse DNA, an enhancer, and said constant region;

wherein said human DNA comprises human intronic DNA downstream of and contiguous with said human JH region,

wherein said mouse DNA comprises mouse intronic DNA downstream of and contiguous with said human/mouse DNA junction and upstream of said constant region,

wherein DNA between said human/mouse DNA junction and said enhancer comprises mouse 129 strain JC intronic DNA

wherein said 3′

JH gene segment is less than 2 Kb upstream of said human/mouse DNA junction,

said mouse being functional to breed to another said mouse to produce progeny having in its germline said homozygous recombinant immunoglobulin heavy chain locus,

said mouse being functional to form rearranged human VH, DH, and JH gene segments and to produce mRNA transcripts encoding immunoglobulin heavy chain polypeptide comprising a human VH region and a mouse Cμ

region,

wherein said mouse comprises IgH mRNA transcripts comprising IgH-VDJCμ

transcripts encoding polypeptide comprising a human VH region and mouse Cμ

,

wherein said IgH-VDJCμ

transcripts comprise transcripts encoding a human variable region comprising a CDR-H3 length of 17 amino acids and transcripts encoding a human variable region comprising a CDR-H3 length of 18 amino acids according to the Kabat numbering system,

wherein the mean frequency of the group consisting of said transcripts encoding CDR-H3 lengths of 17 and 18 amino acids present in said IgH-VDJCμ

transcripts of said mouse is between 5% and 10%.

2. The mouse of claim 1, wherein said mouse comprises IgH-VDJCμ

transcripts encoding IgH polypeptides comprising CDR-H3 lengths of 17, 18 and 19 amino acids, wherein the mean frequency of the group consisting of said transcripts encoding CDR-H3 lengths of 17, 18 and 19 amino acids is between 5% and 10%.

3. The mouse of claim 1, wherein said mouse further comprises IgH-VDJCμ

transcripts encoding IgH polypeptide comprising CDR-H3 lengths selected from the group consisting of;

20, 21, and 22 amino acids.

4. The mouse of claim 1, wherein said mouse further comprises IgH-VDJCμ

transcripts encoding IgH polypeptide comprising CDR-H3 lengths of 19 and 20 amino acids.

5. The mouse of claim 1, wherein said mouse further comprises IgH-VDJCμ

transcripts encoding IgH polypeptide comprising CDR-H3 lengths of 20 and 21 amino acids.

6. The mouse of claim 1 wherein said mouse further comprises IgH-VDJCμ

transcripts encoding IgH polypeptide comprising CDR-H3 lengths of 21 and 22 amino acids.

7. The mouse of claim 1, wherein each said IgH mRNA transcript comprises a transcript region complementary to DNA comprising a rearranged human JH segment, and wherein transcripts comprise regions complementary to rearranged human JH4 and rearranged human JH6 gene segments.

8. A method of obtaining B cells expressing an antigen-specific antibody comprising a chimeric immunoglobulin heavy chain polypeptide comprising a human heavy chain variable (VH) region and a mouse constant (C) region, comprising contacting the mouse of claim 1 with an antigen, and isolating B cells expressing said antigen-specific antibody from said mouse contacted with antigen.

9. The method of claim 8, further comprising the step of culturing said B cells, and isolating antigen-specific antibody from said cultured B cells.

10. The method of claim 8, further comprising the step of isolating from said B cells nucleic acid encoding said human VH region and said mouse C region of said chimeric immunoglobulin heavy chain polypeptide.

11. The method of claim 10, further comprising the step of replacing in said nucleic acid encoding said human VH region and said mouse C region of said chimeric immunoglobulin heavy chain polypeptide, the portion encoding said mouse C region with nucleic acid encoding a human C region, thereby forming a nucleic acid encoding said human VH region and said human C region.

12. The method of claim 8, further comprising isolating an antibody that binds said antigen, comprising

(i) immortalizing said isolated B cells expressing said antigen-specific antibody or progeny thereof, optionally producing hybridomas therefrom;

and

(ii) isolating an antibody expressed by the immortalized B cells of (i).

13. The mouse of claim 1, where each said percentage is determined by analysis of PCR products generated using primers specific for individual human VH gene segments, where the primers are selected from the group consisting of:

SEQ ID NO;

7, SEQ ID NO;

8, SEQ ID NO;

9, SEQ ID NO;

10 and SEQ ID NO;

11.

14. The mouse of claim 1, wherein said mouse is naï

ve.

15. The mouse of claim 1, wherein said plurality of unrearranged human DH gene segments and said plurality of unrearranged human JH gene segments comprises all human D (DH) gene segments and all human heavy chain J (JH) gene segments.

16. The mouse of claim 15, wherein said plurality of unrearranged human VH gene segments comprises all human VH gene segments.