ANIMAL MODELS AND THERAPEUTIC MOLECULES

• US 20140120582A1
• Filed: 11/14/2013
• Published: 05/01/2014
• 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 10,165,763 B2
• Time in Patent Office: Days
• US Class Current: 435/69.6
• 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 mouse comprising in its germline homozygous recombinant immunoglobulin heavy chain loci, each said recombinant immunoglobulin heavy chain locus comprising a human heavy chain variable region positioned at an endogenous mouse heavy chain locus and upstream of a mouse constant region, said human heavy chain variable region comprising one or more unrearranged human heavy chain V (VH) gene segments, one or more human D (DH) gene segments and one or more human heavy chain J (JH) gene segments, said mouse constant region comprising one or more mouse C segments, said mouse being functional to form rearranged human VH, DH, and JH gene segments and to express RNA encoding chimeric immunoglobulin heavy chain polypeptide comprising a human VH region and a mouse C region,

wherein said mouse comprises said expressed RNA, said expressed RNA being selected from the group consisting of:

(i) said expressed RNA being encoded by rearranged human VH-DH-JH gene segments reflecting a deletion or addition of 1-10 nucleotides with reference to nucleotides of said unrearranged human VH and DH gene segments, and

(ii) said expressed RNA encoding a said chimeric polypeptide comprises a human variable region comprising a CDR-H3 length of at least 17 amino acids.

2. The mouse of claim 1, wherein 5% or more of said RNA encoding said chimeric polypeptide encodes a said CDR-H3 comprising at least 17 amino acids in length.

3. The mouse of claim 1 wherein said RNA encodes a chimeric polypeptide comprising said CDR-H3 having a length selected from the group consisting of:

17, 18, and 19 amino acids.

4. The mouse of claim 3, wherein 5% or more of said chimeric RNA encodes a chimeric polypeptide comprising said CDR-H3 having a length selected from the group consisting of:

17, 18, and 19 amino acids.

5. The mouse of claim 3, wherein said chimeric RNA encodes a chimeric polypeptide comprising CDR-H3 length(s) occurring at a frequency of at least 4% of said chimeric RNA.

6. The mouse of claim 3, wherein said chimeric RNA encodes chimeric polypeptide further comprises additional CDR-H3 lengths selected from the group consisting of:

20, 21, and 22 amino acids.

7. The mouse of claim 3, wherein said chimeric RNA encodes polypeptide comprising CDR-H3 length(s) are present in said expressed RNA at a combined frequency of at least 5%.

8. The mouse of claim 3, wherein said chimeric RNA encodes polypeptide further comprising CDR-H3 lengths of 17, 18 and 19 amino acids.

9. The mouse of claim 3, wherein said chimeric RNA encodes polypeptide further comprising additional CDR-H3 lengths of 17, 18, 19 and 20 amino acids.

10. The mouse of claim 3, wherein said chimeric RNA encodes polypeptide further comprising additional CDR-H3 lengths of 17, 18, 19, 20 21 and 22 amino acids.

11. The mouse of claim 3, wherein said chimeric RNA encodes polypeptide comprising CDR-H3 lengths ranging from 10-19 amino acids, inclusive.

12. The mouse of claim 11 wherein said chimeric RNA encoding polypeptide comprising said CDR-H3 lengths comprises at least ten distinct CDR-H3 length-encoding RNAs, each said distinct CDR-H3 length-encoding RNA consisting of one of said 10-19 CDR-H3 encoding lengths, and wherein each said CDR-H3 length encoding RNA, individually, is present in said chimeric RNA at a frequency of at least 5%.

13. The mouse of claim 1, wherein said RNAs of said plurality comprises individual RNAs which comprise a portion complementary to a said rearranged human VH-DH segment reflecting a deletion or addition of 1-20 nucleotides with reference to nucleotides of said unrearranged human VH and DH segments.

14. The mouse of claim 1, wherein said plurality of RNAs comprises RNAs comprising a region complementary to DNA comprising a human JH segment distribution comprising rearranged human JH4 and rearranged human JH6 gene segments.

15. The mouse of claim 1, said germline further comprising a recombinant immunoglobulin light chain locus comprising a human variable region positioned at an endogenous mouse light chain locus and upstream of a mouse constant region, said human variable region comprising one or more unrearranged human light chain V gene segments, and one or more human J light chain gene segments, said mouse constant region comprising one or more mouse C segments, said mouse being functional to express chimeric immunoglobulin light chain polypeptide, said mouse being homozygous for said recombinant immunglobulin light chain locus.

16. The mouse of claim 1, wherein said chimeric RNA encodes heavy chain polypeptide comprising a mouse p constant region.

17. The mouse of claim 1, wherein said mouse comprises a plurality of chimaeric Ig heavy chains, each said chimaeric heavy chain comprising a human V region and a mouse C region.

18. The mouse of claim 1, said mouse being functional to breed to produce progeny comprising said germline homozygous recombinant immunogloublin heavy chain locus.

19. The mouse of claim 1, said mouse being a genetically altered subsequent generation mouse produced by breeding a mouse comprising in its germline said recombinant heavy chain locus

20. The mouse of claim 1, said mouse being produced by breeding a mouse resulting from implantation of a transgenic ES cell comprising a said recombinant heavy chain locus, wherein said ES cell is from a mouse strain selected from the group consisting of C57BL and 129.

21. A method of obtaining B cells expressing an antigen-specific antibody comprising a chimeric immunoglobulin heavy chain polypeptide, comprising immunizing the mouse of claim 1 with an antigen, and isolating B cells expressing said antigen-specific antibody from said immunized mouse.

22. The method of claim 21, further comprising the step of culturing said B cells, and isolating antigen-specific antibody from said cultured B cells.

23. The method of claim 21, further comprising the step of isolating from said B cells nucleic acid encoding said chimeric immunologobulin heavy chain polypeptide, said nucleic acid comprising a human VH coding portion and a mouse C region coding portion.

24. The method of claim 23, further comprising the step of replacing in said nucleic acid said mouse constant C region-coding portion with a human constant region-coding portion to form a nucleic acid comprising a human VH coding portion and a human C region coding portion.