Non-human mammals for the production of chimeric antibodies

• US 10,638,736 B2
• Filed: 04/11/2016
• Issued: 05/05/2020
• Est. Priority Date: 09/30/2008
• Status: Active Grant

• Current Assignee: Ablexis LLC
• Original Assignee: Ablexis LLC
• Inventors: Green, Larry, Shizuya, Hiroaki
• Primary Examiner(s): Leavitt, Maria G
• Application Number:
  US15/095,881
• Publication Number:
  US 20160222092A1
• Time in Patent Office: 1,485 Days
• Field of Search: None
• CPC Class Codes: A01K 2207/15 Humanized animals
• CPC Class Codes: A01K 2217/052 inducing gain of function
• CPC Class Codes: A01K 2217/072 maintaining or altering function, i.e. knock in
• 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 67/0278 Knock-in vertebrates, e.g. humanised vertebrates
• 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 16/283 against Fc-receptors, e.g. CD16, CD32, CD64 CD23 C07K16/2851
• 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 2317/522 CH1 domain
• CPC Class Codes: C07K 2317/524 CH2 domain
• CPC Class Codes: C07K 2317/526 CH3 domain
• CPC Class Codes: C07K 2317/53 Hinge
• CPC Class Codes: C07K 2317/56 : variable (Fv) region, i.e. VH and/or VL
• 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: C07K 2317/94 : Stability, e.g. half-life, pH, temperature or enzyme-resistance
• CPC Class Codes: C12N 15/8509 : for producing genetically modified animals, e.g. transgenic

Abstract

The invention provides knock-in non-human cells and mammals having a genome encoding chimeric antibodies and methods of producing knock-in cells and mammals. Certain aspects of the invention include chimeric antibodies, humanized antibodies, pharmaceutical compositions and kits. Certain aspects of the invention also relate to diagnostic and treatment methods using the antibodies of the invention.

Claims

1. An isolated non-human mammalian cell whose genome comprises a transgene comprising a chimeric Ig heavy chain locus, or a portion thereof, the transgene comprising in operable linkage from 5′

to 3′

an unrearranged DNA sequence comprising one or more human variable heavy (VH) gene segments, one or more diversity heavy (DH) gene segments and one or more human joining heavy (JH) gene segments, operably linked to a constant region gene comprising a human CH1 exon, a human C_{upper hinge} exon, a non-human CH2 exon and a non-human CH3 exon, wherein the human CH1 exon is selected from the group consisting of a human mu constant region (Cμ

) CH1 exon, a human delta constant region (Cδ

) CH1 exon, a human gamma-1 constant region (Cγ

1) CH1 exon, a human gamma-2 constant region (Cγ

2) CH1 exon, a human gamma-4 constant region (Cγ

4) CH1 exon, a human alpha constant region (Cα

) CH1 exon, and a human epsilon constant region (Cε

) CH1 exon, wherein said isolated non-human mammalian cell comprises a genome encoding a chimeric immunoglobulin heavy chain (IgH) comprising an IgH variable domain and a chimeric IgH constant region and wherein the transgene is constructed by genetic engineering, recombineering or synthesis.

2. The isolated non-human mammalian cell according to claim 1, wherein the DH gene segments are selected from the group consisting of human, non-human primate, rabbit, sheep, rat, hamster, and mouse.

3. The isolated non-human mammalian cell according to claim 2, wherein the DH gene segments are human.

4. The isolated non-human mammalian cell according to claim 1, wherein the cell is an embryonic stem cell.

5. The isolated non-human mammalian cell according to claim 1, wherein the cell is a mouse cell.

6. The isolated non-human mammalian cell according to claim 1, wherein the transgene further comprises a 3′

locus control region (LCR).

7. The isolated non-human mammalian cell according to claim 1, wherein the genome further comprises a second transgene, wherein the second transgene comprises a human Ig light chain locus, or a portion thereof, and wherein the second transgene is constructed by genetic engineering, recombineering or synthesis.

8. The isolated non-human mammalian cell according to claim 7, wherein the second transgene comprising the human Ig light chain locus replaces all or a portion of an endogenous Ig light chain locus.

9. The isolated non-human mammalian cell according to claim 7, wherein the human Ig light chain locus comprises a human Igκ

variable region.

10. The isolated non-human mammalian cell according to claim 9, wherein the human Ig light chain locus further comprises a human Igκ

constant region.

11. The isolated non-human mammalian cell according to claim 7, wherein the human Ig light chain locus comprises a human Igλ

variable region.

12. The isolated non-human mammalian cell according to claim 11, wherein the human Ig light chain locus further comprises a human Igλ

constant region.

13. The isolated non-human mammalian cell according to claim 12, wherein the human Ig light chain locus further comprises an Igλ

3′

LCR.

14. The isolated non-human mammalian cell according to claim 12, wherein the human Ig light chain locus comprises the complete human Igλ

light chain locus.

15. The isolated non-human mammalian cell according to claim 12, wherein the human Ig light chain locus comprises one or more human Vλ

a gene segments and 1 to 7 human Jλ

-Cλ

gene segment pairs.

16. The isolated non-human mammalian cell according to claim 12, wherein the human Ig light chain locus comprises one or more human Vλ

a gene segments, 1 to 7 human Jλ

gene segments, and a single human Cλ

gene segment.

17. The isolated non-human mammalian cell according to claim 13, wherein the Igλ

3′

LCR is from a mammal selected from the group consisting of human, non-human primate, and rat.

19. The isolated non-human mammalian cell according to claim 1, wherein the transgene further comprises a human or non-human mammalian C_{middle hinge}.

20. The isolated non-human mammalian cell according to claim 1, wherein the transgene is randomly integrated.

21. The isolated non-human mammalian cell according to claim 1, wherein all or a portion of the endogenous Ig heavy chain locus of the non-human mammalian cell is inactivated.

18. An isolated mouse B cell whose genome comprises a transgene comprising in operable linkage from 5′

to 3′

a DNA sequence comprising a human variable heavy (VH) gene segment, a diversity heavy (DH) gene segment and a human joining heavy (JH) gene segment, operably linked to a constant region gene comprising a human CH1 exon, a human C_{upper hinge} exon, a mouse CH2 exon and a mouse CH3 exon, wherein the human CH1 exon is selected from the group consisting of a human mu constant region (Cμ

) CH1 exon, a human delta constant region (Cδ

) CH1 exon, a human gamma-1 constant region (Cγ

1) CH1 exon, a human gamma-2 constant region (Cγ

2) CH1 exon, a human gamma-4 constant region (Cγ

4) CH1 exon, a human alpha constant region (Cα

) CH1 exon, and a human epsilon constant region (Cε

) CH1 exon, wherein the transgene expresses a chimeric immunoglobulin heavy chain (IgH) comprising an IgH variable domain and a chimeric IgH constant region and wherein the transgene is constructed by genetic engineering, recombineering or synthesis.

22. The isolated mouse B cell according to claim 18, wherein the genome further comprises a second transgene, wherein the second transgene comprises a human Ig light chain locus, or a portion thereof, wherein the second transgene expresses a human Ig light chain, wherein the second transgene is constructed by genetic engineering, recombineering or synthesis and wherein the isolated mouse B cell produces an antibody comprising a chimeric Ig heavy chain and a human Ig light chain.

23. A hybridoma formed by fusing the isolated mouse B cell according to claim 22 with a myeloma cell.