Non-human mammals for the production of chimeric antibodies

• US 9,346,873 B2
• Filed: 09/30/2009
• Issued: 05/24/2016
• 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 GOMEZ
• Application Number:
  US13/121,883
• Publication Number:
  US 20110236378A1
• Time in Patent Office: 2,428 Days
• Field of Search: 536/23.53, 536/23.1, 435/320.1, 800/13, 800/18, 800/24
• US Class Current: 1/1
• 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. A non-human mammalian cell whose genome comprises an immunoglobulin (Ig) heavy chain locus comprising a transgene comprising a portion of a syngeneic Ig heavy chain locus, said portion comprising all or part of constant region genes encoding a chimeric Ig heavy chain, the transgene comprising a DNA fragment comprising in operable linkage from 5′

to 3′

a DNA sequence of a human variable heavy (VH) segment gene operably linked to a constant region gene comprising a human CH1, human C_{upper hinge}, human C_{middle hinge}, syngeneic mammalian non-human CH2 and syngeneic mammalian non-human CH3 exons, wherein the human CH1 exon is selected from the group consisting of a human mu constant region (Cμ

), a human delta constant region (Cδ

), a human gamma-1 constant region (Cγ

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

2) and a human gamma-4 constant region (Cγ

4), wherein the transgene comprising said human VH segment gene and said syngeneic Ig heavy chain locus replaces all or a portion of the endogenous Ig heavy chain locus, thereby said non-human mammalian cell comprises a genome encoding the chimeric Ig heavy chain.

2. A method of producing the non-human mammalian cell of claim 1, comprising the steps of:

producing one or more recombination vectors comprising the desired Ig transgene to be introduced into the endogenous Ig heavy chain locus, the transgene comprising a portion of a syngeneic Ig heavy chain locus, said portion comprising all or part of constant region genes encoding a chimeric Ig heavy chain, the transgene comprising a DNA fragment comprising in operable linkage from 5′

to 3′

a DNA sequence of a human variable heavy (VH) segment gene operably linked to a constant region gene comprising a human CH1, human C_{upper hinge}, human C_{middle hinge}, syngeneic mammalian non-human CH2 and syngeneic mammalian non-human CH3 exons, wherein the human CH1 exon is selected from the group consisting of a human Cμ

, a human Cδ

, a human Cγ

1, a human Cγ

2 and a human Cγ

4, and

introducing said one or more recombination vectors into the non-human mammalian cell and integrating, in operable linkage, said transgene into the genome of the non-human mammalian cell, wherein the transgene comprising said human VH segment gene and said syngeneic Ig heavy chain locus replaces all or a portion of the endogenous Ig heavy chain locus, thereby said non-human mammalian cell comprising a genome encoding the chimeric Ig heavy chain.

3. The cell according to claim 1, wherein the transgene further comprises a diversity heavy (DH) segment gene 3′

of the human VH segment gene and 5′

of the human CH1 segment gene, wherein the DH segment gene is selected from the group consisting of human, non-human primate, rabbit, sheep, rat, hamster, and mouse.

4. The cell according to claim 3, wherein the DH segment gene is human.

5. The non-human mammalian cell according to claim 1, wherein the human upper and middle hinge exons are IgG4 sequences.

6. The non-human mammalian cell according to claim 1, wherein the chimeric Ig heavy chain binds an endogenous Fc receptor.

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

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

9. The non-human mammalian cell according to claim 1, wherein the transgene further comprises a human joining heavy (JH) segment gene 3′

of the human VH segment gene and 5′

of the human CH1 segment gene.

10. A non-human mammalian B cell whose genome comprises an immunoglobulin (Ig) heavy chain locus comprising a transgene encoding a chimeric Ig heavy chain, the transgene comprising a portion of a syngeneic Ig heavy chain locus, said portion comprising all or part of constant region genes encoding a chimeric Ig heavy chain, the transgene comprising a DNA fragment comprising in operable linkage from 5′

to 3′

a DNA sequence of a human variable heavy (VH) segment gene operably linked to a constant region gene comprising a human CH1, human C_{upper hinge}, syngeneic mammalian non-human CH2 and syngeneic mammalian non-human CH3 exons, wherein the human CH1 exon is selected from the group consisting of a human Cμ

, a human Cδ

, a human Cγ

1, a human Cγ

2 and a human Cγ

4, wherein the transgene comprising said human VH segment gene and said syngeneic Ig heavy chain locus replaces all or a portion of the endogenous Ig heavy chain locus, thereby said non-human B cell comprising a genome encoding the chimeric Ig heavy chain.

11. A non-human mammalian hybridoma cell whose genome comprises an immunoglobulin (Ig) heavy chain locus comprising a transgene encoding a chimeric Ig heavy chain, the transgene comprising a portion of a syngeneic Ig heavy chain locus, said portion comprising all or part of constant region genes, the transgene comprising a DNA fragment comprising in operable linkage from 5′

to 3′

a DNA sequence of a human variable heavy (VH) segment gene operably linked to a constant region gene comprising a human CH1, human C_{upper hinge}, syngeneic mammalian non-human CH2 and syngeneic mammalian non-human CH3 exons, wherein the human CH1 exon is selected from the group consisting of a human Cμ

, a human Cδ

, a human Cγ

1, a human Cγ

2 and a human Cγ

4, wherein the transgene comprising said human VH segment gene and said syngeneic Ig heavy chain locus replaces all or a portion of the endogenous Ig heavy chain locus, thereby said non-human hybridoma cell comprising a genome encoding the chimeric Ig heavy chain.