Transgenic non-human animals capable of producing heterologous antibodies of various isotypes

• US 5,877,397 A
• Filed: 09/19/1994
• Issued: 03/02/1999
• Est. Priority Date: 08/29/1990
• Status: Expired due to Term

• Current Assignee: Genpharm Incorporated (Bristol-Myers Squibb Company)
• Original Assignee: Genpharm Incorporated (Bristol-Myers Squibb Company)
• Inventors: Kay, Robert M., Lonberg, Nils
• Primary Examiner(s): Ziska, Suzanne E.
• Application Number:
  US08/308,865
• Time in Patent Office: 1,625 Days
• Field of Search: 800/2, 435/172.3
• US Class Current: 800/18
• CPC Class Codes: A01K 2207/15 Humanized animals
• CPC Class Codes: A01K 2217/00 Genetically modified animals
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: A01K 2217/075 inducing loss of function, i.e. knock out
• CPC Class Codes: A01K 2227/105 Murine
• CPC Class Codes: A01K 2267/01 Animal expressing industrially exogenous proteins
• 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 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: C07K 14/7051 T-cell receptor (TcR)-CD3 complex
• 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 16/2812 against CD4
• CPC Class Codes: C07K 16/3007 Carcino-embryonic Antigens
• CPC Class Codes: C07K 16/4266 against anti-tumor receptor Ig
• CPC Class Codes: C07K 16/4291 against IgE
• CPC Class Codes: C07K 16/44 against material not provided for elsewhere , e.g. haptens, metals, DNA, RNA, amino acids
• 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 2317/52 Constant or Fc region; Isotype
• CPC Class Codes: C07K 2317/56 : variable (Fv) region, i.e. VH and/or VL
• CPC Class Codes: C12N 15/00 : Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor mutants or genetically engineered microorganisms, per se C12N1/00, C12N5/00, C12N7/00; new plants per se A01H; plant reproduction by tissue culture techniques A01H4/00; new animals per se A01K67/00; use of medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases, gene therapy A61K48/00
• CPC Class Codes: C12N 15/85 : for animal cells
• CPC Class Codes: C12N 15/8509 : for producing genetically modified animals, e.g. transgenic
• CPC Class Codes: C12N 15/87 : Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
• CPC Class Codes: C12N 15/90 : Stable introduction of foreign DNA into chromosome
• CPC Class Codes: C12N 15/907 : in mammalian cells
• CPC Class Codes: C12N 2800/206 : of yeast origin, e.g. YAC, 2u
• CPC Class Codes: C12N 2800/70 : Vectors containing special elements for cloning, e.g. topoisomerase, adaptor sites
• CPC Class Codes: C12N 2830/30 : being an enhancer not forming part of the promoter region
• CPC Class Codes: C12N 2830/36 : being a transcription termination element
• CPC Class Codes: C12N 2830/85 : mammalian
• CPC Class Codes: C12N 5/163 : one of the fusion partners being a B or a T lymphocyte

Abstract

The invention relates to transgenic non-human animals capable of producing heterologous antibodies and transgenic non-human animals having inactivated endogenous immunoglobulin genes. In one aspect of the invention, endogenous immunoglobulin genes are suppressed by antisense polynucleotides and/or by antiserum directed against endogenous immunoglobulins. Heterologous antibodies are encoded by immunoglobulin genes not normally found in the genome of that species of non-human animal. In one aspect of the invention, one or more transgenes containing sequences of unrearranged heterologous human immunoglobulin heavy chains are introduced into a non-human animal thereby forming a transgenic animal capable of functionally rearranging transgenic immunoglobulin sequences and producing a repertoire of antibodies of various isotypes encoded by human immunoglobulin genes. Such heterologous human antibodies are produced in B-cells which are thereafter immortalized, e.g., by fusing with an immortalizing cell line such as a myeloma or by manipulating such B-cells by other techniques to perpetuate a cell line capable of producing a monoclonal heterologous antibody. The invention also relates to heavy and light chain immunoglobulin transgenes for making such transgenic non-human animals as well as methods and vectors for disrupting endogenous immunoglobulin loci in the transgenic animal.

Claims

1. A transgenic mouse comprising an inactivated endogenous mouse immunoglobulin gene locus, said transgenic mouse further containing in its genome a transgene comprising in operable linkage a plurality of human V genes, a plurality of human D genes, a plurality of human J genes, a human μ

C_H gene, at least two different non-μ

human C_H genes and associated isotype switching sequences, wherein human μ and

human γ

switch sequences are located in closer proximity to each other than in the naturally occurring human immunoglobulin locus; and

wherein in lymphocytes of said mouse the transgene undergoes productive VDJ rearrangement and μ

to γ

isotype switching by recombination between the human μ and

human γ

switch sequences such that the mouse produces a serum containing immunoglobuline of at least three human heavy chain isotypes in detectable amounts in response to antigenic stimulation.

2. A transgenic mouse of claim 1, wherein the lymphocytes further have integrated a human light chain transgene.

3. A transgenic mouse of claim 2, wherein the serum comprises a population of IgG comprising a subpopulation of IgG composed of human γ

1 and human κ and

a subpopulation of IgG composed of human γ

3 and human κ

.

4. The transgenic mouse of claim 3, wherein the inactivated gene locus is a mouse heavy chain immunoglobulin gene locus.

5. A mouse of claim 4, wherein the inactivated endogenous mouse immunoglobulin heavy chain gene locus has intact V_H and C_H genes and lacks functional J_H genes.

6. A mouse of claim 5, wherein said inactivated endogenous mouse immunoglobulin heavy chain locus further comprises a neo^R gene replacing said J_H genes between the endogenous V_H genes and the endogenous C_H genes of said heavy chain locus.

7. A mouse of claim 6, wherein the inactivated endogenous immunoglobulin heavy chain gene locus is a product of homologous recombination between J_H KO1 and a mouse heavy chain allele of an ES cell of the AB-1 cell line.

8. A transgenic mouse having a germline genome with:

a human heavy chain transgene comprising in operable linkage a plurality of human V genes, a plurality of human D genes, a plurality of human J genes, a human μ

C_H gene, at least two different non-μ

human C_H genes and associated isotype switching sequences, wherein human μ and

human γ

switch sequences are located in closer proximity to each other than in the naturally occurring human immunoglobulin locus; and

wherein in lymphocytes of said mouse the transgene undergoes productive VDJ rearrangement and μ

to γ

isotype switching by recombination between the human μ and

human γ

switch sequences such that the mouse produces a serum containing immunoglobulins of at least three human heavy chain isotypes in detectable amounts in response to antigenic stimulation;

a human kappa chain transgene with a human V.sub.κ

gene segment, human J.sub.κ

gene segments, a human J-Cκ

intronic enhancer, and a human C.sub.κ

coding exon;

a pair of endogenous mouse heavy chain immunoglobulin loci, each of said loci having an inactivated heavy chain gene; and

a pair of endogenous mouse immunoglobulin kappa loci, each of said loci having an inactivated chain gene.

9. A mouse of claim 8, wherein said mouse makes an immune response to an antigen, said immune response comprising an antiserum containing antibodies that specifically bind to the antigen and wherein said antibodies comprise a human μ

or γ

chain and a human κ

chain.

10. A mouse of claim 8, wherein the inactivated κ

chain gene has a deletion spanning Jκ and

Cκ

.