Transgenic non-human animals for producing heterologous antibodies

• US 5,814,318 A
• Filed: 07/22/1993
• Issued: 09/29/1998
• 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:
  US08/096,762
• Time in Patent Office: 1,895 Days
• Field of Search: 800/2, 435/172.3, 424/184.1
• US Class Current: 424/184.1
• 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 16/462 Igs containing a variable region (Fv) from one specie and a constant region (Fc) from another
• 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: C07K 2319/00 : Fusion polypeptide
• 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 15/113 : Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• 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 method for producing a secondary repertoire antibody that is reactive with a predetermined antigen, said method comprising the steps of:

(a) immunizing a transgenic mouse with the predetermined antigen, the transgenic mouse having a genome comprising a homozygous functionally disrupted endogenous heavy chain locus and an unrearranged human heavy chain immunoglobulin transgene having V, D, and J gene segments in operable cis linkage to μ and

γ

switch recombination sequences and associated human μ and

γ

constant regions;

(b) obtaining B cells from said immunized transgenic mouse expressing said secondary repertoire antibody reactive with said predetermined antigen, said antibody having a human γ

heavy chain encoded by joined V, D, and J gene segments from the unrearranged transgene in cis linkage to a recombined switch sequence and a γ

constant region gene segment from the human constant regions in the transgene, the recombined switch sequence having sequences from the μ and

γ

switch recombination sequences;

(c) obtaining, from said B cells, variable region sequences of the human heavy chain; and

(d) expressing, in an immortalized host cell, said antibody having the variable region sequences.

2. A method of claim 1 further comprising the step of purifying said antibody.

3. A method of claim 1, wherein the genome of the transgenic mouse further comprises a homozygous functionally disrupted endogenous light chain locus.

4. A method of claim 3, wherein the light chain locus is the kappa locus.

5. A method of claim 3, wherein the light chain locus lacks functional J gene segments.

6. A method of claim 2, wherein the disrupted heavy chain locus lacks functional J gene segments.

7. A method of claim 2, wherein the disrupted heavy chain locus lacks a functional Cμ

gene segment.

8. A method of claim 2, wherein the transgenic mouse genome further comprises human kappa light chain immunoglobulin V, J and C gene segments capable of rearranging to encode a functional human light chain.

9. A method of claim 2, comprising expressing, in the host cell, the secondary repertoire antibody having full length immunoglobulin sequences.

10. A method of claim 9, further comprising purifying the full length immunoglobulins produced by the host cell.