Transgenic non-human animals capable of producing heterologous antibodies

• US 5,874,299 A
• Filed: 02/14/1997
• Issued: 02/23/1999
• 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: Lonberg, Nils, Kay, Robert M.
• Primary Examiner(s): Chambers, Jasemine C.
• Assistant Examiner(s): HAUDA, KAREN MARIE
• Application Number:
  US08/800,353
• Time in Patent Office: 739 Days
• Field of Search: 800/2, 435/320.1, 435/7.1, 435/69.1, 435/172.3, 935/34, 935/56, 935/71, 536/23.1, 536/23.53
• US Class Current: 435/320.1
• 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 67/0276 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 15/907 : 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, i.e., antibodies encoded by immunoglobulin heavy and light chain genes not normally found in the genome of that species of non-human animal. In one aspect of the invention, transgenes encoding unrearranged heterologous human immunoglobulin heavy and light chains are introduced into a non-human animal thereby forming a transgenic animal capable of producing antibodies 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. The invention also includes methods to generate a synthetic immunoglobulin variable region gene segment repertoire used in transgene construction and methods to induce heterologous antibody production using animals containing heterologous rearranged or unrearranged heavy and light chain immunoglobulin transgenes.

Claims

1. A transgene comprising, in operable linkage for expression of human immunoglobulin heavy chains, a plurality of human heavy chain VH gene segments, a plurality of human heavy chain D gene segments, a plurality of human heavy chain J gene segments, a mu constant region comprised of a mu switch region located upstream from a mu coding segment, and a gamma constant region comprised of a gamma switch region located upstream from a human gamma coding segment, wherein the gamma constant region is in closer proximity to the mu constant region than in a naturally occurring human immunoglobulin heavy chain gene locus.

2. A transgene comprising, in operable linkage for expression of human immunoglobulin heavy chains, a plurality of human VH gene segments, a plurality of human D gene segments, a plurality of human JH gene segments, an immunoglobulin heavy chain enhancer, a mu constant region comprised of a mu switch region located upstream from a mu constant gene segment, a gamma constant region comprised of a gamma switch region located upstream from a human gamma constant gene segment, wherein said transgene has at least one discontinuity of at least 2 kb between said mu and gamma gene segments as compared to a human germline heavy chain locus.

3. The transgene of claim 2, wherein the gamma switch region comprises an approximately 5.3 kb HindIII fragment.

4. The transgene of claim 2, wherein the gamma switch region comprises an approximately 4 kb HindIII fragment.

5. A transgene comprising in operable linkage a plurality of human VH gene segments, a plurality of human D gene segments, a plurality of human JH gene segments, a human mu CH gene, at least two different non-mu human CH genes and associated isotype switching sequences, wherein human mu and human gamma switch sequences are located in closer proximity to each other than in a naturally occurring human immunoglobulin locus.

6. The transgene of claim 5, wherein the gamma switch sequences are contained within an approximately 5.3 kb HindIII fragment.

7. The transgene of claim 5, wherein the gamma switch sequences are contained within an approximately 4 kb HindIII fragment.