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

• US 5,569,825 A
• Filed: 12/17/1991
• Issued: 10/29/1996
• 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:
  US07/810,279
• Time in Patent Office: 1,778 Days
• Field of Search: 800/2, 536/23.53
• 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 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: 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 2830/008 : cell type or tissue specific enhancer/promoter combination
• CPC Class Codes: C12N 2830/15 : chimeric enhancer/promoter combination
• CPC Class Codes: C12N 2830/36 : being a transcription termination element
• CPC Class Codes: C12N 2830/60 : from viruses
• CPC Class Codes: C12N 2840/44 : being a specific part of the splice mechanism, e.g. donor, acceptor
• 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 of multiple isotypes. Heterologous antibodies are encoded by immunoglobulin heavy chain 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 that permit isotype switching of encoded unrearranged heterologous human immunoglobulin heavy chains are introduced into a non-human animal thereby forming a transgenic animal capable of producing 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 having a genome comprising a germline copy of an unrearranged human heavy chain immunoglobulin minilocus transgene comprising 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, and wherein B lymphocytes of said transgenic mouse rearrange said unrearranged human heavy chain transgene by V-D-J joining to produce a V-D-J gene joined in-frame encoding a heavy chain variable region expressed in polypeptide linkage to the constant region encoded by said human gamma constant gene segment on said transgene by intratransgene isotype switching, and wherein said minilocus 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, wherein said mouse expresses human IgM heavy chains, and as a result of isotype switching, human IgG heavy chains.

2. A transgenic mouse of claim 1, wherein said human heavy chain transgene comprises a 5.3 kb HindIII fragment of a human heavy chain gene locus containing the gamma-1 switch region and the first exon of a preswitch sterile transcript, and wherein said B lymphocytes rearrange said human heavy chain transgene forming a V-D-J gene joined in-frame to encode a heavy chain variable region which is expressed in polypeptide linkage to said human gamma chain constant region in B lymphocytes of said transgenic mouse.

3. A transgenic mouse of claim 2, wherein said transgene further comprises a 0.7 kb XbaI/HindIII fragment of a human heavy chain gene locus, said 0.7 kb XbaI/HindIII fragment having sequences immediately upstream of said 5.3 kb gamma-1 fragment and said transgene further comprising a neighboring upstream 3.1 kb XbaI fragment of said human heavy chain gene locus.

4. A transgenic mouse of claim 1, wherein said human heavy chain transgene comprises a human gamma-1 constant region including the associated switch region and sterile transcript associated exons, together with approximately 4 kb flanking sequences upstream of the sterile transcript initiation site, and a rat heavy chain 3'"'"' enhancer that can be PCR amplified with the following oligonucleotide primers:

##STR40##

5. A transgenic mouse of claim 4, wherein said human heavy chain transgene comprises a NotI insert of pHC1.

6. A transgenic mouse of claim 3, wherein said human heavy chain transgene undergoes isotype switching whereby said V-D-J gene joined in-frame encodes a human heavy chain variable region which is initially expressed in peptide linkage to a human mu constant region and subsequently expressed in peptide linkage to a human gamma constant region in B lymphocytes of said transgenic mouse.

7. A transgenic mouse having an intact integrated germline copy of a human heavy chain transgene having a NotI insert of pHC1, wherein said transgenic mouse expresses, in its serum, human mu and human gamma-1 immunoglobulin chains.

8. A transgenic mouse comprising an intact integrated germline copy of a NotI insert of pHC1, wherein said transgenic mouse expresses in serum antibodies having human sequence gamma-1 chains, each human gamma-1 chain comprising a variable region having a polypeptide sequence encoded by a human V_H gene segment, a human D gene segment, and a human J_H gene segment, joined in-frame as a VDJ gene.