Transgenic non-human animals capable of producing heterologous antibodies

• US 5,633,425 A
• Filed: 02/05/1992
• Issued: 05/27/1997
• Est. Priority Date: 08/29/1990
• Status: Expired due to Term

• Current Assignee: Genpharm Incorporated (Bristol-Myers Squibb Company), Genpharm International Inc (Bristol-Myers Squibb Company)
• Original Assignee: Genpharm International Inc (Bristol-Myers Squibb Company)
• Inventors: Kay, Robert M., Lonberg, Nils
• Primary Examiner(s): Ziska, Suzanne E.
• Application Number:
  US07/834,539
• Time in Patent Office: 1,938 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 2217/072 maintaining or altering function, i.e. knock in
• 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: 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 16/44 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

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 transgenic mouse having DNA segments from a human immunoglobulin heavy chain gene locus incorporated into its germline DNA to form a heavy chain immunoglobulin mini-locus such that upon antigenic stimulation the transgenic mouse develops primary response B cells expressing IgM having a mu chain encoded by a rearranged human mini-locus and secondary response B cells expressing somatically mutated IgG having a gamma chain encoded by the rearranged human mini-locus, the germline DNA of said transgenic mouse comprises:

an unrearranged human immunoglobulin mini-locus comprising a plurality of human heavy chain V 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 μ

switch region located upstream from a μ

coding segment, and a gamma constant region comprised of a γ

switch region located upstream from a human γ

coding segment, wherein the γ

constant region is in closer proximity to the μ

constant region that in the human immunoglobulin heavy chain gene locus;

said primary response B cells wherein after said antigenitic expressing IgM stimulation have chromosomal DNA comprised of said mu constant region and said human gamma constant and a rearranged variable region which is comprised of a VDJ rearrangement of said unrearranged human immunoglobulin minilocus, said rearranged variable region having N-region nucleotides at recombination joints between said human heavy chain V gene segments and human D gene segments and between said human D gene segments and said human heavy chain J segments, and wherein FR1, FR2, FR3, CDR1, and CDR2 portions of said rearranged variable region have DNA sequences from said human heavy chain V gene segments of said unrearranged human immunoglobulin mini-locus;

and wherein said secondary response B cells have chromosomal DNA exhibiting a class switch recombination from said mu constant region to said gamma constant region operably linked to said rearranged variable region;

wherein the sequence spanning the FR1, FR2, FR3, CDR1 and CDR2 portions includes a plurality of DNA sequences not identical to corresponding sequences from said unrearranged human immunoglobulin mini-locus.

2. The transgenic mouse of claim 1 wherein the human immunoglobulin mini-locus is comprised of between 2 and human V segments, between 2 and 15 human D segments, and between 2 and 6 human J segments.

3. The transgenic mouse of claim 1 wherein the human γ

switch region is contained within a human Hind III DNA fragment which further comprises a start site for a human γ

pre-switch sterile transcript.

4. The transgenic mouse of claim 3 wherein the Hind III fragment is an approximately 5.3 kb human Hind III fragment containing the γ

₁ switch region and further comprising a start site for a human γ

₁ pre-switch sterile transcript.

5. The transgenic mouse of claim 1 wherein the human immunoglobulin heavy chain mini-locus is further comprised of a heavy chain J-mu intronic enhancer.

6. The transgenic mouse of claim 1 wherein the human immunoglobulin mini-locus is further comprised of an immunoglobulin heavy chain 3 enhancer.

7. The transgenic mouse of claim 1 wherein the secondary response B cells lack a μ

coding region and have said μ

switch region contiguous to said γ

switch region.