Transgenic Non-Human Animals For Producing Chimeric Antibodies

• US 20100287629A1
• Filed: 09/26/2008
• Published: 11/11/2010
• Est. Priority Date: 03/09/1994
• Status: Active Grant

• Current Assignee: Nils Lonberg, Robert M. Kay
• Original Assignee: Nils Lonberg, Robert M. Kay
• Inventors: Lonberg, Nils, Kay, Robert M.
• Granted Patent: US 8,158,419 B2
• Time in Patent Office: Days
• US Class Current: 800/4
• 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 67/0275 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: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C07K 16/462 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: C12N 15/8509 for producing genetically modified animals, e.g. transgenic

Abstract

The invention relates to transgenic non-human animals capable of producing heterologous antibodies and methods for producing human sequence antibodies which bind to human antigens with substantial affinity.

Claims

1-32. -32. (canceled)

33. An isolated B lymphocyte hybridoma clone wherein the B lymphocyte hybridoma clone expresses an antibody comprising a heavy chain consisting of a human immunoglobulin heavy chain variable region and a mouse immunoglobulin heavy chain gamma constant region.

34. A method for identifying a murine IgG isotype immunoglobulin molecule comprising a human variable region that specifically binds to a predetermined antigen, wherein the immunoglobulin molecule is expressed by a B lymphocyte hybridoma clone, which method comprises:

(i) incubating a first antibody mixture containing the murine IgG isotype immunoglobulin molecule with said antigen under conditions sufficient to allow for specific binding,

(ii) modifying the incubation conditions to remove non-specifically bound antibodies,

(iii) incubating a second antibody mixture containing an anti-murine IgG antibody comprising a marker under conditions sufficient to allow for specific binding of the anti-murine IgG antibody to murine IgG antibody,

(iv) modifying the incubation conditions to remove non-specifically bound anti-murine IgG antibodies,

(v) identifying the murine IgG immunoglobulin molecule which specifically binds to the predetermined antigen by identifying the presence of the marker.

35. A trans genic mouse comprising an unrearranged human heavy chain immunoglobulin variable region,

wherein the transgenic mouse has a genome comprising inactivated endogenous murine heavy chain variable gene regions, and further comprising an unrearranged human heavy chain immunoglobulin variable region operably linked to a mu constant region gene segment,

wherein the unrearranged human heavy chain variable region comprises multiple human V_H gene segments, the human V_H gene segments comprising V_H251 and V_H105, multiple human D gene segments, and multiple human J_H gene segments and wherein the mu constant region is selected from the group consisting of:

(i) a human S-mu sequence and a mu coding sequence, and

(ii) a murine S-mu sequence and a mu coding sequence, and

wherein B lymphocytes of the transgenic mouse express an antibody comprising a human immunoglobulin heavy chain variable region.

36. A method for obtaining a somatically mutated antibody that binds to a predetermined antigen, which method comprises:

(a) immunizing a transgenic mouse with a predetermined antigen, wherein the transgenic mouse has a genome comprising an unrearranged human heavy chain immunoglobulin variable region operably linked to a mu constant region gene segment,

wherein the unrearranged human heavy chain variable region comprises multiple human V_H gene segments, multiple human D gene segments, and multiple human J_H gene segments; and

wherein the mu constant region comprises a human S-mu sequence and a human mu coding sequence or a murine S-mu sequence and a murine mu coding sequence,

(b) removing B lymphocytes from the transgenic mouse,

(c) identifying mouse IgG isotype immunoglobulin molecules that bind to the predetermined antigen expressed by the B lymphocytes; and

(d) isolating the IgG isotype immunoglobulin molecules that bind to the predetermined antigen, whereby a somatically mutated antibody that binds to the predetermined antigen is obtained from the B lymphocytes of step (c).

37. A transgenic mouse comprising an unrearranged human heavy chain immunoglobulin variable region,

wherein the variable region comprises human V_H gene segments V_H251 and V_H105, multiple human D gene segments, and multiple human J_H gene segments, operably linked to an Smu sequence, and further comprising a B lymphocyte that expresses a somatically mutated heavy chain immunoglobulin consisting of a human immunoglobulin heavy chain variable region and a mouse immunoglobulin heavy chain gamma or alpha constant region.

38. A method for preparing a human heavy chain variable region containing immunoglobulin, which method comprises culturing a host cell comprising a human immunoglobulin heavy chain recombinant construct comprising a nucleic acid molecule that encodes a human heavy chain variable region from a mouse B lymphocyte operably linked to a human heavy chain constant region,

wherein the mouse B lymphocyte is from a transgenic mouse having an unrearranged human heavy chain immunoglobulin variable region comprising multiple human V_H gene segments, the human V_H gene segments comprising V_H251 and V_H105, multiple human D gene segments and a multiple human J_H gene segments, operably linked to a S_mu sequence, under conditions that permit expression of the human heavy chain variable region.

41. A method for constructing a human heavy chain, which method comprises operably linking a nucleic acid molecule encoding a human heavy chain variable region to a nucleic acid molecule encoding a human heavy chain constant region, wherein the human heavy chain variable region is isolated from a mouse B lymphocyte, wherein the mouse B lymphocyte is from a transgenic mouse having an unrearranged human heavy chain immunoglobulin variable region comprising multiple human V_H gene segments, the human V_H gene segments comprising V_H251 and V_H105, multiple human D gene segments and a multiple human J_H gene segments, operably linked to a S_mu sequence, under conditions that permit expression of the human heavy chain variable region.