Manipulation of immunoglobulin gene diversity and multi-antibody therapeutics

Abstract

The invention provides improved non-human vertebrates and non-vertebrate cells capable of expressing antibodies comprising human variable region sequences. The present invention is directed to the provision of long HCDR3s from non-human vertebrates and cells. The present invention is also directed to the provision of novel V, D and J pairings in immunoglobulin heavy and light chain loci. Novel, biased antibody diversities and potentially expanded diversities are provided. The invention also provides for novel and potentially expanded diversity or diversity that is biased towards variable gene usage common to antibodies useful for treating and/or preventing certain diseases or conditions, such as infectious diseases. The invention also provides methods of generating antibodies using such vertebrates, as well as the antibodies per se, therapeutic compositions thereof and uses.

What is claimed is: 
     
         1 . A method of producing an antigen specific antibody or antigen binding fragment thereof, said antibody comprising a human immunoglobulin heavy (IgH) chain, wherein said human IgH chain comprises a human IgH chain variable region and a human IgH chain constant region, and said fragment comprising a human IgH chain variable region, the method comprising:
 expressing the antibody or antigen binding fragment thereof from a host cell comprising nucleic acid encoding said human IgH chain variable region and said human IgH chain constant region of said antibody, or comprising nucleic acid encoding said human IgH chain variable region of said fragment,   wherein said human IgH chain variable region of said antigen-specific antibody or antigen binding fragment is of a B cell, or a hybridoma thereof, of a transgenic mouse contacted with said antigen;   wherein said transgenic mouse comprises a germline comprising:
 (i) an immunoglobulin heavy (IgH) chain locus comprising a plurality of human VH, one or more human D and one or more human JH gene segments at an endogenous locus upstream of and operably linked to a constant region;
 wherein said plurality of human VH gene segments is selected from at least two of the group consisting of: IGHV3-7*01, IGHV3-9*01, IGHV7-4-1*01, IGHV1-3*01, IGHV4-4*02, IGHV3-13*01, IGHV3-23*04 and VH3-20*d01, and where one or more human D gene segments is selected from the group consisting of: IGHD2-2*01, IGHD3-9*01, IGHD3-10*01, IGHD6-13*01, IGHD4-17*01, IGHD6-19*01, IGHD3-22*01 and IGHD1-26*01, 
 wherein each of said selected human VH gene segments of said plurality of human VH gene segments is capable of joining with a human D gene segment and a human JH gene segment to encode a variable region, 
 wherein each of said selected human VH gene segments of said plurality of human VH gene segments is capable of joining with a human D gene segment and a human JH segment to encode a variable region comprising a HCDR3 of 20 or more amino acids in length in said mouse; 
 
 (ii) an immunoglobulin kappa light (Igκ) chain locus comprising one or more human Igκ variable (Vκ) gene segments and one or more human Igκ joining (Jκ) gene segments upstream of and operatively linked to a constant region; 
 wherein said one or more Vκ gene segments is selected from the group consisting of a VκI gene family member, κ1-15A (KL012), a VκII family member, a VκIII family member, VκII A2, VκII A2a and Vκ A27 (Humkv325), 
 such that said transgenic mouse is capable, upon contact with an antigen, of producing a plurality of antibodies specific for said antigen comprising an immunoglobulin heavy chain comprising a human VH region and a C region; 
 wherein said plurality of antibodies specific for said antigen comprises a human VH region having an HCDR-3 length of at least 20 amino acids. 
   
     
     
         2 . The method of  claim 1 , wherein said one or more human D gene segments comprises IGHD2-2*02, IGHD3-9*01, IGHD3-10*01, IGHD6-13*01, IGHD4-17*01, IGHD6-19*01, IGHD3-22*01 and IGHD1-26*01. 
     
     
         3 . The method of  claim 1 , wherein the C region of the IgH locus is a mouse C region. 
     
     
         4 . The method of  claim 1 , wherein said B cells of said transgenic mouse contacted with antigen are isolated. 
     
     
         5 . The method of  claim 4 , comprising the step of isolating antigen-specific antibody from said isolated B cells. 
     
     
         6 . The method of  claim 4 , further comprising the step of isolating from said B cells nucleic acid encoding said human VH region and said mouse C region of said chimeric immunoglobulin heavy chain. 
     
     
         7 . The method of  claim 6 , further comprising the step of replacing in said nucleic acid encoding said human VH region and said mouse C region of said chimeric immunoglobulin heavy chain polypeptide, the portion encoding said mouse C region with nucleic acid encoding a human C region, thereby forming a nucleic acid encoding said human VH region and said human C region. 
     
     
         8 . The method of  claim 4 , further comprising isolating an antibody that binds said antigen, comprising
 (i) immortalizing said isolated B cells expressing said antigen-specific antibody or progeny thereof, optionally producing hybridomas therefrom; and   (ii) isolating an antibody expressed by the immortalized B cells of (i).   
     
     
         9 . The method of  claim 1 , wherein before said contact with antigen, said mouse is naïve. 
     
     
         10 . The method of  claim 1 , wherein said antibody is an IgG-type antibody. 
     
     
         11 . The method of  claim 1 , wherein said antigen is an antigen of an infectious disease pathogen. 
     
     
         12 . The method of  claim 1 , wherein the antigen is of a viral pathogen. 
     
     
         13 . The method of  claim 1 , wherein expression of mouse Ig heavy chain comprising a mouse IgH variable region and a mouse IgH constant region is inactive in said mouse. 
     
     
         14 . A method of producing an antigen specific antibody or antigen binding fragment thereof, said antibody comprising a human immunoglobulin heavy (IgH) chain, wherein said human IgH chain comprises a human IgH chain variable region and a human IgH chain constant region, and said fragment comprising a human IgH chain variable region, the method comprising expressing the antibody or antigen binding fragment thereof from a host cell comprising nucleic acid encoding said human IgH chain variable region and said human IgH chain constant region of said antibody, or comprising nucleic acid encoding said human IgH chain variable region of said fragment,
 wherein said human IgH chain variable region of said antigen-specific antibody or antigen binding fragment is of a B cell, or a hybridoma thereof, of a transgenic mouse contacted with said antigen;   wherein said transgenic mouse comprises a germline comprising:
 (i) wherein said immunoglobulin heavy (IgH) chain locus comprising a plurality of human VH, one or more human D and one or more human JH gene segments at an endogenous locus upstream of and operably linked to a constant region;
 wherein IGHV3-7*01, IGHV3-9*01, IGHV7-4-1*01, IGHV1-3*01, IGHV4-4*02, IGHV3-13*01, IGHV3-23*04, and VH3-20*d01, and where one or more human D gene segments is selected from the group consisting of: IGHD2-2*01, IGHD3-9*01, IGHD3-10*01, IGHD6-13*01, IGHD4-17*01, IGHD6-19*01, IGHD3-22*01 and IGHD1-26*01, 
 wherein said plurality of human VH gene segments comprise human VH gene segments capable of joining with a human D gene segment and a human JH gene segment to encode a variable region, 
 wherein said plurality of human VH gene segments comprise human VH gene segments capable of joining with a human D gene segment and a human JH segment to encode a variable region comprising a HCDR3 of 20 or more amino acids in length in said mouse; 
 
 (ii) an immunoglobulin kappa light (Igκ) chain locus comprising one or more human Igκ variable (Vκ) gene segments and one or more human Igκ joining (Jκ) gene segments upstream of and operatively linked to a constant region; 
 wherein said one or more Vκ gene segments is selected from the group consisting of a VκI gene family member, κ1-15A (KL012), a VκII family member, a VκIII family member, VκII A2, VκII A2a and Vκ A27 (Humkv325), 
   such that said transgenic mouse is capable, upon stimulation with an antigen, of producing a plurality of antibodies specific for said antigen comprising an immunoglobulin heavy chain comprising a human VH region and a C region;   wherein said plurality of antibodies specific for said antigen comprises a human VH region having an HCDR-3 length of at least 20 amino acids.   
     
     
         15 . The method of  claim 14 , wherein said one or more human D gene segments comprises IGHD2-2*02, IGHD3-9*01, IGHD3-10*01, IGHD6-13*01, IGHD4-17*01, IGHD6-19*01, IGHD3-22*01 and IGHD1-26*01. 
     
     
         16 . The method of  claim 14 , wherein the C region of the IgH locus is a mouse C region. 
     
     
         17 . The method of  claim 14 , wherein said B cells of said transgenic mouse contacted with antigen are isolated. 
     
     
         18 . The method of  claim 17 , further comprising the step of isolating antigen-specific antibody from said isolated B cells. 
     
     
         19 . The method of  claim 17 , further comprising the step of isolating from said B cells nucleic acid encoding said human VH region and said mouse C region of said chimeric immunoglobulin heavy chain polypeptide. 
     
     
         20 . The method of  claim 19 , further comprising the step of replacing in said nucleic acid encoding said human VH region and said mouse C region of said chimeric immunoglobulin heavy chain polypeptide, the portion encoding said mouse C region with nucleic acid encoding a human C region, thereby forming a nucleic acid encoding said human VH region and said human C region. 
     
     
         21 . The method of  claim 17 , further comprising isolating an antibody that binds said antigen, comprising
 (i) immortalizing said isolated B cells expressing said antigen-specific antibody or progeny thereof, optionally producing hybridomas therefrom; and   (ii) isolating an antibody expressed by the immortalized B cells of (i).   
     
     
         22 . The method of  claim 14 , wherein before said contact with antigen, said mouse is naïve. 
     
     
         23 . The method of  claim 14 , wherein said antibody is an IgG-type antibody. 
     
     
         24 . The method of  claim 14 , wherein said antigen is an antigen of an infectious disease pathogen. 
     
     
         25 . The method of  claim 14 , wherein the antigen is of a viral pathogen. 
     
     
         26 . The method of  claim 14 , wherein expression of mouse Ig heavy chain comprising a mouse IgH variable region and a mouse IgH constant region is inactive in said mouse.