Immunoglobulin Variable Region Libraries

Abstract

Antigen-specific immunoglobulin V-regions are identified from a library of nucleic acids amplified using polymerase chain reaction using leader sequence-specific forward primers. The use of leader sequence primers allows all V-region sequences to be amplified (including those with extensive 5′ end mutations) without loss of the original 5′ V gene segment sequence. These libraries can be screened for antigen-specific V-regions using eukaryotic cells engineered to express the amplified V-region-encoding nucleic acids or using bacterial phage display techniques. In the latter, a second V-region library is made using a larger than conventional set of 5′ V-region primers. The sequence errors introduced into the amplification products by this method are corrected using sequence information obtained in the products amplified by the V-region primers to screen the library created using the leader sequence primers. Amino acid sequence information from fragments of donor immunoglobulins can be used to assist in the identification of nucleic acids encoding the heavy and light chains of donor antibodies as well as to design primers to amplify such nucleic acids.

What is claimed is: 
     
         1 . A purified monoclonal antibody comprising a variable region amino acid sequence that promotes binding of the antibody to a target antigen and has somatic hypermutations in its first N-terminal 8 amino acids, wherein the variable region amino acid sequence is determined by screening a library of human immunoglobulin nucleotide sequences made by a method comprising the steps of:
 (a) using a first PCR to amplify human immunoglobulin heavy and light chain nucleotide sequences from a cDNA library made from a sample of human peripheral blood mononuclear cells obtained from a human donor previously determined to possess antibodies specific for target antigens of interest, wherein leader-specific forward primers that amplify at least 90% of the human immunoglobulin V-region genes and reverse primers for amplifying nucleic acid sequences encoding hinge or constant regions are used in the first PCR amplification;   (b) isolating the amplification products from the first PCR step;   (c) creating a first set of constructs by inserting the isolated amplification products from the first PCR step into expression vectors; and   (d) introducing the first set of constructs into a first set of host cells to create the library of human immunoglobulin nucleotide sequences.   
     
     
         2 . The purified monoclonal antibody of  claim 1 , wherein the leader-specific primers comprise a set of ten or more different polynucleotides each of which binds under stringent hybridization conditions to the complement of a different nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-76. 
     
     
         3 . The purified monoclonal antibody of  claim 1 , wherein the antibody comprises an immunoglobulin heavy chain having somatic hypermutations in the first N-terminal 8 amino acids of its variable region covalently linked to an immunoglobulin light chain having somatic hypermutations in the first N-terminal 8 amino acids of its variable region.