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 antibody comprising a variable region amino acid sequence that promotes binding of the immunoglobulin to a target antigen, wherein the variable region amino acid sequence is determined by screening a phage display library comprising variable region-encoding nucleic acid sequences isolated by PCR amplification of a cDNA library made from B lymphocytes using human immunoglobulin variable region leader-specific primers, wherein the human subject was previously determined to possess antibodies specific for the target antigen. 
     
     
         2 . The purified antibody of  claim 1 , wherein the variable region amino acid sequence has somatic hypermutations in its first N-terminal 8 amino acids. 
     
     
         3 . The purified 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.