Novel fab fragment libraries and methods for their use

Abstract

The present invention provides Fab libraries and methods for using the Fab libraries to obtain antibodies against a target. The Fab library of the invention contains at least 10 9 different Fabs, and in some embodiments, at least 10 10 different Fabs. The Fab libraries of the invention are used to isolate polyclonal or monoclonal Fabs that bind with high specificity to targets.

1 . A plurality of polynucleotides encoding a Fab library, the library comprising a plurality of vectors wherein each vector of the plurality of vectors comprises: 
 a first cloning region and a second cloning region, wherein 
 each cloning region comprises at least one, for the vector unique, restriction enzyme cleavage site,  
 each cloning region being 5′ flanked by a ribosome binding site and a signal sequence,  
   a polynucleotide encoding an anchor region, located 3′ of the second cloning region,    a member of a first plurality of variable polynucleotides, said plurality of variable polynucleotides encoding a first plurality of polypeptides, wherein the member of the first plurality is cloned into the first cloning region of the vector, and the member of the first plurality of polypeptides encodes a polypeptide selected from the group consisting of a complete antibody variable region, a complete antibody variable region followed by a complete antibody constant region, a complete antibody variable region followed by a part of an antibody constant region, a part of an antibody variable region, a part of an antibody variable region followed by a complete antibody constant region or a part of an antibody variable region followed by a part of an antibody constant region;    a member of a second plurality of variable polynucleotides, said plurality of variable polynucleotides encoding a second plurality of polypeptides, wherein the member of the second plurality is cloned into the second cloning region of the vector, and the member of the second plurality of polypeptides encodes a polypeptide selected from the group consisting of a complete antibody variable region, a complete antibody variable region followed by a complete antibody constant region, a complete antibody variable region followed by a part of an antibody constant region, a part of an antibody variable region, a part of an antibody variable region followed by a complete antibody constant region or a part of an antibody variable region followed by a part of an antibody constant region; 
 and  
   a polynucleotide encoding a tag;    wherein the plurality of vectors comprises the first plurality of variable polynucleotides and the second plurality of variable polynucleotides.    
     
     
         2 . The plurality of polynucleotides according to  claim 1 , wherein the first plurality of variable polynucleotides are V L  polynucleotides, and the second plurality of variable polynucleotides are V H  polynucleotides.  
     
     
         3 . The plurality of polynucleotides according to any one of the preceding claims, wherein the plurality of polynucleotides encode a Fab library of at least 10 9  different Fabs.  
     
     
         4 . The plurality of polynucleotides according to  claim 3 , wherein the polynucleotides encode a Fab library of at least 10 10  different Fabs.  
     
     
         5 . The plurality of polynucleotides according to  claim 4 , wherein the polynucleotides encode a Fab library of at least 3.7×10 10  different Fabs.  
     
     
         6 . A plurality of vectors, wherein each vector of the plurality of vectors comprises 
 a first cloning region and a second cloning region, wherein 
 each cloning region comprises at least one, for the vector unique, restriction enzyme cleavage site,  
 each cloning region being 5′ flanked by a ribosome binding site and a signal sequence,  
   a polynucleotide encoding an anchor region, located 3′ of the second cloning region,    a member of a first plurality of variable polynucleotides, said plurality of variable polynucleotides encoding a first plurality of polypeptides, wherein the member of the first plurality is cloned into the first cloning region of the vector, and the member of the first plurality of polypeptides encodes a polypeptide selected from the group consisting of a complete antibody variable region, a complete antibody variable region followed by a complete antibody constant region, a complete antibody variable region followed by a part of an antibody constant region, a part of an antibody variable region, a part of an antibody variable region followed by a complete antibody constant region or a part of an antibody variable region followed by a part of an antibody constant region;    a member of a second plurality of variable polynucleotides, said plurality of variable polynucleotides encoding a second plurality of polypeptides, wherein the member of the second plurality is cloned into the second cloning region of the vector, and the member of the second plurality of polypeptides encodes a polypeptide selected from the group consisting of a complete antibody variable region, a complete antibody variable region followed by a complete antibody constant region, a complete antibody variable region followed by a part of an antibody constant region, a part of an antibody variable region, a part of an antibody variable region followed by a complete antibody constant region or a part of an antibody variable region followed by a part of an antibody constant region; 
 and  
   a polynucleotide encoding a tag,    wherein the plurality of vectors comprises the first plurality of polynucleotides and the second plurality of variable polynucleotides.    
     
     
         7 . The plurality of vectors according to  claim 6 , wherein the first plurality of variable polynucleotides are V L  polynucleotides, and the second plurality of variable polynucleotides are V H  polynucleotides.