Heteromeric variable regions with unvaried human framework regions

Abstract

The invention provides a method of conferring donor CDR binding affinity onto an antibody acceptor variable region framework. The method consists of: (a) constructing a population of altered antibody variable region encoding nucleic acids, said population comprising encoding nucleic acids for an acceptor variable region framework containing a plurality of different amino acids at one or more acceptor framework region amino acid positions and donor CDRs containing a plurality of different amino acids at one or more donor CDR amino acid positions; (b) expressing said population of altered variable region encoding nucleic acids, and (c) identifying one or more altered variable regions having binding affinity substantially the same or greater than the donor CDR variable region. The acceptor variable region framework can be a heavy or light chain variable region framework and the populations of heavy and light chain altered variable regions can be expressed alone to identify heavy or light chains having binding affinity substantially the same or greater than the donor CDR variable region. The populations of heavy and light chains additionally can be coexpressed to identify heteromeric altered variable region binding fragments. The invention also provides a method of simultaneously grafting and optimizing the binding affinity of a variable region binding fragment. The method consists of: (a) constructing a population of altered heavy chain variable region encoding nucleic acids comprising an acceptor variable region framework containing donor CDRs and a plurality of different amino acids at one or more framework region and CDR amino acid positions; (b) constructing a population of altered light chain variable region encoding nucleic acids comprising an acceptor variable region framework containing donor CDRs and a plurality of different amino acids at one or more framework regions and CDR amino acid positions; (c) coexpressing said populations of heavy and light chain variable region encoding nucleic acids to produce diverse combinations of heteromeric variable region binding fragments, and (d) identifying one or more heteromeric variable region binding fragments having affinity substantially the same or greater than the donor CDR heteromeric variable region binding fragment. A method of optimizing the binding affinity of an antibody variable region is also provided. The method consists of: (a) constructing a population of antibody variable region encoding nucleic acids, said population comprising two or more CDRs containing a plurality of different amino acids at one or more CDR amino acid positions; (b) expressing said population of variable region encoding nucleic acids, and (c) identifying one or more variable regions having binding affinity substantially the same or greater than the donor CDR variable region. The variable region populations can be heavy or light chains and can be expressed as individual populations or they can be coexpressed to produce heteromeric variable region binding fragments.

1 - 24 . (cancelled).  
     
     
         25 . A heteromeric variable region having higher antigen binding affinity than a donor heteromeric variable region, wherein said donor heteromeric variable region comprises three light chain donor CDRs and three heavy chain donor CDRs, and wherein said heteromeric variable region comprises: 
 a) a light chain altered variable region comprising; 
 i) four unvaried human germline light chain framework regions, and  
 ii) three light chain altered variable region CDRs, wherein at least one of said three light chain altered variable region CDRs is a light chain donor CDR variant, and wherein said light chain donor CDR variant comprises a different amino acid at only one, two, three or four positions compared to one of said three light chain donor CDRs, and  
   b) a heavy chain altered variable region comprising; 
 i) four unvaried human germline heavy chain framework regions, and  
 ii) three heavy chain altered variable region CDRs, wherein at least one of said three heavy chain altered variable region CDRs is a heavy chain donor CDR variant, and wherein said heavy chain donor CDR variant comprises a different amino acid at only one, two, three, or four positions compared to one of said three heavy chain donor CDRs.  
   
     
     
         26 . The heteromeric variable region of  claim 25 , wherein one of said three light chain altered variable region CDRs is identical to one of said three light chain donor CDRs.  
     
     
         27 . The heteromeric variable region of  claim 25 , wherein two of said three light chain altered variable region CDRs are each identical to one of said three light chain donor CDRs.  
     
     
         28 . The heteromeric variable region of  claim 25 , wherein one of said three heavy chain altered variable region CDRs is identical to one of said three heavy chain donor CDRs.  
     
     
         29 . The heteromeric variable region of  claim 25 , wherein two of said three heavy chain altered variable region CDRs are each identical to one of said three light chain donor CDRs.  
     
     
         30 . The heteromeric variable region of  claim 25 , wherein at least two of said three light chain altered variable region CDRs are light chain donor CDR variants.  
     
     
         31 . The heteromeric variable region of  claim 25 , wherein three of said three light chain altered variable region CDRs are light chain donor CDR variants.  
     
     
         32 . The heteromeric variable region of  claim 25 , wherein at least two of said three heavy chain altered variable region CDRs are heavy chain donor CDR variants.  
     
     
         33 . The heteromeric variable region of  claim 25 , wherein three of said three heavy chain altered variable region CDRs are heavy chain donor CDR variants.  
     
     
         34 . The heteromeric variable region of  claim 25 , wherein said donor heteromeric variable region is murine.  
     
     
         35 . The heteromeric variable region of  claim 25 , wherein said higher antigen binding affinity is at least 2-fold higher than the affinity of said donor heteromeric variable region.  
     
     
         36 . The heteromeric variable region of  claim 25 , wherein said higher antigen binding affinity is at least 3-fold higher than the affinity of said donor heteromeric variable region.  
     
     
         37 . A method of expressing a heteromeric variable region having higher antigen binding affinity than a donor heteromeric variable region, wherein said donor heteromeric variable region comprises three light chain donor CDRs and three heavy chain donor CDRs, and wherein said method comprises; 
 a) providing; 
 i) a first oligonucleotide encoding an altered light chain variable region, wherein said altered light chain variable region comprises: 
 A) four unvaried human germline light chain framework regions, and  
 B) three light chain altered variable region CDRs, wherein at least one of said three light chain altered variable region CDRs is a light chain donor CDR variant, and wherein said light chain donor CDR variant comprises a different amino acid at only one, two, three or four positions compared to one of said three light chain donor CDRs, and  
 
 ii) a second oligonucleotide encoding an altered heavy chain variable region, wherein said altered heavy chain variable region comprises; 
 A) four unvaried human germline heavy chain framework regions, and  
 B) three heavy chain altered variable region CDRs, wherein at least one of said three heavy chain altered variable region CDRs is a heavy chain donor CDR variant, and wherein said heavy chain donor CDR variant comprises a different amino acid at only one, two, three, or four positions compared to one of said heavy chain donor CDRs, and  
 
   b) expressing said first and second oligonucleotides under conditions such that a heteromeric variable region binding fragment is generated that exhibits higher antigen binding affinity than said donor heteromeric variable region.    
     
     
         38 . The method of  claim 37 , wherein said expressing is coexpressing.  
     
     
         39 . A method of expressing a heteromeric variable region having higher antigen binding affinity than a donor heteromeric variable region, wherein said donor heteromeric variable region comprises three light chain donor CDRs and three heavy chain donor CDRs, said method comprising; 
 a) providing; 
 i) first oligonucleotides encoding four unvaried human germline light chain framework regions,  
 ii) a population of second oligonucleotides encoding: 
 A) first light chain CDRs, wherein said first light chain CDRs comprise donor CDR variants, wherein said donor CDR variants comprise a different amino acid at only one, two, three or four positions compared to one of said three light chain donor CDRs,  
 B) second light chain CDRs, wherein said second light chain CDRs encode each of said three light chain donor CDRs;  
 
 iii) third oligonucleotides encoding four unvaried human germline heavy chain framework regions, and  
 iv) a population of fourth oligonucleotides encoding: 
 A) first heavy chain CDRs, wherein said first heavy chain CDRs comprise donor CDR variants, wherein said donor CDR variants comprise a different amino acid at only one, two, three or four positions compared to one of said three heavy chain donor CDRs,  
 B) second heavy chain CDRs, wherein said second heavy chain CDRs encode each of said three heavy chain donor CDRs; and  
 
   b) mixing said first oligonucleotides and said population of second oligoncucleotides such that a population of fifth oligonucleotides encoding light chain variable regions is generated, wherein at least one of said light chain variable regions encoded by said population of fifth oligonucleotides comprises i) an unvaried human germline light chain framework, and ii) at least one light chain donor CDR variant;    c) mixing said third oligonucleotides and said population of fourth oligonucleotides such that a population of sixth oligonucleotides encoding heavy chain variable regions is generated, wherein at least one of said heavy chain variable regions encoded by said population of sixth oligonucleotides comprises; i) an unvaried human germline heavy chain framework, and ii) at least one heavy chain donor CDR variant; and    d) expressing said fifth and sixth populations of oligonucleotides to produce combinations of heteromeric variable region binding fragments.    
     
     
         40 . The method of  claim 39 , further comprising step e) identifying at least one heteromeric variable region having higher antigen binding affinity than said donor heteromeric variable region.  
     
     
         41 . The method of  claim 39 , wherein said unvaried human germline light chain framework regions comprises FR1, FR2, FR3 and FR4 regions configured to hybridize to said light chain donor CDRs and said light chain donor CDR variants such that said population of fifth oligonucleotides encoding light chain variable regions is generated.  
     
     
         42 . The method of  claim 39 , wherein said unvaried human germline heavy chain framework regions comprises FR1, FR2, FR3 and FR4 regions configured to hybridize to said heavy chain donor CDRs and said heavy chain donor CDR variants such that said population of fifth oligonucleotides encoding heavy chain variable regions is generated.