Immunoglobulin variable region cassette exchange

Abstract

The invention provides methods for generating human antibodies with the specificity of a reference antibody by replacement of portions of the V H and V L sequences of the reference antibody with sequences from human antibody repertoires. The invention also provides novel compositions comprising hybrid immunoglobulin variable domains containing a combination of frameworks (FRs) and CDRs from different antibody clones.

1 . A method of engineering an antibody that retains the binding specificity of a reference antibody for a target antigen, the method comprising: 
 (a) obtaining a variable region from the reference antibody;    (b) replacing at least one exchange cassette of a V gene segment of the variable region of the reference antibody with a library of corresponding exchange cassettes from human V-gene segments, thereby generating a library of hybrid V-regions, 
 with the proviso that the exchange cassette has less than three framework regions;  
   (c) pairing the library of hybrid V regions of (b) with a complementary V-region; and    (d) selecting an antibody comprising a hybrid V region having at least one exchanged cassette that has a binding affinity for the target antigen.    
     
     
         2 . The method of  claim 1 , wherein at least one of the CDRs of the exchange cassette is a partial CDR sequence.  
     
     
         3 . The method of  claim 1 , wherein at least one of the FRs of the exchange cassette is a partial FR sequence.  
     
     
         4 . The method of  claim 1 , wherein the complementary V-region has a naturally occurring V-segment.  
     
     
         5 . The method of  claim 1 , wherein the complementary V-region has a germline V-segment.  
     
     
         6 . The method of  claim 1 , wherein the complementary V-region is a hybrid V-region.  
     
     
         7 . The method of  claim 1 , wherein the complementary V-region is a hybrid V region that is a member of a library that comprises different hybrid V-regions.  
     
     
         8 . The method of  claim 1 , wherein the exchange cassette is selected from the group consisting of FR1-CDR1, FR1-CDR1-FR2, FR2-CDR2-FR3, and CDR2-FR3.  
     
     
         9 . The method of  claim 1 , further comprising: 
 (e) replacing a second exchange cassette of the V region of the reference antibody with a library of corresponding exchange cassettes from human V-gene segments to create a second hybrid library of hybrid V regions;    (f) pairing the second library of hybrid V regions with a complementary V-region;    (g) selecting an antibody comprising a second hybrid V region, which antibody has a binding affinity for the target antigen; and    (h) combining the human exchange cassette of the engineered antibody of (d) with the second human exchange cassette of the antibody of (g), to obtain an antibody with the binding specificity of the reference antibody, wherein the antibody has a hybrid V-region that comprises at least two human exchange cassettes.    
     
     
         10 . The method of  claim 9 , further comprising a step of replacing the CDR3-FR4 of the hybrid V-region with a library of CDR3-FR4 regions, pairing the variable region with a complementary variable region, and selecting an antibody that retains the binding specificity for the target antigen.  
     
     
         11 . The method of  claim 9 , further comprising a step of replacing the FR4 of the hybrid V-region with a library of FR4 sequences.  
     
     
         12 . The method of  claim 9 , wherein at least one CDR of the exchange cassette of (e) is a partial CDR sequence.  
     
     
         13 . The method of  claim 9 , wherein the complementary V-region of (f) comprises a naturally occurring V-segment.  
     
     
         14 . The method of  claim 9 , wherein the complementary V-region of (f) is a hybrid V-region.  
     
     
         15 . The method of  claim 9 , wherein the complementary V-region of (f) is a hybrid V region that is a member of a library that comprises different hybrid V-regions.  
     
     
         16 . The method of  claim 1 , wherein the variable region is from the heavy chain of the reference antibody.  
     
     
         17 . The method of  claim 1 , wherein the variable region is from the light chain of the reference antibody.  
     
     
         18 . The method of  claim 1 , wherein the antibody is an Fv fragment, an Fab, an Fab′, an F(ab′)2, or an scFv.  
     
     
         19 . The method of  claim 1 , wherein the antibodies are expressed and secreted in soluble form from a host cell and bind to an antigen.  
     
     
         20 . The method of  claim 19 , wherein the host cell is a prokaryotic cell, a yeast, or a mammalian cell.  
     
     
         21 . The method of  claim 1 , wherein the antibodies are displayed on a cell, a spore, or a virus.  
     
     
         22 . A method of engineering an antibody that retains the binding specificity of a reference antibody for a target antigen, the method comprising: 
 (a) obtaining a variable region of a reference antibody having a desired binding specificity;    (b) replacing the FR1-CDR1-FR2 of the variable region of the reference antibody with a library of human FR1-CDR1-FR2 regions to create a library of hybrid variable regions, pairing the hybrid variable regions with a complementary variable region, and selecting an antibody having a detectable affinity for the target antigen;    (c) replacing the FR2-CDR2-FR3 of the variable region of the reference antibody with a library of human FR2-CDR2-FR3 regions to create a library of hybrid variable regions, pairing the hybrid variable regions with a complementary variable region, and selecting an antibody having a detectable affinity for the target antigen;    (d) combining the FR1-CDR1-FR2 of the hybrid variable region of the antibody selected in (b) with the FR2-CDR2-FR3 of the hybrid variable region of the antibody selected in (c) to obtain an antibody with a human variable region V segment, which antibody has the binding specificity of the reference antibody.    
     
     
         23 . The method of  claim 22 , wherein (b) and (c) are performed sequentially.  
     
     
         24 . The method of  claim 22 , wherein the complementary variable region of (b) or (c) comprises a naturally occurring V-segment.  
     
     
         25 . The method of  claim 22 , wherein the complementary variable region of (b) or (c) is a hybrid V-region.  
     
     
         26 . The method of  claim 22 , wherein the complementary variable region of (b) or (c) has a germline V-segment.  
     
     
         27 . The method of  claim 22 , wherein the complementary variable region of (b) or (c) is a member of a library of hybrid V-regions.  
     
     
         28 . The method of  claim 22 , wherein the variable region of the reference antibody is a heavy chain variable region.  
     
     
         29 . The method of  claim 22 , wherein the variable region of the reference antibody is a light chain variable region.  
     
     
         30 . The method of  claim 22 , wherein combining the FR1-CDR1-FR2 with the FR2-CDR2-FR3 comprises combining the FR2 regions in a region of homology.  
     
     
         31 . The method of  claim 22 , wherein combining the FR1-CDR1-FR2 with the FR2-CDR2-FR3 comprises replacing the FR2 from FR1-CDR1-FR2 with the FR2 from FR2-CDR2-FR3, or replacing the FR2 from FR2-CDR2-FR3 with the FR2 from FR1-CDR1-FR2.  
     
     
         32 . The method of  claim 22 , further comprising a step of replacing the CDR3-FR4 of the hybrid variable region with a library of human CDR3-FR4 regions, pairing the variable region with a complementary variable region, and selecting an antibody that binds to the target antigen.  
     
     
         33 . The method of  claim 32 , wherein the CDR3 regions of the library of human CDR3-FR4 regions are partial CDR3 regions.  
     
     
         34 . The method of  claim 22 , further comprising: 
 (v) replacing the -FR3-CDR3-FR4 of the variable region comprising the human V segment of (iv) with a library of FR3-CDR3-FR4 regions, pairing the variable region with a complementary variable region, and selecting an antibody has a detectable affinity for the target antigen.    (vi) combining the FR3-CDR4-FR4 of the hybrid variable region of the antibody selected in (v) with the FR2-CDR2-FR3 of the hybrid variable region of the antibody selected in (iv) to obtain an antibody with a human variable region V segment, which antibody has the binding specificity of the reference antibody.    
     
     
         35 . The method of  claim 22 , wherein one or more of the human exchange cassettes is germline.  
     
     
         36 . The method of  claim 22 , wherein the antibody is an Fv fragment, an Fab, an Fab′, an F(ab′)2, or an scFv.  
     
     
         37 . The method of  claim 22 , wherein the antibodies are expressed and secreted in soluble form from a host cell and bind to an antigen.  
     
     
         38 . The method of  claim 37 , wherein the host cell is a prokaryotic cell, a yeast cell, or a mammalian cell.  
     
     
         39 . The method of  claim 22 , wherein the antibodies are displayed on the surface of a cell, a spore, or a virus.  
     
     
         40 . A method of engineering an antibody that retains the binding specificity of a reference antibody for a target antigen, the method comprising: 
 (a) obtaining a variable region of a reference antibody having a desired binding specificity;    (b) replacing the FR1-CDR1-FR2 of the variable region of the reference antibody with a library of human FR1-CDR1-FR2 regions to create a library of hybrid variable regions,    pairing the hybrid variable regions with a complementary variable region, and selecting an antibody having a detectable affinity for the target antigen;    (c) replacing the CDR2-FR3 of the variable region of the reference antibody with a library of human CDR2-FR3 regions to create a library of hybrid variable regions, wherein the CDR2 of the CDR2-FR3 of the reference antibody is a partial CDR2 and the library of human CDR2-FR3 sequences comprise corresponding partial CDR2-FR3 sequences,    pairing the hybrid variable regions with a complementary variable region, and    selecting an antibody having a detectable affinity for the target antigen,    (d) combining the FR1-CDR1-FR2 of the hybrid variable region of the antibody selected in (b) with the CDR2-FR3 of the hybrid variable region of the antibody selected in (c) to obtain an antibody with a human variable region V segment, which antibody has the binding specificity of the reference antibody.    
     
     
         41 . The method of  claim 40 , further comprising a step of replacing the CDR3-FR4 of the reference antibody a library of human CDR3-FR4 regions, pairing the variable region with a complementary variable region, and selecting an antibody that binds to the target antigen.  
     
     
         42 . The method of  claim 41 , wherein the CDR3 regions of the library of human CDR3-FR4 regions are partial CDR3 regions.  
     
     
         43 . The method of  claim 40 , further comprising: 
 (e) replacing the FR4 of the variable region of the reference antibody c with a library of FR4 regions, pairing the variable region with a complementary variable region, and selecting an antibody has a detectable affinity for the target antigen.    
     
     
         44 . The method of  claim 40 , wherein one or more of the human exchange cassettes is germline.  
     
     
         45 . The method of  claim 40 , wherein the antibody is an Fv fragment, an Fab, an Fab′, an F(ab′)2, or an scFv.  
     
     
         46 . The method of  claim 40 , wherein the antibodies are expressed and secreted in soluble form from a host cell and bind to an antigen.  
     
     
         47 . The method of  claim 46 , wherein the host cell is a prokaryotic cell, a yeast cell, or a mammalian cell.  
     
     
         48 . The method of  claim 40 , wherein the antibodies are displayed on the surface of a cell, a spore, or a virus.  
     
     
         49 . An engineered antibody having the binding specificity of a reference antibody, the engineered antibody comprising: 
 a variable domain comprising a V-gene segment having a human exchange cassette from one human antibody gene and a second exchange cassette from a different human antibody gene, with the proviso that each exchange cassette has less than three framework regions; and    a CDR sequence, or a part of the CDR sequence, from the reference antibody.    
     
     
         50 . The antibody of  claim 49 , wherein the first and second exchange cassettes are human germline sequences.  
     
     
         51 . The antibody of  claim 49 , wherein the CDR sequence, or the part of the CDR sequence, is a CDR3 sequence, or partial CDR3 sequence.  
     
     
         52 . The antibody of  claim 51 , comprising a human FR4 sequence.  
     
     
         53 . The antibody of  claim 51 , wherein the partial CDR3 sequence is an MEBSD from the reference antibody.  
     
     
         54 . The antibody of  claim 49 , wherein the partial CDR3 sequence is the D segment sequence of a CDR3 from the reference antibody.  
     
     
         55 . The antibody of  claim 51 , wherein the CDR3 is a nonhuman CDR3.  
     
     
         56 . An engineered antibody in which at least one FR3 of a reference antibody has been replaced with a human FR3.  
     
     
         57 . The antibody of  claim 56 , wherein the FR3 that is replaced is a heavy chain FR3.  
     
     
         58 . The antibody of  claim 56 , wherein the FR3 that is replaced is a light chain FR3.  
     
     
         59 . The antibody of  claim 56 , wherein both the heavy chain and the light chain FR3 is replaced.  
     
     
         60 . A library of hybrid V-regions comprising members that have a diversity of V-regions, wherein a member has at least a MEBSD of a CDR from a reference antibody and at least one exchange cassette from an antibody repertoire, with the proviso that the exchange cassette has less than three framework regions  
     
     
         61 . The library of  claim 60 , wherein the antibody repertoire is a human repertoire.  
     
     
         62 . The library of  claim 60 , wherein the exchange cassette is selected from the group consisting of FR1-CDR1, FR1-CDR1-FR2, FR2-CDR2-FR3, and CDR2-FR3.  
     
     
         63 . The library of  claim 60 , wherein at least one of the CDRs of the exchange cassette is a partial CDR.  
     
     
         64 . The library of  claim 60 , wherein the member of the library has at least two exchange cassettes from a human repertoire.  
     
     
         65 . The library of  claim 60 , wherein the exchange cassette is a human germline sequence.  
     
     
         66 . The library of  claim 60 , wherein the MEBSD is from the CDR3 of the reference antibody.  
     
     
         67 . The library of  claim 66 , wherein the member of the library has a CDR3 from the reference antibody.  
     
     
         68 . The library of  claim 67 , wherein the member has a CDR3 from the reference antibody and a human FR4.  
     
     
         69 . The library of  claim 67 , wherein the member has a CDR3 in which the D segment is from the reference antibody.