Ion concentration-dependent binding molecule library

Abstract

Disclosed is a library consisting essentially of a plurality of antigen-binding molecules differing in sequence from each other, wherein an antigen-binding domain in each of the antigen-binding molecules comprises at least one amino acid residue that changes the antigen-binding activity of the antigen-binding molecule depending on ion concentration conditions. Also disclosed are a composition comprising a plurality of polynucleotide molecules each encoding the antigen-binding molecules, a composition comprising a plurality of vectors each comprising the polynucleotide molecules, a method for selecting the antigen-binding molecules, a method for isolating the polynucleotide molecules, a method for producing the antigen-binding molecules, and a pharmaceutical composition comprising any of the antigen-binding molecules.

1 .- 82 . (canceled) 
     
     
         83 . A library for screening an antigen-binding molecule, which binds to an antigen of interest, consisting of a plurality of antigen-binding molecules differing in sequence from each other, wherein an antigen-binding domain in each of the antigen-binding molecules is a heavy chain variable region and a light chain variable region of an antibody, and
 an amino acid residue that changes the antigen-binding activity of the antigen-binding molecule depending on pH conditions is comprised in either or both of the heavy chain variable region and the light chain variable region.   
     
     
         84 . The library according to  claim 83 , wherein the light chain variable region of the antigen-binding molecule comprises a germline sequence. 
     
     
         85 . The library according to  claim 83 , wherein a light chain framework region comprises a germline framework sequence. 
     
     
         86 . The library according to  claim 83 , wherein the heavy chain variable region has a naive sequence. 
     
     
         87 . The library according to  claim 83 , wherein the amino acid residue is an amino acid having a side chain pKa of 4.0 to 8.0. 
     
     
         88 . The library according to  claim 87 , wherein the amino acid residue is glutamic acid. 
     
     
         89 . The library according to  claim 83 , wherein the amino acid residue is an amino acid having a side chain pKa of 5.5 to 7.0. 
     
     
         90 . The library according to  claim 89 , wherein the amino acid residue is histidine. 
     
     
         91 . A library consisting of a plurality of fusion polypeptides each comprising antigen-binding molecules according to  claim 83 , wherein each of the fusion polypeptides is a fusion product of the heavy chain variable region of the antigen-binding molecule and at least a portion of a viral coat protein. 
     
     
         92 . The library according to  claim 91 , wherein the viral coat protein is selected from the group consisting of protein pIII, major coat protein pVIII, pVII, pIX, Soc, Hoc, gpD, pv1, and variants thereof. 
     
     
         93 . A composition comprising a plurality of polynucleotide molecules each encoding antigen-binding molecules differing in sequence from each other according to  claim 83 . 
     
     
         94 . A composition comprising a plurality of vectors each comprising a plurality of polynucleotide molecules according to  claim 93  in an operably linked state. 
     
     
         95 . The composition according to  claim 94 , wherein the vectors are replicable expression vectors. 
     
     
         96 . The composition according to  claim 95 , wherein each of the replicable expression vectors is an expression vector in which the polynucleotide is operably linked to a promoter region selected from the group consisting of a lacZ promoter system, an alkaline phosphatase phoA promoter (Ap), a bacteriophage XPL promoter (temperature-sensitive promoter), a tac promoter, a tryptophan promoter, a pBAD promoter, and a bacteriophage T7 promoter. 
     
     
         97 . The composition according to  claim 95 , wherein each of the replicable expression vectors is an M13, f1, fd, or Pf3 phage or a derivative thereof, or a lambdoid phage or a derivative thereof. 
     
     
         98 . A composition comprising a plurality of viruses each comprising vectors according to  claim 94 . 
     
     
         99 . A composition comprising a plurality of viruses each displaying on their surface antigen-binding molecules differing in sequence from each other according to  claim 83 . 
     
     
         100 . A library comprising antigen-binding molecules differing in sequence from each other according to  claim 83  wherein the library has 1×10 6  to 1×10 14  distinct variable region sequences. 
     
     
         101 . The library according to  claim 100 , wherein the library has 1×10 8  or more distinct variable region sequences. 
     
     
         102 . A method for preparing a library for screening an antigen-binding molecule, which binds to an antigen of interest,
 wherein the library is consisting of a plurality of antigen-binding molecules differing in sequence from each other and an antigen-binding domain in each of the antigen-binding molecules is a heavy chain variable region and a light chain variable region of an antibody, and   the method comprising producing a plurality of antigen-binding molecules designed so that the antigen-binding domain in each of the antigen-binding molecules comprises an amino acid residue that changes the antigen-binding activity of the antigen-binding molecule depending on pH conditions in either or both of the heavy chain variable region and the light chain variable region.   
     
     
         103 . (canceled) 
     
     
         104 . The preparation method according to  claim 102 , wherein the heavy chain variable region of each of the antigen-binding molecules is fused with at least a portion of a viral coat protein. 
     
     
         105 . The preparation method according to  claim 102 , wherein the viral coat protein is selected from the group consisting of protein pIII, major coat protein pVIII, pVII, pIX, Soc, Hoc, gpD, pv1, and variants thereof. 
     
     
         106 . A method for selecting an antigen-binding molecule whose antigen-binding activity is changed depending on pH conditions, the method comprising the steps of:
 a) preparing a library consisting of antigen-binding molecules differing in sequence from each other according to  claim 83 ;   b) contacting the library with antigens under two or more different pH conditions;   c) sorting, from the library, a subpopulation of antigen-binding molecules whose antigen-binding activity is changed depending on the pH conditions; and   d) isolating each antigen-binding molecule whose antigen-binding activity is changed depending on the pH conditions from the subpopulation sorted in the step c).   
     
     
         107 . A method for isolating a polynucleotide encoding an antigen-binding molecule whose antigen-binding activity is changed depending on pH conditions, the method comprising the steps of:
 a) preparing a library comprising a plurality of replicable expression vectors each comprising, in an operably linked state, a plurality of polynucleotides each encoding antigen-binding molecules differing in sequence from each other according to  claim 83 ;   b) allowing a plurality of viruses each transformed with the expression vectors contained in the library to express on their surface the antigen-binding molecules or the fusion polypeptides differing in sequence from each other encoded by the polynucleotides;   c) contacting the plurality of viruses with antigens under two or more different pH conditions;   d) sorting, from the library, a subpopulation of viruses whose antigen-binding activity is changed depending on the pH conditions;   e) isolating each virus whose antigen-binding activity is changed depending on the pH conditions from the virus subpopulation sorted in the step d); and   f) isolating the polynucleotides from the isolated virus.   
     
     
         108 . The method according to  claim 107 , wherein the steps c) and d) are additionally repeated at least once. 
     
     
         109 . The method according to  claim 106 , wherein an antigen-binding molecule having lower antigen-binding activity in an acidic pH condition than that in a neutral pH condition is selected. 
     
     
         110 . The method according to  claim 109 , wherein the acidic pH condition is pH 4.0 to 6.5. 
     
     
         111 . The method according to  claim 109 , wherein the neutral pH condition is pH 6.7 to 10.0. 
     
     
         112 . A method for producing an antigen-binding molecule whose antigen-binding activity is changed depending on pH conditions, the method comprising the steps of:
 a) preparing a library comprising a plurality of replicable expression vectors each comprising, in an operably linked state, a plurality of polynucleotides each encoding antigen-binding molecules differing in sequence from each other according to  claim 83 ;   b) allowing a plurality of viruses each transformed with the expression vectors contained in the library to express on their surface the antigen-binding molecules or the fusion polypeptides differing in sequence from each other encoded by the polynucleotides;   c) contacting the plurality of viruses with antigens under two or more different pH conditions;   d) sorting, from the library, a subpopulation of viruses whose antigen-binding activity is changed depending on the pH conditions;   e) isolating each virus whose antigen-binding activity is changed depending on the pH conditions from the virus subpopulation sorted in the step d);   f) isolating the polynucleotides from the isolated virus;   g) culturing a host cell transfected with a vector having an operably linked insert of the isolated polynucleotides; and   h) collecting the antigen-binding molecules from the cultures of the cell cultured in the step g).   
     
     
         113 . A method for producing an antigen-binding molecule whose antigen-binding activity is changed depending on pH conditions, the method comprising the steps of:
 a) preparing a library comprising a plurality of replicable expression vectors each comprising, in an operably linked state, a plurality of polynucleotides each encoding antigen-binding molecules differing in sequence from each other according to  claim 83 ;   b) allowing a plurality of viruses each transformed with the expression vectors contained in the library to express on their surface the antigen-binding molecules or the fusion polypeptides differing in sequence from each other encoded by the polynucleotides;   c) contacting the plurality of viruses with antigens under two or more different pH conditions;   d) sorting, from the library, a subpopulation of viruses whose antigen-binding activity is changed depending on the pH conditions;   e) isolating each virus whose antigen-binding activity is changed depending on the pH conditions from the virus subpopulation sorted in the step d);   f) isolating the polynucleotides from the isolated virus;   g) linking the isolated polynucleotides in frame with a polynucleotide encoding an antibody constant region;   h) culturing a host cell transfected with a vector having an operably linked insert of the polynucleotides linked in the step g); and   i) collecting the antigen-binding molecules from the cultures of the cell cultured in the step h).   
     
     
         114 . The production method according to  claim 112 , wherein the steps c) and d) are additionally repeated at least once. 
     
     
         115 . The production method according to  claim 112 , wherein an antigen-binding molecule having lower antigen-binding activity in an acidic pH condition than that in a neutral pH condition is selected. 
     
     
         116 . The production method according to  claim 115 , wherein the acidic pH condition is pH 4.0 to 6.5. 
     
     
         117 . The production method according to  claim 115 , wherein the neutral pH condition is pH 6.7 to 10.0. 
     
     
         118 . An antigen-binding molecule produced by a production method according to  claim 112 .