Neonatal Fc receptor (FcRn)-binding polypeptide variants, dimeric Fc binding proteins and methods related thereto

Abstract

The compositions and methods of the present invention are based, in part, on our discovery that an effector function mediated by an Fc-containing polypeptide can be altered by modifying one or more amino acid residues within the polypeptide (by, for example, electrostatic optimization). The polypeptides that can be generated according to the methods of the invention are highly variable, and they can include antibodies and fusion proteins that contain an Fc region or a biologically active portion thereof.

1 . An altered polypeptide comprising at least an FcRn binding portion of an Fc region wherein said polypeptide comprises at least one mutation compared to a starting polypeptide and wherein the at least one mutation is selected from the group consisting of: 
 a substitution at EU amino acid position 248 with a charged amino acid;    a substitution at EU amino acid position 249 with a positively charged amino acid;    a substitution at EU amino acid position 251 with a polar amino acid or lysine;    a substitution at EU amino acid position 252 with a polar amino acid;    a substitution at EU amino acid position 255 with a polar amino acid;    a substitution at EU amino acid position 256 with lysine;    a substitution at EU amino acid position 257 with a charged amino acid;    a substitution at EU amino acid position 258 with a polar amino acid or a charged amino acid;    a substitution at EU amino acid position 277;    a substitution at EU amino acid position 279 with a charged amino acid;    a substitution at EU amino acid position 280 with a charged amino acid;    a substitution at EU amino acid position 281 with a charged amino acid or glutamine;    a substitution at EU amino acid position 282 with a charged amino acid;    a substitution at EU amino acid position 284 with a polar amino acid or a charged amino acid;    a substitution at EU amino acid position 285 with a positively charged amino acid, a polar amino acid, or aspartate;    a substitution at EU amino acid position 286 with glutamate, threonine, or methionine;    a substitution at EU amino acid position 287 with a polar amino acid or a charged amino acid;    a substitution at EU amino acid position 288 with a charged amino acid;    a substitution at EU amino acid position 289;    a substitution at EU amino acid position 304 with a polar amino acid or a charged amino acid;    a substitution at EU amino acid position 305 with a polar amino acid or a charged amino acid;    a substitution at EU amino acid position 306;    a substitution at EU amino acid position 307 with a polar or charged amino acid;    a substitution at EU amino acid position 308 with a charged amino acid;    a substitution at EU amino acid position 309 with a charged amino acid;    a substitution at EU amino acid position 310 with a charged amino acid or a polar amino acid;    a substitution at EU amino acid position 311 with a positively charged amino acid;    a substitution at EU amino acid position 312 with a positively charged amino acid or a polar amino acid;    a substitution at EU amino acid position 313 with a charged amino acid;    a substitution at EU amino acid position 315 with a charged amino acid;    a substitution at EU amino acid position 316 with a positively charged amino acid;    a substitution at EU amino acid position 317 with a charged amino acid or a polar amino acid;    a substitution at EU amino acid position 340 with a charged amino acid;    a substitution at EU amino acid position 343 with a polar amino acid or a charged amino acid;    a substitution at EU amino acid position 344 with leucine;    a substitution at EU amino acid position 345 with a polar amino acid or a charged amino acid;    a substitution at EU amino acid position 376 with a polar amino acid or a charged amino acid;    a substitution at EU amino acid position 378 with serine;    a substitution at EU amino acid position 383 with a charged amino acid;    a substitution at EU amino acid position 385 with a charged amino acid;    a substitution at EU amino acid position 389 with a negatively charged amino acid;    a substitution at EU amino acid position 424 with a charged amino acid;    a substitution at EU amino acid position 426 with a charged amino acid;    a substitution at EU amino acid position 430 with a polar amino acid or a charged amino acid;    a substitution at EU amino acid position 431 with a charged amino acid;    a substitution at EU amino acid position 432 with a polar amino acid;    a substitution at EU amino acid position 434 with lysine, arginine, or leucine;    a substitution at EU amino acid position 436 with a negatively charged amino acid; and    a substitution at EU amino acid position 438 with a charged amino acid.    
     
     
         2 . The altered polypeptide of  claim 1 , wherein the amino acid at least one of EU amino acid positions 277, 289, 306, 344, or 378 is replaced with a charged amino acid, a polar amino acid, or a nonpolar amino acid.  
     
     
         3 . The altered polypeptide of  claim 1 , wherein the altered polypeptide is an antibody or fragment thereof.  
     
     
         4 . The altered polypeptide of  claim 1 , wherein the altered polypeptide is a fusion protein.  
     
     
         5 . The altered polypeptide of  claim 1 , wherein the Fc region or the FcRn binding portion thereof is derived from a human antibody.  
     
     
         6 . The altered polypeptide of  claim 1 , which comprises a complete Fc region.  
     
     
         7 . The altered polypeptide of  claim 1 , wherein the starting polypeptide comprises the amino acid sequence of SEQ ID NO:2.  
     
     
         8 . The altered polypeptide of  claim 1 , wherein the polypeptide comprises one or more non-human amino acids residues in a complementarity determining region (CDR) of V L  or V H .  
     
     
         9 . The altered polypeptide of  claim 3 , wherein the polypeptide binds (a) an antigen and (b) an FcR.  
     
     
         10 . The altered polypeptide of  claim 4 , wherein the polypeptide binds (a) a ligand and (b) an FcR.  
     
     
         11 . The altered polypeptide of  claim 1 , wherein the polypeptide binds the FcR with different binding affinity compared to the starting polypeptide that does not contain the mutation.  
     
     
         12 . The altered polypeptide of  claim 11 , wherein the altered polypeptide exhibits one binding affinity for the FcR at a first pH, and exhibits a different binding affinity for the FcR at a second pH.  
     
     
         13 . The altered polypeptide of claims  1 , wherein the altered polypeptide, when administered to a patient, exhibits a circulatory half-life that is different from the starting polypeptide that does not contain the mutation.  
     
     
         14 . The altered polypeptide of  claim 1 , wherein the altered polypeptide binds to Protein A or G.  
     
     
         15 . A pharmaceutical composition comprising the altered polypeptide of  claim 1 .  
     
     
         16 . A nucleic acid molecule comprising a nucleotide sequence encoding the polypeptide of  claim 1 .  
     
     
         17 . The nucleic acid molecule of  claim 16 , which is in an expression vector.  
     
     
         18 . A host cell comprising the expression vector of  claim 17 .  
     
     
         19 . A method for treating a patient suffering from a disorder, the method comprising administering to the patient an altered polypeptide comprising at least an FcRn binding portion of an Fc region comprising at least one mutation selected from the group consisting of: 
 a substitution at EU amino acid position 284 with glutamate;    a substitution at EU amino acid position 285 with glutamate;    a substitution at EU amino acid position 286 with aspartate;    a substitution at EU amino acid position 288 with glutamate or aspartate;    a substitution at EU amino acid position 290 with glutamate; and    a substitution at EU amino acid position 304 with aspartate,    wherein the altered polypeptide exhibits a circulatory half-life than is longer than the starting polypeptide that does not contain the mutation.    
     
     
         20 . A method for treating a patient suffering from a disorder, the method comprising administering to the patient an altered polypeptide comprising at least an FcRn binding portion of an Fc region comprising at least one mutation selected from the group consisting of: 
 a substitution at EU amino acid position 248 with aspartate;    a substitution at EU amino acid position 249 with arginine or lysine;    a substitution at EU amino acid position 250 with arginine or lysine;    a substitution at EU amino acid position 251 with arginine, lysine, or asparagine;    a substitution at EU amino acid position 252 with serine or threonine;    a substitution at EU amino acid position 254 with serine or threonine;    a substitution at EU amino acid position 256 with arginine, glutamate, or lysine;    a substitution at EU amino acid position 255 with leucine, aspartate or methionine;    a substitution at EU amino acid position 260 with lysine;    a substitution at EU amino acid position 257 with arginine, aspartate, glutamate, or lysine;    a substitution at EU amino acid position 277 with arginine, aspartate, glutamine, or lysine;    a substitution at EU amino acid position 279 with glutamate;    a substitution at EU amino acid position 281 with glutamine;    a substitution at EU amino acid position 282 with arginine, aspartate, glutamate, or lysine;    a substitution at EU amino acid position 287 with aspartate, glutamate, lysine, proline, or threonine;    a substitution at EU amino acid position 284 with aspartate;    a substitution at EU amino acid position 285 with aspartate or phenylalanine;    a substitution at EU amino acid position 286 with glutamate or methionine;    a substitution at EU amino acid position 288 with aspartate;    a substitution at EU amino acid position 290 with aspartate;    a substitution at EU amino acid position 304 with aspartate or glutamate;    a substitution at EU amino acid position 305 with arginine;    a substitution at EU amino acid position 306 with arginine, aspartate, glutamate, or lysine;    a substitution at EU amino acid position 307 with arginine, aspartate, or glutamate;    a substitution at EU amino acid position 309 with arginine, aspartate, lysine or glutamate;    a substitution at EU amino acid position 310 with arginine, leucine, lysine or asparagine;    a substitution at EU amino acid position 312 with arginine, asparagine, or lysine; a substitution at EU amino acid position 313 with aspartate, arginine, or lysine;    a substitution at EU amino acid position 315 with aspartate or glutamate;    a substitution at EU amino acid position 343 with glutamine or lysine;    a substitution at EU amino acid position 345 with arginine or glutamine;    a substitution at EU amino acid position 374 with arginine, lysine, or leucine;    a substitution at EU amino acid position 376 with asparagine;    a substitution at EU amino acid position 426 with arginine, aspartate, or glutamate;    a substitution at EU amino acid position 428 with arginine, glutamine, or lysine;    a substitution at EU amino acid position 430 with lysine;    a substitution at EU amino acid position 431 with proline;    a substitution at EU amino acid position 432 with arginine;    a substitution at EU amino acid position 434 with lecuine or lysine; and    a substitution at EU amino acid position 438 with glutamate    wherein the altered polypeptide exhibits a circulatory half-life than is shorter than the starting polypeptide that does not contain the mutation.    
     
     
         21 . A method of producing the altered polypeptide of  claim 1 , the method comprising: 
 (a) transfecting a cell with the nucleic acid molecule comprising a nucleotide sequence that encodes the altered polypeptide; and    (b) purifying the altered polypeptide from the cell or cell supernatant.    
     
     
         22 . A dimeric Fc binding protein comprising a first and second polypeptide chains, wherein the first and the second polypeptide chains each comprise at least one Fc region domain operably linked to an Fc binding domain.  
     
     
         23 . The dimeric Fc binding protein of  claim 22 , wherein said Fc domain is mutated to reduce or eliminate binding to FcRn.  
     
     
         24 . The dimeric Fc binding protein of  claim 22 , wherein said first and second polypeptide chains are covalently linked.  
     
     
         25 . The dimeric Fc binding protein of  claim 22 , wherein the Fc binding domain comprises the extracellular domain of FcRn.  
     
     
         26 . The dimeric Fc binding protein of  claim 22 , wherein the Fc binding domain is bound to beta-2-microglobulin.  
     
     
         27 . The dimeric Fc binding protein of  claim 22 , wherein the Fc binding domain is derived from human FcRn.  
     
     
         28 . The dimeric Fc binding protein of  claim 22 , wherein the binding protein comprises the amino acid sequence shown in SEQ ID NO:10.  
     
     
         29 . A method for measuring binding affinity of polypeptide comprising at least an FcRn binding portion of an Fc region for an FcR, the method comprising contacting a polypeptide comprising at least an FcRn binding portion of an Fc region with the dimeric Fc binding protein of  claim 22  and determining the affinity of the interaction.  
     
     
         30 . A method for screening a library of polypeptides which comprise at least an FcRn binding portion of an Fc region for those polypeptides having an altered binding affinity for FcRn, the method comprising 
 (a) contacting members of the library with the dimeric Fc binding protein of  claim 22;  and    (b) measuring the binding affinity of the polypeptides for the dimeric Fc binding protein; and    (c) selecting those polypeptides which have altered binding affinity for FcRn.    
     
     
         31 . A method for purifying a polypeptide at least an FcRn binding portion of an Fc region from a mixture of polypeptides, the method comprising applying the mixture to an affinity column containing the dimeric Fc binding protein of  claim 22 , eluting the polypeptide comprising at least an FcRn binding portion of an Fc region such that the polypeptide is purified.  
     
     
         32 . A method for identifying a polypeptide with an altered binding affinity for FcRn compared to a starting polypeptide, the method comprising: 
 (a) determining a spatial representation of an optimal charge distribution of the amino acids of the starting polypeptide and an associated change in binding free energy of the starting polypeptide when bound to FcRn in a solvent;    (b) identifying at least one candidate amino acid residue position of the starting polypeptide to be modified to alter the binding free energy of the starting polypeptide when bound to FcRn; and    (c) identifying an elected amino acid at the amino acid position, such that incorporation of the mutation in the starting polypeptide results in an altered polypeptide with an altered binding affinity for FcRn.    
     
     
         33 . The method of  claim 33 , further comprising incorporating the elected amino acid in the starting polypeptide.  
     
     
         34 . The method of  claim 34 , further comprising calculating the change in the free energy of binding of the altered Fc-containing polypeptide when bound to the FcRn, as compared to the starting polypeptide when bound to the FcRn.  
     
     
         35 . A method for identifying an altered Fc-containing polypeptide with an altered binding affinity for FcRn at two different pH levels, the method comprising: 
 (a) determining a spatial representation of an optimal charge distribution of the amino acids of the starting polypeptide and an associated change in binding free energy of the starting polypeptide when bound to FcRn in a solvent at a first pH level;    (b) determining a spatial representation of an optimal charge distribution of the amino acids of the starting polypeptide and an associated change in binding free energy of the starting polypeptide when bound to FcRn in a solvent at a second pH level;    (c) identifying, based on a comparison of the charge distributions, residues that exhibit different charge distributions at the first and second pH levels, at least one candidate amino acid residue position of the starting polypeptide to be modified to alter the binding free energy of the starting polypeptide when bound to FcRn; and    (d) selecting an elected amino acid at said amino acid position, such that incorporation of the elected amino acid in the starting polypeptide results in an altered Fc-containing polypeptide with an altered binding affinity for FcRn.    
     
     
         36 . The method of  claim 35 , wherein the first pH is about 7.4.  
     
     
         37 . The method of  claim 35 , wherein the affinity of a polypeptide is about 1.5-fold to about 100-fold greater at the first pH than at the second pH.  
     
     
         38 . An altered polypeptide that exhibits an affinity for an FcRn at a first pH, and exhibits a different affinity for an FcRn at a second pH, wherein the polypeptide comprises an amino acid sequence predicted by the method of  claim 34 .  
     
     
         39 . A pharmaceutical composition comprising the polypeptide of  claim 38 .  
     
     
         40 . A nucleic acid molecule comprising a nucleotide sequence encoding the polypeptide of  claim 39.