Molecules with altered neonate fc receptor binding having enhanced therapeutic and diagnostic properties

Abstract

The present invention provides molecules, including proteins, more particularly, immunoglobulins whose in vivo half-lives are altered (increased or decreased) by the presence of an IgG, constant domain, or FcRn binding fragment thereof (e.g., an Fc region or hinge-Fc region) (e.g., from a human IgGI, e.g., human IgGI), that have modifications of one or more of amino acid residues in at least the CH3 domain.

What is claimed is: 
     
         1 . A modified IgG comprising an Fc region comprising amino acid substitutions at two or more of positions 432 to 437, numbered according to the EU numbering index of Kabat, relative to a wild-type Fc region; wherein
 (i) at least one of positions 432 and 437 is substituted with cysteine; or   (ii) at least one of positions 432 and 437 is substituted with an amino acid selected from the group consisting of glutamine, glutamic acid, aspartic acid, lysine, arginine, and histidine; and   wherein the modified IgG has an altered half-life compared to the half-life of an IgG having the wild-type Fc region.   
     
     
         2 . The modified IgG of  claim 1 , which is a human or humanized IgG. 
     
     
         3 . The modified IgG of  claim 1  or  2 , wherein
 (i) both of positions 432 and 437 are substituted with cysteine; or 
 (ii) both of positions 432 and 437 are substituted with an amino acid independently selected from the group consisting of glutamine, glutamic acid, aspartic acid, lysine, arginine, and histidine. 
 
     
     
         4 . The modified IgG of any of the preceding claims, further comprising an amino acid insertion after position 437. 
     
     
         5 . The modified IgG of  claim 4 , wherein the amino acid insertion is glutamic acid. 
     
     
         6 . The modified IgG of any of the preceding claims, wherein the binding affinity of the modified IgG for FcRn at pH 6.0 is higher than the binding affinity of the IgG having the wild-type Fc region for FcRn at pH 6. 
     
     
         7 . The modified IgG of any of the preceding claims, wherein the binding affinity of the modified IgG for FcRn at pH 7.4 is higher than the binding affinity of the IgG having the wild-type Fc region for FcRn at pH 7.4. 
     
     
         8 . The modified IgG of any of the preceding claims, wherein the KD of the modified IgG for FcRn at pH 6.0 is less than 500 nM, and the KD at pH 7.4 is at least 1000 nM. 
     
     
         9 . The modified IgG of any of  claims 1  to  7 , wherein the KD at pH 6.0 is less than 500 nM and the KD at pH 7.4 is less than 1000 nM. 
     
     
         10 . The modified IgG of any of  claims 1  to  7 , wherein the KD at pH 6.0 is greater than 500 nM and the KD at pH 7.4 is at least 1000 nM. 
     
     
         11 . The modified IgG of any of the preceding claims, wherein the modified IgG exhibits increased pH dependence of binding affinity for FcRn compared to the IgG having the wild-type Fc region. 
     
     
         12 . The modified IgG of any of  claims 1  to  10 , wherein the modified IgG exhibits decreased pH dependence of binding affinity for FcRn compared to the IgG having the wild-type Fc region. 
     
     
         13 . The modified IgG of any of the preceding claims, wherein the modified IgG retains wild-type levels of at least one attribute selected from the group consisting of (i) binding to at least one Fc gamma receptor, (ii) binding to C1q, or (iii) effector function. 
     
     
         14 . The modified IgG of  claim 13 , wherein the Fc gamma receptor is selected from the group consisting of an FcγRI receptor, an FcγRII receptor and an FcγRIII receptor. 
     
     
         15 . The modified IgG of  claim 13 , wherein the effector function is selected from the group consisting of antibody dependent cellular cytotoxicity (ADCC), complement dependent cytotoxicity (CDC), antibody dependent cellular phagocytosis (ADCP). 
     
     
         16 . The modified IgG of any of the preceding claims, wherein the modified IgG has amino acid substitutions of three or more of positions 432, 433, 434, 435, 436 or 437. 
     
     
         17 . The modified IgG of any of the preceding claims, wherein the modified IgG has amino acid substitutions of four or more of positions 432, 433, 434, 435, 436 or 437. 
     
     
         18 . The modified IgG of any of the preceding claims wherein position 432 and 437 are each substituted with cysteine; position 433 is histidine or is substituted with arginine, proline, threonine, lysine, serine, alanine, methionine, or asparagine; position 434 is asparagine or is substituted with arginine, tryptophan, histidine, phenylalanine, tyrosine, serine, methionine or threonine; position 435 is histidine or is substituted with histidine; and position 436 is tyrosine or phenylalanine or is substituted with leucine, arginine, isoleucine, lysine, methionine, valine, histidine, serine, or threonine. 
     
     
         19 . The modified IgG of  claim 18 , wherein position 433 is histidine. 
     
     
         20 . The modified IgG of  claim 18 , wherein position 433 is substituted with arginine, asparagine, proline, threonine, or lysine. 
     
     
         21 . The modified IgG of any of  claims 18  to  20 , wherein position 434 is substituted with arginine, tryptophan, histidine, phenylalanine, or tyrosine. 
     
     
         22 . The modified IgG of  claim 21 , wherein position 434 is substituted with arginine. 
     
     
         23 . The modified IgG of any of  claims 18  to  21 , wherein position 436 is substituted with leucine, arginine, isoleucine, lysine, methionine, valine or histidine. 
     
     
         24 . The modified IgG of  claim 18 , wherein position 433 is histidine or substituted with arginine, asparagine, proline, threonine, or lysine; position 434 is substituted with arginine, tryptophan, histidine, phenylalanine, or tyrosine; and position 436 is substituted with leucine, arginine, isoleucine, lysine, methionine, valine or histidine. 
     
     
         25 . The modified IgG of  claim 18  comprising the amino acid sequence at positions 432 to 437 of CXRHXC (SEQ ID NO:11), where position 433 is histidine or is substituted with arginine, asparagine, proline, or serine, and position 436 is substituted with arginine, leucine, isoleucine, methionine, or serine. 
     
     
         26 . The modified IgG of  claim 18  comprising the amino acid sequence at positions 432 to 437 of CRRHXC (SEQ ID NO:12) wherein position 436 is substituted with leucine, arginine, isoleucine, lysine, methionine, valine, histidine, serine, or threonine. 
     
     
         27 . The modified IgG of  claim 25 , wherein position 436 is substituted with leucine, isoleucine, serine or threonine. 
     
     
         28 . The modified IgG of  claim 18  comprising the amino acid sequence at positions 432 to 437 of CXRHRC (SEQ ID NO:13) wherein position 433 is arginine, proline, threonine, lysine, serine, alanine, methionine, or asparagine. 
     
     
         29 . The modified IgG of any of  claims 1  to  17  comprising the amino acid sequence at positions 432 to 437 of ZXXHXZ, wherein position 432 is substituted with glutamic acid, glutamine, histidine, or aspartic acid; position 433 is histidine or is substituted with arginine, alanine, lysine, threonine, leucine, proline, serine, or glutamine; position 434 is substituted with tyrosine, phenylalanine, histidine, serine or tryptophan; position 436 is tyrosine or substituted with arginine, histidine, asparagine, lysine, leucine, methionine, threonine, or valine; and position 437 is substituted with glutamine, histidine, glutamic acid, or aspartic acid. 
     
     
         30 . The modified IgG of  claim 27 , wherein position 432 is substituted with glutamic acid or histidine; position 433 substituted with arginine, alanine, lysine, threonine or leucine; position 434 is substituted with phenylalanine or tyrosine; position 436 is substituted with arginine, histidine, asparagine or lysine; and position 437 is substituted with glutamine or glutamic acid. 
     
     
         31 . The modified IgG of any of the preceding claims further comprising mutations at any one or more of positions 251-256, 285-290, 308-314, 385-389, and 428-431, according to the EU numbering index of Kabat. 
     
     
         32 . The modified IgG of  claim 31  further comprising a substitution with tyrosine at position 252, substitution with threonine 254, and substitution with glutamic acid at position 256. 
     
     
         33 . The modified IgG of any of  claims 1  to  3  selected from the group consisting of N3, YC37-YTE, YC56-YTE, YC59-YTE, Y3-YTE, Y31-YTE, Y12-YTE, Y83-YTE, Y37-YTE, and Y9-YTE, as shown in Table 1. 
     
     
         34 . The modified IgG of any of  claims 1  to  3  comprising the amino acid sequence at positions 432 to 437 selected from the group consisting of N3-YTE, N3E-YTE, SerN3-YTE, Y54-YTE, Y74-YTE, Y8-YTE, as shown in Table 1. 
     
     
         35 . The modified IgG of any of the preceding claims, wherein the modified IgG has histidine at amino acid position 435. 
     
     
         36 . The modified IgG of any of the preceding claims comprising the amino acid sequence of E(R/A)(W/S/F)HRQ (SEQ ID NO:15) at positions 432 to 437. 
     
     
         37 . The modified IgG of any of the preceding claims, wherein the modified IgG binds to an antigen selected from the group consisting of viral antigen, a bacterial antigen, a protist antigen, a prion and a mammalian self-antigen. 
     
     
         38 . A polypeptide comprising at least an FcRn-binding portion of an Fc region of an IgG molecule, wherein said FcRn-binding portion comprises amino acid substitutions at two or more of positions 432 to 437, numbered according to the EU numbering index of Kabat, relative to a wild-type FcRn-binding portion; wherein
 (i) at least one of positions 432 and 437 is substituted with cysteine; or   (ii) at least one of positions 432 and 437 is substituted with an amino acid selected from the group consisting of glutamine, glutamic acid, aspartic acid, and histidine.   
     
     
         39 . The polypeptide of  claim 38 , wherein
 (i) both of positions 432 and 437 are substituted with cysteine; or   (ii) both of positions 432 and 437 are substituted with an amino acid independently selected from the group consisting of glutamine, glutamic acid, aspartic acid, and histidine.   
     
     
         40 . The polypeptide of  claim 39 , wherein position 432 and 437 are each substituted with cysteine; position 433 is histidine or is substituted with arginine, proline, threonine, lysine, serine, alanine, methionine, or asparagine; position 434 is asparagine or is substituted with arginine, tryptophan, histidine, phenylalanine, tyrosine, serine, methionine or threonine; position 435 is histidine or is substituted with histidine; and position 436 is tyrosine or phenylalanine or is substituted with leucine, arginine, isoleucine, lysine, methionine, valine, histidine, serine, or threonine. 
     
     
         41 . The polypeptide of  claim 39  comprising the amino acid sequence at positions 432 to 437 of ZXXHXZ, wherein position 432 is substituted with glutamic acid, glutamine, histidine, or aspartic acid; position 433 is histidine or is substituted with arginine, alanine, lysine, threonine, leucine, proline, serine, or glutamine; position 434 is substituted with tyrosine, phenylalanine, histidine, serine or tryptophan; position 436 is tyrosine or substituted with arginine, histidine, asparagine, lysine, leucine, methionine, threonine, or valine; and position 437 is substituted with glutamine, histidine, glutamic acid, or aspartic acid. 
     
     
         42 . The polypeptide of  claim 41  comprising the amino acid sequence of E(R/A)(W/S/F)HRQ (SEQ ID NO:15) at positions 432 to 437. 
     
     
         43 . The polypeptide of any of  claim 41  or  42 , further comprising an amino acid insertion after position 437, wherein the amino acid insertion is glutamic acid. 
     
     
         44 . The polypeptide of any of  claims 38  to  43 , wherein the FcRn binding portion of the Fc region comprises from about amino acid residues 231-446 of an IgG molecule according to the EU numbering index of Kabat. 
     
     
         45 . The polypeptide of any of  claims 38  to  43 , wherein the FcRn binding portion of the Fc region comprises from about amino acid residues 216-446 of an IgG molecule according to the EU numbering index of Kabat. 
     
     
         46 . A fusion protein comprising a non-IgG polypeptide covalently linked to at least an FcRn-binding portion of an Fc region of an IgG molecule, wherein said FcRn-binding portion comprises amino acid substitutions at two or more of positions 432 to 437, numbered according to the EU numbering index of Kabat, relative to a wild-type Fc region; wherein
 (i) at least one of positions 432 and 437 is substituted with cysteine; or   (ii) at least one of positions 432 and 437 is substituted with an amino acid selected from the group consisting of glutamine, glutamic acid, aspartic acid, and histidine;   wherein said fusion protein has a longer or shorter in vivo half life than the non-IgG polypeptide.   
     
     
         47 . The fusion protein of  claim 46 , wherein
 (i) both of positions 432 and 437 are substituted with cysteine; or   (ii) both of positions 432 and 437 are substituted with an amino acid independently selected from the group consisting of glutamine, glutamic acid, aspartic acid, and histidine.   
     
     
         48 . The fusion protein of  claim 47 , wherein position 432 and 437 are each substituted with cysteine; position 433 is histidine or is substituted with arginine, proline, threonine, lysine, serine, alanine, methionine, or asparagine; position 434 is asparagine or is substituted with arginine, tryptophan, histidine, phenylalanine, tyrosine, serine, methionine or threonine; position 435 is histidine or is substituted with histidine; and position 436 is tyrosine or phenylalanine or is substituted with leucine, arginine, isoleucine, lysine, methionine, valine, histidine, serine, or threonine. 
     
     
         49 . The fusion protein of  claim 47  comprising the amino acid sequence at positions 432 to 437 of ZXXHXZ, wherein position 432 is substituted with glutamic acid, glutamine, histidine, or aspartic acid; position 433 is histidine or is substituted with arginine, alanine, lysine, threonine, leucine, proline, serine, or glutamine; position 434 is substituted with tyrosine, phenylalanine, histidine, serine or tryptophan; position 436 is tyrosine or substituted with arginine, histidine, asparagine, lysine, leucine, methionine, threonine, or valine; and position 437 is substituted with glutamine, histidine, glutamic acid, or aspartic acid. 
     
     
         50 . The fusion protein of  claim 49  comprising the amino acid sequence of E(R/A)(W/S/F)HRQ (SEQ ID NO:15) at positions 432 to 437. 
     
     
         51 . The fusion protein of any of  claim 49  or  50 , further comprising an amino acid insertion after position 437, wherein the amino acid insertion is glutamic acid. 
     
     
         52 . The fusion protein of any of  claims 46  to  51 , wherein the FcRn binding portion of the Fc region comprises from about amino acid residues 231-446 of an IgG molecule according to the EU numbering index of Kabat. 
     
     
         53 . The fusion protein of any of  claims 46  to  51 , wherein the FcRn binding portion of the Fc region comprises from about amino acid residues 216-446 of an IgG molecule according to the EU numbering index of Kabat. 
     
     
         54 . The fusion protein of any of  claims 46  to  53  wherein the non-IgG polypeptide is an immunomodulator, a receptor, a hormone or a drug. 
     
     
         55 . A molecule comprising a non-protein agent conjugated to at least an FcRn-binding portion of an Fc region of an IgG molecule, wherein said FcRn-binding portion comprises amino acid substitutions at two or more of positions 432 to 437, numbered according to the EU numbering index of Kabat, relative to a wild-type Fc region; wherein
 (i) at least one of positions 432 and 437 is substituted with cysteine; or   (ii) at least one of positions 432 and 437 is substituted with an amino acid selected from the group consisting of glutamine, glutamic acid, aspartic acid, and histidine;   wherein said molecule has a longer or shorter in vivo half life than the non-protein agent.   
     
     
         56 . The molecule of  claim 55 , wherein
 (i) both of positions 432 and 437 are substituted with cysteine; or   (ii) both of positions 432 and 437 are substituted with an amino acid independently selected from the group consisting of glutamine, glutamic acid, aspartic acid, and histidine.   
     
     
         57 . The molecule of  claim 56 , wherein position 432 and 437 are each substituted with cysteine; position 433 is histidine or is substituted with arginine, proline, threonine, lysine, serine, alanine, methionine, or asparagine; position 434 is asparagine or is substituted with arginine, tryptophan, histidine, phenylalanine, tyrosine, serine, methionine or threonine; position 435 is histidine or is substituted with histidine; and position 436 is tyrosine or phenylalanine or is substituted with leucine, arginine, isoleucine, lysine, methionine, valine, histidine, serine, or threonine. 
     
     
         58 . The molecule of  claim 56  comprising the amino acid sequence at positions 432 to 437 of ZXXHZZ, wherein position 432 is substituted with glutamic acid, glutamine, histidine, or aspartic acidaspartic acid; position 433 is histidine or is substituted with arginine, alanine, lysine, threonine, leucine, proline, serine, or glutamine; position 434 is substituted with tyrosine, phenylalanine, histidine, serine or tryptophan; position 436 is tyrosine or substituted with arginine, histidine, asparagine, lysine, leucine, methionine, threonine, or valine; and position 437 is substituted with glutamine, histidine, glutamic acid, or aspartic acid. 
     
     
         59 . The molecule of  claim 58  comprising the amino acid sequence of E(R/A)(W/S/F)HRQ (SEQ ID NO:15) at positions 432 to 437. 
     
     
         60 . The molecule of any of  claim 58  or  59 , further comprising an amino acid insertion after position 437, wherein the amino acid insertion is glutamic acid. 
     
     
         61 . The molecule of any of  claims 55  to  60 , wherein the FcRn binding portion of the Fc region comprises from about amino acid residues 231-446 of an IgG molecule according to the EU numbering index of Kabat. 
     
     
         62 . The molecule of any of  claims 55  to  60 , wherein the FcRn binding portion of the Fc region comprises from about amino acid residues 216-446 of an IgG molecule according to the EU numbering index of Kabat. 
     
     
         63 . A pharmaceutical composition comprising the modified human or humanized IgG, polypeptide, fusion protein, or molecule according to any of  claims 2  to  62 , and a pharmaceutically acceptable carrier. 
     
     
         64 . A nucleic acid comprising a nucleotide sequence encoding the modified IgG, polypeptide or fusion protein according to any of  claims 1  to  54 , or an FcRn binding fragment thereof. 
     
     
         65 . A vector comprising the nucleic acid of  claim 64 . 
     
     
         66 . The vector of  claim 65  comprising a cloning vector or an expression vector. 
     
     
         67 . A host cell comprising the nucleic acid according to  claim 64 . 
     
     
         68 . A kit comprising the modified human or humanized IgG, polypeptide, fusion protein, or molecule, according to any of  claims 2  to  62 . 
     
     
         69 . A method of treating a disease, disorder or infection comprising administering to a patient in need thereof a therapeutically effective amount of the modified human or humanized IgG, polypeptide, fusion protein or molecule, according to claim any of  claims 2  to  62 . 
     
     
         70 . The method of  claim 69  comprising administering the modified human or humanized IgG, fusion protein or molecule, in a lower dose, less frequently, or with fewer side effects than a comparable unmodified IgG, fusion protein, or molecule. 
     
     
         71 . A method of preventing a disease or disorder comprising administering to a subject a prophylactically effective amount of the modified human or humanized IgG, polypeptide, fusion protein or molecule, according to claim any of  claims 2  to  62 . 
     
     
         72 . A method of vaccinating a subject comprising administering to said subject an amount of the modified human or humanized IgG, polypeptide, fusion protein or molecule, according to claim any of  claims 2  to  62  effective to elicit an immune response. 
     
     
         73 . An in vivo method of diagnosing, monitoring or prognosing a disease, disorder or infection in a subject comprising:
 (a) administering to a subject an effective amount of the modified human or humanized IgG, polypeptide, fusion protein or molecule, according to claim any of  claims 2  to  62 , said modified human or humanized IgG specifically binding to an antigen associated with a disease, disorder or infection;   (b) allowing the modified human or humanized IgG, polypeptide, fusion protein or molecule to concentrate at sites in said subject where said antigen is found; and   (c) detecting said modified human or humanized IgG, polypeptide, fusion protein or molecule;   whereby detection of said modified human or humanized IgG, polypeptide, fusion protein or molecule above a background or standard level indicates that the subject has said disease disorder or infection.   
     
     
         74 . A method for altering the half-life of an IgG comprising introducing amino acid substitutions at two or more of positions 432 to 437, numbered according to the EU numbering index of Kabat; wherein
 (i) at least one of positions 432 and 437 is substituted with cysteine; or   (ii) at least one of positions 432 and 437 is substituted with an amino acid selected from the group consisting of glutamine, glutamic acid, aspartic acid, and histidine.   
     
     
         75 . The method of  claim 74  wherein
 (i) both of positions 432 and 437 are substituted with cysteine; or 
 (ii) both of positions 432 and 437 are substituted with an amino acid independently selected from the group consisting of glutamine, glutamic acid, aspartic acid, and histidine. 
 
     
     
         76 . The method of  claim 75  wherein position 432 and 437 are each substituted with cysteine; position 433 is histidine or is substituted with arginine, proline, threonine, lysine, serine, alanine, methionine, or asparagine; position 434 is asparagine or is substituted with arginine, tryptophan, histidine, phenylalanine, tyrosine, serine, methionine or threonine; position 435 is histidine or is substituted with histidine; and position 436 is tyrosine or phenylalanine or is substituted with leucine, arginine, isoleucine, lysine, methionine, valine, histidine, serine, or threonine. 
     
     
         77 . The method of  claim 75  wherein position 432 is substituted with glutamic acid, glutamine, histidine, or aspartic acid; position 433 is histidine or is substituted with arginine, alanine, lysine, threonine, leucine, proline, serine, or glutamine; position 434 is substituted with tyrosine, phenylalanine, histidine, serine or tryptophan; position 435 is histidine; position 436 is tyrosine or substituted with arginine, histidine, asparagine, lysine, leucine, methionine, threonine, or valine; and position 437 is substituted with glutamine, histidine, glutamic acid, or aspartic acid. 
     
     
         78 . A method for altering the half life of a non-IgG polypeptide or a non-protein agent comprising conjugating the non-IgG polypeptide or a non-protein agent to at least an FcRn-binding portion of an Fc region of an IgG molecule, wherein said FcRn-binding portion comprises amino acid substitutions at two or more of positions 432 to 437, numbered according to the EU numbering index of Kabat, relative to a wild-type Fc region; wherein
 (i) at least one of positions 432 and 437 is substituted with cysteine; or   (ii) at least one of positions 432 and 437 is substituted with an amino acid selected from the group consisting of glutamine, glutamic acid, aspartic acid, and histidine;   wherein said molecule has a longer or shorter in vivo half life than the non-protein agent.   
     
     
         79 . The method of  claim 78 , wherein
 (i) both of positions 432 and 437 are substituted with cysteine; or   (ii) both of positions 432 and 437 are substituted with an amino acid independently selected from the group consisting of glutamine, glutamic acid, aspartic acid, and histidine.   
     
     
         80 . The method of  claim 79 , wherein position 432 and 437 are each substituted with cysteine; position 433 is histidine or is substituted with arginine, proline, threonine, lysine, serine, alanine, methionine, or asparagine; position 434 is asparagine or is substituted with arginine, tryptophan, histidine, phenylalanine, tyrosine, serine, methionine or threonine; position 435 is histidine or is substituted with histidine; and position 436 is tyrosine or phenylalanine or is substituted with leucine, arginine, isoleucine, lysine, methionine, valine, histidine, serine, or threonine. 
     
     
         81 . The method of  claim 79 , wherein position 432 is substituted with glutamic acid, glutamine, histidine, or aspartic acid; position 433 is histidine or is substituted with arginine, alanine, lysine, threonine, leucine, proline, serine, or glutamine; position 434 is substituted with tyrosine, phenylalanine, histidine, serine or tryptophan; position 435 is histidine; position 436 is tyrosine or substituted with arginine, histidine, asparagine, lysine, leucine, methionine, threonine, or valine; and position 437 is substituted with glutamine, histidine, glutamic acid, or aspartic acid.