Antibodies with enhanced or suppressed effector function

Abstract

Rationally designed antibodies and polypeptides that comprise multiple Fc region amino acid substitutions that synergistically provide enhanced selectivity and binding affinity to a target Fc receptor are provided. The polypeptides are mutated at multiple positions to make them more effective when incorporated in antibody therapeutics than those having wild-type Fc components.

What is claimed is: 
     
         1 . A polypeptide comprising a variant Fc region, wherein said variant Fc region comprises three or more amino acid modifications relative to a wild-type Fc region, and has an altered effect relative to a polypeptide comprising a wild-type Fc region or variant Fc region having only one of the three or more amino acid modifications; wherein at least two of the three or more modifications provide a synergistic effect compared to single position modifications at the at least two positions thereby exhibiting a selected binding profile to Fcγ receptors. 
     
     
         2 . The polypeptide of  claim 1  wherein the amino acid modifications produce amino acid interactions and dynamics that result in enhanced binding affinity and/or specificity to a first Fcγ receptor while diminishing binding affinity and/or specificity to a second Fcγ receptor compared to a polypeptide that lacks the at least three or more amino acid modifications. 
     
     
         3 . The polypeptide of  claim 2  wherein the first Fcγ receptor is FcγRIIIa receptor and the second Fcγ receptor is FcγRIIa or FcγRIIb. 
     
     
         4 . The polypeptide of  claim 1  wherein the amino acid modifications produce favorable FcγRIIIa-specific interactions and/or unfavorable interactions with FcγRIIa and/or FcγRIIb receptors. 
     
     
         5 . The polypeptide of  claim 1  wherein the amino acid modifications have minimal impact on the FcγRIIIa receptor while producing detrimental effects on binding of the polypeptide to FcγRIIa and/or FcγRIIb. 
     
     
         6 . The polypeptide of  claim 1  wherein the polypeptide comprises modifications S239E/D265S/I332E. 
     
     
         7 . The polypeptide of  claim 1  wherein the polypeptide comprises modifications H268D/E269L/S298A/K326A/A327H. 
     
     
         8 . The polypeptide of  claim 2  wherein the first Fcγ receptor is FcγRIIa receptor and the second Fcγ receptor is FcγRIIIa or FcγRIIb. 
     
     
         9 . The polypeptide of  claim 1  wherein the amino acid modifications produce favorable FcγRIIa-specific interactions and/or unfavorable interactions with FcγRIIIa and/or FcγRIIb receptors. 
     
     
         10 . The polypeptide of  claim 1  wherein the amino acid modifications have minimal impact on the FcγRIIa receptor while producing detrimental effects on binding of the polypeptide to FcγRIIIa and/or FcγRIIb. 
     
     
         11 . The polypeptide of  claim 2  wherein the first Fcγ receptor is FcγRIIb receptor and the second Fcγ receptor is FcγRIIIa receptor or FcγRIIa receptor. 
     
     
         12 . The polypeptide of  claim 1  wherein the amino acid modifications produce favorable FcγRIIb-specific interactions and/or unfavorable interactions with FcγRIIIa and/or FcγRIIa receptors. 
     
     
         13 . The polypeptide of  claim 1  wherein the amino acid modifications have minimal impact on the FcγRIIb receptor while producing detrimental effects on binding of the polypeptide to FcγRIIIa and/or FcγRIIa. 
     
     
         14 . The polypeptide of  claim 1  wherein the polypeptide comprises modifications D265E/S267D/A330S. 
     
     
         15 . The polypeptide of  claim 1  wherein the polypeptide comprises modifications G237F/A327L/A330I. 
     
     
         16 . The polypeptide of  claim 1  wherein the polypeptide comprises modifications G237F/S239E/H268D. 
     
     
         17 . The polypeptide of  claim 1  wherein the polypeptide comprises modifications S239E/S267E/H268D. 
     
     
         18 . The polypeptide of  claim 1  wherein the polypeptide comprises modifications S239E/S298A/K326A/A327H. 
     
     
         19 . The polypeptide of  claim 1  wherein the polypeptide comprises modifications G237F/S298A/A330L/I332E. 
     
     
         20 . The polypeptide of  claim 1  wherein the polypeptide comprises modifications G237F/S239E/A327H. 
     
     
         21 . The polypeptide of  claim 1  wherein the polypeptide comprises modifications L235A/S239E/D265E/A327H. 
     
     
         22 . The polypeptide of  claim 1  wherein the polypeptide comprises modifications G237F/S239E/D270N. 
     
     
         23 . The polypeptide of  claim 1  wherein binding of the polypeptide comprising a wild-type Fc region to Fcγ receptors is detectable by an in vitro assay. 
     
     
         24 . The polypeptide of  claim 1  wherein the three or more modifications comprise the mutation S239E wherein the polypeptide has higher selectivity in binding to the FcγRIIIa receptor compared to a polypeptide that lacks the S239E mutation. 
     
     
         25 . The polypeptide of  claim 1  wherein the three or more modifications comprise the mutation S298A wherein the polypeptide has reduced binding affinity to FcγRIIa and FcγRIIb receptors compared to a polypeptide that lacks the S298A mutation. 
     
     
         26 . The polypeptide of  claim 1  wherein the polypeptide comprises the amino acid modifications S298A/K326A/A327H wherein the polypeptide has improved binding selectivity to FcγRIIIa receptor as compared to a polypeptide lacking the S298A/K326A/A327H modifications. 
     
     
         27 . The polypeptide of  claim 1  wherein the polypeptide comprises the amino acid modifications S239E/S298A/K326A/A327H wherein the polypeptide has improved binding selectivity to FcγRIIIa receptor as compared to a polypeptide lacking the S239E/S298A/K326A/A327H modifications. 
     
     
         28 . The polypeptide of  claim 1  wherein the polypeptide comprises the amino acid modifications G236A/S239D/D270L/I332E, wherein the polypeptide has improved binding selectivity to FcγRIIIa receptor as compared to a polypeptide lacking the G236A/S239D/D270L/I332E modifications. 
     
     
         29 . The polypeptide of  claim 1  wherein the polypeptide comprises three amino acid modifications, said mutations selected from D270L/Y300L/A330K, G237F/S267G/N325F, G237F/V266L/S267D, L234F/S267G/N325L, and L234F/S267E/N325L. 
     
     
         30 . The polypeptide of  claim 1  wherein the polypeptide comprises modifications of at least three amino acids, said modifications selected from G237F/S239E/A327H, G237F/A327L/A330I, S239E/A327L/A330I, S239E/S267E/H268D, G237F/S239E/D270N, G236E/G237F/S239E, S239E/D265S/I332E, G237F/S239E/D265E, G237F/S239E/H268D, H268E/D270E/S267G, H268D/K326A/A327H, D265E/S267D/A330S, L235A/S239E/D265E, A327H/E269L/K236A, G237F/D270Q/S239E, A330V/I332L/K326, and G236S/A327H/A330I. 
     
     
         31 . The polypeptide of  claim 1  wherein the polypeptide comprises simultaneous modification of at least four amino acids, said modifications selected from L235A/S239E/D265E/A327H, S239E/D265S/H268D/I332E, S239D/D265S/S298A/I332E, S239E/S298A/K326A/A327H, G237F/S298A/A330L/I332E, and G236E/D270N/A327V/I332E, and G236A/S239D/D270L/I332E. 
     
     
         32 . The polypeptide of  claim 1  wherein the polypeptide comprises the amino acid modifications H268D/E269L/S298A/K326A/A327H. 
     
     
         33 . The polypeptide of any of  claims 1 - 32  wherein the polypeptide comprises the antibody trastuzumab or an antigen binding portion thereof. 
     
     
         34 . The polypeptide of  claim 1 , wherein one or more amino acid modifications are located between positions 234-330 according to the EU index. 
     
     
         35 . The polypeptide of  claim 1  wherein at least three amino acid modifications are selected from the group consisting of: L234Q, L234N, L235A, G236E, E236L, E236D, G237F, G237N, S239E, S239D, D265E, D265S, S267E, S267D, S267G, H268D, H268E, E269L, E269L, D270N, D270I, D270E, S298A, K326A, K326D, A327H, A327V, A327L, A327T, A330V, A330L, A330W, A330I, A330S, I332L, I332D, and 1332E. 
     
     
         36 . The polypeptide of  claim 1  wherein said variant Fc region comprises a combination of amino acid modifications wherein said combination is selected from the group consisting of: L235A/S239E/D265E; L235A/G237F/D265E; S239E/E269D/A327H; S239E/G237N/A327H; S239E/G237F/A327V; G237F/D270I/S239E; G237F/A327L/S239E; A327H/E269L/K326A; A330V/I332L/S239E; A327T/E269L/K326A; D270N/A327T/K326A; A330V/I332L/S239E; A330W/I332D/S239E; G236E/D265E/A327H/A330I; D270N/S298A/A327V; G236E/D265E/D270N/A327H/A330I; G236E/D270N/A327H/A330I; G236E/D270N/A327V/I332E; G236E/D270N/A327V/G237F; L234N/S239E/A330I/I332E; L234Q/S239E/A330I/I332E; L234Q/S239E/A330I/I332E/S298A; G237F/S239D/D265E/D270N/S298A; G237F/S239E/D270N/A330L/I332E; G237F/S239E/D270N/A330L/I332E/S298A; S239E/G237F/A327H; G237F/A327L/A330I; S239E/A330I/A327L; D265E/S239E/L235A/A327H; S267E/S239E/H268D; G237F/D270N/S239E; S239E/G237F/G236E; 1332E/D265S/S239E/H268D; I332E/D265S/S239E; D265E/S239E/G237F; S239E/H268D/G237F; S298A/D265S/S239D/I332E; S298A/K326A/A327H/S239E; S298A/G237F/A3330L/I332E; H268E/D270E/S267G; H268D/K326A/A327H; H268D/K326A/A327H/E269L/S298A; and A330S/D265E/S267D. 
     
     
         37 . The polypeptide of  claim 1  wherein said variant Fc region comprises a combination of amino acid modifications wherein said combination is selected from the group consisting of S239E/S267E/H268D; S237F/S239E/D265E and H268E/D270/E/S267G. 
     
     
         38 . The polypeptide of  claim 1 , wherein said variant Fc region comprises amino acid modification H268D and not modification S267E. 
     
     
         39 . The polypeptide of  claim 1 , wherein the Fc region of the parent polypeptide is a human IgG Fc region. 
     
     
         40 . The polypeptide of  claim 39 , wherein the human IgG Fc region is a human IgG1, IgG2, IgG3, or IgG4 Fc region. 
     
     
         41 . The polypeptide of  claim 1  wherein said polypeptide is an antibody. 
     
     
         42 . The antibody of  claim 41 , wherein said antibody is a monoclonal antibody, a humanized antibody, or a human antibody. 
     
     
         43 . A nucleic acid comprising a nucleotide sequence encoding the polypeptide of  claim 1 . 
     
     
         44 . A vector comprising the nucleic acid of  claim 43 . 
     
     
         45 . A method for producing the polypeptide of  claim 1 , said method comprising: (i) culturing in a medium a host cell comprising a nucleic acid encoding said polypeptide, under conditions suitable for the expression of said polypeptide; and (ii) recovering the polypeptide from said medium. 
     
     
         46 . A therapeutic antibody that specifically binds a cancer target antigen, said therapeutic antibody comprising a variant Fc region of  claim 1 . 
     
     
         47 . The therapeutic antibody of  claim 46 , wherein said therapeutic antibody is selected from the group consisting of abagovomab, adalimumab, alemtuzumab, aurograb, bapineuzumab, basiliximab, belimumab, bevacizumab, briakinumab, canakinumab, catumaxomab, certolizumab pegol, cetuximab, daclizumab, denosumab, efalizumab, galiximab, gemtuzumab ozogamicin, golimumab, ibritumomab tiuxetan, infliximab, ipilimumab, lumiliximab, mepolizumab, motavizumab, muromonab, mycograb, natalizumab, nimotuzumab, ocrelizumab, ofatumumab, omalizumab, palivizumab, panitumumab, pertuzumab, ranibizumab, reslizumab, rituximab, teplizumab, tocilizumab/atlizumab, tositumomab, trastuzumab, Proxinium™, Rencarex™, ustekinumab, zalutumumab, and any other antibodies. 
     
     
         48 . The therapeutic antibody of  claim 46 , wherein said target antigen is selected from the group consisting of a-chain (CD25) of IL-2R, Amyloid beta, anti-EpCAM×anti-CD3, BLyS (or BAFF), CD11a, CD20, CD22, CD23, CD3, CD4, CD52, CD80, CTLA-4, EGFR, EpCAM, F protein of RSV, G250, glycoprotein IIb/IIIa R, HER2, HER2/neu R, Hsp90, IgE antibody, IL-12/IL-23, IL-1b, IL-5, IL-6 receptor, Integrin alpha-4/beta-1, Mucin 16/CA-125, RANKL, TNF alpha, VEGF-A, and other therapeutically advantageous targets. 
     
     
         49 . A method of treating cancer in a patient having a cancer characterized by a cancer antigen, said method comprising administering to said patient a therapeutically effective amount of a therapeutic antibody of  claim 46 . 
     
     
         50 . The method of  claim 49 , wherein said patient is human. 
     
     
         51 . A method of treating immune disorders in a patient having an immune disorder characterized by an immune antigen, said method comprising administering to said patient a therapeutically effective amount of a therapeutic antibody of  claim 46 . 
     
     
         52 . A pharmaceutical composition comprising a therapeutically effective amount of the polypeptides of  claim 1 , and a pharmaceutically acceptable carrier. 
     
     
         53 . The polypeptide of  claim 1  wherein the polypeptide comprising a variant Fc region is more effective at mediating ADCC relative to wild type. 
     
     
         54 . The polypeptide of  claim 53  wherein the polypeptide comprising a variant Fc region is about 1.5 to about 100 fold more effective in mediating ADCC relative to wild type. 
     
     
         55 . The polypeptide of  claim 53  wherein the polypeptide comprising a variant Fc region is about 2 to about 50 fold more effective in mediating ADCC relative to wild type. 
     
     
         56 . The polypeptide of  claim 1  wherein the polypeptide comprising a variant Fc region is more effective at mediating inhibition of inflammatory immune responses relative to wild type. 
     
     
         57 . The polypeptide of  claim 56  wherein the polypeptide comprising a variant Fc region is about 2 fold more effective in mediating inhibition of inflammatory immune responses relative to wild type. 
     
     
         58 . The polypeptide of  claim 56  wherein the polypeptide comprising a variant Fc region is about 10 fold more effective in mediating inhibition of inflammatory immune responses relative to wild type. 
     
     
         59 . The polypeptide of  claim 56  wherein the polypeptide comprising a variant Fc region is about 50 fold more effective in mediating inhibition of inflammatory immune responses relative to wild type. 
     
     
         60 . The polypeptide of  claim 56  wherein the polypeptide comprising a variant Fc region is about 100 fold more effective in mediating inhibition of inflammatory immune responses relative to wild type. 
     
     
         61 . A method for identifying Fc variant polypeptides in silico based on calculated binding affinities to FcγRIIa, FcγRIIb and/or FcγRIIIa. 
     
     
         62 . The method of  claim 61  comprising further calculating in silico electrostatics, solvation, packing, packing density, hydrogen binding, and entropic effects of said Fc variant polypeptides. 
     
     
         63 . The method of  claim 61  further comprising constructing said Fc variant polypeptides and expressing said polypeptides in the context of an antibody in mammalian cells. 
     
     
         64 . The polypeptide of  claim 1 , wherein amino acid modifications do not comprise simultaneous modifications at positions 317 and 353 according to the EU index. 
     
     
         65 . The polypeptide of  claim 1 , wherein amino acid modifications do not encompass a substitution at position 332 according to the EU index.