Immunoglobulin fc polypeptides

Abstract

Methods and compositions involving polypeptides having an aglycosylated antibody Fc domain. In certain embodiments, polypeptides have an aglycosylated Fc domain that contains one or more substitutions compared to a native Fc domain. Additionally, some embodiments involve an Fc domain that is binds some Fc receptors but not others. For example, polypeptides are provided with an aglycosylated Fc domain that selectively binds FcγRI at a level within 2-fold of a glycosylated Fc domain, but that is significantly reduced for binding to other Fc receptors. Furthermore, methods and compositions are provided for promoting antibody-dependent cell-mediated toxicity (ADCC) using a polypeptide having a modified aglycosylated Fc domain and a second non-Fc binding domain, which can be an antigen binding region of an antibody or a non-antigen binding region. Some embodiments concern antibodies with such polypeptides, which may have the same or different non-Fc binding domain.

1 .- 67 . (canceled) 
     
     
         68 . A method for screening for an aglycosylated polypeptide having an Fc domain that binds a specific FcγR polypeptides comprising:
 a) obtaining a population of Gram negative bacterial cells, cells of which population express a aglycosylated polypeptide comprising an Fc domain in their periplasm, wherein the population expresses a plurality of different Fc domains; 
 b) contacting the bacterial cells with a first FcR polypeptide under conditions to allow contact between the FcγR polypeptide and the aglycosylated Fc domains, wherein the FcγR polypeptide is FcγRIa, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, FcγRIIIb, or FcαRI; 
 c) selecting at least one bacterial cell based on binding of the aglycosylated Fc domain to the first FcR polypeptide. 
 
     
     
         69 . The method of  claim 68 , further comprising identifying or isolating the aglycosylated polypeptide from the selected bacterial cell. 
     
     
         70 . The method of  claim 68 , further comprising determining whether an aglycosylated polypeptide in selected bacterial cells can bind to other FcR polypeptides. 
     
     
         71 . The method of  claim 70 , wherein determining whether an aglycosylated polypeptide in selected bacterial cells can bind to other FcR polypeptides comprises repeating steps a)-c) with a second FcR polypeptide to determine whether the aglycosylated polypeptide also binds the second FcR polypeptide. 
     
     
         72 . The method of  claim 71 , wherein the steps a)-c) are repeated with more than two different FcR polypeptides. 
     
     
         73 . The method of  claim 72 , wherein the aglycosylated polypeptide binds multiple FcR polypeptides. 
     
     
         74 . The method of  claim 68 , wherein the bacterial cells are  E. coli  cells. 
     
     
         75 . The method of  claim 68 , wherein the Fc domain is an IgG, IgA or IgE Fc domain. 
     
     
         76 . The method of  claim 75 , wherein the IgG Fc domain is an IgG1 Fc domain. 
     
     
         77 . The method of  claim 76 , wherein the IgG1 Fc domain is the Fc domain of an anti-HER2 antibody. 
     
     
         78 . The method of  claim 77 , wherein the IgG1 Fc domain is the Fc domain of the Fc domain of trastuzumab. 
     
     
         79 . The method of  claim 68 , wherein the population of Gram negative bacterial cells comprise a plurality of nucleic acids encoding said plurality of aglycosylated Fc domains. 
     
     
         80 . The method of  claim 79 , wherein the plurality of nucleic acids further encodes a membrane secretion signal fused to said plurality of aglycosylated Fc domains. 
     
     
         81 . The method of  claim 80 , wherein the membrane secretion signal is PelB. 
     
     
         82 . The method of  claim 80 , wherein the membrane secretion signal is DsbA. 
     
     
         83 . The method of  claim 68 , wherein the aglycosylated Fc domain comprises a hinge, CH2 and CH3 region. 
     
     
         84 . The method of  claim 68 , wherein the aglycosylated polypeptide comprises an eukaryotic Fc domain. 
     
     
         85 . The method of  claim 68 , wherein the aglycosylated polypeptide comprises a synthetic Fc domain. 
     
     
         86 . The method of  claim 84 , wherein the FcR polypeptide comprises an antibody-binding domain from one of the polypeptides of Table 1. 
     
     
         87 . The method of  claim 86 , wherein the FcR polypeptide comprises an antibody-binding domain from human FcγRIa, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, FcγRIIIb, FcαRI or C1q. 
     
     
         88 . The method of  claim 87 , wherein the FcR polypeptide comprises an antibody-binding domain from human FcγRIa. 
     
     
         89 . The method of  claim 68 , wherein the FcR polypeptide is labeled. 
     
     
         90 . The method of  claim 89 , wherein the FcR polypeptide is labeled with a fluorophore, a radioisotope or an enzyme. 
     
     
         91 . The method of  claim 90 , wherein the FcR polypeptide is labeled with a fluorophore. 
     
     
         92 . The method of  claim 89 , wherein the FcR polypeptide is fused to a fluorescence protein or an enzyme. 
     
     
         93 . The method of  claim 92 , wherein the FcR polypeptide is fused to a green fluorescence protein GFP. 
     
     
         94 . The method of  claim 68 , wherein the FcR polypeptide is immobilized. 
     
     
         95 . The method of  claim 68 , wherein the selecting of step (c) is further defined as comprising at least two rounds of selection wherein the sub-population of bacterial cells obtained in the first round of selection is subjected to at least a second round of selection based on the binding of the Fc polypeptide to FcR polypeptide. 
     
     
         96 . The method of  claim 95 , comprising two to ten rounds of selection. 
     
     
         97 . The method of  claim 96 , wherein the selecting is carried out by FACS or magnetic separation. 
     
     
         98 . The method of  claim 68 , further comprising contacting the bacterial cells with at least two FcR polypeptides. 
     
     
         99 . The method of  claim 98 , wherein the at least two FcR polypeptides comprise distinct labels. 
     
     
         100 . The method of  claim 99 , further comprising selecting bacterial cells based on binding of the aglycosylated Fc domain to the at least two FcR polypeptides. 
     
     
         101 . The method of  claim 99 , further comprising selecting bacterial cells based on binding of the aglycosylated Fc domain to at least one FcR polypeptide and based on the aglycosylated Fc domain not binding to at least one other FcR polypeptide. 
     
     
         102 . The method of  claim 68 , further comprising disrupting the outer membrane of the bacterial cells before contacting the bacterial cells with an FcR polypeptide. 
     
     
         103 . The method of  claim 102 , wherein disrupting the outer membrane of the bacterial cell comprises treatment with hyperosmotic conditions, treatment with physical stress, infecting the bacterium with a phage, treatment with lysozyme, treatment with EDTA, treatment with a digestive enzyme or treatment with a chemical that disrupts the outer membrane. 
     
     
         104 . The method of  claim 103 , wherein disrupting the outer membrane of the bacterial cell comprises a combination of said methods. 
     
     
         105 . The method of  claim 102 , wherein disrupting the outer membrane of the bacterial cell comprises heating the bacterial cell with a combination of physical, chemical and enzyme disruption of the outer membrane. 
     
     
         106 . The method of  claim 102 , wherein disrupting the outer membrane of the bacterial cell further comprises removing the outer membrane of said bacterium. 
     
     
         107 . The method of  claim 68 , further comprising removing FcR polypeptide not bound to the aglycosylated Fc domain. 
     
     
         108 . The method of  claim 68 , wherein the bacteria are grown in a media comprising sucrose, sorbitol, mannitol or trehalose. 
     
     
         109 . The method of  claim 68 , wherein the bacteria are grown in a media comprising trehalose. 
     
     
         110 . The method of  claim 68 , further defined as a method of producing a nucleic acid sequence encoding an antibody Fc polypeptide having a specific affinity for an FcR polypeptide and further comprising the step of cloning a nucleic acid sequence encoding the Fc polypeptide from the bacterial cell to produce a nucleic acid sequence encoding an antibody Fc polypeptide having a specific affinity for an FcR polypeptide. 
     
     
         111 . The method of  claim 110 , wherein cloning comprises amplification of the nucleic acid sequence. 
     
     
         112 . The method of  claim 110 , further defined as a method of producing an antibody Fc polypeptide having a specific affinity for an FcR polypeptide further comprising the step of expressing a nucleic acid sequence encoding the antibody Fc polypeptide to produce an antibody Fc polypeptide having a specific affinity for an FcR polypeptide. 
     
     
         113 . The method of  claim 112 , wherein the FcR polypeptide is an FcγRI polypeptide. 
     
     
         114 . The method of  claim 68 , wherein the plurality of different Fc domains are mutant variations of one Fc domain. 
     
     
         115 . The method of  claim 114 , wherein the mutant variations were generated randomly. 
     
     
         116 . (canceled)