SUBSTANCE BINDING HUMAN IgG Fc RECEPTOR IIb (FcgammaRIIb)

Abstract

This invention relates to novel immunogens carrying conformationally discriminating epitopes (CDEs) and to immunization methods for producing antibodies that specifically recognize proteins with very closely related homologues. In particular, the invention relates to antibodies which are specific for either FcγRIIb and FcγRIIa.

1 - 53 . (canceled) 
     
     
         54 . An artificial peptide or polypeptide comprising a conformationally discriminating epitope (CDE) in its native conformation, wherein the CDE is structurally stabilized by circularization. 
     
     
         55 . The peptide or polypeptide  claim 54 , comprising artificial or glycosylated amino acids. 
     
     
         56 . The peptide or polypeptide  claim 54 , conjugated to a carrier molecule. 
     
     
         57 . The peptide or polypeptide of  claim 54  comprising a CDE of an Fc receptor. 
     
     
         58 . The peptide or polypeptide of  claim 57  comprising a CDE of FcγRIIb or FcγRIIa, the CDE comprising at least one residue which is unique to either FcγRIIb or FcγRIIa. 
     
     
         59 . The peptide or polypeptide of  claim 58 , wherein the CDE comprises amino acids 27 to 30, or amino acids 127 to 135 or amino acids 160 to 171 of FcγRIIb of SEQ ID NO: 2 or the corresponding amino acids of FcγRIIa of SEQ ID NO: 1, or the amino acid sequence of SEQ ID NO: 3. 
     
     
         60 . The peptide or polypeptide of  claim 57  conjugated to FcγRIIb or FcγRIIa. 
     
     
         61 . A method of producing a peptide carrying a conformationally discriminating epitope (CDE) for the generation of antibodies specifically recognizing a protein of interest carrying such an epitope, comprising:
 (a) providing a protein of interest,   (b) identifying a CDE on said protein,   (c) producing a peptide comprising the sequence of the CDE,   (d) structurally stabilizing the peptide by circularization so that the CDE is present in its native conformation.   
     
     
         62 . The method of  claim 61 , wherein the circularization of the peptide is achieved by generating cysteine bridges, or by bridging amino acid side chains that form a pseudopeptide. 
     
     
         63 . The method of  claim 61 , wherein the peptide is generated using amino acids carrying glycosylation moieties which are present on the protein of interest. 
     
     
         64 . The method of  claim 61 , further comprising:
 (e) conjugating the peptide to a carrier molecule selected from haptens, polypeptides, peptides, and the protein of interest.   
     
     
         65 . A peptide or polypeptide comprising a CDE, obtained by the method of  claim 61 . 
     
     
         66 . A method comprising generating immunomodulatory substances specifically recognizing the CDE in its natural environment by providing the peptide or polypeptide of  claim 54  as an immunogen in a environment suitable to generate the substance. 
     
     
         67 . A method comprising immunizing an animal or a transgenic animal expressing human FcγRIIa by administering an effective amount of the peptide or polypeptide of  claim 59  to a subject in need thereof. 
     
     
         68 . A method comprising generating an antibody that can specifically recognize alleles of the FcγRIIa Arg/His polymorphism at position 131 or the FcγRIIa Val/Phe polymorphism at position 155 by providing an effective amount of the peptide or polypeptide of  claim 59  in an environment effective to a subject to generate the antibody. 
     
     
         69 . A method of producing substances capable of discriminating between an antigen of interest and closely related antigens, comprising immunizing an animal with a peptide or polypeptide according to  claim 59  or with a correctly folded peptide derived from FcγRIIb or FcγRIIa, or both, and isolating the resulting antibodies. 
     
     
         70 . A nucleic acid sequence encoding the peptide of  claim 58  or an antibody or fragment or derivative thereof that specifically binds to human FcγRIIb or FcγRIIa in the natural environment of the Fc receptor. 
     
     
         71 . A nucleic acid vector comprising the nucleic acid sequence according to  claim 70 . 
     
     
         72 . A host cell transfected with a vector according to  claim 71 . 
     
     
         73 . A diagnostic kit for the detection of autoimmune diseases or cancer, comprising the antibody, fragment or derivative thereof that specifically binds to human FcγRIIb or FcγRIIa in the natural environment of the Fc receptor, or a recombinant peptide or polypeptide comprising a conformationally discriminating epitope (CDE) in its native conformation, wherein the CDE is structurally stabilized by circulization. 
     
     
         74 . The method of  claim 69 , further comprising generating recombinant immunomodulatory substances with antibodies.