Methods of identifying glycopeptides recognized by disease-associated auto-antibodies

Abstract

Methods for identifying glycopeptides and more particularly glycopeptide epitopes that are specifically recognized by disease-associated auto-antibodies are provided. In some aspects the auto-antibodies are cancer-associated or autoimmune disease associated. In other aspects, methods of diagnosing a patient with cancer or an autoimmune disease, or for eliciting an immune response in a mammalian host directed to the glycopeptides of the invention are provided.

What is claimed is: 
     
         1 . A method for identifying glycopeptides reactive with cancer-associated auto-antibodies, the method comprising:
 providing a panel comprising peptides immobilized on said panel, at least a plurality of said peptides comprising one or more sites amenable to glycosylation, wherein at least one of said glycosylation sites is modified with a glycan to form a glycopeptide having a peptide portion and a glycan portion;   contacting said panel with an antibody-containing sample from a patient with cancer; and   identifying glycopeptides in said panel that (i) are selectively recognized by antibodies in said sample, but not by antibodies in a control sample and (ii) are recognized by such antibodies in said sample through recognition of both the peptide portion and the glycan portion and not through recognition of either the peptide or the glycan alone.   
     
     
         2 . The method of  claim 1 , wherein said panel comprises peptides having an amino acid sequence comprising at least one serine or threonine residue, wherein said residue is a glycosylation site. 
     
     
         3 . The method of  claim 1 , wherein said control sample contains pooled samples from a plurality of control individuals. 
     
     
         4 . The method of  claim 1 , wherein said panel comprises mutants of said peptides. 
     
     
         5 . The method of  claim 1 , further comprising identifying minimum cancer-associated glycopeptide epitopes. 
     
     
         6 . The method of  claim 5 , further comprising elucidating the epitope structure of said identified glycopeptide epitopes. 
     
     
         7 . The method of  claim 1 , wherein each of said peptides in said panel is about 2 to about 50 amino acid residues in length. 
     
     
         8 . The method of  claim 7 , wherein each of said peptides in said panel is about 4 to about 25 amino acid residues in length. 
     
     
         9 . The method of  claim 1 , wherein said amino acid sequence of said peptides is a fragment found in a protein or variant of said protein or a conservative mutant. 
     
     
         10 . The method of  claim 1 , wherein said identified glycopeptides are found in at least one glycoprotein that is aberrantly glycosylated in cancer cells. 
     
     
         11 . The method of  claim 1 , wherein said identified glycopeptides are found in at least one glycoprotein that is overexpressed in cancer cells. 
     
     
         12 . The method of  claim 1 , wherein the serine or threonine residue is placed at about the center of each of said amino acid sequences. 
     
     
         13 . The method of  claim 1 , wherein said glycopeptides are synthesized synthetically or chemoenzymatically. 
     
     
         14 . The method of  claim 1 , wherein said glycopeptides of said panel are partially glycosylated peptides. 
     
     
         15 . The method of  claim 1  or  14 , wherein said glycopeptides of said panel are glycosylated in situ, in solution, or in vivo by recombinant expression in a host cell. 
     
     
         16 . The method of  claim 1 , wherein said glycopeptides of said panel are treated with one or more exoglycosidases to expose O-glycans. 
     
     
         17 . The method of  claim 1 , wherein said glycan is an O-glycan. 
     
     
         18 . The method of  claim 17 , wherein said O-glycan is a member selected from the group consisting of: Tn, STn, T, Truncated C3, Truncated C2, Truncated C4, non-capped type1-C3, non-capped type2-C2, non-capped type2-C4, GalNAca-3Tn, SA-type1-C3, SLea-C3, LacDiNAc-C3, LacDiNAc-C2, and LacDiNAc-C4. 
     
     
         19 . The method of  claim 1 , wherein said panel is a microarray. 
     
     
         20 . The method of  claim 1 , wherein said identified glycopeptide is selectively recognized by a cancer-associated IgG antibody. 
     
     
         21 . The method of  claim 1 , wherein said panel comprises one or more glycopeptides having an amino acid sequence selected from the group consisting of: SHHSDESDELVTDFPTDLPA (SEQ ID NO: 15); TPTPKEKPEAGTYSVNNGND (SEQ ID NO: 36); SESFPHPGFNMSLLENHTRQ (SEQ ID NO: 49); LAKMYYSAVEPTKDIFTGLI (SEQ ID NO: 86); TDCGGPKDHPLTCDDPRFQA (SEQ ID NO: 109); PGTSTTPSQPNSAGVQDTEM (SEQ ID NO: 116); TKTDASSTHHSTVPPLTSSN (SEQ ID NO: 132); HDVETQFNQYKTEAASRYNL (SEQ ID NO: 134); ASRYNLTISDVSVSDVPFPF (SEQ ID NO: 135); VPVTRPALGSTTPPAHDVTS (SEQ ID NO: 145); and SLASQATDTFSTVPPTPPSI (SEQ ID NO: 146). 
     
     
         22 . A pharmaceutical composition for eliciting an immune response in a patient, comprising one or more isolated glycopeptides consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 86, 109, 116, 132, 134, 135 and 146, and a pharmaceutically acceptable carrier. 
     
     
         23 . The pharmaceutical composition of  claim 22 , wherein the one or more isolated glycopeptides are selected from the group consisting of LAKMYYSAVEPTKDIFTGLI (SEQ ID NO: 86); TDCGGPKDHPLTDPRFQA (SEQ ID NO: 109); TKTDASSTHHSTVPPLTSSN (SEQ ID NO: 132), and SLASQATDTFSTVPPTPPSI (SEQ ID NO: 146). 
     
     
         24 . A pharmaceutical composition for eliciting an immune response in a patient, comprising one or more isolated glycopeptides consisting of an epitope of 5 to 10 amino acid residues contained in a glycopeptide selected from the group consisting of SEQ ID NOs: 86, 109, 116, 132, 134, 135 and 146, and a pharmaceutically acceptable carrier, said epitope having been previously determined (i) to be selectively recognized by a subset of antibodies in sera from cancer patients, which subset recognizes neither (a) the corresponding naked peptides of said panel when not glycosylated; nor (b) the corresponding glycan when not bound to said peptide; and (ii) not to be recognized by antibodies in control sera. 
     
     
         25 . A panel of glycopeptides comprising at least a plurality of isolated glycopeptides immobilized on said panel, each isolated glycopeptide comprising a glycopeptide epitope, said epitope having been previously determined (i) to be selectively recognized by a subset of antibodies in sera from cancer patients, which subset recognizes neither (a) the corresponding naked peptides of said panel when not glycosylated; nor (b) the corresponding glycan when not bound to said peptide; and (ii) not to be recognized by antibodies in control sera, said plurality comprising at least 8 isolated glycopeptides, wherein each epitope is 5 to 10 amino acid residues contained in one of the amino acid sequences of SEQ ID NOs: 86, 109, 116, 132, 134, 135, 145, and 146, respectively.