Tumor specific oligosaccharide epitopes and use thereof

Abstract

The present invention describes oligosaccharide sequences, which are specifically expressed by human tumors. The present invention is related to a method of determining an oligosaccharide sequence, which comprises a tumor specific terminal N-acetylglucosamine residue, in a biological sample, the presence of said sequence in said sample being an indication of the presence of cancer. The present invention provides antigenic substances comprising said oligosaccharide sequences in a polyvalent form and it further provides diagnostic agents, pharmaceutical compositions and cancer vaccines comprising said oligosaccharide sequences or substances binding to said oligosaccharide sequences. The present invention is also related to methods for the treatment of cancer.

What is claimed: 
     
         1 . A pharmaceutical composition comprising a substance binding to a human tumor specific oligosaccharide sequence containing a terminal protein linked GlcNAcβ-structure or a terminal protein linked GlcNAcβ-glycan-structure for the treatment of a human cancer. 
     
     
         2 . The pharmaceutical composition according to  claim 1 , wherein said human cancer is a human tumor and said human tumor specific oligosaccharide sequence is expressed on the cell surface or tissue surface of said human tumor. 
     
     
         3 . The pharmaceutical composition according to  claim 1 , wherein said substance is a human antibody, humanized antibody, or glycosyltransferase enzyme. 
     
     
         4 . The pharmaceutical composition according to  claim 1 , wherein said human tumor is diagnosed to express elevatedly said human tumor specific oligosaccharide sequence when compared to patient's normal tissue. 
     
     
         5 . The pharmaceutical composition according to  claim 1 , wherein said oligosaccharide sequence has the sequence according to Formula
   [GlcNAcβ x /Galβ3] s1 (GlcNAcβ1-6) s2 Sacch
   
       wherein x is 3, when Sacch is GalNAc; or
 x is 2, when Sacch is Man; and wherein 
 s1 and s2 are independently 0 or 1 with the proviso that there is at least one terminal GlcNAc; the structure is branched, when both s1 and s2 are 1; Sacch is GalNAc with the provision that it is not α6-linked to another GalNAc; Sacch is GlcNAcβ with the proviso that s1 and s2 is 0 and said GlcNAcβ is linked to a protein or peptide; [GlcNAcβx/Galβ3] means that terminal residue is either GlcNAcβx or Galβ3. 
 
     
     
         6 . The pharmaceutical composition according to  claim 1 , wherein said substance binding to said oligosaccharide sequence is specific to one or several of the terminal oligosaccharide sequences of a N-glycan type structure according to Formula
   [GNβ2Man] r1 α3([GNβ2Man] r2 α6){Man[β4GN[β4(Fucα6) r3 GN] r4 ] r5 } r6   (I)
   
       wherein r1, r2, r3, r4, r5, and r6 are either 0 or 1 with the proviso that at least r1 is 1 or r2 is 1; GN is GlcNAc, with the proviso that when both r1 and r2 are 1, one GNβMan can be further elongated with one or several other monosaccharide residues, and one GNβ2Man can be truncated to Man, and Manα6 residue and/or Manα3 residue(s) can be further substituted by GNβ6 or GNβ4, and Manβ4 can be further substituted by GNβ4. 
     
     
         7 . The pharmaceutical composition according to  claim 1 , wherein said substance binding to said oligosaccharide sequence is specific to one or several of the terminal oligosaccharide sequences of a N-glycan type structure according to Formula
   [GNβ2Man] r1 α3([GNβ2Man] r2 α6){Man[β4GN] r5 } r6   (II)
   
       wherein r1, r2, r5, and r6 are either 0 or 1, with the proviso that at least r1 is 1 or r2 is 1; GN is GlcNAc, with the proviso that when both r1 and r2 are 1, one GNβMan can be further elongated with one or several other monosaccharide residues, and one GNβ2Man can be truncated to Man, and Manα6 residue and/or Manα3 residue can be further substituted by GNβ6 or GNβ4, and Manβ4 can be further substituted by GNβ4. 
     
     
         8 . The pharmaceutical composition according to  claim 1 , wherein said oligosaccharide sequence is
 GlcNAcβ2Man, GlcNAcβ2Manα3(GlcNAcβ2Manα6)Man, GlcNAcβ2Manα3(GlcNAcβ2Manα6)Manβ4GlcNAc, GlcNAcβ2Manα3 (GlcNAcβ2Manα6)Manβ4GlcNAcβ4GlcNAc, GlcNAcβ2Manα3 (GlcNAc(2Manα6)Manβ4GlcNAcβ4(Fucα6)GlcNAc, GlcNAcβ2Manα3(Manα6)Man, GlcNAcβ2Manα3(Manα6)Manβ4GlcNAc, GlcNAcβ2Manα3(Manα6)Manβ4GlcNAcβ4GlcNAc, GlcNAcβ2Manα3(Manα6)Manβ4GlcNAcβ4(Fucα6)GlcNAc, Manα3(GlcNAcβ2Manα6)Man, Manα3(GlcNAcβ2Manα6)Manβ4GlcNAc, Manα3(GlcNAcβ2Manα6)Manβ4GlcNAcβ4GlcNAc, Manα3(GlcNAcβ2Manα6)Manβ4GlcNAcβ4(Fucα6)GlcNAc, GlcNAcβ2Manα3Man, GlcNAcβ2Manα3Manβ4GlcNAc, GlcNAcβ2Manα3Manβ4GlcNAcβ4GlcNAc, GlcNAcβ2Manα3Manβ4GlcNAcβ4(Fucα6)GlcNAc, GlcNAcβ2Manα6Man, GlcNAcβ2Manα6Manβ4GlcNAc, GlcNAcβ2Manα6Manβ4GlcNAcβ4GlcNAc, or GlcNAcβ2Manα6Manβ4GlcNAcβ4(Fucα6)GlcNAc   
     
     
         9 . The pharmaceutical composition according to  claim 1 , wherein said substance binding to said oligosaccharide sequence is specific to one or several of the terminal oligosaccharide sequences of an O-glycan type structure according to Formula
   [GlcNAcβ3] s1 [Galβ3] s2 (GlcNAcβ6) s5 GalNAc
   
       wherein s1, s2 and s5 are independently 0 or 1, so that the oligosaccharide sequence comprises at least one nonreducing end terminal GlcNAcβ-residue. 
     
     
         10 . The pharmaceutical composition according to  claim 1 , wherein said oligosaccharide sequence is protein linked GlcNAc or a derivative thereof. 
     
     
         11 . The pharmaceutical composition according to  claim 1 , wherein said oligosaccharide sequence is
 GlcNAcβ3Galβ3(Galβ4GlcNAcβ6)GalNAc, GlcNAcβ3Galβ3(GlcNAcβ6)GalNAc, GlcNAcβ3Galβ3GalNAc, Galβ3(GlcNAcβ6)GalNAc, GlcNAcβ3 (GlcNAcβ6)GalNAc, GlcNAcβ6GalNAc, or GlcNAcβ3GalNAc   
     
     
         12 . The pharmaceutical composition according to  claim 1  further comprising a substance binding to one or several of the following terminal oligosaccharide sequences:
 GlcNAcβ3Gal, GlcNAcβ3Galβ4GlcNAc, GlcNAcβ6Gal, GlcNAcβ6Galβ4GlcNAc GlcNAcβ3(GlcNAcβ6)Gal, and GlcNAcβ3(GlcNAcβ6)Galβ4GlcNAc 
 said composition being for the treatment of lung, larynx, colon, gastric or ovarian cancer. 
 
     
     
         13 . The pharmaceutical composition according to  claim 1 , wherein said oligosaccharide sequence is linked by an unnatural glycosidic linkage to other monosaccharide or oligosaccharide structures. 
     
     
         14 . The pharmaceutical composition according to  claim 1 , wherein said substance binding to said oligosaccharide sequence is an aptamer, a peptide or a protein. 
     
     
         15 . The pharmaceutical composition according to  claim 14 , wherein said protein is an antibody, a lectin, or a fragment thereof. 
     
     
         16 . The pharmaceutical composition according to  claim 14 , wherein said protein is an enzyme recognizing the terminal GlcNAc-structures. 
     
     
         17 . The pharmaceutical composition according to  claim 1  comprising a polyvalent conjugate of said oligosaccharide sequence wherein position C1 of the reducing end terminal of the oligosaccharide sequence (OS) comprising the tumor specific terminal sequence of the invention is linked (-L-) to an oligovalent or a polyvalent carrier (Z), via a spacer group (Y), forming the following structure
   [OS—(X) n -L-Y] m -Z
 
 
       where integer m have values m>1 and n is independently 0 or 1; L can be oxygen, nitrogen, sulfur or a carbon atom; X can be lactosyl-, galactosyl-, poly-N-acetyl-lactosaminyl, or part of an O-glycan or an N-glycan oligosaccharide sequence, Y is a spacer group, a terminal conjugate or a linkage to Z. 
     
     
         18 . The pharmaceutical composition according to  claim 1  comprising a pharmaceutically acceptable carrier and/or an adjuvant. 
     
     
         19 . A method for diagnosing cancer or tumor in a biological sample taken from a human patient, the method comprising determining the presence in said sample of an oligosaccharide sequence which comprises a tumor specific terminal protein linked GlcNAcβ-structure or a terminal protein linked GlcNAcβ-glycan-structure. 
     
     
         20 . The method according to  claim 19  wherein the determination comprises
 (a) contacting said biological sample with a substance binding to said oligosaccharide sequence, and 
 determining the presence of a combination of said substance and said sample, the presence of said combination being an indication of cancer present in said sample, or 
 (b) releasing the oligosaccharide structures of said biological sample by enzymatic or chemical methods to form a fraction containing free oligosaccharide structures from said sample, and 
 
       determining the presence of said oligosaccharide sequence in said fraction, the presence of said oligosaccharide sequence in said fraction being an indication of cancer present in said sample. 
     
     
         21 . The method according to  claim 19 , wherein said oligosaccharide sequence is a terminal oligosaccharide sequence has the sequence according to Formula
   [GlcNAcβ x /Galβ3] s1 (GlcNAcβ1-6) s2 Sacch
   
       wherein x is 3, when Sacch is GalNAc; or
 x is 2, when Sacch is Man; and wherein 
 s1 and s2 are independently 0 or 1 with the proviso that there is at least one terminal GlcNAc; the structure is branched, when both s1 and s2 are 1; Sacch is GalNAc with the provision that it is not α6-linked to another GalNAc; Sacch is GlcNAcβ with the proviso that s1 and s2 is 0 and said GlcNAcβ is linked to a protein or peptide; [GlcNAcβx/Galβ3] means that terminal residue is either GlcNAcβx or Galβ3. 
 
     
     
         22 . The method according to  claim 19 , wherein said cancer is a tumor. 
     
     
         23 . The method according to  claim 19 , wherein a cancer or tumor type is determined. 
     
     
         24 . The method according to  claim 19 , wherein normal glycosylation of the tissue containing the cancer is determined. 
     
     
         25 . The method according to  claim 19 , wherein the glycosylations are determined on the surface of cancer or normal tissue. 
     
     
         26 . A human anti-GlcNAc antibody obtainable by:
 passing human serum sample through a column containing immobilized terminal GlcNAcβ epitopes;   washing the column;   eluting the column with a buffer containing high concentration of GlcNAc;   and collecting the antibody.   
     
     
         27 . The antibody according to  claim 26 , wherein said antibody recognizes oligosaccharide sequence GlcNAcβ6GalNAcα-O—CH 2 —R, GlcNAcβ6(Galβ3)GalNAcα-O—CH 2 —R, GlcNAcβ2Man or GlcNAcβ-O—CH 2 —R or GlcNAcβ3GalNAcα-O—CH 2 .