Blood cells having modified antigenicity

Abstract

This invention relates to enzymatic removal of type A and B antigens from blood group A, B, and AB reactive cells in blood products, and thereby converting these to non-A and non-B reactive cells. The invention further relates to using unique alpha N-acetylgalactosaminidases and alpha-galactosidases with superior kinetic properties for removing the immunodominant monosaccharides of the blood group A and B antigens and improved performance in enzymatic conversion of red blood cells. The preferred unique alpha-N-acetylgalactosaminidases and alpha-galactosidases exhibit the following characteristics: (i) exclusive, preferred or no less than 10% substrate specificity for the type A and B branched polysaccharide structures relative to measurable activity with simple mono- and disaccharide structures and aglycon derivatives hereof; (ii) optimal performance at neutral pH with blood group oligosaccharides and in enzymatic conversion of cells; and (iii) a favorable kinetic constant Km with mono- and oligosaccharide substrates. The conversion methods of the invention use significantly lower amounts of recombinant glycosidase enzymes than previous and result in complete sero-conversion of all blood group A and B red cells.

1 . A non-naturally occurring blood cell, the blood cell comprising blood type group A associated H antigens that are substantially free of immunodominant A residues.  
     
     
         2 . The blood cell of  claim 1 , wherein the blood cell is an erythrocyte or a platelet.  
     
     
         3 . The blood cell of  claim 1 , where the blood type group A associated H antigens are substantially free of terminal N-acetylgalactosamine residues.  
     
     
         4 . The blood cell of  claim 1 , where blood type group A associated H antigens are substantially free of terminal repeating N-acetylgalactosamine residues.  
     
     
         5 . The blood cell of  claim 1 , wherein the cell does not cause agglutination, hemolysis or transfusion reactions when introduced into a transfusion recipient having serum antibodies to type A blood group antigens.  
     
     
         6 . The blood cell of  claim 1 , wherein the cell is substantially free of immunodominant A residues, as determined by standard blood bank serological typing.  
     
     
         7 . A non-naturally occurring blood cell, the blood cell comprising blood type group B associated P1 antigens, and further comprising blood type group B associated H antigens that are substantially free of immunodominant B residues  
     
     
         8 . The blood cell of  claim 7 , wherein the blood cell is an erythrocyte or a platelet.  
     
     
         9 . The blood cell of  claim 7 , where the blood type group B associated H antigens are substantially free of terminal galactose residues.  
     
     
         10 . The blood cell of  claim 7 , where the blood type group B associated H antigens are substantially free of terminal alpha1,3 galactose residues.  
     
     
         11 . The blood cell of  claim 7 , wherein the blood type group B associated H antigens comprise linear carbohydrates having alpha-1-3 galactose or alpha 1-4 galactose, but where branched carbohydrates are substantially free of terminal galactose residues.  
     
     
         12 . The blood cell of  claim 7 , wherein the cell does not cause agglutination, hemolysis or transfusion reactions when introduced into a transfusion recipient having serum antibodies to type B blood group antigens.  
     
     
         13 . The blood cell of  claim 7 , wherein the cell is substantially free of immunodominant B residues, as determined by standard blood bank serological typing.  
     
     
         14 . A non-naturally occurring blood cell, the blood cell comprising blood type group B associated P1 antigens, and further comprising blood type group A associated H antigens that are substantially free of immunodominant A residues and blood type group B associated H antigens that are substantially free of immunodominant B residues.  
     
     
         15 . The blood cell of  claim 14 , wherein the blood cell is an erythrocyte or a platelet.  
     
     
         16 . The blood cell of  claim 14 , where the blood type group A associated H antigens are substantially free of terminal N-acetylgalactosamine residues.  
     
     
         17 . The blood cell of  claim 14 , where the blood type group B associated H antigens are substantially free of terminal galactose residues.  
     
     
         18 . The blood cell of  claim 14 , wherein the blood type group B associated H antigens comprise linear carbohydrates having alpha-1-3 galactose or alpha 1-4 galactose, but where branched carbohydrates are substantially free of terminal galactose residues.  
     
     
         19 . The blood cell of  claim 14 , wherein the cell does not cause agglutination, hemolysis or transfusion reactions when introduced into a transfusion recipient having serum antibodies to type A blood group antigens or type B blood group antigens  
     
     
         20 . The blood cell of  claim 14 , wherein the cell is substantially free of immunodominant A residues and immunodominant B residues, as determined by standard blood bank serological typing.  
     
     
         21 . A sero-converted erythrocyte, comprising A associated H structures but no detectable immunodominant A antigens, as determined by standard blood bank serological typing wherein said sero-converted erythrocyte has been converted from a type A or type AB erythrocyte to a non-A erythrocyte by the steps of a) contacting said erythrocyte with an alpha-N-acetylgalactosaminidase enzyme, under neutral pH conditions, for a period sufficient to substantially remove the immunodominant A antigens from the erythrocyte, and b) removing said enzyme from said erythrocytes, wherein said alpha-N-acetylgalactosaminidase enzyme has the following characteristics: (i) the enzyme is capable of removing substantially all detectable immunodominant A antigens from group A and AB reactive cells, (ii) has no less than 10% activity with blood group A oligosaccharides compared to simple alpha-GalNAc monosaccharide derivatives, and (iii) is active in red blood cell conversion at neutral pH.  
     
     
         22 . A sero-converted erythrocyte, comprising P1 antigens and B associated H structures, but no detectable immunodominant B antigens, as determined by standard blood bank serological typing, wherein said sero-converted erythrocyte has been converted from a type B or type AB erythrocyte to a non-B erythrocyte by the steps of a) contacting said erythrocyte with an alpha-galactosidase enzyme, under neutral pH conditions, for a period sufficient to remove the immunodominant B antigens, and b) removing said enzyme from said erythrocytes, wherein said enzyme has the following characteristics: (i) no less than 10% activity with blood group B oligosaccharides compared to simple alpha-Gal monosaccharide derivatives, and (ii) active in red blood cell conversion at neutral pH.