Enzymatic conversion of blood group A, B, and AB red blood cells using alpha-N-acetylgalactosaminidases and alpha-galactosidases with unique substrate specificities and kinetic properties

A method for removing type A antigens from blood group A or AB reactive cells in a blood product, said method comprising the steps of:

• (a) contacting said blood product with an α-N-acetylgalactosamimidase enzyme, under neutral pH conditions, for a period sufficient to remove the A antigens, and
• (b) removing said enzyme from said blood product,
• wherein said α-N-acetylgalactosamimidase enzyme has the following characteristics:
• (i) capable of removing all detectable A antigens from group A and AB reactive cells,
• (ii) no less than 10% activity with blood group A oligosaccharides compared to simple α-GalNAc monosaccharide derivatives, and
• (iii) active in red blood cell conversion at neutral pH.

A method for converting type A or AB erythrocytes to non-A erythrocytes, said method comprising the steps of:

• (a) contacting said erythrocytes with an α-N-acetylgalactosamimidase enzyme, under neutral pH conditions, for a period sufficient to convert the A antigens in said erythrocytes to the H antigens, and
• (b) removing said enzyme from said erythrocytes,
• wherein said α-N-acetylgalactosamimidase enzyme has the following characteristics:
• (i) capable of removing all detectable A antigens from group A and AB reactive cells,
• (ii) no less than 10% activity with blood group A oligosaccharides compared to simple α-GalNAc monosaccharide derivatives, and
• (iii) active in red blood cell conversion at neutral pH.

A method for removing type B antigens from blood group B or AB reactive cells in a blood product, said method comprising the steps of:

• (a) contacting said blood product with an α-galactosidase enzyme, under neutral pH conditions, for a period sufficient to remove the B antigens, and
• (b) removing said enzyme from said blood product,
• wherein said α-galactosidase enzyme has the following characteristics:
• (i) no less than 10% activity with blood group B oligosaccharides compared to simple α-Gal monosaccharide derivatives, and
• (ii) active in red blood cell conversion at neutral pH.

A method for converting type B or AB erythrocytes to non-B erythrocytes, said method comprising the steps of:

• (a) contacting said erythrocytes with an α-galactosidase enzyme, under neutral pH conditions, for a period sufficient to remove the B antigens, and
• (b) removing said enzyme from said erythrocytes,
• wherein said α-galactosidase enzyme has the following characteristics:
• (i) no less than 10% activity with blood group B oligosaccharides compared to simple α-Gal monosaccharide derivatives, and
• (ii) active in red blood cell conversion at neutral pH.

A method for removing type A and type B antigens from blood group A, B, or AB reactive cells in a blood product, said method comprising the steps of:

• (a) contacting said blood product with an α-N-acetylgalactosamimidase enzyme and an α-galactosidase enzyme, under neutral pH conditions, for a period sufficient to remove the A and B antigens, and
• (b) removing said enzyme from said blood product,
• wherein said α-N-acetylgalactosamimidase enzyme has the following characteristics:
• (i) capable of removing all detectable A antigens from group A and AB reactive cells,
• (ii) no less than 10% activity with blood group A oligosaccharides compared to simple α-GalNAc monosaccharide derivatives, and
• (iii) active in red blood cell conversion at neutral pH, and
• wherein said α-galactosidase enzyme has the following characteristics:
• (i) no less than 10% activity with blood group B oligosaccharides compared to simple α-Gal monosaccharide derivatives, and
• (ii) active in red blood cell conversion at neutral pH.

A method for converting type A and type B erythrocytes from blood group A, B, or AB reactive cells in a blood product to non-A, non-B erythrocytes, said method comprising the steps of:

• (a) contacting said erythrocytes with an α-N-acetylgalactosamimidase enzyme and an α-galactosidase enzyme, under neutral pH conditions, for a period sufficient to remove the A and B antigens, and
• (b) removing said enzyme from said erythrocytes,
• wherein said α-N-acetylgalactosamimidase enzyme has the following characteristics:
• (i) capable of removing all detectable A antigens from group A and AB reactive cells,
• (ii) no less than 10% activity with blood group A oligosaccharides compared to simple α-GalNAc monosaccharide derivatives, and
• (iii) active in red blood cell conversion at neutral pH, and
• wherein said α-galactosidase enzyme has the following characteristics:
• (i) no less than 10% activity with blood group B oligosaccharides compared to simple α-Gal monosaccharide derivatives, and
• (ii) active in red blood cell conversion at neutral pH.

A method for screening and selecting an α-galactosidase enzyme useful for removing type B antigens from blood group B and AB reactive cells in blood products under neutral pH conditions, comprising the step of:

• (a) contacting a candidate α-galactosidase enzyme, under neutral pH conditions, with a group B oligosaccharide substrate and measuring the activity of the candidate enzyme with the group B oligosaccharide substrate,
• (b) contacting said candidate α-galactosidase enzyme, under neutral pH conditions, with an α-Gal monosaccharide derivative and measuring the activity of the candidate enzyme with the group B monosaccharide derivative, and
• (c) comparing the activity of the candidate enzyme with the group B oligosaccharide substrate with the activity with the α-Gal monosaccharide derivative,
• wherein the candidate α-galactosidase enzyme having no less than 10% activity with blood group B oligosaceharides compared to simple α-Gal monosaccharide derivatives are selected as useful for removing type B antigens from blood group B and AB reactive cells in blood products under neutral pH conditions.

A method for screening and selecting an α-N-acetylgalactosamimidase enzyme useful for removing type A antigens from blood group A and AB reactive cells in blood products under neutral pH conditions, comprising the step of:

• (a) contacting a candidate α-N-acetylgalactosamimidase enzyme, under neutral pH conditions, with a group A oligosaccharide substrate and measuring the activity of the candidate enzyme with the group A oligosaccharide substrate,
• (b) contacting said candidate α-N-acetylgalactosamimidase enzyme, under neutral pH conditions, with an α-GalNAc monosaccharide derivative and measuring the activity of the candidate enzyme with the group A monosaccharide derivative, and
• (c) comparing the activity of the candidate enzyme with the group A oligosaccharide substrate with the activity with the α-GalNAc monosaccharide derivative,
• wherein the candidate α-N-acetylgalactosamimidase enzyme having no less than 10% activity with blood group A oligosaccharides compared to simple α-GalNAc monosaccharide derivatives are selected as useful for removing type A antigens from blood group A and AB reactive cells in blood products under neutral pH conditions.

A sero-converted erythrocyte, wherein said sero-converted erythrocyte:

• (a) has been converted from a type AB erythrocyte to a non-A, non-B erythrocyte by an α-N-acetylgalactosamimidase and an α-galactosidase,
• (b) has A associated H structures,
• (c) retains P1 antigens, and
• (d) has no detectable A or B antigens, as determined by standard blood bank serological typing.

A blood product comprising sero-converted erythrocytes, wherein said sero-converted erythrocytes:

• (a) have been converted from type AB erythrocytes to non-A, non-B erythrocytes by an α-N-acetylgalactosamimidase and an α-galactosidase, and
• (b) have A associated H structures,
• (c) retains P1 antigens, and
• (d) have no detectable A or B antigens, as determined by standard blood bank serological typing.