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

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 α-N-acetylgalactosaminidases and α-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 α-N-acetylgalactosaminidases and α-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 sera-conversion of all blood group A and B red cells.

What is claimed is: 
     
         1 . 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-acetylgalactosaminidase 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 enzymes from said blood product,   wherein said α-N-acetylgalactosaminidase 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, 
 ii. active in red blood cell conversion at neutral pH, and 
 iii. isolated and purified from a non-recombinant strain of  Streptomyces griseoplamus , wherein said α-galactosidase enzyme is isolatable by a method comprising the steps of:
 A. fermentation culturing of an alpha-galactosidase producing  Streptomyces griseoplanus  strain; 
 B. disrupting the cultured  Streptomyces griseoplanus  strain of step “A.”; 
 C. isolating an α-galactosidase-containing supernatant fraction from the disrupted  Streptomyces griseoplanus  strain of step “B.” by centrifugation; 
 D. treating the α-galactosidase-containing supernatant fraction of step “C.” with ammonium sulfate to yield a 20 to 60 percent ammonium sulfate fraction enriched in the α-galactosidase; 
 E. purifying the α-galactosidase from the 20 to 60 percent ammonium sulfate fraction of step “D.” by anion exchange chromatography followed by cation exchange chromatography to yield an ion exchange purified α-galactosidase; and 
 F. fractionating the ion-exchange purified α-galactosidase of step “E.” by size-exclusion chromatography to yield a purified α-galactosidase which elutes with a molecular weight in the range of 40-80 kD. 
 
   
     
     
         2 . 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-acetylgalactosaminidase 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 enzymes from said erythrocytes,   wherein said α-N-acetylgalactosaminidase 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, 
 ii. active in red blood cell conversion at neutral pH, and 
 iii. isolated and purified from a non-recombinant strain of  Streptomyces griseoplanus , wherein said α-galactosidase enzyme is isolatable by a method comprising the steps of:
 A. fermentation culturing of an alpha-galactosidase producing  Streptomyces griseoplanus  strain; 
 B. disrupting the cultured  Streptomyces griseoplanus  strain of step “A.”; 
 C. isolating an α-galactosidase-containing supernatant fraction from the disrupted  Streptomyces griseoplanus  strain of step “B.” by centrifugation; 
 D. treating the α-galactosidase-containing supernatant fraction of step “C.” with ammonium sulfate to yield a 20 to 60 percent ammonium sulfate fraction enriched in the α-galactosidase; 
 E. purifying the α-galactosidase from the 20 to 60 percent ammonium sulfate fraction of step “D.” by anion exchange chromatography followed by cation exchange chromatography to yield an ion exchange purified α-galactosidase; and 
 F. fractionating the ion-exchange purified α-galactosidase of step “E.” by size-exclusion chromatography to yield a purified α-galactosidase which elutes with a molecular weight in the range of 40-80 kD. 
 
   
     
     
         3 . 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 oligosaccharides 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. 
 
     
     
         4 . A sero-converted erythrocyte, wherein said sero-converted erythrocyte:
 a. has been converted from a type B or type AB erythrocyte to a non-B erythrocyte by an α-galactosidase, wherein the α-galactosidase has the following characteristics:
 i. active in red blood cell conversion at neutral pH, and 
 ii. isolated and purified from a non-recombinant strain of  Streptomyces griseoplanus , wherein said α-galactosidase enzyme is isolatable by a method comprising the steps of:
 A. fermentation culturing of an alpha-galactosidase producing  Streptomyces griseoplanus  strain; 
 B. disrupting the cultured  Streptomyces griseoplanus  strain of step “A.”; 
 C. isolating an α-galactosidase-containing supernatant fraction from the disrupted  Streptomyces griseoplanus  strain of step “B.” by centrifugation; 
 D. treating the α-galactosidase-containing supernatant fraction of step “C.” with ammonium sulfate to yield a 20 to 60 percent ammonium sulfate fraction enriched in the α-galactosidase; 
 E. purifying the α-galactosidase from the 20 to 60 percent ammonium sulfate fraction of step “D.” by anion exchange chromatography followed by cation exchange chromatography to yield an ion exchange purified α-galactosidase; and 
 F. fractionating the ion-exchange purified α-galactosidase of step “E.” by size-exclusion chromatography to yield a purified α-galactosidase which elutes with a molecular weight in the range of 40-80 kD, 
 
   b. retains P 1  antigens, and   c. has no detectable B antigens, as determined by standard blood bank serological typing.   
     
     
         5 . A blood product comprising sero-converted erythrocytes, wherein said sero-converted erythrocytes:
 a. have been converted from type B or AB erythrocytes to a non-B erythrocytes by an α-galactosidase, wherein the α-galactosidase has the following characteristics:
 i. active in red blood cell conversion at neutral pH, and 
 ii. isolated and purified from a non-recombinant strain of  Streptomyces griseoplanus , wherein said α-galactosidase enzyme is isolatable by a method comprising the steps of:
 A. fermentation culturing of an alpha-galactosidase producing  Streptomyces griseoplanus  strain; 
 B. disrupting the cultured  Streptomyces griseoplanus  strain of step “A.”; 
 C. isolating an α-galactosidase-containing supernatant fraction from the disrupted  Streptomyces griseoplanus  strain of step “B.” by centrifugation; 
 D. treating the α-galactosidase-containing supernatant fraction of step “C.” with ammonium sulfate to yield a 20 to 60 percent ammonium sulfate fraction enriched in the α-galactosidase; 
 E. purifying the α-galactosidase from the 20 to 60 percent ammonium sulfate fraction of step “D.” by anion exchange chromatography followed by cation exchange chromatography to yield an ion exchange purified α-galactosidase; and 
 F. fractionating the ion-exchange purified α-galactosidase of step “E.” by size-exclusion chromatography to yield a purified α-galactosidase which elutes with a molecular weight in the range of 40-80 kD, 
 
   b. retains P 1  antigens, and   c. have no detectable B antigens, as determined by standard blood bank serological typing.   
     
     
         6 . 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-acetylgalactosaminidase and an α-galactosidase, wherein the α-galactosidase has the following characteristics:
 i. active in red blood cell conversion at neutral pH, and 
 ii. isolated and purified from a non-recombinant strain of  Streptomyces griseoplanus , wherein said α-galactosidase enzyme is isolatable by a method comprising the steps of:
 A. fermentation culturing of an alpha-galactosidase producing  Streptomyces griseoplanus  strain; 
 B. disrupting the cultured  Streptomyces griseoplanus  strain of step “A.”; 
 C. isolating an α-galactosidase-containing supernatant fraction from the disrupted  Streptomyces griseoplanus  strain of step “B.” by centrifugation; 
 D. treating the α-galactosidase-containing supernatant fraction of step “C.” with ammonium sulfate to yield a 20 to 60 percent ammonium sulfate fraction enriched in the α-galactosidase; 
 E. purifying the α-galactosidase from the 20 to 60 percent ammonium sulfate fraction of step “D.” by anion exchange chromatography followed by cation exchange chromatography to yield an ion exchange purified α-galactosidase; and 
 F. fractionating the ion-exchange purified α-galactosidase of step “E.” by size-exclusion chromatography to yield a purified α-galactosidase which elutes with a molecular weight in the range of 40-80 kD, 
 
   b. has A associated H structures,   c. retains P 1  antigens, and   d. has no detectable A or B antigens, as determined by standard blood bank serological typing.   
     
     
         7 . 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-acetylgalactosaminidase and an α-galactosidase, wherein the α-galactosidase has the following characteristics:
 i. active in red blood cell conversion at neutral pH, and 
 ii. isolated and purified from a non-recombinant strain of  Streptomyces griseoplanus , wherein said α-galactosidase enzyme is isolatable by a method comprising the steps of:
 A. fermentation culturing of an alpha-galactosidase producing  Streptomyces griseoplanus  strain; 
 B. disrupting the cultured  Streptomyces griseoplanus  strain of step “A.”; 
 C. isolating an α-galactosidase-containing supernatant fraction from the disrupted  Streptomyces griseoplanus  strain of step “B.” by centrifugation; 
 D. treating the α-galactosidase-containing supernatant fraction of step “C.” with ammonium sulfate to yield a 20 to 60 percent ammonium sulfate fraction enriched in the α-galactosidase; 
 E. purifying the α-galactosidase from the 20 to 60 percent ammonium sulfate fraction of step “D.” by anion exchange chromatography followed by cation exchange chromatography to yield an ion exchange purified α-galactosidase; and 
 F. fractionating the ion-exchange purified α-galactosidase of step “E.” by size-exclusion chromatography to yield a purified α-galactosidase which elutes with a molecular weight in the range of 40-80 kD, 
 
   b. have A associated H structures,   c. retains P 1  antigens, and   d. have no detectable A or B antigens, as determined by standard blood bank serological typing.   
     
     
         8 . The method of  claim 1 , wherein said α-N-acetylgalactosaminidase is NEB α-N-acetylgalactosaminidase. 
     
     
         9 . The method of  claim 2 , wherein said α-N-acetylgalactosaminidase is NEB α-N-acetylgalactosaminidase. 
     
     
         10 . The method of  claim 6 , wherein said α-N-acetylgalactosaminidase is NEB α-N-acetylgalactosaminidase. 
     
     
         11 . The method of  claim 7 , wherein said α-N-acetylgalactosaminidase is NEB α-N-acetylgalactosaminidase. 
     
     
         12 . The isolated non-recombinant  Streptomyces griseoplanus  α-galactosidase of  claim 1 , wherein the enzyme is isolated from  Streptomyces griseoplanus , ATCC Deposit No: PTA-4077. 
     
     
         13 . The isolated non-recombinant  Streptomyces griseoplanus  α-galactosidase of  claim 2 , wherein the enzyme is isolated from  Streptomyces griseoplanus , ATCC Deposit No: PTA-4077. 
     
     
         14 . The isolated non-recombinant  Streptomyces griseoplanus  α-galactosidase of  claim 4 , wherein the enzyme is isolated from  Streptomyces griseoplanus , ATCC Deposit No: PTA-4077. 
     
     
         15 . The isolated non-recombinant  Streptomyces griseoplanus  α-galactosidase of  claim 5 , wherein the enzyme is isolated from  Streptomyces griseoplanus , ATCC Deposit No: PTA-4077. 
     
     
         16 . The isolated non-recombinant  Streptomyces griseoplanus  α-galactosidase of  claim 6 , wherein the enzyme is isolated from  Streptomyces griseoplanus , ATCC Deposit No: PTA-4077. 
     
     
         17 . The isolated non-recombinant  Streptomyces griseoplanus  α-galactosidase of  claim 7 , wherein the enzyme is isolated from  Streptomyces griseoplanus . ATCC Deposit No: PTA-4077.