Enzymatic compositions for b antigen cleavage, methods, uses, apparatuses and systems associated therewith

Abstract

Provided herein is an enzyme for carbohydrate B-antigen cleavage, methods, uses, apparatuses and systems associated therewith. In particular, the enzyme comprises an alpha-galactosidase and may further comprise a crowding agent. Furthermore, the enzymes described herein were found to have activity a temperatures and pH levels suitable for cell viability.

Description

ENZYMATIC COMPOSITIONS FOR B ANTIGEN CLEAVAGE, METHODS, USES, APPARATUSES AND SYSTEMS ASSOCIATED THEREWITH CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 632,699 filed 11 April 2024 entitled “ENZYMATIC COMPOSITIONS FOR B ANTIGEN CLEAVAGE, METHODS, USES, APPARATUSES AND SYSTEMS ASSOCIATED THEREWITH”. TECHNICAL FIELD

[0002] The present invention relates to the field of enzyme compositions. In particular, the invention relates to enzyme compositions for cleaving antigens, and for providing methods, uses, apparatuses and systems for cleaving antigens using the compositions. BACKGROUND

[0003] The ABO blood group system, first identified by Karl Landsteiner in the early 20th century, plays a crucial role in transfusion medicine1. The ABO system categorizes blood into four groups: A, B, AB, and O, based on the presence of A, B and H antigens on the surface of red blood cells (RBCs) and epithelial organ tissue2. The ABO system is foundational for ensuring compatibility in blood transfusions and organ transplantation, as the presence of foreign antigens can trigger fatal immune responses.

[0004] At the core of this system is the H antigen, a precursor carbohydrate moiety found on the surface of RBCs and organ tissue that, through the action of specific enzymes, is transformed into either A or B antigens. Individuals with type O blood possess the H antigen in its unmodified form, lacking the additional sugar moieties that characterize A and B antigens. This absence of A and B antigens allows type O blood and organs to be universally transfused and transplanted into patients of any ABO blood group, earning it the designation of “universal donor” blood and organs.

[0005] Universal donor blood is especially critical in emergency situations where there is no time to determine the recipient's blood type. However, the scarcity of type O blood poses challenges to blood supply. Similarly, patients with hard-to-match blood types can often spend years longer on waitlists for life-saving organ transplants. Innovations in enzymatic 1  conversion of A and B antigens back to the H antigen offer a promising solution to these issues3. In the past, enzymes have been shown to effectively remove the terminal carbohydrate moiety from the immune-dominant A and B antigens, converting them to the universal O type4,5. However, the low efficiency of these enzymes, especially in the case of B to O conversion, has prevented their practical application in the clinic. High-efficiency enzymatic treatment holds the potential to increase the availability of universal donor blood and organs, thereby enhancing the efficiency and safety of blood transfusions and organ transplants. Liu, Q. P. et al. identified a subfamily of α1,3-Galactosidases GH110 enzymes having two subclasses: subclass A, which is active only on the branched B-antigen substrate, and subclass B, which is active on both the branched and linear substrates10. There are currently three known crystal structures of GH110s, but only two of them have publicly available structures15, 16. The first enzyme in the GH110 group to have the crystal structure solved is from Pseudoalteromonas distincta and this enzyme does not act on the B-antigen15. The second published crystal structure is Akkermansia muciniphila16, which is the same enzyme as Amuc_048012and AM110B described herein. The third crystal structure is from Bifidobacterium bifidum glycoside hydrolase (family 110 α-galactosidase) and is not published, but some details are available in a Biorxiv preprint11. The B. bifidum enzyme is reported to be specific for blood group B antigen and that there is a significance of the fucose recognition site for activity was determined with mutational analysis11. Bakshani et al. describe two (2) Akkermansia muciniphila GH110 α-galactosidase enzymes (Amuc_0480GH110and Amuc_1463GH110)12.

[0006] Although there are known enzymes capable of converting B-antigen to O-antigen, substantial amounts of enzyme are needed for the conversion limiting further development. Enzymes having greater efficiency in cleaving the carbohydrate antigens from cells would be of use. SUMMARY

[0007] The present invention is based in part, on the surprising discovery that a number of GH110 enzymes identified herein proved to be more efficient at converting the B-antigen to the H-antigen, which in turn could enhance the availability of universal donor blood and 2  organs. The approach utilized involved exploring genomic sequence space in assembling a set of enzyme sequences from a GH family known to act on the B-antigen, then focused on sequences having a carbohydrate-binding module (CBM), which is hypothesized to be crucial for efficient activity on RBCs and human tissue. Additionally, we targeted enzymes from bacteria residing in the human gut microbiome, believing this to be a key factor in discovering highly efficient enzymes. After narrowing down the pool of potential candidates, we designed expression constructs in silico and ordered them from a gene synthesis vendor. Subsequent activity analysis of these candidates led to the identification of four (4) enzymes that outperformed the current standards, with the most promising candidate undergoing detailed characterization.

[0008] The present invention is based in part, on the surprising discovery that particular alpha galactosidases, as described herein, are orders of magnitude more efficient than previously identified B-antigen cleaving enzymes. For example, under some conditions some of the alpha galactosidase enzymes may be capable of cleaving all B-antigens using enzyme concentrations at or below 5 µg / ml. Furthermore, the cleavage efficiency of the enzyme combination is maintained at a pH suitable to maintain viability of the erythrocytes (i.e. pH between about 6.5 and about 7.5). Additionally, the enzymes were found to be active at temperatures between 4°C and 37°C, and in whole blood, which is also suitable for blood collection, washing and storage protocols or organ storage and perfusion protocols. Furthermore, the alpha galactosidase enzymes also show compatibility with a range of solutions. It has also been appreciated that the same alpha galactosidase enzymes could be applied to donor organs or tissues.

[0009] In accordance with a first embodiment, there is provided a purified alpha galactosidase selected from one or more of the following: SEQ ID NO.:2; SEQ ID NO.:3; SEQ ID NO.:4; SEQ ID NO.:5; SEQ ID NO.:6; SEQ ID NO.:7; SEQ ID NO.:8; SEQ ID NO.:9; SEQ ID NO.:10; SEQ ID NO.:11; and SEQ ID NO.:12.

[0010] In accordance with one embodiment, there is provided a purified Akkermansia muciniphila alpha galactosidase of SEQ ID NO.:2 or SEQ ID NO.:3.

[0011] In accordance with one embodiment, there is provided a purified Akkermansia muciniphila alpha galactosidase of SEQ ID NO.:1, SEQ ID NO.:2 or SEQ ID NO.:3. 3

[0012] In accordance with a further embodiment, there is provided a purified Luteolibacter ambystomatis alpha galactosidase of SEQ ID NO.:4, SEQ ID NO.:5 or SEQ ID NO.:6.

[0013] In accordance with a further embodiment, there is provided a purified Akkermansia glycaniphila alpha galactosidase of SEQ ID NO.:7, SEQ ID NO.:8 or SEQ ID NO.:9.

[0014] In accordance with a further embodiment, there is provided a purified Akkermansia glycaniphila alpha galactosidase of SEQ ID NO.:10, SEQ ID NO.:11 or SEQ ID NO.:12.

[0015] The purified alpha galactosidase enzyme may be for use in enzymatically cleaving B- antigens from whole blood, erythrocytes, a donor tissue, or a donor organ.

[0016] In accordance with a further embodiment, there is provided a purified alpha galactosidase enzyme of: SEQ ID NO.:1; SEQ ID NO.:2; SEQ ID NO.:4; SEQ ID NO.:5; SEQ ID NO.:7; SEQ ID NO.:8; SEQ ID NO.:10; or SEQ ID NO.:11 and a protein tag.

[0017] The protein tag may be selected from one or more of: Albumin-binding protein (ABP); Alkaline Phosphatase (AP); AU1 epitope; AU5 epitope; AviTag; Bacteriophage T7 epitope (T7- tag); Bacteriophage V5 epitope (V5-tag); Biotin-carboxy carrier protein (BCCP); Bluetongue virus tag (B-tag); single-domain camelid antibody (C-tag); Calmodulin binding peptide (CBP or Calmodulin-tag); Chloramphenicol Acetyl Transferase (CAT); Cellulose binding domain (CBP); Chitin binding domain (CBD); Choline-binding domain (CBD); Dihydrofolate reductase (DHFR); DogTag; E2 epitope; E-tag; FLAG epitope (FLAG-tag); Galactose-binding protein (GBP); Green fluorescent protein (GFP); Glu-Glu (EE-tag); Glutathione S-transferase (GST); heparin-binding affinity tag (HB-tag); Human influenza hemagglutinin (HA); HaloTag™; Alternating histidine and glutamine tags (HQ tag); Alternating histidine and asparagine tags (HN tag); Histidine affinity tag (HAT); Histidine tag; Horseradish Peroxidase (HRP); HSV epitope; Isopeptag (Isopep-tag); Ketosteroid isomerase (KSI); KT3 epitope; LacZ; Luciferase; Maltose-binding protein (MBP); Myc epitope (Myc-tag); NE-tag; NusA; PDZ domain; PDZ ligand; Polyarginine (Arg-tag); Polyaspartate (Asp-tag); Polycysteine (Cys- tag); Polyglutamate (Glu-tag); Polyhistidine (His-tag); Polyphenylalanine (Phe-tag); Profinity eXact; Protein C; Rho1D4-tag; S1-tag; S-tag; Softag 1; Softag 3; SnoopTagJr; SnoopTag; Spot- tag; SpyTag (Spy-tag); Streptavadin-binding peptide (SBP); Staphylococcal protein A (Protein A); Staphylococcal protein G (Protein G); Strep-tag; Streptavadin (SBP-tag); Strep-tag II; Sdy- tag; Small Ubiquitin-like Modifier (SUMO); Tandem Affinity Purification (TAP); T7 epitope; 4  tetracysteine tag (TC tag); Thioredoxin (Trx); TrpE; Ty tag; Ubiquitin; Universal; V5 tag; VSV- G or VSV-tag; and Xpress tag.

[0018] In accordance with a further embodiment, there is provided an isolated nucleic acid sequence encoding alpha galactosidase selected from one or more of: SEQ ID NO.:13; SEQ ID NO.:14; SEQ ID NO.:15; and SEQ ID NO.:16.

[0019] In accordance with a further embodiment, there is provided an isolated nucleic acid sequence encoding alpha galactosidase, wherein the nucleic acid sequence encodes an amino acid sequence selected from one or more of: SEQ ID NOs.:1-12.

[0020] In accordance with a further embodiment, there is provided a vector including the nucleic acid selected from one or more of: SEQ ID NO.:13; SEQ ID NO.:14; SEQ ID NO.:15; and SEQ ID NO.:16 or encoding an amino acid sequence selected from one or more of: SEQ ID NOs.:1-12 and a heterologous nucleic acid sequence.

[0021] The heterologous nucleic acid sequence may be selected from one or more of the following: a protein tag; and a cleavage site.

[0022] In accordance with a further embodiment, there is provided a vector including the nucleic acid selected from one or more of: SEQ ID NO.:13; SEQ ID NO.:14; SEQ ID NO.:15; and SEQ ID NO.:16 or encoding an amino acid sequence selected from one or more of: SEQ ID NOs.:1-12.

[0023] In accordance with a further embodiment, there is provided a method for enzymatically cleaving B-antigens from whole blood, erythrocytes, a donor tissue or a donor organ, the method including: (a) combining a purified alpha galactosidase enzyme as described herein with the whole blood, the erythrocytes, the donor tissue or the donor organ including (i) type B antigen, or (ii) AB type antigen; (b) incubating the purified alpha galactosidase enzyme with the whole blood, the erythrocytes, the donor tissue or the donor organ; for a period of time sufficient to allow the enzymes to cleave B-antigens from the whole blood, the erythrocytes, the donor tissue or the donor organ.

[0024] The purified alpha galactosidase enzyme may be selected from one or more of: SEQ ID NO.:1; SEQ ID NO.:2; SEQ ID NO.:3; SEQ ID NO.:4; SEQ ID NO.:5; SEQ ID NO.:6; SEQ ID NO.:7; SEQ ID NO.:8; SEQ ID NO.:9; SEQ ID NO.:10; SEQ ID NO.:11; and SEQ ID NO.:12. 5

[0025] The purified alpha galactosidase enzyme may be selected from one or more of: SEQ ID NO.:2; SEQ ID NO.:3; SEQ ID NO.:5; SEQ ID NO.:6; SEQ ID NO.:8; SEQ ID NO.:9; SEQ ID NO.:11; and SEQ ID NO.:12.

[0026] The purified alpha galactosidase enzyme may be combined with a Galactosaminidase and a GalNAcDeacetylase, having A-antigen cleaving activity.

[0027] The composition may include: a purified enzyme having alpha galactosidase activity having an amino acid sequence at least 90% identical to the sequence set forth in one of SEQ ID NOs:1-12.

[0028] The method may further include adding a crowding agent. The crowding agent may be selected from one or more of: a dextran; a dextran sulfate; a dextrin; a pullulan; a poly(ethylene glycol); a Ficoll™; a hyper-branched glycerol; hydroxyethyl starch; polyvinyl alcohol; and an inert protein.

[0029] The method further include washing the blood, the erythrocytes, the donor tissue or the donor organ to remove alpha galactosidase and the crowding agent.

[0030] The alpha galactosidase may be capable of cleaving B-antigen using enzyme concentrations at or below 1 µg / ml. The alpha galactosidase may be capable of cleaving B- antigen using enzyme concentrations at or below 1.5 µg / ml. The alpha galactosidase may be capable of cleaving B-antigen using enzyme concentrations at or below 2 µg / ml. The alpha galactosidase may be capable of cleaving B-antigen using enzyme concentrations at or below 2.5 µg / ml. The alpha galactosidase may be capable of cleaving B-antigen using enzyme concentrations at or below 3 µg / ml. The alpha galactosidase may be capable of cleaving B- antigen using enzyme concentrations at or below 3.5 µg / ml. The alpha galactosidase may be capable of cleaving B-antigen using enzyme concentrations at or below 4 µg / ml. The alpha galactosidase may be capable of cleaving B-antigen using enzyme concentrations at or below 4.5 µg / ml. The alpha galactosidase may be capable of cleaving B-antigen using enzyme concentrations at or below 5 µg / ml. The alpha galactosidase may be capable of cleaving B- antigen using enzyme concentrations at or below 5.5 µg / ml. The alpha galactosidase may be capable of cleaving B-antigen using enzyme concentrations at or below 6 µg / ml. The alpha galactosidase may be capable of cleaving B-antigen using enzyme concentrations at or below 6.5 µg / ml. The alpha galactosidase may be capable of cleaving B-antigen using enzyme concentrations at or below 7 µg / ml. The alpha galactosidase may be capable of cleaving B- 6  antigen using enzyme concentrations at or below 7.5 µg / ml. The alpha galactosidase may be capable of cleaving B-antigen using enzyme concentrations at or below 8 µg / ml. The alpha galactosidase may be capable of cleaving B-antigen using enzyme concentrations at or below 8.5 µg / ml. The alpha galactosidase may be capable of cleaving B-antigen using enzyme concentrations at or below 9 µg / ml. The alpha galactosidase may be capable of cleaving B- antigen using enzyme concentrations at or below 9.5 µg / ml. The alpha galactosidase may be capable of cleaving B-antigen using enzyme concentrations at or below 10 µg / ml.

[0031] The alpha galactosidase may have B-antigen cleaving activity at a pH between about 6.0 and about 8.0. The alpha galactosidase may have B-antigen cleaving activity at a pH between about 6.5 and about 8.0. The alpha galactosidase may have B-antigen cleaving activity at a pH between about 6.8 and about 8.0. The alpha galactosidase may have B- antigen cleaving activity at a pH between about 6.0 and about 7.5. The alpha galactosidase may have B-antigen cleaving activity at a pH between about 6.5 and about 7.5. The alpha galactosidase may have B-antigen cleaving activity at a pH between about 6.3 and about 7.8. The alpha galactosidase may have B-antigen cleaving activity at a pH between about 6.4 and about 7.8. The alpha galactosidase may have B-antigen cleaving activity at a pH between about 6.2 and about 7.5. The alpha galactosidase may have B-antigen cleaving activity at a pH between about 6.4 and about 7.5.

[0032] The alpha galactosidase has B-antigen cleaving activity at temperatures between 4°C and 37°C. The alpha galactosidase has B-antigen cleaving activity at temperatures between 3°C and 37°C. The alpha galactosidase has B-antigen cleaving activity at temperatures between 3°C and 38°C. The alpha galactosidase has B-antigen cleaving activity at temperatures between 2°C and 37°C. The alpha galactosidase has B-antigen cleaving activity at temperatures between 2°C and 38°C. The alpha galactosidase has B-antigen cleaving activity at temperatures between 1°C and 37°C. The alpha galactosidase has B-antigen cleaving activity at temperatures between 1°C and 38°C. The alpha galactosidase has B- antigen cleaving activity at temperatures between 5°C and 37°C. The alpha galactosidase has B-antigen cleaving activity at temperatures between 5°C and 38°C.

[0033] The donor organ may be a solid organ. The solid organ may be selected from one of the following: lung; kidney; liver; heart; pancreas; skin; uterus, thymus, and intestine. The solid organ may be selected from one of the following: lung; kidney; liver; heart; pancreas; skin; and intestine. The solid organ may be a lung. The solid organ may be a kidney. The solid 7  organ may be a liver. The solid organ may be a heart. The solid organ may be a pancreas. The solid organ may be skin. The solid organ may be an intestine. The donor tissue may be selected from one or more of the following: bones, tendons, corneae, skin, heart valves, nerves, and veins. The donor tissue may be skin. The donor tissue may be heart. The donor tissue may be lung. The donor tissue may be kidney. The donor tissue may be liver. The donor tissue may be pancreas. The donor tissue may be intestine. The donor tissue may be eye. The donor tissue may be thymus. The donor tissue may be uterus.

[0034] The method of claim 26, wherein the purified enzyme having alpha galactosidase activity may be mixed with an ex vivo lung solution and circulated through the lung, whereby the alpha galactosidase enzyme may be in contact with the vasculature of the donor organ for a period of time sufficient to substantially clear the B-antigens from the vasculature of the lung.

[0035] The time to clear the B-antigens from the vasculature of the lung may be about 1 hour.

[0036] The method may further include washing the donor organ to remove alpha galactosidase enzyme and cleaved B-antigens. The method may further include washing the donor organ to remove GalNAcDeacetylase enzyme, Galactosaminidase enzyme, and cleaved A-antigens. The method may further include washing the donor organ to remove alpha galactosidase enzyme, GalNAcDeacetylase enzyme, Galactosaminidase enzyme, cleaved B- antigen, and cleaved A-antigens.

[0037] In accordance with a further embodiment, there is provided a blood collection and storage system, including: (a) a purified alpha galactosidase enzyme, wherein the purified enzyme having alpha galactosidase activity having an amino acid sequence at least 90% identical to the sequence set forth in one of SEQ ID NOs:1-12; and (b) a surface to which the enzyme may be immobilized.

[0038] The purified alpha galactosidase enzyme may be selected from one or more of: SEQ ID NO.:1; SEQ ID NO.:2; SEQ ID NO.:3; SEQ ID NO.:4; SEQ ID NO.:5; SEQ ID NO.:6; SEQ ID NO.:7; SEQ ID NO.:8; SEQ ID NO.:9; SEQ ID NO.:10; SEQ ID NO.:11; and SEQ ID NO.:12. The purified GalNAcDeacetylase enzyme and / or Galactosaminidase enzyme may be selected from one or more of: SEQ ID NOs.:69-91. The purified GalNAcDeacetylase enzyme and / or Galactosaminidase enzyme may be selected from one or more of: SEQ ID NOs.:69-74. 8

[0039] The surface may be selected from one or more of the following: (a) a bead or microsphere; (b) a container; (c) a tube; (d) a column; or (e) a matrix. The container may be a bag.

[0040] In accordance with a further embodiment, there is provided a blood collection and storage apparatus, the apparatus including: (a) a surface; and (b) a purified alpha galactosidase enzyme, wherein the purified enzyme having alpha galactosidase activity having an amino acid sequence at least 90% identical to the sequence set forth in one of SEQ ID NOs:1-12 immobilized to the surface.

[0041] In accordance with a further embodiment, there is provided a perfusion fluid for enzymatically cleaving B-antigen from a donor organ or donor tissue including: (a) a purified alpha galactosidase enzyme, wherein the purified enzyme having alpha galactosidase activity having an amino acid sequence at least 90% identical to the sequence set forth in one of SEQ ID NOs:1-12; and (b) a solution.

[0042] The purified alpha galactosidase enzyme may be capable of cleaving B-antigen at or below 1 µg / ml. The purified alpha galactosidase enzyme has B-antigen cleaving activity at a pH between about 6.5 and about 7.5. The purified alpha galactosidase enzyme has B-antigen cleaving activity at a temperatures between 4°C and 37°C.

[0043] The solution may be selected from: Steen™; Perfadex™; Perfadex Plus™; EuroCollins solution; Histidine-Tryptophan-Ketoglutarate (HTK) solution; University of Wisconsin solution (UW); Celsior solution; Kidney Perfusion solution (KPS-1); Kyoto University solution; IGL-1 solution; and Citrate solution.

[0044] In accordance with a further embodiment, there is provided a method for enzymatically cleaving B-antigens from whole blood, erythrocytes, a donor tissue or a donor organ, the method including: (a) combining a purified alpha galactosidase enzyme as described herein; and a purified Galactosaminidase and a purified GalNAcDeacetylase, having A-antigen cleaving activity; with whole blood, the erythrocytes, the donor tissue or the donor organ including (i) type B antigen, (ii) type A antigen, or (iii) AB type antigen; (b)incubating the purified alpha galactosidase enzyme with the whole blood, the erythrocytes, the donor tissue or the donor organ; for a period of time sufficient to allow the enzymes to cleave B-antigens and A-antigens from the whole blood, the erythrocytes, the donor tissue or the donor organ. 9

[0045] The purified alpha galactosidase enzyme may be selected from one or more of: SEQ ID NO.:2; SEQ ID NO.:3; SEQ ID NO.:5; SEQ ID NO.:6; SEQ ID NO.:8; SEQ ID NO.:9; SEQ ID NO.:11; and SEQ ID NO.:12.

[0046] In accordance with a further embodiment, there is provided a perfusion fluid for enzymatically cleaving B-antigen and A-antigen from a donor organ or donor tissue including: (a) a purified alpha galactosidase enzyme, wherein the purified enzyme having alpha galactosidase activity including having an amino acid sequence at least 90% identical to the sequence set forth in one of SEQ ID NOs:1-12; (b) a purified Galactosaminidase having an amino acid sequence at least 90% identical to the sequence set forth in one of SEQ ID NOs: 69-71 and a purified GalNAcDeacetylase having an amino acid sequence at least 90% identical to the sequence set forth in one of SEQ ID NOs: 72-74, having A-antigen cleaving activity; and (c) a solution.

[0047] The alpha galactosidase may be capable of cleaving B-antigen at or below 100µg / ml. The alpha galactosidase may be capable of cleaving B-antigen at or below 90µg / ml. The alpha galactosidase may be capable of cleaving B-antigen at or below 80µg / ml. The alpha galactosidase may be capable of cleaving B-antigen at or below 70µg / ml. The alpha galactosidase may be capable of cleaving B-antigen at or below 60µg / ml. The alpha galactosidase may be capable of cleaving B-antigen at or below 50µg / ml. The alpha galactosidase may be capable of cleaving B-antigen at or below 40µg / ml. The alpha galactosidase may be capable of cleaving B-antigen at or below 30µg / ml. The alpha galactosidase may be capable of cleaving B-antigen at or below 20µg / ml. The alpha galactosidase may be capable of cleaving B-antigen at or below 15µg / ml. The alpha galactosidase may be capable of cleaving B-antigen at or below 14µg / ml. The alpha galactosidase may be capable of cleaving B-antigen at or below 13µg / ml. The alpha galactosidase may be capable of cleaving B-antigen at or below 12µg / ml. The alpha galactosidase may be capable of cleaving B-antigen at or below 11µg / ml. The alpha galactosidase may be capable of cleaving B-antigen at or below 10µg / ml. The alpha galactosidase may be capable of cleaving B-antigen at or below 9µg / ml. The alpha galactosidase may be capable of cleaving B-antigen at or below 8µg / ml. The alpha galactosidase may be capable of cleaving B-antigen at or below 7µg / ml. The alpha galactosidase may be capable of cleaving B-antigen at or below 6µg / ml. The alpha galactosidase may be capable of cleaving B-antigen at or below 5µg / ml. The alpha 10  galactosidase may be capable of cleaving B-antigen at or below 4µg / ml. The alpha galactosidase may be capable of cleaving B-antigen at or below 3µg / ml. The alpha galactosidase may be capable of cleaving B-antigen at or below 2µg / ml. The alpha galactosidase may be capable of cleaving B-antigen at or below 1µg / ml. The alpha galactosidase may be capable of cleaving B-antigen at or below 0.9µg / ml. The alpha galactosidase may be capable of cleaving B-antigen at or below 0.8µg / ml. The alpha galactosidase may be capable of cleaving B-antigen at or below 0.7µg / ml. The alpha galactosidase may be capable of cleaving B-antigen at or below 0.6µg / ml. The alpha galactosidase may be capable of cleaving B-antigen at or below 0.5µg / ml. The alpha galactosidase may be capable of cleaving B-antigen at or below 0.4µg / ml. The alpha galactosidase may be capable of cleaving B-antigen at or below 0.3µg / ml. The alpha galactosidase may be capable of cleaving B-antigen at or below 0.2µg / ml. The alpha galactosidase may be capable of cleaving B-antigen at or below 0.1µg / ml. The alpha galactosidase may be capable of cleaving B-antigen at or below 0.09µg / ml. The alpha galactosidase may be capable of cleaving B-antigen at or below 0.08µg / ml. The alpha galactosidase may be capable of cleaving B-antigen at or below 0.07µg / ml. The alpha galactosidase may be capable of cleaving B-antigen at or below 0.06µg / ml. The alpha galactosidase may be capable of cleaving B-antigen at or below 0.05µg / ml. The alpha galactosidase may be capable of cleaving B-antigen at or below 0.04µg / ml. The alpha galactosidase may be capable of cleaving B-antigen at or below 0.03µg / ml. The alpha galactosidase may be capable of cleaving B-antigen at or below 0.02µg / ml. The alpha galactosidase may be capable of cleaving B-antigen at or below 0.01µg / ml.

[0048] The GalNAcDeacetylase and Galactosaminidase may be capable of cleaving A-antigen at or below 100µg / ml. The GalNAcDeacetylase and Galactosaminidase may be capable of cleaving A-antigen at or below 90µg / ml. The GalNAcDeacetylase and Galactosaminidase may be capable of cleaving A-antigen at or below 80µg / ml. The GalNAcDeacetylase and Galactosaminidase may be capable of cleaving A-antigen at or below 70µg / ml. The GalNAcDeacetylase and Galactosaminidase may be capable of cleaving A-antigen at or below 60µg / ml. The GalNAcDeacetylase and Galactosaminidase may be capable of cleaving A- antigen at or below 50µg / ml. The GalNAcDeacetylase and Galactosaminidase may be capable of cleaving A-antigen at or below 40µg / ml. The GalNAcDeacetylase and Galactosaminidase may be capable of cleaving A-antigen at or below 30µg / ml. The GalNAcDeacetylase and 11  Galactosaminidase may be capable of cleaving A-antigen at or below 20µg / ml. The GalNAcDeacetylase and Galactosaminidase may be capable of cleaving A-antigen at or below 15µg / ml. The GalNAcDeacetylase and Galactosaminidase may be capable of cleaving A- antigen at or below 14µg / ml. The GalNAcDeacetylase and Galactosaminidase may be capable of cleaving A-antigen at or below 13µg / ml. The GalNAcDeacetylase and Galactosaminidase may be capable of cleaving A-antigen at or below 12µg / ml. The GalNAcDeacetylase and Galactosaminidase may be capable of cleaving A-antigen at or below 11µg / ml. The GalNAcDeacetylase and Galactosaminidase may be capable of cleaving A-antigen at or below 10µg / ml. The GalNAcDeacetylase and Galactosaminidase may be capable of cleaving A- antigen at or below 9µg / ml. The GalNAcDeacetylase and Galactosaminidase may be capable of cleaving A-antigen at or below 8µg / ml. The GalNAcDeacetylase and Galactosaminidase may be capable of cleaving A-antigen at or below 7µg / ml. The GalNAcDeacetylase and Galactosaminidase may be capable of cleaving A-antigen at or below 6µg / ml. The GalNAcDeacetylase and Galactosaminidase may be capable of cleaving A-antigen at or below 5µg / ml. The GalNAcDeacetylase and Galactosaminidase may be capable of cleaving A- antigen at or below 4µg / ml. The GalNAcDeacetylase and Galactosaminidase may be capable of cleaving A-antigen at or below 3µg / ml. The GalNAcDeacetylase and Galactosaminidase may be capable of cleaving A-antigen at or below 2µg / ml. The GalNAcDeacetylase and Galactosaminidase may be capable of cleaving A-antigen at or below 1µg / ml. The GalNAcDeacetylase and Galactosaminidase may be capable of cleaving A-antigen at or below 0.9µg / ml. The GalNAcDeacetylase and Galactosaminidase may be capable of cleaving A- antigen at or below 0.8µg / ml. The GalNAcDeacetylase and Galactosaminidase may be capable of cleaving A-antigen at or below 0.7µg / ml. The GalNAcDeacetylase and Galactosaminidase may be capable of cleaving A-antigen at or below 0.6µg / ml. The GalNAcDeacetylase and Galactosaminidase may be capable of cleaving A-antigen at or below 0.5µg / ml. The GalNAcDeacetylase and Galactosaminidase may be capable of cleaving A- antigen at or below 0.4µg / ml. The GalNAcDeacetylase and Galactosaminidase may be capable of cleaving A-antigen at or below 0.3µg / ml. The GalNAcDeacetylase and Galactosaminidase may be capable of cleaving A-antigen at or below 0.2µg / ml. The GalNAcDeacetylase and Galactosaminidase may be capable of cleaving A-antigen at or below 0.1µg / ml. The GalNAcDeacetylase and Galactosaminidase may be capable of cleaving A- antigen at or below 0.09µg / ml. The GalNAcDeacetylase and Galactosaminidase may be capable of cleaving A-antigen at or below 0.08µg / ml. The GalNAcDeacetylase and 12  Galactosaminidase may be capable of cleaving A-antigen at or below 0.07µg / ml. The GalNAcDeacetylase and Galactosaminidase may be capable of cleaving A-antigen at or below 0.06µg / ml. The GalNAcDeacetylase and Galactosaminidase may be capable of cleaving A- antigen at or below 0.05µg / ml. The GalNAcDeacetylase and Galactosaminidase may be capable of cleaving A-antigen at or below 0.04µg / ml. The GalNAcDeacetylase and Galactosaminidase may be capable of cleaving A-antigen at or below 0.03µg / ml. The GalNAcDeacetylase and Galactosaminidase may be capable of cleaving A-antigen at or below 0.02µg / ml. The GalNAcDeacetylase and Galactosaminidase may be capable of cleaving A- antigen at or below 0.01µg / ml. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] FIGURE 1 shows a GH110 sequence similarity network, with ~40% sequence ID threshold (EVALUE = 120) Colour coded by meta-node. The lightest nodes indicate the presence of a carbohydrate-binding module (CBM), including Am110B, which is marked with a black arrow.

[0050] FIGURE 2 shows His-tag purification of Am110B, with elution fractions 1 and 2 combined for the in final Am110B preparation.

[0051] FIGURE 3 shows a thin-layer chromatography (TLC) analysis of Am110B with free substrate, where (A) shows chemical structures and GH110 reaction schemes for branched and linear B-antigens (Type-VI), (B) shows enzymatic reactions on branched and linear B- antigen substrates for 30 minutes at 37°C, where activity was determined by comparing the "no enzyme" control lane with the sample lanes. Positive activity is indicated by a light dot, while inactivity is marked with a dark dot. The GH110 subclasses were determined based on each enzyme's activity on the substrates. Am110B was classified into subclass B. Note: Edge effects on the TLC plate caused the mobile phase to run at an accelerated rate on the right side, resulting in a curling effect.

[0052] FIGURE 4 shows a median fluorescence intensity plot for La110B and AgaBb, where the median fluorescence values were derived from the raw data, providing a comparison of enzyme activities based on fluorescence intensity. n = 2.

[0053] FIGURE 5 shows a flow analysis of Am110B, Ag110A and Ag110B. Top: Median fluorescence intensity plot. n = 2. Bottom: Flow cytometry histograms. 13

[0054] FIGURE 6 shows a median fluorescence intensity plot of Am110B at varying concentrations. Top: Bar chart annotated with plotted values. Bottom XY plot. The median fluorescence values were derived from the raw data n = 2.

[0055] FIGURE 7 shows a median fluorescence intensity plot for Am110B at varying concentrations, conditions and solutions, where the median fluorescence values were derived from the raw data. n = 2.

[0056] FIGURE 8 shows a median fluorescence intensity plot for AB RBC conversion at varying conditions and solutions. (A) Anti-A antibody treatment. (B) Anti-B antibody treatment. The median fluorescence values were derived from raw data. n = 2.

[0057] FIGURE 9 shows a median fluorescence intensity plot for AB RBC conversion in whole blood. (top) Anti-A antibody treatment. (bottom) Anti-B antibody treatment. The median fluorescence values were derived from raw data.

[0058] FIGURE 10 shows B-type human kidneys antigen remodelling. (A) Untreated type- B tissue control. (B) Type-B tissue treated with Am110B. (C) Type-B tissue treated with Am110B, EnzAO1 and EnzAO2. (D) Quantification and comparison of anti-B signal. Statistical analysis was by one way analysis of variance (ANOVA) followed by post hoc Tukey’s test. Differences were considered significant for p < 0.0001 (****). ‘ns’ stands for not statistically significant. Error bars indicate standard error. n = 5-7.

[0059] FIGURE 11 shows A-type human kidney antigen remodelling. (A) Untreated type-A tissue control. (B) Type-A tissue treated with Am110B. (C) Type-A tissue treated with Am110B, EnzAO1 and EnzAO2. (D) Quantification and comparison of anti-A signal. Statistical analysis was by one way analysis of variance (ANOVA) followed by post hoc Tukey’s test. Differences were considered significant for p < 0.05 (*), p < 0.01 (**), p < 0.001 (***). ‘ns’ stands for not statistically significant. Error bars indicate standard error. n = 5-7.

[0060] FIGURE 12 shows median fluorescence intensity plot for B and AB RBC conversion with and without dextran-40, wherein the median fluorescence values were derived from raw data (not shown) n = 2. Error bars indicate standard error. 14  DETAILED DESCRIPTION

[0061] The following detailed description will be better understood when read in conjunction with the appended figures. For the purpose of illustrating the invention, the figures demonstrate embodiments of the present invention. However, the invention is not limited to the precise arrangements, examples, and instrumentalities shown.

[0062] Any terms not directly defined herein shall be understood to have the meanings commonly associated with them as understood within the art of the invention.

[0063] An “immobilized enzyme” as used herein is an enzyme attached to surface, which may be an inert, insoluble material. Immobilization of enzymes can provide increased resistance to changes in conditions such as pH, temperature etc. and assist in their removal following use and for enzyme re-use.

[0064] Immobilization of an enzyme may be accomplished in various ways (for example, affinity-tag binding, surface adsorption on glass, resin, alginate beads or matrix, bead, fiber or microsphere entrapment, cross-linking to a surface or other enzymes and covalent binding to a surface).

[0065] As used herein “affinity-tag binding” refers to the immobilization of enzymes to a surface (for example, a porous material, using non-covalent or covalent protein tags). Affinity-tag binding has been used for protein purification and has more recently been used for biocatalysis applications by EziG™ (ENGINZYME AB™, Sweden - for example, PCT / US1992 / 010113; and PCT / SE2015 / 050108). Alternative systems are known in the art for attaching active enzymes to a surface (see for example, US4088538; US4141857; US4206259; US4218363; US4229536; US4239854; US4619897; US4748121; US4749653; US4897352; US4954444; US4978619; US5154808; US5914367; US5962279; US6030933; US6291582; US6254645; US10,016,490; and US10,041,055)13.

[0066] Protein tags are peptide sequences genetically grafted onto a recombinant protein, are often removable by chemical agents or by enzymatic means and are attached to proteins for various purposes. The protein tags set out in TABLE A are intended to be examples and are not intended to be limiting in any way. One type of protein tag is an affinity tag, which are added to proteins or peptide sequences so that they can be purified from a crude biological source using an affinity technique (for example, from expression system organisms) or to facilitate immobilization of the “tagged” protein to a surface. Some examples of affinity tags include chitin binding domain (CBD), maltose binding protein (MBP), heparin-binding 15  affinity tag (HB-tag), Strep-tag, glutathione-S-transferase (GST) and the Polyhistidine (His- tag), which binds to metal matrices. Another type of protein tag is an epitope tag (for example, including V5-tag, Myc-tag, HA-tag, Spot-tag and NE-tag), which are short peptide sequences chosen for the ease of producing high-affinity antibodies and are often derived from viral gene sequences to improve immunoreactivity. Epitope tags are particularly useful for western blotting, immunofluorescence and immunoprecipitation experiments, although they also find use in purification and immobilization of proteins to a surface. Yet another type of protein tag is a chromatography tag (for example, polyanionic amino acids, such as FLAG- tag), which may be used to alter chromatographic properties of the protein to assist with separation and purification or immobilization. Yet further protein tags are solubilization tags (for example, Maltose-binding protein (MBP), Glutathione S-transferase (GST), thioredoxin (TRX) and poly(NANP)) and fluorescence tags (for example, Green fluorescent protein (GFP)). Protein tags may allow specific enzymatic modification, chemical modifications or to connect proteins to other components. However, depending on the type or number of tags added to a protein sequence the native function of the protein, in this case the enzymatic function, may be compromised by the tag. Accordingly, the protein tag would need to be selected to ensure that the activity of the enzyme is not compromised or alternatively, the protein tag may be cleaved from the protein before use.

[0067] TABLE A: Exemplary Protein Tags Tag Name Length (sequence) SEQ Position ID NO: Bacteriophage V5 14 (GKPIPNPLLGLDST) 21 C-term epitope (V5-tag) Glu-Glu (EE-tag) 6 (EYMPME or 29 N-term, or C-term, or internal EFMPME)18  Maltose-binding 396 N-term or C-term protein (MBP)19  Softag 1, for 13 (SLAELLNAGLGGS) 50 mammalian T7 epitope 260 N-term

[0068] The use of a protein tag is exemplified in the current application through the use of Polyhistidine protein tag (His-tag) as shown in SEQ ID NOs: 3, 6, 9 and 12, but a person of skill in the art would readily appreciate that any number of other protein tags may be used to purify the enzymes and / or be used to attach the enzymes to a surface as described herein, depending on the purification method used and / or the surface the enzymes are attached to. Such protein tags may be selected from any one or more of the protein tags listed in TABLE A, but other such protein tags are known in the art.

[0069] Furthermore, the use of one or more cleavage sites (for example, the thrombin cleavage site) may be employed to release the protein tag from the enzyme or to otherwise cleave the enzyme. A cleavage site may be used for the removal of the N-terminal methionine, signal peptide, and / or the conversion of an inactive or non-functional protein to an active one (i.e. zymogens or proenzymes). Alternatively, a cleavage site may be used to separate two or more enzymes that were expressed in the same reading frame. Examples of enzymes that are capable of cleaving proteins or peptides and which would have sequence specific cleavage sites may be selected from one or more of the following: Arg-C proteinase; Asp-N endopeptidase; 21  Asp-N endopeptidase + N-terminal Glu BNPS-Skatole; Caspase 1; Caspase 2; Caspase 3; Caspase 4; Caspase 5 Caspase 6; Caspase 7; Caspase 8; Caspase 9; Caspase 10; Chymotrypsin- high specificity (C-term to [FYW], not before P); Chymotrypsin-low specificity (C-term to [FYWML], not before P); Clostripain (Clostridiopeptidase B); CNBr; Enterokinase; Factor Xa; Formic acid; Glutamyl endopeptidase; GranzymeB; Hydroxylamine; Iodosobenzoic acid; LysC; LysN; NTCB (2-nitro-5-thiocyanobenzoic acid); Neutrophil elastase; Pepsin (pH1.3); Pepsin (pH>2); Proline-endopeptidase; Proteinase K; Staphylococcal peptidase I; Tobacco etch virus protease; Thermolysin; Thrombin; and Trypsin.

[0070] A person of skill in the art would also appreciate that the combination of an active alpha galactosidase described herein with an active Galactosaminidase enzyme and an active GalNAcDeacetylase enzyme to cleave both A and B antigens, capable of efficiently cleaving B- antigen and A-antigen is of importance and that person of skill would also appreciate that the addition of one or more cleavage sites and / or one or more protein tags is optional and that such modifications may be selected based on the particular expression system, purification system and possible surface attachment strategy. Furthermore, other modifications to the alpha galactosidase, Galactosaminidase and the GalNAcDeacetylase sequences are possible, provided that the activity in cleaving A-antigens and B-antigens is not significantly impaired. Additionally, modifications to the alpha galactosidase, the Galactosaminidase and the GalNAcDeacetylase enzymes are possible, provided that the A-antigen and B-antigen cleavage activity is not significantly impaired. The modifications may be a deletion, an insertion and / or a substitution. The substitution may be a conservative substitution or a neutral substitution. For example, the alpha galactosidase sequences may share 90% or more sequence identity with the mature enzymes is possible. For example, the alpha galactosidase sequences may share 85% or more sequence identity with the mature enzymes is possible. For example, the alpha galactosidase sequences may share 75% or more sequence identity with the mature enzymes is possible. Alternatively, the alpha galactosidase sequences may have modifications to 5, 10, 13, 15, 20 or up to 25%, of the amino acids.

[0071] Active Galactosaminidase enzymes and active GalNAcDeacetylase enzyme (i.e. as described in WO 2020 / 034042) are set out in SEQ ID NOs.: 69-91.

[0072] As used herein “adsorption on glass, alginate beads or matrix” refers to the attached of an enzyme to the outside of an inert material. Generally, this type of immobilization does not result from a chemical reaction and the active site of the immobilized enzyme can be 22  blocked by the surface to which it has absorbed, which may reduce the activity of the enzyme being absorbed.

[0073] As used herein “entrapment” refers to the trapping of an enzyme within an insoluble beads or microspheres. However, entrapment may hinder the arrival of the substrate, and the exit of products. One example, is the use of as calcium alginate beads, which may be produced by reacting a mixture of sodium alginate solution and enzyme solution with calcium chloride.

[0074] As used herein “cross-linkage” refers to the covalent bonding of enzymes to each other to create a matrix consisting of almost only enzyme. When a cross-linkage enzyme reaction is designed, the binding site ideally does not cover the enzyme's active site so that the activity of the enzyme is only affected by immobility and not by blockage of the enzyme’s active site. Nevertheless, spacer molecules like poly(ethylene glycol) may be used to reduce the steric hindrance by the substrate.

[0075] As used herein “covalent bonding” refers to the bonding of an enzyme to an insoluble support or surface (for example, a silica gel) via a covalent bond. Due to the strength of the covalent bonds between the enzymes and the support or surface, there is much less likelihood of enzymes detaching from the support or surface.

[0076] As used herein “crowding agent” refers to any polymer or protein that facilitates macromolecular crowding by concentrating enzyme on the cell surface to improve activity of the enzyme. A crowding agent may for example be a dextran, a dextran sulfate, a dextrin, a pullulans, a poly(ethylene glycol), a Ficoll™, a hyper-branched glycerol hydroxyethyl starch, polyvinyl alcohol, and an inert protein14.

[0077] As used herein “dextran” refers to a polysaccharide with molecular weights ≥1,000 Daltons and having a linear backbone of α-linked d-glucopyranosyl repeating units. Dextrans may divided into 3 structural classes (i.e. classes 1-3) based on the pyranose ring structure, which contains five carbon atoms and one oxygen atom. Class 1 dextrans contain the α(1→6)- linked d-glucopyranosyl backbone modified with small side chains of d-glucose branches with α(1→2), α(1→3), and α(1→4)-linkage. The class 1 dextrans vary in their molecular weight, spatial arrangement, type and degree of branching, and length of branch chains, 3-5 depending on the microbial producing strains and cultivation conditions. Isomaltose and isomaltotriose are oligosaccharides with the class 1 dextran backbone structure. Class 2 dextrans (alternans) contain a backbone structure of alternating α(1→3) and α(1→6)-linked d-glucopyranosyl units with α(1→3)-linked branches. Class 3 dextrans (mutans) have a 23  backbone structure of consecutive α(1→3)-linked d-glucopyranosyl units with α(1→6)-linked branches.

[0078] As used herein, “pullulans” are structural polysaccharides primarily produced from starch by the fungus Aureobasidium pullulans and are composed of repeating α(1→6)-linked maltotriose (D-glucopyranosyl-α(1→4)-D-glucopyranosyl-α(1→4)-D-glucose) units with the inclusion of occasional maltotetraose units.

[0079] As used herein, “dextrin” refers to D-glucopyranosyl units with a shorter chain lengths than dextran, which start with a single α(1→6) bond, but continue linearly with α(1→4)-linked D-glucopyranosyl units.

[0080] As used herein, “dextran sulfates” are derived from dextran via sulfation.

[0081] As used herein, “Ficoll™” is a neutral, highly branched, high-mass, hydrophilic polysaccharide, which dissolves readily in aqueous solutions.

[0082] Various alternative embodiments and examples are described herein. These embodiments and examples are illustrative and should not be construed as limiting the scope of the invention.

[0083] As used herein GH110 refers to a prokaryotic α-galactosidase gene family designated GH110 in the carbohydrate-active enzymes (CAZy) data base. GH110 enzymes are also referred to as alpha galactosidases or glycoside hydrolases or alpha 1,3-galactosidase and some members of this gene family are known to cleave galactose from the B-antigen (see FIGURE 3). Of the B-antigen cleaving enzymes, there are two subgroups A and B. The A subgroup only cleaves galactose from branched B-antigen and the B subgroup enzymes are able to cleave galactose from both linear and branched B-antigens.

[0084] As used herein, “perfusion” or “perfusing” refers to permeating an organ with a fluid by circulating the fluid through blood vessels.

[0085] As described herein “solutions” are formed from compositions by combining components with a fluid, from more concentrated solutions by dilution, or from more dilute solutions by concentration. For “organ perfusion solutions” and “organ preservation solutions”, suitable solutions often include an energy source, one or more stimulants to assist the organ in continuing its normal physiologic function prior to and during transplantation, and one or more amino acids (i.e. naturally occurring, non-naturally occurring or modified amino acids, essential amino acids, non-essential and / or semi-essential amino acids) selected and proportioned so that the organ continues its cellular metabolism during perfusion. 24  Cellular metabolism includes, for example requires protein synthesis. Solutions may be aqueous based, non-aqueous based, for example organic solvent-based, ionic-liquid-based, or fatty-acid-based. Solutions may include one or more energy-rich components to assist the organ in conducting its normal physiologic function. Such components may include energy rich materials that are metabolized and / or components that an organ can use to produce energy. Energy-rich molecules may be selected from, one or more carbohydrates (i.e. monosaccharides, disaccharides, oligosaccharides, polysaccharides, or combinations thereof, or precursors or metabolites thereof). Alternative energy sources may be adenosine triphosphate (ATP), co-enzyme A, pyruvate, flavin adenine dinucleotide (FAD), thiamine pyrophosphate chloride (co-carboxylase), β-nicotinamide adenine dinucleotide (NAD), β- nicotinamide adenine dinucleotide phosphate (NADPH), and phosphate derivatives of nucleosides (i.e. nucleotides, including mono-, di-, and tri-phosphates (e.g., UTP, GTP, GDF, and UDP), coenzymes, or other bio-molecules having similar cellular metabolic functions, and / or metabolites or precursors thereof). Carbohydrates may be provided with a phosphate source to produce ATP or other energy sources during perfusion.

[0086] Solutions may also include one or more organ stimulants for assisting the organ's normal physiologic function during perfusion. For example, when the organ is a heart, cardio stimulants may be used to allow the heart to continue functioning during perfusion and transplantation. Such stimulants may include, for example, catecholamines (e.g., as epinephrine and / or norepinephrine), peptides and / or polypeptides (e.g., vasopressin, Anthropleurin-A and Anthropleurin-B), β1 / β2-adrenoreceptor blocking agents (e.g., CGP 12177), buplinarol, pindolol, alprenolol, cardiac glycosides, digitalis (digoxin), palustrin, and / or ferulic acid.

[0087] Solutions may also include electrolytes, (e.g., calcium ions, sodium, potassium, chloride, sulfate, magnesium and other inorganic and organic charged species, or combinations thereof). It should be noted that any component provided hereunder may be provided, where valence and stability permit, in an ionic form, in a protonated or unprotonated form, in salt or free base form, or as ionic or covalent substituents in combination with other components that hydrolyze and make the component available in aqueous solutions, as suitable and appropriate.

[0088] Solutions may also include buffering components. For example, buffer systems may include: 2-morpholinoethanesulfonic acid monohydrate (MES); cacodylic acid; 25  H2CO3 / NaHCO3 (pKa1); citric acid (pKa3); bis(2-hydroxyethyl)-imino-tris-(hydroxymethyl)- methane (Bis-Tris); N-carbamoylmethylimidino acetic acid (ADA); 3- bis[tris(hydroxymethyl)methylamino]propane (Bis-Tris Propane) (pKa1); piperazine-1,4- bis(2-ethanesulfonic acid) (PIPES); N-(2-Acetamido)-2-aminoethanesulfonic acid (ACES); imidazole; N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES); 3-(N- morpholino)propanesulphonic acid (MOPS); NaH2PO4 / Na2HPO4 (pKa2); tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES); N-(2-hydroxyethyl)- piperazine-N′-2-ethanesulfonic acid (HEPES); N-(2-hydroxyethyl)piperazine-N′-(2- hydroxypropanesulfonic acid) (HEPPSO); triethanolamine; N- [tris(hydroxymethyl)methyl]glycine (Tricine); tris hydroxymethylaminoethane (Tris); glycineamide; N,N-bis(2-hydroxyethyl) glycine (Bicine); glycylglycine (pKa2); N- tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS); or a combination thereof. Solutions may also contain sodium bicarbonate, potassium phosphate, or TRIS buffer.

[0089] Solutions may also include other components to help maintain the organ and protect it against ischemia, reperfusion injury and other ill effects during perfusion (for example, hormones, vitamins and / or steroids).

[0090] As used herein “organ perfusion solution” and “organ preservation solution” includes any solution that is suitable for perfusing or preserving an organ, particularly a human organ. Examples of such solutions include: Steen™; Perfadex™; Perfadex Plus™; EuroCollins; Histidine-Tryptophan-Ketoglutarate (HTK) solution; University of Wisconsin Solution (UW); Celsior solution; Kidney Perfusion Solution (KPS-1); Kyoto University Solution; IGL-1 Solution; and Citrate solution17. Many of these are commercially available and variations to these solutions would be apparent to persons of skill in the art.

[0091] An important goal in organ preservation is to increase the number of available transplantable organs. Typically, organs were kept in cold storage, but this has potential diffusional limitations, and thus cold perfusion systems have been developed. Furthermore, near-normothermic systems are also being used to enhance the functional preservation of solid organs including livers, lungs, hearts and kidneys. A number of solutions are used as perfusion solutions or preservation solutions. A number of solutions are known in the art for the preservation of organs and tissues that will range in complexity of components, pH, temperature, pressure, flow rate, oxygenation, nutrients, concentrations, etc. depending on the conditions under which the solutions are being used. 26

[0092] Chemicals and commercial enzymes used in this study were purchased from Sigma- Aldrich™ unless otherwise stated.

[0093] Genomic exploration

[0094] Our objective was to identify GH110 enzymes that possess a carbohydrate-binding module (CBM) and are sourced from organisms found in the human gut microbiome that are capable of efficient conversion of the B-antigen to the H-antigen. To achieve this, a sequence similarity network (SSN) was generated using the Enzyme Function Initiative's Enzyme Similarity Tool (EFI-EST) and Genome Neighborhood Tool (EFI-GNT)6,7, with a sequence identity threshold of approximately 40% (EVALUE = 120) from 665 unique members of the CAZy GH110 family available in the CAZy database (CAZyDB)8 as of November 2023 (FIGURE 1). As part of this process, sequences were annotated with Pfam and InterPro domain IDs, which contain information about protein families and domains that can help identify and categorize sequences based on their functional characteristics. The Pfam and InterPro domain IDs were used to create an SSN filter that identifies any GH110 with a predicted CBM (FIGURE 1: lightest nodes). This filtering yielded 19 unique GH110 members. Of these, four were particularly interesting because they originated from a well- known member of the human gut microbiome, Akkermansia muciniphila (TABLE 1). These four, each representing separate A. muciniphila strains, all shared greater than 97% sequence identity with one another. Therefore, the GH110 from A. muciniphila strain ATCC BAA-835 (Uniprot ID: B2UNU8) was selected as the representative of this group. This enzyme, hereafter referred to as Am110B, contained a CBM at its N-terminus, which had not been previously functionally characterized in the GH110 family. Additionally, Bifidobacterium bifidum is also represented in this list; however, a GH110 from this organism has already been characterized9.

[0095] TABLE 1: GH110s annotated with a CBM Pfam and InterPro domain ID (yellow nodes from FIGURE 1). UniProt ID NCBI ID Organism Pfam InterPro A0A1H6LP70 SEH88108.1 Akkermansia glycaniphila. PF08305 IPR013222 PF08305 IPR013222 A0A1H6MHY7 SEI01258.1 Akkermansia glycaniphila. PF13229 IPR039448 27  Akkermansia muciniphila (strain ATCC BAA-835 / DSM B2UNU8 ACD04318.1 22959 / JCM 33894 / PF08305 IPR013222 BCRC81048 / CCUG 64013 / CIP 107961 / Muc) A0A410S9U7 QAT91028.1 Akkermansia muciniphila. PF08305 IPR013222 A0A857Z5S6 QHV67426.1 Akkermansia muciniphila. PF08305 IPR013222 A0A3R5U9L1 QAA55193.1 Akkermansia muciniphila. PF08305 IPR013222 PF02368 IPR003343 Bifidobacterium bifidum (strain PF08305 IPR013222 A0A0H3EAY7 ADP36352.1 PRL2010) PF12733 IPR025883 PF13229 IPR039448 PF02368 IPR003343 PF08305 IPR013222 I3WJ09 AFL04872.1 Bifidobacterium bifidum BGN4. PF12733 IPR025883 PF13229 IPR039448 PF02368 IPR003343 A0A286TAC1 BBA47302.1 Bifidobacterium bifidum LMG PF08305 IPR013222 13195. PF12733 IPR025883 PF13229 IPR039448 PF02368 IPR003343 PF08305 IPR013222 L8B3G2 BAM76380.1 Bifidobacterium bifidum. PF12733 IPR025883 PF13229 IPR039448 PF02368 IPR003343 PF08305 IPR013222 A0A0M5L0L5 ALE11773.1 Bifidobacterium bifidum. PF12733 IPR025883 PF13229 IPR039448 PF08305 IPR013222 A0A292FV73 BBA55548.1 Bifidobacterium bifidum. PF12733 IPR025883 PF13229 IPR039448 PF02368 IPR003343 Bifidobacterium sp. TKU. PF08305 IPR013222 A0A9E7YH47 UZF00666.1 Bifidobacterium sp. SKU. PF12733 IPR025883 PF13229 IPR039448 A0A975J0H1 QUE51756.1 Luteolibacter ambystomatis. PF08305 IPR013222 A0A858RIC2 QJE96647.1 Luteolibacter luteus. PF08305 IPR013222A0A1S6RI54 AQW50555.1 Streptomyces hygroscopicus. PF08305 IPR013222 PF08305 IPR013222 A0A222SV72 ASQ94414.1 Streptomyces sp.11-1-2.

[0097] Am110B is predicted to be a 737 amino acid enzyme with a MW of 81.5 kDa, after removal of the predicted 57 amino acid N-terminal signal peptide (Am110B_mat). A codon-optimized DNA sequence of Am110B_mat was generated in silico and inserted into a pET29 vector (pET29- Am110B.his). The expressed product was designed to possess a C-terminal hexa-histidine tag. The plasmid was ordered through Twist Biosciences™. The plasmid was transformed into E. coli BL21(DE3). In addition, to Am110B, three other genes from TABLE 1 were ordered from Twist Biosciences™. Predicted signal peptides were removed from the coding region and all were engineered with C-terminal histidine tags. a. Am110B: B2UNU8 (pET29-Am110B.his) b. La110B: A0A975J0H1 (pET29-La110B.his) c. Ag110A: A0A1H6MHY7 (pET29-Ag110A.his) d. Ag110B: A0A1H6LP70 (pET29-Ag110B.his)

[0098] See appendix section below for full DNA and amino acid sequence information.

[0099] Enzyme production

[0100] Am110B was expressed in E. coli BL21(DE3) harbouring the pET29-Am110B.his expression plasmid then purified using a 5 mL Ni-NTA chromatography column. A 2 L culture yielded approximately 40 mg of purified Am110B (FIGURE 2). Elution fractions 1 and 2 were combined, the buffer exchanged into 1x PBS + 10 % glycerol prior to storage at – 70 °C. Ag110A, Ag110B and La110B were expressed and purified in a similar manner.

[0101] Activity testing

[0102] Free substrates method

[0103] Enzymatic activity of Am110B, Ag110A, Ag110B, and La110B was confirmed using a thin-layer chromatography (TLC) method with two fluorescently labeled substrates: the branched type-VI B- 29  antigen and the unbranched linear form (FIGURE 3A). The linear substrate is chemically identical to the branched form, except for the absence of the α-2 linked fucose on the core galactose residue. AgaBb, a previously characterized GH110 enzyme, served as a control. The enzymes were tested with both the linear and branched substrates in separate reactions, using relative concentrations optimized to reveal their specificity towards either or both substrates. If an enzyme is active on one or both substrates, it will hydrolyze the terminal galactose, leading to a reduction in the overall polarity of the resultant product, which still possesses the fluorescent group. This change in polarity will affect the product's migration distance, which can be monitored by analyzing the relative migration distances of the fluorescent signals on TLC (FIGURE 3B). GH110 enzymes are known to have two subclasses: subclass A, which is active only on the branched B-antigen substrate, and subclass B, which is active on both the branched and linear substrates10. The TLC results indicated that all enzymes were active on at least one substrate. Furthermore, the results indicate AgaBb and Ag110A belong to subclass A, while Am110B, Ag110B, and La110B belong to subclass B. These findings are consistent with the phylogenetic analysis of their amino acid sequences, which groups them in the same manner.

[0104] B-antigen conversion on red blood cells

[0105] Am110B, La110B, Ag110A, Ag110B, AgaBb, and BfGH110B (a GH110 enzyme from Bacteroides fragilis lacking a carbohydrate-binding module10) were tested on red blood cells (RBCs). B-type RBCs were incubated with these enzymes in platelet-poor plasma (PPP). After treatment, the remaining B-antigens on the RBC surface were quantified using anti-B antibodies coupled with a fluorescent secondary antibody, and antibody binding was monitored using flow cytometry. MTS™ Anti-IgG gel card testing was also performed, serving as a supplementary method. The MTS system uses antibodies within a gel matrix that bind to B-antigens on the RBCs during centrifugation, enabling visual detection of antigen presence. The degree of agglutination is scored from 0 to 4, with 0 indicating no agglutination—this suggests the effective enzymatic removal of B-antigens, a result indicative of the desired enzyme activity.

[0106] Initially, the activity of La110B on B-antigen-bearing RBCs was compared with that of AgaBb. Following this comparison, the activities of Am110B, Ag110B, and Ag110A were evaluated against those of AgaBb and BfGH110B. Among the tested enzymes, Am110B demonstrated optimal activity on RBCs, and its activity was further characterized in detail.

[0107] La110B activity on RBCs

[0108] First, the activities of La110B and AgaBb were directly compared. The enzymes were used to treat B-type RBCs at concentrations of 20 or 50 µg / mL, with a hematocrit (HCT) level set at 10%. The incubation conditions were 1 hour at 37°C and 18 hours at 4°C. MTS analysis was then conducted to 30  evaluate the enzymes' efficacy in modifying B-antigens. The summarized results in TABLE 2 showed that both AgaBb and La110B performed equally at the 37°C, 1-hour condition. However, under the prolonged 4°C, 18-hour condition, AgaBb outperformed La110B, achieving a score of 0 in the MTS analysis, which signifies no agglutination and indicates superior enzyme performance at lower temperatures over an extended duration.

[0109] TABLE 2: La110B and AgaBb anti-B MTS results. [enzyme] Condition Sample Anti-B MTS score (µg / mL) B-RBC - 0 4 Control 20 3 AgaBb20 0 AgaBb 4 °C 50 0 18 h 20 3 / 2 La110B 50 1 / 0

[0110] For the flow cytometry analysis, the quantification of residual B-antigen on RBCs involved an initial treatment with Novaclone™ anti-B primary antibody at a 1 / 100 dilution for 30 minutes. This was followed by incubation with an anti-mouse IgM-FITC secondary antibody at a 1 / 600 dilution for another 30 minutes, facilitating fluorescence detection. Flow cytometry histograms for each treatment condition were generated (not shown). Subsequently, the median fluorescence intensity (FITC-A) for each treatment was plotted on a bar chart for a clearer comparison of the conditions (FIGURE 4). The bar chart results generally correlated with the MTS scores, indicating that AgaBb and La110B exhibited similar activities at 37°C for 1 hour. However, under the conditions of 4°C for 18 hours, AgaBb performed better.

[0111] Am110B, Ag110A and Ag110B activity on RBCs

[0112] The next step involved analyzing a broader set of GH110 enzymes, including Am110B, Ag110A, Ag110B, AgaBb, and Gf110B, all at a concentration of 50 µg / mL. These enzymes were used to treat B-type RBCs with an HCT level of 10%. The incubation conditions were set at 1 h at 37°C. MTS analysis was then conducted to evaluate the enzymes' efficacy in modifying B-antigens. According to the MTS results (TABLE 3 and FIGURE 4), Ag110A, Ag110B, and Am110B all outperformed AgaBb 31  and Bf110B, with Am110B being the only candidate scoring a 0, indicating no presence of agglutination.

[0113] TABLE 3: Am110B, Ag110A and Ag110B Anti-B MTS results [enzyme] Sample (µg / mL) Anti-B MTS score B-RBC 0 4 Control AgaBb 503 Bf110B 50 3 Ag110A 50 2 Ag110B 50 1 Am110B 50 0

[0114] The flow analysis was conducted using the same protocol described earlier for La110B. The results of the flow analysis correlated with the MTS results, demonstrating that Am110B performed the best, as indicated in FIGURE 5. This includes outperforming the current B to H-antigen converting enzyme standards, AgaBb and Bf110B. While Ag110A and Ag110B also showed promising potential in converting the B-antigen to the H-antigen, we decided to proceed with further characterizing the activity of Am110B on RBCs.

[0115] Characterization of Am110B activity on B-RBCs: concentration dependency

[0116] A concentration dependency trial was performed to understand Am110B’s efficacy over a wide concentration range. B RBCs (10 % HCT) were treated with either 0, 5, 10, 20, 30, 40, 50, 75 or 100 µg / mL Am110B for 2 h at 37 °C. According to the MTS results, no agglutination was observed at concentration as low as 20 µg / mL (TABLE 4).

[0117] TABLE 4: Am110B concentration dependency anti-B MTS results [enzyme] Sample (µg / mL) Anti-B MTS score B-RBC Control 0 4 53 Am110B 10 1 20 0 32  30 0 40 0 50 0 75 0 100 0

[0118] The flow cytometry data for B-RBCs treated with Am110B (FIGURE 6) showed a consistent activity profile. The median fluorescence intensity remained stable at around 82 units until the enzyme concentration decreased below 40 µg / mL, at which point a slight increase in fluorescence intensity was observed. At an enzyme concentration of 5 µg / mL, the fluorescence intensity reached 408 units, which is still two orders of magnitude lower than the untreated B-RBC control (0 µg / mL) with a fluorescence intensity of 11,325 units. This demonstrates the efficiency of Am110B in converting B-RBCs to O-RBCs even at low concentrations.

[0119] Characterization of Am110B activity on B-RBCs: temperature and solution dependency

[0120] Next Am110B activity was on B-RBCs was testing in 4 conditions: 4 °C, 18 h 4 °C, 2 h 22 °C, 2 h 37 °C, 2 h

[0121] In each of those conditions two Am110B concentrations were tested: 25 µg / mL 50 µg / mL

[0122] Three solutions were tested for each of these conditions and concentrations: Platelet poor plasma (PPP) Phosphate buffered saline (PBS) Commercially available University of Wisconsin perfusion solution (UW)

[0123] In total, 24 different combinations were analyzed using anti-B MTS cards and flow cytometry. The MTS analysis (TABLE 5) revealed that Am110B exhibited no agglutination (scored as 0) under all conditions, except for the 2-hour incubation at 4°C in PPP, where 33  scores of 3 and 1 were observed for concentrations of 25 µg / mL and 50 µg / mL, respectively. Notably, no agglutination occurred in PPP when the incubation time was extended to 18 hours at 4°C or when the temperature was increased to either 22°C or 37°C. This demonstrates the enzyme's versatility, as it efficiently converts B-RBCs to O-RBCs under a variety of conditions.

[0124] TABLE 5: Am110B temperature and solution dependency anti-B MTS results. PBS: phosphate buffered saline, PPP: platelet poor plasma, UW: University of Wisconsin profusion solution. Anti-B Anti-B Temp. [Am110B] MTS MTS Anti-B MTS score Time (h) (°C) (µg / mL) score score (UW)(PBS) (PPP) 25 0 0 0 4 18 50 0 0 0 25 0 3 0 4 2 50 0 1 0 25 0 0 0 22 2 50 0 0 0 25 0 0 0 37 2 50 0 0 0

[0125] The flow cytometry results were consistent with the MTS findings (FIGURE 7), further reinforcing the versatility of Am110B. This enzyme efficiently converts B-RBCs to O- RBCs under various conditions, as demonstrated by both flow cytometry and MTS analyses. The collective results highlight Am110B's potential as a highly effective enzyme for blood type conversion, offering promise for its application in transfusion medicine and organ transplantation.

[0126] AB to O conversion

[0127] The subsequent experiment involved testing Am110B alongside an enzyme set that converts the A antigen to the H antigen on AB red blood cells (RBCs). The aim was to transform type-AB blood into the universal type-O blood. This strategy could potentially 34  result in a single enzyme "cocktail" capable of converting blood or organs of any ABO type into universal donor material, regardless of the original blood type.

[0128] In this experiment, AB RBCs, which display both A and B antigens on their surfaces, were treated with Am110B combined with a dual-enzyme system known for its efficiency in converting the A antigen to the H antigen. The efficacy of this combinatorial approach was assessed under various conditions, demonstrating its effectiveness over a range of temperatures and in different solutions. Importantly, it was shown to efficiently convert AB RBCs to type-O in whole blood. This is a crucial advantage, as it means the conversion process can be performed without the need for separating the blood into its components, a process that requires specialized equipment. Consequently, this technology could be particularly useful in settings such as military field hospitals, where access to such equipment might be limited.

[0129] Additionally, the combination approach was tested on human kidney tissue of types A and B, while AB kidney tissue was not available for testing, the results showed that the enzyme cocktail could successfully convert both A and B kidney tissue to type-O when used in combination.

[0130] Characterization of Am110B activity on AB-RBCs: temperature and solution dependency

[0131] In these experiments, a solution containing 10% AB+ red blood cells (RBCs) were incubated with a fixed concentration of 25 µg / mL of enzymes in different solutions, at specified temperatures and durations. The enzyme combinations tested were as follows: 1) EnzAO1 and EnzAO2, 2) Am110B, 3) EnzAO1, EnzAO2, and Am110B together, and 4) EnzAO1 and EnzAO2 first, followed by Am110B. The incubation solutions were phosphate-buffered saline (PBS), AB frozen plasma (PPP), and the University of Wisconsin (UW) solution. The incubation conditions were set at 22°C for 2 hours and at 4°C for 18 hours (FIGURE 8).

[0132] For the flow cytometric analysis of the A antigen, the BD Pharmingen™ Alexa Fluor 647™ Mouse Anti-Human Blood Group A antibody was used at a dilution of 1 / 100 at room temperature (RT) for 30 minutes. For the B antigen analysis, the Novaclone™ anti-B antibody was used at the same dilution and conditions. A secondary antibody, Alexa Fluor 488™ goat anti-mouse IgM, was applied at a dilution of 1 / 300 at RT for 30 minutes for both analyses. Additionally, Immuncor™ Anti-A, B (Murine Monoclonal Blend) Series 1 was used 35  at a dilution of 1 / 200 at RT for 30 minutes, followed by the same secondary antibody under the same conditions. Anti-A and anti-B MTS analysis was also performed.

[0133] MTS testing showed that in all conditions where a combination of all three enzymes were used, the MTS score was 0, indicating no agglutination. When AB RBCs were treated with Am110B alone, the anti-B MTS scored a 0, indicating that Am110B is able to efficiently convert the B to H-antigen on AB-RBCs in addition to B-RBCs.

[0134] TABLE 6: Temperature and solution dependency MTS results. Anti-A and anti-B results are shown for each solution (PBS: phosphate buffered saline, PPP: platelet poor plasma, UW: University of Wisconsin profusion solution). A → O = 25 µg / mL each of EnzAO1 and EnzAO2. B → O = 25 µg / mL of Am110B. Superscript numbering indicates sequence of addition. Enzymes PBS PPP UWAm110B, EnzAO1 and EnzAO2 combinatorial approach is an efficient system for converting AB RBCs to the universal O-type.

[0136] AB to O in whole blood

[0137] In this experiment, whole blood samples were treated with various combinations and concentrations of enzymes. Specifically, the samples included: 1. A mixture of AO1, AO2, and Am110B each at 25 µg / mL in whole blood 36  2. A mixture of AO1, AO2, and Am110B each at 50 µg / mL in whole blood 3. Am110B alone at 25 µg / mL in whole blood 4. Am110B alone at 50 µg / mL in whole blood 5. A combination of AO1 and AO2 at 25 µg / mL in whole blood

[0138] These samples were incubated under two different conditions: at 4°C overnight and at 22°C for 2 hours. For the antigen analysis in whole blood, the procedures outlined above were followed, using the same antibodies and conditions for the detection of A and B antigens.

[0139] Am110B was shown to efficiently convert the B-antigen to the H-antigen on AB-RBCs, as indicated by its anti-B MTS score of 0 at both 25 and 50 µg / mL concentrations. Furthermore, when used in combination with EnzAO1 and EnzAO2, this enzyme cocktail can convert both the A and B-antigens to H, as indicated by a score of 0 for both the anti-A and anti-B MTS tests.

[0140] TABLE 7: Whole blood MTS results with AB RBCs. Anti-A and anti-B results are shown for each condition (22 °C for 2 h or 4 °C for 18 h). A → O = treatment with the indicated concentration of each EnzAO1 and EnzAO2. B → O = treatment with the indicated concentration of Am110B. Sample # column is for MTS card reference (not shown). Enzymes 22 °C, 2 h 4 °C, 18 h

[0141] The flow analysis of AB-RBCs in whole blood aligns with the MTS data (FIGURE 9), demonstrating efficient conversion of the B-antigen in all cases tested at 4°C for 18 hours. However, a slight discrepancy was noted in the detection of the anti-B antigen between the use of Am110B at concentrations of 25 and 50 µg / mL during the 22°C for 2-hour treatment. This difference was also observed regardless of the presence or absence of EnzAO1 37  and EnzAO2. This may suggest that Am110B is less efficient in whole blood compared to its performance in PBS and UW solutions. This finding is consistent with previous observations in FIGURE 7, where the median FITC signal for B-RBCs in PPP (4°C for 2 hours) was slightly higher compared to all other conditions tested. Further investigation is required to understand the underlying reasons for this result, which could be related to the pH of plasma or another factor. Nevertheless, the combination of Am110B with A-type converting enzymes successfully achieved efficient conversion of AB to O-type blood.

[0142] AB to O on human kidney tissue

[0143] Our research into enzymatic treatments for blood type conversion led us to examine the efficacy of Am110B, alone and in combination with EnzAO1 and EnzAO2, in modifying surface A and B antigens on human kidney tissue. Given the importance of blood type compatibility in transplant medicine, kidney tissues were chosen for their relevance to the field. The use of enzymes EnzAO1, EnzAO2, and Am110B aims to modify these antigens, ultimately converting A and B antigens to H. This approach holds promise for overcoming blood type barriers in organ transplantation. To assess the enzymes' effectiveness, we employed immunofluorescence histology assays to detect the presence or absence of A and B antigens before and after enzyme treatment. This method provides a clear visualization and quantification of any changes in antigen expression on the tissue surface.

[0144] Human kidney tissues of types A and B were prepared for immunofluorescence histology assays by Wax It Inc. ™ (Vancouver, BC). The tissues were cryo-embedded and sectioned into slices of 10 µm thickness. To evaluate the expression of antigens A and B before and after enzyme treatment, the tissue samples were treated with the following solutions for 4 hours at 4°C: UW profusion solution as a control 50 µg / mL each of AO1 and AO2 50 µg / mL of Am110B 50 µg / mL each of AO1, AO2, and Am110B

[0145] After enzyme treatment, the tissue slices were washed twice with PBS (pH 7.4) for 5 minutes each and fixed with pre-cooled acetone (-20°C) for 10 minutes at room temperature (RT). To block non-specific binding, the slices were incubated for 1 hour with 10% goat serum (Sigma-Aldrich™) in PBS (pH 7.4). 38

[0146] For antigen detection, the type A tissue slices were incubated with a primary anti-A antibody (MA1-19693, Invitrogen™) at a 1:100 dilution, and the type B tissue slices were incubated with a primary anti-B antibody (Novaclone Anti-B, Immucor™) at a 1:100 dilution, both overnight at 4°C. After this, the tissue slices were washed three times with 0.1% PBST (0.1% Tween 20 in PBS, pH 7.4) for 5 minutes each and then incubated with a secondary antibody labeled with Alexa Fluor 546™ (A-21045, Invitrogen™) for 1 hour at RT for the detection of antigens A or B.

[0147] All tissue slices were counterstained with 5 µg / mL of Hoechst 33342™ (H3570, Invitrogen™) for 10 minutes at RT to visualize cell nuclei and mounted with Fluoromount- G™ (Santa Cruz Biotech Inc. ™). The expression of antigens A and B before and after enzyme treatment was analyzed using ImageJ software™ (NIH, Bethesda) by measuring the integrated densities (in arbitrary units (a.u)) of five to seven fluorescent images taken under the Alexa Fluor 546™ filter using a laser-scanning confocal microscope (Eclipse Ti, Nikon™). Fluorescent images of randomly selected fields were captured under identical parameter settings.

[0148] Treatment of B-type human kidney tissue with Am110B resulted in a statistically significant reduction (p < 0.0001) in anti-B antibody binding (FIGURE 10) compared to the untreated control. Furthermore, similar results were observed when Am110B was used in combination with EnzAO1 and EnzAO2, indicating that the presence of these enzymes does not negatively impact Am110B's ability to modify the B-antigen on kidney tissue. In contrast, treatment of A-type human kidney tissue with Am110B alone showed no statistical difference in anti-B antibody binding (FIGURE 11) compared to the untreated control, as expected, because Am110B does not target the A-antigen. However, when combined with EnzAO1 and EnzAO2, which together efficiently convert the A-antigen to the H-antigen, a statistically significant reduction (p < 0.001) in anti-A antibody binding was observed. These results demonstrate that Am110B can be effectively combined with EnzAO1 and EnzAO2 to convert both A and B antigens on human kidney tissue to the H-antigen without either hindering the effect of the other enzyme(s). This combination enzyme treatment has the potential to convert type-A, B, and AB donor kidneys to the universal donor type-O, while broadening its application beyond kidneys to other organs like hearts and lungs is a possibility. EnzAO1 and EnzAO2 have already been shown to efficiently convert A-type organs to O-type. 39

[0149] Impact of a molecular crowding agent on Am110B activity

[0150] Many organ preservation solutions contain molecular crowding agents, like dextran, which play a crucial role in controlling osmolarity, preventing cell edema, and ensuring the stability of preserved organs. In the context of red blood cell (RBC) antigen remodeling, the inclusion of dextran has been shown to enhance the efficiency of EnzAO1 and EnzAO23, allowing the use of lower enzyme concentrations to achieve the same level of conversion as compared to formulations without crowding agents. Given these findings, we aimed to investigate whether dextran similarly influences the activity of Am110B in converting the B-antigen to the H-antigen.

[0151] To evaluate the potential impact of dextran on Am110B's activity, we incubated A or AB RBCs with a low concentration of 5 µg / mL Am110B, both in the absence and presence of 300 mg / mL dextran-40. This concentration of Am110B was selected based on previous findings that indicated it was not sufficient to achieve maximum conversion (FIGURE 6), allowing us to clearly observe any enhancement in the enzyme's activity due to the addition of dextran.

[0152] Flow cytometry analysis was conducted to assess the efficacy of Am110B in antigen remodeling on both B+ and AB+ red blood cells (RBCs). Cells were adjusted to a 10% hematocrit (HCT) and treated with 5 µg / mL of Am110B in PBS. The treatment included conditions both with and without the addition of 40 kDa dextran at a concentration of 300 mg / mL and was carried out for 2 hours at RT.

[0153] Anti-B MTS analysis revealed differing levels of agglutination in the treated RBCs (2 hours at RT). In the absence of the molecular crowding agent, Am110B-treated B RBCs scored a 1, suggesting a low level of agglutination. However, with the inclusion of the crowding agent, these cells achieved an MTS score of 0, indicating the absence of agglutination and suggesting that the crowding agent significantly enhances Am110B's efficiency. On the other hand, AB RBCs, which typically possess fewer B-antigens (as indicated by the untreated controls), showed maximal conversion with 5 µg / mL Am110B alone, negating the impact of the crowding agent at this concentration.

[0154] Post-treatment, anti-B antigen detection was performed by incubating the RBCs with the Novaclone™ anti-B antibody at a 1 / 100 dilution for 30 minutes at room temperature. This was followed by a wash and incubation with Alexa Fluor 488™-conjugated goat anti- mouse IgM secondary antibody at a 1 / 300 dilution for another 30 minutes, enabling fluorescence detection. 40

[0155] The flow cytometry data correlated with the MTS results, showing improved antigen conversion in the presence of the crowding agent for both B and AB RBCs. Notably, treated AB RBCs exhibited a marginal reduction in the median FITC signal when dextran was included, underscoring the crowding agent's positive impact. These findings confirm Am110B's activity in the presence of dextran and suggest that dextran enhances its activity for antigen remodeling on the RBC surface.

[0156] TABLE 8: Am110B molecular crowding agent dependency anti-B MTS results. Agglutination reactions are scored from 0 to 4, with 0 indicating no agglutination and 4 representing a solid band of agglutinates at the top of the gel. Scores of 1 to 3 reflect increasing degrees of agglutination dispersed within the gel. Am110B  Dextran  Anti‐B  RBC type  (5 µg / mL)  (300 mg / mL)  MTS ‐  4 B +  ‐  1 +  +  0 ‐  ‐  4 AB +  ‐  0 +  +  0

[0157] Summary, significance and conclusion

[0158] This study aimed to identify GH110 enzymes from the human gut microbiome, particularly those with a carbohydrate-binding module, capable of efficiently converting the B-antigen to the H-antigen. Through a sequence similarity network, we identified Am110B from Akkermansia muciniphila as a promising candidate. Am110B demonstrated high efficiency, showing maximum efficacy at concentrations as low as 20 µg / mL. Its versatility was evident in its effectiveness across various media, including blood plasma, phosphate- buffered saline, University of Wisconsin profusion solution, and whole blood. Furthermore, Am110B proved effective at a wide range of temperatures, from 4°C to 37°C, on red blood cells. Importantly, it can be used in combination with EnzAO1 and EnzAO2 to convert AB blood to the universal O-type, showcasing its potential in transfusion medicine. Additionally, its effectiveness in converting the B-antigen to the H-antigen on human kidney tissue indicates its applicability in organ transplantation. 41

[0159] The implications of these findings are significant for addressing the scarcity of universal donor blood and organs. By efficiently converting A, B, and AB-type blood and organs to the universal O-type, we can potentially reduce wait times for organ transplants and improve the availability of universal donor blood. This ability to convert blood types efficiently could revolutionize transfusion medicine and organ transplantation, ultimately saving lives and improving patient outcomes.

[0160] The versatility and effectiveness of Am110B in various conditions, along with its ability to work at low concentrations, in combination with other A-antigen modifying enzymes, and on human tissue, highlight the potential of genomic exploration in discovering novel enzymes for medical applications. The successful combination of Am110B with A-type converting enzymes underscores the potential for a single enzyme cocktail that can convert blood or organs of any ABO type into universal donor material. In conclusion, this research paves the way for practical applications in transfusion medicine and organ transplantation, offering a promising solution to the challenges of blood type compatibility and the availability of universal donor organs.

[0161] Although various embodiments of the invention are disclosed herein, many adaptations and modifications may be made within the scope of the invention in accordance with the common general knowledge of those skilled in this art. Such modifications include the substitution of known equivalents for any aspect of the invention in order to achieve the same result in substantially the same way. Numeric ranges are inclusive of the numbers defining the range. The word “comprising” is used herein as an open-ended term, substantially equivalent to the phrase “including, but not limited to”, and the word “comprises” has a corresponding meaning. As used herein, the singular forms “a”, “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a thing” includes more than one such thing. Citation of references herein is not an admission that such references are prior art to an embodiment of the present invention. The invention includes all embodiments and variations substantially as hereinbefore described and with reference to the examples and drawings. 42  REFERENCES (1) Bertsch, T.; Lüdecke, J.; Antl, W.; Nausch, L. W. M. Karl Landsteiner: The Discovery of the ABO Blood Group System and Its Value for Teaching Medical Students. Clin. Lab.2019, 65 (6). https: / / doi.org / 10.7754 / Clin.Lab.2018.181218. (2) Daniels, G.; Reid, M. E. Blood Groups: The Past 50 Years. Transfusion (Paris) 2010, 50 (2), 281–289. https: / / doi.org / 10.1111 / j.1537-2995.2009.02456.x. (3) Rahfeld, P.; Sim, L.; Moon, H.; Constantinescu, I.; Morgan-Lang, C.; Hallam, S. J.; Kizhakkedathu, J. N.; Withers, S. G. An Enzymatic Pathway in the Human Gut Microbiome That Converts A to Universal O Type Blood. Nat. Microbiol.2019, 4 (9), 1475–1485. https: / / doi.org / 10.1038 / s41564-019-0469-7. (4) Goldstein, J.; Siviglia, G.; Hurst, R.; Lenny, L.; Reich, L. Group B Erythrocytes Enzymatically Converted to Group O Survive Normally in A, B, and O Individuals. Science 1982, 215 (4529), 168–170. https: / / doi.org / 10.1126 / science.6274021. (5) Liu, Q. P.; Sulzenbacher, G.; Yuan, H.; Bennett, E. P.; Pietz, G.; Saunders, K.; Spence, J.; Nudelman, E.; Levery, S. B.; White, T.; Neveu, J. M.; Lane, W. S.; Bourne, Y.; Olsson, M. L.; Henrissat, B.; Clausen, H. Bacterial Glycosidases for the Production of Universal Red Blood Cells. Nat. Biotechnol.2007, 25 (4), 454–464. https: / / doi.org / 10.1038 / nbt1298. (6) Oberg, N.; Zallot, R.; Gerlt, J. A. EFI-EST, EFI-GNT, and EFI-CGFP: Enzyme Function Initiative (EFI) Web Resource for Genomic Enzymology Tools. J. Mol. Biol.2023, 435 (14), 168018. https: / / doi.org / 10.1016 / j.jmb.2023.168018. (7) Zallot, R.; Oberg, N.; Gerlt, J. A. The EFI Web Resource for Genomic Enzymology Tools: Leveraging Protein, Genome, and Metagenome Databases to Discover Novel Enzymes and Metabolic Pathways. Biochemistry 2019, 58 (41), 4169–4182. https: / / doi.org / 10.1021 / acs.biochem.9b00735. (8) Drula, E.; Garron, M.-L.; Dogan, S.; Lombard, V.; Henrissat, B.; Terrapon, N. The Carbohydrate-Active Enzyme Database: Functions and Literature. Nucleic Acids Res.2022, 50 (D1), D571–D577. https: / / doi.org / 10.1093 / nar / gkab1045. (9) Wakinaka, T.; Kiyohara, M.; Kurihara, S.; Hirata, A.; Chaiwangsri, T.; Ohnuma, T.; Fukamizo, T.; Katayama, T.; Ashida, H.; Yamamoto, K. Bifidobacterial α-Galactosidase with Unique Carbohydrate-Binding Module Specifically Acts on Blood Group B Antigen. Glycobiology 2013, 23 (2), 232–240. https: / / doi.org / 10.1093 / glycob / cws142. 43  (10) Liu, Q. P.; Yuan, H.; Bennett, E. P.; Levery, S. B.; Nudelman, E.; Spence, J.; Pietz, G.; Saunders, K.; White, T.; Olsson, M. L.; Henrissat, B.; Sulzenbacher, G.; Clausen, H. Identification of a GH110 Subfamily of Α1,3-Galactosidases: NOVEL ENZYMES FOR REMOVAL OF THE α3GAL XENOTRANSPLANTATION ANTIGEN*. J. Biol. Chem.2008, 283 (13), 8545–8554. https: / / doi.org / 10.1074 / jbc.M709020200. (11) Kashima, T. et al. Crystal structure of Bifidobacterium bifidum glycoside hydrolase family 110 α-galactosidase specific for blood group B antigen (2024) doi: https: / / doi.org / 10.1101 / 2024.03.03.583176). (12) Bakshani, C. R. et al. Carbohydrate-active enzymes from Akkermansia muciniphila breakdown mucin O-glycans to completion (2024) doi: https: / / doi.org / 10.1101 / 2024.03.27.586211. (13) PCT / US1992 / 010113; PCT / SE2015 / 050108; US4088538; US4141857; US4206259; US4218363; US4229536; US4239854; US4619897; US4748121; US4749653; US4897352; US4954444; US4978619; US5154808; US5914367; US5962279; US6030933; US6291582; US6254645; US10,016,490; and US10,041,055. (14) Kuznetsova, I.M et al. Int J Mol Sci. (2014) “What Macromolecular Crowding Can Do to a Protein” 15(12): 23090–23140. (15) McGuire, B.E., Hettle, A.G., Vickers, C., King, D.T., Vocadlo, D.J., Boraston, A.B. The structure of a family 110 glycoside hydrolase provides insight into the hydrolysis of alpha-1,3-galactosidic linkages in lambda-carrageenan and blood group antigens. J Biol Chem (2020) 295: 18426-18435 (P. Distincta https: / / www.rcsb.org / structure / 7JW4). (16) Weikum, J., Jensen, M., Abou Hachem, M., Morth, J.P. Targeting extended blood antigens by Akkermansia muciniphila enzymes unveils a missing link for generating universal donor blood https: / / www.rcsb.org / structure / 8PXV. (17) Guibert, E. E. et al. Transfus Med Hemother. (2011) “Organ Preservation: Current Concepts and New Strategies for the Next Decade” 38(2): 125–142. 44

Claims

What is Claimed is:

1. A purified alpha galactosidase selected from one or more of the following: SEQ ID NO.:2; SEQ ID NO.:3; SEQ ID NO.:4; SEQ ID NO.:5; SEQ ID NO.:6; SEQ ID NO.:7; SEQ ID NO.:8; SEQ ID NO.:9; SEQ ID NO.:10; SEQ ID NO.:11; and SEQ ID NO.:

12.

2. The purified alpha galactosidase of claim 1, wherein the purified alpha galactosidase is a purified Akkermansia muciniphila alpha galactosidase of SEQ ID NO.:2 or SEQ ID NO.:

3.

3. The purified alpha galactosidase of claim 1, wherein the purified alpha galactosidase is a purified Luteolibacter ambystomatis alpha galactosidase of SEQ ID NO.:4, SEQ ID NO.:5 or SEQ ID NO.:

6.

4. The purified alpha galactosidase of claim 1, wherein the purified alpha galactosidase is a purified Akkermansia glycaniphila alpha galactosidase of SEQ ID NO.:7, SEQ ID NO.:8 or SEQ ID NO.:

9.

5. The purified alpha galactosidase of claim 1, wherein the purified alpha galactosidase is a purified Akkermansia glycaniphila alpha galactosidase of SEQ ID NO.:10, SEQ ID NO.:11 or SEQ ID NO.:

12.

6. The purified alpha galactosidase enzyme of any one of claims 1-5, for use in enzymatically cleaving B-antigens from whole blood, erythrocytes, a donor tissue or a donor organ.

7. A purified alpha galactosidase enzyme of: SEQ ID NO.:1; SEQ ID NO.:2; SEQ ID NO.:4; SEQ ID NO.:5; SEQ ID NO.:7; SEQ ID NO.:8; SEQ ID NO.:10; or SEQ ID NO.:11 and a protein tag.

8. The purified alpha galactosidase enzyme of claim 7, wherein the protein tag is selected from one or more of: Albumin-binding protein (ABP); Alkaline Phosphatase (AP); AU1 epitope; AU5 epitope; AviTag; Bacteriophage T7 epitope (T7-tag); Bacteriophage V5 epitope (V5-tag); Biotin-carboxy carrier protein (BCCP); Bluetongue virus tag (B-tag); single-domain camelid antibody (C-tag); Calmodulin binding peptide (CBP or Calmodulin-tag); Chloramphenicol Acetyl Transferase (CAT); Cellulose binding domain (CBP); Chitin binding domain (CBD); Choline-binding domain (CBD); Dihydrofolate reductase (DHFR); DogTag; E2 epitope; E-tag; FLAG epitope (FLAG-tag); Galactose-binding protein (GBP); Green fluorescent protein (GFP); Glu-Glu (EE-tag); Glutathione S-transferase (GST); heparin-binding affinity tag (HB-tag); Human influenza hemagglutinin (HA); HaloTag™; Alternating histidine and glutamine tags (HQ tag); Alternating histidine and asparagine tags (HN tag); Histidine affinity tag (HAT); Histidine tag; Horseradish Peroxidase (HRP); HSV epitope; Isopeptag (Isopep-tag); Ketosteroid isomerase (KSI); KT3 epitope; LacZ; Luciferase; Maltose- binding protein (MBP); Myc epitope (Myc-tag); NE-tag; NusA; PDZ domain; PDZ ligand; Polyarginine (Arg-tag); Polyaspartate (Asp-tag); Polycysteine (Cys-tag); Polyglutamate (Glu- tag); Polyhistidine (His-tag); Polyphenylalanine (Phe-tag); Profinity eXact; Protein C; Rho1D4-tag; S1-tag; S-tag; Softag 1; Softag 3; SnoopTagJr; SnoopTag; Spot-tag; SpyTag (Spy- tag); Streptavadin-binding peptide (SBP); Staphylococcal protein A (Protein A); Staphylococcal protein G (Protein G); Strep-tag; Streptavadin (SBP-tag); Strep-tag II; Sdy- tag; Small Ubiquitin-like Modifier (SUMO); Tandem Affinity Purification (TAP); T7 epitope; tetracysteine tag (TC tag); Thioredoxin (Trx); TrpE; Ty tag; Ubiquitin; Universal; V5 tag; VSV- G or VSV-tag; and Xpress tag.

9. An isolated nucleic acid sequence encoding alpha galactosidase selected from one or more of: SEQ ID NO.:13; SEQ ID NO.:14; SEQ ID NO.:15; and SEQ ID NO.:

16.

10. An isolated nucleic acid sequence encoding alpha galactosidase, wherein the nucleic acid sequence encodes an amino acid sequence selected from one or more of: SEQ ID NOs.:1- 12.

11. A vector comprising the nucleic acid of claim 9 or 10 and a heterologous nucleic acid sequence.

12. The vector of claim 11, wherein the heterologous nucleic acid sequence is selected from one or more of the following: a protein tag; and a cleavage site.

13. The vector of claim 12, wherein the protein tag is selected from one or more of: Albumin-binding protein (ABP); Alkaline Phosphatase (AP); AU1 epitope; AU5 epitope; AviTag; Bacteriophage T7 epitope (T7-tag); Bacteriophage V5 epitope (V5-tag); Biotin- carboxy carrier protein (BCCP); Bluetongue virus tag (B-tag); single-domain camelid antibody (C-tag); Calmodulin binding peptide (CBP or Calmodulin-tag); Chloramphenicol Acetyl Transferase (CAT); Cellulose binding domain (CBP); Chitin binding domain (CBD); Choline- binding domain (CBD); Dihydrofolate reductase (DHFR); DogTag; E2 epitope; E-tag; FLAG epitope (FLAG-tag); Galactose-binding protein (GBP); Green fluorescent protein (GFP); Glu- Glu (EE-tag); Glutathione S-transferase (GST); Human influenza hemagglutinin (HA);heparin-binding affinity tag (HB-tag); HaloTag™; Alternating histidine and glutamine tags (HQ tag); Alternating histidine and asparagine tags (HN tag); Histidine affinity tag (HAT); Histidine tag; Horseradish Peroxidase (HRP); HSV epitope; Isopeptag (Isopep-tag); Ketosteroid isomerase (KSI); KT3 epitope; LacZ; Luciferase; Maltose-binding protein (MBP); Myc epitope (Myc-tag); NE-tag; NusA; PDZ domain; PDZ ligand; Polyarginine (Arg-tag); Polyaspartate (Asp-tag); Polycysteine (Cys-tag); Polyglutamate (Glu-tag); Polyhistidine (His- tag); Polyphenylalanine (Phe-tag); Profinity eXact; Protein C; Rho1D4-tag; S1-tag; S-tag; Softag 1; Softag 3; SnoopTagJr; SnoopTag; Spot-tag; SpyTag (Spy-tag); Streptavadin-binding peptide (SBP); Staphylococcal protein A (Protein A); Staphylococcal protein G (Protein G); Strep-tag; Streptavadin (SBP-tag); Strep-tag II; Sdy-tag; Small Ubiquitin-like Modifier (SUMO); Tandem Affinity Purification (TAP); T7 epitope; tetracysteine tag (TC tag); Thioredoxin (Trx); TrpE; Ty tag; Ubiquitin; Universal; V5 tag; VSV-G or VSV-tag; and Xpress tag.

14. A vector comprising the nucleic acid of claim 8 or 9.

15. A method for enzymatically cleaving B-antigens from whole blood, erythrocytes, a donor tissue or a donor organ, the method comprising: (a) combining a purified alpha galactosidase enzyme of any one of claims 1-8 with the whole blood, the erythrocytes, the donor tissue or the donor organ comprising (i) type B antigen, or (ii) AB type antigen; (b) incubating the purified alpha galactosidase enzyme with the whole blood, the erythrocytes, the donor tissue or the donor organ; for a period of time sufficient to allow the enzymes to cleave B-antigens from the whole blood, the erythrocytes, the donor tissue or the donor organ.

16. The method of claim 15, wherein the purified alpha galactosidase enzyme is selected from one or more of: SEQ ID NO.:1; SEQ ID NO.:2; SEQ ID NO.:3; SEQ ID NO.:4; SEQ ID NO.:5; SEQ ID NO.:6; SEQ ID NO.:7; SEQ ID NO.:8; SEQ ID NO.:9; SEQ ID NO.:10; SEQ ID NO.:11; and SEQ ID NO.:

12.

17. The method of claim 15 or 16, wherein the purified alpha galactosidase enzyme is selected from one or more of: SEQ ID NO.:2; SEQ ID NO.:3; SEQ ID NO.:5; SEQ ID NO.:6; SEQ ID NO.:8; SEQ ID NO.:9; SEQ ID NO.:11; and SEQ ID NO.:12.

18. The method of claim 15, 16, or 17, wherein the purified alpha galactosidase enzyme is combined with a Galactosaminidase and a GalNAcDeacetylase, having A-antigen cleaving activity.

19. The method of claim 15, wherein the composition comprises: a purified enzyme having alpha galactosidase activity consisting essentially of an amino acid sequence at least 90% identical to the sequence set forth in one of SEQ ID NOs:1-12.

20. The method of any one of claims 15-19, the method further comprising adding a crowding agent.

21. The method of claim 20, wherein the crowding agent is selected from one or more of: a dextran; a dextran sulfate; a dextrin; a pullulan; a poly(ethylene glycol); a Ficoll™; a hyper- branched glycerol; hydroxyethyl starch; polyvinyl alcohol; and an inert protein.

22. The method of any one of claims 15-21, the method further comprising washing the blood, the erythrocytes, the donor tissue or the donor organ to remove alpha galactosidase and the crowding agent.

23. The method of any one of claims 15-22, wherein the alpha galactosidase is capable of cleaving B-antigen using enzyme concentrations at or below 1µg / ml.

24. The method of any one of claims 15-23, wherein the alpha galactosidase has B-antigen cleaving activity at a pH between about 6.0 and about 8.

0.

25. The method of any one of claims 15-24, wherein the alpha galactosidase has B-antigen cleaving activity at temperatures between 4°C and 37°C.

26. The method of any one of claims 15-25, wherein the donor organ is a solid organ.

27. The method of claim 26, wherein the solid organ is selected from one of the following: lung; kidney; liver; heart; pancreas; skin; uterus; thymus; and intestine.

28. The method of claim 27, wherein the solid organ is a lung.

29. The method of claim 28, wherein the purified enzyme having alpha galactosidase activity is mixed with an ex vivo lung solution and circulated through the lung, whereby the alpha galactosidase enzyme is in contact with the vasculature of the donor organ for a period of time sufficient to substantially clear the B-antigens from the vasculature of the lung.

30. The method of claim 29, wherein the time to clear the B-antigens from the vasculature of the lung is about 1 hour.

31. The method of any one of claims 15-30, wherein the method further comprises washing the donor organ to remove alpha galactosidase enzyme and cleaved B-antigens.

32. A blood collection and storage system, comprising: (a) a purified alpha galactosidase enzyme, wherein the purified enzyme having alpha galactosidase activity consisting essentially of an amino acid sequence at least 90% identical to the sequence set forth in one of SEQ ID NOs:1-12; and (b) a surface to which the enzyme is immobilized.

33. The system of claim 32, wherein the purified alpha galactosidase enzyme is selected from one or more of: SEQ ID NO.:1; SEQ ID NO.:2; SEQ ID NO.:3; SEQ ID NO.:4; SEQ ID NO.:5; SEQ ID NO.:6; SEQ ID NO.:7; SEQ ID NO.:8; SEQ ID NO.:9; SEQ ID NO.:10; SEQ ID NO.:11; and SEQ ID NO.:

12.

34. The system of claim 32 or 33, wherein the surface is selected from one or more of the following: (a) a bead or microsphere; (b) a container; (c) a tube; (d) a column; or (e) a matrix.

35. A blood collection and storage apparatus, the apparatus comprising: (a) a surface; and (b) a purified alpha galactosidase enzyme, wherein the purified enzyme having alpha galactosidase activity consists essentially of an amino acid sequence at least 90% identical to the sequence set forth in one of SEQ ID NOs:1-12 immobilized to the surface.

36. The apparatus of claim 35, wherein the purified alpha galactosidase enzyme is selected from one or more of: SEQ ID NO.:1; SEQ ID NO.:2; SEQ ID NO.:3; SEQ ID NO.:4; SEQ ID NO.:5; SEQ ID NO.:6; SEQ ID NO.:7; SEQ ID NO.:8; SEQ ID NO.:9; SEQ ID NO.:10; SEQ ID NO.:11; and SEQ ID NO.:12.

37. The apparatus of claim 35 or 36, wherein the surface to which the enzyme is immobilized is selected from one or more of the following: (a) a bead or microsphere; (b) a container; (c) a tube; (d) a column; or (e) a matrix.

38. The apparatus of claim 37, wherein the container is a bag.

39. A perfusion fluid for enzymatically cleaving B-antigen from a donor organ or donor tissue comprising: (a) a purified alpha galactosidase enzyme, wherein the purified enzyme having alpha galactosidase activity consisting essentially of an amino acid sequence at least 90% identical to the sequence set forth in one of SEQ ID NOs:1-12; and (b) a solution.

40. The perfusion fluid of claim 39, wherein the purified alpha galactosidase enzyme is selected from one or more of: SEQ ID NO.:1; SEQ ID NO.:2; SEQ ID NO.:3; SEQ ID NO.:4; SEQ ID NO.:5; SEQ ID NO.:6; SEQ ID NO.:7; SEQ ID NO.:8; SEQ ID NO.:9; SEQ ID NO.:10; SEQ ID NO.:11; and SEQ ID NO.:

12.

41. The perfusion fluid of claim 39 or 40, wherein the purified alpha galactosidase enzyme is capable of cleaving B-antigen at or below 1µg / ml.

42. The perfusion fluid of any one of claims 39-41, wherein the purified alpha galactosidase enzyme has B-antigen cleaving activity at a pH between about 6.5 and about 7.

5.

43. The perfusion fluid of any one of claims 39-42, wherein the purified alpha galactosidase enzyme has B-antigen cleaving activity at a temperatures between 4°C and 37°C.

44. The perfusion fluid of any one of claims 39-43, wherein the solution is selected from: Steen™; Perfadex™; Perfadex Plus™; EuroCollins solution; Histidine-Tryptophan- Ketoglutarate (HTK) solution; University of Wisconsin solution (UW); Celsior solution; Kidney Perfusion solution (KPS-1); Kyoto University solution; IGL-1 solution; and Citrate solution.

45. A method for enzymatically cleaving B-antigens from whole blood, erythrocytes, a donor tissue or a donor organ, the method comprising: (a) combining a purified alpha galactosidase enzyme of any one of claims 1-8; and a purified Galactosaminidase and a purified GalNAcDeacetylase, having A-antigen cleaving activity; with whole blood, the erythrocytes, the donor tissue or the donor organ comprising (i) type B antigen, (ii) type A antigen, or (iii) AB type antigen; (b) incubating the purified alpha galactosidase enzyme with the whole blood, the erythrocytes, the donor tissue or the donor organ; for a period of time sufficient to allow the enzymes to cleave B-antigens and A-antigens from the whole blood, the erythrocytes, the donor tissue or the donor organ.

46. The method of claim 45, wherein the purified alpha galactosidase enzyme is selected from one or more of: SEQ ID NO.:1; SEQ ID NO.:2; SEQ ID NO.:3; SEQ ID NO.:4; SEQ ID NO.:5; SEQ ID NO.:6; SEQ ID NO.:7; SEQ ID NO.:8; SEQ ID NO.:9; SEQ ID NO.:10; SEQ ID NO.:11; and SEQ ID NO.:

12.

47. The method of claim 45 or 46, wherein the purified alpha galactosidase enzyme is selected from one or more of: SEQ ID NO.:2; SEQ ID NO.:3; SEQ ID NO.:5; SEQ ID NO.:6; SEQ ID NO.:8; SEQ ID NO.:9; SEQ ID NO.:11; and SEQ ID NO.:

12.

48. A perfusion fluid for enzymatically cleaving B-antigen and A-antigen from a donor organ or donor tissue comprising: (a) a purified alpha galactosidase enzyme, wherein the purified enzyme having alpha galactosidase activity consisting essentially of an amino acid sequence at least 90% identical to the sequence set forth in one of SEQ ID NOs:1-12; (b) a purified Galactosaminidase consisting essentially of an amino acid sequence at least 90% identical to the sequence set forth in one of SEQ ID NOs: 69-71 and a purified GalNAcDeacetylase consisting essentially of an amino acid sequence at least 90% identical to the sequence set forth in one of SEQ ID NOs: 72-74, having A-antigen cleaving activity; and (c) a solution.

Images