Enzymatic compositions for removal of carbohydrate antigens in donor organs, methods and uses associated therewith

Abstract

To provide perfusion fluids for enzymatically cleaving A-antigens from a donor organ, and methods for enzymatically cleaving A-antigens from a donor organ ex vivo.SOLUTION: The perfusion fluid comprises two enzymes, GalNAc deacetylase and galactosaminidase, and the perfusion fluid may further comprise a buffered extracellular solution and / or a crowding agent. Furthermore, the compositions described herein were found to have activity at temperatures and pH levels suitable for cell viability.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of U.S. Provisional Patent Application No. 62 / 719,272, filed August 17, 2018, entitled "Enzyme Compositions for Carbohydrate Antigen Cleavage, Related Methods, Uses, Devices, and Systems."

[0002] The present invention relates to the field of enzyme compositions. In particular, the present invention relates to an enzyme composition for cleaving antigens in donor organs, as well as methods and uses for cleaving antigens using said composition. [Background technology]

[0003] Accurate matching of blood types is a central requirement in transfusion medicine because the plasma of blood type A individuals contains antibodies against B antigens, and vice versa, which can result in complement activation and red blood cell (RBC) lysis following incompatible transfusions (Daniels 2010). These cell surface antigens are carbohydrate structures terminating in α-1,3-linked N-acetylgalactosamine (GalNAc) or galactose (Gal) for type A and type B blood, respectively. In contrast, type O red blood cells do not contain these terminal sugars and can be transfused universally (Garratty 2008). Therefore, blood banks must have an adequate supply of type O red blood cells for emergencies when a patient's blood type is unknown or unclear. However, supplies are often limited.

[0004] The concept of enzymatically removing GalNAc or Gal structures from A or B RBCs as a means of converting them to O was first proposed and demonstrated by Goldstein (Goldstein 1982; US 4,609,627 and CA 2,272,925). Using α-galactosidase from green coffee beans, B RBCs were successfully converted to O RBCs followed by transfusion (Kruskall 2000). However, the amount of enzyme required made this approach impractical. Conversion of A RBCs is more difficult, primarily because there are many subtypes of A RBCs that differ in their internal linkages (Clausen 1989). Similarly, α-galactosidase has been used to remove B RBC antigens (see, e.g., EP 2,243,793). Significant progress toward practical conversion, including A RBC conversion, was achieved by screening bacterial libraries for both A and B RBC conversion activity using a tetrasaccharide substrate. Two new families of glycosidases that exhibit high antigen-cleaving activity at neutral pH have been discovered (CAZy GH109 α-N-acetylgalactosaminidase and GH110 α-galactosidase (Liu 2007)). Both enzymes completely removed their respective antigens and converted the corresponding RBCs. However, a substantial amount of enzyme is required (60 mg enzyme / blood unit), especially for type A conversion, limiting further development. Enzymes that more efficiently remove carbohydrate antigens from cells would be useful. Summary of the Invention

[0005] The present invention is based, in part, on the surprising discovery that the combination of galactosaminidase and GalNAc deacetylase, as described herein, is orders of magnitude more efficient than previously identified A antigen-cleaving enzymes. For example, under certain conditions, some GalNAc deacetylase and galactosaminidase enzymes may be able to cleave A antigens at concentrations of 1 μg / ml or less. Furthermore, the cleavage efficiency of the enzyme combination is maintained at a pH suitable for maintaining red blood cell viability (i.e., a pH between about 6.5 and about 7.5). Furthermore, the enzymes were found to be active at temperatures between 4°C and 37°C, which is also suitable for blood collection, washing, and storage protocols. Furthermore, the efficiency of the enzymes is further improved by the addition of crowding agents (e.g., dextran). It is also understood that the same two-step removal process can be applied to donor organs.

[0006] However, it will be understood by those skilled in the art that if the donor organ is perfused for a longer period of time, more enzyme can be used to shorten the time the donor organ can be perfused, or less enzyme can be used.

[0007] According to one embodiment, a perfusion fluid for enzymatic cleavage of A antigen from a donor organ is provided, comprising: (a) a purified GalNAc deacetylase protein; and (b) a purified galactosaminidase protein.

[0008] According to further embodiments, a perfusion fluid is provided, the perfusion fluid comprising: (a) a purified GalNAc deacetylase protein selected from one or more of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:17, SEQ ID NO:23, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, and SEQ ID NO:35; and (b) a purified galactosaminidase protein selected from one or more of SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:36, and SEQ ID NO:37.

[0009] According to a further embodiment, a perfusion fluid is provided, the perfusion fluid comprising a purified enzyme having GalNAc deacetylase activity consisting essentially of an amino acid sequence at least 90% identical to one of SEQ ID NOs: 2, 4, 5, 17, 23, 29, 31, and 32-35, and a purified enzyme having galactosaminidase activity consisting essentially of an amino acid sequence at least 90% identical to one of SEQ ID NOs: 7, 9, 10, 19, 21, 36, and 37.

[0010] The enzyme can be selected from one or more of: (a) a purified GalNAc deacetylase protein that is a purified Flavonifractor plautii GalNAc deacetylase protein of SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 5; and (b) a purified galactosaminidase protein that is a purified Flavonifractor plautii galactosaminidase protein of SEQ ID NO: 7, SEQ ID NO: 9, and SEQ ID NO: 10. The enzyme can be selected from one or more of: (a) a purified GalNAc deacetylase protein that is a purified Clostridium tertium GalNAc deacetylase protein of SEQ ID NO: 17 and SEQ ID NO: 32; and (b) a purified galactosaminidase protein that is a purified Clostridium tertium galactosaminidase protein of SEQ ID NO: 19 and SEQ ID NO: 36. The GalNAc deacetylase and galactosaminidase can be capable of cleaving the A antigen at 1 μg / ml or less. The GalNAc deacetylase and galactosaminidase may have A antigen cleavage activity at a pH between about 6.5 and about 7.5. The GalNAc deacetylase and galactosaminidase may have A antigen cleavage activity at a temperature between 4°C and 37°C. The perfusion fluid may further comprise a buffered extracellular solution. The buffered extracellular 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.

[0011] According to further embodiments, there is provided a method for enzymatically cleaving type A antigens from a donor organ ex vivo, comprising the steps of: (a) perfusing a donor organ presenting type A antigens with a fluid comprising GalNAc deacetylase protein and galactosaminidase protein for a time sufficient to allow the enzymes to cleave type A antigens from the donor organ; or (b) incubating a donor organ presenting type A antigens with a fluid comprising GalNAc deacetylase protein and galactosaminidase protein for a time sufficient to allow the enzymes to cleave type A antigens from the donor organ.

[0012] The GalNAc deacetylase can be a purified protein selected from one or more of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:17, SEQ ID NO:23, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, and SEQ ID NO:35. The galactosaminidase can be a purified protein selected from one or more of SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:36, and SEQ ID NO:37.

[0013] A purified enzyme having GalNAc deacetylase activity can essentially comprise an amino acid sequence at least 90% identical to one of SEQ ID NOs: 2, 4, 5, 17, 23, 29, 31, and 32-35, and a purified enzyme having galactosaminidase activity can essentially comprise an amino acid sequence at least 90% identical to one of SEQ ID NOs: 7, 9, 10, 19, 21, 36, and 37.

[0014] The GalNAc deacetylase can be a purified Flavonifractor plautii GalNAc deacetylase protein of SEQ ID NO:4 or SEQ ID NO:5, and the galactosaminidase can be a purified Flavonifractor plautii galactosaminidase protein of SEQ ID NO:9 or SEQ ID NO:10.

[0015] The GalNAc deacetylase protein and the galactosaminidase protein can be present in a buffered extracellular solution. The buffered extracellular solution can 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. The donor organ can be a solid organ. The solid organ can be selected from one of the lung, kidney, liver, heart, pancreas, and intestine. The solid organ can be a lung.

[0016] The GalNAc deacetylase protein and galactosaminidase protein can be mixed with an ex vivo buffered extracellular lung solution and circulated through the lungs, whereby the GalNAc deacetylase protein and galactosaminidase protein contact the donor organ's vasculature for a time sufficient to substantially remove the A antigen from the lung's vasculature. The GalNAc deacetylase protein and galactosaminidase protein can be mixed with an ex vivo buffered extracellular kidney solution and circulated through the kidneys, whereby the GalNAc deacetylase protein and galactosaminidase protein contact the donor organ's vasculature for a time sufficient to substantially remove the A antigen from the kidney's vasculature. The GalNAc deacetylase protein and galactosaminidase protein can be mixed with an ex vivo buffered extracellular liver solution and circulated through the liver, whereby the GalNAc deacetylase protein and galactosaminidase protein contact the donor organ's vasculature for a time sufficient to substantially remove the A antigen from the liver's vasculature. The GalNAc deacetylase protein and galactosaminidase protein can be mixed with a buffered extracellular heart solution ex vivo and circulated through the heart, whereby the GalNAc deacetylase protein and galactosaminidase protein contact the vasculature of the donor organ for a time sufficient to substantially remove the A antigen from the vasculature of the heart.The GalNAc deacetylase protein and galactosaminidase protein can be mixed with a buffered extracellular pancreas solution ex vivo and circulated through the pancreas, whereby the GalNAc deacetylase protein and galactosaminidase protein contact the vasculature of the donor organ for a time sufficient to substantially remove the A antigen from the vasculature of the pancreas. The GalNAc deacetylase protein and the galactosaminidase protein can be mixed with ex vivo buffered extracellular intestinal solution and circulated through the intestine, whereby the GalNAc deacetylase protein and the galactosaminidase protein contact the vasculature of the donor organ for a time sufficient to substantially remove the A antigen from the intestinal vasculature.

[0017] The time to clear the A antigen from the vasculature can be about 1 hour. The time to clear the A antigen from the blood vessels can be less than 1 hour. The time to clear the A antigen from the vasculature is about 2 hours.

[0018] The method may further comprise washing the donor organ to remove GalNAc deacetylase, galactosaminidase, and cleaved A antigen. GalNAc deacetylase and galactosaminidase may cleave A antigen at 1 μg / ml or less. GalNAc deacetylase and galactosaminidase may have A antigen cleavage activity at a pH between about 6.5 and about 7.5. GalNAc deacetylase and galactosaminidase may have A antigen cleavage activity at a temperature between 4° C. and 37° C.

[0019] According to a further embodiment, there is provided a composition comprising a purified enzyme having GalNAc deacetylase activity consisting essentially of an amino acid sequence at least 85% identical to one of SEQ ID NOs: 2, 4, 5, 17, 23, 29, 31, and 32-35, and a purified enzyme having galactosaminidase activity consisting essentially of an amino acid sequence at least 85% identical to one of SEQ ID NOs: 7, 9, 10, 19, 21, 36, and 37.

[0020] According to a further embodiment, there is provided a composition comprising a purified enzyme having GalNAc deacetylase activity consisting essentially of an amino acid sequence at least 80% identical to one of SEQ ID NOs: 2, 4, 5, 17, 23, 29, 31, and 32-35, and a purified enzyme having galactosaminidase activity consisting essentially of an amino acid sequence at least 80% identical to one of SEQ ID NOs: 7, 9, 10, 19, 21, 36, and 37.

[0021] According to a further embodiment, there is provided a composition comprising a purified enzyme having GalNAc deacetylase activity consisting essentially of an amino acid sequence at least 75% identical to one of SEQ ID NOs: 2, 4, 5, 17, 23, 29, 31, and 32-35, and a purified enzyme having galactosaminidase activity consisting essentially of an amino acid sequence at least 75% identical to one of SEQ ID NOs: 7, 9, 10, 19, 21, 36, and 37.

[0022] The composition may comprise: (a) the purified GalNAc deacetylase and the purified galactosaminidase may be immobilized; (b) the purified GalNAc deacetylase may be immobilized; or (c) the purified galactosaminidase may be immobilized.

[0023] The immobilized enzyme may be bound to a surface, which may be selected from one or more of the following: (a) beads or microspheres, (b) containers, (c) tubes, (d) columns, and (e) matrices. The composition may further include a crowding agent, which may be selected from one or more of dextran, dextran sulfate, dextrin, pullulan, poly(ethylene glycol), Ficoll™, and inert proteins.

[0024] In a further embodiment, a purified enzyme is provided comprising the Flavonifractor proutii GalNAc deacetylase of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:5.

[0025] According to a further embodiment, there is provided a purified enzyme comprising the Flavonifracter platii galactosaminidase of SEQ ID NO:7, SEQ ID NO:9 or SEQ ID NO:10.

[0026] In a further embodiment, a purified enzyme is provided comprising the Clostridium tertium GalNAc deacetylase of SEQ ID NO: 17 or SEQ ID NO: 32.

[0027] In a further embodiment, there is provided a purified enzyme comprising the Clostridium tertium galactosaminidase of SEQ ID NO: 19 or SEQ ID NO: 36.

[0028] Protein tags include 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 acetyltransferase (CAT), and 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), HaloTag™, alternating histidine and glutamine tag (HQ-tag), alternating histidine and asparagine tag ( HN-tag), histidine affinity tag (HAT), horseradish peroxidase (HRP), HSV epitope, isopep tag (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), polyaspartic acid (Asp-tag), polycysteine ​​(Cys-tag), polyglutamic acid (Glu-tag), poly Histidine-rich tag (His-tag), polyphenylalanine tag (Phe-tag), Profinity eXact, Protein C, Rho1D4 tag, S1-tag, S-tag, Softag 1, Softag 3, Snoop Tag Jr, Snoop Tag, Spot Tag, SpyTag (Spy-tag), streptavidin-binding peptide (SBP), Staphylococcal protein A (Protein A), Staphylococcal protein G (Protein G), Strep Tag, streptavidin (SBP-tag), Strep Tag II, Sdy-tag, small ubiquitin-like modifier (SUMO),The tag may be selected from one or more of 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.

[0029] According to a further embodiment, there is provided a method for enzymatically cleaving A antigen from a donor organ, the method comprising the steps of: (a) mixing a GalNAc deacetylase protein and a galactosaminidase protein with a donor organ presenting type A antigen; and (b) perfusing the enzyme into the donor organ vasculature for a time sufficient for the enzyme to cleave the A antigen from the vascular lumen of the donor organ.

[0030] The method may further include adding a crowding agent. The crowding agent may be selected from one or more of dextran, dextran sulfate, dextrin, pullulan, poly(ethylene glycol), Ficoll™, hyperbranched glycerol, and inert proteins. The method may include perfusing the donor organ with an organ perfusion solution or organ preservation solution comprising the enzyme composition described herein.

[0031] The method may further comprise washing the donor organ to remove GalNAc deacetylase, galactosaminidase and / or crowding agents.

[0032] The GalNAc deacetylase and galactosaminidase may be capable of cleaving the A antigen at 1 μg / ml or less. The GalNAc deacetylase and galactosaminidase may have A antigen cleavage activity at a pH between about 6.5 and about 7.5. The GalNAc deacetylase and galactosaminidase may have A antigen cleavage activity at a temperature between 4° C. and 37° C.

[0033] GalNAc deacetylase and galactosaminidase may be capable of cleaving the A antigen at 100 μg / ml or less. GalNAc deacetylase and galactosaminidase may be capable of cleaving the A antigen at 90 μg / ml or less. GalNAc deacetylase and galactosaminidase may be capable of cleaving the A antigen at 80 μg / ml or less. GalNAc deacetylase and galactosaminidase may be capable of cleaving the A antigen at 70 μg / ml or less. GalNAc deacetylase and galactosaminidase may be capable of cleaving the A antigen at 60 μg / ml or less. GalNAc deacetylase and galactosaminidase may be capable of cleaving the A antigen at 50 μg / ml or less. GalNAc deacetylase and galactosaminidase may be capable of cleaving the A antigen at 40 μg / ml or less. GalNAc deacetylase and galactosaminidase may be capable of cleaving the A antigen at 30 μg / ml or less. GalNAc deacetylase and galactosaminidase may be capable of cleaving the A antigen at 20 μg / ml or less. GalNAc deacetylase and galactosaminidase may be capable of cleaving the A antigen at 15 μg / ml or less. GalNAc deacetylase and galactosaminidase may be capable of cleaving the A antigen at 14 μg / ml or less. GalNAc deacetylase and galactosaminidase may be capable of cleaving the A antigen at 13 μg / ml or less. GalNAc deacetylase and galactosaminidase may be capable of cleaving the A antigen at 12 μg / ml or less. GalNAc deacetylase and galactosaminidase may be capable of cleaving the A antigen at 11 μg / ml or less. GalNAc deacetylase and galactosaminidase may be capable of cleaving the A antigen at 10 μg / ml or less. GalNAc deacetylase and galactosaminidase may be capable of cleaving the A antigen at 9 μg / ml or less. GalNAc deacetylase and galactosaminidase may be capable of cleaving the A antigen at 8 μg / ml or less. GalNAc deacetylase and galactosaminidase may be capable of cleaving the A antigen at 7 μg / ml or less. GalNAc deacetylase and galactosaminidase may be capable of cleaving the A antigen at 6 μg / ml or less. GalNAc deacetylase and galactosaminidase may be capable of cleaving the A antigen at 5 μg / ml or less.GalNAc deacetylase and galactosaminidase may be capable of cleaving the A antigen at 4 μg / ml or less. GalNAc deacetylase and galactosaminidase may be capable of cleaving the A antigen at 3 μg / ml or less. GalNAc deacetylase and galactosaminidase may be capable of cleaving the A antigen at 2 μg / ml or less. GalNAc deacetylase and galactosaminidase may be capable of cleaving the A antigen at 1 μg / ml or less. GalNAc deacetylase and galactosaminidase may be capable of cleaving the A antigen at 0.9 μg / ml or less. GalNAc deacetylase and galactosaminidase may be capable of cleaving the A antigen at 0.8 μg / ml or less. GalNAc deacetylase and galactosaminidase may be capable of cleaving the A antigen at 0.7 μg / ml or less. GalNAc deacetylase and galactosaminidase may be capable of cleaving the A antigen at 0.6 μg / ml or less. GalNAc deacetylase and galactosaminidase may be capable of cleaving the A antigen at 0.5 μg / ml or less. GalNAc deacetylase and galactosaminidase may be capable of cleaving the A antigen at 0.4 μg / ml or less. GalNAc deacetylase and galactosaminidase may be capable of cleaving the A antigen at 0.3 μg / ml or less. GalNAc deacetylase and galactosaminidase may be capable of cleaving the A antigen at 0.2 μg / ml or less. GalNAc deacetylase and galactosaminidase may be capable of cleaving the A antigen at 0.1 μg / ml or less. GalNAc deacetylase and galactosaminidase may be capable of cleaving the A antigen at 0.09 μg / ml or less. GalNAc deacetylase and galactosaminidase may be capable of cleaving the A antigen at 0.08 μg / ml or less. GalNAc deacetylase and galactosaminidase may be capable of cleaving the A antigen at 0.07 μg / ml or less. GalNAc deacetylase and galactosaminidase may be capable of cleaving the A antigen at 0.06 μg / ml or less. GalNAc deacetylase and galactosaminidase may be capable of cleaving the A antigen at 0.05 μg / ml or less. GalNAc deacetylase and galactosaminidase may be capable of cleaving the A antigen at 0.04 μg / ml or less. GalNAc deacetylase and galactosaminidase may be capable of cleaving the A antigen at 0.03 μg / ml or less.GalNAc deacetylase and galactosaminidase may be able to cleave the A antigen at 0.02 μg / ml or less. GalNAc deacetylase and galactosaminidase may be able to cleave the A antigen at 0.01 μg / ml or less.

[0034] The GalNAc deacetylase and galactosaminidase may have A antigen cleavage activity at a pH between about 6.5 and about 7.5. The GalNAc deacetylase and galactosaminidase may have A antigen cleavage activity at a pH between about 6.0 and about 8.0. The GalNAc deacetylase and galactosaminidase may have A antigen cleavage activity at a pH between about 6.8 and about 7.8. The GalNAc deacetylase and galactosaminidase may have A antigen cleavage activity at a pH between about 6.9 and about 7.9. The GalNAc deacetylase and galactosaminidase may have A antigen cleavage activity at a pH between about 6.4 and about 7.8.

[0035] The GalNAc deacetylase and galactosaminidase may have A antigen cleavage activity at a temperature between 4°C and 37°C. The GalNAc deacetylase and galactosaminidase may have A antigen cleavage activity at a temperature between 3°C and 38°C. The GalNAc deacetylase and galactosaminidase may have A antigen cleavage activity at a temperature between 4°C and 40°C. The GalNAc deacetylase and galactosaminidase may have A antigen cleavage activity at a temperature between 4°C and 37°C. The GalNAc deacetylase and galactosaminidase may have A antigen cleavage activity at a temperature between 5°C and 37°C.

[0036] According to another embodiment, a purified enzyme is provided comprising the Flavonifractor proutii GalNAc deacetylase of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:5.

[0037] According to another embodiment, there is provided a purified enzyme comprising the Flavonifractor platii galactosaminidase of SEQ ID NO:7, SEQ ID NO:9 or SEQ ID NO:10.

[0038] According to another embodiment, a purified enzyme is provided comprising a purified Clostridium tertium GalNAc deacetylase and a galactosaminidase fusion protein of SEQ ID NO:14.

[0039] According to another embodiment, there is provided a vector comprising a nucleic acid described herein and a heterologous nucleic acid sequence.

[0040] According to another embodiment, the method may be performed in vitro or ex vivo. As used herein, ex vivo means that the method is performed outside of an organism. For example, ex vivo includes ex vivo lung perfusion (EVLP) and treatment of donated blood. As used herein, ex vivo refers to an experiment, measurement, or treatment performed in or on tissue or cells from an organism in an external environment with minimal or some change from the condition the tissue or cells (e.g., red blood cells or donor organs) were in when the tissue or cells were in vivo. [Brief explanation of the drawings]

[0041] [Figure 1] Figure 1 shows a schematic representation of the cell surface antigen glycan structures terminating in α-1,3-linked N-acetylgalactosamine (GalNAc) or galactose (Gal) for types A, H, and B, where the triangles indicate the cleavage points of α-acetylgalactosaminidase EmGH109 and α-galactosidase BfGal110.

[0042] [Figure 2]Figure 2 shows the deacetylase pathway for A antigen cleavage along with the corresponding mass spectrometry (MS) analysis, whereby Flavonifracter proutii (Fp) GalNAc deacetylase cleaves the acetyl group from the terminal α-N-acetyl-galactosamine of the A antigen (m / z -42), and the galactosaminide intermediate is then cleaved by Flavonifracter proutii (Fp) galactosaminidase (m / z -161).

[0043] [Figure 3] Figure 3 shows FACS analysis of A+ RBCs treated with different concentrations of EmGH109 or Flavonifractor proutii GalNAc deacetylase (FpGalNAc deacetylase) + Flavonifractor proutii galactosaminidase (FpGalNAc deacetylase), or treated at 37 °C for 1 h. Anti-H antibody (added secondary FITC label) and APC-labeled anti-A antibody were used for visualization. The area of ​​H antigen appearance is in the upper left box. Columns A–D compare EmGH109 and FpGalNAcDeAc + FpGalNAc at 5 μg / ml (A), 10 μg / ml (B), 50 μg / ml (C), and 50 μg / ml + dextran 40k (D).

[0044] [Figure 4] Figure 4 compares EmGH109 and FpGalNAcDeAc+FpGalNase at various enzyme concentrations and at various temperatures (i.e., 4°C, room temperature (RT), and 37°C) in the presence (■) and absence (◆) of dextran.

[0045] [Figure 5] Figure 5 shows HPAE-PAD analysis of A+B+ and O+ erythrocyte cleavage products and a comparison of the full-length Flavonifractor proutii GalNAc deacetylase (FpGalNAcDeAc) + Flavonifractor proutii galactosaminidase (FpGalNase) enzyme with the cleaved FpGalNAcDeAc + FpGalNase enzyme in A+ erythrocytes.

[0046] [Figure 6] FIG. 6 shows the pH profile for each of (A) FpGalNAc deacetylase and (B) Fp galactosaminidase.

[0047] [Figure 7] Figure 7 shows the conversion of A antigen to H antigen on A red blood cells analyzed via FACS for (A) A+ RBC control, (B) Flavonifractor proutii GalNAc deacetylase (FpGalNAcDeAc) + Flavonifractor proutii galactosaminidase (FpGalNase) (10 μg / mL), (C) FpGalNAcDeAc + Clostridium tertium (Ct) Ct5757_GalNase (10 μg / mL), and (D) FpGalNAcDeAc + Robinsoniella peoriensis (Rp) galactosaminidase (Rp1021) GalNase (10 μg / mL).

[0048] [Figure 8] FIG. 8 shows the effect of increasing doses of enzyme on A antigen removal from group A human red blood cells in different perfusion solutions (ie, PBS, Steen™, and Perfadex™).

[0049] [Figure 9] FIG. 9 shows the effect of increasing doses of enzyme in STEEN solution on type A human arteries, where the percentage of type A antigen is quantified from immunohistochemical analysis of biopsies taken from untreated (control), treated (treatment) type A arteries, and type O arteries as a negative control.

[0050] [Figure 10]Figure 10 shows the effect of 1 hour of enzyme treatment on ex vivo perfused human donor lungs. Immunohistochemical staining of biopsied human donor lungs revealed that type A antigen was not present in blood vessels when comparing pre-treatment images with post-treatment images of the right upper dependent (RUD), right upper independent (RUND), right middle independent (RMND), right middle dependent (RMD), right lower independent (RLND), and right lower dependent (RLD) regions of the lung.

[0051] [Figure 11] Figure 11 shows the effect of 3 hours of enzyme treatment on ex vivo perfused human donor lungs, where immunohistochemical staining of biopsied human donor lungs revealed that type A antigen was not present in blood vessels when comparing pre-treatment images with post-treatment images of the right upper dependent (RUD), right upper independent (RUND), right middle independent (RMND), right middle dependent (RMD), right lower independent (RLND), and right lower dependent (RLD) regions of the lung. DETAILED DESCRIPTION OF THE INVENTION

[0052] The following detailed description will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the invention, the drawings show embodiments of the invention. However, the invention is not limited to the precise arrangements, examples, and instrumentalities shown.

[0053] Terms not directly defined herein shall be understood to have the meaning commonly associated with them as understood in the art of the present invention.

[0054] As used herein, an "immobilized enzyme" is an enzyme attached to a surface, which may be an inert, insoluble material. Immobilization of enzymes can increase their resistance to changes in conditions such as pH, temperature, etc., and aid in their removal after use and for reuse of the enzyme.

[0055] Immobilization of enzymes may be achieved by a variety of methods (e.g., affinity tag binding, surface adsorption on glass, entrapment on resins, alginate beads or matrices, beads, fibers or microspheres, cross-linking to surfaces or other enzymes, and covalent binding to surfaces).

[0056] As used herein, "affinity tag binding" refers to the immobilization of an enzyme to a surface (e.g., a porous material using a non-covalent or covalent protein tag). Affinity tag binding has been used in protein purification and, more recently, in biocatalysis applications by EziG™ (ENGINZYME AB™, Sweden (e.g., PCT / US 1992 / 010113) and PCT / SE 2015 / 050108). Alternative systems for attaching active enzymes to surfaces are known in the art (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).

[0057] Protein tags are peptide sequences genetically grafted onto recombinant proteins, often removable by chemical or enzymatic means, and attached to proteins for a variety of purposes. The protein tags listed in Table A are intended to be examples and are in no way limiting. One type of protein tag is an affinity tag, which is added to a protein or peptide sequence to enable purification from crude biological sources (e.g., from expression system organisms) using affinity techniques or to facilitate immobilization of the "tagged" protein to a surface. Examples of affinity tags include chitin-binding domains (CBDs), maltose-binding protein (MBP), strep-tags, glutathione-S-transferase (GST), and polyhistidine (His-tags) that bind to metal matrices. Another type of protein tag is an epitope tag (e.g., V5-tag, Myc-tag, HA-tag, Spot-tag, NE-tag), which is a short peptide sequence selected to facilitate the generation of high-affinity antibodies and is often derived from a viral gene sequence to improve immunoreactivity. Epitope tags are also used to purify proteins and immobilize them on surfaces, but are particularly useful in Western blotting, immunofluorescence, and immunoprecipitation experiments. Yet another type of protein tag is a chromatography tag (e.g., a polyanionic amino acid such as the FLAG tag), which can be used to modify the chromatographic properties of a protein to aid in separation and purification or immobilization. Still other protein tags are solubilization tags (e.g., maltose-binding protein (MBP), glutathione S-transferase (GST), thioredoxin (TRX), and poly(NANP)) and fluorescent tags (e.g., green fluorescent protein (GFP)). Protein tags allow for specific enzymatic modifications, chemical modifications, or the binding of proteins to other components. However, depending on the type or number of tags added to a protein sequence, the original function of the protein, in this case, enzymatic function, may be impaired by the tag.Therefore, the protein tag must be selected to ensure that the activity of the enzyme is not impaired, or alternatively, the protein tag may be cleaved from the protein before use.

[0058] Table A: Representative protein tags JPEG2025131609000002.jpg150165JPEG2025131609000003.jpg180144JPEG20251316090 00004.jpg186138JPEG2025131609000005.jpg176142JPEG2025131609000006.jpg154148

[0059] Although the use of protein tags is exemplified in this application through the use of polyhistidine protein tags (His-tags) such as those set forth in SEQ ID NOs: 5, 10, 15, 17, 19, 21, 23, 25, 27, 29, and 31, one of skill in the art will readily appreciate that any number of other protein tags can be used to purify enzymes and / or bind enzymes to surfaces as described herein, depending on the purification method used and / or the surface to which the enzymes are bound. Such protein tags can be selected from any one or more of the protein tags listed in Table A, although other such protein tags are known in the art.

[0060] Additionally, one or more cleavage sites (e.g., the thrombin cleavage sites used in SEQ ID NOs: 15, 17, 19, 21, 23, 25, 27, 29, and 31) can be used to separate a protein tag from an enzyme or otherwise cleave the enzyme. Cleavage sites can be used to remove an N-terminal methionine, a signal peptide, and / or convert an inactive or non-functional protein to an active protein (i.e., a zymogen or proenzyme). Alternatively, cleavage sites may be used to separate two or more enzymes expressed in the same reading frame. Examples of enzymes that can cleave proteins or peptides and would have sequence-specific cleavage sites can be selected from one or more of the following: Arg-C proteinase, Asp-N endopeptidase, 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 (not before P, but at the C-terminus of [FYW]), chymotrypsin low specificity (not before P, but at the C-terminus of [FYWML]), clostripain (clostridiopeptidase B), CNBr, enterokinase, X a-factor, formic acid, glutamyl endopeptidase, granzyme B, hydroxylamine, iodobenzoate, LvsC, LvsN, NTCB (2-nitro-5-thiocyanbenzoic acid), neutrophil elastase, pepsin (pH 1.3), pepsin (pH > 2), proline endopeptidase, proteinase K, staphylococcal peptidase I, tobacco etch virus protease, thermolysin, thrombin, trypsin.

[0061] Those skilled in the art will understand that the combination of the active galactosaminidase enzyme and active GalNAc deacetylase enzyme described herein, which can efficiently cleave the A antigen, is important. Those skilled in the art will also understand that the addition of one or more cleavage sites and / or one or more protein tags is optional, and such modifications can be selected based on the specific expression system, purification system, and possible surface attachment strategy. Furthermore, other modifications to the galactosaminidase sequence and GalNAc deacetylase sequence are also possible as long as the cleavage activity of the A antigen is not significantly impaired. Furthermore, modifications to the galactosaminidase and GalNAc deacetylase sequence are possible as long as the cleavage activity of the A antigen is not significantly impaired. Modifications to the galactosaminidase and GalNAc deacetylase sequence can be deletions, insertions, and / or substitutions. Substitutions can be conservative or neutral substitutions. For example, the galactosaminidase sequence and GalNAc deacetylase sequence can share 90% or more sequence identity with the mature enzyme. For example, the galactosaminidase and GalNAc deacetylase sequences may share 85% or more sequence identity with the mature enzyme. For example, the galactosaminidase and GalNAc deacetylase sequences may share 75% or more sequence identity with the mature enzyme. Alternatively, the galactosaminidase and GalNAc deacetylase sequences may have up to 5, 10, 13, 15, 20, or 25% amino acid modifications.

[0062] As used herein, "adsorption to glass, alginate beads, or matrices" refers to the attachment of an enzyme to the exterior of an inert material. Generally, this type of immobilization does not occur by chemical reaction, and the active sites of the immobilized enzyme may be blocked by the surface to which it is adsorbed, which may reduce the activity of the adsorbed enzyme.

[0063] As used herein, "entrapment" refers to the entrapment of an enzyme within an insoluble bead or microsphere. However, entrapment can prevent the access of substrates and the egress of products. One example is the use of calcium alginate beads, which can be produced by reacting a mixture of sodium alginate solution and enzyme solution with calcium chloride.

[0064] As used herein, "crosslinking" refers to the covalent bonding of enzymes to one another to create a matrix consisting almost entirely of enzymes. When designing crosslinked enzyme reactions, ideally, the binding sites do not cover the active site of the enzyme, so that the activity of the enzyme is affected only by immobilization, not by blockage of the active site. Nevertheless, spacer molecules such as poly(ethylene glycol) can be used to reduce steric hindrance by the substrate.

[0065] As used herein, "covalently bound" refers to the attachment of an enzyme to an insoluble support or surface (e.g., silica gel) via a covalent bond. Due to the strength of the covalent bond between the enzyme and the support or surface, the enzyme is highly unlikely to detach from the support or surface.

[0066] As used herein, "crowding agent" refers to any polymer or protein that promotes the aggregation of macromolecules by concentrating enzymes on the cell surface to improve enzyme activity. Crowding agents can be, for example, dextran, dextran sulfate, dextrin, pullulan, poly(ethylene glycol), Ficoll™, hyperbranched glycerol, and inert proteins (Kuznetsova, IM et al. Int J Mol Sci. (2014) "What Macromolecular Crowding Can Do to a Protein" 15(12):23090-23140).

[0067] As used herein, "dextran" refers to a polysaccharide with a molecular weight of 1,000 daltons or greater and a linear backbone of α-linked d-glucopyranosyl repeating units. Dextrans are divided into three 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 an 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) linkages. Class 1 dextrans vary in molecular weight, spatial arrangement, type and degree of branching, and branching chain length, depending on the microbial production strain and culture conditions. Isomaltose and isomaltotriose are oligosaccharides with a 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 and α(1→3)-linked branches. Class 3 dextrans (mutans) have a backbone structure of consecutive α(1→3)-linked d-glucopyranosyl units with α(1→6)-linked branches.

[0068] As used herein, "pullulans" are structural polysaccharides produced primarily from starch by the fungus Aureobasidium pullulans, consisting of repeating α(1→6)-linked maltotriose (D-glucopyranosyl-α(1→4)-D-glucopyranosyl-α(1→4)-D-glucose) units, occasionally containing maltotetraose units.

[0069] As used herein, "dextrin" refers to D-glucopyranosyl units that begin with a single α(1→6) linkage and are linearly followed by α(1→4) linked D-glucopyranosyl units, with a chain length shorter than that of dextran.

[0070] As used herein, "Ficoll™" is a neutral, highly branched, high mass hydrophilic polysaccharide that is readily soluble in aqueous solutions.

[0071] As used herein, "perfusion" or "perfusing" refers to the perfusion of a fluid through an organ by circulating the fluid through blood vessels.

[0072] An important goal in organ preservation is to increase the number of available transplantable organs. Organs have typically been stored under refrigeration, which can be diffusion-limited, and therefore hypothermic perfusion systems have been developed. Furthermore, near-normothermic systems have also been used to enhance the functional preservation of solid organs, including the liver, lungs, heart, and kidneys. Numerous buffered extracellular solutions are used as perfusion or preservation solutions. Many buffered extracellular solutions are known, including 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 (Guibert, EE et al. (2011)). Many of these are commercially available, and modifications of these solutions will be apparent to those skilled in the art.

[0073] Various alternative embodiments and examples are described herein, which are illustrative and should not be construed as limiting the scope of the invention.

[0074] (material and method) Chemicals and commercially available enzymes used in this study were purchased from Sigma-Aldrich™ unless otherwise noted. Monosaccharide methylumbelliferyl glycoside was a generous gift from Dr. Hongming Chen, and A antigen subtype 1 penta-MU was a generous gift from Dr. David Kwan (Kwan et al. 2015).

[0075] Human fecal metagenomic library

[0076] Generation of human metagenomics fosmid libraries for blood group AB + Fresh human fecal samples were collected from healthy Asian male volunteers with HIV. Direct DNA extraction and fosmid library construction were performed according to the procedures described in the MoE protocol (Armstrong et al. 2017).

[0077] Fosmid library screening

[0078] 51 x 384 well AB + Blood fosmid library plates were thawed at room temperature and replicated in 384-well plates containing 50 μl of screening LB medium (12.5 μg / mL chloramphenicol, 25 μg / mL kanamycin, 100 μg / mL arabinose, 0.2% (v / v) maltose, 10 mM MgSO). The plates were incubated at 37°C for 18 h in a sealed container containing a water reservoir to prevent excessive evaporation. 45 μl of the reaction mixture (100 mM NaHPO, pH 7.4, 2% (v / v) Triton-X 100, 100 μM GalNAc-α-MU, 100 μM Gal-α-MU) was added onto the grown screening plates using a QFill™ device (Genetix™). The plates were then incubated in a sealed container at 37°C for 24 hours, and the fluorescence of each plate (e.g., 365 nm Em: 435 nm, sweep mode, gain 80) was measured via a Synergy H1 plate reader (BioTek™) at 1, 2, 4, 8, and 24 hours. For every well, a Z-score was calculated, given by the formula: Z-score = (median fluorescence) / standard deviation.

[0079] All positive hits above a certain threshold were rearranged onto a new 384-well plate, designated the "simple substrate hit" plate, and stored at -70°C. Two screening plates were replicated from the "simple substrate hit" plate and rescreened for either GalNAc-α-MU or Gal-α-MU activity to confirm and deconvolute the previously detected activity.

[0080] To determine which of the hits could cleave the A or B antigen structure, a coupled enzyme assay was used to cleave the A antigen subtype 1 tetra-MU at 50 μM or the B antigen subtype 1 tetra-MU at 50 μM. 1tetra- Their activity against MU was measured. A version of this binding assay was previously described by Kwan (Kwan et al. 2015). The assay was improved by using BgaC (Jeong 2009) instead of BgaA (Singh 2014) as the binding enzyme to detect cleavage of subtype 1A antigen. α-N-acetylgalactosaminidase and α-galactosidase cleave the terminal sugars, resulting in the cleavage of H antigen subtype 1A. tri- MU is released. Subsequently, α-fucosidase (AfcA (Katayarna 2004)), β-galactosidase (BgaC (Jeong 2009)), and β-hexosaminidase (SpHex (Williams 2002)) exo-cleave the remaining sugars until 4-methylumbelliferyl alcohol is released, which can be detected as an increase in fluorescence. To achieve this, 50 μg / mL of each enzyme was added to the reaction mixture. All positive hits above a certain threshold were rescreened three times, and a host cell strain containing the vector lacking the insert was used as a negative control. All confirmed hits were stored separately in LB medium (12.5 μg / mL chloramphenicol, 25 μg / mL kanamycin, 15% (v / v) glycerol, 0.2% (v / v) maltose, 10 mM MgSO4) at -70°C.

[0081] Fosmid hit sequencing

[0082] To isolate fosmid DNA for sequencing, a positive-hit fosmid glycerol stock was used to inoculate 5 mL of TB medium (12.5 μg / mL chloramphenicol, 25 μg / mL kanamycin, 100 μg / mL arabinose, 0.2% (v / v) maltose, 10 mM MgSO) and incubated overnight at 37 °C and 220 rpm. Fosmid isolation was performed using a GeneJet™ Plasmid Miniprep Kit (Thermo Fisher™). The isolated plasmid was purified from contaminating linear E. coli DNA using Plasmid-Safe™ ATP-dependent DNase (Epicentre™), followed by another purification using a GeneJet™ PCR Purification Kit (Thermo Fisher™). Concentrations were calculated using the Quant-iT™ dsDNAHS Assay Kit (Invitrogen™) on a Qbit™ fluorometer (ThermoFisher™). Expected DNA sizes were confirmed on a 1% agarose gel. Two nanograms of each fosmid was sent to the UBC Sequencing Center (Vancouver, BC, Canada) for complete fosmid sequencing. Each fosmid was individually barcoded and sequenced using an Illumina MiSeq™ system.

[0083] All raw Illumina MiSeq™ sequencing data was trimmed and assembled using Python scripts available on GitHub™ at https: / / github.com / hallamlab / FabFos. Briefly, reads were purified using Trimmomatic to remove adapters and low-quality sequences (Bolger 2014). These reads were screened for vector and host sequences using BWA (Li 2013) and then filtered to remove contaminants using Samtools™ and the bam2fastq script. These high-quality, purified reads were assembled using MEGAHIT with k-mer values ​​ranging between 71 and 241, increasing by 10 increments (Li 2015). These libraries often have coverage exceeding 20,000-fold, and to prevent the accumulation of sequencing errors that would interfere with proper sequence assembly, a minimum k-mer multiplicity was calculated based on 1% of the estimated fosmid coverage. Scaffolding was performed using minimus2 (Treangen 2011) outside of the Python script assembly, which generates multiple contigs. Parameterized commands can be found both in the documentation on the GitHub™ page and in the Python script itself.

[0084] Fosmid ORF prediction and hit validation

[0085] Fosmid ORFs were identified using the metagenomic version of Prodigal™ (Hyatt 2010) and compared to the CAZy™ database using BLASTP™ as part of the MetaPathways™ v2.5 software package (Konwar 2015). MetaPathways™ parameters were: length > 60, BLAST score > 20, blast score ratio > 0.4, E Value <1×10 -6 is.

[0086] All predicted ORFs annotated for members of the GH or CBM family (with known or suspected α-galactosidase and / or α-N-acetylgalactosaminidase activity) were cloned into the pET16b plasmid using the Golden Gate™ cloning strategy (Engler 2008), with primer sequences listed in Table B. Proteins were expressed in BL21(DE3) cells grown at 37°C and 220 rpm for 20 h in 10 mL of ZY5052 autoinduction medium (Studier 2005). Cells were harvested by centrifugation (4000 × g, 4°C, 10 min) and resuspended in 1 mL of lysis buffer (100 mM NaH2PO4, pH 7.4, 2% (v / v) Triton-X™ 100, 1x protease inhibitor EDTA-free [Pierce™]). Ligation assays (Kwan 2015) were performed using 50 μl of crude cell lysate from the candidate and 50 μl of assay buffer (100 mM NaH2PO4, pH 7.4, 50 μg / mL SpHex, 50 μg / mL AfcA, 50 μg / mL BgaC, 100 μM A antigen subtype 1tetra- The resulting mixture was mixed with 100 μM MU or 100 μM B antigen subtype 1 tetra-MU and incubated at 37°C. All reactions were performed in triplicate in black 96-well plates. Fluorescence (365 / 435 nm) was continuously monitored for 4 h using a Synergy™ H1 plate reader (BioTek™). Assays from crude extracts showing cleavage activity against A or B antigen were repeated, this time without the conjugated enzyme, and reaction products were isolated via HF Bond Elut C18 columns and analyzed by LC-MS and / or TLC. TLC was performed using TLC silica gel 60 F254 TLC plates (EMD Millipore Corp.™, Billerica, MA, USA).

[0087] Table B: Primer sequences JPEG2025131609000007.jpg211139

[0088] HPAE-PAD assay

[0089] The analysis of enzymatic release of galactosamine was performed using an HPAE-PAD (Dionex™) HPLC system. The cleavage activity of various proteins was tested against the following substrates: 7.5 μg / μL porcine stomach type II mucin dissolved in 100 mM NaHPO pH 7.4, 5 mM A antigen subtype 1, and 100 mM NaHPO pH 7.4. penta- MU was dissolved in 100 mM NaH2PO4, pH 7.4, and RBCs (50% hematocrit) from type A+, B+, and O- donors were dissolved in 1x PBS, pH 7.4. Samples containing 10 μg / mL of enzyme were incubated at 37°C for two hours and then stored at -80°C for further analysis. A small aliquot of the reaction (10 μl) was diluted in HO (100 μl) and analyzed on an HPAE-PAD instrument. Separation was performed on a CarboPACPA200™ (150 mm) column with a guard column, and detection was performed using a disposable gold on polytetrafluoroethylene (PTFE) electrode and a quadrupole waveform. Separation conditions were as follows: 100 mM sodium hydroxide and a 70 to 300 mM sodium acetate gradient over the first 10 minutes of separation. The eluent was held at the final gradient conditions for 1 minute and then returned to the initial conditions over the next 1 minute. The flow rate was 1.0 ml / min, and injections were performed every 27 min. Standards of the free sugars GalNAc, Gal, and GalN (10 μM) were applied to HPAE-PAD to determine the peak elution times for reference.

[0090] Kinetic assay

[0091] All kinetic assays using 4-methylumbelliferone as the leaving group were performed by measuring fluorescence. To avoid measurement errors due to the inner filter effect (Palmier 2007), a standard curve was used to verify the linear range of the fluorescent dye.

[0092] Fp galactosaminidase

[0093] Michaelis-Menten parameters for GalN antigen subtype 1 penta-MU and A antigen subtype 1 penta MU was measured in 100 mM NaH2PO4, pH 7.4, 37°C. The reaction mixture consisted of 3.4 nM Fp galactosaminidase (5.31 nM FpGalNase_truncA) and 0.1 mg / mL SpHex, AfcA, 0.2 mg / mL BgaC, and various substrate concentrations (5 μM to 2 mM) in a 100 μl volume. A series of four reactions, including a control (without Fp galactosaminidase), was performed as a duplicate. Fluorescence signals (365 / 435 nm) resulting from MU hydrolysis were monitored using a Synergy H1™ plate reader (BioTek™) and converted to concentrations using a standard MU concentration curve measured under identical reaction conditions. Initial velocities (μM / s) were measured and plotted using Grafit 7.0™ to determine kinetic parameters.

[0094] k cat / K M The parameters were GalN antigen subtype 1 / 2 / 4 tetra- MU and B antigen subtype 1 tetra MU was measured at pH 7.4 and 37°C. Reactions (total volume 100 μL) were performed in black 96-well plates. Binding assays were performed in 100 mM NaH2PO4 (pH 7.4) with 8.63 nM Fp galactosaminidase, 0.1 mg / mL SpHex, BgaC (BgaA for subtype 2), AfcA, and various concentrations of substrate (25 μM, 20 μM, 15 μM, 10 μM, 7.5 μM, 5 μM). A series of four reactions was performed with a control (no Fp galactosaminidase) as a replicate. Fluorescence signals (365 / 435 nm) resulting from MU release by hydrolysis were monitored using a Synergy H1™ plate reader (BioTek™) and converted to concentrations using a MU standard concentration curve measured under identical reaction conditions. Initial velocities (μM / s) were determined and plotted using Grafit 7.0™ to obtain k cat / K M (s -1 *mM -1 ) parameters were determined.

[0095] 863.2 nM Fp galactosaminidase (in 100 mM NaHPO, pH 7.4) or 369.9 nM Fp GH4 (in 50 mM Tris / HCl, pH 7.4, 100 μM NAD) with various substrate concentrations (10 μM to 5 mM) in a volume of 100 μl + Michaelis-Menten parameters were measured for GalN-α-pNP in a clear 96-well plate with 1 mM MnCl2 (in 1 mM MnCl2) at 37°C. Reactions were performed as a series of three reactions with two controls (no enzyme). Absorbance (at 405 nm) resulting from hydrolytic release of pNP was monitored by a Synergy H1™ plate reader (BioTek™) and converted to concentration using a p-nitrophenol standard concentration curve measured under identical reaction conditions. Initial velocities (μM / s) were measured and plotted in Grafit 7.0™ to determine kinetic parameters.

[0096] FpGalNac deacetylase

[0097] A antigen subtype 1 in 100 mM NaH2PO4, pH 7.4, 37°C penta- For MU, we measured the Michaelis-Menten parameters using the previously described binding assay (Kwan 2015). To detect cleavage of subtype 1 (later 4), we used BgaC (Jeong 2009) instead of BgaA (Singh 2014) as the β-galactosidase. Furthermore, we also measured the cleavage of A antigen subtype 1. penta-Because MU contains an additional galactose, the concentration of BgaC was increased to 0.2 mg / mL to compensate for the need to cleave both Gal-β-1,3-β-GlcNAc-β-1,3-Gal-β-MU and Gal-β-MU. Additionally, Fp galactosaminidase was included to allow for cleavage of galactosamine-containing intermediates. The 100 μl reaction setup contained 3 nM FpGalNAc deacetylase (4.52 nM FpGalNAcDeAc_D1ext, 3.55 nM FpGalNAcDeAc_D1+2) and 0.01 mg / mL Fp galactosaminidase, 0.1 mg / mL SpHex, AfcA, 0.2 mg / mL BgaC, and various concentrations of substrate (5 μM to 2.5 mM). A series of four reactions, including a control (without FpGalNAc deacetylase), were performed as replicates. The fluorescence signal (365 / 435 nm) resulting from MU release by hydrolysis was monitored using a Synergy H1™ plate reader (BioTek™) and converted to concentration using a MU standard concentration curve measured under identical reaction conditions. Initial rates (µM / s) were determined and plotted using Grafit 7.0 to determine kinetic parameters.

[0098] k cat / K M The parameters are A antigen subtype 1 / 2 / 4 tetra-MU was determined at pH 7.4 and 37°C. Reactions (100 μL total volume) were performed in black 96-well plates with 12 nM FpGalNAc deacetylase, 0.1 mg / mL SpHex, BgaC (BgaA for subtype II), and AfcA in 100 mM NaH2PO4 (pH 7.4) for binding assays, with varying substrate concentrations (25 μM, 20 μM, 15 μM, 10 μM, 7.5 μM, and 5 μM). A series of four reactions, including a control (no FpGalNAc deacetylase), were performed as replicates. Fluorescence signals (365 / 435 nm) resulting from MU release upon hydrolysis were monitored using a Synergy H1™ plate reader (BioTek™) and converted to concentrations using a MU standard concentration curve measured under identical reaction conditions. Initial velocities (μM / s) were determined and plotted with Grafit™ 7.0 to determine the kcat / KM (s-1*mM-1) parameter.

[0099] GH109 subtype dynamics

[0100] A antigen subtypes 1 / 2 / 4 tetra-The kcat / KM parameters were measured for MU at pH 7.4 and 37°C. Reactions (100 μL total volume) were performed in black 96-well plates. Binding assays were performed in 100 mM NaH2PO4, pH 7.4, with 86.02 nM BvGH109_1, 100.49 nM EmGH109, ​​80.52 nM BvGH109_2, and 87.4 nM BsGH109, ​​5 μM NAD+, 0.1 mg / mL each of SpHex, BgaC (BgaA for subtype 2), and AfcA, and various substrate concentrations (25 μM, 20 μM, 15 μM, 10 μM, 7.5 μM, and 5 μM). A series of four reactions, including a control (without α-N-acetylgalactosaminidase), were performed as replicates. The fluorescence signal (365 / 435 nm) resulting from MU release by hydrolysis was monitored by a Synergy H1™ plate reader (BioTek™) and converted to concentration using a MU standard concentration curve measured under identical reaction conditions. Initial rates (µM / s) were determined and plotted with Grafit 7.0™ to determine the kcat / KM (s-1 * mM-1) parameter.

[0101] Crystallographic analysis

[0102] Prior to crystallization, FpGalNAcDeAc_D1ext was digested with thrombin (Novagen™) at a concentration of 1 mg / mL overnight using the manufacturer's suggested protocol. The protein was then purified on a HisTrap FF column, and the flow-through was collected, buffer-exchanged into 10 mM Tris pH 8.0 + 75 mM NaCl, and concentrated to 12 mg / mL.

[0103] Crystallization

[0104] FpGalNAcDeAc_D1ext (12 mg / mL) was crystallized at a 1:1 protein:reservoir ratio using the hanging drop diffusion deposition method with a reservoir solution consisting of 0.2 M CaCl2, 0.1 M MES pH 6, 18% PEG4000, and 20 mM MnCl2. Rapid bromide immersion was used to derivatize the crystals for phasing, and they were prepared by transferring them to a solution of 1 M NaBr, 25% glycerol, 18% PEG4000, 20 mM CaCl2, and 0.1 M MES pH 6 for 30 seconds and flash-freezing them in liquid nitrogen. Crystal complexes with the blood group B antigen trisaccharide (B_tri) were prepared by preincubating the protein (12 mg / mL) with 10 mM B_tri for 2 hours, followed by setting up drops under the same conditions as above, but omitting MnCl2. Crystals were cryoprotected in a reservoir solution supplemented with 25% glycerol.

[0105] Data collection, phasing, and structure determination

[0106] The dataset was collected at the Canadian Light Source™. Data were integrated using XDS (Kabsch 2010) and scaled with Aimless™ (Evans 2013). Phasing and automated structure solution were performed using CRANK2™ (Skubak 2013) in the CCP4I2™ program suite (Potterton 2018). Structures were checked and refined using alternating cycles of Coot™ (Emsley 2004) and Refmac™ (Vagin 2004). The B_tri structural complex was solved by differential Fourier transform, and the ligands, as well as water and metal ions, were manually built in Coot™. Difference density maps revealed the presence of Mn in the apo structure. 2+ , Ca in the coordination structure 2+The presence of was confirmed. The model was validated by Coot™ and Molprobity™ (Chen 2010). The atomic coordinates and structure factors of the apo and B_tri complexes have been deposited in the Protein Data Bank (PDB) with the following accession numbers: Flavonifructor proutii GalNAc deacetylase protein sequence number: WP_009260926.1, and Flavonifructor proutii galactosaminidase protein sequence number: WP_044942952.1.

[0107] Active site mutagenesis

[0108] Based on structural information (not shown) and sequence alignments (not shown), FpGalNAcDeAc_D1min and FpGalNase_truncA were mutated using the QuickChange™ protocol (Zhang 2004) with the primers listed in Table B. The mutants were purified via NiNTA and HIC columns as described above. The structural integrity of all mutants was confirmed by CD spectroscopy, which showed that all tested enzymes were structurally similar to the wild-type. For mutants with relatively low activity, reactions were performed under the same conditions as those used for full kinetic determination. However, k cat / K M The substrate consumption method was used to measure the values ​​as previously described (Vocadlo 2002). Briefly, [substrate] <K M (K m At low substrate concentrations (equivalent to ~1 / 5-1 / 10 of the k cat / K M The value can be approximated by non-linearly fitting the reaction time course to a first-order curve and dividing by the enzyme concentration.

[0109] GH36 phylogenetic mapping

[0110] Reference sequences for GH36 were downloaded from the CAZy™ database using the SACCHARIS™ cazy_extract.pl script (Jones 2018). Reference trees were constructed and sequences were mapped to these trees using the phylogeny-based protein profiling software, TreeSAPP™ (available at https: / / github.com / hallamlab / TreeSAPP). Briefly, protein family domains were extracted from all full-length sequences downloaded from CAZy™ (Yin 2012) using HMMs from dbCAN. These sequences were then clustered at 70% sequence similarity using UCLUST™ to remove redundant sequence space and reduce the size of the tree (Edgar 2010). RAxML™ version 8.2.0 was used to construct the reference tree, “--autoMRE” determined when to terminate bootstrapping before 1000 replicates were performed, and the best protein model was selected by PROTGAMMAAUTO™ (Stamatakis2006 and Stamatakis2008).

[0111] Query sequences were then mapped to these reference trees using TreeSAPP™. Briefly, protein sequences were aligned to HMMs using hmmsearch™ and aligned regions were extracted (Eddy 1998). hmmalign™ was used to include new query sequences in the reference multiple alignment, and TrimAl™ removed non-conserved positions from the alignment file (Capella-Gutierrez 2009). RAxML™ was used to sort query sequences in the reference tree by insertion. Alignments for each query sequence were filtered and concatenated into a single Jplace™ file before displaying it in iTOL™ (Matsen 2012 and Letunic 2016).

[0112] RBC assay

[0113] Using a protocol approved by the University of British Columbia Clinical Ethics Committee, whole blood from healthy consenting donors was collected into citrate vacutainers. The tubes were spun at 1000 x g for 4 minutes at room temperature to separate RBCs, which were then washed three times with 1x PBS pH 7.4. For assays in the presence of dextran 40k, washed RBCs (200 μL, 10% hematocrit) were placed in the tubes, the supernatant partially removed, and replaced with 1x PBS pH 7.4 (final concentration 300 mg / mL) in the presence and absence of dextran 40k. Additionally, some assays were performed in 1x PBS pH 7.4 + 25% plasma or 100% plasma. The RBCs were carefully mixed and placed on an orbital shaker for 30 seconds. The diluted enzyme solution was then added, bringing the final volume to 200 μL. The tubes were vortexed very gently and placed on an orbital shaker at a set temperature for the desired time.

[0114] MTS Card

[0115] After the reaction, the RBCs were washed three times with excess 1x PBS pH 7.4 and analyzed using a Micro Typing System™ (MTS™) card (MTS™, Florida, USA). RBCs (12 μl, 5% hematocrit) suspended in diluent (MTS, Florida, USA) were carefully added to the minigel column, leaving a space between the blood and the minigel contents. The MTS card was centrifuged at 156 x g for 6 minutes at room temperature using a Beckman Coulter Allegra X-22R™ centrifuge with a modified sample holder as recommended. The degree of antigen removal from the RBC surface was assessed by the position of the RBCs in the minigel after spinning, according to the manufacturer's instructions. RBCs with high concentrations of surface antigens clumped together through interaction with the monoclonal antibodies present in the gel column and were unable to penetrate (MTS™ score 4). RBCs lacking surface antigens did not clump together and migrated to the bottom of the minigel (MTS score 0). RBCs that had undergone partial removal of surface antigens migrated to positions between these and were assigned a score between 0 (absent) and 4 (present) according to the manufacturer's instructions.

[0116] H antigen agglutination assay

[0117] To analyze the conversion of A antigen to H antigen after enzymatic treatment, washed A-ECO-RBCs were mixed with an equal volume of 2 μg / mL anti-H antibody (anti-blood group Hab antigen antibody [97-I]: cat no. ab24213 (Abcam™)), and the appearance of agglutination was monitored within a 30-minute time frame. RBCs that agglutinated with anti-H antibody were assigned a score between 0 (no agglutination within 1800 seconds) and 5 (agglutination within 120 seconds).

[0118] FACS

[0119] The enzyme-treated RBCs were washed twice with 1x PBS, pH 7.4. The 1% hematocrit ECO-RBCs were treated with 1 / 100 APC-anti-A antibody (Alexa Fluor™ 647 mouse anti-human blood group A: cat no. 565384, BD Pharmingen™) and / or anti-H antibody (anti-blood group Hab antigen antibody [97-I]: cat no. ab24213, Abcam™) at room temperature for 30 minutes, followed by washing twice with 1x PBS, pH 7.4. For detection of anti-H antibody, a secondary FITC-labeled antibody (goat F(ab')2 anti-mouse IgM mu chain (FITC): cat no. ab5926, Abcam™) was used at a concentration of 1 / 500. Data were evaluated using a flow cytometer (CytoFLEX™ (Beckman Coulter™)) after reconstitution in 1x PBS pH 7.4 (hematocrit 1%).

[0120] Enzyme adsorption and antigenicity

[0121] To test whether the enzymes could be easily removed from RBCs after treatment, we evaluated their adsorption potential. Pacific Blue-labeled FpGalNAc deacetylase and FpGalNase (F / P = 1) were incubated with RBCs at 37°C for 1 hour, and after several washes, residual fluorescence was measured using a flow cytometer (CytoFLEX™ (Beckman Coulter™)).

[0122] Antigenicity was tested by incubating RBCs with 50 μg / mL of each enzyme and mixing the enzyme-treated RBCs with allogeneic or autologous serum to observe the possibility of agglutination. Additionally, to assess potential anti-IgG-C3d exposure, the treated RBCs were tested with anti-IgG-C3d MTS™ cards (MTS™, Florida, USA). The incubation time was 30 minutes at 37°C.

[0123] Synthesis of antigen subtypes

[0124] The synthesis of A and B antigen subtype 1 / 2 / 4 tetra-MU was performed using a modified protocol described by Kwan (Kwan et al. 2015).

[0125] Two-step H antigen subtype 1 / 2 / 4 tri-MU synthesis

[0126] All three syntheses were performed at a 20 mg GalNAc-α-MU / GlcNAc-α-MU scale in 10 mL of 50 mM Tris / HCl, 200 mM NaCl, pH 7.4, 10 mM MnCl, 50 U alkaline phosphorylase, 1.5 equivalents UDP-Gal, and 1.2 equivalents GDP-Fuc (scaled to the LacNAc-MU product). Depending on the desired product, various glycosyltransferases were added at a concentration of 100 μg / mL: CgtBS42 and Te2FT for subtype I, HP0826 and WbgL for subtype II, and LgtD and Te2FT for subtype IV. The reaction was carried out at 37 °C, and the progress was controlled by TLC (mobile phase EtAc:MeOH:HO ratio 6:2:1). 4-Methylumbelliferone was hydrolyzed from the compound with 10% HSO and detected by UV (360 nm). After no further product increase was observed, the reaction was applied to an HF Bond Elut C18 column, washed with several column volumes of 5% methanol, and the product was eluted with 25% methanol. The solvent was then removed under reduced pressure.

[0127] A antigen subtypes 1 / 2 / 4 tetra- MU synthesis

[0128] The final synthesis step is 10mg H antigen subtype 1 / 2 / 4 tri- A 500-mM scale reaction was performed in 5 mL of 50 mM Tris / HCl, 200 mM NaCl, pH 7.4, 10 mM MnCl2, 25 U alkaline phosphorylase, 1.5 equivalents of UDP-GalNAc, and 100 μg / mL BgtA at 37°C. After no further product growth was observed via TLC, the reaction was applied to an HF Bond Elut C18 column, washed with several column volumes of 5% methanol, and the product was eluted with 25% methanol. The solvent was then removed under reduced pressure. The final product was further purified on a 1.5 x 46 cm HW-40F size-extraction column and then lyophilized.

[0129] B antigen subtypes 1 / 2 / 4 tetra- MU synthesis

[0130] The final synthesis step is 10mgH antigen subtype 1 / 2 / 4 tri- The reaction was carried out at 37°C in 5 mL of 50 mM Tris / HCl, 200 mM NaCl, pH 7.4, 25 U alkaline phosphorylase, 1.5 equivalents UDP-Gal, and 100 μg / mL BoGT6a at MU scale. After no further product growth was observed, progress was followed via TLC. The reaction was applied to an HF Bond Elut C18 column, washed with several column volumes of 5% methanol, and the product was eluted with 25% methanol. The solvent was then removed under reduced pressure. The final product was further purified on a 1.5 x 46 cm HW-40F size-exclusion column and then lyophilized.

[0131] GalN antigen subtype 1 penta- MU synthesis

[0132] 10 mg of A antigen subtype 1 penta-MU was incubated with 1 μg / mL FpGalNAc deacetylase in 5 mL of 100 mM NaH2PO4 for 30 min at 37 °C. The reaction was then stopped by adding 1 mM EDTA. Complete conversion of the substrate was confirmed by TLC. The reaction mixture was applied to an HF Bond Elut C18 column, washed with several column volumes of 2% methanol, and the product was eluted with 10% methanol. The solvent was then removed under reduced pressure.

[0133] Protein purification

[0134] All proteins and truncations were cloned into pET16b or pET28a via Golden Gate™ cloning (Engler 2008) or PIPE cloning (Klock 2008). Primer sequences are listed in Table B.

[0135] Protein production for extended characterization was performed in BL21(DE3) cells grown for 20 h at 37°C and 220 rpm in 200 mL of ZY5052 autoinduction medium (Studier 2005) and inoculated into 100 μl of an overnight LB culture. Cells were harvested by centrifugation (4000 × g, 40 °C, 10 min) and resuspended in 10 mL of lysis buffer (50 mM Tris / HCl, 150 mM NaCl, 1% (v / v) glycerol, 40 mM imidazole, pH 7.4, 2 mM DTT, 1x protease inhibitor EDTA-free (Pierce™), 2 U Benzonase (Novagen™), 0.3 mg / mL lysozyme, 10 mM MgCl2), and then sonicated on ice (3 min pulse time; 5 s pulse, 10 s pause, 35% amplitude). After removal of cell debris by centrifugation (14000 × g, 4 °C, 30 min), the supernatant was collected and loaded onto a nickel affinity chromatography column (5 mL HisTrapHP™ column (GE™)) using a peristaltic pump. Elution was performed on an AEKTApurifier™ system (GE™) and monitored via SDS-PAGE with a 10-75% gradient of 50 mM Tris / HCl, 400 mM imidazole, pH 7.4, 2 mM DTT. Protein-containing fractions were identified and then pooled. Buffer exchange to 50 mM Tris / HCl, 150 mM NaCl, pH 7.4, 2 mM DTT and concentration were performed in Amicon Ultra-15 Centrifugal Filter Units™, MWCO 10 kDa (Millipore™).

[0136] FpGalNAc deacetylase, Fp galactosaminidase, and its cleavage products required a second round of purification. Amicon Ultra-15 Centrifugal Filter Units, MWCO 10 kDa (Millipore™), were used to exchange the buffer before loading the protein onto a hydrophobic interaction chromatography column (Phenyl Sepharose High-Performance Column 10 mL (Pharmacia Biotech™)). Column loading, washing, and elution (gradient 0-100%) were performed through an AEKTA purifier (GE™) using the following buffer conditions: FpGalNAc deacetylase, bind 1x PBS, 800 mM NH2PO4, pH 7.4, and elute 1x PBS, pH 7.4; Fp galactosaminidase, bind 25 mM Tris / HCl, 1 M NaCl, pH 7.4, and elute 25 mM Tris / HCl, pH 7.4. Protein-containing fractions were identified by SDS-PAGE and then pooled. Buffer exchange into 50 mM Tris / HCl, 150 mM NaCl, pH 7.4 and concentration was performed in Amicon Ultra-15 Centrifugal Filter Units™ MWCO 10 kDa (Millipore™).

[0137] Protein characterization

[0138] Optimal pH value

[0139] A antigen subtype 1 penta- MU and GalN antigen subtype 1 penta- The general pH ranges for activity of FpGalNAc deacetylase and Fp-galactosaminidase against MU were determined by the appearance of products on TLC plates at different pH values. Reactions were performed in 100 μl scale at 37°C with 50 μM substrate and 1 μg / mL enzyme in the appropriate buffer system. Buffers for pH 4-6 were based on 50 mM citric acid / sodium citrate buffer, for pH 6-8, 50 mM sodium phosphate buffer, and for pH 8-10, 50 mM glycine / sodium hydroxide buffer.

[0140] To determine the optimal pH value, 5 μg / mL of Fp galactosaminidase was incubated with 200 μM GalN-α-pNP in 100 μl of 50 mM sodium phosphate buffer over a pH range (5.8–8.0) and the absorbance (at 405 nm) resulting from pNP release was monitored by a Synergy H1™ plate reader (BioTek™) at 37°C for 1 h.

[0141] 5 μg / mL FpGalNAc deacetylase and 50 μM A antigen subtype Ipenta-MU were preincubated for 10 min at 37 °C in 25 mM sodium phosphate buffer at various pH ranges (5.8-10.0). The reaction was quenched with 100 mM sodium phosphate buffer pH 7.5, 100 μM EDTA, 5 μg / mL FpGalNAc, 50 μg / mL SpHex, 50 μg / mL AfcA, and 50 μg / mL BgaC in a final volume of 100 μl. The fluorescence signal (365 / 435 nm) resulting from MU release upon hydrolysis was monitored for 30 min at 37 °C using a Synergy H1™ plate reader (BioTek™).

[0142] Protein stability

[0143] FpGalNAc deacetylase and FpGalNase were stored in 1x PBS buffer, pH 7.4, at 4°C. After 2 and 12 weeks, the A antigen subtype 1 penta- As described for the pH optimum for MU, the enzyme activity was tested in a coupled enzymatic reaction of GalN-α-pNP with FpGalNAc deacetylase and FpGalNase.

[0144] FpGalNAc deacetylase inhibition

[0145] FpGalNAc deacetylase was tested against different potential inhibitors in a 96-well plate format as a binding assay. Reactions were performed in 100 μL at 37°C using 50 μM A antigen subtype 1 penta-MU and 5 μg / mL FpGalNAc deacetylase with 10 μg / mL Fp galactosaminidase, 50 μg / mL SpHex, 50 μg / mL AfcA, and 50 μg / mL BgaC in 100 mM NaH2PO4, pH 7.4. The following inhibitors were tested: EDTA (1, 10, 100 μM), Marimastat (1, 10, 100, 1000 μM), DMSO (2%, 4%), and Protease Inhibitor Cocktail EDTA-free (Pierce™) (1x, 2x, and 4x). Fluorescence (365 / 435 nm) was continuously monitored for 1 h using a Synergy H1™ plate reader (BioTek™). Additives showing strong effects were retested without bound enzyme, and product formation was analyzed by TLC.

[0146] Limited decomposition

[0147] To determine whether there is a smaller, more stable subdomain of Fp galactosaminidase, limited proteolysis was performed. Fp galactosaminidase was treated with thermolysin (protein:protease mass ratio 10:1) at various temperatures (20°C, 37°C, 42°C, 50°C, and 65°C) for 1.5 hours. Samples were then run on an SDS-PAGE gel, and a stable fragment was identified migrating at approximately 70 kDa (from the initial 118 kDa), with nearly complete degradation achieved at an incubation temperature of 50°C. This fragment was sent to the UBC Proteomics Core Facility for peptide identification and determined to be a C-terminal truncation of the full-length protein, with a cleavage site between amino acids 690 and 700.

[0148] Glycan array screening

[0149] For glycan array screening, 500 μg of FpGalNAcDeAc_D2ext was labeled with fluorescein isothiocyanate (FITC) at an F / P ratio of 1 using the Fluorotag™ FITC conjugation kit (Sigma™). Screening was performed on CFG's Protein-Glycan Interaction Core Facility™ version 5.3 printed arrays consisting of 600 glycans in six replicates for protein concentrations of 5 and 50 μg / mL. Analysis of binding motifs was performed using a web tool from Emory University (https: / / glycopattern.emory.edu / ).

[0150] Enzyme assay in buffered extracellular solution

[0151] A composition containing purified GalNAc deacetylase enzyme (SEQ ID NO: 5) and purified galactosaminidase enzyme (SEQ ID NO: 10) was used to test compatibility with the buffered extracellular solutions PBS, Steen™, and Perfadex™ at 37°C, 37°C, and 4°C, respectively. Human group A red blood cells (RBCs) were incubated in PBS, Steen™, and Perfadex™ with various doses of the enzyme composition to measure the ability of the enzyme to cleave A antigen from the red blood cells. 1% RBC solutions were treated with various doses of enzyme in PBS, Steen™, and Perfadex™ solutions, and the level of antigen removal at the end of treatment was analyzed by flow cytometry.

[0152] Immunohistochemical analysis of arterial biopsies

[0153] To test the effect of increasing doses of an enzyme composition comprising purified GalNAc deacetylase enzyme (SEQ ID NO: 5) and purified galactosaminidase enzyme (SEQ ID NO: 10), type A human arteries in STEEN™ solution were used to test, and the percentage of type A antigen was quantified by immunohistochemical analysis of biopsies taken from untreated (control), treated (treated) type A arteries, and type O arteries as a negative control. Area quantification software was used and normalized to the control group using the following formula: JPEG2025131609000008.jpg17152The residual positive levels of type A antigen quantified in the type O group explains the artifacts introduced during the process.

[0154] Enzyme treatment of human arteries was tested in human pulmonary arteries (static treatment). The dose was calculated as a unit of enzyme weight relative to the volume of STEEN™ solution. Arteries were biopsied, processed, and analyzed by immunohistochemistry using double staining with CD31 (which stains endothelial cells) and BTA (which stains blood group A antigen). Human arteries were treated with enzyme at both 1 μg / mL and 10 μg / mL for 4 hours. Immunohistochemical staining of arterial biopsies at 20x magnification is shown for arteries treated without enzyme (control) and with enzyme (treated). CD31 indicated the location of endothelial cells (blood vessels), and BTA indicated the location of blood group A antigen. BTA was present along with endothelial cells (CD31 positive) in untreated arteries, but was absent in treated arteries.

[0155] Human donor lung study

[0156] For the effect of 1 hour of enzyme treatment on ex vivo perfused human donor lungs, the expression levels of type A antigen were quantified using immunohistochemical analysis of lung tissue biopsies and area quantification software normalized to the pre-treatment biopsy using the following formula: JPEG2025131609000009.jpg26153

[0157] The effects of 1-hour and 3-hour enzyme treatments (i.e., an enzyme composition containing purified GalNAc deacetylase enzyme (SEQ ID NO: 5) and purified galactosaminidase enzyme (SEQ ID NO: 10)) on ex vivo perfused human donor lungs were tested. Immunohistochemical staining of biopsied human donor lungs was imaged at 20x magnification to measure the effects of lungs treated with the enzyme composition. CD31 indicates the location of endothelial cells (blood vessels). BTA indicates the location of blood group A antigen. Pre-treatment images showed that blood group antigens were localized in both blood vessels and airways. Post-treatment images of the right upper dependent (RUD), right upper independent (RUND), right middle independent (RMND), right middle dependent (RMD), right lower independent (RLND), and right lower dependent (RLD) regions of the lung showed that blood group A antigen was not present in blood vessels.

[0158] In this study, two separate ex vivo perfused human donor lungs were tested, and the results are shown in Figures 10 and 11, corresponding to 1 hour and 3 hours, respectively.

[0159] (Example) Example 1: Construction and screening of a metagenomic library

[0160] AB +We constructed a metagenomic library containing large (35-65 kb) fragments of DNA extracted from fecal samples provided by male donors with the same blood type. Such libraries contain multiple genes per bacterium, increasing the probability of expression of at least some of these genes and allowing expression of small "pathways" of multiple genes. Our library contained approximately 19,500 clones in 51 x 384-well plates, potentially representing approximately 800,000 genes. Therefore, initial screening of such a library with expensive A antigen substrates was impractical. Rather, we screened with the simple and sensitive fluorogenic substrates methylumbelliferyl α-glycosides of galactose and N-acetyl-galactosamine (Gal-α-MU and GalNAc-α-MU). Initial screening using a mixture of these two substrates yielded a subset of 226 hits. These were rescreened against each individual substrate, identifying 44 with GalNAcase and 166 with galactosidase activity. A second round of screening was performed on these hits using the A and B antigen tetrasaccharide glycoside substrates shown in Figure 1, along with a control group in which no substrate was present, using a coupled enzyme assay (Kwan 2015): only when the first Gal or GalNAc is cleaved can the coupled enzyme act and release MU. Eleven of these hits contained A antigen cleavage activity, one of which also cleaved B antigen, while six generated fluorescence in the absence of substrate, thus encoding pathways that generate unrelated fluorescent products.

[0161] Example 2: Sequencing and initial analysis of hits

[0162] Eleven plasmids were sequenced on an Illumina MiSeq™, and ORFs present in the CAZy™ database (http: / / www.cazy.org / ) (Lombard 2014) were identified using Metapathways™ software (Konwar 2015). Due to the considerable depth of currently available human microbiome sequencing, we were able to identify the organisms from which all fosmids originated. Eight of the eleven fosmids were derived from overlapping fragments of two Bacteroides genomes, allowing the sequences to be grouped into five clusters. The only gene common to all fosmids in Cluster B is the GH109 enzyme (B. vulgatus). Cluster A also contains GH109 (B. stercoris), but GH109 is the only CAZy gene found in other Bacteroides-derived fosmids (B. vulgatus). Fosmid N08 from the obligate anaerobe Flavonifracter proutii (Li 2015) contains three ORFs found within CAZy: an apparent glycan-binding module CBM32 and two potential glycoside hydrolases, GH36 and GH4. Finally, fosmid K05 from Collinsella sp., possibly Collinsella tanakaei, does not contain any CAZy-related ORFs. Here, generation of a sublibrary of fosmid K05 allowed the identification of an ORF with A-cleavage activity, which was later identified as GH36 (not shown).

[0163] Example 3: Analysis of GH109 enzymes

[0164] The GH109 family was discovered based on the A antigen cleavage activity of some of its members. These enzymes are unique in that they cleave NAD +The enzyme uses a GH4-dependent mechanism, which was first discovered in the enzyme described in Add Yip Ref (2004) J. Amer. Chem. Soc. 126, 8354-8355 (Varrot 2005 and Liu 2007). The three GH109 genes identified here were cloned with His tags after removal of the signal peptide and expressed in Escherichia coli BL21(DE3). These three proteins, BsGH109, ​​BvGH109_1, and BvGH109_2 (not shown), were purified along with the standard GH109 from Elizabethkingia meningosepticum (EmGH109) (Liu 2007), and kinetic parameters were determined for each. The three novel enzymes showed similar catalytic efficiency with each of the three A-subtype substrates tested, largely reflecting the kinetic parameters of the EmGH109 standard. In contrast, with an approved MTS card, + Unfortunately, only EmGH109 showed significant activity when its A antigen-cleaving activity was tested on RBCs. The test was performed in the presence of dextran 40K as a crowding agent, which has been shown to increase activity by concentrating the enzyme on the cell surface (Chapanian 2014). In its absence, even 150 μg / mL of EmGH109 was ineffective, whereas in the presence of 300 mg / mL of dextran 40K, 15 μg / mL of the enzyme was sufficient (see Figures 3 and 4). Previous studies have shown that low ionic strength also enhances the activity of EmGH109 on cells (Liu 2007). Therefore, EmGH109 is not effective in whole blood.

[0165] Example 4: Analysis of GH36 from fosmid K05 from Collinsella sp.

[0166] The GH36 protein identified in fosmid K05 (designated K05GH36) exhibited activity against GalNAc-α-MU and the A-antigen tetrasaccharide. This is consistent with its membership in the GH36 family, which primarily contains α-galactosidase and α-N-acetylgalactosaminidase enzymes and hydrolyzes the enzyme by a dual-displacement mechanism involving a covalent β-glycosyl-enzyme intermediate (Comfort 2007). Phylogenetic analysis placed the sequence within Cluster 4 of the GH36 subfamily (Fredslund 2011). Interestingly, this cluster also closely contains a characterized GH36 from Clostridium perfringens, which is also known to cleave the A-antigen structure (Calcutt 2002). However, when K05GH36's ability to remove A-antigen from erythrocytes was tested, its activity was disappointing, scoring only 3, even when used in combination with crowding agents.

[0167] Example 5: Analysis of fosmid N08 from Flavonifractor proutii

[0168] Because these novel enzymes offered no advantage, we focused on the N08 fosmid from F. proutii, specifically because its gene product cleaves both A and B antigens. We cloned the three CAZ-related genes, removed their signal peptide sequences, expressed them in E. coli BL21(DE3), and purified the resulting enzyme with yields up to 140 mg / L. Surprisingly, when we tested the individual purified proteins against A and B tetrasaccharide substrates, the only cleavage observed was of the B antigen by N08GH36; no cleavage of the A antigen was observed by either of them. Therefore, we tested pairwise combinations of these enzymes and were surprised to find that a mixture of N08CBM32 and N08GH36 rapidly cleaved the A antigen tetrasaccharide. TLC analysis of the reaction mixtures with the individual enzymes revealed that N08CBM32 catalyzed the conversion of A antigen to a more polar, but still UV-active, product, while subsequent addition of N08GH36 released a sugar product that comigrated with galactosamine along with the H antigen trisaccharide. MS analysis of the reaction mixtures indicated that N08CBM32 was an A antigen deacetylase, thus resulting in the reduction of the 42 m / z and more polar product, while N08GH36 was a galactosaminidase, demonstrating a novel activity in this family (Figure 2). This was further confirmed by high-performance anion-exchange chromatography (HPAE-PAD) analysis of the reactions (Figure 5), which showed that treatment of A antigen with both enzymes, but not the individual enzymes, released galactosamine. Similar results were obtained with a gastric mucin substrate, for which this enzyme likely evolved. Therefore, the two enzymes are hereafter referred to as FpGalNAc deacetylase (FpGalNAcDeAc) and Fp galactosaminidase (FpGalNase).

[0169] This pathway of A antigen degradation has not been characterized previously but, intriguingly, was suggested more than 50 years ago as an explanation for the so-called “acquired” B phenomenon, in which type A patients infected with Clostridium tertium underwent an apparent blood group change to type B (Gerbal 1975), as did forensic samples of human tissue submerged in the River Thames (Ref Judd and Annesley https: / / doi.org / 10.1016 / S0887-7963(96)80087-3,Transfusion medicine reviews(1996)10,111-117). This was probably because the anti-B antibodies used for typing could not distinguish between terminal Gal and GalN.

[0170] The third enzyme in the fosmid, GH4, was examined and found to hydrolyze Gal-α-pNP, GalN-α-pNP, and GlcN-α-pNP but not cleave A-antigen-based substrates. Thus, it does not appear to be directly involved in A-antigen conversion. However, these glycosaminidases represent novel activities within the GH4 family.

[0171] Example 6: Characterization of FpGalNAc deacetylase

[0172] Further bioinformatic analysis of this gene using Phyre2™ (Kelley 2015) revealed a previously unknown ~308 amino acid domain at the N-terminus and a ~145 amino acid CBM32 near the C-terminus, with a linker region in between. Cleavage analysis confirmed this basic structure, as all constructs containing the intact deacetylase domain were indeed catalytically active (Table 2). This protein is therefore classified as the founding member of a new glycosylated esterase family, CExx.

[0173] It has been demonstrated that all acetamido sugar deacetylases are metalloenzymes that require divalent metal ions (Blair 2005). Consistent with this, treatment with 100 μM EDTA almost completely abolished the enzyme activity, but Mn 2+ , Co 2+ , Ni 2+ or Zn 2+ The activity increased with the addition of acetyltransferase (A). Other inhibitors of (non-metallo)amidases were ineffective. The enzyme had a pH optimum near 8 (Figure 6), and its substrate specificity was narrow, restricted to different A subtypes and their shorter versions. However, within these subtypes, it was poorly differentiated, with only an approximately two-fold difference in specific activity between all of these subtypes (Table 2). This pH-dependence and specificity profile makes it ideal for RBC conversion, as all A subtypes are deacetylated, but none are otherwise.

[0174] The specificity of the CBM portion of the protein was investigated using the Consortium for Functional Glycomics (CFG) glycan array. The preferred targets were glycans with repeating N-acetyllactosamine (LacNAc) structures, as seen for the founding member of the CBM32 family, N-acetylglucosaminidase from Clostridium perfringens (Ficko-Blean 2006). However, unlike CBMs, our composition does not exhibit high-affinity binding to blood antigen structures. Repeating LacNAc structures are common components of cell surfaces as common components of complex and hybrid N-glycans, as well as some O-glycans and glycolipids (Cohen 2009). In our case, these likely serve as anchor points for the deacetylase domain to bind. This allows the catalytic domain to be in close proximity to the A antigen without competing with its own substrate. In support of this model, removal of the domain reduced activity in RBCs without affecting the rate of cleavage of soluble substrates (Table 2).

[0175] Example 7: Crystallographic analysis of FpGalNAc deacetylase

[0176] To provide structural insight into this novel enzyme activity, we subjected the truncated proteins to crystallization studies and found that FpGalNAcDeAc_D1ext produced crystals that diffracted to the best resolution. Solution analysis of this structure revealed a catalytic domain adopting a five-fold β-propeller configuration, with the active site containing a divalent metal ion coordinated by D100 and H252. Cocrystallization of the enzyme with the B antigen trisaccharide as an analog of the reaction product revealed its binding mode. At the base of the active site pocket, the non-reducing terminal galactosyl moiety, which distinguishes A and B antigens, forms hydrogen-bonding interactions with H97, E64, and two metal-coordinated water molecules. The remaining ligands are surface-exposed, and polar interactions between the fucosyl group and the side chains of S61 and D121 are identified. Because the C1-OH group of the reducing terminal galactosyl moiety is solvent-exposed, extension to the substrate (i.e., at GlcNAc) is easily accommodated by the enzyme. Modeling the N-acetyl group of the A-trisaccharide on this structure allowed us to rationally mutate neighboring amino acids that may be involved in substrate deacetylation. Residue E64 was found to be important for activity, as both mutants were inactive, suggesting a likely direct role in activating the nucleophilic water molecule (Table 1). Residues coordinating the divalent metal ions D100, Y315, and H252 were also found to be important, with all mutations resulting in approximately 5000-fold rate reductions consistent with their apparent role in divalent metal ion binding. By analogy with other acetamido sugar deacetylases, we propose that FpGalNAc deacetylase hydrolyzes the enzyme by polarizing the carbonyl, activating a water molecule for nucleophilic attack on the carbonyl, and forming a tetrahedral intermediate. Resolution of this intermediate is facilitated by proton donation to the sugar nitrogen atom by His100.

[0177] Table 1|A antigen Type2 tetra Specific activity of FpGalNAcDeAc_D1min and its mutants for cleavage of -MU JPEG2025131609000010.jpg77153

[0178] Example 8: Characterization of FpGalNAcDeAc and FpGalNase

[0179] Phylogenetic analysis of the sequence placed FpGalNase in a new subgroup (5) of the GH36 family (Fredslund 2011). A 390 amino acid catalytic domain is located at the center of this large (1079 amino acid) protein, with a potential carbohydrate-binding domain at the C-terminus. Removal of this C-terminal domain did not affect the kinetic parameters of the enzyme with soluble substrates (Table 2), but did affect the deacetylation of A. + This enzyme is specific for galactosamine-containing sugars and does not cleave GalNAc residues under any of the conditions tested. However, it has a fairly broad specificity for cleaving de-N-acetylated galactosaminides ranging from simple aryl glycosides GalN-α-pNP to higher ones. Indeed, (Table 2) k of the three A subtypes tested was significantly higher than that of the other two. cat / K M The values ​​were all similar to each other and to the values ​​for deacetylase. k for cleavage of B antigen cat / K M Although the specificity for deacetylated α-galactose constitutive substrates was more than 2,000-fold lower than that for the corresponding GalN antigen, it was nevertheless sufficient to generate positive hits in the original screen. This specificity for deacetylated α-galactose constitutive substrates, combined with its pH optimum of approximately 6.5–7.0, makes it suitable for use in blood group conversion with deacetylases (Figure 6).

[0180] Table 2 | Kinetic parameters of FpGalNAcDeAc and FpGalNase constructs toward different antigenic substrates JPEG2025131609000011.jpg149160

[0181] Example 9: Cleavage of A antigen from RBCs

[0182] A + , B + and O + Type A RBCs were incubated with FpGalNAcDeAc and FpGalNase, respectively, and as a mixture, and the released sugars were analyzed by HPAE-PAD ion chromatogram. Neither enzyme used individually released any sugar products. However, when the mixture of both enzymes was used, galactosamine was released from type A RBCs. + It was clearly released from the RBC, but B + or O + The FpGalNase was not released from the RBC surface, and showed high specificity only for the A antigen. This is very significant because it indicates that GalNAc is not released from the RBC surface under other circumstances. A truncated form of FpGalNase was also effective, although with slightly lower activity.

[0183] Next, we tested antigen removal from RBCs using industry-standard MTS™ cards. RBCs were loaded onto these antibody-binding columns and spun in a centrifuge. RBCs lacking the antigen migrated to the bottom of the column and were scored as 0, while untreated RBCs containing the corresponding antigen adhered to the top and were scored as 4, with intermediate scores used to rank the degree of antigen removal. Treatment with FpGalNase alone, at the concentrations shown in Table 3, did not remove A or B antigenicity, consistent with its inactivity toward GalNAc substrates and low activity toward Gal. Incubation with FpGalNAcDeAc removed antigenicity by converting acetamide to amine, weakening the binding of the anti-A antibody used. The minimum amount of enzyme required for complete antigen deacetylation was evaluated using FpGalNAcDeAc alone and in combination with FpGalNase, both with and without 300 mg / ml dextran as a crowding agent. Amounts of FpGalNAc up to 3 μg / ml were sufficient without dextran, but the inclusion of 300 mg / ml dextran reduced the required loading to 0.5 μg / ml (Table 3). The previous best enzyme, EmGH109, ​​was ineffective in the absence of dextran unless a low-salt buffer was used, but in the presence of dextran the minimum effective concentration was 15 μg / ml, a 30-fold higher loading. A version of FpGalNAcDeAc lacking the CBM was much less effective.

[0184] Table 3|A + , B + and A.B. + MTS card results when RBCs were treated with EmGH109, ​​FpGalNAcDeAc, and FpGalNase JPEG2025131609000012.jpg140156

[0185] Because the MTS™ card test does not assess complete conversion of A antigens, and because antibodies to detect GalN antigens were not available, we focused on detecting newly formed H antigens on treated RBCs. FpGalNase was functional at a concentration of only 5 μg / ml, resulting in a concomitant increase in H antigen levels with a loss of A antigens, as confirmed by FACS analysis shown in Figure 3. By measuring the agglutination time in the presence of anti-H antibodies, we were able to detect the agglutination time of several A antigens. + We demonstrated functionality of both enzymes on RBC donors and in whole blood reaction conditions that have not been achieved with other blood converting enzymes. Thus, this enzyme pair demonstrates A activity at enzyme loadings much lower than those required for the best conventional enzymes. + RBCs are converted to type O "universal donor" RBCs. However, before these RBCs are transfused into patients, it is recommended that all traces of the enzyme used in the conversion be removed, most likely by washing the cells after centrifugation, to avoid adverse immune reactions. To confirm that this can be achieved, fluorescently labeled samples of FpGalNAcDeAc and FpGalNase were used. + Processing of RBCs followed by FACS analysis confirmed that simple washing was indeed effective (Figure 3).

[0186] Although further characterization of the produced A-ECO RBCs may be useful to assess their full viability for use in transfusion medicine, the possibility of including the enzyme directly in plasma, potentially during collection of blood donations, may allow for easy and cost-effective implementation into existing automated routines for blood collection and storage. Notably, the stability of the enzyme was tested, as shown in Table 4. Table 4: Storage stability of galactosaminidase and GalNAc deacetylase JPEG2025131609000013.jpg92157

[0187] Example 10: GalNAc deacetylase and galactosaminidase fusion from Clostridium tertium

[0188] In our search for similar enzymes, we identified a novel Clostridium tertium natural fusion of galactosaminidase and GalNAc deacetylase linked by a CBM (GH36 domain-CBM-deacetylation domain). Initial studies showed that this enzyme cleaved A antigen on erythrocytes (using the same mechanism of first deacetylation, then galactosamine cleavage), but not as efficiently (i.e., similar to EmGH109). Although the Clostridium tertium deacetylation domain was not as efficient as the F. platii GalNAc deacetylase, when assisted by the F. platii GalNAc deacetylase, the Clostridium tertium galactosaminidase domain displayed activity on erythrocytes similar to that of F. platii galactosaminidase.

[0189] Example 11: Alternative GalNAc deacetylase and galactosaminidase enzymes

[0190] The data indicate that Clostridium tertium galactosaminidase (Ct5757_GalNAse) and Rp1021 have comparable enzymatic activity for the conversion of GalN antigen to H antigen (second reaction step).

[0191] Data were also collected for alternative GalNAc deacetylase and galactosaminidase enzymes, and the alternative enzymes were compared with Flavonifractor proutii GalNAc deacetylase and Flavonifractor proutii galactosaminidase. As shown in Table 5, MTS scores for anti-A antibodies on processed A RBCs were shown for a Clostridium tertium native fusion of galactosaminidase and GalNAc deacetylase, which requires the presence of dextran to effectively cleave the A antigen, and also shows good activity (Ct5757_DeAcase) when combined with Flavonifractor proutii galactosaminidase (FpGalNase). Also in Table 6, the data show that Robinsonia peoriensis (Rp) Rp3672 and Rp3671 can deacetylate A antigens on RBCs, but less efficiently than FpGalNAcDeAcase, and activity was only achieved in the presence of a crowding agent (i.e., dextran 40k).

[0192] Table 5: MTS scores of anti-A antibodies on processed A RBCs JPEG2025131609000014.jpg53153

[0193] Table 6: MTS scores of Robinsonia peoriensis (Rp) 3671 and 3672 JPEG2025131609000015.jpg36148

[0194] Figure 7 shows the conversion of A antigen to H antigen on A RBCs analyzed by FACS sorting for (A) A RBC control, (B) Flavonifractor proutii GalNAc deacetylase (FpGalNAcDeAc) + Flavonifractor proutii galactosaminidase (FpGalNase) (10 μg / mL), (C) FpGalNAcDeAc + Clostridium tertium (Ct) Ct5757_GalNase (10 μg / mL), and (D) FpGalNAcDeAc + Robinsonia peoriensis (Rp) galactosaminidase (Rp1021) GalNase (10 μg / mL). The data show that Clostridium tertium (Ct) Ct5757_GalNase and Robinsoniaella peoriensis (Rp) galactosaminidase (Rp1021) GalNase exhibit comparable enzymatic activity to Flavonifructor platii galactosaminidase for the conversion of GalN antigen to H antigen (second reaction step).

[0195] Example 12: Compatibility of Enzyme Compositions with Perfusion / Preservation Fluids

[0196] To confirm the compatibility of the enzyme composition with the EVLP system, we first tested the function of the enzymes (purified Flavonifractor proutii GalNAc deacetylase protein of SEQ ID NO: 5 and purified Flavonifractor proutii galactosaminidase protein of SEQ ID NO: 10) in organ perfusion / preservation fluids (STEEN™ and Perfadex™, XVIVO perfusion). Compatibility was assessed based on the ability of the enzyme composition to remove blood group A antigen from red blood cells in STEEN™ at 37°C or Perfadex™ at 4°C. Phosphate-buffered saline (PBS) at 37°C was used as a comparison group because PBS is one of the standard solutions used in blood processing. The temperatures tested for STEEN™ and Perfadex™ were based on the operating temperatures in clinical practice. Antigen removal levels were analyzed by flow cytometry. To help predict appropriate organ doses, dose escalation studies were performed in STEEN™ and Perfadex™ (see Figure 8). The dose units used throughout the study are defined as the weight of enzyme (μg) per volume of solution (mL).

[0197] The enzyme composition was fully compatible with STEEN™ and Perfadex™ perfusion / preservation fluids, and the perfusion / preservation fluids were shown to enhance the efficiency of the enzyme composition compared to PBS. The enzyme composition was able to remove over 90% of the antigens in STEEN™ and Perfadex™ at a total enzyme concentration of 1 μg / mL, while in PBS the same effect was achieved at a dose of 4 μg / mL (Figure 8).

[0198] Example 13: Static treatment of human arteries

[0199] To test the efficacy of the enzymes (purified Flavonifractor proutii GalNAc deacetylase protein of SEQ ID NO: 5 and purified Flavonifractor proutii galactosaminidase protein of SEQ ID NO: 10) at the tissue level, an in vitro model of human artery was used. Pulmonary arteries from the same human donor were divided into a control group (STEEN™ solution) and a treatment group (enzyme composition + STEEN™ solution) and statically incubated at 37°C for 4 hours. Biopsies were taken from both groups at the end of the incubation period. The enzyme composition was administered at 1 μg / mL and 10 μg / mL. Changes in blood group antigens were analyzed immunohistochemically. Serial sections of the biopsies were double-stained with CD31 (a marker for endothelial cells) to demonstrate the location of the vascular lining, and BTA demonstrated the expression of blood group antigens.

[0200] The expression level of blood group A antigen in the treated group was significantly reduced compared to the control group. The dose effects of 1 μg / mL and 10 μg / mL were similar in the treated arteries. The enzyme may also have an effect if the total enzyme concentration (dose) is lower than 1 μg / mL. Comparing the stained image of BTA with CD31 confirmed the disappearance of blood group antigen (Figure 9).

[0201] Example 14: Ex vivo perfusion of human lungs

[0202] The efficacy of enzyme-containing STEEN™ solution in removing histo-blood group antigens in human organs (e.g., lungs) was tested in a Toronto EVLP setting. Human donor lungs were evaluated by clinical ex vivo lung perfusion (EVLP) and determined to be unsuitable for transplantation and therefore suitable for testing the enzyme composition. After lung degradation, treatment was initiated by adding the enzyme composition (purified Flavonifractor prouti GalNAc deacetylase protein of SEQ ID NO: 5 and purified Flavonifractor prouti galactosaminidase protein of SEQ ID NO: 10) to the STEEN™ perfusion fluid. The dose was 1 μg / mL. Biopsies were taken before and after treatment. Changes in blood group antigen expression were analyzed immunohistochemically. Lung function and physiology were also monitored hourly throughout the experiment to ensure there were no acute adverse effects from the treatment.

[0203] Human lungs require 1.5 L of perfusate for single-lung EVLP and 2 L for double-lung EVLP. In the initial study of right single-lung EVLP (Figure 10), 1.5 mg of enzyme composition was added to the perfusion fluid at 1 μg / mL. Lungs were treated for 1 hour. Immunohistochemical analysis showed a significant reduction in blood group A antigen levels after treatment (Figure 10). Comparison of pre-treatment biopsy sections with double staining for blood group antigens and blood vessels revealed that the lung antigens were localized not only on the surface of the blood vessel walls but also in the airways. Comparison of double-stained post-treatment biopsies indicates effective removal of intravascular antigens.

[0204] In the second study (Figure 11), right single lung EVLPs were treated with 1.5 mg of the enzyme composition in STEEN™ perfusion fluid to a concentration of 1 μg / mL. Lungs were treated for 3 hours. Immunohistochemical analysis demonstrated a significant reduction in the expression level of blood group A antigen. Comparison of pre-treatment biopsies with double staining for blood group antigens and blood vessels revealed that pulmonary blood group antigens were localized not only on the vascular surface but also in the airways (Figure 11). Comparison of post-treatment biopsies with double staining demonstrated effective removal of intravascular antigens (Figure 11). No acute adverse effects on lung physiology or function were observed after initiation of enzyme treatment.

[0205] Results show that at a dose of 1 μg / mL, the enzyme is effective within 1 hour in perfused human lungs.

[0206] While various embodiments of the present invention are disclosed herein, many adaptations and modifications may be made within the scope of the present invention, according to the general knowledge of those skilled in the art. Such modifications include the substitution of known equivalents for any aspect of the invention to achieve the same result in substantially the same way. Numerical ranges are inclusive of the values ​​defining the range. As used herein, the term "comprising" is used 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 a plurality of such things. The citation of a reference herein is not an admission that such reference is prior art to embodiments of the present invention. The present invention includes all embodiments and variations substantially as described above with reference to the examples and drawings.

[0207] (array) The Flavonifracter proutii DNA sequence was modified from the naturally occurring sequence (GalNAc deacetylase 2311 / 2319nt / galactosaminidase 3228 / 3237nt). Specifically, the length of the sequence used for protein purification was varied, thereby removing the signal peptide and adding an N-terminal His tag via the vector backbone.

[0208] Informal Sequence Listing

[0209] SEQ ID NO: 2

[0210] Description: Flavonifractor proutii GalNAc deacetylase (protein sequence) MRNRRKAVSLLTGLLVTAQLFPTAALAADSSESALNKAPGYQDFPAYYSDSAHADDQVTHPDVVVLEEPWNGYRYWAVYTPNVMRISIYENPSIVASSDGVHWVEPEGLSNPIEPQPPSTRYHNCDADMVYNAEYDAMMAYWNWADDQGGGVGAEVRLRISYDGVHWGVPVTYDEMTRVWSKPTSDAERQVAD GEDDFITAIASPDRYDMLSPTIVYDDFRDVFILWANNTGDVGYQNGQANFVEMRYSDDGITWGEPVRVNGFLGLDENGQQLAPWHQDVQYVPDLKEFVCISQCFAGRNPDGSVLHLTTSKDGVNWEQVGTKPLLSPGPDGSWDDFQIYRSSFYYEPGSSAGDGTMRVWYSALQKDTNNKMVADSSGNLTIQAK SEDDRIWRIGYAENSFVEMMRVLLDDPGYTTPALVSGNSLMLSAETTSLPTGDVMKLETSFAPVDTSDQVVKYTSSDPDVATVDEFGTITGVSVGSARIMAETREGLSDDLEIAVVENPYTLIPQSNMTATATSVYGGTTEGPASNVLDGNVRTIWHTNYAPKDELPQSITVSFDQPYTVGRFVYTPRQNGTN GIISEYELYAIHQDGSKDLVASGSDWALDAKDKTVSFAPVEAVGLELKAIAGAGGFGTAAELNVYAYGPIEPAPVYVPVDDRDASLVFTGAWNSDSNGSFYEGTARYTNEIGASVEFTFVGTAIRWYGQNDVNFGAAEVYVDGVLAGEVNVYGPAAAQQLLFEADGLAYGKHTIRIVCVSPVVDFDYFSYVGE

[0211] SEQ ID NO:4

[0212] Description: Flavonifractor proutii GalNAc deacetylase (removes signal peptide protein sequence) ADSSESALNKAPGYQDFPAYYSDSAHADDQVTHPDVVVLEEPWNGYRYWAVYTPNVMRISIYENPSIVASSDGVHWVEPEGLSNPIEPQPPSTRYHNCDADMVYNAEYDAMMAYWNWADDQGGGVGAEVRLRISYDGVHWGVPVTYDEMTRVWSKPTSDAERQVADGEDDFITAIASPDRYDMLSP TIVYDDFRDVFILWANNTGDVGYQNGQANFVEMRYSDDGITWGEPVRVNGFLGLDENGQQLAPWHQDVQYVPDLKEFVCISQCFAGRNPDGSVLHLTTSKDGVNWEQVGTKPLLSPGPDGSWDDFQIYRSSFYYEPGSSAGDGTMRVWYSALQKDTNNKMVADSSGNLTIQAKSEDDRIWRIGYAE NSFVEMMRVLLDDPGYTTPALVSGNSLMLSAETTSLPTGDVMKLETSFAPVDTSDQVVKYTSSDPDVATVDEFGTITGVSVGSARIMAETREGLSDDLEIAVVENPYTLIPQSNMTATATSVYGGTTEGPASNVLDGNVRTIWHTNYAPKDELPQSITVSFDQPYTVGRFVYTPRQNGTNGIISEY ELYAIHQDGSKDLVASGSDWALDAKDKTVSFAPVEAVGLELKAIAGAGGFGTAAELNVYAYGPIEPAPVYVPVDDRDASLVFTGAWNSDSNGSFYEGTARYTNEIGASVEFTFVGTAIRWYGQNDVNFGAAEVYVDGVLAGEVNVYGPAAAQQLLFEADGLAYGKHTIRIVCVSPVVDFDYFSYVGE

[0213] SEQ ID NO:5

[0214] explanation: His tag Flavonifractor proutii GalNAc deacetylase (pET16a-protein sequence) MG HHHHHHHHHHSSGADSSESALNKAPGYQDFPAYYSDSAHADDQVTHPDVVVLEEPWNGYRYWAVYTPNVMRISIYENPSIVASSDGVHWVEPEGLSNPIEPQPPSTRYHNCDADMVYNAEYDAMMAYWNWADDQGGGVGAEVRLRISYDGVHWGVPVTYDEMTRVWSKPTSDAERQVADGEDDFITAIASPDRYDML SPTIVYDDFRDVFILWANNTGDVGYQNGQANFVEMRYSDDGITWGEPVRVNGFLGLDENGQQLAPWHQDVQYVPDLKEFVCISQCFAGRNPDGSVLHLTTSKDGVNWEQVGTKPLLSPGPDGSWDDFQIYRSSFYYEPGSSAGDGTMRVWYSALQKDTNNKMVADSSGNLTIQAKSEDDRIWRIGYA ENSFVEMMRVLLDDPGYTTPALVSGNSLMLSAETTSLPTGDVMKLETSFAPVDTSDQVVKYTSSDPDVATVDEFGTITGVSVGSARIMAETREGLSDDLEIAVVENPYTLIPQSNMTATATSVYGGTTEGPASNVLDGNVRTIWHTNYAPKDELPQSITVSFDQPYTVGRFVYTPRQNGTNGIISEY ELYAIHQDGSKDLVASGSDWALDAKDKTVSFAPVEAVGLELKAIAGAGGFGTAAELNVYAYGPIEPAPVYVPVDDRDASLVFTGAWNSDSNGSFYEGTARYTNEIGASVEFTFVGTAIRWYGQNDVNFGAAEVYVDGVLAGEVNVYGPAAAQQLLFEADGLAYGKHTIRIVCVSPVVDFDYFSYVGE

[0215] SEQ ID NO:7

[0216] Description: Flavonifractor plautii galactosaminidase

[0217] SEQ ID NO:9

[0218] Description: Flavonifractor plautii galactosaminidase (removal signal peptide protein sequence)

[0219] SEQ ID NO: 10

[0220] explanation: His tag Flavonifractor plautii galactosaminidase (pET16a-protein sequence) MG HHHHHHHHHH

[0221] SEQ ID NO: 12

[0222] Description: Clostridium tertium isolated protein sequence 099345757.1-Ct5757 (fusion of galactosaminidase and GalNAc deacetylase linked by CBM (original protein sequence))

[0223] SEQ ID NO: 14

[0224] Description: Clostridium tertium 5757 (Ct5757) isolated protein sequence excluding signal peptide (Identification number 099345757.1-Ct5757)

[0225] SEQ ID NO: 15

[0226] explanation: His tag and Clostridium tertium 5757 (Ct5757) fusion protein sequence expression construct (in pET28a vector) with a thrombin cleavage site MGSS HHHHHH

[0227] SEQ ID NO: 17

[0228] explanation: His tag and Clostridium tertium 5757 (Ct5757) GalNAc deacetylase protein sequence expression construct (in pET28a vector) with a thrombin cleavage site. MGSS HHHHHH SSGLVPRGSHSGQYWLVFQPDNDVLQTKTNPSSMKQSANNNPYNYNILPNSFPIGTGYNAYKGDVSFYATFKEASSQAIPQNSWALKYVDSEETTGENGRATNAFDGNNNTIWHTKYSGGNAAPMPHEIQIDLR GVYNINQINYLPRQDGGTNGTIKDYEVYLSLDGVNWGQPISKGTFESNSTEKIVKFNETKSRYVKLKALSEINNKQFTTVADLKVFGWEISKIEKPLQNAETYLNIPTYDGLNQSTHPDVKYFKNGWNGYKYWM IMTPNRTGSSVAENPSILASDDGINWEVPAGVTNPIAPMPQVGHNCDVDMIYNEATDELWVYWVESDDITKGWVKLIKSKDGVNWSSQQVVVDDNRAKYSTLSPSIIFKDNKYYMWSVNTGNSGWNNQSNKVEL RESDGVNWSNPTVVNTLAQDGSQIWHVNVEYIPSKNEYWAIYPAYKNGTGSDKTELYYAKSSDGVNWTTYKNPILSKGTSGKWDDMEIYRSCFVYDEDTNMIKVWYGAVSQNPQIWKIGFTENDYDKFIEGLTQ

[0229] SEQ ID NO: 19

[0230] explanation: His tag and Clostridium tertium 5757 (Ct5757) protein sequence galactosaminidase expression construct (in pET28a vector) with a thrombin cleavage site. MGSS HHHHHHSSGLVPRGSHYNLIDNISVEKLDTDISQANENVFLNGNGIALEVDNRGATCIYLVDENGVKTKATTSLDTADFSGYPIIGGQKIRDFVIISKNLEENINSILGVGNRLTIISKSSSTNLIRKIVFETSNSNPGAIYSTVSYKAESNDLLVDSFHENEYTMSLGQGPFLAYQGCADQQGANTIVNVTNGYNHNSGQNNYSVGVPFSYVYNSVGGIGIDDASTSRREFKLPIIGKDNTVSLGMEWNGQTLKKGAETAIGTSVITTTNGDYYSGLKSYAEVMKDKGISAPASIPDIAYDSRWESWGFEFDFTIEKIVNKLDELKAMGIKQITLDDGWYTYAGDWKLSPQKFPNGNADMKYLTDEIHKRGMTAILWWRPPVDGGINSKLVSEHPEWFIKNSQGNMVRLPGPGGNGGTAGYALCPNSEGSIQHHKDFVTVALEEWGFDGFKEDYVWGIPKCYDSSHKHSSLSDTLENQYKFYEAIYEQSIAINPDTFIELCN CGTPQDFYSTPYVNHAPTADPISRVQTRTRVKAFKAIFGDDFPVTTDHNSVWLPSALGTGSVMITKHTTLSSDRQYNKYFGLARDLELAKGEFIGNLYKYGIDPLESYVIRKGEDIYYSFYKDNSSYSGNIEIKGLDSNATYRIEDYVNNRVIARGVKGPTATINTSFTDNLLVRAIPDDTPAEVTTFDVGNNTILSSTDSGNSKYLNAVSTTLEKTATIDSLSIYIGNNSENGKLQIAIYDDNNGKPGTKKAYVEEFVPTKNSWNTKKVNSVNTLPSGQYWLVFQPDNDVLQTKTNPSSMKQSANNNPYNYNILPNSFPIGTGYNAYKGDVSFYATFKEASSQAIPQNSWALKYVDSEETTGENGRATNAFDGNNTIWHTKYSGGNAAPMPHEIQIDLRGVYNINQINYLPRQDGGTNGTIKDYEVYLSLDGVNWGQPISKGTFESNSTEKIVKFNETKSRYVKLKALSEINNKQFTTVADLKVFGWEISKIEK

[0231] SEQ ID NO: 21

[0232] explanation: His tag and Robinsonia peoriensis Rp1021 galactosaminidase protein expression construct (in pET28a vector) with a thrombin cleavage site. MGSS HHHHHH

[0233] SEQ ID NO: 23

[0234] explanation: His tag and a Ruthenibacterium lactatiformans R18755 GalNAc deacetylase protein sequence expression construct (in pET28a vector) with a thrombin cleavage site. MGSS HHHHHH SSGLVPRGSHEETDLLVNGGFETGDSTGWNWFNNAVVDSAAPHSGNYCAKVAKNSSYEQVVTVSPDTKYVLTGWAKSEGSSVMTLGVKNYGGQETFSATLSADYQQLAVTFTTG PNAQTATIYGYRQNSGSGAGYFDDVELTAVQDFAPYQPLANAIAPQAIPTYDGANQPTHPSVVKFEQPWNGYLYWMAMTPYPFNDGSYENPSIVASNDGENWIVPEGVSNPLAGT PSPGHNCDVDLVYVPASDELRMYYVEADDIISSRVKMISSRDGVHWSEPQVVMQDLVRKYSILSPSIEILPDGTYMMWYVDTGNAGWNSQNNQVKYRTSADGIKWSGAVTCTDF VQPGYQIWHIDVHYDTSSGAYYAVYPAYPNGTDCDHCNLFFAVNRTGKQWETFSRPILKPSTEGGWDDFCIYRSSMLIDDGMLKVWYGAKKQEDSSWHTGLTMRDFSEFMKILER

[0235] SEQ ID NO: 25

[0236] explanation: His tag and Robinsonia peoriensis Rp 3671 GalNAc deacetylase protein expression construct (in pET28a vector) with a thrombin cleavage site. MGSS HHHHHH

[0237] SEQ ID NO: 27

[0238] explanation: His tag and Robinsonia peoriensis Rp 3672 GalNAc deacetylase protein expression construct (in pET28a vector) with a thrombin cleavage site. MGSS HHHHHH

[0239] SEQ ID NO: 29

[0240] explanation: His tag and Robinsonia peoriensis Rp 3671 GalNAc deacetylase protein Rp3671 expression construct (in pET28 a vector) with a thrombin cleavage site. MGSS HHHHHHSSGLVPRGSHSPLSAAAESGTGTRLVKGQTGYLTEEQAIRNQEQTTEEREQKLTGEETAEVLMEGTKDSGIVQTEEVQTKEMQTEDAQTEEVQTEEMQTEDAQTKE VQTEEMQTEDAQTEEVQTKEEPAEETHMKEIQTQGTKKASDRNGKARVTEILEDAQDPANRIVYLSDLQWKSENHTVDSELPTRKDKSFGGGKITLKVDGTVTEFDK GIGTQTDSTIVYDLEGKGYTKFETYVGVDYSQKENIPGEVCDVKFRVKIDDKIVSETGVLDPLSNAVKISVNIPDTAKTLTLYADKVTETWSDHANWADAKFYQALP EPENVAFKKTVVTRKTSDNSEAPVNPDSAVNSSKAVDGVIDSSSYFDFGDQANSGAVRESLYMEVDLKGSYLLSDIQLWRYWKDGRTYAATAIVVAEDENFENAAVI YNSDTTGEIHHLGAGSDMLYAETESGKTFPVPENTKARYIRVYTYGVNGTSGVTNHIVELKVNAYVFGDEILPEKPDDSKIFPNAVNPLKLQGPGTNDQVTHPDVT VFDEPWNGYKYWMAYTPNKPGSSYFENPCIAASNDGVNWEFPAQNPVQPRYDSEIENQNEHNCDTDIVYDPVNDRLIMYWEWAQDEAVNGKTHRSEIRYRVSYDGIN WGVEDKTGVLMTGPTDHGCAIATEGERYSDLSPTVVYDKTEKIYKMWANDAGDVGYENKQNNKVWYRTSQDGISNWSDKTYVENFLGVNEDGLQMYPWHQDIQWVEE FQEYWALQQAFPAGSGPDNSSLRFSKSKDGLHWEPVSEKALITVGAPGTWDAGQIYRSTFWYEPGGAKGNGTFHIWYAALAEGQSHWDIGYTSANYADAMYKLTGSR

[0241] SEQ ID NO: 31

[0242] explanation: His tagand Robinsonia peoriensis Rp 3672 GalNAc deacetylase protein expression construct with a thrombin cleavage site (in pET28a vector) MGSS HHHHHH

[0243] SEQ ID NO: 32

[0244] Description: Clostridium tertium 5757 (Ct5757) GalNAc deacetylase protein sequence HSGQYWLVFQPDNDVLQTKTNPSSMKQSANNNPYNYNILPNSFPIGTGYNAYKGDVSFYATFKEASSQAIPQNSWALKYVDSEETTGENGRATNAFDGNNNTIWHTKYSGGNAAPMPPHEIQIDLRGVYNINQ INYLPRQDGGTNGTIKDYEVYLSLDGVNWGQPISKGTFESNSTEKIVKFNETKSRYVKLKALSEINNKQFTTVADLKVFGWEISKIEKPLQNAETYLNIPTYDGLNQSTHPDVKYFKNGWNGYKYWMIMTPN RTGSSVAENPSILASDDGINWEVPAGVTNPIAPMPQVGHNCDVDMIYNEATDELWVYWVESDDITKGWVKLIKSKDGVNWSSQQVVVDDNRAKYSTLSPSIIFKDNKYYMWSVNTGNSGWNNQSNKVELRES SDGVNWSNPTVVNTLAQDGSQIWHVNVEYIPSKNEYWAIYPAYKNGTGSDKTELYYAKSSDGVNWTTYKNPILSKGTSGKWDDMEIYRSCFVYDEDTNMIKVWYGAVSQNPQIWKIGFTENDYDKFIEGLTQ

[0245] SEQ ID NO: 33

[0246] Description: Ruthenibacterium lactatiformans R18755 GalNAc deacetylase protein sequence HEETDLLVNGGFETGDSTGWNWFNNAVVDSAAPHSGNYCAKVAKNSSYEQVVTVSPDTKYVLTGWAKSEGSSVMTLGVKNYGGQETFSATLSADYQQLAVTFTTGPNAQTAT IYGYRQNSGSGAGYFDDVELTAVQDFAPYQPLANAIAPQAIPTYDGANQPTHPSVVKFEQPWNGYLYWMAMTPYPFNDGSYENPSIVASNDGENWIVPEGVSNPLAGTPSPG HNCDVDLVYVPASDELRMYYVEADDIISSRVKMISSRDGVHWSEPQVVMQDLVRKYSILSPSIEILPDGTYMMWYVDTGNAGWNSQNNQVKYRTSADGIKWSGAVTCTDFVQ PGYQIWHIDVHYDTSSGAYYAVYPAYPNGTDCDHCNLFFAVNRTGKQWETFSRPILKPSTEGGWDDFCIYRSSMLIDDGMLKVWYGAKKQEDSSWHTGLTMRDFSEFMKILER

[0247] SEQ ID NO: 34

[0248] Description: Robinsonia peoriensis Rp3671 GalNAc deacetylase protein HSPLSAAAESGTGTRLVKGQTGYLTEEQAIRNQEQTTEEREQKLTGEETAEVLMEGTKDSGIVQTEEVQTKEMQTEDAQTEEVQTEEMQTEDAQTKEVQTEEMQT EDAQTEEVQTKEEPAEETHMKEIQTQGTKKASDRNGKARVTEILEDAQDPANRIVYLSDLQWKSENHTVDSELPTRKDKSFGGGKITLKVDGTVTEFDKGIGTQTD STIVYDLEGKGYTKFETYVGVDYSQKENIPGEVCDVKFRVKIDDKIVSETGVLDPLSNAVKISVNIPDTAKTLTLYADKVTETWSDHANWADAKFYQALPEPENV AFKKTVVTRKTSDNSEAPVNPDSAVNSSKAVDGVIDSSSYFDFGDQANSGAVRESLYMEVDLKGSYLLSDIQLWRYWKDGRTYAATAIVVAEDENFENAAVIYNSD TTGEIHHLGAGSDMLYAETESGKTFPVPENTKARYIRVYTYGVNGTSGVTNHIVELKVNAYVFGDEILPEKPDDSKIFPNAVNPLKLQGPGTNDQVTHPDVTVFD EPWNGYKYWMAYTPNKPGSSYFENPCIAASNDGVNWEFPAQNPVQPRYDSEIENQNEHNCDTDIVYDPVNDRLIMYWEWAQDEAVNGKTHRSEIRYRVSYDGINWG VEDKTGVLMTGPTDHGCAIATEGERYSDLSPTVVYDKTEKIYKMWANDAGDVGYENKQNNKVWYRTSQDGISNWSDKTYVENFLGVNEDGLQMYPWHQDIQWVEEF QEYWALQQAFPAGSGPDNSSLRFSKSKDGLHWEPVSEKALITVGAPGTWDAGQIYRSTFWYEPGGAKGNGTFHIWYAALAEGQSHWDIGYTSANYADAMYKLTGSR

[0249] SEQ ID NO: 35

[0250] Description: Robinsonia peoriensis Rp3672 GalNAc deacetylase protein

[0251] SEQ ID NO: 36

[0252] Description: Clostridium tertium 5757 (Ct5757) galactosaminidase protein sequence HYNLIDNISVEKLDTDISQANENVFLNGNIALEVDNRGATCIYLVDENGVKTKATTSLDTADFSGYPIIGGQKIRDFVIISKNLEENINSILGVGNRLTIISKSSSTNLIRKIVFETSNSNPGAIYSTVSYKAESNDLLVDSFHENEYTMSLGQGPFLAYQGCADQQGANTIVNVTNGYNHNSGQNNYSVGVPFSYVYNSVGGIGIDDASTSRREFKLPIIGKDNTVSLGMEWNGQTLKKGAETAIGTSVITTTNGDYYSGLKSYAEVMKDKGISAPASIPDIAYDSRWESWGFEFDFTIEKIVNKLDELKAMGIKQITLDDGWYTYAGDWKLSPQKFPNGNADMKYLTDEIHKRGMTAILWWRPPVDGGINSKLVSEHPEWFIKNSQGNMVRLPGPGGNGGTAGYALCPNSEGSIQHHKDFVTVALEEWGFDGFKEDYVWGIPKCYDSSHKHSSLSDTLENQYKFYEAIYEQSIAINPDTFIELCNCGTPQ DFYSTPYVNHAPTADPISRVQTRTRVKAFKAIFGDDFPVTTDHNSVWLPSALGTGSVMITKHTTLSSDRQYNKYFGLARDLELAKGEFIGNLYKYGIDPLESYVIRKGEDIYYSFYKDNSSYSGNIEIKGLDSNATYRIEDYVNNRVIARGVKGPTATINTSFTNLLVRAIPDDTPAEVTTFDVGNNTILSSTDSGNSKYLNAVSTTLEKTATIDSLSIYIGNNSENGKLQIAIYDDNNGKPGTKKAYVEEFVPTKNSWNTKKVNSVNTLPSGQYWLVFQPDNDVLQTKTNPSSMKQSANNNPYNYNILPNSFPIGTGYNAYKGDVSFYATFKEASSQAIPQNSWALKYVDSEETTGENGRATNAFDGNNTIWHTKYSGGNAAPMPHEIQIDLRGVYNINQINYLPRQDGGTNGTIKDYEVYLSLDGVNWGQPISKGTFESNSTEKIVKFNETKSRYVKLKALSEINNKQFTTVADLKVFGWEISKIEK

[0253] SEQ ID NO: 37

[0254] Description: Robinsonia peoriensis Rp1021 galactosaminidase protein sequence

[0255] <References> Kuznetsova, I.M et al. Int J Mol Sci.(2014) “What Macromolecular Crowding Can Do to a Protein” 15(12):23090-23140. Marcus, D,M. et al. Biochem (1964) “Immunochemical Studies on Blood Groups. XXXI. Destruction of Blood Group A Activity by an Enzyme from Clostridium tertium Which Deacetylates N-Acetylgalactosamine in Intact Blood Group Substances” (4)437-443. Daniels, G. and Reid M.E. Transfusion (2010) “Blood groups: the past 50 years.” 50(2):281-9. doi:10.1111 / j.1537-2995.2009.02456.x. Epub 2009 Nov 9 Vox Sang. 2011 Nov;101(4):327-32. doi:10.1111 / j.1423-0410.2011.01540.x. Epub 2011 Sep 6. Garratty, G. Vox Sang.(2008) “Modulating the red cell membrane to produce universal / stealth donor red cells suitable for transfusion.” 94(2):87-95. Epub 2007 Nov 22. Goldstein et al. Science (1982) “Group B erythrocytes enzymatically converted to group O survive normally in A,B,and O individuals.” 215(4529):168-70. US4609627;and CA2272925 Kruskall M.S. et al. Transfusion (2000) “Transfusion to blood group A and O patients of group B RBCs that have been enzymatically converted to group O.” 40(11):1290-8. Clausen, H and Hakomori, S. Vox Sang. (1989) “ABH and related histo-blood group antigens; immunochemical differences in carrier isotypes and their distribution.” 56(1):1-20. EP2243793 Liu, Q.P. et al. J Biol Chem.(2008)“Identification of a GH110 subfamily of alpha 1,3-galactosidases: novel enzymes for removal of the alpha 3Gal xenotransplantation antigen.” 283(13):8545-54. doi:10.1074 / jbc.M709020200. Epub 2008 Jan 28. PCT / US1992 / 010113; and 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 Jeong, J.K. et al. J Bacteriol.(2009) “Characterization of the Streptococcus pneumoniae BgaC protein as a novel surface beta-galactosidase with specific hydrolysis activity for the Galbeta1-3GlcNAc moiety of oligosaccharides.” 191(9):3011-23. doi:10.1128 / JB.01601-08. Epub 2009 Mar 6. Singh, A.K. et al. PLoS Pathog.(2014) “Unravelling the multiple functions of the architecturally intricate Streptococcus pneumoniae β-galactosidase, BgaA.” 10(9):e1004364. doi:10.1371 / journal.ppat.1004364. eCollection 2014 Sep. Katayarna, T. et al. J Bacteriol.(2004) “Molecular cloning and characterization of Bifidobacterium bifidum 1,2-alpha-L-fucosidase (AfcA), a novel inverting glycosidase (glycoside hydrolase family 95).” 186(15):4885-93. Williams, S.J. et al. J Biol Chem.(2002) “Aspartate 313 in the Streptomyces plicatus hexosaminidase plays a critical role in substrate-assisted catalysis by orienting the 2-acetamido group and stabilizing the transition state.” 277(42):40055-65. Epub 2002 Aug 8. Bolger, A.M. et al. Bioinformatics.(2014) “Trimmomatic: a flexible trimmer for Illumina sequence data.” 30(15):2114-20. doi: 10.1093 / bioinformatics / btu170. Epub 2014 Apr 1. Li 2013 Treangen, T.J. et al. Curr Protoc Bioinformatics (2011) “Next generation sequence assembly with AMOS.” Chapter 11:Unit 11.8. doi: 10.1002 / 0471250953.bi1108s33. Hyatt, D. et al. BMC Bioinformatics.(2010) “Prodigal: prokaryotic gene recognition and translation initiation site identification.” 11:119. doi:10.1186 / 1471-2105-11-119. Konwar, K.M. et al. Bioinformatics.(2015) “MetaPathways v2.5: quantitative functional, taxonomic and usability improvements.” 31(20):3345-7. doi:10.1093 / bioinformatics / btv361. Epub 2015 Jun 15. Studier, F.W. Protein Expr Purif.(2005) “Protein production by auto-induction in high density shaking cultures.” 41(1):207-34. Palmier M.O. and Van Doren S.R. Anal Biochem. (2007) “Rapid determination of enzyme kinetics from fluorescence: overcoming the inner filter effect.” 371(1):43-51. Epub 2007 Jul 18. Kabsch, W. Acta Crystallogr D Biol Crystallogr.(2010) “XDS” 66(Pt 2):125-32. doi: 10.1107 / S0907444909047337. Epub 2010 Jan 22. Evans, P.R. and Murshudov, G.N. Acta Crystallogr D Biol Crystallogr.(2013) “How good are my data and what is the resolution?” 69(Pt 7):1204-14. doi: 10.1107 / S0907444913000061. Epub 2013 Jun 13. Skubak, P. and Pannu, N.S. Nat Commun.(2013) “Automatic protein structure solution from weak X-ray data.” 4:2777. doi: 10.1038 / ncomms3777. Potterton, L. et al. Acta Crystallogr D Struct Biol.(2018) “CCP4i2: the new graphical user interface to the CCP4 program suite.” 74(Pt 2):68-84. doi: 10.1107 / S2059798317016035. Epub 2018 Feb 1. Emsley, P. and Cowtan, K. Acta Crystallogr D Biol Crystallogr.(2004) “Coot: model-building tools for molecular graphics.” 60(Pt 12 Pt 1):2126-32. Epub 2004 Nov 26. Vagin, A.A. et al. Acta Crystallogr D Biol Crystallogr.(2004) “REFMAC5 dictionary: organization of prior chemical knowledge and guidelines for its use.” 60(Pt 12 Pt 1):2184-95. Epub 2004 Nov 26. Chen, V.B. et al. Acta Crystallogr D Biol Crystallogr.(2010) “MolProbity: all-atom structure validation for macromolecular crystallography.” 66(Pt 1):12-21. doi: 10.1107 / S0907444909042073. Epub 2009 Dec 21. Zhang 2004 Vocadlo, D.J. et al. Biochemistry.(2002) “A case for reverse protonation: identification of Glu160 as an acid / base catalyst in Thermoanaerobacterium saccharolyticum beta-xylosidase and detailed kinetic analysis of a site-directed mutant.” 41(31):9736-46. Jones, D.R. et al. Biotechnol Biofuels.(2018) “SACCHARIS: an automated pipeline to streamline discovery of carbohydrate active enzyme activities within polyspecific families and de novo sequence datasets.” 11:27. doi: 10.1186 / s13068-018-1027-x. eCollection 2018. Yin, Y. et al. Nucleic Acids Res.(2012) “dbCAN: a web resource for automated carbohydrate-active enzyme annotation.” 40(Web Server issue):W445-51. doi: 10.1093 / nar / gks479. Epub 2012 May 29. Edgar, R.C. Bioinformatics.(2010) “Search and clustering orders of magnitude faster than BLAST.” 26(19):2460-1. doi: 10.1093 / bioinformatics / btq461. Epub 2010 Aug 12. Stamatakis, A. Bioinformatics. (2006) “RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models.” 22:2688-2690. doi:10.1093 / bioinformatics / btl446. Stamatakis, A. and Ott, M. Philos Trans R Soc Lond B Biol Sci. (2008) “Efficient computation of the phylogenetic likelihood function on multi-gene alignments and multi-core architectures.” 363(1512):3977-84. doi: 10.1098 / rstb.2008.0163. Eddy, S.R. Bioinformatics. (1998) “Profile hidden Markov models.” 14(9):755-63. Review. Capella-Gutierrez, S. et al. Bioinformatics. (2009) “trimAl: a tool for automated alignment trimming in large-scale phylogenetic analyses.” 25(15):1972-3. doi: 10.1093 / bioinformatics / btp348. Epub 2009 Jun 8. Matsen, F.A. et al. PLoS One. (2012) “A format for phylogenetic placements.” 7(2):e31009. doi: 10.1371 / journal.pone.0031009. Epub 2012 Feb 22. Letunic, I. and Bork, P. Nucleic Acids Res. (2016) “Interactive tree of life (iTOL) v3: an online tool for the display and annotation of phylogenetic and other trees.” 44(W1):W242-5. doi: 10.1093 / nar / gkw290. Epub 2016 Apr 19. Engler, C. et al. PLoS One. (2008) “A one pot, one step, precision cloning method with high throughput capability.” 3(11):e3647. doi: 10.1371 / journal.pone.0003647. Epub 2008 Nov 5. Kwan, D.H. et al. J Am Chem Soc. (2015) “Toward Efficient Enzymes for the Generation of Universal Blood through Structure-Guided Directed Evolution.” 137(17):5695-705. doi: 10.1021 / ja5116088. Epub 2015 Apr 24. The eleven fosmids were sequenced on an Illumina MiSeq TM and ORFs therein that are present in the CAZy TM database (http: / / www.cazy.org / )(Lombard 2014 Konwar, K.M. et al. Bioinformatics. (2015) “MetaPathways v2.5: quantitative functional, taxonomic and usability improvements.” 31(20):3345-7. doi: 10.1093 / bioinformatics / btv361. Epub 2015 Jun 15. Li, D. et al. Bioinformatics. (2015) “MEGAHIT: an ultra-fast single-node solution for large and complex metagenomics assembly via succinct de Bruijn graph.” 31(10):1674-6. doi: 10.1093 / bioinformatics / btv033. Epub 2015 Jan 20. enzymes from GH4 Add Yip, V.L. and Withers, S.G. J. Amer. Chem. Soc. (2006) “Mechanistic analysis of the unusual redox-elimination sequence employed by Thermotoga maritima BglT: a 6-phospho-beta-glucosidase from glycoside hydrolase family 4.” 126, 8354-8355 Chapanian, R. et al. Nat Commun. (2014) “Enhancement of biological reactions on cell surfaces via macromolecular crowding.” 5:4683. doi: 10.1038 / ncomms5683. Varrot, A. et al. J Mol Biol. (2005) “NAD+ and metal-ion dependent hydrolysis by family 4 glycosidases: structural insight into specificity for phospho-beta-D-glucosides.” 346(2):423-35. Epub 2005 Jan 7. Liu, Q.P. et al. Nat Biotechnol. (2007) “Bacterial glycosidases for the production of universal red blood cells.” 25(4):454-64. Epub 2007 Apr 1. Comfort, D.A. et al. Biochemistry (2007) “Biochemical analysis of Thermotoga maritima GH36 alpha-galactosidase (TmGalA) confirms the mechanistic commonality of clan GH-D glycoside hydrolases.” 46(11):3319-30. Epub 2007 Feb 27. Calcutt, M.J. et al. FEMS Microbiol Lett. (2002) “Identification, molecular cloning and expression of an alpha-N-acetylgalactosaminidase gene from Clostridium perfringens.” 214(1):77-80. Gerbal, A. Maslet, C. and Salmon, C. Vox Sang. (1975) “Immunological aspects of the acquired B antigen.” 28(5):398-403. Judd, W.J. and Annesley, T.M Transfusion medicine reviews (1996) “The acquired-B phenomenon.” 10, 111-117. Kelley, L.A. et al. Nat Protoc. (2015) “The Phyre2 web portal for protein modeling, prediction and analysis.” 10(6):845-58. doi: 10.1038 / nprot.2015.053. Epub 2015 May 7. Ficko-Blean, E. and Boraston, AB. J Biol Chem. (2006) “The interaction of a carbohydrate-binding module from a Clostridium perfringens N-acetyl-beta-hexosaminidase with its carbohydrate receptor.” 281(49):37748-57. Epub 2006 Sep 21. Cohen, M. et al. Blood. (2009) “ABO blood group glycans modulate sialic acid recognition on erythrocytes.” 114(17):3668-76. doi: 10.1182 / blood-2009-06-227041. Epub 2009 Aug 24. Fredslund, F. et al. J Mol Biol. (2011) “Crystal structure of α-galactosidase from Lactobacillus acidophilus NCFM: insight into tetramer formation and substrate binding.” 412(3):466-80. doi: 10.1016 / j.jmb.2011.07.057. Epub 2011 Jul 30. Guibert, E. E. et al. Transfus Med Hemother. (2011) “Organ Preservation: Current Concepts and New Strategies for the Next Decade” 38(2): 125-142.

Claims

1. 1. A perfusion fluid for enzymatic cleavage of A antigen from a donor organ, said perfusion fluid comprising: (a) a purified GalNAc deacetylase protein; (b) purified galactosaminidase protein; and , a perfusion fluid.

2. (a) the GalNAc deacetylase is a purified protein selected from one or more of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:17, SEQ ID NO:23, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, and SEQ ID NO:35; 2. The perfusion fluid of claim 1, wherein (b) the galactosaminidase is a purified protein selected from one or more of SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:36, and SEQ ID NO:

37.

3. 2. The perfusion fluid of claim 1, comprising a purified enzyme having GalNAc deacetylase activity consisting essentially of an amino acid sequence at least 90% identical to one of SEQ ID NOs: 2, 4, 5, 17, 23, 29, 31, and 32-35, and a purified enzyme having galactosaminidase activity consisting essentially of an amino acid sequence at least 90% identical to one of SEQ ID NOs: 7, 9, 10, 19, 21, 36, and 37.

4. 3. The perfusion fluid according to claim 1 or 2, wherein the perfusion fluid comprises: (a) the purified Flavonifractor proutii GalNAc deacetylase proteins of SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:5; (b) the purified galactosaminidase proteins are the purified Flavonifractor plautii galactosaminidase proteins of SEQ ID NO: 7, SEQ ID NO: 9, and SEQ ID NO: 10; A perfusion fluid comprising an enzyme selected from one or more of:

5. 3. The perfusion fluid according to claim 1 or 2, wherein the perfusion fluid comprises: (a) a purified Clostridium tertium GalNAc deacetylase protein of SEQ ID NO: 17 or SEQ ID NO: 32; (b) the purified galactosaminidase protein is a purified Clostridium tertium galactosaminidase protein of SEQ ID NO: 19 or SEQ ID NO: 36; and The perfusion fluid is selected from one or more of:

6. The perfusion fluid according to any one of claims 1 to 5, wherein the GalNAc deacetylase and galactosaminidase are capable of cleaving A antigen at 1 µg / ml or less.

7. 7. The perfusion fluid of claim 1, wherein the GalNAc deacetylase and galactosaminidase have A antigen cleavage activity at a pH between about 6.5 and about 7.

5.

8. 8. The perfusion fluid of claim 1, wherein the GalNAc deacetylase and galactosaminidase have A antigen cleavage activity at a temperature between 4°C and 37°C.

9. The perfusion fluid of any one of claims 1 to 8, wherein the perfusion fluid further comprises a buffered extracellular solution.

10. 10. The perfusion fluid of claim 9, wherein the buffered extracellular 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.

11. 1. A method for enzymatically cleaving A antigen from a donor organ ex vivo, comprising: (a) perfusing a donor organ presenting type A antigens with a fluid containing GalNAc deacetylase protein and galactosaminidase protein for a time sufficient to allow the enzymes to cleave type A antigens from the donor organ; or (b) incubating a donor organ presenting type A antigen with a fluid containing GalNAc deacetylase protein and galactosaminidase protein for a time sufficient to allow the enzymes to cleave the A antigen from the donor organ.

12. The method of claim 11, wherein the GalNAc deacetylase is a purified protein selected from one or more of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:17, SEQ ID NO:23, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, and SEQ ID NO:35, and the galactosaminidase is a purified protein selected from one or more of SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:36, and SEQ ID NO:

37.

13. 12. The method of claim 11, wherein the composition comprises a purified enzyme having GalNAc deacetylase activity consisting essentially of an amino acid sequence at least 90% identical to one of SEQ ID NOs: 2, 4, 5, 17, 23, 29, 31, and 32-35, and a purified enzyme having galactosaminidase activity consisting essentially of an amino acid sequence at least 90% identical to one of SEQ ID NOs: 7, 9, 10, 19, 21, 36, and 37.

14. 12. The method of claim 11, wherein the GalNAc deacetylase is a purified Flavonifractor platii GalNAc deacetylase protein of SEQ ID NO: 4 or SEQ ID NO: 5, and the galactosaminidase is a purified Flavonifractor platii galactosaminidase protein of SEQ ID NO: 9 or SEQ ID NO:

10.

15. The method of any one of claims 11 to 14, wherein the GalNAc deacetylase protein and the galactosaminidase protein are present in a buffered extracellular solution.

16. 16. The method of claim 15, wherein the buffered extracellular 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.

17. The method according to any one of claims 11 to 16, wherein the donor organ is a solid organ.

18. 18. The method of claim 17, wherein the solid organ is selected from one of the lung, kidney, liver, heart, pancreas, and intestine.

19. 19. The method of claim 18, wherein the solid organ is a lung.

20. 18. The method of claim 17, wherein the GalNAc deacetylase protein and the galactosaminidase protein are mixed with ex vivo buffered extracellular lung solution and circulated through the lungs, whereby the GalNAc deacetylase protein and the galactosaminidase protein contact the vasculature of the donor organ for a time sufficient to substantially remove A antigen from the pulmonary vasculature.

21. 21. The method of claim 20, wherein the time for A antigen clearance from the pulmonary vasculature is about 1 hour.

22. The method of any one of claims 11 to 21, further comprising washing the donor organ to remove GalNAc deacetylase, galactosaminidase and cleaved A antigen.

23. The method according to any one of claims 11 to 22, wherein the GalNAc deacetylase and galactosaminidase are capable of cleaving A antigen at 1 µg / ml or less.

24. 24. The method of any one of claims 11 to 23, wherein the GalNAc deacetylase and galactosaminidase have A antigen cleavage activity at a pH between about 6.5 and about 7.

5.

25. 25. The method of any one of claims 11 to 24, wherein the GalNAc deacetylase and galactosaminidase have A antigen cleavage activity at a temperature between 4°C and 37°C.

Images